Oral Presentations - Federation of Clinical Immunology Societies
Oral Presentations - Federation of Clinical Immunology Societies
Oral Presentations - Federation of Clinical Immunology Societies
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<strong>Clinical</strong> <strong>Immunology</strong> (2007) 123, S3–S128<br />
ABSTRACTS<br />
<strong>Oral</strong> <strong>Presentations</strong>: Friday, June 8<br />
#1201- Therapies to Induce Tolerance<br />
Prevention and Treatment <strong>of</strong> Autoimmune Arthritis<br />
Using IL-12 Gene-Silenced Dendritic Cells<br />
Friday, June 8<br />
10:45 am−11:05 am<br />
Wei-Ping Min, Scientist, University <strong>of</strong> Western Ontario,<br />
London, ON, Canada, Xiufen Zheng, Fellow, University <strong>of</strong><br />
Western Ontario, London, ON, Canada, Igor Popov, Fellow,<br />
University <strong>of</strong> Western Ontario, London, ON, Canada, Xusheng<br />
Zhang, Fellow, University <strong>of</strong> Western Ontario, London, ON,<br />
Canada, Mu Li, Fellow, University <strong>of</strong> Western Ontario,<br />
London, ON, Canada, Hongtao Sun, Fellow, University <strong>of</strong><br />
Western Ontario, London, ON, Canada, Motohiko Suzuki,<br />
Fellow, University <strong>of</strong> Western Ontario, London, ON, Canada,<br />
Costin Vladau, Student, University <strong>of</strong> Western Ontario,<br />
London, ON, Canada, Bertha Garcia, Pr<strong>of</strong>essor, University <strong>of</strong><br />
Western Ontario, London, ON, Canada, Robert Inman,<br />
Pr<strong>of</strong>essor, Toronto Western Hospital, Toronto, ON, Canada<br />
We have recently demonstrated that dendritic cell(DC)mediated<br />
immune modulation and deviation can be accomplished<br />
through RNA interference (RNAi), highlighting the<br />
therapeutic potential <strong>of</strong> RNAi-modified DC as antigenspecific<br />
tolerogenic vaccines. To date, an RNAi-based<br />
immune therapy for autoimmune diseases has not been<br />
reported. The current study was designed to develop siRNAmodified<br />
DC as antigen-specific, tolerogenic vaccines for<br />
prevention and intervention <strong>of</strong> autoimmune arthritis. Using<br />
small interfering RNA (siRNA) that specifically targets IL-<br />
12p35 gene (IL-12 siRNA), we have generated a type <strong>of</strong> DC<br />
that exhibits multiple tolerogenic characteristics. After<br />
inducation <strong>of</strong> RNAi in DC using siRNA, the expression <strong>of</strong> IL-<br />
12 was inhibited as determined by real time PCR and flow<br />
cytometry. IL-12 silenced DC suppressed Tcell response in an<br />
MLR, and impaired Th1 differentiation in vitro. Immunization<br />
with IL-12 gene-silenced and type II collagen (CII)-pulsed DC<br />
(CII-pulsed/gene-silenced DC) resulted in antigen-specific<br />
non-responsiveness. Vaccination with CII-pulsed/genesilenced<br />
DC prevented collagen-induced arthritis (CIA)<br />
onset in a murine rheumatoid arthritis model. Furthermore,<br />
administration <strong>of</strong> CII-pulsed/gene-silenced DC was sufficient<br />
doi:10.1016/j.clim.2007.03.179<br />
available at www.sciencedirect.com<br />
www.elsevier.com/locate/yclim<br />
to inhibit progression <strong>of</strong> CIA. The therapeutic effects were<br />
further evidenced by decreased clinical score in CIA,<br />
inhibited inflammatory infiltrates in joints, and suppressed<br />
T cell and B cell responses to CII. In conclusion, this study is<br />
the first to demonstrate the therapeutic utilization <strong>of</strong> RNAimodified<br />
DC as antigen-specific tolerogenic vaccines for<br />
autoimmune arthritis.<br />
doi:10.1016/j.clim.2007.03.180<br />
#1202- Commensal Flora and the Immune Response<br />
Helicobacter Pylori in the Pathogenesis <strong>of</strong> Chronic<br />
Autoimmune Pancreatitis<br />
Friday, June 8<br />
10:45 am−11:05 am<br />
Antonio Puccetti, Pr<strong>of</strong>essor <strong>of</strong> Medicine, Institute G. Gaslini,<br />
Genova, Italy, Claudio Lunardi, Pr<strong>of</strong>essor <strong>of</strong> Medicine,<br />
University <strong>of</strong> Verona-Department <strong>of</strong> Internal Medicine,<br />
Verona, Italy, Luca Frullloni, Pr<strong>of</strong>essor <strong>of</strong> Gastroenterology,<br />
Department <strong>of</strong> Gastroenterology, University <strong>of</strong> Verona,<br />
Verona, Italy, Caterina Bason, Research Fellow, University <strong>of</strong><br />
Verona-Department <strong>of</strong> Internal Medicine, Verona, Italy,<br />
Dimitri Peterlana, PhD Student, University <strong>of</strong> Verona-<br />
Department <strong>of</strong> Internal Medicine, Verona, Italy<br />
Autoimmune chronic pancreatitis (ACP) is a type <strong>of</strong> pancreatitis<br />
characterized by a chronic inflammatory process<br />
with prominent lymphocyte infiltration leading to fibrosis <strong>of</strong><br />
the pancreas and eventually to organ dysfunction. The cause<br />
<strong>of</strong> the disease is still unknown. Current evidence suggests an<br />
autoimmune origin for ACP, based on its frequent association<br />
with other autoimmune diseases (rheumatoid arthritis,<br />
Sjogren’s syndrome, and inflammatory bowel disease) and<br />
on the presence <strong>of</strong> immunological abnormalities, including<br />
hypergammaglobulinemia, elevated serum IgG4 levels and the<br />
presence <strong>of</strong> autoantibodies, such as antibodies against<br />
carbonic anhydrase II. However, little is known about its<br />
actual pathogenesis. In order to clarify the pathogenesis <strong>of</strong> the<br />
disease, we screened a random peptide library with pooled<br />
IgG immunoglobulins derived from 20 patients with ACP.
S4 Abstracts<br />
Among the identified peptides, one was recognized by the<br />
majority <strong>of</strong> patients' sera, but not by sera <strong>of</strong> normal donors and<br />
<strong>of</strong> patients with other autoimmune diseases. The peptide<br />
showed homology with an Helicobacter pylori derived protein<br />
and with carbonic anhydrase 5B, a protein highly expressed in<br />
pancreas, kidney and salivary glands, and with lact<strong>of</strong>errin,<br />
considered an important autoantigen target in ACP. Antipeptide<br />
antibodies, affinity purified from patients' sera<br />
recognize the helicobacter derived protein, carbonic anhydrase<br />
5B and lact<strong>of</strong>errin. Moreover antibodies against the<br />
bacterial epitope can be detected in the vast majority <strong>of</strong><br />
patients with ACP. Our findings suggest that Helicobacter<br />
pylori infection may be linked to the pathogenesis <strong>of</strong> ACP<br />
through a molecular mimicry mechanism and that carbonic<br />
anhydrase 5B can be considered a novel autoantigen in ACP.<br />
doi:10.1016/j.clim.2007.03.181<br />
OR.1 Foxp3 (Scurfy) Mutation Greatly Accelerates<br />
Diabetes in Insulin-specific TCR Transgenic Mice<br />
Jean Jasinski, University <strong>of</strong> Colorado Health Sciences Center,<br />
Aurora, CO, Maki Nakayama, Fellow, University <strong>of</strong> Colorado<br />
Health Sciences Center, Barbara Davis Center, Aurora, CO,<br />
Kelly Johnson, Pr<strong>of</strong>essional Research Assistant, University <strong>of</strong><br />
Colorado Health Sciences Center, Barbara Davis Center,<br />
Aurora, CO, George Eisenbarth, Executive Director, Barbara<br />
Davis Center, Aurora, CO, Liping Yu, Senior Research<br />
Assistant, University <strong>of</strong> Colorado Health Sciences Center,<br />
Barbara Davis Center, Aurora, CO<br />
Previously we developed and described the development<br />
<strong>of</strong> a transgenic mouse expressing an insulin-specific (ins2<br />
B:9–23) T cell receptor mouse model (BDC12-4.1). This<br />
pathogenic model exhibited spontaneous diabetes development<br />
on a backcross-1 (FVB×NOD)×NOD background. Disease<br />
development was heterogeneous and dependent on both I-<br />
Ag7 and RAG-deficient genotypes with relatively slow<br />
development <strong>of</strong> diabetes such that the median age <strong>of</strong> onset<br />
<strong>of</strong> diabetes was 49 weeks, and less than 10% <strong>of</strong> mice<br />
developed diabetes prior to 10 weeks <strong>of</strong> age. To test the<br />
hypothesis that the 12-4.1 transgenic T cells, even on a<br />
RAG−/− background, were heterogeneous with development <strong>of</strong><br />
both regulatory and pathogenic T cells, we crossed NOD<br />
congenic BDC12-4.1 mice with C57BL/6 Foxp3 mutant mice.<br />
Expression <strong>of</strong> the single BDC12-4.1 TCR on a RAG-deficient<br />
background rescues the mouse from the scurfy phenotype at<br />
the BC1 (C57BL/6×NOD)×NOD generation. All RAG-deficient<br />
Foxp3 mutant mice (8/8) expressing the BDC12-4.1 TCR develop<br />
diabetes between 5 and 10 weeks <strong>of</strong> age with aggressive<br />
destruction <strong>of</strong> essentially all beta cells whereas prediabetic<br />
mice exhibit invasive insulitis. Disease develops equally in<br />
homozygous (I-Ag7/g7) and heterozygous (I-Ag7/b) mice<br />
significantly earlier and with greater uniformity than previously<br />
described (Pb0.0001). Disease can be adoptively transferred<br />
from diabetic animals to NOD.scid mice. These studies provide<br />
evidence that a single transgenic TCR generates both pathogenic<br />
and Foxp3-dependent regulatory T cells and provides an<br />
experimental model to test therapies <strong>of</strong> diabetes prevention<br />
without Foxp3 dependent regulatory T cells.<br />
doi:10.1016/j.clim.2007.03.182<br />
OR.2 Regulatory T Cells Require Serum for<br />
Suppression <strong>of</strong> Effector T Cell Proliferation and<br />
Express Stable Membrane-bound CD25<br />
Todd Brusko, Postdoctoral Associate, University <strong>of</strong> Florida,<br />
College <strong>of</strong> Medicine, Gainesville, FL, Clive Wasserfall,<br />
Biological Scientist, University <strong>of</strong> Florida, College <strong>of</strong><br />
Medicine, Department <strong>of</strong> Pathology, Gainesville, FL, Kieran<br />
McGrail, Laboratory Technician, University <strong>of</strong> Florida,<br />
College <strong>of</strong> Medicine, Department <strong>of</strong> Pathology, Gainesville,<br />
FL, Marcus Moore, Laboratory Technician, University <strong>of</strong><br />
Florida, College <strong>of</strong> Medicine, Department <strong>of</strong> Pathology,<br />
Gainesville, FL, Alyssa Huegel, Laboratory Technician,<br />
University <strong>of</strong> Florida, College <strong>of</strong> Medicine, Department <strong>of</strong><br />
Pathology, Gainesville, FL, Desmond Schatz, Pr<strong>of</strong>essor <strong>of</strong><br />
Pediatric Medicine, University <strong>of</strong> Florida, College <strong>of</strong><br />
Medicine, Department <strong>of</strong> Pediatrics, Gainesville, FL, Mark<br />
Atkinson, Pr<strong>of</strong>essor, University <strong>of</strong> Florida, College <strong>of</strong><br />
Medicine, Department <strong>of</strong> Pathology, Gainesville, FL<br />
CD4+CD25+ regulatory T cells (Treg) suppress effector T<br />
cell (Teff) functions and are essential for maintaining<br />
peripheral tolerance. Recent work suggests that IL-2 signaling<br />
is crucial for proper Treg development and function. In this<br />
study, we assessed the production <strong>of</strong> the soluble form <strong>of</strong> CD25<br />
(sCD25) in human Treg or Teff cells, alone and in combination,<br />
following polyclonal activation during an in vitro suppression<br />
assay. We found that surface CD25 is relatively stable on Treg<br />
in vitro, whereas CD25 expression on isolated Teff cells is<br />
upregulated following activation; subsequently resulting in<br />
high levels <strong>of</strong> sCD25 detected in culture supernatants (mean +<br />
SD, 682.2+367.1 vs. 5369.5+ 2575.6 pg/ml for Treg vs. Teff<br />
cells respectively; N=7, P=0.004). The production <strong>of</strong> sCD25<br />
can occur in an autocrine fashion and correlates with T cell<br />
proliferation (r=0.76, Pb0.0001). To eliminate the influence<br />
<strong>of</strong> serum sCD25, our studies were also conducted under<br />
serum-free conditions. Surprisingly, under these conditions,<br />
Treg fail to suppress Teff cell proliferation (% suppression at a<br />
1:1 Treg to Teff ratio; −197.8+ 270.6 for serum-free medium<br />
vs. 70.4+17.3 with 1.0% serum; P=0.04). Despite this<br />
functional defect, Treg cells still maintained their capacity<br />
to inhibit Teff cell cytokine production (e.g., IFN-γ responses<br />
were suppressed 75.6% in co-culture vs. Teff alone, Pb0.05).<br />
These data highlight a mechanism by which Treg may obtain<br />
qualitatively greater IL-2 signals than Teff cells and suggest<br />
that serum is required for full Treg activity.<br />
doi:10.1016/j.clim.2007.03.183<br />
OR.3 TGF-β-induced TCR-Tg/NOD Regulatory T Cells<br />
Suppress Both Diabetes Transfer and Islet Graft<br />
Destruction in an Antigen-dependent Manner<br />
Daniel Tonkin, Graduate Student, University <strong>of</strong> Colorado<br />
Health Sciences Center, Department <strong>of</strong> <strong>Immunology</strong>, Denver,<br />
CO, Brenda Bradley, Senior Pr<strong>of</strong>essional Research Assistant,<br />
University <strong>of</strong> Colorado Health Sciences Center, Department<br />
<strong>of</strong> <strong>Immunology</strong>, Denver, CO, Gene Barbour, Pr<strong>of</strong>essional<br />
Research Assistant, University <strong>of</strong> Colorado Health Sciences<br />
Center, Department <strong>of</strong> <strong>Immunology</strong>, Denver, CO, Kathryn<br />
Haskins, Pr<strong>of</strong>essor, University <strong>of</strong> Colorado Health Sciences<br />
Center, Department <strong>of</strong> <strong>Immunology</strong>, Denver, CO
Abstracts<br />
Regulatory T cells (Tregs) are understood to fall into<br />
two categories: natural Tregs and induced Tregs. Induced<br />
Tregs can be produced by activating CD4 T cells in the<br />
presence <strong>of</strong> TGF- 2 . Such induced Tregs have great promise<br />
for immunotherapy, but it has been unclear if they<br />
require stimulation by antigen in order to suppress in<br />
vivo. We have investigated induced Treg protection in the<br />
NOD mouse model <strong>of</strong> autoimmune diabetes. We produced<br />
TGF- 2 -induced Tregs from the 6.9 TCR-Tg/NOD mouse that<br />
are specific for islet autoantigen. These Tregs express<br />
Foxp3 and produce little IFN- 3 . We have shown that<br />
induced Tregs can prevent transfer <strong>of</strong> diabetes by effector<br />
T cells in NOD mice, where they decrease inflammatory<br />
cytokine production and macrophage infiltration in the<br />
pancreas. In some experiments we transferred cells into a<br />
congenic mouse, the NOD.C6, that specifically lacks the<br />
antigen for the 6.9 TCR-Tg T cells but not for other isletspecific<br />
T cells. The 6.9 TCR-Tg Tregs fail to prevent<br />
diabetes transfer in the NOD.C6 mice, demonstrating that<br />
their function is antigen dependent. In human diabetes,<br />
induced Tregs might protect islet grafts from immune<br />
destruction. We grafted NOD.scid islets into NOD.scid.C6<br />
recipients after streptozotocin treatment, and demonstrated<br />
that induced Tregs can prevent islet graft<br />
destruction by effector T cells. When we grafted NOD.<br />
scid.C6 islets, the 6.9 TCR-Tg islets failed to protect.<br />
With these antigen-free islets, we have demonstrated that<br />
induced Tregs require activation by their antigen in the<br />
islet graft in order to protect the graft from autoimmune<br />
destruction.<br />
doi:10.1016/j.clim.2007.03.184<br />
OR.4 Leptin Modulates Treg Cell Activity in Human<br />
SLE<br />
Antonio La Cava, Associate Pr<strong>of</strong>essor, University <strong>of</strong><br />
California, Los Angeles, Los Angeles, CA, Giuseppe<br />
Matarese, Assistant Researcher, University <strong>of</strong> Naples,<br />
Naples, Bevra Hahn, Pr<strong>of</strong>essor and Chief <strong>of</strong> Rheumatology,<br />
University <strong>of</strong> California, Los Angeles, Los Angeles, CA<br />
Leptin is a cytokine/hormone that links nutritional<br />
status with neuroendocrine and immune functions. We<br />
and others have recently shown that leptin can contribute<br />
to the onset and progression <strong>of</strong> organ-specific autoimmunity.<br />
Here, we measured serum leptin in systemic lupus<br />
erythematosus (SLE) patients (n=66) and healthy controls<br />
(n=50) matched for gender, age and ethnicity. Parallel flow<br />
cytometry studies correlated the numbers <strong>of</strong> CD4+<br />
CD25highFoxp3+ T (Treg) cells with serum leptin in the<br />
SLE patients vs. controls. Serum leptin concentration was<br />
significantly higher in SLE patients than in controls<br />
(Pb0.0001) and did not relate to body mass index and/or<br />
therapy. Interestingly, a reduced number <strong>of</strong> CD4+<br />
CD25highFoxp3+ T cells significantly correlated with the<br />
elevated serum leptin in SLE patients (n=9, P=0.02, r 2 =<br />
0.51) but not in controls (n=10, P=0.3, r 2 =0.2). Because<br />
<strong>of</strong> this inverse correlation between CD4+CD25highFoxp3+ T<br />
cells and serum leptin levels, we tested whether leptin<br />
could influence the number and/or activity <strong>of</strong> human Treg<br />
cells. We found that neutralization <strong>of</strong> leptin with antileptin<br />
Ab reversed the hyporesponsiveness <strong>of</strong> human Tregs<br />
in a dose-dependent fashion and Foxp3-expressing Treg<br />
cells expressed elevated levels <strong>of</strong> leptin receptor. Additionally,<br />
anti-leptin Ab promoted both the upregulation <strong>of</strong><br />
Foxp3 and the suppressive in vitro activity <strong>of</strong> the Tregs<br />
cells on effector CD4+CD25− T cells. These data suggest<br />
that leptin can influence the number and activity <strong>of</strong> Treg<br />
cells and may have implication for leptin-based intervention<br />
on Treg cells in SLE.<br />
doi:10.1016/j.clim.2007.03.185<br />
OR.5 Gene Related to Anergy in Lymphocytes (GRAIL)<br />
Expression in CD4+ CD25+ T Regulatory Cells and<br />
Correlation with T:APC Conjugate Formation<br />
Jill Schartner, Graduate Student, University <strong>of</strong> Wisconsin,<br />
Department <strong>of</strong> Pediatrics, Madison, WI, Amanda Timmel,<br />
Technician, University <strong>of</strong> Wisconsin, Department <strong>of</strong><br />
Pediatrics, Madison, WI, William Simonson, Graduate<br />
Student, University <strong>of</strong> Wisconsin, Madison, WI, Christine<br />
Seroogy, Assistant Pr<strong>of</strong>essor, University <strong>of</strong> Wisconsin,<br />
Department <strong>of</strong> Pediatrics, Madison, WI, Anna Huttenlocher,<br />
Associate Pr<strong>of</strong>essor, University <strong>of</strong> Wisconsin, Department <strong>of</strong><br />
Pediatrics, Madison, WI<br />
It has been reported that CD4+CD25+ regulatory T cells<br />
(CD25+ Tregs) have an anergic phenotype and disrupted actin<br />
cap formation in vitro. Recent reports suggest that strong<br />
activation can abrogate the suppressor activity <strong>of</strong> CD25+<br />
Tregs. We have found that the anergy-related E3 ubiquitin<br />
ligase, GRAIL is differentially expressed in CD25+ Tregs. We<br />
sought to correlate the expression pr<strong>of</strong>ile <strong>of</strong> an anergyrelated<br />
gene, GRAIL, with immunologic synapse formation<br />
and suppressor activity in CD25+ Tregs. GRAIL mRNA was<br />
quantitated by real-time QPCR in CD25+ Tregs activated with<br />
anti-CD3/CD28 or anti-CD3 conjugated beads. DO11.10 CD4+<br />
Tcell lines were transduced with GRAIL-IRES-GFP or GFP-only<br />
expressing retrovirus. The ability <strong>of</strong> these cells to form<br />
conjugates with OVA323–339 loaded antigen presenting cells<br />
(APCs) was evaluated via FACS. Microscopy was used to<br />
evaluate localization <strong>of</strong> LFA-1, actin, and GRAIL during<br />
conjugate formation. Strong activation <strong>of</strong> CD25+ Tregs with<br />
anti-CD3/CD28 beads resulted in a 10-fold reduction in GRAIL<br />
mRNA. In contrast, activation <strong>of</strong> CD25+ Tregs with anti-CD3<br />
beads resulted in stable expression <strong>of</strong> GRAIL mRNA. Forced<br />
expression <strong>of</strong> GRAIL in a DO11.10 T cells sufficiently converts<br />
these cells to a suppressor phenotype and resulted in a 2-fold<br />
reduction in their ability to form conjugates with OVA323–<br />
339 loaded APCs. Following conjugation with anti-CD3coated<br />
beads GRAIL expressing T cells exhibited impaired<br />
LFA-1 localization and disrupted actin cap formation. These<br />
data suggest that expression <strong>of</strong> GRAIL in CD25+ Tregs is<br />
differentially regulated and dependent on the activation<br />
context and may correlate with suppressor function.<br />
Furthermore, ectopic expression <strong>of</strong> GRAIL correlates with a<br />
suppressor phenotype and disrupted synapse formation.<br />
doi:10.1016/j.clim.2007.03.186<br />
S5
S6 Abstracts<br />
OR.6 Islet Antigen Specific Regulatory T Cells<br />
Isolated From Non-Diabetic Individuals Suppress<br />
Pro-Inflammatory Responses via Cytotoxic Mechanisms<br />
Timothy Tree, Lecturer, Department <strong>of</strong> Immunobiology,<br />
King’s College London, London, Jenifer Lawson, Research<br />
Assistant, Department <strong>of</strong> Immunobiology, King’s College<br />
London, London, Bart Roep, Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Immunohaematology and Blood Transfusion, Leiden<br />
University Medical Center, Leiden, Hannah Edwards,<br />
Research Technician, Department <strong>of</strong> Immunobiology,<br />
King’s College London, London, England, Mark Peakman,<br />
Pr<strong>of</strong>essor <strong>of</strong> <strong>Clinical</strong> <strong>Immunology</strong>, Department <strong>of</strong><br />
Immunobiology, King’s College London, London,<br />
England<br />
Autoreactive T cells recognizing islet ? cell antigens<br />
could represent both pathogenic effectors in the development<br />
<strong>of</strong> type 1 diabetes (T1DM) and mediators <strong>of</strong><br />
tolerance in non-diabetic individuals depending on the<br />
quality <strong>of</strong> response they produce. We have previously<br />
demonstrated that, whereas autoreactive T cells in<br />
patients with T1DM exhibit polarization towards a proinflammatory<br />
T helper 1 (Th1) phenotype, the majority <strong>of</strong><br />
non-diabetic subjects also manifest a response against<br />
islet peptides, but one that shows T regulatory cell (Treg),<br />
IL-10 secreting, bias. We therefore proposed that isletspecific<br />
IL-10 producing T cells may be involved in the<br />
maintenance <strong>of</strong> tolerance to islets by the suppression <strong>of</strong><br />
proinflammatory Th1 cells. To test this hypothosis, we<br />
isolated T cell clones from healthy individuals that secrete<br />
high levels <strong>of</strong> IL-10 in response to islet antigens directly<br />
ex vivo. These CD4+CD25+Foxp3+ Tregs are potent<br />
suppressors <strong>of</strong> proinflammatory Th1 effector cells. Suppression<br />
is not mediated by soluble factors but is cellcontact<br />
dependent, and dependent upon presentation <strong>of</strong><br />
stimulating antigens for both the Treg and effector Th1<br />
cells by the same antigen presenting cell (APC), thus<br />
providing exquisite specificity <strong>of</strong> suppression. These Tregs<br />
express the cytotoxic molecules perforin and granzymes A<br />
and B directly ex vivo and mediate their suppression via<br />
the specific lysis <strong>of</strong> APC presenting high levels <strong>of</strong> islet<br />
antigens. This hitherto un-described population <strong>of</strong> antigen<br />
specific Tregs, which appear to be constitutively present<br />
in the majority <strong>of</strong> non-diabetic individuals, may provide a<br />
suitable target to strengthen tolerance to islets by clinical<br />
immunointervention.<br />
doi:10.1016/j.clim.2007.03.187<br />
OR.7 Rapamycin Promotes Induction <strong>of</strong> CD4+Foxp3+<br />
T Cells that Proliferate in Response to Common<br />
Gamma Chain Cytokines<br />
S. Alice Long, Scientist, Benaroya Research Institute,<br />
Seattle, WA, Megan Van Landeghen, RA II, Benaroya<br />
Research Institute, Seattle, WA, Jane Buckner, Pr<strong>of</strong>essor,<br />
Benaroya Research Institute, Seattle, WA<br />
We and others have shown that CD4+CD25+Foxp3+ T<br />
cells can be induced from CD4+CD25−Foxp3− T cells<br />
(iTReg). Using intracellular staining for Foxp3, we consistently<br />
observe that the CD4+CD25+ population that arises<br />
upon activation <strong>of</strong> CD4+CD25− T cells with polyclonal or<br />
antigenic stimulation is composed <strong>of</strong> both Foxp3− and Foxp3+<br />
T cells. Thus, determining factors that influence the<br />
frequency <strong>of</strong> iTReg may lead to development <strong>of</strong> targeted<br />
therapies. IL-2 is a known growth factor for natural Treg.<br />
Others have observed that treatment <strong>of</strong> patients with<br />
Rapamycin leads to an increased number <strong>of</strong> CD4+CD25+<br />
Foxp3+ Tcells. Here, we demonstrate that in vitro activation<br />
<strong>of</strong> CD4+CD25− T cells in the presence <strong>of</strong> IL-2 or Rapamycin<br />
results in generation <strong>of</strong> functional iTReg. However, combination<br />
<strong>of</strong> both Rapamycin and IL-2 results in a significantly<br />
higher frequency and absolute number <strong>of</strong> iTReg. Analysis <strong>of</strong><br />
Foxp3 over time demonstrates that up-regulation <strong>of</strong> Foxp3<br />
occurs prior to cell division and increases during the course<br />
<strong>of</strong> cell division. Foxp3− and Foxp3+ T cells proliferate in<br />
parallel suggesting that Rapamycin is not significantly<br />
inhibiting the proliferation <strong>of</strong> Foxp3− T cells, but instead,<br />
is preferentially inducing Foxp3 expression in a subset <strong>of</strong><br />
CD25− T cells. The addition <strong>of</strong> IL-2, but also IL-4, IL-7, and<br />
IL-15, facilitates proliferation <strong>of</strong> iTReg. Thus, we demonstrate<br />
that Rapamycin plus IL-2 and/or common gamma<br />
chain cytokines promotes induction and proliferation <strong>of</strong><br />
Foxp3+ T cells. These studies provide rationale for using<br />
Rapamycin in combination with IL-2 to enhance expansion<br />
<strong>of</strong> regulatory T cells in vivo.<br />
doi:10.1016/j.clim.2007.03.188<br />
OR.8 IL-2Rβ Links IL-2R Signaling with Foxp3<br />
Expression<br />
David Soper, Graduate Student, University <strong>of</strong> Washington,<br />
Department <strong>of</strong> <strong>Immunology</strong>, Seattle, WA<br />
Immunological tolerance to self-antigens is a tightly<br />
regulated process. Recent work has demonstrated that<br />
the forkhead family member Foxp3 is a critical element in<br />
the differentiation and function <strong>of</strong> mouse CD4+CD25+<br />
regulatory T (TR) cells. Recent work has suggested an<br />
important role for IL-2 in the development and maintenance<br />
<strong>of</strong> TR cells. To directly assess the effect <strong>of</strong> IL-2<br />
signaling on TR development and function, we analyzed<br />
mice that were genetically deficient in components <strong>of</strong> the<br />
IL-2 receptor (IL-2R). Mice lacking CD25 (IL-2Rα) displayed<br />
a slight decrease in TR cells within the thymus, while<br />
peripheral numbers are unchanged. In contrast, we found<br />
that mice deficient in CD122 (IL-2Rβ) had a pr<strong>of</strong>ound<br />
reduction in both thymic and peripheral TR cells,<br />
coinciding with more rapid development <strong>of</strong> a fatal<br />
lymphoproliferative disease. Expression <strong>of</strong> a Foxp3 transgene<br />
restored TR cells and protected against the onset <strong>of</strong><br />
autoimmunity. Thus, a signal mediated by IL-2Rβ is essential<br />
for the development and homeostasis <strong>of</strong> Foxp3+ TR cells<br />
in vivo.<br />
doi:10.1016/j.clim.2007.03.189
Abstracts<br />
OR.9 High Affinity T Cell Receptors as Therapeutic<br />
Agents<br />
Rebecca Ashfield, Senior Research Project Manager, Avidex<br />
Ltd, Abingdon, Oxfordshire, England, Deborah Sutton,<br />
Group Leader, Cell Biology, Avidex Ltd, Abingdon,<br />
Oxfordshire, England, Giovanna Bossi, Senior Scientist,<br />
Avidex Ltd, Abingdon, Oxfordshire, England, Joanna Brewer,<br />
Group Leader, Cell biology, Avidex Ltd, Abingdon,<br />
Oxfordshire, England, Marco Purbhoo, Research Associate,<br />
Division <strong>of</strong> Molecular and Cell Biology, London, Rebecca<br />
Dennis, Senior Scientist, Avidex Ltd, Abingdon, Oxfordshire,<br />
England, Julia Karbach, Researcher, Ludwig Institute,<br />
Frankfurt, Germany, Tara Mahon, Senior Scientist, Avidex<br />
Ltd, Abingdon, Oxfordshire, England, Brian Cameron, Senior<br />
Scientist, Avidex Ltd, Abingdon, Oxfordshire, England, Bent<br />
Jakobsen, Senior Vice President, Research, Avidex Ltd,<br />
Abingdon, Oxfordshire, England<br />
T cell receptors (TCRs) recognise peptides derived from<br />
disease related antigens, many <strong>of</strong> them intracellular in origin,<br />
which are presented on the cell surface by HLA molecules. The<br />
development <strong>of</strong> TCRs as therapeutic agents has until recently<br />
been impeded by their low natural affinity, and the difficulty <strong>of</strong><br />
expressing membrane-bound receptors as soluble molecules.<br />
We have engineered TCRs to produce stable, soluble proteins<br />
that can be made in large quantities by expression in E. coli.<br />
These monoclonal TCRs (mTCRs) can be affinity matured by<br />
phage display technology to sub-nM affinity, and can be fused to<br />
toxins or immuno-modulatory effector functions to produce<br />
therapeutic proteins suitable for targeting tumours, virally<br />
infected cells or tissues under autoimmune attack. Using this<br />
technology, high affinity mTCRs have been produced which<br />
recognise HLA-A2 specific epitopes from HIVgag (amino acids<br />
77–85) and two tumour associated antigens, NY-ESO (amino<br />
acids 157–165) and Melan-A (amino acids 26–35). Each <strong>of</strong> these<br />
mTCRs has sub-nM affinity and a binding half life <strong>of</strong> several<br />
hours, whilst retaining specificity for the target HLA–epitope<br />
complex. Naturally processed epitopes on the surface <strong>of</strong> antigen<br />
presenting cells are targeted by the high affinity mTCRs, and<br />
can be detected using flow cytometry and fluorescence<br />
microscopy. We present data comparing the antigen density <strong>of</strong><br />
the two tumour associated epitopes, NY-ESO157–165 and<br />
Melan-A26–35, on the surface <strong>of</strong> melanoma cell lines. Furthermore,<br />
we show that the NY-ESO specific mTCR binds specifically<br />
to freshly isolated NY-ESO+, HLA-A2+ myeloma cells.<br />
doi:10.1016/j.clim.2007.03.190<br />
Regulatory T Cells I<br />
Friday, June 8<br />
2:45 pm−4:45 pm<br />
OR.10 From EAE to an MS <strong>Clinical</strong> Trial:<br />
DR2/MOG-35−55 Recombinant T Cell Receptor<br />
Ligand (RTL) Treats Relapse <strong>of</strong> Experimental<br />
Encephalomyelitis in DR2 Transgenic Mice<br />
Halina Offner, Pr<strong>of</strong>essor, Oregon Health and Science<br />
University, Neurology, Portland, OR, Gregory Burrows,<br />
Research Associate Pr<strong>of</strong>essor, Oregon Health and Science<br />
University, Neurology, Portland, OR, Arthur Vandenbark,<br />
Pr<strong>of</strong>essor, Portland VA Medical Center, Neuroimmunology<br />
Research, Portland, OR, Jason Link, Associate Investigator,<br />
Portland VA Medical Center, Neuroimmunology Research,<br />
Portland, OR<br />
To evaluate the ability <strong>of</strong> a newly designed monomeric<br />
recombinant TCR ligand, RTL342M, containing HLA-DR2<br />
peptide-binding domains covalently linked to MOG-35–55<br />
peptide, to prevent and treat both the initial episode and<br />
subsequent relapses <strong>of</strong> experimental autoimmune encephalomyelitis<br />
(EAE) in HLA-DR2 transgenic mice, in preparation<br />
for a safety trial in patients with multiple sclerosis (MS),<br />
single or multiple doses <strong>of</strong> RTL342M were given i.v. or s.c. to<br />
HLA-DR2 mice prior to, at onset, or during relapses <strong>of</strong><br />
paralytic EAE. RTL342M produced rapid (within 24 h), dosedependent<br />
reversal <strong>of</strong> clinical signs <strong>of</strong> paralytic EAE; even a<br />
single dose could produce significant treatment effect.<br />
Multiple daily doses were even more effective than the<br />
same total amount <strong>of</strong> RTL given as a single dose. By<br />
establishing the minimal effective dose, RTLs may be 50<br />
times more potent than molar equivalent doses <strong>of</strong> myelin<br />
peptide alone. RTL342M given prior to induction <strong>of</strong> EAE<br />
prevented disease in most mice, and the remainder could be<br />
successfully re-treated with RTL. Most important for clinical<br />
application, RTL342M was highly effective for treating EAE<br />
relapses when given periodically prior to the relapse or even<br />
after relapses had occurred. These data demonstrate rapid<br />
and potent clinical effects <strong>of</strong> RTL342M at disease onset and<br />
during relapses in EAE, establishing important principles<br />
governing application <strong>of</strong> this novel, highly selective therapeutic<br />
approach for regulating pathogenic Tcells. Data from<br />
this study provide key preclinical information for a phase I<br />
safety study in patients with MS (outline to be presented) that<br />
is now in progress.<br />
doi:10.1016/j.clim.2007.03.191<br />
OR.11 Immunological Efficacy <strong>of</strong> hsp60 Peptide<br />
DiaPep277 Therapy in Human Type 1 Diabetes<br />
Bart Roep, Immunologist, Leiden University Medical Center,<br />
Leiden<br />
An immunogenic peptide (p277) from the 60-kDa heat-shock<br />
protein (hsp60) arrested beta-cell destruction in non-obese<br />
diabetic (NOD) mice. A randomised, double blind, phase Ib/II<br />
clinical trial <strong>of</strong> DiaPep277 peptide treatment was performed in<br />
recent onset type 1 diabetes patients with remaining insulin<br />
production. We studied the immunological efficacy <strong>of</strong> this<br />
peptide therapy and correlated this with clinical outcome.<br />
Forty-eight C-peptide positive patients were assigned subcutaneous<br />
injections <strong>of</strong> 0.2, 1.0 or 2.5 mg p277 (n=12perdosage)at<br />
entry, and 1, 6 and 12 months, or four placebo injections (n=12).<br />
T cell autoimmunity to hsp60, DiaPep277, GAD and tetanus<br />
toxoid (recall response control) were assayed by proliferation<br />
and cytokine secretion assays (ELIspot) on regular intervals until<br />
18 months after first injection. All treated patients at each<br />
dosage <strong>of</strong> peptide demonstrated an altered immune response to<br />
DiaPep277 while the majority <strong>of</strong> placebo-treated patient<br />
remained non-responsive to treatment (p=0.00001), indicating<br />
a 100% efficacy <strong>of</strong> immunization. Cytokine production in<br />
S7
S8 Abstracts<br />
response to therapy was dominated by IL-10, but this pr<strong>of</strong>ile did<br />
not correlate with better preserved beta-cell function. Instead,<br />
patients developing immunological tolerance to Diapep277<br />
showed less decreased C-peptide production than patients<br />
that kept a reactive phenotype. Third party control immune<br />
responses were unaffected by therapy. No potentially adverse<br />
immunological side effects were noted.<br />
DiaPep277 is immunogenic in type 1 diabetic subjects and<br />
has immune modulating properties. Immunological monitoring<br />
allowed to distinguish therapy from placebo treatment<br />
and could determine immunological efficacy. Tolerance to<br />
peptide DiaPep277 may serve as immunological biomarker<br />
measure for clinical efficacy.<br />
doi:10.1016/j.clim.2007.03.192<br />
OR.12 Somatic B-Cell Gene Vaccination with<br />
Anti-DNA Ig Consensus Peptide Protects (NZB × NZW)<br />
F1 Lupus Mice From Renal Disease<br />
Francesca Ferrera, Postdoctoral Fellow, University <strong>of</strong><br />
Genova, Genova, Bevra Hahn, Pr<strong>of</strong>essor and Chief <strong>of</strong><br />
Rheumatology, University <strong>of</strong> California, Los Angeles, Los<br />
Angeles, CA, Marta Rizzi, Postdoctoral Fellow, University<br />
<strong>of</strong> Genova, Genova, Gilberto Filaci, Associate Pr<strong>of</strong>essor,<br />
University <strong>of</strong> Genova, Genova, Francesco Indiveri,<br />
Pr<strong>of</strong>essor, University <strong>of</strong> Genova, Genova, Antonio La Cava,<br />
Associate Pr<strong>of</strong>essor, University <strong>of</strong> California, Los Angeles,<br />
Los Angeles, CA, Antonio La Cava, Associate Pr<strong>of</strong>essor<br />
<strong>of</strong> Medicine, University <strong>of</strong> California, Los Angeles,<br />
Los Angeles, CA<br />
Ig molecules that participate in humoral immune responses<br />
contain epitopes that induce T cell-mediated immune<br />
responses. B cells process and present such epitopes and<br />
activate T cells. We hypothesized that T cells recognizing an Ig<br />
consensus sequence presented by B cells will modulate lupuslike<br />
disease in mice. To test this hypothesis, NZB/W F1 lupus<br />
mice received B cell somatic gene transfer <strong>of</strong> DNA plasmid<br />
encoding an artificial consensus sequence mimicking T cell<br />
determinants <strong>of</strong> murine anti-DNA IgG, or control plasmids.<br />
Treated animals were monitored for production <strong>of</strong> antibody,<br />
development <strong>of</strong> renal disease and phenotype, number, and<br />
function <strong>of</strong> consensus gene-induced Tcells. It was found that the<br />
treatment <strong>of</strong> mice with Ig consensus gene induced TGFβproducing<br />
CD8+CD28− T cells that: (1) suppressed antigenspecific<br />
stimulation <strong>of</strong> CD4+ Tcells in a cell-contact independent<br />
manner; (2) reduced autoantibody production; (3) retarded the<br />
development <strong>of</strong> nephritis, and (4) improved survival <strong>of</strong> treated<br />
animals. Significantly, the adoptive transfer <strong>of</strong> CD8+CD28− T<br />
cells from Ig consensus gene-protected mice into hypergammaglobulinemic<br />
NZB/W F1 mice effectively protected the transferred<br />
mice from development <strong>of</strong> renal disease. We conclude<br />
that genetic expression <strong>of</strong> anti-DNA Ig consensus can induce<br />
immunoregulatory circuits capable <strong>of</strong> delaying development <strong>of</strong><br />
lupus nephritis via the suppression <strong>of</strong> hypergammaglobulinemia<br />
and renal disease.<br />
doi:10.1016/j.clim.2007.03.193<br />
OR.13 Therapeutic Vaccination with a Trivalent<br />
T Cell Receptor Peptide Vaccine Induces<br />
Peptide-specific IL-10-secreting T Cells, Restores<br />
Deficient Foxp3 Expression, and Induces Bystander<br />
Immunomodulation in Multiple Sclerosis Subjects<br />
Arthur Vandenbark, Pr<strong>of</strong>essor, Oregon Health and Science<br />
University, Neurology, Portland, OR, Richard Bartholomew,<br />
Executive Director <strong>of</strong> Research and Development, The<br />
Immune Response Corporation, Carlsbad, CA, Halina<br />
Offner, Pr<strong>of</strong>essor, Oregon Health and Science University,<br />
Neurology, Portland, OR, Dennis Bourdette, Pr<strong>of</strong>essor and<br />
Chairman, Oregon Health and Science University,<br />
Neurology, Portland, OR<br />
We are developing a therapeutic vaccine containing<br />
CDR2 peptides from BV5S2, BV6S5, and BV13S1 emulsified<br />
in IFA as a V gene-specific therapy for RR-MS. This TCR<br />
vaccine is currently being tested in a blinded, placebocontrolled,<br />
200 patient study with reduction <strong>of</strong> new<br />
gadolinium enhancing lesions as the primary endpoint. We<br />
here report new mechanistic data from a recently<br />
completed open-label study. Immunological and clinical<br />
parameters were followed in 27 treatment naïve and 9<br />
previously vaccinated subjects with MS who received<br />
monthly injections <strong>of</strong> the trivalent peptide vaccine over<br />
1 year. TCR vaccination induced high frequencies <strong>of</strong> IL-10secreting<br />
T cells specific for the injected TCR peptides as<br />
well as an apparently broader pattern <strong>of</strong> anti-TCR<br />
specificities. These regulatory specificities induced biphasic<br />
immunomodulation that involved an initial rise<br />
after 9 weeks <strong>of</strong> IL-10-secreting neuroantigen-specific T<br />
cells, in conjunction with potent induction <strong>of</strong> Foxp3+ Treg<br />
cells to normal levels after 12 weeks <strong>of</strong> treatment. These<br />
findings are consistent with vaccine-induced stimulation <strong>of</strong><br />
Foxp3+ Treg cells originating from both CD25(−) and<br />
conventional CD25(+) T cells. Restoration <strong>of</strong> previously<br />
deficient Treg cells could explain the broad reduction <strong>of</strong><br />
TCR-dependent responses <strong>of</strong> both cognate T cells expressing<br />
V genes targeted by the vaccine as well as bystander T<br />
cells expressing different V genes, including those specific<br />
for neuroantigens. These findings demonstrate that therapeutic<br />
vaccination using only three commonly expressed<br />
BV gene determinants can induce a broad immunoregulatory<br />
network in vivo that may help control autoreactive<br />
responses that characterize the inflammatory phase <strong>of</strong> MS.<br />
doi:10.1016/j.clim.2007.03.194<br />
OR.14 Mechanistic Insights Into the Differential<br />
Efficacy <strong>of</strong> GAD65 DNA Vaccine and Anti-CD3<br />
Combination Therapy in Treating New-Onset<br />
Autoimmune Diabetes in RIP-LCMV and NOD Mice<br />
Damien Bresson, Postdoctoral Fellow, La Jolla Institute for<br />
Allergy and <strong>Immunology</strong>, La Jolla, CA, Diane Rottembourg,<br />
Postdoctoral Fellow, La Jolla Institute for Allergy and<br />
<strong>Immunology</strong>, La Jolla, CA, Lisa Togher, Technician, La Jolla<br />
Institute for Allergy and <strong>Immunology</strong>, La Jolla, CA, Matthias<br />
von Herrath, Pr<strong>of</strong>essor, La Jolla Institute for Allergy and<br />
<strong>Immunology</strong>, La Jolla, CA
Abstracts<br />
Type 1 diabetes (T1D) results from a progressive<br />
destruction <strong>of</strong> insulin-producing pancreatic beta cells by<br />
autoaggressive T cells. In this study, we evaluated the<br />
efficacy <strong>of</strong> a combination therapy (CT) (non-Fc binding<br />
anti-CD3 and human GAD65-expressing DNA vaccine) to<br />
reverse new-onset T1D in two animal models (NOD and<br />
RIP-LCMV mice). CT enhanced efficacy only in the RIP-<br />
LCMV model (40% improvement compared to anti-CD3 or<br />
GAD65 vaccine given alone). We evaluated three factors<br />
that could account for such a discrepancy: (i) amount <strong>of</strong><br />
GAD65 protein within the islets <strong>of</strong> Langerhans, (ii) level<br />
and functionality <strong>of</strong> toll-like receptor-9 (TLR9) expressed<br />
by immune cells and (iii) epitopes recognized by GAD65specific<br />
Tregs expanded after CT vs. anti-CD3 alone.<br />
Although similar levels <strong>of</strong> pancreatic GAD65 levels were<br />
found in both animal models, TLR9 expression was slighlty<br />
enhanced in antigen-presenting cells derived from RIP-<br />
LCMV vs. NOD mice. By using a peptide library the GAD65specific<br />
Tregs epitopes were mapped in the C-terminal<br />
domain <strong>of</strong> the protein after CT in the RIP-LCMV but not<br />
NOD mice, thus emphasizing the importance <strong>of</strong> genetic<br />
background in the therapeutic efficacy <strong>of</strong> antigen-specific<br />
immuno-interventions. Furthermore, these CD4+CD25+<br />
Foxp3+GITR+ Tregs also expressed the co-stimulatory<br />
molecule OX40 and protected 50% <strong>of</strong> mice when transferred<br />
in vivo into immunocompetent recipients. The<br />
involvement <strong>of</strong> OX40 in this bystander suppression mechanism<br />
will be studied by antibody blockade and siRNA<br />
technology.<br />
This work was supported by a NIH DK51091, AI44451 and a<br />
U-19 prevention center grant to MVH. DB is supported by a<br />
European Marie-Curie Outgoing Fellowship.<br />
doi:10.1016/j.clim.2007.03.195<br />
OR.15 Mimotopes for Active Immunotherapy <strong>of</strong><br />
Tumors: Allergooncology<br />
Erika Jensen-Jarolim, Pr<strong>of</strong>essor, Medical University Vienna,<br />
Vienna, Angelika B. Riemer, Medicine University Vienna,<br />
Vienna, Regina Knittelfelder, MSc, Medicine University Vienna,<br />
Vienna, Panos Karagiannis, Student, Med University <strong>of</strong> Vienna,<br />
Vienna, Josef Singer, Student, Medicine University Vienna,<br />
Vienna, Eva Untersmayr, Medical University Vienna, Vienna,<br />
Kira Braemswig, Medicine University <strong>of</strong> Vienna, Vienna<br />
Antibodies recognize 3D-epitopes and have the great<br />
potential to sense viable, malignant cells and to destroy<br />
them by various mechanisms. Several chimeric or humanized<br />
antibodies are, therefore, in clinical use today.<br />
However, the effects <strong>of</strong> these passive immunotherapy<br />
treatments are limited by the antibody’s halflife, and<br />
repeated applications have to be given to the patient. In<br />
contrast, active induction <strong>of</strong> these desired antibody<br />
specificities in the patient could be pursued by the<br />
generation <strong>of</strong> peptide epitope mimics – mimotopes –<br />
which are suitable tools for vaccination. By virtue <strong>of</strong><br />
molecular mimicry, these mimotopes induced anti-tumor<br />
antibodies in mice able to attack tumors not only in vitro<br />
but also in vivo in murine transgenic, and in syngenic<br />
transplant models. Interestingly, besides IgG also IgE<br />
antibodies could be induced when mimotopes were<br />
gavaged by the oral route. These IgE antibodies sensitized<br />
mast cells and could be triggered by tumor cells in a<br />
specific fashion. Most importantly, the released mediators<br />
were cytotoxic for tumor cells. Indeed, immunohistochemical<br />
analyses <strong>of</strong> human tumor sections showed that IgE<br />
antibodies are present in tumor lesions, pointing towards<br />
a natural surveillance function <strong>of</strong> this antibody class.<br />
Conclusion: Taken together, we suggest that mimotopes<br />
are candidates for active immunization <strong>of</strong> patients for<br />
tumor prophylaxis or therapy, especially in settings <strong>of</strong><br />
minimal residual disease. Studies supported by BioLife-<br />
Science and grant P-18238-B13 <strong>of</strong> the Austrian Science<br />
Fund FWF.<br />
doi:10.1016/j.clim.2007.03.196<br />
OR.16 Gene Expression Analysis by Microarray<br />
Following Anti-CD3 Antibody Therapy <strong>of</strong> NOD Mice<br />
Hideyuki Iwai, Postdoctoral Fellow, Stanford University,<br />
Department <strong>of</strong> Medicine Division <strong>of</strong> <strong>Immunology</strong> and<br />
Rheumatology, Stanford, CA, Keichi Kodama, Postdoctoral<br />
Fellow, Stanford University, Department <strong>of</strong> Medicine<br />
Division <strong>of</strong> <strong>Immunology</strong> and Rheumatology, Stanford, CA,<br />
Demi Dang, Technician, Stanford University, Department <strong>of</strong><br />
Medicine Division <strong>of</strong> <strong>Immunology</strong> and Rheumatology,<br />
Stanford, CA, C. Garrison Fathman, Pr<strong>of</strong>essor, Stanford<br />
University, Department <strong>of</strong> Medicine Division <strong>of</strong> <strong>Immunology</strong><br />
and Rheumatology, Stanford, CA, Jeffrey A. Bluestone,<br />
Pr<strong>of</strong>essor, Diabetes Center, University <strong>of</strong> California,<br />
San Francisco, San Francisco, CA<br />
Anti-CD3-specific antibodies have the demonstrable<br />
capacity to induce long-term remission <strong>of</strong> overt diabetes<br />
in nonobese diabetic (NOD) mice and have had some<br />
success in C1-peptide preservation when used to treat<br />
recent onset human type I diabetics. The underlying<br />
mechanisms <strong>of</strong> action are unclear. To begin to study<br />
mechanism <strong>of</strong> action, we examined gene expression<br />
following 5 days <strong>of</strong> anti-CD3 antibody therapy <strong>of</strong> NOD<br />
mice, using 41K Agilent mouse genome oligo-microarrays.<br />
This treatment successfully restored euglycemia in NOD<br />
mice treated after the onset <strong>of</strong> hyperglycemia (blood<br />
glucose N250) with 10 μg <strong>of</strong> either conventional anti-CD-3,<br />
IgG1 2C11, or FcR nonbinding IgG3 anti-CD3 mAb, which<br />
cannot cross-link surface CD3 molecules on T cells and is<br />
therefore non-mitogenic. Both antibodies had similar<br />
effect in disease remission. RNA from islets and pancreatic<br />
lymph nodes <strong>of</strong> treated and untreated NOD mice was<br />
isolated on day 12. Microarray analysis was performed<br />
comparing treated mouse tissues to untreated control.<br />
Several hundred genes were dysregulated following therapy.<br />
We hypothesized that genes specifically affected by<br />
anti-CD3 antibody would be in overlapping groups among<br />
those dysregulated by these two antibodies. We will<br />
present data on these overlapping genes and discuss<br />
potential mechanism <strong>of</strong> action <strong>of</strong> anti-CD3 therapy.<br />
doi:10.1016/j.clim.2007.03.197<br />
S9
S10 Abstracts<br />
OR.17 C-Peptide Reactive T Cells Induce<br />
Autoimmune Diabetes: A Novel Mechanism <strong>of</strong> Beta<br />
Cell Autoimmunity<br />
Matteo Levisetti, Assistant Pr<strong>of</strong>essor, Washington<br />
University, Saint Louis, MO<br />
There is extensive experimental evidence supporting an<br />
important role for proinsulin as an autoantigen in the<br />
pathogenesis <strong>of</strong> autoimmune diabetes in both humans and<br />
the NOD mouse model <strong>of</strong> the disease. We identified<br />
spontaneously occurring C-peptide reactive T cells in the<br />
islet-infiltrate and peri-pancreatic lymph nodes <strong>of</strong> prediabetic<br />
mice. CD4 Tcells lines were generated by immunization<br />
with C-peptide in adjuvant and characterized. These T cells<br />
are reactive with full length C-peptide 1 and 2 and are<br />
reactive with freshly isolated mouse beta cells. Furthermore,<br />
these T cells are activated by fixed splenocytes,<br />
demonstrating that C-peptide can be presented in the<br />
absence <strong>of</strong> antigen processing by I-Ag7 bearing antigen<br />
presenting cells. C-peptide reactive T cells transferred<br />
disease into NOD.Scid recipient mice and accelerated<br />
disease in neonatal NOD recipients. These data identify a<br />
new T cell epitope within the proinsulin molecule that gives<br />
rise to diabetogenic T cells in the NOD mouse. In addition,<br />
these data define a novel mechanism for the loss <strong>of</strong> tolerance<br />
to secreted self proteins.<br />
doi:10.1016/j.clim.2007.03.198<br />
Autoantigens<br />
Friday, June 8<br />
2:45 pm−4:45 pm<br />
OR.18 Cytotoxic Preproinsulin-specific CD8 T Cells<br />
in Type 1 Diabetes in Man<br />
Anna Skowera, Postdoctoral Fellow, King’s College London,<br />
Department <strong>of</strong> Immunobiology, London, England, Richard<br />
Ellis, Postdoctoral Fellow, King’s College London,<br />
Department <strong>of</strong> Immunobiology, London, England, Timothy<br />
Tree, Lecture, King’s College London, Department <strong>of</strong><br />
Immunobiology, London, England, Mark Peakman, Pr<strong>of</strong>essor<br />
<strong>of</strong> <strong>Clinical</strong> <strong>Immunology</strong>, King’s College London, Department<br />
<strong>of</strong> Immunobiology, London, England, Sefina Arif,<br />
Postdoctoral Fellow, King’s College London, Department <strong>of</strong><br />
Immunobiology, London, England<br />
Type 1 diabetes (T1D) is a T cell mediated autoimmune<br />
disease in which the insulin producing pancreatic islet<br />
beta-cells are selectively eliminated. The mechanism <strong>of</strong><br />
this precisely targeted destruction is unknown, but<br />
cytotoxic T cells (CTLs) recognizing beta cell-specific<br />
targets are good candidates. To date, no beta cellspecific<br />
CTLs have been characterised in T1D. In this<br />
study, we identified novel epitopes originating from<br />
preproinsulin (PPI) that were naturally processed and<br />
presented by HLA-A2.1. Using these epitopes deployed in<br />
sensitive IFN-γ ELISPOTs a significantly increased prevalence<br />
<strong>of</strong> PPI-reactive CD8 T cells in the blood <strong>of</strong> HLA-A2.1<br />
+ T1D patients was detected, compared with A2.1+<br />
healthy controls in which these cells were extremely<br />
rare. CD8 T cell clones were generated from T1D patients<br />
using PPI epitope-loaded HLA-A2.1 tetramers, but could<br />
not be raised in controls subjects. Such clones were<br />
highly cytotoxic against HLA-A2.1+ PPI-transfected target<br />
cells and express NKG2D and CXCR3, which may be<br />
important both in beta cell targeting and migration to<br />
inflamed islets. The identification <strong>of</strong> beta cell restricted<br />
CD8 T cell epitopes and isolation <strong>of</strong> relevant CTLs in T1D<br />
patients suggests that this pathway may be important in<br />
targeted beta cell destruction.<br />
doi:10.1016/j.clim.2007.03.199<br />
OR.19 Anti-Thymocyte Globulin Prevents<br />
Autoimmune Encephalomyelitis by Expanding<br />
Myelin Antigen Specific Foxp3+ Regulatory T Cells<br />
Denise Chung, Postdoctoraltoral Fellow, Brigham and<br />
Women’s Hospital, Boston, MA, Thomas Korn, Research<br />
Fellow, Brigham and Women’s Hospital, Boston, MA, Julie<br />
Richard, Scientist, Genzyme Corporation, Framingham, MA,<br />
Adam Kohm, Research Fellow, Feinberg School <strong>of</strong> Medicine,<br />
Northwestern University, Chicago, IL, Melanie Ruzek,<br />
Scientist, Genzyme Corporation, Framingham, MA,<br />
Mohamed Oukka, Instructor, Brigham and Women’s<br />
hospital, Boston, MA, Stephen Miller, Pr<strong>of</strong>essor, Feinberg<br />
School <strong>of</strong> Medicine, Northwestern University, Chicago, IL,<br />
Sharon Nahill, Director <strong>of</strong> Research, Genzyme Corporation,<br />
Framingham, MA, Vijay Kuchroo, Pr<strong>of</strong>essor <strong>of</strong> Neurology,<br />
Brigham and Women’s Hospital, Boston, MA<br />
The T cell-depleting polyclonal antibody, anti-thymocyte<br />
globulin (ATG) has been used in organ transplantation<br />
to treat acute rejection episodes. However, its mechanism<br />
has not been fully understood. It is assumed that ATG<br />
treatment deletes all T cells equally resulting in generalized<br />
inhibition <strong>of</strong> T cell response. We hypothesized that<br />
ATG might selectively delete effector T cells and spare<br />
regulatory T cells and thereby provide an attractive<br />
option for the treatment <strong>of</strong> autoimmune diseases. In<br />
order to test this, we used Foxp3gfp “knock-in” mice in<br />
combination with a myelin oligodendrocyte glycoprotein<br />
(MOG)35–55/IAb tetramer to study more closely the<br />
effect <strong>of</strong> ATG treatment on antigen-specific T cell<br />
responses in vivo during MOG-induced experimental<br />
autoimmune encephalomyelitis (EAE). Administration <strong>of</strong><br />
ATG in vivo resulted in preferential depletion <strong>of</strong> T-eff<br />
with a marked expansion <strong>of</strong> MOG-specific tetramerreactive<br />
T-reg (CD4+Foxp3+) upon MOG immunization. In<br />
both acute and relapsing remitting disease models, ATG<br />
treatment resulted in the protection from EAE, both in a<br />
preventive and therapeutic setting. Analysis <strong>of</strong> purified Teff<br />
from control and ATG treated animals revealed that<br />
on a per cell basis, the effector function <strong>of</strong> residual T-eff<br />
was not compromised by ATG. Thus, ATG tips the balance<br />
<strong>of</strong> T-eff and T-reg and skews an autoantigen specific<br />
immune reaction from a pathogenic T cell response to a<br />
protective T-reg response that maintains immunological<br />
tolerance and prevents autoimmune inflammation. We<br />
conclude that ATG treatment enforces the development <strong>of</strong>
Abstracts<br />
a dominant immunoregulatory environment which may be<br />
advantageous for the treatment <strong>of</strong> T cell driven autoimmune<br />
diseases.<br />
doi:10.1016/j.clim.2007.03.200<br />
OR.20 Regulation Through Mimicry: The Autologous<br />
Milk Protein Butyrophilin Determines Susceptibility<br />
to Myelin Oligodendrocyte Glycoprotein-induced<br />
Experimental Autoimmune Encephalomyelitis<br />
Kerstin Berer, PhD Student, University <strong>of</strong> Aberdeen,<br />
Department <strong>of</strong> Medicine and Therapeutics, Aberdeen, Maria<br />
Storch, Pr<strong>of</strong>essor, Universitätsklinikum Graz,<br />
Universitätsklinik für Neurologie, Graz, Ian Mather,<br />
Pr<strong>of</strong>essor, University <strong>of</strong> Maryland, Department <strong>of</strong> Animal<br />
and Avian Sciences, Washington, MD, Christopher Linington,<br />
Pr<strong>of</strong>essor, University <strong>of</strong> Aberdeen, Department <strong>of</strong> Medicine<br />
and Therapeutics, Aberdeen<br />
Sensitization to the autologous milk protein butyrophilin<br />
(BTN) expands a pre-existing, regulatory T cell<br />
response which cross-reacts with myelin oligodendrocyte<br />
glycoprotein (MOG), an important candidate autoantigen<br />
in multiple sclerosis (MS). This regulatory T cell population<br />
is associated with the production <strong>of</strong> high levels <strong>of</strong> IL-10<br />
and is able to suppress proliferation <strong>of</strong> MOG-specific T<br />
cells in vitro, and to abrogate disease severity in C57BL/6<br />
mice with MOG-induced experimental autoimmune encephalomyelitis<br />
(EAE). These findings led us to speculate<br />
that post-natal induction <strong>of</strong> regulatory T cell responses by<br />
BTN in the mother’s milk is a factor that may determine<br />
susceptibility to MOG-induced EAE in adulthood. We<br />
investigated this hypothesis using BTN-deficient (BTN−/−)<br />
C57BL/6 mice and wild-type (WT) littermates. Earlier<br />
onset <strong>of</strong> disease and higher disease severity clearly<br />
indicate an increased susceptibility to MOG-induced EAE<br />
in BTN−/− mice. This was associated with higher inflammatory<br />
and demyelinating indices in BTN−/− mice<br />
compared to their WT littermates. Moreover, functional<br />
analysis <strong>of</strong> MOG-specific T cells in BTN−/− mice revealed a<br />
marked increase in the production <strong>of</strong> encephalitogenic<br />
cytokines (IL-17, IFN-γ and IL-6), while the synthesis <strong>of</strong> IL-<br />
10 was consistently decreased. Collectively, our data<br />
suggest that regulatory T cell responses induced by<br />
autologous BTN play an important role in determining<br />
susceptibility to MOG-induced EAE.<br />
doi:10.1016/j.clim.2007.03.201<br />
OR.21 NOD Islet Autoimmunity Dependence on<br />
Single Amino Acid Difference in Insulin B:9−23<br />
Peptide<br />
Maki Nakayama, Fellow, Barbara Davis Center for Childhood<br />
Diabetes, University <strong>of</strong> Colorado Health Sciences Center,<br />
Aurora, CO, Jean Jasinski, Graduate Student, Barbara Davis<br />
Center for Childhood Diabetes, University <strong>of</strong> Colorado<br />
Health Sciences Center, Aurora, CO, Dongmei Miao, PRA,<br />
Barbara Davis Center for Childhood Diabetes, University <strong>of</strong><br />
Colorado Health Sciences Center, Aurora, CO, Kelly<br />
Johnson, PRA, Barbara Davis Center for Childhood Diabetes,<br />
University <strong>of</strong> Colorado Health Sciences Center, Aurora, CO,<br />
Edwin Liu, Assistant Pr<strong>of</strong>essor, Barbara Davis Center for<br />
Childhood Diabetes, University <strong>of</strong> Colorado Health Sciences<br />
Center, Aurora, CO, John Elliott, Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Medical Microbiology and <strong>Immunology</strong>, University <strong>of</strong><br />
Alberta, Edmonton, AB, Canada, Canada, George<br />
Eisenbarth, Executive Director, Barbara Davis Center for<br />
Childhood Diabetes, University <strong>of</strong> Colorado Health Sciences<br />
Center, Aurora, CO<br />
NOD mice lacking native insulin genes but bearing<br />
mutated preproinsulin transgene (alanine [A] rather than<br />
tyrosine [Y] at B chain position 16) do not develop diabetes<br />
(Nakayama et al., Nature 2005). In the current study, we<br />
explore whether the initiation <strong>of</strong> anti-islet autoimmunity is<br />
dependent on this one amino acid difference and demonstrate<br />
that peripheral exposure to native B:9–23 sequence<br />
restores anti-islet autoimmunity. We created multiple<br />
double insulin knockout NOD mice bearing a normal<br />
preproinsulin 2 transgene (B16:Y-dKO mice), instead <strong>of</strong> the<br />
mutated B16:A transgene (B16:A-dKO mice). Eleven out <strong>of</strong><br />
twenty five B16:Y-dKO mice developed insulin autoantibodies,<br />
whereas less than 5% <strong>of</strong> B16:A-dKO mice (1/31) did<br />
(Pb0.01). B16:Y-dKO mice express insulin message in thymus<br />
as high as B16:A-dKO and wild-type NOD mice. These results<br />
indicate that the native B16:Y sequence in a transgene<br />
determines development <strong>of</strong> anti-insulin autoimmunity not<br />
because <strong>of</strong> less thymic insulin expression. We next investigated<br />
where the normal insulin sequence acts to induce<br />
autoimmunity. Transplantation <strong>of</strong> NOD islets, but not bone<br />
marrow, with native insulin sequences [B16:Y] into B16:AdKO<br />
mice rapidly restored development <strong>of</strong> insulin autoantibodies<br />
and insulitis. Splenocytes from B16:A-dKO mice<br />
transplanted with B16:Y islets induced diabetes when<br />
transferred into either wild-type or B16:A dKO NOD.SCID<br />
mice. Splenocytes from mice immunized with native insulin<br />
B:9–23 peptide induced diabetes on transfer if the recipients<br />
expressed the native B:9–23 [B16:Y] in their pancreas. These<br />
studies document dependence upon insulin B16:Tyrosine <strong>of</strong><br />
the insulin B:9–23 peptide for both the initial priming and<br />
effector phase <strong>of</strong> NOD anti-islet autoimmunity.<br />
doi:10.1016/j.clim.2007.03.202<br />
S11<br />
OR.22 Use <strong>of</strong> Random Peptide Display and Novel<br />
Bioinformatics Algorithm to Detect Conformational<br />
Epitopes <strong>of</strong> Jun a 1, The Major Allergen <strong>of</strong> Mountain<br />
Cedar Pollen<br />
Terumi Midoro-Horiuti, Assistant Pr<strong>of</strong>essor, Pediatrics,<br />
Galveston, TX, Ruby Tiwari, Postdoctoraltoral Fellow,<br />
Pediatrics, Galveston, TX, Surendra Negi, Postdoctoral<br />
Fellow, Biochemistry and Molecular Biology, Galveston, TX,<br />
Catherine Schein, Assistant Pr<strong>of</strong>essor, Biochemistry and<br />
Molecular Biology, Galveston, TX, Bo Ning, Research<br />
Assistant, Pediatrics, Galveston, TX, Randall Goldblum,<br />
Pr<strong>of</strong>essor, Pediatrics, Galveston, TX, Werner Braun,<br />
Pr<strong>of</strong>essor, Biochemistry and Molecular Biology, Galveston, TX
S12 Abstracts<br />
Rationale: Pollen hypersensitivity is a major cause <strong>of</strong><br />
seasonal airway disease world wide. We recently resolved<br />
the crystal structure and mapped the linear IgE epitopes <strong>of</strong><br />
Jun a 1. Heat and chemical denaturation <strong>of</strong> Jun a 1<br />
reduced the binding <strong>of</strong> human IgE, suggesting the importance<br />
<strong>of</strong> conformational epitopes for reactivity. Methods:<br />
To identify conformational IgE epitopes, we developed a<br />
panel <strong>of</strong> monoclonal antibodies (mAbs) against Jun a 1 and<br />
selected those that were heat sensitive and inhibited the<br />
binding <strong>of</strong> IgE from patient sera. These mAbs were used to<br />
screen a random peptide, phage display library. After<br />
repeated panning, phage clones were isolated and their<br />
peptide sequences determined. To compare the affinity<br />
and specificity <strong>of</strong> phage-expressed peptides the binding <strong>of</strong><br />
the phage to mAbs and patient IgE, and the ability <strong>of</strong> the<br />
phage to inhibit binding <strong>of</strong> Jun a 1 to the mAb were tested<br />
by ELISA. Consensus amino acids from selected phage were<br />
matched with clusters <strong>of</strong> amino acid residues exposed on<br />
the surface <strong>of</strong> Jun a 1, using GETAREA (http://www.scsb.<br />
utmb.edu/cgi-bin/get_a_form.tcl) and a program specifically<br />
designed to analyze similarity between the specific<br />
phage residues and surface patches on allergens. Results:<br />
Consensus amino acids from phage clones revealed common<br />
patterns <strong>of</strong> amino acids which were identical or<br />
similar to compact patches on the 3-D structure <strong>of</strong> Jun a 1<br />
and represent putative conformational epitopes. Conclusion:<br />
Bioinformatics search tools can play an important role<br />
in identifying the IgE epitopes on allergens with known<br />
structures or reliable 3D models.<br />
doi:10.1016/j.clim.2007.03.203<br />
OR.23 Antibodies Against Human Cytomegalovirus<br />
in the Pathogenesis <strong>of</strong> Atherosclerosis: A Gene Array<br />
Approach<br />
Antonio Puccetti, Pr<strong>of</strong>essor <strong>of</strong> Medicine, Gaslini<br />
Institute-Department <strong>of</strong> <strong>Immunology</strong>, Genova, Marzia Dolci,<br />
England Research Fellow, Gaslini Institute-Department <strong>of</strong><br />
<strong>Immunology</strong>, Genova, Dimitri Peterlana, PhD Student,<br />
University <strong>of</strong> Verona-Department <strong>of</strong> Internal Medicine,<br />
Verona, Riccardo Navone, Research Fellow, University <strong>of</strong><br />
Verona-Department <strong>of</strong> Internal Medicine, Verona, Caterina<br />
Bason, Research Fellow, University <strong>of</strong> Verona-Department <strong>of</strong><br />
Internal Medicine, Verona, Claudio Lunardi, Pr<strong>of</strong>essor <strong>of</strong><br />
Medicine, University <strong>of</strong> Verona-Department <strong>of</strong> Internal<br />
Medicine, Verona<br />
Human cytomegalovirus (hCMV) is involved in the<br />
pathogenesis <strong>of</strong> atherosclerosis. We have shown in patients<br />
with atherosclerosis that antibodies directed against the<br />
hCMV-derived proteins US28 and UL122 are able to induce<br />
endothelial cell damage and apoptosis <strong>of</strong> non-stressed<br />
endothelial cells through cross-rection with normally<br />
expressed surface molecules. Our aim was to dissect the<br />
molecular basis <strong>of</strong> such interaction and to investigate<br />
mechanisms linking innate immunity to atherosclerosis. We<br />
analysed the gene expression pr<strong>of</strong>iles in endothelial cells<br />
stimulated with antibodies affinity purified against either<br />
the UL122 or the US28 peptides. Real-time polymerase<br />
chain reaction was used to validate the microarray results.<br />
Supernatant <strong>of</strong> endothelial cells incubated with antibodies<br />
was analysed for the presence <strong>of</strong> Heat Shock Protein (HSP)<br />
60 and was used to assess stimulation <strong>of</strong> toll-like receptor-<br />
4 (TLR4). Antibodies against UL122 and US28 induced the<br />
expression <strong>of</strong> genes encoding for adhesion molecules,<br />
chemokines, growth factors and molecules involved in the<br />
apoptotic process together with other genes involved in<br />
the initiation and progression <strong>of</strong> atherosclerosis. HSP60 was<br />
released in the medium <strong>of</strong> cells incubated with anti-US28<br />
antibodies and was able to engage TLR4. Antibodies<br />
directed against hCMV modulate the expression <strong>of</strong> genes<br />
coding for molecules involved in the pathogenesis <strong>of</strong><br />
atherosclerosis. Moreover, endothelial cells exposed to<br />
such antibodies express HSP60 on the cell surface and<br />
release HSP60 in the medium able to activate TLR4. These<br />
data confirm that hCMV plays a crucial role in mediating<br />
the atherosclerotic process and that HSP60 is an endogenous<br />
ligand for TLR4 signaling.<br />
doi:10.1016/j.clim.2007.03.204<br />
OR.24 Differential Effects <strong>of</strong> Aire Gene Deficiency<br />
on the Development <strong>of</strong> Autoimmune<br />
Encephalomyelitis Induced by Two Representative<br />
Myelin Peptides<br />
Asako Tagawa, Research Fellow, National Institute <strong>of</strong><br />
Neuroscience, NCNP, Kodaira, Tokyo, Japan, Toshimasa<br />
Aranami, Section chief, Department <strong>of</strong> <strong>Immunology</strong>,<br />
National Institute <strong>of</strong> Neuroscience, NCNP, Kodaira, Tokyo,<br />
Japan, Mitsuru Matsumoto, Director, Division <strong>of</strong> Molecular<br />
<strong>Immunology</strong>, Institute for Enzyme Research, University <strong>of</strong><br />
Tokushima, Japan, Tokushima, Japan, Takashi Yamamura,<br />
Director, Department <strong>of</strong> <strong>Immunology</strong>, National Institute <strong>of</strong><br />
Neuroscience, NCNP, Kodaira, Tokyo, Japan<br />
Hazardous autoimmunity is prevented partly by clonal<br />
deletion <strong>of</strong> autoimmune T cells in the thymus. In the process<br />
referred to as central tolerance, autoimmune regulator<br />
(Aire) gene plays a pivotal role through transcriptional<br />
control <strong>of</strong> ectopic antigen expression. In fact, thymic<br />
expression <strong>of</strong> myelin antigens was shown to be involved in<br />
shaping Tcell repertoire that would mediate development <strong>of</strong><br />
EAE. Here we asked if Aire gene deficiency might alter<br />
manifestations <strong>of</strong> EAE induced with representative, encephalitogenic<br />
peptides myelin oligodendrocyte glycoprotein<br />
(MOG)35–55 and proteolipid protein (PLP)178–191 by using<br />
Aire−/− mice. It is thought that MOG is not expressed in the<br />
thymus (JCI 12: 544, 2003), whereas thymic expression <strong>of</strong><br />
PLP178–191 was previously proven (Nat Med 6:56, 2000).<br />
First, we assessed recall responses to the peptides in the<br />
immunized mice, and found that sensitized T cells from the<br />
Aire−/− produced a higher amount <strong>of</strong> IL-17 than from wildtype<br />
(WT) mice regardless <strong>of</strong> the peptides used for<br />
immunization. Anti-MOG35–55 response was also associated<br />
with a higher IFN-ã production in Aire−/−. However, there<br />
was no significant difference between Aire−/− and WT in<br />
IFN-ã production in response to PLP178–191, which resulted<br />
in an augmented Th17/Th1 ratio in PLP178–191-immunized<br />
mice. PLP178–191 immunization induced more serious EAE in<br />
Aire−/− than WT, whereas such a difference could not be
Abstracts<br />
seen after sensitization with MOG35–55. As such, Aire gene<br />
deficiency would differentially alter autoimmune responses<br />
according to the presence or absence <strong>of</strong> the directed epitope<br />
in the thymus.<br />
doi:10.1016/j.clim.2007.03.205<br />
Stem Cell/Immunodeficiency/HIV<br />
Friday, June 8<br />
2:45 pm−4:45 pm<br />
OR.25 A Novel RAG-1 Null Mutant Causes T-B-NK+<br />
SCID in Native Americans<br />
Morton Cowan, Pr<strong>of</strong>essor, Pediatric Bone Marrow Transplant<br />
Division, University <strong>of</strong> California, San Francisco Children’s<br />
Hospital, San Francisco, CA<br />
Severe combined immunodeficiency disease (SCID) is the<br />
most serious inherited immunological deficit. Ubiquitous<br />
DNA double strand repair factors in the non-homologous<br />
ending joining (NHEJ) pathway resolve DNA double-strand<br />
breaks (DSB) during V(D)J recombination <strong>of</strong> T and B<br />
lymphocyte receptor genes. We have described a null<br />
mutation <strong>of</strong> the Artemis gene that has relatively high<br />
occurrence among Athabascan-speaking Native Americans,<br />
which results in a complete absence <strong>of</strong> T and B lymphocytes<br />
and increased cellular sensitivity to ionizing radiation<br />
causing radiosensitive-SCID (RS-SCID). Recently we identified<br />
a novel RAG-1 null mutation, R776W, in three related<br />
children from the Dine Indian Tribe in the Canadian<br />
Northwest Territories who manifest a classic T-B-NK+ SCID<br />
phenotype. We found a normal pattern <strong>of</strong> radiation<br />
sensitivity in skin fibroblast cell lines from these patients,<br />
indicating that the NHEJ pathway is intact. The impaired<br />
activity <strong>of</strong> this RAG-1 mutant in V(D)J recombination was<br />
revealed by an enhanced green fluorescent protein (EGFP)<br />
based assay. Additional studies <strong>of</strong> the RAG-1 null mutant<br />
will evaluate DNA binding and cleavage activity, hairpin<br />
formation in vitro as well as catalysis <strong>of</strong> other DNA strand<br />
transfer reactions such as transposition. Our current results<br />
suggest that the T-B-NK+ SCID phenotype is triggered by this<br />
novel RAG-1 mutant.<br />
doi:10.1016/j.clim.2007.03.206<br />
OR.26 HIV-1 Infectivity Inhibited by Binding <strong>of</strong> the<br />
Green Tea Catechin, Epigallocatechin Gallate, to<br />
CD4<br />
Christina Nance, Instructor, Baylor College <strong>of</strong> Medicine,<br />
Houston, TX, Edward Siwak, Assistant Pr<strong>of</strong>essor, Baylor<br />
College <strong>of</strong> Medicine, Virology, Houston, TX, Aarthi Ram,<br />
Research Technician, Baylor College <strong>of</strong> Medicine, Pediatrics,<br />
Houston, TX, Van Willis, Research Technician, Baylor College<br />
<strong>of</strong> Medicine, Pediatrics, Houston, TX, Susan Westerfield,<br />
Research Technician, Baylor College <strong>of</strong> Medicine, Pediatrics,<br />
Houston, TX, William Shearer, Pr<strong>of</strong>essor, Baylor College <strong>of</strong><br />
Medicine, Pediatrics, Houston, TX<br />
Binding <strong>of</strong> HIV-1 envelope glycoprotein, gp120, to the CD4<br />
T cell receptor results in HIV-1 infection. Previously, we have<br />
presented evidence <strong>of</strong> high affinity binding <strong>of</strong> the green tea<br />
catechin, epigallocatechin gallate (EGCG), to the CD4<br />
molecule at the gp120 binding pocket. We now present<br />
evidence that EGCG inhibits the binding <strong>of</strong> gp120 on human<br />
CD4+ T cells and PBMC and prevents the HIV-1 infectivity <strong>of</strong><br />
human macrophages and PBMC. Binding studies utilized flow<br />
cytometry. HIV-1 infectivity was assessed by HIV-1 p24 EIA. Mtropic<br />
(R5) (strains SF162, Bal), T-tropic (X4) (strain IIIB), and<br />
dual tropic (R5X4) (strain 89.6) HIV-1 isolates were used.<br />
EGCG markedly inhibited the binding <strong>of</strong> HIV-1-gp120IIIB to<br />
CD4+ T cells in a dose-dependent manner at physiologically<br />
relevant levels (32% at 0.2 μM pb0.05, 42% at 2 μM and 47%<br />
at 20 μM pb0.01) and higher (55% at 50 μM and 71% at 100 μM<br />
pb0.001). EGCG significantly inhibited the infectivity <strong>of</strong><br />
human macrophages by M- and dual-tropic HIV-1 strains at<br />
200–400 TCID50 in a dose-dependent manner. There was<br />
100% inhibition <strong>of</strong> p24 production by EGCG (25–100 μM)<br />
(pb0.0001), 95% at 12 μM, and 79% at 6 μM (pb0.001). The<br />
response by human PBMC was similar. The control catechin<br />
did not alter gp120 binding nor inhibit HIV-1 infectivity. We<br />
conclude that EGCG is able to significantly reduce the<br />
attachment <strong>of</strong> gp120 to CD4, along with a decrease in the<br />
infectivity <strong>of</strong> HIV-1 to target cells. The competitive binding<br />
properties <strong>of</strong> EGCG for the CD4 binding sites by gp120 may<br />
translate to an HIV-1 preventative strategy.<br />
doi:10.1016/j.clim.2007.03.207<br />
S13<br />
OR.27 Impaired Dendritic Cell Function in<br />
Ectodermal Dysplasia with Immune Deficiency is<br />
Linked to Defective NEMO Ubiquitination<br />
Stephan Temmerman, Postdoctoral Fellow, NIH-NIAID-LHD,<br />
Bethesda, MD, Chi Ma, Senior Biologist, NIH-NIAID-LHD,<br />
Bethesda, MD, Ashish Jain, PI, NIH-NIAID-LHD, Bethesda, MD<br />
NF-kappaB essential modulator (NEMO) regulates the<br />
activation <strong>of</strong> the transcription factors NF-kappaB. Alterations<br />
in NEMO cause ectodermal dysplasia with immunodeficiency<br />
(EDI), a disorder that is characterized by<br />
defects in innate and adaptive immunity as well as<br />
abnormal development <strong>of</strong> ectoderm-derived tissues.<br />
Because the biochemical mechanism by which NEMO<br />
mutations cause immune dysfunction remains undefined,<br />
we investigated the effect <strong>of</strong> a cysteine to arginine<br />
substitution found in the NEMO zinc finger domain on<br />
dendritic cell (DC) function. Following CD40 ligand<br />
(CD40L) stimulation <strong>of</strong> DCs from two EDI patients, we<br />
found that lysine 63-linked polyubiquitination <strong>of</strong> NEMO,<br />
which promotes activation <strong>of</strong> the IKK complex, was<br />
absent. We associated this defect with preserved RelA<br />
but absent c-Rel activity. Therefore, CD40L-stimulated EDI<br />
DCs failed to synthesize the c-Rel dependant cytokine IL-<br />
12, displayed reduced cell aggregates and membrane<br />
extensions, and failed to support allogeneic lymphocyte<br />
proliferation. We utilized gene expression pr<strong>of</strong>iling to<br />
compare the genes induced by CD40L in normal DCs but<br />
not in EDI DCs. In this gene set, we identified a number
S14 Abstracts<br />
<strong>of</strong> molecules that are likely to be critical to the<br />
maturation and function <strong>of</strong> DCs. In contrast, downstream<br />
NF-kappaB activity, DC maturation, and NEMO polyubiquitination<br />
were normal in EDI DCs following stimulation with<br />
the TLR4 ligand. These results show for the first time that<br />
CD40 and TLR4 use different signaling pathways to<br />
ubiquitinate NEMO, and <strong>of</strong>fer insight into how a mutation<br />
in the zinc finger domain <strong>of</strong> NEMO leads to pathway<br />
specific defects in NF-kappaB signaling and thus immune<br />
deficiency.<br />
doi:10.1016/j.clim.2007.03.208<br />
OR.28 Treatment <strong>of</strong> HIV-infected Patients with<br />
Vitamin D-binding Protein Derived Macrophage<br />
Activating Factor (GcMAF) Eradicates HIV-Infection<br />
Nobuto Yamamoto, Director, Socrates Institute for<br />
Therapeutic <strong>Immunology</strong>, Philadelphia, PA, Masumi Ueda,<br />
<strong>Immunology</strong>, Chief, Microbiology, Philadelphia, PA,<br />
Charles Benson, Head, Infectious Diseases, School <strong>of</strong><br />
Veterinary Medicine, University <strong>of</strong> Pennsylvania, Kennett<br />
Square, PA<br />
Serum vitamin D-binding protein (known as Gc protein) is<br />
the precursor for the principal macrophage activating factor<br />
(MAF). The MAF precursor activity <strong>of</strong> serum Gc protein <strong>of</strong><br />
HIV-infected patients was lost or reduced because Gc<br />
protein is deglycosylated by serum α-N-acetylgalactosaminidase<br />
(Nagalase) secreted from HIV-infected cells. Since<br />
Nagalase is the intrinsic component <strong>of</strong> gp120, serum<br />
Nagalase activity is the sum <strong>of</strong> enzyme activities expressed<br />
in both HIV virions and envelope proteins released from HIVinfected<br />
cells. Because <strong>of</strong> Nagalase being an HIV viral<br />
protein and immunogenic, serum Nagalase was already<br />
complexed with anti-HIV immunoglobulin G (IgG) in patient<br />
blood stream. These antibodies, however, were largely not<br />
neutralizing antibodies. The IgG-bound viral proteins<br />
retained Nagalase activity that deglycosylates Gc protein.<br />
Therefore, macrophages <strong>of</strong> HIV-infected patients having<br />
deglycosylated Gc protein cannot be activated, leading to<br />
immunosuppression. Stepwise treatment <strong>of</strong> purified Gc<br />
protein with immobilized beta-galactosidase and sialidase<br />
generated the most potent macrophage activating factor<br />
(termed GcMAF), which produces no side effect in humans.<br />
Macrophages activated by administration <strong>of</strong> GcMAF<br />
(100 ng/patient) develop a large amount <strong>of</strong> Fc receptors<br />
as well as enormous variation <strong>of</strong> receptors that recognize<br />
IgG bound and unbound HIV virions. Thus, macrophages<br />
activated by GcMAF preferentially phagocytize IgG-bound<br />
HIV virions via Fc receptor mediation. Cells latently<br />
infected with HIV are unstable and spontaneously release<br />
the virions at a high rate. After less than 18 weekly<br />
administrations <strong>of</strong> 100 ng GcMAF for twenty-one nonanemic<br />
patients, they exhibited low serum Nagalase activities<br />
equivalent to healthy controls, indicating eradication <strong>of</strong> HIV<br />
infection.<br />
doi:10.1016/j.clim.2007.03.209<br />
OR.29 Impaired In Vitro Regulatory T Cell Function<br />
Associated with Wiskott-Aldrich Syndrome<br />
Marsilio Adriani, Visiting Fellow, NIH/NHGRI, Bethesda, MD,<br />
Joseph Aoki, Visiting Fellow, NIH/NHGRI, Bethesda, MD,<br />
Reiko Horai, Postdoctoral Fellow, NIH/NHGRI, Bethesda,<br />
MD, Akihiro Kon, England Postdoctoral, Fellow, Bethesda,<br />
MD, Angela M. Thornton, Staff Scientist, NIH/NIAID,<br />
Bethesda, MD, Martha Kirby, Senior Research Assistant,<br />
NIH/NHGRI, Bethesda, MD, Stacie M. Anderson, Senior<br />
Research Assistant, NIH/NHGRI, Bethesda, MD, Richard M.<br />
Siegel, Investigator, NIH/NIAMS, Bethesda, MD, Pamela L.<br />
Schwartzberg, Investigator, NIH/NHGRI, Bethesda, MD,<br />
Fabio Candotti, Senior Investigator, NIH/NHGRI, Bethesda,<br />
MD<br />
Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency<br />
characterized by thrombocytopenia, eczema, recurrent<br />
infections and high incidence <strong>of</strong> malignancy. This disease is<br />
caused by mutations <strong>of</strong> the WAS protein (WASP) gene, a key<br />
regulator <strong>of</strong> actin polymerization. WAS patients show high<br />
incidence <strong>of</strong> autoimmune disorders (∼40–70%) the occurrence<br />
<strong>of</strong> which, however, does not correlate with the severity <strong>of</strong> the<br />
other classical hallmarks <strong>of</strong> the disease. A central question in<br />
understanding the pathophysiology <strong>of</strong> WAS is why immunodeficient<br />
patients develop symptoms suggestive <strong>of</strong> hyperactivation<br />
<strong>of</strong> immune compartments, such as eczema and autoimmune<br />
diseases. Since defects in CD4+CD25+ regulatory T cells (Treg)<br />
have been associated with autoimmunity, we examined the<br />
presence and function <strong>of</strong> these cells in WAS patients and Waspdeficient<br />
mice. Wasp-deficient patients and mice develop Treg<br />
cells that express Foxp3 with no detectable differences in<br />
frequency compared to controls. However, in vitro suppression<br />
assays showed that these Treg cells have impaired inhibitory<br />
function in vitro,which,inmice,couldonlybepartiallyrescued<br />
by pre-activation with exogenous IL-2. The demonstration <strong>of</strong><br />
impaired in vitro suppressor function in WASp-deficient Treg<br />
cells suggests that defective regulatory Tcell function may be an<br />
important factor contributing to immune dysregulation in WAS.<br />
doi:10.1016/j.clim.2007.03.210<br />
OR.30 Regulatory T Cell Abnormalities Associated<br />
with Aberrant CD4+ T Cell Responses in Patients<br />
with Immune Reconstitution Disease (IRD)<br />
Nabila Seddiki, Research Fellow, Centre For <strong>Immunology</strong><br />
and National Centre in HIV Epidemiology and <strong>Clinical</strong><br />
Research (UNSW), Darlinghurst<br />
Up to 30% <strong>of</strong> patients with HIV commencing antiretroviral<br />
therapy (ART) late in the disease restore a pathogen-specific<br />
cellular immune response that is immunopathological and<br />
causes disease referred as immune reconstitution disease or<br />
IRD. The immunopathogenesis <strong>of</strong> IRD is not well understood and<br />
is likely to be a consequence <strong>of</strong> an aberrant reconstitution <strong>of</strong><br />
certain T cell subsets. In a cross-sectional study, using HIV<br />
patients who developed mycobacterial IRD, we show that in<br />
comparison to those starting ARTand not developing IRD, CD4+ T<br />
cells specific for MTB and M. avium complex (MAC) antigens,<br />
produce high levels <strong>of</strong> IFN-g and IL-2 (Pb0.01) and proliferate<br />
strongly when compared to controls. We postulated that deficits<br />
in regulatory T cells (Tregs) numbers may play a central role in
Abstracts<br />
the pathogenesis <strong>of</strong> IRD. When Tregs numbers were examined by<br />
using our recently described marker CD127 (IL-7R), we found a<br />
significant expansion <strong>of</strong> memory CD127loFoxp3+CD25+ Tregs in<br />
IRD patients compared to controls. To further investigate<br />
abnormalities in T cell responses, we examined a range <strong>of</strong><br />
cytokines in the plasma <strong>of</strong> patients and controls and found<br />
increased levels <strong>of</strong> IL-4, IL-6, IL-7 and IL-21. Interestingly, we<br />
show that some <strong>of</strong> these cytokines inhibit strongly Treg<br />
suppression. Furthermore, we show that suppressors and<br />
responders from these patients present defect to regulate CD4<br />
T cell immune responses. Altogether, these data suggest that<br />
despite the high Treg expansion in IRD, their ability to induce<br />
suppression and turn <strong>of</strong>f these aberrant immune responses is<br />
compromised.<br />
doi:10.1016/j.clim.2007.03.211<br />
OR.31 Liver Tissue Cells Inhibit Immune Responses<br />
by Inducing T Cell Apoptosis: An Application in Islet<br />
Transplantation<br />
Lina Lu, Associate Pr<strong>of</strong>essor, Cleveland Clinic, Cleveland,<br />
OH, Cheng-Hsu Chen, Research Fellow, Department <strong>of</strong><br />
<strong>Immunology</strong>, Cleveland Clinic, Cleveland, OH, Zhenyu Yin,<br />
Research Fellow, Department <strong>of</strong> <strong>Immunology</strong>, Cleveland<br />
Clinic, Cleveland, OH, John J. Fung, Pr<strong>of</strong>essor, Department<br />
<strong>of</strong> General Surgery, Cleveland Clinic, Cleveland, OH,<br />
Liang-Mou Kuo, Research Fellow, Department <strong>of</strong><br />
<strong>Immunology</strong>, Cleveland Clinic, Cleveland, OH, Shiguang<br />
Qian, Associate Pr<strong>of</strong>essor, Department <strong>of</strong> <strong>Immunology</strong>,<br />
General Surgery, Cleveland Clinic, Cleveland, OH<br />
Organ transplantation has been successful for decades, but<br />
outcome <strong>of</strong> cell transplants remains poor. This is true in animal<br />
models. Liver transplants are spontaneously accepted in mice,<br />
but hepatocyte allografts are acutely rejected, suggesting an<br />
immunosuppressive effect <strong>of</strong> liver tissue cells. In this study, we<br />
demonstrated a pr<strong>of</strong>ound in vitro immune inhibitory activity <strong>of</strong><br />
activated (but not quiescent) hepatic stellate cells (HSC) that<br />
are well known to be involved in liver fibrosis. Activation <strong>of</strong> HSC<br />
by exposure to IFN-γ or directly contact with activated T cells<br />
resulted in upregulation <strong>of</strong> B7-H1, inhibitory cytokines (IL-6, IL-<br />
10, TGF-β), and enhanced T cell apoptotic death (annexin<br />
V + 7AAD− ), which is largely mediated by B7-H1, since B7-H1−/− HSC triggered significantly less apoptosis. To explore potential<br />
clinical application, BALB/c (H2d ) islets (300) mixed with<br />
activated HSC (3×10 5 )<strong>of</strong>B6(H2b )miceweretransplanted<br />
under renal capsule <strong>of</strong> chemically diabetic B6 recipients,<br />
showing a marked prolongation <strong>of</strong> islet graft survival [from<br />
median survival time (MST) <strong>of</strong> 13 days in control to N60 days<br />
(pb0.01)] with 55% <strong>of</strong> recipients being euglycemia for N60 days.<br />
None <strong>of</strong> islet-only recipients remained euglycemia for N17 days.<br />
Removal <strong>of</strong> islet-grafted kidney resulted in quick glycemia.<br />
Immunochemistry showed that prolongation <strong>of</strong> islet allografts by<br />
co-transplanted HSC was associated with upregulated apoptotic<br />
activity (TUNEL) and less T cell infiltration (CD3), which<br />
appeared to be mediated by B7-H1, as co-transplant with B7-<br />
H1 −/− HSC markedly reduced their protection to islet allografts<br />
(MST 26 days). These scientific observations may lead to<br />
substantial improvement <strong>of</strong> islet transplantation.<br />
doi:10.1016/j.clim.2007.03.212<br />
OR.32 BAFF Receptor Expression in CVID Patients<br />
Marta Rizzi, MD PhD, <strong>Clinical</strong> Research Unit for<br />
Rheumatology, Freiburg/Br., Ulrich Salzer, PhD, Division <strong>of</strong><br />
Rheumatology and <strong>Clinical</strong> <strong>Immunology</strong>, Freiburg/Br., Klaus<br />
Warnatz, MD PhD, Division <strong>of</strong> Rheumatology and <strong>Clinical</strong><br />
<strong>Immunology</strong>, Freiburg/Br., Sigune Goldacker, MD, Division <strong>of</strong><br />
Rheumatology and <strong>Clinical</strong> <strong>Immunology</strong>, Freiburg/Br.,<br />
Stefanie Hamm, Student, <strong>Clinical</strong> Research Unit for<br />
Rheumatology, Freiburg/Br., Hans Hartmut Peter, Pr<strong>of</strong>essor<br />
MD PhD, Division <strong>of</strong> Rheumatology and <strong>Clinical</strong> <strong>Immunology</strong>,<br />
Freiburg/Br., Hermann Eibel, PhD, <strong>Clinical</strong> Research Unit<br />
for Rheumatology, Freiburg/Br.<br />
BAFF receptor (BAFF-R) is a member <strong>of</strong> the TNF-R superfamily<br />
and is expressed mainly on mature B cells. Common<br />
variable immune deficiency (CVID) is a primary immune<br />
deficiency characterized by hypogammaglobulinemia. Several<br />
genetic defects contributing to this phenotype have been<br />
identified (mutations in ICOS gene, TACI gene, CD19 gene). Our<br />
group recently identified a deletion mutation within the BAFF-<br />
R gene, removing the transmembrane domain <strong>of</strong> the protein.<br />
The homozygous mutation precludes the syntheses <strong>of</strong> functional<br />
BAFF-R. Therefore, B cell development in the BAFF-R<br />
deficient patient is arrested at the transitional B cell stage. As<br />
a result, the patient lacks IgG-producing plasma cells and is<br />
severely B lymphopenic. Searching for additional BAFF-R mutations<br />
we screened a subgroup <strong>of</strong> our cohort <strong>of</strong> CVID patients<br />
with very low number <strong>of</strong> B cells in peripheral blood (b5%) for<br />
BAFF-R expression. We found that 8 out <strong>of</strong> the 13 patients<br />
presented low BAFF-R expression on the B cells surface (mean<br />
fluorescence 21.9 SD 4.3, control mean fluorescence 58.9 SD<br />
3.6), and low BAFF binding activity. Low BAFF-R expression<br />
significantly correlates with a low number <strong>of</strong> detectable<br />
memory B cells (CD19+CD27+). Sequencing <strong>of</strong> the BAFF-R<br />
alleles excluded known mutations in the BAFF-R gene. We then<br />
investigated if low BAFF-R expression might result from<br />
regulatory defects. Stimulation <strong>of</strong> the BCR, TLR 9 and/or<br />
CD40 increased BAFF-R expression. Therefore, low levels <strong>of</strong><br />
BAFF-R expression in CVID patients may result from regulatory<br />
defects and contribute to antibody deficiency.<br />
doi:10.1016/j.clim.2007.03.213<br />
S15
S16 Abstracts<br />
F.1 Thyroid Function and Prevalence <strong>of</strong><br />
Anti-Thyroid Antibodies in Iranian Patients with<br />
Type 1 Diabetes Mellitus: Influences <strong>of</strong> Age and Sex<br />
Faranak Sharifi, Endocrinologist, Zanjan University <strong>of</strong><br />
Medical Sciences, Zanjan, Leila Ghasemi Hashtroodi,<br />
Resident for Internal Medicine, Zanjan University <strong>of</strong> Medical<br />
Sciences, Zanjan, Yahya Jaberi, Assistant Pr<strong>of</strong>essor, Zanjan<br />
University <strong>of</strong> Medical Sciences, Zanjan<br />
Objective: Type 1 diabetes mellitus is frequently associated<br />
with autoimmune thyroid disease (ATD). Genetic<br />
susceptibility to autoantibody formation in association with<br />
ATD and type 1 diabetes mellitus has been described with<br />
varying frequencies, but there is still debate about the<br />
situation in Iran. We have, therefore, investigated the<br />
prevalence <strong>of</strong> anti-thyroid peroxidase (anti-TPO) and antithyroglobulin<br />
(anti-TG) antibodies in type 1 diabetic patients,<br />
and compared the effect <strong>of</strong> age and sex on the thyroid<br />
autoimmunity in patients with type 1 diabetes mellitus in<br />
Iran. Subjects and Methods: Ninety one subjects with type 1<br />
diabetes mellitus and one hundred and sixty three unrelated<br />
normal controls under age thirty were recruited for the<br />
detection <strong>of</strong> anti-TPO and anti-TG. Radioimmunoassay was<br />
used for anti-TPO and anti-TG detection respectively.<br />
Results: Among 91 type 1 diabetic patients, 36 (39.6%)<br />
were positive for anti-TPO and 27 (30%) were positive for<br />
anti-TG. Anti-TPO antibodies were detected only in 6.7% <strong>of</strong><br />
control group. Compared with those without thyroid autoimmunity,<br />
there was a female preponderance for the type 1<br />
diabetic patients with thyroid autoimmunity (female: male,<br />
28:14 vs. 28:20 respectively). Among the type 1 diabetic<br />
patients those with thyroid autoimmunity tended to have<br />
older age (p: 0.04) and higher TSH concentration (p: 0.03).<br />
Patients with high anti-TPO levels had longer duration <strong>of</strong><br />
diabetes (p: 0.02). Conclusion: The presence <strong>of</strong> anti-TPO in<br />
39.6% <strong>of</strong> our type 1 diabetic patients compared with 8.5% <strong>of</strong><br />
normal subjects confirmed the strong association <strong>of</strong> ATD and<br />
type 1 diabetes mellitus.<br />
doi:10.1016/j.clim.2007.03.214<br />
Poster Sessions<br />
Friday, June 8<br />
7:30 am−7:00 pm<br />
Authors Present: 5:45 pm−7:00 pm<br />
F.2 Reduced Foxp3 and Interleukin 10 Expression<br />
During the Partial Remission Phase <strong>of</strong> Type 1<br />
Diabetes in Children<br />
Srinath Sanda, <strong>Clinical</strong> Fellow, University <strong>of</strong> California, San<br />
Diego School <strong>of</strong> Medicine, San Diego, CA, Bart Roep,<br />
Associate Pr<strong>of</strong>essor, Department <strong>of</strong> Immunohematology and<br />
Blood Transfusion Leiden University Medical Center, Leiden,<br />
Matthias von Herrath, Pr<strong>of</strong>essor, Developmental<br />
<strong>Immunology</strong> at the La Jolla Institute for Allergy and<br />
<strong>Immunology</strong>, La Jolla, CA<br />
Background: Some children undergo a reduction in insulin<br />
requirements after diagnosis and initiation <strong>of</strong> insulin<br />
therapy. The exact mechanism underlying this partial<br />
remission phase (also known as the honeymoon phase) <strong>of</strong><br />
type 1 diabetes in children is not known. Study Design: We<br />
conducted a cross-sectional study comparing peripheral<br />
blood T cell responses by ELISPOT and flow cytometry<br />
between a cohort <strong>of</strong> newly diagnosed children with type 1<br />
diabetes at initiation <strong>of</strong> insulin therapy and a cohort <strong>of</strong><br />
children in the honeymoon phase. Children were classified<br />
in the honeymoon phase if they were between 3 and<br />
9 months from diagnosis, had a total daily insulin requirement<br />
<strong>of</strong> b0.5 U/kg/day, and had experienced a N50%<br />
reduction in their total daily insulin dose since diagnosis. Six<br />
children were recruited in each group. Results: Honeymoon<br />
patients were on average 7.4 months from diagnosis and had<br />
significantly reduced insulin doses and hemoglobin A1c<br />
levels compared to new onset patients. Both cohorts had<br />
similar numbers <strong>of</strong> peripheral CD4+, CD8+, and CD4+CD25+<br />
cells. Honeymoon patients had fewer numbers <strong>of</strong> peripheral<br />
CD4+CD25+Foxp3+ cells compared to newly diagnosed<br />
patients (37.3% ±23% vs. 57.8±27.8% p=0.054). ELISPOT<br />
assays using epitopes to GAD65, IA2, and proinsulin,<br />
demonstrated a trend for increased interferon-gamma<br />
production and reduced interleukin-10 production in the<br />
honeymoon patients. Conclusion: Reduced numbers <strong>of</strong><br />
peripheral blood CD4+CD25+Foxp3+ and interleukin 10<br />
producing cells are seen in type 1 diabetes patients during<br />
their honeymoon phase compared to patients at diagnosis.<br />
doi:10.1016/j.clim.2007.03.215<br />
F.3 A Novel Cell-based Therapeutic Approach to<br />
Prevent and Cure Autoimmune Type I Diabetes in<br />
NOD Mice<br />
Dong Zhang, Postdoctoral Research Fellow, Harvard Medical<br />
School, Beth Israel Deaconess Medical Center, Boston, MA,<br />
Yan Tian, Research Assistant, Harvard Medical School, Beth<br />
Israel Deaconess Medical Center, Boston, MA, Nicolas<br />
Degauque, Research Fellow, Harvard Medical School, Beth<br />
Israel Deaconess Medical Center, Boston, MA, Wei Yang,<br />
Research Fellow, Harvard Medical School, Beth Israel<br />
Deaconess Medical Center, Boston, MA, Allison Mikita,<br />
Research Assistant, Harvard Medical School, Beth Israel<br />
Deaconess Medical Center, Boston, MA, Xin Xiao Zheng,<br />
Assistant Pr<strong>of</strong>essor, Harvard Medical School, Beth Israel<br />
Deaconess Medical Center, Boston, MA
Abstracts<br />
Regulatory T cells play vital roles in maintaining peripheral<br />
tolerance to auto- and alloantigens. Further characterization<br />
<strong>of</strong> the function and development <strong>of</strong> these regulatory<br />
T cells will contribute to understanding <strong>of</strong> the intrinsic and<br />
extrinsic mechanisms for peripheral tolerance. Our study<br />
showed that proliferated CD4+ T cells could convert to CD4−<br />
CD8−CD3+ (DN) regulatory T cells after either allogeneic or<br />
syngeneic DC triggered proliferation, and IL-2 and IL-15<br />
enhanced the conversion. Converted DN T cells were<br />
resistant to AICD and expressed unique cell markers and<br />
gene pr<strong>of</strong>ile. Converted DN T cells exerted powerful<br />
inhibition on the same, but not third party, alloantigen<br />
triggered proliferation <strong>of</strong> naive CD4+CD25− T effectors. It<br />
was notable that at a per cell base comparison, converted DN<br />
T cells were more potent in suppressing alloantigen trigged<br />
naive T cells proliferation than naive and proliferated CD4+<br />
CD25+ Tregs. Perforin, a cytotoxic cytokine, which was highly<br />
expressed by DN T cells, played a role in DN T cell mediated<br />
suppression. Adoptive transferring <strong>of</strong> T cells from new onset<br />
diabetic NOD mice precipitated a rapid onset <strong>of</strong> diabetes in<br />
NOD/SCID recipients. Co-transferring <strong>of</strong> DN T cells (in vitro<br />
converted from CD4+ T cells from new onset diabetic NOD)<br />
with diabetogenic T cells significantly delay the onset <strong>of</strong><br />
diabetes in NOD/SCID mice (P=0.0012). Moreover, the small<br />
number <strong>of</strong> converted DN can reverse diabetes after disease<br />
onset in NOD mice. The results <strong>of</strong> using ex vivo CD4+ T cells<br />
converted DN T cells in NOD mouse model support the<br />
concept and the feasibility <strong>of</strong> potentially utilizing this novel<br />
cell-based therapeutic approach clinically for the treatment<br />
<strong>of</strong> autoimmune diseases.<br />
doi:10.1016/j.clim.2007.03.216<br />
F.4 Innate Immune Modulation <strong>of</strong> Type 1 Diabetes<br />
with Glycosphingolipids<br />
Dalam Ly, Graduate Student, University <strong>of</strong> Western Ontario,<br />
Microbiology and <strong>Immunology</strong>, London, ON, Canada, Terry<br />
Delovitch, Senior Scientist/Pr<strong>of</strong>essor, Robarts Research<br />
Institute/University <strong>of</strong> Western Ontario, London, ON,<br />
Canada<br />
The pathogenesis <strong>of</strong> T1D may be mediated by functional<br />
deficiencies in CD4+CD25+Foxp3+ Tregs and iNKT cells. As<br />
self-reactive Tregs and iNKT cells both protect NOD mice<br />
from T1D, we investigated whether iNKT-Treg collaboration<br />
is required for this protection. While α-galactosylceramide<br />
(α-GalCer) therapy does not alter the proliferation, clonal<br />
expansion or function <strong>of</strong> Tregs, protection from T1D by Tregs<br />
does not require iNKT cell activity. However, Treg activity is<br />
required for α-GalCer induced protection from T1D by iNKT<br />
cells. Significant decreases in IFN-γ and IL-4 secretion and<br />
cellular (T, B, NK, DC) transactivation by iNKT cells were<br />
detected in the presence <strong>of</strong> functional Tregs. Thus, during<br />
α-GalCer therapy <strong>of</strong> T1D, activated iNKT cells transactivate<br />
Tregs that may then downregulate iNKT cell responses and<br />
return iNKT cells to a homeostatic level <strong>of</strong> activation. Such<br />
Treg-dependent homeostatic control <strong>of</strong> iNKT cell activity<br />
may explain how Treg/iNKT collaboration protects from<br />
T1D. In contrast, we found that Treg activity is not required<br />
for protection from T1D induced by the C20:2 analog <strong>of</strong> α-<br />
GalCer, which possesses a shortened (C20) fatty acyl chain<br />
and cis-unsaturations at carbons 11 and 14. This may arise<br />
from the ability <strong>of</strong> C20:2 to elicit significantly reduced IFN-γ<br />
and enhanced IL-4 secretion by iNKT cells relative to α-<br />
GalCer. Thus, α-GalCer and C20:2 may differ in their<br />
requirement <strong>of</strong> Tregs for iNKT mediated protection from<br />
T1D. Ongoing studies <strong>of</strong> the mechanism(s) <strong>of</strong> this differential<br />
requirement <strong>of</strong> Tregs for protection from T1D may<br />
have significant implications for innate immunity focused<br />
T1D therapeutic approaches.<br />
doi:10.1016/j.clim.2007.03.217<br />
F.5 Targeted Cellular Gene Therapy Using IL-4<br />
Secreting DCs Corrects Immune Dysregulations and<br />
Prevents Diabetes in NOD Mice<br />
Remi J. Creusot, Postdoctoral Fellow, Stanford University,<br />
Department <strong>of</strong> Medicine, Stanford, CA, Shahriar Yaghoubi,<br />
Research Associate, Stanford University, Department <strong>of</strong><br />
Radiology, Stanford, CA, Keiichi Kodama, Postdoctoral<br />
Fellow, Stanford University, Department <strong>of</strong> Medicine,<br />
Stanford, CA, Sam S. Gambhir, Pr<strong>of</strong>essor, Stanford<br />
University, Department <strong>of</strong> Radiology, Stanford, CA, Demi<br />
Dang, Research Assistant, Stanford University, Department<br />
<strong>of</strong> Medicine, Stanford, CA, C. Garrison Fathman, Pr<strong>of</strong>essor,<br />
Stanford University, Department <strong>of</strong> Medicine, Stanford, CA<br />
A deficit in IL-4 has been associated with the development<br />
<strong>of</strong> type 1 diabetes in man and in the non-obese diabetic<br />
(NOD) mouse. We set out to correct this deficiency by<br />
delivering IL-4 using bone marrow-derived dendritic cells<br />
transduced to express IL-4 (DC/IL-4). DC/IL/4 were injected<br />
into 12-week-old female NOD mice with advanced prediabetic<br />
insulitis. After either intravenous (iv) or intraperitoneal<br />
(ip) injection <strong>of</strong> DC/IL-4, the onset <strong>of</strong> the disease was<br />
delayed for ∼5 weeks and the majority <strong>of</strong> the treated mice<br />
were protected until at least 30 weeks <strong>of</strong> age. Using<br />
bioluminescence imaging and biodistribution assays, we<br />
demonstrated that iv-injected DCs trafficked primarily to<br />
the spleen and pancreatic lymph nodes (PLN), with little or<br />
no homing to other lymph nodes. While equally effective, ipinjected<br />
DCs showed preferential migration to the PLN, with<br />
some homing to the pancreas. We then asked what effect<br />
DC/IL-4 had in the PLN by performing cDNA microarray<br />
analysis. Initial studies demonstrated that many genes were<br />
significantly dysregulated in the PLN <strong>of</strong> 12-week-old female<br />
NOD mice, compared to non-diabetic congenic NOD.B10<br />
mice. Interestingly, the expression level <strong>of</strong> most <strong>of</strong> these<br />
dysregulated genes (N200), in addition to IL-4 itself, was<br />
brought towards normal levels as early as 3 days after DC/IL-<br />
4 treatment. MHC-deficient DC/IL-4 had no protective<br />
effect, demonstrating that interaction <strong>of</strong> the transduced<br />
DCs with T cells was required for the therapeutic effect. In<br />
conclusion, the effects <strong>of</strong> DC/IL-4 were localized, rapid, long<br />
lasting and characterized by the normalization <strong>of</strong> many<br />
dysregulated genes.<br />
doi:10.1016/j.clim.2007.03.218<br />
S17
S18 Abstracts<br />
F.6 Plasmacytoid Dendritic Cells Induction by a<br />
Self-peptide Ep1.B Derived from Apolipoprotein E<br />
Prevent Autoimmune Diabetes<br />
Enayat Nikoopour, Postdoctoral Fellow, Department <strong>of</strong><br />
Microbiology and <strong>Immunology</strong> and Robarts Research<br />
Institute, London, ON, Canada, Tracey A. Stephens,<br />
Graduate Student, Department <strong>of</strong> Microbiology and<br />
<strong>Immunology</strong> and Robarts Research Institute, London, ON,<br />
Canada, Beverley J. Rider, Graduate Student, Department<br />
<strong>of</strong> Microbiology and <strong>Immunology</strong> and Robarts Research<br />
Institute, London, ON, Canada, Edwin Lee-Chan, Lab<br />
Technician/Lab Manager, Department <strong>of</strong> Microbiology and<br />
<strong>Immunology</strong> and Robarts Research Institute, London, ON,<br />
Canada, Bhagirath Singh, Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Microbiology and <strong>Immunology</strong> and Robarts Research<br />
Institute, London, ON, Canada<br />
Dendritic cells (DC) are potent inducers <strong>of</strong> T cell<br />
tolerance. The mechanism by which this is achieved is not<br />
well understood. We postulated that in vivo induction <strong>of</strong><br />
plasmacytoid dendritic cells (PDC) could prevent autoimmunity<br />
through induction <strong>of</strong> T cell tolerance. We report that a<br />
novel self-peptide Ep1.B <strong>of</strong> mouse Apolipoprotein E (ApoE)<br />
induced differentiation <strong>of</strong> bone marrow derived monocytes<br />
(BMM) into PDC. In vitro Ep1.B induced PDCs are B220+, Ly6-<br />
C+, CD11c+, mPDCA+, CD62L+ and CD8a+. Footpad injection<br />
<strong>of</strong> Ep1.B in diabetes prone NOD mice increased the level <strong>of</strong><br />
CD11c+, mPDCA and B220+ cells in the draining lymph nodes<br />
(LN) and these CD11c+ cells have an increased expression <strong>of</strong><br />
tolerogenic programmed death ligand (PD-L1). Since PD-1-<br />
PD-L1 pathway has been shown to be important in maintaining<br />
tolerance, PDC induced by Ep1.B may inhibit the effector<br />
T cells in disease process. In addition, we found increased<br />
number <strong>of</strong> CD4+CD25+ T cells with elevated glucocorticoidinduced<br />
tumor necrosis factor receptor related protein<br />
(GITR) in the LN <strong>of</strong> these animals. We found that injection<br />
<strong>of</strong> Ep1.B in young NOD mice significantly prevented type 1<br />
diabetes development in these mice. In summary, we suggest<br />
that in vivo Ep1.B induced differentiation <strong>of</strong> BMM into PDC<br />
that in turn prevented autoimmunity and induced regulatory<br />
T cells in type 1 diabetes prone NOD mice.<br />
doi:10.1016/j.clim.2007.03.219<br />
F.7 Generation and Characterization <strong>of</strong> Insulin<br />
Peptide-specific Regulatory T Cells<br />
Marianne Martinic, Postdoctoral Fellow, Immune<br />
Regulation Lab DI-3, La Jolla Institute for Allergy and<br />
<strong>Immunology</strong>, La Jolla, CA, Lisa Togher, Technician, Immune<br />
Regulation Lab DI-3, La Jolla Institute for Allergy and<br />
<strong>Immunology</strong>, La Jolla, CA, Christophe Filippi, Postdoctoral<br />
Fellow, Immune Regulation Lab DI-3, La Jolla Institute for<br />
Allergy and <strong>Immunology</strong>, La Jolla, CA, Jean Jasinski, PhD<br />
Student, Barbara Davis Center for Childhood Diabetes,<br />
Aurora, CO, Damien Bresson, Postdoctoral Fellow, Immune<br />
Regulation Lab DI-3, La Jolla Institute for Allergy and<br />
<strong>Immunology</strong>, La Jolla, CA, George Eisenbarth, Executive<br />
Director, Barbara Davis Center for Childhood Diabetes,<br />
Aurora, CO, Matthias von Herrath, Division Head, Immune<br />
Regulation Lab DI-3, La Jolla Institute for Allergy and<br />
<strong>Immunology</strong>, La Jolla, CA<br />
Our goal is to generate insulin peptide-specific regulatory<br />
T cells (Tregs) in vitro, which suppress overt diabetes in<br />
prediabetic and reverse already ongoing disease in diabetic<br />
mice. Furthermore we aim to analyze the suppressive<br />
properties and mechanisms <strong>of</strong> these Tregs in vitro and in<br />
vivo. In order to generate insulin peptide-specific Tregs, we<br />
took advantage <strong>of</strong> insTCR transgenic mice, which express T<br />
cell receptor (TCR) transgenic CD4+ T cells specific for<br />
the insulinB9–23 peptide (insB) presented on MHC class<br />
II H-2IAg7/d molecules. The in vitro generation <strong>of</strong> insBspecific<br />
Tregs involved purification <strong>of</strong> either CD4+CD25+ or<br />
CD4+CD25− insTCR T cells, which were cultured for 1–<br />
2 weeks with the insB peptide, syngeneic antigen presenting<br />
cells and high doses <strong>of</strong> IL-2 yielding 25+ and 25− cultures,<br />
respectively. Using the classical in vitro suppression assay,<br />
only cells derived from the 25+ cultures were able to<br />
suppress while cells from the 25− cultures enhanced<br />
proliferation and cytokine secretion <strong>of</strong> CD8+ effector T<br />
cells. In vivo, however, cells from both cultures were unable<br />
to suppress lymphocytic choriomeningitis virus-induced<br />
diabetes. Interestingly, freshly isolated as well as IL-10cultured<br />
CD4+CD25− but not freshly isolated CD4+CD25+<br />
insTCR T cells suppressed spontaneous diabetes in NOD<br />
females. We are currently investigating the mechanisms<br />
underlying the in vivo suppressive potential <strong>of</strong> these CD4<br />
+CD25− T cells.<br />
doi:10.1016/j.clim.2007.03.220<br />
F.8 The Insulitis Reporter Mouse<br />
Jennifer Ondr, Postdoctoral Fellow, Cincinnati Children’s<br />
Hospital Medical Center, Division <strong>of</strong> Endocrinology, Diabetes<br />
Research Center, Cincinnati, OH, Robert Opoka, Research<br />
Assistant, Cincinnati Children’s Hospital Medical Center,<br />
Division <strong>of</strong> Endocrinology, Diabetes Research Center,<br />
Cincinnati, OH, Sankarannand Vukkadapu, Postdoctoral<br />
Fellow, Cincinnati Children’s Research Foundation,<br />
Cincinnati, OH, Jonathan Katz, Associate Pr<strong>of</strong>essor,<br />
Cincinnati Children’s Hospital Medical Center, Division <strong>of</strong><br />
Endocrinology, Diabetes Research Center, Cincinnati, OH<br />
Type 1 diabetes mellitus (T1DM) is caused by the<br />
autoimmune destruction <strong>of</strong> insulin producing beta cells by<br />
leukocytes that infiltrate the pancreas in a prolonged and<br />
clinically silent process termed insulitis. Inability to detect<br />
insulitis prior to the onset <strong>of</strong> overt disease symptoms stymies<br />
progress in T1DM research and treatment. In humans,<br />
detection <strong>of</strong> antibody to islet cell antigens is the only means<br />
to diagnose pre-diabetic insulitis, however, no relationship<br />
between sero-conversion and insulitis progression is established.<br />
In NOD mice, insulitis can be measured, but only as an<br />
end-stage pancreatectomy. An early, accurate diagnosis <strong>of</strong><br />
insulitis would allow the possibility <strong>of</strong> therapeutic intervention,<br />
thus there remains an urgent need for non-invasive and<br />
real-time measures <strong>of</strong> insulitis. To this end, we are generating<br />
a gene-targeted NOD mouse in which production <strong>of</strong> a
Abstracts<br />
molecular reporter, soluble human alkaline phosphatase<br />
(sHPAP), is controlled by a gene, Reg3γ, expressed exclusively<br />
by islets cells <strong>of</strong> pancreata undergoing insulitis. Our insulitis<br />
reporter (InsuRe) mouse will self-indicate the onset and<br />
severity <strong>of</strong> insulitis when Reg3γ expression, triggered by the<br />
infiltration <strong>of</strong> pancreatic islets, induces rapid and measurable<br />
secretion <strong>of</strong> sHPAP into the blood. We will validate the fidelity<br />
<strong>of</strong> sHPAP expression in the InsuRe mouse with multiple<br />
established NOD mouse models <strong>of</strong> insulitis and diabetes.<br />
The InsuRe mouse will allow the identification <strong>of</strong> biomarkers<br />
that appear in the blood <strong>of</strong> NOD mice throughout insulitis. The<br />
identification <strong>of</strong> these biomarkers will aid in the non-invasive<br />
detection <strong>of</strong> occult insulitis, or diagnosis <strong>of</strong> pre-diabetes, in<br />
both mice and humans.<br />
doi:10.1016/j.clim.2007.03.221<br />
F.9 Identification <strong>of</strong> Candidate IDDM Disease<br />
Susceptibility Genes in the Idd Regions <strong>of</strong> NOD Mice<br />
by Temporal Microarray Gene Expression Data<br />
Analysis<br />
Keiichi Kodama, Postdoctoral Fellow, Stanford University,<br />
Stanford, CA, Demi Dang, Tech Person, Stanford University,<br />
Stanford, CA, Claire Holness, PhD, Stanford University,<br />
Stanford, CA, Hideyuki Iwai, MD. PhD, Stanford<br />
University, Stanford, CA, Mark Hartnett, Engineer,<br />
Agilent Technologies, Inc, Santa Clara, CA, Atul Butte,<br />
Assistant Pr<strong>of</strong>essor, Stanford University, Stanford, CA,<br />
C. Garrison Fathman, Pr<strong>of</strong>essor, Stanford University,<br />
Stanford, CA<br />
Using Agilent 41K mouse genome oligo-microarrays to<br />
study temporal gene expression, we identified candidate<br />
genes in the Idd disease susceptibility regions <strong>of</strong> NOD mice<br />
dysregulated by the pathophysiology <strong>of</strong> disease, not as<br />
mutations or polymorphic variants <strong>of</strong> disease susceptibility<br />
genes. Pancreatic lymph nodes (PLN) and spleen (SPL) were<br />
removed from 10-day, 4-week, 8-week, 12-week, 16-week,<br />
and 20-week-old NOD female mice, or as control from 10 day<br />
and 20-week-old genetically identical (except the MHC),<br />
NOD.B10Sn-H2b/J (NOD.B10) female mice (n¡Ü10). At 10 days<br />
to 4 weeks <strong>of</strong> age, coincident with the initiation <strong>of</strong> disease,<br />
there were ∼500 significantly dysregulated genes detected in<br />
the NOD PLN but none in the spleen, supporting the possibility<br />
that the PLN may be an important site for disease initiation.<br />
∼450 significantly dysregulated genes were seen in the PLN<br />
and ∼400 in splenic tissues from 12- to 20-week-old mice,<br />
suggesting that both tissues may be important in disease<br />
progression. b100 genes were simultaneously dysregulated in<br />
both the PLN and SPL during this period. Two <strong>of</strong> these, Ptpn22<br />
and Chi3l3, are located within the Idd 18.2 region. Expression<br />
changes for these two genes were confirmed by qPCR using<br />
the same mouse RNA samples as were used for the<br />
microarrays. This strategy may allow identification <strong>of</strong> Idd<br />
candidate genes that play critical roles in the induction or<br />
progression <strong>of</strong> autoimmune type 1 diabetes that are the<br />
consequence, not the cause <strong>of</strong> disease.<br />
doi:10.1016/j.clim.2007.03.222<br />
F.10 Peptide-based Immunotherapy for Type 1<br />
Diabetes; The Antigen, the Dose, the Immunization<br />
Route and the State <strong>of</strong> the Disease Can Make a<br />
Difference<br />
Georgia Fousteri, Postdoctoral Fellow, La Jolla Institute<br />
for Allergy and <strong>Immunology</strong>, La Jolla, CA, Amy Dave,<br />
Student, University <strong>of</strong> California, San Diego, La Jolla, CA,<br />
Michael Cr<strong>of</strong>t, Pr<strong>of</strong>essor, La Jolla Institute for Allergy and<br />
<strong>Immunology</strong>, La Jolla, CA, Matthias von Herrath,<br />
Pr<strong>of</strong>essor, La Jolla Institute for Allergy and <strong>Immunology</strong>,<br />
La Jolla, CA<br />
Immunization with beta-cell derived antigens such as<br />
insulin has been under intense investigation for treating or<br />
preventing T1D. This strategy is <strong>of</strong> particular interest since<br />
the administration <strong>of</strong> immunosuppressive drugs may be<br />
circumvented. We compared several islet-antigen derived<br />
peptides (insulin and IGRP) given alone or in combination<br />
by two different routes (intranasally [i.n.] or subcutaneously<br />
[s.c.]) to 10-week-old prediabetic NOD mice.<br />
Surprisingly, and in contrast to previous studies, we found<br />
that neither intranasal nor subcutaneous administration <strong>of</strong><br />
human pro-insulin II peptide B24–C36 (hpII) affected T1D,<br />
whereas insB-chain 9–23 (B9–23) and its altered-peptide<br />
ligand (Ala 16, 19) (APL) accelerated diabetes development<br />
significantly following intranasal administration. Combination<br />
<strong>of</strong> the three CD4 epitopes did not affect T1D when<br />
administered by either route. When insB-chain 15–23 (B15–<br />
23) and mouse insB24–36 (B24–C36) CD8 epitopes were<br />
tested (s.c. route), B15–23 delayed and B24–36 accelerated<br />
diabetes. Overall the diabetes incidence reached<br />
similar levels in both groups. Lastly, when two IGRPderived<br />
altered peptide ligands NRPI4 and NRPV7 were<br />
compared for their protective effect following s.c. immunization,<br />
NRPV7 provided the greatest delay but without a<br />
long-term protection. In contrast to our present findings,<br />
mucosal immunization with whole oral or intranasal insulin<br />
or insB9–23 peptide combined with suitable adjuvants (i.<br />
e., IFA) was able to prevent diabetes efficiently in previous<br />
studies, especially when given at a much earlier stage (4to<br />
6-week-old NODs). It will be important to consider the<br />
issues <strong>of</strong> age, route <strong>of</strong> administration and adjuvants for the<br />
design <strong>of</strong> future clinical trials.<br />
doi:10.1016/j.clim.2007.03.223<br />
S19<br />
F.11 Monocyte Immunophenotypes in Type 1<br />
Diabetes<br />
Erik Fung, DRF Fellow, Department <strong>of</strong> Medical Genetics,<br />
University <strong>of</strong> Cambridge, England, Cambridge, England, John<br />
A. Todd, Pr<strong>of</strong>essor and Director <strong>of</strong> Laboratory, Department<br />
<strong>of</strong> Medical Genetics, University <strong>of</strong> Cambridge, England,<br />
Cambridge, England, Linda S. Wicker, Pr<strong>of</strong>essor and<br />
Co-Director <strong>of</strong> Laboratory, Department <strong>of</strong> Medical Genetics,<br />
University <strong>of</strong> Cambridge, England, Cambridge, England<br />
Type 1 diabetes (T1D) is a complex-trait disease that<br />
results from interaction <strong>of</strong> genes and environmental<br />
exposures leading to immune destruction <strong>of</strong> pancreatic<br />
islet cells. Previous studies have indicated that monocytes
S20 Abstracts<br />
may be altered in chronic T1D. However, it is still unclear if<br />
this immune phenotypic variation is a cause or effect <strong>of</strong><br />
T1D. Using polychromatic flow cytometry to analyze whole<br />
blood, we have extended to pediatric T1D patients the<br />
findings by others that adult T1D cases have increased<br />
expression <strong>of</strong> CD11b on monocytes as compared to<br />
apparently healthy non-T1D adults. We examined 37 T1D<br />
patients (age 9.2–18.3 years, median 14.4 years; median<br />
time past diagnosis for non-newly diagnosed cases=4.3<br />
years) and 63 healthy volunteers (age 9.9–60 years, median<br />
34 years). CD11b expression (MFI) in HLA-DR+ monocyte<br />
subsets were as follows: on CD16+ monocytes, 265±20 vs.<br />
218±10 (pediatric T1D cases vs. non-T1D adults); CD16+<br />
CD14+ monocytes, 1108 ±51 vs. 840±27; CD14++ ‘inflammatory’<br />
monocytes, 1479±38 vs. 1146±54. Expression<br />
levels <strong>of</strong> CCR2 on CD14++ monocytes were lower in T1D<br />
patients than in healthy volunteers (MFI 62±2 vs. 75±2),<br />
consistent with an activated monocyte phenotype. CD11b<br />
and CCR2 expression differences were independent <strong>of</strong> ages<br />
at diagnosis or at immunophenotyping. In order to help<br />
distinguish between cause and effect and begin to explore<br />
the possibility that extremes <strong>of</strong> these phenotypes may be<br />
precursors to disease, we will analyze age-matched<br />
subjects and unaffected siblings, and test the association<br />
<strong>of</strong> alleles <strong>of</strong> known T1D susceptibility genes (e.g., HLA<br />
haplotypes, INS, PTPN22, CD25, IFIH1, CTLA4) with monocyte-subset<br />
phenotypes.<br />
doi:10.1016/j.clim.2007.03.224<br />
F.12 A High Specificity Competitive Europium Based<br />
Assay for Autoantibodies <strong>of</strong> APS1 Patients Reacting<br />
with Interferon Alpha<br />
Li Zhang, Fellow, Barbara Davis Center, Denver, CO,<br />
Raffaele Badolato, MD, PhD, Department <strong>of</strong> Pediatrics,<br />
University <strong>of</strong> Brescia, Brescia, Jennifer Barker, Assistant<br />
Pr<strong>of</strong>essor <strong>of</strong> Pediatrics, Barbara Davis Center for Childhood<br />
Diabetes, Denver, CO, Maureen Su Pra, University <strong>of</strong><br />
California, San Francisco Diabetes Center, San Francisco,<br />
CA, Sunanda Babu, Research Associate, Barbara Davis<br />
Center, Denver, CO, Mickie Cheng, MD, PhD, University <strong>of</strong><br />
California, San Francisco Diabetes Center, San Francisco,<br />
CA, Tony Shum, MD, University <strong>of</strong> California, San Francisco<br />
Diabetes Center, San Francisco, CA, Ehud Zamir, MD, The<br />
Royal Victorian Eye and Ear Hospital, Victoria, BC, Canada,<br />
Adam Law, MD, Ithaca Medical School, Ithaca, NY, George<br />
Eisenbarth, Executive Director, Barbar Davis Center, Denver,<br />
CO, Mark Anderson, Assistant Pr<strong>of</strong>essor, University <strong>of</strong><br />
California, San Francisco Diabetes Center, San Francisco, CA<br />
IFN-α autoantibodies have been described in autoimmune<br />
polyglandular syndrome type 1 (APS1) patients. We have<br />
established a competitive Europium time resolved fluorescence<br />
assay for autoantibodies to IFN-α and have tested it in<br />
a cohort <strong>of</strong> APS1 patients. Methods: The europium-ELISA<br />
method utilizes plate bound IF α incubated with or without<br />
competition with fluid phase IFN-α and sera. Anti-IgG<br />
biotinylated antibody is added, followed by incubation with<br />
streptavidin–europium and detection with time resolved<br />
fluorescence (measured in counts per second, CPS). Seven<br />
APS1 patients, 6 relatives, 71 non-APS1 Addison patients and<br />
141 T1DM patients were tested as well as 100 normal<br />
controls. The APS1 patients had multiple different mutations<br />
in the AIRE gene. Results: Normal control CPS without<br />
competition was 31,237∼17,328 CPS and delta with competition<br />
was −6563∼10,303 CPS. The initial APS1 patient (used<br />
to create the index, index 1.0) gave 394,063 CPS without<br />
competition and a delta <strong>of</strong> 363,662∼31,587 CPS with<br />
competition. Scatchard plot analysis revealed a high avidity<br />
(Kd <strong>of</strong> 0.5 nM). The CPS, delta and index for 6/7 APS1<br />
patients were strongly positive and above 3 standard<br />
deviations <strong>of</strong> controls. Relatives were negative as were 71<br />
Addison disease (non-APS-1) and 141 T1DM patients. The one<br />
negative APS1 patient is presumed APS1 based on clinical<br />
history. The assay has a sensitivity <strong>of</strong> 86% or greater and<br />
specificity greater than 99.5%. Conclusion: IFN-α autoantibodies<br />
are detected specifically in APS1 patients with<br />
specificity greater than 99% using a competitive Europium<br />
time resolved fluorescence assay.<br />
doi:10.1016/j.clim.2007.03.225<br />
F.13 Validation <strong>of</strong> Anti-Idiotypic Bridging ELISA to<br />
Quantitate TRX4 (Anti-Human CD3) Mab Levels in<br />
Human Serum<br />
Francis Carmody, Preclinical Scientist III, Preclinical<br />
Development, Cambridge, MA, Michael Slavonic, Preclinical<br />
Scientist II, Tolerx, Preclinical Development, Cambridge, MA,<br />
Adam O’Shea, Research Scientist II, Tolerx, Applied Research,<br />
Cambridge, MA, Michael Paglia, Senior Manager Manufacturing,<br />
Tolerx, Manufacturing, Cambridge, MA, Reema Gulati,<br />
Research Scientist II, Tolerx, Applied Research, Cambridge, MA,<br />
Sharon Li, Former Tolerx Employee, Applied Research,<br />
Cambridge, MA, Herman Waldmann, Pr<strong>of</strong>essor <strong>of</strong> Pathology,<br />
Head <strong>of</strong> Department, University <strong>of</strong> Oxford, Sir William Dunn<br />
School <strong>of</strong> Pathology, Cambridge, MA, Michale Rosenzweig,<br />
Director Preclinical Development, Tolerx, Preclinical<br />
Development, Cambridge, MA<br />
TRX4 is an aglycosyl anti-human CD3μ monoclonal antibody<br />
(Mab) that is being evaluated as a therapy for autoimmune<br />
disorders such as new-onset type 1 diabetes. Our goal was to<br />
develop a sensitive, high throughput assay to quantitate TRX4<br />
serum levels that could be used as an alternative to a cell-based<br />
assay that had been used in previous studies. Historically, TRX4<br />
was detected with a polyclonal anti-human IgG after it bound to<br />
the human T cell line, HuT 78. This method was both laborious<br />
and lacked sufficient sensitivity. Two different approaches were<br />
used to raise monoclonal antibodies to TRX4 complementarity<br />
determining regions (CDRs). First, transgenic human CD3 mice<br />
were tolerized to human IgG by two 1 mg IV doses and then<br />
subsequently immunized with 100 μg IP <strong>of</strong> TRX4. For the second<br />
strategy, BALB/c mice were administered 100 μg SC <strong>of</strong> TRX4 F<br />
(ab)′2 fragments in incomplete Freund’s adjuvant. These<br />
independent immunization strategies produced two non-competing<br />
anti-idiotypic TRX4 Mabs that satisfied specificity<br />
criteria. Significant serum interference was observed with<br />
both the Mabs when used individually in ELISAs. However,<br />
when used together, these anti-idiotypic antibodies were able to<br />
capture and detect TRX4 in whole sera with no detectable
Abstracts<br />
enhancement or inhibition due to serum alone. Assay validation<br />
has confirmed the accuracy (±20%), precision (±20%), specificity<br />
and robustness necessary to fulfil the ng/mL detection goal<br />
sought. Once validated, the ELISA was utilized as a crossover<br />
analysis between preclinical and clinical programs in support <strong>of</strong><br />
our TRX4 development program.<br />
doi:10.1016/j.clim.2007.03.226<br />
F.14 Regulation <strong>of</strong> Insulin Expression in Thymic<br />
Epithelial Cells<br />
Dina Levi, PhD Candidate, McGill University, Human<br />
Genetics, Pointe-Claire, QC, Canada, Michael Palumbo, PhD<br />
Candidate, McGill University, Experimental Medicine,<br />
Montreal, QC, Canada, Constantin Polychronakos, Principal<br />
Investigator, Montreal Children’s Hospital Research<br />
Institute, Montreal, QC, Canada<br />
Thethymushasanessentialfunctionintheselection<strong>of</strong>T<br />
cells with a properly formed TCR and the elimination <strong>of</strong> selfreactive<br />
ones. It has been found that tissue specific selfantigens<br />
are expressed in thymic epithelial cells, specifically<br />
in the medulla, for the purpose <strong>of</strong> presentation to thymocytes.<br />
Insulin is an important self-antigen that is implicated in the<br />
destruction <strong>of</strong> the pancreatic beta-cells in type 1 diabetes. We<br />
have isolated and cultured insulin-expressing (as well as<br />
insulin-non-expressing) epithelial clones from the medulla <strong>of</strong><br />
the thymus (mTECs), which provide an exclusive in vitro<br />
model to study self-antigen expression in the thymus.<br />
Preliminary studies show a number <strong>of</strong> tissue specific selfantigens<br />
overexpressed by microarray gene expression pr<strong>of</strong>iling,<br />
in both insulin-expressing and non-expressing clones, with<br />
the majority <strong>of</strong> tissues being represented. Stimulation <strong>of</strong><br />
mTECs with IFN- 3 and TNF-± cytokines demonstrates a pattern<br />
<strong>of</strong> decrease in insulin expression by real-time RT-PCR,<br />
indicating a possible regulation mechanism. Furthermore,<br />
co-culturing mTECs with isolated thymocytes results in an<br />
increase in insulin expression when cells are in direct contact.<br />
This interaction is supposed essential for the proper selection<br />
<strong>of</strong> non-autoreactive T lymphocytes in order to prevent<br />
autoimmune disease. Therefore, the regulation <strong>of</strong> insulin<br />
self-antigen expression in the thymus is an essential mechanism<br />
involved in negative selection and central tolerance and<br />
necessitates further studies.<br />
doi:10.1016/j.clim.2007.03.227<br />
F.15 Dysregulation <strong>of</strong> the TIM-3-Galectin-9 Pathway<br />
in Type I Diabetic T Cells<br />
William Hastings, Postdoctoral Fellow, Brigham and<br />
Women’s Hospital/Harvard Medical School, Center for<br />
Neurologic Diseases, Boston, MA, Allison Greer, Research<br />
Technician, Brigham and Women’s Hospital/Harvard<br />
Medical School, Center for Neurologic Diseases, Boston, MA,<br />
Vijay Kuchroo, Pr<strong>of</strong>essor, Brigham and Women’s Hospital/<br />
Harvard Medical School, Center for Neurologic Diseases,<br />
Boston, MA, David Hafler, Pr<strong>of</strong>essor, Brigham and Women’s<br />
Hospital/Harvard Medical School, Center for Neurologic<br />
Diseases, Boston, MA, David Anderson, Instructor, Brigham<br />
and Women’s Hospital/Harvard Medical School, Center for<br />
Neurologic Diseases, Boston, MA, Sally Kent, Pr<strong>of</strong>essor,<br />
Brigham and Women’s Hospital/Harvard Medical School,<br />
Center for Neurologic Diseases, Boston, MA<br />
Our laboratory has generated, in a non-biased manner, a<br />
large panel <strong>of</strong> T cell clones from the pancreatic lymph nodes<br />
(PLN) <strong>of</strong> type 1 diabetic (T1D) subjects and we asked if there<br />
were alterations in function <strong>of</strong> T cells from the PLN <strong>of</strong> T1D<br />
subjects versus those from controls. In this regard, we have<br />
recently shown that TIM-3, a molecule associated with negative<br />
regulation <strong>of</strong> fully differentiated Th1 but not Th2 cells, is<br />
dysregulated on T cell clones isolated from the CSF <strong>of</strong> MS<br />
patients. In light <strong>of</strong> this, we analyzed Tcell clones from the PLN<br />
<strong>of</strong> T1D as well as from normal controls to determine if there are<br />
similar alterations in expression and/or function <strong>of</strong> TIM-3 and its<br />
ligand Galectin-9 in these cells. We report that PLN clones from<br />
T1D subjects express lower levels <strong>of</strong> Galectin-9 but not TIM-3<br />
mRNA than clones from normal controls, as analyzed by Taqman<br />
PCR. In addition, we show that blockade <strong>of</strong> TIM-3 signaling with<br />
antibody increased proliferation and IFNg secretion in PLN Tcell<br />
clones. Interestingly, FACS analysis <strong>of</strong> PLN cells from controls and<br />
T1D subjects showed that TIM-3 is more highly expressed than on<br />
comparable subpopulations <strong>of</strong> T cells present in peripheral<br />
blood. Gene chip analysis has identified key differences in gene<br />
expression between TIM-3+ and TIM-3− T cell populations.<br />
Collectively, these data contribute to our understanding <strong>of</strong> how<br />
TIM-3 may regulate human T cell function and impact type 1<br />
diabetes.<br />
doi:10.1016/j.clim.2007.03.228<br />
S21<br />
F.16 huFlt3-L Treatment Does Not Protect RIP-GP<br />
and RIP-NP C57B1/6 Mice Against LCMV-induced<br />
Diabetes<br />
Tom Van Belle, Postdoctoral Fellow, La Jolla Institute for<br />
Allergy and <strong>Immunology</strong>, La Jolla, CA, Eleanor Ling, PhD, La<br />
Jolla Institute for Allergy and <strong>Immunology</strong>, La Jolla, CA, Lisa<br />
Togher, Research Assistant, La Jolla Institute for Allergy and<br />
<strong>Immunology</strong>, La Jolla, CA, Matthias von Herrath, PI, La Jolla<br />
Institute for Allergy and <strong>Immunology</strong>, La Jolla, CA<br />
Human Fms-like Tyrosine Kinase3-L (huFlt3-L) treatment <strong>of</strong><br />
prediabetic NOD mice has been shown to increase the<br />
intrinsically low numbers <strong>of</strong> myeloid dendritic cells in these<br />
mice, while decreasing insulitis and delaying the progression <strong>of</strong><br />
diabetes. In this study, we investigated the influence <strong>of</strong> huFlt3-L<br />
treatment on insulitis and diabetes onset in the LCMV-induced<br />
RIP-GP (fast onset) or RIP-NP (slow onset) C57Bl/6 mouse models<br />
<strong>of</strong> T1D. We showed that these mice have no intrinsic abnormality<br />
in DC subset development during LCMV infection. As expected,<br />
huFlt3-L treatment dramatically increased the total DC fraction<br />
and numbers in both spleen and pancreatic draining LN, with a<br />
preferential outgrowth <strong>of</strong> myeloid DCs (CD11c+B220−CD11b+)<br />
over plasmacytoid DCs (CD11c+B220+CD11b−). Moreover, NK<br />
cells and NK-DC cells were also increased, but CD4 and CD8 Tcell<br />
fractions were reduced. Strikingly, repeated huFlt3-L administrations,<br />
initiated in the prediabetic phase, did not protect<br />
against diabetes onset in this model. It is possible that this is
S22 Abstracts<br />
correlated with the observed reduced fraction <strong>of</strong> CD4+CD25+ T<br />
cells in the huFlt3-L-treated mice. Translation <strong>of</strong> these data<br />
suggests that huFlt3-L might only be a useful therapeutic agent<br />
for T1D patients with a demonstrated DC (subset) defect.<br />
doi:10.1016/j.clim.2007.03.229<br />
F.17 The BDC 12-4.1 TCR Alpha Chain is Sufficient to<br />
Generate Anti-Insulin Autoantibodies; α+β Chain<br />
for Insulitis<br />
Masakazu Kobayashi, Fellow, Barbara Davis Center, Aurora,<br />
CO, Jean Jasinski, Graduate Student, Barbara Davis Center,<br />
Aurora, CO, Maki Nakayama, Fellow, Barbara Davis Center,<br />
Aurora, CO, Dongmei Miao, PRA, Barbara Davis Center,<br />
Aurora, CO, Marcella Li, PRA, Barbara Davis Center, Aurora,<br />
CO, Liping Yu, PRA, Barbara Davis Center, Aurora, CO, Edwin<br />
Liu, Associate Pr<strong>of</strong>essor, Barbara Davis Center, Aurora, CO,<br />
George Eisenbarth, PI, Barbara Davis Center, Aurora, CO<br />
There is a marked T cell receptor (TCR) α chain restriction<br />
<strong>of</strong> T cell clones that target the insulin peptide B:9–23 in NOD<br />
mice. We are pursuing the hypothesis that this non-stringent<br />
TCR with conserved V α (TRAV5D-4*04) and J α (TRAJ53*01)<br />
chains (not N <strong>of</strong> α or β chain) underlies insulin autoimmunity<br />
and diabetes propensity. To test this hypothesis, we created<br />
TCR transgenic mice bearing only the BDC12-4.1 α chain,<br />
combined with C α knockout. Mice with the BDC 12-4.1 α<br />
chain, regardless <strong>of</strong> the presence or absence <strong>of</strong> the BDC 12-4.1<br />
TCR β chain, produced insulin autoantibodies, whereas mice<br />
bearing BDC 12-4.1 TCR β chain but not α chain did not. In<br />
contrast to production <strong>of</strong> insulin autoantibodies, we found<br />
insulitis in only mice with both 12-4.1 α and β chains (C α TCR<br />
knockout). Our results indicate that provision <strong>of</strong> only the<br />
BDC12-4.1 α chain results in expression <strong>of</strong> insulin autoantibodies<br />
(with endogenous β chains) and the ββ chain<br />
contributes to development <strong>of</strong> insulitis. At present we are<br />
producing mice with BDC12-4.1 α chain (Cα knockout) and β<br />
chain from anti-ovalbumin DO.11.10 T cell clone, which also<br />
produce insulin autoantibodies, but we need to combine Vβ<br />
knockout. A simple TCR motif and single insulin peptide may<br />
be essential for anti-insulin/anti-islet autoimmunity <strong>of</strong> the<br />
NOD mouse.<br />
doi:10.1016/j.clim.2007.03.230<br />
F.18 HLA-DR3/4 Heterozygosity is a Risk Factor for<br />
Autoantibody Conversion and Type 1 Diabetes<br />
Recurrence After Simultaneous Pancreas-Kidney<br />
Transplantation<br />
Stavros Diamantopoulos, Fellow, Diabetes Research<br />
Institute and Department <strong>of</strong> Pediatrics, University <strong>of</strong><br />
Miami, Miami, FL, Gloria Allende, Research Associate,<br />
Diabetes Research Institute, Miller School <strong>of</strong> Medicine,<br />
University <strong>of</strong> Miami, Miami, FL, George W. Burke, Pr<strong>of</strong>essor<br />
and Co-Director, Division <strong>of</strong> Transplantation, Department <strong>of</strong><br />
Surgery, Miller School <strong>of</strong> Medicine, University <strong>of</strong> Miami,<br />
Miami, FL, Gaetano Ciancio, Pr<strong>of</strong>essor and Director <strong>of</strong><br />
Transplant Education, Department <strong>of</strong> Surgery, Miller School<br />
<strong>of</strong> Medicine, University <strong>of</strong> Miami, Miami, FL, Alberto<br />
Pugliese, Director, Division <strong>of</strong> Immunogenetics, Diabetes<br />
Research Institute, Miller School <strong>of</strong> Medicine, University <strong>of</strong><br />
Miami, Miami, FL<br />
We studied 252 patients with type 1 diabetes (T1D) who<br />
received a simultaneous pancreas-kidney (SPK) transplant.<br />
Patients were tested for GAD and IA-2 autoantibodies, wellknown<br />
T1D risk markers. One-hundred-thirty-one patients (52%)<br />
remained GAD/IA-2 autoantibody-negative; 44 (17.4%) converted<br />
to GAD and/or IA-2 positivity while 77 (30.6%) had<br />
autoantibodies before the transplant that persisted on followup.<br />
We then analyzed recipients' HLA-DR types in relation to<br />
autoantibody conversion and T1D recurrence (T1DR) on followup<br />
(range 0.08-15.9 years, mean 5.6+3.6 years) in the 175<br />
patients who remained autoantibody-negative or converted.<br />
Twenty <strong>of</strong> 44 (45%) converters carried the HLA-DR3/4 genotype<br />
vs. 22/131 (17%) autoantibody-negative patients (p=0.0001,<br />
OR=4.5). Conversion occurred in 20/42 (47.6%) patients with the<br />
HLA-DR3/4 genotype vs. 24/133 (18%) patients without it<br />
(p=0.0001, Kaplan-Meier lifetable analysis; p=0.0002. OR=4.2,<br />
Chi-square). The non-heterozygotes included 53 patients with<br />
HLA-DR3 alone, 53 with HLA-DR4 alone and 23 with neither<br />
types. Excluding patients that did not carry HLA-DR3 or HLA-<br />
DR4, HLA-DR3/4 heterozygotes converted more <strong>of</strong>ten than<br />
patients carrying HLA-DR3 or HLA-DR4 alone (p=0.0001, Kaplan-<br />
Meier lifetable analysis; p=0.0002, OR=4.4, Chi-square). Ten<br />
patients developed T1DR on follow-up, <strong>of</strong> whom 60% were HLA-<br />
DR3/4 heterozygotes: 6/42 (14%) patients with the HLA-DR3/4<br />
genotype developed T1DR vs. 4/133 (3%) without it (p=0.01,<br />
Kaplan-Meier lifetables analysis; p=0.01, OR=5.37, Chi-square).<br />
Our data suggest that the HLA-DR3/4 genotype is a risk factor for<br />
autoantibody conversion and for the development <strong>of</strong> disease<br />
recurrence in SPK recipients. Patients with this genotype<br />
represent a high-risk group that should be monitored for<br />
markers <strong>of</strong> recurrent autoimmunity.<br />
doi:10.1016/j.clim.2007.03.231<br />
F.19 Kinetics <strong>of</strong> Regulatory T Cells Upon Specific<br />
Diabetogeneic Activation in a Group <strong>of</strong> High Risk<br />
Relatives <strong>of</strong> Type 1 Diabetes Mellitus Patients in<br />
Comparison with Healthy Controls<br />
Zuzana Vrabelova, PhD Student, University Hospital Motol,<br />
Children’s Neurology Department, Prague, Zuzana<br />
Hladikova, Statist, Czech Agriculture University,<br />
Department <strong>of</strong> Statistics, Prague, Katerina Stechova, Lab<br />
Chief, University Hospital Motol, Pediatric Department,<br />
Prague, Kristyna Bohmova, PhD Student, University Hospital<br />
Motol, Pediatric Department, Prague, Jaroslav Michalek,<br />
Head <strong>of</strong> Cell Immunotherapy Center, Masaryk University Br<br />
England Cell Immunotherapy Center, England<br />
Abnormalities in CD4+CD25+ regulatory Tcells (Tregs) may<br />
contribute to type 1 diabetes (T1D) development. We have<br />
studied immunoregulatory populations <strong>of</strong> CD4+CD25+ T cells<br />
in a group <strong>of</strong> high-risk relatives <strong>of</strong> T1D patients. We have<br />
evaluated kinetics <strong>of</strong> Tregs (using specific markers CD127−<br />
and Foxp3+) after specific stimulation with diabetogenic<br />
autoantigens in 11 high-risk (according to HLA-linked T1D<br />
genetic risk) relatives <strong>of</strong> T1D patients and 14 healthy
Abstracts<br />
controls. For stimulation specific amino acid sequences <strong>of</strong><br />
glutamic acid decarboxylase 65 (GAD65), thyrosine phosphatase<br />
(IA2), beta-proinsulin chain and whole molecule <strong>of</strong><br />
insulin were used. Cell activation was measured by surface<br />
expression <strong>of</strong> interferon gamma (IFN-g) on activated CD4+ T<br />
cells. High-risk relatives <strong>of</strong> T1D patients have significantly<br />
lower pre- and post-stimulatory numbers <strong>of</strong> Tregs than<br />
healthy controls (pb0.05). The autoantigen activation <strong>of</strong><br />
diabetogenic T cells was significantly higher in high-risk<br />
relatives <strong>of</strong> T1D patients than in healthy controls (pb0.02).<br />
In conclusion, defects <strong>of</strong> CD4+CD25+ regulatory T cells have<br />
been proved in individuals at increased genetic risk for T1D.<br />
Supported by IGA MZCR NR 9355-3.<br />
doi:10.1016/j.clim.2007.03.232<br />
F.20 Differential Immune Recognition <strong>of</strong> Heat-Shock<br />
Protein 60 Epitopes in Type 1 Diabetes<br />
Sylvia Kamphuis, Pediatric Immunologist, Department <strong>of</strong><br />
Paediatric <strong>Immunology</strong>, Erasmus MC Sophia Children’sHospital,<br />
Rotterdam,GijsTeklenburg,MD,Pediatric<strong>Immunology</strong>,UMC<br />
Utrecht, Utrecht, Annemarie Verrijn Stuart, Pediatric<br />
Endocrinologist, Department <strong>of</strong> Pediatric Endocrinology, UMC<br />
Utrecht, Utrecht, Irun Cohen, MD PhD, Department <strong>of</strong><br />
<strong>Immunology</strong>, Weizmann Institute <strong>of</strong> Science, Rehovot, Wilco de<br />
Jager, Msc, Department <strong>of</strong> <strong>Immunology</strong>, UMC Utrecht, Utrecht,<br />
Wietse Kuis, MD PhD, Department <strong>of</strong> <strong>Immunology</strong>, UMC Utrecht,<br />
Utrecht, Salvatore Albani, MD PhD, Department <strong>of</strong> Pediatrics<br />
and Medicine, University <strong>of</strong> California, San Diego, CA, Berent<br />
Prakken, MD PhD, Department <strong>of</strong> <strong>Immunology</strong>, UMC Utrecht,<br />
Utrecht, Mark Klein, Msc, Department <strong>of</strong> <strong>Immunology</strong>, UMC<br />
Utrecht, Utrecht<br />
Aim: Evidence is cumulating for an immunoregulatory role <strong>of</strong><br />
heat-shock proteins (HSP) in an increasing number <strong>of</strong> autoimmune<br />
diseases. Previously we identified pan-DR binding<br />
HSP60 epitopes that were recognized in juvenile idiopathic<br />
arthritis, an autoimmune disease characterized by chronic<br />
joint inflammation. The present study was performed to test<br />
whether these HSP60 epitopes were recognized in children<br />
with type 1 diabetes as well. Methods: We analyzed the<br />
pattern <strong>of</strong> HSP60 peptide-specific cytokine and chemokine<br />
responses in peripheral blood mononuclear cells <strong>of</strong> 18 type 1<br />
diabetes patients with primarily longstanding disease. In<br />
addition, a subgroup <strong>of</strong> newly diagnosed patients was followed<br />
over time for HSP60 peptide-specific immune responses.<br />
Results: We found peptide-specific induction <strong>of</strong> 8 cytokines<br />
(IL-1a, IL-1b, IL-6, IL-10, IL-17, IL-18, TNF-a, IFN-g) and 5<br />
chemokines (MIP-1a, MDC, IL-8, IP10, and OSM). Considerable<br />
variation in peptide-induced cytokine and chemokine pr<strong>of</strong>iles<br />
however was seen in the individual patients, irrespective <strong>of</strong><br />
age and disease duration. When following newly diagnosed<br />
patients over time, we found that the presence <strong>of</strong> T cell<br />
proliferative responses was almost exclusively seen in the<br />
patients that experienced a partial remission phase, with<br />
concomitant induction <strong>of</strong> IL-10. Conclusion: The identified<br />
HSP60 epitopes are immunogenic in children with type 1<br />
diabetes. The recorded correlation <strong>of</strong> peptide-specific T cell<br />
responses with (temporary) disease remission underscores the<br />
alleged immunomodulating role <strong>of</strong> HSP in chronic inflammation.<br />
This makes these HSP60 epitopes promising candidates<br />
for antigen-specific immunotherapy in not only juvenile<br />
idiopathic arthritis, but also in type 1 diabetes and possibly<br />
other autoimmune diseases.<br />
doi:10.1016/j.clim.2007.03.233<br />
F.21 Blocking the Toll Like Receptor 9 Signal Inhibits<br />
Activation <strong>of</strong> Diabetogenic CD8 T Cells and Delays<br />
Autoimmune Diabetes in NOD Mice<br />
Yiqun Zhang, Research Associate, Child and Family Research<br />
Institute, University <strong>of</strong> British Columbia, Vancouver, BC,<br />
Canada, Xuan Geng, Research Assistant, Kinesiology, Simon<br />
Fraser University, Burnaby, BC, Canada, Pere Santamaria,<br />
Pr<strong>of</strong>essor, Microbiology and Infectious Diseases, The University<br />
<strong>of</strong> Calgary, Calgary, AB, Canada, Diane Finegood, Pr<strong>of</strong>essor,<br />
Kinesiology, Simon Fraser University, Burnaby, BC, Canada, Jan<br />
P. Dutz, Pr<strong>of</strong>essor, CFRI, University <strong>of</strong> British Columbia,<br />
Vancouver, BC, Canada<br />
Type 1 diabetes is an autoimmune disease in which pancreatic<br />
beta cells are destroyed by CD8 T cells. Dendritic cells<br />
(DC)s are capable <strong>of</strong> priming CD8 Tcells. Toll-like receptor (TLR)<br />
triggering can lead to DC maturation. The contributions <strong>of</strong> TLR<br />
signals to spontaneous diabetes development are still largely<br />
unknown. We found that non-obese diabetic (NOD) mouse bone<br />
marrow derived DCs (BMDC)s pulsed with freeze–thawed NIT1<br />
insulinoma cells in the presence <strong>of</strong> TLR9 agonist CpG and CD40<br />
ligation were able to fully prime 8.3 TCR transgenic and<br />
diabetogenic CD8 T cells. Addition <strong>of</strong> the TLR9 inhibitors ODN<br />
2088 or chloroquine potently inhibited CD8 T cell CD25<br />
upregulation and intracellular IFN-γ production in response to<br />
CPG but not LPS. Furthermore, ODN 2088 or chloroquine<br />
inhibited CPG-induced BMDC CD40 upregulation and IL-12 p70<br />
production. In vivo, CPGaloneorwithanti-CD40induced8.3<br />
CD8 T cell CD69 upregulation and CTL activity that triggered<br />
rapid diabetes development in 8.3 NOD mice. Pre-treatment<br />
with ODN 2088 inhibited CPG-induced diabetes and treatment<br />
with either ODN 2088 or chloroquine delayed spontaneous<br />
diabetes in 8.3 NOD mice. Finally, administration <strong>of</strong> chloroquine<br />
inhibited spontaneous diabetes in NOD mice, down-regulated<br />
CD40 expression on pancreatic DCs, and inhibited serum rises <strong>of</strong><br />
IL-12 p70, IL-6, IFN-γ andMCP-1inducedbyCPGbutnotLPS.<br />
Thus, TLR9 activation may contribute to the spontaneous onset<br />
<strong>of</strong> CD8 T cell autoimmunity in NOD mice and TLR9 family<br />
inhibitors may be used to prevent and delay autoimmune<br />
diabetes in disease-prone animals.<br />
doi:10.1016/j.clim.2007.03.234<br />
S23<br />
F.22 Genome-Wide Association Scan for Type 1<br />
Diabetes Susceptibility Genes in a Danish Population<br />
Caroline Brorsson, PhD Student, Steno Diabetes Center,<br />
Gent<strong>of</strong>te, Elzbieta Swiergala, Lab Manager, Department <strong>of</strong><br />
Medical Genetics, University <strong>of</strong> British Columbia,<br />
Vancouver, BC, Canada, Krist<strong>of</strong>fer Rapacki, Computer<br />
Scientist, Center for Biological Sequence Analysis, Technical<br />
University <strong>of</strong> Denmark, Lyngby, Regine Bergholdt, Research<br />
Fellow, Steno Diabetes Center, Gent<strong>of</strong>te, Shaun Purcell,<br />
Assistant Pr<strong>of</strong>essor, Center for Human Genetic Research,
S24 Abstracts<br />
Massachusetts General Hospital, Boston, MA, Leigh Field,<br />
Pr<strong>of</strong>essor, Department <strong>of</strong> Medical Genetics, University <strong>of</strong><br />
British Columbia, Vancouver, BC, Canada, Flemming Pociot,<br />
Pr<strong>of</strong>essor, Steno Diabetes Center, Gent<strong>of</strong>te<br />
Type 1 diabetes (T1D) is a complex disease believed to<br />
result from interactions between multiple risk-conferring<br />
genes and environmental factors. In order to further<br />
investigate the genetic contribution to T1D we genotyped a<br />
case–control material for 58,000 SNP and tested these for<br />
association to disease. The first phase <strong>of</strong> the study comprised<br />
<strong>of</strong> thorough quality tests <strong>of</strong> the data for issues such as sample<br />
contamination, inbreeding, low genotyping rates, relatedness<br />
and population stratification which led to samples being<br />
excluded. SNPs were removed for failing HWE in controls, low<br />
genotyping rate, differences in missing genotyping rates<br />
between cases and controls and being non-polymorphic,<br />
which contributed to significant false positive findings. After<br />
ensuring the highest quality <strong>of</strong> the data-set the remaining<br />
52,000 SNPs and 605 individuals were analysed using basic<br />
allelic and model-based tests <strong>of</strong> association corrected for<br />
multiple testing and using permutation with empirically<br />
corrected p-values. Furthermore we performed genomewide<br />
sliding-window haplotype tests and full pair-wise test<br />
for epistasis across the genome. We also performed the same<br />
analyses for markers in 12 linkage regions previously reported<br />
in T1D. Every individual test confirmed the importance <strong>of</strong> the<br />
HLA region on chromosome 6p21.3 for developing T1D. In<br />
combination these tests point to other chromosomal regions<br />
contributing to T1D susceptibility. The significant findings<br />
will be followed up in a larger case–control material to<br />
confirm these present results. In addition this study has<br />
demonstrated the importance <strong>of</strong> high quality data for<br />
avoiding false positive findings when performing association<br />
analyses in complex diseases.<br />
doi:10.1016/j.clim.2007.03.235<br />
F.23 iNKT Cell Regulation <strong>of</strong> Type 1 Diabetes<br />
Dalam Ly, PhD Candidate, Robarts Research Institute,<br />
London, ON, Canada, Qing Sheng Mi, Research Associate,<br />
Robarts Research Institute, London, ON, Canada, Shabbir<br />
Hussain, Research Associate, Robarts Reseach Institute,<br />
London, ON, Canada, Terry L. Delovitch, Pr<strong>of</strong>essor, Robarts<br />
Research Institute, London, ON, Canada, Steven A. Porcelli,<br />
Pr<strong>of</strong>essor, Albert Einstein College <strong>of</strong> Medicine, Bronx, NY<br />
The pathogenesis <strong>of</strong> type 1 diabetes (T1D) in NOD mice is<br />
mediated by numerical and functional deficiencies in both<br />
CD4+CD25+FoxP3+ regulatory T cells (Tregs) and invariant<br />
natural killer T (iNKT) cells. Previously, we reported that<br />
protection <strong>of</strong> NOD mice from T1D can be achieved by<br />
activation <strong>of</strong> iNKT cells upon treatment with a multi-dose agalactosylceramide<br />
(a-GalCer) protocol that promotes preferential<br />
IL-4 secretion by iNKT cells. Since activated Tregs<br />
and iNKT cells are both self-reactive and protect NOD mice<br />
from T1D, we investigated whether iNKT-Treg collaboration<br />
is required for this protection. We recently reported that<br />
while a-GalCer therapy does not alter the proliferation or<br />
regulatory activity <strong>of</strong> Tregs, Treg activity is required for a-<br />
GalCer induced protection from T1D. This requirement <strong>of</strong><br />
Treg/iNKT collaboration for iNKT-mediated protection from<br />
T1D may not apply for the therapy <strong>of</strong> T1D provided by certain<br />
analogs <strong>of</strong> a-GalCer. Of particular interest is C20:2, an a-<br />
GalCer analog with a shortened (C20) acyl chain and two<br />
unsaturated bonds. When compared to a-GalCer, C20:2 yields<br />
significantly reduced IFN-g and enhanced IL-4 secretion by<br />
iNKT cells. Interestingly, we found that Treg activity is not<br />
required for C20:2 mediated protection from T1D by iNKT<br />
cells. Thus, our current results indicate that a-GalCer and<br />
C20:2 may differ in their requirement <strong>of</strong> Tregs for iNKT<br />
mediated protection from T1D. Ongoing studies will address<br />
the mechanism(s) that underlies this differential requirement<br />
<strong>of</strong> Tregs for protection from T1D.<br />
doi:10.1016/j.clim.2007.03.236<br />
F.24 Absence <strong>of</strong> Beta Cells in Long-Term Type 1a<br />
Diabetic Patients<br />
Travis Still, Pr<strong>of</strong>essional Research Assistant, Barbara Davis<br />
Center, Aurora, CO, Sally Kent, Instructor, Brigham and<br />
Women’s Hospital, Boston, MA, Alberto Pugliese, Diabetes<br />
Research Institute, University <strong>of</strong> Miami, Miami, FL, Piero<br />
Marchetti, Full Pr<strong>of</strong>essor, Ospedale di Cisanello, Pisa, Italy,<br />
Francesco Dotta, Full Pr<strong>of</strong>essor, University <strong>of</strong> Siena, Siena,<br />
Bernhard Hering, Full Pr<strong>of</strong>essor, Diabetes Institute for<br />
<strong>Immunology</strong> and Transplantation, Department <strong>of</strong> Surgery,<br />
University <strong>of</strong> Minnesota, Minneapolis, MN, Norma Kenyon,<br />
Full Pr<strong>of</strong>essor, Diabetes Research Institute, Cell Transplant<br />
Center, University <strong>of</strong> Miami, Miami, FL, Camillo Ricordi,<br />
Full Pr<strong>of</strong>essor, Diabetes Research Institute, Cell Transplant<br />
Center, University <strong>of</strong> Miami, FL, Miami, FL, Roberto<br />
Gianani, Assistant Pr<strong>of</strong>essor, Barbara Davis Center For<br />
Childhood Diabetes, Aurora, CT<br />
A recent report indicates that residual beta cells can be<br />
identified in the pancreas <strong>of</strong> long-term diabetic subjects by<br />
insulin immunostaining and in a subset <strong>of</strong> individuals by<br />
detection <strong>of</strong> C-peptide. We have analyzed pancreata from 20<br />
normal controls and 4 long-term diabetic subjects (1.5, 5, 22<br />
and 30 years post diagnosis) by immunohistochemistry for<br />
both insulin and the beta cell specific marker VMAT-2. We<br />
confirmed the beta cell specificity <strong>of</strong> VMAT-2 by triple<br />
immunostaining with glucagon, somatostatin and VMAT-2 <strong>of</strong><br />
normal human pancreas.<br />
In the pancreata from long-term diabetic subjects we did<br />
not find any VMAT-2 or insulin positive cells. In contrast<br />
abundant insulin (as expected) and VMAT-2 positive cells<br />
were present in all 20 normal human pancreata, in one new<br />
onset type 1a and in a type 2 diabetic patient. There was<br />
significant heterogeneity <strong>of</strong> staining intensity for VMAT-2<br />
amongst the normal control pancreata suggesting individual<br />
variability in VMAT-2 islet expression. Furthermore, there<br />
was also variability in VMAT-2 expression between different<br />
areas within the same pancreas with a zonal distribution <strong>of</strong><br />
strongly stained islets. These data indicate that in subjects<br />
with long-term type 1A diabetes, there are no residual beta<br />
cells by immunohistochemistry. Further studies <strong>of</strong> a larger<br />
series <strong>of</strong> patients are needed to establish whether beta cells<br />
continue to be present in type 1a diabetic subjects. In
Abstracts<br />
particular, it will be important to study pancreata from longterm<br />
diabetic subjects with documented C-peptide secretion<br />
(not available for these subjects).<br />
doi:10.1016/j.clim.2007.03.237<br />
F.25 Regulation <strong>of</strong> Pathogenicity Through CD40<br />
Expressed on CD4 T Cells<br />
Rocky Baker, Postdoctoral Fellow, Department <strong>of</strong><br />
<strong>Immunology</strong>, University <strong>of</strong> Colorado Health Sciences Center,<br />
Denver, CO, David Wagner, Assistant Pr<strong>of</strong>essor, Department<br />
<strong>of</strong> <strong>Immunology</strong>, University <strong>of</strong> Colorado Health Sciences<br />
Center, Denver, CO, Kathryn Haskins, Pr<strong>of</strong>essor, NJMRC,<br />
Denver, CO<br />
We have reported previously that the costimulatory<br />
molecule CD40 is a marker <strong>of</strong> autoreactive T cells found on<br />
diabetogenic T cell clones and that CD40+ T cells from the<br />
NOD mouse transfer diabetes. Although there is strong<br />
evidence that CD40 is functional on T cells, CD40 expression<br />
on T cells remains controversial because <strong>of</strong> typically low and<br />
variable levels <strong>of</strong> CD40 on primary T cells. We have<br />
investigated the conditions under which T cells express the<br />
CD40 molecule and how CD40 expression contributes to<br />
pathogenicity <strong>of</strong> autoreactive T cells. First, we have<br />
demonstrated that CD40 levels on primary T cells can be<br />
induced on 90% <strong>of</strong> CD4+CD40− T cells from NOD mice upon<br />
activation. Furthermore, comparing naive and primed T cells<br />
from different TCR-Tg NOD mouse models, we have found<br />
that the ability to express CD40 depends on the activation<br />
status <strong>of</strong> the T cell. Second, we investigated how CD40<br />
costimulation influences the effector function <strong>of</strong> CD4 T cells<br />
by comparing CD28 and CD40 costimulation in NOD mice. We<br />
have found that CD40 engagement can replace or synergize<br />
with CD28 costimulation in terms <strong>of</strong> T cell activation and<br />
proliferation. However, there is a major difference when<br />
costimulation occurs through CD40: our results indicate that<br />
CD40 costimulation influences the Th1/Th2 balance differently<br />
and that CD40 engagement on autoreactive T cells<br />
enhances a Th1 effector phenotype. Taken together, the data<br />
indicate that CD40 is an effective alternative pathway to<br />
CD28 costimulation and might regulate the pathogenicity <strong>of</strong><br />
CD4 T cells in T1D.<br />
doi:10.1016/j.clim.2007.03.238<br />
F.26 TRECS and Telomerase Analysis <strong>of</strong> Lymphocytes<br />
from Autoimmune Thyroid Disease Patients Point to<br />
the Migration <strong>of</strong> Recent Thymic Emigrants to the<br />
Thyroid Gland at the First Stages <strong>of</strong> Disease<br />
Ricardo Pujol Borrell, Pr<strong>of</strong>essor, Blood and Tissue Bank<br />
(LIRAD), Health Science Research Institut, Badalona, Marco<br />
Antonio Fernandez, Head Cytometry Facility, Health Science<br />
Research Institut, Badalona, Maria Pilar Armengol,<br />
Posdoctoral Fellow, Blood and Tissue Bank (LIRAD), Health<br />
Science Research Institut, Badalona, Manel Juan, Head HLA<br />
Laboratory, Blood and Tissue Bank (LIRAD), Health Science<br />
Research Institut, Badalona, Lidia Sabater, Research Fellow,<br />
Blood and Tissue Bank (LIRAD), Health Science Research<br />
Institut, Badalona, Ricardo Pujol Borrell, Head <strong>Immunology</strong><br />
Laboratory, Blood and Tissue Bank (LIRAD), Health Science<br />
Research Institut, Badalona<br />
According to the threshold hypothesis <strong>of</strong> autoimmunity,<br />
the abundance and affinity <strong>of</strong> autoreactive T cells in the<br />
repertoire determine the probability for the triggering <strong>of</strong><br />
autoimmune diseases. We have investigated how recent<br />
thymic emigrants may contribute the autoreactive repertoire<br />
in autoimmune thyroid disease (AITD) patients, by measuring<br />
TRECs in PBLs from AITD patients (n=50) and controls (n=55)<br />
and in intrathyroidal lymphocytes from autoimmune thyroid<br />
glands. TRECS were higher in AITD patients, especially in the<br />
N45-year-old patients. The analysis <strong>of</strong> TRECS in intrathyroidal<br />
lymphocytes (ITL) (n=45) in relation to disease duration<br />
revealed a distinct pattern <strong>of</strong> distribution: TRECS were higher<br />
in ITL than in PBL in b30-month-disease-duration patients<br />
(fourfold excess in average), and vice-versa N30-month<br />
patients (tenfold excess). Interestingly there was a correlation<br />
between the levels <strong>of</strong> TRECS in ITLs and PBLs in both<br />
groups but <strong>of</strong> different sign and intensity: it was stronger in<br />
the N30 months (r=0.94) than in the b30 months (r =0.535).<br />
This may indicate that while in the N30 months group the<br />
TRECs in ITLs is dependent on the TRECs in PBLs, this is not the<br />
case in the b30 months. The study <strong>of</strong> the telomere length did<br />
not reveal a relation between TRECs and telomere length in<br />
the T cells but was significantly shorter in ITLs cells than in<br />
PBLs. These results suggest that early in disease there is a<br />
migration to AITD glands <strong>of</strong> T lymphocytes newly generated in<br />
the thymus that then undergoes a local expansion.<br />
doi:10.1016/j.clim.2007.03.239<br />
S25<br />
F.27 Nramp1 Enhances Autoimmune Diabetogenic<br />
T Cell Response by Altering the Processing and<br />
Presentation <strong>of</strong> Pancreatic Islet Antigens in<br />
Dendritic Cells<br />
Yang Dai, Research Scientist, Torrey Pines Institute for<br />
Molecular Studies, San Diego, CA, Babak Shirdel, Student,<br />
Torrey Pines Institute for Molecular Studies, San Diego, CA,<br />
Sandra Chau, Student, Torrey Pines Institute for Molecular<br />
Studies, San Diego, CA, Linda Wicker, Pr<strong>of</strong>essor, Cambridge<br />
Institute for Medical Research, Cambridge, Philippe Gros,<br />
Pr<strong>of</strong>essor, McGill University, Montreal, QC, Canada, Eli<br />
Sercarz, Pr<strong>of</strong>essor, Torrey Pines Institute for Molecular<br />
Studies, San Diego, CA<br />
Dendritic cells are essential for initiating adaptive immune<br />
responses and establishing self tolerance. We hypothesize<br />
that the antigen processing machinery differs between<br />
diabetes-susceptible and -resistant, MHC-matched individuals,<br />
and that this difference contributes to the generation<br />
<strong>of</strong> pathogenic effectors rather than regulatory T cells in<br />
diabetes prone individuals. The Idd5.2 (insulin-dependent<br />
diabetes 5.2) region contributes to the genetic susceptibility<br />
<strong>of</strong> NOD (non-obese diabetes) mice to autoimmune diabetes<br />
and contains the gene encoding Nramp1 (natural resistanceassociated<br />
macrophage protein 1). We found that Nramp1<br />
expression is not restricted to macrophages; importantly,
S26 Abstracts<br />
dendritic cells (DC) can upregulate Nramp1 expression<br />
dramatically after stimulation. Nramp1-expressing DC exhibit<br />
an accelerated pH change (acidification) within endocytotic<br />
vesicles. Interestingly, when Nramp1 function is present, both<br />
bone marrow-derived DC and adherent splenic DC are more<br />
potent in stimulating autoreactive Tcells following processing<br />
<strong>of</strong> a diabetes-associated antigen, GAD (glutamic acid decarboxylase)<br />
65. Furthermore, an islet-infiltrating Tcell clone,<br />
BDC2.5, reacts much more vigorously to the islets when<br />
Nramp1-functional DC are used as antigen presenting cells.<br />
These data suggest that Nramp1 may alter the processing <strong>of</strong><br />
self antigens in antigen processing compartments to increase<br />
the availability <strong>of</strong> determinants involved in inducing pathogenic<br />
T cells (supported by grants to EES from Juvenile<br />
Diabetes Research Foundation and Diabetes National<br />
Research Group).<br />
doi:10.1016/j.clim.2007.03.241<br />
F.28 Perturbations in the Function and Composition<br />
<strong>of</strong> the B Cell Compartment are Present in Type 1<br />
Diabetes<br />
Mary Rieck, Research Technician, Benaroya Research<br />
Institute, Seattle, WA, Adrian Arechiga, Research Associate,<br />
Benaroya Research Institute, Seattle, WA, Catherine<br />
Pihoker, Associate Pr<strong>of</strong>essor, Department <strong>of</strong> Pediatrics,<br />
Seattle, WA, Jane Buckner, Associate Member, Benaroya<br />
Research Institute, Seattle, WA, Carla Greenbaum, Member,<br />
Benaroya Research Institute, Seattle, WA<br />
Disturbances in B cell populations have been identified in<br />
autoimmune diseases where autoantibodies are present. A<br />
role for B cells in the pathogenesis <strong>of</strong> type 1 diabetes (T1D) is<br />
suggested by studies in animal models and by the autoantibodies<br />
present in T1D. To address whether alterations in<br />
the B cell compartment are present in individuals with T1D we<br />
examined PBMC from healthy, T1D, and type 2 diabetic (T2D)<br />
subjects. The relative frequency <strong>of</strong> the naïve, memory and<br />
plasmablast subsets were significantly different between<br />
healthy and T1D subjects, while total B cells were no<br />
different. The plasmablast (CD19+CD27high) population was<br />
increased in T1D subjects compared to controls (pb0.006),<br />
and was most pronounced in subjects within 2 years <strong>of</strong><br />
diagnosis. The memory B cell (CD19+CD27+) subset was<br />
decreased in diabetic subjects (p =0.0006). The B cell<br />
populations in T2D subjects were comparable to healthy<br />
controls; however, first degree relatives <strong>of</strong> T1D subjects, like<br />
individuals with T1D, displayed a decrease in memory B cells<br />
(p=0.02). Thus the decrease in memory B cells is not due to<br />
the metabolic abnormalities associated with T1D but may be<br />
genetically influenced. In addition, we have identified a<br />
defect in BCR signaling within memory B cells <strong>of</strong> T1D subjects.<br />
This suggests that a B cell intrinsic mechanism may contribute<br />
to alterations in the B cell compartment. We hypothesize that<br />
this alteration <strong>of</strong> B cell signal transduction may contribute to<br />
pathogenesis <strong>of</strong> T1D by tipping the balance <strong>of</strong> long-term B cell<br />
memory toward high affinity antibody production.<br />
doi:10.1016/j.clim.2007.03.242<br />
F.30 Tracking Putative Pathogenic Members <strong>of</strong> the<br />
T Cell Repertoire in NOD Mice<br />
Idania Marrero, Postdoctoral Fellow, Torrey Pines Institute<br />
for Molecular Studies, San Diego, CA, Yang Dai, Postdoctoral<br />
Fellow, Torrey Pines Institute for Molecular Studies, San<br />
Diego, CA, Eli Sercarz, Pr<strong>of</strong>essor, Torrey Pines Institute for<br />
Molecular Studies, San Diego, CA<br />
To define the major components <strong>of</strong> the autoreactive Tcell<br />
repertoire that arise spontaneously in the diabetic NOD<br />
mouse, we used CDR3 length spectroscopy to (1) study the<br />
entire Tcell repertoire in pancreatic lymph nodes (PLN) from<br />
pre-diabetic NOD mice and (2) to identify potentially<br />
pathogenic clones in islet-infiltrating cells that persist after<br />
cyclophosphamide (Cyp) treatment <strong>of</strong> female NOD mice,<br />
which is designed to destroy regulatory T cells. Any public or<br />
quasi-public T cell expansions were identified in PLN <strong>of</strong><br />
female NOD mice at 4 and 10 weeks <strong>of</strong> age. We identified two<br />
expansions, Vb5.2/Jb2.5 and Vb10/Jb1.5, which were found<br />
in 60% and 50% <strong>of</strong> the 4-week-old female NOD mice. These<br />
expansions can be useful in the study <strong>of</strong> how the T cell<br />
repertoire can be modified after islet transplantation. After<br />
Cyp treatment, the Vb4, Vb5.2, Vb10 and Vb12 families<br />
showed dominant expansions. Interestingly, we found a<br />
unique Vb4-“195 nucleotide” peak that was found in 10 out<br />
<strong>of</strong> 11 (91%) Cyp-treated mice. In contrast, this peak was not<br />
detected in untreated NOD mice. Another interesting result<br />
was seen in the Vb12 family, in which a Vb12-“200” peak was<br />
prominent in 6 out <strong>of</strong> 10 (60%) Cyp-treated mice. These<br />
expansions may represent the more pathogenic clones.<br />
Currently, we are sequencing these expansions to establish<br />
whether the Cyp-induced expansions represent truly unique<br />
clones. We believe that these Cyp experiments will provide<br />
the possibility <strong>of</strong> distinguishing between regulatory and aggressive<br />
clones. This work was supported by JDRF 3-2005-336.<br />
doi:10.1016/j.clim.2007.03.243<br />
F.31 Reduced CD4+ T Cell-specific Gene Expression<br />
in Human Type 1 Diabetes Mellitus<br />
Tihamer Orban, Researcher, Joslin Diabetes Center, Boston,<br />
MA, Janos Kis, Diabetologist, Department <strong>of</strong> Internal<br />
Medicine and Gastroenterology, Polyclinic <strong>of</strong> the Hospitaller<br />
Brothers, Budapest, Laszlo Szereday, PhD, Reproductive and<br />
Tumor <strong>Immunology</strong> Research Group Hungarian Academy <strong>of</strong><br />
Science, Pecs, Klara Farkas, Joslin Diabetes Center, Boston,<br />
MA, Peter Engelmann, PhD, Department <strong>of</strong> <strong>Immunology</strong> and<br />
Biotechnology, Faculty <strong>of</strong> Medicine, University <strong>of</strong> Pecs,<br />
Pecs, Heyam Jalahej, Joslin Diabetes Center, Boston, MA,<br />
Andras Treszl, Pediatrician, SOTE First Department <strong>of</strong><br />
Paediatrics, Budapest<br />
Type 1 diabetes mellitus (T1DM) in humans is characterized<br />
by the T cell dependent destruction <strong>of</strong> the insulin<br />
producing pancreatic beta cells; however, the precise<br />
pathogenesis <strong>of</strong> the disease, especially the initiation <strong>of</strong><br />
pathologic immune response, is still largely unknown. We<br />
hypothesized that the function <strong>of</strong> human CD4+ T cells is<br />
altered in T1DM and analyzed unstimulated human peripheral<br />
blood CD4+ T cell gene expression. We used a novel<br />
three-way comparison <strong>of</strong> DNA microarray data <strong>of</strong> CD4+ Tcells
Abstracts<br />
isolated from new onset T1DM, patients with long-term type<br />
2 diabetes (T2DM), and from healthy controls in order to<br />
eliminate any possible influence <strong>of</strong> glucose homeostasis on<br />
our findings. We analyzed the T1DM specific gene expression<br />
changes and their functional relevance to T1DM autoimmunity.<br />
Our genetic and functional data show that T1DM CD4+ T<br />
cells are down-regulated specifically affecting key immune<br />
functions and cell cycle. Histone deacetylase gene expression,<br />
a key regulator <strong>of</strong> epigenetic modification, is also<br />
reduced. The CD4+ T cells showed impaired function,<br />
including an abnormal immune response, which may be a<br />
key element that leads to the breakdown <strong>of</strong> self-tolerance.<br />
doi:10.1016/j.clim.2007.03.244<br />
F.32 Protective Versus aggressive T Cell Responses<br />
to the Major Proinsulin73−90 Epitope in Beta-Islet<br />
Cell Autoimmunity<br />
Ivana Durinovic-Bello, Staff Scientist, Visiting Associate<br />
Pr<strong>of</strong>essor, Benaroya Research Institute, Virginia Mason<br />
Research Center, Seattle, WA, Nicol Pratt, Research<br />
Technician, Benaroya Research Institute, Virginia Mason<br />
Research Center, Seattle, WA, Susan A. Masewicz, Research<br />
Technician, Benaroya Research Institute, Virginia Mason<br />
Research Center, Seattle, WA, Gerald Nepom, Director,<br />
Virginia Mason Research Center, Benaroya Research<br />
Institute, Seattle, WA<br />
Proinsulin (P-Ins)73–90 is a major T cell epitope <strong>of</strong><br />
DRB1*0401 subjects with beta-islet cell autoimmunity. Subset<br />
<strong>of</strong> P-Ins73–90 specific T cells <strong>of</strong> the recent onset type 1<br />
diabetic (T1D) expressed Foxp3, a marker for regulatory Tcell<br />
lineages, secreted high IL-5 and IL-10 and low levels <strong>of</strong> IFNg,<br />
and predominantly used Tcell receptor Vb14 (Durinovic-Belló<br />
I et al. PNAS 103; 11,683, 2006). Addition <strong>of</strong> N- or C-terminal<br />
amino acids to P-Ins73–90 drastically changed their reactivity<br />
and cytokine phenotype, suggesting an immunomodulatory<br />
potential for this P-Ins epitope. To test the hypothesis that in<br />
subjects with beta-islet cell autoimmunity subsets <strong>of</strong> P-<br />
Ins73–90 specific T cells with potential down-regulatory<br />
phenotypes still exist, we have isolated P-Ins73–90 specific T<br />
cells <strong>of</strong> additional DRB1*0401 subjects (n=17) by depletion <strong>of</strong><br />
CD25+ Tcells, stimulation with P-Ins73–90 and IL-2, and Vb14<br />
and tetramer sorting. Both effector and down-regulatory<br />
cells show characteristics <strong>of</strong> activated T cells and express<br />
CD25 and OX40; in addition effector cells up-regulate CD154<br />
(CD40L), CD80/CD86, Fas (CD95) and FasL (CD178) and have<br />
high proliferative capacity, and down-regulatory cells<br />
express Foxp3 and suppress cognate and bystander responses.<br />
In T1D patients T cells secreting high levels <strong>of</strong> IFNg and IL-2<br />
predominate, whereas T cells <strong>of</strong> the control individuals and<br />
prediabetic subjects are characterized by high IL-4 and IL-5<br />
secretion and low IFNg and IL-10. Subsets <strong>of</strong> P-Ins73–90<br />
specific Tcells with down-regulatory phenotype may prove as<br />
a suitable targets for therapeutic intervention in DRB1*0401<br />
positive humans with beta-islet cell autoimmunity or recent<br />
onset T1D.<br />
doi:10.1016/j.clim.2007.03.245<br />
F.33 The Programmed Death-1 (pd-1) Pathway<br />
RegulatesPeripheralTCellToleranceDuring<br />
Autoimmune Diabetes in Nonobese Diabetic (NOD)<br />
Mice<br />
Brian T. Fife, Postdoctoral Fellow, University <strong>of</strong> California, San<br />
Francisco, San Francisco, CA, Indira Guleria, PhD,<br />
Transplantation Research Center, Brigham and Women’s<br />
Hospital and Children’s Hospital, Boston, MA, Melanie Bupp,<br />
PhD, University <strong>of</strong> California, San Francisco Diabetes Center,<br />
San Francisco, CA, Qizhi Tang, PhD, University <strong>of</strong> California, San<br />
Francisco Diabetes Center, San Francisco, CA, Todd Eagar, PhD,<br />
University <strong>of</strong> California, San Francisco Diabetes Center, San<br />
Francisco, CA, Helene Bour-Jordan, PhD, University <strong>of</strong><br />
California, San Francisco Diabetes Center, San Francisco, CA,<br />
Hideo Yagita, Pr<strong>of</strong>essor, Department <strong>of</strong> <strong>Immunology</strong>, Juntendo<br />
University School <strong>of</strong> Medicine, Tokyo, Japan, Miyuki Azuma,<br />
Pr<strong>of</strong>essor, Department <strong>of</strong> Molecular <strong>Immunology</strong>, Tokyo Medical<br />
and Dental University, Tokyo, Japan, Mohamed H. Sayegh,<br />
Pr<strong>of</strong>essor, Transplantation Research Center, Brigham and<br />
Women’s Hospital and Children’s Hospital, Boston, MA,<br />
Jeffrey Bluestone, Pr<strong>of</strong>essor, University <strong>of</strong> California,<br />
San Francisco Diabetes Center, San Francisco, CA<br />
Programmed death-1 (PD-1), an inhibitory costimulatory<br />
molecule on activated T cells, down-regulates immune<br />
responses. We investigated the role <strong>of</strong> this pathway in<br />
peripheral tolerance in the NOD mouse model <strong>of</strong> autoimmune<br />
diabetes. In this study, tolerance was induced using<br />
either antigen-coupled ethylene carbodiimide-fixed splenocytes<br />
or FcR non-binding anti-CD3 mAb administration.<br />
Antigen-coupled splenocyte treatment conferred complete<br />
and long lasting protection from diabetes transfer using islet<br />
reactive BDC2.5 TCR transgenic T cells. In addition, insulincoupled<br />
splenocytes were used to treat new onset spontaneously<br />
diabetic mice. Using this approach we demonstrate<br />
that disease remission is associated with inhibition <strong>of</strong><br />
pathogenic T cell proliferation, decreased cytokine production,<br />
and T cell anergy induction. Moreover, we show that<br />
robust long-term tolerance depends on the PD-1/PD-L1<br />
pathway in both systems. Anti-PD-1 and PD-L1, but not anti-<br />
PD-L2, reversed tolerance weeks after tolerogenic therapy<br />
by promoting antigen-specific T cell proliferation and<br />
inflammatory cytokines directly in infiltrated tissues. PD-<br />
1/PD-L1 blockade did not limit Treg activity suggesting<br />
direct effects on pathogenic T cells. Finally, we describe a<br />
critical role for PD-1/PD-L1 in another powerful immunotherapy<br />
model using anti-CD3, currently being tested in<br />
clinical trials. Anti-CD3 treatment <strong>of</strong> new onset spontaneously<br />
diabetic NOD mice results in disease remission. PD-<br />
1/PD-L1 blockade reverses tolerance and rapidly precipitates<br />
diabetes. These findings suggest that PD-1/PD-L1<br />
interactions form a common pathway to selectively maintain<br />
tolerance directly in the target tissues. Taken together,<br />
these results provide important insights in the regulation <strong>of</strong><br />
autoimmune responses and may lead to novel immunotherapeutic<br />
approaches for peripheral tolerance and treatment<br />
<strong>of</strong> autoimmune diabetes. Supported by NIH AI35297 and<br />
JDRF 10-2006-799.<br />
doi:10.1016/j.clim.2007.03.248<br />
S27
S28 Abstracts<br />
F.34 Gleevec: A New Therapeutic for Type I<br />
Diabetes?<br />
Cedric Louvet, Postdoctoral Research Fellow, Diabetes<br />
Center at University <strong>of</strong> California, San Francisco, San<br />
Francisco, CA, Gregory L. Szot, Islet Transplant Facility<br />
Manager, Diabetes Center, San Francisco, CA, Jiena Lang,<br />
Staff Research Associate, Diabetes Center at University <strong>of</strong><br />
California, San Francisco, San Francisco, CA, Jeffrey A.<br />
Bluestone, Diabetes Center Director, Diabetes Center at<br />
University <strong>of</strong> California, San Francisco, San Francisco, CA,<br />
Michael R. Lee, Staff Research Assistant, Diabetes Center at<br />
University <strong>of</strong> California, San Francisco, San Francisco, CA<br />
Gleevec (Imatinib, formerly STI571) is the first FDA-approved<br />
drug to directly turn <strong>of</strong>f a specific protein known to cause a<br />
cancer. Although targeted to the Bcr-Abl kinase, Gleevec has<br />
been shown to modestly affect related kinases including PDGF,<br />
c-fms and c-kit potentially involved in various cell types critical<br />
to the development and progression <strong>of</strong> autoimmune diseases.<br />
Based on these fundamental properties, we embarked on a<br />
study to determine the clinical effects <strong>of</strong> Gleevec in T1D in NOD<br />
mice.<br />
NOD mice were treated by gavage daily with 1.5 mg/<br />
mouse <strong>of</strong> commercially available Gleevec suspended in<br />
peanut oil. Pre-diabetic mice treated with Gleevec, but not<br />
with peanut oil only, were protected from spontaneous<br />
diabetes. More importantly, when Gleevec treatment was<br />
initiated at the time <strong>of</strong> disease onset (blood sugars between<br />
250 and 350 mg/dl), the majority <strong>of</strong> mice went into remission<br />
and b10% relapsed during the course <strong>of</strong> the therapy. When<br />
therapy was discontinued at 2 weeks, the majority <strong>of</strong> the<br />
mice became hyperglycemic within 1–2 weeks. However,<br />
longer therapy (8–10 weeks) resulted in long-term normoglycemia<br />
in a majority <strong>of</strong> treated animals even after drug<br />
cessation suggesting that Gleevec does not have to be given<br />
continuously to promote long-lasting protection. The possibility<br />
that short-term therapy with Gleevec can lead to longterm<br />
effects makes it a very attractive potential therapeutic<br />
for the treatment <strong>of</strong> patients with new onset T1D as well as<br />
potentially patients undergoing an islet transplant.<br />
doi:10.1016/j.clim.2007.03.249<br />
F.38 Characterization <strong>of</strong> Peptide Agonists and<br />
Antagonists for the Interaction <strong>of</strong> CD200 with<br />
CD200R1 and Their Efficacy in Modulation <strong>of</strong><br />
LPS-induced TNFα Production and Mortality In Vivo<br />
Reginald Gorczynski, Pr<strong>of</strong>essor, University Health Network,<br />
Toronto, ON, Canada, Ivo Boudakov, PDF, University Health<br />
Network, Toronto, ON, Canada, Ismat Khatri, PDF,<br />
University Health Network, Toronto, ON, Canada<br />
After engagement <strong>of</strong> the Ig-supergene family members<br />
CD200R1 and CD200, immunosuppressive and/or antiinflammatory<br />
signals augment allograft survival and<br />
decrease production <strong>of</strong> inflammatory cytokines. The primary<br />
domains <strong>of</strong> both CD200 and CD200R1 implicated in<br />
these interactions are found in the NH2-terminal region <strong>of</strong><br />
each molecule. We investigated the capacity <strong>of</strong> 10–15 mer<br />
synthetic peptides defining the three complementaritydetermining<br />
(CDR1–3) regions <strong>of</strong> mouse CD200 to reproduce<br />
the function <strong>of</strong> full-length CD200, or antagonize that<br />
function, assaying the suppression <strong>of</strong> LPS-induced TNFα<br />
production (in vivo/in); suppression <strong>of</strong> MLC responses in<br />
vitro; and LPS-induced mortality (d7) in vivo. Peptides<br />
located in the CDR2 region, but not CDR1 or CDR3 were<br />
potent agonists in vitro and in vivo, recapitulating the<br />
effects seen with the full-length molecule (CD200Fc). These<br />
small molecular weight agonists <strong>of</strong> CD200 might have<br />
efficacy in vivo in models <strong>of</strong> sepsis (and TNFα production).<br />
In a further analysis we explored the ability <strong>of</strong> the same<br />
peptides to block the suppressive and anti-inflammatory<br />
effect <strong>of</strong> the full-length CD200Fc which was seen in MLCs<br />
and after LPS administration both in vitro and in vivo. In<br />
these studies peptides located in the CDR1 and CDR3<br />
regions <strong>of</strong> the NH2-terminal <strong>of</strong> CD200 were found to be<br />
antagonists <strong>of</strong> the biological effects <strong>of</strong> CD200. Our data<br />
confirm that discrete small molecular weight agonists and<br />
antagonists can be defined for the interactions <strong>of</strong> CD200<br />
and the CD200R1 receptor. Since these interactions result in<br />
regulation <strong>of</strong> both acquired immune responses, and<br />
inflammatory responses, these peptides may have clinical<br />
utility.<br />
doi:10.1016/j.clim.2007.03.250<br />
F.39 Calculating the Number <strong>of</strong> Ordered Mutations<br />
Necessary to Cause a Specific Cancer in a Specific<br />
Risk Group and the Fraction <strong>of</strong> the Group that is<br />
Immune to the Cancer<br />
Ivan Kramer, Associate Pr<strong>of</strong>essor, UMBC, Canada Physics,<br />
Catonsville, MD<br />
The series <strong>of</strong> ordered mutations that cause a cell to<br />
become cancerous is modeled so that the fraction <strong>of</strong> a risk<br />
group (e.g., white men) that has developed a specific cancer<br />
(e.g., melanoma) at any age can be computed. The saturated<br />
model constructed here allows the possibility that a fraction<br />
<strong>of</strong> a risk group may be immune to developing a specific<br />
cancer but is otherwise isomorphic to the physical model<br />
describing an ordered chain <strong>of</strong> radioactive nuclear decays.<br />
The model’s cancer incidence function depends on only<br />
three independent parameters: the number <strong>of</strong> mutations<br />
necessary for a cell to become cancerous, the fraction <strong>of</strong> the<br />
group that is immune to developing the cancer, and the time<br />
required for 50% <strong>of</strong> the group to experience an average<br />
mutation (defined as the mutation half-life). These three<br />
parameters are determined by fitting the model’s cancer<br />
incidence function to the cancer incidence data. For<br />
example, the modeling predicts that all white males in the<br />
USA are vulnerable to developing melanoma, five ordered<br />
mutations are required to develop it, and the mutation halflife<br />
is 33.5 years. By contrast, the modeling also predicts that<br />
80.7% <strong>of</strong> white females in the USA are immune to developing<br />
melanoma, three ordered mutations are required to develop<br />
it, and the mutation half-life is 54.7 years. Thus, different<br />
risk groups can develop the same cancer through different<br />
pathways. The mechanism underlying female immunity to<br />
melanoma should become a research priority. Stimulating
Abstracts<br />
the immune system to eliminate mutated, pre-cancerous<br />
cells would prevent cancer.<br />
doi:10.1016/j.clim.2007.03.255<br />
F.41 The State <strong>of</strong> Immune System in the Pregnants<br />
with Untreated Syphilis<br />
Tatiana Yaremtchuk, Doucent, Lviv National Medical<br />
University, Department <strong>of</strong> Obstetrics, Gynecology and<br />
Perinatology <strong>of</strong> PGE, Lviv, Ukraine, Olga Korchynska,<br />
Assistant, Department <strong>of</strong> Obstetrics, Gynecology and<br />
Perinatology <strong>of</strong> PGE <strong>of</strong> Lviv National Medical University,<br />
Lviv, Ukraine, Yaroslav Korinets, Doctor, Scientific<br />
Collaborator, Department <strong>of</strong> Antenatal Diagnostics and<br />
Perinatology <strong>of</strong> Institute <strong>of</strong> Hereditary Pathology, Lviv,<br />
Ukraine, Natalia Prokoptchuk, Genetics and USG Diagnostics<br />
Doctor, Department <strong>of</strong> Antenatal Diagnostics and<br />
Perinatology <strong>of</strong> Institute <strong>of</strong> Hereditary Pathology, Lviv,<br />
Ukraine, Angela Misiura, Assistant, Department <strong>of</strong><br />
Obstetrics, Gynecology and Perinatology <strong>of</strong> PGE <strong>of</strong> Lviv<br />
National Medical University, Lviv, Ukraine<br />
Objective <strong>of</strong> Study: The state <strong>of</strong> immune system in the<br />
pregnants with untreated syphilis. Materials: Vein blood <strong>of</strong> 5<br />
pregnants with untreated early latent syphilis within 26-33<br />
weeks and 30 healthy pregnants in the same terms. Methods:<br />
Designation <strong>of</strong> quantity <strong>of</strong> CD3, CD4, CD8, CD16, CD56, CD19,<br />
CD4/CD8 was carried out with test-systems <strong>of</strong> monoclonal<br />
antibodies. The concentrations <strong>of</strong> IgA, IgM, IgG were<br />
designated by method <strong>of</strong> radial immune diffusion. The level<br />
<strong>of</strong> circulating immune complexes was investigated by<br />
spectrophotometral method. Results: Initial investigations<br />
showed the quantity <strong>of</strong> CD3 composed 59.4 % in comparison<br />
with 62,53+/–1,23 % in control group (P>0,05). The level <strong>of</strong><br />
CD4 trustworthy was lower – 16,2 % contra 35,6+/–0,86 %<br />
(Pb0,01). The quantity <strong>of</strong> CD8, CD16, CD56 correspondingly<br />
had tendency to reduce. These indicators accordingly made<br />
up – 25,0 % in comparison with 27,6+/–1,72 % (P>0.05), 3,0 %<br />
contra 6,51+/–0,88 % (P>0,05), 12,2 % in comparison with<br />
14,68+/–1,71 % (P>0,05). The immuneregulative index was<br />
0,65+/–0,22 in comparison 1,29+/–0,06 (Pb0,05). The concentration<br />
CD19 was trustworthy lower 5,2 % contra 12,83+/–<br />
0,88 % (Pb0,05). The levels <strong>of</strong> IgA and IgG were accordingly<br />
1,56+/–0,2 g/l in comparison with 1,98+/–0,06 g/l (Pb0,05)<br />
and 10,29+/–0,54 g/l contra 12,38+/–0,43 g/l (Pb0,05). The<br />
concentration <strong>of</strong> IgM had tendency to decrease – 1,37+/–0,32<br />
g/l contra 1,76+/–0,11 g/l (P>0,05). The level <strong>of</strong> circulating<br />
immune complexes also was trustworthy lower 59,8+/–11,4<br />
ODU contra 93,5+/–3,58 ODU (Pb0,01). Conclusions: The<br />
suppression <strong>of</strong> immune system has been found out in the<br />
pregnants with untreated early latent syphilis.<br />
doi:10.1016/j.clim.2007.03.256<br />
F.44 Human Norovirus Infection: Genetic and<br />
Immune Determinants <strong>of</strong> Susceptibility and<br />
Resistance<br />
Juan Leon, Postdoctoral Fellow, Emory University, Atlanta,<br />
GA, Lisa Lindesmith, Laboratory Technician, Department <strong>of</strong><br />
Epidemiology, University <strong>of</strong> North Carolina at Chapel Hill,<br />
Chapel Hill, NC, Erin-Joi McNeal, Laboratory Technician,<br />
Department <strong>of</strong> Global Health, Rollins School <strong>of</strong> Public<br />
Health, Atlanta, GA, Ralph Baric, Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Epidemiology, University <strong>of</strong> North Carolina at Chapel Hill,<br />
Chapel Hill, NC, Elizabeth Ailes, Graduate Student,<br />
Department <strong>of</strong> Epidemiology, Emory University, Atlanta,<br />
GA, Christine Moe, Associate Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Global Health, Rollins School <strong>of</strong> Public Health, Atlanta, GA<br />
Infection with Norovirus (NoV) is the main cause <strong>of</strong><br />
epidemic worldwide. NoV are classified by the CDC and NIAID<br />
as Bioterrorism Category B Priority Pathogens based on their<br />
high transmissibility, low infectious dose, and serious<br />
economic impact. Little is known on what factors protect<br />
individuals from NoV infection. To meet this need, our goal is<br />
to identify genetic and immunologic determinants <strong>of</strong><br />
protection from NoV infection. Because animal models are<br />
not available and these viruses do not replicate in culture,<br />
we developed expertise in human challenge models. We<br />
challenged 109 human volunteers with varying doses <strong>of</strong> the<br />
Norwalk virus (NV) strain <strong>of</strong> NoV. We found that gender,<br />
ethnicity, blood group, levels <strong>of</strong> pre-challenge serum NVspecific<br />
IgG or levels <strong>of</strong> pre-challenge saliva NV-specific IgA<br />
were not significant predictors <strong>of</strong> infection. Presence <strong>of</strong><br />
serum NV-specific IgG and presence <strong>of</strong> a putative NV-binding<br />
receptor, H type 1, were significantly associated with<br />
infection. Dose <strong>of</strong> inoculum received and age <strong>of</strong> the<br />
individual may also be predictors <strong>of</strong> infection. We observed<br />
a significant correlation in levels <strong>of</strong> salivary NV-specific IgG<br />
with salivary NV-specific IgA and serum NV-specific IgG.<br />
Lastly, in genetically susceptible individuals, infected individuals<br />
exhibited a rise in NV-specific serum and salivary<br />
antibodies while uninfected individuals did not. These<br />
results suggest that only infected individuals develop<br />
activation <strong>of</strong> the humoral response. These data will be<br />
used to better understand the immune response to NV<br />
infection and design effective vaccines against NoV to<br />
protect high risk populations including children, food<br />
handlers, and the elderly.<br />
doi:10.1016/j.clim.2007.03.257<br />
F.45 Polymorphism <strong>of</strong> IL-6 gene and Its Association<br />
with Susceptibility to Brucellosis<br />
Manoochehr Rasouli, <strong>Immunology</strong> Department, <strong>Clinical</strong><br />
Microbiology Research Center-Shiraz University <strong>of</strong> Medical<br />
Sciences, Shiraz, Simin Kiany, MSc, <strong>Immunology</strong> Department,<br />
<strong>Clinical</strong> Microbiology Research Center-Shiraz University <strong>of</strong><br />
Medical Sciences, Shiraz<br />
S29<br />
Background: Brucellosis is a highly contagious bacterial<br />
zoonosis that affects millions <strong>of</strong> people worldwide. Polymorphonuclear<br />
cells (PMNs) are the most abundant type <strong>of</strong><br />
circulating white blood cells (WBCs) and are the major cell<br />
type mediating acute inflammation response to bacterial<br />
infection. Since IL-6 plays a major role in inflammation and<br />
stimulates production <strong>of</strong> neutrophils from BM progenitors,<br />
this supports the involvement <strong>of</strong> IL-6 in the process and<br />
pathogenesis <strong>of</strong> brucellosis. It has been shown that the
S30 Abstracts<br />
production level <strong>of</strong> IL-6 has been correlated with allele<br />
inherited in individuals. Therefore, the aim <strong>of</strong> current study<br />
was to assess whether IL-6 (−174) polymorphism was<br />
associated with susceptibility to brucellosis. Method: One<br />
hundred and seventy-five patients with brucellosis and 77<br />
healthy animal husbandmen who had infected animals and<br />
consumed their dairy products, joined this study. DNAs<br />
extracted from samples were genotyped for IL-6 (−174 G/C)<br />
polymorphism using AS-PCR method. Results: Our results<br />
showed that IL-6 (−174) gene polymorphism was distributed<br />
similarly in the patient and control groups (P =0.3).<br />
Discussion: Although we found no association between IL-6<br />
(−174) gene polymorphism and susceptibility to brucellosis<br />
we cannot exclude that other polymorphisms within this<br />
gene or its receptor may donate susceptibility to brucellosis.<br />
Moreover these findings need confirmation in other<br />
population groups.<br />
doi:10.1016/j.clim.2007.03.259<br />
F.46 Interleukin-1β(+3953) Gene Polymorphism and<br />
Susceptibility to Helicobacter Pylori Associated<br />
Gastritis<br />
Ayda Hosseinkhani, Pharmacist, <strong>Clinical</strong> Microbiology<br />
Research Center, Shiraz University <strong>of</strong> Medical Sciences,<br />
Shiraz, Simin Kiany, MSc, <strong>Clinical</strong> Microbiology Research<br />
Center, Shiraz University <strong>of</strong> Medical Sciences, Shiraz,<br />
Manoochehr Rasouli, PhD, <strong>Clinical</strong> Microbiology Research<br />
Center, Shiraz University <strong>of</strong> Medical Sciences, Shiraz, Akram<br />
Jamshidzadeh, PhD, Faculty <strong>of</strong> Pharmacy-Shiraz University<br />
<strong>of</strong> Medical Sciences, Shiraz, Shohreh Farshad, PhD, <strong>Clinical</strong><br />
Microbiology Research Center, Shiraz University <strong>of</strong> Medical<br />
Sciences, Shiraz<br />
Introduction: It has been speculated that IL-1 genes<br />
play a crucial role in the genetic predisposition to gastric<br />
problems upon H. pylori infection by modulating the host<br />
immune response. Three biallelic polymorphisms have<br />
been reported in IL-1 β all representing CNT base<br />
transition at position −511, −31 and +3953 from the<br />
transcriptional site. In the present study we attempted to<br />
determine the IL-1 β (+3953) risk genotypes to H. pylori<br />
mediated gastritis. Methods: The subjects were 373<br />
individuals with no gastrointestinal problems and 49 H.<br />
pylori positive patients suffering from gastritis which was<br />
confirmed on the basis <strong>of</strong> endoscopic findings. All subjects<br />
were genotyped for IL-1 β+3953 gene polymorphism using<br />
polymerase chain reaction-restriction fragment length<br />
polymorphism (PCR-RFLP). Results: There was no significant<br />
difference in the frequency <strong>of</strong> IL-1 β +3953 C/T<br />
genotypes between two groups.<br />
Discussion: The lack <strong>of</strong> significant difference in the<br />
frequency <strong>of</strong> IL-1 β+3953 C/T between the studied groups<br />
might be the result <strong>of</strong> our small patient population. Thus we<br />
suggest that this study be performed on larger group <strong>of</strong><br />
patients; also investigation <strong>of</strong> other IL-1 functional gene<br />
polymorphism on Iranian population is recommended.<br />
doi:10.1016/j.clim.2007.03.260<br />
F.48 The −251 AA Genotype <strong>of</strong> the Interleukin-8<br />
Promoter is Associated with Susceptibility to<br />
Brucellosis<br />
Manoochehr Rasouli, PhD, <strong>Immunology</strong> Department, <strong>Clinical</strong><br />
Microbiology Research Center, Shiraz University <strong>of</strong> Medical<br />
Sciences, Shiraz, Simin Kiany, MSc, <strong>Immunology</strong> Department,<br />
<strong>Clinical</strong> Microbiology Research Center, Shiraz University <strong>of</strong><br />
Medical Sciences, Shiraz<br />
Background: Interleukin-8 (IL-8), a member <strong>of</strong> CXC chemokine<br />
family, is a chemoattractant <strong>of</strong> neutrophils and lymphocytes.<br />
IL-8 promoter is estimated to be 1500 bp. Several reports<br />
have shown relationship between IL-8 gene polymorphism and<br />
several human diseases such as bronchial asthma, Parkinson’s<br />
disease and Helicobacter pylori infection. The aim <strong>of</strong> this study<br />
was to evaluate the relationship between IL-8 (−251 A/T)<br />
polymorphism and brucellosis. Methods: One hundred and<br />
eighty eight patients with brucellosis and 77 healthy farmers<br />
who consumed contaminated row milk and dairy products <strong>of</strong><br />
animals with brucellosis were included in this study. All<br />
individuals were genotyped for IL-8 (−251 A/T) promoter<br />
polymorphism using polymerase chain reaction-restriction fragment<br />
length polymorphism (PCR-RFLP). Results: The frequency<br />
<strong>of</strong> AA genotype was significantly higher in patient group than<br />
healthy individuals (14.3% vs. 2.5%, P=0.005). Discussion: As<br />
data revealed that the frequency <strong>of</strong> AA genotype was lower in<br />
healthy controls than patients. Hence, our study provides<br />
evidence that the presence <strong>of</strong> AA genotype is significantly<br />
associated with susceptibility to brucellosis.<br />
doi:10.1016/j.clim.2007.03.261<br />
F.49 Interferon Gamma Producing T Lymphocytes in<br />
Bacterial Bi<strong>of</strong>ilm Infection: Response Modifiers for<br />
Polymorphonuclear Neutrophils (PMN)<br />
Christ<strong>of</strong> Wagner, Associate Pr<strong>of</strong>essor, University <strong>of</strong><br />
Heidelberg, Heidelberg, Germany, Christ<strong>of</strong> Iking-Konert,<br />
Assisant Pr<strong>of</strong>essor, University <strong>of</strong> Duesseldorf, Duesseldorf,<br />
Germany, Andreas Wentzensen, Head <strong>of</strong> Clinic, Klinik fur<br />
Unfall-und Weiderherstellungschirurgie, Ludwigshafen,<br />
Germany, Gertrud Maria Haensch, Full Pr<strong>of</strong>essor, University<br />
<strong>of</strong> Heidelberg Department <strong>of</strong> <strong>Immunology</strong>, Heidelberg,<br />
Germany, Volkmar Heppert, Head <strong>of</strong> Department, Septic<br />
Traumatology, Ludwigshafen, Germany<br />
Bacterial infection with bi<strong>of</strong>ilm formation on orthopaedic<br />
implants causes a progressive, destructive inflammatory<br />
process, causing massive tissue destruction, bone resorption,<br />
and loosening <strong>of</strong> the implant. In most cases, this so-called<br />
implant-associated posttraumatic osteomyelitis requires the<br />
removal <strong>of</strong> the implant, allowing recovery and analysis <strong>of</strong> the<br />
local cellular infiltrate. By cyt<strong>of</strong>luorometry, the majority (60–<br />
85%) <strong>of</strong> the infiltrated cells were identified as highly activated<br />
polymorphonuclear neutrophils (PMN); the next largest cell<br />
population (5–35%) were T-lymphocytes. CD4 and CD8 positive T<br />
cells were found, the latter prevailing compared to the<br />
peripheral blood. The majority <strong>of</strong> the CD8+ cells were CD28<br />
negative; about 26% expressed CD11b, 76% CD57. The latter two<br />
receptors were co-expressed on less than 5% <strong>of</strong> the cells. The
Abstracts<br />
CD11b positive Tcells produced interferon (IFN) gamma. In vitro<br />
experiments provided a link between activated Tcells and PMN:<br />
IFN γ, supernatants <strong>of</strong> the Tcells, or co-incubation <strong>of</strong> PMN with<br />
activated T cells induced expression on PMN <strong>of</strong> CD64, the high<br />
affinity receptor for immunoglobulin, and <strong>of</strong> MHC class II<br />
antigens. The fact that IFN γ is among the few cytokines that<br />
can stimulate PMN to produce CD64 and MHC class II, and the<br />
observation that CD64 and MHC class II positive PMN is found in<br />
the cellular infiltrate suggest that CD8+ T cells via synthesis <strong>of</strong><br />
IFN γ modulatethefunction<strong>of</strong>infiltratedPMNandpointtoa<br />
novel, not yet appreciated role <strong>of</strong> Tcells in the defence against<br />
bacterial bi<strong>of</strong>ilm infections.<br />
doi:10.1016/j.clim.2007.03.262<br />
F.50 Server Hepatitis Related Gene mfgl2 shRNA<br />
Plasmid Construction and Its RNA Interference<br />
In Vitro<br />
Qin Ning, Chief Physician, Department <strong>of</strong> Infectious Disease,<br />
Tongji Hospital Huazhong University <strong>of</strong> Science and<br />
Technology, Wuhan, China, Zhimo Wang, Doctor in Charge,<br />
Department <strong>of</strong> Infectious Disease, Tongji Hospital, Huazhong<br />
University <strong>of</strong> Science and Technology, Wuhan, China, Dong Xi,<br />
Assistant Researcher, Department <strong>of</strong> Infectious Disease, Tongji<br />
Hospital, Huazhong University <strong>of</strong> Science and Technology,<br />
Wuhan, China, Chuanlong Zhu, Doctor in Charge, Department<br />
<strong>of</strong> Infectious Disease, Tongji Hospital, Huazhong University <strong>of</strong><br />
ScienceandTechnology,Wuhan,China,SuiGao,Postgraduate<br />
Student, Department <strong>of</strong> Infectious Disease, Tongji Hospital,<br />
Huazhong University <strong>of</strong> Science and Technology, Wuhan,<br />
China, Weiming Yan, Associate Researcher, Department <strong>of</strong><br />
Infectious Disease, Tongji Hospital, Huazhong University <strong>of</strong><br />
Science and Technology, Wuhan, China, Xiaoping Luo, Chief<br />
Physician, Department <strong>of</strong> Infectious Disease, Tongji Hospital,<br />
Huazhong University <strong>of</strong> Science and Technology, Wuhan, China<br />
Objective: To determine the viral and host factors involved<br />
in the transcription <strong>of</strong> hfgl2 gene which was demonstrated to<br />
play a pivotal role in human and experiment fulminant viral<br />
hepatitis. Methods: HBc, HBs or HBx expression plasmids were<br />
constructed and cotransfected with a hfgl2 luciferase report<br />
construct into eukaryotic cells. Results: Luciferase assay<br />
showed that HBc or HBx protein, but not HBs protein<br />
significantly enhanced hfgl2 transcription in both CHO and<br />
HepG2 cells. Series deletion assay <strong>of</strong> hfgl2 gene promoter<br />
demonstrated that a strong regulatory domain from ¨C 712 to<br />
−568 was responsible for hfgl2 gene transcription in response<br />
to HBc or HBx proteins. By site-directed mutagenesis, the<br />
overlapped cis-elements LEF/c-Ets in domain −712/−568 was<br />
demonstrated to play an important role in the regulation <strong>of</strong><br />
hfgl2 gene in response to HBc protein, while another two ciselements<br />
LEF/c-Ets and HSTF in the same domain were found<br />
to participate in hfgl2 transcription regulation in response to<br />
HBx protein. EMSA assays showed that HBc and HBx proteins<br />
did not directly induce hfgl2 gene activation, while an Ets<br />
family member c-Ets-2 bound to the cognate cis-element in<br />
hfgl2 promoter was responsible for induction <strong>of</strong> hfgl2<br />
activation in response to viral proteins. Conclusion: Our<br />
study provides new insights in understanding the pathology <strong>of</strong><br />
fulminant hepatic failure and the interaction between HBV<br />
virus and host gene expression. This work was supported by<br />
NSFC 30225040, 30571643, 30672380, National Key Basic<br />
Research Program <strong>of</strong> China (2005CB522901, 2005CB522507)<br />
and <strong>Clinical</strong> Subject Key Project from the Ministry <strong>of</strong> Health.<br />
doi:10.1016/j.clim.2007.03.263<br />
F.51 TLR2 and TLR4 Expression on PBMC and Ascites<br />
Fluid from Hepatic Cirrhotic Patients with SPB<br />
Carmen Contreras, MD, <strong>Immunology</strong> Universidad de<br />
Guadalajara, Guadalajara, Mexico, Jorge Segura,<br />
Gastroenterologist, Gastroenterology Hospital Civil,<br />
Guadalajara, Mexico, Cecilia Guillen, Pr<strong>of</strong>essor,<br />
<strong>Immunology</strong> Universidad de Guadalajara, Guadalajara,<br />
Mexico, Mary Fafutis, Immunologist Researcher,<br />
<strong>Immunology</strong>, Universidad de Guadalajara, Guadalajara,<br />
Mexico, Margarita Montoya, Immunologist, <strong>Immunology</strong><br />
Universidad de Guadalajara, Guadalajara, Mexico<br />
Justification: The cirrhosis <strong>of</strong> the liver is a pathology with a<br />
high morbid-mortality, being one <strong>of</strong> the main complication’s<br />
cause <strong>of</strong> spontaneous bacterial peritonitis (SBP) due to an<br />
alteration in the immunologic balance, observed like a<br />
deficiency in the bactericide action <strong>of</strong> serum, opsonins, and<br />
complement; an increase in the proinflammatory cytokines;<br />
changes in the endoplasmatic reticulum system; and a<br />
neutrophil’s functional alteration. It has been demonstrated<br />
that the toll-like receptors (TLR) are essentials for the<br />
pathogenic recognition that starts with the activation <strong>of</strong> NFkB<br />
a transcriptional factor necessary for the biosynthesis <strong>of</strong><br />
proinflammatory cytokines. At the moment the role played by<br />
these membrane receptors in the development <strong>of</strong> SBP is<br />
unknown. The aim <strong>of</strong> this work is to study the TLR2 and 4<br />
expression on PBMC and ascites fluid <strong>of</strong> cirrhotic patients (CP)<br />
with or without an SPB development. Methodology: Venous<br />
peripheral blood and ascites fluid was obtained from CP with<br />
or without SPB, as well as venous peripheral blood from<br />
healthy individuals (HI). The expression <strong>of</strong> TLR2, TLR4 and<br />
CD14 by flow cytometer was measured. Results: The<br />
peripheral blood’s cells from HI show a higher expression <strong>of</strong><br />
TLR2 and TLR4 than CP and SPB, however the differences<br />
were not significant. The TLR2 and TLR4 expression in<br />
patient’s cells with SPB was higher than the cirrhotic patients<br />
(pb0.05) in ascites fluid. Conclusion: In this work it was found<br />
that the TLR2 and TLR4 expression was higher in SPB than<br />
cirrhotic patients in ascites fluid.<br />
doi:10.1016/j.clim.2007.03.264<br />
S31<br />
F.52 Persistently Deficient In Vitro<br />
Anti-Mycobacterial Immunity Despite Highly<br />
Successful Long-Term (>4 years) HAART:<br />
Differential Impact <strong>of</strong> Latent Tuberculosis Infection<br />
and Previous Active Disease<br />
Gil Benard, Medical Researcher, Medical School <strong>of</strong> the<br />
University <strong>of</strong> São Paulo, São Paulo, Brazil, Marcelo<br />
Mendonça, Laboratory <strong>of</strong> Dermatology and<br />
Immunodeficiencies, Medical School <strong>of</strong> the University <strong>of</strong>
S32 Abstracts<br />
São Paulo, São Paulo, Brazil, Léia C. Rodrigues Silva, PhD<br />
Student, Laboratory <strong>of</strong> Dermatology and<br />
Immunodeficiencies, Medical School <strong>of</strong> the University <strong>of</strong><br />
São Paulo, São Paulo, Brazil, Guilherme G. Silveira,<br />
Student, Laboratory <strong>of</strong> Dermatology and<br />
Immunodeficiencies, Medical School <strong>of</strong> the University <strong>of</strong><br />
São Paulo, São Paulo, Brazil, Maury M. Tanji, Laboratory <strong>of</strong><br />
Dermatology and Immunodeficiencies, Medical School <strong>of</strong><br />
the University <strong>of</strong> São Paulo, São Paulo, Brazil, Denise<br />
Schout, Epidemiology Service, Hospital das Clínicas,<br />
Medical School <strong>of</strong> the University <strong>of</strong> São Paulo, São Paulo,<br />
Brazil, Alberto J.S. Duarte, Pr<strong>of</strong>essor, Laboratory <strong>of</strong><br />
Dermatology and Immunodeficiencies, Medical School <strong>of</strong><br />
the University <strong>of</strong> São Paulo, São Paulo, Brazil<br />
Background: The degree Mycobacterium tuberculosis (Mtb)<br />
immune reconstitution provided by long-term (N4 years)<br />
successful HAART and whether a past tuberculosis (TB) history<br />
influences this reconstitution are not known. Methods: We<br />
compared the lymphocyte proliferative response (LPR) and IFNã<br />
secretion to phytohemagglutinin (PHA) and Mtb antigens<br />
(ESAT6, Ag85B and whole Mtb lysate [SMtb]) <strong>of</strong> immunereconstituted<br />
(CD4 N500 cells/ìl) HIV+ patients with a past history <strong>of</strong><br />
cured TB (HIV+ CTB group) or latent infection as presumed by<br />
positive purified protein derivative skin test (PPD+) (HIV+ PPD+<br />
group) with HIV− controls either cured from TB (CTB group) or<br />
PPD reactors (PPD+ group). The databank on TB incidence<br />
among HIV+ and HIV− patients at our services during 1999–2005<br />
was also analyzed. Results: Most HIV+ patients presented normal<br />
PHA responses. However, Mtb immune reconstitution was<br />
incomplete, especially to ESAT6 and Ag85B. SMtb reactivity<br />
was fully restored in the HIV+ CTB group, while in HIV+ PPD+<br />
patients it remained lower than in PPD+ controls. Comparison<br />
between the two HIV groups also suggested better immune<br />
reconstitution in HIV+ CTB patients. Databank analysis revealed<br />
that some HIV+ patients with N360 CD4 cells/ìl still developed<br />
TB, but not those with previous TB history. Conclusions: Mtb<br />
immune reconstitution is incomplete in HIV+ CTB and HIV+ PPD<br />
+ patients even after highly successful HAART. However,<br />
reactivity to whole mycobacteria antigens is restored,<br />
especially in HIV+ CTB patients, possibly due to survival <strong>of</strong><br />
higher numbers <strong>of</strong> mycobacteria-specific T cell clones throughout<br />
immunosuppression, probably allowing sufficient protection<br />
against new Mtb challenges.<br />
doi:10.1016/j.clim.2007.03.266<br />
F.53 Human CD8+ Cytotoxic T Lymphocyte Epitopes<br />
Identified from Herpes Simplex Virus Type 1<br />
Glycoprotein D<br />
Aziz Alami Chentoufi, Assistant Specialist, Cellular and<br />
Molecular <strong>Immunology</strong> Laboratory, The Eye Institute,<br />
University <strong>of</strong> California, Irvine, Orange, CA, Xiuli Zhang,<br />
Postdoctoral Fellow, Cellular and Molecular <strong>Immunology</strong><br />
Laboratory, The Eye Institute, University <strong>of</strong> California,<br />
Irvine, Orange, CA, Kasper Lamberth, Researcher, Institute<br />
<strong>of</strong> Medical Microbiology and <strong>Immunology</strong> (IMMI), University<br />
<strong>of</strong> Copenhagen, 2200 Copenhagen, Denmark, Xiaoming Zhu,<br />
Research Associate, Cellular and Molecular <strong>Immunology</strong><br />
Laboratory, The Eye Institute, University <strong>of</strong> California,<br />
Irvine, Orange, CA, Gargi Dasgupta, Research Associate,<br />
Cellular and Molecular <strong>Immunology</strong> Laboratory, The Eye<br />
Institute, University <strong>of</strong> California, Irvine, orange, CA,<br />
Michele Wu, Student, Cellular and Molecular <strong>Immunology</strong><br />
Laboratory, The Eye Institute, University <strong>of</strong> California,<br />
Irvine, Orange, CA, Alex Nguyen, Bio-199 Student, Cellular<br />
and Molecular <strong>Immunology</strong> Laboratory, The Eye Institute,<br />
University <strong>of</strong> California, Irvine, Orange, CA, Ilham Bettahi,<br />
Postdoctoral Fellow, Cellular and Molecular <strong>Immunology</strong><br />
Laboratory, The Eye Institute, University <strong>of</strong> California,<br />
Irvine, Orange, CA, Søren Buus, Pr<strong>of</strong>essor, Institute <strong>of</strong><br />
Medical Microbiology and <strong>Immunology</strong> (IMMI), University <strong>of</strong><br />
Copenhagen, 2200 Copenhagen, Denmark, Lbachir<br />
BenMohamed, Assistant Pr<strong>of</strong>essor, Cellular and Molecular<br />
<strong>Immunology</strong> Laboratory, The Eye Institute, University <strong>of</strong><br />
California, Irvine, Orange, CA<br />
Cytolystic T cells (CTLs) play a major role in herpes simplex<br />
virus type 1 and type 2 (HSV-1 and HSV-2) infections and<br />
diseases. Among some eleven HSV-1 and HSV-2 coat glycoproteins,<br />
glycoprotein D (gD) induces immunodominant T cells and<br />
is a leading vaccine candidate antigen. However, little is known<br />
about human CD8+ T cell epitopes <strong>of</strong> gD. Based on predictive<br />
computational algorithms and peptide binding affinity to HLA-<br />
A*0201 molecules, we have identified ten regions within the<br />
HSV-1 gD, each 9 to 10 amino acids in length, exhibiting high<br />
affinity CD8+ Tcell epitope(s). T2 cell stabilization assay showed<br />
peptide gD53-61, gD70-78 and gD278-286 significantly, and in<br />
dose-dependent manner, upregulates HLA-A*0201 molecules<br />
expression.Toobtainanobjectiveenumeration<strong>of</strong>CD8+Tcells<br />
induced by each gD peptide epitope, we employed the HLA-<br />
A*0201 tetramer staining procedure. A relatively high percentages<br />
<strong>of</strong> CD8+ Tcells positive for gD-18-10, gD53-61, gD70-78 and<br />
gD278-286/HLA-A*0201 tetramers were detected in PBMC from<br />
HLA-A*0201 seropositive patients, either directly ex vivo or<br />
following a single in vitro stimulation. Accordingly, strong HLA-<br />
A*0201-restricted CD8+ CTLs, assessed by INF-γ-ELISPOT and<br />
CD107a/b cytotoxic assays, were mapped to gD53-61, gD70-78<br />
and gD278-286 peptides. However, only gD53-61 and gD278-286<br />
peptides were able to generate CD8+ T cells that recognize<br />
infected target cells. Collectively, these findings suggest that<br />
high-affinity HLA-A*0201-binding gD53-61 and gD278-286 epitopes<br />
are naturally processed and are recognized by HSVspecific<br />
CD8+ CTLs in the infected human population, providing<br />
important information for the development <strong>of</strong> subunit vaccine<br />
strategies against herpes.<br />
doi:10.1016/j.clim.2007.03.267<br />
F.55 Protection Against SEB-Induced Toxic Shock by<br />
Anti-TCR Vb8 Antibody<br />
Manisha Singh, Postdoctoral Associate, Baylor College <strong>of</strong><br />
Medicine, <strong>Immunology</strong> Department, Houston, TX<br />
Intraperitoneal injection <strong>of</strong> the bacterial superantigen,<br />
Staphylococcus enterotoxin B (SEB) in HLA-DR3 transgenic<br />
mice leads to toxic shock and death within 72 h. In vivo,<br />
this is characterized by a marked expansion <strong>of</strong> splenic TCR<br />
Vb8+ CD4 and CD8 T cells compared to PBS treated mice. It
Abstracts<br />
appears that TCR Vb8-bearing CD4 and CD8 cells and the<br />
cytokines they secrete might be responsible for the<br />
pathogenesis <strong>of</strong> toxic shock caused by SEB. Therefore, we<br />
reasoned that depletion <strong>of</strong> TCR Vb8 bearing T cells by anti-<br />
Vb8 antibody might protect mice from SEB-induced shock.<br />
To test this hypothesis, we administered 200 μg <strong>of</strong> purified<br />
anti-TCR Vb8 antibody (clone 23.1) either prior to or 2 h<br />
after SEB injection. Irrespective <strong>of</strong> timing <strong>of</strong> anti-TCR Vb8<br />
administration, all anti-Vb8-treated mice (six <strong>of</strong> six)<br />
remained healthy while those injected with control antibody<br />
were either sick (n=2) or died (n=3). This salutary<br />
anti-TCR Vb8 effect correlated with markedly diminished<br />
SEB-induced serum levels <strong>of</strong> IL-6 and IL-2; however IFN-g<br />
and TNF release was not affected. Anti-TCR Vb8 Abs<br />
treatment also reduced infiltration in various organs like<br />
liver, lungs and kidney, those normally affected by SEB.<br />
These findings suggest that the pathogenesis <strong>of</strong> SEB induced<br />
toxic shock is primarily mediated by T cells and anti-TCR<br />
Vb8 antibody therapy may have therapeutic implications for<br />
SEB mediated shock.<br />
doi:10.1016/j.clim.2007.03.268<br />
F.56 Anti-Mouse GITR Monoclonal Antibody (Mab)<br />
Augments Humoral and Cellular Responses to H5N1<br />
and H3N2 Avian Influenza Hemagglutinin<br />
Joe Ponte, Senior Scientist, Tolerx, Inc, Cambridge, MA,<br />
Reema Gulati, Scientist, Tolerx, Cambridge, MA, Adam<br />
O’Shea, Scientist, Tolerx, Cambridge, MA, Paul Ponath,<br />
Immunologist, Tolerx, Cambridge, MA, Michael Paglia,<br />
Senior Manager, Tolerx, Cambridge, MA, Michael<br />
Rosenzweig, Senior Director, Tolerx, Cambridge, MA<br />
Adjuvants, including antibodies to tumor necrosis factor<br />
receptor superfamily (TNFRSF) members, augment immune<br />
responses to vaccines. One member <strong>of</strong> this family, glucocorticoid-induced<br />
tumor necrosis factor receptor (GITR), is<br />
expressed on regulatory and effector T cells. The aim <strong>of</strong> this<br />
study was to assess the ability <strong>of</strong> an anti-mouse GITR Mab,<br />
2F8, to stimulate murine humoral and cellular immunity to<br />
H3N2 and H5N1 viral hemagglutanins (HA). 2F8 enhanced<br />
anti-HA titers compared with controls and this enhancement<br />
was equal to or greater than titers obtained with incomplete<br />
Freund’s adjuvant or aluminum hydroxide. 2F8 F(ab(E))2<br />
fragments were also effective in boosting humoral immunity,<br />
suggesting that Fc interactions were not required. Moreover,<br />
the enhanced response was durable and specific for the<br />
antigen administered at the time <strong>of</strong> antibody treatment, and<br />
2F8-treated mice required less antigen to obtain titers<br />
equivalent to IFA-treated mice. 2F8 also enhanced cellular<br />
immunity as measured by EliSPOT. These results were<br />
recapitulated in cynomolgus monkeys using an anti-human<br />
GITR Mab (6C8). Thus, anti-GITR Mabs augment humoral and<br />
cellular immunity in mice and cynomolgus monkeys. Ongoing<br />
studies are evaluating the effect <strong>of</strong> anti-GITR Mabs on<br />
regulatory and effector T cells.<br />
doi:10.1016/j.clim.2007.03.269<br />
F.57 The B Cell Superantigen Peptostreprococcus<br />
Magnus Protein L Induces Lung Inflammation<br />
Through a MyD88-dependent Mechanism<br />
Amy Anderson, Research Associate, University <strong>of</strong> Pennsylvania<br />
School <strong>of</strong> Medicine, Philadelphia, PA, David LaRosa,<br />
Postdoctoral Fellow, University <strong>of</strong> Pennsylvania, Medicine,<br />
Philadelphia, PA, Arnold Levinson, Pr<strong>of</strong>essor <strong>of</strong> Medicine,<br />
University <strong>of</strong> Pennsylvania School <strong>of</strong> Medicine, Medicine,<br />
Philadelphia, PA<br />
Peptostreptococcus magnus protein L functions as a B cell<br />
superantigen by interacting with V kappa light chain<br />
determinants on many human and mouse immunoglobulins,<br />
irrespective <strong>of</strong> their antigen specificities or heavy chain<br />
isotype. We sought to determine if this property endowed<br />
protein L with the capacity to elicit lung inflammation.<br />
Following intratracheal (i.t.) administration <strong>of</strong> recombinant<br />
protein L or BSA to C57/BL6 mice, broncheoalveolar lavage<br />
fluid (BALF) was recovered at varying time points and<br />
analyzed for total neutrophil counts and concentrations <strong>of</strong><br />
MIP-2, KC and TNF. Lung tissue was harvested for histological<br />
examination. Protein L-treated mice accumulated neutrophils<br />
in their BALF as early as 4 h and showed an alveolar and<br />
peribronchial infiltrate <strong>of</strong> inflammatory cells peaking<br />
between 8 and 24 h. MIP-2, TNF, and KC levels in protein L<br />
challenged mice peaked at 4 h. These findings are consistent<br />
with those reported in conventional immune-complex models<br />
<strong>of</strong> lung inflammation. Studies were repeated in JHT mice to<br />
examine the importance <strong>of</strong> B cells/immunoglobulins in these<br />
reactions. Surprisingly, both the BALF response and lung<br />
histopathology were preserved. By contrast, both types <strong>of</strong><br />
reactions were abrogated in MyD88 knockout mice, which<br />
have defective toll-like receptor signaling. These results<br />
indicate that protein L-induced lung inflammation is not<br />
dependent on its immunoglobulin binding (B cell superantigenic)<br />
activity but does appear to rely on signaling<br />
through a toll-like receptor. Thus, the present study reveals a<br />
novel, unanticipated pro-inflammatory mechanism initiated<br />
by a microbial protein with known B cell superantigenic<br />
properties.<br />
doi:10.1016/j.clim.2007.03.272<br />
S33<br />
F.58 Regulation <strong>of</strong> the Epstein−Barr Virus<br />
LMP1-mediated NF-kappaB Signaling Pathway<br />
by a Novel Adaptor Protein STAP-2<br />
Osamu Ikeda, Undergraduate, Department <strong>of</strong> <strong>Immunology</strong>,<br />
Graduate School <strong>of</strong> Pharmaceutical Sciences Hokkaido<br />
University, Sapporo, Japan, Yuichi Sekine, Assistant,<br />
Department <strong>of</strong> <strong>Immunology</strong>, Graduate School <strong>of</strong> Pharmaceutical<br />
Sciences Hokkaido University, Sapporo, Japan, Teruhito Yasui,<br />
Assistant, Department <strong>of</strong> Molecular <strong>Immunology</strong>, Research<br />
Institute for Microbial Diseases, Osaka University, Japan, Suita<br />
Kenji Sugiyma, PhD, Nippon Boehringer Ingelheim Co. Ltd.<br />
Kawanishi Pharma Research Institute, Kawanishi, Japan, Kenji<br />
Oritani, Assistant, Department <strong>of</strong> Hematology and Oncology,<br />
Graduate School <strong>of</strong> Medicine, Osaka University, Japan, Suita<br />
Akihiko Yoshimura, Pr<strong>of</strong>essor, Division <strong>of</strong> Molecular and Cellular<br />
<strong>Immunology</strong>, Medical Institute <strong>of</strong> Bioregulation, Kyushu
S34 Abstracts<br />
University, Maidashi, Japan, Tadashi Matsuda, Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> <strong>Immunology</strong>, Graduate School <strong>of</strong> Pharmaceutical<br />
Sciences Hokkaido University, Sapporo, Japan<br />
Signal transducing adaptor protein-2 (STAP-2) is a recently<br />
identified adaptor protein, which contains pleckstrin homology<br />
(PH) and Src homology 2 (SH2)-like domains as well as a prolinerich<br />
domain in its C-terminal region. Our previous studies have<br />
demonstrated that STAP-2 binds to MyD88 and IKK-α/β, and<br />
modulates NF-kappaB signaling in macrophages. In this study,<br />
STAP-2 was induced by expression <strong>of</strong> the Epstein–Barr virus LMP1<br />
in B cells, while overexpression <strong>of</strong> STAP-2 inhibited LMP1mediated<br />
NF-kappaB signaling. Furthermore, STAP-2 associated<br />
with LMP1 in EBV-positive B cells. These results strongly suggest<br />
that STAP-2 acts as an endogenous negative inhibitor <strong>of</strong> the EBV<br />
LMP1-mediated signaling, which is critical for the EBV persistent<br />
infection and EBV-associated pathogenesis.<br />
doi:10.1016/j.clim.2007.03.273<br />
F.61 Defensins Block Bacterial Toxin-induced<br />
Interleukin-1β Production<br />
Jishu Shi, Assistant Pr<strong>of</strong>essor, Anatomy, Physiology and<br />
Pharmacology, Auburn, AL, Shelly Ao, Research Assistant,<br />
APP, Auburn, AL, Charalabos Pathoulakis, Pr<strong>of</strong>essor,<br />
Gasterenterology, Boston, MA<br />
This study was designed to test the hypothesis that<br />
defensins can block the release <strong>of</strong> IL-1β induced by<br />
Clostridium difficile toxin A (TxA) and Staphylococcus<br />
aureus α-toxin (a-Tox). LPS-activated (20 ng/ml, 2 h)<br />
human monocytes were treated with 3 nM TxA in the<br />
presence or absence <strong>of</strong> human neutrophil defensin HNP-1<br />
(25–50 μg/ml) or human Paneth cell defensin HD-5 (25–50<br />
μg/ml) for 6 h. In the absence <strong>of</strong> defensins, TxAstimulated<br />
monocytes released a large amount <strong>of</strong> mature<br />
IL-1β (17 kDa) into the extracellular milieu. The release<br />
<strong>of</strong> mature IL-1β from TxA-stimulated monocytes was<br />
blocked by HNP-1 and HD-5 in a dose-dependent manner.<br />
Different from ATP, TxA-induced IL-1β release was not<br />
inhibited by KN-62, a P2X7 receptor antagonist. In<br />
contrast to TxA, a-Tox (250 ng/ml) induced the release<br />
<strong>of</strong> both proIL-1β (31 kDa) and mature IL-1β from LPSactivated<br />
monocytes. In the presence <strong>of</strong> 20 μg/ml <strong>of</strong><br />
HNP-1 or HD-5, the amount <strong>of</strong> mature IL-1β released from<br />
a-Tox-treated monocytes was reduced by 70%, while a-<br />
Tox-induced release <strong>of</strong> proIL-1β was completely blocked.<br />
Similar to TxA, a-Tox-induced IL-1β release was not<br />
inhibited by KN-62. Furthermore, the release <strong>of</strong> mature<br />
IL-1β was caspase-1-dependent for both toxins. These<br />
data suggest that TxA- and a-Tox-induced IL-1β release is<br />
independent <strong>of</strong> the ATP/P2X7 receptor pathway, and that<br />
defensins can block bacterial toxin-induced posttranslational<br />
processing and release <strong>of</strong> IL-1β. In addition to their<br />
antimicrobial activity, defensins may play an important<br />
role in host inflammatory response to bacterial infection<br />
by controlling the production <strong>of</strong> IL-1β.<br />
doi:10.1016/j.clim.2007.03.274<br />
F.62 Lack <strong>of</strong> Association <strong>of</strong> Interleukin-8 Gene<br />
Polymorphism with Helicobacter Pylori-induced<br />
Gastritis in Iranian Patients<br />
Ayda Hosseinkhani, Pharmacist, <strong>Clinical</strong> Microbiology<br />
Research Center, Shiraz University <strong>of</strong> Medical Sciences,<br />
Shiraz, Iran, Farshad Shohreh, Microbiologist, <strong>Clinical</strong><br />
Microbiology Research Center, Shiraz University <strong>of</strong> Medical<br />
Sciences, Shiraz, Iran, Akram Jamshidzadeh, PhD, Faculty <strong>of</strong><br />
Pharmacy-Shiraz University <strong>of</strong> Medical Sciences, Shiraz,<br />
Iran, Manoochehr Rasouli, PhD, <strong>Clinical</strong> Microbiology<br />
Research Center, Shiraz University <strong>of</strong> Medical Sciences,<br />
Shiraz, Iran, Simin Kiany, MSc, <strong>Clinical</strong> Microbiology<br />
Research Center, Shiraz University <strong>of</strong> Medical Sciences,<br />
Shiraz, Iran<br />
Background: Helicobacter pylori is a major cause <strong>of</strong><br />
neoplastic and inflammatory gastroduodenal diseases <strong>of</strong> the<br />
stomach. H. pylori infection and associated gastric diseases are<br />
common in developing countries. Cytokine gene polymorphisms<br />
and H. pylori have been linked to gastric disease. We determined<br />
the role <strong>of</strong> host interleukin-8 (−251 A/T) gene polymorphism in<br />
the development <strong>of</strong> gastritis in south Iranian population.<br />
Methods: We genotyped IL-8 (−251 A/T) gene polymorphism in<br />
54 H. pylori infected individuals with gastritis and 337 infected<br />
individuals with normal mucosa using polymerase chain reaction-restriction<br />
fragment length polymorphism (PCR-RFLP). The<br />
diagnosis <strong>of</strong> gastritis was established on the basis <strong>of</strong> endoscopic<br />
findings.<br />
Results: We did not find any significant differences in<br />
allele and genotype frequencies <strong>of</strong> IL-8 (−251A/T) among our<br />
study groups. Discussion: Our analysis did not reveal a<br />
significant difference between the frequencies <strong>of</strong> IL-8<br />
(−251) genotypes and alleles which might be the result <strong>of</strong><br />
our limited number <strong>of</strong> patient population. We suggest this<br />
study be continued on larger population <strong>of</strong> the patients. Also,<br />
analysis <strong>of</strong> polymorphism in other positions <strong>of</strong> IL-8 gene is<br />
recommended.<br />
doi:10.1016/j.clim.2007.03.275<br />
F.63 The Impact <strong>of</strong> HLA Polymorphisms on<br />
Measles Virus-specific T-Cell Memory Responses<br />
in Vaccinated Subjects<br />
Inna Ovsyannikova, Senior Research Associate, Mayo Clinic,<br />
Mayo Vaccine Research Group, Rochester, MN, Jenna Ryan,<br />
Senior Research Technologist, Mayo Clinic, Mayo Vaccine<br />
Research Group, Rochester, MN, Norman Pinsky, Senior<br />
Research Technologist, Mayo Clinic, Mayo Vaccine Research<br />
Group, Rochester, MN, Robert Jacobson, Pr<strong>of</strong>essor <strong>of</strong><br />
Pediatrics, Mayo Clinic, Department <strong>of</strong> Pediatric and<br />
Adolescent Medicine, Rochester, MN, Robert Vierkant,<br />
Senior Statistician, Mayo Clinic, Division <strong>of</strong> Biostatistics,<br />
Rochester, MN, Gregory Poland, Pr<strong>of</strong>essor <strong>of</strong> Medicine, Mayo<br />
Clinic, Department <strong>of</strong> Internal Medicine, Rochester, MN<br />
The humoral and cellular immune responses to measles virus<br />
(MV) are genetically influenced. We hypothesized that the<br />
antigen-presenting function <strong>of</strong> HLA molecules could play a role<br />
in the variability in measles-specific T cell memory responses in
Abstracts<br />
vaccinated subjects. We studied the association between IFN-g<br />
producing T cells specific for MV and the alleles <strong>of</strong> HLA genes<br />
among 336 children (12−18 years <strong>of</strong> age) who previously<br />
received two doses <strong>of</strong> the measles vaccine. PBMCs (2x100,000<br />
cells/well; HLA typed) were cultured with Edmonston strain <strong>of</strong><br />
measles (moi 0.5) and spot-forming cells (SFC) were analyzed by<br />
ELISPOT. Linear-regression analysis was used to study allelic<br />
associations. Measles-induced IFN-g responses (median 6 SFC;<br />
interquartile range 2, 17 SFC per 2x100,000 T cells) were<br />
detected in subjects up to 12 years after vaccination. Univariate<br />
analyses identified that class I HLA-A*1101 (median 6 SFC,<br />
p=0.04) and B*3701 (median 34 SFC, p=0.07) alleles were<br />
suggestive <strong>of</strong> an association with higher memory IFN-g<br />
responses, while the Cw*0802 (median SFC 3, p=0.08) allele<br />
wassuggestive<strong>of</strong>anassociationwithadecreaseintherelative<br />
frequency <strong>of</strong> IFN-g producing T cells. Class II HLA alleles with<br />
potential associations with increased measles-specific T cell<br />
responses included DRB1*1303 (median SFC 17, p=0.01),<br />
DPB1*0301 (median SFC 11, p=0.02) and DPB1*1501 (median<br />
SFC 10, p=0.07). A suggestive association was observed between<br />
DQB1*0303 (median SFC 4, p=0.07) alleles and decreased<br />
frequency <strong>of</strong> MV memory IFN-g response. Thus, the presence<br />
or absence <strong>of</strong> certain HLA alleles may influence long-term<br />
measles immunity and the dynamics <strong>of</strong> virus-specific T cell<br />
memory outcome in vaccinated subjects.<br />
doi:10.1016/j.clim.2007.03.276<br />
F.64 Loss <strong>of</strong> Interferon Gamma Receptor Alpha<br />
(IFNGR α) in Peripheral Blood Mononuclear Cells<br />
from Patients with Chronic Hepatitis C<br />
Lucia Cristina Jamli Abel, Pr<strong>of</strong>essor, Albert Einstein<br />
Research Institute-Albert Einstein Hospital, São Paulo,<br />
Brazil, Mário Pessoa, Albert Einstein Research<br />
Institute-IEP-Albert Einstein Hospital, São Paulo, Brazil,<br />
Hoel Sette Jr., Albert Einstein Research Institute-IEP-<br />
Albert Einstein Hospital, São Paulo, Brazil, Carlos<br />
Moreira-Filho, Albert Einstein Research Institute-IEP-<br />
Albert Einstein Hospital, São Paulo, Brazil, Ben-Hur Ferraz<br />
Neto, Albert Einstein Research Institute-IEP-Albert Einstein<br />
Hospital, São Paulo, Brazil, Luciana Marti, Albert Einstein<br />
Research Institute-IEP-Albert Einstein Hospital, São Paulo,<br />
Brazil, Denise Rodrigues, Infectious Diseases Division,<br />
Federal University <strong>of</strong> São Paulo, São Paulo SP, Brazil, Esper<br />
Kallas, Pr<strong>of</strong>essor, Infectious Diseases Division, Federal<br />
University <strong>of</strong> São Paulo, São Paulo, Brazil, Venancio Alves,<br />
Pr<strong>of</strong>essor, Department <strong>of</strong> Pathology, University <strong>of</strong> São<br />
Paulo School <strong>of</strong> Medicine, São Paulo, Brazil<br />
The mechanisms underlying the pathogenesis <strong>of</strong> chronic liver<br />
disease due to HCV are not fully understood. IFN-γ plays an<br />
important role in viral clearance, where IFN-γ either exogenous<br />
or endogenously produced by virus-specific CD8+ Tcells inhibits<br />
the replication <strong>of</strong> HCV. The alpha chain receptor (IFNGR α) isa<br />
high affinity receptor that binds to IFN-γ, therefore its<br />
expression can be important in the control <strong>of</strong> HCV infection.<br />
Using flow cytometric analysis, we found that the number <strong>of</strong><br />
peripheral lymphocytes expressing alpha chain receptor (IFNGR<br />
α) in patients with chronic hepatitis C was lower than the normal<br />
controls (N) (p=0.018) and significantly decreased in patients<br />
with abnormal ALT (p=0.05). In chronic patients the expression<br />
<strong>of</strong> IFNGR α was inversely correlated to viral load (r=−0.362,<br />
p=0.038). In the liver, 50% <strong>of</strong> T cell lines obtained from biopsy<br />
isolated from patients with end stage liver disease expressed<br />
IFNGR α and when stimulated with staphylococcal enterotoxin B<br />
(SEB)producedhighlevels<strong>of</strong>IFN-γ. No significant difference<br />
was found for IFN-γ production by peripheral blood mononuclear<br />
cells in the presence <strong>of</strong> PHA between chronic HCV patients and<br />
normal controls, but the IFN-γ production was weaker in viremic<br />
chronic hepatitis C patients than in subjects who are able to<br />
clear the virus (p=0.033). The results suggest that HCV infection<br />
may down-regulate the number <strong>of</strong> lymphocytes expressing<br />
IFNGR α in the peripheral blood but not in liver and that the<br />
intrahepatic response contributes at least in part to the control<br />
<strong>of</strong> disease.<br />
doi:10.1016/j.clim.2007.03.277<br />
S35<br />
F.65 Normal Adult Ranges and Diversity <strong>of</strong> Serotype<br />
Specific Anti-Pneumococcal Antibodies (SSAPAbs)<br />
after Immunisation with 23-Valent Polysaccharide<br />
Vaccine<br />
Robert Hallam, Senior Biomedical Scientist, Papworth Hospital/<br />
<strong>Immunology</strong> Department, Cambridgeshire, England, Paul<br />
Balmer, <strong>Clinical</strong> Scientist, HPA, Vaccine Evaluation Department,<br />
Manchester, England, Ray Borrow, <strong>Clinical</strong> Scientist, HPA-<br />
Vaccine Evaluation Department, Manchester, England, Diana<br />
Bilton, Consultant Respiratory Physician, Papworth Hospital,<br />
Respiratory Infection, Inflammation and <strong>Immunology</strong> (RIII)<br />
Department, Cambridgeshire, England, Charles Haworth,<br />
Consultant Respiratory Physician, Papworth Hospital,<br />
Respiratory Infection, Inflammation and <strong>Immunology</strong> (RIII)<br />
Department, Cambridgeshire, England, Andrew Exley,<br />
Consultant Immunologist, Papworth Hospital/<strong>Immunology</strong><br />
Department, Cambridge<br />
Background: <strong>Clinical</strong> and experimental studies identify<br />
SSAPAbs as key elements <strong>of</strong> pneumococcal immunity reflecting<br />
interactions with antigen specific and pathogen recognition<br />
receptors on APCs, B and T cells. New assays for SSAPAbs<br />
incorporating multiplex bead arrays with CPS and 22F<br />
absorption enable analysis <strong>of</strong> large data sets to determine<br />
normal adult ranges and diversity <strong>of</strong> responses. Earlier<br />
studies indicating diversity <strong>of</strong> responses and potential for<br />
immunologic refractoriness caution against standard<br />
approaches in healthy volunteers. Method: We investigated<br />
pneumococcal immunity in 250 adults with recurrent lower<br />
respiratory tract infections, measuring serum SSAPAbs pre<br />
and 4–6 weeks post-immunization with 23-valent polysaccharide<br />
vaccine. We tested the hypothesis that cases include<br />
a mixture <strong>of</strong> normal and low responders to standard<br />
immunization, on a background <strong>of</strong> natural exposure to<br />
antigen through carriage and infection. SSAPAb levels pre,<br />
post, and post/pre were log transformed and examined for<br />
goodness <strong>of</strong> fit using mixture modeling, with censoring <strong>of</strong><br />
outlying data according to post-immunization SSAPAb levels.<br />
We then investigated for hierarchy and diversity <strong>of</strong> SSAPAb<br />
levels after immunization. Results: Mixture modeling indicated<br />
serotype specific normal and low adult ranges.<br />
Confidence intervals were resolved to determine threshold
S36 Abstracts<br />
values for antibody levels post-immunization. Serotypes 14,<br />
9V, and 4 appear high to low in hierarchy <strong>of</strong> responses.<br />
Discussion: Third generation multiplex assays for SSAPAb<br />
levels, without cross-reacting antibodies as confounders,<br />
indicate normal ranges after immunization are serotype<br />
specific with evidence <strong>of</strong> hierarchy and diversity. These data<br />
are consistent with serotype specific and non-specific pathways<br />
in antibody responses to pneumococcal capsular<br />
polysaccharides.<br />
doi:10.1016/j.clim.2007.03.278<br />
F.66 Differential Cytokine Response Induced by<br />
M. Avium and M. Abscessus in Human Macrophages<br />
is Mediated Through p38 MapKinase Signalling<br />
Pathway and Partially Dependent on TLR2<br />
Activation<br />
Elizabeth Sampaio, Senior Visiting Investigator, NIAID,<br />
NIH, Bethesda, MD, Houda Elloumi, Postdoctoral Fellow,<br />
NIH/NIAID, Bethesda, MD, Adrian Zelazny, Staff Scientist,<br />
NIH/NIAID, Bethesda, MD, Yvonne Shea, Laboratory<br />
Supervisor, NIAID/NIH, Bethesda, MD, Li Ding, Lab<br />
Manager, NIAID/NIH, Bethesda, MD, Steven Holland, Lab<br />
Chief, NIH/NIAID, Bethesda, MD<br />
Non-tuberculous mycobacteria (NTM) are ubiquitous<br />
environmental organisms that can cause chronic lung<br />
infection associated with primary or acquired immune<br />
deficiencies or in otherwise apparently normal individuals.<br />
Among NTM, M. avium is the most common cause <strong>of</strong><br />
mycobacterial infection. Moreover, M. abscessus is an<br />
emerging pulmonary pathogen responsible for the majority<br />
<strong>of</strong> infections by rapidly growing mycobacteria. It has been<br />
shown that the ability <strong>of</strong> mycobacteria to induce TNFα<br />
secretion is inversely related to their virulence. In this<br />
work, cytokine response and signaling pathways triggered<br />
by reference and clinical isolates <strong>of</strong> M. abscessus and M.<br />
avium (5 smooth and 5 rough morphotypes) were assessed<br />
in human PBMCs and monocytes. Mycobacteria-induced<br />
TNFα response is enhanced for M. abscessus (8535<br />
±1010 pg/ml) as compared to M. avium (2400±244 pg/<br />
ml, pb0.017) and no major differences were noted when<br />
compared colony morphologies within the same species.<br />
All mycobacterial strains were able to activate p38 MAP<br />
kinase phosphorylation and NF-kB translocation. Induction<br />
<strong>of</strong> TNFα was dependent on p38 MAPK signaling pathway<br />
since pre-incubation <strong>of</strong> cells with the p38 signaling<br />
inhibitor (SB203580) led to N80% reduction in cytokine<br />
secretion. In addition, treatment <strong>of</strong> cells with anti-human<br />
TLR2 antibodies showed TLR2 to be at least partially<br />
involved in signaling for M. abscessus as well. The present<br />
data indicate that M. avium is a more virulent pathogen<br />
than M. abscessus and elicits limited activation <strong>of</strong> cellular<br />
effector mechanisms, thereby escaping elimination.<br />
Accordingly, lung disease induced by NTM in nonpredisposing<br />
individuals may be associated with a yet uncharacterized<br />
underlying genetic defect.<br />
doi:10.1016/j.clim.2007.03.279<br />
F.67 Characterization <strong>of</strong> the Cellular Immunity in<br />
Patients Presenting Extensive Dermatophytoses Due<br />
to Trichophyton Rubrum<br />
Dewton Moraes Vasconcelos, MD, PhD, Department <strong>of</strong><br />
Dermatology, University <strong>of</strong> São Paulo Medical School, São Paulo,<br />
Brazil, Anna Cristina Collanieri, BSc, Department <strong>of</strong><br />
Dermatology, University <strong>of</strong> São Paulo Medical School, São Paulo,<br />
Brazil, Mauricio Domingues Ferreira, MD, PhD, Department <strong>of</strong><br />
Dermatology, University <strong>of</strong> São Paulo Medical School, São Paulo,<br />
Brazil, Tatiana Negri Santi-BSc, Department <strong>of</strong> Dermatology,<br />
University <strong>of</strong> São Paulo Medical School, São Paulo, Brazil, Anete<br />
S. Grumach, MD, PhD, Department <strong>of</strong> Dermatology, University<br />
<strong>of</strong> São Paulo Medical School, São Paulo, Brazil, Alexandre<br />
Almeida, MD, MSc, Department <strong>of</strong> Dermatology, University <strong>of</strong><br />
São Paulo Medical School, São Paulo, Brazil, Alberto Jose da<br />
Silva Duarte, MD, PhD, Department <strong>of</strong> Dermatology, University<br />
<strong>of</strong> São Paulo Medical School, São Paulo, Brazil<br />
Background: Dermatophytes cause infection in humans<br />
independent <strong>of</strong> the immunological status <strong>of</strong> the patient. In<br />
common to other infections the clinical features differ in<br />
immunodeficient patients. Dermatophytoses in cellular immunodeficient<br />
patients are usually less inflammatory, but some<br />
patients present pustular extensive lesions, frequently with<br />
follicular involvement.<br />
Objectives: Obtaining a reliable antigen by growth and<br />
purification <strong>of</strong> T. rubrum and to evaluate the immune<br />
reactivity “in vitro”, and quantify the immune response to<br />
the peptide YIIDTGIDID <strong>of</strong> T. rubrum. Methods: The fungal<br />
samples were obtained from the fungal library <strong>of</strong> the Institute<br />
<strong>of</strong> Tropical Medicine for antigen preparation, and the peptide<br />
was synthesized by EvoQuest. The lymphoproliferation assay<br />
was performed by tritiated thymidine incorporation and the<br />
cytokine quantifications by ELISA <strong>of</strong> culture supernatants.<br />
Results: The antigenic extract was efficient in the stimulation<br />
<strong>of</strong> cell cultures. The response to the peptide was also efficient<br />
and highly specific in sensitized controls. Despite most<br />
patients presented deficient responses, some presented<br />
normal lymphoproliferation. There was no difference<br />
between the cytokine secretion among patients and controls.<br />
doi:10.1016/j.clim.2007.03.280<br />
F.68 Evaluation <strong>of</strong> Cellular Responses to Rotavirus<br />
and NSP4 in Children during Natural Rotavirus<br />
Infection<br />
Jyoti Logani, Research Officer, Department <strong>of</strong> Pediatrics, All<br />
India Institute <strong>of</strong> Medical Sciences, New Delhi, India, Santosh<br />
Gupta, Research Associate, Department <strong>of</strong> Pediatrics, All India<br />
Institute <strong>of</strong> Medical Sciences, New Delhi, India, Shinjini<br />
Bhatnagar, Scientist, Department <strong>of</strong> Pediatrics, All India<br />
Institute <strong>of</strong> Medical Sciences, New Delhi, India, Pratima Ray,<br />
Scientist, Department <strong>of</strong> Pediatrics, All India Institute <strong>of</strong><br />
Medical Sciences, New Delhi, India, M.K. Bhan, Department <strong>of</strong><br />
Pediatrics, All India Institute <strong>of</strong> Medical Sciences, New Delhi,<br />
India<br />
Rotavirus (RV) is a major cause <strong>of</strong> gastroenteritis in young<br />
children leading to ∼611,000 deaths annually. Further
Abstracts<br />
development and evaluation <strong>of</strong> effective vaccines require better<br />
understanding <strong>of</strong> immune effectors involved in protective<br />
immunity. We examined IFN-γ and/or IL-4 responses in PBMC <strong>of</strong><br />
children suffering from RV (n=25), non-RV (n=10) gastroenteritis<br />
and healthy adults (n=10) by ELISPOT assay. We detected IFN-γ<br />
but no IL-4 response to NSP4 and RV in children with RV<br />
gastroenteritis and adults. IFN-γ responses in adults were<br />
persistent and significantly higher than children (pb0.01). The<br />
responses to the RV were stronger in magnitude in comparison to<br />
NSP4, but were positively correlated (pb0.01, rs=0.666). During<br />
RV gastroenteritis, 8/25 children elicited IFN-γ response to NSP4<br />
between 1 and 30 days after onset <strong>of</strong> diarrhea; in the control<br />
uninfected children, no response to NSP4 was observed. The %<br />
responders with positive IFN-γ response to NSP4 detected<br />
between 1 and 6 days <strong>of</strong> illness were 11.1%, 66.6% between 7<br />
and 15 days and 30% between 16 and 30 days <strong>of</strong> illness. No IFN-γ<br />
responses were detected beyond 30 days <strong>of</strong> diarrhea. On<br />
screening <strong>of</strong> three samples from same subject, it was further<br />
validatedthatatransientriseintheIFN-γresponsetoNSP4occurs<br />
between 7 and 25 days <strong>of</strong> symptomatic RV diarrhea. Stool RV+<br />
children depicted similar kinetics <strong>of</strong> IFN-γ response to RV. Thus<br />
significant IFN-γ response to NSP4 and RV indicates Th1 response<br />
duringnaturalRVinfection.IFN-γresponseistransientinchildren<br />
whereas in adults, responses are persistent and may play a role in<br />
protection.<br />
doi:10.1016/j.clim.2007.03.281<br />
F.69 Differential NF-kappaB Responses Induced by<br />
Bordetella Pertussis Filamentous-Hemagglutinin<br />
(FHA) in Macrophages and Bronchial Epithelial Cells<br />
Tzvia Abramson, Pr<strong>of</strong>esor, SJSU, Palo Alto, CA, David<br />
Relman, Pr<strong>of</strong>essor, Stanford University, Palo Alto, CA,<br />
Hassya Kedem, Research Associate, Stanford University,<br />
Palo Alto, CA<br />
Filamentous hemagglutinin (FHA), an adhesin and secreted<br />
factor <strong>of</strong> Bordetella pertussis, induces both inflammatory and<br />
apoptotic responses. Given the role <strong>of</strong> NF-kappaB transcription<br />
factor family in both these functions, we investigated the FHA<br />
interference with this pathway. FHA (5 μg/ml) induces a rapid<br />
degradation <strong>of</strong> IkappaB (within 30 min) in human monocytes<br />
and U-937 macrophages. IkappaB cytosolic levels are restored<br />
and accumulated within 2 h. This activation <strong>of</strong> NF-kappaB<br />
pathway is confirmed by NF-kappaB DNA binding as well as by<br />
inflammatorycytokinesecretion.None<strong>of</strong>thoseactivitieswas<br />
observed in BEAS-2B bronchial epithelial cells. Surprisingly, 2hour<br />
exposure <strong>of</strong> both epithelial cells and macrophages to FHA<br />
blocked the ability <strong>of</strong> TNF-α to induce proteasomal degradation<br />
<strong>of</strong> IkappaB as well as the nuclear translocation <strong>of</strong> p65.<br />
Immunoprecipitation analysis revealed the ubiquitination and<br />
phosphorylation <strong>of</strong> IkappaB, implicating that 2-hour treatment<br />
with FHA interferes with the proteasomal activity rather than<br />
an upstream activity. Attenuated proteasome activity is<br />
revealed at 2–8 h FHA treatment in both cell types. However,<br />
no significant inhibitory activity was observed at less than 1 h<br />
exposure to FHA. These results imply that the accumulated<br />
levels <strong>of</strong> IkappaB at 2 h exposure <strong>of</strong> cells to FHA may result in<br />
proteasomal inhibition. This study suggests that despite the<br />
immediate strong activation <strong>of</strong> inflammatory responses by FHA<br />
in macrophages, longer exposure <strong>of</strong> immune and host cells to<br />
this secreted virulent factor may trigger anti-inflammatory<br />
activity which may interfere with the development <strong>of</strong> an<br />
effective immune response that will resolve the infection<br />
promptly.<br />
doi:10.1016/j.clim.2007.03.282<br />
F.70 Cytokine Activated Human Neutrophils<br />
Simultaneously Express T Cell Co-Stimulatory and<br />
Negative Regulatory Molecules<br />
Paul Bankey, Associate Pr<strong>of</strong>essor, University <strong>of</strong> Rochester<br />
Medical Center; Surgery, Rochester, NY, Mita De, Research<br />
Associate, University <strong>of</strong> Rochester Medical Center; Surgery,<br />
Rochester, NY, Andrea Zucchiatti, Research Fellow,<br />
University <strong>of</strong> Rochester Medical Center; Surgery, Rochester,<br />
NY, Asit De, Assistant Pr<strong>of</strong>essor, University <strong>of</strong> Rochester<br />
Medical Center, Rochester, NY, Carol Miller-Grazia,<br />
Pr<strong>of</strong>essor <strong>of</strong> <strong>Immunology</strong>, University <strong>of</strong> Rochester Medical<br />
Center; Surgery, Rochester, NY<br />
The role <strong>of</strong> neutrophils (PMN) in innate immunity is well<br />
established. However, their role in adaptive immunity is not<br />
well characterized. Initiation <strong>of</strong> adaptive immunity by pr<strong>of</strong>essional<br />
antigen presenting cells (APCs) is critically dependent on<br />
expression <strong>of</strong> MHC class II molecules (e.g., HLA-DR) and costimulatory<br />
molecules (e.g., CD86). APCs also regulate T cell<br />
mediated adaptive immunity through expression <strong>of</strong> negative<br />
regulatory molecules such as program death ligands (PDL) and<br />
immunoglobulin like transcript (ILT) molecules. Our purpose<br />
was to assess the surface expression <strong>of</strong> HLA-DR, CD86, PD-L1<br />
and 2 and ILT-2 and 4 by flow cytometry in fresh and cytokine<br />
(GM-CSF+IFN-γ: G+I) treated neutrophils isolated from healthy<br />
controls (n=22). Untreated neutrophils expressed low levels<br />
(b2%) <strong>of</strong> HLA-DR and CD86; however, in vitro culture <strong>of</strong><br />
neutrophils with cytokines G+I significantly increased the<br />
expression <strong>of</strong> both molecules (HLA-DR: 5.61±1.16% at 24 h<br />
and 17.94±2.95% at 48h; CD86: 2.57±0.45% at 24 h and 8.11±<br />
1.59% at 48 h). Concurrently, cytokines G+I also significantly<br />
induce PMN expression <strong>of</strong> the T cell negative regulatory<br />
molecules: PD-L1 and 2 (PD-L1: 0.11±0.06% in fresh cells,<br />
26.49±2.47% at 24 h and 39.55±4.72% at 48 h; PD-L2: 0.25±<br />
0.0.25% in fresh cells, 6.12±1.37% at 24 h and 14.94±4.58% at<br />
48 h). ILT4 is constitutively expressed on over 90% <strong>of</strong> human<br />
neutrophils and expression is unchanged after culture with G+I.<br />
ILT-2 is not expressed on either fresh or G+I treated neutrophils.<br />
Simultaneous expression <strong>of</strong> T cell negative signaling molecules<br />
(PD-L1 and 2, ILT4) and MHC/co-stimulatory molecule CD86 in<br />
neutrophils supports an expanded role for PMN in both initiation<br />
and regulation <strong>of</strong> adaptive immunity.<br />
doi:10.1016/j.clim.2007.03.283<br />
S37<br />
F.71 An Analysis <strong>of</strong> HCV and CMV Specific Effector T<br />
Cells in Peripheral Blood, Perihepatic Lymph Nodes<br />
and Liver in HCV Infected and Uninfected Patients<br />
Dilip Moonka, Medical Director <strong>of</strong> Liver Transplantation,<br />
Henry Ford Health Systems, Division <strong>of</strong> Gastroenterology,
S38 Abstracts<br />
Detroit, MI, Kimberly Milkovich, Research Associate,<br />
Division <strong>of</strong> Rheumatology, Case Western Reserve University,<br />
Cleveland, OH, Benig Rodriguez, Assistant Pr<strong>of</strong>essor, Center<br />
for AIDS Research, Case Western Reserve University,<br />
Cleveland, OH, Michael Lederman, Pr<strong>of</strong>essor, Case Western<br />
Reserve University, Center for AIDS Research, Cleveland,<br />
OH, Marwan Abouljoud, Director <strong>of</strong> Transplant Institute,<br />
Henry Ford Health Systems, Transplant Institute, Detroit,<br />
MI, Donald Anthony, Assistant Pr<strong>of</strong>essor, Case Western<br />
Reserve University, Division <strong>of</strong> Rheumatology and Infectious<br />
Disease, Cleveland, OH<br />
It is difficult to detect HCV-specific T cells in the peripheral<br />
blood <strong>of</strong> chronic HCV patients, while T cells specific<br />
for other viruses appear intact. One explanation for this is a<br />
compartmentalization <strong>of</strong> the immune response. Here we<br />
performed phenotypic analysis <strong>of</strong> T cells isolated from liver,<br />
perihepatic lymph nodes (LN), and peripheral blood <strong>of</strong> 23<br />
HCV infected and 12 uninfected subjects undergoing liver<br />
transplantation. Results: HCV specific IFN-γ and proliferative<br />
responses were commonly observed in the LN <strong>of</strong> HCV infected<br />
subjects (40% and 46% respectively), while they were<br />
uncommonly observed in the peripheral blood and liver<br />
(IFN-γ 16% and 25% respectively; proliferation 11% and 8%<br />
respectively). In contrast, CMV specific IFN-γ producing<br />
responses were not as commonly observed in the LN (30%)<br />
compared to the periphery (58%) and liver (60%) <strong>of</strong> these<br />
same HCV patients. Conclusions: These data are among the<br />
first to look at HCV-specific responses in perihepatic lymph<br />
nodes. The data indicate a selective defect in HCV, but not<br />
CMV specific, T cell effector function in the periphery <strong>of</strong><br />
chronic HCV patients. The relatively common presence <strong>of</strong><br />
HCV-reactive T cells in the LN is consistent with T cell<br />
activation at this site. Finally, we do not detect HCVspecific<br />
T cells more readily in the liver compared to the<br />
periphery or LN, indicating that hepatic lymphocytes are<br />
defective in effector function similar to those in the<br />
peripheral blood. The frequency <strong>of</strong> HCV-specific T cells did<br />
not clearly correlate with the severity <strong>of</strong> recurrent HCV<br />
after transplant.<br />
doi:10.1016/j.clim.2007.03.284<br />
F.72 Carbohydrate Analysis <strong>of</strong> HIV Envelope<br />
Glycoprotein<br />
Ingrid D. Cruzado-Park, Senior Development Scientist,<br />
Beckman Coulter, Inc. Fullerton, CA, Munir Alam, Duke<br />
University Medical Center, Durham, NC, Hua-Xin Liao, Duke<br />
University Medical Center, Durham, NC, Barton Haynes,<br />
Director, Duke University Medical Center, Human Vaccine<br />
Institute and CHAVI, Durham, NC, Heather Desaire, Duke<br />
University Medical Center, Durham, NC, Enrique Rabelli,<br />
Director, Custom BioPharma Solutions, Beckman Coulter,<br />
Inc. Miami, FL, Sybil D’Costa, Staff Advanced Research<br />
Scientist, Beckman Coulter, Inc. Miami, FL, Michael H.<br />
Simonian, Manager, Biomarker Discovery and Automation<br />
Center, Beckman Coulter, Inc. Fullerton, CA, Chitra K.<br />
Ratnayake, Team Leader, Beckman Coulter, Inc. Biomarker<br />
Applications and Chemistry Development, Fullerton, CA<br />
To date over 65 million people have been infected with the<br />
human immunodeficiency virus (HIV) which continues to<br />
claim several million lives a year. There is an urgent need to<br />
design and develop vaccines that generate broad neutralizing<br />
antibody enabling preventative vaccination strategies.<br />
Although the virus has evolved to circumvent the immune<br />
response, for example, carbohydrates on the envelope<br />
glycoprotein <strong>of</strong> HIV serve as a strong defense against antibody<br />
mediated responses, there is evidence that these oligomannose<br />
components can also serve as ideal vaccine candidates<br />
due to their ability to generate efficacious neutralizing<br />
antibodies in HIV infected individuals (2G12 antibody) albeit<br />
infrequently. To address the role <strong>of</strong> glycosylation in HIV<br />
pathogenesis and immunogenicity, carbohydrate pr<strong>of</strong>iles<br />
from wild type and consensus modeled, genetically engineered<br />
envelope glycoproteins were compared. We present<br />
data showing the N-linked oligosaccharide pr<strong>of</strong>iles for two<br />
types <strong>of</strong> HIV gp140 envelope glycoproteins; JRFL is a primary<br />
isolate and the other from a genetically engineered strain<br />
containing a consensus sequence (ConS), which is more<br />
effective at eliciting neutralizing antibodies. Following<br />
treatment with PNGase F, released N-linked oligosaccharides<br />
were labeled with fluorescent 8-aminopyrene-1,3,6-trisulfonate<br />
and pr<strong>of</strong>iled using the ProteomeLab PA 800 Protein<br />
Characterization System with laser-induced fluorescence<br />
(LIF) detection (488 nm excitation, 520 nm emission). We<br />
observed significant differences in the carbohydrate pr<strong>of</strong>iles<br />
<strong>of</strong> these gp140 envelopes and will present and discuss those<br />
differences. This strategy for oligosaccharide characterization<br />
and subsequent identification may help evaluate role <strong>of</strong><br />
glycosylation in generating broadly neutralizing antibody<br />
responses and enable intelligent design <strong>of</strong> carbohydratebased<br />
epitope vaccines.<br />
doi:10.1016/j.clim.2007.03.285<br />
F.73 Defining the Role <strong>of</strong> BmIL-5 in Tropical<br />
Pulmonary Eosinophilia<br />
Sung (Steve) Kwon, University <strong>of</strong> Illinois College <strong>of</strong> Medicine<br />
at Rockford, Rockford, IL, Gnanasekar Munirathinam,<br />
University <strong>of</strong> Illinois College <strong>of</strong> Medicine at Rockford,<br />
Rockford, IL, Anand Setty Balakrishnan, University <strong>of</strong> Illinois<br />
College <strong>of</strong> Medicine at Rockford, Rockford, IL, Ramaswamy<br />
Kalyanasundaram, University <strong>of</strong> Illinois College <strong>of</strong> Medicine<br />
at Rockford, Rockford, IL<br />
This study describes characterization <strong>of</strong> a novel pathogenderived<br />
human IL-5 receptor binding molecule designated<br />
BmIL-5. This molecule was identified in our laboratory by<br />
screening a phage displayed cDNA expression library <strong>of</strong> the<br />
infective larvae <strong>of</strong> the human lymphatic filarial parasite,<br />
Brugia malayi. Soluble human IL-5 receptor (huIL-5R) was<br />
used as the bait. This screening procedure yielded a single<br />
clone and sequence analyses <strong>of</strong> the insert from this clone<br />
showed a novel protein designated as “B. malayi-derived IL-<br />
5-like molecule” (BmIL-5). Recombinant BmIL-5 was then<br />
expressed and purified. ELISA-based binding assays confirmed<br />
binding <strong>of</strong> rBmIL-5 to huIL-5R. Functional significance<br />
<strong>of</strong> this binding was then evaluated using a human eosinophil<br />
cell line, named Hs 454.T. Initial studies showed that BmIL-5
Abstracts<br />
binds to eosinophil surface. To test whether this binding<br />
initiates any signaling events, we evaluated STAT1 phosphorylation<br />
status in the eosinophil. We also performed a series <strong>of</strong><br />
experiments to identify the receptor binding region <strong>of</strong> BmIL-5<br />
by peptide mapping. In these studies, we designed overlapping<br />
20 mer peptides and evaluated the binding pattern <strong>of</strong><br />
these peptides to huIL-5R. These studies identified peptide 1<br />
and peptide 11 as the domains binding to huIL-5R. An<br />
interesting observation was that both these peptides interfered<br />
with the binding <strong>of</strong> huIL-5 to huIL-5R in vitro. Thus, we<br />
believe that BmIL-5 interferes with IL-5 function by preventing<br />
IL-5 binding to IL-5R. Our findings demonstrate a novel<br />
method <strong>of</strong> how pathogens potentially manipulate the human<br />
immune system.<br />
doi:10.1016/j.clim.2007.03.286<br />
F.74 What Triggers Transient AIDS in the Acute<br />
Phase <strong>of</strong> HIV Infection and Chronic AIDS at the End<br />
<strong>of</strong> the Incubation Period?<br />
Ivan Kramer, Associate Pr<strong>of</strong>essor, UMBC, Canada Physics,<br />
Catonsville, MD<br />
Novel dynamical models are introduced demonstrating<br />
that the T helper cell (THC) density drops in the acute<br />
infection phase <strong>of</strong> HIV infection, sometimes causing<br />
transient AIDS, and at the end <strong>of</strong> the incubation period<br />
causing chronic AIDS have a common dynamical cause. The<br />
immune systems’ inability to produce enough uninfected<br />
THCs to replace the infected ones it is destroying causes a<br />
drop in the THC density at any stage <strong>of</strong> HIV infection.<br />
Increases in viral infectivity, probably caused by random<br />
mutation <strong>of</strong> HIV, are shown to drive the progression <strong>of</strong> the<br />
infection. The existence <strong>of</strong> transient AIDS and this model<br />
which explains it are inconsistent with the antigenic<br />
diversity thresholds model <strong>of</strong> AIDS. The minimum incubation<br />
period for the long-term non-progressors (LTNPs) was<br />
calculated from a novel physical model: 0.3% <strong>of</strong> infected<br />
people have incubation periods <strong>of</strong> 23.1 years or more, and<br />
there is no biomedical difference between LTNPs and<br />
progressors. If this model is correct, then there is no<br />
unique, special anti-viral substance secreted by the CD8+ T<br />
cells <strong>of</strong> LTNPs that accounts for their unusually long<br />
incubation periods; indeed, to date, no such substance has<br />
been found although researchers have looked for it for<br />
years. The model also predicts that chronic AIDS results<br />
from 3 random transitions linking 4 clinically distinct stages<br />
<strong>of</strong> HIV infection following seroconversion, a result that<br />
agrees with the World Health Organization’s staging system<br />
based on clinical manifestations <strong>of</strong> the disease.<br />
doi:10.1016/j.clim.2007.03.287<br />
F.75 Characterization <strong>of</strong> a CD21low B Cell<br />
Subpopulation in Patients with CVID<br />
Mirzokhid Rakhmanov, Postdoctoral Fellow, Department <strong>of</strong><br />
Rheumatology and <strong>Clinical</strong> <strong>Immunology</strong>, University Hospital<br />
Freiburg, Freiburg i. Breisgau, Germany, Sylvia Gutenberger,<br />
Research Assistant, Department <strong>of</strong> Rheumatology and<br />
<strong>Clinical</strong> <strong>Immunology</strong>, University Hospital Freiburg, Freiburg i.<br />
Breisgau, Germany, Baerbel Keller, Graduate Student,<br />
Department <strong>of</strong> Rheumatology and <strong>Clinical</strong> <strong>Immunology</strong>,<br />
University Hospital Freiburg, Freiburg i. Breisgau, Germany,<br />
Klaus Warnatz, Assistant Medical Director, Department <strong>of</strong><br />
Rheumatology and <strong>Clinical</strong> <strong>Immunology</strong>, University Hospital<br />
Freiburg, Freiburg, Germany, Hans-Hartmut Peter, Director<br />
<strong>of</strong> the Department, Department <strong>of</strong> Rheumatology and<br />
<strong>Clinical</strong> <strong>Immunology</strong>, University Hospital Freiburg, Freiburg,<br />
Germany<br />
Common variable immunodeficiency (CVID) is a primary<br />
immune deficiency disorder characterized by hypogammaglobulinemia<br />
and recurrent bacterial infections. Analyzing B<br />
cell homeostasis in a subgroup <strong>of</strong> CVID patients with<br />
splenomegaly and autoimmune cytopenia revealed a B cell<br />
population with unusually low expression <strong>of</strong> CD21. We<br />
therefore termed these cells “CD21low” B cells. The low<br />
expression <strong>of</strong> CD21 is the effect <strong>of</strong> a reduced transcription.<br />
However, coexisting B cells expressing normal levels <strong>of</strong> CD21<br />
in these patients suggest that the low expression is not due to<br />
genetic dysregulation <strong>of</strong> CD21 expression, but rather the<br />
result <strong>of</strong> an unusual cellular differentiation. The phenotyping<br />
<strong>of</strong> these cells did not identify a stage along the current<br />
understanding <strong>of</strong> B cell differentiation, but the increased<br />
cell size and expression <strong>of</strong> CD86 suggest rather a recent<br />
activation status. The frequency <strong>of</strong> somatic hypermutations<br />
was increased compared to naïve CD21+ B cells, but below<br />
memory B cells <strong>of</strong> the same patient. We and others had<br />
previously described a similar B cell population in patients<br />
with SLE or chronic HIV infection. Interestingly, CD21low B<br />
cells express increased levels <strong>of</strong> MxA message, suggesting<br />
recent exposure to type I interferons. The expansion <strong>of</strong><br />
CD21low B cells is associated with severe shifts within the<br />
CD4+ T cell compartment <strong>of</strong> these patients. Therefore, we<br />
suggest an inflammatory dysregulation <strong>of</strong> the immune system<br />
underlying the disturbed B and T cell homeostasis in CVID<br />
patients with Freiburg type Ia.<br />
doi:10.1016/j.clim.2007.03.288<br />
S39<br />
F.76 Use <strong>of</strong> Prophylactic Antibiotics and Adjunctive<br />
Therapies in Primary Immunodeficiency Diseases<br />
Pierre Yong, Fellow, Hospital <strong>of</strong> the University <strong>of</strong><br />
Pennsylvania, Division <strong>of</strong> Allergy and <strong>Immunology</strong>,<br />
Philadelphia, PA, John Boyle, Founder, Immune Deficiency<br />
Foundation, Towson, MD, Jordan Orange, Assistant<br />
Pr<strong>of</strong>essor, Children’s Hospital <strong>of</strong> Philadelphia, Division <strong>of</strong><br />
Allergy and <strong>Immunology</strong>, Philadelphia, PA<br />
Background: There is limited data supporting the use <strong>of</strong><br />
prophylactic antibiotics and other adjunctive therapies in<br />
primary immunodeficiency diseases (PIDD). Methods: A webbased<br />
survey regarding therapy in PIDD was designed jointly by<br />
the Immune Deficiency Foundation and the American Academy<br />
<strong>of</strong> Allergy, Asthma & <strong>Immunology</strong> (AAAAI), and <strong>of</strong>fered to AAAAI<br />
members. The response rate was 13.5%, n = 405. Responses<br />
were analyzed with a basic statistical program. Those devoting
S40 Abstracts<br />
more than 10% <strong>of</strong> their clinical practice to PIDD were defined as<br />
experts. Results: Use <strong>of</strong> prophylactic antibiotics in PIDD is<br />
widespread and perceived to be efficacious. Significant<br />
differences, however, between expert and non-expert immunologists<br />
were found in frequency <strong>of</strong> use as well as in perceived<br />
effectiveness in multiple PIDD. Experts find prophylaxis more<br />
useful and use it more <strong>of</strong>ten. They also use prophylaxis in<br />
conjunction with IVIG more frequently. Live viral vaccines were<br />
most frequently avoided in severe combined immunodeficiency<br />
and DiGeorge syndrome. Additionally, differences in each<br />
group’s vaccine use were seen in several PIDD, including<br />
hyper-IgM syndrome. The only hygiene intervention felt<br />
beneficial was hand-washing with soap and water. Complementary<br />
and alternative medicine (CAM) was not believed to<br />
enhance immunity or prevent infections. Discussion: These<br />
results highlight the significant use <strong>of</strong> prophylactic antibiotics<br />
for PIDD by immunologists. Important differences were found<br />
between experts and non-experts. Surprisingly, many immunologists<br />
do not avoid live viral vaccines in many PIDD despite<br />
labeling recommending avoidance. Most hygiene and CAM<br />
interventions were not seen as beneficial, although some were<br />
more popular than others.<br />
doi:10.1016/j.clim.2007.03.289<br />
F.77 Common Variable Immunodeficiency:<br />
Presentation <strong>of</strong> 7 Cases and Review <strong>of</strong> Literature<br />
Carlos Torres-Loza, England Immunoallergist, West National<br />
Medical Center and Department <strong>of</strong> Allergy and <strong>Clinical</strong><br />
<strong>Immunology</strong>, Guadalajara, Jalisco, Mexico<br />
Aim <strong>of</strong> our work is to present a global view <strong>of</strong> this syndrome<br />
and analyze our experience <strong>of</strong> 7 adult patients bearing this<br />
syndrome which were seen at our department. It is a<br />
retrospective work for which it was designed a sheet to<br />
recollect data and also we reviewed literature <strong>of</strong> all possible<br />
electronic ways from the last 7 years, six are alive and one is<br />
now dead. The patients were diagnosed in most cases through<br />
air view infections such as pneumonia, chronic sinusitis and<br />
chronic middle otitis. In one <strong>of</strong> the cases an autoimmune<br />
process was the clinical problem before we made the<br />
diagnosis <strong>of</strong> this syndrome. During their evolution in our<br />
department, only three <strong>of</strong> them have had intestinal clinical<br />
manifestations characterized by several periods <strong>of</strong> chronic<br />
diarrhea and in three we found evidence <strong>of</strong> autoimmunity. All<br />
<strong>of</strong> the cases had low levels <strong>of</strong> serum immunoglobulin, neither<br />
<strong>of</strong> them had high serum level <strong>of</strong> IgE and all <strong>of</strong> our patients have<br />
shown normal lymphocytes subpopulations except in two<br />
where we found low levels <strong>of</strong> CD4.<br />
doi:10.1016/j.clim.2007.03.290<br />
F.78 Severe Agammaglobulinemia in an Adult Male<br />
with Nomal Number <strong>of</strong> Circulating B Cells<br />
Daniel Suez, Assistant <strong>Clinical</strong> Pr<strong>of</strong>essor at UTSW Dallas, TX,<br />
Daniel Suez, MD, AAIC, PA, Irving, TX, Hans Ochs, Pr<strong>of</strong>essor<br />
<strong>of</strong> Pediatrics, University <strong>of</strong> Washington School <strong>of</strong> Medicine,<br />
Seattle, WA<br />
We report here a case <strong>of</strong> a 34-year-old male with a history<br />
<strong>of</strong> recurrent pneumonia since early childhood with no other<br />
significant family history who presented to our clinic with<br />
severe agammaglobulinemia: IgG b7 mg/dl, IgM b5 mg/dl,<br />
IgA b13 mg/dl. Flow cytometry revealed normal numbers <strong>of</strong><br />
circulating B cells −9.5% <strong>of</strong> CD20 (221/mm 3 ) and 12.4% <strong>of</strong><br />
CD19 (322/mm 3 ) respectively. <strong>Clinical</strong> examination was<br />
remarkable for the paucity <strong>of</strong> lymphoid tissue. Further<br />
evaluation revealed normal numbers <strong>of</strong> memory B cells by<br />
CD27 expression yet very few switched memory B cells<br />
(1.36% vs. 23.7 in controls). The patient had normal BTK<br />
expression in B cells and had normal sequence analysis <strong>of</strong><br />
gDNA for CD40L. Flow cytometry analysis <strong>of</strong> CD3 lymphocyte<br />
subsets revealed CD4/CD8 inversion with total CD4 16.9%<br />
(439/mm 3 ) and CD8 <strong>of</strong> 85.5% (2220/mm 3 ) while NK cell<br />
number was at 2% (52/mm 3 ). Other laboratory findings<br />
included normal kidney and liver functions. There is no<br />
history <strong>of</strong> significant gut disease and the patient’s ability to<br />
maintain stable through IgG levels on intravenous immunoglobulin<br />
therapy rules out the possibility <strong>of</strong> protein loss or<br />
high catabolic rate to explain the severe agammaglobulinemia.<br />
The differential diagnosis considered: all hyper-IgM<br />
syndromes can be ruled out due to the severe IgM deficiency,<br />
XLP syndrome may be considered yet the patient has no<br />
history <strong>of</strong> mononucleosis. To rule out XLP the patient will be<br />
sequenced for SAP and EBV by PCR will be requested.<br />
Autosomal recessive agammaglobulinemia can be also considered<br />
yet unlikely with normal circulating B cell numbers.<br />
doi:10.1016/j.clim.2007.03.291<br />
F.79 Severe, Steroid Resistant, Numular Atopic<br />
Dermatitis with Hypogammaglobulinemia<br />
Daniel Suez, Assistant <strong>Clinical</strong> Pr<strong>of</strong>essor at UTSW Dallas,<br />
Daniel Suez, MD, AAIC, PA, Irving, TX, Hans Ochs, Pr<strong>of</strong>essor<br />
<strong>of</strong> Pediatrics, University <strong>of</strong> Washington School <strong>of</strong> Medicine,<br />
Seattle, WA<br />
We report here the case <strong>of</strong> a 26-month-old white male with<br />
severe, steroid resistant numular atopic dermatitis since early<br />
infancy. Allergy evaluation revealed severe IgE mediated<br />
sensitivities to food and perennial allergens with elevated<br />
total serum IgE N8900 IU/ml. The patient failed to respond to<br />
numerous attempts <strong>of</strong> aggressive steroid therapy, both topical<br />
(under closed dressings) and systemic treatment along with<br />
comprehensive supportive therapy. Laboratory evaluation<br />
revealed hypogammaglobulinemia with a serum IgG level <strong>of</strong><br />
342 mg/dl (normal range for age 453–916 mg/dl), and normal<br />
IgA (40 mg/dl) and IgM (28 mg/dl). CBC and differential revealed<br />
eosinophilia (N20%) and flow cytometry analysis was normal<br />
(CD19, CD3, CD4, CD8, CD16/56, CD45RO, and CD45RA). The<br />
patient was placed on high dose intravenous immunoglobulin<br />
therapy with dramatic improvement. Differential diagnosis<br />
considered is: Netherton syndrome (currently being evaluated<br />
by sequence analysis), Wiskott-Aldrich syndrome (which can be<br />
ruled out with normal platelet count), Omenn syndrome (yet not<br />
likely because he has normal numbers <strong>of</strong> circulating B cells),<br />
IPEX syndrome (yet not likely since the patient does not have any<br />
evidence for autoimmune disease such as polyendocrinopathy,
Abstracts<br />
neutropenia, or hemolytic anemia), and hyper IgE syndrome<br />
(yet unlikely as well since he does not have any <strong>of</strong> the clinical<br />
features).<br />
doi:10.1016/j.clim.2007.03.292<br />
F.80 Lymphocytic Interstitial Pneumonitis in<br />
Common Variable Immunodeficiency Disorders:<br />
A Comparison with HIV<br />
Dilani Arnold, Specialist Registrar, Department <strong>of</strong><br />
<strong>Immunology</strong>, John Radcliffe Hospital, Oxford, England,<br />
Gleeson Fergus, Consultant Radiologist, Department <strong>of</strong><br />
Radiology, Churchill Hospital, Oxford, England, Helen<br />
Chapel, Consultant in <strong>Immunology</strong>, Department <strong>of</strong><br />
<strong>Immunology</strong>, John Radcliffe Hospital, Oxford, England,<br />
Robert Davies, Consultant in Chest Medicine, Department<br />
<strong>of</strong> Chest Medicine, Churchill Hospital, Oxford, England,<br />
Siraj Misbah, Consultant in <strong>Immunology</strong>, Department <strong>of</strong><br />
<strong>Immunology</strong>, John Radcliffe Hospital, Oxford, England<br />
Background: The objective <strong>of</strong> this study was to assess the<br />
presentation <strong>of</strong> Lymphocytic Interstitial Pneumonitis (LIP) in<br />
Common Variable Immunodeficiency Disorders (CVID). Methods:<br />
The medical records <strong>of</strong> all CVID patients followed up in<br />
our department were examined retrospectively. Results: In<br />
our institution, between 1970 and 2006, 96 patients met the<br />
diagnostic criteria for CVID. Of these, six (four women, two<br />
men, mean age, 45 years, range 42−65) had biopsy-proven<br />
LIP (microscopic evidence <strong>of</strong> polyclonal lymphoid cell<br />
infiltrate). HIV PCR was negative in all patients. Two patients<br />
were asymptomatic; four presented with cough, fatigue and<br />
exertional dyspnoea (mean time 2.75 months (range 1−7)<br />
from initial presentation to LIP diagnosis). Five patients had<br />
elevated C-reactive protein (11−31); four had raised beta-2<br />
microglobulin (3.8−4.7). Two had T-cell lymphocytosis (2.5−<br />
8.09) with elevated CD4 counts (1.82−3.43). One patient had<br />
an elevated CD8 count <strong>of</strong> 5.14 (0.2−0.7) and another had B<br />
cell lymphopenia (CD19 0.06). Two had falling IgG levels<br />
(4.8−5.87) despite immunoglobulin therapy. The predominant<br />
radiological abnormalities on CT were ground-glass<br />
opacification and mediastinal lymphadenopathy (present<br />
in four patients). Two had subpleural nodules. Immunohistology<br />
showed either an excess <strong>of</strong> CD4 cells or CD8 cells in a minority <strong>of</strong><br />
patients. No viral antigen was identified. Conclusion: The<br />
presentation <strong>of</strong> LIP is highly variable and diagnostically<br />
challenging. Comparison with LIP in immunodeficiency states<br />
such as HIV shows similar clinical and radiological features. The<br />
aetiology <strong>of</strong> LIP in CVID and HIV is unknown but is presumed to be<br />
viral, though no specific virus was identified in our small cohort.<br />
doi:10.1016/j.clim.2007.03.293<br />
F.81 Defective CD107a Surface Expression Heralds<br />
Munc13-4 Defect and Discriminates Between<br />
Genetic Subtypes <strong>of</strong> Familial Hemophagocytic<br />
Lymphohistiocytosis (FHL)<br />
Daniela Pende, Senior Investigator, Istituto Nazionale per la<br />
Ricerca sul Cancro, Genova, Stefania Marcenaro, Fellow,<br />
DIMES, Genova, Stefania Martini, Technician, Istituto<br />
Nazionale per la Ricerca sul Cancro, Genova, Alessandra<br />
Santoro, Fellow, Ospedale “G. Di Cristina”, Palermo, Gillian<br />
Griffiths, Senior Investigator, Sir William Dunn School <strong>of</strong><br />
Pathology, Oxford, England, Maurizio Aricò, Physician,<br />
Ospedale dei Bambini “G. Di Cristina”, Palermo, Lorenzo<br />
Moretta, Full Pr<strong>of</strong>essor, Istituto G. Gaslini, Genova<br />
Familial hemophagocytic lymphohistiocytosis (FHL) is a<br />
rare, heterogeneous fatal disease <strong>of</strong> early infancy characterized<br />
by an hyperinflammatory syndrome. Defective cellular<br />
cytotoxicity results in pathogen persistence and hypercytokinemia<br />
with disseminated infiltration <strong>of</strong> lymphocytes and<br />
histiocytes and hemophagocytosis. Differential diagnosis<br />
between FHL due to PRF1 (FHL2) or Munc13-4 (FHL3) and<br />
additional still unclassified genetic subtypes is not easy<br />
unless mutation analysis is performed. We investigated<br />
natural killer (NK) cells function and phenotype in patients<br />
with FHL2 (n=5) or FHL3 (n=8). NK cells were cultured in IL-2<br />
prior to their use in the various assays. Here we report on the<br />
surface CD107a expression as a novel rapid tool for<br />
identification <strong>of</strong> patients with Munc13-4 defect. Upon target<br />
interaction and degranulation, FHL3 NK cells displayed low<br />
levels <strong>of</strong> surface CD107a staining, in contrast to normal<br />
controls or perforin-deficient NK cells. B-EBV cell lines and<br />
dendritic cell targets reveal the FHL3 NK cell defect while<br />
highly susceptible tumor targets were partially lysed by FHL3<br />
NK cells expressing only trace amounts <strong>of</strong> Munc13-4 protein.<br />
Perforin-deficient NK cells were completely devoid <strong>of</strong> any<br />
ability to lyse target cells. Cytokine production induced by<br />
mAb-crosslinking <strong>of</strong> triggering receptors was comparable in<br />
patients and normal controls. However, when cytokine<br />
production was induced by co-culture with 721.221 B-EBV<br />
cells, FHL NK cells resulted high producers, whereas control<br />
cells were almost ineffective. This could reflect survival<br />
versus elimination <strong>of</strong> B-EBV cells i.e., the source <strong>of</strong> NK cell<br />
stimulation, in patients vs. healthy controls, thus mimicking<br />
the pathophysiological scenario <strong>of</strong> FHL.<br />
doi:10.1016/j.clim.2007.03.294<br />
S41<br />
F.82 Autoimmune Polyendocrinopathy-Candidiasis-<br />
Ectodermal Dystrophy Syndrome (APECED) Caused<br />
by Homozygous P326L AIRE Mutations in a Brazilian<br />
Family<br />
Patrícia de Campos Pieri, Biologist, Laboratory <strong>of</strong> Medical<br />
Investigation 36, Pediatrics Department FMUSP, São Paulo,<br />
Brazil, Joao B. Oliveira, Associate Researcher, Molecular<br />
<strong>Immunology</strong> and Genetics Section, Laboratory <strong>of</strong> Medical<br />
Investigation #56, USP, São Paulo, Brazil, Cristina Miuki Abe<br />
Jacob, Head <strong>of</strong> Unit, Department <strong>of</strong> Pediatrics, Allergy and<br />
<strong>Immunology</strong> Unit FMUSP, São Paulo, Brazil, Alberto J.S.<br />
Duarte, Lab Director, Laboratory <strong>of</strong> Medical Investigation<br />
#56 (LIM-56), USP, São Paulo, Brazil, Antonio Carlos Pastori,<br />
Assistant Pr<strong>of</strong>essor, Pediatrics Department, Allergy and<br />
<strong>Immunology</strong> Unit FMUSP, São Paulo, Brazil, Magda Maria<br />
Carneiro-Sampaio, Head <strong>of</strong> Department, Department <strong>of</strong><br />
Pediatrics, Allergy and <strong>Immunology</strong> Unit, FMUSP, São Paulo,<br />
Brazil
S42 Abstracts<br />
Introduction: APECED is characterized by the early onset<br />
<strong>of</strong> multiple organ directed autoimmunity, chronic mucocutaneous<br />
candidiasis and ectodermal dystrophy, and caused<br />
by recessive mutations in AIRE gene. The vast majority <strong>of</strong><br />
affected patients are <strong>of</strong> northern European descent,<br />
Sardinian or Iranian Jews. We describe here a Brazilian<br />
family affected by the disease. Material and methods: All<br />
14 exons and flanking intronic regions <strong>of</strong> AIRE were<br />
amplified by PCR, purified and sequenced using an<br />
automated capillary sequencer. Results: The index patient,<br />
LGM, is a 15-year-old boy from São Paulo. At 7 months he<br />
developed recurrent thrush and at the age <strong>of</strong> 2 years<br />
presented ungueal candidiasis. At 9 years his height was<br />
119 cm (pb3), levels <strong>of</strong> IGF1 and IGFBP3 were low, and a<br />
diagnosis <strong>of</strong> GH deficiency was made. At 12 years he<br />
developed anti-adrenal antibodies (1/40), and at 15 years<br />
he showed high levels <strong>of</strong> ACTH and PTH. His mother<br />
presents oral thrush, megaloblastic anemia and hypoparathyroidism,<br />
and his sister has recurrent candidiasis, without<br />
signs <strong>of</strong> autoimmunity. Parents deny consanguinity.<br />
Genomic DNA sequencing <strong>of</strong> AIRE identified a homozygous<br />
missense mutation in exon 8 (Pro326Leu). This mutation<br />
affects the first PHD finger domain, probably interfering<br />
with adequate subcellular protein localization and functioning.<br />
Conclusion: This is to our knowledge the first report<br />
<strong>of</strong> AIRE mutations causing APECED in a Brazilian family, and<br />
<strong>of</strong> a P326L AIRE mutation in a homozygous state. Molecular<br />
diagnosis is important for genetic counseling and institution<br />
<strong>of</strong> adequate therapy early in life.<br />
doi:10.1016/j.clim.2007.03.295<br />
F.83 NOD2 Polymorphisms in CVID: Investigation<br />
into Associations with Gastrointestinal Pathology<br />
and Granulomatous Disease<br />
Kerri Packwood, Grade A <strong>Clinical</strong> Scientist in<br />
<strong>Immunology</strong>, <strong>Clinical</strong> <strong>Immunology</strong>, Oxford Radcliffe<br />
Hospitals NHS Trust, Oxford, OXON, England, Berne Ferry,<br />
Principle Scientist, <strong>Clinical</strong> <strong>Immunology</strong>, Oxford Radcliffe<br />
Hospitals NHS Trust, Oxford, OXON, England, Divya<br />
Punwani, PhD Student, Wetherall Institute <strong>of</strong> Molecular<br />
Medicine, Human <strong>Immunology</strong> Unit, Oxford, OXON,<br />
England, Eduardo Lopez-Granados, Consultant<br />
Immunologist, <strong>Clinical</strong> <strong>Immunology</strong>, Oxford Radcliffe<br />
Hospitals NHS Trust, Oxford, OXON, England, Helen<br />
Chapel, Consultant Immunologist, <strong>Clinical</strong> <strong>Immunology</strong>,<br />
Oxford Radcliffe Hospitals NHS Trust, Oxford, OXON,<br />
England<br />
Common Variable Immunodeficiency Disorders (CVID)<br />
are a heterogeneous group <strong>of</strong> diseases characterised by<br />
hypogammaglobulinaemia. Associated complications<br />
include enteropathy and granulomatous disease. These<br />
conditions have diverse immunopathogeneses including<br />
primary defects in B cells, T cells, macrophages, dendritic<br />
cells, NK cells and cytokine production yet <strong>of</strong>ten the<br />
underlying aetiology is unknown. Prediction <strong>of</strong> complications<br />
would aid clinical management and we are<br />
investigating possible disease modifier genes as a potentially<br />
powerful tool. Classification <strong>of</strong> heterophenotypic<br />
CVID subgroups would also enable more focused and<br />
productive research studies. Mutations <strong>of</strong> the NOD2 gene<br />
including gly908arg, leu1007finsc and arg702trp polymorphisms<br />
are associated with Crohn'€s disease. We<br />
hypothesised that NOD2 may also be a disease modifier<br />
gene towards an enteropathic or granulomatous CVID<br />
phenotype. Methods to examine the above three polymorphisms<br />
were established and investigated in a cohort<br />
<strong>of</strong> 52 CVID patients and 26 non-patient controls. Five<br />
heterozygous NOD2 leu1007finsc mutations, two heterozygous<br />
NOD2 gly908arg mutations and four heterozygous<br />
NOD2 arg702trp mutations were found amongst CVID<br />
patients giving allele frequencies <strong>of</strong> 0.048, 0.019 and<br />
0.038 respectively. No polymorphisms were found amongst<br />
non-patient controls. Although to date we are unable to<br />
demonstrate a significant genotype/phenotype association,<br />
results from this study show this as reliable<br />
screening method for gly908arg, leu1007finsc and<br />
arg702trp NOD2 polymorphisms. More patients are<br />
required to conclude whether these and/or additional<br />
NOD2 polymorphisms can define a clinical subgroup <strong>of</strong><br />
CVID patients likely to develop enteropathic or granulomatous<br />
complications.<br />
doi:10.1016/j.clim.2007.03.296<br />
F.84 Development <strong>of</strong> Regulatory T Cells After<br />
Thymus Transplantation in Subjects with Complete<br />
DiGeorge Syndrome<br />
Ivan Chinn, Fellow-In-Training, Duke University Medical<br />
Center, Pediatrics, Durham, NC, Blythe Devlin, Assistant<br />
Research Pr<strong>of</strong>essor, Duke University Medical Center,<br />
Pediatrics, Durham, NC, M. Louise Markert, Associate<br />
Pr<strong>of</strong>essor, Duke University Medical Center, Pediatrics,<br />
Durham, NC<br />
Purpose: Characterize the development <strong>of</strong> CD4+Foxp3+<br />
regulatory Tcells in subjects with complete DiGeorge syndrome<br />
after thymus transplantation. Background: Forty-four subjects<br />
with complete DiGeorge syndrome, including athymia (b50<br />
naïveTcells/cumm),havereceivedculturedpostnatalallogeneic<br />
HLA nonmatched thymus grafts. Twenty-seven <strong>of</strong> 32<br />
survivors are currently at 1-year or greater post-transplantation.<br />
All 1-year survivors have naïve T cells with normal<br />
proliferative responses to phytohemagglutinin and normal T<br />
cell receptor repertoires. We now report the percentages <strong>of</strong><br />
Foxp3+ CD4+ T cells in the peripheral blood <strong>of</strong> 11 subjects who<br />
are 1-year survivors after thymus transplantation. Five <strong>of</strong> the<br />
subjects received peri-transplantation immunosuppression.<br />
Five <strong>of</strong> the 11 subjects have autoimmune thyroid disease,<br />
including one subject in the group who received immunosuppression.<br />
Results: All eleven subjects had percentages <strong>of</strong> CD4<br />
+Foxp3+ Tcells in the expected range compared to healthy adult<br />
controls at various time points post-transplantation. Absolute<br />
numbers <strong>of</strong> CD4+Foxp3+ T cells were low (12−65 cells/cumm),<br />
reflecting the subjects’ low total T cell counts for age. Similar<br />
percentages <strong>of</strong> regulatory T cells were found in subjects who<br />
received immunosuppression (median 3.85%, range 3.48−5.33%)<br />
compared with those who did not (median 4.58%, range 2.96<br />
−7.35%). The percentages <strong>of</strong> CD4+Foxp3+ Tcells did not appear
Abstracts<br />
to correlate with the development <strong>of</strong> autoimmune thyroid<br />
disease (median 5.2% [range 3.05−6.96%] in subjects with<br />
thyroid disease vs. 3.8% [range 2.96−7.35%] without thyroid<br />
disease). Discussion: Subjects with complete DiGeorge syndrome<br />
develop normal percentages <strong>of</strong> regulatory T cells in the<br />
peripheral blood after thymus transplantation.<br />
doi:10.1016/j.clim.2007.03.297<br />
F.85 Identification <strong>of</strong> a Mutation in the Gene Encoding<br />
the Endosomal Adaptor Protein p14 (MAPBPIP) in a<br />
Novel Primary Immunodeficiency Syndrome<br />
Jens Thiel, MD, University Hospital Freiburg, Freiburg,<br />
Germany<br />
The study <strong>of</strong> human primary immunodeficiency disorders<br />
has greatly advanced our understanding <strong>of</strong> basic<br />
host defense. We have investigated the molecular etiology<br />
<strong>of</strong> a novel autosomal recessive primary immunodeficiency<br />
syndrome. In a pedigree <strong>of</strong> 15 members <strong>of</strong> a Caucasian<br />
Mennonite family, four children suffered from congenital<br />
neutropenia, B cell and cytotoxic T cell deficiency. These<br />
children additionally showed a characteristic clinical<br />
phenotype associating small stature, hypopigmented<br />
skin, coarse facial features, and recurrent bronchopulmonary<br />
infections. In an attempt to identify a genetic<br />
basis for this novel immunodeficiency syndrome, we<br />
carried out a genetic linkage study. Since the family<br />
belongs to a religious isolate <strong>of</strong> Mennonite background,<br />
we expected that the disease would be associated with a<br />
homozygous mutation in a single gene. We identified four<br />
adjacent, perfectly segregating markers on chromosome<br />
1q21: D1S498, D1S2346, D1S305, and D1S1153. Within this<br />
minimal critical region <strong>of</strong> the linkage interval, there were<br />
a total <strong>of</strong> 87 genes or predicted genes and within the<br />
maximal possible linkage region, there were an additional<br />
105 genes or predicted genes. Combining the results <strong>of</strong><br />
the linkage analysis with a transcriptional pr<strong>of</strong>iling<br />
approach, only one gene located within the critical region<br />
on chromosome 1q21 was significantly underexpressed in<br />
the patients, namely MAPBPIP. By genomic sequencing a<br />
homozygous mutation in the 3′-UTR <strong>of</strong> MAPBPIP, a recently<br />
identified adaptor molecule controlling late endosomal<br />
signaling by the MP1–MAPK complex was identified in the<br />
four index family members. Neutrophils from all patients<br />
showed decreased RNA and protein levels <strong>of</strong> p14.<br />
doi:10.1016/j.clim.2007.03.298<br />
F.86 Foxp3 Expression Following Bone Marrow<br />
Transplantation for IPEX Syndrome After<br />
Reduced-Intensity Conditioning<br />
Morna J. Dorsey, Assistant Pr<strong>of</strong>essor, University <strong>of</strong> South<br />
Florida, St. Petersburg, FL, Aleksandra Petrovic, Assistant<br />
Pr<strong>of</strong>essor, All Children’s Hospital, St. Petersburg, FL,<br />
Matthew R. Morrow, Director, Flow Cytometry Core Facility,<br />
University <strong>of</strong> South Florida, Pediatrics, St. Petersburg, FL,<br />
John W. Sleasman, Pr<strong>of</strong>essor, University <strong>of</strong> South Florida,<br />
Pediatrics, St. Petersburg, FL, Larry J. Dishaw, Instructor,<br />
All Children’s Hospital, St. Petersburg, FL<br />
Objectives: To determine if immune reconstitution <strong>of</strong> Foxp3+<br />
regulatory T cells correlates with clinical improvement <strong>of</strong> IPEX<br />
syndrome following allogeneic HSCT. Methods: 8-month-old<br />
male infant with a mutation in the polyadenylation site <strong>of</strong> Foxp3<br />
gene, absence <strong>of</strong> Foxp3 protein expression and clinical<br />
manifestations <strong>of</strong> IPEX syndrome, including eczema, colitis,<br />
failure to thrive, TPN requirement, and elevated serum IgE,<br />
underwent matched unrelated HSCT following reduced-intensity<br />
conditioning regimen. Donor chimerism was determined by<br />
molecular analysis <strong>of</strong> blood or bone marrow. Blood samples were<br />
collected pre- and post-transplant. Regulatory Tcell population<br />
was determined by flow cytometry staining for Foxp3+<br />
CD25bright CD4+ T cells. Semi-quantitative PCR <strong>of</strong> Foxp3+<br />
mRNA was conducted as well. Results: Patient underwent<br />
reduced-intensity conditioning with alemtuzumab followed by<br />
fludarabine and melphalan. GVHD prophylaxis included tacrolimus,<br />
methotrexate, and prednisone. Neutrophils engrafted<br />
day +15, and platelets day+29. Patient was a full donor chimera<br />
day +28 and +60. Foxp3 protein expression was absent pre-<br />
HSCT. Post-transplantation, percentage CD4+ T cells expressing<br />
Foxp3+ CD25bright phenotype increased from 4.5% (day +29) to<br />
23% (day +90) and continued in this trend. Foxp3 mRNA<br />
expression confirmed flow cytometry data. Serum IgE levels<br />
decreased from 5000 IU/mL pre-transplant to 6 IU/mL on day<br />
+90, eczema resolved, and secretory diarrhea and feeding<br />
intolerance improved. Conclusions: Regulatory T cell reconstitution<br />
is evident soon after HSCT following reduced-intensity<br />
conditioning correlating with development <strong>of</strong> full donor<br />
chimerism. Increased Foxp3 expression correlates with correction<br />
<strong>of</strong> clinical and laboratory manifestations <strong>of</strong> IPEX syndrome<br />
providing evidence that HSCT is a curative procedure for this<br />
disorder.<br />
doi:10.1016/j.clim.2007.03.299<br />
S43<br />
F.87 Monoclonal Gammapathy Following Cord Blood<br />
Transplantation for Chediak−Higashi Syndrome<br />
Kevin YehSheng Wang, FIT, Pediatrics, University <strong>of</strong><br />
California, Los Angeles, Los Angeles, CA, Robert Roberts,<br />
Pr<strong>of</strong>essor <strong>of</strong> Pediatrics, Department <strong>of</strong> Pediatrics,<br />
University <strong>of</strong> California, Los Angeles, Los Angeles, CA<br />
Introduction: Chediak–Higashi syndrome (CHS) is a rare<br />
autosomal recessive disorder which <strong>of</strong>ten undergoes progression<br />
to a life-threatening accelerated phase with<br />
lymphohistiocytic infiltration in multiple organs. We report<br />
a case <strong>of</strong> CHS in the accelerated phase which was successfully<br />
treated with IVIG and high dose corticosteroids. He<br />
underwent cord blood transplantation and then developed a<br />
monoclonal gammapathy. Case report: Our patient was<br />
diagnosed with CHS at 16 months <strong>of</strong> age by peripheral<br />
blood smear which revealed giant granules in his white blood<br />
cells. At 6 years <strong>of</strong> age, he presented to us with<br />
pancytopenia, fever, respiratory stress, mental status<br />
changes, and seizure activity. On physical examination, he
S44 Abstracts<br />
had significant hepatosplenomegaly. Bone marrow biopsy<br />
showed cytoplasmic inclusions granule and hemophagocytic<br />
cells. For treatment, he received 3 courses <strong>of</strong> IVIG (4.5 g/kg<br />
total). He was also given methlyprednisolone. He was then<br />
placed on dexamethasone until the time <strong>of</strong> transplantation.<br />
Over the course <strong>of</strong> several weeks, his mental status returned<br />
to baseline, seizure activity ceased, hepatomegaly resolved,<br />
and pancytopenia improved. Therefore his accelerated<br />
phase had undergone remission. He then received a 10/10<br />
match cord blood transplantation. The patient appeared to<br />
engraft successfully following the transplantation and<br />
routine infusions <strong>of</strong> IVIG were stopped. Approximately<br />
15 months after transplantation, the patient was clinically<br />
stable but serum IgG level was found to be 4000 mg/dL and<br />
his CD 4/8 ratio was 0.55. Immunoelectrophoresis revealed<br />
that he had developed a monoclonal IgG gammapathy. Bone<br />
marrow and repeat RFLP studies are being done to further<br />
investigate this abnormality.<br />
doi:10.1016/j.clim.2007.03.300<br />
F.88 Distribution, Infections, Treatments and<br />
Molecular Analysis in a Large Cohort <strong>of</strong> Patients with<br />
Primary Immunodeficiency Diseases (PIDD) in<br />
Taiwan<br />
Wen-I Lee, Assistant Pr<strong>of</strong>essor, Immunodeficiency Diagnosis<br />
and Research Institute, Chang Gung University and<br />
Children’s Hospital, Taoyuan, China, Tang-Her Jaing,<br />
Assistant Pr<strong>of</strong>essor, Department <strong>of</strong> Pediatric Hematology<br />
and Oncology, Chung Gung Children’s Hospital, Taoyuan,<br />
China, Meng-Ying Hsieh, MD, Department <strong>of</strong> Pediatric<br />
Neurology, Chang Gung University and Children’s Hospital,<br />
Taoyuan, China, Syh-Jae Lin, Associated Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> Pediatric Allergy, <strong>Immunology</strong> and<br />
Rheumatology, Chang Gung University, Taoyuan, China,<br />
Jing-Long Huang, Pr<strong>of</strong>essor, Department <strong>of</strong> Pediatric<br />
Allergy, <strong>Immunology</strong> and Rheumatology, Chang Gung<br />
University, Taoyuan, China, Li-Chen Chen, Assistant<br />
Pr<strong>of</strong>essor, Department <strong>of</strong> Pediatric Allergy, <strong>Immunology</strong> and<br />
Rheumatology, Chang Gung University, Taoyuan, China,<br />
Ming-Ling Kuo, Pr<strong>of</strong>essor, Department <strong>of</strong> Microbiology and<br />
<strong>Immunology</strong>, Graduate Institute <strong>of</strong> Basic Medical Sciences,<br />
Taoyuan, China<br />
One hundred and twenty-four patients (from 120<br />
families) to date diagnosed as primary immunodeficiency<br />
diseases were enrolled from five tertiary medical centers.<br />
The distribution by an update eight categories showed 45<br />
patients (13 females/32 males; 36.3%) with predominant<br />
antibody deficiencies, 27 patients (6/21; 21.8%) with T and<br />
B cell immunodeficiency, 25 patients (9/16; 20.2%) with<br />
congenital defects <strong>of</strong> phagocyte, 25 patients (4/21; 20.2%)<br />
with other well-defined immunodeficiency syndromes, one<br />
boy (0.8%) with disease in immune deregulation (Chediak–<br />
Higashi syndrome) and another with complement 3 deficiency.<br />
None had defects in innate immunity or auto<br />
inflammatory disorders. Pseudomonas and Salmonella spp.<br />
were the two most identified microorganisms in septicemia<br />
(39.7%; 27/68 episodes). Twenty-three patients (18.5%) had<br />
mortality. Stem cell transplantation succeeded in 7 <strong>of</strong> 12<br />
patients. In addition to nine patients with DiGerge<br />
syndrome recognized by FISH, direct sequencing identified<br />
12 unique mutations from 20 families, reflecting distinct<br />
Taiwan geography, although a selection bias may exist. The<br />
study is still ongoing.<br />
doi:10.1016/j.clim.2007.03.301<br />
F.89 Expression <strong>of</strong> FoxP3 and CD25 in T Cells is<br />
Altered in ICOS-/- Common Variable<br />
Immunodeficiency (CVID) patients<br />
Hans Hartmut Peter, Head <strong>of</strong> Department,University<br />
Hospital Freiburg, Freiburg, Germany, Julia Horn, Assistant<br />
Doctor, University Hospital Freiburg, Department <strong>of</strong> <strong>Clinical</strong><br />
<strong>Immunology</strong> and Rheumatology, Freiburg, Germany, Ulrich<br />
Salzer, Senior Postdoctoral Fellow, University Hospital<br />
Freiburg, Department <strong>of</strong> <strong>Clinical</strong> <strong>Immunology</strong> and<br />
Rheumatology, Freiburg, Germany, Jennifer Birmelin, MTA,<br />
Royal Free Hospital Department <strong>of</strong> <strong>Immunology</strong>, London,<br />
England, Klaus Warnatz, Consultant, University Hospital<br />
Freiburg, Department <strong>of</strong> <strong>Clinical</strong> <strong>Immunology</strong> and<br />
Rheumatology, Freiburg, Germany, Michael Schlesier, Head<br />
<strong>of</strong> Laboratory, University Hospital Freiburg, Department <strong>of</strong><br />
<strong>Clinical</strong> Immunolgy and Rheumatology, Freiburg,<br />
Germany, Bodo Grimbacher, Consultant, Royal Free<br />
Hospital, Department <strong>of</strong> <strong>Immunology</strong>,<br />
London, England<br />
Objective: FoxP3+CD25+ high regulatory T cells play a<br />
central role in suppressing immune responses to self<br />
antigens. To determine whether CVID patients have an<br />
altered frequency <strong>of</strong> FoxP3+CD25+ high regulatory T cells<br />
(Tregs), we studied this cell type in 48 CVID patients<br />
including four ICOS−/− CVID patients from the Freiburg<br />
CVID cohort by FACS analysis. Results: First, we found that<br />
the frequency <strong>of</strong> FoxP3+CD25+ Tregs (median +/− SD; p>=<br />
Wilcoxon rank sum test) was not significantly different in<br />
CVID patients (1.9% +/− 0.9%, n = 48; p>= 0.05) as compared<br />
to healthy controls (3.41% +/− 0.9%, n = 17). Second, CVID<br />
patients with autoimmune phenomena (n = 17 <strong>of</strong> 48) do not<br />
have lower frequencies <strong>of</strong> Tregs than CVID patients without<br />
autoimmune phenomena except one patient who presents<br />
with immune thrombocytopenia (ITP). Interestingly, ICOS-/-<br />
CVID patients (n = 4 <strong>of</strong> 48) have normal or increased Treg<br />
frequencies. In addition, in ICOS−/− CVID patients an<br />
increased FoxP3+CD25− T cell population in comparison to<br />
other CVID patients and healthy controls is observed.<br />
However, culturing ICOS−/− CD4+ T cells with IL-2 for 72 h<br />
results in decreased FoxP3 expression (16.4% to 4.68%) <strong>of</strong><br />
CD25- T cells due to activation. Conclusion: Our data suggest<br />
that CVID patients with autoimmune phenomena, do not<br />
have reduced numbers <strong>of</strong> Treg cells. However, ICOS−/− CVID<br />
patients who have no autoimmune phenomena show an<br />
increased FoxP3+CD25+ T cell population and an increased<br />
Foxp3 expression <strong>of</strong> CD25− T cells.<br />
doi:10.1016/j.clim.2007.03.302
Abstracts<br />
F.90 Clusters Differentiation in HIV Patients<br />
According to Measurements <strong>of</strong> T Cells<br />
Subpopulations in Whole Blood After In Vitro<br />
Activation with Mitogen<br />
Marie-Paule Guillaume, Physician, MD, Brugmann Hospital,<br />
Brussels<br />
Absolute counts <strong>of</strong> CD4+ lymphocytes are considered as the<br />
most important surrogate marker for the risk <strong>of</strong> AIDS. However,<br />
contrasts between patient evolutions and their presumed<br />
risk category are not unusual. We formulated the hypothesis<br />
that underlying regulation patterns <strong>of</strong> T cell responses affect<br />
the actual personal outcome despite similar levels <strong>of</strong><br />
circulating CD4+ T cells. We used a standardized PHA stimulation<br />
<strong>of</strong> whole blood cultures as a simplified model <strong>of</strong> T cell<br />
interactions. Relative proportions <strong>of</strong> different subpopulations<br />
were assessed by cyt<strong>of</strong>luorometry, characterized by combined<br />
expressions <strong>of</strong> CD4, CD25, CD8, CD28, and CD69 molecules.<br />
Prospective data from 175 HIV+ patients with different disease<br />
status, and 235 blood donors as controls, were evaluated.<br />
Three different clusters were found: controls and 2 subgroups<br />
<strong>of</strong> patients. Hierarchical analysis <strong>of</strong> these parameters allowed<br />
the provisional definition <strong>of</strong> an algorithm classifying over 95%<br />
<strong>of</strong> the patients according to their cluster. This was based on<br />
levels <strong>of</strong> CD4+25++CD69+ under PHA stimulation, basal levels<br />
<strong>of</strong> CD4+CD25+CD69+ and CD8+CD28negCD69+. Involvement <strong>of</strong><br />
regulatory or suppressive cells was independently confirmed<br />
by expression <strong>of</strong> GITR markers. Repeated measures on 735<br />
successive observations show that patients tend to stick to<br />
their cluster despite changes in CD4 absolute levels or PHA<br />
response in vitro. Confirmation <strong>of</strong> these data would support<br />
the existence <strong>of</strong> different patterns <strong>of</strong> functional immune<br />
regulations in HIV patients.<br />
doi:10.1016/j.clim.2007.03.303<br />
F.91 HIV Treatment Reveals<br />
Hypogammaglobulinemia in an Individual with<br />
Common Variable Immune Deficiency<br />
Lulu Sun, Immunodeficiency Treatment Centre, McGill<br />
University Health Centre, Montreal, QC, Canada,<br />
Christos M Tsoukas, Immunodeficiency Treatment Centre,<br />
McGill University Health Centre, Montreal, QC, Canada<br />
While common variable immune deficiency (CVID) is<br />
characterized by hypogammaglobulinemia, HIV-1 infection<br />
is associated with elevated immunoglobulin levels. There<br />
have been four CVID patients reported in the literature who<br />
recovered antibody production after contracting HIV-1. Here<br />
we describe an HIV patient with concomitant CVID whose<br />
immunoglobulin levels dramatically decreased upon successful<br />
antiretroviral therapy (ART), and investigate a plausible<br />
mechanism by which HIV is able to modulate circulating<br />
immunoglobulin through regulation <strong>of</strong> the B cell activation<br />
factor (BAFF)/BAFF-receptor pathway. Four cohorts were<br />
compared: our HIV/CVID patient, HIV patients, CVID<br />
patients, and healthy controls. Phenotypic and functional<br />
analyses <strong>of</strong> the Tand B cells <strong>of</strong> these cohorts were performed<br />
using flow cytometry, and BAFF serum levels were measured<br />
by ELISA. In addition to marked hypogammaglobulinemia,<br />
the patient has a complete defect <strong>of</strong> memory IgD+/−<br />
CD27+CD19+ B cells that was not altered by ART, consistent<br />
with severe CVID. When viral load briefly increased, a<br />
transient normalization <strong>of</strong> immunoglobulins was also<br />
observed. At the same time, the percentage <strong>of</strong> BAFF-R+ B<br />
cells was boosted from low to normal levels. Plasma BAFF<br />
levels were high compared to controls during viral suppression,<br />
due primarily to the lack <strong>of</strong> memory B cells. Finally, T<br />
cell phenotypes remained abnormal, with a low CD4+ T cell<br />
count that is characteristic <strong>of</strong> rigorous HIV disease, and<br />
impaired in vitro cellular proliferation. Our findings suggest<br />
that high HIV viraemia was required to maintain normal<br />
immunoglobulin levels in the CVID patient, possibly through<br />
upregulating BAFF-R.<br />
doi:10.1016/j.clim.2007.03.304<br />
S45<br />
F.92 Novel IL12RB1 Compound Heterozygous<br />
Mutation in a Brazilian Family with Disseminated<br />
Bacille Calmette-Guérin (BCG) Infection<br />
Joao B. Oliveira, Associate Researcher, Molecular<br />
<strong>Immunology</strong> and Genetics Section, LIM56, USP, São Paulo,<br />
Brazil, Ana Nishiya, Technician, Molecular <strong>Immunology</strong> and<br />
Genetics Section, LIM-56, USP, São Paulo, Brazil, Ludovic de<br />
Beaucoudrey, Researcher, Laboratory <strong>of</strong> Human Genetics <strong>of</strong><br />
Infectious Diseases, University <strong>of</strong> Paris Rene Descartes,<br />
INSERM U550, Paris, Jean-Laurent Casanova, Director,<br />
Laboratoire de Genetique Humaine des Maladies<br />
Infectieuses, INSERM Unite 550, Paris, Anete Grumach,<br />
Associate Pr<strong>of</strong>essor, Primary Immunodeficiency Outpatient<br />
Unit ADEE-3003, LIM-56, USP, São Paulo, Brazil, Alberto J.S.<br />
Duarte, Lab Director, Laboratory <strong>of</strong> Medical Investigation<br />
Unit 56 (LIM-56), University <strong>of</strong> São Paulo, Brazil, São Paulo,<br />
Brazil, Dewton Moraes-Vasconcelos, Associate Pr<strong>of</strong>essor,<br />
2Primary Immunodeficiency Outpatient Unit ADEE-3003,<br />
LIM-56, USP, São Paulo, Brazil<br />
Introduction. Mendelian inheritance to mycobacterial<br />
disease (MSMD) refers to a group <strong>of</strong> rare diseases characterized<br />
by predisposition to clinical disease caused by<br />
environmental non-tuberculous or poorly virulent mycobaterial<br />
species such as Bacille Calmette-Guérin (BCG)<br />
vaccine. Mutations in 5 components <strong>of</strong> the IFN-γ/IL-12 axis<br />
underlie MSMD: IFNR1, IFNR2, STAT1, IL12B and IL12RB1. We<br />
describe here a Brazilian family with a novel compound<br />
heterozygous mutation in IL12RB1. Material and methods.<br />
The 17 exons <strong>of</strong> IL12RB1 and flanking intronic regions were<br />
amplified by PCR, purified and sequenced using an automated<br />
capillary sequencer. Results. LMS, a 2.7-year-old girl,<br />
presented disseminated BCG infection 1 month after<br />
vaccination. Therapeutics was instituted 4 times with<br />
partial response, with relapse <strong>of</strong> the disease immediately<br />
after withdrawal <strong>of</strong> the drugs. She had normal blood cell<br />
counts and lymphoproliferation to mitogens and Candida,<br />
but low response to PPD. Surface expression <strong>of</strong> IL-12 RB1, IL-<br />
12 p40 and IFN-γ R1 was normal. IFN-γ production was low,<br />
with no improvement after stimulation by BCG plus IL-12.<br />
Genomic DNA sequencing <strong>of</strong> IL12RB1 demonstrated a<br />
compound heterozygous pattern, with a substitution in
S46 Abstracts<br />
exon 5 (P173W) and a deletion <strong>of</strong> 17 bp in exon 9. Analysis <strong>of</strong><br />
parents and relatives showed the deletion to be <strong>of</strong> paternal<br />
origin and the substitution <strong>of</strong> maternal origin, but present<br />
also in a maternal uncle. Conclusion. A novel heterozygous<br />
mutation in IL12RB1 causes susceptibility to BCG infection<br />
after vaccination. In suspected cases, mutational screening<br />
should be performed even with normal expression <strong>of</strong> the<br />
IL12Rb1 receptor.<br />
doi:10.1016/j.clim.2007.03.305<br />
F.93 Modulation <strong>of</strong> In Vivo T Cell Activation by an<br />
Acetylcholine-Esterase Inhibitor in Patients<br />
Chronically Infected with HIV<br />
Carlos Cantu-Brito, Investigator, Department <strong>of</strong> Neurology,<br />
Instituto Nacional de Ciencias Medicas y Nutricion SZ,<br />
Mexico City, Mexico, Sergio Ivan Valdes-Ferrer, Resident,<br />
Department <strong>of</strong> Neurology, Instituto Nacional de Ciencias<br />
Medicas y Nutricion SZ, Mexico City, Mexico, Jose Crispin,<br />
PhD Student, Department <strong>of</strong> <strong>Immunology</strong> and<br />
Rheumatology, Instituto Nacional de Ciencias Medicas y<br />
Nutricion SZ, Mexico City, Mexico, Francisco Belaunzaran,<br />
Resident, Department <strong>of</strong> Infectious Diseases, Instituto<br />
Nacional de Ciencias Medicas y Nutricion SZ, Mexico City,<br />
Mexico, Maria Ines Vargas Rojas, PhD Student, Department<br />
<strong>of</strong> <strong>Immunology</strong> and Rheumatology, Instituto Nacional de<br />
Ciencias Medicas y Nutricion SZ, Mexico City, Mexico, Juan<br />
Sierra, Investigator, Department <strong>of</strong> Infectious Diseases,<br />
Instituto Nacional de Ciencias Medicas y Nutricion SZ,<br />
Mexico City, Mexico, Jorge Alcocer Varela, Investigator,<br />
Department <strong>of</strong> <strong>Immunology</strong> and Rheumatology, Instituto<br />
Nacional de Ciencias Medicas y Nutricion SZ, Mexico City,<br />
Mexico<br />
The immune system <strong>of</strong> patients with HIV is overstimulated.<br />
An increased fraction <strong>of</strong> in vivo activated CD4+<br />
Tcells coupled with a decrease in regulatory Tcell numbers is<br />
a cardinal feature commonly observed. Vagal stimulation can<br />
modulate inflammatory response through T cell and macrophages<br />
by down-regulating their production <strong>of</strong> pro-inflammatory<br />
cytokines, namely TNF-α, IL-1, and IL-6. The aim <strong>of</strong><br />
this work was to analyze if the administration <strong>of</strong> an<br />
acetylcholine-esterase (Ach-E) inhibitor is capable <strong>of</strong> diminishing<br />
immune over-stimulation in patients chronically<br />
infected with HIV. Methods: Nineteen HIV-infected patients<br />
with more than 250 CD4+ T cells, devoid <strong>of</strong> antiretroviral<br />
therapy, were assigned to pyridostigmine sulfate 90 mg/day<br />
or matching placebo. In every case, two peripheral blood<br />
samples were obtained: one before and one after 1 week <strong>of</strong><br />
treatment. In vivo activated CD4+ T cells (HLA-DR+, CD69+)<br />
and regulatory T cells (CD4+CD25+Foxp3+) were quantified<br />
with flow cytometry. CD4+ Tcell proliferation was quantified<br />
after stimulation with a polyclonal stimulus (either PHA or<br />
PMA+ionomycin). Results: Nine patients received pyridostigmine,<br />
10 placebo. There were no basal differences<br />
between treatment and placebo groups. In vivo activated<br />
CD4+ T cells diminished significantly (p=0.025) in patients<br />
who received pyridostigmine. Conversely, regulatory T cells<br />
increased in treated patients. Finally, CD4+ T cell proliferation<br />
was significantly lower in patients who received<br />
pyridostigmine (p=0.026). Discussion: We found that the<br />
administration <strong>of</strong> a low dose <strong>of</strong> Ach-E inhibitors can<br />
successfully diminish CD4+ Tcell over-stimulation in patients<br />
with chronic HIV infection.<br />
doi:10.1016/j.clim.2007.03.306<br />
F.94 CD4/CD8 T Cell Ratio Predicts HIV Infection in<br />
Infants: The NHLBI P2C2 HIV Study<br />
William Shearer, Pr<strong>of</strong>essor, Baylor College <strong>of</strong> Medicine,<br />
Texas Children’s Hospital, Pediatrics, Houston, TX, Savita<br />
Pahwa, Pr<strong>of</strong>essor, University <strong>of</strong> Miami, Miller School <strong>of</strong><br />
Medicine, Miami, FL, Jennifer Read, Medical Officer,<br />
National Institute <strong>of</strong> Child Health and Human Development,<br />
Bethesda, MD, Sameera Wijayawardana, Biostatistician<br />
Student, Emory University Rollins School <strong>of</strong> Medicine,<br />
Atlanta, GA, Jing Chen, Assistant Pr<strong>of</strong>essor, Emory<br />
University Rollins School <strong>of</strong> Medicine, Antanta, GA, Kirk<br />
Easley, Senior Associate, Emory University Rollins School <strong>of</strong><br />
Medicine, Atlanta, GA, Paul Palumbo, Pr<strong>of</strong>essor, Dartmouth<br />
Medical School, Hanover, NH, Elaine Abrams, Associate<br />
Pr<strong>of</strong>essor, Columbia University, New York, NY, Steven<br />
Nesheim, Associate Pr<strong>of</strong>essor, Emory University School <strong>of</strong><br />
Medicine, Atlanta, GA<br />
Background: Although CD4 and CD8 T cell counts are<br />
available in many resource-poor settings, HIV DNA PCR tests<br />
are not. We analyzed data from the P2C2 HIV and CDC PACTS<br />
studies (overall HIV transmission rates: 17% and 18%,<br />
respectively) to evaluate the CD4/CD8 T cell ratio as a<br />
predictor <strong>of</strong> HIV infection among HIV-exposed infants.<br />
Methods: Data from the 3-month visit (45–150 days) for<br />
infants born to HIV-infected mothers enrolled in the P2C2<br />
HIV study (79 HIV-infected, 409 uninfected) were analyzed.<br />
The CDC PACTS cohort (224 HIV-infected, 1015<br />
uninfected) was used for validation. Results: The area<br />
under the ROC curve was higher for the CD4/CD8 ratio<br />
compared to the CD4 cell count (AUC=0.83 and 0.75,<br />
P=0.03). The mean CD4/CD8 ratio at the 3-month visit<br />
was 1.7 for HIV-infected infants and 3.0 for uninfected<br />
infants. A CD4/CD8 ratio <strong>of</strong> 2.4 was almost 2.5 times (95%<br />
CI for the likelihood ratio: 2.1–2.9) more likely to occur in<br />
an HIV-infected infant compared to an uninfected infant (test<br />
sensitivity 81%; posttest probability <strong>of</strong> HIV 33%). Model<br />
performance in the CDC PACTS validation sample was equally<br />
good (AUC=0.78 for the CD4/CD8 ratio; good agreement<br />
between the predicted and observed risk <strong>of</strong> HIV). Conclusion:<br />
We conclude that the CD4/CD8 Tcell ratio is a more sensitive<br />
predictor <strong>of</strong> HIV infection in infants than the CD4 Tcell count<br />
and, when necessary, the CD4/CD8 T cell ratio may be used<br />
with caution to diagnose HIV infection.<br />
doi:10.1016/j.clim.2007.03.307<br />
F.95 First Case <strong>of</strong> Human CD21 Deficiency—<br />
Association with Hypogammaglobulinemia<br />
Jens Thiel, MD, University Hospital Freiburg, Freiburg
Abstracts<br />
Complement receptor type 2 (CR2/CD21) is mainly<br />
expressed by mature B cells and FDCs. CD21 functions as<br />
receptor for C3d-opsonized immune complexes and forms a<br />
coreceptor signalling complex together with CD19, and CD81.<br />
We report a case <strong>of</strong> human CD21 deficiency associated with<br />
hypogammaglobulinemia. The 28-year-old patient presented<br />
with flu-like symptoms and reduced serum IgG and IgA.<br />
Leucocytes and lymphocytes were within normal range, as<br />
were the cell counts for T cell subpopulations and CD19+ B<br />
cells. However, class switched CD27+ memory B cells were<br />
reduced and CD21 expression was absent from all patient B<br />
cells. Moreover, no soluble CD21 was detectable in the<br />
patient’s serum, ruling out increased CD21 shedding from the<br />
B cell surface. Complement receptor type 1 was expressed<br />
normally and an immature B cell phenotype could be ruled<br />
out. Thus, the hypogammaglobulinemia and mild immunodeficiency<br />
in this patient may be caused by insufficient<br />
formation <strong>of</strong> switched memory B cell and antibodies. Looking<br />
for the underlying molecular defect, no mutation could be<br />
identified within the coding regions <strong>of</strong> CD21 by genomic<br />
sequencing. CD21 mRNA levels in the patient’s B cells were<br />
not reduced. Sequencing <strong>of</strong> intronic regions adjacent to the<br />
19 exons <strong>of</strong> CD21 revealed a heterozygous point mutation<br />
within the 3′ splice site <strong>of</strong> CD21 exon 6 leading to one<br />
shortened mRNA allele that lacks exon 6. Further experiments<br />
are necessary to test whether this heterozygous splice<br />
site mutation is sufficient to explain the absence <strong>of</strong> CD21 on<br />
the patient’s B cells.<br />
doi:10.1016/j.clim.2007.03.308<br />
F.96 Warning Signs for Primary Immunodeficiences<br />
Investigation in Hospitalized Patients<br />
Anete Sevciovic Grumach, Department <strong>of</strong> Dermatology,<br />
University <strong>of</strong> São Paulo, Brazil, São Paulo, Brazil, Dewton<br />
Moraes-Vasconcelos, Department <strong>of</strong> Dermatology,<br />
University <strong>of</strong> São Paulo, Brazil, São Paulo, Brazil, Joao Bosco<br />
Oliveira, Department <strong>of</strong> Dermatology, University de São<br />
Paulo, Brazil, São Paulo, Brazil, Alexandre Correa, BSc,<br />
Department <strong>of</strong> Dermatology, University <strong>of</strong> São Paulo, Brazil,<br />
São Paulo, Brazil, Rosemeire Navickas Constantino-Silva,<br />
BSc, Department <strong>of</strong> Dermatology, University <strong>of</strong> São Paulo,<br />
Brazil, São Paulo, Brazil, Noemia Orii, BSc, Department <strong>of</strong><br />
Dermatology, University <strong>of</strong> São Paulo, Brazil, São Paulo,<br />
Brazil, Noac Chuffi Barros, Department <strong>of</strong> Dermatology,<br />
University <strong>of</strong> São Paulo, Brazil, São Paulo, Brazil, Maria do<br />
Socorro Ferrao, Department <strong>of</strong> Dermatology, University <strong>of</strong><br />
São Paulo, Brazil, São Paulo, Brazil, Marinella Dellanegra,<br />
Instituto de Infectologia Emilio Ribas, São Paulo, Brazil,<br />
Alberto Jose da Silva Duarte, Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Dermatology, University <strong>of</strong> São Paulo, Brazil, São Paulo,<br />
Brazil<br />
Background: It has been published a higher prevalence <strong>of</strong><br />
PID in hospitalized patients. Nevertheless, most <strong>of</strong> the<br />
recommendations for the identification <strong>of</strong> PID are based on<br />
retrospective reports or in outpatients groups. Objective: To<br />
select the main signs dealing with PID diagnosis based on a<br />
prospective evaluation <strong>of</strong> hospitalized patients. Methodology:<br />
A prospective evaluation <strong>of</strong> inpatients was performed<br />
for a 6 year period. All the patients with clinical symptomatology<br />
not well elucidated were referred to the immunologist.<br />
A comprehensive immunological evaluation was<br />
available and the laboratorial tests were performed according<br />
to the clinical manifestations. Results: 462 patients (1.4<br />
M:1 F) were evaluated and the main symptoms were:<br />
prolonged fever (3.5%), hepatosplenomegaly (2%), uncommon<br />
infections (67%), recurrent infections (4.3%), unexpected<br />
complications (18.2%), suggestive auto-immunity<br />
(3.7%), complications due to vaccines (0.7%), and allergy<br />
(2.2%). PIDs were identified in 18 patients. No PID was<br />
diagnosed among patients complaining <strong>of</strong> prolonged fever<br />
and hepatosplenomegaly. Conclusions: The following criteria<br />
could be used as warning signs for hospitalized patients:<br />
recurrent infections, meningococcal meningitis above 5<br />
years old, complicated infections caused by common pathogens,<br />
uncommon pathogens in HIV negative patients and<br />
vaccinal complications.<br />
doi:10.1016/j.clim.2007.03.309<br />
S47<br />
F.97 Single Cell Evaluation <strong>of</strong> the Production <strong>of</strong><br />
T Cell Cytokines by Chronic Mucocutaneous<br />
Candidiasis Patients<br />
Dewton Moraes Vasconcelos, MD, PhD, Department <strong>of</strong><br />
Dermatology, University <strong>of</strong> São Paulo Medical School, São<br />
Paulo, Brazil, Alexandre de Almeida, MD, MSc, Department<br />
<strong>of</strong> Dermatology, University <strong>of</strong> São Paulo Medical School, São<br />
Paulo, Brazil, Dewton Moraes Vasconcelos, MD, PhD,<br />
Department <strong>of</strong> Dermatology, University <strong>of</strong> São Paulo<br />
Medical School, São Paulo, Brazil, Anete S. Grumach, MD,<br />
PhD, Department <strong>of</strong> Dermatology, University <strong>of</strong> São Paulo<br />
Medical School, São Paulo, Brazil, Mauricio Domingues<br />
Ferreira, MD, PhD, Department <strong>of</strong> Dermatology, University<br />
<strong>of</strong> São Paulo Medical School, São Paulo, Brazil, Tatiana Negri<br />
Santi, BSc, Department <strong>of</strong> Dermatology, University <strong>of</strong> São<br />
Paulo Medical School, São Paulo, Brazil, Alberto Jose da<br />
Silva Duarte, MD, PhD, Department <strong>of</strong> Dermatology,<br />
University <strong>of</strong> São Paulo Medical School, São Paulo, Brazil<br />
Background: Chronic mucocutaneous candidiasis (CMC) is<br />
a rare primary immune deficiency characterized by chronic<br />
or recurrent infection by Candida in mucous membranes,<br />
nails and skin. Impaired cell mediated immunity to Candida<br />
as well as dysregulation <strong>of</strong> the production <strong>of</strong> Th1 and Th2<br />
cytokines is an important mechanism in CMC. Objective:<br />
Evaluate the number <strong>of</strong> Th1 (IL-2, IFN-γ) and Th2 (IL-10, IL-<br />
4) cytokines producing cells <strong>of</strong> a group <strong>of</strong> patients with CMC<br />
to understand the relationship <strong>of</strong> these cytokines and CMC.<br />
Methods: We studied the production <strong>of</strong> cytokines <strong>of</strong> 15<br />
patients with CMC and 13 controls. We evaluated the<br />
production <strong>of</strong> IL-2, IL-4, IL-10 and IFN-γ by total T cells, CD4<br />
+ and CD8+ subsets by intracellular cytokine flow cytometry.<br />
Results: The patients presented lower number <strong>of</strong> IL-2, IFNγ,<br />
IL-10 and IL-4 producing CD4+ cells than controls, when<br />
stimulated with Candida and tetanus toxoid. The patients’<br />
presented less CD8+ T cells producing Th1 cytokines when<br />
compared to controls. There were two distinct subgroups <strong>of</strong><br />
patients. One presented less IL-2 and IFN-γ producing T cells<br />
than the other. The second subgroup presented a lower
S48 Abstracts<br />
number <strong>of</strong> IFN-γ and a similar number <strong>of</strong> IL-2 producing T<br />
cells compared to control group. Conclusion: Our patients<br />
presented a decreased number <strong>of</strong> T cells producing both Th1<br />
and Th2 cytokines. Despite the presence <strong>of</strong> 2 different<br />
groups in relation to cytokine production there was no<br />
correlation to the clinical features.<br />
doi:10.1016/j.clim.2007.03.310<br />
F.98 Immunomodulation by Cytokines in Chronic<br />
Mucocutaneous Candidiasis Patients: Evaluation <strong>of</strong><br />
the Production <strong>of</strong> T Cell Cytokines at a Single Cell<br />
Level<br />
Mauricio Domingues Ferreira, MD, PhD, Department <strong>of</strong><br />
Dermatology, University <strong>of</strong> São Paulo Medical School, São<br />
Paulo, Brazil, Alberto Jose da Silva Duarte, MD, PhD,<br />
Department <strong>of</strong> Dermatology, University <strong>of</strong> São Paulo<br />
Medical School, São Paulo, Brazil, Dewton Moraes<br />
Vasconcelos, MD, PhD, Department <strong>of</strong> Dermatology,<br />
University <strong>of</strong> São Paulo Medical School, São Paulo, Brazil,<br />
Tatiana Negri Santi-BSc, Department <strong>of</strong> Dermatology,<br />
University <strong>of</strong> São Paulo Medical School, São Paulo, Brazil,<br />
Anete S. Grumach, MD, PhD, Department <strong>of</strong> Dermatology,<br />
University <strong>of</strong> São Paulo Medical School, São Paulo, Brazil,<br />
Alexandre Almeida, MD, MSc, Department <strong>of</strong> Dermatology,<br />
University <strong>of</strong> São Paulo Medical School, São Paulo, Brazil<br />
Background: Chronic mucocutaneous candidiasis (CMC)<br />
is a rare primary immune deficiency characterized by<br />
chronic or recurrent Candida infection in mucous membranes,<br />
nails and skin. Impaired cell mediated response<br />
and dysregulation <strong>of</strong> the production <strong>of</strong> Th1 and Th2<br />
cytokines to Candida are important in CMC. We previously<br />
found a lower number <strong>of</strong> IL-2, IL-10, IL-4 and IFN-γ<br />
producing T cells and its CD4+ and CD8+ subsets compared<br />
to the control group when stimulated with Candida and<br />
tetanus toxoid. Objective: Modulate the T cell response by<br />
adding to the cell culture medium IL-2, or IL-12, or anti-IL-<br />
10. Methods: We studied the production <strong>of</strong> cytokines <strong>of</strong> 15<br />
patients with CMC and 13 controls stimulated by Candida<br />
and tetanus toxoid. We evaluated IL-2, IL-4, IL-10 and IFN-γ<br />
in total T cells, as well as CD4+ and CD8+ subsets by an<br />
intracellular cytokine flow cytometric assay. We tried to<br />
immunomodulate with recombinant human IL-2, or IL-12, or<br />
a monoclonal antibody anti-IL-10. Results: We did not find a<br />
significant response to the immunomodulation trials, but<br />
there was a trend <strong>of</strong> improvement in the synthesis <strong>of</strong> IL-2<br />
from the CD4+ cells <strong>of</strong> patients when stimulated by Candida<br />
and modulated by IL-2, IL-12 or anti-IL-10. Conclusion:<br />
There was no significant improvement by the immunomodulation<br />
by cytokines, but there was a tendency <strong>of</strong><br />
improvement. It is possible that if we have used a<br />
combination <strong>of</strong> cytokines, the results could be better. Our<br />
data suggest a new possible way <strong>of</strong> treatment to patients<br />
with CMC.<br />
doi:10.1016/j.clim.2007.03.311<br />
F.99 Hereditary Angiodema (HAE) in Brazil: Registry<br />
<strong>of</strong> 100 Cases<br />
Anete Sevciovic Grumach, Department <strong>of</strong> Dermatology,<br />
University <strong>of</strong> São Paulo, Brazil, São Paulo, Brazil, Jorge<br />
Andrade Pinto, Department <strong>of</strong> Pediatrics, Federal<br />
University <strong>of</strong> Minas Gerais, Belo Horizonte, Alexandre Pires<br />
Correa, BSc, Department <strong>of</strong> Dermatology, University <strong>of</strong><br />
São Paulo, Brazil, São Paulo, Brazil, Dewton<br />
Moraes-Vasconcelos, MD, PhD, Department <strong>of</strong> Dermatology,<br />
University <strong>of</strong> São Paulo, Brazil, São Paulo, Brazil, Rosemeire<br />
Navickas Constabtino-Silva, BSc, Department <strong>of</strong><br />
Dermatology, University <strong>of</strong> São Paulo, Brazil, São Paulo,<br />
Brazil, Sollange Valle, Federal University <strong>of</strong> Rio de Janeiro,<br />
Rio de Janeiro, Eli Mansour, Department <strong>of</strong> Pediatrics,<br />
University <strong>of</strong> Campinas, Campinas, Maria Marluce Vilela,<br />
Department <strong>of</strong> Pediatrics, University <strong>of</strong> Campinas,<br />
Campinas, Ricardo Zollner, <strong>Clinical</strong> Medicine, University <strong>of</strong><br />
Campinas, Campinas, Alberto Jose da Silva Duarte,<br />
Pr<strong>of</strong>essor, Department <strong>of</strong> Dermatology, University <strong>of</strong> São<br />
Paulo, Brazil, São Paulo, Brazil, Evandro Rivitti, Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> Dermatology, University <strong>of</strong> São Paulo, Brazil,<br />
São Paulo, Brazil<br />
HAE is a primary quantitative or functional defect <strong>of</strong> C1<br />
inhibitor. <strong>Clinical</strong> manifestations include subcutaneous,<br />
respiratory and gastrointestinal edema. Asphyxia could<br />
occur in 25–40% <strong>of</strong> the non-treated cases. There was no<br />
registry in Brazil until now. Objective: Report the clinical and<br />
laboratorial characteristics <strong>of</strong> HAE in Brazilians. Methods:<br />
Collaborative work groups were established among specialized<br />
services. The following parameters were evaluated:<br />
gender, age, age <strong>of</strong> diagnosis, main clinical manifestations,<br />
time for diagnosis, triggering factor, treatment, familial<br />
history and C1 inhibitor and C4 levels and CH50. Laboratorial<br />
diagnosis <strong>of</strong> C1 INH deficiency was confirmed in all patients.<br />
Results: HAE affected 67 F:33 M, 1–70 years <strong>of</strong> age and the<br />
first symptoms were reported during childhood in most <strong>of</strong> the<br />
cases. One episode <strong>of</strong> laryngeal edema was present in 17%<br />
and the triggering factors were trauma (31/100), stress (11/<br />
100), and menses (6/100). Familial history was positive in<br />
53/100. Conclusion: Familial history was determinant for<br />
diagnosis in half <strong>of</strong> the cases. Late diagnosis had been<br />
established but the first symptoms occurred during childhood.<br />
HAE diagnosis is still not well recognized in our<br />
country.<br />
doi:10.1016/j.clim.2007.03.312<br />
F.100 Autosomal Dominant Combined Immune<br />
Deficiency: A Unique Family with Absent B-Cells and<br />
Decreased T-Cells<br />
Michael Land, Physician, University <strong>of</strong> California, Los<br />
Angeles, Pediatrics, Los Angeles, CA, Raffi Tachdjian,<br />
Physician, University <strong>of</strong> California, Los Angeles, Pediatrics,<br />
Los Angeles, CA, Erina Lin, Physician, University <strong>of</strong><br />
California, Los Angeles, Pediatrics, Los Angeles, CA, Robert<br />
Roberts, Physician, University <strong>of</strong> California, Los Angeles,<br />
Pediatrics, Los Angeles, CA, Marc Riedl, Physician,<br />
University <strong>of</strong> California, Los Angeles, Department <strong>of</strong><br />
Medicine, Los Angeles, CA
Abstracts<br />
Rationale: We present a family <strong>of</strong> four individuals with<br />
immunodeficiency. The father and daughter were initially<br />
diagnosed with Common Variable Immune Deficiency (CVID)<br />
and were found to have absent B cells and low T cells. The<br />
father’s brother and mother also had immunodeficiency but<br />
died years ago. This may represent a unique form <strong>of</strong><br />
Combined Immunodeficiency.<br />
Methods/Results: The father is a 35-year-old man with a<br />
history <strong>of</strong> CVID diagnosed at age 12 following multiple upper<br />
respiratory infections. He was started on intravenous<br />
immunoglobulin (IVIG) for hypogammaglobulinemia, but<br />
never had any severe infections. His mother and older<br />
brother also received gamma globulin for presumed CVID,<br />
but both died from infections at age 36. He had one episode<br />
<strong>of</strong> pneumonia at age 19, with occasional sinus infections. His<br />
trough IgG level recently was 162, but increased to 724 with<br />
higher dosing. His IgA was b7, IgM b0.5, IgE b0.1. The<br />
patient’s 3-year-old daughter has been receiving IVIG since<br />
age 8 months for hypogammaglobulinemia. She has a history<br />
<strong>of</strong> sinusitis and has never been hospitalized. Her IgA is b7,<br />
IgM 6, IgE 0.1. Interestingly, both father and daughter have<br />
undetectable B cells (CD19 b1%) and CD4 lymphopenia<br />
(father: 202, daughter: 608). Both patients also have low<br />
CD8 counts (father: 162, daughter: 189). They have normal<br />
responses to mitogens and antigens. Conclusion: This family,<br />
along with the deceased relatives, exhibits an unusual<br />
pattern <strong>of</strong> inheritance for B cell and T cell deficiency<br />
consistent with combined immunodeficiency, suggesting<br />
autosomal dominance. Further characterization and investigation<br />
<strong>of</strong> this disease are warranted.<br />
doi:10.1016/j.clim.2007.03.313<br />
F.101 Circulating Class-Switched Memory B Cells<br />
are Markedly Reduced in Some Patients with<br />
Specific Antibody Deficiency (SAD) and/or Mild IgG<br />
Subclass Deficiency<br />
Lily E. Leiva, Associate Pr<strong>of</strong>essor, Louisiana State University<br />
Health Sciences Center and Children’s Hospital, Department<br />
<strong>of</strong> Pediatrics, New Orleans, LA, Kenneth Paris, Assistant<br />
Pr<strong>of</strong>essor, Louisiana State University Health Sciences Center<br />
and Children’s Hospital, Department <strong>of</strong> Pediatrics, New<br />
Orleans, LA, Hanh Monjure, Research Associate, Louisiana<br />
State University Health Sciences Center and Children’s<br />
Hospital, Department <strong>of</strong> Pediatrics, New Orleans, LA,<br />
Ricardo U. Sorensen, Pr<strong>of</strong>essor and Chair, Louisiana State<br />
University Health Sciences Center and Children’s Hospital,<br />
Department <strong>of</strong> Pediatrics, New Orleans, LA, Dana Minor,<br />
Research Technician, Research Institute for Children,<br />
Children’s Hospital, New Orleans, LA<br />
Some patients with recurrent respiratory infections are<br />
affected by specific antibody deficiency (SAD) and/or IgG<br />
subclass deficiencies. In this study we investigated if the<br />
inability <strong>of</strong> patients with SAD and/or IgG subclass deficiency to<br />
respond to pneumococcal polysaccharide antigens (PP) was<br />
associated with reduced numbers <strong>of</strong> circulating class-switched<br />
memory B cells. We studied 7 children with recurrent respiratory<br />
infections with SAD and/or mild IgG subclass deficiency and 8<br />
healthy controls. All patients were evaluated with total<br />
immunoglobulin and IgG subclass concentrations. We measured<br />
IgG and IgM anti-PP antibody levels by a standardized ELISA test.<br />
Purified PBMC were stained for the expression <strong>of</strong> CD27 and IgD<br />
on CD19+ B cells. Percentages <strong>of</strong> naïve (CD27NIgD+), nonswitched<br />
memory (CD27+IgD+) and class-switched memory<br />
(CD27+IgDN) B cells were determined by flow cytometry.<br />
Results: In patients in whom IgG anti-PP antibody levels were<br />
low, IgM levels were within the normal ranges for each <strong>of</strong> the 8<br />
serotypes tested. The B cell compartment in these patients<br />
showed a marked reduction in the percentage <strong>of</strong> class-switched<br />
memory B cells in 2 patients (6% and 2%, respectively) in<br />
comparison to the other 5 patients (range: 32%–90%) and to<br />
controls (range: 22%–87%). These results suggest that a deficient<br />
IgM to IgG antibody switch is associated with a lack <strong>of</strong> circulating<br />
class-switched memory B cells in some patients, and that this<br />
may be the explanation for the failure <strong>of</strong> some patients to<br />
respond to PP. Studies are in progress to determine the<br />
prognostic implications <strong>of</strong> these differences.<br />
doi:10.1016/j.clim.2007.03.314<br />
F.102 HIV Treatment Reveals<br />
Hypogammaglobulinemia in an Individual with<br />
Common Variable Immune Deficiency<br />
Lulu Sun, Immunodeficiency Treatment Centre, McGill<br />
University Health Centre, Montreal, QC, Canada, Christos<br />
M. Tsoukas, Immunodeficiency Treatment Centre, McGill<br />
University Health Centre, Montreal, QC, Canada<br />
While common variable immune deficiency (CVID) is characterized<br />
by hypogammaglobulinemia, HIV-1 infection is associated<br />
with elevated immunoglobulin levels. There have been<br />
four CVID patients reported in the literature who recovered<br />
antibody production after contracting HIV-1. Here we describe<br />
an HIV patient with concomitant CVID whose immunoglobulin<br />
levels dramatically decreased upon successful antiretroviral<br />
therapy (ART), and investigate a plausible mechanism by which<br />
HIV is able to modulate circulating immunoglobulin through<br />
regulation <strong>of</strong> the B cell activation factor (BAFF)/BAFF-receptor<br />
pathway. Four cohorts were compared: our HIV/CVID patient,<br />
HIV patients, CVID patients, and healthy controls. Phenotypic<br />
and functional analyses <strong>of</strong> the Tand B cells <strong>of</strong> these cohorts were<br />
performed using flow cytometry, and BAFF serum levels were<br />
measured by ELISA. In addition to marked hypogammaglobulinemia,<br />
the patient has a complete defect <strong>of</strong> memory IgD+/−<br />
CD27+ CD19+ B cells that was not altered by ART, consistent with<br />
severe CVID. When viral load briefly increased, a transient<br />
normalization <strong>of</strong> immunoglobulins was also observed. At the<br />
same time, the percentage <strong>of</strong> BAFF-R+B cells was boosted from<br />
low to normal levels. Plasma BAFF levels were high compared to<br />
controls during viral suppression, due primarily to the lack <strong>of</strong><br />
memory B cells. Finally, T cell phenotypes remained abnormal,<br />
with a low CD4+ Tcell count that is characteristic <strong>of</strong> rigorous HIV<br />
disease, and impaired in vitro cellular proliferation. Our findings<br />
suggest that high HIV viraemia was required to maintain normal<br />
immunoglobulin levels in the CVID patient, possibly through<br />
upregulating BAFF-R.<br />
doi:10.1016/j.clim.2007.03.315<br />
S49
S50 Abstracts<br />
F.103 Differences in Telomerase Expression by the<br />
CD1a+ Cells in Langerhans Cell Histiocytosis Reflects<br />
the Diverse <strong>Clinical</strong> Presentation <strong>of</strong> the Disease<br />
Cristiana Costa, PhD Student, Leiden University Medical<br />
Center, Department <strong>of</strong> Pediatrics, Leiden, The Netherlands,<br />
Maarten Egeler, MD PhD, Leiden University Medical Center,<br />
Leiden, The Netherlands, Manja Hoogeboom, Leiden<br />
University Medical Center, Leiden, The Netherlands, Ramses<br />
Forsyth, N. Goormaghtigh Institute <strong>of</strong> Pathology, University<br />
Hospital Gent, Gent, Karoly Szuhai, PhD, Leiden University<br />
Medical Center, Leiden, The Netherlands, Marieke Niesters,<br />
Leiden University Medical Center, Department <strong>of</strong> Pediatrics,<br />
Leiden, The Netherlands, Ronald de Krijger, Erasmus<br />
Medical Center, Department <strong>of</strong> Pathology, Rotterdam,<br />
Abdellatif Tati, Service de Pneumonologie, Hospital Saint<br />
Louis, Paris, Pancras Hogendoorn, Leiden University Medical<br />
Center, Department <strong>of</strong> Pathology, Leiden, The Netherlands,<br />
Nicola Annels, PhD, Leiden University Medical Center,<br />
Department <strong>of</strong> Pediatrics, Leiden, The Netherlands<br />
Langerhans cell histiocytosis (LCH) is a disease characterised<br />
by an uncontrolled clonal proliferation <strong>of</strong> Langerhans<br />
cells, whose aetiology is still unclear. The clonal nature <strong>of</strong><br />
LCH could support the hypothesis that it is a neoplastic<br />
disease with unlimited growth potential. One requirement<br />
for unlimited proliferation is the maintenance <strong>of</strong> telomere<br />
length. In a group <strong>of</strong> 70 patients we investigated whether a<br />
telomere maintenance mechanism is active by LCH cells.<br />
Results showed that LCH cells from all restricted skin LCH<br />
lesions (6/6) expressed telomerase as assessed by human<br />
telomere reverse transcriptase (hTERT) immunohistochemistry,<br />
whereas LCH cells from the majority <strong>of</strong> bone lesions<br />
analysed did not (26/34). Interestingly, in contrast to solitary<br />
bone lesions, LCH cells from lesions <strong>of</strong> multisystem patients<br />
always expressed telomerase (11/11), regardless <strong>of</strong> the<br />
lesional site. In situ telomeric repeat amplification protocol<br />
(TRAP) assay performed on different lesional sites showed<br />
that telomerase was active. In addition, the telomere length<br />
<strong>of</strong> LCH cells from a hTERT positive skin multisystem lesion<br />
was long and homogeneous when compared to the LCH cells<br />
from hTERT negative bone single system LCH lesions, which<br />
were heterogeneous in length. No evidence for an alternative<br />
lengthening <strong>of</strong> telomeres mechanism was found in<br />
hTERT negative lesions. The difference in telomerase<br />
expression and telomere length in the different lesional<br />
sites and in biopsies from patients with solitary versus<br />
multisystem disease appears to be reflective <strong>of</strong> the diverse<br />
clinical presentation and course <strong>of</strong> LCH. The results from this<br />
study have important implications for understanding the<br />
nature <strong>of</strong> this disease.<br />
doi:10.1016/j.clim.2007.03.316<br />
F.104 Multiplex Capabilities for Serological<br />
Immunoassays Using Luminex xMAP ® Technology<br />
Harold Baker, Senior Scientist, Luminex Corporation,<br />
Austin, TX<br />
The Luminex xMAP technology incorporates fluorescently<br />
encoded microspheres (xMAP microspheres) to perform<br />
multiplexed bioassays in conjunction with advanced digital<br />
signal processing and proprietary identification techniques.<br />
The advantage <strong>of</strong> the multiplexing technology is the<br />
capability to measure several antigens within one sample.<br />
The combination <strong>of</strong> multiple results and reduced sample<br />
volume increases the efficiency for testing and sample<br />
consumption. Another advantage is that the serological<br />
assays can be multiplexed with other immunoassays. Five<br />
specific antigens were coupled to xMAP microspheres<br />
representing several individual regions. BSA coated microspheres<br />
were prepared to function as internal assay controls<br />
for non-specific reactivity. A total <strong>of</strong> five regions were used<br />
with each antigen to develop a 30-plex assay. The xMAP<br />
serological assay format we used was a 60 minute, no wash<br />
assay. Diluted patient serum, 10 ƒÝL, was combined with the<br />
microsphere mixture, 50 ƒÝL, and incubated 30 min. Diluted<br />
PE conjugated antibody, 150 ƒÝL, was added and incubated<br />
30 min before being analyzed with the Luminex system.<br />
Equivalent results were obtained with the replicate antigen<br />
coated regions. Analytical sensitivity was defined by the<br />
maximum dilution before an appreciable decrement in MFI<br />
response was observed. This was observed to be at a dilution<br />
<strong>of</strong> 1:1000 representing the use <strong>of</strong> 0.01 ƒÝL serum for the<br />
measurement <strong>of</strong> antibodies in the serum sample. These<br />
results demonstrate that the Luminex xMAP technology can<br />
be used to develop multiplex assays such as those required<br />
for serological assessment <strong>of</strong> autoimmune diseases.<br />
doi:10.1016/j.clim.2007.03.319<br />
F.105 Immun<strong>of</strong>luorometrical Exploring <strong>of</strong> FSH<br />
Levels in the Serum <strong>of</strong> Patients with Histologically<br />
Verified Macrocellular Lung Cancer<br />
Ivan Milosevic, Primary Care Physician, Dispensary 2, ZZZZR,<br />
Kragujevac<br />
It is already known in that the cells <strong>of</strong> macrocellular lung<br />
cancer can produce gonadotropins. By following the levels <strong>of</strong><br />
FSH as one <strong>of</strong> gonadotropins, in the serum <strong>of</strong> macrocellular<br />
lung cancer patients, we could make certain statistical<br />
conclusions how significant these levels would be in eventual<br />
future early diagnostic procedures, besides already existing<br />
tumor markers and other methods. This work would connect<br />
the Oncology, Endocrinology and <strong>Immunology</strong> fields, containing<br />
very interesting immun<strong>of</strong>luorometrical procedures,<br />
hormonal theories and statistical estimates. As an immunological<br />
part in this theoretical model, it is represented<br />
through figure, time-resolved fluoroimmunoassay for quantitative<br />
detection <strong>of</strong> FSH. By following <strong>of</strong> FSH levels in the<br />
serum <strong>of</strong> microcellular lung cancer patients and making<br />
determinate statistical conclusions about its importance,<br />
that procedure could be eventually used as one <strong>of</strong> early or<br />
advanced diagnostic methods concerning mentioned disease.<br />
What would we get with that? The histological verification is<br />
very slow and painful, when the macrocellular lung cancer is<br />
in the question. This histological type gives the metastases<br />
rapidly and it needs to be diagnosed in the fastest possible<br />
way in order to be prevented by adequate reaction and<br />
therapy. At the same time the biopsy as a very invasive<br />
method would be avoided. When the tumor markers are in
Abstracts<br />
question as a diagnostic method supplemented and supported<br />
by the FSH levels exploring could be more reliable.<br />
Concerning all above presented, in future establishing <strong>of</strong> this<br />
procedure, we would get a rapid, comfortable and safe<br />
diagnostic method.<br />
doi:10.1016/j.clim.2007.03.320<br />
F.108 Distinct Regulation <strong>of</strong> Individual Tyrosine<br />
Phosphorylation <strong>of</strong> the Cytoplasmic Domain <strong>of</strong> CD19<br />
Nobuko Ishiura, MD, Department <strong>of</strong> Dermatology, Graduate<br />
School <strong>of</strong> Medicine, University <strong>of</strong> Tokyo, Tokyo, Japan,<br />
Manabu Fujimoto, PhD, Department <strong>of</strong> Dermatology,<br />
Kanazawa University Graduate School <strong>of</strong> Medical Science,<br />
Kanazawa Ishikawa, Japan, Kunihiko Tamaki, PhD,<br />
Department <strong>of</strong> Dermatology, Graduate School <strong>of</strong> Medicine,<br />
University <strong>of</strong> Tokyo, Tokyo, Japan<br />
Cell surface molecules called co-receptors on lymphocytes<br />
positively or negatively modulate the antigen receptor<br />
signaling. CD19 is a B lymphocyte-specific co-receptor which<br />
regulates constitutive thresholds and enhances antigen<br />
receptor-induced signaling through its cytoplasmic domain.<br />
The cytoplasmic region <strong>of</strong> CD19 contains nine conserved<br />
tyrosine residues, which become phosphorylated upon B cell<br />
activation. Tyrosine phosphorylation <strong>of</strong> CD19 mediates<br />
transmembrane signals by recruiting multiple signaling<br />
molecules including Src family protein tyrosine kinases<br />
(PTKs), especially Lyn, Vav, and PI 3-kinase. Of nine tyrosine<br />
residues, Y513, Y482, and Y391 are supposed to play critical<br />
roles in CD19-mediated signal transduction, although the<br />
precise regulation <strong>of</strong> individual tyrosine phosphorylation<br />
remains unknown. Herein, we have developed phosphospecific<br />
antibodies against these tyrosine residues, CD19pY513,<br />
CD19-pY482 and CD19-pY391, and have assessed the<br />
regulation <strong>of</strong> tyrosine phosphorylation <strong>of</strong> CD19 following<br />
various stimulations. BCR ligation induced Y513 phosphorylation<br />
preceded by Y482 and Y391 phosphorylation, suggesting<br />
Y513 as the primary phosphorylation site.<br />
Phosphorylations <strong>of</strong> three tyrosine residues were much<br />
enhanced and prolonged by simultaneous stimulation <strong>of</strong><br />
BCR and CD40 ligation. Intriguingly, CD19 containing phosphorylated<br />
Y513 was exclusively located within the lipid rafts<br />
following the BCR ligation, while majority <strong>of</strong> CD19 containing<br />
phosphorylated Y482 and Y391 stayed out <strong>of</strong> the lipid rafts.<br />
Therefore, individual tyrosines <strong>of</strong> CD19 undergo distinct<br />
regulation <strong>of</strong> phosphorylation, which in turn determines<br />
different distribution on the cell surface.<br />
doi:10.1016/j.clim.2007.03.321<br />
F.109 Vitamin D Receptor Activators Calcitriol and<br />
Paricalcitol Modulate Dendritic Cell Phenotype and<br />
Function In Vitro Study<br />
Klara Sochorova, Pharm Department <strong>of</strong> <strong>Immunology</strong>, 2nd<br />
Medical School, Charles University Prague, Prague, Vit<br />
Budinsky, Manager. Department <strong>of</strong> immunology, 2nd Medical<br />
School, Charles University, Prague, Zuzana Tobiasova,<br />
Manager. Department <strong>of</strong> <strong>Immunology</strong>, 2nd Medical School,<br />
Charles University, Prague, Jirina Bartunkova, Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> <strong>Immunology</strong>, 2nd Medical School, Charles<br />
University, Prague, Sylva Dusilova Sulkova, Pr<strong>of</strong>essor,<br />
Departments <strong>of</strong> Gerontology and Metabolism, Medical<br />
School and Teaching Hospital, Hradec Kralove<br />
Vitamin D is known as an important immunomodulatory<br />
agent. Its potential to suppress immune response in autoimmunity<br />
is intensively studied. Use <strong>of</strong> active metabolite <strong>of</strong><br />
vit. D3, calcitriol, in vivo, however, is limited due to its<br />
hypercalcemic effect. Paricalcitol, the synthetic analogue <strong>of</strong><br />
calcitriol, is characterized by reduced hypercalcemic effect,<br />
but its immunosupressive properties have not been evaluated<br />
yet. In our study we compared the effect <strong>of</strong> calcitriol and<br />
paricalcitol on morphological and functional characteristics<br />
<strong>of</strong> dendritic cells (DC) in vitro. DC were differentiated from<br />
monocytes with IL4 and GM-CSF for 5 days and then induced to<br />
maturate with viral or bacterial products (poly-IC, LPS).<br />
Calcitriol or paricalcitol were added during the period <strong>of</strong><br />
differentiation or maturation. We studied the expression <strong>of</strong><br />
DC-relevant surface molecules. DC function was tested by<br />
endocytosis, T-stimulatory capacity and cytokine production.<br />
DC differentiated in the presence <strong>of</strong> calcitriol or paricalcitol<br />
remain at an immature state (low expression <strong>of</strong> CD80, CD83,<br />
CD86 and HLA-DR after stimulation by both poly-IC and LPS,<br />
high expression <strong>of</strong> CD14 and PD-L1, high endocytic activity,<br />
low T cell activation and low IL-6 and TNF production). DC<br />
maturated in the presence <strong>of</strong> both drugs were also impaired,<br />
but less than in previous case. The immunosuppressive effect<br />
<strong>of</strong> both calcitriol and paricalcitol on DC is comparable.<br />
Paricalcitol thus seems to be a perspective drug for<br />
influencing the pathological immune response in autoimmunity.<br />
This project was supported by MSM 0021620812.<br />
doi:10.1016/j.clim.2007.03.322<br />
S51<br />
F.110 Significance <strong>of</strong> IgG4 in the Diagnosis <strong>of</strong> Mucous<br />
Membrane Pemphigoid<br />
Vijay Kumar, Director, IMMCO Diagnostics Inc., Buffalo, NY,<br />
Lakshmanan Suresh, IMMCO Diagnostics Inc., Buffalo, NY<br />
Background: Mucous membrane pemphigoid (MMP) is an<br />
immune mediated vesiculo-bullous disorder predominantly<br />
affecting the mucous membranes <strong>of</strong> the oropharynx. Direct<br />
immun<strong>of</strong>luorescence (IF) studies <strong>of</strong> the presence <strong>of</strong> immunoglobulins<br />
and/or complement are considered as the gold<br />
standard in the diagnosis <strong>of</strong> MMP. The tissue-bound IgG and<br />
other immunoreactants are seen as a linear band in the<br />
basement membrane zone (BMZ). However immunodeposition<br />
<strong>of</strong> IgG and/or C3 is not observed in all cases <strong>of</strong> MMP. One <strong>of</strong> the<br />
reasons <strong>of</strong> this lack <strong>of</strong> immune deposits may be that certain IgG<br />
subclass <strong>of</strong> immunoglobulins may be the autoreactants and that<br />
the total IgG conjugates fail to detect this subclass <strong>of</strong> IgG.<br />
Objective: The purpose <strong>of</strong> our study was to determine the<br />
diagnostic value and frequency <strong>of</strong> tissue deposition <strong>of</strong> IgG4 in<br />
comparison to polyclonal IgG, other immunoglobulins and C3.<br />
Methods: <strong>Oral</strong> mucosal biopsies <strong>of</strong> 82 patients clinically<br />
suspected to have MMP were analyzed by direct IF using<br />
polyclonal anti-human IgG, IgM, IgA, fibrin, complement C3,<br />
and anti-human IgG4 subclass monoclonal antibodies. Results:
S52 Abstracts<br />
Based on H&E and direct IF studies, 34 cases were diagnosed as<br />
MMP. The most common antibody deposited was IgG (90%)<br />
followed by C3 (82%) and IgG4 (71%). Strikingly, IgG4 was the<br />
sole antibody detected in two cases (6%). Conclusion: Our<br />
results suggest that the use <strong>of</strong> monoclonal IgG4 is important in<br />
the diagnosis <strong>of</strong> MMP. We suggest adding monoclonal IgG4 to the<br />
routine panel <strong>of</strong> antibodies used in studies <strong>of</strong> cases suspected to<br />
have MMP to avoid false-negatives.<br />
doi:10.1016/j.clim.2007.03.324<br />
F.111 Immuophenotyping <strong>of</strong> PBL and B.M Markers in<br />
Patients with Leukemia by Flowcytometry<br />
Fereshteh Alesahebfosoul, Academic Member <strong>of</strong> Isfahan<br />
University, Isfahan University Medical School, <strong>Immunology</strong><br />
Department, Isfahan<br />
Introduction: Peripheral blood lymphocytes (PBL) and bone<br />
marrow (B.M.) immunophenotyping is widely used for diagnosis<br />
and prognosis <strong>of</strong> leukemia. Flow cytometry technique provided<br />
facilities to assess the frequency <strong>of</strong> malignant cell population<br />
and the protein expression in each group <strong>of</strong> cells which have<br />
been gated. Method and material: Fresh peripheral blood and<br />
bone marrow <strong>of</strong> patient were collected and tested for following<br />
examination: total white blood cells and leukocyte percentage<br />
rate were determined by H1. Machine: The samples were<br />
treatedwithconjugatedMoAbwithFITCand/orphycoErythrine<br />
and then dual staining <strong>of</strong> cells was performed. The following<br />
monoclonal antibodies were used: CD10, CD19, CD20, CD5,<br />
CD3, CD22, CD7, CD33, anti-HLA DR, and CD13. After direct<br />
staining <strong>of</strong> cell samples, 30,000 cells were analyzed by flow<br />
cytometry. Result and discussion: The results <strong>of</strong> the first case <strong>of</strong><br />
the study were WBC=780 and LUC=2.2%, CD7=60%, CD=57%,<br />
CD5=56%<strong>of</strong>thelymphocytepopulation<strong>of</strong>peripheralblood.But<br />
the same markers in bone marrow sample were determined for<br />
D7=5%, CD3=5%, and CD5=6.5%. The results <strong>of</strong> the second case<br />
<strong>of</strong> the study were CD19=90%, CD20=89%, CD22=82%, and<br />
CD5=95% <strong>of</strong> the lymphocyte population <strong>of</strong> peripheral blood. In<br />
addition the same markers in bone marrow samples were<br />
determined for CD19=76%, CD20==85%, CD22=75.9%, and<br />
CD5=93%. HLA-DR marker was shown to be over 80% in both<br />
peripheral blood and bone marrow for this case. The result <strong>of</strong><br />
cell counting for the third case <strong>of</strong> the study showed WBC=4800<br />
and LUC=6.3%, CD19=41.5%, CD20=29.5%, CD22=39% and<br />
CD5=43.12%, CD3=40.3%, and CD7=41.1% in peripheral blood.<br />
But the same markers from bone marrow sample were<br />
determined for CD19=85.6%, CD22=81.2% and CD5=2.08%<br />
and CD3=1.88%. The results <strong>of</strong> this study show that the<br />
lymphocyte protein expression in leukemic samples is the value<br />
in diagnostic and classification <strong>of</strong> leukemia.<br />
doi:10.1016/j.clim.2007.03.325<br />
F.113 An All-Human Culture Model Supporting<br />
Human B Lymphopoesis<br />
Mercy Kagoda, Graduate Research Assistant/Student, Loma<br />
Linda University School <strong>of</strong> Medicine Department <strong>of</strong> Pathology<br />
and Human Anatomy, Loma Linda, CA, Yasmin Parrish,<br />
Research Specialist II, Childrens Hospital Los Angeles Research<br />
<strong>Immunology</strong> Bone Marrow Transplant, Los Angeles, CA,<br />
Jaqueline Rogerio, Post Doctoral Fellow, Childrens Hospital Los<br />
Angeles Research <strong>Immunology</strong> Bone Marrow Transplant, Los<br />
Angeles, CA, Ineavely Baez, Senior Research Assistant, Loma<br />
Linda University School <strong>of</strong> Medicine Department <strong>of</strong> Pathology<br />
and Human Anatomy, Loma Linda, CA, Qian-Lin Hao, Research<br />
Specialist IV, Childrens Hospital Los Angeles Research<br />
<strong>Immunology</strong> Bone Marrow Transplant, Los Angeles, CA, Lora<br />
Barsky, Flow Technologist, Childrens Hospital Los Angeles<br />
Research <strong>Immunology</strong> Bone Marrow Transplant, Los Angeles,<br />
CA, Ewa Zielinska, Flow Technologist, Childrens Hospital Los<br />
Angeles Research <strong>Immunology</strong> Bone Marrow Transplant, Los<br />
Angeles, CA, Monika Smogorzewska, Flow Technologist,<br />
Childrens Hospital Los Angeles Research <strong>Immunology</strong> Bone<br />
Marrow Transplant, Los Angeles, CA, Kimberly Payne, Assistant<br />
Pr<strong>of</strong>essor, Loma Linda University School <strong>of</strong> Medicine<br />
Department <strong>of</strong> Pathology and Human Anatomy, Loma Linda,<br />
CA<br />
Traditionally, nonfetal models <strong>of</strong> human B cell development<br />
have been based on co-culturing hematopoietic stem cells<br />
(HSCs) from cord blood (CB) and bone marrow (BM) on murine<br />
stromal cell lines. Here we describe the progressive replacement<br />
<strong>of</strong> murine components to generate a totally human<br />
culture model that supports human hematopoiesis, including B<br />
cell development. First, we replaced murine stromal cell lines<br />
with primary human BM stroma, next human serum was<br />
substituted for nonhuman sera. It has been reported that<br />
human B cell precursors selectively interact with CD10+ stromal<br />
cells during in vivo B cell development. In our cultures, human<br />
stroma express CD10 and secrete IL-7 at levels comparable to<br />
primary murine BM stroma. We found that human stroma bind<br />
IL-7 independently <strong>of</strong> the IL-7 receptor thereby having the<br />
potential for presenting IL-7 to developing B cell precursors.<br />
This is important as we have recently shown that IL-7 expands<br />
human B cell precursors by ∼50 fold and that IL-7 is essential for<br />
production <strong>of</strong> B cells from HSCs in adult human BM. While<br />
human stroma in our cultures express the human pre-BCR<br />
ligand, galectin-1 (Gal-1), the presence <strong>of</strong> Gal-1 was not<br />
required for IL-7-induced expansion <strong>of</strong> human B cell precursors.<br />
HSCs in co-cultures with human stroma and serum showed a<br />
generative capacity for CD19+ and CD19− progeny that was at<br />
least equal to that seen on murine stroma with nonhuman sera.<br />
Planned experiments will assess the ability <strong>of</strong> this culture model<br />
to support primary B cell malignancies.<br />
doi:10.1016/j.clim.2007.03.326<br />
F.114 Expression <strong>of</strong> CD3 and T Cell Receptor (TCR)<br />
on a Minor Population <strong>of</strong> Human CD14 Positive Cells<br />
Daron Forman, Senior Scientist, Tolerx, Cambridge, MA,<br />
Michael Rosenzweig, Senior Director <strong>of</strong> Preclinical<br />
Research, Tolerx, Cambridge, MA, Lou Vaickus, Chief<br />
Medical Officer, Tolerx, Cambridge, MA<br />
The TCR associates with CD3 polypeptides including CD3<br />
delta, gamma, epsilon, and zeta. It was commonly believed<br />
that TCR and CD3 expression occur exclusively on T cells.<br />
Recently however, TCR expression was reported on a minor<br />
population <strong>of</strong> both mouse and human neutrophils indicating
Abstracts<br />
that other populations may express CD3 and/or TCR. We<br />
evaluated whether CD3 and TCR-ab expression could be<br />
detected on non-T cell lineages in human peripheral<br />
blood. Cells were analyzed for CD3 and TCR-ab expression<br />
using monoclonal antibodies (Mabs) and flow cytometry. In<br />
addition to the expected distribution on lymphocytes, CD3<br />
and TCR-ab were detected on 0.3–1.0% population <strong>of</strong><br />
cells that expressed CD11b, CD11c, and CD14. This<br />
population expressed CD3 and TCR with an intensity<br />
similar to CD4+ and CD8+ T cells. CD14 is expressed at<br />
high levels on Fc-receptor (FcR) bearing monocytes. To<br />
exclude FcR binding as an explanation for the detection<br />
<strong>of</strong> anti-CD3 and anti-TCR-ab Mabs on CD14+ cells, studies<br />
were repeated with human serum or with pre-incubation<br />
with anti-FcR Mabs (anti-CD16 and anti-CD32). Staining<br />
was equivalent with and without FcR block, suggesting<br />
that anti-CD3 and anti-TCR-ab Mab binding was specific.<br />
These data suggest that a minor population <strong>of</strong> nonlymphoid<br />
cells that express CD11b, CD11c, and CD14 also<br />
express CD3 and TCR-ab. Further studies are ongoing to<br />
fully characterize this population.<br />
doi:10.1016/j.clim.2007.03.327<br />
F.115 Haplotype-specific Use <strong>of</strong> Lysosomal<br />
Proteases Can Modulate the Class II MHC Peptide<br />
Repertoire but Does Not Impair Invariant Chain<br />
Degradation in Human Antigen Presenting Cells<br />
Cristina Costanti-England PhD Candidate, Harvard Medical<br />
School/Brigham and Women’s Hospital, Center for<br />
Neurologic Diseases, Boston, MA, Howard Hang,<br />
Postdoctoral Fellow, Whitehead Institute, Massachusetts<br />
Institute <strong>of</strong> Technology, Cambridge, MA, David Hafler,<br />
Jack, Sadie and David Breakstone, Pr<strong>of</strong>essor <strong>of</strong> Neurology,<br />
Harvard Medical School/Brigham and Women’s Hospital,<br />
Center for Neurologic Diseases, Boston, MA, Hidde Ploegh,<br />
Pr<strong>of</strong>essor <strong>of</strong> Biology, Whitehead Institute, Massachusetts<br />
institute <strong>of</strong> Technology, Cambridge, MA<br />
The lysosomal protease asparagine endopeptidase (AEP)<br />
has been implicated in both the destructive processing <strong>of</strong><br />
self-antigen and the regulation <strong>of</strong> class II MHC maturation.<br />
In this study, we explore the role <strong>of</strong> human AEP in<br />
invariant chain processing in the maturation <strong>of</strong> several<br />
distinct allotypes <strong>of</strong> human class II MHC products. We find<br />
that, as is the case in mice, AEP activity is not necessary<br />
for class II maturation in human antigen-presenting cells.<br />
Our data demonstrate that Ii cleavage is regulated by<br />
many lysosomal proteases, including both aspartyl and<br />
cysteine proteases. Furthermore, we find that the<br />
repertoire <strong>of</strong> active proteases in EBV-transformed B cells<br />
differs between lines generated from different donors.<br />
These results suggest a unique pr<strong>of</strong>ile <strong>of</strong> lysosomal<br />
protease activity within each antigen-presenting cell<br />
that regulates Ii degradation. Our findings underscore<br />
the complexity <strong>of</strong> the class II MHC-restricted pathway <strong>of</strong><br />
antigen presentation. While allelic variation in MHC<br />
products is a decisive factor in determining the repertoire<br />
<strong>of</strong> presented peptides, the identity and level <strong>of</strong> activity<br />
<strong>of</strong> processing proteases present in antigen-presenting cells<br />
are contributing factors as well. As these proteases also<br />
generate the peptide cargo loaded into the binding<br />
pocket <strong>of</strong> class II MHC, it is certain that the repertoire<br />
<strong>of</strong> self and antigenic peptides presented to CD4+ T cells<br />
differs between individuals. Thus, altered presentation <strong>of</strong><br />
immunogenic self-peptides due to alternate protease<br />
pr<strong>of</strong>iles may be a factor contributing to the susceptibility<br />
to and severity <strong>of</strong> autoimmune diseases such as type 1<br />
diabetes and multiple sclerosis.<br />
doi:10.1016/j.clim.2007.03.330<br />
S53<br />
F.116 Induction <strong>of</strong> Antigen-specific <strong>Oral</strong> Tolerance<br />
by Genetically Modified Lactococcus Lactis<br />
Delivering DQ8-specific Immunodominant Gliadin<br />
Epitopes to Gluten-sensitized Class II Transgenic<br />
Mice<br />
Inge Huibregtse, Academic Medical Center, Amsterdam,<br />
Eric Marietta, Mayo Clinic, Department <strong>of</strong> <strong>Immunology</strong>,<br />
Rochester, MN, Shadi Rashtak, Drs, Mayo Clinic,<br />
Department <strong>of</strong> <strong>Immunology</strong>, Rochester, MN, Chella David,<br />
Mayo Clinic, Department <strong>of</strong> <strong>Immunology</strong>, Rochester, MN,<br />
Pieter Rottiers, VIB, Department for Molecular Biomedical<br />
Research, Zwijnaarde, Ghent, Sander van Deventer,<br />
Pr<strong>of</strong>essor Academic Medical Center, Center for<br />
Experimental and Molecular Medicine, Amsterdam,<br />
Joseph Murray, Mayo Clinic, Department <strong>of</strong> <strong>Immunology</strong>,<br />
Rochester, MN<br />
Antigen-specific immune suppression is an attractive<br />
therapy for the treatment <strong>of</strong> several gastro-intestinal<br />
diseases especially celiac disease. Active delivery <strong>of</strong><br />
recombinant autoantigens or allergens at the intestinal<br />
mucosa by genetically modified Lactococcus lactis (LL)<br />
provides a novel therapeutic approach for the induction<br />
<strong>of</strong> tolerance. For this purpose we genetically engineered<br />
deamidated DQ8 epitope secreting LL (LL-eDQ8d) and<br />
evaluated local and systemic immune responses in glutensensitized<br />
NOD ABo DQ8 class II transgenic mice after oral<br />
supplementation. Mice were sensitized with either 10 1/4g<br />
deamidated DQ8 epitopes or 50 ng pertussis toxin and 100<br />
1/4 g crude gluten and fed for 10 or 21 days, respectively<br />
with LL-pT1NX (empty vector) or LL-eDQ8d (1×10 9 CFU/<br />
day). Tolerance induction was assessed by DTH responses,<br />
ex vivo proliferation assays and cytokine measurements.<br />
Daily intragastric administration <strong>of</strong> LL-eDQ8d in sensitized<br />
transgenic mice led to a significant decrease in DTH response<br />
and proliferative capacity <strong>of</strong> the splenocytes and inguinal<br />
lymph node cells compared to the negative controls. LLpT1NX<br />
was also able to reduce the DTH response and the<br />
proliferation <strong>of</strong> splenocytes although not as much as the LLeDQ8d.<br />
Moreover, LL-eDQ8d significantly reduced the IFN- 3<br />
production in the draining lymph nodes and the spleen<br />
compared to the LL-pT1NX. Mucosal delivery <strong>of</strong> immunodominant<br />
gliadin epitopes by genetically modified LL induces<br />
suppression <strong>of</strong> local and systemic specific T cell responses in<br />
NOD ABo DQ8 transgenic mice. Moreover our data provide<br />
promise for the development <strong>of</strong> effective therapeutics for<br />
treatment <strong>of</strong> several common autoimmune, inflammatory
S54 Abstracts<br />
and/or allergic diseases by autoantigen- and/or allergensecreting<br />
L. lactis.<br />
doi:10.1016/j.clim.2007.03.331<br />
F.120 Evaluation <strong>of</strong> an Enhanced-sensitivity<br />
Interferon-γ ELISpot Assay Kit Using Cryopreserved<br />
PBMC Donor Bank Panels<br />
Keith Moskowitz, Director, SeraCare Life Sciences,<br />
Gaithersburg, MD, Janet Lathey, Director, Virology/<br />
<strong>Immunology</strong>, Gaithersburg, MD, Lisa Jeffrey, Research<br />
Associate, Virology/<strong>Immunology</strong>, Gaithersburg, MD, Scott<br />
Hickman, Product Manager, Marketing, Gaithersburg, MD,<br />
Rodshawn Branch, Research Associate, SeraCare Life<br />
Sciences, Gaithersburg, MD, Dmitry Moshk<strong>of</strong>f, Project<br />
Scientist, Virology/<strong>Immunology</strong>, Gaiithersburg, MD, Mark<br />
Manak, CSO, SeraCare, Gaithersburg, MD<br />
Human interferon-gamma (IFN-γ) ELISpot assays detect<br />
single IFN-γ secreting cells. We compared a novel ELISpot<br />
(SC) to a commercial kit (CK). Three lots <strong>of</strong> SC and one <strong>of</strong> CK<br />
were evaluated using PHA, CEF, CMV and mock stimulation <strong>of</strong><br />
cryopreserved PBMC from 24 independent donors. In<br />
response to PHA, SC and CK showed significant differences<br />
between frequency distributions (KS 0.36). SC median was<br />
479, 25th–75th percentiles (quartiles), and 275–975 SFC/<br />
well. The CK median was 200 (quartiles 45–343). Means were<br />
also different: SC=474, CK=152, Pb0.0001. Average CVs<br />
were 11% for SC and 18% for CK. For CEF stimulus, SC median<br />
was 64 (quartiles 27–179), mean was 129, CV 20%; CK median<br />
was 54 (quartiles 23–174), mean 139, and CV 28%. CMV<br />
stimulus median for SC was 100 (quartiles 12–200), mean<br />
149, and CV 27%; CK median was 87 (quartiles 7–222), mean<br />
112, and CV 38%. All backgrounds averaged b2. SC lots<br />
performed equivalently with all agonists. Some CK wells<br />
were unreadable (30/216), while 100% <strong>of</strong> 648 SC wells gave<br />
SFC values, Pb0.000001. CK averaged ∼14% failures, with no<br />
readings in 4 <strong>of</strong> 24 subjects. Thus, SeraCare has validated an<br />
IFN-γ ELISpot kit demonstrating superior performance in<br />
response to PHA, lower CV to all agonists and statistically<br />
fewer well failures relative to a commercial kit.<br />
doi:10.1016/j.clim.2007.03.332<br />
F.121 Involvement <strong>of</strong> Hsp90β but Not Hsp90α in<br />
Anti-Apoptotic Effect <strong>of</strong> CpG-B ODN<br />
Cheng-Chin Kuo, Postdoctoral Fellow, Agricultural<br />
Biotechnology Research Center, Academia Sinica, Taipei,<br />
Chi-Ming Liang, Pr<strong>of</strong>essor, Institute <strong>of</strong> Biological Chemistry,<br />
Academia Sinica, Taipei, Chen-Yen Lai, Research Assistant,<br />
Agricultural Biotechnology Research Center, Academia<br />
Sinica, Taipei, Shu-Mei Liang, Pr<strong>of</strong>essor, Agricultural<br />
Biotechnology Research Center, Academia Sinica, Taipei<br />
Unmethylated CpG oligodeoxynucleotides (CpG ODNs)<br />
activate immune responses in a toll-like receptor 9 (TLR9)dependent<br />
manner. In this study, we found that stimulation<br />
<strong>of</strong> mouse macrophages and dendritic cells with B-type CpG<br />
ODN (CpG-B ODN) increased cellular heat shock protein<br />
(Hsp) 90β but not Hsp90α and prevented apoptosis induced<br />
by serum starvation or staurosporine treatment. The CpG-B<br />
ODN-induced Hsp90β expression depended on TLR9, myeloid<br />
differentiation factor 88 and PI3K. Inhibition <strong>of</strong> Hsp90β<br />
by expressing small interfering RNA suppressed not only<br />
Hsp90β expression but also PI3K-dependent phosphorylation<br />
<strong>of</strong> Akt and CpG-B ODN-mediated anti-apoptosis. Additional<br />
studies demonstrated that as described by other group,<br />
Hsp90β but not Hsp90α was associated with Bcl-2. Inhibition<br />
<strong>of</strong> Hsp90β suppressed the CpG-B ODN-induced association <strong>of</strong><br />
Hsp90β with Bcl-2 and impaired the inhibitory effect <strong>of</strong><br />
CpG-B ODN on the release <strong>of</strong> cytochrome c as well as the<br />
activation <strong>of</strong> caspase-3. This study thus reveals the<br />
involvement <strong>of</strong> Hsp90β but not Hsp90α in CpG-B ODNmediated<br />
anti-apoptotic responses and shows that Hsp90β is<br />
distinct from Hsp90α in its regulation <strong>of</strong> the cellular<br />
function <strong>of</strong> immune cells.<br />
doi:10.1016/j.clim.2007.03.333<br />
F.122 Immunoassays for Detection <strong>of</strong> Soluble Fc<br />
Alpha Receptor (CD89) in Plasma <strong>of</strong> IgA<br />
Nephropathy Patients<br />
Mirjana Hahn-Zoric, Research and Development Engineer,<br />
Sahlgrenska University Hospital, <strong>Clinical</strong> <strong>Immunology</strong>,<br />
Gothenburg, Sweden, Neda Tahmasebifar, Student,<br />
Sahlgrenska University Hospital, <strong>Clinical</strong> <strong>Immunology</strong>,<br />
Gothenburg, Sweden, Cees van Kooten, Department <strong>of</strong><br />
Nephrology, Leiden, The Netherlands, Sweden, Jarl Ahlmen,<br />
Nephrology Clinics, Skövde, Per-Anton Westerberg,<br />
Nephrology Clinics, Skövde, Sweden, Svante Swerkersson,<br />
Pediatric Uro-Nephrologic Center, Gothenburg, Sweden,<br />
Sverker Hansson, Pediatric Uro-Nephrologic Center,<br />
Gothenburg, Sweden, Ulla Berg, Karolinska University<br />
Hospital, Stockholm, Sweden, Lars Åke Hanson, Pr<strong>of</strong>essor<br />
Emeritus, Sahlgrenska Academy, Gothenburg University,<br />
<strong>Clinical</strong> <strong>Immunology</strong>, Gothenburg, Sweden, Leonid<br />
Padyukov, Senior Researcher, Rheumatology Unit,<br />
Department <strong>of</strong> Medicine, Stockholm, Sweden, Stefan H.<br />
Jacobson, Department <strong>of</strong> Nephrology, Stockholm, Sweden<br />
Background: Fc±RI, or CD89, is a receptor binding the Fc<br />
portion <strong>of</strong> IgA. Recently it has been shown that CD89 is involved<br />
in IgA–immune complex formation. CD89–IgA complexes were<br />
suggested to be pathogenic in several diseases, one <strong>of</strong> which is<br />
IgA nephropathy (IgAN). IgAN is characterized by increased<br />
serum IgA levels paralleled by the deposition <strong>of</strong> IgA in renal<br />
tissue. IgAN is the most common form <strong>of</strong> glomerulonephritis in<br />
the Western world and is the second most common cause <strong>of</strong><br />
dialysis or kidney transplantation after diabetes. The pathogenesis<br />
and the mechanisms <strong>of</strong> this disease are not well understood<br />
and there are no reliable prognostic factors that can predict<br />
which patients are <strong>of</strong> high risk to develop severe kidney damage.<br />
Aim <strong>of</strong> this study was to develop immunoassay(s) for detection <strong>of</strong><br />
soluble IgA receptor (sCD89) in human plasma. Methods and<br />
material: By using CD89-specific MIP8a and A3 monoclonal<br />
antibodies and polyclonal anti-CD89 IgG, a Western blot method<br />
as well as a direct and an indirect ELISA for quantification <strong>of</strong><br />
sCD89 were developed. 45 IgAN patients (32 adults and 13
Abstracts<br />
teenagers) and 45 matched healthy controls were analyzed.<br />
Results: A single sCD89 band <strong>of</strong> around 30 kDa was identified in<br />
Western blot with polyclonal IgG. Significantly higher levels <strong>of</strong><br />
sCD89 were found in adult IgAN patients compared to controls in<br />
monoclonal antibody-based ELISA. Conclusion: We developed<br />
assays that provide helpful tools for detection <strong>of</strong> sCD89, which<br />
may be a useful diagnostic marker for further studies <strong>of</strong> IgAN.<br />
doi:10.1016/j.clim.2007.03.335<br />
F.124 Resistin Like Molecule Beta Regulates<br />
Intestinal Inflammation in Chronic Colitis<br />
Luqman Seidu, Allergy/<strong>Immunology</strong> Fellow, Cincinnati<br />
Children’s Hospital, Cincinnati, OH, Elizabeth Forbes, PhD<br />
Student, Cincinnati Children’s Hospital, Cincinnati, OH,<br />
Richard Aherns, Rheumatoid Arthritis, Cincinnati Children’s<br />
Hospital, Cincinnati, OH, Margaret Karow, PhD, Regeneron<br />
Pharma, Tarrytown, NY, Carine Blanchard, PhD, Cincinnati<br />
Children’s Hospital, Cincinnati, OH, Andrew Murphy, PhD,<br />
Regeneron Pharma, Tarrytown, NY, David Valenzuela, PhD,<br />
Regeneron Pharma, Tarrytown, NY, George Yancopoulos,<br />
MD/PhD, Regeneron Pharma, Tarrytown, NY, Marc<br />
Rothenberg, MD/PhD, Cincinnati Children’s Hospital,<br />
Cincinnati, OH, Simon Hogan, PhD, Cincinnati Children’s<br />
Hospital, Cincinnati, OH<br />
ResistinlikemoleculeRELM-β, a member <strong>of</strong> resistin family,<br />
has been implicated in glucose metabolism and intestinal<br />
immunity. We demonstrate that RELM-β is constitutively<br />
expressed in the colonic epithelium and is upregulated during<br />
chronic intestinal inflammation. To examine the contribution<br />
<strong>of</strong> RELM-β to chronic intestinal inflammation, we utilized<br />
RELM-β deficient mice and models <strong>of</strong> colitis. We show that<br />
RELM-β deficient mice have decreased susceptibility to<br />
chemical (DSS [dextran sodium sulfate])-induced colitis<br />
(disease activity index, 1.53±1.53 vs. 7.37±2.4 respectively<br />
mean±SE; survival at day 8, 100% vs. 75% respectively n=4–<br />
5 mice per group). We show that the link between colitis<br />
susceptibility and RELM β was associated with the expression<br />
<strong>of</strong> the NFkappaB signaling inhibitor, REG3β. Moreover,<br />
REG3β mRNA expression was increased 500-fold in DSStreated<br />
RELM-β deficient mice vs. wild type mice (616.8±<br />
138.1 vs. 26.35±11.69, fold increase [REG3β expression/<br />
GAPDH ratio], respectively n=4–5 mice per group, mean±<br />
SE). Collectively this work suggests an interaction among<br />
RELM-β, REG3β and intestinal inflammation.<br />
doi:10.1016/j.clim.2007.03.336<br />
F.125 Immune Parameters in Normal Humans with<br />
Different Behavior Patterns: Type A and B Behavior<br />
Pattern<br />
Fereshteh Alesahebfosoul, Academic Member <strong>of</strong> Isfahan<br />
University, Medical School, <strong>Immunology</strong> DEP, Isfahan,<br />
Bahareh Zzarkeshfard, Food Science Engineering, Health<br />
Faculty, Isfahan, Farzad Oreizi, Immunologist, Medical<br />
School, <strong>Immunology</strong> DEP, Isfahan, Minoo Adib, Pr<strong>of</strong>essor,<br />
medical school, <strong>Immunology</strong> DEP, Isfahan<br />
Introduction: Numerous studies have described various<br />
aspects <strong>of</strong> behavioral influences on immune function. Behavior<br />
and emotions modulate immune function. In this study<br />
distinctive immunological findings in normal humans with type<br />
A and B behavior pattern were described. Method: In cross<br />
sectional study 105 participants (P) admitted to be in the first<br />
phase (20–30 years old). 6 Ps were excluded because <strong>of</strong><br />
abnormal CBC. We used standard questioner (Bortne scale) to<br />
separate type A and B behavior patterns. The samples <strong>of</strong><br />
peripheral blood were referred to immunology laboratory for<br />
measurement <strong>of</strong> immune factors. SPSS (ver.10) and t test were<br />
used in statistical analysis. Results: 40 Ps were type A and 59<br />
type B. There were no significant differences between two<br />
groups on serum B cell, T cell, and NK cell. There was a<br />
significant increase (P valueb0.05) on serum CD4 positive Tcells<br />
in type A but no significant increase was viewed in CD8 positive T<br />
cells. There were significant increases on lymphocytes and<br />
granulocytes in type A. It was significant in male Ps with type A.<br />
CD4 positive T cells increased in counter female with type B,<br />
however no significant difference was seen in male with type A<br />
in contrast to female. Discussion: The finding <strong>of</strong> this study was<br />
that type A behavior pattern with specific characteristics (time<br />
emergency) has no significant effect on immune system and the<br />
effect <strong>of</strong> gender <strong>of</strong> immune parameters has to be considered.<br />
doi:10.1016/j.clim.2007.03.337<br />
S55<br />
F.126 Multi-Parameter Analysis (MPA) <strong>of</strong> Cells and<br />
Serum Using a Standard Flow Cytometer<br />
Robert Hallam, Senior Biomedical Scientist, Papworth<br />
Hospital, <strong>Immunology</strong> Department, Cambridgeshire,<br />
England, Paul Balmer, <strong>Clinical</strong> Scientist, HPA, Vaccine<br />
Evaluation Department, Manchester, England, Ray Borrow,<br />
<strong>Clinical</strong> Scientist, HPA, Vaccine Evaluation Department,<br />
Manchester, England, Andrew Exley, Consultant<br />
Immunologist, Papworth Hospital/<strong>Immunology</strong> Department,<br />
Cambridge, England<br />
Background: Flow cytometric peripheral blood MPA identifies<br />
cell populations by size, granularity, and expression pattern<br />
<strong>of</strong> cell surface or intracellular antigens using fluorophore<br />
conjugated Abs. Alternative platforms for serum MPA measure<br />
concentrations <strong>of</strong> secreted proteins including cytokines and<br />
pathogen specific Abs in multiplex, liquid phase capture assays<br />
using antigen or antibody coated beads identified by size,<br />
fluorescence intensity and wavelength. Fresh blood samples<br />
preferred for cellular analysis generate peaks and troughs in<br />
workload contrasting with batch analysis <strong>of</strong> sera. Methods: B<br />
cell populations were defined by CD45/ss pan-leucogating, then<br />
differential expression <strong>of</strong> CD19, CD27, IgD or IgM using a<br />
Beckman-Coulter FC-500 flow cytometer. 10-plex serotype<br />
specific anti-pneumococcal antibody assays were established<br />
using carboxylated microspheres coated with pneumococcal<br />
capsular polysaccharide, CPS and 22F absorption, PE-conjugated<br />
mouse anti-human IgG/IgM, and reference standard 89SF.<br />
Results: These serum MPAs have 2–3 log dynamic linear ranges,<br />
b6% intraassay and 16% interassay CVs, 1.88 ng/ml median<br />
limits <strong>of</strong> quantification, and correlate well with Bioplex systems<br />
and ELISAs. The complex data set is presented as an assay
S56 Abstracts<br />
report highlighted by a “traffic light system” and pictorial<br />
patient reports in MS-WORD, after analysis by especially<br />
designed s<strong>of</strong>tware incorporating MS-EXCEL macros enhanced<br />
by Visual Basic programming. B cell phenotyping correlated<br />
with pre versus post-immunisation serum pneumococcal antibody<br />
levels in cases investigated for primary and secondary<br />
immunodeficiency. Discussion: MPA <strong>of</strong> cells and serum on a<br />
single standard flow cytometer with powerful s<strong>of</strong>tware applies<br />
existing expertise and resources in a flexible cost-effective<br />
approach suitable for high-throughput testing and clinical<br />
laboratories.<br />
doi:10.1016/j.clim.2007.03.338<br />
F.127 Catalytic Anti-dsDNA Activity from SLE Sera is<br />
Associated with Remission Phases <strong>of</strong> the Disease<br />
Genevieve Servais, Responsible Autoimmunity LAB, Canada,<br />
Canada, CHU Brugmann, <strong>Immunology</strong>, Bruxelles, Belgium,<br />
Mimouna El Baz, Pharmacist, CHU Brugmann <strong>Immunology</strong>,<br />
Bruxelles, Belgium, Rafik Karmali, MD, CHU Brugmann<br />
Internal Medicine, Bruxelles, Belgium, Marie Paule<br />
Guillaume, MD, CHU Brugmann Internal Medicine,<br />
Bruxelles, Belgium, Jean Duchateau, MD PHD, CHU<br />
Brugmann <strong>Immunology</strong>, Bruxelles, Belgium<br />
SLE is characterized by the presence <strong>of</strong> circulating anti-<br />
DNA antibodies, one <strong>of</strong> the eleven ACR criteria for the<br />
classification <strong>of</strong> SLE, and they play an important role in the<br />
pathogenesis <strong>of</strong> SLE. Anti-DNA antibodies display different<br />
specificities, some being restricted to nuclear double<br />
stranded DNA (dsDNA), or single stranded DNA (ssDNA), or<br />
membrane associated DNA (mDNA) or nucleosomes. Some<br />
are sharing dual specificities. Autoantibodies displaying a<br />
catalytic activity, abzymes, were reported in patient’s sera,<br />
including catalytic anti-DNA antibodies. They have not been<br />
reported in healthy control subjects. Catalytic activity varies<br />
in different pathologies and changes during the different<br />
phases <strong>of</strong> the disease. The biological role <strong>of</strong> the catalytic<br />
anti-DNA present in serum <strong>of</strong> SLE is complex, as it seems that<br />
some may play positive and others negative roles, from a<br />
clearance role to a lowering <strong>of</strong> DNAse activity in SLE<br />
serum. We show here that circulating catalytic anti-DNA is<br />
found in SLE and RA patients but also in healthy controls<br />
though with a higher percentage in SLE than in controls,<br />
where they can be unraveled when the sera are diluted<br />
at 1/10. Their presence is inversely correlated with the<br />
clinical activity <strong>of</strong> the disease and with the titer <strong>of</strong><br />
circulating autoantibodies to dsDNA, mDNA but not antinucleosomes<br />
antibodies.<br />
doi:10.1016/j.clim.2007.03.339<br />
F.128 Fungal Hypersensitivity is Not Type I (IgE)<br />
Allergy<br />
Vincent A. Marinkovich, MD, Inc., Redwood City, CA<br />
One hundred adult patients undergoing clinical evaluations<br />
for symptoms developing during chronic exposure to<br />
high ambient fungal antigen levels in water damaged<br />
homes were tested for specific IgE (RAST) and specific IgG<br />
(ELISA) levels to a number <strong>of</strong> fungi commonly found in<br />
mold-amplified environments. The tests were standard<br />
runs in a State licensed Physician’s Office Laboratory<br />
using test materials provided by Hitachi Chemical Diagnostics<br />
for specific IgE and specific IgG in a multi antigen<br />
panel format. The panels contain aspergillus, penicillium,<br />
alternaria, cladosporium and mucor. Results read as<br />
positive when greater than 2 standard deviations greater<br />
than the mean using reference ranges previously determined<br />
by Hitachi. Seven patients had elevated specific<br />
IgE levels to at least one <strong>of</strong> the fungi tested (7%). Eighty<br />
nine patients showed elevated IgG titers to at least one<br />
<strong>of</strong> the molds. The sera were then exposed to an<br />
additional eight common molds, botrytis, candida, epicoccum,<br />
fusarium, helminthosporium, pullularia, rhizopus<br />
and stemphylium, on an IgG panel and all patients (100%)<br />
showed elevated titers to at least one <strong>of</strong> the molds. The<br />
common symptoms reported were remarkably consistent<br />
among patients: fatigue, nasal congestion, headaches,<br />
cough, memory loss and chronic flu-like symptoms. All the<br />
patients showed signs <strong>of</strong> chronic rhinosinusitis suggesting<br />
fungal colonization and eosinophilic degranulation. Systemic<br />
symptoms are likely the result <strong>of</strong> circulating<br />
immune complexes under conditions <strong>of</strong> functional asplenia<br />
(i.e., IC overload). The neurological symptoms may be the<br />
result <strong>of</strong> local uptake <strong>of</strong> eosinophilic neurotoxin via the<br />
cribriform plate.<br />
doi:10.1016/j.clim.2007.03.340<br />
F.129 Automation for Isolation <strong>of</strong> Peripheral Blood<br />
Mononuclear Cells (PBMC)<br />
Carlos Aparicio, Scientist, Beckman Coulter, Inc., Miami, FL,<br />
Enrique Rabelli, Director, Beckman Coulter, Inc., Miami, FL,<br />
Edward Jachimowicz, Scientist, Beckman Coulter, Inc.,<br />
Miami, FL, Sybil D’Costa, Scientist, Beckman Coulter, Inc.,<br />
Miami, FL, Mahsa Aspsater, Scientist, Beckman Coulter,<br />
Inc., Miami, FL, Julie Wilkinson, Scientist, Beckman<br />
Coulter, Inc., Miami, FL, Wade Bolton, VP, Beckman<br />
Coulter, Inc., Miami, FL, Enrique Rabelli, Director,<br />
Beckman Coulter, Inc., Miami, FL<br />
Peripheral blood mononuclear cells (PBMC) are considered<br />
appropriate target populations to immunologically<br />
assess the various subsets <strong>of</strong> blood lymphocytes. Purification<br />
<strong>of</strong> PBMC by density gradient centrifugation is a<br />
laborious and time consuming manual process and, thus,<br />
limiting factor for processing large number specimens.<br />
Generally, the media–gradient interface is generated by<br />
carefully pipetting the blood cell suspension onto a<br />
density gradient solution without disturbing the interface.<br />
Disruption <strong>of</strong> the interface can jeopardize the overall<br />
yield, purity and viability <strong>of</strong> the mononuclear cell<br />
preparation. The study described herein focuses on an<br />
alternative semi-automated strategy to harvest PBMC<br />
using the Ficoll-Paque density gradient. This process<br />
integrates the use <strong>of</strong> an automated liquid handler and a<br />
centrifuge to minimize the number <strong>of</strong> steps and time
Abstracts<br />
requiring operator intervention in current laboratory<br />
procedures. PBMC harvested by this automated process<br />
had a viability over 95%, and when compared to the<br />
manual procedure showed: (1) cell recovery (N95%) with<br />
no selective loss as judged by phenotyping and functional<br />
response by the various lymphocytes. The goal <strong>of</strong> this<br />
work is to provide a fully automated and standardized<br />
method for preparing PBMC that can facilitate support<br />
cell based-assay testing strategies in clinical trials by<br />
improving overall quality and efficiency <strong>of</strong> the process as<br />
well as reducing labor and cost.<br />
doi:10.1016/j.clim.2007.03.341<br />
F.130 Interleukin 32 Expression is Associated with a<br />
Unique Subset <strong>of</strong> Human Dendritic Cells Receptive<br />
to the Venezuelan Equine Encephalitis<br />
Virus-derived Replicon Vector<br />
Kevin Nishimoto, Graduate student, University <strong>of</strong> California<br />
Irvine, Department <strong>of</strong> Microbiology and Biochemistry,<br />
Irvine, CA, Charles Dinarello, Pr<strong>of</strong>essor <strong>of</strong> Medicine,<br />
University <strong>of</strong> Colorado Health Sciences Center, Department<br />
<strong>of</strong> Medicine, Denver, CO, Stephen Hou, PhD, University <strong>of</strong><br />
California Irvine, Department <strong>of</strong> Medicine, Division <strong>of</strong><br />
Hematology/Oncology, School <strong>of</strong>, Irvine, CA, Edward<br />
Nelson, Assistant Pr<strong>of</strong>essor <strong>of</strong> Medicine, University <strong>of</strong><br />
California Irvine Department <strong>of</strong> Medicine, Division <strong>of</strong><br />
Hematology/Oncology, School <strong>of</strong> Medicine, Irvine, CA<br />
Establishing successful immunotherapeutic strategies<br />
utilizing dendritic cells (DC), the most potent antigen<br />
presenting cell, largely depends on understanding the<br />
requirements necessary for optimal DC function. We and<br />
others have reported that the Venezuelan Equine Encephalitis<br />
(VEE) replicon particle (VRP) vector system has<br />
conserved tropism for murine, rat, and human DCs.<br />
However the DC subset(s) and functional capacities are<br />
not fully understood. The VRP vector system has in vitro<br />
tropism for a subset <strong>of</strong> human immature monocytederived<br />
DCs (M-DCs); therefore, we developed a novel<br />
technique to isolate this VRP receptive DC subset for<br />
further characterization. An evaluation between VRP<br />
receptive and non-receptive M-DC subsets by microarray<br />
revealed distinct differences in gene expression pr<strong>of</strong>iles<br />
with N700 significantly expressed genes (0.001 e P value,<br />
0.98 d PPDE) between cellular subsets. Notably in the<br />
receptive DC population, the NK4 transcript was significantly<br />
increased and accompanied by protein expression.<br />
The NK4 gene has recently been identified as encoding a<br />
recently described cytokine, interleukin 32. These data<br />
provide the initial characterization <strong>of</strong> a new subset <strong>of</strong><br />
myeloid DCs targeted by this vector, and provide insights<br />
into human DC biology allowing explicit study <strong>of</strong> the<br />
mechanisms employed by this exceptionally potent immunotherapeutic<br />
vector system.<br />
doi:10.1016/j.clim.2007.03.342<br />
F.131 Retinoic Acid Promotes Development <strong>of</strong><br />
Human Macrophages Over Dendritic Cell<br />
Differentiation<br />
Juliana Sousa-Canavez, Researcher, Genoa Biotech, São<br />
Paulo, Brazil, Cristina Massoco, Researcher, Genoa<br />
Biotech, São Paulo, Brazil, Elaine Corneta, Student,<br />
Genoa Biotech, São Paulo, Brazil, Katia Leite, Pathologist,<br />
Genoa Biotech, São Paulo, Brazil, Tatiane Marisis, Student,<br />
Genoa Biotech, São Paulo, Brazil, Luiz Camara-Lopes,<br />
Pathologist, Genoa Biotech, São Paulo, Brazil, Antonio<br />
Misiara, MD, Genoa Biotech, São Paulo, Brazil, Dewton<br />
Moraes-Vasconcelos, Pr<strong>of</strong>essor, Genoa Biotech, São Paulo,<br />
Brazil<br />
Previous studies showed that all vitamins including<br />
vitamin A are fundamental for immune system properly<br />
functioning. All trans retinoic acid (atRA) at physiological<br />
or near-physiological concentrations appears to affect<br />
Th1–Th2 differentiation and it is believed that effects<br />
<strong>of</strong> retinoids on immune responses might also be mediated<br />
by dendritic cells (DC). Nonetheless, effects <strong>of</strong> atRA in DC<br />
differentiation have been showing contradictory results<br />
since it was observed either induction or inhibition <strong>of</strong><br />
differentiation. Our work shows effects <strong>of</strong> atRA on human<br />
monocyte derived DC by phenotype and phagocytosis<br />
studies. DC (positive control group) was differentiated<br />
with cytokines (GM-CSF and IL-4) and maturation induced<br />
by TNF-α. We have tested pharmacological (10 1/4M and<br />
1 1/4M) and physiological (10 nM) doses <strong>of</strong> atRA with or<br />
without cytokines. Cell phenotype was analyzed by flow<br />
cytometry using CD80, CD86 and CD14 antibodies. We also<br />
investigated functional performance analyzing phagocytosis<br />
and respiratory burst (ROS) in all culture conditions<br />
mentioned above. Our data demonstrate that effects <strong>of</strong><br />
atRA depend on the dose used, as pharmacological doses<br />
inhibited expression <strong>of</strong> all phenotypic markers tested,<br />
while a physiological one caused cell differentiation.<br />
Physiologic concentration <strong>of</strong> atRA especially in combination<br />
with GM-CSF blocked differentiation <strong>of</strong> DC population<br />
corroborating a macrophage preferable transformation.<br />
Moreover phagocytosis and ROS were approximately three<br />
times greater in cells cultivated with atRA compared to<br />
positive control group. On the other hand, IL-4 and atRA<br />
apparently induced differentiation to an immature DC<br />
phenotype when TNF-α stimulus was added.<br />
doi:10.1016/j.clim.2007.03.343<br />
S57<br />
F.132 OTC Allergy Testing<br />
Chris Brown, ImmuneTech, Inc., Menlo Park, CA, Vincent A.<br />
Marinkovich, MD, Inc. Redwood City, CA<br />
Quantitative, comprehensive in vitro, specific IgE tests<br />
for allergy have not hitherto been introduced into the<br />
over the counter (OTC) market because <strong>of</strong> their high costs<br />
and the need for a large sample <strong>of</strong> blood requiring a<br />
venapuncture. Recently the U.S. Food and Drug Administration<br />
cleared for OTC distribution a new allergenspecific<br />
IgE test which has two major advantages over
S58 Abstracts<br />
its predecessors: low blood volume and economy. This<br />
new system, utilizing a flow cytometry platform, is<br />
currently configured to provide quantitative specific IgE<br />
levels to each <strong>of</strong> ten allergens simultaneously. It utilizes<br />
just 5 μl <strong>of</strong> capillary or venous serum in a homogenous<br />
assay, and can be completed in under 3 h. The system<br />
compares favorably to the current laboratory testing<br />
methods:<br />
Sensitivity/specificity/accuracy data in %:<br />
– Mountain cedar 88/93/90<br />
– Timothy grass 94/97/90<br />
– Bermuda grass 86/97/91<br />
– Short ragweed 90/95/92<br />
– House dust mite 96/93/95<br />
– Cat hair 96/89/93<br />
– Cow’s milk 81/92/89<br />
– Egg 80/94/92<br />
– Wheat 85/93/90<br />
– Alternaria 87/94/90<br />
The coefficient <strong>of</strong> variation between test runs on<br />
different days ranged from 3.4 to 7.2% and within the<br />
same day from 2.9 to 8.3%. The test’s lowest level <strong>of</strong><br />
detection is 0.35 IU/ml. This accurate, low cost, low blood<br />
volume test with fingerstick convenience can expand the<br />
availability <strong>of</strong> accurate allergy diagnosis to a patient<br />
population not currently served by existing in vitro systems<br />
or by skin testing.<br />
doi:10.1016/j.clim.2007.03.344<br />
F.133 Standardization and Quality Assurance <strong>of</strong><br />
Cytokine Flow Cytometry Assays<br />
Maria Jaimes, Scientist, BD Biosciences, San Jose, CA,<br />
Janice Darden, CRS Team Leader, DAIDS/NIAID/NIH,<br />
Bethesda, MD, Patricia D’Souza, Program Officer, DAIDS/<br />
NIAID/NIH, Bethesda, MD, Holden Maecker, Group<br />
Manager-Immune Function, BD Biosciences, San Jose, CA<br />
Cytokine flow cytometry (CFC) is used to measure<br />
antigen-specific T cell responses in settings such as<br />
experimental HIV vaccination. Standardization <strong>of</strong> CFC<br />
among different laboratories would provide a common<br />
endpoint assay for comparing the immunogenicity <strong>of</strong><br />
vaccine candidates in clinical trials. For this purpose, a<br />
Quality Assurance Program has been developed in a<br />
collaborative effort between BD Biosciences, the Division<br />
<strong>of</strong> AIDS, NIAID, NIH and SeraCare BioServices, in which 15<br />
sites currently participate. Three rounds <strong>of</strong> the program<br />
have been completed. In each round, IFN-ƒ× and IL-2<br />
responses in CD4+ and CD8+ T cells to CEF or CMV pp65<br />
lyophilized peptide mixes were tested using cryopreserved<br />
PBMC from CMV+ donors. Each site analyzed its data<br />
independently and provided, through the program’s<br />
website (http://bdicsqa.webbasix.com), their raw data<br />
for centralized analysis. In addition, the data generated<br />
by each laboratory were compared to a standard. The<br />
first two rounds <strong>of</strong> the program yielded inter-laboratory<br />
variability from 12 to 60%, for cytokine+ responses higher<br />
than 0.2%. Instrument setup and analysis (gating strategy)<br />
played a critical role in this variation. This variability was<br />
decreased in the third round by providing feedback to the<br />
laboratories, guidelines for data analysis, and a protocol<br />
and reagents for instrument setup. Of note, the interlaboratory<br />
variability (18–32%) was comparable to the<br />
intra-laboratory variability (4–25%) in the third round.<br />
Critical to success was the provision <strong>of</strong> standard operating<br />
procedures, centrally supplied reagents, and laboratory<br />
adherence to GCLP. The implementation <strong>of</strong> this Quality<br />
Assurance Program will ensure greater uniformity <strong>of</strong> data<br />
from multiple laboratories.<br />
doi:10.1016/j.clim.2007.03.345<br />
F.134 A Novel Cell Contact-dependent Requirement<br />
for Human CD40 Ligand Expression<br />
Jack Ragheb, Senior <strong>Clinical</strong> Investigator, Laboratory <strong>of</strong><br />
<strong>Immunology</strong>, NEI, NIH, Bethesda, MD, James Snyder,<br />
Research Fellow, NIH, Bethesda, MD, Jijia Shen, Research<br />
Fellow, NIH, Bethesda, MD, Paul Hu, Research Fellow, nIH,<br />
Bethesda, MD, Hooman Azmi, Research Fellow, NIH,<br />
Bethesda, MD, Qian Chen, Research Fellow, NIH, Bethesda,<br />
MD<br />
CD40L (CD154), an inducible costimulatory molecule<br />
expressed on CD4+ T cells, plays an essential role in the<br />
activation <strong>of</strong> B cells. Through the CD40L mediated<br />
induction <strong>of</strong> IL-12 and upregulation <strong>of</strong> MHC class II,<br />
CD80/86, and other accessory molecules on antigen<br />
presenting cells (APC), CD40L also catalyzes a positive<br />
feedback loop for T cell activation. Despite the pivotal<br />
juxtaposition <strong>of</strong> CD40L between APC and T cell activation,<br />
it is thought that only a TCR stimulus is required to<br />
initiate early CD40L expression. We demonstrate that<br />
unlike its mouse counterpart, human CD40L is not<br />
constitutively expressed on circulating T cells, nor do<br />
human CD4+ T cells contain an intracellular pool <strong>of</strong> CD40L<br />
protein. While a TCR stimulus is necessary for CD40L<br />
expression on primary human T cells, we show for the<br />
first time that expression <strong>of</strong> CD40L is highly dependent<br />
upon a cell–cell interaction with CD14hi monocytes that<br />
does not require LFA-3, ICAM-1, or CD80/86. Like CD28dependent<br />
IL-2 expression, this monocyte signal upregulates<br />
the transcription <strong>of</strong> CD40L. Thus the inducible<br />
expression <strong>of</strong> CD40L on CD4+ T cells, which provides a<br />
critical costimulatory signal for monocyte and B cell<br />
activation, is itself dependent upon a costimulatory signal<br />
delivered by monocytes but not B cells. The latter<br />
finding implies that a human B cell cannot activate its<br />
cognate helper T cell to deliver CD40L-mediated help.<br />
Therapeutics targeted at this monocyte costimulatory<br />
factor for CD40L may lead to new means <strong>of</strong> modulating<br />
the immune response and inducing long-term graft<br />
acceptance.<br />
doi:10.1016/j.clim.2007.03.346
Abstracts<br />
F.135 Endocytosis <strong>of</strong> Hsp-chaperoned Proteins and<br />
Peptides is Essential for MHC II Dependent Peptide<br />
Presentation<br />
Irena Straub, Medical Candidate, University Children’s<br />
Hospital Tübingen, Tuebingen, Markus Haug, University<br />
Children’s Hospital Tübingen, Tuebingen, Dorothee Wernet,<br />
Pr<strong>of</strong>essor, Department <strong>of</strong> Transfusion Medicine University<br />
Hospital Tübingen, Tuebingen, Ursula Holzer, Dr. Medicine,<br />
University Children’s Hospital Tübingen, Tuebingen,<br />
Guenther E. Dannecker, Pr<strong>of</strong>essor, Olgahospital Stuttgart,<br />
Stuttgart<br />
Heat shock protein Hsp70 complexed to antigenic peptides<br />
enhances the activation and proliferation <strong>of</strong> CD8+ and CD4+ T<br />
cells in vitro. However the mechanism for HSP70-dependent<br />
antigen presentation by MHC II molecules is not yet clear. It is<br />
thought that after internalization <strong>of</strong> the HSP–peptide<br />
complexes by APCs the complex is trafficking into the<br />
cytoplasm or endosomal vesicles to be represented by MHC I<br />
and/or MHC II, although the necessity <strong>of</strong> an uptake for<br />
presentation by MHC II is not proven. Here we investigated<br />
the need <strong>of</strong> endocytosis <strong>of</strong> HSP–peptide complexes for<br />
sufficient antigen presentation in a human antigen-specific<br />
system. PBMCs <strong>of</strong> healthy donors immunized against tetanus<br />
or influenza with known HLA-DR haplotype (DRB*1101, 0401)<br />
were used. Stimulation <strong>of</strong> Tcells was induced by tetanus toxin<br />
C-fragment (TT944-966), influenza hemagglutinin (HA307–<br />
319) or the whole tetanus toxid protein either with peptide/<br />
protein alone or with the peptide/protein complexed to<br />
Hsp70. Antigen specificity was detected by HLA-DR-tetramers.<br />
For blocking the endocytosis mechanism <strong>of</strong> CD4− APCs,<br />
the cells were pre-treated with Cytochalasin, Genistein or<br />
Chloroquine, resulting in a marked decline in generating<br />
specific T cells. These results argue for an internalization <strong>of</strong><br />
the complexes by APCs and uptake into the endosomal<br />
vesicles for presentation by MHC II. Furthermore HSP–protein<br />
complexes are able to enhance the proliferation <strong>of</strong> antigenspecific<br />
T cells affirming an intracellular pathway <strong>of</strong> HSPmediated<br />
peptide presentation. Taken together, we conclude<br />
that for MHC class II dependent activation <strong>of</strong> CD4+ antigenspecific<br />
T cells uptake <strong>of</strong> Hsp-chaperoned proteins and<br />
peptides into APCs and intracellular processing is necessary.<br />
doi:10.1016/j.clim.2007.03.347<br />
F.136 Development and Validation <strong>of</strong> a Novel Ig<br />
Isotyping Assay on Magnetic Microspheres<br />
Candice Reyes, Scientist, Bio-Rad Laboratories Protein<br />
Function Division, Research and Development, Hercules,<br />
CA, Joe Fedynyshyn, Staff Scientist, Bio-Rad Laboratories<br />
Protein Function Division, Research and Development,<br />
Hercules, CA, Sophie Allauzen, Research and<br />
Development Manager, Bio-Rad Laboratories Protein<br />
Function Division, Research and Development,<br />
Hercules, CA<br />
The Bio-Plex Suspension Array system provides a<br />
flexible and efficient platform for multiplex analysis <strong>of</strong><br />
biological samples. Monitoring the levels <strong>of</strong> immunoglobulins<br />
is a valuable tool for evaluating the immune<br />
response in many different fields <strong>of</strong> research. Bio-Rad<br />
has developed a new multiplex immunoglobulin isotyping<br />
assay on magnetic beads that simultaneously quantifies<br />
the levels <strong>of</strong> IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE in<br />
human serum and plasma. The levels <strong>of</strong> immunoglobulins<br />
can vary for each class and subclass depending on factors<br />
like genetics, disease state, and drug treatment. Using<br />
the Bio-Plex Pro Human Ig Isotyping assay kit, a complete<br />
pr<strong>of</strong>ile <strong>of</strong> isotype levels can be created from as little as<br />
5 μL <strong>of</strong> sample in about 3 h. Sensitivity and upper and<br />
lower limits <strong>of</strong> quantitation (LLOQ and ULLOQ) will be<br />
discussed. Additional validation data obtained from control<br />
human serum samples and World Health Organization<br />
(WHO) Reference Standards will also be presented.<br />
doi:10.1016/j.clim.2007.03.348<br />
F.137 Utility <strong>of</strong> Lyophilized PMA and Ionomycin to<br />
Stimulate Lymphocytes in Whole Blood for<br />
Immunological Assays<br />
Shelley Belouski, Senior Scientist, Amgen <strong>Clinical</strong><br />
<strong>Immunology</strong>, Thousand Oaks, CA, John Ferbas, Director<br />
Medical Sciences, Amgen <strong>Clinical</strong> <strong>Immunology</strong>, Thousand<br />
Oaks, CA, Keith Kelley, Senior Associate Scientist, Amgen<br />
<strong>Clinical</strong> <strong>Immunology</strong>, Thousand Oaks, CA, Julie Wilkinson,<br />
Staff Adv Research Scientist, Beckman Coulter, Inc., Custom<br />
BioPharma Solutions, Miami, FL, Sid Suggs, Director, Amgen<br />
Medical Sciences, Thousand Oaks, CA, John Thomas, Senior<br />
Associate Scientist, Amgen <strong>Clinical</strong> <strong>Immunology</strong>, Thousand<br />
Oaks, CA, Shen-Wu Wang, Senior Associate Scientist, Amgen<br />
Molecular Sciences, Thousand Oaks, CA<br />
Background: The need to implement robust biomarkers in<br />
clinical trials has never been greater, and such efforts can be<br />
easily compromised by reagent instability or simple human<br />
error during assay set-up. Many biotechnology and pharmaceutical<br />
companies are introducing efforts to conduct<br />
biomarker studies under more rigorous settings, and use <strong>of</strong><br />
plates or tubes pre-loaded with stimulation or staining<br />
reagents could be <strong>of</strong> value for studies that involve flow<br />
cytometry. Results: Our laboratory took a biomarker analytical<br />
method that relied on liquid formulations <strong>of</strong> phorbol 12myristate<br />
13-acetate (PMA) and ionomycin and demonstrates<br />
here that tubes pre-loaded with lyophilized versions <strong>of</strong> the<br />
liquid reagents can provide equivalent results. The value <strong>of</strong><br />
this approach is that it safeguards against omission or<br />
erroneous addition <strong>of</strong> bulk liquid formulations <strong>of</strong> PMA and<br />
ionomycin to the reaction vessel (i.e., plate or tube) and also<br />
lends itself to extended stability/shelf-life <strong>of</strong> these<br />
reagents. Conclusions: On the basis <strong>of</strong> this initial success,<br />
we plan to expand our evaluation <strong>of</strong> lyophilized reagents so<br />
that they can be incorporated into our clinical biomarker<br />
campaigns as appropriate.<br />
doi:10.1016/j.clim.2007.03.349<br />
S59
S60 Abstracts<br />
#2201- Inflammation Good or Bad for Tregs<br />
Saturday, June 9<br />
10:45 am−11:05 am<br />
Viral Infection Enables CD4+CD25+ Regulatory T<br />
Cells to Protect From Autoimmune Diabetes<br />
Christophe Filippi, Postdoctoral Fellow, La Jolla Institute<br />
for Allergy and <strong>Immunology</strong>, La Jolla, CA, Janine Oldham,<br />
Research Assistant, La Jolla Institute for Allergy and<br />
<strong>Immunology</strong>, La Jolla, CA, Tom Wolfe, Research Assistant,<br />
La Jolla Institute for Allergy and <strong>Immunology</strong>, La Jolla,<br />
CA, Evelyn Rodrigo, Research Assistant, La Jolla Institute<br />
for Allergy and <strong>Immunology</strong>, La Jolla, CA, Urs Christen,<br />
Research Scientist, La Jolla Institute for Allergy and<br />
<strong>Immunology</strong>, La Jolla, CA, Lisa Togher, Research Assistant,<br />
La Jolla Institute for Allergy and <strong>Immunology</strong>, La Jolla,<br />
CA, Marianne Martinic, Postdoctoral Fellow, La Jolla<br />
Institute for Allergy and <strong>Immunology</strong>, La Jolla, CA,<br />
Matthias von Herrath, Pr<strong>of</strong>essor, La Jolla Institute for<br />
Allergy and <strong>Immunology</strong>, La Jolla, CA<br />
Type 1 Diabetes (T1D) is an autoimmune disease that<br />
results from the selective destruction <strong>of</strong> insulin-producing<br />
beta cells in the pancreas. While viral infections may be<br />
capable <strong>of</strong> triggering autoimmunity in genetically susceptible<br />
individuals, accumulating evidence indicates that viruses<br />
can also prevent T1D. Furthermore, viruses have been<br />
suggested to be potent inducers <strong>of</strong> CD4+CD25+ regulatory T<br />
cells (Tregs), which are known to play a crucial role in the<br />
prevention <strong>of</strong> autoimmunity. However, how viral infections<br />
may protect from autoimmune diabetes is still under<br />
investigation. Here, we show that activation <strong>of</strong> CD4+CD25+<br />
Tregs by acute lymphocytic choriomeningitis virus (LCMV)<br />
infection provides these cells with the capacity to prevent<br />
both spontaneous and virally induced T1D in the mouse. We<br />
found that CD4+CD25+ Tregs activated by LCMV in vivo or in<br />
vitro produce the regulatory cytokine TGF-β and are capable<br />
<strong>of</strong> protecting NOD and RIP-LCMV mice from spontaneous and<br />
LCMV-induced diabetes, respectively. Our results suggest<br />
that LCMV-modulated CD4+CD25+ Tregs act in a bystander<br />
fashion independent <strong>of</strong> beta-cell antigens and suppress TNFα<br />
production by autoreactive CD8+ T cells, leading to<br />
prevention <strong>of</strong> T1D. These findings provide a novel mechanistic<br />
explanation for the previously reported ability <strong>of</strong> viral<br />
infections to prevent autoimmune diabetes. Furthermore,<br />
our results support a model explaining the differential ability<br />
<strong>of</strong> viral infections to modulate diabetes. Activation <strong>of</strong> Tregs<br />
during viral infections may be a general feature accounting<br />
for the ability <strong>of</strong> viruses to prevent T1D as well as other<br />
autoimmune disorders.<br />
doi:10.1016/j.clim.2007.03.350<br />
<strong>Oral</strong> <strong>Presentations</strong>: Saturday, June 9<br />
Saturday, June 9<br />
2:45 pm−4:45 pm<br />
OR.33 Cytotoxic Human IL-22-expressing Th17<br />
Lymphocytes Promote Immune Cell Migration Into<br />
the Central Nervous System<br />
Hania Kebir, PhD Student, CHUM-Notre-Dame Hospital,<br />
Neuroimmunology Department, Montreal, QC, Canada, Igal<br />
Ifergan, PhD Student, CHUM-Notre-Dame Hospital,<br />
Neuroimmunology Department, Montreal, QC, Canada,<br />
Aurore Dodelet-Devillers, M.Sc. Student, CHUM-Notre-Dame<br />
Hospital, Neuroimmunolgy Department, Montreal, QC,<br />
Canada, Fabrizio Giuliani, Assistant Pr<strong>of</strong>essor <strong>of</strong> Medicine,<br />
University <strong>of</strong> Alberta, Department <strong>of</strong> Medicine, Edmonton,<br />
AB, Canada, Romain Cayrol, PhD Student, CHUM-Notre-Dame<br />
Hospital, Neuroimmunolgy Department, Montreal, QC,<br />
Canada, Nathalie Arbour, Assistant Pr<strong>of</strong>essor <strong>of</strong> Medicine,<br />
CHUM-Notre-Dame Hospital, Neuroimmunology<br />
Department, Montreal, QC, Canada, Alexandre Prat,<br />
Assistant Pr<strong>of</strong>essor <strong>of</strong> Medicine and Doctor,<br />
CHUM-Notre-Dame Hospital, Neuroimmunolgy Department<br />
and MS Clinic, Montreal, QC, Canada<br />
Interleukin (IL)-17-secreting lymphocytes (Th17) appear<br />
essential in the pathogenesis <strong>of</strong> numerous models <strong>of</strong><br />
inflammatory diseases, including experimental autoimmune<br />
encephalomyelitis (EAE). As survival <strong>of</strong> Th17 cells<br />
is attributed to IL-23, recent evidence suggests that<br />
transforming growth factor (TGF)-β is required for the<br />
expression <strong>of</strong> IL-23 receptor (R) by lymphocytes, thereby<br />
acting upstream in the development <strong>of</strong> Th17. However,<br />
while IL-23 and TGF-β R null animals are resistant to<br />
autoimmune diseases, IL-17 neutralization only partially<br />
protects against EAE and colitis, suggesting that additional<br />
Th17-derived factors contribute to tissue inflammation. We<br />
demonstrate that human Th17 lymphocytes originate from<br />
memory CD4+CD45RO+ cells and express IL-22 under the<br />
influence <strong>of</strong> IL-23, while TGF-β acts as a negative regulator<br />
<strong>of</strong> IL-22 production. We show that IL-17R and IL-22R are upregulated<br />
on blood–brain barrier (BBB)-endothelial cells<br />
(ECs) in situ during inflammation, and that IL-17 and IL-22<br />
disrupt the human BBB and promote CD4+ T lymphocyte<br />
recruitment in vitro. Furthermore, Th17 lymphocytes transmigrate<br />
more readily across brain-ECs than Th1 and<br />
express high levels <strong>of</strong> granzyme B. Thus, IL-23 induces<br />
the formation <strong>of</strong> a unique Th subset expressing cytotoxic<br />
factors and promoting leukocyte recruitment across the<br />
BBB, through the concerted and redundant effects <strong>of</strong> IL-17<br />
and IL-22.<br />
doi:10.1016/j.clim.2007.03.351
Abstracts<br />
OR.34 CNS Myeloid Dendritic Cells Drive Epitope<br />
Spreading and Th-17 Differentiation in Relapsing<br />
Experimental Autoimmune Encephalomyelitis<br />
(R-EAE)<br />
Stephen Miller, Pr<strong>of</strong>essor, Northwestern University Medical<br />
School, Department <strong>of</strong> Microbiology-<strong>Immunology</strong>, Chicago,<br />
IL, Samantha Bailey, Postdoctoral Fellow, Northwestern<br />
University Medical School, Department <strong>of</strong><br />
Microbiology-<strong>Immunology</strong>, Chicago, IL, Bettina Schreiner,<br />
Postdoctoral Fellow, Northwestern University Medical<br />
School, Department <strong>of</strong> Microbiology-<strong>Immunology</strong>, Chicago, IL<br />
Chronic progression <strong>of</strong> relapsing experimental autoimmune<br />
encephalomyelitis (R-EAE) is dependent on the activation<br />
<strong>of</strong> T cells to released endogenous myelin epitopes, i.e.,<br />
epitope spreading. Using transfer <strong>of</strong> naive CFSE-labeled TCR<br />
transgenic T cells, we have shown that activation <strong>of</strong> naive<br />
PLP139–151-specific T cells in SJL mice undergoing PLP178-<br />
191-induced R-EAE occurs directly in the CNS. Flow cytometric<br />
and histologic examination <strong>of</strong> the R-EAE CNS revealed<br />
the infiltration <strong>of</strong> significant numbers <strong>of</strong> CD11c+ dendritic<br />
cells (DCs) (including myeloid, lymphoid and plasmacytoid<br />
subsets) which are not seen in the healthy CNS. Functional<br />
examination <strong>of</strong> the antigen presentation capacity <strong>of</strong> CNS APC<br />
populations shows that only F4/80-CD11c+CD45hi DCs, not<br />
infiltrating macrophages or resident microglia, efficiently<br />
present endogenous antigen resulting in the activation <strong>of</strong><br />
naive PLP139–151-specific Tg T cells. Bone marrow chimeras<br />
indicate that the vast majority <strong>of</strong> CNS DCs are <strong>of</strong> peripheral<br />
origin. Among the DC subsets, CD11b+ CD11c+ myeloid DCs<br />
(mDCs) cluster with CNS-resident PLP139–151-specific Tg T<br />
cells and are by far the most efficient endogenous activators<br />
<strong>of</strong> naumlautve T cells both in vivo and in vitro. In contrast,<br />
lymphoid and plasmacytoid DCs are less effective. mDCs<br />
uniquely biased Th-17 and not Th1 differentiation, correlating<br />
with their enhanced expression <strong>of</strong> TGF- 2 1 and IL-6 and IL-<br />
23. These findings indicate that the inflamed CNS can<br />
function as a neo-lymphoid organ and inhibition <strong>of</strong> DC<br />
migration may be a viable target for treatment <strong>of</strong> MS<br />
(supported by NIH Grant NS-030871, NMSS Grant RG-3793-A-<br />
7 and a grant from the Myelin Repair Foundation).<br />
doi:10.1016/j.clim.2007.03.352<br />
OR.35 IL-23 and IL-17 in Pathogenesis <strong>of</strong><br />
Experimental Ocular Autoimmunity: Requirement<br />
for IL-23 May Extend Beyond Its Role in Sustaining<br />
the IL-17 Effector Response<br />
Dror Luger, Postdoctoral Fellow, National Institutes <strong>of</strong><br />
Health, NEI Laboratory, Phyllis Silver, Biologist, National<br />
Institutes <strong>of</strong> Health, NEI Laboratory, Daniel Cua, Senior<br />
Principal Scientist, Schering Plough Biopharma, DNAX, Palo<br />
Alto, CA, Zoe Chen, Scientist, Schering Plough Biopharma,<br />
Palo Alto, CA, Yoichiro Iwakura, Pr<strong>of</strong>essor, University <strong>of</strong><br />
Tokyo, Tokyo, Japan, Edward Bowman, Principal Scientist,<br />
Schering-Plough Biopharma, DNAX, Palo Alto, CA, Chi-Chao<br />
Chan, Senior Scientist, National Institutes <strong>of</strong> Health, NEI,<br />
Laboratory <strong>Immunology</strong>, Bethesda, MD, Rachel Caspi, Senior<br />
Investigator, National Institutes <strong>of</strong> Health, NEI, Laboratory<br />
<strong>Immunology</strong>, Bethesda, MD<br />
Experimental autoimmune uveitis (EAU) represents autoimmune<br />
uveitis in humans. Here we examined the role <strong>of</strong> the<br />
IL-23/IL-17 pathway in EAU. We immunized IL-23p19, IL-<br />
12p35, IL-12p40, IFN-g and IL-17 KO mice with the<br />
uveitogenic Ag IRBP. Alternatively, we treated wild type<br />
mice immunized for EAU with Abs to IL-23p19 or to IL-17<br />
throughout the disease course (prevention), or only during<br />
the effector phase (reversal). IL-23p19 KO were resistant to<br />
EAU, showing reduced Ag-specific DTH, IL-2, IFN-g, IL-17, IL-<br />
1b, IL-6 and IL-5. In contrast, IL-12p35 and IFN-g KO mice<br />
developed exacerbated EAU, DTH and IL-17 responses.<br />
Treatment with anti-IL-23p19 prevented EAU and reduced<br />
immunological responses, but was unable to reverse an<br />
ongoing disease process, indicating an inductive role for IL-<br />
23. In contrast, anti-IL-17 prevented as well as reversed EAU,<br />
indicating a role for IL-17 in effector mechanisms. Interestingly,<br />
severe EAU could be induced with a Th1 cell line that<br />
does not produce IL-17. EAU in recipients <strong>of</strong> the line could be<br />
inhibited with anti-IFN-g or anti-TNF-a, but not with anti-IL-<br />
17, speaking against a need for host-derived IL-17 and<br />
suggesting that under some conditions an antigen-specific<br />
Th1 effector is sufficient to induce EAU. In keeping with this,<br />
IL-17 KO mice were still susceptible to disease. These data<br />
raise the possibility that the nonredundant need for IL-23 in<br />
EAU may extend beyond its role in promoting the Th17<br />
effector response and point to IL-23 and IL-17 as new<br />
therapeutic targets for uveitis.<br />
doi:10.1016/j.clim.2007.03.353<br />
S61<br />
OR.36 The Role <strong>of</strong> IL-17 and IFN-γ in Defining<br />
Pathogenic Potential <strong>of</strong> T Cells in EAE<br />
Ingunn Stromnes, Graduate Student, University <strong>of</strong><br />
Washington, Department <strong>of</strong> <strong>Immunology</strong>, Seattle, WA, Joan<br />
Goverman, Pr<strong>of</strong>essor, University <strong>of</strong> Washington,<br />
Department <strong>of</strong> <strong>Immunology</strong>, Seattle, WA, Denny Liggitt,<br />
Chair, University <strong>of</strong> Washington, Department <strong>of</strong><br />
Comparative Medicine, Seattle, WA<br />
Multiple sclerosis (MS) is a demyelinating disease <strong>of</strong> the<br />
central nervous system (CNS) in which inflammatory infiltrates<br />
localize in the white matter <strong>of</strong> the brain and spinal<br />
cord. The mechanisms that determine specific sites <strong>of</strong><br />
inflammation are not understood. We developed a novel<br />
model <strong>of</strong> experimental allergic encephalomyelitis, an animal<br />
model for MS, in which T cells specific for one epitope <strong>of</strong> a<br />
myelin oligodendrocyte glycoprotein (MOG) preferentially<br />
induce inflammation in the brain, while T cells specific for<br />
two different MOG epitopes preferentially induce inflammation<br />
in the spinal cord, resulting in pr<strong>of</strong>oundly distinct<br />
clinical signs. T cells responding to all three epitopes<br />
generate both IL-17- and IFN-γ-producing non-overlapping<br />
populations, and both the IL-17- and IFN-γ-producing T cells<br />
induce disease upon adoptive transfer. We found that the<br />
ability to induce inflammation preferentially in the brain or<br />
spinal cord is not determined by the absolute number <strong>of</strong><br />
either IL-17- or IFN-γ-producing T cells, but rather by the<br />
ratio <strong>of</strong> the two populations. Changing the IL-17 to IFN-γ<br />
ratio for Tcells <strong>of</strong> each specificity by culturing them in either<br />
IL-12 or IL-23 prior to adoptive transfer altered the
S62 Abstracts<br />
inflammation sites in the CNS, which resulted in different<br />
clinical signs in recipient mice. These data show that the<br />
encephalitogenic potential <strong>of</strong> Tcells is not defined solely by a<br />
production <strong>of</strong> a single cytokine, but by the relative frequency<br />
<strong>of</strong> Tcells producing either IFN-γ or IL-17. These findings have<br />
important implications for cytokine-targeted therapies <strong>of</strong><br />
CNS autoimmune disease.<br />
doi:10.1016/j.clim.2007.03.354<br />
OR.37 The Role <strong>of</strong> IL-22, a TH17 Cytokine, in<br />
Autoimmune Diseases and Bacteria Infection<br />
Yan Zheng, Postdoctoral Research Fellow, Department <strong>of</strong><br />
<strong>Immunology</strong>, Genentech Inc, South San Francisco, CA,<br />
Dimitry Danilenko, Pathologist, Department <strong>of</strong> Pathology,<br />
Genentech Inc, South San Francisco, CA, Patricia Valdez,<br />
Senior Research Associate, Department <strong>of</strong> <strong>Immunology</strong>,<br />
Genentech Inc, South San Francisco, CA, Jeffrey<br />
Eastham-Anderson, Department <strong>of</strong> Pathology, Genentech<br />
Inc, South San Francisco, CA, Ian Kasman, Department <strong>of</strong><br />
Pathology, Genentech Inc, South San Francisco, CA, Jianfeng<br />
Wu, Research Associate, Department <strong>of</strong> <strong>Immunology</strong>,<br />
Genentech Inc, South San Francisco, CA, Wenjun Ouyang,<br />
Scientist, Department <strong>of</strong> <strong>Immunology</strong>, Genentech Inc, South<br />
San Francisco, CA<br />
Recently, interleukin (IL)-23, a cytokine involved in the<br />
development <strong>of</strong> IL-17-producing T helper cells (TH17 cells),<br />
was found to have a potential function in the pathogenesis <strong>of</strong><br />
autoimmune diseases and the host defense against infectious<br />
agents. We identified that IL-22 is preferentially produced by<br />
TH17 cells. However, the production <strong>of</strong> IL-22 and IL-17 from<br />
TH17 cells is differentially regulated. Transforming growth<br />
factor-beta (TGF-β), although crucial for IL-17 production,<br />
actually inhibits IL-22 production. Furthermore, IL-22 mediates<br />
IL-23-induced acanthosis and dermal inflammation<br />
through the activation <strong>of</strong> Stat3 (signal transduction and<br />
activators <strong>of</strong> transcription 3) in vivo. Our preliminary data<br />
also showed a protective role <strong>of</strong> IL-22 in host defense against<br />
bacteria infection. Our results suggest that TH17 cells,<br />
through the production <strong>of</strong> both IL-22 and IL-17, might have<br />
essential functions both in host defense and in the pathogenesis<br />
<strong>of</strong> autoimmune diseases such as psoriasis. IL-22, as an<br />
effector cytokine produced by Tcells, mediates the crosstalk<br />
between the immune system and the tissues.<br />
doi:10.1016/j.clim.2007.03.355<br />
OR.38 Endogenous IL-6 is Required for Inducing and<br />
Sustaining an Antigen-specific IL-17 Memory<br />
Response in Experimental Autoimmune Uveitis<br />
(EAU), but Not for Innate IL-17 Production<br />
Aleksandra V. Rachitskaya, Medical Student, Laboratory <strong>of</strong><br />
<strong>Immunology</strong>, National Eye Institute, NIH; HHMI-NIH<br />
Research Scholars Program, Bethesda, MD, Anna M. Hansen,<br />
Postdoctoraltoral Fellow, Laboratory <strong>of</strong> <strong>Immunology</strong>,<br />
National Eye Institute, National Institutes <strong>of</strong> Health (NIH),<br />
Bethesda, MD, Rajeev K. Agarwal, Staff Scientist,<br />
Laboratory <strong>of</strong> <strong>Immunology</strong>, National Eye Institute, National<br />
Institutes <strong>of</strong> Health (NIH), Bethesda, MD, Dror Luger,<br />
Postdoctoraltoral Fellow, Laboratory <strong>of</strong> <strong>Immunology</strong>,<br />
National Eye Institute, National Institutes <strong>of</strong> Health (NIH),<br />
Bethesda, MD, Phyllis B. Silver, Biologist, Laboratory <strong>of</strong><br />
<strong>Immunology</strong>, National Eye Institute, National Institutes <strong>of</strong><br />
Health (NIH), Bethesda, MD, Chi-Chao Chan, Senior<br />
Investigator, Laboratory <strong>of</strong> <strong>Immunology</strong>, National Eye<br />
Institute, National Institutes <strong>of</strong> Health (NIH), Bethesda, MD,<br />
Rachel R. Caspi, Senior Investigator, Laboratory <strong>of</strong><br />
<strong>Immunology</strong>, National Eye Institute, National Institutes <strong>of</strong><br />
Health (NIH), Bethesda, MD<br />
We examined the role <strong>of</strong> IL-6 in production <strong>of</strong> the<br />
proinflammatory cytokine IL-17 under conditions <strong>of</strong> a primary<br />
immune response, as represented by T cell receptor ligation<br />
in vitro, and under conditions <strong>of</strong> antigen-specific recall<br />
response in the model <strong>of</strong> experimental autoimmune uveitis<br />
(EAU) in vivo. To mimic a primary response, naive splenocytes<br />
from IL-6 deficient mice (IL-6 KO) or from their C57BL/6 wildtype<br />
counterparts (WT) were stimulated with anti-CD3 and IL-<br />
23. EAU was induced in IL-6 KO and WT mice by immunization<br />
with the retinal antigen IRBP in complete Freund’s adjuvant<br />
(CFA). EAU development was evaluated by fundus examination<br />
and confirmed by eye histology. Eyes and lymphoid organs<br />
were harvested on day 21. Responses were examined by IL-17<br />
ELISA, intracellular cytokine staining combined with immunophenotyping,<br />
DTH and lymphocyte proliferation. IRBPimmunized<br />
IL-6 KO mice were completely resistant to EAU and<br />
had significantly reduced DTH and proliferative responses to<br />
IRBP as compared to their WT counterparts. Notably, their<br />
IRBP-specific IL-17 production was conspicuously decreased<br />
as compared to WT. In contrast, during an in vitro<br />
primary response, IL-17 production, observed at approximately<br />
24 hours after stimulation, was the same in both<br />
genotypes and required IL-23, but not IL-6. Our results<br />
indicate that IL-6 plays an essential role in inducing and<br />
sustaining the IL-17 memory and recall responses. However,<br />
an initial innate IL-17 production occurs early during<br />
a primary response and is IL-6 independent. By immunophenotyping,<br />
the cellular source <strong>of</strong> this early IL-17<br />
response appears to be NK/NKT cells.<br />
doi:10.1016/j.clim.2007.03.356<br />
OR.39 Evidence for a Role <strong>of</strong> the Interleukin-23<br />
Pathway in the Pathogenesis <strong>of</strong> Psoriasis<br />
Frank Nestle, Pr<strong>of</strong>essor, Kings College London, London,<br />
England, Francesca Capon, Doctor, Department <strong>of</strong> Genetics,<br />
King’s College London School <strong>of</strong> Medicine, London, England,<br />
Jonathan Barker, Pr<strong>of</strong>essor, St. Johns Institute <strong>of</strong><br />
Dermatology, London, England, Robert Kastelein, Staff,<br />
Schering-Plough Biopharma, Palo Alto, CA, Richard<br />
Trembath, Pr<strong>of</strong>essor, Division <strong>of</strong> Genetics and Molecular<br />
Medicine, London, England, Giulia Tonel, PhD Student,<br />
Department <strong>of</strong> Dermatology (F14), University <strong>of</strong> Zurich,<br />
Zurich, Switzerland, Paola Di Meglio, Research Associate,<br />
Cutaneous Medicine and Immunotherapy Unit, St. John’s<br />
Institute <strong>of</strong> Dermatology, London, England, Elizabeth<br />
Oldham, Staff, Schering-Plough Biopharma, Palo Alto, CA,<br />
Jerry Lanchbury, Staff, Myriad Genetics, Salt Lake City, UT
Abstracts<br />
To identify genes relevant to disease pathogenesis we<br />
performed a genome-wide association study in psoriasis, one<br />
<strong>of</strong> the most common chronic inflammatory disorders. We<br />
detected a highly significant association between psoriasis<br />
and genetic markers in the interleukin-23 receptor (IL-23R)<br />
gene on chromosome 1p31, a finding replicated in an independent<br />
dataset. The most significantly associated polymorphism<br />
(combined p value=1.6 ×10 −5 ) results in an amino<br />
acid substitution (Arg381Gln) located in the IL-23R cytoplasmic<br />
domain. The same variant has recently been implicated<br />
in the pathogenesis <strong>of</strong> inflammatory bowel disease, supporting<br />
a critical role <strong>of</strong> IL-23 signaling in epithelial inflammation.<br />
As a necessary step to determine the importance <strong>of</strong> this<br />
pathway in the pathogenesis <strong>of</strong> psoriasis, we undertook functional<br />
investigations using patient samples and a clinically<br />
relevant model system. We found an increased expression <strong>of</strong><br />
IL-23R on psoriatic helper T cells compared to normal controls.<br />
Functional dissection <strong>of</strong> the IL-23 pathway using immunosuppressed<br />
AGR mice grafted with psoriatic skin<br />
revealed a key role for the IL-23 pathways in the disease<br />
process. Administration <strong>of</strong> a neutralizing anti-human IL-23<br />
antibody inhibited psoriasis development comparable to the<br />
use <strong>of</strong> anti-TNF-α blockers. These data provide genetic and<br />
functional evidence for a crucial role <strong>of</strong> the IL-23 pathway<br />
in cutaneous inflammation and lay the foundation for new<br />
treatment strategies in psoriasis and potentially other chronic<br />
epithelial inflammatory disorders.<br />
doi:10.1016/j.clim.2007.03.357<br />
OR.40 The Fully Human Antibody CNTO1275<br />
Effectively Inhibits Human IL-12 and IL-23 Mediated<br />
Th1 and Th17 Cell Function and Provides <strong>Clinical</strong><br />
Benefit to Patients with Plaque Psoriasis<br />
Jacqueline Benson, Assistant Director, Centocor Research<br />
and Development, Discovery Research, Radnor, PA,<br />
Yevgeniya Orlovsky, Senior Associate Scientist, Centocor<br />
Research and Development, Radnor, PA, Kim Staquet,<br />
Manager, Centocor Research and Development, Radnor,<br />
PA, Jinquan Luo, Principal Research Scientist, Centocor<br />
Research and Development, Radnor, PA, Patrick Branigan,<br />
Research Scientist, Centocor Research and Development,<br />
Radnor, PA, Roberta Lamb, Senior Associate Scientist,<br />
Centocor Research and Development, Radnor, PA, Jian Li,<br />
Research Scientist, Centocor Research and Development,<br />
Radnor, PA, Renold Capocasale, Research Scientist,<br />
Centocor, Radnor, PA, Janine Huber, Senior Associate<br />
Scientist, Therakos, Exton, PA, Bernie Scallon, Sr<br />
Research Fellow, Centocor, Radnor, PA, David Peritt,<br />
Director, Therakos, Exton, PA, David Shealy, Senior<br />
Research Fellow, Centocor Research and Development,<br />
Radnor, PA, George Heavner, Distinguished Research<br />
Fellow, Centocor Research and Development, Radnor, PA,<br />
Jill Giles-Komar, Director, Centocor Research and<br />
Development, Radnor, PA, Tom Nesspor, Senior Associate<br />
Scientist, Centocor, Radnor, PA<br />
Interleukins (IL)-12 and IL-23 are potent contributors to<br />
CD4+ T cell differentiation to Th1 and Th17 cell types which<br />
are associated with several immune-mediated human dis-<br />
eases, including psoriasis, Crohn’s disease, rheumatoid<br />
arthritis, multiple sclerosis, and others. Unique TLR agonist<br />
combinations will elicit IL-12 versus IL-23 production from<br />
antigen presenting cells and these cytokines demonstrate<br />
distinct effects on human and mouse T cell activation.<br />
CNTO1275 is a fully human monoclonal antibody that<br />
potently inhibits the bioactivity <strong>of</strong> human IL-12 and IL-23<br />
by binding to the shared p40 subunit and preventing<br />
interaction with the IL-12Rb1 receptor on the surface on<br />
NK and CD4+ T cells. Thus, CNTO1275 inhibits IL-12 and IL-23<br />
mediated activation and cytokine production <strong>of</strong> receptorbearing<br />
cells. CNTO1275 binds human and primate, but not<br />
rodent, IL-12 and IL-23 and suppressed disease in a primate<br />
model <strong>of</strong> multiple sclerosis and in a human skin transplant<br />
mouse model <strong>of</strong> psoriasis. CNTO1275 has also provided a<br />
significant level <strong>of</strong> efficacy in the majority <strong>of</strong> subjects in<br />
phase I and II clinical studies <strong>of</strong> plaque psoriasis. These data<br />
support a central role for IL-12/23p40 in psoriasis pathogenesis.<br />
CNTO1275 was generally well-tolerated and adverse<br />
events observed in these studies did not indicate dose–<br />
response safety relationships. Similarly, studies in cynomolgus<br />
monkeys have shown that even high doses <strong>of</strong> CNTO1275<br />
are generally well-tolerated. Collectively, these results<br />
suggest that CNTO1275 may represent an exciting first-inclass<br />
therapy with significant therapeutic potential for<br />
immune-mediated diseases such as psoriasis.<br />
doi:10.1016/j.clim.2007.03.358<br />
Therapy- From Gene Silencing to Costimulation<br />
Saturday, June 9<br />
2:45 pm–4:45 pm<br />
S63<br />
OR.41 Effects <strong>of</strong> Long-Term Fingolimod Therapy on<br />
Circulating Mononuclear Cell Populations in Multiple<br />
Sclerosis<br />
Amit Bar-Or, Associate Pr<strong>of</strong>essor, Montreal Neurological<br />
Institute, McGill University, Montreal, QC, Canada, Nathalie<br />
Arbour, Associate Researcher, University <strong>of</strong> Montreal,<br />
Faculty <strong>of</strong> Medicine, Montreal, QC, Canada, Ellie McCrae,<br />
Technician, Montreal Neurological Institute, Montreal, QC,<br />
Canada, Jack Antel, Pr<strong>of</strong>essor, Montreal Neurological<br />
Institute, Montreal, QC, Canada, Tarik Touil, Postdoctoral<br />
Fellow, Montreal Neurological Institute, Montreal, QC,<br />
Canada, Karen Newell, Visiting Pr<strong>of</strong>essor, Montreal<br />
Neurological Institute, Montreal, QC, Canada<br />
Fingolimod (FTY720) is an orally administered sphingosine-1-phosphate<br />
(S1P) receptor modulator. In animal and<br />
short-term human studies it down-regulates lymphocyte (but<br />
not myeloid cell) receptor expression. Fingolimod reduced<br />
clinical and MRI activity in relapsing MS patients. Objectives:<br />
To examine the pr<strong>of</strong>ile <strong>of</strong> circulating mononuclear cells<br />
(MNC) in MS patients (n=6) who continue daily use <strong>of</strong> 1.25 mg<br />
<strong>of</strong> oral fingolimod for N18 months (open label extension<br />
phase <strong>of</strong> original phase II trial). This represents the longest<br />
experience using fingolimod as a single agent in humans.<br />
Methods: Whole peripheral blood samples obtained from the<br />
patient cohort were immunostained for leukocyte markers
S64 Abstracts<br />
and analyzed by flow cytometry. Results: Absolute lymphocyte<br />
number (mean 0.48+0.06 ×10 3 per microliter) was<br />
reduced with treatment (normal range 0.91–4.28×103);<br />
while CD14+ monocyte counts (0.47 +0.07×103) were within<br />
normal range (0.12–0.92×103). CD4+ T cells were relatively<br />
more depleted (2.3+0.8% <strong>of</strong> total MNCs) (control 28.0+6.4%,<br />
n=6) than CD8+ T cells (10.4 +2.5%) (control 22.0+4.2%),<br />
resulting in reversed CD4:CD8 ratio (0.2). CD19+ B cells were<br />
also markedly reduced (1.1+0.2% <strong>of</strong> total MNCs) (control<br />
11.1+2.1%). The proportion <strong>of</strong> monocytes (29.4+4.4%) and<br />
CD56+ NK cells (32.2+9.3%) among MNC was increased in<br />
treated patients compared to controls (7.0+1.8%) and (14.5 +<br />
4.3%) respectively — although their absolute number did not.<br />
Conclusions: MS patients receiving fingolimod monotherapy<br />
show sustained decreases in circulating B cells and CD4 T<br />
cells, relative less depletion <strong>of</strong> CD8 T cells, and maintained<br />
numbers <strong>of</strong> innate immune cells. Correlations with clinical<br />
measures will help define the basis for the efficacy, and<br />
safety pr<strong>of</strong>ile <strong>of</strong> this type <strong>of</strong> immuno-modulation.<br />
doi:10.1016/j.clim.2007.03.359<br />
OR.42 In Vivo Dendritic Cell-specific Gene Silencing<br />
Using a Novel and Selective siRNA Delivery Method<br />
to Induce Immune Modulation<br />
Costin Vladau, Graduate Student, University <strong>of</strong> Western<br />
Ontario, Department <strong>of</strong> Microbiology and <strong>Immunology</strong>,<br />
London, ON, Canada, Dong Chen, Postdoctoral Fellow,<br />
University <strong>of</strong> Western Ontario, Department <strong>of</strong> Microbiology<br />
and <strong>Immunology</strong>, London, ON, Canada, Motohiko Suzuki,<br />
Postdoctoral Fellow, University <strong>of</strong> Western Ontario,<br />
Department <strong>of</strong> Microbiology and <strong>Immunology</strong>, London,<br />
England, Xusheng Zhang, Postdoctoral Fellow, University <strong>of</strong><br />
Western Ontario, Department <strong>of</strong> Microbiology and<br />
<strong>Immunology</strong>, London, ON, Canada, Xiufen Zheng,<br />
Postdoctoral Fellow, London Health Sciences Centre,<br />
University Hospital, Department <strong>of</strong> Surgery, London, ON,<br />
Canada, Wei-Ping Min, Principle Investigator, University <strong>of</strong><br />
Western Ontario, Department <strong>of</strong> Microbiology and<br />
<strong>Immunology</strong>, London, ON, Canada<br />
Silencing immune molecules, such as the costimulatory<br />
molecule CD40, using siRNA was shown to have therapeutic<br />
potential and promise in immune modulation. However, a<br />
major barrier to the clinical application <strong>of</strong> siRNA is the current<br />
lack <strong>of</strong> an effective and cell-specific delivery system. Herein,<br />
we present a new method <strong>of</strong> selectively delivering siRNA to DC<br />
in vivo using stealth immunoliposomes (SILs). CD40 siRNAcontaining<br />
SILs were generated using 4 types <strong>of</strong> lipids and<br />
decorated with surface-bound, DC-specific mAbs. DC-specific<br />
binding capacity <strong>of</strong> SILs was demonstrated by fluorescence<br />
microscopy and flow cytometry. Upon treatment with CD40<br />
siRNA-SILs, DC expression <strong>of</strong> CD40 was successfully suppressed<br />
in vitro. In vivo administration <strong>of</strong> CD40 siRNA-SILs resulted in<br />
DC-specific tissue targeting, as evidenced by increased uptake<br />
<strong>of</strong> fluorescence-tagged siRNA. Additionally, DC from mice<br />
treated with CD40 siRNA-SILs exhibited CD40-specific gene<br />
silencing in vivo. Tolerogenic properties <strong>of</strong> DC treated with<br />
CD40 siRNA-SILs were determined by inhibition <strong>of</strong> allogeneic T<br />
cell proliferation in MLR. Furthermore, a strong in vivo<br />
immune modulation was observed in mice treated with CD40<br />
siRNA-SILs. In conclusion, this is the first demonstration <strong>of</strong> DCspecific<br />
siRNA delivery and gene silencing in vivo, which<br />
highlights the potential <strong>of</strong> DC-mediated immune modulation<br />
and the feasibility <strong>of</strong> siRNA based clinical therapy.<br />
doi:10.1016/j.clim.2007.03.360<br />
OR.43 Immunotherapy <strong>of</strong> Solid Tumors Utilizing<br />
PLGA/Tumor Lysate Nanoparticles to Stimulate TIL<br />
and Circulating CD8+ Tumor-specific T Cells<br />
Douglas Hanlon, Associate Research Scientist, Yale<br />
Dermatology, New Haven, CT, Virginia Cody, Research<br />
Assistant, Yale Dermatology, New Haven, CT, Shashi Prasad,<br />
Postdoctoraltoral Fellow, Yale Dermatology, New Haven, CT,<br />
Mark Saltzman, Pr<strong>of</strong>essor, Yale Biomedical Engineering, New<br />
Haven, CT, Camille Solbrig, Associate Research Scientist,<br />
Yale Biomedical Engineering, New Haven, CT<br />
Dendritic cell (DC)-based therapies for solid tumors have<br />
failed to achieve large-scale clinical utilization because at<br />
present no unified technology exists to overcome two fundamental<br />
obstacles. These are the lack <strong>of</strong> known tumor-associated<br />
antigens (TAA) from most solid malignancies and the lack <strong>of</strong><br />
efficient methodologies to transfer antigen from tumor to DC.<br />
Here we describe the development <strong>of</strong> an alternative technology<br />
for antigen delivery that appears to transfer a broad spectrum <strong>of</strong><br />
tumor antigens to DC with extremely high efficiency. We used<br />
this methodology, termed “nanoparticle (NP)-mediated tumor<br />
delivery,” to encapsulate whole melanoma protein lysates into<br />
PLGA biodegradable microspheres and targeted these conjugates<br />
to DC. Optimization <strong>of</strong> Ag delivery was achieved by<br />
experimental determination <strong>of</strong> which polymer formulation, Ag<br />
source and release characteristics led to most potent DC antigen<br />
presentation. In both murine and human T cell stimulation<br />
assays, DC loaded with NP/tumor conjugates were superior to<br />
other Ag forms in stimulating anti-tumor T cells — including<br />
murine gp100-specific naive T cells from pmel transgenics and<br />
human TIL and blood CD8+ T cells from melanoma patients.<br />
Analysis <strong>of</strong> cytokine production by DC and T cell by cytometric<br />
bead array (CBA) assays indicated that NP-loaded DC drove<br />
increased production <strong>of</strong> Th1 and proinflammatory cytokines<br />
such as IFN-g and IL-2. Although melanoma Ags were utilized as<br />
controls, a major potential advantage <strong>of</strong> NP-based antigen<br />
delivery is the fact that antigens need not be defined prior to DC<br />
loading, opening the door to vaccine targeting <strong>of</strong> virtually any<br />
solid tumor.<br />
doi:10.1016/j.clim.2007.03.361<br />
OR.44 Safety and In Vivo Efficacy <strong>of</strong> Innate (Class R)<br />
and Broadly Reactive (Class B) Inhibitory<br />
Oligonucleotides (INH-ODN) in MRL-Fas/lpr Lupus<br />
Mice<br />
Petar Lenert, Assistant Pr<strong>of</strong>essor, Department <strong>of</strong> Internal<br />
Medicine, The University <strong>of</strong> Iowa, Iowa City, IA, Sarah<br />
Seyfer, BS, Internal Medicine, Iowa City, IA, Elizabeth R.<br />
Pareigis, BS, Internal Medicine, The University <strong>of</strong> Iowa, Iowa<br />
City, IA, Robert F. Ashman, Pr<strong>of</strong>essor, Internal Medicine, The<br />
University <strong>of</strong> Iowa, Iowa City, IA
Abstracts<br />
Several lines <strong>of</strong> evidence indicate that intracytoplasmic<br />
TLR receptors 7 and 9 play an important role in the<br />
pathogenesis <strong>of</strong> SLE, both in vitro and in vivo. For example,<br />
MRL-Fas/lpr mice lacking TLR9 have a shorter life span and<br />
develop more aggressive kidney disease. On contrary, mice<br />
lacking TLR7 have a milder disease. INH-ODNs containing<br />
nuclease-resistant phosphorothioate backbone (PS) were<br />
recently developed that can block TLR7 and/or TLR9<br />
activation at low nanomolar concentrations. Here we show<br />
that under certain stimulatory conditions prototypic TLR9specific<br />
INH-ODNs can also block TLR7 activation <strong>of</strong> mouse<br />
macrophages induced by several (but not all) TLR7 ligands<br />
like imiquimod, CL075, CL097, CL087 and loxoribine. Interestingly,<br />
in primary B cells, certain TLR7/8 ligands, but not<br />
phosphodiester (PO) CpG-ODNs, could escape inhibition<br />
when INH-ODNs were made with the natural PO backbone.<br />
Such INH-ODNs could actually enhance B cell G1–M entry,<br />
survival and polyclonal IgM secretion induced by suboptimal<br />
TLR7/8 stimulation. Both TLR7/8 inhibition and enhancement<br />
were independent <strong>of</strong> the TLR9 expression. We have<br />
recently modified these INH-ODNs to allow formation <strong>of</strong><br />
stable secondary structures. In vitro, these modified innate<br />
INH-ODNs (class R) showed higher potency for macrophages,<br />
dendritic cells and marginal zone B cells over follicular B<br />
cells. However, in vivo, both linear and Class R INH-ODNs<br />
promoted better survival and ameliorated spontaneous lupus<br />
disease in MRL-Fas/lpr mice. It remains to be determined<br />
whether this effect required TLR7/8 or TLR9. This work was<br />
supported by the NIH grant 1R56AI064736-01A2 and by a<br />
grant from the Carver Trust fund.<br />
doi:10.1016/j.clim.2007.03.362<br />
OR.45 Tumor Cell Lysate-specific DTH Reaction<br />
Correlates with Longer Survival and Disease<br />
Stability in Vaccinated Melanoma Patients<br />
Flavio Salazar-Onfray, PhD, Universidad de Chile, Santiago<br />
de Chile, Chile<br />
Dendritic cell (DCs)-based therapy has proven to be<br />
effective in patients with malignancies. However, an optimal<br />
immunization protocol using DCs and the best means for<br />
delivering antigens has not been yet described. In this study, 37<br />
patients with malignant melanoma in stage IV were vaccinated<br />
with autologous DCs pulsed with a melanoma cell lysate, to<br />
assess toxicity, immunological and clinical responses. The<br />
responses <strong>of</strong> the patients were analyzed by ELISPOT and<br />
Delayed type IV hypersensitivity reaction (DTH) against the<br />
melanoma cell lysate. After vaccination, 50% <strong>of</strong> tested<br />
patients (18 out <strong>of</strong> 37) showed a positive DTH against the<br />
melanoma cell lysate. All patients showed a positive DTH<br />
reaction against the used adjuvant KLH demonstrating that all<br />
patients were immunocompetents. Significant correlations<br />
were found between positive DTH responses against tumor<br />
lysate and both disease stability and post-vaccination survival<br />
on stage IV patients. In fact, the mean <strong>of</strong> post-vaccination<br />
survival was 15 months for all the vaccinated patients, double<br />
than the historical survival for stage IV melanoma patients.<br />
More important, positive DTH patients showed a postvaccination<br />
survival <strong>of</strong> 25 months compared to 8 months<br />
observed for the negative DTH group <strong>of</strong> patients. There<br />
were no toxicities associated with the vaccines or evidence<br />
<strong>of</strong> autoimmunity including vitiligo. Our data suggest that<br />
autologous DCs pulsed with tumour lysate may provide a<br />
standardized and widely applicable source <strong>of</strong> melanoma<br />
specific antigens for clinical use, it is safe and causes no<br />
significant side effects, demonstrating to be partially efficient<br />
at triggering effective anti-melanoma immunity.<br />
doi:10.1016/j.clim.2007.03.363<br />
OR.46 In Vivo CTLA4Ig Treatment Substantially<br />
Inhibits CD4+CD25+ Regulatory T Cell Turnover but<br />
Not Function<br />
Anita Tang, Surgical Resident, Division <strong>of</strong> Transplantation,<br />
Department <strong>of</strong> Surgery, University <strong>of</strong> Maryland School <strong>of</strong><br />
Medicine, Baltimore, MD, Modesta Ndejembi, Graduate<br />
Student, Department <strong>of</strong> Microbiology and <strong>Immunology</strong>,<br />
University <strong>of</strong> Maryland School <strong>of</strong> Medicine, Baltimore, MD,<br />
Donna Farber, Associate Pr<strong>of</strong>essor, Division <strong>of</strong><br />
Transplantation, Department <strong>of</strong> Surgery, University <strong>of</strong><br />
Maryland School <strong>of</strong> Medicine, Baltimore, MD<br />
CTLA4Ig and its variant, LEA29Y, target the CD28/B7<br />
costimulatory pathway and are highly effective treatments<br />
in rheumatoid arthritis and transplantation, respectively.<br />
Since CD28/B7 signaling has been implicated in regulatory T<br />
cell (Treg) generation, we investigated the effects <strong>of</strong><br />
CTLA4Ig on Treg phenotype, proliferation and function.<br />
BALB/c mice were administered CTLA4Ig (250 μg/dose)<br />
every other day for 3 doses, with IgG2a and PBS as controls.<br />
Splenic CD4 T cells were purified 3–14 days later and<br />
analyzed for CD25, CTLA4 and Foxp3 expression. Following<br />
CTLA4Ig treatment, CD4+Foxp3+ Tregs decreased 60% and<br />
CTLA4 expression by 30% compared to controls, with the<br />
most significant depletion (80%) in the CTLA4+Foxp3+ subset.<br />
Additionally, CD25 expression and CD25+Foxp3+ Tregs<br />
dropped 50%. Similar findings were observed in thymectomized<br />
mice, suggesting that CTLA4Ig mediates its effects on<br />
peripheral Tregs. To assess the mechanistic effects <strong>of</strong><br />
CTLA4Ig, CFSE-labeled CD4+CD25+ and CD4+ CD25− T cells<br />
were transferred into syngeneic mice receiving CTLA4Ig and<br />
harvested 6 days later. Interestingly, we noted extensive and<br />
rapid in vivo proliferation <strong>of</strong> CD4+CD25+ Tregs (62%)<br />
compared to the CD4+CD25− subset (10%) in controltreated<br />
mice, suggesting that Treg proliferation is an<br />
antigen-driven phenomenon. Moreover, we observed that<br />
this rapid turnover <strong>of</strong> Tregs was almost completely blocked<br />
by CTLA4Ig. Despite a decreased yield, expression <strong>of</strong> CTLA4<br />
and turnover after CTLA4Ig treatment, the remaining Tregs<br />
were functionally competent and suppressed CD4+CD25− T<br />
cell proliferation comparable to controls in co-culture<br />
assays. These findings may have important implications<br />
regarding the constitution <strong>of</strong> Treg subsets and their balance<br />
within non-regulatory populations in patients receiving<br />
CTLA4Ig and LEA29Y.<br />
doi:10.1016/j.clim.2007.03.364<br />
S65
S66 Abstracts<br />
OR.47 A Novel Anti-Tumor Vaccine Using IDO<br />
Silenced DC<br />
Xiufen Zheng, Postdoctoraltoral Fellow, University <strong>of</strong><br />
Western Ontario, London, ON, Canada, Bertha Garcia,<br />
Pr<strong>of</strong>essor, Departments <strong>of</strong> Surgery, Microbiology, Pathology,<br />
University <strong>of</strong> Western Ontario, London, ON, Canada, Costin<br />
Vladau, MS Student, Departments <strong>of</strong> Microbiology and<br />
<strong>Immunology</strong>, University <strong>of</strong> Western Ontario, London, ON,<br />
Canada, James Koropatnick, Pr<strong>of</strong>essor, Departments <strong>of</strong><br />
Microbiology and <strong>Immunology</strong>, Pathology, Oncology,<br />
University <strong>of</strong> Western Ontario, London, ON, Canada, Dong<br />
Chen, PDF, Departments <strong>of</strong> Surgery, University <strong>of</strong> Western<br />
Ontario, London, ON, Canada, Motohiko Suzuki1, PDF,<br />
Departments <strong>of</strong> Surgery, University <strong>of</strong> Western Ontario,<br />
London, ON, Canada, Mu Li, PDF, Departments <strong>of</strong> Surgery,<br />
University <strong>of</strong> Western Ontario, London, ON, Canada,<br />
Wei-ping Min, Pr<strong>of</strong>essor, Departments <strong>of</strong> Surgery,<br />
Microbiology and <strong>Immunology</strong>, Pathology, Oncology,<br />
University <strong>of</strong> Western Ontario, London, ON, Canada, Xusheng<br />
Zhang, Postdoctoral Fellow, Department <strong>of</strong> Surgery,<br />
University <strong>of</strong> Western Ontario, London, Canada<br />
Hyporesponsiveness is a major hallmark in dendritic cell<br />
(DC)-mediated anticancer immunotherapy. Indoleanime 2,3dioxygenase<br />
(IDO), an immunosuppressive molecule<br />
expressed by DC, is a critical factor mediating hyporesponsiveness<br />
to cancer immune therapy. We hypothesized that<br />
antisense silencing <strong>of</strong> IDO in DCs would enhance anticancer<br />
therapy. In this study, DC were cultured in vitro, exposed to<br />
melanoma B16 lysate, transfected with IDO siRNA, and<br />
injected into tail veins <strong>of</strong> C57/BL6 mice. Mice were then<br />
challenged with B16 tumor cells. The anticancer effects <strong>of</strong><br />
IDO-silenced DC therapy, as compared with non-silenced,<br />
conventional control DC vaccine, were evidenced by (i)<br />
postponed melanoma tumor onset time, and (ii) decreased<br />
tumor size. In addition, after immunization with IDOsilenced<br />
DC, the number <strong>of</strong> CD8+ T cells was significantly<br />
greater than in animals immunized with control RNA-treated<br />
DC and CD4+ and CD8+ T cell apoptosis in draining lymph<br />
nodes was significantly lower. Furthermore, immunization<br />
with IDO-silenced DC enhanced tumor antigen-specific T cell<br />
proliferation and CTL activity and decreased numbers <strong>of</strong> CD4<br />
+CD25+ Foxp3+ regulatory T cells (Treg). In conclusion, this<br />
study is the first to demonstrate a novel anti-tumor vaccine<br />
by silencing an immunosuppressive gene (IDO) in DC, which<br />
enhanced anti-tumor immunity, reduced T cell apoptosis and<br />
Treg cell formation, and prevented tumor growth. IDOsilenced<br />
DC has clinical potential as an immune-based therapy.<br />
doi:10.1016/j.clim.2007.03.365<br />
OR.48 Post-Transcriptional Regulation <strong>of</strong> T Cell<br />
Receptor zeta Chain in Systemic Lupus<br />
Erythematosus<br />
Vaishali R. Moulton, Postdoctoral Fellow, Beth Israel<br />
Deaconess Medical Center, Medicine, Boston, MA, Vasileios<br />
C. Kyttaris, Instructor, Beth Israel Deaconess Medical<br />
Center, Medicine, Boston, MA, Yuang-Taung Juang,<br />
Instructor, Beth Israel Deaconess Medical Center, Medicine,<br />
Boston, MA, George C. Tsokos, Pr<strong>of</strong>essor, Beth Israel<br />
Deaconess Medical Center, Medicine, Boston, MA<br />
Systemic lupus erythematosus (SLE) is a complex autoimmune<br />
disease characterized by the production <strong>of</strong> autoantibodies,<br />
immune complex deposition, T cell infiltration<br />
and abnormal production <strong>of</strong> cytokines all leading to<br />
inflammatory damage <strong>of</strong> target organs (joints, kidneys<br />
and brain). SLE T cells are deficient in their expression <strong>of</strong><br />
the T cell receptor (TCR)/CD3 zeta chain protein (CD3Z), a<br />
critical molecule for the initiation <strong>of</strong> intracellular signaling<br />
upon antigen encounter. This deficiency is partially due to<br />
differential splicing <strong>of</strong> the CD3Z mRNA; SLE T cells express<br />
an alternatively spliced form <strong>of</strong> CD3Z mRNA which is missing<br />
562 nucleotides (672 to 1233) within its 3′ untranslated<br />
region (UTR). Studies by our group have shown that this<br />
alternatively spliced CD3Z mRNA is unstable and has<br />
reduced translation efficiency. We have also demonstrated<br />
that two adenosine-uridine-rich elements (AREs) at positions<br />
705 and 985 in the 3′ UTR are independently essential<br />
for its stability and translation. We hypothesized that<br />
proteins binding to these two AREs may stabilize the CD3Z<br />
mRNA. Electrophoretic mobility shift assays showed several<br />
protein complexes in jurkat cell nuclear extracts binding to<br />
32P-labeled oligonucleotides containing the ARE (705)<br />
sequence, which were outcompeted in the presence <strong>of</strong><br />
similar unlabeled oligonucleotides. Using an oligonucleotide<br />
pulldown assay, mass spectrophotometry and peptide<br />
sequencing, we identified HuR as one <strong>of</strong> the putative<br />
proteins binding to the ARE (705) oligonucleotide. Immunoblotting<br />
with specific antibody confirmed this finding.<br />
These results reveal molecular mechanisms that may<br />
contribute to CD3Z mRNA stability and altered TCRZ chain<br />
expression in lupus.<br />
doi:10.1016/j.clim.2007.03.366<br />
Biomarkers & New Technologies<br />
Saturday, June 9<br />
2:45 pm–4:45 pm<br />
OR.49 Autoimmune Development in Phenotypic<br />
Type 2 Diabetes Patients<br />
Barbara Brooks-Worrell, Research Scientist, University <strong>of</strong><br />
Washington/VA Puget Sound Health Care System, Seattle,<br />
WA, Heba Ismail, Research Fellow, VA Puget Sound Health<br />
Care System, Seattle, WA, Michael Wotring, Research<br />
Scientist, University <strong>of</strong> Washington, Seattle, WA, A.U.<br />
Liemin, Data Processor, University <strong>of</strong> Washington/VA Puget<br />
Sound Health Care System, Seattle, WA, Crystal Kimmie,<br />
Research Scientist, VA Puget Sound Health Care System,<br />
Seattle, WA, Jamie Felton, Research Associate, VA Puget<br />
Sound Health Care System, Seattle, WA, Jerry Palmer,<br />
Pr<strong>of</strong>essor <strong>of</strong> Medicine, University <strong>of</strong> Washington/VA Puget<br />
Sound Health Care System, Seattle, WA<br />
Type 1 (T1DM) diabetes is an autoimmune disease,<br />
whereas type 2 (T2DM) diabetes is considered nonautoimmune.<br />
However, cross-sectional studies have identified<br />
autoimmune diabetes in 10–15% <strong>of</strong> phenotypic T2DM<br />
patients. In this study, we evaluated phenotypic autoimmune<br />
negative T2DM patients prospectively to determine<br />
the importance <strong>of</strong> repeated testing to detect autoimmune
Abstracts<br />
diabetes. In 40 phenotypic T2DM patients we determined<br />
autoantibody (IA-2/IAA/GAD), T cell status (cellular immunoblotting),<br />
cytokine responses (ELISPOT), fasting C-peptide,<br />
and glucagon-stimulated C-peptide responses at<br />
baseline and every 3 months for an average follow-up <strong>of</strong><br />
23.3 months (range 6–36 months). Of the 40 patients, 14/<br />
40 (35%) were positive for autoantibody (Ab) and/or T cell<br />
(T) reactivity at baseline while 26/40 (65%) were Ab-T−. Of<br />
the 26 patients Ab-T− at baseline, 8/26 (30.7%) remained<br />
Ab-T− stable (persistently negative), 4/26 patients demonstrated<br />
transient Ab or T cell positivity (1 positive time<br />
point during follow-up), while 14/26 developed stable<br />
(persistent positivity after conversion) Ab+ and/or T+. We<br />
have categorized the patients with stable negative or<br />
transient Ab and/or T reactivity as non-autoimmune nonprogressors<br />
(NANP) and the patients developing stable Ab<br />
and/or T cell positivity as progressors. We observed that<br />
the mean fasting and glucagon-stimulated C-peptide<br />
responses were significantly (pb0.0001) lower, the INF-g<br />
responses to sonicated islets and GAD were significantly<br />
higher (pb0.05), and the IL-10 responses (pb0.05) to islets<br />
were significantly lower in the progressors compared to<br />
NANP. These results demonstrate that continued follow-up<br />
<strong>of</strong> T2DM patients is needed to identify development <strong>of</strong><br />
autoimmunity in patients previously classified as nonautoimmune.<br />
doi:10.1016/j.clim.2007.03.367<br />
OR.50 Derangements in Cytokine-induced STAT<br />
Protein Phosphorylation in Human Systemic Lupus<br />
Erythematosus<br />
Veronika Sharp, Fellow, Stanford University Medical School,<br />
Palo Alto, CA, Paul J. Utz, Stanford University Medical<br />
School, Palo Alto, CA, G.P. Nolan, The Baxter Laboratory for<br />
Genetic Pharmacology, CA, OD Perez, The Baxter Laboratory<br />
for Genetic Pharmacology, CA<br />
Systemic lupus erythematosus (SLE) is an autoimmune<br />
disease with variable clinical manifestations, many <strong>of</strong> which<br />
are devastating to patients. Currently it is difficult to<br />
predict disease onset, severity, or response to therapy. New<br />
and better markers are needed in clinical practice for<br />
improved predictability. Phospho-specific flow cytometry is<br />
a powerful technique that allows for the analysis <strong>of</strong> several<br />
intracellular signaling networks in multiple cellular subsets<br />
simultaneously in response to functionally relevant stimuli.<br />
Using this technique we interrogated signaling through<br />
different members <strong>of</strong> the Jak-STAT and MAPK pathways in<br />
response to cytokines (including IFN α, IFN γ, IL-4, IL-6 and<br />
IL-10) in peripheral blood mononuclear cells (PBMC)<br />
isolated from 14 patients with SLE and 11 healthy controls.<br />
CD3, CD4, CD8 and CD33 were used as surface markers to<br />
differentiate T cells subsets, B cells and monocytes. While<br />
PBMC from healthy and diseased individuals had similar<br />
phosphorylation pr<strong>of</strong>iles in the resting state, there were<br />
remarkable differences in cellular response once cells were<br />
stimulated. Among other differential responses, hyperphosphorylation<br />
<strong>of</strong> some STAT proteins was observed in<br />
response to IFN α. This is consistent with prior studies<br />
implicating a role for type I interferons in SLE pathogenesis.<br />
Our data demonstrate the feasibility <strong>of</strong> analyzing several<br />
signaling networks simultaneously in viable PBMC subsets in<br />
response to an array <strong>of</strong> cytokines. These differences may<br />
not be visible when only whole and resting PBMC are used.<br />
This study takes us one step closer to defining individual<br />
biosignatures for SLE patients that would allow for more<br />
customized immunotherapy.<br />
doi:10.1016/j.clim.2007.03.368<br />
OR.51 Evidence for an Increase in the Functional<br />
Avidity <strong>of</strong> the GAD-reactive CD4+ T Cell Population<br />
Prior to Diabetes Onset<br />
Nathan Standifer, Postdoctoral Research Associate,<br />
Benaroya Research Institute, Seattle, WA, Gerald Nepom,<br />
Director, Benaroya Research Institute, Seattle, WA<br />
Type 1 diabetes (T1D) is an autoimmune disease characterized<br />
by the cell-mediated destruction <strong>of</strong> insulin-producing<br />
β cells <strong>of</strong> the pancreatic islets. Immunologically, islet<br />
destruction is marked by increased auto-antibody titers and<br />
the expansion <strong>of</strong> glutamic acid decarboxylase (GAD)-reactive<br />
CD4+ T cells in many pre-diabetic subjects. We hypothesized<br />
that the functional avidity <strong>of</strong> the GAD-reactive T cell<br />
population increases over time in pre-diabetic subjects as<br />
the islet β cell mass decreases. To test this, we cultured<br />
longitudinal peripheral blood lymphocyte samples drawn<br />
from auto-antibody+, at-risk, pre-diabetic or diabetic subjects<br />
and calculated the GAD-reactive CD4+ Tcell population<br />
ED50 values: the concentration <strong>of</strong> peptide that induces onehalf<br />
maximal interferon-γ secretion. Longitudinal samples<br />
from an at-risk subject exhibited the highest Tcell population<br />
ED50 values (4.26 1/4M and 8.76 1/4M) while the ED50 values<br />
<strong>of</strong> samples from a T1D patient drawn 4 or 5 years after<br />
diagnosis were substantially lower (2.24 nM and 48.30 nM<br />
respectively). Interestingly, the ED50 value <strong>of</strong> the GADreactive<br />
T cell population cultured from a pre-diabetic<br />
subject 6 years prior to diagnosis was significantly higher<br />
than that <strong>of</strong> a sample drawn 2 years prior to diagnosis (5.04<br />
¼M and 15.38 nM respectively). We also observed that the<br />
frequency <strong>of</strong> GAD-reactive, central-memory cells increased<br />
over time in the pre-diabetic samples. Our data imply that, as<br />
the islet β cell mass decreases, the GAD-reactive T cell<br />
population undergoes a phenotypic shift from effector to<br />
central memory cells concomitant with an increase in the<br />
functional avidity as measured by interferon-γ secretion.<br />
doi:10.1016/j.clim.2007.03.369<br />
S67<br />
OR.52 Peripheral Blood Flow Cytometric Evaluation<br />
<strong>of</strong> a Large Cohort <strong>of</strong> Hyper Immunoglobulin E<br />
Syndrome Patients<br />
Nina N. Brodsky, Researcher, NIH/NIAID/LCID, Bethesda,<br />
MD, Margaret R. Brown, Biologist, NIH/CC/DLM, Bethesda,<br />
MD, Steven M. Holland, Chief, LCID, NIH/NIAID/LCID,<br />
Bethesda, MD, Gulbu Uzel, Staff Clinician, NIH/NIAID/LCID,<br />
Bethesda, MD
S68 Abstracts<br />
Job’s Syndrome, or the Hyper-IgE Syndrome (HIES), is an<br />
autosomal dominant disorder characterized by elevated<br />
serum IgE levels, eosinophilia, dermatitis, skeletal abnormalities,<br />
staphylococcal skin infections, and lung cysts. An in<br />
vitro immunologic phenotype has not been established, and<br />
earlier findings have included only a small number <strong>of</strong> HIES<br />
patients. We have performed flow cytometric analysis <strong>of</strong><br />
lymphocyte subsets and eosinophils by using relevant cell<br />
surface markers in a cohort <strong>of</strong> thirty HIES patients and<br />
compared them to healthy controls using a non-parametric<br />
t test (Mann–Whitney U). Absolute lymphocyte counts did<br />
not differ between the two groups (pN0.3). We have found<br />
that patients’ eosinophils have significantly higher expression<br />
<strong>of</strong> CD23, HLA-DR, and CD69 compared to controls<br />
(p=0.0002, 0.0001 and b0.0001, respectively). We have<br />
also found that percentages <strong>of</strong> natural killer cells<br />
(pb0.0001) and CD3+ CD54+ T cells (p b0.0001) were<br />
lower in HIES. We have detected significantly smaller<br />
number <strong>of</strong> CD8 and CD4 memory T cells (CD8+CD45RO+ and<br />
CD4+CD45RO+) (pb0.0001 and pb0.04 respectively), more<br />
naive CD4 T cells (CD4+ CD45RA+) (p=0.001) and CD8+CD57+<br />
cells (p=0.0007) in patients. HIES patients had significantly<br />
more T cells expressing CD147 (a plasma membrane glycoprotein<br />
shown to induce extracellular matrix metalloproteinases<br />
[MMPs], pb0.04). Our findings <strong>of</strong> decreased number <strong>of</strong><br />
memory T cells in HIES, similar to Hyper IgM syndrome and<br />
ectodermal dysplasia with immunodeficiency (EDID), suggest a<br />
potential defect in the NFkB pathway. On the other hand,<br />
increased CD147 expression points at pathways responsible for<br />
regulation <strong>of</strong> MMPs, which may be critical for tissue<br />
remodeling and regeneration defects in HIES.<br />
doi:10.1016/j.clim.2007.03.370<br />
OR.53 Global Assessment <strong>of</strong> B Cell Alloimmunity<br />
Using Human Protein Microarrays<br />
Persis P. Wadia, Postdoctoral Fellow, Stanford University,<br />
Miklos Lab, Stanford, CA, Marc Coram, Stanford University,<br />
Stanford, CA, Marina Sirota, Stanford University, Stanford,<br />
CA, David B. Miklos, Assistant Pr<strong>of</strong>essor, Stanford<br />
University, Stanford, CA, Atul Butte, Stanford University,<br />
Stanford, CA<br />
Allogeneic hematopoietic cell transplantation (HCT) can<br />
cure hematologic malignancies through beneficial graft-vleukemia<br />
(GVL) allo-immune responses, but is limited by<br />
graft-versus-host disease (GVHD). Previous studies demonstrate<br />
that allogeneic antibodies (Ab) develop against minor<br />
histocompatibility antigens (mHA) encoded on the Y-chromosome<br />
(H-Y antigens) after sex-mismatch HCT in association<br />
with chronic GVHD and persistent disease remission. We<br />
hypothesize that novel mHA can be serologically identified as<br />
targets <strong>of</strong> allo-Ab responses that develop post-transplant.<br />
ProtoArray displays 5000 full-length human proteins with Nterminal<br />
GSTepitopes. Technical replicates <strong>of</strong> plasma measured<br />
at 1:50, 1:150, 1:500 confirmed technical feasibility with R 2<br />
N0.95. In this study, plasma collected from four AML patients<br />
1 year post-transplant, pre-transplant, and their donors were<br />
detected for antibody specificity against 5000 human proteins<br />
displayed on the ProtoArray. Pre-post fluorescence differences<br />
ranging from 0.5 to 3 logs identified 60–75 targets <strong>of</strong> allo-Ab.<br />
Over 90% <strong>of</strong> allo-Ab targets have known non-synonymous<br />
SNPs. Chromatin Assembly Factor 1, subunit B (p60)<br />
(CHAF1B), and Nucleolar and Spindle Associated Protein 1<br />
(NuSAP1) were recognized by two <strong>of</strong> the four patients after<br />
HCT. These allo-Abs were absent pre and 2 months post-HCT,<br />
developed in association with cGVHD at 11 months, and<br />
persisted through 16 months. CHAF1B and NuSAP have been<br />
recombinantly expressed and specifically detected by post-<br />
HCT patient sera by western blot validating their discovery as<br />
allo-antigens. Ongoing studies are characterizing CHAF1B and<br />
NuSAP1 Ab by ELISA in HCT patient samples, and normals. In<br />
summary, protein microarrays are innovative tools for highthroughput<br />
global assessment <strong>of</strong> B cell alloimmunity and mHA<br />
discovery.<br />
doi:10.1016/j.clim.2007.03.371<br />
OR.54 Improving Targeted Immunotherapy Through<br />
Direct Validation <strong>of</strong> Peptide–MHC Epitopes Using<br />
TCRm Antibodies<br />
Tiffany Nguyen, Senior Research Technician, Receptor<br />
Logic, Ltd, Amarillo, TX, Tito Woodburn, Research<br />
Associate, Receptor Logic, Ltd, Amarillo, TX, Francisca<br />
Neethling, Scientist, Receptor Logic, Ltd, Amarillo, TX,<br />
Jon A. Weidanz, Assistant Pr<strong>of</strong>essor, Texas Tech<br />
University Health Sciences Center, Amarillo, TX<br />
Adoptive T cell immunotherapy represents a promising<br />
approach for the treatment <strong>of</strong> cancer and chronic infections.<br />
Improved response rates using this approach might occur if<br />
the specific peptide–MHC targets on the diseased cells were<br />
known. We have developed technology to directly assess the<br />
presentation <strong>of</strong> specific peptide–MHC complexes on tumor<br />
cells using antibodies that mimic the binding specificity <strong>of</strong> T<br />
cell receptors designated as TCRmimics (TCRms). A pro<strong>of</strong>-<strong>of</strong>concept<br />
validation assay was developed to directly examine<br />
the presentation and density <strong>of</strong> MHC class I peptides derived<br />
from the processing <strong>of</strong> a model tumor antigen, hCGb, on the<br />
surface <strong>of</strong> various human tumor cell lines. In this study we<br />
used previously characterized TCRm to three peptide–HLA-<br />
A*0201 epitopes derived from hCGb and designated as TMT<br />
(60–68), VLQ (64–72), and GVL (67–75). For the assessment<br />
<strong>of</strong> antigen presentation function, hCGb positive tumor cell<br />
lines were stained for detection and quantitation <strong>of</strong> each<br />
peptide–HLA epitope. To our surprise, presentation <strong>of</strong> each<br />
peptide–epitope varied widely with no clear dominant<br />
peptide–epitope being expressed on the different tumor<br />
cell lines. A human breast carcinoma cell line expressed all<br />
three peptide–HLA-A2 epitopes with GVL peptide being<br />
dominant. In contrast, only GVL peptide–epitope was<br />
detected on an ovarian carcinoma cell line while only TMT<br />
peptide–epitope was found expressed on a colon carcinoma<br />
cell line. Taken together, these findings illustrate a heterogeneous<br />
pattern <strong>of</strong> peptide–HLA expression and signify the<br />
need for development <strong>of</strong> reagents that can directly assess<br />
specific peptide–HLA expression and lead to better outcomes<br />
with T cell immunotherapy.<br />
doi:10.1016/j.clim.2007.03.372
Abstracts<br />
OR.55 Transcription Pr<strong>of</strong>iles <strong>of</strong> Rheumatoid Arthritis<br />
Patients Reveal Genes Characterizing Different<br />
Response to Anti-TNF Therapy<br />
Franak Batliwalla, Assistant Investigator, Feinstein Institute<br />
for Medical Research, Manhasset, NY, Peter Gregersen,<br />
Investigator, Feinstein Institute for Medical Research,<br />
Manhasset, NY, Normand Allaire, Scientist, BiogenIdec, Drug<br />
Discovery, Cambridge, NY, Wentian Li, Assistant Investigator,<br />
Feinstein Institute for Medical Research, Manhasset, NY,<br />
Houman Khalili, Steve Perrin, Associate Director, BiogenIdec,<br />
Drug Discovery, Cambridge, MA, Marlena Kern, Research<br />
Associate, Feinstein Institute for Medical Research,<br />
Manhasset, NY, Aarti Damle, Research Associate, Feinstein<br />
Institute for Medical Research, Manhasset, NY, John Carulli,<br />
Principal Scientist, BiogenIdec, Drug Discovery, Cambridge,<br />
MA, Jadwiga Bienkowska, Principal Scientist, Biogenidec,<br />
Drug Discovery, Cambridge, MA<br />
The Autoimmune Biomarkers Collaborative Network<br />
(ABCoN) has enrolled a longitudinal cohort <strong>of</strong> RA patients<br />
beginning anti-TNF treatment in order to identify biomarkers<br />
influencing response to anti-TNF therapy. 116 patients<br />
beginning anti-TNF therapy (54 etaneracept, 25 adalimumab,<br />
37 infliximab) were followed for 14 weeks, with DAS28<br />
measurements and RNA collection at three time points: pretreatment,<br />
6 weeks and 14 weeks post-treatment. Using the<br />
hgu133plus2 Affymetrix chips we have completed genomewide<br />
transcript pr<strong>of</strong>iling for these 116 patients as well as 65<br />
healthy controls. Defining response as a DDAS28 N40% and<br />
non-response as DDAS28 b20%, analysis <strong>of</strong> the gene expression<br />
differences between patients and controls indicates that the<br />
overall number <strong>of</strong> differentially expressed genes is different<br />
for responders and non-responders. Furthermore, the responders<br />
are characterized by a unique list <strong>of</strong> genes differentially<br />
expressed as compared to controls at 14 weeks posttreatment.<br />
This observation suggests that anti-TNF therapy<br />
recruits specific biological pathways in responders. In order<br />
to identify the biomarkers that predict the response to anti-<br />
TNF therapy we have analyzed the gene expression pr<strong>of</strong>iles <strong>of</strong><br />
responders and non-responders using blood collected at the<br />
pre-treatment visit. Using the machine learning technique<br />
Random Forest we have identified a set <strong>of</strong> over 100 genes that<br />
are predictive <strong>of</strong> the anti-TNF response. Using a selected set<br />
<strong>of</strong> 25 candidate biomarkers we can distinguish the responders<br />
versus non-responders with 75% accuracy. We are validating<br />
the proposed biomarkers using an independent cohort <strong>of</strong><br />
patients as well as low-density RT-PCR arrays.<br />
doi:10.1016/j.clim.2007.03.373<br />
New Animal Models <strong>of</strong> Disease<br />
Saturday, June 9<br />
2:45 pm−4:45 pm<br />
OR.56 Development <strong>of</strong> Latest-generation<br />
HU-PBMC-NOD/SCID Mice to Study Human Islet<br />
Allo-reactivity<br />
Todd Pearson, Postdoctoral Fellow, University <strong>of</strong> Massachusetts<br />
Medical School, Diabetes Division, Worcester, MA, Marie King,<br />
MD/PhD Student, University <strong>of</strong> Massachusetts Medical School,<br />
Diabetes Division, Worcester, MA, Leonard Shultz, Senior Staff<br />
Scientist, The Jackson Laboratory, Bar Harbor, ME, Jean Leif,<br />
Lab Manager, University <strong>of</strong> Massachusetts Medical School,<br />
Diabetes Division, Worcester, MA, Dale Greiner, Pr<strong>of</strong>essor,<br />
University <strong>of</strong> Massachusetts Medical School, Diabetes Division,<br />
Worcester, MA, John Mordes, Pr<strong>of</strong>essor, University <strong>of</strong><br />
Massachusetts Medical School, Diabetes Division, Worcester,<br />
MA, Aldo Rossini, Pr<strong>of</strong>essor, University <strong>of</strong> Massachusetts<br />
Medical School, Diabetes Division, Worcester, MA, Mark<br />
Atkinson, Pr<strong>of</strong>essor, University <strong>of</strong> Florida College <strong>of</strong> Medicine,<br />
Department <strong>of</strong> Pathology, <strong>Immunology</strong> and Laboratory<br />
Medicine, Gainesville, FL, Clive Wasserfall, Assistant in<br />
Pathology, University <strong>of</strong> Florida College <strong>of</strong> Medicine,<br />
Department <strong>of</strong> Pathology, <strong>Immunology</strong> and Laboratory<br />
Medicine, Gainesville, FL, Massimo Trucco, Pr<strong>of</strong>essor,<br />
University <strong>of</strong> Pittsburgh School <strong>of</strong> Medicine, Pediatrics,<br />
Pittsburgh, PA, Kevan Herold, Pr<strong>of</strong>essor, Yale University School<br />
<strong>of</strong> Medicine, Department <strong>of</strong> Internal Medicine, New Haven, CT,<br />
Rita Botti, Assistant Pr<strong>of</strong>essor, University <strong>of</strong> Pittsburgh,<br />
Pediatrics, Pittsburgh, PA<br />
Small animal models have been used to study a number <strong>of</strong><br />
human diseases, including autoimmune diseases such as type<br />
1 diabetes (T1D). Unfortunately, translating therapies from<br />
animal models to human patients has been hindered by<br />
differences in rodent and human immune systems. “Humanized”<br />
mouse models hold great promise in the development<br />
<strong>of</strong> efficacious therapies to treat a wide array <strong>of</strong> human<br />
immune-mediated conditions, without putting human subjects<br />
at risk during protocol development. However, developing<br />
a system that faithfully recapitulates human immunity<br />
in a murine host has proven difficult. Recently, the generation<br />
<strong>of</strong> a new stock <strong>of</strong> immunodeficient hosts, the NOD.Cg-<br />
Prkdcscid Il2rgtm1Wjl/Sz (NOD-scid Il2rgnull) strain, has<br />
overcome many <strong>of</strong> the previous limitations <strong>of</strong> humanized<br />
mice. We have characterized the engraftment <strong>of</strong> human<br />
PBMC into NOD-scid Il2rgnull hosts and document that this<br />
stock supports higher human cell engraftment at lower cell<br />
input levels. Importantly, we further demonstrate that<br />
human PBMC-engrafted NOD-scid Il2rgnull mice allow for<br />
allogeneic rejection <strong>of</strong> transplanted HLA-mismatched human<br />
islets, even when the islets are allowed to heal-in prior to<br />
PBMC engraftment. Collectively, these data suggest that<br />
humanized NOD-scid Il2rgnull mice may be superior to<br />
previous immunodeficient recipients for generation <strong>of</strong><br />
humanized mice for studies <strong>of</strong> in vivo human immune<br />
function.<br />
doi:10.1016/j.clim.2007.03.374<br />
S69<br />
OR.57 TSLP-dependent Induction <strong>of</strong> Airway<br />
Inflammatory Disease is Antigen-driven in an Acute<br />
Model <strong>of</strong> Allergic Airway Inflammation<br />
Mark Headley, Graduate Student, University <strong>of</strong> Washington<br />
<strong>Immunology</strong>, Seattle, WA, Baohua Zhou, Post Doctoral<br />
Fellow, Benaroya Research Institute, Ziegler Lab, Seattle,<br />
WA, Steve Ziegler, Director <strong>of</strong> <strong>Immunology</strong>, Benaroya<br />
Research Institute, Ziegler Lab, Seattle, WA
S70 Abstracts<br />
The cytokine, Thymic Stromal Lymphopoietin (TSLP), has<br />
been shown to play an important role in the mediation <strong>of</strong> Th2biased<br />
inflammatory diseases in particular the atopic diseases:<br />
asthma and atopic dermatitis. Mice expressing a TSLP transgene<br />
driven by the lung-specific Surfactant Protein C promoter (SPC-<br />
TSLP) develop a severe and chronic asthma-like disease<br />
characterized by airway hyperresponsiveness, leukocytic infiltration<br />
<strong>of</strong> the lung, eosinophilia, goblet cell hyperplasia, subepithelial<br />
fibrosis, and elevated serum IgE. Previous studies have<br />
indicated that TSLP alone is sufficient to induce all <strong>of</strong> the<br />
hallmark features <strong>of</strong> asthma in mice. Here we show that an<br />
acute airway inflammatory disease, similar both to that seen in<br />
asthmatics as well as SPC-TSLP mice, can be induced in BALB/c<br />
mice through direct intranasal administration <strong>of</strong> TSLP in<br />
conjunction with a foreign antigen, sans adjuvant. This has<br />
been observed with multiple antigens, including ovalbumin,<br />
chicken gamma globulin, and bovine serum albumin. In contrast,<br />
mice treated with either TSLP alone, the antigens alone, or TSLP<br />
in conjunction with the self-protein, mouse serum albumin, fail<br />
to develop disease. These data demonstrate for the first time<br />
that in an acute model <strong>of</strong> TSLP-mediated airway inflammation,<br />
both thymic stromal lymphopoietin and antigen are required in<br />
order to develop full disease symptoms.<br />
doi:10.1016/j.clim.2007.03.375<br />
OR.58 Disrupting Mer Receptor Tyrosine Kinase<br />
Expression Prevents Autoimmune Chronic<br />
Graft-versus-host Disease<br />
Wenha Shao, Postdoctoral Research Associate,<br />
Rheumatology Division, Department <strong>of</strong> Medicine,<br />
Philadelphia, PA, Robert A. Eisenberg, Pr<strong>of</strong>essor <strong>of</strong><br />
Medicine, Rheumatology Division, Department <strong>of</strong> Medicine,<br />
University <strong>of</strong> Pennsylvania, Philadelphia, PA, Philip L.<br />
Cohen, Pr<strong>of</strong>essor <strong>of</strong> Medicine, Rheumatology Division,<br />
Department <strong>of</strong> Medicine, University <strong>of</strong> Pennsylvania,<br />
Philadelphia, PA<br />
The Mer receptor tyrosine kinase mediates apoptotic cell<br />
phagocytosis and modulates macrophage cytokine production.<br />
Mer knockout mice (Mer-KO) have defective clearance <strong>of</strong><br />
apoptotic debris and develop SLE-like autoimmune disease.<br />
Because <strong>of</strong> their spontaneous autoimmune propensity, we<br />
wondered if Mer-KO mice (backcrossed 10 generations onto<br />
C57BL/6) might be particularly susceptible to the SLE-like<br />
autoimmunity induced by chronic GVH. Using the wellestablished<br />
cGVH model (bm12′B6 mice), we were surprised<br />
to observe that the Mer-KO mice were protected from the<br />
development <strong>of</strong> GVHD. Mer-KO recipients <strong>of</strong> bm12 spleen cells<br />
failed to develop detectable anti-dsDNA and anti-chromatin<br />
autoantibodies, while the B6 hosts produced significant<br />
amounts <strong>of</strong> these antibodies that peaked at week 2. Slightly<br />
increased levels <strong>of</strong> rheumatoid factor were found in Mer-KO<br />
mice, though much lower than B6 control hosts. Mer-KO mice<br />
did not develop splenomegaly. The lack <strong>of</strong> autoantibody<br />
formation was not due to absence <strong>of</strong> alloreactivity because<br />
spleen cells from bm12 mice proliferated equally in response<br />
to irradiated WT and Mer-KO irradiated spleen cells. These<br />
findings indicate a hitherto unrecognized requirement for Mer<br />
in the genesis <strong>of</strong> alloreactivity-induced autoimmunity. Future<br />
experiments will determine whether this reflects the role <strong>of</strong><br />
this kinase in recognition and phagocytosis <strong>of</strong> apoptotic cells,<br />
its function in regulating cytokine production, or perhaps a<br />
new function in the immune response.<br />
doi:10.1016/j.clim.2007.03.376<br />
OR.59 In Vivo and In Silico Modeling the Dynamics <strong>of</strong><br />
Autoimmune Diabetes<br />
Qizhi Tang, Assistant Adjunct Pr<strong>of</strong>essor, University <strong>of</strong><br />
California, San Francisco Diabetes Center, San Francisco,<br />
CA, Yanan Zheng, Engineer, Biological Systems, Entelos, In<br />
Silico Research and Development, Foster City, CA, Jason<br />
Adams, MD, Stanford University, Stanford, CA, Huub<br />
Kreuwel, Scientist, Immunologic Diseases, Entelos, In Silico<br />
Research and Development, Foster City, CA, Kapil Gadkar,<br />
Biosystems Engineer, Entelos, In Silico Research and<br />
Development, Foster City, CA, Cristina Penaranda,<br />
Graduate Student, University <strong>of</strong> California, San Francisco<br />
Diabetes Center, San Francisco, CA, Lisl Shoda, Senior<br />
Scientist, Entelos, In Silico Research and Development,<br />
Foster City, CA, Chan C. Whiting, Scientist, Immunologic<br />
Diseases, Entelos, In Silico Research and Development,<br />
Foster City, CA, Daniel Young, Dynamics Engineer, Entelos,<br />
In Silico Research and Development, Foster City, CA, Jeffrey<br />
Bluestone, Pr<strong>of</strong>essor, University <strong>of</strong> California, San Francisco<br />
Diabetes Center, San Francisco, CA<br />
Progression <strong>of</strong> autoimmune diabetes results as a consequence<br />
<strong>of</strong> an imbalance <strong>of</strong> Teff and suppressive regulatory T<br />
cells (Tregs). A novel mathematical model <strong>of</strong> type 1 diabetes<br />
(T1D) in the non-obese diabetes (NOD) mouse (T1D Physio-<br />
Lab® platform) was developed that encompasses the<br />
dynamics <strong>of</strong> key immune components in the pancreatic<br />
lymph nodes (PLNs) and islets, and beta cell physiology.<br />
Research using this platform predicted that autoimmunity<br />
was controlled in the PLN, as shown by an increase in Tregs<br />
over the course <strong>of</strong> disease. This unexpected behavior was<br />
confirmed experimentally by functional and histological<br />
data. Additionally, laboratory analysis <strong>of</strong> individual islets<br />
demonstrated an inverse relationship between Teff and Treg<br />
infiltration <strong>of</strong> the tissue. Specifically, as Teff infiltration<br />
increases, the Treg fraction decreases. However, differential<br />
proliferation <strong>of</strong> Teffs and Tregs in vivo was found to be<br />
insufficient to explain this relationship although evidence <strong>of</strong><br />
reduced expression <strong>of</strong> CD25 on the resident pancreatic Tregs<br />
could alter Treg survival accounting for the observed effects.<br />
Based on these in vivo results, the relative turnover and<br />
recruitment <strong>of</strong> Tregs and Teffs were tested in the T1D<br />
platform. Results <strong>of</strong> these in silico investigations demonstrated<br />
that the ultimate determination <strong>of</strong> disease activity<br />
depends on dynamic changes in intra-islet Treg turnover and<br />
changes in the Teff/Treg balance. These combined experimental<br />
and mathematical modeling results emphasize the<br />
central importance <strong>of</strong> both PLN and intra-islet Treg function<br />
in regulating disease pathogenesis and the power <strong>of</strong><br />
integrating in vivo and in silico modeling to dissect mechanisms<br />
leading to disease pathogenesis.<br />
doi:10.1016/j.clim.2007.03.377
Abstracts<br />
OR.60 Antigen Identification in a Novel Model <strong>of</strong><br />
Spontaneous Autoimmune Ovarian Disease<br />
Mickie Cheng, <strong>Clinical</strong> Fellow in Endocrinology, University <strong>of</strong><br />
California, San Francisco Diabetes Center, San Francisco,<br />
CA, Kellsey Johannes, Staff Research Associate, University<br />
<strong>of</strong> California, San Francisco Diabetes Center, San Francisco,<br />
CA, Wen Lu, Staff Research Associate, University <strong>of</strong><br />
California, San Francisco Diabetes Center, San Francisco,<br />
CA, Mark Anderson, Assistant Pr<strong>of</strong>essor <strong>of</strong> Medicine in<br />
Residence, University <strong>of</strong> California, San Francisco Diabetes<br />
Center, San Francisco, CA<br />
Autoimmune ovarian disease (AOD) is a poorly understood<br />
clinical entity wherein immune-mediated destruction <strong>of</strong><br />
oocytes leads to premature ovarian failure and infertility.<br />
We have generated a novel spontaneous mouse model to<br />
study AOD using mice deficient for the AutoImmune<br />
REgulator (aire), a gene first identified in the human<br />
Autoimmune Polyglandular Syndrome (APS) type 1. Like<br />
patients with APS 1, aire-deficient mice develop disease <strong>of</strong><br />
multiple organs, including the ovary, due to a breakdown in<br />
central tolerance. In aire KO mice, thymic epithelial cells are<br />
defective in their ability to tolerize developing Tcells to selfantigens,<br />
thus allowing autoreactive cells to escape into the<br />
periphery and induce self tissue destruction. Autoimmunity<br />
is manifested by the development <strong>of</strong> antigen-specific<br />
autoantibodies, organ infiltration, and transfer <strong>of</strong> disease<br />
by lymphocytes from affected aire KO mice. aire KO females<br />
exhibit 100% incidence <strong>of</strong> histologic oophoritis as well as<br />
circulating autoantibodies to ovarian tissue in a strain<br />
specific manner, presenting a valuable spontaneous disease<br />
model to identify ovarian antigens. Initial characterization <strong>of</strong><br />
AOD in these mice implicates a central role for T cell<br />
mediated disease with predominantly Th1 CD4+ T cell<br />
infiltrate. Furthermore, autoantibodies from aire KO females<br />
target the oocyte cytoplasm and follicular layer and<br />
recognize a 70 kDa autoantigen in ovarian lysates. Identification<br />
<strong>of</strong> the disease-inciting antigen could enable improved<br />
testing for the diagnosis <strong>of</strong> autoimmune ovarian disease and<br />
may provide a potential therapeutic target for treatment<br />
and prevention <strong>of</strong> a significant cause <strong>of</strong> human ovarian<br />
failure and infertility.<br />
doi:10.1016/j.clim.2007.03.378<br />
OR.61 Delineating the Roles <strong>of</strong> CD4+ and CD8+ T Cell<br />
Subsets in the Pathogenesis <strong>of</strong> Spontaneous<br />
Autoimmune Diabetes Using HLA-DQ8 Transgenic<br />
Mice<br />
Govindarajan Rajagopalan, Department <strong>of</strong> <strong>Immunology</strong>,<br />
Mayo Clinic, Rochester, MN, Ashutosh Mangalam,<br />
Department <strong>of</strong> <strong>Immunology</strong>, Mayo Clinic, Rochester, MN,<br />
Yogish Kudva, Department <strong>of</strong> Endocrinology, Mayo Clinic,<br />
Rochester, MN, Chella David, Department <strong>of</strong> <strong>Immunology</strong>,<br />
Mayo Clinic, Rochester, MN<br />
Certain MHC class II molecules show a strong association<br />
with T1D implying an important role for CD4+ T cells. MHC<br />
class I-restricted CD8+ T cells also play a crucial effector<br />
role. The roles <strong>of</strong> CD4+ and CD8+ T cell subsets in the<br />
pathogenesis <strong>of</strong> spontaneous autoimmune diabetes were<br />
studied using HLA-DQ8 transgenic mouse models. CD8sufficient<br />
or CD8-deficient Abo.DQ8 transgenic mice expressing<br />
either the proinflammatory cytokine, tumor necrosis<br />
factor (TNF)-α or the costimulatory molecule, B7.1 or both<br />
in the pancreatic beta cells were used in this study. In the<br />
RIP-TNF model, the incidence <strong>of</strong> diabetes was class II or<br />
CD4+ T cell independent as 75% <strong>of</strong> mice lacking HLA-DQ8<br />
(and there by CD4+ T cells) developed T1D. However, it was<br />
absolutely class I or CD8+ T cell dependent as none <strong>of</strong> the<br />
CD8-deficient mice developed T1D. In the RIP-B7 model, the<br />
incidence <strong>of</strong> diabetes was class II or CD4+ T cell dependent,<br />
as none <strong>of</strong> the mice lacking HLA-DQ8 (and there by CD4+ T<br />
cells) developed T1D when compared to 35% incidence<br />
when HLA-DQ8 (and there by CD4+ T cells) is expressed.<br />
Nonetheless, in RIP-B7 model, CD8-deficient HLA-DQ8<br />
transgenic mice were still susceptible to T1D albeit at a<br />
lower rate (Incidence ¡V 17%). In the RIP-B7.RIP-TNF double<br />
transgenic mouse model, 100% <strong>of</strong> the animals developed<br />
diabetes irrespective <strong>of</strong> whether they lacked CD4+ or CD8+<br />
T cells. Thus, CD4+ and CD8+ T cells contribute differentially<br />
to the pathogenesis <strong>of</strong> T1D according to the local immunological<br />
insult.<br />
doi:10.1016/j.clim.2007.03.379<br />
S71<br />
OR.62 The CYP450 Mouse Model for Autoimmune<br />
Hepatitis: Breaking <strong>of</strong> Self-Tolerance in Transgenic<br />
CYP2D6 and Wildtype FVB-Mice by Viral Infection<br />
Urs Christen, Assistant Pr<strong>of</strong>essor, Pharmazentrum, JWG<br />
University Frankfurt, Frankfurt am Main, Germany,<br />
Martin Holdener, PhD Student, Pharmazentrum, JWG<br />
University Frankfurt, Frankfurt am Main, Germany,<br />
Edith Hintermann, Senior Postdoctoral Fellow,<br />
Pharmazentrum, JWG University Frankfurt, Frankfurt<br />
am Main, Germany, Matthias von Herrath, Pr<strong>of</strong>essor, La<br />
Jolla Institute for Allergy and <strong>Immunology</strong>, La Jolla,<br />
CA, Michael Manns, Pr<strong>of</strong>essor, Hannover Medical School,<br />
Hannover, Germany<br />
The etiology <strong>of</strong> autoimmune hepatitis (AIH) is poorly<br />
understood although the major autoantigen, cytochrome<br />
P450 2D6 (CYP2D6), has been identified. We generated an<br />
animal model for human AIH using the natural autoantigen<br />
CYP2D6. We infected transgenic mice expressing human<br />
CYP2D6 in the liver with an Adenovirus-CYP2D6 vector (Ad-<br />
2D6) to break self-tolerance. Upon infection with Ad-2D6<br />
not only transgenic CYP2D6 mice but also wildtype FVB<br />
mice showed persistent features <strong>of</strong> severe liver damage<br />
associated with AIH (hepatic fibrosis, fused liver lobules,<br />
cellular infiltrations, elevated serum ALT levels, confluent<br />
necrosis). Ad-2D6-infected mice (CYP2D6 and FVB) generated<br />
high titers <strong>of</strong> anti-CYP2D6 antibodies. Epitope mapping<br />
revealed that anti-CYP2D6 antibodies predominantly<br />
recognized the same immunodominant linear epitope<br />
recognized by sera <strong>of</strong> AIH patients (AQPPRD aa 265–270).<br />
In contrast, mice infected with a control Adenovirus<br />
expressing green fluorescence protein did neither develop
S72 Abstracts<br />
liver damage nor generated anti-CYP2D6 antibodies. The<br />
severity <strong>of</strong> liver damage as well as antibody formation was<br />
enhanced in FVB mice compared to transgenic CYP2D6<br />
mice indicating a stronger tolerance to human CYP2D6 in<br />
CYP2D6 mice. In FVB mice, due to the homology <strong>of</strong> the<br />
mouse isoenzymes <strong>of</strong> the CYP superfamily to human<br />
CYP2D6, autoimmune liver damage by Ad-2D6 infection<br />
was possibly induced via true molecular mimicry.<br />
doi:10.1016/j.clim.2007.03.380<br />
Immunoregulatory Mechanisms<br />
Saturday, June 9<br />
2:45 pm−4:45 pm<br />
OR.63 Modification <strong>of</strong> Host Cellular DNA by<br />
Parvovirus B19 Nonstructural Protein 1 (NS1):<br />
Implications for Induction <strong>of</strong> Autoimmunity by DNA<br />
Viruses<br />
Leona Gilbert, University <strong>of</strong> Jyvaskala, University <strong>of</strong><br />
Jyvaskala, Jyvaskala, Finland, Brian D. Poole, Oklahoma<br />
Medical Research Foundation, Oklahoma Medical Research<br />
Foundation, Oklahoma City, OK, Violetta Kivovich,<br />
University <strong>of</strong> Jyvaskala, University <strong>of</strong> Jyvaskala, Jyvaskala,<br />
Finland, Stanley Naides, Medical Director, <strong>Immunology</strong>,<br />
Quest Diagnostics Nichols Institute, San Juan Capistra, CA,<br />
Jing Zhou, Pennsylvania State University College <strong>of</strong><br />
Medicine/Milton S. Hershey Medical Center, Pennsylvania<br />
State University College <strong>of</strong> Medicine/Milton S. Hershey<br />
Medical Center, Hershey, PA<br />
Autoantibodies cross-reactive to EBV proteins precede<br />
SLE onset in a subset <strong>of</strong> patients. However, the event that<br />
links viral infection to autoimmunity, the “original sin,”<br />
remains unknown. We studied the role <strong>of</strong> parvovirus B19<br />
infection in host DNA modification. B19 infection in nonerythroid<br />
cells (e.g., Hep G2 and primary hepatocytes) is<br />
restricted to expression <strong>of</strong> NS1. NS1 is a superfamily 3 (SF3)<br />
helicase, typical <strong>of</strong> small DNA viruses, that plays a key role<br />
in viral genome replication. In hepatocytes, B19 induces<br />
apoptosis through mitochondrial stress pathways. We<br />
examined NS1 expression in Hep G2 cells using an enhanced<br />
green fluorescent fusion protein (eGFP/NS1). Expressed<br />
eGFP/NS1 localized to the nucleus. The majority <strong>of</strong> cells<br />
expressing eGFP/NS1 underwent apoptosis with PARP<br />
activation. Inhibitors <strong>of</strong> PARP or ATR abrogated apoptosis.<br />
eGFP/NS1 colocalized with RPA and PCNA. Western blot<br />
analysis and autoradiographs demonstrated that NS1<br />
formed dimers under reducing conditions and covalently<br />
bound to cellular DNA. NS1 was polyADP ribosylated.<br />
Apoptotic subcellular particles from cells expressing<br />
eGFP/NS1 fluoresced. Our data demonstrated that B19<br />
NS1 forms bulky adducts with cellular DNA and nicks<br />
cellular DNA, initiating pathways that lead to mitochondrial<br />
stress and apoptosis. NS1 with covalently linked self-DNA is<br />
extruded into the extracellular milieu in apoptotic bodies.<br />
We propose that modification <strong>of</strong> cellular DNA by viral SF3<br />
helicase breaks tolerance to self DNA when presented to<br />
the immune system during apoptosis. These findings suggest<br />
a general model in which modification <strong>of</strong> host DNA by viral<br />
SF3 helicase induces or accelerates autoimmunity, the<br />
“original sin.”<br />
doi:10.1016/j.clim.2007.03.381<br />
OR.64 Long-term Deposition <strong>of</strong> Inhaled Antigen in<br />
Lung Resident CD11b−CD11c+ Cells<br />
Kate E. Matthews, Student, Malaghan Institute, Wellington<br />
South, New Zealand, Adela Karabeg, Student, Medical<br />
University <strong>of</strong> Vienna, Department Dermatology, Vienna,<br />
Austria, Gerhard Dekan, Pr<strong>of</strong>essor, Medical University <strong>of</strong><br />
Vienna, <strong>Clinical</strong> Pathology, Vienna, Austria, Franca<br />
Roncheses, Principal Investigator, Malaghan Institute,<br />
Wellington South, New Zealand, Michelle Epstein,<br />
Laboratory Leader, Medical University <strong>of</strong> Vienna,<br />
Department <strong>of</strong> Dermatology, Vienna, Austria<br />
We report the characterization <strong>of</strong> a population <strong>of</strong> lung<br />
resident CD11b−CD11c+ cells that are able to take up inhaled<br />
antigen and retain it for extended periods <strong>of</strong> time.<br />
Ovalbumin conjugated to fluorescein isothiocyanate administered<br />
intranasally to mice was taken up by two main<br />
populations <strong>of</strong> cells in the lung, a migratory CD11c+CD11b+<br />
population consisting <strong>of</strong> DC, which rapidly transported<br />
antigen to the draining lymph node (LN), and a resident<br />
CD11b−CD11c+ population that retained engulfed antigen<br />
without apparently degrading it for up to 8 weeks after<br />
administration. The FITC+CD11b−CD11c+ cells did not<br />
migrate to draining LN and did not upregulate expression <strong>of</strong><br />
costimulatory molecules in response to LPS treatment.<br />
FITC+CD11b−CD11c+ cells found in the airway were large<br />
and were consistent with macrophages. Additionally, a<br />
population <strong>of</strong> FITC+CD11b−CD11c+ seen in lung sections<br />
were situated along alveolar walls with a distribution also<br />
consistent with macrophages. Although FITC+CD11b−<br />
CD11c+ cells expressed molecules associated with APC<br />
function, they did not induce proliferation <strong>of</strong> antigenspecific<br />
CD4+ T cells in vitro or acute cytokine production<br />
by activated CD4+ Tcells in vivo. We propose that these longlived,<br />
lung resident cells might provide a depot <strong>of</strong> antigen for<br />
the indirect reactivation or retention <strong>of</strong> antigen-specific T<br />
cells in the lung.<br />
doi:10.1016/j.clim.2007.03.382<br />
OR.65 Proteinase-activated Receptor-2 (PAR-2)<br />
Activation Promotes Allergic Sensitization and<br />
Prevents the Development <strong>of</strong> Immune Tolerance to<br />
Inhaled Antigens Through a TNF Mediated Pathway<br />
Cory Ebeling, PhD candidate, University <strong>of</strong> Alberta,<br />
Department <strong>of</strong> Medicine, Edmonton, AB, Canada, Tong Lam,<br />
Medical Student, University <strong>of</strong> Alberta, Edmonton, AB,<br />
Canada, John Gordon, Associate Pr<strong>of</strong>essor, University <strong>of</strong><br />
Saskatchewan, Department <strong>of</strong> Veterinary Microbiology,<br />
Saskatoon, SK, Canada, Harissios Vliag<strong>of</strong>tis, Associate<br />
Pr<strong>of</strong>essor, University <strong>of</strong> Alberta, Department <strong>of</strong> Medicine,<br />
Edmonton, AB, Canada, Morley Hollenberg, Associate
Abstracts<br />
Pr<strong>of</strong>essor, University <strong>of</strong> Calgary, Department <strong>of</strong><br />
Pharmacology and Therapeutics, Calgary, AB, Canada<br />
The reason why particular inhaled antigens induce<br />
allergic sensitization and prevent the development <strong>of</strong><br />
immune tolerance is unclear. Intrinsic characteristics <strong>of</strong><br />
these antigens must be <strong>of</strong> importance. A common<br />
characteristic <strong>of</strong> many potent allergens is that they either<br />
possess serine proteinase activity or are inhaled in particles<br />
rich in serine proteinases. Many allergens, such as house<br />
dust mite, have the potential to activate the Proteinaseactivated<br />
Receptor-2 (PAR-2). We hypothesized that PAR-2<br />
activation by such allergens is crucial to facilitate our<br />
allergic sensitization to them. To test our hypothesis we<br />
used a Balb/c murine system with mucosal exposure to<br />
OVA, as an antigen, with a PAR-2 activating peptide (PAR-<br />
2AP) to mimic the potential <strong>of</strong> a proteolytic allergen, or<br />
other inhaled proteinases, to activate PAR-2. Upon allergen<br />
re-exposure mice initially administered OVA with the PAR-<br />
2AP developed airway inflammation, airway hyperresponsiveness<br />
(AHR), produced OVA-specific IgE as well as OVAspecific<br />
T cells with a Th2 cytokine pr<strong>of</strong>ile. Conversely,<br />
mice given OVA alone or with a control peptide (PAR-2CP)<br />
developed tolerance. These immune tolerant mice did not<br />
develop airway inflammation, AHR, or OVA-specific IgE, but<br />
developed OVA-specific T cells that secreted high levels <strong>of</strong><br />
IL-10 indicative <strong>of</strong> Treg cell function. Finally, we showed<br />
that this PAR-2-mediated allergic sensitization was TNF<br />
dependent. Thus, PAR-2 activation in the airways could be<br />
a critical factor in the development <strong>of</strong> allergic sensitization<br />
following mucosal exposure to allergens with serine<br />
proteinase activity. Interfering with this pathway may<br />
prove to be useful for the prevention or treatment <strong>of</strong><br />
asthma.<br />
doi:10.1016/j.clim.2007.03.383<br />
OR.66 Downregulation <strong>of</strong> Innate B Cells, B-1a and<br />
MZB, by iNKT Cells<br />
Xiangshu Wen, Postdoctoral Fellow, University <strong>of</strong> California,<br />
Los Angeles, Medicine, Los Angeles, CA, Jun-Qi Yang,<br />
Assistant Research Pr<strong>of</strong>essor, UC, Medicine, Cincinnati, OH,<br />
Ram Raj Singh, Pr<strong>of</strong>essor, University <strong>of</strong> California, Los<br />
Angeles, Medicine, Los Angeles, CA<br />
B cells are essential for the development <strong>of</strong> autoimmune<br />
diseases such as lupus. Although all mature B cell subsets<br />
can produce autoantibodies, several lines <strong>of</strong> evidence<br />
implicate innate B cells, namely B-1a and marginal zone B<br />
(MZB) cells, in the development <strong>of</strong> such diseases. Here, we<br />
investigated the role <strong>of</strong> interaction between innate B cells<br />
that express high levels <strong>of</strong> CD1d and CD1d-reactive iNKT<br />
cells in the regulation <strong>of</strong> autoantibodies. We found that<br />
whereas activated iNKT cells increase activation markers<br />
CD69 and CD86 on all B cell subsets, they selectively reduce<br />
the frequency <strong>of</strong> B-1a and MZB cells. In resonance with such<br />
regulatory role, the proportions <strong>of</strong> MZB cells are increased<br />
in iNKT cell deficient (Ja18−/−) mice, whereas MZB cells<br />
are reduced in iNKT cell transgenic (Va14Tg) mice.<br />
Furthermore, whereas iNKT cells increase expression <strong>of</strong><br />
activation markers and production <strong>of</strong> normal Ig via<br />
cytokines secreted by iNKT cells in transwell cultures,<br />
they selectively suppress the production <strong>of</strong> IgG anti-DNA<br />
antibody and rheumatoid factor and IL-10 by B cells in a<br />
contact-dependent manner. Such regulation <strong>of</strong> B cells by<br />
iNKT cells must be important for in vivo regulation <strong>of</strong><br />
autoantibodies and lupus disease, as the in vivo transfer <strong>of</strong><br />
iNKT cells in B cell-reconstituted SCID mice or in Ja18−/−<br />
mice suppresses autoantibody production. Treatment with<br />
an iNKT cell ligand also delays the onset <strong>of</strong> lupus in BWF1<br />
mice. Thus, iNKT cells suppress autoreactive B cells and can<br />
prevent systemic autoimmunity. They do so via regulating<br />
innate B cells.<br />
doi:10.1016/j.clim.2007.03.384<br />
OR.67 CD40 Is<strong>of</strong>orm Differences and Microdomain<br />
Localization Explain Preferential Survival <strong>of</strong><br />
CD4+CD40+ T Cells in Autoimmunity<br />
Gisela M. Vaitaitis, Pr<strong>of</strong>essional Research Assistant,<br />
University <strong>of</strong> Colorado Health Sciences Center, Webb-<br />
Waring, Denver, CO, David H. Wagner, Assistant Pr<strong>of</strong>essor,<br />
University <strong>of</strong> Colorado Health Sciences Center, Webb-<br />
Waring, Denver, CO<br />
CD40 plays a critical role in autoimmunity. CD40expressing<br />
CD4 T cells (TCD40) are expanded in autoimmunity<br />
and are necessary and sufficient in transferring disease<br />
in mouse type I diabetes. CD40 on TCD40 can have<br />
costimulatory effect and induces Nf-kB and recombinase<br />
protein expression with subsequent alteration <strong>of</strong> TCR. CD40<br />
exists as five is<strong>of</strong>orms in mice with little known-about<br />
differences in is<strong>of</strong>orm expression between cell types,<br />
between autoimmune and non-autoimmune conditions and<br />
differences in outcome <strong>of</strong> CD40 is<strong>of</strong>orm signaling. Here we<br />
show that the major is<strong>of</strong>orm expressed in TCD40 is is<strong>of</strong>orm-<br />
V with is<strong>of</strong>orm-I expressed at lower levels. This differs<br />
distinctly from antigen presenting cells which predominantly<br />
express is<strong>of</strong>orm-I. In autoimmune NOD the ratio <strong>of</strong><br />
TCD40 is<strong>of</strong>orm-V to -I is exaggerated and overall expression<br />
is greater compared to non-autoimmune BALB/c. It was<br />
shown in lymphocyte cell lines that CD40 and TRAF2 are not<br />
associated with detergent-insoluble microdomains (rafts)<br />
until CD40 is engaged. However, untreated NOD TCD40 have<br />
the CD40 is<strong>of</strong>orms and TRAF2 associated with this fraction<br />
while BALB/c TCD40 do not. A functional outcome <strong>of</strong> CD40<br />
signals to NOD TCD40 is increased survival through induction<br />
<strong>of</strong> survival proteins Bcl-XL and cFLIPp43. In addition, CD40<br />
signals to NOD TCD40 alter the outcome <strong>of</strong> Fas engagement<br />
such that Fas instead <strong>of</strong> inducing apoptosis induces<br />
increased survival beyond that <strong>of</strong> CD40 alone. Therefore<br />
TCD40 from NOD may be poised for survival even when<br />
encountering death promoting conditions explaining how<br />
this population gains the upper hand in autoimmune<br />
conditions to thwart T cell homeostasis.<br />
doi:10.1016/j.clim.2007.03.385<br />
S73
S74 Abstracts<br />
OR.68 An Immunoregulatory Network <strong>of</strong> Natural<br />
Killer T Cells and CD4+CD25+Foxp3+ T Cells<br />
Protects Against Graft-versus-host Disease<br />
Asha Pillai, Postdoctoraltoral Research Fellow, Stanford,<br />
Stanford, CA, Suparna Dutt, Research Associate,<br />
Department <strong>of</strong> Medicine, Stanford, CA, Tracy George,<br />
Assistant Pr<strong>of</strong>essor, Department <strong>of</strong> Pathology, Stanford, CA,<br />
Samuel Strober, Pr<strong>of</strong>essor <strong>of</strong> Medicine, Stanford University,<br />
Department <strong>of</strong> Medicine, Stanford, CA<br />
To investigate acute graft-versus-host disease (GVHD)<br />
protection in the murine regimen <strong>of</strong> total lymphoid irradiation<br />
and anti-thymocyte serum (TLI/ATS), we performed transplants<br />
<strong>of</strong> 50×10 6 bone marrow cells and 60×10 6 splenocytes (BMT)<br />
from wild-type (WT) C57BL/6 donors into WT BALB/c hosts<br />
treated with TLI/ATS or control BALB/c hosts given 800 cGy total<br />
body irradiation (TBI) and ATS. TLI/ATS-conditioned WT hosts<br />
given BMT from WT donors all survived 100 days without GVHD.<br />
BMT from WT donors into TBI/ATS treated WT hosts resulted in<br />
lethal GVHD. TLI/ATS-treated natural killer (NK) Tcell deficient<br />
Jα18−/− hosts also died <strong>of</strong> GVHD. TLI/ATS conditioned WT hosts<br />
receiving WT BMT demonstrated a significant (pb0.01) increase<br />
in the absolute number <strong>of</strong> donor CD4+CD25+Foxp3+ cells (CD4+<br />
Tregs) in the spleen at day 6 after BMT relative to TBI/ATS<br />
controls or TLI/ATS-treated Jα18−/− hosts. All BMT groups<br />
developing GVHD demonstrated dramatic donor CD8+ T cell<br />
accumulation in the mesenteric lymph nodes (MLN) and colon at<br />
day 6 after BMT. Adoptive transfer <strong>of</strong> sorted BALB/c NK T cells<br />
protected TLI/ATS-treated Jα18−/− hosts from donor CD8+ T<br />
cell accumulation, with a significant (pb0.001) increase in the<br />
absolute number <strong>of</strong> donor CD4+ Tregs in the host spleen at day 6.<br />
Depletion <strong>of</strong> CD25+ T cells before BMT into TLI/ATS-treated WT<br />
hosts resulted in loss <strong>of</strong> donor CD4+ Tregs in the host spleen and<br />
lethal GVHD. The present studies show that both host NK Tcells<br />
and donor CD4+CD25+Foxp3+ Tregs are required to protect<br />
against GVHD after TLI/ATS conditioning and allogeneic BMT.<br />
doi:10.1016/j.clim.2007.03.386<br />
OR.69 Peroxisome Poliferator-activated Receptor-α<br />
(PPARα) Expression in T Cells Mediates Gender<br />
Differences in Development <strong>of</strong> T Cell-mediated<br />
Autoimmunity<br />
Shannon Dunn, Postdoctoral Fellow, Stanford University,<br />
Department <strong>of</strong> Neurology, Stanford, CA, Shalina Ousman,<br />
Postdoctoral Fellow, Stanford University, Department <strong>of</strong><br />
Neurology, Stanford, CA, Raymond Sobel, Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> Pathology, Stanford, CA, Sergio Baranzini,<br />
Research Associate, University <strong>of</strong> California, San Francisco,<br />
Department <strong>of</strong> Neurology, San Francisco, CA, Luis Zuniga,<br />
Student, Stanford University, Stanford, CA, Sawsan Youssef,<br />
Postdoctoral Fellow, Stanford University, Department <strong>of</strong><br />
Neurology, Stanford, CA, Jorge Oksenberg, Associate<br />
Pr<strong>of</strong>essor, University <strong>of</strong> California, San Francisco,<br />
Department <strong>of</strong> Neurology, San Francisco, CA, Lawrence<br />
Steinman, Pr<strong>of</strong>essor, Departmental Chair in <strong>Immunology</strong>,<br />
Stanford University, Department <strong>of</strong> Neurology,<br />
Stanford, CA<br />
PPAR is a nuclear receptor that mediates gender<br />
differences in lipid metabolism. PPAR also functions to<br />
control inflammatory responses by repressing the activity <strong>of</strong><br />
NFkappaB and c-jun in immune cells. Since PPAR is situated<br />
at the crossroads <strong>of</strong> gender and immune regulation, we<br />
hypothesized that this gene may mediate sex differences in<br />
the development <strong>of</strong> T cell-mediated autoimmune disease.<br />
We show that PPAR is more abundant in male as compared<br />
to female CD4 + cells and is sensitive to androgen levels.<br />
Genetic ablation <strong>of</strong> this gene selectively removed the brake<br />
on NFkappaB and c-jun activity in male T lymphocytes<br />
resulting in higher production <strong>of</strong> IFN-γ and TNF (but not IL-<br />
17), and lower production <strong>of</strong> Th2 cytokines. Upon induction<br />
<strong>of</strong> experimental autoimmune encephalomyelitis (EAE), male<br />
but not PPAR −/− mice developed more severe clinical signs<br />
that were restricted to the acute phase <strong>of</strong> disease. To<br />
determine the extent to which changes in PPAR expression<br />
explain the androgen sensitivity <strong>of</strong> T helper responses, we<br />
contrasted the effects <strong>of</strong> castration on male WT versus<br />
PPAR −/− mice. This surgical intervention lowered PPAR<br />
expression in T cells, and enhanced the production <strong>of</strong> IFNγ,<br />
TNF, and IL-17 by WT male T cells in response to anti-CD3<br />
and anti-CD28 stimulation. Castration also enhanced the<br />
severity <strong>of</strong> acute EAE in male WT mice. These effects <strong>of</strong><br />
castration were significantly blunted in PPAR −/− mice. These<br />
results suggest that males may be less prone to develop<br />
Th1-mediated autoimmunity because they have higher<br />
levels <strong>of</strong> PPAR.<br />
doi:10.1016/j.clim.2007.03.390
Abstracts<br />
Sa.1 Nitric Oxide Metabolites and Nitrotyrosine in<br />
Asthma and COPD<br />
Jenny Garmendia, <strong>Clinical</strong> Immunologist and Internal<br />
Medicine, Instituto de Inmunología, UCV, Caracas,<br />
Venezuela, Dolores Moreno, Pulmonologist, Servicio de<br />
Neumonología, Hospital Universitario de Caracas, Caracas,<br />
Venezuela, Nancy Larocca, <strong>Clinical</strong> Immunologist and<br />
Pediatrician, Instituto de Inmunología, UCV, Caracas,<br />
Venezuela, Juan De Sanctis, Biochemistry, Instituto de<br />
Inmunología, UCV, Caracas, Venezuela, Carlos Tálamo,<br />
Pulmonologist, Servicio de Neumonología, Hospital<br />
Universitario de Caracas, Caracas, Venezuela<br />
Asthma and chronic obstructive pulmonary disease (COPD)<br />
are two different inflammatory airway diseases. Serum nitric<br />
oxide (NO) metabolites and nitrotyrosine may be useful<br />
parameters <strong>of</strong> inflammation. We studied 150 Asthma patients<br />
(24 ± 4.5 years), 185 COPD patients (62 ± 9 years), and 300<br />
healthy subjects (45 ± 19 years). The controls were divided in<br />
twogroups<strong>of</strong>150individualseach(meanage25±5and62±6<br />
years, respectively). Serum nitrites, nitrates (NO metabolites)<br />
and nitrotyrosine levels were determined by colorimetric,<br />
enzymatic assay and ELISA, respectively. No significant differences<br />
were recorded among the two groups <strong>of</strong> controls and thus<br />
we used the whole group for comparison. Nitrite levels were<br />
significantly reduced (pb 0.001) only en COPD patients (COPD<br />
4.1±1.9, Asthma 6.1 ± 1.3 vs. 6.5 ± 1.7 µM). Nitrates levels were<br />
significantly reduced in both groups <strong>of</strong> patients (COPD 16.2 ±<br />
4.1, Asthma 18.6 ± 3.2 vs controls 24.9 ± 3.5 µM, p b 0.001).<br />
Nevertheless, nitrotyrosine levels were significantly increased<br />
in the patient groups (controls: 4.2 ± 2.5; COPD: 11.3 ± 8.5<br />
pb0.001; Asthma: 6.7 ± 4.3 uM pb0.01). Nitrotyrosine levels<br />
were higher in COPD patients as compared to asthmatics (pb<br />
0.01). Moreover, we observed an inverse correlation between<br />
nitrates and nitrotyrosine only in patients (r = 0.62, n = 335). We<br />
conclude that metabolism <strong>of</strong> NO is modified in patients with<br />
asthma and COPD with a bypass <strong>of</strong> normal NO metabolites to a<br />
reactive species <strong>of</strong> nitrogen as peroxynitrites assessed partially<br />
by nitrotyrosine. FONACIT G2005000389.<br />
doi:10.1016/j.clim.2007.03.391<br />
Poster Session<br />
Saturday, June 9<br />
7:30 am−7:00 pm<br />
Authors Present: 5:45 pm−7:00 pm<br />
Sa.5 Effect <strong>of</strong> Omalizumab on Pulmonary Function<br />
and Asthma-related Outcome Measures in Patients<br />
Allergic to Cat Dander<br />
Marc Massanari, Associate Medical Director, Novartis<br />
Pharmaceuticals Corporation, East Hanover, NJ, Robert<br />
Maykut, Medical Director, Novartis Pharmaceuticals<br />
Corporation, East Hanover, NJ, Robert Zeldin, Executive<br />
Medical Director, Novartis Pharmaceuticals Corporation,<br />
East Hanover, NJ, Farid Kianifard, Statistician, Novartis<br />
Pharmaceuticals Corporation, East Hanover, NJ, Gregory<br />
Geba, Therapeutic Area Head, Novartis Pharmaceuticals<br />
Corporation, Verona, NJ<br />
Rationale: Exposure to cat dander, which if <strong>of</strong>ten<br />
unexpected, can worsen control in susceptible asthmatic<br />
patients. Add-on omalizumab (OMA) has been shown to help<br />
protect patients with perennial allergen sensitivity from<br />
exacerbations. We evaluated the effect <strong>of</strong> OMA on FEV1 and<br />
asthma-related outcome measures in patients with asthma<br />
and cat dander sensitivity. Methods: Data were pooled from 5<br />
randomized, double-blind, 28–32 week, placebo (PBO)controlled<br />
studies <strong>of</strong> patients with moderate–severe persistent<br />
IgE-mediated asthma and perennial allergen sensitivity<br />
inadequately controlled on inhaled corticosteroids (ICS). Cat<br />
sensitivity was determined by skin prick or RAST testing. The<br />
effect <strong>of</strong> add-on OMA on mean changes from baseline in FEV1,<br />
total rescue puffs <strong>of</strong> beta-agonist, and total asthma symptom<br />
score were analyzed using analysis <strong>of</strong> covariance; least<br />
squares means (LSM) and 95% confidence intervals (CI) were<br />
calculated. Results: 1589 patients (811 OMA, 778 PBO) were<br />
sensitive to cat dander. Mean age =39.7 years, 60% were<br />
female, mean IgE=219.1 IU/mL and FEV1 percent predicted<br />
was 60–79% in 46% and b60% in 26% <strong>of</strong> patients at baseline.<br />
End-<strong>of</strong>-study mean FEV1 increased by 100.3 mL (5.4%) and<br />
33.1 mL (2.5%) in OMA- and PBO-treated patients, respectively.<br />
LSM treatment difference was 63.11 (26.88, 99.35;<br />
pb0.001) in favor <strong>of</strong> OMA. The LSM difference (OMA-PBO)<br />
for change in rescue beta-agonist puffs was −0.47 (95% CI<br />
−0.72, −0.22; pb0.001) and was −0.40 (−0.55, −0.25;<br />
pb0.001) for change in total asthma symptom score. Conclusion:<br />
Omalizumab improved pulmonary function and asthmarelated<br />
outcome measures in cat allergic patients with<br />
moderate–severe persistent asthma inadequately controlled<br />
with ICS.<br />
doi:10.1016/j.clim.2007.03.392<br />
S75<br />
Sa.6 Hypersensitivity Pneumonitis <strong>of</strong> Unidentifiable<br />
Cause<br />
Viktor Hanak, MD, Mayo Clinic Foundation, Rochester, MN,<br />
Jason Golbin, MD, Mayo Clinic Foundation, Rochester,<br />
MN, Jay Ryu, MD, Mayo Clinic Foundation, Rochester, MN<br />
Identification <strong>of</strong> a specific antigen is important in disease<br />
management since antigen avoidance is necessary. Frequency<br />
<strong>of</strong> HP cases where causative antigen is not identifiable<br />
despite a thorough medical evaluation is not known.<br />
Objective: To assess the proportion <strong>of</strong> HP cases with no<br />
identifiable inciting antigen. Methods: Consecutive cases <strong>of</strong><br />
HP diagnosed at Mayo Clinic Rochester, MN between 1997 and
S76 Abstracts<br />
2002 were reviewed. Diagnostic criteria included: (1)<br />
respiratory symptoms, (2) compatible radiologic changes,<br />
(3) known exposure or a positive serologic test to an inciting<br />
antigen, and (4) no other identifiable cause for the lung<br />
disease. If there was no identifiable inciting antigen, one <strong>of</strong><br />
following two criteria was required: (1) diagnostic lung biopsy<br />
or (2) bronchoalveolar lavage lymphocytosis and compatible<br />
chest CTchanges. Results: We identified 85 consecutive cases<br />
<strong>of</strong> HP. The mean age (±SD) was 53±14 years; 53 (62%) patients<br />
were women. Cause was identified in 64 patients (75%). In 21<br />
patients (25%) the inciting antigen could not be identified by<br />
exposure history or serologic testing. In all 21 <strong>of</strong> these<br />
patients the diagnosis <strong>of</strong> HP was supported by histopathologic<br />
findings with diagnostic samples obtained in 18 patients by<br />
surgical and in 3 patients by bronchoscopic lung biopsy.<br />
Conclusions: The inciting antigen was not identifiable in 25%<br />
<strong>of</strong> patients evaluated for HP at a tertiary referral care center<br />
in the Midwest region <strong>of</strong> U.S.<br />
doi:10.1016/j.clim.2007.03.393<br />
Sa.7 Comparison <strong>of</strong> Influences <strong>of</strong> Three Types <strong>of</strong><br />
Music (Western, Classical, Traditional Persian and<br />
Pop) on Immunological Parameters<br />
Alireza Salekmoghaddam, Pr<strong>of</strong>essor, Iran University <strong>of</strong><br />
Medical Sciences, <strong>Immunology</strong>, Tehran, Iran, Alireza Salek<br />
Moghddam, Pr<strong>of</strong>essor, <strong>Immunology</strong>, Tehran, Iran, Saba Arshi,<br />
Assistant Pr<strong>of</strong>essor, <strong>Immunology</strong>, Tehran, Iran, Mohammad<br />
Kamali, Assistant Pr<strong>of</strong>essor, Statistics, Tehran, Iran, Hassan<br />
Ashayeri, Pr<strong>of</strong>essor, Neuropsychology, Tehran, Iran,<br />
Gholomreza Azadi, Technologist, <strong>Immunology</strong>, Tehran, Iran<br />
28 allergic rhinitis patients who had inclusion, exclusion<br />
criteria for this intervention were divided in four groups, three<br />
interventional groups and one control group. In each there were<br />
seven subjects related to their favorite music. Methods: Blood<br />
samples were taken before and after 1 month interventional<br />
period. Samples were examined immediately by flow cytometry<br />
and six cellular parameters (CD3, CD4, CD8, CD16, CD19 and<br />
HLA-DR) were measured and were analyzed by t test and<br />
between group by ANOVA. Conclusion: Evaluation <strong>of</strong> immune<br />
cells in atopic patients that listened to pop music showed<br />
significant increases in CD3 T cells (p=0.03) and CD4+ cells<br />
(p=0.03), CD8+ cells (p=0.05), and NK cells (p=0.014) that show<br />
immune response shifts to Th1 immune responses. As a result<br />
pop music had positive effects on atopic subjects. Possibly<br />
regular listening to favorite music can modulate immune<br />
parameters and improve signs and symptoms <strong>of</strong> allergic rhinitis<br />
in atopic patients.<br />
doi:10.1016/j.clim.2007.03.394<br />
Sa.8 Emergency Department (ED) Evaluation;<br />
Treatment, Hospital Admission and sIgE Levels in<br />
African American (AA) and Hispanic (H) Pediatric<br />
Asthmatics<br />
Ray Mun Loo, Resident in Pediatrics, Nassau University<br />
Medical Center, East Meadow, NY, Katarzyna Koscielna,<br />
Resident, Nassau University Medical Center, East Meadow,<br />
NY, Krishan Kumar, Attending in Pediatric Emergency<br />
Medicine, Nassau University Medical Center, East Meadow,<br />
NY, Marianne Frieri, Attending in Allergy <strong>Immunology</strong>,<br />
Nassau University Medical Center, East Meadow, NY<br />
Introduction: Asthma is higher in AA and 20–25% higher in<br />
the ED. We evaluated 66 ED adult asthmatics with class II<br />
allergens to cat, dust mite, mold and cockroach. This study<br />
evaluated pediatric patients on either nebulized Budesonide (B)<br />
or normal saline (NS) prior to admission. Methods: Eleven<br />
moderately severe asthmatic patients, age 8–16 years (9 AA; 2<br />
H) were clinically evaluated in the ED over 4 months and<br />
randomized to receive either 1 mg <strong>of</strong> nebulized (B) or (NS) as<br />
control. Improvement was based on pulmonary index score<br />
(PIS), peak flow (PF), and spirometry. FENO (Aerocrine) was<br />
measured in 3 patients. All non-responsive patients were<br />
admitted; sIgE levels were determined by sensitive ELISA’s<br />
(Quest; Hitachi Chemical Diagnostics). Results: Six received (B)<br />
with higher PSI <strong>of</strong> 6–9 vs.5on(NS).PF=59–267L/min. FEV1<br />
levels reversed post bronchodilation in 9 <strong>of</strong> 10, and B increased<br />
FEV1 relative to baseline (P=0.024). Increased FNO levels in 2<br />
had PSI <strong>of</strong> 8. sIgE levels in 9 <strong>of</strong> 11 tested=class III–IV to mite in 8;<br />
IV–Vtocockroach;II–IV to animal dander; II–IV to grass in 7; I–<br />
IV to mold; II–IV to rice and wheat in 6; I–III to ragweed in 5; II–<br />
VI to trees; II–IV to fish in 4. Conclusion: Polysensitization<br />
developed in 89% to inhalants and various foods but higher<br />
levels compared to our adult ED population. Education <strong>of</strong><br />
allergenic exposure, environmental and dietary control is<br />
essential to prevent hospitalization <strong>of</strong> pediatric asthmatics.<br />
doi:10.1016/j.clim.2007.03.395<br />
Sa.9 Association <strong>of</strong> the Polymorphisms <strong>of</strong> IL-4 Gene<br />
Promoter (−590CNT), IL-13 Coding Region (r130q)<br />
and IL-16 Gene Promoter (−295TNC) with Allergic<br />
Asthma<br />
Sara Hosseini-Farahabadi, Researcher, Bu-Ali Research<br />
Institute (BARI), Department <strong>of</strong> Immunogenetics, Mashhad,<br />
Iran, Jalil Tavakkol-Afshari, Vice President <strong>of</strong> Research,<br />
Head, Department <strong>of</strong> Immunogenetics and Cell Culture,<br />
Bu-Ali Research Institute (BARI), Department <strong>of</strong><br />
Immunogenetics and Cell Culture, Mashhad, Iran, Houshang<br />
Rafatpanah, Ghaem Medical Center, Department <strong>of</strong> Allergy<br />
and <strong>Immunology</strong>, Mashhad, Iran, Mohammad Khaje Daluei,<br />
Ghaem Medical Center, Department <strong>of</strong> Epidemiology and<br />
Statistics, Mashhad, Iran, Rez Farid-Hosseini, Head,<br />
Department <strong>of</strong> Allergy and <strong>Immunology</strong>, Ghaem Medical<br />
Center, Department <strong>of</strong> Allergy and <strong>Immunology</strong>, Mashhad<br />
Allergic asthma is a multifactorial disease, influenced by<br />
genetic and environmental factors. Recent family-based studies<br />
have revealed evidence for linkage <strong>of</strong> human chromosomes<br />
5q31–33, 12q15–24, 11q13 and 15q23.6 as regions likely to<br />
contain genes related to asthma. Among the candidate genes in<br />
these regions are the genes encoding for human interleukin-4,<br />
interleukin-13 and interleukin-16. To test this linkage, we<br />
examined an Iranian population <strong>of</strong> patients with asthma. A total<br />
<strong>of</strong> 30 patients with allergic asthma and 50 normal subjects were<br />
studied. Allergic asthma was confirmed using skin prick test and
Abstracts<br />
spirometry. DNA was extracted from whole blood and IL-4<br />
(−590CNT), IL-13 (R130Q) and IL-16 (−295TNC) polymorphisms<br />
were determined by PCR-RFLP method. Out <strong>of</strong> 30 patients with<br />
allergic asthma, 17 (56.7%) had CC, 8 (26.7%) had CT and 5<br />
(16.7%) had TT genotypes for IL-4 polymorphism, 11 (36.7%) had<br />
GG, 13 (43.3%) had GA and 6 (20%) had AA genotypes for IL-13<br />
polymorphism, 23 (76.7%) had TT, 7 (23.3%) had CT and no<br />
patient had CC genotypes for IL-16 polymorphism. A higher<br />
proportion <strong>of</strong> case subjects with the C allele for the IL-4, G<br />
allele for the IL-13 and T allele for the IL-16 polymorphisms<br />
were found compared with the T, A and C alleles respectively.<br />
These results do suggest an influence <strong>of</strong> genetic variability at<br />
the promoter <strong>of</strong> IL-4 gene (−590CNT) and a coding region <strong>of</strong> IL-<br />
13gene(R130Q)ontheoccurrence<strong>of</strong>allergicasthmaandno<br />
relationship between IL-16 promoter polymorphism (−295TNC)<br />
and this disease.<br />
doi:10.1016/j.clim.2007.03.396<br />
Sa.10 Lymphotactin Improves Treg Function in<br />
Asthmatics via the STAT5 Pathway<br />
Alison Fohner, Undergraduate Student, Stanford University,<br />
Pediatrics and <strong>Immunology</strong>, Stanford, CA, Khoa Nguyen, Lab<br />
Specialist, Stanford University, Pediatrics and <strong>Immunology</strong>,<br />
Stanford, CA, Alan M. Krensky, Doctor, Stanford University,<br />
Department <strong>of</strong> Pediatrics and <strong>Immunology</strong>, Stanford, CA,<br />
Kari C. Nadeau, Doctor, Stanford University, Department <strong>of</strong><br />
Pediatrics and <strong>Immunology</strong>, Stanford, CA<br />
Lymphotactin is a chemokine (XCL1) associated with the<br />
function <strong>of</strong> regulatory T cells (Treg) and with allergic airway<br />
inflammation. Compared to healthy controls (HC) (n=10),<br />
allergic asthma patients (AA) (n=10) have fewer Tregs and<br />
more invariant natural killer T cells (iNKT) in the periphery,<br />
and iNKT kill Treg. We reported that Treg in asthmatics are<br />
dysfunctional but that XCL1 increases their suppressive<br />
function in vitro. We hypothesized that incubation <strong>of</strong> Treg<br />
with XCL1 could increase Treg absolute numbers and/or<br />
improve Treg function in AA subjects. Therefore, we added<br />
200 ng/mL XCL1 to HC Treg (Foxp3+CD127−CD25+CD4+) vs.<br />
non-Treg (Foxp3−CD127−CD25−CD4+) and AA Treg vs. non-<br />
Treg. Incubation with XCL1 for 4 days increased the ratio <strong>of</strong><br />
Treg to non-Treg by 2–3×. Via a CFSE uptake assay,<br />
increased proliferation was not detected, suggesting that<br />
non-Treg were becoming Treg. We examined biochemical<br />
differences that could cause this conversion, including<br />
STAT1, STAT3, STAT5, and granzyme B expression since they<br />
have been implicated in Treg function. STAT5 phosphorylation<br />
increased in AA Treg and non-Treg, but not in HC or<br />
non-allergic asthmatics (NA). However, after XCL1 stimulation<br />
for 10 min, phosphorylated STAT5 increased significantly<br />
in non-Treg, but not Treg, <strong>of</strong> AA compared to HC<br />
(pb0.02) and NA (pb0.02). Since STAT5 phosphorylation<br />
plays a role in normal Treg function, these data support the<br />
conversion <strong>of</strong> non-Treg into Treg after incubation with<br />
XCL1. Thus, XCL1 functions through a STAT5 pathway to<br />
enhance Treg function in asthmatics. STAT5 could be a<br />
target for asthma therapy in the future.<br />
doi:10.1016/j.clim.2007.03.397<br />
Sa.11 Long-Term Tolerance in a Murine Model <strong>of</strong><br />
Type I Allergy Through Transplantation <strong>of</strong><br />
Genetically Modified Hematopoietic Stem Cells<br />
Ulrike Baranyi, PhD, Medical University <strong>of</strong> Vienna,<br />
Department <strong>of</strong> Surgery, Division <strong>of</strong> Transplantation, Vienna,<br />
Austria, Birgit Linhart, PhD, Division <strong>of</strong> Immunopathology,<br />
Department <strong>of</strong> Pathophysiology, Center <strong>of</strong> Physiology and<br />
Pathophysiology, Vienna, Austria, Nina Pilat, Division <strong>of</strong><br />
Transplantation, Department <strong>of</strong> Surgery, Medical University<br />
<strong>of</strong> Vienna, Vienna, Austria, John Iacomini, PhD,<br />
Transplantation Research Center, Brigham and Women’s<br />
Hospital and Women’s Hospital and Children’s Hospital,<br />
Boston, MA, Jessamyn Bagely, PhD, Transplantation<br />
Research Center, Brigham and Women’s Hospital and<br />
Children’s Hospital, Harvard Medical School, Boston, MA,<br />
Rudolf Valenta, MD, Division <strong>of</strong> Immunopathology, Center <strong>of</strong><br />
Physiology and Pathophysiology, Vienna, Austria, Thomas<br />
Wekerle, MD, Division <strong>of</strong> Transplantation, Department <strong>of</strong><br />
Surgery, Vienna, Austria<br />
Introduction: Current therapies for treating allergy are<br />
associated with various limitations. We thus investigated<br />
whether tolerance can be induced through transplantation <strong>of</strong><br />
syngeneic bone marrow retrovirally transduced to express an<br />
allergen. Methods: Balb/c bone marrow cells (BMC) were<br />
retrovirally transduced in vitro to express Phl p 5 (a major<br />
grass pollen allergen) in a membrane-anchored fashion<br />
(transduction efficiency: 35–55%). Myeloablated Balb/c<br />
mice received 2–4×10 6 transduced BMC iv and were thereafter<br />
repeatedly injected sc with recombinant Phl p 5 and<br />
rBet v 1 (an unrelated control allergen) (0.5 μg plus aluminum<br />
hydroxide, weeks 6, 9, 12 and 22). Allergen-specific antibody<br />
levels were determined in sera by ELISA, Phl p 5 expression by<br />
FACS. Results: All mice (n=10) transplanted with Phl p 5transduced<br />
BMC developed high levels <strong>of</strong> multi-lineage<br />
molecular chimerism which remained stable for the length<br />
<strong>of</strong> follow-up (up to 9 months) (e.g., 11 mean% Phl p 5+B cells<br />
and 22% T cells, 25 weeks post-BMT). Serum levels <strong>of</strong> Phl p 5specific<br />
IgE and IgG1 remained undetectable in all chimeras<br />
throughout follow-up while high levels <strong>of</strong> Bet v 1-specific IgE<br />
and IgG1 were measured. Basophil degranulation assays<br />
revealed the absence <strong>of</strong> Phl p 5-specific degranulation in<br />
chimeras, while in contrast Bet v 1-specific degranulation was<br />
preserved. In T cell proliferation assays chimeras showed<br />
specific non-responsiveness to Phl p 5 (n=3; pb0.01 vs. nontransduced<br />
mice). Conclusion: These pro<strong>of</strong>-<strong>of</strong>-principle<br />
experiments demonstra5.5e for the first time that molecular<br />
chimerism induces robust and long-lasting tolerance in<br />
allergy at the B cell, T cell and effector cell levels.<br />
doi:10.1016/j.clim.2007.03.398<br />
S77<br />
Sa.12 Lichen Planus (LP) Associated with Xolair<br />
Administration<br />
Filiz Seeborg, Instructor, Baylor College <strong>of</strong> Medicine and<br />
Texas Children’s Hospital, Section <strong>of</strong> Allergy and<br />
<strong>Immunology</strong>, Houston, TX, Pardeep S. Rihal, MD,<br />
West Houston Allergy and Asthma, Katy, TX, Adam Czelusta,<br />
MD, Katy Dermatology, PA, Katy, TX, Imelda C. Hanson,
S78 Abstracts<br />
Pr<strong>of</strong>essor, Baylor College <strong>of</strong> Medicine and Texas Childrens<br />
Hospital, Section <strong>of</strong> Allergy and <strong>Immunology</strong>, Houston, TX<br />
Background: Omalizumab (Xolair) is a recombinant<br />
monoclonal anti-immunoglobulin E (IgE) antibody that<br />
binds to free IgE, clearing it from the circulation. Xolair is<br />
approved for moderate to severe persistent asthma in<br />
adolescents and adults. Contraindication to use includes<br />
prior anaphylaxis to Xolair. Case report: We describe a 63year-old<br />
female who developed LP following Xolair administration.<br />
She had steroid-dependent asthma, allergic rhinitis,<br />
osteoarthritis, and hypertension. Her serum IgE level was<br />
55 KU/L. She developed a brief, generalized pruritus<br />
following first Xolair administration (300 μg/month). With<br />
second dosing, she had a generalized pruritic rash with lacy,<br />
violaceous facial papules lasting 2 weeks. Xolair and<br />
Dicl<strong>of</strong>enac were discontinued. A skin biopsy revealed LP.<br />
Following third dosing (6 months later), she developed white<br />
oral lesions in addition to the previously experienced pruritic<br />
rash. Discussion: LP is a T cell mediated inflammatory<br />
mucocutaneous condition with pruritic violaceous papules<br />
and plaques. <strong>Oral</strong> LP may predispose to development <strong>of</strong><br />
squamous cell carcinoma. LP may develop secondary to<br />
certain systemic medications and hepatitis C infection. No<br />
association between Xolair and LP is in the published<br />
literature. Malignant neoplasms have been reported during<br />
Xolair treatment, but a causal relationship has not been<br />
documented. Our case demonstrates a clear temporal<br />
association between Xolair use/cessation and the development/disappearance<br />
<strong>of</strong> LP. Per the manufacturer, malignancy<br />
risk for Xolair is unknown in individuals with existing<br />
malignant risk (oral LP). Conclusion: Repeated exposure to<br />
immunoregulatory therapy using Xolair may have potential<br />
to produce a T cell mediated immune activation leading to<br />
LP.<br />
doi:10.1016/j.clim.2007.03.401<br />
Sa.13 Inducible Expression <strong>of</strong> the Proallergic<br />
Cytokine TSLP in Airway Epithelial Cells is<br />
Controlled by NFkappaB<br />
Hai-Chon Lee, Postdoctoral Fellow, <strong>Immunology</strong> Program,<br />
Benaroya Research Institute, Seattle, WA, Steven F. Ziegler,<br />
Program Reader, <strong>Immunology</strong> Program, Benaroya Research<br />
Institute, Seattle, WA<br />
The epithelial-derived cytokine thymic stromal lymphopoietin<br />
(TSLP) is important for the initiation <strong>of</strong> allergic<br />
airway inflammation through a dendritic cell-mediated Th2<br />
response. To identify the factors that control TSLP expression,<br />
we examined the ability <strong>of</strong> inflammatory mediators to<br />
regulate TSLP production in human airway epithelial cells.<br />
We found that both IL-12 and TNF-± were capable <strong>of</strong><br />
inducing rapid TSLP production in primary human bronchial<br />
airway epithelial cells. We further characterized the human<br />
TSLP gene promoter using two human epithelial cell lines,<br />
16HBEo and A549, and showed that IL-12- and TNF-±mediated<br />
human TSLP promoter activation in these cells<br />
was mediated by an upstream NFkB site. Mutation <strong>of</strong> this<br />
NFkB site completely abolished activation, as did overexpression<br />
<strong>of</strong> a dominant-negative version <strong>of</strong> IKK 2 . Interestingly,<br />
human TSLP mRNA levels were also increased<br />
following exposure to TLR2, 8, and 9 ligands, further<br />
supporting an important role for NFkB in TSLP gene<br />
regulation. In a similar manner, analysis <strong>of</strong> the mouse<br />
TSLP gene promoter revealed the presence <strong>of</strong> a similar<br />
situated NFkB site that was also critical for IL-12-inducible<br />
expression <strong>of</strong> mouse TSLP. Taken together, these results<br />
demonstrate that the inflammatory mediators IL-12 and<br />
TNF-± regulate human TSLP gene expression in an NFkBdependent<br />
manner.<br />
doi:10.1016/j.clim.2007.03.402<br />
Sa.16 CpG Induces Th1 Response in Neonatal Mice<br />
but Does Not Suppress Anaphylactic IgG1 Response:<br />
Possible Role <strong>of</strong> Low TLR-9 Expression<br />
Cyro Alves Brito, PharmD, University <strong>of</strong> São Paulo, Brazil,<br />
Department <strong>of</strong> Dermatology, São Paulo, Brazil, Ana Elisa<br />
Fusaro, PharmD, University <strong>of</strong> São Paulo, Brazil,<br />
Department <strong>of</strong> Dermatology, São Paulo, Brazil, Jeferson<br />
Russo Victor, BSc, University <strong>of</strong> São Paulo, Brazil,<br />
Department <strong>of</strong> Dermatology, São Paulo, Brazil, Adriana<br />
Leticia Goldoni, BSc, University <strong>of</strong> São Paulo, Brazil,<br />
Department <strong>of</strong> Dermatology, São Paulo, Brazil, Paula<br />
Ordonhez Rigato, BSc, University <strong>of</strong> São Paulo, Brazil,<br />
Department <strong>of</strong> Dermatology, São Paulo, Brazil, Alberto Jose<br />
Silva Duarte, MD, University <strong>of</strong> São Paulo, Brazil,<br />
Department <strong>of</strong> Dermatology, São Paulo, Brazil, Maria<br />
Notomi Sato, BSc, University <strong>of</strong> São Paulo, Brazil,<br />
Department <strong>of</strong> Dermatology, São Paulo, Brazil<br />
It has been described that there are several differences<br />
between immune responses in adults and newborns. The<br />
aim <strong>of</strong> this work was to evaluate in early life and adult<br />
stage <strong>of</strong> mice, the immune modulatory effects <strong>of</strong> oligodeoxynucleotides<br />
containing CpG motif (CpG-ODN) in<br />
immunization with ovalbumin (OVA) and Blomia tropicalis<br />
(Bt). Three-day-old and 8–10-week-old A/Sn mice were<br />
immunized with OVA or Bt in alum, associated with CpG-<br />
ODN or control-ODN. Spleen cells from non-immunized mice<br />
were cultured with CpG or control-ODN for TLR-9 expression<br />
analysis. Neonate and adult co-administration <strong>of</strong> CpG<br />
with OVA or Bt was able to significantly decrease IgE<br />
response in parallel to an enhancement <strong>of</strong> IgG2a antibody<br />
levels compared to control mice. Furthermore, spleen cells<br />
from newborns immunized with OVA associated to CpG<br />
showed increased IFN-g production after anti-CD3 or LPS<br />
stimuli compared to control group. However, the negative<br />
regulation <strong>of</strong> IgE response was more evident in adult- than<br />
in neonate-immunized mice, also CpG down-regulates<br />
anaphylactic anti-OVA IgG1 production only in adult mice.<br />
Next we investigated whether an altered TLR-9 expression<br />
could be involved in impaired CpG effect in neonates. It<br />
was observed that after 48 hours <strong>of</strong> CpG stimulus TLR-9<br />
expression was already up-regulated in adult but not in<br />
neonate B cells. The pattern <strong>of</strong> antibody and cytokine<br />
production suggests a Th1-type response induced by CpG-<br />
ODN. Administration <strong>of</strong> CpG in newborns may be less
Abstracts<br />
effective than in adults due, in part, to a decreased B cell<br />
sensitivity to CpG stimulus. Supported by FAPESP, LIM-56,<br />
and HC-FMUSP.<br />
doi:10.1016/j.clim.2007.03.403<br />
Sa.17 IgE-reactive 60S Ribosomal Protein P2 in<br />
Almond (Prunus Dulcis) and Walnut (Juglans Regia),<br />
a New Class <strong>of</strong> Food Allergen Cross-reactive with<br />
Fungal Aeroallergens<br />
Pallavi Tawde, PhD Candidate, Florida State University,<br />
Department <strong>of</strong> Biological Science and Institute <strong>of</strong> Molecular<br />
Biophysics, Tallahassee, FL, Mohsen Abolhassani, Visiting<br />
Scholar, Florida State University, Department <strong>of</strong> Biology,<br />
Tallahassee, FL, Vanessa Seamon, Research Technician,<br />
Florida State University, Department <strong>of</strong> Biology,<br />
Tallahassee, FL, Suzanne Teuber, Associate Pr<strong>of</strong>essor,<br />
University <strong>of</strong> California, Davis, Internal Medicine,<br />
Rheumatology and Allergy, Davis, CA, Fang Wang,<br />
Postdoctoraltoral Research Fellow, Florida State University,<br />
Department <strong>of</strong> Biology, Tallahassee, FL, Sandra Uratsu,<br />
Staff Research Associate, University <strong>of</strong> California,<br />
Davis, Department <strong>of</strong> Plant Sciences, Davis, CA, Abhya<br />
Dandekar, Pr<strong>of</strong>essor, University <strong>of</strong> California, Davis,<br />
Department <strong>of</strong> Plant Sciences, Davis, CA, Stefan Vieths,<br />
Pr<strong>of</strong>essor, Paul-Ehrlich-Institut, Department <strong>of</strong><br />
Allergology, Langen, Germany, Shridhar Sathe,<br />
Pr<strong>of</strong>essor, Florida State University, Department <strong>of</strong><br />
Food Science, Tallahassee, FL, Kenneth Roux, Pr<strong>of</strong>essor,<br />
Florida State University, Department <strong>of</strong> Biological<br />
Science and Institute <strong>of</strong> Molecular Biophysics,<br />
Tallahassee, FL<br />
Introduction: Tree nuts are a source <strong>of</strong> food allergens<br />
<strong>of</strong>ten associated with life-threatening reactions in susceptible<br />
individuals. Molecular characterization <strong>of</strong> the protein<br />
allergens is essential for a better understanding <strong>of</strong> the<br />
pathogenesis <strong>of</strong> atopic diseases, and in the development<br />
<strong>of</strong> more efficacious diagnostic and therapeutic modalities.<br />
We employed molecular cloning and expression to identify<br />
the 60S acidic ribosomal protein P2 (60S RP) as IgEreactive<br />
proteins in almonds and walnuts. 60S RPs have<br />
been described as aeroallergens in some molds. Results:<br />
Immunoscreening identified IgE-reactive almond and walnut<br />
cDNA clones, each encoding an 11,450 Da 60S acidic<br />
ribosomal protein P2 (60S RP), designated as Pru du 5 and<br />
Jug r 5, respectively, and sharing 91% sequence similarity.<br />
Of 27 sera from patients reporting allergy to almond and/<br />
or walnut, 9 (33%) had IgE reactive with the recombinant<br />
proteins: 8 reacted to both proteins and 1 only with<br />
walnut Jug r 5. The 9 positive patient sera, but no<br />
others, also reacted with, and were inhibited by,<br />
recombinant Fus c 1, a fungal (Fusarium culmorum) 60S<br />
RP aeroallergen. Native 60S RP was detected in both<br />
almond and walnut crude extracts by reaction with rabbit<br />
anti-Pru du 5 antibodies. Conclusions: 60S RP represents a<br />
new class <strong>of</strong> food allergen in plants. Over a third <strong>of</strong><br />
tested patients had IgE that bound almond (Pru du 5)<br />
and/or walnut (Jug r 5) homologues in in vitro assays.<br />
The data indicate considerable cross-reactivity between<br />
the almond, walnut, and fungal homologues.<br />
doi:10.1016/j.clim.2007.03.404<br />
Sa.18 Pistachio Vicilin, Pis v 1, is Allergenic and<br />
Cross-reactive with the Homologous Cashew<br />
Allergen, Ana o 1<br />
LeAnna N. Willison, Graduate Student, Florida State<br />
University, Tallahasee, FL, Pallavi Tawde, Graduate<br />
Student, Florida State University, Department <strong>of</strong> Biological<br />
Sciences, Tallahasee, FL, Jason M. Robotham,<br />
Postdoctoraltoral Fellow, Florida State University,<br />
Department <strong>of</strong> Biological Sciences, Tallahasee, FL, Suzanne<br />
S. Teuber, Associate Pr<strong>of</strong>essor, University <strong>of</strong> California<br />
Davis, Internal Medicine, Rheumatology and Allergy, Davis,<br />
CA, Richard Penny, Graduate Student, Florida State<br />
University, Department <strong>of</strong> Biological Sciences, Tallahasee,<br />
FL, Shridhar K. Sathe, Pr<strong>of</strong>essor, Florida State University,<br />
Food Sciences and Nutrition Department, Tallahasee, FL,<br />
Kenneth H. Roux, Pr<strong>of</strong>essor, Florida State University,<br />
Department <strong>of</strong> Biological Sciences, Tallahasee, FL<br />
Introduction: Several studies have noted potential crossreactivity<br />
between certain tree nuts, especially those<br />
within the same botanical family. A potential example<br />
includes pistachio and cashew, which are both from the<br />
Anacardiaceae family. Results: A pistachio cDNA library was<br />
screened to detect potential pistachio allergens. An isolate<br />
was sequenced, cloned, and expressed in E. coli. The<br />
isolate coded for a 7S vicilin-like protein designated Pis v 1.<br />
Reactivity to the allergen was screened using 18 patients’<br />
sera: 9 cashew- and pistachio-allergic, 6 cashew-allergic<br />
but had never eaten pistachios, 1 pistachio-allergic only,<br />
and 2 cashew-allergic only. IgE reactivity to Pis v 1 was<br />
found in the serum <strong>of</strong> 5 patients, all <strong>of</strong> which had histories<br />
<strong>of</strong> allergy to both tree nuts or had never eaten pistachio.<br />
Cross-reactivity between Pis v 1 and the cashew homologue,<br />
Ana o 1, was investigated using an inhibition dot blot<br />
assay. Of the tested sera, the 5 (28%) patients that<br />
recognized Pis v 1 also recognized Ana o 1 and IgE binding<br />
was completely inhibited after pre-incubation with either<br />
cloned allergen. Conclusion: Patients allergic to cashew<br />
<strong>of</strong>ten report allergy to pistachio, which could be a result <strong>of</strong><br />
cross-reactivity between the two. This study revealed that<br />
28% <strong>of</strong> tested patients recognized the pistachio allergen Pis<br />
v 1 and these same patients also showed complete crossreactivity<br />
between the vicilin-like allergens from pistachio<br />
and cashew. Therefore, the pistachio allergen Pis v 1 is a<br />
likely contributor to the observed co-sensitivity to pistachio<br />
and cashew in some patients.<br />
doi:10.1016/j.clim.2007.03.405<br />
Sa.19 Infusion Reactions from Human<br />
Plasma-derived Products<br />
Hon-Sum Ko, Medical Officer, Food and Drug<br />
Administration/Division <strong>of</strong> Hematology, Potomac, MD<br />
S79
S80 Abstracts<br />
Introduction: Although human plasma-derived products<br />
are generally considered to have a favorable safety<br />
pr<strong>of</strong>ile, intravenous infusion <strong>of</strong> these products is occasionally<br />
associated with local or systemic reactions,<br />
varying from local discomfort to anaphylaxis-like manifestations.<br />
This study is an attempt to identify the factors<br />
that may affect the risks <strong>of</strong> such reactions. Materials and<br />
methods: A review was made in the clinical and<br />
laboratory data found in the package inserts or license<br />
applications <strong>of</strong> fifteen randomly chosen human plasmaderived<br />
products available in the U.S. The products<br />
included albumin, plasma protein fraction, intravenous<br />
immunoglobulins, coagulation factors, and enzyme inhibitors<br />
in plasma. The adverse effects from administration <strong>of</strong><br />
the products, including the frequency, severity, and<br />
potential attribution <strong>of</strong> local and systemic reactions<br />
were correlated with patient age, sex, ethnicity, allergic<br />
history, concomitant medications, previous infusion reactions,<br />
infusion rate, product class, product manufacture<br />
process, serum immunoglobulin levels, and other immune<br />
parameters whenever available. Results: Aside from<br />
infusion rate, which appears to correlate positively with<br />
the frequency <strong>of</strong> systemic reactions upon administration<br />
<strong>of</strong> immunoglobulin products, no consistent relationship<br />
between reaction frequency or severity and the above<br />
clinical/laboratory parameters was observed. A protective<br />
effect <strong>of</strong> premedication use before infusion was not<br />
observed. Conclusions: Much remains to be learned<br />
concerning the risk factors for infusion reactions to<br />
human plasma-derived products. Although current clinical<br />
and laboratory parameters may not be adequate to<br />
predict such risks, the development <strong>of</strong> newer biomarkers<br />
might <strong>of</strong>fer an opportunity to help mitigate potentially<br />
serious infusion reactions.<br />
doi:10.1016/j.clim.2007.03.406<br />
Sa.20 Tolerance-Inducing Capacity is Preserved in<br />
Dendritic Cells from Patients with SLE<br />
Jose Crispin, PhD Student, Department <strong>of</strong> <strong>Immunology</strong> and<br />
Rheumatology, Instituto Nacional de Ciencias Medicas y<br />
Nutricion SZ, Mexico City, Mexico, Leonardo Limon,<br />
Resident, Department <strong>of</strong> <strong>Immunology</strong> and Rheumatology,<br />
Instituto Nacional de Ciencias Medicas y Nutricion SZ,<br />
Mexico City, Mexico, Maria Ines Vargas Rojas, PhD Student,<br />
Department <strong>of</strong> <strong>Immunology</strong> and Rheumatology, Instituto<br />
Nacional de Ciencias Medicas y Nutricion SZ, Mexico City,<br />
Mexico, Jorge Alcocer Varela, Investigator, Department <strong>of</strong><br />
<strong>Immunology</strong> and Rheumatology, Instituto Nacional de<br />
Ciencias Medicas y Nutricion SZ, Mexico City, Mexico<br />
Granted with the faculty <strong>of</strong> inducing T cell activation<br />
and regulation, dendritic cell (DC) function is pivotal for<br />
the maintenance <strong>of</strong> tolerance. DC are able to promote<br />
anti-nuclear oriented responses and worsen SLE in animal<br />
models. The aim <strong>of</strong> this project was to analyze the<br />
phenotype and function <strong>of</strong> SLE-derived DC and explore<br />
their capacity to induce T cell tolerance. 23 patients with<br />
SLE and 11 controls were included. The phenotype <strong>of</strong><br />
circulating DC was analyzed by flow cytometry. Phenotype<br />
and cytokine production <strong>of</strong> monocyte-derived DC (MD-DC)<br />
was studied at the end <strong>of</strong> the differentiation period (basal)<br />
and after stimulation (LPS, TNF + PGE2 or anti-CD40).<br />
Ability to stimulate T cell proliferation was tested in<br />
allogeneic MLR. Phenotype and function <strong>of</strong> tolerogenic DC<br />
(grown in the presence <strong>of</strong> IL-10) was analyzed. The<br />
capacity <strong>of</strong> DC to induce tolerance towards allogeneic<br />
antigens, was analyzed in a recall assay. A higher<br />
proportion <strong>of</strong> DC from patients with SLE displayed<br />
costimulatory molecules in vivo (Pb0.05). Conversely,<br />
phenotype and T cell stimulatory capacity did not differ<br />
between MD-DC from patients and controls, neither in<br />
basal conditions, nor after stimulation. However, DC from<br />
patients with SLE showed a diminished response to<br />
stimulation via CD40. MD-DC from patients with SLE<br />
exhibited a normal capacity to induce tolerance towards<br />
alloantigens. DC from patients with SLE have an overstimulated<br />
phenotype when studied in vivo. However,such<br />
abnormality is absent when cells are grown in vitro.<br />
Tolerogenic MD-DC from SLE patients are capable <strong>of</strong><br />
inducing T cell tolerance.<br />
doi:10.1016/j.clim.2007.03.407<br />
Sa.21 The Largest International Collaborative<br />
Studies on Ocular Lesions in Behcet Disease<br />
Nobuyoshi Kitaichi, Ophthalmologist, Department <strong>of</strong><br />
Ophthalmology and Visual Sciences, Hokkaido University<br />
Graduate School <strong>of</strong> Medicine, Sapporo, Japan,<br />
Shigeaki Oh, Ophthalmologist, Department <strong>of</strong><br />
Ophthalmology and Visual Sciences, Hokkaido University<br />
Graduate School <strong>of</strong> Medicine, Sapporo, Japan<br />
Purpose: Behcet disease is predominantly found<br />
between East Asia and Mediterranean basin along the<br />
ancient Silk Road. In order to investigate the clinical<br />
features <strong>of</strong> ocular lesions in Behcet disease, international<br />
collaborative studies were performed by utilizing the<br />
same questionnaire. Methods: Descriptive questionnaires<br />
were sent to 132 ophthalmology centers in the world. Answers<br />
<strong>of</strong> 1465 patients were collected from 25 eye centers in 14<br />
countries; Australia, Germany, Greece, India, Iran, Italy,<br />
Japan, Jordan, Morocco, Portugal, Turkey, Saudi Arabia,<br />
Tunisia, and United Kingdom. Results: Men were seen in<br />
68.3% <strong>of</strong> the patients. The average age <strong>of</strong> onset was 27.5 years<br />
old. The frequency <strong>of</strong> HLA-B51 was detected in 62.0%.<br />
Recurrent oral aphthous ulcers were observed in 94.5%, skin<br />
lesions in 69.5% and genital ulcers in 61.4%. Allergic diseases<br />
were recognized in only 4.3% <strong>of</strong> the patients. Bilaterality and<br />
recurrency <strong>of</strong> intraocular inflammation were seen in 85.5%<br />
and 95.7%, respectively. Regarding visual prognosis, 23.3% had<br />
poor vision less than 0.1 (20/200) at the final visit. The<br />
percentage <strong>of</strong> poor vision was significantly higher in India,<br />
Iran, and Japan, and the lowest in Italy (pb0.01). Combined<br />
anterior and posterior segment intraocular inflammation<br />
(CAPSII), more serious than anterior segment intraocular<br />
inflammation, was seen more frequently in men (95.4%) than<br />
in women (89.9%, pb0.01). Conclusions: We performed the<br />
largest world-scale investigation <strong>of</strong> ocular lesions in Behcet
Abstracts<br />
disease. Th2-dominant allergic diseases seem quite rare in<br />
Th1-dominant Behcet disease. Men tend to have worse visual<br />
prognosis than women. <strong>Clinical</strong> pictures <strong>of</strong> Behcet disease<br />
vary regionally.<br />
doi:10.1016/j.clim.2007.03.408<br />
Sa.22 Splenic Macrophages Promotes Responses to<br />
Nucleosomes in NZB/W F1 Mice<br />
Akiko Okamoto, Department <strong>of</strong> Allergy and Rheumatology,<br />
School <strong>of</strong> Medicine, The University <strong>of</strong> Tokyo, Tokyo, Japan,<br />
Keishi Fujio, Department <strong>of</strong> Allergy and Rheumatology,<br />
School <strong>of</strong> Medicine, The University <strong>of</strong> Tokyo, Tokyo, Japan,<br />
Nico van Rooijen, Department <strong>of</strong> Cell biology and<br />
<strong>Immunology</strong>, Faculty <strong>of</strong> Medicine, Free University,<br />
Amsterdam, Netherlands, Kazuhiko Yamamoto, Pr<strong>of</strong>essor<br />
and Chairman <strong>of</strong> the Department <strong>of</strong> Allergy and<br />
Rheumatology, Department <strong>of</strong> Allergy and Rheumatology,<br />
School <strong>of</strong> Medicine, The University <strong>of</strong> Tokyo, Tokyo, Japan,<br />
Keith B. Elkon, Pr<strong>of</strong>essor <strong>of</strong> Medicine, Head, Division <strong>of</strong><br />
Rheumatology, Division <strong>of</strong> Rheumatology, University <strong>of</strong><br />
Washington, Seattle, WA<br />
Systemic lupus erythematosus (SLE) is characterized by<br />
autoantibody production and organ damage. Autoantigen<br />
presentation to T cells is important for the development <strong>of</strong><br />
lupus. Nucleosomes are major autoantigens in SLE. In this<br />
study, we examined nucleosome presentation in lupus-prone<br />
NZB×NZW F1 (NZB/W F1) mice. Nucleosome-specific T cells<br />
were generated by retroviral transfer <strong>of</strong> nucleosome-specific<br />
TCR. We found that nucleosome-specific T cells were<br />
stimulated dominantly in the spleen, compared to lymph<br />
nodes, lung, and thymus. Among splenic antigen-presenting<br />
cells (APCs), F4/80+ macrophages and CD11c+CD11b+<br />
dendritic cells stimulated nucleosome-specific T cells<br />
strongly. F4/80+ macrophage was the most abundant APC<br />
subset in the spleen. Then we investigated whether splenic<br />
phagocytes were responsible for nucleosome presentation<br />
and disease progression in vivo. Splenic phagocytes, mostly<br />
F4/80+ macrophages, were depleted in NZB/W F1 mice by<br />
intravenous injection <strong>of</strong> Cl2MDP (dichloromethylene diphosphonate)-liposomes.<br />
Cl2MDP-liposome treatment dramatically<br />
suppressed nucleosome presentation in the spleen.<br />
Moreover, Cl2MDP-liposome treatment at 20 and 22 weeks <strong>of</strong><br />
age significantly suppressed anti-nucleosome antibody (Ab)<br />
and anti-double stranded DNA (dsDNA) Ab production.<br />
Proteinuria progression was delayed and survival was<br />
prolonged in phagocyte-depleted mice. The numbers <strong>of</strong><br />
autoantibody secreting cells were decreased in the spleen<br />
from phagocyte-depleted mice. Multiple injections <strong>of</strong><br />
splenic F4/80+ macrophages, not CD11c+ dendritic cells,<br />
induced autoantibody production and proteinuria progression<br />
in NZB/W F1 mice. These results indicate that<br />
autoantigen presentation by splenic macrophages significantly<br />
contributes to autoantibody production and disease<br />
progression in lupus-prone mice.<br />
doi:10.1016/j.clim.2007.03.409<br />
Sa.23 A Comparison <strong>of</strong> TNF Dependent and<br />
Independent Features <strong>of</strong> Murine<br />
Spondyloarthropathy<br />
Peggy Jacques, MD, Ugent, University Hospital, Internal<br />
Medicine, Ghent, Belgium, Rik Lories, MD, PhD, University<br />
Hospitals Leuven, Internal Medicine, Leuven, Belgium, Ken<br />
Coppieters, M. Sc, Ugent, University Hospital, Internal<br />
Medicine, Ghent, Belgium, Katrien Van Beneden, PhD,<br />
Ugent, University Hospital, Internal Medicine, Ghent,<br />
Belgium, Herman Mielants, MD, PhD, Ugent, University<br />
Hospital, Internal Medicine, Gent, Belgium, Sylvie Seeuws,<br />
PhD, Ugent, University Hospital, Internal Medicine, Ghent,<br />
Belgium, Gust Verbruggen, MD, PhD, Ugent, University<br />
Hospital, Internal Medicine, Gent, Belgium, Martine De Vos,<br />
MD, PhD, Ugent, University Hospital, Internal Medicine,<br />
Gent, Belgium, Dirk Elewaut, MD, PhD, Ugent, University<br />
Hospital, Internal Medicine, Gent, Belgium<br />
Rheumatoid arthritis (RA) and spondyloarthropathies<br />
(SpA) are frequently occurring inflammatory disorders<br />
with distinct clinical features in which tumour necrosis<br />
factor (TNF) plays a predominant role. The focus <strong>of</strong> this<br />
study was to compare the typical features <strong>of</strong> three distinct<br />
arthritis models with special reference to synovitis and<br />
enthesitis. Collagen induced arthritis (CIA) is a well<br />
established model for RA in which peripheral joints are<br />
characterized by inflamed synovium and exudate in the<br />
synovial cavity. Proteoglycan depletion, cartilage destruction<br />
and bone loss are other features <strong>of</strong> this disease, which<br />
can be restored by TNF neutralisation. We also evaluated<br />
the TNF&DeltaARE mouse model, characterized by an<br />
increased TNF mRNA stability, where Crohn's disease is<br />
accompanied by peripheral arthritis. We demonstrated that<br />
this model, unlike CIA, is also characterized by dactylitis,<br />
enthesitis and axial involvement with sacroiliitis and<br />
spondylitis (histologically and by in vivo imaging (MRI)).<br />
Strikingly, severe bone demineralisation was observed but<br />
no signs <strong>of</strong> new bone formation, a common feature <strong>of</strong><br />
human SpA, were evident. Spontaneous ankylosing dactylitis<br />
(SpAD) in ageing male DBA/1 mice, which develop<br />
ankylosing enthesitis with enthesial cartilage and bone<br />
formation is an established SpA model characterized by<br />
dactylitis, onychoperiostitis, and joint space bridging,<br />
without axial involvement or bowel inflammation. Contrary<br />
to the inflammatory features in the first models, the new<br />
bone formation was not found to be enhanced by TNF<br />
neutralisation. Conclusion: These findings indicate that<br />
inflammatory features <strong>of</strong> murine SpA are largely dependent<br />
upon TNF, but other mechanisms control bone new<br />
formation frequently occurring in SpA.<br />
doi:10.1016/j.clim.2007.03.410<br />
S81<br />
Sa.24 The Presence <strong>of</strong> Anti-La Autoantibody is<br />
Associated with a Lower Risk <strong>of</strong> Nephritis and<br />
Seizures in Lupus Patients<br />
Sanober Malik, Medicine Resident, Department <strong>of</strong> Medicine,<br />
Oklahoma University Health Sciences Center, Oklahoma<br />
City, OK, Gail R. Bruner, <strong>Clinical</strong> Coordinator, Arthritis and
S82 Abstracts<br />
<strong>Immunology</strong>/Oklahoma Medical Research Foundation,<br />
Oklahoma City, OK, Lourdes Feo, Recruiter, Oklahoma<br />
Medical Research Foundation, Oklahoma City, OK, Courtney<br />
Williams-Weese, Recruiter, Oklahoma Medical Research<br />
Foundation, Oklahoma City, OK, R. Hal Sc<strong>of</strong>ield, Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> Medicine, Oklahoma University Health<br />
Sciences Center/VA Medical Center/Oklahoma Medical<br />
Research Foundation, Oklahoma City, OK, John B. Harley,<br />
Pr<strong>of</strong>essor and Section Chief, Program Head, Department <strong>of</strong><br />
Medicine, Oklahoma University Health Sciences Center/VA<br />
Medical Center/Oklahoma Medical Research Foundation,<br />
Oklahoma City, OK, Amr H. Sawalha, Assistant Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> Medicine, Arthritis and <strong>Immunology</strong><br />
Program, Oklahoma University Health Sciences Center/VA<br />
Medical Center/Oklahoma Medical Research Foundation,<br />
Oklahoma City, OK<br />
Background: Previous reports suggest a protective role for<br />
anti-La autoantibody against the development <strong>of</strong> lupus<br />
nephritis. We studied the effect <strong>of</strong> anti-La on the prevalence<br />
<strong>of</strong> nephritis in a large cohort <strong>of</strong> lupus patients. In addition, we<br />
determined the association between anti-La and the presence<br />
<strong>of</strong> the various lupus manifestations. Methods: We<br />
studied 1100 lupus patients enrolled in the Lupus Multiplex<br />
Registry and Repository. Only one lupus patient per family<br />
was selected to exclude intrafamilial correlation. Since anti-<br />
La is present in patients who also have anti-Ro autoantibody,<br />
we compared anti-Ro positive lupus patients in the presence<br />
or absence <strong>of</strong> anti-La. <strong>Clinical</strong> data were obtained from<br />
medical records, interviews, and participant questionnaires.<br />
Tests for autoantibodies against extractable nuclear antigens<br />
were performed using immunodiffussion assays. Results:<br />
There is no difference in the age, sex, or race between the<br />
anti-La positive and anti-La negative lupus patients. The<br />
presence <strong>of</strong> anti-La is associated with a significantly reduced<br />
risk <strong>of</strong> lupus nephritis (proteinuria: 29.3% versus 46.3%,<br />
OR=0.48, p =0.023; cellular casts: 8.6% versus 20.6%,<br />
OR=0.36, p=0.038). In addition, lupus patients with anti-La<br />
have a reduced risk for seizures (0% versus 11%, p=0.0096).<br />
The presence <strong>of</strong> anti-nRNP autoantibody is significantly<br />
reduced in anti-La positive compared to anti-La negative<br />
lupus patients (10.3% versus 27.4%, OR=0.30, p=0.0075).<br />
Conclusion: Anti-La autoantibody is associated with less<br />
severe lupus. Patients with anti-La have a lower risk <strong>of</strong> renal<br />
and CNS involvement compared to anti-La negative patients.<br />
doi:10.1016/j.clim.2007.03.411<br />
Sa.25 Establishment <strong>of</strong> an Animal Model for Allergic<br />
Granulomatous Vasculitis by IgE-mediated<br />
Cutaneous Reverse Passive Arthus Reaction:<br />
Contribution <strong>of</strong> Selectins and ICAM-1<br />
Minoru Hasegawa, Assistant Pr<strong>of</strong>essor, Dermatology,<br />
Kanazawa University, Kanazawa, Tomoyuki Fujita, Graduate<br />
Student, Dermatology, Kanazawa University, Kanazawa,<br />
Manabu Fujimoto, Associate Pr<strong>of</strong>essor, Dermatology,<br />
Kanazawa University, Kanazawa, Shinichi Sato, Pr<strong>of</strong>essor,<br />
Dermatology, Nagasaki University, Nagasaki, Kazuhiko<br />
Takehara, Pr<strong>of</strong>essor, Dermatology, Kanazawa University,<br />
Kanazawa<br />
Allergic granulomatous vasculitis (Churg–Strauss syndrome)<br />
occurs in patients with a history <strong>of</strong> asthma and is<br />
characterized by necrotizing vasculitis <strong>of</strong> small arteries and<br />
veins, with extravascular granulomas and a marked eosinophilia<br />
in the perivascular tissue <strong>of</strong> multiple organs and in the<br />
peripheral blood. IgE deposition in the lesion has been<br />
demonstrated, which suggests the involvement <strong>of</strong> IgE-containing<br />
immune complex (IC) in the pathomechanism. The classical<br />
and standard animal model for the inflammatory response in<br />
IC-mediated vasculitis is the reverse Arthus reaction. In the<br />
current study, we have established a mouse model mimicking<br />
allergic granulomatous vasculitis using cutaneous reverse<br />
passive Arthus reaction by IgE injection. IgE-mediated IC<br />
challenge induced hemorrhage and the accumulation <strong>of</strong><br />
eosinophil, mast cell, and macrophage, which similar to<br />
allergic granulomatous vasculitis. Remarkably, infiltration <strong>of</strong><br />
eosinophils and mast cells was observed, which was associated<br />
with the increased expression <strong>of</strong> MCP-3 and TNF-α. Furthermore,<br />
to assess the relative contribution <strong>of</strong> adhesion molecules,<br />
including selectins and ICAM-1, this model was<br />
examined in mice lacking E-selectin, P-selectin, L-selectin or<br />
ICAM-1. Eosinophil and mast cell numbers were significantly<br />
reduced in all mutant mice compared with wild type mice.<br />
Furthermore, both E- and P-selectin blockade in mice lacking<br />
for both L-selectin and ICAM-1 completely abrogated hemorrhage.<br />
These results indicate that E-, P-, L-selectin and ICAM-1<br />
contribute to the IgE-mediated cutaneous Arthus reaction by<br />
regulating eosinophil and mast cell recruitment and suggest<br />
that these adhesion molecules would be potential therapeutic<br />
targets for allergic granulomatous vasculitis.<br />
doi:10.1016/j.clim.2007.03.412<br />
Sa.26 Innate Immune Signal in New Murine Models <strong>of</strong><br />
Autoimmunity Induced by U1 RNA and the RNA<br />
Binding Domain <strong>of</strong> the U1-70KD Ribonucleoprotein<br />
Antigen<br />
Annette Abril, Resident in Medicine, University <strong>of</strong> Miami/<br />
Department <strong>of</strong> Medicine/Division <strong>of</strong> Rheumatology, Miami,<br />
FL, Kimberly Jaimes, Research Specialist, University <strong>of</strong><br />
Miami/Department <strong>of</strong> Medicine/Division <strong>of</strong> Rheumatology,<br />
Miami, FL, Robert W. H<strong>of</strong>fman, Pr<strong>of</strong>essor and Chief,<br />
University <strong>of</strong> Miami/Department <strong>of</strong> Medicine, Division<br />
Rheumatology and Department Microbiology and<br />
<strong>Immunology</strong>, Miami, FL<br />
We have recently developed two complementary murine<br />
models <strong>of</strong> systemic autoimmunity that are induced following<br />
a single exposure to U1 RNA and a polypeptide<br />
encompassing the RNA binding domain <strong>of</strong> the 70 kDa<br />
polypeptide <strong>of</strong> U1 RNP. In mice deficient for the toll-like<br />
receptor (TLR3−/−), tissue targeting in the model is shifted<br />
from the lungs to the kidneys, and anti-double stranded<br />
DNA (dsDNA) antibodies emerge. This study examines the<br />
influence <strong>of</strong> TLR3 and innate immune signaling on the<br />
induction <strong>of</strong> antiphospholipid antibodies in these new<br />
models. Methods: Mice transgenic for the human major<br />
histocompatibility complex gene HLA-DR4 and TLR3−/−<br />
mice were immunized with U1 RNA and U1-70kDa
Abstracts<br />
polypeptide, control fusion protein, with or without U1-<br />
RNA, or saline. Sera from control mice <strong>of</strong> the background<br />
strains C57BL/6 or B6/129 were also studied. Complete<br />
serologic and histologic analyses were performed. Anticardiolipin,<br />
anti-dsDNA and anti-U1-70kDa antibodies were<br />
measured by ELISA. Results: We found that the mice<br />
deficient for TLR3 failed to produce anticardiolipin antibodies<br />
despite the development <strong>of</strong> high titers <strong>of</strong> antidsDNA,<br />
anti-U1-70kDa and other antibodies, as well as<br />
developing proteinuria and proliferative glomerulonephritis.<br />
In fact, TLR3 deficient mice had reduced levels <strong>of</strong><br />
anticardiolipin reactivity in their sera as measured by ELISA<br />
compared to control mice (pb0.05). Conclusions: We have<br />
developed two new models <strong>of</strong> systemic autoimmunity that<br />
are induced following a single exposure to U1-RNA and its<br />
associated RNA binding protein. These models allow for<br />
genetic dissection <strong>of</strong> differences in tissue targeting,<br />
autoantibody development and the influences <strong>of</strong> innate<br />
immune signaling.<br />
doi:10.1016/j.clim.2007.03.413<br />
Sa.27 Urinary Lipocalin-2 is Upregulated in Murine<br />
and Human Lupus Nephritis<br />
Milena Pitashny, Posdoctoral Fellow, Albert Einstein College<br />
<strong>of</strong> Medicine, Division <strong>of</strong> Rheumatology, Bronx, NY<br />
Lipocalin-2, a member <strong>of</strong> the lipocalin family <strong>of</strong> iron<br />
binding proteins, is upregulated in the kidney following a<br />
variety <strong>of</strong> insults that lead to renal injury. We recently<br />
reported that in vitro exposure <strong>of</strong> kidney cells to monoclonal<br />
anti-double stranded (ds) DNA antibodies significantly upregulates<br />
Lipocalin-2. We hypothesized that polyclonal pathogenic<br />
anti-dsDNA antibodies induce kidney overexpression <strong>of</strong><br />
Lipocalin-2, and the latter may represent a marker <strong>of</strong> renal<br />
involvement in lupus-prone mice and SLE patients. Kidney<br />
and serum Lipocalin-2 from old lupus-prone MRL lpr/lpr<br />
(MRL/lpr), NZB×NZW F1 (B/W) and non-autoimmune control<br />
mouse strains were studied. B/W and MRL/lpr mice had<br />
significantly higher kidney RNA expression <strong>of</strong> Lipocalin-2 than<br />
age-matched BALB/c mice (pb0.01). Furthermore, MRL/lpr<br />
had higher levels <strong>of</strong> serum Lipocalin-2 than MRL+/+ and<br />
BALB/c mice (pb0.05). Kidney Lipocalin-2 correlated with<br />
serum Lipocalin-2 (pb0.005), proteinuria (pb0.05) and antidsDNA<br />
antibody titers (pb0.05). We measured Lipocalin-2<br />
levels in urine from patients with or without active lupus<br />
nephritis (LN). SLE patients had significantly higher levels <strong>of</strong><br />
urinary (u) Lipocalin-2 than normal controls (pb0.005).<br />
Moreover, patients with active LN had significantly higher<br />
levels <strong>of</strong> uLipocalin-2 than patients without LN (pb0.05).<br />
Finally, in LN patients we found a significant correlation<br />
between uLipocalin-2 levels and the renal SLE disease<br />
activity index (SLEDAI) (r=0.45, pb0.01). Upregulation <strong>of</strong><br />
Lipocalin-2 in the kidneys <strong>of</strong> lupus prone mice and SLE<br />
patients with nephritis strongly suggests that Lipocalin-2 may<br />
serve as a valuable marker <strong>of</strong> nephritis in SLE, although its<br />
role in the pathogenesis <strong>of</strong> LN has yet to be determined.<br />
doi:10.1016/j.clim.2007.03.414<br />
Sa.28 CD4+ T Cells Reactive with U1-70kD<br />
Autoantigen Can Transfer Disease and are Central to<br />
Pathogenesis in New Murine Model <strong>of</strong> Lupus<br />
Robert W. H<strong>of</strong>fman, Pr<strong>of</strong>essor and Chief, University <strong>of</strong><br />
Miami/Department <strong>of</strong> Medicine, Division Rheumatology and<br />
Department Microbiology and <strong>Immunology</strong>, Miami, FL, Eric<br />
L. Greidinger, Assistant Pr<strong>of</strong>essor, University <strong>of</strong> Miami/<br />
Department <strong>of</strong> Medicine, Division Rheumatology and<br />
Department Microbiology and <strong>Immunology</strong>, Miami, FL, Yun<br />
Zang, Research Scientist, University <strong>of</strong> Miami/Department<br />
<strong>of</strong> Medicine, Division Rheumatology and Department<br />
Microbiology and <strong>Immunology</strong>, Miami, FL<br />
C57BL/6 mice transgenic for the human major histocompatibility<br />
complex gene HLA-DR4 (DR4-Tg) were immunized<br />
with U1 RNA and the U1-70kDa polypeptide, control fusion<br />
protein, with or without U1-RNA, or saline. Complete serologic<br />
and histologic analyses were performed on all mice. CD4+ T<br />
cells and APCs were isolated from immunized mice using<br />
immunoaffinity columns and tested for proliferation. Either<br />
freshly isolated CD4+ T cells from immunized mice or T cell<br />
lines were adoptively transferred to unmanipulated HLA-DR4<br />
Tg or RAG−/− mice. Freshly isolated CD4+ T cell from<br />
immunized DR4 Tg mice proliferated in response to U1-<br />
70kDa and to synthetic peptides from the RNA binding domain<br />
region <strong>of</strong> the protein in the presence <strong>of</strong> irradiated autologous<br />
APCs. We found that CD4+ T cells specific for U1-70kDa were<br />
able to transfer disease to unmanipulated syngeneic mice.<br />
Furthermore, using purified CD4+ Tcells or Tcell lines specific<br />
for U1-70kDa we were able to transfer disease to RAG−/− mice<br />
in the absence <strong>of</strong> detectable B cells or autoantibodies. In<br />
conclusion, we have developed a new model <strong>of</strong> systemic<br />
autoimmunity that is induced following a single exposure to<br />
U1-RNA and its associated RNA binding protein. Anti-U1-70kDa<br />
autoantigen-specific CD4+ T cells appear to medicate pathogenesis<br />
<strong>of</strong> disease in this model and can do so in the absence <strong>of</strong><br />
detectable B cells or autoantibodies. This work was supported<br />
by NIH award AR43308 (RWH), the Lupus Foundation <strong>of</strong><br />
American (RWH) and Department <strong>of</strong> Veterans Affairs Merit<br />
Review awards (RWH and ELG).<br />
doi:10.1016/j.clim.2007.03.415<br />
S83<br />
Sa.29 Nasal Delivery <strong>of</strong> Anti-CD3 Antibody Suppresses<br />
Murine Lupus by Inducing IL-10 Producing<br />
Foxp3+CD4+CD25−LAP+ Regulatory T Cells<br />
Henry Yim Wu, Instructor, Harvard Medical School, Boston, MA,<br />
Howard Weiner, Pr<strong>of</strong>essor, Harvard Medical School, Boston, MA<br />
One <strong>of</strong> the major contributors to the development <strong>of</strong> SLE<br />
is a functionally defective T regulatory cell pool in the<br />
periphery <strong>of</strong> susceptible individuals. Therefore the induction<br />
<strong>of</strong> novel T regulatory cells that can control autoaggressive<br />
immune responses in patients holds promise as<br />
immunotherapy for SLE. We previously reported that oral<br />
administration <strong>of</strong> anti-CD3 monoclonal antibody suppresses<br />
experimental autoimmune encephalomyelitis in a TGF-?<br />
dependent fashion both in vitro and in vivo. We have now<br />
discovered that nasally administered murine anti-CD3 F(ab′)
S84 Abstracts<br />
2 monoclonal antibody (clone 2C-11) is biologically active<br />
and reduced the incidence <strong>of</strong> lupus nephritis and autoantibody<br />
production in the SNF1 accelerated lupus model. In<br />
addition nasal anti-CD3 arrested on-going lupus in NZB strain<br />
<strong>of</strong> lupus prone mice. Animals were given 5 treatments per<br />
week every other week for 12 weeks. Nasal administration<br />
<strong>of</strong> anti-CD3 antibody induced Foxp3+CD4+CD25−LAP+ T<br />
regulatory cells that mediate suppression in an IL-10<br />
dependent but contact independent manor in vitro.<br />
Adoptive transfer studies demonstrated that LAP+ T<br />
regulatory cells required IL-10 and TGF-? to suppress disease<br />
in vivo. We hypothesize that LAP+ T regulatory cells control<br />
the activation CD4+ICOS+CXCR5+ T follicular helper cells<br />
which in turn downregulate the differentiation <strong>of</strong> B cells<br />
into IgG+CD38+ plasma cells and inhibit the production <strong>of</strong><br />
anti-nuclear autoantibodies. Our findings identify a novel<br />
immunotherapeutic approach for the treatment <strong>of</strong> SLE that<br />
is applicable to humans.<br />
doi:10.1016/j.clim.2007.03.416<br />
Sa.30 Reverse Phase Protein Lysate Microarrays in<br />
the Analysis <strong>of</strong> Activated B Lymphocytes<br />
Alvina D. Chu, Rheumatology Fellow, Stanford University,<br />
Division <strong>of</strong> <strong>Immunology</strong> and Rheumatology, Palo Alto, CA,<br />
Andrzej J. Chruscinski, Cardiology Fellow, Stanford<br />
University, Division <strong>of</strong> Cardiovascular Medicine, Stanford,<br />
CA, Harvir Singh, Research Assistant, Stanford University,<br />
Division <strong>of</strong> <strong>Immunology</strong> and Rheumatology, Stanford, CA,<br />
Paul J. Utz, Associate Pr<strong>of</strong>essor, Stanford University,<br />
Division <strong>of</strong> <strong>Immunology</strong> and Rheumatology, Stanford, CA<br />
Systemic lupus erythematosus (SLE) is an autoimmune<br />
inflammatory disease characterized by destruction <strong>of</strong> multiple<br />
organ systems. The pathophysiology <strong>of</strong> SLE involves<br />
activated B cells that produce antibodies directed against<br />
self-antigens. Our laboratory has previously demonstrated<br />
that reverse phase protein (RPP) lysate microarrays can be<br />
used to detect multiple intracellular phosphorylation events<br />
from T cell populations maintained in cell culture in vitro.<br />
Here we show a novel application <strong>of</strong> RPP lysate microarray<br />
technology to analyze activated B lymphocytes taken ex<br />
vivo from MRL/MpJ-Faslpr (MRL/lpr) mice. Splenocytes<br />
were harvested from MRL/lpr mice at ∼25 weeks <strong>of</strong> age,<br />
toward the end <strong>of</strong> their lifespan. B lymphocytes were<br />
isolated by negative selection using magnetic beads and<br />
then briefly stimulated with B cell activating factors,<br />
including phorbol 12-myristate 13-acetate (PMA). Using a<br />
robotic microarrayer, nanoliter amounts <strong>of</strong> lysate were<br />
deposited onto nitrocellulose-coated slides. Slides were<br />
probed with phosphorylation state-dependent antibodies<br />
targeting key intracellular phosphorylation events involved<br />
with B cell activation, including phosphorylation <strong>of</strong> ERK,<br />
Akt, Jak, and STATs. Bound antibodies were detected using<br />
secondary antibodies conjugated to fluorophores, and mean<br />
fluorescence intensity for each sample was calculated. We<br />
found differential activation <strong>of</strong> kinase substrates within<br />
stimulated cells compared to basal levels within unstimulated<br />
B cell populations. Results were validated by Western<br />
blot analysis. Reverse phase protein microarrays provide a<br />
powerful tool for quantifying and comparing the level <strong>of</strong><br />
phosphorylation events in activated B cells isolated from<br />
secondary lymphoid organs, and show promise for elucidating<br />
signal transduction pathways involved in the pathogenesis<br />
<strong>of</strong> SLE.<br />
doi:10.1016/j.clim.2007.03.417<br />
Sa.31 Complement C4d Deposition on Circulating<br />
Blood Cells in Systemic Lupus Erythematosus (SLE)<br />
Chau-Ching Liu, Assistant Pr<strong>of</strong>essor, University <strong>of</strong> Pittsburgh<br />
School <strong>of</strong> Medicine, Department <strong>of</strong> Medicine, Pittsburgh,<br />
PA, Jean Lin, Medical Student, University <strong>of</strong> Pittsburgh<br />
School <strong>of</strong> Medicine, Pittsburgh, PA, Jeannine S. Navratil,<br />
Research Associate, University <strong>of</strong> Pittsburgh School <strong>of</strong><br />
Medicine, Department <strong>of</strong> Medicine, Pittsburgh, PA, Susan<br />
Manzi, Associate Pr<strong>of</strong>essor, University <strong>of</strong> Pittsburgh School<br />
<strong>of</strong> Medicine, Department <strong>of</strong> Medicine, Pittsburgh, PA, Amy<br />
H. Kao, Assistant Pr<strong>of</strong>essor, University <strong>of</strong> Pittsburgh School<br />
<strong>of</strong> Medicine, Department <strong>of</strong> Medicine, Pittsburgh, PA,<br />
Joseph M. Ahearn, Associate Pr<strong>of</strong>essor, University <strong>of</strong><br />
Pittsburgh School <strong>of</strong> Medicine, Department <strong>of</strong> Medicine,<br />
Pittsburgh, PA<br />
Immune dysregulation in SLE is characterized by polyclonal<br />
lymphocyte activation, autoantibody production, and<br />
complement activation. Recently, we discovered that significant<br />
levels <strong>of</strong> complement C4d are present on surfaces <strong>of</strong><br />
erythrocytes, platelets, and lymphocytes <strong>of</strong> SLE patients and<br />
that C4d-bearing T lymphocytes exhibited functional<br />
abnormalities. These observations led to the current study<br />
in which circulating blood cells from SLE patients were<br />
investigated for C4d deposition and for the underlying<br />
mechanism(s). Specifically, we performed flow cytometry<br />
to measure C4d levels on various circulating cells from 307<br />
SLE patients, 187 patients with other inflammatory diseases,<br />
and 95 healthy controls. The results demonstrated that<br />
significantly elevated levels <strong>of</strong> C4d were present on T<br />
lymphocytes, B lymphocytes, and monocytes <strong>of</strong> SLE patients<br />
(mean fluorescence intensity (MFI) =13.5, 50.6, and 9.7,<br />
respectively), compared to patients with other diseases<br />
(MFI=3.2, 6.9, 4.7, respectively; pb0.0001) and healthy<br />
controls (MFI=1.8, 8.0, and 3.7, respectively; pb0.0001).<br />
However, only modest levels <strong>of</strong> C4d were detected on<br />
granulocytes from a small number <strong>of</strong> SLE patients (MFI=2.9).<br />
Furthermore, high C4d levels were not necessarily concurrently<br />
present on erythrocytes, platelets, lymphocytes, and<br />
monocytes <strong>of</strong> a given SLE patient on a particular day. These<br />
findings, combined, indicate that the C4d-bearing circulating<br />
cell phenotype is mediated by a patient-dependent, cell<br />
lineage-specific mechanism rather than the result <strong>of</strong><br />
complement activation that simply affects all circulating<br />
cells simultaneously. Ongoing studies investigating the<br />
association <strong>of</strong> the differential leukocyte-C4d binding patterns<br />
with clinical features <strong>of</strong> SLE may lead to additional<br />
clues to SLE pathogenesis and may identify targets for<br />
therapeutic intervention.<br />
doi:10.1016/j.clim.2007.03.418
Abstracts<br />
Sa.32 Cell-Type and Stimulus Determine the Impact<br />
<strong>of</strong> the Serine/Threonine Kinase Cot/Tpl2 in Acute<br />
and Chronic Inflammation<br />
Anwar Murtaza, Senior Scientist, Abbott Bioresearch<br />
Center, Pharmacology, Worcester, MA, Shaughn Bryant,<br />
Research Associate, Abbott Bioresearch Center,<br />
Pharmacology, Worcester, MA, Suzanne Mathieu, Research<br />
Associate, Abbott Bioresearch Center, Pharmacology,<br />
Worcester, MA, Hamish Allen, Director, Abbott Bioresearch<br />
Center, Molecular and Cellular Biology, Worcester, MA,<br />
Catherine Tripp, Senior Director, Abbott Bioresearch<br />
Center, Worcester, MA, Neil Wishart, Associate Director,<br />
Abbott Bioresearch Center, Chemistry, Worcester, MA<br />
TNF has been identified as a key pro-inflammatory<br />
cytokine in the pathogenesis <strong>of</strong> acute and chronic inflammatory<br />
diseases such as septic shock and rheumatoid<br />
arthritis (RA). Anti-TNF therapy has been effective in the<br />
treatments <strong>of</strong> RA, Crohn’s disease (CD) and psoriasis (Ps).<br />
Binding <strong>of</strong> TNF to its receptors induces a number <strong>of</strong> signaling<br />
cascades leading to the activation <strong>of</strong> MAP kinases and NF-kB.<br />
Targeting Cot/Tpl2 a serine/threonine kinase in the MEK/ERK<br />
MAP kinase pathway <strong>of</strong>fers an attractive approach for<br />
developing an oral therapeutic that inhibits TNF production<br />
and hence an alternative strategy to anti-TNF therapy. Here<br />
we have used Tpl2 deficient mice to elucidate its role in<br />
acute and chronic inflammation. Our results demonstrate<br />
that Tpl2 deficiency decreases the production <strong>of</strong> a panel <strong>of</strong><br />
pro-inflammatory cytokines such as TNF, IL-1, IFN-g IL-6, and<br />
IL-18 following LPS challenge in vivo. We further demonstrate<br />
for the first time that Tpl2 knockout mice are only<br />
partially protected to collagen-induced arthritis (CIA).<br />
Moreover, the treatment <strong>of</strong> Tpl2 deficient mice with an<br />
anti-TNF antibody dramatically inhibited the progression and<br />
severity <strong>of</strong> disease suggesting a Tpl2 independent production<br />
<strong>of</strong> TNF in rodent arthritis. Moreover, we show that the impact<br />
<strong>of</strong> Tpl2 deficiency in LPS and arthritis models may be due to a<br />
differential activity <strong>of</strong> the kinase in different cell types.<br />
Thus, our data demonstrate that the Cot/Tpl2 pathway is<br />
critical in the response to endotoxin but may have a limited<br />
role in arthritis.<br />
doi:10.1016/j.clim.2007.03.419<br />
Sa.33 Bosentan Synergizes with Iloprost in Treating<br />
Pulmonary Hypertension in Scleroderma Patients<br />
Marta Sessarego, Doctor, Switzerland, Department <strong>of</strong><br />
Internal Medicine-University <strong>of</strong> Genova, Genova,<br />
Switzerland, Marta Rizzi, Department <strong>of</strong> Internal<br />
Medicine-University <strong>of</strong> Genova, Genova, Switzerland,<br />
Massimo Ghio, Department <strong>of</strong> Internal Medicine-University<br />
<strong>of</strong> Genova, Genova, Francesco Indiveri, Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> Internal Medicine-University <strong>of</strong> Genova,<br />
Genova, Switzerland<br />
Pulmonary arterial hypertension (PAH) is a devastating<br />
complication <strong>of</strong> systemic sclerosis (SSc) characterized by<br />
endothelial dysfunction (lesser production <strong>of</strong> prostacyclin<br />
associated with increased secretion <strong>of</strong> endotelin), for which<br />
no curative treatments are available. On this basis it has<br />
been postulated that the oral dual endothelin receptors<br />
antagonist, bosentan, might strengthen the activity <strong>of</strong> prostacyclin<br />
therapy. Patients and methods: 8 patients affected<br />
by SSc with increased PAH (PA systolic pressure PAPS ¡Ý 45 mm<br />
Hg by echocardiogram or PA pressure ¡Ý 35 mm Hg at rest by<br />
cardiac catheterism, WHO functional classes III–IV) were<br />
reclutated, all <strong>of</strong> them had shown a poor response to 1 year<br />
treatment with prostacyclin e.v. infusion (iloprost, 0.5–2 ng/<br />
kg/min 6 h a day — 5 days a month). Bosentan (62.5 mg bid<br />
for 1 month, then 125 mg bid) and iloprost (0.5–2 ng/kg/min)<br />
in continuous (24h/24 h) were administered. The Doppler<br />
echocardiogram, the six-minute walking test (6MWT) and the<br />
complete pulmonary tests were performed at baseline and<br />
after 3, 6 and 12 months. Results: After 3 months PAPS was<br />
decreased and 6MWT was improved at least 25 min in all<br />
patients; at the end <strong>of</strong> the study an improvement <strong>of</strong> WHO<br />
class and pulmonary function was observed in 7 and 3<br />
patients respectively. All the subjects tolerated the therapy,<br />
thereafter no one discontinued therapy. Conclusion: The<br />
association <strong>of</strong> bosentan with iloprost infused continuously<br />
may improve PAP and hemodynamics in SSc patients.<br />
doi:10.1016/j.clim.2007.03.421<br />
S85<br />
Sa.35 Anti-lymphocyte Autoantibodies in Systemic<br />
Lupus Erythematosus (SLE) Revisited<br />
Jean Lin, Medical Student, University <strong>of</strong> Pittsburgh,<br />
Pittsburgh, PA, Joseph M. Ahearn, Associate Pr<strong>of</strong>essor,<br />
University <strong>of</strong> Pittsburgh School <strong>of</strong> Medicine, Department <strong>of</strong><br />
Medicine, Pittsburgh, PA, Susan Manzi, Associate Pr<strong>of</strong>essor,<br />
University <strong>of</strong> Pittsburgh School <strong>of</strong> Medicine, Department <strong>of</strong><br />
Medicine, Pittsburgh, PA, Chau-Ching Liu, Assistant<br />
Pr<strong>of</strong>essor, University <strong>of</strong> Pittsburgh School <strong>of</strong> Medicine,<br />
Department <strong>of</strong> Medicine, Pittsburgh, PA<br />
Complement, anti-lymphocyte autoantibodies (ALA), and<br />
lymphocyte dysfunction have previously been implicated in<br />
the pathogenesis <strong>of</strong> SLE. However, the causal relationships<br />
between complement, ALA, and lymphocyte function are<br />
largely unexplored. Our recent identification <strong>of</strong> complement<br />
C4d on T cells <strong>of</strong> SLE patients has led us to hypothesize that<br />
binding <strong>of</strong> ALA to T cells may trigger complement activation<br />
and C4d deposition on these cells, which in turn causes<br />
functional dysregulation <strong>of</strong> these cells and contributes to SLE<br />
pathogenesis. The current study was performed to characterize<br />
C4d deposition on T cells (T-C4d) and to investigate<br />
the role <strong>of</strong> ALA in T-C4d deposition. Briefly, T-C4d and ALA<br />
were determined by flow cytometry in 225 SLE patients, 135<br />
patients with other inflammatory diseases, and 15 healthy<br />
controls. Significantly elevated levels <strong>of</strong> C4d were detected<br />
on T cells <strong>of</strong> SLE patients (median fluorescence intensity<br />
(MFI)=14.8), compared with T cells from patients with other<br />
diseases (MFI=3.4; pb0.001) or healthy controls (MFI=0.8;<br />
pb0.001). IgM and/or IgG were detected concomitantly<br />
with C4d on T cells in a significant fraction <strong>of</strong> SLE patients<br />
(73/225), but not in healthy controls or patients with other<br />
diseases. Moreover, high levels <strong>of</strong> T-C4d could be induced on<br />
normal T cells after incubation with selected SLE plasma or<br />
immunoglobulins. Such in vitro T-C4d inducing activity could<br />
be eliminated by pre-absorption <strong>of</strong> SLE plasma or
S86 Abstracts<br />
immunoglobulins with lymphocytes, indicating that ALA is<br />
primarily responsible for triggering C4d deposition on Tcells.<br />
These results, together, support a previously unrecognized<br />
ALA-mediated pathogenic mechanism in SLE.<br />
doi:10.1016/j.clim.2007.422<br />
Sa.36 Treg Cells Directly Suppress Anti-DNA<br />
Ig-producing B Cells Through TGFβ-mediated<br />
Mechanisms in Murine Lupus<br />
Antonio La Cava, Associate Pr<strong>of</strong>essor, University <strong>of</strong><br />
California, Los Angeles, Los Angeles, CA, Chung Lee,<br />
Pr<strong>of</strong>essor, Northwestern University, Chicago, IL, Bevra<br />
Hahn, Pr<strong>of</strong>essor and Chief <strong>of</strong> Rheumatology, University <strong>of</strong><br />
California, Los Angeles, Los Angeles, CA<br />
In SLE, production <strong>of</strong> autoantibodies (autoAb) including<br />
anti-DNA Ig is central to pathogenesis <strong>of</strong> the disease. We<br />
reported that regulatory Foxp3+CD4+CD25+ T (Treg) cells in<br />
(NZB×NZW) F1 (NZB/W F1) lupus mice suppress the production<br />
<strong>of</strong> anti-DNA Ig by B cells in vitro. To discriminate between<br />
the possibility that Treg cells suppressed Tcells providing help<br />
to Ig-producing B cells and the possibility that Treg cells<br />
directly suppressed autoAb-producing B cells, we sublethally<br />
irradiated NZB/W F1 mice for adoptive transfer with<br />
syngeneic Treg cells together with B cells only or with B<br />
cells plus helper T cells. Additional experiments consisted <strong>of</strong><br />
transfer <strong>of</strong> these combinations but membrane expression <strong>of</strong><br />
TGFβ receptor II (TBR) in B cells was disrupted using a<br />
retrovirally based vector that expresses a dominant negative<br />
(DN) TBR. It was found that Treg suppression <strong>of</strong> anti-DNA Ig in<br />
vivo occurred without requirement <strong>of</strong> intermediate regulation<br />
<strong>of</strong> CD4+ Tcells; suppression <strong>of</strong> anti-DNA Ab was observed<br />
in the absence <strong>of</strong> CD4+ T cells (Pb0.03 vs. anti-DNA Ab from<br />
transfer <strong>of</strong> B cells only). Importantly, an intact TGFβ signaling<br />
pathway was required for B cell suppression by Treg.<br />
Disruption <strong>of</strong> B cell surface expression <strong>of</strong> TGFβ receptor via<br />
the dominant negative (DN) TBR permitted escape <strong>of</strong> B cells<br />
from the Treg-controlled suppression and led to dysregulated<br />
production <strong>of</strong> autoAb in vitro and in vivo. The findings<br />
identify events controlling production <strong>of</strong> autoAb in SLE and<br />
envision new possibilities for targeted intervention.<br />
doi:10.1016/j.clim.2007.03.424<br />
Sa.38 Agalactosyl Immunoglobulins are a Useful<br />
Marker for <strong>Clinical</strong> Progression and Successful<br />
Therapy <strong>of</strong> Collagen-induced Arthritis<br />
Katrien Van Beneden, PhD, Ghent University, Ghent,<br />
Belgium, Ken Coppieters, PhD Student, Ghent University,<br />
Ghent, Belgium, Wouter Laroy, PhD, Ghent University and<br />
Flanders Interuniversity Institute for Biotechnology,<br />
Zwijnaarde, Belgium, Pieter Rottiers, PhD, Ghent<br />
University and Flanders Interuniversity Institute for<br />
Biotechnology, Zwijnaarde, Belgium, Gust Verbruggen, MD<br />
PhD, Ghent University, Ghent, Belgium, Nico Callewaert,<br />
PhD, Ghent University and Flanders Interuniversity Institute<br />
for Biotechnology, Zwijnaarde, Belgium, Dirk Elewaut, MD<br />
PhD, Ghent University, Ghent, Belgium<br />
The purpose <strong>of</strong> this study was to evaluate the validity <strong>of</strong><br />
serum agalactosyl Ig as a biomarker for murine collagen-induced<br />
arthritis(CIA).Wesoughttoclarifythecorrelationbetween<br />
arthritic index and agalactosyl Ig fraction, the kinetics <strong>of</strong><br />
agalactosyl Ig levels upon development and progression <strong>of</strong> CIA,<br />
and the effect <strong>of</strong> antigen specific versus non-specific treatment.<br />
CIA was induced in susceptible mice, serum was collected from<br />
several arthritic and non-arthritic animals and agalactosyl Ig<br />
content was determined using a sensitive, improved technology<br />
for DNA sequencer aided analysis <strong>of</strong> N-linked glycans. Prophylactic<br />
tolerization with collagen type II was performed and the<br />
clinical effect was monitored; agalactosyl IgG was analyzed in<br />
serum derived from endpoint bleeding. Semi-therapeutic<br />
treatment with either dexamethasone or etanercept was<br />
performed and compared with PBS controls. All mice were<br />
sequentially bled at various time points during treatment.<br />
Agalactosyl Ig ratios were quantified and compared with agematched,<br />
unimmunized DBA/1 mice. Arthritic animals could be<br />
distinguished from non-arthritic animals with high sensitivity<br />
and specificity. Improvement <strong>of</strong> clinical arthritis scores by<br />
antigen specific tolerance induction correlated with downregulated<br />
levels <strong>of</strong> agalactosyl Ig. Finally, increased serum<br />
agalactosyl Ig levels correlate with CIA progression and are<br />
reversed by successful semi-therapeutic treatment. These<br />
findings indicate that agalactosyl determination may be a very<br />
useful serum marker for monitoring the clinical outcome <strong>of</strong><br />
experimental treatment in CIA, by use <strong>of</strong> a sensitive albeit<br />
simple methodology. This observation may be valuable for<br />
longitudinal CIA studies or for a more detailed judgment <strong>of</strong><br />
outcome upon treatment.<br />
doi:10.1016/j.clim.2007.03.426<br />
Sa.40 NKT Cells are Novel Accelerator and Producer<br />
<strong>of</strong> IL-17 in the Pathogenesis <strong>of</strong> Collagen-induced<br />
Arthritis<br />
Yohei Yoshiga, Graduate Student, Tsukuba University,<br />
Tsukuba, Japan, Goto Daisuke, MD, PhD, Tsukuba<br />
University, Tsukuba, Japan, Mizuko Mamura, MD, PhD,<br />
Tsukuba University, Tsukuba, Satoshi Ito, MD, PhD, Tsukuba<br />
University, Tsukuba, Japan, Isao Matsumoto, MD, PhD,<br />
Tsukuba University, Tsukuba, Japan, Akito Tsutsumi, MD,<br />
PhD, Tsukuba University, Tsukuba, Japan, Takayuki Sumida,<br />
MD, PhD, Tsukuba University, Tsukuba, Japan<br />
Objective: To clarify the role <strong>of</strong> NKTcells in CIA. Methods: (1)<br />
CIA was induced in C57BL/6 (B6) and NKT-KO (Jƒ¿281−/−)mice.<br />
Mice were immunized with type II collagen (CII) emulsified in<br />
CFA intradermally at day 0 and day 21, and then the incidence<br />
and severity <strong>of</strong> arthritis were evaluated. (2) To analyze the CIIspecific<br />
T cell response, splenocytes and draining lymph node<br />
(DLN) cells from B6 and NKT-KO mice immunized with CII/CFA<br />
were stimulated with CII. The culture supernatants were used<br />
for cytokine ELISA, and these cells were used for intracellular<br />
cytokine staining. (3) To identify the association <strong>of</strong> NKTcells and<br />
IL-17 production, splenocytes from naive B6 and NKT-KO mice<br />
were stimulated with ƒ¿-GalCer, ligand for NKT cells. The IL-17<br />
production was assayed by intracellular cytokine staining.<br />
Results: (1) The incidence <strong>of</strong> arthritis in NKT-KO mice was<br />
suppressed compared with that in B6 mice. The severity <strong>of</strong>
Abstracts<br />
arthritis in NKT-KO mice was also milder than that in B6 mice.<br />
(2) The production <strong>of</strong> IFN-ƒÁ and IL-4 was not significantly<br />
different between NKT-KO and B6 mice, but IL-17 production<br />
was reduced in NKT-KO mice. IL-17-producing T helper (Th17)<br />
cells were reduced in DLN cells from NKT-KO mice. (3) NKTcells<br />
produced IL-17 in B6 splenocytes stimulated with ƒ¿-GalCer,<br />
although there are no IL-17-producing cells in NKT-KO<br />
splenocytes. Conclusion: These findings support the notion<br />
that NKT cells are a novel producer and accelerator <strong>of</strong> IL-17,<br />
indicating NKTcells might play a crucial role in the pathogenesis<br />
<strong>of</strong> CIA via IL-17.<br />
doi:10.1016/j.clim.2007.03.427<br />
Sa.41 Endogenous CD4 T Cells Program B Cells to<br />
Respond in the Chronic GVH Model <strong>of</strong> SLE<br />
Arpita Choudhury, Post Doctoral Fellow, Division <strong>of</strong><br />
Rheumatology, School <strong>of</strong> Medicine, University <strong>of</strong><br />
Pennsylvania, Philadelphia, PA, Philip Cohen, Pr<strong>of</strong>essor,<br />
Division <strong>of</strong> Rheumatology, School <strong>of</strong> Medicine, University <strong>of</strong><br />
Pennsylvania, Philadelphia, PA, Robert Eisenberg, Pr<strong>of</strong>essor,<br />
Division <strong>of</strong> Rheumatology, School <strong>of</strong> Medicine, University <strong>of</strong><br />
Pennsylvania, Philadelphia, PA<br />
The murine chronic GVH (cGVH) model <strong>of</strong> SLE is induced by<br />
allo-recognition <strong>of</strong> foreign major histocompatibility complex<br />
(MHC) class II determinants. Our previous studies suggested that<br />
B cells in CD4KO mice have intrinsic defects that prevent them<br />
from responding to allo-help (JI 174, 7600, 2005). We have<br />
further explored the role <strong>of</strong> host CD4 T cells in this model by<br />
using post-irradiation auto-reconstitution and cell transfer<br />
experiments. C57Bl/6 (B6)-CD4KO mice were irradiated (5<br />
Gy), and 3–5 million CD4 Tcells from various B6 congenic strains<br />
were transferred. After allowing the B cells to auto-reconstitute<br />
over 3 weeks, 10 8 bm12 spleen cells were injected to initiate<br />
the cGVH. Purified CD4 T cells from IL-6KO, IFN- 3 KO and IL-10<br />
KO, but not IL-4 KO, mice were able to restore B cell response to<br />
cGVH in CD4 KO mice, signifying a critical role for IL-4.<br />
Treatment with recombinant IL-4 (rIL-4) also restored the<br />
ability <strong>of</strong> B cells from CD4 KO mice to secrete auto-abs in cGVH.<br />
Furthermore, the treatment <strong>of</strong> CD4KO recipients with an<br />
agonistic anti-CD40 (FGK-115, rat IgG2a) mAb also restored B<br />
cell function in cGVH. Our studies show that the endogenous<br />
CD4 T cell help needed for B cells to mount an effective cGVH<br />
response depends on IL-4, but not IL-6, IL-10 or IFN- 3 .Thus,<br />
autoreactive B cells need to have developed in a particular<br />
cytokine milieu in order to be receptive to alloreactive stimuli.<br />
Understanding the intracellular changes that condition B cells<br />
to become activated in cGVH may yield clues to their function<br />
in SLE-like autoimmunity.<br />
doi:10.1016/j.clim.2007.03.428<br />
Sa.42 The Mer Receptor Tyrosine Kinase Influences<br />
Toll-like Receptor Stimulation <strong>of</strong> Dendritic Cells<br />
Benjamin Scott, Postdoctoral Fellow, University <strong>of</strong><br />
Pennsylvania, Division <strong>of</strong> Rheumatology, Philadelphia, PA,<br />
Joudy-Ann Dinnall, Research Associate, Children’s Hospital,<br />
Department <strong>of</strong> Pediatrics, Philadelphia, PA, Stefania<br />
Gallucci, Assistant Pr<strong>of</strong>essor, Children’s Hospital <strong>of</strong><br />
Philadelphia, Department <strong>of</strong> Pediatrics, Philadelphia, PA,<br />
Philip Cohen, Pr<strong>of</strong>essor, University <strong>of</strong> Pennsylvania, Division<br />
<strong>of</strong> Rheumatology, Philadelphia, PA, Robert Eisenberg,<br />
Pr<strong>of</strong>essor, University <strong>of</strong> Pennsylvania, Division <strong>of</strong><br />
Rheumatology, Philadelphia, PA<br />
The mer receptor tyrosine kinase, which is expressed on<br />
macrophages and dendritic cells (DC), is known to promote<br />
apoptotic cell disposal and inhibit inflammatory cytokine<br />
production by macrophages, but its role in DCs is unclear.<br />
Mice lacking functional mer (mer kd mice) develop autoimmunity<br />
characterized by antibodies to DNA and chromatin,<br />
presumably due to their accumulation <strong>of</strong> apoptotic cells. To<br />
explore the role <strong>of</strong> mer on splenic DCs, mer kd and B6 mice<br />
were injected i.p. with toll-like receptor (TLR) ligands, and<br />
the mice were sacrificed 1 day later for analysis by flow<br />
cytometry. The numbers <strong>of</strong> splenic DCs and degree <strong>of</strong><br />
activation <strong>of</strong> splenic DCs in control mer kd mice are comparable<br />
to control B6 mice. However, following LPS (TLR-4<br />
ligand) injection, merkd splenic DCs displayed an abnormally<br />
activated pr<strong>of</strong>ile compared to B6 controls, characterized by<br />
the increased expression <strong>of</strong> the costimulatory molecules<br />
CD40, CD80, and CD86. This activated phenotype was highly<br />
pronounced in the plasmacytoid subset <strong>of</strong> DC (pDC) and was<br />
present to a lesser extent in the CD8α + and myeloid subsets.<br />
In contrast, after poly(I:C) (TLR-3 ligand) injection, mer kd DC<br />
appeared to have suppressed costimulatory molecule expression<br />
compared to B6 controls, whereas CpG-ODN (TLR-9<br />
ligand) stimulation <strong>of</strong> DC did not seem affected by the mer<br />
deficiency. Thus, mer may differentially regulate DC TLR 3<br />
and 4 signals and may negatively regulate pDC during<br />
inflammation. Further studies may provide important insight<br />
into SLE pathogenesis since pDC have been linked to lupus in<br />
humans and animal models.<br />
doi:10.1016/j.clim.2007.03.429<br />
S87<br />
Sa.43 Membranous Glomerulopathy Associated with<br />
Rheumatoid Arthritis may Respond to Rituximab<br />
Bryan J. Wolf, Resident Physician, NV, Medical Center/<br />
University <strong>of</strong> Nevada, Reno Department <strong>of</strong> Internal<br />
Medicine, NV, William M. O’Neil, <strong>Clinical</strong> Pr<strong>of</strong>essor,<br />
University <strong>of</strong> Nevada School <strong>of</strong> Medicine, V, John S. Pixley,<br />
Associate Pr<strong>of</strong>essor <strong>of</strong> Medicine, VA Medical Center/<br />
University <strong>of</strong> Nevada School <strong>of</strong> Medicine, NV<br />
Membranous glomerulonephritis is an uncommon extraarticular<br />
manifestation <strong>of</strong> rheumatoid arthritis, which need<br />
not be associated with prior DMARD (disease-modifying<br />
antirheumatic drug) therapies (Clin Rheum 1996, 15, 385).<br />
We observed a patient with an 8-year history <strong>of</strong> seropositive<br />
erosive rheumatoid arthritis complicated by the development<br />
<strong>of</strong> refractory nephrotic syndrome secondary to<br />
biopsy-proven membranous glomerulonephritis. Signs and<br />
symptoms <strong>of</strong> his disabling kidney disease included renal<br />
insufficiency, massive proteinuria (25 g), edema and a<br />
hypercoaguable state (deep venous thrombosis and pulmonary<br />
emboli). Alkylating agents, antimetabolites and
S88 Abstracts<br />
glucocorticoids were ineffective in reducing his proteinuria<br />
over a 2 year period. The patient subsequently received 2<br />
courses (1 g twice over a 2 week period) <strong>of</strong> rituximab (a<br />
monoclonal antibody to CD20 that specifically depletes B<br />
cells), 6 months apart. Serum albumin at initial treatment<br />
was 2.0 g/dl and normalized (3.7 g/dl) 1 month after the<br />
second course <strong>of</strong> therapy. This was accompanied by a<br />
corresponding decrease excretion <strong>of</strong> urinary protein (22 g<br />
to 7.4 g/24 h) and a reduction in diuretic requirements. B<br />
cell directed therapy may prove useful in treating membranous<br />
glomerulopathies.<br />
doi:10.1016/j.clim.2007.03.430<br />
Sa.44 Design and Elaboration <strong>of</strong> Truncated Mutants<br />
<strong>of</strong> the Autoantigen hSRP72, Present in<br />
Dermatomyositis, in Order to Identify the Sites <strong>of</strong><br />
Phosphorylation<br />
Victor Ermilo Arana-Argaez, Master in Science, <strong>Immunology</strong>,<br />
University <strong>of</strong> Guadalajara, Institute <strong>of</strong> Rheumatology,<br />
Guadalajara, Mexico, Victor Ermilo Arana-Argaez, Master in<br />
Science, University <strong>of</strong> Guadalajara, Guadalajara, Mexico,<br />
Beatriz Martin-Marquez, Master in Science, University <strong>of</strong><br />
Guadalajara, Guadalajara, Mexico, Muñoz-Valle Jose<br />
Francisco, PhD, PhD, Guadalajara, Mexico, Erika<br />
Martínez-Garcia, Master in Science, University <strong>of</strong><br />
Guadalajara, Institute <strong>of</strong> Rheumatology, Guadalajara,<br />
Mexico, Oscar Leon-Murguia, MD, Institute <strong>of</strong><br />
Rheumatology, Guadalajara, Mexico, Monica Vazquez-Del<br />
Mercado, PhD, MD, University <strong>of</strong> Guadalajara, Guadalajara,<br />
Mexico<br />
Dermatomyositis (DM) is an idiopathic inflammatory<br />
myopathy (IIM) with characteristic muscle-cutaneous findings.<br />
Using sera from patients with DM the SRP72<br />
component <strong>of</strong> the signal recognition particle (SRP) as an<br />
autoantigen phosphorylated on serine (S) residues was<br />
identified. The mechanisms to explain the lost <strong>of</strong> tolerance<br />
against the hSRP72 remain without explanation. To determine<br />
whether the phosphorylation state <strong>of</strong> this particle<br />
could affect the antigenicity, we cloned the autoantigen<br />
hSRP72 into a pRS314-TRP shuttle vector and we designed<br />
truncated mutants to identify the possible site/sites <strong>of</strong><br />
phosphorylation <strong>of</strong> the serine residues and its further<br />
implication on the break <strong>of</strong> tolerance against this protein.<br />
Aim: To truncate mutants <strong>of</strong> the hSRP72 to further identify<br />
the site or sites <strong>of</strong> phosphorylation for the application on<br />
the study <strong>of</strong> the induction mechanisms <strong>of</strong> autoantigenicity<br />
in DM. Material and methods: Based on the map <strong>of</strong> serine<br />
residues in the full length SRP72 sequence, we cloned the<br />
autoantigen hSRP72, designed and elaborated 13 truncated<br />
mutants to identify the region <strong>of</strong> the protein where<br />
phosphorylation might occur. We designed primers to<br />
amplify 13 mutants truncated by means <strong>of</strong> PCR using as a<br />
template the construct pRS314hSRP72. Next, in vitro<br />
translation and metabolic labeling with 35 S and 32P-g ATP<br />
will be done. Results and conclusions: We cloned successfully<br />
the autoantigen hSRP72 and the design and elaboration<br />
<strong>of</strong> truncated mutants according to the phosphorylation<br />
prediction map was correct based on its PCR product length<br />
for later use in in vitro translation phosphorylation tests<br />
and studies on antigenicity induction.<br />
doi:10.1016/j.clim.2007.03.431<br />
Sa.45 Activated Interstitial Macrophages are<br />
Important Mediators <strong>of</strong> SLE Nephritis<br />
Meera Ramanujam, Research Scientist, Feinstein Medical<br />
Research Institute, Manhasset, NY, Ramalingam<br />
Bethunaickan, Postdoctoral Fellow, Feinstein Medical<br />
Research Institute, Manhasset, NY, Anne Davidson,<br />
Pr<strong>of</strong>essor <strong>of</strong> Medicine, Feinstein Medical Research Center,<br />
Manhasset, NY<br />
SLE is an autoimmune disease that is complicated by<br />
autoantibody mediated glomerulonephritis. In NZB/W SLEprone<br />
mice recruitment <strong>of</strong> inflammatory cells and mediators<br />
is associated with early expression <strong>of</strong> a limited panel <strong>of</strong><br />
chemokines followed by spreading to involve multiple other<br />
inflammatory molecules. Furthermore, inflammatory cell<br />
subsets home to different areas <strong>of</strong> the NZB/W kidney. B<br />
cells, T cells and dendritic cells are found in disorganized<br />
aggregates in the perihilar region and around blood vessels,<br />
whereas macrophages invade the interstitium <strong>of</strong> the kidney<br />
and form a layer around the glomeruli. In NZW/BXSB mice,<br />
despite a similar expression pr<strong>of</strong>ile <strong>of</strong> inflammatory genes<br />
in the kidney, there is marked invasion <strong>of</strong> the interstitium<br />
by sheets <strong>of</strong> F4/80 macrophages whereas dendritic cells are<br />
found in large numbers only inside the glomeruli. NZM2410<br />
mice that develop a sclerotic form <strong>of</strong> glomerulonephritis<br />
express few inflammatory mediators in the kidney; the<br />
onset <strong>of</strong> glomerulonephritis is associated with invasion only<br />
by macrophages that are confined to the renal medulla.<br />
Both NZB/W and NZW/BXSB mice have large numbers <strong>of</strong><br />
circulating CD11bhi/Gr1lo monocytes; the kidneys <strong>of</strong> both<br />
these strains contain numerous F4/80+/Gr1lo/Ly6Clo/<br />
CD62L− macrophages. These cells appear concomitant<br />
with proteinuria onset and produce TNF-±, IL-1, BAFF and<br />
CXCL13; thus although phenotypically they resemble alternatively<br />
activated macrophages, they migrate to inflamed<br />
tissue and produce pro-inflammatory mediators. Further<br />
study <strong>of</strong> these cells should help us understand what directs<br />
traffic and local differentiation <strong>of</strong> monocytes to macrophages<br />
and dendritic cells, and how interactions between<br />
the differentiated macrophages and the kidney microenvironment<br />
promote injury.<br />
doi:10.1016/j.clim.2007.03.432<br />
Sa.46 Optimization <strong>of</strong> Autoantigen Microarrays to<br />
Study Autoimmunity<br />
Imelda Balboni, Postdoctoral Fellow, Stanford University,<br />
Medicine and Pediatrics, Stanford, CA, Cindy Limb,<br />
Research Assistant, Stanford University, Medicine, Stanford,<br />
CA, Paul Utz, Associate Pr<strong>of</strong>essor, Stanford University,<br />
Medicine, Stanford, CA, Jessica Tenenbaum, Graduate<br />
Student, Stanford University, Medicine, Stanford, CA
Abstracts<br />
Autoantigen microarrays are used to study autoimmune<br />
diseases including systemic lupus erythematosus, rheumatoid<br />
arthritis, and multiple sclerosis. Hundreds <strong>of</strong> autoantigens<br />
can be analyzed with microliter volumes <strong>of</strong><br />
serum in a high throughput manner. Various slide surfaces,<br />
printing methods and arraying conditions have been<br />
reported, and we sought to determine the optimal<br />
conditions for printing microarrays based on the initial<br />
protocol developed by the Robinson and Utz laboratories.<br />
We analyzed 22 slide surfaces for overall background,<br />
uniformity, streaking, and smearing <strong>of</strong> features. Based on<br />
these characteristics, coefficient <strong>of</strong> variance (CV) was<br />
determined for eight surfaces, and ultimately FAST®,<br />
poly-L-lysine, and SuperEpoxy slides were chosen for<br />
further studies. Several histidine-tagged autoantigens<br />
were spotted onto microarrays using a robotic microarrayer.<br />
Slides were probed with a histidine-specific<br />
monoclonal antibody and fluorophore-labeled secondary<br />
antibody. Arrays were scanned with a GenePix 4000<br />
scanner and analyzed using GenePix Pro 6.0 s<strong>of</strong>tware.<br />
CV was calculated based on the fluorescence intensity<br />
minus background for each feature. Similar studies using<br />
human sera were performed using the FAST® surface to<br />
demonstrate applicability to the study <strong>of</strong> human autoimmune<br />
disease. FAST® slides had the lowest CV in all<br />
studies, with interslide CV as low as 9% and intraslide CV<br />
as low as 15% when probing with a histidine-specific<br />
monoclonal antibody. CV was more variable when probing<br />
with human autoimmune sera, ranging from 5 to 36% with<br />
a median <strong>of</strong> 15%. Additional parameters for printing,<br />
probing, and analyzing microarrays were also investigated,<br />
and optimal conditions have been established to study<br />
autoantibody pr<strong>of</strong>iles in autoimmune disease.<br />
doi:10.1016/j.clim.2007.03.434<br />
Sa.47 Over-expression <strong>of</strong> Interferon Alpha in Lupus<br />
Families: Evidence for a Complex Heritable Trait<br />
Timothy Niewold, Fellow, Mary Kirkland Center for Lupus<br />
Research, Hospital for Special Surgery, New York, NY, John<br />
Harley, Pr<strong>of</strong>essor, Oklahoma Medical Research Foundation,<br />
Oklahoma City, OK, Jing Hua, Research Fellow, Mary<br />
Kirkland Center for Lupus Research, Hospital for Special<br />
Surgery, New York, NY, Mary Crow, Pr<strong>of</strong>essor, Mary Kirkland<br />
Center for Lupus Research, Hospital for Special Surgery,<br />
New York, NY, Thomas Lehman, Pr<strong>of</strong>essor, Mary Kirkland<br />
Center for Lupus Research, Hospital for Special Surgery,<br />
New York, NY<br />
Background: Interferon alpha (IFN-α) levels are elevated<br />
in many patients with systemic lupus erythematosus<br />
(SLE), however it is not known whether increased IFN-α is<br />
a cause or a result <strong>of</strong> SLE. A haplotype <strong>of</strong> the IFN-α<br />
pathway gene IRF5 has been associated with risk <strong>of</strong> SLE.<br />
Methods: Serum IFN-α activity was measured in samples<br />
from the Hospital for Special Surgery Family Lupus<br />
Registry (n=226, 111 SLE) and the Lupus Multiplex Registry<br />
and Repository (n=445, 155 SLE) using a reporter cell<br />
assay and normalized to unrelated healthy donor samples<br />
(n=106). The rs2004640 and rs2070197 SNPs in IRF5 were<br />
genotyped using ABI Taqman probes. Results: 47% <strong>of</strong> SLE<br />
patients had high serum IFN-a activity, and 18% <strong>of</strong> healthy<br />
relatives <strong>of</strong> SLE patients also had high IFN-α activity<br />
compared to unrelated donors (p=0.0002). High IFN-α<br />
expression is clustered in certain families. 54% <strong>of</strong> high<br />
IFN-α SLE patients have a healthy first degree relative<br />
with high IFN-α (p=2x10-7), and SLE-affected sib pairs had<br />
concordant IFN-α expression in most cases (p=0.023).<br />
There was a trend toward higher IFN-α expression in SLE<br />
patients with the IRF5 SLE risk haplotype. There were no<br />
ethnic differences in IFN-α expression, but the IRF5 risk<br />
haplotype was much more common in European American<br />
and Hispanic populations (p=0.0018). Conclusions: Many<br />
SLE families demonstrate abnormally high IFN-α expression<br />
in both healthy and affected individuals compared to<br />
healthy unrelated donors, suggesting that high endogenous<br />
IFN-α expression is a heritable SLE risk factor. It is<br />
likely that many genetic factors underlie this complex<br />
trait.<br />
doi:10.1016/j.clim.2007.03.435<br />
Sa.48 Evaluation <strong>of</strong> the Polymorphism <strong>of</strong> IL-1RN<br />
Gene in Patients with SLE (Systemic Lupus<br />
Erythematosus)<br />
Rashin Ganjali, Researcher, Immunogenetics Department,<br />
Bu-Ali Research Institute, Mashhad University <strong>of</strong> Medical<br />
Sciences, Mashhad, Iran, Jalil Tavakkol Afshari, Vice<br />
President for Research, Immunogenetics Department,<br />
Bu-Ali Research Institute, Mashhad University <strong>of</strong> Medical<br />
Sciences, Mashhad, Iran, Maryam Mazhani, Researcher,<br />
Immunogenetics Department, Bu-Ali Research Institute,<br />
Mashhad University <strong>of</strong> Medical Sciences, Mashhad, Iran<br />
Systemic lupus erythematosus (SLE) is an autoimmune<br />
disease with unknown etiology. SLE is characterized by<br />
malregulation <strong>of</strong> the immune system, with hyperactive B cells<br />
synthesizing excessive amounts <strong>of</strong> different autoantibodies.<br />
Enhanced IL-1 gene expression and IL-1 protein secretion are<br />
detected in the pathogenesis <strong>of</strong> SLE. IL-1RN has a downregulatory<br />
effect; therefore it is supposed that its gene<br />
polymorphisms may contribute to the pathogenesis <strong>of</strong> SLE. A<br />
total <strong>of</strong> 34 patients with SLE and 26 healthy subjects were<br />
studied. DNA was extracted from whole blood by salting out<br />
method and IL-1RN genotype was determined after PCR. The<br />
data were analyzed by Chi-square and Fisher’s exact tests.<br />
There were no statistical differences between genotype<br />
frequencies in both groups. Except A4 and A5 alleles, there<br />
were no statistically differences between allele frequencies,<br />
too; while A4 and A5 had been increased in healthy subjects<br />
(p=0.07). In the present population, polymorphic genotypes <strong>of</strong><br />
IL-1RN did not increase the susceptibility to SLE; while<br />
increased two polymorphic alleles in healthy subjects confirm<br />
this idea. However for/against relations between gene polymorphisms<br />
and such diseases require more studies with more<br />
individuals.<br />
doi:10.1016/j.clim.2007.03.436<br />
S89
S90 Abstracts<br />
Sa.49 TNF Receptor-associated Periodic Fever<br />
Syndrome (TRAPS) Caused by a Novel C30F Mutation<br />
Associated with the Common R92Q Low-Penetrance<br />
Mutation in TNFRSF1A<br />
Joao B. Oliveira, Associate Researcher, Molecular<br />
<strong>Immunology</strong> and Genetics Section, Laboratory <strong>of</strong> Medical<br />
Investigation #56, USP, São Paulo, Brazil, Adriana Jesus,<br />
Pediatric Rheumatology Second Year Fellow, Pediatrics<br />
Department, University <strong>of</strong> São Paulo, Brazil, São Paulo,<br />
Brazil, Ivona Aksentijevich, Staff Scientist, NIH/NIAMS/<br />
GGB, Bethesda, MD, Clovis Silva, Head <strong>of</strong> Pediatric<br />
Rheumatology Unit, Pediatrics Department, University <strong>of</strong><br />
São Paulo, Brazil, São Paulo, Brazil, Alberto J.S. Duarte,<br />
Lab Director, Laboratory <strong>of</strong> Medical Investigation #56<br />
(LIM_56), USP, São Paulo, Brazil<br />
Introduction: TRAPS is the most common <strong>of</strong> the autosomal<br />
dominant periodic fever syndromes. It is caused by<br />
mutations in the TNFRSF1A gene, which encodes for the<br />
type 1 TNF-receptor (TNFR1). We describe here a Brazilian<br />
patient with TRAPS and a novel TNFRSF1A mutation.<br />
Material and methods: The exons 2 to 5 and flanking<br />
intronic regions were amplified by PCR, purified and<br />
sequenced using an automated capillary sequencer. Results.<br />
The patient is a 9-year-old girl from São Paulo, Brazil. She<br />
presents recurrent fevers since age <strong>of</strong> 3 years, usually<br />
lasting 3 to 7 days, and recurring every other week. These<br />
episodes are associated with a mild abdominal pain, nausea,<br />
vomiting and generalized myalgia. Recurrent conjunctivitis<br />
and erysipela-like skin lesions in lower limbs are also<br />
present. Laboratory studies show persistent normocytic<br />
normochromic anemia, thrombocytosis, elevated erythrocyte<br />
sedimentation rate and C-reactive protein. Antinuclear<br />
antibody is positive (1:80). Physical examination is unremarkable,<br />
except for height in percentile 3 and weight in<br />
percentile 10–25. IgD level is within normal range. Genetic<br />
testing for Familial Mediterranean Fever (FMF) excluded five<br />
most common FMF mutations (M680I, M694V, M694I, V726A,<br />
E148Q). Mutational screening <strong>of</strong> TNFRSF1A revealed the<br />
novel C30F mutation and the common R92Q low-penetrance<br />
mutation. The R92Q is seen in 5% <strong>of</strong> the general population,<br />
and is associated with atypical inflammatory phenotypes.<br />
Conclusion: We report a TRAPS patient with the novel<br />
TNFRSF1 mutation, C30F, in conjunction with the low-penetrance<br />
mutation R92Q. Our findings expand the diversity <strong>of</strong><br />
TNFRSF1A mutations associated with TRAPS.<br />
doi:10.1016/j.clim.2007.03.437<br />
Sa.50 Cryopyrin-associated Periodic Syndromes<br />
(CAPS): Report <strong>of</strong> Two Brazilian Cases, Including a<br />
Rare Non-Exon 3 CIAS1 Mutation<br />
Adriana Jesus, Second Year Pediatric Rheumatology Fellow,<br />
Pediatrics Department, University <strong>of</strong> São Paulo, Brazil, São<br />
Paulo, Brazil, Brazil Joao Oliveira, Associate Researcher,<br />
Molecular <strong>Immunology</strong> and Genetics Section, Laboratory <strong>of</strong><br />
Medical Investigation #56, USP, São Paulo, Brazil, Ivona<br />
Aksentijevich, Staff Scientist, NIH/NIAMS/GGB, Bethesda,<br />
MD, Clovis Silva, Head <strong>of</strong> Pediatric Rheumatology Unit,<br />
Pediatrics Department, São Paulo, Brazil, Alberto J.S.<br />
Duarte, Lab Director, Laboratory <strong>of</strong> Medical Investigation<br />
#56 (LIM-56), USP, São Paulo, Brazil<br />
Introduction: Cryopyrin-associated periodic syndromes<br />
(CAPS) are autoinflammatory diseases that include three<br />
clinical syndromes linked to mutations in CIAS1 gene: familial<br />
cold autoinflammatory syndrome (FCAS), Muckle–Wells syndrome<br />
(MWS), and neonatal onset multisystem inflammatory<br />
disease (NOMID). We describe here 2 CAPS patients with the<br />
corresponding molecular defect. Material and methods: Exons<br />
and flanking intronic regions <strong>of</strong> CIAS1 were amplified by PCR,<br />
purified and sequenced using an automated capillary sequencer.<br />
Results: Patient 1 is an 8 year-old girl who presented at<br />
20 days <strong>of</strong> age with an erythematous and macular rash<br />
involving face, chest, abdomen, limbs, palms and soles. At<br />
3 months <strong>of</strong> age, she developed recurrent fever, exacerbated<br />
by cold weather. Elevated acute phase reactants were present.<br />
Arthritis <strong>of</strong> large joints and unilateral anterior uveitis developed<br />
during follow-up. DNA sequencing identified the T436I<br />
CIAS1 mutation. Patient 2 is a 7-year-old boy who soon after<br />
birth developed a papular exanthema involving chest, abdomen<br />
and extremities with daily fever episodes. Persistent<br />
anemia, leukocytosis, thrombocytosis, and high acute phase<br />
reactants were seen. Later, he presented with growth failure,<br />
frontal bossing and marked patellae enlargement. Cognitive<br />
and motor impairments and chronic intracranial hypertension<br />
were noted. Molecular studies identified a rare non-exon 3<br />
mutation, G755R, located in exon 4 <strong>of</strong> CIAS1. Conclusion: Two<br />
Brazilian patients with clinically and genetically related<br />
entities known as FCAS and NOMID were found to have<br />
disease-causing mutations in the CIAS1 gene, including one<br />
outside exon 3. Our result reinforces that CAPS patients should<br />
be screened for mutations in all exons <strong>of</strong> CIAS1.<br />
doi:10.1016/j.clim.2007.03.438<br />
Sa.51 Dickkopf-1 is a Link Between the Immune<br />
System and Bone Formation<br />
Georg Schett, Pr<strong>of</strong>essor, University <strong>of</strong> Erlangen-Nuremberg,<br />
Erlangen, Danielle Diarra, Fellow, Medical University <strong>of</strong><br />
Vienna, Vienna, Austria, William Richards, PhD, Amgen,<br />
Thousand Oaks, CA, Marina Stolina, PhD, Amgen, Thousand<br />
Oaks, CA<br />
Inflammatory joint disease can either lead to bone<br />
erosion or to bone formation. In rheumatoid arthritis,<br />
immune-mediated inflammation leads to joint destruction<br />
with virtually no signs <strong>of</strong> bone repair. In contrast, diseases<br />
such as ankylosing spondylitis are characterized by dramatic<br />
bone formation. The molecular signals determine whether<br />
inflammatory tissue induces bone loss or bone formation. By<br />
inhibiting Dickkopf-1 (DKK-1), a regulatory molecule <strong>of</strong> the<br />
Wnt pathway, we show that a bone destructive pattern <strong>of</strong><br />
arthritis can be reversed into a bone-forming pattern. Thus,<br />
treatment <strong>of</strong> tumor necrosis factor transgenic mice by an<br />
antibody against DKK-1 completely inhibited bone erosions<br />
and induced the formation <strong>of</strong> bony nodules called osteophytes.<br />
Tumor necrosis factor α (TNF) was identified as key<br />
inducer <strong>of</strong> DKK-1 in mouse inflammatory arthritis as well as
Abstracts<br />
human rheumatoid arthritis. This suggests that the Wnt<br />
pathway is a key regulator <strong>of</strong> joint remodeling and a link<br />
between the immune system and bone formation.<br />
doi:10.1016/j.clim.2007.03.439<br />
Sa.52 Biosimulations Predict that Rituximab is More<br />
Efficacious than Other Standards <strong>of</strong> Care in<br />
Rheumatoid Arthritis Patients with Severe Disease<br />
Kosmas Kretsos, Engineer, Entelos, Inc., Foster City, CA,<br />
Daniel L. Young, Engineer, Entelos Inc., Foster City, CA,<br />
Chan C. Whiting, Scientist, Entelos, Inc., Foster City, CA,<br />
Katherine Kudrycki, Scientist, Entelos, Inc., Foster City, CA,<br />
Sirid-Aimee Kellermann, Senior Scientist, Entelos, Inc.,<br />
Foster City, CA, Nadine A. Derfanoux, Senior Scientist,<br />
Entelos, Inc., Foster City, CA<br />
The clinical efficacy <strong>of</strong> the B cell-depleting therapy<br />
rituximab in patients with rheumatoid arthritis (RA) highlights<br />
the critical contribution <strong>of</strong> B cells to the disease<br />
process. To explore the efficacy <strong>of</strong> rituximab in RA patients<br />
with different phenotypes, the Entelos® RA PhysioLab®<br />
platform, a mathematical model representing an inflamed<br />
joint in an RA patient, was used to simulate the effects <strong>of</strong><br />
rituximab and other therapeutic approaches on key clinical<br />
outcomes. The platform dynamically integrates the contributions<br />
<strong>of</strong> multiple cell types and mediators involved in synovial<br />
hyperplasia and joint structural damage. B cell development,<br />
recruitment, activation and effector processes (e.g., antigen<br />
presentation, cytokine synthesis and autoantibody production)<br />
are also represented in the platform. Biosimulation in a<br />
virtual patient with severe RA predicts an ACR response<br />
almost identical (within a 95% confidence interval) to that<br />
reported in rituximab clinical trials. Furthermore, the<br />
predicted level <strong>of</strong> B cell depletion in the virtual patient is<br />
consistent with published reports. These results suggest that<br />
rituximab will be a highly effective single-agent treatment<br />
for severe RA. However, similar analyses in a virtual patient<br />
with moderate RA show that rituximab is less effective than<br />
anti-TNF-α therapies. In both phenotypes, biosimulations<br />
predict that rituximab induces sustained benefits in joint<br />
structure, namely decreases in cartilage degradation and<br />
bone erosion, which persist for months after cessation <strong>of</strong><br />
rituximab treatment, even after joint inflammation returns.<br />
Findings such as these can be used to help develop more<br />
targeted RA therapies and improve drug discovery by<br />
identifying prospective responder patient phenotypes.<br />
doi:10.1016/j.clim.2007.03.440<br />
Sa.53 Gene-environment Interactions Between<br />
Heavy Cigarette Smoking and HLA-DRB1 Shared<br />
Epitope in Predicting Incident RA, Data from a Two<br />
Large Prospective Cohort Studies<br />
Elizabeth W. Karlson, Associate Pr<strong>of</strong>essor <strong>of</strong> Medicine,<br />
Brigham and Women’s Hospital, Department <strong>of</strong> Medicine,<br />
Dover, MA, Shun-Chiao Chang, Doctoral Student, Harvard<br />
School <strong>of</strong> Public Health, Epidemiology, Boston, MA, Karen H.<br />
Costenbader, Assistant Pr<strong>of</strong>essor <strong>of</strong> Medicine, Brigham and<br />
Women’s Hospital, Department <strong>of</strong> Medicine, Boston, MA,<br />
Lori B. Chibnik, Statistician, Brigham and Women’s Hospital,<br />
Medicine, Boston, MA, Patricia Fraser, Medical Director,<br />
Pharmacovigilance/Medical Affairs, Cambridge, MA<br />
Background: Heavy smoking (N10 pack years) increases<br />
risk <strong>of</strong> RA. HLA-DRB1 demonstrates gene–environment<br />
interaction (GEI) with smoking when comparing current to<br />
never smokers. We studied this GEI in the Nurses’ Health<br />
Studies. Methods: RA diagnosis was validated by chart review<br />
for ACR criteria. Controls were matched on age, menopausal<br />
status, and postmenopausal hormone use. High resolution<br />
HLA-DRB1 genotyping was performed for SE alleles. HLA-SE,<br />
smoking, and HLA-SE×smoking interaction, and risk <strong>of</strong> RA<br />
were assessed using conditional logistic regression models,<br />
adjusted for age and reproductive factors. We tested for<br />
multiplicative and additive interaction. Results: 244 Caucasian<br />
matched pairs were included. Mean age at diagnosis was<br />
54 years; 59% <strong>of</strong> cases were RF+. There was no interaction<br />
between never/ever smoking and HLA-SE and risk <strong>of</strong> RA. We<br />
found no multiplicative interaction (p=0.10) but a strong<br />
additive interaction (p=0.006, attributable proportion (AP)<br />
0.49) with smoking categorized as b10 pack years vs. N10<br />
pack years. The highest risk was in heavy smokers with<br />
double copy HLA-SE (OR, 21.5; 95% CI, 2.8–167.9). There was<br />
evidence for additive interaction in RF+ (p=0.01, AP 0.47)<br />
and RF− cases (p=0.04, AP 0.53), each compared with<br />
controls. Models using pack years smoked as a continuous<br />
variable had no evidence for multiplicative interaction<br />
(p=0.33). Conclusion: GEI between HLA-SE and smoking in<br />
RA risk is strongest with N10 pack years smoked. Future<br />
studies should assess pack years smoked when testing for<br />
GEI. Additive interaction suggests a pathway that requires<br />
involvement <strong>of</strong> both risk factors.<br />
doi:10.1016/j.clim.2007.03.441<br />
S91<br />
Sa.54 Anti-fibrinbogen Autoimmunity in<br />
Rheumatoid Arthritis<br />
William Robinson, Assistant Pr<strong>of</strong>essor, Division <strong>of</strong><br />
<strong>Immunology</strong> and Rheumatology, Stanford University, Palo<br />
Alto, CA, Wolfgang Hueber, Research Associate, Division <strong>of</strong><br />
<strong>Immunology</strong> and Rheumatology, Stanford University, Palo<br />
Alto, CA, Peggy P. Ho, Research Associate, Department<br />
Neurology, Stanford University, Stanford, CA, Xiaoyan Zhao,<br />
Postdoctoral Fellow, Division <strong>of</strong> <strong>Immunology</strong> and<br />
Rheumatology, Stanford University, Palo Alto, CA<br />
Rheumatoid arthritis (RA) is an autoimmune synovitis<br />
affecting 0.5% <strong>of</strong> the world population, yet the mechanisms<br />
underlying the initiation and perpetuation <strong>of</strong> RA remain<br />
elusive. A subset <strong>of</strong> RA patients exhibit anti-citrulline<br />
autoimmunity and citrullinated proteins are generated<br />
through post-transnational conversion <strong>of</strong> arginine to citrulline<br />
by peptidyl-arginine deiminase. The pathogenesis <strong>of</strong> RA<br />
involves the deposition and excessive local generation <strong>of</strong><br />
fibrin in the inflamed joint, and recent studies identified<br />
citrullinated fibrin as a candidate autoantigen in RA. We<br />
performed direct immunoprecipitation <strong>of</strong> immune
S92 Abstracts<br />
complexes from RA and control synovial fluid and tissue<br />
lysates, followed by mass spectrometry (MS) analysis. MS/<br />
MS analysis revealed previously described and novel<br />
candidate autoantigens, including native and citrullinated<br />
forms <strong>of</strong> fibrinogen, fibronectin, HSPs, vimentin, and<br />
elongation factor. We added peptides and proteins representing<br />
these candidates to synovial antigen arrays to<br />
screen samples derived from RA and control patient<br />
cohorts. Synovial mircroarray and ELISA analyses demonstrated<br />
specific autoreactive B cell targeting <strong>of</strong> citrullinated<br />
fibrinogen peptides and proteins in approximately<br />
30% <strong>of</strong> RA patients. Anti-citrullinated fibrinogen antibodies<br />
were present almost exclusively in patients possessing anti-<br />
CCP antibodies. We also detect fibrinogen-containing<br />
immune complexes in blood derived from patients possessing<br />
anti-fibrinogen autoantibodies. Based on these results<br />
we developed a fibrinogen-induced arthritis (FIA) mouse<br />
model using human fibrinogen as the immunizing antigen<br />
(see abstract presented by Ho et al.). These data support<br />
our hypothesis that citrullinated fibrinogen is a pathogenic<br />
autoantigen in RA, and that autoimmunity against fibrinogen<br />
defines a molecular subtype representing approximately<br />
30% <strong>of</strong> RA patients.<br />
doi:10.1016/j.clim.2007.03.442<br />
Sa.55 Genome-Wide SNP Scan Identifies<br />
Polymorphisms Associated with Differential<br />
Response to Anti-TNF Treatment Among<br />
Rheumatoid Arthritis Patients in the ABCoN Cohort<br />
John Carulli, Principal Scientist, BiogenIdec, Cambridge,<br />
MA, Franak Batliwalla, Assistant Investigator, Feinstein<br />
Institute for Medical Research, Manhasset, NY, Chunyu Liu,<br />
Scientist, BiogenIdec, Drug Discovery, Cambridge, MA, Aarti<br />
Damle, Assistant Investigator, Feinstein Institute for<br />
Medical Research, Manhasset, NY, Jadwiga Bienkowska,<br />
Principal Scientist, Biogenidec, Drug Discovery, Cambridge,<br />
MA, Annette Lee, Assistant Investigator, Feinstein Institute<br />
for Medical Research, Manhasset, NY, Wentian Li, Assistant<br />
Investigator, Feinstein Institute for Medical Research,<br />
Manhasset, NY, Peter Gregersen, Investigator, Feinstein<br />
Institute for Medical Research, Manhasset, NY<br />
The Autoimmune Biomarkers Collaborative Network<br />
(ABCoN) has enrolled a longitudinal cohort <strong>of</strong> RA patients<br />
beginning anti-TNF treatment in order to identify biomarkers<br />
influencing response to anti-TNF therapy. Genomewide<br />
(Illumina 317K) SNP data were collected on 116 <strong>of</strong><br />
these patients (54 etaneracept, 25 adalimumab, 37<br />
infliximab). DAS28 scores for these 116 patients ranged<br />
from 2.72 to 7.08 (mean =5.23) prior to starting anti-TNF<br />
therapy, decreasing to 1.4 to 6.68 (mean=3.77) at 14 weeks<br />
after enrollment. SNP genotypes were tested for association<br />
with the change in DAS28 score at 6 weeks and<br />
14 weeks after initiation <strong>of</strong> anti-TNF therapy. While no<br />
individual SNPs show genome-wide significance (pb10 −7 )in<br />
this small data set, preliminary analysis shows 36 SNPs<br />
associated (pb10 −4 ) with DDAS28 at 6 weeks, and 41 SNPs<br />
associated with DDAS28 at 14 weeks. Nine regions <strong>of</strong> the<br />
genome have two or more SNPs within 100 kb with pb10 −5 ,<br />
and genes in these regions represent candidates for anti-<br />
TNF pharmacogenetic markers. Permutation tests on these<br />
data indicate a low type I error rate. Haplotype analysis<br />
may reveal additional evidence for association. RNA<br />
samples (PaxGene) from peripheral blood <strong>of</strong> the same<br />
patients were analyzed by genome-wide transcript pr<strong>of</strong>iling.<br />
Cross-referencing <strong>of</strong> genes comprising expression-based<br />
classifiers for anti-TNF responders and non-responders (see<br />
FOCIS 2007 abstract by Batliwalla et al.) with genes showing<br />
nominal genetic association with anti-TNF response represents<br />
a powerful approach to identifying pharmacogenomic<br />
markers for anti-TNF response among rheumatoid arthritis<br />
patients.<br />
doi:10.1016/j.clim.2007.03.443<br />
Sa.56 Oncogenic Properties <strong>of</strong> Rheumatoid Synovial<br />
Cells<br />
Toshihiro Nakajima, Pr<strong>of</strong>essor, Institute <strong>of</strong> Medical Science,<br />
St. Marianna University School <strong>of</strong> Medicine, Kawasaki,<br />
Japan, Naoko Yagishita, Senior Assistant Pr<strong>of</strong>essor, Institute<br />
<strong>of</strong> Medical Science, St. Marianna University School <strong>of</strong><br />
Medicine, Kawasaki, Japan, Satoshi Yamasaki, Senior<br />
Assistant Pr<strong>of</strong>essor, Institute <strong>of</strong> Medical Science, St.<br />
Marianna University School <strong>of</strong> Medicine, Kawasaki, Japan,<br />
Kusuki Nishioka, Pr<strong>of</strong>essor, I, Kawasaki, Japan<br />
Rheumatoid arthritis (RA) is one <strong>of</strong> the most common<br />
and critical articular diseases with synovial hyperplasia.<br />
However, the mechanism that regulates synovial cell outgrowth<br />
is not fully understood. To clarify the mechanism,<br />
we carried out immunoscreening using anti-rheumatoid<br />
synovial cell antibody and cloned a putative RING finger<br />
protein, designated Synoviolin (G&D, 2003). Synoviolin is an<br />
E3 ubiquitin ligase and located in endoreticulum. It was<br />
highly expressed in the rheumatoid synovium, and mice<br />
overexpressing this molecule developed spontaneous<br />
arthropathy. In this regard, like rheumatoid synovial cells,<br />
the anti-apoptotic effect <strong>of</strong> Synoviolin was observed even<br />
for its expressed ectopically in NIH3T3 cells, which resulted<br />
in enhanced cell overgrowth in these cells (MCB, 2005).<br />
These results were confirmed also in fly system. Next, to<br />
identify the substrates for Synoviolin that may be involved<br />
in cell growth, we analyzed the comprehensive pr<strong>of</strong>ile <strong>of</strong><br />
protein expression in Synoviolin null cells (JBC, 2005).<br />
Then, we found that Synoviolin targets tumor suppressor<br />
gene p53 for ubiquitination (EMBO J., 2007). Synoviolin<br />
sequestrated and metabolized p53 in the cytoplasm and<br />
negatively regulated its cellular level and biological<br />
functions, including transcription, cell cycle regulation<br />
and apoptosis. Furthermore, these p53 regulatory functions<br />
<strong>of</strong> Synoviolin were irrelevant to other E3 ubiquitin ligases<br />
for p53, such as MDM2, Pirh2 and Cop1, which form<br />
autoregulatory feedback loops. Our results provide novel<br />
insights into p53 signaling mediated by Synoviolin and<br />
clarify the molecular basis <strong>of</strong> synovial hyperplasia in RA.<br />
doi:10.1016/j.clim.2007.03.444
Abstracts<br />
Sa.57 T Lymphocyte Clonal Alterations in<br />
Anti-Citrullinated Protein Antibody Positive<br />
Synovitis<br />
Tineke Cantaert, PhD Student, University <strong>of</strong> Amsterdam,<br />
Amsterdam, The Netherlands, Sophie Brouard, PhD, INSERM<br />
U643, Nantes, France, Cristophe Braud, PhD, INSERM U643,<br />
Nantes, France, Jean-Paul Soulillou, Pr<strong>of</strong>essor, INSERM<br />
U643, Nantes, France, Niek de Vries, PhD, <strong>Clinical</strong><br />
<strong>Immunology</strong> and Rheumatology, Academic Medical Center,<br />
University <strong>of</strong> Amsterdam, Amsterdam, The Netherlands,<br />
Paul-Peter Tak, Pr<strong>of</strong>essor, <strong>Clinical</strong> <strong>Immunology</strong> and<br />
Rheumatology, Academic Medical Center, University <strong>of</strong><br />
Amsterdam, Amsterdam, The Netherlands, Dominique<br />
Baeten, Pr<strong>of</strong>essor, <strong>Clinical</strong> <strong>Immunology</strong> and Rheumatology,<br />
Academic Medical Center/University <strong>of</strong> Amsterdam,<br />
Amsterdam, The Netherlands<br />
Objective: Increasing evidence suggests that the pathophysiology<br />
<strong>of</strong> rheumatoid arthritis (RA) is fundamentally<br />
different in patients with or without anti-citrullinated<br />
protein antibodies (ACPA). The association <strong>of</strong> RA with HLA-<br />
DR shared epitope exclusively in the ACPA+ subset supports<br />
this concept and suggests that T cell help may be involved in<br />
the ACPA response. We assessed the potential involvement<br />
<strong>of</strong> T lymphocytes in ACPA+ synovitis. Methods: Synovial<br />
biopsies were obtained from inflamed knee joints <strong>of</strong> 54 RA<br />
patients. 37 were ACPA+ (anti-CCP2 ELISA). mRNA was<br />
isolated from 7 ACPA+ RA synovia, 4 ACPA− RA synovia, and<br />
10 control spondyloarthritis (SpA) synovia with similar levels<br />
<strong>of</strong> T and B cell infiltration. T lymphocyte clonality in the<br />
synovium was studied by combined quantitative and<br />
qualitative TCR CDR3 analysis by the TcLandscape technology.<br />
Results: Comparing ACPA+ with the ACPA− RA patients,<br />
there were no significant differences in clinical and<br />
histological parameters. Alterations <strong>of</strong> the normal TCR<br />
length distribution were analyzed for all Vbeta families.<br />
TCR analysis showed a significantly higher degree <strong>of</strong><br />
alteration <strong>of</strong> the TCR length distribution in ACPA+ (31.2±<br />
3.7%) compared to ACPA− (18.5±1.4%; p=0.012) and to SpA<br />
synovitis (22.4 ±1.3%; p=0.014). These alterations reflecting<br />
oligo- or monoclonal expansions were observed in several<br />
Vbeta families and were different in each individual sample.<br />
Conclusion: We demonstrate an increased alteration <strong>of</strong> the<br />
TCR length distribution in ACPA+ but not in ACPA− RA or<br />
control synovitis. These data suggest a specific contribution<br />
<strong>of</strong> T lymphocytes in the local disease process and possibly<br />
the ACPA response in this RA subset.<br />
doi:10.1016/j.clim.2007.03.445<br />
Sa.58 Synovial T/B Cell Lymphoid Aggregates<br />
Regulate the Production <strong>of</strong> Rheumatoid<br />
Arthritis-specific Autoantibodies<br />
Tineke Cantaert, PhD Student, University <strong>of</strong> Amsterdam,<br />
Amsterdam, The Netherlands, Trieneke Timmer, PhD<br />
Student, Molecular Cell Biology and <strong>Immunology</strong>, VU<br />
Medical Center, Amsterdam, The Netherlands, Bernard<br />
Vandooren, PhD Student, Rheumatology, Ghent University<br />
Hospital, Ghent, Belgium, Carla Wijbrandts, PhD Student,<br />
<strong>Clinical</strong> <strong>Immunology</strong> and Rheumatology, Academic Medical<br />
Center/University <strong>of</strong> Amsterdam, Amsterdam, The<br />
Netherlands, Rogier Thurlings, PhD Student, <strong>Clinical</strong><br />
<strong>Immunology</strong> and Rheumatology, Academic Medical Center/<br />
University <strong>of</strong> Amsterdam, Amsterdam, The Netherlands,<br />
Leen De Rycke, PhD, <strong>Clinical</strong> <strong>Immunology</strong> and<br />
Rheumatology, Academic Medical Center, University <strong>of</strong><br />
Amsterdam, Amsterdam, The Netherlands, Tineke van der<br />
Pouw-Kraan, PhD, Molecular Cell Biology and <strong>Immunology</strong>,<br />
VU Medical Center, Amsterdam, The Netherlands, Theo Out,<br />
PhD, Experimental <strong>Immunology</strong>, Academic Medical Center/<br />
University <strong>of</strong> Amsterdam, The Netherlands, Paul-Peter Tak,<br />
Pr<strong>of</strong>essor, <strong>Clinical</strong> <strong>Immunology</strong> and Rheumatology, Academic<br />
Medical Center, University <strong>of</strong> Amsterdam, Amsterdam, The<br />
Netherlands, Dominique Baeten, Pr<strong>of</strong>essor, <strong>Clinical</strong><br />
<strong>Immunology</strong> and Rheumatology, Academic Medical Center,<br />
University <strong>of</strong> Amsterdam, Amsterdam, The Netherlands<br />
Ectopic lymphoid neogenesis occurs in synovial membrane<br />
in rheumatoid arthritis (RA) and inflamed tissues.<br />
Partial structural resemblance with follicles in secondary<br />
lymphoid organs suggests a role in the maturation <strong>of</strong><br />
humoral immune and autoimmune responses. We investigated<br />
the relation between synovial T/B cell lymphoid<br />
aggregates, inflammation, and autoantibody production.<br />
Synovial biopsies were obtained from inflamed joint <strong>of</strong> 130<br />
patients with RA or spondyloarthritis (SpA), a chronic<br />
arthritis devoid <strong>of</strong> known autoantibodies. B–T cell aggregates<br />
were analyzed by immunohistochemistry and realtime<br />
PCR. Autoantibodies were measured in synovial fluid by<br />
ELISA, corrected for total IgG and IgM. The presence <strong>of</strong> large<br />
lymphoid aggregates was observed in 1/3 <strong>of</strong> the rheumatoid<br />
synovia, but also in SpA. This was associated with synovial<br />
inflammation in both diseases, with infiltrating memory B<br />
lymphocytes observed almost exclusively within these<br />
aggregates. TNFα blockade induced the disintegration <strong>of</strong><br />
the follicles (pb0.05). Levels <strong>of</strong> CXCL13 and CCL19 were<br />
elevated in follicular versus diffuse synovitis (pb0.05). We<br />
could not observe a dark and light zone nor detect naive B<br />
cells by CD23/IgD staining. These structures occasionally<br />
displayed germinal centres characterized by high endothelial<br />
venules, T/B cell segregation, and follicular dendritic<br />
cells (b10%). This was confirmed by the low amount <strong>of</strong><br />
CD21L mRNA. Both rheumatoid factor (p=0.033) and anticitrullinated<br />
protein antibodies (p=0.09) were lower in<br />
follicular versus diffuse RA synovitis.<br />
Ectopic lymphoid neogenesis is an inducible and reversible<br />
phenomenon in chronic synovitis. Germinal centre reactions<br />
can occasionally be observed in synovial follicles, which<br />
regulate rather than support local humoral autoimmunity in<br />
RA.<br />
doi:10.1016/j.clim.2007.03.447<br />
S93<br />
Sa.59 Non-Pathogenic Antinuclear Antibodies<br />
Contribute to the Clearance <strong>of</strong> Apoptotic Antigens<br />
Released During TNFα Blockade in Spondyloarthritis<br />
(SpA)<br />
Tineke Cantaert, PhD Student, University <strong>of</strong> Amsterdam,<br />
Amsterdam, The Netherlands, Leen De Rycke, PhD, <strong>Clinical</strong>
S94 Abstracts<br />
<strong>Immunology</strong> and Rheumatology, Academic Medical Center/<br />
University <strong>of</strong> Amsterdam, Amsterdam, The Netherlands,<br />
Bernard Vandooren, PhD Student, Rheumatology, Ghent<br />
University Hospital, Ghent, Belgium, Carla Wijbrandts, PhD<br />
Student, <strong>Clinical</strong> <strong>Immunology</strong> and Rheumatology, Academic<br />
Medical Center, University <strong>of</strong> Amsterdam, Amsterdam, The<br />
Netherlands, Elli Kruith<strong>of</strong>, PhD, Rheumatology, Ghent<br />
University Hospital, Ghent, Belgium, Filip De Keyser,<br />
Pr<strong>of</strong>essor, Rheumatology, Ghent University Hospital, Ghent,<br />
Belgium, Eric M. Veys, Pr<strong>of</strong>essor, Rheumatology, Ghent<br />
University Hospital, Ghent, Belgium, Paul-Peter Tak,<br />
Pr<strong>of</strong>essor, <strong>Clinical</strong> <strong>Immunology</strong> and Rheumatology,<br />
Academic Medical Center, University <strong>of</strong> Amsterdam,<br />
Amsterdam, The Netherlands, Dominique Baeten, Pr<strong>of</strong>essor,<br />
<strong>Clinical</strong> <strong>Immunology</strong> and Rheumatology, Academic Medical<br />
Center, University <strong>of</strong> Amsterdam, Amsterdam, The<br />
Netherlands<br />
Infliximab, not etanercept, treatment in arthritis induces<br />
non-pathogenic, IgM antinuclear antibodies. In SLE, antinuclear<br />
antibodies have been related to increased BAFF and<br />
type I-interferon signaling and increased presence <strong>of</strong><br />
apoptotic blebs. We assessed the mechanisms leading to<br />
autoantibody induction by TNFα blockade in spondyloarthritis<br />
(SpA), a disease without humoral autoimmunity.<br />
Serum and synovial biopsies were obtained from SpA<br />
patients treated with infliximab (n=20) or etanercept<br />
(n=20). Serum levels <strong>of</strong> BAFF, IFNα, nucleosomes, and<br />
anti-nucleosomes were assessed (ELISA). Synovial apoptosis<br />
was assessed by TUNEL and active caspase3. Nucleosome<br />
levels increased at 2 (+89%; p =0.002) and 6 (+26%;<br />
p=0.014) weeks <strong>of</strong> infliximab but not etanercept treatment.<br />
This was not related to cell death in the synovium at<br />
weeks 1 and 12 <strong>of</strong> treatment. Infliximab induced an<br />
upregulation <strong>of</strong> IFNα at 2 (p=0.090) and 6 (p=0.042)<br />
weeks. BAFF levels remained stable during treatment with<br />
both biologicals. Anti-nucleosome antibodies were<br />
increased at week 12 (+94%; pb0.001) and 34 (+124%;<br />
p b0.001) <strong>of</strong> infliximab treatment. This increase was<br />
restricted to IgM isotype and correlated with the rise <strong>of</strong><br />
nucleosomes at week 12 (r =0.383;p =0.008) and 34<br />
(r =0.453; p=0.014). The continuous requirement <strong>of</strong> nuclear<br />
antigens for the autoantibody response was indicated by the<br />
disappearance <strong>of</strong> the antibodies upon interruption <strong>of</strong> anti-<br />
TNFα. However, nucleosome levels dropped to normal upon<br />
appearance <strong>of</strong> the autoantibodies. Infliximab induces nonpathogenic<br />
IgM antinuclear antibodies by upregulation <strong>of</strong><br />
serum nucleosomes, suggesting induction <strong>of</strong> apoptosis in<br />
vivo, and the associated increase in type I IFN. This antibody<br />
response could represent a physiological mechanism which<br />
contributes to the normal clearance <strong>of</strong> nuclear antigens. TC<br />
and LDR contributed equally.<br />
doi:10.1016/j.clim.2007.03.448<br />
Sa.61 Proteomic Signatures <strong>of</strong> Secreted Proteins for<br />
the Prediction <strong>of</strong> Treatment Response to TNF<br />
Blockers in RA<br />
Wolfgang Hueber, Postdoctoral Fellow, Stanford University,<br />
Medicine, Palo Alto, CA, Beren Tomooka, Research<br />
Assistant, Stanford University, Medicine, Palo Alto, CA,<br />
Franak Batliwalla, Research Scientist, North Shore Long<br />
Island Jewish Health Care, Manhasset, NY, Robert<br />
Tibshirani, Pr<strong>of</strong>essor, Stanford University, Stanford, CA,<br />
Peter Gregersen, Pr<strong>of</strong>essor, North Shore Long Island Jewish<br />
Health Care System, Manhasset, NY, Lars Klareskog,<br />
Pr<strong>of</strong>essor, Karolinska Institutet, Stockholm, Sweden,<br />
William Robinson, Pr<strong>of</strong>essor, Stanford University, Medicine,<br />
Palo Alto, CA<br />
We pr<strong>of</strong>iled autoantibodies and cytokines in pre-treatment<br />
and 3-month post-treatment sera from RA patients to<br />
delineate secreted proteins predictive <strong>of</strong> response to<br />
therapy with etanercept. Subgroups from two cohorts <strong>of</strong><br />
Enbrel-treated RA patients were analyzed: 29 patients<br />
enrolled in the ABCoN cohort, and 28 patients treated at a<br />
Swedish tertiary care hospital. Serum autoantibodies were<br />
determined using synovial antigen microarrays, and 14<br />
cytokines were measured using a bead-based multiplex<br />
assay. Serum was collected before and 3 months after<br />
initiation <strong>of</strong> therapy. Differential expression <strong>of</strong> autoantibodies<br />
and cytokines in serum from responders and nonresponders<br />
was analyzed. Prediction analysis <strong>of</strong> microarrays<br />
(PAM) was applied to identify panels <strong>of</strong> secreted molecules<br />
predictive <strong>of</strong> response. Pre-treatment samples from etanercept<br />
responders with ACR 50 or greater response but not<br />
non-responders demonstrated higher reactivity against<br />
peptides derived from RA candidate autoantigens including<br />
fibrinogen, calpastatin, heat shock proteins, proteoglycans,<br />
and histones (FDRb0.1). The cytokines IL-6, IL12p40 and<br />
Eotaxin showed a trend towards higher mean levels in pretreatment<br />
samples (pb0.1) in the Swedish but not in the<br />
ABCoN cohort. Pair-wise comparisons <strong>of</strong> pre-treatment with<br />
post-treatment pr<strong>of</strong>iles demonstrated that decreases in<br />
certain autoantibody reactivities and the cytokine IL-6 were<br />
associated with ACR 50 or greater responses. PAM identified<br />
a panel <strong>of</strong> secreted molecules that correctly predicted 73%<br />
<strong>of</strong> patients who subsequently responded to etanercept<br />
therapy with ACR 50 responses or greater. Our data suggest<br />
that pr<strong>of</strong>iles <strong>of</strong> secreted molecules in pre-treatment sera<br />
are associated with RA patients more likely to respond to<br />
etanercept.<br />
doi:10.1016/j.clim.2007.03.449<br />
Sa.62 Distinct Molecular and Cellular Aspects <strong>of</strong><br />
Systemic Juvenile Idiopathic Arthritis (SJIA) and<br />
Polyarticular (PolyJIA)<br />
Claudia Macaubas, Research Associate, Stanford University<br />
Department <strong>of</strong> Pediatrics, Stanford, CA, Khoa Nguyen, BS,<br />
Stanford University Department <strong>of</strong> Pediatrics, Stanford, CA,<br />
Kuang-Hung Pan, PhD Student, Stanford University<br />
Department <strong>of</strong> Genetics, Stanford, CA, Tzielan Lee, <strong>Clinical</strong><br />
Assistant Pr<strong>of</strong>essor, Stanford University Department <strong>of</strong><br />
Pediatrics, Stanford, CA, Chetan Deshpande, Lab Manager,<br />
Stanford University Department <strong>of</strong> Pediatrics, Stanford, CA,<br />
Christy Sandborg, Pr<strong>of</strong>essor and Chief, Stanford<br />
University Department <strong>of</strong> Pediatrics, Stanford, CA,<br />
Stanley Cohen, Pr<strong>of</strong>essor, Stanford University Department<br />
<strong>of</strong> Genetics, Stanford, CA, Elizabeth Mellins, Associate
Abstracts<br />
Pr<strong>of</strong>essor, Stanford University Department <strong>of</strong> Pediatrics,<br />
Stanford, CA<br />
Juvenile idiopathic arthritis (JIA) is a family <strong>of</strong> chronic<br />
inflammatory arthritides that includes distinct subtypes:<br />
systemic (SJIA), polyarticular (polyJIA) and pauciarticular.<br />
The subtypes differ in epidemiology, genetic risk factors,<br />
clinical features, prognosis and response to therapy.<br />
However, the specific molecular mechanisms that distinguish<br />
these diseases are not well understood. We performed<br />
a comparative analysis <strong>of</strong> molecular and cellular aspects <strong>of</strong><br />
SJIA and polyJIA. Blood samples and associated clinical<br />
information were obtained from children with SJIA (n=24),<br />
polyJIA (n=17), and age-matched children without immunological<br />
health problems (n =14). Gene expression in<br />
freshly isolated PBMCs was studied by quantitative PCR<br />
(qPCR) <strong>of</strong> 81 immune related genes. Cell types were<br />
enumerated by FACS. Using GABRIEL analysis s<strong>of</strong>tware, we<br />
identified genes whose expression correlates with erythrocyte<br />
sedimentation rate (ESR). In SJIA, ESR is positively<br />
correlated with transcripts expressed by activated monocytes,<br />
whereas in polyJIA, ESR correlates with transcripts<br />
for cytolytic proteins and Th1-related genes. Expression <strong>of</strong><br />
distinct genes correlates with number <strong>of</strong> active joints in<br />
SJIA and polyJIA, including IL2R and MIF vs. IL-12,<br />
respectively. At the cellular level, we observed expansion<br />
<strong>of</strong> monocytes and altered monocyte subset distribution<br />
(CD14hi vs. CD16hi) in SJIA, but not polyJIA subjects,<br />
compared to controls. Samples from poly JIA subjects<br />
showed a reduced percentage <strong>of</strong> NK cells, and lower<br />
percentage <strong>of</strong> T regulatory cells. Our findings argue that<br />
SJIA is associated with dysregulation <strong>of</strong> innate cells,<br />
whereas poly JIA is associated with altered adaptive<br />
immunity.<br />
doi:10.1016/j.clim.2007.03.450<br />
Sa.63 The Alternative Pathway is Necessary and<br />
Sufficient for Complement Involvement in the<br />
Effector Phase <strong>of</strong> Anti-Collagen Antibody Induced<br />
Arthritis (ACAIA)<br />
Nirmal Banda, Associate Pr<strong>of</strong>essor, University <strong>of</strong> Colorado at<br />
Denver Health Sciences Center, Aurora, CO, Allyson Wood,<br />
Research Associate, University <strong>of</strong> Colorado at Denver Health<br />
Sciences Center, Aurora, CO, Kazue Takahashi, Assistant<br />
Pr<strong>of</strong>essor, Pediatrics, Massachusetts General Hospital,<br />
Boston, MA, William P. Arend, Pr<strong>of</strong>essor <strong>of</strong> Medicine,<br />
University <strong>of</strong> Colorado at Denver Health Sciences Center,<br />
Aurora, CO, Alan Ezekowitz, Pr<strong>of</strong>essor, Pediatrics,<br />
Massachusetts General Hospital, Boston, MA, V. Michael<br />
Holers, Pr<strong>of</strong>essor <strong>of</strong> Medicine and <strong>Immunology</strong>, University <strong>of</strong><br />
Colorado at Denver and Health Sciences Center, Aurora, CO<br />
We reported that ACAIA is unchanged in classical/lectin<br />
pathway C4-deficient, mice but 90% decreased in alternative<br />
pathway factor-B-deficient mice (J. Immunol. 2006;<br />
177: 1904). We have further examined the role <strong>of</strong><br />
complement pathways in ACAIA using C57BL/6 mice<br />
genetically deficient in components C3 (C3−/−) or C1q<br />
(C1q−/−), deficient in both mannose-binding lectins A and<br />
C (MBL−/−), and doubly deficient in both C1q and MBL-A/C<br />
(C1q−/−/MBL−/−), with only the alternative pathway<br />
intact. Arthritis was induced by a mixture <strong>of</strong> 4 anti-type II<br />
collagen monoclonal antibodies, with an IP injection at day<br />
3 <strong>of</strong> LPS to cycle arthritis development. Mice were scored<br />
daily for arthritis in the paws using a scale <strong>of</strong> 0–3<br />
(maximum score <strong>of</strong> 12 per mouse) and sacrificed on day<br />
10. There were no significant differences in the clinical<br />
disease activity scores (CDAS) between C1q−/− mice and<br />
wild type (WT, normal C57BL/6 mice), MBL−/− and WT<br />
mice, or C1q−/−/MBL−/− and WT mice. In contrast, similar<br />
to factor B-deficient mice, CDAS in C3−/− mice were 66%<br />
decreased in comparison to WT mice (3.75 ±1.03 and 11.75<br />
±0.25, mean±SEM, n=4, respectively). The histological<br />
scores for inflammation, pannus formation, bone damage<br />
and cartilage damage, as well as immunohistochemical<br />
scores for the deposition <strong>of</strong> C3, were all consistent with the<br />
CDAS. These results indicate that the alternative pathway<br />
<strong>of</strong> complement is unique in being both necessary and<br />
sufficient for complement involvement in the effector<br />
phase <strong>of</strong> ACAIA. Potential mechanism(s) underlying this key<br />
role are under investigation.<br />
doi:10.1016/j.clim.2007.03.451<br />
S95<br />
Sa.64 Anti-Inflammatory Effect <strong>of</strong> Modified-<br />
Extracellular Matrix Proteins (MECMP) in CIA<br />
Perla Macip-Rodríguez, Physician, Instituto Nacional de<br />
Ciencias Medicas y Nutricion SZ, Department <strong>of</strong><br />
<strong>Immunology</strong> and Rheumatology, Mexico, Janette Furuzawa-<br />
Carballeda, Chemistry, Instituto Nacional de Ciencias<br />
Medicas y Nutricion SZ, Department <strong>of</strong> <strong>Immunology</strong> and<br />
Rheumatology, Mexico, Ma. Inés Vargas-Rojas, MD, Instituto<br />
Nacional de Ciencias Medicas y Nutricion SZ, Department <strong>of</strong><br />
<strong>Immunology</strong> and Rheumatology, Mexico, Virginia<br />
Soto-Abraham, Physician, Instituto Nacional de Cardiología<br />
ICh. Department <strong>of</strong> Pathology, Mexico, David Cruz-Robles,<br />
Biologist, Instituto Nacional de Cardiología ICh,<br />
Department <strong>of</strong> Pathology, Mexico, Leonardo Limón-<br />
Camacho, Physician, Instituto Nacional de Ciencias Medicas<br />
y Nutricion SZ, Department <strong>of</strong> <strong>Immunology</strong> and<br />
Rheumatology, Mexico<br />
The aim <strong>of</strong> this study was to evaluate the effect <strong>of</strong><br />
intradermal MECMP administration on a type II collagen<br />
(CII)-induced arthritis (CIA). Age-matched mice were<br />
immunized intradermally at the base <strong>of</strong> the tail with 100<br />
μg <strong>of</strong> chicken CII emulsified in complete Freund’s adjuvant.<br />
Mice were then boosted with 100 μg <strong>of</strong> CII in incomplete<br />
Freund’s adjuvant at 21 days. Mice were treated 1 week<br />
after boost with 100 μl <strong>of</strong> (a) placebo, (b) matrikines<br />
(hydrolyzed-collagen and elastin), (c) collagen-PVP, (d) b+c<br />
and (e) methotrexate (2.5 mg/kg) and every week during<br />
1 month. <strong>Clinical</strong> scores were assessed immediately before<br />
immunization (day 0) and thereafter weekly. Inflammation<br />
<strong>of</strong> the four paws was cored as follows: 0, no inflammation;<br />
1, swelling or redness <strong>of</strong> one joint; 2, swelling or redness <strong>of</strong><br />
more than one joint or mild inflammation <strong>of</strong> the whole<br />
paw; 3, severe inflammation <strong>of</strong> whole paw or ankylosis. The<br />
incidence <strong>of</strong> CIA was 100% by day 28 in CII challenged mice.
S96 Abstracts<br />
Weight and temperature were also determined. Macroscopical<br />
analysis showed a down-regulation <strong>of</strong> inflammation<br />
post administration <strong>of</strong> MECMP and methotrexate treatments.<br />
The histological analysis showed that CIA-mice<br />
group had an extensive bone erosion, pannus and severe<br />
focal inflammatory infiltrates. MECMP mice-treated group<br />
ameliorated the clinical signs. Bone erosion and inflammation<br />
were moderate compared to CIA-mice group. Concluding,<br />
MECMP exerts a down-regulation <strong>of</strong> inflammation on<br />
established inflammation and is able to ameliorate the<br />
tissue damage associated with CIA.<br />
doi:10.1016/j.clim.2007.03.452<br />
Sa.65 Established Murine Collagen-induced Arthritis<br />
and IL-17 Production are Specifically Inhibited by a<br />
Single Inoculation <strong>of</strong> Lipopolysaccharide-stimulated<br />
Dendritic Cells<br />
Juan C. Aguillon, Biochemist, Universidad de Chile,<br />
Santiago, Chile, Lorena Salazar, Biochemist, Universidad<br />
de Chile, <strong>Immunology</strong> Program, Santiago, Chile, Octavio<br />
Aravena, Biochemist, Universidad de Chile, <strong>Immunology</strong><br />
Program, Santiago, Chile, Carlos Gonzalez, DVM,<br />
Universidad de Chile, Facultad de Ciencias Veterinarias,<br />
Santiago, Chile, Paula Abello, DVM, Universidad de Chile,<br />
<strong>Immunology</strong> Program, Santiago, Juan Contreras-Levicoy,<br />
MD, Universidad de Chile, <strong>Immunology</strong> Program, Santiago,<br />
Chile, Barbara Pesce, Biochemist, Universidad de Chile,<br />
<strong>Immunology</strong> Program, Santiago, Chile, Flavi Salazar-<br />
Onfray, Biologist, Universidad de Chile, <strong>Immunology</strong><br />
Program, Santiago, Chile, Miguel Cuchacovich, MD,<br />
Hospital Clinico Universidad de Chile, Santiago, Chile<br />
Dendritic cells (DCs), crucial in immune responses, are<br />
also involved in peripheral tolerance induction. TNFmodulated<br />
DCs have demonstrated to restore tolerance<br />
in experimental multiple sclerosis and collagen induced<br />
arthritis (CIA). We investigated the capacity <strong>of</strong> short-term<br />
lipopolysaccharide (LPS)-stimulated DCs pulsed with type II<br />
collagen (CII) to induce tolerance against established CIA.<br />
Bone marrow-derived DCs were generated in the presence<br />
<strong>of</strong> GM-CSF. After CIA induction with CII, mice were<br />
injected at day 35 with a single dose <strong>of</strong> immature DCs, 4<br />
or 24 h LPS-stimulated DCs that had been loaded with CII<br />
(CII/DCs, 4 h LPS/CII/DCs, or 24 h LPS/CII/DCs). Arthritis<br />
progression was monitored by clinical and histologic<br />
evaluations. Flow cytometry <strong>of</strong> 4 h LPS/CII/DCs showed<br />
intermediate CD40 and CD86 expression, lower than that<br />
<strong>of</strong> 24 h LPS/CII/DCs (fully mature), and higher than that <strong>of</strong><br />
CII/DCs (immature). A functional assay showed that 4 h<br />
LPS/CII/DCs display increased endocytosis ability with<br />
respect to 24 h LPS/CII/DCs, indicating a semi-mature<br />
state. The single inoculation <strong>of</strong> 4 h LPS/CII/DCs in mice<br />
with established CIA reduced significantly disease severity<br />
at day 70. Histologic evaluation <strong>of</strong> mice treated with 4 h<br />
LPS/CII/DCs revealed diminished inflammatory synovitis,<br />
cartilage damage, and fibrosis. Unlike CII-stimulated<br />
splenocytes from CIA control mice, those from mice<br />
inoculated with 4 h LPS/CII/DCs showed diminished IL-17<br />
and increased IL-10 levels in culture supernatants. The<br />
high IL-10 production is consistent with the induction <strong>of</strong><br />
TR1 regulatory T cells responsible for inhibiting to effector<br />
IL-17-producing CD4+ T cells. Conclusion: Short-term LPSmodulated<br />
DCs inoculation interferes with CIA progression<br />
and IL-17 production when loaded with CII. Supported by<br />
Fondecyt-1070553, Millennium Nucleus-P04/030-F and<br />
Mecesup-UCH 0115.<br />
doi:10.1016/j.clim.2007.03.453<br />
Sa.66 Kinetics <strong>of</strong> Immune Tolerization in a <strong>Clinical</strong><br />
Trial on Epitope-specific Therapy in Rheumatoid<br />
Arthritis (RA)<br />
Eva K<strong>of</strong>feman, University <strong>of</strong> California San Diego,<br />
Department <strong>of</strong> Medicine and Pediatrics, Utrecht, The<br />
Netherlands, Diane Amox, University <strong>of</strong> California San<br />
Diego, Department <strong>of</strong> Medicine and Pediatrics, La Jolla, CA,<br />
Adriana Tremoulet, University <strong>of</strong> California San Diego,<br />
Department <strong>of</strong> Medicine and Pediatrics, La Jolla, CA, Elissa<br />
Keogh, University <strong>of</strong> California San Diego, Department <strong>of</strong><br />
Medicine and Pediatrics, La Jolla, CA, Peter Zeiseniss,<br />
University <strong>of</strong> California San Diego, Department <strong>of</strong> Medicine<br />
and Pediatrics, La Jolla, CA, Courtney Gosch, University <strong>of</strong><br />
California San Diego, Department <strong>of</strong> Medicine and<br />
Pediatrics, La Jolla, CA, Samantha Friend, University <strong>of</strong><br />
California San Diego, Department <strong>of</strong> Medicine and<br />
Pediatrics, La Jolla, CA, Xiao Zhang, University <strong>of</strong> Alabama<br />
at Birmingham, School <strong>of</strong> Public Health Department <strong>of</strong><br />
Biostatistics, Birmingham, AL, Gary Cutter, University <strong>of</strong><br />
Alabama at Birmingham, School <strong>of</strong> Public Health,<br />
Department <strong>of</strong> Biostatistics, Birmingham, AL, Salvatore<br />
Albani, Pr<strong>of</strong>essor, University <strong>of</strong> California San Diego,<br />
Department <strong>of</strong> Medicine and Pediatrics, La Jolla, CA<br />
Background: In a phase II trial on mucosal tolerization<br />
in 160 RA-patients, dnaJp1 (E. coli heat shock protein<br />
dnaJ-peptide) 25 mg 1dd for 168 days was safe and<br />
clinically effective. Objectives: (i) Study immunological<br />
kinetics underlying clinical response. (ii) Identify biological<br />
markers predicting clinical response. Methods: PBMCs<br />
collected on days 0, 112 and 168 were cultured with<br />
dnaJP1, Tetanus Toxoid and controls and FACS-stained for<br />
CD3 and intracellular cytokines. <strong>Clinical</strong> response is ≥1<br />
ACR20 (American College <strong>of</strong> Rheumatology) response<br />
between day 112 and 1-month follow-up. Results: High<br />
CD69 expression upon dnaJp1 in vitro on day 0 associated<br />
with later clinical amelioration (ACR20 112–168 p=0.06,<br />
ACR50 p=0.02). TNFa production to dnaJp1 decreased<br />
before day 112 (0–112 pb0.0001) in the dnaJp1 group,<br />
remaining low on day 168. In the clinical responders<br />
subgroup, trends were seen <strong>of</strong> an increase in IL10 (0–168<br />
p=0.09) and initial decrease (0–112 p=0.17) and late<br />
increase in IFNg (112–168 p=0.15). IFNg increase was<br />
associated with the IL10 increase in dnaJP1 patients<br />
(p=0.10). Reactions to Tetanus Toxoid and reactions in<br />
the placebo group did not change. Discussion: CD69<br />
expression to dnaJp1 in vitro may be a predictor <strong>of</strong><br />
clinical effect. This suggests that antigen-specific T cells<br />
play an important role in mucosal tolerization. Cytokine<br />
data suggest that tolerization started with deviation <strong>of</strong>
Abstracts<br />
antigen-specific T-effectors (TNFa decrease IFNg decrease,<br />
IL10 increase), followed by induction <strong>of</strong> regulatory T cell<br />
mechanisms (IL10+/IFNg+ Tr1) and clinical amelioration.<br />
doi:10.1016/j.clim.2007.03.454<br />
Sa.67 Nasal Co-Administration <strong>of</strong> ISS Increases the<br />
Arthritis-Protective Effect <strong>of</strong> an HSP60-derived<br />
Peptide<br />
Evelien Zonneveld-Huijssoon, University Medical Center<br />
Utrecht, Pediatric <strong>Immunology</strong>, Utrecht, The Netherlands,<br />
Femke van Wijk, University Medical Center Utrecht,<br />
Pediatric <strong>Immunology</strong>, Utrecht, The Netherlands, Wilco de<br />
Jager, Mr, University Medical Center Utrecht, Pediatric<br />
<strong>Immunology</strong>, Utrecht, The Netherlands, Sarah Roord,<br />
University Medical Center Utrecht, Pediatric <strong>Immunology</strong>,<br />
Utrecht, The Netherlands, Salvatore Albani, University <strong>of</strong><br />
California San Diego, Medicine and Pediatrics, La Jolla, CA,<br />
Wietse Kuis, University Medical Center Utrecht, Pediatric<br />
<strong>Immunology</strong>, Utrecht, The Netherlands, Berent Prakken,<br />
University Medical Center Utrecht, Pediatric <strong>Immunology</strong>,<br />
Utrecht, The Netherlands<br />
Peptide-based immunotherapy is a promising way to treat<br />
autoimmune diseases. In earlier studies we identified T cell<br />
epitopes derived from human and mycobacterial HSP60 that<br />
are recognized by a majority <strong>of</strong> patients with rheumatoid or<br />
juvenile idiopathic arthritis. Nasal administration <strong>of</strong> one <strong>of</strong><br />
these epitopes (p1) partially prevented adjuvant arthritis in<br />
the rat. To further enhance this arthritis-protective effect we<br />
tested two types <strong>of</strong> ISS and peptidoglycan (PGN) as adjuvants.<br />
Rats were treated with three nasal doses <strong>of</strong> p1, adjuvant or a<br />
combination <strong>of</strong> p1 and adjuvant prior to arthritis induction<br />
with CFA. Sixty days after arthritis induction, mandibular and<br />
inguinal lymph nodes and spleens were harvested. We<br />
determined phenotype, proliferative responses and cytokine<br />
production <strong>of</strong> fresh and in vitro stimulated cells. P1+ISS cotreatment<br />
enhanced the arthritis-protective effect <strong>of</strong> p1. ISS<br />
treatment alone did not have any effect on arthritis scores,<br />
nor did P1+PGN co-treatment or PGN alone. P1+ISS cotreatment<br />
led to increased p1-induced IFNg production by<br />
spleen cells. Co-treatment with one <strong>of</strong> the tested ISS led to<br />
increased IL-10 production as well. No clear differences could<br />
be detected between treatment groups in percentage <strong>of</strong> CD4<br />
+IL10+IFNg+ Tcells or CD4+CD25+Foxp3+ Tcells. <strong>Clinical</strong> data<br />
so far imply that ISS may be a suitable adjuvant for peptidespecific<br />
immune therapy in arthritis. Future plans include<br />
adoptive transfer <strong>of</strong> p1+ISS treated spleen cells and a further<br />
analysis <strong>of</strong> the induced p1 specific T cells and dendritic cells.<br />
doi:10.1016/j.clim.2007.03.455<br />
Sa.68 Abrogation <strong>of</strong> Autoimmune-mediated<br />
Inflammation by the Natural Plant Product,<br />
Honokiol via GABA(A)-mediated Alteration <strong>of</strong><br />
CD40 and LMP1 Signaling in B Cells<br />
Melissa E. Munroe, Postdoctoral Research Fellow, University<br />
<strong>of</strong> Iowa Department <strong>of</strong> Microbiology, Iowa City, IA, Gail A.<br />
Bishop, Pr<strong>of</strong>essor, University <strong>of</strong> Iowa Department <strong>of</strong> Internal<br />
Medicine and Microbiology and the VAMC, Iowa City, IA<br />
Our lab has extensively studied signaling in B cells by<br />
CD40 and its viral mimic, LMP1, which signals in an<br />
amplified and sustained manner, and has been implicated<br />
in pathogenesis <strong>of</strong> lymphoma and autoimmune disease.<br />
Honokiol (HNK), a phenolic compound isolated and purified<br />
from magnolia, has been found to have a number <strong>of</strong><br />
pharmacologic benefits, including anti-inflammatory properties,<br />
without the toxicity found in vivo with current<br />
treatments. HNK stabilized already established inflammatory<br />
arthritis, with a decrease in TNF-α, IL-6, and collagenspecific,<br />
pro-inflammatory mediated, antibodies. We evaluated<br />
potential molecular mechanisms leading to the antiinflammatory<br />
effects <strong>of</strong> HNK using B cell lines expressing<br />
either hCD40 or the hCD40-LMP1 chimeric receptor. CD40<br />
and LMP1 mediated NFκB and AP-1 pathways were<br />
abrogated, but not eliminated by HNK, with a dosedependent<br />
decrease in nuclear translocation <strong>of</strong> NFκB1,<br />
NFκB2, and AP-1 associated proteins. Furthermore, the<br />
anti-inflammatory effects <strong>of</strong> HNK could be reversed using<br />
inhibitors <strong>of</strong> the neurotransmitter GABA(A), a previously<br />
reported target for HNK interaction. We are currently<br />
investigating potential downstream mediators <strong>of</strong> GABA<br />
activation which may affect CD40 and LMP1 signaling,<br />
including kinases and proteosome function. These findings<br />
are particularly exciting and suggest that the nontoxic antiinflammatory<br />
properties <strong>of</strong> HNK could be a means for<br />
blocking the autoimmune response.<br />
doi:10.1016/j.clim.2007.03.456<br />
S97<br />
Sa.69 Reversal <strong>of</strong> CD4:CD8 Ratio in Pediatric<br />
Patients with Macrophage Activation Syndrome/<br />
Reactive Hemophagocytic Lymphohistiocytosis<br />
(MAS/HLH): A Potential Marker for Active Disease<br />
Process<br />
Paivi Miettunen, Doctor, Division <strong>of</strong> Pediatric Rheumatology<br />
and University <strong>of</strong> Calgary, Calgary, Alberta, AB, Canada<br />
Background: MAS/HLH in pediatric rheumatology refers<br />
to excessive activation and proliferation <strong>of</strong> mature macrophages<br />
leading to an overwhelming inflammatory reaction.<br />
While abnormal T cell activation (TCA) has been postulated<br />
to be at the center <strong>of</strong> this process, the details <strong>of</strong> the<br />
immunological mechanisms that initiate and maintain the<br />
abnormal macrophage functions are unclear. Objective: To<br />
test the hypothesis that alterations in CD4:CD8 ratios<br />
would provide an early marker for abnormal TCA in MAS/<br />
HLH. Methods: All patients met Histiocytosis Society’s 2004<br />
criteria for HLH. Peripheral blood was collected at the time<br />
<strong>of</strong> diagnosis and at remission (n=7, 4 F; 3 M: ages 4 to 16<br />
years). Blood from age and sex matched healthy children<br />
was used as controls. Peripheral blood CD4:CD8 ratios were<br />
analyzed by flow cytometry. Results: Significant reversal <strong>of</strong><br />
CD4:CD8 ratio was seen in four <strong>of</strong> seven patients with<br />
active MAS/HLH. An infectious process was ruled out<br />
(negative HIV, EBV and CMV serology). At the time <strong>of</strong>
S98 Abstracts<br />
remission CD4:CD8 ratio had normalized. In one patient<br />
with reactivation <strong>of</strong> MAS/HLH, a reversal <strong>of</strong> the CD4:CD8<br />
ratio recurred, and normalized with cyclosporin, corticosteroids<br />
and interleukin-1 antagonist, anakinra treatment.<br />
Conclusions: Our preliminary studies indicate that reversal<br />
<strong>of</strong> CD4:CD8 can be an important marker for active MAS/<br />
HLH in a significant proportion <strong>of</strong> patients, supporting role<br />
<strong>of</strong> abnormal T cell activation. CD4:CD8 ratio can also be<br />
used to follow treatment response in selected patients.<br />
Studies are in progress to identify additional immunological<br />
markers to diagnose this condition accurately and to<br />
delineate the potential mechanisms involved in this<br />
process.<br />
doi:10.1016/j.clim.2007.03.457<br />
Sa.70 Quantitative Biomarker Analysis <strong>of</strong> Anti-CCP,<br />
Rheumatoid Factor (RF) and Immunoglobulin Levels<br />
in Synovial Biopsies Indicate Enrichment and Local<br />
Production in Rheumatoid Arthritis (RA)<br />
Sanna Rosengren, Associate Project Scientist, University <strong>of</strong><br />
California, San Diego, Medicine, La Jolla, CA, Nathan Wei,<br />
<strong>Clinical</strong> Director, Arthritis and Osteoporosis Center,<br />
Frederick, MD, David Boyle, Associate Research<br />
Immunologist, University <strong>of</strong> California, San Diego,<br />
Medicine, La Jolla, CA, Nathan Zvaifler, Pr<strong>of</strong>essor Emeritus,<br />
University <strong>of</strong> California, San Diego, Medicine, La Jolla, CA<br />
Circulating autoantibodies such as RF and anti-CCP are<br />
associated with RA. B cells and plasma cells accumulate in RA<br />
synovium, yet their contribution to autoantibody production<br />
is unclear. We developed ELISA-based methods to quantify<br />
autoantibodies and total Ig (immunoglobulin) G and IgM in<br />
extracts <strong>of</strong> RA and osteoarthritis synovial tissues prepared<br />
using 1% Brij-35 in PBS. Synovial tissue and matched serum<br />
were obtained at arthroplasty or arthroscopic biopsy from RA<br />
(n=11) and OA (n=6) patients. RF-IgM, anti-CCP and antitetanus<br />
IgG, total IgM and IgG, and albumin were determined<br />
by ELISA in synovial extracts and serum. Specific activity was<br />
calculated as the ratio <strong>of</strong> synovial to serum antibody/<br />
albumin. IgG1 and IgM heavy constant mRNA levels were<br />
determined by qPCR as a measure <strong>of</strong> synovial Ig synthesis.<br />
Interestingly, anti-CCP IgG, but not RF-IgM, was significantly<br />
enriched in RA synovia compared to serum. OA synovial<br />
extracts contained no detectable autoantibodies. Total IgG<br />
specific activity was significantly higher in RA synovia than in<br />
OA (median (range): RA 2.58 (1.22–2.99); OA 0.76 (0.73–<br />
0.87), p=0.0077). Similar results were obtained for total IgM<br />
and tetanus IgG. Notably, mRNA content and specific activity<br />
correlated significantly for both IgM and IgG. Synovia<br />
containing lymphocyte aggregates (n=3) contained significantly<br />
higher total IgG but not total IgM. In conclusion,<br />
correlations between immunoglobulin message and protein<br />
indicate that local synthesis contributes to synovial Ig<br />
content. Reliable determinations <strong>of</strong> autoantibodies, antitetanus<br />
IgG, and total IgM and IgG in synovial biopsy extracts<br />
will be <strong>of</strong> value in pro<strong>of</strong>-<strong>of</strong>-concept clinical trials.<br />
doi:10.1016/j.clim.2007.03.458<br />
Sa.71 Thrombin-activatable Carboxypeptidase B<br />
Prevents Anti-Collagen Antibody Induced Arthritis<br />
Jason Song, Rheumatology Fellow, Department <strong>of</strong> Medicine<br />
Division <strong>of</strong> Rheumatology Stanford University, Palo Alto,<br />
CA, Toshihiko Nishimura, Life Science Research Assistant,<br />
Department <strong>of</strong> Medicine, Division <strong>of</strong> Pulmonary and Critical<br />
Care Medicine, Stanford University, Menlo Park, CA, Michael<br />
Garcia, Undergraduate Student, Department <strong>of</strong> Medicine<br />
Division <strong>of</strong> Rheumatology Stanford University, Palo Alto,<br />
CA, Timothy Myles, Senior Research Scientist, Department<br />
<strong>of</strong> Medicine, Division <strong>of</strong> Hematology, Stanford University,<br />
Stanford, CA, Peggy Ho, Basic Life Science Research<br />
Associate, Department <strong>of</strong> Neurology, Stanford University,<br />
Stanford, CA, Xiaoyan Du, Post Doc, Department <strong>of</strong><br />
Medicine, Division <strong>of</strong> Hematology, Stanford University,<br />
Stanford, CA, Shadi Sharif, Life Science Research Assistant,<br />
Department <strong>of</strong> Medicine, Division <strong>of</strong> Hematology, Stanford<br />
University, Stanford, CA, Lawrence Leung, Pr<strong>of</strong>essor <strong>of</strong><br />
Medicine, Department <strong>of</strong> Medicine, Division <strong>of</strong> Hematology,<br />
Stanford University, Stanford, CA, William Robinson,<br />
Assistant Pr<strong>of</strong>essor in Medicine, Department <strong>of</strong> Medicine<br />
Division <strong>of</strong> Rheumatology Stanford University, Palo Alto, CA<br />
Thrombin-activatable carboxypeptidase B (CPB) is well<br />
established to play an anti-fibrinolytic role by removing Cterminal<br />
lysine residues from fibrin, thereby preventing its<br />
cleavage by plasmin. Recently, C3a, C5a, thrombin-cleaved<br />
osteopontin and bradykinin have been identified as additional<br />
substrates <strong>of</strong> CPB. CPB removes arginine residues from<br />
the C-terminus <strong>of</strong> these inflammatory mediators, thereby<br />
altering the biologic functions. We investigated the role <strong>of</strong><br />
CPB in arthritis using proCPB deficient mice. Arthritis was<br />
induced by an intravenous injection <strong>of</strong> anti-collagen antibodies<br />
on day 0, followed by intraperitoneal injection <strong>of</strong><br />
lipopolysaccharide on day 3. Arthritis was assessed by visual<br />
score and paw thickness. On day 10 post-injection, joints<br />
were collected for histology. Arthritis was also studied in<br />
bradykinin receptor−/− mice and C5−/− mice. As compared<br />
to controls, proCPB−/− mice exhibited significantly more<br />
severe arthritis (on day 10 arthritis score 12.3 vs. 0.4*; paw<br />
thickness 3.02 vs. 2.21 mm*, *pb0.01). Histology, graded 0–<br />
4, demonstrated severe arthritis in proCPB −/− mice versus<br />
controls (synovitis 3.7 vs. 1.2*, pannus 2.8 vs. 1.1*, bone<br />
erosion 3.2 vs. 0.7*, *pb0.01). C5−/− mice were resistant to<br />
induction <strong>of</strong> arthritis, while bradykinin receptor−/− mice<br />
exhibited a similar severity <strong>of</strong> arthritis to controls. These<br />
results suggest that CPB plays a critical role in regulating<br />
inflammation in anti-collagen antibody induced arthritis,<br />
and that this effect could be in part mediated by its downregulation<br />
<strong>of</strong> C5a activity. Studies are underway to determine<br />
the impact <strong>of</strong> CPB on C5a, C3a, and thrombin-cleaved<br />
osteopontin in the pathogenesis <strong>of</strong> this arthritis model.<br />
doi:10.1016/j.clim.2007.03.459<br />
Sa.72 Kinetics <strong>of</strong> Cytokine Gene Expression After<br />
Antigen Stimulation in Frozen Human PBMCs Using<br />
Quantitative Real-Time PCR and Flow Cytometry<br />
Annick van de Ven, MD Student, University <strong>of</strong> California, San<br />
Diego, La Jolla, CA, Pediatrics/University <strong>of</strong> Utrecht, The
Abstracts<br />
Netherlands, Elissa Keogh, Senior Research Associate,<br />
Supervisor, University <strong>of</strong> California, San Diego, La Jolla, CA,<br />
Negar Ghahramani, Staff Research Associate, Pediatrics,<br />
University <strong>of</strong> California, San Diego, La Jolla, CA, Salvatore<br />
Albani, Pr<strong>of</strong>essor <strong>of</strong> Pediatrics, University <strong>of</strong> California, San<br />
Diego, Department <strong>of</strong> Pediatrics, La Jolla, CA<br />
Background: Understanding <strong>of</strong> antigen-induced kinetics <strong>of</strong><br />
gene expression facilitates the analysis <strong>of</strong> immune responses<br />
using RT-PCR and DNA microarrays. Therefore, cytokine<br />
expression was investigated using 2 distinct antigenic stimuli:<br />
a common recall antigen (influenza peptide HA307–319) and<br />
the peptide epitope dnaJP1 associated with the pathogenesis<br />
<strong>of</strong> rheumatoid arthritis. Method: Frozen PBMCs from 5<br />
dnaJP1-responsive RA patients and fresh and frozen PBMCs<br />
from one healthy, influenza-vaccinated donor were stimulated<br />
for up to 96 hours with or without dnaJP1 and HA307–<br />
319, respectively. At designated intervals, aliquots were<br />
removed for measurement <strong>of</strong> cytokines by TaqMan. Additionally,<br />
intracellular IL-2 and TNFα were measured by FACS.<br />
Results: Following dnaJP1 stimulation, both IL-2 and IFNγ<br />
expressions were detectable by TaqMan at 12–36 hours while<br />
TNFα was undetectable. Intracellular IL-2 exhibited a<br />
biphasic pr<strong>of</strong>ile (48 and 96 hours) and TNFα expression was<br />
detected at 48 hours. It should be noted that gene expression<br />
at 6 hours was highly nonspecific. When fresh PBMCs were<br />
stimulated with HA307–319, IL-2 expression was biphasic (24<br />
and 48 hours) while TNFα and INFγ peaked at 12 and 48 hours,<br />
respectively. Gene expression in frozen PBMCs appeared to be<br />
delayed by about 12 hours. Conclusions: Peptide antigen<br />
stimulation induces cytokine expression more slowly than<br />
that observed for mitogen stimulation. Gene expression also<br />
depends, to some extent, on the gene <strong>of</strong> interest. As<br />
expected, intracellular detection follows gene expression<br />
by 24–36 hours. Additionally, PBMCs require a 12 hour<br />
recovery period after cryopreservation.<br />
doi:10.1016/j.clim.2007.03.460<br />
Sa.73 Epitope-specific Immune Tolerance Can<br />
Maintain the Disease Control Induced by<br />
Conventional Methotrexate—Etanercept<br />
Combination Therapy in Rat Adjuvant Arthritis<br />
Negar Ghahramani, Staff Research Associate, University <strong>of</strong><br />
California, San Diego, Department <strong>of</strong> Pediatrics, La Jolla,<br />
CA, Elissa Keogh, Staff Research Associate, Supervisor,<br />
University <strong>of</strong> California, San Diego Department <strong>of</strong><br />
Pediatrics, La Jolla, CA, Annick van de Ven, MD Student,<br />
University <strong>of</strong> California, San Diego Pediatrics/University <strong>of</strong><br />
Utrecht, The Netherlands, La Jolla, CA, Salvatore Albani,<br />
Pr<strong>of</strong>essor, University <strong>of</strong> California, San Diego, Department<br />
<strong>of</strong> Pediatrics, La Jolla, CA<br />
Tools to maintain remission that allow tapering from<br />
aggressive therapies are still lacking for the majority <strong>of</strong><br />
patients with rheumatoid arthritis. The success for longterm<br />
disease control with minimal background therapy<br />
could lie in the ability to establish tolerance. This approach<br />
can be complementary to the blend <strong>of</strong> blockade and<br />
suppression which characterizes current therapies. We<br />
have demonstrated that in rats with adjuvant arthritis<br />
(AA), mucosal tolerization to an arthritogenic peptide<br />
(hsp60 p180–188) can lead to significant improvement and<br />
immune deviation. In this study, the effect <strong>of</strong> nasal hsp60<br />
p180–188 peptide treatment (0.1 mg, days 10, 13 and 16) in<br />
combination with oral methotrexate (0.3 mg/kg, days 9 and<br />
13) and s.c. etanercept (0.3 mg/kg, day 9) was compared<br />
with standard etanercept–methotrexate treatment regimen.<br />
Results clearly indicate that the combination <strong>of</strong><br />
mucosal tolerization to hsp60 p180–188 with single dose<br />
<strong>of</strong> etanercept can lead to significant disease control, to a<br />
degree comparable to full dose etanercept and tangibly<br />
better than the other treatment regimens (average clinical<br />
improvement <strong>of</strong> 42.3% for hsp60 p180–188 +single dose<br />
etanercept+methotrexate, 36.7% for hsp60 p180–188+<br />
methotrexate; and 14% for full dose etanercept–methotrexate<br />
therapy). The marked increase in the percentage <strong>of</strong> CD4<br />
+ CD25hiFoxp3+ T cells following epitope-specific combination<br />
therapy suggested induction <strong>of</strong> Treg cells and restoration<br />
<strong>of</strong> tolerance. Such changes were restricted to the<br />
draining site <strong>of</strong> the nasal mucosa <strong>of</strong> animals receiving the<br />
combined treatment.<br />
doi:10.1016/j.clim.2007.03.461<br />
S99<br />
Sa.74 Independent Signals Within the MHC are<br />
Associated with Systemic Lupus Erythematosus—A<br />
Family-based SNP Study<br />
Michelle Fernando, ARC <strong>Clinical</strong> Research Fellow, Imperial<br />
College London, Section <strong>of</strong> Molecular Genetics and<br />
Rheumatology, London, England, Christine Stevens, Project<br />
Coordinator, The Broad Institute <strong>of</strong> MIT and Harvard,<br />
Inflammatory Genetics Group, Cambridge, MA, Emily Walsh,<br />
Cancer Research Institute Postdoctoral Fellow, Broad<br />
Institute <strong>of</strong> MIT and Harvard, Cambridge, MA, Todd Green,<br />
Project Coordinator, Program in Medical and Population<br />
Genetics, Broad Institute <strong>of</strong> Harvard and MIT, Cambridge,<br />
MA, Pardis Sabeti, Postdoctoral Fellow, Infectious Disease<br />
Initiative, The Broad Institute <strong>of</strong> MIT and Harvard,<br />
Cambridge, MA, John Rioux, Canada Research Chair in<br />
Genetics and Genomic Medicine <strong>of</strong> Inflammation, Universite<br />
de Montreal, Montreal Heart Institute, Montreal, QC,<br />
Canada, Tim Vyse, Reader and Honorary Consultant in<br />
Rheumatology/Medicine, Imperial College London, Section<br />
<strong>of</strong> Molecular Genetics and Rheumatology, London, England<br />
The major histocompatibility complex (MHC) has been<br />
associated with SLE in numerous case–control studies. The<br />
main associated alleles are HLA-DR3 (0301) and HLA-DR2<br />
(1501) and their respective haplotypes in white Caucasian<br />
populations. MHC class III alleles have also shown association<br />
with lupus but extensive linkage disequilibrium (LD) in<br />
the region has prevented the discovery <strong>of</strong> the causal<br />
alleles. We therefore set out to investigate whether we<br />
could delineate separate class II and class III signals in our<br />
UK SLE cohort using a family-based SNP association study.<br />
67 SNPs were successfully typed using the Sequenom<br />
MassARRAYTM platform across 1.7 mb <strong>of</strong> genome (HLA-B<br />
to HLA-DPA2) in 314 Caucasian UK SLE trios. 4-digit HLA-<br />
DRB1 typing was performed using oligo-hybridization. Our
S100 Abstracts<br />
findings confirm previously published data <strong>of</strong> an association<br />
with HLA-DRB1*0301 (p=2.1×10 −8 ). We find no association<br />
with HLA-DRB1*1501 or HLA-DRB1*0801. 15 out <strong>of</strong> 67 SNP<br />
markers show evidence <strong>of</strong> association with WHAP TDT<br />
(permutated pb0.05, 10,000 permutations). Conditional<br />
analysis (WHAP) reveals association signals independent <strong>of</strong><br />
HLA-DRB1*0301 in class I (HLA-B) and class III (BAT3,<br />
SLC44A4, RDBP) with the latter signal being the strongest.<br />
These independent markers are not in LD with any other<br />
HLA-DRB1 allele (TRANSMIT) but do arise from the B8-<br />
DRB1*0301 extended haplotype shown by Extended Haplotype<br />
Homozygosity analysis (SWEEP).<br />
HLA-DRB1*0301 is associated with SLE in our UK cohort.<br />
We have also demonstrated a separate class III signal arising<br />
from recombination on the B8-DRB1*0301 extended haplotype.<br />
The haplotype block from which the class III signal<br />
arises encompasses the complement C4-containing RCCX<br />
module.<br />
doi:10.1016/j.clim.2007.03.462<br />
Sa.75 Quantitation <strong>of</strong> Total and Unoccupied BAFF-R<br />
in SLE<br />
Seth Knight, University <strong>of</strong> Alabama, Birmingham,<br />
Department <strong>of</strong> Medicine, Birmingham, AL, Hong Zhao,<br />
University <strong>of</strong> Alabama, Birmingham, Department <strong>of</strong><br />
Medicine, Birmingham, AL, Tong Zhou, University <strong>of</strong><br />
Alabama, Birmingham, Department <strong>of</strong> Medicine,<br />
Birmingham, AL, Robert Carter, University <strong>of</strong> Alabama,<br />
Birmingham, Department <strong>of</strong> Medicine, Birmingham, AL<br />
Background: Systemic lupus erythematosus (SLE) is an<br />
autoimmune disease characterized by abnormally active B<br />
lymphocytes. B lymphocyte stimulator (BLyS) has been<br />
shown to be an important regulator <strong>of</strong> B cell activity in SLE.<br />
The serum concentration <strong>of</strong> BLyS is increased in SLE<br />
patients, and in mice, increased BLyS induces a lupus-like<br />
syndrome. We have previously demonstrated that BAFF-R,<br />
the primary receptor for BlyS on B cells, is occupied to a<br />
greater extent on PBMCs in SLE patients than in healthy<br />
controls. We have now developed a robust assay for<br />
measuring total BAFF-R and occupied BAFF-R using quantitative<br />
flow cytometry. Methods/Results: Anti-BAFF-R monoclonal<br />
Ab-producing hybridoma clones were screened by<br />
ELISA. We identified clone 4A4 and performed a BLyS<br />
competition assay on tonsillar B cells and show that BlyS<br />
and 4A4 Ab competitively bind to BAFF-R. 4A4 Ab and a<br />
second anti-BAFF-R Ab (11C1) were then used to quantitate<br />
total and occupied BAFF-R on peripheral blood B cells in SLE<br />
and control cohorts using bead-based, quantitative flow<br />
cytometry to derive absolute values for antibody binding<br />
based on a standard curve. We show that both total and<br />
unoccupied BAFF-R are decreased on peripheral blood B<br />
cells in SLE patients, in some cases to 10% <strong>of</strong> healthy<br />
controls. These data demonstrate a more robust system for<br />
BAFF-R analysis in SLE and confirm the engagement <strong>of</strong><br />
BAFF-R in most lupus subjects.<br />
doi:10.1016/j.clim.2007.03.464<br />
Sa.76 Role <strong>of</strong> CD8+ Ti cells in Suppression <strong>of</strong><br />
Autoimmunity Depends on Foxp3 Expression<br />
Ram Singh, Assistant Pr<strong>of</strong>essor, David Geffen School <strong>of</strong><br />
Medicine at University <strong>of</strong> California, Los Angeles, Los<br />
Angeles, CA, Antonio La Cava, Associate Pr<strong>of</strong>essor, David<br />
Geffen School <strong>of</strong> Medicine at University <strong>of</strong> California, Los<br />
Angeles, Medicine, Los Angeles, CA, Bevra Hahn, Pr<strong>of</strong>essor<br />
and Chief, David Geffen School <strong>of</strong> Medicine at University <strong>of</strong><br />
California, Los Angeles (Medicine), Los Angeles, CA<br />
Systemic lupus erythematosus (SLE) is an autoimmune<br />
disease caused by autoantibodies including IgG anti-DNA.<br />
NZB/NZW Fl female (BWF1) mice, a model <strong>of</strong> spontaneous<br />
polygenic SLE, tolerized with an artificial peptide (pCONSEN-<br />
SUS, pCons) based on anti-DNA IgG sequences containing MHC<br />
class I and class iI T cell determinants, develop regulatory<br />
CD4+CD25+ T cells and inhibitory CD8+ T cells (CD8+ Ti), both<br />
<strong>of</strong> which suppress autoAb production. In the present study,<br />
using various cellular and molecular approaches, we show that<br />
inhibitory function <strong>of</strong> CD8+ T cells from tolerized mice is<br />
sustained for up to 8 weeks and at all times depends on<br />
expression <strong>of</strong> Foxp3. Both CD28-positive and -negative CD8+ T<br />
cells contain inhibitory cells, but the expression <strong>of</strong> Foxp3 and<br />
<strong>of</strong> mRNA for TGFb is higher and lasts longer in the CD28−<br />
subset. In vitro addition <strong>of</strong> TGFb (in the presence <strong>of</strong> IL-2)<br />
induces Foxp3 expression in a dose–response manner. Gene<br />
inhibition or blockade with siRNA <strong>of</strong> Foxp3 abrogates the<br />
ability <strong>of</strong> the CD8+ Ti to inhibit anti-DNA production and the<br />
proliferation <strong>of</strong> CD4+ helper T cells. Moreover, we found a<br />
significant correlation between expression <strong>of</strong> Foxp3 and<br />
ability <strong>of</strong> CD8+ Ti cells to secrete TGFb. Therefore, CD8+ Ti<br />
in this system <strong>of</strong> tolerance is dependent on expression <strong>of</strong><br />
Foxp3, and there may be a bi-directional Foxp3/TGFb<br />
autocrine loop that determines the ability <strong>of</strong> the CD8+ T<br />
cells to control autoimmunity. Supported by NIH grants.<br />
R37 AI 46776, AI63515AR53239, Tina C Foundation, and<br />
gifts from Jeanne Rappaport and the Horchow Family<br />
Trust.<br />
doi:10.1016/j.clim.2007.03.465<br />
Sa.78 T Cell Epitope Molecular Mimicry Can Initiate<br />
Immune Response Against Lupus-associated SmD<br />
Autoantigen<br />
Davis Sim, MD/PhD Student, University <strong>of</strong> Virginia Division<br />
<strong>of</strong> Rheumatology and <strong>Immunology</strong>, Charlottesville, VA,<br />
Govind Rajagopolan, Research Associate, Mayo Clinic<br />
Department <strong>of</strong> <strong>Immunology</strong>, Rochester, MN, Shu Man Fu,<br />
Pr<strong>of</strong>essor, University <strong>of</strong> Virginia Division <strong>of</strong> Rheumatology<br />
and <strong>Immunology</strong>, Charlottesville, VA, Chella David,<br />
Pr<strong>of</strong>essor, Mayo Clinic Department <strong>of</strong> <strong>Immunology</strong>,<br />
Rochester, MN, Umesh Deshmukh, Assistant Pr<strong>of</strong>essor,<br />
University <strong>of</strong> Virginia Division <strong>of</strong> Rheumatology and<br />
<strong>Immunology</strong>, Charlottesville, VA<br />
Autoantibodies reactive with Sm proteins are specific<br />
for systemic lupus erythematosus. One <strong>of</strong> the hypotheses<br />
behind the initiation <strong>of</strong> an immune response against Sm<br />
autoantigens is molecular mimicry between foreign and<br />
self proteins. However, clear evidence for this at T cell
Abstracts<br />
level is lacking. This study was designed to determine the<br />
role <strong>of</strong> T cell epitope mimicry in the initiation <strong>of</strong> immune<br />
response against SmD1.Using HLA-DR3 transgenic mice as<br />
experimental model system, a dominant T cell epitope<br />
was identified between amino acid SmD71 and 95. Using a<br />
T cell hybridoma (C1P2) reactive against this epitope,<br />
critical amino acid residues were localized within SmD81–<br />
88. Screening <strong>of</strong> databases with pattern recognition and<br />
MHC binding s<strong>of</strong>tware identified 20 putative peptide<br />
mimics. Among these, a peptide from galactoside ABC<br />
transporter protein from Vibrio activated C1P2. Immunization<br />
<strong>of</strong> HLA-DR3 mice with this peptide induced both T and<br />
B cell responses against SmD. To further characterize T<br />
cell responses against this mimic, additional T cell<br />
hybridomas were generated. Interestingly a T hybridoma<br />
P20 not only reacted with SmD79–93 and the Vibrio<br />
peptide but also with peptides from a human La-related<br />
protein and methyltransferase protein from Streptococcus<br />
agalactiae. TCR characterization <strong>of</strong> both C1P2 and P20<br />
revealed different V 2 usage, V 2 8.3 and V 2 8.1 respectively.<br />
Our study demonstrates the potential <strong>of</strong> T cell<br />
epitope mimicry to initiate autoimmune responses against<br />
lupus-associated antigens. In addition our data suggest<br />
that based on the TCR usage, different T cells reactive<br />
against the same region <strong>of</strong> an autoantigen can be<br />
activated by multiple molecular mimics.<br />
doi:10.1016/j.clim.2007.03.466<br />
Sa.79 Autoantibody Onset Prior to SLE Diagnosis<br />
Follows Predictable Patterns <strong>of</strong> Development<br />
Latisha Heinlen, Graduate Student, Oklahoma Medical<br />
Research Foundation, Oklahoma City, OK, Micah McClain,<br />
Scientist, Oklahoma Medical Research Foundation,<br />
Oklahoma City, OK, Tony Prestigiacomo, Scientist, BioRad<br />
Laboratories, Benecia, CA, Ben Bruner, Graduate Student,<br />
Oklahoma Medical Research Foundation, Oklahoma City,<br />
OK, Steven Binder, Scientist, Bio-Rad Laboratories,<br />
Benecia, CA, Colin Edgerton, Chief, Rheumatology Service,<br />
US Army Center for Health Promotion and Prevention,<br />
Washington, DC, Mark Rubertone, Active Duty Military, US<br />
Army Center for Health Promotion and Preventive<br />
Medicine, Washington, DC, Judith James, Member,<br />
Oklahoma Medical Research Foundation, Oklahoma City,<br />
OK, John Harley, Member, Oklahoma Medical Research<br />
Foundation, Oklahoma City, OK<br />
Systemic lupus erythematosus (SLE) is a clinically<br />
diverse humoral autoimmune disorder. We sought to<br />
determine the earliest known protein autoantigens targeted<br />
prior to SLE diagnosis, and to define the order in<br />
which specific protein autoantibodies arise. Previously<br />
collected and stored serum samples were obtained from<br />
the United States Department <strong>of</strong> Defense Serum Repository.<br />
All available patient and selected control samples were<br />
tested for autoantibodies to lupus autoantigens. Using<br />
these prospective samples we identified patterns <strong>of</strong><br />
autoantibody onset. In 33 patients the initial single<br />
autoantibody specificities were identified and were most<br />
commonly against 60 kDa Ro, and nRNP A. Surprisingly,<br />
none <strong>of</strong> these patients initiated lupus autoimmunity with<br />
anti-nRNP 70K or anti-Sm. Also, no patient had antibodies to<br />
nRNP 70K without antibodies to nRNP A. Anti-A appeared<br />
before or simultaneously with anti-70K in 96% <strong>of</strong> patients that<br />
developed both under observation. Anti-60 kDa Ro antibodies<br />
appeared before or simultaneously with anti-La or anti-52kD<br />
Ro in nearly all patients (98% and 95% respectively). These<br />
data suggest that the autoantibody response in SLE begins<br />
simply, <strong>of</strong>ten with a single protein years before disease onset,<br />
followed by the accumulation <strong>of</strong> additional autoantigenic<br />
specificities in partly predictable patterns.<br />
doi:10.1016/j.clim.2007.03.467<br />
Sa.80 Osteopontin Gene Polymorphism and<br />
Systemic Lupus Erythematosus in Chinese<br />
Maida Wong, <strong>Clinical</strong> Fellow, University <strong>of</strong> California, Los<br />
Angeles, School <strong>of</strong> Medicine, Los Angeles, CA, Joseph Yeung,<br />
Research Associate, University <strong>of</strong> Hong Kong, Faculty <strong>of</strong><br />
Medicine, Hong Kong, Chak-Sing Lau, Pr<strong>of</strong>essor, University<br />
<strong>of</strong> Hong Kong, Faculty <strong>of</strong> Medicine, Hong Kong<br />
Osteopontin (OPN) is a secreted glycosylated and phosphorylated<br />
extracellular matrix protein that influences autoimmune<br />
disease. Its serum level is known to be elevated in<br />
patients with systemic lupus erythematosus (SLE). In this<br />
study, we analyzed a minor Tallele (C707T SNP, corresponding<br />
to rs1126616) <strong>of</strong> OPN in Chinese SLE patients and healthy<br />
controls by polymerase chain reaction. Serum OPN levels were<br />
measured by ELISA, and its correlation with OPN polymorphism<br />
was analyzed statistically based on their genotypes, renal<br />
involvement, and disease activity (active disease defined as<br />
SLEDAI = or > 4). 162/253 SLE patients and 91/168 controls had<br />
one or more allelic mutations at 707T <strong>of</strong> the OPN gene. OPN<br />
mutation is associated with the development <strong>of</strong> SLE, OR = 1.51<br />
(95% CI 1.012-2.253). The OPN genotype is also associated with<br />
SLE (p=0.0003). Serum OPN is elevated in SLE patients with<br />
history <strong>of</strong> renal disease, irrespective <strong>of</strong> their disease activity<br />
(pb0.005 by ANOVA and post-hoc test, p= 0.013 by Mann-<br />
Whitney U test). There was no association between the allelic<br />
mutation and CNS disease, hematological disorders, malar or<br />
discoid rash, low complements, or elevated dsDNA antibody.<br />
The C707Tallelic frequency <strong>of</strong> the control population followed<br />
the Hardy-Weinberg equilibrium. Although the allelic frequencies<br />
between SLE patients (0.340) and controls (0.327) were<br />
not significantly different, they were significantly higher than<br />
the healthy Caucasian controls as published by Forton et al<br />
(p=0.004), which may explain the higher prevalence <strong>of</strong> SLE<br />
in the Chinese population, especially those with renal<br />
involvement.<br />
doi:10.1016/j.clim.2007.03.468<br />
S101<br />
Sa.81 Quantitative and Qualitatively Normal<br />
Regulatory T Cells are Not Capable <strong>of</strong> Inducing<br />
Suppression in SLE Patients Due to T Cell Resistance<br />
María Inés Vargas-Rojas, Medical Doctor, Instituto Nacional<br />
de Ciencias Médicas y Nutrición Salvador Zubiran/
S102 Abstracts<br />
Rheumatology and <strong>Immunology</strong>, Mexico, José Carlos Crispín,<br />
Medical Doctor, Instituto Nacional de Ciencias Médicas y<br />
Nutrición Salvador Zubiran/Rheumatology and <strong>Immunology</strong>,<br />
Mexico, Jorge Alcocer-Varela, Medical Doctor, Instituto<br />
Nacional de Ciencias Médicas y Nutrición Salvador Zubiran/<br />
Rheumatology and <strong>Immunology</strong>, Mexico<br />
Regulatory T cells (Treg) are considered an essential<br />
component <strong>of</strong> the mechanisms that protect and maintain<br />
peripheral tolerance. Their absence leads inevitably to lifethreatening<br />
autoimmune disease. Numerous works have<br />
studied their numbers and behavior in patients with<br />
autoimmune conditions; several defects that include diminished<br />
numbers and lack <strong>of</strong> suppressive capacity have been<br />
documented in various conditions including systemic lupus<br />
erythematosus. The aim <strong>of</strong> this study was to further<br />
characterize the regulatory defect documented in Treg <strong>of</strong><br />
patients with SLE. Twenty patients with SLE (10 with active<br />
disease) and 20 age- and sex-matched controls were<br />
included. Patients were not receiving treatment. Treg<br />
cells, defined as CD4+Foxp3+, were quantified by flow<br />
cytometry. CD4+CD25+ and CD4+CD25− cells were isolated<br />
by magnetic beads. Suppressive capacity was quantified in<br />
autologous and allogeneic co-cultures. No quantitative<br />
difference was found in Treg numbers between patients<br />
and controls (2.8±2.1 vs. 3.2±2.2, respectively). Suppression<br />
<strong>of</strong> cell proliferation was not observed in autologous cocultures<br />
<strong>of</strong> SLE cells. Conversely, normal Treg greatly<br />
suppressed autologous T cell proliferation (∼80%). Surprisingly,<br />
when SLE-derived Treg were added to healthy T cell<br />
cultures suppression was observed. In contrast, T cells<br />
obtained from SLE patients were not susceptible to suppression<br />
by normal Treg. Our findings suggest that even though a<br />
T cell suppression defect is present in patients with SLE, it<br />
cannot be explained by defects in Treg. Our data indicate<br />
that SLE-derived T cells are resistant to Treg-mediated<br />
suppression.<br />
doi:10.1016/j.clim.2007.03.469<br />
Sa.82 Increased Regulatory T Cell Prevalence and<br />
Retained Capacity to Suppress Effector T Cells<br />
During Disease in NZB/W Lupus Mice<br />
Kenneth Scalapi, Adjunct Assistant Pr<strong>of</strong>essor <strong>of</strong> Medicine,<br />
University <strong>of</strong> California, San Francisco and San Francisco VA<br />
Medical Center, San Francisco, CA, David Daikh, Associate<br />
Pr<strong>of</strong>essor <strong>of</strong> Medicine, University <strong>of</strong> California, San<br />
Francisco and San Francisco VA Medical Center, San<br />
Francisco, CA<br />
Purpose: SLE is characterized by loss <strong>of</strong> tolerance to<br />
multiple self-antigens. Regulatory T cells (Tregs) serve to<br />
maintain peripheral tolerance by inhibiting autoreactive<br />
effector Tcells (Teff). Studies have correlated low peripheral<br />
blood Treg prevalence and/or abnormal Treg function with<br />
several autoimmune diseases including SLE. To assess Treg<br />
frequency and function in lupus prone NZB/W (B/W) mice,<br />
we evaluated CD4+Foxp3+ T cells during the course <strong>of</strong><br />
disease. Cellular function in young and old B/W mice was<br />
assessed utilizing the CD4+CD25+ Treg subset, young and old<br />
Teffs and antigen-presenting cells (APCs) in mixed suppression<br />
assays. Methods: CD4+Foxp3+ Treg prevalence was<br />
measured by FACS. Function <strong>of</strong> purified Tregs, Teffs and<br />
APCs was evaluated in mixed suppression assays. Results:<br />
Frequency <strong>of</strong> CD4+Foxp3+ Tregs as a percentage <strong>of</strong> CD4+ cells<br />
is low only in pre-diseased B/W mice. During active lupus,<br />
Tregs prevalence significantly increases such that it equals or<br />
exceeds that <strong>of</strong> age-matched control mice. A significant<br />
fraction <strong>of</strong> Tregs in sick mice loses CD25− expression. The<br />
Treg prevalence in peripheral blood did not correlate with<br />
tissue prevalence or disease state. Mixed suppression assays<br />
demonstrated that the function <strong>of</strong> CD4+CD25+ Tregs is not<br />
affected by disease state and Teffs do not become resistant<br />
to Treg suppression as mice age and develop disease.<br />
Conclusion: A significant expansion <strong>of</strong> functional Tregs, not<br />
evident in peripheral blood, occurs in lupus prone B/W with<br />
disease onset, emphasizing the unreliability <strong>of</strong> peripheral<br />
blood to assess these cells. Furthermore, Tregs retain the<br />
capacity to suppress Teffs in mice with active lupus.<br />
doi:10.1016/j.clim.2007.03.470<br />
Sa.84 Immune Opsonins Modulate BLyS/BAFF<br />
Release in a Receptor-specific Fashion<br />
Xinrui Li, Graduate Student, Division <strong>of</strong> <strong>Clinical</strong> <strong>Immunology</strong><br />
and Rheumatology, University <strong>of</strong> Alabama at Birmingham,<br />
Birmingham, AL, Jeffrey Edberg, Associate Pr<strong>of</strong>essor,<br />
Division <strong>of</strong> <strong>Clinical</strong> <strong>Immunology</strong> and Rheumatology,<br />
University <strong>of</strong> Alabama at Birmingham, Birmingham, AL,<br />
Kaihong Su, Assistant Pr<strong>of</strong>essor, Division <strong>of</strong> <strong>Clinical</strong><br />
<strong>Immunology</strong> and Rheumatology, University <strong>of</strong> Alabama at<br />
Birmingham, Birmingham, AL, Tong Zhou, Associate<br />
Pr<strong>of</strong>essor, Division <strong>of</strong> <strong>Clinical</strong> <strong>Immunology</strong> and<br />
Rheumatology, University <strong>of</strong> Alabama at Birmingham,<br />
Birmingham, AL, Alexander Szalai, Associate Pr<strong>of</strong>essor,<br />
Division <strong>of</strong> <strong>Clinical</strong> <strong>Immunology</strong> and Rheumatology,<br />
University <strong>of</strong> Alabama at Birmingham, Birmingham, AL,<br />
Robert Kimberly, Pr<strong>of</strong>essor, Division <strong>of</strong> <strong>Clinical</strong> <strong>Immunology</strong><br />
and Rheumatology, University <strong>of</strong> Alabama at Birmingham,<br />
Birmingham, AL<br />
TNF ligand superfamily member 13B (B lymphocyte<br />
stimulator (BLyS), B cell activating factor (BAFF)) promotes<br />
primary B cell proliferation and immunoglobulin production.<br />
It exists in both membrane-bound and soluble forms. Because<br />
the soluble form is thought to be the primary biologically<br />
active form, we investigated factors that might regulate its<br />
cleavage and processing. In both myeloid cell lines (U937,<br />
THP-1and HL-60) and primary human monocytes, the<br />
shedding <strong>of</strong> membrane BLyS can be regulated by CRP and<br />
IgG. Within 10 minites, both <strong>of</strong> these opsonins trigger<br />
significant shedding <strong>of</strong> BLyS (up to 52.2+/-3.0%, p=0.003)<br />
by engaging FcgRs. In U937 cells, a primary role <strong>of</strong> FcgRI is<br />
demonstrated both by direct receptor cross-linking (FcgRI<br />
45.69+/-2.05% verses FcgRIIa 2.11+/-1.33%) and by the lack<br />
<strong>of</strong> enhanced cleavage when FcgRI and FcgRIIa are cocrosslinked.<br />
The shed BLyS is biologically functional since it<br />
promotes B cell survival. The generation <strong>of</strong> a B cell<br />
proliferation and survival factor by Fc gamma receptor<br />
engagement, especially FcgRI, which can also enhance
Abstracts<br />
antigen uptake and presentation mediated by ligands <strong>of</strong> both<br />
innate and adaptive immune systems, provides a unique<br />
opportunity to facilitate antibody production. These functions<br />
suggest that Fc gamma receptors may provide a link between<br />
immune complex mediated autoimmune diseases and elevations<br />
in circulating BLyS in autoimmune disease patients.<br />
doi:10.1016/j.clim.2007.03.471<br />
Sa.85 The CD4+CD25+Foxp3+ T Cells in Lupus<br />
Nephritis<br />
Mercedes Zabaleta-Lanz, Immunologist, Institute <strong>of</strong><br />
<strong>Immunology</strong>, Central University School <strong>of</strong> Medicine,<br />
Caracas, Venezuela, Ronak Seyeddi, MD, Institute <strong>of</strong><br />
<strong>Immunology</strong>, Caracas, Venezuela, Maria Elena Marquez,<br />
Bbiologist, Institute <strong>of</strong> <strong>Immunology</strong>, Caracas, Venezuela,<br />
Perla Chirinos, Bioanalist, Instituto de Inmunologia, UCV,<br />
Caracas, Venezuela, Isaac Blanca, Biologist, Institute <strong>of</strong><br />
<strong>Immunology</strong>, Caracas, Venezuela, Nicolás Bianco,<br />
Immunologist, Institute <strong>of</strong> <strong>Immunology</strong>, Caracas, Venezuela,<br />
Mercedes Zabaleta-Lanz, Immunologist, Institute <strong>of</strong><br />
<strong>Immunology</strong>, Caracas, Venezuela<br />
The role <strong>of</strong> CD4+CD25+Foxp3+ regulatory T cells (Tregs)<br />
are critical in maintaining self tolerance. The levels <strong>of</strong> Treg<br />
in patient with systemic lupus erythematosus (SLE), a<br />
chronic autoimmune disorder, have been reported<br />
depressed. Here we have determined the levels <strong>of</strong> Treg<br />
and the CTLA-4 CD4 T cells in 21 patients with SLE, 7 with<br />
active disease and 15 with inactive disease and 25 healthy<br />
controls. Treg and CTLA-4 were evaluated in highly purified<br />
CD4 T cells from patients and controls by flow cytometry<br />
using a tri-color analysis protocol. The 7 SLE patients with<br />
active disease, 4 with overt lupus nephritis (OLN) and 3<br />
with silent lupus nephritis (SLN) show a significant reduction<br />
<strong>of</strong> Treg in comparison to controls (SLN, 0.23±0.18%,<br />
OLN 1.35±1.21%, controls, 3,69±3%¸ pb0.05 and pb0.02<br />
respectively). A significant reduction <strong>of</strong> Treg was also<br />
observed between the inactive SLE patients versus controls<br />
(pb0.02). A higher expression <strong>of</strong> Foxp3 was observed in<br />
Treg cells from patients with active SLE disease (88.74 ±<br />
5.92%) vs. inactive disease (71.39 ±20.15%; p=0.008). Nonsignificant<br />
differences were observed in the levels <strong>of</strong> CD4<br />
+CD25+CTLA-4+ T cells, and no correlation between Tregs<br />
and serological parameters (anti-DNA, anti-C1q, antinucleosome,<br />
C3 and C4) was observed. Ours results suggest<br />
that Tregs are significantly decreased in active lupus even<br />
before the installation <strong>of</strong> an OLN. However, the increased<br />
expression <strong>of</strong> Foxp3 in the Tregs cells from patients with<br />
active disease suggests a possible mechanism <strong>of</strong> compensatory<br />
function <strong>of</strong> these molecules.<br />
doi:10.1016/j.clim.2007.03.472<br />
Sa.86 Rapamycin Induces Tolerance by Increasing<br />
Regulatory T Cells in Chronic Graft Versus Host<br />
Disease Model<br />
Jeanne Palmer, Postdoctoral Fellow, Duke University<br />
Medical Center, Department <strong>of</strong> Hematology, Oncology and<br />
Cellular Therapy, Durham, NC, Benny Chen, Assistant<br />
Pr<strong>of</strong>essor <strong>of</strong> Medicine, Duke University Medical Center,<br />
Division <strong>of</strong> Cellular Therapy/BMT, Durham, NC, Nelson<br />
Chao, Director, Division <strong>of</strong> Cellular Therapy/BMT, Pr<strong>of</strong>essor<br />
<strong>of</strong> Medicine and <strong>Immunology</strong>, Duke University Medical<br />
Center, Durham, NC, Ngocdiep Le, Medical Sciences<br />
Director, Amgen Inc., Thousand Oaks, CA<br />
Rapamycin is an immunosuppressant that has been found<br />
to prevent/treat acute and chronic graft versus host disease<br />
after allogeneic hematopoeitic stem cell transplant (HSCT)<br />
in both murine models and humans. One possible mechanism<br />
is through induction <strong>of</strong> tolerance, which may be due to<br />
increased number or enhanced survival <strong>of</strong> regulatory T cells.<br />
In two experiments B10.D2 bone marrow and splenocytes<br />
were injected into lethally irradiated BALB/c mice. They<br />
were then given IP injections from days 1-28 with rapamycin<br />
1.5 mg/kg or 5 mg/kg, which induced tolerance in this<br />
chronic graft versus host disease model. We noted however a<br />
dense cellular infiltrate in the animals who did not have<br />
clinical GVHD. We have documented many these infiltrating<br />
cells as in situ regulatory T cells by intracellular foxP3<br />
staining. These T regs are increased in tissues <strong>of</strong> rapamycin<br />
treated mice. This effect appears to decrease following<br />
discontinuation <strong>of</strong> rapamycin. To understand the kinetics <strong>of</strong><br />
this effect, we will repeat the experiments, and sacrifice the<br />
mice at 2, 4, and 6 weeks. At that point, serum cytokine<br />
analysis, histologic evaluation <strong>of</strong> multiple organs and FACS<br />
analysis on splenocytes will be performed.<br />
doi:10.1016/j.clim.2007.03.473<br />
S103<br />
Sa.87 Elimination <strong>of</strong> Insulitis and Augmentation <strong>of</strong><br />
Islet β Cell Regeneration via Induction <strong>of</strong> Chimerism<br />
in Overtly Diabetic NOD Mice<br />
Chunyan Zhang, Postdoctoral Research Fellow, The Beckman<br />
Research Institute, City <strong>of</strong> Hope National Medical Center,<br />
Duarte, CA, Fouad Kandeel, Physician, The Beckman<br />
Research Institute, City <strong>of</strong> Hope National Medical Center,<br />
Duarte, CA, Ivan Todorov, Associate Research Scientist, The<br />
Beckman Research Institute, City <strong>of</strong> Hope National Medical<br />
Center, Duarte, CA, Mark Atkinson, Pr<strong>of</strong>essor, University <strong>of</strong><br />
Florida College <strong>of</strong> Medicine, Gainesville, FL, Chia-Lei Lin,<br />
Research Associate I, The Beckman Research Institute, City<br />
<strong>of</strong> Hope National Medical Center, Duarte, CA, Stephen<br />
Forman, Physician, The Beckman Research Institute, City <strong>of</strong><br />
Hope National Medical Center, Duarte, CO, Defu Zeng,<br />
Assistant Pr<strong>of</strong>essor, The Beckman Research Institute, City <strong>of</strong><br />
Hope National Medical Center, Duarte, CA<br />
Type 1 diabetes in both humans and NOD mice results from<br />
autoreactive T cell destruction <strong>of</strong> insulin-producing beta<br />
cells. Cure <strong>of</strong> type 1 diabetes may require both reversal <strong>of</strong><br />
autoimmunity and regeneration <strong>of</strong> beta cells. Induction <strong>of</strong><br />
chimerism via allogeneic hematopoietic transplantation<br />
(HCT) has been shown to re-establish tolerance in both<br />
prediabetic and diabetic NOD mice. However, it is unclear<br />
whether this therapy augments beta cell regeneration.<br />
Furthermore, this procedure usually requires total body
S104 Abstracts<br />
irradiation (TBI) conditioning <strong>of</strong> recipients. The toxicity <strong>of</strong><br />
TBI conditioning and potential for graft-versus-host disease<br />
(GVHD) limit the application <strong>of</strong> allogeneic HCT for treating<br />
type 1 diabetes. Here, we report that injection <strong>of</strong> donor bone<br />
marrow and CD4+ T-depleted spleen cells induced chimerism<br />
without causing GVHD in overtly diabetic NOD mice conditioned<br />
with anti-CD3/CD8, and that induction <strong>of</strong> chimerism<br />
in new-onset diabetic NOD mice led to elimination <strong>of</strong><br />
insulitis, replication <strong>of</strong> host beta cells, and reversal <strong>of</strong><br />
hyperglycemia. Although induction <strong>of</strong> chimerism in latestage<br />
<strong>of</strong> diabetic NOD mice did not reverse diabetes, the<br />
chimeras were tolerant to donor islets, and donor islet<br />
transplantation normalized blood glucose. Furthermore, we<br />
found that induction <strong>of</strong> mixed chimerism in autoreactive<br />
BDC2.5 TCR transgenic NOD mice deleted 99% <strong>of</strong> the BDC2.5<br />
T cells among host CD4+CD8+ thymocytes. Therefore, this<br />
radiation-free GVHD preventive approach for induction <strong>of</strong><br />
chimerism may represent a viable means for reversing type 1<br />
diabetes.<br />
doi:10.1016/j.clim.2007.03.474<br />
Sa.88 Purified MHC-matched Hematopoietic Stem<br />
Cell Transplantation Following Total Lymphoid<br />
Irradiation and Anti-thymocyte Globulin (TLI+ATG)<br />
Blocks EAE Pathogenesis<br />
Li-Fen Lee, Instructor, Blood and Marrow Transplantation,<br />
Stanford, CA, Matthew Burge, Research Assistant, Blood<br />
and Marrow Transplantation, Stanford, CA, Rosa Landa,<br />
Research Assistant, Blood and Marrow Transplantation,<br />
Stanford, CA, Raymond Sobels, Pr<strong>of</strong>essor, Pathology,<br />
Stanford, CA, Peggy Ho, Basic Life Science Research<br />
Associate, Neurology, Stanford, CA, Judith Shizuru,<br />
Associate Pr<strong>of</strong>essor, Blood and Marrow Transplantation,<br />
Stanford, CA<br />
In prior studies, we demonstrated that transplantation <strong>of</strong><br />
major histocompatibility complex (MHC) matched and mismatched<br />
purified hematopoietic cells (HSCs) can block<br />
diabetes pathogenesis in pre-diabetic NOD mice. Here we<br />
applied this treatment to experimental allergic encephalomyelitis<br />
(EAE), an animal model for the human autoimmune<br />
disease multiple sclerosis (MS). C57BL/6 (B6) strains congenic<br />
for the CD45 locus were immunized with MOG 35–55 to induce<br />
disease. Similar to our studies in NOD mice we observed that<br />
disease response after transplantation <strong>of</strong> MHC-mismatched<br />
hematopoietic cells in EAE affected mice was superior to those<br />
that received congenic cells. Importantly, mice transplanted<br />
with MHC-matched HSC or BM showed no significant improvement<br />
in disease symptoms when compared with congenic<br />
recipients or disease controls. This result was markedly<br />
different from the positive response observed in NOD mice<br />
transplanted with MHC-matched HSC. We then sought to<br />
optimize translation <strong>of</strong> this approach by giving total lymphoid<br />
irradiation (TLI) plus anti-thymocyte globulin (ATG), a nonmyeloablative<br />
regimen, and permitting allogeneic hematopoietic<br />
cell engraftment with significant protective effect<br />
against GVHD. We found that TLI/ATG treatment in wild type<br />
C57BL/6 mice permits engraftment <strong>of</strong> purified HSC isolated<br />
from MHC-matched AKR/B donors and improves disease<br />
severity significantly. The level <strong>of</strong> stable mixed chimerism<br />
correlated with the clinical score. A predominance <strong>of</strong> host<br />
NK1.1 T cells and CD4+CD25+ regulatory T cells was observed<br />
after TLI/ATG regimen. We hope an MHC-matched hematopoietic<br />
graft in conjunction with TLI/ATG conditioning shown<br />
here to successfully ameliorate EAE can be translated to the<br />
treatment <strong>of</strong> human autoimmune disease.<br />
doi:10.1016/j.clim.2007.03.475<br />
Sa.89 Two Types <strong>of</strong> Immune Regulatory Mechanisms<br />
are Developed In Vivo for the Control <strong>of</strong> Chronic<br />
Graft-versus-Host Disease<br />
Magali Noval Rivas, PhD Student, Institute for Medical<br />
<strong>Immunology</strong> (IMI), Gosselies, Brussels, Marc Hazzan, Doctor,<br />
Department <strong>of</strong> Nephrology and Hemodialysis, CHR Lille,<br />
Lille, France, Michel Braun, Institute for Medical<br />
<strong>Immunology</strong> (IMI), Gosselies, Brussels<br />
We developed a graft-versus-host disease (GVHD) model<br />
based on the adoptive transfer <strong>of</strong> RAG1 −/− TcR-trangenic H-Yspecific<br />
Marilyn T cells (1×10 6 cells) into male recipients.<br />
Adoptive transfer into sub-lethally irradiated RAG1 +/+ IL-<br />
2Rgc +/+ , RAG1 −/− IL-2Rgc +/+ or RAG1 −/− IL-2Rgc −/− male<br />
recipients induced 100% mortality after 1 week. On the<br />
contrary, non-irradiated mice <strong>of</strong> the three types survived<br />
indefinitely after transfer and did not develop clinical sign <strong>of</strong><br />
acute GVHD. Marilyn T cells transferred into non-irradiated<br />
RAG1 +/+ IL-2Rgc +/+ or RAG1 −/− IL-2Rgc +/+ did not expand or<br />
were rapidly eliminated (b45×10 3 in the spleen after<br />
30 days). Remarkably, transgenic T cells transferred into<br />
non-irradiated RAG1 −/− IL-2Rgc −/− hosts expanded extensively<br />
(N15×10 6 in the spleen after 30 days) and were present<br />
in many organs (spleen, lung, liver, kidney, lymph nodes).<br />
Histology revealed their capacity to mediate chronic GVHD<br />
lesions (grade 1 after 30 days) in these hosts. The T cells<br />
did not develop a regulatory phenotype since they<br />
remained Foxp3-negative and produced high amounts <strong>of</strong><br />
IFN-g when stimulated in vitro. However, they were unable<br />
to promote acute GVHD after transfer into irradiated<br />
recipients. Our model suggests that, in vivo, there are two<br />
types <strong>of</strong> mechanisms involved into the regulation <strong>of</strong> T cell<br />
responses to persistent antigenic stimulation: one is selfcontained<br />
by the T cells and involves partial anergy,<br />
probably maintained by antigenic persistence. The other<br />
one involves the activity <strong>of</strong> existing populations <strong>of</strong><br />
regulatory cells. Both <strong>of</strong> them appear to be necessary for<br />
full T cell tolerance.<br />
doi:10.1016/j.clim.2007.03.476<br />
Sa.90 NK1.1+ Cells Promote Human Cell<br />
Repopulation via an NKG2D Dependent Mechanism<br />
Hye-Youn Son, Research Pr<strong>of</strong>essor, Samsung Biomedical<br />
Research Institute, Seoul, Republic <strong>of</strong> Korea, Sung-Yeon Joo,<br />
MS, Samsung Biomedical Research Institute, Seoul, Republic<br />
<strong>of</strong> Korea, Mi Jin Kang, MS, Samsung Biomedical Research<br />
Institute, Seoul, Republic <strong>of</strong> Korea, Sung-Joo Kim,<br />
Prodessor, Samsung Medical Center, Seoul, Republic <strong>of</strong>
Abstracts<br />
Korea, Bong-Kum Choi, MS, Samsung Biomedical Research<br />
Institute, Seoul, Republic <strong>of</strong> Korea<br />
To achieve donor-specific immune tolerance in Rag2−/−<br />
f×C−/− mice to xenogeneic human CD34+ hematopoietic<br />
stem cells (HSCs), it is imperative to understand the cell<br />
types involved in xenograft rejection. In this study, we<br />
examined whether NK1.1+ cells in Rag2−/−f×C−/− mice play<br />
a direct role in the tolerance against transplanted HSCs, if<br />
thus which molecules are important. We used CD34+<br />
hematopoietic stem cells to reconstitute human cells from<br />
either cord blood or fetal liver. From 2 weeks posttransplantation,<br />
we have monitored human cells repopulation<br />
(referred as % <strong>of</strong> CD45) in peripheral blood every 2 or<br />
4 weeks. Besides, anti-mouse CD3, CD19, NK1.1, Gr-1, and<br />
NKG2D were used to determine the mouse cells involved<br />
in tolerance induction. In our system, hCD45 was<br />
significantly increased following human HSC’s transplantation<br />
as well as successfully developed multi-lineage. We<br />
found that NK1.1+ cells, especially Gr-1− subset, were<br />
directly co-related with human cell reconstitution. Interestingly,<br />
NKG2D expression on NK1.1+ cells was significantly<br />
increased in the group highly repopulated with<br />
human cells. Finally, CD3 population appeared after<br />
transplantation was shown to be inversely related with<br />
NKG2D expression. Taken together, our data strikingly<br />
demonstrate that NK1.1+ cells play a direct role to regulate<br />
human cell reconstitution as well as HSCs engraftment in<br />
Rag2−/−f×C−/− mice. Moreover, our data show that NKG2D<br />
expression significantly contributes to tolerance induction<br />
which could be involved in donor-specific cytotoxic cell<br />
killing mechanism. We are planning to down-regulate and/<br />
or delete specific NKG2D+ subsets in mixed chimeras in<br />
vivo to clear the results.<br />
doi:10.1016/j.clim.2007.03.477<br />
Sa.91 Human Cell Reconstitution from Umbilical<br />
Cord Blood-derived Stem Cells in Rag1−/−gC−/− Mice<br />
by Co-Transplantation <strong>of</strong> Unrelated Fetal<br />
Liver/Thymus Tissues<br />
Mi Jin Kang, MS, Samsung Biomedical Research Institute,<br />
Seoul, Republic <strong>of</strong> Korea, Hye-Youn Son, Research pr<strong>of</strong>essor,<br />
Samsung Biomedical Research Institute, Seoul, Republic <strong>of</strong><br />
Korea, Bong-Kum Choi, MS, Samsung Biomedical Research<br />
Institute, Seoul, Republic <strong>of</strong> Korea, Sung-Joo Kim,<br />
Pr<strong>of</strong>essor, Samsung Medical Center, Seoul, Republic <strong>of</strong><br />
Korea, Sung-Yeon Joo, MS, Samsung Biomedical Research<br />
Institute, Seoul, Republic <strong>of</strong> Korea<br />
To establish human immune and hematopoietic development<br />
and function in vivo, a number <strong>of</strong> studies have<br />
been tried to reproduce human hematopoiesis as human<br />
hematopoietic tissue and cell transplantation in immunodeficient<br />
mice. We report a model co-transplanted <strong>of</strong><br />
human fetal thymus/liver (Thy/Liv) tissues with cord blood<br />
(CB) CD34+ cells into Rag2−/−gC−/− mice. To solve the<br />
limitation <strong>of</strong> cell dose, we also used ex vivo expansion with<br />
cytokines combination; Flt-3 ligand (FL), thrombopoietin<br />
(TPO), stem cell factor (SCF), erythropoietin (EPO), G-CSF,<br />
GM-CSF or interleukin-3 (IL-3). Rag2−/−gC−/− mice were<br />
transplanted with CB CD34+ hematopoietic progenitor cells<br />
with HLA unrelated fetal Thy/Liv. MHC-mismatched CB<br />
CD34+ cell transplantation with fetal tissues resulted in<br />
measurable engraftment levels (about 5% <strong>of</strong> human CD45)<br />
in PBL more than 4 months after transplantation. Also, we<br />
observed that implanted human fetal liver and thymus<br />
under the kidney capsule were significantly grown. We<br />
found T cell and NK cell activity after in vitro restimulation<br />
at 20 weeks after transplantation. Especially, CD3+ T cell<br />
development was remarkable in a part <strong>of</strong> ex vivo cultured<br />
cell transplanted group (included with FL, TPO, and SCF).<br />
We also found human IgG in serum 20 weeks after<br />
transplantation even though we have never seen reasonable<br />
CD19+ cells in PBL, BM, or spleen. These results<br />
demonstrate that functional human T cell can be reconstituted<br />
by transfusion <strong>of</strong> MHC mismatched CB derived<br />
CD34+ cells with co-implantation <strong>of</strong> fetal Thy/Liv and that<br />
might provide a useful tool to study human disease like<br />
viral infection.<br />
doi:10.1016/j.clim.2007.03.478<br />
S105<br />
Sa.92 Fetal Liver Cells as Corrector <strong>of</strong> Immune and<br />
Hemopoietic Activity <strong>of</strong> Allogeneic Bone Marrow<br />
Anatoliy Goltsev, Pr<strong>of</strong>essor, Institute for Problems <strong>of</strong><br />
Cryobiology and Cryomedicine <strong>of</strong> the National Academy <strong>of</strong><br />
Science <strong>of</strong> Ukraine, Kharkov, Ukraine, Irina Matsevitaya,<br />
Institute for Problems <strong>of</strong> Cryobiology and Cryomedicine <strong>of</strong><br />
the National Academcy <strong>of</strong> Science <strong>of</strong> Ukraine, Kharkov,<br />
Ukraine, Elena Lutsenko, Institute for Problems <strong>of</strong><br />
Cryobiology and Cryomedicine <strong>of</strong> the National Academy <strong>of</strong><br />
Science <strong>of</strong> Ukraine, Kharkov, Ukraine, Yulia Kozlova,<br />
Institute for Problems <strong>of</strong> Cryobiology and Cryomedicine <strong>of</strong><br />
the National Academy <strong>of</strong> Science <strong>of</strong> Ukraine, Kharkov,<br />
Ukraine, Tatyana Dubrava, Institute for Problems <strong>of</strong><br />
Cryobiology and Cryomedicine <strong>of</strong> the National Academy <strong>of</strong><br />
Science <strong>of</strong> Ukraine, Kharkov, Ukraine, Maxim Ostankov,<br />
Institute for Problems <strong>of</strong> Cryobiology and Cryomedicine <strong>of</strong><br />
the National Academy <strong>of</strong> Science <strong>of</strong> Ukraine, Kharkov,<br />
Ukraine<br />
We have shown the possibility to reduce immune reactivity <strong>of</strong><br />
allogeneic bone marrow (BM) as graft-versus-host reaction<br />
(GVHR) by the change <strong>of</strong> the content <strong>of</strong> myelotransplant<br />
hemopoietic microenvironment cells. These changes may be<br />
achieved by additional introduction with BM <strong>of</strong> cells possessing<br />
regulatory activity. There is accumulated information about<br />
manifested immune modulating ability <strong>of</strong> fetal liver cells (FLCs).<br />
Research aim was the substantiation <strong>of</strong> the possible use <strong>of</strong> FLCs<br />
as the preparation: modulators <strong>of</strong> immune and hemopoietic<br />
activity <strong>of</strong> allogeneic bone marrow and a rise in its protective<br />
potential. Lethally irradiated Balb/c mice were intravenously<br />
injected BM <strong>of</strong> CBA ones (5×10 6 /mouse) together with FLC<br />
(5×10 5 /mouse). GVHR development intensity was assessed<br />
according to traditional parameters. Content <strong>of</strong> stem hemopoietic<br />
cells in BM <strong>of</strong> recipients was examined by cloning <strong>of</strong><br />
hemopoietic precursors in vivo (CFUs). Obtained results testify<br />
to the fact that co-transplanted FLCs rendered manifested<br />
regulatory effect on immune reactivity and hemopoietic
S106 Abstracts<br />
function <strong>of</strong> cryopreserved allogeneic BM. Character and extent<br />
<strong>of</strong> its manifestation have dose-dependent effect. FLC cotransplantation<br />
contributed to minimization <strong>of</strong> hypoplasia <strong>of</strong><br />
recipient’s thymus, increase <strong>of</strong> content in BM <strong>of</strong> recipients <strong>of</strong><br />
CFUs and finally to a rise in the survival percentage <strong>of</strong> the<br />
animals to the 70th day. Therefore the effect <strong>of</strong> FLC may be<br />
stipulated not only by the limitation <strong>of</strong> the extent <strong>of</strong><br />
allomyelotransplant GVHR activity but also by an increase in<br />
its hemopoietic potential. Mechanisms <strong>of</strong> FLC modulating<br />
activity in respect to co-transplanted BM are under discussion.<br />
doi:10.1016/j.clim.2007.03.479<br />
Sa.93 Suppression <strong>of</strong> Acute Graft-versus-host<br />
Disease by Regulatory T Cells Induced by Toxic<br />
Shock Syndrome Toxin-1<br />
Haowei Li, PhD Candidate, University <strong>of</strong> British Columbia,<br />
Department <strong>of</strong> Medicine, Vancouver, BC, Canada, Anthony<br />
W. Chow, Pr<strong>of</strong>essor Emeritus, University <strong>of</strong> British<br />
Columbia, Department <strong>of</strong> Medicine, Vancouver, BC, Canada<br />
Allogeneic bone marrow transplantation can cure multiple<br />
hematopoietic malignancies, but acute graft-versus-host<br />
disease (aGVHD) is a major complication. Previous studies in<br />
our laboratory have shown that chronic exposure to the<br />
staphylococcal superantigen, toxic shock syndrome toxin-1<br />
(TSST-1), can induce regulatory T cells (Tregs) in C57BL/6<br />
(B6) mice. These TSST-1-induced Tregs have potent suppressive<br />
ability to inhibit TSST-1 or SEA-induced pro-inflammatory<br />
cytokine responses in vitro and in vivo. However, it is<br />
unclear if TSST-1-induced Tregs can be applied clinically to<br />
control aGVHD. In this report, using a murine aGVHD model<br />
(B6 to B6D2F1), we showed that transplantation <strong>of</strong> an<br />
allogeneic graft containing TSST-1-induced Tregs alone did<br />
not confer protection against aGVHD. However, reactivation<br />
<strong>of</strong> TSST-1-induced Tregs by post-transplant exposure to TSST-<br />
1 led to attenuation <strong>of</strong> aGVHD. While one dose <strong>of</strong> TSST-1 only<br />
prolonged the survival time but not the survival rate <strong>of</strong><br />
recipients <strong>of</strong> TSST-1-induced Tregs, two doses <strong>of</strong> TSST-1 both<br />
prolonged the survival time and increased the survival rate <strong>of</strong><br />
the recipients. Activation <strong>of</strong> TSST-1-induced Tregs did not<br />
reduce engraftment or expansion <strong>of</strong> donor T cells, nor<br />
enhance the elimination <strong>of</strong> host antigen presenting cells, but<br />
was associated with decreased production <strong>of</strong> pro-inflammatory<br />
cytokines. Blockade <strong>of</strong> IL-10 did not reverse the<br />
suppression. More importantly, control <strong>of</strong> aGVHD by activation<br />
<strong>of</strong> TSST-1-induced Tregs did not interfere with graft-vs.tumor<br />
(GVT) effects. In summary, we conceptually demonstrated<br />
that Tregs induced by TSST-1 were able to control<br />
aGVHD in vivo via bystander suppression while sparing GVT<br />
effects.<br />
doi:10.1016/j.clim.2007.03.480<br />
Sa.94 Bone Marrow Derived Mesenchymal Stromal<br />
Cells Promote Treg Cell Development Ex Vivo<br />
Qingping Yao, Rheumatology Fellow, Medicine, University <strong>of</strong><br />
California, Los Angeles David Geffen School <strong>of</strong> Medicine, Los<br />
Angeles, CA, James Wang, Researcher, Medicine, University<br />
<strong>of</strong> California, Los Angeles David Geffen School <strong>of</strong> Medicine,<br />
Los Angeles, CA, Ram Raj Singh, Pr<strong>of</strong>essor, Medicine,<br />
University <strong>of</strong> California, Los Angeles David Geffen School <strong>of</strong><br />
Medicine, Los Angeles, CA<br />
Objective: Bone marrow derived mesenchymal stromal<br />
cells (BMSCs) provide niche for the development <strong>of</strong> hematopoietic<br />
cells. The BMSCs likely accomplish this via multiple<br />
cytokines. Here, we asked whether BMSCs affect the<br />
development <strong>of</strong> regulatory T (Treg) cells that can downregulate<br />
autoimmune diseases. Methods: Bone marrow cells<br />
isolated from lupus-prone (NZB×NZW) F1 (BWF1) and normal<br />
BALB/c mice were cultured in 24 well plates for 6 days. After<br />
removal <strong>of</strong> nonadherent cells in these cultures, adherent<br />
BMSCs were co-incubated with freshly isolated splenocytes<br />
from either BWF1 or BALB/c mice for an additional 6 days.<br />
Splenic cells harvested from these cultures were analyzed by<br />
flow cytometry (FACS) for CD4+CD25+ T lymphocytes. Cell<br />
culture supernatants were assayed for cytokines by ELISA.<br />
Results: We found a significantly higher number <strong>of</strong> CD4+<br />
CD25+ T lymphocytes in co-cultures <strong>of</strong> splenocytes plus<br />
BMSCs than in cultures <strong>of</strong> splenocyte alone. CD4+CD25+ T<br />
cells were found by FACS to be 8.5 and 178 fold higher in cocultures<br />
<strong>of</strong> splenocytes +BMSCs from BWF1 and BALB/c mice,<br />
respectively, over cultures <strong>of</strong> splenocytes alone. The<br />
increase in Treg cells was associated with increased IL-10<br />
production. Conclusion: BMSCs promote the development <strong>of</strong><br />
Treg cells, which is more efficient in normal mice than in<br />
autoimmune-prone mice. Thus, BMSC impairments might<br />
contribute to abnormalities in Treg cells in autoimmune<br />
conditions. Ongoing study will further characterize the Treg<br />
cell functions in our model, determine mechanisms by which<br />
BMSCs interact with Treg cells and examine the in vivo<br />
effects <strong>of</strong> BMSCs on Treg cells.<br />
doi:10.1016/j.clim.2007.03.481<br />
Sa.95 Selective Depletion <strong>of</strong> Alloreactive T Cells and<br />
Study <strong>of</strong> Anti-Tumor Activity <strong>of</strong> Specific T Cell<br />
Clones in Patients with Leukemia and Renal<br />
Carcinoma<br />
Eva Matejkova, PhD Student, Masaryk University, Zuzana<br />
Hrotekova, MSc., Cell Immunotherapy Center, Drahomira<br />
Kyjovska, Medical Laboratory Technician, Masaryk<br />
University, Czech Republic, Jaroslav Michalek, Head <strong>of</strong> Cell<br />
Immunotherapy Center, Masaryk University, Czech<br />
Republic, Petra Vidlakova, Lab Chief, Masaryk University,<br />
Czech Republic<br />
A major challenge in the field <strong>of</strong> hematopoietic stem<br />
cell transplantation (HSCT) is to prevent the alloreactivity<br />
<strong>of</strong> donor T cells which leads to acute graft-versus-host<br />
disease (GVHD) while preserving a graft-versus-tumor (GVT)<br />
effect. Selective depletion using anti-CD25 immunotoxin<br />
(IT) can eliminate harmful alloreactive T cells while<br />
preserving other donor T cells with antileukemic/antitumor<br />
reactivity. We have used irradiated peripheral blood<br />
mononuclear cells (PBMC) from cancer patients and healthy
Abstracts<br />
donor PBMC as responder cells in primary mixed leukocyte<br />
reaction (MLR). To prepare GVL/GVT-specific T cells,<br />
alloreactive T cells in primary MLR were depleted with<br />
anti-CD25 IT. The remaining T cells had insignificant<br />
alloreactivity in secondary MLR. Allodepleted donor cells<br />
were then repeatedly stimulated using purified leukemia/<br />
tumor cells from the same cancer patient. Leukemia/<br />
tumor-reactive donor T cells were purified by immunomagnetic<br />
separation on the basis <strong>of</strong> INF-g production. 17 MLRs<br />
(10 with leukemic and 7 with renal carcinoma cells) were<br />
performed. Selective depletion <strong>of</strong> alloreactive donor T cells<br />
with anti-CD25 IT led to more than 2log depletion. Graftversus-leukemia<br />
(GVL) effect <strong>of</strong> donor T cells was well<br />
preserved while the graft-versus-host (GVH) reactivation <strong>of</strong><br />
donor cells was negligible even after repeated stimulation<br />
with patient’s non-leukemic PBMC. In the case <strong>of</strong> renal<br />
carcinoma GVT effect was less dominant and GVH<br />
reactivation <strong>of</strong> donor cells was significant. In conclusion,<br />
it is possible to selectively deplete donor alloreactive T<br />
cells with anti-CD25 IT. In the case <strong>of</strong> patients with<br />
leukemia, the GVL effect can be separated from GVHD, but<br />
in case <strong>of</strong> renal carcinoma severe GVHD effect reappeared.<br />
Supported by the Ministry <strong>of</strong> Education <strong>of</strong> the<br />
Czech Republic, NPVII-2B06058.<br />
doi:10.1016/j.clim.2007.03.482<br />
Sa.96 CD28 Costimulates Suppression in an In Vitro<br />
Model <strong>of</strong> the Tumor Microenvironment<br />
Daniel Silberman, Student, Biology Department, Rider<br />
University, Lawrenceville, NJ, Amanda Bucknum, Student,<br />
Biology Department, Rider University, Lawrenceville, NJ,<br />
Tiffany Bloomfield, Student, Biology Department, Rider<br />
University, Lawrenceville, NJ, John Somerville, Associate<br />
Scientist, Bristol-Myers Squibb Pharmaceutical Research<br />
Institute, Princeton, NJ, Jessica Reid, Student, Biology<br />
Department, Rider University, Lawrenceville, NJ, James<br />
Riggs, Pr<strong>of</strong>essor <strong>of</strong> Biology, Biology Department, Rider<br />
University, Lawrenceville, NJ<br />
Although the cellular components essential for an<br />
effective immune response are <strong>of</strong>ten found in the tumor<br />
microenvironment they fail to prevent cancer progression.<br />
T cell activation, dependent upon costimulation provided<br />
by antigen-presenting cells (APCs), is restrained in tumors.<br />
Macrophages are <strong>of</strong>ten found in large numbers in tumors.<br />
An unbalanced macrophage:T cell ratio fosters T cell<br />
suppression. We have developed an in vitro model that<br />
mimics this characteristic <strong>of</strong> the tumor microenvironment.<br />
Murine peritoneal cavity (PerC) macrophages suppress T<br />
cell activation by indoleamine 2,3-dioxygenase (IDO) and<br />
inducible nitric oxide synthase (iNOS) catalyzed consumption<br />
<strong>of</strong> tryptophan and arginine. 1-Methyl tryptophan and<br />
NG-monomethyl-L-arginine block these enzymes and permit<br />
T cell activation. We are testing various costimulatory<br />
ligand–receptor combinations to determine if T cell<br />
activation can be rescued by treatment with an immunobiologic.<br />
Rather than promoting T cell activation, CD28<br />
ligation was found to promote macrophage-mediated<br />
suppression. The suppression was triggered by T cell<br />
production <strong>of</strong> IFN 3 . Treatment with CD28-Ig did not release<br />
suppression. These data will be discussed in the context <strong>of</strong><br />
strategies to curb the suppressive activity evident in tissues<br />
with aberrant macrophage:T cell ratios. Supported by NIH<br />
AREA grant R15-AI060356-01.<br />
doi:10.1016/j.clim.2007.03.483<br />
Sa.97 Development and In Vitro Validation <strong>of</strong><br />
Anti-Mesothelin Biobodies that Prevent CA125/<br />
Mesothelin-dependent Cell Attachment<br />
Nathalie Scholler, Senior Staff Scientist, Fred Hutchinson<br />
Cancer Research Center, Seattle, WA, Lindsay Bergan,<br />
Research Technician, Fred Hutchinson Cancer Research<br />
Center, Seattle, WA, Jennifer Gross, Research Technician,<br />
Fred Hutchinson Cancer Research Center, Seattle, WA, Barry<br />
Nevin, Research Technician, Fred Hutchinson Cancer<br />
Research Center, Seattle, WA, Barbara Garvik, Research<br />
Technician, Fred Hutchinson Cancer Research Center,<br />
Seattle, WA, Nicole Urban, Department head, Fred<br />
Hutchinson Cancer Research Center, Seattle, WA<br />
Preventing peritoneal implantation <strong>of</strong> carcinoma cells<br />
could prolong ovarian cancer patient remission and<br />
survival. Peritoneal cells constitutively express mesothelin,<br />
a ligand for CA125 that is expressed by tumor cells.<br />
Blocking CA125/mesothelin-dependent cell attachment<br />
may then prevent or delay peritoneal metastatic recurrence.<br />
We previously developed an in vitro cell adhesion<br />
assay combining cells expressing CA125 and mesothelintransfected<br />
cells. We demonstrated that CA125/mesothelin-dependent<br />
cell adhesion could be blocked with<br />
mesothelin recombinant protein fused to an Ig domain<br />
(meso-Ig) and cross-linked with anti-Ig antibodies, or with<br />
anti-CA125 mouse monoclonal antibodies (mAb) which<br />
suggests that new therapies based upon affinity agents<br />
able to compete with or to block the CA125/mesothelin<br />
interaction could prevent or delay the development <strong>of</strong><br />
peritoneal metastasis. However, mAbs are highly immunogenic<br />
in vivo. Recombinant antibodies are emerging as an<br />
attractive alternative to mouse antibodies for in vivo<br />
imaging and therapy. We have developed a new class <strong>of</strong><br />
recombinant antibodies, referred to as biobodies (Bbs)<br />
that are secreted by yeast as in vivo biotinylated proteins.<br />
Building on our previous work, we generated Bbs that<br />
specifically bind to mesothelin. Anti-mesothelin recognition<br />
sequences were isolated from a yeast-display scFv<br />
library by magnetic and flow sorting, using an in vivo<br />
biotinylated mesothelin recombinant protein also secreted<br />
by yeast. Anti-mesothelin yeast-display scFv were transformed<br />
into Bbs and validated by ELISA and flow<br />
cytometry. We demonstrated that the anti-mesothelin<br />
Bbs corresponding to the yeast-display scFv pool <strong>of</strong> highest<br />
affinity could recognize both membrane-bound and soluble<br />
mesothelins, and block CA125/mesothelin-dependent cell<br />
adhesion.<br />
doi:10.1016/j.clim.2007.03.484<br />
S107
S108 Abstracts<br />
Sa.98 Visualization and Characterisation <strong>of</strong> Melan A<br />
Epitope (25–35)/HLA-A2 Complex by High Affinity<br />
Soluble Monoclonal T Cell Receptors (mTCRs)<br />
Samantha Paston, Avidex Ltd, Abingdon, Oxfordshire,<br />
England, Katherine Adams, Ms, Avidex Ltd, Abingdon,<br />
Oxfordshire, England, Giovanna Bossi, Avidex Ltd,<br />
Abingdon, Oxfordshire, England, Nathaniel Liddy, Avidex<br />
Ltd, Abingdon, Oxfordshire, England, Deborah Sutton,<br />
Avidex Ltd, Abingdon, Oxfordshire, England, Tara Mahon,<br />
Avidex Ltd, Abingdon, Oxfordshire, England, Peter Molloy,<br />
Avidex Ltd, Abingdon, Oxfordshire, England, Emma<br />
Gostick, Avidex Ltd, Abingdon, Oxfordshire, England, Andy<br />
Sewell, Department <strong>of</strong> Medical Biochemistry and<br />
<strong>Immunology</strong>, Cardiff, England, Bent Jakobsen, Avidex Ltd,<br />
Abingdon, Oxfordshire, England<br />
Cytotoxic T cells (CTL) use TCRs to detect tumor and viral<br />
antigens. Soluble TCRs are potential tools for therapeutic<br />
treatment <strong>of</strong> cancer, viral infections and autoimmune diseases.<br />
The Melan A protein (MART-1) was one <strong>of</strong> the first tumour<br />
associated antigens to be identified, and is over-expressed in<br />
80–100% <strong>of</strong> melanomas. CTL’s recognising the heterolytic<br />
ELAGIGILTV peptide can be found in both healthy donors and<br />
melanoma patients. Adoptive therapy and vaccination trials<br />
using the ELAGIGILTV peptide have resulted in some good<br />
clinical responses with partial or complete regression <strong>of</strong> some<br />
metastates. In healthy donors, up to 0.1% <strong>of</strong> Tcells in peripheral<br />
blood can be stained with the Melan A tetramer; patients have a<br />
higher frequency <strong>of</strong> Melan A specific Tcells where 1–15% <strong>of</strong> the T<br />
cellscanbestainedwiththetetramer.TheMelanAmTCRwas<br />
affinity matured to bind HLA-A2 presenting the heteroclytic<br />
ELAGIGILTVpeptidewithakDa<strong>of</strong>10pMandahalflife<strong>of</strong>67h.<br />
Here we describe the generation <strong>of</strong> a Tcell clone specific for the<br />
HLA-A2/Melan A epitope (26–35) derived from a healthy blood<br />
donor. Using this Melan A (26–35) mTCR we can specifically block<br />
cytokineproductionfromaMelanATcellclone.Inadditionwe<br />
are also able to detect and quantify the endogenous peptide<br />
processed and presented by melanoma cell lines using 3D<br />
fluoresence microscopy and flow cytometry. Visualisation and<br />
quantification <strong>of</strong> these specific antigen/MHC complexes enable<br />
the potential <strong>of</strong> mTCRs recognising these complexes as anticancer<br />
therapeutics to be evaluated.<br />
doi:10.1016/j.clim.2007.03.486<br />
Sa.100 AdCD40L Immunogene Therapy for Bladder<br />
Carcinoma<br />
Moa Fransson, PhD Student, <strong>Clinical</strong> <strong>Immunology</strong> Division,<br />
Uppsala, Sweden, Angelica Loskog, Scientist, <strong>Clinical</strong><br />
<strong>Immunology</strong> Division, Uppsala, Sweden, Thomas Tötterman,<br />
Pr<strong>of</strong>esor, <strong>Clinical</strong> <strong>Immunology</strong> Division, Uppsala, Sweden<br />
Recently regulatory T cells (Treg) have made a comeback in<br />
the immunological arena and the role <strong>of</strong> these cells in cancer is<br />
in focus. <strong>Clinical</strong> protocols that effectively counteract Tregmediated<br />
immunosuppression are in high demand. We developed<br />
an immunostimulatory gene therapy for urinary bladder<br />
cancer based on CD40L gene transfer into the tumor site. The<br />
efficacy <strong>of</strong> this therapy was evaluated in mouse and human<br />
experimental models <strong>of</strong> bladder cancer. The CD40L gene was<br />
transferred to the bladder wall <strong>of</strong> tumor-bearing mice by<br />
adenoviral vector instillation. AdCD40L gene therapy cured 60%<br />
<strong>of</strong> mice with pre-established aggressive tumors. Cured mice<br />
were completely resistant to s.c. challenge with wt tumor. The<br />
mRNA levels <strong>of</strong> the Treg transcription factor Foxp3 were<br />
measured in tumor biopsies and lymph nodes. There were no<br />
differences within the tumors <strong>of</strong> the different treatment groups.<br />
However, Foxp3 mRNA levels were down-regulated in the lymph<br />
nodes <strong>of</strong> AdCD40L treated mice. Human bladder cancer cell line<br />
cells were transduced with AdCD40L vector and used to<br />
stimulate immune cells. Cytokine and immune cell pr<strong>of</strong>iling<br />
indicated a Th1 type response. The AdCD40L-modified cell lines<br />
stimulated maturation <strong>of</strong> dendritic cells and cytotoxic Tcells as<br />
opposed to wt tumor cells. In conclusion, CD40L gene transfer<br />
evokes Th1 cytokine responses and counteracts Tregulatory cell<br />
development and/or function in the preclinical setting. We are<br />
currently conducting a phase I/II trial with AdCD40L in patients<br />
with bladder cancer.<br />
doi:10.1016/j.clim.2007.03.487<br />
Sa.101 Functional T Cell Responses to Tumor<br />
Antigens in Breast Cancer Patients Have a Unique<br />
Phenotype and Cytokine Signature<br />
Margaret Inokuma, Scientist, BD Biosciences, Immune<br />
Function, San Jose, CA, Corazon dela Rosa, Research<br />
Associate, University <strong>of</strong> Washington, Seattle, WA, Perry<br />
Haaland, BD Fellow, BD Technologies, Research Triangle<br />
Park, NC, Douglas Petry, patent attorney, BD Biosciences,<br />
San Jose, CA, Janet Siebert, president, CytoAnalytics,<br />
Denver, CO, MengXiang Tang, Senior Algorithm Developer,<br />
BD Biosciences, San Jose, CA, John Dunne, Associate<br />
Director, BD Biosciences, San Jose, CA, Vernon Mai, Director,<br />
BD Biosciences, San Jose, CA, Mary Disis, Associate<br />
Pr<strong>of</strong>essor, University <strong>of</strong> Washington, Department <strong>of</strong><br />
Oncology, Seattle, WA, Holden Maecker, Research Grp<br />
Manager, BD Biosciences, San Jose, CA<br />
The prevalence with which endogenous tumor antigens<br />
induce host T cell responses is unclear. Even when such<br />
responses are detected, they do not usually result in<br />
spontaneous remission <strong>of</strong> the cancer. We hypothesized that<br />
this might be associated with a predominant phenotype and/or<br />
cytokine pr<strong>of</strong>ile <strong>of</strong> tumor-specific responses that is different<br />
from protective Tcell responses to other chronic antigens, such<br />
as cytomegalovirus (CMV). We detected significant T cell<br />
responses to CEA, HER-2/neu, and/or MAGE-3 in 17 <strong>of</strong> 21<br />
breast cancer patients naive to immunotherapy. The pattern <strong>of</strong><br />
T cell cytokines produced in response to tumor-associated<br />
antigens (TAAs) in breast cancer patients was significantly<br />
different from that produced in response to CMV in the same<br />
patients. Specifically, there was a higher proportion <strong>of</strong> IL-2<br />
producing CD8+ T cells, and a lower proportion <strong>of</strong> IFN γproducing<br />
CD4+ and CD8+ T cells responding to TAAs compared<br />
to CMV antigens. Finally, the phenotype <strong>of</strong> TAA-responsive CD8+<br />
T cells in breast cancer patients was almost completely CD28<br />
+CD45RA− (central memory phenotype). CMV-responsive CD8+<br />
Tcellsinthesamepatientswerebroadlydistributedamong<br />
phenotypes, and contained a high proportion <strong>of</strong> terminal
Abstracts<br />
effector cells (CD27−CD28−CD45RA+) that were absent in the<br />
TAA responses. Taken together, these results suggest that TAAresponsive<br />
T cells are induced in breast cancer patients, but<br />
those Tcells are phenotypically and functionally different from<br />
CMV-responsive Tcells. Immunotherapies directed against TAAs<br />
may need to alter these T cell signatures in order to be<br />
effective.<br />
doi:10.1016/j.clim.2007.03.488<br />
Sa.102 Dendritic Cell-based Therapy for Mantle Cell<br />
Lymphoma<br />
Corey Munger, Graduate Student, Nebraska Medical Center,<br />
Genetics, Cell Biology, and Anatomy, Omaha, NE, Ganapati<br />
Hegde, Postdoctoral Fellow, Nebraska Medical Center,<br />
Genetics, Cell Biology, and Anatomy, Omaha, NE, Julie Vose,<br />
Pr<strong>of</strong>essor, Internal Medicine, Omaha, NE, Shantaram Joshi,<br />
Pr<strong>of</strong>essor, Nebraska Medical Center, Genetics Cell Biology,<br />
and Anatomy, Omaha, NE, Dennis Weisenburger, Pr<strong>of</strong>essor,<br />
Pathology and Microbiology, Omaha, NE<br />
Mantle cell lymphoma (MCL) is an incurable B cell<br />
malignancy due to the development <strong>of</strong> therapy-resistant<br />
cells. The ability <strong>of</strong> dendritic cells (DC) to stimulate tumorspecific<br />
cytotoxic T lymphocytes (CTL) makes DC-based<br />
therapy a promising strategy to treat therapy-resistant MCL.<br />
Therefore, we investigated the efficacy <strong>of</strong> DC-based immunotherapy<br />
to treat resistant MCL. Specifically, DCs loaded<br />
with MCL lysate, DCs fused with MCL cells, and DCs<br />
electroporated with MCL RNA were used to generate<br />
tumor-specific CTLs against the MCL cell line, Granta 519.<br />
CTL-mediated cytotoxicities at a 50:1 effector-to-target<br />
ratio using each method <strong>of</strong> DC priming are as follows: 58.4<br />
±12.7% (MCL lysate-pulsed), 54.0±8.3% (MCL RNA-transfection),<br />
and 59.3±8.4% (DC-MCL hybrid). Cytotoxicity toward<br />
the irrelevant HLA-matched cell line MDA-231 was 28.7±<br />
3.6%. To assess the in vivo cytotoxic abilities <strong>of</strong> CTLs<br />
stimulated by DC-MCL hybrids, NOD/SCID mice transplanted<br />
intravenously with 1×106 Granta 519 cells were treated with<br />
four weekly injections <strong>of</strong> 3×106 HLA-matched CTLs. Histological<br />
analysis revealed that 66% <strong>of</strong> the control mice at week<br />
five displayed significant tumor growth in the liver and<br />
kidneys versus only 16% in the treatment group. In addition to<br />
reducing the number <strong>of</strong> tumor nodules, the average size <strong>of</strong><br />
the nodules was decreased in treated mice versus the<br />
untreated control mice. These studies demonstrate each<br />
method <strong>of</strong> DC priming stimulated tumor-specific CTLs in vitro<br />
and administration <strong>of</strong> DC-MCL hybrid-stimulated CTLs inhibited<br />
tumor formation in vivo. This study was supported by<br />
the Lymphoma Research Foundation, New York, NY.<br />
doi:10.1016/j.clim.2007.03.489<br />
Sa.103 Targetting Sonic Hedgehog-GLI Signaling for<br />
the Treatment <strong>of</strong> Mantle Cell Lymphoma<br />
Ganapati Hegde, Postdoctoral Research Associate,<br />
Department <strong>of</strong> Genetics, Cell Biology and Anatomy, Omaha,<br />
NE, Corey Munger, Graduate Student, Department <strong>of</strong><br />
Genetics, Cell Biology and Anatomy, Omaha, NE, Katy<br />
Emanuel, Summer Student, Department <strong>of</strong> Genetics, Cell<br />
Biology and Anatomy, Omaha, NE, Dennis Weisenburger,<br />
Pr<strong>of</strong>essor, Pathology and Microbiology, Omaha, NE, Avadhut<br />
Joshi, Graduate student, Department <strong>of</strong> Genetics, Cell<br />
Biology and Anatomy, Omaha, NE, Julie Vose, Pr<strong>of</strong>essor,<br />
Internal Medicine, Oncology/Hematology, Omaha, NE,<br />
Shantaram Joshi, Pr<strong>of</strong>essor, Department <strong>of</strong> Genetics, Cell<br />
Biology and Anatomy, Omaha, NE<br />
Mantle cell lymphoma (MCL) has one <strong>of</strong> the worst clinical<br />
outcomes among the B cell lymphomas with a median<br />
survival <strong>of</strong> only 3–4 years. Particularly, the blastoid variant<br />
has a poorer prognosis compared to classical MCL. Therefore,<br />
a better understanding <strong>of</strong> the underlying mechanisms that<br />
regulate MCL proliferation and survival is clearly needed.<br />
Since, sonic hedgehog (Shh)-GLI signaling has been shown to<br />
be important in the proliferation/survival <strong>of</strong> several cancers,<br />
and no such information is available in any B cell malignancies<br />
including MCL, this study was undertaken. Our<br />
results demonstrate that the molecules associated with Shh<br />
signaling (PTCH, SMO, GLI) are considerably expressed in<br />
classical, blastoid and patients’ primary MCL. Overexpression<br />
<strong>of</strong> PTCH and GLI1, the Shh-GLI signaling target genes, in<br />
blastoid versus classical MCL suggests constitutive expression<br />
<strong>of</strong> these genes in blastoid variant. Classical, but not the<br />
blastoid MCL cells responded (pb0.01) to perturbation <strong>of</strong> Shh<br />
signaling in the presence <strong>of</strong> exogenous Shh/cyclopamine.<br />
Furthermore, down-regulation <strong>of</strong> GLI transcription factors<br />
using anti-sense oligonucleotides resulted in significantly<br />
(pb0.001) decreased proliferation <strong>of</strong> both blastoid and<br />
classical MCL, and significantly (pb0.001) increased their<br />
susceptibility to chemotherapy. In addition, down-regulation<br />
<strong>of</strong> GLI by anti-sense ODN decreased Cyclin D1 (CCND1) and<br />
BCL2 transcript levels, which suggests that these key<br />
molecules might be regulated by GLI in MCL. Thus, these<br />
results suggest a significant role for Shh-GLI signaling in the<br />
proliferation <strong>of</strong> MCL, and targeting GLI as therapeutic<br />
approach to treat MCL effectively is very promising (this<br />
study was supported by the Lymphoma Research Foundation,<br />
New York).<br />
doi:10.1016/j.clim.2007.03.490<br />
S109<br />
Sa.104 The Role <strong>of</strong> Thrombospondin-1 in Driving<br />
Regulatory T Cell Generation Following<br />
Extracorporeal Photopheresis<br />
Amy Krutsick, Senior Scientist, Therakos, Inc. RandCD,<br />
Exton, PA, Peter O’Brien, Principal Scientist, Therakos, Inc.<br />
RandCD, Exton, PA, Ryan Gailey, Research Associate I,<br />
Therakos, Inc. RandCD, Exton, PA, Kim Campbell, Principal<br />
Research Scientist, Therakos, Inc. RandCD, Exton, PA, Frank<br />
Strobl, Director <strong>of</strong> Scientific Affairs, Therakos, Inc. RandCD,<br />
Exton, PA<br />
Extracorporeal photopheresis (ECP) is an immune cell<br />
therapy approved for the palliative treatment <strong>of</strong> cutaneous<br />
T cell lymphoma and has demonstrated activity in<br />
immune-mediated inflammatory diseases. During ECP,
S110 Abstracts<br />
peripheral blood leukocytes are treated ex vivo with 8methoxypsoralen<br />
and UVA light, rendering cells apoptotic.<br />
Reinfusion <strong>of</strong> ECP-treated cells modulates immune<br />
responses through the generation <strong>of</strong> tolerogenic dendritic<br />
cells and T regulatory cells (Tregs). We previously<br />
demonstrated that ECP-treated peripheral blood mononuclear<br />
cells (PBMCs) co-cultured directly with T cells<br />
result in the generation <strong>of</strong> a regulatory T cell population.<br />
ECP-generated Tregs can suppress a syngeneic T cell<br />
response in a contact-dependent fashion by greater than<br />
70%. These Tregs demonstrate a modest increase in Foxp3<br />
expression and exhibit an anergic phenotype upon in vitro<br />
re-stimulation. We found that monocytes are required for<br />
ECP-mediated Treg generation and apoptotic ECP-treated<br />
monocytes secrete a significant level (N500 ng/ml) <strong>of</strong><br />
Thrombospondin-1 (TSP-1). TSP-1 is an extracellular<br />
matrix protein with anti-angiogenic properties shown by<br />
others to have immunomodulatory effects on T cells<br />
through interactions with CD47. We hypothesized that<br />
TSP-1 is involved in the generation <strong>of</strong> Tregs via ECP cells.<br />
To confirm this hypothesis, TSP-1 activity was blocked<br />
through addition <strong>of</strong> anti-CD47 to cultures containing ECPtreated<br />
PBMCs and T cells. Blocking this TSP-1 receptor on<br />
T cells inhibited ECP-mediated Treg generation. Additional<br />
studies are in progress to further evaluate the role <strong>of</strong> TSP-<br />
1 in the mechanism <strong>of</strong> action <strong>of</strong> ECP. TSP-1 may be<br />
important for the clinical benefit <strong>of</strong> ECP in a variety <strong>of</strong><br />
pathophysiological disorders.<br />
doi:10.1016/j.clim.2007.03.491<br />
Sa.105 Cytotocix Effect <strong>of</strong> Total Saffron Extract on<br />
Human Hepatocell Carcinoma (HCC)<br />
Jalil Tavakkol-Afshari, Vice President for Research, Head,<br />
Department <strong>of</strong> Immunogenetics and Cell Culture, Bu-Ali<br />
Research Institute (BARI), Department <strong>of</strong> Immunogenetics<br />
and Cell Culture, Mashhad, Iran, Khadijeh Nejad<br />
Shahrokhabadi, Researcher, Bu-Ali Research Institute<br />
(BARI), Department <strong>of</strong> Immunogenetics, Mashhad, Iran,<br />
Hassan Rakhshandeh, Pharmacologist, School <strong>of</strong><br />
Pharmocology, Mashhad University <strong>of</strong> Medical Sciences,<br />
Mashhad, Iran, Azam Brook, Researcher, Bu-Ali Research<br />
Institute (BARI), Department <strong>of</strong> Immunogenetics, Mashhad,<br />
Iran<br />
Saffron – dried stigma <strong>of</strong> Crocus Sativus L. – is used as a<br />
nutritional spice and dry additive in cooking. The antitumor<br />
effect <strong>of</strong> saffron extract has been proved both in vitro and<br />
in vivo during recent years. In this research, cytotoxic<br />
effect <strong>of</strong> total saffron aqueous extract on human Hepatocell<br />
Carcinoma (HepG2) and mouse fibroblastic cells (L929)<br />
(as controls) in vitro has been investigated. Effects <strong>of</strong><br />
extract on quantitative proliferation <strong>of</strong> each cell line were<br />
determined using (MTT) colorimetric assay. MTT assay is a<br />
fast, sensitive and quantitative method for all kinds <strong>of</strong> cell<br />
proliferation by spectrophotometry. Different amounts <strong>of</strong><br />
extract were used and results showed that the 50%<br />
inhibition occurred in tumoral cells at the concentration<br />
<strong>of</strong> 400 microgram/milliliter that had no inhibitory effects<br />
on normal cells. According to our findings, the effect <strong>of</strong><br />
saffron extract may be useful in vivo against hepatic tumoral<br />
cells.<br />
doi:10.1016/j.clim.2007.03.492<br />
Sa.106 Optimizing Peptide Loading <strong>of</strong> Dendritic<br />
Cells Using TCRm Antibodies<br />
Tffany Nguyen, Senior Research Technician, Receptor Logic,<br />
Ltd. Amarillo, TX, Olivier Faure, Scientist, IDM, Inc. Irvine,<br />
CA, Francisca Neethling, Scientist, Receptor Logic, Ltd.<br />
Amarillo, TX, Roy Guillermo, Scientist, IDM, Inc. Irvine, CA,<br />
Sun min Lee, Scientist, IDM, Inc. Irvine, CA, James Bender,<br />
Scientist, IDM, Inc. Irvine, CA, Jon A. Weidanz, Assistant<br />
Pr<strong>of</strong>essor, Texas Tech University Health Sciences Center,<br />
Amarillo, TX<br />
Dendritic cells (DCs) represent potential vaccine vehicles<br />
for the treatment <strong>of</strong> cancer. Peptide-loaded DC’s require no<br />
protein processing and are amendable for development <strong>of</strong><br />
“personalized” vaccines. Conditions for peptide-loading DC<br />
are poorly described and have not yet been optimized. We<br />
examined peptide-loading conditions for DC using a TCRm<br />
antibody that specifically recognizes a peptide<br />
(TMTRVLQGV40-48) presented by HLA-A*0201 derived from<br />
the tumor-associated antigen human chorionic gonadotropin<br />
beta (hCGb). Characterization <strong>of</strong> the 3F9 TCRm included<br />
ELISA and flow cytometric analysis and demonstrated specific<br />
binding to peptide–HLA-A2 tetramer and T2 cells loaded with<br />
the TMT peptide. Moreover, the 3F9 TCRm displayed<br />
detection sensitivity in the picomolar range. TCRm staining<br />
<strong>of</strong> DCs was shown to be dependent on peptide concentration<br />
with maximum and minimum mean fluorescence intensity<br />
(MFI) reported at 80 μM and 10 μM peptide, respectively. We<br />
next examined the relationship between pulsing time and<br />
peptide presentation. Immature DCs were pulsed with 20 μM<br />
peptide for 1, 2, 4, 6, 8 and 22 h followed by cytokine-induced<br />
maturation during the final six hours. DC pulsed for shorter<br />
periods <strong>of</strong> time displayed TCRm staining with higher MFI<br />
values, suggesting that longer pulse times decrease peptide-<br />
HLA presentation. DCs were then pulsed with a peptide<br />
cocktail (TMT+6 other peptides) to determine TMT peptide<br />
loading efficiency. Similar TCRm staining intensities were<br />
observed for DC pulsed with TMT peptide alone and cells<br />
pulsed with the peptide cocktail. These findings suggest that<br />
TCRm may be useful tools for the development and<br />
standardization <strong>of</strong> peptide-pulsed DC vaccines.<br />
doi:10.1016/j.clim.2007.03.493<br />
Sa.107 Phase I Study <strong>of</strong> the Anti-MUC-1 Antibody,<br />
huHMFG1 (AS1402), in Patients with Advanced<br />
Breast Cancer<br />
Nigel Courtenay-Luck, Chief Scientific Officer, Antisoma<br />
Research Ltd, London, England, Mark Pegram, Associate<br />
Pr<strong>of</strong>essor <strong>of</strong> Medicine, David Geffen School <strong>of</strong> Medicine, Los<br />
Angeles, CA<br />
Background: A 20 amino acid MUC-1 core tandem repeat<br />
is aberrantly glycosylated in N90% <strong>of</strong> epithelial tumors,
Abstracts<br />
exposing the peptide sequence PDTRP. Humanized IgG1 K<br />
monoclonal antibody huHMFG1 (AS1402, formerly R1550),<br />
binds to PDTRP (Kd ∼3 nM) and, in vitro, induces potent<br />
antibody dependent cellular cytotoxicity (ADCC). This study<br />
was designed to evaluate the safety, tolerability and<br />
recommended dose <strong>of</strong> huHMFG1 in patients with advanced<br />
breast cancer. Methods: 26 patients were enrolled with<br />
locally advanced or metastatic breast cancer, which was<br />
progressive following up to 3 prior chemotherapy regimens.<br />
Patients were randomly assigned to 4 cohorts, each<br />
allocated to receive huHMFG1 at a dose <strong>of</strong> 1, 3, 9, or<br />
16 mg/kg, which was administered via I.V. infusion with<br />
repeated weekly dosing until disease progression. <strong>Clinical</strong><br />
and laboratory analysis was performed regularly. Results:<br />
The maximum tolerated dose (MTD) <strong>of</strong> huHMFG1 was not<br />
reached in this study and no serious adverse events were<br />
reported. No anti-huHMFG1 antibodies were detected.<br />
Response data are available from 22 patients with the<br />
best recorded clinical response, using RECIST criteria, being<br />
stable disease. Time to tumor progression ranged between<br />
28 and 119 days. Conclusion: huHMFG1 appears to be well<br />
tolerated with the MTD exceeding the doses tested. This<br />
study showed cases <strong>of</strong> prolonged stable disease among<br />
breast cancer patients who had relapsed following chemotherapy,<br />
suggesting that evaluation <strong>of</strong> huHMFG1 in phase<br />
II trials is warranted.<br />
doi:10.1016/j.clim.2007.03.494<br />
Sa.108 30 Years from Creation <strong>of</strong> the First<br />
Cytochemical Method Analysing RNP, DNP and<br />
Cationic Proteins in Circulating Leukocytes <strong>of</strong><br />
Cancer Patients as a Tool for Early Diagnostics <strong>of</strong><br />
Malignancies<br />
Elissaveta Borissova Zvetkova, Associated Pr<strong>of</strong>essor, IEMAM,<br />
S<strong>of</strong>ia, Bulgaria, Georgi Stefanov Kostov, Head <strong>of</strong> Surgery<br />
Department, Oncological Hospital, Veliko Tarnovo, Yordanka<br />
Georgieva Gluhcheva, Research Associate, IEMAM, Cell<br />
Differentiation, S<strong>of</strong>ia, Bulgaria<br />
Background: In our efforts to analyse cellular features <strong>of</strong><br />
peripheral blood leukocytes in cancer patients and tumorbearing<br />
animals, we developed (30 years ago) an accurate<br />
method for early detection <strong>of</strong> malignancies (1–4). Methods:<br />
We combined our cytological/cytochemical method for<br />
simultaneous visualization <strong>of</strong> nucleoproteins (RNP and DNP)<br />
and cationic proteins with ultrastructural and cytophotometrical<br />
techniques. Results and Discussion: Examining a panel<br />
<strong>of</strong> activation/deactivation cell markers in peripheral blood<br />
leukocytes <strong>of</strong> cancer and precancerous patients, we observed<br />
specific abnormalities: (1) Quantitative reduction <strong>of</strong> the<br />
cytoplasmic (ribosomal) RNP-content in the peripheral blood<br />
mononuclears T-lymphocytes and monocytes, as an additional<br />
tool for early cancer diagnostics; (2) Changes in the<br />
quantity, condensation and distribution <strong>of</strong> DNP in the nuclear<br />
chromatin <strong>of</strong> mono-and polymorphonuclear leukocytes as<br />
cytological signs for tumor-microenvironment-induced transcriptional<br />
arrest and apoptosis. (3) Polymorphism in size,<br />
staining properties and high extractibility <strong>of</strong> cytoplasmic<br />
(cationic proteins’ containing) granules <strong>of</strong> circulating poly-<br />
morphonuclear granulocytes—neutrophils and eosinophils.<br />
Today, 30 years later, our old hypothesis that cancer is not<br />
local, but systemic nuclear chromatin disease altering<br />
peripheral blood cell genome expression is in very good<br />
agreement with recent data based on gene expression<br />
patterns in peripheral blood cells <strong>of</strong> cancer patients (5) as<br />
monitors for early detection <strong>of</strong> malignant disease elsewhere<br />
in the body. References: (1) Acta histochem., 57, 1976, 1. (2)<br />
Folia Haematol., 106/2, 1979, 205. (3) Arch. Union Med Balk.,<br />
XX/1–2, 1984, 140. (4) Acta morphol. and anthropol., 5,<br />
2000, 3. (5) Breast Cancer Res., 7/5, 2005, R634.<br />
doi:10.1016/j.clim.2007.03.495<br />
Sa.109 Human Lung Tumor Cells Modifies Rates <strong>of</strong><br />
CD4+CD25+ T Regulatory, CD19+CD23+ B Regulatory<br />
Cells, CD3+CD95+ Cells, NK Cells and B Lymphocytes<br />
Bayram Kiran, Tip Fakultesi, Temel Bilimler Binasi, Istanbul,<br />
Turkey, Akif Turna, Yedikule Hospital for Chest Diseases and<br />
Thoracic Surgery, Kadikoy, Istanbul, Turkey, Alper Yener,<br />
Istanbul Tip Fakultesi, Istanbul, Turkey, Atilla Gürses,<br />
Yedikule Gogus Hastaliklari Hastanesi, Istanbul, Turkey,<br />
Andac Salman, Istanbul Tip Fakultesi, Istanbul, Turkey,<br />
Selim Badur, Istanbul Tip Fakultesi, Temel Bilimler Binasi,<br />
Istanbul, Turkey<br />
The role <strong>of</strong> T regulatory cells and NK cells in human lung<br />
cancer has not yet been clarified. Our aim was to evaluate<br />
how the microenvironment <strong>of</strong> a tumor mass induced phenotypical<br />
changes in lymphocyte subsets. Our subjects were<br />
10 patients with resectable non-small cell lung cancer. We<br />
evaluated 47 different phenotypically different lymphocyte<br />
subsets in blood samples taken from the pulmonary artery,<br />
pulmonary vein <strong>of</strong> a tumor bearing pulmonary lobe and<br />
peripheral blood during pulmonary resectional surgery. We<br />
showed that, tumor cells boosted CD25high+CD4high+T<br />
lymphocytes (Treg cells), B lymphocytes, NK and CD19+<br />
CD23+ cells (Breg cells) and CD3+CD95+ (FasL+T lymphocytes)<br />
(p=0.015, p=0.001, p=0.02, p=0.04, p=0.03 respectively).<br />
However, Mac-1 cells and HLADR+T lymphocytes<br />
were found to be decreased by tumor mass (p=0.04 and<br />
p=0.04), whereas only Breg, NK cell and B lymphocyte<br />
subsets were found to be expansed. In addition, the patients<br />
showed expansions <strong>of</strong> CD45R0+ activated T lymphocytes and<br />
CD18+CD11b+(Mac-1) cell subsets without significant alteration<br />
by tumor microenvironment. In conclusion, subsets <strong>of</strong><br />
Treg, Breg cells, FasL+ T lymphocytes, B lymphocytes were<br />
modified by lung cancer microenvironment itself. This study<br />
also showed that, peripheral blood might not be good source<br />
for investigating the anti-tumor immunity since only Breg, NK<br />
cell and Treg cell subsets were found expansed peripherally.<br />
T lymphocytes and Mac-1 cells may be modified by systemic<br />
antitumor response rather than by local tumoral microenvironment.<br />
This study provides important insight into how<br />
tumor infiltrating lymphocytes and peripheral immune<br />
systems may be different in terms <strong>of</strong> lymphocyte subset<br />
expansion dynamics.<br />
doi:10.1016/j.clim.2007.03.496<br />
S111
S112 Abstracts<br />
Sa.110 ABCB5 Gene is Expressed in Acral Melanoma<br />
<strong>of</strong> Poor Prognosis<br />
Norma Herrera-Gonzàlez, Pharmaceutical Industrial<br />
Chemest, Postgraduate Division, Mexico, Ismael Vasquez,<br />
Medical Doctor, Postgraduate Division, Mexico, Marco<br />
Antonio Meraz-Rios, Pharmacobiology Chemist, Biomedicine<br />
Department, CINVESTAV, Mexico, Cleva Villanueva, Medical<br />
Doctor, Postgraduate Division, Mexico<br />
Cutaneous melanomais a malignant neoplasm characterized<br />
by a high risk <strong>of</strong> metastasis disease and death.<br />
Metastatic melanoma displays strong resistance against<br />
antineoplastic drugs. Combination <strong>of</strong> chemotherapy and<br />
radiotherapy has been tried in patients and the best response<br />
rates have been less than 20%. The dramatic increase in the<br />
incidence and mortality <strong>of</strong> melanoma highlights the need for<br />
improved methods <strong>of</strong> diagnosis as well as a better understanding<br />
<strong>of</strong> basic concepts <strong>of</strong> some types <strong>of</strong> melanoma that<br />
have been scarcely studied as Acral Melanoma (AM), this<br />
work describes a molecular screen for differentially<br />
expressed genes in AM, the most common melanoma in<br />
Hispanic population. We used differential display reverse<br />
transcription-polymerase chain reaction and compared the<br />
gene expression pattern between primary melanomas and<br />
normal skin. In this work we fond an overexpression <strong>of</strong> the<br />
ABCB5 gene in melanoma tissue. ABCB5 has been recently<br />
characterized in normal human melanocytes and has been<br />
implicated in the regulation <strong>of</strong> progenitor cell fusion. ABCB5<br />
amplification bands were detected in nine <strong>of</strong> ten melanoma<br />
tissues by using RT-PCR. In situ hybridization using specific<br />
probes showed the presence <strong>of</strong> this gene in paraffin<br />
melanoma tissues. We suggest that this event might be<br />
related to metastatic melanoma behavior.<br />
doi:10.1016/j.clim.2007.03.497<br />
Sa.111 MHC Class I and MICA Expressions in Liver<br />
Fluke Associated Cholangiocarcinoma<br />
Wachanan Wongsena, Lecturer, Biomedical Sciences<br />
Program, Graduate School, Khon Kaen University, Khon<br />
Kaen, Thailand, Solda Ferrone, Educator/Research Director,<br />
Roswell Park Cancer Institute, Buffalo, NY, Richard Cheney,<br />
Pathologist/Dermatopathologist, Roswell Park Cancer<br />
Institute, Buffalo, NY, Chanvit Leelayuwat, Educator, The<br />
Centre for Research and Development <strong>of</strong> Medical Diagnostic<br />
Laboratories, Khon Kaen University, Khon Kaen, Thailand,<br />
Banchob Sripa, Educator, Liver Fluke and<br />
Cholangiocarcinoma Research Center, Khon Kaen, Thailand<br />
Alterations <strong>of</strong> HLA class I antigens and MHC class I chain<br />
related gene A (MICA), a ligand for activating receptor,<br />
NKG2D, have been described in several types <strong>of</strong> tumors. Such<br />
a study in cholangiocarcinoma has not been reported. A total<br />
<strong>of</strong> 102 paraffin-embedded cholangiocarcinoma lesions were<br />
stained by mouse anti-HLA class I heavy chain, anti-β-2microglobulin,<br />
anti-Tapasin, anti-TAP1, anti-TAP2, anti-<br />
LMP2, anti-LMP7, and three <strong>of</strong> anti-MICA monoclonal antibodies<br />
using immunoperoxidase reaction. HLA class I heavy<br />
chain, β-2-microglobulin, Tapasin, TAP1, TAP2, LMP2 and<br />
LMP7 were lost in 12.7, 5.9, 6.9, 2.0, 1.0, 6.9 and 8.8% <strong>of</strong> the<br />
lesions tested, respectively. Although most <strong>of</strong> the lesions<br />
expressed HLA class I heavy chain and β-2-microglobulin, the<br />
expression was restricted to the cytoplasm in 68.5 and 64.6%<br />
<strong>of</strong> the lesions, respectively. Loss <strong>of</strong> HLA class I heavy chain<br />
expression was associated with late stage (p=0.034) and<br />
poor differentiation <strong>of</strong> tumors (p=0.014). Loss <strong>of</strong> LMP7<br />
expression was also associated with poor differentiation <strong>of</strong><br />
tumors and Adenosquamous/squamous type (p =0.038).<br />
Multivariate analysis showed that Tapasin and TAP1 losses<br />
were associated with a high risk <strong>of</strong> death. Although 85% <strong>of</strong><br />
CCA lesions were stained with anti-MICA, most <strong>of</strong> them were<br />
restricted in the cytoplasm. Thus, MICA expression was not<br />
associated with any parameters even in cases <strong>of</strong> HLA class I<br />
loss. Our results demonstrated several abnormalities in HLA<br />
class I antigens and MICA expressions suggesting the potential<br />
mechanisms utilized by cholangiocarcinoma for immune<br />
evasion.<br />
doi:10.1016/j.clim.2007.03.498<br />
Sa.112 CD8+CD28− T Suppressor Lymphocytes<br />
Inhibiting T Cell Proliferative and Cytotoxic<br />
Functions Infiltrate Human Cancers<br />
Gilberto Filaci, Associate Pr<strong>of</strong>essor <strong>of</strong> Internal Medicine,<br />
University <strong>of</strong> Genoa, Department <strong>of</strong> Internal Medicine and<br />
Centre <strong>of</strong> Excellence for Biomedical Research, Genoa, Italy,<br />
Daniela Fenoglio, Assistant Pr<strong>of</strong>essor, University <strong>of</strong> Genoa,<br />
Centre <strong>of</strong> Excellence for Biomedical Research, Genoa, Italy,<br />
Marco Fravega, Fellow, University <strong>of</strong> Genoa, Centre <strong>of</strong><br />
Excellence for Biomedical Research, Genoa, Italy, Giacomo<br />
Borgonovo, Associate Pr<strong>of</strong>essor, University <strong>of</strong> Genoa,<br />
Department <strong>of</strong> Surgical and Morphological Disciplines and<br />
Integrated Methodologi, Genoa, Italy, Gianluca Ansaldo,<br />
Assistant Pr<strong>of</strong>essor, University <strong>of</strong> Genoa, Department <strong>of</strong><br />
Surgical and Morphological Disciplines and Integrated<br />
Methodologi, Genoa, Italy, Paolo Traverso, Assistant<br />
Pr<strong>of</strong>essor, University <strong>of</strong> Genoa, Urology Department, Genoa,<br />
Barbara Villaggio, Technician, University <strong>of</strong> Genoa,<br />
Department <strong>of</strong> Internal Medicine, Genoa, Italy, Jean Louis<br />
Ravetti, Director <strong>of</strong> Anatomic Pathology, San Martino<br />
Hospital, Anatomic Pathology, Genoa, Italy, Giorgio<br />
Carmignani, Full Pr<strong>of</strong>essor, University <strong>of</strong> Genoa, Urology<br />
Department, Genoa, Giancarlo Torre, Full pr<strong>of</strong>essor,<br />
Department <strong>of</strong> Surgical and Morphological Disciplines and<br />
Integrated Methodologies, University <strong>of</strong> Gen, Genoa, Italy,<br />
Francesco Indiveri, Full Pr<strong>of</strong>essor <strong>of</strong> Internal Medicine,<br />
University <strong>of</strong> Genoa, Department <strong>of</strong> Internal Medicine and<br />
Centre <strong>of</strong> Excellence for Biomedical Research, Genoa, Italy<br />
Since an important role in determining tumor evasion<br />
from immune control might be played by tumor infiltrating<br />
regulatory lymphocytes we performed a study aimed at<br />
characterizing phenotype and function <strong>of</strong> CD8+CD28− T<br />
suppressor cells infiltrating human cancer. Lymphocytes<br />
infiltrating primitive tumor lesion and/or satellite lymph<br />
node from a series <strong>of</strong> 42 human cancers were phenotypically<br />
studied and functionally analyzed by suppressor assays. The<br />
unprecedented observation was made that CD8+CD28− T<br />
suppressor lymphocytes are almost constantly present and<br />
functional in human tumors, being able to inhibit both T cell
Abstracts<br />
proliferation and cytotoxicity. CD4+CD25+ T regulatory<br />
lymphocytes associate with CD8+CD28− T suppressor cells<br />
so that the immunosuppressive activity <strong>of</strong> tumor infiltrating<br />
regulatory T cell subsets, altogether considered, may<br />
become predominant. The infiltration <strong>of</strong> regulatory T cells<br />
seems tumor-related, being present in metastatic but not in<br />
metastasis free satellite lymph nodes; it likely depends on<br />
both in situ generation (via cytokine production) and<br />
recruitment from the periphery (via chemokine secretion).<br />
Collectively, these results have pathogenic relevance and<br />
implication for immunotherapy <strong>of</strong> cancer.<br />
doi:10.1016/j.clim.2007.03.499<br />
Sa.113 GCN2 Kinase: An Evolutionarily Conserved<br />
Mechanism <strong>of</strong> Human T Lymphocyte Suppression<br />
Upon Arginine Depletion<br />
Markus Munder, Instructor in Medicine, University <strong>of</strong><br />
Heidelberg. Department <strong>of</strong> Hematology, Oncology and<br />
Rheumatology, Heidelberg, Germany, Michelle Bhairo,<br />
Research Assistant, University <strong>of</strong> Heidelberg. Institute <strong>of</strong><br />
<strong>Immunology</strong>, Heidelberg, Germany, Thomas Giese, Group<br />
Leader, University <strong>of</strong> Heidelberg. Institute <strong>of</strong> <strong>Immunology</strong>,<br />
Heidelberg, Germany, Claudia Luckner, Research Assistant,<br />
University <strong>of</strong> Heidelberg, Department <strong>of</strong> Hematology,<br />
Oncology and Rheumatology, Heidelberg, Germany, Anthony<br />
Ho, Head <strong>of</strong> Department, University <strong>of</strong> Heidelberg.<br />
Department <strong>of</strong> Hematology, Oncology and Rheumatology,<br />
Heidelberg, Germany, Stefan Meuer, Head <strong>of</strong> Department,<br />
University <strong>of</strong> Heidelberg. Institute <strong>of</strong> <strong>Immunology</strong>,<br />
Heidelberg, Germany<br />
Impaired T cell immunity is a cardinal feature <strong>of</strong> chronic<br />
inflammation and tumor growth. We have recently demonstrated<br />
that human polymorphonuclear leukocytes (PMN)<br />
constitutively express high activities <strong>of</strong> the arginine-metabolising<br />
enzyme arginase. Upon PMN cell death (e.g., during<br />
purulent inflammation) this enzyme is liberated and metabolizes<br />
arginine to ornithine and urea in the microenvironment.<br />
This arginine depletion completely inhibits<br />
proliferation and effector functions <strong>of</strong> activated human T<br />
lymphocytes while their viability remains unaltered. Specifically,<br />
the T cell cytokine response is impaired at a<br />
posttranscriptional level by arginine depletion. Arginasemediated<br />
arginine depletion is likely a key mechanism <strong>of</strong> T<br />
cell immunosuppression in the inflammatory or tumor<br />
environment. We now wanted to further clarify how T cell<br />
reactivity is turned <strong>of</strong>f in the face <strong>of</strong> arginine depletion. Here<br />
we demonstrate for the first time that human T lymphocytes<br />
respond to arginine depletion by phosphorylation <strong>of</strong> the<br />
kinase GCN2. This is analogous to the general control<br />
response <strong>of</strong> lower eukaryotes upon amino acid depletion.<br />
We also analysed phosphorylation <strong>of</strong> the α-subunit <strong>of</strong><br />
eukaryotic translation initiation factor 2 (eIF2α) and ATF-2<br />
as well as expression <strong>of</strong> transcription factors ATF-4 and CHOP,<br />
known elements in lower eukaryotes in their response to<br />
amino acid depletion. We show that these mammalian<br />
homologues are less stringently regulated in human lymphocytes<br />
upon arginine depletion than phosphorylation <strong>of</strong><br />
GCN2K. In summary, our data demonstrate an evolutionarily<br />
conserved mechanism <strong>of</strong> cellular response towards amino<br />
acid depletion in human T lymphocytes.<br />
doi:10.1016/j.clim.2007.03.500<br />
Sa.114 Characterization <strong>of</strong> the B Cell Infiltrate<br />
Within Intracranial Germinomas<br />
Shannon L. McArdel, Research Technician, Brigham and<br />
Women’s Hospital, Boston, MA, Katharine Cronk,<br />
Neurosurgery Resident, Columbia Medical School, New York,<br />
NY, Kimberly L. Shampain, Student Researcher, Brigham and<br />
Women’s Hospital, Boston, MA, Richard C.E. Anderson,<br />
Assistant Pr<strong>of</strong>essor <strong>of</strong> Neurosurgery and Pediatric<br />
Neurosurgery, Columbia University Medical Center, New<br />
York, NY, David E. Anderson, Instructor in Neurology,<br />
Harvard Medical School, Boston, MA, Jeffrey N. Bruce,<br />
Pr<strong>of</strong>essor <strong>of</strong> Neurosurgery, Columbia University Medical<br />
Center, New York, NY, David A. Hafler, Pr<strong>of</strong>essor <strong>of</strong><br />
Neurology, Harvard Medical School, Brigham and Women’s<br />
Hospital, Boston, MA, Kevin C. O’Connor, Instructor in<br />
Neurology, Harvard Medical School, Boston, MA<br />
Intracranial germinomas arise primarily in children, from<br />
neoplastic transformation <strong>of</strong> embryonic germ cells in the<br />
CNS, most <strong>of</strong>ten in the pineal and suprasellar regions. These<br />
malignant tumors are currently treated with surgery followed<br />
by radiation therapy. Many children however, are too<br />
young to receive radiation therapy or present with tumor<br />
disseminated throughout the cerebrospinal fluid pathways,<br />
making treatment difficult. Thus, germinomas present an<br />
attractive target for antibody-based immunotherapy. Intracranial<br />
germinomas commonly include an immune cell<br />
infiltrate, including a prominent population <strong>of</strong> B cell subsets<br />
that have not yet been characterized. We began to<br />
investigate these B cells by examining their antibody<br />
repertoire. We constructed immunoglobulin variable region<br />
(V) gene libraries from consecutive sections and from laser<br />
capture microdissected B cells from resected germinoma<br />
tumors. Comparison <strong>of</strong> the V-gene sequences to their<br />
corresponding germline sequences revealed that somatic<br />
mutation, oligoclonal expansion and isotype switching were<br />
prominent within the tumor infiltrate. These are cardinal<br />
features <strong>of</strong> an antigen driven B cell response, indicating that<br />
a local inflammatory response may be occurring within the<br />
CNS. This response is likely to be driven by tumor-associated<br />
antigens, the identification <strong>of</strong> which will provide insight into<br />
the immune-related tumor microenvironment.<br />
doi:10.1016/j.clim.2007.03.501<br />
S113<br />
Sa.115 Wnt5a Regulates Melanoma Antigen<br />
Recognized by T Cells 1 (MART-1), a Predominant<br />
Antigen in Melanoma Cells<br />
Samudra Dissanayake, Post doctoral Fellow, National<br />
Institute on Aging, National Institute <strong>of</strong> Health, Baltimore,<br />
MD, Devin Rosenthal, Student, Laboratory <strong>of</strong> <strong>Immunology</strong>,<br />
National Institute on Aging, National Institute <strong>of</strong> Health,<br />
Baltimore, MD, Kyle Hewitt, Student, Laboratory <strong>of</strong>
S114 Abstracts<br />
<strong>Immunology</strong>, National Institute on Aging, National Institute<br />
<strong>of</strong> Health, Baltimore, MD, Michael Wade, Student,<br />
Laboratory <strong>of</strong> <strong>Immunology</strong>, National Institute on Aging,<br />
National Institute <strong>of</strong> Health, Baltimore, MD, Sherry Yang,<br />
Student, Laboratory <strong>of</strong> <strong>Immunology</strong>, National Institute on<br />
Aging, National Institute <strong>of</strong> Health, Baltimore, MD, Poloko<br />
Leotlela, Post doctoral Fellow, Laboratory <strong>of</strong> <strong>Immunology</strong>,<br />
National Institute on Aging National Institute <strong>of</strong> Health,<br />
Baltimore, MD, Dennis Taub, Chief, Laboratory <strong>of</strong><br />
<strong>Immunology</strong>, Laboratory <strong>of</strong> <strong>Immunology</strong>, National Institute<br />
on Aging, National Institute <strong>of</strong> Health, Baltimore, MD, Adam<br />
Riker, Chief <strong>of</strong> Surgical Oncology, Mitchell cancer Institute,<br />
University <strong>of</strong> South Alabama, North Mobile, AL, Brian<br />
Nickol<strong>of</strong>f, Pr<strong>of</strong>essor pathology, Loyola University Medical<br />
Center, Maywood, IL, Ashani Weeraratna, Staff Scientist,<br />
Laboratory <strong>of</strong> <strong>Immunology</strong>, National Institute on Aging,<br />
National Institute <strong>of</strong> Health, Baltimore, MD<br />
Wnt5a increases melanoma cell motility via activation <strong>of</strong><br />
Protein Kinase C (PKC). Microarray analysis showed an<br />
inverse relationship between Wnt5a and MART-1. Using PKC<br />
activation and inhibition studies, as well as recombinant<br />
Wnt5a and Wnt5a RNAi, we demonstrate here that Wnt5aactivated<br />
PKC affects the expression <strong>of</strong> MART-1 via the<br />
phosphorylation <strong>of</strong> STAT3. STAT3 is a transcriptional factor<br />
known to inhibit the binding <strong>of</strong> MART-1 transcriptional<br />
activators MITF, SOX10 and PAX3. These effects are mediated<br />
by Wnt5a upstream <strong>of</strong> PKC, as overexpression <strong>of</strong> Wnt5a<br />
increases pSTAT3 and Wnt5a RNAi treatment decreases<br />
pSTAT3 levels. The decrease in pSTAT3 corresponds to an<br />
increase in MART1 and vice versa. Translocation <strong>of</strong> pSTAT3<br />
from the cytosol to the nucleus upon transfection <strong>of</strong> Wnt5a,<br />
indicated activation <strong>of</strong> STAT3 by Wnt5a, while the reverse<br />
was observed for cells knocked down for Wnt5a. Further,<br />
steady increases in PAX3 and MART-1 protein levels were<br />
observed in cells treated with Wnt5a RNAi, possibly due to<br />
suppressing the effects <strong>of</strong> STAT3. MART1 positive melanoma<br />
cells are able to activate CTL, while addition <strong>of</strong> recombinant<br />
Wnt5a to these cells could not, presumably due to the downregulation<br />
<strong>of</strong> MART-1. RNA from human melanoma biopsies<br />
analyzed for Wnt5a by real-time PCR shows an inverse<br />
relationship between Wnt5a levels and patient survival time,<br />
suggesting that cells with higher MART-1 have a better<br />
outcome. Collectively, our data suggest that Wnt5a, via PKC,<br />
results in the phosphorylation <strong>of</strong> STAT-3 and a subsequent<br />
down-regulation MART1, thereby rendering melanoma cells<br />
able to escape immune detection.<br />
doi:10.1016/j.clim.2007.03.502<br />
Sa.116 Discovery <strong>of</strong> Cancer and Autoimmune<br />
Biomarkers Utilizing Serum Antibody Pr<strong>of</strong>iling on<br />
Protein Microarrays<br />
Jennifer Cannon, Scientist, Invitrogen Corporation,<br />
Carlsbad, CA, Michael Snyder, Pr<strong>of</strong>essor, Yale University,<br />
New Haven, CT, Michael Hudson, Scientist, Yale University,<br />
New Haven, CT, Gregory Michaud, Scientist, Invitrogen<br />
Corporation, Carlsbad, CA, Michael Smith, Scientist,<br />
Invitrogen Corporation, Carlsbad, CA, Barry Schweitzer,<br />
Research and Development Director, Invitrogen<br />
Corporation, Carlsbad, CA, Guillermo Mor, Associate<br />
Pr<strong>of</strong>esor, OB/GYN, New Haven, CT<br />
It has been well established that serum autoantibodies<br />
have important diagnostic value for many diseases, including<br />
cancer and autoimmune diseases. Identifying the antigens<br />
which elicit an autoimmune response can yield sets <strong>of</strong><br />
biomarkers that provide classifiers for particular diseases<br />
and disease stages as well as predictors <strong>of</strong> patient outcomes<br />
and patient responses to therapeutics. Protein microarrays<br />
are valuable tools that have been successfully applied to<br />
investigate the circulating antibody pr<strong>of</strong>ile in several disease<br />
states. To discover potential autoantibody biomarkers<br />
specific to ovarian cancer, sera from 30 ovarian cancer<br />
patients and 30 healthy patients were pr<strong>of</strong>iled on protein<br />
microarrays. Patient and control sera were diluted and<br />
applied to protein microarrays containing over 5000 purified<br />
proteins expressed in a baculovirus expression system. Over<br />
95 candidate biomarkers were identified that exhibited<br />
enhanced reactivity in sera from cancer patients relative to<br />
that <strong>of</strong> the control individuals. Antibodies to four antigens<br />
were tested for differential reactivity in tissue samples <strong>of</strong><br />
cancer patients relative to healthy patients using immunoblot<br />
analysis and tissue microarrays. Three biomarkers<br />
exhibited elevated expression in the cancer tissue relative<br />
to controls. Signal observed for the biomarker antigens<br />
appeared significantly stronger than signal corresponding to<br />
the existing ovarian cancer antigen, CA-125. Additional<br />
studies focused on identifying biomarkers associated with<br />
autoimmune diseases, such as Rheumatoid Arthritis and<br />
Systemic Lupus Erythematosus, have recently utilized a<br />
higher content protein microarray including over 8000<br />
unique human proteins. The application <strong>of</strong> protein microarray<br />
technology for serum antibody pr<strong>of</strong>iling will be<br />
discussed along with data from on-going validation studies.<br />
doi:10.1016/j.clim.2007.03.503<br />
Sa.117 Phenotype and Function Analysis <strong>of</strong><br />
Dendritic Cells From Patients with Prostate<br />
Carcinoma After Radical Prostatectomy<br />
Ivo Minarik, PhD Student, Department <strong>of</strong> <strong>Immunology</strong>, 2nd<br />
Medical School, Charles University, Prague 5, Czech<br />
Republic, Zuzana Tobiasova, PhD Student, Department <strong>of</strong><br />
<strong>Immunology</strong>, 2nd Medical School, Charles University, Prague<br />
5, Czech Republic, Jirina Bartunkova, Head <strong>of</strong> Department,<br />
Department <strong>of</strong> <strong>Immunology</strong>, 2nd Medical School, Charles<br />
University, Prague 5, Czech Republic, Ivan Kawaciuk, Head<br />
<strong>of</strong> Department, Department <strong>of</strong> Urology, 2nd Medical School,<br />
Charles University, Prague 5, Czech Republic<br />
Prostate carcinoma represents the 3rd most frequent<br />
malignancy in the Czech male population with the<br />
incidence <strong>of</strong> 68.3/100000 and mortality 28.2/100000.<br />
About 35% <strong>of</strong> patients experience biochemical relapse<br />
(PSA increase) any time after radical prostatectomy.<br />
Hence, alternative treatment is highly required. Dendritic<br />
cell (DC)-based immunotherapy seems to be a promising<br />
approach for the elimination <strong>of</strong> relapse. In our study we aim<br />
to compare function and phenotype <strong>of</strong> dendritic cells from
Abstracts<br />
patients with prostate carcinoma, who underwent radical<br />
prostatectomy, to controls without tumor. We included 10<br />
patients (age 53-76) with prostate carcinoma who fulfilled<br />
at least one <strong>of</strong> the following risk factors <strong>of</strong> biochemical<br />
relapse: preoperative PSA> 10ng/ml, postoperative Gleason<br />
score >7 or penetration through prostate capsule. The<br />
control group comprised 10 patients (age 53-73) with<br />
benign prostate hyperplasia. We examined the phenotype<br />
(HLA-DR, CD11c, CD83, CD80, and CD86) and functionality<br />
(endocytosis, production <strong>of</strong> IL-12, IL-6, IL-1beta, IL-10, and<br />
TNF-alpha) <strong>of</strong> dendritic cells (DC) and compared the same<br />
features on DC from controls. Immature DCs were prepared<br />
from monocytes after 6 days <strong>of</strong> cultivation in the presence<br />
<strong>of</strong> GM-CSF and IL-4. Their maturation was achieved by 24<br />
hours co-incubation with either poly (I:C), or LPS. Immature<br />
DCs demonstrated higher phagocytic activity, while mature<br />
DCs produced more inflammatory cytokines and expressed<br />
CD83, HLA-DR and costimulatory molecules in higher<br />
amounts. The difference between patients and healthy<br />
donors was insignificant. In conclusion, dendritic cells<br />
prepared from monocytes <strong>of</strong> patients are functionally and<br />
phenotypically competent and so can be used for development<br />
<strong>of</strong> DC vaccines.<br />
doi:10.1016/j.clim.2007.03.505<br />
Sa.119 Parallel Mechanisms <strong>of</strong> Activation by Human<br />
and Mouse Dendritic Cells Treated with the IgM<br />
Immune Modulator B7-DC XAb<br />
Larry Pease, Pr<strong>of</strong>essor, Mayo Clinic College <strong>of</strong> Medicine,<br />
<strong>Immunology</strong>, Rochester, MN, Erin Schenk, Graduate Student,<br />
Mayo Clinic College <strong>of</strong> Medicine, Department <strong>of</strong><br />
<strong>Immunology</strong>, Rochester, MN, Svetomir Markovic, Associate<br />
Pr<strong>of</strong>essor, Mayo Clinic College <strong>of</strong> Medicine, Rochester, MN<br />
Systemic treatment <strong>of</strong> mice with the human antibody B7-<br />
DC XAb modulates dendritic cell (DC) function, protecting<br />
animals from experimental inflammatory airway disease and<br />
established syngeneic tumor implants. Protection in both<br />
models involves modulation <strong>of</strong> T cell function by activated<br />
DCs. B7-DC XAb (1) activates mouse and human DCs in a B7-<br />
DC XAb dependent fashion, (2) induces very similar macromolecular<br />
complexes on the cell surface <strong>of</strong> mouse and human<br />
DCs, and (3) initiates similar signaling cascades, patterns <strong>of</strong><br />
gene expression, and DC functions in these antigen presenting<br />
cells derived from the two species. Furthermore, human<br />
DCs activated with B7-DC XAb are strong inducers <strong>of</strong> antigenspecific<br />
T cell immune responses by peripheral blood T cells<br />
in vitro. The similar response pattern found in mouse and<br />
human DCs is even more remarkable since bone marrowderived<br />
DCs from the mouse and monocyte-derived DCs from<br />
human peripheral blood do not represent closely related<br />
differentiation states prior to activation with the immune<br />
modulator. Nonetheless, these strong parallels in mouse and<br />
human responsiveness to B7-DC XAb treatment provide<br />
justification for our ongoing efforts to develop this human<br />
antibody for use in clinical trials.<br />
doi:10.1016/j.clim.2007.03.506<br />
Sa.120 Human Glioblastoma Multiforme (GBM)<br />
Tumors are Enriched for Strongly Suppressive,<br />
Apoptosis Resistant, DR+ Tregs<br />
Charles Ashley, Research Technician, Harvard Medical<br />
School, Brigham and Women’s Hospital, Department <strong>of</strong><br />
Neurology, Boston, MA, Richard Anderson, Assistant<br />
Pr<strong>of</strong>essor, Columbia University, Department <strong>of</strong> Pediatrics,<br />
New York, NY, Jeffrey Bruce, Pr<strong>of</strong>essor, Neurological<br />
Institute <strong>of</strong> New York, New York, NY, David Anderson,<br />
Instructor <strong>of</strong> Neurology, Harvard Medical School, Brigham<br />
and Women’s Hospital, Department <strong>of</strong> Neurology, Boston,<br />
MA, David Hafler, Breakstone Chair <strong>of</strong> Neurology, Harvard<br />
Medical School, Brigham and Women’s Hospital,<br />
Department <strong>of</strong> Neurology, Boston, MA, Clare Baecher-Allan,<br />
Instructor <strong>of</strong> Neurology, Harvard Medical School, Brigham<br />
and Women’s Hospital, Department <strong>of</strong> Neurology,<br />
Boston, MA<br />
Glioblastoma Multiforme (GBM) is a CNS tumor <strong>of</strong> astrocytic<br />
origin, that is highly refractory to current therapies<br />
and associated with survival <strong>of</strong> less than 15 months. Using<br />
post surgical resection GBM samples to study the pr<strong>of</strong>ile <strong>of</strong><br />
the infiltrating regulatory T cells, we have found marked<br />
alterations in subset frequency, activation state, and gene<br />
expression in GBM-derived Tregs. In GBM, not only are<br />
there a large number <strong>of</strong> tumor-infiltrating Tregs, but<br />
importantly, these Tregs are strikingly enriched for the<br />
most suppressive Treg subset that are classified by HLA-DR<br />
expression. In a large panel <strong>of</strong> samples, flow cytometric<br />
analyses indicated that GBM tumors contained a significantly<br />
greater Treg infiltrate than meningiomas, or<br />
metastases <strong>of</strong> non-CNS tumors. Subsequently, using six<br />
color FACS sorting, specific DR+ and DR− Treg cell subsets<br />
were quantified and isolated from matched tumor and<br />
peripheral blood samples from the same individual with<br />
either GBM or other CNS tumors, and from blood <strong>of</strong> healthy<br />
controls. Not only did these analyses demonstrate an<br />
overwhelming prevalence <strong>of</strong> DR+ Tregs in the GBM tumor,<br />
but also that these tumor derived DR+ Tregs exhibit<br />
extremely high levels <strong>of</strong> Foxp3 and extremely low levels<br />
<strong>of</strong> the pro-apoptotic gene, Bax, as compared to peripheral<br />
blood derived DR+ Tregs. These data indicate that the GBM<br />
associated increase in Tregs may be due to increased Treg<br />
survival, possibly due to the actions <strong>of</strong> IL-10 which we have<br />
found reduces Treg expression <strong>of</strong> both Bax protein and<br />
mRNA.<br />
doi:10.1016/j.clim.2007.03.507<br />
S115<br />
Sa.121 Therapeutic Dendritic Cell Vaccine<br />
Preparation Using Tumor RNA Transfection<br />
Juliana Sousa-Canavez, Researcher, Genoa Biotech, São<br />
Paulo, Brazil, Flavio Canavez, Researcher, Genoa Biotech,<br />
São Paulo, Brazil, Cristina Massoco, Researcher, Genoa<br />
Biotech, São Paulo, Brazil, Katia Leite, Pathologist,<br />
Genoa Biotech, São Paulo, Brazil, Luiz Camara-Lopes,<br />
Pathologist, Genoa Biotech, São Paulo, Brazil, Dewton<br />
Moraes Vasconcelos, Pr<strong>of</strong>essor, Genoa Biotech, São Paulo,<br />
Brazil
S116 Abstracts<br />
Dendritic cells (DC) are potent antigen-presenting cells<br />
that induce and maintain primary immune responses. In the<br />
last years, many studies have used this property to initiate<br />
antigen T cell responses against neoplasic cells. This was<br />
achieved by vaccinating patients with DC carrying tumorassociated<br />
antigens prepared in vitro. A promising method<br />
for DC vaccine preparation is the transfection <strong>of</strong> tumor<br />
cells RNA. Preliminary clinical studies using RNA transfection<br />
have been shown encouraging results and minor side<br />
effects. Many aspects <strong>of</strong> this method, however, are not<br />
understood and further investigations are needed, especially<br />
regarding vaccine preparation. Here, we present our<br />
data obtained by DC transfection with total RNA isolated<br />
from a prostate tumor lineage LNCAP that overexpresses<br />
PSA antigen. PBMC were collected from healthy volunteers<br />
through apheresis and mononuclear cells were separated in<br />
Ficoll gradient. DC was differentiated with GM-CSF and IL-<br />
4, and maturation induced by TNFα DC phenotypes were<br />
confirmed by flow-cytometry using CD80, CD86, CD14,<br />
CD11c, CD1a and CCR7 antibodies. Immature cells were<br />
transfected with 4 1/4 and 16 1/4 g <strong>of</strong> RNA using either<br />
300 V or 400 V pulses. Mature cells were transfected with 4<br />
1/4 μg and 5 1/4 μg <strong>of</strong> RNA using 300 V, 400 V or 500 V<br />
pulses. In all conditions, we detected PSA expression by<br />
immunohistochemistry indicating that RNA penetrated in<br />
DC. For mature cells, we have also co-incubated total RNA<br />
(4 1/4 μg and 5 1/4 μg) and DC with poor results. Our<br />
experiments point to RNA transfection (5 1/4 μg total RNA)<br />
in mature DC using 400 V pulse as the most efficient<br />
condition.<br />
doi:10.1016/j.clim.2007.03.508<br />
Sa.122 Regulatory T Cells Affect the DC-mediated<br />
Immunotherapy Outcome in Melanoma Patients<br />
Mercedes N. López, Medical Doctor, Research Support<br />
Office, <strong>Clinical</strong> Hospital, University <strong>of</strong> Chile, Santiago de<br />
Chile, Chile, Gabriela Segal, Biotech, Program <strong>of</strong><br />
<strong>Immunology</strong>, Institute <strong>of</strong> Biomedical Sciences, University <strong>of</strong><br />
Chile, Santiago de Chile, Chile, Cristian Pereda, Biotech,<br />
Program <strong>of</strong> <strong>Immunology</strong>, Institute <strong>of</strong> Biomedical Sciences,<br />
University <strong>of</strong> Chile, Santiago de Chile, Chile, Raquel<br />
Aguilera, Medical Doctor, Program <strong>of</strong> <strong>Immunology</strong>, Institute<br />
<strong>of</strong> Biomedical Sciences, University <strong>of</strong> Chile, Santiago de<br />
Chile, Chile, Alexandra Ginesta, Medical Doctor, Program<br />
<strong>of</strong> <strong>Immunology</strong>, Institute <strong>of</strong> Biomedical Sciences, University<br />
<strong>of</strong> Chile, Santiago de Chile, Chile, Diego Reyes, Medical<br />
Doctor, Program <strong>of</strong> <strong>Immunology</strong>, Institute <strong>of</strong> Biomedical<br />
Sciences, University <strong>of</strong> Chile, Santiago de Chile, Chile,<br />
Carlos Ferrada, Medical Doctor, Surgery department,<br />
<strong>Clinical</strong> Hospital, University <strong>of</strong> Chile, Santiago de Chile,<br />
Chile, Flavio Salazar-Onfray, PhD <strong>of</strong> Sciences, Program <strong>of</strong><br />
<strong>Immunology</strong>, Institute <strong>of</strong> Biomedical Sciences, University <strong>of</strong><br />
Chile, Santiago de Chile, Chile<br />
Dendritic cell (DCs)-based therapy has proven to be<br />
effective in patients with malignancies. A Chilean phase I<br />
clinical study for the treatment <strong>of</strong> advanced malignant<br />
melanoma performed in our laboratory, indicates that<br />
autologous human dendritic cells (hDCs) pulsed with a<br />
melanoma cell lysate acquired a mature phenotype and<br />
were able to trigger specific immune responses to tumor<br />
antigens in vivo. In this study, 37 patients with malignant<br />
melanoma stage IV were vaccinated with autologous DCs<br />
pulsed with a melanoma cell lysate. After vaccination, 50%<br />
tested patients (18 out <strong>of</strong> 37) showed a positive Delayed type<br />
IV hypersensitivity reaction (DTH) against the melanoma cell<br />
lysate. All patients showed a positive DTH against the used<br />
adjuvant KLH demonstrating that all patients were immunocompetents.<br />
Significant correlations were found between<br />
DTH positive responses against tumour cell lysate and both<br />
disease stability and post-vaccination survival on the stage IV<br />
patients. However, there is a group <strong>of</strong> patients that did not<br />
show immunological responses. We compared a number <strong>of</strong><br />
DTH responder patients (n=4) with a group <strong>of</strong> DTH noresponder<br />
patients (n=4) and analyzed the number and<br />
proportion <strong>of</strong> regulatory T cells (Treg). Our results showed<br />
that DTH non-responder patients showed an increase <strong>of</strong> Treg<br />
cells after the DC vaccination compared with DTH responder<br />
patients that did not show changes in the number or<br />
proportion <strong>of</strong> Treg cells. These observations should be<br />
important in the study <strong>of</strong> the role <strong>of</strong> regulatory T cells in<br />
the clinical responses <strong>of</strong> cancer patients treated with<br />
immunotherapy.<br />
doi:10.1016/j.clim.2007.03.509<br />
Sa.123 Dendritic Cell-Tumor Cell Fusion Vaccine for<br />
the Treatment <strong>of</strong> Advanced Tumors: Evaluation <strong>of</strong><br />
the Quality <strong>of</strong> Life by Questionnaires<br />
Dewton Moraes Vasconcelos, MD, PhD, Genoa Biotecnologia,<br />
São Paulo, Brazil, Antonio Carlos Misiara, MD, PhD, Genoa<br />
Biotecnologia S.A., São Paulo, Brazil, Juliana Moreira<br />
Sousa-Canavez, BSc, PhD, Genoa Biotecnologia S.A., São<br />
Paulo, Brazil, Paloma Aguiar, BSc, Genoa Biotecnologia S.A.,<br />
São Paulo, Brazil, Elaine Cristina Corneta, MSc, Genoa<br />
Biotecnologia S.A., São Paulo, Brazil, Katia Ramos Moreira<br />
Leite, MD, PhD, Genoa Biotecnologia S.A., São Paulo, Brazil,<br />
Luiz Heraldo Arouche Camara Lopes, MD, PhD, Genoa<br />
Biotecnologia S.A., São Paulo, Brazil<br />
Dendritic cells are the most potent antigen-presenting<br />
cells, and the possibility <strong>of</strong> their use for cancer vaccination<br />
has renewed the interest in this therapeutic modality.<br />
Nevertheless, the ideal immunization protocol with these<br />
cells has not been described yet. In this report we describe<br />
the preliminary results <strong>of</strong> a protocol using autologous tumor<br />
and allogeneic dendritic hybrid cell vaccination every<br />
4 weeks, for metastatic melanoma and renal cell carcinoma<br />
(RCC) patients, and a few patients with other types <strong>of</strong><br />
neoplasia. Thirty-six patients were evaluated by a questionnaire<br />
searching for information <strong>of</strong> the quality <strong>of</strong> life<br />
(QoL) during the treatment with the vaccine. Though all<br />
patients included presented with large tumor burdens and<br />
progressive diseases, 77% <strong>of</strong> them experienced stability after<br />
vaccination, 9% related improvement and 14% presented<br />
deterioration <strong>of</strong> the clinical features. Among RCC patients 3/<br />
10 (30%) presented deterioration, 1/10 improvement (10%)<br />
and 6/10 (60%) stabilization. Among melanoma patients 1/15<br />
(6.6%) deteriorated, 1/15 (6.6%) improved and 13/15 (86.6%)
Abstracts<br />
stabilized the disease. These data indicate that dendritic<br />
cell-tumor cell hybrid vaccination affects the natural history<br />
<strong>of</strong> advanced cancer and provides support for its study in less<br />
advanced patients, who should, more likely, benefit even<br />
more from this approach.<br />
doi:10.1016/j.clim.2007.03.510<br />
Sa.124 Rice Bran Supplement (MGN-3/Biobran)<br />
Suppresses Tumor Growth via Modulating Cytokine<br />
Production and Increasing Apoptotic Level in Ehrlich<br />
Carcinoma-bearing Mice<br />
Nariman Badr El-Din, Pr<strong>of</strong>essor, University <strong>of</strong> Mansoura,<br />
Faculty <strong>of</strong> Science, Department <strong>of</strong> Zoology, Mansoura, Eman<br />
Noaman, Pr<strong>of</strong>essor, National Center for Radiation and<br />
Technology, Department <strong>of</strong> Radiation Biology, Cairo, Alia<br />
Ghoneum, Student, Charles R. Drew University <strong>of</strong> Medicine<br />
and Science, Department <strong>of</strong> Otolaryngology, Los Angeles,<br />
CA, Mamdooh Ghoneum, Associate Pr<strong>of</strong>essor III, Charles R.<br />
Drew University <strong>of</strong> Medicine and Science, Department <strong>of</strong><br />
Otolaryngology, Los Angeles, CA<br />
This study was undertaken to investigate the anti-tumor<br />
activity in vivo by MGN-3/Biobran and the mechanisms<br />
underlying its effect. MGN-3 is an arabinoxylan extracted<br />
from rice bran that is treated with hydrolyzing enzymes from<br />
shiitake mushrooms. Female Swiss albino mice were inoculated<br />
intramuscularly in the right thigh with Ehrlich Ascites<br />
Carcinoma (EAC) cells. At day 8, mice bearing Solid Ehrlich<br />
Carcinoma tumor (SEC) were treated with MGN-3 (40 mg/kg<br />
body weight) via intraperitoneal injection, 3 times/week,<br />
until day 35. Tumor growth (volume and weight), plasma<br />
cytokine production, and apoptotic effect <strong>of</strong> MGN-3 were<br />
examined. Injection with MGN-3 caused a highly significant<br />
delay in both tumor volume (63.27%) and tumor weight<br />
(45.2%) from controls (pb0.01). The mechanisms by which<br />
MGN-3 exerts its anti-tumor effect seem to involve its ability<br />
to influence plasma cytokine production via increasing the<br />
levels <strong>of</strong> IFNγ (184.4%, pb0.01) and TNFα (11%, pb0.01)<br />
over control mice bearing SEC, while down-regulating levels<br />
<strong>of</strong> the immune suppressing cytokine IL-10. In addition, MGN-3<br />
induced 1.8 fold increase in the percentage <strong>of</strong> apoptotic SEC<br />
cells as determined by flow cytometry and the histopathological<br />
examination. No adverse side effects due to MGN-3<br />
treatment were observed; all animals displayed normal<br />
feeding/drinking and life activity patterns with a significant<br />
body weight gain <strong>of</strong> 7.32%, pb0.025. These data may have<br />
clinical implications for the treatment <strong>of</strong> solid cancers. MGN-<br />
3/Biobran was <strong>of</strong>fered by Daiwa Pharmaceuticals Co., Ltd.,<br />
Tokyo, Japan.<br />
doi:10.1016/j.clim.2007.03.511<br />
Sa.125 Therapeutic Efficacy <strong>of</strong> the Most Potent<br />
Macrophage Activating Factor (GcMAF) for Prostate<br />
and Breast Cancers<br />
Nobuto Yamamoto, Director, Socrates Institute for<br />
Therapeutic <strong>Immunology</strong>, Philadelphia, PA, Masumi Ueda,<br />
Chief, Microbiology Department, Socrates Institute for<br />
Therapeutic <strong>Immunology</strong>, Philadelphia, PA, Charles Benson,<br />
Head, Infectious Diseases, School <strong>of</strong> Veterinary Medicine,<br />
University <strong>of</strong> Pennsylvania, Kennett Square, PA<br />
Inflammation-primed macrophage activation is the principal<br />
macrophage activation process that requires serum<br />
vitamin D-binding protein (known as Gc protein) and<br />
participation <strong>of</strong> B and T lymphocytes. A trisaccharide<br />
composed <strong>of</strong> N-acetylgalactosamine with dibranched galactose<br />
and sialic acid termini at 420 threonine residue <strong>of</strong> Gc<br />
protein is hydrolyzed by the β-galactosidase (Bgl) <strong>of</strong><br />
inflammation-primed B cells and the Neu-1 sialidase <strong>of</strong> T<br />
cells to yield the macrophage activating factor (MAF). Thus,<br />
Gc protein is the precursor for the principal MAF. However,<br />
the MAF precursor activity <strong>of</strong> serum Gc protein <strong>of</strong> cancer<br />
patients was lost or reduced because Gc protein is<br />
deglycosylated by serum α-N-acetylgalactosaminidase<br />
(Nagalase) secreted from cancerous cells. Serum Nagalase<br />
activity is proportional to tumor burden and serves as a<br />
prognostic index. Stepwise treatment <strong>of</strong> highly purified Gc<br />
protein with immobilized β-galactosidase and sialidase<br />
generated the most potent macrophage activating factor<br />
(GcMAF) that produces no side effect in humans. When<br />
human macrophages were treated in vitro with GcMAF (100<br />
pg/ml) for 3 h, the macrophages developed enormous<br />
variation <strong>of</strong> receptors that recognize a variety <strong>of</strong> cellular<br />
abnormalities, i.e., tumor associated antigens, in cancerous<br />
cell surface. Thus, the activated macrophages were<br />
highly tumoricidal to a variety <strong>of</strong> cancerous cells. Administration<br />
<strong>of</strong> 100 ng GcMAF/human results in the maximal<br />
activation <strong>of</strong> systemic macrophages. When nonanemic<br />
prostate and breast cancer patients (total <strong>of</strong> 36 patients)<br />
were treated with 14–25 weekly administrations <strong>of</strong> GcMAF<br />
(100 ng/week), all cancer patients exhibited healthy<br />
control levels <strong>of</strong> the serum Nagalase activity, indicating<br />
eradication <strong>of</strong> tumors.<br />
doi:10.1016/j.clim.2007.03.512<br />
S117<br />
Sa.126 Activation <strong>of</strong> Hepatic Stellate Cells During<br />
Fibrosis: Comparison <strong>of</strong> the CYP2D6 Model for<br />
Autoimmune Hepatits and CCl4 Injection<br />
Urs Christen, Assistant Pr<strong>of</strong>essor, Pharmazentrum, JWG<br />
University Frankfurt, Frankfurt am Main, Germany, Edith<br />
Hintermann, Senior Postdoctoral Fellow, Pharmazentrum,<br />
JWG University Frankfurt, Frankfurt am Main, Germany,<br />
Monika Bayer, Res Tech, Pharmazentrum, JWG University<br />
Frankfurt, Frankfurt am Main, Germany, Selina Christen,<br />
PhD Student, Pharmazentrum, JWG University Frankfurt,<br />
Frankfurt am Main, Germany<br />
Only little is known about the mechanisms <strong>of</strong> periportal<br />
fibrosis in the pathogenesis <strong>of</strong> human autoimmune hepatitis.<br />
We used the virus induced CYP2D6 model system to<br />
investigate the activation <strong>of</strong> hepatic stellate cells (HSC)<br />
and the kinetics <strong>of</strong> fibrosis in comparison with the CCl4induced<br />
fibrosis model. CYP2D6 transgenic mice express the<br />
human Cytochrome P450 in the liver and develop liver<br />
damage upon Adenovirus-CYP2D6 infection. In the CYP2D6
S118 Abstracts<br />
model we found mostly subcapsular fibrosis resulting in the<br />
fusion <strong>of</strong> individual lobules (Sirius Red, Collagen I). At 10–<br />
12 weeks post-infection, weak periportal fibrosis became<br />
apparent. In contrast, in CCl4-treated mice the kinetic <strong>of</strong><br />
extracellular matrix deposition was accelerated resulting in<br />
periportal fibrosis after 3–4 week <strong>of</strong> CCl4 administration. At<br />
later times, fibrosis was much more pronounced in CCl4treated<br />
mice compared to virus-infected CYP2D6 mice but<br />
subcapsular fibrosis was not as dominant. Activation <strong>of</strong> HSCs<br />
could be detected by staining for a-smooth muscle actin<br />
(aSMA) in liver sections <strong>of</strong> both CCl4-treated mice and virusinfected<br />
CYP2D6 mice. In addition, isolation <strong>of</strong> HSCs<br />
revealed an enhanced activation status (decreased amount<br />
<strong>of</strong> oil droplets, de novo aSMA expression) in CCl4-treated<br />
mice and virus-infected CYP2D6 mice. Our data indicate<br />
that virus-infected CYP2D6 mice display subcapsular and<br />
periportal fibrosis that correlates with an activation <strong>of</strong> HSCs<br />
similar to CCl4-induced fibrosis. Thus, the CYP2D6 mouse is<br />
a good model system to further investigate the molecular<br />
mechanisms involved in fibrotic events during autoimmune<br />
hepatitis.<br />
doi:10.1016/j.clim.2007.03.513<br />
Sa.127 Bone-Marrow Derived DCs From CD200R1<br />
KO Mice Stimulated with the Ligand CD200Fc<br />
Preferentially Induce Populations <strong>of</strong><br />
CD4+CD25+ Treg<br />
Reg Gorczynski, Pr<strong>of</strong>essor, University Health Network,<br />
Toronto, ON, Canada, Ivo Boudakov, PDF, University Health<br />
Network, Toronto, ON, Canada<br />
CD200: CD200R1 interaction causes suppression <strong>of</strong><br />
inflammation and graft rejection. The functional activity<br />
induced by alternate CD200R is<strong>of</strong>orms remains unclear. We<br />
generated a BL/6 mouse with deletion <strong>of</strong> the gene for<br />
CD200R1. Marrow cells from KO and wt mice were<br />
cultured for 8 days in the presence <strong>of</strong> (GMCSF +IL-4),<br />
with/without addition <strong>of</strong> a soluble form <strong>of</strong> CD200<br />
(CD200Fc). DCs were stimulated with LPS and used to<br />
activate C3H splenocytes. CTL for BL/6 targets was<br />
assayed at 5 days, or cells were harvested at 72 h,<br />
enriched for CD4+CD25+ cells, and added to fresh cultures<br />
<strong>of</strong> C3H splenocytes stimulated with either BL/6 or BALB/c<br />
mitomycin-c treated DCs. CTL specific for H2b or H2d<br />
targets was assayed 5d later.<br />
DCs <strong>of</strong> wt or CD200R1KO mice cultured without<br />
CD200Fc induced allospecific (anti-H2b) CTL in vitro.<br />
After culture in the presence <strong>of</strong> CD200Fc, DCs from<br />
CD200R1KO mice were inefficient inducers <strong>of</strong> CTL, but<br />
promoted development <strong>of</strong> CD4+CD25+Foxp3+ Treg which<br />
suppressed C3H anti-BL/6 CTL induction in fresh responder/stimulator<br />
cells. To characterize an in vivo equivalent<br />
<strong>of</strong> this phenomenon CD200Fc or mouseIgG2a was infused<br />
iv into wt or CD200R1KO mice for 14 days. Splenic DCs<br />
were isolated by conventional techniques. DCs from<br />
control mice (wt or CD200R1KO) receiving IgG2a only<br />
were equivalent in induction <strong>of</strong> CTL. DCs isolated after<br />
infusion <strong>of</strong> CD200Fc into CD200R1KO mice were impaired<br />
for CTL induction, but induced antigen-specific Treg in<br />
culture. We conclude that immunoregulation induced by<br />
CD200 binding <strong>of</strong> non-CD200R1 is<strong>of</strong>orms <strong>of</strong> the CD200R<br />
receptor family reflects the preferential induction <strong>of</strong><br />
“tolerogenic” DCs.<br />
doi:10.1016/j.clim.2007.03.514<br />
Sa.128 High Molecular Weight Hyaluronan Promotes<br />
the Suppressive Effects <strong>of</strong> CD4+CD25+ Treg<br />
Paul Bollyky, Benaroya Research Institute, Seattle, WA,<br />
Gerald Nepom, Benaroya Research Institute, Seattle, WA,<br />
Jane Buckner, Benaroya Research Institute, Seattle, WA,<br />
Susan Masewicz, Benaroya Research Institute, Seattle, WA,<br />
James Lord, Benaroya Research Institute, Seattle, WA,<br />
Stephen Evanko, Benaroya Research Institute, Seattle,<br />
WA, Thomas Wight, Benaroya Research Institute, Seattle,<br />
WA<br />
Hyaluronan (HA) is a glycosaminoglycan present in the<br />
extra-cellular matrix. When HA is degraded during infection<br />
and injury low-molecular-weight (LMW-HA) forms are<br />
generated whose interactions influence inflammation and<br />
angiogenesis. Intact high-molecular-weight hyaluronan<br />
(HMW-HA), conversely, has been reported to convey antiinflammatory<br />
signals. Here we demonstrate that HMW-HA<br />
acts on human CD4+CD25+ regulatory T cells (Treg) to<br />
promote their suppressive effects while LMW-HA does not.<br />
HMW-HA enhances regulatory T cell functional suppression<br />
<strong>of</strong> responder cell proliferation and up-regulates the<br />
transcription factor Foxp3 on Treg. These effects are only<br />
seen with activated Treg and are associated with expression<br />
<strong>of</strong> CD44 isomers which more highly bind HMW-HA. At<br />
higher concentrations HMW-HA also has direct suppressive<br />
effects on T cells. We propose that the state <strong>of</strong> HA in the<br />
matrix environment provides contextual cues to Treg and T<br />
cells, thereby providing a link between the innate<br />
inflammatory network and the regulation <strong>of</strong> adaptive<br />
immune responses.<br />
doi:10.1016/j.clim.2007.03.515<br />
Sa.129 The Forkhead Transcription Factor, Foxq1,<br />
Enhances NF-kB Activity and is Critical for Robust<br />
T Cell Activation and Autoimmunity<br />
Melanie Gubbels Bupp, Postdoctoral Fellow, Roche Palo<br />
Alto, Palo Alto, CA, Stanford Peng, Director, Arthritis<br />
Research, Roche Palo Alto, Palo Alto, CA<br />
Essential immunoregulatory functions have recently<br />
been ascribed to several members <strong>of</strong> the forkhead<br />
transcription factor family. For example, Foxo3a and<br />
Foxj1 inhibit NF-kB activity and Foxp3 regulates lineage<br />
commitment and function <strong>of</strong> regulatory T cells. Here, we<br />
report that another family member, Foxq1, is critical for<br />
robust helper T cell activation. CD4+ T cells from mice<br />
bearing a mutant allele <strong>of</strong> Foxq1 displayed a significantly<br />
reduced proliferative response with impaired secretion <strong>of</strong><br />
IL-2 and IFN-gamma after TCR ligation as compared to
Abstracts<br />
wild-type (WT) T cells. This defect is largely rescued by<br />
the addition <strong>of</strong> recombinant human IL-2, suggesting that<br />
Foxq1 is required for optimal production <strong>of</strong> IL-2 and<br />
consequently, robust T cell proliferation. Accordingly, in in<br />
vitro luciferase assays, Foxq1 augmented transcriptional<br />
activity <strong>of</strong> the IL-2 transactivator NF-kB while mutant<br />
Foxq1 did not retain this function. Finally, Foxq1-mutant<br />
mice exhibited reduced susceptibility to the progression <strong>of</strong><br />
experimental autoimmune encephalomyelitis than their<br />
WT counterparts. Thus, Foxq1 is essential for helper T cell<br />
activation and autoaggressive T cell responses in vivo,<br />
likely via its ability to potentiate NF-kB activity and IL-2<br />
production.<br />
doi:10.1016/j.clim.2007.03.516<br />
Sa.130 Human Bone Marrow Mesenchymal Stem<br />
Cells Support Polyclonal Stimulation <strong>of</strong> Human B<br />
Cells<br />
Elisabetta Traggiai, PhD, Institute G Gaslini, Genova, Italy,<br />
Alberto Martini, MD/PhD, Institute G Gaslini, Genova, Italy,<br />
Antonio Uccelli, MD/PhD, Department <strong>of</strong> Neurosciences,<br />
Ophthalmology and Genetics, Genova, Italy, Lorenzo<br />
Moretta, MD/PhD, Institute G Gaslini, Genova, Italy,<br />
Federica Benvenuto, PhD, Department <strong>of</strong> Neurosciences,<br />
Ophthalmology and Genetics, Genova, Italy, Marco<br />
Gattorno, MD, Institute G Gaslini, Genova, Italy<br />
To investigate the possible application <strong>of</strong> bone marrow<br />
(BM) mesenchymal stem cells (MSCs) in Systemic Lupus<br />
Erythematosus (SLE), an autoimmune disease in which B<br />
cells play a pivotal role, we studied the interaction <strong>of</strong> B<br />
cell subsets isolated from healthy donor and pediatric SLE<br />
patients with human BM MSCs. We isolated from peripheral<br />
blood <strong>of</strong> healthy donors: a) immature transitional B<br />
cells, b) naive B cells, c) IgM memory and d) switch<br />
memory B cells. BM MSCs promoted proliferation and<br />
differentiation into immunoglobulin secreting cells <strong>of</strong><br />
transitional and naive B cells stimulated with CpG 2006<br />
in the absence <strong>of</strong> BCR triggering, and strongly enhanced<br />
proliferation and differentiation <strong>of</strong> both memory B cell<br />
populations. Furthermore, both proliferation and differentiation<br />
into plasma cells <strong>of</strong> CD19+ B cells isolated from<br />
pediatric SLE patients were enhanced by BM MSCs upon<br />
polyclonal stimulation. Inhibition <strong>of</strong> T cell proliferation by<br />
MSCs was suggested to be dependent on INF-g€ . To test<br />
whether INF-g€ could have an effect on the B cell<br />
response under the influence <strong>of</strong> MSCs, we co-cultured B<br />
cell subsets with MSC in the presence <strong>of</strong> human<br />
recombinant INF-ï §ï€®ï€ After 4 days <strong>of</strong> culture both<br />
proliferation and differentiation into plasma cells <strong>of</strong> all B<br />
cell subsets stimulated with CpG, soluble CD40L and anti-<br />
Ig were inhibited in both healthy donors and SLE patients.<br />
These data show the complexity <strong>of</strong> the cross-talk between<br />
MSCs and B cell and how the microenviroment in which<br />
the interaction take place could influence the outcome <strong>of</strong><br />
the immune response.<br />
doi:10.1016/j.clim.2007.03.517<br />
Sa.131 Examination <strong>of</strong> a Unique T Cell Subset TCD40<br />
in Type 1 Diabetes<br />
David Wagner, Assistant Pr<strong>of</strong>essor, Webb-Waring Institute,<br />
Denver, CO<br />
The identification <strong>of</strong> autoaggressive T cells in human<br />
disease has proven very difficult. We have discovered a<br />
unique T cell subset described as CD4lo and expressing the<br />
CD40 receptor termed TCD40. These T cells are significantly<br />
expanded in T1D but not T2D or control subjects. A portion<br />
<strong>of</strong> TCD40 cells respond to pre-pro-insulin, GAD peptides,<br />
and human islets. These T cells are expanded in diabetes<br />
high susceptibility HLA (DR3, DR4, DQ8) subjects, but are<br />
also expanded when T1D subjects do not possess any <strong>of</strong> the<br />
high susceptibility HLAs. TCD40 cells remain at expanded<br />
percentages in long-term diabetic subjects, suggesting that<br />
CD40 is not merely a T cell activation marker. Interestingly,<br />
non-autoimmune subjects carrying DR3, DR4 or DQ8 do not<br />
have expanded TCD40 cells. Mechanistic differences in<br />
induction <strong>of</strong> RAG1 and RAG2 are seen between T1D and<br />
control subjects. Examination <strong>of</strong> TRAV usage in T1D and<br />
controls demonstrate prominent differences including the<br />
detection <strong>of</strong> TRAV8 in the periphery. TCD40 cells achieve<br />
effector status synthesizing predominantly Th1 cytokines.<br />
These data suggest a unique T cell population that may be<br />
predictive <strong>of</strong> autoimmunity.<br />
doi:10.1016/j.clim.2007.03.518<br />
S119<br />
Sa.132 Effects <strong>of</strong> Class II/CLIP Affinity on the Class II<br />
Antigen Presentation Pathway in the Context <strong>of</strong><br />
Autoimmunity<br />
Cornelia Rinderknecht, Graduate Student, Stanford<br />
University, Department <strong>of</strong> Pediatrics, San Carlos, CA,<br />
Tatiana Catanzarite, Student, Stanford University,<br />
Department <strong>of</strong> Pediatrics, Stanford, CA, Michael Belmares,<br />
Scientist, Arbor Vita Corporation, Sunnyvale, CA, Elizabeth<br />
Mellins, Associate Pr<strong>of</strong>essor, Stanford University,<br />
Department <strong>of</strong> Pediatrics, Stanford, CA, Susan Kovats,<br />
Assistant Member, Oklahoma Medical Research Foundation,<br />
Arthritis and <strong>Immunology</strong> Research Program, Oklahoma<br />
City, OK<br />
Several MHC class II alleles linked with autoimmune<br />
diseases form unusually low-stability complexes with class IIassociated<br />
invariant chain peptides (CLIP), leading us to<br />
hypothesize that this is an important feature contributing to<br />
disease pathogenesis. To investigate cellular consequences<br />
<strong>of</strong> altering class II/CLIP affinity, we evaluated invariant chain<br />
(Ii) mutants with increased CLIP affinity for two murine class<br />
II alleles, I-Ed and I-Ag7, which have low affinity for wt CLIP.<br />
I-Ed is associated with a mouse model <strong>of</strong> spontaneous,<br />
autoimmune joint inflammation, and I-Ag7 is associated with<br />
models <strong>of</strong> diabetes (NOD) and arthritis (KRN). Single amino<br />
acid mutations were made in murine Ii. Pulse-chase/coimmunoprecipitation<br />
analysis <strong>of</strong> I-Ed or I-Ag7 and CLIP was<br />
used to identify physiologically relevant CLIP mutants that<br />
are <strong>of</strong> high affinity but remain DM-sensitive. An increase in<br />
CLIP affinity for I-Ed or I-Ag7 resulted in increased cell-
S120 Abstracts<br />
surface and total cellular levels <strong>of</strong> class II, suggesting post-ER<br />
chaperoning by Ii via its CLIP peptides. Quantitative effects<br />
on class II were less robust in H-2M-expressing cells,<br />
suggesting complementary effects mediated by Ii and H-<br />
2M, and implying that the impact <strong>of</strong> varied CLIP affinity on<br />
immune responses will be highest in cells with limited H-2M<br />
activity. Functionally, cell lines expressing wt or high CLIPaffinity<br />
mutant Ii have differential capacity to present<br />
peptide or whole antigen to a T cell hybridoma, suggesting<br />
a model in which increased CLIP affinity for class II serves to<br />
restrict peptide loading to H-2M-containing compartments,<br />
ensuring proper editing <strong>of</strong> antigenic peptides.<br />
doi:10.1016/j.clim.2007.03.519<br />
Sa.133 Treg Potency Assay: Suppression <strong>of</strong> T Cell<br />
Cytokine Expression in Response to T Cell Specific<br />
Antigenic Stimulation <strong>of</strong> Autologous PBMC<br />
Smita Ghanekar, Senior Research Scientist, BD Biosciences,<br />
Research and Development, San Jose, CA, Joyce Ruitenberg,<br />
RAII, BD Biosciences, Research and Development, San Jose,<br />
CA<br />
CD4+CD25+ regulatory Tcells (Tregs) play a critical role in<br />
the induction and maintenance <strong>of</strong> peripheral immune<br />
tolerance. Naturally occurring Tregs are crucial for the<br />
control <strong>of</strong> auto-reactive T cells and <strong>of</strong> the effector function<br />
<strong>of</strong> allo-reactive CD4+ and CD8+ T cells in transplantation<br />
models in vivo. Tregs have been shown to be potent<br />
suppressors <strong>of</strong> activated T cells in vitro. Phenotypically,<br />
Tregs are characterized as T cells that are CD4+, CD25++,<br />
CD127− and Foxp3+. Suppressive ability <strong>of</strong> Tregs is usually<br />
assessed by applying long-term (5 days) assays such as<br />
proliferation, but the therapeutic potential <strong>of</strong> these cells<br />
demands a quicker analysis method. Therefore, in this study,<br />
the ability <strong>of</strong> Tregs from healthy donors to inhibit or suppress<br />
cytokine expression in short-term assays has been examined.<br />
PBMC enriched for CD4+ cells were purified as CD4+CD25++<br />
Tregs by sorting with a BD FACSAria. These highly purified<br />
Tregs were then cultured with rIL2 in vitro for 5–7 days and<br />
used in 6-h intracellular cytokine expression assays <strong>of</strong><br />
autologous PBMC. Activation <strong>of</strong> PBMC with SEB and/or<br />
CD3+CD28 results in the expression <strong>of</strong> inflammatory cytokines<br />
(IFNg, TNFa, and IL2) by CD4+ and CD8+ T cells. The<br />
results suggest that the addition <strong>of</strong> Tregs to the cytokine<br />
assay <strong>of</strong> autologous PBMC reduces the frequency <strong>of</strong> cytokine<br />
expressing T cells. Since the intracellular T cell cytokine<br />
expression assay has been validated extensively, this shortterm<br />
assay would be a valuable tool to qualify Tregs for<br />
immunotherapy.<br />
doi:10.1016/j.clim.2007.03.520<br />
Sa.134 β-Tubulin-induced Autoimmune<br />
Hearing Loss Exacerbates in IL-10-deficient Mice<br />
and Restores by IL-10 Gene Transfer<br />
Bin Zhou, Research Associate, University <strong>of</strong> Tennessee,<br />
Department <strong>of</strong> Medicine, Memphis, TN, Mohammad Habiby<br />
Kermany, Postdoctoral Fellow, University <strong>of</strong> Tennessee,<br />
Department <strong>of</strong> Medicine, Memphis, TN, Yixuan Zhou,<br />
Postdoctoral Fellow, University <strong>of</strong> Tennessee, Department<br />
<strong>of</strong> Medicine, Memphis, TN, Qing Cai, Postdoctoral Fellow,<br />
University <strong>of</strong> Tennessee, Department <strong>of</strong> Medicine, Memphis,<br />
TN, Chun Cai, Postdoctoral Fellow, University <strong>of</strong> Tennessee,<br />
Department <strong>of</strong> Medicine, Memphis, TN, Junwoo Kim,<br />
Postdoctoral Fellow, University <strong>of</strong> Tennessee, Department<br />
<strong>of</strong> Medicine, Memphis, TN, Patrick Kim, Postdoctoral<br />
Fellow, University <strong>of</strong> Tennessee, Department <strong>of</strong> Medicine,<br />
Memphis, TN, Wenxi Liu, Postdoctoral Fellow, University <strong>of</strong><br />
Tennessee, Department <strong>of</strong> Medicine, Memphis, TN, Tai June<br />
Yoo, Pr<strong>of</strong>essor, University <strong>of</strong> Tennessee, Department <strong>of</strong><br />
Medicine, Memphis, TN<br />
We hypothesized that endogenous levels <strong>of</strong> IL-10 function<br />
to regulate the severity <strong>of</strong> βtubulin-induced experimental<br />
autoimmune hearing loss (EAHL), and exogenous <strong>of</strong><br />
IL-10 would abrogate βtubulin-induced EAHL. BALB/c wildtype<br />
(WT) and homozygous (IL-10−/−) IL-10-deficient mice<br />
underwent βtubulin immunization, development <strong>of</strong> EAHL<br />
was monitored over time. Additional groups <strong>of</strong> IL-10deficient<br />
mice immunized βtubulin to induce EAHL; IL-10deficient<br />
mice were intramuscularly injected with plasmid<br />
DNA encoding IL-10 on the climax <strong>of</strong> EAHL. Auditory<br />
brainstem responses (ABR) were examined over time.<br />
EAHL developed progressively in both BALB/c WT and IL-<br />
10-deficient mice immunized with βtubulin. However, the<br />
severity <strong>of</strong> hearing loss in the IL-10−/− mice was significantly<br />
greater than that in WT animals. Disease severity was<br />
associated with significantly enhanced IFNγ and TNFα level<br />
from stimulated splenocyte cultures, increased IgG2a antiβtubulin<br />
antibody responses and degeneration <strong>of</strong> Spiral<br />
ganglion cells <strong>of</strong> the Cochlea. αtubulin immunized IL-10−/−<br />
mice receiving intramuscular injection <strong>of</strong> plasmid DNA<br />
encoding IL-10 developed high serum levels <strong>of</strong> IL-10 yet<br />
the hearing function was significantly improved in the IL-10treated<br />
groups than that in the vector control group.<br />
Moreover, the ABR thresholds in the IL-10-treated groups<br />
are equivalent to naive controls and the histological<br />
analysis <strong>of</strong> the cochlear cross section was similar to those<br />
<strong>of</strong> naive controls. Furthermore, IL-10-treated mice exhibited<br />
significant suppression <strong>of</strong> βtubulin-induced IFNγ and<br />
TNFγ, but increased IgG1 production compared to levels<br />
exhibited by control mice. Our data demonstrate that lack<br />
<strong>of</strong> IL-10 exacerbates hearing loss and exogenous administration<br />
<strong>of</strong> IL-10 improved hearing function.<br />
doi:10.1016/j.clim.2007.03.521<br />
Sa.135 Galectin-1 Selectively Suppresses<br />
Plasmacytoid DC<br />
James Chen, Student, University <strong>of</strong> California, Los Angeles,<br />
Medicine, Los Angeles, CA, Xiangshu Wen, Postdoctoral<br />
Fellow, University <strong>of</strong> California, Los Angeles, Medicine, Los<br />
Angeles, CA, Jennifer Fulcher, MD, PhD Student, University<br />
<strong>of</strong> California, Los Angeles, Microbiology <strong>Immunology</strong> and<br />
Molecular Genetics, Los Angeles, CA, Benhur Lee, Assistant<br />
Pr<strong>of</strong>essor, University <strong>of</strong> California, Los Angeles,<br />
Microbiology <strong>Immunology</strong> and Molecular Genetics, Los
Abstracts<br />
Angeles, CA, Anna Eriksson, Postdoctoral Fellow, University<br />
<strong>of</strong> California, Los Angeles, Medicine, Los Angeles, CA, Ram<br />
Raj Singh, Pr<strong>of</strong>essor, University <strong>of</strong> California, Los Angeles,<br />
Medicine, Los Angeles, CA<br />
We have recently reported that galectin-1, a β-galactoside-binding<br />
lectin with a broad range <strong>of</strong> immunomodulatory<br />
properties, binds to and activates human monocyte-derived<br />
dendritic cells (DC). To evaluate the implications <strong>of</strong> this<br />
finding in vivo, we have investigated the role <strong>of</strong> galectin-1 in<br />
the modulation <strong>of</strong> DC in mice. We first show that galectin-1<br />
strongly binds murine DC. To examine the effect <strong>of</strong> galectin-1<br />
on DC subsets, we cultured spleen cells from BALB/c mice<br />
with LPS or galectin-1. We found that whereas LPS induces<br />
CD40 expression on all DC subsets including myeloid<br />
(CD11c+CD11b+), lymphoid (CD11c+CD8a+) and plasmacytoid<br />
(pDC; CD11c+B220+) DC, galectin-1 induces CD40 expression<br />
only in a fraction <strong>of</strong> DC. Strikingly, galectin-1 reduces the<br />
percentage and numbers <strong>of</strong> pDC and their activation as<br />
determined by CD40 expression, whereas LPS causes their<br />
expansion and activation. To assess the effect <strong>of</strong> galectin-1 on<br />
pDC in vivo, we used autoimmune-prone MRL-lpr mice that<br />
have increased numbers <strong>of</strong> pDC accumulating in inflamed<br />
organs. A single s.c. injection <strong>of</strong> galectin-1 in these mice<br />
reduced the proportion <strong>of</strong> pDC in cutaneous lymph nodes.<br />
These data suggest a role <strong>of</strong> galectin-1 in the regulation <strong>of</strong><br />
pDC. B220 is a spliced is<strong>of</strong>orm <strong>of</strong> CD45 and thus, our data are<br />
consistent with the known ability <strong>of</strong> galectin-1 to trigger<br />
apoptotic cell death in T cells via cross-linking <strong>of</strong> CD45.<br />
Ongoing studies will investigate the implication <strong>of</strong> this finding<br />
on the development <strong>of</strong> systemic inflammation in MRL-lpr<br />
mice, and the possible therapeutic consequences <strong>of</strong> galecin-1<br />
administration in the murine lupus model.<br />
doi:10.1016/j.clim.2007.03.522<br />
Sa.136 T Cell Responsiveness to Complementary<br />
PR3 Protein Supports a Pathogenic Role <strong>of</strong><br />
Autoantigen Complementarity in PR3-ANCA<br />
Autoimmune Disease<br />
Jiajin Yang, Assistant Pr<strong>of</strong>essor, University <strong>of</strong> North Carolina<br />
at Chapel Hill Kidney Center, Chapel Hill, NC, David Bautz,<br />
Graduate Student, University <strong>of</strong> North Carolina at Chapel<br />
Hill Kidney Center, Chapel Hill, NY, John Smitz, Associate<br />
Pr<strong>of</strong>essor, University <strong>of</strong> North Carolina at Chapel Hill<br />
Department <strong>of</strong> Pathology and Laboratory Medicine, Chapel<br />
Hill, NC, Hyunsook Chin, Statistician II, University <strong>of</strong> North<br />
Carolina at Chapel Hill Kidney Center, Chapel Hill, NC,<br />
Barrrak Pressler, Assistant Pr<strong>of</strong>essor <strong>of</strong> Internal Medicine,<br />
Purdue University, Lafayette, IN, Susan Hogan, Research<br />
Assistant Pr<strong>of</strong>essor, Medicine, University <strong>of</strong> North Carolina<br />
at Chapel Hill Kidney Center, Chapel Hill, NC, Roland Tisch,<br />
Assoc Pr<strong>of</strong>essor, Microbiology and <strong>Immunology</strong>, Chapel Hill,<br />
NC, J. Charles Jennette, Distinguished Pr<strong>of</strong>essor,<br />
Department Chair, Pathology and Laboratory Medicine,<br />
Chapel Hill, NC, Ronald Falk, Distinguished Pr<strong>of</strong>essor/<br />
Division Chief/Director, University <strong>of</strong> North Carolina at<br />
Chapel Hill Kidney Center, Chapel Hill, NC, Gloria Preston,<br />
Pr<strong>of</strong>essor, University <strong>of</strong> North Carolina at Chapel Hill Kidney<br />
Center, Chapel Hill, NC<br />
We discovered that patients with PR3-ANCA vasculitis<br />
who have autoantibodies against the autoantigen proteinase<br />
3 (PR3) also have a subset <strong>of</strong> antibodies reactive<br />
against a protein complementary – or antisense – to the<br />
autoantigen, as detected using recombinant, complementary-PR3<br />
protein, cPR3. Investigations into the etiology and<br />
consequences <strong>of</strong> these anti-cPR3 antibodies led to the<br />
proposal that complementary proteins are involved in<br />
inciting autoantibody production, as detailed in the theory<br />
<strong>of</strong> autoantigen complementarity (Nat Med 2004, 10: 72–79).<br />
The present studies indicate that CD4+TH1 cells from<br />
patients (n=26) with PR3-ANCA vasculitis versus healthy<br />
controls (n=34) exhibit increased proliferation (Wilcoxon<br />
rank sum test, p=0.0014) and IFN-γ expression (p=b0.0001)<br />
when stimulated with cPR3-peptide (32 aa, 138–169) but<br />
not a scrambled peptide (p=0.60). These recall responses<br />
indicate that the responding T cells were activated prior in<br />
vivo. Reactivity to smaller, overlapping fragments <strong>of</strong> cPR3<br />
ruled out the possibility that cPR3-peptide functions as a<br />
superantigen. Interestingly, the HLA-DRB1*15 allele group<br />
was overrepresented in the patient group, as compared to<br />
frequencies from a Caucasian population US database<br />
(Fisher’s exact test, p=0.006). This allele group is predicted<br />
to bind cPR3 peptide with high affinity (based on the<br />
Immune Epitope and Analysis Resource database). Ranked<br />
linear regression analysis indicated a likelihood <strong>of</strong> p=0.009<br />
that anti-cPR3 antibodies and cPR3-specific T cells coexist<br />
within an individual. The T cell responses observed are<br />
consistent with a history <strong>of</strong> complementary-protein encounter<br />
in these patients. Consideration <strong>of</strong> potential contributions<br />
<strong>of</strong> complementary protein pairs in autoimmune<br />
diseases could revolutionize the approach for exploring<br />
pathogenic mechanisms.<br />
doi:10.1016/j.clim.2007.03.523<br />
S121<br />
Sa.137 Enhanced CD16 Mediated Depletion <strong>of</strong><br />
Activated Lymphocytes by a Non-Fucosylated Fully<br />
Human Anti-CD70 IgG1<br />
Hadia Lemar, Research Associate III, Medarex, Inc., Milpitas,<br />
CA, Diane Feingersh, Scientist I, Medarex, Inc., Milpitas,<br />
CA, Alison Witte, Scientist III, Medarex, Inc., Milpitas, CA,<br />
Jon Terrett, Director, Medarex, Inc., Milpitas, CA, Ben<br />
Preston, Research Assotciate III, Medarex, Inc., Milpitas,<br />
CA, Mark Selby, Director, Medarex, Inc., Milpitas, CA, Alan<br />
Korman, VP, Medarex, Inc., Milpitas, CA, Marco Coccia,<br />
Senior Scientist II, Medarex, Inc., Milpitas, CA<br />
CD70 is the only known ligand for CD27 mediated<br />
costimulation <strong>of</strong> B and T memory and effector responses.<br />
CD70 expression in normal tissues is restricted to activated<br />
lymphocytes and dendritic cells. Patients with autoimmune<br />
disorders express high levels <strong>of</strong> CD70 on chronically activated<br />
T cells and lymphocytes. Furthermore, CD70 blocking<br />
antibodies were shown to delay onset <strong>of</strong> experimental<br />
autoimmune encephalomyelitis and cardiac allograft rejection<br />
in mice. We demonstrate here that antibody dependent<br />
cellular cytotoxicity (ADCC) mediated depletion <strong>of</strong> hyperactivated<br />
CD70+ leukocytes may be an effective therapeutic<br />
strategy for autoimmunity. We previously reported that a
S122 Abstracts<br />
fully human, non-fucoslyated anti-human CD70 IgG1 (anti-<br />
CD70nf) mediated superior ADCC <strong>of</strong> CD70+ tumor cells<br />
compared to parental fucosylated IgG1 (anti-CD70p). Anti-<br />
CD70nf also stimulated ADCC <strong>of</strong> purified T cells activated by<br />
anti-CD3/anti-CD28 coated beads with ∼10-fold greater<br />
potency and ∼50% greater efficacy than anti-CD70p.<br />
Stimulation <strong>of</strong> PBMC with cytomegalovirus (CMV) peptide<br />
induced CD70 expression on responsive CD8+/CMV pentamer+<br />
cells. Anti-CD70p and anti-CD70nf decreased CD8+/CMV<br />
pentamer+ cells but depletion by anti-CD70nf was both<br />
more potent and efficacious. Anti-CD16 blocking was effective<br />
in reversing depletion by both antibodies, but 1000-fold<br />
greater concentration <strong>of</strong> anti-CD16 was required to inhibit<br />
depletion by anti-CD70nf. CD8+/CMV pentamer− cells were<br />
unaffected by either CD70 antibody. These CD70 antibodies<br />
also inhibited CD70-Fc binding to CD27+/CD70− RL cells and<br />
CD70 costimulation <strong>of</strong> T cells activated by anti-CD3 on CD32/<br />
CD70 CHO transfectants. Further evaluation <strong>of</strong> enhanced<br />
ADCC mediated depletion <strong>of</strong> activated leukocytes and anti-<br />
CD70 functional blockade as therapeutic strategies for<br />
autoimmunity is in progress.<br />
doi:10.1016/j.clim.2007.03.524<br />
Sa.138 Evidence for DC:Treg Paracrine IL-2 Delivery<br />
Katarina Kulhankova, Postdoctoral Scholar, Carver College<br />
<strong>of</strong> Medicine and Veterans Affairs Medical Center,<br />
Department <strong>of</strong> Internal Medicine, Iowa City, IA, Mohamed<br />
Nasr, Research Scientist, Carver College <strong>of</strong> Medicine and<br />
Veterans Affairs Medical Center, Department <strong>of</strong> Internal<br />
Medicine, Iowa City, IA, Michael E. Dailey, Pr<strong>of</strong>essor,<br />
University <strong>of</strong> Iowa, Department <strong>of</strong> Biological Sciences, Iowa<br />
City, IA, Todd Rouse, Research Assistant, Carver College <strong>of</strong><br />
Medicine and Veterans Affairs Medical Center, Department<br />
<strong>of</strong> Internal Medicine, Iowa City, IA, Elizabeth H. Field,<br />
Pr<strong>of</strong>essor, Carver College <strong>of</strong> Medicine and Veterans Affairs<br />
Medical Center, Department <strong>of</strong> Internal Medicine,<br />
Iowa City, IA<br />
Regulatory CD4+CD25+ Tcells (Tregs) play a key role in the<br />
maintenance <strong>of</strong> immune tolerance, and understanding the<br />
precise mechanism <strong>of</strong> their function can help guide<br />
therapeutic strategies for achieving transplant tolerance.<br />
Once activated, Tregs inhibit CD4+ cell proliferation and IL-2<br />
production in vitro. Treg cell function is blocked by culturing<br />
Tregs and effectors in the presence <strong>of</strong> anti-CD25 or<br />
separately in transwell system. This indicates that both IL-<br />
2 and cell:cell contact is necessary for the gain or execution<br />
<strong>of</strong> Treg-mediated suppression. However, the source for IL-2<br />
and the context, in which it is delivered, remains unknown.<br />
To address this question we generated the CD4+DC25+<br />
hybridoma RD6 from a mouse with acquired tolerance to<br />
foreign MHCs. Like natural Tregs, RD6 inhibition <strong>of</strong> CD4+<br />
effectors requires CD25 and cell:cell contact. Neither Treg<br />
nor RD6 showed inhibition when IL-2KO DCs were substituted<br />
for WT DCs in Treg assays. We utilized four-dimensional<br />
multi-channel fluorescent live-cell confocal microscopy to<br />
define the CD25 expression on RD6 cells during cell:cell<br />
interactions between RD6 and semi-allogeneic DCs or<br />
allogeneic membrane-labeled DC cells, JAWSII. RD6 engaged<br />
themselves and DCs in a variety <strong>of</strong> cell:cell interactions that<br />
included long-lasting conjugate formation as well as formation<br />
<strong>of</strong> stable cell:cell membrane bridges <strong>of</strong> various lengths.<br />
RD6 CD25 expression localized to points <strong>of</strong> contact with<br />
dendritic processes over 24 hours. RD6 cells acquired patches<br />
<strong>of</strong> DC membranes and internalized them. These data are<br />
consistent with a model, in which DCs deliver IL-2 to Tregs via<br />
tight membrane contacts.<br />
doi:10.1016/j.clim.2007.03.525<br />
Sa.139 Admixture Mapping, a New Tool for Disease<br />
Gene Finding in Autoimmune and Other Diseases<br />
Alicja Waliszewska, Research Specialist, Broad Institute,<br />
Cambridge, MA, Phillip De Jager, Assistant Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> Genetics, Harvard Medical School, Boston<br />
MA, David Hafler, Pr<strong>of</strong>essor, Department <strong>of</strong> Genetics,<br />
Harvard Medical School, Boston MA, David Reich, Assistant<br />
Pr<strong>of</strong>essor, Department <strong>of</strong> Genetics, Harvard Medical School,<br />
Boston, MA<br />
Admixture mapping is a powerful technique for finding<br />
genes for complex diseases. The practical requirements<br />
include (a) a set <strong>of</strong> markers spanning the genome with large<br />
allele-frequency differences between the parental ethnicities<br />
contributing to the admixed population and (b) an<br />
understanding <strong>of</strong> the extent <strong>of</strong> admixture in the study<br />
population. African American and Latino American populations<br />
are the product <strong>of</strong> gene flow (genetic admixture) during<br />
the last 500 years, which has produced new genotypes from<br />
once-isolated ancestral groups. Admixture mapping attempts<br />
to associate African, European, or Native American to disease<br />
risk. We have performed admixture scans in African Americans<br />
to find genes affecting Multiple Sclerosis (MS),<br />
Rheumatoid Arthritis (RA), markers <strong>of</strong> inflammation, and<br />
prostate cancer. We have also been building resources for<br />
admixture mapping in Latinos. We have detected loci that are<br />
significantly associated to MS on chromosome 1 (LOD=5.2), to<br />
prostate cancer on chromosome 8 (LOD=7.1), and to<br />
interleukin 6 soluble receptor (IL6 SR) on chromosome 1<br />
(LOD=4.59). For IL6 SR and prostate cancer this has led to<br />
cloning <strong>of</strong> disease risk alleles. In African Americans with<br />
rheumatoid arthritis, the scan was negative, and identified<br />
no loci for disease risk.<br />
doi:10.1016/j.clim.2007.03.526<br />
Sa.140 Comparison <strong>of</strong> Membrane-bound and<br />
Secreted TGFβ by IL-2 Activated Natural and<br />
Adaptive CD4+CD25+ Regulatory Cells from Healthy<br />
and Lupus-prone Mice<br />
Song Guo Zheng, Assistant Pr<strong>of</strong>essor <strong>of</strong> Research Medicine,<br />
University <strong>of</strong> Southern California Keck School <strong>of</strong> Medicine,<br />
Los Angeles, CA, Julie Wang, Research Lab Specialist,<br />
University <strong>of</strong> Southern California Keck School <strong>of</strong> Medicine,<br />
Los Angeles, CA, Shuang Ye, Postdoctoral Fellow, University<br />
<strong>of</strong> Southern California Keck School <strong>of</strong> Medicine, Los Angeles,<br />
CA, David Sehy, Director, New Technologies, eBioscience/<br />
Department <strong>of</strong> New Technologies, San Diego, CA, Beth
Abstracts<br />
Palian, Postdoctoral Student, University <strong>of</strong> Southern<br />
California Keck School <strong>of</strong> Medicine, Los Angeles, CA, David<br />
A. Horwitz, Pr<strong>of</strong>essor Chief, Division <strong>of</strong> Rheumatology and<br />
<strong>Immunology</strong>, University <strong>of</strong> Southern California Keck School<br />
<strong>of</strong> Medicine, Los Angeles, CA<br />
CD4+CD25+Foxp3+ regulatory T cells can be divided into<br />
natural, thymus-derived cells and adaptive Tregs induced<br />
from CD25− precursors in the periphery. Although TGFβ<br />
induced, adaptive Tregs produce TGFβ, whether natural<br />
CD4+CD25+ Tregs also produce this cytokine has been<br />
controversial. Mouse CD4+CD25+ cells were stimulated via<br />
TCR and/or IL-2 and naive CD4+CD25− cells were stimulated<br />
with TGFβ to induce adaptive Th3 cells. Although<br />
freshly isolated murine CD4+CD25+ cells did not express<br />
mature TGFβ, when TCR activated with IL-2, these Foxp3+<br />
Treg cells displayed similar numbers <strong>of</strong> membrane-bound<br />
TGFβ under appropriate staining conditions as Th3 cells<br />
induced with TGFβ ex vivo. Both natural and adaptive Treg<br />
subsets also secreted similar amounts <strong>of</strong> active TGFβ. Since<br />
TGFβ induces CD8+ cells to express α-E integrin (CD103),<br />
we found the activated CD25+ Tregs induced CD103<br />
expression on CD8+ cells to levels equivalent to that <strong>of</strong><br />
activated Th3 cells. ALK5 (TGFβ receptor I inhibitor), but<br />
not anti-TGFβ almost completely abolished this CD103<br />
expression. Transwell experiments revealed that surface<br />
contact was not required. Thus, activated natural CD4+<br />
CD25+ Treg cells can produce similar levels <strong>of</strong> mature TGFβ<br />
as adaptive TGFβ induced Tregs. Finally, we studied<br />
activated CD4+CD25+ cells from lupus-prone (NZBxNZW)<br />
F1 young and old mice and found that cells from old mice<br />
with established disease expressed decreased membranebound<br />
and produced less soluble TGFβ. Thus, defective<br />
TGFβ production by CD4+CD25+ Tregs may contribute to<br />
their decreased functional activity and to the development<br />
and progression <strong>of</strong> systemic lupus.<br />
doi:10.1016/j.clim.2007.03.527<br />
Sa.141 Thymic Organogenesis Controlled by<br />
NF-kappaB Activating Factors<br />
Masashi Ya, Graduate Student, Institute for Enzyme<br />
Research, University <strong>of</strong> Tokushima, Tokushima, Japan,<br />
Yasuhiro Mouri, Graduate Student, Institute for Enzyme<br />
Research, University <strong>of</strong> Tokushima, Tokushima, Japan,<br />
Mitsuru Matsumoto, Pr<strong>of</strong>essor, Institute for Enzyme<br />
Research, University <strong>of</strong> Tokushima, Tokushima, Japan<br />
Thymic epithelial cells (TEC) play pivotal roles in the<br />
establishment <strong>of</strong> self-tolerance through critical dialogue<br />
with developing thymocytes. Unique actions <strong>of</strong> two NFkappaB<br />
activating factors within TECs, NF-kappaB-inducing<br />
kinase (NIK) and IkappaB kinase α (IKKα), for the establishment<br />
<strong>of</strong> self-tolerance have recently been highlighted by<br />
studies using a strain <strong>of</strong> mouse bearing a natural mutation<br />
<strong>of</strong> the NIK gene (aly mice) and gene-targeted mice,<br />
respectively. Previous studies have demonstrated essential<br />
roles <strong>of</strong> NIK-IKKα downstream <strong>of</strong> the lymphotoxin-beta<br />
receptor (LTβR), which is essential for the development <strong>of</strong><br />
secondary lymphoid organs; aly mice lack all lymph nodes<br />
and Peyer’s patches because <strong>of</strong> the defective LTβR<br />
signaling. Now additional roles <strong>of</strong> NIK-IKKα in thymic<br />
organogenesis, mainly through the developmental regulation<br />
<strong>of</strong> TECs, have emerged, although the corresponding<br />
upstream receptor(s) and ligand(s) participating in this<br />
action have not been fully characterized. Previous studies<br />
have suggested that LTβR regulates thymic organogenesis.<br />
In order to investigate the contribution <strong>of</strong> LTβR signaling for<br />
the thymic organogenesis in NIK-IKKα-dependent pathways,<br />
we have analyzed thymic architecture together with Aire<br />
expression from both LTα-deficient mice and LTβR-deficient<br />
mice. We found that Aire-expressing cells were largely<br />
retained from those mice, whereas they were dramatically<br />
reduced from both NIK-mutant mice and IKKα-deficient<br />
mice. Because NIK-mutant mice and IKKα-deficient mice<br />
show more pr<strong>of</strong>ound thymic disorganization <strong>of</strong> TECs than<br />
that from LTβR-deficient mice, it is likely that NIK-IKKα is<br />
additionally acting downstream on other receptor(s) beyond<br />
LTβR in this process.<br />
doi:10.1016/j.clim.2007.03.528<br />
S123<br />
Sa.142 A FLIPR-based Assay to Assess Potency <strong>of</strong><br />
Inhibitors <strong>of</strong> the TEC Family Kinases Itk and Btk<br />
John Douhan III, Principal Research Scientist, Wyeth<br />
Research, Inflammation, Cambridge, MA, Joy Miyashiro,<br />
Research Scientist, Wyeth Research, Inflammation,<br />
Cambridge, MA, Xiaochuan Zhou, Research Scientist, Wyeth<br />
Research, Inflammation, Cambridge, MA, Paul Wu, Research<br />
Scientist, Wyeth Research, Inflammation, Cambridge, MA,<br />
Derek Cole, Principal Research Scientist, Wyeth Research,<br />
Chemical and Screening Sciences, Pearl River, NY, Mary<br />
Collins, Vice President, Wyeth Research, Inflammation,<br />
Cambridge, MA, Kyriaki Dunussi-Joannopoulos, Associate<br />
Director, Wyeth Research, Inflammation, Cambridge, MA<br />
Bruton’s tyrosine kinase (Btk) and interleukin-2-inducible<br />
Tcell kinase (Itk), members <strong>of</strong> the TEC family <strong>of</strong> nonreceptor<br />
protein tyrosine kinases, are expressed primarily in B and T<br />
cells respectively and are critically involved in lymphocyte<br />
development and signal transduction. In particular, both Btk<br />
and Itk regulate calcium mobilization subsequent to antigen<br />
receptor stimulation which in turn regulates downstream<br />
effector functions such as proliferation, antibody secretion<br />
and cytokine production. Small molecule antagonists <strong>of</strong> Btk<br />
and Itk may therefore be useful in treating inflammatory and<br />
autoimmune conditions. We have developed a FLIPR-based<br />
assay which takes advantage <strong>of</strong> Btk-deficient DT40 chicken B<br />
cells. It has been reported that these cells are unable to<br />
mobilize calcium in response to cross-linking <strong>of</strong> their B cell<br />
receptor and that ectopic expression <strong>of</strong> human Btk or Itk can<br />
restore signaling upon B cell receptor stimulation. Here we<br />
report that not only the expression <strong>of</strong> wild type human Btk,<br />
but also the expression <strong>of</strong> a chimeric Btk–Itk kinase molecule<br />
can restore calcium responses in Btk-deficient DT40 cells. We<br />
have generated stable cell lines expressing either full length<br />
human Btk or a chimeric human Btk–Itk molecule—a Btk<br />
protein whose kinase domain has been replaced by that <strong>of</strong><br />
Itk. Calcium responses in both cell lines can be inhibited by<br />
the tyrosine kinase inhibitor staurosporine, but only the
S124 Abstracts<br />
chimeric Btk-Itk DT40 line is inhibited by an Itk-specific<br />
kinase inhibitor. We demonstrate that potency and selectivity<br />
<strong>of</strong> inhibitors <strong>of</strong> Itk and Btk can be assessed in this FLIPR<br />
cell-based assay.<br />
doi:10.1016/j.clim.2007.03.529<br />
Sa.143 Anti-Inflammatory Activities <strong>of</strong> Stilbene<br />
Analogs for Targeting Autoimmune Diseases<br />
Liren Tang, Scientist, Welichem Biotech Inc., Burnaby, BC,<br />
Canada, Genhui Chen, Celestial Pharmaceuticals Ltd,<br />
Shenzhen, China, Bin Li, Scientist, Welichem Biotech Inc.,<br />
Burnaby, BC, Canada, Quanhai Liu, Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Pharmacology, Shanghai Instititue <strong>of</strong> Pharmaceutical<br />
Industry, Shanghai, China, Michael Lyle, Postdoctoral<br />
Fellow, Welichem Biotech Inc., Burnaby, BC, Canada, John<br />
Webster, Pr<strong>of</strong>essor and CSO, Welichem Biotech Inc., Simon<br />
Fraser University, Burnaby, BC, Canada<br />
Certain species <strong>of</strong> symbiotic bacteria (Enterobacteriaceae),<br />
when released into an insect by their nematode<br />
vector, released metabolites that modulate the insect’s<br />
innate immune response. These metabolites (stilbene<br />
analogs, in the WBI-1000 series) possess unique antiinflammatory<br />
properties. In general, they are weakly<br />
cytotoxic to mammalian cells, being more active on<br />
activated than on quiescent T cells or other normal cells.<br />
When tested in vitro, the WBI-1000 series selectively<br />
inhibited IL-2 and IFN-γ production in human T cells, and<br />
significantly inhibited TNF-α production in mice, but IL-4 was<br />
not affected. As well, they significantly inhibited T cell<br />
migration towards LTB4 in vitro. When topically applied in<br />
the TPA-induced ear edema mouse model, WBI-1000 compounds<br />
significantly reduced both skin redness and thickness.<br />
To further explore the clinical applications <strong>of</strong> these<br />
compounds one <strong>of</strong> them, WBI-1001 was tested for its efficacy<br />
on different animal models for autoimmune diseases. WBI-<br />
1001 showed dose-related efficacy in both the vaginal<br />
mucosa and mouse tail models for psoriasis as a topical<br />
treatment, significant clinical improvement in dextran<br />
sodium sulphate (DSS)-induced inflammatory bowel disease<br />
(IBD) in mice and decreased inflammation-induced arthritis<br />
in rats when compared with indomathecin. In conclusion,<br />
due to its unique anti-inflammatory properties, including<br />
selectivity on activated T cells, strong inhibition <strong>of</strong> proinflammatory<br />
cytokines and T cell migration, the WBI-1000<br />
series <strong>of</strong> compounds could be developed into effective, novel<br />
treatment modalities for autoimmune diseases.<br />
doi:10.1016/j.clim.2007.03.551<br />
Sa.145 Functional Knockout <strong>of</strong> Kv1.3 Channels<br />
Suppresses Effector Memory Phenotype Acquisition<br />
in Dominant Negative Kv1.3-expressing Human<br />
CD4+ T Cells<br />
Lina Hu, Research Associate, Department <strong>of</strong> Neurology,<br />
Johns Hopkins School <strong>of</strong> Medicine, Baltimore, MD, Katharine<br />
Whartenby, Assistant Pr<strong>of</strong>essor, Sidney Kimmel Cancer<br />
Center, Johns Hopkins School <strong>of</strong> Medicine, Baltimore, MD,<br />
Rameeza Allie, Research Specialist, Department <strong>of</strong><br />
Neurology, Johns Hopkins School <strong>of</strong> Medicine Baltimore, MD,<br />
Peter Calabresi, Associate Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Neurology, Johns Hopkins School <strong>of</strong> Medicine, Baltimore, MD<br />
Activated effector memory T cells (TEM) that highly<br />
express the voltage-gated potassium channel Kv1.3 have<br />
the potential <strong>of</strong> migrating to the CNS and are hypothesized<br />
to play a pathogenic role in multiple sclerosis (MS). The<br />
pharmacological effects <strong>of</strong> Kv1.3 channel blockade on TEM<br />
cell activation and proliferation are well documented.<br />
However, the functional relevance <strong>of</strong> Kv1.3 in the homeostatic<br />
maintenance <strong>of</strong> TEM is poorly understood. Herein,<br />
using a system in which Kv1.3-channel function was blocked<br />
by transduction <strong>of</strong> CD4+ T cells with a GFP-tagged,<br />
lentiviral vector expressing dominant-negative Kv1.x<br />
sequence, we studied the kinetic changes in TEM subset<br />
within dominant negative Kv1.3-expressing CD4+ T cells<br />
following anti-CD3/CD28 stimulation. The interference <strong>of</strong><br />
endogenous Kv1.3 by misexpression <strong>of</strong> dominant-negative<br />
Kv1.x markedly attenuated the wild-type current. Analysis<br />
<strong>of</strong> GFP expression in CD4+ T cell subsets demonstrated that<br />
while a substantial reservoir <strong>of</strong> TCM was maintained, TEM<br />
cells expressing the dominant-negative Kv1.3 product were<br />
significantly less abundant than that infected with GFP<br />
control viruses at 2, 3 and 4 weeks after transduction. In<br />
contrast, the ratio between TEM and TCM cells in GFP<br />
control transfected cells is strongly biased toward the TEM.<br />
Thus, the generation and maintenance <strong>of</strong> TEM cells are<br />
markedly impaired by the inactivation <strong>of</strong> Kv1.3 channel<br />
function. These results <strong>of</strong>fer new insights into the<br />
functional dependence <strong>of</strong> TEM cells on Kv1.3, and have<br />
important clinical implications for using Kv1.3 blockers to<br />
prevent the generation <strong>of</strong> TEM cells, thereby abrogating<br />
the pathologic consequences <strong>of</strong> this subset in autoimmune<br />
diseases, such as MS.<br />
doi:10.1016/j.clim.2007.03.532<br />
Sa.146 Tim-4 Expressed on Antigen-presenting Cells<br />
Induces T Cell Expansion and Survival<br />
Roselynn Rodriguez Manzanet, Graduate Student, Brigham<br />
and Women’s Hospital, Boston, MA, Jennifer Hartt-Meyers,<br />
Post doctoral Fellow, LIR/NIAID/NIH, Bethesda, MD,<br />
Jacqueline Slavik, Administrative Director, Brigham and<br />
Women’s Hospital, Biomedical Research Institute, Boston,<br />
MA, Valerie Dardalhon, Post doctoral Fellow, Brigham and<br />
Women’s Hospital, Department <strong>of</strong> Neurology, Boston, MA,<br />
Nasim Kassam, Research specialist, Brigham and Women’s<br />
Hospital, Department <strong>of</strong> Neurology, Boston, MA, Edward A.<br />
Greenfield, Consultant, Brigham and Women’s Hospital,<br />
Department <strong>of</strong> Neurology, Boston, MA, Raymond A. Sobel,<br />
Pr<strong>of</strong>essor, Stanford School <strong>of</strong> Medicine, Department <strong>of</strong><br />
Pathology, Palo Alto, CA, Terry B. Strom, Physician<br />
Pr<strong>of</strong>essor, Beth Israel Deaconess Medical Center, Boston,<br />
MA, David A. Hafler, Pr<strong>of</strong>essor, Brigham and Women’s<br />
Hospital, Department <strong>of</strong> Neurology, Boston, MA, Vijay K.<br />
Kuchroo, Pr<strong>of</strong>essor, Brigham and Women’s Hospital,<br />
Department <strong>of</strong> Neurology, Boston, MA
Abstracts<br />
The TIM (T cell, immunoglobulin, mucin) proteins characterized<br />
to date have been shown to regulate Tcell immune<br />
responses. Tim-4 has been shown to be a ligand for Tim-1 and<br />
Tim-4.Ig fusion protein could both inhibit and activate T cell<br />
proliferation in in vitro assays. Further studies <strong>of</strong> this<br />
molecule have been hindered by the lack <strong>of</strong> a specific<br />
monoclonal antibody. Thus, we have generated three<br />
monoclonal anti-Tim-4 antibodies and show that Tim-4<br />
protein expression is restricted to the antigen-presenting<br />
cell population, specifically CD11c+ and CD11b+ cells. We<br />
demonstrate that Tim-4 expression is upregulated upon<br />
cellular activation, and that ectopic expression <strong>of</strong> Tim-4 on<br />
artificial APCs induces massive T cell proliferation. Additionally,<br />
in vitro crosslinking <strong>of</strong> Tim-4 ligand on the surface <strong>of</strong> T<br />
cells directly induced cell division and anti-apoptotic protein<br />
Bcl-2. We conclude that Tim-4 is a costimulatory molecule<br />
capable <strong>of</strong> promoting T cell expansion and show that it can<br />
enhance both T cell proliferation and survival.<br />
doi:10.1016/j.clim.2007.03.533<br />
Sa.147 Interleukin-2 Mastering Regulation in Cancer<br />
and Autoimmunity<br />
Enrique Montero, MD, PhD, Center <strong>of</strong> Molecular<br />
<strong>Immunology</strong>, Department <strong>of</strong> Experimental Immunotherapy,<br />
Havana, Cuba, Livan Alonso, BSc in Physics, Center <strong>of</strong><br />
Molecular <strong>Immunology</strong>, Department <strong>of</strong> Experimental<br />
Immunotherapy, Havana, Cuba, Rolando Perez, PhD, Center<br />
<strong>of</strong> Molecular <strong>Immunology</strong>, Havana, Cuba, Agustin Lage, MD,<br />
PhD, Center <strong>of</strong> Molecular <strong>Immunology</strong>, Havana, Cuba<br />
Autoimmunity and tumor immunity have evolved as two<br />
well distant arenas in immunological research. However, the<br />
identification <strong>of</strong> self-antigens as the major components <strong>of</strong><br />
malignant cells may define a central role for autoimmunity in<br />
cancer control tuned by peripheral immunoregulatory<br />
mechanisms avoiding self-aggression. Interleukin-2 (IL-2)<br />
paradoxical effects after in vitro or in vivo manipulations<br />
may reflect the complex interplay between immunoregulatory<br />
mechanisms. Emerging evidences support the contribution<br />
<strong>of</strong> IL-2 to the maintenance <strong>of</strong> natural immunological<br />
tolerance. It encourages the systematic evaluation in vivo <strong>of</strong><br />
formulations with IL-2 neutralization capacity compared<br />
with IL-2 exogenous administration to explore their influence<br />
on immunobiology in cancer and autoimmunity. IL-2 chemically<br />
conjugated to the carrier protein P64k from Neisseria<br />
Meningitides in Montanide ISA51 adjuvant was used for active<br />
immunization. We found that BALB/c, C57BL/6 and NMRI<br />
mice developed a long-lasting immunoresponse to IL-2 after<br />
immunization, without signs <strong>of</strong> autoimmune diseases.<br />
Immune sera had a similar IL-2 blocking effect in vitro to a<br />
specific anti-IL-2 monoclonal antibody (mAb). Interestingly,<br />
IL-2 deprivation did not affect the immunoresponse to<br />
therapeutic vaccines; however, it restores the response to<br />
nominal antigens in immunosuppressed hosts. Surprisingly,<br />
we found a differential effect <strong>of</strong> anti-IL-2 and anti-CD25 mAb<br />
on anti-tumor immunoresponse that may represent a broader<br />
impact <strong>of</strong> IL-2 on immunoregulation beyond its effect on<br />
CD4+CD25+ regulatory T cells. Moreover, the main effect <strong>of</strong><br />
IL-2 administration was associated with an enhancement <strong>of</strong><br />
immunoregulation. We conclude that IL-2 plays a major role<br />
in the interplay between cancer and autoimmunity with<br />
therapeutic implications.<br />
doi:10.1016/j.clim.2007.03.534<br />
Sa.148 Decrease in Memory B Cell Frequency and<br />
Activation is Associated with PTPN22 1858T Variant<br />
in Healthy Subjects<br />
Mary Rieck, Research Technician II, Benaroya Research<br />
Institute, Seattle, WA, Patric Concannon, Pr<strong>of</strong>essor, Center<br />
for Public Health Genomic, University <strong>of</strong> Virginia,<br />
Charlotte, SC, Adrian Arechiga, Research Associate,<br />
Benaroya Research Institute, Seattle, WA, Jane Buckner,<br />
Associate Member, Benaroya Research Institute, Seattle,<br />
WA, Suna Onengut-Gumuscu, Research Associate, Center for<br />
Health Genomic, Charlotte, SC<br />
It is known that the 1858T variant <strong>of</strong> the PTPN22 gene is<br />
associated with multiple autoimmune disorders, yet the<br />
functional consequences <strong>of</strong> this variant and how they<br />
contribute to autoimmunity are still unknown. PTPN22<br />
encodes the protein tyrosine phosphatase, Lyp, which<br />
participates in the proximal events <strong>of</strong> TCR and BCR signaling.<br />
The 1858T mutations results in an R and #61664;W amino acid<br />
change at residue 620 <strong>of</strong> Lyp. In Tcells Lyp620W dampens TCR<br />
signaling, however, its effects on the B cell compartment are<br />
not known. In this study we address this question by<br />
examining B cell function and phenotype in control subjects<br />
possessing the 1858T allele (1858C/T) as compared to<br />
subjects without the variant (1858C/C). These studies reveal<br />
no difference in percentage or number <strong>of</strong> total B cells in the<br />
periphery. However, within the B cell pool there was a<br />
decrease in the percent <strong>of</strong> memory B cells, defined as<br />
CD19+CD27+, in 1858C/T versus 1858C/C subjects (p=0.01).<br />
This decrease in the memory pool was even more pr<strong>of</strong>ound in<br />
autoimmune subjects homozygous for the 1858T variant<br />
(p=0.002). In addition, we find that the response <strong>of</strong> B cells to<br />
BCR stimulation, as measured by calcium flux is diminished in<br />
subjects carrying the 1858T allele, this was most pr<strong>of</strong>ound in<br />
the memory subset. These studies demonstrate that the<br />
PTPN22 1858T variant leads to altered B cell subsets and<br />
function. This suggests the possibility that 1858Tacts through<br />
B cell intrinsic mechanisms that contribute to the pathogenesis<br />
<strong>of</strong> autoimmunity in individuals with this variant.<br />
doi:10.1016/j.clim.2007.03.535<br />
S125<br />
Sa.149 Deconvolution <strong>of</strong> Blood Microarray Data<br />
Elucidates Cellular Activation Patterns in SLE<br />
Alexander R. Abbas, Genentech Inc., South San Francisco,<br />
CA, Kristen Wolslegel, Genentech Inc., South San Francisco,<br />
CA, Dhaya Seshasayee, Genentech Inc., South San Francisco,<br />
CA, Hilary F. Clark, Genentech Inc., South San Francisco, CA<br />
Understanding the biology <strong>of</strong> a disease <strong>of</strong>ten depends on<br />
knowing the different types and states <strong>of</strong> cells in biological
S126 Abstracts<br />
samples from relevant organs. The proportions <strong>of</strong> cells in<br />
blood or tissue are traditionally determined using methods<br />
that rely on one or two genes' expression or protein products,<br />
such as FACS or immunohistochemistry. These techniques<br />
detect only a few cell types at once and <strong>of</strong>ten cannot<br />
determine the state <strong>of</strong> cells. Here we present a method for<br />
using gene expression pr<strong>of</strong>iles <strong>of</strong> purified leukocyte cell types<br />
to probe the levels <strong>of</strong> cell types and cell states in whole blood<br />
samples. We demonstrate that this method accurately<br />
deconvolves mixed samples <strong>of</strong> known composition. We<br />
deconvolve blood samples from healthy donors and systemic<br />
lupus erythematosus patients to reveal patterns <strong>of</strong> cellular<br />
activation that apparently underlie this disease's prominent<br />
interferon signature.<br />
doi:10.1016/j.clim.2007.03.536<br />
Sa.150 Characterization <strong>of</strong> the Psoriasis-associated<br />
IL12B and IL23R Genes<br />
Ann Begovich, Director, Celera, Alameda, CA, Monica<br />
Chang, Senior Associate Scientist, Celera, Alameda, CA,<br />
Mark Leppert, Pr<strong>of</strong>essor, Department <strong>of</strong> Human Genetics,<br />
University <strong>of</strong> Utah, Salt Lake City, UT, Steven Schrodi,<br />
Senior Scientist, Celera, Alameda, CA, Gerald Krueger,<br />
Pr<strong>of</strong>essor, Department <strong>of</strong> Dermatology, University <strong>of</strong> Utah,<br />
Salt Lake City, UT<br />
Common genetic variants in IL12B and IL23R have been<br />
associated with psoriasis risk. To further investigate the<br />
role <strong>of</strong> these genes in susceptibility to psoriasis and other<br />
autoimmune diseases, we have resequenced both genes in<br />
96 individuals with psoriasis, identified novel SNPs and<br />
genotyped a subset <strong>of</strong> these and others from public<br />
databases in three independent white North American<br />
case–control sample sets (1446 cases and 1432 controls).<br />
To date, allelic and genotypic data for 51 IL12B-region and<br />
46 IL23R-region SNPs have been analyzed and preliminary<br />
haplotype analyses have been performed. The data suggest<br />
that association <strong>of</strong> IL12B with psoriasis is driven by two<br />
SNPs, rs3212227 and rs6887695, or SNPs in significant LD<br />
with these two markers. A sliding-window <strong>of</strong> haplotype<br />
association co-localizes psoriasis susceptibility within the<br />
boundaries <strong>of</strong> IL23R and not IL12RB2, which lies directly<br />
adjacent. Further genotyping in this region is ongoing to<br />
better define the IL23R genetic variants responsible for the<br />
association with psoriasis. We have also assessed whether<br />
the two psoriasis-associated IL12B SNPs are associated with<br />
specific clinical characteristics including age <strong>of</strong> onset,<br />
family history, psoriatic arthritis, and the effect <strong>of</strong><br />
infection on psoriasis severity in the two sample sets<br />
with complete phenotype data. The only interesting<br />
replicated finding was that the effect <strong>of</strong> rs6887695 may<br />
be significantly different when stratifying by response to<br />
infection (Breslow-Day P=0.04 and 0.043). Finally, the role<br />
<strong>of</strong> these SNPs in susceptibility to rheumatoid arthritis and<br />
multiple sclerosis will be addressed.<br />
doi:10.1016/j.clim.2007.03.537<br />
Sa.151 Elifacs: A Multiplex Assay to Detect Antigen<br />
Specific T Cells and to Monitor the Immune<br />
Response<br />
Khadir Raddassi, Research Instructor, Brigham and Women’s<br />
Hospital, Neurology, Boston, MA, Kasia Bouchier, Scientist,<br />
Immune Tolerance Network, San Francisco, CA, Jose<br />
Estevam, Research Assistant, Brigham and Women’s<br />
Hospital, Neurology, Boston, MA, Vicki Seyfert-Margolis,<br />
Scientist, Immune Tolerance Network, San Francisco, CA,<br />
David Hafler, Lab Director, Brigham and Women’s Hospital,<br />
Neurology, Boston, MA<br />
As part <strong>of</strong> the Immune Tolerance Network assay development<br />
group, we optimized a multiplex cell based assay to<br />
detect antigen reactive T cells. Cell assays to detect antigen<br />
specific T cells require large cell numbers. The Elispot assay<br />
is fast and sensitive but provides limited information<br />
regarding individual cells. In contrast, flow cytometry allows<br />
better characterization <strong>of</strong> antigen reactive T cells. Similarly,<br />
measuring thymidine incorporation provides no information<br />
regarding T cell function. Here, we combined the three to<br />
monitor T cell response to antigens using the same sample.<br />
Fresh or cryopreserved PBMCs were stained with CFSE, and<br />
exposed to tetanus toxoid or myelin antigens. After 42 h the<br />
cells were transferred to Elispot plates for 16 h; the Elispot<br />
plates were developed for cytokine expression, and the cells<br />
were collected and cultured for an additional 5 days before<br />
measuring intracellular cytokines and proliferation by flow<br />
cytometry. The combination <strong>of</strong> Elispot and flow cytometry<br />
data did not alter the specificity or sensitivity <strong>of</strong> these<br />
techniques. The recovery and viability <strong>of</strong> the cells from the<br />
Elispot plate were above 90%. There was a tight correlation<br />
(r 2 =0.960) between these data obtained by Elispot (cytokine<br />
positive cells) and by flow cytometry (CFSE and the cytokine<br />
production). We were able to measure IFNg, IL-10, IL-4,<br />
TGFβ and proliferative responses from the same sample. This<br />
technique allows us to extract more information from the<br />
same sample and thus provides a useful tool in monitoring<br />
the immune response.<br />
doi:10.1016/j.clim.2007.03.538<br />
Sa.152 Population-based Studies <strong>of</strong> Risk Factors in<br />
Autoimmunity: The Kaiser Permanente<br />
Autoimmune Disease Research Group (KPADRG)<br />
Lisa Barcellos, Assistant Pr<strong>of</strong>essor, UC Berkeley, School <strong>of</strong><br />
Public Health, Berkeley, CA, Ling Shen, Programmer/<br />
Analyst, Kaiser Permanente Division <strong>of</strong> Research, Oakland,<br />
CA, Lisa Herrinton, Epidemiologist/Senior Researcher,<br />
Kaiser Permanente Division <strong>of</strong> Research, Oakland, CA, Allan<br />
L. Bernstein, Chief <strong>of</strong> Neurology, Kaiser Permanente Santa<br />
Rosa, Kaiser Permanente Division <strong>of</strong> Research, Oakland, CA,<br />
James E. Allison, Adjunct Investigator, Kaiser Permanente<br />
Division <strong>of</strong> Research, Oakland, CA, John Citron,<br />
Endocrinologist, Kaiser Permanente Walnut Creek, Kaiser<br />
Permanente Division <strong>of</strong> Research, Oakland, CA, Stanford<br />
Shoor, Kaiser Permanente Santa Clara, Kaiser Permanente<br />
Division <strong>of</strong> Research, Oakland, CA, Andrew Karter,<br />
Epidemiologist/Senior Researcher, Kaiser Permanente<br />
Division <strong>of</strong> Research, Oakland, CA, De-Kun Li,
Abstracts<br />
Epidemiologist/Senior Researcher, Kaiser Permanente<br />
Division <strong>of</strong> Research, Oakland, CA, Catherine Schaefer,<br />
Epidemiologist/Senior Researcher, Kaiser Permanente<br />
Division <strong>of</strong> Research, Oakland, CA, Erica Gunderson,<br />
Epidemiologist/Senior Researcher, Kaiser Permanente<br />
Division <strong>of</strong> Research, Oakland, CA, Joe Selby, Director,<br />
Kaiser Permanente Division <strong>of</strong> Research, Oakland, CA, Lisa<br />
Croen, Epidemiologist/Senior Researcher, Kaiser<br />
Permanente Division <strong>of</strong> Research, Oakland, CA, Neil Risch,<br />
Adjunct Investigator, Kaiser Permanente Division <strong>of</strong><br />
Research, Oakland, CA<br />
Autoimmune disorders, collectively, include more than<br />
60 chronic and <strong>of</strong>ten disabling conditions that result from<br />
underlying defects in the immune system. As a group they<br />
affect approximately 15–20 million people in the United<br />
States, resulting in significant morbidity and mortality.<br />
Large, well-characterized, clinical populations in an environment<br />
that supports a broad research program are needed<br />
to advance our understanding <strong>of</strong> disease etiology. The<br />
Kaiser Permanente Medical Care Program (Northern California<br />
Region) or KPNC membership includes 3.3 million<br />
people, representing about 30% <strong>of</strong> the population <strong>of</strong><br />
northern California. The membership is sociodemographically<br />
and culturally diverse, while highly representative <strong>of</strong><br />
the general population. Comprehensive electronic medical<br />
records, including outpatient, pharmacy, laboratory, imaging<br />
and hospitalization data maintained since 1995 were<br />
used to construct a large registry <strong>of</strong> over 200,000<br />
individuals with diagnoses <strong>of</strong> one or more autoimmune<br />
conditions. Conditions for the study were derived from<br />
over 50 ICD-9 codes, broadly categorized into the following<br />
groups: endocrine, gastrointestinal, hematologic, kidney,<br />
liver, skin, neurologic/muscular, vascular, and connective<br />
tissue/joints. Planned additions to this registry include<br />
biospecimens and survey data on environmental and<br />
behavioral risk factors. This resource will be used to<br />
conduct the large epidemiologic investigations needed to<br />
advance our understanding <strong>of</strong> autoimmunity, including<br />
studies <strong>of</strong> genetic, social, and environmental contributions<br />
to risk, progression, and response to treatment. Results<br />
from our current investigations <strong>of</strong> autoimmune disease<br />
incidence/prevalence rates and patterns <strong>of</strong> co-morbidity<br />
will be presented, including results from our focused<br />
research efforts in multiple sclerosis and inflammatory<br />
bowel disease.<br />
doi:10.1016/j.clim.2007.03.539<br />
Sa.153 Functional Characterization <strong>of</strong> the<br />
Autoimmune-associated LYP-W620 Variant<br />
Lei Zhao, PhD Student, University <strong>of</strong> Southern California,<br />
Los Angeles, CA, Yingge Liu, Postdoctoral Fellow, University<br />
<strong>of</strong> Southern California, Los Angeles, CA, Edoardo Fiorillo,<br />
Postdoctoral Fellow, University <strong>of</strong> Southern California, Los<br />
Angeles, CA, Stephanie Stanford, PhD Student, University <strong>of</strong><br />
Southern California, Los Angeles, CA, Valeria Orru,<br />
Postdoctoral Fellow, University <strong>of</strong> Southern California, Los<br />
Angeles, CA, Nunzio Bottini, Assistant Pr<strong>of</strong>essor, University<br />
<strong>of</strong> Southern California, Los Angeles, CA<br />
A missense single-nucleotide polymorphism, C1858T in<br />
the PTPN22 gene is associated with multiple human<br />
autoimmune diseases, including type 1 diabetes, rheumatoid<br />
arthritis, systemic lupus erythematosus, Graves disease,<br />
juvenile idiopathic arthritis, generalized vitiligo, and<br />
others. Genetic studies have shown that the PTPN22<br />
C1858T polymorphism is primarily associated with autoimmunity<br />
in different populations. The PTPN22 gene<br />
encodes the lymphoid tyrosine phosphatase LYP, which is<br />
expressed only in white blood cells and acts as a<br />
gatekeeper <strong>of</strong> T lymphocyte activation. The molecular<br />
mechanism by which LYP tempers T lymphocyte activation<br />
involves the formation <strong>of</strong> a complex between LYP and the<br />
negative regulatory kinase Csk. When compared to the<br />
common LYP-R620 variant, the autoimmune-predisposing<br />
LYP-W620 variant shows reduced binding to Csk, and more<br />
recently we found that LYP-W620 is a gain-<strong>of</strong>-function form<br />
<strong>of</strong> the phosphatase. In order to analyze the molecular<br />
mechanism <strong>of</strong> the gain-<strong>of</strong>-function phenotype <strong>of</strong> LYP-W620,<br />
we isolated recombinant Csk-free LYP-R620 and W620 using<br />
an insect cell expression system. When we reconstituted in<br />
vitro the complex between LYP and Csk, we found that<br />
recombinant LYP-R620 binds Csk more efficiently than LYP-<br />
W620. We also analyzed the effect <strong>of</strong> Csk on the enzymatic<br />
activity <strong>of</strong> LYP. Our data obtained on in vitro reconstituted<br />
LYP–Csk complexes suggest that reduced binding to Csk<br />
cannot fully explain the gain-<strong>of</strong>-function phenotype <strong>of</strong> the<br />
LYP-W620 variant.<br />
doi:10.1016/j.clim.2007.03.540<br />
S127<br />
Sa.154 Spontaneous Myocarditis in Transgenic Mice<br />
Needs Appropriate MHC and Non-MHC Genes<br />
Veena Taneja, Assistant Pr<strong>of</strong>essor, Department <strong>of</strong><br />
<strong>Immunology</strong>, Mayo Clinic, Rochester, MN, Marshall Behrens,<br />
Senior Technician, Department <strong>of</strong> <strong>Immunology</strong>, Mayo Clinic,<br />
Rochester, MN, Leslie Cooper, Consultant, Mayo Clinic,<br />
Department <strong>of</strong> Cardiovascular Disease, Rochester, MN,<br />
Chella David, Pr<strong>of</strong>essor, Department <strong>of</strong> <strong>Immunology</strong>, Mayo<br />
Clinic, Rochester, MN<br />
Viral infection has been associated with the development<br />
<strong>of</strong> myocarditis in humans. Myocarditis is characterized by<br />
mononuclear infiltrate with attendant myocyte necrosis.<br />
Animal models <strong>of</strong> induced myocarditis have been generated<br />
by using coxsackie virus and immunization with cardiac<br />
myosin. We have recently generated a spontaneous model <strong>of</strong><br />
myocarditis. HLA-DQ8 and DR3 were expressed as transgenes<br />
in NOD mice lacking endogenous class II molecules, Abo.DQ8.<br />
NOD and Abo.DR3.NOD. Also mice expressing both transgenes<br />
in C57\B10 mice lacking endogenous class II molecules were<br />
generated. All mice were bred to congenic backgrounds. Only<br />
mice expressing HLA-DQ8 in NOD background developed<br />
spontaneous myocarditis. Abo.DR3.NOD, Abo.DQ8.B10 and<br />
Abo.DR3.B10 and transgene negative littermates did develop<br />
any gross or microscopic cardiac pathology. The autoimmunity<br />
was associated with the presence <strong>of</strong> spontaneous<br />
autoreactive T cells and antibodies to cardiac myosin. Only<br />
DQ8.NOD mice mounted a strong spontaneous in vitro T cell<br />
response and in vivo DTH response to cardiac myosin. These
S128 Abstracts<br />
data suggest that DQ8.NOD mice have lost tolerance to the<br />
self-cardiac myosin leading to spontaneous myocarditis and<br />
dilated cardiomyopathy. HLA-DQ8 is required for predisposition<br />
to the spontaneous autoreactivity while NOD background<br />
influences onset and progression <strong>of</strong> disease. This model <strong>of</strong><br />
myocarditis occurs predominantly in female mice and may<br />
provide insight into the pathogenesis <strong>of</strong> heart disease in<br />
women.<br />
doi:10.1016/j.clim.2007.03.554<br />
Sa.155 Human Lung Tumor Cells Modifies Rates <strong>of</strong><br />
CD4+CD25+ T Regulatory, CD19+CD23+ B Regulatory<br />
Cells, CD3+CD95+ Cells, NK Cells and B Lymphocytes<br />
Bayram Kiran, Tip Fakultesi, Temel Bilimler Binasi, Istanbul,<br />
Turkey, Akif Turna, Yedikule Hospital for Chest Diseases and<br />
Thoracic Surgery, Kadikoy, Istanbul, Turkey, Alper Yener,<br />
Istanbul Tip Fakultesi, Istanbul, Turkey, Atilla Gürses,<br />
Yedikule Gogus Hastaliklari Hastanesi, Istanbul, Turkey,<br />
Andac Salman, Istanbul Tip Fakultesi, Istanbul, Turkey,<br />
Selim Badur, Istanbul Tip Fakultesi, Temel Bilimler Binasi,<br />
Istanbul, Turkey<br />
The role <strong>of</strong> T regulatory cells and NK cells in human lung<br />
cancer has not yet been clarified. Our aim was to evaluate<br />
how the microenvironment <strong>of</strong> a tumor mass induced<br />
phenotypical changes in lymphocyte subsets. Our subjects<br />
were 10 patients with resectable non-small cell lung cancer.<br />
We evaluated 47 different phenotypically different lymphocyte<br />
subsets in blood samples taken from the pulmonary<br />
artery, pulmonary vein <strong>of</strong> a tumor bearing pulmonary lobe<br />
and peripheral blood during pulmonary resectional surgery.<br />
We showed that, tumor cells boosted CD25high+CD4high+T<br />
lymphocytes (Treg cells), B lymphocytes, NK and CD19+CD23<br />
+ cells (Breg cells) and CD3+CD95+ (FasL+T lymphocytes)<br />
(p=0.015, p=0.001, P=0.02, p=0.04, p=0.03 respectively).<br />
However, Mac-1 cells and HLADR+T lymphocytes were found<br />
to be decreased by tumor mass(p=0.04 and p=0.04), whereas<br />
only Breg , NK cell and B lymphocyte subsets were found to<br />
be expansed. In addition, the patients showed expansions <strong>of</strong><br />
CD45R0+ activated T lymphocytes and CD18+CD11b+(Mac-1)<br />
cell subsets without significant alteration by tumor microenvironment.<br />
In conclusion, subsets <strong>of</strong> Treg, Breg cells, FasL+<br />
T lymphocytes, B lymphocytes were modified by lung cancer<br />
microenvironment itself. This study also showed that,<br />
peripheral blood might not be good source for investigating<br />
the anti-tumor immunity since only Breg, NK cell and Treg<br />
cell subsets were found expansed peripherally. T lymphocytes<br />
and Mac-1 cells may be modified by systemic antitumor<br />
response rather than by local tumoral microenvironment.<br />
This study provides important insight into how tumor<br />
infiltrating lymphocytes and peripheral immune systems<br />
may be different in terms <strong>of</strong> lymphocyte subset expansion<br />
dynamics.<br />
doi:10.1016/j.clim.2007.03.552
<strong>Clinical</strong> <strong>Immunology</strong> (2007) 123, S129–S188<br />
ABSTRACTS<br />
<strong>Oral</strong> <strong>Presentations</strong>: Sunday, June 10<br />
#3201- From Genes to Treatment<br />
Sunday, June 10<br />
10:45 am−11:05 am<br />
Immune Reconstitution in X-Linked Hyper-IgM<br />
Syndrome With Recombinant CD40 Ligand<br />
Ashish Jain, Tenure- Track Investigator, Laboratory<br />
<strong>of</strong> Host Defenses, NIAID, NIH, Bethesda, MD, David<br />
Nelson, Senior Investigator, NCI, Bethesda, MD, Jospeh<br />
Kovacs, Senior Investigator, <strong>Clinical</strong> Center, Bethesda,<br />
MD, Shuying Liu, Study Coordinator, NIAID, Bethesda, MD,<br />
Thomas Fleisher, Senior Investigator, <strong>Clinical</strong> Center,<br />
Bethesda, MD, William Fanslow, Group Leader, Amgen<br />
Seattle, Seattle, WA, Hans Ochs, Pr<strong>of</strong>essor, Department<br />
<strong>of</strong> Pediatrics, Seattle, WA, Warren Strober, Senior<br />
Investigator, Laboratory <strong>of</strong> Host Defenses, NIAID, NIH,<br />
Bethesda, MD<br />
Purpose: X-linked hyper IgM syndrome (XHIM) is a<br />
combined immune deficiency disorder caused by mutations<br />
in the gene encoding CD40 ligand. The purpose <strong>of</strong><br />
this study was to investigate the safety and efficacy<br />
recombinant human CD40 ligand (rCD40L) for patients<br />
with XHIM. Methods: The study was designed as a phase I/<br />
II, open-label, single-dose study <strong>of</strong> rCD40L administered<br />
subcutaneously three times per week for 24 weeks in<br />
three children with XHIM. Adverse effects and efficacy <strong>of</strong><br />
rCD40L were evaluated during 6- month trial period.<br />
Results: The results <strong>of</strong> more than 230 subcutaneous<br />
injections showed that rCD40L was generally well tolerated.<br />
With rCD40L treatment, patients developed for the<br />
first time delayed type hypersensitivity reactions on skin<br />
testing that disappeared during the drug free follow up<br />
period. Analysis <strong>of</strong> patients’ T cells demonstrated first<br />
time capacity to synthesize IFN-g and TNF-a when<br />
stimulated with anti-CD3, or SEB, or SEA in vitro. Studies<br />
<strong>of</strong> cytokine production by intracellular staining demonstrate<br />
that rCD40L was able to prime both the CD4 and<br />
CD8 T cell populations with in vivo administration.<br />
Improvements <strong>of</strong> lymph node size and architecture were<br />
also noted; patients showed the development <strong>of</strong> primary<br />
follicles and follicular dendritic cells, as well as an<br />
doi:10.1016/j.clim.2007.03.006<br />
available at www.sciencedirect.com<br />
www.elsevier.com/locate/yclim<br />
expansion <strong>of</strong> B and T cell populations. Conclusions: This<br />
phase I/II first study <strong>of</strong> recombinant human CD40 ligand<br />
showed that rCD40L is capable <strong>of</strong> restoring immune<br />
functions in patients with XHIM. Further study will be<br />
needed to assess the overall potential <strong>of</strong> this therapy.<br />
doi:10.1016/j.clim.2007.03.007<br />
#3202- Antigen Specific Immune Modulation<br />
Sunday, June 10<br />
10:45 am−11:05 am<br />
Neur<strong>of</strong>ascin-specific Autoantibodies Mediate Axonal<br />
Injury in Inflammatory Demyelinating Diseases <strong>of</strong><br />
the Central Nervous System<br />
Christopher Linington, Pr<strong>of</strong>essor <strong>of</strong> Immunobiology,<br />
University <strong>of</strong> Aberdeen, Aberdeen, UK, Emily Mathey,<br />
Research Associate, University <strong>of</strong> Aberdeen, Aberdeen,<br />
UK, Tobias Derfuss, Max Planck Institute for<br />
Neurobiology, Martinsried, Germany, Matthew Rasband,<br />
Assistant Pr<strong>of</strong>essor, University <strong>of</strong> Connecticut Health<br />
Center, Farmington, CT, Maria Storch, Pr<strong>of</strong>essor, Medical<br />
University Graz, General Neurology, Graz, Germany,<br />
Reinhard Hohlfeld, Pr<strong>of</strong>essor, LMU University Hospital,<br />
<strong>Clinical</strong> Neuroimmunology, Munich, Germany, Edgar<br />
Meinl, Pr<strong>of</strong>essor, Max Planck Institute for Neurobiology,<br />
Martinsried, Germany<br />
We report that multiple sclerosis is associated with<br />
elevated autoantibody responses to neur<strong>of</strong>ascin, as<br />
compared to other inflammatory neurological diseases.<br />
The two is<strong>of</strong>orms <strong>of</strong> neur<strong>of</strong>ascin play essential roles in<br />
maintaining the molecular organization <strong>of</strong> myelinated<br />
fibers: NF186 is a neuronal protein located at the node<br />
<strong>of</strong> Ranvier and axonal initial segment where it interacts<br />
with voltage gated sodium channels, whilst NF155 is an<br />
oligodendroglial product sequestered at junctional complexes<br />
formed where paranodal loops <strong>of</strong> the myelin<br />
sheath contact the axonal surface. Analysis <strong>of</strong> neur<strong>of</strong>ascin-specific<br />
autoantibodies immunopurified from
S130 Abstracts<br />
seropositive donors demonstrates that they recognise the<br />
extracellular domain <strong>of</strong> both is<strong>of</strong>orms <strong>of</strong> the native<br />
protein indicating that this response may participate in<br />
disease pathogenesis. We investigated this hypothesis in<br />
experimental autoimmune encephalomyelitis using a pan<br />
NF155/186 mouse mAb to mimic the human autoantibody<br />
response. Passive transfer (i.p.) <strong>of</strong> mAb induced a rapid<br />
exacerbation <strong>of</strong> disease that was associated with a<br />
corresponding increase in acute axonal injury. Intriguing<br />
this occurred in the absence <strong>of</strong> any concomitant increase<br />
in the local inflammatory response or demyelination.<br />
Immun<strong>of</strong>luorescence microscopy revealed that binding <strong>of</strong><br />
the transferred neur<strong>of</strong>ascin-specific antibody was<br />
restricted nodes <strong>of</strong> Ranvier where it co-localised with<br />
voltage gated sodium channels. These results identify<br />
NF186 as a primary target for neur<strong>of</strong>ascin-specific autoantibodies<br />
and indicate antibody-dependent effector<br />
mechanisms are involved in the pathogenesis <strong>of</strong> axonal<br />
injury and loss in multiple sclerosis.<br />
doi:10.1016/j.clim.2007.03.008<br />
#3203- Innate <strong>Immunology</strong><br />
Sunday, June 10<br />
10:45 am−11:05 am<br />
IL-23 is Required for Induction <strong>of</strong> an Innate Immune<br />
Response to Citrobacter Rodentium<br />
Darrell O’Quinn, Postdoctoral Fellow, University <strong>of</strong><br />
Alabama, Department <strong>of</strong> Pathology, Birmingham, AL,<br />
Trenton Schoeb, Pr<strong>of</strong>essor, University <strong>of</strong> Alabama,<br />
Department <strong>of</strong> Genetics, Birmingham, AL, Casey Weaver,<br />
Pr<strong>of</strong>essor, University <strong>of</strong> Alabama, Department <strong>of</strong><br />
Pathology, Birmingham, AL<br />
We have previously shown that infection <strong>of</strong> IL-23deficient<br />
mice with the enteric murine pathogen Citrobacter<br />
rodentium results in complete mortality within 7-10<br />
days following inoculation despite the presence <strong>of</strong> CD4+ IL-<br />
17-competent cells. Using a mutant strain <strong>of</strong> bioluminescent<br />
C. rodentium, we here demonstrate that colonization<br />
<strong>of</strong> the cecum and mid- and distal colon <strong>of</strong> IL-23-deficient<br />
mice occurs earlier and in greater numbers than in IFNγ-,<br />
IFNγR-, IL-12-deficient, or wild-type controls. Histopathological<br />
examination <strong>of</strong> the lesions in IL-23-deficient mice<br />
confirms the presence <strong>of</strong> massive numbers <strong>of</strong> bacteria<br />
attached to intestinal epithelial cells <strong>of</strong> the mid- and distal<br />
colon, and multi-focal areas <strong>of</strong> ulcerative necrosis indicate<br />
a limited or absent inflammatory response. Analysis <strong>of</strong> C.<br />
rodentium colonization in IL-17R-/- mice reveal no significant<br />
differences from wild-type controls suggesting that<br />
IL-23, not IL-17, may be critical for the maintenance <strong>of</strong><br />
innate defenses in the distal colon.<br />
doi:10.1016/j.clim.2007.03.009<br />
OR.70 Intestinal Epithelial Cell Derived Thymic<br />
Stromal Lymphopoeitin Controls Dendritic<br />
Cell Function and T Regulatory Cell<br />
Homeostasis<br />
Iliyan D. Iliev, PhD Student, European Institute <strong>of</strong><br />
Oncology, Milan, Italy, Erika Mileti, Laboratory Assistant,<br />
European Institute <strong>of</strong> Oncology, Milan, Italy, Maria<br />
Rescig, Group Leader, European Institute <strong>of</strong> Oncology,<br />
Milan, Italy<br />
The mucosal immune system has developed mechanisms<br />
to tolerate beneficial micr<strong>of</strong>lora, but in the same time to<br />
initiate immunity against invading pathogens. How these<br />
mechanisms function is still not completely understood. In<br />
our current study we investigated the possible role <strong>of</strong><br />
Epithelial Cells (EC) as a “tolerance keepers” in the gut<br />
driving the development <strong>of</strong> non-inflammatory Dendritic<br />
Cells (DC) able to promote CD4+CD25+Foxp3+ T regulatory<br />
cells (Treg) development. We developed a co-culture<br />
system, in which EC (Caco2 or primary intestinal EC)<br />
supernatants were used to condition monocyte-derived DC.<br />
We found that in both, human and mice, EC-conditioned DC<br />
were producing less inflammatory cytokines in response to<br />
bacterial stimuli and favored the development <strong>of</strong> functionally<br />
active Treg. These tolerogenic DC were involved in<br />
driving Treg also in vivo in mice. Thymic stromal<br />
lymphopoeitin (TSLP) produced by EC, together with<br />
additional factors, was involved in this process since DC<br />
conditioned with supernatants from Caco2 cell line<br />
“silenced” for TSLP, were unable to expand Treg. Finally,<br />
mice immunized with Salmonella loaded EC-conditioned DC<br />
were less resistant to lethal Salmonella dose than control<br />
mice, confirming their tolerogenic potential in vivo.<br />
Therefore, intestinal EC released TSLP, can influence gut<br />
DC function and can control the homeostasis <strong>of</strong> peripheral<br />
Treg. We propose that failure in this mechanism can<br />
participate in the pathology <strong>of</strong> Inflammatory Bowel<br />
Disease.<br />
doi:10.1016/j.clim.2007.03.010<br />
Regulatory T Cells II<br />
Sunday, June 10<br />
2:45 pm−4:45 pm<br />
OR.71 Critical Role <strong>of</strong> IFNg in Allograft Tolerance<br />
Mediated by Foxp3+CD4+CD25+ Regulatory T Cells<br />
and the Production <strong>of</strong> Indoleamine 2, 3-dioxygenase<br />
by Graft Endothelial Cells<br />
Pamela Thebault, PhD, INSERM U643, Nantes, France,<br />
Michele Heslan, IE, INSERM U643, Nantes, France, Thomas<br />
Condamine, PhD, INSERM U643, Nantes, France, Abdelhadi<br />
Saoudi, CR1, INSERM U563, Toulouse, France, Marcello Hill,<br />
Postdoctoral Fellow, INSERM U643, Nantes, France, Regis<br />
Josien, CR1, INSERM U643, Nantes, France, Ignacio Anegon,<br />
DR2, INSERM U643, Nantes, France, Maria-Cristina Cuturi,<br />
DR2, INSERM U643, Nantes, France, Elise Chiffoleau, CR 2,<br />
INSERM U643, Nantes, France
Abstracts<br />
Functionally specialized subsets <strong>of</strong> regulatory CD4+T cells<br />
have been shown to play an important role in the regulation <strong>of</strong><br />
both T cell-mediated and innate immune responses. In particular,<br />
the CD4+CD25+ subpopulation <strong>of</strong> T cells has been<br />
shown to be crucial in self-tolerance and also to prevent<br />
allograft rejection. As CD25 is not uniquely expressed by<br />
regulatory T cells, but can also be expressed by activated<br />
effector cells, the identification <strong>of</strong> Foxp3 has provided an<br />
opportunity to specifically identify regulatory T cells and to<br />
track them in vivo so as to better define the mechanisms<br />
involved in their regulation <strong>of</strong> immune responses. We previously<br />
demonstrated, in a fully MHC mismatched rat cardiac<br />
allograft combination, that a short-term treatment with a<br />
deoxyspergualine analogue, LF15-0195, induces long-term<br />
allograft tolerance with a specific expansion <strong>of</strong> regulatory<br />
CD4+CD25+T cells that accumulate within the graft. In this<br />
study, we show that following transfer to a secondary irradiated<br />
recipient, regulatory CD4+CD25+T cells are able to<br />
rapidly home to the graft, induce accumulation <strong>of</strong> Foxp3+ and<br />
Tr1/IL10+ regulatory CD4+Tcells and induce graft endothelial<br />
cell expression <strong>of</strong> Indoleamine 2, 3-dioxygenase (IDO),<br />
inducible Nitric Oxide synthase (iNOS) and Heme-Oxygenase-1<br />
(HO-1). Moreover, in vivo transfer <strong>of</strong> tolerance can be<br />
abrogated by blocking IFNg or IDO and in vitro, anti-IFNg<br />
reduces the survival/function <strong>of</strong> alloantigen-induced Foxp3+<br />
CD4+ regulatory T cells. Together, our results demonstrate<br />
interrelated mechanisms between regulatory CD4+Tcells and<br />
the graft endothelial cells in this local immune privilege and a<br />
key role for IFNg and IDO in this process.<br />
doi:10.1016/j.clim.2007.03.011<br />
OR.72 Amino-Terminal Region <strong>of</strong> Foxp3 Performs<br />
Multiple Functions in Regulating Transcription<br />
Jared E. Lopes, Graduate Students, University <strong>of</strong><br />
Washington, Seattle, WA, Jianguang Du, Postdoctoral Fellow,<br />
Benaroya Research Institute, Seattle, WA, Xiaocui Sun,<br />
Research Technician, Benaroya Research Institute, Seattle,<br />
WA, Mark Chong, Postdoctoral Fellow, Molecular<br />
Pathogenesis Program, Skirball Institute <strong>of</strong> Biomolecular<br />
Medicine, New York, NY, Liang Zhou, Fellow, Molecular<br />
Pathogenesis Program, Skirball Institute <strong>of</strong> Biomolecular<br />
Medicine, New York, NY, Dan R. Littman, Director <strong>of</strong><br />
Molecular Pathogenesis Program, Skirball Institute <strong>of</strong><br />
Biomolecular Medicine and Howard Hughes Medical<br />
Institute, New York, NY, Steven F. Ziegler, <strong>Immunology</strong><br />
Program Director, Benaroya Research Institute, <strong>Immunology</strong><br />
Program, Seattle, WA<br />
Foxp3 has been shown to be both necessary and sufficient<br />
for the development and function <strong>of</strong> naturally-arising CD4+<br />
CD25+ regulatory Tcells (Tregs) in mice. Mutation <strong>of</strong> Foxp3 in<br />
scurfy mice and Foxp3 in humans with IPEX results in fatal,<br />
early onset autoimmune disease and demonstrates the<br />
critical role <strong>of</strong> Foxp3 in maintaining immune homeostasis.<br />
The Foxp3 protein encodes several functional domains<br />
including a C2H2 zinc finger, a leucine zipper, and a wingedhelix/forkhead<br />
(FKH) domain. We have previously shown that<br />
Foxp3 functions as a transcriptional repressor and inhibits<br />
activation-induced IL-2 gene transcription. We recently<br />
identified a novel functional domain within the aminoterminal<br />
half <strong>of</strong> Foxp3, which is required for Foxp3-mediated<br />
repression <strong>of</strong> transcription from both a constitutively active<br />
and an NFAT-inducible promoter. In order to determine the<br />
mechanism by which Foxp3 regulates transcription, we<br />
performed a yeast-2-hybrid analysis searching for proteins<br />
that interact with the amino-terminal region <strong>of</strong> Foxp3. One<br />
target was RORgammat (RORgt), which is required for the<br />
differentiation <strong>of</strong> naïve CD4+ T cells into pro-inflammatory,<br />
IL-17-producing T cells (Th17). Interestingly, CD4+ T cells<br />
express Foxp3 and RORgt in response to TCR stimulation in the<br />
presence <strong>of</strong> TGF-β and differentiate into either Tregs or<br />
Th17 cells, depending on the presence <strong>of</strong> IL-6. We show that<br />
Foxp3 directly inhibits RORgt-mediated transcription. Future<br />
studies will address the mechanism by which Foxp3 inhibits<br />
RORgt function and how this process is regulated by IL-6.<br />
doi:10.1016/j.clim.2007.03.012<br />
S131<br />
OR.73 Phenotypic and Functional Characteristics <strong>of</strong><br />
Different Regulatory T Cell Subsets (Tregs) in<br />
Patients with Cancer<br />
Christoph Bergmann, Postdoctoral Fellow, University <strong>of</strong><br />
Pittsburgh Cancer Institute, Department <strong>of</strong> Pathology,<br />
Pittsburgh, PA, Laura Strauss, Postdoctoral Fellow,<br />
University <strong>of</strong> Pittsburgh Cancer Institute, Department <strong>of</strong><br />
Pathology, Pittsburgh, PA, Jonas Johnson, Pr<strong>of</strong>essor and<br />
Chair, University <strong>of</strong> Pittsburgh Cancer Institute, Department<br />
<strong>of</strong> Otolaryngology, Pittsburgh, PA, Theresa L. Whiteside,<br />
Director, University <strong>of</strong> Pittsburgh Cancer Institute,<br />
Department <strong>of</strong> Pathology, Pittsburgh, PA<br />
Immune suppression mediated by Tregs is a feature <strong>of</strong><br />
cancer. To compare phenotype and function <strong>of</strong> Tregs present<br />
in the tumor (TIL) and blood (PBMC) <strong>of</strong> patients with head and<br />
neck cancer (HNC), we obtained samples from 40 patients and<br />
15 normal controls (NC). The phenotype and frequency <strong>of</strong><br />
CD4 + CD25 high and CD4 + CD25 − T cell subsets were evaluated.<br />
These cells were separated by single-cell sorting, tested for<br />
suppressor function and also cultured +/− cytokines and/or<br />
rapamycin (1 nM). IL-10 or TGF- 2 1 mAbs were used to block<br />
suppression <strong>of</strong> responder cell (R) proliferation. TIL were<br />
enriched in CD4 + CD25 high (13%±3) cells (Foxp3 + GITR + IL-<br />
10 + TGF− 2 1 + ). In PBMC, Tregs (CD25 high Foxp3 + CD62L + CCR7 + GI-<br />
TR + IL-10 + TGF− 2 1 + ) represented 5%±3 among CD4 + cells. In<br />
contrast, PBMC in NC contained fewer (pb0.001) Tregs (2%±<br />
1.5). In PBMC <strong>of</strong> patients and NC, CD4 + CD25 - IL-10 + TGF- 2 1 +<br />
(Tr1) cells were detected. In TIL, Tr1 cells represented 23%±3<br />
<strong>of</strong> CD4 + cells. Tr1 cells expanded from PBMC and TIL were<br />
CD25 − IL-10 + IL2R 3+ TGF- 2 1 + IL-4 − with low levels <strong>of</strong> Foxp3 and<br />
CTLA-4 expression. TIL Tregs mediated stronger suppression<br />
(p b0.001) than PBMC Tregs. NC Tregs mediated weak<br />
suppression. Neutralizing mAbs to IL-10 or TGF- 2 1 abolished<br />
suppression <strong>of</strong> R proliferation by Tregs. Cell contact <strong>of</strong> Tregs,<br />
but not Tr1, with R enhanced suppression. Rapamycin<br />
promoted selective expansion <strong>of</strong> Tregs and Tr1 cells by<br />
eliminating non-Tregs in patients and NC. Characteristics <strong>of</strong><br />
rapamycin-expanded Tregs and Tregs in freshly-isolated<br />
lymphocytes were similar. Tregs mediating suppression in<br />
the tumor and circulation <strong>of</strong> HNC patients are heterogeneous
S132 Abstracts<br />
populations distinct from Tregs in NC. To mediate immune<br />
escape, tumor activates and/or induces unique Treg subsets.<br />
doi:10.1016/j.clim.2007.03.013<br />
OR.74 Hepatic Stellate Cells Expand<br />
CD4 + CD25 + Foxp3 + Tregs and Prevent Rejection <strong>of</strong><br />
Allogeneic Islet Transplants<br />
Shiguang Qian, Associate Pr<strong>of</strong>essor, Cleveland Clinic,<br />
Cleveland, OH, Guoping Jiang, Research Fellow,<br />
Department <strong>of</strong> <strong>Immunology</strong>, Cleveland Clinic, Cleveland,<br />
OH, Zhenyu Yin, Research Fellow, Department <strong>of</strong><br />
<strong>Immunology</strong>, Cleveland Clinic, Cleveland, OH, John J. Fung,<br />
Pr<strong>of</strong>essor, Department <strong>of</strong> General Surgery, Cleveland, OH,<br />
Liang-Mou Kuo, Research Fellow, Department <strong>of</strong><br />
<strong>Immunology</strong>, Cleveland Clinic, Cleveland, OH, Lina Lu,<br />
Associate Pr<strong>of</strong>essor, Department <strong>of</strong> <strong>Immunology</strong>, General<br />
Surgery, Cleveland Clinic, Cleveland, OH<br />
Liver tolerance was demonstrated by spontaneous acceptance<br />
<strong>of</strong> liver allografts in many species, but hepatocyte<br />
transplants were acutely rejected, suggesting an immunosuppressive<br />
effect <strong>of</strong> liver tissue cells. In this study, we<br />
demonstrated immunosuppressive activity <strong>of</strong> hepatic stellate<br />
cells (HSC) through expansion <strong>of</strong> CD4 + CD25 + Foxp3 +<br />
Tregs. HSC isolated from B6 mice (H2 b ) constitutively expressed<br />
low MHC class I and CD80. Expression <strong>of</strong> MHC class I, II<br />
and B7-H1 was markedly upregulated in IFN-γ activated-HSC<br />
(aHSC), which rendered them to stimulate allogeneic T cell<br />
proliferation in both CD4 + and CD8 + cells (CFSE). aHSC<br />
cultured with allogenetic CD4 + T cells at 1:10 ratio for 5 days<br />
markedly expanded CD4 + CD25+ + Foxp3 + cells (from 2.5% to<br />
10.5%), which were obviously from naïve CD4 + CD25 + population,<br />
since depletion <strong>of</strong> CD4 + CD25 + , only small number <strong>of</strong><br />
CD4 + CD25 + cells were expanded, and were all Foxp3 − .<br />
Addition <strong>of</strong> aHSC expanded CD4 + CD25 + cells into CD4 + cells<br />
either stimulated by anti-CD3 or allo-antigens, resulted in<br />
marked inhibition <strong>of</strong> T cell proliferation in a dose dependent<br />
manner. To explore clinical application, 300 BALB/c (H2 d )<br />
islets mixed with aHSC (3×10 5 ) transplanted under renal<br />
capsule <strong>of</strong> chemically diabetic B6 recipients, achieving<br />
markedly prolonged islet graft survival [median survival<br />
time (MST) from 13 days to N60 days (pb0.01)] with 55%<br />
being euglycemia for N60d. None <strong>of</strong> islet-only recipients<br />
remained euglycemia for N17d. Removal <strong>of</strong> islet-grafted<br />
kidney resulted in quick glycemia. Prolongation <strong>of</strong> islet<br />
allografts by co-transplanted HSC was associated with<br />
upregulated apoptotic activity (TUNEL) and less T cell<br />
infiltration (CD3), indicating potential application in improving<br />
islet transplantation.<br />
doi:10.1016/j.clim.2007.03.014<br />
OR.75 Polyclonal CD4+CD25+ Regulatory T Cells<br />
Induced With TGF-β Prevent a Stimulatory<br />
Graft-Versus-Host Disease With a Lupus-Like Syndrome<br />
Song Guo Zheng, Assistant Pr<strong>of</strong>essor <strong>of</strong> Research Medicine,<br />
University <strong>of</strong> Southern California Keck School <strong>of</strong> Medicine, Los<br />
Angeles, CA, Julie Wang, Research Lab Specialist, University <strong>of</strong><br />
Southern California Keck School <strong>of</strong> Medicine, Los Angeles, CA,<br />
Shuang Ye, Postdoctoraltoral Fellow, University <strong>of</strong> Southern<br />
California Keck School <strong>of</strong> Medicine, Los Angeles, CA, David A.<br />
Horwitz, Pr<strong>of</strong>essor, University <strong>of</strong> Southern California Keck<br />
School <strong>of</strong> Medicine, Los Angeles, CA<br />
We have previously reported that the adoptive transfer <strong>of</strong><br />
antigen-specific Tregs generated by allogeneic antigen stimulation<br />
with TGF-β prevents the appearance <strong>of</strong> lupus syndrome<br />
induced by injection <strong>of</strong> DBA/2 mouse spleen cells into (DBA/<br />
2×C57BL/6) F1 mice. Here we have considered whether<br />
polyclonal CD4+ Tregs induced ex vivo may have similar<br />
suppressive effects in this model. Naïve CD4+CD25- cells<br />
isolated from DBA/2 mice were stimulated with anti-CD3/<br />
CD28 coated beads+TGF-β without APC. TGF-β was able to<br />
directly induce the majority <strong>of</strong> activated CD25+ cells to express<br />
Foxp3, develop cell markers characteristic <strong>of</strong> Tregs, and acquire<br />
suppressive activity in vitro. The remaining CD25- cells did not<br />
express Foxp3 or develop suppressive activity. TGF-β -induced<br />
CD4+ Tregs not only suppressed the Tcell proliferative response<br />
to anti-CD3, but also inhibited the T cell response to allogeneic<br />
cells. Substituting soluble anti-CD3 and antigen-presenting cells<br />
(APCs) for anti-CD3/28 beads resulted in decreased Foxp3+<br />
expression, an effect that was reversed by the addition <strong>of</strong> anti-<br />
IL-6. These levels <strong>of</strong> IL-6 in cultures containing TGF-β did not<br />
result in the production <strong>of</strong> IL-17. Finally, (D2xB6)F1 mice<br />
injected with D2 cells and 5 million TGF-β -induced CD4+ CD25+<br />
Tregs did not develop increased polyclonal IgG, anti-dsDNA<br />
autoantibodies, or nephritis. Since SLE is a generalized<br />
autoimmune disease with many autoantibodies, and production<br />
<strong>of</strong> IL-2 and TGF-β is decreased in this disease, the<br />
generation <strong>of</strong> large numbers <strong>of</strong> autologous, polyclonal CD4+<br />
CD25+Foxp3+ Treg cells with these cytokines ex vivo may have<br />
considerable therapeutic potential.<br />
doi:10.1016/j.clim.2007.03.015<br />
OR.76 Low Dose Antigen Promotes Induction <strong>of</strong><br />
Both Foreign and Self Reactive Human<br />
CD25+Foxp3+ Regulatory T Cells from<br />
CD4+CD25-Foxp3- Peripheral T Cells<br />
S. Alice Long, Benaroya Research Institute, Seattle, WA,<br />
Mindi Walker, Scientist, Therakos, Exton, PA, Isabelle<br />
Mueller, Graduate Student, Benaroya Research Institute,<br />
Seattle, WA, Jane Buckner, Pr<strong>of</strong>essor, Benaroya Research<br />
Institute, Seattle, WA, Mary Rieck, RA II, Benaroya Research<br />
Institute, Seattle, WA<br />
Upon activation, a subpopulation <strong>of</strong> human CD4+CD25-<br />
Foxp3- T cells upregulate Foxp3 and become regulatory. These<br />
cells are referred to as induced TR (iTR) and have similar<br />
properties to TR isolated directly from peripheral blood. We<br />
focus here on properties <strong>of</strong> stimulation that influence generation<br />
<strong>of</strong> iTR and characteristics <strong>of</strong> the CD4+ CD25- T cells from<br />
which they are derived. We consistently observe that stimulation<br />
<strong>of</strong> CD4+CD25- T cells with foreign antigen results in two<br />
populations <strong>of</strong> antigen specific tetramer positive (Tmr+) Tcells;<br />
Tmr+Foxp3+ and Tmr− Foxp3− cells. The Foxp3+ population can<br />
arise from cells which express low or high levels <strong>of</strong> CD127, with<br />
the greatest number <strong>of</strong> which originate from CD127mid/hi
Abstracts<br />
populations. We demonstrate similar results with islet antigens<br />
in frequency, source and potency. Generation <strong>of</strong> iTR is enhanced<br />
in the presence <strong>of</strong> low dose antigen relative to high dose. Thus,<br />
low level TCR stimulation results in enhanced frequency <strong>of</strong> Tmr+<br />
CD25+Foxp3+ T cells. These results demonstrate that iTR are<br />
generated as a consequence <strong>of</strong> normal immune responses in<br />
settings where antigen is in limited quantities, which may, in<br />
part, explain the phenomenon <strong>of</strong> low dose tolerance. Studies<br />
with T1D samples demonstrate the induction <strong>of</strong> islet and<br />
foreign iTR with similar functional potency. However, dose <strong>of</strong><br />
antigen has no effect on the induction <strong>of</strong> iTR in T1D. Thus, T1D<br />
subjects have the ability to generate islet specific iTR in the<br />
periphery, but defects in the response to activation or the<br />
frequency <strong>of</strong> iTR, may contribute to disease progression.<br />
doi:10.1016/j.clim.2007.03.016<br />
OR.77 IL-10 Both Inhibits Treg Suppressive Activity<br />
and is a Survival Factor for Human Natural Tregs<br />
Clare Baecher-Allan, Instructor <strong>of</strong> Neurology, Harvard<br />
Medical School, Brigham and Women’s Hospital,<br />
Department <strong>of</strong> Neurology, Boston, MA, Charles Ashley,<br />
Research Technician, Harvard Medical School, Brigham and<br />
Women's Hospital, Department <strong>of</strong> Neurology, Boston, MA,<br />
David Hafler, Breakstone Chair <strong>of</strong> Neurology, Harvard<br />
Medical School, Brigham and Women’s Hospital,<br />
Department <strong>of</strong> Neurology, Boston, MA<br />
Human natural Tregs (CD4+CD25high) are comprised <strong>of</strong><br />
two functionally distinct populations that differentially<br />
express HLA Class II proteins directly ex vivo. The MHC<br />
class II+ Tregs, referred to as ‘DR+Tregs’, are a highly suppressive<br />
population that rapidly inhibit the activation <strong>of</strong> cocultured<br />
responder T cells. The DRnegTregs, on the other<br />
hand, exhibit a delayed effector function as they allow an<br />
initial period <strong>of</strong> responder T cell proliferation before they<br />
shut down the response. While the DR+Tregs suppress solely<br />
by cell contact and do not produce IL-10, IL-10 is <strong>of</strong>ten (but<br />
not always) produced by the responder T cells and by the<br />
DR-Tregs in these accessory-cell-free assays. Interestingly,<br />
in those experiments in which the responder T cells did<br />
produce IL-10, the DR+Treg co-cultures exhibit less suppression<br />
than the duplicate co-cultures that had been given<br />
neutralizing IL-10 antibody from the outset. Thus, DR+Treg<br />
rapid suppression proceeds unimpeded in the absence <strong>of</strong> IL-<br />
10, while the presence <strong>of</strong> IL-10 inhibits their function. In<br />
order to understand this effect <strong>of</strong> IL-10, the two different<br />
cellular components <strong>of</strong> the co-culture were assayed to<br />
determine if the presence <strong>of</strong> IL-10 modulated the expression<br />
<strong>of</strong> regulatory (Foxp3, IL-10, TGFb), or apoptosis (BAX,<br />
BCL-2, and BCL-XL) related products. The results indicate<br />
that blocking IL-10 in DR+Treg co-cultures increases the<br />
frequency <strong>of</strong> AnnexinV+ responder T cells, reflecting the<br />
increase in suppression. Within the DR+Treg, however,<br />
blocking IL-10 triggered a reproducible decrease in BCL-XL<br />
and an increase in BAX, indicating increased Treg<br />
sensitivity to apoptosis that may affect long-term maintenance<br />
<strong>of</strong> suppression.<br />
doi:10.1016/j.clim.2007.03.017<br />
OR.78 High Density SNP Analysis <strong>of</strong> the MHC Region<br />
Reveals Multiple Loci for Type 1A Diabetes<br />
Theresa A. Aly, Graduate Student, University <strong>of</strong> Colorado<br />
Health Sciences Center, Barbara Davis Center, Aurora, CO,<br />
Erin E. Baschal, Graduate Student, University <strong>of</strong> Colorado<br />
Health Sciences Center, Barbara Davis Center, Aurora, CO,<br />
Mohamed M. Jahromi, Fellow, University <strong>of</strong> Colorado Health<br />
Sciences Center, Barbara Davis Center for Childhood<br />
Diabetes, Aurora, CO, Adam Kretowski, Physician,<br />
University <strong>of</strong> Colorado Health Sciences Center, Barbara<br />
Davis Center for Childhood Diabetes, Aurora, CO, Sunanda<br />
R. Babu, Research Associate, University <strong>of</strong> Colorado Health<br />
Sciences Center, Barbara Davis Center for Childhood<br />
Diabetes, Aurora, CO, Marian J. Rewers, Physician,<br />
University <strong>of</strong> Colorado Health Sciences Center, Barbara<br />
Davis Center for Childhood Diabetes, Aurora, CO, George S.<br />
Eisenbarth, Physician-Scientist, University <strong>of</strong> Colorado<br />
Health Sciences Center, Barbara Davis Center, Aurora, CO<br />
Haplotype sharing studies combined with HLA genotyping<br />
demonstrate that loci linked to the MHC region in addition to<br />
HLA-DR/DQ loci contribute major risk for type 1A diabetes. The<br />
hunt for these additional major loci is underway. In our survey<br />
study, we analyzed 656 single nucleotide polymorphisms (SNPs)<br />
in the MHC region and compared their allele frequencies in case<br />
versus control chromosomes from 45 families participating in<br />
the Diabetes Autoimmunity Study <strong>of</strong> the Young (DAISY). We<br />
included significant SNPs (pb0.01) from our survey study and<br />
additional SNPs extending 6 Mb further telomeric <strong>of</strong> the MHC in<br />
a follow-up study <strong>of</strong> 342 additional DAISY, HBDI, and Polish<br />
families. The most significant SNPs with distinct diabetesassociated<br />
peaks centromeric or within the MHC were at the<br />
ITPR3 (inositol-triphosphate receptor 3), HLA-DPB1, -DQB1, -<br />
DRB1, -DRA, and MICA loci. In addition to confirming these<br />
associations, our follow-up studies revealed a strong association<br />
<strong>of</strong> a 4 SNP DRA haplotype in 422/461 case chromosomes (92%)<br />
versus 437/579 control chromosomes (75%, p=1.8×10–11). The<br />
DRA haplotype remained significant when specific high-risk<br />
(DR3, DR4) and protective (DR2) chromosomes were excluded<br />
(p=0.05). The most significant regions telomeric <strong>of</strong> the MHC<br />
were at the Mas1L/UBD (Mas-related G protein coupled<br />
receptor/diubiquitin, p=0.00001) and PRSS16 loci (thymusspecific<br />
serine protease, p=0.00005). An HLA-DRA haplotype is<br />
strongly associated with type 1A diabetes; however, the<br />
biological basis for this association may be due to linked<br />
genes (e.g. HLA-DRB1-DQB1). Two novel regions 3100 kb and<br />
5400 kb telomeric <strong>of</strong> the HLA-DR and -DQ loci are also<br />
associated with diabetes.<br />
doi:10.1016/j.clim.2007.03.018<br />
Genetics<br />
Sunday, June 10<br />
2:45 pm−4:45 pm<br />
OR.79 Uniquely Designed Candidate Gene<br />
Identification Platform Discovers Novels Genes in<br />
Childhood Onset SLE<br />
Don Armstrong, FOCIS Center, University <strong>of</strong> Southern<br />
California School <strong>of</strong> Medicine, Los Angeles, CA, Andreas<br />
S133
S134 Abstracts<br />
Reiff, Children’s Hospital <strong>of</strong> Los Angeles and University <strong>of</strong><br />
Southern California School <strong>of</strong> Medicine, Los Angeles, CA,<br />
Chaim Jacob, FOCIS Center, University <strong>of</strong> Southern<br />
California School <strong>of</strong> Medicine, Los Angeles, CA, Raphael<br />
Zidovetzki, FOCIS Center, University <strong>of</strong> Southern California<br />
School <strong>of</strong> Medicine, Los Angeles, CA<br />
We present a novel methodology to maximize the ability <strong>of</strong><br />
microarray based single nucleotide polymorphism (SNP) typing<br />
platforms to discover genes which are associated with complex<br />
polygenic diseases when complete genomic coverage is infeasible.<br />
This methodology was used to identify genetic variants<br />
which are associated with childhood onset SLE. A platform <strong>of</strong><br />
9412 SNPs corresponding to 1204 genes was selected using<br />
bioinformatics and expert knowledge and validated. Molecular<br />
inversion probes and high throughput techniques were used to<br />
type SNP alleles. A cohort <strong>of</strong> 753 subjects, corresponding to 251<br />
full trios (both parents and affected SLE child) were genotyped.<br />
Utilizing the Transmission Disequilibrium Test (TDT) and appropriate<br />
multiple comparison corrections, a panel <strong>of</strong> several novel<br />
childhood onset SLE genes were determined to be significant.<br />
Among these, the N673S polymorphism in SELP (P-Selectin) and<br />
the C203S polymorphism in IRAK1 (Interleukin 1 receptorassociated<br />
kinase 1) were found with a high degree <strong>of</strong><br />
significance. These results suggest new directions in which to<br />
expand the understanding <strong>of</strong> the pathogenesis <strong>of</strong> SLE. The<br />
results <strong>of</strong> this study demonstrate that the novel methodology<br />
provides a powerful approach which is generally applicable to<br />
the identification <strong>of</strong> the genetic foundations <strong>of</strong> complex<br />
diseases.<br />
doi:10.1016/j.clim.2007.03.019<br />
OR.80 The Molecular Basis by Which Polymorphism<br />
in the OX40L Gene Predisposes to SLE<br />
Harinder Manku, Research Fellow, Imperial College London,<br />
Molecular Genetics and Rheumatology, London, England,<br />
Deborah Cunninghame Graham, Research Fellow, Imperial<br />
College London, Molecular Genetics and Rheumatology,<br />
London, England, Robert R. Graham, Research Fellow, Broad<br />
Institute <strong>of</strong> Harvard and MIT, Cambridge, MA, Timothy W.<br />
Behrens, Genentech Inc., San Francisco, CA, Timothy W.<br />
Behrens, Adjunct Pr<strong>of</strong>essor, University <strong>of</strong> Minnesota,<br />
Department <strong>of</strong> Medicine, Minneapolis, MN, David<br />
Althshuler, Associate Pr<strong>of</strong>essor, Broad Institute <strong>of</strong> Harvard<br />
and MIT, Cambridge, MA, Timothy J. Vyse, Reader and<br />
Honorary Consultant, Imperial College London, Department<br />
<strong>of</strong> Molecular Genetics and Rheumatology, London, England<br />
SLE is a complex polygenic disease where immune dysregulation<br />
results in chronic activation <strong>of</strong> B lymphocytes and<br />
autoantibody production against a variety <strong>of</strong> nuclear antigens.<br />
OX40L, a type II membrane glycoprotein found on the surface <strong>of</strong><br />
activated B lymphocytes, interacts with OX40 on T lymphocytes<br />
to amplify T-dependent B cell proliferation and differentiation.<br />
A family-based association analysis using a set <strong>of</strong> 45 SNP<br />
markers across OX40L was conducted in 472 UK and 429 US SLE<br />
parent-<strong>of</strong>fspring trios. We identified an associated promoter<br />
haplotype block (32 kb) that was preferentially transmitted to<br />
affected <strong>of</strong>fspring (P=0.003 UK, P=0.0005 US, P=6.7×10 −6<br />
combined). A tagging SNP (rs2205960) from this haplotype<br />
showed significant association with SLE in an independent set <strong>of</strong><br />
310UKcasesand642controls(P= 1.3×10 −5 OR=1.55, 95%<br />
CI=1.27–1.89). In order to investigate the molecular basis <strong>of</strong> the<br />
genetic association, we studied OX40L expression in individuals<br />
selected on the basis <strong>of</strong> allelic composition at the OX40L<br />
promoter. The promoter polymorphisms associated with SLE<br />
correlated with increased expression levels <strong>of</strong> OX40L. This was<br />
shown by three-colour flow cytometry <strong>of</strong> 16 EBV-transformed<br />
cell lines: those homozygous for the over-transmitted haplotype<br />
exhibited increased expression relative to the under-transmitted<br />
homozygote. We further show that the disease susceptibility<br />
alleles in the OX40L promoter were associated with an<br />
eight-fold increase in RNA expression (P=0.008). The mechanism<br />
by which increased OX40L expression increases the<br />
susceptibility to SLE remains to be established, but is the<br />
subject <strong>of</strong> ongoing investigation.<br />
doi:10.1016/j.clim.2007.03.020<br />
OR.81 Genetic Association <strong>of</strong> IL-21 Polymorphisms<br />
with Systemic Lupus Erythematosus<br />
Amr H. Sawalha, Assistant Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Medicine, Oklahoma University Health Sciences Center, VA<br />
Medical Center, Oklahoma Medical Research Foundation,<br />
Oklahoma City, OK, Kenneth Kaufman, Senior Research<br />
Scientist, Oklahoma Medical Research Foundation,<br />
Oklahoma City, OK, Jennifer Kelly, Senior Research<br />
Assistant, Oklahoma Medical Research Foundation,<br />
Oklahoma City, OK, Teresa Aberle, PA-C, Oklahoma Medical<br />
Research Foundation, Oklahoma City, OK, Adam Adler,<br />
Research Associate, Oklahoma Medical Research<br />
Foundation, Oklahoma City, OK, Jeff Kilpatrick, Computer<br />
Programmer/Data Analyst, Oklahoma Medical Research<br />
Foundation, Oklahoma City, OK, Edward Wakeland,<br />
Pr<strong>of</strong>essor, UT Southwestern, Center <strong>of</strong> <strong>Immunology</strong>, Dallas,<br />
TX, Quan-Zhen Li, Assistant Pr<strong>of</strong>essor, Center for<br />
<strong>Immunology</strong>, University <strong>of</strong> Texas Southwestern Medical<br />
Center, Dallas, TX, Amy Wandstrat, Instructor, UT<br />
Southwestern, Rheumatology, Dallas, TX, Judith James,<br />
Pr<strong>of</strong>essor/Member, Oklahoma Medical Research<br />
Foundation/ Arthritis and <strong>Immunology</strong>, Oklahoma City, OK,<br />
Joan Merrill, Member, Oklahoma Medical Research<br />
Foundation, <strong>Clinical</strong> Pharmacology, Oklahoma City, OK,<br />
Peter Lipsky, Chief, NIAMS, Bethesda, MD, John B. Harley,<br />
Pr<strong>of</strong>essor and Department Head, Oklahoma University<br />
Health Sciences Center/Oklahoma Medical Research<br />
Foundation, Oklahoma City, OK<br />
Purpose: The etiology <strong>of</strong> systemic lupus erythematosus (SLE)<br />
is incompletely understood. Both genetic and environmental<br />
factors are implicated in the pathogenesis <strong>of</strong> the disease.<br />
Herein, we describe genetic association between SLE and<br />
polymorphisms in the IL-21 gene. The reported effects <strong>of</strong> IL-21<br />
on B cell differentiation into plasma cells, as well as its effect<br />
on dendritic cell maturation and T cell response, make IL-21 an<br />
attractive SLE candidate gene. Methods: A genetic association<br />
study was performed by genotyping three single nucleotide<br />
polymorphisms (SNPs) in the IL-21 gene in a total <strong>of</strong> 2636<br />
samples (1318 independent SLE cases and 1318 unrelated<br />
controls matched for age, sex and race). Genotyping was<br />
performed on the Illumina BeadStation 500GX system at the
Abstracts<br />
University <strong>of</strong> Texas Southwestern Microarray Core Facility<br />
(Dallas, TX). Population-based case-control association designs<br />
were employed. Results: Two SNPs located within intronal<br />
regions <strong>of</strong> IL-21 are associated with SLE (rs907715: chi2=11.55,<br />
p=0.00068; rs2221903: chi2=5.49, p=0.019). Furthermore,<br />
genotypes homozygous for the risk alleles were more frequent<br />
than genotypes homozygous for the non-risk alleles in<br />
European-American patients as compared to controls<br />
(rs907715 (GG versus AA): Odds ratio=1.66, p=0.0049;<br />
rs2221903 (GG versus AA): Odds ratio=1.60, p=0.025). Conclusion:<br />
We report an association between polymorphisms in the<br />
IL-21 gene and SLE. The functional effects <strong>of</strong> IL-21 polymorphisms,<br />
when revealed, might improve our understanding <strong>of</strong> SLE<br />
and provide new therapeutic targets.<br />
doi:10.1016/j.clim.2007.03.021<br />
OR.82 Both Shared and Strain-specific Genetic Loci<br />
Control Susceptibility to Virus-induced Autoimmune<br />
Diabetes in Two Rat Models<br />
Elizabeth Blankenhorn, Pr<strong>of</strong>essor, Drexel University College<br />
<strong>of</strong> Medicine, Philadelphia, PA, Laura Cort, Research<br />
Assistant, Department <strong>of</strong> Microbiology and <strong>Immunology</strong>,<br />
Philadelphia, PA, Daniel Cheeran, Research Assistant,<br />
Department <strong>of</strong> Microbiology and <strong>Immunology</strong>, Philadelphia,<br />
PA, Rebecca Tirabasi, Research Scientist, BRM, Inc.,<br />
Worcester, MA, Elaine Norowski, Research Assistant,<br />
Diabetes Division, Worcester, MA, Dennis Guberski,<br />
President, BRM, Inc., Worcester, MA, John Mordes,<br />
Pr<strong>of</strong>essor, Diabetes Division, Worcester, MA<br />
Viral infection may be important in the pathogenesis <strong>of</strong><br />
human type 1 diabetes (T1DM). MHC congenic (RT1u/u/a)<br />
LEW.1WR1 rats develop spontaneous T1DM at a low<br />
frequency (∼2%) and are also susceptible to the induction<br />
<strong>of</strong> diabetes in response to infection with a parvovirus, Kilham<br />
rat virus (KRV). An injection <strong>of</strong> 1×10 7 PFU KRV induces<br />
diabetes in ∼40% <strong>of</strong> animals, and co-injection <strong>of</strong> a low dose<br />
<strong>of</strong> poly I:C with the KRV increases disease penetrance to<br />
100%. KRV does not infect beta cells. In their susceptibility to<br />
KRV-T1DM, LEW.1WR1 rats resemble BBDR (RT1u/u/u) rats,<br />
but WF strain rats (also RT1u/u/u) are resistant to the<br />
induction <strong>of</strong> diabetes by this virus. In crosses <strong>of</strong> (BBDR×WF)<br />
rats, we have mapped two quantitative trait loci (QTL) that<br />
determine susceptibility to KRV-poly I:C induced T1DM,<br />
Iddm14 and Iddm20. We have now mapped the QTL required<br />
for diabetes in (LEW.1WR1 ×WF) rats. We show that Iddm14<br />
allele from the diabetes-susceptible LEW.1WR1 strain is<br />
required for both insulitis and diabetes (46/47 rats with<br />
diabetes had Iddm14d/−). However, Iddm20 is not a determinant<br />
<strong>of</strong> either insulitis or diabetes in this strain combination.<br />
Instead, markers on chromosome 20 near RT1 show<br />
significance. These data demonstrate that virus-specific<br />
diabetogenic responses differ between LEW.1WR1 and BBDR<br />
rats, even though both are susceptible to KRV. A genome scan<br />
for LEW.1WR1-specific QTL will be reported. These data shed<br />
light on light on the factors responsible for the variable<br />
penetrance <strong>of</strong> T1D in high risk human populations.<br />
doi:10.1016/j.clim.2007.03.022<br />
OR.83 An Association Between the Autoimmune<br />
Susceptibility Locus CTLA-4 and Alterations in<br />
Proximal TCR Signaling in Healthy Human<br />
Volunteers<br />
Lisa Maier, Neurology Fellow, Center for Neurologic<br />
Diseases, Boston, MA, David Anderson, Instructor, Center<br />
for Neurologic Diseases, Boston, MA, Philip de Jager,<br />
Assistant Pr<strong>of</strong>essor, Center for Neurologic Diseases, Boston,<br />
MA, David Hafler, Pr<strong>of</strong>essor, Center for Neurologic Diseases,<br />
Boston, MA, Linda Wicker, Pr<strong>of</strong>essor, JDRF/WT Diabetes and<br />
Inflammation Laboratory, Cambridge<br />
With the rapid advancement in high-throughput genotyping<br />
technologies, an increasing number <strong>of</strong> genetic variants have<br />
been associated with susceptibility to human autoimmune<br />
disease. However, little is known about the functional effects <strong>of</strong><br />
susceptibility variants on human immune cells. The single<br />
nucleotide polymorphism (SNP) called CT60 (rs3087243),<br />
located in the 3′ untranslated region <strong>of</strong> CTLA-4, has been<br />
associated with many autoimmune diseases, including type 1<br />
diabetes. The susceptible genotype at this SNP has previously<br />
been shown to result in a 2-fold lower production <strong>of</strong> the soluble<br />
CTLA-4mRNAis<strong>of</strong>orminnaïveCD4+Tcells.Wewerethus<br />
interested in whether this or other CT60-associated functional<br />
consequences would affect TCR signaling, given that CTLA-4 is a<br />
negative T cell regulator. We have applied a flow-cytometric<br />
technique that measures phosphoproteins to the study <strong>of</strong><br />
proximal TCR signaling in naïve and memory CD4+ T cells using<br />
fresh whole blood stimulated with anti-CD3 monoclonal antibody,<br />
and have measured CD3z (pY142), LAT (pY171), LCK<br />
(pY505), ZAP70 (pY292), ZAP70 (pY319/Syk (pY352)) and SLP-76<br />
(pY128) in healthy human volunteers. By categorizing results<br />
obtained from TCR stimulation into the three genotype classes;<br />
AA, AG and GG, we observed no differences in TCR responses<br />
with CD4+CD45RA+ T cells. However, we observed that relative<br />
to the CD45RA+ subset, CD4+CD45RA- T cells from individuals<br />
with the susceptibility allele are less responsive in all TCR<br />
signaling molecules examined. These data contribute to our<br />
understanding <strong>of</strong> the link between the susceptibility genotype<br />
at CTLA4 and the mechanisms involved that may result in<br />
autoimmunity.<br />
doi:10.1016/j.clim.2007.03.023<br />
S135<br />
OR.84 Variants <strong>of</strong> CARD15/NOD2 Associated with<br />
Crohn’s Disease and Blau Syndrome Differ from Wild<br />
Type NOD2 in Regulating Cytokine Secretion<br />
Induced by TLR2 and TLR4 Agonists in a Transduced<br />
Macrophage Cell Line<br />
Michael Davey, Pr<strong>of</strong>essor <strong>of</strong> Medicine, Portland VA Medical<br />
Center and OHSU, Portland, OR, Zili Zhang, Assistant<br />
Pr<strong>of</strong>essor, OHSU, Pediatrics, Portland, OR, Tammy Martin,<br />
Associate Pr<strong>of</strong>essor, OHSU and CEI, Portland, OR, Holly<br />
Rosenzweig, Postdoctoral Fellow, OHSU, Casey Eye<br />
Institute, Portland, OR, Steven Planck, Pr<strong>of</strong>essor, OHSU and<br />
CEI, Portland, OR, James Rosenbaum, Pr<strong>of</strong>essor, OHSU and<br />
CEI, Portland, OR<br />
NOD2 mutations are associated with Crohn's disease and<br />
Blau syndrome. NOD2 senses muramyl dipeptide (MDP) and
S136 Abstracts<br />
activates NF-kB. Studies in NOD2 deficient mice found that<br />
NOD2 suppresses TLR2 cytokine responses. Using cells overexpressing<br />
NOD2, this study further explored the regulatory<br />
role <strong>of</strong> NOD2. Murine mononuclear cells (J774 line) were<br />
transduced with constructs containing murine wild type (WT)<br />
NOD2, the murine equivalent <strong>of</strong> common Blau (BL) (R314Q)<br />
and Crohn's mutations (frame shift [fs] at L980) or an empty<br />
retroviral vector control (VC). Cells were stimulated with<br />
TLR2 (PAM3CSK4) or TLR4 (LPS) agonists with or without MDP,<br />
and secretion <strong>of</strong> IL-6, IL-10, IL-12p40 and TNF-α was<br />
measured. Over-expression <strong>of</strong> WT, BL and fs variants <strong>of</strong><br />
NOD2 suppressed IL-10 secretion and enhanced TNF-α<br />
secretion in response to TLR2 and TLR4 agonists. Both<br />
agonists suppressed IL-6 and IL-12p40 secretion in WT cells<br />
but enhanced secretion from BL and fs cells. MDP and TLR<br />
ligands given simultaneously to VC cells enhanced secretion<br />
<strong>of</strong> all cytokines studied. Synergy did not occur or was<br />
suppressed in WT cells, while it was enhanced in BL and fs<br />
cells. These data confirm the regulatory role <strong>of</strong> NOD2 on TLR2<br />
signaling and show TLR4 pathways can also be regulated. The<br />
differences observed between suppression and enhancement<br />
<strong>of</strong> cytokine secretion with WTcompared to BL and fs variants<br />
<strong>of</strong> NOD2 may be in vitro correlates <strong>of</strong> processes linking these<br />
mutations with disease. Elucidating mechanisms responsible<br />
for these different patterns may enhance our understanding<br />
<strong>of</strong> Crohn's disease and Blau syndrome.<br />
doi:10.1016/j.clim.2007.03.024<br />
OR.85 A Genome-wide Assessment <strong>of</strong> the Genetic<br />
Basis <strong>of</strong> Type 1 Diabetes<br />
Vincent Plagnol, Mr, JDRF/WT DIL, University <strong>of</strong> Cambridge,<br />
Cambridge, England, David Clayton, Pr<strong>of</strong>essor, JDRF/WT<br />
Diabetes and Inflammation Laboratory, Cambridge,<br />
England, David Dunger, Department <strong>of</strong> Pediatric, University<br />
<strong>of</strong> Cambridge, Cambridge, England, Kate Downes, JDRF/WT<br />
Diabetes and Inflammation Laboratory, Cambridge,<br />
England, Hin-Tak Leung, JDRF/WT Diabetes and<br />
Inflammation Laboratory, Cambridge, England, Deborah<br />
Smyth, JDRF/WT Diabetes and Inflammation Laboratory,<br />
Cambridge, England, Helen Stevens, JDRF/WT Diabetes and<br />
Inflammation Laboratory, Cambridge, England, Neil Walker,<br />
Mr, JDRF/WT Diabetes and Inflammation Laboratory,<br />
Cambridge, England, WTCCC, Wellcome Trust Case Control<br />
Consortium, http://www.wtccc.org.uk/, Wellcome Trust,<br />
Cambridge, England, John Todd, Pr<strong>of</strong>essor, JDRF/WT<br />
Diabetes and Inflammation Laboratory, Cambridge, England<br />
The strong familial clustering <strong>of</strong> autoimmune type 1 diabetes<br />
(T1D) in families remains only partially explained. The six<br />
confirmed genes or chromosome regions with convincing<br />
statistical support in large, and multiple, populations, namely<br />
the major histocompatibility complex (MHC), the insulin gene<br />
(INS), CTLA-4, PTPN22, IL2RA/CD25, and IFIH1/MDA5 can<br />
explain only about 50% <strong>of</strong> familial aggregation. Nonetheless,<br />
their identification has provided many insights into the biology<br />
and pathways <strong>of</strong> T1D. Recent expansions in sample collections,<br />
including the Juvenile Diabetes Research Foundation/Wellcome<br />
Trust Diabetes and Inflammation Laboratory (JDRF/WT DIL)<br />
British T1D case sample (n= 8000), and advances in affordable,<br />
genome-wide, high throughput single nucleotide polymorphism<br />
(SNP) genotyping technologies (Affymetrix), has allowed an<br />
association analysis <strong>of</strong> 500,000 SNPs across the genome in 2000<br />
T1D cases and 3000 controls as part <strong>of</strong> the Wellcome Trust Case-<br />
Control Consortium (WTCCC). Attesting to the quality <strong>of</strong> the<br />
SNP map, positive results were obtained for all six susceptibility<br />
loci reported previously. However, follow-up genotyping studies<br />
<strong>of</strong> 14 other chromosome regions showing Pb10 −5 in the WTCCC<br />
scan in over 6000 cases and 6000 controls, and in approximately<br />
2000 families, supported in a convincing way (Pb10 −15 and odds<br />
ratios approximately 1.2) four regions associated with T1D in<br />
both the family and case-control samples, on chromosomes<br />
16p13, 18p11, 12q13 and 12q24. These results illustrate the<br />
power <strong>of</strong> genome-wide association approach to characterise<br />
the genetic basis <strong>of</strong> T1D.<br />
doi:10.1016/j.clim.2007.03.025<br />
Cytokine/Chemocine Regulators <strong>of</strong><br />
Inflammation & Allergy<br />
Sunday, June 10<br />
2:45 pm−4:45 pm<br />
OR.86 Human B Cell Cytokine Regulation and<br />
Multiple Sclerosis (MS)<br />
Christine Ghorayeb, McGill University/<strong>Immunology</strong>,<br />
Montreal, QC, Masaaki Nii Hokkaido, University Hospital/<br />
Neurology, Sapporo, Japan, Claudia Calder, McGill<br />
University/Neuroimmunology, Montreal, QC, Canada, Amit<br />
Bar-Or, McGill University/Neurology and Neurosurgery,<br />
Montreal, QC, Canada<br />
B cells are increasingly recognized for roles in both normal<br />
immune responses and in autoimmune diseases including MS.<br />
We recently reported that normal human B cells can produce<br />
either pro-inflammatory (TNFá; Lymphotoxin/LT) or regulatory<br />
(IL-10) effector cytokines, depending on their sequence<br />
<strong>of</strong> activation. We further identified that MS patient B cells<br />
exhibit deficient expression <strong>of</strong> IL-10, implicating a B cell<br />
abnormality in failed immune regulation. Here, we asked (i)<br />
whether and how the local cytokine milieu may modulate the<br />
pr<strong>of</strong>ile <strong>of</strong> the normal effector B cell cytokine network, and<br />
(ii) whether such modulation is different for MS B cells.<br />
Specifically, we added either IFNγ (Th1 milieu) or IL-4 (Th2<br />
milieu) during our established ex vivo paradigm <strong>of</strong> B cell<br />
activation. The presence <strong>of</strong> IFNγ induced normal B cells to<br />
secrete increased levels <strong>of</strong> LT (429±44; versus 259±28 pg/<br />
ml; n=15; p=0.0002) and TNFá (274±44 versus 144±30 pg/<br />
ml; p=0.0003). IL-4 also induced increased levels <strong>of</strong> LT (419±<br />
47 pg/ml, p=0.0001) and <strong>of</strong> TNFá (366±51, pb0.0001),<br />
while significantly suppressing secretion <strong>of</strong> IL-10 (37±11<br />
versus 145 ±26 pg/ml; pb0.0001). We further discovered<br />
that in the ‘Th1 milieu’, MS B cells produced significantly<br />
higher levels <strong>of</strong> LT (average increase 28%; n=22, p=0.0069)<br />
and TNFá (26%, p=0.0049), compared to normal B cells. We<br />
demonstrate that a novel regulatory network <strong>of</strong> effector<br />
cytokines in normal B cells is significantly modulated<br />
depending on the local cytokine milieu. Furthermore, in a<br />
'Th1 milieu', MS B cells secrete abnormally high levels <strong>of</strong> TNFá
Abstracts<br />
and LT, which would contribute to the local inflammatory<br />
response.<br />
doi:10.1016/j.clim.2007.03.026<br />
OR.87 Immune Mediated Protection in Multiple<br />
Sclerosis: A Role for Neurokines in Oligodendrocyte<br />
Survival and Macrophage Modulation<br />
Niels Hellings, PhD, Hasselt University, Diepenbeek,<br />
Belgium, Niels Hellings, PhD, Hasselt University, BIOMED,<br />
Diepenbeek, Belgium, Jerome JJA Hendriks, PhD, Hasselt<br />
University, BIOMED, Diepenbeek, Belgium, S<strong>of</strong>ie Carmans,<br />
MsC, Hasselt University, BIOMED, Diepenbeek, Belgium,<br />
Leen Slaets, MsC, Hasselt University, BIOMED, Diepenbeek,<br />
Belgium, Debora Dumont, PhD, Hasselt University, BIOMED,<br />
Diepenbeek, Belgium, Joris Vanderlocht, PhD, Hasselt<br />
University, BIOMED, Diepenbeek, Belgium, Piet Stinissen,<br />
PhD, Hasselt University, BIOMED, Diepenbeek, Belgium<br />
In multiple sclerosis (MS), immune mediated destruction<br />
<strong>of</strong> the myelin sheath, oligodendrocytes and axons leads to<br />
irreversible neurological deficits. Recent data show that<br />
immune responses in the central nervous system (CNS) may<br />
also confer protective effects. We recently demonstrated<br />
that autoreactive T cells and macrophages produce<br />
leukemia inhibitory factor (LIF), a member <strong>of</strong> the IL-6<br />
family <strong>of</strong> neurokines. CD4+ T cells from relapsing remitting<br />
MS-patients show a reduced LIF production. Still, LIF<br />
immunoreactivity is detected among T cells and perivascular<br />
macrophages in MS lesions. In rat oligodendrocyte<br />
cultures, LIF protects against TNF-α and IFN-γ induced<br />
apoptosis, but not against other cell death mediators<br />
(oxidative stress, staurosporin). LIF receptor signalling in<br />
oligodendrocytes does not induce the expression <strong>of</strong><br />
suppressors <strong>of</strong> cytokine signalling (SOCS) or the antiapoptotic<br />
molecules Bcl-2/Bcl-XL. Using quantitative proteomics,<br />
we demonstrate that LIF leads to upregulation <strong>of</strong><br />
a panel <strong>of</strong> proteins that have pro-survival functions. Future<br />
experiments are needed to study their role in LIF mediated<br />
oligodendrocyte protection. Since macrophages, but not T<br />
cells, express functional LIF-receptor complexes we studied<br />
whether LIF also exerts immunomodulatory functions.<br />
LIF significantly upregulates myelin phagocytosis and<br />
reduces the production <strong>of</strong> reactive oxygen species and<br />
TNF-α in vitro. In summary, neurokines may act on various<br />
levels during CNS inflammation: they may modulate<br />
immune responses and protect CNS resident cells under<br />
attack. Further clarification in the actions <strong>of</strong> different<br />
neurokine members during neuroinflammation may lead to<br />
new therapeutic strategies for MS.<br />
doi:10.1016/j.clim.2007.03.027<br />
OR.88 ALCAM is a Novel Adhesion Molecule <strong>of</strong> the<br />
Inflammed Endothelium Involved in Leukocyte<br />
Trafficking to the Central Nervous System<br />
Romain Cayrol, PhD Student, Université de Montreal,<br />
Montreal, QC, Canada, Aurore Dodelet-Devillers, M.Sc.<br />
Student, Université de Montreal, Montreal, QC, Canada, Igal<br />
Ifergan, PhD Student, Université de Montreal, Montreal, QC,<br />
Canada, Arsalan Haqqani, PhD Student, National Research<br />
Council, Institute for Biological Sciences, Ottawa, ON,<br />
Canada, Hania Kebir, PhD Student, Université de Montreal,<br />
Montreal, QC, Canada, Danica Stanimirovic, Pr<strong>of</strong>essor,<br />
Institute for Biological Sciences; National Research Council <strong>of</strong><br />
Canada, Ottawa, ON, Canada, Alexandre Prat, Clinician and<br />
Pr<strong>of</strong>essor, Université de Montreal, Montreal, QC, Canada<br />
Leukocyte trafficking from the blood to local inflammatory<br />
sites is essential for the initiation and maintenance <strong>of</strong> tissue<br />
specific immune responses. In autoimmune diseases such as<br />
multiple sclerosis (MS), leukocyte transmigration from the<br />
blood to the target organ is dependent on intercellular cell<br />
adhesion molecule 1 (ICAM-1) and vascular cell adhesion<br />
molecule 1 (VCAM-1) expressed by the endothelial cells (ECs).<br />
In this study we describe CD166/activated leukocyte cell<br />
adhesion molecule (ALCAM) as a novel adhesion molecule <strong>of</strong><br />
the human blood–brain barrier (BBB). We demonstrate that<br />
inflammatory cytokines up-regulate the expression <strong>of</strong> ALCAM<br />
on the surface <strong>of</strong> BBB-ECs in vitro and that endothelial ALCAM<br />
co-localizes with leukocyte expressed-CD6 in the transmigratory<br />
cup during migration. We further show that ALCAM<br />
blocking antibody restricts the transmigration <strong>of</strong> CD4, CD14<br />
and CD19 immune cells across human BBB-ECs and that ALCAM<br />
expression is increased on ECs within active MS lesions, as<br />
compared to normal appearing white matter and to non-MS<br />
brains. These results suggest an important role for ALCAM in<br />
the recruitment <strong>of</strong> leukocytes to the CNS and identify ALCAM<br />
as a potential target to modulate CNS inflammatory reactions.<br />
doi:10.1016/j.clim.2007.03.028<br />
S137<br />
OR.89 Circulation <strong>of</strong> Membrane-bound TNFa by Way<br />
<strong>of</strong> Plasma-derived Exosomes<br />
Nicole Bianco, Postdoctoral Scholar, University <strong>of</strong><br />
Pittsburgh, Molecular Genetics and Biochemistry,<br />
Pittsburgh, PA, Seon-Hee Kim, Director <strong>of</strong> Research, Seoul<br />
National University, Seoul, South Korea, Paul Robbins,<br />
Pr<strong>of</strong>essor, University <strong>of</strong> Pittsburgh, Molecular Genetics and<br />
Biochemistry, Pittsburgh, PA, Teresa Hennon, Research<br />
Fellow, University <strong>of</strong> Pittsburgh, Molecular Genetics and<br />
Biochemistry, Pittsburgh, PA<br />
Exosomes are membrane nanovesicles (50–100 nm diameter)<br />
generated by reverse budding <strong>of</strong> the limiting membrane<br />
<strong>of</strong> multivesicular late endosomes. Exosomes originating<br />
from immune cells, such as B cells, T cells, DC, and mast<br />
cells, are thought to play a role in communicating immunoregulatory<br />
signals to other cells in either an immuno-stimulating<br />
or suppressive manner. Since we have demonstrated<br />
the presence <strong>of</strong> FasL, a TNF family member, in exosomes, we<br />
examined whether exosomes also can carry TNFa. To establish<br />
this, we first generated exosomes from a murine tumor<br />
cell line overexpressing murine TNFa and demonstrated the<br />
presence <strong>of</strong> membrane-bound TNFa (mbTNFa) in exosomes by<br />
FACS and Western blot analysis. The mbTNFa in exosomes was<br />
also functional in the L929 cytotoxicity assay. To determine<br />
whether exosomes containing mbTNFa might circulate<br />
systemically during active disease, we collected serum from<br />
WT or IL10−/− mice with inflammatory bowel disease. There
S138 Abstracts<br />
was significantly more mbTNFa in IL10−/− serum exosomes.<br />
Mice with collagen-induced arthritis also contained elevated<br />
levels <strong>of</strong> mbTNFa in their serum exosomes. This research<br />
implies that mbTNFa, in addition to functioning in a local<br />
manner by cell-to-cell contact, can also travel either locally<br />
or systemically to regulate inflammation at distant sites. This<br />
research also suggests that the bioactive mbTNFa in the<br />
vesicles may play a role in the pathology <strong>of</strong> various TNFdependent<br />
autoimmune diseases and that anti-TNF therapies<br />
might target mbTNFa carried on microvesicles. Furthermore,<br />
the level <strong>of</strong> TNFa in the serum exosomes might be useful as a<br />
diagnostic marker <strong>of</strong> certain autoimmune diseases.<br />
doi:10.1016/j.clim.2007.03.029<br />
OR.90 Ccr2 Deficiency Impairs Microglial<br />
Accumulation and Accelerates Progression <strong>of</strong><br />
Alzheimer’s Disease<br />
Joseph El Khoury, Assistant Pr<strong>of</strong>essor <strong>of</strong> Medicine, Harvard<br />
Medical School, Charlestown, MA, Michelle T<strong>of</strong>t, Laboratory<br />
Technician, Massachusetts General Hospital, Charlestown,<br />
MA, Suzanne Hickman, Scientist, Massachusetts General<br />
Hospital, Charlestown, MA, Changiz Geula, Pr<strong>of</strong>essor,<br />
Harvard Medical School, Charlestown, MA, Terry Means,<br />
Instructor in Medicine, Massachusetts General Hospital,<br />
Charlestown, MA, Andrew Luster, Pr<strong>of</strong>essor <strong>of</strong> Medicine,<br />
Harvard Medical School, Charelstown, MA<br />
Microglia are the principal immune cell <strong>of</strong> the brain. In<br />
Alzheimer's disease (AD), microglia accumulate in senile<br />
plaques and may in part be recruited from peripheral blood.<br />
The role <strong>of</strong> microglia in AD pathogenesis has not been<br />
resolved. Microglia may play a neuroprotective role by phagocytosing<br />
beta amyloid, a pathogenic protein deposited in<br />
AD brains. But their activation and subsequent secretion <strong>of</strong><br />
neurotoxins may also lead to neurodegeneration. Chemokine<br />
receptor 2 (Ccr2) is a chemokine receptor expressed on<br />
microglia that mediates accumulation <strong>of</strong> leukocytes at sites<br />
<strong>of</strong> inflammation. Transgenic mice expressing a mutated form<br />
<strong>of</strong> the human amyloid precursor protein (APP) are a robust<br />
model <strong>of</strong> AD used to study the role <strong>of</strong> microglia in AD. To<br />
determine the role <strong>of</strong> Ccr2 in microglial accumulation in AD,<br />
we generated transgenic APP mice deficient in Ccr2 and<br />
analyzed these mice for AD-like pathology. We found that<br />
Ccr2 deficiency accelerates early disease progression and<br />
markedly reduces microglial recruitment and accumulation<br />
to baseline in this transgenic mouse model <strong>of</strong> AD. APP mice<br />
deficient in Ccr2 died prematurely in correlation with Ccr2<br />
gene dosage, and accumulated beta amyloid in their brains<br />
much earlier than APP mice with normal Ccr2 levels,<br />
indicating that absence <strong>of</strong> early microglial recruitment<br />
leads to decreased beta amyloid clearance. Furthermore,<br />
Ccr2-deficient mice microinjected with beta amyloid into<br />
their brains failed to recruit microglia to the sites <strong>of</strong><br />
injection. Thus, Ccr2-dependent microglial accumulation<br />
plays a protective role in the early stages <strong>of</strong> AD by promoting<br />
clearance <strong>of</strong> beta amyloid.<br />
doi:10.1016/j.clim.2007.03.030<br />
OR.91 Chemokine-like Receptor-1 (CMKLR1) Regulates<br />
Experimental Autoimmune Encephalomyelitis<br />
Kareem L. Graham, Postdoctoral Scholar, Department <strong>of</strong><br />
Pathology, Stanford, CA, Luis A. Zuniga, Graduate Student,<br />
Department <strong>of</strong> Pathology, Stanford, CA, Brian A. Zabel,<br />
Postdoctoral Fellow, Department <strong>of</strong> Pathology, Stanford,<br />
CA, Eugene C. Butcher, Pr<strong>of</strong>essor <strong>of</strong> Pathology, Department<br />
<strong>of</strong> Pathology, Stanford, CA, Raymond A. Sobel, Pr<strong>of</strong>essor <strong>of</strong><br />
Pathology, Department <strong>of</strong> Pathology, Stanford, CA<br />
The pathology <strong>of</strong> multiple sclerosis (MS) involves leukocyte<br />
extravasation <strong>of</strong> the blood–brain barrier and associated myelin<br />
damage, which leads to impaired nerve function and paralysis.<br />
Chemokines, adhesion molecules, and their receptors have been<br />
implicated in recruitment <strong>of</strong> inflammatory cells to the central<br />
nervous system (CNS) during MS. However, the mechanisms that<br />
regulate migration <strong>of</strong> various leukocyte subsets to the CNS<br />
remain poorly understood. Chemokine-like receptor-1 (CMKLR1,<br />
also known as ChemR23 or Dez) is a recently de-orphaned<br />
chemoattractant receptor that has emerging roles in macrophage<br />
migration during inflammatory processes. In this study,<br />
we examined the role <strong>of</strong> CMKLR1 in experimental autoimmune<br />
encephalomyelitis (EAE), a mouse model <strong>of</strong> human MS. We found<br />
that mice deficient in CMKLR1 are resistant to progressive EAE.<br />
CMKLR1-deficient mice had reduced inflammatory infiltrates in<br />
the brain and spinal cord, with especially marked differences in<br />
the CNS parenchyma. While transcripts for CMKRL1 have been<br />
detected in a variety <strong>of</strong> tissues, experiments with bone marrow<br />
chimeric mice indicate that CMKLR1 expression on cells <strong>of</strong><br />
hematopoietic origin is required for maximal expression <strong>of</strong> EAE.<br />
Together, the data demonstrate that CMKLR1 regulates a model<br />
<strong>of</strong> autoimmune demyelinating disease and suggest that CMKLR1<br />
may be a potential target in blocking development <strong>of</strong><br />
progressive EAE/MS.<br />
doi:10.1016/j.clim.2007.03.031<br />
OR.92 Cell-type Specific Changes in Cytokine-STAT<br />
Signal Transduction Stage Systemic Lupus<br />
Erythematosus<br />
Matthew, Postdoctoral Fellow, Stanford University,<br />
Department <strong>of</strong> Microbiology and <strong>Immunology</strong>, Stanford, CA,<br />
Peter Krutzik, Research Associate, Stanford University,<br />
Department <strong>of</strong> Microbiology and <strong>Immunology</strong>, Stanford, CA,<br />
Garry Nolan, Pr<strong>of</strong>essor, Stanford University, Department <strong>of</strong><br />
Microbiology and <strong>Immunology</strong>, Stanford, CA<br />
The regulation <strong>of</strong> cytokine signal transduction is thought to<br />
be critical in shaping the character and duration <strong>of</strong> immune<br />
responses, but little is known <strong>of</strong> its role in autoimmune disease.<br />
We applied phospho-specific flow cytometry to generate a<br />
highly multiplexed view <strong>of</strong> how Systemic Lupus Erythematosus<br />
(SLE) disturbs cytokine signaling through the STAT family <strong>of</strong><br />
transcription factors. Using a panel <strong>of</strong> 10 cytokines previously<br />
suggested to have causal roles in the disease, we measured<br />
signaling responses at the single-cell level in multiple immune<br />
cell types from human SLE patients with a range <strong>of</strong> disease<br />
activity, as well as followed disease progression in the MRLlpr<br />
murine model. Unexpected changes in subset responses to IFNα,<br />
IFNγ, IL-4, IL-6, IL-15 and IL-21 were observed. Suppressor <strong>of</strong><br />
cytokine signaling 1 (SOCS1) levels were elevated in PBMC from
Abstracts<br />
human patients as well as in lymphocytes from the murine model<br />
where SOCS1 was implicated in pathway-specific inhibition <strong>of</strong><br />
responses to IL-6 in T cell subsets. Not only did aberrant signal<br />
transduction distinguish SLE patients from normal donors, but<br />
robustly significant differences in certain subsets discriminated<br />
patients experiencing flare from those with low levels <strong>of</strong> disease<br />
activity. Importantly, cell-type specific regulation <strong>of</strong> cytokine<br />
signaling may mark an important transition between flare and<br />
remission in humans. These results provide unique mechanistic<br />
insights into the signaling disruptions that occur during SLE,<br />
demonstrate that this approach can rapidly relate disease status<br />
to signaling network changes, and suggest it will be possible to<br />
stratify patients with greater accuracy.<br />
doi:10.1016/j.clim.2007.03.032<br />
OR.93 Accelerated Development <strong>of</strong><br />
Glomerulonephritis in TNF Receptor 1 and 2<br />
Double-knockout Lupus-mice<br />
Noam Jacob, Research Fellow, University <strong>of</strong> Southern<br />
California Keck School <strong>of</strong> Medicine, Los Angeles, CA,<br />
Luminita Pricop, Associate Pr<strong>of</strong>essor <strong>of</strong> Medicine, Hospital<br />
for Special Surgery, New York, NY, Chaim Putterman,<br />
Associate Pr<strong>of</strong>essor, Albert Einstein College <strong>of</strong> Medicine/<br />
Department <strong>of</strong> Medicine, Bronx, NY, Chaim O. Jacob,<br />
Associate Pr<strong>of</strong>essor <strong>of</strong> Medicine, University <strong>of</strong> Southern<br />
California Keck School <strong>of</strong> Medicine/Department <strong>of</strong><br />
Medicine, Los Angeles, CA, Michael Koss, Pr<strong>of</strong>essor <strong>of</strong><br />
Pathology, University <strong>of</strong> Southern California Keck School <strong>of</strong><br />
Medicine/Department <strong>of</strong> Pathology, Los Angeles, CA<br />
We have developed lupus-prone (NZB×NZW)F1-derived<br />
congenic New Zealand Mixed (NZM) 2328 lines that are deficient<br />
in TNF receptor 1 (TNFR1), TNF receptor 2 (TNFR2) or in both<br />
TNFR1 and TNFR2. While the development <strong>of</strong> lupus-nephritis in<br />
TNFR2 deficient mice was very similar to wild type, TNFR1<br />
deficient mice had only a slightly delayed disease development<br />
compared to TNF receptors intact NZM 2328 controls. On the<br />
other hand, mice that lacked both TNFR1 and TNFR2 developed<br />
a significantly accelerated lupus-nephritis and increased<br />
mortality associated with increased levels <strong>of</strong> IgG anti-doublestranded<br />
DNA autoantibodies both in the periphery and in the<br />
glomerular deposits. Double knockout mice have significantly<br />
enlarged spleens compared to all other lines, which is<br />
associated with increased number <strong>of</strong> B and T lymphocytes<br />
mostly <strong>of</strong> CD4+ subsets. Most notably, double KO mice have a 3–<br />
5 fold increase in the number <strong>of</strong> activated memory Tcells (CD4+<br />
CD25− CD44high CD62Llow) while TNFR1 deficient and TNFR2<br />
deficient are not different from each other. Immunostaining <strong>of</strong><br />
spleens shows spontaneous germinal center formation in wild<br />
type NZM 2328 and TNFR2 deficient mice. TNFR1 deficient mice<br />
have reduced germinal center formation at 2 months and still at<br />
6 months <strong>of</strong> age, while double KO mice have reduced GC<br />
formation at 2 months but by 6 months have as much or more<br />
than the control mice. These results suggest a fine and complex<br />
balance between the two TNF receptors and underscore the<br />
need for both receptors in the immune development <strong>of</strong><br />
autoimmune kidney disease.<br />
doi:10.1016/j.clim.2007.03.033<br />
OR.94 Protective and Therapeutic Role for α<br />
B-Crystallin in Autoimmune Demyelination<br />
Shalina Ousman, Stanford University, Stanford, CA, Beren<br />
Tomooka, Mr. Stanford University, Division <strong>of</strong> <strong>Immunology</strong><br />
and Rheumatology, Palo Alto, CA, Johannes Van Noort,<br />
Department <strong>of</strong> Biosciences, Leiden, The Netherlands, Kevin<br />
O’Conner, Harvard Medical School, Brigham and Women’s<br />
Hospital, Boston, MA, Eric Wawrousek, National Eye<br />
Institute, Bethesda, MD, David Hafler, Harvard Medical<br />
School, Brigham and Women's Hospital, Boston, MA,<br />
Raymond Sobel, Department <strong>of</strong> Pathology, Stanford<br />
University, Palo Alto, CA, William Robinson, Division <strong>of</strong><br />
<strong>Immunology</strong> and Rheumatology, Stanford University,<br />
Stanford, Palo Alto, CA, Lawrence Steinman, Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> Neurology and Neurological Studies,<br />
Stanford University, Stanford, CA<br />
Alpha B-crystallin (±BC) is the major target <strong>of</strong> the immune<br />
response to myelin from multiple sclerosis (MS) and control<br />
brain. It is the most abundant transcript uniquely present in<br />
MS lesions and, recent studies have identified anti-apoptotic<br />
and anti-inflammatory roles for±BC. We hypothesize that<br />
this crystallin plays a key protective role in demyelinating<br />
disease. ±BC−/− mice displayed worse symptoms <strong>of</strong> experimental<br />
allergic encephalomyelitis (EAE), the animal model <strong>of</strong><br />
MS, with higher Th1 and Th-17 cytokine secretion, proliferation<br />
and p38 signaling by their T cells and macrophages and,<br />
more intense CNS inflammation and demyelination compared<br />
to their WT counterparts. Glial cells from ±BC−/− mice also<br />
expressed more cleaved caspase-3, TUNEL staining and<br />
enhanced ERK and NFkB signaling. These results indicated<br />
that ±BC plays a protective role in curbing both glial cell<br />
death and immune cell activation. Of interest, myelin antigen<br />
arrays identified antibodies to native ±BC and to p21–40<br />
and p116–135 <strong>of</strong> ±BC in the cerebrospinal fluid <strong>of</strong> patients<br />
with relapsing remitting MS (RRMS). These antibodies could<br />
neutralize ±BC function. We then assessed whether recombinant<br />
±BC itself could resolve disease in mice with ongoing<br />
EAE. Remarkably, clinical disease was ameliorated in ±BCtreated<br />
animals along with decreased Th1 and Th17 cytokine<br />
production and glial cell death. The immune response against<br />
a negative regulator <strong>of</strong> inflammation in demyelinating<br />
disease, would promote inflammation and demyelination,<br />
and this can be countered therapeutically by giving ±BC itself.<br />
Supported by NIH, National Multiple Sclerosis Society and<br />
Multiple Sclerosis Society <strong>of</strong> Canada.<br />
doi:10.1016/j.clim.2007.03.034<br />
Regulating Inflammation<br />
Sunday, June 10<br />
2:45 pm−4:45 pm<br />
S139<br />
OR.95 Defect in TIM-3 Regulation <strong>of</strong> CD4+ T Cells in<br />
a Human Autoimmune Disease<br />
Li Yang, Postdoctoral Fellow, Center for Neurologic<br />
Diseases, Harvard Medical School, Boston, MA, David E.<br />
Anderson, Instructor, Center for Neurologic Diseases,<br />
Harvard Medical School, Boston, MA, Vijay K. Kuchroo,<br />
Pr<strong>of</strong>essor, Center for Neurologic Diseases, Harvard Medical
S140 Abstracts<br />
School, Boston, MA, David A. Hafler, Pr<strong>of</strong>essor, Center for<br />
Neurologic Diseases, Harvard Medical school, Boston, MA<br />
T cell immunoglobulin- and mucin-domain-containing molecules-3<br />
(Tim-3), a T helper type 1 (Th1)-specific cell surface<br />
molecule, functions as an inhibitory molecule in mice. However,<br />
lessisknownaboutthefunction<strong>of</strong>TIM-3inhumans.Wehave<br />
previously shown that reduction <strong>of</strong> TIM-3 expression on ex vivo<br />
human CD4+ T cells by small interfering RNA (siRNA) oligos<br />
increases cell proliferation and IFN-γ secretion, which confirms<br />
a negative role <strong>of</strong> TIM-3 in regulation <strong>of</strong> human T cell function.<br />
We now report the results <strong>of</strong> a study that interrogated the<br />
function <strong>of</strong> TIM-3 regulation <strong>of</strong> CD4+ T cells in the autoimmune<br />
diseasemultiplesclerosis(MS).Westimulatedex vivo CD4+ T<br />
cellsfrompatientswithMS(n=54) and healthy subjects (n=37)<br />
with anti-CD3/CD28 monoclonal antibodies in the presence or<br />
absence <strong>of</strong> anti-TIM-3 blocking antibody. Our data indicate that<br />
in the presence <strong>of</strong> anti-TIM-3 monoclonal antibody treatment<br />
there is a significant increase <strong>of</strong> IFN-γ secretion from CD4+ T<br />
cells in healthy subjects but not in untreated patients with MS.<br />
Analysis <strong>of</strong> the kinetics <strong>of</strong> TIM-3 expression demonstrates lower<br />
steady state levels <strong>of</strong> TIM-3 and delayed kinetics <strong>of</strong> upregulation<br />
on T cells present in patients with MS relative to<br />
healthy subjects. Interestingly, T cells obtained from patents<br />
treated with interferons restored TIM-3 function, as IFN-γ<br />
secretion in interferons-treated patients is increased with TIM3<br />
blockade when activated at lower doses <strong>of</strong> anti-CD3. Taken<br />
together, our results demonstrate that there is a significant<br />
defect in TIM-3 regulation <strong>of</strong> CD4 Tcells in a human autoimmune<br />
disease.<br />
doi:10.1016/j.clim.2007.03.035<br />
OR.96 Increasing GABA Activity Prevents<br />
Autoimmune Neuroinflammation<br />
Roopa Bhat, Post Doctoral Fellow, Stanford University,<br />
Neurology and Neurological Sciences, Stanford, CA,<br />
Christopher Lock, Vice President, Research and Development,<br />
Carantech BioSciences, Inc. San Francisco, CA, Lawrence<br />
Steinman, Pr<strong>of</strong>essor and Chair, Stanford University,<br />
Interdepartmental Program in <strong>Immunology</strong>, Stanford, CA<br />
Gamma-amino butyric acid (GABA) is the principal inhibitory<br />
neurotransmitter in the central nervous system (CNS). GABA is<br />
made by glutamic acid decarboxylase (GAD) and catabolized by<br />
GABA transaminase (GABAT). Using transcriptional microarrays,<br />
we showed that, in acute/active inflammatory brain lesions in<br />
multiple sclerosis (MS) patients, GABAT is upregulated 38-fold<br />
while specific GABA receptor subunits and GAD are downregulated<br />
(Lock et al., Nat Med 8: 500–508, 2002). GABA<br />
inhibits the development <strong>of</strong> autoimmunity, pro-inflammatory T<br />
cell responses, and disease progression in the mouse model <strong>of</strong><br />
autoimmune type1 diabetes (Tian et al., JImmunol173: 5298–<br />
5304, 2004). We, therefore, hypothesized that GABA ameliorates<br />
autoimmune inflammation in MS. To test this hypothesis,<br />
we used the mouse model <strong>of</strong> MS, experimental autoimmune<br />
encephalomyelitis, and pre-treated daily with vigabatrin,<br />
which irreversibly inhibits GABAT. We found that vigabatrin<br />
ameliorated paralysis significantly: disease incidence<br />
decreased by 64%, while the severity and number <strong>of</strong> relapses<br />
diminished by 10-fold and 72±14% (SEM) respectively. Topi-<br />
ramate, which binds the GABA-A receptor, also prevented CNS<br />
autoimmunity in a similar manner. This indicates a common<br />
mechanism that acts by increasing GABA-ergic function<br />
whether that be through the GABA metabolic or signaling<br />
pathway. Both agents suppressed myelin-specific T cell<br />
proliferation and inhibited the production <strong>of</strong> TNF, IFN- 3 ,IL-17<br />
and IL-6. Inherent abnormalities in the GABA metabolic and<br />
signaling pathway may play a previously unrecognized role in<br />
the pathogenesis <strong>of</strong> MS. Increasing GABA-ergic function using<br />
agents such as those in our study could be beneficial in the<br />
prevention <strong>of</strong> autoimmune disorders such as MS.<br />
doi:10.1016/j.clim.2007.03.036<br />
OR.97 Cyclooxygenase-2 Deficiency has a Protective<br />
Function in Liver Ischemia/Reperfusion Injury<br />
Takashi Hamada, Postdoctoraltoral Fellow, The<br />
Dumont-University <strong>of</strong> California, Los Angeles Transplant<br />
Center, Los Angeles, CA, Armine Avanesyan, Medical<br />
Student, The Dumont-University <strong>of</strong> California, Los Angeles<br />
Transplant Center, Los Angeles, CA, Sei-ichiro Tsuchihashi,<br />
Postdoctoral Fellow, The Dumont-University <strong>of</strong> California,<br />
Los Angeles Transplant Center, Los Angeles, CA, Carolina<br />
Moore, Student, The Dumont-University <strong>of</strong> California, Los<br />
Angeles Transplant Center, Los Angeles, CA, Ana Coito,<br />
Associate Pr<strong>of</strong>essor, The Dumont University <strong>of</strong> California,<br />
Los Angeles Transplant Center, Los Angeles, CA<br />
Cyclooxygenase-2 (COX-2) is a potent mediator <strong>of</strong> inflammation.<br />
In liver ischemia/reperfusion (I/R) injury, COX-2<br />
expression is highly correlated with the degree <strong>of</strong> tissue<br />
damage. This work tests the hypothesis that COX-2 expression<br />
has a critical function in the pathogenesis <strong>of</strong><br />
hepatic I/R injury. Methods and Results: COX-2 −/− mice<br />
(KO) and matched wild-type (WT) (COX-2 +/+) controls<br />
(n=8/g) were submitted to partial warm ischemia in the<br />
left/medium hepatic lobes for 90 minutes, followed by<br />
reperfusion. H&E staining <strong>of</strong> liver sections in COX-2 −/−<br />
mice at 24 h post-reperfusion revealed overall good<br />
histological preservation with modest signs <strong>of</strong> vascular<br />
congestion and necrosis, contrasting with significant<br />
edema, centrilobular ballooning, hepatocellular necrosis<br />
(N60%), and sinusoidal congestion observed in the WT<br />
counterparts (score: 1.5 ±1 vs. 3.5 ±0.6; pb0.03). Liver<br />
function, as evidenced by sGOT levels (IU/L), was improved<br />
in COX-2 (−/−) mice as compared with WT mice (206±45 vs.<br />
1244 ±290; pb0.01). MPO activity (U/gm) was also significantly<br />
lower in COX-2 (−/−) mice (1.2±0.4 vs. 3.4 ±1.5;<br />
pb0.01). Moreover, TUNEL staining showed reduced levels<br />
<strong>of</strong> apoptosis in COX-2 KO livers as compared with controls<br />
(16.2 ±3.3 vs. 73.6± 7.9, pb0.01). This was correlated with<br />
significant higher levels <strong>of</strong> BclXL, an anti-apoptotic member<br />
<strong>of</strong> the Bcl-2 family, in the COX-2 (−/−) livers. Conclusion:<br />
Our findings evidence an important function for COX-2 in<br />
the pathogenesis <strong>of</strong> hepatic I/R injury. They provide also<br />
the rationale for identification <strong>of</strong> improved therapeutic<br />
approaches to prevent hepatic I/R injury, and consequently<br />
for improving the outcome <strong>of</strong> liver transplantation.<br />
doi:10.1016/j.clim.2007.03.037
Abstracts<br />
OR.98 Crosstalk Between Leptin and LPS Signaling<br />
Pathways to Upregulate CD40 Expression via Akt in<br />
Dendritic Cells<br />
Queenie Lai Kwan Lam, PhD Candidate, University <strong>of</strong> Hong<br />
Kong, Department <strong>of</strong> Pathology, Hong Kong, Liwei Lu,<br />
Pr<strong>of</strong>essor, University <strong>of</strong> Hong Kong, Department <strong>of</strong><br />
Pathology, Hong Kong<br />
A body <strong>of</strong> evidence has demonstrated the regulatory role <strong>of</strong><br />
leptin in the immune system over the past decade but little is<br />
clear about how it regulates dendritic cell (DC) function. CD40, a<br />
cell-surface receptor and a costimulatory molecule, is a key<br />
determinant for the activity <strong>of</strong> DC in immunity and tolerance.<br />
We have previously shown that leptin signaling is critically<br />
involved in DC maturation and survival. In this study, we showed<br />
that leptin induced CD40 expression synergistically with LPS. In<br />
an Akt-dependent manner, both leptin and LPS activated the<br />
downstream transcription factors Nuclear Factor (NF)-kappaBp65<br />
and Signal Transducer and Activator <strong>of</strong> Transcription-<br />
1α (STAT-1α) in DC. Using dominant negative forms <strong>of</strong> Akt and<br />
IKK, as well as small interfering RNA (siRNA) for STAT-1α, we<br />
showed that Akt, STAT-1α and NF-kappaBp65 were important for<br />
CD40 expression induced by leptin, LPS and their synergistic<br />
action. Blocking studies demonstrated a crucial role for Akt in<br />
the translocation <strong>of</strong> STAT-1α and NF-kappaBp65 to the nucleus<br />
and activation <strong>of</strong> the CD40 promoter. Further analysis with<br />
chromatin immunoprecipitation assay confirmed that leptin<br />
recruited STAT-1α, NF-kappaBp65, acetylated histone 4 and RNA<br />
polymerase II to the CD40 promoter in a time-dependent<br />
manner. Administration <strong>of</strong> leptin into normal mice significantly<br />
augmented LPS-induced CD40 expression in DC in the lymph<br />
nodes. Thus, this study identifies a novel function for leptin to<br />
act an adjuvant to modulate DC function and provides new<br />
insight into the new role <strong>of</strong> leptin in modulating inflammatory<br />
immune response.<br />
doi:10.1016/j.clim.2007.03.038<br />
OR.99 The prolyl isomerase Pin1 regulates ECM<br />
mRNA expression in primary lung fibroblasts<br />
Zhong-Jian Shen, PhD, University <strong>of</strong> Wisconsin Madison,<br />
Madison, WI, James Malter, Pr<strong>of</strong>essor, University <strong>of</strong><br />
Wisconsin Madison, Department <strong>of</strong> Pathology and<br />
Laboratory Medicine, Madison, WI<br />
A key pathological feature <strong>of</strong> asthma is excessive airway and<br />
parenchymal deposition <strong>of</strong> extracellular matrix (ECM) protein,<br />
possibly resulting from reduced catabolism or increased<br />
production or both. TGF-β1 playsacentralroleintheairway<br />
remodeling, mediating ECM gene expression by resident<br />
fibroblasts. The molecular mechanisms underlying this dynamic<br />
process remain poorly understood. Here, we examined the role<br />
<strong>of</strong> the peptidyl-prolyl isomerase (PPIase) Pin1 in ECM expression<br />
using lung fibroblasts from Pin1 knockout mice. Cells were<br />
starved before TGF-β1 treatment, and the level <strong>of</strong> ECM mRNA<br />
was measured by RT/qPCR. Treatment with TGF-β1 consistently<br />
increased the level <strong>of</strong> collagen I, III and V in wild type cells.<br />
Knockout cells opposed to TGF-β1 however were unable to<br />
upregulate collagen III mRNA. As matrix metalloproteinase<br />
(MMP)2and3aremajoris<strong>of</strong>ormsinthelung,whichdegrade<br />
collagen I/V and collagen III/V, respectively, the level <strong>of</strong> MMPs<br />
was also evaluated. In the presence <strong>of</strong> TGF-β1, the level <strong>of</strong><br />
MMP2 and 3 was considerably increased in Pin1 knockout cells.<br />
Among the three tissue inhibitor <strong>of</strong> matrix metalloproteinase<br />
(TIMP) is<strong>of</strong>orms (1, 2 and 3), TIMP1 levels were significantly<br />
reduced in Pin1 knockout cells despite TGF-β1 stimulation.<br />
Therefore, the decreased expression <strong>of</strong> type III collagen and<br />
TIMP1 along with increased MMP2 and 3 in Pin1 knockout<br />
fibroblasts support our previous finding that in vivo administration<br />
<strong>of</strong> Pin1 inhibitors reduced airway fibrosis and suggest that<br />
Pin1 may be a target for the treatment <strong>of</strong> asthma and other<br />
fibrotic disorders as well.<br />
doi:10.1016/j.clim.2007.03.039<br />
OR.100 Genetic Evidence for CD8 + T Cell<br />
Immunoregulation in Murine CD4 + T Cell Colitis<br />
Bo Wei, Assistant Pr<strong>of</strong>essor, Department Pathology and Lab<br />
Medicine, University <strong>of</strong> California, Los Angeles David Geffen<br />
School <strong>of</strong> Medicine, Los Angeles, CA, Michael Macpherson,<br />
Graduate Student, Department Pathology and Lab Medicine,<br />
University <strong>of</strong> California, Los Angeles David Geffen School <strong>of</strong><br />
Medicine, Los Angeles, CA, Daisuke Fujiwara, Postgraduate<br />
Researcher, Department Pathology and Lab Medicine, University<br />
<strong>of</strong> California, Los Angeles David Geffen School <strong>of</strong> Medicine, Los<br />
Angeles, CA, Sarah Brewer, Graduate Student, University <strong>of</strong><br />
California, Los Angeles David Geffen School <strong>of</strong> Medicine, Los<br />
Angeles, CA, Jonathan Braun, Pr<strong>of</strong>essor, Department Pathology<br />
and Lab Medicine, University <strong>of</strong> California, Los Angeles David<br />
Geffen School <strong>of</strong> Medicine, Los Angeles, CA<br />
Abnormal CD4 + Tcell activity and consequent tissue damage<br />
are major pathogenic features <strong>of</strong> inflammatory bowel disease<br />
(IBD) and many systemic and tissue restricted autoimmune<br />
diseases. Disease susceptibility may involve deficiency <strong>of</strong> normal<br />
mucosal immunoregulation that attenuates CD4 + T cell activity<br />
targeting the mucosa. CD8 + T cells have emerged as one such<br />
regulatory population, but their modes <strong>of</strong> induction and<br />
targeting are poorly understood. We have demonstrated that<br />
B cells attenuate immune colitis through the interaction with<br />
CD8 + Tcells.Inthisstudy,weevaluatedthegenetics<strong>of</strong>Bcell<br />
traits involved in immunoregulation <strong>of</strong> G±i2−/− Tcellsinduced<br />
colitis. Transfer <strong>of</strong> G±i2−/− Tcells from 4 to 5 weeks mice (prior<br />
to colitis onset) induced progressive colitis, with additional<br />
features <strong>of</strong> systemic immune inflammation. Mesenteric node B<br />
cells from wild type mice inhibited colitis and systemic<br />
inflammation, and blocked the attendant CD4 + Tcellexpansion<br />
expressing TNF- 2 ,IFN- 3 and IL-17. Surprisingly, the protective<br />
function <strong>of</strong> B cells did not require genetic sufficiency for MHC<br />
class II, indicating that they did not directly interact with<br />
colitigenic or regulatory CD4 + T cells. However, protection<br />
required both MHC class I and TAP1 sufficiency, indicating that<br />
they augmented immunoregulation through direct interaction<br />
with classical peptide-restricted CD8 + T cells. These findings<br />
provide new evidence for CD8 + T cell immunoregulation in<br />
colitis, and suggest that functional or numerical deficiencies <strong>of</strong><br />
cooperative B or CD8 + Tcell subpopulations may be susceptibility<br />
traits, and targets for compensatory therapy, in inflammatory<br />
bowel disease. Supported by NIH DK4673 and DK69434.<br />
doi:10.1016/j.clim.2007.03.040<br />
S141
S142 Abstracts<br />
OR.101 The Galectin-9/TIM-3 Pathway and Human<br />
Glioma Pathogenesis<br />
Chengjie Zheng, Student, Harvard University, Boston, MA,<br />
Juhi Kuchroo, Student, Brigham and Women’s Hospital and<br />
Harvard Medical School, Boston, MA, David E. Anderson,<br />
Instructor in Neurology, Brigham and Women's Hospital and<br />
Harvard Medical School, Boston, MA<br />
Approximately 15,000 patients die each year in the United<br />
States from GBMs. Two well-documented aspects <strong>of</strong> malignant<br />
gliomas are that they are highly invasive and that immunity<br />
directed against them is ineffective. We hypothesize that<br />
glioma expression <strong>of</strong> galectin-9 (Gal-9) regulates both <strong>of</strong> these<br />
aspects <strong>of</strong> glioma pathogenesis. Gal-9 expression by peripheral<br />
human tumors has a favorable clinical prognosis and limits<br />
tumor cell metastasis/invasiveness. Thus, one prediction is<br />
that down-modulation <strong>of</strong> Gal-9 in gliomas may contribute to<br />
tumor cell invasiveness. Indeed, using FACS to isolate purified<br />
tumor cells or reactive astrocytes from ex vivo tumor speci-<br />
mens and quantitative RT-PCR, we have found that GBM tumor<br />
cells express little/no Gal-9 whereas Gal-9 is readily detected<br />
in reactive astrocytes. Inhibition <strong>of</strong> TGF-β signaling using<br />
soluble receptor or siRNA approaches indicates that TGF-<br />
β is responsible for down-modulation <strong>of</strong> Gal-9, which<br />
provides a molecular explanation for the long-standing<br />
observation that levels <strong>of</strong> TGF-β increase with grade <strong>of</strong><br />
malignancy and invasiveness. On the other hand, Gal-9 has<br />
recently been identified as a ligand <strong>of</strong> TIM-3 and been shown to<br />
kill IFN-γ-secreting Th1 cells. To this end, co-culture <strong>of</strong><br />
ex vivo CD4+ Tcells with a primary GBM cell line in the presence<br />
<strong>of</strong> blocking anti-TIM-3 monoclonal antibody enhances T cell<br />
proliferation and IFN-γ secretion. Collectively, the data<br />
suggest a paradigm in which Gal-9 expression by low-grade<br />
gliomas inactivates infiltrating TIM-3+ CD4+ and CD8+ T cells,<br />
and that with increasing stages <strong>of</strong> malignancy, down-modulation<br />
<strong>of</strong> Gal-9 promotes tumor cell invasiveness.<br />
doi:10.1016/j.clim.2007.03.042
Abstracts<br />
Su.1 Multiple Sclerosis Exacerbations:<br />
Simultaneous Activation <strong>of</strong> the Immune System by<br />
Multiple Immunogens<br />
Frederick Westall, President and Research Pr<strong>of</strong>essor,<br />
Institute for Disease Research, Temecula, CA<br />
Multiple Sclerosis (MS)is considered to be an autoimmune<br />
response against central nervous system myelin components.<br />
Immunogenic mimics on a surface protein <strong>of</strong> a microorganism<br />
initiate the immune process. Because <strong>of</strong> recent data indicating<br />
that such mimics are plentiful, it is suggested that in MS<br />
multiple mimics actually are simultaneously processed. Multiple<br />
simultaneous mimic processing can explain much <strong>of</strong> the<br />
confusing immunological data in MS and would parallel what is<br />
observed in chronic relapsing allergic encephalomyelitis, a<br />
clinical and histological model for MS.<br />
doi:10.1016/j.clim.2007.03.043<br />
Su.2 Comprehensive Molecular Explanation <strong>of</strong><br />
Recent MRI Multiple Sclerosis Studies<br />
Frederick Westall, President and Research Pr<strong>of</strong>essor,<br />
Institute for Disease Research, Temecula, CA<br />
Recent MRI studies have indicated that MS is a two part<br />
disease. First, diffuse lesions, apparently initiated by inflammatory<br />
processes, appear. Some <strong>of</strong> these then develop into the<br />
classical immunologically involved focal events. Inflammatory<br />
agents appear in the CSF and serum as much as a month before<br />
the focal lesions are seen. One <strong>of</strong> the earliest <strong>of</strong> these substances<br />
observed is metalloproteinase 9. This is followed by caspases and<br />
plasminogen and eventually various cytokines. Since there is no<br />
evidence <strong>of</strong> a chronic microbiological invasion <strong>of</strong> the CNS in MS,<br />
what is initiating this early inflammation? The immunological<br />
component <strong>of</strong> MS is thought to be caused by a microorganism<br />
possessing a protein sequence, which mimics an immunological<br />
active region in myelin. These mimics are not rare. Many<br />
potential mimics appear in the proteins <strong>of</strong> normal gut bacteria.<br />
In fact gut bacteria also contain the adjuvant molecules shown<br />
essential for autoimmune disease. The initiation <strong>of</strong> clinical<br />
autoimmune disease takes weeks. However, it has been clearly<br />
shown that the adjuvant molecules themselves can quickly<br />
inititate an inflammatory response. Many <strong>of</strong> these adjuvant<br />
molecules, for example lipopolysaccharides, can pass the gut<br />
barrier. An abnormal digestion in the gut could initiate the<br />
production <strong>of</strong> both immunogens and adjuvant molecules. These<br />
adjuvants could initially cause a general inflammatory response<br />
and later a specific autoimmune response in the CNS. These<br />
actions would explain the MRI studies, i.e. a diffuse inflammatory<br />
response with an occasional autoimmune involvement.<br />
doi:10.1016/j.clim.2007.03.044<br />
Poster Session<br />
Sunday, June 10<br />
7:30 am−7:00 pm<br />
Authors Present: 5:45 pm−7:00 pm<br />
Su.3 <strong>Oral</strong> Therapy with FTY720 Inhibits<br />
Angiogenesis in the Spinal Cord <strong>of</strong> Lewis Rats in the<br />
Relapse Phase <strong>of</strong> Experimental Autoimmune<br />
Encephalomyelitis<br />
Peter Hiestand, Senior Research Scientist, Novartis<br />
Institutes for BioMedical Research, Autoimmune Diseases<br />
and Transplantation, Basel, Switzerland, Christian Schnell,<br />
Senior Research Investigator, Novartis Institutes for<br />
BioMedical Research, Autoimmune Diseases and<br />
Transplantation, Basel, Switzerland<br />
Fingolimod (FTY720) is a sphingosine-1-phosphate receptor<br />
modulator that retards the egress <strong>of</strong> lymphocytes from<br />
peripheral lymph nodes. FTY720 has been shown to affect all<br />
stages <strong>of</strong> experimental autoimmune encephalomyelitis (EAE)<br />
in rats as well as in mice either induced with neuronal tissue<br />
or specific myelin protein antigens. While we have demonstrated<br />
that monocyte migration into the CNS <strong>of</strong> rats<br />
affected by EAE is blocked in the acute phase, the pronounced<br />
effects observed with FTY720 in the relapse phase<br />
<strong>of</strong> EAE seem to be linked to additional mechanisms. One <strong>of</strong><br />
the proposed factors involved in the induction <strong>of</strong> relapses <strong>of</strong><br />
EAE in the Lewis rats could be an increase in blood vessel<br />
density in the spinal cord as it has been described in EAE in<br />
guinea pigs. Using a casting procedure <strong>of</strong> blood vessels <strong>of</strong> the<br />
brain and spinal cord <strong>of</strong> Lewis rats going through acute and<br />
relapsing phases <strong>of</strong> EAE, we demonstrate an increase in<br />
vessel density induced in relapsing EAE and a FTY720mediated<br />
blockade <strong>of</strong> the induced angiogenesis. A similar<br />
protective effect was seen using a specific VEGF-receptor<br />
kinase inhibitor, demonstrating that a blockade <strong>of</strong> diseaseinduced<br />
angiogenesis leads to complete amelioration <strong>of</strong><br />
clinical symptoms <strong>of</strong> relapsing EAE.<br />
This neo-angiogenesis was mainly observed in the lumbar<br />
section <strong>of</strong> the spinal cord suggesting that this region be <strong>of</strong><br />
particular importance to the paralytic episodes observed in<br />
EAE in rats.<br />
doi:10.1016/j.clim.2007.03.045<br />
S143<br />
Su.5 Enhanced Glucocorticoid Signaling Impacts<br />
Thymocyte Apoptosis and Adaptive Immune<br />
Responses<br />
Fred Lühder, PhD, Institute for Multiple Sclerosis Research,<br />
Göttingen, Germany, Jens van den Brandt, PhD, Department<br />
<strong>of</strong> Cellular and Molecular <strong>Immunology</strong>, Göttingen, Germany,<br />
Holger Reichardt, PhD, Department <strong>of</strong> Cellular and<br />
Molecular <strong>Immunology</strong>, Göttingen, Germany, Ralf Gold, MD,<br />
St. Josef-Hospital/Ruhr-University Bochum, Bochum,<br />
Germany<br />
Glucocorticoids (GC) are synthesized by the adrenal gland<br />
and exert pleiotropic effects ranging from the regulation <strong>of</strong>
S144 Abstracts<br />
energy metabolism and the control <strong>of</strong> cognitive functions to the<br />
modulation <strong>of</strong> the immune system. Therapeutically they are<br />
used as potent anti-inflammatory and immunosuppressive drugs<br />
in a variety <strong>of</strong> conditions, but endogenous GCs rather exert<br />
positive as well as negative effects on the immune system. To<br />
study the effects <strong>of</strong> enhanced GC signaling on T cells we<br />
generated transgenic rats expressing a glucocorticoid receptor<br />
(GR) with increased ligand affinity. This causes massive<br />
thymocyte apoptosis resulting in a dramatic loss <strong>of</strong> thymic<br />
cellularity, which could be reverted by adrenalectomy. In the<br />
periphery, mature T lymphocytes accumulate that respond<br />
normally to costimulation and whose expansion is probably due<br />
to homeostatic proliferation, but have a skewed Tcell receptor<br />
repertoire. The transgenic rats are partially protected from<br />
Experimental Autoimmune Encephalomyelitis (EAE) induced by<br />
immunization with gpMBP, a predominantly TH1 mediated<br />
autoimmune model. The onset <strong>of</strong> EAE in transgenic rats was<br />
delayed by 5 days and the disease course was significantly<br />
milder. This difference appears to be the consequence <strong>of</strong> an<br />
impaired leukocyte infiltration into the CNS and a distinct<br />
cytokine pr<strong>of</strong>ile. In contrast, the ability <strong>of</strong> the transgenic rats to<br />
mount a predominantly TH2 mediated allergic airway response<br />
to ovalbumin was unaffected, although isotype switching <strong>of</strong><br />
antigen-specific immunoglobulins was altered. Collectively, our<br />
findings suggest that endogenous glucocorticoids impact T cell<br />
development and favor the selection <strong>of</strong> TH2 over TH1 dominated<br />
adaptive immune responses.<br />
doi:10.1016/j.clim.2007.03.046<br />
Su.6 Microarray Analysis Identifies<br />
Interferon-β-responsive Genes in Human Microglia<br />
Hiroko Tabunoki, Assistant, Meiji Pharmaceutical<br />
University, Bioinformatics, Kiyose, Japan, Tamako Misawa,<br />
Graduate Student, Meiji Pharmaceutical University,<br />
Bioinformatics, Kiyose, Japan, Jun-ichi Sato, Pr<strong>of</strong>essor,<br />
Meiji Pharmaceutical University, Bioinformatics, Kiyose,<br />
Japan<br />
Objective/Background: MS is mediated by autoreactive Th1<br />
cells. When activated Th1 cells enter the CNS across the blood–<br />
brain barrier (BBB), they are reactivated by microglia (MCG), the<br />
pr<strong>of</strong>essional antigen-presenting cell type in the CNS. Consequently,<br />
MCG activated by Th1 cytokines release mediators <strong>of</strong><br />
demyelination. Although interferon-beta (IFNB) reduces the<br />
frequency <strong>of</strong> relapses in MS patients, the molecular mechanism<br />
responsible for beneficial effects <strong>of</strong> IFNB in MS remains<br />
unknown. Because IFNB could pass through the BBB disrupted<br />
in active lesions <strong>of</strong> MS, it might directly modulate the biological<br />
function <strong>of</strong> MCG. Methods: To identify IFNB-responsive genes<br />
(IRGs) in human MCG, we studied the gene expression pr<strong>of</strong>ile <strong>of</strong> a<br />
human MCG cell line HMO6 (Neurobiol Dis 8:1057, 2001)<br />
following treatment with 50 ng/ml IFNB for 3 hours, by using<br />
an oligonucleotide microarray (Hitach), verified by Northern<br />
blot and real-time RT-PCR. Results: Among total 1606 genes on<br />
the array, IFNB elevated the expression <strong>of</strong> 39 genes. Top10<br />
included IFI56, IFI60, MX1, MX2, ISG15, IFI44, IRF7, STAT1, IFI6,<br />
and TAP1, all <strong>of</strong> which were known IRGs. The common upstream<br />
search <strong>of</strong> 39 genes on KeyMolnet (Curr Drug Discov Technol 2:89,<br />
2005), the knowledge-based data mining tool <strong>of</strong> bioinformatics,<br />
indicated a molecular network most relevant to gene regulation<br />
by IRF and NF-kB. Conclusions: IFNB induces the expression <strong>of</strong> a<br />
battery <strong>of</strong> IRGs in human microglia, including IRF7 and ISG15, a<br />
central regulator <strong>of</strong> innate and adaptive immunity (Nature Rev<br />
Immunol 6:644, 2006), suggesting an immunomodulatory effect<br />
<strong>of</strong> IFNB on human microglia.<br />
doi:10.1016/j.clim.2007.03.047<br />
Su.7 The Complement-scavenging Capacity <strong>of</strong> IVIG<br />
Products<br />
Martin O. Spycher, PhD, CSL Behring AG, Research and<br />
Development, Bern 22, Switzerland, Katja Matozan,<br />
University <strong>of</strong> Bern, Department <strong>of</strong> <strong>Clinical</strong> Research, Bern,<br />
Switzerland, Kathrin Minnig, PhD, CSL Behring AG, QA, Bern<br />
22, Switzerland, Sylvia Miescher, PhD, CSL Behring AG,<br />
Research and Development, Bern 22, Switzerland, Roland<br />
Zehnder, CSL Behring AG, Research and Development, Bern<br />
22, Switzerland, Liane Hoefferer, PhD, CSL Behring AG,<br />
Research and Development, Bern 22, Switzerland, Robert<br />
Rieben, PhD, University <strong>of</strong> Bern, Department <strong>of</strong> <strong>Clinical</strong><br />
Research, Bern, Switzerland<br />
IVIG induced complement scavenging describes one antiinflammatory<br />
effect <strong>of</strong> IVIG. This mechanism <strong>of</strong> action is<br />
considered important in inflammatory autoimmune disorders<br />
such as multifocal motor neuropathy or dematomyositis. The<br />
complement scavenging properties <strong>of</strong> eight IVIG products were<br />
compared. Constant amounts <strong>of</strong> aggregated human IgG coated<br />
to microtiterplates served as complement activators. They were<br />
incubated with constant amounts <strong>of</strong> human serum as a<br />
complementsourceinthepresenceorabsence<strong>of</strong>increasing<br />
concentrations <strong>of</strong> IVIGs. Complement activation was quantified<br />
by measuring bound activation products C3b/iC3b/C3c using a<br />
peroxidase-conjugated anti-C3c antibody.<br />
All IVIG tested exhibited dose-dependent inhibition <strong>of</strong> C3<br />
deposition. This effect was associated with reduced C3a<br />
generation, confirming that it was due to complement inhibition,<br />
not complement activation and consumption. Inhibition <strong>of</strong><br />
complement deposition was paralleled by an increase <strong>of</strong> C3<br />
activation product binding to IgG in the fluid phase. The extent<br />
<strong>of</strong> complement inhibition was dependent on the IVIG tested,<br />
with up to 3.5 fold differences between products. An IVIG<br />
currently in development, named IgPro10, manufactured with<br />
gentle procedures was the most potent product. Differences <strong>of</strong><br />
inhibition between products did not correlate with differences<br />
in C3 activation product binding to IgG in the fluid phase at given<br />
IVIG concentrations suggesting additional mechanisms being<br />
functional besides complement deviation. Complement scavenging<br />
is a common feature <strong>of</strong> IVIGs, however the extent <strong>of</strong><br />
complement scavenging depends on the product. IgPro10, a new<br />
IVIG product in development and manufactured with a gentle<br />
chromatographic process, is a more potent complement<br />
scavenger than currently used IVIGs.<br />
doi:10.1016/j.clim.2007.03.048<br />
Su.8 Influence <strong>of</strong> Interferon-β Therapy Switching on<br />
Neutralizing Antibody Titers: Results from the<br />
Austrian Switch Study (ASS)<br />
Florian Deisenhammer, Pr<strong>of</strong>essor, Department <strong>of</strong> Neurology,
Abstracts<br />
Innsbruck Medical University, Innsbruck, Austria, Alban<br />
Millonig, Assistant, Department <strong>of</strong> Neurology, Innsbruck<br />
Medical University, Innsbruck, Austria, Claudia Gneiss,<br />
Assistant, Department <strong>of</strong> Neurology, Innsbruck Medical<br />
University, Innsbruck, Austria<br />
Continuous treatment with interferon beta (IFNb) leads<br />
to neutralizing antibody (NAb) negativity in many previously<br />
NAb positive patients. To date no strategies are<br />
available to accelerate NAb reversion. We investigated the<br />
effect <strong>of</strong> switching between IFNb products on the development<br />
<strong>of</strong> NAb titers. Twenty-four NAb positive MS patients<br />
on IFNb-1a s.c. or IFNb-1b were included. At baseline IFNb<br />
treatment was interrupted for 3 months in all patients and<br />
2 infusions with 1 g methylprednisolone were given. Then<br />
patients were randomized either to their previous treatment<br />
or to IFNb-1a im. Twelve months later NAb titers were<br />
measured. Twelve patients were switched to IFNb-1a im<br />
30 μg (7 from IFNb-1a sc, 5 from IFNb-1b). Currently, final<br />
NAb titers (given in neutralizing units) are available in 18<br />
patients. The median baseline NAb titer was 846, 293 after<br />
corticosteroids, 1239 3 months after switching, and 393 at<br />
the end <strong>of</strong> study. The median change <strong>of</strong> NAb titers between<br />
baseline and end <strong>of</strong> study was −317 in patients who<br />
switched and −94 in patients who did not switch therapy<br />
(not significant). Baseline and end <strong>of</strong> study NAb titers<br />
correlated significantly (r=0.73). NAb can be regarded as a<br />
model <strong>of</strong> autoimmunity by breaking and re-inducing<br />
immune tolerance. The main influencing factor on the<br />
final NAb titer was the baseline titer. Many factors must be<br />
considered when choosing therapy for an individual patient.<br />
However, as far as NAb are concerned no strategy for<br />
reversion could be established leaving prevention the only<br />
option as yet.<br />
doi:10.1016/j.clim.2007.03.049<br />
Su.9 IL-21 Receptor Modulates Autoimmune<br />
Responses and Expression <strong>of</strong> Experimental<br />
Autoimmune Encephalomyelitis<br />
Ruolan Liu, Postdoctoral, Barrow Neurological Institute,<br />
Neurology, Phoenix, AZ, Antoni La Cava, Associate<br />
Pr<strong>of</strong>essor, David Geffen School <strong>of</strong> Medicine, Los Angeles,<br />
CA, Debbie Young, Pr<strong>of</strong>essor, Wyeth Research,<br />
Inflammation, Cambridge, MA, Mary Collins, Scientist,<br />
Wyeth Research, Cambridge, MA, Denise Campagnolo,<br />
Neurologist, Barrow Neurological Institute, Neurology,<br />
Phoenix, AZ, Timothy Vollmer, Neurologist, Barrow<br />
Neurological Institute, Neurology, Phoenix, MA, Fu-Dong<br />
Shi, Scientist, Barrow Neurological Institute, Neurology,<br />
Phoenix, AZ<br />
IL-21 receptor (IL-21R) consists <strong>of</strong> a unique IL-21R<br />
subunit and also shares the common gamma chain (γ c).<br />
IL-21R ligand IL-21 plays a plethora <strong>of</strong> roles in innate and<br />
adaptive immune responses. In this study we examined<br />
the influence <strong>of</strong> IL-21R deficiency in the development <strong>of</strong><br />
myelin oligodendrocyte glycoprotein peptide 35–55<br />
(MOG35–55)-induced experimental autoimmune encephalomyelitis<br />
(EAE), an animal model for human multiple<br />
sclerosis (MS). Upon immunization, IL-21R−/− mice devel-<br />
oped an exacerbated form <strong>of</strong> EAE with marked inflammatory<br />
infiltration into the central nervous system (CNS).<br />
Both Th1 and Th2 responses to MOG were augmented in<br />
IL-21R−/− mice. Although IL-21R deficiency did not<br />
dramatically alter the numbers <strong>of</strong> NK cells, NKT cells,<br />
CD11c, CD11b cells, and CD4+CD25+ regulatory T cells<br />
(Treg) during EAE, the expression <strong>of</strong> Foxp3 on Treg cells<br />
was reduced in MOG-primed IL-21R−/− mice. Our results<br />
suggest that IL-21R/ligand pathways may affect Treg cells,<br />
which may subsequently affect the magnitude <strong>of</strong> CNS<br />
autoimmunity.<br />
doi:10.1016/j.clim.2007.03.050<br />
S145<br />
Su.10 Targeted Delivery <strong>of</strong> Immunomodulating<br />
Agents to Dendritic Cells for Treatment <strong>of</strong><br />
Autoimmune Disease Using Biodegradable<br />
Microspheres<br />
Tali Brunner, PhD Student, Biotechnology Engineering,<br />
Ben-Gurion University <strong>of</strong> the Negev, Beer Sheva, Israel,<br />
Alon Monsonego, Researcher, Department <strong>of</strong> Microbiology<br />
and <strong>Immunology</strong>, Faculty <strong>of</strong> Health Sciences and The<br />
National Institute, Beer Sheva, Israel, Smadar Cohen,<br />
Researcher, Department <strong>of</strong> Biotechnology Engineering,<br />
Ben Gurion University <strong>of</strong> the Negev,<br />
Beer Sheva, Israel<br />
Background: Dendritic cells (DCs) are a central element<br />
for drug targeting in autoimmune diseases, due to their<br />
function in the onset and progression <strong>of</strong> destructive<br />
autoimmunity and in maintaining the Treg repertoire.<br />
When injected intradermally, microspheres sized 1–10<br />
microns are preferentially uptaken by DCs. Use <strong>of</strong> such<br />
microspheres has been investigated for delivery <strong>of</strong> antigens<br />
and DNA to DCs for vaccination; however this delivery<br />
system has not been implemented for delivery <strong>of</strong> siRNAs<br />
capable <strong>of</strong> regulating autoimmunity. In vivo delivery <strong>of</strong><br />
siRNA is a great challenge, since these RNA duplexes are<br />
rapidly degraded and require appropriate stabilization.<br />
Goal: To use biodegradable microspheres for delivering<br />
siRNA and other immunomodulating agents to DCs, for<br />
treatment <strong>of</strong> autoimmune disease. Results: Microspheres<br />
composed <strong>of</strong> poly-lactic-acid better target CD11c positive<br />
cells in a bone marrow DC (BMDC) culture, in comparison to<br />
a poly-lactic-co-glycolic composition. siRNA, stabilized<br />
using poly-ethylene-imine and encapsulated in microspheres<br />
using a double-emulsion procedure, remained<br />
active after release from microspheres in BMDCs. Importantly,<br />
the microspheres did not have an adjuvant effect<br />
upon the BMDCs compared with LPS-induced DC-mediated<br />
T cell proliferation, meaning that the BMDCs maintained<br />
their immature state, which is crucial for creating an<br />
immunoregulatory environment which can ameliorate the<br />
course <strong>of</strong> autoimmune disease.<br />
Summary: This work combines biotechnology engineering,<br />
assessing microsphere behavior and distribution, with<br />
basic immunological research, assessing activity <strong>of</strong> immunomodulating<br />
agents. We believe that this delivery platform<br />
can be used for delivering not only various targeting<br />
siRNAs, but also other immunomodulating agents such as
S146 Abstracts<br />
peptides which can interfere with intracellular signaling<br />
pathways.<br />
doi:10.1016/j.clim.2007.03.051<br />
Su.11 Neur<strong>of</strong>ascin-specific Autoantibodies Mediate<br />
Axonal Injury in Inflammatory Demyelinating<br />
Diseases <strong>of</strong> the Central Nervous System<br />
Christopher Linington, Pr<strong>of</strong>essor <strong>of</strong> Immunobiology,<br />
University <strong>of</strong> Aberdeen, Aberdeen, UK, Emily Mathey,<br />
Research Associate, University <strong>of</strong> Aberdeen, Aberdeen, UK,<br />
Tobias Derfuss, Max Planck Institute for Neurobiology,<br />
Martinsried, Germany, Matthew Rasband, Assistant<br />
Pr<strong>of</strong>essor, University <strong>of</strong> Connecticut Health Center,<br />
Farmington, CT, Maria Storch, Pr<strong>of</strong>essor, Medical University<br />
Graz, General Neurology, Graz, Germany, Reinhard<br />
Hohlfeld, Pr<strong>of</strong>essor, LMU University Hospital, <strong>Clinical</strong><br />
Neuroimmunology, Munich, Germany, Edgar Meinl,<br />
Pr<strong>of</strong>essor, Max Planck Institute for Neurobiology,<br />
Martinsried, Germany<br />
We report that multiple sclerosis is associated with elevated<br />
autoantibody responses to neur<strong>of</strong>ascin, as compared to other<br />
inflammatory neurological diseases. The two is<strong>of</strong>orms <strong>of</strong><br />
neur<strong>of</strong>ascin play essential roles in maintaining the molecular<br />
organization <strong>of</strong> myelinated fibers: NF186 is a neuronal protein<br />
located at the node <strong>of</strong> Ranvier and axonal initial segment where<br />
it interacts with voltage gated sodium channels, whilst NF155 is<br />
an oligodendroglial product sequestered at junctional complexes<br />
formed where paranodal loops <strong>of</strong> the myelin sheath<br />
contact the axonal surface. Analysis <strong>of</strong> neur<strong>of</strong>ascin-specific<br />
autoantibodies immunopurified from seropositive donors<br />
demonstrates that they recognise the extracellular domain <strong>of</strong><br />
both is<strong>of</strong>orms <strong>of</strong> the native protein indicating that this response<br />
may participate in disease pathogenesis. We investigated this<br />
hypothesis in experimental autoimmune encephalomyelitis<br />
using a pan NF155/186 mouse mAb to mimic the human<br />
autoantibody response. Passive transfer (i.p.) <strong>of</strong> mAb induced<br />
a rapid exacerbation <strong>of</strong> disease that was associated with a<br />
corresponding increase in acute axonal injury. Intriguing this<br />
occurred in the absence <strong>of</strong> any concomitant increase in the local<br />
inflammatory response or demyelination. Immun<strong>of</strong>luorescence<br />
microscopy revealed that binding <strong>of</strong> the transferred neur<strong>of</strong>ascin-specific<br />
antibody was restricted nodes <strong>of</strong> Ranvier where it<br />
co-localised with voltage gated sodium channels. These results<br />
identify NF186 as a primary target for neur<strong>of</strong>ascin-specific<br />
autoantibodies and indicate antibody-dependent effector<br />
mechanisms are involved in the pathogenesis <strong>of</strong> axonal injury<br />
andlossinmultiplesclerosis.<br />
doi:10.1016/j.clim.2007.03.052<br />
Su.12 CD11c on NK Cells Mirrors the Temporal<br />
Disease Activity <strong>of</strong> Multiple Sclerosis<br />
Toshimasa Aranami, Section Chief, Department <strong>of</strong><br />
<strong>Immunology</strong>, National Institute <strong>of</strong> Neuroscience, NCNP,<br />
Kodaira, Japan, Sachiko Miyake, Section Chief, Department<br />
<strong>of</strong> <strong>Immunology</strong>, National Institute <strong>of</strong> Neuroscience, NCNP,<br />
Kodaira, Japan, Masafumi Ogawa, Section Chief,<br />
Department <strong>of</strong> Neurology, National Center Hospital for<br />
Mental Nervous and Muscular Disorders, NCNP, Kodaira,<br />
Japan, Takashi Yamamura, Director, Department <strong>of</strong><br />
<strong>Immunology</strong>, National Institute <strong>of</strong> Neuroscience, NCNP,<br />
Kodaira, Japan<br />
Multiple sclerosis (MS) is an autoimmune disease, showing a<br />
greatdegree<strong>of</strong>varianceindiseaseactivity.Wehaverecently<br />
demonstrated that NK cells biased for producing IL-5 (bias<br />
towards type2 NK cells, NK2 bias) are associated with remission<br />
state <strong>of</strong> MS. Here we report that upregulation <strong>of</strong> CD11c on NK<br />
cells would characterize a subgroup <strong>of</strong> MS in remission whose NK<br />
cells are lacking NK2 bias. When we compared this group<br />
(CD11chigh) with the other group <strong>of</strong> patients (CD11clow)<br />
concerning NK cell expression <strong>of</strong> IL-5 and GATA-3, we found<br />
NK2 bias to be a selective character <strong>of</strong> CD11clow patients. In<br />
contrast, CD11chigh group showed an obvious trend for a larger<br />
proportion <strong>of</strong> HLA-DR+ cells within NK cells. Since NK cell<br />
stimulatory cytokine such as IL-15 was found to upregulate<br />
CD11c expression on NK cells in vitro, we postulate that<br />
inflammatory cytokine milieu may play a role in inducing<br />
CD11chigh NK cell phenotype. Although there was no clinical<br />
difference between CD11chigh and CD11clow at blood sampling,<br />
CD11chigh MS showed significantly higher relapsing rate than<br />
CD11clow during 120 days follow-up. These data point to an<br />
interpretation that CD11chigh patients are at a higher risk for<br />
developing relapse, which is probably due to the loss <strong>of</strong> NK2<br />
phenotype. Thus, CD11c on NK cells mirrors the temporal<br />
disease activity <strong>of</strong> MS and may be used as a disease biomarker.<br />
doi:10.1016/j.clim.2007.03.053<br />
Su.13 Functional Involvement <strong>of</strong> NR4A2 in the<br />
Pathogenesis <strong>of</strong> Multiple Sclerosis<br />
Yoshimitsu Doi, Physician, Department <strong>of</strong> <strong>Immunology</strong>,<br />
National Institute <strong>of</strong> Neuroscience, NCNP, Kodaira City,<br />
Tokyo, Japan, Shinji Oki, Scientist, Department <strong>of</strong><br />
<strong>Immunology</strong>, National Institute <strong>of</strong> Neuroscience, NCNP,<br />
Kodaira City, Tokyo, Japan, Sachiko Miyake, Physician,<br />
Department <strong>of</strong> <strong>Immunology</strong>, National Institute <strong>of</strong><br />
Neuroscience, NCNP, Kodaira City, Tokyo, Japan, Takashi<br />
Yamamura, Physician, Department <strong>of</strong> <strong>Immunology</strong>, National<br />
Institute <strong>of</strong> Neuroscience, NCNP, Kodaira City, Tokyo, Japan<br />
Objective: To study a role <strong>of</strong> the NR4A2 in T cells <strong>of</strong><br />
multiple sclerosis (MS). Background: NR4A2, a transcription<br />
factor <strong>of</strong> the steroid/thyroid receptor superfamily, is rapidly<br />
induced by exposure to growth factors and cytokines,<br />
suggesting a biological relevance to various cell functions.<br />
Through a cDNA microarray analysis, higher expression <strong>of</strong><br />
NR4A2 was observed in CD3+ T cells <strong>of</strong> 57 MS patients<br />
compared with 19 healthy control subjects (Satoh et al.<br />
Neurobiol Dis 18:537–50, 2005). However, the functional<br />
involvement <strong>of</strong> NR4A2 expression and its transcriptional<br />
targets in MS Tcells remain unknown. Methods: Expression <strong>of</strong><br />
NR4A2 mRNA was analyzed by quantitative RT-PCR in PBMCderived<br />
human T cells or murine T cells infiltrated in CNS<br />
after EAE induction. Activities <strong>of</strong> cytokine gene promoters<br />
were analyzed by reporter gene analysis. Cytokine production<br />
in primary T cells was measured after retroviral<br />
transduction <strong>of</strong> NR4A2 gene or silencing <strong>of</strong> NR4A2 gene by<br />
using siRNA. Results: Higher expression <strong>of</strong> NR4A2 was
Abstracts<br />
demonstrated both in MS Tcells and in CNS-infiltrating Tcells<br />
in EAE mice. NR4A2 augmented promoter activities <strong>of</strong><br />
inflammatory cytokine genes. Overexpression <strong>of</strong> NR4A2<br />
induced up-regulation <strong>of</strong> IL-17 and IFN-f× production. Meanwhile,<br />
silencing <strong>of</strong> NR4A2 expression by siRNA resulted in<br />
down-regulation <strong>of</strong> the inflammatory cytokines. Conclusions:<br />
NR4A2 was upregulated in T cells <strong>of</strong> MS patients and in CNSinfiltrating<br />
T cells <strong>of</strong> EAE mice. Inflammatory cytokines<br />
including IL-17 and IFN-f× were the possible targets <strong>of</strong> NR4A2<br />
in T cells, suggesting that NR4A2 may play an important role<br />
in immunopathogenesis <strong>of</strong> MS.<br />
doi:10.1016/j.clim.2007.03.054<br />
Su.14 Functional Analysis <strong>of</strong> Carcinoembryonic<br />
Antigen-related Cellular Adhesion Molecule 1 in<br />
Experimental Autoimmune Encephalomyelitis<br />
Shinji Oki, Section Chief, Department <strong>of</strong> <strong>Immunology</strong>,<br />
National Institute <strong>of</strong> Neuroscience, NCNP, Tokyo, Japan,<br />
Mayumi Fujita, Postdoctoral Researcher, Department <strong>of</strong><br />
<strong>Immunology</strong>, National Institute <strong>of</strong> Neuroscience, NCNP,<br />
Tokyo, Japan, Takao Ootsuka, Graduate Student, Department<br />
<strong>of</strong> <strong>Immunology</strong>, National Institute <strong>of</strong> Neuroscience, NCNP,<br />
Tokyo, Japan, Chiharu Tomi, Researcher, Department <strong>of</strong><br />
<strong>Immunology</strong>, National Institute <strong>of</strong> Neuroscience, NCNP,<br />
Tokyo, Japan, Miho Mizu, Researcher, Department <strong>of</strong><br />
<strong>Immunology</strong>, National Institute <strong>of</strong> Neuroscience, NCNP,<br />
Tokyo, Japan, Shinjiro Kaieda, Graduate Student,<br />
Department <strong>of</strong> <strong>Immunology</strong>, National Institute <strong>of</strong><br />
Neuroscience, NCNP, Tokyo, Japan, Takashi Yamamura,<br />
Director, Department <strong>of</strong> <strong>Immunology</strong>, National Institute <strong>of</strong><br />
Neuroscience, NCNP, Tokyo, Japan, Sachiko Miyake, Section<br />
Chief, Department <strong>of</strong> <strong>Immunology</strong>, National Institute <strong>of</strong><br />
Neuroscience, NCNP, Tokyo, Japan<br />
Introduction: Carcinoembryonic antigen-related cellular<br />
adhesion molecule 1 (CEACAM1) is one <strong>of</strong> the CEA-family<br />
members and is demonstrated to play an important role in<br />
immune regulation. In this study, we investigated the role <strong>of</strong><br />
CEACAM1 in experimental autoimmune encephalomyelitis<br />
(EAE). [Methods] C57BL/6J mice immunized with MOG peptide<br />
were treated with anti-CEACAM1 mAb (AgB10) or CEACAM1-Fc<br />
fusion protein twice a week. The clinical signs <strong>of</strong> each group<br />
were scored to evaluate the effect <strong>of</strong> CEACAM1-mediated<br />
signals in EAE. MOG-specific cytokine production by inguinal<br />
lymph node cells derived from AgB10-treated or CEACAM1-Fctreated<br />
mice were analyzed by ELISA 10 days after the<br />
immunization. MOG-specific antibody production in serum<br />
was analyzed for AgB10-treated mice. Results and Discussion:<br />
The repetitive treatment with AgB10 enhanced the clinical sign<br />
<strong>of</strong> EAE, whereas the treatment with CEACAM1-Fc suppressed<br />
EAE as compared with control animals, indicating that CEACAM1<br />
exerts suppressive signal for the development <strong>of</strong> EAE. The<br />
production <strong>of</strong> inflammatory cytokines such as IFN-fÁ andIL-17<br />
in T cells derived from AgB10-treated mice was significantly<br />
enhanced, whereas the production <strong>of</strong> these cytokines from<br />
CEACAM1-Fc-treated mice was significantly decreased. These<br />
results suggest that CEACAM1-mediated signal is inhibitory for<br />
MOG-specific Th1 or Th17 responses. Accordingly, MOG-specific<br />
antibody production from AgB10-treated mice showed the<br />
reduced level <strong>of</strong> IgG1 and enhanced level <strong>of</strong> IgG2a compared<br />
with control mice. Taken together, CEACAM1 signal holds an<br />
important suppressive role in EAE by inhibiting both Th1 and<br />
Th17 responses to tissue-specific antigens. Mechanistic analysis<br />
for the role <strong>of</strong> CEACAM1 signal is currently under investigation.<br />
doi:10.1016/j.clim.2007.03.055<br />
Su.15 Gene Expression Pr<strong>of</strong>ile Modulated by IVIG in<br />
Healthy Donors and Multiple Sclerosis Patients<br />
Christian Jacobi, MD, University <strong>of</strong> Heidelberg, Department<br />
<strong>of</strong> Neurology, Institute <strong>of</strong> <strong>Immunology</strong>, Heidelberg,<br />
Germany, Jürgen, Römisch, PhD, Octapharma PPmbH,<br />
Vienna, Austria, Stefan, Meuer, MD, University <strong>of</strong><br />
Heidelberg, Institute <strong>of</strong> <strong>Immunology</strong>, Heidelberg, Germany,<br />
Thomas Giese, MD, University <strong>of</strong> Heidelberg, Institute <strong>of</strong><br />
<strong>Immunology</strong>, Heidelberg, Germany<br />
Intravenous immunoglobulin G (IVIG) has become an<br />
established first- and second-line line treatment in a<br />
number <strong>of</strong> immune mediated diseases during recent years.<br />
IVIG is now proposed to modulate a wide range <strong>of</strong> molecules<br />
(e.g. bacterial/viral antigens, toxins, Fc receptors, complement,<br />
autoantibodies), to act on different cell types (e.<br />
g. B cells, T cells, macrophages, dendritic cells, endothelial<br />
cells), and to modulate various immunological pathways at<br />
both the humoral and cellular levels including inflammation,<br />
antigen presentation, cell growth and apoptosis. Using<br />
a variety <strong>of</strong> specific assays many distinct effects <strong>of</strong> IVIG<br />
have been demonstrated in vitro. To provide further insight<br />
into the mechanisms involved in IVIG-dependent immunmodulation<br />
under physiological conditions, we have established<br />
a human whole blood gene expression assay in vitro.<br />
There, we analyzed the effect <strong>of</strong> IVIG on CD2-, CD3-, LPSand<br />
PMA/Ionomycin stimulated leukocytes in whole blood <strong>of</strong><br />
20 healthy donors and 12 untreated MS patients. IVIG<br />
induces among others the expression and release <strong>of</strong><br />
Interferon-γ, MCP1 and IP10. Interestingly, IVIG reduces<br />
the MCP1 and IP10 expression and release upon LPS<br />
stimulation, whereas Interferon-γ expression is further<br />
enhanced. Since both chemokines are physiologically<br />
induced by Interferon, this effect <strong>of</strong> IVIG is unanticipated<br />
and deserves further investigation for its possible role in<br />
anti-inflammatory properties <strong>of</strong> IVIG. No significant differences<br />
in the IVIG induced expression pr<strong>of</strong>ile between<br />
healthy donors and MS patients could be observed.<br />
doi:10.1016/j.clim.2007.03.056<br />
S147<br />
Su.16 IL-15 and IL-15Rα Expressed in Human<br />
Central Nervous System by Astrocytes Contribute to<br />
CD8 T Lymphocyte Activation and Persistence:<br />
Implications for Multiple Sclerosis<br />
Nathalie Arbour, Assistant Pr<strong>of</strong>essor, University <strong>of</strong><br />
Montreal-Medicine, Montreal, QC, Canada, Philippe Saikali,<br />
PhD Student, McGill University-Neurology, Montreal, QC,<br />
Canada, Jack Antel, Pr<strong>of</strong>essor<br />
Increasing experimental evidences suggest that CD8 T<br />
lymphocytes partake in multiple sclerosis (MS) related central<br />
nervous system (CNS) damage. CD8 T lymphocytes are
S148 Abstracts<br />
prominent cells in MS brain infiltrates and could injure<br />
oligodendrocytes and axons, both damaged in MS, as they are<br />
observed touching these structures. Activated memory CD8 T<br />
cells were shown to clonally expand and persist in the CNS <strong>of</strong> MS<br />
patients for years. Interleukin-15 (IL-15) is pivotal in the<br />
generation and maintenance <strong>of</strong> memory CD8 T cells. IL-15<br />
bound to IL-15Rα onitscell<strong>of</strong>originefficientlyactivatesIL-<br />
15Rβ/γ bearing cells. We determined whether a local source <strong>of</strong><br />
IL-15 and IL-15Rα can supply this cytokine to infiltrating CD8 T<br />
lymphocytes in the MS CNS. Human astrocytes expressed surface<br />
bound IL-15 and IL-15Rα in vitro; such expression was upregulated<br />
in response to pro-inflammatory cytokines. We<br />
detected astrocytes expressing IL-15 in MS brain supporting its<br />
in vivo presence. Human CD8 T cells cultured in the presence<br />
<strong>of</strong> IL-15 were more activated and cytotoxic (high amounts <strong>of</strong><br />
cytolytic enzymes) and acquired a memory phenotype that was<br />
not observed in the presence <strong>of</strong> IL-2. CD8 T lymphocytes bearing<br />
a similar activated memory phenotype were observed in the<br />
cerebrospinal fluid <strong>of</strong> MS patients. The localization <strong>of</strong> astrocytes<br />
within the brain architecture suggests that infiltrating CD8 T<br />
cells would readily encounter this source <strong>of</strong> IL-15+IL15Rα.<br />
These results underline a novel potential role for IL-15/IL-15Rα<br />
in the survival <strong>of</strong> memory CD8 T cells in the CNS during<br />
inflammatory diseases such as MS.<br />
doi:10.1016/j.clim.2007.03.057<br />
Su.18 CD4+CD28null T Cells in Autoimmune Disease:<br />
Pathogenic Features and Decreased Susceptibility to<br />
Immunoregulation<br />
Marielle Thewissen, Hasselt University, Biomedical Research<br />
Institute, Diepenbeek, Belgium, Veerle Somers, Hasselt<br />
University, Biomedical Research Institute, Diepenbeek,<br />
Belgium, Niels Hellings, Hasselt University, Biomedical<br />
Research Institute, Diepenbeek, Belgium, Piet Stinissen,<br />
Director, Hasselt University, Diepenbeek, Belgium, Koen<br />
Venken, Hasselt University, Biomedical Research Institute,<br />
Diepenbeek, Belgium<br />
CD4+CD28null T cells have been described to be<br />
generated as a consequence <strong>of</strong> persistent immune stimulation.<br />
These CD4+ T cells are incapable <strong>of</strong> providing help for<br />
B cell proliferation and antibody production, but have<br />
acquired cytolytic function. To date, the triggers <strong>of</strong><br />
activation <strong>of</strong> CD4+CD28null T cells have not been fully<br />
elucidated. An expansion <strong>of</strong> this cell subset has been<br />
described in several pathological conditions including<br />
multiple sclerosis (MS) and rheumatoid arthritis (RA). In<br />
this study the role <strong>of</strong> the expanded CD4+CD28null T cell<br />
subset in RA and MS pathology was further investigated.<br />
CD4+CD28null T cells were found to be terminally differentiated<br />
effector memory cells. They are equipped to<br />
infiltrate tissues and could be detected in the synovial<br />
tissue <strong>of</strong> RA patients. Whereas no reactivity to candidate<br />
autoantigens (myelin, collagen) was observed within the<br />
CD4+CD28null T cell subset, cytomegalovirus (CMV) reactivity<br />
was prominent in 4/4 HC, 4/4 RA patients and 3/4<br />
MS patients. Of note was that the intensity <strong>of</strong> the CMV<br />
induced proliferative response was related to the number<br />
<strong>of</strong> CMV epitopes recognized. Interestingly, our results<br />
illustrated that CD4+CD28null T cells were not susceptible<br />
to the suppressive actions <strong>of</strong> CD4+CD25 high regulatory T<br />
cells. In conclusion, this study provides several indications<br />
for a role <strong>of</strong> CD4+CD28null T cells in autoimmune pathology.<br />
These cells display pathogenic features, fill up immunological<br />
space and are less vulnerable to regulatory mechanisms.<br />
However, our results do not support a direct<br />
autoreactive role <strong>of</strong> CD4+CD28null T cells in the pathogenesis<br />
<strong>of</strong> autoimmune diseases.<br />
doi:10.1016/j.clim.2007.03.058<br />
Su.19 Intravenous TCV-CD4 Treatment for Multiple<br />
Sclerosis<br />
Gustavo A. Moviglia, MD, Regina Mater Foundation and<br />
Maimonides University, Buenos Aires, Argentina, Gabriela<br />
Varela, PhD, Regina Mater Foundation, Buenos Aires,<br />
Argentina, J. Hirsch, MD, Regina Mater Foundation, Buenos<br />
Aires, Argentina, J.A. Brizuela, MD, Regina Mater<br />
Foundation and Maimonides University, Buenos Aires,<br />
Argentina, C.A. Palleros, Regina Mater Foundation, Buenos<br />
Aires, Argentina, R. Moya, MD, Regina Mater Foundation,<br />
Buenos Aires, Argentina, Fabiana Bastos, MD, Regina Mater<br />
Foundation and Maimonides University, Buenos Aires,<br />
Argentina, M.T. Moviglia, MD, Regina Mater Foundation and<br />
Maimonides University, Buenos Aires, Argentina<br />
Background: T cell vaccine (TCV) is a proven pre clinical and<br />
clinical cell therapy for MS. The original technique, developed<br />
by Irum Cohen and collaborators, was as monoclonal or<br />
oligoclonal antimyelin and selective cell suspension to be<br />
activated, irradiated and administered to MS stable patients.<br />
Recent clinical trials showed that 92% <strong>of</strong> treated patients were<br />
stabilized. Methods: 82 CPMS patients were divided into 3<br />
groups: Group 1 (42 patients) received 10 subcutaneous<br />
immunizations <strong>of</strong> polyclonal TCV (1 every 4 weeks). Group 2<br />
(24 patients) received 10 IV polyclonal TCV. Group 3 (16<br />
patients) received 10 IV polyclonal TCV-CD4. Experienced<br />
neurologist evaluated the EDSS index from the beginning up to<br />
3 years <strong>of</strong> follow-up. The TCV-CD4 vaccine was prepared<br />
selecting only CD4+ cells from polyclonal TCV (Stem Sep for<br />
CD4, Vancouver) Results: Group 1, 19% <strong>of</strong> patients improved an<br />
average <strong>of</strong> 1 point <strong>of</strong> original EDSS index, 65% remained stable<br />
and 16% deteriorated only 0.5 points. 83% developed local<br />
aseptic abscesses at immunization site that took 1–6monthsto<br />
be solved. Group 2, 50% improved 2 points; 29% improved 1 point<br />
and 21% remained stable. No abscesses on the 24 patients but all<br />
developed fever (39.5 C) during first 24 hours after immunization.<br />
Group 3, 69% improved 2 points, 25%, 1 point and 6%<br />
remained stable. No fever nor abscesses observed. Conclusion:<br />
IV TCV-CD4 treatment is safe and shows therapeutic efficacy for<br />
CPMS. The spleen seems to be where antiergotype regulator<br />
cells are produced.<br />
doi:10.1016/j.clim.2007.03.059<br />
Su.20 Ingested (<strong>Oral</strong>) Soluble Immune Response<br />
Suppressor (SIRS) Peptide 1−21 Inhibits Acute EAE<br />
Staley Brod, Pr<strong>of</strong>essor, University <strong>of</strong> Texas Houston, MSRG,<br />
Houston, TX, Zach Hood, Technician, University <strong>of</strong> Texas<br />
Houston, MSRG, Houston, TX
Abstracts<br />
Background: Ingested type I IFN inhibits clinical attacks,<br />
relapses and inflammation in murine EAE. Type I IFN activate<br />
human suppressor T cells that produce SIRS. Objectives: We<br />
examined whether SIRS peptide would have similar effects to<br />
ingested IFN-α in EAE. Methods: C57BL/6 mice were actively<br />
immunized with myelin oligodendroglial (MOG) peptide 35–<br />
55 (MEVGWYRSPFSRVVHLYRNGK) in IFA and followed for<br />
disease. <strong>Clinical</strong> severity was graded daily. For parenteral<br />
treatment, B6 mice were injected with control saline<br />
(mock), 0.1, 1, or 10 mg SIRS peptide 1–21 (NH–MET–Thr–<br />
Glu–Glu–Asn–Gln–Gln–Ser–Ser–Gln–Pro–Lys–Thr–Thr–Ile–<br />
Asn–Asn–Ala–Gly–Asp–Ser–CysOH). For oral treatment, B6<br />
mice were fed with 0.1 ml <strong>of</strong> saline (mock) or 1, 10 or 100 mg<br />
SIRS peptide 1– 21 daily. Following sacrifice, spinal cords<br />
were evaluated for foci <strong>of</strong> inflammation. Splenocytes from<br />
grouped saline (mock) fed or 100 mcg SIRS peptide fed mice<br />
were stimulated with Con A and examined using mouse<br />
cytokine inflammatory antibody array. Results: S.C. 10 mcg<br />
SIRS peptide 1–21 showed significant inhibition <strong>of</strong> EAE<br />
(pb0.001). Ingested SIRS peptide at 10 and 100 mcg SIRS<br />
peptide inhibited EAE compared to placebo (pb0.001).<br />
There were significantly less inflammatory foci in the SIRS<br />
peptide fed group compared to the control mock saline group<br />
(pb0.03). Splenocytes from SIRS peptide 1–21 fed mice<br />
showed increased production <strong>of</strong> CD30L, IL-6, IL-13, I-TAC,<br />
TCA-3/CCL1, TNF-α. Conclusion: Ingested SIRS peptide<br />
inhibits both clinical EAE and inflammation via counterregulatory<br />
type 2-like cytokines and chemokines IL-13,<br />
CD30L and TCA-3.<br />
doi:10.1016/j.clim.2007.03.060<br />
Su.21 Identifying New Immunodominant Myelin<br />
Peptides in Relapsing Remitting Multiple Sclerosis<br />
Patients<br />
Brian Newsom, Senior Director <strong>of</strong> Project Development,<br />
Opexa Therapeutics, Inc. The Woodlands, TX, Kathrin von<br />
Gynz Rekowski, Research Scientist, Opexa Therapeutics,<br />
Inc. The Woodlands, TX, Mitzi Montgomery, Consultant,<br />
Opexa Therapeutics, Inc. The Woodlands, TX, Jim Williams,<br />
Chief Operating Officer, Opexa Therapeutics, The<br />
Woodlands, TX, Edward Fox, <strong>Clinical</strong> Assistant Pr<strong>of</strong>essor,<br />
University <strong>of</strong> Texas Medical Branch, MS Clinic <strong>of</strong> Central<br />
Texas, Round Rock, TX<br />
T cell reactivity to peptides found to be instrumental in<br />
Multiple Sclerosis (MS) pathology and experimental animal<br />
models has centered on the major myelin proteins; Myelin<br />
Basic Protein (MBP), Myelin Proteolipid Protein (PLP), and<br />
Myelin Oligodendrocyte Glycoprotein (MOG). With recent<br />
structural studies on these proteins and discoveries <strong>of</strong><br />
additional myelin proteins, there are questions about<br />
potential interactions between these proteins and T cells in<br />
MS. Using synthetic peptides <strong>of</strong> 16 amino acid residues and<br />
overlaps <strong>of</strong> 12 residues, we have surveyed the resulting 163<br />
peptides from these three proteins for their proliferative<br />
reactivity in 16 healthy subjects and 63 MS patients,<br />
including 22 from our Phase I trials <strong>of</strong> the T cell vaccine<br />
Tovaxin. Using a Tcell Epitope Analysis Assay (EAA), peptides<br />
were screened using peripheral blood mononuclear cells and<br />
a tritiated thymidine incorporation assay. The results from<br />
this assay generated a pattern <strong>of</strong> individual, patient specific<br />
reactivity by calculating the stimulation index <strong>of</strong> T cells in<br />
the presence <strong>of</strong> peptide antigens when compared to media<br />
controls. Each assay also included controls <strong>of</strong> non-specific or<br />
superantigenic stimuli, such as phytohemagglutin (PHA). The<br />
results <strong>of</strong> 173 assays have allowed us to identify several<br />
peptides as being immunodominant, including some never<br />
before reported, and others as being non-reactive. The EAA<br />
can be utilized to screen additional peptides or combinations<br />
that have biological significance. The results have permitted<br />
us to reduce the number <strong>of</strong> peptides to 109, in the screening<br />
assay for Tovaxin vaccine production, to qualify subjects for<br />
our current Phase IIb clinical trial.<br />
doi:10.1016/j.clim.2007.03.061<br />
Su.22 Differential Gene Expression Identifies Novel<br />
Markers <strong>of</strong> Myelin-Specific Murine Memory CD4+<br />
T Cells<br />
Wassim Elyaman, Research Fellow, Brigham and Women’s<br />
Hospital-Harvard, Boston, MA, Pia Kivisakk, Research<br />
Fellow, Brigham and Women's Hospital, Boston, MA, Jaime<br />
Imitola, Instructor, Brigham and Women’s Hospital, Boston,<br />
MA, Samia Khoury, Associate Pr<strong>of</strong>essor, Brigham and<br />
Women’s Hospital -Harvard, Boston, MA, Mohamed Sayegh,<br />
Pr<strong>of</strong>essor, Brigham and Women's Hospital -Children's<br />
Hospital-Harvard, Boston, MA<br />
Memory CD4+ T cells function as a dynamic repository <strong>of</strong><br />
antigen-experienced T lymphocytes that tend to resist<br />
immunomodulatory and tolerance strategies. Their activation<br />
in response to a secondary antigenic stimulation involves<br />
changes in the expression level <strong>of</strong> a large number <strong>of</strong> genes.<br />
Recently, we have shown that myelin specific CD4 memory<br />
cells express high levels <strong>of</strong> ICOS and lower amounts <strong>of</strong> CD28 in<br />
comparison to effector T cells. This was associated with<br />
differential response to costimulation blockade in a mouse<br />
model <strong>of</strong> autoimmune encephalomyelitis. Here, we have<br />
used cDNA array technology to characterize the differences<br />
in gene expression between murine myelin-specific memory<br />
and effector CD4+ T cells. To generate myelin-specific<br />
memory CD4+ T cells, MOG-TCR transgenic mice were<br />
immunized with MOG/CFA for 2 weeks and CD4+ T cells<br />
were parked for 3 months in Wild-type naïve recipients.<br />
Effector CD4+ T cells were generated by immunizing MOG<br />
transgenic mice for 2 weeks. The gene expression pr<strong>of</strong>iles <strong>of</strong><br />
mouse donors <strong>of</strong> CD4+ T cells <strong>of</strong> each group (n=3) were<br />
analyzed. 204 genes were consistently differentially<br />
expressed (3 fold change) between memory and effector T<br />
cells, which included 23 genes that are only expressed in<br />
CD4+ memory cells. These differentially expressed genes<br />
include a combination <strong>of</strong> well-known, previously characterized<br />
genes with a range <strong>of</strong> biological functions and unknown<br />
in silico predicted hypothetical genes. These observed<br />
differences in the gene expression pr<strong>of</strong>ile <strong>of</strong> autoantigen<br />
memory and effector CD4+ T cells have important clinical<br />
implications for designing new therapies for autoimmune<br />
diseases in humans.<br />
doi:10.1016/j.clim.2007.03.063<br />
S149
S150 Abstracts<br />
Su.23 Reduced Number <strong>of</strong> Blood Circulating Foxp3+<br />
CD25highCD4+ Regulatory T Cells and a Decreased<br />
Foxp3 Expression at the Single-cell Level in Patients<br />
with Relapsing-remitting Multiple Sclerosis<br />
Koen Venken, MSc, Hasselt University, Biomedisch<br />
Onderzoeksinstituut, Diepenbeek, Belgium, Niels Hellings,<br />
PhD, Hasselt University, Biomedisch Onderzoeksinstituut,<br />
Diepenbeek, Belgium, Veerle Somers, PhD, Hasselt<br />
University, Biomedisch Onderzoeksinstituut, Diepenbeek,<br />
Belgium, Pieter Meuwissen, Student, Hasselt University,<br />
Biomedisch Onderzoeksinstituut, Diepenbeek, Belgium,<br />
Marielle Thewissen, MSc, Hasselt University, Biomedisch<br />
Onderzoeksinstituut, Diepenbeek, Belgium, Karen Hensen,<br />
PhD, <strong>Clinical</strong> Laboratory <strong>of</strong> Experimental Hematology, Virga<br />
Jesse Hospital, Hasselt, Belgium, Jean-Luc Rummens, MD,<br />
<strong>Clinical</strong> Laboratory <strong>of</strong> Experimental Hematology, Virga<br />
Jesse Hospital, Hasselt, Belgium, Piet Stinissen, PhD,<br />
Hasselt University, Biomedisch Onderzoeksinstituut,<br />
Diepenbeek, Belgium<br />
CD4+CD25high regulatory T cells (Tregs) <strong>of</strong> patients with<br />
relapsing–remitting (RR), but not secondary-progressive (SP)<br />
multiple sclerosis (MS), show a reduced suppressive function<br />
and FoxP3 mRNA expression. In this study we analyzed FoxP3<br />
protein expression by means <strong>of</strong> flow cytometry in PBMC <strong>of</strong> 55<br />
RR-MS, 15 SP-MS patients and 40 healthy controls (HC). RR-MS<br />
patients displayed a significant reduced number <strong>of</strong> CD4+<br />
CD25highFoxp3+ T cells as compared to HC and SP-MS. In<br />
addition, a significant lower Foxp3 expression per cell was<br />
detected in RR-MS patients. Interestingly, IFN-β treated RR-<br />
MS patients (n=15) showed a higher frequency <strong>of</strong> CD4+ CD25<br />
highFoxp3+ cells as compared to untreated RR-MS patients<br />
(n=40). Furthermore, a correlation was observed between<br />
Foxp3 levels and suppressive capacity <strong>of</strong> Tregs as measured in<br />
Treg-Tresponder coculture experiments. We further phenotypically<br />
analyzed Tregs <strong>of</strong> HC and untreated MS patients by<br />
measuring the expression <strong>of</strong> adhesion molecules by flow<br />
cytometry. Whereas no differences were detected in percentage<br />
<strong>of</strong> L-selectin+ (CD62L) and hyaluronate receptor+ (CD44)<br />
cells, a significant higher percentage <strong>of</strong> integrin αE+ (CD103)<br />
and α4+ (CD49d, VLA-4) cells was observed within CD4+<br />
CD25high Foxp3+ Tregs <strong>of</strong> RR-MS as compared to HC and SP-<br />
MS. Taken together, a lower number <strong>of</strong> CD4+ CD25highFoxp3+<br />
T cells and a reduced Foxp3 expression level was detected in<br />
RR-MS patients further demonstrating Treg dysfunction in<br />
these patients. The detection <strong>of</strong> a higher percentage <strong>of</strong><br />
CD103+ and VLA-4+ in the Treg population in RR-MS patients<br />
could reflect an increased migration capacity <strong>of</strong> Tregs<br />
towards inflammatory lesions in the central nervous system.<br />
doi:10.1016/j.clim.2007.03.064<br />
Su.24 Upregulation <strong>of</strong> Water Channel Aquaporin-4<br />
in Experimental Autoimmune Encephalomyeritis<br />
Katsuichi Miyamoto, MD, PhD, Kinki University School <strong>of</strong><br />
Medicine, Osaka-sayama, Japan, Kazuo Kataoka, MD, PhD,<br />
Department <strong>of</strong> Neurosurgery, Kinki University School <strong>of</strong><br />
Medicine, Osaka-sayama, Japan, Susumu Kusunoki, MD,<br />
PhD, Department <strong>of</strong> Neurology, Kinki University School <strong>of</strong><br />
Medicine, Osaka-sayama, Japan<br />
Aquaporin-4 (AQP4) is a water channel protein, which plays<br />
an important role in water movement in the central nervous<br />
system. Involvement <strong>of</strong> AQP4 in the pathogenesis <strong>of</strong> brain edema<br />
in the acute ischemic brain model was reported. Recently<br />
presence <strong>of</strong> the antibody against AQP4 has been reported in the<br />
patients affected with neuromyelitis optica (NMO) or opticospinal<br />
type multiple sclerosis (MS). AQP4 therefore may be<br />
involved in the pathophysiological mechanism <strong>of</strong> inflammatory<br />
demyelinating disorder. Here we analyzed AQP4 expression in<br />
the central nervous system <strong>of</strong> experimental autoimmune<br />
encephalomyelitis (EAE), an animal model <strong>of</strong> MS. We performed<br />
semi-quantitative analysis <strong>of</strong> AQP4-mRNA in brain and spinal<br />
cord from EAE-induced mice by using real-time PCR. Furthermore<br />
immuno-histochemical analysis was performed. The brain<br />
and spinal cord from myelin oligodendrocyte glycoprotein<br />
(MOG)-induced EAE mice were removed on 7, 14, 21, 28 and<br />
35 days after first inoculation. The increased levels <strong>of</strong> AQP4mRNA<br />
expression were seen; that in the spinal cord reached the<br />
peak on 14 days, and that in the brain on 21 days after first<br />
inoculation, respectively. The histopathological analysis showed<br />
that demyelination and cell infiltration were most severe on<br />
21 days after first inoculation, when the clinical manifestations<br />
also were most severe. This is the first study to show the<br />
upregulation <strong>of</strong> AQP4 in the inflammatory demyelinating disease<br />
<strong>of</strong> CNS. Further investigation is necessary to investigate the<br />
possibility that anti-AQP4 antibody binds to the upregulated<br />
AQP4 within CNS and is directly involved in the pathogenesis <strong>of</strong><br />
NMO or optico-spinal type MS.<br />
doi:10.1016/j.clim.2007.03.065<br />
Su.26 Role <strong>of</strong> IFN-γ in Virus-induced Demyelinating<br />
Disease<br />
Julie Olson, Assistant Pr<strong>of</strong>essor, Department <strong>of</strong> Neurological<br />
Surgery, University <strong>of</strong> Wisconsin-Madison, Madison, WI<br />
Multiple sclerosis is a demyelinating disease in humans<br />
which has been associated with an inflammatory component.<br />
Epidemiological evidence has suggested that a virus infection<br />
may be involved in disease initiation and disease exacerbation.<br />
Theiler's murine encephalomyelitis virus (TMEV) serves as a<br />
mouse model <strong>of</strong> MS. Susceptible mice (SJL strain) infected with<br />
TMEV, BeAn strain, develop demyelinating disease beginning<br />
with clinical signs <strong>of</strong> disease around 35 days post infection.<br />
TMEV induces a chronic progressive demyelinating disease<br />
associated with a myelin-specific CD4+ Tcell response which is<br />
a Th1 type Tcell response. However, some strains <strong>of</strong> mice, such<br />
as C57BL6, are resistant to development <strong>of</strong> TMEV- induced<br />
demyelinating disease. IFN-γ is a proinflammatory cytokine<br />
associated with demyelinating disease and the myelin- specific<br />
autoimmune response. In these studies, the role <strong>of</strong> IFN-γ was<br />
examined during the initiation and progression <strong>of</strong> demyelinating<br />
disease using resistant C57BL6 mice deficient in IFN-γ.<br />
Interestingly, the IFN-γ deficient mice infected with TMEV<br />
developed demyelinating disease with clinical disease similar<br />
to the susceptible (SJL) mice. TMEV- infected IFN γ deficient<br />
mice developed a virus- specific CD4+ T cell response and a<br />
myelin- specific T cell response late during demyelinating<br />
disease. The demyelinating disease was associated with<br />
infiltration <strong>of</strong> immune cells into the CNS, most interestingly,<br />
CD4+ T cells and activated macrophage. These results suggest
Abstracts<br />
that IFN-γ was protective against development <strong>of</strong> virusinduced<br />
demyelinating disease and that IFN-γ was not required<br />
for development <strong>of</strong> autoimmune demyelinating disease. This<br />
research was supported by the National Multiple Sclerosis<br />
Society RG3625.<br />
doi:10.1016/j.clim.2007.03.066<br />
Su.27 Large Scale Immunopr<strong>of</strong>iling Uncovers<br />
Population Structure in Untreated MS Subjects and<br />
Highlights the Role <strong>of</strong> NK Cells<br />
Elizabeth Rossin, Data Analyst, The Broad Institute <strong>of</strong> MIT<br />
and Harvard, The Program in Medical and Population<br />
Genetics, Cambridge, MA, Vissia Viglietta, Instructer,<br />
Brigham and Women’s Hospital, Department <strong>of</strong> Neurology,<br />
Boston, MA, Dulce Soler-Ferran, Research Scientist,<br />
Millenium Pharmaceuticals, Cambridge, MA, Alex Parker,<br />
Research Scientist, Millenium Pharmaceuticals, Cambridge,<br />
MA, Mira Weiner, Project Manager, Brigham and Women’s<br />
Hospital, Department <strong>of</strong> Neurology, Boston, MA, Guy<br />
Buckle, Associate Neurologist, Department <strong>of</strong> Neurology,<br />
Brigham and Women's Hospital, Brookline, MA, Samia<br />
Khoury, Associate Pr<strong>of</strong>essor, Brigham and Women’s Hospital,<br />
Department <strong>of</strong> Neurology, Boston, MA, David Hafler,<br />
Pr<strong>of</strong>essor, Brigham and Women’s Hospital, Department <strong>of</strong><br />
Neurology, Boston, MA, Howard Weiner, Pr<strong>of</strong>essor, Brigham<br />
and Women’s Hospital, Department <strong>of</strong> Neurology, Boston,<br />
MA, Philip De Jager, Assistant Pr<strong>of</strong>essor, Brigham and<br />
Women’s Hospital, Department <strong>of</strong> Neurology, Boston, MA<br />
As part <strong>of</strong> the Multiple Sclerosis Registry, we acquired an<br />
immunological pr<strong>of</strong>ile from healthy subjects and subjects<br />
with remitting relapsing multiple sclerosis (RRMS) who were<br />
untreated. Fresh blood from each subject was screened ex<br />
vivo using a panel <strong>of</strong> 50 fluorescently labeled monoclonal<br />
antibodies (Ab) distributed amongst 56 pools; flow cytometry<br />
was performed to quantitate the binding <strong>of</strong> each Ab.<br />
Longitudinal analysis <strong>of</strong> 15 healthy control subjects identified<br />
the “% positive” parameters as the most robust to<br />
fluctuation over time; after further quality control processing,<br />
1018 individual “% positive” parameters or “features”<br />
were selected for the analysis. The exploratory screen<br />
consisted <strong>of</strong> 17 healthy control subjects and 23 untreated<br />
RRMS subjects. It was followed by an extension analysis in<br />
which 15 additional healthy controls and 15 additional RRMS<br />
subjects were added to the dataset. This analysis identified a<br />
CD8lowCD56+CD3− population (NK cells) as being reduced in<br />
frequency in untreated RRMS subjects (P=10–4). In analyzing<br />
the 38 cases by themselves, clustering analysis revealed that<br />
there were 3 populations <strong>of</strong> RRMS subjects with different<br />
immunological pr<strong>of</strong>iles and that disease duration (range 3–<br />
42 years) might explain some <strong>of</strong> this population structure<br />
(P=0.03). In prediction analyses, our Ab panel was unable to<br />
accurately predict control and RRMS subjects but was able to<br />
accurately segregate 11 cases <strong>of</strong> clinically isolated syndrome<br />
from controls. These data suggest that disease duration is an<br />
important factor to consider in the analysis and future<br />
development <strong>of</strong> immunological pr<strong>of</strong>iling in MS.<br />
doi:10.1016/j.clim.2007.03.068<br />
Su.28 Treatment with a Fullerene Derivative<br />
(ABS-75) Reduces Disease Progression and Axonal<br />
Loss in MOG-induced Progressive EAE in NOD Mice<br />
Alexandre S. Basso, Research Fellow, Center for Neurologic<br />
Disease, Harvard Medical School, Boston, MA, Dan Frenkel,<br />
Research Fellow, Center for Neurologic Diseases, Harvard<br />
Medical School, Boston, MA, Sanja Petrovic-Stojkovic,<br />
Research Assistant, Center for Neurologic Diseases, Harvard<br />
Medical School, Boston, MA, Alon Monsonego, Research<br />
Fellow, Center for Neurologic Diseases, Harvard Medical<br />
School, Boston, MA, Lindsay Puckett, Research Assistant,<br />
Center for Neurologic Diseases, Harvard Medical School,<br />
Boston, MA, Michael Gozin, Pr<strong>of</strong>essor, School <strong>of</strong> Chemistry,<br />
Faculty <strong>of</strong> Exact Sciences, Tel Aviv University, Tel Aviv,<br />
Israel, Howard Weiner, Robert L. Kroc Pr<strong>of</strong>essor <strong>of</strong><br />
Neurology, Center for Neurologic Diseases, Harvard Medical<br />
School, Boston, MA<br />
Inflammation-induced oxidative stress can lead to axonal<br />
degeneration, which is felt to be a major determinant <strong>of</strong><br />
progressive neurological disability in Multiple Sclerosis (MS).<br />
Water-soluble derivatives <strong>of</strong> fullerenes are a unique class <strong>of</strong><br />
allotropic form <strong>of</strong> carbon compounds with potent antioxidant<br />
properties. 10 week old mice were immunized with 150 μg<strong>of</strong><br />
MOG 35–55 peptide in CFA followed by pertussis toxin.<br />
Disease is characterized by an attack followed by a<br />
progressive phase with chronic clinical impairment. Following<br />
the first attack, animals were distributed into different<br />
groups with similar disease courses and treated intraperitoneally<br />
either with 200 μl<strong>of</strong>a1μM fullerene solution or vehicle<br />
every day until termination <strong>of</strong> the experiment (day 63). We<br />
tested three different fullerene derivatives (ABS-75, ABS-16,<br />
and the C60 fullerene core) and found that ABS-75 treatment<br />
initiated after disease onset reduced the clinical progression<br />
<strong>of</strong> chronic EAE in NOD mice (treated vs. control, pb0.05).<br />
Fullerene ABS-75 consists <strong>of</strong> a C60 carbon fullerene core to<br />
which a known NMDA receptor ligand was attached. ABS-75<br />
treatment also reduced axonal loss, demyelination (as<br />
evaluated by silver and Luxol fast blue staining, respectively),<br />
and CD11b+ infiltration in the white matter <strong>of</strong> mice.<br />
In vitro, ABS-75 was able to protect neurons from glutamateinduced<br />
toxicity and to reverse reduced glutamine synthetase<br />
expression in astrocytes under inflammatory insult. Our<br />
data demonstrate a neuroprotective effect <strong>of</strong> a treatment<br />
with a fullerene compound combined with a NMDA receptor<br />
ligand that may have applicability in the treatment <strong>of</strong><br />
progressive MS and other neurodegenerative diseases.<br />
doi:10.1016/j.clim.2007.03.069<br />
S151<br />
Su.29 Human Blood−brain Barrier-associated DCs<br />
Originate from Blood Monocytes and Polarize CD4+<br />
Lymphocytes into Th17 or Th1<br />
Igal Ifergan, PhD Student, CHUM-Hopital Notre-Dame,<br />
Neuroimmunology Department, Montreal, QC, Canada,<br />
Hania, Kebir, PhD student, CHUM-Hopital Notre-Dame,<br />
Neuroimmunology Department, Montreal, QC, Canada,<br />
Nathalie Arbour, PhD, CHUM-Hopital Notre-Dame,<br />
Neuroimmunology Department, Montreal, QC, Canada,<br />
Alexandre Prat, MD, PhD, CHUM-Hopital Notre-Dame,<br />
Neuroimmunology Department, Montreal, QC, Canada
S152 Abstracts<br />
Trafficking <strong>of</strong> antigen presenting cells (APC) into the<br />
central nervous system (CNS) is essential for lymphocyte<br />
reactivation within the CNS compartment. The origin and<br />
function <strong>of</strong> the APC found in CNS autoimmune or inflammatory<br />
lesions remain, however, controversial. We demonstrate<br />
that blood CD14+ monocytes migrate across the<br />
inflammed blood–brain barrier (BBB) and differentiate into<br />
CD209+CD83+ (myeloid) or CD123+ (plasmacytoid) dendritic<br />
cells (DCs) under the influence <strong>of</strong> CNS endothelial-secreted<br />
transforming growth factor-beta (TGF-β) and granulocyte<br />
macrophage colony stimulating factor (GM-CSF). We also<br />
demonstrate that while CD123+ DCs secrete interleukin-12<br />
p70 (IL-12p70) and promote the proliferation and expansion<br />
<strong>of</strong> interferon-γ-secreting Th1 CD4+ lymphocytes, CD209+<br />
CD83+ DCs secrete high levels <strong>of</strong> TGF-β and IL-6 and skew<br />
CD4+ lymphocytes towards a Th17 phenotype. Using multiple<br />
sclerosis as a prototypical human CNS autoimmune<br />
disease, we confirmed by immunohistochemistry the abundance<br />
<strong>of</strong> such perivascular CD83+ and CD123+ DCs within<br />
acute lesions, closely associated with microvascular BBBendothelial<br />
cells. Our data support the notion that<br />
functional perivascular myeloid and plasmacytoid CNS DCs<br />
arise as a consequence <strong>of</strong> migration <strong>of</strong> blood CD14+<br />
monocytes across the human BBB, through the concerted<br />
actions <strong>of</strong> TGF-β and GM-CSF.<br />
doi:10.1016/j.clim.2007.03.071<br />
Su.31 Characterization <strong>of</strong> Single B Cell<br />
Immunoglobulin Variable Region Genes Derived<br />
from Infiltrated Muscle Tissue <strong>of</strong> Subjects with<br />
Inflammatory Myopathies<br />
Elizabeth Bradshaw, Postdoctoral Fellow, Laboratory <strong>of</strong><br />
Molecular <strong>Immunology</strong>, Center for Neurologic Diseases,<br />
Brigham and Women’s Hospital, Boston, MA, Ana Orihuela,<br />
Research Technician, Children’s Hospital Informatics<br />
Program at the Harvard-MIT Division <strong>of</strong> Health Sciences and<br />
Technology, Boston, MA, Shannon McArdel, Research<br />
Technician, Laboratory <strong>of</strong> Molecular <strong>Immunology</strong>, Center for<br />
Neurologic Diseases, Brigham and Women’s Hospital,<br />
Boston, MA, David Hafler, Pr<strong>of</strong>essor, Laboratory <strong>of</strong><br />
Molecular <strong>Immunology</strong>, Center for Neurologic Diseases,<br />
Brigham and Women’s Hospital, Boston, MA, Anthony<br />
Amato, Associate Pr<strong>of</strong>essor, Department <strong>of</strong> Neurology,<br />
Division <strong>of</strong> Neuromuscular Disease, Brigham and Women’s<br />
Hospital and Harvard, Boston, MA, Kevin O’Connor,<br />
Assistant Pr<strong>of</strong>essor, Laboratory <strong>of</strong> Molecular <strong>Immunology</strong>,<br />
Center for Neurologic Diseases, Brigham and Women's<br />
Hospital, Boston, MA<br />
The inflammatory myopathies (IM) are putative autoimmune<br />
disorders characterized by muscle weakness and the<br />
presence <strong>of</strong> inflammatory infiltrates in skeletal muscle.<br />
Varying numbers <strong>of</strong> both B cells and plasma cells are among<br />
the muscle tissue infiltrate found in these diseases. Through<br />
examination <strong>of</strong> immunoglobulin heavy chain variable regions,<br />
we previously demonstrated that there was significant<br />
oligoclonal expansion <strong>of</strong> B cells found in IM muscle. To extend<br />
these findings, we isolated single B cells from IM muscle by<br />
laser capture microdissection or FACS, then examined the<br />
paired immunoglobulin heavy and light chain variable region<br />
sequences. B cell immunoglobulin variable region cDNAs were<br />
sequenced affording identification <strong>of</strong> the isotype, CDR3 and<br />
the degree <strong>of</strong> somatic mutation in the entire V-region. A<br />
pattern <strong>of</strong> B cell affinity maturation was evident in clones<br />
that had accumulated varying numbers <strong>of</strong> common and<br />
unique somatic mutations. These data suggested that a<br />
local inflammatory response occurs within the muscle tissue<br />
<strong>of</strong> patients with IM that may indicate the presence <strong>of</strong> a B cell<br />
antigen-specific response.<br />
doi:10.1016/j.clim.2007.03.072<br />
Su.32 Myelin-specific Regulatory T Cells Accumulate<br />
in the Central Nervous System, but Fail to Suppress<br />
Pathogenic Effector T Cells at the Peak <strong>of</strong><br />
Autoimmune Inflammation<br />
Thomas Korn, Postdoctoral Fellow, Harvard Medical School,<br />
Center for Neurologic Diseases, Boston, MA, Mohamed<br />
Oukka, Instructor, Harvard Medical School, Center for<br />
Neurologic Diseases, Boston, MA, Jay, Reddy, Instructor,<br />
Harvard Medical School, Center for Neurologic Diseases,<br />
Boston, MA, Estelle Bettelli, Instructor, Harvard Medical<br />
School, Center for Neurologic Diseases, Boston, MA, Amit<br />
Awasthi, Postdoctoral Fellow, Harvard Medical School,<br />
Center for Neurologic Diseases, Boston, MA, Raymond Sobel,<br />
Associate Pr<strong>of</strong>essor <strong>of</strong> Pathology, Stanford University,<br />
Department <strong>of</strong> Pathology, Stanford, CA, Kai Wucherpfennig,<br />
Associate Pr<strong>of</strong>essor <strong>of</strong> Neurology, Harvard Medical School,<br />
Dana Farber Cancer Institute, Department <strong>of</strong> Cancer<br />
<strong>Immunology</strong> and AIDS, Boston, MA, Vijay Kuchroo, Pr<strong>of</strong>essor<br />
<strong>of</strong> Neurology, Harvard Medical School, Center for Neurologic<br />
Diseases, Boston, MA<br />
Treatment with ex vivo generated regulatory T cells (Treg)<br />
has been regarded as highly attractive therapeutic<br />
approach for autoimmune diseases. However, the dynamics<br />
and function <strong>of</strong> T-reg in autoimmunity are not well understood.<br />
Thus, we developed Foxp3gfp “knock-in” mice and<br />
myelin oligodendrocyte glycoprotein (MOG)35–55/IAb tetramers<br />
to track autoantigen-specific effector T cells (T-eff)<br />
and T-reg in vivo during experimental autoimmune encephalomyelitis,<br />
an animal model for Multiple Sclerosis.<br />
Following immunization with the encephalitogenic peptide<br />
MOG35–55 emulsified in complete Freund's adjuvant,<br />
MOG35–55-tetramer-reactive, Foxp3+ T-reg expanded in<br />
the peripheral lymphoid compartment and readily accumulated<br />
in the central nervous system (CNS), but did not<br />
prevent the onset <strong>of</strong> disease. During disease onset, the MOGtetramer+<br />
T-eff population in the CNS increased faster than<br />
the population <strong>of</strong> antigen-specific T-reg. At the peak <strong>of</strong><br />
disease, the ratio <strong>of</strong> T-reg vs. T-eff was 1:17 which dramatically<br />
changed into 1:2 at the beginning <strong>of</strong> recovery.<br />
Foxp3+ T-reg isolated from the CNS were fully competent in<br />
suppressing naive MOG-specific T cells. However, Foxp3+ Treg<br />
failed to control encephalitogenic T-eff which in contrast<br />
to T-eff from the peripheral immune compartment, secreted<br />
IL-6 and TNF when they were isolated from the CNS at the<br />
peak <strong>of</strong> disease. Our data suggest that in the face <strong>of</strong> inflammation,<br />
the regulation <strong>of</strong> autoimmunity by CD4+Foxp3+<br />
T-reg in situ may not be accomplished simply by changing<br />
the numerical balance <strong>of</strong> antigen-specific pathogenic vs.
Abstracts<br />
regulatory T cells, but may require the control <strong>of</strong> tissue<br />
inflammation as well.<br />
doi:10.1016/j.clim.2007.03.073<br />
Su.34 Proteomic Pr<strong>of</strong>iling <strong>of</strong> Multiple Sclerosis<br />
Lesions Identify Potential Targets for Therapy<br />
May H. Han, Postdoctoral Fellow, Department <strong>of</strong> Neurology<br />
and Neurological Sciences Stanford University, Stanford,<br />
CA, Sunil Hwang, Postdoctoral Fellow, Department <strong>of</strong><br />
Physiology, University <strong>of</strong> Conneticut Health Center,<br />
Farmington, CT, Dolly Roy, Neurologist, Department <strong>of</strong><br />
Neurology and Neurological Sciences, Stanford University,<br />
Stanford, CA, Cedric Raine, Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Pathology, Albert Einstein College <strong>of</strong> Medicine, Bronx, NY,<br />
William H. Robinson, Assistant Pr<strong>of</strong>essor, Departments <strong>of</strong><br />
Medicine and <strong>Immunology</strong>, Stanford University, Stanford,<br />
CA, Raymond A. Sobel, Pr<strong>of</strong>essor <strong>of</strong> Pathology, Department<br />
<strong>of</strong> Pathology, Stanford University, Stanford, CA, David Han,<br />
Associate Pr<strong>of</strong>essor, Department <strong>of</strong> Physiology, University <strong>of</strong><br />
Conneticut Health Center, Farmington, CT, Lawrence<br />
Steinman, Pr<strong>of</strong>essor, Department <strong>of</strong> Neurology and<br />
Neurological Sciences, Stanford, CA<br />
Demyelinating lesions or plaques in multiple sclerosis (MS)<br />
have distinct histological features that reflect their stage <strong>of</strong><br />
evolution. To identify key molecules involved at different stages<br />
during lesion progression, we performed proteomic pr<strong>of</strong>iling <strong>of</strong><br />
three types <strong>of</strong> MS plaques using mass spectrometry and bioinformatics.<br />
Fresh frozen autopsy brain samples from MS<br />
patients (n=6) and age-matched controls (n=2)wereclassified<br />
as 1) acute plaque (AP, characterized by presence <strong>of</strong> inflammatory<br />
cells, ongoing demyelination), 2) chronic active plaque<br />
(CAP, characterized by chronic demyelination, astrogliosis with<br />
active rim) or 3) chronic plaque (CP, characterized by lack <strong>of</strong><br />
inflammatory cells, dense astrogliosis). Samples from plaques<br />
were isolated by laser capture microdissection and global<br />
protein identification was carried out by SDS-PAGE, in-gel<br />
protease digestion, liquid chromatography and mass spectrometry.<br />
A total <strong>of</strong> 3894 proteins were identified out <strong>of</strong> which 2184<br />
proteins were unique to MS lesions. Then we identified proteins<br />
uniquetoeachtype<strong>of</strong>lesion(AP(N=172), CAP (N=248) and CP<br />
(N=252), and proteins common to all three types (N=89) using<br />
bio-informatics s<strong>of</strong>tware INTERSECT. Analysis by s<strong>of</strong>tware<br />
PROTEOME 3D identified proteins <strong>of</strong> coagulation cascade and<br />
neural regeneration which may be potential targets for therapy.<br />
These results provide the first and most comprehensive<br />
information on global protein expression in MS plaques, enhance<br />
our understanding <strong>of</strong> the molecular pathogenesis <strong>of</strong> MS lesions<br />
and identify potential key molecules involved in disease<br />
progression that <strong>of</strong>fer possibilities for therapeutic targets.<br />
doi:10.1016/j.clim.2007.03.075<br />
Su.35 A Genome-wide Screen for Genetic Variants<br />
Affecting the Expression <strong>of</strong> Immunologically<br />
Relevant Cell Surface Markers<br />
Philip De Jager, Assistant Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Neurology, Brigham and Women’s Hospital, Boston, MA,<br />
Roman Yelensky, Student, Medical and Population Genetics<br />
Program, Broad Institute, Cambridge, MA, Elizabeth Rossin,<br />
Data Analyst, Medical and Population Genetics Program,<br />
Broad Institute, Boston, MA, Sasha Bonadkar, Research<br />
Assistant, Medical and Population Genetics Program, Broad<br />
Institute, Cambridge, MA, Edwin Choy, Director, Sarcoma<br />
Research, Medical and Population Genetics Program, Broad<br />
Institute, Cambridge, MA, Mark Daly, Assistant Pr<strong>of</strong>essor,<br />
Medical and Population Genetics Program, Broad Institute,<br />
Cambridge, MA, David Altshuler, Associate Pr<strong>of</strong>essor,<br />
Medical and Population Genetics Program, Broad Institute,<br />
Cambridge, MA, David Hafler, Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Neurology, Brigham and Women's Hospital, Boston, MA<br />
While a few alleles affecting the expression level <strong>of</strong> immunologically<br />
relevant molecules have been identified and<br />
validated, our knowledge <strong>of</strong> human polymorphisms associated<br />
with differences in immune function remains very<br />
limited. We have therefore evaluated the use <strong>of</strong> the HapMap<br />
lymphoblastic cell lines (LCLs) as a platform to discover such<br />
polymorphisms on a large scale using a test panel <strong>of</strong> cell<br />
surface molecules that includes 15 cell-surface markers:<br />
CD19, CD20, CD21, CD40, CD58, CD80, CD86, CD95, CD227,<br />
HLA DQ, HLA DR, IgD, IgG, IgM, and IL6R. We then correlated<br />
the mean expression level <strong>of</strong> a particular molecule in each<br />
LCL with the over 2 million genotypes available in the<br />
HapMap database. Amongst several positive results, this<br />
analysis reveals that certain SNPs in the vicinity <strong>of</strong> the HLA<br />
DQA1 gene are correlated with both the level <strong>of</strong> expression <strong>of</strong><br />
HLA DQ on the LCL surface and the level <strong>of</strong> mRNA expression<br />
for HLA DQA1. One <strong>of</strong> these SNPs, rs9272346, is a null allele<br />
<strong>of</strong> HLA DQA1 and is associated to this immunophenotype at a<br />
level that exceeds our predetermined level <strong>of</strong> genome-wide<br />
significance (P b2.6×10 −8 ), validating our approach. In<br />
exploring the mRNA data further within the MHC class I and<br />
class II clusters, it is clear that several different polymorphisms<br />
exist that affect the level <strong>of</strong> expression <strong>of</strong> these<br />
molecules. We are currently assessing the role <strong>of</strong> these<br />
functional variants in the association <strong>of</strong> the MHC with MS<br />
susceptibility.<br />
doi:10.1016/j.clim.2007.03.076<br />
S153<br />
Su.36 Novel Computational Methods to Enhance the<br />
Analysis <strong>of</strong> Cytometric Immunophenotypes<br />
Philip De Jager, Assistant Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Neurology, Brigham and Women’s Hospital, Boston, MA,<br />
Saumyadipta Pyne, Postdoctoral Fellow, Cancer Program,<br />
Broad Institute, Cambridge, MA, Elizabeth Rossin, Data<br />
Analyst, Medical and Population Genetics Program, Broad<br />
Institute, Cambridge, MA, Jill Mesirov, Director, Cancer<br />
Program, Broad Institute, Cambridge, MA, Pablo Tamayo,<br />
Staff Scientist, Cancer Program, Broad Institute,<br />
Cambridge, MA, David Hafler, Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Neurology, Brigham and Women’s Hospital,<br />
Boston, MA<br />
The current method for analyzing raw flow cytometry<br />
information involves gating, the process <strong>of</strong> first visualizing<br />
cell-surface marker expression values for a population <strong>of</strong><br />
cells and then manually selecting distinct cell populations on
S154 Abstracts<br />
which to report statistics. Thus, manual processing is timeconsuming<br />
and is limited to 2-dimensional projections. Furthermore,<br />
these cell populations are described by statistics<br />
that fail to characterize their full distribution and shape. We<br />
propose two automated methods to improve these shortcomings:<br />
(1) histogram mean shape comparison: this method<br />
creates an n+1-dimensional histogram for n-dimensional<br />
data. Expression data is then parsed into n+1-dimensional<br />
bins, and individual bin values can be compared across<br />
categories <strong>of</strong> subjects or samples to discover cell populations<br />
that differ across sample categories. (2) Hierarchical mixture<br />
model: this method characterizes the expression values <strong>of</strong><br />
each cell population in n dimensions using a hierarchical<br />
mixture model. The mixture model clusters individual cells<br />
by assigning them to distributions with maximum likelihood.<br />
A mixture <strong>of</strong> distributions that make up a model thus<br />
characterizes the n dimensional data. This method allows<br />
the cells to exist in distinct distributions rather than be<br />
collapsed to a statistic, and the distribution <strong>of</strong> cells can then<br />
be compared across samples. Both methods have been<br />
implemented to characterize the expression <strong>of</strong> 15 molecules<br />
on the 269 HapMap lymphoblastic cell lines. We present a<br />
comparison <strong>of</strong> both methods against manually processed<br />
data to demonstrate the most robust pipeline with which to<br />
characterize cell population structure and calculate expression<br />
levels using cytometric data.<br />
doi:10.1016/j.clim.2007.03.077<br />
Su.37 Improving Elispot to Detect Antigen Specific<br />
T Cells<br />
Khadir Raddassi, Research Instructor, Brigham and Women’s<br />
Hospital, Neurology, Boston, MA, Kasia Bourcier, Scientist,<br />
Immune Tolerance Network, San Francisco, CA, Jose<br />
Estevam, Research Assistant, Brigham and Women's<br />
Hospital, Neurology, Boston, MA, Vicki, Seyfert-Margolis,<br />
Scientist, Immune Tolerance Network, San Francisco, CA,<br />
David Hafler, Lab Director, Brigham and Women’s Hospital,<br />
Neurology, Boston, MA<br />
A major interest <strong>of</strong> our Immune Tolerance Network assay<br />
group is to develop and validate assays to detect auto- and<br />
allo-antigen reactive cells. In collaboration with Dr. Peakman<br />
(Kings College, London) we have developed a modified<br />
elispot protocol. We tested the sensitivity <strong>of</strong> this improved<br />
elispot to detect myelin-reactive T cells in multiple sclerosis<br />
patients (MS) and healthy controls (HC). Fresh or cryopreserved<br />
PBMCs were exposed to myelin antigens or tetanus<br />
toxoid (TT). After 42 hours the cells were transferred to<br />
elispot plates for 16 hours; the elispot plates were developed<br />
to detect the frequency <strong>of</strong> IFNg secreting cells. Comparing<br />
side by side elispot and flow cytometry regarding IFNg<br />
production in response to TT showed similar results, but<br />
elispot was more sensitive at low doses stimulation. The<br />
detection <strong>of</strong> antigen reactive cells by elispot could be<br />
performed on fresh and cryopreserved PBMCs. We validated<br />
this method between different labs, the data were well<br />
correlated (r2=0.987). Using IL-7 enhanced the response to<br />
weak stimuli. Taking advantage <strong>of</strong> this improved elispot and<br />
IL-7, we examined the reactivity <strong>of</strong> PBMCs from 13 HC and 11<br />
MS. In the presence <strong>of</strong> IL-7 PBMCs from MS showed high<br />
reactivity to human MBP (30±5 spots for MS vs. 17±4 for HC)<br />
and to a pool <strong>of</strong> myelin peptides (46 ±8 spots for MS vs. 15±4<br />
for HC). This modified elispot would provide a quick method<br />
to monitor immune reaction in clinical research. The fast<br />
read out allows the cells to be recovered and used for further<br />
testing.<br />
doi:10.1016/j.clim.2007.03.078<br />
Su.38 TIM-3 is a Negative Regulator <strong>of</strong> Human Th1<br />
Cells<br />
David E. Anderson, PhD, Brigham and Women’s Hospital and<br />
Harvard Medical School, Boston, MA, Juhi Kuchroo, Womens<br />
Hospital and Harvard Medical School, Boston, MA, Nasim<br />
Kassam, Brigham and Women’s Hospital and Harvard<br />
Medical School, Boston, MA, David Hafler, Jack, Sadie and<br />
David Breakstone Pr<strong>of</strong>essor <strong>of</strong> Neurology (Neuroscience),<br />
Brigham and Womens Hospital and Harvard Medical School,<br />
Boston, MA<br />
The relevance and utility <strong>of</strong> the Th1/Th2 paradigm has<br />
been well established in murine models <strong>of</strong> a many human<br />
immune-mediated diseases. However, data on the role and<br />
regulation <strong>of</strong> Th1 and Th2 cells in modulating immune<br />
responses in humans is less clear. TIM-3 is a molecule<br />
selectively expressed on a subset <strong>of</strong> murine IFN- 3 -secreting<br />
Th1 cells but not Th2 cells, and regulates Th1 immunity and<br />
tolerance in vivo. To determine if TIM-3 similarly identifies<br />
and regulates Th1 cells in humans, we generated a panel <strong>of</strong><br />
monoclonal antibodies specific for human TIM-3. We report<br />
that TIM-3 is preferentially expressed on the surface <strong>of</strong> in<br />
vitro polarized human Th1 cells. TIM-3 is not readily<br />
apparent on the surface <strong>of</strong> CD4+ T cells examined ex vivo,<br />
but T cell activation rapidly induces cell surface expression<br />
<strong>of</strong> TIM-3 on a subset <strong>of</strong> IFN- 3 -secreting CD4+ T cells. More<br />
specifically, highest expression <strong>of</strong> TIM-3 is present on T cells<br />
secreting the greatest amounts <strong>of</strong> IFN- 3 . Functionally,<br />
human T cells secrete elevated levels <strong>of</strong> IFN- 3 when<br />
stimulated in the presence <strong>of</strong> TIM-3-specific antibodies,<br />
with no effect on other cytokines including TNF-α and IL-<br />
13. These results demonstrate that TIM-3 preferentially<br />
regulates IFN- 3 -secretion from human Th1 cells and may<br />
influence a variety <strong>of</strong> human diseases including cancer and<br />
autoimmunity.<br />
doi:10.1016/j.clim.2007.03.079<br />
Su.39 Rapamycin Inhibits Autocrine Signaling<br />
(IL-6, IL-10) in Viral Lymphomas<br />
Sang-Hoon Sin, Postdoctoral Research Associate, University<br />
<strong>of</strong> North Carolina Chapel Hill, Chapel Hill, NC, Debasmita<br />
Roy, Graduate Student, University <strong>of</strong> North Carolina Chapel<br />
Hill, Microbiology & <strong>Immunology</strong>, Chapel Hill, NC,<br />
Dhavalkumar Patel, Pr<strong>of</strong>essor, University <strong>of</strong> North Carolina,<br />
Chapel Hill, Chapel Hill, NC, Dirk Dittmer, Assistant<br />
Pr<strong>of</strong>essor, University <strong>of</strong> North Carolina, Chapel Hill,<br />
Microbiology & <strong>Immunology</strong>, Chapel Hill, NC<br />
The mTOR inhibitor, rapamycin (sirolimus) is known for its<br />
immunosuppressive activity and more recently has been
Abstracts<br />
subject <strong>of</strong> intense investigations as an anti-cancer agent.<br />
Here we show that a group <strong>of</strong> viral malignancies, which<br />
critically dependent in inflammatory cytokines (and thus<br />
have sometimes be described as viral inflammatory lesions)<br />
are susceptible to rapamycin. We found that rapamycin was<br />
efficacious against primary effusion lymphoma in culture and<br />
in a murine xenograft model; that rapamycin inhibited mTOR<br />
signaling and that this correlated with inhibition <strong>of</strong> IL-10, IL-<br />
6, IFNgamma, IP-10, and IL-12p40 secretion (as measured by<br />
Luminex and ELISA). Moreover, addition <strong>of</strong> exogenous IL-10 or<br />
IL-6 could be reversed the rapamycin growth arrest. This<br />
validates sirolimus as a new treatment option for viral<br />
malignancies that depend on inflammatory cytokines.<br />
doi:10.1016/j.clim.2007.03.080<br />
Su.40 The Transcription Factor GATA-3 Regulates<br />
IL-4 Responses in B cells<br />
Dee Aud, Research Scientist, Roche Palo Alto, Palo Alto, CA,<br />
Sung-Yun Pai, Instructor, Dana Farber Cancer Institute,<br />
Boston, MA, Stanford Peng, Director, Arthritis Research,<br />
Roche Palo Alto, Palo Alto, CA, I-Cheng Ho, Associate<br />
Pr<strong>of</strong>essor, Brigham and Women's Hospital, Boston, MA<br />
The GATA-3 transcription factor plays a central role in T<br />
helper type 2 development, but its role in B cells has not been<br />
well described, even though GATA-3 is present in naïve B cells.<br />
To study the role <strong>of</strong> GATA-3 in B cells, mice with a floxed Gata3<br />
allele (Gata3fl/fl) mice were crossed to CD19-Cre transgenic<br />
mice, allowing B cell-specific deletion <strong>of</strong> GATA-3. B cell<br />
development was grossly normal in Gata3fl/fl CD19-Cre, but<br />
surprisingly, Gata3fl/fl CD19-Cre B cells showed a decreased<br />
ability to proliferate (in an IL-4 dose dependent manner) but<br />
an increased ability to produce IgG1 and IgE in response to<br />
anti-CD40 and/or IL-4 in vitro. These findings may reflect an<br />
interaction between GATA-3 and type 2 Ig-regulating transcription<br />
factors such as NF-kB or STAT6, because GATA-3 can<br />
modulate the activities <strong>of</strong> these factors in vitro. These<br />
findings demonstrate a critical yet surprising role for GATA-3<br />
in the regulation <strong>of</strong> IL-4-mediated responses in B cells.<br />
doi:10.1016/j.clim.2007.03.081<br />
Su.41 Natural and Synthetic TLR7 Ligands Inhibit<br />
CpG-A- and CpG-C-ODN-induced IFN-α Production<br />
by Plasmacytoid Dendritic Cells<br />
Holger Hackstein, Resident, University <strong>of</strong> Giessen, Giessen,<br />
Germany, Beate Berghöfer, PhD Student, University <strong>of</strong><br />
Giessen, Giessen, Germany, Gregor Bein, Pr<strong>of</strong>essor,<br />
University <strong>of</strong> Giessen, Giessen, Germany<br />
Plasmacytoid dendritic cells (pDC) are unique with respect<br />
to their capacity to produce unsurpassed amounts <strong>of</strong><br />
IFN-α and coexpress TLR7 and TLR9, mediating IFN-α<br />
production. Although TLRs are critical receptors <strong>of</strong> innate<br />
immunity, little is known about the immunological effects <strong>of</strong><br />
TLR7/TLR9 costimulation. We have analyzed the effects <strong>of</strong><br />
TLR7/TLR9 costimulation on IFN-α production by leukocytes<br />
and pDCs. Our experiments revealed that both synthetic<br />
(resiquimod, loxoribine) and natural (ssRNA40) TLR7 ligands<br />
abrogate CpG-A- and CpG-C-ODN-induced IFN-α production<br />
by human leukocytes. Since TLR7 ligands themselves<br />
represent important IFN-α inducers, we demonstrated that<br />
substimulatory TLR7 ligand concentrations significantly<br />
inhibited CpG-A-induced IFN-α. Delayed addition <strong>of</strong> TLR7<br />
ligands still resulted in complete suppression <strong>of</strong> CpG-A-ODNinduced<br />
IFN-α production, suggesting that the inhibition is<br />
unlikely to be caused by a kinetic uptake advantage. Unlike<br />
for CpG-A and CpG-C, TLR7 ligands did not inhibit CpG-B-<br />
ODN-induced IFN-α production. Experiments with purified<br />
human pDCs demonstrated that the inhibitory effects <strong>of</strong><br />
TLR7/TLR9 costimulation were mediated directly by pDCs.<br />
Suppression <strong>of</strong> IFN-α production was not related to increased<br />
cell death and was also detectable in enriched mouse pDCs.<br />
Analyses <strong>of</strong> pDCs suggested that the TLR7 signal regulates the<br />
outcome <strong>of</strong> TLR7 ligand/CpG-A-ODN costimulation and can<br />
either inhibit (IFN-α) or promote (IL-8/CD40) cytokine and<br />
surface marker expression. Our data reveal for the first time<br />
a strong inhibitory effect <strong>of</strong> TLR7 stimulation on IFN-α<br />
production induced by CpG-A- and CpG-C-ODNs. These<br />
findings provide novel insight into the effects <strong>of</strong> TLR7/TLR9<br />
costimulation and may support the development <strong>of</strong> novel<br />
TLR9 inhibitors.<br />
doi:10.1016/j.clim.2007.03.082<br />
S155<br />
Su.42 Association <strong>of</strong> MHC Class II Haplotypes and<br />
Immunogenicity <strong>of</strong> a Therapeutic Biological Protein<br />
Danika Wullner, Senior Associate, Amgen, Thousand Oaks,<br />
CA, Erica Yue, Associate, <strong>Clinical</strong> <strong>Immunology</strong>, Thousand<br />
Oaks, CA, Steven J. Swanson, Executive Director, <strong>Clinical</strong><br />
<strong>Immunology</strong>, Thousand Oaks, CA, Vibha Jawa, Senior<br />
Scientist, <strong>Clinical</strong> <strong>Immunology</strong>, Thousand Oaks, CA<br />
A mature high affinity antibody response that is T cell<br />
driven can be elicited during administration <strong>of</strong> biological<br />
therapeutics in humans. The processing <strong>of</strong> the protein<br />
antigen by antigen presenting cells and their binding to<br />
Class II HLA molecules is necessary for the antibody response<br />
to mature. In this study, the predisposition <strong>of</strong> certain MHC<br />
haplotypes to present peptides derived after processing a<br />
recombinant protein (FPX-protein) is discussed. Ex vivo T<br />
cell priming assays were used to predict immunogenicity <strong>of</strong><br />
Class II restricted T cell epitopes contained within the FPXprotein.<br />
The immunogenic, promiscuous epitope(s) are<br />
contained within the 14-amino acid carboxy-terminal region<br />
<strong>of</strong> the FPX-peptide, but none are found in the aminoterminal<br />
region comprising 10 amino acids as observed by in<br />
silico prediction and in vivo data. Peripheral blood mononuclear<br />
cells (PBMCs) from haplotyped donors were challenged<br />
with multiple rounds <strong>of</strong> FPX peptides (C-terminal and<br />
N-terminal) for 7–14 days followed by ELISPOT assays for T<br />
lymphocyte activation. HLA haplotype DRB1*0701/1301 was<br />
associated with an increased number <strong>of</strong> spot forming cells<br />
when challenged with the immunogenic C-terminal FPXpeptide<br />
(10.15 fold increase) and negligible with the nonimmunogenic<br />
N-terminal (1.38 fold increase). The same<br />
haplotype was associated with a high antibody response and<br />
a memory T cell response when the protein was administered<br />
in vivo as well as by in silico prediction. Ex vivo T cell<br />
priming assays can be potential tools for prediction <strong>of</strong>
S156 Abstracts<br />
immunogenicity <strong>of</strong> T cell epitopes. These assays enable<br />
selection <strong>of</strong> the right therapeutic in early development and<br />
determine which MHC haplotypes are predisposed to an<br />
immune response.<br />
doi:10.1016/j.clim.2007.03.083<br />
Su.43 Hepatitis B Virus Proteins Induce Activation <strong>of</strong><br />
hfgl2 Prothrombinase Gene Through c-Ets-2<br />
Transcription Factors<br />
Meifang Han, Doctor in Charge, Tongji Hospital, Huazhong<br />
University <strong>of</strong> Science and Technology, Department <strong>of</strong><br />
Infectious Disease, Wuhan, China, Dong Xi, Assistant<br />
Researcher, Department <strong>of</strong> Infectious Disease, Tongji<br />
Hospital, Huazhong University <strong>of</strong> Science and Technology,<br />
Wuhan, China, Weiming Yan, Assistant Researcher,<br />
Department <strong>of</strong> Infectious Disease, Tongji Hospital,<br />
Huazhong University <strong>of</strong> Science and Technology, Wuhan,<br />
China, Gary Levy, Pr<strong>of</strong>essor, Chief Physician, University<br />
Health Network, University <strong>of</strong> Toronto, Toronto, ON,<br />
Canada, Mingfeng Liu, Postdoctoral Researcher and<br />
Associate, University Health Network, University <strong>of</strong><br />
Toronto, Toronto, ON, Canada, Xiaoping Luo, Chief<br />
Physician, Department <strong>of</strong> Pediatrics <strong>of</strong> Tongji Hospital,<br />
Huazhong University <strong>of</strong> Science and Technology, Wuhan,<br />
China, Qin Ning, Chief Physician, Department <strong>of</strong> Infectious<br />
Disease, Tongji Hospital, Huazhong University <strong>of</strong> Science<br />
and Technology, Wuhan, China<br />
Objective: To determine the viral and host factors<br />
involved in the transcription <strong>of</strong> hfgl2 gene which was<br />
demonstrated to play pivoral role in human and experiment<br />
fulminant viral hepatitis. Methods. HBc, HBs or HBx expression<br />
plasmids were constructed and cotransfected with a<br />
hfgl2 luciferase report construct into eukaryotic cells.<br />
Results. Luciferase assay showed that HBc or HBx protein,<br />
but not HBs protein significantly enhanced hfgl2 transcription<br />
in both CHO and HepG2 cells. Series deletion assay <strong>of</strong><br />
hfgl2 gene promoter demonstrated that a strong regulatory<br />
domain from −712 to −568 was responsible for hfgl2 gene<br />
transcription in response to HBc or HBx proteins. By sitedirected<br />
mutagenesis, the overlapped cis-elements LEF/c-<br />
Ets in domain −712/−568 was demonstrated to play an<br />
important role in the regulation <strong>of</strong> hfgl2 gene in response<br />
to HBc protein, while another two cis-elements LEF/c-Ets<br />
and HSTF in the same domain were found to participate in<br />
hfgl2 transcription regulation in response to HBx protein.<br />
EMSA assays showed that HBc and HBx proteins did not<br />
directly induce hfgl2 gene activation, while an Ets family<br />
member c-Ets-2 bound the cognate cis-element in hfgl2<br />
promoter was responsible for induction <strong>of</strong> hfgl2 activation<br />
in response to viral proteins. Conclusion. Our study provides<br />
new insights in understanding the pathology <strong>of</strong> fulminant<br />
hepatic failure and the interaction between HBV virus and<br />
host gene expression. This work was supported by NSFC<br />
30225040, 30571643, 30672380, National Key Basic Research<br />
Program <strong>of</strong> China (2005CB522901, 2005CB522507) and <strong>Clinical</strong><br />
Subject Key Project from the Ministry <strong>of</strong> Health.<br />
doi:10.1016/j.clim.2007.03.084<br />
Su.44 Arthritogenic Simulation <strong>of</strong> Human Synovial<br />
Fibroblasts by TWEAK<br />
Timothy Zheng, Senior Scientist, Biogen Idec, Inc.,<br />
Cambridge, MA, Shawn Weng, Associate Scientist, Biogen<br />
Idec, Inc., Cambridge, MA, Suzanne Szak, Scientist II, Biogen<br />
Idec, Inc., Cambridge, MA, Linda Burkly, Distinguished<br />
Investigator, Biogen Idec, Inc., Cambridge, MA<br />
Synovial fibroblasts are critically involved in propagating<br />
joint inflammation in rheumatoid arthritis (RA) through the<br />
production <strong>of</strong> numerous arthritogenic mediators. The proinflammatory<br />
TNF ligand superfamily cytokine TWEAK has<br />
previously been shown to induce several chemokine/cytokines<br />
in human fibroblast cell types including synovciocytes. In this<br />
study, we seek to understand the involvement <strong>of</strong> TWEAK in<br />
human RA pathogenesis by studying its broad effect on human<br />
synovial fibroblasts. We found that synovial fluid TWEAK levels<br />
were elevated in RA patients when compared to those in OA<br />
patients, consistent with the notion that TWEAK may play a role<br />
in driving inflammatory response in the joints <strong>of</strong> RA patients.<br />
Using transcription pr<strong>of</strong>iling, we demonstrated that TWEAK<br />
consistently induced a large panel <strong>of</strong> genes, including many<br />
known arthritogenic mediators such as IL-1, IL-6, RANTES, ENA-<br />
78, IP-10 and MMPs. Interestingly, the potency <strong>of</strong> gene<br />
regulation by TWEAK when compared to TNF varied greatly<br />
amongst the four different donor cells, perhaps reflecting<br />
patient heterogeneity regarding the involvement <strong>of</strong> TWEAK visa-vie<br />
TNF in driving synoviocyte-mediated joint inflammation.<br />
Importantly, the production <strong>of</strong> TWEAK and TNF appeared to be<br />
independent <strong>of</strong> each other as neither TWEAK nor TNF induced<br />
or inhibited the expression <strong>of</strong> the other. When combined<br />
however, TWEAK and TNF synergically or additively induced the<br />
production <strong>of</strong> many other well-known arthritogenic mediators.<br />
Taken together, these results suggest that TWEAK and TNF may<br />
represent two concurrent upstream cytokines that regulate<br />
joint inflammation and damage in RA patients.<br />
doi:10.1016/j.clim.2007.03.085<br />
Su.45 IL-6/TH-17 Axis Plays a Crucial Role in the<br />
Generation <strong>of</strong> GPI-induced Arthritis<br />
Keiichi Iwanami, PhD Student, Graduate School <strong>of</strong><br />
Comprehensive Human Science, University <strong>of</strong> Tsukuba,<br />
Tsukuba, Japan, Asuka Inoue, Master Student, Graduate<br />
School <strong>of</strong> Comprehensive Human Science, University <strong>of</strong><br />
Tsukuba, Tsukuba, Japan, Isao Matsumoto, Instructor,<br />
Graduate School <strong>of</strong> Comprehensive Human Science,<br />
University <strong>of</strong> Tsukuba, Tsukuba, Japan, Mizuko Mamura,<br />
Research Fellow, Graduate School <strong>of</strong> Comprehensive Human<br />
Science, University <strong>of</strong> Tsukuba, Tsukuba, Japan, Yoko<br />
Watanabe, PhD Student, Graduate School <strong>of</strong> Comprehensive<br />
Human Science, University <strong>of</strong> Tsukuba, Tsukuba, Japan,<br />
Daisuke Goto, Instructor, Graduate School <strong>of</strong><br />
Comprehensive Human Science, University <strong>of</strong> Tsukuba,<br />
Tsukuba, Japan, Satoshi Ito, Instructor, Graduate School <strong>of</strong><br />
Comprehensive Human Science, University <strong>of</strong> Tsukuba,<br />
Tsukuba, Japan, Akito Tsutsumi, Assistant Pr<strong>of</strong>essor,<br />
Graduate School <strong>of</strong> Comprehensive Human Science,<br />
University <strong>of</strong> Tsukuba, Tsukuba, Japan, Takayuki Sumida,<br />
Pr<strong>of</strong>essor, Graduate School <strong>of</strong> Comprehensive Human<br />
Science, University <strong>of</strong> Tsukuba, Tsukuba, Japan
Abstracts<br />
Backgrounds: Glucose-6-phosphate isomerase (GPI)induced<br />
arthritis is a murine model <strong>of</strong> rheumatoid arthritis<br />
(RA). Although anti-CD4 antibodies (Abs) had therapeutic<br />
effect in this model, the pathogenicity <strong>of</strong> each CD4+ T cell<br />
lineage was uncertain. Here we undertook the present study<br />
to clarify GPI-specific T cell lineages and investigate their<br />
pathological and regulatory roles in the generation <strong>of</strong><br />
arthritis. [Methods] DBA/1 mice were immunized with<br />
300fÊg <strong>of</strong> GPI to induce arthritis. 1) CD4+ T cells and APCs<br />
were co-cultured with GPI for 24 h, and the supernatant was<br />
analyzed by cytokine ELISA. 2) Anti-IFNfÁ mAb or anti IL-17<br />
mAb was injected on day 7 to investigate arthritis development.<br />
3) A mAb to murine IL-6 receptor (MR16-1) was<br />
injected on days 0, 3 or 8 to investigate arthritis development.<br />
4) After the injection <strong>of</strong> MR16-1 on days 0 or 3,<br />
draining lymph nodes (DLNs) were harvested on day 7, and<br />
TH-17 cells were analyzed by flow cytometry. Results: 1)<br />
IFNfÁ and IL-17 were produced by GPI-specific CD4+ T cells.<br />
2) The administration <strong>of</strong> anti-IL-17 mAb on day 7<br />
significantly ameliorated arthritis (Pb0.01), whereas that<br />
<strong>of</strong> anti-IFNfÁ mAb exacerbated. 3) The administration <strong>of</strong><br />
MR16-1 on day 0 or 3 protected the induction <strong>of</strong> arthritis,<br />
and that <strong>of</strong> MR16-1 on day 8 significantly ameliorated<br />
arthritis (Pb0.05). 4) The differentiation <strong>of</strong> TH-17 in DLNs<br />
was markedly suppressed by MR16-1. Conclusions: IL-6 and<br />
TH-17 play an essential role in GPI-induced arthritis. Our<br />
study suggests that IL-6/TH-17 axis might be also involved<br />
in RA.<br />
doi:10.1016/j.clim.2007.03.086<br />
Su.46 The Serum Cytokine Pr<strong>of</strong>ile in Low-Stage<br />
Chronic Lymphocytic Leukemia is a 7th Hallmark <strong>of</strong><br />
Cancer<br />
Catherine Spier, Associate Pr<strong>of</strong>essor, Pathology, University<br />
<strong>of</strong> Arizona College <strong>of</strong> Medicine, Department <strong>of</strong> Pathology,<br />
Tucson, AZ, Christopher Riley, Graduate Student, University<br />
<strong>of</strong> Arizona College <strong>of</strong> Medicine, Arizona Cancer Center,<br />
Tucson, AZ, James Choi, Fellow, University <strong>of</strong> Arizona<br />
College <strong>of</strong> Medicine, Arizona Cancer Center, Tucson, AZ,<br />
Lawrence Cooke, Research Specialist, University <strong>of</strong> Arizona<br />
College <strong>of</strong> Medicine, Arizona Cancer Center, Tucson, AZ,<br />
Thomas Klinkhammer, Fellow, University <strong>of</strong> Arizona College<br />
<strong>of</strong> Medicine, Arizona Cancer Center, Tucson, AZ, Daruka<br />
Mahadevan, Associate Pr<strong>of</strong>essor, Medicine, University <strong>of</strong><br />
Arizona College <strong>of</strong> Medicine, Arizona Cancer Center,<br />
Tucson, AZ<br />
Background: In B cell Chronic Lymphocytic Leukemia (B-<br />
CLL) there are no validated screening, diagnostic or<br />
prognostic serum markers for early detection. Several<br />
aberrant cytokines have previously been identified individually<br />
in patients with B-CLL that are thought to enhance<br />
malignant cell survival. Methods: An IRB approved protocol<br />
was used to collect and examine 120 serum cytokines from<br />
26 Rai Stage 0/1 patients who were treatment naive and<br />
also from 4 normal volunteers (RayBiotech Cytokine Array,<br />
#AAH-CYT-G1000). Fold change was calculated for each<br />
patient, corrected for absolute lymphocyte count and a<br />
mean ± SD obtained for each cytokine for all patients.<br />
Results: This cytokine pr<strong>of</strong>ile identified all <strong>of</strong> the<br />
published cytokines associated with B-CLL cell survival<br />
(IL-1B, IL-2, IL-4, IL-6, IL-8, IL-10, TNFa, INFa or g, G-CSF<br />
or GM-CSF) or inhibition (IL-5, TGF-B). In this pr<strong>of</strong>ile the<br />
1st ranked is INF-g (14.46 fold) which is known to prevent<br />
apoptosis <strong>of</strong> B-CLL cells. The 2nd ranked is IGFBP-4 (14.22<br />
fold) and 3rd ranked is G-CSF (10.89 fold) which is known<br />
to decrease B-CLL cell apoptosis. Other cytokines <strong>of</strong> note<br />
are Flt-3 ligand (7.82 fold), SCF (6.76 fold) and SDF-1<br />
(6.54 fold). Conclusion: Thus, our cytokine pr<strong>of</strong>ile, that<br />
sampled 120 cytokines simultaneously, validates the existing<br />
data, lists the ctyokines by order <strong>of</strong> expression from<br />
highest to lowest and significantly adds to the list <strong>of</strong><br />
cytokines identified as important for B-CLL cell survival in<br />
low stage patients.<br />
doi:10.1016/j.clim.2007.03.087<br />
Su.47 High Sensitivity Multiplexed Immunoassays<br />
for Simultaneous Quantification <strong>of</strong> Key Cytokines in<br />
Human Samples<br />
Jehangir Mistry, PhD, LINCO (now part <strong>of</strong> Millipore), St.<br />
Charles, MO, Jiaxin Dong, PhD, LINCO (now part <strong>of</strong><br />
Millipore), St. Charles, MO, Terry Whitehead, NA, LINCO<br />
(now part <strong>of</strong> Millipore), St. Charles, MO, Shaoquan Ji,<br />
Research and Development Manager, LINCO (now part <strong>of</strong><br />
Millipore), St. Charles, MO, Hank Hwang, PhD, LINCO (now<br />
part <strong>of</strong> Millipore), St. Charles, MO<br />
Low levels <strong>of</strong> inflammation play a key role in etiology <strong>of</strong><br />
many disease states (e.g. atherosclerosis, allergy, cancer,<br />
diabetes). Understanding the role <strong>of</strong> inflammation in pathogenesis<br />
has been impeded because currently available cytokine<br />
assays are not sensitive enough to detect the<br />
differences between normal and subclinically inflammed<br />
states. Using the Luminex xMAP technology, we developed a<br />
highly sensitive, multiplexed immunoassay panel for simultaneously<br />
quantifying 13 human cytokines commonly involved<br />
in Th1/Th2 and inflammatory responses (IL-1β, IL-2,<br />
IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, GMCSF,<br />
IFNγ and TNFα). The assays follow the typical bead-based<br />
sandwich format and use a simple protocol. Each antibody<br />
pair is highly specific, with no or negligible cross-reactivity<br />
to other analytes within the panel. All assays in the panel<br />
have a common dynamic range <strong>of</strong> 0.128 to 2000 pg/ml,<br />
wide enough for almost all sample types. Sensitivities <strong>of</strong> the<br />
assays are between 0.01 to 0.48 pg/ml with most analytes<br />
at approximately 0.1 pg/ml. Normal serum samples are 80<br />
to 100% detectable for most cytokines. The assay robustness<br />
is demonstrated by acceptable precisions (CV=2.2–<br />
14.3% for inter-assay; CV=3.1–5.9% for intra-assay), accuracy<br />
(93.0 to 112%), and linearity <strong>of</strong> dilution (102 to 113%)<br />
for serum or plasma samples. No serum/plasma dilution is<br />
required. The assays may be used for other sample types (e.<br />
g. cell culture supernatant, tissue/cell lysate from human<br />
origin). This novel, robust and highly sensitive assay panel<br />
serves as an accurate, reproducible and economic tool for<br />
simultaneously quantifying multiple cytokines in human<br />
samples.<br />
doi:10.1016/j.clim.2007.03.088<br />
S157
S158 Abstracts<br />
Su.48 Use <strong>of</strong> a 10-Cytokine Multiplex RT-PCR in<br />
Conjunction with a Corresponding 10-plex<br />
Fluorescent Bead Quantitative Protein<br />
Immunoassay to Reveal Expression Patterns in<br />
Mitogen-Stimulated Human Peripheral Leukocytes<br />
Cynthia Boardman, Development Scientist, Beckman<br />
Coulter Inc. Nucleic Acid Testing Business, Fullerton, CA,<br />
Cynthia Boardman, Development Scientist, Beckman<br />
Coulter Inc. Nucleic Acid Testing Business, Fullerton, CA, Yu<br />
Suen, Staff Development Scientist, Beckman Coulter Inc.<br />
Biomarker Discovery and Automation Center, Fullerton, CA,<br />
Scott Boyer, Genomics Group Manager, Beckman Coulter Inc.<br />
Nucleic Acid Testing Business, Fullerton, CA, Keith Roby,<br />
Product Manager, Beckman Coulter Inc. Biomarker Discovery<br />
and Automation Center, Fullerton, CA<br />
The ability to monitor the expression <strong>of</strong> mediators <strong>of</strong><br />
inflammation and immune response is critical to the study <strong>of</strong><br />
disease and pharmacodynamics. Quantitative protein immunoassays<br />
are one <strong>of</strong> the standard tools for the examination<br />
<strong>of</strong> cellular responses but reveal only part <strong>of</strong> the cellular<br />
story. We show here how the GenomeLab GeXP Genetic<br />
Analysis System can be used to corroborate protein studies<br />
and provide a more complete picture <strong>of</strong> cellular responses<br />
to effectors. Specifically, we have developed a multiplexed<br />
RT-PCR† assay which can analyze the mRNA expression<br />
pr<strong>of</strong>ile <strong>of</strong> ten Th1/Th2 cytokines simultaneously. The<br />
eXpress Designer s<strong>of</strong>tware included in the GeXP System<br />
was used to design multiplex PCR primers for the following:<br />
IFN-g, IL-1B, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-α and<br />
TNF-B; and the resulting multiplex was used to analyze<br />
changes in the mRNA expression pr<strong>of</strong>iles for these ten<br />
products in human peripheral leukocytes upon stimulation<br />
with LPS or PMA/PHA. The data were then used to corroborate<br />
results <strong>of</strong> a quantitative protein analysis performed<br />
on the matching cell supernatant samples using the<br />
FlowCytomix Multiplex Human Th1/Th2 10-Plex fluorescent<br />
bead immunoassay kit and analyzed on the Cytomics FC500<br />
MPL flow cytometry system. This dual-platform approach<br />
can be used to monitor the immune response effected by<br />
diseases and treatments at the level <strong>of</strong> both mRNA and<br />
protein expression.<br />
doi:10.1016/j.clim.2007.03.089<br />
Su.49 Dysregulated Inflammatory Cytokine<br />
Response in Lipopolysaccharide- and<br />
Peptidoglycan-Stimulated Monouclear Cells <strong>of</strong> a<br />
Patient with Blau Syndrome<br />
Sang Wook Son, Pr<strong>of</strong>essor, Department <strong>of</strong> Dermatology,<br />
Korea University College <strong>of</strong> Medicine, Ansan-si, Korea, Jin<br />
Lee, Doctor, Department <strong>of</strong> Pediatrics, Korea University<br />
College <strong>of</strong> Medicine, Ansan-si, Korea, Jae Eun Choi, Doctor,<br />
Department <strong>of</strong> Dermatology, Korea University College <strong>of</strong><br />
Medicine, Ansan-si, Korea, Il Hwan Kim, Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> Dermatology, Korea University College <strong>of</strong><br />
Medicine, Ansan-si, Korea, Jeongwon Sohn, Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> Biochemistry, Korea University College <strong>of</strong><br />
Medicine, Seoul, South Korea, Young Chul Kye, Pr<strong>of</strong>esor,<br />
Department <strong>of</strong> Dermatology, Korea University College <strong>of</strong><br />
Medicine, Ansan-si, Korea, Kwang Chul Lee, Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> Pediatrics, Korea University College <strong>of</strong><br />
Medicine, Ansan-si, Korea, JungHwa Lee, Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> Pediatrics, Korea University College <strong>of</strong><br />
Medicine, Ansan-si, Korea<br />
Inheritable Blau syndrome (BS) and sporadic early-onset<br />
sarcoidosis (EOS) share characteristic clinical features <strong>of</strong><br />
multi-organ granulomatous inflammation involving joints,<br />
skin and eyes. Multiple mutations in the caspase recruitment<br />
domain gene CARD15 were described in BS. EOS was<br />
diagnosed in a 5-month-old girl who initially had biopsyproven<br />
chronic non-caseating granulomatous papules on a<br />
whole body and later developed joint abnormalities and<br />
steroid-responsive hypercalcemia. Her 32-year-old mother<br />
was blind since she was about 5 years old and showed<br />
camptodactyly and eczematous skin lesions. All <strong>of</strong> the other<br />
mother side family members were healthy with no evidence<br />
<strong>of</strong> any skin, joint and eye abnormalities. DNA analysis<br />
demonstrated a missense mutation, 1000CNT (R334W), in<br />
the CARD15 gene in both the mother and the child,<br />
suggesting sporadic incidence <strong>of</strong> the mutation in the mother<br />
and its inheritance to her child. When peripheral blood<br />
mononuculear cells <strong>of</strong> the mother were stimulated by<br />
lipopolysaccharide and peptidoglycan, production <strong>of</strong> an<br />
anti-inflammatory cytokine, IL-10 was reduced compared<br />
to the normal control. In addition, induction <strong>of</strong> TNF-α<br />
production was reduced when stimulated with peptidoglycan<br />
alone. Basal levels <strong>of</strong> both cytokines in the mother were not<br />
different compared to the normal control. These findings<br />
indicate that BS and EOS are diseases <strong>of</strong> the same spectrum<br />
sharing the common etiology <strong>of</strong> CARD15 mutation and that a<br />
dysregulated anti- and pro-inflammatory cytokine response<br />
to the stimulation may lead to the chronic inflammatory<br />
manifestations in BS.<br />
doi:10.1016/j.clim.2007.03.090<br />
Su.50 Quantitative Evaluation <strong>of</strong> Interleukin-6,<br />
Interleukin-11 and Interleukin-17 in Healthy and<br />
Inflammatory Tissues <strong>of</strong> Gingival<br />
Hamid Reza, ArAB, Dentist, Periodontology Department,<br />
School <strong>of</strong> Dentistry and Mashhad Dental Research Center,<br />
Mashhad, Iran, Jalil Tavakkol-Afshari, Vice President for<br />
Research; Head, Department <strong>of</strong> Immunogenetics and Cell<br />
Culture, Bu-Ali Research Institute (BARI), Department <strong>of</strong><br />
Immunogenetics and Cell Culture, Mashhad, Iran, Zahra<br />
Baghani, Dentist, Periodontology Department, School <strong>of</strong><br />
Dentistry and Mashhad Dental Research Center, Mashhad,<br />
Iran, Nasrin Moheghi, Researcher, Bu-Ali Research Institute<br />
(BARI), Department <strong>of</strong> Immunogenetics, Mashhad, Iran<br />
Interleukin 11(IL-11), IL-17 and IL-6 are cytokines that<br />
modulate the inflammatory process and have not been<br />
assessed within normal or inflammed gingival tissues. Our<br />
purpose was comparing the concentrations <strong>of</strong> human IL-6, IL-<br />
11 and IL-17 within healthy and diseased human gingival<br />
tissues to determine their possible role in the initiation or<br />
progression <strong>of</strong> periodontal disease. Biopsies from healthy<br />
(non-hemorrhagic gingiva adjacent to the 3 mm, 4–5 mm and<br />
≥ 6 mm periodontal pockets) were studied. Interleukin-<br />
11, IL-17 and IL-6 concentrations were assessed within
Abstracts<br />
solubilized gingival biopsies by enzyme-linked immunosorbent<br />
assay. Data were analyzed by Kruskal–Wallis and Mann–<br />
Whitney U tests for finding significant correlation between<br />
groups. Interleukin 11, IL-17 and IL-6 concentrations were<br />
decreased within gingival adjacent to 4–5 mm sulcular depth<br />
but later increased within gingiva adjacent to ≥ 6mm<br />
pocket depths. The amount <strong>of</strong> IL-17 between healthy tissue<br />
and gingiva adjacent to ≥ 6 mm pockets and 4–5 mm and â<br />
‰¥ 6 mm pockets was significant (pb0.05). Interleukin-11,<br />
IL-6 and IL-17 concentrations were greatly correlated with<br />
sulcular depth. However, in most cases this difference was<br />
not statistically significant. Interleukin-6, IL-17, IL-11 concentrations<br />
were increased in the inflammatory gingival<br />
tissue adjacent to ≥ 6 mm pockets in comparison to<br />
healthy gingiva but only the concentration <strong>of</strong> IL-17 was<br />
significant (p=0.03).<br />
doi:10.1016/j.clim.2007.03.091<br />
Su.51 Inhibition <strong>of</strong> Experimental Tumor Metastasis<br />
by CpG Oligodeoxynucleotide-Pulsed Dendritic Cell<br />
Han-A Kim, Assistant, Department <strong>of</strong> Biological Sciences,<br />
Kwangju, South Korea, Hyun-Mi Ko, Department <strong>of</strong><br />
Biological Sciences, Kwangju, South Korea, Hern-Ku Lee,<br />
Pr<strong>of</strong>essor, Department <strong>of</strong> <strong>Immunology</strong>, Chonju, South Korea,<br />
Suhn-young Im, Pr<strong>of</strong>essor, Department <strong>of</strong> Biological<br />
Sciences, Kwangju, South Korea<br />
Dendritic cell (DC) pulsed with tumor antigen induced<br />
tumor-specific immune response. CpG ODN has been recognized<br />
as a powerful stimulant <strong>of</strong> DC. This study was<br />
designed to investigate whether CpG oligodeoxynucleotide<br />
(ODN)-pulsed DC could inhibit the experimental pulmonary<br />
metastasis <strong>of</strong> B16F10 murine melanoma. Treatment <strong>of</strong> CpG<br />
ODN intraperitoneally either before or after B16F10, it<br />
inhibited lung colonization <strong>of</strong> B16F10. Bone marrow-derived<br />
DC pulsed with B16F10 lysate inhibited the tumor metastasis.<br />
In this case, the inhibitory effect <strong>of</strong> mature DC was<br />
much more prominent than <strong>of</strong> immature DC. In addition, the<br />
inhibitory effect DC was further augmented when DC was<br />
stimulated with CpG ODN. The magnitude <strong>of</strong> experimental<br />
pulmonary metastasis was increased in IL-12 knock out<br />
(IL-12−/−) mice compared with their littermates. CpG ODN<br />
did not exert its metastasis-inhibiting activity in IL-12−/−<br />
mice. Moreover, DC originated from IL-12−/− mice do not<br />
inhibited metastasis even when the cells were stimulated<br />
with B16F10 lysate plus CpG ODN. In vitro, CpG ODN induced<br />
the gene expression and protein synthesis <strong>of</strong> IL-12 in DC.<br />
Tumor metastasis was inhibited by adoptive transfer <strong>of</strong><br />
splenic T cells from CpG ODN-treated mice. These results<br />
suggest that CpG ODN inhibited tumor metastasis through<br />
activating DC to produce IL-12, which resulted in the<br />
development <strong>of</strong> effector T cells.<br />
doi:10.1016/j.clim.2007.03.092<br />
Su.52 Regulation <strong>of</strong> the STAT3-Mediated Signaling by<br />
Daxx<br />
Kenji Sugiyama, Researcher, Department Molecular Biology,<br />
Nippon Boehringer Ingelheim Co. Kawanishi, Japan, Ryuta<br />
Muromoto, Postdoctoral Fellow, Department <strong>Immunology</strong>,<br />
Graduate School Pharmaceutical Science, Hokkaido<br />
University, Sapporo, Japan, Masato Ishida, Researcher,<br />
Department Molecular Biology, Nippon Boehringer<br />
Ingelheim Co, Kawanishi, Japan, Yuichi Sekine, Assosiate<br />
Pr<strong>of</strong>essor, Department <strong>Immunology</strong>, Graduate School<br />
Pharmaceutical Science, Hokkaido University, Sapporo,<br />
Japan, Kenji Oritani, Assistant Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Hematol. and Oncology, Graduate School Med, Osaka<br />
University, Suita, Japan, Tadashi Matsuda, Pr<strong>of</strong>essor,<br />
Department <strong>Immunology</strong>, Graduate, School Pharmaceutical<br />
Science, Hokkaido University, Sapporo, Japan<br />
Signal transducer and activator <strong>of</strong> transcription 3 (STAT3)<br />
plays key roles in the intracellular signaling pathways <strong>of</strong> the<br />
interleukin (IL)-6 family <strong>of</strong> cytokines, which exhibits a<br />
diverse set <strong>of</strong> cellular responses, including cell proliferation<br />
and differentiation. Dysregulated IL-6/STAT3 signaling is<br />
involved in the pathogenesis <strong>of</strong> several diseases, for<br />
examples autoimmune diseases and tumors. Type I interferon<br />
(IFN) induces the expression <strong>of</strong> proapoptotic genes<br />
and has been used in the clinical treatment <strong>of</strong> several<br />
tumors. In the present study, we found that type I IFN<br />
suppressed IL-6/STAT3-mediated transcription and gene<br />
expression. Furthermore, a type I IFN-induced protein,<br />
Daxx, also suppressed STAT3-mediated transcriptional activation,<br />
while overexpression <strong>of</strong> Daxx inhibited IL-6/STAT3mediated<br />
gene expression. Importantly, small-interfering<br />
RNA-mediated reduction <strong>of</strong> Daxx expression enhanced IL-6/<br />
leukemia inhibitory factor (LIF)-induced STAT3-dependent<br />
transcription. Co-immunoprecipitation studies revealed a<br />
physical interaction between Daxx and STAT3 in transiently<br />
transfected 293T cells. We further found that Daxx and<br />
STAT3 were co-localized in the nucleus. These results<br />
indicate that Daxx may serve as a transcriptional regulator<br />
<strong>of</strong> type I IFN-mediated suppression <strong>of</strong> the IL-6/STAT3<br />
signaling pathway.<br />
doi:10.1016/j.clim.2007.03.093<br />
S159<br />
Su.53 Immunological Relationships between Heart<br />
Tissue and Non-Cardiac Organs in Murine<br />
Eosinophilc and Lymphocytic Myocarditis<br />
Masao Hirasawa, Doctor <strong>of</strong> Medicine, Third Division <strong>of</strong><br />
Internal Medicine, Osaka Medical College, Takatsuki-city,<br />
Osaka, Japan, Minoru Miyashita, Assistant, Department <strong>of</strong><br />
Neurosurgery, Osaka Medical College, Takatsuki-city, Osaka,<br />
Japan<br />
Background: Eosinophilic disorders usually develop into<br />
one <strong>of</strong> the factors leading to myocarditis. However,<br />
immunological interactions between heart tissue and noncardiac<br />
organs in such diseases are still unclear. In this study,<br />
the JAK/STAT signaling pathway in acute myocarditis was<br />
investigated, considering the immunological mechanism in<br />
non-cardiac organs. Methods: Adoptive transfer and Coxsackie<br />
B3 viral infection were performed to initiate<br />
eosinophilic and lymphocytic myocarditis in DBA/2 mice.<br />
Eosinophils, T cells, and JAK3/STAT6 mRNAs were examined<br />
by histological methods and RT-PCR. In the spleen, the ratios<br />
<strong>of</strong> CD4 to CD8 were compared between OVA-sensitized and
S160 Abstracts<br />
control mice. In the bone marrow, we attempted to detect<br />
CD34 by FACS analysis. Results: Eosinophilic myocarditis was<br />
observed in adoptive transferred mice, and marked lymphocytic<br />
myocarditis was developed in virus-infected mice. In<br />
both cases, JAK3s were usually seen in inflammatory heart<br />
lesions; in particular, the parallel expression <strong>of</strong> JAK3 and<br />
STAT6 mRNAs was seen in eosinophilic myocarditis. In the<br />
spleen, CD4/CD8 ratios were elevated in OVA-sensitized<br />
cases compared with controls (pb0.05), and the quantity <strong>of</strong><br />
JAK3 mRNA was higher in adoptive transfer cases than<br />
controls (pb0.05). Bone marrow cell analysis showed the<br />
role <strong>of</strong> CD34 in OVA-sensitized and normal control mice.<br />
Conclusion: These results suggested that (1) JAK/STAT<br />
signaling plays a role in myocardial damage in eosinophilic<br />
and viral myocarditis, (2) JAK3 also conveys immunological<br />
tolerance within a CD4-dominant environment in noncardiac<br />
organs, and (3) bone marrow cells <strong>of</strong> donor mice<br />
may have potential for regeneration therapy.<br />
doi:10.1016/j.clim.2007.03.094<br />
Su.54 Combined Pharmacodynamic and<br />
Transcriptome Pr<strong>of</strong>iling <strong>of</strong> Recombinant Human<br />
Interleukin-21 Responses in Patients with Stage IV<br />
Malignant Melanoma<br />
Dorthe Lundsgaard, Research Scientist, Project Manager,<br />
Novo Nordisk A/S, Maaloev, Denmark, Klaus Stensgaard<br />
Frederiksen, Senior Scientist, Novo Nordisk A/S, Bagsvaerd,<br />
Denmark, Lasse Tengbjerg Hansen, <strong>Clinical</strong> Scientist, Novo<br />
Nordisk A/S, Bagsvaerd, Denmark, Birte K. Skrumsager,<br />
Senior <strong>Clinical</strong> Pharmacologist, Novo Nordisk A/S,<br />
Bagsvaerd, Denmark, Ulrik Mouritzen, International Medical<br />
Officer, Novo Nordisk A/S, Bagsvaerd, Denmark, Grant<br />
McArthur, Pr<strong>of</strong>essor, Cancer Trials Australia, Peter<br />
MacCallum Cancer Centre, Melbourne, Australia, Ian D.<br />
Davis, Pr<strong>of</strong>essor, Cancer Trials Australia, Austin Hospital,<br />
Melbourne, Australia, Kresten Skak, Senior Scientist, Novo<br />
Nordisk A/S, Bagsvaerd, Denmark<br />
Interleukin-21 is a pleiotropic class I cytokine that<br />
activates CD8+ T cells and NK cells. In a phase 1 dose<br />
escalation trial <strong>of</strong> intravenous IL-21 administered in two<br />
different dose regimens, the pharmacodynamic effects<br />
were monitored by multi-analyte pr<strong>of</strong>iling <strong>of</strong> 90 serum<br />
proteins and global transcriptional pr<strong>of</strong>iling <strong>of</strong> peripheral<br />
CD8+ T cells. A total <strong>of</strong> 17 serum proteins were observed to<br />
increase at least 2-fold upon IL-21 treatment whereas 4<br />
proteins were down-regulated. Each dose regimen displayed<br />
a distinct pattern <strong>of</strong> regulation, reflecting differences<br />
in pharmacodynamic effects. Among the up-regulated<br />
proteins were acute-phase proteins, chemokines, and<br />
cytokines, e.g. CRP, TNF-α, MCP-1, and IL-18. In CD8+ T<br />
cells, isolated within the first week <strong>of</strong> treatment, a total <strong>of</strong><br />
471 probe sets were found to be significantly differentially<br />
regulated. In particular, effector genes such as granzyme A<br />
and interferon-gamma as well as chemokine receptor<br />
genes, such as CXCR3 and CXCR6, were clearly up-regulated<br />
upon IL-21 treatment. These observations may indicate<br />
stimulation <strong>of</strong> both effector functions and motility <strong>of</strong> CD8+<br />
T cells. Moreover, suggestive <strong>of</strong> increased proliferation <strong>of</strong><br />
CD8+ T cells, levels <strong>of</strong> several DNA replication and cell cycle<br />
related genes, including PCNA and Ki-67, were induced in a<br />
dose-dependent manner. The presented data demonstrate<br />
that administration <strong>of</strong> IL-21 in patients with stage IV<br />
malignant melanoma induces a variety <strong>of</strong> pharmacodynamic<br />
responses related to immunological activation that warrants<br />
further clinical investigations <strong>of</strong> this cytokine.<br />
doi:10.1016/j.clim.2007.03.095<br />
Su.55 Differential Expression on the TNF-RI, IL-1RI,<br />
IL-6R Membrane and Soluble Receptors and NF-kB,<br />
AP-1 Activation by In Vitro Proteasome Inhibition in<br />
U937 cells<br />
Alejandro Bravo-Cuéllar, MD, Instituto Mexicano del Seguro<br />
Social, Centro de Investigacion Biomedica de Occidente<br />
Division de Inmunologia, Guadalajara, Mexico, Pablo C.<br />
Ortiz-Lazare, Chemistry Biologist and Pharmacology,<br />
Instituto Mexicano del Seguro Social, Centro de<br />
Investigacion Biomedica de Occidente Division de<br />
Inmunologia, Guadalajara, Mexico, Jorge R. Dominguez-<br />
Rodriguez, Biologist, Instituto Mexicano del Seguro Social,<br />
Centro de Investigacion Biomedica de Occidente Division de<br />
Inmunologia, G, Mexico, Jose M. Lerma-Diaz, MD,<br />
Universidad de Guadalajara, CUALTOS, Tepatitlan de<br />
Morelos, Mexico, Georgina Hernandez-Flores, Biologist,<br />
Instituto Mexicano del Seguro Social, Centro de<br />
Investigacion Biomedica de Occidente Division de<br />
Inmunologia, Guadalajara, Mexico, Piedad C. Gomez-<br />
Contreras, Chemistry Biologist and Pharmacology, Instituto<br />
Mexicano del Seguro Social, Centro de Investigacion<br />
Biomedica de Occidente Division de Inmunologia,<br />
Guadalajara, Mexico, Martha Barba-Barajas, Chemistry<br />
Biologist and Pharmacology, Instituto Mexicano del Seguro<br />
Social, Centro de Investigacion Biomedica de Occidente<br />
Division de Inmunologia, Guadalajara, Mexico,<br />
Luis F. Jave-Suarez, Biologist, Instituto Mexicano del Seguro<br />
Social, Centro de Investigacion Biomedica de Occidente<br />
Division de Inmunologia, Guadalajara, Mexico, Adriana C.<br />
Aguilar-Lemarrroy, Biologist, Instituto Mexicano del Seguro<br />
Social, Centro de Investigacion Biomedica de Occidente<br />
Division de Inmunologia, Guadalajara, Mexico<br />
Proteasome is a large multimeric protease complex,<br />
which plays a vital role in several cellular functions, such<br />
as: proteins degradation, regulation <strong>of</strong> cyclins and transcription<br />
factors, its also related with antigens degradation. On<br />
the other hand several stimuli such as proinflammatory<br />
cytokines, free oxygen radicals, components <strong>of</strong> the bacteria<br />
cellular wall, can trigger NF-kB activation, which normally<br />
exits as an inactive form, characterized by its union at the<br />
trimolecular complex called IkB. NF-kB activation begins by<br />
phosphorylation, ubiquitination and degradation <strong>of</strong> IkB<br />
complex in the proteasome. Once NF-kB transcriptional<br />
factor is liberated from its natural IkB inhibitor, it can reach<br />
the nucleus and link to promoters regions <strong>of</strong> genes that codify<br />
for TNF-α, IL-1β, IL-6, membrane receptors, and adhesion<br />
molecules. Monocyte/macrophages, lymphocytes and others<br />
cells generate these cytokines that increase the inflammatory<br />
process. TNF-α, IL-1β, IL-6 have been associated with<br />
different pathologies such as rheumatoid arthritis, inflammatory<br />
intestinal disease, shock septic. In this study, we
Abstracts<br />
investigated the in vitro effects <strong>of</strong> the proteasome inhibitor<br />
MG132 on TNF-RI, IL-1RI and IL-6R, the activation <strong>of</strong> NF-kB<br />
and AP-1 in U937 cells. Cell viability was evaluated by flow<br />
cytometry using cellular annexin V binding. Proteasome<br />
inhibition increases the release <strong>of</strong> TNF-RI and IL-1RI from<br />
U937 cells and decreases their cell surface expression. In<br />
other way, the MG132 increases the cell surface expression <strong>of</strong><br />
IL-6R and decreases their liberation. The proteasome<br />
inhibitor, also, led to increased LPS+PMA-induced AP-1<br />
activation, and attenuated LPS+PMA-induced IkB degradation<br />
resulting in abolished NF-kB activation.<br />
doi:10.1016/j.clim.2007.03.096<br />
Su.56 Mucosal and Systemic T Cell Responses to<br />
Cry1Ac Protoxin from Bacillus Thuringiensis<br />
Aldo Arturo Reséndiz Albor, Cell Biology, Universidad<br />
Nacional Autonoma de Mexico, Mexico, Leticia<br />
Moreno-Fierros, Cell Biology, Inmunidad en Mucosas<br />
UBIMED, FES-Iztacala, Universidad Nacional Autonoma de<br />
Mexico, Mexico<br />
Cry1Ac protoxin (pCry1Ac) from Bacillus thuringiensis is a<br />
potent immunogen with mucosal and systemic adjuvant<br />
properties when administered to mice by systemic or<br />
mucosal routes although its mechanism <strong>of</strong> action remains<br />
unknown. Thus, in this work, we investigated in vivo and in<br />
vitro the proportion <strong>of</strong> T cells producing IL-2, IL-4, IL-10,<br />
and IFN-g by intracellular cytokine staining on CD3+ T<br />
lymphocytes freshly isolated from spleen and Peyer's<br />
patches (PP) following intraperitoneal immunization <strong>of</strong><br />
pCry1Ac. Furthermore, we evaluated the induction <strong>of</strong><br />
CD69 and CD25 in T cell subsets after a single intraperitoneal<br />
administration <strong>of</strong> pCry1Ac in spleen and PP as a<br />
parameter for detecting T cell activation following by in<br />
vitro stimuli with pCry1Ac. In control mice we observed that<br />
PP present T cells spontaneously producing IFN-g while<br />
cytokine production was not detected in spleen lymphocytes.<br />
Addition <strong>of</strong> pCry1Ac to splenic and PP T cell cultures<br />
promoted a Th1 pr<strong>of</strong>ile <strong>of</strong> cytokines with high levels <strong>of</strong> IL-2<br />
and IFN-g without IL-4. We also found that intraperitoneal<br />
immunization with pCry1Ac increase <strong>of</strong> T lymphocytes<br />
expressing CD69, IL-4 in the spleen and CD69, IL-2 and<br />
IFN-g in PP. pCry1Ac stimulation led to rapid expression <strong>of</strong><br />
CD69 on both TCD4+ and B cells between 4 h post incubation<br />
in spleen and PP which remained stable throughout the 72 h<br />
culture period. The effects elicited by pCry1Ac on cytokine<br />
pr<strong>of</strong>iles are different between lymphocytes from the spleen<br />
and PP suggesting that their phenotypic and functional<br />
differences may influence the immunomodulatory effects <strong>of</strong><br />
the protein.<br />
doi:10.1016/j.clim.2007.03.097<br />
Su.57 Effects <strong>of</strong> a Novel Mucosal Adjuvant AT-1002<br />
on Immune Responses in Mice Immunized<br />
Intranasally with Ovalbumin<br />
Amir Tamiz, Director <strong>of</strong> Chemistry, Alba Therapeutics,<br />
Baltimore, MD, Niranjan Pandey, Director <strong>of</strong> Biology, Alba<br />
Therapeutics, Baltimore, MD, Blake Paterson, CEO, Alba<br />
Therapeutics, Baltimore, MD, Sefik Alkan, Vice President,<br />
Alba Therapeutics, Baltimore, MD<br />
Antigens, adjuvants and delivery systems are the main<br />
essential components to the development <strong>of</strong> efficient<br />
vaccines. The purpose <strong>of</strong> this study was to evaluate AT-<br />
1002 HCl, a six-mer synthetic peptide, FCIGRL, derived<br />
from Zonula Occludens Toxin (cholera's second toxin) as<br />
an effective mucosal delivery agent that is expected to<br />
enhance antigen passage through the mucosal/endothelial<br />
barriers and hence stimulate antigen-specific immune<br />
responses. In this study, female Balb/c mice were<br />
immunized intranasally with Ova and AT-1002 simultaneously,<br />
four times in 21-day intervals and complete<br />
antibody responses to Ova were measured in the serum<br />
and in vaginal washes using quantitative Ova-specific<br />
ELISA. Effects <strong>of</strong> AT-1002 stimulation on IgG subclasses<br />
(IgG1, IgG2a, IgG2b, IgA and IgE) were studied. Ovaspecific<br />
T cell responses were also measured at 5–8 days<br />
after the last immunization in both the spleen and the<br />
lung <strong>of</strong> all mice. We found that mice immunized with Ova<br />
in the presence <strong>of</strong> AT-1002 exhibit significantly higher<br />
antibody titers than those induced by immunization with<br />
antigen alone. In addition, we found that AT-1002 and<br />
Cholera Toxin induced similar patterns <strong>of</strong> Ova-specific IgG<br />
subclasses. In summary, our study suggests that the tight<br />
junction modulator AT-1002 might provide a novel<br />
approach to vaccination.<br />
doi:10.1016/j.clim.2007.03.098<br />
S161<br />
Su.58 Dual Targeting <strong>of</strong> CCR2 and CX3CR1 in an<br />
Arterial Injury Model <strong>of</strong> Vascular Inflammation<br />
Maya Jerath, Fellow, University <strong>of</strong> North Carolina,<br />
Department <strong>of</strong> Medicine, Chapel Hill, NC, Peng Liu,<br />
Research Associate, University <strong>of</strong> North Carolina, Thurston<br />
Arthritis Research Center, Chapel Hill, NC, Mauricio Rojas,<br />
Research Instructor, University <strong>of</strong> North Carolina,<br />
Department <strong>of</strong> Medicine, Chapel Hill, NC, Mary Struthers,<br />
Research Fellow, Merck Research Laboratories, Department<br />
<strong>of</strong> <strong>Immunology</strong>, Rahway, NJ, Julie Demartino, Senior<br />
Director, Merck Research Laboratories, Department <strong>of</strong><br />
<strong>Immunology</strong>, Rahway, NJ, Kathryn Lyons, Senior Research<br />
Associate, Merck Research Laboratories, Department <strong>of</strong><br />
Medicinal Chemistry, Rahway, NJ, Ruoqi Peng, Senior<br />
Research Immunologist, Merck Research Laboratories,<br />
Department <strong>of</strong> <strong>Immunology</strong>, Rahway, NJ, Lihu Yang,<br />
Director, Merck Research Laboratories, Department <strong>of</strong><br />
Medicinal Chemistry, Rahway, NJ, Dhaval Patel, Pr<strong>of</strong>essor,<br />
The University <strong>of</strong> North Carolina, Department <strong>of</strong> Medicine,<br />
Chapel Hill, NC<br />
Rationale: CCR2 and CX3CR1 are chemokine receptors<br />
differentially expressed on monocyte subsets in diverse<br />
species from rodents to man, and each receptor has been<br />
individually hypothesized to play a role in inflammatory<br />
cell trafficking to atherosclerotic lesions. We hypothesized<br />
that CCR2 and CX3CR1 had non-redundant effects in<br />
vascular inflammation, and sought to determine their<br />
combined effect. Methods: We used a murine vascular<br />
injury model in which both CCR2 KO and CX3CR1 KO have
S162 Abstracts<br />
shown a partial block in the injury response. Experimental<br />
arms consisted <strong>of</strong> C57Bl/6 WT mice, CX3CR1 KO mice, WT<br />
mice given a small molecule CCR2 antagonist (MRL-677,<br />
Merck & Co.) and CX3CR1 KO mice given the same<br />
antagonist. Vessels were harvested at 14 days analyzed<br />
by histology, morphometry and immunohistochemistry.<br />
Results: Blocking CCR2 with MRL-677 resulted in a 39%<br />
decrease in the vascular injury response (n=10, p=NS).<br />
CX3CR1 deficient mice had similar injury to WT animals<br />
(n=8, p=NS). Mice in which both CCR2 and CX3CR1<br />
pathways were targeted (CX3CR1 KO mice given MRL-677)<br />
had an 86% decrease in the injury response (n=6, p=0.002).<br />
Conclusions: In this study, we have shown that blocking<br />
CCR2 with a low molecular weight antagonist ameliorates<br />
the inflammatory response to vascular injury. Simultaneous<br />
targeting <strong>of</strong> both CCR2 and CX3CR1 has complementary and<br />
perhaps synergistic effects on the development <strong>of</strong> the<br />
vascular inflammatory response. Our results imply that<br />
CCR2 and CX3CR1 have independent, non-overlapping roles<br />
in vascular inflammation and that approaches to target<br />
both may have more beneficial effects than targeting each<br />
one separately.<br />
doi:10.1016/j.clim.2007.03.099<br />
Su.59 Cytokine and Chemokine Production by NK<br />
Cells Activated by Monokines IL-12, IL-18<br />
Following Crosslinking <strong>of</strong> CD16 and Modulation<br />
by KIR<br />
Zaheed Husain, Junior Investigator/Instructor, CBR<br />
Institute for Biomedical Research, Harvard Medical<br />
School, Boston, MA, Devendra Dubey, Investigator, CBR<br />
Institute for Biomedical Research, Harvard Medical<br />
School, Boston, MA, Shampa Das, Research Fellow, CBR<br />
Institute for Biomedical Research, Harvard Medical<br />
School, Boston, MA, Chester Alper, Pr<strong>of</strong>essor, CBR<br />
Institute for Biomedical Research, Harvard Medical<br />
School, Boston, MA<br />
NK cells express both activation and inhibitory receptors.<br />
Activating receptors play a central role in cytokine and<br />
chemokine production that is modulated by inhibitory<br />
receptors. We used high throughput, antibody-based cytokine<br />
chips to study the levels <strong>of</strong> secreted cytokines/<br />
chemokines. Data was collected to study the rate <strong>of</strong><br />
cytokines/chemokines production and secretion by human<br />
NK cells activated by monokines IL-12 and IL-18 following<br />
crosslinking <strong>of</strong> CD16. We also determined the effects <strong>of</strong> KIR<br />
on the production <strong>of</strong> these cytokines/chemokines. Several<br />
characteristics were observed: (1) IFN-g and IL-8 showed a<br />
linear increase with time, (2) the level <strong>of</strong> chemokines<br />
peaked within 2 hrs and reverted back to control levels<br />
within 24 hrs. The synthesis <strong>of</strong> chemokines was significantly<br />
higher (3.5 to 4.5 fold) than cytokines such as IFN-γ. Several<br />
cytokines and growth factors were inhibited by KIR3DL1. The<br />
mechanism <strong>of</strong> the increase in the levels <strong>of</strong> IFN-γ and IL-8 was<br />
further investigated by analyzing intracellular cytokine<br />
production using 4-color FACS analysis. Exposure <strong>of</strong> IFN-γ<br />
to NK cells induced a rapid increase in the intracellular<br />
expression <strong>of</strong> IL-8 suggesting that increase in IL-8 production<br />
could be due to the de novo synthesis <strong>of</strong> IL-8 by NK cells. This<br />
study shows that IL-12/IL-18/CD16 induced activation <strong>of</strong> NK<br />
cells triggers several signaling pathways that regulate<br />
adaptive immunity.<br />
doi:10.1016/j.clim.2007.03.100<br />
Su.60 Differential Effect <strong>of</strong> NK Activation Molecules<br />
CD16 and 2B4 on Cytokine Production: Significant<br />
Inhibition <strong>of</strong> GM-CSF but not IFN-g, TNF-α or<br />
Cytotoxic Activity<br />
Zaheed Husain, Junior Investigator/Instructor, CBR Institute<br />
for Biomedical Research, Harvard Medical School, Boston,<br />
MA, Shampa Das, Research Fellow, CBR Institute for<br />
Biomedical Research, Harvard Medical School, Boston, MA,<br />
Chester Alper, Pr<strong>of</strong>essor, CBR Institute for Biomedical<br />
Research, Harvard Medical School, Boston, MA, Devendra<br />
Dubey, Investigator, CBR Institute for Biomedical Research,<br />
Harvard Medical School, Boston, MA<br />
NK cells express several activation and inhibitory<br />
receptors which are involved in cytokine, chemokine and<br />
growth factor production and cytotoxic activity against<br />
virus infected cells, tumor or cells deficient in HLA class I<br />
expression. Crosslinking <strong>of</strong> CD16 or 2B4 significantly<br />
increases IFN-γ, TNF-α and GM-CSF production, and<br />
cytotoxic activity in redirected lysis assay. It is not known<br />
how co-crosslinking <strong>of</strong> these two receptors affect cytokine<br />
production and cytotoxic activity. To investigate this<br />
question we used antibody mediated crosslinking <strong>of</strong> CD16<br />
and 2B4 molecules on IL-2 activated NK cells activity in<br />
redirected lysis assay. Separate engagement <strong>of</strong> CD16 or 2B4<br />
strongly enhanced cytotoxic activity, IFN-γ, TNF-α and GM-<br />
CSF production. These are inhibited by co-crosslinking <strong>of</strong><br />
KIR receptors. Co-engagement <strong>of</strong> these activation receptors<br />
strongly inhibited the production <strong>of</strong> GM-CSF (N90%) but not<br />
IFN-γ or TNF-α production or cytotoxic activity suggesting<br />
that the pathway for GM-CSF production is separate from<br />
IFN-γ or TNF-α production. This further suggests that CD16<br />
and 2B4 may function together to optimize cytokine<br />
production.<br />
doi:10.1016/j.clim.2007.03.101<br />
Su.61 Augmenting CD8+ T Cell Responses with<br />
IL-15/sIL-15Rα Complexes<br />
Mark Rubinstein, Postdoctoral Fellow, University <strong>of</strong><br />
California, San Diego, La Jolla, CA, Jonathan Sprent,<br />
Pr<strong>of</strong>essor, Garvan Institute <strong>of</strong> Medical Research,<br />
Darlinghurst, Australia, Ananda Goldrath, Assistant<br />
Pr<strong>of</strong>essor, University <strong>of</strong> California, San Diego,<br />
La Jolla, CA<br />
Administration <strong>of</strong> IL-15 has shown efficacy in augmenting<br />
CD8+ T cell responses following vaccination. However,<br />
soluble IL-15 is limited in vivo by half life and poor biological<br />
activity. Recently, we showed that the biological activity <strong>of</strong><br />
soluble IL-15 is much improved after interaction with<br />
recombinant soluble IL-15Rα. After injection, soluble IL-<br />
15/IL-15Rα complexes rapidly induce strong and selective<br />
expansion <strong>of</strong> memory CD8+ cells and natural killer cells.
Abstracts<br />
Surprisingly, we also find that soluble IL-15/IL-15Rα complexes<br />
can drive conversion <strong>of</strong> naïve Tcells to memory Tcells<br />
in vivo, without administration <strong>of</strong> antigen. This conversion is<br />
associated with an acquisition <strong>of</strong> effector function. Our<br />
results suggest the therapeutic potential <strong>of</strong> IL-15 could be<br />
considerably enhanced by administration <strong>of</strong> preformed<br />
soluble IL-15/IL-15Rα complexes.<br />
doi:10.1016/j.clim.2007.03.102<br />
Su.62 Evaluation <strong>of</strong> IL-1 Beta Secretion Level <strong>of</strong><br />
Human Osteoblasts and Morphology <strong>of</strong> These Cells<br />
Adjunct to Gray MTA, White MTA, Portland Cement<br />
and IRM as Retr<strong>of</strong>illing<br />
Jalil Tavakkol Afshari, Vice President for Research,<br />
Immunogenetics Department, Bu-Ali Research Institute,<br />
Mashhad University <strong>of</strong> Medical Sciences, Mashhad, Iran,<br />
Navid Aghasizadeh, Dentist, School <strong>of</strong> Dentistry, Mashhad<br />
University <strong>of</strong> Medical Sciences, Mashhad, Iran, Maryam<br />
Bidar, Dentist, School <strong>of</strong> Dentistry, Mashhad University<br />
<strong>of</strong> Medical Sciences, Mashhad, Iran, Mohammad Ali<br />
Fahmidekar, Researcher, Immunogenetics Department,<br />
Bu-Ali Research Institute, Mashhad University <strong>of</strong><br />
Medical Sciences, Mashhad, Iran, Mohammad Zarabi,<br />
Dentist, School <strong>of</strong> Dentistry, Mashhad University <strong>of</strong><br />
Medical Sciences, Mashhad, Iran, Azam Brook,<br />
Researcher, Immunogenetics Department, Bu-Ali Research<br />
Institute, Mashhad University <strong>of</strong> Medical Sciences,<br />
Mashhad, Iran<br />
Ostetoblasts and ligament periodontal cells are the<br />
essential cells for wound repair after root-end resections<br />
and perforation repair. Cell attachment and spread on the<br />
surface materials and evaluation <strong>of</strong> the cell secretory<br />
function are primary phases in normal cell function. The<br />
aim <strong>of</strong> this study was evaluating osteoblasts (MG-63) function<br />
morphologically and IL-1 β level adjacent to gray MTA, white<br />
MTA, Portland cement and IRM, as filling materials for rootend<br />
fillings and repairing perforations. Human osteoblasts<br />
(MG-63) were grown in RPMI-1640 medium. Materials were<br />
mixed and seeded in appropriate plates. Cells were added<br />
after primary setting <strong>of</strong> materials. They were surveyed in<br />
days 1, 3 and 7. The amount <strong>of</strong> IL-1 β in each specimen was<br />
measured by ELISA. Morphological outcomes showed that<br />
after 7 days, a large amount <strong>of</strong> osteoblasts adjacent to gray<br />
and white MTA had a nice attachment. Cells adjacent to<br />
Portland cement were round and mostly separated from the<br />
surface. All cells were round and separated from the plate<br />
surface adjacent to IRM. Levels <strong>of</strong> IL-1 β adjacent to gray and<br />
white MTA were significantly more than to IRM and Portland<br />
cement (P=0.00). Amount <strong>of</strong> IL-1 β was not significantly<br />
different adjacent to gray and white MTA. Osteoblasts<br />
adjacent to gray and white MTA perform more appropriate<br />
response in comparison to Portland cement and IRM. Therefore,<br />
noticing the color <strong>of</strong> white MTA, it can be substituted by<br />
gray MTA in places with more cosmetic effects. It also<br />
appears that Portland cement is not a good substitute for<br />
MTA.<br />
doi:10.1016/j.clim.2007.03.103<br />
Su.63 Blockade <strong>of</strong> CD40−CD40 Ligand Interactions<br />
Attenuates Skin Fibrosis and Autoimmunity in the<br />
Tight-skin Mouse<br />
Kazuhiro Komura, MD, Department <strong>of</strong> Dermatology,<br />
Nagasaki University Graduate School <strong>of</strong> Biomedical<br />
Sciences, Nagasaki, Japan, Manabu Fujimoto, MD,<br />
Department <strong>of</strong> Dermatology, Kanazawa University Graduate<br />
School <strong>of</strong> Medical Science, Kanazawa, Japan, Minoru<br />
Hasegawa, MD, Department <strong>of</strong> Dermatology, Kanazawa<br />
University Graduate School <strong>of</strong> Medical Science, Kanazawa,<br />
Japan, Shinichi Sato, MD, Department <strong>of</strong> Dermatology,<br />
Nagasaki University Graduate School <strong>of</strong> Biomedical<br />
Sciences, Nagasaki, Japan<br />
The tight-skin (TSK/+) mouse, a mouse model for human<br />
systemic sclerosis (SSc), develops cutaneous fibrosis and<br />
autoimmunity. Molecular mechanisms <strong>of</strong> fibrosis and autoimmune<br />
responses in both TSK/+ mice and SSc remain<br />
unknown. In this study, we investigated the role <strong>of</strong> CD40/<br />
CD40 ligand (CD40L) interactions in TSK/+ mice and SSc. The<br />
blockade <strong>of</strong> CD40/CD40L interactions by anti-CD40L mAb<br />
reduced the development <strong>of</strong> cutaneous fibrosis (65%) and<br />
autoantibody production in TSK/+ mice. Furthermore, abnormal<br />
CD40/CD40L interactions in TSK/+ mice was suggested<br />
by up-regulated CD40L expression on CD4+ T<br />
lymphocytes, elevated plasma CD40L levels, and hyperresponsiveness<br />
to CD40 stimulation <strong>of</strong> B cells and fibroblasts. In<br />
addition, augmented CD40/CD40L signaling in human SSc was<br />
also suggested by elevated levels <strong>of</strong> circulating soluble<br />
CD40L and circulating soluble CD40. In addition to the<br />
reports from other groups, the results <strong>of</strong> the current studies<br />
suggest that CD40/CD40L interactions can be therapeutical<br />
targets in SSc.<br />
doi:10.1016/j.clim.2007.03.104<br />
S163<br />
Su.64 Intracellular Expression <strong>of</strong> TNF-α and IFN-γ<br />
by Peripheral Blood Mononuclear Cells in a Family<br />
with Rosacea<br />
Gabriel Andres Luna-Baca, MD, Institute <strong>of</strong> Ophthalmology<br />
Conde de Valenciana, Mexico City, Mexico, Concepcion<br />
Santacruz-Valdes, MD, Cornea and Refractive Surgery<br />
Department, Institute <strong>of</strong> Ophthalmology Conde de<br />
Valenciana, Mexico City, Mexico, Maria Carmen<br />
Jimenez-Martinez, MD, PhD, Research Unit, Institute <strong>of</strong><br />
Ophthalmology Conde de Valenciana, Mexico City, Mexico<br />
Introduction: Rosacea is a chronic dermatologic condition<br />
that predominantly affects the central aspect <strong>of</strong> the face.<br />
Although the pathophysiology <strong>of</strong> rosacea remains unknown,<br />
there is increasing agreement that inflammatory mediators<br />
are operative in this disorder. Recent studies have shown the<br />
role <strong>of</strong> oxidative stress and antioxidative system disorders in<br />
Rosacea. Despite IFN-γ and TNF-α have been involved in the<br />
pathogenesis <strong>of</strong> many dermatologic diseases driving to the<br />
exacerbation <strong>of</strong> inflammatory and prooxidative responses,<br />
there is no evidence that these mediators are involved in<br />
rosacea. Objective: To evaluate the expression <strong>of</strong> IFN-γ and<br />
TNF-α by peripheral blood mononuclear cells (PBMC) in a<br />
family with Rosacea (RF). Methods: CD4, CD8, CD19, CD56,<br />
IFN-γ, TNF-α, IL-1β, IL-4 and IL-6 expression was determined
S164 Abstracts<br />
by flow cytometry on PBMC from RF and also on PBMC from<br />
healthy controls (HC). Results. IFN-γ+cells in RF-1=38.5 vs.<br />
15 HC (p=0.0016); TNF-α+cells in RF-1=55 vs. 3.2 HC (p=<br />
0.0015); IFN-γ+cells in RF-2=32.6 vs. 8.1 HC (p=0.026);<br />
TNF-α+cells in RF-2=34.6 vs. 1.5 HC (p=0.0056); IFN-γ+cells<br />
in RF-2=32.6 vs. 8.1 HC (p=0.026); TNF-α+cells in RF-2=34.6<br />
vs. 1.5 HC (p=0.0056); IFN-γ+cells in RF-3=24.2 vs. 8.3 HC<br />
(p=0.04); TNF-α+cells in RF-3=32.2 vs. 3.1 HC (p=0.0046).<br />
CONCLUSIONS. Peripheral blood mononuclear cells from a<br />
Rosacea family have significant increase in the expression <strong>of</strong><br />
IFN-γ+ and TNF-α+ cells. IFN-γ and TNF-α may have a role in<br />
the inflammatory process <strong>of</strong> rosacea stimulating the release<br />
<strong>of</strong> reactive oxygen species in macrophages and neutrophils<br />
causing damage to facial follicles in persons with Rosacea.<br />
doi:10.1016/j.clim.2007.03.105<br />
Su.65 Does Quality <strong>of</strong> Life in Cutaneous Lupus<br />
Erythematosus Correlate with Disease Severity?<br />
Elizabeth Gaines, Pre-doctoral Research Fellow,<br />
University <strong>of</strong> Pennsylvania, Department <strong>of</strong> Dermatology,<br />
Philadelphia, PA<br />
The purpose <strong>of</strong> this study was to assess the correlation<br />
between cutaneous lupus erythematosus (CLE) disease<br />
severity, as measured by the CLASI, and quality-<strong>of</strong>-life<br />
(QoL), as measured by the Skindex-29. This was a prospective,<br />
longitudinal study from baseline (Day 0) to Day 56, done<br />
at a University hospital cutaneous autoimmunity clinic. Eight<br />
patients with biopsy-confirmed CLE (4 DLE, 2 SCLE, 2 DLE/<br />
SLE) completed the study. At each visit, patients completed<br />
the CLE-modified Skindex-29. Using scales from 0-10, they<br />
also assessed their general and skin health, and their pain,<br />
itch, and fatigue. The PI evaluated disease severity, with the<br />
CLASI, and the patient s global skin health using a scale from<br />
0−10. There was a significant improvement in total Skindex<br />
score over time (t=2.36, pb0.05). There was a moderate<br />
correlation (r=0.59, p=0.12, n=8) between change in activity<br />
(CLASI), and change in skin symptoms (Skindex). Neither<br />
change in emotion or function correlated with change in<br />
activity. There was no correlation between any Skindex<br />
subscale with damage. Skindex scores for patients with SCLE<br />
were, on average, lower and changed less than Skindex<br />
scores for patients with DLE. While both the CLASI activity<br />
score and the modified Skindex total score improved<br />
significantly over time, the correlation between the two<br />
scores was poor. Different elements <strong>of</strong> skin health may be<br />
captured by each index. Given the different disease courses<br />
and likelihood <strong>of</strong> damage, skin-related QoL may differ across<br />
different subtypes <strong>of</strong> CLE. Preliminary analysis <strong>of</strong> data from<br />
25 additional CLE patients supports these conclusions.<br />
doi:10.1016/j.clim.2007.03.106<br />
Su.66 Association Between the Polymorphism <strong>of</strong><br />
TGF-β1 Gene Promoter (−509CNT) and Idiopathic<br />
Chronic Urticaria<br />
Sara Hosseini-Farahabadi, Dr.; Researcher, Bu-Ali Research<br />
Institute (BARI), Department <strong>of</strong> Immunogenetics, Mashhad,<br />
Iran, Jalil Tavakkol-Afshari, Vice President <strong>of</strong> Research;<br />
Head, Department <strong>of</strong> Immunogenetics and Cell Culture,<br />
Bu-Ali Research Institute (BARI), Department <strong>of</strong><br />
Immunogenetics and Cell Culture, Mashhad, Iran, Rashin<br />
Ganjali, Researcher, Bu-Ali Research Institute (BARI),<br />
Department <strong>of</strong> Immunogenetics, Mashhad, Iran, Javad<br />
Ghaffari, Mazandaran University <strong>of</strong> Medical Sciences,<br />
Department <strong>of</strong> Pediatrics, Sari, Iran, Houshang Rafatpanah,<br />
Ghaem Medical Center, Department <strong>of</strong> Allergy and<br />
<strong>Immunology</strong>, Mashhad, Iran, Reza Farid-Hosseini, Head,<br />
Department <strong>of</strong> Allergy and <strong>Immunology</strong>, Ghaem Medical<br />
Center, Department <strong>of</strong> Allergy and <strong>Immunology</strong>,<br />
Mashhad, Iran<br />
Background: Idiopathic Chronic Urticaria (ICU), the most<br />
common form (70–80%) <strong>of</strong> chronic urticaria is supposed to<br />
have immune basis causes. It is speculated that the promoter<br />
polymorphism <strong>of</strong> TGF-β1 gene may be involved in ICU. This<br />
condition is thought to affect at least 0.1% <strong>of</strong> the population<br />
and <strong>of</strong>ten it can be severe and difficult to treat. Materials<br />
and Methods: A total <strong>of</strong> 40 patients with ICU and 41 normal<br />
subjects were studied. DNA was extracted from whole blood<br />
and TGF-β1 promoter −509CNT polymorphism was determined<br />
by PCR-RFLP method. Results: Out <strong>of</strong> the 40 patients<br />
with ICU, 11 (27.5%) had CC, 26 (65%) had CTand 3 (7.5%) had<br />
TT genotypes. A higher proportion <strong>of</strong> case subjects with the C<br />
allele (CT type or CC type) was found compared with the T<br />
allele. Conclusion: These results do suggest an influence <strong>of</strong><br />
genetic variability at the promoter <strong>of</strong> TGF-β1 gene<br />
(−509CNT) on the occurrence <strong>of</strong> ICU. This polymorphism<br />
has been shown as a useful genetic change in our study.<br />
Further work is required to confirm this result.<br />
doi:10.1016/j.clim.2007.03.107<br />
Su.67 Investigation <strong>of</strong> the Mechanisms <strong>of</strong> Pressure<br />
Ulcers Using a Cyclical Cutaneous<br />
Ischemic−reperfusion Injury Model<br />
Yuki Saito, Graduate Student, Kanazawa University,<br />
Dermatology, Kanazawa, Japan, Minoru Hasegawa, Assistant<br />
pr<strong>of</strong>essor, Kanazawa University, Dermatology, Kanazawa,<br />
Japan, Manabu Fujimoto, Associate pr<strong>of</strong>essor, Kanazawa<br />
university, Dermatology, Kanazawa, Japan, Kazuhiko<br />
Takehara, Pr<strong>of</strong>essor, Kanazawa university, Dermatology,<br />
Kanazawa, Japan<br />
The formation <strong>of</strong> pressure ulcers is considered to be<br />
derived from multifactorial factors. Among them, the occurrence<br />
<strong>of</strong> cycles <strong>of</strong> ischemic–reperfusion is an especially<br />
significant contributing factor. This study assessed immunological<br />
mechanism <strong>of</strong> a reproducible murine model <strong>of</strong><br />
ischemic–reperfusion injury by the external application <strong>of</strong><br />
magnets. Dorsal skin was gently pulled and placed between<br />
two round ceramic magnetic plates. A single ischemic–<br />
reperfusion cycle (I–R cycle) consisted <strong>of</strong> a 12 hours <strong>of</strong><br />
magnet placement followed by a 12 hours <strong>of</strong> release or rest.<br />
Three cycles were performed to induce pressure ulcer formation.<br />
A marked edema was observed after an I–R cycle (day<br />
1) and skin ulcer developed until day 6 (3 days post<br />
treatment). Then, skin ulcers were healed until day 20 in<br />
all mice. In the lesional skin, the infiltration <strong>of</strong> neutrophils<br />
and macrophages, and tumor necrosis factor-alpha expression
Abstracts<br />
were most prominent at day 2. Then, inducible nitric oxide<br />
synthase (iNOS) expression was augmented at day 3. While<br />
nitric oxide is considered to be involved in ischemic–<br />
reperfusion injury, iNOS is a critical enzyme for the synthesis<br />
<strong>of</strong> nitric oxide. iNOS is produced by various cells including<br />
neutrophils and macrophages stimulated with proinflammatory<br />
cytokines such as tumor necrosis factor-alpha. Therefore,<br />
our findings suggest that iNOS production from<br />
infiltrated neutrophils and macrophages stimulated by<br />
tumor necrosis factor-alpha contributes to the development<br />
<strong>of</strong> skin ulcer in this model. We propose that this cyclical<br />
cutaneous ischemic–reperfusion injury model is useful for<br />
investigating the mechanism or developing novel therapies <strong>of</strong><br />
pressure ulcers.<br />
doi:10.1016/j.clim.2007.03.109<br />
Su.68 CD19 Expression in B cells is Important for<br />
Suppression <strong>of</strong> Contact Hypersensitivity<br />
Manabu Fujimoto, Associate Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Dermatology, Kanzawa University, Kanazawa Ishikawa,<br />
Japan, Rei Watanabe, Student, Department <strong>of</strong> Dermatology,<br />
University <strong>of</strong> Tokyo, Bunkyo Tokyo, Japan, Thomas Tedder,<br />
Pr<strong>of</strong>essor, Department <strong>of</strong> <strong>Immunology</strong>, Duke University<br />
Medical Center, Durham, NC, Kunihiko Tamaki, Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> Dermatology, University <strong>of</strong> Tokyo, Bunkyo<br />
Tokyo, Japan<br />
Contact hypersensitivity (CHS) is a cutaneous immune<br />
reaction mediated mainly by Ag-specific effector Tcells, and<br />
regarded as a model for Th1/Tc1-mediated inflammation.<br />
However, recent reports have suggested pivotal roles <strong>of</strong> B<br />
cells in CHS. CD19 is a member <strong>of</strong> the Ig superfamily expressed<br />
on B cells and follicular dendritic cells, and serves as a<br />
positive B cell response regulator which defines signaling<br />
thresholds critical for B cell responses. In the current study,<br />
we assessed the role <strong>of</strong> the B cell-specific surface molecule<br />
CD19 on the development <strong>of</strong> CHS through examining CD19deficient<br />
mice. Although CD19-deficient mice are hyposensitive<br />
to a variety <strong>of</strong> transmembrane signals, CD19 loss resulted<br />
in increased and prolonged reaction <strong>of</strong> CHS, suggesting an<br />
inhibitory role <strong>of</strong> CD19 expression in the etiology <strong>of</strong> CHS.<br />
Sensitized draining lymph nodes and elicited ear lesions from<br />
CD19-deficient mice exhibited Th1/Tc1-shifted cytokine<br />
pr<strong>of</strong>ile with increased IFN-fÁ expression and decreased IL-<br />
10 expression. Adoptive transfer experiments revealed that<br />
CD19 expression in recipient mice was required for optimal<br />
suppression <strong>of</strong> CHS response, indicating its role in elicitation<br />
phase. Furthermore, spleen B-2 cells, especially marginal<br />
zone B cells, from wild type mice were able to normalize<br />
exaggerated CHS reactions in CD19-deficient mice. Thus,<br />
CD19 expression in B cells is critical for termination <strong>of</strong> CHS<br />
responses, possibly through the function <strong>of</strong> regulatory B cells.<br />
doi:10.1016/j.clim.2007.03.111<br />
Su.69 Automated Enumeration <strong>of</strong> Skin Mast Cells by<br />
Laser Scanning Cytometry<br />
Esther Trueblood, Director <strong>of</strong> Pathology, Amgen, Inc./<br />
Pathology, Seattle, WA, Stephen Z., Scientist, Amgen, Inc./<br />
<strong>Clinical</strong> <strong>Immunology</strong>, Thousand Oaks, CA, Andrea Ita, Senior<br />
Scientist, Amgen, Inc./Inflammation, Thousand Oaks, CA,<br />
John Ferbas, Director Medical Sciences, Amgen, Inc./<br />
<strong>Clinical</strong> <strong>Immunology</strong>, Thousand Oaks, CA, James Chung,<br />
Medical Sciences Director, Amgen, Inc./Early development,<br />
Thousand Oaks, CA, Gordon Ng, Scientific Director, Amgen,<br />
Inc./Inflammation, Thousand Oaks, CA, Gloria Juan,<br />
Senior Scientist, Amgen, Inc./<strong>Clinical</strong> <strong>Immunology</strong>,<br />
Thousand Oaks, CA<br />
Mast cells (MCs) are bone marrow-derived immune competent<br />
cells dependent on stem cell factor for survival,<br />
chemotaxis, functional activation, and adhesion to connective<br />
tissue matrix. After activation, MCs can release a<br />
variety <strong>of</strong> mediators including histamine, tryptase, leukotrienes,<br />
prostaglandins, contributing to anaphylactoid,<br />
allergic and inflammatory processes. MCs are also associated<br />
with wound repair together with fibroblasts and<br />
other immune cells. The current effort presents a novel<br />
application <strong>of</strong> Laser Scanning Cytometry (LSC) to study the<br />
time course <strong>of</strong> mast cell recruitment in a skin woundhealing<br />
model. A full-thickness incisional wound model was<br />
developed in the mouse. A skin biopsy was taken on day 8<br />
after wounding in animals treated with control or an antimurine<br />
c-Kit antibody (ACK2) that blocks SCF. A two-step<br />
protocol on the LSC was created to automate quantitation<br />
<strong>of</strong> wound-adjacent MCs. The first step executed a lowresolution<br />
optical scan that defined the position and area <strong>of</strong><br />
each biopsy on the slide. The second step consisted <strong>of</strong> a<br />
high-resolution scan that captured sequential 20× image<br />
fields <strong>of</strong> the entire biopsy and stitched the images together<br />
into a mosaic. Computer analysis segregated stored image<br />
events into discrete populations, and ultimately MCs. MCs<br />
counts were obtained from a defined region around the<br />
wound site. ACK2 treatment reduced wound-activated Mast<br />
Cell expansion as measured by LSC (T-test; P=0.0094) and<br />
consistent with the manual counts. This method <strong>of</strong><br />
quantitation allows implementation <strong>of</strong> a more efficient,<br />
objective and precise method to score effects on MCs which<br />
are tissue resident and difficult to analyze.<br />
doi:10.1016/j.clim.2007.03.112<br />
S165<br />
Su.70 Kawasaki Disease: Presentation <strong>of</strong> One Case<br />
Carlos Torres-Loza, Immunoallergist, West National Medical<br />
Center, UMAE-IMSS, Guadalajara, Mexico<br />
Kawasaki disease (KD) is the most common vasculitis <strong>of</strong><br />
childhood and may well be the most common vasculitis at any<br />
age. The aim <strong>of</strong> this work is to present a case with an atypical<br />
clinical presentation or perhaps to consider other immunoinflamatory<br />
syndrome and to share this clinical experience<br />
with other immunologists. Ketzalli is a girl <strong>of</strong> 5 years old who<br />
started abruptly with abdominal pain, remittent fever <strong>of</strong>ten<br />
up 38.5 °C and scarlatiniform and multiform cutaneous rash<br />
and irritability by 2–3 days. After that acute period, we<br />
treated with metamizole as antithermic. After that period,<br />
we realized that our patient started to feel better but, with<br />
changes in her lips characterized by dry, swollen and vertically<br />
cracked lips and diffusely without strawberry tongue.<br />
Actually, by that time we realized both erythema <strong>of</strong> palms
S166 Abstracts<br />
and soles and skin desquamation. However, after 1–2 days<br />
more we see how her fingertips begin to peel and how one<br />
typical lesion <strong>of</strong> vasculitis appear on skin <strong>of</strong> her right<br />
fingertip. Ketzalli was seen by several specialist in pediatrics<br />
including, infectologist, dermatologist, immunoallergist and<br />
not all <strong>of</strong> them were in agreement about diagnosis <strong>of</strong><br />
Kawasaki disease. Giving benefit <strong>of</strong> the doubt, Ketzalli was<br />
treated with IVIgG to a doses <strong>of</strong> 1.5 g/kg in one doses. After<br />
two weeks <strong>of</strong> such clinical event a normal echocardiogram<br />
was reported. As far as we know does not exist other clinical<br />
problem with such skin desquamation and peeling hence to<br />
share this case with other immunologists.<br />
doi:10.1016/j.clim.2007.03.113<br />
Su.72 CD4+CD25+ Regulatory T Cells Abrogate<br />
Psoriatic-like Skin Lesions in a Murine Model <strong>of</strong><br />
Psoriasis, Whereas TNFa Blockade Exacerbates the<br />
Lesions<br />
Hak-Ling Ma, Senior Research Scientist II, Wyeth Research,<br />
Inflammation Department, Cambridge, MA<br />
Psoriasis is a chronic inflammatory skin disease that is due<br />
to rapid epidermal proliferation with mononuclear cell infiltrates.<br />
Recent studies demonstrated that transferring<br />
naïve CD4+ T cells into scid/scid mice was able to induce<br />
psoriatic like lesions in the recipient mice. This indicates<br />
that T cells play a central role in the pathogenesis <strong>of</strong> the<br />
disease. By adoptive transferring naïve T cells that lacked<br />
Tregs, we found that recipient mice experienced close to<br />
100% incidence <strong>of</strong> disease with clinical symptoms (i.e. skin<br />
erythema and scaling) and histological features (i.e. epidermal<br />
thickening, rete pegs and hyperplasia <strong>of</strong> epidermis and<br />
keratinocytes) resembling that <strong>of</strong> human psoriasis. We also<br />
found that co-administration <strong>of</strong> Tregs with naïve T cells<br />
significantly decreased the incidence and severity <strong>of</strong><br />
psoriatic-like skin lesions. It has been shown that cytokines<br />
such as IL-12, TNFa, IL-22, and IL-17 play important roles in<br />
disease progression. TNFa antagonists have been used in<br />
treatment <strong>of</strong> psoriasis, however, they can also exacerbate<br />
skin lesions in some individuals. Interestingly, in our studies,<br />
soluble murine TNFRIIFc exacerbated disease in comparison<br />
to isotype control treated mice. Overall, our results suggest<br />
that Tregs inhibit whereas soluble murine TNFRIIFc exacerbates<br />
skin inflammation in this model.<br />
doi:10.1016/j.clim.2007.03.114<br />
Su.74 Investigator-Initiated Trial <strong>of</strong> Efalizumab for<br />
Atopic Dermatitis: A Pro<strong>of</strong>-<strong>of</strong>-Concept Study in<br />
Adults<br />
Melissa Magliocco, Assistant Pr<strong>of</strong>essor <strong>of</strong> Medicine,<br />
UMDNJ-Robert Wood Johnson Medical School, Department<br />
<strong>of</strong> Medicine, New Brunswick, NJ, Irina Lipets, Assistant<br />
Program Manager, UMDNJ-Robert Wood Johnson Medical<br />
School, New Brunswick, NJ, Viktor Dombrovskiy, Assistant<br />
Pr<strong>of</strong>essor <strong>of</strong> Medicine, UMDNJ-Robert Wood Johnson<br />
Medical School, New Brunswick, NJ, Alice Gottlieb,<br />
Pr<strong>of</strong>essor and Chair <strong>of</strong> the Department <strong>of</strong> Dermatology,<br />
Tufts-New England Medical Center, Boston, MA<br />
Background: Atopic dermatitis (AD) is an autoimmune<br />
skin disorder mediated acutely by predominantly activated<br />
Th2 T cells and in chronic stages by activated Th1 cells.<br />
There is a need for safe, effective AD treatment. Efalizumab<br />
is a humanized monoclonal IgG1 antibody that binds the<br />
CD11a subunit <strong>of</strong> LFA-1, inhibiting T cell activation,<br />
reactivation, and emigration into skin. Its FDA approval for<br />
psoriasis suggests that it would be effective and safe for AD,<br />
because both diseases exhibit Th1 cell predominance in<br />
lesional skin. Objective: To determine whether targeting<br />
CD11a on T cells decreases the clinical activity <strong>of</strong> AD.<br />
Methods: This was a single-arm, open-label, pilot study.<br />
Fifteen subjects with at least a “moderate” Investigator's<br />
Global Assessment (IGA) score were recruited. Subjects<br />
were excluded if they received photo/systemic therapy<br />
within 1 week <strong>of</strong> baseline. Subjects received a subcutaneous<br />
injection <strong>of</strong> efalizumab weekly. Efficacy was assessed<br />
monthly for 6 months using EASI (Eczema Area and Severity<br />
Index), IGA, a subjective pruritus assessment scale, and<br />
photography. Safety was evaluated by physical examination<br />
and laboratory tests. Results: Three subjects completed the<br />
study. Changes in EASI, IGA, and pruritus scores at 6 months<br />
compared with baseline were analyzed. The mean decrease<br />
in EASI was 15.3% (95% CI −3.4%–34.0%), whereas that <strong>of</strong> the<br />
IGA was 10.6% (95% CI 1.0%–20.2%). Reduction in mean<br />
pruritus score was 29.5% (95% CI 45.0%–104.1%) Few adverse<br />
events were noted, including infection. Conclusion: Treatment<br />
<strong>of</strong> AD with efalizumab resulted in a significant<br />
improvement in IGA, but did not significantly affect EASI<br />
or pruritus.<br />
doi:10.1016/j.clim.2007.03.115<br />
Su.75 Demodex Folliculorum and Chronic<br />
Granulomatous Disease<br />
Anete Sevciovic Grumach, University <strong>of</strong> Sao Paulo, São<br />
Paulo, Brazil, Rosemeire Navickas Constantino-Silva, BSc,<br />
Department <strong>of</strong> Dermatology, São Paulo, Brazil, Marcelo<br />
Mentha Nico, Department <strong>of</strong> Dermatology, University <strong>of</strong> São<br />
Paulo, São Paulo, Brazil, Alexandre Correa, BSc,<br />
Department <strong>of</strong> Dermatology, University <strong>of</strong> Sao Paulo, Sao<br />
Paulo, MRF, Correa, Hospital Mandaqui, Sao Paulo, Brazil,<br />
Israel Zeckcer, Department <strong>of</strong> Dermatology, University <strong>of</strong><br />
Sao Paulo, São Paulo, Brazil, Joao Bosco Oliveira,<br />
Department <strong>of</strong> Dermatology, University <strong>of</strong> Sao Paulo, São<br />
Paulo, Brazil, Alberto Jose da Silva Duarte, Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> Dermatology, São Paulo, Brazil, Dewton<br />
Moraes-Vasconcelos, Department <strong>of</strong> Dermatology, São<br />
Paulo, Brazil<br />
Background: Chronic Granulomatous Disease (CGD) is a<br />
primary immunodeficiency with a defect on oxidative<br />
metabolism <strong>of</strong> phagocytes. Demodex are also recognized as<br />
mites <strong>of</strong> the face and they live in the human pillous follicles.<br />
They affect aging people or immunosuppressed patients. No<br />
previous report has found those mites in primary immunodeficiencies.<br />
Objective: To report the clinical and laboratorial<br />
manifestations <strong>of</strong> a patient with CGD and Demodex<br />
folliculorum. Case report: A 1 year and 4 months old, male,<br />
born to a non-consanguineous family presenting with facial<br />
“suppurative” lesions was referred to our outpatient group in
Abstracts<br />
order to evaluate the recurrent localized lesions on his face.<br />
He was treated with several antibiotics with no clinical<br />
improvement. He had been hospitalized twice for bronchopneumonia<br />
(2X) and severe anemia receiving blood transfusions.<br />
The facial secretion was evaluated in the microscope<br />
and Demodex was identified. Considering the cause <strong>of</strong><br />
referral, DHR was performed and CGD was confirmed.<br />
Conclusion: The follicular pitiriasis or demodicidosis was<br />
first described in secondary immunodeficiencies such as<br />
leukemia or after kidney transplantation. This patient<br />
represents the first case report associating Demodex with a<br />
primary immunodeficiency (CGD).<br />
doi:10.1016/j.clim.2007.03.116<br />
Su.76 Immune Response Using Dialyzable Leukocyte<br />
Extract in the Treatment <strong>of</strong> Herpetic Keratitis<br />
Gabriel Andres Luna-Baca, MD, Research Unit, Institute <strong>of</strong><br />
Ophthalmology Conde de Valenciana, Mexico City, Mexico,<br />
Marisela Linares, Master <strong>of</strong> Science, Research Unit, Institute<br />
<strong>of</strong> Ophthalmology Conde de Valenciana, Mexico City,<br />
Mexico, Concepcion Santacruz-Valdes, MD, Cornea and<br />
Refractive Surgery Department, Institute <strong>of</strong> Ophthalmology<br />
Conde de Valenciana, Mexico City, Mexico, Raul Chavez, MD,<br />
PhD, Laboratorio de Inmunologia, Departamento de<br />
Bioquimica, Universidad Nacional Autonoma de Mexico,<br />
Mexico City, Mexico, Gustavo Aguilar-Velazquez, MD,<br />
Research Unit, Institute <strong>of</strong> Ophthalmology Conde de<br />
Valenciana, Mexico City, Mexico, Mayra Perez-Tapia, PhD,<br />
Department <strong>of</strong> <strong>Immunology</strong>, National School <strong>of</strong> Biological<br />
Sciences, ENCB-IPN, Mexico City, Mexico, Iris Estrada-<br />
Garcia, PhD, Department <strong>of</strong> <strong>Immunology</strong>, National School <strong>of</strong><br />
Biological Sciences, ENCB-IPN, Mexico City, Mexico, Sergio<br />
Estrada-Parra, PhD, Department <strong>of</strong> <strong>Immunology</strong>, National<br />
School <strong>of</strong> Biological Sciences, ENCB-IPN, Mexico City,<br />
Mexico, Maria Carmen Jimenez-Martinez, MD, PhD,<br />
Research Unit, Institute <strong>of</strong> Ophthalmology Conde de<br />
Valenciana, Mexico City, Mexico<br />
Introduction: T lymphocytes are involved in herpetic<br />
keratitis (HK) producing IFNg. Transfer factor (TF) has the<br />
ability to modulate the immune response, probably due to<br />
IFNg production. TF has been used in the treatment <strong>of</strong> viral<br />
infections like herpes simplex and zoster. Objective: To<br />
evaluate the cytokine expression by peripheral blood mononuclear<br />
cells (PBMC) from HK patients (p) treated with TF.<br />
Methods. CD4, CD8, IFNg, IL-4, perforin and granzyme expression<br />
was determined by flow cytometry on PBMC from<br />
HK-p, before (BTF) and after (ATF) treatment with TF and<br />
also from healthy controls (HC). Results: Percentage (%) CD4+<br />
IFNg+T cells in HK-p BTF=15 vs. 9 HC (p=0.028) vs. 33 ATF; %<br />
CD4+IL-4+Tcells in HK-p BTF=20 vs. 12 HC vs. 6.5 ATF; %CD8+<br />
IFNg+T cells in HK-p BTF=23 vs. 25 HC vs. 50 ATF (p=0.0002<br />
vs. ATF, p=0.005 vs. HC); %CD8+IL-4+T cells in HK-p BTF=7<br />
vs. 10 HC vs. 10 ATF; %CD8+IL-4+T cells=7 vs. 23 CD8+IFNg+T<br />
cells in HK-p BTF (p=0.005); %CD8+IL-4+T cells=10 vs. 50<br />
CD8+IFNg+T cells in HK-p ATF (p=0.003); %CD4+Granzyme+<br />
T cells in HK-p BTF=9.5 vs. 9.4 HC vs. 10.7 ATF; %CD4+<br />
Perforin+T cells in HK-p BTF=5.7 vs. 1.1 HC vs. 9 ATF; %CD4+<br />
Granzyme+Perforin+T cells in HK-p BTF=8.1 vs. 2.7 HC vs.<br />
10.8 ATF; %CD8+Granzyme+T cells in HK-p BTF=12 vs. 26 HC<br />
(p=0.012) vs. 10.7 ATF (p=0.008 vs. IS); %CD8+Perforin+Tcells<br />
in HK-p BTF=8.5 vs. 4.8 HC vs. 13.2 ATF; %CD8+ Granzyme+<br />
Perforin+T cells in HK-p BTF=32.8 vs. 33.5 HC vs. 37.1 ATF.<br />
Conclusions: CD8+lymphocytes from HK-p predominantly produce<br />
IFNg after treatment with TF. This could explain the better<br />
outcomes by a cytotoxic Tcell response in the studied patients.<br />
doi:10.1016/j.clim.2007.03.117<br />
Su.78 Gene Expression Pr<strong>of</strong>iling <strong>of</strong> Non-infectious<br />
Uveitis Patients Using Pathway Specific cDNA<br />
Microarray Analysis<br />
Zhuqing Li, Staff Scientist, Lab <strong>Immunology</strong>, NEI, NIH,<br />
Bethesda, MD, Sankaranarayana Mahesh, Postdoctoral<br />
Fellow, Laboratory <strong>of</strong> <strong>Immunology</strong>, NEI, NIH, Bethesda, MD,<br />
Baoying Liu, Fellow, <strong>Immunology</strong>, NEI, NIH, Bethesda, MD,<br />
Grace Clarke, Physician, <strong>Immunology</strong> Lab, NEI, NIH,<br />
Bethesda, MD, Wee Kiak Lim, Fellow, <strong>Immunology</strong>, NEI, NIH,<br />
Bethesda, MD, Robert Nussenblatt, Physician, <strong>Immunology</strong><br />
Lab, NEI, NIH, Bethesda, MD<br />
To study gene expression pr<strong>of</strong>iling <strong>of</strong> leukocytes from<br />
peripheral blood <strong>of</strong> patients with non-infectious uveitis,<br />
peripheral blood mononuclear cells (PBMCs) were isolated<br />
from 50 patients with clinically characterized non-infectious<br />
intermediate uveitis. Total RNA from PBMCs were extracted<br />
and quantified. Biotinylated probes were generated from<br />
total RNAs by incorporating biotinylated dUTP into synthesized<br />
cDNAs using a T7-polymerase linear amplification<br />
strategy. RNAs isolated from 20 healthy donors were pooled<br />
and served as normal control. Probes were hybridized to a<br />
pathway-specific cDNA microarray chip that contains some<br />
400 autoimmune and inflammatory response related genes.<br />
Signals for specific binding were recorded by a CCD camera<br />
and gene expression pr<strong>of</strong>iling was analyzed using GEArray<br />
Suite s<strong>of</strong>tware. Despite tremendous heterogeneity <strong>of</strong> gene<br />
expression among patients and normal controls, there were<br />
clear differential gene expression patterns comparing those<br />
from patients to those from normal control. Although there<br />
was more than 80% <strong>of</strong> overlap <strong>of</strong> gene expression, some 15% <strong>of</strong><br />
the genes were differentially expressed among patients<br />
compared to the normal donors. Several cytokine, chemokine<br />
or chemokine receptor genes were consistently more highly<br />
expressed among uveitis patients compared to the normal<br />
control. There seems less consistency in terms <strong>of</strong> downregulated<br />
genes among patients when compared to normal<br />
control. We show here that pathway specific cDNA microarray<br />
can be an efficient and economic strategy to pr<strong>of</strong>iling uveitis<br />
patients at the genomic level. Although preliminary and<br />
needing further confirmation, our data revealed several<br />
immune response genes that may be implicated in presumably<br />
autoimmune originated non-infectious uveitis.<br />
doi:10.1016/j.clim.2007.03.118<br />
S167<br />
Su.79 A Novel Model <strong>of</strong> MDP-induced Ocular<br />
Inflammation is Dependent on CARD15/NOD2 But<br />
Not on Interleukin-1<br />
Holly Rosenzweig, Postdoctoral Fellow, Oregon Health and<br />
Science University, Portland, OR, Tammy Martin, Research
S168 Abstracts<br />
Assistant Pr<strong>of</strong>essor, Oregon Health and Science University,<br />
Department <strong>of</strong> Ophthalmology, Portland, OR, Michael<br />
Davey, Pr<strong>of</strong>essor, VA Medical Center, Portland, OR, Monica<br />
Jann, Research Assistant, VA Medical Center, Portland, OR,<br />
Steve Planck, Research Associate Pr<strong>of</strong>essor, Oregon Health<br />
and Science University, Department <strong>of</strong> Ophthalmology,<br />
Portland, OR, Kelley Goodwin, Student Research Assistant,<br />
Oregon Health and Science University, Department <strong>of</strong><br />
Ophthalmology, Portland, OR, Koichi Kobayashi, Assistant<br />
Pr<strong>of</strong>essor, Harvard Medical School, Department <strong>of</strong><br />
Pathology, Boston, MA, Richard Flavell, Pr<strong>of</strong>essor, Yale<br />
University School Med, HHMI, New Haven, CT, James<br />
Rosenbaum, Pr<strong>of</strong>essor, Oregon Health and Science<br />
University, Department <strong>of</strong> Ophthalmology, Portland, OR<br />
Mutations in the human CARD15/NOD2 gene are associated<br />
with Crohn's disease or Blau syndrome, an autosomal<br />
dominant form <strong>of</strong> uveitis, arthritis, and dermatitis. NOD2 is<br />
responsible for sensing the bacterial product, muramyl<br />
dipeptide (MDP). Interestingly, mutations in a related gene,<br />
CIAS1, are associated with autoinflammatory syndromes in<br />
which interleukin (IL)-1 β plays a critical role in disease<br />
pathogenesis. Since the in vivo effects <strong>of</strong> MDP are<br />
incompletely studied and the susceptibility <strong>of</strong> the eye to<br />
mutations in NOD2 is unexplained, we developed a novel<br />
murine model <strong>of</strong> MDP-dependent uveitis. Intraocularly<br />
injected MDP induced a significant, transient inflammatory<br />
response. We characterized several salient features <strong>of</strong> this<br />
model such as the effect <strong>of</strong> dose and timing on the rolling,<br />
sticking and infiltrating leukocytes within the iris vasculature<br />
and tissue by intravital microscopy and histology. The<br />
increase in rolling leukocytes in MDP treated mice was<br />
dependent on the adhesion molecule L-selectin, as Lselectin<br />
knockout mice showed a significant decrease in the<br />
number <strong>of</strong> rolling cells within the iris vasculature in<br />
response to MDP. Importantly, NOD2 plays an essential role<br />
in ocular inflammation induced by MDP, as NOD2 knockout<br />
mice failed to develop uveitis in response to MDP.<br />
Unexpectedly, IL-1 receptor knockout mice did not show<br />
reduced MDP-induced uveitis. Thus, NOD2 may participate<br />
in the development <strong>of</strong> uveitis in response to bacterial<br />
products through its ability to enhance intravascular<br />
interactions between leukocytes and the endothelium in<br />
the eye in an IL-1 receptor independent fashion.<br />
doi:10.1016/j.clim.2007.03.119<br />
Su.81 Aut<strong>of</strong>luorescence Can be Used to<br />
Differentiate Between Normal, Activated, or<br />
Malignant Lymphocyte Populations<br />
Seth Pantanelli, Guest Researcher, National Institutes <strong>of</strong><br />
Health, NEI, Bethesda, MD, Zhuqing Li, Staff Scientist,<br />
National Institutes <strong>of</strong> Health, NEI, Bethesda, MD, Rober<br />
Fariss, Researcher, National Institutes <strong>of</strong> Health, NEI,<br />
Bethesda, MD, Matthew Cunningham, Guest Researcher,<br />
National Institutes <strong>of</strong> Health, NEI, Bethesda, MD, Baoying<br />
Liu, Researcher, National Institutes <strong>of</strong> Health, NEI,<br />
Bethesda, MD, Sankara Mahesh, Researcher, National<br />
Institutes <strong>of</strong> Health, NEI, Bethesda, MD, Robert<br />
Nussenblatt, Principal Investigator, National Institutes <strong>of</strong><br />
Health, NEI, Bethesda, MD<br />
The goal <strong>of</strong> this study was to investigate whether aut<strong>of</strong>luorescent<br />
properties <strong>of</strong> normal, activated or malignant<br />
lymphocyte populations could facilitate clinical diagnosis <strong>of</strong><br />
ocular diseases. Peripheral blood mononuclear cells<br />
(PBMCs) were isolated from normal human donor buffy<br />
coat blood products. T cells were purified from PBMCs and<br />
cultured with or without anti-CD3 and anti-CD28 stimulation.<br />
A B-lymphoma cell line (CA46) was cultured separately.<br />
Five experimental groups were prepared:<br />
unstimulated T cell, stimulated T cell, CA46 cell, stimulated<br />
T cell mixed with CA46 cell at a ratio <strong>of</strong> 1:4, or mixed<br />
at a ratio <strong>of</strong> 4:1. At 0, 1, and 3 days after culturing,<br />
0.6 ×106 cells from each <strong>of</strong> the five cell groups were cytospun<br />
onto a slide and imaged with a confocal microscope.<br />
Samples were excited with six excitation wavelengths: 351,<br />
364, 458, 476, 488, and 514 nm. For each excitation<br />
condition, the aut<strong>of</strong>luorescence intensity emitted from the<br />
sample was measured over a broad range <strong>of</strong> wavelengths.<br />
Pure T and B lymphoma populations were clearly distinguishable<br />
based on aut<strong>of</strong>luorescence intensity spectra. B<br />
lymphoma cells were the least fluorescent when excited<br />
with 351 nm light, but most fluorescent when excited with<br />
longer wavelengths like 488 nm. Mixed populations <strong>of</strong> T and<br />
B lymphoma cells had emission intensities that fell inbetween<br />
those <strong>of</strong> the pure populations. We conclude that<br />
normal, activated and malignant lymphocyte populations<br />
can be distinguished based on their aut<strong>of</strong>luorescent<br />
properties in vitro. Future work with in vivo models may<br />
prove useful in facilitating the diagnosis <strong>of</strong> uveitis and<br />
other ocular diseases.<br />
doi:10.1016/j.clim.2007.03.120<br />
Su.82 Developmental Expression <strong>of</strong> PD1 in the<br />
Retina<br />
Ling Chen, PhD Student, Jules Stein Eye Institute, Los<br />
Angeles, CA, Vicky Chang, Medical Student, Jules Stein Eye<br />
Institute, Los Angeles, CA, Ralph Levinson, Assistant<br />
Pr<strong>of</strong>essor, Jules Stein Eye Institute, Los Angeles, CA, Lynn<br />
Gordon, Associate Pr<strong>of</strong>essor, Jules Stein Eye Institute, Los<br />
Angeles, CA, Jonathan Braun, Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Pathology and Laboratory Medicine, Los Angeles, CA<br />
Purpose: Programmed cell death 1 (PD-1) has a major<br />
function as a negative immune regulator. We recently<br />
identified PD-1 expression in neuronal cells <strong>of</strong> the retina.<br />
One potential role for PD-1 expression is in retinal<br />
maturation. The purpose <strong>of</strong> this study was to identify<br />
neuronal cell types that express retinal PD-1 and determine<br />
the time course <strong>of</strong> expression in the developing<br />
retina. Methods: PD-1 protein expression in the mouse<br />
retina was detected by immunohistochemistry at multiple<br />
embryonic and adult stages. Co-localization experiments<br />
were performed to detect PD-1 and Brn3a, Thy-1, AP2,<br />
GFAP, calbindin, or islet. RT-PCR and in situ hybridization<br />
were performed to quantify PD-1 mRNA expression.<br />
Results: Constitutive expression <strong>of</strong> PD-1 protein and<br />
mRNA was observed in the retinal ganglion cell. Confocal<br />
microscopy revealed that 68% <strong>of</strong> the PD-1 cells were <strong>of</strong> the<br />
retinal ganglion cell lineage and 27% were amacrine cells.<br />
PD-1 expression levels changed markedly during development.
Abstracts<br />
PD-1 expression was first observed at E14, 2 days after the<br />
first retinal ganglion cells appear in the retina. Expression<br />
levels <strong>of</strong> PD-1 significantly increased during development,<br />
reaching a peak at P13 with a subsequent decrease to an<br />
intermediate expression level at P24. Conclusions: PD-1 is<br />
expressed in retinal ganglion cells and amacrine cells and<br />
its expression dynamically changes during retinal development.<br />
Maximal PD-1 expression is noted at the most critical<br />
time period in postnatal visual plasticity. This observation<br />
raises the possibility <strong>of</strong> a developmental role for PD-1 in<br />
maturation <strong>of</strong> the ganglion cell layer and retinal remodeling<br />
process<br />
doi:10.1016/j.clim.2007.03.121<br />
Su.83 Human Lung Tumor Cells Modifies Rates <strong>of</strong><br />
CD4+CD25+ T Regulatory, CD19+CD23+ B Regulatory<br />
Cells, CD3+CD95+ Cells, NK Cells and B Lymphocytes<br />
Bayram Kiran, Tip Fakultesi, Temel Bilimler Binasi, Istanbul,<br />
Turkey, Akif Turna, Yedikule Hospital for Chest Diseases and<br />
Thoracic Surgery, Kadikoy, Istanbul, Turkey, Alper Yener,<br />
Istanbul Tip Fakultesi, Istanbul, Turkey, Atilla Gürses,<br />
Yedikule Gogus Hastaliklari Hastanesi, Istanbul, Turkey,<br />
Andac Salman, Istanbul Tip Fakultesi, Istanbul, Turkey,<br />
Selim Badur, Istanbul Tip Fakultesi, Temel Bilimler Binasi,<br />
Istanbul, Turkey<br />
The role <strong>of</strong> T regulatory cells and NK cells in human lung<br />
cancer has not yet been clarified. Our aim was to evaluate<br />
how the microenvironment <strong>of</strong> a tumor mass induced phenotypical<br />
changes in lymphocyte subsets. Our subjects were<br />
10 patients with resectable non-small cell lung cancer. We<br />
evaluated 47 different phenotypically different lymphocyte<br />
subsets in blood samples taken from the pulmonary artery,<br />
pulmonary vein <strong>of</strong> a tumor bearing pulmonary lobe and<br />
peripheral blood during pulmonary resectional surgery. We<br />
showed that, tumor cells boosted CD25high+CD4high+T<br />
lymphocytes (Treg cells), B lymphocytes, NK and CD19+<br />
CD23+ cells (Breg cells) and CD3+CD95+ (FasL+T lymphocytes)<br />
(p=0.015, p=0.001, p=0.02, p=0.04, p=0.03 respectively).<br />
However, Mac-1 cells and HLADR+T lymphocytes<br />
were found to be decreased by tumor mass (p=0.04 and<br />
p=0.04), whereas only Breg, NK cell and B lymphocyte<br />
subsets were found to be expansed. In addition, the patients<br />
showed expansions <strong>of</strong> CD45R0+ activated T lymphocytes and<br />
CD18+CD11b+(Mac-1) cell subsets without significant alteration<br />
by tumor microenvironment. In conclusion, subsets <strong>of</strong><br />
Treg, Breg cells, FasL+ T lymphocytes, B lymphocytes were<br />
modified by lung cancer microenvironment itself. This study<br />
also showed that, peripheral blood might not be good source<br />
for investigating the anti-tumor immunity since only Breg, NK<br />
cell and Treg cell subsets were found expansed peripherally.<br />
T lymphocytes and Mac-1 cells may be modified by systemic<br />
antitumor response rather than by local tumoral microenvironment.<br />
This study provides important insight into how<br />
tumor infiltrating lymphocytes and peripheral immune<br />
systems may be different in terms <strong>of</strong> lymphocyte subset<br />
expansion dynamics.<br />
doi:10.1016/j.clim.2007.03.122<br />
Su.84 Gene Expression Patterns <strong>of</strong> Intestinal<br />
Epithelial Cells in Murine Models <strong>of</strong> Colitis<br />
Ken Flanagan, Postdoctoral Researcher, Genentech,<br />
Department <strong>of</strong> Pathology, South San Francisco, CA, Jennine<br />
Cornelius, Research Associate, Genentech, Department <strong>of</strong><br />
Pathology, South San Francisco, CA, Zora Modrusan,<br />
Scientist, Genentech, Department <strong>of</strong> Molecular Biology,<br />
South San Francisco, CA, Lian Mo, Senior Research<br />
Associate, Genentech, Department <strong>of</strong> Pathology, South San<br />
Francisco, CA, Arvind Chavali, Intern, Genentech,<br />
Department <strong>of</strong> Pathology, South San Francisco, CA, Ian<br />
Kasman, Research Associate, Genentech, Department <strong>of</strong><br />
Pathology, South San Francisco, CA, Laszlo Komuves,<br />
Scientist, Genentech, Department <strong>of</strong> Pathology, South San<br />
Francisco, CA, Lauri Diehl, Scientist, Genentech,<br />
Department <strong>of</strong> Pathology, South San Francisco, CA<br />
In the healthy colon, intestinal epithelial cells (IEC) form<br />
a physical barrier separating the myriad <strong>of</strong> gut antigens from<br />
the cells <strong>of</strong> the immune system. Simultaneously, IEC employ<br />
several mechanisms to actively maintain immunologic<br />
tolerance to non-pathogenic antigens, including commensal<br />
bacteria. However, during inflammatory bowel disease (IBD)<br />
the line <strong>of</strong> defense provided by IEC is breached, resulting in<br />
uncontrolled immune responses. As IEC are a principal mediator<br />
<strong>of</strong> immune responses in the gut, we were interested in<br />
discerning the gene expression pattern <strong>of</strong> IEC during<br />
development and progression <strong>of</strong> IBD. Laser capture microdissection<br />
and microarray analysis were combined to identify<br />
genes with altered expression patterns in two models <strong>of</strong><br />
murine colitis. Genes involved in processes <strong>of</strong> the immune<br />
system, including lymphocyte migration, apoptosis and<br />
antigen presentation were affected in the IEC <strong>of</strong> mice with<br />
colitis indicating that IEC function to attract and activate<br />
cells <strong>of</strong> the immune system.<br />
doi:10.1016/j.clim.2007.03.123<br />
S169<br />
Su.85 TNF Binding by Certolizumab Pegol,<br />
Adalimumab, and Infliximab-Stoichiometry,<br />
Complex Formation, and the Biologic Effects <strong>of</strong><br />
Complexes<br />
Alistair Henry, Senior Principal Scientist, UCB Celltech,<br />
Research Biologicals, Slough, UK, Jeff Kennedy, Senior<br />
Scientist, UCB Celltech, Enzymology, Slough, UK, Gianluca<br />
Fossati, Principal Scientist, UCB Celltech, Slough, UK,<br />
Andrew Nesbitt, Senior Group Leader, UCB Celltech,<br />
Inflammation Discovery, Slough, UK<br />
Immune complexes can have various unwanted consequences.<br />
We determined the binding stoichiometry <strong>of</strong> certolizumab<br />
pegol, adalimumab, and infliximab to the tumor<br />
necrosis factor (TNF) trimer, and the size and in vitro biologic<br />
consequences <strong>of</strong> the resulting immune complexes. Isothermal<br />
titration calorimetry was used to assess the stoichiometry <strong>of</strong><br />
binding through calculation <strong>of</strong> the number <strong>of</strong> anti-TNF<br />
molecules bound to a TNF trimer when no more heat <strong>of</strong><br />
binding was released. Dynamic light scatter assessed the size<br />
<strong>of</strong> complexes formed over a range <strong>of</strong> antigen:antibody ratios.<br />
Effect <strong>of</strong> the complexes on peripheral blood neutrophil<br />
degranulation (myeloperoxidase release) and superoxide
S170 Abstracts<br />
production (oxidation <strong>of</strong> cytochrome c) were measured. At<br />
optimum antigen:antibody ratio, adalimumab and infliximab<br />
formed huge complexes N30 nm in diameter, suggesting that<br />
these bivalent antibodies crosslink TNF trimers. Certolizumab<br />
pegol complexes were b20 nm in diameter, consistent<br />
with its univalent structure preventing crosslinking. Certolizumab<br />
pegol bound 2.9 monomers at saturation, whereas<br />
infliximab and adalimumab both bound around 2.5 monomers.<br />
This suggests that steric or allosteric restrictions<br />
prevent the bivalent antibodies binding to all monomers in<br />
a trimer. The large immune complexes formed by adalimumab<br />
and infliximab caused degranulation and superoxide<br />
production by neutrophils. The smaller certolizumab pegol<br />
complexes had only marginal effects. The bivalent structures<br />
<strong>of</strong> infliximab and adalimumab form enormous complexes with<br />
TNF trimers, which have proinflammatory effects on neutrophils<br />
in vitro. Certolizumab pegol does not form large<br />
complexes with TNF trimers due to its univalent structure<br />
preventing crosslinking, giving it a unique mode <strong>of</strong> action in<br />
this regard.<br />
doi:10.1016/j.clim.2007.03.124<br />
Su.86 Certolizumab Pegol and Adalimumab<br />
Accumulation in the Inflammed Paws <strong>of</strong> Mice with<br />
Collagen-induced Arthritis Compared to<br />
Noninflammed Tissue In Vivo Bi<strong>of</strong>luorescence<br />
Imaging <strong>of</strong> Alexa 680-labeled Antibodies<br />
Roger Palframan, Senior Group Leader, UCB Celltech,<br />
Pharmacology, Slough, UK, Alex Vugler, Research Scientist,<br />
UCB Celltech, Pharmacology, Slough, UK, Adrian Moore,<br />
Senior Group Leader, UCB Celltech, Pharmacology, Slough,<br />
UK, Mark Baker, Research Scientist, UCB Celltech, <strong>Clinical</strong><br />
Pharmacology and Experimental Medicine, Slough, UK,<br />
Daniel Lightwood, Group Leader, UCB Celltech, Antibody<br />
Biology, Slough, UK, Andrew Nesbitt, Senior Group Leader,<br />
UCB Celltech, Inflammation Discovery, Slough, UK, Roly<br />
Foulkes, Director, UCB Celltech, Non-clinical Development/<br />
Inflammation Therapeutic Area, Slough, UK, Neil Gozzard,<br />
Director, UCB Celltech, Pharmacology, Slough, UK<br />
Variability in disposition <strong>of</strong> monoclonal antibodies results<br />
in different exposure time courses in inflammed and noninflammed<br />
tissues. We investigated the disposition <strong>of</strong> certolizumab<br />
pegol and adalimumab in normal and inflammed<br />
tissue in vivo in mice, using a novel noninvasive bi<strong>of</strong>luorescence<br />
technique. Certolizumab pegol or adalimumab labeled<br />
with the low-molecular weight dye Alexa 680 was administered<br />
intravenously (2 mg/kg) in naïve DBA/1 mice and in<br />
DBA/1 mice with ongoing collagen-induced arthritis. The<br />
accumulation <strong>of</strong> certolizumab pegol and adalimumab was<br />
measured in the hind paws at multiple points up to 26 hours<br />
using a Xenogen IVIS200 bi<strong>of</strong>luorescence imager. Tail blood<br />
samples were taken for determination <strong>of</strong> serum levels <strong>of</strong> the<br />
reagents by ELISA. Both reagents penetrated inflammed<br />
tissue more than noninflammed tissue. The penetration <strong>of</strong><br />
certolizumab pegol into inflammed arthritic paws was<br />
greater and more prolonged than for adalimumab (inflammed:noninflammed<br />
tissue ratios were 3.9:1 for certolizumab<br />
pegol and 1.9:1 for adalimumab; elimination half-lives<br />
were 27.8 and 5.5 hours, respectively). The plasma time<br />
courses reflected known differences in exposure, with<br />
certolizumab pegol maintaining higher plasma concentrations<br />
than adalimumab. In this model, certolizumab pegol<br />
disposition was more responsive to inflammation, with<br />
prolonged tissue infiltration occurring primarily in inflammed<br />
tissue. Certolizumab pegol, in contrast to adalimumab,<br />
appeared to readily enter inflammed tissue but not noninflammed<br />
tissue. This feature <strong>of</strong> certolizumab pegol may be<br />
conferred on the molecule by PEGylation. Increased drug<br />
exposure at the site <strong>of</strong> inflammation might be an important<br />
consideration for the treatment <strong>of</strong> inflammatory disorders<br />
such as rheumatoid arthritis and Crohn's disease.<br />
doi:10.1016/j.clim.2007.03.126<br />
Su.87 Comparison <strong>of</strong> Naturally Occurring Human<br />
Immunoglobulin Sequences with the Anti-TNF<br />
Agents Certolizumab Pegol, Adalimumab, and<br />
Infliximab<br />
Andrew Martin, Senior Lecturer, University College London,<br />
Biochemistry and Molecular Biology, London, UK, Kerry<br />
Tyson, Principal Scientist, UCB Celltech, Antibody Biology,<br />
Slough, UK, Matt Page, Research Associate Informatics, UCB<br />
Celltech, Therapeutic Project Informatics, Slough, UK,<br />
Gianluca Fossati, Principal Scientist, UCB Celltech,<br />
Inflammation Discovery, Slough, UK, James Snowden, Senior<br />
Scientist, UCB Celltech, Informatics, Slough, UK, Raghavan<br />
Abhinandan, Lecturer, University College London,<br />
Biochemistry and Molecular Biology, London, UK, Alastair<br />
Lawson, Director <strong>of</strong> Antibody Biology, UCB Celltech,<br />
Antibody Biology, Slough, UK, Andrew Nesbitt, Senior Group<br />
Leader, UCB Celltech, Inflammation Discovery, Slough, UK,<br />
Andrew Popplewell, Associate Director, UCB Celltech,<br />
Antibody Biology, Slough, UK<br />
V-region sequences <strong>of</strong> certolizumab pegol, adalimumab,<br />
and infliximab were compared with a database <strong>of</strong> normal<br />
human IgG V-region sequences. A database <strong>of</strong> rearranged<br />
human variable heavy [VH] and variable kappa light [VK]<br />
amino acid sequences was obtained by sequencing 243<br />
unique VH chains and 312 unique VK chains from healthy<br />
donors. Each sequence was compared to the closest human<br />
“germline” sequence and also scored against every other<br />
sequence <strong>of</strong> the same chain type, using percentage identity<br />
to measure sequence similarity. Z-scores were calculated<br />
from a frequency distribution plot and compared between<br />
agents. A positive Z-score indicates that a sequence is more<br />
typical than average <strong>of</strong> a human sequence. The mean<br />
number <strong>of</strong> nongermline framework residues in rearranged<br />
human antibodies was 4.3% for VK and 8.8% for VH. Of the<br />
three agents, certolizumab pegol had a V-region most typical<br />
<strong>of</strong> the rearranged human sequences with 3.8% nongermline<br />
residues for VK and 8.5% for VH. Adalimumab had 1.3% and<br />
1.2% and infliximab 31.3% and 22.2%, respectively. Positive Zscores<br />
were obtained for the VK and VH sequences <strong>of</strong><br />
certolizumab pegol (0.15 and 1.46) and adalimumab (0.67<br />
and 1.34). However, infliximab V-region sequences gave<br />
negative Z-scores (−2.37 and −0.58) indicating they are less<br />
typical <strong>of</strong> human sequences. The certolizumab pegol Vregion<br />
framework sequence has a similar mean number <strong>of</strong><br />
nongermline residues as rearranged human IgG V-region
Abstracts<br />
frameworks. The complete VH and VK sequences were also<br />
more human-like than average. The certolizumab pegol Fab'<br />
sequence can be considered typical <strong>of</strong> naturally occurring<br />
human IgG.<br />
doi:10.1016/j.clim.2007.03.127<br />
Su.89 Role <strong>of</strong> Enteric Lumenal TLR Ligand Sampling<br />
in the Formation <strong>of</strong> Novel Resident Mucosal<br />
Dendritic Cells<br />
Daisuke Fujiwara, Postdoctoral Fellow, Department <strong>of</strong><br />
Pathology and Laboratory Medicine, David Geffen School <strong>of</strong><br />
Medicine at University <strong>of</strong> California, Los Angeles, Los<br />
Angeles, CA, Bo Wei, Assistant Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Pathology and Laboratory Medicine, David Geffen School <strong>of</strong><br />
Medicine at University <strong>of</strong> California, Los Angeles, Los<br />
Angeles, CA, Michael McPherson, PhD Student, Department<br />
<strong>of</strong> Pathology and Laboratory Medicine, David Geffen School<br />
<strong>of</strong> Medicine at University <strong>of</strong> California, Los Angeles, Los<br />
Angeles, CA, Jonathan Braun, Pr<strong>of</strong>essor and Chair,<br />
Department <strong>of</strong> Pathology and Laboratory Medicine, David<br />
Geffen School <strong>of</strong> Medicine at University <strong>of</strong> California, Los<br />
Angeles, Los Angeles, CA<br />
Resident intestinal dendritic cells (DCs) play important<br />
immunoregulatory roles through sensing and antigen handling<br />
<strong>of</strong> enteric microbiota. However, intestinal DCs have not been<br />
well characterized. In this study, we found that CD11c+ cells<br />
isolated from the small intestine superficial and deep lamina<br />
propria were unusual (compared to lymph node and spleen<br />
DC's) due to their graduated expression <strong>of</strong> CD11b or B220, and<br />
the predominance <strong>of</strong> B220+CD11b+ double-positive (DP) DC's.<br />
Since lamina propria DC's engage in lumenal sampling and thus<br />
encounter TLR ligands, we wondered whether TLR signaling<br />
might induce the unusual DP phenotype. Using bone marrowderived<br />
DC's differentiated with Flt-3L, we evaluated the<br />
effect <strong>of</strong> co-culture with various TLR ligands. Whereas Flt-3L<br />
alone induced single-positive CD11b+ or B220+ DCs, DP DCs<br />
predominated after TLR2, TLR4, and TLR9 ligands. The<br />
response was abrogated in myd88−/− mice, suggesting that<br />
DP DC's are induced through TLR-MyD88 pathway. Fecal<br />
extracts also induced DP DC formation; because this response<br />
was absent in TLR4−/− mice, it appeared that the predominant<br />
enteric bioactivity for DP DC induction was LPS. Among<br />
lamina propria DCs, we also observed a probable immature<br />
mDC subset (CD11c-B220-CD11blow). This subset was deficient<br />
in germ-free (GF) mice or cd8 knockout mice, suggesting an<br />
important TLRL role in differentiation stage <strong>of</strong> DCs in vivo,and<br />
a novel requirement for CD8+ T cells. These findings indicate<br />
that lumenal TLR4 ligands shape the differentiation <strong>of</strong> mucosal<br />
resident DCs, resulting in a novel phenotype associated with<br />
distinct immunoregulatory traits. Supported by NIH DK46763<br />
and DK69434.<br />
doi:10.1016/j.clim.2007.03.128<br />
Su.90 Up-Regulation <strong>of</strong> the PI3-Kinase Pathway in<br />
Lamina Propria T Lymphocytes<br />
Jutta Braunstein, Research Scientist, University Hospital<br />
Heidelberg, Heidelberg, Germany, Frank Autschbach,<br />
Deputy Head <strong>of</strong> Pathology, University Hospital Heidelberg,<br />
Heidelberg, Germany, Felix Lasitschka, Postdoctoral Fellow,<br />
University Hospital Heidelberg, Heidelberg, Germany,<br />
Thomas Giese, Group Leader, University Hospital<br />
Heidelberg, Heidelberg, Germany, Antje Heidtmann,<br />
Technician, University Hospital Heidelberg, Heidelberg,<br />
Germany, Bernd Sido, Senior Physician, University Hospital<br />
Heidelberg, Heidelberg, Germany, Benjamin Funke,<br />
Physician, University Hospital Heidelberg, Heidelberg,<br />
Germany, Andreas Schroeder, Medical Advisor, Merck Sero,<br />
Darmstadt, Germany, Yvonne Samstag, Group Leader,<br />
University Hospital Heidelberg, Heidelberg, Germany,<br />
Stefan Meuer, Head <strong>of</strong> Institute <strong>of</strong> <strong>Immunology</strong>, University<br />
Hospital Heidelberg, Heidelberg, Germany<br />
Understanding the regulation <strong>of</strong> immune responses in the<br />
normal intestinal mucosa is critical for defining its alterations<br />
in inflammatory bowel disease. Importantly, intestinal lamina<br />
propria T lymphocytes (LPT) when investigated ex vivo exhibit<br />
functional properties pr<strong>of</strong>oundly different from those <strong>of</strong><br />
peripheral blood T lymphocytes (PBT). In particular, following<br />
CD2 stimulation they are able to produce markedly higher<br />
levels <strong>of</strong> cytokines than PBT. This study provides insight into<br />
signaling events associated with the high CD2 responsiveness <strong>of</strong><br />
LPT. CD2 stimulation results in enhanced phosphorylation <strong>of</strong><br />
the PI3-kinase dependent kinase Akt and its down-stream<br />
target GSK-3 in LPTwhen compared to PBT. That up-regulation<br />
<strong>of</strong> PI3-kinase pathway activation in LPTcontributes to the high<br />
IL-2 expression in response to CD2 stimulation is demonstrated<br />
by the fact that inhibition <strong>of</strong> this pathway by the PI3-kinase<br />
specific inhibitor Ly294002 to levels induced in PBT clearly<br />
reduces IL-2 gene expression (and TNF-α, CD40L gene<br />
expression) in LPT. Immunohistochemical analysis reveals<br />
that Akt phosphorylation occurs in few lamina propria mononuclear<br />
cells in the normal mucosa in vivo; moreintense<br />
phospho-Akt staining <strong>of</strong> larger numbers <strong>of</strong> lamina propria<br />
mononuclear cells is detectable in the inflammed intestine<br />
(ulcerative colitis) indicating that activation <strong>of</strong> the PI3-kinase<br />
pathway plays an important role in intestinal inflammation.<br />
Enhanced responsiveness <strong>of</strong> these pathways to costimulatory<br />
stimuli may allow for rapid and vigorous T cell responses and<br />
could therefore be fundamental to intestinal mucosal immune<br />
responses and homeostasis.<br />
doi:10.1016/j.clim.2007.03.129<br />
S171<br />
Su.91 Differential Levels <strong>of</strong> Intercellular Glutathion<br />
(GSH) Dictate the Shift From Adaptive to Innate T<br />
Cell Function in the Human Intestinal Mucosa<br />
Bernd Sido, Senior Physician, Department <strong>of</strong> Surgery,<br />
Heidelberg, Germany, Frank Autschbach, Senior Physician,<br />
Institute <strong>of</strong> Pathology, Heidelberg, Germany, Jutta<br />
Schroeder-Braunstein, Scientist, Institute <strong>of</strong> <strong>Immunology</strong>,<br />
Heidelberg, Germany, Thomas Giese, Group Leader,<br />
Institute <strong>of</strong> <strong>Immunology</strong>, Heidelberg, Germany, Felix<br />
Lasitschka, Postdoctoral Fellow, Institute <strong>of</strong> <strong>Immunology</strong>,<br />
Heidelberg, Germany, Stefan Meuer, Director, Institute <strong>of</strong><br />
<strong>Immunology</strong>, Heidelberg, Germany<br />
Isolated human Tcells from the healthy human mucosa are<br />
non-responsive to T cell receptor engagement yet mount
S172 Abstracts<br />
vigorous responses to non-specific activation through CD2 or<br />
CD28 triggering with regard to both proliferation and cytokine<br />
secretion. Since resting T lymphocytes do not express<br />
the cystine transporter (xCT), which is critical for the import<br />
<strong>of</strong> cystine across the plasma membrane and since cystine is a<br />
critical precursor for GSH synthesis, they are dependent on an<br />
extracellular source <strong>of</strong> cysteine. Under physiologic conditions,<br />
cysteine is not available to T cells in the intestinal<br />
mucosa since the local myeloid cell population is unable to<br />
secrete cysteine, unlike circulating monocytes from peripheral<br />
blood. As a consequence, mucosal T lymphocytes contain<br />
significantly lower GSH concentrations than blood T lymphocytes.<br />
Enhancement <strong>of</strong> GSH levels in mucosal T cells by<br />
providing cysteine either directly or by adding blood<br />
monocytes restores their antigen receptor reactivity. This<br />
mechanism appears to be relevant for their behaviour in<br />
inflammatory bowel diseases where GSH levels in mucosal T<br />
cells from inflammatory sites are at least as high as in<br />
circulating blood T cells. Here, myeloid cells with the capacity<br />
to secrete cysteine are frequently detected. Importantly,<br />
this is not only due to their influx from blood but, at least in<br />
part, to a functional and phenotypic change under altered<br />
microenvironmental conditions. Supported by a grant from<br />
DFG/SFB405/B6.<br />
doi:10.1016/j.clim.2007.03.130<br />
Su.92 Whole Genome Association Identifies Novel<br />
Susceptibility Genes for Crohn's Disease and<br />
Implicates a Crucial Role for Autophagy<br />
John Rioux, Associate Pr<strong>of</strong>essor, Universite de Montreal,<br />
Department <strong>of</strong> Medicine, Montreal, QC, Canada, Ramnik<br />
Xavier, Associate Pr<strong>of</strong>essor, Massachusetts General Hospital,<br />
Harvard Medical School, Boston, MA, Kent Taylor, Associate<br />
Pr<strong>of</strong>essor, Cedars Sinai Medical Center, Los Angeles, CA,<br />
Philippe Goyette, Scientist, Montreal Heart Institute,<br />
Montreal, QC, Canada, Mark Silverberg, Associate Pr<strong>of</strong>essor,<br />
Mount Sinai Hospital, Toronto, ON, Canada, Alan Huett,<br />
Postdoctoral Fellow, Massachusetts General Hospital,<br />
Harvard Medical School, Boston, MA, Todd Green, Project<br />
Manager, Broad Institute <strong>of</strong> MIT and Harvard, Cambridge,<br />
MA, Petric Kuballa, Postdoctoral Fellow, Massachusetts<br />
General Hospital, Harvard Medical School, Boston, MA,<br />
Michael Barmada, Associate Pr<strong>of</strong>essor, University <strong>of</strong><br />
Pittsburgh, Pittsburgh, PA, Lisa Datta, John Hopkins<br />
University School <strong>of</strong> Medicine, Baltimore, MD, Yin Yao<br />
Shugart, Associate Pr<strong>of</strong>essor, John Hopkins University,<br />
Bloomberg School <strong>of</strong> Public Health, Baltimore, MD, Edmond<br />
Jean Bernard, Assistant Pr<strong>of</strong>essor, Universite de Montreal,<br />
Montreal, QC, Canada, Ling Mei, Cedars Sinai Medical<br />
Center, Los Angeles, CA, Dan Nicolae, Associate Pr<strong>of</strong>essor,<br />
University <strong>of</strong> Chicago, Departments <strong>of</strong> Medicine and<br />
Statistics, Chicago, IL, Hillary Steinhart, Associate<br />
Pr<strong>of</strong>essor, Mount Sinai Hospital and University <strong>of</strong> Toronto,<br />
Toronto, ON, Canada, Jerome Rotter, Pr<strong>of</strong>essor, Cedars Sinai<br />
Medical Center, Los Angeles, CA, Judy Cho, Yale University,<br />
New Haven, CT, Mark Daly, The Broad Institute <strong>of</strong> MIT and<br />
Harvard, Cambridge, MA, Miguel Regueiro, Associate<br />
Pr<strong>of</strong>essor <strong>of</strong> Medicine, University <strong>of</strong> Pittsburgh, Division<br />
<strong>of</strong> Gastroenterology, Hepatology and Nutrition, Pittsburgh,<br />
PA, Philip Schumm, University <strong>of</strong> Chicago, Department<br />
<strong>of</strong> Health Studies, Chicago, IL, Richard Duerr, Associate<br />
Pr<strong>of</strong>essor, University <strong>of</strong> Pittsburgh, School <strong>of</strong> Medicine,<br />
Pittsburgh, PA, Steven Brant, Meyerh<strong>of</strong>f Inflammatory<br />
Bowel Disease Center, John Hopkins University School<br />
<strong>of</strong> Medicine, Baltimore, MD<br />
In 2002, the NIDDK IBD Genetics Consortium was founded<br />
by six Genetic Research Centers located in Canada and the<br />
US. Our first stage genome-wide association (GWA) study <strong>of</strong><br />
308,332 autosomal single nucleotide polymorphisms (SNPs)<br />
in 567 ileal CD samples and 571 controls identified genetic<br />
variants within the IL23R gene that influence an individual's<br />
risk for developing IBD (Duerr et al. Science 2006). In the<br />
current study, we typed an independent set 401 patients with<br />
ileal CD and 433 controls for the exact same set <strong>of</strong> SNPs. We<br />
have since performed a combined analysis <strong>of</strong> all cases and<br />
controls as well as an independent replication study that<br />
establish five regions <strong>of</strong> unambiguously confirmed association<br />
<strong>of</strong> genome-wide significance. Specifically, in addition to<br />
the established CARD15 and IL23R associations, we report<br />
three novel confirmed associations with variation in the<br />
PHOX2B and ATG16L1 genes and in an intergenic region on<br />
10q21.1. Moreover, we also identify a statistically significant<br />
excess <strong>of</strong> additional regions showing nominal replication <strong>of</strong><br />
association that likely contain additional gene(s). We further<br />
demonstrate that the ATG16L1 gene is expressed in intestinal<br />
epithelial cells and that functional knock down <strong>of</strong> this gene<br />
abrogates autophagy <strong>of</strong> Salmonella typhimurium. Together<br />
these findings implicate that autophagy and other host<br />
responses to intra-cellular microbes are involved in the pathogenesis<br />
<strong>of</strong> CD.<br />
doi:10.1016/j.clim.2007.03.131<br />
Su.93 A High Density Association Study <strong>of</strong> the<br />
Extended Major Histocompatibility Locus in<br />
Ulcerative Colitis<br />
Philippe Goyette, Research Associate, Montreal Heart<br />
Institute, Montreal, QC, Canada, Todd Green, The Broad<br />
Institute <strong>of</strong> MIT & Harvard, Cambridge, MA, Paul de Bakker,<br />
The Broad Institute <strong>of</strong> MIT & Harvard, Cambridge, MA,<br />
Daniel Mirel, The Broad Institute <strong>of</strong> MIT & Harvard,<br />
Cambridge, MA, Christine Stevens, The Board Institute<br />
<strong>of</strong> MIT & Harvard, Cambridge, MA, Anna Latiano, IRCCS<br />
Casa Sollievo della S<strong>of</strong>ferenza, Italy, Jorge Oksenberg,<br />
University <strong>of</strong> California, San Francisco, San Francisco, CA,<br />
S. Hauser, University <strong>of</strong> California, San Francisco, San<br />
Francisco, CA, Vitto Annese, Medical School and University<br />
<strong>of</strong> Foggia, Italy, John Rioux, Montreal, QC, Canada,<br />
Mark Daly, The Broad Institute <strong>of</strong> MIT & Harvard,<br />
Cambridge, MA<br />
Ulcerative colitis (UC) and Crohn's disease (CD) are chronic<br />
inflammatory diseases <strong>of</strong> the gastrointestinal tract. While<br />
the study <strong>of</strong> genetic risk factors in CD has led to the identification<br />
<strong>of</strong> multiple risk factors, few have been convincingly<br />
identified in UC. Several independent genome-wide<br />
linkage scans, however, have suggested that the major<br />
histocompatibility complex (MHC) region plays a role in the<br />
genetic susceptibility to UC. While several putative association<br />
results have been reported for UC in this region, findings
Abstracts<br />
have not been consistent due to the limited number <strong>of</strong> alleles<br />
tested, to small sample sizes and to extensive LD in the<br />
region. We have recently reported the creation <strong>of</strong> a high<br />
density genetic map, including 7500 SNPs and DIPs, as well as<br />
the classical HLA genes. In the current study, we have<br />
selected 1500 <strong>of</strong> the most informative SNPs in order to<br />
capture the common variability in this region. Using this<br />
panel, we performed an association study in a cohort <strong>of</strong> 643<br />
UC cases and 1056 controls. Single marker association testing<br />
identified 35 SNPs with significant association (b4.5×10 − 5 )<br />
to UC. The peak <strong>of</strong> association, supported by multiple SNPs,<br />
is localized in the HLA-DRA and HLA-DQA gene regions. HLA<br />
tagging SNPs also identify this region as containing a UC<br />
susceptibility locus. Our initial analyses suggest that a<br />
common variant (∼30% frequency) in this region can explain<br />
the observed association. Higher density association mapping<br />
<strong>of</strong> this region and replication in an independent cohort<br />
will be necessary to identify causal allele(s).<br />
doi:10.1016/j.clim.2007.03.132<br />
Su.94 TRAM-34, a Blocker <strong>of</strong> the Calcium-activated<br />
Potassium Channel KCa3.1, Prevents Acute<br />
Transplant Rejection<br />
Heike Wulff, Assistant Pr<strong>of</strong>essor, University <strong>of</strong> California,<br />
Davis, Davis, CA, Yi-Je Chen, Graduate Student, University<br />
<strong>of</strong> California, Davis, Surgical and Radiological Sciences,<br />
Davis, CA, Girija Raman, Postdoctoral Researcher,<br />
University <strong>of</strong> California, Davis, Medical Pharmacology,<br />
Davis, CA, Clare Gregory, Pr<strong>of</strong>essor, University <strong>of</strong> California,<br />
Davis, Surgical and Radiological Sciences, Davis, CA<br />
Rejection <strong>of</strong> transplanted organs is caused by an immune<br />
response <strong>of</strong> recipient T cells against the donor MHC. One<br />
emerging new target for the suppression <strong>of</strong> T cell activation<br />
is the calcium-activated potassium channel KCa3.1. In<br />
addition to T and B cells, KCa3.1 is also expressed in<br />
macrophages, proliferating vascular smooth muscle and<br />
endothelial cells and has been shown to play an important<br />
role in the proliferation and migration <strong>of</strong> these cells. In<br />
2000, we designed a selective small molecule KCa3.1<br />
blocker called TRAM-34 (EC50=20 nM) and showed that it<br />
suppressed T cell proliferation and synergized with cyclosporine<br />
(PNAS 2000, 97: 8151). TRAM-34 has a half-life <strong>of</strong><br />
1.8 hours in rats and <strong>of</strong> 2.1 hours in rhesus macaques,<br />
prevents restenosis in rats and exhibits no signs <strong>of</strong> acute <strong>of</strong><br />
long-term toxicity. Encouraged by these results we are here<br />
testing TRAM-34 alone or in combination with cyclosporine<br />
in a model <strong>of</strong> acute transplant rejection. Hearts are<br />
heterotopically transplanted from male Brown Norway to<br />
male Lewis rats. While transplanted hearts in vehicle<br />
treated animals failed within 9 days (n=6), transplanted<br />
hearts in TRAM-34 treated rats (10 mg/kg twice daily)<br />
remained functional for an average <strong>of</strong> 25 days (n=6). In<br />
comparison, treatment with low dose cyclosporine (2 mg/kg<br />
Neoral®) resulted in a graft survival time <strong>of</strong> 18 days (n=6).<br />
Combinations <strong>of</strong> TRAM-34 with low dose cyclosporine are<br />
currently being tested. Supported by NIH and UC Davis.<br />
doi:10.1016/j.clim.2007.03.133<br />
Su.95 Low Doses IVIG with Plasmapheresis and<br />
Rituximab in Positive Cross Match Patients<br />
Miguel Rodriguez, Immunoallergist, Hospital<br />
Especialidades, IMSS, Guadalajara, Mexico, Luis Vales-<br />
Alberto, MD, Hospital Especialidades IMSS, Departmento de<br />
Nefrologia, Guadalajara, Mexico, Felicia Castro-Ramos,<br />
Biologist, Hospital Especialidades IMSS, Nephrology Unit,<br />
Guadalajara, Mexico, Francisco Monteon-Ramos,<br />
Nephrologist, Hospital de Especialidades IMSS, Nephrology<br />
Unit, Guadalajara, Efrain Montano-Gonzales,<br />
Immunoallergologist, Hospital Especialidades UMAE, IMSS<br />
Department <strong>of</strong> <strong>Immunology</strong> and Allergy, Guadalajara,<br />
Mexico, Alejandro Bautista-Vazquez, Nephrologist, Hospital<br />
de Especialidades, CMENGLANDUMAE, IMSS, Unit <strong>of</strong><br />
Nephrology, Guadalajara, Mexico, Salvador Chavez, MD,<br />
Hospital Especialidades IMSS, Departamento de Nefrologia,<br />
Guadalajara, Mexico<br />
Introduction: Renal Transplant (RT) is the best option for<br />
End Stage Renal Disease (ESRD) Positive Cross Match (+XM) is<br />
formal contraindication to RT. Protocols have been developed<br />
to overcome this problem. Methods and Materials: Simple<br />
not ramdomized sample. Patients ABO compatible with<br />
+XM (up to 30%) by FlowCytometry (FC) to living related<br />
donor. Patients scheme was: PP after that IVIG 100 mg/kg 3<br />
times every other day. If XM becomes negative we administrated<br />
Rituximab and go to RT. After transplant 3 more<br />
sessions <strong>of</strong> PP and IVIG were given. Results: We treated 6<br />
patients, 3 in 2nd RT. One persisted positive after scheme<br />
and was not transplanted. Average PRA Class I 91.83% (SD<br />
6.1) Class II 24.14 (SD 31.7. Five transplanted patients with<br />
satisfactory evolution after 7.6 Mo (SD 3.9 Mo) with excellent<br />
renal function: creatinine 0.9 (SD 0.07 mg/dl) and creatinine<br />
depuration 84 (SD 10 ml/min/1.73 m2. One case presented<br />
cellular rejection (percutaneous renal biopsy with negative<br />
c4d) 10 weeks after RT that respond to IV steroids. We do not<br />
detect any infectious complication. Discussion: It is possible<br />
to desensitize positive cross match patients with this<br />
schedule <strong>of</strong> very low IVIG doses, diminishing costs and<br />
without side effects. Until now results are very satisfactory,<br />
we need more cases to obtain experience.<br />
doi:10.1016/j.clim.2007.03.135<br />
S173<br />
Su.96 Semi-mature Bone Marrow Derived DC Expand<br />
Foxp3+CD4+CD25+CD127- Regulatory T Cells with<br />
Suppressive Activity in the Macaque<br />
Aureli Moreau, PhD student, INSERM U643- ITERT/U649,<br />
Nantes, France, Gaelle Beriou, PhD, Center <strong>of</strong> Neurologic<br />
Diseases, Boston, MA, Jack-Yves Deschamps, DVM, ENVN,<br />
Nantes, France, Joanna Ashton-Chess, PhD, INSERM<br />
U643- ITERT, Nantes, France, Heslan Michele, Engineer,<br />
INSERM U643- ITERT, Nantes, France, Elise Chiffoleau, CR2<br />
INSERM, INSERM U643- ITERT, Nantes, France, Regis Josien,<br />
CR1, INSERM U643- ITERT, Nantes, France, Philippe Moullier,<br />
DR2 INSERM, U649, Nantes, France, Brigitte Alliot-Licht,<br />
PhD, INSERM U643- ITERT, Nantes, France, Maria-Cristina<br />
Cuturi, DR2, INSERM U643- ITERT, Nantes, France<br />
Over the past 10 years, several types <strong>of</strong> tolerogenic dendritic<br />
cells (DC) and T regulatory cells (Treg) have been
S174 Abstracts<br />
identified that play a pivotal role in the control <strong>of</strong> autoimmunity<br />
and transplantation tolerance in rodents. Recent<br />
studies have shown that immature DC and ex vivo expanded<br />
Treg could be potential reagents to promote antigen-specific<br />
tolerance in vivo. To date, these works have been carried out<br />
mostly in rodents and will need to be validated in primates<br />
before being applied in the clinic. In order to better<br />
characterise tolerogenic DC and Treg in primates we first<br />
generated and characterised different types <strong>of</strong> DC from<br />
macaque bone marrow (BMDC). Among these cells, the most<br />
immature BMDC (adherent cells resulting from culture in the<br />
presence <strong>of</strong> GM-CSF alone) could be good candidates for in<br />
vivo testing in protocols <strong>of</strong> tolerance induction. We were also<br />
able to demonstrate that in the presence <strong>of</strong> IL-2, semimature<br />
BMDC (cultured with GM-CSF and IL-4) have the<br />
ability to expand CD4+CD25+CD127-Foxp3+ macaque Treg<br />
cells (25 fold in 7 days) and additionally increase their suppressive<br />
activity. These results represent an important step<br />
towards the potential use <strong>of</strong> tolerogenic DC and/or Treg as an<br />
adoptive cell therapy to induce antigen-specific tolerance in<br />
pre-clinical, non-human primate models.<br />
doi:10.1016/j.clim.2007.03.136<br />
Su.98 Low Doses <strong>of</strong> Intravenous Immunoglobulin in<br />
Renal Transplant Patients with High Levels <strong>of</strong><br />
Anti-HLA Panel Reactive Antibodies (PRA)<br />
Miguel Rodriguez, Immunologist, Hospital Especialidades,<br />
IMSS Department Inmunologia, Guadalajara, Mexico,<br />
Alejandro Bautista, Nephrologist, Hospital Especialidades<br />
IMSS, Department Nephrology, Guadalajara, Mexico,<br />
Francisco Monteon, MD, Hospital Especialidades Division <strong>of</strong><br />
Transplantes, Guadalajara, Mexico, Felicia Castro,<br />
Pharmacobiology Chemist, Hospital Especialidades, Division<br />
<strong>of</strong> Transplantes, Guadalajara, Mexico, Efrain Montaño, MD,<br />
Hospital Especialidades IMSS, Department Inmunologia<br />
Clínica, Guadalajara, Mexico, Antonio Flores, MD, Hospital<br />
Especialidades, IMSS Department Nefrologia, Guadalajara,<br />
Mexico<br />
Introduction: The renal transplant (RT) is the best<br />
treatment for the patients with End Stage Renal Disease<br />
(ESRD). Patient with anti-HLA antibodies has a short graft life.<br />
Different studies shown that intravenous immunoglobulin<br />
(IVIG) diminishes anti-HLA antibodies. Methods and materials:<br />
An open, not randomized clinical trial. We used 100 mg/kg<br />
<strong>of</strong> IVIG in patient with PRA higher than 305 with negative<br />
crossmatch to donor (XM−) by flow cytometry (FC). We<br />
administered 3 doses <strong>of</strong> IVIG 100 mg/kg every other day. One<br />
week after the PRA was repeated. Patients with PRA lower<br />
than 30%, were considered desensitized and proceeded to RT,<br />
monitoring the occurrence <strong>of</strong> rejections and graft survival.<br />
Results: We reported 8 patients (3M/5F), age <strong>of</strong> 22.8 SD<br />
2.4 years. Four patients with second RT (50%). PRA previous<br />
88.65% (SD 5.65%) Class I and 50.09% (SD 40.32%) Class II. After<br />
treatment 7.66% (SD 12.32%) Class I and 11.39% (SD25.37%)<br />
Class II. PRA diminished in 7 patients and in 1 patient<br />
remained elevated. Five received a RT from living related<br />
donor, ABO compatible (71.4%) and two <strong>of</strong> them from cadaver<br />
(28.6). The monitoring <strong>of</strong> this group is 46+–25.7 weeks. One<br />
patient (from cadaver) presented two rejection crises both<br />
resolved favorably. Creatinine being 0.95 mg/dl (SD 0.4).<br />
Creatinine depuration (MDRD)88.6 SD 22.05 ml/min/1.73m2.<br />
We did not document any infectious process within the group.<br />
Discussion: IVIG in low doses confer a protective effect, in<br />
patients with high PRA. We need more monitoring to evaluate<br />
the impact in the long term.<br />
doi:10.1016/j.clim.2007.03.137<br />
Su.99 Efficient Ex Vivo Priming <strong>of</strong> Naïve Precursors<br />
into EBV-specific Type-1 CD8+ T Cell Responses from<br />
EBV Seronegative Pediatric HTx Patients Requires<br />
IL-12 and IL-27<br />
Diana Metes, Assistant Pr<strong>of</strong>essor, University <strong>of</strong> Pittsburgh,<br />
Transplantation Surgery, Pittsburgh, PA, Camila Macedo,<br />
Postdoctoral Associate, University <strong>of</strong> Pittsburgh,<br />
Transplantation Surgery, Pittsburgh, PA, Walter Storkus,<br />
Pr<strong>of</strong>essor, University <strong>of</strong> Pittsburgh, Dermatology,<br />
Pittsburgh, PA, Iulia Popescu, Research Instructor,<br />
University <strong>of</strong> Pittsburgh, Transplantation Surgery,<br />
Pittsburgh, PA, Steve Webber, Pr<strong>of</strong>essor, University <strong>of</strong><br />
Pittsburgh, Pediatrics, Pittsburgh, PA<br />
The risk <strong>of</strong> post-transplant lymphoproliferative disorders<br />
(PTLD) is increased in pediatric recipients, and cellular<br />
immunotherapy with EBV specific cytotoxic T lymphocytes<br />
(CTLs) represents a promising therapeutic strategy to restore<br />
cellular immunity in PTLD patients. Although we were<br />
effective at priming strong EBV-specific CTL responses from<br />
EBV-seronegative healthy adults using DC-based protocols,<br />
similar attempts were unsuccessful for EBV-seronegative<br />
pediatric Tx patients. Peripheral blood lymphocytes were<br />
obtained from 5 EBV seronegative HTx patients and were<br />
stimulated ex vivo for 10 days with autologous mature DC<br />
pulsed with a cocktail <strong>of</strong> MHC class I-restricted EBV peptides<br />
as follows: (i)DC/peptide only (ii)DC/peptide +IL-12 (an<br />
adjuvant cytokine for promoting Type-1 immunity-IFNgama<br />
secretion) (iii)DC/peptide +IL-27 (a potent early cytokine<br />
regulator <strong>of</strong> Type-1 commitment) (IV)DC/peptide+IL-12+IL-27.<br />
EBV-specific CD8+ T cell responses were screened by flow<br />
cytometry and IFNgama ELISPOT assays. The DC/peptide +<br />
IL-12 +IL-27 approach yielded the best yields (1.7X), the<br />
optimal EBV-specific CD8+ T cell expansion (from undetectable<br />
to 1.5% +0.5 EBV-tetramer+ CD8 T cells), and optimal<br />
EBV specificity (from 3+2 spots/105 to 45+15 spots/105<br />
CD8+ T cells in ELISPOT assays). DC/peptide (9+2 spots/105)<br />
or the DC/peptide+IL-12 (12 +4 spots/105) protocols were<br />
unsuccessful in this setting. These results suggest that<br />
priming <strong>of</strong> functional EBV-specific CD8+ Tcells from pediatric<br />
EBV seronegative HTx patients is feasible, but EBV-Ag presentation<br />
by DC may require IL-27 in addition to IL-12 to<br />
promote Tc1 cells from naive CD8+ T cell precursors ex vivo.<br />
doi:10.1016/j.clim.2007.03.138<br />
Su.100 Pharmacodynamic Controlled Tapering <strong>of</strong><br />
Cyclosporine A: A New Step Towards Individualized<br />
Immunosuppressive Therapy in Transplanted Patients<br />
Thomas Giese, Group Leader, Institute <strong>of</strong> <strong>Immunology</strong>,<br />
Heidelberg, Germany, Claudia Sommerer, Physician,
Abstracts<br />
Department <strong>of</strong> Nephrology, Heidelberg, Germany, Martin<br />
Zeier, Medical Director, Department <strong>of</strong> Nephrology,<br />
Heidelberg, Germany, Stefan Meuer, Director, Institute <strong>of</strong><br />
<strong>Immunology</strong>, Heidelberg, Germany<br />
Background: At present it is unclear which dose <strong>of</strong> Cyclosporine<br />
A (CsA) is optimal with respect to immunosuppressive<br />
efficacy and drug specific side effects at the level <strong>of</strong> the<br />
individual patient. Recently we proposed the quantitative<br />
assessment <strong>of</strong> NFAT regulated gene expression in peripheral<br />
lymphocytes as a new tool to measure the individual degree<br />
<strong>of</strong> immunosuppression by CsA and demonstrated that a<br />
significant correlation exists between suppression <strong>of</strong> NFATregulated<br />
genes and clinical complications such as infections<br />
and malignancies. The reduction <strong>of</strong> CsA dosage under close<br />
pharmacodynamic monitoring may lead to reduced drug<br />
specific side effects while maintaining optimal graft protection.<br />
Methods: Eight stable renal transplant recipients were<br />
enrolled and longitudinally followed for 27 (12–44) months.<br />
Median CsA dose was 1.83 mg/kg/day at the time <strong>of</strong><br />
enrolment and was tapered to 1.19 mg/kg/day. NFATregulated<br />
gene expression was measured in peripheral<br />
blood lymphocytes stimulated with PMA and ionomycin ex<br />
vivo. All patients had an ultrasound guided allograft biopsy.<br />
Results: The stepwise reduction <strong>of</strong> CsA was inversely<br />
correlated to the residual NFAT-regulated gene expression.<br />
In seven patients there were no signs <strong>of</strong> acute rejection in<br />
the whole study period. One patient had a BANFF Ia<br />
rejection. In this patient the mean residual NFAT-regulated<br />
gene expression had increased up to 47% in 6 months prior to<br />
rejection. All patients without rejection had a median NFATregulated<br />
gene expression <strong>of</strong> 25%. Conclusion: The measurement<br />
<strong>of</strong> residual NFAT-related gene expression is a helpful<br />
and reliable tool in individualizing the immunosuppressive<br />
therapy in long-term allograft recipients.<br />
doi:10.1016/j.clim.2007.03.139<br />
Su.101 A Non-allogeneic Stimulus Triggers the<br />
Production <strong>of</strong> De Novo HLA Antibodies in Healthy<br />
Adults<br />
Jose Crispin, PhD Student, Instituto Nacional de Ciencias<br />
Medicas y Nutricion SZ, Mexico City, Mexico, Luis Morales<br />
Buenrostro, Investigator, Department <strong>of</strong> Nephrology,<br />
Instituto Nacional de Ciencias Medicas y Nutricion SZ,<br />
Mexico City, Mexico, Maria Ines Vargas Rojas, PhD Student,<br />
Department <strong>of</strong> <strong>Immunology</strong> and Rheumatology, Instituto<br />
Nacional de Ciencias Medicas y Nutricion SZ, Mexico City,<br />
Mexico, Lluvia Marino Vazquez, Undergraduate Student,<br />
Department <strong>of</strong> Nephrology, Instituto Nacional de Ciencias<br />
Medicas y Nutricion SZ, Mexico City, Mexico, Claudia de Leo,<br />
Chemist, Department <strong>of</strong> Transplantation, Instituto Nacional<br />
de Ciencias Medicas y Nutricion SZ, Mexico City, Mexico,<br />
Josefina Alberu, Head <strong>of</strong> Department, Department <strong>of</strong><br />
Transplantation, Instituto Nacional de Ciencias Medicas y<br />
Nutricion SZ, Mexico City, Mexico<br />
Transplant recipients develop antibodies against a wide<br />
array <strong>of</strong> HLA specificities, and not only against the antigens<br />
to which they have been exposed. The aim <strong>of</strong> the present<br />
study was to establish if HLA-unrelated immune stimulation<br />
is capable <strong>of</strong> triggering the production <strong>of</strong> HLA antibodies. We<br />
determined the presence <strong>of</strong> HLA antibodies in 20 healthy<br />
adults before and after the administration <strong>of</strong> an immune<br />
stimulus (hepatitis B vaccine plus tuberculin skin test). HLA<br />
antibodies were assessed with a flow-cytometry based<br />
system. At baseline, HBsAg antibodies were detected in<br />
protective titers in 75% <strong>of</strong> the subjects; HLA antibodies were<br />
negative in all but one. One week after the antigenic<br />
stimulus, HBsAg antibody titers increased significantly,<br />
without any detectable changes in HLA antibodies. Surprisingly,<br />
HLA antibodies developed in 8 participants 1 month<br />
after the application <strong>of</strong> the stimulus. One additional subject<br />
developed HLA antibodies 1 month later. Therefore 9/20<br />
subjects became PRA positive during the observation period.<br />
Antibody specificities were identified by single-antigen<br />
assay. The alloantibody response elicited by the immune<br />
stimulus was not consistent with memory cell stimulation.<br />
The findings <strong>of</strong> this study demonstrate that a non-HLA<br />
antigenic stimulus is capable <strong>of</strong> eliciting the development <strong>of</strong><br />
HLA antibodies in healthy adults.<br />
doi:10.1016/j.clim.2007.03.140<br />
S175<br />
Su.102 Activation <strong>of</strong> Transcription Factor<br />
PPARγ In Vivo Protects Against Alloreactive Vascular<br />
Remodeling <strong>of</strong> Human Artery<br />
Ivana Kawikova, Associate Research Scientist, Yale School <strong>of</strong><br />
Medicine, New Haven, CT, Yi Tai, Associate Research<br />
Scientist, Transplantation Surgery, New Haven, CT, Marc<br />
Lorber, Pr<strong>of</strong>essor, Transplantation Surgery, Yale School <strong>of</strong><br />
Medicine, New Haven, CT, George Tellides, Associate<br />
Pr<strong>of</strong>essor, Cardiothoracic Surgery, New Haven, CT, Jordan<br />
Pober, Pr<strong>of</strong>essor, Immunobiology, Yale School <strong>of</strong> Medicine,<br />
New Haven, CT, Alfred Bothwell, Pr<strong>of</strong>essor, Immunobiology,<br />
New Haven, CT<br />
PPARγ is a ligand-activated transcription factor that<br />
controls lipogenesis and plays a role in regulation <strong>of</strong> immune<br />
responses. PPARγ ligands inhibited inflammation in several<br />
animal disease models. Here we tested effects <strong>of</strong> PPARγ<br />
ligands in the in vivo model <strong>of</strong> human graft arteriosclerosis<br />
(GA) in humanized (SCID)/beige mice. Human artery was<br />
inserted into mouse abdominal aorta, allowed to heal and<br />
then the mice were injected with alloreactive PBMC (3×108<br />
ip/mouse). This led to the development <strong>of</strong> GA in human<br />
artery within 4 weeks, while mouse vessels remained intact.<br />
Treatment with endogenous PPARγ ligands, 15-deoxy-prostaglandin<br />
J2(15-d-PGJ2; 50 μg/kg ip for 4 weeks) or<br />
pharmacological PPARγ ligand, ciglitazone (2 mg/kg for 4<br />
weeks ip.) prevents the vascular remodeling. PPARN=antagonist,<br />
GW9662 (300 μg/kg ip for 4 weeks), significantly<br />
reduced the protective effects <strong>of</strong> PPARγ ligands suggesting<br />
that the protection is mainly mediated via PPARγ activation.<br />
Since T cells infiltrating GA lesion produce IFNγ that can on<br />
its own induce GA <strong>of</strong> human artery, we tested the effects <strong>of</strong><br />
15-d-PGJ2(50 μg/kg ip. for 4 weeks) also in this model and<br />
found that the treatment inhibited also IFNγ-induced GA. In<br />
summary, activation <strong>of</strong> PPARN=protects human artery<br />
against alloreactive GA and the mechanisms involve effects<br />
on pathways downstream <strong>of</strong> IFNγ. These studies may open<br />
new possibilities for the treatment <strong>of</strong> human graft rejection
S176 Abstracts<br />
since glitazons are PPARγ ligands that are already FDA<br />
approved drugs used for treatment <strong>of</strong> diabetes type 2.<br />
doi:10.1016/j.clim.2007.03.141<br />
Su.103 Potential Role <strong>of</strong> Donor-derived Regulatory<br />
T Cells in the Suppression <strong>of</strong> Alloimmune Responses<br />
Fleur Samantha Benghiat, MD, Institute for Medical<br />
<strong>Immunology</strong>, Gosselies, Belgium<br />
Naturally occurring CD4+CD25+FoxP3+ regulatory cells<br />
(Tregs) are essential for the regulation <strong>of</strong> allogeneic responses.<br />
In view <strong>of</strong> the small numbers <strong>of</strong> circulating Treg cells,<br />
many protocols nowadays attempt to expand recipient Tregs<br />
ex vivo in order to achieve allograft tolerance by infusing<br />
them back to the recipient. But these are tedious procedures.<br />
We instead hypothesized that Tregs coming from the<br />
same donor as the allograft could be easily extracted in large<br />
numbers and could target the graft by recognizing selfantigens<br />
on the graft. For this purpose, in vitro experiments<br />
were performed with C57BL/6 CD4+CD25neg cells as<br />
responders and bm12 bone marrow derived immature<br />
dendritic cells as stimulators. The efficacy <strong>of</strong> syngeneic<br />
(bm12) versus allogeneic (C57BL/6) Tregs in regulating Th1<br />
and Th2 alloresponses were compared. Our results show that<br />
syngeneic Tregs are as efficient as allogeneic Tregs to<br />
suppress IL-2, IFN-g and IL-13 in a dose dependent manner.<br />
Nevertheless, the more Tregs were added, the more the<br />
proliferation – tested by thymidine incorporation – was<br />
enhanced. Using CFSE staining and Thy1.1–Thy1.2 congenic<br />
markers, it was confirmed that proliferating cells were<br />
Foxp3+ Tregs originally coming from naturally occurring Tregs<br />
added to the culture. Treg proliferation was still observed<br />
when CD4+CD25neg alloreactive cells were substituted by<br />
recombinant IL-2. Similar results were observed with human<br />
cells. In conclusion, allogeneic and syngeneic Tregs are<br />
equally efficient in regulating Th1 and Th2 alloresponses in<br />
vitro and proliferate under the cover <strong>of</strong> IL-2. Ongoing experiments<br />
are investigating the role <strong>of</strong> donor Tregs in allograft<br />
acceptance.<br />
doi:10.1016/j.clim.2007.03.142<br />
Su.104 HMGB1 and IL-1 are Endogenous Mediators<br />
Linking Cell Injury to the Adaptive Immune<br />
Response<br />
Deepak A. Rao, MD/PhD Student, Immunobiology, Yale<br />
University School <strong>of</strong> Medicine, New Haven, CT, Jordan S.<br />
Pober, Pr<strong>of</strong>essor and Vice-Chair, Immunobiology, Yale<br />
University School <strong>of</strong> Medicine, New Haven, CT<br />
Pre- or peri-operative allograft injury predisposes to<br />
acute and chronic allograft rejection; however, the mediators<br />
released by injured tissues that promote rejection<br />
remain unclear. We and others have demonstrated that IFNgamma-producing<br />
T cells play a key role in the development<br />
<strong>of</strong> chronic vascular rejection. Here, we report that freezethaw<br />
lysates <strong>of</strong> human umbilical vein endothelial cells (EC)<br />
increase the amount <strong>of</strong> IFN-gamma produced by T cells when<br />
added to co-cultures <strong>of</strong> CD4+ T cells with HLA-DR+ allogeneic<br />
EC. Immunoadsorption <strong>of</strong> HMGB1, a mediator released by<br />
necrotic cells, partly removes activity from the lysates.<br />
Recombinant HMGB1 has no effect on EC but increases IFNgamma<br />
production by CD4+ Tcells activated by allogeneic EC<br />
or by anti-CD3 and anti-CD28 mAbs. However, HMGB1enhanced,<br />
but not basal, IFN-gamma production is markedly<br />
reduced in co-cultures thoroughly depleted <strong>of</strong> monocytes.<br />
HMGB1 preparations rigorously depleted <strong>of</strong> endotoxin act on<br />
monocytes via TLR4 and CD14 to induce IL-1beta release,<br />
and neutralization <strong>of</strong> IL-1beta significantly reduces the<br />
effect <strong>of</strong> HMGB1 on EC-T cell co-cultures containing<br />
monocytes. EC lysates enhance IFN-gamma production in<br />
the absence <strong>of</strong> monocytes, and neutralization <strong>of</strong> IL-1alpha<br />
blocks almost all <strong>of</strong> this activity. Memory T cells express IL-<br />
1R1, and both IL-1alpha and IL-1beta increase IFN-gamma<br />
production from memory CD4+ T cells activated by allogeneic<br />
EC or anti-CD3 and anti-CD28 mAbs. We conclude that<br />
damaged EC may release HMGB1 and IL-1alpha, both <strong>of</strong><br />
which directly co-stimulate T cell production <strong>of</strong> IFN-gamma.<br />
In addition, HMGB1 induces monocyte secretion <strong>of</strong> IL-1beta,<br />
further promoting IFN-gamma production. Supported by the<br />
NIH.<br />
doi:10.1016/j.clim.2007.03.144<br />
Su.105 Alloantigen-specific Treg: Repertoire<br />
Selection, TH17 Cell Differentiation and<br />
Prolongation <strong>of</strong> Organ Allograft Survival<br />
Giorgio Raimondi, Postdoctoral Scholar, Starzl<br />
Transplantation Institute, University <strong>of</strong> Pittsburgh,<br />
Department <strong>of</strong> Surgery, Pittsburgh, PA, Tokita Daisuke,<br />
Postdoctoral Associate, Starzl Transplantation Institute,<br />
University <strong>of</strong> Pittsburgh, Department <strong>of</strong> Surgery,<br />
Pittsburgh, PA, Zhiliang Wang, Research Assistant Pr<strong>of</strong>essor,<br />
Starzl Transplantation Institute, University <strong>of</strong> Pittsburgh,<br />
Department <strong>of</strong> Surgery, Pittsburgh, PA, Tina Supter,<br />
Postdoctoral Associate, Starzl Transplantation Institute,<br />
University <strong>of</strong> Pittsburgh, Department <strong>of</strong> Surgery,<br />
Pittsburgh, PA, Angus Thomson, Pr<strong>of</strong>essor <strong>of</strong> Surgery, Starzl<br />
Transplantation Institute, University <strong>of</strong> Pittsburgh,<br />
Department <strong>of</strong> Surgery, Pittsburgh, PA<br />
Little information exists regarding the selection and<br />
functional evaluation <strong>of</strong> CD4+CD25+Foxp3+ naturally-arising<br />
regulatory T cells (Treg) for promotion <strong>of</strong> transplant<br />
tolerance. We have investigated/optimized procedures for<br />
the selection <strong>of</strong> murine alloantigen (Ag)-specific Treg<br />
(AAsTreg), and tested their function in a MHC-mismatched<br />
heart allograft model. When naïve Treg were stimulated with<br />
mature (LPS-stimulated), allogeneic DC, with a high IL-2<br />
concentration (500 U/ml), significant numbers <strong>of</strong> Foxp3cells<br />
quickly accumulated. Intracellular flow analysis identified<br />
these Foxp3- cells as IL-17 producers (TH17). The<br />
expansion <strong>of</strong> Treg during an MLR supported then the use <strong>of</strong><br />
immature allogeneic DC as stimulators. However, determination<br />
<strong>of</strong> Treg responder frequency (based on CFSE dilution<br />
analysis) during the MLR (immature DC:CD4+ T cells),<br />
revealed that an unexpected high frequency <strong>of</strong> Treg was<br />
induced to proliferate (approximately 27% <strong>of</strong> Foxp3+ cells,<br />
compared with the anticipated 5–8% <strong>of</strong> non-Treg). The high<br />
levels <strong>of</strong> IL-2 accounted for most <strong>of</strong> what we determined to
Abstracts<br />
be an ‘Ag-non-specific’ stimulation, causing the high<br />
precursor frequency measured. Based on these results, we<br />
used immature allogeneic DC and a mixture <strong>of</strong> stimulatory<br />
cytokines (low concentration) to select AAsTreg that proved<br />
extremely powerful Ag-specific inhibitors <strong>of</strong> alloreactive T<br />
cells proliferation (several-fold superior to freshly-isolated<br />
Treg). Moreover, 50% <strong>of</strong> heart transplants in normal animals<br />
given a short course <strong>of</strong> sub-therapeutic rapamycin, and<br />
injected with AAsTreg on day 7 post-transplant, are currently<br />
beating N70 days. Accounting for <strong>of</strong> all these observations<br />
and further optimization <strong>of</strong> the AAsTreg adoptive transfer<br />
strategy will be key to successful implementation <strong>of</strong> this<br />
clinically relevant regimen.<br />
doi:10.1016/j.clim.2007.03.145<br />
Su.107 Polyclonal Anti-thymocyte Globulin Exhibits<br />
Consistent Immunosuppressive Capabilities Beyond<br />
Cell Depletion<br />
Gina LaCorcia, Principal Research Associate, Genzyme Corp.<br />
IMD Biology and Transplant Research, Framingham, MA,<br />
Mark Swistak, Senior Research Associate, Genzyme Corp.<br />
IMD Biology and Transplant Research, Framingham, MA,<br />
Carla Lawendowski, Principal Research Associate, Genzyme<br />
Corp. IMD Biology and Transplant Research, Framingham,<br />
MA, Tim Weeden, Senior Research Associate, Genzyme Corp.<br />
IMD Biology and Transplant Research, Framingham, MA, Su<br />
Duan, Principal Research Associate, Genzyme Corp. IMD<br />
Biology and Transplant Research, Framingham, MA, Sharon<br />
Nahill, Scientific Director, Genzyme Corp. IMD Biology and<br />
Transplant Research, Framingham, MA, John Williams, Vice<br />
President, Genzyme Corp. IMD Biology and Transplant<br />
Research, Framingham, MA, John Dzuris, Senior Scientist,<br />
Genzyme Corp. IMD Biology and Transplant Research,<br />
Framingham, MA<br />
Thymoglobulin® [Anti-thymocyte Globulin (Rabbit)] is a<br />
purified, gamma globulin produced by immunization <strong>of</strong><br />
rabbits with human thymocytes. It is an FDA approved therapy<br />
for acute organ transplant rejection. Although the<br />
mechanism <strong>of</strong> action <strong>of</strong> Thymoglobulin® is not fully understood,<br />
its efficacy is thought to be the result <strong>of</strong> lymphocyte<br />
depletion via cytotoxic antibodies directed against antigens<br />
expressed on human T lymphocytes. Thymoglobulin® also<br />
contains antigen reactivities that may account for downmodulation<br />
<strong>of</strong> T cell activation and lymphocyte trafficking.<br />
The in vitro studies presented here demonstrate some <strong>of</strong> the<br />
immunosuppressive effects <strong>of</strong> different manufactured<br />
batches <strong>of</strong> Thymoglobulin®. We show that culture <strong>of</strong><br />
human PBMC with Thymoglobulin® results in the emergence<br />
<strong>of</strong> an increased population <strong>of</strong> Foxp3+ regulatory T cells (T<br />
reg) which are functional in that they potently suppress in<br />
vitro T cell activation. All 14 retained batches <strong>of</strong> manufactured<br />
Thymoglobulin® tested induced T reg that were<br />
immunosuppressive. Thymoglobulin® also interacts with<br />
many different cell surface receptors in a consistent and<br />
reproducible manner. Flow cytometric analysis demonstrated<br />
that all batches <strong>of</strong> Thymoglobulin® tested were<br />
comparable in binding ability to several cell surface<br />
receptors. One receptor to which Thymoglobulin® has<br />
reactivity is the chemokine receptor CXCR4. We demonstrate<br />
that 14 manufactured batches <strong>of</strong> Thymoglobulin® tested<br />
inhibit chemotaxis <strong>of</strong> the Jurkat Tcell line in response to SDF-1.<br />
These studies provide additional insights into the mechanisms<br />
by which Thymoglobulin® contributes to prevention and<br />
treatment <strong>of</strong> transplant rejection episodes and underscores<br />
the overall consistency <strong>of</strong> the immunosuppressive capability<br />
among different manufactured batches <strong>of</strong> Thymoglobulin®.<br />
doi:10.1016/j.clim.2007.03.146<br />
Su.108 KIR Genotyping <strong>of</strong> the Venezuelan Mestizo<br />
Population by PCR-SSP<br />
Angela Conesa, Bioanalist, Institute <strong>of</strong> <strong>Immunology</strong>,<br />
Caracas, Venezuela, Felix Toro, Biologist, Institute <strong>of</strong><br />
<strong>Immunology</strong>, Caracas, Venezuela, Nubia Silva, Biologist,<br />
Institute <strong>of</strong> <strong>Immunology</strong>, Caracas, Venezuela, Maria-Elena<br />
Marquez, Biologist, Institute <strong>of</strong> <strong>Immunology</strong>, Caracas,<br />
Venezuela, Paolo Tassinari, MD, Institute <strong>of</strong> <strong>Immunology</strong>,<br />
Caracas, Venezuela, Isaac Blanca, Biologist, Institute <strong>of</strong><br />
<strong>Immunology</strong>, Caracas, Venezuela<br />
Killer-cell-Immunoglobulin-like Receptors (KIR) belong to a<br />
family <strong>of</strong> NK cell receptors that recognize MHC-I molecules. 17<br />
KIR genes have been identified and clustered in 2 major<br />
haplotypes (A and B). These haplotypes show variations in<br />
geographical distribution among different human populations.<br />
Goals: a) To standardize the PCR technique for KIR genotyping<br />
in DNA samples, b) To determine KIR gene frequency in the<br />
Venezuelan mestizo population. Methods: Genomic DNA was<br />
isolated from Peripheral Blood Leucocytes. KIR genotyping was<br />
determined by PCR-SSP technique (1). Different experimental<br />
conditions were assessed: primers and DNA sample concentration,<br />
time and temperature <strong>of</strong> annealing and buffer composition.<br />
A commercial kit for KIR genotyping (Invitrogen) was used<br />
as reference assay. Results: We accomplished gene amplification<br />
<strong>of</strong> 14 KIR genes and the pseudogenes 2DP1 and 3DP1A/B.<br />
From 40 DNA samples analyzed, 100% (f=1) were positive for<br />
KIR2DL1, 2DL4, 2DS4, 3DL1 and pseudogenes 2DP1 and 3DP1.<br />
Other KIR showed a gene frequency b1: 2DL2(.28), 2DL3(.78),<br />
2DL5(.5), 2DS1(.39), 2DS2(.28), 2DS3(.18), 2DS5(.29),<br />
3DL2(.78), 3DL3(.73) and 3DS1(.33). Conclusions: a) Our<br />
preliminary results showed that frequency <strong>of</strong> inhibitory KIR<br />
genes was N0,73, except for KIR2DL2 and 2DL5, whereas<br />
frequency <strong>of</strong> the activating KIR2DS4 gene was equal to 1,<br />
showing an A haplotype similar to the Caucasian population,<br />
b) The PCR-SSP technique represents an efficient and low<br />
cost method for KIR genotyping. Key words, KIR, ADN, PCR-<br />
SSP. Supported by FONACIT G-2005000395. (1)Tissue Antigen<br />
2002; 59:184–193.<br />
doi:10.1016/j.clim.2007.03.147<br />
S177<br />
Su.109 Immunological Properties <strong>of</strong> Human<br />
Embryonic Stem Cell-derived Oligodendrocyte<br />
Progenitor Cells<br />
Ross Okamura, Scientist, Geron Corporation, Cell Biology,<br />
Menlo Park, CA, Melinda Au, Senior Research Associate,<br />
Geron Corporation, Cell Biology, Menlo Park, CA, Catherine<br />
Priest, Associate Director, Geron Corporation, Cell Biology,<br />
Menlo Park, CA, Anish Majumdar, Senior Director, Geron
S178 Abstracts<br />
Corporation, Cell Biology, Menlo Park, CA, Jerrod Denham,<br />
Senior Manager, Geron Corporation, Product Development,<br />
Menlo Park, CA<br />
A major concern in the use <strong>of</strong> allotransplantation <strong>of</strong> human<br />
embryonic stem cell (hESC)-derived cell therapies is the<br />
possibility <strong>of</strong> rejection by the host's immune system. We have<br />
previously reported that the undifferentiated hESC lines H1,<br />
H7 and H9 possess unique immunological properties which<br />
may make them less susceptible to rejection. To determine if<br />
differentiation alters the immunogenicity <strong>of</strong> hESCs, we chose<br />
to study hESC-derived oligodendrocyte progenitor cells (OPC)<br />
that have the potential for clinical application to treat<br />
patients with spinal cord injury. Undifferentiated H1 and H1derived<br />
OPCs express low to moderate levels <strong>of</strong> surface HLA-A,<br />
B,C antigens and lack surface expression <strong>of</strong> HLA-DR,DP,DQ<br />
antigens. In an in vitro mixed lymphocyte reaction (MLR)<br />
assay, both hESCs and OPCs stimulated weak T cell proliferation<br />
compared to allogeneic dendritic cells. Both undifferentiated<br />
hESCs (uhESCs) and their differentiated OPC<br />
derivatives were largely resistant to NK cell-mediated lysis.<br />
We have also investigated whether hESC-derived OPCs are<br />
susceptible to the presence <strong>of</strong> anti-Neu5Gc antibodies in<br />
normal human sera. We observed that <strong>of</strong> ten sera from normal<br />
healthy individuals representing all four blood groups, uhESCs<br />
showed no susceptibility to lysis and eight sera did not show<br />
measurable cytotoxicity against OPCs. The same sera when<br />
tested against murine embryonic fibroblasts (MEFs) demonstrated<br />
significant complement-dependent lysis. Taken<br />
together, these data show that the hESC-derived OPCs retain<br />
some <strong>of</strong> the unique immunological properties <strong>of</strong> the parental<br />
cell line from which they were differentiated.<br />
doi:10.1016/j.clim.2007.03.148<br />
Su.110 Adhesion Molecules in Pancreatitisassociated<br />
Lung Injury<br />
Roman Vatseba, Medical University, Lviv, Ukraine, Serge<br />
Chooklin, Medical University, Lviv, Ukraine, Myroslav Lyba,<br />
Medical University, Lviv, Ukraine<br />
Respiratory failure is typical for acute necrotizing pancreatitis.<br />
Unfortunately, the pathogenesis <strong>of</strong> these changes is<br />
fully unknown. The role <strong>of</strong> the endothelium in the human<br />
acute pancreatitis is still unclear. Applying the ELISA<br />
technique, plasma levels <strong>of</strong> soluble E-selectin (sE-selectin)<br />
and sICAM-1 were studied in 51 patients with acute<br />
pancreatitis. Twenty-five <strong>of</strong> them had respiratory complications.<br />
In this study enrolled patients who admitted to clinic<br />
within 48 hours after disease onset. The control group<br />
compiled 10 healthy volunteers. Already after admission the<br />
increased levels <strong>of</strong> adhesion molecules were noted in all<br />
patients. By that, the highest levels were observed in<br />
patients with lung injury. During first 7 days plasma levels<br />
<strong>of</strong> sE-selectin and sICAM-1 practically did not change in<br />
patients with necrotizing pancreatitis, while in patients with<br />
the pancreatitis-associated lung injury its levels gradually<br />
increased starting from the third day. There was a clear<br />
correlation between sE-selectin, sICAM-1 and severity <strong>of</strong><br />
hemodynamic compromise was established. The intensity <strong>of</strong><br />
endothelial activation, measured by adhesion molecules<br />
concentration, considered to be key features <strong>of</strong> systemic<br />
inflammation in severe pancreatitis. These data pointed on<br />
the role <strong>of</strong> adhesion molecules in the pathogenesis <strong>of</strong><br />
pancreatitis-associated lung injury. Blockade <strong>of</strong> ICAM-1 and<br />
E-selectin synthesis and its receptors may be a novel target<br />
in the management <strong>of</strong> pancreatitis-associated lung injury.<br />
doi:10.1016/j.clim.2007.03.149<br />
Su.111 Aire Deficient Mice—A Model for<br />
Autoimmune Lung Disease<br />
Anthony Shum, <strong>Clinical</strong> Fellow, University <strong>of</strong> California, San<br />
Francisco Department <strong>of</strong> Medicine, San Francisco, CA, Jason<br />
DeVoss, Postdoctoral Fellow, University <strong>of</strong> California, San<br />
Francisco Diabetes Center, San Francisco, CA, Mark<br />
Anderson, Assistant Pr<strong>of</strong>essor, University <strong>of</strong> California, San<br />
Francisco Diabetes Center and Department <strong>of</strong> Medicine, San<br />
Francisco, CA<br />
Aire deficient mice have a mutation for the gene encoding<br />
AIRE, a transcription factor that drives the ectopic expression<br />
<strong>of</strong> organ-specific antigens in the thymus. A deficiency in AIRE<br />
results in a breakdown <strong>of</strong> central tolerance toward selfantigens<br />
resulting in organ-specific autoimmune disease. The<br />
AIRE knockout mouse is a model for Autoimmune Polyglandular<br />
Syndrome Type 1, a disorder characterized by polyglandular<br />
autoimmune disease. Pulmonary manifestations,<br />
while rare, have been reported, including lymphocytic<br />
interstitial pneumonitis and bronchiolitis obliterans organizing<br />
pneumonia. Our goals are to 1) understand the mechanism<br />
<strong>of</strong> autoimmune-mediated attack and its relationship to the<br />
thymus 2) identify lung specific auto-antigens. Methods: AIRE<br />
knockout mice were examined for lung histology and<br />
immunohistochemistry. Serum from knockout mice was used<br />
for immunoblotting and indirect immun<strong>of</strong>luorescence. Flow<br />
cytometry and intracellular cytokine staining was performed<br />
on lymphocytes isolated from AIRE knockout lungs. Results:<br />
Lung histology reveals perivascular and peribronchiolar<br />
mononuclear inflammation. Immunoblots reveal a predominant<br />
antigen at 84 kilodaltons. The presence <strong>of</strong> autoantibody<br />
to this antigen correlates with the presence <strong>of</strong> histologic<br />
infiltrates. Indirect immun<strong>of</strong>luorescence on lung sections<br />
from unaffected mice show staining <strong>of</strong> bronchiolar epithelium.<br />
Immune cell subtypes visualized on sections <strong>of</strong> knockout<br />
mice reveal a predominance <strong>of</strong> CD4+ cells. Flow cytometric<br />
analysis <strong>of</strong> lymphocytes from lungs <strong>of</strong> knockout mice confirm<br />
the predominance <strong>of</strong> CD4+ T cells. Intracellular cytokine<br />
staining <strong>of</strong> lymphocytes reveal the predominance <strong>of</strong> the TH1<br />
cytokine IFN-γ. Conclusions: AIRE deficient mice are a<br />
promising model <strong>of</strong> spontaneous autoimmune lung disease<br />
<strong>of</strong>fering the potential to identify lung auto-antigens.<br />
doi:10.1016/j.clim.2007.03.150<br />
Su.112 The XX Sex Chromosome Complement, as<br />
compared to the XY, Confers Greater Susceptibility to<br />
Experimental Autoimmune Diseases — EAE and SLE<br />
Deborah Smith, Doctoral Candidate, University <strong>of</strong> California,<br />
Los Angeles Neuroscience, Los Angeles, CA, Sienmi Du,<br />
Research Assistant, University <strong>of</strong> California, Los Angeles
Abstracts<br />
Neurology, Los Angeles, CA, Seema Tiwari-Woodruff,<br />
Associate Pr<strong>of</strong>essor, University <strong>of</strong> California, Los Angeles<br />
Neurology, Los Angeles, CA, Arthur P. Arnold, Chair,<br />
Distinguished Pr<strong>of</strong>essor, University <strong>of</strong> California, Los Angeles<br />
Physiological Science, Los Angeles, CA, Jennifer K. King,<br />
Post-Residency Fellow, University <strong>of</strong> California, Los Angeles,<br />
Rheumatology, Los Angeles, CA, Ram Raj Singh, Pr<strong>of</strong>essor<br />
<strong>of</strong> Medicine, University <strong>of</strong> California, Los Angeles Medicine-<br />
Rheumatology, Los Angeles, CA, Rhonda R. Voskuhl,<br />
Pr<strong>of</strong>essor in Residence, University <strong>of</strong> California, Los<br />
Angeles Neurology, Los Angeles, CA<br />
The majority <strong>of</strong> autoimmune diseases are more common<br />
in women as compared to men. This may be attributed to<br />
differences in sex hormones, sex chromosomes or both. To<br />
determine the contribution <strong>of</strong> sex chromosomes to sex<br />
differences in susceptibility to autoimmune disease, we used<br />
animal models characterized by a known female preponderance,<br />
experimental autoimmune encephalomyelitis (EAE)<br />
and pristane-induced lupus, each in SJL mice. Mice which<br />
have Sry, the gene that encodes for testicular development,<br />
deleted from the Y chromosome were backcrossed onto the<br />
SJL strain, resulting in EAE and lupus-susceptible mice which<br />
differ in their complement <strong>of</strong> sex chromosomes, while having<br />
the same gonadal type, thereby permitting assessment <strong>of</strong><br />
sex chromosome effects not confounded by differences in<br />
sex hormones (ovary bearing XX vs. XY- mice). When<br />
comparing EAE in ovariectomized XX vs. XY- SJL mice,<br />
disease was more severe and there was more inflammation in<br />
the spinal cords <strong>of</strong> mice with the XX sex chromosome<br />
complement. Also, when lupus was induced in ovariectomized<br />
XX vs. XY- SJL mice, mortality, nephritis and autoantibody<br />
levels were each greater in mice with the XX sex<br />
chromosome complement. The differences in susceptibility<br />
to EAE and lupus also occurred when comparing castrated XX<br />
Sry versus XY- Sry mice (testis bearing mice). This is the first<br />
evidence that XX sex chromosome complement, as compared<br />
to XY, confers greater susceptibility to two immunopathologically<br />
distinct diseases. This may be relevant to the<br />
increased susceptibility <strong>of</strong> women to a variety <strong>of</strong> distinct<br />
autoimmune diseases.<br />
doi:10.1016/j.clim.2007.03.151<br />
Su.113 Expression <strong>of</strong> the Renal Antigen Neprhin by<br />
Human Medullary Thymic Epithelial Cells<br />
Michael Tasch, Senior Fellow, University <strong>of</strong> Washington,<br />
Department <strong>of</strong> Medicine, Seattle, WA, Kimberly Muczynski,<br />
Associate Pr<strong>of</strong>essor, University <strong>of</strong> Washington, Department<br />
<strong>of</strong> Medicine, Seattle, WA, Anne Stevens, Assistant Pr<strong>of</strong>essor,<br />
University <strong>of</strong> Washington, Department <strong>of</strong> Pediatrics,<br />
Seattle, WA, J. Lee Nelson, Pr<strong>of</strong>essor, Fred Hutchinson<br />
Cancer Research Center, Division <strong>of</strong> <strong>Clinical</strong> Research,<br />
Seattle, WA<br />
Idiopathic nephrotic syndrome, a set <strong>of</strong> glomerular pathologies<br />
<strong>of</strong>ten leading to end-stage renal disease, is associated<br />
with disturbances in Tcell function. This has led to the<br />
view that these diseases are a consequence <strong>of</strong> T cell<br />
mediated autoimmunity, though the relevant autoantigen(s)<br />
remain unidentified. Nephrin is a possible target for<br />
autoimmunity in the kidney. The protein product <strong>of</strong> the<br />
NPHS1 gene and an essential component <strong>of</strong> the glomerular<br />
filtration barrier, nephrin is expressed proximal to autoimmune<br />
renal lesions and conforms to the definition <strong>of</strong> a<br />
tissue restricted antigen (TRA). Since intrathymic expression<br />
<strong>of</strong> other TRAs has been implicated in the establishment <strong>of</strong><br />
central tolerance and protection from organ-specific autoimmunity,<br />
we hypothesized that nephrin is expressed by cells<br />
<strong>of</strong> the human thymus. RT-PCR analysis showed that NPHS1<br />
mRNA is present in all human thymi (N=12) tested. Using<br />
primer sets flanking sequences encoding immunoglobulinlike<br />
domains, we showed that the thymic-specific transcript<br />
appears identical to that from kidney. FACS-purified CD326+,<br />
HLA-DR+ medullary thymic epithelial cells (mTEC) exhibited<br />
the highest concentration <strong>of</strong> NPHS1 transcripts. Immun<strong>of</strong>luorescence<br />
analysis showed that nephrin protein is<br />
expressed in thymus sections. Nephrin exhibited a predominantly<br />
medullary pattern <strong>of</strong> expression, co-localizing with<br />
HLA-DR and cytokeratin, consistent with mTEC, although<br />
infrequent nephrin-positive cells were seen in the cortex and<br />
capsule also. Nephrin staining also showed distinct intracellular<br />
patterns including surface, intracellular granules and<br />
punctate structures. These data provide a strong rationale<br />
for exploring nephrin's role in negative selection and the role<br />
<strong>of</strong> nephrin-specific T cells in human nephrotic syndrome.<br />
doi:10.1016/j.clim.2007.03.153<br />
S179<br />
Su.114 Low-dose <strong>of</strong> Local Radiation Treatment Can<br />
Inhibit the Progression <strong>of</strong> Experimental<br />
Autoimmune Nephritis by Promoting Apoptosis<br />
M. S. Razzaque, Research Scientist, Department <strong>of</strong><br />
Pathology, Nagasaki University, Nagasaki, Japan, L. Diange,<br />
Research Scientist, Department <strong>of</strong> Pathology, Nagasaki<br />
University, Nagasaki, Japan, A. Nazneen, Research<br />
Scientist, Department <strong>of</strong> Pathology, Nagasaki University,<br />
Nagasaki, Japan, T. Taguchi, Pr<strong>of</strong>essor, Department <strong>of</strong><br />
Pathology, Nagasaki University, Nagasaki, Japan<br />
Autoimmune crescentic glomerulonephritis (CrGN) is a<br />
rapidly progressive form <strong>of</strong> nephritis and usually resistant to<br />
therapeutic intervention. Apoptosis plays important role in<br />
resolution <strong>of</strong> CrGN. We investigated the effects <strong>of</strong> local<br />
radiation treatment in the progression <strong>of</strong> CrGN in rats. Three<br />
experimental groups were studied; Group-I: sham-operated<br />
control (n=12); Group-II: intravenous injection <strong>of</strong> rabbit<br />
anti-rat GBM antibody (nephrotoxic serum, NTS) (n=23); and<br />
Group-III: a single low-dose <strong>of</strong> 0.5 Gy X-ray radiation<br />
treatment to local bilateral kidneys at 6, 13, 20, and<br />
27 days after NTS injection (n=55). In Group-III, the level<br />
<strong>of</strong> BUN and serum creatinine was significantly decreased<br />
after radiation treatment compared with age-matched<br />
Group-II nephritic rats (pN0.05 or 0.001). Glomerular<br />
hypercellularity, crescents, global sclerosis and tubulointerstitial<br />
damage gradually developed throughout the time<br />
course in Group-II rats, were significantly less (PN 0.05 or<br />
0.001) after radiation treatment in Group-III. The expression<br />
<strong>of</strong> active caspases-3 and -7 was increased for irradiated<br />
kidneys, compared with their corresponding Group-II rats.<br />
Western blot analysis showed that 17 kDa active caspase-3<br />
and 35 kDa active caspase-7 expressions markedly increased
S180 Abstracts<br />
in Group-III kidneys, compared with the Group-II. Increased<br />
expression <strong>of</strong> p53 protein was also observed in radiation<br />
treated kidneys compared with groups Group-II. Low-dose <strong>of</strong><br />
local radiation to the kidneys, can delay the progression <strong>of</strong><br />
CrGN in rats by p53-dependent radiation-induced apoptosis.<br />
Caspases-3 and -7 are the key mediators in such apoptosis.<br />
This study shows that radiation treatment is effective in<br />
preventing acute glomerular inflammation and crescent<br />
formation in the rat model <strong>of</strong> autoimmune CrGN.<br />
doi:10.1016/j.clim.2007.03.154<br />
Su.116 IL-1beta, MMP-1, and MMP-3 Transcript<br />
Levels are Associated with CD3delta Expression in<br />
the Osteoarthritic Synovial Membrane<br />
Carla Scanzello, Rheumatology Fellow, Department <strong>of</strong><br />
Rheumatology, Hospital for Special Surgery, New York, NY,<br />
Andrew Pearle, Assistant Attending Orthopedic Surgeon,<br />
Department <strong>of</strong> Orthopedics, Hospital for Special Surgery,<br />
New York, NY, Mary Crow, Attending Physician, Department<br />
<strong>of</strong> Rheumatology, Hospital for Special Surgery, New York,<br />
NY, Edward DiCarlo, Associate Attending Pathologist,<br />
Department <strong>of</strong> Laboratory Medicine, Hospital for Special<br />
Surgery, New York, NY<br />
Many patients with osteoarthritis (OA) develop inflammatory<br />
infiltrates containing T lymphocytes within the<br />
synovial membrane (SM). These infiltrates have the<br />
potential to augment cytokine (i.e.IL-1beta and TNFalpha)<br />
and degradative enzyme (i.e. MMPs) production by<br />
macrophages or synovial fibroblasts. Purpose: The aim <strong>of</strong><br />
this study was to determine if lymphocytic synovial<br />
infiltration in patients with OA is associated with increased<br />
cytokines and enzyme expression implicated in cartilage<br />
catabolism. Methods: SM specimens from 37 OA patients<br />
undergoing total hip or knee arthroplasty (n=31) or<br />
arthroscopy (n=6) were obtained. Total RNA was extracted<br />
and cDNA synthesized. Expression levels <strong>of</strong> CD3delta, IL-<br />
1beta, TNF-alpha, IL-15, IL-6, IL-10, TGF-beta, MMP-1, and<br />
MMP-3, were measured by real-time PCR. Synovial inflammation<br />
was graded in H&E sections on a four-point scale.<br />
Correlation coefficients were calculated to determine if<br />
cytokine and enzyme mRNA levels varied with CD3delta<br />
expression or histologic score. Results: CD3delta transcript<br />
levels correlated most significantly with IL-1beta (r=0.677,<br />
pb0.0001), but were also associated with TNF-alpha<br />
(r=0.487, p=0.003), IL-15 (r=0.524, p=0.001), IL-10<br />
(r=0.570, p=0.0004), and TGF-beta (r=0.381, p=0.022).<br />
CD3delta levels were also associated with MMP-1<br />
(r=0.432, p=0.008) and MMP-3 (r=0.506, r=0.0014) transcripts.<br />
In addition, MMP-1 expression was significantly<br />
associated with increasing histologic inflammatory score<br />
(r=0.412, p=0.011). Conclusions: mRNA levels <strong>of</strong> IL-1beta,<br />
MMP-1 and MMP-3 correlated with levels <strong>of</strong> CD3delta<br />
transcripts within the SM <strong>of</strong> patients with both knee and<br />
hip OA.These relationships suggest that synovial infiltrating<br />
T cells may contribute to disease pathogenesis in OA by<br />
promoting synthesis <strong>of</strong> these mediators <strong>of</strong> joint damage.<br />
doi:10.1016/j.clim.2007.03.155<br />
Su.117 An In Silico Model <strong>of</strong> Regulatory T Cell<br />
Function<br />
Paul Taylor, Postdoctoral Researcher, La Jolla Institute for<br />
Allergy and <strong>Immunology</strong>, San Diego, CA, Matthias von<br />
Herrath, Primary Investigator, La Jolla Institute for Allergy<br />
and <strong>Immunology</strong>, San Diego, CA<br />
Regulatory T (Treg) cells participate in response to<br />
infection reported so far involve chronic or persistent viral<br />
infections. The findings that Treg cells influence the functional<br />
immunity during viral infections however, might<br />
indicate that, in some cases, virus-specific Treg cells not<br />
only influence immune pathology or prevent pathogen elimination<br />
but also can promote a generalized state <strong>of</strong><br />
immuno-suppression in vivo such that the host is more<br />
susceptible to secondary infections with other pathogens or<br />
has reduced tumour resistance. The regulatory mechanism by<br />
which Tregs act has yet to be definitively characterized.<br />
There is opposing evidence to suggest either cell-to-cell<br />
contact being required with the cell being suppressed or a<br />
cytokine mediated mechanism, either through immunosuppressive<br />
cytokines or by acting as a growth factor ‘sink’. We<br />
have developed a cellular automaton that is an in silico representation<br />
<strong>of</strong> the cells <strong>of</strong> the immune system. The<br />
automaton aims to faithfully simulate these cells and models<br />
their interaction with cognate rule sets derived from in vitro<br />
and in vivo experimentation. The automaton has been<br />
developed to provide a stochastic model <strong>of</strong> a Treg suppression<br />
assay which can be used to investigate the possible mechanisms<br />
behind their immuno-suppressive effect. The model can<br />
rapidly simulate a large number <strong>of</strong> assays under a wide range<br />
<strong>of</strong> parameters and the detailed results allow for the analysis<br />
<strong>of</strong> the parameters at a detail level currently impossible in<br />
vitro. The insights obtained by using the automaton provide a<br />
powerful tool for directing in vitro research into Treg<br />
function.<br />
doi:10.1016/j.clim.2007.03.156<br />
Su.118 Data Analysis Protocols for Identifying<br />
Cytokine Signatures in Breast Cancer and Type 1<br />
Diabetes<br />
Janet Siebert, Data Architect, CytoAnalytics, Denver, CO,<br />
Margaret Inokuma, Scientist, BD Biosciences, San Jose, CA,<br />
Nathan Pennock, Pr<strong>of</strong>essional Research Assistant, Webb-<br />
Waring Institute, The University <strong>of</strong> Colorado Denver and<br />
Health Sciences Center, Denver, CO, Dan Waid, Researcher,<br />
Webb-Waring Institute, Denver, CO, Corazon dela Rosa,<br />
Research Scientist, Medicine/Oncology/Tumor Vaccine<br />
Group, University <strong>of</strong> Washington, Seattle, WA, Mary Disis,<br />
Pr<strong>of</strong>essor, Medicine/Oncology/Tumor Vaccine Group,<br />
University <strong>of</strong> Washington, Seattle, WA, Jack Dunne,<br />
Research Scientist, BD Biosciences, San Jose, CA, David<br />
Wagner, Assistant Pr<strong>of</strong>essor, Webb-Waring Institute, The<br />
University <strong>of</strong> Colorado Denver and Health Sciences Center,<br />
Denver, CO, Holden Maecker, Research Scientist, BD<br />
Biosciences, San Jose, CA<br />
Large clinical studies <strong>of</strong> cytokine expression generate<br />
complex data sets which can be difficult to analyze. With the<br />
identification <strong>of</strong> cytokine signatures and biomarkers a goal <strong>of</strong>
Abstracts<br />
much immunodiagnostic research, fast and effective methods<br />
for extracting meaning from the data are needed. This<br />
interdisciplinary study presents several data analysis protocols<br />
for identfying cytokine signatures. Two different data<br />
sets are considered. The first examines cytokine signatures<br />
<strong>of</strong> T cell responses to tumor antigens in breast cancer<br />
patients (IFNγ, IL-2, and TNFα as expressed by CD4+ and<br />
CD8+ T cells). The second explores cytokine signatures <strong>of</strong><br />
Type 1 diabetes patients (IFNγ, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-<br />
8, IL-10, TNFα and TNFβ; on T cells). In the breast cancer<br />
study, a successful response to a foreign antigen (CMV<br />
antigen in CMV seropositive donors) was compared to an<br />
unsuccessful response to a self antigen (CEA, Her2/neu, and<br />
MAGE-3). The differing magnitude <strong>of</strong> the cytokine responses<br />
required data normalization techniques to support visual and<br />
statistical comparison <strong>of</strong> the cytokine responses. In the Type<br />
1 diabetes study, measurements were collected for 10<br />
cytokines and 4 activation environments, for a total <strong>of</strong> 40<br />
different measurements per donor. High-throughput data<br />
analysis techniques allowed quick identification <strong>of</strong> significant<br />
differences between healthy and diseased cohorts, and<br />
the exploration <strong>of</strong> differing donor-level signature patterns.<br />
Taken together, these protocols demonstrate effective<br />
methods for identifying signature patterns <strong>of</strong> multiple<br />
cytokines in complex data sets.<br />
doi:10.1016/j.clim.2007.03.157<br />
Su.119 Carbon Monoxide Generated by Heme<br />
Oxygenase-1 Activity Confers Tolerogenic Capacity<br />
to Dendritic Cells<br />
Christine Chauveau, Postdoctoral Fellow, ITERT-INSERM<br />
U643, Nantes, France, Régis Brion, Technician, ITERT-INSERM<br />
U643, New York, AK, Séverine Remy, Engineer, ITERT-INSERM<br />
U643, Nantes, France, Marcelo Hill, Postdoctoral Fellow,<br />
ITERT-INSERM U643, Nantes, France, Pierre-Joseph Royer,<br />
Postdoctoral Fellow, INSERM U601, Nantes, France, Séverine<br />
Tanguy-Royer, PhD, ITERT-INSERM U643, Nantes, France,<br />
Laurent Tesson, Engineer, ITERT-INSERM U643, Nantes,<br />
France, Roberto Motterlini, GroupLeader, Northwick Park<br />
Hospital, Harrow, Roberta Foresti, Group Leader, Northwick<br />
Park Hospital, Harrow, Ignacio Anegon, Group Leader,<br />
ITERT-INSERM U643, Nantes, France<br />
Heme oxygenase -1 (HO-1) is the inducible heme oxygenase<br />
that catabolizes the degradation <strong>of</strong> heme into<br />
biliverdin Fe 2+ and carbon monoxide (CO). Biliverdin is<br />
subsequently reduced into bilirubin by biliverdin reductase,<br />
and Fe2+ induces the expression <strong>of</strong> ferritin. We have<br />
recently described that immature DCs express HO-1 and<br />
that the expression <strong>of</strong> HO-1 confers tolerogenic capacity to<br />
DCs. We now demonstrate that treatment <strong>of</strong> human<br />
monocyte-derived DCs by exogenous CO blocks LPS-induced<br />
increases in MHC II, CD83, CD80, CD86 expression and proinflammatory<br />
IL-12 cytokine production associated with DC<br />
maturation, while preserving the expression <strong>of</strong> the antiinflammatory<br />
cytokine IL-10. CO treated DCs also display a<br />
reduced capacity to induce T cell allogeneic proliferation in<br />
vitro. In contrast, treatment <strong>of</strong> DCs with bilirubin, biliverdin<br />
and deferoxamine (which mimics the action <strong>of</strong> ferritin) had<br />
no effect on DC phenotype or cytokine production. These<br />
observations indicate that the effect <strong>of</strong> HO-1 on DC function<br />
is mediated through CO. HO-1 induced the expression <strong>of</strong> IL-<br />
10 and the inhibition <strong>of</strong> DC maturation by HO-1 was partially<br />
prevented by anti-IL-10 antibodies. Preliminary results show<br />
that HO-1 expression by DCs inhibits autoimmune diabetes<br />
in a transgenic model. In conclusion, we show the capacity<br />
<strong>of</strong> CO generated by HO-1 activity to block DC maturation<br />
and to inhibit pro-inflammatory and allogeneic immune<br />
responses while preserving IL-10 production. This novel<br />
immune function for CO may be <strong>of</strong> interest for the inhibition<br />
<strong>of</strong> immune responses in autoimmune diseases, transplantation,<br />
and other conditions involving activation <strong>of</strong> the<br />
immune system.<br />
doi:10.1016/j.clim.2007.03.158<br />
Su.120 Real-time Characterization <strong>of</strong> Antibodies<br />
and Biomarker Detection in the Study <strong>of</strong><br />
Immune-based Disease<br />
Jean-Francois Houle, Director, Axela Biosensors, Toronto,<br />
ON, Canada<br />
We describe a versatile platform for more informed decisions<br />
in assay development for biomarker detection and<br />
characterization <strong>of</strong> the etiological agents <strong>of</strong> some immune<br />
disorders and monitoring responses to therapeutic interventions.<br />
Diffraction-based sensing is a simple, sensitive technique<br />
for detecting real-time biomolecular interactions in<br />
complex media. Briefly, capture molecules or substrates are<br />
immobilized on a flat surface in a specific optimized grating<br />
that produces a strong diffraction pattern when illuminated.<br />
Binding <strong>of</strong> biomolecules to the patterned capture molecules<br />
causes a change in the diffractive properties <strong>of</strong> the pattern,<br />
which increases the diffracted signal intensity. Conversely,<br />
release <strong>of</strong> the substrate or interacting species leads to a<br />
measurable decrease in signal. We have used this ability to<br />
continuously observe antibody–antigen interactions as a<br />
means to significantly accelerate immunoassay development.<br />
Representative examples include antibody characterization,<br />
activity measurements, antibody mapping and<br />
pairing studies, buffer optimization for assays as well as<br />
quantitative analyte measurements. Furthermore, we have<br />
demonstrated simultaneous measurement <strong>of</strong> biomarkers<br />
from high micromolar through single-digit picomolar levels<br />
in a single sample.<br />
doi:10.1016/j.clim.2007.03.159<br />
S181<br />
Su.121 Inhibition <strong>of</strong> CXCR2 Expression in HL60 Cells<br />
by CXCR2 Antisense RNA<br />
Lingjie Liao, Postdoctoral Researcher, Department <strong>of</strong><br />
Pediatrics, Tongji Hospital, Huazhong University <strong>of</strong> Science<br />
and Technology, Wuhan, China, Wei Wang, Postgraduate<br />
Student, Department <strong>of</strong> Pediatrics, Tongji Hospital,<br />
Huazhong University <strong>of</strong> Science and Technology, Wuhan,<br />
China, Qin Ning, Chief Physician, Department <strong>of</strong> Infectious<br />
Disease, Tongji Hospital, Huazhong University <strong>of</strong> Science<br />
and Technology, Wuhan, China, Xiaoping Luo, Chief<br />
Physician, Department <strong>of</strong> Pediatrics, Tongji Hospital,<br />
Huazhong University <strong>of</strong> Science and Technology, Wuhan,
S182 Abstracts<br />
China, Guanxin Shen, Pr<strong>of</strong>essor, Department <strong>of</strong> <strong>Immunology</strong>,<br />
Tongji Medical College, Huazhong University <strong>of</strong> Science and<br />
Technology, Wuhan, China<br />
Summary: CXC chemokine receptor 2 (CXCR2) is the<br />
common receptor <strong>of</strong> several potent chemokines, which plays<br />
an important role in the neutrophil-mediated inflammation.<br />
In this study, we investigated the effect <strong>of</strong> CXCR2 antisense<br />
RNA on the gene expression and chemotactic activity in<br />
promyelocytic HL60 cells. A human CXCR2 antisense RNA<br />
recombinant vector, pcDNA-ASCXCR2 complementary to nt<br />
−21∼ nt 1080 <strong>of</strong> CXCR2 was constructed and transfected into<br />
HL60 cells by electroporation. The stable transfected clones<br />
were screened by G418. The expression <strong>of</strong> CXCR2 gene was<br />
measured by RT-PCR and immunohistochemistry. Chemotactic<br />
response to IL-8 was determined using Boyden chamber<br />
procedure. CXCR2 expression in pcDNA-ASCXCR2 transfected<br />
HL60 cells was significantly decreased at mRNA and protein<br />
levels, as compared with those <strong>of</strong> untransfected cells.<br />
Furthermore, chemotactic indexes <strong>of</strong> HL60 cells were<br />
markedly decreased by pcDNA-ASCXCR2 transfection. In<br />
conclusion, CXCR2 antisense RNA can suppress chemotactic<br />
activity through depleting CXCR2 expression on plasma<br />
membrane <strong>of</strong> targeted cells, which would be a new<br />
therapeutic strategy for inflammatory diseases. This work<br />
was supported by the NSFC National Science Fund<br />
£¨No.30571643£No.30672380£©, and National Key Basic<br />
Research Program <strong>of</strong> China (2005CB522901, 2005CB522507)<br />
and <strong>Clinical</strong> Subject Key Project from the Ministry <strong>of</strong> Health.<br />
doi:10.1016/j.clim.2007.03.160<br />
Su.122 Functional Polymorphisms <strong>of</strong> MICA and MICB<br />
in NK Cell Activation<br />
Amonrat Jumnainsong, PhD Candidate, Faculty <strong>of</strong> Medical<br />
Technology, Mahidol University, Bangkok, Thailand, Khon<br />
Kaen, Thailand, Hugh Reyburn, Department <strong>of</strong> Pathology,<br />
University <strong>of</strong> Cambridge, Cambridge, MA, Mar Vales-Gomez,<br />
Department <strong>of</strong> Pathology, University <strong>of</strong> Cambridge,<br />
Cambridge, MA, Chanvit Leelayuwat, Assistant Pr<strong>of</strong>essor,<br />
Department <strong>of</strong> <strong>Clinical</strong> <strong>Immunology</strong>, Khon Kaen University,<br />
Khon Kaen, Thailand<br />
MICA and MICB are ligands <strong>of</strong> NKG2D that expressed on the<br />
cell surface <strong>of</strong> many immune cells. The MIC expressions are<br />
increased in response to cellular stress including tumor<br />
transformation and viral infection. Although MICA and MICB<br />
genes are highly polymorphic and associated with some<br />
diseases, the evidence demonstrating different capability <strong>of</strong><br />
diverse alleles to activate NK cells is rare. In this study, we<br />
investigated the effect <strong>of</strong> various MICA and MICB alleles in NK<br />
cell activation. C1R stable transfected cells expressing<br />
different MICA or MICB alleles were tested for NK cell<br />
activation by chromium releasing assay and IFN-f× production.<br />
MICA*004 transfectants expressing MICA at a higher level<br />
could induce NK cells less than transfectants expressing<br />
MICA*01801 and 045. It is known that MICA amino acid at<br />
position 129 affects NKG2D binding. The methionine at this<br />
position can bind to NKG2D stronger than that <strong>of</strong> valine.<br />
MICA*004 carries valine whereas MICA*01801 and 045 carry<br />
methionine at this position. Thus, it is conceivable that this<br />
position may also affect NK cell activation. MICB*002 and<br />
MICB*004 transfectants could express MICB at the same level<br />
but MICB*002 expressing cells could activate NK cells<br />
stronger than those <strong>of</strong> MICB*004. From crystal structure <strong>of</strong><br />
NKG2D-MIC binding, position 52 in the alpha 1 domain is<br />
located near the NKG2D binding site. Thus, the alteration <strong>of</strong><br />
charge between aspartic acid and asparagine (in MICB*004)<br />
may affect NK cell activation. These results suggested that<br />
the MICA/B polymorphisms affecting NK cell activation<br />
could be relevant to diseases and transplantations.<br />
doi:10.1016/j.clim.2007.03.161<br />
Su.123 The Role <strong>of</strong> Regulatory T Cells in the<br />
Pathogenesis <strong>of</strong> Experimental Biliary Atresia<br />
Alexander Miethke, <strong>Clinical</strong> Fellow, Cincinnati Children’s<br />
Hospital Medical Center, Pediatric Gastroenterology,<br />
Cincinnati, OH, Pranavkumar Shivakumar, Research<br />
Associate, Cincinnati Children’s Hospital Medical Center,<br />
Division <strong>of</strong> Pediatric Gastroenterology, Cincinnati, OH,<br />
Jorge Bezerra, Pr<strong>of</strong>essor <strong>of</strong> Pediatrics, Cincinnati Children’s<br />
Hospital Medical Center, Division <strong>of</strong> Pediatric<br />
Gastroenterology, Cincinnati, OH<br />
In rotavirus induced experimental biliary atresia (BA) the<br />
extraheaptic bile ducts <strong>of</strong> newborn mice undergo a Th1regulated<br />
fibroinflammatory obstruction similar to BA in<br />
children. Here we investigate the role <strong>of</strong> regulatory T (Treg)<br />
cells in the pathogenesis <strong>of</strong> experimental BA. Using 3 color<br />
flow cytometry identifying CD4+CD25+Foxp3+, CD4+CD25+<br />
CD103+ and CD4+CD25+CTLA-4+ populations, we found that<br />
Treg cells had low abundance in livers <strong>of</strong> 3-day-old mice as<br />
compared to adult mice. Infection <strong>of</strong> neonatal mice with<br />
rotavirus did not induce an emergence <strong>of</strong> Treg cells in the liver<br />
within 3 days post infection. However, 7 days after infection,<br />
at a time when neonatal mice develop jaundice, acholic<br />
stools and bilirubinuria, CD4+CD25+ lymphocytes co-staining<br />
for Foxp3+, CD103+ and CTLA-4+ increased by 3-, 10- and 43fold,<br />
respectively in the liver when compared to age-matched<br />
uninfected controls. Real-time PCR analysis <strong>of</strong> purified<br />
lymphocyte subsets revealed a 970 fold increase <strong>of</strong> IL-10<br />
mRNA expression in CD4+CD25+ Treg cells from livers <strong>of</strong> RRV<br />
infected mice compared to saline controls; however, similar<br />
increase in TGF β1 expression was not found. In conclusion,<br />
postnatal viral challenge did not induce a rise in Treg cells<br />
within 3 days <strong>of</strong> life. While there was an increased expression<br />
<strong>of</strong> functional markers (CTLA-4 and IL-10), activated Treg cells<br />
failed to surge TGF β1 mRNA expression at the time <strong>of</strong> duct<br />
obstruction. We speculate that a deficit in number and<br />
selective function <strong>of</strong> neonatal Treg cells may be responsible<br />
for the susceptibility <strong>of</strong> neonates to biliary atresia.<br />
doi:10.1016/j.clim.2007.03.162<br />
Su.124 Association <strong>of</strong> HLA DRB1*04 and HLA<br />
DQB1*06 with Severe Early Childhood Caries<br />
Hossein Neamatollahi, Dentist, Pediatric Department,<br />
School <strong>of</strong> Dentistry, Mashhad, University <strong>of</strong> Medical<br />
Sciences, Mashhad, Iran, Jalil Tavakkol-Afshari, Vice<br />
President for Research, Head, Department <strong>of</strong>
Abstracts<br />
Immunogenetics and Cell Culture, Bu-Ali Research Institute<br />
(BARI), Department <strong>of</strong> Immunogenetics and Cell Culture,<br />
Mashhad, Iran, Alireza Sarraf, Dentist, Pediatric<br />
Department, School <strong>of</strong> Dentistry, Mashhad University <strong>of</strong><br />
Medical Sciences, Mashhad, Iran, Ali Bagherian, Dentist,<br />
School <strong>of</strong> Dentistry, Mashhad University <strong>of</strong> Medical<br />
Sciences, Mashhad, Iran, Habibollah Esmaili,<br />
Epidemiologist, Epidemiology and Statistics Department,<br />
Ghaem Medical Center, Mashhad University <strong>of</strong> Medical<br />
Sciences, Mashhad, Iran, Nasrin Moheghi, Researcher, Bu-<br />
Ali Research Institute (BARI), Department <strong>of</strong><br />
Immunogenetics, Mashhad, Iran<br />
Severe early childhood caries (SECC) is one <strong>of</strong> the most<br />
common diseases in childhood. Etiology <strong>of</strong> SECC is multifactorial<br />
and both genetic and environmental factors play<br />
important roles in the pathogenesis <strong>of</strong> disease. Genetic<br />
variation <strong>of</strong> the host may contribute to the different risks <strong>of</strong><br />
dental caries. Genetic factors such as the Human Leukocyte<br />
Antigen (HLA) have been recently introduced as predisposing<br />
factors. The aim <strong>of</strong> this study was looking for an association<br />
between HLA-DRB1*04 and HLADQB1*06 with SECC for early<br />
diagnosis as well as prevention <strong>of</strong> the disease. In this study<br />
we extracted the genomic DNAs from the whole blood<br />
samples <strong>of</strong> 44 patients with SECC and 35 caries-free children<br />
(control group) by salting out method. We amplified the<br />
genomic DNA by PCR Sequence Specific Primer (PCR-SSP) and<br />
HLA-typing was performed for both alleles. The results<br />
revealed a significant increase in the frequency <strong>of</strong><br />
HLADRB1*04 in the patient group (P-Value =0.019). The<br />
odds ratio for this allele was detected to be 10. Frequency<br />
<strong>of</strong> HLA-DQB1*06 allele was not significantly different<br />
between two groups (P-Value=0.37).<br />
The above results suggest that HLA-DRB1*04 is associated<br />
with the susceptibility to SECC. Therefore, the detection <strong>of</strong><br />
HLA-DRB1*04 allele as a molecular marker for early diagnosis<br />
<strong>of</strong> SECC is recommended.<br />
doi:10.1016/j.clim.2007.03.163<br />
Su.125 The Genetic Relationship Among Eleven<br />
Persian Ethnic Groups: An Anthropological View<br />
Based on HLA Class II Gene Polymorphism<br />
Shirin Farjadian, Assistant Pr<strong>of</strong>essor, Shiraz University<br />
Medical Sciences, <strong>Immunology</strong> Department, Shiraz, Iran,<br />
Abbas Ghaderi, Full Pr<strong>of</strong>essor, <strong>Immunology</strong> Department,<br />
Shiraz, Iran<br />
Besides being considered by immunologists for its pivotal<br />
role in the immune response, highly polymorphic HLA genes<br />
are still regarded as useful markers by anthropologists for<br />
determination <strong>of</strong> genetic relationship and interaction among<br />
different populations. Knowledge <strong>of</strong> the HLA allele distributions<br />
in various populations is also critical for establishing<br />
bone marrow donor registries; study <strong>of</strong> HLA associated<br />
disease and designing <strong>of</strong> peptide vaccines against infections,<br />
tumors, or autoimmune diseases. In this study, HLA class II<br />
pr<strong>of</strong>iles were determined by molecular methods in 816 DNA<br />
samples from eleven ethnic groups <strong>of</strong> Persia and the genetic<br />
relationship <strong>of</strong> Persians to Asians and Europeans were<br />
investigated. DRB1*1103=04, DQA1*0501, and DQB1*0301<br />
were the most frequent alleles and DRB1*1103=04-<br />
DQA1*0501-DQB1*0301 was the most common haplotype in<br />
Persia. Six samples typed as DRB1*1605 by direct sequencing<br />
<strong>of</strong> exon 2 and all <strong>of</strong> them were heterozygote for DRB1 locus<br />
and belonged to DRB1*1605-DQA1*0101/2-DQB1*0502 haplotype.<br />
The results <strong>of</strong> neighbor-joining and correspondence<br />
analysis revealed a close genetic relationship among Persian<br />
subpopulations that were well separated from other Asians<br />
and European populations. The results <strong>of</strong> AMOVA also<br />
revealed that the main variation component (96.9%) was<br />
contributed to by the within-population level and genetic<br />
differentiation (FST) was 0.03 when all Persian subpopulations<br />
considered as one group.<br />
doi:10.1016/j.clim.2007.03.164<br />
Su.126 IFN-g Increases Neurogenesis in Aging and<br />
Alzheimer's-like Disease<br />
Shai Abutbul, PhD Student, The National Institute <strong>of</strong><br />
Biotechnology in the Negev, Department <strong>of</strong> Microbiology<br />
and <strong>Immunology</strong>, Beer-Sheva, Israel, Rona Baron, MSc, The<br />
National Institute <strong>of</strong> Biotechnology in the Negev,<br />
Department <strong>of</strong> Microbiology and <strong>Immunology</strong>, Beer Sheva,<br />
Israel, Anna Nemirovsky, MSc, The National Institute <strong>of</strong><br />
Biotechnology in the Negev, Department <strong>of</strong> Microbiology<br />
and <strong>Immunology</strong>, Beer sheva, Alon Monsonego, PhD, The<br />
National Institute <strong>of</strong> Biotechnology in the Negev,<br />
Department <strong>of</strong> Microbiology and <strong>Immunology</strong>, Beer Sheva,<br />
Israel, Yair Fisher, BSc, The National Institute <strong>of</strong><br />
Biotechnology in the Negev, Department <strong>of</strong> Microbiology<br />
and <strong>Immunology</strong>, Beer Sheva, Israel<br />
The birth <strong>of</strong> new neurons is maintained in the adult central<br />
nervous system (CNS) within restricted areas in the brain,<br />
primarily the dentate gyrus (DG) and the subventricular zone<br />
(SVZ). Neurogenesis significantly declines with aging, possibly<br />
to a greater extent with the progression <strong>of</strong> Alzheimer's disease<br />
(AD). The primary cause <strong>of</strong> this decline is unclear, but brainendogenous<br />
and peripheral immune mechanisms associated<br />
with aging and the progression <strong>of</strong> AD appear to affect stem cell<br />
proliferation and differentiation in the brain. In contrast to<br />
the suppression <strong>of</strong> neurogenesis by proinflammatory cytokines<br />
(such as IL-1b, TNF-a, and IL-6) associated with chronic glial<br />
activation and neurotoxicity in the aging or AD brain, our<br />
studies in IFNg-transgenic mice with AD-like pathology show<br />
that the T cell-derived cytokine IFN-g, expressed under the<br />
myelin basic protein promoter, induces an approximately<br />
tw<strong>of</strong>old increase in the quantity <strong>of</strong> neuronal progenitor cells in<br />
the DG <strong>of</strong> aged, but not young, mice. Limited expression <strong>of</strong><br />
IFN-g in the periphery also resulted in a significant decrease in<br />
the portion <strong>of</strong> CD4+CD25+ and CD4+Foxp3+ regulatory T cells<br />
among the CD4+ T cell population in the spleens <strong>of</strong> the<br />
transgenic mice (vs. control). IFN-g has <strong>of</strong>ten been implicated<br />
in brain inflammation and neuronal damage, although recent<br />
studies suggest that it plays a neuroprotective role. Our<br />
findings also demonstrate a novel role for IFN-g in neuronal<br />
recovery by acting directly on brain endogenous cells and/or<br />
indirectly by increasing peripheral immunity.<br />
doi:10.1016/j.clim.2007.03.165<br />
S183
S184 Abstracts<br />
Su.127 Astrocyte-Mediated Regulation <strong>of</strong> Human<br />
CNS Inflammation<br />
Alex Kostianovsky, MD, Center for Neurologic Diseases,<br />
Harvard Medical School, Boston, MA, David E. Anderson,<br />
PhD, Center for Neurologic Diseases, Harvard Medical<br />
School, Boston, MA<br />
Microglial activation, or a lack there<strong>of</strong>, is associated with<br />
numerous central nervous system (CNS) neurodegenerative<br />
and inflammatory diseases, including Alzheimer's disease,<br />
multiple sclerosis, and CNS tumors. We are interested in<br />
better understanding the extent to which astrocytes modulate<br />
microglial and T cell activation within the CNS. Using<br />
primary human astrocytes co-cultured with ex vivo human<br />
monocytes or primary human microglia, we report that<br />
astrocytes potently inhibit monocyte/microglial activation<br />
induced with pathogen-derived (TLR agonists) but not T cellderived<br />
(CD40L) inflammatory signals. More specifically,<br />
human astrocytes inhibit TNF-± secretion and induce IL-10<br />
secretion. Comparisons between primary human astrocytes<br />
and primary human malignant glioma cell lines (transformed<br />
astrocytes) have demonstrated that gliomas inhibit TNF-±<br />
and induce IL-10 to greater extents than do primary astrocytes;<br />
moreover, they can inhibit monocyte activation<br />
induced with both TLR and CD40L signals. Trans-well assays<br />
demonstrate that inhibition <strong>of</strong> TNF-± secretion is mediated<br />
by both soluble and contact-dependent mechanisms, whereas<br />
induction <strong>of</strong> IL-10 is contact-dependent. Candidate molecules<br />
such as TGF- 2 , IL-10, COX2, and CD200 do not appear to<br />
be responsible for the astrocyte-associated suppression <strong>of</strong><br />
TNF-±. Preliminary data suggest indicate that up-regulation<br />
<strong>of</strong> members <strong>of</strong> the STAT gene family in transformed<br />
astrocytes is associated with the immunosuppressive phenotype.<br />
Collectively, these results indicate that astrocytes<br />
serve an immunoregulatory role within the CNS, which may<br />
be amplified or suppressed in different CNS diseases. In the<br />
context <strong>of</strong> malignant gliomas, amplification <strong>of</strong> the normal<br />
immunoregulatory function <strong>of</strong> astrocytes may explain microglial<br />
defects and impaired T cell immunity associated with<br />
these tumors.<br />
doi:10.1016/j.clim.2007.03.166<br />
Su.128 Peripheral Markers <strong>of</strong> Encephalitis on<br />
Monocytes from SIV-infected Rhesus Monkeys<br />
Maria Cecilia Marcondes, Staff Scientist, The Scripps<br />
Research Institute, Molecular and Integrative Neurosciences<br />
Department, La Jolla, CA, Tricia Burdo, Research Associate,<br />
The Scripps Research Institute, Molecular and Integrative<br />
Neurosciences Department, La Jolla, CA, Caroline Lanigan,<br />
Scientific Associate, The Scripps Research Institute,<br />
Molecular and Integrative Neurosciences Department, La<br />
Jolla, CA, Howard Fox, Associate Pr<strong>of</strong>essor, The Scripps<br />
Research Institute, Molecular and Integrative Neurosciences<br />
Department, La Jolla, CA, Debbie Watry, Research<br />
Assistant, The Scripps Research Institute, Molecular and<br />
Integrative Neurosciences Department, La Jolla, CA<br />
In HIV-1 infected people, the accumulation <strong>of</strong> macrophages<br />
(MØs) in the brain correlates with dementia and<br />
encephalitis. We hypothesized that a pattern <strong>of</strong> monocyte<br />
surface marker expression may serve as a peripheral marker<br />
for CNS disease. Using the SIV/rhesus monkey model, we first<br />
analyzed functionally relevant surface markers on monocytes<br />
and macrophages from blood and organs upon<br />
termination in groups <strong>of</strong> animals that later did (SIVE) or did<br />
not (SIVnoE) develop encephalitis. Cluster analysis revealed<br />
that the pattern <strong>of</strong> staining <strong>of</strong> cells from the brain was<br />
closest to that found in blood, compared to spleen, liver,<br />
bone marrow and bronchioalveolar lavage. In the blood, the<br />
predominant (CD16low) population <strong>of</strong> monocytes from SIVE<br />
animals had higher levels <strong>of</strong> both CD44v6 and CCR5 compared<br />
to SIVnoE animals. Furthermore the inflammatory CD16+<br />
subpopulation <strong>of</strong> blood monocytes from SIVE animals<br />
expressed high levels <strong>of</strong> CD44v6, but not CCR5, compared<br />
to those from SIVnoE animals. In the brain, only CCR5<br />
expression on CD16+ macrophages correlated with encephalitis.<br />
A longitudinal analysis <strong>of</strong> blood monocyte markers<br />
revealed that, beginning at 3 weeks prior to termination,<br />
both CD16low and CD16+ monocytes indeed upregulated<br />
CD44v6. This provides a potential biomarker to determine<br />
individuals who may develop the central nervous system<br />
complications <strong>of</strong> HIV infection. Furthermore, given its role in<br />
cellular adhesion and as a receptor for osteopontin, CD44v6<br />
upregulation on monocytes <strong>of</strong>fers functional clues to the<br />
pathogenesis <strong>of</strong> such complications, and provides a target for<br />
preventative and therapeutic measures. (NIH Grant 1R01<br />
NS045534-04).<br />
doi:10.1016/j.clim.2007.03.169<br />
Su.129 Polymerized-Type I Collagen a Biodrug for<br />
the Treatment <strong>of</strong> Patients with Knee Osteoarthritis<br />
Janette Furuzawa-Carballeda, Chemistry, Instituto Nacional<br />
de Ciencias Medicas y Nutricion SZ, Department <strong>of</strong><br />
<strong>Immunology</strong> and Rheumatology, Mexico, Olga Muñoz-<br />
Chable, Physician, Instituto Nacional de Ciencias Medicas y<br />
Nutricion SZ, Department <strong>of</strong> <strong>Immunology</strong> and<br />
Rheumatology, Mexico, Israel Macias-Hernandez, Physician,<br />
Instituto Nacional de Ciencias Medicas y Nutricion SZ,<br />
Department <strong>of</strong> <strong>Immunology</strong> and Rheumatology, Mexico,<br />
Andres Agualimpia-Janning, Physician, Instituto Nacional de<br />
Ciencias Medicas y Nutricion SZ, Department <strong>of</strong> <strong>Immunology</strong><br />
and Rheumatology, Mexico<br />
Polymerized-Type I Collagen (Collagen-PVP) is an antiinflammatory<br />
and a tissue regenerator biodrug. In vivo<br />
study: Patients (n=53) were treated with 12 intraarticular<br />
injections <strong>of</strong> 2 ml <strong>of</strong> collagen-PVP (16.6 mg <strong>of</strong> collagen)<br />
(n=27) or 2 ml <strong>of</strong> placebo (n=26) during 6 months. The<br />
follow up was done during the next 6 months. The primary<br />
endpoints included WOMAC, Lequesne index and pain<br />
intensity on a VAS. Secondary outcomes were patient and<br />
investigator global score and biodrug evaluation. <strong>Clinical</strong><br />
improvement was determined if the decrease in pain<br />
exceeds 20 mm on VAS and patients achieved at least<br />
20% <strong>of</strong> improvement in primary endpoints from baseline to<br />
week 24. In vitro study: Cartilage and synovial tissue cocultures<br />
from 5 patients with OA were performed. Tissues<br />
were cultured during 7 days with/without 1% Collagen-PVP<br />
and they were stained with Alcian Blue technique. IL-1β,<br />
IL-8, IL-10, IL-12, TNF-α and IFN-γ were measured in
Abstracts<br />
supernatants by ELISA. COMP, type II collagen, TNF-α, IL-10<br />
and Ki-67 expression was determined by immunohistochemistry.<br />
Patients had a statistically significant improvement<br />
(pb0.05) in Collagen-PVP-treated versus baseline and versus<br />
placebo in Lequesne Index, WOMAC, VAS, patient global score<br />
and physician drug evaluation. The histological analysis<br />
showed that Collagen-PVP increased 3 to 6-fold proteoglycans,<br />
Ki-67, COMP and type II collagen. IL-1β and TNF-α<br />
production was inhibited; meanwhile IL-10 levels were upregulated<br />
treated versus untreated-cultures. Collagen-PVP<br />
was safe and more effective than placebo for the treatment<br />
<strong>of</strong> knee OA, probably due to the induction <strong>of</strong> tissue<br />
regeneration and the down-regulation <strong>of</strong> inflammation.<br />
doi:10.1016/j.clim.2007.03.170<br />
Su.132 Antibody-oligo Arrays for Multiplex Surface<br />
Marker Pr<strong>of</strong>iling <strong>of</strong> Immune Cells Subsets<br />
Michael Kattah, MSTP Student, Stanford University<br />
<strong>Immunology</strong> and Rheumatology, Stanford, CA, John Coller,<br />
Research and Development Engineer, School <strong>of</strong> Medicine,<br />
MDRP’S, Stanford Functional Genomics Facility, Stanford,<br />
CA, Golnaz Alemi, Medical Student, Drexel University,<br />
Stanford, CA, Neekaan Oshidary, Undergraduate Student,<br />
Stanford University <strong>Immunology</strong> and Rheumatology,<br />
Stanford, CA, Paul J. Utz, Associate Pr<strong>of</strong>essor <strong>of</strong> Medicine<br />
(<strong>Immunology</strong> and Rheumatology), Medicine, <strong>Immunology</strong><br />
and Rheumatology and Center for <strong>Clinical</strong> <strong>Immunology</strong> at<br />
Stanford, Stanford, CA<br />
While the field <strong>of</strong> proteomics holds great promise for the<br />
study <strong>of</strong> immune-related disorders, methods for analyzing<br />
specific proteins in a high-throughput manner are still needed.<br />
In an effort to develop a multi-analyte proteomics platform<br />
using monoclonal antibodies, we are developing a new assay<br />
termed antibody-oligo arrays (AOAs). This method employs a<br />
panel <strong>of</strong> monoclonal antibodies, each tagged with a unique<br />
oligonucleotide sequence identifier. After staining a sample,<br />
the complex mixture <strong>of</strong> tags is amplified and hybridized to a<br />
custom DNA microarray, providing an indirect measure <strong>of</strong> the<br />
relative levels <strong>of</strong> each analyte in the original sample. In an<br />
effort to identify cell-surface markers that are either up- or<br />
down-regulated in a given cell population, we have developed<br />
a cell-surface AOA to pr<strong>of</strong>ile 1–2×106 cells with a panel <strong>of</strong> 90<br />
surface markers and 4 isotype controls in two 48-plex<br />
reactions. Although there are many potential applications for<br />
this technology, we have performed pilot experiments using<br />
both cancer cell lines and primary human lymphocytes.<br />
Preliminary results have identified changes on the surface <strong>of</strong><br />
primary human naïve CD4+CD45RA+CD45RO-CD25- Tcells upon<br />
polyclonal activation in vitro with anti-CD3 and anti-CD28<br />
coated beads. The markers identified in this screen agree with<br />
previously described activation markers and were all validated<br />
by conventional flow cytometry. In the future we hope to<br />
pr<strong>of</strong>ile cell-surface markers on Tregulatory and IL-17 secreting<br />
Th17 cells with our newly developed cell-surface AOA in order<br />
to identify targets for future functional studies and for<br />
potential therapeutic interventions.<br />
doi:10.1016/j.clim.2007.03.171<br />
Su.133 Industrial Science: Biomarker Discovery<br />
from Plasma: Maximizing Proteomic Breath and<br />
Depth Using Protein Partitioning and Fractionation<br />
Followed by Shotgun Mass Spectrometry<br />
Shijun Sheng, Senior Research Technologist, Johns Hopkins<br />
Bayview NHLBI Proteomics Center, Baltimore, MD, Qin Fu,<br />
Research Associate, Johns Hopkins Bayview NHLBI<br />
Proteomics Center, Baltimore, MD, Jeff Chapman, Director,<br />
Beckman Coulter, Biomarker Discovery and Automation<br />
Business Center, Fullerton, CA, Jennifer Van Eyk, Associate<br />
Pr<strong>of</strong>essor Medicine, Johns Hopkins University-Bayview<br />
Campus, Baltimore, MD, Jerald Feitelson, Manager<br />
Strategic Marketing, Beckman Coulter, Biomarker Discovery<br />
and Automation Business Center, Fullerton, CA<br />
The potential <strong>of</strong> proteomics to identify new diagnostic<br />
markers was widely touted but has met with limited success<br />
due to technical issues related to reproducibility, sample<br />
requirements, availability <strong>of</strong> well characterized clinical<br />
cohorts for discovery and validation, and ability to obtain<br />
sufficient proteome depth and coverage. To discover novel<br />
biomarkers while overcoming previous limitations in proteomic<br />
methodology, we have developed a robust biomarker<br />
discovery pipeline that allows the detection and quantification<br />
<strong>of</strong> proteins below 100 pg/ml. The proteomic pipeline<br />
involves two key sample preparation steps at a large scale:<br />
(a) partitioning (removal) <strong>of</strong> the top 12 most abundant<br />
plasma proteins using immunoaffinity chromatography, and<br />
(b) subsequent two-dimensional liquid chromatography that<br />
fractionates intact proteins based on two <strong>of</strong> their key<br />
intrinsic properties, pI and hydrophobicity. Fractions are<br />
split allowing the third intrinsic protein parameter, intact<br />
mass, to be obtained using MALDI-TOF mass spectrometry.<br />
When combined with analysis <strong>of</strong> proteome maps based on<br />
aligned second dimension chromatograms, we can identify<br />
fractions that differ between individual samples in large data<br />
sets. To push the proteomics envelope, we carried out<br />
multiple analyses on each individual sample prior to pooling<br />
and quantitative shotgun LC-MS/MS to identify and characterize<br />
proteins. This biomarker discovery pipeline is being<br />
applied to unprecedented broad and deep analyses <strong>of</strong> 100<br />
individuals attempting to obtain quantitative protein characterization<br />
(is<strong>of</strong>orm and PTM) data. We have shown that this<br />
scale <strong>of</strong> discovery proteomics (1 mL plasma per patient, 100<br />
patients and controls) was able to identify candidate<br />
biomarkers <strong>of</strong> a crucially important disease.<br />
doi:10.1016/j.clim.2007.03.173<br />
S185<br />
Su.134 Human C-Reactive Protein Augments the Gut<br />
Ischemia/Reperfusion Injury in Mice<br />
Xinyue Lu, Research Fellow, Department Cellular Injury,<br />
Walter Reed Army Institute <strong>of</strong> Research, Silver Spring, MD,<br />
Russell Peckham, Principle Investigator, Corresponding<br />
Author, Department Cellular Injury, Walter Reed Army<br />
Institute <strong>of</strong> Research, Silver Spring, MD, Michael Falabella,<br />
Research Assistant, Department Cellular Injury, Walter Reed<br />
Army Institute <strong>of</strong> Research, Silver Spring, MD, George C.<br />
Tsokos, Principle Investigator, Pr<strong>of</strong>essor, Department<br />
Cellular Injury, Walter Reed Army Institute <strong>of</strong> Research,<br />
Silver Spring, MD
S186 Abstracts<br />
C-reactive protein (CRP) has been shown to increase<br />
ischemia/reperfusion (IR) injury in models <strong>of</strong> acute myocardial<br />
infarction and stroke. The role <strong>of</strong> CRP on intestinal IR which<br />
results in serious local and systemic inflammatory reactions is<br />
unknown. We adapted a murine superior mesenteric artery IR<br />
model which we used to study the effect <strong>of</strong> CRP. Human<br />
purified CRP (250, 500, and 1000 μg per mouse) was<br />
administered prior to the ischemia period <strong>of</strong> 30 min and tissue<br />
injury was evaluated after 2 hours <strong>of</strong> reperfusion. Intestinal<br />
injury damage, estimated using an established score, was<br />
statistically significantly higher in all CRP-treated groups<br />
compared to non-treated animals. The increased injury score<br />
was associated with increased intestinal myeloperoxidase<br />
activity only in the CRP-treated animals. In addition, the levels<br />
<strong>of</strong> the proinflammatory cytokines IL-6, IL-1α,TNF-α mRNA, as<br />
determined by real-time RT-PCR, were increased in the<br />
intestines <strong>of</strong> CRP-treated animals compared to the nontreated<br />
animals. Immun<strong>of</strong>luorescent staining revealed that<br />
human CRP was deposited in the intestinal tissues and that it<br />
co-localized with complement C3, factor H, MBL-A and C5b-9.<br />
Our results indicate CRP enhances intestinal IR injury probably<br />
by recruiting and activating the complement pathway.<br />
doi:10.1016/j.clim.2007.03.174<br />
Su.136 Expression, Histological Localization <strong>of</strong> hfgl2<br />
and Its Gene Regulation in Cancer<br />
Kai Su, Postgraduate Student, Department <strong>of</strong> Infectious<br />
Disease, Tongji Hospital, Huazhong University <strong>of</strong> Science<br />
and Technology, Wuhan, China, Fang Chen, Resident,<br />
Department <strong>of</strong> Infectious Disease, Tongji Hospital,<br />
Huazhong University <strong>of</strong> Science and Technology, Wuhan,<br />
China, Ping Zhang, Graduate Student, Department <strong>of</strong><br />
Infectious Disease, Tongji Hospital, Huazhong University <strong>of</strong><br />
Science and Technology, Wuhan, China, Xiaomin Qin, Doctor<br />
in Charge, Department <strong>of</strong> Infectious Disease, Tongji<br />
Hospital, Huazhong University <strong>of</strong> Science and Technology,<br />
Wuhan, China, Meifang Han, Doctor in Charge, Department<br />
<strong>of</strong> Infectious Disease, Tongji Hospital, Huazhong University<br />
<strong>of</strong> Science and Technology, Wuhan, China, Weiming Yan,<br />
Assistant Researcher, Department <strong>of</strong> Infectious Disease,<br />
Tongji Hospital, Huazhong University <strong>of</strong> Science and<br />
Technology, Wuhan, China, Bin Pi, Assistant Researcher,<br />
Department <strong>of</strong> Infectious Disease, Tongji Hospital,<br />
Huazhong University <strong>of</strong> Science and Technology, Wuhan,<br />
China, Dong Xi, Assistant Research, Department <strong>of</strong><br />
Infectious Disease, Tongji Hospital, Huazhong University <strong>of</strong><br />
Science and Technology, Wuhan, China, Xiaoping Luo, Chief<br />
Physician, Department <strong>of</strong> Pediatrics <strong>of</strong> Tongji Hospital,<br />
Huazhong University <strong>of</strong> Science and Technology, Wuhan,<br />
China, Qin Ning, Chief Physician, Department <strong>of</strong> Infectious<br />
Disease, Tongji Hospital, Huazhong University <strong>of</strong> Science<br />
and Technology, Wuhan, China<br />
To study the role <strong>of</strong> fibrinogen-like protein 2/fibroleukin<br />
(fgl2) in tumor development, human malignant tumor tissues<br />
from 133 patients with various kinds <strong>of</strong> cancers and the<br />
animal tumor tissues from human hepatocellular carcinoma<br />
(HCC) model on nude mice established by using different HCC<br />
cell lines were used to detect the fgl2 expression through an<br />
immunohistological method. Antibodies against different<br />
cellular markers (CD8, CD57, CD68 and vWF) were used to<br />
determine the cellular types in which the fgl2 was expressed.<br />
Fgl2 was detected in 127 out <strong>of</strong> 133 patients' samples and<br />
human HCC nude mice model. Fibrin (nogen) co-localization<br />
with fgl2 expression was determined by double staining.<br />
Particularly, it was evidenced that fgl2 was highly expressed<br />
not just in cancer cells, but also in interstitial infiltrated cells<br />
including macrophages, NK cells, CD8+T lymphocytes and<br />
vascular endothelial cells. In situ hybridization shows fgl2<br />
mRNA stained in associated location. Furthermore, IL-2, IFN-γ<br />
and TNF-a can dramatically up-regulate fgl2 mRNA (increased<br />
within the range <strong>of</strong> 10–100 fold) and protein expression in<br />
both THP-1 and HUVEC cell lines. One-stage clotting assay<br />
demonstrated that the significantly increased procoagulant<br />
activity after cytokines stimulation <strong>of</strong> both cell lines.<br />
The present study shows that fg12 contribute to the<br />
hypercoagulability in cancer, and thus in turn induces tumor<br />
angiogenesis and metastasis. This work was supported by<br />
Natural Science Foundation <strong>of</strong> China (NSFC 30225040,<br />
30571643, 30672380), National Key Basic Research Program<br />
<strong>of</strong> China (2005CB522901, 2005CB522507) and <strong>Clinical</strong> Subject<br />
Key Project from the Ministry <strong>of</strong> Health.<br />
doi:10.1016/j.clim.2007.03.175<br />
Su.137 CpG ODN Reduced Amyloid-β<br />
Accumulation Through Up-Regulated F-Spondin on<br />
Mouse BV2 Microglial Cell<br />
Yung Chih Cheng, PhD Student, Institute <strong>of</strong> Biochemical<br />
Science, National Taiwan University, Taipei, Taiwan, Cheng-<br />
Chin Kuo, Postdoctoral Fellowtor, Agricultural<br />
Biotechnology Research Center, Academia Sinica, Taipei,<br />
Taiwan, Shu-Mei Liang, Pr<strong>of</strong>essor, Agricultural<br />
Biotechnology Research Center, Academia Sinica, Taipei,<br />
Taiwan<br />
Alzheimer's disease, the most common senile dementia, is<br />
characterized by amyloid plaques, vascular amyloid, neur<strong>of</strong>ibrillary<br />
tangles, and progressive neurodegeneration. Amyloid<br />
plaques are mainly composed <strong>of</strong> amyloid-beta (A-β)<br />
peptides, which are derived from processing <strong>of</strong> the betaamyloid<br />
precursor protein (APP) by secretases. In this study,<br />
we found that BV2 microglial cells stimulated with unmethylated<br />
CpG-ODN not only increased the level <strong>of</strong> F-spondin but<br />
also resistant to A-β formation. Similar results were observed<br />
in CpG ODN stimulated RAW264.7 cells. Results from both real<br />
time PCR and western blotting demonstrated that CpG ODN<br />
regulated F-spondin expression at both transcriptional and<br />
translational level. Inhibition <strong>of</strong> TLR9 and MyD88 by inhibitors<br />
or dominant negative mutants indicated that CpG ODN upregulated<br />
F-spondin was TLR9/MyD88 dependent. In addition,<br />
we also found that a downstream kinase <strong>of</strong> TLR9, PI3K,<br />
was essential for CpG ODN-induced F-spondin expression.<br />
Depletion <strong>of</strong> F-spondin by small interfering RNA attenuated<br />
the prevention <strong>of</strong> CpG ODN on APP degradation by secretases,<br />
thereby increasing the level <strong>of</strong> A-β and soluble APP in culture<br />
medium. Collectively, these findings suggest that CpG ODN<br />
increases F-spondin via a TLR9/MyD88/PI3K pathway. The<br />
enhancement <strong>of</strong> F-spondin plays a critical role in the<br />
reduction <strong>of</strong> A-β formation mediated by CpG ODN, thereby<br />
reducing Alzheimer disease occurrence. These results may
Abstracts<br />
provide an insight for developing a potential drug for the<br />
treatment <strong>of</strong> Alzheimer's disease.<br />
doi:10.1016/j.clim.2007.03.176<br />
Su.138 Loss <strong>of</strong> Th1 and Effector T Cell Function in<br />
Chronic Versus Subacute Hypersensitivity<br />
Pneumonitis<br />
Lourdes Barrera, Biological Sciences Doctor, National<br />
Institute <strong>of</strong> Respiratory Diseases, Mexico City, Mexico,<br />
Felipe Mendoza, MsC, National Institute <strong>of</strong> Respiratory<br />
Diseases, Mexico City, Mexico, Moises Selman, National<br />
Insitute <strong>of</strong> Respiratory Diseases, Mexico City, Mexico<br />
Hypersensitivity pneumonitis (HP) represents an immunologically-mediated<br />
inflammation caused by a variety <strong>of</strong><br />
antigens. 21 patients with Sub-HP, 20 with Chr-HP and 8<br />
healthy subjects were studied. T cells from bronchoalveolar<br />
lavage (BAL) were analyzed by flow cytometry. Cytokine<br />
expression and memory functional pr<strong>of</strong>ile were evaluated in<br />
cell cultures. Chr-HP patients showed lower BAL lymphocytosis,<br />
higher CD4+/CD8+ ratio (3.5–3.3 versus 1.2–0.4 and<br />
1.7–1.5 from controls and Sub-HP; pb0.05), and a decrease<br />
<strong>of</strong> f× fÔT cells (2.5+0.9 versus 13.3+6.3 and 10.4+5.6 from<br />
controls and Sub-HP; pb0.01). Chr-HP had an increase in the<br />
terminally differentiated memory CD4+ and CD8+ T cells<br />
subsets compared with the subacute group (p b0.05).<br />
Memory cells from Chr-HP showed lower IFN-f× production,<br />
and decreased cytotoxicity <strong>of</strong> CD8+ T-lymphocytes. Chr-HP<br />
displayed a Th2-like phenotype with decreased CXCR3/CCR4<br />
ratio on T cells (2.5–1.8 versus 14.8–8.8; pb0.01) and lower<br />
levels <strong>of</strong> the CXCR3 ligands, IP-10/CXCL10 and MIG/CXCL9 in<br />
BAL supernatants. BAL lymphocytes from Chr-HP, stimulated<br />
with the specific antigen expressed lower levels <strong>of</strong> Th1<br />
cytokines and higher levels <strong>of</strong> Th2 cytokines (IFN-f× /IL-4<br />
ratio: 0.7–0.3% versus 2.1–0.6% from Sub-HP; pb0.01).<br />
Differences between chronic and subacute patients include<br />
an increase in CD4+/CD8+ ratio, a decrease <strong>of</strong> f× fÔT cells, a<br />
skewing towards Th2 as opposed to Th1 and exhaustion <strong>of</strong><br />
effector CD8+ T cells. These findings may provide insight in<br />
the development <strong>of</strong> new therapeutic strategies e.g. the<br />
inhibition <strong>of</strong> GATA-3 transcription factor to modify the Th1/<br />
Th2 cell balance and the blocking <strong>of</strong> PD-1 receptor to restore<br />
the Tcell function may be useful in the treatment for chronic HP.<br />
doi:10.1016/j.clim.2007.03.558<br />
Su.139 Analysis <strong>of</strong> Natural Killer Cells Isolated from<br />
Human Decidua: Evidence that 2B4 (CD244)<br />
Functions as an Inhibitory Receptor and Blocks NK<br />
Cell Function<br />
Gabriella Pietra, PhD, DIMES-University <strong>of</strong> Genoa,<br />
Genova, Italy, Paola Vacca, PhD Student, DIMES-University<br />
<strong>of</strong> Genoa, Genova, Italy, Lorenzo Moretta, MD, G Gaslini<br />
Institute, Genova, Italy, Federico Prefumo, MD, G<br />
Gaslini Institute, Genova, Italy, Maria Cristina Mingari,<br />
PhD, National Cancer Institute, Genova, Italy<br />
During the first trimester <strong>of</strong> pregnancy, CD56bright NK<br />
cells constitute more than 50% <strong>of</strong> the lymphocytes present<br />
in the decidua, a tissue in which B and T lymphocytes are<br />
rare. The role they have at this site is still unclear. It is<br />
currently believed that in the early stages <strong>of</strong> gestation,<br />
decidual NK (dNK) cells possess the ability to regulate<br />
trophoblastic differentiation and invasion. In this study we<br />
analyzed dNK cells for their phenotype and functional<br />
capability. We show that dNK cells express normal surface<br />
levels <strong>of</strong> certain activating receptors, including NKp46,<br />
NKG2D and 2B4, as well as <strong>of</strong> killer-cell immunoglobulin-like<br />
receptors (KIRs) and CD94/NKG2A receptor. In addition,<br />
high levels <strong>of</strong> cytoplasmic granules despite their CD56bright<br />
CD16- surface phenotype characterize them. Moreover, we<br />
provide evidence that in dNK cells, activating NK receptors<br />
display normal triggering capability while 2B4 functions as<br />
an inhibitory receptor. Thus, crosslinking <strong>of</strong> 2B4 resulted in<br />
inhibition <strong>of</strong> both cytolytic activity and interferon-gamma<br />
production. Clonal analysis revealed that, in the majority <strong>of</strong><br />
dNK cell clones, the 2B4 inhibitory function is related to the<br />
deficient expression <strong>of</strong> SLAM-associated protein (SAP)<br />
mRNA. Moreover biochemical analysis revealed low levels<br />
<strong>of</strong> SAP protein in dNK polyclonal population. Thus, in the<br />
absence <strong>of</strong> SAP, SHP-1 phosphatase can bind to 2B4 and<br />
mediate inhibition <strong>of</strong> the dNK-mediated cytolysis CD48+<br />
target cells (eg EBV+ B cells). This suggests that dNK cells,<br />
although potentially capable <strong>of</strong> killing, are inhibited in their<br />
function when interacting with cells expressing CD48 (the<br />
2B4 ligand).<br />
doi:10.1016/j.clim.2007.03.555<br />
S187<br />
Su.140 Population <strong>of</strong> T, T Reg, NK Cells and Their<br />
Activation and Inhibitory Receptors in Peripheral<br />
Blood <strong>of</strong> Healthy Women During the Menstrual<br />
Cycle<br />
Erika Aurora Martínez García, Master in Science<br />
(<strong>Immunology</strong>), University <strong>of</strong> Guadalajara, Guadalajara,<br />
Mexico, Victor Ermilo Arana Argaez, Master in Science<br />
(<strong>Immunology</strong>), University <strong>of</strong> Guadalajara, Guadalajara,<br />
Mexico, Beatriz Teresita Martín Márquez, Master in<br />
Science (<strong>Immunology</strong>), University <strong>of</strong> Guadalajara,<br />
Guadalajara, Mexico, Pedro Ernesto Sánchez Hernández,<br />
PhD, University <strong>of</strong> Guadalajara, Guadalajara, Mexico, Luz<br />
Maria Adriana Balderas Peña, PhD, Unidad de<br />
Investigación Medica en Epidemiología Clínica, UMAE, HE,<br />
CMNO Instituto Mexicano del Segu, Guadalajara, Mexico,<br />
Trinidad García Iglesias, PhD, University <strong>of</strong> Guadalajara,<br />
Guadalajara, Mexico, Alicia Del Toro Arreola, Master in<br />
Science, University <strong>of</strong> Guadalajara, Guadalajara, Mexico,<br />
Oscar Javier López León Murguía, MD, University <strong>of</strong><br />
Guadalajara, Guadalajara, Mexico, José Francisco Muñoz<br />
Valle, PhD, University <strong>of</strong> Guadalajara, Guadalajara,<br />
Mexico, Adrián Daneri Navarro, PhD, University <strong>of</strong><br />
Guadalajara, Guadalajara, Mexico, Mónica Vázquez Del<br />
Mercado Espin, PhD, University <strong>of</strong> Guadalajara,<br />
Guadalajara, Mexico<br />
There is evidence that the maternal immune system is<br />
influence by changes in the hormonal levels during the<br />
menstrual cycle. So far, the information related to the levels<br />
<strong>of</strong> T, T reg, NK cells and their activation and inhibitory<br />
receptors is scarce. Aim: To analyze the populations <strong>of</strong> T, T
S188 Abstracts<br />
reg, NK cells and their activation and inhibitory receptors <strong>of</strong><br />
peripheral blood <strong>of</strong> healthy women during the menstrual<br />
cycle. Material and Methods: We studied 10 women not using<br />
hormonal contraceptives in the day 5 <strong>of</strong> the follicular phase<br />
and 21st <strong>of</strong> the luteal phase <strong>of</strong> the menstrual cycle.<br />
Peripheral blood lymphocyte subsets and their receptors<br />
were determined by flow cytometry. Results: We do not<br />
observe changes in the percentages <strong>of</strong> CD4+ and CD8+ Tcells.<br />
With respect to ILT-2 and CD94/NKG2 receptors expressed in<br />
CD4+ T cells and at the levels <strong>of</strong> T reg cells (CD4+CD25+<br />
Foxp3+) we noted an increased in the follicular phase. On<br />
the other hand, the levels <strong>of</strong> NK cells remain constant in the<br />
two menstrual phases as well as their receptors NKG2D and<br />
ILT-2. According to the receptors NKp30 and NKp46 we<br />
identified major fluctuations in the follicular phase and the<br />
opposite for CD94/NKG2. Conclusions: We observed an<br />
increase in day 5 and decay in day 21 <strong>of</strong> the menstrual<br />
cycle in the majority <strong>of</strong> the cellular populations and their<br />
receptors. Perhaps these results are product <strong>of</strong> the necessary<br />
mechanisms in menstrual cycle to prepare the<br />
processes <strong>of</strong> implantation in early pregnancy.<br />
doi:10.1016/j.clim.2007.03.556
<strong>Clinical</strong> <strong>Immunology</strong> (2007) 123, S189–S206<br />
FOCIS 2007 Abstract Index by Author<br />
Abbas, Alexander R. Sa.149<br />
Abel, Lucia Cristina Jamli F.64<br />
Abello, Paula Sa.65<br />
Aberle, Teresa Sa.27<br />
Abhinandan, Raghavan Su.87<br />
Abolhassani, Mohsen Sa.17<br />
Abouljoud, Marwan F.71<br />
Abrams, Elaine F.94<br />
Abramson, Tzvia F.69<br />
Abril, Annette Sa.26<br />
Abutbul, Shai Su.126<br />
Adams, Jason OR.59<br />
Adams, Katherine Sa.98<br />
Adib, Minoo F.125<br />
Adler, Adam OR.81<br />
Adriani, Marsilio OR.29<br />
Agarwal, Rajeev K. OR.38<br />
Aghasizadeh, Navid Su.62<br />
Agualimpia-Janning, Andres Su.129<br />
Aguiar, Paloma Sa.134<br />
Aguilar-Lemarrroy, Adriana C. Su.55<br />
Aguilar-Velazquez, Gustavo Su.76<br />
Aguilera, Raquel Sa.133<br />
Aguillon, Juan C. Sa.65<br />
Ahearn, Joseph M. Sa.31, Sa.35<br />
Aherns, Richard F.124<br />
Ahlmen, Jarl F.129<br />
Ailes, Elizabeth F.44<br />
Aksentijevich, Ivona Sa.49, Sa.50<br />
Alam, Munir F.72<br />
Alami Chentoufi, Aziz F.53<br />
Albani, Salvatore F.20, Sa.66, Sa.67, Sa.72,<br />
Sa.73<br />
Alberu, Josefina Su.101<br />
Alcocer Varela, Jorge F.93, Sa.81<br />
Alemi, Golnaz<br />
Alesahebfosoul, Fereshteh F.111, F.125<br />
Alkan, Sefik Su.57<br />
Allaire, Normand OR.55<br />
Allauzen, Sophie F.136<br />
Allen, Hamish Sa.40<br />
Allende, Gloria F.18<br />
Allie, Rameeza Sa.145<br />
Alliot-Licht, Brigitte Su.96<br />
doi:10.1016/j.clim.2007.03.541<br />
available at www.sciencedirect.com<br />
www.elsevier.com/locate/yclim<br />
Allison, James E. Sa.152<br />
Almeida, Alexandre F.67, F.98<br />
Alonso, Liv an Sa.147<br />
Alper, Chester Su.59, Su.60<br />
Althshuler, David OR.80<br />
Altshuler, David Su.35<br />
Alves, Venancio F.64<br />
Aly, Theresa A. OR.78<br />
Amato, Anthony Su.31<br />
Amox, Diane Sa.66<br />
Anderson, Amy F.57<br />
Anderson, David E. OR.95, OR.101, Sa.114,<br />
Su.127, Su.38<br />
Anderson, David F.15, OR.83, Sa.131<br />
Anderson, Mark F.12, OR.60, Su.111<br />
Anderson, Richard C.E. OR.58<br />
Anderson, Richard Sa.120<br />
Anderson, Stacie M. OR.29<br />
Anegon, Ignacio OR.71, Su.119<br />
Annels, Nicola F.103<br />
Annese, Vitto Su.93<br />
Ansaldo, Gianluca Sa.112<br />
Antel, Jack Su.10, Su.16<br />
Anthony, Donald F.71<br />
Aoki, Joseph OR.29<br />
Aono, Shelly F.61<br />
Aparicio, Carlos F.129<br />
Arab, Hamid Reza Su.50<br />
Arana Argaez, Victor Ermilo Su.140, Sa.44<br />
Aranami, Toshimasa OR.24, Su.12<br />
Aravena, Octavio Sa.65<br />
Arbour, Nathalie OR.33, OR.41, Su.16,<br />
Su.29<br />
Arechiga, Adrian F.28, Sa.148<br />
Arend, William P. Sa.71<br />
Aricò, Maurizio F.81<br />
Arif, Sefina OR.18<br />
Armengol, Maria Pilar F.26<br />
Armstrong, Don OR.79<br />
Arnold, Arthur P. Su.134<br />
Arnold, Dilani F.80<br />
Arouche Camara Lopes,<br />
Luiz Heraldo<br />
Sa.123<br />
Arshi, Saba Sa.7
S190 FOCIS 2007 Abstract Index by Author<br />
Ashayeri, Hassan Sa.7<br />
Ashfield, Rebecca OR.9<br />
Ashley, Charles OR.77, Sa.120<br />
Ashman, Robert F. OR.44<br />
Ashton-Chess, Joanna Su.113<br />
Ashutosh, Mangalam OR.61<br />
Aspsater, Mahsa F.129<br />
Atkinson, Mark OR.15, OR.2, OR.56<br />
Au, Liemin OR.49<br />
Au, Melinda Su.109<br />
Aud, Dee Su.40<br />
Autschbach, Frank Su.90, Su.91<br />
Avanesyan, Armine OR.97<br />
Awasthi, Amit Su.32<br />
Azadi, Gholomreza Sa.7<br />
Azmi, Hooman F.134<br />
Azuma, Miyuki F.33<br />
Azzari, Chiara F.91<br />
B. Elkon, Keith Sa.22<br />
Babu, Sunanda R. OR.78, F.12<br />
Bacchetta, Rosa F.91<br />
Badolato, Raffaele F.12, F.91<br />
Badr El-Din, Nariman Sa.124<br />
Badur, Selim Sa.109<br />
Baecher-Allan, Clare OR.77, Sa.120<br />
Baeten, Dominique Sa.57, Sa.58, Sa.59<br />
Baez, Ineavely F.113<br />
Bagely, Jessamyn Sa.11<br />
Baghani, Zahra<br />
Bagherian, Ali:<br />
Su.50<br />
Bailey, Samantha OR.34<br />
Baker, Harold F.104<br />
Baker, Mark Su.101<br />
Baker, Rocky F.25<br />
Balboni, Imelda Sa.46<br />
Balderas Peña, Luz Maria Su.140<br />
Adriana<br />
Balmer, Paul F.126, F.65<br />
Banda, Nirmal Sa.63<br />
Bankey, Paul F.70<br />
Baranyi, Ulrike Sa.11<br />
Baranzini, Sergio Su.15<br />
Barba-Barajas, Martha Su.55<br />
Barbour, Gene OR.3<br />
Barcellos, Lisa Sa.152<br />
Baric, Ralph F.46<br />
Barker, Jennifer F.12<br />
Barker, Jonathan OR.39<br />
Barmada, Michael Su.92<br />
Baron, Rona Su.126<br />
Bar-Or, Amit OR.41, OR.86<br />
Barrera, Lourdes Su.138<br />
Barros, Noac Chuffi F.96<br />
Barsky, Lora F.113<br />
Bartholomew, Richard OR.13<br />
Bartunkova, Jirina F.116, Sa.117<br />
Baschal, Erin E. OR.78<br />
Bason, Caterina F.65, OR.23, 1202<br />
Basso, Alexandre S. Su.28<br />
Bastos, Fabiana Su.19<br />
Batliwalla, Franak OR.55, Sa.55, Sa.61<br />
Bautista, Alejandro Su.98<br />
Bautista-Vazquez, Alejandro Su.95<br />
Bautz, David Sa.136<br />
Bayer, Monika Sa.126<br />
Begovich, Ann Sa.150<br />
Behrens, Marshall Sa.154<br />
Behrens, Timothy W. OR.80<br />
Bein, Gregor Su.41<br />
Belaunzaran, Francisco F.97<br />
Belmares, Michael Sa.132<br />
Belouski, Shelley F.137<br />
Benard, Gil F.52<br />
Bender, James Sa.106<br />
Benghiat, Fleur Samantha Su.103, Su.130<br />
BenMohamed, Lbachir F.53<br />
Benson, Charles OR.28, Sa.125<br />
Benson, Jacqueline OR.40<br />
Benvenuti, Luiz A. Su.124<br />
Benvenuto, Federica Sa.130<br />
Berer, Kerstin OR.20<br />
Berg, Ulla F.122<br />
Bergan, Lindsay Sa.97<br />
Berghöfer, Beate Su.41<br />
Bergholdt, Regine F.22<br />
Bergmann, Christoph OR.73<br />
Beriou, Gaelle Su.96<br />
Bernstein, Allan L. Su.2<br />
Bethunaickan, Ramalingam Sa.45<br />
Bettahi, Ilham F.53<br />
Bettelli, Estelle Su.42<br />
Bezerra, Jorge Su.123<br />
Bhairo, Michelle Sa.113<br />
Bhan, M.K. F.68<br />
Bhat, Roopa OR.96<br />
Bhatnagar, Shinjini F.68<br />
Bianchi, Lucia F.91<br />
Bianco, Nicolás Sa.85<br />
Bianco, Nicole OR.89<br />
Bidar, Maryam Su.62<br />
Biedermann, Tilo Sa.153<br />
Bienkowska, Jadwiga OR.55, Sa.55<br />
Bilton, Diana F.67<br />
Binder, Steven Sa.79<br />
Birmelin, Jennifer F.89<br />
Bishop, Gail A. Sa.68<br />
Blanca, Isaac Sa.85, Su.108<br />
Blanchard, Carine F.124<br />
Blankenhorn, Elizabeth OR.82<br />
Bloomfield, Tiffany Sa.96<br />
Bluestone, Jeffrey A. OR.16, OR.59, F.33, F.34<br />
Boardman, Cynthia Su.48<br />
Bohmova, Kristyna F.19<br />
Bollyky, Paul Sa.128<br />
Bolton, Wade F.129<br />
Bonadkar, Sasha Su.45<br />
Borgonovo, Giacomo OR.62<br />
Borrow, Ray F.126, F.65<br />
Bossi, Giovanna OR.9, Sa.98<br />
Bothwell, Alfred Su.102<br />
Bottini, Nunzio Sa.153<br />
Boudakov, Ivo F.38, Sa.127<br />
Bourcier, Kasia Sa.151, Su.37
FOCIS 2007 Abstract Index by Author<br />
Bourdette, Dennis OR.13<br />
Bour-Jordan, Helene F.33<br />
Bowman, Edward Su.91<br />
Boyer, Scott Su.63<br />
Boyle, John F.76<br />
Bradley, Brenda OR.3<br />
Bradshaw, Elizabeth Su.31<br />
Braemswig, Kira OR.15<br />
Branch, Rodshawn F.120<br />
Branigan, Patrick OR.40<br />
Braud, Cristophe Sa.57<br />
Braun, Jonathan OR.100, Su.82, Su.89<br />
Braun, Michel Sa.89<br />
Braun, Werner OR.22<br />
Braunstein, Jutta Su.90<br />
Bravo-Cuéllar, Alejandro Su.55<br />
Bresson, Damien OR.14, F.7<br />
Brewer, Joanna Sa.107<br />
Brion, Régis Su.119<br />
Brito, Cyro Alves Sa.16<br />
Brizuela, J.A. Su.32<br />
Brod, Staley Su.20<br />
Brodsky, Nina N. OR.52<br />
Brook, Azam Sa.105, Su.62<br />
Brooks-Worrell, Barbara OR.49<br />
Brorsson, Caroline F.22<br />
Brouard, Sophie Sa.57<br />
Brown, Chris F.132<br />
Brown, Margaret R. OR.52<br />
Bruce, Jeffrey N. Sa.114<br />
Bruce, Jeffrey Sa.120<br />
Bruner, Ben OR.89<br />
Bruner, Gail R. Sa.24<br />
Brunner, Tali Su.10<br />
Brusko, Todd OR.2<br />
Bryant, Shaughn Sa.32<br />
Buckle, Guy Su.27<br />
Buckner, Jane OR.19, OR.7, OR.76, F.28,<br />
Sa.128<br />
Bucknum, Amanda Sa.96<br />
Budinsky, Vit F.109<br />
Bupp, Melanie F.33<br />
Burdo, Tricia Su.128<br />
Burge, Matthew Sa.88<br />
Burke, George W. F.18<br />
Burrows, Gregory OR.10<br />
Butcher, Eugene C. OR.37<br />
Butte, Atul OR.53, F.9<br />
Buus, Søren F.53<br />
Cai, Chun Sa.134<br />
Cai, Qing Sa.150<br />
Calabresi, Peter Sa.145<br />
Calder, Claudia OR.86<br />
Callewaert, Nico Sa.38<br />
Camara-Lopes, Luiz F.131, Sa.121<br />
Cameron, Brian OR.9<br />
Campagnolo, Denise Su.9<br />
Canavez, Flavio Sa.121<br />
Candotti, Fabio OR.29<br />
Cannon, Jennifer Sa.116<br />
Cantaert, Tineke Sa.57, Sa.58, Sa.59<br />
Cantu-Brito, Carlos F.93<br />
Capocasale, Renold OR.40<br />
Capon, Francesca OR.39<br />
Carmans, S<strong>of</strong>ie OR.94<br />
Carmignani, Giorgio Sa.112<br />
Carmody, Francis F.13<br />
Carneiro-Sampaio, Magda Maria F.82<br />
Carter, Robert Sa.75<br />
Carulli, John OR.55, Sa.55<br />
Casanova, Jean-Laurent F.96<br />
Caspi, Rachel R. OR.38, OR.35<br />
Castro-Ramos, Feliciano Su.95<br />
Catanzarite, Tatiana Sa.132<br />
Cayrol, Romain OR.33, OR.88<br />
Chaim, Jacob OR.79<br />
Chan, Chi-Chao OR.35, OR.38<br />
Chang, Monica Sa.150<br />
Chang, Shun-Chiao Sa.53<br />
Chang, Vicky Su.82<br />
Chao, Nelson Sa.86<br />
Chapel, Helen F.80, F.83<br />
Chapman, Jeff Su.133<br />
Chau, Sandra F.27<br />
Chauveau, Christine Su.119<br />
Chavali, Arvind Su.84<br />
Chavez, Raul Su.93<br />
Chavez, Salvador Su.95<br />
Cheeran, Daniel OR.82<br />
Chella, David OR.61<br />
Chen, Benny Sa.86<br />
Chen, Cheng-Hsu OR.31<br />
Chen, Dong OR.42, OR.47<br />
Chen, Fang Su.136<br />
Chen, Genhui Sa.143<br />
Chen, James Sa.135<br />
Chen, Jing F.94<br />
Chen, Li-Chen F.88<br />
Chen, Ling Su.82<br />
Chen, Qian F.134<br />
Chen, Yi-Je Su.94<br />
Chen, Zoe OR.35<br />
Cheney, Richard Sa.111<br />
Cheng, Mickie OR.60, F.12<br />
Cheng, Yung Chih Su.137<br />
Chiffoleau, Elise OR.71, Su.96<br />
Chin, Hyunsook Sa.152<br />
Chini, Loredana F.93<br />
Chinn, Ivan F.84<br />
Chirinos, Perla Sa.90<br />
Cho, Judy Su.92<br />
Choi, Bong-Kum Sa.90, Sa.91<br />
Choi, Jae Eun Su.49<br />
Choi, James Su.62<br />
Chong, Mark Su.3<br />
Chooklin, Serge Su.110<br />
Choudhury, Arpita Sa.41<br />
Chow, Anthony W. Sa.93<br />
Choy, Edwin Su.35<br />
Christen, Selina Sa.126<br />
Christen, Urs 2201, OR.62, Sa.126<br />
Chruscinski, Andrzej J. Sa.30<br />
Chu, Alvina D. Sa.30<br />
S191
S192 FOCIS 2007 Abstract Index by Author<br />
Chung, Denise OR.19<br />
Chung, James Su.69<br />
Ciancio, Gaetano F.18<br />
Citron, John Sa.152<br />
Ciullini Mannurita, Sara F.91<br />
Clarke, Grace Su.94<br />
Clayton, David OR.85<br />
Coccia, Marco Sa.137<br />
Cody, Virginia OR.43<br />
Cohen, Irun F.20<br />
Cohen, Philip L. OR.58<br />
Cohen, Philip Sa.41, Sa.50<br />
Cohen, Smadar Su.10<br />
Cohen, Stanley Sa.62<br />
Coito, Ana OR.97<br />
Cole, Derek Sa.142<br />
Collanieri, Anna Cristina F.67<br />
Coller, John Su.132<br />
Collins, Mary Sa.142, Su.9<br />
Concannon, Patric Sa.148<br />
Condamine, Thomas OR.71<br />
Conesa, Angela Su.108<br />
Constabtino-Silva, Rosemeire F.99, F.96, Su.75<br />
Navickas<br />
Contreras, Carmen F.51<br />
Contreras-Levicoy, Juan Sa.65<br />
Cooke, Lawrence Su.46<br />
Cooper, Leslie Sa.154<br />
Coppieters, Ken Sa.38, Sa.95, Sa.23<br />
Coram, Marc OR.53<br />
Cornelius, Jennine Su.84<br />
Corneta, Elaine Cristina Sa.123<br />
Corneta, Elaine F.131<br />
Correa, Alexandre OR.100, F.100<br />
Correa, MRF Su.75<br />
Cort, Laura OR.82<br />
Costa, Cristiana F.103<br />
Costantino, Cristina F.115<br />
Costenbader, Karen H. Sa.53<br />
Courtenay-Luck, Nigel Sa.107<br />
Cowan, Morton OR.25<br />
Creusot, Remi J. F.5<br />
Crispín, José Carlos Sa.81<br />
Crispin, Jose F.93, Su.101, Sa.20<br />
Croen, Lisa Sa.152<br />
Cr<strong>of</strong>t, Michael F.10<br />
Cronk, Katharine Sa.114<br />
Crow, Mary Sa.47, Sa.92, Su.116<br />
Cruzado-Park, Ingrid D. F.72<br />
Cruz-Robles, David Sa.64<br />
Cua, Daniel OR.35<br />
Cuchacovich, Miguel Sa.65<br />
Cunningham, Matthew Su.96<br />
Cunninghame Graham, Deborah OR.80<br />
Cutter, Gary Sa.66<br />
Cuturi, Maria-Cristina OR.71, Su.96<br />
Czelusta, Adam Sa.12<br />
da Silva Duarte, Alberto Jose F.67, F.97, F.98<br />
Dai, Yang F.27, F.30<br />
Daikh, David Sa.82<br />
Dailey, Michael E. Sa.138, Su.5<br />
Daisuke, Goto Sa.40<br />
Daisuke, Tokita Su.105<br />
Daly, Mark Su.35, Su.93<br />
Damle, Aarti OR.55, Sa.63<br />
Dan, Nicolae Su.92<br />
Dandekar, Abhya Sa.17<br />
Daneri Navarro, Adrián Su.140<br />
Dang, Demi F.5, F.9, OR.16<br />
Danilenko, Dimitry OR.37<br />
Dannecker, Guenther E. F.135<br />
Dardalhon, Valerie OR.34<br />
Darden, Janice F.133<br />
Das, Shampa Su.59, Su.60<br />
Dasgupta, Gargi F.53<br />
Datta, Lisa Su.92<br />
Dave, Amy F.10<br />
Davey, Michael OR.84, Su.79<br />
David, Chella Sa.154, Sa.78<br />
Davidson, Anne Sa.45<br />
Davies, Robert F.80<br />
Davis, Ian D. Su.54<br />
D’Costa, Sybil F.136, F.72<br />
De, Asit F.72<br />
De, Mita F.70<br />
de Almeida, Alexandre F.97<br />
de Bakker, Paul Su.93<br />
de Beaucoudrey, Ludovic F.92<br />
De Jager, Philip OR.83, Su.27, Su.35,<br />
Su.36, Sa.139<br />
de Jager, Wilco F.20, Sa.75<br />
De Keyser, Filip Sa.59<br />
de Krijger, Ronald F.103<br />
de Leo, Claudia Su.101<br />
De Rycke, Leen Sa.58, Sa.59<br />
De Sanctis, Juan Sa.1, OR.81<br />
De Vos, Martine Sa.23<br />
de Vries, Niek Sa.57<br />
Degauque, Nicolas F.3<br />
Deisenhammer, Florian Su.8<br />
Dekan, Gerhard OR.64<br />
Del Toro Arreola, Alicia Su.140<br />
dela Rosa, Corazon Sa.101, Su.118<br />
Dellanegra, Marinella F.96<br />
Delovitch, Terry L. OR.30, F.4, F.23<br />
Demartino, Julie Su.58<br />
Denham, Jerrod Su.109<br />
Dennis, Rebecca OR.9<br />
Derfanoux, Nadine A. Sa.52<br />
Derfuss, Tobias 3202<br />
Desaire, Heather F.72<br />
Deschamps, Jack-Yves Su.96<br />
Deshmukh, Umesh Sa.78<br />
Deshpande, Chetan Sa.62<br />
Devlin, Blythe F.84<br />
DeVoss, Jason Su.111<br />
Di Meglio, Paola OR.39<br />
Di Mitri, Diletta Su.43<br />
Diamantopoulos, Stavros F.18<br />
Diange, L. Su.114<br />
Diarra, Danielle Sa.51<br />
DiCarlo, Edward Su.116<br />
Diehl, Lauri Su.84
FOCIS 2007 Abstract Index by Author<br />
Dinarello, Charles F.130<br />
Ding, Li F.66<br />
Dinnall, Joudy-Ann Sa.42<br />
Dishaw, Larry J. F.86<br />
Disis, Mary Sa.101, Su.118<br />
Dissanayake, Samudra Sa.115<br />
Dittmer, Dirk Su.39<br />
Dodelet-Devillers, Aurore OR.33, OR.88<br />
Doi, Yoshimitsu Su.13<br />
Dolcino, Marzia OR.23<br />
Dombrovskiy, Viktor Su.74<br />
Domingues Ferreira, Mauricio F.67, F.97, F.98<br />
Dominguez-Rodriguez, Su.55<br />
Jorge R.<br />
Dong, Jiaxin Su.47<br />
Dorsey, Morna J. F.86<br />
Dotta, Francesco F.24<br />
Douhan III, John Sa.142<br />
Downes, Kate OR.85<br />
D'Souza, Patricia F.133<br />
Du, Jianguang OR.72<br />
Du, Sienmi Su.112<br />
Du, Xiaoyan Sa.71<br />
Duan, Su Su.107<br />
Duarte, Alberto J.S. Sa.49, F.52, F.86, Sa.50,<br />
F.96, F.99, Su.75, Sa.16,<br />
F.92<br />
Dubey, Devendra Su.59, Su.60<br />
Dubrava, Tatyana Sa.92<br />
Duchateau, Jean<br />
Dugast, Anne-Sophie<br />
F.127<br />
Dumont, Debora OR.87<br />
Dunger, David OR.85<br />
Dunn, Shannon OR.69<br />
Dunne, Jack Su.118<br />
Dunne, John Sa.101<br />
Dunussi-Joannopoulos, Sa.142<br />
Kyriaki<br />
Durinovic-Bello, Ivana F.32<br />
Dusilova Sulkova, Sylva F.109<br />
Dutt, Suparna OR.68<br />
Dutz, Jan P. F.21<br />
Dzuris, John Su.107<br />
Eagar, Todd F.33<br />
Easley, Kirk F.94<br />
Eastham-Anderson, Jeffrey Su.55<br />
Ebeling, Cory OR.65<br />
Edberg, Jeffrey Sa.84<br />
Edgerton, Colin Sa.79<br />
Edmond, Jean Su.92<br />
Edwards, Hannah OR.6<br />
Egeler, Maarten F.103<br />
Eibel, Hermann OR.32<br />
Eisenbarth, George S. OR.78, F.12, F.17, F.7,<br />
OR.1, OR.21<br />
Eisenberg, Robert A. OR.58, Sa.41, Sa.42<br />
El Baz, Mimouna F.127<br />
El Khoury, Joseph OR.90<br />
Elewaut, Dirk Sa.23, Sa.38<br />
Elliott, John OR.21<br />
Ellis, Richard OR.18<br />
Elloumi, Houda F.66<br />
Elmets, Craig Su.89<br />
Elyaman, Wassim Su.22<br />
Emanuel, Katy Sa.103<br />
Engelmann, Peter F.31<br />
Epstein, Michelle OR.64<br />
Eriksson, Anna Sa.135<br />
Esmaili, Habibollah Su.124<br />
Estevam, Jose Sa.151, Su.37<br />
Estrada-Garcia, Iris Su.76<br />
Estrada-Parra, Sergio Su.76<br />
Evanko, Stephen Sa.128<br />
Exley, Andrew F.126, F.65<br />
Ezekowitz, Alan Sa.63<br />
Fafutis, Mary F.53<br />
Fahmidekar, Mohammad Ali Su.78<br />
Falabella, Michael Su.134<br />
Falk, Ronald Sa.136<br />
Fanslow, William 3201<br />
Farber, Donna OR.46<br />
Farid-Hosseini, Reza Sa.9, Su.66<br />
Fariss, Robert Su.81<br />
Farjadian, Shirin Su.125<br />
Farkas, Klara F.31<br />
Farshad, Shohreh F.46<br />
Fathman, C. Garrison F.5, F.9, OR.16<br />
Faure, Olivier Sa.106<br />
Fedynyshyn, Joe F.136<br />
Feingersh, Diane Sa.137<br />
Feitelson, Jerald Su.133<br />
Felton, Jamie OR.49<br />
Fenoglio, Daniela Sa.112<br />
Feo, Lourdes Sa.32<br />
Ferbas, John F.137, Su.86<br />
Fergus, Gleeson F.80<br />
Fernandez, Marco Antonio F.26<br />
Fernando, Michelle Sa.74<br />
Ferrada, Carlos Sa.122<br />
Ferrao, Maria do Socorro F.96<br />
Ferraz Neto, Ben-Hur F.64<br />
Ferrera, Francesca OR.12<br />
Ferrone, Soldano Sa.111<br />
Ferry, Berne F.83<br />
Field, Elizabeth H. Sa.138<br />
Field, Leigh F.22<br />
Fife, Brian T. F.33<br />
Filaci, Gilberto Sa.112<br />
Filippi, Christophe 2201, F.7<br />
Finegood, Diane F.21<br />
Fiorillo, Edoardo Sa.153<br />
Fisher, Yair Su.126<br />
Flanagan, Ken Su.84<br />
Flavell, Richard Su.79<br />
Fleisher, Thomas 3201<br />
Flores, Antonio Su.98<br />
Fohner, Alison Sa.10<br />
Forbes, Elizabeth F.124<br />
Foresti, Roberta Su.119<br />
Forman, Daron F.114<br />
Forman, Stephen Sa.87<br />
Forsyth, Ramses F.105<br />
S193
S194 FOCIS 2007 Abstract Index by Author<br />
Fossati, Gianluca Su.102, Su.85<br />
Foulkes, Roly Su.86<br />
Fousteri, Georgia F.10<br />
Fox, Edward Su.21<br />
Fransson, Moa Sa.100<br />
Fraser, Patricia Sa.53<br />
Fravega, Marco Sa.112<br />
Frederiksen,<br />
Su.54<br />
Klaus Stensgaard<br />
Frenkel, Dan Su.28<br />
Friend, Samantha Sa.66<br />
Frieri, Marianne Sa.8<br />
Frulloni, Luca 1202<br />
Fu, Qin Su.133<br />
Fujimoto, Manabu F.108, Sa.25, Su.63,<br />
Su.67, Su.68, Su.68<br />
Fujio, Keishi Sa.22<br />
Fujita, Mayumi Su.14<br />
Fujita, Tomoyuki Sa.25<br />
Fujiwara, Daisuke OR.100, Su.89<br />
Fulcher, Jennifer Sa.135<br />
Fung, Erik F.11<br />
Fung, John J. OR.31, Su.121<br />
Funke, Benjamin Su.90<br />
Furuzawa-Carballeda, Janette Sa.64, Su.129<br />
Fusaro, Ana Elisa Sa.16<br />
Gadkar, Kapil OR.59<br />
Gailey, Ryan Sa.104<br />
Gaines, Elizabeth Su.65<br />
Gallucci, Stefania Sa.42<br />
Gambhir, Sam S. F.5<br />
Gambineri, Eleonora F.91<br />
Ganjali, Rashin Sa.48, Su.66<br />
Gao, Sui F.50<br />
García Iglesias, Trinidad Su.140<br />
Garcia, Bertha OR.47, 1201<br />
Garcia, Michael Sa.71<br />
Garmendia, Jenny Sa.1<br />
Garvik, Barbara Sa.97<br />
Gattorno, Marco Sa.130<br />
Geba, Gregory Sa.5<br />
Gelli, Anna Maria Grazia<br />
Geng, Xuan F.21<br />
George, Tracy OR.68<br />
George, Heavner OR.40<br />
Gailey, Ryan Sa.104<br />
Ghaderi, Abbas Su.125<br />
Ghaffari, Javad Sa.109<br />
Ghahramani, Negar Sa.72, Sa.73<br />
Ghanekar, Smita Sa.133<br />
Ghasemi Hashtroodi, Leila F.1<br />
Ghio, Massimo Sa.33<br />
Ghoneum, Alia Sa.124<br />
Ghoneum, Mamdooh Sa.124<br />
Ghorayeb, Christine OR.86<br />
Giese, Thomas OR.46, Su.100, Su.107,<br />
Su.15, Su.90<br />
Gilbert, Leona OR.63<br />
Ginesta, Alexandra Sa.122<br />
Giuliani, Fabrizio Su.14<br />
Gluhcheva, Yordanka Georgieva Sa.108<br />
Gneiss, Claudia Su.8<br />
Golbin, Jason Sa.6<br />
Gold, Ralf Su.5<br />
Goldblum, Randall OR.22<br />
Goldoni, Adriana Leticia OR.48<br />
Goldrath, Ananda Su.61<br />
Goltsev, Anatoliy Sa.92<br />
Gomez-Contreras, Piedad C. Su.55<br />
Gonzalez, Carlos Sa.73<br />
Goodwin, Kelley Su.79<br />
Gorczynski, Reg Sa.127<br />
Gorczynski, Reginald F.38<br />
Gordon, John OR.65<br />
Gorelik, Elieser Sa.123<br />
Gosch, Courtney Sa.66<br />
Gostick, Emma Sa.98<br />
Goto, Daisuke Su.45<br />
Gottlieb, Alice Su.74<br />
Goverman, Joan OR.36<br />
Govindarajan, Rajagopalan OR.61<br />
Goyette, Philippe Su.108, Su.93<br />
Gozin, Michael Su.28<br />
Gozzard, Neil Su.86<br />
Graham, Kareem L. OR.91<br />
Graham, Robert R. OR.80<br />
Green, Todd Su.92, Su.93<br />
Greenbaum, Carla F.28<br />
Greenfield, Edward A. Sa.146<br />
Greer, Allison F.15<br />
Gregersen, Peter OR.55, Sa.55, Sa.61<br />
Gregory, Clare Su.94<br />
Greidinger, Eric L. Sa.28<br />
Greiner, Dale OR.56<br />
Griffiths, Gillian F.81<br />
Grimbacher, Bodo F.89<br />
Gros, Philippe F.27<br />
Gross, Jennifer Sa.97<br />
Grumach, Anete Sevciovic F.96, F.99, Su.75, F.92<br />
Gubbels Bupp, Melanie Sa.129<br />
Guberski, Dennis OR.82<br />
Guillaume, Marie Paule F.90, F.134<br />
Guillen, Cecilia F.51<br />
Guillermo, Roy Sa.120<br />
Gulati, Reema F.13, F.56<br />
Guleria, Indira F.33<br />
Gunderson, Erica Sa.152<br />
Gupta, Santosh F.68<br />
Gürses, Atilla Su.98<br />
Gutenberger, Sylvia F.75<br />
Haaland, Perry Sa.101<br />
Habiby Kermany,<br />
Sa.134<br />
Mohammad<br />
Hackstein, Holger Su.41<br />
Haensch, Gertrud Maria OR.98<br />
Hafler, David A. OR.77, OR.83, OR.94,<br />
OR.95, F.15, F.115,<br />
Sa.114, Sa.120, Sa.139,<br />
Sa.146, Sa.151, Su.27,<br />
Su.35, Su.36, Su.37,<br />
Su.38, Su.41<br />
Hahn, Bevra OR.4, OR.12, Sa.36, Sa.76
FOCIS 2007 Abstract Index by Author<br />
Hahn-Zoric, Mirjana F.122<br />
Hale, Matthew OR.92<br />
Hallam, Robert F.65, F.126<br />
Hamada, Takashi OR.97<br />
Hamm, Stefanie OR.32<br />
Han, David Su.34<br />
Han, May H. Su.34<br />
Han, Meifang Su.136, Su.43<br />
Hanak, Viktor Sa.6<br />
Hang, Howard F.115<br />
Hanlon, Douglas OR.43<br />
Hansen, Anna M. OR.38<br />
Hansen, Lasse Tengbjerg Su.54<br />
Hanson, Imelda C. Sa.12<br />
Hanson, Lars Åke F.122<br />
Hansson, Sverker F.122<br />
Hao, Qian-Lin F.113<br />
Haqqani, Arsalan OR.2<br />
Harley, John B. Sa.24, Sa.79<br />
Harley, John Sa.47, OR.81<br />
Hartnett, Mark F.9<br />
Hartt-Meyers, Jennifer Sa.146<br />
Hasegawa, Minoru Sa.25, Su.63, Su.67<br />
Haskins, Kathryn F.25, OR.3<br />
Hastings, William F.15<br />
Haug, Markus F.135<br />
Hauser, S. Su.93<br />
Haworth, Charles F.65<br />
Haynes, Barton OR.25<br />
Hazzan, Marc Sa.89<br />
Headley, Mark OR.57<br />
Hegde, Ganapati Sa.102, Sa.103<br />
Heidtmann, Antje Su.90<br />
Heinlen, Latisha Sa.79<br />
Hellings, Niels OR.87, Su.18, Su.23<br />
Hendriks, Jerome JJA OR.87<br />
Hennon, Teresa OR.89<br />
Henry, Alistair Su.85<br />
Hensen, Karen Su.23<br />
Heppert, Volkmar F.49<br />
Hering, Bernhard F.24<br />
Hernandez-Flores, Georgina Su.55<br />
Herold, Kevan OR.56<br />
Herrera-Gonzàlez, Norma Sa.110<br />
Herrinton, Lisa Sa.152<br />
Heslan, Michele OR.71, Su.96<br />
Hewitt, Kyle Sa.115<br />
Hickman, Scott F.127<br />
Hickman, Suzanne OR.90<br />
Hiestand, Peter Su.3<br />
Highton, John Sa.88<br />
Hill, Marcello OR.71<br />
Hillary, Steinhart Su.92<br />
Hintermann, Edith OR.62, Sa.126<br />
Hirasawa, Masao Su.53<br />
Hirsch, J. Su.19<br />
Hladikova, Zuzana F.19<br />
Ho, Anthony Sa.113<br />
Ho, I-Cheng Su.40<br />
Ho, Peggy P. Sa.54, Sa.71, Sa.88<br />
Hoefferer, Liane Su.7<br />
H<strong>of</strong>fman, Robert W. Sa.26, Sa.28<br />
Hogan, Simon F.124<br />
Hogan, Susan Sa.136<br />
Hogendoorn, Pancras F.103<br />
3202<br />
Hojaili, Bernard OR.9<br />
Holdener, Martin OR.62<br />
Holers, V Michael Sa.63<br />
Holland, Steven M. OR.52, F.66<br />
Hollenberg, Morley OR.65<br />
Holness, Claire F.9<br />
Holzer, Ursula OR.65<br />
Hood, Zach Su.20<br />
Hoogeboom, Manja F.103<br />
Horai, Reiko OR.29<br />
Horn, Julia F.89<br />
Horwitz, David A. OR.75, Sa.140<br />
Hosseini-Farahabadi, Sara Sa.9, Su.66<br />
Hosseinkhani, Ayda F.46, F.62<br />
Hou, Stephen F.130<br />
Houle, Jean-Francois Su.120<br />
Hrotekova, Zuzana F.19, Sa.95<br />
Hsieh, Meng-Ying F.88<br />
Hu, Lina Sa.145<br />
Hu, Paul OR.64<br />
Hua, Jing Sa.47<br />
Huang, Jing-Long F.88<br />
Huber, Janine OR.40<br />
Hudson, Michael Sa.116<br />
Hueber, Wolfgang Sa.54, Sa.61<br />
Huegel, Alyssa OR.2<br />
Huett, Alan Su.92<br />
Huibregtse, Inge F.116<br />
Husain, Zaheed Su.59, Su.60<br />
Hussain, Shabbir F.23<br />
Huttenlocher, Anna OR.5<br />
Hwang, Hank Su.47<br />
Hwang, Sunil Su.34<br />
Iacomini, John Sa.18<br />
Ifergan, Igal OR.33, OR.88, Su.29<br />
Ikeda, Osamu F.58<br />
Iking-Konert, Christ<strong>of</strong> F.49<br />
Iliev, Iliyan D. OR.70<br />
Im, Suhn-young Su.51<br />
Imitola, Jaime Su.22<br />
Indiveri, Francesco OR.12, Sa.112, Sa.33<br />
Inman, Robert Sa.47, 1201<br />
Inokuma, Margaret Sa.101, Su.118<br />
Inoue, Asuka Su.45<br />
Ishida, Masato Su.52<br />
Ishiura, Nobuko F.108<br />
Ismail, Heba OR.49<br />
Israel, David OR.74<br />
Itano, Andrea Su.69<br />
Ito, Satoshi Sa.48, Su.45<br />
Iwai, Hideyuki F.9, OR.16<br />
Iwakura, Yoichiro OR.35<br />
Iwanami, Keiichi Su.45<br />
Jaberi, Yahya F.1<br />
Jachimowicz, Edward F.129<br />
Jacob, Chaim O. Sa.94<br />
S195
S196 FOCIS 2007 Abstract Index by Author<br />
Jacob, Cristina Miuki Abe F.82<br />
Jacob, Noam OR.93<br />
Jacobi, Christian Su.15<br />
Jacobson, Robert OR.26, F.63<br />
Jacobson, Stefan H. F.122<br />
Jacques, Peggy Sa.23<br />
Jahromi, Mohamed M. OR.78<br />
Jaimes, Kimberly Sa.26<br />
Jaimes, Maria F.133<br />
Jain, Ashish 3201, OR.27<br />
Jaing, Tang-Her F.88<br />
Jakobsen, Bent OR.9, Sa.98<br />
Jalahej, Heyam F.31<br />
James, Judith OR.81, Sa.79<br />
Jamshidzadeh, Akram F.48, F.62<br />
Jann, Monica OR.71<br />
Jasinski, Jean F.17, F.7, OR.1, OR.21<br />
Jave-Suarez, Luis F. Su.55<br />
Jawa, Vibha Su.42<br />
Jeffrey, Lisa F.120<br />
Jennette, J. Charles Sa.136<br />
Jensen-Jarolim, Erika OR.15<br />
Jerath, Maya Su.58<br />
Jerome, Rotter Su.92<br />
Jesus, Adriana Sa.49, Sa.50<br />
Ji, Shaoquan OR.39, Su.47<br />
Jiang, Guoping OR.74<br />
Jill, Giles OR.40<br />
Jimenez-Martinez, Maria Su.64, Su.76<br />
Carmen<br />
Joe, Selby Sa.152<br />
Johannes, Kellsey OR.60<br />
John, Eigth Author Su.107<br />
Johnson, Jonas OR.73<br />
Johnson, Kelly OR.1, OR.21<br />
Joo, Sung-Yeon Sa.90, Sa.91<br />
Jose Francisco,<br />
Sa.52<br />
Muñoz-Valle<br />
Joshi, Avadhut Sa.103<br />
Joshi, Shantaram Sa.103, Sa.116<br />
Josien, Regis OR.71, Su.96<br />
Juan, Gloria Su.69<br />
Juan, Manel F.26<br />
Juang, Yuang-Taung OR.48<br />
Jumnainsong, Amonrat Su.122<br />
Kagoda, Mercy F.113<br />
Kaieda, Shinjiro Su.14<br />
Kallas, Esper F.64<br />
Kalyanasundaram,<br />
F.73<br />
Ramaswamy<br />
Kamphuis, Sylvia F.20<br />
Kandeel, Fouad Sa.87<br />
Kang, Mi Jin Sa.90, Sa.91<br />
Kao, Amy H. Sa.31<br />
Karabeg, Adela OR.64<br />
Karagiannis, Panos Sa.112<br />
Karbach, Julia OR.9<br />
Karlson, Elizabeth W. Sa.53<br />
Karmali, Rafik F.127<br />
Karow, Margaret F.131<br />
Karter, Andrew Sa.152<br />
Kasman, Ian OR.37, Su.84<br />
Kassam, Nasim Sa.146, Su.38<br />
Kastelein, Robert Su.79<br />
Kataoka, Kazuo Su.24<br />
Kattah, Michael Su.132<br />
Katz, Jonathan F.8<br />
Kaufman, Kenneth OR.81<br />
Kawaciuk, Ivan Sa.117<br />
Kawikova, Ivana Su.102<br />
Kebir, Hania OR.33, OR.88, Su.29<br />
Kedem, Hassya F.69<br />
Keller, Baerbel F.75<br />
Kellermann, Sirid-Aimee Sa.52<br />
Kelley, Keith F.137<br />
Kelly, Jennifer OR.81<br />
Kennedy, Jeff Su.85<br />
Kent, Sally F.15, F.24<br />
Kenyon, Norma F.24<br />
Keogh, Elissa Sa.66, Sa.72, Sa.73<br />
Kern, Marlena OR.55<br />
Khaje Daluei, Mohammad Sa.16<br />
Khalili, Houman OR.55<br />
Khatri, Ismat F.38<br />
Khoury, Samia Su.27, Su.35<br />
Kianifard, Farid Sa.5<br />
Kiany, Simin F.45, F.46, F.48, F.62<br />
Kilpatrick, Jeff OR.81<br />
Kim, Han-A Su.51<br />
Kim, Il Hwan Su.64<br />
Kim, Junwoo Sa.134<br />
Kim, Patrick Sa.134<br />
Kim, Seon-Hee OR.89<br />
Kim, Sung-Joo Sa.100<br />
Kimberly, Robert Sa.84<br />
Kimmie, Crystal OR.49<br />
King, Jennifer K. Su.112<br />
King, Marie OR.56<br />
Kiran, Bayram Sa.109<br />
Kirby, Martha OR.29<br />
Kis, Janos F.31<br />
Kitaichi, Nobuyoshi Sa.21<br />
Kivisakk, Pia Su.22<br />
Kivovich, Violetta OR.63<br />
Klareskog, Lars Sa.61<br />
Klein, Mark F.20<br />
Klinkhammer, Thomas Su.46<br />
Knight, Seth Sa.75<br />
Knittelfelder, Regina OR.15<br />
Ko, Hon-Sum Sa.19<br />
Ko, Hyun-Mi Su.51<br />
Kobayashi, Koichi Su.79<br />
Kobayashi, Masakazu F.17<br />
Kodama, Keichi OR.16, F.5, F.9<br />
K<strong>of</strong>feman, Eva Sa.66<br />
Kohlmoos, Cassidy OR.93<br />
Kohm, Adam Su.13<br />
Komura, Kazuhiro Su.63<br />
Komuves, Laszlo Su.84<br />
Korchynska, Olga F.41<br />
Korinets, Yaroslav F.41<br />
Korman, Alan Sa.137<br />
Korn, Thomas OR.19, Su.32
FOCIS 2007 Abstract Index by Author<br />
Koscielna, Katarzyna Sa.8<br />
Koss, Michael OR.93<br />
Kostianovsky, Alex Su.127<br />
Kostov, Georgi Stefanov Sa.108<br />
Kovacs, Jospeh 3201<br />
Kovats, Susan Sa.132<br />
Kozlova, Yulia Sa.101<br />
Kramer, Ivan F.39, F.74<br />
Krensky, Alan M. Sa.10<br />
Kretowski, Adam OR.78<br />
Kretsos, Kosmas Sa.52<br />
Kreuwel, Huub OR.59<br />
Krienke, Stefan Sa.47, 1201<br />
Krueger, Gerald Sa.150<br />
Kruith<strong>of</strong>, Elli Sa.59<br />
Krutsick, Amy Sa.104<br />
Krutzik, Peter OR.92<br />
Kuballa, Petric Su.92<br />
Kuchroo, Juhi OR.101, Su.38<br />
Kuchroo, Vijay K. OR.19, OR.95, F.15,<br />
Sa.146, Su.32<br />
Kudrycki, Katherine Sa.59<br />
Kudva, Yogish OR.61<br />
Kühne, Ronald Sa.129<br />
Kuis, Wietse F.20, Sa.67<br />
Kumar, Krishan Sa.8<br />
Kumar, Vijay F.110<br />
Kulhankova, Katarina Sa.138<br />
Kuo, Cheng-Chin F.121, Su.137<br />
Kuo, Liang-Mou OR.31, OR.74<br />
Kuo, Ming-Ling F.88<br />
Kusunoki, Susumu Su.24<br />
Kwon, Sung(Steve) F.73<br />
Kye, Young Chul Su.49<br />
Kyjovska, Drahomira Sa.95<br />
Kyttaris, Vasileios C. OR.48<br />
La Cava, Antonio OR.12, OR.4, Sa.36,<br />
Sa.76, Su.9<br />
LaCorcia, Gina Su.107<br />
Lage, Agustin Sa.147<br />
Lai, Chen-Yen F.121<br />
Lam, Queenie Lai Kwan OR.98<br />
Lam, Tong OR.65<br />
Lamb, Roberta OR.40<br />
Lamberth, Kasper F.53<br />
Lanchbury, Jerry OR.39<br />
Land, Michael F.100<br />
Landa, Rosa Sa.88<br />
Lang, Jiena F.34<br />
Lanigan, Caroline Su.128<br />
Larocca, Nancy Sa.1<br />
LaRosa, David F.57<br />
Laroy, Wouter Sa.38<br />
Lasitschka, Felix Su.105, Su.91<br />
Lathey, Janet F.120<br />
Latiano, A. Su.93<br />
Lau, Chak-Sing Sa.80<br />
Law, Adam F.12<br />
Lawendowski, Carla Su.107<br />
Lawson, Alastair Su.87<br />
Lawson, Jenifer OR.6<br />
Lederman, Michael F.73<br />
Le, Ngocdiep Sa.86<br />
Lee, Annette Sa.55<br />
Lee, Benhur Sa.151<br />
Lee, Chung Sa.36<br />
Lee, Hai-Chon Sa.13<br />
Lee, Hern-Ku Su.51<br />
Lee, Jin Su.49<br />
Lee, JungHwa Su.49<br />
Lee, Kwang Chul Su.49<br />
Lee, Li-Fen Sa.88<br />
Lee, Michael R. F.34<br />
Lee, Sun min Sa.106<br />
Lee, Tzielan Sa.70<br />
Lee, Wen-I F.88<br />
Lee-Chan, Edwin F.6<br />
Leelayuwat, Chanvit Sa.125, Su.122<br />
Lehman, Thomas Sa.47<br />
Leif, Jean OR.97<br />
Leite, Katia Sa.132<br />
Leiva, Lily E. F.101<br />
Lemar, Hadia Sa.137<br />
Lenert, Petar OR.44<br />
Leon, Juan F.44<br />
Leon-Murguia, Oscar Sa.44<br />
Leotlela, Poloko Sa.115<br />
Leppert, Mark Sa.150<br />
Lerma-Diaz, Jose M. Su.70<br />
Leung, Hin-Tak OR.85<br />
Leung, Lawrence Sa.71<br />
Levi, Dina F.14<br />
Levinson, Arnold F.57<br />
Levinson, Ralph Su.82<br />
Levisetti, Matteo OR.17<br />
Levy, Gary Su.59<br />
Li, Bin Sa.143<br />
Li, De-Kun Sa.152<br />
Li, Haowei Sa.93<br />
Li, Jian OR.40<br />
Li, Marcella F.17<br />
Li, Mu OR.12, OR.47, 1201<br />
Li, Quan-Zhen OR.81<br />
Li, Sharon F.13<br />
Li, Wentian Sa.25, Sa.55<br />
Li, Xinrui Sa.84<br />
Li, Zhuqing Su.78, Su.81<br />
Liang, Chi-Ming F.121<br />
Liang, Shu-Mei F.121, Su.137<br />
Liao, Hua-Xin F.72<br />
Liao, Lingjie Su.121<br />
Liddy, Nathaniel Sa.114<br />
Lierl, Michelle B. Su.131<br />
Liggitt, Denny OR.36<br />
Lightwood, Daniel Su.86<br />
Lim, Wee Kiak Su.78<br />
Limb, Cindy Sa.46<br />
Limón-Camacho, Leonardo Sa.20, Sa.64<br />
Lin, Chia-Lei Sa.87<br />
Lin, Erina F.100<br />
Lin, Jean Sa.31, Sa.35<br />
Lin, Syh-Jae F.92<br />
Linares, Marisela Su.76<br />
S197
S198 FOCIS 2007 Abstract Index by Author<br />
Lindesmith, Lisa F.44<br />
Ling, Eleanor F.16<br />
Ling, Mei Su.92<br />
Linhart, Birgit Sa.11<br />
Linington, Christopher OR.20, 3202<br />
Link, Jason OR.10<br />
Lipets, Irina Su.74<br />
Littman, Dan R. OR.72<br />
Liu, Baoying Su.78, Su.81<br />
Liu, Chau-Ching Sa.31, Sa.35<br />
Liu, Chunyu Sa.55<br />
Liu, Edwin F.17, OR.21<br />
Liu, Mingfeng Su.43<br />
Liu, Peng Su.58<br />
Liu, Quanhai OR.41<br />
Liu, Ruolan Su.9, Su.9<br />
Liu, Shuying F.87, 3201<br />
Liu, Wenxia Sa.134<br />
Liu, Yingge Sa.153<br />
Lock, Christopher OR.96<br />
Logani, Jyoti F.68<br />
Long, S. Alice OR.7, OR.76<br />
Loo, Ray Mun Sa.8<br />
Lopes, Jared E. OR.72<br />
Lopez-Granados, Eduardo F.83<br />
López León Murguía,<br />
Su.140<br />
Oscar Javier<br />
López, Mercedes N. Sa.122<br />
Lorber, Marc Su.102<br />
Lord, James Sa.128<br />
Lories, Rik Sa.23<br />
Loskog, Angelica Sa.100<br />
Louvet, Cedric F.34<br />
Lu, Lina OR.31, OR.74<br />
Lu, Liwei OR.98<br />
Lu, Wen OR.60<br />
Lu, Xinyue Su.134<br />
Luckner, Claudia Sa.113<br />
Luger, Dror OR.35, Su.54<br />
Lühder, Fred Su.5, Su.5<br />
Luna-Baca, Gabriel Andres Su.64, Su.76<br />
Lunardi, Claudio 1202, OR.23<br />
Lundsgaard, Dorthe Su.54<br />
Luo, Jinquan Sa.141<br />
Luo, Xiaoping OR.86, F.50, Su.43,<br />
Su.136<br />
Luster, Andrew OR.90<br />
Lutsenko, Elena Sa.92<br />
Ly, Dalam F.4, F.23<br />
Lyba, Myroslav Su.110<br />
Lyle, Michael Sa.143<br />
Lyons, Kathryn Su.58<br />
Ma, Chi OR.27<br />
Ma, Hak-Ling Su.72<br />
Macaubas, Claudia Sa.62<br />
Macedo, Camila Su.99<br />
Macias-Hernandez, Israel Su.129<br />
Macip-Rodríguez, Perla Sa.64<br />
Macpherson, Michael OR.100<br />
Maecker, Holden F.133, Sa.101, Su.118<br />
Magliocco, Melissa Su.74<br />
Mahadevan, Daruka Su.46<br />
Mahesh, Sankara Su.81<br />
Mahesh, Sankaranarayana Su.78<br />
Mahon, Tara OR.9, Sa.98<br />
Maier, Lisa OR.83<br />
Maino, Vernon Sa.101<br />
Majumdar, Anish Su.127<br />
Malik, Sanober Sa.24<br />
Malter, James OR.99<br />
Mamura, Mizuko Sa.40, Su.61<br />
Manak, Mark F.120<br />
Manku, Harinder OR.80<br />
Manns, Michael OR.62<br />
Mansour, Eli F.99<br />
Manzi, Susan Sa.35<br />
Marcenaro, Stefania F.81<br />
Marchetti, Piero F.24<br />
Marcondes, Maria Cecilia Su.128<br />
Marietta, Eric F.116<br />
Marinkovich, Vincent A. F.128, F.132<br />
Marino Vazquez, Lluvia Su.118<br />
Marisis, Tatiane F.131<br />
Mark, Daly Su.92<br />
Markert, Louise F.84<br />
Markovic, Svetomir Sa.119<br />
Marquez, Maria Elena Sa.85, Su.126<br />
Marrero, Idania F.30<br />
Marti, Luciana F.64<br />
Martín Márquez, Beatriz Teresita Sa.44, Su.140<br />
Martin, Andrew Su.87<br />
Martin, Tammy Su.79<br />
Martínez García, Erika Su.140, Sa.44<br />
Martini, Alberto Sa.130, Su.137<br />
Martini, Stefania F.85<br />
Martinic, Marianne 2201, F.7<br />
Maryam, Tadayon F.106<br />
Masewicz, Susan A. F.32<br />
Massanari, Marc Sa.5<br />
Massoco, Cristina F.131, Sa.121<br />
Matarese, Giuseppe OR.4<br />
Matejkova, Eva Sa.95<br />
Mather, Ian OR.20<br />
Mathey, Emily 3202<br />
Mathieu, Suzanne Sa.32<br />
Matozan, Katja Su.7<br />
Matsevitaya, Irina Sa.92<br />
Matsuda, Tadashi F.58, Su.52<br />
Matsumoto, Isao Sa.40, Su.45<br />
Matsumoto, Mitsuru OR.24, Sa.141<br />
Matthews, Kate OR.64<br />
Maykut, Robert Sa.5<br />
Mazhani, Maryam Sa.48<br />
McArdel, Shannon Sa.114, Su.31<br />
McArthur, Grant Su.54<br />
McClain, Micah Sa.79<br />
McCrae, Ellie OR.41<br />
McGrail, Kieran OR.2<br />
McNeal, Erin-Joi F.44<br />
McPherson, Michael Su.89<br />
Means, Terry OR.90<br />
Meinl, Edgar 3202<br />
Mellins, Elizabeth Sa.148, Sa.62
FOCIS 2007 Abstract Index by Author<br />
Mendonça, Marcelo F.52<br />
Mendoza, Felipe Su.138<br />
Meraz-Rios, Marco Antonio Sa.110<br />
Merrill, Joan OR.81<br />
Mesirov, Jill Su.46<br />
Metes, Diana Su.99<br />
Meuer, Stefan Sa.113, Su.15, Su.90,<br />
Su.91, Su.100<br />
Meuwissen, Pieter Su.36<br />
Miao, Dongmei OR.21, F.17<br />
Michalek, Jaroslav F.19, Sa.104<br />
Michaud, Gregory Sa.127<br />
Midoro-Horiuti, Terumi OR.22<br />
Mielants, Herman Sa.23<br />
Miescher, Sylvia Su.22<br />
Miethke, Alexander Su.123<br />
Miettunen, Paivi Sa.69<br />
Miguel, Regueiro Su.92<br />
Mikita, Allison F.3<br />
Miklos, David B. OR.43<br />
Mileti, Erika OR.70<br />
Milkovich, Kimberly F.71<br />
Miller, Stephen OR.19, OR.34<br />
Miller-Graziano, Carol F.70<br />
Millonig, Alban Su.8<br />
Milosevic, Ivan F.105<br />
Min, Wei-Ping OR.42, OR.47, 1201<br />
Minarik, Ivo Sa.117<br />
Mingari, Maria Cristina Su.139<br />
Minnig, Kathrin Su.7<br />
Minor, Dana F.101<br />
Mi Qing, Sheng F.23<br />
Mirel, Daniel Su.109<br />
Misawa, Tamako Su.6<br />
Misbah, Siraj F.80<br />
Misiara, Antonio Sa.123, F.131<br />
Misiura, Angela F.41<br />
Mistry, Jehangir Su.47<br />
Miyake, Sachiko Su.12, Su.13, Su.14<br />
Miyamoto, Katsuichi Su.24<br />
Miyashiro, Joy Sa.142<br />
Miyashita, Minoru Su.53<br />
Mizuno, Miho Su.14<br />
Mo, Lian Su.99<br />
Modrusan, Zora Su.84<br />
Moe, Christine F.44<br />
Moheghi, Nasrin Su.50, Su.124<br />
Molloy, Peter Sa.98<br />
Monjure, Hanh F.101<br />
Monsonego, Alon OR.38, Su.10<br />
Montaño, Efrain Su.98<br />
Montano-Gonzales, Efrain Su.95<br />
Monteon, Francisco Su.98<br />
Monteon-Ramos, Francisco Su.112<br />
Montero, Enrique Sa.147<br />
Montgomery, Mitzi Su.21<br />
Montoya, Margarita F.51<br />
Moonka, Dilip F.71<br />
Moore, Adrian Su.86<br />
Moore, Carolina OR.97<br />
Moore, Marcus Su.52<br />
Mor, Guillermo Sa.116<br />
Moraes Vasconcelos, Dewton F.67, F.92, F.96, F.97,<br />
F.98, F.103, F.131, Sa.121,<br />
Sa.123, Su.75<br />
Morales Buenrostro, Luis Su.101<br />
Mordes, John OR.56, OR.82<br />
Moreau, Aurelie Su.96<br />
Moreira-Filho, Carlos F.66<br />
Moreno, Dolores Sa.1<br />
Moreno-Fierros, Leticia Su.56<br />
Moretta, Lorenzo F.81, Sa.130, Su.139<br />
Morrow, Matthew R. F.86<br />
Moshk<strong>of</strong>f, Dmitry F.120<br />
Moskowitz, Keith F.120<br />
Motterlini, Roberto Su.119<br />
Moullier, Philippe Su.96<br />
Moulton, Vaishali R. OR.48<br />
Mouri, Yasuhiro Sa.141<br />
Mouritzen, Ulrik Su.54<br />
Moviglia, Gustavo A. Su.19<br />
Moviglia, M.T. Su.19<br />
Moya, R. Su.19<br />
Muczynski, Kimberly Su.113<br />
Mueller, Isabelle OR.76<br />
Munder, Markus Sa.113<br />
Munger, Corey Sa.102, Sa.103<br />
Munirathinam, Gnanasekar F.73<br />
Muñoz Valle, José Francisco Su.140<br />
Muñoz-Chable, Olga Su.129<br />
Munroe, Melissa E. Sa.68<br />
Muromoto, Ryuta Su.52<br />
Murphy, Andrew F.124<br />
Murray, Joseph F.116<br />
Murtaza, Anwar Sa.32<br />
Myles, Timothy Sa.78<br />
Nadeau, Kari C. Sa.10<br />
Nahill, Sharon OR.19, Su.107<br />
Nakajima, Toshihiro Sa.56<br />
Nakayama, Maki OR.1, OR.21, F.17<br />
Nance, Christina OR.26<br />
Nasr, Mohamed Sa.138<br />
Navratil, Jeannine S. Sa.31<br />
Nazneen, A. Su.114<br />
Ndejembi, Modesta OR.46<br />
Neamatollahi, Hossein Su.124<br />
Neethling, Francisca OR.54, Sa.106<br />
Negi, Surendra OR.22<br />
Negri Santi, Tatiana F.69, F.97<br />
Neil, Risch Sa.152<br />
Nejad Shahrokhabadi, Khadijeh Sa.105<br />
Nelson, David 3201<br />
Nelson, Edward F.130<br />
Nelson, J. Lee Su.113<br />
Nemirovsky, Anna Su.126<br />
Nepom, Gerald OR.51, F.32, Sa.128<br />
Nesbitt, Andrew Su.85, Su.86<br />
Nesheim, Steven F.94<br />
Nesspor, Tom OR.40<br />
Nestle, Frank OR.39<br />
Nevin, Barry Sa.113<br />
Newell, Karen OR.41<br />
Newsom, Brian Su.21<br />
S199
S200 FOCIS 2007 Abstract Index by Author<br />
Ng, Gordon Su.69<br />
Nguyen, Alex F.53<br />
Nguyen, Khoa Sa.10, Sa.62<br />
Nguyen, Tffany OR.54, Sa.106<br />
Nickol<strong>of</strong>f, Brian Sa.115<br />
Nico, Marcelo Mentha Su.75<br />
Niesters, Marieke F.103<br />
Niewold, Timothy Sa.47<br />
Nikoopour, Enayat F.6<br />
Ning, Bo OR.22<br />
Ning, Qin F.50, Su.43, Su.121,<br />
Su.136<br />
Nishimoto, Kevin F.130<br />
Nishimura, Toshihiko Sa.71<br />
Nishioka, Kusuki Sa.56<br />
Nishiya, Ana F.92<br />
Noaman, Eman Sa.124<br />
Nolan, Garry OR.92<br />
Nolan, GP OR.50<br />
Norowski, Elaine OR.82<br />
Notarangelo, Luigi F.91<br />
Noval Rivas, Magali Sa.89<br />
Nussenblatt, Robert Su.78, Su.81<br />
O’Brien, Peter Sa.104<br />
Ochs, Hans F.78, F.79, 3201<br />
O’Conner, Kevin Sa.114, Su.31, Su.47<br />
Offner, Halina OR.10, OR.13<br />
Ogawa, Masafumi Su.12<br />
Ohno, Shigeaki Sa.21<br />
Okamoto, Akiko Sa.22<br />
Okamura, Ross Su.109<br />
Oki, Shinji Su.13, Su.14<br />
Oksenberg, Jorge OR.69, Su.93<br />
Oldham, Elizabeth OR.39<br />
Oldham, Janine 2201<br />
Oliveira, Joao B. F.82, F.92, Sa.49, F.96,<br />
Su.75, Sa.50<br />
Olson, Julie Su.26<br />
Ondr, Jennifer F.8<br />
O’Neil, William M. Sa.43<br />
Onengut-Gumuscu, Suna Sa.148<br />
Ootsuka, Takao Su.14<br />
Opoka, Robert F.8<br />
O’Quinn, Darrell 3203<br />
Orange, Jordan F.76<br />
Orban, Tihamer F.31<br />
Oreizi, Farzad F.125<br />
Orihuela, Ana Su.31<br />
Orii, Noemia F.96<br />
Oritani, Kenji F.58, Su.52<br />
Orlovsky, Yevgeniya OR.40<br />
Orru, Valeria Sa.153<br />
Ortiz-Lazareno, Pablo C. Su.55<br />
O’Shea, Adam F.13, F.56<br />
Oshidary, Neekaan Su.132<br />
Ostankov, Maxim Sa.92<br />
Oukka, Mohamed OR.19, Su.32<br />
Ousman, Shalina OR.69, OR.94<br />
Out, Theo Sa.58<br />
Ouyang, Wenjun OR.37<br />
Ovsyannikova, Inna F.63<br />
Packwood, Kerri F.83<br />
Padyukov, Leonid F.122<br />
Page, Matt Su.87<br />
Paglia, Michael F.13, F.56<br />
Pahwa, Savita F.94<br />
Pai, Sung-Yun Su.40<br />
Palframan, Roger Su.86<br />
Palian, Beth Sa.140<br />
Palleros, C.A. Su.19<br />
Palmer, Jeanne Sa.86<br />
Palmer, Jerry OR.49<br />
Palumbo, Michael F.14<br />
Palumbo, Paul F.94<br />
Pan, Kuang-Hung Sa.62<br />
Pandey, Niranjan Su.57<br />
Pantanelli, Seth Su.81<br />
Pareigis, Elizabeth R. OR.44<br />
Paris, Kenneth F.101<br />
Parker, Alex OR.8<br />
Parrish, Yasmin F.113<br />
Pasalar, Mehdi F.114<br />
Passerini, Laura F.91<br />
Paston, Samantha Sa.98<br />
Pastorino, Antonio Carlos F.82<br />
Patel, Dhavalkumar Su.39, Su.58<br />
Paterson, Blake Su.57<br />
Pathoulakis, Charalabos F.61<br />
Patil, Tanvi F.38<br />
Payne, Kimberly F.113<br />
Peakman, Mark OR.6, OR.18<br />
Pearle, Andrew Su.116<br />
Pearson, Todd OR.56<br />
Pease, Larry Sa.119<br />
Peckham, Russell Su.134<br />
Pegram, Mark Sa.107<br />
Penaranda, Cristina OR.59<br />
Pende, Daniela F.81<br />
Peng, Stanford Sa.129, Su.57<br />
Peng, Ruoqi Su.58<br />
Penny, Richard Sa.18<br />
Pereda, Cristian Sa.122<br />
Perez, OD OR.50<br />
Perez, Rolando Sa.147<br />
Perez-Tapia, Mayra Su.76<br />
Peritt, David OR.40<br />
Perkins, Izabella F.44<br />
Perrin, Steve OR.55<br />
Perroni, Lucia Beatrice F.91<br />
Pesce, Barbara Sa.65<br />
Pessoa, Mário F.64<br />
Peter, Hans Hartmut OR.32, F.75, F.89<br />
Peter, Lipsky OR.81<br />
Peterlana, Dimitri OR.23, 1202<br />
Petrovic, Aleksandra F.86<br />
Petrovic-Stojkovic, Sanja Su.28<br />
Petry, Douglas OR.101<br />
Pi, Bin Su.136<br />
Pieri, Patrícia de Campos F.82<br />
Pietra, Gabriella Su.139<br />
Pihoker, Catherine F.28<br />
Pilat, Nina Sa.11<br />
Pillai, Asha OR.68
FOCIS 2007 Abstract Index by Author<br />
Pinsky, Norman F.63<br />
Pinto, Jorge Andrade F.99<br />
Pires Correa, Alexandre F.99<br />
Pitashny, Milena Sa.27<br />
Pixley, John S. Sa.43<br />
Plagnol, Vincent OR.85<br />
Planck, Steve Su.79<br />
Planck, Steven OR.84<br />
Ploegh, Hidde F.115<br />
Pober, Jordan Su.102, Su.104<br />
Pociot, Flemming F.22<br />
Poland, Gregory F.63<br />
Polychronakos, Constantin F.14<br />
Ponath, Paul F.58<br />
Ponte, Joe F.56<br />
Poole, Brian D. OR.63<br />
Popescu, Iulia Su.99<br />
Popov, Igor Sa.47, 1201<br />
Popplewell, Andrew Su.87<br />
Porcelli, A. F.23<br />
Prakken, Berent F.20, Sa.67<br />
Prasad, Shashi OR.43<br />
Prat, Alexandre OR.33, OR.88, Su.29<br />
Pratt, Nicol F.32<br />
Prefumo, Federico Su.139<br />
Pressler, Barrrak Sa.136<br />
Prestigiacomo, Tony Sa.79<br />
Preston, Ben Sa.137<br />
Preston, Gloria Sa.136<br />
Pricop, Luminita OR.93<br />
Priest, Catherine Su.109<br />
Prokoptchuk, Natalia F.41<br />
Puccetti, Antonio OR.23, 1202<br />
Puckett, Lindsay Su.28<br />
Pugliese, Alberto F.18, F.24<br />
Pujol Borrell, Ricardo F.26<br />
Punwani, Divya F.83<br />
Purbhoo, Marco OR.9<br />
Purcell, Shaun F.22<br />
Putterman, Chaim OR.93<br />
Pyne, Saumyadipta Su.36<br />
Qian, Shiguang OR.31, OR.74<br />
Rabellino, Enrique F.72, F.129<br />
Rachitskaya, Aleksandra V. OR.38<br />
Rafatpanah, Houshang Sa.9, Su.66<br />
Ragheb, Jack F.134<br />
Raimondi, Giorgio Su.105<br />
Rajagopolan, Govind Sa.78<br />
Rakhmanov, Mirzokhid F.75<br />
Rakhshandeh, Hassan Sa.105<br />
Ram, Aarthi OR.26<br />
Raman, Girija Su.94<br />
Ramanujam, Meera Sa.45<br />
Ramos Moreira Leite, Katia Sa.123<br />
Rao, Deepak A. Su.104<br />
Rapacki, Krist<strong>of</strong>fer F.22<br />
Rasband, Matthew Su.26, 3202<br />
Rashtak, Shadi F.116<br />
Rasouli, Manoochehr F.45, F.46, F.48, F.64<br />
Rassassi, Khadir Sa.151, Su.37<br />
Ratnayake, Chitra K. F.72<br />
Ravetti, Jean Louis Sa.112<br />
Ray, Pratima F.70<br />
Razzaque, M.S. Su.114<br />
Read, Jennifer F.94<br />
Reddy, Jay Su.32<br />
Reich, David Sa.139<br />
Reichardt, Holger Su.20<br />
Reid, Jessica Sa.96<br />
Reiff, Andreas OR.79<br />
Relman, David F.69<br />
Remy, Séverine Su.119<br />
Rescigno, Maria OR.70<br />
Reséndiz Albor, Aldo Arturo Su.56<br />
Rewers, Marian J. OR.78<br />
Reyburn, Hugh Su.122<br />
Reyes, Candice F.136<br />
Reyes, Diego Sa.122<br />
Richard, Julie OR.19<br />
Richards, William Sa.51<br />
Ricordi, Camillo F.24<br />
Rider, Beverley J. OR.23, F.6<br />
Rieben, Robert Su.7<br />
Rieck, Mary F.28, OR.76, Sa.148<br />
Riedl, Marc F.100<br />
Riemer, Angelika B. OR.15<br />
Rigato, Paula Ordonhez Sa.16<br />
Riggs, James Sa.96<br />
Rihal, Pardeep S. Sa.12<br />
Riker, Adam Sa.115<br />
Riley, Christopher Su.46<br />
Rinderknecht, Cornelia Sa.132<br />
Rioux, John Sa.74, Su.92, Su.93<br />
Rita, Bottino OR.56<br />
Rivitti, Evandro F.99<br />
Rizzi, Marta OR.12, OR.32, Sa.33<br />
Robbins, Paul Sa.91<br />
Roberto, Eigth Author F.24<br />
Roberts, Robert OR.42, F.87<br />
Robinson, William OR.94, Sa.54, Sa.61,<br />
Sa.71, Su.34<br />
Robotham, Jason M. Sa.18<br />
Roby, Keith Su.48<br />
Rodrigo, Evelyn 2201<br />
Rodrigues, Denise F.64<br />
Rodriguez Manzanet, Roselynn Sa.146<br />
Rodriguez, Benigno F.71<br />
Rodriguez, Miguel Su.95, Su.98<br />
Roep, Bart F.2, OR.11, OR.6<br />
Rogerio, Jaqueline F.120<br />
Rojas, Mauricio Su.58<br />
Romeo, Elisa<br />
Römisch, Jürgen Su.15<br />
Roncarolo, Maria Grazia F.91<br />
Ronchese, Franca Sa.6<br />
Roord, Sarah Sa.67<br />
Rosenbaum, James OR.84, Su.79<br />
Rosengren, Sanna Sa.70<br />
Rosenthal, Devin Sa.115<br />
Rosenzweig, Holly OR.84, Su.79<br />
Rosenzweig, Michael F.114, F.56, F.13<br />
Rossin, Elizabeth Su.27, Su.35, Su.36<br />
S201
S202 FOCIS 2007 Abstract Index by Author<br />
Rossini, Aldo OR.56<br />
Rothenberg, Marc F.124<br />
Rottembourg, Diane OR.14<br />
Rottiers, Pieter F.116, Sa.46<br />
Rouse, Todd Sa.138<br />
Roux, Kenneth H. Sa.17, Sa.18<br />
Roy, Debasmita Su.39<br />
Roy, Dolly Su.34<br />
Royer, Pierre-Joseph Su.119<br />
Rubertone, Mark Sa.79<br />
Rubinstein, Mark Su.61<br />
Ruitenberg, Joyce Sa.133<br />
Rummens, Jean-Luc Su.23<br />
Ruzek, Melanie OR.19<br />
Ryan, Jenna F.63<br />
Ryu, Jay Sa.6<br />
S. Grumach, Anete F.67, F.98, F.101<br />
Sabater, Lidia F.26<br />
Sabeti, Pardis Sa.74<br />
Saikali, Philippe Su.16<br />
Saito, Yuki Su.67<br />
Salazar, Lorena Sa.65<br />
Salazar-Onfray, Flavio OR.45, Sa.65, Sa.122<br />
Salek Moghddam, Alireza Sa.7<br />
Salman, Andac Sa.109<br />
Saltzman, Mark Sa.110<br />
Salzer, Ulrich OR.32, F.89<br />
Sampaio, Elizabeth F.66<br />
Samstag, Yvonne<br />
Sánchez Hernández,<br />
Pedro Ernesto<br />
Su.90<br />
Sanda, Srinath F.2<br />
Sandborg, Christy Sa.62<br />
Santacruz-Valdes,<br />
Su.64, Su.76<br />
Concepcion<br />
Santamaria, Pere F.21<br />
Santoro, Alessandra F.81<br />
Saoudi, Abdelhadi Su.111<br />
Sarraf, Alireza Su.124<br />
Sathe, Shridhar Sa.17, Sa.18<br />
Sato, Jun-ichi Su.6<br />
Sato, Maria Notomi Sa.16<br />
Sato, Shinichi Sa.33, Su.63<br />
Sawalha, Amr H. OR.81, Sa.24<br />
Sayegh, Mohamed Su.22<br />
Scalapino, Kenneth Sa.82<br />
Scallon, Bernie OR.40<br />
Scanzello, Carla Su.116<br />
Schaefer, Catherine Sa.152<br />
Schartner, Jill OR.5<br />
Schatz, Desmond OR.2<br />
Schein, Catherine Sa.5<br />
Schenk, Erin Sa.119<br />
Schett, Georg Sa.51<br />
Schlesier, Michael F.89<br />
Schnell, Christian Su.3<br />
Schoeb, Trenton 3203<br />
Scholler, Nathalie Sa.97<br />
Schout, Denise F.52<br />
Schreiner, Bettina OR.34<br />
Schrodi, Steven OR.69<br />
Schroeder, Andreas Su.90<br />
Schroeder-Braunstein, Jutta Su.91<br />
Schwartzberg, Pamela L. OR.29<br />
Schweitzer, Barry Sa.116<br />
Sc<strong>of</strong>ield, R Hal Sa.24<br />
Scott, Benjamin Sa.42<br />
Seamon, Vanessa Sa.17<br />
Seddiki, Nabila OR.30<br />
Seeborg, Filiz Sa.12<br />
Seeuws, Sylvie Sa.23<br />
Segal, Gabriela Sa.122<br />
Segura, Jorge F.51<br />
Seidu, Luqman F.124<br />
Sekine, Yuichi F.58, Su.67<br />
Selby, Mark Sa.137<br />
Selman, Moises Su.138<br />
Sercarz, Eli F.27, F.30<br />
Seroogy, Christine F.110<br />
Servais, Genevieve F.127<br />
Seshasayee, Dhaya Sa.149<br />
Sessarego, Marta Sa.33<br />
Sette, Jr., Hoel F.64<br />
Setty Balakrishnan, Anand F.73<br />
Sewell, Andy Sa.98<br />
Seyeddi, Ronak Sa.85<br />
Seyfer, Sarah OR.44<br />
Seyfert-Margolis, Vicki Su.1, Su.50<br />
Shampain, Kimberly L. Sa.114<br />
Shao, Wenhai OR.58<br />
Sharif, Shadi Sa.71<br />
Sharifi, Faranak F.1<br />
Sharma, Meera F.79<br />
Sharp, Veronika OR.50<br />
Shea, Yvonne F.68<br />
Shealy, David OR.40<br />
Shearer, William OR.26, F.94<br />
Shen, Guanxin Su.121<br />
Shen, Jijia F.134<br />
Shen, Ling Sa.152<br />
Shen, Zhong-Jian OR.99<br />
Sheng, Shijun Su.133<br />
Shi, Fu-Dong Su.9<br />
Shi, Jishu F.61<br />
Shirdel, Babak F.27<br />
Shivakumar, Pranavkumar Su.123<br />
Shizuru, Judith Sa.88<br />
Shoda, Lisl OR.59<br />
Shohreh, Farshad F.62<br />
Shoor, Stanford Sa.152<br />
Shugart, Yin Yao Su.92<br />
Shultz, Leonard OR.56<br />
Shum, Anthony Su.111<br />
Shum, Tony F.12<br />
Sido, Bernd Su.90, Su.91<br />
Siebert, Janet Sa.101, Su.118<br />
Siegel, Richard M. OR.29<br />
Sierra, Juan F.93<br />
Silberman, Daniel Sa.96<br />
Silva, Clovis Sa.56, Sa.57<br />
Silva, Léia C. Rodrigues F.52<br />
Silva, Nubia Su.108<br />
Silver, Phyllis OR.35, OR.38
FOCIS 2007 Abstract Index by Author<br />
Silverberg, Mark Su.92<br />
Sim, Davis Sa.78<br />
Simonian, Michael H. F.72<br />
Simonson, William OR.5<br />
Sims, Martin Sa.42<br />
Sin, Sang-Hoon Su.39<br />
Singer, Josef OR.15<br />
Singh, Bhagirath F.6<br />
Singh, Harvir Sa.30<br />
Singh, Manisha F.55<br />
Singh, Ram Raj OR.66, Sa.76, Sa.94,<br />
Su.112, Sa.135<br />
Sirota, Marina OR.53<br />
Siwak, Edward OR.26<br />
Skak, Kresten Su.54<br />
Skowera, Anna OR.18<br />
Skrumsager, Birte K. Su.69<br />
Slaets, Leen OR.87<br />
Slavik, Jacqueline Sa.146<br />
Slavonic, Michael F.13<br />
Sleasman, John W. F.90<br />
Smit, Helga Su.114<br />
Smith, Deborah Su.112<br />
Smith, Michael Sa.116<br />
Smitz, John Sa.136<br />
Smogorzewska, Monika F.113<br />
Smyth, Deborah OR.85<br />
Snowden, James Su.87<br />
Snyder, James F.134<br />
Snyder, Michael Sa.116<br />
Sobel, Raymond A. Sa.146, OR.69, OR.91,<br />
OR.94, Sa.97, Su.32,<br />
Su.34<br />
Sochorova, Klara F.109<br />
Sohn, Jeongwon Su.49<br />
Solbrig, Camille OR.43<br />
Soler-Ferran, Dulce Su.27<br />
Somers, Veerle Su.18, Su.23<br />
Somerville, John Sa.106<br />
Sommerer, Claudia Su.100<br />
Son, Hye-Youn Sa.90, Sa.91<br />
Son, Sang Wook Su.49<br />
Song, Jason Sa.71<br />
Soper, David OR.8<br />
Sorensen, Ricardo U. OR.5<br />
Soto-Abraham, Virginia Sa.72<br />
Soulillou, Jean-Paul Sa.65<br />
Sousa-Canavez, Juliana F.131, Sa.121, Sa.123<br />
Spier, Catherine Su.46<br />
Sprent, Jonathan Su.61<br />
Spycher, Martin O. Su.7<br />
Sripa, Banchob Sa.111<br />
Standifer, Nathan OR.51<br />
Stanimirovic, Danica OR.88<br />
Staquet, Kim OR.40<br />
Stechova, Katerina F.19<br />
Steinman, Lawrence OR.69, OR.94, OR.96,<br />
Su.34<br />
Stephens, Tracey A. F.6<br />
Sternjak, Alexander OR.10<br />
Stevens, Anne Su.113<br />
Stevens, Christine Sa.74, Su.93<br />
Stevens, Helen OR.85<br />
Still, Travis F.24<br />
Stinissen, Piet OR.87, Su.18, Su.23,<br />
Su.31<br />
Stolina, Marina Sa.51<br />
Storch, Maria OR.20, 3202<br />
Stourac, Petr F.62<br />
Straub, Irena F.135<br />
Strauss, Laura OR.73<br />
Strober, Samuel OR.68<br />
Strober, Warren 3201<br />
Strobl, Frank Sa.104<br />
Strom, Terry B. Sa.146<br />
Stromnes, Ingunn OR.36<br />
Struthers, Mary Su.58<br />
Su, Kai Su.136<br />
Su, Kaihong Sa.84<br />
Su, Maureen F.12<br />
Suen, Yu Su.48<br />
Suez, Daniel F.78, F.79<br />
Suggs, Sid F.137<br />
Sugiyama, Kenji Su.52<br />
Sumida, Takayuki Sa.40, Su.45<br />
Sun, Hongtao 1201<br />
Sun, Lulu F.102<br />
Sun, Xiaocui OR.72<br />
Supter, Tina Su.105<br />
Suresh, Lakshmanan F.110<br />
Sutton, Deborah OR.9, Sa.98<br />
Suzuki, Motohiko OR.42, OR.47, 1201<br />
Swanson, Steven J. Su.42<br />
Swerkersson, Svante F.122<br />
Swiergala, Elzbieta F.22<br />
Swistak, Mark Su.107<br />
Szalai, Alexander Sa.84<br />
Szereday, Laszlo F.31<br />
Szot, Gregory L. F.34<br />
Szuhai, Karoly F.103<br />
Tabunoki, Hiroko Su.6<br />
Tachdjian, Raffi F.100<br />
Tagawa, Asako OR.24<br />
Taguchi, T. Su.114<br />
Tahmasebifar, Neda F.122<br />
Tai, Yi Su.102<br />
Tak, Paul-Peter Sa.57, Sa.58, Sa.59<br />
Takahashi, Kazue Sa.63<br />
Takehara, Kazuhiko Sa.25, Su.67<br />
Tálamo, Carlos Sa.1<br />
Tamaki, Kunihiko F.108, Su.68<br />
Tamayo, Pablo Su.36<br />
Tamiz, Amir Su.57<br />
Taneja, Veena Sa.154<br />
Tang, Anita OR.46<br />
Tang, Liren Sa.143<br />
Tang, MengXiang Sa.101<br />
Tang, Qizhi OR.59, F.33<br />
Tanguy-Royer, Séverine Su.119<br />
Tanji, Maury M. F.52<br />
Tasch, Michael Su.113<br />
Tassinari, Paolo Su.108<br />
Tati, Abdellatif F.103<br />
S203
S204 FOCIS 2007 Abstract Index by Author<br />
Taub, Dennis Sa.115<br />
Tavakkol Afshari, Jalil Sa.9, Sa.48, Sa.105,<br />
Su.50, Su.62, Su.66,<br />
Su.124<br />
Tavares, R.C. F.104<br />
Tawde, Pallavi Sa.17, Sa.18<br />
Taylor, Kent Su.92<br />
Taylor, Paul Su.117<br />
Tedder, Thomas Su.85<br />
Teklenburg, Gijs F.20<br />
Tellides, George Su.119, Su.129<br />
Temmerman, Stephane OR.27<br />
Tenenbaum, Jessica Sa.46<br />
Terrett, Jon OR.72<br />
Tesson, Laurent Su.119<br />
Teuber, Suzanne OR.50, OR.63<br />
Thebault, Pamela OR.71<br />
Thewissen, Marielle Su.18, Su.23<br />
Thiel, Jens F.85, F.95<br />
Thomas, John F.137<br />
Thomson, Angus Su.105<br />
Thornton, Angela M. OR.29<br />
Thurlings, Rogier Sa.58<br />
Tian, Yan F.3<br />
Tibshirani, Robert Sa.69<br />
Timmel, Amanda OR.5<br />
Timmer, Trieneke Sa.58<br />
Tirabasi, Rebecca OR.82<br />
Tisch, Roland Sa.136<br />
Tiwari, Ruby OR.22<br />
Tiwari-Woodruff, Seema Su.112<br />
Tobiasova, Zuzana F.109, Sa.117<br />
Todd, John A. F.11<br />
Todd, John OR.85<br />
Todorov, Ivan Sa.87<br />
T<strong>of</strong>t, Michelle OR.90<br />
Togher, Lisa OR.14, 2201, F.7, F.16<br />
Tomi, Chiharu Su.28<br />
Tommasini, Alberto F.95<br />
Tomooka, Beren OR.94, Sa.61<br />
Tonel, Giulia OR.39<br />
Tonkin, Daniel OR.3<br />
Toro, Felix Su.108<br />
Torre, Giancarlo Sa.112<br />
Torres-Lozano, Carlos F.77, Su.70<br />
Tötterman, Thomas Sa.100<br />
Touil, Tarik OR.41<br />
Traggiai, Elisabetta Sa.130<br />
Traverso, Paolo Sa.112<br />
Tree, Timothy OR.6, OR.18<br />
Trembath, Richard OR.39<br />
Tremoulet, Adriana Sa.74<br />
Treszl, Andras F.31<br />
Tripp, Catherine Sa.32<br />
Trucco, Massimo OR.56<br />
Trueblood, Esther Su.69<br />
Tsokos, George C. OR.48, Su.134<br />
Tsoukas, Christos M. F.91<br />
Tsuchihashi, Sei-ichiro Su.128<br />
Tsutsumi, Akito Sa.40, Su.45<br />
Turna, Akif Sa.109<br />
Tyson, Kerry Su.87<br />
Uccelli, Antonio Sa.130<br />
Ueda, Masumi OR.28, Sa.125<br />
Untersmayr, Eva OR.15<br />
Uratsu, Sandra Sa.17<br />
Urban, Nicole Sa.97<br />
Utz, Paul J. OR.50, OR.75, Sa.30,<br />
Su.132<br />
Uzel, Gulbu OR.52<br />
Vacca, Paola Su.139<br />
Vaickus, Lou F.114<br />
Vaitaitis, Gisela M. OR.67<br />
Valdes-Ferrer, Sergio Ivan F.93<br />
Valdez, Patricia OR.37<br />
Valenta, Rudolf Sa.11<br />
Valenzuela, David F.124<br />
Vales-Alberto, Luis Su.95<br />
Vales-Gomez, Mar Su.122<br />
Valle, Sollange F.99<br />
Van Belle, Tom F.16<br />
Van Beneden, Katrien Sa.23, Sa.38<br />
van de Ven, Annick Sa.72, Sa.73<br />
van den Brandt, Jens Su.5<br />
van der Pouw-Kraan, Tineke Sa.58<br />
van Deventer, Sander<br />
Van Eyk, Jennifer<br />
F.116<br />
van Kooten, Cees F.122<br />
Van Landeghen, Megan OR.7<br />
Van Noort, Johannes OR.94<br />
van Rooijen, Nico Sa.22<br />
van Wijk, Femke Sa.67<br />
Vandenbark, Arthur OR.10, OR.13<br />
Vanderlocht, Joris OR.87<br />
Vandooren, Bernard Sa.58, Sa.59<br />
Varela, Gabriela Su.19<br />
Varela, Jorge Alcocer Sa.20<br />
Vargas Rojas, Maria Ines F.93, Sa.20, Sa.64, Sa.81,<br />
Su.101<br />
Vasquez, Ismael Sa.110<br />
Vatseba, Roman Su.110<br />
Vázquez Del Mercado Espin, Sa.44, Su.140<br />
Mónica<br />
Venken, Koen Su.18, Su.23<br />
Verbruggen, Gust Sa.23, Sa.38<br />
Verrijn Stuart, Annemarie F.20<br />
Veys, Eric M. Sa.59<br />
Victor, Jeferson Russo Sa.16<br />
Vidlakova, Petra Sa.95<br />
Vierkant, Robert F.63<br />
Vieths, Stefan Sa.17<br />
Viglietta, Vissia Su.27<br />
Vilela, Maria Marluce F.99<br />
Villaggio, Barbara Sa.112<br />
Villanueva, Cleva Sa.110<br />
Vladau, Costin F.124, OR.42, OR.47, 1201<br />
Vliag<strong>of</strong>tis, Harissios Sa.7<br />
Vollmer, Timothy Su.9<br />
von Gynz Rekowski, Kathrin Su.21<br />
von Herrath, Matthias OR.14, F.2, 2201, F.7, F.10,<br />
F.16, Sa.137, Su.117<br />
Vose, Julie Sa.102, Sa.103<br />
Voskuhl, Rhonda R. Su.112
FOCIS 2007 Abstract Index by Author<br />
Vrabelova, Zuzana F.19<br />
Vugler, Alex Su.86<br />
Vyse, Timothy J. OR.80, Sa.74<br />
Wade, Michael OR.7<br />
Wadia, Persis P. OR.53<br />
Wagner, Christ<strong>of</strong> F.49<br />
Wagner, David OR.67, F.25, Sa.131,<br />
Su.118<br />
Waid, Dan Su.118<br />
Wakeland, Edward OR.81<br />
Waldmann, Herman F.13<br />
Waliszewska, Alicja Sa.139<br />
Walker, Mindi OR.76<br />
Walker, Neil OR.85<br />
Walsh, Emily Sa.74<br />
Wandstrat, Amy OR.81<br />
Wang, Fang Sa.17<br />
Wang, James Sa.94<br />
Wang, Julie OR.75, Sa.140<br />
Wang, Kevin YehSheng F.87<br />
Wang, Shen-Wu F.137<br />
Wang, Wei Su.121<br />
Wang, Zhiliang Su.123<br />
Wang, Zhimo F.50<br />
Warnatz, Klaus OR.32, F.75, F.89<br />
Wasserfall, Clive OR.2, OR.56<br />
Watanabe, Rei Su.68<br />
Watanabe, Yoko Su.45<br />
Watry, Debbie Su.128<br />
Wawrousek, Eric OR.94<br />
Weaver, Casey 3203<br />
Webber, Steve Su.99<br />
Webster, John Sa.143<br />
Weeraratna, Ashani Sa.115<br />
Wei, Bo OR.100, Su.89<br />
Wei, Nathan Sa.70<br />
Weidanz, Jon A. OR.54, Sa.106, Sa.108<br />
Weiner, Howard Su.27, Su.28<br />
Weiner, Mira Su.27<br />
Weisenburger, Dennis Sa.102<br />
Wekerle, Thomas Sa.11<br />
Wellcome Trust Case Contr OR.85<br />
Wen, Xiangshu OR.66, Sa.135<br />
Wentzensen, Andreas F.49<br />
Wernet, Dorothee F.135<br />
Westall, Frederick Su.1, Su.2<br />
Westerberg, Per-Anton F.122<br />
Westerfield, Susan OR.26<br />
Whartenby, Katharine Sa.145<br />
Whitehead, Terry Su.47<br />
Whiteside, Theresa L. OR.73<br />
Whiting, Chan C. OR.59, Sa.52<br />
Wicker, Linda F.11, F.27, OR.83<br />
Wight, Thomas Sa.128<br />
Wijayawardana, Sameera F.98<br />
Wijbrandts, Carla Sa.66, Sa.67<br />
Wilkinson, Julie F.129, Sa.2<br />
Williams, Jim Su.34<br />
Williams, John Su.107<br />
Williams-Weese, Courtney Sa.24<br />
Willis, Van OR.52<br />
Willison, LeAnna N. Sa.18<br />
Wishart, Neil Sa.32<br />
Witte, Alison Sa.137<br />
Wolf, Bryan J. Sa.43<br />
Wolslegel, Kristen Sa.149<br />
2201<br />
Wong, Maida Sa.80<br />
Wongsena, Wachanan Sa.111<br />
Wood, Allyson Sa.63<br />
Woodburn, Tito OR.54<br />
Wotring, Michael OR.49<br />
Wu, Jianfeng OR.37<br />
Wu, Michele F.53<br />
Wu, Paul OR.13<br />
Wucherpfennig, Kai Su.32<br />
Wulff, Heike Su.94<br />
Wullner, Danika Su.42<br />
Xavier, Ramnik Su.92<br />
Xi, Dong F.50, Su.43, Su.136<br />
Yaghoubi, Shahriar F.5<br />
Yagishita, Naoko Sa.56<br />
Yagita, Hideo F.33<br />
Yamamoto, Kazuhiko Sa.30<br />
Yamamoto, Nobuto OR.28, Sa.125<br />
Yamamura, Takashi OR.24, Su.12, Su.13,<br />
Su.14<br />
Yamasaki, Satoshi Sa.56<br />
Yan, Weiming F.50, Su.43, Su.136<br />
Yancopoulos, George F.124<br />
Yang, Jiajin Sa.136<br />
Yang, Jun-Qi OR.66<br />
Yang, Li OR.95<br />
Yang, Lihu Su.58<br />
Yang, Sherry Sa.115<br />
Yang, Wei F.3<br />
Yano, Masashi Sa.141<br />
Yao, Qingping Sa.94<br />
Yaremtchuk, Tatiana F.41<br />
Yasui, Teruhito F.58<br />
Ye, Shuang OR.75, Sa.140<br />
Yelensky, Roman Su.35<br />
Yener, Alper Sa.109<br />
Yeung, Joseph Sa.80<br />
Yin, Zhenyu OR.31, OR.74<br />
Yong, Pierre F.76<br />
Yoo, Tai June Sa.134<br />
Yoshiga, Yohei Sa.40<br />
Yoshimura, Akihiko F.58<br />
Young, Daniel OR.59, Sa.52<br />
Young, Debbie OR.87<br />
Youssef, Sawsan OR.69<br />
Yu, Liping OR.1, F.17<br />
Yue, Erica Su.42<br />
Z, Stephen Su.69<br />
Zabaleta-Lanz, Mercedes Sa.85<br />
Zabel, Brian A. OR.91<br />
Zamir, Ehud F.12<br />
Zang, Yun Sa.28<br />
Zarabi, Mohammad Su.62<br />
S205
S206 FOCIS 2007 Abstract Index by Author<br />
Zarkeshfard, Bahareh F.125<br />
Zeckcer, Israel Su.75<br />
Zehnder, Roland Su.7<br />
Zeier, Martin Su.100<br />
Zeiseniss, Peter Sa.66<br />
Zelazny, Adrian F.66<br />
Zeldin, Robert Sa.12<br />
Zeng, Defu Sa.87<br />
Zhang, Chunyan Sa.87<br />
Zhang, Dong F.3<br />
Zhang, Li F.12<br />
Zhang, Ping Su.136<br />
Zhang, Xiao Sa.66<br />
Zhang, Xiuli F.53<br />
Zhang, Xusheng OR.47, F.109, Sa.54<br />
Zhang, Yiqun F.21<br />
Zhang, Zili OR.84<br />
Zhao, Hong Sa.75<br />
Zhao, Lei Sa.153<br />
Zhao, Xiaoyan Sa.54<br />
Zheng, Chengjie OR.101<br />
Zheng, Song Guo OR.75, Sa.140<br />
Zheng, Xin Xiao F.3<br />
Zheng, Xiufen OR.42, OR.47, 1201<br />
Zheng, Yan OR.37<br />
Zheng, Yanan OR.59<br />
Zhou, Baohua OR.57<br />
Zhou, Bin Sa.134<br />
Zhou, Jing OR.63<br />
Zhou, Liang OR.72<br />
Zhou, Tong OR.47, Sa.75, Sa.84<br />
Zhou, Xiaochuan Sa.142<br />
Zhou, Yixuan Sa.134<br />
Zhu, Chuanlong F.51<br />
Zhu, Xiaoming F.55, OR.57<br />
Zidovetzki, Raphael OR.79<br />
Ziegler, Steven F. OR.72, Sa.13<br />
Zielinska, Ewa F.113<br />
Zollner, Ricardo F.99<br />
Zonneveld-Huijssoon, Evelien Sa.67<br />
Zucchiatti, Andrea F.70<br />
Zuniga, Luis OR.91, OR.69<br />
Zvaifler, Nathan Sa.70<br />
Zvetkova, Elissaveta Borissova Sa.108
<strong>Clinical</strong> <strong>Immunology</strong> (2007) 123, S207–S208<br />
Abstract Index by Category<br />
Diabetes and other autoimmune endocrine diseases<br />
2201, OR.1, OR.3, OR.6, OR.11, OR.14, OR.16, OR.17, OR.18, OR.21, OR.31, OR.49, OR.51, OR.59, OR.61, OR.67, OR.76,<br />
OR.78, OR.82, OR.83, OR.85, F.1, F.2, F.3, F.4, F.5, F.6, F.7, F.8, F.9, F.10, F.11, F.12, F.13, F.14, F.15, F.16, F.17, F.18, F.19,<br />
F.20, F.21, F.22, F.23, F.24, F.25, F.26, F.27, F.28, F.30, F.31, F.32, F.33, F.34<br />
Immunity and infection<br />
1202, 3203, OR.23, OR.30, OR.98, F.38, F.39, F.44, F.45, F.46, F.48, F.49, F.50, F.51, F.52, F.53, F.55, F.56, F.57, F.58, F.61,<br />
F.62, F.63; F.64, F.65, F.66, F.67, F.68, F.69, F.70, F.71, F.72, F.73<br />
Immunodeficiency: primary or acquired<br />
2201, 3201, OR.25, OR.26, OR.27, OR.28, OR.29, OR.32, OR.52, F.74, F.75, F.76, F.77, F.78, F.79, F.80, F.81, F.82, F.83, F.84,<br />
F.85, F.86, F.87, F.88, F.89, F.90, F.91, F.92, F.93, F.94, F.95, F.96, F.97, F.98, F.99, F.100, F.101, F.102, F.103<br />
Laboratory immunology<br />
OR.5, OR.42, F.104, F.105, F.108, F.109, F.110, F.111, F.113, F.114, F.115, F.116, F.120, F.121, F.122, F.124, F.125, F.126, F.127,<br />
F.128, F.129, F.130, F.131, F.132, F.133, F.134, F.135, F.136, F.137<br />
Allergy/Asthma<br />
OR.22, OR.57, OR.64, OR.65, OR.99, Sa.1, Sa.5, Sa.6, Sa.7, Sa.8, Sa.9, Sa.10, Sa.11, Sa.12, Sa.13, Sa.16, Sa.17, Sa.18, Sa.19<br />
Autoimmune rheumatologic diseases<br />
1201, OR.12, OR.44, OR.48, OR.50, OR.55, OR.63, OR.66, OR.75, OR. 80, OR.81, OR.89, OR.92, OR.93, Sa.20, Sa.21, Sa.22,<br />
Sa.23, Sa.24, Sa.25, Sa.26, Sa.27, Sa.28, Sa.29, Sa.30, Sa.31, Sa.32, Sa.33, Sa.35, Sa.36, Sa.38, Sa.40, Sa.41, Sa.42, Sa.43,<br />
Sa.44, Sa.45, Sa.46, Sa.47, Sa.48, Sa.49, Sa.50, Sa.51, Sa.52, Sa.53, Sa.54, Sa.55, Sa.56, Sa.57, Sa.58, Sa.59, Sa.61, Sa.62,<br />
Sa.63, Sa.64, Sa.65, Sa.66, Sa.67, Sa.68, Sa.69, Sa.70, Sa.71, Sa.72, Sa.73, Sa.74, Sa.75, Sa.76, Sa.78, Sa.79, Sa.80, Sa.81,<br />
Sa.82, Sa.84, Sa.85, Sa.86<br />
Bone marrow or stem cell transplantation<br />
OR.53, OR.68, Sa.87, Sa.88, Sa.89, Sa.90, Sa.91, Sa.92, Sa.93, Sa.94, Sa.95<br />
Immuno-oncology<br />
OR.9, OR.15, OR.43, OR.45, OR.47, OR.54, OR.73, OR.101, Sa.96, Sa.97, Sa.98, Sa.100, Sa.101, Sa.102, Sa.103, Sa.104,<br />
Sa.105, Sa.106, Sa.107, Sa.108, Sa.109, Sa.110, Sa.111, Sa.112, Sa.113, Sa.114, Sa.115, Sa.116, Sa.117, Sa.119, Sa.120,<br />
Sa.121, Sa.122, Sa.123, Sa.124, Sa.125, Su.83<br />
General autoimmunity<br />
OR.7, OR.40, OR.46, OR.58, OR.62, OR.72, OR.79, Sa.126, Sa.127, Sa.128, Sa.129, Sa.130, Sa.131, Sa.132, Sa.133, Sa.134,<br />
Sa.135, Sa.136, Sa.137, Sa.138, Sa.139, Sa.140, Sa.141, Sa.142, Sa.143, Sa.145, Sa.146, Sa.147, Sa.148, Sa.149, Sa.150,<br />
Sa.151, Sa.152, Sa.153, Sa.154, Sa.155<br />
doi:10.1016/j.clim.2007.03.542<br />
available at www.sciencedirect.com<br />
www.elsevier.com/locate/yclim
S208 Abstract Index by Category<br />
Autoimmune neurologic diseases<br />
3202, OR.10, OR.13, OR.19, OR.20, OR.24, OR.33, OR.34, OR.36, OR.41, OR.69, OR.77, OR.86, OR.87, OR.88, OR.90, OR.91,<br />
OR.94, OR.95, OR.96, Su. 1, Su. 2, Su. 3, Su. 5, Su. 6, Su. 7, Su. 8, Su. 9, Su. 10, Su.11, Su.12, Su.13, Su.14, Su.15, Su.16,<br />
Su.18, Su.19, Su.20, Su.21, Su.22, Su.23, Su.24, Su.26, Su.27, Su.28, Su.29, Su.31, Su.32, Su.34, Su.35, Su.36, Su.37, Su.38<br />
Cytokines/Chemokines<br />
OR.2, OR.4, OR.8, OR.37, OR.38, OR.84, Su.39, Su.40, Su.41, Su.42, Su.43, Su.44, Su.45, Su.46, Su.47, Su.48, Su.49, Su.50,<br />
Su.51, Su.52, Su.53, Su.54, Su.55, Su.56, Su.57, Su.58, Su.59, Su.60, Su.61, Su.62<br />
Immuno-dermatology<br />
OR.39, Su.63, Su.64, Su.65, Su.66, Su.67, Su.68, Su.69, Su.70, Su.72, Su.74, Su.75<br />
<strong>Immunology</strong> <strong>of</strong> the eye<br />
OR.35, Su.76, Su.78, Su.79, Su.81, Su.82, Su.83<br />
Inflammatory bowel disease<br />
OR.70, OR.100, Su.84, Su.85, Su.86, Su.87, Su.89, Su.90, Su.91, Su.92, Su.93<br />
Organ transplantation<br />
OR.56, OR.71, OR.74, OR.97, Su.94, Su.95, Su.96, Su.98, Su.99, Su.100, Su.101, Su.102, Su.103, Su.104, Su.105, Su.107,<br />
Su.108, Su.109<br />
Other<br />
Su.110, Su.111, Su.112, Su.113, Su.114, Su.116, Su.117, Su.118, Su.119, Su.120, Su.121, Su.122, Su.123, Su.124, Su.125,<br />
Su.126, Su.127, Su.128, Su.129, Su.132, Su.133, Su.134, Su.136, Su.137, Su.138<br />
Reproductive immunology<br />
OR.60, Su.139, Su.140