Oral Presentations - Federation of Clinical Immunology Societies

Clinical Immunology (2007) 123, S3–S128 

ABSTRACTS 

Oral Presentations: Friday, June 8 

#1201- Therapies to Induce Tolerance 

Prevention and Treatment of Autoimmune Arthritis 

Using IL-12 Gene-Silenced Dendritic Cells 

Friday, June 8 

10:45 am−11:05 am 

Wei-Ping Min, Scientist, University of Western Ontario, 

London, ON, Canada, Xiufen Zheng, Fellow, University of 

Western Ontario, London, ON, Canada, Igor Popov, Fellow, 

University of Western Ontario, London, ON, Canada, Xusheng 

Zhang, Fellow, University of Western Ontario, London, ON, 

Canada, Mu Li, Fellow, University of Western Ontario, 

London, ON, Canada, Hongtao Sun, Fellow, University of 

Western Ontario, London, ON, Canada, Motohiko Suzuki, 

Fellow, University of Western Ontario, London, ON, Canada, 

Costin Vladau, Student, University of Western Ontario, 

London, ON, Canada, Bertha Garcia, Professor, University of 

Western Ontario, London, ON, Canada, Robert Inman, 

Professor, Toronto Western Hospital, Toronto, ON, Canada 

We have recently demonstrated that dendritic cell(DC)mediated 

immune modulation and deviation can be accomplished 

through RNA interference (RNAi), highlighting the 

therapeutic potential of RNAi-modified DC as antigenspecific 

tolerogenic vaccines. To date, an RNAi-based 

immune therapy for autoimmune diseases has not been 

reported. The current study was designed to develop siRNAmodified 

DC as antigen-specific, tolerogenic vaccines for 

prevention and intervention of autoimmune arthritis. Using 

small interfering RNA (siRNA) that specifically targets IL- 

12p35 gene (IL-12 siRNA), we have generated a type of DC 

that exhibits multiple tolerogenic characteristics. After 

inducation of RNAi in DC using siRNA, the expression of IL- 

12 was inhibited as determined by real time PCR and flow 

cytometry. IL-12 silenced DC suppressed Tcell response in an 

MLR, and impaired Th1 differentiation in vitro. Immunization 

with IL-12 gene-silenced and type II collagen (CII)-pulsed DC 

(CII-pulsed/gene-silenced DC) resulted in antigen-specific 

non-responsiveness. Vaccination with CII-pulsed/genesilenced 

DC prevented collagen-induced arthritis (CIA) 

onset in a murine rheumatoid arthritis model. Furthermore, 

administration of CII-pulsed/gene-silenced DC was sufficient 

doi:10.1016/j.clim.2007.03.179 

available at www.sciencedirect.com 

www.elsevier.com/locate/yclim 

to inhibit progression of CIA. The therapeutic effects were 

further evidenced by decreased clinical score in CIA, 

inhibited inflammatory infiltrates in joints, and suppressed 

T cell and B cell responses to CII. In conclusion, this study is 

the first to demonstrate the therapeutic utilization of RNAimodified 

DC as antigen-specific tolerogenic vaccines for 

autoimmune arthritis. 

doi:10.1016/j.clim.2007.03.180 

#1202- Commensal Flora and the Immune Response 

Helicobacter Pylori in the Pathogenesis of Chronic 

Autoimmune Pancreatitis 


10:45 am−11:05 am 

Antonio Puccetti, Professor of Medicine, Institute G. Gaslini, 

Genova, Italy, Claudio Lunardi, Professor of Medicine, 

University of Verona-Department of Internal Medicine, 

Verona, Italy, Luca Frullloni, Professor of Gastroenterology, 

Department of Gastroenterology, University of Verona, 

Verona, Italy, Caterina Bason, Research Fellow, University of 

Verona-Department of Internal Medicine, Verona, Italy, 

Dimitri Peterlana, PhD Student, University of Verona- 

Department of Internal Medicine, Verona, Italy 

Autoimmune chronic pancreatitis (ACP) is a type of pancreatitis 

characterized by a chronic inflammatory process 

with prominent lymphocyte infiltration leading to fibrosis of 

the pancreas and eventually to organ dysfunction. The cause 

of the disease is still unknown. Current evidence suggests an 

autoimmune origin for ACP, based on its frequent association 

with other autoimmune diseases (rheumatoid arthritis, 

Sjogren’s syndrome, and inflammatory bowel disease) and 

on the presence of immunological abnormalities, including 

hypergammaglobulinemia, elevated serum IgG4 levels and the 

presence of autoantibodies, such as antibodies against 

carbonic anhydrase II. However, little is known about its 

actual pathogenesis. In order to clarify the pathogenesis of the 

disease, we screened a random peptide library with pooled 

IgG immunoglobulins derived from 20 patients with ACP.

S4 Abstracts 

Among the identified peptides, one was recognized by the 

majority of patients' sera, but not by sera of normal donors and 

of patients with other autoimmune diseases. The peptide 

showed homology with an Helicobacter pylori derived protein 

and with carbonic anhydrase 5B, a protein highly expressed in 

pancreas, kidney and salivary glands, and with lactoferrin, 

considered an important autoantigen target in ACP. Antipeptide 

antibodies, affinity purified from patients' sera 

recognize the helicobacter derived protein, carbonic anhydrase 

5B and lactoferrin. Moreover antibodies against the 

bacterial epitope can be detected in the vast majority of 

patients with ACP. Our findings suggest that Helicobacter 

pylori infection may be linked to the pathogenesis of ACP 

through a molecular mimicry mechanism and that carbonic 

anhydrase 5B can be considered a novel autoantigen in ACP. 

doi:10.1016/j.clim.2007.03.181 

OR.1 Foxp3 (Scurfy) Mutation Greatly Accelerates 

Diabetes in Insulin-specific TCR Transgenic Mice 

Jean Jasinski, University of Colorado Health Sciences Center, 

Aurora, CO, Maki Nakayama, Fellow, University of Colorado 

Health Sciences Center, Barbara Davis Center, Aurora, CO, 

Kelly Johnson, Professional Research Assistant, University of 

Colorado Health Sciences Center, Barbara Davis Center, 

Aurora, CO, George Eisenbarth, Executive Director, Barbara 

Davis Center, Aurora, CO, Liping Yu, Senior Research 

Assistant, University of Colorado Health Sciences Center, 

Barbara Davis Center, Aurora, CO 

Previously we developed and described the development 

of a transgenic mouse expressing an insulin-specific (ins2 

B:9–23) T cell receptor mouse model (BDC12-4.1). This 

pathogenic model exhibited spontaneous diabetes development 

on a backcross-1 (FVB×NOD)×NOD background. Disease 

development was heterogeneous and dependent on both I- 

Ag7 and RAG-deficient genotypes with relatively slow 

development of diabetes such that the median age of onset 

of diabetes was 49 weeks, and less than 10% of mice 

developed diabetes prior to 10 weeks of age. To test the 

hypothesis that the 12-4.1 transgenic T cells, even on a 

RAG−/− background, were heterogeneous with development of 

both regulatory and pathogenic T cells, we crossed NOD 

congenic BDC12-4.1 mice with C57BL/6 Foxp3 mutant mice. 

Expression of the single BDC12-4.1 TCR on a RAG-deficient 

background rescues the mouse from the scurfy phenotype at 

the BC1 (C57BL/6×NOD)×NOD generation. All RAG-deficient 

Foxp3 mutant mice (8/8) expressing the BDC12-4.1 TCR develop 

diabetes between 5 and 10 weeks of age with aggressive 

destruction of essentially all beta cells whereas prediabetic 

mice exhibit invasive insulitis. Disease develops equally in 

homozygous (I-Ag7/g7) and heterozygous (I-Ag7/b) mice 

significantly earlier and with greater uniformity than previously 

described (Pb0.0001). Disease can be adoptively transferred 

from diabetic animals to NOD.scid mice. These studies provide 

evidence that a single transgenic TCR generates both pathogenic 

and Foxp3-dependent regulatory T cells and provides an 

experimental model to test therapies of diabetes prevention 

without Foxp3 dependent regulatory T cells. 

doi:10.1016/j.clim.2007.03.182 

OR.2 Regulatory T Cells Require Serum for 

Suppression of Effector T Cell Proliferation and 

Express Stable Membrane-bound CD25 

Todd Brusko, Postdoctoral Associate, University of Florida, 

College of Medicine, Gainesville, FL, Clive Wasserfall, 

Biological Scientist, University of Florida, College of 

Medicine, Department of Pathology, Gainesville, FL, Kieran 

McGrail, Laboratory Technician, University of Florida, 

College of Medicine, Department of Pathology, Gainesville, 

FL, Marcus Moore, Laboratory Technician, University of 

Florida, College of Medicine, Department of Pathology, 

Gainesville, FL, Alyssa Huegel, Laboratory Technician, 

University of Florida, College of Medicine, Department of 

Pathology, Gainesville, FL, Desmond Schatz, Professor of 

Pediatric Medicine, University of Florida, College of 

Medicine, Department of Pediatrics, Gainesville, FL, Mark 

Atkinson, Professor, University of Florida, College of 

Medicine, Department of Pathology, Gainesville, FL 

CD4+CD25+ regulatory T cells (Treg) suppress effector T 

cell (Teff) functions and are essential for maintaining 

peripheral tolerance. Recent work suggests that IL-2 signaling 

is crucial for proper Treg development and function. In this 

study, we assessed the production of the soluble form of CD25 

(sCD25) in human Treg or Teff cells, alone and in combination, 

following polyclonal activation during an in vitro suppression 

assay. We found that surface CD25 is relatively stable on Treg 

in vitro, whereas CD25 expression on isolated Teff cells is 

upregulated following activation; subsequently resulting in 

high levels of sCD25 detected in culture supernatants (mean + 

SD, 682.2+367.1 vs. 5369.5+ 2575.6 pg/ml for Treg vs. Teff 

cells respectively; N=7, P=0.004). The production of sCD25 

can occur in an autocrine fashion and correlates with T cell 

proliferation (r=0.76, Pb0.0001). To eliminate the influence 

of serum sCD25, our studies were also conducted under 

serum-free conditions. Surprisingly, under these conditions, 

Treg fail to suppress Teff cell proliferation (% suppression at a 

1:1 Treg to Teff ratio; −197.8+ 270.6 for serum-free medium 

vs. 70.4+17.3 with 1.0% serum; P=0.04). Despite this 

functional defect, Treg cells still maintained their capacity 

to inhibit Teff cell cytokine production (e.g., IFN-γ responses 

were suppressed 75.6% in co-culture vs. Teff alone, Pb0.05). 

These data highlight a mechanism by which Treg may obtain 

qualitatively greater IL-2 signals than Teff cells and suggest 

that serum is required for full Treg activity. 

doi:10.1016/j.clim.2007.03.183 

OR.3 TGF-β-induced TCR-Tg/NOD Regulatory T Cells 

Suppress Both Diabetes Transfer and Islet Graft 

Destruction in an Antigen-dependent Manner 

Daniel Tonkin, Graduate Student, University of Colorado 

Health Sciences Center, Department of Immunology, Denver, 

CO, Brenda Bradley, Senior Professional Research Assistant, 

University of Colorado Health Sciences Center, Department 

of Immunology, Denver, CO, Gene Barbour, Professional 

Research Assistant, University of Colorado Health Sciences 

Center, Department of Immunology, Denver, CO, Kathryn 

Haskins, Professor, University of Colorado Health Sciences 

Center, Department of Immunology, Denver, CO

Abstracts 

Regulatory T cells (Tregs) are understood to fall into 

two categories: natural Tregs and induced Tregs. Induced 

Tregs can be produced by activating CD4 T cells in the 

presence of TGF- 2 . Such induced Tregs have great promise 

for immunotherapy, but it has been unclear if they 

require stimulation by antigen in order to suppress in 

vivo. We have investigated induced Treg protection in the 

NOD mouse model of autoimmune diabetes. We produced 

TGF- 2 -induced Tregs from the 6.9 TCR-Tg/NOD mouse that 

are specific for islet autoantigen. These Tregs express 

Foxp3 and produce little IFN- 3 . We have shown that 

induced Tregs can prevent transfer of diabetes by effector 

T cells in NOD mice, where they decrease inflammatory 

cytokine production and macrophage infiltration in the 

pancreas. In some experiments we transferred cells into a 

congenic mouse, the NOD.C6, that specifically lacks the 

antigen for the 6.9 TCR-Tg T cells but not for other isletspecific 

T cells. The 6.9 TCR-Tg Tregs fail to prevent 

diabetes transfer in the NOD.C6 mice, demonstrating that 

their function is antigen dependent. In human diabetes, 

induced Tregs might protect islet grafts from immune 

destruction. We grafted NOD.scid islets into NOD.scid.C6 

recipients after streptozotocin treatment, and demonstrated 

that induced Tregs can prevent islet graft 

destruction by effector T cells. When we grafted NOD. 

scid.C6 islets, the 6.9 TCR-Tg islets failed to protect. 

With these antigen-free islets, we have demonstrated that 

induced Tregs require activation by their antigen in the 

islet graft in order to protect the graft from autoimmune 

destruction. 

doi:10.1016/j.clim.2007.03.184 

OR.4 Leptin Modulates Treg Cell Activity in Human 

SLE 

Antonio La Cava, Associate Professor, University of 

California, Los Angeles, Los Angeles, CA, Giuseppe 

Matarese, Assistant Researcher, University of Naples, 

Naples, Bevra Hahn, Professor and Chief of Rheumatology, 

University of California, Los Angeles, Los Angeles, CA 

Leptin is a cytokine/hormone that links nutritional 

status with neuroendocrine and immune functions. We 

and others have recently shown that leptin can contribute 

to the onset and progression of organ-specific autoimmunity. 

Here, we measured serum leptin in systemic lupus 

erythematosus (SLE) patients (n=66) and healthy controls 

(n=50) matched for gender, age and ethnicity. Parallel flow 

cytometry studies correlated the numbers of CD4+ 

CD25highFoxp3+ T (Treg) cells with serum leptin in the 

SLE patients vs. controls. Serum leptin concentration was 

significantly higher in SLE patients than in controls 

(Pb0.0001) and did not relate to body mass index and/or 

therapy. Interestingly, a reduced number of CD4+ 

CD25highFoxp3+ T cells significantly correlated with the 

elevated serum leptin in SLE patients (n=9, P=0.02, r 2 = 

0.51) but not in controls (n=10, P=0.3, r 2 =0.2). Because 

of this inverse correlation between CD4+CD25highFoxp3+ T 

cells and serum leptin levels, we tested whether leptin 

could influence the number and/or activity of human Treg 

cells. We found that neutralization of leptin with antileptin 

Ab reversed the hyporesponsiveness of human Tregs 

in a dose-dependent fashion and Foxp3-expressing Treg 

cells expressed elevated levels of leptin receptor. Additionally, 

anti-leptin Ab promoted both the upregulation of 

Foxp3 and the suppressive in vitro activity of the Tregs 

cells on effector CD4+CD25− T cells. These data suggest 

that leptin can influence the number and activity of Treg 

cells and may have implication for leptin-based intervention 

on Treg cells in SLE. 

doi:10.1016/j.clim.2007.03.185 

OR.5 Gene Related to Anergy in Lymphocytes (GRAIL) 

Expression in CD4+ CD25+ T Regulatory Cells and 

Correlation with T:APC Conjugate Formation 

Jill Schartner, Graduate Student, University of Wisconsin, 

Department of Pediatrics, Madison, WI, Amanda Timmel, 

Technician, University of Wisconsin, Department of 

Pediatrics, Madison, WI, William Simonson, Graduate 

Student, University of Wisconsin, Madison, WI, Christine 

Seroogy, Assistant Professor, University of Wisconsin, 

Department of Pediatrics, Madison, WI, Anna Huttenlocher, 

Associate Professor, University of Wisconsin, Department of 

Pediatrics, Madison, WI 

It has been reported that CD4+CD25+ regulatory T cells 

(CD25+ Tregs) have an anergic phenotype and disrupted actin 

cap formation in vitro. Recent reports suggest that strong 

activation can abrogate the suppressor activity of CD25+ 

Tregs. We have found that the anergy-related E3 ubiquitin 

ligase, GRAIL is differentially expressed in CD25+ Tregs. We 

sought to correlate the expression profile of an anergyrelated 

gene, GRAIL, with immunologic synapse formation 

and suppressor activity in CD25+ Tregs. GRAIL mRNA was 

quantitated by real-time QPCR in CD25+ Tregs activated with 

anti-CD3/CD28 or anti-CD3 conjugated beads. DO11.10 CD4+ 

Tcell lines were transduced with GRAIL-IRES-GFP or GFP-only 

expressing retrovirus. The ability of these cells to form 

conjugates with OVA323–339 loaded antigen presenting cells 

(APCs) was evaluated via FACS. Microscopy was used to 

evaluate localization of LFA-1, actin, and GRAIL during 

conjugate formation. Strong activation of CD25+ Tregs with 

anti-CD3/CD28 beads resulted in a 10-fold reduction in GRAIL 

mRNA. In contrast, activation of CD25+ Tregs with anti-CD3 

beads resulted in stable expression of GRAIL mRNA. Forced 

expression of GRAIL in a DO11.10 T cells sufficiently converts 

these cells to a suppressor phenotype and resulted in a 2-fold 

reduction in their ability to form conjugates with OVA323– 

339 loaded APCs. Following conjugation with anti-CD3coated 

beads GRAIL expressing T cells exhibited impaired 

LFA-1 localization and disrupted actin cap formation. These 

data suggest that expression of GRAIL in CD25+ Tregs is 

differentially regulated and dependent on the activation 

context and may correlate with suppressor function. 

Furthermore, ectopic expression of GRAIL correlates with a 

suppressor phenotype and disrupted synapse formation. 

doi:10.1016/j.clim.2007.03.186 

S5

S6 Abstracts 

OR.6 Islet Antigen Specific Regulatory T Cells 

Isolated From Non-Diabetic Individuals Suppress 

Pro-Inflammatory Responses via Cytotoxic Mechanisms 

Timothy Tree, Lecturer, Department of Immunobiology, 

King’s College London, London, Jenifer Lawson, Research 

Assistant, Department of Immunobiology, King’s College 

London, London, Bart Roep, Professor, Department of 

Immunohaematology and Blood Transfusion, Leiden 

University Medical Center, Leiden, Hannah Edwards, 

Research Technician, Department of Immunobiology, 

King’s College London, London, England, Mark Peakman, 

Professor of Clinical Immunology, Department of 

Immunobiology, King’s College London, London, 

England 

Autoreactive T cells recognizing islet ? cell antigens 

could represent both pathogenic effectors in the development 

of type 1 diabetes (T1DM) and mediators of 

tolerance in non-diabetic individuals depending on the 

quality of response they produce. We have previously 

demonstrated that, whereas autoreactive T cells in 

patients with T1DM exhibit polarization towards a proinflammatory 

T helper 1 (Th1) phenotype, the majority of 

non-diabetic subjects also manifest a response against 

islet peptides, but one that shows T regulatory cell (Treg), 

IL-10 secreting, bias. We therefore proposed that isletspecific 

IL-10 producing T cells may be involved in the 

maintenance of tolerance to islets by the suppression of 

proinflammatory Th1 cells. To test this hypothosis, we 

isolated T cell clones from healthy individuals that secrete 

high levels of IL-10 in response to islet antigens directly 

ex vivo. These CD4+CD25+Foxp3+ Tregs are potent 

suppressors of proinflammatory Th1 effector cells. Suppression 

is not mediated by soluble factors but is cellcontact 

dependent, and dependent upon presentation of 

stimulating antigens for both the Treg and effector Th1 

cells by the same antigen presenting cell (APC), thus 

providing exquisite specificity of suppression. These Tregs 

express the cytotoxic molecules perforin and granzymes A 

and B directly ex vivo and mediate their suppression via 

the specific lysis of APC presenting high levels of islet 

antigens. This hitherto un-described population of antigen 

specific Tregs, which appear to be constitutively present 

in the majority of non-diabetic individuals, may provide a 

suitable target to strengthen tolerance to islets by clinical 

immunointervention. 

doi:10.1016/j.clim.2007.03.187 

OR.7 Rapamycin Promotes Induction of CD4+Foxp3+ 

T Cells that Proliferate in Response to Common 

Gamma Chain Cytokines 

S. Alice Long, Scientist, Benaroya Research Institute, 

Seattle, WA, Megan Van Landeghen, RA II, Benaroya 

Research Institute, Seattle, WA, Jane Buckner, Professor, 

Benaroya Research Institute, Seattle, WA 

We and others have shown that CD4+CD25+Foxp3+ T 

cells can be induced from CD4+CD25−Foxp3− T cells 

(iTReg). Using intracellular staining for Foxp3, we consistently 

observe that the CD4+CD25+ population that arises 

upon activation of CD4+CD25− T cells with polyclonal or 

antigenic stimulation is composed of both Foxp3− and Foxp3+ 

T cells. Thus, determining factors that influence the 

frequency of iTReg may lead to development of targeted 

therapies. IL-2 is a known growth factor for natural Treg. 

Others have observed that treatment of patients with 

Rapamycin leads to an increased number of CD4+CD25+ 

Foxp3+ Tcells. Here, we demonstrate that in vitro activation 

of CD4+CD25− T cells in the presence of IL-2 or Rapamycin 

results in generation of functional iTReg. However, combination 

of both Rapamycin and IL-2 results in a significantly 

higher frequency and absolute number of iTReg. Analysis of 

Foxp3 over time demonstrates that up-regulation of Foxp3 

occurs prior to cell division and increases during the course 

of cell division. Foxp3− and Foxp3+ T cells proliferate in 

parallel suggesting that Rapamycin is not significantly 

inhibiting the proliferation of Foxp3− T cells, but instead, 

is preferentially inducing Foxp3 expression in a subset of 

CD25− T cells. The addition of IL-2, but also IL-4, IL-7, and 

IL-15, facilitates proliferation of iTReg. Thus, we demonstrate 

that Rapamycin plus IL-2 and/or common gamma 

chain cytokines promotes induction and proliferation of 

Foxp3+ T cells. These studies provide rationale for using 

Rapamycin in combination with IL-2 to enhance expansion 

of regulatory T cells in vivo. 

doi:10.1016/j.clim.2007.03.188 

OR.8 IL-2Rβ Links IL-2R Signaling with Foxp3 

Expression 

David Soper, Graduate Student, University of Washington, 

Department of Immunology, Seattle, WA 

Immunological tolerance to self-antigens is a tightly 

regulated process. Recent work has demonstrated that 

the forkhead family member Foxp3 is a critical element in 

the differentiation and function of mouse CD4+CD25+ 

regulatory T (TR) cells. Recent work has suggested an 

important role for IL-2 in the development and maintenance 

of TR cells. To directly assess the effect of IL-2 

signaling on TR development and function, we analyzed 

mice that were genetically deficient in components of the 

IL-2 receptor (IL-2R). Mice lacking CD25 (IL-2Rα) displayed 

a slight decrease in TR cells within the thymus, while 

peripheral numbers are unchanged. In contrast, we found 

that mice deficient in CD122 (IL-2Rβ) had a profound 

reduction in both thymic and peripheral TR cells, 

coinciding with more rapid development of a fatal 

lymphoproliferative disease. Expression of a Foxp3 transgene 

restored TR cells and protected against the onset of 

autoimmunity. Thus, a signal mediated by IL-2Rβ is essential 

for the development and homeostasis of Foxp3+ TR cells 

in vivo. 

doi:10.1016/j.clim.2007.03.189

Abstracts 

OR.9 High Affinity T Cell Receptors as Therapeutic 

Agents 

Rebecca Ashfield, Senior Research Project Manager, Avidex 

Ltd, Abingdon, Oxfordshire, England, Deborah Sutton, 

Group Leader, Cell Biology, Avidex Ltd, Abingdon, 

Oxfordshire, England, Giovanna Bossi, Senior Scientist, 

Avidex Ltd, Abingdon, Oxfordshire, England, Joanna Brewer, 

Group Leader, Cell biology, Avidex Ltd, Abingdon, 

Oxfordshire, England, Marco Purbhoo, Research Associate, 

Division of Molecular and Cell Biology, London, Rebecca 

Dennis, Senior Scientist, Avidex Ltd, Abingdon, Oxfordshire, 

England, Julia Karbach, Researcher, Ludwig Institute, 

Frankfurt, Germany, Tara Mahon, Senior Scientist, Avidex 

Ltd, Abingdon, Oxfordshire, England, Brian Cameron, Senior 

Scientist, Avidex Ltd, Abingdon, Oxfordshire, England, Bent 

Jakobsen, Senior Vice President, Research, Avidex Ltd, 

Abingdon, Oxfordshire, England 

T cell receptors (TCRs) recognise peptides derived from 

disease related antigens, many of them intracellular in origin, 

which are presented on the cell surface by HLA molecules. The 

development of TCRs as therapeutic agents has until recently 

been impeded by their low natural affinity, and the difficulty of 

expressing membrane-bound receptors as soluble molecules. 

We have engineered TCRs to produce stable, soluble proteins 

that can be made in large quantities by expression in E. coli. 

These monoclonal TCRs (mTCRs) can be affinity matured by 

phage display technology to sub-nM affinity, and can be fused to 

toxins or immuno-modulatory effector functions to produce 

therapeutic proteins suitable for targeting tumours, virally 

infected cells or tissues under autoimmune attack. Using this 

technology, high affinity mTCRs have been produced which 

recognise HLA-A2 specific epitopes from HIVgag (amino acids 

77–85) and two tumour associated antigens, NY-ESO (amino 

acids 157–165) and Melan-A (amino acids 26–35). Each of these 

mTCRs has sub-nM affinity and a binding half life of several 

hours, whilst retaining specificity for the target HLA–epitope 

complex. Naturally processed epitopes on the surface of antigen 

presenting cells are targeted by the high affinity mTCRs, and 

can be detected using flow cytometry and fluorescence 

microscopy. We present data comparing the antigen density of 

the two tumour associated epitopes, NY-ESO157–165 and 

Melan-A26–35, on the surface of melanoma cell lines. Furthermore, 

we show that the NY-ESO specific mTCR binds specifically 

to freshly isolated NY-ESO+, HLA-A2+ myeloma cells. 

doi:10.1016/j.clim.2007.03.190 

Regulatory T Cells I 


2:45 pm−4:45 pm 

OR.10 From EAE to an MS Clinical Trial: 

DR2/MOG-35−55 Recombinant T Cell Receptor 

Ligand (RTL) Treats Relapse of Experimental 

Encephalomyelitis in DR2 Transgenic Mice 

Halina Offner, Professor, Oregon Health and Science 

University, Neurology, Portland, OR, Gregory Burrows, 

Research Associate Professor, Oregon Health and Science 

University, Neurology, Portland, OR, Arthur Vandenbark, 

Professor, Portland VA Medical Center, Neuroimmunology 

Research, Portland, OR, Jason Link, Associate Investigator, 

Portland VA Medical Center, Neuroimmunology Research, 

Portland, OR 

To evaluate the ability of a newly designed monomeric 

recombinant TCR ligand, RTL342M, containing HLA-DR2 

peptide-binding domains covalently linked to MOG-35–55 

peptide, to prevent and treat both the initial episode and 

subsequent relapses of experimental autoimmune encephalomyelitis 

(EAE) in HLA-DR2 transgenic mice, in preparation 

for a safety trial in patients with multiple sclerosis (MS), 

single or multiple doses of RTL342M were given i.v. or s.c. to 

HLA-DR2 mice prior to, at onset, or during relapses of 

paralytic EAE. RTL342M produced rapid (within 24 h), dosedependent 

reversal of clinical signs of paralytic EAE; even a 

single dose could produce significant treatment effect. 

Multiple daily doses were even more effective than the 

same total amount of RTL given as a single dose. By 

establishing the minimal effective dose, RTLs may be 50 

times more potent than molar equivalent doses of myelin 

peptide alone. RTL342M given prior to induction of EAE 

prevented disease in most mice, and the remainder could be 

successfully re-treated with RTL. Most important for clinical 

application, RTL342M was highly effective for treating EAE 

relapses when given periodically prior to the relapse or even 

after relapses had occurred. These data demonstrate rapid 

and potent clinical effects of RTL342M at disease onset and 

during relapses in EAE, establishing important principles 

governing application of this novel, highly selective therapeutic 

approach for regulating pathogenic Tcells. Data from 

this study provide key preclinical information for a phase I 

safety study in patients with MS (outline to be presented) that 

is now in progress. 

doi:10.1016/j.clim.2007.03.191 

OR.11 Immunological Efficacy of hsp60 Peptide 

DiaPep277 Therapy in Human Type 1 Diabetes 

Bart Roep, Immunologist, Leiden University Medical Center, 

Leiden 

An immunogenic peptide (p277) from the 60-kDa heat-shock 

protein (hsp60) arrested beta-cell destruction in non-obese 

diabetic (NOD) mice. A randomised, double blind, phase Ib/II 

clinical trial of DiaPep277 peptide treatment was performed in 

recent onset type 1 diabetes patients with remaining insulin 

production. We studied the immunological efficacy of this 

peptide therapy and correlated this with clinical outcome. 

Forty-eight C-peptide positive patients were assigned subcutaneous 

injections of 0.2, 1.0 or 2.5 mg p277 (n=12perdosage)at 

entry, and 1, 6 and 12 months, or four placebo injections (n=12). 

T cell autoimmunity to hsp60, DiaPep277, GAD and tetanus 

toxoid (recall response control) were assayed by proliferation 

and cytokine secretion assays (ELIspot) on regular intervals until 

18 months after first injection. All treated patients at each 

dosage of peptide demonstrated an altered immune response to 

DiaPep277 while the majority of placebo-treated patient 

remained non-responsive to treatment (p=0.00001), indicating 

a 100% efficacy of immunization. Cytokine production in 

S7

S8 Abstracts 

response to therapy was dominated by IL-10, but this profile did 

not correlate with better preserved beta-cell function. Instead, 

patients developing immunological tolerance to Diapep277 

showed less decreased C-peptide production than patients 

that kept a reactive phenotype. Third party control immune 

responses were unaffected by therapy. No potentially adverse 

immunological side effects were noted. 

DiaPep277 is immunogenic in type 1 diabetic subjects and 

has immune modulating properties. Immunological monitoring 

allowed to distinguish therapy from placebo treatment 

and could determine immunological efficacy. Tolerance to 

peptide DiaPep277 may serve as immunological biomarker 

measure for clinical efficacy. 

doi:10.1016/j.clim.2007.03.192 

OR.12 Somatic B-Cell Gene Vaccination with 

Anti-DNA Ig Consensus Peptide Protects (NZB × NZW) 

F1 Lupus Mice From Renal Disease 

Francesca Ferrera, Postdoctoral Fellow, University of 

Genova, Genova, Bevra Hahn, Professor and Chief of 

Rheumatology, University of California, Los Angeles, Los 

Angeles, CA, Marta Rizzi, Postdoctoral Fellow, University 

of Genova, Genova, Gilberto Filaci, Associate Professor, 

University of Genova, Genova, Francesco Indiveri, 

Professor, University of Genova, Genova, Antonio La Cava, 

Associate Professor, University of California, Los Angeles, 

Los Angeles, CA, Antonio La Cava, Associate Professor 

of Medicine, University of California, Los Angeles, 

Los Angeles, CA 

Ig molecules that participate in humoral immune responses 

contain epitopes that induce T cell-mediated immune 

responses. B cells process and present such epitopes and 

activate T cells. We hypothesized that T cells recognizing an Ig 

consensus sequence presented by B cells will modulate lupuslike 

disease in mice. To test this hypothesis, NZB/W F1 lupus 

mice received B cell somatic gene transfer of DNA plasmid 

encoding an artificial consensus sequence mimicking T cell 

determinants of murine anti-DNA IgG, or control plasmids. 

Treated animals were monitored for production of antibody, 

development of renal disease and phenotype, number, and 

function of consensus gene-induced Tcells. It was found that the 

treatment of mice with Ig consensus gene induced TGFβproducing 

CD8+CD28− T cells that: (1) suppressed antigenspecific 

stimulation of CD4+ Tcells in a cell-contact independent 

manner; (2) reduced autoantibody production; (3) retarded the 

development of nephritis, and (4) improved survival of treated 

animals. Significantly, the adoptive transfer of CD8+CD28− T 

cells from Ig consensus gene-protected mice into hypergammaglobulinemic 

NZB/W F1 mice effectively protected the transferred 

mice from development of renal disease. We conclude 

that genetic expression of anti-DNA Ig consensus can induce 

immunoregulatory circuits capable of delaying development of 

lupus nephritis via the suppression of hypergammaglobulinemia 

and renal disease. 

doi:10.1016/j.clim.2007.03.193 

OR.13 Therapeutic Vaccination with a Trivalent 

T Cell Receptor Peptide Vaccine Induces 

Peptide-specific IL-10-secreting T Cells, Restores 

Deficient Foxp3 Expression, and Induces Bystander 

Immunomodulation in Multiple Sclerosis Subjects 

Arthur Vandenbark, Professor, Oregon Health and Science 

University, Neurology, Portland, OR, Richard Bartholomew, 

Executive Director of Research and Development, The 

Immune Response Corporation, Carlsbad, CA, Halina 

Offner, Professor, Oregon Health and Science University, 

Neurology, Portland, OR, Dennis Bourdette, Professor and 

Chairman, Oregon Health and Science University, 

Neurology, Portland, OR 

We are developing a therapeutic vaccine containing 

CDR2 peptides from BV5S2, BV6S5, and BV13S1 emulsified 

in IFA as a V gene-specific therapy for RR-MS. This TCR 

vaccine is currently being tested in a blinded, placebocontrolled, 

200 patient study with reduction of new 

gadolinium enhancing lesions as the primary endpoint. We 

here report new mechanistic data from a recently 

completed open-label study. Immunological and clinical 

parameters were followed in 27 treatment naïve and 9 

previously vaccinated subjects with MS who received 

monthly injections of the trivalent peptide vaccine over 

1 year. TCR vaccination induced high frequencies of IL-10secreting 

T cells specific for the injected TCR peptides as 

well as an apparently broader pattern of anti-TCR 

specificities. These regulatory specificities induced biphasic 

immunomodulation that involved an initial rise 

after 9 weeks of IL-10-secreting neuroantigen-specific T 

cells, in conjunction with potent induction of Foxp3+ Treg 

cells to normal levels after 12 weeks of treatment. These 

findings are consistent with vaccine-induced stimulation of 

Foxp3+ Treg cells originating from both CD25(−) and 

conventional CD25(+) T cells. Restoration of previously 

deficient Treg cells could explain the broad reduction of 

TCR-dependent responses of both cognate T cells expressing 

V genes targeted by the vaccine as well as bystander T 

cells expressing different V genes, including those specific 

for neuroantigens. These findings demonstrate that therapeutic 

vaccination using only three commonly expressed 

BV gene determinants can induce a broad immunoregulatory 

network in vivo that may help control autoreactive 

responses that characterize the inflammatory phase of MS. 

doi:10.1016/j.clim.2007.03.194 

OR.14 Mechanistic Insights Into the Differential 

Efficacy of GAD65 DNA Vaccine and Anti-CD3 

Combination Therapy in Treating New-Onset 

Autoimmune Diabetes in RIP-LCMV and NOD Mice 

Damien Bresson, Postdoctoral Fellow, La Jolla Institute for 

Allergy and Immunology, La Jolla, CA, Diane Rottembourg, 

Postdoctoral Fellow, La Jolla Institute for Allergy and 

Immunology, La Jolla, CA, Lisa Togher, Technician, La Jolla 

Institute for Allergy and Immunology, La Jolla, CA, Matthias 

von Herrath, Professor, La Jolla Institute for Allergy and 

Immunology, La Jolla, CA

Abstracts 

Type 1 diabetes (T1D) results from a progressive 

destruction of insulin-producing pancreatic beta cells by 

autoaggressive T cells. In this study, we evaluated the 

efficacy of a combination therapy (CT) (non-Fc binding 

anti-CD3 and human GAD65-expressing DNA vaccine) to 

reverse new-onset T1D in two animal models (NOD and 

RIP-LCMV mice). CT enhanced efficacy only in the RIP- 

LCMV model (40% improvement compared to anti-CD3 or 

GAD65 vaccine given alone). We evaluated three factors 

that could account for such a discrepancy: (i) amount of 

GAD65 protein within the islets of Langerhans, (ii) level 

and functionality of toll-like receptor-9 (TLR9) expressed 

by immune cells and (iii) epitopes recognized by GAD65specific 

Tregs expanded after CT vs. anti-CD3 alone. 

Although similar levels of pancreatic GAD65 levels were 

found in both animal models, TLR9 expression was slighlty 

enhanced in antigen-presenting cells derived from RIP- 

LCMV vs. NOD mice. By using a peptide library the GAD65specific 

Tregs epitopes were mapped in the C-terminal 

domain of the protein after CT in the RIP-LCMV but not 

NOD mice, thus emphasizing the importance of genetic 

background in the therapeutic efficacy of antigen-specific 

immuno-interventions. Furthermore, these CD4+CD25+ 

Foxp3+GITR+ Tregs also expressed the co-stimulatory 

molecule OX40 and protected 50% of mice when transferred 

in vivo into immunocompetent recipients. The 

involvement of OX40 in this bystander suppression mechanism 

will be studied by antibody blockade and siRNA 

technology. 

This work was supported by a NIH DK51091, AI44451 and a 

U-19 prevention center grant to MVH. DB is supported by a 

European Marie-Curie Outgoing Fellowship. 

doi:10.1016/j.clim.2007.03.195 

OR.15 Mimotopes for Active Immunotherapy of 

Tumors: Allergooncology 

Erika Jensen-Jarolim, Professor, Medical University Vienna, 

Vienna, Angelika B. Riemer, Medicine University Vienna, 

Vienna, Regina Knittelfelder, MSc, Medicine University Vienna, 

Vienna, Panos Karagiannis, Student, Med University of Vienna, 

Vienna, Josef Singer, Student, Medicine University Vienna, 

Vienna, Eva Untersmayr, Medical University Vienna, Vienna, 

Kira Braemswig, Medicine University of Vienna, Vienna 

Antibodies recognize 3D-epitopes and have the great 

potential to sense viable, malignant cells and to destroy 

them by various mechanisms. Several chimeric or humanized 

antibodies are, therefore, in clinical use today. 

However, the effects of these passive immunotherapy 

treatments are limited by the antibody’s halflife, and 

repeated applications have to be given to the patient. In 

contrast, active induction of these desired antibody 

specificities in the patient could be pursued by the 

generation of peptide epitope mimics – mimotopes – 

which are suitable tools for vaccination. By virtue of 

molecular mimicry, these mimotopes induced anti-tumor 

antibodies in mice able to attack tumors not only in vitro 

but also in vivo in murine transgenic, and in syngenic 

transplant models. Interestingly, besides IgG also IgE 

antibodies could be induced when mimotopes were 

gavaged by the oral route. These IgE antibodies sensitized 

mast cells and could be triggered by tumor cells in a 

specific fashion. Most importantly, the released mediators 

were cytotoxic for tumor cells. Indeed, immunohistochemical 

analyses of human tumor sections showed that IgE 

antibodies are present in tumor lesions, pointing towards 

a natural surveillance function of this antibody class. 

Conclusion: Taken together, we suggest that mimotopes 

are candidates for active immunization of patients for 

tumor prophylaxis or therapy, especially in settings of 

minimal residual disease. Studies supported by BioLife- 

Science and grant P-18238-B13 of the Austrian Science 

Fund FWF. 

doi:10.1016/j.clim.2007.03.196 

OR.16 Gene Expression Analysis by Microarray 

Following Anti-CD3 Antibody Therapy of NOD Mice 

Hideyuki Iwai, Postdoctoral Fellow, Stanford University, 

Department of Medicine Division of Immunology and 

Rheumatology, Stanford, CA, Keichi Kodama, Postdoctoral 

Fellow, Stanford University, Department of Medicine 

Division of Immunology and Rheumatology, Stanford, CA, 

Demi Dang, Technician, Stanford University, Department of 

Medicine Division of Immunology and Rheumatology, 

Stanford, CA, C. Garrison Fathman, Professor, Stanford 

University, Department of Medicine Division of Immunology 

and Rheumatology, Stanford, CA, Jeffrey A. Bluestone, 

Professor, Diabetes Center, University of California, 

San Francisco, San Francisco, CA 

Anti-CD3-specific antibodies have the demonstrable 

capacity to induce long-term remission of overt diabetes 

in nonobese diabetic (NOD) mice and have had some 

success in C1-peptide preservation when used to treat 

recent onset human type I diabetics. The underlying 

mechanisms of action are unclear. To begin to study 

mechanism of action, we examined gene expression 

following 5 days of anti-CD3 antibody therapy of NOD 

mice, using 41K Agilent mouse genome oligo-microarrays. 

This treatment successfully restored euglycemia in NOD 

mice treated after the onset of hyperglycemia (blood 

glucose N250) with 10 μg of either conventional anti-CD-3, 

IgG1 2C11, or FcR nonbinding IgG3 anti-CD3 mAb, which 

cannot cross-link surface CD3 molecules on T cells and is 

therefore non-mitogenic. Both antibodies had similar 

effect in disease remission. RNA from islets and pancreatic 

lymph nodes of treated and untreated NOD mice was 

isolated on day 12. Microarray analysis was performed 

comparing treated mouse tissues to untreated control. 

Several hundred genes were dysregulated following therapy. 

We hypothesized that genes specifically affected by 

anti-CD3 antibody would be in overlapping groups among 

those dysregulated by these two antibodies. We will 

present data on these overlapping genes and discuss 

potential mechanism of action of anti-CD3 therapy. 

doi:10.1016/j.clim.2007.03.197 

S9

S10 Abstracts 

OR.17 C-Peptide Reactive T Cells Induce 

Autoimmune Diabetes: A Novel Mechanism of Beta 

Cell Autoimmunity 

Matteo Levisetti, Assistant Professor, Washington 

University, Saint Louis, MO 

There is extensive experimental evidence supporting an 

important role for proinsulin as an autoantigen in the 

pathogenesis of autoimmune diabetes in both humans and 

the NOD mouse model of the disease. We identified 

spontaneously occurring C-peptide reactive T cells in the 

islet-infiltrate and peri-pancreatic lymph nodes of prediabetic 

mice. CD4 Tcells lines were generated by immunization 

with C-peptide in adjuvant and characterized. These T cells 

are reactive with full length C-peptide 1 and 2 and are 

reactive with freshly isolated mouse beta cells. Furthermore, 

these T cells are activated by fixed splenocytes, 

demonstrating that C-peptide can be presented in the 

absence of antigen processing by I-Ag7 bearing antigen 

presenting cells. C-peptide reactive T cells transferred 

disease into NOD.Scid recipient mice and accelerated 

disease in neonatal NOD recipients. These data identify a 

new T cell epitope within the proinsulin molecule that gives 

rise to diabetogenic T cells in the NOD mouse. In addition, 

these data define a novel mechanism for the loss of tolerance 

to secreted self proteins. 

doi:10.1016/j.clim.2007.03.198 

Autoantigens 


2:45 pm−4:45 pm 

OR.18 Cytotoxic Preproinsulin-specific CD8 T Cells 

in Type 1 Diabetes in Man 

Anna Skowera, Postdoctoral Fellow, King’s College London, 

Department of Immunobiology, London, England, Richard 

Ellis, Postdoctoral Fellow, King’s College London, 

Department of Immunobiology, London, England, Timothy 

Tree, Lecture, King’s College London, Department of 

Immunobiology, London, England, Mark Peakman, Professor 

of Clinical Immunology, King’s College London, Department 

of Immunobiology, London, England, Sefina Arif, 

Postdoctoral Fellow, King’s College London, Department of 

Immunobiology, London, England 

Type 1 diabetes (T1D) is a T cell mediated autoimmune 

disease in which the insulin producing pancreatic islet 

beta-cells are selectively eliminated. The mechanism of 

this precisely targeted destruction is unknown, but 

cytotoxic T cells (CTLs) recognizing beta cell-specific 

targets are good candidates. To date, no beta cellspecific 

CTLs have been characterised in T1D. In this 

study, we identified novel epitopes originating from 

preproinsulin (PPI) that were naturally processed and 

presented by HLA-A2.1. Using these epitopes deployed in 

sensitive IFN-γ ELISPOTs a significantly increased prevalence 

of PPI-reactive CD8 T cells in the blood of HLA-A2.1 

+ T1D patients was detected, compared with A2.1+ 

healthy controls in which these cells were extremely 

rare. CD8 T cell clones were generated from T1D patients 

using PPI epitope-loaded HLA-A2.1 tetramers, but could 

not be raised in controls subjects. Such clones were 

highly cytotoxic against HLA-A2.1+ PPI-transfected target 

cells and express NKG2D and CXCR3, which may be 

important both in beta cell targeting and migration to 

inflamed islets. The identification of beta cell restricted 

CD8 T cell epitopes and isolation of relevant CTLs in T1D 

patients suggests that this pathway may be important in 

targeted beta cell destruction. 

doi:10.1016/j.clim.2007.03.199 

OR.19 Anti-Thymocyte Globulin Prevents 

Autoimmune Encephalomyelitis by Expanding 

Myelin Antigen Specific Foxp3+ Regulatory T Cells 

Denise Chung, Postdoctoraltoral Fellow, Brigham and 

Women’s Hospital, Boston, MA, Thomas Korn, Research 

Fellow, Brigham and Women’s Hospital, Boston, MA, Julie 

Richard, Scientist, Genzyme Corporation, Framingham, MA, 

Adam Kohm, Research Fellow, Feinberg School of Medicine, 

Northwestern University, Chicago, IL, Melanie Ruzek, 

Scientist, Genzyme Corporation, Framingham, MA, 

Mohamed Oukka, Instructor, Brigham and Women’s 

hospital, Boston, MA, Stephen Miller, Professor, Feinberg 

School of Medicine, Northwestern University, Chicago, IL, 

Sharon Nahill, Director of Research, Genzyme Corporation, 

Framingham, MA, Vijay Kuchroo, Professor of Neurology, 

Brigham and Women’s Hospital, Boston, MA 

The T cell-depleting polyclonal antibody, anti-thymocyte 

globulin (ATG) has been used in organ transplantation 

to treat acute rejection episodes. However, its mechanism 

has not been fully understood. It is assumed that ATG 

treatment deletes all T cells equally resulting in generalized 

inhibition of T cell response. We hypothesized that 

ATG might selectively delete effector T cells and spare 

regulatory T cells and thereby provide an attractive 

option for the treatment of autoimmune diseases. In 

order to test this, we used Foxp3gfp “knock-in” mice in 

combination with a myelin oligodendrocyte glycoprotein 

(MOG)35–55/IAb tetramer to study more closely the 

effect of ATG treatment on antigen-specific T cell 

responses in vivo during MOG-induced experimental 

autoimmune encephalomyelitis (EAE). Administration of 

ATG in vivo resulted in preferential depletion of T-eff 

with a marked expansion of MOG-specific tetramerreactive 

T-reg (CD4+Foxp3+) upon MOG immunization. In 

both acute and relapsing remitting disease models, ATG 

treatment resulted in the protection from EAE, both in a 

preventive and therapeutic setting. Analysis of purified Teff 

from control and ATG treated animals revealed that 

on a per cell basis, the effector function of residual T-eff 

was not compromised by ATG. Thus, ATG tips the balance 

of T-eff and T-reg and skews an autoantigen specific 

immune reaction from a pathogenic T cell response to a 

protective T-reg response that maintains immunological 

tolerance and prevents autoimmune inflammation. We 

conclude that ATG treatment enforces the development of

Abstracts 

a dominant immunoregulatory environment which may be 

advantageous for the treatment of T cell driven autoimmune 

diseases. 

doi:10.1016/j.clim.2007.03.200 

OR.20 Regulation Through Mimicry: The Autologous 

Milk Protein Butyrophilin Determines Susceptibility 

to Myelin Oligodendrocyte Glycoprotein-induced 

Experimental Autoimmune Encephalomyelitis 

Kerstin Berer, PhD Student, University of Aberdeen, 

Department of Medicine and Therapeutics, Aberdeen, Maria 

Storch, Professor, Universitätsklinikum Graz, 

Universitätsklinik für Neurologie, Graz, Ian Mather, 

Professor, University of Maryland, Department of Animal 

and Avian Sciences, Washington, MD, Christopher Linington, 

Professor, University of Aberdeen, Department of Medicine 

and Therapeutics, Aberdeen 

Sensitization to the autologous milk protein butyrophilin 

(BTN) expands a pre-existing, regulatory T cell 

response which cross-reacts with myelin oligodendrocyte 

glycoprotein (MOG), an important candidate autoantigen 

in multiple sclerosis (MS). This regulatory T cell population 

is associated with the production of high levels of IL-10 

and is able to suppress proliferation of MOG-specific T 

cells in vitro, and to abrogate disease severity in C57BL/6 

mice with MOG-induced experimental autoimmune encephalomyelitis 

(EAE). These findings led us to speculate 

that post-natal induction of regulatory T cell responses by 

BTN in the mother’s milk is a factor that may determine 

susceptibility to MOG-induced EAE in adulthood. We 

investigated this hypothesis using BTN-deficient (BTN−/−) 

C57BL/6 mice and wild-type (WT) littermates. Earlier 

onset of disease and higher disease severity clearly 

indicate an increased susceptibility to MOG-induced EAE 

in BTN−/− mice. This was associated with higher inflammatory 

and demyelinating indices in BTN−/− mice 

compared to their WT littermates. Moreover, functional 

analysis of MOG-specific T cells in BTN−/− mice revealed a 

marked increase in the production of encephalitogenic 

cytokines (IL-17, IFN-γ and IL-6), while the synthesis of IL- 

10 was consistently decreased. Collectively, our data 

suggest that regulatory T cell responses induced by 

autologous BTN play an important role in determining 

susceptibility to MOG-induced EAE. 

doi:10.1016/j.clim.2007.03.201 

OR.21 NOD Islet Autoimmunity Dependence on 

Single Amino Acid Difference in Insulin B:9−23 

Peptide 

Maki Nakayama, Fellow, Barbara Davis Center for Childhood 

Diabetes, University of Colorado Health Sciences Center, 

Aurora, CO, Jean Jasinski, Graduate Student, Barbara Davis 

Center for Childhood Diabetes, University of Colorado 

Health Sciences Center, Aurora, CO, Dongmei Miao, PRA, 

Barbara Davis Center for Childhood Diabetes, University of 

Colorado Health Sciences Center, Aurora, CO, Kelly 

Johnson, PRA, Barbara Davis Center for Childhood Diabetes, 

University of Colorado Health Sciences Center, Aurora, CO, 

Edwin Liu, Assistant Professor, Barbara Davis Center for 

Childhood Diabetes, University of Colorado Health Sciences 

Center, Aurora, CO, John Elliott, Professor, Department of 

Medical Microbiology and Immunology, University of 

Alberta, Edmonton, AB, Canada, Canada, George 

Eisenbarth, Executive Director, Barbara Davis Center for 

Childhood Diabetes, University of Colorado Health Sciences 

Center, Aurora, CO 

NOD mice lacking native insulin genes but bearing 

mutated preproinsulin transgene (alanine [A] rather than 

tyrosine [Y] at B chain position 16) do not develop diabetes 

(Nakayama et al., Nature 2005). In the current study, we 

explore whether the initiation of anti-islet autoimmunity is 

dependent on this one amino acid difference and demonstrate 

that peripheral exposure to native B:9–23 sequence 

restores anti-islet autoimmunity. We created multiple 

double insulin knockout NOD mice bearing a normal 

preproinsulin 2 transgene (B16:Y-dKO mice), instead of the 

mutated B16:A transgene (B16:A-dKO mice). Eleven out of 

twenty five B16:Y-dKO mice developed insulin autoantibodies, 

whereas less than 5% of B16:A-dKO mice (1/31) did 

(Pb0.01). B16:Y-dKO mice express insulin message in thymus 

as high as B16:A-dKO and wild-type NOD mice. These results 

indicate that the native B16:Y sequence in a transgene 

determines development of anti-insulin autoimmunity not 

because of less thymic insulin expression. We next investigated 

where the normal insulin sequence acts to induce 

autoimmunity. Transplantation of NOD islets, but not bone 

marrow, with native insulin sequences [B16:Y] into B16:AdKO 

mice rapidly restored development of insulin autoantibodies 

and insulitis. Splenocytes from B16:A-dKO mice 

transplanted with B16:Y islets induced diabetes when 

transferred into either wild-type or B16:A dKO NOD.SCID 

mice. Splenocytes from mice immunized with native insulin 

B:9–23 peptide induced diabetes on transfer if the recipients 

expressed the native B:9–23 [B16:Y] in their pancreas. These 

studies document dependence upon insulin B16:Tyrosine of 

the insulin B:9–23 peptide for both the initial priming and 

effector phase of NOD anti-islet autoimmunity. 

doi:10.1016/j.clim.2007.03.202 

S11 

OR.22 Use of Random Peptide Display and Novel 

Bioinformatics Algorithm to Detect Conformational 

Epitopes of Jun a 1, The Major Allergen of Mountain 

Cedar Pollen 

Terumi Midoro-Horiuti, Assistant Professor, Pediatrics, 

Galveston, TX, Ruby Tiwari, Postdoctoraltoral Fellow, 

Pediatrics, Galveston, TX, Surendra Negi, Postdoctoral 

Fellow, Biochemistry and Molecular Biology, Galveston, TX, 

Catherine Schein, Assistant Professor, Biochemistry and 

Molecular Biology, Galveston, TX, Bo Ning, Research 

Assistant, Pediatrics, Galveston, TX, Randall Goldblum, 

Professor, Pediatrics, Galveston, TX, Werner Braun, 

Professor, Biochemistry and Molecular Biology, Galveston, TX

S12 Abstracts 

Rationale: Pollen hypersensitivity is a major cause of 

seasonal airway disease world wide. We recently resolved 

the crystal structure and mapped the linear IgE epitopes of 

Jun a 1. Heat and chemical denaturation of Jun a 1 

reduced the binding of human IgE, suggesting the importance 

of conformational epitopes for reactivity. Methods: 

To identify conformational IgE epitopes, we developed a 

panel of monoclonal antibodies (mAbs) against Jun a 1 and 

selected those that were heat sensitive and inhibited the 

binding of IgE from patient sera. These mAbs were used to 

screen a random peptide, phage display library. After 

repeated panning, phage clones were isolated and their 

peptide sequences determined. To compare the affinity 

and specificity of phage-expressed peptides the binding of 

the phage to mAbs and patient IgE, and the ability of the 

phage to inhibit binding of Jun a 1 to the mAb were tested 

by ELISA. Consensus amino acids from selected phage were 

matched with clusters of amino acid residues exposed on 

the surface of Jun a 1, using GETAREA (http://www.scsb. 

utmb.edu/cgi-bin/get_a_form.tcl) and a program specifically 

designed to analyze similarity between the specific 

phage residues and surface patches on allergens. Results: 

Consensus amino acids from phage clones revealed common 

patterns of amino acids which were identical or 

similar to compact patches on the 3-D structure of Jun a 1 

and represent putative conformational epitopes. Conclusion: 

Bioinformatics search tools can play an important role 

in identifying the IgE epitopes on allergens with known 

structures or reliable 3D models. 

doi:10.1016/j.clim.2007.03.203 

OR.23 Antibodies Against Human Cytomegalovirus 

in the Pathogenesis of Atherosclerosis: A Gene Array 

Approach 

Antonio Puccetti, Professor of Medicine, Gaslini 

Institute-Department of Immunology, Genova, Marzia Dolci, 

England Research Fellow, Gaslini Institute-Department of 

Immunology, Genova, Dimitri Peterlana, PhD Student, 

University of Verona-Department of Internal Medicine, 

Verona, Riccardo Navone, Research Fellow, University of 

Verona-Department of Internal Medicine, Verona, Caterina 

Bason, Research Fellow, University of Verona-Department of 

Internal Medicine, Verona, Claudio Lunardi, Professor of 

Medicine, University of Verona-Department of Internal 

Medicine, Verona 

Human cytomegalovirus (hCMV) is involved in the 

pathogenesis of atherosclerosis. We have shown in patients 

with atherosclerosis that antibodies directed against the 

hCMV-derived proteins US28 and UL122 are able to induce 

endothelial cell damage and apoptosis of non-stressed 

endothelial cells through cross-rection with normally 

expressed surface molecules. Our aim was to dissect the 

molecular basis of such interaction and to investigate 

mechanisms linking innate immunity to atherosclerosis. We 

analysed the gene expression profiles in endothelial cells 

stimulated with antibodies affinity purified against either 

the UL122 or the US28 peptides. Real-time polymerase 

chain reaction was used to validate the microarray results. 

Supernatant of endothelial cells incubated with antibodies 

was analysed for the presence of Heat Shock Protein (HSP) 

60 and was used to assess stimulation of toll-like receptor- 

4 (TLR4). Antibodies against UL122 and US28 induced the 

expression of genes encoding for adhesion molecules, 

chemokines, growth factors and molecules involved in the 

apoptotic process together with other genes involved in 

the initiation and progression of atherosclerosis. HSP60 was 

released in the medium of cells incubated with anti-US28 

antibodies and was able to engage TLR4. Antibodies 

directed against hCMV modulate the expression of genes 

coding for molecules involved in the pathogenesis of 

atherosclerosis. Moreover, endothelial cells exposed to 

such antibodies express HSP60 on the cell surface and 

release HSP60 in the medium able to activate TLR4. These 

data confirm that hCMV plays a crucial role in mediating 

the atherosclerotic process and that HSP60 is an endogenous 

ligand for TLR4 signaling. 

doi:10.1016/j.clim.2007.03.204 

OR.24 Differential Effects of Aire Gene Deficiency 

on the Development of Autoimmune 

Encephalomyelitis Induced by Two Representative 

Myelin Peptides 

Asako Tagawa, Research Fellow, National Institute of 

Neuroscience, NCNP, Kodaira, Tokyo, Japan, Toshimasa 

Aranami, Section chief, Department of Immunology, 

National Institute of Neuroscience, NCNP, Kodaira, Tokyo, 

Japan, Mitsuru Matsumoto, Director, Division of Molecular 

Immunology, Institute for Enzyme Research, University of 

Tokushima, Japan, Tokushima, Japan, Takashi Yamamura, 

Director, Department of Immunology, National Institute of 

Neuroscience, NCNP, Kodaira, Tokyo, Japan 

Hazardous autoimmunity is prevented partly by clonal 

deletion of autoimmune T cells in the thymus. In the process 

referred to as central tolerance, autoimmune regulator 

(Aire) gene plays a pivotal role through transcriptional 

control of ectopic antigen expression. In fact, thymic 

expression of myelin antigens was shown to be involved in 

shaping Tcell repertoire that would mediate development of 

EAE. Here we asked if Aire gene deficiency might alter 

manifestations of EAE induced with representative, encephalitogenic 

peptides myelin oligodendrocyte glycoprotein 

(MOG)35–55 and proteolipid protein (PLP)178–191 by using 

Aire−/− mice. It is thought that MOG is not expressed in the 

thymus (JCI 12: 544, 2003), whereas thymic expression of 

PLP178–191 was previously proven (Nat Med 6:56, 2000). 

First, we assessed recall responses to the peptides in the 

immunized mice, and found that sensitized T cells from the 

Aire−/− produced a higher amount of IL-17 than from wildtype 

(WT) mice regardless of the peptides used for 

immunization. Anti-MOG35–55 response was also associated 

with a higher IFN-ã production in Aire−/−. However, there 

was no significant difference between Aire−/− and WT in 

IFN-ã production in response to PLP178–191, which resulted 

in an augmented Th17/Th1 ratio in PLP178–191-immunized 

mice. PLP178–191 immunization induced more serious EAE in 

Aire−/− than WT, whereas such a difference could not be

Abstracts 

seen after sensitization with MOG35–55. As such, Aire gene 

deficiency would differentially alter autoimmune responses 

according to the presence or absence of the directed epitope 

in the thymus. 

doi:10.1016/j.clim.2007.03.205 

Stem Cell/Immunodeficiency/HIV 


2:45 pm−4:45 pm 

OR.25 A Novel RAG-1 Null Mutant Causes T-B-NK+ 

SCID in Native Americans 

Morton Cowan, Professor, Pediatric Bone Marrow Transplant 

Division, University of California, San Francisco Children’s 

Hospital, San Francisco, CA 

Severe combined immunodeficiency disease (SCID) is the 

most serious inherited immunological deficit. Ubiquitous 

DNA double strand repair factors in the non-homologous 

ending joining (NHEJ) pathway resolve DNA double-strand 

breaks (DSB) during V(D)J recombination of T and B 

lymphocyte receptor genes. We have described a null 

mutation of the Artemis gene that has relatively high 

occurrence among Athabascan-speaking Native Americans, 

which results in a complete absence of T and B lymphocytes 

and increased cellular sensitivity to ionizing radiation 

causing radiosensitive-SCID (RS-SCID). Recently we identified 

a novel RAG-1 null mutation, R776W, in three related 

children from the Dine Indian Tribe in the Canadian 

Northwest Territories who manifest a classic T-B-NK+ SCID 

phenotype. We found a normal pattern of radiation 

sensitivity in skin fibroblast cell lines from these patients, 

indicating that the NHEJ pathway is intact. The impaired 

activity of this RAG-1 mutant in V(D)J recombination was 

revealed by an enhanced green fluorescent protein (EGFP) 

based assay. Additional studies of the RAG-1 null mutant 

will evaluate DNA binding and cleavage activity, hairpin 

formation in vitro as well as catalysis of other DNA strand 

transfer reactions such as transposition. Our current results 

suggest that the T-B-NK+ SCID phenotype is triggered by this 

novel RAG-1 mutant. 

doi:10.1016/j.clim.2007.03.206 

OR.26 HIV-1 Infectivity Inhibited by Binding of the 

Green Tea Catechin, Epigallocatechin Gallate, to 

CD4 

Christina Nance, Instructor, Baylor College of Medicine, 

Houston, TX, Edward Siwak, Assistant Professor, Baylor 

College of Medicine, Virology, Houston, TX, Aarthi Ram, 

Research Technician, Baylor College of Medicine, Pediatrics, 

Houston, TX, Van Willis, Research Technician, Baylor College 

of Medicine, Pediatrics, Houston, TX, Susan Westerfield, 

Research Technician, Baylor College of Medicine, Pediatrics, 

Houston, TX, William Shearer, Professor, Baylor College of 

Medicine, Pediatrics, Houston, TX 

Binding of HIV-1 envelope glycoprotein, gp120, to the CD4 

T cell receptor results in HIV-1 infection. Previously, we have 

presented evidence of high affinity binding of the green tea 

catechin, epigallocatechin gallate (EGCG), to the CD4 

molecule at the gp120 binding pocket. We now present 

evidence that EGCG inhibits the binding of gp120 on human 

CD4+ T cells and PBMC and prevents the HIV-1 infectivity of 

human macrophages and PBMC. Binding studies utilized flow 

cytometry. HIV-1 infectivity was assessed by HIV-1 p24 EIA. Mtropic 

(R5) (strains SF162, Bal), T-tropic (X4) (strain IIIB), and 

dual tropic (R5X4) (strain 89.6) HIV-1 isolates were used. 

EGCG markedly inhibited the binding of HIV-1-gp120IIIB to 

CD4+ T cells in a dose-dependent manner at physiologically 

relevant levels (32% at 0.2 μM pb0.05, 42% at 2 μM and 47% 

at 20 μM pb0.01) and higher (55% at 50 μM and 71% at 100 μM 

pb0.001). EGCG significantly inhibited the infectivity of 

human macrophages by M- and dual-tropic HIV-1 strains at 

200–400 TCID50 in a dose-dependent manner. There was 

100% inhibition of p24 production by EGCG (25–100 μM) 

(pb0.0001), 95% at 12 μM, and 79% at 6 μM (pb0.001). The 

response by human PBMC was similar. The control catechin 

did not alter gp120 binding nor inhibit HIV-1 infectivity. We 

conclude that EGCG is able to significantly reduce the 

attachment of gp120 to CD4, along with a decrease in the 

infectivity of HIV-1 to target cells. The competitive binding 

properties of EGCG for the CD4 binding sites by gp120 may 

translate to an HIV-1 preventative strategy. 

doi:10.1016/j.clim.2007.03.207 

S13 

OR.27 Impaired Dendritic Cell Function in 

Ectodermal Dysplasia with Immune Deficiency is 

Linked to Defective NEMO Ubiquitination 

Stephan Temmerman, Postdoctoral Fellow, NIH-NIAID-LHD, 

Bethesda, MD, Chi Ma, Senior Biologist, NIH-NIAID-LHD, 

Bethesda, MD, Ashish Jain, PI, NIH-NIAID-LHD, Bethesda, MD 

NF-kappaB essential modulator (NEMO) regulates the 

activation of the transcription factors NF-kappaB. Alterations 

in NEMO cause ectodermal dysplasia with immunodeficiency 

(EDI), a disorder that is characterized by 

defects in innate and adaptive immunity as well as 

abnormal development of ectoderm-derived tissues. 

Because the biochemical mechanism by which NEMO 

mutations cause immune dysfunction remains undefined, 

we investigated the effect of a cysteine to arginine 

substitution found in the NEMO zinc finger domain on 

dendritic cell (DC) function. Following CD40 ligand 

(CD40L) stimulation of DCs from two EDI patients, we 

found that lysine 63-linked polyubiquitination of NEMO, 

which promotes activation of the IKK complex, was 

absent. We associated this defect with preserved RelA 

but absent c-Rel activity. Therefore, CD40L-stimulated EDI 

DCs failed to synthesize the c-Rel dependant cytokine IL- 

12, displayed reduced cell aggregates and membrane 

extensions, and failed to support allogeneic lymphocyte 

proliferation. We utilized gene expression profiling to 

compare the genes induced by CD40L in normal DCs but 

not in EDI DCs. In this gene set, we identified a number

S14 Abstracts 

of molecules that are likely to be critical to the 

maturation and function of DCs. In contrast, downstream 

NF-kappaB activity, DC maturation, and NEMO polyubiquitination 

were normal in EDI DCs following stimulation with 

the TLR4 ligand. These results show for the first time that 

CD40 and TLR4 use different signaling pathways to 

ubiquitinate NEMO, and offer insight into how a mutation 

in the zinc finger domain of NEMO leads to pathway 

specific defects in NF-kappaB signaling and thus immune 

deficiency. 

doi:10.1016/j.clim.2007.03.208 

OR.28 Treatment of HIV-infected Patients with 

Vitamin D-binding Protein Derived Macrophage 

Activating Factor (GcMAF) Eradicates HIV-Infection 

Nobuto Yamamoto, Director, Socrates Institute for 

Therapeutic Immunology, Philadelphia, PA, Masumi Ueda, 

Immunology, Chief, Microbiology, Philadelphia, PA, 

Charles Benson, Head, Infectious Diseases, School of 

Veterinary Medicine, University of Pennsylvania, Kennett 

Square, PA 

Serum vitamin D-binding protein (known as Gc protein) is 

the precursor for the principal macrophage activating factor 

(MAF). The MAF precursor activity of serum Gc protein of 

HIV-infected patients was lost or reduced because Gc 

protein is deglycosylated by serum α-N-acetylgalactosaminidase 

(Nagalase) secreted from HIV-infected cells. Since 

Nagalase is the intrinsic component of gp120, serum 

Nagalase activity is the sum of enzyme activities expressed 

in both HIV virions and envelope proteins released from HIVinfected 

cells. Because of Nagalase being an HIV viral 

protein and immunogenic, serum Nagalase was already 

complexed with anti-HIV immunoglobulin G (IgG) in patient 

blood stream. These antibodies, however, were largely not 

neutralizing antibodies. The IgG-bound viral proteins 

retained Nagalase activity that deglycosylates Gc protein. 

Therefore, macrophages of HIV-infected patients having 

deglycosylated Gc protein cannot be activated, leading to 

immunosuppression. Stepwise treatment of purified Gc 

protein with immobilized beta-galactosidase and sialidase 

generated the most potent macrophage activating factor 

(termed GcMAF), which produces no side effect in humans. 

Macrophages activated by administration of GcMAF 

(100 ng/patient) develop a large amount of Fc receptors 

as well as enormous variation of receptors that recognize 

IgG bound and unbound HIV virions. Thus, macrophages 

activated by GcMAF preferentially phagocytize IgG-bound 

HIV virions via Fc receptor mediation. Cells latently 

infected with HIV are unstable and spontaneously release 

the virions at a high rate. After less than 18 weekly 

administrations of 100 ng GcMAF for twenty-one nonanemic 

patients, they exhibited low serum Nagalase activities 

equivalent to healthy controls, indicating eradication of HIV 

infection. 

doi:10.1016/j.clim.2007.03.209 

OR.29 Impaired In Vitro Regulatory T Cell Function 

Associated with Wiskott-Aldrich Syndrome 

Marsilio Adriani, Visiting Fellow, NIH/NHGRI, Bethesda, MD, 

Joseph Aoki, Visiting Fellow, NIH/NHGRI, Bethesda, MD, 

Reiko Horai, Postdoctoral Fellow, NIH/NHGRI, Bethesda, 

MD, Akihiro Kon, England Postdoctoral, Fellow, Bethesda, 

MD, Angela M. Thornton, Staff Scientist, NIH/NIAID, 

Bethesda, MD, Martha Kirby, Senior Research Assistant, 

NIH/NHGRI, Bethesda, MD, Stacie M. Anderson, Senior 

Research Assistant, NIH/NHGRI, Bethesda, MD, Richard M. 

Siegel, Investigator, NIH/NIAMS, Bethesda, MD, Pamela L. 

Schwartzberg, Investigator, NIH/NHGRI, Bethesda, MD, 

Fabio Candotti, Senior Investigator, NIH/NHGRI, Bethesda, 

MD 

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency 

characterized by thrombocytopenia, eczema, recurrent 

infections and high incidence of malignancy. This disease is 

caused by mutations of the WAS protein (WASP) gene, a key 

regulator of actin polymerization. WAS patients show high 

incidence of autoimmune disorders (∼40–70%) the occurrence 

of which, however, does not correlate with the severity of the 

other classical hallmarks of the disease. A central question in 

understanding the pathophysiology of WAS is why immunodeficient 

patients develop symptoms suggestive of hyperactivation 

of immune compartments, such as eczema and autoimmune 

diseases. Since defects in CD4+CD25+ regulatory T cells (Treg) 

have been associated with autoimmunity, we examined the 

presence and function of these cells in WAS patients and Waspdeficient 

mice. Wasp-deficient patients and mice develop Treg 

cells that express Foxp3 with no detectable differences in 

frequency compared to controls. However, in vitro suppression 

assays showed that these Treg cells have impaired inhibitory 

function in vitro,which,inmice,couldonlybepartiallyrescued 

by pre-activation with exogenous IL-2. The demonstration of 

impaired in vitro suppressor function in WASp-deficient Treg 

cells suggests that defective regulatory Tcell function may be an 

important factor contributing to immune dysregulation in WAS. 

doi:10.1016/j.clim.2007.03.210 

OR.30 Regulatory T Cell Abnormalities Associated 

with Aberrant CD4+ T Cell Responses in Patients 

with Immune Reconstitution Disease (IRD) 

Nabila Seddiki, Research Fellow, Centre For Immunology 

and National Centre in HIV Epidemiology and Clinical 

Research (UNSW), Darlinghurst 

Up to 30% of patients with HIV commencing antiretroviral 

therapy (ART) late in the disease restore a pathogen-specific 

cellular immune response that is immunopathological and 

causes disease referred as immune reconstitution disease or 

IRD. The immunopathogenesis of IRD is not well understood and 

is likely to be a consequence of an aberrant reconstitution of 

certain T cell subsets. In a cross-sectional study, using HIV 

patients who developed mycobacterial IRD, we show that in 

comparison to those starting ARTand not developing IRD, CD4+ T 

cells specific for MTB and M. avium complex (MAC) antigens, 

produce high levels of IFN-g and IL-2 (Pb0.01) and proliferate 

strongly when compared to controls. We postulated that deficits 

in regulatory T cells (Tregs) numbers may play a central role in

Abstracts 

the pathogenesis of IRD. When Tregs numbers were examined by 

using our recently described marker CD127 (IL-7R), we found a 

significant expansion of memory CD127loFoxp3+CD25+ Tregs in 

IRD patients compared to controls. To further investigate 

abnormalities in T cell responses, we examined a range of 

cytokines in the plasma of patients and controls and found 

increased levels of IL-4, IL-6, IL-7 and IL-21. Interestingly, we 

show that some of these cytokines inhibit strongly Treg 

suppression. Furthermore, we show that suppressors and 

responders from these patients present defect to regulate CD4 

T cell immune responses. Altogether, these data suggest that 

despite the high Treg expansion in IRD, their ability to induce 

suppression and turn off these aberrant immune responses is 

compromised. 

doi:10.1016/j.clim.2007.03.211 

OR.31 Liver Tissue Cells Inhibit Immune Responses 

by Inducing T Cell Apoptosis: An Application in Islet 

Transplantation 

Lina Lu, Associate Professor, Cleveland Clinic, Cleveland, 

OH, Cheng-Hsu Chen, Research Fellow, Department of 

Immunology, Cleveland Clinic, Cleveland, OH, Zhenyu Yin, 

Research Fellow, Department of Immunology, Cleveland 

Clinic, Cleveland, OH, John J. Fung, Professor, Department 

of General Surgery, Cleveland Clinic, Cleveland, OH, 

Liang-Mou Kuo, Research Fellow, Department of 

Immunology, Cleveland Clinic, Cleveland, OH, Shiguang 

Qian, Associate Professor, Department of Immunology, 

General Surgery, Cleveland Clinic, Cleveland, OH 

Organ transplantation has been successful for decades, but 

outcome of cell transplants remains poor. This is true in animal 

models. Liver transplants are spontaneously accepted in mice, 

but hepatocyte allografts are acutely rejected, suggesting an 

immunosuppressive effect of liver tissue cells. In this study, we 

demonstrated a profound in vitro immune inhibitory activity of 

activated (but not quiescent) hepatic stellate cells (HSC) that 

are well known to be involved in liver fibrosis. Activation of HSC 

by exposure to IFN-γ or directly contact with activated T cells 

resulted in upregulation of B7-H1, inhibitory cytokines (IL-6, IL- 

10, TGF-β), and enhanced T cell apoptotic death (annexin 

V + 7AAD− ), which is largely mediated by B7-H1, since B7-H1−/− HSC triggered significantly less apoptosis. To explore potential 

clinical application, BALB/c (H2d ) islets (300) mixed with 

activated HSC (3×10 5 )ofB6(H2b )miceweretransplanted 

under renal capsule of chemically diabetic B6 recipients, 

showing a marked prolongation of islet graft survival [from 

median survival time (MST) of 13 days in control to N60 days 

(pb0.01)] with 55% of recipients being euglycemia for N60 days. 

None of islet-only recipients remained euglycemia for N17 days. 

Removal of islet-grafted kidney resulted in quick glycemia. 

Immunochemistry showed that prolongation of islet allografts by 

co-transplanted HSC was associated with upregulated apoptotic 

activity (TUNEL) and less T cell infiltration (CD3), which 

appeared to be mediated by B7-H1, as co-transplant with B7- 

H1 −/− HSC markedly reduced their protection to islet allografts 

(MST 26 days). These scientific observations may lead to 

substantial improvement of islet transplantation. 

doi:10.1016/j.clim.2007.03.212 

OR.32 BAFF Receptor Expression in CVID Patients 

Marta Rizzi, MD PhD, Clinical Research Unit for 

Rheumatology, Freiburg/Br., Ulrich Salzer, PhD, Division of 

Rheumatology and Clinical Immunology, Freiburg/Br., Klaus 

Warnatz, MD PhD, Division of Rheumatology and Clinical 

Immunology, Freiburg/Br., Sigune Goldacker, MD, Division of 

Rheumatology and Clinical Immunology, Freiburg/Br., 

Stefanie Hamm, Student, Clinical Research Unit for 

Rheumatology, Freiburg/Br., Hans Hartmut Peter, Professor 

MD PhD, Division of Rheumatology and Clinical Immunology, 

Freiburg/Br., Hermann Eibel, PhD, Clinical Research Unit 

for Rheumatology, Freiburg/Br. 

BAFF receptor (BAFF-R) is a member of the TNF-R superfamily 

and is expressed mainly on mature B cells. Common 

variable immune deficiency (CVID) is a primary immune 

deficiency characterized by hypogammaglobulinemia. Several 

genetic defects contributing to this phenotype have been 

identified (mutations in ICOS gene, TACI gene, CD19 gene). Our 

group recently identified a deletion mutation within the BAFF- 

R gene, removing the transmembrane domain of the protein. 

The homozygous mutation precludes the syntheses of functional 

BAFF-R. Therefore, B cell development in the BAFF-R 

deficient patient is arrested at the transitional B cell stage. As 

a result, the patient lacks IgG-producing plasma cells and is 

severely B lymphopenic. Searching for additional BAFF-R mutations 

we screened a subgroup of our cohort of CVID patients 

with very low number of B cells in peripheral blood (b5%) for 

BAFF-R expression. We found that 8 out of the 13 patients 

presented low BAFF-R expression on the B cells surface (mean 

fluorescence 21.9 SD 4.3, control mean fluorescence 58.9 SD 

3.6), and low BAFF binding activity. Low BAFF-R expression 

significantly correlates with a low number of detectable 

memory B cells (CD19+CD27+). Sequencing of the BAFF-R 

alleles excluded known mutations in the BAFF-R gene. We then 

investigated if low BAFF-R expression might result from 

regulatory defects. Stimulation of the BCR, TLR 9 and/or 

CD40 increased BAFF-R expression. Therefore, low levels of 

BAFF-R expression in CVID patients may result from regulatory 

defects and contribute to antibody deficiency. 

doi:10.1016/j.clim.2007.03.213 

S15

S16 Abstracts 

F.1 Thyroid Function and Prevalence of 

Anti-Thyroid Antibodies in Iranian Patients with 

Type 1 Diabetes Mellitus: Influences of Age and Sex 

Faranak Sharifi, Endocrinologist, Zanjan University of 

Medical Sciences, Zanjan, Leila Ghasemi Hashtroodi, 

Resident for Internal Medicine, Zanjan University of Medical 

Sciences, Zanjan, Yahya Jaberi, Assistant Professor, Zanjan 

University of Medical Sciences, Zanjan 

Objective: Type 1 diabetes mellitus is frequently associated 

with autoimmune thyroid disease (ATD). Genetic 

susceptibility to autoantibody formation in association with 

ATD and type 1 diabetes mellitus has been described with 

varying frequencies, but there is still debate about the 

situation in Iran. We have, therefore, investigated the 

prevalence of anti-thyroid peroxidase (anti-TPO) and antithyroglobulin 

(anti-TG) antibodies in type 1 diabetic patients, 

and compared the effect of age and sex on the thyroid 

autoimmunity in patients with type 1 diabetes mellitus in 

Iran. Subjects and Methods: Ninety one subjects with type 1 

diabetes mellitus and one hundred and sixty three unrelated 

normal controls under age thirty were recruited for the 

detection of anti-TPO and anti-TG. Radioimmunoassay was 

used for anti-TPO and anti-TG detection respectively. 

Results: Among 91 type 1 diabetic patients, 36 (39.6%) 

were positive for anti-TPO and 27 (30%) were positive for 

anti-TG. Anti-TPO antibodies were detected only in 6.7% of 

control group. Compared with those without thyroid autoimmunity, 

there was a female preponderance for the type 1 

diabetic patients with thyroid autoimmunity (female: male, 

28:14 vs. 28:20 respectively). Among the type 1 diabetic 

patients those with thyroid autoimmunity tended to have 

older age (p: 0.04) and higher TSH concentration (p: 0.03). 

Patients with high anti-TPO levels had longer duration of 

diabetes (p: 0.02). Conclusion: The presence of anti-TPO in 

39.6% of our type 1 diabetic patients compared with 8.5% of 

normal subjects confirmed the strong association of ATD and 

type 1 diabetes mellitus. 

doi:10.1016/j.clim.2007.03.214 

Poster Sessions 


7:30 am−7:00 pm 

Authors Present: 5:45 pm−7:00 pm 

F.2 Reduced Foxp3 and Interleukin 10 Expression 

During the Partial Remission Phase of Type 1 

Diabetes in Children 

Srinath Sanda, Clinical Fellow, University of California, San 

Diego School of Medicine, San Diego, CA, Bart Roep, 

Associate Professor, Department of Immunohematology and 

Blood Transfusion Leiden University Medical Center, Leiden, 

Matthias von Herrath, Professor, Developmental 

Immunology at the La Jolla Institute for Allergy and 

Immunology, La Jolla, CA 

Background: Some children undergo a reduction in insulin 

requirements after diagnosis and initiation of insulin 

therapy. The exact mechanism underlying this partial 

remission phase (also known as the honeymoon phase) of 

type 1 diabetes in children is not known. Study Design: We 

conducted a cross-sectional study comparing peripheral 

blood T cell responses by ELISPOT and flow cytometry 

between a cohort of newly diagnosed children with type 1 

diabetes at initiation of insulin therapy and a cohort of 

children in the honeymoon phase. Children were classified 

in the honeymoon phase if they were between 3 and 

9 months from diagnosis, had a total daily insulin requirement 

of b0.5 U/kg/day, and had experienced a N50% 

reduction in their total daily insulin dose since diagnosis. Six 

children were recruited in each group. Results: Honeymoon 

patients were on average 7.4 months from diagnosis and had 

significantly reduced insulin doses and hemoglobin A1c 

levels compared to new onset patients. Both cohorts had 

similar numbers of peripheral CD4+, CD8+, and CD4+CD25+ 

cells. Honeymoon patients had fewer numbers of peripheral 

CD4+CD25+Foxp3+ cells compared to newly diagnosed 

patients (37.3% ±23% vs. 57.8±27.8% p=0.054). ELISPOT 

assays using epitopes to GAD65, IA2, and proinsulin, 

demonstrated a trend for increased interferon-gamma 

production and reduced interleukin-10 production in the 

honeymoon patients. Conclusion: Reduced numbers of 

peripheral blood CD4+CD25+Foxp3+ and interleukin 10 

producing cells are seen in type 1 diabetes patients during 

their honeymoon phase compared to patients at diagnosis. 

doi:10.1016/j.clim.2007.03.215 

F.3 A Novel Cell-based Therapeutic Approach to 

Prevent and Cure Autoimmune Type I Diabetes in 

NOD Mice 

Dong Zhang, Postdoctoral Research Fellow, Harvard Medical 

School, Beth Israel Deaconess Medical Center, Boston, MA, 

Yan Tian, Research Assistant, Harvard Medical School, Beth 

Israel Deaconess Medical Center, Boston, MA, Nicolas 

Degauque, Research Fellow, Harvard Medical School, Beth 

Israel Deaconess Medical Center, Boston, MA, Wei Yang, 

Research Fellow, Harvard Medical School, Beth Israel 

Deaconess Medical Center, Boston, MA, Allison Mikita, 

Research Assistant, Harvard Medical School, Beth Israel 

Deaconess Medical Center, Boston, MA, Xin Xiao Zheng, 

Assistant Professor, Harvard Medical School, Beth Israel 

Deaconess Medical Center, Boston, MA

Abstracts 

Regulatory T cells play vital roles in maintaining peripheral 

tolerance to auto- and alloantigens. Further characterization 

of the function and development of these regulatory 

T cells will contribute to understanding of the intrinsic and 

extrinsic mechanisms for peripheral tolerance. Our study 

showed that proliferated CD4+ T cells could convert to CD4− 

CD8−CD3+ (DN) regulatory T cells after either allogeneic or 

syngeneic DC triggered proliferation, and IL-2 and IL-15 

enhanced the conversion. Converted DN T cells were 

resistant to AICD and expressed unique cell markers and 

gene profile. Converted DN T cells exerted powerful 

inhibition on the same, but not third party, alloantigen 

triggered proliferation of naive CD4+CD25− T effectors. It 

was notable that at a per cell base comparison, converted DN 

T cells were more potent in suppressing alloantigen trigged 

naive T cells proliferation than naive and proliferated CD4+ 

CD25+ Tregs. Perforin, a cytotoxic cytokine, which was highly 

expressed by DN T cells, played a role in DN T cell mediated 

suppression. Adoptive transferring of T cells from new onset 

diabetic NOD mice precipitated a rapid onset of diabetes in 

NOD/SCID recipients. Co-transferring of DN T cells (in vitro 

converted from CD4+ T cells from new onset diabetic NOD) 

with diabetogenic T cells significantly delay the onset of 

diabetes in NOD/SCID mice (P=0.0012). Moreover, the small 

number of converted DN can reverse diabetes after disease 

onset in NOD mice. The results of using ex vivo CD4+ T cells 

converted DN T cells in NOD mouse model support the 

concept and the feasibility of potentially utilizing this novel 

cell-based therapeutic approach clinically for the treatment 

of autoimmune diseases. 

doi:10.1016/j.clim.2007.03.216 

F.4 Innate Immune Modulation of Type 1 Diabetes 

with Glycosphingolipids 

Dalam Ly, Graduate Student, University of Western Ontario, 

Microbiology and Immunology, London, ON, Canada, Terry 

Delovitch, Senior Scientist/Professor, Robarts Research 

Institute/University of Western Ontario, London, ON, 

Canada 

The pathogenesis of T1D may be mediated by functional 

deficiencies in CD4+CD25+Foxp3+ Tregs and iNKT cells. As 

self-reactive Tregs and iNKT cells both protect NOD mice 

from T1D, we investigated whether iNKT-Treg collaboration 

is required for this protection. While α-galactosylceramide 

(α-GalCer) therapy does not alter the proliferation, clonal 

expansion or function of Tregs, protection from T1D by Tregs 

does not require iNKT cell activity. However, Treg activity is 

required for α-GalCer induced protection from T1D by iNKT 

cells. Significant decreases in IFN-γ and IL-4 secretion and 

cellular (T, B, NK, DC) transactivation by iNKT cells were 

detected in the presence of functional Tregs. Thus, during 

α-GalCer therapy of T1D, activated iNKT cells transactivate 

Tregs that may then downregulate iNKT cell responses and 

return iNKT cells to a homeostatic level of activation. Such 

Treg-dependent homeostatic control of iNKT cell activity 

may explain how Treg/iNKT collaboration protects from 

T1D. In contrast, we found that Treg activity is not required 

for protection from T1D induced by the C20:2 analog of α- 

GalCer, which possesses a shortened (C20) fatty acyl chain 

and cis-unsaturations at carbons 11 and 14. This may arise 

from the ability of C20:2 to elicit significantly reduced IFN-γ 

and enhanced IL-4 secretion by iNKT cells relative to α- 

GalCer. Thus, α-GalCer and C20:2 may differ in their 

requirement of Tregs for iNKT mediated protection from 

T1D. Ongoing studies of the mechanism(s) of this differential 

requirement of Tregs for protection from T1D may 

have significant implications for innate immunity focused 

T1D therapeutic approaches. 

doi:10.1016/j.clim.2007.03.217 

F.5 Targeted Cellular Gene Therapy Using IL-4 

Secreting DCs Corrects Immune Dysregulations and 

Prevents Diabetes in NOD Mice 

Remi J. Creusot, Postdoctoral Fellow, Stanford University, 

Department of Medicine, Stanford, CA, Shahriar Yaghoubi, 

Research Associate, Stanford University, Department of 

Radiology, Stanford, CA, Keiichi Kodama, Postdoctoral 

Fellow, Stanford University, Department of Medicine, 

Stanford, CA, Sam S. Gambhir, Professor, Stanford 

University, Department of Radiology, Stanford, CA, Demi 

Dang, Research Assistant, Stanford University, Department 

of Medicine, Stanford, CA, C. Garrison Fathman, Professor, 

Stanford University, Department of Medicine, Stanford, CA 

A deficit in IL-4 has been associated with the development 

of type 1 diabetes in man and in the non-obese diabetic 

(NOD) mouse. We set out to correct this deficiency by 

delivering IL-4 using bone marrow-derived dendritic cells 

transduced to express IL-4 (DC/IL-4). DC/IL/4 were injected 

into 12-week-old female NOD mice with advanced prediabetic 

insulitis. After either intravenous (iv) or intraperitoneal 

(ip) injection of DC/IL-4, the onset of the disease was 

delayed for ∼5 weeks and the majority of the treated mice 

were protected until at least 30 weeks of age. Using 

bioluminescence imaging and biodistribution assays, we 

demonstrated that iv-injected DCs trafficked primarily to 

the spleen and pancreatic lymph nodes (PLN), with little or 

no homing to other lymph nodes. While equally effective, ipinjected 

DCs showed preferential migration to the PLN, with 

some homing to the pancreas. We then asked what effect 

DC/IL-4 had in the PLN by performing cDNA microarray 

analysis. Initial studies demonstrated that many genes were 

significantly dysregulated in the PLN of 12-week-old female 

NOD mice, compared to non-diabetic congenic NOD.B10 

mice. Interestingly, the expression level of most of these 

dysregulated genes (N200), in addition to IL-4 itself, was 

brought towards normal levels as early as 3 days after DC/IL- 

4 treatment. MHC-deficient DC/IL-4 had no protective 

effect, demonstrating that interaction of the transduced 

DCs with T cells was required for the therapeutic effect. In 

conclusion, the effects of DC/IL-4 were localized, rapid, long 

lasting and characterized by the normalization of many 

dysregulated genes. 

doi:10.1016/j.clim.2007.03.218 

S17

S18 Abstracts 

F.6 Plasmacytoid Dendritic Cells Induction by a 

Self-peptide Ep1.B Derived from Apolipoprotein E 

Prevent Autoimmune Diabetes 

Enayat Nikoopour, Postdoctoral Fellow, Department of 

Microbiology and Immunology and Robarts Research 

Institute, London, ON, Canada, Tracey A. Stephens, 

Graduate Student, Department of Microbiology and 

Immunology and Robarts Research Institute, London, ON, 

Canada, Beverley J. Rider, Graduate Student, Department 

of Microbiology and Immunology and Robarts Research 

Institute, London, ON, Canada, Edwin Lee-Chan, Lab 

Technician/Lab Manager, Department of Microbiology and 

Immunology and Robarts Research Institute, London, ON, 

Canada, Bhagirath Singh, Professor, Department of 

Microbiology and Immunology and Robarts Research 

Institute, London, ON, Canada 

Dendritic cells (DC) are potent inducers of T cell 

tolerance. The mechanism by which this is achieved is not 

well understood. We postulated that in vivo induction of 

plasmacytoid dendritic cells (PDC) could prevent autoimmunity 

through induction of T cell tolerance. We report that a 

novel self-peptide Ep1.B of mouse Apolipoprotein E (ApoE) 

induced differentiation of bone marrow derived monocytes 

(BMM) into PDC. In vitro Ep1.B induced PDCs are B220+, Ly6- 

C+, CD11c+, mPDCA+, CD62L+ and CD8a+. Footpad injection 

of Ep1.B in diabetes prone NOD mice increased the level of 

CD11c+, mPDCA and B220+ cells in the draining lymph nodes 

(LN) and these CD11c+ cells have an increased expression of 

tolerogenic programmed death ligand (PD-L1). Since PD-1- 

PD-L1 pathway has been shown to be important in maintaining 

tolerance, PDC induced by Ep1.B may inhibit the effector 

T cells in disease process. In addition, we found increased 

number of CD4+CD25+ T cells with elevated glucocorticoidinduced 

tumor necrosis factor receptor related protein 

(GITR) in the LN of these animals. We found that injection 

of Ep1.B in young NOD mice significantly prevented type 1 

diabetes development in these mice. In summary, we suggest 

that in vivo Ep1.B induced differentiation of BMM into PDC 

that in turn prevented autoimmunity and induced regulatory 

T cells in type 1 diabetes prone NOD mice. 

doi:10.1016/j.clim.2007.03.219 

F.7 Generation and Characterization of Insulin 

Peptide-specific Regulatory T Cells 

Marianne Martinic, Postdoctoral Fellow, Immune 

Regulation Lab DI-3, La Jolla Institute for Allergy and 

Immunology, La Jolla, CA, Lisa Togher, Technician, Immune 


Immunology, La Jolla, CA, Christophe Filippi, Postdoctoral 

Fellow, Immune Regulation Lab DI-3, La Jolla Institute for 

Allergy and Immunology, La Jolla, CA, Jean Jasinski, PhD 

Student, Barbara Davis Center for Childhood Diabetes, 

Aurora, CO, Damien Bresson, Postdoctoral Fellow, Immune 


Immunology, La Jolla, CA, George Eisenbarth, Executive 

Director, Barbara Davis Center for Childhood Diabetes, 

Aurora, CO, Matthias von Herrath, Division Head, Immune 


Immunology, La Jolla, CA 

Our goal is to generate insulin peptide-specific regulatory 

T cells (Tregs) in vitro, which suppress overt diabetes in 

prediabetic and reverse already ongoing disease in diabetic 

mice. Furthermore we aim to analyze the suppressive 

properties and mechanisms of these Tregs in vitro and in 

vivo. In order to generate insulin peptide-specific Tregs, we 

took advantage of insTCR transgenic mice, which express T 

cell receptor (TCR) transgenic CD4+ T cells specific for 

the insulinB9–23 peptide (insB) presented on MHC class 

II H-2IAg7/d molecules. The in vitro generation of insBspecific 

Tregs involved purification of either CD4+CD25+ or 

CD4+CD25− insTCR T cells, which were cultured for 1– 

2 weeks with the insB peptide, syngeneic antigen presenting 

cells and high doses of IL-2 yielding 25+ and 25− cultures, 

respectively. Using the classical in vitro suppression assay, 

only cells derived from the 25+ cultures were able to 

suppress while cells from the 25− cultures enhanced 

proliferation and cytokine secretion of CD8+ effector T 

cells. In vivo, however, cells from both cultures were unable 

to suppress lymphocytic choriomeningitis virus-induced 

diabetes. Interestingly, freshly isolated as well as IL-10cultured 

CD4+CD25− but not freshly isolated CD4+CD25+ 

insTCR T cells suppressed spontaneous diabetes in NOD 

females. We are currently investigating the mechanisms 

underlying the in vivo suppressive potential of these CD4 

+CD25− T cells. 

doi:10.1016/j.clim.2007.03.220 

F.8 The Insulitis Reporter Mouse 

Jennifer Ondr, Postdoctoral Fellow, Cincinnati Children’s 

Hospital Medical Center, Division of Endocrinology, Diabetes 

Research Center, Cincinnati, OH, Robert Opoka, Research 

Assistant, Cincinnati Children’s Hospital Medical Center, 

Division of Endocrinology, Diabetes Research Center, 

Cincinnati, OH, Sankarannand Vukkadapu, Postdoctoral 

Fellow, Cincinnati Children’s Research Foundation, 

Cincinnati, OH, Jonathan Katz, Associate Professor, 

Cincinnati Children’s Hospital Medical Center, Division of 

Endocrinology, Diabetes Research Center, Cincinnati, OH 

Type 1 diabetes mellitus (T1DM) is caused by the 

autoimmune destruction of insulin producing beta cells by 

leukocytes that infiltrate the pancreas in a prolonged and 

clinically silent process termed insulitis. Inability to detect 

insulitis prior to the onset of overt disease symptoms stymies 

progress in T1DM research and treatment. In humans, 

detection of antibody to islet cell antigens is the only means 

to diagnose pre-diabetic insulitis, however, no relationship 

between sero-conversion and insulitis progression is established. 

In NOD mice, insulitis can be measured, but only as an 

end-stage pancreatectomy. An early, accurate diagnosis of 

insulitis would allow the possibility of therapeutic intervention, 

thus there remains an urgent need for non-invasive and 

real-time measures of insulitis. To this end, we are generating 

a gene-targeted NOD mouse in which production of a

Abstracts 

molecular reporter, soluble human alkaline phosphatase 

(sHPAP), is controlled by a gene, Reg3γ, expressed exclusively 

by islets cells of pancreata undergoing insulitis. Our insulitis 

reporter (InsuRe) mouse will self-indicate the onset and 

severity of insulitis when Reg3γ expression, triggered by the 

infiltration of pancreatic islets, induces rapid and measurable 

secretion of sHPAP into the blood. We will validate the fidelity 

of sHPAP expression in the InsuRe mouse with multiple 

established NOD mouse models of insulitis and diabetes. 

The InsuRe mouse will allow the identification of biomarkers 

that appear in the blood of NOD mice throughout insulitis. The 

identification of these biomarkers will aid in the non-invasive 

detection of occult insulitis, or diagnosis of pre-diabetes, in 

both mice and humans. 

doi:10.1016/j.clim.2007.03.221 

F.9 Identification of Candidate IDDM Disease 

Susceptibility Genes in the Idd Regions of NOD Mice 

by Temporal Microarray Gene Expression Data 

Analysis 

Keiichi Kodama, Postdoctoral Fellow, Stanford University, 

Stanford, CA, Demi Dang, Tech Person, Stanford University, 

Stanford, CA, Claire Holness, PhD, Stanford University, 

Stanford, CA, Hideyuki Iwai, MD. PhD, Stanford 

University, Stanford, CA, Mark Hartnett, Engineer, 

Agilent Technologies, Inc, Santa Clara, CA, Atul Butte, 

Assistant Professor, Stanford University, Stanford, CA, 

C. Garrison Fathman, Professor, Stanford University, 

Stanford, CA 

Using Agilent 41K mouse genome oligo-microarrays to 

study temporal gene expression, we identified candidate 

genes in the Idd disease susceptibility regions of NOD mice 

dysregulated by the pathophysiology of disease, not as 

mutations or polymorphic variants of disease susceptibility 

genes. Pancreatic lymph nodes (PLN) and spleen (SPL) were 

removed from 10-day, 4-week, 8-week, 12-week, 16-week, 

and 20-week-old NOD female mice, or as control from 10 day 

and 20-week-old genetically identical (except the MHC), 

NOD.B10Sn-H2b/J (NOD.B10) female mice (n¡Ü10). At 10 days 

to 4 weeks of age, coincident with the initiation of disease, 

there were ∼500 significantly dysregulated genes detected in 

the NOD PLN but none in the spleen, supporting the possibility 

that the PLN may be an important site for disease initiation. 

∼450 significantly dysregulated genes were seen in the PLN 

and ∼400 in splenic tissues from 12- to 20-week-old mice, 

suggesting that both tissues may be important in disease 

progression. b100 genes were simultaneously dysregulated in 

both the PLN and SPL during this period. Two of these, Ptpn22 

and Chi3l3, are located within the Idd 18.2 region. Expression 

changes for these two genes were confirmed by qPCR using 

the same mouse RNA samples as were used for the 

microarrays. This strategy may allow identification of Idd 

candidate genes that play critical roles in the induction or 

progression of autoimmune type 1 diabetes that are the 

consequence, not the cause of disease. 

doi:10.1016/j.clim.2007.03.222 

F.10 Peptide-based Immunotherapy for Type 1 

Diabetes; The Antigen, the Dose, the Immunization 

Route and the State of the Disease Can Make a 

Difference 

Georgia Fousteri, Postdoctoral Fellow, La Jolla Institute 

for Allergy and Immunology, La Jolla, CA, Amy Dave, 

Student, University of California, San Diego, La Jolla, CA, 

Michael Croft, Professor, La Jolla Institute for Allergy and 

Immunology, La Jolla, CA, Matthias von Herrath, 

Professor, La Jolla Institute for Allergy and Immunology, 

La Jolla, CA 

Immunization with beta-cell derived antigens such as 

insulin has been under intense investigation for treating or 

preventing T1D. This strategy is of particular interest since 

the administration of immunosuppressive drugs may be 

circumvented. We compared several islet-antigen derived 

peptides (insulin and IGRP) given alone or in combination 

by two different routes (intranasally [i.n.] or subcutaneously 

[s.c.]) to 10-week-old prediabetic NOD mice. 

Surprisingly, and in contrast to previous studies, we found 

that neither intranasal nor subcutaneous administration of 

human pro-insulin II peptide B24–C36 (hpII) affected T1D, 

whereas insB-chain 9–23 (B9–23) and its altered-peptide 

ligand (Ala 16, 19) (APL) accelerated diabetes development 

significantly following intranasal administration. Combination 

of the three CD4 epitopes did not affect T1D when 

administered by either route. When insB-chain 15–23 (B15– 

23) and mouse insB24–36 (B24–C36) CD8 epitopes were 

tested (s.c. route), B15–23 delayed and B24–36 accelerated 

diabetes. Overall the diabetes incidence reached 

similar levels in both groups. Lastly, when two IGRPderived 

altered peptide ligands NRPI4 and NRPV7 were 

compared for their protective effect following s.c. immunization, 

NRPV7 provided the greatest delay but without a 

long-term protection. In contrast to our present findings, 

mucosal immunization with whole oral or intranasal insulin 

or insB9–23 peptide combined with suitable adjuvants (i. 

e., IFA) was able to prevent diabetes efficiently in previous 

studies, especially when given at a much earlier stage (4to 

6-week-old NODs). It will be important to consider the 

issues of age, route of administration and adjuvants for the 

design of future clinical trials. 

doi:10.1016/j.clim.2007.03.223 

S19 

F.11 Monocyte Immunophenotypes in Type 1 

Diabetes 

Erik Fung, DRF Fellow, Department of Medical Genetics, 

University of Cambridge, England, Cambridge, England, John 

A. Todd, Professor and Director of Laboratory, Department 

of Medical Genetics, University of Cambridge, England, 

Cambridge, England, Linda S. Wicker, Professor and 

Co-Director of Laboratory, Department of Medical Genetics, 

University of Cambridge, England, Cambridge, England 

Type 1 diabetes (T1D) is a complex-trait disease that 

results from interaction of genes and environmental 

exposures leading to immune destruction of pancreatic 

islet cells. Previous studies have indicated that monocytes

S20 Abstracts 

may be altered in chronic T1D. However, it is still unclear if 

this immune phenotypic variation is a cause or effect of 

T1D. Using polychromatic flow cytometry to analyze whole 

blood, we have extended to pediatric T1D patients the 

findings by others that adult T1D cases have increased 

expression of CD11b on monocytes as compared to 

apparently healthy non-T1D adults. We examined 37 T1D 

patients (age 9.2–18.3 years, median 14.4 years; median 

time past diagnosis for non-newly diagnosed cases=4.3 

years) and 63 healthy volunteers (age 9.9–60 years, median 

34 years). CD11b expression (MFI) in HLA-DR+ monocyte 

subsets were as follows: on CD16+ monocytes, 265±20 vs. 

218±10 (pediatric T1D cases vs. non-T1D adults); CD16+ 

CD14+ monocytes, 1108 ±51 vs. 840±27; CD14++ ‘inflammatory’ 

monocytes, 1479±38 vs. 1146±54. Expression 

levels of CCR2 on CD14++ monocytes were lower in T1D 

patients than in healthy volunteers (MFI 62±2 vs. 75±2), 

consistent with an activated monocyte phenotype. CD11b 

and CCR2 expression differences were independent of ages 

at diagnosis or at immunophenotyping. In order to help 

distinguish between cause and effect and begin to explore 

the possibility that extremes of these phenotypes may be 

precursors to disease, we will analyze age-matched 

subjects and unaffected siblings, and test the association 

of alleles of known T1D susceptibility genes (e.g., HLA 

haplotypes, INS, PTPN22, CD25, IFIH1, CTLA4) with monocyte-subset 

phenotypes. 

doi:10.1016/j.clim.2007.03.224 

F.12 A High Specificity Competitive Europium Based 

Assay for Autoantibodies of APS1 Patients Reacting 

with Interferon Alpha 

Li Zhang, Fellow, Barbara Davis Center, Denver, CO, 

Raffaele Badolato, MD, PhD, Department of Pediatrics, 

University of Brescia, Brescia, Jennifer Barker, Assistant 

Professor of Pediatrics, Barbara Davis Center for Childhood 

Diabetes, Denver, CO, Maureen Su Pra, University of 

California, San Francisco Diabetes Center, San Francisco, 

CA, Sunanda Babu, Research Associate, Barbara Davis 

Center, Denver, CO, Mickie Cheng, MD, PhD, University of 


CA, Tony Shum, MD, University of California, San Francisco 

Diabetes Center, San Francisco, CA, Ehud Zamir, MD, The 

Royal Victorian Eye and Ear Hospital, Victoria, BC, Canada, 

Adam Law, MD, Ithaca Medical School, Ithaca, NY, George 

Eisenbarth, Executive Director, Barbar Davis Center, Denver, 

CO, Mark Anderson, Assistant Professor, University of 

California, San Francisco Diabetes Center, San Francisco, CA 

IFN-α autoantibodies have been described in autoimmune 

polyglandular syndrome type 1 (APS1) patients. We have 

established a competitive Europium time resolved fluorescence 

assay for autoantibodies to IFN-α and have tested it in 

a cohort of APS1 patients. Methods: The europium-ELISA 

method utilizes plate bound IF α incubated with or without 

competition with fluid phase IFN-α and sera. Anti-IgG 

biotinylated antibody is added, followed by incubation with 

streptavidin–europium and detection with time resolved 

fluorescence (measured in counts per second, CPS). Seven 

APS1 patients, 6 relatives, 71 non-APS1 Addison patients and 

141 T1DM patients were tested as well as 100 normal 

controls. The APS1 patients had multiple different mutations 

in the AIRE gene. Results: Normal control CPS without 

competition was 31,237∼17,328 CPS and delta with competition 

was −6563∼10,303 CPS. The initial APS1 patient (used 

to create the index, index 1.0) gave 394,063 CPS without 

competition and a delta of 363,662∼31,587 CPS with 

competition. Scatchard plot analysis revealed a high avidity 

(Kd of 0.5 nM). The CPS, delta and index for 6/7 APS1 

patients were strongly positive and above 3 standard 

deviations of controls. Relatives were negative as were 71 

Addison disease (non-APS-1) and 141 T1DM patients. The one 

negative APS1 patient is presumed APS1 based on clinical 

history. The assay has a sensitivity of 86% or greater and 

specificity greater than 99.5%. Conclusion: IFN-α autoantibodies 

are detected specifically in APS1 patients with 

specificity greater than 99% using a competitive Europium 

time resolved fluorescence assay. 

doi:10.1016/j.clim.2007.03.225 

F.13 Validation of Anti-Idiotypic Bridging ELISA to 

Quantitate TRX4 (Anti-Human CD3) Mab Levels in 

Human Serum 

Francis Carmody, Preclinical Scientist III, Preclinical 

Development, Cambridge, MA, Michael Slavonic, Preclinical 

Scientist II, Tolerx, Preclinical Development, Cambridge, MA, 

Adam O’Shea, Research Scientist II, Tolerx, Applied Research, 

Cambridge, MA, Michael Paglia, Senior Manager Manufacturing, 

Tolerx, Manufacturing, Cambridge, MA, Reema Gulati, 

Research Scientist II, Tolerx, Applied Research, Cambridge, MA, 

Sharon Li, Former Tolerx Employee, Applied Research, 

Cambridge, MA, Herman Waldmann, Professor of Pathology, 

Head of Department, University of Oxford, Sir William Dunn 

School of Pathology, Cambridge, MA, Michale Rosenzweig, 

Director Preclinical Development, Tolerx, Preclinical 

Development, Cambridge, MA 

TRX4 is an aglycosyl anti-human CD3μ monoclonal antibody 

(Mab) that is being evaluated as a therapy for autoimmune 

disorders such as new-onset type 1 diabetes. Our goal was to 

develop a sensitive, high throughput assay to quantitate TRX4 

serum levels that could be used as an alternative to a cell-based 

assay that had been used in previous studies. Historically, TRX4 

was detected with a polyclonal anti-human IgG after it bound to 

the human T cell line, HuT 78. This method was both laborious 

and lacked sufficient sensitivity. Two different approaches were 

used to raise monoclonal antibodies to TRX4 complementarity 

determining regions (CDRs). First, transgenic human CD3 mice 

were tolerized to human IgG by two 1 mg IV doses and then 

subsequently immunized with 100 μg IP of TRX4. For the second 

strategy, BALB/c mice were administered 100 μg SC of TRX4 F 

(ab)′2 fragments in incomplete Freund’s adjuvant. These 

independent immunization strategies produced two non-competing 

anti-idiotypic TRX4 Mabs that satisfied specificity 

criteria. Significant serum interference was observed with 

both the Mabs when used individually in ELISAs. However, 

when used together, these anti-idiotypic antibodies were able to 

capture and detect TRX4 in whole sera with no detectable

Abstracts 

enhancement or inhibition due to serum alone. Assay validation 

has confirmed the accuracy (±20%), precision (±20%), specificity 

and robustness necessary to fulfil the ng/mL detection goal 

sought. Once validated, the ELISA was utilized as a crossover 

analysis between preclinical and clinical programs in support of 

our TRX4 development program. 

doi:10.1016/j.clim.2007.03.226 

F.14 Regulation of Insulin Expression in Thymic 

Epithelial Cells 

Dina Levi, PhD Candidate, McGill University, Human 

Genetics, Pointe-Claire, QC, Canada, Michael Palumbo, PhD 

Candidate, McGill University, Experimental Medicine, 

Montreal, QC, Canada, Constantin Polychronakos, Principal 

Investigator, Montreal Children’s Hospital Research 

Institute, Montreal, QC, Canada 

ThethymushasanessentialfunctionintheselectionofT 

cells with a properly formed TCR and the elimination of selfreactive 

ones. It has been found that tissue specific selfantigens 

are expressed in thymic epithelial cells, specifically 

in the medulla, for the purpose of presentation to thymocytes. 

Insulin is an important self-antigen that is implicated in the 

destruction of the pancreatic beta-cells in type 1 diabetes. We 

have isolated and cultured insulin-expressing (as well as 

insulin-non-expressing) epithelial clones from the medulla of 

the thymus (mTECs), which provide an exclusive in vitro 

model to study self-antigen expression in the thymus. 

Preliminary studies show a number of tissue specific selfantigens 

overexpressed by microarray gene expression profiling, 

in both insulin-expressing and non-expressing clones, with 

the majority of tissues being represented. Stimulation of 

mTECs with IFN- 3 and TNF-± cytokines demonstrates a pattern 

of decrease in insulin expression by real-time RT-PCR, 

indicating a possible regulation mechanism. Furthermore, 

co-culturing mTECs with isolated thymocytes results in an 

increase in insulin expression when cells are in direct contact. 

This interaction is supposed essential for the proper selection 

of non-autoreactive T lymphocytes in order to prevent 

autoimmune disease. Therefore, the regulation of insulin 

self-antigen expression in the thymus is an essential mechanism 

involved in negative selection and central tolerance and 

necessitates further studies. 

doi:10.1016/j.clim.2007.03.227 

F.15 Dysregulation of the TIM-3-Galectin-9 Pathway 

in Type I Diabetic T Cells 

William Hastings, Postdoctoral Fellow, Brigham and 

Women’s Hospital/Harvard Medical School, Center for 

Neurologic Diseases, Boston, MA, Allison Greer, Research 

Technician, Brigham and Women’s Hospital/Harvard 

Medical School, Center for Neurologic Diseases, Boston, MA, 

Vijay Kuchroo, Professor, Brigham and Women’s Hospital/ 

Harvard Medical School, Center for Neurologic Diseases, 

Boston, MA, David Hafler, Professor, Brigham and Women’s 

Hospital/Harvard Medical School, Center for Neurologic 

Diseases, Boston, MA, David Anderson, Instructor, Brigham 

and Women’s Hospital/Harvard Medical School, Center for 

Neurologic Diseases, Boston, MA, Sally Kent, Professor, 

Brigham and Women’s Hospital/Harvard Medical School, 

Center for Neurologic Diseases, Boston, MA 

Our laboratory has generated, in a non-biased manner, a 

large panel of T cell clones from the pancreatic lymph nodes 

(PLN) of type 1 diabetic (T1D) subjects and we asked if there 

were alterations in function of T cells from the PLN of T1D 

subjects versus those from controls. In this regard, we have 

recently shown that TIM-3, a molecule associated with negative 

regulation of fully differentiated Th1 but not Th2 cells, is 

dysregulated on T cell clones isolated from the CSF of MS 

patients. In light of this, we analyzed Tcell clones from the PLN 

of T1D as well as from normal controls to determine if there are 

similar alterations in expression and/or function of TIM-3 and its 

ligand Galectin-9 in these cells. We report that PLN clones from 

T1D subjects express lower levels of Galectin-9 but not TIM-3 

mRNA than clones from normal controls, as analyzed by Taqman 

PCR. In addition, we show that blockade of TIM-3 signaling with 

antibody increased proliferation and IFNg secretion in PLN Tcell 

clones. Interestingly, FACS analysis of PLN cells from controls and 

T1D subjects showed that TIM-3 is more highly expressed than on 

comparable subpopulations of T cells present in peripheral 

blood. Gene chip analysis has identified key differences in gene 

expression between TIM-3+ and TIM-3− T cell populations. 

Collectively, these data contribute to our understanding of how 

TIM-3 may regulate human T cell function and impact type 1 

diabetes. 

doi:10.1016/j.clim.2007.03.228 

S21 

F.16 huFlt3-L Treatment Does Not Protect RIP-GP 

and RIP-NP C57B1/6 Mice Against LCMV-induced 

Diabetes 

Tom Van Belle, Postdoctoral Fellow, La Jolla Institute for 

Allergy and Immunology, La Jolla, CA, Eleanor Ling, PhD, La 

Jolla Institute for Allergy and Immunology, La Jolla, CA, Lisa 

Togher, Research Assistant, La Jolla Institute for Allergy and 

Immunology, La Jolla, CA, Matthias von Herrath, PI, La Jolla 

Institute for Allergy and Immunology, La Jolla, CA 

Human Fms-like Tyrosine Kinase3-L (huFlt3-L) treatment of 

prediabetic NOD mice has been shown to increase the 

intrinsically low numbers of myeloid dendritic cells in these 

mice, while decreasing insulitis and delaying the progression of 

diabetes. In this study, we investigated the influence of huFlt3-L 

treatment on insulitis and diabetes onset in the LCMV-induced 

RIP-GP (fast onset) or RIP-NP (slow onset) C57Bl/6 mouse models 

of T1D. We showed that these mice have no intrinsic abnormality 

in DC subset development during LCMV infection. As expected, 

huFlt3-L treatment dramatically increased the total DC fraction 

and numbers in both spleen and pancreatic draining LN, with a 

preferential outgrowth of myeloid DCs (CD11c+B220−CD11b+) 

over plasmacytoid DCs (CD11c+B220+CD11b−). Moreover, NK 

cells and NK-DC cells were also increased, but CD4 and CD8 Tcell 

fractions were reduced. Strikingly, repeated huFlt3-L administrations, 

initiated in the prediabetic phase, did not protect 

against diabetes onset in this model. It is possible that this is

S22 Abstracts 

correlated with the observed reduced fraction of CD4+CD25+ T 

cells in the huFlt3-L-treated mice. Translation of these data 

suggests that huFlt3-L might only be a useful therapeutic agent 

for T1D patients with a demonstrated DC (subset) defect. 

doi:10.1016/j.clim.2007.03.229 

F.17 The BDC 12-4.1 TCR Alpha Chain is Sufficient to 

Generate Anti-Insulin Autoantibodies; α+β Chain 

for Insulitis 

Masakazu Kobayashi, Fellow, Barbara Davis Center, Aurora, 

CO, Jean Jasinski, Graduate Student, Barbara Davis Center, 

Aurora, CO, Maki Nakayama, Fellow, Barbara Davis Center, 

Aurora, CO, Dongmei Miao, PRA, Barbara Davis Center, 

Aurora, CO, Marcella Li, PRA, Barbara Davis Center, Aurora, 

CO, Liping Yu, PRA, Barbara Davis Center, Aurora, CO, Edwin 

Liu, Associate Professor, Barbara Davis Center, Aurora, CO, 

George Eisenbarth, PI, Barbara Davis Center, Aurora, CO 

There is a marked T cell receptor (TCR) α chain restriction 

of T cell clones that target the insulin peptide B:9–23 in NOD 

mice. We are pursuing the hypothesis that this non-stringent 

TCR with conserved V α (TRAV5D-4*04) and J α (TRAJ53*01) 

chains (not N of α or β chain) underlies insulin autoimmunity 

and diabetes propensity. To test this hypothesis, we created 

TCR transgenic mice bearing only the BDC12-4.1 α chain, 

combined with C α knockout. Mice with the BDC 12-4.1 α 

chain, regardless of the presence or absence of the BDC 12-4.1 

TCR β chain, produced insulin autoantibodies, whereas mice 

bearing BDC 12-4.1 TCR β chain but not α chain did not. In 

contrast to production of insulin autoantibodies, we found 

insulitis in only mice with both 12-4.1 α and β chains (C α TCR 

knockout). Our results indicate that provision of only the 

BDC12-4.1 α chain results in expression of insulin autoantibodies 

(with endogenous β chains) and the ββ chain 

contributes to development of insulitis. At present we are 

producing mice with BDC12-4.1 α chain (Cα knockout) and β 

chain from anti-ovalbumin DO.11.10 T cell clone, which also 

produce insulin autoantibodies, but we need to combine Vβ 

knockout. A simple TCR motif and single insulin peptide may 

be essential for anti-insulin/anti-islet autoimmunity of the 

NOD mouse. 

doi:10.1016/j.clim.2007.03.230 

F.18 HLA-DR3/4 Heterozygosity is a Risk Factor for 

Autoantibody Conversion and Type 1 Diabetes 

Recurrence After Simultaneous Pancreas-Kidney 

Transplantation 

Stavros Diamantopoulos, Fellow, Diabetes Research 

Institute and Department of Pediatrics, University of 

Miami, Miami, FL, Gloria Allende, Research Associate, 

Diabetes Research Institute, Miller School of Medicine, 

University of Miami, Miami, FL, George W. Burke, Professor 

and Co-Director, Division of Transplantation, Department of 

Surgery, Miller School of Medicine, University of Miami, 

Miami, FL, Gaetano Ciancio, Professor and Director of 

Transplant Education, Department of Surgery, Miller School 

of Medicine, University of Miami, Miami, FL, Alberto 

Pugliese, Director, Division of Immunogenetics, Diabetes 

Research Institute, Miller School of Medicine, University of 

Miami, Miami, FL 

We studied 252 patients with type 1 diabetes (T1D) who 

received a simultaneous pancreas-kidney (SPK) transplant. 

Patients were tested for GAD and IA-2 autoantibodies, wellknown 

T1D risk markers. One-hundred-thirty-one patients (52%) 

remained GAD/IA-2 autoantibody-negative; 44 (17.4%) converted 

to GAD and/or IA-2 positivity while 77 (30.6%) had 

autoantibodies before the transplant that persisted on followup. 

We then analyzed recipients' HLA-DR types in relation to 

autoantibody conversion and T1D recurrence (T1DR) on followup 

(range 0.08-15.9 years, mean 5.6+3.6 years) in the 175 

patients who remained autoantibody-negative or converted. 

Twenty of 44 (45%) converters carried the HLA-DR3/4 genotype 

vs. 22/131 (17%) autoantibody-negative patients (p=0.0001, 

OR=4.5). Conversion occurred in 20/42 (47.6%) patients with the 

HLA-DR3/4 genotype vs. 24/133 (18%) patients without it 

(p=0.0001, Kaplan-Meier lifetable analysis; p=0.0002. OR=4.2, 

Chi-square). The non-heterozygotes included 53 patients with 

HLA-DR3 alone, 53 with HLA-DR4 alone and 23 with neither 

types. Excluding patients that did not carry HLA-DR3 or HLA- 

DR4, HLA-DR3/4 heterozygotes converted more often than 

patients carrying HLA-DR3 or HLA-DR4 alone (p=0.0001, Kaplan- 

Meier lifetable analysis; p=0.0002, OR=4.4, Chi-square). Ten 

patients developed T1DR on follow-up, of whom 60% were HLA- 

DR3/4 heterozygotes: 6/42 (14%) patients with the HLA-DR3/4 

genotype developed T1DR vs. 4/133 (3%) without it (p=0.01, 

Kaplan-Meier lifetables analysis; p=0.01, OR=5.37, Chi-square). 

Our data suggest that the HLA-DR3/4 genotype is a risk factor for 

autoantibody conversion and for the development of disease 

recurrence in SPK recipients. Patients with this genotype 

represent a high-risk group that should be monitored for 

markers of recurrent autoimmunity. 

doi:10.1016/j.clim.2007.03.231 

F.19 Kinetics of Regulatory T Cells Upon Specific 

Diabetogeneic Activation in a Group of High Risk 

Relatives of Type 1 Diabetes Mellitus Patients in 

Comparison with Healthy Controls 

Zuzana Vrabelova, PhD Student, University Hospital Motol, 

Children’s Neurology Department, Prague, Zuzana 

Hladikova, Statist, Czech Agriculture University, 

Department of Statistics, Prague, Katerina Stechova, Lab 

Chief, University Hospital Motol, Pediatric Department, 

Prague, Kristyna Bohmova, PhD Student, University Hospital 

Motol, Pediatric Department, Prague, Jaroslav Michalek, 

Head of Cell Immunotherapy Center, Masaryk University Br 

England Cell Immunotherapy Center, England 

Abnormalities in CD4+CD25+ regulatory Tcells (Tregs) may 

contribute to type 1 diabetes (T1D) development. We have 

studied immunoregulatory populations of CD4+CD25+ T cells 

in a group of high-risk relatives of T1D patients. We have 

evaluated kinetics of Tregs (using specific markers CD127− 

and Foxp3+) after specific stimulation with diabetogenic 

autoantigens in 11 high-risk (according to HLA-linked T1D 

genetic risk) relatives of T1D patients and 14 healthy

Abstracts 

controls. For stimulation specific amino acid sequences of 

glutamic acid decarboxylase 65 (GAD65), thyrosine phosphatase 

(IA2), beta-proinsulin chain and whole molecule of 

insulin were used. Cell activation was measured by surface 

expression of interferon gamma (IFN-g) on activated CD4+ T 

cells. High-risk relatives of T1D patients have significantly 

lower pre- and post-stimulatory numbers of Tregs than 

healthy controls (pb0.05). The autoantigen activation of 

diabetogenic T cells was significantly higher in high-risk 

relatives of T1D patients than in healthy controls (pb0.02). 

In conclusion, defects of CD4+CD25+ regulatory T cells have 

been proved in individuals at increased genetic risk for T1D. 

Supported by IGA MZCR NR 9355-3. 

doi:10.1016/j.clim.2007.03.232 

F.20 Differential Immune Recognition of Heat-Shock 

Protein 60 Epitopes in Type 1 Diabetes 

Sylvia Kamphuis, Pediatric Immunologist, Department of 

Paediatric Immunology, Erasmus MC Sophia Children’sHospital, 

Rotterdam,GijsTeklenburg,MD,PediatricImmunology,UMC 

Utrecht, Utrecht, Annemarie Verrijn Stuart, Pediatric 

Endocrinologist, Department of Pediatric Endocrinology, UMC 

Utrecht, Utrecht, Irun Cohen, MD PhD, Department of 

Immunology, Weizmann Institute of Science, Rehovot, Wilco de 

Jager, Msc, Department of Immunology, UMC Utrecht, Utrecht, 

Wietse Kuis, MD PhD, Department of Immunology, UMC Utrecht, 

Utrecht, Salvatore Albani, MD PhD, Department of Pediatrics 

and Medicine, University of California, San Diego, CA, Berent 

Prakken, MD PhD, Department of Immunology, UMC Utrecht, 

Utrecht, Mark Klein, Msc, Department of Immunology, UMC 

Utrecht, Utrecht 

Aim: Evidence is cumulating for an immunoregulatory role of 

heat-shock proteins (HSP) in an increasing number of autoimmune 

diseases. Previously we identified pan-DR binding 

HSP60 epitopes that were recognized in juvenile idiopathic 

arthritis, an autoimmune disease characterized by chronic 

joint inflammation. The present study was performed to test 

whether these HSP60 epitopes were recognized in children 

with type 1 diabetes as well. Methods: We analyzed the 

pattern of HSP60 peptide-specific cytokine and chemokine 

responses in peripheral blood mononuclear cells of 18 type 1 

diabetes patients with primarily longstanding disease. In 

addition, a subgroup of newly diagnosed patients was followed 

over time for HSP60 peptide-specific immune responses. 

Results: We found peptide-specific induction of 8 cytokines 

(IL-1a, IL-1b, IL-6, IL-10, IL-17, IL-18, TNF-a, IFN-g) and 5 

chemokines (MIP-1a, MDC, IL-8, IP10, and OSM). Considerable 

variation in peptide-induced cytokine and chemokine profiles 

however was seen in the individual patients, irrespective of 

age and disease duration. When following newly diagnosed 

patients over time, we found that the presence of T cell 

proliferative responses was almost exclusively seen in the 

patients that experienced a partial remission phase, with 

concomitant induction of IL-10. Conclusion: The identified 

HSP60 epitopes are immunogenic in children with type 1 

diabetes. The recorded correlation of peptide-specific T cell 

responses with (temporary) disease remission underscores the 

alleged immunomodulating role of HSP in chronic inflammation. 

This makes these HSP60 epitopes promising candidates 

for antigen-specific immunotherapy in not only juvenile 

idiopathic arthritis, but also in type 1 diabetes and possibly 

other autoimmune diseases. 

doi:10.1016/j.clim.2007.03.233 

F.21 Blocking the Toll Like Receptor 9 Signal Inhibits 

Activation of Diabetogenic CD8 T Cells and Delays 

Autoimmune Diabetes in NOD Mice 

Yiqun Zhang, Research Associate, Child and Family Research 

Institute, University of British Columbia, Vancouver, BC, 

Canada, Xuan Geng, Research Assistant, Kinesiology, Simon 

Fraser University, Burnaby, BC, Canada, Pere Santamaria, 

Professor, Microbiology and Infectious Diseases, The University 

of Calgary, Calgary, AB, Canada, Diane Finegood, Professor, 

Kinesiology, Simon Fraser University, Burnaby, BC, Canada, Jan 

P. Dutz, Professor, CFRI, University of British Columbia, 

Vancouver, BC, Canada 

Type 1 diabetes is an autoimmune disease in which pancreatic 

beta cells are destroyed by CD8 T cells. Dendritic cells 

(DC)s are capable of priming CD8 Tcells. Toll-like receptor (TLR) 

triggering can lead to DC maturation. The contributions of TLR 

signals to spontaneous diabetes development are still largely 

unknown. We found that non-obese diabetic (NOD) mouse bone 

marrow derived DCs (BMDC)s pulsed with freeze–thawed NIT1 

insulinoma cells in the presence of TLR9 agonist CpG and CD40 

ligation were able to fully prime 8.3 TCR transgenic and 

diabetogenic CD8 T cells. Addition of the TLR9 inhibitors ODN 

2088 or chloroquine potently inhibited CD8 T cell CD25 

upregulation and intracellular IFN-γ production in response to 

CPG but not LPS. Furthermore, ODN 2088 or chloroquine 

inhibited CPG-induced BMDC CD40 upregulation and IL-12 p70 

production. In vivo, CPGaloneorwithanti-CD40induced8.3 

CD8 T cell CD69 upregulation and CTL activity that triggered 

rapid diabetes development in 8.3 NOD mice. Pre-treatment 

with ODN 2088 inhibited CPG-induced diabetes and treatment 

with either ODN 2088 or chloroquine delayed spontaneous 

diabetes in 8.3 NOD mice. Finally, administration of chloroquine 

inhibited spontaneous diabetes in NOD mice, down-regulated 

CD40 expression on pancreatic DCs, and inhibited serum rises of 

IL-12 p70, IL-6, IFN-γ andMCP-1inducedbyCPGbutnotLPS. 

Thus, TLR9 activation may contribute to the spontaneous onset 

of CD8 T cell autoimmunity in NOD mice and TLR9 family 

inhibitors may be used to prevent and delay autoimmune 

diabetes in disease-prone animals. 

doi:10.1016/j.clim.2007.03.234 

S23 

F.22 Genome-Wide Association Scan for Type 1 

Diabetes Susceptibility Genes in a Danish Population 

Caroline Brorsson, PhD Student, Steno Diabetes Center, 

Gentofte, Elzbieta Swiergala, Lab Manager, Department of 

Medical Genetics, University of British Columbia, 

Vancouver, BC, Canada, Kristoffer Rapacki, Computer 

Scientist, Center for Biological Sequence Analysis, Technical 

University of Denmark, Lyngby, Regine Bergholdt, Research 

Fellow, Steno Diabetes Center, Gentofte, Shaun Purcell, 

Assistant Professor, Center for Human Genetic Research,

S24 Abstracts 

Massachusetts General Hospital, Boston, MA, Leigh Field, 

Professor, Department of Medical Genetics, University of 

British Columbia, Vancouver, BC, Canada, Flemming Pociot, 

Professor, Steno Diabetes Center, Gentofte 

Type 1 diabetes (T1D) is a complex disease believed to 

result from interactions between multiple risk-conferring 

genes and environmental factors. In order to further 

investigate the genetic contribution to T1D we genotyped a 

case–control material for 58,000 SNP and tested these for 

association to disease. The first phase of the study comprised 

of thorough quality tests of the data for issues such as sample 

contamination, inbreeding, low genotyping rates, relatedness 

and population stratification which led to samples being 

excluded. SNPs were removed for failing HWE in controls, low 

genotyping rate, differences in missing genotyping rates 

between cases and controls and being non-polymorphic, 

which contributed to significant false positive findings. After 

ensuring the highest quality of the data-set the remaining 

52,000 SNPs and 605 individuals were analysed using basic 

allelic and model-based tests of association corrected for 

multiple testing and using permutation with empirically 

corrected p-values. Furthermore we performed genomewide 

sliding-window haplotype tests and full pair-wise test 

for epistasis across the genome. We also performed the same 

analyses for markers in 12 linkage regions previously reported 

in T1D. Every individual test confirmed the importance of the 

HLA region on chromosome 6p21.3 for developing T1D. In 

combination these tests point to other chromosomal regions 

contributing to T1D susceptibility. The significant findings 

will be followed up in a larger case–control material to 

confirm these present results. In addition this study has 

demonstrated the importance of high quality data for 

avoiding false positive findings when performing association 

analyses in complex diseases. 

doi:10.1016/j.clim.2007.03.235 

F.23 iNKT Cell Regulation of Type 1 Diabetes 

Dalam Ly, PhD Candidate, Robarts Research Institute, 

London, ON, Canada, Qing Sheng Mi, Research Associate, 

Robarts Research Institute, London, ON, Canada, Shabbir 

Hussain, Research Associate, Robarts Reseach Institute, 

London, ON, Canada, Terry L. Delovitch, Professor, Robarts 

Research Institute, London, ON, Canada, Steven A. Porcelli, 

Professor, Albert Einstein College of Medicine, Bronx, NY 

The pathogenesis of type 1 diabetes (T1D) in NOD mice is 

mediated by numerical and functional deficiencies in both 

CD4+CD25+FoxP3+ regulatory T cells (Tregs) and invariant 

natural killer T (iNKT) cells. Previously, we reported that 

protection of NOD mice from T1D can be achieved by 

activation of iNKT cells upon treatment with a multi-dose agalactosylceramide 

(a-GalCer) protocol that promotes preferential 

IL-4 secretion by iNKT cells. Since activated Tregs 

and iNKT cells are both self-reactive and protect NOD mice 

from T1D, we investigated whether iNKT-Treg collaboration 

is required for this protection. We recently reported that 

while a-GalCer therapy does not alter the proliferation or 

regulatory activity of Tregs, Treg activity is required for a- 

GalCer induced protection from T1D. This requirement of 

Treg/iNKT collaboration for iNKT-mediated protection from 

T1D may not apply for the therapy of T1D provided by certain 

analogs of a-GalCer. Of particular interest is C20:2, an a- 

GalCer analog with a shortened (C20) acyl chain and two 

unsaturated bonds. When compared to a-GalCer, C20:2 yields 

significantly reduced IFN-g and enhanced IL-4 secretion by 

iNKT cells. Interestingly, we found that Treg activity is not 

required for C20:2 mediated protection from T1D by iNKT 

cells. Thus, our current results indicate that a-GalCer and 

C20:2 may differ in their requirement of Tregs for iNKT 

mediated protection from T1D. Ongoing studies will address 

the mechanism(s) that underlies this differential requirement 

of Tregs for protection from T1D. 

doi:10.1016/j.clim.2007.03.236 

F.24 Absence of Beta Cells in Long-Term Type 1a 

Diabetic Patients 

Travis Still, Professional Research Assistant, Barbara Davis 

Center, Aurora, CO, Sally Kent, Instructor, Brigham and 

Women’s Hospital, Boston, MA, Alberto Pugliese, Diabetes 

Research Institute, University of Miami, Miami, FL, Piero 

Marchetti, Full Professor, Ospedale di Cisanello, Pisa, Italy, 

Francesco Dotta, Full Professor, University of Siena, Siena, 

Bernhard Hering, Full Professor, Diabetes Institute for 

Immunology and Transplantation, Department of Surgery, 

University of Minnesota, Minneapolis, MN, Norma Kenyon, 

Full Professor, Diabetes Research Institute, Cell Transplant 

Center, University of Miami, Miami, FL, Camillo Ricordi, 

Full Professor, Diabetes Research Institute, Cell Transplant 

Center, University of Miami, FL, Miami, FL, Roberto 

Gianani, Assistant Professor, Barbara Davis Center For 

Childhood Diabetes, Aurora, CT 

A recent report indicates that residual beta cells can be 

identified in the pancreas of long-term diabetic subjects by 

insulin immunostaining and in a subset of individuals by 

detection of C-peptide. We have analyzed pancreata from 20 

normal controls and 4 long-term diabetic subjects (1.5, 5, 22 

and 30 years post diagnosis) by immunohistochemistry for 

both insulin and the beta cell specific marker VMAT-2. We 

confirmed the beta cell specificity of VMAT-2 by triple 

immunostaining with glucagon, somatostatin and VMAT-2 of 

normal human pancreas. 

In the pancreata from long-term diabetic subjects we did 

not find any VMAT-2 or insulin positive cells. In contrast 

abundant insulin (as expected) and VMAT-2 positive cells 

were present in all 20 normal human pancreata, in one new 

onset type 1a and in a type 2 diabetic patient. There was 

significant heterogeneity of staining intensity for VMAT-2 

amongst the normal control pancreata suggesting individual 

variability in VMAT-2 islet expression. Furthermore, there 

was also variability in VMAT-2 expression between different 

areas within the same pancreas with a zonal distribution of 

strongly stained islets. These data indicate that in subjects 

with long-term type 1A diabetes, there are no residual beta 

cells by immunohistochemistry. Further studies of a larger 

series of patients are needed to establish whether beta cells 

continue to be present in type 1a diabetic subjects. In

Abstracts 

particular, it will be important to study pancreata from longterm 

diabetic subjects with documented C-peptide secretion 

(not available for these subjects). 

doi:10.1016/j.clim.2007.03.237 

F.25 Regulation of Pathogenicity Through CD40 

Expressed on CD4 T Cells 

Rocky Baker, Postdoctoral Fellow, Department of 

Immunology, University of Colorado Health Sciences Center, 

Denver, CO, David Wagner, Assistant Professor, Department 

of Immunology, University of Colorado Health Sciences 

Center, Denver, CO, Kathryn Haskins, Professor, NJMRC, 

Denver, CO 

We have reported previously that the costimulatory 

molecule CD40 is a marker of autoreactive T cells found on 

diabetogenic T cell clones and that CD40+ T cells from the 

NOD mouse transfer diabetes. Although there is strong 

evidence that CD40 is functional on T cells, CD40 expression 

on T cells remains controversial because of typically low and 

variable levels of CD40 on primary T cells. We have 

investigated the conditions under which T cells express the 

CD40 molecule and how CD40 expression contributes to 

pathogenicity of autoreactive T cells. First, we have 

demonstrated that CD40 levels on primary T cells can be 

induced on 90% of CD4+CD40− T cells from NOD mice upon 

activation. Furthermore, comparing naive and primed T cells 

from different TCR-Tg NOD mouse models, we have found 

that the ability to express CD40 depends on the activation 

status of the T cell. Second, we investigated how CD40 

costimulation influences the effector function of CD4 T cells 

by comparing CD28 and CD40 costimulation in NOD mice. We 

have found that CD40 engagement can replace or synergize 

with CD28 costimulation in terms of T cell activation and 

proliferation. However, there is a major difference when 

costimulation occurs through CD40: our results indicate that 

CD40 costimulation influences the Th1/Th2 balance differently 

and that CD40 engagement on autoreactive T cells 

enhances a Th1 effector phenotype. Taken together, the data 

indicate that CD40 is an effective alternative pathway to 

CD28 costimulation and might regulate the pathogenicity of 

CD4 T cells in T1D. 

doi:10.1016/j.clim.2007.03.238 

F.26 TRECS and Telomerase Analysis of Lymphocytes 

from Autoimmune Thyroid Disease Patients Point to 

the Migration of Recent Thymic Emigrants to the 

Thyroid Gland at the First Stages of Disease 

Ricardo Pujol Borrell, Professor, Blood and Tissue Bank 

(LIRAD), Health Science Research Institut, Badalona, Marco 

Antonio Fernandez, Head Cytometry Facility, Health Science 

Research Institut, Badalona, Maria Pilar Armengol, 

Posdoctoral Fellow, Blood and Tissue Bank (LIRAD), Health 

Science Research Institut, Badalona, Manel Juan, Head HLA 

Laboratory, Blood and Tissue Bank (LIRAD), Health Science 

Research Institut, Badalona, Lidia Sabater, Research Fellow, 

Blood and Tissue Bank (LIRAD), Health Science Research 

Institut, Badalona, Ricardo Pujol Borrell, Head Immunology 

Laboratory, Blood and Tissue Bank (LIRAD), Health Science 

Research Institut, Badalona 

According to the threshold hypothesis of autoimmunity, 

the abundance and affinity of autoreactive T cells in the 

repertoire determine the probability for the triggering of 

autoimmune diseases. We have investigated how recent 

thymic emigrants may contribute the autoreactive repertoire 

in autoimmune thyroid disease (AITD) patients, by measuring 

TRECs in PBLs from AITD patients (n=50) and controls (n=55) 

and in intrathyroidal lymphocytes from autoimmune thyroid 

glands. TRECS were higher in AITD patients, especially in the 

N45-year-old patients. The analysis of TRECS in intrathyroidal 

lymphocytes (ITL) (n=45) in relation to disease duration 

revealed a distinct pattern of distribution: TRECS were higher 

in ITL than in PBL in b30-month-disease-duration patients 

(fourfold excess in average), and vice-versa N30-month 

patients (tenfold excess). Interestingly there was a correlation 

between the levels of TRECS in ITLs and PBLs in both 

groups but of different sign and intensity: it was stronger in 

the N30 months (r=0.94) than in the b30 months (r =0.535). 

This may indicate that while in the N30 months group the 

TRECs in ITLs is dependent on the TRECs in PBLs, this is not the 

case in the b30 months. The study of the telomere length did 

not reveal a relation between TRECs and telomere length in 

the T cells but was significantly shorter in ITLs cells than in 

PBLs. These results suggest that early in disease there is a 

migration to AITD glands of T lymphocytes newly generated in 

the thymus that then undergoes a local expansion. 

doi:10.1016/j.clim.2007.03.239 

S25 

F.27 Nramp1 Enhances Autoimmune Diabetogenic 

T Cell Response by Altering the Processing and 

Presentation of Pancreatic Islet Antigens in 

Dendritic Cells 

Yang Dai, Research Scientist, Torrey Pines Institute for 

Molecular Studies, San Diego, CA, Babak Shirdel, Student, 

Torrey Pines Institute for Molecular Studies, San Diego, CA, 

Sandra Chau, Student, Torrey Pines Institute for Molecular 

Studies, San Diego, CA, Linda Wicker, Professor, Cambridge 

Institute for Medical Research, Cambridge, Philippe Gros, 

Professor, McGill University, Montreal, QC, Canada, Eli 

Sercarz, Professor, Torrey Pines Institute for Molecular 

Studies, San Diego, CA 

Dendritic cells are essential for initiating adaptive immune 

responses and establishing self tolerance. We hypothesize 

that the antigen processing machinery differs between 

diabetes-susceptible and -resistant, MHC-matched individuals, 

and that this difference contributes to the generation 

of pathogenic effectors rather than regulatory T cells in 

diabetes prone individuals. The Idd5.2 (insulin-dependent 

diabetes 5.2) region contributes to the genetic susceptibility 

of NOD (non-obese diabetes) mice to autoimmune diabetes 

and contains the gene encoding Nramp1 (natural resistanceassociated 

macrophage protein 1). We found that Nramp1 

expression is not restricted to macrophages; importantly,

S26 Abstracts 

dendritic cells (DC) can upregulate Nramp1 expression 

dramatically after stimulation. Nramp1-expressing DC exhibit 

an accelerated pH change (acidification) within endocytotic 

vesicles. Interestingly, when Nramp1 function is present, both 

bone marrow-derived DC and adherent splenic DC are more 

potent in stimulating autoreactive Tcells following processing 

of a diabetes-associated antigen, GAD (glutamic acid decarboxylase) 

65. Furthermore, an islet-infiltrating Tcell clone, 

BDC2.5, reacts much more vigorously to the islets when 

Nramp1-functional DC are used as antigen presenting cells. 

These data suggest that Nramp1 may alter the processing of 

self antigens in antigen processing compartments to increase 

the availability of determinants involved in inducing pathogenic 

T cells (supported by grants to EES from Juvenile 

Diabetes Research Foundation and Diabetes National 

Research Group). 

doi:10.1016/j.clim.2007.03.241 

F.28 Perturbations in the Function and Composition 

of the B Cell Compartment are Present in Type 1 

Diabetes 

Mary Rieck, Research Technician, Benaroya Research 

Institute, Seattle, WA, Adrian Arechiga, Research Associate, 

Benaroya Research Institute, Seattle, WA, Catherine 

Pihoker, Associate Professor, Department of Pediatrics, 

Seattle, WA, Jane Buckner, Associate Member, Benaroya 

Research Institute, Seattle, WA, Carla Greenbaum, Member, 

Benaroya Research Institute, Seattle, WA 

Disturbances in B cell populations have been identified in 

autoimmune diseases where autoantibodies are present. A 

role for B cells in the pathogenesis of type 1 diabetes (T1D) is 

suggested by studies in animal models and by the autoantibodies 

present in T1D. To address whether alterations in 

the B cell compartment are present in individuals with T1D we 

examined PBMC from healthy, T1D, and type 2 diabetic (T2D) 

subjects. The relative frequency of the naïve, memory and 

plasmablast subsets were significantly different between 

healthy and T1D subjects, while total B cells were no 

different. The plasmablast (CD19+CD27high) population was 

increased in T1D subjects compared to controls (pb0.006), 

and was most pronounced in subjects within 2 years of 

diagnosis. The memory B cell (CD19+CD27+) subset was 

decreased in diabetic subjects (p =0.0006). The B cell 

populations in T2D subjects were comparable to healthy 

controls; however, first degree relatives of T1D subjects, like 

individuals with T1D, displayed a decrease in memory B cells 

(p=0.02). Thus the decrease in memory B cells is not due to 

the metabolic abnormalities associated with T1D but may be 

genetically influenced. In addition, we have identified a 

defect in BCR signaling within memory B cells of T1D subjects. 

This suggests that a B cell intrinsic mechanism may contribute 

to alterations in the B cell compartment. We hypothesize that 

this alteration of B cell signal transduction may contribute to 

pathogenesis of T1D by tipping the balance of long-term B cell 

memory toward high affinity antibody production. 

doi:10.1016/j.clim.2007.03.242 

F.30 Tracking Putative Pathogenic Members of the 

T Cell Repertoire in NOD Mice 

Idania Marrero, Postdoctoral Fellow, Torrey Pines Institute 

for Molecular Studies, San Diego, CA, Yang Dai, Postdoctoral 

Fellow, Torrey Pines Institute for Molecular Studies, San 

Diego, CA, Eli Sercarz, Professor, Torrey Pines Institute for 

Molecular Studies, San Diego, CA 

To define the major components of the autoreactive Tcell 

repertoire that arise spontaneously in the diabetic NOD 

mouse, we used CDR3 length spectroscopy to (1) study the 

entire Tcell repertoire in pancreatic lymph nodes (PLN) from 

pre-diabetic NOD mice and (2) to identify potentially 

pathogenic clones in islet-infiltrating cells that persist after 

cyclophosphamide (Cyp) treatment of female NOD mice, 

which is designed to destroy regulatory T cells. Any public or 

quasi-public T cell expansions were identified in PLN of 

female NOD mice at 4 and 10 weeks of age. We identified two 

expansions, Vb5.2/Jb2.5 and Vb10/Jb1.5, which were found 

in 60% and 50% of the 4-week-old female NOD mice. These 

expansions can be useful in the study of how the T cell 

repertoire can be modified after islet transplantation. After 

Cyp treatment, the Vb4, Vb5.2, Vb10 and Vb12 families 

showed dominant expansions. Interestingly, we found a 

unique Vb4-“195 nucleotide” peak that was found in 10 out 

of 11 (91%) Cyp-treated mice. In contrast, this peak was not 

detected in untreated NOD mice. Another interesting result 

was seen in the Vb12 family, in which a Vb12-“200” peak was 

prominent in 6 out of 10 (60%) Cyp-treated mice. These 

expansions may represent the more pathogenic clones. 

Currently, we are sequencing these expansions to establish 

whether the Cyp-induced expansions represent truly unique 

clones. We believe that these Cyp experiments will provide 

the possibility of distinguishing between regulatory and aggressive 

clones. This work was supported by JDRF 3-2005-336. 

doi:10.1016/j.clim.2007.03.243 

F.31 Reduced CD4+ T Cell-specific Gene Expression 

in Human Type 1 Diabetes Mellitus 

Tihamer Orban, Researcher, Joslin Diabetes Center, Boston, 

MA, Janos Kis, Diabetologist, Department of Internal 

Medicine and Gastroenterology, Polyclinic of the Hospitaller 

Brothers, Budapest, Laszlo Szereday, PhD, Reproductive and 

Tumor Immunology Research Group Hungarian Academy of 

Science, Pecs, Klara Farkas, Joslin Diabetes Center, Boston, 

MA, Peter Engelmann, PhD, Department of Immunology and 

Biotechnology, Faculty of Medicine, University of Pecs, 

Pecs, Heyam Jalahej, Joslin Diabetes Center, Boston, MA, 

Andras Treszl, Pediatrician, SOTE First Department of 

Paediatrics, Budapest 

Type 1 diabetes mellitus (T1DM) in humans is characterized 

by the T cell dependent destruction of the insulin 

producing pancreatic beta cells; however, the precise 

pathogenesis of the disease, especially the initiation of 

pathologic immune response, is still largely unknown. We 

hypothesized that the function of human CD4+ T cells is 

altered in T1DM and analyzed unstimulated human peripheral 

blood CD4+ T cell gene expression. We used a novel 

three-way comparison of DNA microarray data of CD4+ Tcells

Abstracts 

isolated from new onset T1DM, patients with long-term type 

2 diabetes (T2DM), and from healthy controls in order to 

eliminate any possible influence of glucose homeostasis on 

our findings. We analyzed the T1DM specific gene expression 

changes and their functional relevance to T1DM autoimmunity. 

Our genetic and functional data show that T1DM CD4+ T 

cells are down-regulated specifically affecting key immune 

functions and cell cycle. Histone deacetylase gene expression, 

a key regulator of epigenetic modification, is also 

reduced. The CD4+ T cells showed impaired function, 

including an abnormal immune response, which may be a 

key element that leads to the breakdown of self-tolerance. 

doi:10.1016/j.clim.2007.03.244 

F.32 Protective Versus aggressive T Cell Responses 

to the Major Proinsulin73−90 Epitope in Beta-Islet 

Cell Autoimmunity 

Ivana Durinovic-Bello, Staff Scientist, Visiting Associate 

Professor, Benaroya Research Institute, Virginia Mason 

Research Center, Seattle, WA, Nicol Pratt, Research 

Technician, Benaroya Research Institute, Virginia Mason 

Research Center, Seattle, WA, Susan A. Masewicz, Research 

Technician, Benaroya Research Institute, Virginia Mason 

Research Center, Seattle, WA, Gerald Nepom, Director, 

Virginia Mason Research Center, Benaroya Research 

Institute, Seattle, WA 

Proinsulin (P-Ins)73–90 is a major T cell epitope of 

DRB1*0401 subjects with beta-islet cell autoimmunity. Subset 

of P-Ins73–90 specific T cells of the recent onset type 1 

diabetic (T1D) expressed Foxp3, a marker for regulatory Tcell 

lineages, secreted high IL-5 and IL-10 and low levels of IFNg, 

and predominantly used Tcell receptor Vb14 (Durinovic-Belló 

I et al. PNAS 103; 11,683, 2006). Addition of N- or C-terminal 

amino acids to P-Ins73–90 drastically changed their reactivity 

and cytokine phenotype, suggesting an immunomodulatory 

potential for this P-Ins epitope. To test the hypothesis that in 

subjects with beta-islet cell autoimmunity subsets of P- 

Ins73–90 specific T cells with potential down-regulatory 

phenotypes still exist, we have isolated P-Ins73–90 specific T 

cells of additional DRB1*0401 subjects (n=17) by depletion of 

CD25+ Tcells, stimulation with P-Ins73–90 and IL-2, and Vb14 

and tetramer sorting. Both effector and down-regulatory 

cells show characteristics of activated T cells and express 

CD25 and OX40; in addition effector cells up-regulate CD154 

(CD40L), CD80/CD86, Fas (CD95) and FasL (CD178) and have 

high proliferative capacity, and down-regulatory cells 

express Foxp3 and suppress cognate and bystander responses. 

In T1D patients T cells secreting high levels of IFNg and IL-2 

predominate, whereas T cells of the control individuals and 

prediabetic subjects are characterized by high IL-4 and IL-5 

secretion and low IFNg and IL-10. Subsets of P-Ins73–90 

specific Tcells with down-regulatory phenotype may prove as 

a suitable targets for therapeutic intervention in DRB1*0401 

positive humans with beta-islet cell autoimmunity or recent 

onset T1D. 

doi:10.1016/j.clim.2007.03.245 

F.33 The Programmed Death-1 (pd-1) Pathway 

RegulatesPeripheralTCellToleranceDuring 

Autoimmune Diabetes in Nonobese Diabetic (NOD) 

Mice 

Brian T. Fife, Postdoctoral Fellow, University of California, San 

Francisco, San Francisco, CA, Indira Guleria, PhD, 

Transplantation Research Center, Brigham and Women’s 

Hospital and Children’s Hospital, Boston, MA, Melanie Bupp, 

PhD, University of California, San Francisco Diabetes Center, 

San Francisco, CA, Qizhi Tang, PhD, University of California, San 

Francisco Diabetes Center, San Francisco, CA, Todd Eagar, PhD, 

University of California, San Francisco Diabetes Center, San 

Francisco, CA, Helene Bour-Jordan, PhD, University of 

California, San Francisco Diabetes Center, San Francisco, CA, 

Hideo Yagita, Professor, Department of Immunology, Juntendo 

University School of Medicine, Tokyo, Japan, Miyuki Azuma, 

Professor, Department of Molecular Immunology, Tokyo Medical 

and Dental University, Tokyo, Japan, Mohamed H. Sayegh, 

Professor, Transplantation Research Center, Brigham and 

Women’s Hospital and Children’s Hospital, Boston, MA, 

Jeffrey Bluestone, Professor, University of California, 

San Francisco Diabetes Center, San Francisco, CA 

Programmed death-1 (PD-1), an inhibitory costimulatory 

molecule on activated T cells, down-regulates immune 

responses. We investigated the role of this pathway in 

peripheral tolerance in the NOD mouse model of autoimmune 

diabetes. In this study, tolerance was induced using 

either antigen-coupled ethylene carbodiimide-fixed splenocytes 

or FcR non-binding anti-CD3 mAb administration. 

Antigen-coupled splenocyte treatment conferred complete 

and long lasting protection from diabetes transfer using islet 

reactive BDC2.5 TCR transgenic T cells. In addition, insulincoupled 

splenocytes were used to treat new onset spontaneously 

diabetic mice. Using this approach we demonstrate 

that disease remission is associated with inhibition of 

pathogenic T cell proliferation, decreased cytokine production, 

and T cell anergy induction. Moreover, we show that 

robust long-term tolerance depends on the PD-1/PD-L1 

pathway in both systems. Anti-PD-1 and PD-L1, but not anti- 

PD-L2, reversed tolerance weeks after tolerogenic therapy 

by promoting antigen-specific T cell proliferation and 

inflammatory cytokines directly in infiltrated tissues. PD- 

1/PD-L1 blockade did not limit Treg activity suggesting 

direct effects on pathogenic T cells. Finally, we describe a 

critical role for PD-1/PD-L1 in another powerful immunotherapy 

model using anti-CD3, currently being tested in 

clinical trials. Anti-CD3 treatment of new onset spontaneously 

diabetic NOD mice results in disease remission. PD- 

1/PD-L1 blockade reverses tolerance and rapidly precipitates 

diabetes. These findings suggest that PD-1/PD-L1 

interactions form a common pathway to selectively maintain 

tolerance directly in the target tissues. Taken together, 

these results provide important insights in the regulation of 

autoimmune responses and may lead to novel immunotherapeutic 

approaches for peripheral tolerance and treatment 

of autoimmune diabetes. Supported by NIH AI35297 and 

JDRF 10-2006-799. 

doi:10.1016/j.clim.2007.03.248 

S27

S28 Abstracts 

F.34 Gleevec: A New Therapeutic for Type I 

Diabetes? 

Cedric Louvet, Postdoctoral Research Fellow, Diabetes 

Center at University of California, San Francisco, San 

Francisco, CA, Gregory L. Szot, Islet Transplant Facility 

Manager, Diabetes Center, San Francisco, CA, Jiena Lang, 

Staff Research Associate, Diabetes Center at University of 

California, San Francisco, San Francisco, CA, Jeffrey A. 

Bluestone, Diabetes Center Director, Diabetes Center at 

University of California, San Francisco, San Francisco, CA, 

Michael R. Lee, Staff Research Assistant, Diabetes Center at 

University of California, San Francisco, San Francisco, CA 

Gleevec (Imatinib, formerly STI571) is the first FDA-approved 

drug to directly turn off a specific protein known to cause a 

cancer. Although targeted to the Bcr-Abl kinase, Gleevec has 

been shown to modestly affect related kinases including PDGF, 

c-fms and c-kit potentially involved in various cell types critical 

to the development and progression of autoimmune diseases. 

Based on these fundamental properties, we embarked on a 

study to determine the clinical effects of Gleevec in T1D in NOD 

mice. 

NOD mice were treated by gavage daily with 1.5 mg/ 

mouse of commercially available Gleevec suspended in 

peanut oil. Pre-diabetic mice treated with Gleevec, but not 

with peanut oil only, were protected from spontaneous 

diabetes. More importantly, when Gleevec treatment was 

initiated at the time of disease onset (blood sugars between 

250 and 350 mg/dl), the majority of mice went into remission 

and b10% relapsed during the course of the therapy. When 

therapy was discontinued at 2 weeks, the majority of the 

mice became hyperglycemic within 1–2 weeks. However, 

longer therapy (8–10 weeks) resulted in long-term normoglycemia 

in a majority of treated animals even after drug 

cessation suggesting that Gleevec does not have to be given 

continuously to promote long-lasting protection. The possibility 

that short-term therapy with Gleevec can lead to longterm 

effects makes it a very attractive potential therapeutic 

for the treatment of patients with new onset T1D as well as 

potentially patients undergoing an islet transplant. 

doi:10.1016/j.clim.2007.03.249 

F.38 Characterization of Peptide Agonists and 

Antagonists for the Interaction of CD200 with 

CD200R1 and Their Efficacy in Modulation of 

LPS-induced TNFα Production and Mortality In Vivo 

Reginald Gorczynski, Professor, University Health Network, 

Toronto, ON, Canada, Ivo Boudakov, PDF, University Health 

Network, Toronto, ON, Canada, Ismat Khatri, PDF, 

University Health Network, Toronto, ON, Canada 

After engagement of the Ig-supergene family members 

CD200R1 and CD200, immunosuppressive and/or antiinflammatory 

signals augment allograft survival and 

decrease production of inflammatory cytokines. The primary 

domains of both CD200 and CD200R1 implicated in 

these interactions are found in the NH2-terminal region of 

each molecule. We investigated the capacity of 10–15 mer 

synthetic peptides defining the three complementaritydetermining 

(CDR1–3) regions of mouse CD200 to reproduce 

the function of full-length CD200, or antagonize that 

function, assaying the suppression of LPS-induced TNFα 

production (in vivo/in); suppression of MLC responses in 

vitro; and LPS-induced mortality (d7) in vivo. Peptides 

located in the CDR2 region, but not CDR1 or CDR3 were 

potent agonists in vitro and in vivo, recapitulating the 

effects seen with the full-length molecule (CD200Fc). These 

small molecular weight agonists of CD200 might have 

efficacy in vivo in models of sepsis (and TNFα production). 

In a further analysis we explored the ability of the same 

peptides to block the suppressive and anti-inflammatory 

effect of the full-length CD200Fc which was seen in MLCs 

and after LPS administration both in vitro and in vivo. In 

these studies peptides located in the CDR1 and CDR3 

regions of the NH2-terminal of CD200 were found to be 

antagonists of the biological effects of CD200. Our data 

confirm that discrete small molecular weight agonists and 

antagonists can be defined for the interactions of CD200 

and the CD200R1 receptor. Since these interactions result in 

regulation of both acquired immune responses, and 

inflammatory responses, these peptides may have clinical 

utility. 

doi:10.1016/j.clim.2007.03.250 

F.39 Calculating the Number of Ordered Mutations 

Necessary to Cause a Specific Cancer in a Specific 

Risk Group and the Fraction of the Group that is 

Immune to the Cancer 

Ivan Kramer, Associate Professor, UMBC, Canada Physics, 

Catonsville, MD 

The series of ordered mutations that cause a cell to 

become cancerous is modeled so that the fraction of a risk 

group (e.g., white men) that has developed a specific cancer 

(e.g., melanoma) at any age can be computed. The saturated 

model constructed here allows the possibility that a fraction 

of a risk group may be immune to developing a specific 

cancer but is otherwise isomorphic to the physical model 

describing an ordered chain of radioactive nuclear decays. 

The model’s cancer incidence function depends on only 

three independent parameters: the number of mutations 

necessary for a cell to become cancerous, the fraction of the 

group that is immune to developing the cancer, and the time 

required for 50% of the group to experience an average 

mutation (defined as the mutation half-life). These three 

parameters are determined by fitting the model’s cancer 

incidence function to the cancer incidence data. For 

example, the modeling predicts that all white males in the 

USA are vulnerable to developing melanoma, five ordered 

mutations are required to develop it, and the mutation halflife 

is 33.5 years. By contrast, the modeling also predicts that 

80.7% of white females in the USA are immune to developing 

melanoma, three ordered mutations are required to develop 

it, and the mutation half-life is 54.7 years. Thus, different 

risk groups can develop the same cancer through different 

pathways. The mechanism underlying female immunity to 

melanoma should become a research priority. Stimulating

Abstracts 

the immune system to eliminate mutated, pre-cancerous 

cells would prevent cancer. 

doi:10.1016/j.clim.2007.03.255 

F.41 The State of Immune System in the Pregnants 

with Untreated Syphilis 

Tatiana Yaremtchuk, Doucent, Lviv National Medical 

University, Department of Obstetrics, Gynecology and 

Perinatology of PGE, Lviv, Ukraine, Olga Korchynska, 

Assistant, Department of Obstetrics, Gynecology and 

Perinatology of PGE of Lviv National Medical University, 

Lviv, Ukraine, Yaroslav Korinets, Doctor, Scientific 

Collaborator, Department of Antenatal Diagnostics and 

Perinatology of Institute of Hereditary Pathology, Lviv, 

Ukraine, Natalia Prokoptchuk, Genetics and USG Diagnostics 

Doctor, Department of Antenatal Diagnostics and 

Perinatology of Institute of Hereditary Pathology, Lviv, 

Ukraine, Angela Misiura, Assistant, Department of 

Obstetrics, Gynecology and Perinatology of PGE of Lviv 

National Medical University, Lviv, Ukraine 

Objective of Study: The state of immune system in the 

pregnants with untreated syphilis. Materials: Vein blood of 5 

pregnants with untreated early latent syphilis within 26-33 

weeks and 30 healthy pregnants in the same terms. Methods: 

Designation of quantity of CD3, CD4, CD8, CD16, CD56, CD19, 

CD4/CD8 was carried out with test-systems of monoclonal 

antibodies. The concentrations of IgA, IgM, IgG were 

designated by method of radial immune diffusion. The level 

of circulating immune complexes was investigated by 

spectrophotometral method. Results: Initial investigations 

showed the quantity of CD3 composed 59.4 % in comparison 

with 62,53+/–1,23 % in control group (P>0,05). The level of 

CD4 trustworthy was lower – 16,2 % contra 35,6+/–0,86 % 

(Pb0,01). The quantity of CD8, CD16, CD56 correspondingly 

had tendency to reduce. These indicators accordingly made 

up – 25,0 % in comparison with 27,6+/–1,72 % (P>0.05), 3,0 % 

contra 6,51+/–0,88 % (P>0,05), 12,2 % in comparison with 

14,68+/–1,71 % (P>0,05). The immuneregulative index was 

0,65+/–0,22 in comparison 1,29+/–0,06 (Pb0,05). The concentration 

CD19 was trustworthy lower 5,2 % contra 12,83+/– 

0,88 % (Pb0,05). The levels of IgA and IgG were accordingly 

1,56+/–0,2 g/l in comparison with 1,98+/–0,06 g/l (Pb0,05) 

and 10,29+/–0,54 g/l contra 12,38+/–0,43 g/l (Pb0,05). The 

concentration of IgM had tendency to decrease – 1,37+/–0,32 

g/l contra 1,76+/–0,11 g/l (P>0,05). The level of circulating 

immune complexes also was trustworthy lower 59,8+/–11,4 

ODU contra 93,5+/–3,58 ODU (Pb0,01). Conclusions: The 

suppression of immune system has been found out in the 

pregnants with untreated early latent syphilis. 

doi:10.1016/j.clim.2007.03.256 

F.44 Human Norovirus Infection: Genetic and 

Immune Determinants of Susceptibility and 

Resistance 

Juan Leon, Postdoctoral Fellow, Emory University, Atlanta, 

GA, Lisa Lindesmith, Laboratory Technician, Department of 

Epidemiology, University of North Carolina at Chapel Hill, 

Chapel Hill, NC, Erin-Joi McNeal, Laboratory Technician, 

Department of Global Health, Rollins School of Public 

Health, Atlanta, GA, Ralph Baric, Professor, Department of 

Epidemiology, University of North Carolina at Chapel Hill, 

Chapel Hill, NC, Elizabeth Ailes, Graduate Student, 

Department of Epidemiology, Emory University, Atlanta, 

GA, Christine Moe, Associate Professor, Department of 

Global Health, Rollins School of Public Health, Atlanta, GA 

Infection with Norovirus (NoV) is the main cause of 

epidemic worldwide. NoV are classified by the CDC and NIAID 

as Bioterrorism Category B Priority Pathogens based on their 

high transmissibility, low infectious dose, and serious 

economic impact. Little is known on what factors protect 

individuals from NoV infection. To meet this need, our goal is 

to identify genetic and immunologic determinants of 

protection from NoV infection. Because animal models are 

not available and these viruses do not replicate in culture, 

we developed expertise in human challenge models. We 

challenged 109 human volunteers with varying doses of the 

Norwalk virus (NV) strain of NoV. We found that gender, 

ethnicity, blood group, levels of pre-challenge serum NVspecific 

IgG or levels of pre-challenge saliva NV-specific IgA 

were not significant predictors of infection. Presence of 

serum NV-specific IgG and presence of a putative NV-binding 

receptor, H type 1, were significantly associated with 

infection. Dose of inoculum received and age of the 

individual may also be predictors of infection. We observed 

a significant correlation in levels of salivary NV-specific IgG 

with salivary NV-specific IgA and serum NV-specific IgG. 

Lastly, in genetically susceptible individuals, infected individuals 

exhibited a rise in NV-specific serum and salivary 

antibodies while uninfected individuals did not. These 

results suggest that only infected individuals develop 

activation of the humoral response. These data will be 

used to better understand the immune response to NV 

infection and design effective vaccines against NoV to 

protect high risk populations including children, food 

handlers, and the elderly. 

doi:10.1016/j.clim.2007.03.257 

F.45 Polymorphism of IL-6 gene and Its Association 

with Susceptibility to Brucellosis 

Manoochehr Rasouli, Immunology Department, Clinical 

Microbiology Research Center-Shiraz University of Medical 

Sciences, Shiraz, Simin Kiany, MSc, Immunology Department, 

Clinical Microbiology Research Center-Shiraz University of 

Medical Sciences, Shiraz 

S29 

Background: Brucellosis is a highly contagious bacterial 

zoonosis that affects millions of people worldwide. Polymorphonuclear 

cells (PMNs) are the most abundant type of 

circulating white blood cells (WBCs) and are the major cell 

type mediating acute inflammation response to bacterial 

infection. Since IL-6 plays a major role in inflammation and 

stimulates production of neutrophils from BM progenitors, 

this supports the involvement of IL-6 in the process and 

pathogenesis of brucellosis. It has been shown that the

S30 Abstracts 

production level of IL-6 has been correlated with allele 

inherited in individuals. Therefore, the aim of current study 

was to assess whether IL-6 (−174) polymorphism was 

associated with susceptibility to brucellosis. Method: One 

hundred and seventy-five patients with brucellosis and 77 

healthy animal husbandmen who had infected animals and 

consumed their dairy products, joined this study. DNAs 

extracted from samples were genotyped for IL-6 (−174 G/C) 

polymorphism using AS-PCR method. Results: Our results 

showed that IL-6 (−174) gene polymorphism was distributed 

similarly in the patient and control groups (P =0.3). 

Discussion: Although we found no association between IL-6 

(−174) gene polymorphism and susceptibility to brucellosis 

we cannot exclude that other polymorphisms within this 

gene or its receptor may donate susceptibility to brucellosis. 

Moreover these findings need confirmation in other 

population groups. 

doi:10.1016/j.clim.2007.03.259 

F.46 Interleukin-1β(+3953) Gene Polymorphism and 

Susceptibility to Helicobacter Pylori Associated 

Gastritis 

Ayda Hosseinkhani, Pharmacist, Clinical Microbiology 

Research Center, Shiraz University of Medical Sciences, 

Shiraz, Simin Kiany, MSc, Clinical Microbiology Research 

Center, Shiraz University of Medical Sciences, Shiraz, 

Manoochehr Rasouli, PhD, Clinical Microbiology Research 

Center, Shiraz University of Medical Sciences, Shiraz, Akram 

Jamshidzadeh, PhD, Faculty of Pharmacy-Shiraz University 

of Medical Sciences, Shiraz, Shohreh Farshad, PhD, Clinical 

Microbiology Research Center, Shiraz University of Medical 

Sciences, Shiraz 

Introduction: It has been speculated that IL-1 genes 

play a crucial role in the genetic predisposition to gastric 

problems upon H. pylori infection by modulating the host 

immune response. Three biallelic polymorphisms have 

been reported in IL-1 β all representing CNT base 

transition at position −511, −31 and +3953 from the 

transcriptional site. In the present study we attempted to 

determine the IL-1 β (+3953) risk genotypes to H. pylori 

mediated gastritis. Methods: The subjects were 373 

individuals with no gastrointestinal problems and 49 H. 

pylori positive patients suffering from gastritis which was 

confirmed on the basis of endoscopic findings. All subjects 

were genotyped for IL-1 β+3953 gene polymorphism using 

polymerase chain reaction-restriction fragment length 

polymorphism (PCR-RFLP). Results: There was no significant 

difference in the frequency of IL-1 β +3953 C/T 

genotypes between two groups. 

Discussion: The lack of significant difference in the 

frequency of IL-1 β+3953 C/T between the studied groups 

might be the result of our small patient population. Thus we 

suggest that this study be performed on larger group of 

patients; also investigation of other IL-1 functional gene 

polymorphism on Iranian population is recommended. 

doi:10.1016/j.clim.2007.03.260 

F.48 The −251 AA Genotype of the Interleukin-8 

Promoter is Associated with Susceptibility to 

Brucellosis 

Manoochehr Rasouli, PhD, Immunology Department, Clinical 


Sciences, Shiraz, Simin Kiany, MSc, Immunology Department, 

Clinical Microbiology Research Center, Shiraz University of 

Medical Sciences, Shiraz 

Background: Interleukin-8 (IL-8), a member of CXC chemokine 

family, is a chemoattractant of neutrophils and lymphocytes. 

IL-8 promoter is estimated to be 1500 bp. Several reports 

have shown relationship between IL-8 gene polymorphism and 

several human diseases such as bronchial asthma, Parkinson’s 

disease and Helicobacter pylori infection. The aim of this study 

was to evaluate the relationship between IL-8 (−251 A/T) 

polymorphism and brucellosis. Methods: One hundred and 

eighty eight patients with brucellosis and 77 healthy farmers 

who consumed contaminated row milk and dairy products of 

animals with brucellosis were included in this study. All 

individuals were genotyped for IL-8 (−251 A/T) promoter 

polymorphism using polymerase chain reaction-restriction fragment 

length polymorphism (PCR-RFLP). Results: The frequency 

of AA genotype was significantly higher in patient group than 

healthy individuals (14.3% vs. 2.5%, P=0.005). Discussion: As 

data revealed that the frequency of AA genotype was lower in 

healthy controls than patients. Hence, our study provides 

evidence that the presence of AA genotype is significantly 

associated with susceptibility to brucellosis. 

doi:10.1016/j.clim.2007.03.261 

F.49 Interferon Gamma Producing T Lymphocytes in 

Bacterial Biofilm Infection: Response Modifiers for 

Polymorphonuclear Neutrophils (PMN) 

Christof Wagner, Associate Professor, University of 

Heidelberg, Heidelberg, Germany, Christof Iking-Konert, 

Assisant Professor, University of Duesseldorf, Duesseldorf, 

Germany, Andreas Wentzensen, Head of Clinic, Klinik fur 

Unfall-und Weiderherstellungschirurgie, Ludwigshafen, 

Germany, Gertrud Maria Haensch, Full Professor, University 

of Heidelberg Department of Immunology, Heidelberg, 

Germany, Volkmar Heppert, Head of Department, Septic 

Traumatology, Ludwigshafen, Germany 

Bacterial infection with biofilm formation on orthopaedic 

implants causes a progressive, destructive inflammatory 

process, causing massive tissue destruction, bone resorption, 

and loosening of the implant. In most cases, this so-called 

implant-associated posttraumatic osteomyelitis requires the 

removal of the implant, allowing recovery and analysis of the 

local cellular infiltrate. By cytofluorometry, the majority (60– 

85%) of the infiltrated cells were identified as highly activated 

polymorphonuclear neutrophils (PMN); the next largest cell 

population (5–35%) were T-lymphocytes. CD4 and CD8 positive T 

cells were found, the latter prevailing compared to the 

peripheral blood. The majority of the CD8+ cells were CD28 

negative; about 26% expressed CD11b, 76% CD57. The latter two 

receptors were co-expressed on less than 5% of the cells. The

Abstracts 

CD11b positive Tcells produced interferon (IFN) gamma. In vitro 

experiments provided a link between activated Tcells and PMN: 

IFN γ, supernatants of the Tcells, or co-incubation of PMN with 

activated T cells induced expression on PMN of CD64, the high 

affinity receptor for immunoglobulin, and of MHC class II 

antigens. The fact that IFN γ is among the few cytokines that 

can stimulate PMN to produce CD64 and MHC class II, and the 

observation that CD64 and MHC class II positive PMN is found in 

the cellular infiltrate suggest that CD8+ T cells via synthesis of 

IFN γ modulatethefunctionofinfiltratedPMNandpointtoa 

novel, not yet appreciated role of Tcells in the defence against 

bacterial biofilm infections. 

doi:10.1016/j.clim.2007.03.262 

F.50 Server Hepatitis Related Gene mfgl2 shRNA 

Plasmid Construction and Its RNA Interference 

In Vitro 

Qin Ning, Chief Physician, Department of Infectious Disease, 

Tongji Hospital Huazhong University of Science and 

Technology, Wuhan, China, Zhimo Wang, Doctor in Charge, 

Department of Infectious Disease, Tongji Hospital, Huazhong 

University of Science and Technology, Wuhan, China, Dong Xi, 

Assistant Researcher, Department of Infectious Disease, Tongji 

Hospital, Huazhong University of Science and Technology, 

Wuhan, China, Chuanlong Zhu, Doctor in Charge, Department 

of Infectious Disease, Tongji Hospital, Huazhong University of 

ScienceandTechnology,Wuhan,China,SuiGao,Postgraduate 

Student, Department of Infectious Disease, Tongji Hospital, 

Huazhong University of Science and Technology, Wuhan, 

China, Weiming Yan, Associate Researcher, Department of 

Infectious Disease, Tongji Hospital, Huazhong University of 

Science and Technology, Wuhan, China, Xiaoping Luo, Chief 

Physician, Department of Infectious Disease, Tongji Hospital, 

Huazhong University of Science and Technology, Wuhan, China 

Objective: To determine the viral and host factors involved 

in the transcription of hfgl2 gene which was demonstrated to 

play a pivotal role in human and experiment fulminant viral 

hepatitis. Methods: HBc, HBs or HBx expression plasmids were 

constructed and cotransfected with a hfgl2 luciferase report 

construct into eukaryotic cells. Results: Luciferase assay 

showed that HBc or HBx protein, but not HBs protein 

significantly enhanced hfgl2 transcription in both CHO and 

HepG2 cells. Series deletion assay of hfgl2 gene promoter 

demonstrated that a strong regulatory domain from ¨C 712 to 

−568 was responsible for hfgl2 gene transcription in response 

to HBc or HBx proteins. By site-directed mutagenesis, the 

overlapped cis-elements LEF/c-Ets in domain −712/−568 was 

demonstrated to play an important role in the regulation of 

hfgl2 gene in response to HBc protein, while another two ciselements 

LEF/c-Ets and HSTF in the same domain were found 

to participate in hfgl2 transcription regulation in response to 

HBx protein. EMSA assays showed that HBc and HBx proteins 

did not directly induce hfgl2 gene activation, while an Ets 

family member c-Ets-2 bound to the cognate cis-element in 

hfgl2 promoter was responsible for induction of hfgl2 

activation in response to viral proteins. Conclusion: Our 

study provides new insights in understanding the pathology of 

fulminant hepatic failure and the interaction between HBV 

virus and host gene expression. This work was supported by 

NSFC 30225040, 30571643, 30672380, National Key Basic 

Research Program of China (2005CB522901, 2005CB522507) 

and Clinical Subject Key Project from the Ministry of Health. 

doi:10.1016/j.clim.2007.03.263 

F.51 TLR2 and TLR4 Expression on PBMC and Ascites 

Fluid from Hepatic Cirrhotic Patients with SPB 

Carmen Contreras, MD, Immunology Universidad de 

Guadalajara, Guadalajara, Mexico, Jorge Segura, 

Gastroenterologist, Gastroenterology Hospital Civil, 

Guadalajara, Mexico, Cecilia Guillen, Professor, 

Immunology Universidad de Guadalajara, Guadalajara, 

Mexico, Mary Fafutis, Immunologist Researcher, 

Immunology, Universidad de Guadalajara, Guadalajara, 

Mexico, Margarita Montoya, Immunologist, Immunology 

Universidad de Guadalajara, Guadalajara, Mexico 

Justification: The cirrhosis of the liver is a pathology with a 

high morbid-mortality, being one of the main complication’s 

cause of spontaneous bacterial peritonitis (SBP) due to an 

alteration in the immunologic balance, observed like a 

deficiency in the bactericide action of serum, opsonins, and 

complement; an increase in the proinflammatory cytokines; 

changes in the endoplasmatic reticulum system; and a 

neutrophil’s functional alteration. It has been demonstrated 

that the toll-like receptors (TLR) are essentials for the 

pathogenic recognition that starts with the activation of NFkB 

a transcriptional factor necessary for the biosynthesis of 

proinflammatory cytokines. At the moment the role played by 

these membrane receptors in the development of SBP is 

unknown. The aim of this work is to study the TLR2 and 4 

expression on PBMC and ascites fluid of cirrhotic patients (CP) 

with or without an SPB development. Methodology: Venous 

peripheral blood and ascites fluid was obtained from CP with 

or without SPB, as well as venous peripheral blood from 

healthy individuals (HI). The expression of TLR2, TLR4 and 

CD14 by flow cytometer was measured. Results: The 

peripheral blood’s cells from HI show a higher expression of 

TLR2 and TLR4 than CP and SPB, however the differences 

were not significant. The TLR2 and TLR4 expression in 

patient’s cells with SPB was higher than the cirrhotic patients 

(pb0.05) in ascites fluid. Conclusion: In this work it was found 

that the TLR2 and TLR4 expression was higher in SPB than 

cirrhotic patients in ascites fluid. 

doi:10.1016/j.clim.2007.03.264 

S31 

F.52 Persistently Deficient In Vitro 

Anti-Mycobacterial Immunity Despite Highly 

Successful Long-Term (>4 years) HAART: 

Differential Impact of Latent Tuberculosis Infection 

and Previous Active Disease 

Gil Benard, Medical Researcher, Medical School of the 

University of São Paulo, São Paulo, Brazil, Marcelo 

Mendonça, Laboratory of Dermatology and 

Immunodeficiencies, Medical School of the University of

S32 Abstracts 

São Paulo, São Paulo, Brazil, Léia C. Rodrigues Silva, PhD 

Student, Laboratory of Dermatology and 

Immunodeficiencies, Medical School of the University of 

São Paulo, São Paulo, Brazil, Guilherme G. Silveira, 

Student, Laboratory of Dermatology and 

Immunodeficiencies, Medical School of the University of 

São Paulo, São Paulo, Brazil, Maury M. Tanji, Laboratory of 

Dermatology and Immunodeficiencies, Medical School of 

the University of São Paulo, São Paulo, Brazil, Denise 

Schout, Epidemiology Service, Hospital das Clínicas, 

Medical School of the University of São Paulo, São Paulo, 

Brazil, Alberto J.S. Duarte, Professor, Laboratory of 

Dermatology and Immunodeficiencies, Medical School of 

the University of São Paulo, São Paulo, Brazil 

Background: The degree Mycobacterium tuberculosis (Mtb) 

immune reconstitution provided by long-term (N4 years) 

successful HAART and whether a past tuberculosis (TB) history 

influences this reconstitution are not known. Methods: We 

compared the lymphocyte proliferative response (LPR) and IFNã 

secretion to phytohemagglutinin (PHA) and Mtb antigens 

(ESAT6, Ag85B and whole Mtb lysate [SMtb]) of immunereconstituted 

(CD4 N500 cells/ìl) HIV+ patients with a past history of 

cured TB (HIV+ CTB group) or latent infection as presumed by 

positive purified protein derivative skin test (PPD+) (HIV+ PPD+ 

group) with HIV− controls either cured from TB (CTB group) or 

PPD reactors (PPD+ group). The databank on TB incidence 

among HIV+ and HIV− patients at our services during 1999–2005 

was also analyzed. Results: Most HIV+ patients presented normal 

PHA responses. However, Mtb immune reconstitution was 

incomplete, especially to ESAT6 and Ag85B. SMtb reactivity 

was fully restored in the HIV+ CTB group, while in HIV+ PPD+ 

patients it remained lower than in PPD+ controls. Comparison 

between the two HIV groups also suggested better immune 

reconstitution in HIV+ CTB patients. Databank analysis revealed 

that some HIV+ patients with N360 CD4 cells/ìl still developed 

TB, but not those with previous TB history. Conclusions: Mtb 

immune reconstitution is incomplete in HIV+ CTB and HIV+ PPD 

+ patients even after highly successful HAART. However, 

reactivity to whole mycobacteria antigens is restored, 

especially in HIV+ CTB patients, possibly due to survival of 

higher numbers of mycobacteria-specific T cell clones throughout 

immunosuppression, probably allowing sufficient protection 

against new Mtb challenges. 

doi:10.1016/j.clim.2007.03.266 

F.53 Human CD8+ Cytotoxic T Lymphocyte Epitopes 

Identified from Herpes Simplex Virus Type 1 

Glycoprotein D 

Aziz Alami Chentoufi, Assistant Specialist, Cellular and 

Molecular Immunology Laboratory, The Eye Institute, 

University of California, Irvine, Orange, CA, Xiuli Zhang, 

Postdoctoral Fellow, Cellular and Molecular Immunology 

Laboratory, The Eye Institute, University of California, 

Irvine, Orange, CA, Kasper Lamberth, Researcher, Institute 

of Medical Microbiology and Immunology (IMMI), University 

of Copenhagen, 2200 Copenhagen, Denmark, Xiaoming Zhu, 

Research Associate, Cellular and Molecular Immunology 


Irvine, Orange, CA, Gargi Dasgupta, Research Associate, 

Cellular and Molecular Immunology Laboratory, The Eye 

Institute, University of California, Irvine, orange, CA, 

Michele Wu, Student, Cellular and Molecular Immunology 


Irvine, Orange, CA, Alex Nguyen, Bio-199 Student, Cellular 

and Molecular Immunology Laboratory, The Eye Institute, 

University of California, Irvine, Orange, CA, Ilham Bettahi, 

Postdoctoral Fellow, Cellular and Molecular Immunology 


Irvine, Orange, CA, Søren Buus, Professor, Institute of 

Medical Microbiology and Immunology (IMMI), University of 

Copenhagen, 2200 Copenhagen, Denmark, Lbachir 

BenMohamed, Assistant Professor, Cellular and Molecular 

Immunology Laboratory, The Eye Institute, University of 

California, Irvine, Orange, CA 

Cytolystic T cells (CTLs) play a major role in herpes simplex 

virus type 1 and type 2 (HSV-1 and HSV-2) infections and 

diseases. Among some eleven HSV-1 and HSV-2 coat glycoproteins, 

glycoprotein D (gD) induces immunodominant T cells and 

is a leading vaccine candidate antigen. However, little is known 

about human CD8+ T cell epitopes of gD. Based on predictive 

computational algorithms and peptide binding affinity to HLA- 

A*0201 molecules, we have identified ten regions within the 

HSV-1 gD, each 9 to 10 amino acids in length, exhibiting high 

affinity CD8+ Tcell epitope(s). T2 cell stabilization assay showed 

peptide gD53-61, gD70-78 and gD278-286 significantly, and in 

dose-dependent manner, upregulates HLA-A*0201 molecules 

expression.ToobtainanobjectiveenumerationofCD8+Tcells 

induced by each gD peptide epitope, we employed the HLA- 

A*0201 tetramer staining procedure. A relatively high percentages 

of CD8+ Tcells positive for gD-18-10, gD53-61, gD70-78 and 

gD278-286/HLA-A*0201 tetramers were detected in PBMC from 

HLA-A*0201 seropositive patients, either directly ex vivo or 

following a single in vitro stimulation. Accordingly, strong HLA- 

A*0201-restricted CD8+ CTLs, assessed by INF-γ-ELISPOT and 

CD107a/b cytotoxic assays, were mapped to gD53-61, gD70-78 

and gD278-286 peptides. However, only gD53-61 and gD278-286 

peptides were able to generate CD8+ T cells that recognize 

infected target cells. Collectively, these findings suggest that 

high-affinity HLA-A*0201-binding gD53-61 and gD278-286 epitopes 

are naturally processed and are recognized by HSVspecific 

CD8+ CTLs in the infected human population, providing 

important information for the development of subunit vaccine 

strategies against herpes. 

doi:10.1016/j.clim.2007.03.267 

F.55 Protection Against SEB-Induced Toxic Shock by 

Anti-TCR Vb8 Antibody 

Manisha Singh, Postdoctoral Associate, Baylor College of 

Medicine, Immunology Department, Houston, TX 

Intraperitoneal injection of the bacterial superantigen, 

Staphylococcus enterotoxin B (SEB) in HLA-DR3 transgenic 

mice leads to toxic shock and death within 72 h. In vivo, 

this is characterized by a marked expansion of splenic TCR 

Vb8+ CD4 and CD8 T cells compared to PBS treated mice. It

Abstracts 

appears that TCR Vb8-bearing CD4 and CD8 cells and the 

cytokines they secrete might be responsible for the 

pathogenesis of toxic shock caused by SEB. Therefore, we 

reasoned that depletion of TCR Vb8 bearing T cells by anti- 

Vb8 antibody might protect mice from SEB-induced shock. 

To test this hypothesis, we administered 200 μg of purified 

anti-TCR Vb8 antibody (clone 23.1) either prior to or 2 h 

after SEB injection. Irrespective of timing of anti-TCR Vb8 

administration, all anti-Vb8-treated mice (six of six) 

remained healthy while those injected with control antibody 

were either sick (n=2) or died (n=3). This salutary 

anti-TCR Vb8 effect correlated with markedly diminished 

SEB-induced serum levels of IL-6 and IL-2; however IFN-g 

and TNF release was not affected. Anti-TCR Vb8 Abs 

treatment also reduced infiltration in various organs like 

liver, lungs and kidney, those normally affected by SEB. 

These findings suggest that the pathogenesis of SEB induced 

toxic shock is primarily mediated by T cells and anti-TCR 

Vb8 antibody therapy may have therapeutic implications for 

SEB mediated shock. 

doi:10.1016/j.clim.2007.03.268 

F.56 Anti-Mouse GITR Monoclonal Antibody (Mab) 

Augments Humoral and Cellular Responses to H5N1 

and H3N2 Avian Influenza Hemagglutinin 

Joe Ponte, Senior Scientist, Tolerx, Inc, Cambridge, MA, 

Reema Gulati, Scientist, Tolerx, Cambridge, MA, Adam 

O’Shea, Scientist, Tolerx, Cambridge, MA, Paul Ponath, 

Immunologist, Tolerx, Cambridge, MA, Michael Paglia, 

Senior Manager, Tolerx, Cambridge, MA, Michael 

Rosenzweig, Senior Director, Tolerx, Cambridge, MA 

Adjuvants, including antibodies to tumor necrosis factor 

receptor superfamily (TNFRSF) members, augment immune 

responses to vaccines. One member of this family, glucocorticoid-induced 

tumor necrosis factor receptor (GITR), is 

expressed on regulatory and effector T cells. The aim of this 

study was to assess the ability of an anti-mouse GITR Mab, 

2F8, to stimulate murine humoral and cellular immunity to 

H3N2 and H5N1 viral hemagglutanins (HA). 2F8 enhanced 

anti-HA titers compared with controls and this enhancement 

was equal to or greater than titers obtained with incomplete 

Freund’s adjuvant or aluminum hydroxide. 2F8 F(ab(E))2 

fragments were also effective in boosting humoral immunity, 

suggesting that Fc interactions were not required. Moreover, 

the enhanced response was durable and specific for the 

antigen administered at the time of antibody treatment, and 

2F8-treated mice required less antigen to obtain titers 

equivalent to IFA-treated mice. 2F8 also enhanced cellular 

immunity as measured by EliSPOT. These results were 

recapitulated in cynomolgus monkeys using an anti-human 

GITR Mab (6C8). Thus, anti-GITR Mabs augment humoral and 

cellular immunity in mice and cynomolgus monkeys. Ongoing 

studies are evaluating the effect of anti-GITR Mabs on 

regulatory and effector T cells. 

doi:10.1016/j.clim.2007.03.269 

F.57 The B Cell Superantigen Peptostreprococcus 

Magnus Protein L Induces Lung Inflammation 

Through a MyD88-dependent Mechanism 

Amy Anderson, Research Associate, University of Pennsylvania 

School of Medicine, Philadelphia, PA, David LaRosa, 

Postdoctoral Fellow, University of Pennsylvania, Medicine, 

Philadelphia, PA, Arnold Levinson, Professor of Medicine, 

University of Pennsylvania School of Medicine, Medicine, 

Philadelphia, PA 

Peptostreptococcus magnus protein L functions as a B cell 

superantigen by interacting with V kappa light chain 

determinants on many human and mouse immunoglobulins, 

irrespective of their antigen specificities or heavy chain 

isotype. We sought to determine if this property endowed 

protein L with the capacity to elicit lung inflammation. 

Following intratracheal (i.t.) administration of recombinant 

protein L or BSA to C57/BL6 mice, broncheoalveolar lavage 

fluid (BALF) was recovered at varying time points and 

analyzed for total neutrophil counts and concentrations of 

MIP-2, KC and TNF. Lung tissue was harvested for histological 

examination. Protein L-treated mice accumulated neutrophils 

in their BALF as early as 4 h and showed an alveolar and 

peribronchial infiltrate of inflammatory cells peaking 

between 8 and 24 h. MIP-2, TNF, and KC levels in protein L 

challenged mice peaked at 4 h. These findings are consistent 

with those reported in conventional immune-complex models 

of lung inflammation. Studies were repeated in JHT mice to 

examine the importance of B cells/immunoglobulins in these 

reactions. Surprisingly, both the BALF response and lung 

histopathology were preserved. By contrast, both types of 

reactions were abrogated in MyD88 knockout mice, which 

have defective toll-like receptor signaling. These results 

indicate that protein L-induced lung inflammation is not 

dependent on its immunoglobulin binding (B cell superantigenic) 

activity but does appear to rely on signaling 

through a toll-like receptor. Thus, the present study reveals a 

novel, unanticipated pro-inflammatory mechanism initiated 

by a microbial protein with known B cell superantigenic 

properties. 

doi:10.1016/j.clim.2007.03.272 

S33 

F.58 Regulation of the Epstein−Barr Virus 

LMP1-mediated NF-kappaB Signaling Pathway 

by a Novel Adaptor Protein STAP-2 

Osamu Ikeda, Undergraduate, Department of Immunology, 

Graduate School of Pharmaceutical Sciences Hokkaido 

University, Sapporo, Japan, Yuichi Sekine, Assistant, 

Department of Immunology, Graduate School of Pharmaceutical 

Sciences Hokkaido University, Sapporo, Japan, Teruhito Yasui, 

Assistant, Department of Molecular Immunology, Research 

Institute for Microbial Diseases, Osaka University, Japan, Suita 

Kenji Sugiyma, PhD, Nippon Boehringer Ingelheim Co. Ltd. 

Kawanishi Pharma Research Institute, Kawanishi, Japan, Kenji 

Oritani, Assistant, Department of Hematology and Oncology, 

Graduate School of Medicine, Osaka University, Japan, Suita 

Akihiko Yoshimura, Professor, Division of Molecular and Cellular 

Immunology, Medical Institute of Bioregulation, Kyushu

S34 Abstracts 

University, Maidashi, Japan, Tadashi Matsuda, Professor, 

Department of Immunology, Graduate School of Pharmaceutical 

Sciences Hokkaido University, Sapporo, Japan 

Signal transducing adaptor protein-2 (STAP-2) is a recently 

identified adaptor protein, which contains pleckstrin homology 

(PH) and Src homology 2 (SH2)-like domains as well as a prolinerich 

domain in its C-terminal region. Our previous studies have 

demonstrated that STAP-2 binds to MyD88 and IKK-α/β, and 

modulates NF-kappaB signaling in macrophages. In this study, 

STAP-2 was induced by expression of the Epstein–Barr virus LMP1 

in B cells, while overexpression of STAP-2 inhibited LMP1mediated 

NF-kappaB signaling. Furthermore, STAP-2 associated 

with LMP1 in EBV-positive B cells. These results strongly suggest 

that STAP-2 acts as an endogenous negative inhibitor of the EBV 

LMP1-mediated signaling, which is critical for the EBV persistent 

infection and EBV-associated pathogenesis. 

doi:10.1016/j.clim.2007.03.273 

F.61 Defensins Block Bacterial Toxin-induced 

Interleukin-1β Production 

Jishu Shi, Assistant Professor, Anatomy, Physiology and 

Pharmacology, Auburn, AL, Shelly Ao, Research Assistant, 

APP, Auburn, AL, Charalabos Pathoulakis, Professor, 

Gasterenterology, Boston, MA 

This study was designed to test the hypothesis that 

defensins can block the release of IL-1β induced by 

Clostridium difficile toxin A (TxA) and Staphylococcus 

aureus α-toxin (a-Tox). LPS-activated (20 ng/ml, 2 h) 

human monocytes were treated with 3 nM TxA in the 

presence or absence of human neutrophil defensin HNP-1 

(25–50 μg/ml) or human Paneth cell defensin HD-5 (25–50 

μg/ml) for 6 h. In the absence of defensins, TxAstimulated 

monocytes released a large amount of mature 

IL-1β (17 kDa) into the extracellular milieu. The release 

of mature IL-1β from TxA-stimulated monocytes was 

blocked by HNP-1 and HD-5 in a dose-dependent manner. 

Different from ATP, TxA-induced IL-1β release was not 

inhibited by KN-62, a P2X7 receptor antagonist. In 

contrast to TxA, a-Tox (250 ng/ml) induced the release 

of both proIL-1β (31 kDa) and mature IL-1β from LPSactivated 

monocytes. In the presence of 20 μg/ml of 

HNP-1 or HD-5, the amount of mature IL-1β released from 

a-Tox-treated monocytes was reduced by 70%, while a- 

Tox-induced release of proIL-1β was completely blocked. 

Similar to TxA, a-Tox-induced IL-1β release was not 

inhibited by KN-62. Furthermore, the release of mature 

IL-1β was caspase-1-dependent for both toxins. These 

data suggest that TxA- and a-Tox-induced IL-1β release is 

independent of the ATP/P2X7 receptor pathway, and that 

defensins can block bacterial toxin-induced posttranslational 

processing and release of IL-1β. In addition to their 

antimicrobial activity, defensins may play an important 

role in host inflammatory response to bacterial infection 

by controlling the production of IL-1β. 

doi:10.1016/j.clim.2007.03.274 

F.62 Lack of Association of Interleukin-8 Gene 

Polymorphism with Helicobacter Pylori-induced 

Gastritis in Iranian Patients 

Ayda Hosseinkhani, Pharmacist, Clinical Microbiology 


Shiraz, Iran, Farshad Shohreh, Microbiologist, Clinical 


Sciences, Shiraz, Iran, Akram Jamshidzadeh, PhD, Faculty of 

Pharmacy-Shiraz University of Medical Sciences, Shiraz, 

Iran, Manoochehr Rasouli, PhD, Clinical Microbiology 


Shiraz, Iran, Simin Kiany, MSc, Clinical Microbiology 


Shiraz, Iran 

Background: Helicobacter pylori is a major cause of 

neoplastic and inflammatory gastroduodenal diseases of the 

stomach. H. pylori infection and associated gastric diseases are 

common in developing countries. Cytokine gene polymorphisms 

and H. pylori have been linked to gastric disease. We determined 

the role of host interleukin-8 (−251 A/T) gene polymorphism in 

the development of gastritis in south Iranian population. 

Methods: We genotyped IL-8 (−251 A/T) gene polymorphism in 

54 H. pylori infected individuals with gastritis and 337 infected 

individuals with normal mucosa using polymerase chain reaction-restriction 

fragment length polymorphism (PCR-RFLP). The 

diagnosis of gastritis was established on the basis of endoscopic 

findings. 

Results: We did not find any significant differences in 

allele and genotype frequencies of IL-8 (−251A/T) among our 

study groups. Discussion: Our analysis did not reveal a 

significant difference between the frequencies of IL-8 

(−251) genotypes and alleles which might be the result of 

our limited number of patient population. We suggest this 

study be continued on larger population of the patients. Also, 

analysis of polymorphism in other positions of IL-8 gene is 

recommended. 

doi:10.1016/j.clim.2007.03.275 

F.63 The Impact of HLA Polymorphisms on 

Measles Virus-specific T-Cell Memory Responses 

in Vaccinated Subjects 

Inna Ovsyannikova, Senior Research Associate, Mayo Clinic, 

Mayo Vaccine Research Group, Rochester, MN, Jenna Ryan, 

Senior Research Technologist, Mayo Clinic, Mayo Vaccine 

Research Group, Rochester, MN, Norman Pinsky, Senior 

Research Technologist, Mayo Clinic, Mayo Vaccine Research 

Group, Rochester, MN, Robert Jacobson, Professor of 

Pediatrics, Mayo Clinic, Department of Pediatric and 

Adolescent Medicine, Rochester, MN, Robert Vierkant, 

Senior Statistician, Mayo Clinic, Division of Biostatistics, 

Rochester, MN, Gregory Poland, Professor of Medicine, Mayo 

Clinic, Department of Internal Medicine, Rochester, MN 

The humoral and cellular immune responses to measles virus 

(MV) are genetically influenced. We hypothesized that the 

antigen-presenting function of HLA molecules could play a role 

in the variability in measles-specific T cell memory responses in

Abstracts 

vaccinated subjects. We studied the association between IFN-g 

producing T cells specific for MV and the alleles of HLA genes 

among 336 children (12−18 years of age) who previously 

received two doses of the measles vaccine. PBMCs (2x100,000 

cells/well; HLA typed) were cultured with Edmonston strain of 

measles (moi 0.5) and spot-forming cells (SFC) were analyzed by 

ELISPOT. Linear-regression analysis was used to study allelic 

associations. Measles-induced IFN-g responses (median 6 SFC; 

interquartile range 2, 17 SFC per 2x100,000 T cells) were 

detected in subjects up to 12 years after vaccination. Univariate 

analyses identified that class I HLA-A*1101 (median 6 SFC, 

p=0.04) and B*3701 (median 34 SFC, p=0.07) alleles were 

suggestive of an association with higher memory IFN-g 

responses, while the Cw*0802 (median SFC 3, p=0.08) allele 

wassuggestiveofanassociationwithadecreaseintherelative 

frequency of IFN-g producing T cells. Class II HLA alleles with 

potential associations with increased measles-specific T cell 

responses included DRB1*1303 (median SFC 17, p=0.01), 

DPB1*0301 (median SFC 11, p=0.02) and DPB1*1501 (median 

SFC 10, p=0.07). A suggestive association was observed between 

DQB1*0303 (median SFC 4, p=0.07) alleles and decreased 

frequency of MV memory IFN-g response. Thus, the presence 

or absence of certain HLA alleles may influence long-term 

measles immunity and the dynamics of virus-specific T cell 

memory outcome in vaccinated subjects. 

doi:10.1016/j.clim.2007.03.276 

F.64 Loss of Interferon Gamma Receptor Alpha 

(IFNGR α) in Peripheral Blood Mononuclear Cells 

from Patients with Chronic Hepatitis C 

Lucia Cristina Jamli Abel, Professor, Albert Einstein 

Research Institute-Albert Einstein Hospital, São Paulo, 

Brazil, Mário Pessoa, Albert Einstein Research 

Institute-IEP-Albert Einstein Hospital, São Paulo, Brazil, 

Hoel Sette Jr., Albert Einstein Research Institute-IEP- 

Albert Einstein Hospital, São Paulo, Brazil, Carlos 

Moreira-Filho, Albert Einstein Research Institute-IEP- 

Albert Einstein Hospital, São Paulo, Brazil, Ben-Hur Ferraz 

Neto, Albert Einstein Research Institute-IEP-Albert Einstein 

Hospital, São Paulo, Brazil, Luciana Marti, Albert Einstein 

Research Institute-IEP-Albert Einstein Hospital, São Paulo, 

Brazil, Denise Rodrigues, Infectious Diseases Division, 

Federal University of São Paulo, São Paulo SP, Brazil, Esper 

Kallas, Professor, Infectious Diseases Division, Federal 

University of São Paulo, São Paulo, Brazil, Venancio Alves, 

Professor, Department of Pathology, University of São 

Paulo School of Medicine, São Paulo, Brazil 

The mechanisms underlying the pathogenesis of chronic liver 

disease due to HCV are not fully understood. IFN-γ plays an 

important role in viral clearance, where IFN-γ either exogenous 

or endogenously produced by virus-specific CD8+ Tcells inhibits 

the replication of HCV. The alpha chain receptor (IFNGR α) isa 

high affinity receptor that binds to IFN-γ, therefore its 

expression can be important in the control of HCV infection. 

Using flow cytometric analysis, we found that the number of 

peripheral lymphocytes expressing alpha chain receptor (IFNGR 

α) in patients with chronic hepatitis C was lower than the normal 

controls (N) (p=0.018) and significantly decreased in patients 

with abnormal ALT (p=0.05). In chronic patients the expression 

of IFNGR α was inversely correlated to viral load (r=−0.362, 

p=0.038). In the liver, 50% of T cell lines obtained from biopsy 

isolated from patients with end stage liver disease expressed 

IFNGR α and when stimulated with staphylococcal enterotoxin B 

(SEB)producedhighlevelsofIFN-γ. No significant difference 

was found for IFN-γ production by peripheral blood mononuclear 

cells in the presence of PHA between chronic HCV patients and 

normal controls, but the IFN-γ production was weaker in viremic 

chronic hepatitis C patients than in subjects who are able to 

clear the virus (p=0.033). The results suggest that HCV infection 

may down-regulate the number of lymphocytes expressing 

IFNGR α in the peripheral blood but not in liver and that the 

intrahepatic response contributes at least in part to the control 

of disease. 

doi:10.1016/j.clim.2007.03.277 

S35 

F.65 Normal Adult Ranges and Diversity of Serotype 

Specific Anti-Pneumococcal Antibodies (SSAPAbs) 

after Immunisation with 23-Valent Polysaccharide 

Vaccine 

Robert Hallam, Senior Biomedical Scientist, Papworth Hospital/ 

Immunology Department, Cambridgeshire, England, Paul 

Balmer, Clinical Scientist, HPA, Vaccine Evaluation Department, 

Manchester, England, Ray Borrow, Clinical Scientist, HPA- 

Vaccine Evaluation Department, Manchester, England, Diana 

Bilton, Consultant Respiratory Physician, Papworth Hospital, 

Respiratory Infection, Inflammation and Immunology (RIII) 

Department, Cambridgeshire, England, Charles Haworth, 

Consultant Respiratory Physician, Papworth Hospital, 

Respiratory Infection, Inflammation and Immunology (RIII) 

Department, Cambridgeshire, England, Andrew Exley, 

Consultant Immunologist, Papworth Hospital/Immunology 

Department, Cambridge 

Background: Clinical and experimental studies identify 

SSAPAbs as key elements of pneumococcal immunity reflecting 

interactions with antigen specific and pathogen recognition 

receptors on APCs, B and T cells. New assays for SSAPAbs 

incorporating multiplex bead arrays with CPS and 22F 

absorption enable analysis of large data sets to determine 

normal adult ranges and diversity of responses. Earlier 

studies indicating diversity of responses and potential for 

immunologic refractoriness caution against standard 

approaches in healthy volunteers. Method: We investigated 

pneumococcal immunity in 250 adults with recurrent lower 

respiratory tract infections, measuring serum SSAPAbs pre 

and 4–6 weeks post-immunization with 23-valent polysaccharide 

vaccine. We tested the hypothesis that cases include 

a mixture of normal and low responders to standard 

immunization, on a background of natural exposure to 

antigen through carriage and infection. SSAPAb levels pre, 

post, and post/pre were log transformed and examined for 

goodness of fit using mixture modeling, with censoring of 

outlying data according to post-immunization SSAPAb levels. 

We then investigated for hierarchy and diversity of SSAPAb 

levels after immunization. Results: Mixture modeling indicated 

serotype specific normal and low adult ranges. 

Confidence intervals were resolved to determine threshold

S36 Abstracts 

values for antibody levels post-immunization. Serotypes 14, 

9V, and 4 appear high to low in hierarchy of responses. 

Discussion: Third generation multiplex assays for SSAPAb 

levels, without cross-reacting antibodies as confounders, 

indicate normal ranges after immunization are serotype 

specific with evidence of hierarchy and diversity. These data 

are consistent with serotype specific and non-specific pathways 

in antibody responses to pneumococcal capsular 

polysaccharides. 

doi:10.1016/j.clim.2007.03.278 

F.66 Differential Cytokine Response Induced by 

M. Avium and M. Abscessus in Human Macrophages 

is Mediated Through p38 MapKinase Signalling 

Pathway and Partially Dependent on TLR2 

Activation 

Elizabeth Sampaio, Senior Visiting Investigator, NIAID, 

NIH, Bethesda, MD, Houda Elloumi, Postdoctoral Fellow, 

NIH/NIAID, Bethesda, MD, Adrian Zelazny, Staff Scientist, 

NIH/NIAID, Bethesda, MD, Yvonne Shea, Laboratory 

Supervisor, NIAID/NIH, Bethesda, MD, Li Ding, Lab 

Manager, NIAID/NIH, Bethesda, MD, Steven Holland, Lab 

Chief, NIH/NIAID, Bethesda, MD 

Non-tuberculous mycobacteria (NTM) are ubiquitous 

environmental organisms that can cause chronic lung 

infection associated with primary or acquired immune 

deficiencies or in otherwise apparently normal individuals. 

Among NTM, M. avium is the most common cause of 

mycobacterial infection. Moreover, M. abscessus is an 

emerging pulmonary pathogen responsible for the majority 

of infections by rapidly growing mycobacteria. It has been 

shown that the ability of mycobacteria to induce TNFα 

secretion is inversely related to their virulence. In this 

work, cytokine response and signaling pathways triggered 

by reference and clinical isolates of M. abscessus and M. 

avium (5 smooth and 5 rough morphotypes) were assessed 

in human PBMCs and monocytes. Mycobacteria-induced 

TNFα response is enhanced for M. abscessus (8535 

±1010 pg/ml) as compared to M. avium (2400±244 pg/ 

ml, pb0.017) and no major differences were noted when 

compared colony morphologies within the same species. 

All mycobacterial strains were able to activate p38 MAP 

kinase phosphorylation and NF-kB translocation. Induction 

of TNFα was dependent on p38 MAPK signaling pathway 

since pre-incubation of cells with the p38 signaling 

inhibitor (SB203580) led to N80% reduction in cytokine 

secretion. In addition, treatment of cells with anti-human 

TLR2 antibodies showed TLR2 to be at least partially 

involved in signaling for M. abscessus as well. The present 

data indicate that M. avium is a more virulent pathogen 

than M. abscessus and elicits limited activation of cellular 

effector mechanisms, thereby escaping elimination. 

Accordingly, lung disease induced by NTM in nonpredisposing 

individuals may be associated with a yet uncharacterized 

underlying genetic defect. 

doi:10.1016/j.clim.2007.03.279 

F.67 Characterization of the Cellular Immunity in 

Patients Presenting Extensive Dermatophytoses Due 

to Trichophyton Rubrum 

Dewton Moraes Vasconcelos, MD, PhD, Department of 

Dermatology, University of São Paulo Medical School, São Paulo, 

Brazil, Anna Cristina Collanieri, BSc, Department of 


Brazil, Mauricio Domingues Ferreira, MD, PhD, Department of 


Brazil, Tatiana Negri Santi-BSc, Department of Dermatology, 

University of São Paulo Medical School, São Paulo, Brazil, Anete 

S. Grumach, MD, PhD, Department of Dermatology, University 

of São Paulo Medical School, São Paulo, Brazil, Alexandre 

Almeida, MD, MSc, Department of Dermatology, University of 

São Paulo Medical School, São Paulo, Brazil, Alberto Jose da 

Silva Duarte, MD, PhD, Department of Dermatology, University 

of São Paulo Medical School, São Paulo, Brazil 

Background: Dermatophytes cause infection in humans 

independent of the immunological status of the patient. In 

common to other infections the clinical features differ in 

immunodeficient patients. Dermatophytoses in cellular immunodeficient 

patients are usually less inflammatory, but some 

patients present pustular extensive lesions, frequently with 

follicular involvement. 

Objectives: Obtaining a reliable antigen by growth and 

purification of T. rubrum and to evaluate the immune 

reactivity “in vitro”, and quantify the immune response to 

the peptide YIIDTGIDID of T. rubrum. Methods: The fungal 

samples were obtained from the fungal library of the Institute 

of Tropical Medicine for antigen preparation, and the peptide 

was synthesized by EvoQuest. The lymphoproliferation assay 

was performed by tritiated thymidine incorporation and the 

cytokine quantifications by ELISA of culture supernatants. 

Results: The antigenic extract was efficient in the stimulation 

of cell cultures. The response to the peptide was also efficient 

and highly specific in sensitized controls. Despite most 

patients presented deficient responses, some presented 

normal lymphoproliferation. There was no difference 

between the cytokine secretion among patients and controls. 

doi:10.1016/j.clim.2007.03.280 

F.68 Evaluation of Cellular Responses to Rotavirus 

and NSP4 in Children during Natural Rotavirus 

Infection 

Jyoti Logani, Research Officer, Department of Pediatrics, All 

India Institute of Medical Sciences, New Delhi, India, Santosh 

Gupta, Research Associate, Department of Pediatrics, All India 

Institute of Medical Sciences, New Delhi, India, Shinjini 

Bhatnagar, Scientist, Department of Pediatrics, All India 

Institute of Medical Sciences, New Delhi, India, Pratima Ray, 

Scientist, Department of Pediatrics, All India Institute of 

Medical Sciences, New Delhi, India, M.K. Bhan, Department of 

Pediatrics, All India Institute of Medical Sciences, New Delhi, 

India 

Rotavirus (RV) is a major cause of gastroenteritis in young 

children leading to ∼611,000 deaths annually. Further

Abstracts 

development and evaluation of effective vaccines require better 

understanding of immune effectors involved in protective 

immunity. We examined IFN-γ and/or IL-4 responses in PBMC of 

children suffering from RV (n=25), non-RV (n=10) gastroenteritis 

and healthy adults (n=10) by ELISPOT assay. We detected IFN-γ 

but no IL-4 response to NSP4 and RV in children with RV 

gastroenteritis and adults. IFN-γ responses in adults were 

persistent and significantly higher than children (pb0.01). The 

responses to the RV were stronger in magnitude in comparison to 

NSP4, but were positively correlated (pb0.01, rs=0.666). During 

RV gastroenteritis, 8/25 children elicited IFN-γ response to NSP4 

between 1 and 30 days after onset of diarrhea; in the control 

uninfected children, no response to NSP4 was observed. The % 

responders with positive IFN-γ response to NSP4 detected 

between 1 and 6 days of illness were 11.1%, 66.6% between 7 

and 15 days and 30% between 16 and 30 days of illness. No IFN-γ 

responses were detected beyond 30 days of diarrhea. On 

screening of three samples from same subject, it was further 

validatedthatatransientriseintheIFN-γresponsetoNSP4occurs 

between 7 and 25 days of symptomatic RV diarrhea. Stool RV+ 

children depicted similar kinetics of IFN-γ response to RV. Thus 

significant IFN-γ response to NSP4 and RV indicates Th1 response 

duringnaturalRVinfection.IFN-γresponseistransientinchildren 

whereas in adults, responses are persistent and may play a role in 

protection. 

doi:10.1016/j.clim.2007.03.281 

F.69 Differential NF-kappaB Responses Induced by 

Bordetella Pertussis Filamentous-Hemagglutinin 

(FHA) in Macrophages and Bronchial Epithelial Cells 

Tzvia Abramson, Profesor, SJSU, Palo Alto, CA, David 

Relman, Professor, Stanford University, Palo Alto, CA, 

Hassya Kedem, Research Associate, Stanford University, 

Palo Alto, CA 

Filamentous hemagglutinin (FHA), an adhesin and secreted 

factor of Bordetella pertussis, induces both inflammatory and 

apoptotic responses. Given the role of NF-kappaB transcription 

factor family in both these functions, we investigated the FHA 

interference with this pathway. FHA (5 μg/ml) induces a rapid 

degradation of IkappaB (within 30 min) in human monocytes 

and U-937 macrophages. IkappaB cytosolic levels are restored 

and accumulated within 2 h. This activation of NF-kappaB 

pathway is confirmed by NF-kappaB DNA binding as well as by 

inflammatorycytokinesecretion.Noneofthoseactivitieswas 

observed in BEAS-2B bronchial epithelial cells. Surprisingly, 2hour 

exposure of both epithelial cells and macrophages to FHA 

blocked the ability of TNF-α to induce proteasomal degradation 

of IkappaB as well as the nuclear translocation of p65. 

Immunoprecipitation analysis revealed the ubiquitination and 

phosphorylation of IkappaB, implicating that 2-hour treatment 

with FHA interferes with the proteasomal activity rather than 

an upstream activity. Attenuated proteasome activity is 

revealed at 2–8 h FHA treatment in both cell types. However, 

no significant inhibitory activity was observed at less than 1 h 

exposure to FHA. These results imply that the accumulated 

levels of IkappaB at 2 h exposure of cells to FHA may result in 

proteasomal inhibition. This study suggests that despite the 

immediate strong activation of inflammatory responses by FHA 

in macrophages, longer exposure of immune and host cells to 

this secreted virulent factor may trigger anti-inflammatory 

activity which may interfere with the development of an 

effective immune response that will resolve the infection 

promptly. 

doi:10.1016/j.clim.2007.03.282 

F.70 Cytokine Activated Human Neutrophils 

Simultaneously Express T Cell Co-Stimulatory and 

Negative Regulatory Molecules 

Paul Bankey, Associate Professor, University of Rochester 

Medical Center; Surgery, Rochester, NY, Mita De, Research 

Associate, University of Rochester Medical Center; Surgery, 

Rochester, NY, Andrea Zucchiatti, Research Fellow, 

University of Rochester Medical Center; Surgery, Rochester, 

NY, Asit De, Assistant Professor, University of Rochester 

Medical Center, Rochester, NY, Carol Miller-Grazia, 

Professor of Immunology, University of Rochester Medical 

Center; Surgery, Rochester, NY 

The role of neutrophils (PMN) in innate immunity is well 

established. However, their role in adaptive immunity is not 

well characterized. Initiation of adaptive immunity by professional 

antigen presenting cells (APCs) is critically dependent on 

expression of MHC class II molecules (e.g., HLA-DR) and costimulatory 

molecules (e.g., CD86). APCs also regulate T cell 

mediated adaptive immunity through expression of negative 

regulatory molecules such as program death ligands (PDL) and 

immunoglobulin like transcript (ILT) molecules. Our purpose 

was to assess the surface expression of HLA-DR, CD86, PD-L1 

and 2 and ILT-2 and 4 by flow cytometry in fresh and cytokine 

(GM-CSF+IFN-γ: G+I) treated neutrophils isolated from healthy 

controls (n=22). Untreated neutrophils expressed low levels 

(b2%) of HLA-DR and CD86; however, in vitro culture of 

neutrophils with cytokines G+I significantly increased the 

expression of both molecules (HLA-DR: 5.61±1.16% at 24 h 

and 17.94±2.95% at 48h; CD86: 2.57±0.45% at 24 h and 8.11± 

1.59% at 48 h). Concurrently, cytokines G+I also significantly 

induce PMN expression of the T cell negative regulatory 

molecules: PD-L1 and 2 (PD-L1: 0.11±0.06% in fresh cells, 

26.49±2.47% at 24 h and 39.55±4.72% at 48 h; PD-L2: 0.25± 

0.0.25% in fresh cells, 6.12±1.37% at 24 h and 14.94±4.58% at 

48 h). ILT4 is constitutively expressed on over 90% of human 

neutrophils and expression is unchanged after culture with G+I. 

ILT-2 is not expressed on either fresh or G+I treated neutrophils. 

Simultaneous expression of T cell negative signaling molecules 

(PD-L1 and 2, ILT4) and MHC/co-stimulatory molecule CD86 in 

neutrophils supports an expanded role for PMN in both initiation 

and regulation of adaptive immunity. 

doi:10.1016/j.clim.2007.03.283 

S37 

F.71 An Analysis of HCV and CMV Specific Effector T 

Cells in Peripheral Blood, Perihepatic Lymph Nodes 

and Liver in HCV Infected and Uninfected Patients 

Dilip Moonka, Medical Director of Liver Transplantation, 

Henry Ford Health Systems, Division of Gastroenterology,

S38 Abstracts 

Detroit, MI, Kimberly Milkovich, Research Associate, 

Division of Rheumatology, Case Western Reserve University, 

Cleveland, OH, Benig Rodriguez, Assistant Professor, Center 

for AIDS Research, Case Western Reserve University, 

Cleveland, OH, Michael Lederman, Professor, Case Western 

Reserve University, Center for AIDS Research, Cleveland, 

OH, Marwan Abouljoud, Director of Transplant Institute, 

Henry Ford Health Systems, Transplant Institute, Detroit, 

MI, Donald Anthony, Assistant Professor, Case Western 

Reserve University, Division of Rheumatology and Infectious 

Disease, Cleveland, OH 

It is difficult to detect HCV-specific T cells in the peripheral 

blood of chronic HCV patients, while T cells specific 

for other viruses appear intact. One explanation for this is a 

compartmentalization of the immune response. Here we 

performed phenotypic analysis of T cells isolated from liver, 

perihepatic lymph nodes (LN), and peripheral blood of 23 

HCV infected and 12 uninfected subjects undergoing liver 

transplantation. Results: HCV specific IFN-γ and proliferative 

responses were commonly observed in the LN of HCV infected 

subjects (40% and 46% respectively), while they were 

uncommonly observed in the peripheral blood and liver 

(IFN-γ 16% and 25% respectively; proliferation 11% and 8% 

respectively). In contrast, CMV specific IFN-γ producing 

responses were not as commonly observed in the LN (30%) 

compared to the periphery (58%) and liver (60%) of these 

same HCV patients. Conclusions: These data are among the 

first to look at HCV-specific responses in perihepatic lymph 

nodes. The data indicate a selective defect in HCV, but not 

CMV specific, T cell effector function in the periphery of 

chronic HCV patients. The relatively common presence of 

HCV-reactive T cells in the LN is consistent with T cell 

activation at this site. Finally, we do not detect HCVspecific 

T cells more readily in the liver compared to the 

periphery or LN, indicating that hepatic lymphocytes are 

defective in effector function similar to those in the 

peripheral blood. The frequency of HCV-specific T cells did 

not clearly correlate with the severity of recurrent HCV 

after transplant. 

doi:10.1016/j.clim.2007.03.284 

F.72 Carbohydrate Analysis of HIV Envelope 

Glycoprotein 

Ingrid D. Cruzado-Park, Senior Development Scientist, 

Beckman Coulter, Inc. Fullerton, CA, Munir Alam, Duke 

University Medical Center, Durham, NC, Hua-Xin Liao, Duke 

University Medical Center, Durham, NC, Barton Haynes, 

Director, Duke University Medical Center, Human Vaccine 

Institute and CHAVI, Durham, NC, Heather Desaire, Duke 

University Medical Center, Durham, NC, Enrique Rabelli, 

Director, Custom BioPharma Solutions, Beckman Coulter, 

Inc. Miami, FL, Sybil D’Costa, Staff Advanced Research 

Scientist, Beckman Coulter, Inc. Miami, FL, Michael H. 

Simonian, Manager, Biomarker Discovery and Automation 

Center, Beckman Coulter, Inc. Fullerton, CA, Chitra K. 

Ratnayake, Team Leader, Beckman Coulter, Inc. Biomarker 

Applications and Chemistry Development, Fullerton, CA 

To date over 65 million people have been infected with the 

human immunodeficiency virus (HIV) which continues to 

claim several million lives a year. There is an urgent need to 

design and develop vaccines that generate broad neutralizing 

antibody enabling preventative vaccination strategies. 

Although the virus has evolved to circumvent the immune 

response, for example, carbohydrates on the envelope 

glycoprotein of HIV serve as a strong defense against antibody 

mediated responses, there is evidence that these oligomannose 

components can also serve as ideal vaccine candidates 

due to their ability to generate efficacious neutralizing 

antibodies in HIV infected individuals (2G12 antibody) albeit 

infrequently. To address the role of glycosylation in HIV 

pathogenesis and immunogenicity, carbohydrate profiles 

from wild type and consensus modeled, genetically engineered 

envelope glycoproteins were compared. We present 

data showing the N-linked oligosaccharide profiles for two 

types of HIV gp140 envelope glycoproteins; JRFL is a primary 

isolate and the other from a genetically engineered strain 

containing a consensus sequence (ConS), which is more 

effective at eliciting neutralizing antibodies. Following 

treatment with PNGase F, released N-linked oligosaccharides 

were labeled with fluorescent 8-aminopyrene-1,3,6-trisulfonate 

and profiled using the ProteomeLab PA 800 Protein 

Characterization System with laser-induced fluorescence 

(LIF) detection (488 nm excitation, 520 nm emission). We 

observed significant differences in the carbohydrate profiles 

of these gp140 envelopes and will present and discuss those 

differences. This strategy for oligosaccharide characterization 

and subsequent identification may help evaluate role of 

glycosylation in generating broadly neutralizing antibody 

responses and enable intelligent design of carbohydratebased 

epitope vaccines. 

doi:10.1016/j.clim.2007.03.285 

F.73 Defining the Role of BmIL-5 in Tropical 

Pulmonary Eosinophilia 

Sung (Steve) Kwon, University of Illinois College of Medicine 

at Rockford, Rockford, IL, Gnanasekar Munirathinam, 

University of Illinois College of Medicine at Rockford, 

Rockford, IL, Anand Setty Balakrishnan, University of Illinois 

College of Medicine at Rockford, Rockford, IL, Ramaswamy 

Kalyanasundaram, University of Illinois College of Medicine 

at Rockford, Rockford, IL 

This study describes characterization of a novel pathogenderived 

human IL-5 receptor binding molecule designated 

BmIL-5. This molecule was identified in our laboratory by 

screening a phage displayed cDNA expression library of the 

infective larvae of the human lymphatic filarial parasite, 

Brugia malayi. Soluble human IL-5 receptor (huIL-5R) was 

used as the bait. This screening procedure yielded a single 

clone and sequence analyses of the insert from this clone 

showed a novel protein designated as “B. malayi-derived IL- 

5-like molecule” (BmIL-5). Recombinant BmIL-5 was then 

expressed and purified. ELISA-based binding assays confirmed 

binding of rBmIL-5 to huIL-5R. Functional significance 

of this binding was then evaluated using a human eosinophil 

cell line, named Hs 454.T. Initial studies showed that BmIL-5

Abstracts 

binds to eosinophil surface. To test whether this binding 

initiates any signaling events, we evaluated STAT1 phosphorylation 

status in the eosinophil. We also performed a series of 

experiments to identify the receptor binding region of BmIL-5 

by peptide mapping. In these studies, we designed overlapping 

20 mer peptides and evaluated the binding pattern of 

these peptides to huIL-5R. These studies identified peptide 1 

and peptide 11 as the domains binding to huIL-5R. An 

interesting observation was that both these peptides interfered 

with the binding of huIL-5 to huIL-5R in vitro. Thus, we 

believe that BmIL-5 interferes with IL-5 function by preventing 

IL-5 binding to IL-5R. Our findings demonstrate a novel 

method of how pathogens potentially manipulate the human 

immune system. 

doi:10.1016/j.clim.2007.03.286 

F.74 What Triggers Transient AIDS in the Acute 

Phase of HIV Infection and Chronic AIDS at the End 

of the Incubation Period? 

Ivan Kramer, Associate Professor, UMBC, Canada Physics, 

Catonsville, MD 

Novel dynamical models are introduced demonstrating 

that the T helper cell (THC) density drops in the acute 

infection phase of HIV infection, sometimes causing 

transient AIDS, and at the end of the incubation period 

causing chronic AIDS have a common dynamical cause. The 

immune systems’ inability to produce enough uninfected 

THCs to replace the infected ones it is destroying causes a 

drop in the THC density at any stage of HIV infection. 

Increases in viral infectivity, probably caused by random 

mutation of HIV, are shown to drive the progression of the 

infection. The existence of transient AIDS and this model 

which explains it are inconsistent with the antigenic 

diversity thresholds model of AIDS. The minimum incubation 

period for the long-term non-progressors (LTNPs) was 

calculated from a novel physical model: 0.3% of infected 

people have incubation periods of 23.1 years or more, and 

there is no biomedical difference between LTNPs and 

progressors. If this model is correct, then there is no 

unique, special anti-viral substance secreted by the CD8+ T 

cells of LTNPs that accounts for their unusually long 

incubation periods; indeed, to date, no such substance has 

been found although researchers have looked for it for 

years. The model also predicts that chronic AIDS results 

from 3 random transitions linking 4 clinically distinct stages 

of HIV infection following seroconversion, a result that 

agrees with the World Health Organization’s staging system 

based on clinical manifestations of the disease. 

doi:10.1016/j.clim.2007.03.287 

F.75 Characterization of a CD21low B Cell 

Subpopulation in Patients with CVID 

Mirzokhid Rakhmanov, Postdoctoral Fellow, Department of 

Rheumatology and Clinical Immunology, University Hospital 

Freiburg, Freiburg i. Breisgau, Germany, Sylvia Gutenberger, 

Research Assistant, Department of Rheumatology and 

Clinical Immunology, University Hospital Freiburg, Freiburg i. 

Breisgau, Germany, Baerbel Keller, Graduate Student, 

Department of Rheumatology and Clinical Immunology, 

University Hospital Freiburg, Freiburg i. Breisgau, Germany, 

Klaus Warnatz, Assistant Medical Director, Department of 

Rheumatology and Clinical Immunology, University Hospital 

Freiburg, Freiburg, Germany, Hans-Hartmut Peter, Director 

of the Department, Department of Rheumatology and 

Clinical Immunology, University Hospital Freiburg, Freiburg, 

Germany 

Common variable immunodeficiency (CVID) is a primary 

immune deficiency disorder characterized by hypogammaglobulinemia 

and recurrent bacterial infections. Analyzing B 

cell homeostasis in a subgroup of CVID patients with 

splenomegaly and autoimmune cytopenia revealed a B cell 

population with unusually low expression of CD21. We 

therefore termed these cells “CD21low” B cells. The low 

expression of CD21 is the effect of a reduced transcription. 

However, coexisting B cells expressing normal levels of CD21 

in these patients suggest that the low expression is not due to 

genetic dysregulation of CD21 expression, but rather the 

result of an unusual cellular differentiation. The phenotyping 

of these cells did not identify a stage along the current 

understanding of B cell differentiation, but the increased 

cell size and expression of CD86 suggest rather a recent 

activation status. The frequency of somatic hypermutations 

was increased compared to naïve CD21+ B cells, but below 

memory B cells of the same patient. We and others had 

previously described a similar B cell population in patients 

with SLE or chronic HIV infection. Interestingly, CD21low B 

cells express increased levels of MxA message, suggesting 

recent exposure to type I interferons. The expansion of 

CD21low B cells is associated with severe shifts within the 

CD4+ T cell compartment of these patients. Therefore, we 

suggest an inflammatory dysregulation of the immune system 

underlying the disturbed B and T cell homeostasis in CVID 

patients with Freiburg type Ia. 

doi:10.1016/j.clim.2007.03.288 

S39 

F.76 Use of Prophylactic Antibiotics and Adjunctive 

Therapies in Primary Immunodeficiency Diseases 

Pierre Yong, Fellow, Hospital of the University of 

Pennsylvania, Division of Allergy and Immunology, 

Philadelphia, PA, John Boyle, Founder, Immune Deficiency 

Foundation, Towson, MD, Jordan Orange, Assistant 

Professor, Children’s Hospital of Philadelphia, Division of 

Allergy and Immunology, Philadelphia, PA 

Background: There is limited data supporting the use of 

prophylactic antibiotics and other adjunctive therapies in 

primary immunodeficiency diseases (PIDD). Methods: A webbased 

survey regarding therapy in PIDD was designed jointly by 

the Immune Deficiency Foundation and the American Academy 

of Allergy, Asthma & Immunology (AAAAI), and offered to AAAAI 

members. The response rate was 13.5%, n = 405. Responses 

were analyzed with a basic statistical program. Those devoting

S40 Abstracts 

more than 10% of their clinical practice to PIDD were defined as 

experts. Results: Use of prophylactic antibiotics in PIDD is 

widespread and perceived to be efficacious. Significant 

differences, however, between expert and non-expert immunologists 

were found in frequency of use as well as in perceived 

effectiveness in multiple PIDD. Experts find prophylaxis more 

useful and use it more often. They also use prophylaxis in 

conjunction with IVIG more frequently. Live viral vaccines were 

most frequently avoided in severe combined immunodeficiency 

and DiGeorge syndrome. Additionally, differences in each 

group’s vaccine use were seen in several PIDD, including 

hyper-IgM syndrome. The only hygiene intervention felt 

beneficial was hand-washing with soap and water. Complementary 

and alternative medicine (CAM) was not believed to 

enhance immunity or prevent infections. Discussion: These 

results highlight the significant use of prophylactic antibiotics 

for PIDD by immunologists. Important differences were found 

between experts and non-experts. Surprisingly, many immunologists 

do not avoid live viral vaccines in many PIDD despite 

labeling recommending avoidance. Most hygiene and CAM 

interventions were not seen as beneficial, although some were 

more popular than others. 

doi:10.1016/j.clim.2007.03.289 

F.77 Common Variable Immunodeficiency: 

Presentation of 7 Cases and Review of Literature 

Carlos Torres-Loza, England Immunoallergist, West National 

Medical Center and Department of Allergy and Clinical 

Immunology, Guadalajara, Jalisco, Mexico 

Aim of our work is to present a global view of this syndrome 

and analyze our experience of 7 adult patients bearing this 

syndrome which were seen at our department. It is a 

retrospective work for which it was designed a sheet to 

recollect data and also we reviewed literature of all possible 

electronic ways from the last 7 years, six are alive and one is 

now dead. The patients were diagnosed in most cases through 

air view infections such as pneumonia, chronic sinusitis and 

chronic middle otitis. In one of the cases an autoimmune 

process was the clinical problem before we made the 

diagnosis of this syndrome. During their evolution in our 

department, only three of them have had intestinal clinical 

manifestations characterized by several periods of chronic 

diarrhea and in three we found evidence of autoimmunity. All 

of the cases had low levels of serum immunoglobulin, neither 

of them had high serum level of IgE and all of our patients have 

shown normal lymphocytes subpopulations except in two 

where we found low levels of CD4. 

doi:10.1016/j.clim.2007.03.290 

F.78 Severe Agammaglobulinemia in an Adult Male 

with Nomal Number of Circulating B Cells 

Daniel Suez, Assistant Clinical Professor at UTSW Dallas, TX, 

Daniel Suez, MD, AAIC, PA, Irving, TX, Hans Ochs, Professor 

of Pediatrics, University of Washington School of Medicine, 

Seattle, WA 

We report here a case of a 34-year-old male with a history 

of recurrent pneumonia since early childhood with no other 

significant family history who presented to our clinic with 

severe agammaglobulinemia: IgG b7 mg/dl, IgM b5 mg/dl, 

IgA b13 mg/dl. Flow cytometry revealed normal numbers of 

circulating B cells −9.5% of CD20 (221/mm 3 ) and 12.4% of 

CD19 (322/mm 3 ) respectively. Clinical examination was 

remarkable for the paucity of lymphoid tissue. Further 

evaluation revealed normal numbers of memory B cells by 

CD27 expression yet very few switched memory B cells 

(1.36% vs. 23.7 in controls). The patient had normal BTK 

expression in B cells and had normal sequence analysis of 

gDNA for CD40L. Flow cytometry analysis of CD3 lymphocyte 

subsets revealed CD4/CD8 inversion with total CD4 16.9% 

(439/mm 3 ) and CD8 of 85.5% (2220/mm 3 ) while NK cell 

number was at 2% (52/mm 3 ). Other laboratory findings 

included normal kidney and liver functions. There is no 

history of significant gut disease and the patient’s ability to 

maintain stable through IgG levels on intravenous immunoglobulin 

therapy rules out the possibility of protein loss or 

high catabolic rate to explain the severe agammaglobulinemia. 

The differential diagnosis considered: all hyper-IgM 

syndromes can be ruled out due to the severe IgM deficiency, 

XLP syndrome may be considered yet the patient has no 

history of mononucleosis. To rule out XLP the patient will be 

sequenced for SAP and EBV by PCR will be requested. 

Autosomal recessive agammaglobulinemia can be also considered 

yet unlikely with normal circulating B cell numbers. 

doi:10.1016/j.clim.2007.03.291 

F.79 Severe, Steroid Resistant, Numular Atopic 

Dermatitis with Hypogammaglobulinemia 

Daniel Suez, Assistant Clinical Professor at UTSW Dallas, 

Daniel Suez, MD, AAIC, PA, Irving, TX, Hans Ochs, Professor 

of Pediatrics, University of Washington School of Medicine, 

Seattle, WA 

We report here the case of a 26-month-old white male with 

severe, steroid resistant numular atopic dermatitis since early 

infancy. Allergy evaluation revealed severe IgE mediated 

sensitivities to food and perennial allergens with elevated 

total serum IgE N8900 IU/ml. The patient failed to respond to 

numerous attempts of aggressive steroid therapy, both topical 

(under closed dressings) and systemic treatment along with 

comprehensive supportive therapy. Laboratory evaluation 

revealed hypogammaglobulinemia with a serum IgG level of 

342 mg/dl (normal range for age 453–916 mg/dl), and normal 

IgA (40 mg/dl) and IgM (28 mg/dl). CBC and differential revealed 

eosinophilia (N20%) and flow cytometry analysis was normal 

(CD19, CD3, CD4, CD8, CD16/56, CD45RO, and CD45RA). The 

patient was placed on high dose intravenous immunoglobulin 

therapy with dramatic improvement. Differential diagnosis 

considered is: Netherton syndrome (currently being evaluated 

by sequence analysis), Wiskott-Aldrich syndrome (which can be 

ruled out with normal platelet count), Omenn syndrome (yet not 

likely because he has normal numbers of circulating B cells), 

IPEX syndrome (yet not likely since the patient does not have any 

evidence for autoimmune disease such as polyendocrinopathy,

Abstracts 

neutropenia, or hemolytic anemia), and hyper IgE syndrome 

(yet unlikely as well since he does not have any of the clinical 

features). 

doi:10.1016/j.clim.2007.03.292 

F.80 Lymphocytic Interstitial Pneumonitis in 

Common Variable Immunodeficiency Disorders: 

A Comparison with HIV 

Dilani Arnold, Specialist Registrar, Department of 

Immunology, John Radcliffe Hospital, Oxford, England, 

Gleeson Fergus, Consultant Radiologist, Department of 

Radiology, Churchill Hospital, Oxford, England, Helen 

Chapel, Consultant in Immunology, Department of 

Immunology, John Radcliffe Hospital, Oxford, England, 

Robert Davies, Consultant in Chest Medicine, Department 

of Chest Medicine, Churchill Hospital, Oxford, England, 

Siraj Misbah, Consultant in Immunology, Department of 

Immunology, John Radcliffe Hospital, Oxford, England 

Background: The objective of this study was to assess the 

presentation of Lymphocytic Interstitial Pneumonitis (LIP) in 

Common Variable Immunodeficiency Disorders (CVID). Methods: 

The medical records of all CVID patients followed up in 

our department were examined retrospectively. Results: In 

our institution, between 1970 and 2006, 96 patients met the 

diagnostic criteria for CVID. Of these, six (four women, two 

men, mean age, 45 years, range 42−65) had biopsy-proven 

LIP (microscopic evidence of polyclonal lymphoid cell 

infiltrate). HIV PCR was negative in all patients. Two patients 

were asymptomatic; four presented with cough, fatigue and 

exertional dyspnoea (mean time 2.75 months (range 1−7) 

from initial presentation to LIP diagnosis). Five patients had 

elevated C-reactive protein (11−31); four had raised beta-2 

microglobulin (3.8−4.7). Two had T-cell lymphocytosis (2.5− 

8.09) with elevated CD4 counts (1.82−3.43). One patient had 

an elevated CD8 count of 5.14 (0.2−0.7) and another had B 

cell lymphopenia (CD19 0.06). Two had falling IgG levels 

(4.8−5.87) despite immunoglobulin therapy. The predominant 

radiological abnormalities on CT were ground-glass 

opacification and mediastinal lymphadenopathy (present 

in four patients). Two had subpleural nodules. Immunohistology 

showed either an excess of CD4 cells or CD8 cells in a minority of 

patients. No viral antigen was identified. Conclusion: The 

presentation of LIP is highly variable and diagnostically 

challenging. Comparison with LIP in immunodeficiency states 

such as HIV shows similar clinical and radiological features. The 

aetiology of LIP in CVID and HIV is unknown but is presumed to be 

viral, though no specific virus was identified in our small cohort. 

doi:10.1016/j.clim.2007.03.293 

F.81 Defective CD107a Surface Expression Heralds 

Munc13-4 Defect and Discriminates Between 

Genetic Subtypes of Familial Hemophagocytic 

Lymphohistiocytosis (FHL) 

Daniela Pende, Senior Investigator, Istituto Nazionale per la 

Ricerca sul Cancro, Genova, Stefania Marcenaro, Fellow, 

DIMES, Genova, Stefania Martini, Technician, Istituto 

Nazionale per la Ricerca sul Cancro, Genova, Alessandra 

Santoro, Fellow, Ospedale “G. Di Cristina”, Palermo, Gillian 

Griffiths, Senior Investigator, Sir William Dunn School of 

Pathology, Oxford, England, Maurizio Aricò, Physician, 

Ospedale dei Bambini “G. Di Cristina”, Palermo, Lorenzo 

Moretta, Full Professor, Istituto G. Gaslini, Genova 

Familial hemophagocytic lymphohistiocytosis (FHL) is a 

rare, heterogeneous fatal disease of early infancy characterized 

by an hyperinflammatory syndrome. Defective cellular 

cytotoxicity results in pathogen persistence and hypercytokinemia 

with disseminated infiltration of lymphocytes and 

histiocytes and hemophagocytosis. Differential diagnosis 

between FHL due to PRF1 (FHL2) or Munc13-4 (FHL3) and 

additional still unclassified genetic subtypes is not easy 

unless mutation analysis is performed. We investigated 

natural killer (NK) cells function and phenotype in patients 

with FHL2 (n=5) or FHL3 (n=8). NK cells were cultured in IL-2 

prior to their use in the various assays. Here we report on the 

surface CD107a expression as a novel rapid tool for 

identification of patients with Munc13-4 defect. Upon target 

interaction and degranulation, FHL3 NK cells displayed low 

levels of surface CD107a staining, in contrast to normal 

controls or perforin-deficient NK cells. B-EBV cell lines and 

dendritic cell targets reveal the FHL3 NK cell defect while 

highly susceptible tumor targets were partially lysed by FHL3 

NK cells expressing only trace amounts of Munc13-4 protein. 

Perforin-deficient NK cells were completely devoid of any 

ability to lyse target cells. Cytokine production induced by 

mAb-crosslinking of triggering receptors was comparable in 

patients and normal controls. However, when cytokine 

production was induced by co-culture with 721.221 B-EBV 

cells, FHL NK cells resulted high producers, whereas control 

cells were almost ineffective. This could reflect survival 

versus elimination of B-EBV cells i.e., the source of NK cell 

stimulation, in patients vs. healthy controls, thus mimicking 

the pathophysiological scenario of FHL. 

doi:10.1016/j.clim.2007.03.294 

S41 

F.82 Autoimmune Polyendocrinopathy-Candidiasis- 

Ectodermal Dystrophy Syndrome (APECED) Caused 

by Homozygous P326L AIRE Mutations in a Brazilian 

Family 

Patrícia de Campos Pieri, Biologist, Laboratory of Medical 

Investigation 36, Pediatrics Department FMUSP, São Paulo, 

Brazil, Joao B. Oliveira, Associate Researcher, Molecular 

Immunology and Genetics Section, Laboratory of Medical 

Investigation #56, USP, São Paulo, Brazil, Cristina Miuki Abe 

Jacob, Head of Unit, Department of Pediatrics, Allergy and 

Immunology Unit FMUSP, São Paulo, Brazil, Alberto J.S. 

Duarte, Lab Director, Laboratory of Medical Investigation 

#56 (LIM-56), USP, São Paulo, Brazil, Antonio Carlos Pastori, 

Assistant Professor, Pediatrics Department, Allergy and 

Immunology Unit FMUSP, São Paulo, Brazil, Magda Maria 

Carneiro-Sampaio, Head of Department, Department of 

Pediatrics, Allergy and Immunology Unit, FMUSP, São Paulo, 

Brazil

S42 Abstracts 

Introduction: APECED is characterized by the early onset 

of multiple organ directed autoimmunity, chronic mucocutaneous 

candidiasis and ectodermal dystrophy, and caused 

by recessive mutations in AIRE gene. The vast majority of 

affected patients are of northern European descent, 

Sardinian or Iranian Jews. We describe here a Brazilian 

family affected by the disease. Material and methods: All 

14 exons and flanking intronic regions of AIRE were 

amplified by PCR, purified and sequenced using an 

automated capillary sequencer. Results: The index patient, 

LGM, is a 15-year-old boy from São Paulo. At 7 months he 

developed recurrent thrush and at the age of 2 years 

presented ungueal candidiasis. At 9 years his height was 

119 cm (pb3), levels of IGF1 and IGFBP3 were low, and a 

diagnosis of GH deficiency was made. At 12 years he 

developed anti-adrenal antibodies (1/40), and at 15 years 

he showed high levels of ACTH and PTH. His mother 

presents oral thrush, megaloblastic anemia and hypoparathyroidism, 

and his sister has recurrent candidiasis, without 

signs of autoimmunity. Parents deny consanguinity. 

Genomic DNA sequencing of AIRE identified a homozygous 

missense mutation in exon 8 (Pro326Leu). This mutation 

affects the first PHD finger domain, probably interfering 

with adequate subcellular protein localization and functioning. 

Conclusion: This is to our knowledge the first report 

of AIRE mutations causing APECED in a Brazilian family, and 

of a P326L AIRE mutation in a homozygous state. Molecular 

diagnosis is important for genetic counseling and institution 

of adequate therapy early in life. 

doi:10.1016/j.clim.2007.03.295 

F.83 NOD2 Polymorphisms in CVID: Investigation 

into Associations with Gastrointestinal Pathology 

and Granulomatous Disease 

Kerri Packwood, Grade A Clinical Scientist in 

Immunology, Clinical Immunology, Oxford Radcliffe 

Hospitals NHS Trust, Oxford, OXON, England, Berne Ferry, 

Principle Scientist, Clinical Immunology, Oxford Radcliffe 

Hospitals NHS Trust, Oxford, OXON, England, Divya 

Punwani, PhD Student, Wetherall Institute of Molecular 

Medicine, Human Immunology Unit, Oxford, OXON, 

England, Eduardo Lopez-Granados, Consultant 

Immunologist, Clinical Immunology, Oxford Radcliffe 

Hospitals NHS Trust, Oxford, OXON, England, Helen 

Chapel, Consultant Immunologist, Clinical Immunology, 

Oxford Radcliffe Hospitals NHS Trust, Oxford, OXON, 

England 

Common Variable Immunodeficiency Disorders (CVID) 

are a heterogeneous group of diseases characterised by 

hypogammaglobulinaemia. Associated complications 

include enteropathy and granulomatous disease. These 

conditions have diverse immunopathogeneses including 

primary defects in B cells, T cells, macrophages, dendritic 

cells, NK cells and cytokine production yet often the 

underlying aetiology is unknown. Prediction of complications 

would aid clinical management and we are 

investigating possible disease modifier genes as a potentially 

powerful tool. Classification of heterophenotypic 

CVID subgroups would also enable more focused and 

productive research studies. Mutations of the NOD2 gene 

including gly908arg, leu1007finsc and arg702trp polymorphisms 

are associated with Crohn'€s disease. We 

hypothesised that NOD2 may also be a disease modifier 

gene towards an enteropathic or granulomatous CVID 

phenotype. Methods to examine the above three polymorphisms 

were established and investigated in a cohort 

of 52 CVID patients and 26 non-patient controls. Five 

heterozygous NOD2 leu1007finsc mutations, two heterozygous 

NOD2 gly908arg mutations and four heterozygous 

NOD2 arg702trp mutations were found amongst CVID 

patients giving allele frequencies of 0.048, 0.019 and 

0.038 respectively. No polymorphisms were found amongst 

non-patient controls. Although to date we are unable to 

demonstrate a significant genotype/phenotype association, 

results from this study show this as reliable 

screening method for gly908arg, leu1007finsc and 

arg702trp NOD2 polymorphisms. More patients are 

required to conclude whether these and/or additional 

NOD2 polymorphisms can define a clinical subgroup of 

CVID patients likely to develop enteropathic or granulomatous 

complications. 

doi:10.1016/j.clim.2007.03.296 

F.84 Development of Regulatory T Cells After 

Thymus Transplantation in Subjects with Complete 

DiGeorge Syndrome 

Ivan Chinn, Fellow-In-Training, Duke University Medical 

Center, Pediatrics, Durham, NC, Blythe Devlin, Assistant 

Research Professor, Duke University Medical Center, 

Pediatrics, Durham, NC, M. Louise Markert, Associate 

Professor, Duke University Medical Center, Pediatrics, 

Durham, NC 

Purpose: Characterize the development of CD4+Foxp3+ 

regulatory Tcells in subjects with complete DiGeorge syndrome 

after thymus transplantation. Background: Forty-four subjects 

with complete DiGeorge syndrome, including athymia (b50 

naïveTcells/cumm),havereceivedculturedpostnatalallogeneic 

HLA nonmatched thymus grafts. Twenty-seven of 32 

survivors are currently at 1-year or greater post-transplantation. 

All 1-year survivors have naïve T cells with normal 

proliferative responses to phytohemagglutinin and normal T 

cell receptor repertoires. We now report the percentages of 

Foxp3+ CD4+ T cells in the peripheral blood of 11 subjects who 

are 1-year survivors after thymus transplantation. Five of the 

subjects received peri-transplantation immunosuppression. 

Five of the 11 subjects have autoimmune thyroid disease, 

including one subject in the group who received immunosuppression. 

Results: All eleven subjects had percentages of CD4 

+Foxp3+ Tcells in the expected range compared to healthy adult 

controls at various time points post-transplantation. Absolute 

numbers of CD4+Foxp3+ T cells were low (12−65 cells/cumm), 

reflecting the subjects’ low total T cell counts for age. Similar 

percentages of regulatory T cells were found in subjects who 

received immunosuppression (median 3.85%, range 3.48−5.33%) 

compared with those who did not (median 4.58%, range 2.96 

−7.35%). The percentages of CD4+Foxp3+ Tcells did not appear

Abstracts 

to correlate with the development of autoimmune thyroid 

disease (median 5.2% [range 3.05−6.96%] in subjects with 

thyroid disease vs. 3.8% [range 2.96−7.35%] without thyroid 

disease). Discussion: Subjects with complete DiGeorge syndrome 

develop normal percentages of regulatory T cells in the 

peripheral blood after thymus transplantation. 

doi:10.1016/j.clim.2007.03.297 

F.85 Identification of a Mutation in the Gene Encoding 

the Endosomal Adaptor Protein p14 (MAPBPIP) in a 

Novel Primary Immunodeficiency Syndrome 

Jens Thiel, MD, University Hospital Freiburg, Freiburg, 

Germany 

The study of human primary immunodeficiency disorders 

has greatly advanced our understanding of basic 

host defense. We have investigated the molecular etiology 

of a novel autosomal recessive primary immunodeficiency 

syndrome. In a pedigree of 15 members of a Caucasian 

Mennonite family, four children suffered from congenital 

neutropenia, B cell and cytotoxic T cell deficiency. These 

children additionally showed a characteristic clinical 

phenotype associating small stature, hypopigmented 

skin, coarse facial features, and recurrent bronchopulmonary 

infections. In an attempt to identify a genetic 

basis for this novel immunodeficiency syndrome, we 

carried out a genetic linkage study. Since the family 

belongs to a religious isolate of Mennonite background, 

we expected that the disease would be associated with a 

homozygous mutation in a single gene. We identified four 

adjacent, perfectly segregating markers on chromosome 

1q21: D1S498, D1S2346, D1S305, and D1S1153. Within this 

minimal critical region of the linkage interval, there were 

a total of 87 genes or predicted genes and within the 

maximal possible linkage region, there were an additional 

105 genes or predicted genes. Combining the results of 

the linkage analysis with a transcriptional profiling 

approach, only one gene located within the critical region 

on chromosome 1q21 was significantly underexpressed in 

the patients, namely MAPBPIP. By genomic sequencing a 

homozygous mutation in the 3′-UTR of MAPBPIP, a recently 

identified adaptor molecule controlling late endosomal 

signaling by the MP1–MAPK complex was identified in the 

four index family members. Neutrophils from all patients 

showed decreased RNA and protein levels of p14. 

doi:10.1016/j.clim.2007.03.298 

F.86 Foxp3 Expression Following Bone Marrow 

Transplantation for IPEX Syndrome After 

Reduced-Intensity Conditioning 

Morna J. Dorsey, Assistant Professor, University of South 

Florida, St. Petersburg, FL, Aleksandra Petrovic, Assistant 

Professor, All Children’s Hospital, St. Petersburg, FL, 

Matthew R. Morrow, Director, Flow Cytometry Core Facility, 

University of South Florida, Pediatrics, St. Petersburg, FL, 

John W. Sleasman, Professor, University of South Florida, 

Pediatrics, St. Petersburg, FL, Larry J. Dishaw, Instructor, 

All Children’s Hospital, St. Petersburg, FL 

Objectives: To determine if immune reconstitution of Foxp3+ 

regulatory T cells correlates with clinical improvement of IPEX 

syndrome following allogeneic HSCT. Methods: 8-month-old 

male infant with a mutation in the polyadenylation site of Foxp3 

gene, absence of Foxp3 protein expression and clinical 

manifestations of IPEX syndrome, including eczema, colitis, 

failure to thrive, TPN requirement, and elevated serum IgE, 

underwent matched unrelated HSCT following reduced-intensity 

conditioning regimen. Donor chimerism was determined by 

molecular analysis of blood or bone marrow. Blood samples were 

collected pre- and post-transplant. Regulatory Tcell population 

was determined by flow cytometry staining for Foxp3+ 

CD25bright CD4+ T cells. Semi-quantitative PCR of Foxp3+ 

mRNA was conducted as well. Results: Patient underwent 

reduced-intensity conditioning with alemtuzumab followed by 

fludarabine and melphalan. GVHD prophylaxis included tacrolimus, 

methotrexate, and prednisone. Neutrophils engrafted 

day +15, and platelets day+29. Patient was a full donor chimera 

day +28 and +60. Foxp3 protein expression was absent pre- 

HSCT. Post-transplantation, percentage CD4+ T cells expressing 

Foxp3+ CD25bright phenotype increased from 4.5% (day +29) to 

23% (day +90) and continued in this trend. Foxp3 mRNA 

expression confirmed flow cytometry data. Serum IgE levels 

decreased from 5000 IU/mL pre-transplant to 6 IU/mL on day 

+90, eczema resolved, and secretory diarrhea and feeding 

intolerance improved. Conclusions: Regulatory T cell reconstitution 

is evident soon after HSCT following reduced-intensity 

conditioning correlating with development of full donor 

chimerism. Increased Foxp3 expression correlates with correction 

of clinical and laboratory manifestations of IPEX syndrome 

providing evidence that HSCT is a curative procedure for this 

disorder. 

doi:10.1016/j.clim.2007.03.299 

S43 

F.87 Monoclonal Gammapathy Following Cord Blood 

Transplantation for Chediak−Higashi Syndrome 

Kevin YehSheng Wang, FIT, Pediatrics, University of 

California, Los Angeles, Los Angeles, CA, Robert Roberts, 

Professor of Pediatrics, Department of Pediatrics, 

University of California, Los Angeles, Los Angeles, CA 

Introduction: Chediak–Higashi syndrome (CHS) is a rare 

autosomal recessive disorder which often undergoes progression 

to a life-threatening accelerated phase with 

lymphohistiocytic infiltration in multiple organs. We report 

a case of CHS in the accelerated phase which was successfully 

treated with IVIG and high dose corticosteroids. He 

underwent cord blood transplantation and then developed a 

monoclonal gammapathy. Case report: Our patient was 

diagnosed with CHS at 16 months of age by peripheral 

blood smear which revealed giant granules in his white blood 

cells. At 6 years of age, he presented to us with 

pancytopenia, fever, respiratory stress, mental status 

changes, and seizure activity. On physical examination, he

S44 Abstracts 

had significant hepatosplenomegaly. Bone marrow biopsy 

showed cytoplasmic inclusions granule and hemophagocytic 

cells. For treatment, he received 3 courses of IVIG (4.5 g/kg 

total). He was also given methlyprednisolone. He was then 

placed on dexamethasone until the time of transplantation. 

Over the course of several weeks, his mental status returned 

to baseline, seizure activity ceased, hepatomegaly resolved, 

and pancytopenia improved. Therefore his accelerated 

phase had undergone remission. He then received a 10/10 

match cord blood transplantation. The patient appeared to 

engraft successfully following the transplantation and 

routine infusions of IVIG were stopped. Approximately 

15 months after transplantation, the patient was clinically 

stable but serum IgG level was found to be 4000 mg/dL and 

his CD 4/8 ratio was 0.55. Immunoelectrophoresis revealed 

that he had developed a monoclonal IgG gammapathy. Bone 

marrow and repeat RFLP studies are being done to further 

investigate this abnormality. 

doi:10.1016/j.clim.2007.03.300 

F.88 Distribution, Infections, Treatments and 

Molecular Analysis in a Large Cohort of Patients with 

Primary Immunodeficiency Diseases (PIDD) in 

Taiwan 

Wen-I Lee, Assistant Professor, Immunodeficiency Diagnosis 

and Research Institute, Chang Gung University and 

Children’s Hospital, Taoyuan, China, Tang-Her Jaing, 

Assistant Professor, Department of Pediatric Hematology 

and Oncology, Chung Gung Children’s Hospital, Taoyuan, 

China, Meng-Ying Hsieh, MD, Department of Pediatric 

Neurology, Chang Gung University and Children’s Hospital, 

Taoyuan, China, Syh-Jae Lin, Associated Professor, 

Department of Pediatric Allergy, Immunology and 

Rheumatology, Chang Gung University, Taoyuan, China, 

Jing-Long Huang, Professor, Department of Pediatric 

Allergy, Immunology and Rheumatology, Chang Gung 

University, Taoyuan, China, Li-Chen Chen, Assistant 

Professor, Department of Pediatric Allergy, Immunology and 

Rheumatology, Chang Gung University, Taoyuan, China, 

Ming-Ling Kuo, Professor, Department of Microbiology and 

Immunology, Graduate Institute of Basic Medical Sciences, 

Taoyuan, China 

One hundred and twenty-four patients (from 120 

families) to date diagnosed as primary immunodeficiency 

diseases were enrolled from five tertiary medical centers. 

The distribution by an update eight categories showed 45 

patients (13 females/32 males; 36.3%) with predominant 

antibody deficiencies, 27 patients (6/21; 21.8%) with T and 

B cell immunodeficiency, 25 patients (9/16; 20.2%) with 

congenital defects of phagocyte, 25 patients (4/21; 20.2%) 

with other well-defined immunodeficiency syndromes, one 

boy (0.8%) with disease in immune deregulation (Chediak– 

Higashi syndrome) and another with complement 3 deficiency. 

None had defects in innate immunity or auto 

inflammatory disorders. Pseudomonas and Salmonella spp. 

were the two most identified microorganisms in septicemia 

(39.7%; 27/68 episodes). Twenty-three patients (18.5%) had 

mortality. Stem cell transplantation succeeded in 7 of 12 

patients. In addition to nine patients with DiGerge 

syndrome recognized by FISH, direct sequencing identified 

12 unique mutations from 20 families, reflecting distinct 

Taiwan geography, although a selection bias may exist. The 

study is still ongoing. 

doi:10.1016/j.clim.2007.03.301 

F.89 Expression of FoxP3 and CD25 in T Cells is 

Altered in ICOS-/- Common Variable 

Immunodeficiency (CVID) patients 

Hans Hartmut Peter, Head of Department,University 

Hospital Freiburg, Freiburg, Germany, Julia Horn, Assistant 

Doctor, University Hospital Freiburg, Department of Clinical 

Immunology and Rheumatology, Freiburg, Germany, Ulrich 

Salzer, Senior Postdoctoral Fellow, University Hospital 

Freiburg, Department of Clinical Immunology and 

Rheumatology, Freiburg, Germany, Jennifer Birmelin, MTA, 

Royal Free Hospital Department of Immunology, London, 

England, Klaus Warnatz, Consultant, University Hospital 

Freiburg, Department of Clinical Immunology and 

Rheumatology, Freiburg, Germany, Michael Schlesier, Head 

of Laboratory, University Hospital Freiburg, Department of 

Clinical Immunolgy and Rheumatology, Freiburg, 

Germany, Bodo Grimbacher, Consultant, Royal Free 

Hospital, Department of Immunology, 

London, England 

Objective: FoxP3+CD25+ high regulatory T cells play a 

central role in suppressing immune responses to self 

antigens. To determine whether CVID patients have an 

altered frequency of FoxP3+CD25+ high regulatory T cells 

(Tregs), we studied this cell type in 48 CVID patients 

including four ICOS−/− CVID patients from the Freiburg 

CVID cohort by FACS analysis. Results: First, we found that 

the frequency of FoxP3+CD25+ Tregs (median +/− SD; p>= 

Wilcoxon rank sum test) was not significantly different in 

CVID patients (1.9% +/− 0.9%, n = 48; p>= 0.05) as compared 

to healthy controls (3.41% +/− 0.9%, n = 17). Second, CVID 

patients with autoimmune phenomena (n = 17 of 48) do not 

have lower frequencies of Tregs than CVID patients without 

autoimmune phenomena except one patient who presents 

with immune thrombocytopenia (ITP). Interestingly, ICOS-/- 

CVID patients (n = 4 of 48) have normal or increased Treg 

frequencies. In addition, in ICOS−/− CVID patients an 

increased FoxP3+CD25− T cell population in comparison to 

other CVID patients and healthy controls is observed. 

However, culturing ICOS−/− CD4+ T cells with IL-2 for 72 h 

results in decreased FoxP3 expression (16.4% to 4.68%) of 

CD25- T cells due to activation. Conclusion: Our data suggest 

that CVID patients with autoimmune phenomena, do not 

have reduced numbers of Treg cells. However, ICOS−/− CVID 

patients who have no autoimmune phenomena show an 

increased FoxP3+CD25+ T cell population and an increased 

Foxp3 expression of CD25− T cells. 

doi:10.1016/j.clim.2007.03.302

Abstracts 

F.90 Clusters Differentiation in HIV Patients 

According to Measurements of T Cells 

Subpopulations in Whole Blood After In Vitro 

Activation with Mitogen 

Marie-Paule Guillaume, Physician, MD, Brugmann Hospital, 

Brussels 

Absolute counts of CD4+ lymphocytes are considered as the 

most important surrogate marker for the risk of AIDS. However, 

contrasts between patient evolutions and their presumed 

risk category are not unusual. We formulated the hypothesis 

that underlying regulation patterns of T cell responses affect 

the actual personal outcome despite similar levels of 

circulating CD4+ T cells. We used a standardized PHA stimulation 

of whole blood cultures as a simplified model of T cell 

interactions. Relative proportions of different subpopulations 

were assessed by cytofluorometry, characterized by combined 

expressions of CD4, CD25, CD8, CD28, and CD69 molecules. 

Prospective data from 175 HIV+ patients with different disease 

status, and 235 blood donors as controls, were evaluated. 

Three different clusters were found: controls and 2 subgroups 

of patients. Hierarchical analysis of these parameters allowed 

the provisional definition of an algorithm classifying over 95% 

of the patients according to their cluster. This was based on 

levels of CD4+25++CD69+ under PHA stimulation, basal levels 

of CD4+CD25+CD69+ and CD8+CD28negCD69+. Involvement of 

regulatory or suppressive cells was independently confirmed 

by expression of GITR markers. Repeated measures on 735 

successive observations show that patients tend to stick to 

their cluster despite changes in CD4 absolute levels or PHA 

response in vitro. Confirmation of these data would support 

the existence of different patterns of functional immune 

regulations in HIV patients. 

doi:10.1016/j.clim.2007.03.303 

F.91 HIV Treatment Reveals 

Hypogammaglobulinemia in an Individual with 

Common Variable Immune Deficiency 

Lulu Sun, Immunodeficiency Treatment Centre, McGill 

University Health Centre, Montreal, QC, Canada, 

Christos M Tsoukas, Immunodeficiency Treatment Centre, 

McGill University Health Centre, Montreal, QC, Canada 

While common variable immune deficiency (CVID) is 

characterized by hypogammaglobulinemia, HIV-1 infection 

is associated with elevated immunoglobulin levels. There 

have been four CVID patients reported in the literature who 

recovered antibody production after contracting HIV-1. Here 

we describe an HIV patient with concomitant CVID whose 

immunoglobulin levels dramatically decreased upon successful 

antiretroviral therapy (ART), and investigate a plausible 

mechanism by which HIV is able to modulate circulating 

immunoglobulin through regulation of the B cell activation 

factor (BAFF)/BAFF-receptor pathway. Four cohorts were 

compared: our HIV/CVID patient, HIV patients, CVID 

patients, and healthy controls. Phenotypic and functional 

analyses of the Tand B cells of these cohorts were performed 

using flow cytometry, and BAFF serum levels were measured 

by ELISA. In addition to marked hypogammaglobulinemia, 

the patient has a complete defect of memory IgD+/− 

CD27+CD19+ B cells that was not altered by ART, consistent 

with severe CVID. When viral load briefly increased, a 

transient normalization of immunoglobulins was also 

observed. At the same time, the percentage of BAFF-R+ B 

cells was boosted from low to normal levels. Plasma BAFF 

levels were high compared to controls during viral suppression, 

due primarily to the lack of memory B cells. Finally, T 

cell phenotypes remained abnormal, with a low CD4+ T cell 

count that is characteristic of rigorous HIV disease, and 

impaired in vitro cellular proliferation. Our findings suggest 

that high HIV viraemia was required to maintain normal 

immunoglobulin levels in the CVID patient, possibly through 

upregulating BAFF-R. 

doi:10.1016/j.clim.2007.03.304 

S45 

F.92 Novel IL12RB1 Compound Heterozygous 

Mutation in a Brazilian Family with Disseminated 

Bacille Calmette-Guérin (BCG) Infection 

Joao B. Oliveira, Associate Researcher, Molecular 

Immunology and Genetics Section, LIM56, USP, São Paulo, 

Brazil, Ana Nishiya, Technician, Molecular Immunology and 

Genetics Section, LIM-56, USP, São Paulo, Brazil, Ludovic de 

Beaucoudrey, Researcher, Laboratory of Human Genetics of 

Infectious Diseases, University of Paris Rene Descartes, 

INSERM U550, Paris, Jean-Laurent Casanova, Director, 

Laboratoire de Genetique Humaine des Maladies 

Infectieuses, INSERM Unite 550, Paris, Anete Grumach, 

Associate Professor, Primary Immunodeficiency Outpatient 

Unit ADEE-3003, LIM-56, USP, São Paulo, Brazil, Alberto J.S. 


Unit 56 (LIM-56), University of São Paulo, Brazil, São Paulo, 

Brazil, Dewton Moraes-Vasconcelos, Associate Professor, 

2Primary Immunodeficiency Outpatient Unit ADEE-3003, 

LIM-56, USP, São Paulo, Brazil 

Introduction. Mendelian inheritance to mycobacterial 

disease (MSMD) refers to a group of rare diseases characterized 

by predisposition to clinical disease caused by 

environmental non-tuberculous or poorly virulent mycobaterial 

species such as Bacille Calmette-Guérin (BCG) 

vaccine. Mutations in 5 components of the IFN-γ/IL-12 axis 

underlie MSMD: IFNR1, IFNR2, STAT1, IL12B and IL12RB1. We 

describe here a Brazilian family with a novel compound 

heterozygous mutation in IL12RB1. Material and methods. 

The 17 exons of IL12RB1 and flanking intronic regions were 

amplified by PCR, purified and sequenced using an automated 

capillary sequencer. Results. LMS, a 2.7-year-old girl, 

presented disseminated BCG infection 1 month after 

vaccination. Therapeutics was instituted 4 times with 

partial response, with relapse of the disease immediately 

after withdrawal of the drugs. She had normal blood cell 

counts and lymphoproliferation to mitogens and Candida, 

but low response to PPD. Surface expression of IL-12 RB1, IL- 

12 p40 and IFN-γ R1 was normal. IFN-γ production was low, 

with no improvement after stimulation by BCG plus IL-12. 

Genomic DNA sequencing of IL12RB1 demonstrated a 

compound heterozygous pattern, with a substitution in

S46 Abstracts 

exon 5 (P173W) and a deletion of 17 bp in exon 9. Analysis of 

parents and relatives showed the deletion to be of paternal 

origin and the substitution of maternal origin, but present 

also in a maternal uncle. Conclusion. A novel heterozygous 

mutation in IL12RB1 causes susceptibility to BCG infection 

after vaccination. In suspected cases, mutational screening 

should be performed even with normal expression of the 

IL12Rb1 receptor. 

doi:10.1016/j.clim.2007.03.305 

F.93 Modulation of In Vivo T Cell Activation by an 

Acetylcholine-Esterase Inhibitor in Patients 

Chronically Infected with HIV 

Carlos Cantu-Brito, Investigator, Department of Neurology, 

Instituto Nacional de Ciencias Medicas y Nutricion SZ, 

Mexico City, Mexico, Sergio Ivan Valdes-Ferrer, Resident, 

Department of Neurology, Instituto Nacional de Ciencias 

Medicas y Nutricion SZ, Mexico City, Mexico, Jose Crispin, 

PhD Student, Department of Immunology and 

Rheumatology, Instituto Nacional de Ciencias Medicas y 

Nutricion SZ, Mexico City, Mexico, Francisco Belaunzaran, 

Resident, Department of Infectious Diseases, Instituto 

Nacional de Ciencias Medicas y Nutricion SZ, Mexico City, 

Mexico, Maria Ines Vargas Rojas, PhD Student, Department 

of Immunology and Rheumatology, Instituto Nacional de 

Ciencias Medicas y Nutricion SZ, Mexico City, Mexico, Juan 

Sierra, Investigator, Department of Infectious Diseases, 


Mexico City, Mexico, Jorge Alcocer Varela, Investigator, 

Department of Immunology and Rheumatology, Instituto 


Mexico 

The immune system of patients with HIV is overstimulated. 

An increased fraction of in vivo activated CD4+ 

Tcells coupled with a decrease in regulatory Tcell numbers is 

a cardinal feature commonly observed. Vagal stimulation can 

modulate inflammatory response through T cell and macrophages 

by down-regulating their production of pro-inflammatory 

cytokines, namely TNF-α, IL-1, and IL-6. The aim of 

this work was to analyze if the administration of an 

acetylcholine-esterase (Ach-E) inhibitor is capable of diminishing 

immune over-stimulation in patients chronically 

infected with HIV. Methods: Nineteen HIV-infected patients 

with more than 250 CD4+ T cells, devoid of antiretroviral 

therapy, were assigned to pyridostigmine sulfate 90 mg/day 

or matching placebo. In every case, two peripheral blood 

samples were obtained: one before and one after 1 week of 

treatment. In vivo activated CD4+ T cells (HLA-DR+, CD69+) 

and regulatory T cells (CD4+CD25+Foxp3+) were quantified 

with flow cytometry. CD4+ Tcell proliferation was quantified 

after stimulation with a polyclonal stimulus (either PHA or 

PMA+ionomycin). Results: Nine patients received pyridostigmine, 

10 placebo. There were no basal differences 

between treatment and placebo groups. In vivo activated 

CD4+ T cells diminished significantly (p=0.025) in patients 

who received pyridostigmine. Conversely, regulatory T cells 

increased in treated patients. Finally, CD4+ T cell proliferation 

was significantly lower in patients who received 

pyridostigmine (p=0.026). Discussion: We found that the 

administration of a low dose of Ach-E inhibitors can 

successfully diminish CD4+ Tcell over-stimulation in patients 

with chronic HIV infection. 

doi:10.1016/j.clim.2007.03.306 

F.94 CD4/CD8 T Cell Ratio Predicts HIV Infection in 

Infants: The NHLBI P2C2 HIV Study 

William Shearer, Professor, Baylor College of Medicine, 

Texas Children’s Hospital, Pediatrics, Houston, TX, Savita 

Pahwa, Professor, University of Miami, Miller School of 

Medicine, Miami, FL, Jennifer Read, Medical Officer, 

National Institute of Child Health and Human Development, 

Bethesda, MD, Sameera Wijayawardana, Biostatistician 

Student, Emory University Rollins School of Medicine, 

Atlanta, GA, Jing Chen, Assistant Professor, Emory 

University Rollins School of Medicine, Antanta, GA, Kirk 

Easley, Senior Associate, Emory University Rollins School of 

Medicine, Atlanta, GA, Paul Palumbo, Professor, Dartmouth 

Medical School, Hanover, NH, Elaine Abrams, Associate 

Professor, Columbia University, New York, NY, Steven 

Nesheim, Associate Professor, Emory University School of 

Medicine, Atlanta, GA 

Background: Although CD4 and CD8 T cell counts are 

available in many resource-poor settings, HIV DNA PCR tests 

are not. We analyzed data from the P2C2 HIV and CDC PACTS 

studies (overall HIV transmission rates: 17% and 18%, 

respectively) to evaluate the CD4/CD8 T cell ratio as a 

predictor of HIV infection among HIV-exposed infants. 

Methods: Data from the 3-month visit (45–150 days) for 

infants born to HIV-infected mothers enrolled in the P2C2 

HIV study (79 HIV-infected, 409 uninfected) were analyzed. 

The CDC PACTS cohort (224 HIV-infected, 1015 

uninfected) was used for validation. Results: The area 

under the ROC curve was higher for the CD4/CD8 ratio 

compared to the CD4 cell count (AUC=0.83 and 0.75, 

P=0.03). The mean CD4/CD8 ratio at the 3-month visit 

was 1.7 for HIV-infected infants and 3.0 for uninfected 

infants. A CD4/CD8 ratio of 2.4 was almost 2.5 times (95% 

CI for the likelihood ratio: 2.1–2.9) more likely to occur in 

an HIV-infected infant compared to an uninfected infant (test 

sensitivity 81%; posttest probability of HIV 33%). Model 

performance in the CDC PACTS validation sample was equally 

good (AUC=0.78 for the CD4/CD8 ratio; good agreement 

between the predicted and observed risk of HIV). Conclusion: 

We conclude that the CD4/CD8 Tcell ratio is a more sensitive 

predictor of HIV infection in infants than the CD4 Tcell count 

and, when necessary, the CD4/CD8 T cell ratio may be used 

with caution to diagnose HIV infection. 

doi:10.1016/j.clim.2007.03.307 

F.95 First Case of Human CD21 Deficiency— 

Association with Hypogammaglobulinemia 

Jens Thiel, MD, University Hospital Freiburg, Freiburg

Abstracts 

Complement receptor type 2 (CR2/CD21) is mainly 

expressed by mature B cells and FDCs. CD21 functions as 

receptor for C3d-opsonized immune complexes and forms a 

coreceptor signalling complex together with CD19, and CD81. 

We report a case of human CD21 deficiency associated with 

hypogammaglobulinemia. The 28-year-old patient presented 

with flu-like symptoms and reduced serum IgG and IgA. 

Leucocytes and lymphocytes were within normal range, as 

were the cell counts for T cell subpopulations and CD19+ B 

cells. However, class switched CD27+ memory B cells were 

reduced and CD21 expression was absent from all patient B 

cells. Moreover, no soluble CD21 was detectable in the 

patient’s serum, ruling out increased CD21 shedding from the 

B cell surface. Complement receptor type 1 was expressed 

normally and an immature B cell phenotype could be ruled 

out. Thus, the hypogammaglobulinemia and mild immunodeficiency 

in this patient may be caused by insufficient 

formation of switched memory B cell and antibodies. Looking 

for the underlying molecular defect, no mutation could be 

identified within the coding regions of CD21 by genomic 

sequencing. CD21 mRNA levels in the patient’s B cells were 

not reduced. Sequencing of intronic regions adjacent to the 

19 exons of CD21 revealed a heterozygous point mutation 

within the 3′ splice site of CD21 exon 6 leading to one 

shortened mRNA allele that lacks exon 6. Further experiments 

are necessary to test whether this heterozygous splice 

site mutation is sufficient to explain the absence of CD21 on 

the patient’s B cells. 

doi:10.1016/j.clim.2007.03.308 

F.96 Warning Signs for Primary Immunodeficiences 

Investigation in Hospitalized Patients 

Anete Sevciovic Grumach, Department of Dermatology, 

University of São Paulo, Brazil, São Paulo, Brazil, Dewton 

Moraes-Vasconcelos, Department of Dermatology, 

University of São Paulo, Brazil, São Paulo, Brazil, Joao Bosco 

Oliveira, Department of Dermatology, University de São 

Paulo, Brazil, São Paulo, Brazil, Alexandre Correa, BSc, 

Department of Dermatology, University of São Paulo, Brazil, 

São Paulo, Brazil, Rosemeire Navickas Constantino-Silva, 

BSc, Department of Dermatology, University of São Paulo, 

Brazil, São Paulo, Brazil, Noemia Orii, BSc, Department of 

Dermatology, University of São Paulo, Brazil, São Paulo, 

Brazil, Noac Chuffi Barros, Department of Dermatology, 

University of São Paulo, Brazil, São Paulo, Brazil, Maria do 

Socorro Ferrao, Department of Dermatology, University of 

São Paulo, Brazil, São Paulo, Brazil, Marinella Dellanegra, 

Instituto de Infectologia Emilio Ribas, São Paulo, Brazil, 

Alberto Jose da Silva Duarte, Professor, Department of 


Brazil 

Background: It has been published a higher prevalence of 

PID in hospitalized patients. Nevertheless, most of the 

recommendations for the identification of PID are based on 

retrospective reports or in outpatients groups. Objective: To 

select the main signs dealing with PID diagnosis based on a 

prospective evaluation of hospitalized patients. Methodology: 

A prospective evaluation of inpatients was performed 

for a 6 year period. All the patients with clinical symptomatology 

not well elucidated were referred to the immunologist. 

A comprehensive immunological evaluation was 

available and the laboratorial tests were performed according 

to the clinical manifestations. Results: 462 patients (1.4 

M:1 F) were evaluated and the main symptoms were: 

prolonged fever (3.5%), hepatosplenomegaly (2%), uncommon 

infections (67%), recurrent infections (4.3%), unexpected 

complications (18.2%), suggestive auto-immunity 

(3.7%), complications due to vaccines (0.7%), and allergy 

(2.2%). PIDs were identified in 18 patients. No PID was 

diagnosed among patients complaining of prolonged fever 

and hepatosplenomegaly. Conclusions: The following criteria 

could be used as warning signs for hospitalized patients: 

recurrent infections, meningococcal meningitis above 5 

years old, complicated infections caused by common pathogens, 

uncommon pathogens in HIV negative patients and 

vaccinal complications. 

doi:10.1016/j.clim.2007.03.309 

S47 

F.97 Single Cell Evaluation of the Production of 

T Cell Cytokines by Chronic Mucocutaneous 

Candidiasis Patients 

Dewton Moraes Vasconcelos, MD, PhD, Department of 

Dermatology, University of São Paulo Medical School, São 

Paulo, Brazil, Alexandre de Almeida, MD, MSc, Department 

of Dermatology, University of São Paulo Medical School, São 

Paulo, Brazil, Dewton Moraes Vasconcelos, MD, PhD, 

Department of Dermatology, University of São Paulo 

Medical School, São Paulo, Brazil, Anete S. Grumach, MD, 

PhD, Department of Dermatology, University of São Paulo 

Medical School, São Paulo, Brazil, Mauricio Domingues 

Ferreira, MD, PhD, Department of Dermatology, University 

of São Paulo Medical School, São Paulo, Brazil, Tatiana Negri 

Santi, BSc, Department of Dermatology, University of São 

Paulo Medical School, São Paulo, Brazil, Alberto Jose da 

Silva Duarte, MD, PhD, Department of Dermatology, 

University of São Paulo Medical School, São Paulo, Brazil 

Background: Chronic mucocutaneous candidiasis (CMC) is 

a rare primary immune deficiency characterized by chronic 

or recurrent infection by Candida in mucous membranes, 

nails and skin. Impaired cell mediated immunity to Candida 

as well as dysregulation of the production of Th1 and Th2 

cytokines is an important mechanism in CMC. Objective: 

Evaluate the number of Th1 (IL-2, IFN-γ) and Th2 (IL-10, IL- 

4) cytokines producing cells of a group of patients with CMC 

to understand the relationship of these cytokines and CMC. 

Methods: We studied the production of cytokines of 15 

patients with CMC and 13 controls. We evaluated the 

production of IL-2, IL-4, IL-10 and IFN-γ by total T cells, CD4 

+ and CD8+ subsets by intracellular cytokine flow cytometry. 

Results: The patients presented lower number of IL-2, IFNγ, 

IL-10 and IL-4 producing CD4+ cells than controls, when 

stimulated with Candida and tetanus toxoid. The patients’ 

presented less CD8+ T cells producing Th1 cytokines when 

compared to controls. There were two distinct subgroups of 

patients. One presented less IL-2 and IFN-γ producing T cells 

than the other. The second subgroup presented a lower

S48 Abstracts 

number of IFN-γ and a similar number of IL-2 producing T 

cells compared to control group. Conclusion: Our patients 

presented a decreased number of T cells producing both Th1 

and Th2 cytokines. Despite the presence of 2 different 

groups in relation to cytokine production there was no 

correlation to the clinical features. 

doi:10.1016/j.clim.2007.03.310 

F.98 Immunomodulation by Cytokines in Chronic 

Mucocutaneous Candidiasis Patients: Evaluation of 

the Production of T Cell Cytokines at a Single Cell 

Level 

Mauricio Domingues Ferreira, MD, PhD, Department of 

Dermatology, University of São Paulo Medical School, São 

Paulo, Brazil, Alberto Jose da Silva Duarte, MD, PhD, 

Department of Dermatology, University of São Paulo 

Medical School, São Paulo, Brazil, Dewton Moraes 

Vasconcelos, MD, PhD, Department of Dermatology, 

University of São Paulo Medical School, São Paulo, Brazil, 

Tatiana Negri Santi-BSc, Department of Dermatology, 


Anete S. Grumach, MD, PhD, Department of Dermatology, 


Alexandre Almeida, MD, MSc, Department of Dermatology, 

University of São Paulo Medical School, São Paulo, Brazil 

Background: Chronic mucocutaneous candidiasis (CMC) 

is a rare primary immune deficiency characterized by 

chronic or recurrent Candida infection in mucous membranes, 

nails and skin. Impaired cell mediated response 

and dysregulation of the production of Th1 and Th2 

cytokines to Candida are important in CMC. We previously 

found a lower number of IL-2, IL-10, IL-4 and IFN-γ 

producing T cells and its CD4+ and CD8+ subsets compared 

to the control group when stimulated with Candida and 

tetanus toxoid. Objective: Modulate the T cell response by 

adding to the cell culture medium IL-2, or IL-12, or anti-IL- 

10. Methods: We studied the production of cytokines of 15 

patients with CMC and 13 controls stimulated by Candida 

and tetanus toxoid. We evaluated IL-2, IL-4, IL-10 and IFN-γ 

in total T cells, as well as CD4+ and CD8+ subsets by an 

intracellular cytokine flow cytometric assay. We tried to 

immunomodulate with recombinant human IL-2, or IL-12, or 

a monoclonal antibody anti-IL-10. Results: We did not find a 

significant response to the immunomodulation trials, but 

there was a trend of improvement in the synthesis of IL-2 

from the CD4+ cells of patients when stimulated by Candida 

and modulated by IL-2, IL-12 or anti-IL-10. Conclusion: 

There was no significant improvement by the immunomodulation 

by cytokines, but there was a tendency of 

improvement. It is possible that if we have used a 

combination of cytokines, the results could be better. Our 

data suggest a new possible way of treatment to patients 

with CMC. 

doi:10.1016/j.clim.2007.03.311 

F.99 Hereditary Angiodema (HAE) in Brazil: Registry 

of 100 Cases 

Anete Sevciovic Grumach, Department of Dermatology, 

University of São Paulo, Brazil, São Paulo, Brazil, Jorge 

Andrade Pinto, Department of Pediatrics, Federal 

University of Minas Gerais, Belo Horizonte, Alexandre Pires 

Correa, BSc, Department of Dermatology, University of 

São Paulo, Brazil, São Paulo, Brazil, Dewton 

Moraes-Vasconcelos, MD, PhD, Department of Dermatology, 

University of São Paulo, Brazil, São Paulo, Brazil, Rosemeire 

Navickas Constabtino-Silva, BSc, Department of 


Brazil, Sollange Valle, Federal University of Rio de Janeiro, 

Rio de Janeiro, Eli Mansour, Department of Pediatrics, 

University of Campinas, Campinas, Maria Marluce Vilela, 

Department of Pediatrics, University of Campinas, 

Campinas, Ricardo Zollner, Clinical Medicine, University of 

Campinas, Campinas, Alberto Jose da Silva Duarte, 

Professor, Department of Dermatology, University of São 

Paulo, Brazil, São Paulo, Brazil, Evandro Rivitti, Professor, 

Department of Dermatology, University of São Paulo, Brazil, 

São Paulo, Brazil 

HAE is a primary quantitative or functional defect of C1 

inhibitor. Clinical manifestations include subcutaneous, 

respiratory and gastrointestinal edema. Asphyxia could 

occur in 25–40% of the non-treated cases. There was no 

registry in Brazil until now. Objective: Report the clinical and 

laboratorial characteristics of HAE in Brazilians. Methods: 

Collaborative work groups were established among specialized 

services. The following parameters were evaluated: 

gender, age, age of diagnosis, main clinical manifestations, 

time for diagnosis, triggering factor, treatment, familial 

history and C1 inhibitor and C4 levels and CH50. Laboratorial 

diagnosis of C1 INH deficiency was confirmed in all patients. 

Results: HAE affected 67 F:33 M, 1–70 years of age and the 

first symptoms were reported during childhood in most of the 

cases. One episode of laryngeal edema was present in 17% 

and the triggering factors were trauma (31/100), stress (11/ 

100), and menses (6/100). Familial history was positive in 

53/100. Conclusion: Familial history was determinant for 

diagnosis in half of the cases. Late diagnosis had been 

established but the first symptoms occurred during childhood. 

HAE diagnosis is still not well recognized in our 

country. 

doi:10.1016/j.clim.2007.03.312 

F.100 Autosomal Dominant Combined Immune 

Deficiency: A Unique Family with Absent B-Cells and 

Decreased T-Cells 

Michael Land, Physician, University of California, Los 

Angeles, Pediatrics, Los Angeles, CA, Raffi Tachdjian, 

Physician, University of California, Los Angeles, Pediatrics, 

Los Angeles, CA, Erina Lin, Physician, University of 

California, Los Angeles, Pediatrics, Los Angeles, CA, Robert 

Roberts, Physician, University of California, Los Angeles, 

Pediatrics, Los Angeles, CA, Marc Riedl, Physician, 

University of California, Los Angeles, Department of 

Medicine, Los Angeles, CA

Abstracts 

Rationale: We present a family of four individuals with 

immunodeficiency. The father and daughter were initially 

diagnosed with Common Variable Immune Deficiency (CVID) 

and were found to have absent B cells and low T cells. The 

father’s brother and mother also had immunodeficiency but 

died years ago. This may represent a unique form of 

Combined Immunodeficiency. 

Methods/Results: The father is a 35-year-old man with a 

history of CVID diagnosed at age 12 following multiple upper 

respiratory infections. He was started on intravenous 

immunoglobulin (IVIG) for hypogammaglobulinemia, but 

never had any severe infections. His mother and older 

brother also received gamma globulin for presumed CVID, 

but both died from infections at age 36. He had one episode 

of pneumonia at age 19, with occasional sinus infections. His 

trough IgG level recently was 162, but increased to 724 with 

higher dosing. His IgA was b7, IgM b0.5, IgE b0.1. The 

patient’s 3-year-old daughter has been receiving IVIG since 

age 8 months for hypogammaglobulinemia. She has a history 

of sinusitis and has never been hospitalized. Her IgA is b7, 

IgM 6, IgE 0.1. Interestingly, both father and daughter have 

undetectable B cells (CD19 b1%) and CD4 lymphopenia 

(father: 202, daughter: 608). Both patients also have low 

CD8 counts (father: 162, daughter: 189). They have normal 

responses to mitogens and antigens. Conclusion: This family, 

along with the deceased relatives, exhibits an unusual 

pattern of inheritance for B cell and T cell deficiency 

consistent with combined immunodeficiency, suggesting 

autosomal dominance. Further characterization and investigation 

of this disease are warranted. 

doi:10.1016/j.clim.2007.03.313 

F.101 Circulating Class-Switched Memory B Cells 

are Markedly Reduced in Some Patients with 

Specific Antibody Deficiency (SAD) and/or Mild IgG 

Subclass Deficiency 

Lily E. Leiva, Associate Professor, Louisiana State University 

Health Sciences Center and Children’s Hospital, Department 

of Pediatrics, New Orleans, LA, Kenneth Paris, Assistant 

Professor, Louisiana State University Health Sciences Center 

and Children’s Hospital, Department of Pediatrics, New 

Orleans, LA, Hanh Monjure, Research Associate, Louisiana 

State University Health Sciences Center and Children’s 

Hospital, Department of Pediatrics, New Orleans, LA, 

Ricardo U. Sorensen, Professor and Chair, Louisiana State 

University Health Sciences Center and Children’s Hospital, 

Department of Pediatrics, New Orleans, LA, Dana Minor, 

Research Technician, Research Institute for Children, 

Children’s Hospital, New Orleans, LA 

Some patients with recurrent respiratory infections are 

affected by specific antibody deficiency (SAD) and/or IgG 

subclass deficiencies. In this study we investigated if the 

inability of patients with SAD and/or IgG subclass deficiency to 

respond to pneumococcal polysaccharide antigens (PP) was 

associated with reduced numbers of circulating class-switched 

memory B cells. We studied 7 children with recurrent respiratory 

infections with SAD and/or mild IgG subclass deficiency and 8 

healthy controls. All patients were evaluated with total 

immunoglobulin and IgG subclass concentrations. We measured 

IgG and IgM anti-PP antibody levels by a standardized ELISA test. 

Purified PBMC were stained for the expression of CD27 and IgD 

on CD19+ B cells. Percentages of naïve (CD27NIgD+), nonswitched 

memory (CD27+IgD+) and class-switched memory 

(CD27+IgDN) B cells were determined by flow cytometry. 

Results: In patients in whom IgG anti-PP antibody levels were 

low, IgM levels were within the normal ranges for each of the 8 

serotypes tested. The B cell compartment in these patients 

showed a marked reduction in the percentage of class-switched 

memory B cells in 2 patients (6% and 2%, respectively) in 

comparison to the other 5 patients (range: 32%–90%) and to 

controls (range: 22%–87%). These results suggest that a deficient 

IgM to IgG antibody switch is associated with a lack of circulating 

class-switched memory B cells in some patients, and that this 

may be the explanation for the failure of some patients to 

respond to PP. Studies are in progress to determine the 

prognostic implications of these differences. 

doi:10.1016/j.clim.2007.03.314 

F.102 HIV Treatment Reveals 

Hypogammaglobulinemia in an Individual with 

Common Variable Immune Deficiency 

Lulu Sun, Immunodeficiency Treatment Centre, McGill 

University Health Centre, Montreal, QC, Canada, Christos 

M. Tsoukas, Immunodeficiency Treatment Centre, McGill 

University Health Centre, Montreal, QC, Canada 

While common variable immune deficiency (CVID) is characterized 

by hypogammaglobulinemia, HIV-1 infection is associated 

with elevated immunoglobulin levels. There have been 

four CVID patients reported in the literature who recovered 

antibody production after contracting HIV-1. Here we describe 

an HIV patient with concomitant CVID whose immunoglobulin 

levels dramatically decreased upon successful antiretroviral 

therapy (ART), and investigate a plausible mechanism by which 

HIV is able to modulate circulating immunoglobulin through 

regulation of the B cell activation factor (BAFF)/BAFF-receptor 

pathway. Four cohorts were compared: our HIV/CVID patient, 

HIV patients, CVID patients, and healthy controls. Phenotypic 

and functional analyses of the Tand B cells of these cohorts were 

performed using flow cytometry, and BAFF serum levels were 

measured by ELISA. In addition to marked hypogammaglobulinemia, 

the patient has a complete defect of memory IgD+/− 

CD27+ CD19+ B cells that was not altered by ART, consistent with 

severe CVID. When viral load briefly increased, a transient 

normalization of immunoglobulins was also observed. At the 

same time, the percentage of BAFF-R+B cells was boosted from 

low to normal levels. Plasma BAFF levels were high compared to 

controls during viral suppression, due primarily to the lack of 

memory B cells. Finally, T cell phenotypes remained abnormal, 

with a low CD4+ Tcell count that is characteristic of rigorous HIV 

disease, and impaired in vitro cellular proliferation. Our findings 

suggest that high HIV viraemia was required to maintain normal 

immunoglobulin levels in the CVID patient, possibly through 

upregulating BAFF-R. 

doi:10.1016/j.clim.2007.03.315 

S49

S50 Abstracts 

F.103 Differences in Telomerase Expression by the 

CD1a+ Cells in Langerhans Cell Histiocytosis Reflects 

the Diverse Clinical Presentation of the Disease 

Cristiana Costa, PhD Student, Leiden University Medical 

Center, Department of Pediatrics, Leiden, The Netherlands, 

Maarten Egeler, MD PhD, Leiden University Medical Center, 

Leiden, The Netherlands, Manja Hoogeboom, Leiden 

University Medical Center, Leiden, The Netherlands, Ramses 

Forsyth, N. Goormaghtigh Institute of Pathology, University 

Hospital Gent, Gent, Karoly Szuhai, PhD, Leiden University 

Medical Center, Leiden, The Netherlands, Marieke Niesters, 

Leiden University Medical Center, Department of Pediatrics, 

Leiden, The Netherlands, Ronald de Krijger, Erasmus 

Medical Center, Department of Pathology, Rotterdam, 

Abdellatif Tati, Service de Pneumonologie, Hospital Saint 

Louis, Paris, Pancras Hogendoorn, Leiden University Medical 

Center, Department of Pathology, Leiden, The Netherlands, 

Nicola Annels, PhD, Leiden University Medical Center, 

Department of Pediatrics, Leiden, The Netherlands 

Langerhans cell histiocytosis (LCH) is a disease characterised 

by an uncontrolled clonal proliferation of Langerhans 

cells, whose aetiology is still unclear. The clonal nature of 

LCH could support the hypothesis that it is a neoplastic 

disease with unlimited growth potential. One requirement 

for unlimited proliferation is the maintenance of telomere 

length. In a group of 70 patients we investigated whether a 

telomere maintenance mechanism is active by LCH cells. 

Results showed that LCH cells from all restricted skin LCH 

lesions (6/6) expressed telomerase as assessed by human 

telomere reverse transcriptase (hTERT) immunohistochemistry, 

whereas LCH cells from the majority of bone lesions 

analysed did not (26/34). Interestingly, in contrast to solitary 

bone lesions, LCH cells from lesions of multisystem patients 

always expressed telomerase (11/11), regardless of the 

lesional site. In situ telomeric repeat amplification protocol 

(TRAP) assay performed on different lesional sites showed 

that telomerase was active. In addition, the telomere length 

of LCH cells from a hTERT positive skin multisystem lesion 

was long and homogeneous when compared to the LCH cells 

from hTERT negative bone single system LCH lesions, which 

were heterogeneous in length. No evidence for an alternative 

lengthening of telomeres mechanism was found in 

hTERT negative lesions. The difference in telomerase 

expression and telomere length in the different lesional 

sites and in biopsies from patients with solitary versus 

multisystem disease appears to be reflective of the diverse 

clinical presentation and course of LCH. The results from this 

study have important implications for understanding the 

nature of this disease. 

doi:10.1016/j.clim.2007.03.316 

F.104 Multiplex Capabilities for Serological 

Immunoassays Using Luminex xMAP ® Technology 

Harold Baker, Senior Scientist, Luminex Corporation, 

Austin, TX 

The Luminex xMAP technology incorporates fluorescently 

encoded microspheres (xMAP microspheres) to perform 

multiplexed bioassays in conjunction with advanced digital 

signal processing and proprietary identification techniques. 

The advantage of the multiplexing technology is the 

capability to measure several antigens within one sample. 

The combination of multiple results and reduced sample 

volume increases the efficiency for testing and sample 

consumption. Another advantage is that the serological 

assays can be multiplexed with other immunoassays. Five 

specific antigens were coupled to xMAP microspheres 

representing several individual regions. BSA coated microspheres 

were prepared to function as internal assay controls 

for non-specific reactivity. A total of five regions were used 

with each antigen to develop a 30-plex assay. The xMAP 

serological assay format we used was a 60 minute, no wash 

assay. Diluted patient serum, 10 ƒÝL, was combined with the 

microsphere mixture, 50 ƒÝL, and incubated 30 min. Diluted 

PE conjugated antibody, 150 ƒÝL, was added and incubated 

30 min before being analyzed with the Luminex system. 

Equivalent results were obtained with the replicate antigen 

coated regions. Analytical sensitivity was defined by the 

maximum dilution before an appreciable decrement in MFI 

response was observed. This was observed to be at a dilution 

of 1:1000 representing the use of 0.01 ƒÝL serum for the 

measurement of antibodies in the serum sample. These 

results demonstrate that the Luminex xMAP technology can 

be used to develop multiplex assays such as those required 

for serological assessment of autoimmune diseases. 

doi:10.1016/j.clim.2007.03.319 

F.105 Immunofluorometrical Exploring of FSH 

Levels in the Serum of Patients with Histologically 

Verified Macrocellular Lung Cancer 

Ivan Milosevic, Primary Care Physician, Dispensary 2, ZZZZR, 

Kragujevac 

It is already known in that the cells of macrocellular lung 

cancer can produce gonadotropins. By following the levels of 

FSH as one of gonadotropins, in the serum of macrocellular 

lung cancer patients, we could make certain statistical 

conclusions how significant these levels would be in eventual 

future early diagnostic procedures, besides already existing 

tumor markers and other methods. This work would connect 

the Oncology, Endocrinology and Immunology fields, containing 

very interesting immunofluorometrical procedures, 

hormonal theories and statistical estimates. As an immunological 

part in this theoretical model, it is represented 

through figure, time-resolved fluoroimmunoassay for quantitative 

detection of FSH. By following of FSH levels in the 

serum of microcellular lung cancer patients and making 

determinate statistical conclusions about its importance, 

that procedure could be eventually used as one of early or 

advanced diagnostic methods concerning mentioned disease. 

What would we get with that? The histological verification is 

very slow and painful, when the macrocellular lung cancer is 

in the question. This histological type gives the metastases 

rapidly and it needs to be diagnosed in the fastest possible 

way in order to be prevented by adequate reaction and 

therapy. At the same time the biopsy as a very invasive 

method would be avoided. When the tumor markers are in

Abstracts 

question as a diagnostic method supplemented and supported 

by the FSH levels exploring could be more reliable. 

Concerning all above presented, in future establishing of this 

procedure, we would get a rapid, comfortable and safe 

diagnostic method. 

doi:10.1016/j.clim.2007.03.320 

F.108 Distinct Regulation of Individual Tyrosine 

Phosphorylation of the Cytoplasmic Domain of CD19 

Nobuko Ishiura, MD, Department of Dermatology, Graduate 

School of Medicine, University of Tokyo, Tokyo, Japan, 

Manabu Fujimoto, PhD, Department of Dermatology, 

Kanazawa University Graduate School of Medical Science, 

Kanazawa Ishikawa, Japan, Kunihiko Tamaki, PhD, 

Department of Dermatology, Graduate School of Medicine, 

University of Tokyo, Tokyo, Japan 

Cell surface molecules called co-receptors on lymphocytes 

positively or negatively modulate the antigen receptor 

signaling. CD19 is a B lymphocyte-specific co-receptor which 

regulates constitutive thresholds and enhances antigen 

receptor-induced signaling through its cytoplasmic domain. 

The cytoplasmic region of CD19 contains nine conserved 

tyrosine residues, which become phosphorylated upon B cell 

activation. Tyrosine phosphorylation of CD19 mediates 

transmembrane signals by recruiting multiple signaling 

molecules including Src family protein tyrosine kinases 

(PTKs), especially Lyn, Vav, and PI 3-kinase. Of nine tyrosine 

residues, Y513, Y482, and Y391 are supposed to play critical 

roles in CD19-mediated signal transduction, although the 

precise regulation of individual tyrosine phosphorylation 

remains unknown. Herein, we have developed phosphospecific 

antibodies against these tyrosine residues, CD19pY513, 

CD19-pY482 and CD19-pY391, and have assessed the 

regulation of tyrosine phosphorylation of CD19 following 

various stimulations. BCR ligation induced Y513 phosphorylation 

preceded by Y482 and Y391 phosphorylation, suggesting 

Y513 as the primary phosphorylation site. 

Phosphorylations of three tyrosine residues were much 

enhanced and prolonged by simultaneous stimulation of 

BCR and CD40 ligation. Intriguingly, CD19 containing phosphorylated 

Y513 was exclusively located within the lipid rafts 

following the BCR ligation, while majority of CD19 containing 

phosphorylated Y482 and Y391 stayed out of the lipid rafts. 

Therefore, individual tyrosines of CD19 undergo distinct 

regulation of phosphorylation, which in turn determines 

different distribution on the cell surface. 

doi:10.1016/j.clim.2007.03.321 

F.109 Vitamin D Receptor Activators Calcitriol and 

Paricalcitol Modulate Dendritic Cell Phenotype and 

Function In Vitro Study 

Klara Sochorova, Pharm Department of Immunology, 2nd 

Medical School, Charles University Prague, Prague, Vit 

Budinsky, Manager. Department of immunology, 2nd Medical 

School, Charles University, Prague, Zuzana Tobiasova, 

Manager. Department of Immunology, 2nd Medical School, 

Charles University, Prague, Jirina Bartunkova, Professor, 

Department of Immunology, 2nd Medical School, Charles 

University, Prague, Sylva Dusilova Sulkova, Professor, 

Departments of Gerontology and Metabolism, Medical 

School and Teaching Hospital, Hradec Kralove 

Vitamin D is known as an important immunomodulatory 

agent. Its potential to suppress immune response in autoimmunity 

is intensively studied. Use of active metabolite of 

vit. D3, calcitriol, in vivo, however, is limited due to its 

hypercalcemic effect. Paricalcitol, the synthetic analogue of 

calcitriol, is characterized by reduced hypercalcemic effect, 

but its immunosupressive properties have not been evaluated 

yet. In our study we compared the effect of calcitriol and 

paricalcitol on morphological and functional characteristics 

of dendritic cells (DC) in vitro. DC were differentiated from 

monocytes with IL4 and GM-CSF for 5 days and then induced to 

maturate with viral or bacterial products (poly-IC, LPS). 

Calcitriol or paricalcitol were added during the period of 

differentiation or maturation. We studied the expression of 

DC-relevant surface molecules. DC function was tested by 

endocytosis, T-stimulatory capacity and cytokine production. 

DC differentiated in the presence of calcitriol or paricalcitol 

remain at an immature state (low expression of CD80, CD83, 

CD86 and HLA-DR after stimulation by both poly-IC and LPS, 

high expression of CD14 and PD-L1, high endocytic activity, 

low T cell activation and low IL-6 and TNF production). DC 

maturated in the presence of both drugs were also impaired, 

but less than in previous case. The immunosuppressive effect 

of both calcitriol and paricalcitol on DC is comparable. 

Paricalcitol thus seems to be a perspective drug for 

influencing the pathological immune response in autoimmunity. 

This project was supported by MSM 0021620812. 

doi:10.1016/j.clim.2007.03.322 

S51 

F.110 Significance of IgG4 in the Diagnosis of Mucous 

Membrane Pemphigoid 

Vijay Kumar, Director, IMMCO Diagnostics Inc., Buffalo, NY, 

Lakshmanan Suresh, IMMCO Diagnostics Inc., Buffalo, NY 

Background: Mucous membrane pemphigoid (MMP) is an 

immune mediated vesiculo-bullous disorder predominantly 

affecting the mucous membranes of the oropharynx. Direct 

immunofluorescence (IF) studies of the presence of immunoglobulins 

and/or complement are considered as the gold 

standard in the diagnosis of MMP. The tissue-bound IgG and 

other immunoreactants are seen as a linear band in the 

basement membrane zone (BMZ). However immunodeposition 

of IgG and/or C3 is not observed in all cases of MMP. One of the 

reasons of this lack of immune deposits may be that certain IgG 

subclass of immunoglobulins may be the autoreactants and that 

the total IgG conjugates fail to detect this subclass of IgG. 

Objective: The purpose of our study was to determine the 

diagnostic value and frequency of tissue deposition of IgG4 in 

comparison to polyclonal IgG, other immunoglobulins and C3. 

Methods: Oral mucosal biopsies of 82 patients clinically 

suspected to have MMP were analyzed by direct IF using 

polyclonal anti-human IgG, IgM, IgA, fibrin, complement C3, 

and anti-human IgG4 subclass monoclonal antibodies. Results:

S52 Abstracts 

Based on H&E and direct IF studies, 34 cases were diagnosed as 

MMP. The most common antibody deposited was IgG (90%) 

followed by C3 (82%) and IgG4 (71%). Strikingly, IgG4 was the 

sole antibody detected in two cases (6%). Conclusion: Our 

results suggest that the use of monoclonal IgG4 is important in 

the diagnosis of MMP. We suggest adding monoclonal IgG4 to the 

routine panel of antibodies used in studies of cases suspected to 

have MMP to avoid false-negatives. 

doi:10.1016/j.clim.2007.03.324 

F.111 Immuophenotyping of PBL and B.M Markers in 

Patients with Leukemia by Flowcytometry 

Fereshteh Alesahebfosoul, Academic Member of Isfahan 

University, Isfahan University Medical School, Immunology 

Department, Isfahan 

Introduction: Peripheral blood lymphocytes (PBL) and bone 

marrow (B.M.) immunophenotyping is widely used for diagnosis 

and prognosis of leukemia. Flow cytometry technique provided 

facilities to assess the frequency of malignant cell population 

and the protein expression in each group of cells which have 

been gated. Method and material: Fresh peripheral blood and 

bone marrow of patient were collected and tested for following 

examination: total white blood cells and leukocyte percentage 

rate were determined by H1. Machine: The samples were 

treatedwithconjugatedMoAbwithFITCand/orphycoErythrine 

and then dual staining of cells was performed. The following 

monoclonal antibodies were used: CD10, CD19, CD20, CD5, 

CD3, CD22, CD7, CD33, anti-HLA DR, and CD13. After direct 

staining of cell samples, 30,000 cells were analyzed by flow 

cytometry. Result and discussion: The results of the first case of 

the study were WBC=780 and LUC=2.2%, CD7=60%, CD=57%, 

CD5=56%ofthelymphocytepopulationofperipheralblood.But 

the same markers in bone marrow sample were determined for 

D7=5%, CD3=5%, and CD5=6.5%. The results of the second case 

of the study were CD19=90%, CD20=89%, CD22=82%, and 

CD5=95% of the lymphocyte population of peripheral blood. In 

addition the same markers in bone marrow samples were 

determined for CD19=76%, CD20==85%, CD22=75.9%, and 

CD5=93%. HLA-DR marker was shown to be over 80% in both 

peripheral blood and bone marrow for this case. The result of 

cell counting for the third case of the study showed WBC=4800 

and LUC=6.3%, CD19=41.5%, CD20=29.5%, CD22=39% and 

CD5=43.12%, CD3=40.3%, and CD7=41.1% in peripheral blood. 

But the same markers from bone marrow sample were 

determined for CD19=85.6%, CD22=81.2% and CD5=2.08% 

and CD3=1.88%. The results of this study show that the 

lymphocyte protein expression in leukemic samples is the value 

in diagnostic and classification of leukemia. 

doi:10.1016/j.clim.2007.03.325 

F.113 An All-Human Culture Model Supporting 

Human B Lymphopoesis 

Mercy Kagoda, Graduate Research Assistant/Student, Loma 

Linda University School of Medicine Department of Pathology 

and Human Anatomy, Loma Linda, CA, Yasmin Parrish, 

Research Specialist II, Childrens Hospital Los Angeles Research 

Immunology Bone Marrow Transplant, Los Angeles, CA, 

Jaqueline Rogerio, Post Doctoral Fellow, Childrens Hospital Los 

Angeles Research Immunology Bone Marrow Transplant, Los 

Angeles, CA, Ineavely Baez, Senior Research Assistant, Loma 

Linda University School of Medicine Department of Pathology 

and Human Anatomy, Loma Linda, CA, Qian-Lin Hao, Research 

Specialist IV, Childrens Hospital Los Angeles Research 

Immunology Bone Marrow Transplant, Los Angeles, CA, Lora 

Barsky, Flow Technologist, Childrens Hospital Los Angeles 

Research Immunology Bone Marrow Transplant, Los Angeles, 

CA, Ewa Zielinska, Flow Technologist, Childrens Hospital Los 

Angeles Research Immunology Bone Marrow Transplant, Los 

Angeles, CA, Monika Smogorzewska, Flow Technologist, 

Childrens Hospital Los Angeles Research Immunology Bone 

Marrow Transplant, Los Angeles, CA, Kimberly Payne, Assistant 

Professor, Loma Linda University School of Medicine 

Department of Pathology and Human Anatomy, Loma Linda, 

CA 

Traditionally, nonfetal models of human B cell development 

have been based on co-culturing hematopoietic stem cells 

(HSCs) from cord blood (CB) and bone marrow (BM) on murine 

stromal cell lines. Here we describe the progressive replacement 

of murine components to generate a totally human 

culture model that supports human hematopoiesis, including B 

cell development. First, we replaced murine stromal cell lines 

with primary human BM stroma, next human serum was 

substituted for nonhuman sera. It has been reported that 

human B cell precursors selectively interact with CD10+ stromal 

cells during in vivo B cell development. In our cultures, human 

stroma express CD10 and secrete IL-7 at levels comparable to 

primary murine BM stroma. We found that human stroma bind 

IL-7 independently of the IL-7 receptor thereby having the 

potential for presenting IL-7 to developing B cell precursors. 

This is important as we have recently shown that IL-7 expands 

human B cell precursors by ∼50 fold and that IL-7 is essential for 

production of B cells from HSCs in adult human BM. While 

human stroma in our cultures express the human pre-BCR 

ligand, galectin-1 (Gal-1), the presence of Gal-1 was not 

required for IL-7-induced expansion of human B cell precursors. 

HSCs in co-cultures with human stroma and serum showed a 

generative capacity for CD19+ and CD19− progeny that was at 

least equal to that seen on murine stroma with nonhuman sera. 

Planned experiments will assess the ability of this culture model 

to support primary B cell malignancies. 

doi:10.1016/j.clim.2007.03.326 

F.114 Expression of CD3 and T Cell Receptor (TCR) 

on a Minor Population of Human CD14 Positive Cells 

Daron Forman, Senior Scientist, Tolerx, Cambridge, MA, 

Michael Rosenzweig, Senior Director of Preclinical 

Research, Tolerx, Cambridge, MA, Lou Vaickus, Chief 

Medical Officer, Tolerx, Cambridge, MA 

The TCR associates with CD3 polypeptides including CD3 

delta, gamma, epsilon, and zeta. It was commonly believed 

that TCR and CD3 expression occur exclusively on T cells. 

Recently however, TCR expression was reported on a minor 

population of both mouse and human neutrophils indicating

Abstracts 

that other populations may express CD3 and/or TCR. We 

evaluated whether CD3 and TCR-ab expression could be 

detected on non-T cell lineages in human peripheral 

blood. Cells were analyzed for CD3 and TCR-ab expression 

using monoclonal antibodies (Mabs) and flow cytometry. In 

addition to the expected distribution on lymphocytes, CD3 

and TCR-ab were detected on 0.3–1.0% population of 

cells that expressed CD11b, CD11c, and CD14. This 

population expressed CD3 and TCR with an intensity 

similar to CD4+ and CD8+ T cells. CD14 is expressed at 

high levels on Fc-receptor (FcR) bearing monocytes. To 

exclude FcR binding as an explanation for the detection 

of anti-CD3 and anti-TCR-ab Mabs on CD14+ cells, studies 

were repeated with human serum or with pre-incubation 

with anti-FcR Mabs (anti-CD16 and anti-CD32). Staining 

was equivalent with and without FcR block, suggesting 

that anti-CD3 and anti-TCR-ab Mab binding was specific. 

These data suggest that a minor population of nonlymphoid 

cells that express CD11b, CD11c, and CD14 also 

express CD3 and TCR-ab. Further studies are ongoing to 

fully characterize this population. 

doi:10.1016/j.clim.2007.03.327 

F.115 Haplotype-specific Use of Lysosomal 

Proteases Can Modulate the Class II MHC Peptide 

Repertoire but Does Not Impair Invariant Chain 

Degradation in Human Antigen Presenting Cells 

Cristina Costanti-England PhD Candidate, Harvard Medical 

School/Brigham and Women’s Hospital, Center for 

Neurologic Diseases, Boston, MA, Howard Hang, 

Postdoctoral Fellow, Whitehead Institute, Massachusetts 

Institute of Technology, Cambridge, MA, David Hafler, 

Jack, Sadie and David Breakstone, Professor of Neurology, 

Harvard Medical School/Brigham and Women’s Hospital, 

Center for Neurologic Diseases, Boston, MA, Hidde Ploegh, 

Professor of Biology, Whitehead Institute, Massachusetts 

institute of Technology, Cambridge, MA 

The lysosomal protease asparagine endopeptidase (AEP) 

has been implicated in both the destructive processing of 

self-antigen and the regulation of class II MHC maturation. 

In this study, we explore the role of human AEP in 

invariant chain processing in the maturation of several 

distinct allotypes of human class II MHC products. We find 

that, as is the case in mice, AEP activity is not necessary 

for class II maturation in human antigen-presenting cells. 

Our data demonstrate that Ii cleavage is regulated by 

many lysosomal proteases, including both aspartyl and 

cysteine proteases. Furthermore, we find that the 

repertoire of active proteases in EBV-transformed B cells 

differs between lines generated from different donors. 

These results suggest a unique profile of lysosomal 

protease activity within each antigen-presenting cell 

that regulates Ii degradation. Our findings underscore 

the complexity of the class II MHC-restricted pathway of 

antigen presentation. While allelic variation in MHC 

products is a decisive factor in determining the repertoire 

of presented peptides, the identity and level of activity 

of processing proteases present in antigen-presenting cells 

are contributing factors as well. As these proteases also 

generate the peptide cargo loaded into the binding 

pocket of class II MHC, it is certain that the repertoire 

of self and antigenic peptides presented to CD4+ T cells 

differs between individuals. Thus, altered presentation of 

immunogenic self-peptides due to alternate protease 

profiles may be a factor contributing to the susceptibility 

to and severity of autoimmune diseases such as type 1 

diabetes and multiple sclerosis. 

doi:10.1016/j.clim.2007.03.330 

S53 

F.116 Induction of Antigen-specific Oral Tolerance 

by Genetically Modified Lactococcus Lactis 

Delivering DQ8-specific Immunodominant Gliadin 

Epitopes to Gluten-sensitized Class II Transgenic 

Mice 

Inge Huibregtse, Academic Medical Center, Amsterdam, 

Eric Marietta, Mayo Clinic, Department of Immunology, 

Rochester, MN, Shadi Rashtak, Drs, Mayo Clinic, 

Department of Immunology, Rochester, MN, Chella David, 

Mayo Clinic, Department of Immunology, Rochester, MN, 

Pieter Rottiers, VIB, Department for Molecular Biomedical 

Research, Zwijnaarde, Ghent, Sander van Deventer, 

Professor Academic Medical Center, Center for 

Experimental and Molecular Medicine, Amsterdam, 

Joseph Murray, Mayo Clinic, Department of Immunology, 

Rochester, MN 

Antigen-specific immune suppression is an attractive 

therapy for the treatment of several gastro-intestinal 

diseases especially celiac disease. Active delivery of 

recombinant autoantigens or allergens at the intestinal 

mucosa by genetically modified Lactococcus lactis (LL) 

provides a novel therapeutic approach for the induction 

of tolerance. For this purpose we genetically engineered 

deamidated DQ8 epitope secreting LL (LL-eDQ8d) and 

evaluated local and systemic immune responses in glutensensitized 

NOD ABo DQ8 class II transgenic mice after oral 

supplementation. Mice were sensitized with either 10 1/4g 

deamidated DQ8 epitopes or 50 ng pertussis toxin and 100 

1/4 g crude gluten and fed for 10 or 21 days, respectively 

with LL-pT1NX (empty vector) or LL-eDQ8d (1×10 9 CFU/ 

day). Tolerance induction was assessed by DTH responses, 

ex vivo proliferation assays and cytokine measurements. 

Daily intragastric administration of LL-eDQ8d in sensitized 

transgenic mice led to a significant decrease in DTH response 

and proliferative capacity of the splenocytes and inguinal 

lymph node cells compared to the negative controls. LLpT1NX 

was also able to reduce the DTH response and the 

proliferation of splenocytes although not as much as the LLeDQ8d. 

Moreover, LL-eDQ8d significantly reduced the IFN- 3 

production in the draining lymph nodes and the spleen 

compared to the LL-pT1NX. Mucosal delivery of immunodominant 

gliadin epitopes by genetically modified LL induces 

suppression of local and systemic specific T cell responses in 

NOD ABo DQ8 transgenic mice. Moreover our data provide 

promise for the development of effective therapeutics for 

treatment of several common autoimmune, inflammatory

S54 Abstracts 

and/or allergic diseases by autoantigen- and/or allergensecreting 

L. lactis. 

doi:10.1016/j.clim.2007.03.331 

F.120 Evaluation of an Enhanced-sensitivity 

Interferon-γ ELISpot Assay Kit Using Cryopreserved 

PBMC Donor Bank Panels 

Keith Moskowitz, Director, SeraCare Life Sciences, 

Gaithersburg, MD, Janet Lathey, Director, Virology/ 

Immunology, Gaithersburg, MD, Lisa Jeffrey, Research 

Associate, Virology/Immunology, Gaithersburg, MD, Scott 

Hickman, Product Manager, Marketing, Gaithersburg, MD, 

Rodshawn Branch, Research Associate, SeraCare Life 

Sciences, Gaithersburg, MD, Dmitry Moshkoff, Project 

Scientist, Virology/Immunology, Gaiithersburg, MD, Mark 

Manak, CSO, SeraCare, Gaithersburg, MD 

Human interferon-gamma (IFN-γ) ELISpot assays detect 

single IFN-γ secreting cells. We compared a novel ELISpot 

(SC) to a commercial kit (CK). Three lots of SC and one of CK 

were evaluated using PHA, CEF, CMV and mock stimulation of 

cryopreserved PBMC from 24 independent donors. In 

response to PHA, SC and CK showed significant differences 

between frequency distributions (KS 0.36). SC median was 

479, 25th–75th percentiles (quartiles), and 275–975 SFC/ 

well. The CK median was 200 (quartiles 45–343). Means were 

also different: SC=474, CK=152, Pb0.0001. Average CVs 

were 11% for SC and 18% for CK. For CEF stimulus, SC median 

was 64 (quartiles 27–179), mean was 129, CV 20%; CK median 

was 54 (quartiles 23–174), mean 139, and CV 28%. CMV 

stimulus median for SC was 100 (quartiles 12–200), mean 

149, and CV 27%; CK median was 87 (quartiles 7–222), mean 

112, and CV 38%. All backgrounds averaged b2. SC lots 

performed equivalently with all agonists. Some CK wells 

were unreadable (30/216), while 100% of 648 SC wells gave 

SFC values, Pb0.000001. CK averaged ∼14% failures, with no 

readings in 4 of 24 subjects. Thus, SeraCare has validated an 

IFN-γ ELISpot kit demonstrating superior performance in 

response to PHA, lower CV to all agonists and statistically 

fewer well failures relative to a commercial kit. 

doi:10.1016/j.clim.2007.03.332 

F.121 Involvement of Hsp90β but Not Hsp90α in 

Anti-Apoptotic Effect of CpG-B ODN 

Cheng-Chin Kuo, Postdoctoral Fellow, Agricultural 

Biotechnology Research Center, Academia Sinica, Taipei, 

Chi-Ming Liang, Professor, Institute of Biological Chemistry, 

Academia Sinica, Taipei, Chen-Yen Lai, Research Assistant, 

Agricultural Biotechnology Research Center, Academia 

Sinica, Taipei, Shu-Mei Liang, Professor, Agricultural 

Biotechnology Research Center, Academia Sinica, Taipei 

Unmethylated CpG oligodeoxynucleotides (CpG ODNs) 

activate immune responses in a toll-like receptor 9 (TLR9)dependent 

manner. In this study, we found that stimulation 

of mouse macrophages and dendritic cells with B-type CpG 

ODN (CpG-B ODN) increased cellular heat shock protein 

(Hsp) 90β but not Hsp90α and prevented apoptosis induced 

by serum starvation or staurosporine treatment. The CpG-B 

ODN-induced Hsp90β expression depended on TLR9, myeloid 

differentiation factor 88 and PI3K. Inhibition of Hsp90β 

by expressing small interfering RNA suppressed not only 

Hsp90β expression but also PI3K-dependent phosphorylation 

of Akt and CpG-B ODN-mediated anti-apoptosis. Additional 

studies demonstrated that as described by other group, 

Hsp90β but not Hsp90α was associated with Bcl-2. Inhibition 

of Hsp90β suppressed the CpG-B ODN-induced association of 

Hsp90β with Bcl-2 and impaired the inhibitory effect of 

CpG-B ODN on the release of cytochrome c as well as the 

activation of caspase-3. This study thus reveals the 

involvement of Hsp90β but not Hsp90α in CpG-B ODNmediated 

anti-apoptotic responses and shows that Hsp90β is 

distinct from Hsp90α in its regulation of the cellular 

function of immune cells. 

doi:10.1016/j.clim.2007.03.333 

F.122 Immunoassays for Detection of Soluble Fc 

Alpha Receptor (CD89) in Plasma of IgA 

Nephropathy Patients 

Mirjana Hahn-Zoric, Research and Development Engineer, 

Sahlgrenska University Hospital, Clinical Immunology, 

Gothenburg, Sweden, Neda Tahmasebifar, Student, 

Sahlgrenska University Hospital, Clinical Immunology, 

Gothenburg, Sweden, Cees van Kooten, Department of 

Nephrology, Leiden, The Netherlands, Sweden, Jarl Ahlmen, 

Nephrology Clinics, Skövde, Per-Anton Westerberg, 

Nephrology Clinics, Skövde, Sweden, Svante Swerkersson, 

Pediatric Uro-Nephrologic Center, Gothenburg, Sweden, 

Sverker Hansson, Pediatric Uro-Nephrologic Center, 

Gothenburg, Sweden, Ulla Berg, Karolinska University 

Hospital, Stockholm, Sweden, Lars Åke Hanson, Professor 

Emeritus, Sahlgrenska Academy, Gothenburg University, 

Clinical Immunology, Gothenburg, Sweden, Leonid 

Padyukov, Senior Researcher, Rheumatology Unit, 

Department of Medicine, Stockholm, Sweden, Stefan H. 

Jacobson, Department of Nephrology, Stockholm, Sweden 

Background: Fc±RI, or CD89, is a receptor binding the Fc 

portion of IgA. Recently it has been shown that CD89 is involved 

in IgA–immune complex formation. CD89–IgA complexes were 

suggested to be pathogenic in several diseases, one of which is 

IgA nephropathy (IgAN). IgAN is characterized by increased 

serum IgA levels paralleled by the deposition of IgA in renal 

tissue. IgAN is the most common form of glomerulonephritis in 

the Western world and is the second most common cause of 

dialysis or kidney transplantation after diabetes. The pathogenesis 

and the mechanisms of this disease are not well understood 

and there are no reliable prognostic factors that can predict 

which patients are of high risk to develop severe kidney damage. 

Aim of this study was to develop immunoassay(s) for detection of 

soluble IgA receptor (sCD89) in human plasma. Methods and 

material: By using CD89-specific MIP8a and A3 monoclonal 

antibodies and polyclonal anti-CD89 IgG, a Western blot method 

as well as a direct and an indirect ELISA for quantification of 

sCD89 were developed. 45 IgAN patients (32 adults and 13

Abstracts 

teenagers) and 45 matched healthy controls were analyzed. 

Results: A single sCD89 band of around 30 kDa was identified in 

Western blot with polyclonal IgG. Significantly higher levels of 

sCD89 were found in adult IgAN patients compared to controls in 

monoclonal antibody-based ELISA. Conclusion: We developed 

assays that provide helpful tools for detection of sCD89, which 

may be a useful diagnostic marker for further studies of IgAN. 

doi:10.1016/j.clim.2007.03.335 

F.124 Resistin Like Molecule Beta Regulates 

Intestinal Inflammation in Chronic Colitis 

Luqman Seidu, Allergy/Immunology Fellow, Cincinnati 

Children’s Hospital, Cincinnati, OH, Elizabeth Forbes, PhD 

Student, Cincinnati Children’s Hospital, Cincinnati, OH, 

Richard Aherns, Rheumatoid Arthritis, Cincinnati Children’s 

Hospital, Cincinnati, OH, Margaret Karow, PhD, Regeneron 

Pharma, Tarrytown, NY, Carine Blanchard, PhD, Cincinnati 

Children’s Hospital, Cincinnati, OH, Andrew Murphy, PhD, 

Regeneron Pharma, Tarrytown, NY, David Valenzuela, PhD, 

Regeneron Pharma, Tarrytown, NY, George Yancopoulos, 

MD/PhD, Regeneron Pharma, Tarrytown, NY, Marc 

Rothenberg, MD/PhD, Cincinnati Children’s Hospital, 

Cincinnati, OH, Simon Hogan, PhD, Cincinnati Children’s 

Hospital, Cincinnati, OH 

ResistinlikemoleculeRELM-β, a member of resistin family, 

has been implicated in glucose metabolism and intestinal 

immunity. We demonstrate that RELM-β is constitutively 

expressed in the colonic epithelium and is upregulated during 

chronic intestinal inflammation. To examine the contribution 

of RELM-β to chronic intestinal inflammation, we utilized 

RELM-β deficient mice and models of colitis. We show that 

RELM-β deficient mice have decreased susceptibility to 

chemical (DSS [dextran sodium sulfate])-induced colitis 

(disease activity index, 1.53±1.53 vs. 7.37±2.4 respectively 

mean±SE; survival at day 8, 100% vs. 75% respectively n=4– 

5 mice per group). We show that the link between colitis 

susceptibility and RELM β was associated with the expression 

of the NFkappaB signaling inhibitor, REG3β. Moreover, 

REG3β mRNA expression was increased 500-fold in DSStreated 

RELM-β deficient mice vs. wild type mice (616.8± 

138.1 vs. 26.35±11.69, fold increase [REG3β expression/ 

GAPDH ratio], respectively n=4–5 mice per group, mean± 

SE). Collectively this work suggests an interaction among 

RELM-β, REG3β and intestinal inflammation. 

doi:10.1016/j.clim.2007.03.336 

F.125 Immune Parameters in Normal Humans with 

Different Behavior Patterns: Type A and B Behavior 

Pattern 

Fereshteh Alesahebfosoul, Academic Member of Isfahan 

University, Medical School, Immunology DEP, Isfahan, 

Bahareh Zzarkeshfard, Food Science Engineering, Health 

Faculty, Isfahan, Farzad Oreizi, Immunologist, Medical 

School, Immunology DEP, Isfahan, Minoo Adib, Professor, 

medical school, Immunology DEP, Isfahan 

Introduction: Numerous studies have described various 

aspects of behavioral influences on immune function. Behavior 

and emotions modulate immune function. In this study 

distinctive immunological findings in normal humans with type 

A and B behavior pattern were described. Method: In cross 

sectional study 105 participants (P) admitted to be in the first 

phase (20–30 years old). 6 Ps were excluded because of 

abnormal CBC. We used standard questioner (Bortne scale) to 

separate type A and B behavior patterns. The samples of 

peripheral blood were referred to immunology laboratory for 

measurement of immune factors. SPSS (ver.10) and t test were 

used in statistical analysis. Results: 40 Ps were type A and 59 

type B. There were no significant differences between two 

groups on serum B cell, T cell, and NK cell. There was a 

significant increase (P valueb0.05) on serum CD4 positive Tcells 

in type A but no significant increase was viewed in CD8 positive T 

cells. There were significant increases on lymphocytes and 

granulocytes in type A. It was significant in male Ps with type A. 

CD4 positive T cells increased in counter female with type B, 

however no significant difference was seen in male with type A 

in contrast to female. Discussion: The finding of this study was 

that type A behavior pattern with specific characteristics (time 

emergency) has no significant effect on immune system and the 

effect of gender of immune parameters has to be considered. 

doi:10.1016/j.clim.2007.03.337 

S55 

F.126 Multi-Parameter Analysis (MPA) of Cells and 

Serum Using a Standard Flow Cytometer 

Robert Hallam, Senior Biomedical Scientist, Papworth 

Hospital, Immunology Department, Cambridgeshire, 

England, Paul Balmer, Clinical Scientist, HPA, Vaccine 

Evaluation Department, Manchester, England, Ray Borrow, 

Clinical Scientist, HPA, Vaccine Evaluation Department, 

Manchester, England, Andrew Exley, Consultant 

Immunologist, Papworth Hospital/Immunology Department, 

Cambridge, England 

Background: Flow cytometric peripheral blood MPA identifies 

cell populations by size, granularity, and expression pattern 

of cell surface or intracellular antigens using fluorophore 

conjugated Abs. Alternative platforms for serum MPA measure 

concentrations of secreted proteins including cytokines and 

pathogen specific Abs in multiplex, liquid phase capture assays 

using antigen or antibody coated beads identified by size, 

fluorescence intensity and wavelength. Fresh blood samples 

preferred for cellular analysis generate peaks and troughs in 

workload contrasting with batch analysis of sera. Methods: B 

cell populations were defined by CD45/ss pan-leucogating, then 

differential expression of CD19, CD27, IgD or IgM using a 

Beckman-Coulter FC-500 flow cytometer. 10-plex serotype 

specific anti-pneumococcal antibody assays were established 

using carboxylated microspheres coated with pneumococcal 

capsular polysaccharide, CPS and 22F absorption, PE-conjugated 

mouse anti-human IgG/IgM, and reference standard 89SF. 

Results: These serum MPAs have 2–3 log dynamic linear ranges, 

b6% intraassay and 16% interassay CVs, 1.88 ng/ml median 

limits of quantification, and correlate well with Bioplex systems 

and ELISAs. The complex data set is presented as an assay

S56 Abstracts 

report highlighted by a “traffic light system” and pictorial 

patient reports in MS-WORD, after analysis by especially 

designed software incorporating MS-EXCEL macros enhanced 

by Visual Basic programming. B cell phenotyping correlated 

with pre versus post-immunisation serum pneumococcal antibody 

levels in cases investigated for primary and secondary 

immunodeficiency. Discussion: MPA of cells and serum on a 

single standard flow cytometer with powerful software applies 

existing expertise and resources in a flexible cost-effective 

approach suitable for high-throughput testing and clinical 

laboratories. 

doi:10.1016/j.clim.2007.03.338 

F.127 Catalytic Anti-dsDNA Activity from SLE Sera is 

Associated with Remission Phases of the Disease 

Genevieve Servais, Responsible Autoimmunity LAB, Canada, 

Canada, CHU Brugmann, Immunology, Bruxelles, Belgium, 

Mimouna El Baz, Pharmacist, CHU Brugmann Immunology, 

Bruxelles, Belgium, Rafik Karmali, MD, CHU Brugmann 

Internal Medicine, Bruxelles, Belgium, Marie Paule 

Guillaume, MD, CHU Brugmann Internal Medicine, 

Bruxelles, Belgium, Jean Duchateau, MD PHD, CHU 

Brugmann Immunology, Bruxelles, Belgium 

SLE is characterized by the presence of circulating anti- 

DNA antibodies, one of the eleven ACR criteria for the 

classification of SLE, and they play an important role in the 

pathogenesis of SLE. Anti-DNA antibodies display different 

specificities, some being restricted to nuclear double 

stranded DNA (dsDNA), or single stranded DNA (ssDNA), or 

membrane associated DNA (mDNA) or nucleosomes. Some 

are sharing dual specificities. Autoantibodies displaying a 

catalytic activity, abzymes, were reported in patient’s sera, 

including catalytic anti-DNA antibodies. They have not been 

reported in healthy control subjects. Catalytic activity varies 

in different pathologies and changes during the different 

phases of the disease. The biological role of the catalytic 

anti-DNA present in serum of SLE is complex, as it seems that 

some may play positive and others negative roles, from a 

clearance role to a lowering of DNAse activity in SLE 

serum. We show here that circulating catalytic anti-DNA is 

found in SLE and RA patients but also in healthy controls 

though with a higher percentage in SLE than in controls, 

where they can be unraveled when the sera are diluted 

at 1/10. Their presence is inversely correlated with the 

clinical activity of the disease and with the titer of 

circulating autoantibodies to dsDNA, mDNA but not antinucleosomes 

antibodies. 

doi:10.1016/j.clim.2007.03.339 

F.128 Fungal Hypersensitivity is Not Type I (IgE) 

Allergy 

Vincent A. Marinkovich, MD, Inc., Redwood City, CA 

One hundred adult patients undergoing clinical evaluations 

for symptoms developing during chronic exposure to 

high ambient fungal antigen levels in water damaged 

homes were tested for specific IgE (RAST) and specific IgG 

(ELISA) levels to a number of fungi commonly found in 

mold-amplified environments. The tests were standard 

runs in a State licensed Physician’s Office Laboratory 

using test materials provided by Hitachi Chemical Diagnostics 

for specific IgE and specific IgG in a multi antigen 

panel format. The panels contain aspergillus, penicillium, 

alternaria, cladosporium and mucor. Results read as 

positive when greater than 2 standard deviations greater 

than the mean using reference ranges previously determined 

by Hitachi. Seven patients had elevated specific 

IgE levels to at least one of the fungi tested (7%). Eighty 

nine patients showed elevated IgG titers to at least one 

of the molds. The sera were then exposed to an 

additional eight common molds, botrytis, candida, epicoccum, 

fusarium, helminthosporium, pullularia, rhizopus 

and stemphylium, on an IgG panel and all patients (100%) 

showed elevated titers to at least one of the molds. The 

common symptoms reported were remarkably consistent 

among patients: fatigue, nasal congestion, headaches, 

cough, memory loss and chronic flu-like symptoms. All the 

patients showed signs of chronic rhinosinusitis suggesting 

fungal colonization and eosinophilic degranulation. Systemic 

symptoms are likely the result of circulating 

immune complexes under conditions of functional asplenia 

(i.e., IC overload). The neurological symptoms may be the 

result of local uptake of eosinophilic neurotoxin via the 

cribriform plate. 

doi:10.1016/j.clim.2007.03.340 

F.129 Automation for Isolation of Peripheral Blood 

Mononuclear Cells (PBMC) 

Carlos Aparicio, Scientist, Beckman Coulter, Inc., Miami, FL, 

Enrique Rabelli, Director, Beckman Coulter, Inc., Miami, FL, 

Edward Jachimowicz, Scientist, Beckman Coulter, Inc., 

Miami, FL, Sybil D’Costa, Scientist, Beckman Coulter, Inc., 

Miami, FL, Mahsa Aspsater, Scientist, Beckman Coulter, 

Inc., Miami, FL, Julie Wilkinson, Scientist, Beckman 

Coulter, Inc., Miami, FL, Wade Bolton, VP, Beckman 

Coulter, Inc., Miami, FL, Enrique Rabelli, Director, 

Beckman Coulter, Inc., Miami, FL 

Peripheral blood mononuclear cells (PBMC) are considered 

appropriate target populations to immunologically 

assess the various subsets of blood lymphocytes. Purification 

of PBMC by density gradient centrifugation is a 

laborious and time consuming manual process and, thus, 

limiting factor for processing large number specimens. 

Generally, the media–gradient interface is generated by 

carefully pipetting the blood cell suspension onto a 

density gradient solution without disturbing the interface. 

Disruption of the interface can jeopardize the overall 

yield, purity and viability of the mononuclear cell 

preparation. The study described herein focuses on an 

alternative semi-automated strategy to harvest PBMC 

using the Ficoll-Paque density gradient. This process 

integrates the use of an automated liquid handler and a 

centrifuge to minimize the number of steps and time

Abstracts 

requiring operator intervention in current laboratory 

procedures. PBMC harvested by this automated process 

had a viability over 95%, and when compared to the 

manual procedure showed: (1) cell recovery (N95%) with 

no selective loss as judged by phenotyping and functional 

response by the various lymphocytes. The goal of this 

work is to provide a fully automated and standardized 

method for preparing PBMC that can facilitate support 

cell based-assay testing strategies in clinical trials by 

improving overall quality and efficiency of the process as 

well as reducing labor and cost. 

doi:10.1016/j.clim.2007.03.341 

F.130 Interleukin 32 Expression is Associated with a 

Unique Subset of Human Dendritic Cells Receptive 

to the Venezuelan Equine Encephalitis 

Virus-derived Replicon Vector 

Kevin Nishimoto, Graduate student, University of California 

Irvine, Department of Microbiology and Biochemistry, 

Irvine, CA, Charles Dinarello, Professor of Medicine, 

University of Colorado Health Sciences Center, Department 

of Medicine, Denver, CO, Stephen Hou, PhD, University of 

California Irvine, Department of Medicine, Division of 

Hematology/Oncology, School of, Irvine, CA, Edward 

Nelson, Assistant Professor of Medicine, University of 

California Irvine Department of Medicine, Division of 

Hematology/Oncology, School of Medicine, Irvine, CA 

Establishing successful immunotherapeutic strategies 

utilizing dendritic cells (DC), the most potent antigen 

presenting cell, largely depends on understanding the 

requirements necessary for optimal DC function. We and 

others have reported that the Venezuelan Equine Encephalitis 

(VEE) replicon particle (VRP) vector system has 

conserved tropism for murine, rat, and human DCs. 

However the DC subset(s) and functional capacities are 

not fully understood. The VRP vector system has in vitro 

tropism for a subset of human immature monocytederived 

DCs (M-DCs); therefore, we developed a novel 

technique to isolate this VRP receptive DC subset for 

further characterization. An evaluation between VRP 

receptive and non-receptive M-DC subsets by microarray 

revealed distinct differences in gene expression profiles 

with N700 significantly expressed genes (0.001 e P value, 

0.98 d PPDE) between cellular subsets. Notably in the 

receptive DC population, the NK4 transcript was significantly 

increased and accompanied by protein expression. 

The NK4 gene has recently been identified as encoding a 

recently described cytokine, interleukin 32. These data 

provide the initial characterization of a new subset of 

myeloid DCs targeted by this vector, and provide insights 

into human DC biology allowing explicit study of the 

mechanisms employed by this exceptionally potent immunotherapeutic 

vector system. 

doi:10.1016/j.clim.2007.03.342 

F.131 Retinoic Acid Promotes Development of 

Human Macrophages Over Dendritic Cell 

Differentiation 

Juliana Sousa-Canavez, Researcher, Genoa Biotech, São 

Paulo, Brazil, Cristina Massoco, Researcher, Genoa 

Biotech, São Paulo, Brazil, Elaine Corneta, Student, 

Genoa Biotech, São Paulo, Brazil, Katia Leite, Pathologist, 

Genoa Biotech, São Paulo, Brazil, Tatiane Marisis, Student, 

Genoa Biotech, São Paulo, Brazil, Luiz Camara-Lopes, 

Pathologist, Genoa Biotech, São Paulo, Brazil, Antonio 

Misiara, MD, Genoa Biotech, São Paulo, Brazil, Dewton 

Moraes-Vasconcelos, Professor, Genoa Biotech, São Paulo, 

Brazil 

Previous studies showed that all vitamins including 

vitamin A are fundamental for immune system properly 

functioning. All trans retinoic acid (atRA) at physiological 

or near-physiological concentrations appears to affect 

Th1–Th2 differentiation and it is believed that effects 

of retinoids on immune responses might also be mediated 

by dendritic cells (DC). Nonetheless, effects of atRA in DC 

differentiation have been showing contradictory results 

since it was observed either induction or inhibition of 

differentiation. Our work shows effects of atRA on human 

monocyte derived DC by phenotype and phagocytosis 

studies. DC (positive control group) was differentiated 

with cytokines (GM-CSF and IL-4) and maturation induced 

by TNF-α. We have tested pharmacological (10 1/4M and 

1 1/4M) and physiological (10 nM) doses of atRA with or 

without cytokines. Cell phenotype was analyzed by flow 

cytometry using CD80, CD86 and CD14 antibodies. We also 

investigated functional performance analyzing phagocytosis 

and respiratory burst (ROS) in all culture conditions 

mentioned above. Our data demonstrate that effects of 

atRA depend on the dose used, as pharmacological doses 

inhibited expression of all phenotypic markers tested, 

while a physiological one caused cell differentiation. 

Physiologic concentration of atRA especially in combination 

with GM-CSF blocked differentiation of DC population 

corroborating a macrophage preferable transformation. 

Moreover phagocytosis and ROS were approximately three 

times greater in cells cultivated with atRA compared to 

positive control group. On the other hand, IL-4 and atRA 

apparently induced differentiation to an immature DC 

phenotype when TNF-α stimulus was added. 

doi:10.1016/j.clim.2007.03.343 

S57 

F.132 OTC Allergy Testing 

Chris Brown, ImmuneTech, Inc., Menlo Park, CA, Vincent A. 

Marinkovich, MD, Inc. Redwood City, CA 

Quantitative, comprehensive in vitro, specific IgE tests 

for allergy have not hitherto been introduced into the 

over the counter (OTC) market because of their high costs 

and the need for a large sample of blood requiring a 

venapuncture. Recently the U.S. Food and Drug Administration 

cleared for OTC distribution a new allergenspecific 

IgE test which has two major advantages over

S58 Abstracts 

its predecessors: low blood volume and economy. This 

new system, utilizing a flow cytometry platform, is 

currently configured to provide quantitative specific IgE 

levels to each of ten allergens simultaneously. It utilizes 

just 5 μl of capillary or venous serum in a homogenous 

assay, and can be completed in under 3 h. The system 

compares favorably to the current laboratory testing 

methods: 

Sensitivity/specificity/accuracy data in %: 

– Mountain cedar 88/93/90 

– Timothy grass 94/97/90 

– Bermuda grass 86/97/91 

– Short ragweed 90/95/92 

– House dust mite 96/93/95 

– Cat hair 96/89/93 

– Cow’s milk 81/92/89 

– Egg 80/94/92 

– Wheat 85/93/90 

– Alternaria 87/94/90 

The coefficient of variation between test runs on 

different days ranged from 3.4 to 7.2% and within the 

same day from 2.9 to 8.3%. The test’s lowest level of 

detection is 0.35 IU/ml. This accurate, low cost, low blood 

volume test with fingerstick convenience can expand the 

availability of accurate allergy diagnosis to a patient 

population not currently served by existing in vitro systems 

or by skin testing. 

doi:10.1016/j.clim.2007.03.344 

F.133 Standardization and Quality Assurance of 

Cytokine Flow Cytometry Assays 

Maria Jaimes, Scientist, BD Biosciences, San Jose, CA, 

Janice Darden, CRS Team Leader, DAIDS/NIAID/NIH, 

Bethesda, MD, Patricia D’Souza, Program Officer, DAIDS/ 

NIAID/NIH, Bethesda, MD, Holden Maecker, Group 

Manager-Immune Function, BD Biosciences, San Jose, CA 

Cytokine flow cytometry (CFC) is used to measure 

antigen-specific T cell responses in settings such as 

experimental HIV vaccination. Standardization of CFC 

among different laboratories would provide a common 

endpoint assay for comparing the immunogenicity of 

vaccine candidates in clinical trials. For this purpose, a 

Quality Assurance Program has been developed in a 

collaborative effort between BD Biosciences, the Division 

of AIDS, NIAID, NIH and SeraCare BioServices, in which 15 

sites currently participate. Three rounds of the program 

have been completed. In each round, IFN-ƒ× and IL-2 

responses in CD4+ and CD8+ T cells to CEF or CMV pp65 

lyophilized peptide mixes were tested using cryopreserved 

PBMC from CMV+ donors. Each site analyzed its data 

independently and provided, through the program’s 

website (http://bdicsqa.webbasix.com), their raw data 

for centralized analysis. In addition, the data generated 

by each laboratory were compared to a standard. The 

first two rounds of the program yielded inter-laboratory 

variability from 12 to 60%, for cytokine+ responses higher 

than 0.2%. Instrument setup and analysis (gating strategy) 

played a critical role in this variation. This variability was 

decreased in the third round by providing feedback to the 

laboratories, guidelines for data analysis, and a protocol 

and reagents for instrument setup. Of note, the interlaboratory 

variability (18–32%) was comparable to the 

intra-laboratory variability (4–25%) in the third round. 

Critical to success was the provision of standard operating 

procedures, centrally supplied reagents, and laboratory 

adherence to GCLP. The implementation of this Quality 

Assurance Program will ensure greater uniformity of data 

from multiple laboratories. 

doi:10.1016/j.clim.2007.03.345 

F.134 A Novel Cell Contact-dependent Requirement 

for Human CD40 Ligand Expression 

Jack Ragheb, Senior Clinical Investigator, Laboratory of 

Immunology, NEI, NIH, Bethesda, MD, James Snyder, 

Research Fellow, NIH, Bethesda, MD, Jijia Shen, Research 

Fellow, NIH, Bethesda, MD, Paul Hu, Research Fellow, nIH, 

Bethesda, MD, Hooman Azmi, Research Fellow, NIH, 

Bethesda, MD, Qian Chen, Research Fellow, NIH, Bethesda, 

MD 

CD40L (CD154), an inducible costimulatory molecule 

expressed on CD4+ T cells, plays an essential role in the 

activation of B cells. Through the CD40L mediated 

induction of IL-12 and upregulation of MHC class II, 

CD80/86, and other accessory molecules on antigen 

presenting cells (APC), CD40L also catalyzes a positive 

feedback loop for T cell activation. Despite the pivotal 

juxtaposition of CD40L between APC and T cell activation, 

it is thought that only a TCR stimulus is required to 

initiate early CD40L expression. We demonstrate that 

unlike its mouse counterpart, human CD40L is not 

constitutively expressed on circulating T cells, nor do 

human CD4+ T cells contain an intracellular pool of CD40L 

protein. While a TCR stimulus is necessary for CD40L 

expression on primary human T cells, we show for the 

first time that expression of CD40L is highly dependent 

upon a cell–cell interaction with CD14hi monocytes that 

does not require LFA-3, ICAM-1, or CD80/86. Like CD28dependent 

IL-2 expression, this monocyte signal upregulates 

the transcription of CD40L. Thus the inducible 

expression of CD40L on CD4+ T cells, which provides a 

critical costimulatory signal for monocyte and B cell 

activation, is itself dependent upon a costimulatory signal 

delivered by monocytes but not B cells. The latter 

finding implies that a human B cell cannot activate its 

cognate helper T cell to deliver CD40L-mediated help. 

Therapeutics targeted at this monocyte costimulatory 

factor for CD40L may lead to new means of modulating 

the immune response and inducing long-term graft 

acceptance. 

doi:10.1016/j.clim.2007.03.346

Abstracts 

F.135 Endocytosis of Hsp-chaperoned Proteins and 

Peptides is Essential for MHC II Dependent Peptide 

Presentation 

Irena Straub, Medical Candidate, University Children’s 

Hospital Tübingen, Tuebingen, Markus Haug, University 

Children’s Hospital Tübingen, Tuebingen, Dorothee Wernet, 

Professor, Department of Transfusion Medicine University 

Hospital Tübingen, Tuebingen, Ursula Holzer, Dr. Medicine, 

University Children’s Hospital Tübingen, Tuebingen, 

Guenther E. Dannecker, Professor, Olgahospital Stuttgart, 

Stuttgart 

Heat shock protein Hsp70 complexed to antigenic peptides 

enhances the activation and proliferation of CD8+ and CD4+ T 

cells in vitro. However the mechanism for HSP70-dependent 

antigen presentation by MHC II molecules is not yet clear. It is 

thought that after internalization of the HSP–peptide 

complexes by APCs the complex is trafficking into the 

cytoplasm or endosomal vesicles to be represented by MHC I 

and/or MHC II, although the necessity of an uptake for 

presentation by MHC II is not proven. Here we investigated 

the need of endocytosis of HSP–peptide complexes for 

sufficient antigen presentation in a human antigen-specific 

system. PBMCs of healthy donors immunized against tetanus 

or influenza with known HLA-DR haplotype (DRB*1101, 0401) 

were used. Stimulation of Tcells was induced by tetanus toxin 

C-fragment (TT944-966), influenza hemagglutinin (HA307– 

319) or the whole tetanus toxid protein either with peptide/ 

protein alone or with the peptide/protein complexed to 

Hsp70. Antigen specificity was detected by HLA-DR-tetramers. 

For blocking the endocytosis mechanism of CD4− APCs, 

the cells were pre-treated with Cytochalasin, Genistein or 

Chloroquine, resulting in a marked decline in generating 

specific T cells. These results argue for an internalization of 

the complexes by APCs and uptake into the endosomal 

vesicles for presentation by MHC II. Furthermore HSP–protein 

complexes are able to enhance the proliferation of antigenspecific 

T cells affirming an intracellular pathway of HSPmediated 

peptide presentation. Taken together, we conclude 

that for MHC class II dependent activation of CD4+ antigenspecific 

T cells uptake of Hsp-chaperoned proteins and 

peptides into APCs and intracellular processing is necessary. 

doi:10.1016/j.clim.2007.03.347 

F.136 Development and Validation of a Novel Ig 

Isotyping Assay on Magnetic Microspheres 

Candice Reyes, Scientist, Bio-Rad Laboratories Protein 

Function Division, Research and Development, Hercules, 

CA, Joe Fedynyshyn, Staff Scientist, Bio-Rad Laboratories 

Protein Function Division, Research and Development, 

Hercules, CA, Sophie Allauzen, Research and 

Development Manager, Bio-Rad Laboratories Protein 

Function Division, Research and Development, 

Hercules, CA 

The Bio-Plex Suspension Array system provides a 

flexible and efficient platform for multiplex analysis of 

biological samples. Monitoring the levels of immunoglobulins 

is a valuable tool for evaluating the immune 

response in many different fields of research. Bio-Rad 

has developed a new multiplex immunoglobulin isotyping 

assay on magnetic beads that simultaneously quantifies 

the levels of IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE in 

human serum and plasma. The levels of immunoglobulins 

can vary for each class and subclass depending on factors 

like genetics, disease state, and drug treatment. Using 

the Bio-Plex Pro Human Ig Isotyping assay kit, a complete 

profile of isotype levels can be created from as little as 

5 μL of sample in about 3 h. Sensitivity and upper and 

lower limits of quantitation (LLOQ and ULLOQ) will be 

discussed. Additional validation data obtained from control 

human serum samples and World Health Organization 

(WHO) Reference Standards will also be presented. 

doi:10.1016/j.clim.2007.03.348 

F.137 Utility of Lyophilized PMA and Ionomycin to 

Stimulate Lymphocytes in Whole Blood for 

Immunological Assays 

Shelley Belouski, Senior Scientist, Amgen Clinical 

Immunology, Thousand Oaks, CA, John Ferbas, Director 

Medical Sciences, Amgen Clinical Immunology, Thousand 

Oaks, CA, Keith Kelley, Senior Associate Scientist, Amgen 

Clinical Immunology, Thousand Oaks, CA, Julie Wilkinson, 

Staff Adv Research Scientist, Beckman Coulter, Inc., Custom 

BioPharma Solutions, Miami, FL, Sid Suggs, Director, Amgen 

Medical Sciences, Thousand Oaks, CA, John Thomas, Senior 

Associate Scientist, Amgen Clinical Immunology, Thousand 

Oaks, CA, Shen-Wu Wang, Senior Associate Scientist, Amgen 

Molecular Sciences, Thousand Oaks, CA 

Background: The need to implement robust biomarkers in 

clinical trials has never been greater, and such efforts can be 

easily compromised by reagent instability or simple human 

error during assay set-up. Many biotechnology and pharmaceutical 

companies are introducing efforts to conduct 

biomarker studies under more rigorous settings, and use of 

plates or tubes pre-loaded with stimulation or staining 

reagents could be of value for studies that involve flow 

cytometry. Results: Our laboratory took a biomarker analytical 

method that relied on liquid formulations of phorbol 12myristate 

13-acetate (PMA) and ionomycin and demonstrates 

here that tubes pre-loaded with lyophilized versions of the 

liquid reagents can provide equivalent results. The value of 

this approach is that it safeguards against omission or 

erroneous addition of bulk liquid formulations of PMA and 

ionomycin to the reaction vessel (i.e., plate or tube) and also 

lends itself to extended stability/shelf-life of these 

reagents. Conclusions: On the basis of this initial success, 

we plan to expand our evaluation of lyophilized reagents so 

that they can be incorporated into our clinical biomarker 

campaigns as appropriate. 

doi:10.1016/j.clim.2007.03.349 

S59

S60 Abstracts 

#2201- Inflammation Good or Bad for Tregs 

Saturday, June 9 

10:45 am−11:05 am 

Viral Infection Enables CD4+CD25+ Regulatory T 

Cells to Protect From Autoimmune Diabetes 

Christophe Filippi, Postdoctoral Fellow, La Jolla Institute 

for Allergy and Immunology, La Jolla, CA, Janine Oldham, 

Research Assistant, La Jolla Institute for Allergy and 

Immunology, La Jolla, CA, Tom Wolfe, Research Assistant, 

La Jolla Institute for Allergy and Immunology, La Jolla, 

CA, Evelyn Rodrigo, Research Assistant, La Jolla Institute 

for Allergy and Immunology, La Jolla, CA, Urs Christen, 

Research Scientist, La Jolla Institute for Allergy and 

Immunology, La Jolla, CA, Lisa Togher, Research Assistant, 

La Jolla Institute for Allergy and Immunology, La Jolla, 

CA, Marianne Martinic, Postdoctoral Fellow, La Jolla 

Institute for Allergy and Immunology, La Jolla, CA, 

Matthias von Herrath, Professor, La Jolla Institute for 

Allergy and Immunology, La Jolla, CA 

Type 1 Diabetes (T1D) is an autoimmune disease that 

results from the selective destruction of insulin-producing 

beta cells in the pancreas. While viral infections may be 

capable of triggering autoimmunity in genetically susceptible 

individuals, accumulating evidence indicates that viruses 

can also prevent T1D. Furthermore, viruses have been 

suggested to be potent inducers of CD4+CD25+ regulatory T 

cells (Tregs), which are known to play a crucial role in the 

prevention of autoimmunity. However, how viral infections 

may protect from autoimmune diabetes is still under 

investigation. Here, we show that activation of CD4+CD25+ 

Tregs by acute lymphocytic choriomeningitis virus (LCMV) 

infection provides these cells with the capacity to prevent 

both spontaneous and virally induced T1D in the mouse. We 

found that CD4+CD25+ Tregs activated by LCMV in vivo or in 

vitro produce the regulatory cytokine TGF-β and are capable 

of protecting NOD and RIP-LCMV mice from spontaneous and 

LCMV-induced diabetes, respectively. Our results suggest 

that LCMV-modulated CD4+CD25+ Tregs act in a bystander 

fashion independent of beta-cell antigens and suppress TNFα 

production by autoreactive CD8+ T cells, leading to 

prevention of T1D. These findings provide a novel mechanistic 

explanation for the previously reported ability of viral 

infections to prevent autoimmune diabetes. Furthermore, 

our results support a model explaining the differential ability 

of viral infections to modulate diabetes. Activation of Tregs 

during viral infections may be a general feature accounting 

for the ability of viruses to prevent T1D as well as other 

autoimmune disorders. 

doi:10.1016/j.clim.2007.03.350 

Oral Presentations: Saturday, June 9 


2:45 pm−4:45 pm 

OR.33 Cytotoxic Human IL-22-expressing Th17 

Lymphocytes Promote Immune Cell Migration Into 

the Central Nervous System 

Hania Kebir, PhD Student, CHUM-Notre-Dame Hospital, 

Neuroimmunology Department, Montreal, QC, Canada, Igal 

Ifergan, PhD Student, CHUM-Notre-Dame Hospital, 

Neuroimmunology Department, Montreal, QC, Canada, 

Aurore Dodelet-Devillers, M.Sc. Student, CHUM-Notre-Dame 

Hospital, Neuroimmunolgy Department, Montreal, QC, 

Canada, Fabrizio Giuliani, Assistant Professor of Medicine, 

University of Alberta, Department of Medicine, Edmonton, 

AB, Canada, Romain Cayrol, PhD Student, CHUM-Notre-Dame 

Hospital, Neuroimmunolgy Department, Montreal, QC, 

Canada, Nathalie Arbour, Assistant Professor of Medicine, 

CHUM-Notre-Dame Hospital, Neuroimmunology 

Department, Montreal, QC, Canada, Alexandre Prat, 

Assistant Professor of Medicine and Doctor, 

CHUM-Notre-Dame Hospital, Neuroimmunolgy Department 

and MS Clinic, Montreal, QC, Canada 

Interleukin (IL)-17-secreting lymphocytes (Th17) appear 

essential in the pathogenesis of numerous models of 

inflammatory diseases, including experimental autoimmune 

encephalomyelitis (EAE). As survival of Th17 cells 

is attributed to IL-23, recent evidence suggests that 

transforming growth factor (TGF)-β is required for the 

expression of IL-23 receptor (R) by lymphocytes, thereby 

acting upstream in the development of Th17. However, 

while IL-23 and TGF-β R null animals are resistant to 

autoimmune diseases, IL-17 neutralization only partially 

protects against EAE and colitis, suggesting that additional 

Th17-derived factors contribute to tissue inflammation. We 

demonstrate that human Th17 lymphocytes originate from 

memory CD4+CD45RO+ cells and express IL-22 under the 

influence of IL-23, while TGF-β acts as a negative regulator 

of IL-22 production. We show that IL-17R and IL-22R are upregulated 

on blood–brain barrier (BBB)-endothelial cells 

(ECs) in situ during inflammation, and that IL-17 and IL-22 

disrupt the human BBB and promote CD4+ T lymphocyte 

recruitment in vitro. Furthermore, Th17 lymphocytes transmigrate 

more readily across brain-ECs than Th1 and 

express high levels of granzyme B. Thus, IL-23 induces 

the formation of a unique Th subset expressing cytotoxic 

factors and promoting leukocyte recruitment across the 

BBB, through the concerted and redundant effects of IL-17 

and IL-22. 

doi:10.1016/j.clim.2007.03.351

Abstracts 

OR.34 CNS Myeloid Dendritic Cells Drive Epitope 

Spreading and Th-17 Differentiation in Relapsing 


(R-EAE) 

Stephen Miller, Professor, Northwestern University Medical 

School, Department of Microbiology-Immunology, Chicago, 

IL, Samantha Bailey, Postdoctoral Fellow, Northwestern 

University Medical School, Department of 

Microbiology-Immunology, Chicago, IL, Bettina Schreiner, 

Postdoctoral Fellow, Northwestern University Medical 

School, Department of Microbiology-Immunology, Chicago, IL 

Chronic progression of relapsing experimental autoimmune 

encephalomyelitis (R-EAE) is dependent on the activation 

of T cells to released endogenous myelin epitopes, i.e., 

epitope spreading. Using transfer of naive CFSE-labeled TCR 

transgenic T cells, we have shown that activation of naive 

PLP139–151-specific T cells in SJL mice undergoing PLP178- 

191-induced R-EAE occurs directly in the CNS. Flow cytometric 

and histologic examination of the R-EAE CNS revealed 

the infiltration of significant numbers of CD11c+ dendritic 

cells (DCs) (including myeloid, lymphoid and plasmacytoid 

subsets) which are not seen in the healthy CNS. Functional 

examination of the antigen presentation capacity of CNS APC 

populations shows that only F4/80-CD11c+CD45hi DCs, not 

infiltrating macrophages or resident microglia, efficiently 

present endogenous antigen resulting in the activation of 

naive PLP139–151-specific Tg T cells. Bone marrow chimeras 

indicate that the vast majority of CNS DCs are of peripheral 

origin. Among the DC subsets, CD11b+ CD11c+ myeloid DCs 

(mDCs) cluster with CNS-resident PLP139–151-specific Tg T 

cells and are by far the most efficient endogenous activators 

of naumlautve T cells both in vivo and in vitro. In contrast, 

lymphoid and plasmacytoid DCs are less effective. mDCs 

uniquely biased Th-17 and not Th1 differentiation, correlating 

with their enhanced expression of TGF- 2 1 and IL-6 and IL- 

23. These findings indicate that the inflamed CNS can 

function as a neo-lymphoid organ and inhibition of DC 

migration may be a viable target for treatment of MS 

(supported by NIH Grant NS-030871, NMSS Grant RG-3793-A- 

7 and a grant from the Myelin Repair Foundation). 

doi:10.1016/j.clim.2007.03.352 

OR.35 IL-23 and IL-17 in Pathogenesis of 

Experimental Ocular Autoimmunity: Requirement 

for IL-23 May Extend Beyond Its Role in Sustaining 

the IL-17 Effector Response 

Dror Luger, Postdoctoral Fellow, National Institutes of 

Health, NEI Laboratory, Phyllis Silver, Biologist, National 

Institutes of Health, NEI Laboratory, Daniel Cua, Senior 

Principal Scientist, Schering Plough Biopharma, DNAX, Palo 

Alto, CA, Zoe Chen, Scientist, Schering Plough Biopharma, 

Palo Alto, CA, Yoichiro Iwakura, Professor, University of 

Tokyo, Tokyo, Japan, Edward Bowman, Principal Scientist, 

Schering-Plough Biopharma, DNAX, Palo Alto, CA, Chi-Chao 

Chan, Senior Scientist, National Institutes of Health, NEI, 

Laboratory Immunology, Bethesda, MD, Rachel Caspi, Senior 

Investigator, National Institutes of Health, NEI, Laboratory 

Immunology, Bethesda, MD 

Experimental autoimmune uveitis (EAU) represents autoimmune 

uveitis in humans. Here we examined the role of the 

IL-23/IL-17 pathway in EAU. We immunized IL-23p19, IL- 

12p35, IL-12p40, IFN-g and IL-17 KO mice with the 

uveitogenic Ag IRBP. Alternatively, we treated wild type 

mice immunized for EAU with Abs to IL-23p19 or to IL-17 

throughout the disease course (prevention), or only during 

the effector phase (reversal). IL-23p19 KO were resistant to 

EAU, showing reduced Ag-specific DTH, IL-2, IFN-g, IL-17, IL- 

1b, IL-6 and IL-5. In contrast, IL-12p35 and IFN-g KO mice 

developed exacerbated EAU, DTH and IL-17 responses. 

Treatment with anti-IL-23p19 prevented EAU and reduced 

immunological responses, but was unable to reverse an 

ongoing disease process, indicating an inductive role for IL- 

23. In contrast, anti-IL-17 prevented as well as reversed EAU, 

indicating a role for IL-17 in effector mechanisms. Interestingly, 

severe EAU could be induced with a Th1 cell line that 

does not produce IL-17. EAU in recipients of the line could be 

inhibited with anti-IFN-g or anti-TNF-a, but not with anti-IL- 

17, speaking against a need for host-derived IL-17 and 

suggesting that under some conditions an antigen-specific 

Th1 effector is sufficient to induce EAU. In keeping with this, 

IL-17 KO mice were still susceptible to disease. These data 

raise the possibility that the nonredundant need for IL-23 in 

EAU may extend beyond its role in promoting the Th17 

effector response and point to IL-23 and IL-17 as new 

therapeutic targets for uveitis. 

doi:10.1016/j.clim.2007.03.353 

S61 

OR.36 The Role of IL-17 and IFN-γ in Defining 

Pathogenic Potential of T Cells in EAE 

Ingunn Stromnes, Graduate Student, University of 

Washington, Department of Immunology, Seattle, WA, Joan 

Goverman, Professor, University of Washington, 

Department of Immunology, Seattle, WA, Denny Liggitt, 

Chair, University of Washington, Department of 

Comparative Medicine, Seattle, WA 

Multiple sclerosis (MS) is a demyelinating disease of the 

central nervous system (CNS) in which inflammatory infiltrates 

localize in the white matter of the brain and spinal 

cord. The mechanisms that determine specific sites of 

inflammation are not understood. We developed a novel 

model of experimental allergic encephalomyelitis, an animal 

model for MS, in which T cells specific for one epitope of a 

myelin oligodendrocyte glycoprotein (MOG) preferentially 

induce inflammation in the brain, while T cells specific for 

two different MOG epitopes preferentially induce inflammation 

in the spinal cord, resulting in profoundly distinct 

clinical signs. T cells responding to all three epitopes 

generate both IL-17- and IFN-γ-producing non-overlapping 

populations, and both the IL-17- and IFN-γ-producing T cells 

induce disease upon adoptive transfer. We found that the 

ability to induce inflammation preferentially in the brain or 

spinal cord is not determined by the absolute number of 

either IL-17- or IFN-γ-producing T cells, but rather by the 

ratio of the two populations. Changing the IL-17 to IFN-γ 

ratio for Tcells of each specificity by culturing them in either 

IL-12 or IL-23 prior to adoptive transfer altered the

S62 Abstracts 

inflammation sites in the CNS, which resulted in different 

clinical signs in recipient mice. These data show that the 

encephalitogenic potential of Tcells is not defined solely by a 

production of a single cytokine, but by the relative frequency 

of Tcells producing either IFN-γ or IL-17. These findings have 

important implications for cytokine-targeted therapies of 

CNS autoimmune disease. 

doi:10.1016/j.clim.2007.03.354 

OR.37 The Role of IL-22, a TH17 Cytokine, in 

Autoimmune Diseases and Bacteria Infection 

Yan Zheng, Postdoctoral Research Fellow, Department of 

Immunology, Genentech Inc, South San Francisco, CA, 

Dimitry Danilenko, Pathologist, Department of Pathology, 

Genentech Inc, South San Francisco, CA, Patricia Valdez, 

Senior Research Associate, Department of Immunology, 

Genentech Inc, South San Francisco, CA, Jeffrey 

Eastham-Anderson, Department of Pathology, Genentech 

Inc, South San Francisco, CA, Ian Kasman, Department of 

Pathology, Genentech Inc, South San Francisco, CA, Jianfeng 

Wu, Research Associate, Department of Immunology, 

Genentech Inc, South San Francisco, CA, Wenjun Ouyang, 

Scientist, Department of Immunology, Genentech Inc, South 

San Francisco, CA 

Recently, interleukin (IL)-23, a cytokine involved in the 

development of IL-17-producing T helper cells (TH17 cells), 

was found to have a potential function in the pathogenesis of 

autoimmune diseases and the host defense against infectious 

agents. We identified that IL-22 is preferentially produced by 

TH17 cells. However, the production of IL-22 and IL-17 from 

TH17 cells is differentially regulated. Transforming growth 

factor-beta (TGF-β), although crucial for IL-17 production, 

actually inhibits IL-22 production. Furthermore, IL-22 mediates 

IL-23-induced acanthosis and dermal inflammation 

through the activation of Stat3 (signal transduction and 

activators of transcription 3) in vivo. Our preliminary data 

also showed a protective role of IL-22 in host defense against 

bacteria infection. Our results suggest that TH17 cells, 

through the production of both IL-22 and IL-17, might have 

essential functions both in host defense and in the pathogenesis 

of autoimmune diseases such as psoriasis. IL-22, as an 

effector cytokine produced by Tcells, mediates the crosstalk 

between the immune system and the tissues. 

doi:10.1016/j.clim.2007.03.355 

OR.38 Endogenous IL-6 is Required for Inducing and 

Sustaining an Antigen-specific IL-17 Memory 

Response in Experimental Autoimmune Uveitis 

(EAU), but Not for Innate IL-17 Production 

Aleksandra V. Rachitskaya, Medical Student, Laboratory of 

Immunology, National Eye Institute, NIH; HHMI-NIH 

Research Scholars Program, Bethesda, MD, Anna M. Hansen, 

Postdoctoraltoral Fellow, Laboratory of Immunology, 

National Eye Institute, National Institutes of Health (NIH), 

Bethesda, MD, Rajeev K. Agarwal, Staff Scientist, 

Laboratory of Immunology, National Eye Institute, National 

Institutes of Health (NIH), Bethesda, MD, Dror Luger, 

Postdoctoraltoral Fellow, Laboratory of Immunology, 

National Eye Institute, National Institutes of Health (NIH), 

Bethesda, MD, Phyllis B. Silver, Biologist, Laboratory of 

Immunology, National Eye Institute, National Institutes of 

Health (NIH), Bethesda, MD, Chi-Chao Chan, Senior 

Investigator, Laboratory of Immunology, National Eye 

Institute, National Institutes of Health (NIH), Bethesda, MD, 

Rachel R. Caspi, Senior Investigator, Laboratory of 

Immunology, National Eye Institute, National Institutes of 

Health (NIH), Bethesda, MD 

We examined the role of IL-6 in production of the 

proinflammatory cytokine IL-17 under conditions of a primary 

immune response, as represented by T cell receptor ligation 

in vitro, and under conditions of antigen-specific recall 

response in the model of experimental autoimmune uveitis 

(EAU) in vivo. To mimic a primary response, naive splenocytes 

from IL-6 deficient mice (IL-6 KO) or from their C57BL/6 wildtype 

counterparts (WT) were stimulated with anti-CD3 and IL- 

23. EAU was induced in IL-6 KO and WT mice by immunization 

with the retinal antigen IRBP in complete Freund’s adjuvant 

(CFA). EAU development was evaluated by fundus examination 

and confirmed by eye histology. Eyes and lymphoid organs 

were harvested on day 21. Responses were examined by IL-17 

ELISA, intracellular cytokine staining combined with immunophenotyping, 

DTH and lymphocyte proliferation. IRBPimmunized 

IL-6 KO mice were completely resistant to EAU and 

had significantly reduced DTH and proliferative responses to 

IRBP as compared to their WT counterparts. Notably, their 

IRBP-specific IL-17 production was conspicuously decreased 

as compared to WT. In contrast, during an in vitro 

primary response, IL-17 production, observed at approximately 

24 hours after stimulation, was the same in both 

genotypes and required IL-23, but not IL-6. Our results 

indicate that IL-6 plays an essential role in inducing and 

sustaining the IL-17 memory and recall responses. However, 

an initial innate IL-17 production occurs early during 

a primary response and is IL-6 independent. By immunophenotyping, 

the cellular source of this early IL-17 

response appears to be NK/NKT cells. 

doi:10.1016/j.clim.2007.03.356 

OR.39 Evidence for a Role of the Interleukin-23 

Pathway in the Pathogenesis of Psoriasis 

Frank Nestle, Professor, Kings College London, London, 

England, Francesca Capon, Doctor, Department of Genetics, 

King’s College London School of Medicine, London, England, 

Jonathan Barker, Professor, St. Johns Institute of 

Dermatology, London, England, Robert Kastelein, Staff, 

Schering-Plough Biopharma, Palo Alto, CA, Richard 

Trembath, Professor, Division of Genetics and Molecular 

Medicine, London, England, Giulia Tonel, PhD Student, 

Department of Dermatology (F14), University of Zurich, 

Zurich, Switzerland, Paola Di Meglio, Research Associate, 

Cutaneous Medicine and Immunotherapy Unit, St. John’s 

Institute of Dermatology, London, England, Elizabeth 

Oldham, Staff, Schering-Plough Biopharma, Palo Alto, CA, 

Jerry Lanchbury, Staff, Myriad Genetics, Salt Lake City, UT

Abstracts 

To identify genes relevant to disease pathogenesis we 

performed a genome-wide association study in psoriasis, one 

of the most common chronic inflammatory disorders. We 

detected a highly significant association between psoriasis 

and genetic markers in the interleukin-23 receptor (IL-23R) 

gene on chromosome 1p31, a finding replicated in an independent 

dataset. The most significantly associated polymorphism 

(combined p value=1.6 ×10 −5 ) results in an amino 

acid substitution (Arg381Gln) located in the IL-23R cytoplasmic 

domain. The same variant has recently been implicated 

in the pathogenesis of inflammatory bowel disease, supporting 

a critical role of IL-23 signaling in epithelial inflammation. 

As a necessary step to determine the importance of this 

pathway in the pathogenesis of psoriasis, we undertook functional 

investigations using patient samples and a clinically 

relevant model system. We found an increased expression of 

IL-23R on psoriatic helper T cells compared to normal controls. 

Functional dissection of the IL-23 pathway using immunosuppressed 

AGR mice grafted with psoriatic skin 

revealed a key role for the IL-23 pathways in the disease 

process. Administration of a neutralizing anti-human IL-23 

antibody inhibited psoriasis development comparable to the 

use of anti-TNF-α blockers. These data provide genetic and 

functional evidence for a crucial role of the IL-23 pathway 

in cutaneous inflammation and lay the foundation for new 

treatment strategies in psoriasis and potentially other chronic 

epithelial inflammatory disorders. 

doi:10.1016/j.clim.2007.03.357 

OR.40 The Fully Human Antibody CNTO1275 

Effectively Inhibits Human IL-12 and IL-23 Mediated 

Th1 and Th17 Cell Function and Provides Clinical 

Benefit to Patients with Plaque Psoriasis 

Jacqueline Benson, Assistant Director, Centocor Research 

and Development, Discovery Research, Radnor, PA, 

Yevgeniya Orlovsky, Senior Associate Scientist, Centocor 

Research and Development, Radnor, PA, Kim Staquet, 

Manager, Centocor Research and Development, Radnor, 

PA, Jinquan Luo, Principal Research Scientist, Centocor 

Research and Development, Radnor, PA, Patrick Branigan, 

Research Scientist, Centocor Research and Development, 

Radnor, PA, Roberta Lamb, Senior Associate Scientist, 

Centocor Research and Development, Radnor, PA, Jian Li, 

Research Scientist, Centocor Research and Development, 

Radnor, PA, Renold Capocasale, Research Scientist, 

Centocor, Radnor, PA, Janine Huber, Senior Associate 

Scientist, Therakos, Exton, PA, Bernie Scallon, Sr 

Research Fellow, Centocor, Radnor, PA, David Peritt, 

Director, Therakos, Exton, PA, David Shealy, Senior 

Research Fellow, Centocor Research and Development, 

Radnor, PA, George Heavner, Distinguished Research 

Fellow, Centocor Research and Development, Radnor, PA, 

Jill Giles-Komar, Director, Centocor Research and 

Development, Radnor, PA, Tom Nesspor, Senior Associate 

Scientist, Centocor, Radnor, PA 

Interleukins (IL)-12 and IL-23 are potent contributors to 

CD4+ T cell differentiation to Th1 and Th17 cell types which 

are associated with several immune-mediated human dis- 

eases, including psoriasis, Crohn’s disease, rheumatoid 

arthritis, multiple sclerosis, and others. Unique TLR agonist 

combinations will elicit IL-12 versus IL-23 production from 

antigen presenting cells and these cytokines demonstrate 

distinct effects on human and mouse T cell activation. 

CNTO1275 is a fully human monoclonal antibody that 

potently inhibits the bioactivity of human IL-12 and IL-23 

by binding to the shared p40 subunit and preventing 

interaction with the IL-12Rb1 receptor on the surface on 

NK and CD4+ T cells. Thus, CNTO1275 inhibits IL-12 and IL-23 

mediated activation and cytokine production of receptorbearing 

cells. CNTO1275 binds human and primate, but not 

rodent, IL-12 and IL-23 and suppressed disease in a primate 

model of multiple sclerosis and in a human skin transplant 

mouse model of psoriasis. CNTO1275 has also provided a 

significant level of efficacy in the majority of subjects in 

phase I and II clinical studies of plaque psoriasis. These data 

support a central role for IL-12/23p40 in psoriasis pathogenesis. 

CNTO1275 was generally well-tolerated and adverse 

events observed in these studies did not indicate dose– 

response safety relationships. Similarly, studies in cynomolgus 

monkeys have shown that even high doses of CNTO1275 

are generally well-tolerated. Collectively, these results 

suggest that CNTO1275 may represent an exciting first-inclass 

therapy with significant therapeutic potential for 

immune-mediated diseases such as psoriasis. 

doi:10.1016/j.clim.2007.03.358 

Therapy- From Gene Silencing to Costimulation 


2:45 pm–4:45 pm 

S63 

OR.41 Effects of Long-Term Fingolimod Therapy on 

Circulating Mononuclear Cell Populations in Multiple 

Sclerosis 

Amit Bar-Or, Associate Professor, Montreal Neurological 

Institute, McGill University, Montreal, QC, Canada, Nathalie 

Arbour, Associate Researcher, University of Montreal, 

Faculty of Medicine, Montreal, QC, Canada, Ellie McCrae, 

Technician, Montreal Neurological Institute, Montreal, QC, 

Canada, Jack Antel, Professor, Montreal Neurological 

Institute, Montreal, QC, Canada, Tarik Touil, Postdoctoral 

Fellow, Montreal Neurological Institute, Montreal, QC, 

Canada, Karen Newell, Visiting Professor, Montreal 

Neurological Institute, Montreal, QC, Canada 

Fingolimod (FTY720) is an orally administered sphingosine-1-phosphate 

(S1P) receptor modulator. In animal and 

short-term human studies it down-regulates lymphocyte (but 

not myeloid cell) receptor expression. Fingolimod reduced 

clinical and MRI activity in relapsing MS patients. Objectives: 

To examine the profile of circulating mononuclear cells 

(MNC) in MS patients (n=6) who continue daily use of 1.25 mg 

of oral fingolimod for N18 months (open label extension 

phase of original phase II trial). This represents the longest 

experience using fingolimod as a single agent in humans. 

Methods: Whole peripheral blood samples obtained from the 

patient cohort were immunostained for leukocyte markers

S64 Abstracts 

and analyzed by flow cytometry. Results: Absolute lymphocyte 

number (mean 0.48+0.06 ×10 3 per microliter) was 

reduced with treatment (normal range 0.91–4.28×103); 

while CD14+ monocyte counts (0.47 +0.07×103) were within 

normal range (0.12–0.92×103). CD4+ T cells were relatively 

more depleted (2.3+0.8% of total MNCs) (control 28.0+6.4%, 

n=6) than CD8+ T cells (10.4 +2.5%) (control 22.0+4.2%), 

resulting in reversed CD4:CD8 ratio (0.2). CD19+ B cells were 

also markedly reduced (1.1+0.2% of total MNCs) (control 

11.1+2.1%). The proportion of monocytes (29.4+4.4%) and 

CD56+ NK cells (32.2+9.3%) among MNC was increased in 

treated patients compared to controls (7.0+1.8%) and (14.5 + 

4.3%) respectively — although their absolute number did not. 

Conclusions: MS patients receiving fingolimod monotherapy 

show sustained decreases in circulating B cells and CD4 T 

cells, relative less depletion of CD8 T cells, and maintained 

numbers of innate immune cells. Correlations with clinical 

measures will help define the basis for the efficacy, and 

safety profile of this type of immuno-modulation. 

doi:10.1016/j.clim.2007.03.359 

OR.42 In Vivo Dendritic Cell-specific Gene Silencing 

Using a Novel and Selective siRNA Delivery Method 

to Induce Immune Modulation 

Costin Vladau, Graduate Student, University of Western 

Ontario, Department of Microbiology and Immunology, 

London, ON, Canada, Dong Chen, Postdoctoral Fellow, 

University of Western Ontario, Department of Microbiology 

and Immunology, London, ON, Canada, Motohiko Suzuki, 

Postdoctoral Fellow, University of Western Ontario, 

Department of Microbiology and Immunology, London, 

England, Xusheng Zhang, Postdoctoral Fellow, University of 

Western Ontario, Department of Microbiology and 

Immunology, London, ON, Canada, Xiufen Zheng, 

Postdoctoral Fellow, London Health Sciences Centre, 

University Hospital, Department of Surgery, London, ON, 

Canada, Wei-Ping Min, Principle Investigator, University of 

Western Ontario, Department of Microbiology and 

Immunology, London, ON, Canada 

Silencing immune molecules, such as the costimulatory 

molecule CD40, using siRNA was shown to have therapeutic 

potential and promise in immune modulation. However, a 

major barrier to the clinical application of siRNA is the current 

lack of an effective and cell-specific delivery system. Herein, 

we present a new method of selectively delivering siRNA to DC 

in vivo using stealth immunoliposomes (SILs). CD40 siRNAcontaining 

SILs were generated using 4 types of lipids and 

decorated with surface-bound, DC-specific mAbs. DC-specific 

binding capacity of SILs was demonstrated by fluorescence 

microscopy and flow cytometry. Upon treatment with CD40 

siRNA-SILs, DC expression of CD40 was successfully suppressed 

in vitro. In vivo administration of CD40 siRNA-SILs resulted in 

DC-specific tissue targeting, as evidenced by increased uptake 

of fluorescence-tagged siRNA. Additionally, DC from mice 

treated with CD40 siRNA-SILs exhibited CD40-specific gene 

silencing in vivo. Tolerogenic properties of DC treated with 

CD40 siRNA-SILs were determined by inhibition of allogeneic T 

cell proliferation in MLR. Furthermore, a strong in vivo 

immune modulation was observed in mice treated with CD40 

siRNA-SILs. In conclusion, this is the first demonstration of DCspecific 

siRNA delivery and gene silencing in vivo, which 

highlights the potential of DC-mediated immune modulation 

and the feasibility of siRNA based clinical therapy. 

doi:10.1016/j.clim.2007.03.360 

OR.43 Immunotherapy of Solid Tumors Utilizing 

PLGA/Tumor Lysate Nanoparticles to Stimulate TIL 

and Circulating CD8+ Tumor-specific T Cells 

Douglas Hanlon, Associate Research Scientist, Yale 

Dermatology, New Haven, CT, Virginia Cody, Research 

Assistant, Yale Dermatology, New Haven, CT, Shashi Prasad, 

Postdoctoraltoral Fellow, Yale Dermatology, New Haven, CT, 

Mark Saltzman, Professor, Yale Biomedical Engineering, New 

Haven, CT, Camille Solbrig, Associate Research Scientist, 

Yale Biomedical Engineering, New Haven, CT 

Dendritic cell (DC)-based therapies for solid tumors have 

failed to achieve large-scale clinical utilization because at 

present no unified technology exists to overcome two fundamental 

obstacles. These are the lack of known tumor-associated 

antigens (TAA) from most solid malignancies and the lack of 

efficient methodologies to transfer antigen from tumor to DC. 

Here we describe the development of an alternative technology 

for antigen delivery that appears to transfer a broad spectrum of 

tumor antigens to DC with extremely high efficiency. We used 

this methodology, termed “nanoparticle (NP)-mediated tumor 

delivery,” to encapsulate whole melanoma protein lysates into 

PLGA biodegradable microspheres and targeted these conjugates 

to DC. Optimization of Ag delivery was achieved by 

experimental determination of which polymer formulation, Ag 

source and release characteristics led to most potent DC antigen 

presentation. In both murine and human T cell stimulation 

assays, DC loaded with NP/tumor conjugates were superior to 

other Ag forms in stimulating anti-tumor T cells — including 

murine gp100-specific naive T cells from pmel transgenics and 

human TIL and blood CD8+ T cells from melanoma patients. 

Analysis of cytokine production by DC and T cell by cytometric 

bead array (CBA) assays indicated that NP-loaded DC drove 

increased production of Th1 and proinflammatory cytokines 

such as IFN-g and IL-2. Although melanoma Ags were utilized as 

controls, a major potential advantage of NP-based antigen 

delivery is the fact that antigens need not be defined prior to DC 

loading, opening the door to vaccine targeting of virtually any 

solid tumor. 

doi:10.1016/j.clim.2007.03.361 

OR.44 Safety and In Vivo Efficacy of Innate (Class R) 

and Broadly Reactive (Class B) Inhibitory 

Oligonucleotides (INH-ODN) in MRL-Fas/lpr Lupus 

Mice 

Petar Lenert, Assistant Professor, Department of Internal 

Medicine, The University of Iowa, Iowa City, IA, Sarah 

Seyfer, BS, Internal Medicine, Iowa City, IA, Elizabeth R. 

Pareigis, BS, Internal Medicine, The University of Iowa, Iowa 

City, IA, Robert F. Ashman, Professor, Internal Medicine, The 

University of Iowa, Iowa City, IA

Abstracts 

Several lines of evidence indicate that intracytoplasmic 

TLR receptors 7 and 9 play an important role in the 

pathogenesis of SLE, both in vitro and in vivo. For example, 

MRL-Fas/lpr mice lacking TLR9 have a shorter life span and 

develop more aggressive kidney disease. On contrary, mice 

lacking TLR7 have a milder disease. INH-ODNs containing 

nuclease-resistant phosphorothioate backbone (PS) were 

recently developed that can block TLR7 and/or TLR9 

activation at low nanomolar concentrations. Here we show 

that under certain stimulatory conditions prototypic TLR9specific 

INH-ODNs can also block TLR7 activation of mouse 

macrophages induced by several (but not all) TLR7 ligands 

like imiquimod, CL075, CL097, CL087 and loxoribine. Interestingly, 

in primary B cells, certain TLR7/8 ligands, but not 

phosphodiester (PO) CpG-ODNs, could escape inhibition 

when INH-ODNs were made with the natural PO backbone. 

Such INH-ODNs could actually enhance B cell G1–M entry, 

survival and polyclonal IgM secretion induced by suboptimal 

TLR7/8 stimulation. Both TLR7/8 inhibition and enhancement 

were independent of the TLR9 expression. We have 

recently modified these INH-ODNs to allow formation of 

stable secondary structures. In vitro, these modified innate 

INH-ODNs (class R) showed higher potency for macrophages, 

dendritic cells and marginal zone B cells over follicular B 

cells. However, in vivo, both linear and Class R INH-ODNs 

promoted better survival and ameliorated spontaneous lupus 

disease in MRL-Fas/lpr mice. It remains to be determined 

whether this effect required TLR7/8 or TLR9. This work was 

supported by the NIH grant 1R56AI064736-01A2 and by a 

grant from the Carver Trust fund. 

doi:10.1016/j.clim.2007.03.362 

OR.45 Tumor Cell Lysate-specific DTH Reaction 

Correlates with Longer Survival and Disease 

Stability in Vaccinated Melanoma Patients 

Flavio Salazar-Onfray, PhD, Universidad de Chile, Santiago 

de Chile, Chile 

Dendritic cell (DCs)-based therapy has proven to be 

effective in patients with malignancies. However, an optimal 

immunization protocol using DCs and the best means for 

delivering antigens has not been yet described. In this study, 37 

patients with malignant melanoma in stage IV were vaccinated 

with autologous DCs pulsed with a melanoma cell lysate, to 

assess toxicity, immunological and clinical responses. The 

responses of the patients were analyzed by ELISPOT and 

Delayed type IV hypersensitivity reaction (DTH) against the 

melanoma cell lysate. After vaccination, 50% of tested 

patients (18 out of 37) showed a positive DTH against the 

melanoma cell lysate. All patients showed a positive DTH 

reaction against the used adjuvant KLH demonstrating that all 

patients were immunocompetents. Significant correlations 

were found between positive DTH responses against tumor 

lysate and both disease stability and post-vaccination survival 

on stage IV patients. In fact, the mean of post-vaccination 

survival was 15 months for all the vaccinated patients, double 

than the historical survival for stage IV melanoma patients. 

More important, positive DTH patients showed a postvaccination 

survival of 25 months compared to 8 months 

observed for the negative DTH group of patients. There 

were no toxicities associated with the vaccines or evidence 

of autoimmunity including vitiligo. Our data suggest that 

autologous DCs pulsed with tumour lysate may provide a 

standardized and widely applicable source of melanoma 

specific antigens for clinical use, it is safe and causes no 

significant side effects, demonstrating to be partially efficient 

at triggering effective anti-melanoma immunity. 

doi:10.1016/j.clim.2007.03.363 

OR.46 In Vivo CTLA4Ig Treatment Substantially 

Inhibits CD4+CD25+ Regulatory T Cell Turnover but 

Not Function 

Anita Tang, Surgical Resident, Division of Transplantation, 

Department of Surgery, University of Maryland School of 

Medicine, Baltimore, MD, Modesta Ndejembi, Graduate 

Student, Department of Microbiology and Immunology, 

University of Maryland School of Medicine, Baltimore, MD, 

Donna Farber, Associate Professor, Division of 

Transplantation, Department of Surgery, University of 

Maryland School of Medicine, Baltimore, MD 

CTLA4Ig and its variant, LEA29Y, target the CD28/B7 

costimulatory pathway and are highly effective treatments 

in rheumatoid arthritis and transplantation, respectively. 

Since CD28/B7 signaling has been implicated in regulatory T 

cell (Treg) generation, we investigated the effects of 

CTLA4Ig on Treg phenotype, proliferation and function. 

BALB/c mice were administered CTLA4Ig (250 μg/dose) 

every other day for 3 doses, with IgG2a and PBS as controls. 

Splenic CD4 T cells were purified 3–14 days later and 

analyzed for CD25, CTLA4 and Foxp3 expression. Following 

CTLA4Ig treatment, CD4+Foxp3+ Tregs decreased 60% and 

CTLA4 expression by 30% compared to controls, with the 

most significant depletion (80%) in the CTLA4+Foxp3+ subset. 

Additionally, CD25 expression and CD25+Foxp3+ Tregs 

dropped 50%. Similar findings were observed in thymectomized 

mice, suggesting that CTLA4Ig mediates its effects on 

peripheral Tregs. To assess the mechanistic effects of 

CTLA4Ig, CFSE-labeled CD4+CD25+ and CD4+ CD25− T cells 

were transferred into syngeneic mice receiving CTLA4Ig and 

harvested 6 days later. Interestingly, we noted extensive and 

rapid in vivo proliferation of CD4+CD25+ Tregs (62%) 

compared to the CD4+CD25− subset (10%) in controltreated 

mice, suggesting that Treg proliferation is an 

antigen-driven phenomenon. Moreover, we observed that 

this rapid turnover of Tregs was almost completely blocked 

by CTLA4Ig. Despite a decreased yield, expression of CTLA4 

and turnover after CTLA4Ig treatment, the remaining Tregs 

were functionally competent and suppressed CD4+CD25− T 

cell proliferation comparable to controls in co-culture 

assays. These findings may have important implications 

regarding the constitution of Treg subsets and their balance 

within non-regulatory populations in patients receiving 

CTLA4Ig and LEA29Y. 

doi:10.1016/j.clim.2007.03.364 

S65

S66 Abstracts 

OR.47 A Novel Anti-Tumor Vaccine Using IDO 

Silenced DC 

Xiufen Zheng, Postdoctoraltoral Fellow, University of 

Western Ontario, London, ON, Canada, Bertha Garcia, 

Professor, Departments of Surgery, Microbiology, Pathology, 

University of Western Ontario, London, ON, Canada, Costin 

Vladau, MS Student, Departments of Microbiology and 

Immunology, University of Western Ontario, London, ON, 

Canada, James Koropatnick, Professor, Departments of 

Microbiology and Immunology, Pathology, Oncology, 

University of Western Ontario, London, ON, Canada, Dong 

Chen, PDF, Departments of Surgery, University of Western 

Ontario, London, ON, Canada, Motohiko Suzuki1, PDF, 

Departments of Surgery, University of Western Ontario, 

London, ON, Canada, Mu Li, PDF, Departments of Surgery, 

University of Western Ontario, London, ON, Canada, 

Wei-ping Min, Professor, Departments of Surgery, 

Microbiology and Immunology, Pathology, Oncology, 

University of Western Ontario, London, ON, Canada, Xusheng 

Zhang, Postdoctoral Fellow, Department of Surgery, 

University of Western Ontario, London, Canada 

Hyporesponsiveness is a major hallmark in dendritic cell 

(DC)-mediated anticancer immunotherapy. Indoleanime 2,3dioxygenase 

(IDO), an immunosuppressive molecule 

expressed by DC, is a critical factor mediating hyporesponsiveness 

to cancer immune therapy. We hypothesized that 

antisense silencing of IDO in DCs would enhance anticancer 

therapy. In this study, DC were cultured in vitro, exposed to 

melanoma B16 lysate, transfected with IDO siRNA, and 

injected into tail veins of C57/BL6 mice. Mice were then 

challenged with B16 tumor cells. The anticancer effects of 

IDO-silenced DC therapy, as compared with non-silenced, 

conventional control DC vaccine, were evidenced by (i) 

postponed melanoma tumor onset time, and (ii) decreased 

tumor size. In addition, after immunization with IDOsilenced 

DC, the number of CD8+ T cells was significantly 

greater than in animals immunized with control RNA-treated 

DC and CD4+ and CD8+ T cell apoptosis in draining lymph 

nodes was significantly lower. Furthermore, immunization 

with IDO-silenced DC enhanced tumor antigen-specific T cell 

proliferation and CTL activity and decreased numbers of CD4 

+CD25+ Foxp3+ regulatory T cells (Treg). In conclusion, this 

study is the first to demonstrate a novel anti-tumor vaccine 

by silencing an immunosuppressive gene (IDO) in DC, which 

enhanced anti-tumor immunity, reduced T cell apoptosis and 

Treg cell formation, and prevented tumor growth. IDOsilenced 

DC has clinical potential as an immune-based therapy. 

doi:10.1016/j.clim.2007.03.365 

OR.48 Post-Transcriptional Regulation of T Cell 

Receptor zeta Chain in Systemic Lupus 

Erythematosus 

Vaishali R. Moulton, Postdoctoral Fellow, Beth Israel 

Deaconess Medical Center, Medicine, Boston, MA, Vasileios 

C. Kyttaris, Instructor, Beth Israel Deaconess Medical 

Center, Medicine, Boston, MA, Yuang-Taung Juang, 

Instructor, Beth Israel Deaconess Medical Center, Medicine, 

Boston, MA, George C. Tsokos, Professor, Beth Israel 

Deaconess Medical Center, Medicine, Boston, MA 

Systemic lupus erythematosus (SLE) is a complex autoimmune 

disease characterized by the production of autoantibodies, 

immune complex deposition, T cell infiltration 

and abnormal production of cytokines all leading to 

inflammatory damage of target organs (joints, kidneys 

and brain). SLE T cells are deficient in their expression of 

the T cell receptor (TCR)/CD3 zeta chain protein (CD3Z), a 

critical molecule for the initiation of intracellular signaling 

upon antigen encounter. This deficiency is partially due to 

differential splicing of the CD3Z mRNA; SLE T cells express 

an alternatively spliced form of CD3Z mRNA which is missing 

562 nucleotides (672 to 1233) within its 3′ untranslated 

region (UTR). Studies by our group have shown that this 

alternatively spliced CD3Z mRNA is unstable and has 

reduced translation efficiency. We have also demonstrated 

that two adenosine-uridine-rich elements (AREs) at positions 

705 and 985 in the 3′ UTR are independently essential 

for its stability and translation. We hypothesized that 

proteins binding to these two AREs may stabilize the CD3Z 

mRNA. Electrophoretic mobility shift assays showed several 

protein complexes in jurkat cell nuclear extracts binding to 

32P-labeled oligonucleotides containing the ARE (705) 

sequence, which were outcompeted in the presence of 

similar unlabeled oligonucleotides. Using an oligonucleotide 

pulldown assay, mass spectrophotometry and peptide 

sequencing, we identified HuR as one of the putative 

proteins binding to the ARE (705) oligonucleotide. Immunoblotting 

with specific antibody confirmed this finding. 

These results reveal molecular mechanisms that may 

contribute to CD3Z mRNA stability and altered TCRZ chain 

expression in lupus. 

doi:10.1016/j.clim.2007.03.366 

Biomarkers & New Technologies 


2:45 pm–4:45 pm 

OR.49 Autoimmune Development in Phenotypic 

Type 2 Diabetes Patients 

Barbara Brooks-Worrell, Research Scientist, University of 

Washington/VA Puget Sound Health Care System, Seattle, 

WA, Heba Ismail, Research Fellow, VA Puget Sound Health 

Care System, Seattle, WA, Michael Wotring, Research 

Scientist, University of Washington, Seattle, WA, A.U. 

Liemin, Data Processor, University of Washington/VA Puget 

Sound Health Care System, Seattle, WA, Crystal Kimmie, 

Research Scientist, VA Puget Sound Health Care System, 

Seattle, WA, Jamie Felton, Research Associate, VA Puget 

Sound Health Care System, Seattle, WA, Jerry Palmer, 

Professor of Medicine, University of Washington/VA Puget 

Sound Health Care System, Seattle, WA 

Type 1 (T1DM) diabetes is an autoimmune disease, 

whereas type 2 (T2DM) diabetes is considered nonautoimmune. 

However, cross-sectional studies have identified 

autoimmune diabetes in 10–15% of phenotypic T2DM 

patients. In this study, we evaluated phenotypic autoimmune 

negative T2DM patients prospectively to determine 

the importance of repeated testing to detect autoimmune

Abstracts 

diabetes. In 40 phenotypic T2DM patients we determined 

autoantibody (IA-2/IAA/GAD), T cell status (cellular immunoblotting), 

cytokine responses (ELISPOT), fasting C-peptide, 

and glucagon-stimulated C-peptide responses at 

baseline and every 3 months for an average follow-up of 

23.3 months (range 6–36 months). Of the 40 patients, 14/ 

40 (35%) were positive for autoantibody (Ab) and/or T cell 

(T) reactivity at baseline while 26/40 (65%) were Ab-T−. Of 

the 26 patients Ab-T− at baseline, 8/26 (30.7%) remained 

Ab-T− stable (persistently negative), 4/26 patients demonstrated 

transient Ab or T cell positivity (1 positive time 

point during follow-up), while 14/26 developed stable 

(persistent positivity after conversion) Ab+ and/or T+. We 

have categorized the patients with stable negative or 

transient Ab and/or T reactivity as non-autoimmune nonprogressors 

(NANP) and the patients developing stable Ab 

and/or T cell positivity as progressors. We observed that 

the mean fasting and glucagon-stimulated C-peptide 

responses were significantly (pb0.0001) lower, the INF-g 

responses to sonicated islets and GAD were significantly 

higher (pb0.05), and the IL-10 responses (pb0.05) to islets 

were significantly lower in the progressors compared to 

NANP. These results demonstrate that continued follow-up 

of T2DM patients is needed to identify development of 

autoimmunity in patients previously classified as nonautoimmune. 

doi:10.1016/j.clim.2007.03.367 

OR.50 Derangements in Cytokine-induced STAT 

Protein Phosphorylation in Human Systemic Lupus 

Erythematosus 

Veronika Sharp, Fellow, Stanford University Medical School, 

Palo Alto, CA, Paul J. Utz, Stanford University Medical 

School, Palo Alto, CA, G.P. Nolan, The Baxter Laboratory for 

Genetic Pharmacology, CA, OD Perez, The Baxter Laboratory 

for Genetic Pharmacology, CA 

Systemic lupus erythematosus (SLE) is an autoimmune 

disease with variable clinical manifestations, many of which 

are devastating to patients. Currently it is difficult to 

predict disease onset, severity, or response to therapy. New 

and better markers are needed in clinical practice for 

improved predictability. Phospho-specific flow cytometry is 

a powerful technique that allows for the analysis of several 

intracellular signaling networks in multiple cellular subsets 

simultaneously in response to functionally relevant stimuli. 

Using this technique we interrogated signaling through 

different members of the Jak-STAT and MAPK pathways in 

response to cytokines (including IFN α, IFN γ, IL-4, IL-6 and 

IL-10) in peripheral blood mononuclear cells (PBMC) 

isolated from 14 patients with SLE and 11 healthy controls. 

CD3, CD4, CD8 and CD33 were used as surface markers to 

differentiate T cells subsets, B cells and monocytes. While 

PBMC from healthy and diseased individuals had similar 

phosphorylation profiles in the resting state, there were 

remarkable differences in cellular response once cells were 

stimulated. Among other differential responses, hyperphosphorylation 

of some STAT proteins was observed in 

response to IFN α. This is consistent with prior studies 

implicating a role for type I interferons in SLE pathogenesis. 

Our data demonstrate the feasibility of analyzing several 

signaling networks simultaneously in viable PBMC subsets in 

response to an array of cytokines. These differences may 

not be visible when only whole and resting PBMC are used. 

This study takes us one step closer to defining individual 

biosignatures for SLE patients that would allow for more 

customized immunotherapy. 

doi:10.1016/j.clim.2007.03.368 

OR.51 Evidence for an Increase in the Functional 

Avidity of the GAD-reactive CD4+ T Cell Population 

Prior to Diabetes Onset 

Nathan Standifer, Postdoctoral Research Associate, 

Benaroya Research Institute, Seattle, WA, Gerald Nepom, 

Director, Benaroya Research Institute, Seattle, WA 

Type 1 diabetes (T1D) is an autoimmune disease characterized 

by the cell-mediated destruction of insulin-producing 

β cells of the pancreatic islets. Immunologically, islet 

destruction is marked by increased auto-antibody titers and 

the expansion of glutamic acid decarboxylase (GAD)-reactive 

CD4+ T cells in many pre-diabetic subjects. We hypothesized 

that the functional avidity of the GAD-reactive T cell 

population increases over time in pre-diabetic subjects as 

the islet β cell mass decreases. To test this, we cultured 

longitudinal peripheral blood lymphocyte samples drawn 

from auto-antibody+, at-risk, pre-diabetic or diabetic subjects 

and calculated the GAD-reactive CD4+ Tcell population 

ED50 values: the concentration of peptide that induces onehalf 

maximal interferon-γ secretion. Longitudinal samples 

from an at-risk subject exhibited the highest Tcell population 

ED50 values (4.26 1/4M and 8.76 1/4M) while the ED50 values 

of samples from a T1D patient drawn 4 or 5 years after 

diagnosis were substantially lower (2.24 nM and 48.30 nM 

respectively). Interestingly, the ED50 value of the GADreactive 

T cell population cultured from a pre-diabetic 

subject 6 years prior to diagnosis was significantly higher 

than that of a sample drawn 2 years prior to diagnosis (5.04 

¼M and 15.38 nM respectively). We also observed that the 

frequency of GAD-reactive, central-memory cells increased 

over time in the pre-diabetic samples. Our data imply that, as 

the islet β cell mass decreases, the GAD-reactive T cell 

population undergoes a phenotypic shift from effector to 

central memory cells concomitant with an increase in the 

functional avidity as measured by interferon-γ secretion. 

doi:10.1016/j.clim.2007.03.369 

S67 

OR.52 Peripheral Blood Flow Cytometric Evaluation 

of a Large Cohort of Hyper Immunoglobulin E 

Syndrome Patients 

Nina N. Brodsky, Researcher, NIH/NIAID/LCID, Bethesda, 

MD, Margaret R. Brown, Biologist, NIH/CC/DLM, Bethesda, 

MD, Steven M. Holland, Chief, LCID, NIH/NIAID/LCID, 

Bethesda, MD, Gulbu Uzel, Staff Clinician, NIH/NIAID/LCID, 

Bethesda, MD

S68 Abstracts 

Job’s Syndrome, or the Hyper-IgE Syndrome (HIES), is an 

autosomal dominant disorder characterized by elevated 

serum IgE levels, eosinophilia, dermatitis, skeletal abnormalities, 

staphylococcal skin infections, and lung cysts. An in 

vitro immunologic phenotype has not been established, and 

earlier findings have included only a small number of HIES 

patients. We have performed flow cytometric analysis of 

lymphocyte subsets and eosinophils by using relevant cell 

surface markers in a cohort of thirty HIES patients and 

compared them to healthy controls using a non-parametric 

t test (Mann–Whitney U). Absolute lymphocyte counts did 

not differ between the two groups (pN0.3). We have found 

that patients’ eosinophils have significantly higher expression 

of CD23, HLA-DR, and CD69 compared to controls 

(p=0.0002, 0.0001 and b0.0001, respectively). We have 

also found that percentages of natural killer cells 

(pb0.0001) and CD3+ CD54+ T cells (p b0.0001) were 

lower in HIES. We have detected significantly smaller 

number of CD8 and CD4 memory T cells (CD8+CD45RO+ and 

CD4+CD45RO+) (pb0.0001 and pb0.04 respectively), more 

naive CD4 T cells (CD4+ CD45RA+) (p=0.001) and CD8+CD57+ 

cells (p=0.0007) in patients. HIES patients had significantly 

more T cells expressing CD147 (a plasma membrane glycoprotein 

shown to induce extracellular matrix metalloproteinases 

[MMPs], pb0.04). Our findings of decreased number of 

memory T cells in HIES, similar to Hyper IgM syndrome and 

ectodermal dysplasia with immunodeficiency (EDID), suggest a 

potential defect in the NFkB pathway. On the other hand, 

increased CD147 expression points at pathways responsible for 

regulation of MMPs, which may be critical for tissue 

remodeling and regeneration defects in HIES. 

doi:10.1016/j.clim.2007.03.370 

OR.53 Global Assessment of B Cell Alloimmunity 

Using Human Protein Microarrays 

Persis P. Wadia, Postdoctoral Fellow, Stanford University, 

Miklos Lab, Stanford, CA, Marc Coram, Stanford University, 

Stanford, CA, Marina Sirota, Stanford University, Stanford, 

CA, David B. Miklos, Assistant Professor, Stanford 

University, Stanford, CA, Atul Butte, Stanford University, 

Stanford, CA 

Allogeneic hematopoietic cell transplantation (HCT) can 

cure hematologic malignancies through beneficial graft-vleukemia 

(GVL) allo-immune responses, but is limited by 

graft-versus-host disease (GVHD). Previous studies demonstrate 

that allogeneic antibodies (Ab) develop against minor 

histocompatibility antigens (mHA) encoded on the Y-chromosome 

(H-Y antigens) after sex-mismatch HCT in association 

with chronic GVHD and persistent disease remission. We 

hypothesize that novel mHA can be serologically identified as 

targets of allo-Ab responses that develop post-transplant. 

ProtoArray displays 5000 full-length human proteins with Nterminal 

GSTepitopes. Technical replicates of plasma measured 

at 1:50, 1:150, 1:500 confirmed technical feasibility with R 2 

N0.95. In this study, plasma collected from four AML patients 

1 year post-transplant, pre-transplant, and their donors were 

detected for antibody specificity against 5000 human proteins 

displayed on the ProtoArray. Pre-post fluorescence differences 

ranging from 0.5 to 3 logs identified 60–75 targets of allo-Ab. 

Over 90% of allo-Ab targets have known non-synonymous 

SNPs. Chromatin Assembly Factor 1, subunit B (p60) 

(CHAF1B), and Nucleolar and Spindle Associated Protein 1 

(NuSAP1) were recognized by two of the four patients after 

HCT. These allo-Abs were absent pre and 2 months post-HCT, 

developed in association with cGVHD at 11 months, and 

persisted through 16 months. CHAF1B and NuSAP have been 

recombinantly expressed and specifically detected by post- 

HCT patient sera by western blot validating their discovery as 

allo-antigens. Ongoing studies are characterizing CHAF1B and 

NuSAP1 Ab by ELISA in HCT patient samples, and normals. In 

summary, protein microarrays are innovative tools for highthroughput 

global assessment of B cell alloimmunity and mHA 

discovery. 

doi:10.1016/j.clim.2007.03.371 

OR.54 Improving Targeted Immunotherapy Through 

Direct Validation of Peptide–MHC Epitopes Using 

TCRm Antibodies 

Tiffany Nguyen, Senior Research Technician, Receptor 

Logic, Ltd, Amarillo, TX, Tito Woodburn, Research 

Associate, Receptor Logic, Ltd, Amarillo, TX, Francisca 

Neethling, Scientist, Receptor Logic, Ltd, Amarillo, TX, 

Jon A. Weidanz, Assistant Professor, Texas Tech 

University Health Sciences Center, Amarillo, TX 

Adoptive T cell immunotherapy represents a promising 

approach for the treatment of cancer and chronic infections. 

Improved response rates using this approach might occur if 

the specific peptide–MHC targets on the diseased cells were 

known. We have developed technology to directly assess the 

presentation of specific peptide–MHC complexes on tumor 

cells using antibodies that mimic the binding specificity of T 

cell receptors designated as TCRmimics (TCRms). A proof-ofconcept 

validation assay was developed to directly examine 

the presentation and density of MHC class I peptides derived 

from the processing of a model tumor antigen, hCGb, on the 

surface of various human tumor cell lines. In this study we 

used previously characterized TCRm to three peptide–HLA- 

A*0201 epitopes derived from hCGb and designated as TMT 

(60–68), VLQ (64–72), and GVL (67–75). For the assessment 

of antigen presentation function, hCGb positive tumor cell 

lines were stained for detection and quantitation of each 

peptide–HLA epitope. To our surprise, presentation of each 

peptide–epitope varied widely with no clear dominant 

peptide–epitope being expressed on the different tumor 

cell lines. A human breast carcinoma cell line expressed all 

three peptide–HLA-A2 epitopes with GVL peptide being 

dominant. In contrast, only GVL peptide–epitope was 

detected on an ovarian carcinoma cell line while only TMT 

peptide–epitope was found expressed on a colon carcinoma 

cell line. Taken together, these findings illustrate a heterogeneous 

pattern of peptide–HLA expression and signify the 

need for development of reagents that can directly assess 

specific peptide–HLA expression and lead to better outcomes 

with T cell immunotherapy. 

doi:10.1016/j.clim.2007.03.372

Abstracts 

OR.55 Transcription Profiles of Rheumatoid Arthritis 

Patients Reveal Genes Characterizing Different 

Response to Anti-TNF Therapy 

Franak Batliwalla, Assistant Investigator, Feinstein Institute 

for Medical Research, Manhasset, NY, Peter Gregersen, 

Investigator, Feinstein Institute for Medical Research, 

Manhasset, NY, Normand Allaire, Scientist, BiogenIdec, Drug 

Discovery, Cambridge, NY, Wentian Li, Assistant Investigator, 

Feinstein Institute for Medical Research, Manhasset, NY, 

Houman Khalili, Steve Perrin, Associate Director, BiogenIdec, 

Drug Discovery, Cambridge, MA, Marlena Kern, Research 

Associate, Feinstein Institute for Medical Research, 

Manhasset, NY, Aarti Damle, Research Associate, Feinstein 

Institute for Medical Research, Manhasset, NY, John Carulli, 

Principal Scientist, BiogenIdec, Drug Discovery, Cambridge, 

MA, Jadwiga Bienkowska, Principal Scientist, Biogenidec, 

Drug Discovery, Cambridge, MA 

The Autoimmune Biomarkers Collaborative Network 

(ABCoN) has enrolled a longitudinal cohort of RA patients 

beginning anti-TNF treatment in order to identify biomarkers 

influencing response to anti-TNF therapy. 116 patients 

beginning anti-TNF therapy (54 etaneracept, 25 adalimumab, 

37 infliximab) were followed for 14 weeks, with DAS28 

measurements and RNA collection at three time points: pretreatment, 

6 weeks and 14 weeks post-treatment. Using the 

hgu133plus2 Affymetrix chips we have completed genomewide 

transcript profiling for these 116 patients as well as 65 

healthy controls. Defining response as a DDAS28 N40% and 

non-response as DDAS28 b20%, analysis of the gene expression 

differences between patients and controls indicates that the 

overall number of differentially expressed genes is different 

for responders and non-responders. Furthermore, the responders 

are characterized by a unique list of genes differentially 

expressed as compared to controls at 14 weeks posttreatment. 

This observation suggests that anti-TNF therapy 

recruits specific biological pathways in responders. In order 

to identify the biomarkers that predict the response to anti- 

TNF therapy we have analyzed the gene expression profiles of 

responders and non-responders using blood collected at the 

pre-treatment visit. Using the machine learning technique 

Random Forest we have identified a set of over 100 genes that 

are predictive of the anti-TNF response. Using a selected set 

of 25 candidate biomarkers we can distinguish the responders 

versus non-responders with 75% accuracy. We are validating 

the proposed biomarkers using an independent cohort of 

patients as well as low-density RT-PCR arrays. 

doi:10.1016/j.clim.2007.03.373 

New Animal Models of Disease 


2:45 pm−4:45 pm 

OR.56 Development of Latest-generation 

HU-PBMC-NOD/SCID Mice to Study Human Islet 

Allo-reactivity 

Todd Pearson, Postdoctoral Fellow, University of Massachusetts 

Medical School, Diabetes Division, Worcester, MA, Marie King, 

MD/PhD Student, University of Massachusetts Medical School, 

Diabetes Division, Worcester, MA, Leonard Shultz, Senior Staff 

Scientist, The Jackson Laboratory, Bar Harbor, ME, Jean Leif, 

Lab Manager, University of Massachusetts Medical School, 

Diabetes Division, Worcester, MA, Dale Greiner, Professor, 

University of Massachusetts Medical School, Diabetes Division, 

Worcester, MA, John Mordes, Professor, University of 

Massachusetts Medical School, Diabetes Division, Worcester, 

MA, Aldo Rossini, Professor, University of Massachusetts 

Medical School, Diabetes Division, Worcester, MA, Mark 

Atkinson, Professor, University of Florida College of Medicine, 

Department of Pathology, Immunology and Laboratory 

Medicine, Gainesville, FL, Clive Wasserfall, Assistant in 

Pathology, University of Florida College of Medicine, 

Department of Pathology, Immunology and Laboratory 

Medicine, Gainesville, FL, Massimo Trucco, Professor, 

University of Pittsburgh School of Medicine, Pediatrics, 

Pittsburgh, PA, Kevan Herold, Professor, Yale University School 

of Medicine, Department of Internal Medicine, New Haven, CT, 

Rita Botti, Assistant Professor, University of Pittsburgh, 

Pediatrics, Pittsburgh, PA 

Small animal models have been used to study a number of 

human diseases, including autoimmune diseases such as type 

1 diabetes (T1D). Unfortunately, translating therapies from 

animal models to human patients has been hindered by 

differences in rodent and human immune systems. “Humanized” 

mouse models hold great promise in the development 

of efficacious therapies to treat a wide array of human 

immune-mediated conditions, without putting human subjects 

at risk during protocol development. However, developing 

a system that faithfully recapitulates human immunity 

in a murine host has proven difficult. Recently, the generation 

of a new stock of immunodeficient hosts, the NOD.Cg- 

Prkdcscid Il2rgtm1Wjl/Sz (NOD-scid Il2rgnull) strain, has 

overcome many of the previous limitations of humanized 

mice. We have characterized the engraftment of human 

PBMC into NOD-scid Il2rgnull hosts and document that this 

stock supports higher human cell engraftment at lower cell 

input levels. Importantly, we further demonstrate that 

human PBMC-engrafted NOD-scid Il2rgnull mice allow for 

allogeneic rejection of transplanted HLA-mismatched human 

islets, even when the islets are allowed to heal-in prior to 

PBMC engraftment. Collectively, these data suggest that 

humanized NOD-scid Il2rgnull mice may be superior to 

previous immunodeficient recipients for generation of 

humanized mice for studies of in vivo human immune 

function. 

doi:10.1016/j.clim.2007.03.374 

S69 

OR.57 TSLP-dependent Induction of Airway 

Inflammatory Disease is Antigen-driven in an Acute 

Model of Allergic Airway Inflammation 

Mark Headley, Graduate Student, University of Washington 

Immunology, Seattle, WA, Baohua Zhou, Post Doctoral 

Fellow, Benaroya Research Institute, Ziegler Lab, Seattle, 

WA, Steve Ziegler, Director of Immunology, Benaroya 

Research Institute, Ziegler Lab, Seattle, WA

S70 Abstracts 

The cytokine, Thymic Stromal Lymphopoietin (TSLP), has 

been shown to play an important role in the mediation of Th2biased 

inflammatory diseases in particular the atopic diseases: 

asthma and atopic dermatitis. Mice expressing a TSLP transgene 

driven by the lung-specific Surfactant Protein C promoter (SPC- 

TSLP) develop a severe and chronic asthma-like disease 

characterized by airway hyperresponsiveness, leukocytic infiltration 

of the lung, eosinophilia, goblet cell hyperplasia, subepithelial 

fibrosis, and elevated serum IgE. Previous studies have 

indicated that TSLP alone is sufficient to induce all of the 

hallmark features of asthma in mice. Here we show that an 

acute airway inflammatory disease, similar both to that seen in 

asthmatics as well as SPC-TSLP mice, can be induced in BALB/c 

mice through direct intranasal administration of TSLP in 

conjunction with a foreign antigen, sans adjuvant. This has 

been observed with multiple antigens, including ovalbumin, 

chicken gamma globulin, and bovine serum albumin. In contrast, 

mice treated with either TSLP alone, the antigens alone, or TSLP 

in conjunction with the self-protein, mouse serum albumin, fail 

to develop disease. These data demonstrate for the first time 

that in an acute model of TSLP-mediated airway inflammation, 

both thymic stromal lymphopoietin and antigen are required in 

order to develop full disease symptoms. 

doi:10.1016/j.clim.2007.03.375 

OR.58 Disrupting Mer Receptor Tyrosine Kinase 

Expression Prevents Autoimmune Chronic 

Graft-versus-host Disease 

Wenha Shao, Postdoctoral Research Associate, 

Rheumatology Division, Department of Medicine, 

Philadelphia, PA, Robert A. Eisenberg, Professor of 

Medicine, Rheumatology Division, Department of Medicine, 

University of Pennsylvania, Philadelphia, PA, Philip L. 

Cohen, Professor of Medicine, Rheumatology Division, 

Department of Medicine, University of Pennsylvania, 


The Mer receptor tyrosine kinase mediates apoptotic cell 

phagocytosis and modulates macrophage cytokine production. 

Mer knockout mice (Mer-KO) have defective clearance of 

apoptotic debris and develop SLE-like autoimmune disease. 

Because of their spontaneous autoimmune propensity, we 

wondered if Mer-KO mice (backcrossed 10 generations onto 

C57BL/6) might be particularly susceptible to the SLE-like 

autoimmunity induced by chronic GVH. Using the wellestablished 

cGVH model (bm12′B6 mice), we were surprised 

to observe that the Mer-KO mice were protected from the 

development of GVHD. Mer-KO recipients of bm12 spleen cells 

failed to develop detectable anti-dsDNA and anti-chromatin 

autoantibodies, while the B6 hosts produced significant 

amounts of these antibodies that peaked at week 2. Slightly 

increased levels of rheumatoid factor were found in Mer-KO 

mice, though much lower than B6 control hosts. Mer-KO mice 

did not develop splenomegaly. The lack of autoantibody 

formation was not due to absence of alloreactivity because 

spleen cells from bm12 mice proliferated equally in response 

to irradiated WT and Mer-KO irradiated spleen cells. These 

findings indicate a hitherto unrecognized requirement for Mer 

in the genesis of alloreactivity-induced autoimmunity. Future 

experiments will determine whether this reflects the role of 

this kinase in recognition and phagocytosis of apoptotic cells, 

its function in regulating cytokine production, or perhaps a 

new function in the immune response. 

doi:10.1016/j.clim.2007.03.376 

OR.59 In Vivo and In Silico Modeling the Dynamics of 

Autoimmune Diabetes 

Qizhi Tang, Assistant Adjunct Professor, University of 


CA, Yanan Zheng, Engineer, Biological Systems, Entelos, In 

Silico Research and Development, Foster City, CA, Jason 

Adams, MD, Stanford University, Stanford, CA, Huub 

Kreuwel, Scientist, Immunologic Diseases, Entelos, In Silico 

Research and Development, Foster City, CA, Kapil Gadkar, 

Biosystems Engineer, Entelos, In Silico Research and 

Development, Foster City, CA, Cristina Penaranda, 

Graduate Student, University of California, San Francisco 

Diabetes Center, San Francisco, CA, Lisl Shoda, Senior 

Scientist, Entelos, In Silico Research and Development, 

Foster City, CA, Chan C. Whiting, Scientist, Immunologic 

Diseases, Entelos, In Silico Research and Development, 

Foster City, CA, Daniel Young, Dynamics Engineer, Entelos, 

In Silico Research and Development, Foster City, CA, Jeffrey 

Bluestone, Professor, University of California, San Francisco 

Diabetes Center, San Francisco, CA 

Progression of autoimmune diabetes results as a consequence 

of an imbalance of Teff and suppressive regulatory T 

cells (Tregs). A novel mathematical model of type 1 diabetes 

(T1D) in the non-obese diabetes (NOD) mouse (T1D Physio- 

Lab® platform) was developed that encompasses the 

dynamics of key immune components in the pancreatic 

lymph nodes (PLNs) and islets, and beta cell physiology. 

Research using this platform predicted that autoimmunity 

was controlled in the PLN, as shown by an increase in Tregs 

over the course of disease. This unexpected behavior was 

confirmed experimentally by functional and histological 

data. Additionally, laboratory analysis of individual islets 

demonstrated an inverse relationship between Teff and Treg 

infiltration of the tissue. Specifically, as Teff infiltration 

increases, the Treg fraction decreases. However, differential 

proliferation of Teffs and Tregs in vivo was found to be 

insufficient to explain this relationship although evidence of 

reduced expression of CD25 on the resident pancreatic Tregs 

could alter Treg survival accounting for the observed effects. 

Based on these in vivo results, the relative turnover and 

recruitment of Tregs and Teffs were tested in the T1D 

platform. Results of these in silico investigations demonstrated 

that the ultimate determination of disease activity 

depends on dynamic changes in intra-islet Treg turnover and 

changes in the Teff/Treg balance. These combined experimental 

and mathematical modeling results emphasize the 

central importance of both PLN and intra-islet Treg function 

in regulating disease pathogenesis and the power of 

integrating in vivo and in silico modeling to dissect mechanisms 

leading to disease pathogenesis. 

doi:10.1016/j.clim.2007.03.377

Abstracts 

OR.60 Antigen Identification in a Novel Model of 

Spontaneous Autoimmune Ovarian Disease 

Mickie Cheng, Clinical Fellow in Endocrinology, University of 


CA, Kellsey Johannes, Staff Research Associate, University 

of California, San Francisco Diabetes Center, San Francisco, 

CA, Wen Lu, Staff Research Associate, University of 


CA, Mark Anderson, Assistant Professor of Medicine in 

Residence, University of California, San Francisco Diabetes 

Center, San Francisco, CA 

Autoimmune ovarian disease (AOD) is a poorly understood 

clinical entity wherein immune-mediated destruction of 

oocytes leads to premature ovarian failure and infertility. 

We have generated a novel spontaneous mouse model to 

study AOD using mice deficient for the AutoImmune 

REgulator (aire), a gene first identified in the human 

Autoimmune Polyglandular Syndrome (APS) type 1. Like 

patients with APS 1, aire-deficient mice develop disease of 

multiple organs, including the ovary, due to a breakdown in 

central tolerance. In aire KO mice, thymic epithelial cells are 

defective in their ability to tolerize developing Tcells to selfantigens, 

thus allowing autoreactive cells to escape into the 

periphery and induce self tissue destruction. Autoimmunity 

is manifested by the development of antigen-specific 

autoantibodies, organ infiltration, and transfer of disease 

by lymphocytes from affected aire KO mice. aire KO females 

exhibit 100% incidence of histologic oophoritis as well as 

circulating autoantibodies to ovarian tissue in a strain 

specific manner, presenting a valuable spontaneous disease 

model to identify ovarian antigens. Initial characterization of 

AOD in these mice implicates a central role for T cell 

mediated disease with predominantly Th1 CD4+ T cell 

infiltrate. Furthermore, autoantibodies from aire KO females 

target the oocyte cytoplasm and follicular layer and 

recognize a 70 kDa autoantigen in ovarian lysates. Identification 

of the disease-inciting antigen could enable improved 

testing for the diagnosis of autoimmune ovarian disease and 

may provide a potential therapeutic target for treatment 

and prevention of a significant cause of human ovarian 

failure and infertility. 

doi:10.1016/j.clim.2007.03.378 

OR.61 Delineating the Roles of CD4+ and CD8+ T Cell 

Subsets in the Pathogenesis of Spontaneous 

Autoimmune Diabetes Using HLA-DQ8 Transgenic 

Mice 

Govindarajan Rajagopalan, Department of Immunology, 

Mayo Clinic, Rochester, MN, Ashutosh Mangalam, 

Department of Immunology, Mayo Clinic, Rochester, MN, 

Yogish Kudva, Department of Endocrinology, Mayo Clinic, 

Rochester, MN, Chella David, Department of Immunology, 

Mayo Clinic, Rochester, MN 

Certain MHC class II molecules show a strong association 

with T1D implying an important role for CD4+ T cells. MHC 

class I-restricted CD8+ T cells also play a crucial effector 

role. The roles of CD4+ and CD8+ T cell subsets in the 

pathogenesis of spontaneous autoimmune diabetes were 

studied using HLA-DQ8 transgenic mouse models. CD8sufficient 

or CD8-deficient Abo.DQ8 transgenic mice expressing 

either the proinflammatory cytokine, tumor necrosis 

factor (TNF)-α or the costimulatory molecule, B7.1 or both 

in the pancreatic beta cells were used in this study. In the 

RIP-TNF model, the incidence of diabetes was class II or 

CD4+ T cell independent as 75% of mice lacking HLA-DQ8 

(and there by CD4+ T cells) developed T1D. However, it was 

absolutely class I or CD8+ T cell dependent as none of the 

CD8-deficient mice developed T1D. In the RIP-B7 model, the 

incidence of diabetes was class II or CD4+ T cell dependent, 

as none of the mice lacking HLA-DQ8 (and there by CD4+ T 

cells) developed T1D when compared to 35% incidence 

when HLA-DQ8 (and there by CD4+ T cells) is expressed. 

Nonetheless, in RIP-B7 model, CD8-deficient HLA-DQ8 

transgenic mice were still susceptible to T1D albeit at a 

lower rate (Incidence ¡V 17%). In the RIP-B7.RIP-TNF double 

transgenic mouse model, 100% of the animals developed 

diabetes irrespective of whether they lacked CD4+ or CD8+ 

T cells. Thus, CD4+ and CD8+ T cells contribute differentially 

to the pathogenesis of T1D according to the local immunological 

insult. 

doi:10.1016/j.clim.2007.03.379 

S71 

OR.62 The CYP450 Mouse Model for Autoimmune 

Hepatitis: Breaking of Self-Tolerance in Transgenic 

CYP2D6 and Wildtype FVB-Mice by Viral Infection 

Urs Christen, Assistant Professor, Pharmazentrum, JWG 

University Frankfurt, Frankfurt am Main, Germany, 

Martin Holdener, PhD Student, Pharmazentrum, JWG 

University Frankfurt, Frankfurt am Main, Germany, 

Edith Hintermann, Senior Postdoctoral Fellow, 

Pharmazentrum, JWG University Frankfurt, Frankfurt 

am Main, Germany, Matthias von Herrath, Professor, La 

Jolla Institute for Allergy and Immunology, La Jolla, 

CA, Michael Manns, Professor, Hannover Medical School, 

Hannover, Germany 

The etiology of autoimmune hepatitis (AIH) is poorly 

understood although the major autoantigen, cytochrome 

P450 2D6 (CYP2D6), has been identified. We generated an 

animal model for human AIH using the natural autoantigen 

CYP2D6. We infected transgenic mice expressing human 

CYP2D6 in the liver with an Adenovirus-CYP2D6 vector (Ad- 

2D6) to break self-tolerance. Upon infection with Ad-2D6 

not only transgenic CYP2D6 mice but also wildtype FVB 

mice showed persistent features of severe liver damage 

associated with AIH (hepatic fibrosis, fused liver lobules, 

cellular infiltrations, elevated serum ALT levels, confluent 

necrosis). Ad-2D6-infected mice (CYP2D6 and FVB) generated 

high titers of anti-CYP2D6 antibodies. Epitope mapping 

revealed that anti-CYP2D6 antibodies predominantly 

recognized the same immunodominant linear epitope 

recognized by sera of AIH patients (AQPPRD aa 265–270). 

In contrast, mice infected with a control Adenovirus 

expressing green fluorescence protein did neither develop

S72 Abstracts 

liver damage nor generated anti-CYP2D6 antibodies. The 

severity of liver damage as well as antibody formation was 

enhanced in FVB mice compared to transgenic CYP2D6 

mice indicating a stronger tolerance to human CYP2D6 in 

CYP2D6 mice. In FVB mice, due to the homology of the 

mouse isoenzymes of the CYP superfamily to human 

CYP2D6, autoimmune liver damage by Ad-2D6 infection 

was possibly induced via true molecular mimicry. 

doi:10.1016/j.clim.2007.03.380 

Immunoregulatory Mechanisms 


2:45 pm−4:45 pm 

OR.63 Modification of Host Cellular DNA by 

Parvovirus B19 Nonstructural Protein 1 (NS1): 

Implications for Induction of Autoimmunity by DNA 

Viruses 

Leona Gilbert, University of Jyvaskala, University of 

Jyvaskala, Jyvaskala, Finland, Brian D. Poole, Oklahoma 

Medical Research Foundation, Oklahoma Medical Research 

Foundation, Oklahoma City, OK, Violetta Kivovich, 

University of Jyvaskala, University of Jyvaskala, Jyvaskala, 

Finland, Stanley Naides, Medical Director, Immunology, 

Quest Diagnostics Nichols Institute, San Juan Capistra, CA, 

Jing Zhou, Pennsylvania State University College of 

Medicine/Milton S. Hershey Medical Center, Pennsylvania 

State University College of Medicine/Milton S. Hershey 

Medical Center, Hershey, PA 

Autoantibodies cross-reactive to EBV proteins precede 

SLE onset in a subset of patients. However, the event that 

links viral infection to autoimmunity, the “original sin,” 

remains unknown. We studied the role of parvovirus B19 

infection in host DNA modification. B19 infection in nonerythroid 

cells (e.g., Hep G2 and primary hepatocytes) is 

restricted to expression of NS1. NS1 is a superfamily 3 (SF3) 

helicase, typical of small DNA viruses, that plays a key role 

in viral genome replication. In hepatocytes, B19 induces 

apoptosis through mitochondrial stress pathways. We 

examined NS1 expression in Hep G2 cells using an enhanced 

green fluorescent fusion protein (eGFP/NS1). Expressed 

eGFP/NS1 localized to the nucleus. The majority of cells 

expressing eGFP/NS1 underwent apoptosis with PARP 

activation. Inhibitors of PARP or ATR abrogated apoptosis. 

eGFP/NS1 colocalized with RPA and PCNA. Western blot 

analysis and autoradiographs demonstrated that NS1 

formed dimers under reducing conditions and covalently 

bound to cellular DNA. NS1 was polyADP ribosylated. 

Apoptotic subcellular particles from cells expressing 

eGFP/NS1 fluoresced. Our data demonstrated that B19 

NS1 forms bulky adducts with cellular DNA and nicks 

cellular DNA, initiating pathways that lead to mitochondrial 

stress and apoptosis. NS1 with covalently linked self-DNA is 

extruded into the extracellular milieu in apoptotic bodies. 

We propose that modification of cellular DNA by viral SF3 

helicase breaks tolerance to self DNA when presented to 

the immune system during apoptosis. These findings suggest 

a general model in which modification of host DNA by viral 

SF3 helicase induces or accelerates autoimmunity, the 

“original sin.” 

doi:10.1016/j.clim.2007.03.381 

OR.64 Long-term Deposition of Inhaled Antigen in 

Lung Resident CD11b−CD11c+ Cells 

Kate E. Matthews, Student, Malaghan Institute, Wellington 

South, New Zealand, Adela Karabeg, Student, Medical 

University of Vienna, Department Dermatology, Vienna, 

Austria, Gerhard Dekan, Professor, Medical University of 

Vienna, Clinical Pathology, Vienna, Austria, Franca 

Roncheses, Principal Investigator, Malaghan Institute, 

Wellington South, New Zealand, Michelle Epstein, 

Laboratory Leader, Medical University of Vienna, 

Department of Dermatology, Vienna, Austria 

We report the characterization of a population of lung 

resident CD11b−CD11c+ cells that are able to take up inhaled 

antigen and retain it for extended periods of time. 

Ovalbumin conjugated to fluorescein isothiocyanate administered 

intranasally to mice was taken up by two main 

populations of cells in the lung, a migratory CD11c+CD11b+ 

population consisting of DC, which rapidly transported 

antigen to the draining lymph node (LN), and a resident 

CD11b−CD11c+ population that retained engulfed antigen 

without apparently degrading it for up to 8 weeks after 

administration. The FITC+CD11b−CD11c+ cells did not 

migrate to draining LN and did not upregulate expression of 

costimulatory molecules in response to LPS treatment. 

FITC+CD11b−CD11c+ cells found in the airway were large 

and were consistent with macrophages. Additionally, a 

population of FITC+CD11b−CD11c+ seen in lung sections 

were situated along alveolar walls with a distribution also 

consistent with macrophages. Although FITC+CD11b− 

CD11c+ cells expressed molecules associated with APC 

function, they did not induce proliferation of antigenspecific 

CD4+ T cells in vitro or acute cytokine production 

by activated CD4+ Tcells in vivo. We propose that these longlived, 

lung resident cells might provide a depot of antigen for 

the indirect reactivation or retention of antigen-specific T 

cells in the lung. 

doi:10.1016/j.clim.2007.03.382 

OR.65 Proteinase-activated Receptor-2 (PAR-2) 

Activation Promotes Allergic Sensitization and 

Prevents the Development of Immune Tolerance to 

Inhaled Antigens Through a TNF Mediated Pathway 

Cory Ebeling, PhD candidate, University of Alberta, 

Department of Medicine, Edmonton, AB, Canada, Tong Lam, 

Medical Student, University of Alberta, Edmonton, AB, 

Canada, John Gordon, Associate Professor, University of 

Saskatchewan, Department of Veterinary Microbiology, 

Saskatoon, SK, Canada, Harissios Vliagoftis, Associate 

Professor, University of Alberta, Department of Medicine, 

Edmonton, AB, Canada, Morley Hollenberg, Associate

Abstracts 

Professor, University of Calgary, Department of 

Pharmacology and Therapeutics, Calgary, AB, Canada 

The reason why particular inhaled antigens induce 

allergic sensitization and prevent the development of 

immune tolerance is unclear. Intrinsic characteristics of 

these antigens must be of importance. A common 

characteristic of many potent allergens is that they either 

possess serine proteinase activity or are inhaled in particles 

rich in serine proteinases. Many allergens, such as house 

dust mite, have the potential to activate the Proteinaseactivated 

Receptor-2 (PAR-2). We hypothesized that PAR-2 

activation by such allergens is crucial to facilitate our 

allergic sensitization to them. To test our hypothesis we 

used a Balb/c murine system with mucosal exposure to 

OVA, as an antigen, with a PAR-2 activating peptide (PAR- 

2AP) to mimic the potential of a proteolytic allergen, or 

other inhaled proteinases, to activate PAR-2. Upon allergen 

re-exposure mice initially administered OVA with the PAR- 

2AP developed airway inflammation, airway hyperresponsiveness 

(AHR), produced OVA-specific IgE as well as OVAspecific 

T cells with a Th2 cytokine profile. Conversely, 

mice given OVA alone or with a control peptide (PAR-2CP) 

developed tolerance. These immune tolerant mice did not 

develop airway inflammation, AHR, or OVA-specific IgE, but 

developed OVA-specific T cells that secreted high levels of 

IL-10 indicative of Treg cell function. Finally, we showed 

that this PAR-2-mediated allergic sensitization was TNF 

dependent. Thus, PAR-2 activation in the airways could be 

a critical factor in the development of allergic sensitization 

following mucosal exposure to allergens with serine 

proteinase activity. Interfering with this pathway may 

prove to be useful for the prevention or treatment of 

asthma. 

doi:10.1016/j.clim.2007.03.383 

OR.66 Downregulation of Innate B Cells, B-1a and 

MZB, by iNKT Cells 

Xiangshu Wen, Postdoctoral Fellow, University of California, 

Los Angeles, Medicine, Los Angeles, CA, Jun-Qi Yang, 

Assistant Research Professor, UC, Medicine, Cincinnati, OH, 

Ram Raj Singh, Professor, University of California, Los 

Angeles, Medicine, Los Angeles, CA 

B cells are essential for the development of autoimmune 

diseases such as lupus. Although all mature B cell subsets 

can produce autoantibodies, several lines of evidence 

implicate innate B cells, namely B-1a and marginal zone B 

(MZB) cells, in the development of such diseases. Here, we 

investigated the role of interaction between innate B cells 

that express high levels of CD1d and CD1d-reactive iNKT 

cells in the regulation of autoantibodies. We found that 

whereas activated iNKT cells increase activation markers 

CD69 and CD86 on all B cell subsets, they selectively reduce 

the frequency of B-1a and MZB cells. In resonance with such 

regulatory role, the proportions of MZB cells are increased 

in iNKT cell deficient (Ja18−/−) mice, whereas MZB cells 

are reduced in iNKT cell transgenic (Va14Tg) mice. 

Furthermore, whereas iNKT cells increase expression of 

activation markers and production of normal Ig via 

cytokines secreted by iNKT cells in transwell cultures, 

they selectively suppress the production of IgG anti-DNA 

antibody and rheumatoid factor and IL-10 by B cells in a 

contact-dependent manner. Such regulation of B cells by 

iNKT cells must be important for in vivo regulation of 

autoantibodies and lupus disease, as the in vivo transfer of 

iNKT cells in B cell-reconstituted SCID mice or in Ja18−/− 

mice suppresses autoantibody production. Treatment with 

an iNKT cell ligand also delays the onset of lupus in BWF1 

mice. Thus, iNKT cells suppress autoreactive B cells and can 

prevent systemic autoimmunity. They do so via regulating 

innate B cells. 

doi:10.1016/j.clim.2007.03.384 

OR.67 CD40 Isoform Differences and Microdomain 

Localization Explain Preferential Survival of 

CD4+CD40+ T Cells in Autoimmunity 

Gisela M. Vaitaitis, Professional Research Assistant, 

University of Colorado Health Sciences Center, Webb- 

Waring, Denver, CO, David H. Wagner, Assistant Professor, 

University of Colorado Health Sciences Center, Webb- 

Waring, Denver, CO 

CD40 plays a critical role in autoimmunity. CD40expressing 

CD4 T cells (TCD40) are expanded in autoimmunity 

and are necessary and sufficient in transferring disease 

in mouse type I diabetes. CD40 on TCD40 can have 

costimulatory effect and induces Nf-kB and recombinase 

protein expression with subsequent alteration of TCR. CD40 

exists as five isoforms in mice with little known-about 

differences in isoform expression between cell types, 

between autoimmune and non-autoimmune conditions and 

differences in outcome of CD40 isoform signaling. Here we 

show that the major isoform expressed in TCD40 is isoform- 

V with isoform-I expressed at lower levels. This differs 

distinctly from antigen presenting cells which predominantly 

express isoform-I. In autoimmune NOD the ratio of 

TCD40 isoform-V to -I is exaggerated and overall expression 

is greater compared to non-autoimmune BALB/c. It was 

shown in lymphocyte cell lines that CD40 and TRAF2 are not 

associated with detergent-insoluble microdomains (rafts) 

until CD40 is engaged. However, untreated NOD TCD40 have 

the CD40 isoforms and TRAF2 associated with this fraction 

while BALB/c TCD40 do not. A functional outcome of CD40 

signals to NOD TCD40 is increased survival through induction 

of survival proteins Bcl-XL and cFLIPp43. In addition, CD40 

signals to NOD TCD40 alter the outcome of Fas engagement 

such that Fas instead of inducing apoptosis induces 

increased survival beyond that of CD40 alone. Therefore 

TCD40 from NOD may be poised for survival even when 

encountering death promoting conditions explaining how 

this population gains the upper hand in autoimmune 

conditions to thwart T cell homeostasis. 

doi:10.1016/j.clim.2007.03.385 

S73

S74 Abstracts 

OR.68 An Immunoregulatory Network of Natural 

Killer T Cells and CD4+CD25+Foxp3+ T Cells 

Protects Against Graft-versus-host Disease 

Asha Pillai, Postdoctoraltoral Research Fellow, Stanford, 

Stanford, CA, Suparna Dutt, Research Associate, 

Department of Medicine, Stanford, CA, Tracy George, 

Assistant Professor, Department of Pathology, Stanford, CA, 

Samuel Strober, Professor of Medicine, Stanford University, 

Department of Medicine, Stanford, CA 

To investigate acute graft-versus-host disease (GVHD) 

protection in the murine regimen of total lymphoid irradiation 

and anti-thymocyte serum (TLI/ATS), we performed transplants 

of 50×10 6 bone marrow cells and 60×10 6 splenocytes (BMT) 

from wild-type (WT) C57BL/6 donors into WT BALB/c hosts 

treated with TLI/ATS or control BALB/c hosts given 800 cGy total 

body irradiation (TBI) and ATS. TLI/ATS-conditioned WT hosts 

given BMT from WT donors all survived 100 days without GVHD. 

BMT from WT donors into TBI/ATS treated WT hosts resulted in 

lethal GVHD. TLI/ATS-treated natural killer (NK) Tcell deficient 

Jα18−/− hosts also died of GVHD. TLI/ATS conditioned WT hosts 

receiving WT BMT demonstrated a significant (pb0.01) increase 

in the absolute number of donor CD4+CD25+Foxp3+ cells (CD4+ 

Tregs) in the spleen at day 6 after BMT relative to TBI/ATS 

controls or TLI/ATS-treated Jα18−/− hosts. All BMT groups 

developing GVHD demonstrated dramatic donor CD8+ T cell 

accumulation in the mesenteric lymph nodes (MLN) and colon at 

day 6 after BMT. Adoptive transfer of sorted BALB/c NK T cells 

protected TLI/ATS-treated Jα18−/− hosts from donor CD8+ T 

cell accumulation, with a significant (pb0.001) increase in the 

absolute number of donor CD4+ Tregs in the host spleen at day 6. 

Depletion of CD25+ T cells before BMT into TLI/ATS-treated WT 

hosts resulted in loss of donor CD4+ Tregs in the host spleen and 

lethal GVHD. The present studies show that both host NK Tcells 

and donor CD4+CD25+Foxp3+ Tregs are required to protect 

against GVHD after TLI/ATS conditioning and allogeneic BMT. 

doi:10.1016/j.clim.2007.03.386 

OR.69 Peroxisome Poliferator-activated Receptor-α 

(PPARα) Expression in T Cells Mediates Gender 

Differences in Development of T Cell-mediated 

Autoimmunity 

Shannon Dunn, Postdoctoral Fellow, Stanford University, 

Department of Neurology, Stanford, CA, Shalina Ousman, 

Postdoctoral Fellow, Stanford University, Department of 

Neurology, Stanford, CA, Raymond Sobel, Professor, 

Department of Pathology, Stanford, CA, Sergio Baranzini, 

Research Associate, University of California, San Francisco, 

Department of Neurology, San Francisco, CA, Luis Zuniga, 

Student, Stanford University, Stanford, CA, Sawsan Youssef, 

Postdoctoral Fellow, Stanford University, Department of 

Neurology, Stanford, CA, Jorge Oksenberg, Associate 

Professor, University of California, San Francisco, 

Department of Neurology, San Francisco, CA, Lawrence 

Steinman, Professor, Departmental Chair in Immunology, 

Stanford University, Department of Neurology, 

Stanford, CA 

PPAR is a nuclear receptor that mediates gender 

differences in lipid metabolism. PPAR also functions to 

control inflammatory responses by repressing the activity of 

NFkappaB and c-jun in immune cells. Since PPAR is situated 

at the crossroads of gender and immune regulation, we 

hypothesized that this gene may mediate sex differences in 

the development of T cell-mediated autoimmune disease. 

We show that PPAR is more abundant in male as compared 

to female CD4 + cells and is sensitive to androgen levels. 

Genetic ablation of this gene selectively removed the brake 

on NFkappaB and c-jun activity in male T lymphocytes 

resulting in higher production of IFN-γ and TNF (but not IL- 

17), and lower production of Th2 cytokines. Upon induction 

of experimental autoimmune encephalomyelitis (EAE), male 

but not PPAR −/− mice developed more severe clinical signs 

that were restricted to the acute phase of disease. To 

determine the extent to which changes in PPAR expression 

explain the androgen sensitivity of T helper responses, we 

contrasted the effects of castration on male WT versus 

PPAR −/− mice. This surgical intervention lowered PPAR 

expression in T cells, and enhanced the production of IFNγ, 

TNF, and IL-17 by WT male T cells in response to anti-CD3 

and anti-CD28 stimulation. Castration also enhanced the 

severity of acute EAE in male WT mice. These effects of 

castration were significantly blunted in PPAR −/− mice. These 

results suggest that males may be less prone to develop 

Th1-mediated autoimmunity because they have higher 

levels of PPAR. 

doi:10.1016/j.clim.2007.03.390

Abstracts 

Sa.1 Nitric Oxide Metabolites and Nitrotyrosine in 

Asthma and COPD 

Jenny Garmendia, Clinical Immunologist and Internal 

Medicine, Instituto de Inmunología, UCV, Caracas, 

Venezuela, Dolores Moreno, Pulmonologist, Servicio de 

Neumonología, Hospital Universitario de Caracas, Caracas, 

Venezuela, Nancy Larocca, Clinical Immunologist and 

Pediatrician, Instituto de Inmunología, UCV, Caracas, 

Venezuela, Juan De Sanctis, Biochemistry, Instituto de 

Inmunología, UCV, Caracas, Venezuela, Carlos Tálamo, 

Pulmonologist, Servicio de Neumonología, Hospital 

Universitario de Caracas, Caracas, Venezuela 

Asthma and chronic obstructive pulmonary disease (COPD) 

are two different inflammatory airway diseases. Serum nitric 

oxide (NO) metabolites and nitrotyrosine may be useful 

parameters of inflammation. We studied 150 Asthma patients 

(24 ± 4.5 years), 185 COPD patients (62 ± 9 years), and 300 

healthy subjects (45 ± 19 years). The controls were divided in 

twogroupsof150individualseach(meanage25±5and62±6 

years, respectively). Serum nitrites, nitrates (NO metabolites) 

and nitrotyrosine levels were determined by colorimetric, 

enzymatic assay and ELISA, respectively. No significant differences 

were recorded among the two groups of controls and thus 

we used the whole group for comparison. Nitrite levels were 

significantly reduced (pb 0.001) only en COPD patients (COPD 

4.1±1.9, Asthma 6.1 ± 1.3 vs. 6.5 ± 1.7 µM). Nitrates levels were 

significantly reduced in both groups of patients (COPD 16.2 ± 

4.1, Asthma 18.6 ± 3.2 vs controls 24.9 ± 3.5 µM, p b 0.001). 

Nevertheless, nitrotyrosine levels were significantly increased 

in the patient groups (controls: 4.2 ± 2.5; COPD: 11.3 ± 8.5 

pb0.001; Asthma: 6.7 ± 4.3 uM pb0.01). Nitrotyrosine levels 

were higher in COPD patients as compared to asthmatics (pb 

0.01). Moreover, we observed an inverse correlation between 

nitrates and nitrotyrosine only in patients (r = 0.62, n = 335). We 

conclude that metabolism of NO is modified in patients with 

asthma and COPD with a bypass of normal NO metabolites to a 

reactive species of nitrogen as peroxynitrites assessed partially 

by nitrotyrosine. FONACIT G2005000389. 

doi:10.1016/j.clim.2007.03.391 

Poster Session 


7:30 am−7:00 pm 


Sa.5 Effect of Omalizumab on Pulmonary Function 

and Asthma-related Outcome Measures in Patients 

Allergic to Cat Dander 

Marc Massanari, Associate Medical Director, Novartis 

Pharmaceuticals Corporation, East Hanover, NJ, Robert 

Maykut, Medical Director, Novartis Pharmaceuticals 

Corporation, East Hanover, NJ, Robert Zeldin, Executive 

Medical Director, Novartis Pharmaceuticals Corporation, 

East Hanover, NJ, Farid Kianifard, Statistician, Novartis 

Pharmaceuticals Corporation, East Hanover, NJ, Gregory 

Geba, Therapeutic Area Head, Novartis Pharmaceuticals 

Corporation, Verona, NJ 

Rationale: Exposure to cat dander, which if often 

unexpected, can worsen control in susceptible asthmatic 

patients. Add-on omalizumab (OMA) has been shown to help 

protect patients with perennial allergen sensitivity from 

exacerbations. We evaluated the effect of OMA on FEV1 and 

asthma-related outcome measures in patients with asthma 

and cat dander sensitivity. Methods: Data were pooled from 5 

randomized, double-blind, 28–32 week, placebo (PBO)controlled 

studies of patients with moderate–severe persistent 

IgE-mediated asthma and perennial allergen sensitivity 

inadequately controlled on inhaled corticosteroids (ICS). Cat 

sensitivity was determined by skin prick or RAST testing. The 

effect of add-on OMA on mean changes from baseline in FEV1, 

total rescue puffs of beta-agonist, and total asthma symptom 

score were analyzed using analysis of covariance; least 

squares means (LSM) and 95% confidence intervals (CI) were 

calculated. Results: 1589 patients (811 OMA, 778 PBO) were 

sensitive to cat dander. Mean age =39.7 years, 60% were 

female, mean IgE=219.1 IU/mL and FEV1 percent predicted 

was 60–79% in 46% and b60% in 26% of patients at baseline. 

End-of-study mean FEV1 increased by 100.3 mL (5.4%) and 

33.1 mL (2.5%) in OMA- and PBO-treated patients, respectively. 

LSM treatment difference was 63.11 (26.88, 99.35; 

pb0.001) in favor of OMA. The LSM difference (OMA-PBO) 

for change in rescue beta-agonist puffs was −0.47 (95% CI 

−0.72, −0.22; pb0.001) and was −0.40 (−0.55, −0.25; 

pb0.001) for change in total asthma symptom score. Conclusion: 

Omalizumab improved pulmonary function and asthmarelated 

outcome measures in cat allergic patients with 

moderate–severe persistent asthma inadequately controlled 

with ICS. 

doi:10.1016/j.clim.2007.03.392 

S75 

Sa.6 Hypersensitivity Pneumonitis of Unidentifiable 

Cause 

Viktor Hanak, MD, Mayo Clinic Foundation, Rochester, MN, 

Jason Golbin, MD, Mayo Clinic Foundation, Rochester, 

MN, Jay Ryu, MD, Mayo Clinic Foundation, Rochester, MN 

Identification of a specific antigen is important in disease 

management since antigen avoidance is necessary. Frequency 

of HP cases where causative antigen is not identifiable 

despite a thorough medical evaluation is not known. 

Objective: To assess the proportion of HP cases with no 

identifiable inciting antigen. Methods: Consecutive cases of 

HP diagnosed at Mayo Clinic Rochester, MN between 1997 and

S76 Abstracts 

2002 were reviewed. Diagnostic criteria included: (1) 

respiratory symptoms, (2) compatible radiologic changes, 

(3) known exposure or a positive serologic test to an inciting 

antigen, and (4) no other identifiable cause for the lung 

disease. If there was no identifiable inciting antigen, one of 

following two criteria was required: (1) diagnostic lung biopsy 

or (2) bronchoalveolar lavage lymphocytosis and compatible 

chest CTchanges. Results: We identified 85 consecutive cases 

of HP. The mean age (±SD) was 53±14 years; 53 (62%) patients 

were women. Cause was identified in 64 patients (75%). In 21 

patients (25%) the inciting antigen could not be identified by 

exposure history or serologic testing. In all 21 of these 

patients the diagnosis of HP was supported by histopathologic 

findings with diagnostic samples obtained in 18 patients by 

surgical and in 3 patients by bronchoscopic lung biopsy. 

Conclusions: The inciting antigen was not identifiable in 25% 

of patients evaluated for HP at a tertiary referral care center 

in the Midwest region of U.S. 

doi:10.1016/j.clim.2007.03.393 

Sa.7 Comparison of Influences of Three Types of 

Music (Western, Classical, Traditional Persian and 

Pop) on Immunological Parameters 

Alireza Salekmoghaddam, Professor, Iran University of 

Medical Sciences, Immunology, Tehran, Iran, Alireza Salek 

Moghddam, Professor, Immunology, Tehran, Iran, Saba Arshi, 

Assistant Professor, Immunology, Tehran, Iran, Mohammad 

Kamali, Assistant Professor, Statistics, Tehran, Iran, Hassan 

Ashayeri, Professor, Neuropsychology, Tehran, Iran, 

Gholomreza Azadi, Technologist, Immunology, Tehran, Iran 

28 allergic rhinitis patients who had inclusion, exclusion 

criteria for this intervention were divided in four groups, three 

interventional groups and one control group. In each there were 

seven subjects related to their favorite music. Methods: Blood 

samples were taken before and after 1 month interventional 

period. Samples were examined immediately by flow cytometry 

and six cellular parameters (CD3, CD4, CD8, CD16, CD19 and 

HLA-DR) were measured and were analyzed by t test and 

between group by ANOVA. Conclusion: Evaluation of immune 

cells in atopic patients that listened to pop music showed 

significant increases in CD3 T cells (p=0.03) and CD4+ cells 

(p=0.03), CD8+ cells (p=0.05), and NK cells (p=0.014) that show 

immune response shifts to Th1 immune responses. As a result 

pop music had positive effects on atopic subjects. Possibly 

regular listening to favorite music can modulate immune 

parameters and improve signs and symptoms of allergic rhinitis 

in atopic patients. 

doi:10.1016/j.clim.2007.03.394 

Sa.8 Emergency Department (ED) Evaluation; 

Treatment, Hospital Admission and sIgE Levels in 

African American (AA) and Hispanic (H) Pediatric 

Asthmatics 

Ray Mun Loo, Resident in Pediatrics, Nassau University 

Medical Center, East Meadow, NY, Katarzyna Koscielna, 

Resident, Nassau University Medical Center, East Meadow, 

NY, Krishan Kumar, Attending in Pediatric Emergency 

Medicine, Nassau University Medical Center, East Meadow, 

NY, Marianne Frieri, Attending in Allergy Immunology, 

Nassau University Medical Center, East Meadow, NY 

Introduction: Asthma is higher in AA and 20–25% higher in 

the ED. We evaluated 66 ED adult asthmatics with class II 

allergens to cat, dust mite, mold and cockroach. This study 

evaluated pediatric patients on either nebulized Budesonide (B) 

or normal saline (NS) prior to admission. Methods: Eleven 

moderately severe asthmatic patients, age 8–16 years (9 AA; 2 

H) were clinically evaluated in the ED over 4 months and 

randomized to receive either 1 mg of nebulized (B) or (NS) as 

control. Improvement was based on pulmonary index score 

(PIS), peak flow (PF), and spirometry. FENO (Aerocrine) was 

measured in 3 patients. All non-responsive patients were 

admitted; sIgE levels were determined by sensitive ELISA’s 

(Quest; Hitachi Chemical Diagnostics). Results: Six received (B) 

with higher PSI of 6–9 vs.5on(NS).PF=59–267L/min. FEV1 

levels reversed post bronchodilation in 9 of 10, and B increased 

FEV1 relative to baseline (P=0.024). Increased FNO levels in 2 

had PSI of 8. sIgE levels in 9 of 11 tested=class III–IV to mite in 8; 

IV–Vtocockroach;II–IV to animal dander; II–IV to grass in 7; I– 

IV to mold; II–IV to rice and wheat in 6; I–III to ragweed in 5; II– 

VI to trees; II–IV to fish in 4. Conclusion: Polysensitization 

developed in 89% to inhalants and various foods but higher 

levels compared to our adult ED population. Education of 

allergenic exposure, environmental and dietary control is 

essential to prevent hospitalization of pediatric asthmatics. 

doi:10.1016/j.clim.2007.03.395 

Sa.9 Association of the Polymorphisms of IL-4 Gene 

Promoter (−590CNT), IL-13 Coding Region (r130q) 

and IL-16 Gene Promoter (−295TNC) with Allergic 

Asthma 

Sara Hosseini-Farahabadi, Researcher, Bu-Ali Research 

Institute (BARI), Department of Immunogenetics, Mashhad, 

Iran, Jalil Tavakkol-Afshari, Vice President of Research, 

Head, Department of Immunogenetics and Cell Culture, 

Bu-Ali Research Institute (BARI), Department of 

Immunogenetics and Cell Culture, Mashhad, Iran, Houshang 

Rafatpanah, Ghaem Medical Center, Department of Allergy 

and Immunology, Mashhad, Iran, Mohammad Khaje Daluei, 

Ghaem Medical Center, Department of Epidemiology and 

Statistics, Mashhad, Iran, Rez Farid-Hosseini, Head, 

Department of Allergy and Immunology, Ghaem Medical 

Center, Department of Allergy and Immunology, Mashhad 

Allergic asthma is a multifactorial disease, influenced by 

genetic and environmental factors. Recent family-based studies 

have revealed evidence for linkage of human chromosomes 

5q31–33, 12q15–24, 11q13 and 15q23.6 as regions likely to 

contain genes related to asthma. Among the candidate genes in 

these regions are the genes encoding for human interleukin-4, 

interleukin-13 and interleukin-16. To test this linkage, we 

examined an Iranian population of patients with asthma. A total 

of 30 patients with allergic asthma and 50 normal subjects were 

studied. Allergic asthma was confirmed using skin prick test and

Abstracts 

spirometry. DNA was extracted from whole blood and IL-4 

(−590CNT), IL-13 (R130Q) and IL-16 (−295TNC) polymorphisms 

were determined by PCR-RFLP method. Out of 30 patients with 

allergic asthma, 17 (56.7%) had CC, 8 (26.7%) had CT and 5 

(16.7%) had TT genotypes for IL-4 polymorphism, 11 (36.7%) had 

GG, 13 (43.3%) had GA and 6 (20%) had AA genotypes for IL-13 

polymorphism, 23 (76.7%) had TT, 7 (23.3%) had CT and no 

patient had CC genotypes for IL-16 polymorphism. A higher 

proportion of case subjects with the C allele for the IL-4, G 

allele for the IL-13 and T allele for the IL-16 polymorphisms 

were found compared with the T, A and C alleles respectively. 

These results do suggest an influence of genetic variability at 

the promoter of IL-4 gene (−590CNT) and a coding region of IL- 

13gene(R130Q)ontheoccurrenceofallergicasthmaandno 

relationship between IL-16 promoter polymorphism (−295TNC) 

and this disease. 

doi:10.1016/j.clim.2007.03.396 

Sa.10 Lymphotactin Improves Treg Function in 

Asthmatics via the STAT5 Pathway 

Alison Fohner, Undergraduate Student, Stanford University, 

Pediatrics and Immunology, Stanford, CA, Khoa Nguyen, Lab 

Specialist, Stanford University, Pediatrics and Immunology, 

Stanford, CA, Alan M. Krensky, Doctor, Stanford University, 

Department of Pediatrics and Immunology, Stanford, CA, 

Kari C. Nadeau, Doctor, Stanford University, Department of 

Pediatrics and Immunology, Stanford, CA 

Lymphotactin is a chemokine (XCL1) associated with the 

function of regulatory T cells (Treg) and with allergic airway 

inflammation. Compared to healthy controls (HC) (n=10), 

allergic asthma patients (AA) (n=10) have fewer Tregs and 

more invariant natural killer T cells (iNKT) in the periphery, 

and iNKT kill Treg. We reported that Treg in asthmatics are 

dysfunctional but that XCL1 increases their suppressive 

function in vitro. We hypothesized that incubation of Treg 

with XCL1 could increase Treg absolute numbers and/or 

improve Treg function in AA subjects. Therefore, we added 

200 ng/mL XCL1 to HC Treg (Foxp3+CD127−CD25+CD4+) vs. 

non-Treg (Foxp3−CD127−CD25−CD4+) and AA Treg vs. non- 

Treg. Incubation with XCL1 for 4 days increased the ratio of 

Treg to non-Treg by 2–3×. Via a CFSE uptake assay, 

increased proliferation was not detected, suggesting that 

non-Treg were becoming Treg. We examined biochemical 

differences that could cause this conversion, including 

STAT1, STAT3, STAT5, and granzyme B expression since they 

have been implicated in Treg function. STAT5 phosphorylation 

increased in AA Treg and non-Treg, but not in HC or 

non-allergic asthmatics (NA). However, after XCL1 stimulation 

for 10 min, phosphorylated STAT5 increased significantly 

in non-Treg, but not Treg, of AA compared to HC 

(pb0.02) and NA (pb0.02). Since STAT5 phosphorylation 

plays a role in normal Treg function, these data support the 

conversion of non-Treg into Treg after incubation with 

XCL1. Thus, XCL1 functions through a STAT5 pathway to 

enhance Treg function in asthmatics. STAT5 could be a 

target for asthma therapy in the future. 

doi:10.1016/j.clim.2007.03.397 

Sa.11 Long-Term Tolerance in a Murine Model of 

Type I Allergy Through Transplantation of 

Genetically Modified Hematopoietic Stem Cells 

Ulrike Baranyi, PhD, Medical University of Vienna, 

Department of Surgery, Division of Transplantation, Vienna, 

Austria, Birgit Linhart, PhD, Division of Immunopathology, 

Department of Pathophysiology, Center of Physiology and 

Pathophysiology, Vienna, Austria, Nina Pilat, Division of 

Transplantation, Department of Surgery, Medical University 

of Vienna, Vienna, Austria, John Iacomini, PhD, 

Transplantation Research Center, Brigham and Women’s 

Hospital and Women’s Hospital and Children’s Hospital, 

Boston, MA, Jessamyn Bagely, PhD, Transplantation 

Research Center, Brigham and Women’s Hospital and 

Children’s Hospital, Harvard Medical School, Boston, MA, 

Rudolf Valenta, MD, Division of Immunopathology, Center of 

Physiology and Pathophysiology, Vienna, Austria, Thomas 

Wekerle, MD, Division of Transplantation, Department of 

Surgery, Vienna, Austria 

Introduction: Current therapies for treating allergy are 

associated with various limitations. We thus investigated 

whether tolerance can be induced through transplantation of 

syngeneic bone marrow retrovirally transduced to express an 

allergen. Methods: Balb/c bone marrow cells (BMC) were 

retrovirally transduced in vitro to express Phl p 5 (a major 

grass pollen allergen) in a membrane-anchored fashion 

(transduction efficiency: 35–55%). Myeloablated Balb/c 

mice received 2–4×10 6 transduced BMC iv and were thereafter 

repeatedly injected sc with recombinant Phl p 5 and 

rBet v 1 (an unrelated control allergen) (0.5 μg plus aluminum 

hydroxide, weeks 6, 9, 12 and 22). Allergen-specific antibody 

levels were determined in sera by ELISA, Phl p 5 expression by 

FACS. Results: All mice (n=10) transplanted with Phl p 5transduced 

BMC developed high levels of multi-lineage 

molecular chimerism which remained stable for the length 

of follow-up (up to 9 months) (e.g., 11 mean% Phl p 5+B cells 

and 22% T cells, 25 weeks post-BMT). Serum levels of Phl p 5specific 

IgE and IgG1 remained undetectable in all chimeras 

throughout follow-up while high levels of Bet v 1-specific IgE 

and IgG1 were measured. Basophil degranulation assays 

revealed the absence of Phl p 5-specific degranulation in 

chimeras, while in contrast Bet v 1-specific degranulation was 

preserved. In T cell proliferation assays chimeras showed 

specific non-responsiveness to Phl p 5 (n=3; pb0.01 vs. nontransduced 

mice). Conclusion: These proof-of-principle 

experiments demonstra5.5e for the first time that molecular 

chimerism induces robust and long-lasting tolerance in 

allergy at the B cell, T cell and effector cell levels. 

doi:10.1016/j.clim.2007.03.398 

S77 

Sa.12 Lichen Planus (LP) Associated with Xolair 

Administration 

Filiz Seeborg, Instructor, Baylor College of Medicine and 

Texas Children’s Hospital, Section of Allergy and 

Immunology, Houston, TX, Pardeep S. Rihal, MD, 

West Houston Allergy and Asthma, Katy, TX, Adam Czelusta, 

MD, Katy Dermatology, PA, Katy, TX, Imelda C. Hanson,

S78 Abstracts 

Professor, Baylor College of Medicine and Texas Childrens 

Hospital, Section of Allergy and Immunology, Houston, TX 

Background: Omalizumab (Xolair) is a recombinant 

monoclonal anti-immunoglobulin E (IgE) antibody that 

binds to free IgE, clearing it from the circulation. Xolair is 

approved for moderate to severe persistent asthma in 

adolescents and adults. Contraindication to use includes 

prior anaphylaxis to Xolair. Case report: We describe a 63year-old 

female who developed LP following Xolair administration. 

She had steroid-dependent asthma, allergic rhinitis, 

osteoarthritis, and hypertension. Her serum IgE level was 

55 KU/L. She developed a brief, generalized pruritus 

following first Xolair administration (300 μg/month). With 

second dosing, she had a generalized pruritic rash with lacy, 

violaceous facial papules lasting 2 weeks. Xolair and 

Diclofenac were discontinued. A skin biopsy revealed LP. 

Following third dosing (6 months later), she developed white 

oral lesions in addition to the previously experienced pruritic 

rash. Discussion: LP is a T cell mediated inflammatory 

mucocutaneous condition with pruritic violaceous papules 

and plaques. Oral LP may predispose to development of 

squamous cell carcinoma. LP may develop secondary to 

certain systemic medications and hepatitis C infection. No 

association between Xolair and LP is in the published 

literature. Malignant neoplasms have been reported during 

Xolair treatment, but a causal relationship has not been 

documented. Our case demonstrates a clear temporal 

association between Xolair use/cessation and the development/disappearance 

of LP. Per the manufacturer, malignancy 

risk for Xolair is unknown in individuals with existing 

malignant risk (oral LP). Conclusion: Repeated exposure to 

immunoregulatory therapy using Xolair may have potential 

to produce a T cell mediated immune activation leading to 

LP. 

doi:10.1016/j.clim.2007.03.401 

Sa.13 Inducible Expression of the Proallergic 

Cytokine TSLP in Airway Epithelial Cells is 

Controlled by NFkappaB 

Hai-Chon Lee, Postdoctoral Fellow, Immunology Program, 

Benaroya Research Institute, Seattle, WA, Steven F. Ziegler, 

Program Reader, Immunology Program, Benaroya Research 


The epithelial-derived cytokine thymic stromal lymphopoietin 

(TSLP) is important for the initiation of allergic 

airway inflammation through a dendritic cell-mediated Th2 

response. To identify the factors that control TSLP expression, 

we examined the ability of inflammatory mediators to 

regulate TSLP production in human airway epithelial cells. 

We found that both IL-12 and TNF-± were capable of 

inducing rapid TSLP production in primary human bronchial 

airway epithelial cells. We further characterized the human 

TSLP gene promoter using two human epithelial cell lines, 

16HBEo and A549, and showed that IL-12- and TNF-±mediated 

human TSLP promoter activation in these cells 

was mediated by an upstream NFkB site. Mutation of this 

NFkB site completely abolished activation, as did overexpression 

of a dominant-negative version of IKK 2 . Interestingly, 

human TSLP mRNA levels were also increased 

following exposure to TLR2, 8, and 9 ligands, further 

supporting an important role for NFkB in TSLP gene 

regulation. In a similar manner, analysis of the mouse 

TSLP gene promoter revealed the presence of a similar 

situated NFkB site that was also critical for IL-12-inducible 

expression of mouse TSLP. Taken together, these results 

demonstrate that the inflammatory mediators IL-12 and 

TNF-± regulate human TSLP gene expression in an NFkBdependent 

manner. 

doi:10.1016/j.clim.2007.03.402 

Sa.16 CpG Induces Th1 Response in Neonatal Mice 

but Does Not Suppress Anaphylactic IgG1 Response: 

Possible Role of Low TLR-9 Expression 

Cyro Alves Brito, PharmD, University of São Paulo, Brazil, 

Department of Dermatology, São Paulo, Brazil, Ana Elisa 

Fusaro, PharmD, University of São Paulo, Brazil, 

Department of Dermatology, São Paulo, Brazil, Jeferson 

Russo Victor, BSc, University of São Paulo, Brazil, 

Department of Dermatology, São Paulo, Brazil, Adriana 

Leticia Goldoni, BSc, University of São Paulo, Brazil, 

Department of Dermatology, São Paulo, Brazil, Paula 

Ordonhez Rigato, BSc, University of São Paulo, Brazil, 

Department of Dermatology, São Paulo, Brazil, Alberto Jose 

Silva Duarte, MD, University of São Paulo, Brazil, 

Department of Dermatology, São Paulo, Brazil, Maria 

Notomi Sato, BSc, University of São Paulo, Brazil, 

Department of Dermatology, São Paulo, Brazil 

It has been described that there are several differences 

between immune responses in adults and newborns. The 

aim of this work was to evaluate in early life and adult 

stage of mice, the immune modulatory effects of oligodeoxynucleotides 

containing CpG motif (CpG-ODN) in 

immunization with ovalbumin (OVA) and Blomia tropicalis 

(Bt). Three-day-old and 8–10-week-old A/Sn mice were 

immunized with OVA or Bt in alum, associated with CpG- 

ODN or control-ODN. Spleen cells from non-immunized mice 

were cultured with CpG or control-ODN for TLR-9 expression 

analysis. Neonate and adult co-administration of CpG 

with OVA or Bt was able to significantly decrease IgE 

response in parallel to an enhancement of IgG2a antibody 

levels compared to control mice. Furthermore, spleen cells 

from newborns immunized with OVA associated to CpG 

showed increased IFN-g production after anti-CD3 or LPS 

stimuli compared to control group. However, the negative 

regulation of IgE response was more evident in adult- than 

in neonate-immunized mice, also CpG down-regulates 

anaphylactic anti-OVA IgG1 production only in adult mice. 

Next we investigated whether an altered TLR-9 expression 

could be involved in impaired CpG effect in neonates. It 

was observed that after 48 hours of CpG stimulus TLR-9 

expression was already up-regulated in adult but not in 

neonate B cells. The pattern of antibody and cytokine 

production suggests a Th1-type response induced by CpG- 

ODN. Administration of CpG in newborns may be less

Abstracts 

effective than in adults due, in part, to a decreased B cell 

sensitivity to CpG stimulus. Supported by FAPESP, LIM-56, 

and HC-FMUSP. 

doi:10.1016/j.clim.2007.03.403 

Sa.17 IgE-reactive 60S Ribosomal Protein P2 in 

Almond (Prunus Dulcis) and Walnut (Juglans Regia), 

a New Class of Food Allergen Cross-reactive with 

Fungal Aeroallergens 

Pallavi Tawde, PhD Candidate, Florida State University, 

Department of Biological Science and Institute of Molecular 

Biophysics, Tallahassee, FL, Mohsen Abolhassani, Visiting 

Scholar, Florida State University, Department of Biology, 

Tallahassee, FL, Vanessa Seamon, Research Technician, 

Florida State University, Department of Biology, 

Tallahassee, FL, Suzanne Teuber, Associate Professor, 

University of California, Davis, Internal Medicine, 

Rheumatology and Allergy, Davis, CA, Fang Wang, 

Postdoctoraltoral Research Fellow, Florida State University, 

Department of Biology, Tallahassee, FL, Sandra Uratsu, 

Staff Research Associate, University of California, 

Davis, Department of Plant Sciences, Davis, CA, Abhya 

Dandekar, Professor, University of California, Davis, 

Department of Plant Sciences, Davis, CA, Stefan Vieths, 

Professor, Paul-Ehrlich-Institut, Department of 

Allergology, Langen, Germany, Shridhar Sathe, 

Professor, Florida State University, Department of 

Food Science, Tallahassee, FL, Kenneth Roux, Professor, 

Florida State University, Department of Biological 

Science and Institute of Molecular Biophysics, 

Tallahassee, FL 

Introduction: Tree nuts are a source of food allergens 

often associated with life-threatening reactions in susceptible 

individuals. Molecular characterization of the protein 

allergens is essential for a better understanding of the 

pathogenesis of atopic diseases, and in the development 

of more efficacious diagnostic and therapeutic modalities. 

We employed molecular cloning and expression to identify 

the 60S acidic ribosomal protein P2 (60S RP) as IgEreactive 

proteins in almonds and walnuts. 60S RPs have 

been described as aeroallergens in some molds. Results: 

Immunoscreening identified IgE-reactive almond and walnut 

cDNA clones, each encoding an 11,450 Da 60S acidic 

ribosomal protein P2 (60S RP), designated as Pru du 5 and 

Jug r 5, respectively, and sharing 91% sequence similarity. 

Of 27 sera from patients reporting allergy to almond and/ 

or walnut, 9 (33%) had IgE reactive with the recombinant 

proteins: 8 reacted to both proteins and 1 only with 

walnut Jug r 5. The 9 positive patient sera, but no 

others, also reacted with, and were inhibited by, 

recombinant Fus c 1, a fungal (Fusarium culmorum) 60S 

RP aeroallergen. Native 60S RP was detected in both 

almond and walnut crude extracts by reaction with rabbit 

anti-Pru du 5 antibodies. Conclusions: 60S RP represents a 

new class of food allergen in plants. Over a third of 

tested patients had IgE that bound almond (Pru du 5) 

and/or walnut (Jug r 5) homologues in in vitro assays. 

The data indicate considerable cross-reactivity between 

the almond, walnut, and fungal homologues. 

doi:10.1016/j.clim.2007.03.404 

Sa.18 Pistachio Vicilin, Pis v 1, is Allergenic and 

Cross-reactive with the Homologous Cashew 

Allergen, Ana o 1 

LeAnna N. Willison, Graduate Student, Florida State 

University, Tallahasee, FL, Pallavi Tawde, Graduate 

Student, Florida State University, Department of Biological 

Sciences, Tallahasee, FL, Jason M. Robotham, 

Postdoctoraltoral Fellow, Florida State University, 

Department of Biological Sciences, Tallahasee, FL, Suzanne 

S. Teuber, Associate Professor, University of California 

Davis, Internal Medicine, Rheumatology and Allergy, Davis, 

CA, Richard Penny, Graduate Student, Florida State 

University, Department of Biological Sciences, Tallahasee, 

FL, Shridhar K. Sathe, Professor, Florida State University, 

Food Sciences and Nutrition Department, Tallahasee, FL, 

Kenneth H. Roux, Professor, Florida State University, 

Department of Biological Sciences, Tallahasee, FL 

Introduction: Several studies have noted potential crossreactivity 

between certain tree nuts, especially those 

within the same botanical family. A potential example 

includes pistachio and cashew, which are both from the 

Anacardiaceae family. Results: A pistachio cDNA library was 

screened to detect potential pistachio allergens. An isolate 

was sequenced, cloned, and expressed in E. coli. The 

isolate coded for a 7S vicilin-like protein designated Pis v 1. 

Reactivity to the allergen was screened using 18 patients’ 

sera: 9 cashew- and pistachio-allergic, 6 cashew-allergic 

but had never eaten pistachios, 1 pistachio-allergic only, 

and 2 cashew-allergic only. IgE reactivity to Pis v 1 was 

found in the serum of 5 patients, all of which had histories 

of allergy to both tree nuts or had never eaten pistachio. 

Cross-reactivity between Pis v 1 and the cashew homologue, 

Ana o 1, was investigated using an inhibition dot blot 

assay. Of the tested sera, the 5 (28%) patients that 

recognized Pis v 1 also recognized Ana o 1 and IgE binding 

was completely inhibited after pre-incubation with either 

cloned allergen. Conclusion: Patients allergic to cashew 

often report allergy to pistachio, which could be a result of 

cross-reactivity between the two. This study revealed that 

28% of tested patients recognized the pistachio allergen Pis 

v 1 and these same patients also showed complete crossreactivity 

between the vicilin-like allergens from pistachio 

and cashew. Therefore, the pistachio allergen Pis v 1 is a 

likely contributor to the observed co-sensitivity to pistachio 

and cashew in some patients. 

doi:10.1016/j.clim.2007.03.405 

Sa.19 Infusion Reactions from Human 

Plasma-derived Products 

Hon-Sum Ko, Medical Officer, Food and Drug 

Administration/Division of Hematology, Potomac, MD 

S79

S80 Abstracts 

Introduction: Although human plasma-derived products 

are generally considered to have a favorable safety 

profile, intravenous infusion of these products is occasionally 

associated with local or systemic reactions, 

varying from local discomfort to anaphylaxis-like manifestations. 

This study is an attempt to identify the factors 

that may affect the risks of such reactions. Materials and 

methods: A review was made in the clinical and 

laboratory data found in the package inserts or license 

applications of fifteen randomly chosen human plasmaderived 

products available in the U.S. The products 

included albumin, plasma protein fraction, intravenous 

immunoglobulins, coagulation factors, and enzyme inhibitors 

in plasma. The adverse effects from administration of 

the products, including the frequency, severity, and 

potential attribution of local and systemic reactions 

were correlated with patient age, sex, ethnicity, allergic 

history, concomitant medications, previous infusion reactions, 

infusion rate, product class, product manufacture 

process, serum immunoglobulin levels, and other immune 

parameters whenever available. Results: Aside from 

infusion rate, which appears to correlate positively with 

the frequency of systemic reactions upon administration 

of immunoglobulin products, no consistent relationship 

between reaction frequency or severity and the above 

clinical/laboratory parameters was observed. A protective 

effect of premedication use before infusion was not 

observed. Conclusions: Much remains to be learned 

concerning the risk factors for infusion reactions to 

human plasma-derived products. Although current clinical 

and laboratory parameters may not be adequate to 

predict such risks, the development of newer biomarkers 

might offer an opportunity to help mitigate potentially 

serious infusion reactions. 

doi:10.1016/j.clim.2007.03.406 

Sa.20 Tolerance-Inducing Capacity is Preserved in 

Dendritic Cells from Patients with SLE 

Jose Crispin, PhD Student, Department of Immunology and 

Rheumatology, Instituto Nacional de Ciencias Medicas y 

Nutricion SZ, Mexico City, Mexico, Leonardo Limon, 

Resident, Department of Immunology and Rheumatology, 


Mexico City, Mexico, Maria Ines Vargas Rojas, PhD Student, 



Mexico, Jorge Alcocer Varela, Investigator, Department of 

Immunology and Rheumatology, Instituto Nacional de 

Ciencias Medicas y Nutricion SZ, Mexico City, Mexico 

Granted with the faculty of inducing T cell activation 

and regulation, dendritic cell (DC) function is pivotal for 

the maintenance of tolerance. DC are able to promote 

anti-nuclear oriented responses and worsen SLE in animal 

models. The aim of this project was to analyze the 

phenotype and function of SLE-derived DC and explore 

their capacity to induce T cell tolerance. 23 patients with 

SLE and 11 controls were included. The phenotype of 

circulating DC was analyzed by flow cytometry. Phenotype 

and cytokine production of monocyte-derived DC (MD-DC) 

was studied at the end of the differentiation period (basal) 

and after stimulation (LPS, TNF + PGE2 or anti-CD40). 

Ability to stimulate T cell proliferation was tested in 

allogeneic MLR. Phenotype and function of tolerogenic DC 

(grown in the presence of IL-10) was analyzed. The 

capacity of DC to induce tolerance towards allogeneic 

antigens, was analyzed in a recall assay. A higher 

proportion of DC from patients with SLE displayed 

costimulatory molecules in vivo (Pb0.05). Conversely, 

phenotype and T cell stimulatory capacity did not differ 

between MD-DC from patients and controls, neither in 

basal conditions, nor after stimulation. However, DC from 

patients with SLE showed a diminished response to 

stimulation via CD40. MD-DC from patients with SLE 

exhibited a normal capacity to induce tolerance towards 

alloantigens. DC from patients with SLE have an overstimulated 

phenotype when studied in vivo. However,such 

abnormality is absent when cells are grown in vitro. 

Tolerogenic MD-DC from SLE patients are capable of 

inducing T cell tolerance. 

doi:10.1016/j.clim.2007.03.407 

Sa.21 The Largest International Collaborative 

Studies on Ocular Lesions in Behcet Disease 

Nobuyoshi Kitaichi, Ophthalmologist, Department of 

Ophthalmology and Visual Sciences, Hokkaido University 

Graduate School of Medicine, Sapporo, Japan, 

Shigeaki Oh, Ophthalmologist, Department of 

Ophthalmology and Visual Sciences, Hokkaido University 

Graduate School of Medicine, Sapporo, Japan 

Purpose: Behcet disease is predominantly found 

between East Asia and Mediterranean basin along the 

ancient Silk Road. In order to investigate the clinical 

features of ocular lesions in Behcet disease, international 

collaborative studies were performed by utilizing the 

same questionnaire. Methods: Descriptive questionnaires 

were sent to 132 ophthalmology centers in the world. Answers 

of 1465 patients were collected from 25 eye centers in 14 

countries; Australia, Germany, Greece, India, Iran, Italy, 

Japan, Jordan, Morocco, Portugal, Turkey, Saudi Arabia, 

Tunisia, and United Kingdom. Results: Men were seen in 

68.3% of the patients. The average age of onset was 27.5 years 

old. The frequency of HLA-B51 was detected in 62.0%. 

Recurrent oral aphthous ulcers were observed in 94.5%, skin 

lesions in 69.5% and genital ulcers in 61.4%. Allergic diseases 

were recognized in only 4.3% of the patients. Bilaterality and 

recurrency of intraocular inflammation were seen in 85.5% 

and 95.7%, respectively. Regarding visual prognosis, 23.3% had 

poor vision less than 0.1 (20/200) at the final visit. The 

percentage of poor vision was significantly higher in India, 

Iran, and Japan, and the lowest in Italy (pb0.01). Combined 

anterior and posterior segment intraocular inflammation 

(CAPSII), more serious than anterior segment intraocular 

inflammation, was seen more frequently in men (95.4%) than 

in women (89.9%, pb0.01). Conclusions: We performed the 

largest world-scale investigation of ocular lesions in Behcet

Abstracts 

disease. Th2-dominant allergic diseases seem quite rare in 

Th1-dominant Behcet disease. Men tend to have worse visual 

prognosis than women. Clinical pictures of Behcet disease 

vary regionally. 

doi:10.1016/j.clim.2007.03.408 

Sa.22 Splenic Macrophages Promotes Responses to 

Nucleosomes in NZB/W F1 Mice 

Akiko Okamoto, Department of Allergy and Rheumatology, 

School of Medicine, The University of Tokyo, Tokyo, Japan, 

Keishi Fujio, Department of Allergy and Rheumatology, 


Nico van Rooijen, Department of Cell biology and 

Immunology, Faculty of Medicine, Free University, 

Amsterdam, Netherlands, Kazuhiko Yamamoto, Professor 

and Chairman of the Department of Allergy and 

Rheumatology, Department of Allergy and Rheumatology, 


Keith B. Elkon, Professor of Medicine, Head, Division of 

Rheumatology, Division of Rheumatology, University of 

Washington, Seattle, WA 

Systemic lupus erythematosus (SLE) is characterized by 

autoantibody production and organ damage. Autoantigen 

presentation to T cells is important for the development of 

lupus. Nucleosomes are major autoantigens in SLE. In this 

study, we examined nucleosome presentation in lupus-prone 

NZB×NZW F1 (NZB/W F1) mice. Nucleosome-specific T cells 

were generated by retroviral transfer of nucleosome-specific 

TCR. We found that nucleosome-specific T cells were 

stimulated dominantly in the spleen, compared to lymph 

nodes, lung, and thymus. Among splenic antigen-presenting 

cells (APCs), F4/80+ macrophages and CD11c+CD11b+ 

dendritic cells stimulated nucleosome-specific T cells 

strongly. F4/80+ macrophage was the most abundant APC 

subset in the spleen. Then we investigated whether splenic 

phagocytes were responsible for nucleosome presentation 

and disease progression in vivo. Splenic phagocytes, mostly 

F4/80+ macrophages, were depleted in NZB/W F1 mice by 

intravenous injection of Cl2MDP (dichloromethylene diphosphonate)-liposomes. 

Cl2MDP-liposome treatment dramatically 

suppressed nucleosome presentation in the spleen. 

Moreover, Cl2MDP-liposome treatment at 20 and 22 weeks of 

age significantly suppressed anti-nucleosome antibody (Ab) 

and anti-double stranded DNA (dsDNA) Ab production. 

Proteinuria progression was delayed and survival was 

prolonged in phagocyte-depleted mice. The numbers of 

autoantibody secreting cells were decreased in the spleen 

from phagocyte-depleted mice. Multiple injections of 

splenic F4/80+ macrophages, not CD11c+ dendritic cells, 

induced autoantibody production and proteinuria progression 

in NZB/W F1 mice. These results indicate that 

autoantigen presentation by splenic macrophages significantly 

contributes to autoantibody production and disease 

progression in lupus-prone mice. 

doi:10.1016/j.clim.2007.03.409 

Sa.23 A Comparison of TNF Dependent and 

Independent Features of Murine 

Spondyloarthropathy 

Peggy Jacques, MD, Ugent, University Hospital, Internal 

Medicine, Ghent, Belgium, Rik Lories, MD, PhD, University 

Hospitals Leuven, Internal Medicine, Leuven, Belgium, Ken 

Coppieters, M. Sc, Ugent, University Hospital, Internal 

Medicine, Ghent, Belgium, Katrien Van Beneden, PhD, 

Ugent, University Hospital, Internal Medicine, Ghent, 

Belgium, Herman Mielants, MD, PhD, Ugent, University 

Hospital, Internal Medicine, Gent, Belgium, Sylvie Seeuws, 

PhD, Ugent, University Hospital, Internal Medicine, Ghent, 

Belgium, Gust Verbruggen, MD, PhD, Ugent, University 

Hospital, Internal Medicine, Gent, Belgium, Martine De Vos, 

MD, PhD, Ugent, University Hospital, Internal Medicine, 

Gent, Belgium, Dirk Elewaut, MD, PhD, Ugent, University 

Hospital, Internal Medicine, Gent, Belgium 

Rheumatoid arthritis (RA) and spondyloarthropathies 

(SpA) are frequently occurring inflammatory disorders 

with distinct clinical features in which tumour necrosis 

factor (TNF) plays a predominant role. The focus of this 

study was to compare the typical features of three distinct 

arthritis models with special reference to synovitis and 

enthesitis. Collagen induced arthritis (CIA) is a well 

established model for RA in which peripheral joints are 

characterized by inflamed synovium and exudate in the 

synovial cavity. Proteoglycan depletion, cartilage destruction 

and bone loss are other features of this disease, which 

can be restored by TNF neutralisation. We also evaluated 

the TNF&DeltaARE mouse model, characterized by an 

increased TNF mRNA stability, where Crohn's disease is 

accompanied by peripheral arthritis. We demonstrated that 

this model, unlike CIA, is also characterized by dactylitis, 

enthesitis and axial involvement with sacroiliitis and 

spondylitis (histologically and by in vivo imaging (MRI)). 

Strikingly, severe bone demineralisation was observed but 

no signs of new bone formation, a common feature of 

human SpA, were evident. Spontaneous ankylosing dactylitis 

(SpAD) in ageing male DBA/1 mice, which develop 

ankylosing enthesitis with enthesial cartilage and bone 

formation is an established SpA model characterized by 

dactylitis, onychoperiostitis, and joint space bridging, 

without axial involvement or bowel inflammation. Contrary 

to the inflammatory features in the first models, the new 

bone formation was not found to be enhanced by TNF 

neutralisation. Conclusion: These findings indicate that 

inflammatory features of murine SpA are largely dependent 

upon TNF, but other mechanisms control bone new 

formation frequently occurring in SpA. 

doi:10.1016/j.clim.2007.03.410 

S81 

Sa.24 The Presence of Anti-La Autoantibody is 

Associated with a Lower Risk of Nephritis and 

Seizures in Lupus Patients 

Sanober Malik, Medicine Resident, Department of Medicine, 

Oklahoma University Health Sciences Center, Oklahoma 

City, OK, Gail R. Bruner, Clinical Coordinator, Arthritis and

S82 Abstracts 

Immunology/Oklahoma Medical Research Foundation, 

Oklahoma City, OK, Lourdes Feo, Recruiter, Oklahoma 

Medical Research Foundation, Oklahoma City, OK, Courtney 

Williams-Weese, Recruiter, Oklahoma Medical Research 

Foundation, Oklahoma City, OK, R. Hal Scofield, Professor, 

Department of Medicine, Oklahoma University Health 

Sciences Center/VA Medical Center/Oklahoma Medical 

Research Foundation, Oklahoma City, OK, John B. Harley, 

Professor and Section Chief, Program Head, Department of 

Medicine, Oklahoma University Health Sciences Center/VA 

Medical Center/Oklahoma Medical Research Foundation, 

Oklahoma City, OK, Amr H. Sawalha, Assistant Professor, 

Department of Medicine, Arthritis and Immunology 

Program, Oklahoma University Health Sciences Center/VA 

Medical Center/Oklahoma Medical Research Foundation, 

Oklahoma City, OK 

Background: Previous reports suggest a protective role for 

anti-La autoantibody against the development of lupus 

nephritis. We studied the effect of anti-La on the prevalence 

of nephritis in a large cohort of lupus patients. In addition, we 

determined the association between anti-La and the presence 

of the various lupus manifestations. Methods: We 

studied 1100 lupus patients enrolled in the Lupus Multiplex 

Registry and Repository. Only one lupus patient per family 

was selected to exclude intrafamilial correlation. Since anti- 

La is present in patients who also have anti-Ro autoantibody, 

we compared anti-Ro positive lupus patients in the presence 

or absence of anti-La. Clinical data were obtained from 

medical records, interviews, and participant questionnaires. 

Tests for autoantibodies against extractable nuclear antigens 

were performed using immunodiffussion assays. Results: 

There is no difference in the age, sex, or race between the 

anti-La positive and anti-La negative lupus patients. The 

presence of anti-La is associated with a significantly reduced 

risk of lupus nephritis (proteinuria: 29.3% versus 46.3%, 

OR=0.48, p =0.023; cellular casts: 8.6% versus 20.6%, 

OR=0.36, p=0.038). In addition, lupus patients with anti-La 

have a reduced risk for seizures (0% versus 11%, p=0.0096). 

The presence of anti-nRNP autoantibody is significantly 

reduced in anti-La positive compared to anti-La negative 

lupus patients (10.3% versus 27.4%, OR=0.30, p=0.0075). 

Conclusion: Anti-La autoantibody is associated with less 

severe lupus. Patients with anti-La have a lower risk of renal 

and CNS involvement compared to anti-La negative patients. 

doi:10.1016/j.clim.2007.03.411 

Sa.25 Establishment of an Animal Model for Allergic 

Granulomatous Vasculitis by IgE-mediated 

Cutaneous Reverse Passive Arthus Reaction: 

Contribution of Selectins and ICAM-1 

Minoru Hasegawa, Assistant Professor, Dermatology, 

Kanazawa University, Kanazawa, Tomoyuki Fujita, Graduate 

Student, Dermatology, Kanazawa University, Kanazawa, 

Manabu Fujimoto, Associate Professor, Dermatology, 

Kanazawa University, Kanazawa, Shinichi Sato, Professor, 

Dermatology, Nagasaki University, Nagasaki, Kazuhiko 

Takehara, Professor, Dermatology, Kanazawa University, 

Kanazawa 

Allergic granulomatous vasculitis (Churg–Strauss syndrome) 

occurs in patients with a history of asthma and is 

characterized by necrotizing vasculitis of small arteries and 

veins, with extravascular granulomas and a marked eosinophilia 

in the perivascular tissue of multiple organs and in the 

peripheral blood. IgE deposition in the lesion has been 

demonstrated, which suggests the involvement of IgE-containing 

immune complex (IC) in the pathomechanism. The classical 

and standard animal model for the inflammatory response in 

IC-mediated vasculitis is the reverse Arthus reaction. In the 

current study, we have established a mouse model mimicking 

allergic granulomatous vasculitis using cutaneous reverse 

passive Arthus reaction by IgE injection. IgE-mediated IC 

challenge induced hemorrhage and the accumulation of 

eosinophil, mast cell, and macrophage, which similar to 

allergic granulomatous vasculitis. Remarkably, infiltration of 

eosinophils and mast cells was observed, which was associated 

with the increased expression of MCP-3 and TNF-α. Furthermore, 

to assess the relative contribution of adhesion molecules, 

including selectins and ICAM-1, this model was 

examined in mice lacking E-selectin, P-selectin, L-selectin or 

ICAM-1. Eosinophil and mast cell numbers were significantly 

reduced in all mutant mice compared with wild type mice. 

Furthermore, both E- and P-selectin blockade in mice lacking 

for both L-selectin and ICAM-1 completely abrogated hemorrhage. 

These results indicate that E-, P-, L-selectin and ICAM-1 

contribute to the IgE-mediated cutaneous Arthus reaction by 

regulating eosinophil and mast cell recruitment and suggest 

that these adhesion molecules would be potential therapeutic 

targets for allergic granulomatous vasculitis. 

doi:10.1016/j.clim.2007.03.412 

Sa.26 Innate Immune Signal in New Murine Models of 

Autoimmunity Induced by U1 RNA and the RNA 

Binding Domain of the U1-70KD Ribonucleoprotein 

Antigen 

Annette Abril, Resident in Medicine, University of Miami/ 

Department of Medicine/Division of Rheumatology, Miami, 

FL, Kimberly Jaimes, Research Specialist, University of 

Miami/Department of Medicine/Division of Rheumatology, 

Miami, FL, Robert W. Hoffman, Professor and Chief, 

University of Miami/Department of Medicine, Division 

Rheumatology and Department Microbiology and 

Immunology, Miami, FL 

We have recently developed two complementary murine 

models of systemic autoimmunity that are induced following 

a single exposure to U1 RNA and a polypeptide 

encompassing the RNA binding domain of the 70 kDa 

polypeptide of U1 RNP. In mice deficient for the toll-like 

receptor (TLR3−/−), tissue targeting in the model is shifted 

from the lungs to the kidneys, and anti-double stranded 

DNA (dsDNA) antibodies emerge. This study examines the 

influence of TLR3 and innate immune signaling on the 

induction of antiphospholipid antibodies in these new 

models. Methods: Mice transgenic for the human major 

histocompatibility complex gene HLA-DR4 and TLR3−/− 

mice were immunized with U1 RNA and U1-70kDa

Abstracts 

polypeptide, control fusion protein, with or without U1- 

RNA, or saline. Sera from control mice of the background 

strains C57BL/6 or B6/129 were also studied. Complete 

serologic and histologic analyses were performed. Anticardiolipin, 

anti-dsDNA and anti-U1-70kDa antibodies were 

measured by ELISA. Results: We found that the mice 

deficient for TLR3 failed to produce anticardiolipin antibodies 

despite the development of high titers of antidsDNA, 

anti-U1-70kDa and other antibodies, as well as 

developing proteinuria and proliferative glomerulonephritis. 

In fact, TLR3 deficient mice had reduced levels of 

anticardiolipin reactivity in their sera as measured by ELISA 

compared to control mice (pb0.05). Conclusions: We have 

developed two new models of systemic autoimmunity that 

are induced following a single exposure to U1-RNA and its 

associated RNA binding protein. These models allow for 

genetic dissection of differences in tissue targeting, 

autoantibody development and the influences of innate 

immune signaling. 

doi:10.1016/j.clim.2007.03.413 

Sa.27 Urinary Lipocalin-2 is Upregulated in Murine 

and Human Lupus Nephritis 

Milena Pitashny, Posdoctoral Fellow, Albert Einstein College 

of Medicine, Division of Rheumatology, Bronx, NY 

Lipocalin-2, a member of the lipocalin family of iron 

binding proteins, is upregulated in the kidney following a 

variety of insults that lead to renal injury. We recently 

reported that in vitro exposure of kidney cells to monoclonal 

anti-double stranded (ds) DNA antibodies significantly upregulates 

Lipocalin-2. We hypothesized that polyclonal pathogenic 

anti-dsDNA antibodies induce kidney overexpression of 

Lipocalin-2, and the latter may represent a marker of renal 

involvement in lupus-prone mice and SLE patients. Kidney 

and serum Lipocalin-2 from old lupus-prone MRL lpr/lpr 

(MRL/lpr), NZB×NZW F1 (B/W) and non-autoimmune control 

mouse strains were studied. B/W and MRL/lpr mice had 

significantly higher kidney RNA expression of Lipocalin-2 than 

age-matched BALB/c mice (pb0.01). Furthermore, MRL/lpr 

had higher levels of serum Lipocalin-2 than MRL+/+ and 

BALB/c mice (pb0.05). Kidney Lipocalin-2 correlated with 

serum Lipocalin-2 (pb0.005), proteinuria (pb0.05) and antidsDNA 

antibody titers (pb0.05). We measured Lipocalin-2 

levels in urine from patients with or without active lupus 

nephritis (LN). SLE patients had significantly higher levels of 

urinary (u) Lipocalin-2 than normal controls (pb0.005). 

Moreover, patients with active LN had significantly higher 

levels of uLipocalin-2 than patients without LN (pb0.05). 

Finally, in LN patients we found a significant correlation 

between uLipocalin-2 levels and the renal SLE disease 

activity index (SLEDAI) (r=0.45, pb0.01). Upregulation of 

Lipocalin-2 in the kidneys of lupus prone mice and SLE 

patients with nephritis strongly suggests that Lipocalin-2 may 

serve as a valuable marker of nephritis in SLE, although its 

role in the pathogenesis of LN has yet to be determined. 

doi:10.1016/j.clim.2007.03.414 

Sa.28 CD4+ T Cells Reactive with U1-70kD 

Autoantigen Can Transfer Disease and are Central to 

Pathogenesis in New Murine Model of Lupus 

Robert W. Hoffman, Professor and Chief, University of 

Miami/Department of Medicine, Division Rheumatology and 

Department Microbiology and Immunology, Miami, FL, Eric 

L. Greidinger, Assistant Professor, University of Miami/ 

Department of Medicine, Division Rheumatology and 

Department Microbiology and Immunology, Miami, FL, Yun 

Zang, Research Scientist, University of Miami/Department 

of Medicine, Division Rheumatology and Department 

Microbiology and Immunology, Miami, FL 

C57BL/6 mice transgenic for the human major histocompatibility 

complex gene HLA-DR4 (DR4-Tg) were immunized 

with U1 RNA and the U1-70kDa polypeptide, control fusion 

protein, with or without U1-RNA, or saline. Complete serologic 

and histologic analyses were performed on all mice. CD4+ T 

cells and APCs were isolated from immunized mice using 

immunoaffinity columns and tested for proliferation. Either 

freshly isolated CD4+ T cells from immunized mice or T cell 

lines were adoptively transferred to unmanipulated HLA-DR4 

Tg or RAG−/− mice. Freshly isolated CD4+ T cell from 

immunized DR4 Tg mice proliferated in response to U1- 

70kDa and to synthetic peptides from the RNA binding domain 

region of the protein in the presence of irradiated autologous 

APCs. We found that CD4+ T cells specific for U1-70kDa were 

able to transfer disease to unmanipulated syngeneic mice. 

Furthermore, using purified CD4+ Tcells or Tcell lines specific 

for U1-70kDa we were able to transfer disease to RAG−/− mice 

in the absence of detectable B cells or autoantibodies. In 

conclusion, we have developed a new model of systemic 

autoimmunity that is induced following a single exposure to 

U1-RNA and its associated RNA binding protein. Anti-U1-70kDa 

autoantigen-specific CD4+ T cells appear to medicate pathogenesis 

of disease in this model and can do so in the absence of 

detectable B cells or autoantibodies. This work was supported 

by NIH award AR43308 (RWH), the Lupus Foundation of 

American (RWH) and Department of Veterans Affairs Merit 

Review awards (RWH and ELG). 

doi:10.1016/j.clim.2007.03.415 

S83 

Sa.29 Nasal Delivery of Anti-CD3 Antibody Suppresses 

Murine Lupus by Inducing IL-10 Producing 

Foxp3+CD4+CD25−LAP+ Regulatory T Cells 

Henry Yim Wu, Instructor, Harvard Medical School, Boston, MA, 

Howard Weiner, Professor, Harvard Medical School, Boston, MA 

One of the major contributors to the development of SLE 

is a functionally defective T regulatory cell pool in the 

periphery of susceptible individuals. Therefore the induction 

of novel T regulatory cells that can control autoaggressive 

immune responses in patients holds promise as 

immunotherapy for SLE. We previously reported that oral 

administration of anti-CD3 monoclonal antibody suppresses 

experimental autoimmune encephalomyelitis in a TGF-? 

dependent fashion both in vitro and in vivo. We have now 

discovered that nasally administered murine anti-CD3 F(ab′)

S84 Abstracts 

2 monoclonal antibody (clone 2C-11) is biologically active 

and reduced the incidence of lupus nephritis and autoantibody 

production in the SNF1 accelerated lupus model. In 

addition nasal anti-CD3 arrested on-going lupus in NZB strain 

of lupus prone mice. Animals were given 5 treatments per 

week every other week for 12 weeks. Nasal administration 

of anti-CD3 antibody induced Foxp3+CD4+CD25−LAP+ T 

regulatory cells that mediate suppression in an IL-10 

dependent but contact independent manor in vitro. 

Adoptive transfer studies demonstrated that LAP+ T 

regulatory cells required IL-10 and TGF-? to suppress disease 

in vivo. We hypothesize that LAP+ T regulatory cells control 

the activation CD4+ICOS+CXCR5+ T follicular helper cells 

which in turn downregulate the differentiation of B cells 

into IgG+CD38+ plasma cells and inhibit the production of 

anti-nuclear autoantibodies. Our findings identify a novel 

immunotherapeutic approach for the treatment of SLE that 

is applicable to humans. 

doi:10.1016/j.clim.2007.03.416 

Sa.30 Reverse Phase Protein Lysate Microarrays in 

the Analysis of Activated B Lymphocytes 

Alvina D. Chu, Rheumatology Fellow, Stanford University, 

Division of Immunology and Rheumatology, Palo Alto, CA, 

Andrzej J. Chruscinski, Cardiology Fellow, Stanford 

University, Division of Cardiovascular Medicine, Stanford, 

CA, Harvir Singh, Research Assistant, Stanford University, 

Division of Immunology and Rheumatology, Stanford, CA, 

Paul J. Utz, Associate Professor, Stanford University, 

Division of Immunology and Rheumatology, Stanford, CA 


inflammatory disease characterized by destruction of multiple 

organ systems. The pathophysiology of SLE involves 

activated B cells that produce antibodies directed against 

self-antigens. Our laboratory has previously demonstrated 

that reverse phase protein (RPP) lysate microarrays can be 

used to detect multiple intracellular phosphorylation events 

from T cell populations maintained in cell culture in vitro. 

Here we show a novel application of RPP lysate microarray 

technology to analyze activated B lymphocytes taken ex 

vivo from MRL/MpJ-Faslpr (MRL/lpr) mice. Splenocytes 

were harvested from MRL/lpr mice at ∼25 weeks of age, 

toward the end of their lifespan. B lymphocytes were 

isolated by negative selection using magnetic beads and 

then briefly stimulated with B cell activating factors, 

including phorbol 12-myristate 13-acetate (PMA). Using a 

robotic microarrayer, nanoliter amounts of lysate were 

deposited onto nitrocellulose-coated slides. Slides were 

probed with phosphorylation state-dependent antibodies 

targeting key intracellular phosphorylation events involved 

with B cell activation, including phosphorylation of ERK, 

Akt, Jak, and STATs. Bound antibodies were detected using 

secondary antibodies conjugated to fluorophores, and mean 

fluorescence intensity for each sample was calculated. We 

found differential activation of kinase substrates within 

stimulated cells compared to basal levels within unstimulated 

B cell populations. Results were validated by Western 

blot analysis. Reverse phase protein microarrays provide a 

powerful tool for quantifying and comparing the level of 

phosphorylation events in activated B cells isolated from 

secondary lymphoid organs, and show promise for elucidating 

signal transduction pathways involved in the pathogenesis 

of SLE. 

doi:10.1016/j.clim.2007.03.417 

Sa.31 Complement C4d Deposition on Circulating 

Blood Cells in Systemic Lupus Erythematosus (SLE) 

Chau-Ching Liu, Assistant Professor, University of Pittsburgh 

School of Medicine, Department of Medicine, Pittsburgh, 

PA, Jean Lin, Medical Student, University of Pittsburgh 

School of Medicine, Pittsburgh, PA, Jeannine S. Navratil, 

Research Associate, University of Pittsburgh School of 

Medicine, Department of Medicine, Pittsburgh, PA, Susan 

Manzi, Associate Professor, University of Pittsburgh School 

of Medicine, Department of Medicine, Pittsburgh, PA, Amy 

H. Kao, Assistant Professor, University of Pittsburgh School 

of Medicine, Department of Medicine, Pittsburgh, PA, 

Joseph M. Ahearn, Associate Professor, University of 

Pittsburgh School of Medicine, Department of Medicine, 

Pittsburgh, PA 

Immune dysregulation in SLE is characterized by polyclonal 

lymphocyte activation, autoantibody production, and 

complement activation. Recently, we discovered that significant 

levels of complement C4d are present on surfaces of 

erythrocytes, platelets, and lymphocytes of SLE patients and 

that C4d-bearing T lymphocytes exhibited functional 

abnormalities. These observations led to the current study 

in which circulating blood cells from SLE patients were 

investigated for C4d deposition and for the underlying 

mechanism(s). Specifically, we performed flow cytometry 

to measure C4d levels on various circulating cells from 307 

SLE patients, 187 patients with other inflammatory diseases, 

and 95 healthy controls. The results demonstrated that 

significantly elevated levels of C4d were present on T 

lymphocytes, B lymphocytes, and monocytes of SLE patients 

(mean fluorescence intensity (MFI) =13.5, 50.6, and 9.7, 

respectively), compared to patients with other diseases 

(MFI=3.2, 6.9, 4.7, respectively; pb0.0001) and healthy 

controls (MFI=1.8, 8.0, and 3.7, respectively; pb0.0001). 

However, only modest levels of C4d were detected on 

granulocytes from a small number of SLE patients (MFI=2.9). 

Furthermore, high C4d levels were not necessarily concurrently 

present on erythrocytes, platelets, lymphocytes, and 

monocytes of a given SLE patient on a particular day. These 

findings, combined, indicate that the C4d-bearing circulating 

cell phenotype is mediated by a patient-dependent, cell 

lineage-specific mechanism rather than the result of 

complement activation that simply affects all circulating 

cells simultaneously. Ongoing studies investigating the 

association of the differential leukocyte-C4d binding patterns 

with clinical features of SLE may lead to additional 

clues to SLE pathogenesis and may identify targets for 

therapeutic intervention. 

doi:10.1016/j.clim.2007.03.418

Abstracts 

Sa.32 Cell-Type and Stimulus Determine the Impact 

of the Serine/Threonine Kinase Cot/Tpl2 in Acute 

and Chronic Inflammation 

Anwar Murtaza, Senior Scientist, Abbott Bioresearch 

Center, Pharmacology, Worcester, MA, Shaughn Bryant, 

Research Associate, Abbott Bioresearch Center, 

Pharmacology, Worcester, MA, Suzanne Mathieu, Research 

Associate, Abbott Bioresearch Center, Pharmacology, 

Worcester, MA, Hamish Allen, Director, Abbott Bioresearch 

Center, Molecular and Cellular Biology, Worcester, MA, 

Catherine Tripp, Senior Director, Abbott Bioresearch 

Center, Worcester, MA, Neil Wishart, Associate Director, 

Abbott Bioresearch Center, Chemistry, Worcester, MA 

TNF has been identified as a key pro-inflammatory 

cytokine in the pathogenesis of acute and chronic inflammatory 

diseases such as septic shock and rheumatoid 

arthritis (RA). Anti-TNF therapy has been effective in the 

treatments of RA, Crohn’s disease (CD) and psoriasis (Ps). 

Binding of TNF to its receptors induces a number of signaling 

cascades leading to the activation of MAP kinases and NF-kB. 

Targeting Cot/Tpl2 a serine/threonine kinase in the MEK/ERK 

MAP kinase pathway offers an attractive approach for 

developing an oral therapeutic that inhibits TNF production 

and hence an alternative strategy to anti-TNF therapy. Here 

we have used Tpl2 deficient mice to elucidate its role in 

acute and chronic inflammation. Our results demonstrate 

that Tpl2 deficiency decreases the production of a panel of 

pro-inflammatory cytokines such as TNF, IL-1, IFN-g IL-6, and 

IL-18 following LPS challenge in vivo. We further demonstrate 

for the first time that Tpl2 knockout mice are only 

partially protected to collagen-induced arthritis (CIA). 

Moreover, the treatment of Tpl2 deficient mice with an 

anti-TNF antibody dramatically inhibited the progression and 

severity of disease suggesting a Tpl2 independent production 

of TNF in rodent arthritis. Moreover, we show that the impact 

of Tpl2 deficiency in LPS and arthritis models may be due to a 

differential activity of the kinase in different cell types. 

Thus, our data demonstrate that the Cot/Tpl2 pathway is 

critical in the response to endotoxin but may have a limited 

role in arthritis. 

doi:10.1016/j.clim.2007.03.419 

Sa.33 Bosentan Synergizes with Iloprost in Treating 

Pulmonary Hypertension in Scleroderma Patients 

Marta Sessarego, Doctor, Switzerland, Department of 

Internal Medicine-University of Genova, Genova, 

Switzerland, Marta Rizzi, Department of Internal 

Medicine-University of Genova, Genova, Switzerland, 

Massimo Ghio, Department of Internal Medicine-University 

of Genova, Genova, Francesco Indiveri, Professor, 

Department of Internal Medicine-University of Genova, 

Genova, Switzerland 

Pulmonary arterial hypertension (PAH) is a devastating 

complication of systemic sclerosis (SSc) characterized by 

endothelial dysfunction (lesser production of prostacyclin 

associated with increased secretion of endotelin), for which 

no curative treatments are available. On this basis it has 

been postulated that the oral dual endothelin receptors 

antagonist, bosentan, might strengthen the activity of prostacyclin 

therapy. Patients and methods: 8 patients affected 

by SSc with increased PAH (PA systolic pressure PAPS ¡Ý 45 mm 

Hg by echocardiogram or PA pressure ¡Ý 35 mm Hg at rest by 

cardiac catheterism, WHO functional classes III–IV) were 

reclutated, all of them had shown a poor response to 1 year 

treatment with prostacyclin e.v. infusion (iloprost, 0.5–2 ng/ 

kg/min 6 h a day — 5 days a month). Bosentan (62.5 mg bid 

for 1 month, then 125 mg bid) and iloprost (0.5–2 ng/kg/min) 

in continuous (24h/24 h) were administered. The Doppler 

echocardiogram, the six-minute walking test (6MWT) and the 

complete pulmonary tests were performed at baseline and 

after 3, 6 and 12 months. Results: After 3 months PAPS was 

decreased and 6MWT was improved at least 25 min in all 

patients; at the end of the study an improvement of WHO 

class and pulmonary function was observed in 7 and 3 

patients respectively. All the subjects tolerated the therapy, 

thereafter no one discontinued therapy. Conclusion: The 

association of bosentan with iloprost infused continuously 

may improve PAP and hemodynamics in SSc patients. 

doi:10.1016/j.clim.2007.03.421 

S85 

Sa.35 Anti-lymphocyte Autoantibodies in Systemic 

Lupus Erythematosus (SLE) Revisited 

Jean Lin, Medical Student, University of Pittsburgh, 

Pittsburgh, PA, Joseph M. Ahearn, Associate Professor, 

University of Pittsburgh School of Medicine, Department of 

Medicine, Pittsburgh, PA, Susan Manzi, Associate Professor, 

University of Pittsburgh School of Medicine, Department of 

Medicine, Pittsburgh, PA, Chau-Ching Liu, Assistant 

Professor, University of Pittsburgh School of Medicine, 

Department of Medicine, Pittsburgh, PA 

Complement, anti-lymphocyte autoantibodies (ALA), and 

lymphocyte dysfunction have previously been implicated in 

the pathogenesis of SLE. However, the causal relationships 

between complement, ALA, and lymphocyte function are 

largely unexplored. Our recent identification of complement 

C4d on T cells of SLE patients has led us to hypothesize that 

binding of ALA to T cells may trigger complement activation 

and C4d deposition on these cells, which in turn causes 

functional dysregulation of these cells and contributes to SLE 

pathogenesis. The current study was performed to characterize 

C4d deposition on T cells (T-C4d) and to investigate 

the role of ALA in T-C4d deposition. Briefly, T-C4d and ALA 

were determined by flow cytometry in 225 SLE patients, 135 

patients with other inflammatory diseases, and 15 healthy 

controls. Significantly elevated levels of C4d were detected 

on T cells of SLE patients (median fluorescence intensity 

(MFI)=14.8), compared with T cells from patients with other 

diseases (MFI=3.4; pb0.001) or healthy controls (MFI=0.8; 

pb0.001). IgM and/or IgG were detected concomitantly 

with C4d on T cells in a significant fraction of SLE patients 

(73/225), but not in healthy controls or patients with other 

diseases. Moreover, high levels of T-C4d could be induced on 

normal T cells after incubation with selected SLE plasma or 

immunoglobulins. Such in vitro T-C4d inducing activity could 

be eliminated by pre-absorption of SLE plasma or

S86 Abstracts 

immunoglobulins with lymphocytes, indicating that ALA is 

primarily responsible for triggering C4d deposition on Tcells. 

These results, together, support a previously unrecognized 

ALA-mediated pathogenic mechanism in SLE. 

doi:10.1016/j.clim.2007.422 

Sa.36 Treg Cells Directly Suppress Anti-DNA 

Ig-producing B Cells Through TGFβ-mediated 

Mechanisms in Murine Lupus 

Antonio La Cava, Associate Professor, University of 

California, Los Angeles, Los Angeles, CA, Chung Lee, 

Professor, Northwestern University, Chicago, IL, Bevra 

Hahn, Professor and Chief of Rheumatology, University of 

California, Los Angeles, Los Angeles, CA 

In SLE, production of autoantibodies (autoAb) including 

anti-DNA Ig is central to pathogenesis of the disease. We 

reported that regulatory Foxp3+CD4+CD25+ T (Treg) cells in 

(NZB×NZW) F1 (NZB/W F1) lupus mice suppress the production 

of anti-DNA Ig by B cells in vitro. To discriminate between 

the possibility that Treg cells suppressed Tcells providing help 

to Ig-producing B cells and the possibility that Treg cells 

directly suppressed autoAb-producing B cells, we sublethally 

irradiated NZB/W F1 mice for adoptive transfer with 

syngeneic Treg cells together with B cells only or with B 

cells plus helper T cells. Additional experiments consisted of 

transfer of these combinations but membrane expression of 

TGFβ receptor II (TBR) in B cells was disrupted using a 

retrovirally based vector that expresses a dominant negative 

(DN) TBR. It was found that Treg suppression of anti-DNA Ig in 

vivo occurred without requirement of intermediate regulation 

of CD4+ Tcells; suppression of anti-DNA Ab was observed 

in the absence of CD4+ T cells (Pb0.03 vs. anti-DNA Ab from 

transfer of B cells only). Importantly, an intact TGFβ signaling 

pathway was required for B cell suppression by Treg. 

Disruption of B cell surface expression of TGFβ receptor via 

the dominant negative (DN) TBR permitted escape of B cells 

from the Treg-controlled suppression and led to dysregulated 

production of autoAb in vitro and in vivo. The findings 

identify events controlling production of autoAb in SLE and 

envision new possibilities for targeted intervention. 

doi:10.1016/j.clim.2007.03.424 

Sa.38 Agalactosyl Immunoglobulins are a Useful 

Marker for Clinical Progression and Successful 

Therapy of Collagen-induced Arthritis 

Katrien Van Beneden, PhD, Ghent University, Ghent, 

Belgium, Ken Coppieters, PhD Student, Ghent University, 

Ghent, Belgium, Wouter Laroy, PhD, Ghent University and 

Flanders Interuniversity Institute for Biotechnology, 

Zwijnaarde, Belgium, Pieter Rottiers, PhD, Ghent 

University and Flanders Interuniversity Institute for 

Biotechnology, Zwijnaarde, Belgium, Gust Verbruggen, MD 

PhD, Ghent University, Ghent, Belgium, Nico Callewaert, 

PhD, Ghent University and Flanders Interuniversity Institute 

for Biotechnology, Zwijnaarde, Belgium, Dirk Elewaut, MD 

PhD, Ghent University, Ghent, Belgium 

The purpose of this study was to evaluate the validity of 

serum agalactosyl Ig as a biomarker for murine collagen-induced 

arthritis(CIA).Wesoughttoclarifythecorrelationbetween 

arthritic index and agalactosyl Ig fraction, the kinetics of 

agalactosyl Ig levels upon development and progression of CIA, 

and the effect of antigen specific versus non-specific treatment. 

CIA was induced in susceptible mice, serum was collected from 

several arthritic and non-arthritic animals and agalactosyl Ig 

content was determined using a sensitive, improved technology 

for DNA sequencer aided analysis of N-linked glycans. Prophylactic 

tolerization with collagen type II was performed and the 

clinical effect was monitored; agalactosyl IgG was analyzed in 

serum derived from endpoint bleeding. Semi-therapeutic 

treatment with either dexamethasone or etanercept was 

performed and compared with PBS controls. All mice were 

sequentially bled at various time points during treatment. 

Agalactosyl Ig ratios were quantified and compared with agematched, 

unimmunized DBA/1 mice. Arthritic animals could be 

distinguished from non-arthritic animals with high sensitivity 

and specificity. Improvement of clinical arthritis scores by 

antigen specific tolerance induction correlated with downregulated 

levels of agalactosyl Ig. Finally, increased serum 

agalactosyl Ig levels correlate with CIA progression and are 

reversed by successful semi-therapeutic treatment. These 

findings indicate that agalactosyl determination may be a very 

useful serum marker for monitoring the clinical outcome of 

experimental treatment in CIA, by use of a sensitive albeit 

simple methodology. This observation may be valuable for 

longitudinal CIA studies or for a more detailed judgment of 

outcome upon treatment. 

doi:10.1016/j.clim.2007.03.426 

Sa.40 NKT Cells are Novel Accelerator and Producer 

of IL-17 in the Pathogenesis of Collagen-induced 

Arthritis 

Yohei Yoshiga, Graduate Student, Tsukuba University, 

Tsukuba, Japan, Goto Daisuke, MD, PhD, Tsukuba 

University, Tsukuba, Japan, Mizuko Mamura, MD, PhD, 

Tsukuba University, Tsukuba, Satoshi Ito, MD, PhD, Tsukuba 

University, Tsukuba, Japan, Isao Matsumoto, MD, PhD, 

Tsukuba University, Tsukuba, Japan, Akito Tsutsumi, MD, 

PhD, Tsukuba University, Tsukuba, Japan, Takayuki Sumida, 

MD, PhD, Tsukuba University, Tsukuba, Japan 

Objective: To clarify the role of NKTcells in CIA. Methods: (1) 

CIA was induced in C57BL/6 (B6) and NKT-KO (Jƒ¿281−/−)mice. 

Mice were immunized with type II collagen (CII) emulsified in 

CFA intradermally at day 0 and day 21, and then the incidence 

and severity of arthritis were evaluated. (2) To analyze the CIIspecific 

T cell response, splenocytes and draining lymph node 

(DLN) cells from B6 and NKT-KO mice immunized with CII/CFA 

were stimulated with CII. The culture supernatants were used 

for cytokine ELISA, and these cells were used for intracellular 

cytokine staining. (3) To identify the association of NKTcells and 

IL-17 production, splenocytes from naive B6 and NKT-KO mice 

were stimulated with ƒ¿-GalCer, ligand for NKT cells. The IL-17 

production was assayed by intracellular cytokine staining. 

Results: (1) The incidence of arthritis in NKT-KO mice was 

suppressed compared with that in B6 mice. The severity of

Abstracts 

arthritis in NKT-KO mice was also milder than that in B6 mice. 

(2) The production of IFN-ƒÁ and IL-4 was not significantly 

different between NKT-KO and B6 mice, but IL-17 production 

was reduced in NKT-KO mice. IL-17-producing T helper (Th17) 

cells were reduced in DLN cells from NKT-KO mice. (3) NKTcells 

produced IL-17 in B6 splenocytes stimulated with ƒ¿-GalCer, 

although there are no IL-17-producing cells in NKT-KO 

splenocytes. Conclusion: These findings support the notion 

that NKT cells are a novel producer and accelerator of IL-17, 

indicating NKTcells might play a crucial role in the pathogenesis 

of CIA via IL-17. 

doi:10.1016/j.clim.2007.03.427 

Sa.41 Endogenous CD4 T Cells Program B Cells to 

Respond in the Chronic GVH Model of SLE 

Arpita Choudhury, Post Doctoral Fellow, Division of 

Rheumatology, School of Medicine, University of 

Pennsylvania, Philadelphia, PA, Philip Cohen, Professor, 

Division of Rheumatology, School of Medicine, University of 

Pennsylvania, Philadelphia, PA, Robert Eisenberg, Professor, 

Division of Rheumatology, School of Medicine, University of 

Pennsylvania, Philadelphia, PA 

The murine chronic GVH (cGVH) model of SLE is induced by 

allo-recognition of foreign major histocompatibility complex 

(MHC) class II determinants. Our previous studies suggested that 

B cells in CD4KO mice have intrinsic defects that prevent them 

from responding to allo-help (JI 174, 7600, 2005). We have 

further explored the role of host CD4 T cells in this model by 

using post-irradiation auto-reconstitution and cell transfer 

experiments. C57Bl/6 (B6)-CD4KO mice were irradiated (5 

Gy), and 3–5 million CD4 Tcells from various B6 congenic strains 

were transferred. After allowing the B cells to auto-reconstitute 

over 3 weeks, 10 8 bm12 spleen cells were injected to initiate 

the cGVH. Purified CD4 T cells from IL-6KO, IFN- 3 KO and IL-10 

KO, but not IL-4 KO, mice were able to restore B cell response to 

cGVH in CD4 KO mice, signifying a critical role for IL-4. 

Treatment with recombinant IL-4 (rIL-4) also restored the 

ability of B cells from CD4 KO mice to secrete auto-abs in cGVH. 

Furthermore, the treatment of CD4KO recipients with an 

agonistic anti-CD40 (FGK-115, rat IgG2a) mAb also restored B 

cell function in cGVH. Our studies show that the endogenous 

CD4 T cell help needed for B cells to mount an effective cGVH 

response depends on IL-4, but not IL-6, IL-10 or IFN- 3 .Thus, 

autoreactive B cells need to have developed in a particular 

cytokine milieu in order to be receptive to alloreactive stimuli. 

Understanding the intracellular changes that condition B cells 

to become activated in cGVH may yield clues to their function 

in SLE-like autoimmunity. 

doi:10.1016/j.clim.2007.03.428 

Sa.42 The Mer Receptor Tyrosine Kinase Influences 

Toll-like Receptor Stimulation of Dendritic Cells 

Benjamin Scott, Postdoctoral Fellow, University of 

Pennsylvania, Division of Rheumatology, Philadelphia, PA, 

Joudy-Ann Dinnall, Research Associate, Children’s Hospital, 

Department of Pediatrics, Philadelphia, PA, Stefania 

Gallucci, Assistant Professor, Children’s Hospital of 

Philadelphia, Department of Pediatrics, Philadelphia, PA, 

Philip Cohen, Professor, University of Pennsylvania, Division 

of Rheumatology, Philadelphia, PA, Robert Eisenberg, 

Professor, University of Pennsylvania, Division of 

Rheumatology, Philadelphia, PA 

The mer receptor tyrosine kinase, which is expressed on 

macrophages and dendritic cells (DC), is known to promote 

apoptotic cell disposal and inhibit inflammatory cytokine 

production by macrophages, but its role in DCs is unclear. 

Mice lacking functional mer (mer kd mice) develop autoimmunity 

characterized by antibodies to DNA and chromatin, 

presumably due to their accumulation of apoptotic cells. To 

explore the role of mer on splenic DCs, mer kd and B6 mice 

were injected i.p. with toll-like receptor (TLR) ligands, and 

the mice were sacrificed 1 day later for analysis by flow 

cytometry. The numbers of splenic DCs and degree of 

activation of splenic DCs in control mer kd mice are comparable 

to control B6 mice. However, following LPS (TLR-4 

ligand) injection, merkd splenic DCs displayed an abnormally 

activated profile compared to B6 controls, characterized by 

the increased expression of the costimulatory molecules 

CD40, CD80, and CD86. This activated phenotype was highly 

pronounced in the plasmacytoid subset of DC (pDC) and was 

present to a lesser extent in the CD8α + and myeloid subsets. 

In contrast, after poly(I:C) (TLR-3 ligand) injection, mer kd DC 

appeared to have suppressed costimulatory molecule expression 

compared to B6 controls, whereas CpG-ODN (TLR-9 

ligand) stimulation of DC did not seem affected by the mer 

deficiency. Thus, mer may differentially regulate DC TLR 3 

and 4 signals and may negatively regulate pDC during 

inflammation. Further studies may provide important insight 

into SLE pathogenesis since pDC have been linked to lupus in 

humans and animal models. 

doi:10.1016/j.clim.2007.03.429 

S87 

Sa.43 Membranous Glomerulopathy Associated with 

Rheumatoid Arthritis may Respond to Rituximab 

Bryan J. Wolf, Resident Physician, NV, Medical Center/ 

University of Nevada, Reno Department of Internal 

Medicine, NV, William M. O’Neil, Clinical Professor, 

University of Nevada School of Medicine, V, John S. Pixley, 

Associate Professor of Medicine, VA Medical Center/ 

University of Nevada School of Medicine, NV 

Membranous glomerulonephritis is an uncommon extraarticular 

manifestation of rheumatoid arthritis, which need 

not be associated with prior DMARD (disease-modifying 

antirheumatic drug) therapies (Clin Rheum 1996, 15, 385). 

We observed a patient with an 8-year history of seropositive 

erosive rheumatoid arthritis complicated by the development 

of refractory nephrotic syndrome secondary to 

biopsy-proven membranous glomerulonephritis. Signs and 

symptoms of his disabling kidney disease included renal 

insufficiency, massive proteinuria (25 g), edema and a 

hypercoaguable state (deep venous thrombosis and pulmonary 

emboli). Alkylating agents, antimetabolites and

S88 Abstracts 

glucocorticoids were ineffective in reducing his proteinuria 

over a 2 year period. The patient subsequently received 2 

courses (1 g twice over a 2 week period) of rituximab (a 

monoclonal antibody to CD20 that specifically depletes B 

cells), 6 months apart. Serum albumin at initial treatment 

was 2.0 g/dl and normalized (3.7 g/dl) 1 month after the 

second course of therapy. This was accompanied by a 

corresponding decrease excretion of urinary protein (22 g 

to 7.4 g/24 h) and a reduction in diuretic requirements. B 

cell directed therapy may prove useful in treating membranous 

glomerulopathies. 

doi:10.1016/j.clim.2007.03.430 

Sa.44 Design and Elaboration of Truncated Mutants 

of the Autoantigen hSRP72, Present in 

Dermatomyositis, in Order to Identify the Sites of 

Phosphorylation 

Victor Ermilo Arana-Argaez, Master in Science, Immunology, 

University of Guadalajara, Institute of Rheumatology, 

Guadalajara, Mexico, Victor Ermilo Arana-Argaez, Master in 

Science, University of Guadalajara, Guadalajara, Mexico, 

Beatriz Martin-Marquez, Master in Science, University of 

Guadalajara, Guadalajara, Mexico, Muñoz-Valle Jose 

Francisco, PhD, PhD, Guadalajara, Mexico, Erika 

Martínez-Garcia, Master in Science, University of 

Guadalajara, Institute of Rheumatology, Guadalajara, 

Mexico, Oscar Leon-Murguia, MD, Institute of 

Rheumatology, Guadalajara, Mexico, Monica Vazquez-Del 

Mercado, PhD, MD, University of Guadalajara, Guadalajara, 

Mexico 

Dermatomyositis (DM) is an idiopathic inflammatory 

myopathy (IIM) with characteristic muscle-cutaneous findings. 

Using sera from patients with DM the SRP72 

component of the signal recognition particle (SRP) as an 

autoantigen phosphorylated on serine (S) residues was 

identified. The mechanisms to explain the lost of tolerance 

against the hSRP72 remain without explanation. To determine 

whether the phosphorylation state of this particle 

could affect the antigenicity, we cloned the autoantigen 

hSRP72 into a pRS314-TRP shuttle vector and we designed 

truncated mutants to identify the possible site/sites of 

phosphorylation of the serine residues and its further 

implication on the break of tolerance against this protein. 

Aim: To truncate mutants of the hSRP72 to further identify 

the site or sites of phosphorylation for the application on 

the study of the induction mechanisms of autoantigenicity 

in DM. Material and methods: Based on the map of serine 

residues in the full length SRP72 sequence, we cloned the 

autoantigen hSRP72, designed and elaborated 13 truncated 

mutants to identify the region of the protein where 

phosphorylation might occur. We designed primers to 

amplify 13 mutants truncated by means of PCR using as a 

template the construct pRS314hSRP72. Next, in vitro 

translation and metabolic labeling with 35 S and 32P-g ATP 

will be done. Results and conclusions: We cloned successfully 

the autoantigen hSRP72 and the design and elaboration 

of truncated mutants according to the phosphorylation 

prediction map was correct based on its PCR product length 

for later use in in vitro translation phosphorylation tests 

and studies on antigenicity induction. 

doi:10.1016/j.clim.2007.03.431 

Sa.45 Activated Interstitial Macrophages are 

Important Mediators of SLE Nephritis 

Meera Ramanujam, Research Scientist, Feinstein Medical 

Research Institute, Manhasset, NY, Ramalingam 

Bethunaickan, Postdoctoral Fellow, Feinstein Medical 

Research Institute, Manhasset, NY, Anne Davidson, 

Professor of Medicine, Feinstein Medical Research Center, 

Manhasset, NY 

SLE is an autoimmune disease that is complicated by 

autoantibody mediated glomerulonephritis. In NZB/W SLEprone 

mice recruitment of inflammatory cells and mediators 

is associated with early expression of a limited panel of 

chemokines followed by spreading to involve multiple other 

inflammatory molecules. Furthermore, inflammatory cell 

subsets home to different areas of the NZB/W kidney. B 

cells, T cells and dendritic cells are found in disorganized 

aggregates in the perihilar region and around blood vessels, 

whereas macrophages invade the interstitium of the kidney 

and form a layer around the glomeruli. In NZW/BXSB mice, 

despite a similar expression profile of inflammatory genes 

in the kidney, there is marked invasion of the interstitium 

by sheets of F4/80 macrophages whereas dendritic cells are 

found in large numbers only inside the glomeruli. NZM2410 

mice that develop a sclerotic form of glomerulonephritis 

express few inflammatory mediators in the kidney; the 

onset of glomerulonephritis is associated with invasion only 

by macrophages that are confined to the renal medulla. 

Both NZB/W and NZW/BXSB mice have large numbers of 

circulating CD11bhi/Gr1lo monocytes; the kidneys of both 

these strains contain numerous F4/80+/Gr1lo/Ly6Clo/ 

CD62L− macrophages. These cells appear concomitant 

with proteinuria onset and produce TNF-±, IL-1, BAFF and 

CXCL13; thus although phenotypically they resemble alternatively 

activated macrophages, they migrate to inflamed 

tissue and produce pro-inflammatory mediators. Further 

study of these cells should help us understand what directs 

traffic and local differentiation of monocytes to macrophages 

and dendritic cells, and how interactions between 

the differentiated macrophages and the kidney microenvironment 

promote injury. 

doi:10.1016/j.clim.2007.03.432 

Sa.46 Optimization of Autoantigen Microarrays to 

Study Autoimmunity 

Imelda Balboni, Postdoctoral Fellow, Stanford University, 

Medicine and Pediatrics, Stanford, CA, Cindy Limb, 

Research Assistant, Stanford University, Medicine, Stanford, 

CA, Paul Utz, Associate Professor, Stanford University, 

Medicine, Stanford, CA, Jessica Tenenbaum, Graduate 

Student, Stanford University, Medicine, Stanford, CA

Abstracts 

Autoantigen microarrays are used to study autoimmune 

diseases including systemic lupus erythematosus, rheumatoid 

arthritis, and multiple sclerosis. Hundreds of autoantigens 

can be analyzed with microliter volumes of 

serum in a high throughput manner. Various slide surfaces, 

printing methods and arraying conditions have been 

reported, and we sought to determine the optimal 

conditions for printing microarrays based on the initial 

protocol developed by the Robinson and Utz laboratories. 

We analyzed 22 slide surfaces for overall background, 

uniformity, streaking, and smearing of features. Based on 

these characteristics, coefficient of variance (CV) was 

determined for eight surfaces, and ultimately FAST®, 

poly-L-lysine, and SuperEpoxy slides were chosen for 

further studies. Several histidine-tagged autoantigens 

were spotted onto microarrays using a robotic microarrayer. 

Slides were probed with a histidine-specific 

monoclonal antibody and fluorophore-labeled secondary 

antibody. Arrays were scanned with a GenePix 4000 

scanner and analyzed using GenePix Pro 6.0 software. 

CV was calculated based on the fluorescence intensity 

minus background for each feature. Similar studies using 

human sera were performed using the FAST® surface to 

demonstrate applicability to the study of human autoimmune 

disease. FAST® slides had the lowest CV in all 

studies, with interslide CV as low as 9% and intraslide CV 

as low as 15% when probing with a histidine-specific 

monoclonal antibody. CV was more variable when probing 

with human autoimmune sera, ranging from 5 to 36% with 

a median of 15%. Additional parameters for printing, 

probing, and analyzing microarrays were also investigated, 

and optimal conditions have been established to study 

autoantibody profiles in autoimmune disease. 

doi:10.1016/j.clim.2007.03.434 

Sa.47 Over-expression of Interferon Alpha in Lupus 

Families: Evidence for a Complex Heritable Trait 

Timothy Niewold, Fellow, Mary Kirkland Center for Lupus 

Research, Hospital for Special Surgery, New York, NY, John 

Harley, Professor, Oklahoma Medical Research Foundation, 

Oklahoma City, OK, Jing Hua, Research Fellow, Mary 

Kirkland Center for Lupus Research, Hospital for Special 

Surgery, New York, NY, Mary Crow, Professor, Mary Kirkland 

Center for Lupus Research, Hospital for Special Surgery, 

New York, NY, Thomas Lehman, Professor, Mary Kirkland 

Center for Lupus Research, Hospital for Special Surgery, 

New York, NY 

Background: Interferon alpha (IFN-α) levels are elevated 

in many patients with systemic lupus erythematosus 

(SLE), however it is not known whether increased IFN-α is 

a cause or a result of SLE. A haplotype of the IFN-α 

pathway gene IRF5 has been associated with risk of SLE. 

Methods: Serum IFN-α activity was measured in samples 

from the Hospital for Special Surgery Family Lupus 

Registry (n=226, 111 SLE) and the Lupus Multiplex Registry 

and Repository (n=445, 155 SLE) using a reporter cell 

assay and normalized to unrelated healthy donor samples 

(n=106). The rs2004640 and rs2070197 SNPs in IRF5 were 

genotyped using ABI Taqman probes. Results: 47% of SLE 

patients had high serum IFN-a activity, and 18% of healthy 

relatives of SLE patients also had high IFN-α activity 

compared to unrelated donors (p=0.0002). High IFN-α 

expression is clustered in certain families. 54% of high 

IFN-α SLE patients have a healthy first degree relative 

with high IFN-α (p=2x10-7), and SLE-affected sib pairs had 

concordant IFN-α expression in most cases (p=0.023). 

There was a trend toward higher IFN-α expression in SLE 

patients with the IRF5 SLE risk haplotype. There were no 

ethnic differences in IFN-α expression, but the IRF5 risk 

haplotype was much more common in European American 

and Hispanic populations (p=0.0018). Conclusions: Many 

SLE families demonstrate abnormally high IFN-α expression 

in both healthy and affected individuals compared to 

healthy unrelated donors, suggesting that high endogenous 

IFN-α expression is a heritable SLE risk factor. It is 

likely that many genetic factors underlie this complex 

trait. 

doi:10.1016/j.clim.2007.03.435 

Sa.48 Evaluation of the Polymorphism of IL-1RN 

Gene in Patients with SLE (Systemic Lupus 

Erythematosus) 

Rashin Ganjali, Researcher, Immunogenetics Department, 

Bu-Ali Research Institute, Mashhad University of Medical 

Sciences, Mashhad, Iran, Jalil Tavakkol Afshari, Vice 

President for Research, Immunogenetics Department, 

Bu-Ali Research Institute, Mashhad University of Medical 

Sciences, Mashhad, Iran, Maryam Mazhani, Researcher, 

Immunogenetics Department, Bu-Ali Research Institute, 

Mashhad University of Medical Sciences, Mashhad, Iran 


disease with unknown etiology. SLE is characterized by 

malregulation of the immune system, with hyperactive B cells 

synthesizing excessive amounts of different autoantibodies. 

Enhanced IL-1 gene expression and IL-1 protein secretion are 

detected in the pathogenesis of SLE. IL-1RN has a downregulatory 

effect; therefore it is supposed that its gene 

polymorphisms may contribute to the pathogenesis of SLE. A 

total of 34 patients with SLE and 26 healthy subjects were 

studied. DNA was extracted from whole blood by salting out 

method and IL-1RN genotype was determined after PCR. The 

data were analyzed by Chi-square and Fisher’s exact tests. 

There were no statistical differences between genotype 

frequencies in both groups. Except A4 and A5 alleles, there 

were no statistically differences between allele frequencies, 

too; while A4 and A5 had been increased in healthy subjects 

(p=0.07). In the present population, polymorphic genotypes of 

IL-1RN did not increase the susceptibility to SLE; while 

increased two polymorphic alleles in healthy subjects confirm 

this idea. However for/against relations between gene polymorphisms 

and such diseases require more studies with more 

individuals. 

doi:10.1016/j.clim.2007.03.436 

S89

S90 Abstracts 

Sa.49 TNF Receptor-associated Periodic Fever 

Syndrome (TRAPS) Caused by a Novel C30F Mutation 

Associated with the Common R92Q Low-Penetrance 

Mutation in TNFRSF1A 

Joao B. Oliveira, Associate Researcher, Molecular 

Immunology and Genetics Section, Laboratory of Medical 

Investigation #56, USP, São Paulo, Brazil, Adriana Jesus, 

Pediatric Rheumatology Second Year Fellow, Pediatrics 

Department, University of São Paulo, Brazil, São Paulo, 

Brazil, Ivona Aksentijevich, Staff Scientist, NIH/NIAMS/ 

GGB, Bethesda, MD, Clovis Silva, Head of Pediatric 

Rheumatology Unit, Pediatrics Department, University of 

São Paulo, Brazil, São Paulo, Brazil, Alberto J.S. Duarte, 

Lab Director, Laboratory of Medical Investigation #56 

(LIM_56), USP, São Paulo, Brazil 

Introduction: TRAPS is the most common of the autosomal 

dominant periodic fever syndromes. It is caused by 

mutations in the TNFRSF1A gene, which encodes for the 

type 1 TNF-receptor (TNFR1). We describe here a Brazilian 

patient with TRAPS and a novel TNFRSF1A mutation. 

Material and methods: The exons 2 to 5 and flanking 

intronic regions were amplified by PCR, purified and 

sequenced using an automated capillary sequencer. Results. 

The patient is a 9-year-old girl from São Paulo, Brazil. She 

presents recurrent fevers since age of 3 years, usually 

lasting 3 to 7 days, and recurring every other week. These 

episodes are associated with a mild abdominal pain, nausea, 

vomiting and generalized myalgia. Recurrent conjunctivitis 

and erysipela-like skin lesions in lower limbs are also 

present. Laboratory studies show persistent normocytic 

normochromic anemia, thrombocytosis, elevated erythrocyte 

sedimentation rate and C-reactive protein. Antinuclear 

antibody is positive (1:80). Physical examination is unremarkable, 

except for height in percentile 3 and weight in 

percentile 10–25. IgD level is within normal range. Genetic 

testing for Familial Mediterranean Fever (FMF) excluded five 

most common FMF mutations (M680I, M694V, M694I, V726A, 

E148Q). Mutational screening of TNFRSF1A revealed the 

novel C30F mutation and the common R92Q low-penetrance 

mutation. The R92Q is seen in 5% of the general population, 

and is associated with atypical inflammatory phenotypes. 

Conclusion: We report a TRAPS patient with the novel 

TNFRSF1 mutation, C30F, in conjunction with the low-penetrance 

mutation R92Q. Our findings expand the diversity of 

TNFRSF1A mutations associated with TRAPS. 

doi:10.1016/j.clim.2007.03.437 

Sa.50 Cryopyrin-associated Periodic Syndromes 

(CAPS): Report of Two Brazilian Cases, Including a 

Rare Non-Exon 3 CIAS1 Mutation 

Adriana Jesus, Second Year Pediatric Rheumatology Fellow, 

Pediatrics Department, University of São Paulo, Brazil, São 

Paulo, Brazil, Brazil Joao Oliveira, Associate Researcher, 

Molecular Immunology and Genetics Section, Laboratory of 

Medical Investigation #56, USP, São Paulo, Brazil, Ivona 

Aksentijevich, Staff Scientist, NIH/NIAMS/GGB, Bethesda, 

MD, Clovis Silva, Head of Pediatric Rheumatology Unit, 

Pediatrics Department, São Paulo, Brazil, Alberto J.S. 


#56 (LIM-56), USP, São Paulo, Brazil 

Introduction: Cryopyrin-associated periodic syndromes 

(CAPS) are autoinflammatory diseases that include three 

clinical syndromes linked to mutations in CIAS1 gene: familial 

cold autoinflammatory syndrome (FCAS), Muckle–Wells syndrome 

(MWS), and neonatal onset multisystem inflammatory 

disease (NOMID). We describe here 2 CAPS patients with the 

corresponding molecular defect. Material and methods: Exons 

and flanking intronic regions of CIAS1 were amplified by PCR, 

purified and sequenced using an automated capillary sequencer. 

Results: Patient 1 is an 8 year-old girl who presented at 

20 days of age with an erythematous and macular rash 

involving face, chest, abdomen, limbs, palms and soles. At 

3 months of age, she developed recurrent fever, exacerbated 

by cold weather. Elevated acute phase reactants were present. 

Arthritis of large joints and unilateral anterior uveitis developed 

during follow-up. DNA sequencing identified the T436I 

CIAS1 mutation. Patient 2 is a 7-year-old boy who soon after 

birth developed a papular exanthema involving chest, abdomen 

and extremities with daily fever episodes. Persistent 

anemia, leukocytosis, thrombocytosis, and high acute phase 

reactants were seen. Later, he presented with growth failure, 

frontal bossing and marked patellae enlargement. Cognitive 

and motor impairments and chronic intracranial hypertension 

were noted. Molecular studies identified a rare non-exon 3 

mutation, G755R, located in exon 4 of CIAS1. Conclusion: Two 

Brazilian patients with clinically and genetically related 

entities known as FCAS and NOMID were found to have 

disease-causing mutations in the CIAS1 gene, including one 

outside exon 3. Our result reinforces that CAPS patients should 

be screened for mutations in all exons of CIAS1. 

doi:10.1016/j.clim.2007.03.438 

Sa.51 Dickkopf-1 is a Link Between the Immune 

System and Bone Formation 

Georg Schett, Professor, University of Erlangen-Nuremberg, 

Erlangen, Danielle Diarra, Fellow, Medical University of 

Vienna, Vienna, Austria, William Richards, PhD, Amgen, 

Thousand Oaks, CA, Marina Stolina, PhD, Amgen, Thousand 

Oaks, CA 

Inflammatory joint disease can either lead to bone 

erosion or to bone formation. In rheumatoid arthritis, 

immune-mediated inflammation leads to joint destruction 

with virtually no signs of bone repair. In contrast, diseases 

such as ankylosing spondylitis are characterized by dramatic 

bone formation. The molecular signals determine whether 

inflammatory tissue induces bone loss or bone formation. By 

inhibiting Dickkopf-1 (DKK-1), a regulatory molecule of the 

Wnt pathway, we show that a bone destructive pattern of 

arthritis can be reversed into a bone-forming pattern. Thus, 

treatment of tumor necrosis factor transgenic mice by an 

antibody against DKK-1 completely inhibited bone erosions 

and induced the formation of bony nodules called osteophytes. 

Tumor necrosis factor α (TNF) was identified as key 

inducer of DKK-1 in mouse inflammatory arthritis as well as

Abstracts 

human rheumatoid arthritis. This suggests that the Wnt 

pathway is a key regulator of joint remodeling and a link 

between the immune system and bone formation. 

doi:10.1016/j.clim.2007.03.439 

Sa.52 Biosimulations Predict that Rituximab is More 

Efficacious than Other Standards of Care in 

Rheumatoid Arthritis Patients with Severe Disease 

Kosmas Kretsos, Engineer, Entelos, Inc., Foster City, CA, 

Daniel L. Young, Engineer, Entelos Inc., Foster City, CA, 

Chan C. Whiting, Scientist, Entelos, Inc., Foster City, CA, 

Katherine Kudrycki, Scientist, Entelos, Inc., Foster City, CA, 

Sirid-Aimee Kellermann, Senior Scientist, Entelos, Inc., 

Foster City, CA, Nadine A. Derfanoux, Senior Scientist, 

Entelos, Inc., Foster City, CA 

The clinical efficacy of the B cell-depleting therapy 

rituximab in patients with rheumatoid arthritis (RA) highlights 

the critical contribution of B cells to the disease 

process. To explore the efficacy of rituximab in RA patients 

with different phenotypes, the Entelos® RA PhysioLab® 

platform, a mathematical model representing an inflamed 

joint in an RA patient, was used to simulate the effects of 

rituximab and other therapeutic approaches on key clinical 

outcomes. The platform dynamically integrates the contributions 

of multiple cell types and mediators involved in synovial 

hyperplasia and joint structural damage. B cell development, 

recruitment, activation and effector processes (e.g., antigen 

presentation, cytokine synthesis and autoantibody production) 

are also represented in the platform. Biosimulation in a 

virtual patient with severe RA predicts an ACR response 

almost identical (within a 95% confidence interval) to that 

reported in rituximab clinical trials. Furthermore, the 

predicted level of B cell depletion in the virtual patient is 

consistent with published reports. These results suggest that 

rituximab will be a highly effective single-agent treatment 

for severe RA. However, similar analyses in a virtual patient 

with moderate RA show that rituximab is less effective than 

anti-TNF-α therapies. In both phenotypes, biosimulations 

predict that rituximab induces sustained benefits in joint 

structure, namely decreases in cartilage degradation and 

bone erosion, which persist for months after cessation of 

rituximab treatment, even after joint inflammation returns. 

Findings such as these can be used to help develop more 

targeted RA therapies and improve drug discovery by 

identifying prospective responder patient phenotypes. 

doi:10.1016/j.clim.2007.03.440 

Sa.53 Gene-environment Interactions Between 

Heavy Cigarette Smoking and HLA-DRB1 Shared 

Epitope in Predicting Incident RA, Data from a Two 

Large Prospective Cohort Studies 

Elizabeth W. Karlson, Associate Professor of Medicine, 

Brigham and Women’s Hospital, Department of Medicine, 

Dover, MA, Shun-Chiao Chang, Doctoral Student, Harvard 

School of Public Health, Epidemiology, Boston, MA, Karen H. 

Costenbader, Assistant Professor of Medicine, Brigham and 

Women’s Hospital, Department of Medicine, Boston, MA, 

Lori B. Chibnik, Statistician, Brigham and Women’s Hospital, 

Medicine, Boston, MA, Patricia Fraser, Medical Director, 

Pharmacovigilance/Medical Affairs, Cambridge, MA 

Background: Heavy smoking (N10 pack years) increases 

risk of RA. HLA-DRB1 demonstrates gene–environment 

interaction (GEI) with smoking when comparing current to 

never smokers. We studied this GEI in the Nurses’ Health 

Studies. Methods: RA diagnosis was validated by chart review 

for ACR criteria. Controls were matched on age, menopausal 

status, and postmenopausal hormone use. High resolution 

HLA-DRB1 genotyping was performed for SE alleles. HLA-SE, 

smoking, and HLA-SE×smoking interaction, and risk of RA 

were assessed using conditional logistic regression models, 

adjusted for age and reproductive factors. We tested for 

multiplicative and additive interaction. Results: 244 Caucasian 

matched pairs were included. Mean age at diagnosis was 

54 years; 59% of cases were RF+. There was no interaction 

between never/ever smoking and HLA-SE and risk of RA. We 

found no multiplicative interaction (p=0.10) but a strong 

additive interaction (p=0.006, attributable proportion (AP) 

0.49) with smoking categorized as b10 pack years vs. N10 

pack years. The highest risk was in heavy smokers with 

double copy HLA-SE (OR, 21.5; 95% CI, 2.8–167.9). There was 

evidence for additive interaction in RF+ (p=0.01, AP 0.47) 

and RF− cases (p=0.04, AP 0.53), each compared with 

controls. Models using pack years smoked as a continuous 

variable had no evidence for multiplicative interaction 

(p=0.33). Conclusion: GEI between HLA-SE and smoking in 

RA risk is strongest with N10 pack years smoked. Future 

studies should assess pack years smoked when testing for 

GEI. Additive interaction suggests a pathway that requires 

involvement of both risk factors. 

doi:10.1016/j.clim.2007.03.441 

S91 

Sa.54 Anti-fibrinbogen Autoimmunity in 

Rheumatoid Arthritis 

William Robinson, Assistant Professor, Division of 

Immunology and Rheumatology, Stanford University, Palo 

Alto, CA, Wolfgang Hueber, Research Associate, Division of 

Immunology and Rheumatology, Stanford University, Palo 

Alto, CA, Peggy P. Ho, Research Associate, Department 

Neurology, Stanford University, Stanford, CA, Xiaoyan Zhao, 

Postdoctoral Fellow, Division of Immunology and 

Rheumatology, Stanford University, Palo Alto, CA 

Rheumatoid arthritis (RA) is an autoimmune synovitis 

affecting 0.5% of the world population, yet the mechanisms 

underlying the initiation and perpetuation of RA remain 

elusive. A subset of RA patients exhibit anti-citrulline 

autoimmunity and citrullinated proteins are generated 

through post-transnational conversion of arginine to citrulline 

by peptidyl-arginine deiminase. The pathogenesis of RA 

involves the deposition and excessive local generation of 

fibrin in the inflamed joint, and recent studies identified 

citrullinated fibrin as a candidate autoantigen in RA. We 

performed direct immunoprecipitation of immune

S92 Abstracts 

complexes from RA and control synovial fluid and tissue 

lysates, followed by mass spectrometry (MS) analysis. MS/ 

MS analysis revealed previously described and novel 

candidate autoantigens, including native and citrullinated 

forms of fibrinogen, fibronectin, HSPs, vimentin, and 

elongation factor. We added peptides and proteins representing 

these candidates to synovial antigen arrays to 

screen samples derived from RA and control patient 

cohorts. Synovial mircroarray and ELISA analyses demonstrated 

specific autoreactive B cell targeting of citrullinated 

fibrinogen peptides and proteins in approximately 

30% of RA patients. Anti-citrullinated fibrinogen antibodies 

were present almost exclusively in patients possessing anti- 

CCP antibodies. We also detect fibrinogen-containing 

immune complexes in blood derived from patients possessing 

anti-fibrinogen autoantibodies. Based on these results 

we developed a fibrinogen-induced arthritis (FIA) mouse 

model using human fibrinogen as the immunizing antigen 

(see abstract presented by Ho et al.). These data support 

our hypothesis that citrullinated fibrinogen is a pathogenic 

autoantigen in RA, and that autoimmunity against fibrinogen 

defines a molecular subtype representing approximately 

30% of RA patients. 

doi:10.1016/j.clim.2007.03.442 

Sa.55 Genome-Wide SNP Scan Identifies 

Polymorphisms Associated with Differential 

Response to Anti-TNF Treatment Among 

Rheumatoid Arthritis Patients in the ABCoN Cohort 

John Carulli, Principal Scientist, BiogenIdec, Cambridge, 

MA, Franak Batliwalla, Assistant Investigator, Feinstein 

Institute for Medical Research, Manhasset, NY, Chunyu Liu, 

Scientist, BiogenIdec, Drug Discovery, Cambridge, MA, Aarti 

Damle, Assistant Investigator, Feinstein Institute for 

Medical Research, Manhasset, NY, Jadwiga Bienkowska, 

Principal Scientist, Biogenidec, Drug Discovery, Cambridge, 

MA, Annette Lee, Assistant Investigator, Feinstein Institute 

for Medical Research, Manhasset, NY, Wentian Li, Assistant 

Investigator, Feinstein Institute for Medical Research, 

Manhasset, NY, Peter Gregersen, Investigator, Feinstein 

Institute for Medical Research, Manhasset, NY 

The Autoimmune Biomarkers Collaborative Network 

(ABCoN) has enrolled a longitudinal cohort of RA patients 

beginning anti-TNF treatment in order to identify biomarkers 

influencing response to anti-TNF therapy. Genomewide 

(Illumina 317K) SNP data were collected on 116 of 

these patients (54 etaneracept, 25 adalimumab, 37 

infliximab). DAS28 scores for these 116 patients ranged 

from 2.72 to 7.08 (mean =5.23) prior to starting anti-TNF 

therapy, decreasing to 1.4 to 6.68 (mean=3.77) at 14 weeks 

after enrollment. SNP genotypes were tested for association 

with the change in DAS28 score at 6 weeks and 

14 weeks after initiation of anti-TNF therapy. While no 

individual SNPs show genome-wide significance (pb10 −7 )in 

this small data set, preliminary analysis shows 36 SNPs 

associated (pb10 −4 ) with DDAS28 at 6 weeks, and 41 SNPs 

associated with DDAS28 at 14 weeks. Nine regions of the 

genome have two or more SNPs within 100 kb with pb10 −5 , 

and genes in these regions represent candidates for anti- 

TNF pharmacogenetic markers. Permutation tests on these 

data indicate a low type I error rate. Haplotype analysis 

may reveal additional evidence for association. RNA 

samples (PaxGene) from peripheral blood of the same 

patients were analyzed by genome-wide transcript profiling. 

Cross-referencing of genes comprising expression-based 

classifiers for anti-TNF responders and non-responders (see 

FOCIS 2007 abstract by Batliwalla et al.) with genes showing 

nominal genetic association with anti-TNF response represents 

a powerful approach to identifying pharmacogenomic 

markers for anti-TNF response among rheumatoid arthritis 

patients. 

doi:10.1016/j.clim.2007.03.443 

Sa.56 Oncogenic Properties of Rheumatoid Synovial 

Cells 

Toshihiro Nakajima, Professor, Institute of Medical Science, 

St. Marianna University School of Medicine, Kawasaki, 

Japan, Naoko Yagishita, Senior Assistant Professor, Institute 

of Medical Science, St. Marianna University School of 

Medicine, Kawasaki, Japan, Satoshi Yamasaki, Senior 

Assistant Professor, Institute of Medical Science, St. 

Marianna University School of Medicine, Kawasaki, Japan, 

Kusuki Nishioka, Professor, I, Kawasaki, Japan 

Rheumatoid arthritis (RA) is one of the most common 

and critical articular diseases with synovial hyperplasia. 

However, the mechanism that regulates synovial cell outgrowth 

is not fully understood. To clarify the mechanism, 

we carried out immunoscreening using anti-rheumatoid 

synovial cell antibody and cloned a putative RING finger 

protein, designated Synoviolin (G&D, 2003). Synoviolin is an 

E3 ubiquitin ligase and located in endoreticulum. It was 

highly expressed in the rheumatoid synovium, and mice 

overexpressing this molecule developed spontaneous 

arthropathy. In this regard, like rheumatoid synovial cells, 

the anti-apoptotic effect of Synoviolin was observed even 

for its expressed ectopically in NIH3T3 cells, which resulted 

in enhanced cell overgrowth in these cells (MCB, 2005). 

These results were confirmed also in fly system. Next, to 

identify the substrates for Synoviolin that may be involved 

in cell growth, we analyzed the comprehensive profile of 

protein expression in Synoviolin null cells (JBC, 2005). 

Then, we found that Synoviolin targets tumor suppressor 

gene p53 for ubiquitination (EMBO J., 2007). Synoviolin 

sequestrated and metabolized p53 in the cytoplasm and 

negatively regulated its cellular level and biological 

functions, including transcription, cell cycle regulation 

and apoptosis. Furthermore, these p53 regulatory functions 

of Synoviolin were irrelevant to other E3 ubiquitin ligases 

for p53, such as MDM2, Pirh2 and Cop1, which form 

autoregulatory feedback loops. Our results provide novel 

insights into p53 signaling mediated by Synoviolin and 

clarify the molecular basis of synovial hyperplasia in RA. 

doi:10.1016/j.clim.2007.03.444

Abstracts 

Sa.57 T Lymphocyte Clonal Alterations in 

Anti-Citrullinated Protein Antibody Positive 

Synovitis 

Tineke Cantaert, PhD Student, University of Amsterdam, 

Amsterdam, The Netherlands, Sophie Brouard, PhD, INSERM 

U643, Nantes, France, Cristophe Braud, PhD, INSERM U643, 

Nantes, France, Jean-Paul Soulillou, Professor, INSERM 

U643, Nantes, France, Niek de Vries, PhD, Clinical 

Immunology and Rheumatology, Academic Medical Center, 

University of Amsterdam, Amsterdam, The Netherlands, 

Paul-Peter Tak, Professor, Clinical Immunology and 

Rheumatology, Academic Medical Center, University of 

Amsterdam, Amsterdam, The Netherlands, Dominique 

Baeten, Professor, Clinical Immunology and Rheumatology, 

Academic Medical Center/University of Amsterdam, 

Amsterdam, The Netherlands 

Objective: Increasing evidence suggests that the pathophysiology 

of rheumatoid arthritis (RA) is fundamentally 

different in patients with or without anti-citrullinated 

protein antibodies (ACPA). The association of RA with HLA- 

DR shared epitope exclusively in the ACPA+ subset supports 

this concept and suggests that T cell help may be involved in 

the ACPA response. We assessed the potential involvement 

of T lymphocytes in ACPA+ synovitis. Methods: Synovial 

biopsies were obtained from inflamed knee joints of 54 RA 

patients. 37 were ACPA+ (anti-CCP2 ELISA). mRNA was 

isolated from 7 ACPA+ RA synovia, 4 ACPA− RA synovia, and 

10 control spondyloarthritis (SpA) synovia with similar levels 

of T and B cell infiltration. T lymphocyte clonality in the 

synovium was studied by combined quantitative and 

qualitative TCR CDR3 analysis by the TcLandscape technology. 

Results: Comparing ACPA+ with the ACPA− RA patients, 

there were no significant differences in clinical and 

histological parameters. Alterations of the normal TCR 

length distribution were analyzed for all Vbeta families. 

TCR analysis showed a significantly higher degree of 

alteration of the TCR length distribution in ACPA+ (31.2± 

3.7%) compared to ACPA− (18.5±1.4%; p=0.012) and to SpA 

synovitis (22.4 ±1.3%; p=0.014). These alterations reflecting 

oligo- or monoclonal expansions were observed in several 

Vbeta families and were different in each individual sample. 

Conclusion: We demonstrate an increased alteration of the 

TCR length distribution in ACPA+ but not in ACPA− RA or 

control synovitis. These data suggest a specific contribution 

of T lymphocytes in the local disease process and possibly 

the ACPA response in this RA subset. 

doi:10.1016/j.clim.2007.03.445 

Sa.58 Synovial T/B Cell Lymphoid Aggregates 

Regulate the Production of Rheumatoid 

Arthritis-specific Autoantibodies 


Amsterdam, The Netherlands, Trieneke Timmer, PhD 

Student, Molecular Cell Biology and Immunology, VU 

Medical Center, Amsterdam, The Netherlands, Bernard 

Vandooren, PhD Student, Rheumatology, Ghent University 

Hospital, Ghent, Belgium, Carla Wijbrandts, PhD Student, 

Clinical Immunology and Rheumatology, Academic Medical 

Center/University of Amsterdam, Amsterdam, The 

Netherlands, Rogier Thurlings, PhD Student, Clinical 

Immunology and Rheumatology, Academic Medical Center/ 


Leen De Rycke, PhD, Clinical Immunology and 

Rheumatology, Academic Medical Center, University of 

Amsterdam, Amsterdam, The Netherlands, Tineke van der 

Pouw-Kraan, PhD, Molecular Cell Biology and Immunology, 

VU Medical Center, Amsterdam, The Netherlands, Theo Out, 

PhD, Experimental Immunology, Academic Medical Center/ 

University of Amsterdam, The Netherlands, Paul-Peter Tak, 

Professor, Clinical Immunology and Rheumatology, Academic 

Medical Center, University of Amsterdam, Amsterdam, The 

Netherlands, Dominique Baeten, Professor, Clinical 

Immunology and Rheumatology, Academic Medical Center, 

University of Amsterdam, Amsterdam, The Netherlands 

Ectopic lymphoid neogenesis occurs in synovial membrane 

in rheumatoid arthritis (RA) and inflamed tissues. 

Partial structural resemblance with follicles in secondary 

lymphoid organs suggests a role in the maturation of 

humoral immune and autoimmune responses. We investigated 

the relation between synovial T/B cell lymphoid 

aggregates, inflammation, and autoantibody production. 

Synovial biopsies were obtained from inflamed joint of 130 

patients with RA or spondyloarthritis (SpA), a chronic 

arthritis devoid of known autoantibodies. B–T cell aggregates 

were analyzed by immunohistochemistry and realtime 

PCR. Autoantibodies were measured in synovial fluid by 

ELISA, corrected for total IgG and IgM. The presence of large 

lymphoid aggregates was observed in 1/3 of the rheumatoid 

synovia, but also in SpA. This was associated with synovial 

inflammation in both diseases, with infiltrating memory B 

lymphocytes observed almost exclusively within these 

aggregates. TNFα blockade induced the disintegration of 

the follicles (pb0.05). Levels of CXCL13 and CCL19 were 

elevated in follicular versus diffuse synovitis (pb0.05). We 

could not observe a dark and light zone nor detect naive B 

cells by CD23/IgD staining. These structures occasionally 

displayed germinal centres characterized by high endothelial 

venules, T/B cell segregation, and follicular dendritic 

cells (b10%). This was confirmed by the low amount of 

CD21L mRNA. Both rheumatoid factor (p=0.033) and anticitrullinated 

protein antibodies (p=0.09) were lower in 

follicular versus diffuse RA synovitis. 

Ectopic lymphoid neogenesis is an inducible and reversible 

phenomenon in chronic synovitis. Germinal centre reactions 

can occasionally be observed in synovial follicles, which 

regulate rather than support local humoral autoimmunity in 

RA. 

doi:10.1016/j.clim.2007.03.447 

S93 

Sa.59 Non-Pathogenic Antinuclear Antibodies 

Contribute to the Clearance of Apoptotic Antigens 

Released During TNFα Blockade in Spondyloarthritis 

(SpA) 


Amsterdam, The Netherlands, Leen De Rycke, PhD, Clinical

S94 Abstracts 

Immunology and Rheumatology, Academic Medical Center/ 


Bernard Vandooren, PhD Student, Rheumatology, Ghent 

University Hospital, Ghent, Belgium, Carla Wijbrandts, PhD 

Student, Clinical Immunology and Rheumatology, Academic 

Medical Center, University of Amsterdam, Amsterdam, The 

Netherlands, Elli Kruithof, PhD, Rheumatology, Ghent 

University Hospital, Ghent, Belgium, Filip De Keyser, 

Professor, Rheumatology, Ghent University Hospital, Ghent, 

Belgium, Eric M. Veys, Professor, Rheumatology, Ghent 

University Hospital, Ghent, Belgium, Paul-Peter Tak, 

Professor, Clinical Immunology and Rheumatology, 

Academic Medical Center, University of Amsterdam, 

Amsterdam, The Netherlands, Dominique Baeten, Professor, 

Clinical Immunology and Rheumatology, Academic Medical 

Center, University of Amsterdam, Amsterdam, The 

Netherlands 

Infliximab, not etanercept, treatment in arthritis induces 

non-pathogenic, IgM antinuclear antibodies. In SLE, antinuclear 

antibodies have been related to increased BAFF and 

type I-interferon signaling and increased presence of 

apoptotic blebs. We assessed the mechanisms leading to 

autoantibody induction by TNFα blockade in spondyloarthritis 

(SpA), a disease without humoral autoimmunity. 

Serum and synovial biopsies were obtained from SpA 

patients treated with infliximab (n=20) or etanercept 

(n=20). Serum levels of BAFF, IFNα, nucleosomes, and 

anti-nucleosomes were assessed (ELISA). Synovial apoptosis 

was assessed by TUNEL and active caspase3. Nucleosome 

levels increased at 2 (+89%; p =0.002) and 6 (+26%; 

p=0.014) weeks of infliximab but not etanercept treatment. 

This was not related to cell death in the synovium at 

weeks 1 and 12 of treatment. Infliximab induced an 

upregulation of IFNα at 2 (p=0.090) and 6 (p=0.042) 

weeks. BAFF levels remained stable during treatment with 

both biologicals. Anti-nucleosome antibodies were 

increased at week 12 (+94%; pb0.001) and 34 (+124%; 

p b0.001) of infliximab treatment. This increase was 

restricted to IgM isotype and correlated with the rise of 

nucleosomes at week 12 (r =0.383;p =0.008) and 34 

(r =0.453; p=0.014). The continuous requirement of nuclear 

antigens for the autoantibody response was indicated by the 

disappearance of the antibodies upon interruption of anti- 

TNFα. However, nucleosome levels dropped to normal upon 

appearance of the autoantibodies. Infliximab induces nonpathogenic 

IgM antinuclear antibodies by upregulation of 

serum nucleosomes, suggesting induction of apoptosis in 

vivo, and the associated increase in type I IFN. This antibody 

response could represent a physiological mechanism which 

contributes to the normal clearance of nuclear antigens. TC 

and LDR contributed equally. 

doi:10.1016/j.clim.2007.03.448 

Sa.61 Proteomic Signatures of Secreted Proteins for 

the Prediction of Treatment Response to TNF 

Blockers in RA 

Wolfgang Hueber, Postdoctoral Fellow, Stanford University, 

Medicine, Palo Alto, CA, Beren Tomooka, Research 

Assistant, Stanford University, Medicine, Palo Alto, CA, 

Franak Batliwalla, Research Scientist, North Shore Long 

Island Jewish Health Care, Manhasset, NY, Robert 

Tibshirani, Professor, Stanford University, Stanford, CA, 

Peter Gregersen, Professor, North Shore Long Island Jewish 

Health Care System, Manhasset, NY, Lars Klareskog, 

Professor, Karolinska Institutet, Stockholm, Sweden, 

William Robinson, Professor, Stanford University, Medicine, 

Palo Alto, CA 

We profiled autoantibodies and cytokines in pre-treatment 

and 3-month post-treatment sera from RA patients to 

delineate secreted proteins predictive of response to 

therapy with etanercept. Subgroups from two cohorts of 

Enbrel-treated RA patients were analyzed: 29 patients 

enrolled in the ABCoN cohort, and 28 patients treated at a 

Swedish tertiary care hospital. Serum autoantibodies were 

determined using synovial antigen microarrays, and 14 

cytokines were measured using a bead-based multiplex 

assay. Serum was collected before and 3 months after 

initiation of therapy. Differential expression of autoantibodies 

and cytokines in serum from responders and nonresponders 

was analyzed. Prediction analysis of microarrays 

(PAM) was applied to identify panels of secreted molecules 

predictive of response. Pre-treatment samples from etanercept 

responders with ACR 50 or greater response but not 

non-responders demonstrated higher reactivity against 

peptides derived from RA candidate autoantigens including 

fibrinogen, calpastatin, heat shock proteins, proteoglycans, 

and histones (FDRb0.1). The cytokines IL-6, IL12p40 and 

Eotaxin showed a trend towards higher mean levels in pretreatment 

samples (pb0.1) in the Swedish but not in the 

ABCoN cohort. Pair-wise comparisons of pre-treatment with 

post-treatment profiles demonstrated that decreases in 

certain autoantibody reactivities and the cytokine IL-6 were 

associated with ACR 50 or greater responses. PAM identified 

a panel of secreted molecules that correctly predicted 73% 

of patients who subsequently responded to etanercept 

therapy with ACR 50 responses or greater. Our data suggest 

that profiles of secreted molecules in pre-treatment sera 

are associated with RA patients more likely to respond to 

etanercept. 

doi:10.1016/j.clim.2007.03.449 

Sa.62 Distinct Molecular and Cellular Aspects of 

Systemic Juvenile Idiopathic Arthritis (SJIA) and 

Polyarticular (PolyJIA) 

Claudia Macaubas, Research Associate, Stanford University 

Department of Pediatrics, Stanford, CA, Khoa Nguyen, BS, 

Stanford University Department of Pediatrics, Stanford, CA, 

Kuang-Hung Pan, PhD Student, Stanford University 

Department of Genetics, Stanford, CA, Tzielan Lee, Clinical 

Assistant Professor, Stanford University Department of 

Pediatrics, Stanford, CA, Chetan Deshpande, Lab Manager, 

Stanford University Department of Pediatrics, Stanford, CA, 

Christy Sandborg, Professor and Chief, Stanford 

University Department of Pediatrics, Stanford, CA, 

Stanley Cohen, Professor, Stanford University Department 

of Genetics, Stanford, CA, Elizabeth Mellins, Associate

Abstracts 

Professor, Stanford University Department of Pediatrics, 

Stanford, CA 

Juvenile idiopathic arthritis (JIA) is a family of chronic 

inflammatory arthritides that includes distinct subtypes: 

systemic (SJIA), polyarticular (polyJIA) and pauciarticular. 

The subtypes differ in epidemiology, genetic risk factors, 

clinical features, prognosis and response to therapy. 

However, the specific molecular mechanisms that distinguish 

these diseases are not well understood. We performed 

a comparative analysis of molecular and cellular aspects of 

SJIA and polyJIA. Blood samples and associated clinical 

information were obtained from children with SJIA (n=24), 

polyJIA (n=17), and age-matched children without immunological 

health problems (n =14). Gene expression in 

freshly isolated PBMCs was studied by quantitative PCR 

(qPCR) of 81 immune related genes. Cell types were 

enumerated by FACS. Using GABRIEL analysis software, we 

identified genes whose expression correlates with erythrocyte 

sedimentation rate (ESR). In SJIA, ESR is positively 

correlated with transcripts expressed by activated monocytes, 

whereas in polyJIA, ESR correlates with transcripts 

for cytolytic proteins and Th1-related genes. Expression of 

distinct genes correlates with number of active joints in 

SJIA and polyJIA, including IL2R and MIF vs. IL-12, 

respectively. At the cellular level, we observed expansion 

of monocytes and altered monocyte subset distribution 

(CD14hi vs. CD16hi) in SJIA, but not polyJIA subjects, 

compared to controls. Samples from poly JIA subjects 

showed a reduced percentage of NK cells, and lower 

percentage of T regulatory cells. Our findings argue that 

SJIA is associated with dysregulation of innate cells, 

whereas poly JIA is associated with altered adaptive 

immunity. 

doi:10.1016/j.clim.2007.03.450 

Sa.63 The Alternative Pathway is Necessary and 

Sufficient for Complement Involvement in the 

Effector Phase of Anti-Collagen Antibody Induced 

Arthritis (ACAIA) 

Nirmal Banda, Associate Professor, University of Colorado at 

Denver Health Sciences Center, Aurora, CO, Allyson Wood, 

Research Associate, University of Colorado at Denver Health 

Sciences Center, Aurora, CO, Kazue Takahashi, Assistant 

Professor, Pediatrics, Massachusetts General Hospital, 

Boston, MA, William P. Arend, Professor of Medicine, 

University of Colorado at Denver Health Sciences Center, 

Aurora, CO, Alan Ezekowitz, Professor, Pediatrics, 

Massachusetts General Hospital, Boston, MA, V. Michael 

Holers, Professor of Medicine and Immunology, University of 

Colorado at Denver and Health Sciences Center, Aurora, CO 

We reported that ACAIA is unchanged in classical/lectin 

pathway C4-deficient, mice but 90% decreased in alternative 

pathway factor-B-deficient mice (J. Immunol. 2006; 

177: 1904). We have further examined the role of 

complement pathways in ACAIA using C57BL/6 mice 

genetically deficient in components C3 (C3−/−) or C1q 

(C1q−/−), deficient in both mannose-binding lectins A and 

C (MBL−/−), and doubly deficient in both C1q and MBL-A/C 

(C1q−/−/MBL−/−), with only the alternative pathway 

intact. Arthritis was induced by a mixture of 4 anti-type II 

collagen monoclonal antibodies, with an IP injection at day 

3 of LPS to cycle arthritis development. Mice were scored 

daily for arthritis in the paws using a scale of 0–3 

(maximum score of 12 per mouse) and sacrificed on day 

10. There were no significant differences in the clinical 

disease activity scores (CDAS) between C1q−/− mice and 

wild type (WT, normal C57BL/6 mice), MBL−/− and WT 

mice, or C1q−/−/MBL−/− and WT mice. In contrast, similar 

to factor B-deficient mice, CDAS in C3−/− mice were 66% 

decreased in comparison to WT mice (3.75 ±1.03 and 11.75 

±0.25, mean±SEM, n=4, respectively). The histological 

scores for inflammation, pannus formation, bone damage 

and cartilage damage, as well as immunohistochemical 

scores for the deposition of C3, were all consistent with the 

CDAS. These results indicate that the alternative pathway 

of complement is unique in being both necessary and 

sufficient for complement involvement in the effector 

phase of ACAIA. Potential mechanism(s) underlying this key 

role are under investigation. 

doi:10.1016/j.clim.2007.03.451 

S95 

Sa.64 Anti-Inflammatory Effect of Modified- 

Extracellular Matrix Proteins (MECMP) in CIA 

Perla Macip-Rodríguez, Physician, Instituto Nacional de 

Ciencias Medicas y Nutricion SZ, Department of 

Immunology and Rheumatology, Mexico, Janette Furuzawa- 

Carballeda, Chemistry, Instituto Nacional de Ciencias 

Medicas y Nutricion SZ, Department of Immunology and 

Rheumatology, Mexico, Ma. Inés Vargas-Rojas, MD, Instituto 

Nacional de Ciencias Medicas y Nutricion SZ, Department of 

Immunology and Rheumatology, Mexico, Virginia 

Soto-Abraham, Physician, Instituto Nacional de Cardiología 

ICh. Department of Pathology, Mexico, David Cruz-Robles, 

Biologist, Instituto Nacional de Cardiología ICh, 

Department of Pathology, Mexico, Leonardo Limón- 

Camacho, Physician, Instituto Nacional de Ciencias Medicas 

y Nutricion SZ, Department of Immunology and 

Rheumatology, Mexico 

The aim of this study was to evaluate the effect of 

intradermal MECMP administration on a type II collagen 

(CII)-induced arthritis (CIA). Age-matched mice were 

immunized intradermally at the base of the tail with 100 

μg of chicken CII emulsified in complete Freund’s adjuvant. 

Mice were then boosted with 100 μg of CII in incomplete 

Freund’s adjuvant at 21 days. Mice were treated 1 week 

after boost with 100 μl of (a) placebo, (b) matrikines 

(hydrolyzed-collagen and elastin), (c) collagen-PVP, (d) b+c 

and (e) methotrexate (2.5 mg/kg) and every week during 

1 month. Clinical scores were assessed immediately before 

immunization (day 0) and thereafter weekly. Inflammation 

of the four paws was cored as follows: 0, no inflammation; 

1, swelling or redness of one joint; 2, swelling or redness of 

more than one joint or mild inflammation of the whole 

paw; 3, severe inflammation of whole paw or ankylosis. The 

incidence of CIA was 100% by day 28 in CII challenged mice.

S96 Abstracts 

Weight and temperature were also determined. Macroscopical 

analysis showed a down-regulation of inflammation 

post administration of MECMP and methotrexate treatments. 

The histological analysis showed that CIA-mice 

group had an extensive bone erosion, pannus and severe 

focal inflammatory infiltrates. MECMP mice-treated group 

ameliorated the clinical signs. Bone erosion and inflammation 

were moderate compared to CIA-mice group. Concluding, 

MECMP exerts a down-regulation of inflammation on 

established inflammation and is able to ameliorate the 

tissue damage associated with CIA. 

doi:10.1016/j.clim.2007.03.452 

Sa.65 Established Murine Collagen-induced Arthritis 

and IL-17 Production are Specifically Inhibited by a 

Single Inoculation of Lipopolysaccharide-stimulated 


Juan C. Aguillon, Biochemist, Universidad de Chile, 

Santiago, Chile, Lorena Salazar, Biochemist, Universidad 

de Chile, Immunology Program, Santiago, Chile, Octavio 

Aravena, Biochemist, Universidad de Chile, Immunology 

Program, Santiago, Chile, Carlos Gonzalez, DVM, 

Universidad de Chile, Facultad de Ciencias Veterinarias, 

Santiago, Chile, Paula Abello, DVM, Universidad de Chile, 

Immunology Program, Santiago, Juan Contreras-Levicoy, 

MD, Universidad de Chile, Immunology Program, Santiago, 

Chile, Barbara Pesce, Biochemist, Universidad de Chile, 

Immunology Program, Santiago, Chile, Flavi Salazar- 

Onfray, Biologist, Universidad de Chile, Immunology 

Program, Santiago, Chile, Miguel Cuchacovich, MD, 

Hospital Clinico Universidad de Chile, Santiago, Chile 

Dendritic cells (DCs), crucial in immune responses, are 

also involved in peripheral tolerance induction. TNFmodulated 

DCs have demonstrated to restore tolerance 

in experimental multiple sclerosis and collagen induced 

arthritis (CIA). We investigated the capacity of short-term 

lipopolysaccharide (LPS)-stimulated DCs pulsed with type II 

collagen (CII) to induce tolerance against established CIA. 

Bone marrow-derived DCs were generated in the presence 

of GM-CSF. After CIA induction with CII, mice were 

injected at day 35 with a single dose of immature DCs, 4 

or 24 h LPS-stimulated DCs that had been loaded with CII 

(CII/DCs, 4 h LPS/CII/DCs, or 24 h LPS/CII/DCs). Arthritis 

progression was monitored by clinical and histologic 

evaluations. Flow cytometry of 4 h LPS/CII/DCs showed 

intermediate CD40 and CD86 expression, lower than that 

of 24 h LPS/CII/DCs (fully mature), and higher than that of 

CII/DCs (immature). A functional assay showed that 4 h 

LPS/CII/DCs display increased endocytosis ability with 

respect to 24 h LPS/CII/DCs, indicating a semi-mature 

state. The single inoculation of 4 h LPS/CII/DCs in mice 

with established CIA reduced significantly disease severity 

at day 70. Histologic evaluation of mice treated with 4 h 

LPS/CII/DCs revealed diminished inflammatory synovitis, 

cartilage damage, and fibrosis. Unlike CII-stimulated 

splenocytes from CIA control mice, those from mice 

inoculated with 4 h LPS/CII/DCs showed diminished IL-17 

and increased IL-10 levels in culture supernatants. The 

high IL-10 production is consistent with the induction of 

TR1 regulatory T cells responsible for inhibiting to effector 

IL-17-producing CD4+ T cells. Conclusion: Short-term LPSmodulated 

DCs inoculation interferes with CIA progression 

and IL-17 production when loaded with CII. Supported by 

Fondecyt-1070553, Millennium Nucleus-P04/030-F and 

Mecesup-UCH 0115. 

doi:10.1016/j.clim.2007.03.453 

Sa.66 Kinetics of Immune Tolerization in a Clinical 

Trial on Epitope-specific Therapy in Rheumatoid 

Arthritis (RA) 

Eva Koffeman, University of California San Diego, 

Department of Medicine and Pediatrics, Utrecht, The 

Netherlands, Diane Amox, University of California San 

Diego, Department of Medicine and Pediatrics, La Jolla, CA, 

Adriana Tremoulet, University of California San Diego, 

Department of Medicine and Pediatrics, La Jolla, CA, Elissa 

Keogh, University of California San Diego, Department of 

Medicine and Pediatrics, La Jolla, CA, Peter Zeiseniss, 

University of California San Diego, Department of Medicine 

and Pediatrics, La Jolla, CA, Courtney Gosch, University of 

California San Diego, Department of Medicine and 

Pediatrics, La Jolla, CA, Samantha Friend, University of 

California San Diego, Department of Medicine and 

Pediatrics, La Jolla, CA, Xiao Zhang, University of Alabama 

at Birmingham, School of Public Health Department of 

Biostatistics, Birmingham, AL, Gary Cutter, University of 

Alabama at Birmingham, School of Public Health, 

Department of Biostatistics, Birmingham, AL, Salvatore 

Albani, Professor, University of California San Diego, 

Department of Medicine and Pediatrics, La Jolla, CA 

Background: In a phase II trial on mucosal tolerization 

in 160 RA-patients, dnaJp1 (E. coli heat shock protein 

dnaJ-peptide) 25 mg 1dd for 168 days was safe and 

clinically effective. Objectives: (i) Study immunological 

kinetics underlying clinical response. (ii) Identify biological 

markers predicting clinical response. Methods: PBMCs 

collected on days 0, 112 and 168 were cultured with 

dnaJP1, Tetanus Toxoid and controls and FACS-stained for 

CD3 and intracellular cytokines. Clinical response is ≥1 

ACR20 (American College of Rheumatology) response 

between day 112 and 1-month follow-up. Results: High 

CD69 expression upon dnaJp1 in vitro on day 0 associated 

with later clinical amelioration (ACR20 112–168 p=0.06, 

ACR50 p=0.02). TNFa production to dnaJp1 decreased 

before day 112 (0–112 pb0.0001) in the dnaJp1 group, 

remaining low on day 168. In the clinical responders 

subgroup, trends were seen of an increase in IL10 (0–168 

p=0.09) and initial decrease (0–112 p=0.17) and late 

increase in IFNg (112–168 p=0.15). IFNg increase was 

associated with the IL10 increase in dnaJP1 patients 

(p=0.10). Reactions to Tetanus Toxoid and reactions in 

the placebo group did not change. Discussion: CD69 

expression to dnaJp1 in vitro may be a predictor of 

clinical effect. This suggests that antigen-specific T cells 

play an important role in mucosal tolerization. Cytokine 

data suggest that tolerization started with deviation of

Abstracts 

antigen-specific T-effectors (TNFa decrease IFNg decrease, 

IL10 increase), followed by induction of regulatory T cell 

mechanisms (IL10+/IFNg+ Tr1) and clinical amelioration. 

doi:10.1016/j.clim.2007.03.454 

Sa.67 Nasal Co-Administration of ISS Increases the 

Arthritis-Protective Effect of an HSP60-derived 

Peptide 

Evelien Zonneveld-Huijssoon, University Medical Center 

Utrecht, Pediatric Immunology, Utrecht, The Netherlands, 

Femke van Wijk, University Medical Center Utrecht, 

Pediatric Immunology, Utrecht, The Netherlands, Wilco de 

Jager, Mr, University Medical Center Utrecht, Pediatric 

Immunology, Utrecht, The Netherlands, Sarah Roord, 

University Medical Center Utrecht, Pediatric Immunology, 

Utrecht, The Netherlands, Salvatore Albani, University of 

California San Diego, Medicine and Pediatrics, La Jolla, CA, 

Wietse Kuis, University Medical Center Utrecht, Pediatric 

Immunology, Utrecht, The Netherlands, Berent Prakken, 

University Medical Center Utrecht, Pediatric Immunology, 

Utrecht, The Netherlands 

Peptide-based immunotherapy is a promising way to treat 

autoimmune diseases. In earlier studies we identified T cell 

epitopes derived from human and mycobacterial HSP60 that 

are recognized by a majority of patients with rheumatoid or 

juvenile idiopathic arthritis. Nasal administration of one of 

these epitopes (p1) partially prevented adjuvant arthritis in 

the rat. To further enhance this arthritis-protective effect we 

tested two types of ISS and peptidoglycan (PGN) as adjuvants. 

Rats were treated with three nasal doses of p1, adjuvant or a 

combination of p1 and adjuvant prior to arthritis induction 

with CFA. Sixty days after arthritis induction, mandibular and 

inguinal lymph nodes and spleens were harvested. We 

determined phenotype, proliferative responses and cytokine 

production of fresh and in vitro stimulated cells. P1+ISS cotreatment 

enhanced the arthritis-protective effect of p1. ISS 

treatment alone did not have any effect on arthritis scores, 

nor did P1+PGN co-treatment or PGN alone. P1+ISS cotreatment 

led to increased p1-induced IFNg production by 

spleen cells. Co-treatment with one of the tested ISS led to 

increased IL-10 production as well. No clear differences could 

be detected between treatment groups in percentage of CD4 

+IL10+IFNg+ Tcells or CD4+CD25+Foxp3+ Tcells. Clinical data 

so far imply that ISS may be a suitable adjuvant for peptidespecific 

immune therapy in arthritis. Future plans include 

adoptive transfer of p1+ISS treated spleen cells and a further 

analysis of the induced p1 specific T cells and dendritic cells. 

doi:10.1016/j.clim.2007.03.455 

Sa.68 Abrogation of Autoimmune-mediated 

Inflammation by the Natural Plant Product, 

Honokiol via GABA(A)-mediated Alteration of 

CD40 and LMP1 Signaling in B Cells 

Melissa E. Munroe, Postdoctoral Research Fellow, University 

of Iowa Department of Microbiology, Iowa City, IA, Gail A. 

Bishop, Professor, University of Iowa Department of Internal 

Medicine and Microbiology and the VAMC, Iowa City, IA 

Our lab has extensively studied signaling in B cells by 

CD40 and its viral mimic, LMP1, which signals in an 

amplified and sustained manner, and has been implicated 

in pathogenesis of lymphoma and autoimmune disease. 

Honokiol (HNK), a phenolic compound isolated and purified 

from magnolia, has been found to have a number of 

pharmacologic benefits, including anti-inflammatory properties, 

without the toxicity found in vivo with current 

treatments. HNK stabilized already established inflammatory 

arthritis, with a decrease in TNF-α, IL-6, and collagenspecific, 

pro-inflammatory mediated, antibodies. We evaluated 

potential molecular mechanisms leading to the antiinflammatory 

effects of HNK using B cell lines expressing 

either hCD40 or the hCD40-LMP1 chimeric receptor. CD40 

and LMP1 mediated NFκB and AP-1 pathways were 

abrogated, but not eliminated by HNK, with a dosedependent 

decrease in nuclear translocation of NFκB1, 

NFκB2, and AP-1 associated proteins. Furthermore, the 

anti-inflammatory effects of HNK could be reversed using 

inhibitors of the neurotransmitter GABA(A), a previously 

reported target for HNK interaction. We are currently 

investigating potential downstream mediators of GABA 

activation which may affect CD40 and LMP1 signaling, 

including kinases and proteosome function. These findings 

are particularly exciting and suggest that the nontoxic antiinflammatory 

properties of HNK could be a means for 

blocking the autoimmune response. 

doi:10.1016/j.clim.2007.03.456 

S97 

Sa.69 Reversal of CD4:CD8 Ratio in Pediatric 

Patients with Macrophage Activation Syndrome/ 

Reactive Hemophagocytic Lymphohistiocytosis 

(MAS/HLH): A Potential Marker for Active Disease 

Process 

Paivi Miettunen, Doctor, Division of Pediatric Rheumatology 

and University of Calgary, Calgary, Alberta, AB, Canada 

Background: MAS/HLH in pediatric rheumatology refers 

to excessive activation and proliferation of mature macrophages 

leading to an overwhelming inflammatory reaction. 

While abnormal T cell activation (TCA) has been postulated 

to be at the center of this process, the details of the 

immunological mechanisms that initiate and maintain the 

abnormal macrophage functions are unclear. Objective: To 

test the hypothesis that alterations in CD4:CD8 ratios 

would provide an early marker for abnormal TCA in MAS/ 

HLH. Methods: All patients met Histiocytosis Society’s 2004 

criteria for HLH. Peripheral blood was collected at the time 

of diagnosis and at remission (n=7, 4 F; 3 M: ages 4 to 16 

years). Blood from age and sex matched healthy children 

was used as controls. Peripheral blood CD4:CD8 ratios were 

analyzed by flow cytometry. Results: Significant reversal of 

CD4:CD8 ratio was seen in four of seven patients with 

active MAS/HLH. An infectious process was ruled out 

(negative HIV, EBV and CMV serology). At the time of

S98 Abstracts 

remission CD4:CD8 ratio had normalized. In one patient 

with reactivation of MAS/HLH, a reversal of the CD4:CD8 

ratio recurred, and normalized with cyclosporin, corticosteroids 

and interleukin-1 antagonist, anakinra treatment. 

Conclusions: Our preliminary studies indicate that reversal 

of CD4:CD8 can be an important marker for active MAS/ 

HLH in a significant proportion of patients, supporting role 

of abnormal T cell activation. CD4:CD8 ratio can also be 

used to follow treatment response in selected patients. 

Studies are in progress to identify additional immunological 

markers to diagnose this condition accurately and to 

delineate the potential mechanisms involved in this 

process. 

doi:10.1016/j.clim.2007.03.457 

Sa.70 Quantitative Biomarker Analysis of Anti-CCP, 

Rheumatoid Factor (RF) and Immunoglobulin Levels 

in Synovial Biopsies Indicate Enrichment and Local 

Production in Rheumatoid Arthritis (RA) 

Sanna Rosengren, Associate Project Scientist, University of 

California, San Diego, Medicine, La Jolla, CA, Nathan Wei, 

Clinical Director, Arthritis and Osteoporosis Center, 

Frederick, MD, David Boyle, Associate Research 

Immunologist, University of California, San Diego, 

Medicine, La Jolla, CA, Nathan Zvaifler, Professor Emeritus, 

University of California, San Diego, Medicine, La Jolla, CA 

Circulating autoantibodies such as RF and anti-CCP are 

associated with RA. B cells and plasma cells accumulate in RA 

synovium, yet their contribution to autoantibody production 

is unclear. We developed ELISA-based methods to quantify 

autoantibodies and total Ig (immunoglobulin) G and IgM in 

extracts of RA and osteoarthritis synovial tissues prepared 

using 1% Brij-35 in PBS. Synovial tissue and matched serum 

were obtained at arthroplasty or arthroscopic biopsy from RA 

(n=11) and OA (n=6) patients. RF-IgM, anti-CCP and antitetanus 

IgG, total IgM and IgG, and albumin were determined 

by ELISA in synovial extracts and serum. Specific activity was 

calculated as the ratio of synovial to serum antibody/ 

albumin. IgG1 and IgM heavy constant mRNA levels were 

determined by qPCR as a measure of synovial Ig synthesis. 

Interestingly, anti-CCP IgG, but not RF-IgM, was significantly 

enriched in RA synovia compared to serum. OA synovial 

extracts contained no detectable autoantibodies. Total IgG 

specific activity was significantly higher in RA synovia than in 

OA (median (range): RA 2.58 (1.22–2.99); OA 0.76 (0.73– 

0.87), p=0.0077). Similar results were obtained for total IgM 

and tetanus IgG. Notably, mRNA content and specific activity 

correlated significantly for both IgM and IgG. Synovia 

containing lymphocyte aggregates (n=3) contained significantly 

higher total IgG but not total IgM. In conclusion, 

correlations between immunoglobulin message and protein 

indicate that local synthesis contributes to synovial Ig 

content. Reliable determinations of autoantibodies, antitetanus 

IgG, and total IgM and IgG in synovial biopsy extracts 

will be of value in proof-of-concept clinical trials. 

doi:10.1016/j.clim.2007.03.458 

Sa.71 Thrombin-activatable Carboxypeptidase B 

Prevents Anti-Collagen Antibody Induced Arthritis 

Jason Song, Rheumatology Fellow, Department of Medicine 

Division of Rheumatology Stanford University, Palo Alto, 

CA, Toshihiko Nishimura, Life Science Research Assistant, 

Department of Medicine, Division of Pulmonary and Critical 

Care Medicine, Stanford University, Menlo Park, CA, Michael 

Garcia, Undergraduate Student, Department of Medicine 

Division of Rheumatology Stanford University, Palo Alto, 

CA, Timothy Myles, Senior Research Scientist, Department 

of Medicine, Division of Hematology, Stanford University, 

Stanford, CA, Peggy Ho, Basic Life Science Research 

Associate, Department of Neurology, Stanford University, 

Stanford, CA, Xiaoyan Du, Post Doc, Department of 

Medicine, Division of Hematology, Stanford University, 

Stanford, CA, Shadi Sharif, Life Science Research Assistant, 

Department of Medicine, Division of Hematology, Stanford 

University, Stanford, CA, Lawrence Leung, Professor of 

Medicine, Department of Medicine, Division of Hematology, 

Stanford University, Stanford, CA, William Robinson, 

Assistant Professor in Medicine, Department of Medicine 

Division of Rheumatology Stanford University, Palo Alto, CA 

Thrombin-activatable carboxypeptidase B (CPB) is well 

established to play an anti-fibrinolytic role by removing Cterminal 

lysine residues from fibrin, thereby preventing its 

cleavage by plasmin. Recently, C3a, C5a, thrombin-cleaved 

osteopontin and bradykinin have been identified as additional 

substrates of CPB. CPB removes arginine residues from 

the C-terminus of these inflammatory mediators, thereby 

altering the biologic functions. We investigated the role of 

CPB in arthritis using proCPB deficient mice. Arthritis was 

induced by an intravenous injection of anti-collagen antibodies 

on day 0, followed by intraperitoneal injection of 

lipopolysaccharide on day 3. Arthritis was assessed by visual 

score and paw thickness. On day 10 post-injection, joints 

were collected for histology. Arthritis was also studied in 

bradykinin receptor−/− mice and C5−/− mice. As compared 

to controls, proCPB−/− mice exhibited significantly more 

severe arthritis (on day 10 arthritis score 12.3 vs. 0.4*; paw 

thickness 3.02 vs. 2.21 mm*, *pb0.01). Histology, graded 0– 

4, demonstrated severe arthritis in proCPB −/− mice versus 

controls (synovitis 3.7 vs. 1.2*, pannus 2.8 vs. 1.1*, bone 

erosion 3.2 vs. 0.7*, *pb0.01). C5−/− mice were resistant to 

induction of arthritis, while bradykinin receptor−/− mice 

exhibited a similar severity of arthritis to controls. These 

results suggest that CPB plays a critical role in regulating 

inflammation in anti-collagen antibody induced arthritis, 

and that this effect could be in part mediated by its downregulation 

of C5a activity. Studies are underway to determine 

the impact of CPB on C5a, C3a, and thrombin-cleaved 

osteopontin in the pathogenesis of this arthritis model. 

doi:10.1016/j.clim.2007.03.459 

Sa.72 Kinetics of Cytokine Gene Expression After 

Antigen Stimulation in Frozen Human PBMCs Using 

Quantitative Real-Time PCR and Flow Cytometry 

Annick van de Ven, MD Student, University of California, San 

Diego, La Jolla, CA, Pediatrics/University of Utrecht, The

Abstracts 

Netherlands, Elissa Keogh, Senior Research Associate, 

Supervisor, University of California, San Diego, La Jolla, CA, 

Negar Ghahramani, Staff Research Associate, Pediatrics, 

University of California, San Diego, La Jolla, CA, Salvatore 

Albani, Professor of Pediatrics, University of California, San 

Diego, Department of Pediatrics, La Jolla, CA 

Background: Understanding of antigen-induced kinetics of 

gene expression facilitates the analysis of immune responses 

using RT-PCR and DNA microarrays. Therefore, cytokine 

expression was investigated using 2 distinct antigenic stimuli: 

a common recall antigen (influenza peptide HA307–319) and 

the peptide epitope dnaJP1 associated with the pathogenesis 

of rheumatoid arthritis. Method: Frozen PBMCs from 5 

dnaJP1-responsive RA patients and fresh and frozen PBMCs 

from one healthy, influenza-vaccinated donor were stimulated 

for up to 96 hours with or without dnaJP1 and HA307– 

319, respectively. At designated intervals, aliquots were 

removed for measurement of cytokines by TaqMan. Additionally, 

intracellular IL-2 and TNFα were measured by FACS. 

Results: Following dnaJP1 stimulation, both IL-2 and IFNγ 

expressions were detectable by TaqMan at 12–36 hours while 

TNFα was undetectable. Intracellular IL-2 exhibited a 

biphasic profile (48 and 96 hours) and TNFα expression was 

detected at 48 hours. It should be noted that gene expression 

at 6 hours was highly nonspecific. When fresh PBMCs were 

stimulated with HA307–319, IL-2 expression was biphasic (24 

and 48 hours) while TNFα and INFγ peaked at 12 and 48 hours, 

respectively. Gene expression in frozen PBMCs appeared to be 

delayed by about 12 hours. Conclusions: Peptide antigen 

stimulation induces cytokine expression more slowly than 

that observed for mitogen stimulation. Gene expression also 

depends, to some extent, on the gene of interest. As 

expected, intracellular detection follows gene expression 

by 24–36 hours. Additionally, PBMCs require a 12 hour 

recovery period after cryopreservation. 

doi:10.1016/j.clim.2007.03.460 

Sa.73 Epitope-specific Immune Tolerance Can 

Maintain the Disease Control Induced by 

Conventional Methotrexate—Etanercept 

Combination Therapy in Rat Adjuvant Arthritis 

Negar Ghahramani, Staff Research Associate, University of 

California, San Diego, Department of Pediatrics, La Jolla, 

CA, Elissa Keogh, Staff Research Associate, Supervisor, 

University of California, San Diego Department of 

Pediatrics, La Jolla, CA, Annick van de Ven, MD Student, 

University of California, San Diego Pediatrics/University of 

Utrecht, The Netherlands, La Jolla, CA, Salvatore Albani, 

Professor, University of California, San Diego, Department 

of Pediatrics, La Jolla, CA 

Tools to maintain remission that allow tapering from 

aggressive therapies are still lacking for the majority of 

patients with rheumatoid arthritis. The success for longterm 

disease control with minimal background therapy 

could lie in the ability to establish tolerance. This approach 

can be complementary to the blend of blockade and 

suppression which characterizes current therapies. We 

have demonstrated that in rats with adjuvant arthritis 

(AA), mucosal tolerization to an arthritogenic peptide 

(hsp60 p180–188) can lead to significant improvement and 

immune deviation. In this study, the effect of nasal hsp60 

p180–188 peptide treatment (0.1 mg, days 10, 13 and 16) in 

combination with oral methotrexate (0.3 mg/kg, days 9 and 

13) and s.c. etanercept (0.3 mg/kg, day 9) was compared 

with standard etanercept–methotrexate treatment regimen. 

Results clearly indicate that the combination of 

mucosal tolerization to hsp60 p180–188 with single dose 

of etanercept can lead to significant disease control, to a 

degree comparable to full dose etanercept and tangibly 

better than the other treatment regimens (average clinical 

improvement of 42.3% for hsp60 p180–188 +single dose 

etanercept+methotrexate, 36.7% for hsp60 p180–188+ 

methotrexate; and 14% for full dose etanercept–methotrexate 

therapy). The marked increase in the percentage of CD4 

+ CD25hiFoxp3+ T cells following epitope-specific combination 

therapy suggested induction of Treg cells and restoration 

of tolerance. Such changes were restricted to the 

draining site of the nasal mucosa of animals receiving the 

combined treatment. 

doi:10.1016/j.clim.2007.03.461 

S99 

Sa.74 Independent Signals Within the MHC are 

Associated with Systemic Lupus Erythematosus—A 

Family-based SNP Study 

Michelle Fernando, ARC Clinical Research Fellow, Imperial 

College London, Section of Molecular Genetics and 

Rheumatology, London, England, Christine Stevens, Project 

Coordinator, The Broad Institute of MIT and Harvard, 

Inflammatory Genetics Group, Cambridge, MA, Emily Walsh, 

Cancer Research Institute Postdoctoral Fellow, Broad 

Institute of MIT and Harvard, Cambridge, MA, Todd Green, 

Project Coordinator, Program in Medical and Population 

Genetics, Broad Institute of Harvard and MIT, Cambridge, 

MA, Pardis Sabeti, Postdoctoral Fellow, Infectious Disease 

Initiative, The Broad Institute of MIT and Harvard, 

Cambridge, MA, John Rioux, Canada Research Chair in 

Genetics and Genomic Medicine of Inflammation, Universite 

de Montreal, Montreal Heart Institute, Montreal, QC, 

Canada, Tim Vyse, Reader and Honorary Consultant in 

Rheumatology/Medicine, Imperial College London, Section 

of Molecular Genetics and Rheumatology, London, England 

The major histocompatibility complex (MHC) has been 

associated with SLE in numerous case–control studies. The 

main associated alleles are HLA-DR3 (0301) and HLA-DR2 

(1501) and their respective haplotypes in white Caucasian 

populations. MHC class III alleles have also shown association 

with lupus but extensive linkage disequilibrium (LD) in 

the region has prevented the discovery of the causal 

alleles. We therefore set out to investigate whether we 

could delineate separate class II and class III signals in our 

UK SLE cohort using a family-based SNP association study. 

67 SNPs were successfully typed using the Sequenom 

MassARRAYTM platform across 1.7 mb of genome (HLA-B 

to HLA-DPA2) in 314 Caucasian UK SLE trios. 4-digit HLA- 

DRB1 typing was performed using oligo-hybridization. Our

S100 Abstracts 

findings confirm previously published data of an association 

with HLA-DRB1*0301 (p=2.1×10 −8 ). We find no association 

with HLA-DRB1*1501 or HLA-DRB1*0801. 15 out of 67 SNP 

markers show evidence of association with WHAP TDT 

(permutated pb0.05, 10,000 permutations). Conditional 

analysis (WHAP) reveals association signals independent of 

HLA-DRB1*0301 in class I (HLA-B) and class III (BAT3, 

SLC44A4, RDBP) with the latter signal being the strongest. 

These independent markers are not in LD with any other 

HLA-DRB1 allele (TRANSMIT) but do arise from the B8- 

DRB1*0301 extended haplotype shown by Extended Haplotype 

Homozygosity analysis (SWEEP). 

HLA-DRB1*0301 is associated with SLE in our UK cohort. 

We have also demonstrated a separate class III signal arising 

from recombination on the B8-DRB1*0301 extended haplotype. 

The haplotype block from which the class III signal 

arises encompasses the complement C4-containing RCCX 

module. 

doi:10.1016/j.clim.2007.03.462 

Sa.75 Quantitation of Total and Unoccupied BAFF-R 

in SLE 

Seth Knight, University of Alabama, Birmingham, 

Department of Medicine, Birmingham, AL, Hong Zhao, 

University of Alabama, Birmingham, Department of 

Medicine, Birmingham, AL, Tong Zhou, University of 

Alabama, Birmingham, Department of Medicine, 

Birmingham, AL, Robert Carter, University of Alabama, 

Birmingham, Department of Medicine, Birmingham, AL 

Background: Systemic lupus erythematosus (SLE) is an 

autoimmune disease characterized by abnormally active B 

lymphocytes. B lymphocyte stimulator (BLyS) has been 

shown to be an important regulator of B cell activity in SLE. 

The serum concentration of BLyS is increased in SLE 

patients, and in mice, increased BLyS induces a lupus-like 

syndrome. We have previously demonstrated that BAFF-R, 

the primary receptor for BlyS on B cells, is occupied to a 

greater extent on PBMCs in SLE patients than in healthy 

controls. We have now developed a robust assay for 

measuring total BAFF-R and occupied BAFF-R using quantitative 

flow cytometry. Methods/Results: Anti-BAFF-R monoclonal 

Ab-producing hybridoma clones were screened by 

ELISA. We identified clone 4A4 and performed a BLyS 

competition assay on tonsillar B cells and show that BlyS 

and 4A4 Ab competitively bind to BAFF-R. 4A4 Ab and a 

second anti-BAFF-R Ab (11C1) were then used to quantitate 

total and occupied BAFF-R on peripheral blood B cells in SLE 

and control cohorts using bead-based, quantitative flow 

cytometry to derive absolute values for antibody binding 

based on a standard curve. We show that both total and 

unoccupied BAFF-R are decreased on peripheral blood B 

cells in SLE patients, in some cases to 10% of healthy 

controls. These data demonstrate a more robust system for 

BAFF-R analysis in SLE and confirm the engagement of 

BAFF-R in most lupus subjects. 

doi:10.1016/j.clim.2007.03.464 

Sa.76 Role of CD8+ Ti cells in Suppression of 

Autoimmunity Depends on Foxp3 Expression 

Ram Singh, Assistant Professor, David Geffen School of 

Medicine at University of California, Los Angeles, Los 

Angeles, CA, Antonio La Cava, Associate Professor, David 

Geffen School of Medicine at University of California, Los 

Angeles, Medicine, Los Angeles, CA, Bevra Hahn, Professor 

and Chief, David Geffen School of Medicine at University of 

California, Los Angeles (Medicine), Los Angeles, CA 


disease caused by autoantibodies including IgG anti-DNA. 

NZB/NZW Fl female (BWF1) mice, a model of spontaneous 

polygenic SLE, tolerized with an artificial peptide (pCONSEN- 

SUS, pCons) based on anti-DNA IgG sequences containing MHC 

class I and class iI T cell determinants, develop regulatory 

CD4+CD25+ T cells and inhibitory CD8+ T cells (CD8+ Ti), both 

of which suppress autoAb production. In the present study, 

using various cellular and molecular approaches, we show that 

inhibitory function of CD8+ T cells from tolerized mice is 

sustained for up to 8 weeks and at all times depends on 

expression of Foxp3. Both CD28-positive and -negative CD8+ T 

cells contain inhibitory cells, but the expression of Foxp3 and 

of mRNA for TGFb is higher and lasts longer in the CD28− 

subset. In vitro addition of TGFb (in the presence of IL-2) 

induces Foxp3 expression in a dose–response manner. Gene 

inhibition or blockade with siRNA of Foxp3 abrogates the 

ability of the CD8+ Ti to inhibit anti-DNA production and the 

proliferation of CD4+ helper T cells. Moreover, we found a 

significant correlation between expression of Foxp3 and 

ability of CD8+ Ti cells to secrete TGFb. Therefore, CD8+ Ti 

in this system of tolerance is dependent on expression of 

Foxp3, and there may be a bi-directional Foxp3/TGFb 

autocrine loop that determines the ability of the CD8+ T 

cells to control autoimmunity. Supported by NIH grants. 

R37 AI 46776, AI63515AR53239, Tina C Foundation, and 

gifts from Jeanne Rappaport and the Horchow Family 

Trust. 

doi:10.1016/j.clim.2007.03.465 

Sa.78 T Cell Epitope Molecular Mimicry Can Initiate 

Immune Response Against Lupus-associated SmD 

Autoantigen 

Davis Sim, MD/PhD Student, University of Virginia Division 

of Rheumatology and Immunology, Charlottesville, VA, 

Govind Rajagopolan, Research Associate, Mayo Clinic 

Department of Immunology, Rochester, MN, Shu Man Fu, 

Professor, University of Virginia Division of Rheumatology 

and Immunology, Charlottesville, VA, Chella David, 

Professor, Mayo Clinic Department of Immunology, 

Rochester, MN, Umesh Deshmukh, Assistant Professor, 

University of Virginia Division of Rheumatology and 

Immunology, Charlottesville, VA 

Autoantibodies reactive with Sm proteins are specific 

for systemic lupus erythematosus. One of the hypotheses 

behind the initiation of an immune response against Sm 

autoantigens is molecular mimicry between foreign and 

self proteins. However, clear evidence for this at T cell

Abstracts 

level is lacking. This study was designed to determine the 

role of T cell epitope mimicry in the initiation of immune 

response against SmD1.Using HLA-DR3 transgenic mice as 

experimental model system, a dominant T cell epitope 

was identified between amino acid SmD71 and 95. Using a 

T cell hybridoma (C1P2) reactive against this epitope, 

critical amino acid residues were localized within SmD81– 

88. Screening of databases with pattern recognition and 

MHC binding software identified 20 putative peptide 

mimics. Among these, a peptide from galactoside ABC 

transporter protein from Vibrio activated C1P2. Immunization 

of HLA-DR3 mice with this peptide induced both T and 

B cell responses against SmD. To further characterize T 

cell responses against this mimic, additional T cell 

hybridomas were generated. Interestingly a T hybridoma 

P20 not only reacted with SmD79–93 and the Vibrio 

peptide but also with peptides from a human La-related 

protein and methyltransferase protein from Streptococcus 

agalactiae. TCR characterization of both C1P2 and P20 

revealed different V 2 usage, V 2 8.3 and V 2 8.1 respectively. 

Our study demonstrates the potential of T cell 

epitope mimicry to initiate autoimmune responses against 

lupus-associated antigens. In addition our data suggest 

that based on the TCR usage, different T cells reactive 

against the same region of an autoantigen can be 

activated by multiple molecular mimics. 

doi:10.1016/j.clim.2007.03.466 

Sa.79 Autoantibody Onset Prior to SLE Diagnosis 

Follows Predictable Patterns of Development 

Latisha Heinlen, Graduate Student, Oklahoma Medical 

Research Foundation, Oklahoma City, OK, Micah McClain, 

Scientist, Oklahoma Medical Research Foundation, 

Oklahoma City, OK, Tony Prestigiacomo, Scientist, BioRad 

Laboratories, Benecia, CA, Ben Bruner, Graduate Student, 

Oklahoma Medical Research Foundation, Oklahoma City, 

OK, Steven Binder, Scientist, Bio-Rad Laboratories, 

Benecia, CA, Colin Edgerton, Chief, Rheumatology Service, 

US Army Center for Health Promotion and Prevention, 

Washington, DC, Mark Rubertone, Active Duty Military, US 

Army Center for Health Promotion and Preventive 

Medicine, Washington, DC, Judith James, Member, 

Oklahoma Medical Research Foundation, Oklahoma City, 

OK, John Harley, Member, Oklahoma Medical Research 

Foundation, Oklahoma City, OK 

Systemic lupus erythematosus (SLE) is a clinically 

diverse humoral autoimmune disorder. We sought to 

determine the earliest known protein autoantigens targeted 

prior to SLE diagnosis, and to define the order in 

which specific protein autoantibodies arise. Previously 

collected and stored serum samples were obtained from 

the United States Department of Defense Serum Repository. 

All available patient and selected control samples were 

tested for autoantibodies to lupus autoantigens. Using 

these prospective samples we identified patterns of 

autoantibody onset. In 33 patients the initial single 

autoantibody specificities were identified and were most 

commonly against 60 kDa Ro, and nRNP A. Surprisingly, 

none of these patients initiated lupus autoimmunity with 

anti-nRNP 70K or anti-Sm. Also, no patient had antibodies to 

nRNP 70K without antibodies to nRNP A. Anti-A appeared 

before or simultaneously with anti-70K in 96% of patients that 

developed both under observation. Anti-60 kDa Ro antibodies 

appeared before or simultaneously with anti-La or anti-52kD 

Ro in nearly all patients (98% and 95% respectively). These 

data suggest that the autoantibody response in SLE begins 

simply, often with a single protein years before disease onset, 

followed by the accumulation of additional autoantigenic 

specificities in partly predictable patterns. 

doi:10.1016/j.clim.2007.03.467 

Sa.80 Osteopontin Gene Polymorphism and 

Systemic Lupus Erythematosus in Chinese 

Maida Wong, Clinical Fellow, University of California, Los 

Angeles, School of Medicine, Los Angeles, CA, Joseph Yeung, 

Research Associate, University of Hong Kong, Faculty of 

Medicine, Hong Kong, Chak-Sing Lau, Professor, University 

of Hong Kong, Faculty of Medicine, Hong Kong 

Osteopontin (OPN) is a secreted glycosylated and phosphorylated 

extracellular matrix protein that influences autoimmune 

disease. Its serum level is known to be elevated in 

patients with systemic lupus erythematosus (SLE). In this 

study, we analyzed a minor Tallele (C707T SNP, corresponding 

to rs1126616) of OPN in Chinese SLE patients and healthy 

controls by polymerase chain reaction. Serum OPN levels were 

measured by ELISA, and its correlation with OPN polymorphism 

was analyzed statistically based on their genotypes, renal 

involvement, and disease activity (active disease defined as 

SLEDAI = or > 4). 162/253 SLE patients and 91/168 controls had 

one or more allelic mutations at 707T of the OPN gene. OPN 

mutation is associated with the development of SLE, OR = 1.51 

(95% CI 1.012-2.253). The OPN genotype is also associated with 

SLE (p=0.0003). Serum OPN is elevated in SLE patients with 

history of renal disease, irrespective of their disease activity 

(pb0.005 by ANOVA and post-hoc test, p= 0.013 by Mann- 

Whitney U test). There was no association between the allelic 

mutation and CNS disease, hematological disorders, malar or 

discoid rash, low complements, or elevated dsDNA antibody. 

The C707Tallelic frequency of the control population followed 

the Hardy-Weinberg equilibrium. Although the allelic frequencies 

between SLE patients (0.340) and controls (0.327) were 

not significantly different, they were significantly higher than 

the healthy Caucasian controls as published by Forton et al 

(p=0.004), which may explain the higher prevalence of SLE 

in the Chinese population, especially those with renal 

involvement. 

doi:10.1016/j.clim.2007.03.468 

S101 

Sa.81 Quantitative and Qualitatively Normal 

Regulatory T Cells are Not Capable of Inducing 

Suppression in SLE Patients Due to T Cell Resistance 

María Inés Vargas-Rojas, Medical Doctor, Instituto Nacional 

de Ciencias Médicas y Nutrición Salvador Zubiran/


Rheumatology and Immunology, Mexico, José Carlos Crispín, 

Medical Doctor, Instituto Nacional de Ciencias Médicas y 

Nutrición Salvador Zubiran/Rheumatology and Immunology, 

Mexico, Jorge Alcocer-Varela, Medical Doctor, Instituto 

Nacional de Ciencias Médicas y Nutrición Salvador Zubiran/ 

Rheumatology and Immunology, Mexico 

Regulatory T cells (Treg) are considered an essential 

component of the mechanisms that protect and maintain 

peripheral tolerance. Their absence leads inevitably to lifethreatening 

autoimmune disease. Numerous works have 

studied their numbers and behavior in patients with 

autoimmune conditions; several defects that include diminished 

numbers and lack of suppressive capacity have been 

documented in various conditions including systemic lupus 

erythematosus. The aim of this study was to further 

characterize the regulatory defect documented in Treg of 

patients with SLE. Twenty patients with SLE (10 with active 

disease) and 20 age- and sex-matched controls were 

included. Patients were not receiving treatment. Treg 

cells, defined as CD4+Foxp3+, were quantified by flow 

cytometry. CD4+CD25+ and CD4+CD25− cells were isolated 

by magnetic beads. Suppressive capacity was quantified in 

autologous and allogeneic co-cultures. No quantitative 

difference was found in Treg numbers between patients 

and controls (2.8±2.1 vs. 3.2±2.2, respectively). Suppression 

of cell proliferation was not observed in autologous cocultures 

of SLE cells. Conversely, normal Treg greatly 

suppressed autologous T cell proliferation (∼80%). Surprisingly, 

when SLE-derived Treg were added to healthy T cell 

cultures suppression was observed. In contrast, T cells 

obtained from SLE patients were not susceptible to suppression 

by normal Treg. Our findings suggest that even though a 

T cell suppression defect is present in patients with SLE, it 

cannot be explained by defects in Treg. Our data indicate 

that SLE-derived T cells are resistant to Treg-mediated 

suppression. 

doi:10.1016/j.clim.2007.03.469 

Sa.82 Increased Regulatory T Cell Prevalence and 

Retained Capacity to Suppress Effector T Cells 

During Disease in NZB/W Lupus Mice 

Kenneth Scalapi, Adjunct Assistant Professor of Medicine, 

University of California, San Francisco and San Francisco VA 

Medical Center, San Francisco, CA, David Daikh, Associate 

Professor of Medicine, University of California, San 

Francisco and San Francisco VA Medical Center, San 

Francisco, CA 

Purpose: SLE is characterized by loss of tolerance to 

multiple self-antigens. Regulatory T cells (Tregs) serve to 

maintain peripheral tolerance by inhibiting autoreactive 

effector Tcells (Teff). Studies have correlated low peripheral 

blood Treg prevalence and/or abnormal Treg function with 

several autoimmune diseases including SLE. To assess Treg 

frequency and function in lupus prone NZB/W (B/W) mice, 

we evaluated CD4+Foxp3+ T cells during the course of 

disease. Cellular function in young and old B/W mice was 

assessed utilizing the CD4+CD25+ Treg subset, young and old 

Teffs and antigen-presenting cells (APCs) in mixed suppression 

assays. Methods: CD4+Foxp3+ Treg prevalence was 

measured by FACS. Function of purified Tregs, Teffs and 

APCs was evaluated in mixed suppression assays. Results: 

Frequency of CD4+Foxp3+ Tregs as a percentage of CD4+ cells 

is low only in pre-diseased B/W mice. During active lupus, 

Tregs prevalence significantly increases such that it equals or 

exceeds that of age-matched control mice. A significant 

fraction of Tregs in sick mice loses CD25− expression. The 

Treg prevalence in peripheral blood did not correlate with 

tissue prevalence or disease state. Mixed suppression assays 

demonstrated that the function of CD4+CD25+ Tregs is not 

affected by disease state and Teffs do not become resistant 

to Treg suppression as mice age and develop disease. 

Conclusion: A significant expansion of functional Tregs, not 

evident in peripheral blood, occurs in lupus prone B/W with 

disease onset, emphasizing the unreliability of peripheral 

blood to assess these cells. Furthermore, Tregs retain the 

capacity to suppress Teffs in mice with active lupus. 

doi:10.1016/j.clim.2007.03.470 

Sa.84 Immune Opsonins Modulate BLyS/BAFF 

Release in a Receptor-specific Fashion 

Xinrui Li, Graduate Student, Division of Clinical Immunology 

and Rheumatology, University of Alabama at Birmingham, 

Birmingham, AL, Jeffrey Edberg, Associate Professor, 

Division of Clinical Immunology and Rheumatology, 

University of Alabama at Birmingham, Birmingham, AL, 

Kaihong Su, Assistant Professor, Division of Clinical 

Immunology and Rheumatology, University of Alabama at 

Birmingham, Birmingham, AL, Tong Zhou, Associate 

Professor, Division of Clinical Immunology and 

Rheumatology, University of Alabama at Birmingham, 

Birmingham, AL, Alexander Szalai, Associate Professor, 

Division of Clinical Immunology and Rheumatology, 

University of Alabama at Birmingham, Birmingham, AL, 

Robert Kimberly, Professor, Division of Clinical Immunology 

and Rheumatology, University of Alabama at Birmingham, 

Birmingham, AL 

TNF ligand superfamily member 13B (B lymphocyte 

stimulator (BLyS), B cell activating factor (BAFF)) promotes 

primary B cell proliferation and immunoglobulin production. 

It exists in both membrane-bound and soluble forms. Because 

the soluble form is thought to be the primary biologically 

active form, we investigated factors that might regulate its 

cleavage and processing. In both myeloid cell lines (U937, 

THP-1and HL-60) and primary human monocytes, the 

shedding of membrane BLyS can be regulated by CRP and 

IgG. Within 10 minites, both of these opsonins trigger 

significant shedding of BLyS (up to 52.2+/-3.0%, p=0.003) 

by engaging FcgRs. In U937 cells, a primary role of FcgRI is 

demonstrated both by direct receptor cross-linking (FcgRI 

45.69+/-2.05% verses FcgRIIa 2.11+/-1.33%) and by the lack 

of enhanced cleavage when FcgRI and FcgRIIa are cocrosslinked. 

The shed BLyS is biologically functional since it 

promotes B cell survival. The generation of a B cell 

proliferation and survival factor by Fc gamma receptor 

engagement, especially FcgRI, which can also enhance

Abstracts 

antigen uptake and presentation mediated by ligands of both 

innate and adaptive immune systems, provides a unique 

opportunity to facilitate antibody production. These functions 

suggest that Fc gamma receptors may provide a link between 

immune complex mediated autoimmune diseases and elevations 

in circulating BLyS in autoimmune disease patients. 

doi:10.1016/j.clim.2007.03.471 

Sa.85 The CD4+CD25+Foxp3+ T Cells in Lupus 

Nephritis 

Mercedes Zabaleta-Lanz, Immunologist, Institute of 

Immunology, Central University School of Medicine, 

Caracas, Venezuela, Ronak Seyeddi, MD, Institute of 

Immunology, Caracas, Venezuela, Maria Elena Marquez, 

Bbiologist, Institute of Immunology, Caracas, Venezuela, 

Perla Chirinos, Bioanalist, Instituto de Inmunologia, UCV, 

Caracas, Venezuela, Isaac Blanca, Biologist, Institute of 

Immunology, Caracas, Venezuela, Nicolás Bianco, 

Immunologist, Institute of Immunology, Caracas, Venezuela, 

Mercedes Zabaleta-Lanz, Immunologist, Institute of 

Immunology, Caracas, Venezuela 

The role of CD4+CD25+Foxp3+ regulatory T cells (Tregs) 

are critical in maintaining self tolerance. The levels of Treg 

in patient with systemic lupus erythematosus (SLE), a 

chronic autoimmune disorder, have been reported 

depressed. Here we have determined the levels of Treg 

and the CTLA-4 CD4 T cells in 21 patients with SLE, 7 with 

active disease and 15 with inactive disease and 25 healthy 

controls. Treg and CTLA-4 were evaluated in highly purified 

CD4 T cells from patients and controls by flow cytometry 

using a tri-color analysis protocol. The 7 SLE patients with 

active disease, 4 with overt lupus nephritis (OLN) and 3 

with silent lupus nephritis (SLN) show a significant reduction 

of Treg in comparison to controls (SLN, 0.23±0.18%, 

OLN 1.35±1.21%, controls, 3,69±3%¸ pb0.05 and pb0.02 

respectively). A significant reduction of Treg was also 

observed between the inactive SLE patients versus controls 

(pb0.02). A higher expression of Foxp3 was observed in 

Treg cells from patients with active SLE disease (88.74 ± 

5.92%) vs. inactive disease (71.39 ±20.15%; p=0.008). Nonsignificant 

differences were observed in the levels of CD4 

+CD25+CTLA-4+ T cells, and no correlation between Tregs 

and serological parameters (anti-DNA, anti-C1q, antinucleosome, 

C3 and C4) was observed. Ours results suggest 

that Tregs are significantly decreased in active lupus even 

before the installation of an OLN. However, the increased 

expression of Foxp3 in the Tregs cells from patients with 

active disease suggests a possible mechanism of compensatory 

function of these molecules. 

doi:10.1016/j.clim.2007.03.472 

Sa.86 Rapamycin Induces Tolerance by Increasing 

Regulatory T Cells in Chronic Graft Versus Host 

Disease Model 

Jeanne Palmer, Postdoctoral Fellow, Duke University 

Medical Center, Department of Hematology, Oncology and 

Cellular Therapy, Durham, NC, Benny Chen, Assistant 

Professor of Medicine, Duke University Medical Center, 

Division of Cellular Therapy/BMT, Durham, NC, Nelson 

Chao, Director, Division of Cellular Therapy/BMT, Professor 

of Medicine and Immunology, Duke University Medical 

Center, Durham, NC, Ngocdiep Le, Medical Sciences 

Director, Amgen Inc., Thousand Oaks, CA 

Rapamycin is an immunosuppressant that has been found 

to prevent/treat acute and chronic graft versus host disease 

after allogeneic hematopoeitic stem cell transplant (HSCT) 

in both murine models and humans. One possible mechanism 

is through induction of tolerance, which may be due to 

increased number or enhanced survival of regulatory T cells. 

In two experiments B10.D2 bone marrow and splenocytes 

were injected into lethally irradiated BALB/c mice. They 

were then given IP injections from days 1-28 with rapamycin 

1.5 mg/kg or 5 mg/kg, which induced tolerance in this 

chronic graft versus host disease model. We noted however a 

dense cellular infiltrate in the animals who did not have 

clinical GVHD. We have documented many these infiltrating 

cells as in situ regulatory T cells by intracellular foxP3 

staining. These T regs are increased in tissues of rapamycin 

treated mice. This effect appears to decrease following 

discontinuation of rapamycin. To understand the kinetics of 

this effect, we will repeat the experiments, and sacrifice the 

mice at 2, 4, and 6 weeks. At that point, serum cytokine 

analysis, histologic evaluation of multiple organs and FACS 

analysis on splenocytes will be performed. 

doi:10.1016/j.clim.2007.03.473 

S103 

Sa.87 Elimination of Insulitis and Augmentation of 

Islet β Cell Regeneration via Induction of Chimerism 

in Overtly Diabetic NOD Mice 

Chunyan Zhang, Postdoctoral Research Fellow, The Beckman 

Research Institute, City of Hope National Medical Center, 

Duarte, CA, Fouad Kandeel, Physician, The Beckman 

Research Institute, City of Hope National Medical Center, 

Duarte, CA, Ivan Todorov, Associate Research Scientist, The 

Beckman Research Institute, City of Hope National Medical 

Center, Duarte, CA, Mark Atkinson, Professor, University of 

Florida College of Medicine, Gainesville, FL, Chia-Lei Lin, 

Research Associate I, The Beckman Research Institute, City 

of Hope National Medical Center, Duarte, CA, Stephen 

Forman, Physician, The Beckman Research Institute, City of 

Hope National Medical Center, Duarte, CO, Defu Zeng, 

Assistant Professor, The Beckman Research Institute, City of 

Hope National Medical Center, Duarte, CA 

Type 1 diabetes in both humans and NOD mice results from 

autoreactive T cell destruction of insulin-producing beta 

cells. Cure of type 1 diabetes may require both reversal of 

autoimmunity and regeneration of beta cells. Induction of 

chimerism via allogeneic hematopoietic transplantation 

(HCT) has been shown to re-establish tolerance in both 

prediabetic and diabetic NOD mice. However, it is unclear 

whether this therapy augments beta cell regeneration. 

Furthermore, this procedure usually requires total body


irradiation (TBI) conditioning of recipients. The toxicity of 

TBI conditioning and potential for graft-versus-host disease 

(GVHD) limit the application of allogeneic HCT for treating 

type 1 diabetes. Here, we report that injection of donor bone 

marrow and CD4+ T-depleted spleen cells induced chimerism 

without causing GVHD in overtly diabetic NOD mice conditioned 

with anti-CD3/CD8, and that induction of chimerism 

in new-onset diabetic NOD mice led to elimination of 

insulitis, replication of host beta cells, and reversal of 

hyperglycemia. Although induction of chimerism in latestage 

of diabetic NOD mice did not reverse diabetes, the 

chimeras were tolerant to donor islets, and donor islet 

transplantation normalized blood glucose. Furthermore, we 

found that induction of mixed chimerism in autoreactive 

BDC2.5 TCR transgenic NOD mice deleted 99% of the BDC2.5 

T cells among host CD4+CD8+ thymocytes. Therefore, this 

radiation-free GVHD preventive approach for induction of 

chimerism may represent a viable means for reversing type 1 

diabetes. 

doi:10.1016/j.clim.2007.03.474 

Sa.88 Purified MHC-matched Hematopoietic Stem 

Cell Transplantation Following Total Lymphoid 

Irradiation and Anti-thymocyte Globulin (TLI+ATG) 

Blocks EAE Pathogenesis 

Li-Fen Lee, Instructor, Blood and Marrow Transplantation, 

Stanford, CA, Matthew Burge, Research Assistant, Blood 

and Marrow Transplantation, Stanford, CA, Rosa Landa, 

Research Assistant, Blood and Marrow Transplantation, 

Stanford, CA, Raymond Sobels, Professor, Pathology, 

Stanford, CA, Peggy Ho, Basic Life Science Research 

Associate, Neurology, Stanford, CA, Judith Shizuru, 

Associate Professor, Blood and Marrow Transplantation, 

Stanford, CA 

In prior studies, we demonstrated that transplantation of 

major histocompatibility complex (MHC) matched and mismatched 

purified hematopoietic cells (HSCs) can block 

diabetes pathogenesis in pre-diabetic NOD mice. Here we 

applied this treatment to experimental allergic encephalomyelitis 

(EAE), an animal model for the human autoimmune 

disease multiple sclerosis (MS). C57BL/6 (B6) strains congenic 

for the CD45 locus were immunized with MOG 35–55 to induce 

disease. Similar to our studies in NOD mice we observed that 

disease response after transplantation of MHC-mismatched 

hematopoietic cells in EAE affected mice was superior to those 

that received congenic cells. Importantly, mice transplanted 

with MHC-matched HSC or BM showed no significant improvement 

in disease symptoms when compared with congenic 

recipients or disease controls. This result was markedly 

different from the positive response observed in NOD mice 

transplanted with MHC-matched HSC. We then sought to 

optimize translation of this approach by giving total lymphoid 

irradiation (TLI) plus anti-thymocyte globulin (ATG), a nonmyeloablative 

regimen, and permitting allogeneic hematopoietic 

cell engraftment with significant protective effect 

against GVHD. We found that TLI/ATG treatment in wild type 

C57BL/6 mice permits engraftment of purified HSC isolated 

from MHC-matched AKR/B donors and improves disease 

severity significantly. The level of stable mixed chimerism 

correlated with the clinical score. A predominance of host 

NK1.1 T cells and CD4+CD25+ regulatory T cells was observed 

after TLI/ATG regimen. We hope an MHC-matched hematopoietic 

graft in conjunction with TLI/ATG conditioning shown 

here to successfully ameliorate EAE can be translated to the 

treatment of human autoimmune disease. 

doi:10.1016/j.clim.2007.03.475 

Sa.89 Two Types of Immune Regulatory Mechanisms 

are Developed In Vivo for the Control of Chronic 

Graft-versus-Host Disease 

Magali Noval Rivas, PhD Student, Institute for Medical 

Immunology (IMI), Gosselies, Brussels, Marc Hazzan, Doctor, 

Department of Nephrology and Hemodialysis, CHR Lille, 

Lille, France, Michel Braun, Institute for Medical 

Immunology (IMI), Gosselies, Brussels 

We developed a graft-versus-host disease (GVHD) model 

based on the adoptive transfer of RAG1 −/− TcR-trangenic H-Yspecific 

Marilyn T cells (1×10 6 cells) into male recipients. 

Adoptive transfer into sub-lethally irradiated RAG1 +/+ IL- 

2Rgc +/+ , RAG1 −/− IL-2Rgc +/+ or RAG1 −/− IL-2Rgc −/− male 

recipients induced 100% mortality after 1 week. On the 

contrary, non-irradiated mice of the three types survived 

indefinitely after transfer and did not develop clinical sign of 

acute GVHD. Marilyn T cells transferred into non-irradiated 

RAG1 +/+ IL-2Rgc +/+ or RAG1 −/− IL-2Rgc +/+ did not expand or 

were rapidly eliminated (b45×10 3 in the spleen after 

30 days). Remarkably, transgenic T cells transferred into 

non-irradiated RAG1 −/− IL-2Rgc −/− hosts expanded extensively 

(N15×10 6 in the spleen after 30 days) and were present 

in many organs (spleen, lung, liver, kidney, lymph nodes). 

Histology revealed their capacity to mediate chronic GVHD 

lesions (grade 1 after 30 days) in these hosts. The T cells 

did not develop a regulatory phenotype since they 

remained Foxp3-negative and produced high amounts of 

IFN-g when stimulated in vitro. However, they were unable 

to promote acute GVHD after transfer into irradiated 

recipients. Our model suggests that, in vivo, there are two 

types of mechanisms involved into the regulation of T cell 

responses to persistent antigenic stimulation: one is selfcontained 

by the T cells and involves partial anergy, 

probably maintained by antigenic persistence. The other 

one involves the activity of existing populations of 

regulatory cells. Both of them appear to be necessary for 

full T cell tolerance. 

doi:10.1016/j.clim.2007.03.476 

Sa.90 NK1.1+ Cells Promote Human Cell 

Repopulation via an NKG2D Dependent Mechanism 

Hye-Youn Son, Research Professor, Samsung Biomedical 

Research Institute, Seoul, Republic of Korea, Sung-Yeon Joo, 

MS, Samsung Biomedical Research Institute, Seoul, Republic 

of Korea, Mi Jin Kang, MS, Samsung Biomedical Research 

Institute, Seoul, Republic of Korea, Sung-Joo Kim, 

Prodessor, Samsung Medical Center, Seoul, Republic of

Abstracts 

Korea, Bong-Kum Choi, MS, Samsung Biomedical Research 

Institute, Seoul, Republic of Korea 

To achieve donor-specific immune tolerance in Rag2−/− 

f×C−/− mice to xenogeneic human CD34+ hematopoietic 

stem cells (HSCs), it is imperative to understand the cell 

types involved in xenograft rejection. In this study, we 

examined whether NK1.1+ cells in Rag2−/−f×C−/− mice play 

a direct role in the tolerance against transplanted HSCs, if 

thus which molecules are important. We used CD34+ 

hematopoietic stem cells to reconstitute human cells from 

either cord blood or fetal liver. From 2 weeks posttransplantation, 

we have monitored human cells repopulation 

(referred as % of CD45) in peripheral blood every 2 or 

4 weeks. Besides, anti-mouse CD3, CD19, NK1.1, Gr-1, and 

NKG2D were used to determine the mouse cells involved 

in tolerance induction. In our system, hCD45 was 

significantly increased following human HSC’s transplantation 

as well as successfully developed multi-lineage. We 

found that NK1.1+ cells, especially Gr-1− subset, were 

directly co-related with human cell reconstitution. Interestingly, 

NKG2D expression on NK1.1+ cells was significantly 

increased in the group highly repopulated with 

human cells. Finally, CD3 population appeared after 

transplantation was shown to be inversely related with 

NKG2D expression. Taken together, our data strikingly 

demonstrate that NK1.1+ cells play a direct role to regulate 

human cell reconstitution as well as HSCs engraftment in 

Rag2−/−f×C−/− mice. Moreover, our data show that NKG2D 

expression significantly contributes to tolerance induction 

which could be involved in donor-specific cytotoxic cell 

killing mechanism. We are planning to down-regulate and/ 

or delete specific NKG2D+ subsets in mixed chimeras in 

vivo to clear the results. 

doi:10.1016/j.clim.2007.03.477 

Sa.91 Human Cell Reconstitution from Umbilical 

Cord Blood-derived Stem Cells in Rag1−/−gC−/− Mice 

by Co-Transplantation of Unrelated Fetal 

Liver/Thymus Tissues 

Mi Jin Kang, MS, Samsung Biomedical Research Institute, 

Seoul, Republic of Korea, Hye-Youn Son, Research professor, 

Samsung Biomedical Research Institute, Seoul, Republic of 

Korea, Bong-Kum Choi, MS, Samsung Biomedical Research 

Institute, Seoul, Republic of Korea, Sung-Joo Kim, 

Professor, Samsung Medical Center, Seoul, Republic of 

Korea, Sung-Yeon Joo, MS, Samsung Biomedical Research 

Institute, Seoul, Republic of Korea 

To establish human immune and hematopoietic development 

and function in vivo, a number of studies have 

been tried to reproduce human hematopoiesis as human 

hematopoietic tissue and cell transplantation in immunodeficient 

mice. We report a model co-transplanted of 

human fetal thymus/liver (Thy/Liv) tissues with cord blood 

(CB) CD34+ cells into Rag2−/−gC−/− mice. To solve the 

limitation of cell dose, we also used ex vivo expansion with 

cytokines combination; Flt-3 ligand (FL), thrombopoietin 

(TPO), stem cell factor (SCF), erythropoietin (EPO), G-CSF, 

GM-CSF or interleukin-3 (IL-3). Rag2−/−gC−/− mice were 

transplanted with CB CD34+ hematopoietic progenitor cells 

with HLA unrelated fetal Thy/Liv. MHC-mismatched CB 

CD34+ cell transplantation with fetal tissues resulted in 

measurable engraftment levels (about 5% of human CD45) 

in PBL more than 4 months after transplantation. Also, we 

observed that implanted human fetal liver and thymus 

under the kidney capsule were significantly grown. We 

found T cell and NK cell activity after in vitro restimulation 

at 20 weeks after transplantation. Especially, CD3+ T cell 

development was remarkable in a part of ex vivo cultured 

cell transplanted group (included with FL, TPO, and SCF). 

We also found human IgG in serum 20 weeks after 

transplantation even though we have never seen reasonable 

CD19+ cells in PBL, BM, or spleen. These results 

demonstrate that functional human T cell can be reconstituted 

by transfusion of MHC mismatched CB derived 

CD34+ cells with co-implantation of fetal Thy/Liv and that 

might provide a useful tool to study human disease like 

viral infection. 

doi:10.1016/j.clim.2007.03.478 

S105 

Sa.92 Fetal Liver Cells as Corrector of Immune and 

Hemopoietic Activity of Allogeneic Bone Marrow 

Anatoliy Goltsev, Professor, Institute for Problems of 

Cryobiology and Cryomedicine of the National Academy of 

Science of Ukraine, Kharkov, Ukraine, Irina Matsevitaya, 

Institute for Problems of Cryobiology and Cryomedicine of 

the National Academcy of Science of Ukraine, Kharkov, 

Ukraine, Elena Lutsenko, Institute for Problems of 


Science of Ukraine, Kharkov, Ukraine, Yulia Kozlova, 


the National Academy of Science of Ukraine, Kharkov, 

Ukraine, Tatyana Dubrava, Institute for Problems of 


Science of Ukraine, Kharkov, Ukraine, Maxim Ostankov, 


the National Academy of Science of Ukraine, Kharkov, 

Ukraine 

We have shown the possibility to reduce immune reactivity of 

allogeneic bone marrow (BM) as graft-versus-host reaction 

(GVHR) by the change of the content of myelotransplant 

hemopoietic microenvironment cells. These changes may be 

achieved by additional introduction with BM of cells possessing 

regulatory activity. There is accumulated information about 

manifested immune modulating ability of fetal liver cells (FLCs). 

Research aim was the substantiation of the possible use of FLCs 

as the preparation: modulators of immune and hemopoietic 

activity of allogeneic bone marrow and a rise in its protective 

potential. Lethally irradiated Balb/c mice were intravenously 

injected BM of CBA ones (5×10 6 /mouse) together with FLC 

(5×10 5 /mouse). GVHR development intensity was assessed 

according to traditional parameters. Content of stem hemopoietic 

cells in BM of recipients was examined by cloning of 

hemopoietic precursors in vivo (CFUs). Obtained results testify 

to the fact that co-transplanted FLCs rendered manifested 

regulatory effect on immune reactivity and hemopoietic


function of cryopreserved allogeneic BM. Character and extent 

of its manifestation have dose-dependent effect. FLC cotransplantation 

contributed to minimization of hypoplasia of 

recipient’s thymus, increase of content in BM of recipients of 

CFUs and finally to a rise in the survival percentage of the 

animals to the 70th day. Therefore the effect of FLC may be 

stipulated not only by the limitation of the extent of 

allomyelotransplant GVHR activity but also by an increase in 

its hemopoietic potential. Mechanisms of FLC modulating 

activity in respect to co-transplanted BM are under discussion. 

doi:10.1016/j.clim.2007.03.479 

Sa.93 Suppression of Acute Graft-versus-host 

Disease by Regulatory T Cells Induced by Toxic 

Shock Syndrome Toxin-1 

Haowei Li, PhD Candidate, University of British Columbia, 

Department of Medicine, Vancouver, BC, Canada, Anthony 

W. Chow, Professor Emeritus, University of British 

Columbia, Department of Medicine, Vancouver, BC, Canada 

Allogeneic bone marrow transplantation can cure multiple 

hematopoietic malignancies, but acute graft-versus-host 

disease (aGVHD) is a major complication. Previous studies in 

our laboratory have shown that chronic exposure to the 

staphylococcal superantigen, toxic shock syndrome toxin-1 

(TSST-1), can induce regulatory T cells (Tregs) in C57BL/6 

(B6) mice. These TSST-1-induced Tregs have potent suppressive 

ability to inhibit TSST-1 or SEA-induced pro-inflammatory 

cytokine responses in vitro and in vivo. However, it is 

unclear if TSST-1-induced Tregs can be applied clinically to 

control aGVHD. In this report, using a murine aGVHD model 

(B6 to B6D2F1), we showed that transplantation of an 

allogeneic graft containing TSST-1-induced Tregs alone did 

not confer protection against aGVHD. However, reactivation 

of TSST-1-induced Tregs by post-transplant exposure to TSST- 

1 led to attenuation of aGVHD. While one dose of TSST-1 only 

prolonged the survival time but not the survival rate of 

recipients of TSST-1-induced Tregs, two doses of TSST-1 both 

prolonged the survival time and increased the survival rate of 

the recipients. Activation of TSST-1-induced Tregs did not 

reduce engraftment or expansion of donor T cells, nor 

enhance the elimination of host antigen presenting cells, but 

was associated with decreased production of pro-inflammatory 

cytokines. Blockade of IL-10 did not reverse the 

suppression. More importantly, control of aGVHD by activation 

of TSST-1-induced Tregs did not interfere with graft-vs.tumor 

(GVT) effects. In summary, we conceptually demonstrated 

that Tregs induced by TSST-1 were able to control 

aGVHD in vivo via bystander suppression while sparing GVT 

effects. 

doi:10.1016/j.clim.2007.03.480 

Sa.94 Bone Marrow Derived Mesenchymal Stromal 

Cells Promote Treg Cell Development Ex Vivo 

Qingping Yao, Rheumatology Fellow, Medicine, University of 

California, Los Angeles David Geffen School of Medicine, Los 

Angeles, CA, James Wang, Researcher, Medicine, University 

of California, Los Angeles David Geffen School of Medicine, 

Los Angeles, CA, Ram Raj Singh, Professor, Medicine, 

University of California, Los Angeles David Geffen School of 

Medicine, Los Angeles, CA 

Objective: Bone marrow derived mesenchymal stromal 

cells (BMSCs) provide niche for the development of hematopoietic 

cells. The BMSCs likely accomplish this via multiple 

cytokines. Here, we asked whether BMSCs affect the 

development of regulatory T (Treg) cells that can downregulate 

autoimmune diseases. Methods: Bone marrow cells 

isolated from lupus-prone (NZB×NZW) F1 (BWF1) and normal 

BALB/c mice were cultured in 24 well plates for 6 days. After 

removal of nonadherent cells in these cultures, adherent 

BMSCs were co-incubated with freshly isolated splenocytes 

from either BWF1 or BALB/c mice for an additional 6 days. 

Splenic cells harvested from these cultures were analyzed by 

flow cytometry (FACS) for CD4+CD25+ T lymphocytes. Cell 

culture supernatants were assayed for cytokines by ELISA. 

Results: We found a significantly higher number of CD4+ 

CD25+ T lymphocytes in co-cultures of splenocytes plus 

BMSCs than in cultures of splenocyte alone. CD4+CD25+ T 

cells were found by FACS to be 8.5 and 178 fold higher in cocultures 

of splenocytes +BMSCs from BWF1 and BALB/c mice, 

respectively, over cultures of splenocytes alone. The 

increase in Treg cells was associated with increased IL-10 

production. Conclusion: BMSCs promote the development of 

Treg cells, which is more efficient in normal mice than in 

autoimmune-prone mice. Thus, BMSC impairments might 

contribute to abnormalities in Treg cells in autoimmune 

conditions. Ongoing study will further characterize the Treg 

cell functions in our model, determine mechanisms by which 

BMSCs interact with Treg cells and examine the in vivo 

effects of BMSCs on Treg cells. 

doi:10.1016/j.clim.2007.03.481 

Sa.95 Selective Depletion of Alloreactive T Cells and 

Study of Anti-Tumor Activity of Specific T Cell 

Clones in Patients with Leukemia and Renal 

Carcinoma 

Eva Matejkova, PhD Student, Masaryk University, Zuzana 

Hrotekova, MSc., Cell Immunotherapy Center, Drahomira 

Kyjovska, Medical Laboratory Technician, Masaryk 

University, Czech Republic, Jaroslav Michalek, Head of Cell 

Immunotherapy Center, Masaryk University, Czech 

Republic, Petra Vidlakova, Lab Chief, Masaryk University, 

Czech Republic 

A major challenge in the field of hematopoietic stem 

cell transplantation (HSCT) is to prevent the alloreactivity 

of donor T cells which leads to acute graft-versus-host 

disease (GVHD) while preserving a graft-versus-tumor (GVT) 

effect. Selective depletion using anti-CD25 immunotoxin 

(IT) can eliminate harmful alloreactive T cells while 

preserving other donor T cells with antileukemic/antitumor 

reactivity. We have used irradiated peripheral blood 

mononuclear cells (PBMC) from cancer patients and healthy

Abstracts 

donor PBMC as responder cells in primary mixed leukocyte 

reaction (MLR). To prepare GVL/GVT-specific T cells, 

alloreactive T cells in primary MLR were depleted with 

anti-CD25 IT. The remaining T cells had insignificant 

alloreactivity in secondary MLR. Allodepleted donor cells 

were then repeatedly stimulated using purified leukemia/ 

tumor cells from the same cancer patient. Leukemia/ 

tumor-reactive donor T cells were purified by immunomagnetic 

separation on the basis of INF-g production. 17 MLRs 

(10 with leukemic and 7 with renal carcinoma cells) were 

performed. Selective depletion of alloreactive donor T cells 

with anti-CD25 IT led to more than 2log depletion. Graftversus-leukemia 

(GVL) effect of donor T cells was well 

preserved while the graft-versus-host (GVH) reactivation of 

donor cells was negligible even after repeated stimulation 

with patient’s non-leukemic PBMC. In the case of renal 

carcinoma GVT effect was less dominant and GVH 

reactivation of donor cells was significant. In conclusion, 

it is possible to selectively deplete donor alloreactive T 

cells with anti-CD25 IT. In the case of patients with 

leukemia, the GVL effect can be separated from GVHD, but 

in case of renal carcinoma severe GVHD effect reappeared. 

Supported by the Ministry of Education of the 

Czech Republic, NPVII-2B06058. 

doi:10.1016/j.clim.2007.03.482 

Sa.96 CD28 Costimulates Suppression in an In Vitro 

Model of the Tumor Microenvironment 

Daniel Silberman, Student, Biology Department, Rider 

University, Lawrenceville, NJ, Amanda Bucknum, Student, 

Biology Department, Rider University, Lawrenceville, NJ, 

Tiffany Bloomfield, Student, Biology Department, Rider 

University, Lawrenceville, NJ, John Somerville, Associate 

Scientist, Bristol-Myers Squibb Pharmaceutical Research 

Institute, Princeton, NJ, Jessica Reid, Student, Biology 

Department, Rider University, Lawrenceville, NJ, James 

Riggs, Professor of Biology, Biology Department, Rider 

University, Lawrenceville, NJ 

Although the cellular components essential for an 

effective immune response are often found in the tumor 

microenvironment they fail to prevent cancer progression. 

T cell activation, dependent upon costimulation provided 

by antigen-presenting cells (APCs), is restrained in tumors. 

Macrophages are often found in large numbers in tumors. 

An unbalanced macrophage:T cell ratio fosters T cell 

suppression. We have developed an in vitro model that 

mimics this characteristic of the tumor microenvironment. 

Murine peritoneal cavity (PerC) macrophages suppress T 

cell activation by indoleamine 2,3-dioxygenase (IDO) and 

inducible nitric oxide synthase (iNOS) catalyzed consumption 

of tryptophan and arginine. 1-Methyl tryptophan and 

NG-monomethyl-L-arginine block these enzymes and permit 

T cell activation. We are testing various costimulatory 

ligand–receptor combinations to determine if T cell 

activation can be rescued by treatment with an immunobiologic. 

Rather than promoting T cell activation, CD28 

ligation was found to promote macrophage-mediated 

suppression. The suppression was triggered by T cell 

production of IFN 3 . Treatment with CD28-Ig did not release 

suppression. These data will be discussed in the context of 

strategies to curb the suppressive activity evident in tissues 

with aberrant macrophage:T cell ratios. Supported by NIH 

AREA grant R15-AI060356-01. 

doi:10.1016/j.clim.2007.03.483 

Sa.97 Development and In Vitro Validation of 

Anti-Mesothelin Biobodies that Prevent CA125/ 

Mesothelin-dependent Cell Attachment 

Nathalie Scholler, Senior Staff Scientist, Fred Hutchinson 

Cancer Research Center, Seattle, WA, Lindsay Bergan, 

Research Technician, Fred Hutchinson Cancer Research 

Center, Seattle, WA, Jennifer Gross, Research Technician, 

Fred Hutchinson Cancer Research Center, Seattle, WA, Barry 

Nevin, Research Technician, Fred Hutchinson Cancer 

Research Center, Seattle, WA, Barbara Garvik, Research 

Technician, Fred Hutchinson Cancer Research Center, 

Seattle, WA, Nicole Urban, Department head, Fred 

Hutchinson Cancer Research Center, Seattle, WA 

Preventing peritoneal implantation of carcinoma cells 

could prolong ovarian cancer patient remission and 

survival. Peritoneal cells constitutively express mesothelin, 

a ligand for CA125 that is expressed by tumor cells. 

Blocking CA125/mesothelin-dependent cell attachment 

may then prevent or delay peritoneal metastatic recurrence. 

We previously developed an in vitro cell adhesion 

assay combining cells expressing CA125 and mesothelintransfected 

cells. We demonstrated that CA125/mesothelin-dependent 

cell adhesion could be blocked with 

mesothelin recombinant protein fused to an Ig domain 

(meso-Ig) and cross-linked with anti-Ig antibodies, or with 

anti-CA125 mouse monoclonal antibodies (mAb) which 

suggests that new therapies based upon affinity agents 

able to compete with or to block the CA125/mesothelin 

interaction could prevent or delay the development of 

peritoneal metastasis. However, mAbs are highly immunogenic 

in vivo. Recombinant antibodies are emerging as an 

attractive alternative to mouse antibodies for in vivo 

imaging and therapy. We have developed a new class of 

recombinant antibodies, referred to as biobodies (Bbs) 

that are secreted by yeast as in vivo biotinylated proteins. 

Building on our previous work, we generated Bbs that 

specifically bind to mesothelin. Anti-mesothelin recognition 

sequences were isolated from a yeast-display scFv 

library by magnetic and flow sorting, using an in vivo 

biotinylated mesothelin recombinant protein also secreted 

by yeast. Anti-mesothelin yeast-display scFv were transformed 

into Bbs and validated by ELISA and flow 

cytometry. We demonstrated that the anti-mesothelin 

Bbs corresponding to the yeast-display scFv pool of highest 

affinity could recognize both membrane-bound and soluble 

mesothelins, and block CA125/mesothelin-dependent cell 

adhesion. 

doi:10.1016/j.clim.2007.03.484 

S107


Sa.98 Visualization and Characterisation of Melan A 

Epitope (25–35)/HLA-A2 Complex by High Affinity 

Soluble Monoclonal T Cell Receptors (mTCRs) 

Samantha Paston, Avidex Ltd, Abingdon, Oxfordshire, 

England, Katherine Adams, Ms, Avidex Ltd, Abingdon, 

Oxfordshire, England, Giovanna Bossi, Avidex Ltd, 

Abingdon, Oxfordshire, England, Nathaniel Liddy, Avidex 

Ltd, Abingdon, Oxfordshire, England, Deborah Sutton, 

Avidex Ltd, Abingdon, Oxfordshire, England, Tara Mahon, 

Avidex Ltd, Abingdon, Oxfordshire, England, Peter Molloy, 

Avidex Ltd, Abingdon, Oxfordshire, England, Emma 

Gostick, Avidex Ltd, Abingdon, Oxfordshire, England, Andy 

Sewell, Department of Medical Biochemistry and 

Immunology, Cardiff, England, Bent Jakobsen, Avidex Ltd, 

Abingdon, Oxfordshire, England 

Cytotoxic T cells (CTL) use TCRs to detect tumor and viral 

antigens. Soluble TCRs are potential tools for therapeutic 

treatment of cancer, viral infections and autoimmune diseases. 

The Melan A protein (MART-1) was one of the first tumour 

associated antigens to be identified, and is over-expressed in 

80–100% of melanomas. CTL’s recognising the heterolytic 

ELAGIGILTV peptide can be found in both healthy donors and 

melanoma patients. Adoptive therapy and vaccination trials 

using the ELAGIGILTV peptide have resulted in some good 

clinical responses with partial or complete regression of some 

metastates. In healthy donors, up to 0.1% of Tcells in peripheral 

blood can be stained with the Melan A tetramer; patients have a 

higher frequency of Melan A specific Tcells where 1–15% of the T 

cellscanbestainedwiththetetramer.TheMelanAmTCRwas 

affinity matured to bind HLA-A2 presenting the heteroclytic 

ELAGIGILTVpeptidewithakDaof10pMandahalflifeof67h. 

Here we describe the generation of a Tcell clone specific for the 

HLA-A2/Melan A epitope (26–35) derived from a healthy blood 

donor. Using this Melan A (26–35) mTCR we can specifically block 

cytokineproductionfromaMelanATcellclone.Inadditionwe 

are also able to detect and quantify the endogenous peptide 

processed and presented by melanoma cell lines using 3D 

fluoresence microscopy and flow cytometry. Visualisation and 

quantification of these specific antigen/MHC complexes enable 

the potential of mTCRs recognising these complexes as anticancer 

therapeutics to be evaluated. 

doi:10.1016/j.clim.2007.03.486 

Sa.100 AdCD40L Immunogene Therapy for Bladder 

Carcinoma 

Moa Fransson, PhD Student, Clinical Immunology Division, 

Uppsala, Sweden, Angelica Loskog, Scientist, Clinical 

Immunology Division, Uppsala, Sweden, Thomas Tötterman, 

Profesor, Clinical Immunology Division, Uppsala, Sweden 

Recently regulatory T cells (Treg) have made a comeback in 

the immunological arena and the role of these cells in cancer is 

in focus. Clinical protocols that effectively counteract Tregmediated 

immunosuppression are in high demand. We developed 

an immunostimulatory gene therapy for urinary bladder 

cancer based on CD40L gene transfer into the tumor site. The 

efficacy of this therapy was evaluated in mouse and human 

experimental models of bladder cancer. The CD40L gene was 

transferred to the bladder wall of tumor-bearing mice by 

adenoviral vector instillation. AdCD40L gene therapy cured 60% 

of mice with pre-established aggressive tumors. Cured mice 

were completely resistant to s.c. challenge with wt tumor. The 

mRNA levels of the Treg transcription factor Foxp3 were 

measured in tumor biopsies and lymph nodes. There were no 

differences within the tumors of the different treatment groups. 

However, Foxp3 mRNA levels were down-regulated in the lymph 

nodes of AdCD40L treated mice. Human bladder cancer cell line 

cells were transduced with AdCD40L vector and used to 

stimulate immune cells. Cytokine and immune cell profiling 

indicated a Th1 type response. The AdCD40L-modified cell lines 

stimulated maturation of dendritic cells and cytotoxic Tcells as 

opposed to wt tumor cells. In conclusion, CD40L gene transfer 

evokes Th1 cytokine responses and counteracts Tregulatory cell 

development and/or function in the preclinical setting. We are 

currently conducting a phase I/II trial with AdCD40L in patients 

with bladder cancer. 

doi:10.1016/j.clim.2007.03.487 

Sa.101 Functional T Cell Responses to Tumor 

Antigens in Breast Cancer Patients Have a Unique 

Phenotype and Cytokine Signature 

Margaret Inokuma, Scientist, BD Biosciences, Immune 

Function, San Jose, CA, Corazon dela Rosa, Research 

Associate, University of Washington, Seattle, WA, Perry 

Haaland, BD Fellow, BD Technologies, Research Triangle 

Park, NC, Douglas Petry, patent attorney, BD Biosciences, 

San Jose, CA, Janet Siebert, president, CytoAnalytics, 

Denver, CO, MengXiang Tang, Senior Algorithm Developer, 

BD Biosciences, San Jose, CA, John Dunne, Associate 

Director, BD Biosciences, San Jose, CA, Vernon Mai, Director, 

BD Biosciences, San Jose, CA, Mary Disis, Associate 

Professor, University of Washington, Department of 

Oncology, Seattle, WA, Holden Maecker, Research Grp 

Manager, BD Biosciences, San Jose, CA 

The prevalence with which endogenous tumor antigens 

induce host T cell responses is unclear. Even when such 

responses are detected, they do not usually result in 

spontaneous remission of the cancer. We hypothesized that 

this might be associated with a predominant phenotype and/or 

cytokine profile of tumor-specific responses that is different 

from protective Tcell responses to other chronic antigens, such 

as cytomegalovirus (CMV). We detected significant T cell 

responses to CEA, HER-2/neu, and/or MAGE-3 in 17 of 21 

breast cancer patients naive to immunotherapy. The pattern of 

T cell cytokines produced in response to tumor-associated 

antigens (TAAs) in breast cancer patients was significantly 

different from that produced in response to CMV in the same 

patients. Specifically, there was a higher proportion of IL-2 

producing CD8+ T cells, and a lower proportion of IFN γproducing 

CD4+ and CD8+ T cells responding to TAAs compared 

to CMV antigens. Finally, the phenotype of TAA-responsive CD8+ 

T cells in breast cancer patients was almost completely CD28 

+CD45RA− (central memory phenotype). CMV-responsive CD8+ 

Tcellsinthesamepatientswerebroadlydistributedamong 

phenotypes, and contained a high proportion of terminal

Abstracts 

effector cells (CD27−CD28−CD45RA+) that were absent in the 

TAA responses. Taken together, these results suggest that TAAresponsive 

T cells are induced in breast cancer patients, but 

those Tcells are phenotypically and functionally different from 

CMV-responsive Tcells. Immunotherapies directed against TAAs 

may need to alter these T cell signatures in order to be 

effective. 

doi:10.1016/j.clim.2007.03.488 

Sa.102 Dendritic Cell-based Therapy for Mantle Cell 

Lymphoma 

Corey Munger, Graduate Student, Nebraska Medical Center, 

Genetics, Cell Biology, and Anatomy, Omaha, NE, Ganapati 

Hegde, Postdoctoral Fellow, Nebraska Medical Center, 

Genetics, Cell Biology, and Anatomy, Omaha, NE, Julie Vose, 

Professor, Internal Medicine, Omaha, NE, Shantaram Joshi, 

Professor, Nebraska Medical Center, Genetics Cell Biology, 

and Anatomy, Omaha, NE, Dennis Weisenburger, Professor, 

Pathology and Microbiology, Omaha, NE 

Mantle cell lymphoma (MCL) is an incurable B cell 

malignancy due to the development of therapy-resistant 

cells. The ability of dendritic cells (DC) to stimulate tumorspecific 

cytotoxic T lymphocytes (CTL) makes DC-based 

therapy a promising strategy to treat therapy-resistant MCL. 

Therefore, we investigated the efficacy of DC-based immunotherapy 

to treat resistant MCL. Specifically, DCs loaded 

with MCL lysate, DCs fused with MCL cells, and DCs 

electroporated with MCL RNA were used to generate 

tumor-specific CTLs against the MCL cell line, Granta 519. 

CTL-mediated cytotoxicities at a 50:1 effector-to-target 

ratio using each method of DC priming are as follows: 58.4 

±12.7% (MCL lysate-pulsed), 54.0±8.3% (MCL RNA-transfection), 

and 59.3±8.4% (DC-MCL hybrid). Cytotoxicity toward 

the irrelevant HLA-matched cell line MDA-231 was 28.7± 

3.6%. To assess the in vivo cytotoxic abilities of CTLs 

stimulated by DC-MCL hybrids, NOD/SCID mice transplanted 

intravenously with 1×106 Granta 519 cells were treated with 

four weekly injections of 3×106 HLA-matched CTLs. Histological 

analysis revealed that 66% of the control mice at week 

five displayed significant tumor growth in the liver and 

kidneys versus only 16% in the treatment group. In addition to 

reducing the number of tumor nodules, the average size of 

the nodules was decreased in treated mice versus the 

untreated control mice. These studies demonstrate each 

method of DC priming stimulated tumor-specific CTLs in vitro 

and administration of DC-MCL hybrid-stimulated CTLs inhibited 

tumor formation in vivo. This study was supported by 

the Lymphoma Research Foundation, New York, NY. 

doi:10.1016/j.clim.2007.03.489 

Sa.103 Targetting Sonic Hedgehog-GLI Signaling for 

the Treatment of Mantle Cell Lymphoma 

Ganapati Hegde, Postdoctoral Research Associate, 

Department of Genetics, Cell Biology and Anatomy, Omaha, 

NE, Corey Munger, Graduate Student, Department of 

Genetics, Cell Biology and Anatomy, Omaha, NE, Katy 

Emanuel, Summer Student, Department of Genetics, Cell 

Biology and Anatomy, Omaha, NE, Dennis Weisenburger, 

Professor, Pathology and Microbiology, Omaha, NE, Avadhut 

Joshi, Graduate student, Department of Genetics, Cell 

Biology and Anatomy, Omaha, NE, Julie Vose, Professor, 

Internal Medicine, Oncology/Hematology, Omaha, NE, 

Shantaram Joshi, Professor, Department of Genetics, Cell 

Biology and Anatomy, Omaha, NE 

Mantle cell lymphoma (MCL) has one of the worst clinical 

outcomes among the B cell lymphomas with a median 

survival of only 3–4 years. Particularly, the blastoid variant 

has a poorer prognosis compared to classical MCL. Therefore, 

a better understanding of the underlying mechanisms that 

regulate MCL proliferation and survival is clearly needed. 

Since, sonic hedgehog (Shh)-GLI signaling has been shown to 

be important in the proliferation/survival of several cancers, 

and no such information is available in any B cell malignancies 

including MCL, this study was undertaken. Our 

results demonstrate that the molecules associated with Shh 

signaling (PTCH, SMO, GLI) are considerably expressed in 

classical, blastoid and patients’ primary MCL. Overexpression 

of PTCH and GLI1, the Shh-GLI signaling target genes, in 

blastoid versus classical MCL suggests constitutive expression 

of these genes in blastoid variant. Classical, but not the 

blastoid MCL cells responded (pb0.01) to perturbation of Shh 

signaling in the presence of exogenous Shh/cyclopamine. 

Furthermore, down-regulation of GLI transcription factors 

using anti-sense oligonucleotides resulted in significantly 

(pb0.001) decreased proliferation of both blastoid and 

classical MCL, and significantly (pb0.001) increased their 

susceptibility to chemotherapy. In addition, down-regulation 

of GLI by anti-sense ODN decreased Cyclin D1 (CCND1) and 

BCL2 transcript levels, which suggests that these key 

molecules might be regulated by GLI in MCL. Thus, these 

results suggest a significant role for Shh-GLI signaling in the 

proliferation of MCL, and targeting GLI as therapeutic 

approach to treat MCL effectively is very promising (this 

study was supported by the Lymphoma Research Foundation, 

New York). 

doi:10.1016/j.clim.2007.03.490 

S109 

Sa.104 The Role of Thrombospondin-1 in Driving 

Regulatory T Cell Generation Following 

Extracorporeal Photopheresis 

Amy Krutsick, Senior Scientist, Therakos, Inc. RandCD, 

Exton, PA, Peter O’Brien, Principal Scientist, Therakos, Inc. 

RandCD, Exton, PA, Ryan Gailey, Research Associate I, 

Therakos, Inc. RandCD, Exton, PA, Kim Campbell, Principal 

Research Scientist, Therakos, Inc. RandCD, Exton, PA, Frank 

Strobl, Director of Scientific Affairs, Therakos, Inc. RandCD, 

Exton, PA 

Extracorporeal photopheresis (ECP) is an immune cell 

therapy approved for the palliative treatment of cutaneous 

T cell lymphoma and has demonstrated activity in 

immune-mediated inflammatory diseases. During ECP,


peripheral blood leukocytes are treated ex vivo with 8methoxypsoralen 

and UVA light, rendering cells apoptotic. 

Reinfusion of ECP-treated cells modulates immune 

responses through the generation of tolerogenic dendritic 

cells and T regulatory cells (Tregs). We previously 

demonstrated that ECP-treated peripheral blood mononuclear 

cells (PBMCs) co-cultured directly with T cells 

result in the generation of a regulatory T cell population. 

ECP-generated Tregs can suppress a syngeneic T cell 

response in a contact-dependent fashion by greater than 

70%. These Tregs demonstrate a modest increase in Foxp3 

expression and exhibit an anergic phenotype upon in vitro 

re-stimulation. We found that monocytes are required for 

ECP-mediated Treg generation and apoptotic ECP-treated 

monocytes secrete a significant level (N500 ng/ml) of 

Thrombospondin-1 (TSP-1). TSP-1 is an extracellular 

matrix protein with anti-angiogenic properties shown by 

others to have immunomodulatory effects on T cells 

through interactions with CD47. We hypothesized that 

TSP-1 is involved in the generation of Tregs via ECP cells. 

To confirm this hypothesis, TSP-1 activity was blocked 

through addition of anti-CD47 to cultures containing ECPtreated 

PBMCs and T cells. Blocking this TSP-1 receptor on 

T cells inhibited ECP-mediated Treg generation. Additional 

studies are in progress to further evaluate the role of TSP- 

1 in the mechanism of action of ECP. TSP-1 may be 

important for the clinical benefit of ECP in a variety of 

pathophysiological disorders. 

doi:10.1016/j.clim.2007.03.491 

Sa.105 Cytotocix Effect of Total Saffron Extract on 

Human Hepatocell Carcinoma (HCC) 

Jalil Tavakkol-Afshari, Vice President for Research, Head, 

Department of Immunogenetics and Cell Culture, Bu-Ali 

Research Institute (BARI), Department of Immunogenetics 

and Cell Culture, Mashhad, Iran, Khadijeh Nejad 

Shahrokhabadi, Researcher, Bu-Ali Research Institute 

(BARI), Department of Immunogenetics, Mashhad, Iran, 

Hassan Rakhshandeh, Pharmacologist, School of 

Pharmocology, Mashhad University of Medical Sciences, 

Mashhad, Iran, Azam Brook, Researcher, Bu-Ali Research 


Iran 

Saffron – dried stigma of Crocus Sativus L. – is used as a 

nutritional spice and dry additive in cooking. The antitumor 

effect of saffron extract has been proved both in vitro and 

in vivo during recent years. In this research, cytotoxic 

effect of total saffron aqueous extract on human Hepatocell 

Carcinoma (HepG2) and mouse fibroblastic cells (L929) 

(as controls) in vitro has been investigated. Effects of 

extract on quantitative proliferation of each cell line were 

determined using (MTT) colorimetric assay. MTT assay is a 

fast, sensitive and quantitative method for all kinds of cell 

proliferation by spectrophotometry. Different amounts of 

extract were used and results showed that the 50% 

inhibition occurred in tumoral cells at the concentration 

of 400 microgram/milliliter that had no inhibitory effects 

on normal cells. According to our findings, the effect of 

saffron extract may be useful in vivo against hepatic tumoral 

cells. 

doi:10.1016/j.clim.2007.03.492 

Sa.106 Optimizing Peptide Loading of Dendritic 

Cells Using TCRm Antibodies 

Tffany Nguyen, Senior Research Technician, Receptor Logic, 

Ltd. Amarillo, TX, Olivier Faure, Scientist, IDM, Inc. Irvine, 

CA, Francisca Neethling, Scientist, Receptor Logic, Ltd. 

Amarillo, TX, Roy Guillermo, Scientist, IDM, Inc. Irvine, CA, 

Sun min Lee, Scientist, IDM, Inc. Irvine, CA, James Bender, 

Scientist, IDM, Inc. Irvine, CA, Jon A. Weidanz, Assistant 

Professor, Texas Tech University Health Sciences Center, 

Amarillo, TX 

Dendritic cells (DCs) represent potential vaccine vehicles 

for the treatment of cancer. Peptide-loaded DC’s require no 

protein processing and are amendable for development of 

“personalized” vaccines. Conditions for peptide-loading DC 

are poorly described and have not yet been optimized. We 

examined peptide-loading conditions for DC using a TCRm 

antibody that specifically recognizes a peptide 

(TMTRVLQGV40-48) presented by HLA-A*0201 derived from 

the tumor-associated antigen human chorionic gonadotropin 

beta (hCGb). Characterization of the 3F9 TCRm included 

ELISA and flow cytometric analysis and demonstrated specific 

binding to peptide–HLA-A2 tetramer and T2 cells loaded with 

the TMT peptide. Moreover, the 3F9 TCRm displayed 

detection sensitivity in the picomolar range. TCRm staining 

of DCs was shown to be dependent on peptide concentration 

with maximum and minimum mean fluorescence intensity 

(MFI) reported at 80 μM and 10 μM peptide, respectively. We 

next examined the relationship between pulsing time and 

peptide presentation. Immature DCs were pulsed with 20 μM 

peptide for 1, 2, 4, 6, 8 and 22 h followed by cytokine-induced 

maturation during the final six hours. DC pulsed for shorter 

periods of time displayed TCRm staining with higher MFI 

values, suggesting that longer pulse times decrease peptide- 

HLA presentation. DCs were then pulsed with a peptide 

cocktail (TMT+6 other peptides) to determine TMT peptide 

loading efficiency. Similar TCRm staining intensities were 

observed for DC pulsed with TMT peptide alone and cells 

pulsed with the peptide cocktail. These findings suggest that 

TCRm may be useful tools for the development and 

standardization of peptide-pulsed DC vaccines. 

doi:10.1016/j.clim.2007.03.493 

Sa.107 Phase I Study of the Anti-MUC-1 Antibody, 

huHMFG1 (AS1402), in Patients with Advanced 

Breast Cancer 

Nigel Courtenay-Luck, Chief Scientific Officer, Antisoma 

Research Ltd, London, England, Mark Pegram, Associate 

Professor of Medicine, David Geffen School of Medicine, Los 

Angeles, CA 

Background: A 20 amino acid MUC-1 core tandem repeat 

is aberrantly glycosylated in N90% of epithelial tumors,

Abstracts 

exposing the peptide sequence PDTRP. Humanized IgG1 K 

monoclonal antibody huHMFG1 (AS1402, formerly R1550), 

binds to PDTRP (Kd ∼3 nM) and, in vitro, induces potent 

antibody dependent cellular cytotoxicity (ADCC). This study 

was designed to evaluate the safety, tolerability and 

recommended dose of huHMFG1 in patients with advanced 

breast cancer. Methods: 26 patients were enrolled with 

locally advanced or metastatic breast cancer, which was 

progressive following up to 3 prior chemotherapy regimens. 

Patients were randomly assigned to 4 cohorts, each 

allocated to receive huHMFG1 at a dose of 1, 3, 9, or 

16 mg/kg, which was administered via I.V. infusion with 

repeated weekly dosing until disease progression. Clinical 

and laboratory analysis was performed regularly. Results: 

The maximum tolerated dose (MTD) of huHMFG1 was not 

reached in this study and no serious adverse events were 

reported. No anti-huHMFG1 antibodies were detected. 

Response data are available from 22 patients with the 

best recorded clinical response, using RECIST criteria, being 

stable disease. Time to tumor progression ranged between 

28 and 119 days. Conclusion: huHMFG1 appears to be well 

tolerated with the MTD exceeding the doses tested. This 

study showed cases of prolonged stable disease among 

breast cancer patients who had relapsed following chemotherapy, 

suggesting that evaluation of huHMFG1 in phase 

II trials is warranted. 

doi:10.1016/j.clim.2007.03.494 

Sa.108 30 Years from Creation of the First 

Cytochemical Method Analysing RNP, DNP and 

Cationic Proteins in Circulating Leukocytes of 

Cancer Patients as a Tool for Early Diagnostics of 

Malignancies 

Elissaveta Borissova Zvetkova, Associated Professor, IEMAM, 

Sofia, Bulgaria, Georgi Stefanov Kostov, Head of Surgery 

Department, Oncological Hospital, Veliko Tarnovo, Yordanka 

Georgieva Gluhcheva, Research Associate, IEMAM, Cell 

Differentiation, Sofia, Bulgaria 

Background: In our efforts to analyse cellular features of 

peripheral blood leukocytes in cancer patients and tumorbearing 

animals, we developed (30 years ago) an accurate 

method for early detection of malignancies (1–4). Methods: 

We combined our cytological/cytochemical method for 

simultaneous visualization of nucleoproteins (RNP and DNP) 

and cationic proteins with ultrastructural and cytophotometrical 

techniques. Results and Discussion: Examining a panel 

of activation/deactivation cell markers in peripheral blood 

leukocytes of cancer and precancerous patients, we observed 

specific abnormalities: (1) Quantitative reduction of the 

cytoplasmic (ribosomal) RNP-content in the peripheral blood 

mononuclears T-lymphocytes and monocytes, as an additional 

tool for early cancer diagnostics; (2) Changes in the 

quantity, condensation and distribution of DNP in the nuclear 

chromatin of mono-and polymorphonuclear leukocytes as 

cytological signs for tumor-microenvironment-induced transcriptional 

arrest and apoptosis. (3) Polymorphism in size, 

staining properties and high extractibility of cytoplasmic 

(cationic proteins’ containing) granules of circulating poly- 

morphonuclear granulocytes—neutrophils and eosinophils. 

Today, 30 years later, our old hypothesis that cancer is not 

local, but systemic nuclear chromatin disease altering 

peripheral blood cell genome expression is in very good 

agreement with recent data based on gene expression 

patterns in peripheral blood cells of cancer patients (5) as 

monitors for early detection of malignant disease elsewhere 

in the body. References: (1) Acta histochem., 57, 1976, 1. (2) 

Folia Haematol., 106/2, 1979, 205. (3) Arch. Union Med Balk., 

XX/1–2, 1984, 140. (4) Acta morphol. and anthropol., 5, 

2000, 3. (5) Breast Cancer Res., 7/5, 2005, R634. 

doi:10.1016/j.clim.2007.03.495 

Sa.109 Human Lung Tumor Cells Modifies Rates of 

CD4+CD25+ T Regulatory, CD19+CD23+ B Regulatory 

Cells, CD3+CD95+ Cells, NK Cells and B Lymphocytes 

Bayram Kiran, Tip Fakultesi, Temel Bilimler Binasi, Istanbul, 

Turkey, Akif Turna, Yedikule Hospital for Chest Diseases and 

Thoracic Surgery, Kadikoy, Istanbul, Turkey, Alper Yener, 

Istanbul Tip Fakultesi, Istanbul, Turkey, Atilla Gürses, 

Yedikule Gogus Hastaliklari Hastanesi, Istanbul, Turkey, 

Andac Salman, Istanbul Tip Fakultesi, Istanbul, Turkey, 

Selim Badur, Istanbul Tip Fakultesi, Temel Bilimler Binasi, 

Istanbul, Turkey 

The role of T regulatory cells and NK cells in human lung 

cancer has not yet been clarified. Our aim was to evaluate 

how the microenvironment of a tumor mass induced phenotypical 

changes in lymphocyte subsets. Our subjects were 

10 patients with resectable non-small cell lung cancer. We 

evaluated 47 different phenotypically different lymphocyte 

subsets in blood samples taken from the pulmonary artery, 

pulmonary vein of a tumor bearing pulmonary lobe and 

peripheral blood during pulmonary resectional surgery. We 

showed that, tumor cells boosted CD25high+CD4high+T 

lymphocytes (Treg cells), B lymphocytes, NK and CD19+ 

CD23+ cells (Breg cells) and CD3+CD95+ (FasL+T lymphocytes) 

(p=0.015, p=0.001, p=0.02, p=0.04, p=0.03 respectively). 

However, Mac-1 cells and HLADR+T lymphocytes 

were found to be decreased by tumor mass (p=0.04 and 

p=0.04), whereas only Breg, NK cell and B lymphocyte 

subsets were found to be expansed. In addition, the patients 

showed expansions of CD45R0+ activated T lymphocytes and 

CD18+CD11b+(Mac-1) cell subsets without significant alteration 

by tumor microenvironment. In conclusion, subsets of 

Treg, Breg cells, FasL+ T lymphocytes, B lymphocytes were 

modified by lung cancer microenvironment itself. This study 

also showed that, peripheral blood might not be good source 

for investigating the anti-tumor immunity since only Breg, NK 

cell and Treg cell subsets were found expansed peripherally. 

T lymphocytes and Mac-1 cells may be modified by systemic 

antitumor response rather than by local tumoral microenvironment. 

This study provides important insight into how 

tumor infiltrating lymphocytes and peripheral immune 

systems may be different in terms of lymphocyte subset 

expansion dynamics. 

doi:10.1016/j.clim.2007.03.496 

S111


Sa.110 ABCB5 Gene is Expressed in Acral Melanoma 

of Poor Prognosis 

Norma Herrera-Gonzàlez, Pharmaceutical Industrial 

Chemest, Postgraduate Division, Mexico, Ismael Vasquez, 

Medical Doctor, Postgraduate Division, Mexico, Marco 

Antonio Meraz-Rios, Pharmacobiology Chemist, Biomedicine 

Department, CINVESTAV, Mexico, Cleva Villanueva, Medical 

Doctor, Postgraduate Division, Mexico 

Cutaneous melanomais a malignant neoplasm characterized 

by a high risk of metastasis disease and death. 

Metastatic melanoma displays strong resistance against 

antineoplastic drugs. Combination of chemotherapy and 

radiotherapy has been tried in patients and the best response 

rates have been less than 20%. The dramatic increase in the 

incidence and mortality of melanoma highlights the need for 

improved methods of diagnosis as well as a better understanding 

of basic concepts of some types of melanoma that 

have been scarcely studied as Acral Melanoma (AM), this 

work describes a molecular screen for differentially 

expressed genes in AM, the most common melanoma in 

Hispanic population. We used differential display reverse 

transcription-polymerase chain reaction and compared the 

gene expression pattern between primary melanomas and 

normal skin. In this work we fond an overexpression of the 

ABCB5 gene in melanoma tissue. ABCB5 has been recently 

characterized in normal human melanocytes and has been 

implicated in the regulation of progenitor cell fusion. ABCB5 

amplification bands were detected in nine of ten melanoma 

tissues by using RT-PCR. In situ hybridization using specific 

probes showed the presence of this gene in paraffin 

melanoma tissues. We suggest that this event might be 

related to metastatic melanoma behavior. 

doi:10.1016/j.clim.2007.03.497 

Sa.111 MHC Class I and MICA Expressions in Liver 

Fluke Associated Cholangiocarcinoma 

Wachanan Wongsena, Lecturer, Biomedical Sciences 

Program, Graduate School, Khon Kaen University, Khon 

Kaen, Thailand, Solda Ferrone, Educator/Research Director, 

Roswell Park Cancer Institute, Buffalo, NY, Richard Cheney, 

Pathologist/Dermatopathologist, Roswell Park Cancer 

Institute, Buffalo, NY, Chanvit Leelayuwat, Educator, The 

Centre for Research and Development of Medical Diagnostic 

Laboratories, Khon Kaen University, Khon Kaen, Thailand, 

Banchob Sripa, Educator, Liver Fluke and 

Cholangiocarcinoma Research Center, Khon Kaen, Thailand 

Alterations of HLA class I antigens and MHC class I chain 

related gene A (MICA), a ligand for activating receptor, 

NKG2D, have been described in several types of tumors. Such 

a study in cholangiocarcinoma has not been reported. A total 

of 102 paraffin-embedded cholangiocarcinoma lesions were 

stained by mouse anti-HLA class I heavy chain, anti-β-2microglobulin, 

anti-Tapasin, anti-TAP1, anti-TAP2, anti- 

LMP2, anti-LMP7, and three of anti-MICA monoclonal antibodies 

using immunoperoxidase reaction. HLA class I heavy 

chain, β-2-microglobulin, Tapasin, TAP1, TAP2, LMP2 and 

LMP7 were lost in 12.7, 5.9, 6.9, 2.0, 1.0, 6.9 and 8.8% of the 

lesions tested, respectively. Although most of the lesions 

expressed HLA class I heavy chain and β-2-microglobulin, the 

expression was restricted to the cytoplasm in 68.5 and 64.6% 

of the lesions, respectively. Loss of HLA class I heavy chain 

expression was associated with late stage (p=0.034) and 

poor differentiation of tumors (p=0.014). Loss of LMP7 

expression was also associated with poor differentiation of 

tumors and Adenosquamous/squamous type (p =0.038). 

Multivariate analysis showed that Tapasin and TAP1 losses 

were associated with a high risk of death. Although 85% of 

CCA lesions were stained with anti-MICA, most of them were 

restricted in the cytoplasm. Thus, MICA expression was not 

associated with any parameters even in cases of HLA class I 

loss. Our results demonstrated several abnormalities in HLA 

class I antigens and MICA expressions suggesting the potential 

mechanisms utilized by cholangiocarcinoma for immune 

evasion. 

doi:10.1016/j.clim.2007.03.498 

Sa.112 CD8+CD28− T Suppressor Lymphocytes 

Inhibiting T Cell Proliferative and Cytotoxic 

Functions Infiltrate Human Cancers 

Gilberto Filaci, Associate Professor of Internal Medicine, 

University of Genoa, Department of Internal Medicine and 

Centre of Excellence for Biomedical Research, Genoa, Italy, 

Daniela Fenoglio, Assistant Professor, University of Genoa, 

Centre of Excellence for Biomedical Research, Genoa, Italy, 

Marco Fravega, Fellow, University of Genoa, Centre of 

Excellence for Biomedical Research, Genoa, Italy, Giacomo 

Borgonovo, Associate Professor, University of Genoa, 

Department of Surgical and Morphological Disciplines and 

Integrated Methodologi, Genoa, Italy, Gianluca Ansaldo, 

Assistant Professor, University of Genoa, Department of 

Surgical and Morphological Disciplines and Integrated 

Methodologi, Genoa, Italy, Paolo Traverso, Assistant 

Professor, University of Genoa, Urology Department, Genoa, 

Barbara Villaggio, Technician, University of Genoa, 

Department of Internal Medicine, Genoa, Italy, Jean Louis 

Ravetti, Director of Anatomic Pathology, San Martino 

Hospital, Anatomic Pathology, Genoa, Italy, Giorgio 

Carmignani, Full Professor, University of Genoa, Urology 

Department, Genoa, Giancarlo Torre, Full professor, 

Department of Surgical and Morphological Disciplines and 

Integrated Methodologies, University of Gen, Genoa, Italy, 

Francesco Indiveri, Full Professor of Internal Medicine, 

University of Genoa, Department of Internal Medicine and 

Centre of Excellence for Biomedical Research, Genoa, Italy 

Since an important role in determining tumor evasion 

from immune control might be played by tumor infiltrating 

regulatory lymphocytes we performed a study aimed at 

characterizing phenotype and function of CD8+CD28− T 

suppressor cells infiltrating human cancer. Lymphocytes 

infiltrating primitive tumor lesion and/or satellite lymph 

node from a series of 42 human cancers were phenotypically 

studied and functionally analyzed by suppressor assays. The 

unprecedented observation was made that CD8+CD28− T 

suppressor lymphocytes are almost constantly present and 

functional in human tumors, being able to inhibit both T cell

Abstracts 

proliferation and cytotoxicity. CD4+CD25+ T regulatory 

lymphocytes associate with CD8+CD28− T suppressor cells 

so that the immunosuppressive activity of tumor infiltrating 

regulatory T cell subsets, altogether considered, may 

become predominant. The infiltration of regulatory T cells 

seems tumor-related, being present in metastatic but not in 

metastasis free satellite lymph nodes; it likely depends on 

both in situ generation (via cytokine production) and 

recruitment from the periphery (via chemokine secretion). 

Collectively, these results have pathogenic relevance and 

implication for immunotherapy of cancer. 

doi:10.1016/j.clim.2007.03.499 

Sa.113 GCN2 Kinase: An Evolutionarily Conserved 

Mechanism of Human T Lymphocyte Suppression 

Upon Arginine Depletion 

Markus Munder, Instructor in Medicine, University of 

Heidelberg. Department of Hematology, Oncology and 

Rheumatology, Heidelberg, Germany, Michelle Bhairo, 

Research Assistant, University of Heidelberg. Institute of 

Immunology, Heidelberg, Germany, Thomas Giese, Group 

Leader, University of Heidelberg. Institute of Immunology, 

Heidelberg, Germany, Claudia Luckner, Research Assistant, 

University of Heidelberg, Department of Hematology, 

Oncology and Rheumatology, Heidelberg, Germany, Anthony 

Ho, Head of Department, University of Heidelberg. 

Department of Hematology, Oncology and Rheumatology, 

Heidelberg, Germany, Stefan Meuer, Head of Department, 

University of Heidelberg. Institute of Immunology, 

Heidelberg, Germany 

Impaired T cell immunity is a cardinal feature of chronic 

inflammation and tumor growth. We have recently demonstrated 

that human polymorphonuclear leukocytes (PMN) 

constitutively express high activities of the arginine-metabolising 

enzyme arginase. Upon PMN cell death (e.g., during 

purulent inflammation) this enzyme is liberated and metabolizes 

arginine to ornithine and urea in the microenvironment. 

This arginine depletion completely inhibits 

proliferation and effector functions of activated human T 

lymphocytes while their viability remains unaltered. Specifically, 

the T cell cytokine response is impaired at a 

posttranscriptional level by arginine depletion. Arginasemediated 

arginine depletion is likely a key mechanism of T 

cell immunosuppression in the inflammatory or tumor 

environment. We now wanted to further clarify how T cell 

reactivity is turned off in the face of arginine depletion. Here 

we demonstrate for the first time that human T lymphocytes 

respond to arginine depletion by phosphorylation of the 

kinase GCN2. This is analogous to the general control 

response of lower eukaryotes upon amino acid depletion. 

We also analysed phosphorylation of the α-subunit of 

eukaryotic translation initiation factor 2 (eIF2α) and ATF-2 

as well as expression of transcription factors ATF-4 and CHOP, 

known elements in lower eukaryotes in their response to 

amino acid depletion. We show that these mammalian 

homologues are less stringently regulated in human lymphocytes 

upon arginine depletion than phosphorylation of 

GCN2K. In summary, our data demonstrate an evolutionarily 

conserved mechanism of cellular response towards amino 

acid depletion in human T lymphocytes. 

doi:10.1016/j.clim.2007.03.500 

Sa.114 Characterization of the B Cell Infiltrate 

Within Intracranial Germinomas 

Shannon L. McArdel, Research Technician, Brigham and 

Women’s Hospital, Boston, MA, Katharine Cronk, 

Neurosurgery Resident, Columbia Medical School, New York, 

NY, Kimberly L. Shampain, Student Researcher, Brigham and 

Women’s Hospital, Boston, MA, Richard C.E. Anderson, 

Assistant Professor of Neurosurgery and Pediatric 

Neurosurgery, Columbia University Medical Center, New 

York, NY, David E. Anderson, Instructor in Neurology, 

Harvard Medical School, Boston, MA, Jeffrey N. Bruce, 

Professor of Neurosurgery, Columbia University Medical 

Center, New York, NY, David A. Hafler, Professor of 

Neurology, Harvard Medical School, Brigham and Women’s 

Hospital, Boston, MA, Kevin C. O’Connor, Instructor in 

Neurology, Harvard Medical School, Boston, MA 

Intracranial germinomas arise primarily in children, from 

neoplastic transformation of embryonic germ cells in the 

CNS, most often in the pineal and suprasellar regions. These 

malignant tumors are currently treated with surgery followed 

by radiation therapy. Many children however, are too 

young to receive radiation therapy or present with tumor 

disseminated throughout the cerebrospinal fluid pathways, 

making treatment difficult. Thus, germinomas present an 

attractive target for antibody-based immunotherapy. Intracranial 

germinomas commonly include an immune cell 

infiltrate, including a prominent population of B cell subsets 

that have not yet been characterized. We began to 

investigate these B cells by examining their antibody 

repertoire. We constructed immunoglobulin variable region 

(V) gene libraries from consecutive sections and from laser 

capture microdissected B cells from resected germinoma 

tumors. Comparison of the V-gene sequences to their 

corresponding germline sequences revealed that somatic 

mutation, oligoclonal expansion and isotype switching were 

prominent within the tumor infiltrate. These are cardinal 

features of an antigen driven B cell response, indicating that 

a local inflammatory response may be occurring within the 

CNS. This response is likely to be driven by tumor-associated 

antigens, the identification of which will provide insight into 

the immune-related tumor microenvironment. 

doi:10.1016/j.clim.2007.03.501 

S113 

Sa.115 Wnt5a Regulates Melanoma Antigen 

Recognized by T Cells 1 (MART-1), a Predominant 

Antigen in Melanoma Cells 

Samudra Dissanayake, Post doctoral Fellow, National 

Institute on Aging, National Institute of Health, Baltimore, 

MD, Devin Rosenthal, Student, Laboratory of Immunology, 

National Institute on Aging, National Institute of Health, 

Baltimore, MD, Kyle Hewitt, Student, Laboratory of


Immunology, National Institute on Aging, National Institute 

of Health, Baltimore, MD, Michael Wade, Student, 

Laboratory of Immunology, National Institute on Aging, 

National Institute of Health, Baltimore, MD, Sherry Yang, 

Student, Laboratory of Immunology, National Institute on 

Aging, National Institute of Health, Baltimore, MD, Poloko 

Leotlela, Post doctoral Fellow, Laboratory of Immunology, 

National Institute on Aging National Institute of Health, 

Baltimore, MD, Dennis Taub, Chief, Laboratory of 

Immunology, Laboratory of Immunology, National Institute 

on Aging, National Institute of Health, Baltimore, MD, Adam 

Riker, Chief of Surgical Oncology, Mitchell cancer Institute, 

University of South Alabama, North Mobile, AL, Brian 

Nickoloff, Professor pathology, Loyola University Medical 

Center, Maywood, IL, Ashani Weeraratna, Staff Scientist, 

Laboratory of Immunology, National Institute on Aging, 

National Institute of Health, Baltimore, MD 

Wnt5a increases melanoma cell motility via activation of 

Protein Kinase C (PKC). Microarray analysis showed an 

inverse relationship between Wnt5a and MART-1. Using PKC 

activation and inhibition studies, as well as recombinant 

Wnt5a and Wnt5a RNAi, we demonstrate here that Wnt5aactivated 

PKC affects the expression of MART-1 via the 

phosphorylation of STAT3. STAT3 is a transcriptional factor 

known to inhibit the binding of MART-1 transcriptional 

activators MITF, SOX10 and PAX3. These effects are mediated 

by Wnt5a upstream of PKC, as overexpression of Wnt5a 

increases pSTAT3 and Wnt5a RNAi treatment decreases 

pSTAT3 levels. The decrease in pSTAT3 corresponds to an 

increase in MART1 and vice versa. Translocation of pSTAT3 

from the cytosol to the nucleus upon transfection of Wnt5a, 

indicated activation of STAT3 by Wnt5a, while the reverse 

was observed for cells knocked down for Wnt5a. Further, 

steady increases in PAX3 and MART-1 protein levels were 

observed in cells treated with Wnt5a RNAi, possibly due to 

suppressing the effects of STAT3. MART1 positive melanoma 

cells are able to activate CTL, while addition of recombinant 

Wnt5a to these cells could not, presumably due to the downregulation 

of MART-1. RNA from human melanoma biopsies 

analyzed for Wnt5a by real-time PCR shows an inverse 

relationship between Wnt5a levels and patient survival time, 

suggesting that cells with higher MART-1 have a better 

outcome. Collectively, our data suggest that Wnt5a, via PKC, 

results in the phosphorylation of STAT-3 and a subsequent 

down-regulation MART1, thereby rendering melanoma cells 

able to escape immune detection. 

doi:10.1016/j.clim.2007.03.502 

Sa.116 Discovery of Cancer and Autoimmune 

Biomarkers Utilizing Serum Antibody Profiling on 

Protein Microarrays 

Jennifer Cannon, Scientist, Invitrogen Corporation, 

Carlsbad, CA, Michael Snyder, Professor, Yale University, 

New Haven, CT, Michael Hudson, Scientist, Yale University, 

New Haven, CT, Gregory Michaud, Scientist, Invitrogen 

Corporation, Carlsbad, CA, Michael Smith, Scientist, 

Invitrogen Corporation, Carlsbad, CA, Barry Schweitzer, 

Research and Development Director, Invitrogen 

Corporation, Carlsbad, CA, Guillermo Mor, Associate 

Profesor, OB/GYN, New Haven, CT 

It has been well established that serum autoantibodies 

have important diagnostic value for many diseases, including 

cancer and autoimmune diseases. Identifying the antigens 

which elicit an autoimmune response can yield sets of 

biomarkers that provide classifiers for particular diseases 

and disease stages as well as predictors of patient outcomes 

and patient responses to therapeutics. Protein microarrays 

are valuable tools that have been successfully applied to 

investigate the circulating antibody profile in several disease 

states. To discover potential autoantibody biomarkers 

specific to ovarian cancer, sera from 30 ovarian cancer 

patients and 30 healthy patients were profiled on protein 

microarrays. Patient and control sera were diluted and 

applied to protein microarrays containing over 5000 purified 

proteins expressed in a baculovirus expression system. Over 

95 candidate biomarkers were identified that exhibited 

enhanced reactivity in sera from cancer patients relative to 

that of the control individuals. Antibodies to four antigens 

were tested for differential reactivity in tissue samples of 

cancer patients relative to healthy patients using immunoblot 

analysis and tissue microarrays. Three biomarkers 

exhibited elevated expression in the cancer tissue relative 

to controls. Signal observed for the biomarker antigens 

appeared significantly stronger than signal corresponding to 

the existing ovarian cancer antigen, CA-125. Additional 

studies focused on identifying biomarkers associated with 

autoimmune diseases, such as Rheumatoid Arthritis and 

Systemic Lupus Erythematosus, have recently utilized a 

higher content protein microarray including over 8000 

unique human proteins. The application of protein microarray 

technology for serum antibody profiling will be 

discussed along with data from on-going validation studies. 

doi:10.1016/j.clim.2007.03.503 

Sa.117 Phenotype and Function Analysis of 

Dendritic Cells From Patients with Prostate 

Carcinoma After Radical Prostatectomy 

Ivo Minarik, PhD Student, Department of Immunology, 2nd 

Medical School, Charles University, Prague 5, Czech 

Republic, Zuzana Tobiasova, PhD Student, Department of 

Immunology, 2nd Medical School, Charles University, Prague 

5, Czech Republic, Jirina Bartunkova, Head of Department, 

Department of Immunology, 2nd Medical School, Charles 

University, Prague 5, Czech Republic, Ivan Kawaciuk, Head 

of Department, Department of Urology, 2nd Medical School, 

Charles University, Prague 5, Czech Republic 

Prostate carcinoma represents the 3rd most frequent 

malignancy in the Czech male population with the 

incidence of 68.3/100000 and mortality 28.2/100000. 

About 35% of patients experience biochemical relapse 

(PSA increase) any time after radical prostatectomy. 

Hence, alternative treatment is highly required. Dendritic 

cell (DC)-based immunotherapy seems to be a promising 

approach for the elimination of relapse. In our study we aim 

to compare function and phenotype of dendritic cells from

Abstracts 

patients with prostate carcinoma, who underwent radical 

prostatectomy, to controls without tumor. We included 10 

patients (age 53-76) with prostate carcinoma who fulfilled 

at least one of the following risk factors of biochemical 

relapse: preoperative PSA> 10ng/ml, postoperative Gleason 

score >7 or penetration through prostate capsule. The 

control group comprised 10 patients (age 53-73) with 

benign prostate hyperplasia. We examined the phenotype 

(HLA-DR, CD11c, CD83, CD80, and CD86) and functionality 

(endocytosis, production of IL-12, IL-6, IL-1beta, IL-10, and 

TNF-alpha) of dendritic cells (DC) and compared the same 

features on DC from controls. Immature DCs were prepared 

from monocytes after 6 days of cultivation in the presence 

of GM-CSF and IL-4. Their maturation was achieved by 24 

hours co-incubation with either poly (I:C), or LPS. Immature 

DCs demonstrated higher phagocytic activity, while mature 

DCs produced more inflammatory cytokines and expressed 

CD83, HLA-DR and costimulatory molecules in higher 

amounts. The difference between patients and healthy 

donors was insignificant. In conclusion, dendritic cells 

prepared from monocytes of patients are functionally and 

phenotypically competent and so can be used for development 

of DC vaccines. 

doi:10.1016/j.clim.2007.03.505 

Sa.119 Parallel Mechanisms of Activation by Human 

and Mouse Dendritic Cells Treated with the IgM 

Immune Modulator B7-DC XAb 

Larry Pease, Professor, Mayo Clinic College of Medicine, 

Immunology, Rochester, MN, Erin Schenk, Graduate Student, 

Mayo Clinic College of Medicine, Department of 

Immunology, Rochester, MN, Svetomir Markovic, Associate 

Professor, Mayo Clinic College of Medicine, Rochester, MN 

Systemic treatment of mice with the human antibody B7- 

DC XAb modulates dendritic cell (DC) function, protecting 

animals from experimental inflammatory airway disease and 

established syngeneic tumor implants. Protection in both 

models involves modulation of T cell function by activated 

DCs. B7-DC XAb (1) activates mouse and human DCs in a B7- 

DC XAb dependent fashion, (2) induces very similar macromolecular 

complexes on the cell surface of mouse and human 

DCs, and (3) initiates similar signaling cascades, patterns of 

gene expression, and DC functions in these antigen presenting 

cells derived from the two species. Furthermore, human 

DCs activated with B7-DC XAb are strong inducers of antigenspecific 

T cell immune responses by peripheral blood T cells 

in vitro. The similar response pattern found in mouse and 

human DCs is even more remarkable since bone marrowderived 

DCs from the mouse and monocyte-derived DCs from 

human peripheral blood do not represent closely related 

differentiation states prior to activation with the immune 

modulator. Nonetheless, these strong parallels in mouse and 

human responsiveness to B7-DC XAb treatment provide 

justification for our ongoing efforts to develop this human 

antibody for use in clinical trials. 

doi:10.1016/j.clim.2007.03.506 

Sa.120 Human Glioblastoma Multiforme (GBM) 

Tumors are Enriched for Strongly Suppressive, 

Apoptosis Resistant, DR+ Tregs 

Charles Ashley, Research Technician, Harvard Medical 

School, Brigham and Women’s Hospital, Department of 

Neurology, Boston, MA, Richard Anderson, Assistant 

Professor, Columbia University, Department of Pediatrics, 

New York, NY, Jeffrey Bruce, Professor, Neurological 

Institute of New York, New York, NY, David Anderson, 

Instructor of Neurology, Harvard Medical School, Brigham 

and Women’s Hospital, Department of Neurology, Boston, 

MA, David Hafler, Breakstone Chair of Neurology, Harvard 

Medical School, Brigham and Women’s Hospital, 

Department of Neurology, Boston, MA, Clare Baecher-Allan, 

Instructor of Neurology, Harvard Medical School, Brigham 

and Women’s Hospital, Department of Neurology, 

Boston, MA 

Glioblastoma Multiforme (GBM) is a CNS tumor of astrocytic 

origin, that is highly refractory to current therapies 

and associated with survival of less than 15 months. Using 

post surgical resection GBM samples to study the profile of 

the infiltrating regulatory T cells, we have found marked 

alterations in subset frequency, activation state, and gene 

expression in GBM-derived Tregs. In GBM, not only are 

there a large number of tumor-infiltrating Tregs, but 

importantly, these Tregs are strikingly enriched for the 

most suppressive Treg subset that are classified by HLA-DR 

expression. In a large panel of samples, flow cytometric 

analyses indicated that GBM tumors contained a significantly 

greater Treg infiltrate than meningiomas, or 

metastases of non-CNS tumors. Subsequently, using six 

color FACS sorting, specific DR+ and DR− Treg cell subsets 

were quantified and isolated from matched tumor and 

peripheral blood samples from the same individual with 

either GBM or other CNS tumors, and from blood of healthy 

controls. Not only did these analyses demonstrate an 

overwhelming prevalence of DR+ Tregs in the GBM tumor, 

but also that these tumor derived DR+ Tregs exhibit 

extremely high levels of Foxp3 and extremely low levels 

of the pro-apoptotic gene, Bax, as compared to peripheral 

blood derived DR+ Tregs. These data indicate that the GBM 

associated increase in Tregs may be due to increased Treg 

survival, possibly due to the actions of IL-10 which we have 

found reduces Treg expression of both Bax protein and 

mRNA. 

doi:10.1016/j.clim.2007.03.507 

S115 

Sa.121 Therapeutic Dendritic Cell Vaccine 

Preparation Using Tumor RNA Transfection 

Juliana Sousa-Canavez, Researcher, Genoa Biotech, São 

Paulo, Brazil, Flavio Canavez, Researcher, Genoa Biotech, 

São Paulo, Brazil, Cristina Massoco, Researcher, Genoa 

Biotech, São Paulo, Brazil, Katia Leite, Pathologist, 

Genoa Biotech, São Paulo, Brazil, Luiz Camara-Lopes, 

Pathologist, Genoa Biotech, São Paulo, Brazil, Dewton 

Moraes Vasconcelos, Professor, Genoa Biotech, São Paulo, 

Brazil


Dendritic cells (DC) are potent antigen-presenting cells 

that induce and maintain primary immune responses. In the 

last years, many studies have used this property to initiate 

antigen T cell responses against neoplasic cells. This was 

achieved by vaccinating patients with DC carrying tumorassociated 

antigens prepared in vitro. A promising method 

for DC vaccine preparation is the transfection of tumor 

cells RNA. Preliminary clinical studies using RNA transfection 

have been shown encouraging results and minor side 

effects. Many aspects of this method, however, are not 

understood and further investigations are needed, especially 

regarding vaccine preparation. Here, we present our 

data obtained by DC transfection with total RNA isolated 

from a prostate tumor lineage LNCAP that overexpresses 

PSA antigen. PBMC were collected from healthy volunteers 

through apheresis and mononuclear cells were separated in 

Ficoll gradient. DC was differentiated with GM-CSF and IL- 

4, and maturation induced by TNFα DC phenotypes were 

confirmed by flow-cytometry using CD80, CD86, CD14, 

CD11c, CD1a and CCR7 antibodies. Immature cells were 

transfected with 4 1/4 and 16 1/4 g of RNA using either 

300 V or 400 V pulses. Mature cells were transfected with 4 

1/4 μg and 5 1/4 μg of RNA using 300 V, 400 V or 500 V 

pulses. In all conditions, we detected PSA expression by 

immunohistochemistry indicating that RNA penetrated in 

DC. For mature cells, we have also co-incubated total RNA 

(4 1/4 μg and 5 1/4 μg) and DC with poor results. Our 

experiments point to RNA transfection (5 1/4 μg total RNA) 

in mature DC using 400 V pulse as the most efficient 

condition. 

doi:10.1016/j.clim.2007.03.508 

Sa.122 Regulatory T Cells Affect the DC-mediated 

Immunotherapy Outcome in Melanoma Patients 

Mercedes N. López, Medical Doctor, Research Support 

Office, Clinical Hospital, University of Chile, Santiago de 

Chile, Chile, Gabriela Segal, Biotech, Program of 

Immunology, Institute of Biomedical Sciences, University of 

Chile, Santiago de Chile, Chile, Cristian Pereda, Biotech, 

Program of Immunology, Institute of Biomedical Sciences, 

University of Chile, Santiago de Chile, Chile, Raquel 

Aguilera, Medical Doctor, Program of Immunology, Institute 

of Biomedical Sciences, University of Chile, Santiago de 

Chile, Chile, Alexandra Ginesta, Medical Doctor, Program 

of Immunology, Institute of Biomedical Sciences, University 

of Chile, Santiago de Chile, Chile, Diego Reyes, Medical 

Doctor, Program of Immunology, Institute of Biomedical 

Sciences, University of Chile, Santiago de Chile, Chile, 

Carlos Ferrada, Medical Doctor, Surgery department, 

Clinical Hospital, University of Chile, Santiago de Chile, 

Chile, Flavio Salazar-Onfray, PhD of Sciences, Program of 

Immunology, Institute of Biomedical Sciences, University of 

Chile, Santiago de Chile, Chile 

Dendritic cell (DCs)-based therapy has proven to be 

effective in patients with malignancies. A Chilean phase I 

clinical study for the treatment of advanced malignant 

melanoma performed in our laboratory, indicates that 

autologous human dendritic cells (hDCs) pulsed with a 

melanoma cell lysate acquired a mature phenotype and 

were able to trigger specific immune responses to tumor 

antigens in vivo. In this study, 37 patients with malignant 

melanoma stage IV were vaccinated with autologous DCs 

pulsed with a melanoma cell lysate. After vaccination, 50% 

tested patients (18 out of 37) showed a positive Delayed type 

IV hypersensitivity reaction (DTH) against the melanoma cell 

lysate. All patients showed a positive DTH against the used 

adjuvant KLH demonstrating that all patients were immunocompetents. 

Significant correlations were found between 

DTH positive responses against tumour cell lysate and both 

disease stability and post-vaccination survival on the stage IV 

patients. However, there is a group of patients that did not 

show immunological responses. We compared a number of 

DTH responder patients (n=4) with a group of DTH noresponder 

patients (n=4) and analyzed the number and 

proportion of regulatory T cells (Treg). Our results showed 

that DTH non-responder patients showed an increase of Treg 

cells after the DC vaccination compared with DTH responder 

patients that did not show changes in the number or 

proportion of Treg cells. These observations should be 

important in the study of the role of regulatory T cells in 

the clinical responses of cancer patients treated with 

immunotherapy. 

doi:10.1016/j.clim.2007.03.509 

Sa.123 Dendritic Cell-Tumor Cell Fusion Vaccine for 

the Treatment of Advanced Tumors: Evaluation of 

the Quality of Life by Questionnaires 

Dewton Moraes Vasconcelos, MD, PhD, Genoa Biotecnologia, 

São Paulo, Brazil, Antonio Carlos Misiara, MD, PhD, Genoa 

Biotecnologia S.A., São Paulo, Brazil, Juliana Moreira 

Sousa-Canavez, BSc, PhD, Genoa Biotecnologia S.A., São 

Paulo, Brazil, Paloma Aguiar, BSc, Genoa Biotecnologia S.A., 

São Paulo, Brazil, Elaine Cristina Corneta, MSc, Genoa 

Biotecnologia S.A., São Paulo, Brazil, Katia Ramos Moreira 

Leite, MD, PhD, Genoa Biotecnologia S.A., São Paulo, Brazil, 

Luiz Heraldo Arouche Camara Lopes, MD, PhD, Genoa 

Biotecnologia S.A., São Paulo, Brazil 

Dendritic cells are the most potent antigen-presenting 

cells, and the possibility of their use for cancer vaccination 

has renewed the interest in this therapeutic modality. 

Nevertheless, the ideal immunization protocol with these 

cells has not been described yet. In this report we describe 

the preliminary results of a protocol using autologous tumor 

and allogeneic dendritic hybrid cell vaccination every 

4 weeks, for metastatic melanoma and renal cell carcinoma 

(RCC) patients, and a few patients with other types of 

neoplasia. Thirty-six patients were evaluated by a questionnaire 

searching for information of the quality of life 

(QoL) during the treatment with the vaccine. Though all 

patients included presented with large tumor burdens and 

progressive diseases, 77% of them experienced stability after 

vaccination, 9% related improvement and 14% presented 

deterioration of the clinical features. Among RCC patients 3/ 

10 (30%) presented deterioration, 1/10 improvement (10%) 

and 6/10 (60%) stabilization. Among melanoma patients 1/15 

(6.6%) deteriorated, 1/15 (6.6%) improved and 13/15 (86.6%)

Abstracts 

stabilized the disease. These data indicate that dendritic 

cell-tumor cell hybrid vaccination affects the natural history 

of advanced cancer and provides support for its study in less 

advanced patients, who should, more likely, benefit even 

more from this approach. 

doi:10.1016/j.clim.2007.03.510 

Sa.124 Rice Bran Supplement (MGN-3/Biobran) 

Suppresses Tumor Growth via Modulating Cytokine 

Production and Increasing Apoptotic Level in Ehrlich 

Carcinoma-bearing Mice 

Nariman Badr El-Din, Professor, University of Mansoura, 

Faculty of Science, Department of Zoology, Mansoura, Eman 

Noaman, Professor, National Center for Radiation and 

Technology, Department of Radiation Biology, Cairo, Alia 

Ghoneum, Student, Charles R. Drew University of Medicine 

and Science, Department of Otolaryngology, Los Angeles, 

CA, Mamdooh Ghoneum, Associate Professor III, Charles R. 

Drew University of Medicine and Science, Department of 

Otolaryngology, Los Angeles, CA 

This study was undertaken to investigate the anti-tumor 

activity in vivo by MGN-3/Biobran and the mechanisms 

underlying its effect. MGN-3 is an arabinoxylan extracted 

from rice bran that is treated with hydrolyzing enzymes from 

shiitake mushrooms. Female Swiss albino mice were inoculated 

intramuscularly in the right thigh with Ehrlich Ascites 

Carcinoma (EAC) cells. At day 8, mice bearing Solid Ehrlich 

Carcinoma tumor (SEC) were treated with MGN-3 (40 mg/kg 

body weight) via intraperitoneal injection, 3 times/week, 

until day 35. Tumor growth (volume and weight), plasma 

cytokine production, and apoptotic effect of MGN-3 were 

examined. Injection with MGN-3 caused a highly significant 

delay in both tumor volume (63.27%) and tumor weight 

(45.2%) from controls (pb0.01). The mechanisms by which 

MGN-3 exerts its anti-tumor effect seem to involve its ability 

to influence plasma cytokine production via increasing the 

levels of IFNγ (184.4%, pb0.01) and TNFα (11%, pb0.01) 

over control mice bearing SEC, while down-regulating levels 

of the immune suppressing cytokine IL-10. In addition, MGN-3 

induced 1.8 fold increase in the percentage of apoptotic SEC 

cells as determined by flow cytometry and the histopathological 

examination. No adverse side effects due to MGN-3 

treatment were observed; all animals displayed normal 

feeding/drinking and life activity patterns with a significant 

body weight gain of 7.32%, pb0.025. These data may have 

clinical implications for the treatment of solid cancers. MGN- 

3/Biobran was offered by Daiwa Pharmaceuticals Co., Ltd., 

Tokyo, Japan. 

doi:10.1016/j.clim.2007.03.511 

Sa.125 Therapeutic Efficacy of the Most Potent 

Macrophage Activating Factor (GcMAF) for Prostate 

and Breast Cancers 

Nobuto Yamamoto, Director, Socrates Institute for 

Therapeutic Immunology, Philadelphia, PA, Masumi Ueda, 

Chief, Microbiology Department, Socrates Institute for 

Therapeutic Immunology, Philadelphia, PA, Charles Benson, 

Head, Infectious Diseases, School of Veterinary Medicine, 

University of Pennsylvania, Kennett Square, PA 

Inflammation-primed macrophage activation is the principal 

macrophage activation process that requires serum 

vitamin D-binding protein (known as Gc protein) and 

participation of B and T lymphocytes. A trisaccharide 

composed of N-acetylgalactosamine with dibranched galactose 

and sialic acid termini at 420 threonine residue of Gc 

protein is hydrolyzed by the β-galactosidase (Bgl) of 

inflammation-primed B cells and the Neu-1 sialidase of T 

cells to yield the macrophage activating factor (MAF). Thus, 

Gc protein is the precursor for the principal MAF. However, 

the MAF precursor activity of serum Gc protein of cancer 

patients was lost or reduced because Gc protein is 

deglycosylated by serum α-N-acetylgalactosaminidase 

(Nagalase) secreted from cancerous cells. Serum Nagalase 

activity is proportional to tumor burden and serves as a 

prognostic index. Stepwise treatment of highly purified Gc 

protein with immobilized β-galactosidase and sialidase 

generated the most potent macrophage activating factor 

(GcMAF) that produces no side effect in humans. When 

human macrophages were treated in vitro with GcMAF (100 

pg/ml) for 3 h, the macrophages developed enormous 

variation of receptors that recognize a variety of cellular 

abnormalities, i.e., tumor associated antigens, in cancerous 

cell surface. Thus, the activated macrophages were 

highly tumoricidal to a variety of cancerous cells. Administration 

of 100 ng GcMAF/human results in the maximal 

activation of systemic macrophages. When nonanemic 

prostate and breast cancer patients (total of 36 patients) 

were treated with 14–25 weekly administrations of GcMAF 

(100 ng/week), all cancer patients exhibited healthy 

control levels of the serum Nagalase activity, indicating 

eradication of tumors. 

doi:10.1016/j.clim.2007.03.512 

S117 

Sa.126 Activation of Hepatic Stellate Cells During 

Fibrosis: Comparison of the CYP2D6 Model for 

Autoimmune Hepatits and CCl4 Injection 

Urs Christen, Assistant Professor, Pharmazentrum, JWG 

University Frankfurt, Frankfurt am Main, Germany, Edith 

Hintermann, Senior Postdoctoral Fellow, Pharmazentrum, 

JWG University Frankfurt, Frankfurt am Main, Germany, 

Monika Bayer, Res Tech, Pharmazentrum, JWG University 

Frankfurt, Frankfurt am Main, Germany, Selina Christen, 

PhD Student, Pharmazentrum, JWG University Frankfurt, 

Frankfurt am Main, Germany 

Only little is known about the mechanisms of periportal 

fibrosis in the pathogenesis of human autoimmune hepatitis. 

We used the virus induced CYP2D6 model system to 

investigate the activation of hepatic stellate cells (HSC) 

and the kinetics of fibrosis in comparison with the CCl4induced 

fibrosis model. CYP2D6 transgenic mice express the 

human Cytochrome P450 in the liver and develop liver 

damage upon Adenovirus-CYP2D6 infection. In the CYP2D6


model we found mostly subcapsular fibrosis resulting in the 

fusion of individual lobules (Sirius Red, Collagen I). At 10– 

12 weeks post-infection, weak periportal fibrosis became 

apparent. In contrast, in CCl4-treated mice the kinetic of 

extracellular matrix deposition was accelerated resulting in 

periportal fibrosis after 3–4 week of CCl4 administration. At 

later times, fibrosis was much more pronounced in CCl4treated 

mice compared to virus-infected CYP2D6 mice but 

subcapsular fibrosis was not as dominant. Activation of HSCs 

could be detected by staining for a-smooth muscle actin 

(aSMA) in liver sections of both CCl4-treated mice and virusinfected 

CYP2D6 mice. In addition, isolation of HSCs 

revealed an enhanced activation status (decreased amount 

of oil droplets, de novo aSMA expression) in CCl4-treated 

mice and virus-infected CYP2D6 mice. Our data indicate 

that virus-infected CYP2D6 mice display subcapsular and 

periportal fibrosis that correlates with an activation of HSCs 

similar to CCl4-induced fibrosis. Thus, the CYP2D6 mouse is 

a good model system to further investigate the molecular 

mechanisms involved in fibrotic events during autoimmune 

hepatitis. 

doi:10.1016/j.clim.2007.03.513 

Sa.127 Bone-Marrow Derived DCs From CD200R1 

KO Mice Stimulated with the Ligand CD200Fc 

Preferentially Induce Populations of 

CD4+CD25+ Treg 

Reg Gorczynski, Professor, University Health Network, 

Toronto, ON, Canada, Ivo Boudakov, PDF, University Health 

Network, Toronto, ON, Canada 

CD200: CD200R1 interaction causes suppression of 

inflammation and graft rejection. The functional activity 

induced by alternate CD200R isoforms remains unclear. We 

generated a BL/6 mouse with deletion of the gene for 

CD200R1. Marrow cells from KO and wt mice were 

cultured for 8 days in the presence of (GMCSF +IL-4), 

with/without addition of a soluble form of CD200 

(CD200Fc). DCs were stimulated with LPS and used to 

activate C3H splenocytes. CTL for BL/6 targets was 

assayed at 5 days, or cells were harvested at 72 h, 

enriched for CD4+CD25+ cells, and added to fresh cultures 

of C3H splenocytes stimulated with either BL/6 or BALB/c 

mitomycin-c treated DCs. CTL specific for H2b or H2d 

targets was assayed 5d later. 

DCs of wt or CD200R1KO mice cultured without 

CD200Fc induced allospecific (anti-H2b) CTL in vitro. 

After culture in the presence of CD200Fc, DCs from 

CD200R1KO mice were inefficient inducers of CTL, but 

promoted development of CD4+CD25+Foxp3+ Treg which 

suppressed C3H anti-BL/6 CTL induction in fresh responder/stimulator 

cells. To characterize an in vivo equivalent 

of this phenomenon CD200Fc or mouseIgG2a was infused 

iv into wt or CD200R1KO mice for 14 days. Splenic DCs 

were isolated by conventional techniques. DCs from 

control mice (wt or CD200R1KO) receiving IgG2a only 

were equivalent in induction of CTL. DCs isolated after 

infusion of CD200Fc into CD200R1KO mice were impaired 

for CTL induction, but induced antigen-specific Treg in 

culture. We conclude that immunoregulation induced by 

CD200 binding of non-CD200R1 isoforms of the CD200R 

receptor family reflects the preferential induction of 

“tolerogenic” DCs. 

doi:10.1016/j.clim.2007.03.514 

Sa.128 High Molecular Weight Hyaluronan Promotes 

the Suppressive Effects of CD4+CD25+ Treg 

Paul Bollyky, Benaroya Research Institute, Seattle, WA, 

Gerald Nepom, Benaroya Research Institute, Seattle, WA, 

Jane Buckner, Benaroya Research Institute, Seattle, WA, 

Susan Masewicz, Benaroya Research Institute, Seattle, WA, 

James Lord, Benaroya Research Institute, Seattle, WA, 

Stephen Evanko, Benaroya Research Institute, Seattle, 

WA, Thomas Wight, Benaroya Research Institute, Seattle, 

WA 

Hyaluronan (HA) is a glycosaminoglycan present in the 

extra-cellular matrix. When HA is degraded during infection 

and injury low-molecular-weight (LMW-HA) forms are 

generated whose interactions influence inflammation and 

angiogenesis. Intact high-molecular-weight hyaluronan 

(HMW-HA), conversely, has been reported to convey antiinflammatory 

signals. Here we demonstrate that HMW-HA 

acts on human CD4+CD25+ regulatory T cells (Treg) to 

promote their suppressive effects while LMW-HA does not. 

HMW-HA enhances regulatory T cell functional suppression 

of responder cell proliferation and up-regulates the 

transcription factor Foxp3 on Treg. These effects are only 

seen with activated Treg and are associated with expression 

of CD44 isomers which more highly bind HMW-HA. At 

higher concentrations HMW-HA also has direct suppressive 

effects on T cells. We propose that the state of HA in the 

matrix environment provides contextual cues to Treg and T 

cells, thereby providing a link between the innate 

inflammatory network and the regulation of adaptive 

immune responses. 

doi:10.1016/j.clim.2007.03.515 

Sa.129 The Forkhead Transcription Factor, Foxq1, 

Enhances NF-kB Activity and is Critical for Robust 

T Cell Activation and Autoimmunity 

Melanie Gubbels Bupp, Postdoctoral Fellow, Roche Palo 

Alto, Palo Alto, CA, Stanford Peng, Director, Arthritis 

Research, Roche Palo Alto, Palo Alto, CA 

Essential immunoregulatory functions have recently 

been ascribed to several members of the forkhead 

transcription factor family. For example, Foxo3a and 

Foxj1 inhibit NF-kB activity and Foxp3 regulates lineage 

commitment and function of regulatory T cells. Here, we 

report that another family member, Foxq1, is critical for 

robust helper T cell activation. CD4+ T cells from mice 

bearing a mutant allele of Foxq1 displayed a significantly 

reduced proliferative response with impaired secretion of 

IL-2 and IFN-gamma after TCR ligation as compared to

Abstracts 

wild-type (WT) T cells. This defect is largely rescued by 

the addition of recombinant human IL-2, suggesting that 

Foxq1 is required for optimal production of IL-2 and 

consequently, robust T cell proliferation. Accordingly, in in 

vitro luciferase assays, Foxq1 augmented transcriptional 

activity of the IL-2 transactivator NF-kB while mutant 

Foxq1 did not retain this function. Finally, Foxq1-mutant 

mice exhibited reduced susceptibility to the progression of 

experimental autoimmune encephalomyelitis than their 

WT counterparts. Thus, Foxq1 is essential for helper T cell 

activation and autoaggressive T cell responses in vivo, 

likely via its ability to potentiate NF-kB activity and IL-2 

production. 

doi:10.1016/j.clim.2007.03.516 

Sa.130 Human Bone Marrow Mesenchymal Stem 

Cells Support Polyclonal Stimulation of Human B 

Cells 

Elisabetta Traggiai, PhD, Institute G Gaslini, Genova, Italy, 

Alberto Martini, MD/PhD, Institute G Gaslini, Genova, Italy, 

Antonio Uccelli, MD/PhD, Department of Neurosciences, 

Ophthalmology and Genetics, Genova, Italy, Lorenzo 

Moretta, MD/PhD, Institute G Gaslini, Genova, Italy, 

Federica Benvenuto, PhD, Department of Neurosciences, 

Ophthalmology and Genetics, Genova, Italy, Marco 

Gattorno, MD, Institute G Gaslini, Genova, Italy 

To investigate the possible application of bone marrow 

(BM) mesenchymal stem cells (MSCs) in Systemic Lupus 

Erythematosus (SLE), an autoimmune disease in which B 

cells play a pivotal role, we studied the interaction of B 

cell subsets isolated from healthy donor and pediatric SLE 

patients with human BM MSCs. We isolated from peripheral 

blood of healthy donors: a) immature transitional B 

cells, b) naive B cells, c) IgM memory and d) switch 

memory B cells. BM MSCs promoted proliferation and 

differentiation into immunoglobulin secreting cells of 

transitional and naive B cells stimulated with CpG 2006 

in the absence of BCR triggering, and strongly enhanced 

proliferation and differentiation of both memory B cell 

populations. Furthermore, both proliferation and differentiation 

into plasma cells of CD19+ B cells isolated from 

pediatric SLE patients were enhanced by BM MSCs upon 

polyclonal stimulation. Inhibition of T cell proliferation by 

MSCs was suggested to be dependent on INF-g€ . To test 

whether INF-g€ could have an effect on the B cell 

response under the influence of MSCs, we co-cultured B 

cell subsets with MSC in the presence of human 

recombinant INF-ï §ï€®ï€ After 4 days of culture both 

proliferation and differentiation into plasma cells of all B 

cell subsets stimulated with CpG, soluble CD40L and anti- 

Ig were inhibited in both healthy donors and SLE patients. 

These data show the complexity of the cross-talk between 

MSCs and B cell and how the microenviroment in which 

the interaction take place could influence the outcome of 

the immune response. 

doi:10.1016/j.clim.2007.03.517 

Sa.131 Examination of a Unique T Cell Subset TCD40 

in Type 1 Diabetes 

David Wagner, Assistant Professor, Webb-Waring Institute, 

Denver, CO 

The identification of autoaggressive T cells in human 

disease has proven very difficult. We have discovered a 

unique T cell subset described as CD4lo and expressing the 

CD40 receptor termed TCD40. These T cells are significantly 

expanded in T1D but not T2D or control subjects. A portion 

of TCD40 cells respond to pre-pro-insulin, GAD peptides, 

and human islets. These T cells are expanded in diabetes 

high susceptibility HLA (DR3, DR4, DQ8) subjects, but are 

also expanded when T1D subjects do not possess any of the 

high susceptibility HLAs. TCD40 cells remain at expanded 

percentages in long-term diabetic subjects, suggesting that 

CD40 is not merely a T cell activation marker. Interestingly, 

non-autoimmune subjects carrying DR3, DR4 or DQ8 do not 

have expanded TCD40 cells. Mechanistic differences in 

induction of RAG1 and RAG2 are seen between T1D and 

control subjects. Examination of TRAV usage in T1D and 

controls demonstrate prominent differences including the 

detection of TRAV8 in the periphery. TCD40 cells achieve 

effector status synthesizing predominantly Th1 cytokines. 

These data suggest a unique T cell population that may be 

predictive of autoimmunity. 

doi:10.1016/j.clim.2007.03.518 

S119 

Sa.132 Effects of Class II/CLIP Affinity on the Class II 

Antigen Presentation Pathway in the Context of 

Autoimmunity 

Cornelia Rinderknecht, Graduate Student, Stanford 

University, Department of Pediatrics, San Carlos, CA, 

Tatiana Catanzarite, Student, Stanford University, 

Department of Pediatrics, Stanford, CA, Michael Belmares, 

Scientist, Arbor Vita Corporation, Sunnyvale, CA, Elizabeth 

Mellins, Associate Professor, Stanford University, 

Department of Pediatrics, Stanford, CA, Susan Kovats, 

Assistant Member, Oklahoma Medical Research Foundation, 

Arthritis and Immunology Research Program, Oklahoma 

City, OK 

Several MHC class II alleles linked with autoimmune 

diseases form unusually low-stability complexes with class IIassociated 

invariant chain peptides (CLIP), leading us to 

hypothesize that this is an important feature contributing to 

disease pathogenesis. To investigate cellular consequences 

of altering class II/CLIP affinity, we evaluated invariant chain 

(Ii) mutants with increased CLIP affinity for two murine class 

II alleles, I-Ed and I-Ag7, which have low affinity for wt CLIP. 

I-Ed is associated with a mouse model of spontaneous, 

autoimmune joint inflammation, and I-Ag7 is associated with 

models of diabetes (NOD) and arthritis (KRN). Single amino 

acid mutations were made in murine Ii. Pulse-chase/coimmunoprecipitation 

analysis of I-Ed or I-Ag7 and CLIP was 

used to identify physiologically relevant CLIP mutants that 

are of high affinity but remain DM-sensitive. An increase in 

CLIP affinity for I-Ed or I-Ag7 resulted in increased cell-


surface and total cellular levels of class II, suggesting post-ER 

chaperoning by Ii via its CLIP peptides. Quantitative effects 

on class II were less robust in H-2M-expressing cells, 

suggesting complementary effects mediated by Ii and H- 

2M, and implying that the impact of varied CLIP affinity on 

immune responses will be highest in cells with limited H-2M 

activity. Functionally, cell lines expressing wt or high CLIPaffinity 

mutant Ii have differential capacity to present 

peptide or whole antigen to a T cell hybridoma, suggesting 

a model in which increased CLIP affinity for class II serves to 

restrict peptide loading to H-2M-containing compartments, 

ensuring proper editing of antigenic peptides. 

doi:10.1016/j.clim.2007.03.519 

Sa.133 Treg Potency Assay: Suppression of T Cell 

Cytokine Expression in Response to T Cell Specific 

Antigenic Stimulation of Autologous PBMC 

Smita Ghanekar, Senior Research Scientist, BD Biosciences, 

Research and Development, San Jose, CA, Joyce Ruitenberg, 

RAII, BD Biosciences, Research and Development, San Jose, 

CA 

CD4+CD25+ regulatory Tcells (Tregs) play a critical role in 

the induction and maintenance of peripheral immune 

tolerance. Naturally occurring Tregs are crucial for the 

control of auto-reactive T cells and of the effector function 

of allo-reactive CD4+ and CD8+ T cells in transplantation 

models in vivo. Tregs have been shown to be potent 

suppressors of activated T cells in vitro. Phenotypically, 

Tregs are characterized as T cells that are CD4+, CD25++, 

CD127− and Foxp3+. Suppressive ability of Tregs is usually 

assessed by applying long-term (5 days) assays such as 

proliferation, but the therapeutic potential of these cells 

demands a quicker analysis method. Therefore, in this study, 

the ability of Tregs from healthy donors to inhibit or suppress 

cytokine expression in short-term assays has been examined. 

PBMC enriched for CD4+ cells were purified as CD4+CD25++ 

Tregs by sorting with a BD FACSAria. These highly purified 

Tregs were then cultured with rIL2 in vitro for 5–7 days and 

used in 6-h intracellular cytokine expression assays of 

autologous PBMC. Activation of PBMC with SEB and/or 

CD3+CD28 results in the expression of inflammatory cytokines 

(IFNg, TNFa, and IL2) by CD4+ and CD8+ T cells. The 

results suggest that the addition of Tregs to the cytokine 

assay of autologous PBMC reduces the frequency of cytokine 

expressing T cells. Since the intracellular T cell cytokine 

expression assay has been validated extensively, this shortterm 

assay would be a valuable tool to qualify Tregs for 

immunotherapy. 

doi:10.1016/j.clim.2007.03.520 

Sa.134 β-Tubulin-induced Autoimmune 

Hearing Loss Exacerbates in IL-10-deficient Mice 

and Restores by IL-10 Gene Transfer 

Bin Zhou, Research Associate, University of Tennessee, 

Department of Medicine, Memphis, TN, Mohammad Habiby 

Kermany, Postdoctoral Fellow, University of Tennessee, 

Department of Medicine, Memphis, TN, Yixuan Zhou, 

Postdoctoral Fellow, University of Tennessee, Department 

of Medicine, Memphis, TN, Qing Cai, Postdoctoral Fellow, 

University of Tennessee, Department of Medicine, Memphis, 

TN, Chun Cai, Postdoctoral Fellow, University of Tennessee, 

Department of Medicine, Memphis, TN, Junwoo Kim, 

Postdoctoral Fellow, University of Tennessee, Department 

of Medicine, Memphis, TN, Patrick Kim, Postdoctoral 

Fellow, University of Tennessee, Department of Medicine, 

Memphis, TN, Wenxi Liu, Postdoctoral Fellow, University of 

Tennessee, Department of Medicine, Memphis, TN, Tai June 

Yoo, Professor, University of Tennessee, Department of 

Medicine, Memphis, TN 

We hypothesized that endogenous levels of IL-10 function 

to regulate the severity of βtubulin-induced experimental 

autoimmune hearing loss (EAHL), and exogenous of 

IL-10 would abrogate βtubulin-induced EAHL. BALB/c wildtype 

(WT) and homozygous (IL-10−/−) IL-10-deficient mice 

underwent βtubulin immunization, development of EAHL 

was monitored over time. Additional groups of IL-10deficient 

mice immunized βtubulin to induce EAHL; IL-10deficient 

mice were intramuscularly injected with plasmid 

DNA encoding IL-10 on the climax of EAHL. Auditory 

brainstem responses (ABR) were examined over time. 

EAHL developed progressively in both BALB/c WT and IL- 

10-deficient mice immunized with βtubulin. However, the 

severity of hearing loss in the IL-10−/− mice was significantly 

greater than that in WT animals. Disease severity was 

associated with significantly enhanced IFNγ and TNFα level 

from stimulated splenocyte cultures, increased IgG2a antiβtubulin 

antibody responses and degeneration of Spiral 

ganglion cells of the Cochlea. αtubulin immunized IL-10−/− 

mice receiving intramuscular injection of plasmid DNA 

encoding IL-10 developed high serum levels of IL-10 yet 

the hearing function was significantly improved in the IL-10treated 

groups than that in the vector control group. 

Moreover, the ABR thresholds in the IL-10-treated groups 

are equivalent to naive controls and the histological 

analysis of the cochlear cross section was similar to those 

of naive controls. Furthermore, IL-10-treated mice exhibited 

significant suppression of βtubulin-induced IFNγ and 

TNFγ, but increased IgG1 production compared to levels 

exhibited by control mice. Our data demonstrate that lack 

of IL-10 exacerbates hearing loss and exogenous administration 

of IL-10 improved hearing function. 

doi:10.1016/j.clim.2007.03.521 

Sa.135 Galectin-1 Selectively Suppresses 

Plasmacytoid DC 

James Chen, Student, University of California, Los Angeles, 

Medicine, Los Angeles, CA, Xiangshu Wen, Postdoctoral 

Fellow, University of California, Los Angeles, Medicine, Los 

Angeles, CA, Jennifer Fulcher, MD, PhD Student, University 

of California, Los Angeles, Microbiology Immunology and 

Molecular Genetics, Los Angeles, CA, Benhur Lee, Assistant 

Professor, University of California, Los Angeles, 

Microbiology Immunology and Molecular Genetics, Los

Abstracts 

Angeles, CA, Anna Eriksson, Postdoctoral Fellow, University 

of California, Los Angeles, Medicine, Los Angeles, CA, Ram 

Raj Singh, Professor, University of California, Los Angeles, 

Medicine, Los Angeles, CA 

We have recently reported that galectin-1, a β-galactoside-binding 

lectin with a broad range of immunomodulatory 

properties, binds to and activates human monocyte-derived 

dendritic cells (DC). To evaluate the implications of this 

finding in vivo, we have investigated the role of galectin-1 in 

the modulation of DC in mice. We first show that galectin-1 

strongly binds murine DC. To examine the effect of galectin-1 

on DC subsets, we cultured spleen cells from BALB/c mice 

with LPS or galectin-1. We found that whereas LPS induces 

CD40 expression on all DC subsets including myeloid 

(CD11c+CD11b+), lymphoid (CD11c+CD8a+) and plasmacytoid 

(pDC; CD11c+B220+) DC, galectin-1 induces CD40 expression 

only in a fraction of DC. Strikingly, galectin-1 reduces the 

percentage and numbers of pDC and their activation as 

determined by CD40 expression, whereas LPS causes their 

expansion and activation. To assess the effect of galectin-1 on 

pDC in vivo, we used autoimmune-prone MRL-lpr mice that 

have increased numbers of pDC accumulating in inflamed 

organs. A single s.c. injection of galectin-1 in these mice 

reduced the proportion of pDC in cutaneous lymph nodes. 

These data suggest a role of galectin-1 in the regulation of 

pDC. B220 is a spliced isoform of CD45 and thus, our data are 

consistent with the known ability of galectin-1 to trigger 

apoptotic cell death in T cells via cross-linking of CD45. 

Ongoing studies will investigate the implication of this finding 

on the development of systemic inflammation in MRL-lpr 

mice, and the possible therapeutic consequences of galecin-1 

administration in the murine lupus model. 

doi:10.1016/j.clim.2007.03.522 

Sa.136 T Cell Responsiveness to Complementary 

PR3 Protein Supports a Pathogenic Role of 

Autoantigen Complementarity in PR3-ANCA 

Autoimmune Disease 

Jiajin Yang, Assistant Professor, University of North Carolina 

at Chapel Hill Kidney Center, Chapel Hill, NC, David Bautz, 

Graduate Student, University of North Carolina at Chapel 

Hill Kidney Center, Chapel Hill, NY, John Smitz, Associate 

Professor, University of North Carolina at Chapel Hill 

Department of Pathology and Laboratory Medicine, Chapel 

Hill, NC, Hyunsook Chin, Statistician II, University of North 

Carolina at Chapel Hill Kidney Center, Chapel Hill, NC, 

Barrrak Pressler, Assistant Professor of Internal Medicine, 

Purdue University, Lafayette, IN, Susan Hogan, Research 

Assistant Professor, Medicine, University of North Carolina 

at Chapel Hill Kidney Center, Chapel Hill, NC, Roland Tisch, 

Assoc Professor, Microbiology and Immunology, Chapel Hill, 

NC, J. Charles Jennette, Distinguished Professor, 

Department Chair, Pathology and Laboratory Medicine, 

Chapel Hill, NC, Ronald Falk, Distinguished Professor/ 

Division Chief/Director, University of North Carolina at 

Chapel Hill Kidney Center, Chapel Hill, NC, Gloria Preston, 

Professor, University of North Carolina at Chapel Hill Kidney 

Center, Chapel Hill, NC 

We discovered that patients with PR3-ANCA vasculitis 

who have autoantibodies against the autoantigen proteinase 

3 (PR3) also have a subset of antibodies reactive 

against a protein complementary – or antisense – to the 

autoantigen, as detected using recombinant, complementary-PR3 

protein, cPR3. Investigations into the etiology and 

consequences of these anti-cPR3 antibodies led to the 

proposal that complementary proteins are involved in 

inciting autoantibody production, as detailed in the theory 

of autoantigen complementarity (Nat Med 2004, 10: 72–79). 

The present studies indicate that CD4+TH1 cells from 

patients (n=26) with PR3-ANCA vasculitis versus healthy 

controls (n=34) exhibit increased proliferation (Wilcoxon 

rank sum test, p=0.0014) and IFN-γ expression (p=b0.0001) 

when stimulated with cPR3-peptide (32 aa, 138–169) but 

not a scrambled peptide (p=0.60). These recall responses 

indicate that the responding T cells were activated prior in 

vivo. Reactivity to smaller, overlapping fragments of cPR3 

ruled out the possibility that cPR3-peptide functions as a 

superantigen. Interestingly, the HLA-DRB1*15 allele group 

was overrepresented in the patient group, as compared to 

frequencies from a Caucasian population US database 

(Fisher’s exact test, p=0.006). This allele group is predicted 

to bind cPR3 peptide with high affinity (based on the 

Immune Epitope and Analysis Resource database). Ranked 

linear regression analysis indicated a likelihood of p=0.009 

that anti-cPR3 antibodies and cPR3-specific T cells coexist 

within an individual. The T cell responses observed are 

consistent with a history of complementary-protein encounter 

in these patients. Consideration of potential contributions 

of complementary protein pairs in autoimmune 

diseases could revolutionize the approach for exploring 

pathogenic mechanisms. 

doi:10.1016/j.clim.2007.03.523 

S121 

Sa.137 Enhanced CD16 Mediated Depletion of 

Activated Lymphocytes by a Non-Fucosylated Fully 

Human Anti-CD70 IgG1 

Hadia Lemar, Research Associate III, Medarex, Inc., Milpitas, 

CA, Diane Feingersh, Scientist I, Medarex, Inc., Milpitas, 

CA, Alison Witte, Scientist III, Medarex, Inc., Milpitas, CA, 

Jon Terrett, Director, Medarex, Inc., Milpitas, CA, Ben 

Preston, Research Assotciate III, Medarex, Inc., Milpitas, 

CA, Mark Selby, Director, Medarex, Inc., Milpitas, CA, Alan 

Korman, VP, Medarex, Inc., Milpitas, CA, Marco Coccia, 

Senior Scientist II, Medarex, Inc., Milpitas, CA 

CD70 is the only known ligand for CD27 mediated 

costimulation of B and T memory and effector responses. 

CD70 expression in normal tissues is restricted to activated 

lymphocytes and dendritic cells. Patients with autoimmune 

disorders express high levels of CD70 on chronically activated 

T cells and lymphocytes. Furthermore, CD70 blocking 

antibodies were shown to delay onset of experimental 

autoimmune encephalomyelitis and cardiac allograft rejection 

in mice. We demonstrate here that antibody dependent 

cellular cytotoxicity (ADCC) mediated depletion of hyperactivated 

CD70+ leukocytes may be an effective therapeutic 

strategy for autoimmunity. We previously reported that a


fully human, non-fucoslyated anti-human CD70 IgG1 (anti- 

CD70nf) mediated superior ADCC of CD70+ tumor cells 

compared to parental fucosylated IgG1 (anti-CD70p). Anti- 

CD70nf also stimulated ADCC of purified T cells activated by 

anti-CD3/anti-CD28 coated beads with ∼10-fold greater 

potency and ∼50% greater efficacy than anti-CD70p. 

Stimulation of PBMC with cytomegalovirus (CMV) peptide 

induced CD70 expression on responsive CD8+/CMV pentamer+ 

cells. Anti-CD70p and anti-CD70nf decreased CD8+/CMV 

pentamer+ cells but depletion by anti-CD70nf was both 

more potent and efficacious. Anti-CD16 blocking was effective 

in reversing depletion by both antibodies, but 1000-fold 

greater concentration of anti-CD16 was required to inhibit 

depletion by anti-CD70nf. CD8+/CMV pentamer− cells were 

unaffected by either CD70 antibody. These CD70 antibodies 

also inhibited CD70-Fc binding to CD27+/CD70− RL cells and 

CD70 costimulation of T cells activated by anti-CD3 on CD32/ 

CD70 CHO transfectants. Further evaluation of enhanced 

ADCC mediated depletion of activated leukocytes and anti- 

CD70 functional blockade as therapeutic strategies for 

autoimmunity is in progress. 

doi:10.1016/j.clim.2007.03.524 

Sa.138 Evidence for DC:Treg Paracrine IL-2 Delivery 

Katarina Kulhankova, Postdoctoral Scholar, Carver College 

of Medicine and Veterans Affairs Medical Center, 

Department of Internal Medicine, Iowa City, IA, Mohamed 

Nasr, Research Scientist, Carver College of Medicine and 

Veterans Affairs Medical Center, Department of Internal 

Medicine, Iowa City, IA, Michael E. Dailey, Professor, 

University of Iowa, Department of Biological Sciences, Iowa 

City, IA, Todd Rouse, Research Assistant, Carver College of 

Medicine and Veterans Affairs Medical Center, Department 

of Internal Medicine, Iowa City, IA, Elizabeth H. Field, 

Professor, Carver College of Medicine and Veterans Affairs 

Medical Center, Department of Internal Medicine, 

Iowa City, IA 

Regulatory CD4+CD25+ Tcells (Tregs) play a key role in the 

maintenance of immune tolerance, and understanding the 

precise mechanism of their function can help guide 

therapeutic strategies for achieving transplant tolerance. 

Once activated, Tregs inhibit CD4+ cell proliferation and IL-2 

production in vitro. Treg cell function is blocked by culturing 

Tregs and effectors in the presence of anti-CD25 or 

separately in transwell system. This indicates that both IL- 

2 and cell:cell contact is necessary for the gain or execution 

of Treg-mediated suppression. However, the source for IL-2 

and the context, in which it is delivered, remains unknown. 

To address this question we generated the CD4+DC25+ 

hybridoma RD6 from a mouse with acquired tolerance to 

foreign MHCs. Like natural Tregs, RD6 inhibition of CD4+ 

effectors requires CD25 and cell:cell contact. Neither Treg 

nor RD6 showed inhibition when IL-2KO DCs were substituted 

for WT DCs in Treg assays. We utilized four-dimensional 

multi-channel fluorescent live-cell confocal microscopy to 

define the CD25 expression on RD6 cells during cell:cell 

interactions between RD6 and semi-allogeneic DCs or 

allogeneic membrane-labeled DC cells, JAWSII. RD6 engaged 

themselves and DCs in a variety of cell:cell interactions that 

included long-lasting conjugate formation as well as formation 

of stable cell:cell membrane bridges of various lengths. 

RD6 CD25 expression localized to points of contact with 

dendritic processes over 24 hours. RD6 cells acquired patches 

of DC membranes and internalized them. These data are 

consistent with a model, in which DCs deliver IL-2 to Tregs via 

tight membrane contacts. 

doi:10.1016/j.clim.2007.03.525 

Sa.139 Admixture Mapping, a New Tool for Disease 

Gene Finding in Autoimmune and Other Diseases 

Alicja Waliszewska, Research Specialist, Broad Institute, 

Cambridge, MA, Phillip De Jager, Assistant Professor, 

Department of Genetics, Harvard Medical School, Boston 

MA, David Hafler, Professor, Department of Genetics, 

Harvard Medical School, Boston MA, David Reich, Assistant 

Professor, Department of Genetics, Harvard Medical School, 

Boston, MA 

Admixture mapping is a powerful technique for finding 

genes for complex diseases. The practical requirements 

include (a) a set of markers spanning the genome with large 

allele-frequency differences between the parental ethnicities 

contributing to the admixed population and (b) an 

understanding of the extent of admixture in the study 

population. African American and Latino American populations 

are the product of gene flow (genetic admixture) during 

the last 500 years, which has produced new genotypes from 

once-isolated ancestral groups. Admixture mapping attempts 

to associate African, European, or Native American to disease 

risk. We have performed admixture scans in African Americans 

to find genes affecting Multiple Sclerosis (MS), 

Rheumatoid Arthritis (RA), markers of inflammation, and 

prostate cancer. We have also been building resources for 

admixture mapping in Latinos. We have detected loci that are 

significantly associated to MS on chromosome 1 (LOD=5.2), to 

prostate cancer on chromosome 8 (LOD=7.1), and to 

interleukin 6 soluble receptor (IL6 SR) on chromosome 1 

(LOD=4.59). For IL6 SR and prostate cancer this has led to 

cloning of disease risk alleles. In African Americans with 

rheumatoid arthritis, the scan was negative, and identified 

no loci for disease risk. 

doi:10.1016/j.clim.2007.03.526 

Sa.140 Comparison of Membrane-bound and 

Secreted TGFβ by IL-2 Activated Natural and 

Adaptive CD4+CD25+ Regulatory Cells from Healthy 

and Lupus-prone Mice 

Song Guo Zheng, Assistant Professor of Research Medicine, 

University of Southern California Keck School of Medicine, 

Los Angeles, CA, Julie Wang, Research Lab Specialist, 

University of Southern California Keck School of Medicine, 

Los Angeles, CA, Shuang Ye, Postdoctoral Fellow, University 

of Southern California Keck School of Medicine, Los Angeles, 

CA, David Sehy, Director, New Technologies, eBioscience/ 

Department of New Technologies, San Diego, CA, Beth

Abstracts 

Palian, Postdoctoral Student, University of Southern 

California Keck School of Medicine, Los Angeles, CA, David 

A. Horwitz, Professor Chief, Division of Rheumatology and 

Immunology, University of Southern California Keck School 

of Medicine, Los Angeles, CA 

CD4+CD25+Foxp3+ regulatory T cells can be divided into 

natural, thymus-derived cells and adaptive Tregs induced 

from CD25− precursors in the periphery. Although TGFβ 

induced, adaptive Tregs produce TGFβ, whether natural 

CD4+CD25+ Tregs also produce this cytokine has been 

controversial. Mouse CD4+CD25+ cells were stimulated via 

TCR and/or IL-2 and naive CD4+CD25− cells were stimulated 

with TGFβ to induce adaptive Th3 cells. Although 

freshly isolated murine CD4+CD25+ cells did not express 

mature TGFβ, when TCR activated with IL-2, these Foxp3+ 

Treg cells displayed similar numbers of membrane-bound 

TGFβ under appropriate staining conditions as Th3 cells 

induced with TGFβ ex vivo. Both natural and adaptive Treg 

subsets also secreted similar amounts of active TGFβ. Since 

TGFβ induces CD8+ cells to express α-E integrin (CD103), 

we found the activated CD25+ Tregs induced CD103 

expression on CD8+ cells to levels equivalent to that of 

activated Th3 cells. ALK5 (TGFβ receptor I inhibitor), but 

not anti-TGFβ almost completely abolished this CD103 

expression. Transwell experiments revealed that surface 

contact was not required. Thus, activated natural CD4+ 

CD25+ Treg cells can produce similar levels of mature TGFβ 

as adaptive TGFβ induced Tregs. Finally, we studied 

activated CD4+CD25+ cells from lupus-prone (NZBxNZW) 

F1 young and old mice and found that cells from old mice 

with established disease expressed decreased membranebound 

and produced less soluble TGFβ. Thus, defective 

TGFβ production by CD4+CD25+ Tregs may contribute to 

their decreased functional activity and to the development 

and progression of systemic lupus. 

doi:10.1016/j.clim.2007.03.527 

Sa.141 Thymic Organogenesis Controlled by 

NF-kappaB Activating Factors 

Masashi Ya, Graduate Student, Institute for Enzyme 

Research, University of Tokushima, Tokushima, Japan, 

Yasuhiro Mouri, Graduate Student, Institute for Enzyme 

Research, University of Tokushima, Tokushima, Japan, 

Mitsuru Matsumoto, Professor, Institute for Enzyme 

Research, University of Tokushima, Tokushima, Japan 

Thymic epithelial cells (TEC) play pivotal roles in the 

establishment of self-tolerance through critical dialogue 

with developing thymocytes. Unique actions of two NFkappaB 

activating factors within TECs, NF-kappaB-inducing 

kinase (NIK) and IkappaB kinase α (IKKα), for the establishment 

of self-tolerance have recently been highlighted by 

studies using a strain of mouse bearing a natural mutation 

of the NIK gene (aly mice) and gene-targeted mice, 

respectively. Previous studies have demonstrated essential 

roles of NIK-IKKα downstream of the lymphotoxin-beta 

receptor (LTβR), which is essential for the development of 

secondary lymphoid organs; aly mice lack all lymph nodes 

and Peyer’s patches because of the defective LTβR 

signaling. Now additional roles of NIK-IKKα in thymic 

organogenesis, mainly through the developmental regulation 

of TECs, have emerged, although the corresponding 

upstream receptor(s) and ligand(s) participating in this 

action have not been fully characterized. Previous studies 

have suggested that LTβR regulates thymic organogenesis. 

In order to investigate the contribution of LTβR signaling for 

the thymic organogenesis in NIK-IKKα-dependent pathways, 

we have analyzed thymic architecture together with Aire 

expression from both LTα-deficient mice and LTβR-deficient 

mice. We found that Aire-expressing cells were largely 

retained from those mice, whereas they were dramatically 

reduced from both NIK-mutant mice and IKKα-deficient 

mice. Because NIK-mutant mice and IKKα-deficient mice 

show more profound thymic disorganization of TECs than 

that from LTβR-deficient mice, it is likely that NIK-IKKα is 

additionally acting downstream on other receptor(s) beyond 

LTβR in this process. 

doi:10.1016/j.clim.2007.03.528 

S123 

Sa.142 A FLIPR-based Assay to Assess Potency of 

Inhibitors of the TEC Family Kinases Itk and Btk 

John Douhan III, Principal Research Scientist, Wyeth 

Research, Inflammation, Cambridge, MA, Joy Miyashiro, 

Research Scientist, Wyeth Research, Inflammation, 

Cambridge, MA, Xiaochuan Zhou, Research Scientist, Wyeth 

Research, Inflammation, Cambridge, MA, Paul Wu, Research 

Scientist, Wyeth Research, Inflammation, Cambridge, MA, 

Derek Cole, Principal Research Scientist, Wyeth Research, 

Chemical and Screening Sciences, Pearl River, NY, Mary 

Collins, Vice President, Wyeth Research, Inflammation, 

Cambridge, MA, Kyriaki Dunussi-Joannopoulos, Associate 

Director, Wyeth Research, Inflammation, Cambridge, MA 

Bruton’s tyrosine kinase (Btk) and interleukin-2-inducible 

Tcell kinase (Itk), members of the TEC family of nonreceptor 

protein tyrosine kinases, are expressed primarily in B and T 

cells respectively and are critically involved in lymphocyte 

development and signal transduction. In particular, both Btk 

and Itk regulate calcium mobilization subsequent to antigen 

receptor stimulation which in turn regulates downstream 

effector functions such as proliferation, antibody secretion 

and cytokine production. Small molecule antagonists of Btk 

and Itk may therefore be useful in treating inflammatory and 

autoimmune conditions. We have developed a FLIPR-based 

assay which takes advantage of Btk-deficient DT40 chicken B 

cells. It has been reported that these cells are unable to 

mobilize calcium in response to cross-linking of their B cell 

receptor and that ectopic expression of human Btk or Itk can 

restore signaling upon B cell receptor stimulation. Here we 

report that not only the expression of wild type human Btk, 

but also the expression of a chimeric Btk–Itk kinase molecule 

can restore calcium responses in Btk-deficient DT40 cells. We 

have generated stable cell lines expressing either full length 

human Btk or a chimeric human Btk–Itk molecule—a Btk 

protein whose kinase domain has been replaced by that of 

Itk. Calcium responses in both cell lines can be inhibited by 

the tyrosine kinase inhibitor staurosporine, but only the


chimeric Btk-Itk DT40 line is inhibited by an Itk-specific 

kinase inhibitor. We demonstrate that potency and selectivity 

of inhibitors of Itk and Btk can be assessed in this FLIPR 

cell-based assay. 

doi:10.1016/j.clim.2007.03.529 

Sa.143 Anti-Inflammatory Activities of Stilbene 

Analogs for Targeting Autoimmune Diseases 

Liren Tang, Scientist, Welichem Biotech Inc., Burnaby, BC, 

Canada, Genhui Chen, Celestial Pharmaceuticals Ltd, 

Shenzhen, China, Bin Li, Scientist, Welichem Biotech Inc., 

Burnaby, BC, Canada, Quanhai Liu, Professor, Department of 

Pharmacology, Shanghai Instititue of Pharmaceutical 

Industry, Shanghai, China, Michael Lyle, Postdoctoral 

Fellow, Welichem Biotech Inc., Burnaby, BC, Canada, John 

Webster, Professor and CSO, Welichem Biotech Inc., Simon 

Fraser University, Burnaby, BC, Canada 

Certain species of symbiotic bacteria (Enterobacteriaceae), 

when released into an insect by their nematode 

vector, released metabolites that modulate the insect’s 

innate immune response. These metabolites (stilbene 

analogs, in the WBI-1000 series) possess unique antiinflammatory 

properties. In general, they are weakly 

cytotoxic to mammalian cells, being more active on 

activated than on quiescent T cells or other normal cells. 

When tested in vitro, the WBI-1000 series selectively 

inhibited IL-2 and IFN-γ production in human T cells, and 

significantly inhibited TNF-α production in mice, but IL-4 was 

not affected. As well, they significantly inhibited T cell 

migration towards LTB4 in vitro. When topically applied in 

the TPA-induced ear edema mouse model, WBI-1000 compounds 

significantly reduced both skin redness and thickness. 

To further explore the clinical applications of these 

compounds one of them, WBI-1001 was tested for its efficacy 

on different animal models for autoimmune diseases. WBI- 

1001 showed dose-related efficacy in both the vaginal 

mucosa and mouse tail models for psoriasis as a topical 

treatment, significant clinical improvement in dextran 

sodium sulphate (DSS)-induced inflammatory bowel disease 

(IBD) in mice and decreased inflammation-induced arthritis 

in rats when compared with indomathecin. In conclusion, 

due to its unique anti-inflammatory properties, including 

selectivity on activated T cells, strong inhibition of proinflammatory 

cytokines and T cell migration, the WBI-1000 

series of compounds could be developed into effective, novel 

treatment modalities for autoimmune diseases. 

doi:10.1016/j.clim.2007.03.551 

Sa.145 Functional Knockout of Kv1.3 Channels 

Suppresses Effector Memory Phenotype Acquisition 

in Dominant Negative Kv1.3-expressing Human 

CD4+ T Cells 

Lina Hu, Research Associate, Department of Neurology, 

Johns Hopkins School of Medicine, Baltimore, MD, Katharine 

Whartenby, Assistant Professor, Sidney Kimmel Cancer 

Center, Johns Hopkins School of Medicine, Baltimore, MD, 

Rameeza Allie, Research Specialist, Department of 

Neurology, Johns Hopkins School of Medicine Baltimore, MD, 

Peter Calabresi, Associate Professor, Department of 

Neurology, Johns Hopkins School of Medicine, Baltimore, MD 

Activated effector memory T cells (TEM) that highly 

express the voltage-gated potassium channel Kv1.3 have 

the potential of migrating to the CNS and are hypothesized 

to play a pathogenic role in multiple sclerosis (MS). The 

pharmacological effects of Kv1.3 channel blockade on TEM 

cell activation and proliferation are well documented. 

However, the functional relevance of Kv1.3 in the homeostatic 

maintenance of TEM is poorly understood. Herein, 

using a system in which Kv1.3-channel function was blocked 

by transduction of CD4+ T cells with a GFP-tagged, 

lentiviral vector expressing dominant-negative Kv1.x 

sequence, we studied the kinetic changes in TEM subset 

within dominant negative Kv1.3-expressing CD4+ T cells 

following anti-CD3/CD28 stimulation. The interference of 

endogenous Kv1.3 by misexpression of dominant-negative 

Kv1.x markedly attenuated the wild-type current. Analysis 

of GFP expression in CD4+ T cell subsets demonstrated that 

while a substantial reservoir of TCM was maintained, TEM 

cells expressing the dominant-negative Kv1.3 product were 

significantly less abundant than that infected with GFP 

control viruses at 2, 3 and 4 weeks after transduction. In 

contrast, the ratio between TEM and TCM cells in GFP 

control transfected cells is strongly biased toward the TEM. 

Thus, the generation and maintenance of TEM cells are 

markedly impaired by the inactivation of Kv1.3 channel 

function. These results offer new insights into the 

functional dependence of TEM cells on Kv1.3, and have 

important clinical implications for using Kv1.3 blockers to 

prevent the generation of TEM cells, thereby abrogating 

the pathologic consequences of this subset in autoimmune 

diseases, such as MS. 

doi:10.1016/j.clim.2007.03.532 

Sa.146 Tim-4 Expressed on Antigen-presenting Cells 

Induces T Cell Expansion and Survival 

Roselynn Rodriguez Manzanet, Graduate Student, Brigham 

and Women’s Hospital, Boston, MA, Jennifer Hartt-Meyers, 

Post doctoral Fellow, LIR/NIAID/NIH, Bethesda, MD, 

Jacqueline Slavik, Administrative Director, Brigham and 

Women’s Hospital, Biomedical Research Institute, Boston, 

MA, Valerie Dardalhon, Post doctoral Fellow, Brigham and 

Women’s Hospital, Department of Neurology, Boston, MA, 

Nasim Kassam, Research specialist, Brigham and Women’s 

Hospital, Department of Neurology, Boston, MA, Edward A. 

Greenfield, Consultant, Brigham and Women’s Hospital, 

Department of Neurology, Boston, MA, Raymond A. Sobel, 

Professor, Stanford School of Medicine, Department of 

Pathology, Palo Alto, CA, Terry B. Strom, Physician 

Professor, Beth Israel Deaconess Medical Center, Boston, 

MA, David A. Hafler, Professor, Brigham and Women’s 

Hospital, Department of Neurology, Boston, MA, Vijay K. 

Kuchroo, Professor, Brigham and Women’s Hospital, 

Department of Neurology, Boston, MA

Abstracts 

The TIM (T cell, immunoglobulin, mucin) proteins characterized 

to date have been shown to regulate Tcell immune 

responses. Tim-4 has been shown to be a ligand for Tim-1 and 

Tim-4.Ig fusion protein could both inhibit and activate T cell 

proliferation in in vitro assays. Further studies of this 

molecule have been hindered by the lack of a specific 

monoclonal antibody. Thus, we have generated three 

monoclonal anti-Tim-4 antibodies and show that Tim-4 

protein expression is restricted to the antigen-presenting 

cell population, specifically CD11c+ and CD11b+ cells. We 

demonstrate that Tim-4 expression is upregulated upon 

cellular activation, and that ectopic expression of Tim-4 on 

artificial APCs induces massive T cell proliferation. Additionally, 

in vitro crosslinking of Tim-4 ligand on the surface of T 

cells directly induced cell division and anti-apoptotic protein 

Bcl-2. We conclude that Tim-4 is a costimulatory molecule 

capable of promoting T cell expansion and show that it can 

enhance both T cell proliferation and survival. 

doi:10.1016/j.clim.2007.03.533 

Sa.147 Interleukin-2 Mastering Regulation in Cancer 

and Autoimmunity 

Enrique Montero, MD, PhD, Center of Molecular 

Immunology, Department of Experimental Immunotherapy, 

Havana, Cuba, Livan Alonso, BSc in Physics, Center of 

Molecular Immunology, Department of Experimental 

Immunotherapy, Havana, Cuba, Rolando Perez, PhD, Center 

of Molecular Immunology, Havana, Cuba, Agustin Lage, MD, 

PhD, Center of Molecular Immunology, Havana, Cuba 

Autoimmunity and tumor immunity have evolved as two 

well distant arenas in immunological research. However, the 

identification of self-antigens as the major components of 

malignant cells may define a central role for autoimmunity in 

cancer control tuned by peripheral immunoregulatory 

mechanisms avoiding self-aggression. Interleukin-2 (IL-2) 

paradoxical effects after in vitro or in vivo manipulations 

may reflect the complex interplay between immunoregulatory 

mechanisms. Emerging evidences support the contribution 

of IL-2 to the maintenance of natural immunological 

tolerance. It encourages the systematic evaluation in vivo of 

formulations with IL-2 neutralization capacity compared 

with IL-2 exogenous administration to explore their influence 

on immunobiology in cancer and autoimmunity. IL-2 chemically 

conjugated to the carrier protein P64k from Neisseria 

Meningitides in Montanide ISA51 adjuvant was used for active 

immunization. We found that BALB/c, C57BL/6 and NMRI 

mice developed a long-lasting immunoresponse to IL-2 after 

immunization, without signs of autoimmune diseases. 

Immune sera had a similar IL-2 blocking effect in vitro to a 

specific anti-IL-2 monoclonal antibody (mAb). Interestingly, 

IL-2 deprivation did not affect the immunoresponse to 

therapeutic vaccines; however, it restores the response to 

nominal antigens in immunosuppressed hosts. Surprisingly, 

we found a differential effect of anti-IL-2 and anti-CD25 mAb 

on anti-tumor immunoresponse that may represent a broader 

impact of IL-2 on immunoregulation beyond its effect on 

CD4+CD25+ regulatory T cells. Moreover, the main effect of 

IL-2 administration was associated with an enhancement of 

immunoregulation. We conclude that IL-2 plays a major role 

in the interplay between cancer and autoimmunity with 

therapeutic implications. 

doi:10.1016/j.clim.2007.03.534 

Sa.148 Decrease in Memory B Cell Frequency and 

Activation is Associated with PTPN22 1858T Variant 

in Healthy Subjects 

Mary Rieck, Research Technician II, Benaroya Research 

Institute, Seattle, WA, Patric Concannon, Professor, Center 

for Public Health Genomic, University of Virginia, 

Charlotte, SC, Adrian Arechiga, Research Associate, 

Benaroya Research Institute, Seattle, WA, Jane Buckner, 

Associate Member, Benaroya Research Institute, Seattle, 

WA, Suna Onengut-Gumuscu, Research Associate, Center for 

Health Genomic, Charlotte, SC 

It is known that the 1858T variant of the PTPN22 gene is 

associated with multiple autoimmune disorders, yet the 

functional consequences of this variant and how they 

contribute to autoimmunity are still unknown. PTPN22 

encodes the protein tyrosine phosphatase, Lyp, which 

participates in the proximal events of TCR and BCR signaling. 

The 1858T mutations results in an R and #61664;W amino acid 

change at residue 620 of Lyp. In Tcells Lyp620W dampens TCR 

signaling, however, its effects on the B cell compartment are 

not known. In this study we address this question by 

examining B cell function and phenotype in control subjects 

possessing the 1858T allele (1858C/T) as compared to 

subjects without the variant (1858C/C). These studies reveal 

no difference in percentage or number of total B cells in the 

periphery. However, within the B cell pool there was a 

decrease in the percent of memory B cells, defined as 

CD19+CD27+, in 1858C/T versus 1858C/C subjects (p=0.01). 

This decrease in the memory pool was even more profound in 

autoimmune subjects homozygous for the 1858T variant 

(p=0.002). In addition, we find that the response of B cells to 

BCR stimulation, as measured by calcium flux is diminished in 

subjects carrying the 1858T allele, this was most profound in 

the memory subset. These studies demonstrate that the 

PTPN22 1858T variant leads to altered B cell subsets and 

function. This suggests the possibility that 1858Tacts through 

B cell intrinsic mechanisms that contribute to the pathogenesis 

of autoimmunity in individuals with this variant. 

doi:10.1016/j.clim.2007.03.535 

S125 

Sa.149 Deconvolution of Blood Microarray Data 

Elucidates Cellular Activation Patterns in SLE 

Alexander R. Abbas, Genentech Inc., South San Francisco, 

CA, Kristen Wolslegel, Genentech Inc., South San Francisco, 

CA, Dhaya Seshasayee, Genentech Inc., South San Francisco, 

CA, Hilary F. Clark, Genentech Inc., South San Francisco, CA 

Understanding the biology of a disease often depends on 

knowing the different types and states of cells in biological


samples from relevant organs. The proportions of cells in 

blood or tissue are traditionally determined using methods 

that rely on one or two genes' expression or protein products, 

such as FACS or immunohistochemistry. These techniques 

detect only a few cell types at once and often cannot 

determine the state of cells. Here we present a method for 

using gene expression profiles of purified leukocyte cell types 

to probe the levels of cell types and cell states in whole blood 

samples. We demonstrate that this method accurately 

deconvolves mixed samples of known composition. We 

deconvolve blood samples from healthy donors and systemic 

lupus erythematosus patients to reveal patterns of cellular 

activation that apparently underlie this disease's prominent 

interferon signature. 

doi:10.1016/j.clim.2007.03.536 

Sa.150 Characterization of the Psoriasis-associated 

IL12B and IL23R Genes 

Ann Begovich, Director, Celera, Alameda, CA, Monica 

Chang, Senior Associate Scientist, Celera, Alameda, CA, 

Mark Leppert, Professor, Department of Human Genetics, 

University of Utah, Salt Lake City, UT, Steven Schrodi, 

Senior Scientist, Celera, Alameda, CA, Gerald Krueger, 

Professor, Department of Dermatology, University of Utah, 

Salt Lake City, UT 

Common genetic variants in IL12B and IL23R have been 

associated with psoriasis risk. To further investigate the 

role of these genes in susceptibility to psoriasis and other 

autoimmune diseases, we have resequenced both genes in 

96 individuals with psoriasis, identified novel SNPs and 

genotyped a subset of these and others from public 

databases in three independent white North American 

case–control sample sets (1446 cases and 1432 controls). 

To date, allelic and genotypic data for 51 IL12B-region and 

46 IL23R-region SNPs have been analyzed and preliminary 

haplotype analyses have been performed. The data suggest 

that association of IL12B with psoriasis is driven by two 

SNPs, rs3212227 and rs6887695, or SNPs in significant LD 

with these two markers. A sliding-window of haplotype 

association co-localizes psoriasis susceptibility within the 

boundaries of IL23R and not IL12RB2, which lies directly 

adjacent. Further genotyping in this region is ongoing to 

better define the IL23R genetic variants responsible for the 

association with psoriasis. We have also assessed whether 

the two psoriasis-associated IL12B SNPs are associated with 

specific clinical characteristics including age of onset, 

family history, psoriatic arthritis, and the effect of 

infection on psoriasis severity in the two sample sets 

with complete phenotype data. The only interesting 

replicated finding was that the effect of rs6887695 may 

be significantly different when stratifying by response to 

infection (Breslow-Day P=0.04 and 0.043). Finally, the role 

of these SNPs in susceptibility to rheumatoid arthritis and 

multiple sclerosis will be addressed. 

doi:10.1016/j.clim.2007.03.537 

Sa.151 Elifacs: A Multiplex Assay to Detect Antigen 

Specific T Cells and to Monitor the Immune 

Response 

Khadir Raddassi, Research Instructor, Brigham and Women’s 

Hospital, Neurology, Boston, MA, Kasia Bouchier, Scientist, 

Immune Tolerance Network, San Francisco, CA, Jose 

Estevam, Research Assistant, Brigham and Women’s 

Hospital, Neurology, Boston, MA, Vicki Seyfert-Margolis, 

Scientist, Immune Tolerance Network, San Francisco, CA, 

David Hafler, Lab Director, Brigham and Women’s Hospital, 

Neurology, Boston, MA 

As part of the Immune Tolerance Network assay development 

group, we optimized a multiplex cell based assay to 

detect antigen reactive T cells. Cell assays to detect antigen 

specific T cells require large cell numbers. The Elispot assay 

is fast and sensitive but provides limited information 

regarding individual cells. In contrast, flow cytometry allows 

better characterization of antigen reactive T cells. Similarly, 

measuring thymidine incorporation provides no information 

regarding T cell function. Here, we combined the three to 

monitor T cell response to antigens using the same sample. 

Fresh or cryopreserved PBMCs were stained with CFSE, and 

exposed to tetanus toxoid or myelin antigens. After 42 h the 

cells were transferred to Elispot plates for 16 h; the Elispot 

plates were developed for cytokine expression, and the cells 

were collected and cultured for an additional 5 days before 

measuring intracellular cytokines and proliferation by flow 

cytometry. The combination of Elispot and flow cytometry 

data did not alter the specificity or sensitivity of these 

techniques. The recovery and viability of the cells from the 

Elispot plate were above 90%. There was a tight correlation 

(r 2 =0.960) between these data obtained by Elispot (cytokine 

positive cells) and by flow cytometry (CFSE and the cytokine 

production). We were able to measure IFNg, IL-10, IL-4, 

TGFβ and proliferative responses from the same sample. This 

technique allows us to extract more information from the 

same sample and thus provides a useful tool in monitoring 

the immune response. 

doi:10.1016/j.clim.2007.03.538 

Sa.152 Population-based Studies of Risk Factors in 

Autoimmunity: The Kaiser Permanente 

Autoimmune Disease Research Group (KPADRG) 

Lisa Barcellos, Assistant Professor, UC Berkeley, School of 

Public Health, Berkeley, CA, Ling Shen, Programmer/ 

Analyst, Kaiser Permanente Division of Research, Oakland, 

CA, Lisa Herrinton, Epidemiologist/Senior Researcher, 

Kaiser Permanente Division of Research, Oakland, CA, Allan 

L. Bernstein, Chief of Neurology, Kaiser Permanente Santa 

Rosa, Kaiser Permanente Division of Research, Oakland, CA, 

James E. Allison, Adjunct Investigator, Kaiser Permanente 

Division of Research, Oakland, CA, John Citron, 

Endocrinologist, Kaiser Permanente Walnut Creek, Kaiser 

Permanente Division of Research, Oakland, CA, Stanford 

Shoor, Kaiser Permanente Santa Clara, Kaiser Permanente 

Division of Research, Oakland, CA, Andrew Karter, 

Epidemiologist/Senior Researcher, Kaiser Permanente 

Division of Research, Oakland, CA, De-Kun Li,

Abstracts 


Division of Research, Oakland, CA, Catherine Schaefer, 


Division of Research, Oakland, CA, Erica Gunderson, 


Division of Research, Oakland, CA, Joe Selby, Director, 

Kaiser Permanente Division of Research, Oakland, CA, Lisa 

Croen, Epidemiologist/Senior Researcher, Kaiser 

Permanente Division of Research, Oakland, CA, Neil Risch, 

Adjunct Investigator, Kaiser Permanente Division of 

Research, Oakland, CA 

Autoimmune disorders, collectively, include more than 

60 chronic and often disabling conditions that result from 

underlying defects in the immune system. As a group they 

affect approximately 15–20 million people in the United 

States, resulting in significant morbidity and mortality. 

Large, well-characterized, clinical populations in an environment 

that supports a broad research program are needed 

to advance our understanding of disease etiology. The 

Kaiser Permanente Medical Care Program (Northern California 

Region) or KPNC membership includes 3.3 million 

people, representing about 30% of the population of 

northern California. The membership is sociodemographically 

and culturally diverse, while highly representative of 

the general population. Comprehensive electronic medical 

records, including outpatient, pharmacy, laboratory, imaging 

and hospitalization data maintained since 1995 were 

used to construct a large registry of over 200,000 

individuals with diagnoses of one or more autoimmune 

conditions. Conditions for the study were derived from 

over 50 ICD-9 codes, broadly categorized into the following 

groups: endocrine, gastrointestinal, hematologic, kidney, 

liver, skin, neurologic/muscular, vascular, and connective 

tissue/joints. Planned additions to this registry include 

biospecimens and survey data on environmental and 

behavioral risk factors. This resource will be used to 

conduct the large epidemiologic investigations needed to 

advance our understanding of autoimmunity, including 

studies of genetic, social, and environmental contributions 

to risk, progression, and response to treatment. Results 

from our current investigations of autoimmune disease 

incidence/prevalence rates and patterns of co-morbidity 

will be presented, including results from our focused 

research efforts in multiple sclerosis and inflammatory 

bowel disease. 

doi:10.1016/j.clim.2007.03.539 

Sa.153 Functional Characterization of the 

Autoimmune-associated LYP-W620 Variant 

Lei Zhao, PhD Student, University of Southern California, 

Los Angeles, CA, Yingge Liu, Postdoctoral Fellow, University 

of Southern California, Los Angeles, CA, Edoardo Fiorillo, 

Postdoctoral Fellow, University of Southern California, Los 

Angeles, CA, Stephanie Stanford, PhD Student, University of 

Southern California, Los Angeles, CA, Valeria Orru, 

Postdoctoral Fellow, University of Southern California, Los 

Angeles, CA, Nunzio Bottini, Assistant Professor, University 

of Southern California, Los Angeles, CA 

A missense single-nucleotide polymorphism, C1858T in 

the PTPN22 gene is associated with multiple human 

autoimmune diseases, including type 1 diabetes, rheumatoid 

arthritis, systemic lupus erythematosus, Graves disease, 

juvenile idiopathic arthritis, generalized vitiligo, and 

others. Genetic studies have shown that the PTPN22 

C1858T polymorphism is primarily associated with autoimmunity 

in different populations. The PTPN22 gene 

encodes the lymphoid tyrosine phosphatase LYP, which is 

expressed only in white blood cells and acts as a 

gatekeeper of T lymphocyte activation. The molecular 

mechanism by which LYP tempers T lymphocyte activation 

involves the formation of a complex between LYP and the 

negative regulatory kinase Csk. When compared to the 

common LYP-R620 variant, the autoimmune-predisposing 

LYP-W620 variant shows reduced binding to Csk, and more 

recently we found that LYP-W620 is a gain-of-function form 

of the phosphatase. In order to analyze the molecular 

mechanism of the gain-of-function phenotype of LYP-W620, 

we isolated recombinant Csk-free LYP-R620 and W620 using 

an insect cell expression system. When we reconstituted in 

vitro the complex between LYP and Csk, we found that 

recombinant LYP-R620 binds Csk more efficiently than LYP- 

W620. We also analyzed the effect of Csk on the enzymatic 

activity of LYP. Our data obtained on in vitro reconstituted 

LYP–Csk complexes suggest that reduced binding to Csk 

cannot fully explain the gain-of-function phenotype of the 

LYP-W620 variant. 

doi:10.1016/j.clim.2007.03.540 

S127 

Sa.154 Spontaneous Myocarditis in Transgenic Mice 

Needs Appropriate MHC and Non-MHC Genes 

Veena Taneja, Assistant Professor, Department of 

Immunology, Mayo Clinic, Rochester, MN, Marshall Behrens, 

Senior Technician, Department of Immunology, Mayo Clinic, 

Rochester, MN, Leslie Cooper, Consultant, Mayo Clinic, 

Department of Cardiovascular Disease, Rochester, MN, 

Chella David, Professor, Department of Immunology, Mayo 

Clinic, Rochester, MN 

Viral infection has been associated with the development 

of myocarditis in humans. Myocarditis is characterized by 

mononuclear infiltrate with attendant myocyte necrosis. 

Animal models of induced myocarditis have been generated 

by using coxsackie virus and immunization with cardiac 

myosin. We have recently generated a spontaneous model of 

myocarditis. HLA-DQ8 and DR3 were expressed as transgenes 

in NOD mice lacking endogenous class II molecules, Abo.DQ8. 

NOD and Abo.DR3.NOD. Also mice expressing both transgenes 

in C57\B10 mice lacking endogenous class II molecules were 

generated. All mice were bred to congenic backgrounds. Only 

mice expressing HLA-DQ8 in NOD background developed 

spontaneous myocarditis. Abo.DR3.NOD, Abo.DQ8.B10 and 

Abo.DR3.B10 and transgene negative littermates did develop 

any gross or microscopic cardiac pathology. The autoimmunity 

was associated with the presence of spontaneous 

autoreactive T cells and antibodies to cardiac myosin. Only 

DQ8.NOD mice mounted a strong spontaneous in vitro T cell 

response and in vivo DTH response to cardiac myosin. These


data suggest that DQ8.NOD mice have lost tolerance to the 

self-cardiac myosin leading to spontaneous myocarditis and 

dilated cardiomyopathy. HLA-DQ8 is required for predisposition 

to the spontaneous autoreactivity while NOD background 

influences onset and progression of disease. This model of 

myocarditis occurs predominantly in female mice and may 

provide insight into the pathogenesis of heart disease in 

women. 

doi:10.1016/j.clim.2007.03.554 

Sa.155 Human Lung Tumor Cells Modifies Rates of 













how the microenvironment of a tumor mass induced 

phenotypical changes in lymphocyte subsets. Our subjects 

were 10 patients with resectable non-small cell lung cancer. 

We evaluated 47 different phenotypically different lymphocyte 

subsets in blood samples taken from the pulmonary 

artery, pulmonary vein of a tumor bearing pulmonary lobe 

and peripheral blood during pulmonary resectional surgery. 

We showed that, tumor cells boosted CD25high+CD4high+T 

lymphocytes (Treg cells), B lymphocytes, NK and CD19+CD23 

+ cells (Breg cells) and CD3+CD95+ (FasL+T lymphocytes) 

(p=0.015, p=0.001, P=0.02, p=0.04, p=0.03 respectively). 

However, Mac-1 cells and HLADR+T lymphocytes were found 

to be decreased by tumor mass(p=0.04 and p=0.04), whereas 

only Breg , NK cell and B lymphocyte subsets were found to 

be expansed. In addition, the patients showed expansions of 

CD45R0+ activated T lymphocytes and CD18+CD11b+(Mac-1) 

cell subsets without significant alteration by tumor microenvironment. 

In conclusion, subsets of Treg, Breg cells, FasL+ 

T lymphocytes, B lymphocytes were modified by lung cancer 

microenvironment itself. This study also showed that, 

peripheral blood might not be good source for investigating 

the anti-tumor immunity since only Breg, NK cell and Treg 

cell subsets were found expansed peripherally. T lymphocytes 

and Mac-1 cells may be modified by systemic antitumor 

response rather than by local tumoral microenvironment. 

This study provides important insight into how tumor 

infiltrating lymphocytes and peripheral immune systems 

may be different in terms of lymphocyte subset expansion 

dynamics. 

doi:10.1016/j.clim.2007.03.552


ABSTRACTS 

Oral Presentations: Sunday, June 10 

#3201- From Genes to Treatment 

Sunday, June 10 

10:45 am−11:05 am 

Immune Reconstitution in X-Linked Hyper-IgM 

Syndrome With Recombinant CD40 Ligand 

Ashish Jain, Tenure- Track Investigator, Laboratory 

of Host Defenses, NIAID, NIH, Bethesda, MD, David 

Nelson, Senior Investigator, NCI, Bethesda, MD, Jospeh 

Kovacs, Senior Investigator, Clinical Center, Bethesda, 

MD, Shuying Liu, Study Coordinator, NIAID, Bethesda, MD, 

Thomas Fleisher, Senior Investigator, Clinical Center, 

Bethesda, MD, William Fanslow, Group Leader, Amgen 

Seattle, Seattle, WA, Hans Ochs, Professor, Department 

of Pediatrics, Seattle, WA, Warren Strober, Senior 

Investigator, Laboratory of Host Defenses, NIAID, NIH, 

Bethesda, MD 

Purpose: X-linked hyper IgM syndrome (XHIM) is a 

combined immune deficiency disorder caused by mutations 

in the gene encoding CD40 ligand. The purpose of 

this study was to investigate the safety and efficacy 

recombinant human CD40 ligand (rCD40L) for patients 

with XHIM. Methods: The study was designed as a phase I/ 

II, open-label, single-dose study of rCD40L administered 

subcutaneously three times per week for 24 weeks in 

three children with XHIM. Adverse effects and efficacy of 

rCD40L were evaluated during 6- month trial period. 

Results: The results of more than 230 subcutaneous 

injections showed that rCD40L was generally well tolerated. 

With rCD40L treatment, patients developed for the 

first time delayed type hypersensitivity reactions on skin 

testing that disappeared during the drug free follow up 

period. Analysis of patients’ T cells demonstrated first 

time capacity to synthesize IFN-g and TNF-a when 

stimulated with anti-CD3, or SEB, or SEA in vitro. Studies 

of cytokine production by intracellular staining demonstrate 

that rCD40L was able to prime both the CD4 and 

CD8 T cell populations with in vivo administration. 

Improvements of lymph node size and architecture were 

also noted; patients showed the development of primary 

follicles and follicular dendritic cells, as well as an 

doi:10.1016/j.clim.2007.03.006 



expansion of B and T cell populations. Conclusions: This 

phase I/II first study of recombinant human CD40 ligand 

showed that rCD40L is capable of restoring immune 

functions in patients with XHIM. Further study will be 

needed to assess the overall potential of this therapy. 

doi:10.1016/j.clim.2007.03.007 

#3202- Antigen Specific Immune Modulation 


10:45 am−11:05 am 

Neurofascin-specific Autoantibodies Mediate Axonal 

Injury in Inflammatory Demyelinating Diseases of 

the Central Nervous System 

Christopher Linington, Professor of Immunobiology, 

University of Aberdeen, Aberdeen, UK, Emily Mathey, 

Research Associate, University of Aberdeen, Aberdeen, 

UK, Tobias Derfuss, Max Planck Institute for 

Neurobiology, Martinsried, Germany, Matthew Rasband, 

Assistant Professor, University of Connecticut Health 

Center, Farmington, CT, Maria Storch, Professor, Medical 

University Graz, General Neurology, Graz, Germany, 

Reinhard Hohlfeld, Professor, LMU University Hospital, 

Clinical Neuroimmunology, Munich, Germany, Edgar 

Meinl, Professor, Max Planck Institute for Neurobiology, 

Martinsried, Germany 

We report that multiple sclerosis is associated with 

elevated autoantibody responses to neurofascin, as 

compared to other inflammatory neurological diseases. 

The two isoforms of neurofascin play essential roles in 

maintaining the molecular organization of myelinated 

fibers: NF186 is a neuronal protein located at the node 

of Ranvier and axonal initial segment where it interacts 

with voltage gated sodium channels, whilst NF155 is an 

oligodendroglial product sequestered at junctional complexes 

formed where paranodal loops of the myelin 

sheath contact the axonal surface. Analysis of neurofascin-specific 

autoantibodies immunopurified from


seropositive donors demonstrates that they recognise the 

extracellular domain of both isoforms of the native 

protein indicating that this response may participate in 

disease pathogenesis. We investigated this hypothesis in 

experimental autoimmune encephalomyelitis using a pan 

NF155/186 mouse mAb to mimic the human autoantibody 

response. Passive transfer (i.p.) of mAb induced a rapid 

exacerbation of disease that was associated with a 

corresponding increase in acute axonal injury. Intriguing 

this occurred in the absence of any concomitant increase 

in the local inflammatory response or demyelination. 

Immunofluorescence microscopy revealed that binding of 

the transferred neurofascin-specific antibody was 

restricted nodes of Ranvier where it co-localised with 

voltage gated sodium channels. These results identify 

NF186 as a primary target for neurofascin-specific autoantibodies 

and indicate antibody-dependent effector 

mechanisms are involved in the pathogenesis of axonal 

injury and loss in multiple sclerosis. 

doi:10.1016/j.clim.2007.03.008 

#3203- Innate Immunology 


10:45 am−11:05 am 

IL-23 is Required for Induction of an Innate Immune 

Response to Citrobacter Rodentium 

Darrell O’Quinn, Postdoctoral Fellow, University of 

Alabama, Department of Pathology, Birmingham, AL, 

Trenton Schoeb, Professor, University of Alabama, 

Department of Genetics, Birmingham, AL, Casey Weaver, 

Professor, University of Alabama, Department of 

Pathology, Birmingham, AL 

We have previously shown that infection of IL-23deficient 

mice with the enteric murine pathogen Citrobacter 

rodentium results in complete mortality within 7-10 

days following inoculation despite the presence of CD4+ IL- 

17-competent cells. Using a mutant strain of bioluminescent 

C. rodentium, we here demonstrate that colonization 

of the cecum and mid- and distal colon of IL-23-deficient 

mice occurs earlier and in greater numbers than in IFNγ-, 

IFNγR-, IL-12-deficient, or wild-type controls. Histopathological 

examination of the lesions in IL-23-deficient mice 

confirms the presence of massive numbers of bacteria 

attached to intestinal epithelial cells of the mid- and distal 

colon, and multi-focal areas of ulcerative necrosis indicate 

a limited or absent inflammatory response. Analysis of C. 

rodentium colonization in IL-17R-/- mice reveal no significant 

differences from wild-type controls suggesting that 

IL-23, not IL-17, may be critical for the maintenance of 

innate defenses in the distal colon. 

doi:10.1016/j.clim.2007.03.009 

OR.70 Intestinal Epithelial Cell Derived Thymic 

Stromal Lymphopoeitin Controls Dendritic 

Cell Function and T Regulatory Cell 

Homeostasis 

Iliyan D. Iliev, PhD Student, European Institute of 

Oncology, Milan, Italy, Erika Mileti, Laboratory Assistant, 

European Institute of Oncology, Milan, Italy, Maria 

Rescig, Group Leader, European Institute of Oncology, 

Milan, Italy 

The mucosal immune system has developed mechanisms 

to tolerate beneficial microflora, but in the same time to 

initiate immunity against invading pathogens. How these 

mechanisms function is still not completely understood. In 

our current study we investigated the possible role of 

Epithelial Cells (EC) as a “tolerance keepers” in the gut 

driving the development of non-inflammatory Dendritic 

Cells (DC) able to promote CD4+CD25+Foxp3+ T regulatory 

cells (Treg) development. We developed a co-culture 

system, in which EC (Caco2 or primary intestinal EC) 

supernatants were used to condition monocyte-derived DC. 

We found that in both, human and mice, EC-conditioned DC 

were producing less inflammatory cytokines in response to 

bacterial stimuli and favored the development of functionally 

active Treg. These tolerogenic DC were involved in 

driving Treg also in vivo in mice. Thymic stromal 

lymphopoeitin (TSLP) produced by EC, together with 

additional factors, was involved in this process since DC 

conditioned with supernatants from Caco2 cell line 

“silenced” for TSLP, were unable to expand Treg. Finally, 

mice immunized with Salmonella loaded EC-conditioned DC 

were less resistant to lethal Salmonella dose than control 

mice, confirming their tolerogenic potential in vivo. 

Therefore, intestinal EC released TSLP, can influence gut 

DC function and can control the homeostasis of peripheral 

Treg. We propose that failure in this mechanism can 

participate in the pathology of Inflammatory Bowel 

Disease. 

doi:10.1016/j.clim.2007.03.010 

Regulatory T Cells II 


2:45 pm−4:45 pm 

OR.71 Critical Role of IFNg in Allograft Tolerance 

Mediated by Foxp3+CD4+CD25+ Regulatory T Cells 

and the Production of Indoleamine 2, 3-dioxygenase 

by Graft Endothelial Cells 

Pamela Thebault, PhD, INSERM U643, Nantes, France, 

Michele Heslan, IE, INSERM U643, Nantes, France, Thomas 

Condamine, PhD, INSERM U643, Nantes, France, Abdelhadi 

Saoudi, CR1, INSERM U563, Toulouse, France, Marcello Hill, 

Postdoctoral Fellow, INSERM U643, Nantes, France, Regis 

Josien, CR1, INSERM U643, Nantes, France, Ignacio Anegon, 

DR2, INSERM U643, Nantes, France, Maria-Cristina Cuturi, 

DR2, INSERM U643, Nantes, France, Elise Chiffoleau, CR 2, 

INSERM U643, Nantes, France

Abstracts 

Functionally specialized subsets of regulatory CD4+T cells 

have been shown to play an important role in the regulation of 

both T cell-mediated and innate immune responses. In particular, 

the CD4+CD25+ subpopulation of T cells has been 

shown to be crucial in self-tolerance and also to prevent 

allograft rejection. As CD25 is not uniquely expressed by 

regulatory T cells, but can also be expressed by activated 

effector cells, the identification of Foxp3 has provided an 

opportunity to specifically identify regulatory T cells and to 

track them in vivo so as to better define the mechanisms 

involved in their regulation of immune responses. We previously 

demonstrated, in a fully MHC mismatched rat cardiac 

allograft combination, that a short-term treatment with a 

deoxyspergualine analogue, LF15-0195, induces long-term 

allograft tolerance with a specific expansion of regulatory 

CD4+CD25+T cells that accumulate within the graft. In this 

study, we show that following transfer to a secondary irradiated 

recipient, regulatory CD4+CD25+T cells are able to 

rapidly home to the graft, induce accumulation of Foxp3+ and 

Tr1/IL10+ regulatory CD4+Tcells and induce graft endothelial 

cell expression of Indoleamine 2, 3-dioxygenase (IDO), 

inducible Nitric Oxide synthase (iNOS) and Heme-Oxygenase-1 

(HO-1). Moreover, in vivo transfer of tolerance can be 

abrogated by blocking IFNg or IDO and in vitro, anti-IFNg 

reduces the survival/function of alloantigen-induced Foxp3+ 

CD4+ regulatory T cells. Together, our results demonstrate 

interrelated mechanisms between regulatory CD4+Tcells and 

the graft endothelial cells in this local immune privilege and a 

key role for IFNg and IDO in this process. 

doi:10.1016/j.clim.2007.03.011 

OR.72 Amino-Terminal Region of Foxp3 Performs 

Multiple Functions in Regulating Transcription 

Jared E. Lopes, Graduate Students, University of 

Washington, Seattle, WA, Jianguang Du, Postdoctoral Fellow, 

Benaroya Research Institute, Seattle, WA, Xiaocui Sun, 

Research Technician, Benaroya Research Institute, Seattle, 

WA, Mark Chong, Postdoctoral Fellow, Molecular 

Pathogenesis Program, Skirball Institute of Biomolecular 

Medicine, New York, NY, Liang Zhou, Fellow, Molecular 

Pathogenesis Program, Skirball Institute of Biomolecular 

Medicine, New York, NY, Dan R. Littman, Director of 

Molecular Pathogenesis Program, Skirball Institute of 

Biomolecular Medicine and Howard Hughes Medical 

Institute, New York, NY, Steven F. Ziegler, Immunology 

Program Director, Benaroya Research Institute, Immunology 

Program, Seattle, WA 

Foxp3 has been shown to be both necessary and sufficient 

for the development and function of naturally-arising CD4+ 

CD25+ regulatory Tcells (Tregs) in mice. Mutation of Foxp3 in 

scurfy mice and Foxp3 in humans with IPEX results in fatal, 

early onset autoimmune disease and demonstrates the 

critical role of Foxp3 in maintaining immune homeostasis. 

The Foxp3 protein encodes several functional domains 

including a C2H2 zinc finger, a leucine zipper, and a wingedhelix/forkhead 

(FKH) domain. We have previously shown that 

Foxp3 functions as a transcriptional repressor and inhibits 

activation-induced IL-2 gene transcription. We recently 

identified a novel functional domain within the aminoterminal 

half of Foxp3, which is required for Foxp3-mediated 

repression of transcription from both a constitutively active 

and an NFAT-inducible promoter. In order to determine the 

mechanism by which Foxp3 regulates transcription, we 

performed a yeast-2-hybrid analysis searching for proteins 

that interact with the amino-terminal region of Foxp3. One 

target was RORgammat (RORgt), which is required for the 

differentiation of naïve CD4+ T cells into pro-inflammatory, 

IL-17-producing T cells (Th17). Interestingly, CD4+ T cells 

express Foxp3 and RORgt in response to TCR stimulation in the 

presence of TGF-β and differentiate into either Tregs or 

Th17 cells, depending on the presence of IL-6. We show that 

Foxp3 directly inhibits RORgt-mediated transcription. Future 

studies will address the mechanism by which Foxp3 inhibits 

RORgt function and how this process is regulated by IL-6. 

doi:10.1016/j.clim.2007.03.012 

S131 

OR.73 Phenotypic and Functional Characteristics of 

Different Regulatory T Cell Subsets (Tregs) in 

Patients with Cancer 

Christoph Bergmann, Postdoctoral Fellow, University of 

Pittsburgh Cancer Institute, Department of Pathology, 

Pittsburgh, PA, Laura Strauss, Postdoctoral Fellow, 

University of Pittsburgh Cancer Institute, Department of 

Pathology, Pittsburgh, PA, Jonas Johnson, Professor and 

Chair, University of Pittsburgh Cancer Institute, Department 

of Otolaryngology, Pittsburgh, PA, Theresa L. Whiteside, 

Director, University of Pittsburgh Cancer Institute, 

Department of Pathology, Pittsburgh, PA 

Immune suppression mediated by Tregs is a feature of 

cancer. To compare phenotype and function of Tregs present 

in the tumor (TIL) and blood (PBMC) of patients with head and 

neck cancer (HNC), we obtained samples from 40 patients and 

15 normal controls (NC). The phenotype and frequency of 

CD4 + CD25 high and CD4 + CD25 − T cell subsets were evaluated. 

These cells were separated by single-cell sorting, tested for 

suppressor function and also cultured +/− cytokines and/or 

rapamycin (1 nM). IL-10 or TGF- 2 1 mAbs were used to block 

suppression of responder cell (R) proliferation. TIL were 

enriched in CD4 + CD25 high (13%±3) cells (Foxp3 + GITR + IL- 

10 + TGF− 2 1 + ). In PBMC, Tregs (CD25 high Foxp3 + CD62L + CCR7 + GI- 

TR + IL-10 + TGF− 2 1 + ) represented 5%±3 among CD4 + cells. In 

contrast, PBMC in NC contained fewer (pb0.001) Tregs (2%± 

1.5). In PBMC of patients and NC, CD4 + CD25 - IL-10 + TGF- 2 1 + 

(Tr1) cells were detected. In TIL, Tr1 cells represented 23%±3 

of CD4 + cells. Tr1 cells expanded from PBMC and TIL were 

CD25 − IL-10 + IL2R 3+ TGF- 2 1 + IL-4 − with low levels of Foxp3 and 

CTLA-4 expression. TIL Tregs mediated stronger suppression 

(p b0.001) than PBMC Tregs. NC Tregs mediated weak 

suppression. Neutralizing mAbs to IL-10 or TGF- 2 1 abolished 

suppression of R proliferation by Tregs. Cell contact of Tregs, 

but not Tr1, with R enhanced suppression. Rapamycin 

promoted selective expansion of Tregs and Tr1 cells by 

eliminating non-Tregs in patients and NC. Characteristics of 

rapamycin-expanded Tregs and Tregs in freshly-isolated 

lymphocytes were similar. Tregs mediating suppression in 

the tumor and circulation of HNC patients are heterogeneous


populations distinct from Tregs in NC. To mediate immune 

escape, tumor activates and/or induces unique Treg subsets. 

doi:10.1016/j.clim.2007.03.013 

OR.74 Hepatic Stellate Cells Expand 

CD4 + CD25 + Foxp3 + Tregs and Prevent Rejection of 

Allogeneic Islet Transplants 

Shiguang Qian, Associate Professor, Cleveland Clinic, 

Cleveland, OH, Guoping Jiang, Research Fellow, 

Department of Immunology, Cleveland Clinic, Cleveland, 

OH, Zhenyu Yin, Research Fellow, Department of 

Immunology, Cleveland Clinic, Cleveland, OH, John J. Fung, 

Professor, Department of General Surgery, Cleveland, OH, 

Liang-Mou Kuo, Research Fellow, Department of 

Immunology, Cleveland Clinic, Cleveland, OH, Lina Lu, 

Associate Professor, Department of Immunology, General 

Surgery, Cleveland Clinic, Cleveland, OH 

Liver tolerance was demonstrated by spontaneous acceptance 

of liver allografts in many species, but hepatocyte 

transplants were acutely rejected, suggesting an immunosuppressive 

effect of liver tissue cells. In this study, we 

demonstrated immunosuppressive activity of hepatic stellate 

cells (HSC) through expansion of CD4 + CD25 + Foxp3 + 

Tregs. HSC isolated from B6 mice (H2 b ) constitutively expressed 

low MHC class I and CD80. Expression of MHC class I, II 

and B7-H1 was markedly upregulated in IFN-γ activated-HSC 

(aHSC), which rendered them to stimulate allogeneic T cell 

proliferation in both CD4 + and CD8 + cells (CFSE). aHSC 

cultured with allogenetic CD4 + T cells at 1:10 ratio for 5 days 

markedly expanded CD4 + CD25+ + Foxp3 + cells (from 2.5% to 

10.5%), which were obviously from naïve CD4 + CD25 + population, 

since depletion of CD4 + CD25 + , only small number of 

CD4 + CD25 + cells were expanded, and were all Foxp3 − . 

Addition of aHSC expanded CD4 + CD25 + cells into CD4 + cells 

either stimulated by anti-CD3 or allo-antigens, resulted in 

marked inhibition of T cell proliferation in a dose dependent 

manner. To explore clinical application, 300 BALB/c (H2 d ) 

islets mixed with aHSC (3×10 5 ) transplanted under renal 

capsule of chemically diabetic B6 recipients, achieving 

markedly prolonged islet graft survival [median survival 

time (MST) from 13 days to N60 days (pb0.01)] with 55% 

being euglycemia for N60d. None of islet-only recipients 

remained euglycemia for N17d. Removal of islet-grafted 

kidney resulted in quick glycemia. Prolongation of islet 

allografts by co-transplanted HSC was associated with 

upregulated apoptotic activity (TUNEL) and less T cell 

infiltration (CD3), indicating potential application in improving 

islet transplantation. 

doi:10.1016/j.clim.2007.03.014 

OR.75 Polyclonal CD4+CD25+ Regulatory T Cells 

Induced With TGF-β Prevent a Stimulatory 

Graft-Versus-Host Disease With a Lupus-Like Syndrome 

Song Guo Zheng, Assistant Professor of Research Medicine, 

University of Southern California Keck School of Medicine, Los 

Angeles, CA, Julie Wang, Research Lab Specialist, University of 

Southern California Keck School of Medicine, Los Angeles, CA, 

Shuang Ye, Postdoctoraltoral Fellow, University of Southern 

California Keck School of Medicine, Los Angeles, CA, David A. 

Horwitz, Professor, University of Southern California Keck 

School of Medicine, Los Angeles, CA 

We have previously reported that the adoptive transfer of 

antigen-specific Tregs generated by allogeneic antigen stimulation 

with TGF-β prevents the appearance of lupus syndrome 

induced by injection of DBA/2 mouse spleen cells into (DBA/ 

2×C57BL/6) F1 mice. Here we have considered whether 

polyclonal CD4+ Tregs induced ex vivo may have similar 

suppressive effects in this model. Naïve CD4+CD25- cells 

isolated from DBA/2 mice were stimulated with anti-CD3/ 

CD28 coated beads+TGF-β without APC. TGF-β was able to 

directly induce the majority of activated CD25+ cells to express 

Foxp3, develop cell markers characteristic of Tregs, and acquire 

suppressive activity in vitro. The remaining CD25- cells did not 

express Foxp3 or develop suppressive activity. TGF-β -induced 

CD4+ Tregs not only suppressed the Tcell proliferative response 

to anti-CD3, but also inhibited the T cell response to allogeneic 

cells. Substituting soluble anti-CD3 and antigen-presenting cells 

(APCs) for anti-CD3/28 beads resulted in decreased Foxp3+ 

expression, an effect that was reversed by the addition of anti- 

IL-6. These levels of IL-6 in cultures containing TGF-β did not 

result in the production of IL-17. Finally, (D2xB6)F1 mice 

injected with D2 cells and 5 million TGF-β -induced CD4+ CD25+ 

Tregs did not develop increased polyclonal IgG, anti-dsDNA 

autoantibodies, or nephritis. Since SLE is a generalized 

autoimmune disease with many autoantibodies, and production 

of IL-2 and TGF-β is decreased in this disease, the 

generation of large numbers of autologous, polyclonal CD4+ 

CD25+Foxp3+ Treg cells with these cytokines ex vivo may have 

considerable therapeutic potential. 

doi:10.1016/j.clim.2007.03.015 

OR.76 Low Dose Antigen Promotes Induction of 

Both Foreign and Self Reactive Human 

CD25+Foxp3+ Regulatory T Cells from 

CD4+CD25-Foxp3- Peripheral T Cells 

S. Alice Long, Benaroya Research Institute, Seattle, WA, 

Mindi Walker, Scientist, Therakos, Exton, PA, Isabelle 

Mueller, Graduate Student, Benaroya Research Institute, 

Seattle, WA, Jane Buckner, Professor, Benaroya Research 

Institute, Seattle, WA, Mary Rieck, RA II, Benaroya Research 


Upon activation, a subpopulation of human CD4+CD25- 

Foxp3- T cells upregulate Foxp3 and become regulatory. These 

cells are referred to as induced TR (iTR) and have similar 

properties to TR isolated directly from peripheral blood. We 

focus here on properties of stimulation that influence generation 

of iTR and characteristics of the CD4+ CD25- T cells from 

which they are derived. We consistently observe that stimulation 

of CD4+CD25- T cells with foreign antigen results in two 

populations of antigen specific tetramer positive (Tmr+) Tcells; 

Tmr+Foxp3+ and Tmr− Foxp3− cells. The Foxp3+ population can 

arise from cells which express low or high levels of CD127, with 

the greatest number of which originate from CD127mid/hi

Abstracts 

populations. We demonstrate similar results with islet antigens 

in frequency, source and potency. Generation of iTR is enhanced 

in the presence of low dose antigen relative to high dose. Thus, 

low level TCR stimulation results in enhanced frequency of Tmr+ 

CD25+Foxp3+ T cells. These results demonstrate that iTR are 

generated as a consequence of normal immune responses in 

settings where antigen is in limited quantities, which may, in 

part, explain the phenomenon of low dose tolerance. Studies 

with T1D samples demonstrate the induction of islet and 

foreign iTR with similar functional potency. However, dose of 

antigen has no effect on the induction of iTR in T1D. Thus, T1D 

subjects have the ability to generate islet specific iTR in the 

periphery, but defects in the response to activation or the 

frequency of iTR, may contribute to disease progression. 

doi:10.1016/j.clim.2007.03.016 

OR.77 IL-10 Both Inhibits Treg Suppressive Activity 

and is a Survival Factor for Human Natural Tregs 

Clare Baecher-Allan, Instructor of Neurology, Harvard 


Department of Neurology, Boston, MA, Charles Ashley, 

Research Technician, Harvard Medical School, Brigham and 

Women's Hospital, Department of Neurology, Boston, MA, 

David Hafler, Breakstone Chair of Neurology, Harvard 


Department of Neurology, Boston, MA 

Human natural Tregs (CD4+CD25high) are comprised of 

two functionally distinct populations that differentially 

express HLA Class II proteins directly ex vivo. The MHC 

class II+ Tregs, referred to as ‘DR+Tregs’, are a highly suppressive 

population that rapidly inhibit the activation of cocultured 

responder T cells. The DRnegTregs, on the other 

hand, exhibit a delayed effector function as they allow an 

initial period of responder T cell proliferation before they 

shut down the response. While the DR+Tregs suppress solely 

by cell contact and do not produce IL-10, IL-10 is often (but 

not always) produced by the responder T cells and by the 

DR-Tregs in these accessory-cell-free assays. Interestingly, 

in those experiments in which the responder T cells did 

produce IL-10, the DR+Treg co-cultures exhibit less suppression 

than the duplicate co-cultures that had been given 

neutralizing IL-10 antibody from the outset. Thus, DR+Treg 

rapid suppression proceeds unimpeded in the absence of IL- 

10, while the presence of IL-10 inhibits their function. In 

order to understand this effect of IL-10, the two different 

cellular components of the co-culture were assayed to 

determine if the presence of IL-10 modulated the expression 

of regulatory (Foxp3, IL-10, TGFb), or apoptosis (BAX, 

BCL-2, and BCL-XL) related products. The results indicate 

that blocking IL-10 in DR+Treg co-cultures increases the 

frequency of AnnexinV+ responder T cells, reflecting the 

increase in suppression. Within the DR+Treg, however, 

blocking IL-10 triggered a reproducible decrease in BCL-XL 

and an increase in BAX, indicating increased Treg 

sensitivity to apoptosis that may affect long-term maintenance 

of suppression. 

doi:10.1016/j.clim.2007.03.017 

OR.78 High Density SNP Analysis of the MHC Region 

Reveals Multiple Loci for Type 1A Diabetes 

Theresa A. Aly, Graduate Student, University of Colorado 


Erin E. Baschal, Graduate Student, University of Colorado 


Mohamed M. Jahromi, Fellow, University of Colorado Health 

Sciences Center, Barbara Davis Center for Childhood 

Diabetes, Aurora, CO, Adam Kretowski, Physician, 

University of Colorado Health Sciences Center, Barbara 

Davis Center for Childhood Diabetes, Aurora, CO, Sunanda 

R. Babu, Research Associate, University of Colorado Health 

Sciences Center, Barbara Davis Center for Childhood 

Diabetes, Aurora, CO, Marian J. Rewers, Physician, 

University of Colorado Health Sciences Center, Barbara 

Davis Center for Childhood Diabetes, Aurora, CO, George S. 

Eisenbarth, Physician-Scientist, University of Colorado 

Health Sciences Center, Barbara Davis Center, Aurora, CO 

Haplotype sharing studies combined with HLA genotyping 

demonstrate that loci linked to the MHC region in addition to 

HLA-DR/DQ loci contribute major risk for type 1A diabetes. The 

hunt for these additional major loci is underway. In our survey 

study, we analyzed 656 single nucleotide polymorphisms (SNPs) 

in the MHC region and compared their allele frequencies in case 

versus control chromosomes from 45 families participating in 

the Diabetes Autoimmunity Study of the Young (DAISY). We 

included significant SNPs (pb0.01) from our survey study and 

additional SNPs extending 6 Mb further telomeric of the MHC in 

a follow-up study of 342 additional DAISY, HBDI, and Polish 

families. The most significant SNPs with distinct diabetesassociated 

peaks centromeric or within the MHC were at the 

ITPR3 (inositol-triphosphate receptor 3), HLA-DPB1, -DQB1, - 

DRB1, -DRA, and MICA loci. In addition to confirming these 

associations, our follow-up studies revealed a strong association 

of a 4 SNP DRA haplotype in 422/461 case chromosomes (92%) 

versus 437/579 control chromosomes (75%, p=1.8×10–11). The 

DRA haplotype remained significant when specific high-risk 

(DR3, DR4) and protective (DR2) chromosomes were excluded 

(p=0.05). The most significant regions telomeric of the MHC 

were at the Mas1L/UBD (Mas-related G protein coupled 

receptor/diubiquitin, p=0.00001) and PRSS16 loci (thymusspecific 

serine protease, p=0.00005). An HLA-DRA haplotype is 

strongly associated with type 1A diabetes; however, the 

biological basis for this association may be due to linked 

genes (e.g. HLA-DRB1-DQB1). Two novel regions 3100 kb and 

5400 kb telomeric of the HLA-DR and -DQ loci are also 

associated with diabetes. 

doi:10.1016/j.clim.2007.03.018 

Genetics 


2:45 pm−4:45 pm 

OR.79 Uniquely Designed Candidate Gene 

Identification Platform Discovers Novels Genes in 

Childhood Onset SLE 

Don Armstrong, FOCIS Center, University of Southern 

California School of Medicine, Los Angeles, CA, Andreas 

S133


Reiff, Children’s Hospital of Los Angeles and University of 

Southern California School of Medicine, Los Angeles, CA, 

Chaim Jacob, FOCIS Center, University of Southern 

California School of Medicine, Los Angeles, CA, Raphael 

Zidovetzki, FOCIS Center, University of Southern California 

School of Medicine, Los Angeles, CA 

We present a novel methodology to maximize the ability of 

microarray based single nucleotide polymorphism (SNP) typing 

platforms to discover genes which are associated with complex 

polygenic diseases when complete genomic coverage is infeasible. 

This methodology was used to identify genetic variants 

which are associated with childhood onset SLE. A platform of 

9412 SNPs corresponding to 1204 genes was selected using 

bioinformatics and expert knowledge and validated. Molecular 

inversion probes and high throughput techniques were used to 

type SNP alleles. A cohort of 753 subjects, corresponding to 251 

full trios (both parents and affected SLE child) were genotyped. 

Utilizing the Transmission Disequilibrium Test (TDT) and appropriate 

multiple comparison corrections, a panel of several novel 

childhood onset SLE genes were determined to be significant. 

Among these, the N673S polymorphism in SELP (P-Selectin) and 

the C203S polymorphism in IRAK1 (Interleukin 1 receptorassociated 

kinase 1) were found with a high degree of 

significance. These results suggest new directions in which to 

expand the understanding of the pathogenesis of SLE. The 

results of this study demonstrate that the novel methodology 

provides a powerful approach which is generally applicable to 

the identification of the genetic foundations of complex 

diseases. 

doi:10.1016/j.clim.2007.03.019 

OR.80 The Molecular Basis by Which Polymorphism 

in the OX40L Gene Predisposes to SLE 

Harinder Manku, Research Fellow, Imperial College London, 

Molecular Genetics and Rheumatology, London, England, 

Deborah Cunninghame Graham, Research Fellow, Imperial 

College London, Molecular Genetics and Rheumatology, 

London, England, Robert R. Graham, Research Fellow, Broad 

Institute of Harvard and MIT, Cambridge, MA, Timothy W. 

Behrens, Genentech Inc., San Francisco, CA, Timothy W. 

Behrens, Adjunct Professor, University of Minnesota, 

Department of Medicine, Minneapolis, MN, David 

Althshuler, Associate Professor, Broad Institute of Harvard 

and MIT, Cambridge, MA, Timothy J. Vyse, Reader and 

Honorary Consultant, Imperial College London, Department 

of Molecular Genetics and Rheumatology, London, England 

SLE is a complex polygenic disease where immune dysregulation 

results in chronic activation of B lymphocytes and 

autoantibody production against a variety of nuclear antigens. 

OX40L, a type II membrane glycoprotein found on the surface of 

activated B lymphocytes, interacts with OX40 on T lymphocytes 

to amplify T-dependent B cell proliferation and differentiation. 

A family-based association analysis using a set of 45 SNP 

markers across OX40L was conducted in 472 UK and 429 US SLE 

parent-offspring trios. We identified an associated promoter 

haplotype block (32 kb) that was preferentially transmitted to 

affected offspring (P=0.003 UK, P=0.0005 US, P=6.7×10 −6 

combined). A tagging SNP (rs2205960) from this haplotype 

showed significant association with SLE in an independent set of 

310UKcasesand642controls(P= 1.3×10 −5 OR=1.55, 95% 

CI=1.27–1.89). In order to investigate the molecular basis of the 

genetic association, we studied OX40L expression in individuals 

selected on the basis of allelic composition at the OX40L 

promoter. The promoter polymorphisms associated with SLE 

correlated with increased expression levels of OX40L. This was 

shown by three-colour flow cytometry of 16 EBV-transformed 

cell lines: those homozygous for the over-transmitted haplotype 

exhibited increased expression relative to the under-transmitted 

homozygote. We further show that the disease susceptibility 

alleles in the OX40L promoter were associated with an 

eight-fold increase in RNA expression (P=0.008). The mechanism 

by which increased OX40L expression increases the 

susceptibility to SLE remains to be established, but is the 

subject of ongoing investigation. 

doi:10.1016/j.clim.2007.03.020 

OR.81 Genetic Association of IL-21 Polymorphisms 

with Systemic Lupus Erythematosus 

Amr H. Sawalha, Assistant Professor, Department of 

Medicine, Oklahoma University Health Sciences Center, VA 

Medical Center, Oklahoma Medical Research Foundation, 

Oklahoma City, OK, Kenneth Kaufman, Senior Research 

Scientist, Oklahoma Medical Research Foundation, 

Oklahoma City, OK, Jennifer Kelly, Senior Research 

Assistant, Oklahoma Medical Research Foundation, 

Oklahoma City, OK, Teresa Aberle, PA-C, Oklahoma Medical 

Research Foundation, Oklahoma City, OK, Adam Adler, 

Research Associate, Oklahoma Medical Research 

Foundation, Oklahoma City, OK, Jeff Kilpatrick, Computer 

Programmer/Data Analyst, Oklahoma Medical Research 

Foundation, Oklahoma City, OK, Edward Wakeland, 

Professor, UT Southwestern, Center of Immunology, Dallas, 

TX, Quan-Zhen Li, Assistant Professor, Center for 

Immunology, University of Texas Southwestern Medical 

Center, Dallas, TX, Amy Wandstrat, Instructor, UT 

Southwestern, Rheumatology, Dallas, TX, Judith James, 

Professor/Member, Oklahoma Medical Research 

Foundation/ Arthritis and Immunology, Oklahoma City, OK, 

Joan Merrill, Member, Oklahoma Medical Research 

Foundation, Clinical Pharmacology, Oklahoma City, OK, 

Peter Lipsky, Chief, NIAMS, Bethesda, MD, John B. Harley, 

Professor and Department Head, Oklahoma University 

Health Sciences Center/Oklahoma Medical Research 

Foundation, Oklahoma City, OK 

Purpose: The etiology of systemic lupus erythematosus (SLE) 

is incompletely understood. Both genetic and environmental 

factors are implicated in the pathogenesis of the disease. 

Herein, we describe genetic association between SLE and 

polymorphisms in the IL-21 gene. The reported effects of IL-21 

on B cell differentiation into plasma cells, as well as its effect 

on dendritic cell maturation and T cell response, make IL-21 an 

attractive SLE candidate gene. Methods: A genetic association 

study was performed by genotyping three single nucleotide 

polymorphisms (SNPs) in the IL-21 gene in a total of 2636 

samples (1318 independent SLE cases and 1318 unrelated 

controls matched for age, sex and race). Genotyping was 

performed on the Illumina BeadStation 500GX system at the

Abstracts 

University of Texas Southwestern Microarray Core Facility 

(Dallas, TX). Population-based case-control association designs 

were employed. Results: Two SNPs located within intronal 

regions of IL-21 are associated with SLE (rs907715: chi2=11.55, 

p=0.00068; rs2221903: chi2=5.49, p=0.019). Furthermore, 

genotypes homozygous for the risk alleles were more frequent 

than genotypes homozygous for the non-risk alleles in 

European-American patients as compared to controls 

(rs907715 (GG versus AA): Odds ratio=1.66, p=0.0049; 

rs2221903 (GG versus AA): Odds ratio=1.60, p=0.025). Conclusion: 

We report an association between polymorphisms in the 

IL-21 gene and SLE. The functional effects of IL-21 polymorphisms, 

when revealed, might improve our understanding of SLE 

and provide new therapeutic targets. 

doi:10.1016/j.clim.2007.03.021 

OR.82 Both Shared and Strain-specific Genetic Loci 

Control Susceptibility to Virus-induced Autoimmune 

Diabetes in Two Rat Models 

Elizabeth Blankenhorn, Professor, Drexel University College 

of Medicine, Philadelphia, PA, Laura Cort, Research 

Assistant, Department of Microbiology and Immunology, 

Philadelphia, PA, Daniel Cheeran, Research Assistant, 

Department of Microbiology and Immunology, Philadelphia, 

PA, Rebecca Tirabasi, Research Scientist, BRM, Inc., 

Worcester, MA, Elaine Norowski, Research Assistant, 

Diabetes Division, Worcester, MA, Dennis Guberski, 

President, BRM, Inc., Worcester, MA, John Mordes, 

Professor, Diabetes Division, Worcester, MA 

Viral infection may be important in the pathogenesis of 

human type 1 diabetes (T1DM). MHC congenic (RT1u/u/a) 

LEW.1WR1 rats develop spontaneous T1DM at a low 

frequency (∼2%) and are also susceptible to the induction 

of diabetes in response to infection with a parvovirus, Kilham 

rat virus (KRV). An injection of 1×10 7 PFU KRV induces 

diabetes in ∼40% of animals, and co-injection of a low dose 

of poly I:C with the KRV increases disease penetrance to 

100%. KRV does not infect beta cells. In their susceptibility to 

KRV-T1DM, LEW.1WR1 rats resemble BBDR (RT1u/u/u) rats, 

but WF strain rats (also RT1u/u/u) are resistant to the 

induction of diabetes by this virus. In crosses of (BBDR×WF) 

rats, we have mapped two quantitative trait loci (QTL) that 

determine susceptibility to KRV-poly I:C induced T1DM, 

Iddm14 and Iddm20. We have now mapped the QTL required 

for diabetes in (LEW.1WR1 ×WF) rats. We show that Iddm14 

allele from the diabetes-susceptible LEW.1WR1 strain is 

required for both insulitis and diabetes (46/47 rats with 

diabetes had Iddm14d/−). However, Iddm20 is not a determinant 

of either insulitis or diabetes in this strain combination. 

Instead, markers on chromosome 20 near RT1 show 

significance. These data demonstrate that virus-specific 

diabetogenic responses differ between LEW.1WR1 and BBDR 

rats, even though both are susceptible to KRV. A genome scan 

for LEW.1WR1-specific QTL will be reported. These data shed 

light on light on the factors responsible for the variable 

penetrance of T1D in high risk human populations. 

doi:10.1016/j.clim.2007.03.022 

OR.83 An Association Between the Autoimmune 

Susceptibility Locus CTLA-4 and Alterations in 

Proximal TCR Signaling in Healthy Human 

Volunteers 

Lisa Maier, Neurology Fellow, Center for Neurologic 

Diseases, Boston, MA, David Anderson, Instructor, Center 

for Neurologic Diseases, Boston, MA, Philip de Jager, 

Assistant Professor, Center for Neurologic Diseases, Boston, 

MA, David Hafler, Professor, Center for Neurologic Diseases, 

Boston, MA, Linda Wicker, Professor, JDRF/WT Diabetes and 

Inflammation Laboratory, Cambridge 

With the rapid advancement in high-throughput genotyping 

technologies, an increasing number of genetic variants have 

been associated with susceptibility to human autoimmune 

disease. However, little is known about the functional effects of 

susceptibility variants on human immune cells. The single 

nucleotide polymorphism (SNP) called CT60 (rs3087243), 

located in the 3′ untranslated region of CTLA-4, has been 

associated with many autoimmune diseases, including type 1 

diabetes. The susceptible genotype at this SNP has previously 

been shown to result in a 2-fold lower production of the soluble 

CTLA-4mRNAisoforminnaïveCD4+Tcells.Wewerethus 

interested in whether this or other CT60-associated functional 

consequences would affect TCR signaling, given that CTLA-4 is a 

negative T cell regulator. We have applied a flow-cytometric 

technique that measures phosphoproteins to the study of 

proximal TCR signaling in naïve and memory CD4+ T cells using 

fresh whole blood stimulated with anti-CD3 monoclonal antibody, 

and have measured CD3z (pY142), LAT (pY171), LCK 

(pY505), ZAP70 (pY292), ZAP70 (pY319/Syk (pY352)) and SLP-76 

(pY128) in healthy human volunteers. By categorizing results 

obtained from TCR stimulation into the three genotype classes; 

AA, AG and GG, we observed no differences in TCR responses 

with CD4+CD45RA+ T cells. However, we observed that relative 

to the CD45RA+ subset, CD4+CD45RA- T cells from individuals 

with the susceptibility allele are less responsive in all TCR 

signaling molecules examined. These data contribute to our 

understanding of the link between the susceptibility genotype 

at CTLA4 and the mechanisms involved that may result in 

autoimmunity. 

doi:10.1016/j.clim.2007.03.023 

S135 

OR.84 Variants of CARD15/NOD2 Associated with 

Crohn’s Disease and Blau Syndrome Differ from Wild 

Type NOD2 in Regulating Cytokine Secretion 

Induced by TLR2 and TLR4 Agonists in a Transduced 

Macrophage Cell Line 

Michael Davey, Professor of Medicine, Portland VA Medical 

Center and OHSU, Portland, OR, Zili Zhang, Assistant 

Professor, OHSU, Pediatrics, Portland, OR, Tammy Martin, 

Associate Professor, OHSU and CEI, Portland, OR, Holly 

Rosenzweig, Postdoctoral Fellow, OHSU, Casey Eye 

Institute, Portland, OR, Steven Planck, Professor, OHSU and 

CEI, Portland, OR, James Rosenbaum, Professor, OHSU and 

CEI, Portland, OR 

NOD2 mutations are associated with Crohn's disease and 

Blau syndrome. NOD2 senses muramyl dipeptide (MDP) and


activates NF-kB. Studies in NOD2 deficient mice found that 

NOD2 suppresses TLR2 cytokine responses. Using cells overexpressing 

NOD2, this study further explored the regulatory 

role of NOD2. Murine mononuclear cells (J774 line) were 

transduced with constructs containing murine wild type (WT) 

NOD2, the murine equivalent of common Blau (BL) (R314Q) 

and Crohn's mutations (frame shift [fs] at L980) or an empty 

retroviral vector control (VC). Cells were stimulated with 

TLR2 (PAM3CSK4) or TLR4 (LPS) agonists with or without MDP, 

and secretion of IL-6, IL-10, IL-12p40 and TNF-α was 

measured. Over-expression of WT, BL and fs variants of 

NOD2 suppressed IL-10 secretion and enhanced TNF-α 

secretion in response to TLR2 and TLR4 agonists. Both 

agonists suppressed IL-6 and IL-12p40 secretion in WT cells 

but enhanced secretion from BL and fs cells. MDP and TLR 

ligands given simultaneously to VC cells enhanced secretion 

of all cytokines studied. Synergy did not occur or was 

suppressed in WT cells, while it was enhanced in BL and fs 

cells. These data confirm the regulatory role of NOD2 on TLR2 

signaling and show TLR4 pathways can also be regulated. The 

differences observed between suppression and enhancement 

of cytokine secretion with WTcompared to BL and fs variants 

of NOD2 may be in vitro correlates of processes linking these 

mutations with disease. Elucidating mechanisms responsible 

for these different patterns may enhance our understanding 

of Crohn's disease and Blau syndrome. 

doi:10.1016/j.clim.2007.03.024 

OR.85 A Genome-wide Assessment of the Genetic 

Basis of Type 1 Diabetes 

Vincent Plagnol, Mr, JDRF/WT DIL, University of Cambridge, 

Cambridge, England, David Clayton, Professor, JDRF/WT 

Diabetes and Inflammation Laboratory, Cambridge, 

England, David Dunger, Department of Pediatric, University 

of Cambridge, Cambridge, England, Kate Downes, JDRF/WT 

Diabetes and Inflammation Laboratory, Cambridge, 

England, Hin-Tak Leung, JDRF/WT Diabetes and 

Inflammation Laboratory, Cambridge, England, Deborah 

Smyth, JDRF/WT Diabetes and Inflammation Laboratory, 

Cambridge, England, Helen Stevens, JDRF/WT Diabetes and 

Inflammation Laboratory, Cambridge, England, Neil Walker, 

Mr, JDRF/WT Diabetes and Inflammation Laboratory, 

Cambridge, England, WTCCC, Wellcome Trust Case Control 

Consortium, http://www.wtccc.org.uk/, Wellcome Trust, 

Cambridge, England, John Todd, Professor, JDRF/WT 

Diabetes and Inflammation Laboratory, Cambridge, England 

The strong familial clustering of autoimmune type 1 diabetes 

(T1D) in families remains only partially explained. The six 

confirmed genes or chromosome regions with convincing 

statistical support in large, and multiple, populations, namely 

the major histocompatibility complex (MHC), the insulin gene 

(INS), CTLA-4, PTPN22, IL2RA/CD25, and IFIH1/MDA5 can 

explain only about 50% of familial aggregation. Nonetheless, 

their identification has provided many insights into the biology 

and pathways of T1D. Recent expansions in sample collections, 

including the Juvenile Diabetes Research Foundation/Wellcome 

Trust Diabetes and Inflammation Laboratory (JDRF/WT DIL) 

British T1D case sample (n= 8000), and advances in affordable, 

genome-wide, high throughput single nucleotide polymorphism 

(SNP) genotyping technologies (Affymetrix), has allowed an 

association analysis of 500,000 SNPs across the genome in 2000 

T1D cases and 3000 controls as part of the Wellcome Trust Case- 

Control Consortium (WTCCC). Attesting to the quality of the 

SNP map, positive results were obtained for all six susceptibility 

loci reported previously. However, follow-up genotyping studies 

of 14 other chromosome regions showing Pb10 −5 in the WTCCC 

scan in over 6000 cases and 6000 controls, and in approximately 

2000 families, supported in a convincing way (Pb10 −15 and odds 

ratios approximately 1.2) four regions associated with T1D in 

both the family and case-control samples, on chromosomes 

16p13, 18p11, 12q13 and 12q24. These results illustrate the 

power of genome-wide association approach to characterise 

the genetic basis of T1D. 

doi:10.1016/j.clim.2007.03.025 

Cytokine/Chemocine Regulators of 

Inflammation & Allergy 


2:45 pm−4:45 pm 

OR.86 Human B Cell Cytokine Regulation and 

Multiple Sclerosis (MS) 

Christine Ghorayeb, McGill University/Immunology, 

Montreal, QC, Masaaki Nii Hokkaido, University Hospital/ 

Neurology, Sapporo, Japan, Claudia Calder, McGill 

University/Neuroimmunology, Montreal, QC, Canada, Amit 

Bar-Or, McGill University/Neurology and Neurosurgery, 

Montreal, QC, Canada 

B cells are increasingly recognized for roles in both normal 

immune responses and in autoimmune diseases including MS. 

We recently reported that normal human B cells can produce 

either pro-inflammatory (TNFá; Lymphotoxin/LT) or regulatory 

(IL-10) effector cytokines, depending on their sequence 

of activation. We further identified that MS patient B cells 

exhibit deficient expression of IL-10, implicating a B cell 

abnormality in failed immune regulation. Here, we asked (i) 

whether and how the local cytokine milieu may modulate the 

profile of the normal effector B cell cytokine network, and 

(ii) whether such modulation is different for MS B cells. 

Specifically, we added either IFNγ (Th1 milieu) or IL-4 (Th2 

milieu) during our established ex vivo paradigm of B cell 

activation. The presence of IFNγ induced normal B cells to 

secrete increased levels of LT (429±44; versus 259±28 pg/ 

ml; n=15; p=0.0002) and TNFá (274±44 versus 144±30 pg/ 

ml; p=0.0003). IL-4 also induced increased levels of LT (419± 

47 pg/ml, p=0.0001) and of TNFá (366±51, pb0.0001), 

while significantly suppressing secretion of IL-10 (37±11 

versus 145 ±26 pg/ml; pb0.0001). We further discovered 

that in the ‘Th1 milieu’, MS B cells produced significantly 

higher levels of LT (average increase 28%; n=22, p=0.0069) 

and TNFá (26%, p=0.0049), compared to normal B cells. We 

demonstrate that a novel regulatory network of effector 

cytokines in normal B cells is significantly modulated 

depending on the local cytokine milieu. Furthermore, in a 

'Th1 milieu', MS B cells secrete abnormally high levels of TNFá

Abstracts 

and LT, which would contribute to the local inflammatory 

response. 

doi:10.1016/j.clim.2007.03.026 

OR.87 Immune Mediated Protection in Multiple 

Sclerosis: A Role for Neurokines in Oligodendrocyte 

Survival and Macrophage Modulation 

Niels Hellings, PhD, Hasselt University, Diepenbeek, 

Belgium, Niels Hellings, PhD, Hasselt University, BIOMED, 

Diepenbeek, Belgium, Jerome JJA Hendriks, PhD, Hasselt 

University, BIOMED, Diepenbeek, Belgium, Sofie Carmans, 

MsC, Hasselt University, BIOMED, Diepenbeek, Belgium, 

Leen Slaets, MsC, Hasselt University, BIOMED, Diepenbeek, 

Belgium, Debora Dumont, PhD, Hasselt University, BIOMED, 

Diepenbeek, Belgium, Joris Vanderlocht, PhD, Hasselt 

University, BIOMED, Diepenbeek, Belgium, Piet Stinissen, 

PhD, Hasselt University, BIOMED, Diepenbeek, Belgium 

In multiple sclerosis (MS), immune mediated destruction 

of the myelin sheath, oligodendrocytes and axons leads to 

irreversible neurological deficits. Recent data show that 

immune responses in the central nervous system (CNS) may 

also confer protective effects. We recently demonstrated 

that autoreactive T cells and macrophages produce 

leukemia inhibitory factor (LIF), a member of the IL-6 

family of neurokines. CD4+ T cells from relapsing remitting 

MS-patients show a reduced LIF production. Still, LIF 

immunoreactivity is detected among T cells and perivascular 

macrophages in MS lesions. In rat oligodendrocyte 

cultures, LIF protects against TNF-α and IFN-γ induced 

apoptosis, but not against other cell death mediators 

(oxidative stress, staurosporin). LIF receptor signalling in 

oligodendrocytes does not induce the expression of 

suppressors of cytokine signalling (SOCS) or the antiapoptotic 

molecules Bcl-2/Bcl-XL. Using quantitative proteomics, 

we demonstrate that LIF leads to upregulation of 

a panel of proteins that have pro-survival functions. Future 

experiments are needed to study their role in LIF mediated 

oligodendrocyte protection. Since macrophages, but not T 

cells, express functional LIF-receptor complexes we studied 

whether LIF also exerts immunomodulatory functions. 

LIF significantly upregulates myelin phagocytosis and 

reduces the production of reactive oxygen species and 

TNF-α in vitro. In summary, neurokines may act on various 

levels during CNS inflammation: they may modulate 

immune responses and protect CNS resident cells under 

attack. Further clarification in the actions of different 

neurokine members during neuroinflammation may lead to 

new therapeutic strategies for MS. 

doi:10.1016/j.clim.2007.03.027 

OR.88 ALCAM is a Novel Adhesion Molecule of the 

Inflammed Endothelium Involved in Leukocyte 

Trafficking to the Central Nervous System 

Romain Cayrol, PhD Student, Université de Montreal, 

Montreal, QC, Canada, Aurore Dodelet-Devillers, M.Sc. 

Student, Université de Montreal, Montreal, QC, Canada, Igal 

Ifergan, PhD Student, Université de Montreal, Montreal, QC, 

Canada, Arsalan Haqqani, PhD Student, National Research 

Council, Institute for Biological Sciences, Ottawa, ON, 

Canada, Hania Kebir, PhD Student, Université de Montreal, 

Montreal, QC, Canada, Danica Stanimirovic, Professor, 

Institute for Biological Sciences; National Research Council of 

Canada, Ottawa, ON, Canada, Alexandre Prat, Clinician and 

Professor, Université de Montreal, Montreal, QC, Canada 

Leukocyte trafficking from the blood to local inflammatory 

sites is essential for the initiation and maintenance of tissue 

specific immune responses. In autoimmune diseases such as 

multiple sclerosis (MS), leukocyte transmigration from the 

blood to the target organ is dependent on intercellular cell 

adhesion molecule 1 (ICAM-1) and vascular cell adhesion 

molecule 1 (VCAM-1) expressed by the endothelial cells (ECs). 

In this study we describe CD166/activated leukocyte cell 

adhesion molecule (ALCAM) as a novel adhesion molecule of 

the human blood–brain barrier (BBB). We demonstrate that 

inflammatory cytokines up-regulate the expression of ALCAM 

on the surface of BBB-ECs in vitro and that endothelial ALCAM 

co-localizes with leukocyte expressed-CD6 in the transmigratory 

cup during migration. We further show that ALCAM 

blocking antibody restricts the transmigration of CD4, CD14 

and CD19 immune cells across human BBB-ECs and that ALCAM 

expression is increased on ECs within active MS lesions, as 

compared to normal appearing white matter and to non-MS 

brains. These results suggest an important role for ALCAM in 

the recruitment of leukocytes to the CNS and identify ALCAM 

as a potential target to modulate CNS inflammatory reactions. 

doi:10.1016/j.clim.2007.03.028 

S137 

OR.89 Circulation of Membrane-bound TNFa by Way 

of Plasma-derived Exosomes 

Nicole Bianco, Postdoctoral Scholar, University of 

Pittsburgh, Molecular Genetics and Biochemistry, 

Pittsburgh, PA, Seon-Hee Kim, Director of Research, Seoul 

National University, Seoul, South Korea, Paul Robbins, 

Professor, University of Pittsburgh, Molecular Genetics and 

Biochemistry, Pittsburgh, PA, Teresa Hennon, Research 

Fellow, University of Pittsburgh, Molecular Genetics and 

Biochemistry, Pittsburgh, PA 

Exosomes are membrane nanovesicles (50–100 nm diameter) 

generated by reverse budding of the limiting membrane 

of multivesicular late endosomes. Exosomes originating 

from immune cells, such as B cells, T cells, DC, and mast 

cells, are thought to play a role in communicating immunoregulatory 

signals to other cells in either an immuno-stimulating 

or suppressive manner. Since we have demonstrated 

the presence of FasL, a TNF family member, in exosomes, we 

examined whether exosomes also can carry TNFa. To establish 

this, we first generated exosomes from a murine tumor 

cell line overexpressing murine TNFa and demonstrated the 

presence of membrane-bound TNFa (mbTNFa) in exosomes by 

FACS and Western blot analysis. The mbTNFa in exosomes was 

also functional in the L929 cytotoxicity assay. To determine 

whether exosomes containing mbTNFa might circulate 

systemically during active disease, we collected serum from 

WT or IL10−/− mice with inflammatory bowel disease. There


was significantly more mbTNFa in IL10−/− serum exosomes. 

Mice with collagen-induced arthritis also contained elevated 

levels of mbTNFa in their serum exosomes. This research 

implies that mbTNFa, in addition to functioning in a local 

manner by cell-to-cell contact, can also travel either locally 

or systemically to regulate inflammation at distant sites. This 

research also suggests that the bioactive mbTNFa in the 

vesicles may play a role in the pathology of various TNFdependent 

autoimmune diseases and that anti-TNF therapies 

might target mbTNFa carried on microvesicles. Furthermore, 

the level of TNFa in the serum exosomes might be useful as a 

diagnostic marker of certain autoimmune diseases. 

doi:10.1016/j.clim.2007.03.029 

OR.90 Ccr2 Deficiency Impairs Microglial 

Accumulation and Accelerates Progression of 

Alzheimer’s Disease 

Joseph El Khoury, Assistant Professor of Medicine, Harvard 

Medical School, Charlestown, MA, Michelle Toft, Laboratory 

Technician, Massachusetts General Hospital, Charlestown, 

MA, Suzanne Hickman, Scientist, Massachusetts General 

Hospital, Charlestown, MA, Changiz Geula, Professor, 

Harvard Medical School, Charlestown, MA, Terry Means, 

Instructor in Medicine, Massachusetts General Hospital, 

Charlestown, MA, Andrew Luster, Professor of Medicine, 

Harvard Medical School, Charelstown, MA 

Microglia are the principal immune cell of the brain. In 

Alzheimer's disease (AD), microglia accumulate in senile 

plaques and may in part be recruited from peripheral blood. 

The role of microglia in AD pathogenesis has not been 

resolved. Microglia may play a neuroprotective role by phagocytosing 

beta amyloid, a pathogenic protein deposited in 

AD brains. But their activation and subsequent secretion of 

neurotoxins may also lead to neurodegeneration. Chemokine 

receptor 2 (Ccr2) is a chemokine receptor expressed on 

microglia that mediates accumulation of leukocytes at sites 

of inflammation. Transgenic mice expressing a mutated form 

of the human amyloid precursor protein (APP) are a robust 

model of AD used to study the role of microglia in AD. To 

determine the role of Ccr2 in microglial accumulation in AD, 

we generated transgenic APP mice deficient in Ccr2 and 

analyzed these mice for AD-like pathology. We found that 

Ccr2 deficiency accelerates early disease progression and 

markedly reduces microglial recruitment and accumulation 

to baseline in this transgenic mouse model of AD. APP mice 

deficient in Ccr2 died prematurely in correlation with Ccr2 

gene dosage, and accumulated beta amyloid in their brains 

much earlier than APP mice with normal Ccr2 levels, 

indicating that absence of early microglial recruitment 

leads to decreased beta amyloid clearance. Furthermore, 

Ccr2-deficient mice microinjected with beta amyloid into 

their brains failed to recruit microglia to the sites of 

injection. Thus, Ccr2-dependent microglial accumulation 

plays a protective role in the early stages of AD by promoting 

clearance of beta amyloid. 

doi:10.1016/j.clim.2007.03.030 

OR.91 Chemokine-like Receptor-1 (CMKLR1) Regulates 


Kareem L. Graham, Postdoctoral Scholar, Department of 

Pathology, Stanford, CA, Luis A. Zuniga, Graduate Student, 

Department of Pathology, Stanford, CA, Brian A. Zabel, 

Postdoctoral Fellow, Department of Pathology, Stanford, 

CA, Eugene C. Butcher, Professor of Pathology, Department 

of Pathology, Stanford, CA, Raymond A. Sobel, Professor of 

Pathology, Department of Pathology, Stanford, CA 

The pathology of multiple sclerosis (MS) involves leukocyte 

extravasation of the blood–brain barrier and associated myelin 

damage, which leads to impaired nerve function and paralysis. 

Chemokines, adhesion molecules, and their receptors have been 

implicated in recruitment of inflammatory cells to the central 

nervous system (CNS) during MS. However, the mechanisms that 

regulate migration of various leukocyte subsets to the CNS 

remain poorly understood. Chemokine-like receptor-1 (CMKLR1, 

also known as ChemR23 or Dez) is a recently de-orphaned 

chemoattractant receptor that has emerging roles in macrophage 

migration during inflammatory processes. In this study, 

we examined the role of CMKLR1 in experimental autoimmune 

encephalomyelitis (EAE), a mouse model of human MS. We found 

that mice deficient in CMKLR1 are resistant to progressive EAE. 

CMKLR1-deficient mice had reduced inflammatory infiltrates in 

the brain and spinal cord, with especially marked differences in 

the CNS parenchyma. While transcripts for CMKRL1 have been 

detected in a variety of tissues, experiments with bone marrow 

chimeric mice indicate that CMKLR1 expression on cells of 

hematopoietic origin is required for maximal expression of EAE. 

Together, the data demonstrate that CMKLR1 regulates a model 

of autoimmune demyelinating disease and suggest that CMKLR1 

may be a potential target in blocking development of 

progressive EAE/MS. 

doi:10.1016/j.clim.2007.03.031 

OR.92 Cell-type Specific Changes in Cytokine-STAT 

Signal Transduction Stage Systemic Lupus 

Erythematosus 

Matthew, Postdoctoral Fellow, Stanford University, 

Department of Microbiology and Immunology, Stanford, CA, 

Peter Krutzik, Research Associate, Stanford University, 

Department of Microbiology and Immunology, Stanford, CA, 

Garry Nolan, Professor, Stanford University, Department of 

Microbiology and Immunology, Stanford, CA 

The regulation of cytokine signal transduction is thought to 

be critical in shaping the character and duration of immune 

responses, but little is known of its role in autoimmune disease. 

We applied phospho-specific flow cytometry to generate a 

highly multiplexed view of how Systemic Lupus Erythematosus 

(SLE) disturbs cytokine signaling through the STAT family of 

transcription factors. Using a panel of 10 cytokines previously 

suggested to have causal roles in the disease, we measured 

signaling responses at the single-cell level in multiple immune 

cell types from human SLE patients with a range of disease 

activity, as well as followed disease progression in the MRLlpr 

murine model. Unexpected changes in subset responses to IFNα, 

IFNγ, IL-4, IL-6, IL-15 and IL-21 were observed. Suppressor of 

cytokine signaling 1 (SOCS1) levels were elevated in PBMC from

Abstracts 

human patients as well as in lymphocytes from the murine model 

where SOCS1 was implicated in pathway-specific inhibition of 

responses to IL-6 in T cell subsets. Not only did aberrant signal 

transduction distinguish SLE patients from normal donors, but 

robustly significant differences in certain subsets discriminated 

patients experiencing flare from those with low levels of disease 

activity. Importantly, cell-type specific regulation of cytokine 

signaling may mark an important transition between flare and 

remission in humans. These results provide unique mechanistic 

insights into the signaling disruptions that occur during SLE, 

demonstrate that this approach can rapidly relate disease status 

to signaling network changes, and suggest it will be possible to 

stratify patients with greater accuracy. 

doi:10.1016/j.clim.2007.03.032 

OR.93 Accelerated Development of 

Glomerulonephritis in TNF Receptor 1 and 2 

Double-knockout Lupus-mice 

Noam Jacob, Research Fellow, University of Southern 

California Keck School of Medicine, Los Angeles, CA, 

Luminita Pricop, Associate Professor of Medicine, Hospital 

for Special Surgery, New York, NY, Chaim Putterman, 

Associate Professor, Albert Einstein College of Medicine/ 

Department of Medicine, Bronx, NY, Chaim O. Jacob, 

Associate Professor of Medicine, University of Southern 

California Keck School of Medicine/Department of 

Medicine, Los Angeles, CA, Michael Koss, Professor of 

Pathology, University of Southern California Keck School of 

Medicine/Department of Pathology, Los Angeles, CA 

We have developed lupus-prone (NZB×NZW)F1-derived 

congenic New Zealand Mixed (NZM) 2328 lines that are deficient 

in TNF receptor 1 (TNFR1), TNF receptor 2 (TNFR2) or in both 

TNFR1 and TNFR2. While the development of lupus-nephritis in 

TNFR2 deficient mice was very similar to wild type, TNFR1 

deficient mice had only a slightly delayed disease development 

compared to TNF receptors intact NZM 2328 controls. On the 

other hand, mice that lacked both TNFR1 and TNFR2 developed 

a significantly accelerated lupus-nephritis and increased 

mortality associated with increased levels of IgG anti-doublestranded 

DNA autoantibodies both in the periphery and in the 

glomerular deposits. Double knockout mice have significantly 

enlarged spleens compared to all other lines, which is 

associated with increased number of B and T lymphocytes 

mostly of CD4+ subsets. Most notably, double KO mice have a 3– 

5 fold increase in the number of activated memory Tcells (CD4+ 

CD25− CD44high CD62Llow) while TNFR1 deficient and TNFR2 

deficient are not different from each other. Immunostaining of 

spleens shows spontaneous germinal center formation in wild 

type NZM 2328 and TNFR2 deficient mice. TNFR1 deficient mice 

have reduced germinal center formation at 2 months and still at 

6 months of age, while double KO mice have reduced GC 

formation at 2 months but by 6 months have as much or more 

than the control mice. These results suggest a fine and complex 

balance between the two TNF receptors and underscore the 

need for both receptors in the immune development of 

autoimmune kidney disease. 

doi:10.1016/j.clim.2007.03.033 

OR.94 Protective and Therapeutic Role for α 

B-Crystallin in Autoimmune Demyelination 

Shalina Ousman, Stanford University, Stanford, CA, Beren 

Tomooka, Mr. Stanford University, Division of Immunology 

and Rheumatology, Palo Alto, CA, Johannes Van Noort, 

Department of Biosciences, Leiden, The Netherlands, Kevin 

O’Conner, Harvard Medical School, Brigham and Women’s 

Hospital, Boston, MA, Eric Wawrousek, National Eye 

Institute, Bethesda, MD, David Hafler, Harvard Medical 

School, Brigham and Women's Hospital, Boston, MA, 

Raymond Sobel, Department of Pathology, Stanford 

University, Palo Alto, CA, William Robinson, Division of 

Immunology and Rheumatology, Stanford University, 

Stanford, Palo Alto, CA, Lawrence Steinman, Professor, 

Department of Neurology and Neurological Studies, 

Stanford University, Stanford, CA 

Alpha B-crystallin (±BC) is the major target of the immune 

response to myelin from multiple sclerosis (MS) and control 

brain. It is the most abundant transcript uniquely present in 

MS lesions and, recent studies have identified anti-apoptotic 

and anti-inflammatory roles for±BC. We hypothesize that 

this crystallin plays a key protective role in demyelinating 

disease. ±BC−/− mice displayed worse symptoms of experimental 

allergic encephalomyelitis (EAE), the animal model of 

MS, with higher Th1 and Th-17 cytokine secretion, proliferation 

and p38 signaling by their T cells and macrophages and, 

more intense CNS inflammation and demyelination compared 

to their WT counterparts. Glial cells from ±BC−/− mice also 

expressed more cleaved caspase-3, TUNEL staining and 

enhanced ERK and NFkB signaling. These results indicated 

that ±BC plays a protective role in curbing both glial cell 

death and immune cell activation. Of interest, myelin antigen 

arrays identified antibodies to native ±BC and to p21–40 

and p116–135 of ±BC in the cerebrospinal fluid of patients 

with relapsing remitting MS (RRMS). These antibodies could 

neutralize ±BC function. We then assessed whether recombinant 

±BC itself could resolve disease in mice with ongoing 

EAE. Remarkably, clinical disease was ameliorated in ±BCtreated 

animals along with decreased Th1 and Th17 cytokine 

production and glial cell death. The immune response against 

a negative regulator of inflammation in demyelinating 

disease, would promote inflammation and demyelination, 

and this can be countered therapeutically by giving ±BC itself. 

Supported by NIH, National Multiple Sclerosis Society and 

Multiple Sclerosis Society of Canada. 

doi:10.1016/j.clim.2007.03.034 

Regulating Inflammation 


2:45 pm−4:45 pm 

S139 

OR.95 Defect in TIM-3 Regulation of CD4+ T Cells in 

a Human Autoimmune Disease 

Li Yang, Postdoctoral Fellow, Center for Neurologic 

Diseases, Harvard Medical School, Boston, MA, David E. 

Anderson, Instructor, Center for Neurologic Diseases, 

Harvard Medical School, Boston, MA, Vijay K. Kuchroo, 

Professor, Center for Neurologic Diseases, Harvard Medical


School, Boston, MA, David A. Hafler, Professor, Center for 

Neurologic Diseases, Harvard Medical school, Boston, MA 

T cell immunoglobulin- and mucin-domain-containing molecules-3 

(Tim-3), a T helper type 1 (Th1)-specific cell surface 

molecule, functions as an inhibitory molecule in mice. However, 

lessisknownaboutthefunctionofTIM-3inhumans.Wehave 

previously shown that reduction of TIM-3 expression on ex vivo 

human CD4+ T cells by small interfering RNA (siRNA) oligos 

increases cell proliferation and IFN-γ secretion, which confirms 

a negative role of TIM-3 in regulation of human T cell function. 

We now report the results of a study that interrogated the 

function of TIM-3 regulation of CD4+ T cells in the autoimmune 

diseasemultiplesclerosis(MS).Westimulatedex vivo CD4+ T 

cellsfrompatientswithMS(n=54) and healthy subjects (n=37) 

with anti-CD3/CD28 monoclonal antibodies in the presence or 

absence of anti-TIM-3 blocking antibody. Our data indicate that 

in the presence of anti-TIM-3 monoclonal antibody treatment 

there is a significant increase of IFN-γ secretion from CD4+ T 

cells in healthy subjects but not in untreated patients with MS. 

Analysis of the kinetics of TIM-3 expression demonstrates lower 

steady state levels of TIM-3 and delayed kinetics of upregulation 

on T cells present in patients with MS relative to 

healthy subjects. Interestingly, T cells obtained from patents 

treated with interferons restored TIM-3 function, as IFN-γ 

secretion in interferons-treated patients is increased with TIM3 

blockade when activated at lower doses of anti-CD3. Taken 

together, our results demonstrate that there is a significant 

defect in TIM-3 regulation of CD4 Tcells in a human autoimmune 

disease. 

doi:10.1016/j.clim.2007.03.035 

OR.96 Increasing GABA Activity Prevents 

Autoimmune Neuroinflammation 

Roopa Bhat, Post Doctoral Fellow, Stanford University, 

Neurology and Neurological Sciences, Stanford, CA, 

Christopher Lock, Vice President, Research and Development, 

Carantech BioSciences, Inc. San Francisco, CA, Lawrence 

Steinman, Professor and Chair, Stanford University, 

Interdepartmental Program in Immunology, Stanford, CA 

Gamma-amino butyric acid (GABA) is the principal inhibitory 

neurotransmitter in the central nervous system (CNS). GABA is 

made by glutamic acid decarboxylase (GAD) and catabolized by 

GABA transaminase (GABAT). Using transcriptional microarrays, 

we showed that, in acute/active inflammatory brain lesions in 

multiple sclerosis (MS) patients, GABAT is upregulated 38-fold 

while specific GABA receptor subunits and GAD are downregulated 

(Lock et al., Nat Med 8: 500–508, 2002). GABA 

inhibits the development of autoimmunity, pro-inflammatory T 

cell responses, and disease progression in the mouse model of 

autoimmune type1 diabetes (Tian et al., JImmunol173: 5298– 

5304, 2004). We, therefore, hypothesized that GABA ameliorates 

autoimmune inflammation in MS. To test this hypothesis, 

we used the mouse model of MS, experimental autoimmune 

encephalomyelitis, and pre-treated daily with vigabatrin, 

which irreversibly inhibits GABAT. We found that vigabatrin 

ameliorated paralysis significantly: disease incidence 

decreased by 64%, while the severity and number of relapses 

diminished by 10-fold and 72±14% (SEM) respectively. Topi- 

ramate, which binds the GABA-A receptor, also prevented CNS 

autoimmunity in a similar manner. This indicates a common 

mechanism that acts by increasing GABA-ergic function 

whether that be through the GABA metabolic or signaling 

pathway. Both agents suppressed myelin-specific T cell 

proliferation and inhibited the production of TNF, IFN- 3 ,IL-17 

and IL-6. Inherent abnormalities in the GABA metabolic and 

signaling pathway may play a previously unrecognized role in 

the pathogenesis of MS. Increasing GABA-ergic function using 

agents such as those in our study could be beneficial in the 

prevention of autoimmune disorders such as MS. 

doi:10.1016/j.clim.2007.03.036 

OR.97 Cyclooxygenase-2 Deficiency has a Protective 

Function in Liver Ischemia/Reperfusion Injury 

Takashi Hamada, Postdoctoraltoral Fellow, The 

Dumont-University of California, Los Angeles Transplant 

Center, Los Angeles, CA, Armine Avanesyan, Medical 

Student, The Dumont-University of California, Los Angeles 

Transplant Center, Los Angeles, CA, Sei-ichiro Tsuchihashi, 

Postdoctoral Fellow, The Dumont-University of California, 

Los Angeles Transplant Center, Los Angeles, CA, Carolina 

Moore, Student, The Dumont-University of California, Los 

Angeles Transplant Center, Los Angeles, CA, Ana Coito, 

Associate Professor, The Dumont University of California, 

Los Angeles Transplant Center, Los Angeles, CA 

Cyclooxygenase-2 (COX-2) is a potent mediator of inflammation. 

In liver ischemia/reperfusion (I/R) injury, COX-2 

expression is highly correlated with the degree of tissue 

damage. This work tests the hypothesis that COX-2 expression 

has a critical function in the pathogenesis of 

hepatic I/R injury. Methods and Results: COX-2 −/− mice 

(KO) and matched wild-type (WT) (COX-2 +/+) controls 

(n=8/g) were submitted to partial warm ischemia in the 

left/medium hepatic lobes for 90 minutes, followed by 

reperfusion. H&E staining of liver sections in COX-2 −/− 

mice at 24 h post-reperfusion revealed overall good 

histological preservation with modest signs of vascular 

congestion and necrosis, contrasting with significant 

edema, centrilobular ballooning, hepatocellular necrosis 

(N60%), and sinusoidal congestion observed in the WT 

counterparts (score: 1.5 ±1 vs. 3.5 ±0.6; pb0.03). Liver 

function, as evidenced by sGOT levels (IU/L), was improved 

in COX-2 (−/−) mice as compared with WT mice (206±45 vs. 

1244 ±290; pb0.01). MPO activity (U/gm) was also significantly 

lower in COX-2 (−/−) mice (1.2±0.4 vs. 3.4 ±1.5; 

pb0.01). Moreover, TUNEL staining showed reduced levels 

of apoptosis in COX-2 KO livers as compared with controls 

(16.2 ±3.3 vs. 73.6± 7.9, pb0.01). This was correlated with 

significant higher levels of BclXL, an anti-apoptotic member 

of the Bcl-2 family, in the COX-2 (−/−) livers. Conclusion: 

Our findings evidence an important function for COX-2 in 

the pathogenesis of hepatic I/R injury. They provide also 

the rationale for identification of improved therapeutic 

approaches to prevent hepatic I/R injury, and consequently 

for improving the outcome of liver transplantation. 

doi:10.1016/j.clim.2007.03.037

Abstracts 

OR.98 Crosstalk Between Leptin and LPS Signaling 

Pathways to Upregulate CD40 Expression via Akt in 


Queenie Lai Kwan Lam, PhD Candidate, University of Hong 

Kong, Department of Pathology, Hong Kong, Liwei Lu, 

Professor, University of Hong Kong, Department of 

Pathology, Hong Kong 

A body of evidence has demonstrated the regulatory role of 

leptin in the immune system over the past decade but little is 

clear about how it regulates dendritic cell (DC) function. CD40, a 

cell-surface receptor and a costimulatory molecule, is a key 

determinant for the activity of DC in immunity and tolerance. 

We have previously shown that leptin signaling is critically 

involved in DC maturation and survival. In this study, we showed 

that leptin induced CD40 expression synergistically with LPS. In 

an Akt-dependent manner, both leptin and LPS activated the 

downstream transcription factors Nuclear Factor (NF)-kappaBp65 

and Signal Transducer and Activator of Transcription- 

1α (STAT-1α) in DC. Using dominant negative forms of Akt and 

IKK, as well as small interfering RNA (siRNA) for STAT-1α, we 

showed that Akt, STAT-1α and NF-kappaBp65 were important for 

CD40 expression induced by leptin, LPS and their synergistic 

action. Blocking studies demonstrated a crucial role for Akt in 

the translocation of STAT-1α and NF-kappaBp65 to the nucleus 

and activation of the CD40 promoter. Further analysis with 

chromatin immunoprecipitation assay confirmed that leptin 

recruited STAT-1α, NF-kappaBp65, acetylated histone 4 and RNA 

polymerase II to the CD40 promoter in a time-dependent 

manner. Administration of leptin into normal mice significantly 

augmented LPS-induced CD40 expression in DC in the lymph 

nodes. Thus, this study identifies a novel function for leptin to 

act an adjuvant to modulate DC function and provides new 

insight into the new role of leptin in modulating inflammatory 

immune response. 

doi:10.1016/j.clim.2007.03.038 

OR.99 The prolyl isomerase Pin1 regulates ECM 

mRNA expression in primary lung fibroblasts 

Zhong-Jian Shen, PhD, University of Wisconsin Madison, 

Madison, WI, James Malter, Professor, University of 

Wisconsin Madison, Department of Pathology and 

Laboratory Medicine, Madison, WI 

A key pathological feature of asthma is excessive airway and 

parenchymal deposition of extracellular matrix (ECM) protein, 

possibly resulting from reduced catabolism or increased 

production or both. TGF-β1 playsacentralroleintheairway 

remodeling, mediating ECM gene expression by resident 

fibroblasts. The molecular mechanisms underlying this dynamic 

process remain poorly understood. Here, we examined the role 

of the peptidyl-prolyl isomerase (PPIase) Pin1 in ECM expression 

using lung fibroblasts from Pin1 knockout mice. Cells were 

starved before TGF-β1 treatment, and the level of ECM mRNA 

was measured by RT/qPCR. Treatment with TGF-β1 consistently 

increased the level of collagen I, III and V in wild type cells. 

Knockout cells opposed to TGF-β1 however were unable to 

upregulate collagen III mRNA. As matrix metalloproteinase 

(MMP)2and3aremajorisoformsinthelung,whichdegrade 

collagen I/V and collagen III/V, respectively, the level of MMPs 

was also evaluated. In the presence of TGF-β1, the level of 

MMP2 and 3 was considerably increased in Pin1 knockout cells. 

Among the three tissue inhibitor of matrix metalloproteinase 

(TIMP) isoforms (1, 2 and 3), TIMP1 levels were significantly 

reduced in Pin1 knockout cells despite TGF-β1 stimulation. 

Therefore, the decreased expression of type III collagen and 

TIMP1 along with increased MMP2 and 3 in Pin1 knockout 

fibroblasts support our previous finding that in vivo administration 

of Pin1 inhibitors reduced airway fibrosis and suggest that 

Pin1 may be a target for the treatment of asthma and other 

fibrotic disorders as well. 

doi:10.1016/j.clim.2007.03.039 

OR.100 Genetic Evidence for CD8 + T Cell 

Immunoregulation in Murine CD4 + T Cell Colitis 

Bo Wei, Assistant Professor, Department Pathology and Lab 

Medicine, University of California, Los Angeles David Geffen 

School of Medicine, Los Angeles, CA, Michael Macpherson, 

Graduate Student, Department Pathology and Lab Medicine, 

University of California, Los Angeles David Geffen School of 

Medicine, Los Angeles, CA, Daisuke Fujiwara, Postgraduate 

Researcher, Department Pathology and Lab Medicine, University 

of California, Los Angeles David Geffen School of Medicine, Los 

Angeles, CA, Sarah Brewer, Graduate Student, University of 

California, Los Angeles David Geffen School of Medicine, Los 

Angeles, CA, Jonathan Braun, Professor, Department Pathology 

and Lab Medicine, University of California, Los Angeles David 

Geffen School of Medicine, Los Angeles, CA 

Abnormal CD4 + Tcell activity and consequent tissue damage 

are major pathogenic features of inflammatory bowel disease 

(IBD) and many systemic and tissue restricted autoimmune 

diseases. Disease susceptibility may involve deficiency of normal 

mucosal immunoregulation that attenuates CD4 + T cell activity 

targeting the mucosa. CD8 + T cells have emerged as one such 

regulatory population, but their modes of induction and 

targeting are poorly understood. We have demonstrated that 

B cells attenuate immune colitis through the interaction with 

CD8 + Tcells.Inthisstudy,weevaluatedthegeneticsofBcell 

traits involved in immunoregulation of G±i2−/− Tcellsinduced 

colitis. Transfer of G±i2−/− Tcells from 4 to 5 weeks mice (prior 

to colitis onset) induced progressive colitis, with additional 

features of systemic immune inflammation. Mesenteric node B 

cells from wild type mice inhibited colitis and systemic 

inflammation, and blocked the attendant CD4 + Tcellexpansion 

expressing TNF- 2 ,IFN- 3 and IL-17. Surprisingly, the protective 

function of B cells did not require genetic sufficiency for MHC 

class II, indicating that they did not directly interact with 

colitigenic or regulatory CD4 + T cells. However, protection 

required both MHC class I and TAP1 sufficiency, indicating that 

they augmented immunoregulation through direct interaction 

with classical peptide-restricted CD8 + T cells. These findings 

provide new evidence for CD8 + T cell immunoregulation in 

colitis, and suggest that functional or numerical deficiencies of 

cooperative B or CD8 + Tcell subpopulations may be susceptibility 

traits, and targets for compensatory therapy, in inflammatory 

bowel disease. Supported by NIH DK4673 and DK69434. 

doi:10.1016/j.clim.2007.03.040 

S141


OR.101 The Galectin-9/TIM-3 Pathway and Human 

Glioma Pathogenesis 

Chengjie Zheng, Student, Harvard University, Boston, MA, 

Juhi Kuchroo, Student, Brigham and Women’s Hospital and 

Harvard Medical School, Boston, MA, David E. Anderson, 

Instructor in Neurology, Brigham and Women's Hospital and 

Harvard Medical School, Boston, MA 

Approximately 15,000 patients die each year in the United 

States from GBMs. Two well-documented aspects of malignant 

gliomas are that they are highly invasive and that immunity 

directed against them is ineffective. We hypothesize that 

glioma expression of galectin-9 (Gal-9) regulates both of these 

aspects of glioma pathogenesis. Gal-9 expression by peripheral 

human tumors has a favorable clinical prognosis and limits 

tumor cell metastasis/invasiveness. Thus, one prediction is 

that down-modulation of Gal-9 in gliomas may contribute to 

tumor cell invasiveness. Indeed, using FACS to isolate purified 

tumor cells or reactive astrocytes from ex vivo tumor speci- 

mens and quantitative RT-PCR, we have found that GBM tumor 

cells express little/no Gal-9 whereas Gal-9 is readily detected 

in reactive astrocytes. Inhibition of TGF-β signaling using 

soluble receptor or siRNA approaches indicates that TGF- 

β is responsible for down-modulation of Gal-9, which 

provides a molecular explanation for the long-standing 

observation that levels of TGF-β increase with grade of 

malignancy and invasiveness. On the other hand, Gal-9 has 

recently been identified as a ligand of TIM-3 and been shown to 

kill IFN-γ-secreting Th1 cells. To this end, co-culture of 

ex vivo CD4+ Tcells with a primary GBM cell line in the presence 

of blocking anti-TIM-3 monoclonal antibody enhances T cell 

proliferation and IFN-γ secretion. Collectively, the data 

suggest a paradigm in which Gal-9 expression by low-grade 

gliomas inactivates infiltrating TIM-3+ CD4+ and CD8+ T cells, 

and that with increasing stages of malignancy, down-modulation 

of Gal-9 promotes tumor cell invasiveness. 

doi:10.1016/j.clim.2007.03.042

Abstracts 

Su.1 Multiple Sclerosis Exacerbations: 

Simultaneous Activation of the Immune System by 

Multiple Immunogens 

Frederick Westall, President and Research Professor, 

Institute for Disease Research, Temecula, CA 

Multiple Sclerosis (MS)is considered to be an autoimmune 

response against central nervous system myelin components. 

Immunogenic mimics on a surface protein of a microorganism 

initiate the immune process. Because of recent data indicating 

that such mimics are plentiful, it is suggested that in MS 

multiple mimics actually are simultaneously processed. Multiple 

simultaneous mimic processing can explain much of the 

confusing immunological data in MS and would parallel what is 

observed in chronic relapsing allergic encephalomyelitis, a 

clinical and histological model for MS. 

doi:10.1016/j.clim.2007.03.043 

Su.2 Comprehensive Molecular Explanation of 

Recent MRI Multiple Sclerosis Studies 

Frederick Westall, President and Research Professor, 

Institute for Disease Research, Temecula, CA 

Recent MRI studies have indicated that MS is a two part 

disease. First, diffuse lesions, apparently initiated by inflammatory 

processes, appear. Some of these then develop into the 

classical immunologically involved focal events. Inflammatory 

agents appear in the CSF and serum as much as a month before 

the focal lesions are seen. One of the earliest of these substances 

observed is metalloproteinase 9. This is followed by caspases and 

plasminogen and eventually various cytokines. Since there is no 

evidence of a chronic microbiological invasion of the CNS in MS, 

what is initiating this early inflammation? The immunological 

component of MS is thought to be caused by a microorganism 

possessing a protein sequence, which mimics an immunological 

active region in myelin. These mimics are not rare. Many 

potential mimics appear in the proteins of normal gut bacteria. 

In fact gut bacteria also contain the adjuvant molecules shown 

essential for autoimmune disease. The initiation of clinical 

autoimmune disease takes weeks. However, it has been clearly 

shown that the adjuvant molecules themselves can quickly 

inititate an inflammatory response. Many of these adjuvant 

molecules, for example lipopolysaccharides, can pass the gut 

barrier. An abnormal digestion in the gut could initiate the 

production of both immunogens and adjuvant molecules. These 

adjuvants could initially cause a general inflammatory response 

and later a specific autoimmune response in the CNS. These 

actions would explain the MRI studies, i.e. a diffuse inflammatory 

response with an occasional autoimmune involvement. 

doi:10.1016/j.clim.2007.03.044 

Poster Session 


7:30 am−7:00 pm 


Su.3 Oral Therapy with FTY720 Inhibits 

Angiogenesis in the Spinal Cord of Lewis Rats in the 

Relapse Phase of Experimental Autoimmune 

Encephalomyelitis 

Peter Hiestand, Senior Research Scientist, Novartis 

Institutes for BioMedical Research, Autoimmune Diseases 

and Transplantation, Basel, Switzerland, Christian Schnell, 

Senior Research Investigator, Novartis Institutes for 

BioMedical Research, Autoimmune Diseases and 

Transplantation, Basel, Switzerland 

Fingolimod (FTY720) is a sphingosine-1-phosphate receptor 

modulator that retards the egress of lymphocytes from 

peripheral lymph nodes. FTY720 has been shown to affect all 

stages of experimental autoimmune encephalomyelitis (EAE) 

in rats as well as in mice either induced with neuronal tissue 

or specific myelin protein antigens. While we have demonstrated 

that monocyte migration into the CNS of rats 

affected by EAE is blocked in the acute phase, the pronounced 

effects observed with FTY720 in the relapse phase 

of EAE seem to be linked to additional mechanisms. One of 

the proposed factors involved in the induction of relapses of 

EAE in the Lewis rats could be an increase in blood vessel 

density in the spinal cord as it has been described in EAE in 

guinea pigs. Using a casting procedure of blood vessels of the 

brain and spinal cord of Lewis rats going through acute and 

relapsing phases of EAE, we demonstrate an increase in 

vessel density induced in relapsing EAE and a FTY720mediated 

blockade of the induced angiogenesis. A similar 

protective effect was seen using a specific VEGF-receptor 

kinase inhibitor, demonstrating that a blockade of diseaseinduced 

angiogenesis leads to complete amelioration of 

clinical symptoms of relapsing EAE. 

This neo-angiogenesis was mainly observed in the lumbar 

section of the spinal cord suggesting that this region be of 

particular importance to the paralytic episodes observed in 

EAE in rats. 

doi:10.1016/j.clim.2007.03.045 

S143 

Su.5 Enhanced Glucocorticoid Signaling Impacts 

Thymocyte Apoptosis and Adaptive Immune 

Responses 

Fred Lühder, PhD, Institute for Multiple Sclerosis Research, 

Göttingen, Germany, Jens van den Brandt, PhD, Department 

of Cellular and Molecular Immunology, Göttingen, Germany, 

Holger Reichardt, PhD, Department of Cellular and 

Molecular Immunology, Göttingen, Germany, Ralf Gold, MD, 

St. Josef-Hospital/Ruhr-University Bochum, Bochum, 

Germany 

Glucocorticoids (GC) are synthesized by the adrenal gland 

and exert pleiotropic effects ranging from the regulation of


energy metabolism and the control of cognitive functions to the 

modulation of the immune system. Therapeutically they are 

used as potent anti-inflammatory and immunosuppressive drugs 

in a variety of conditions, but endogenous GCs rather exert 

positive as well as negative effects on the immune system. To 

study the effects of enhanced GC signaling on T cells we 

generated transgenic rats expressing a glucocorticoid receptor 

(GR) with increased ligand affinity. This causes massive 

thymocyte apoptosis resulting in a dramatic loss of thymic 

cellularity, which could be reverted by adrenalectomy. In the 

periphery, mature T lymphocytes accumulate that respond 

normally to costimulation and whose expansion is probably due 

to homeostatic proliferation, but have a skewed Tcell receptor 

repertoire. The transgenic rats are partially protected from 

Experimental Autoimmune Encephalomyelitis (EAE) induced by 

immunization with gpMBP, a predominantly TH1 mediated 

autoimmune model. The onset of EAE in transgenic rats was 

delayed by 5 days and the disease course was significantly 

milder. This difference appears to be the consequence of an 

impaired leukocyte infiltration into the CNS and a distinct 

cytokine profile. In contrast, the ability of the transgenic rats to 

mount a predominantly TH2 mediated allergic airway response 

to ovalbumin was unaffected, although isotype switching of 

antigen-specific immunoglobulins was altered. Collectively, our 

findings suggest that endogenous glucocorticoids impact T cell 

development and favor the selection of TH2 over TH1 dominated 

adaptive immune responses. 

doi:10.1016/j.clim.2007.03.046 

Su.6 Microarray Analysis Identifies 

Interferon-β-responsive Genes in Human Microglia 

Hiroko Tabunoki, Assistant, Meiji Pharmaceutical 

University, Bioinformatics, Kiyose, Japan, Tamako Misawa, 

Graduate Student, Meiji Pharmaceutical University, 

Bioinformatics, Kiyose, Japan, Jun-ichi Sato, Professor, 

Meiji Pharmaceutical University, Bioinformatics, Kiyose, 

Japan 

Objective/Background: MS is mediated by autoreactive Th1 

cells. When activated Th1 cells enter the CNS across the blood– 

brain barrier (BBB), they are reactivated by microglia (MCG), the 

professional antigen-presenting cell type in the CNS. Consequently, 

MCG activated by Th1 cytokines release mediators of 

demyelination. Although interferon-beta (IFNB) reduces the 

frequency of relapses in MS patients, the molecular mechanism 

responsible for beneficial effects of IFNB in MS remains 

unknown. Because IFNB could pass through the BBB disrupted 

in active lesions of MS, it might directly modulate the biological 

function of MCG. Methods: To identify IFNB-responsive genes 

(IRGs) in human MCG, we studied the gene expression profile of a 

human MCG cell line HMO6 (Neurobiol Dis 8:1057, 2001) 

following treatment with 50 ng/ml IFNB for 3 hours, by using 

an oligonucleotide microarray (Hitach), verified by Northern 

blot and real-time RT-PCR. Results: Among total 1606 genes on 

the array, IFNB elevated the expression of 39 genes. Top10 

included IFI56, IFI60, MX1, MX2, ISG15, IFI44, IRF7, STAT1, IFI6, 

and TAP1, all of which were known IRGs. The common upstream 

search of 39 genes on KeyMolnet (Curr Drug Discov Technol 2:89, 

2005), the knowledge-based data mining tool of bioinformatics, 

indicated a molecular network most relevant to gene regulation 

by IRF and NF-kB. Conclusions: IFNB induces the expression of a 

battery of IRGs in human microglia, including IRF7 and ISG15, a 

central regulator of innate and adaptive immunity (Nature Rev 

Immunol 6:644, 2006), suggesting an immunomodulatory effect 

of IFNB on human microglia. 

doi:10.1016/j.clim.2007.03.047 

Su.7 The Complement-scavenging Capacity of IVIG 

Products 

Martin O. Spycher, PhD, CSL Behring AG, Research and 

Development, Bern 22, Switzerland, Katja Matozan, 

University of Bern, Department of Clinical Research, Bern, 

Switzerland, Kathrin Minnig, PhD, CSL Behring AG, QA, Bern 

22, Switzerland, Sylvia Miescher, PhD, CSL Behring AG, 

Research and Development, Bern 22, Switzerland, Roland 

Zehnder, CSL Behring AG, Research and Development, Bern 

22, Switzerland, Liane Hoefferer, PhD, CSL Behring AG, 

Research and Development, Bern 22, Switzerland, Robert 

Rieben, PhD, University of Bern, Department of Clinical 

Research, Bern, Switzerland 

IVIG induced complement scavenging describes one antiinflammatory 

effect of IVIG. This mechanism of action is 

considered important in inflammatory autoimmune disorders 

such as multifocal motor neuropathy or dematomyositis. The 

complement scavenging properties of eight IVIG products were 

compared. Constant amounts of aggregated human IgG coated 

to microtiterplates served as complement activators. They were 

incubated with constant amounts of human serum as a 

complementsourceinthepresenceorabsenceofincreasing 

concentrations of IVIGs. Complement activation was quantified 

by measuring bound activation products C3b/iC3b/C3c using a 

peroxidase-conjugated anti-C3c antibody. 

All IVIG tested exhibited dose-dependent inhibition of C3 

deposition. This effect was associated with reduced C3a 

generation, confirming that it was due to complement inhibition, 

not complement activation and consumption. Inhibition of 

complement deposition was paralleled by an increase of C3 

activation product binding to IgG in the fluid phase. The extent 

of complement inhibition was dependent on the IVIG tested, 

with up to 3.5 fold differences between products. An IVIG 

currently in development, named IgPro10, manufactured with 

gentle procedures was the most potent product. Differences of 

inhibition between products did not correlate with differences 

in C3 activation product binding to IgG in the fluid phase at given 

IVIG concentrations suggesting additional mechanisms being 

functional besides complement deviation. Complement scavenging 

is a common feature of IVIGs, however the extent of 

complement scavenging depends on the product. IgPro10, a new 

IVIG product in development and manufactured with a gentle 

chromatographic process, is a more potent complement 

scavenger than currently used IVIGs. 

doi:10.1016/j.clim.2007.03.048 

Su.8 Influence of Interferon-β Therapy Switching on 

Neutralizing Antibody Titers: Results from the 

Austrian Switch Study (ASS) 

Florian Deisenhammer, Professor, Department of Neurology,

Abstracts 

Innsbruck Medical University, Innsbruck, Austria, Alban 

Millonig, Assistant, Department of Neurology, Innsbruck 

Medical University, Innsbruck, Austria, Claudia Gneiss, 

Assistant, Department of Neurology, Innsbruck Medical 

University, Innsbruck, Austria 

Continuous treatment with interferon beta (IFNb) leads 

to neutralizing antibody (NAb) negativity in many previously 

NAb positive patients. To date no strategies are 

available to accelerate NAb reversion. We investigated the 

effect of switching between IFNb products on the development 

of NAb titers. Twenty-four NAb positive MS patients 

on IFNb-1a s.c. or IFNb-1b were included. At baseline IFNb 

treatment was interrupted for 3 months in all patients and 

2 infusions with 1 g methylprednisolone were given. Then 

patients were randomized either to their previous treatment 

or to IFNb-1a im. Twelve months later NAb titers were 

measured. Twelve patients were switched to IFNb-1a im 

30 μg (7 from IFNb-1a sc, 5 from IFNb-1b). Currently, final 

NAb titers (given in neutralizing units) are available in 18 

patients. The median baseline NAb titer was 846, 293 after 

corticosteroids, 1239 3 months after switching, and 393 at 

the end of study. The median change of NAb titers between 

baseline and end of study was −317 in patients who 

switched and −94 in patients who did not switch therapy 

(not significant). Baseline and end of study NAb titers 

correlated significantly (r=0.73). NAb can be regarded as a 

model of autoimmunity by breaking and re-inducing 

immune tolerance. The main influencing factor on the 

final NAb titer was the baseline titer. Many factors must be 

considered when choosing therapy for an individual patient. 

However, as far as NAb are concerned no strategy for 

reversion could be established leaving prevention the only 

option as yet. 

doi:10.1016/j.clim.2007.03.049 

Su.9 IL-21 Receptor Modulates Autoimmune 

Responses and Expression of Experimental 

Autoimmune Encephalomyelitis 

Ruolan Liu, Postdoctoral, Barrow Neurological Institute, 

Neurology, Phoenix, AZ, Antoni La Cava, Associate 

Professor, David Geffen School of Medicine, Los Angeles, 

CA, Debbie Young, Professor, Wyeth Research, 

Inflammation, Cambridge, MA, Mary Collins, Scientist, 

Wyeth Research, Cambridge, MA, Denise Campagnolo, 

Neurologist, Barrow Neurological Institute, Neurology, 

Phoenix, AZ, Timothy Vollmer, Neurologist, Barrow 

Neurological Institute, Neurology, Phoenix, MA, Fu-Dong 

Shi, Scientist, Barrow Neurological Institute, Neurology, 

Phoenix, AZ 

IL-21 receptor (IL-21R) consists of a unique IL-21R 

subunit and also shares the common gamma chain (γ c). 

IL-21R ligand IL-21 plays a plethora of roles in innate and 

adaptive immune responses. In this study we examined 

the influence of IL-21R deficiency in the development of 

myelin oligodendrocyte glycoprotein peptide 35–55 

(MOG35–55)-induced experimental autoimmune encephalomyelitis 

(EAE), an animal model for human multiple 

sclerosis (MS). Upon immunization, IL-21R−/− mice devel- 

oped an exacerbated form of EAE with marked inflammatory 

infiltration into the central nervous system (CNS). 

Both Th1 and Th2 responses to MOG were augmented in 

IL-21R−/− mice. Although IL-21R deficiency did not 

dramatically alter the numbers of NK cells, NKT cells, 

CD11c, CD11b cells, and CD4+CD25+ regulatory T cells 

(Treg) during EAE, the expression of Foxp3 on Treg cells 

was reduced in MOG-primed IL-21R−/− mice. Our results 

suggest that IL-21R/ligand pathways may affect Treg cells, 

which may subsequently affect the magnitude of CNS 

autoimmunity. 

doi:10.1016/j.clim.2007.03.050 

S145 

Su.10 Targeted Delivery of Immunomodulating 

Agents to Dendritic Cells for Treatment of 

Autoimmune Disease Using Biodegradable 

Microspheres 

Tali Brunner, PhD Student, Biotechnology Engineering, 

Ben-Gurion University of the Negev, Beer Sheva, Israel, 

Alon Monsonego, Researcher, Department of Microbiology 

and Immunology, Faculty of Health Sciences and The 

National Institute, Beer Sheva, Israel, Smadar Cohen, 

Researcher, Department of Biotechnology Engineering, 

Ben Gurion University of the Negev, 

Beer Sheva, Israel 

Background: Dendritic cells (DCs) are a central element 

for drug targeting in autoimmune diseases, due to their 

function in the onset and progression of destructive 

autoimmunity and in maintaining the Treg repertoire. 

When injected intradermally, microspheres sized 1–10 

microns are preferentially uptaken by DCs. Use of such 

microspheres has been investigated for delivery of antigens 

and DNA to DCs for vaccination; however this delivery 

system has not been implemented for delivery of siRNAs 

capable of regulating autoimmunity. In vivo delivery of 

siRNA is a great challenge, since these RNA duplexes are 

rapidly degraded and require appropriate stabilization. 

Goal: To use biodegradable microspheres for delivering 

siRNA and other immunomodulating agents to DCs, for 

treatment of autoimmune disease. Results: Microspheres 

composed of poly-lactic-acid better target CD11c positive 

cells in a bone marrow DC (BMDC) culture, in comparison to 

a poly-lactic-co-glycolic composition. siRNA, stabilized 

using poly-ethylene-imine and encapsulated in microspheres 

using a double-emulsion procedure, remained 

active after release from microspheres in BMDCs. Importantly, 

the microspheres did not have an adjuvant effect 

upon the BMDCs compared with LPS-induced DC-mediated 

T cell proliferation, meaning that the BMDCs maintained 

their immature state, which is crucial for creating an 

immunoregulatory environment which can ameliorate the 

course of autoimmune disease. 

Summary: This work combines biotechnology engineering, 

assessing microsphere behavior and distribution, with 

basic immunological research, assessing activity of immunomodulating 

agents. We believe that this delivery platform 

can be used for delivering not only various targeting 

siRNAs, but also other immunomodulating agents such as


peptides which can interfere with intracellular signaling 

pathways. 

doi:10.1016/j.clim.2007.03.051 

Su.11 Neurofascin-specific Autoantibodies Mediate 

Axonal Injury in Inflammatory Demyelinating 

Diseases of the Central Nervous System 

Christopher Linington, Professor of Immunobiology, 

University of Aberdeen, Aberdeen, UK, Emily Mathey, 

Research Associate, University of Aberdeen, Aberdeen, UK, 

Tobias Derfuss, Max Planck Institute for Neurobiology, 

Martinsried, Germany, Matthew Rasband, Assistant 

Professor, University of Connecticut Health Center, 

Farmington, CT, Maria Storch, Professor, Medical University 

Graz, General Neurology, Graz, Germany, Reinhard 

Hohlfeld, Professor, LMU University Hospital, Clinical 

Neuroimmunology, Munich, Germany, Edgar Meinl, 

Professor, Max Planck Institute for Neurobiology, 

Martinsried, Germany 

We report that multiple sclerosis is associated with elevated 

autoantibody responses to neurofascin, as compared to other 

inflammatory neurological diseases. The two isoforms of 

neurofascin play essential roles in maintaining the molecular 

organization of myelinated fibers: NF186 is a neuronal protein 

located at the node of Ranvier and axonal initial segment where 

it interacts with voltage gated sodium channels, whilst NF155 is 

an oligodendroglial product sequestered at junctional complexes 

formed where paranodal loops of the myelin sheath 

contact the axonal surface. Analysis of neurofascin-specific 

autoantibodies immunopurified from seropositive donors 

demonstrates that they recognise the extracellular domain of 

both isoforms of the native protein indicating that this response 

may participate in disease pathogenesis. We investigated this 

hypothesis in experimental autoimmune encephalomyelitis 

using a pan NF155/186 mouse mAb to mimic the human 

autoantibody response. Passive transfer (i.p.) of mAb induced 

a rapid exacerbation of disease that was associated with a 

corresponding increase in acute axonal injury. Intriguing this 

occurred in the absence of any concomitant increase in the local 

inflammatory response or demyelination. Immunofluorescence 

microscopy revealed that binding of the transferred neurofascin-specific 

antibody was restricted nodes of Ranvier where it 

co-localised with voltage gated sodium channels. These results 

identify NF186 as a primary target for neurofascin-specific 

autoantibodies and indicate antibody-dependent effector 

mechanisms are involved in the pathogenesis of axonal injury 

andlossinmultiplesclerosis. 

doi:10.1016/j.clim.2007.03.052 

Su.12 CD11c on NK Cells Mirrors the Temporal 

Disease Activity of Multiple Sclerosis 

Toshimasa Aranami, Section Chief, Department of 

Immunology, National Institute of Neuroscience, NCNP, 

Kodaira, Japan, Sachiko Miyake, Section Chief, Department 

of Immunology, National Institute of Neuroscience, NCNP, 

Kodaira, Japan, Masafumi Ogawa, Section Chief, 

Department of Neurology, National Center Hospital for 

Mental Nervous and Muscular Disorders, NCNP, Kodaira, 

Japan, Takashi Yamamura, Director, Department of 


Kodaira, Japan 

Multiple sclerosis (MS) is an autoimmune disease, showing a 

greatdegreeofvarianceindiseaseactivity.Wehaverecently 

demonstrated that NK cells biased for producing IL-5 (bias 

towards type2 NK cells, NK2 bias) are associated with remission 

state of MS. Here we report that upregulation of CD11c on NK 

cells would characterize a subgroup of MS in remission whose NK 

cells are lacking NK2 bias. When we compared this group 

(CD11chigh) with the other group of patients (CD11clow) 

concerning NK cell expression of IL-5 and GATA-3, we found 

NK2 bias to be a selective character of CD11clow patients. In 

contrast, CD11chigh group showed an obvious trend for a larger 

proportion of HLA-DR+ cells within NK cells. Since NK cell 

stimulatory cytokine such as IL-15 was found to upregulate 

CD11c expression on NK cells in vitro, we postulate that 

inflammatory cytokine milieu may play a role in inducing 

CD11chigh NK cell phenotype. Although there was no clinical 

difference between CD11chigh and CD11clow at blood sampling, 

CD11chigh MS showed significantly higher relapsing rate than 

CD11clow during 120 days follow-up. These data point to an 

interpretation that CD11chigh patients are at a higher risk for 

developing relapse, which is probably due to the loss of NK2 

phenotype. Thus, CD11c on NK cells mirrors the temporal 

disease activity of MS and may be used as a disease biomarker. 

doi:10.1016/j.clim.2007.03.053 

Su.13 Functional Involvement of NR4A2 in the 

Pathogenesis of Multiple Sclerosis 

Yoshimitsu Doi, Physician, Department of Immunology, 

National Institute of Neuroscience, NCNP, Kodaira City, 

Tokyo, Japan, Shinji Oki, Scientist, Department of 


Kodaira City, Tokyo, Japan, Sachiko Miyake, Physician, 

Department of Immunology, National Institute of 

Neuroscience, NCNP, Kodaira City, Tokyo, Japan, Takashi 

Yamamura, Physician, Department of Immunology, National 

Institute of Neuroscience, NCNP, Kodaira City, Tokyo, Japan 

Objective: To study a role of the NR4A2 in T cells of 

multiple sclerosis (MS). Background: NR4A2, a transcription 

factor of the steroid/thyroid receptor superfamily, is rapidly 

induced by exposure to growth factors and cytokines, 

suggesting a biological relevance to various cell functions. 

Through a cDNA microarray analysis, higher expression of 

NR4A2 was observed in CD3+ T cells of 57 MS patients 

compared with 19 healthy control subjects (Satoh et al. 

Neurobiol Dis 18:537–50, 2005). However, the functional 

involvement of NR4A2 expression and its transcriptional 

targets in MS Tcells remain unknown. Methods: Expression of 

NR4A2 mRNA was analyzed by quantitative RT-PCR in PBMCderived 

human T cells or murine T cells infiltrated in CNS 

after EAE induction. Activities of cytokine gene promoters 

were analyzed by reporter gene analysis. Cytokine production 

in primary T cells was measured after retroviral 

transduction of NR4A2 gene or silencing of NR4A2 gene by 

using siRNA. Results: Higher expression of NR4A2 was

Abstracts 

demonstrated both in MS Tcells and in CNS-infiltrating Tcells 

in EAE mice. NR4A2 augmented promoter activities of 

inflammatory cytokine genes. Overexpression of NR4A2 

induced up-regulation of IL-17 and IFN-f× production. Meanwhile, 

silencing of NR4A2 expression by siRNA resulted in 

down-regulation of the inflammatory cytokines. Conclusions: 

NR4A2 was upregulated in T cells of MS patients and in CNSinfiltrating 

T cells of EAE mice. Inflammatory cytokines 

including IL-17 and IFN-f× were the possible targets of NR4A2 

in T cells, suggesting that NR4A2 may play an important role 

in immunopathogenesis of MS. 

doi:10.1016/j.clim.2007.03.054 

Su.14 Functional Analysis of Carcinoembryonic 

Antigen-related Cellular Adhesion Molecule 1 in 


Shinji Oki, Section Chief, Department of Immunology, 

National Institute of Neuroscience, NCNP, Tokyo, Japan, 

Mayumi Fujita, Postdoctoral Researcher, Department of 


Tokyo, Japan, Takao Ootsuka, Graduate Student, Department 

of Immunology, National Institute of Neuroscience, NCNP, 

Tokyo, Japan, Chiharu Tomi, Researcher, Department of 


Tokyo, Japan, Miho Mizu, Researcher, Department of 


Tokyo, Japan, Shinjiro Kaieda, Graduate Student, 

Department of Immunology, National Institute of 

Neuroscience, NCNP, Tokyo, Japan, Takashi Yamamura, 

Director, Department of Immunology, National Institute of 

Neuroscience, NCNP, Tokyo, Japan, Sachiko Miyake, Section 

Chief, Department of Immunology, National Institute of 

Neuroscience, NCNP, Tokyo, Japan 

Introduction: Carcinoembryonic antigen-related cellular 

adhesion molecule 1 (CEACAM1) is one of the CEA-family 

members and is demonstrated to play an important role in 

immune regulation. In this study, we investigated the role of 

CEACAM1 in experimental autoimmune encephalomyelitis 

(EAE). [Methods] C57BL/6J mice immunized with MOG peptide 

were treated with anti-CEACAM1 mAb (AgB10) or CEACAM1-Fc 

fusion protein twice a week. The clinical signs of each group 

were scored to evaluate the effect of CEACAM1-mediated 

signals in EAE. MOG-specific cytokine production by inguinal 

lymph node cells derived from AgB10-treated or CEACAM1-Fctreated 

mice were analyzed by ELISA 10 days after the 

immunization. MOG-specific antibody production in serum 

was analyzed for AgB10-treated mice. Results and Discussion: 

The repetitive treatment with AgB10 enhanced the clinical sign 

of EAE, whereas the treatment with CEACAM1-Fc suppressed 

EAE as compared with control animals, indicating that CEACAM1 

exerts suppressive signal for the development of EAE. The 

production of inflammatory cytokines such as IFN-fÁ andIL-17 

in T cells derived from AgB10-treated mice was significantly 

enhanced, whereas the production of these cytokines from 

CEACAM1-Fc-treated mice was significantly decreased. These 

results suggest that CEACAM1-mediated signal is inhibitory for 

MOG-specific Th1 or Th17 responses. Accordingly, MOG-specific 

antibody production from AgB10-treated mice showed the 

reduced level of IgG1 and enhanced level of IgG2a compared 

with control mice. Taken together, CEACAM1 signal holds an 

important suppressive role in EAE by inhibiting both Th1 and 

Th17 responses to tissue-specific antigens. Mechanistic analysis 

for the role of CEACAM1 signal is currently under investigation. 

doi:10.1016/j.clim.2007.03.055 

Su.15 Gene Expression Profile Modulated by IVIG in 

Healthy Donors and Multiple Sclerosis Patients 

Christian Jacobi, MD, University of Heidelberg, Department 

of Neurology, Institute of Immunology, Heidelberg, 

Germany, Jürgen, Römisch, PhD, Octapharma PPmbH, 

Vienna, Austria, Stefan, Meuer, MD, University of 

Heidelberg, Institute of Immunology, Heidelberg, Germany, 

Thomas Giese, MD, University of Heidelberg, Institute of 

Immunology, Heidelberg, Germany 

Intravenous immunoglobulin G (IVIG) has become an 

established first- and second-line line treatment in a 

number of immune mediated diseases during recent years. 

IVIG is now proposed to modulate a wide range of molecules 

(e.g. bacterial/viral antigens, toxins, Fc receptors, complement, 

autoantibodies), to act on different cell types (e. 

g. B cells, T cells, macrophages, dendritic cells, endothelial 

cells), and to modulate various immunological pathways at 

both the humoral and cellular levels including inflammation, 

antigen presentation, cell growth and apoptosis. Using 

a variety of specific assays many distinct effects of IVIG 

have been demonstrated in vitro. To provide further insight 

into the mechanisms involved in IVIG-dependent immunmodulation 

under physiological conditions, we have established 

a human whole blood gene expression assay in vitro. 

There, we analyzed the effect of IVIG on CD2-, CD3-, LPSand 

PMA/Ionomycin stimulated leukocytes in whole blood of 

20 healthy donors and 12 untreated MS patients. IVIG 

induces among others the expression and release of 

Interferon-γ, MCP1 and IP10. Interestingly, IVIG reduces 

the MCP1 and IP10 expression and release upon LPS 

stimulation, whereas Interferon-γ expression is further 

enhanced. Since both chemokines are physiologically 

induced by Interferon, this effect of IVIG is unanticipated 

and deserves further investigation for its possible role in 

anti-inflammatory properties of IVIG. No significant differences 

in the IVIG induced expression profile between 

healthy donors and MS patients could be observed. 

doi:10.1016/j.clim.2007.03.056 

S147 

Su.16 IL-15 and IL-15Rα Expressed in Human 

Central Nervous System by Astrocytes Contribute to 

CD8 T Lymphocyte Activation and Persistence: 

Implications for Multiple Sclerosis 

Nathalie Arbour, Assistant Professor, University of 

Montreal-Medicine, Montreal, QC, Canada, Philippe Saikali, 

PhD Student, McGill University-Neurology, Montreal, QC, 

Canada, Jack Antel, Professor 

Increasing experimental evidences suggest that CD8 T 

lymphocytes partake in multiple sclerosis (MS) related central 

nervous system (CNS) damage. CD8 T lymphocytes are


prominent cells in MS brain infiltrates and could injure 

oligodendrocytes and axons, both damaged in MS, as they are 

observed touching these structures. Activated memory CD8 T 

cells were shown to clonally expand and persist in the CNS of MS 

patients for years. Interleukin-15 (IL-15) is pivotal in the 

generation and maintenance of memory CD8 T cells. IL-15 

bound to IL-15Rα onitscelloforiginefficientlyactivatesIL- 

15Rβ/γ bearing cells. We determined whether a local source of 

IL-15 and IL-15Rα can supply this cytokine to infiltrating CD8 T 

lymphocytes in the MS CNS. Human astrocytes expressed surface 

bound IL-15 and IL-15Rα in vitro; such expression was upregulated 

in response to pro-inflammatory cytokines. We 

detected astrocytes expressing IL-15 in MS brain supporting its 

in vivo presence. Human CD8 T cells cultured in the presence 

of IL-15 were more activated and cytotoxic (high amounts of 

cytolytic enzymes) and acquired a memory phenotype that was 

not observed in the presence of IL-2. CD8 T lymphocytes bearing 

a similar activated memory phenotype were observed in the 

cerebrospinal fluid of MS patients. The localization of astrocytes 

within the brain architecture suggests that infiltrating CD8 T 

cells would readily encounter this source of IL-15+IL15Rα. 

These results underline a novel potential role for IL-15/IL-15Rα 

in the survival of memory CD8 T cells in the CNS during 

inflammatory diseases such as MS. 

doi:10.1016/j.clim.2007.03.057 

Su.18 CD4+CD28null T Cells in Autoimmune Disease: 

Pathogenic Features and Decreased Susceptibility to 

Immunoregulation 

Marielle Thewissen, Hasselt University, Biomedical Research 

Institute, Diepenbeek, Belgium, Veerle Somers, Hasselt 

University, Biomedical Research Institute, Diepenbeek, 

Belgium, Niels Hellings, Hasselt University, Biomedical 

Research Institute, Diepenbeek, Belgium, Piet Stinissen, 

Director, Hasselt University, Diepenbeek, Belgium, Koen 

Venken, Hasselt University, Biomedical Research Institute, 

Diepenbeek, Belgium 

CD4+CD28null T cells have been described to be 

generated as a consequence of persistent immune stimulation. 

These CD4+ T cells are incapable of providing help for 

B cell proliferation and antibody production, but have 

acquired cytolytic function. To date, the triggers of 

activation of CD4+CD28null T cells have not been fully 

elucidated. An expansion of this cell subset has been 

described in several pathological conditions including 

multiple sclerosis (MS) and rheumatoid arthritis (RA). In 

this study the role of the expanded CD4+CD28null T cell 

subset in RA and MS pathology was further investigated. 

CD4+CD28null T cells were found to be terminally differentiated 

effector memory cells. They are equipped to 

infiltrate tissues and could be detected in the synovial 

tissue of RA patients. Whereas no reactivity to candidate 

autoantigens (myelin, collagen) was observed within the 

CD4+CD28null T cell subset, cytomegalovirus (CMV) reactivity 

was prominent in 4/4 HC, 4/4 RA patients and 3/4 

MS patients. Of note was that the intensity of the CMV 

induced proliferative response was related to the number 

of CMV epitopes recognized. Interestingly, our results 

illustrated that CD4+CD28null T cells were not susceptible 

to the suppressive actions of CD4+CD25 high regulatory T 

cells. In conclusion, this study provides several indications 

for a role of CD4+CD28null T cells in autoimmune pathology. 

These cells display pathogenic features, fill up immunological 

space and are less vulnerable to regulatory mechanisms. 

However, our results do not support a direct 

autoreactive role of CD4+CD28null T cells in the pathogenesis 

of autoimmune diseases. 

doi:10.1016/j.clim.2007.03.058 

Su.19 Intravenous TCV-CD4 Treatment for Multiple 

Sclerosis 

Gustavo A. Moviglia, MD, Regina Mater Foundation and 

Maimonides University, Buenos Aires, Argentina, Gabriela 

Varela, PhD, Regina Mater Foundation, Buenos Aires, 

Argentina, J. Hirsch, MD, Regina Mater Foundation, Buenos 

Aires, Argentina, J.A. Brizuela, MD, Regina Mater 

Foundation and Maimonides University, Buenos Aires, 

Argentina, C.A. Palleros, Regina Mater Foundation, Buenos 

Aires, Argentina, R. Moya, MD, Regina Mater Foundation, 

Buenos Aires, Argentina, Fabiana Bastos, MD, Regina Mater 

Foundation and Maimonides University, Buenos Aires, 

Argentina, M.T. Moviglia, MD, Regina Mater Foundation and 

Maimonides University, Buenos Aires, Argentina 

Background: T cell vaccine (TCV) is a proven pre clinical and 

clinical cell therapy for MS. The original technique, developed 

by Irum Cohen and collaborators, was as monoclonal or 

oligoclonal antimyelin and selective cell suspension to be 

activated, irradiated and administered to MS stable patients. 

Recent clinical trials showed that 92% of treated patients were 

stabilized. Methods: 82 CPMS patients were divided into 3 

groups: Group 1 (42 patients) received 10 subcutaneous 

immunizations of polyclonal TCV (1 every 4 weeks). Group 2 

(24 patients) received 10 IV polyclonal TCV. Group 3 (16 

patients) received 10 IV polyclonal TCV-CD4. Experienced 

neurologist evaluated the EDSS index from the beginning up to 

3 years of follow-up. The TCV-CD4 vaccine was prepared 

selecting only CD4+ cells from polyclonal TCV (Stem Sep for 

CD4, Vancouver) Results: Group 1, 19% of patients improved an 

average of 1 point of original EDSS index, 65% remained stable 

and 16% deteriorated only 0.5 points. 83% developed local 

aseptic abscesses at immunization site that took 1–6monthsto 

be solved. Group 2, 50% improved 2 points; 29% improved 1 point 

and 21% remained stable. No abscesses on the 24 patients but all 

developed fever (39.5 C) during first 24 hours after immunization. 

Group 3, 69% improved 2 points, 25%, 1 point and 6% 

remained stable. No fever nor abscesses observed. Conclusion: 

IV TCV-CD4 treatment is safe and shows therapeutic efficacy for 

CPMS. The spleen seems to be where antiergotype regulator 

cells are produced. 

doi:10.1016/j.clim.2007.03.059 

Su.20 Ingested (Oral) Soluble Immune Response 

Suppressor (SIRS) Peptide 1−21 Inhibits Acute EAE 

Staley Brod, Professor, University of Texas Houston, MSRG, 

Houston, TX, Zach Hood, Technician, University of Texas 

Houston, MSRG, Houston, TX

Abstracts 

Background: Ingested type I IFN inhibits clinical attacks, 

relapses and inflammation in murine EAE. Type I IFN activate 

human suppressor T cells that produce SIRS. Objectives: We 

examined whether SIRS peptide would have similar effects to 

ingested IFN-α in EAE. Methods: C57BL/6 mice were actively 

immunized with myelin oligodendroglial (MOG) peptide 35– 

55 (MEVGWYRSPFSRVVHLYRNGK) in IFA and followed for 

disease. Clinical severity was graded daily. For parenteral 

treatment, B6 mice were injected with control saline 

(mock), 0.1, 1, or 10 mg SIRS peptide 1–21 (NH–MET–Thr– 

Glu–Glu–Asn–Gln–Gln–Ser–Ser–Gln–Pro–Lys–Thr–Thr–Ile– 

Asn–Asn–Ala–Gly–Asp–Ser–CysOH). For oral treatment, B6 

mice were fed with 0.1 ml of saline (mock) or 1, 10 or 100 mg 

SIRS peptide 1– 21 daily. Following sacrifice, spinal cords 

were evaluated for foci of inflammation. Splenocytes from 

grouped saline (mock) fed or 100 mcg SIRS peptide fed mice 

were stimulated with Con A and examined using mouse 

cytokine inflammatory antibody array. Results: S.C. 10 mcg 

SIRS peptide 1–21 showed significant inhibition of EAE 

(pb0.001). Ingested SIRS peptide at 10 and 100 mcg SIRS 

peptide inhibited EAE compared to placebo (pb0.001). 

There were significantly less inflammatory foci in the SIRS 

peptide fed group compared to the control mock saline group 

(pb0.03). Splenocytes from SIRS peptide 1–21 fed mice 

showed increased production of CD30L, IL-6, IL-13, I-TAC, 

TCA-3/CCL1, TNF-α. Conclusion: Ingested SIRS peptide 

inhibits both clinical EAE and inflammation via counterregulatory 

type 2-like cytokines and chemokines IL-13, 

CD30L and TCA-3. 

doi:10.1016/j.clim.2007.03.060 

Su.21 Identifying New Immunodominant Myelin 

Peptides in Relapsing Remitting Multiple Sclerosis 

Patients 

Brian Newsom, Senior Director of Project Development, 

Opexa Therapeutics, Inc. The Woodlands, TX, Kathrin von 

Gynz Rekowski, Research Scientist, Opexa Therapeutics, 

Inc. The Woodlands, TX, Mitzi Montgomery, Consultant, 

Opexa Therapeutics, Inc. The Woodlands, TX, Jim Williams, 

Chief Operating Officer, Opexa Therapeutics, The 

Woodlands, TX, Edward Fox, Clinical Assistant Professor, 

University of Texas Medical Branch, MS Clinic of Central 

Texas, Round Rock, TX 

T cell reactivity to peptides found to be instrumental in 

Multiple Sclerosis (MS) pathology and experimental animal 

models has centered on the major myelin proteins; Myelin 

Basic Protein (MBP), Myelin Proteolipid Protein (PLP), and 

Myelin Oligodendrocyte Glycoprotein (MOG). With recent 

structural studies on these proteins and discoveries of 

additional myelin proteins, there are questions about 

potential interactions between these proteins and T cells in 

MS. Using synthetic peptides of 16 amino acid residues and 

overlaps of 12 residues, we have surveyed the resulting 163 

peptides from these three proteins for their proliferative 

reactivity in 16 healthy subjects and 63 MS patients, 

including 22 from our Phase I trials of the T cell vaccine 

Tovaxin. Using a Tcell Epitope Analysis Assay (EAA), peptides 

were screened using peripheral blood mononuclear cells and 

a tritiated thymidine incorporation assay. The results from 

this assay generated a pattern of individual, patient specific 

reactivity by calculating the stimulation index of T cells in 

the presence of peptide antigens when compared to media 

controls. Each assay also included controls of non-specific or 

superantigenic stimuli, such as phytohemagglutin (PHA). The 

results of 173 assays have allowed us to identify several 

peptides as being immunodominant, including some never 

before reported, and others as being non-reactive. The EAA 

can be utilized to screen additional peptides or combinations 

that have biological significance. The results have permitted 

us to reduce the number of peptides to 109, in the screening 

assay for Tovaxin vaccine production, to qualify subjects for 

our current Phase IIb clinical trial. 

doi:10.1016/j.clim.2007.03.061 

Su.22 Differential Gene Expression Identifies Novel 

Markers of Myelin-Specific Murine Memory CD4+ 

T Cells 

Wassim Elyaman, Research Fellow, Brigham and Women’s 

Hospital-Harvard, Boston, MA, Pia Kivisakk, Research 

Fellow, Brigham and Women's Hospital, Boston, MA, Jaime 

Imitola, Instructor, Brigham and Women’s Hospital, Boston, 

MA, Samia Khoury, Associate Professor, Brigham and 

Women’s Hospital -Harvard, Boston, MA, Mohamed Sayegh, 

Professor, Brigham and Women's Hospital -Children's 

Hospital-Harvard, Boston, MA 

Memory CD4+ T cells function as a dynamic repository of 

antigen-experienced T lymphocytes that tend to resist 

immunomodulatory and tolerance strategies. Their activation 

in response to a secondary antigenic stimulation involves 

changes in the expression level of a large number of genes. 

Recently, we have shown that myelin specific CD4 memory 

cells express high levels of ICOS and lower amounts of CD28 in 

comparison to effector T cells. This was associated with 

differential response to costimulation blockade in a mouse 

model of autoimmune encephalomyelitis. Here, we have 

used cDNA array technology to characterize the differences 

in gene expression between murine myelin-specific memory 

and effector CD4+ T cells. To generate myelin-specific 

memory CD4+ T cells, MOG-TCR transgenic mice were 

immunized with MOG/CFA for 2 weeks and CD4+ T cells 

were parked for 3 months in Wild-type naïve recipients. 

Effector CD4+ T cells were generated by immunizing MOG 

transgenic mice for 2 weeks. The gene expression profiles of 

mouse donors of CD4+ T cells of each group (n=3) were 

analyzed. 204 genes were consistently differentially 

expressed (3 fold change) between memory and effector T 

cells, which included 23 genes that are only expressed in 

CD4+ memory cells. These differentially expressed genes 

include a combination of well-known, previously characterized 

genes with a range of biological functions and unknown 

in silico predicted hypothetical genes. These observed 

differences in the gene expression profile of autoantigen 

memory and effector CD4+ T cells have important clinical 

implications for designing new therapies for autoimmune 

diseases in humans. 

doi:10.1016/j.clim.2007.03.063 

S149


Su.23 Reduced Number of Blood Circulating Foxp3+ 

CD25highCD4+ Regulatory T Cells and a Decreased 

Foxp3 Expression at the Single-cell Level in Patients 

with Relapsing-remitting Multiple Sclerosis 

Koen Venken, MSc, Hasselt University, Biomedisch 

Onderzoeksinstituut, Diepenbeek, Belgium, Niels Hellings, 

PhD, Hasselt University, Biomedisch Onderzoeksinstituut, 

Diepenbeek, Belgium, Veerle Somers, PhD, Hasselt 

University, Biomedisch Onderzoeksinstituut, Diepenbeek, 

Belgium, Pieter Meuwissen, Student, Hasselt University, 

Biomedisch Onderzoeksinstituut, Diepenbeek, Belgium, 

Marielle Thewissen, MSc, Hasselt University, Biomedisch 

Onderzoeksinstituut, Diepenbeek, Belgium, Karen Hensen, 

PhD, Clinical Laboratory of Experimental Hematology, Virga 

Jesse Hospital, Hasselt, Belgium, Jean-Luc Rummens, MD, 

Clinical Laboratory of Experimental Hematology, Virga 

Jesse Hospital, Hasselt, Belgium, Piet Stinissen, PhD, 

Hasselt University, Biomedisch Onderzoeksinstituut, 

Diepenbeek, Belgium 

CD4+CD25high regulatory T cells (Tregs) of patients with 

relapsing–remitting (RR), but not secondary-progressive (SP) 

multiple sclerosis (MS), show a reduced suppressive function 

and FoxP3 mRNA expression. In this study we analyzed FoxP3 

protein expression by means of flow cytometry in PBMC of 55 

RR-MS, 15 SP-MS patients and 40 healthy controls (HC). RR-MS 

patients displayed a significant reduced number of CD4+ 

CD25highFoxp3+ T cells as compared to HC and SP-MS. In 

addition, a significant lower Foxp3 expression per cell was 

detected in RR-MS patients. Interestingly, IFN-β treated RR- 

MS patients (n=15) showed a higher frequency of CD4+ CD25 

highFoxp3+ cells as compared to untreated RR-MS patients 

(n=40). Furthermore, a correlation was observed between 

Foxp3 levels and suppressive capacity of Tregs as measured in 

Treg-Tresponder coculture experiments. We further phenotypically 

analyzed Tregs of HC and untreated MS patients by 

measuring the expression of adhesion molecules by flow 

cytometry. Whereas no differences were detected in percentage 

of L-selectin+ (CD62L) and hyaluronate receptor+ (CD44) 

cells, a significant higher percentage of integrin αE+ (CD103) 

and α4+ (CD49d, VLA-4) cells was observed within CD4+ 

CD25high Foxp3+ Tregs of RR-MS as compared to HC and SP- 

MS. Taken together, a lower number of CD4+ CD25highFoxp3+ 

T cells and a reduced Foxp3 expression level was detected in 

RR-MS patients further demonstrating Treg dysfunction in 

these patients. The detection of a higher percentage of 

CD103+ and VLA-4+ in the Treg population in RR-MS patients 

could reflect an increased migration capacity of Tregs 

towards inflammatory lesions in the central nervous system. 

doi:10.1016/j.clim.2007.03.064 

Su.24 Upregulation of Water Channel Aquaporin-4 

in Experimental Autoimmune Encephalomyeritis 

Katsuichi Miyamoto, MD, PhD, Kinki University School of 

Medicine, Osaka-sayama, Japan, Kazuo Kataoka, MD, PhD, 

Department of Neurosurgery, Kinki University School of 

Medicine, Osaka-sayama, Japan, Susumu Kusunoki, MD, 

PhD, Department of Neurology, Kinki University School of 

Medicine, Osaka-sayama, Japan 

Aquaporin-4 (AQP4) is a water channel protein, which plays 

an important role in water movement in the central nervous 

system. Involvement of AQP4 in the pathogenesis of brain edema 

in the acute ischemic brain model was reported. Recently 

presence of the antibody against AQP4 has been reported in the 

patients affected with neuromyelitis optica (NMO) or opticospinal 

type multiple sclerosis (MS). AQP4 therefore may be 

involved in the pathophysiological mechanism of inflammatory 

demyelinating disorder. Here we analyzed AQP4 expression in 

the central nervous system of experimental autoimmune 

encephalomyelitis (EAE), an animal model of MS. We performed 

semi-quantitative analysis of AQP4-mRNA in brain and spinal 

cord from EAE-induced mice by using real-time PCR. Furthermore 

immuno-histochemical analysis was performed. The brain 

and spinal cord from myelin oligodendrocyte glycoprotein 

(MOG)-induced EAE mice were removed on 7, 14, 21, 28 and 

35 days after first inoculation. The increased levels of AQP4mRNA 

expression were seen; that in the spinal cord reached the 

peak on 14 days, and that in the brain on 21 days after first 

inoculation, respectively. The histopathological analysis showed 

that demyelination and cell infiltration were most severe on 

21 days after first inoculation, when the clinical manifestations 

also were most severe. This is the first study to show the 

upregulation of AQP4 in the inflammatory demyelinating disease 

of CNS. Further investigation is necessary to investigate the 

possibility that anti-AQP4 antibody binds to the upregulated 

AQP4 within CNS and is directly involved in the pathogenesis of 

NMO or optico-spinal type MS. 

doi:10.1016/j.clim.2007.03.065 

Su.26 Role of IFN-γ in Virus-induced Demyelinating 

Disease 

Julie Olson, Assistant Professor, Department of Neurological 

Surgery, University of Wisconsin-Madison, Madison, WI 

Multiple sclerosis is a demyelinating disease in humans 

which has been associated with an inflammatory component. 

Epidemiological evidence has suggested that a virus infection 

may be involved in disease initiation and disease exacerbation. 

Theiler's murine encephalomyelitis virus (TMEV) serves as a 

mouse model of MS. Susceptible mice (SJL strain) infected with 

TMEV, BeAn strain, develop demyelinating disease beginning 

with clinical signs of disease around 35 days post infection. 

TMEV induces a chronic progressive demyelinating disease 

associated with a myelin-specific CD4+ Tcell response which is 

a Th1 type Tcell response. However, some strains of mice, such 

as C57BL6, are resistant to development of TMEV- induced 

demyelinating disease. IFN-γ is a proinflammatory cytokine 

associated with demyelinating disease and the myelin- specific 

autoimmune response. In these studies, the role of IFN-γ was 

examined during the initiation and progression of demyelinating 

disease using resistant C57BL6 mice deficient in IFN-γ. 

Interestingly, the IFN-γ deficient mice infected with TMEV 

developed demyelinating disease with clinical disease similar 

to the susceptible (SJL) mice. TMEV- infected IFN γ deficient 

mice developed a virus- specific CD4+ T cell response and a 

myelin- specific T cell response late during demyelinating 

disease. The demyelinating disease was associated with 

infiltration of immune cells into the CNS, most interestingly, 

CD4+ T cells and activated macrophage. These results suggest

Abstracts 

that IFN-γ was protective against development of virusinduced 

demyelinating disease and that IFN-γ was not required 

for development of autoimmune demyelinating disease. This 

research was supported by the National Multiple Sclerosis 

Society RG3625. 

doi:10.1016/j.clim.2007.03.066 

Su.27 Large Scale Immunoprofiling Uncovers 

Population Structure in Untreated MS Subjects and 

Highlights the Role of NK Cells 

Elizabeth Rossin, Data Analyst, The Broad Institute of MIT 

and Harvard, The Program in Medical and Population 

Genetics, Cambridge, MA, Vissia Viglietta, Instructer, 

Brigham and Women’s Hospital, Department of Neurology, 

Boston, MA, Dulce Soler-Ferran, Research Scientist, 

Millenium Pharmaceuticals, Cambridge, MA, Alex Parker, 

Research Scientist, Millenium Pharmaceuticals, Cambridge, 

MA, Mira Weiner, Project Manager, Brigham and Women’s 

Hospital, Department of Neurology, Boston, MA, Guy 

Buckle, Associate Neurologist, Department of Neurology, 

Brigham and Women's Hospital, Brookline, MA, Samia 

Khoury, Associate Professor, Brigham and Women’s Hospital, 

Department of Neurology, Boston, MA, David Hafler, 

Professor, Brigham and Women’s Hospital, Department of 

Neurology, Boston, MA, Howard Weiner, Professor, Brigham 

and Women’s Hospital, Department of Neurology, Boston, 

MA, Philip De Jager, Assistant Professor, Brigham and 

Women’s Hospital, Department of Neurology, Boston, MA 

As part of the Multiple Sclerosis Registry, we acquired an 

immunological profile from healthy subjects and subjects 

with remitting relapsing multiple sclerosis (RRMS) who were 

untreated. Fresh blood from each subject was screened ex 

vivo using a panel of 50 fluorescently labeled monoclonal 

antibodies (Ab) distributed amongst 56 pools; flow cytometry 

was performed to quantitate the binding of each Ab. 

Longitudinal analysis of 15 healthy control subjects identified 

the “% positive” parameters as the most robust to 

fluctuation over time; after further quality control processing, 

1018 individual “% positive” parameters or “features” 

were selected for the analysis. The exploratory screen 

consisted of 17 healthy control subjects and 23 untreated 

RRMS subjects. It was followed by an extension analysis in 

which 15 additional healthy controls and 15 additional RRMS 

subjects were added to the dataset. This analysis identified a 

CD8lowCD56+CD3− population (NK cells) as being reduced in 

frequency in untreated RRMS subjects (P=10–4). In analyzing 

the 38 cases by themselves, clustering analysis revealed that 

there were 3 populations of RRMS subjects with different 

immunological profiles and that disease duration (range 3– 

42 years) might explain some of this population structure 

(P=0.03). In prediction analyses, our Ab panel was unable to 

accurately predict control and RRMS subjects but was able to 

accurately segregate 11 cases of clinically isolated syndrome 

from controls. These data suggest that disease duration is an 

important factor to consider in the analysis and future 

development of immunological profiling in MS. 

doi:10.1016/j.clim.2007.03.068 

Su.28 Treatment with a Fullerene Derivative 

(ABS-75) Reduces Disease Progression and Axonal 

Loss in MOG-induced Progressive EAE in NOD Mice 

Alexandre S. Basso, Research Fellow, Center for Neurologic 

Disease, Harvard Medical School, Boston, MA, Dan Frenkel, 

Research Fellow, Center for Neurologic Diseases, Harvard 

Medical School, Boston, MA, Sanja Petrovic-Stojkovic, 

Research Assistant, Center for Neurologic Diseases, Harvard 

Medical School, Boston, MA, Alon Monsonego, Research 

Fellow, Center for Neurologic Diseases, Harvard Medical 

School, Boston, MA, Lindsay Puckett, Research Assistant, 

Center for Neurologic Diseases, Harvard Medical School, 

Boston, MA, Michael Gozin, Professor, School of Chemistry, 

Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, 

Israel, Howard Weiner, Robert L. Kroc Professor of 

Neurology, Center for Neurologic Diseases, Harvard Medical 

School, Boston, MA 

Inflammation-induced oxidative stress can lead to axonal 

degeneration, which is felt to be a major determinant of 

progressive neurological disability in Multiple Sclerosis (MS). 

Water-soluble derivatives of fullerenes are a unique class of 

allotropic form of carbon compounds with potent antioxidant 

properties. 10 week old mice were immunized with 150 μgof 

MOG 35–55 peptide in CFA followed by pertussis toxin. 

Disease is characterized by an attack followed by a 

progressive phase with chronic clinical impairment. Following 

the first attack, animals were distributed into different 

groups with similar disease courses and treated intraperitoneally 

either with 200 μlofa1μM fullerene solution or vehicle 

every day until termination of the experiment (day 63). We 

tested three different fullerene derivatives (ABS-75, ABS-16, 

and the C60 fullerene core) and found that ABS-75 treatment 

initiated after disease onset reduced the clinical progression 

of chronic EAE in NOD mice (treated vs. control, pb0.05). 

Fullerene ABS-75 consists of a C60 carbon fullerene core to 

which a known NMDA receptor ligand was attached. ABS-75 

treatment also reduced axonal loss, demyelination (as 

evaluated by silver and Luxol fast blue staining, respectively), 

and CD11b+ infiltration in the white matter of mice. 

In vitro, ABS-75 was able to protect neurons from glutamateinduced 

toxicity and to reverse reduced glutamine synthetase 

expression in astrocytes under inflammatory insult. Our 

data demonstrate a neuroprotective effect of a treatment 

with a fullerene compound combined with a NMDA receptor 

ligand that may have applicability in the treatment of 

progressive MS and other neurodegenerative diseases. 

doi:10.1016/j.clim.2007.03.069 

S151 

Su.29 Human Blood−brain Barrier-associated DCs 

Originate from Blood Monocytes and Polarize CD4+ 

Lymphocytes into Th17 or Th1 

Igal Ifergan, PhD Student, CHUM-Hopital Notre-Dame, 


Hania, Kebir, PhD student, CHUM-Hopital Notre-Dame, 


Nathalie Arbour, PhD, CHUM-Hopital Notre-Dame, 


Alexandre Prat, MD, PhD, CHUM-Hopital Notre-Dame, 

Neuroimmunology Department, Montreal, QC, Canada


Trafficking of antigen presenting cells (APC) into the 

central nervous system (CNS) is essential for lymphocyte 

reactivation within the CNS compartment. The origin and 

function of the APC found in CNS autoimmune or inflammatory 

lesions remain, however, controversial. We demonstrate 

that blood CD14+ monocytes migrate across the 

inflammed blood–brain barrier (BBB) and differentiate into 

CD209+CD83+ (myeloid) or CD123+ (plasmacytoid) dendritic 

cells (DCs) under the influence of CNS endothelial-secreted 

transforming growth factor-beta (TGF-β) and granulocyte 

macrophage colony stimulating factor (GM-CSF). We also 

demonstrate that while CD123+ DCs secrete interleukin-12 

p70 (IL-12p70) and promote the proliferation and expansion 

of interferon-γ-secreting Th1 CD4+ lymphocytes, CD209+ 

CD83+ DCs secrete high levels of TGF-β and IL-6 and skew 

CD4+ lymphocytes towards a Th17 phenotype. Using multiple 

sclerosis as a prototypical human CNS autoimmune 

disease, we confirmed by immunohistochemistry the abundance 

of such perivascular CD83+ and CD123+ DCs within 

acute lesions, closely associated with microvascular BBBendothelial 

cells. Our data support the notion that 

functional perivascular myeloid and plasmacytoid CNS DCs 

arise as a consequence of migration of blood CD14+ 

monocytes across the human BBB, through the concerted 

actions of TGF-β and GM-CSF. 

doi:10.1016/j.clim.2007.03.071 

Su.31 Characterization of Single B Cell 

Immunoglobulin Variable Region Genes Derived 

from Infiltrated Muscle Tissue of Subjects with 

Inflammatory Myopathies 

Elizabeth Bradshaw, Postdoctoral Fellow, Laboratory of 

Molecular Immunology, Center for Neurologic Diseases, 

Brigham and Women’s Hospital, Boston, MA, Ana Orihuela, 

Research Technician, Children’s Hospital Informatics 

Program at the Harvard-MIT Division of Health Sciences and 

Technology, Boston, MA, Shannon McArdel, Research 

Technician, Laboratory of Molecular Immunology, Center for 

Neurologic Diseases, Brigham and Women’s Hospital, 

Boston, MA, David Hafler, Professor, Laboratory of 

Molecular Immunology, Center for Neurologic Diseases, 

Brigham and Women’s Hospital, Boston, MA, Anthony 

Amato, Associate Professor, Department of Neurology, 

Division of Neuromuscular Disease, Brigham and Women’s 

Hospital and Harvard, Boston, MA, Kevin O’Connor, 

Assistant Professor, Laboratory of Molecular Immunology, 

Center for Neurologic Diseases, Brigham and Women's 

Hospital, Boston, MA 

The inflammatory myopathies (IM) are putative autoimmune 

disorders characterized by muscle weakness and the 

presence of inflammatory infiltrates in skeletal muscle. 

Varying numbers of both B cells and plasma cells are among 

the muscle tissue infiltrate found in these diseases. Through 

examination of immunoglobulin heavy chain variable regions, 

we previously demonstrated that there was significant 

oligoclonal expansion of B cells found in IM muscle. To extend 

these findings, we isolated single B cells from IM muscle by 

laser capture microdissection or FACS, then examined the 

paired immunoglobulin heavy and light chain variable region 

sequences. B cell immunoglobulin variable region cDNAs were 

sequenced affording identification of the isotype, CDR3 and 

the degree of somatic mutation in the entire V-region. A 

pattern of B cell affinity maturation was evident in clones 

that had accumulated varying numbers of common and 

unique somatic mutations. These data suggested that a 

local inflammatory response occurs within the muscle tissue 

of patients with IM that may indicate the presence of a B cell 

antigen-specific response. 

doi:10.1016/j.clim.2007.03.072 

Su.32 Myelin-specific Regulatory T Cells Accumulate 

in the Central Nervous System, but Fail to Suppress 

Pathogenic Effector T Cells at the Peak of 

Autoimmune Inflammation 

Thomas Korn, Postdoctoral Fellow, Harvard Medical School, 

Center for Neurologic Diseases, Boston, MA, Mohamed 

Oukka, Instructor, Harvard Medical School, Center for 

Neurologic Diseases, Boston, MA, Jay, Reddy, Instructor, 

Harvard Medical School, Center for Neurologic Diseases, 

Boston, MA, Estelle Bettelli, Instructor, Harvard Medical 

School, Center for Neurologic Diseases, Boston, MA, Amit 

Awasthi, Postdoctoral Fellow, Harvard Medical School, 

Center for Neurologic Diseases, Boston, MA, Raymond Sobel, 

Associate Professor of Pathology, Stanford University, 

Department of Pathology, Stanford, CA, Kai Wucherpfennig, 

Associate Professor of Neurology, Harvard Medical School, 

Dana Farber Cancer Institute, Department of Cancer 

Immunology and AIDS, Boston, MA, Vijay Kuchroo, Professor 

of Neurology, Harvard Medical School, Center for Neurologic 

Diseases, Boston, MA 

Treatment with ex vivo generated regulatory T cells (Treg) 

has been regarded as highly attractive therapeutic 

approach for autoimmune diseases. However, the dynamics 

and function of T-reg in autoimmunity are not well understood. 

Thus, we developed Foxp3gfp “knock-in” mice and 

myelin oligodendrocyte glycoprotein (MOG)35–55/IAb tetramers 

to track autoantigen-specific effector T cells (T-eff) 

and T-reg in vivo during experimental autoimmune encephalomyelitis, 

an animal model for Multiple Sclerosis. 

Following immunization with the encephalitogenic peptide 

MOG35–55 emulsified in complete Freund's adjuvant, 

MOG35–55-tetramer-reactive, Foxp3+ T-reg expanded in 

the peripheral lymphoid compartment and readily accumulated 

in the central nervous system (CNS), but did not 

prevent the onset of disease. During disease onset, the MOGtetramer+ 

T-eff population in the CNS increased faster than 

the population of antigen-specific T-reg. At the peak of 

disease, the ratio of T-reg vs. T-eff was 1:17 which dramatically 

changed into 1:2 at the beginning of recovery. 

Foxp3+ T-reg isolated from the CNS were fully competent in 

suppressing naive MOG-specific T cells. However, Foxp3+ Treg 

failed to control encephalitogenic T-eff which in contrast 

to T-eff from the peripheral immune compartment, secreted 

IL-6 and TNF when they were isolated from the CNS at the 

peak of disease. Our data suggest that in the face of inflammation, 

the regulation of autoimmunity by CD4+Foxp3+ 

T-reg in situ may not be accomplished simply by changing 

the numerical balance of antigen-specific pathogenic vs.

Abstracts 

regulatory T cells, but may require the control of tissue 

inflammation as well. 

doi:10.1016/j.clim.2007.03.073 

Su.34 Proteomic Profiling of Multiple Sclerosis 

Lesions Identify Potential Targets for Therapy 

May H. Han, Postdoctoral Fellow, Department of Neurology 

and Neurological Sciences Stanford University, Stanford, 

CA, Sunil Hwang, Postdoctoral Fellow, Department of 

Physiology, University of Conneticut Health Center, 

Farmington, CT, Dolly Roy, Neurologist, Department of 

Neurology and Neurological Sciences, Stanford University, 

Stanford, CA, Cedric Raine, Professor, Department of 

Pathology, Albert Einstein College of Medicine, Bronx, NY, 

William H. Robinson, Assistant Professor, Departments of 

Medicine and Immunology, Stanford University, Stanford, 

CA, Raymond A. Sobel, Professor of Pathology, Department 

of Pathology, Stanford University, Stanford, CA, David Han, 

Associate Professor, Department of Physiology, University of 

Conneticut Health Center, Farmington, CT, Lawrence 

Steinman, Professor, Department of Neurology and 

Neurological Sciences, Stanford, CA 

Demyelinating lesions or plaques in multiple sclerosis (MS) 

have distinct histological features that reflect their stage of 

evolution. To identify key molecules involved at different stages 

during lesion progression, we performed proteomic profiling of 

three types of MS plaques using mass spectrometry and bioinformatics. 

Fresh frozen autopsy brain samples from MS 

patients (n=6) and age-matched controls (n=2)wereclassified 

as 1) acute plaque (AP, characterized by presence of inflammatory 

cells, ongoing demyelination), 2) chronic active plaque 

(CAP, characterized by chronic demyelination, astrogliosis with 

active rim) or 3) chronic plaque (CP, characterized by lack of 

inflammatory cells, dense astrogliosis). Samples from plaques 

were isolated by laser capture microdissection and global 

protein identification was carried out by SDS-PAGE, in-gel 

protease digestion, liquid chromatography and mass spectrometry. 

A total of 3894 proteins were identified out of which 2184 

proteins were unique to MS lesions. Then we identified proteins 

uniquetoeachtypeoflesion(AP(N=172), CAP (N=248) and CP 

(N=252), and proteins common to all three types (N=89) using 

bio-informatics software INTERSECT. Analysis by software 

PROTEOME 3D identified proteins of coagulation cascade and 

neural regeneration which may be potential targets for therapy. 

These results provide the first and most comprehensive 

information on global protein expression in MS plaques, enhance 

our understanding of the molecular pathogenesis of MS lesions 

and identify potential key molecules involved in disease 

progression that offer possibilities for therapeutic targets. 

doi:10.1016/j.clim.2007.03.075 

Su.35 A Genome-wide Screen for Genetic Variants 

Affecting the Expression of Immunologically 

Relevant Cell Surface Markers 

Philip De Jager, Assistant Professor, Department of 

Neurology, Brigham and Women’s Hospital, Boston, MA, 

Roman Yelensky, Student, Medical and Population Genetics 

Program, Broad Institute, Cambridge, MA, Elizabeth Rossin, 

Data Analyst, Medical and Population Genetics Program, 

Broad Institute, Boston, MA, Sasha Bonadkar, Research 

Assistant, Medical and Population Genetics Program, Broad 

Institute, Cambridge, MA, Edwin Choy, Director, Sarcoma 

Research, Medical and Population Genetics Program, Broad 

Institute, Cambridge, MA, Mark Daly, Assistant Professor, 

Medical and Population Genetics Program, Broad Institute, 

Cambridge, MA, David Altshuler, Associate Professor, 

Medical and Population Genetics Program, Broad Institute, 

Cambridge, MA, David Hafler, Professor, Department of 

Neurology, Brigham and Women's Hospital, Boston, MA 

While a few alleles affecting the expression level of immunologically 

relevant molecules have been identified and 

validated, our knowledge of human polymorphisms associated 

with differences in immune function remains very 

limited. We have therefore evaluated the use of the HapMap 

lymphoblastic cell lines (LCLs) as a platform to discover such 

polymorphisms on a large scale using a test panel of cell 

surface molecules that includes 15 cell-surface markers: 

CD19, CD20, CD21, CD40, CD58, CD80, CD86, CD95, CD227, 

HLA DQ, HLA DR, IgD, IgG, IgM, and IL6R. We then correlated 

the mean expression level of a particular molecule in each 

LCL with the over 2 million genotypes available in the 

HapMap database. Amongst several positive results, this 

analysis reveals that certain SNPs in the vicinity of the HLA 

DQA1 gene are correlated with both the level of expression of 

HLA DQ on the LCL surface and the level of mRNA expression 

for HLA DQA1. One of these SNPs, rs9272346, is a null allele 

of HLA DQA1 and is associated to this immunophenotype at a 

level that exceeds our predetermined level of genome-wide 

significance (P b2.6×10 −8 ), validating our approach. In 

exploring the mRNA data further within the MHC class I and 

class II clusters, it is clear that several different polymorphisms 

exist that affect the level of expression of these 

molecules. We are currently assessing the role of these 

functional variants in the association of the MHC with MS 

susceptibility. 

doi:10.1016/j.clim.2007.03.076 

S153 

Su.36 Novel Computational Methods to Enhance the 

Analysis of Cytometric Immunophenotypes 

Philip De Jager, Assistant Professor, Department of 

Neurology, Brigham and Women’s Hospital, Boston, MA, 

Saumyadipta Pyne, Postdoctoral Fellow, Cancer Program, 

Broad Institute, Cambridge, MA, Elizabeth Rossin, Data 

Analyst, Medical and Population Genetics Program, Broad 

Institute, Cambridge, MA, Jill Mesirov, Director, Cancer 

Program, Broad Institute, Cambridge, MA, Pablo Tamayo, 

Staff Scientist, Cancer Program, Broad Institute, 

Cambridge, MA, David Hafler, Professor, Department of 

Neurology, Brigham and Women’s Hospital, 

Boston, MA 

The current method for analyzing raw flow cytometry 

information involves gating, the process of first visualizing 

cell-surface marker expression values for a population of 

cells and then manually selecting distinct cell populations on


which to report statistics. Thus, manual processing is timeconsuming 

and is limited to 2-dimensional projections. Furthermore, 

these cell populations are described by statistics 

that fail to characterize their full distribution and shape. We 

propose two automated methods to improve these shortcomings: 

(1) histogram mean shape comparison: this method 

creates an n+1-dimensional histogram for n-dimensional 

data. Expression data is then parsed into n+1-dimensional 

bins, and individual bin values can be compared across 

categories of subjects or samples to discover cell populations 

that differ across sample categories. (2) Hierarchical mixture 

model: this method characterizes the expression values of 

each cell population in n dimensions using a hierarchical 

mixture model. The mixture model clusters individual cells 

by assigning them to distributions with maximum likelihood. 

A mixture of distributions that make up a model thus 

characterizes the n dimensional data. This method allows 

the cells to exist in distinct distributions rather than be 

collapsed to a statistic, and the distribution of cells can then 

be compared across samples. Both methods have been 

implemented to characterize the expression of 15 molecules 

on the 269 HapMap lymphoblastic cell lines. We present a 

comparison of both methods against manually processed 

data to demonstrate the most robust pipeline with which to 

characterize cell population structure and calculate expression 

levels using cytometric data. 

doi:10.1016/j.clim.2007.03.077 

Su.37 Improving Elispot to Detect Antigen Specific 

T Cells 

Khadir Raddassi, Research Instructor, Brigham and Women’s 

Hospital, Neurology, Boston, MA, Kasia Bourcier, Scientist, 

Immune Tolerance Network, San Francisco, CA, Jose 

Estevam, Research Assistant, Brigham and Women's 

Hospital, Neurology, Boston, MA, Vicki, Seyfert-Margolis, 

Scientist, Immune Tolerance Network, San Francisco, CA, 

David Hafler, Lab Director, Brigham and Women’s Hospital, 

Neurology, Boston, MA 

A major interest of our Immune Tolerance Network assay 

group is to develop and validate assays to detect auto- and 

allo-antigen reactive cells. In collaboration with Dr. Peakman 

(Kings College, London) we have developed a modified 

elispot protocol. We tested the sensitivity of this improved 

elispot to detect myelin-reactive T cells in multiple sclerosis 

patients (MS) and healthy controls (HC). Fresh or cryopreserved 

PBMCs were exposed to myelin antigens or tetanus 

toxoid (TT). After 42 hours the cells were transferred to 

elispot plates for 16 hours; the elispot plates were developed 

to detect the frequency of IFNg secreting cells. Comparing 

side by side elispot and flow cytometry regarding IFNg 

production in response to TT showed similar results, but 

elispot was more sensitive at low doses stimulation. The 

detection of antigen reactive cells by elispot could be 

performed on fresh and cryopreserved PBMCs. We validated 

this method between different labs, the data were well 

correlated (r2=0.987). Using IL-7 enhanced the response to 

weak stimuli. Taking advantage of this improved elispot and 

IL-7, we examined the reactivity of PBMCs from 13 HC and 11 

MS. In the presence of IL-7 PBMCs from MS showed high 

reactivity to human MBP (30±5 spots for MS vs. 17±4 for HC) 

and to a pool of myelin peptides (46 ±8 spots for MS vs. 15±4 

for HC). This modified elispot would provide a quick method 

to monitor immune reaction in clinical research. The fast 

read out allows the cells to be recovered and used for further 

testing. 

doi:10.1016/j.clim.2007.03.078 

Su.38 TIM-3 is a Negative Regulator of Human Th1 

Cells 

David E. Anderson, PhD, Brigham and Women’s Hospital and 

Harvard Medical School, Boston, MA, Juhi Kuchroo, Womens 

Hospital and Harvard Medical School, Boston, MA, Nasim 

Kassam, Brigham and Women’s Hospital and Harvard 

Medical School, Boston, MA, David Hafler, Jack, Sadie and 

David Breakstone Professor of Neurology (Neuroscience), 

Brigham and Womens Hospital and Harvard Medical School, 

Boston, MA 

The relevance and utility of the Th1/Th2 paradigm has 

been well established in murine models of a many human 

immune-mediated diseases. However, data on the role and 

regulation of Th1 and Th2 cells in modulating immune 

responses in humans is less clear. TIM-3 is a molecule 

selectively expressed on a subset of murine IFN- 3 -secreting 

Th1 cells but not Th2 cells, and regulates Th1 immunity and 

tolerance in vivo. To determine if TIM-3 similarly identifies 

and regulates Th1 cells in humans, we generated a panel of 

monoclonal antibodies specific for human TIM-3. We report 

that TIM-3 is preferentially expressed on the surface of in 

vitro polarized human Th1 cells. TIM-3 is not readily 

apparent on the surface of CD4+ T cells examined ex vivo, 

but T cell activation rapidly induces cell surface expression 

of TIM-3 on a subset of IFN- 3 -secreting CD4+ T cells. More 

specifically, highest expression of TIM-3 is present on T cells 

secreting the greatest amounts of IFN- 3 . Functionally, 

human T cells secrete elevated levels of IFN- 3 when 

stimulated in the presence of TIM-3-specific antibodies, 

with no effect on other cytokines including TNF-α and IL- 

13. These results demonstrate that TIM-3 preferentially 

regulates IFN- 3 -secretion from human Th1 cells and may 

influence a variety of human diseases including cancer and 

autoimmunity. 

doi:10.1016/j.clim.2007.03.079 

Su.39 Rapamycin Inhibits Autocrine Signaling 

(IL-6, IL-10) in Viral Lymphomas 

Sang-Hoon Sin, Postdoctoral Research Associate, University 

of North Carolina Chapel Hill, Chapel Hill, NC, Debasmita 

Roy, Graduate Student, University of North Carolina Chapel 

Hill, Microbiology & Immunology, Chapel Hill, NC, 

Dhavalkumar Patel, Professor, University of North Carolina, 

Chapel Hill, Chapel Hill, NC, Dirk Dittmer, Assistant 

Professor, University of North Carolina, Chapel Hill, 

Microbiology & Immunology, Chapel Hill, NC 

The mTOR inhibitor, rapamycin (sirolimus) is known for its 

immunosuppressive activity and more recently has been

Abstracts 

subject of intense investigations as an anti-cancer agent. 

Here we show that a group of viral malignancies, which 

critically dependent in inflammatory cytokines (and thus 

have sometimes be described as viral inflammatory lesions) 

are susceptible to rapamycin. We found that rapamycin was 

efficacious against primary effusion lymphoma in culture and 

in a murine xenograft model; that rapamycin inhibited mTOR 

signaling and that this correlated with inhibition of IL-10, IL- 

6, IFNgamma, IP-10, and IL-12p40 secretion (as measured by 

Luminex and ELISA). Moreover, addition of exogenous IL-10 or 

IL-6 could be reversed the rapamycin growth arrest. This 

validates sirolimus as a new treatment option for viral 

malignancies that depend on inflammatory cytokines. 

doi:10.1016/j.clim.2007.03.080 

Su.40 The Transcription Factor GATA-3 Regulates 

IL-4 Responses in B cells 

Dee Aud, Research Scientist, Roche Palo Alto, Palo Alto, CA, 

Sung-Yun Pai, Instructor, Dana Farber Cancer Institute, 

Boston, MA, Stanford Peng, Director, Arthritis Research, 

Roche Palo Alto, Palo Alto, CA, I-Cheng Ho, Associate 

Professor, Brigham and Women's Hospital, Boston, MA 

The GATA-3 transcription factor plays a central role in T 

helper type 2 development, but its role in B cells has not been 

well described, even though GATA-3 is present in naïve B cells. 

To study the role of GATA-3 in B cells, mice with a floxed Gata3 

allele (Gata3fl/fl) mice were crossed to CD19-Cre transgenic 

mice, allowing B cell-specific deletion of GATA-3. B cell 

development was grossly normal in Gata3fl/fl CD19-Cre, but 

surprisingly, Gata3fl/fl CD19-Cre B cells showed a decreased 

ability to proliferate (in an IL-4 dose dependent manner) but 

an increased ability to produce IgG1 and IgE in response to 

anti-CD40 and/or IL-4 in vitro. These findings may reflect an 

interaction between GATA-3 and type 2 Ig-regulating transcription 

factors such as NF-kB or STAT6, because GATA-3 can 

modulate the activities of these factors in vitro. These 

findings demonstrate a critical yet surprising role for GATA-3 

in the regulation of IL-4-mediated responses in B cells. 

doi:10.1016/j.clim.2007.03.081 

Su.41 Natural and Synthetic TLR7 Ligands Inhibit 

CpG-A- and CpG-C-ODN-induced IFN-α Production 

by Plasmacytoid Dendritic Cells 

Holger Hackstein, Resident, University of Giessen, Giessen, 

Germany, Beate Berghöfer, PhD Student, University of 

Giessen, Giessen, Germany, Gregor Bein, Professor, 

University of Giessen, Giessen, Germany 

Plasmacytoid dendritic cells (pDC) are unique with respect 

to their capacity to produce unsurpassed amounts of 

IFN-α and coexpress TLR7 and TLR9, mediating IFN-α 

production. Although TLRs are critical receptors of innate 

immunity, little is known about the immunological effects of 

TLR7/TLR9 costimulation. We have analyzed the effects of 

TLR7/TLR9 costimulation on IFN-α production by leukocytes 

and pDCs. Our experiments revealed that both synthetic 

(resiquimod, loxoribine) and natural (ssRNA40) TLR7 ligands 

abrogate CpG-A- and CpG-C-ODN-induced IFN-α production 

by human leukocytes. Since TLR7 ligands themselves 

represent important IFN-α inducers, we demonstrated that 

substimulatory TLR7 ligand concentrations significantly 

inhibited CpG-A-induced IFN-α. Delayed addition of TLR7 

ligands still resulted in complete suppression of CpG-A-ODNinduced 

IFN-α production, suggesting that the inhibition is 

unlikely to be caused by a kinetic uptake advantage. Unlike 

for CpG-A and CpG-C, TLR7 ligands did not inhibit CpG-B- 

ODN-induced IFN-α production. Experiments with purified 

human pDCs demonstrated that the inhibitory effects of 

TLR7/TLR9 costimulation were mediated directly by pDCs. 

Suppression of IFN-α production was not related to increased 

cell death and was also detectable in enriched mouse pDCs. 

Analyses of pDCs suggested that the TLR7 signal regulates the 

outcome of TLR7 ligand/CpG-A-ODN costimulation and can 

either inhibit (IFN-α) or promote (IL-8/CD40) cytokine and 

surface marker expression. Our data reveal for the first time 

a strong inhibitory effect of TLR7 stimulation on IFN-α 

production induced by CpG-A- and CpG-C-ODNs. These 

findings provide novel insight into the effects of TLR7/TLR9 

costimulation and may support the development of novel 

TLR9 inhibitors. 

doi:10.1016/j.clim.2007.03.082 

S155 

Su.42 Association of MHC Class II Haplotypes and 

Immunogenicity of a Therapeutic Biological Protein 

Danika Wullner, Senior Associate, Amgen, Thousand Oaks, 

CA, Erica Yue, Associate, Clinical Immunology, Thousand 

Oaks, CA, Steven J. Swanson, Executive Director, Clinical 

Immunology, Thousand Oaks, CA, Vibha Jawa, Senior 

Scientist, Clinical Immunology, Thousand Oaks, CA 

A mature high affinity antibody response that is T cell 

driven can be elicited during administration of biological 

therapeutics in humans. The processing of the protein 

antigen by antigen presenting cells and their binding to 

Class II HLA molecules is necessary for the antibody response 

to mature. In this study, the predisposition of certain MHC 

haplotypes to present peptides derived after processing a 

recombinant protein (FPX-protein) is discussed. Ex vivo T 

cell priming assays were used to predict immunogenicity of 

Class II restricted T cell epitopes contained within the FPXprotein. 

The immunogenic, promiscuous epitope(s) are 

contained within the 14-amino acid carboxy-terminal region 

of the FPX-peptide, but none are found in the aminoterminal 

region comprising 10 amino acids as observed by in 

silico prediction and in vivo data. Peripheral blood mononuclear 

cells (PBMCs) from haplotyped donors were challenged 

with multiple rounds of FPX peptides (C-terminal and 

N-terminal) for 7–14 days followed by ELISPOT assays for T 

lymphocyte activation. HLA haplotype DRB1*0701/1301 was 

associated with an increased number of spot forming cells 

when challenged with the immunogenic C-terminal FPXpeptide 

(10.15 fold increase) and negligible with the nonimmunogenic 

N-terminal (1.38 fold increase). The same 

haplotype was associated with a high antibody response and 

a memory T cell response when the protein was administered 

in vivo as well as by in silico prediction. Ex vivo T cell 

priming assays can be potential tools for prediction of


immunogenicity of T cell epitopes. These assays enable 

selection of the right therapeutic in early development and 

determine which MHC haplotypes are predisposed to an 

immune response. 

doi:10.1016/j.clim.2007.03.083 

Su.43 Hepatitis B Virus Proteins Induce Activation of 

hfgl2 Prothrombinase Gene Through c-Ets-2 

Transcription Factors 

Meifang Han, Doctor in Charge, Tongji Hospital, Huazhong 

University of Science and Technology, Department of 

Infectious Disease, Wuhan, China, Dong Xi, Assistant 

Researcher, Department of Infectious Disease, Tongji 


Wuhan, China, Weiming Yan, Assistant Researcher, 

Department of Infectious Disease, Tongji Hospital, 


China, Gary Levy, Professor, Chief Physician, University 

Health Network, University of Toronto, Toronto, ON, 

Canada, Mingfeng Liu, Postdoctoral Researcher and 

Associate, University Health Network, University of 

Toronto, Toronto, ON, Canada, Xiaoping Luo, Chief 

Physician, Department of Pediatrics of Tongji Hospital, 


China, Qin Ning, Chief Physician, Department of Infectious 

Disease, Tongji Hospital, Huazhong University of Science 

and Technology, Wuhan, China 

Objective: To determine the viral and host factors 

involved in the transcription of hfgl2 gene which was 

demonstrated to play pivoral role in human and experiment 

fulminant viral hepatitis. Methods. HBc, HBs or HBx expression 

plasmids were constructed and cotransfected with a 

hfgl2 luciferase report construct into eukaryotic cells. 

Results. Luciferase assay showed that HBc or HBx protein, 

but not HBs protein significantly enhanced hfgl2 transcription 

in both CHO and HepG2 cells. Series deletion assay of 

hfgl2 gene promoter demonstrated that a strong regulatory 

domain from −712 to −568 was responsible for hfgl2 gene 

transcription in response to HBc or HBx proteins. By sitedirected 

mutagenesis, the overlapped cis-elements LEF/c- 

Ets in domain −712/−568 was demonstrated to play an 

important role in the regulation of hfgl2 gene in response 

to HBc protein, while another two cis-elements LEF/c-Ets 

and HSTF in the same domain were found to participate in 

hfgl2 transcription regulation in response to HBx protein. 

EMSA assays showed that HBc and HBx proteins did not 

directly induce hfgl2 gene activation, while an Ets family 

member c-Ets-2 bound the cognate cis-element in hfgl2 

promoter was responsible for induction of hfgl2 activation 

in response to viral proteins. Conclusion. Our study provides 

new insights in understanding the pathology of fulminant 

hepatic failure and the interaction between HBV virus and 

host gene expression. This work was supported by NSFC 

30225040, 30571643, 30672380, National Key Basic Research 

Program of China (2005CB522901, 2005CB522507) and Clinical 

Subject Key Project from the Ministry of Health. 

doi:10.1016/j.clim.2007.03.084 

Su.44 Arthritogenic Simulation of Human Synovial 

Fibroblasts by TWEAK 

Timothy Zheng, Senior Scientist, Biogen Idec, Inc., 

Cambridge, MA, Shawn Weng, Associate Scientist, Biogen 

Idec, Inc., Cambridge, MA, Suzanne Szak, Scientist II, Biogen 

Idec, Inc., Cambridge, MA, Linda Burkly, Distinguished 

Investigator, Biogen Idec, Inc., Cambridge, MA 

Synovial fibroblasts are critically involved in propagating 

joint inflammation in rheumatoid arthritis (RA) through the 

production of numerous arthritogenic mediators. The proinflammatory 

TNF ligand superfamily cytokine TWEAK has 

previously been shown to induce several chemokine/cytokines 

in human fibroblast cell types including synovciocytes. In this 

study, we seek to understand the involvement of TWEAK in 

human RA pathogenesis by studying its broad effect on human 

synovial fibroblasts. We found that synovial fluid TWEAK levels 

were elevated in RA patients when compared to those in OA 

patients, consistent with the notion that TWEAK may play a role 

in driving inflammatory response in the joints of RA patients. 

Using transcription profiling, we demonstrated that TWEAK 

consistently induced a large panel of genes, including many 

known arthritogenic mediators such as IL-1, IL-6, RANTES, ENA- 

78, IP-10 and MMPs. Interestingly, the potency of gene 

regulation by TWEAK when compared to TNF varied greatly 

amongst the four different donor cells, perhaps reflecting 

patient heterogeneity regarding the involvement of TWEAK visa-vie 

TNF in driving synoviocyte-mediated joint inflammation. 

Importantly, the production of TWEAK and TNF appeared to be 

independent of each other as neither TWEAK nor TNF induced 

or inhibited the expression of the other. When combined 

however, TWEAK and TNF synergically or additively induced the 

production of many other well-known arthritogenic mediators. 

Taken together, these results suggest that TWEAK and TNF may 

represent two concurrent upstream cytokines that regulate 

joint inflammation and damage in RA patients. 

doi:10.1016/j.clim.2007.03.085 

Su.45 IL-6/TH-17 Axis Plays a Crucial Role in the 

Generation of GPI-induced Arthritis 

Keiichi Iwanami, PhD Student, Graduate School of 

Comprehensive Human Science, University of Tsukuba, 

Tsukuba, Japan, Asuka Inoue, Master Student, Graduate 

School of Comprehensive Human Science, University of 

Tsukuba, Tsukuba, Japan, Isao Matsumoto, Instructor, 

Graduate School of Comprehensive Human Science, 

University of Tsukuba, Tsukuba, Japan, Mizuko Mamura, 

Research Fellow, Graduate School of Comprehensive Human 

Science, University of Tsukuba, Tsukuba, Japan, Yoko 

Watanabe, PhD Student, Graduate School of Comprehensive 

Human Science, University of Tsukuba, Tsukuba, Japan, 

Daisuke Goto, Instructor, Graduate School of 


Tsukuba, Japan, Satoshi Ito, Instructor, Graduate School of 


Tsukuba, Japan, Akito Tsutsumi, Assistant Professor, 

Graduate School of Comprehensive Human Science, 

University of Tsukuba, Tsukuba, Japan, Takayuki Sumida, 

Professor, Graduate School of Comprehensive Human 

Science, University of Tsukuba, Tsukuba, Japan

Abstracts 

Backgrounds: Glucose-6-phosphate isomerase (GPI)induced 

arthritis is a murine model of rheumatoid arthritis 

(RA). Although anti-CD4 antibodies (Abs) had therapeutic 

effect in this model, the pathogenicity of each CD4+ T cell 

lineage was uncertain. Here we undertook the present study 

to clarify GPI-specific T cell lineages and investigate their 

pathological and regulatory roles in the generation of 

arthritis. [Methods] DBA/1 mice were immunized with 

300fÊg of GPI to induce arthritis. 1) CD4+ T cells and APCs 

were co-cultured with GPI for 24 h, and the supernatant was 

analyzed by cytokine ELISA. 2) Anti-IFNfÁ mAb or anti IL-17 

mAb was injected on day 7 to investigate arthritis development. 

3) A mAb to murine IL-6 receptor (MR16-1) was 

injected on days 0, 3 or 8 to investigate arthritis development. 

4) After the injection of MR16-1 on days 0 or 3, 

draining lymph nodes (DLNs) were harvested on day 7, and 

TH-17 cells were analyzed by flow cytometry. Results: 1) 

IFNfÁ and IL-17 were produced by GPI-specific CD4+ T cells. 

2) The administration of anti-IL-17 mAb on day 7 

significantly ameliorated arthritis (Pb0.01), whereas that 

of anti-IFNfÁ mAb exacerbated. 3) The administration of 

MR16-1 on day 0 or 3 protected the induction of arthritis, 

and that of MR16-1 on day 8 significantly ameliorated 

arthritis (Pb0.05). 4) The differentiation of TH-17 in DLNs 

was markedly suppressed by MR16-1. Conclusions: IL-6 and 

TH-17 play an essential role in GPI-induced arthritis. Our 

study suggests that IL-6/TH-17 axis might be also involved 

in RA. 

doi:10.1016/j.clim.2007.03.086 

Su.46 The Serum Cytokine Profile in Low-Stage 

Chronic Lymphocytic Leukemia is a 7th Hallmark of 

Cancer 

Catherine Spier, Associate Professor, Pathology, University 

of Arizona College of Medicine, Department of Pathology, 

Tucson, AZ, Christopher Riley, Graduate Student, University 

of Arizona College of Medicine, Arizona Cancer Center, 

Tucson, AZ, James Choi, Fellow, University of Arizona 

College of Medicine, Arizona Cancer Center, Tucson, AZ, 

Lawrence Cooke, Research Specialist, University of Arizona 

College of Medicine, Arizona Cancer Center, Tucson, AZ, 

Thomas Klinkhammer, Fellow, University of Arizona College 

of Medicine, Arizona Cancer Center, Tucson, AZ, Daruka 

Mahadevan, Associate Professor, Medicine, University of 

Arizona College of Medicine, Arizona Cancer Center, 

Tucson, AZ 

Background: In B cell Chronic Lymphocytic Leukemia (B- 

CLL) there are no validated screening, diagnostic or 

prognostic serum markers for early detection. Several 

aberrant cytokines have previously been identified individually 

in patients with B-CLL that are thought to enhance 

malignant cell survival. Methods: An IRB approved protocol 

was used to collect and examine 120 serum cytokines from 

26 Rai Stage 0/1 patients who were treatment naive and 

also from 4 normal volunteers (RayBiotech Cytokine Array, 

#AAH-CYT-G1000). Fold change was calculated for each 

patient, corrected for absolute lymphocyte count and a 

mean ± SD obtained for each cytokine for all patients. 

Results: This cytokine profile identified all of the 

published cytokines associated with B-CLL cell survival 

(IL-1B, IL-2, IL-4, IL-6, IL-8, IL-10, TNFa, INFa or g, G-CSF 

or GM-CSF) or inhibition (IL-5, TGF-B). In this profile the 

1st ranked is INF-g (14.46 fold) which is known to prevent 

apoptosis of B-CLL cells. The 2nd ranked is IGFBP-4 (14.22 

fold) and 3rd ranked is G-CSF (10.89 fold) which is known 

to decrease B-CLL cell apoptosis. Other cytokines of note 

are Flt-3 ligand (7.82 fold), SCF (6.76 fold) and SDF-1 

(6.54 fold). Conclusion: Thus, our cytokine profile, that 

sampled 120 cytokines simultaneously, validates the existing 

data, lists the ctyokines by order of expression from 

highest to lowest and significantly adds to the list of 

cytokines identified as important for B-CLL cell survival in 

low stage patients. 

doi:10.1016/j.clim.2007.03.087 

Su.47 High Sensitivity Multiplexed Immunoassays 

for Simultaneous Quantification of Key Cytokines in 

Human Samples 

Jehangir Mistry, PhD, LINCO (now part of Millipore), St. 

Charles, MO, Jiaxin Dong, PhD, LINCO (now part of 

Millipore), St. Charles, MO, Terry Whitehead, NA, LINCO 

(now part of Millipore), St. Charles, MO, Shaoquan Ji, 

Research and Development Manager, LINCO (now part of 

Millipore), St. Charles, MO, Hank Hwang, PhD, LINCO (now 

part of Millipore), St. Charles, MO 

Low levels of inflammation play a key role in etiology of 

many disease states (e.g. atherosclerosis, allergy, cancer, 

diabetes). Understanding the role of inflammation in pathogenesis 

has been impeded because currently available cytokine 

assays are not sensitive enough to detect the 

differences between normal and subclinically inflammed 

states. Using the Luminex xMAP technology, we developed a 

highly sensitive, multiplexed immunoassay panel for simultaneously 

quantifying 13 human cytokines commonly involved 

in Th1/Th2 and inflammatory responses (IL-1β, IL-2, 

IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, GMCSF, 

IFNγ and TNFα). The assays follow the typical bead-based 

sandwich format and use a simple protocol. Each antibody 

pair is highly specific, with no or negligible cross-reactivity 

to other analytes within the panel. All assays in the panel 

have a common dynamic range of 0.128 to 2000 pg/ml, 

wide enough for almost all sample types. Sensitivities of the 

assays are between 0.01 to 0.48 pg/ml with most analytes 

at approximately 0.1 pg/ml. Normal serum samples are 80 

to 100% detectable for most cytokines. The assay robustness 

is demonstrated by acceptable precisions (CV=2.2– 

14.3% for inter-assay; CV=3.1–5.9% for intra-assay), accuracy 

(93.0 to 112%), and linearity of dilution (102 to 113%) 

for serum or plasma samples. No serum/plasma dilution is 

required. The assays may be used for other sample types (e. 

g. cell culture supernatant, tissue/cell lysate from human 

origin). This novel, robust and highly sensitive assay panel 

serves as an accurate, reproducible and economic tool for 

simultaneously quantifying multiple cytokines in human 

samples. 

doi:10.1016/j.clim.2007.03.088 

S157


Su.48 Use of a 10-Cytokine Multiplex RT-PCR in 

Conjunction with a Corresponding 10-plex 

Fluorescent Bead Quantitative Protein 

Immunoassay to Reveal Expression Patterns in 

Mitogen-Stimulated Human Peripheral Leukocytes 

Cynthia Boardman, Development Scientist, Beckman 

Coulter Inc. Nucleic Acid Testing Business, Fullerton, CA, 

Cynthia Boardman, Development Scientist, Beckman 

Coulter Inc. Nucleic Acid Testing Business, Fullerton, CA, Yu 

Suen, Staff Development Scientist, Beckman Coulter Inc. 

Biomarker Discovery and Automation Center, Fullerton, CA, 

Scott Boyer, Genomics Group Manager, Beckman Coulter Inc. 

Nucleic Acid Testing Business, Fullerton, CA, Keith Roby, 

Product Manager, Beckman Coulter Inc. Biomarker Discovery 

and Automation Center, Fullerton, CA 

The ability to monitor the expression of mediators of 

inflammation and immune response is critical to the study of 

disease and pharmacodynamics. Quantitative protein immunoassays 

are one of the standard tools for the examination 

of cellular responses but reveal only part of the cellular 

story. We show here how the GenomeLab GeXP Genetic 

Analysis System can be used to corroborate protein studies 

and provide a more complete picture of cellular responses 

to effectors. Specifically, we have developed a multiplexed 

RT-PCR† assay which can analyze the mRNA expression 

profile of ten Th1/Th2 cytokines simultaneously. The 

eXpress Designer software included in the GeXP System 

was used to design multiplex PCR primers for the following: 

IFN-g, IL-1B, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-α and 

TNF-B; and the resulting multiplex was used to analyze 

changes in the mRNA expression profiles for these ten 

products in human peripheral leukocytes upon stimulation 

with LPS or PMA/PHA. The data were then used to corroborate 

results of a quantitative protein analysis performed 

on the matching cell supernatant samples using the 

FlowCytomix Multiplex Human Th1/Th2 10-Plex fluorescent 

bead immunoassay kit and analyzed on the Cytomics FC500 

MPL flow cytometry system. This dual-platform approach 

can be used to monitor the immune response effected by 

diseases and treatments at the level of both mRNA and 

protein expression. 

doi:10.1016/j.clim.2007.03.089 

Su.49 Dysregulated Inflammatory Cytokine 

Response in Lipopolysaccharide- and 

Peptidoglycan-Stimulated Monouclear Cells of a 

Patient with Blau Syndrome 

Sang Wook Son, Professor, Department of Dermatology, 

Korea University College of Medicine, Ansan-si, Korea, Jin 

Lee, Doctor, Department of Pediatrics, Korea University 

College of Medicine, Ansan-si, Korea, Jae Eun Choi, Doctor, 

Department of Dermatology, Korea University College of 

Medicine, Ansan-si, Korea, Il Hwan Kim, Professor, 


Medicine, Ansan-si, Korea, Jeongwon Sohn, Professor, 

Department of Biochemistry, Korea University College of 

Medicine, Seoul, South Korea, Young Chul Kye, Profesor, 


Medicine, Ansan-si, Korea, Kwang Chul Lee, Professor, 

Department of Pediatrics, Korea University College of 

Medicine, Ansan-si, Korea, JungHwa Lee, Professor, 

Department of Pediatrics, Korea University College of 

Medicine, Ansan-si, Korea 

Inheritable Blau syndrome (BS) and sporadic early-onset 

sarcoidosis (EOS) share characteristic clinical features of 

multi-organ granulomatous inflammation involving joints, 

skin and eyes. Multiple mutations in the caspase recruitment 

domain gene CARD15 were described in BS. EOS was 

diagnosed in a 5-month-old girl who initially had biopsyproven 

chronic non-caseating granulomatous papules on a 

whole body and later developed joint abnormalities and 

steroid-responsive hypercalcemia. Her 32-year-old mother 

was blind since she was about 5 years old and showed 

camptodactyly and eczematous skin lesions. All of the other 

mother side family members were healthy with no evidence 

of any skin, joint and eye abnormalities. DNA analysis 

demonstrated a missense mutation, 1000CNT (R334W), in 

the CARD15 gene in both the mother and the child, 

suggesting sporadic incidence of the mutation in the mother 

and its inheritance to her child. When peripheral blood 

mononuculear cells of the mother were stimulated by 

lipopolysaccharide and peptidoglycan, production of an 

anti-inflammatory cytokine, IL-10 was reduced compared 

to the normal control. In addition, induction of TNF-α 

production was reduced when stimulated with peptidoglycan 

alone. Basal levels of both cytokines in the mother were not 

different compared to the normal control. These findings 

indicate that BS and EOS are diseases of the same spectrum 

sharing the common etiology of CARD15 mutation and that a 

dysregulated anti- and pro-inflammatory cytokine response 

to the stimulation may lead to the chronic inflammatory 

manifestations in BS. 

doi:10.1016/j.clim.2007.03.090 

Su.50 Quantitative Evaluation of Interleukin-6, 

Interleukin-11 and Interleukin-17 in Healthy and 

Inflammatory Tissues of Gingival 

Hamid Reza, ArAB, Dentist, Periodontology Department, 

School of Dentistry and Mashhad Dental Research Center, 

Mashhad, Iran, Jalil Tavakkol-Afshari, Vice President for 

Research; Head, Department of Immunogenetics and Cell 

Culture, Bu-Ali Research Institute (BARI), Department of 

Immunogenetics and Cell Culture, Mashhad, Iran, Zahra 

Baghani, Dentist, Periodontology Department, School of 

Dentistry and Mashhad Dental Research Center, Mashhad, 

Iran, Nasrin Moheghi, Researcher, Bu-Ali Research Institute 

(BARI), Department of Immunogenetics, Mashhad, Iran 

Interleukin 11(IL-11), IL-17 and IL-6 are cytokines that 

modulate the inflammatory process and have not been 

assessed within normal or inflammed gingival tissues. Our 

purpose was comparing the concentrations of human IL-6, IL- 

11 and IL-17 within healthy and diseased human gingival 

tissues to determine their possible role in the initiation or 

progression of periodontal disease. Biopsies from healthy 

(non-hemorrhagic gingiva adjacent to the 3 mm, 4–5 mm and 

â‰¥ 6 mm periodontal pockets) were studied. Interleukin- 

11, IL-17 and IL-6 concentrations were assessed within

Abstracts 

solubilized gingival biopsies by enzyme-linked immunosorbent 

assay. Data were analyzed by Kruskal–Wallis and Mann– 

Whitney U tests for finding significant correlation between 

groups. Interleukin 11, IL-17 and IL-6 concentrations were 

decreased within gingival adjacent to 4–5 mm sulcular depth 

but later increased within gingiva adjacent to â‰¥ 6mm 

pocket depths. The amount of IL-17 between healthy tissue 

and gingiva adjacent to â‰¥ 6 mm pockets and 4–5 mm and â 

‰¥ 6 mm pockets was significant (pb0.05). Interleukin-11, 

IL-6 and IL-17 concentrations were greatly correlated with 

sulcular depth. However, in most cases this difference was 

not statistically significant. Interleukin-6, IL-17, IL-11 concentrations 

were increased in the inflammatory gingival 

tissue adjacent to â‰¥ 6 mm pockets in comparison to 

healthy gingiva but only the concentration of IL-17 was 

significant (p=0.03). 

doi:10.1016/j.clim.2007.03.091 

Su.51 Inhibition of Experimental Tumor Metastasis 

by CpG Oligodeoxynucleotide-Pulsed Dendritic Cell 

Han-A Kim, Assistant, Department of Biological Sciences, 

Kwangju, South Korea, Hyun-Mi Ko, Department of 

Biological Sciences, Kwangju, South Korea, Hern-Ku Lee, 

Professor, Department of Immunology, Chonju, South Korea, 

Suhn-young Im, Professor, Department of Biological 

Sciences, Kwangju, South Korea 

Dendritic cell (DC) pulsed with tumor antigen induced 

tumor-specific immune response. CpG ODN has been recognized 

as a powerful stimulant of DC. This study was 

designed to investigate whether CpG oligodeoxynucleotide 

(ODN)-pulsed DC could inhibit the experimental pulmonary 

metastasis of B16F10 murine melanoma. Treatment of CpG 

ODN intraperitoneally either before or after B16F10, it 

inhibited lung colonization of B16F10. Bone marrow-derived 

DC pulsed with B16F10 lysate inhibited the tumor metastasis. 

In this case, the inhibitory effect of mature DC was 

much more prominent than of immature DC. In addition, the 

inhibitory effect DC was further augmented when DC was 

stimulated with CpG ODN. The magnitude of experimental 

pulmonary metastasis was increased in IL-12 knock out 

(IL-12−/−) mice compared with their littermates. CpG ODN 

did not exert its metastasis-inhibiting activity in IL-12−/− 

mice. Moreover, DC originated from IL-12−/− mice do not 

inhibited metastasis even when the cells were stimulated 

with B16F10 lysate plus CpG ODN. In vitro, CpG ODN induced 

the gene expression and protein synthesis of IL-12 in DC. 

Tumor metastasis was inhibited by adoptive transfer of 

splenic T cells from CpG ODN-treated mice. These results 

suggest that CpG ODN inhibited tumor metastasis through 

activating DC to produce IL-12, which resulted in the 

development of effector T cells. 

doi:10.1016/j.clim.2007.03.092 

Su.52 Regulation of the STAT3-Mediated Signaling by 

Daxx 

Kenji Sugiyama, Researcher, Department Molecular Biology, 

Nippon Boehringer Ingelheim Co. Kawanishi, Japan, Ryuta 

Muromoto, Postdoctoral Fellow, Department Immunology, 

Graduate School Pharmaceutical Science, Hokkaido 

University, Sapporo, Japan, Masato Ishida, Researcher, 

Department Molecular Biology, Nippon Boehringer 

Ingelheim Co, Kawanishi, Japan, Yuichi Sekine, Assosiate 

Professor, Department Immunology, Graduate School 

Pharmaceutical Science, Hokkaido University, Sapporo, 

Japan, Kenji Oritani, Assistant Professor, Department of 

Hematol. and Oncology, Graduate School Med, Osaka 

University, Suita, Japan, Tadashi Matsuda, Professor, 

Department Immunology, Graduate, School Pharmaceutical 

Science, Hokkaido University, Sapporo, Japan 

Signal transducer and activator of transcription 3 (STAT3) 

plays key roles in the intracellular signaling pathways of the 

interleukin (IL)-6 family of cytokines, which exhibits a 

diverse set of cellular responses, including cell proliferation 

and differentiation. Dysregulated IL-6/STAT3 signaling is 

involved in the pathogenesis of several diseases, for 

examples autoimmune diseases and tumors. Type I interferon 

(IFN) induces the expression of proapoptotic genes 

and has been used in the clinical treatment of several 

tumors. In the present study, we found that type I IFN 

suppressed IL-6/STAT3-mediated transcription and gene 

expression. Furthermore, a type I IFN-induced protein, 

Daxx, also suppressed STAT3-mediated transcriptional activation, 

while overexpression of Daxx inhibited IL-6/STAT3mediated 

gene expression. Importantly, small-interfering 

RNA-mediated reduction of Daxx expression enhanced IL-6/ 

leukemia inhibitory factor (LIF)-induced STAT3-dependent 

transcription. Co-immunoprecipitation studies revealed a 

physical interaction between Daxx and STAT3 in transiently 

transfected 293T cells. We further found that Daxx and 

STAT3 were co-localized in the nucleus. These results 

indicate that Daxx may serve as a transcriptional regulator 

of type I IFN-mediated suppression of the IL-6/STAT3 

signaling pathway. 

doi:10.1016/j.clim.2007.03.093 

S159 

Su.53 Immunological Relationships between Heart 

Tissue and Non-Cardiac Organs in Murine 

Eosinophilc and Lymphocytic Myocarditis 

Masao Hirasawa, Doctor of Medicine, Third Division of 

Internal Medicine, Osaka Medical College, Takatsuki-city, 

Osaka, Japan, Minoru Miyashita, Assistant, Department of 

Neurosurgery, Osaka Medical College, Takatsuki-city, Osaka, 

Japan 

Background: Eosinophilic disorders usually develop into 

one of the factors leading to myocarditis. However, 

immunological interactions between heart tissue and noncardiac 

organs in such diseases are still unclear. In this study, 

the JAK/STAT signaling pathway in acute myocarditis was 

investigated, considering the immunological mechanism in 

non-cardiac organs. Methods: Adoptive transfer and Coxsackie 

B3 viral infection were performed to initiate 

eosinophilic and lymphocytic myocarditis in DBA/2 mice. 

Eosinophils, T cells, and JAK3/STAT6 mRNAs were examined 

by histological methods and RT-PCR. In the spleen, the ratios 

of CD4 to CD8 were compared between OVA-sensitized and


control mice. In the bone marrow, we attempted to detect 

CD34 by FACS analysis. Results: Eosinophilic myocarditis was 

observed in adoptive transferred mice, and marked lymphocytic 

myocarditis was developed in virus-infected mice. In 

both cases, JAK3s were usually seen in inflammatory heart 

lesions; in particular, the parallel expression of JAK3 and 

STAT6 mRNAs was seen in eosinophilic myocarditis. In the 

spleen, CD4/CD8 ratios were elevated in OVA-sensitized 

cases compared with controls (pb0.05), and the quantity of 

JAK3 mRNA was higher in adoptive transfer cases than 

controls (pb0.05). Bone marrow cell analysis showed the 

role of CD34 in OVA-sensitized and normal control mice. 

Conclusion: These results suggested that (1) JAK/STAT 

signaling plays a role in myocardial damage in eosinophilic 

and viral myocarditis, (2) JAK3 also conveys immunological 

tolerance within a CD4-dominant environment in noncardiac 

organs, and (3) bone marrow cells of donor mice 

may have potential for regeneration therapy. 

doi:10.1016/j.clim.2007.03.094 

Su.54 Combined Pharmacodynamic and 

Transcriptome Profiling of Recombinant Human 

Interleukin-21 Responses in Patients with Stage IV 

Malignant Melanoma 

Dorthe Lundsgaard, Research Scientist, Project Manager, 

Novo Nordisk A/S, Maaloev, Denmark, Klaus Stensgaard 

Frederiksen, Senior Scientist, Novo Nordisk A/S, Bagsvaerd, 

Denmark, Lasse Tengbjerg Hansen, Clinical Scientist, Novo 

Nordisk A/S, Bagsvaerd, Denmark, Birte K. Skrumsager, 

Senior Clinical Pharmacologist, Novo Nordisk A/S, 

Bagsvaerd, Denmark, Ulrik Mouritzen, International Medical 

Officer, Novo Nordisk A/S, Bagsvaerd, Denmark, Grant 

McArthur, Professor, Cancer Trials Australia, Peter 

MacCallum Cancer Centre, Melbourne, Australia, Ian D. 

Davis, Professor, Cancer Trials Australia, Austin Hospital, 

Melbourne, Australia, Kresten Skak, Senior Scientist, Novo 

Nordisk A/S, Bagsvaerd, Denmark 

Interleukin-21 is a pleiotropic class I cytokine that 

activates CD8+ T cells and NK cells. In a phase 1 dose 

escalation trial of intravenous IL-21 administered in two 

different dose regimens, the pharmacodynamic effects 

were monitored by multi-analyte profiling of 90 serum 

proteins and global transcriptional profiling of peripheral 

CD8+ T cells. A total of 17 serum proteins were observed to 

increase at least 2-fold upon IL-21 treatment whereas 4 

proteins were down-regulated. Each dose regimen displayed 

a distinct pattern of regulation, reflecting differences 

in pharmacodynamic effects. Among the up-regulated 

proteins were acute-phase proteins, chemokines, and 

cytokines, e.g. CRP, TNF-α, MCP-1, and IL-18. In CD8+ T 

cells, isolated within the first week of treatment, a total of 

471 probe sets were found to be significantly differentially 

regulated. In particular, effector genes such as granzyme A 

and interferon-gamma as well as chemokine receptor 

genes, such as CXCR3 and CXCR6, were clearly up-regulated 

upon IL-21 treatment. These observations may indicate 

stimulation of both effector functions and motility of CD8+ 

T cells. Moreover, suggestive of increased proliferation of 

CD8+ T cells, levels of several DNA replication and cell cycle 

related genes, including PCNA and Ki-67, were induced in a 

dose-dependent manner. The presented data demonstrate 

that administration of IL-21 in patients with stage IV 

malignant melanoma induces a variety of pharmacodynamic 

responses related to immunological activation that warrants 

further clinical investigations of this cytokine. 

doi:10.1016/j.clim.2007.03.095 

Su.55 Differential Expression on the TNF-RI, IL-1RI, 

IL-6R Membrane and Soluble Receptors and NF-kB, 

AP-1 Activation by In Vitro Proteasome Inhibition in 

U937 cells 

Alejandro Bravo-Cuéllar, MD, Instituto Mexicano del Seguro 

Social, Centro de Investigacion Biomedica de Occidente 

Division de Inmunologia, Guadalajara, Mexico, Pablo C. 

Ortiz-Lazare, Chemistry Biologist and Pharmacology, 

Instituto Mexicano del Seguro Social, Centro de 

Investigacion Biomedica de Occidente Division de 

Inmunologia, Guadalajara, Mexico, Jorge R. Dominguez- 

Rodriguez, Biologist, Instituto Mexicano del Seguro Social, 

Centro de Investigacion Biomedica de Occidente Division de 

Inmunologia, G, Mexico, Jose M. Lerma-Diaz, MD, 

Universidad de Guadalajara, CUALTOS, Tepatitlan de 

Morelos, Mexico, Georgina Hernandez-Flores, Biologist, 

Instituto Mexicano del Seguro Social, Centro de 

Investigacion Biomedica de Occidente Division de 

Inmunologia, Guadalajara, Mexico, Piedad C. Gomez- 

Contreras, Chemistry Biologist and Pharmacology, Instituto 

Mexicano del Seguro Social, Centro de Investigacion 

Biomedica de Occidente Division de Inmunologia, 

Guadalajara, Mexico, Martha Barba-Barajas, Chemistry 

Biologist and Pharmacology, Instituto Mexicano del Seguro 


Division de Inmunologia, Guadalajara, Mexico, 

Luis F. Jave-Suarez, Biologist, Instituto Mexicano del Seguro 


Division de Inmunologia, Guadalajara, Mexico, Adriana C. 

Aguilar-Lemarrroy, Biologist, Instituto Mexicano del Seguro 


Division de Inmunologia, Guadalajara, Mexico 

Proteasome is a large multimeric protease complex, 

which plays a vital role in several cellular functions, such 

as: proteins degradation, regulation of cyclins and transcription 

factors, its also related with antigens degradation. On 

the other hand several stimuli such as proinflammatory 

cytokines, free oxygen radicals, components of the bacteria 

cellular wall, can trigger NF-kB activation, which normally 

exits as an inactive form, characterized by its union at the 

trimolecular complex called IkB. NF-kB activation begins by 

phosphorylation, ubiquitination and degradation of IkB 

complex in the proteasome. Once NF-kB transcriptional 

factor is liberated from its natural IkB inhibitor, it can reach 

the nucleus and link to promoters regions of genes that codify 

for TNF-α, IL-1β, IL-6, membrane receptors, and adhesion 

molecules. Monocyte/macrophages, lymphocytes and others 

cells generate these cytokines that increase the inflammatory 

process. TNF-α, IL-1β, IL-6 have been associated with 

different pathologies such as rheumatoid arthritis, inflammatory 

intestinal disease, shock septic. In this study, we

Abstracts 

investigated the in vitro effects of the proteasome inhibitor 

MG132 on TNF-RI, IL-1RI and IL-6R, the activation of NF-kB 

and AP-1 in U937 cells. Cell viability was evaluated by flow 

cytometry using cellular annexin V binding. Proteasome 

inhibition increases the release of TNF-RI and IL-1RI from 

U937 cells and decreases their cell surface expression. In 

other way, the MG132 increases the cell surface expression of 

IL-6R and decreases their liberation. The proteasome 

inhibitor, also, led to increased LPS+PMA-induced AP-1 

activation, and attenuated LPS+PMA-induced IkB degradation 

resulting in abolished NF-kB activation. 

doi:10.1016/j.clim.2007.03.096 

Su.56 Mucosal and Systemic T Cell Responses to 

Cry1Ac Protoxin from Bacillus Thuringiensis 

Aldo Arturo Reséndiz Albor, Cell Biology, Universidad 

Nacional Autonoma de Mexico, Mexico, Leticia 

Moreno-Fierros, Cell Biology, Inmunidad en Mucosas 

UBIMED, FES-Iztacala, Universidad Nacional Autonoma de 

Mexico, Mexico 

Cry1Ac protoxin (pCry1Ac) from Bacillus thuringiensis is a 

potent immunogen with mucosal and systemic adjuvant 

properties when administered to mice by systemic or 

mucosal routes although its mechanism of action remains 

unknown. Thus, in this work, we investigated in vivo and in 

vitro the proportion of T cells producing IL-2, IL-4, IL-10, 

and IFN-g by intracellular cytokine staining on CD3+ T 

lymphocytes freshly isolated from spleen and Peyer's 

patches (PP) following intraperitoneal immunization of 

pCry1Ac. Furthermore, we evaluated the induction of 

CD69 and CD25 in T cell subsets after a single intraperitoneal 

administration of pCry1Ac in spleen and PP as a 

parameter for detecting T cell activation following by in 

vitro stimuli with pCry1Ac. In control mice we observed that 

PP present T cells spontaneously producing IFN-g while 

cytokine production was not detected in spleen lymphocytes. 

Addition of pCry1Ac to splenic and PP T cell cultures 

promoted a Th1 profile of cytokines with high levels of IL-2 

and IFN-g without IL-4. We also found that intraperitoneal 

immunization with pCry1Ac increase of T lymphocytes 

expressing CD69, IL-4 in the spleen and CD69, IL-2 and 

IFN-g in PP. pCry1Ac stimulation led to rapid expression of 

CD69 on both TCD4+ and B cells between 4 h post incubation 

in spleen and PP which remained stable throughout the 72 h 

culture period. The effects elicited by pCry1Ac on cytokine 

profiles are different between lymphocytes from the spleen 

and PP suggesting that their phenotypic and functional 

differences may influence the immunomodulatory effects of 

the protein. 

doi:10.1016/j.clim.2007.03.097 

Su.57 Effects of a Novel Mucosal Adjuvant AT-1002 

on Immune Responses in Mice Immunized 

Intranasally with Ovalbumin 

Amir Tamiz, Director of Chemistry, Alba Therapeutics, 

Baltimore, MD, Niranjan Pandey, Director of Biology, Alba 

Therapeutics, Baltimore, MD, Blake Paterson, CEO, Alba 

Therapeutics, Baltimore, MD, Sefik Alkan, Vice President, 

Alba Therapeutics, Baltimore, MD 

Antigens, adjuvants and delivery systems are the main 

essential components to the development of efficient 

vaccines. The purpose of this study was to evaluate AT- 

1002 HCl, a six-mer synthetic peptide, FCIGRL, derived 

from Zonula Occludens Toxin (cholera's second toxin) as 

an effective mucosal delivery agent that is expected to 

enhance antigen passage through the mucosal/endothelial 

barriers and hence stimulate antigen-specific immune 

responses. In this study, female Balb/c mice were 

immunized intranasally with Ova and AT-1002 simultaneously, 

four times in 21-day intervals and complete 

antibody responses to Ova were measured in the serum 

and in vaginal washes using quantitative Ova-specific 

ELISA. Effects of AT-1002 stimulation on IgG subclasses 

(IgG1, IgG2a, IgG2b, IgA and IgE) were studied. Ovaspecific 

T cell responses were also measured at 5–8 days 

after the last immunization in both the spleen and the 

lung of all mice. We found that mice immunized with Ova 

in the presence of AT-1002 exhibit significantly higher 

antibody titers than those induced by immunization with 

antigen alone. In addition, we found that AT-1002 and 

Cholera Toxin induced similar patterns of Ova-specific IgG 

subclasses. In summary, our study suggests that the tight 

junction modulator AT-1002 might provide a novel 

approach to vaccination. 

doi:10.1016/j.clim.2007.03.098 

S161 

Su.58 Dual Targeting of CCR2 and CX3CR1 in an 

Arterial Injury Model of Vascular Inflammation 

Maya Jerath, Fellow, University of North Carolina, 

Department of Medicine, Chapel Hill, NC, Peng Liu, 

Research Associate, University of North Carolina, Thurston 

Arthritis Research Center, Chapel Hill, NC, Mauricio Rojas, 

Research Instructor, University of North Carolina, 

Department of Medicine, Chapel Hill, NC, Mary Struthers, 

Research Fellow, Merck Research Laboratories, Department 

of Immunology, Rahway, NJ, Julie Demartino, Senior 

Director, Merck Research Laboratories, Department of 

Immunology, Rahway, NJ, Kathryn Lyons, Senior Research 

Associate, Merck Research Laboratories, Department of 

Medicinal Chemistry, Rahway, NJ, Ruoqi Peng, Senior 

Research Immunologist, Merck Research Laboratories, 

Department of Immunology, Rahway, NJ, Lihu Yang, 

Director, Merck Research Laboratories, Department of 

Medicinal Chemistry, Rahway, NJ, Dhaval Patel, Professor, 

The University of North Carolina, Department of Medicine, 

Chapel Hill, NC 

Rationale: CCR2 and CX3CR1 are chemokine receptors 

differentially expressed on monocyte subsets in diverse 

species from rodents to man, and each receptor has been 

individually hypothesized to play a role in inflammatory 

cell trafficking to atherosclerotic lesions. We hypothesized 

that CCR2 and CX3CR1 had non-redundant effects in 

vascular inflammation, and sought to determine their 

combined effect. Methods: We used a murine vascular 

injury model in which both CCR2 KO and CX3CR1 KO have


shown a partial block in the injury response. Experimental 

arms consisted of C57Bl/6 WT mice, CX3CR1 KO mice, WT 

mice given a small molecule CCR2 antagonist (MRL-677, 

Merck & Co.) and CX3CR1 KO mice given the same 

antagonist. Vessels were harvested at 14 days analyzed 

by histology, morphometry and immunohistochemistry. 

Results: Blocking CCR2 with MRL-677 resulted in a 39% 

decrease in the vascular injury response (n=10, p=NS). 

CX3CR1 deficient mice had similar injury to WT animals 

(n=8, p=NS). Mice in which both CCR2 and CX3CR1 

pathways were targeted (CX3CR1 KO mice given MRL-677) 

had an 86% decrease in the injury response (n=6, p=0.002). 

Conclusions: In this study, we have shown that blocking 

CCR2 with a low molecular weight antagonist ameliorates 

the inflammatory response to vascular injury. Simultaneous 

targeting of both CCR2 and CX3CR1 has complementary and 

perhaps synergistic effects on the development of the 

vascular inflammatory response. Our results imply that 

CCR2 and CX3CR1 have independent, non-overlapping roles 

in vascular inflammation and that approaches to target 

both may have more beneficial effects than targeting each 

one separately. 

doi:10.1016/j.clim.2007.03.099 

Su.59 Cytokine and Chemokine Production by NK 

Cells Activated by Monokines IL-12, IL-18 

Following Crosslinking of CD16 and Modulation 

by KIR 

Zaheed Husain, Junior Investigator/Instructor, CBR 

Institute for Biomedical Research, Harvard Medical 

School, Boston, MA, Devendra Dubey, Investigator, CBR 


School, Boston, MA, Shampa Das, Research Fellow, CBR 


School, Boston, MA, Chester Alper, Professor, CBR 



NK cells express both activation and inhibitory receptors. 

Activating receptors play a central role in cytokine and 

chemokine production that is modulated by inhibitory 

receptors. We used high throughput, antibody-based cytokine 

chips to study the levels of secreted cytokines/ 

chemokines. Data was collected to study the rate of 

cytokines/chemokines production and secretion by human 

NK cells activated by monokines IL-12 and IL-18 following 

crosslinking of CD16. We also determined the effects of KIR 

on the production of these cytokines/chemokines. Several 

characteristics were observed: (1) IFN-g and IL-8 showed a 

linear increase with time, (2) the level of chemokines 

peaked within 2 hrs and reverted back to control levels 

within 24 hrs. The synthesis of chemokines was significantly 

higher (3.5 to 4.5 fold) than cytokines such as IFN-γ. Several 

cytokines and growth factors were inhibited by KIR3DL1. The 

mechanism of the increase in the levels of IFN-γ and IL-8 was 

further investigated by analyzing intracellular cytokine 

production using 4-color FACS analysis. Exposure of IFN-γ 

to NK cells induced a rapid increase in the intracellular 

expression of IL-8 suggesting that increase in IL-8 production 

could be due to the de novo synthesis of IL-8 by NK cells. This 

study shows that IL-12/IL-18/CD16 induced activation of NK 

cells triggers several signaling pathways that regulate 

adaptive immunity. 

doi:10.1016/j.clim.2007.03.100 

Su.60 Differential Effect of NK Activation Molecules 

CD16 and 2B4 on Cytokine Production: Significant 

Inhibition of GM-CSF but not IFN-g, TNF-α or 

Cytotoxic Activity 

Zaheed Husain, Junior Investigator/Instructor, CBR Institute 

for Biomedical Research, Harvard Medical School, Boston, 

MA, Shampa Das, Research Fellow, CBR Institute for 

Biomedical Research, Harvard Medical School, Boston, MA, 

Chester Alper, Professor, CBR Institute for Biomedical 

Research, Harvard Medical School, Boston, MA, Devendra 

Dubey, Investigator, CBR Institute for Biomedical Research, 

Harvard Medical School, Boston, MA 

NK cells express several activation and inhibitory 

receptors which are involved in cytokine, chemokine and 

growth factor production and cytotoxic activity against 

virus infected cells, tumor or cells deficient in HLA class I 

expression. Crosslinking of CD16 or 2B4 significantly 

increases IFN-γ, TNF-α and GM-CSF production, and 

cytotoxic activity in redirected lysis assay. It is not known 

how co-crosslinking of these two receptors affect cytokine 

production and cytotoxic activity. To investigate this 

question we used antibody mediated crosslinking of CD16 

and 2B4 molecules on IL-2 activated NK cells activity in 

redirected lysis assay. Separate engagement of CD16 or 2B4 

strongly enhanced cytotoxic activity, IFN-γ, TNF-α and GM- 

CSF production. These are inhibited by co-crosslinking of 

KIR receptors. Co-engagement of these activation receptors 

strongly inhibited the production of GM-CSF (N90%) but not 

IFN-γ or TNF-α production or cytotoxic activity suggesting 

that the pathway for GM-CSF production is separate from 

IFN-γ or TNF-α production. This further suggests that CD16 

and 2B4 may function together to optimize cytokine 

production. 

doi:10.1016/j.clim.2007.03.101 

Su.61 Augmenting CD8+ T Cell Responses with 

IL-15/sIL-15Rα Complexes 

Mark Rubinstein, Postdoctoral Fellow, University of 

California, San Diego, La Jolla, CA, Jonathan Sprent, 

Professor, Garvan Institute of Medical Research, 

Darlinghurst, Australia, Ananda Goldrath, Assistant 

Professor, University of California, San Diego, 

La Jolla, CA 

Administration of IL-15 has shown efficacy in augmenting 

CD8+ T cell responses following vaccination. However, 

soluble IL-15 is limited in vivo by half life and poor biological 

activity. Recently, we showed that the biological activity of 

soluble IL-15 is much improved after interaction with 

recombinant soluble IL-15Rα. After injection, soluble IL- 

15/IL-15Rα complexes rapidly induce strong and selective 

expansion of memory CD8+ cells and natural killer cells.

Abstracts 

Surprisingly, we also find that soluble IL-15/IL-15Rα complexes 

can drive conversion of naïve Tcells to memory Tcells 

in vivo, without administration of antigen. This conversion is 

associated with an acquisition of effector function. Our 

results suggest the therapeutic potential of IL-15 could be 

considerably enhanced by administration of preformed 

soluble IL-15/IL-15Rα complexes. 

doi:10.1016/j.clim.2007.03.102 

Su.62 Evaluation of IL-1 Beta Secretion Level of 

Human Osteoblasts and Morphology of These Cells 

Adjunct to Gray MTA, White MTA, Portland Cement 

and IRM as Retrofilling 

Jalil Tavakkol Afshari, Vice President for Research, 

Immunogenetics Department, Bu-Ali Research Institute, 

Mashhad University of Medical Sciences, Mashhad, Iran, 

Navid Aghasizadeh, Dentist, School of Dentistry, Mashhad 

University of Medical Sciences, Mashhad, Iran, Maryam 

Bidar, Dentist, School of Dentistry, Mashhad University 

of Medical Sciences, Mashhad, Iran, Mohammad Ali 

Fahmidekar, Researcher, Immunogenetics Department, 

Bu-Ali Research Institute, Mashhad University of 

Medical Sciences, Mashhad, Iran, Mohammad Zarabi, 

Dentist, School of Dentistry, Mashhad University of 

Medical Sciences, Mashhad, Iran, Azam Brook, 

Researcher, Immunogenetics Department, Bu-Ali Research 

Institute, Mashhad University of Medical Sciences, 

Mashhad, Iran 

Ostetoblasts and ligament periodontal cells are the 

essential cells for wound repair after root-end resections 

and perforation repair. Cell attachment and spread on the 

surface materials and evaluation of the cell secretory 

function are primary phases in normal cell function. The 

aim of this study was evaluating osteoblasts (MG-63) function 

morphologically and IL-1 β level adjacent to gray MTA, white 

MTA, Portland cement and IRM, as filling materials for rootend 

fillings and repairing perforations. Human osteoblasts 

(MG-63) were grown in RPMI-1640 medium. Materials were 

mixed and seeded in appropriate plates. Cells were added 

after primary setting of materials. They were surveyed in 

days 1, 3 and 7. The amount of IL-1 β in each specimen was 

measured by ELISA. Morphological outcomes showed that 

after 7 days, a large amount of osteoblasts adjacent to gray 

and white MTA had a nice attachment. Cells adjacent to 

Portland cement were round and mostly separated from the 

surface. All cells were round and separated from the plate 

surface adjacent to IRM. Levels of IL-1 β adjacent to gray and 

white MTA were significantly more than to IRM and Portland 

cement (P=0.00). Amount of IL-1 β was not significantly 

different adjacent to gray and white MTA. Osteoblasts 

adjacent to gray and white MTA perform more appropriate 

response in comparison to Portland cement and IRM. Therefore, 

noticing the color of white MTA, it can be substituted by 

gray MTA in places with more cosmetic effects. It also 

appears that Portland cement is not a good substitute for 

MTA. 

doi:10.1016/j.clim.2007.03.103 

Su.63 Blockade of CD40−CD40 Ligand Interactions 

Attenuates Skin Fibrosis and Autoimmunity in the 

Tight-skin Mouse 

Kazuhiro Komura, MD, Department of Dermatology, 

Nagasaki University Graduate School of Biomedical 

Sciences, Nagasaki, Japan, Manabu Fujimoto, MD, 

Department of Dermatology, Kanazawa University Graduate 

School of Medical Science, Kanazawa, Japan, Minoru 

Hasegawa, MD, Department of Dermatology, Kanazawa 

University Graduate School of Medical Science, Kanazawa, 

Japan, Shinichi Sato, MD, Department of Dermatology, 

Nagasaki University Graduate School of Biomedical 

Sciences, Nagasaki, Japan 

The tight-skin (TSK/+) mouse, a mouse model for human 

systemic sclerosis (SSc), develops cutaneous fibrosis and 

autoimmunity. Molecular mechanisms of fibrosis and autoimmune 

responses in both TSK/+ mice and SSc remain 

unknown. In this study, we investigated the role of CD40/ 

CD40 ligand (CD40L) interactions in TSK/+ mice and SSc. The 

blockade of CD40/CD40L interactions by anti-CD40L mAb 

reduced the development of cutaneous fibrosis (65%) and 

autoantibody production in TSK/+ mice. Furthermore, abnormal 

CD40/CD40L interactions in TSK/+ mice was suggested 

by up-regulated CD40L expression on CD4+ T 

lymphocytes, elevated plasma CD40L levels, and hyperresponsiveness 

to CD40 stimulation of B cells and fibroblasts. In 

addition, augmented CD40/CD40L signaling in human SSc was 

also suggested by elevated levels of circulating soluble 

CD40L and circulating soluble CD40. In addition to the 

reports from other groups, the results of the current studies 

suggest that CD40/CD40L interactions can be therapeutical 

targets in SSc. 

doi:10.1016/j.clim.2007.03.104 

S163 

Su.64 Intracellular Expression of TNF-α and IFN-γ 

by Peripheral Blood Mononuclear Cells in a Family 

with Rosacea 

Gabriel Andres Luna-Baca, MD, Institute of Ophthalmology 

Conde de Valenciana, Mexico City, Mexico, Concepcion 

Santacruz-Valdes, MD, Cornea and Refractive Surgery 

Department, Institute of Ophthalmology Conde de 

Valenciana, Mexico City, Mexico, Maria Carmen 

Jimenez-Martinez, MD, PhD, Research Unit, Institute of 

Ophthalmology Conde de Valenciana, Mexico City, Mexico 

Introduction: Rosacea is a chronic dermatologic condition 

that predominantly affects the central aspect of the face. 

Although the pathophysiology of rosacea remains unknown, 

there is increasing agreement that inflammatory mediators 

are operative in this disorder. Recent studies have shown the 

role of oxidative stress and antioxidative system disorders in 

Rosacea. Despite IFN-γ and TNF-α have been involved in the 

pathogenesis of many dermatologic diseases driving to the 

exacerbation of inflammatory and prooxidative responses, 

there is no evidence that these mediators are involved in 

rosacea. Objective: To evaluate the expression of IFN-γ and 

TNF-α by peripheral blood mononuclear cells (PBMC) in a 

family with Rosacea (RF). Methods: CD4, CD8, CD19, CD56, 

IFN-γ, TNF-α, IL-1β, IL-4 and IL-6 expression was determined


by flow cytometry on PBMC from RF and also on PBMC from 

healthy controls (HC). Results. IFN-γ+cells in RF-1=38.5 vs. 

15 HC (p=0.0016); TNF-α+cells in RF-1=55 vs. 3.2 HC (p= 

0.0015); IFN-γ+cells in RF-2=32.6 vs. 8.1 HC (p=0.026); 

TNF-α+cells in RF-2=34.6 vs. 1.5 HC (p=0.0056); IFN-γ+cells 

in RF-2=32.6 vs. 8.1 HC (p=0.026); TNF-α+cells in RF-2=34.6 

vs. 1.5 HC (p=0.0056); IFN-γ+cells in RF-3=24.2 vs. 8.3 HC 

(p=0.04); TNF-α+cells in RF-3=32.2 vs. 3.1 HC (p=0.0046). 

CONCLUSIONS. Peripheral blood mononuclear cells from a 

Rosacea family have significant increase in the expression of 

IFN-γ+ and TNF-α+ cells. IFN-γ and TNF-α may have a role in 

the inflammatory process of rosacea stimulating the release 

of reactive oxygen species in macrophages and neutrophils 

causing damage to facial follicles in persons with Rosacea. 

doi:10.1016/j.clim.2007.03.105 

Su.65 Does Quality of Life in Cutaneous Lupus 

Erythematosus Correlate with Disease Severity? 

Elizabeth Gaines, Pre-doctoral Research Fellow, 

University of Pennsylvania, Department of Dermatology, 


The purpose of this study was to assess the correlation 

between cutaneous lupus erythematosus (CLE) disease 

severity, as measured by the CLASI, and quality-of-life 

(QoL), as measured by the Skindex-29. This was a prospective, 

longitudinal study from baseline (Day 0) to Day 56, done 

at a University hospital cutaneous autoimmunity clinic. Eight 

patients with biopsy-confirmed CLE (4 DLE, 2 SCLE, 2 DLE/ 

SLE) completed the study. At each visit, patients completed 

the CLE-modified Skindex-29. Using scales from 0-10, they 

also assessed their general and skin health, and their pain, 

itch, and fatigue. The PI evaluated disease severity, with the 

CLASI, and the patient s global skin health using a scale from 

0−10. There was a significant improvement in total Skindex 

score over time (t=2.36, pb0.05). There was a moderate 

correlation (r=0.59, p=0.12, n=8) between change in activity 

(CLASI), and change in skin symptoms (Skindex). Neither 

change in emotion or function correlated with change in 

activity. There was no correlation between any Skindex 

subscale with damage. Skindex scores for patients with SCLE 

were, on average, lower and changed less than Skindex 

scores for patients with DLE. While both the CLASI activity 

score and the modified Skindex total score improved 

significantly over time, the correlation between the two 

scores was poor. Different elements of skin health may be 

captured by each index. Given the different disease courses 

and likelihood of damage, skin-related QoL may differ across 

different subtypes of CLE. Preliminary analysis of data from 

25 additional CLE patients supports these conclusions. 

doi:10.1016/j.clim.2007.03.106 

Su.66 Association Between the Polymorphism of 

TGF-β1 Gene Promoter (−509CNT) and Idiopathic 

Chronic Urticaria 

Sara Hosseini-Farahabadi, Dr.; Researcher, Bu-Ali Research 


Iran, Jalil Tavakkol-Afshari, Vice President of Research; 

Head, Department of Immunogenetics and Cell Culture, 

Bu-Ali Research Institute (BARI), Department of 

Immunogenetics and Cell Culture, Mashhad, Iran, Rashin 

Ganjali, Researcher, Bu-Ali Research Institute (BARI), 

Department of Immunogenetics, Mashhad, Iran, Javad 

Ghaffari, Mazandaran University of Medical Sciences, 

Department of Pediatrics, Sari, Iran, Houshang Rafatpanah, 

Ghaem Medical Center, Department of Allergy and 

Immunology, Mashhad, Iran, Reza Farid-Hosseini, Head, 

Department of Allergy and Immunology, Ghaem Medical 

Center, Department of Allergy and Immunology, 

Mashhad, Iran 

Background: Idiopathic Chronic Urticaria (ICU), the most 

common form (70–80%) of chronic urticaria is supposed to 

have immune basis causes. It is speculated that the promoter 

polymorphism of TGF-β1 gene may be involved in ICU. This 

condition is thought to affect at least 0.1% of the population 

and often it can be severe and difficult to treat. Materials 

and Methods: A total of 40 patients with ICU and 41 normal 

subjects were studied. DNA was extracted from whole blood 

and TGF-β1 promoter −509CNT polymorphism was determined 

by PCR-RFLP method. Results: Out of the 40 patients 

with ICU, 11 (27.5%) had CC, 26 (65%) had CTand 3 (7.5%) had 

TT genotypes. A higher proportion of case subjects with the C 

allele (CT type or CC type) was found compared with the T 

allele. Conclusion: These results do suggest an influence of 

genetic variability at the promoter of TGF-β1 gene 

(−509CNT) on the occurrence of ICU. This polymorphism 

has been shown as a useful genetic change in our study. 

Further work is required to confirm this result. 

doi:10.1016/j.clim.2007.03.107 

Su.67 Investigation of the Mechanisms of Pressure 

Ulcers Using a Cyclical Cutaneous 

Ischemic−reperfusion Injury Model 

Yuki Saito, Graduate Student, Kanazawa University, 

Dermatology, Kanazawa, Japan, Minoru Hasegawa, Assistant 

professor, Kanazawa University, Dermatology, Kanazawa, 

Japan, Manabu Fujimoto, Associate professor, Kanazawa 

university, Dermatology, Kanazawa, Japan, Kazuhiko 

Takehara, Professor, Kanazawa university, Dermatology, 

Kanazawa, Japan 

The formation of pressure ulcers is considered to be 

derived from multifactorial factors. Among them, the occurrence 

of cycles of ischemic–reperfusion is an especially 

significant contributing factor. This study assessed immunological 

mechanism of a reproducible murine model of 

ischemic–reperfusion injury by the external application of 

magnets. Dorsal skin was gently pulled and placed between 

two round ceramic magnetic plates. A single ischemic– 

reperfusion cycle (I–R cycle) consisted of a 12 hours of 

magnet placement followed by a 12 hours of release or rest. 

Three cycles were performed to induce pressure ulcer formation. 

A marked edema was observed after an I–R cycle (day 

1) and skin ulcer developed until day 6 (3 days post 

treatment). Then, skin ulcers were healed until day 20 in 

all mice. In the lesional skin, the infiltration of neutrophils 

and macrophages, and tumor necrosis factor-alpha expression

Abstracts 

were most prominent at day 2. Then, inducible nitric oxide 

synthase (iNOS) expression was augmented at day 3. While 

nitric oxide is considered to be involved in ischemic– 

reperfusion injury, iNOS is a critical enzyme for the synthesis 

of nitric oxide. iNOS is produced by various cells including 

neutrophils and macrophages stimulated with proinflammatory 

cytokines such as tumor necrosis factor-alpha. Therefore, 

our findings suggest that iNOS production from 

infiltrated neutrophils and macrophages stimulated by 

tumor necrosis factor-alpha contributes to the development 

of skin ulcer in this model. We propose that this cyclical 

cutaneous ischemic–reperfusion injury model is useful for 

investigating the mechanism or developing novel therapies of 

pressure ulcers. 

doi:10.1016/j.clim.2007.03.109 

Su.68 CD19 Expression in B cells is Important for 

Suppression of Contact Hypersensitivity 

Manabu Fujimoto, Associate Professor, Department of 

Dermatology, Kanzawa University, Kanazawa Ishikawa, 

Japan, Rei Watanabe, Student, Department of Dermatology, 

University of Tokyo, Bunkyo Tokyo, Japan, Thomas Tedder, 

Professor, Department of Immunology, Duke University 

Medical Center, Durham, NC, Kunihiko Tamaki, Professor, 

Department of Dermatology, University of Tokyo, Bunkyo 

Tokyo, Japan 

Contact hypersensitivity (CHS) is a cutaneous immune 

reaction mediated mainly by Ag-specific effector Tcells, and 

regarded as a model for Th1/Tc1-mediated inflammation. 

However, recent reports have suggested pivotal roles of B 

cells in CHS. CD19 is a member of the Ig superfamily expressed 

on B cells and follicular dendritic cells, and serves as a 

positive B cell response regulator which defines signaling 

thresholds critical for B cell responses. In the current study, 

we assessed the role of the B cell-specific surface molecule 

CD19 on the development of CHS through examining CD19deficient 

mice. Although CD19-deficient mice are hyposensitive 

to a variety of transmembrane signals, CD19 loss resulted 

in increased and prolonged reaction of CHS, suggesting an 

inhibitory role of CD19 expression in the etiology of CHS. 

Sensitized draining lymph nodes and elicited ear lesions from 

CD19-deficient mice exhibited Th1/Tc1-shifted cytokine 

profile with increased IFN-fÁ expression and decreased IL- 

10 expression. Adoptive transfer experiments revealed that 

CD19 expression in recipient mice was required for optimal 

suppression of CHS response, indicating its role in elicitation 

phase. Furthermore, spleen B-2 cells, especially marginal 

zone B cells, from wild type mice were able to normalize 

exaggerated CHS reactions in CD19-deficient mice. Thus, 

CD19 expression in B cells is critical for termination of CHS 

responses, possibly through the function of regulatory B cells. 

doi:10.1016/j.clim.2007.03.111 

Su.69 Automated Enumeration of Skin Mast Cells by 

Laser Scanning Cytometry 

Esther Trueblood, Director of Pathology, Amgen, Inc./ 

Pathology, Seattle, WA, Stephen Z., Scientist, Amgen, Inc./ 

Clinical Immunology, Thousand Oaks, CA, Andrea Ita, Senior 

Scientist, Amgen, Inc./Inflammation, Thousand Oaks, CA, 

John Ferbas, Director Medical Sciences, Amgen, Inc./ 

Clinical Immunology, Thousand Oaks, CA, James Chung, 

Medical Sciences Director, Amgen, Inc./Early development, 

Thousand Oaks, CA, Gordon Ng, Scientific Director, Amgen, 

Inc./Inflammation, Thousand Oaks, CA, Gloria Juan, 

Senior Scientist, Amgen, Inc./Clinical Immunology, 

Thousand Oaks, CA 

Mast cells (MCs) are bone marrow-derived immune competent 

cells dependent on stem cell factor for survival, 

chemotaxis, functional activation, and adhesion to connective 

tissue matrix. After activation, MCs can release a 

variety of mediators including histamine, tryptase, leukotrienes, 

prostaglandins, contributing to anaphylactoid, 

allergic and inflammatory processes. MCs are also associated 

with wound repair together with fibroblasts and 

other immune cells. The current effort presents a novel 

application of Laser Scanning Cytometry (LSC) to study the 

time course of mast cell recruitment in a skin woundhealing 

model. A full-thickness incisional wound model was 

developed in the mouse. A skin biopsy was taken on day 8 

after wounding in animals treated with control or an antimurine 

c-Kit antibody (ACK2) that blocks SCF. A two-step 

protocol on the LSC was created to automate quantitation 

of wound-adjacent MCs. The first step executed a lowresolution 

optical scan that defined the position and area of 

each biopsy on the slide. The second step consisted of a 

high-resolution scan that captured sequential 20× image 

fields of the entire biopsy and stitched the images together 

into a mosaic. Computer analysis segregated stored image 

events into discrete populations, and ultimately MCs. MCs 

counts were obtained from a defined region around the 

wound site. ACK2 treatment reduced wound-activated Mast 

Cell expansion as measured by LSC (T-test; P=0.0094) and 

consistent with the manual counts. This method of 

quantitation allows implementation of a more efficient, 

objective and precise method to score effects on MCs which 

are tissue resident and difficult to analyze. 

doi:10.1016/j.clim.2007.03.112 

S165 

Su.70 Kawasaki Disease: Presentation of One Case 

Carlos Torres-Loza, Immunoallergist, West National Medical 

Center, UMAE-IMSS, Guadalajara, Mexico 

Kawasaki disease (KD) is the most common vasculitis of 

childhood and may well be the most common vasculitis at any 

age. The aim of this work is to present a case with an atypical 

clinical presentation or perhaps to consider other immunoinflamatory 

syndrome and to share this clinical experience 

with other immunologists. Ketzalli is a girl of 5 years old who 

started abruptly with abdominal pain, remittent fever often 

up 38.5 °C and scarlatiniform and multiform cutaneous rash 

and irritability by 2–3 days. After that acute period, we 

treated with metamizole as antithermic. After that period, 

we realized that our patient started to feel better but, with 

changes in her lips characterized by dry, swollen and vertically 

cracked lips and diffusely without strawberry tongue. 

Actually, by that time we realized both erythema of palms


and soles and skin desquamation. However, after 1–2 days 

more we see how her fingertips begin to peel and how one 

typical lesion of vasculitis appear on skin of her right 

fingertip. Ketzalli was seen by several specialist in pediatrics 

including, infectologist, dermatologist, immunoallergist and 

not all of them were in agreement about diagnosis of 

Kawasaki disease. Giving benefit of the doubt, Ketzalli was 

treated with IVIgG to a doses of 1.5 g/kg in one doses. After 

two weeks of such clinical event a normal echocardiogram 

was reported. As far as we know does not exist other clinical 

problem with such skin desquamation and peeling hence to 

share this case with other immunologists. 

doi:10.1016/j.clim.2007.03.113 

Su.72 CD4+CD25+ Regulatory T Cells Abrogate 

Psoriatic-like Skin Lesions in a Murine Model of 

Psoriasis, Whereas TNFa Blockade Exacerbates the 

Lesions 

Hak-Ling Ma, Senior Research Scientist II, Wyeth Research, 

Inflammation Department, Cambridge, MA 

Psoriasis is a chronic inflammatory skin disease that is due 

to rapid epidermal proliferation with mononuclear cell infiltrates. 

Recent studies demonstrated that transferring 

naïve CD4+ T cells into scid/scid mice was able to induce 

psoriatic like lesions in the recipient mice. This indicates 

that T cells play a central role in the pathogenesis of the 

disease. By adoptive transferring naïve T cells that lacked 

Tregs, we found that recipient mice experienced close to 

100% incidence of disease with clinical symptoms (i.e. skin 

erythema and scaling) and histological features (i.e. epidermal 

thickening, rete pegs and hyperplasia of epidermis and 

keratinocytes) resembling that of human psoriasis. We also 

found that co-administration of Tregs with naïve T cells 

significantly decreased the incidence and severity of 

psoriatic-like skin lesions. It has been shown that cytokines 

such as IL-12, TNFa, IL-22, and IL-17 play important roles in 

disease progression. TNFa antagonists have been used in 

treatment of psoriasis, however, they can also exacerbate 

skin lesions in some individuals. Interestingly, in our studies, 

soluble murine TNFRIIFc exacerbated disease in comparison 

to isotype control treated mice. Overall, our results suggest 

that Tregs inhibit whereas soluble murine TNFRIIFc exacerbates 

skin inflammation in this model. 

doi:10.1016/j.clim.2007.03.114 

Su.74 Investigator-Initiated Trial of Efalizumab for 

Atopic Dermatitis: A Proof-of-Concept Study in 

Adults 

Melissa Magliocco, Assistant Professor of Medicine, 

UMDNJ-Robert Wood Johnson Medical School, Department 

of Medicine, New Brunswick, NJ, Irina Lipets, Assistant 

Program Manager, UMDNJ-Robert Wood Johnson Medical 

School, New Brunswick, NJ, Viktor Dombrovskiy, Assistant 

Professor of Medicine, UMDNJ-Robert Wood Johnson 

Medical School, New Brunswick, NJ, Alice Gottlieb, 

Professor and Chair of the Department of Dermatology, 

Tufts-New England Medical Center, Boston, MA 

Background: Atopic dermatitis (AD) is an autoimmune 

skin disorder mediated acutely by predominantly activated 

Th2 T cells and in chronic stages by activated Th1 cells. 

There is a need for safe, effective AD treatment. Efalizumab 

is a humanized monoclonal IgG1 antibody that binds the 

CD11a subunit of LFA-1, inhibiting T cell activation, 

reactivation, and emigration into skin. Its FDA approval for 

psoriasis suggests that it would be effective and safe for AD, 

because both diseases exhibit Th1 cell predominance in 

lesional skin. Objective: To determine whether targeting 

CD11a on T cells decreases the clinical activity of AD. 

Methods: This was a single-arm, open-label, pilot study. 

Fifteen subjects with at least a “moderate” Investigator's 

Global Assessment (IGA) score were recruited. Subjects 

were excluded if they received photo/systemic therapy 

within 1 week of baseline. Subjects received a subcutaneous 

injection of efalizumab weekly. Efficacy was assessed 

monthly for 6 months using EASI (Eczema Area and Severity 

Index), IGA, a subjective pruritus assessment scale, and 

photography. Safety was evaluated by physical examination 

and laboratory tests. Results: Three subjects completed the 

study. Changes in EASI, IGA, and pruritus scores at 6 months 

compared with baseline were analyzed. The mean decrease 

in EASI was 15.3% (95% CI −3.4%–34.0%), whereas that of the 

IGA was 10.6% (95% CI 1.0%–20.2%). Reduction in mean 

pruritus score was 29.5% (95% CI 45.0%–104.1%) Few adverse 

events were noted, including infection. Conclusion: Treatment 

of AD with efalizumab resulted in a significant 

improvement in IGA, but did not significantly affect EASI 

or pruritus. 

doi:10.1016/j.clim.2007.03.115 

Su.75 Demodex Folliculorum and Chronic 

Granulomatous Disease 

Anete Sevciovic Grumach, University of Sao Paulo, São 

Paulo, Brazil, Rosemeire Navickas Constantino-Silva, BSc, 

Department of Dermatology, São Paulo, Brazil, Marcelo 

Mentha Nico, Department of Dermatology, University of São 

Paulo, São Paulo, Brazil, Alexandre Correa, BSc, 

Department of Dermatology, University of Sao Paulo, Sao 

Paulo, MRF, Correa, Hospital Mandaqui, Sao Paulo, Brazil, 

Israel Zeckcer, Department of Dermatology, University of 

Sao Paulo, São Paulo, Brazil, Joao Bosco Oliveira, 

Department of Dermatology, University of Sao Paulo, São 

Paulo, Brazil, Alberto Jose da Silva Duarte, Professor, 

Department of Dermatology, São Paulo, Brazil, Dewton 

Moraes-Vasconcelos, Department of Dermatology, São 

Paulo, Brazil 

Background: Chronic Granulomatous Disease (CGD) is a 

primary immunodeficiency with a defect on oxidative 

metabolism of phagocytes. Demodex are also recognized as 

mites of the face and they live in the human pillous follicles. 

They affect aging people or immunosuppressed patients. No 

previous report has found those mites in primary immunodeficiencies. 

Objective: To report the clinical and laboratorial 

manifestations of a patient with CGD and Demodex 

folliculorum. Case report: A 1 year and 4 months old, male, 

born to a non-consanguineous family presenting with facial 

“suppurative” lesions was referred to our outpatient group in

Abstracts 

order to evaluate the recurrent localized lesions on his face. 

He was treated with several antibiotics with no clinical 

improvement. He had been hospitalized twice for bronchopneumonia 

(2X) and severe anemia receiving blood transfusions. 

The facial secretion was evaluated in the microscope 

and Demodex was identified. Considering the cause of 

referral, DHR was performed and CGD was confirmed. 

Conclusion: The follicular pitiriasis or demodicidosis was 

first described in secondary immunodeficiencies such as 

leukemia or after kidney transplantation. This patient 

represents the first case report associating Demodex with a 

primary immunodeficiency (CGD). 

doi:10.1016/j.clim.2007.03.116 

Su.76 Immune Response Using Dialyzable Leukocyte 

Extract in the Treatment of Herpetic Keratitis 

Gabriel Andres Luna-Baca, MD, Research Unit, Institute of 

Ophthalmology Conde de Valenciana, Mexico City, Mexico, 

Marisela Linares, Master of Science, Research Unit, Institute 

of Ophthalmology Conde de Valenciana, Mexico City, 

Mexico, Concepcion Santacruz-Valdes, MD, Cornea and 

Refractive Surgery Department, Institute of Ophthalmology 

Conde de Valenciana, Mexico City, Mexico, Raul Chavez, MD, 

PhD, Laboratorio de Inmunologia, Departamento de 

Bioquimica, Universidad Nacional Autonoma de Mexico, 

Mexico City, Mexico, Gustavo Aguilar-Velazquez, MD, 

Research Unit, Institute of Ophthalmology Conde de 

Valenciana, Mexico City, Mexico, Mayra Perez-Tapia, PhD, 

Department of Immunology, National School of Biological 

Sciences, ENCB-IPN, Mexico City, Mexico, Iris Estrada- 

Garcia, PhD, Department of Immunology, National School of 

Biological Sciences, ENCB-IPN, Mexico City, Mexico, Sergio 

Estrada-Parra, PhD, Department of Immunology, National 

School of Biological Sciences, ENCB-IPN, Mexico City, 

Mexico, Maria Carmen Jimenez-Martinez, MD, PhD, 

Research Unit, Institute of Ophthalmology Conde de 

Valenciana, Mexico City, Mexico 

Introduction: T lymphocytes are involved in herpetic 

keratitis (HK) producing IFNg. Transfer factor (TF) has the 

ability to modulate the immune response, probably due to 

IFNg production. TF has been used in the treatment of viral 

infections like herpes simplex and zoster. Objective: To 

evaluate the cytokine expression by peripheral blood mononuclear 

cells (PBMC) from HK patients (p) treated with TF. 

Methods. CD4, CD8, IFNg, IL-4, perforin and granzyme expression 

was determined by flow cytometry on PBMC from 

HK-p, before (BTF) and after (ATF) treatment with TF and 

also from healthy controls (HC). Results: Percentage (%) CD4+ 

IFNg+T cells in HK-p BTF=15 vs. 9 HC (p=0.028) vs. 33 ATF; % 

CD4+IL-4+Tcells in HK-p BTF=20 vs. 12 HC vs. 6.5 ATF; %CD8+ 

IFNg+T cells in HK-p BTF=23 vs. 25 HC vs. 50 ATF (p=0.0002 

vs. ATF, p=0.005 vs. HC); %CD8+IL-4+T cells in HK-p BTF=7 

vs. 10 HC vs. 10 ATF; %CD8+IL-4+T cells=7 vs. 23 CD8+IFNg+T 

cells in HK-p BTF (p=0.005); %CD8+IL-4+T cells=10 vs. 50 

CD8+IFNg+T cells in HK-p ATF (p=0.003); %CD4+Granzyme+ 

T cells in HK-p BTF=9.5 vs. 9.4 HC vs. 10.7 ATF; %CD4+ 

Perforin+T cells in HK-p BTF=5.7 vs. 1.1 HC vs. 9 ATF; %CD4+ 

Granzyme+Perforin+T cells in HK-p BTF=8.1 vs. 2.7 HC vs. 

10.8 ATF; %CD8+Granzyme+T cells in HK-p BTF=12 vs. 26 HC 

(p=0.012) vs. 10.7 ATF (p=0.008 vs. IS); %CD8+Perforin+Tcells 

in HK-p BTF=8.5 vs. 4.8 HC vs. 13.2 ATF; %CD8+ Granzyme+ 

Perforin+T cells in HK-p BTF=32.8 vs. 33.5 HC vs. 37.1 ATF. 

Conclusions: CD8+lymphocytes from HK-p predominantly produce 

IFNg after treatment with TF. This could explain the better 

outcomes by a cytotoxic Tcell response in the studied patients. 

doi:10.1016/j.clim.2007.03.117 

Su.78 Gene Expression Profiling of Non-infectious 

Uveitis Patients Using Pathway Specific cDNA 

Microarray Analysis 

Zhuqing Li, Staff Scientist, Lab Immunology, NEI, NIH, 

Bethesda, MD, Sankaranarayana Mahesh, Postdoctoral 

Fellow, Laboratory of Immunology, NEI, NIH, Bethesda, MD, 

Baoying Liu, Fellow, Immunology, NEI, NIH, Bethesda, MD, 

Grace Clarke, Physician, Immunology Lab, NEI, NIH, 

Bethesda, MD, Wee Kiak Lim, Fellow, Immunology, NEI, NIH, 

Bethesda, MD, Robert Nussenblatt, Physician, Immunology 

Lab, NEI, NIH, Bethesda, MD 

To study gene expression profiling of leukocytes from 

peripheral blood of patients with non-infectious uveitis, 

peripheral blood mononuclear cells (PBMCs) were isolated 

from 50 patients with clinically characterized non-infectious 

intermediate uveitis. Total RNA from PBMCs were extracted 

and quantified. Biotinylated probes were generated from 

total RNAs by incorporating biotinylated dUTP into synthesized 

cDNAs using a T7-polymerase linear amplification 

strategy. RNAs isolated from 20 healthy donors were pooled 

and served as normal control. Probes were hybridized to a 

pathway-specific cDNA microarray chip that contains some 

400 autoimmune and inflammatory response related genes. 

Signals for specific binding were recorded by a CCD camera 

and gene expression profiling was analyzed using GEArray 

Suite software. Despite tremendous heterogeneity of gene 

expression among patients and normal controls, there were 

clear differential gene expression patterns comparing those 

from patients to those from normal control. Although there 

was more than 80% of overlap of gene expression, some 15% of 

the genes were differentially expressed among patients 

compared to the normal donors. Several cytokine, chemokine 

or chemokine receptor genes were consistently more highly 

expressed among uveitis patients compared to the normal 

control. There seems less consistency in terms of downregulated 

genes among patients when compared to normal 

control. We show here that pathway specific cDNA microarray 

can be an efficient and economic strategy to profiling uveitis 

patients at the genomic level. Although preliminary and 

needing further confirmation, our data revealed several 

immune response genes that may be implicated in presumably 

autoimmune originated non-infectious uveitis. 

doi:10.1016/j.clim.2007.03.118 

S167 

Su.79 A Novel Model of MDP-induced Ocular 

Inflammation is Dependent on CARD15/NOD2 But 

Not on Interleukin-1 

Holly Rosenzweig, Postdoctoral Fellow, Oregon Health and 

Science University, Portland, OR, Tammy Martin, Research


Assistant Professor, Oregon Health and Science University, 

Department of Ophthalmology, Portland, OR, Michael 

Davey, Professor, VA Medical Center, Portland, OR, Monica 

Jann, Research Assistant, VA Medical Center, Portland, OR, 

Steve Planck, Research Associate Professor, Oregon Health 

and Science University, Department of Ophthalmology, 

Portland, OR, Kelley Goodwin, Student Research Assistant, 

Oregon Health and Science University, Department of 

Ophthalmology, Portland, OR, Koichi Kobayashi, Assistant 

Professor, Harvard Medical School, Department of 

Pathology, Boston, MA, Richard Flavell, Professor, Yale 

University School Med, HHMI, New Haven, CT, James 

Rosenbaum, Professor, Oregon Health and Science 

University, Department of Ophthalmology, Portland, OR 

Mutations in the human CARD15/NOD2 gene are associated 

with Crohn's disease or Blau syndrome, an autosomal 

dominant form of uveitis, arthritis, and dermatitis. NOD2 is 

responsible for sensing the bacterial product, muramyl 

dipeptide (MDP). Interestingly, mutations in a related gene, 

CIAS1, are associated with autoinflammatory syndromes in 

which interleukin (IL)-1 β plays a critical role in disease 

pathogenesis. Since the in vivo effects of MDP are 

incompletely studied and the susceptibility of the eye to 

mutations in NOD2 is unexplained, we developed a novel 

murine model of MDP-dependent uveitis. Intraocularly 

injected MDP induced a significant, transient inflammatory 

response. We characterized several salient features of this 

model such as the effect of dose and timing on the rolling, 

sticking and infiltrating leukocytes within the iris vasculature 

and tissue by intravital microscopy and histology. The 

increase in rolling leukocytes in MDP treated mice was 

dependent on the adhesion molecule L-selectin, as Lselectin 

knockout mice showed a significant decrease in the 

number of rolling cells within the iris vasculature in 

response to MDP. Importantly, NOD2 plays an essential role 

in ocular inflammation induced by MDP, as NOD2 knockout 

mice failed to develop uveitis in response to MDP. 

Unexpectedly, IL-1 receptor knockout mice did not show 

reduced MDP-induced uveitis. Thus, NOD2 may participate 

in the development of uveitis in response to bacterial 

products through its ability to enhance intravascular 

interactions between leukocytes and the endothelium in 

the eye in an IL-1 receptor independent fashion. 

doi:10.1016/j.clim.2007.03.119 

Su.81 Autofluorescence Can be Used to 

Differentiate Between Normal, Activated, or 

Malignant Lymphocyte Populations 

Seth Pantanelli, Guest Researcher, National Institutes of 

Health, NEI, Bethesda, MD, Zhuqing Li, Staff Scientist, 

National Institutes of Health, NEI, Bethesda, MD, Rober 

Fariss, Researcher, National Institutes of Health, NEI, 

Bethesda, MD, Matthew Cunningham, Guest Researcher, 

National Institutes of Health, NEI, Bethesda, MD, Baoying 

Liu, Researcher, National Institutes of Health, NEI, 

Bethesda, MD, Sankara Mahesh, Researcher, National 

Institutes of Health, NEI, Bethesda, MD, Robert 

Nussenblatt, Principal Investigator, National Institutes of 

Health, NEI, Bethesda, MD 

The goal of this study was to investigate whether autofluorescent 

properties of normal, activated or malignant 

lymphocyte populations could facilitate clinical diagnosis of 

ocular diseases. Peripheral blood mononuclear cells 

(PBMCs) were isolated from normal human donor buffy 

coat blood products. T cells were purified from PBMCs and 

cultured with or without anti-CD3 and anti-CD28 stimulation. 

A B-lymphoma cell line (CA46) was cultured separately. 

Five experimental groups were prepared: 

unstimulated T cell, stimulated T cell, CA46 cell, stimulated 

T cell mixed with CA46 cell at a ratio of 1:4, or mixed 

at a ratio of 4:1. At 0, 1, and 3 days after culturing, 

0.6 ×106 cells from each of the five cell groups were cytospun 

onto a slide and imaged with a confocal microscope. 

Samples were excited with six excitation wavelengths: 351, 

364, 458, 476, 488, and 514 nm. For each excitation 

condition, the autofluorescence intensity emitted from the 

sample was measured over a broad range of wavelengths. 

Pure T and B lymphoma populations were clearly distinguishable 

based on autofluorescence intensity spectra. B 

lymphoma cells were the least fluorescent when excited 

with 351 nm light, but most fluorescent when excited with 

longer wavelengths like 488 nm. Mixed populations of T and 

B lymphoma cells had emission intensities that fell inbetween 

those of the pure populations. We conclude that 

normal, activated and malignant lymphocyte populations 

can be distinguished based on their autofluorescent 

properties in vitro. Future work with in vivo models may 

prove useful in facilitating the diagnosis of uveitis and 

other ocular diseases. 

doi:10.1016/j.clim.2007.03.120 

Su.82 Developmental Expression of PD1 in the 

Retina 

Ling Chen, PhD Student, Jules Stein Eye Institute, Los 

Angeles, CA, Vicky Chang, Medical Student, Jules Stein Eye 

Institute, Los Angeles, CA, Ralph Levinson, Assistant 

Professor, Jules Stein Eye Institute, Los Angeles, CA, Lynn 

Gordon, Associate Professor, Jules Stein Eye Institute, Los 

Angeles, CA, Jonathan Braun, Professor, Department of 

Pathology and Laboratory Medicine, Los Angeles, CA 

Purpose: Programmed cell death 1 (PD-1) has a major 

function as a negative immune regulator. We recently 

identified PD-1 expression in neuronal cells of the retina. 

One potential role for PD-1 expression is in retinal 

maturation. The purpose of this study was to identify 

neuronal cell types that express retinal PD-1 and determine 

the time course of expression in the developing 

retina. Methods: PD-1 protein expression in the mouse 

retina was detected by immunohistochemistry at multiple 

embryonic and adult stages. Co-localization experiments 

were performed to detect PD-1 and Brn3a, Thy-1, AP2, 

GFAP, calbindin, or islet. RT-PCR and in situ hybridization 

were performed to quantify PD-1 mRNA expression. 

Results: Constitutive expression of PD-1 protein and 

mRNA was observed in the retinal ganglion cell. Confocal 

microscopy revealed that 68% of the PD-1 cells were of the 

retinal ganglion cell lineage and 27% were amacrine cells. 

PD-1 expression levels changed markedly during development.

Abstracts 

PD-1 expression was first observed at E14, 2 days after the 

first retinal ganglion cells appear in the retina. Expression 

levels of PD-1 significantly increased during development, 

reaching a peak at P13 with a subsequent decrease to an 

intermediate expression level at P24. Conclusions: PD-1 is 

expressed in retinal ganglion cells and amacrine cells and 

its expression dynamically changes during retinal development. 

Maximal PD-1 expression is noted at the most critical 

time period in postnatal visual plasticity. This observation 

raises the possibility of a developmental role for PD-1 in 

maturation of the ganglion cell layer and retinal remodeling 

process 

doi:10.1016/j.clim.2007.03.121 

Su.83 Human Lung Tumor Cells Modifies Rates of 













how the microenvironment of a tumor mass induced phenotypical 

changes in lymphocyte subsets. Our subjects were 

10 patients with resectable non-small cell lung cancer. We 

evaluated 47 different phenotypically different lymphocyte 

subsets in blood samples taken from the pulmonary artery, 

pulmonary vein of a tumor bearing pulmonary lobe and 

peripheral blood during pulmonary resectional surgery. We 

showed that, tumor cells boosted CD25high+CD4high+T 

lymphocytes (Treg cells), B lymphocytes, NK and CD19+ 

CD23+ cells (Breg cells) and CD3+CD95+ (FasL+T lymphocytes) 

(p=0.015, p=0.001, p=0.02, p=0.04, p=0.03 respectively). 

However, Mac-1 cells and HLADR+T lymphocytes 

were found to be decreased by tumor mass (p=0.04 and 

p=0.04), whereas only Breg, NK cell and B lymphocyte 

subsets were found to be expansed. In addition, the patients 

showed expansions of CD45R0+ activated T lymphocytes and 

CD18+CD11b+(Mac-1) cell subsets without significant alteration 

by tumor microenvironment. In conclusion, subsets of 

Treg, Breg cells, FasL+ T lymphocytes, B lymphocytes were 

modified by lung cancer microenvironment itself. This study 

also showed that, peripheral blood might not be good source 

for investigating the anti-tumor immunity since only Breg, NK 

cell and Treg cell subsets were found expansed peripherally. 

T lymphocytes and Mac-1 cells may be modified by systemic 

antitumor response rather than by local tumoral microenvironment. 

This study provides important insight into how 

tumor infiltrating lymphocytes and peripheral immune 

systems may be different in terms of lymphocyte subset 

expansion dynamics. 

doi:10.1016/j.clim.2007.03.122 

Su.84 Gene Expression Patterns of Intestinal 

Epithelial Cells in Murine Models of Colitis 

Ken Flanagan, Postdoctoral Researcher, Genentech, 

Department of Pathology, South San Francisco, CA, Jennine 

Cornelius, Research Associate, Genentech, Department of 

Pathology, South San Francisco, CA, Zora Modrusan, 

Scientist, Genentech, Department of Molecular Biology, 

South San Francisco, CA, Lian Mo, Senior Research 

Associate, Genentech, Department of Pathology, South San 

Francisco, CA, Arvind Chavali, Intern, Genentech, 

Department of Pathology, South San Francisco, CA, Ian 

Kasman, Research Associate, Genentech, Department of 

Pathology, South San Francisco, CA, Laszlo Komuves, 

Scientist, Genentech, Department of Pathology, South San 

Francisco, CA, Lauri Diehl, Scientist, Genentech, 

Department of Pathology, South San Francisco, CA 

In the healthy colon, intestinal epithelial cells (IEC) form 

a physical barrier separating the myriad of gut antigens from 

the cells of the immune system. Simultaneously, IEC employ 

several mechanisms to actively maintain immunologic 

tolerance to non-pathogenic antigens, including commensal 

bacteria. However, during inflammatory bowel disease (IBD) 

the line of defense provided by IEC is breached, resulting in 

uncontrolled immune responses. As IEC are a principal mediator 

of immune responses in the gut, we were interested in 

discerning the gene expression pattern of IEC during 

development and progression of IBD. Laser capture microdissection 

and microarray analysis were combined to identify 

genes with altered expression patterns in two models of 

murine colitis. Genes involved in processes of the immune 

system, including lymphocyte migration, apoptosis and 

antigen presentation were affected in the IEC of mice with 

colitis indicating that IEC function to attract and activate 

cells of the immune system. 

doi:10.1016/j.clim.2007.03.123 

S169 

Su.85 TNF Binding by Certolizumab Pegol, 

Adalimumab, and Infliximab-Stoichiometry, 

Complex Formation, and the Biologic Effects of 

Complexes 

Alistair Henry, Senior Principal Scientist, UCB Celltech, 

Research Biologicals, Slough, UK, Jeff Kennedy, Senior 

Scientist, UCB Celltech, Enzymology, Slough, UK, Gianluca 

Fossati, Principal Scientist, UCB Celltech, Slough, UK, 

Andrew Nesbitt, Senior Group Leader, UCB Celltech, 

Inflammation Discovery, Slough, UK 

Immune complexes can have various unwanted consequences. 

We determined the binding stoichiometry of certolizumab 

pegol, adalimumab, and infliximab to the tumor 

necrosis factor (TNF) trimer, and the size and in vitro biologic 

consequences of the resulting immune complexes. Isothermal 

titration calorimetry was used to assess the stoichiometry of 

binding through calculation of the number of anti-TNF 

molecules bound to a TNF trimer when no more heat of 

binding was released. Dynamic light scatter assessed the size 

of complexes formed over a range of antigen:antibody ratios. 

Effect of the complexes on peripheral blood neutrophil 

degranulation (myeloperoxidase release) and superoxide


production (oxidation of cytochrome c) were measured. At 

optimum antigen:antibody ratio, adalimumab and infliximab 

formed huge complexes N30 nm in diameter, suggesting that 

these bivalent antibodies crosslink TNF trimers. Certolizumab 

pegol complexes were b20 nm in diameter, consistent 

with its univalent structure preventing crosslinking. Certolizumab 

pegol bound 2.9 monomers at saturation, whereas 

infliximab and adalimumab both bound around 2.5 monomers. 

This suggests that steric or allosteric restrictions 

prevent the bivalent antibodies binding to all monomers in 

a trimer. The large immune complexes formed by adalimumab 

and infliximab caused degranulation and superoxide 

production by neutrophils. The smaller certolizumab pegol 

complexes had only marginal effects. The bivalent structures 

of infliximab and adalimumab form enormous complexes with 

TNF trimers, which have proinflammatory effects on neutrophils 

in vitro. Certolizumab pegol does not form large 

complexes with TNF trimers due to its univalent structure 

preventing crosslinking, giving it a unique mode of action in 

this regard. 

doi:10.1016/j.clim.2007.03.124 

Su.86 Certolizumab Pegol and Adalimumab 

Accumulation in the Inflammed Paws of Mice with 

Collagen-induced Arthritis Compared to 

Noninflammed Tissue In Vivo Biofluorescence 

Imaging of Alexa 680-labeled Antibodies 

Roger Palframan, Senior Group Leader, UCB Celltech, 

Pharmacology, Slough, UK, Alex Vugler, Research Scientist, 

UCB Celltech, Pharmacology, Slough, UK, Adrian Moore, 

Senior Group Leader, UCB Celltech, Pharmacology, Slough, 

UK, Mark Baker, Research Scientist, UCB Celltech, Clinical 

Pharmacology and Experimental Medicine, Slough, UK, 

Daniel Lightwood, Group Leader, UCB Celltech, Antibody 

Biology, Slough, UK, Andrew Nesbitt, Senior Group Leader, 

UCB Celltech, Inflammation Discovery, Slough, UK, Roly 

Foulkes, Director, UCB Celltech, Non-clinical Development/ 

Inflammation Therapeutic Area, Slough, UK, Neil Gozzard, 

Director, UCB Celltech, Pharmacology, Slough, UK 

Variability in disposition of monoclonal antibodies results 

in different exposure time courses in inflammed and noninflammed 

tissues. We investigated the disposition of certolizumab 

pegol and adalimumab in normal and inflammed 

tissue in vivo in mice, using a novel noninvasive biofluorescence 

technique. Certolizumab pegol or adalimumab labeled 

with the low-molecular weight dye Alexa 680 was administered 

intravenously (2 mg/kg) in naïve DBA/1 mice and in 

DBA/1 mice with ongoing collagen-induced arthritis. The 

accumulation of certolizumab pegol and adalimumab was 

measured in the hind paws at multiple points up to 26 hours 

using a Xenogen IVIS200 biofluorescence imager. Tail blood 

samples were taken for determination of serum levels of the 

reagents by ELISA. Both reagents penetrated inflammed 

tissue more than noninflammed tissue. The penetration of 

certolizumab pegol into inflammed arthritic paws was 

greater and more prolonged than for adalimumab (inflammed:noninflammed 

tissue ratios were 3.9:1 for certolizumab 

pegol and 1.9:1 for adalimumab; elimination half-lives 

were 27.8 and 5.5 hours, respectively). The plasma time 

courses reflected known differences in exposure, with 

certolizumab pegol maintaining higher plasma concentrations 

than adalimumab. In this model, certolizumab pegol 

disposition was more responsive to inflammation, with 

prolonged tissue infiltration occurring primarily in inflammed 

tissue. Certolizumab pegol, in contrast to adalimumab, 

appeared to readily enter inflammed tissue but not noninflammed 

tissue. This feature of certolizumab pegol may be 

conferred on the molecule by PEGylation. Increased drug 

exposure at the site of inflammation might be an important 

consideration for the treatment of inflammatory disorders 

such as rheumatoid arthritis and Crohn's disease. 

doi:10.1016/j.clim.2007.03.126 

Su.87 Comparison of Naturally Occurring Human 

Immunoglobulin Sequences with the Anti-TNF 

Agents Certolizumab Pegol, Adalimumab, and 

Infliximab 

Andrew Martin, Senior Lecturer, University College London, 

Biochemistry and Molecular Biology, London, UK, Kerry 

Tyson, Principal Scientist, UCB Celltech, Antibody Biology, 

Slough, UK, Matt Page, Research Associate Informatics, UCB 

Celltech, Therapeutic Project Informatics, Slough, UK, 

Gianluca Fossati, Principal Scientist, UCB Celltech, 

Inflammation Discovery, Slough, UK, James Snowden, Senior 

Scientist, UCB Celltech, Informatics, Slough, UK, Raghavan 

Abhinandan, Lecturer, University College London, 

Biochemistry and Molecular Biology, London, UK, Alastair 

Lawson, Director of Antibody Biology, UCB Celltech, 

Antibody Biology, Slough, UK, Andrew Nesbitt, Senior Group 

Leader, UCB Celltech, Inflammation Discovery, Slough, UK, 

Andrew Popplewell, Associate Director, UCB Celltech, 

Antibody Biology, Slough, UK 

V-region sequences of certolizumab pegol, adalimumab, 

and infliximab were compared with a database of normal 

human IgG V-region sequences. A database of rearranged 

human variable heavy [VH] and variable kappa light [VK] 

amino acid sequences was obtained by sequencing 243 

unique VH chains and 312 unique VK chains from healthy 

donors. Each sequence was compared to the closest human 

“germline” sequence and also scored against every other 

sequence of the same chain type, using percentage identity 

to measure sequence similarity. Z-scores were calculated 

from a frequency distribution plot and compared between 

agents. A positive Z-score indicates that a sequence is more 

typical than average of a human sequence. The mean 

number of nongermline framework residues in rearranged 

human antibodies was 4.3% for VK and 8.8% for VH. Of the 

three agents, certolizumab pegol had a V-region most typical 

of the rearranged human sequences with 3.8% nongermline 

residues for VK and 8.5% for VH. Adalimumab had 1.3% and 

1.2% and infliximab 31.3% and 22.2%, respectively. Positive Zscores 

were obtained for the VK and VH sequences of 

certolizumab pegol (0.15 and 1.46) and adalimumab (0.67 

and 1.34). However, infliximab V-region sequences gave 

negative Z-scores (−2.37 and −0.58) indicating they are less 

typical of human sequences. The certolizumab pegol Vregion 

framework sequence has a similar mean number of 

nongermline residues as rearranged human IgG V-region

Abstracts 

frameworks. The complete VH and VK sequences were also 

more human-like than average. The certolizumab pegol Fab' 

sequence can be considered typical of naturally occurring 

human IgG. 

doi:10.1016/j.clim.2007.03.127 

Su.89 Role of Enteric Lumenal TLR Ligand Sampling 

in the Formation of Novel Resident Mucosal 


Daisuke Fujiwara, Postdoctoral Fellow, Department of 

Pathology and Laboratory Medicine, David Geffen School of 


Angeles, CA, Bo Wei, Assistant Professor, Department of 

Pathology and Laboratory Medicine, David Geffen School of 


Angeles, CA, Michael McPherson, PhD Student, Department 

of Pathology and Laboratory Medicine, David Geffen School 

of Medicine at University of California, Los Angeles, Los 

Angeles, CA, Jonathan Braun, Professor and Chair, 

Department of Pathology and Laboratory Medicine, David 

Geffen School of Medicine at University of California, Los 

Angeles, Los Angeles, CA 

Resident intestinal dendritic cells (DCs) play important 

immunoregulatory roles through sensing and antigen handling 

of enteric microbiota. However, intestinal DCs have not been 

well characterized. In this study, we found that CD11c+ cells 

isolated from the small intestine superficial and deep lamina 

propria were unusual (compared to lymph node and spleen 

DC's) due to their graduated expression of CD11b or B220, and 

the predominance of B220+CD11b+ double-positive (DP) DC's. 

Since lamina propria DC's engage in lumenal sampling and thus 

encounter TLR ligands, we wondered whether TLR signaling 

might induce the unusual DP phenotype. Using bone marrowderived 

DC's differentiated with Flt-3L, we evaluated the 

effect of co-culture with various TLR ligands. Whereas Flt-3L 

alone induced single-positive CD11b+ or B220+ DCs, DP DCs 

predominated after TLR2, TLR4, and TLR9 ligands. The 

response was abrogated in myd88−/− mice, suggesting that 

DP DC's are induced through TLR-MyD88 pathway. Fecal 

extracts also induced DP DC formation; because this response 

was absent in TLR4−/− mice, it appeared that the predominant 

enteric bioactivity for DP DC induction was LPS. Among 

lamina propria DCs, we also observed a probable immature 

mDC subset (CD11c-B220-CD11blow). This subset was deficient 

in germ-free (GF) mice or cd8 knockout mice, suggesting an 

important TLRL role in differentiation stage of DCs in vivo,and 

a novel requirement for CD8+ T cells. These findings indicate 

that lumenal TLR4 ligands shape the differentiation of mucosal 

resident DCs, resulting in a novel phenotype associated with 

distinct immunoregulatory traits. Supported by NIH DK46763 

and DK69434. 

doi:10.1016/j.clim.2007.03.128 

Su.90 Up-Regulation of the PI3-Kinase Pathway in 

Lamina Propria T Lymphocytes 

Jutta Braunstein, Research Scientist, University Hospital 

Heidelberg, Heidelberg, Germany, Frank Autschbach, 

Deputy Head of Pathology, University Hospital Heidelberg, 

Heidelberg, Germany, Felix Lasitschka, Postdoctoral Fellow, 

University Hospital Heidelberg, Heidelberg, Germany, 

Thomas Giese, Group Leader, University Hospital 

Heidelberg, Heidelberg, Germany, Antje Heidtmann, 

Technician, University Hospital Heidelberg, Heidelberg, 

Germany, Bernd Sido, Senior Physician, University Hospital 

Heidelberg, Heidelberg, Germany, Benjamin Funke, 

Physician, University Hospital Heidelberg, Heidelberg, 

Germany, Andreas Schroeder, Medical Advisor, Merck Sero, 

Darmstadt, Germany, Yvonne Samstag, Group Leader, 

University Hospital Heidelberg, Heidelberg, Germany, 

Stefan Meuer, Head of Institute of Immunology, University 

Hospital Heidelberg, Heidelberg, Germany 

Understanding the regulation of immune responses in the 

normal intestinal mucosa is critical for defining its alterations 

in inflammatory bowel disease. Importantly, intestinal lamina 

propria T lymphocytes (LPT) when investigated ex vivo exhibit 

functional properties profoundly different from those of 

peripheral blood T lymphocytes (PBT). In particular, following 

CD2 stimulation they are able to produce markedly higher 

levels of cytokines than PBT. This study provides insight into 

signaling events associated with the high CD2 responsiveness of 

LPT. CD2 stimulation results in enhanced phosphorylation of 

the PI3-kinase dependent kinase Akt and its down-stream 

target GSK-3 in LPTwhen compared to PBT. That up-regulation 

of PI3-kinase pathway activation in LPTcontributes to the high 

IL-2 expression in response to CD2 stimulation is demonstrated 

by the fact that inhibition of this pathway by the PI3-kinase 

specific inhibitor Ly294002 to levels induced in PBT clearly 

reduces IL-2 gene expression (and TNF-α, CD40L gene 

expression) in LPT. Immunohistochemical analysis reveals 

that Akt phosphorylation occurs in few lamina propria mononuclear 

cells in the normal mucosa in vivo; moreintense 

phospho-Akt staining of larger numbers of lamina propria 

mononuclear cells is detectable in the inflammed intestine 

(ulcerative colitis) indicating that activation of the PI3-kinase 

pathway plays an important role in intestinal inflammation. 

Enhanced responsiveness of these pathways to costimulatory 

stimuli may allow for rapid and vigorous T cell responses and 

could therefore be fundamental to intestinal mucosal immune 

responses and homeostasis. 

doi:10.1016/j.clim.2007.03.129 

S171 

Su.91 Differential Levels of Intercellular Glutathion 

(GSH) Dictate the Shift From Adaptive to Innate T 

Cell Function in the Human Intestinal Mucosa 

Bernd Sido, Senior Physician, Department of Surgery, 

Heidelberg, Germany, Frank Autschbach, Senior Physician, 

Institute of Pathology, Heidelberg, Germany, Jutta 

Schroeder-Braunstein, Scientist, Institute of Immunology, 

Heidelberg, Germany, Thomas Giese, Group Leader, 

Institute of Immunology, Heidelberg, Germany, Felix 

Lasitschka, Postdoctoral Fellow, Institute of Immunology, 

Heidelberg, Germany, Stefan Meuer, Director, Institute of 


Isolated human Tcells from the healthy human mucosa are 

non-responsive to T cell receptor engagement yet mount


vigorous responses to non-specific activation through CD2 or 

CD28 triggering with regard to both proliferation and cytokine 

secretion. Since resting T lymphocytes do not express 

the cystine transporter (xCT), which is critical for the import 

of cystine across the plasma membrane and since cystine is a 

critical precursor for GSH synthesis, they are dependent on an 

extracellular source of cysteine. Under physiologic conditions, 

cysteine is not available to T cells in the intestinal 

mucosa since the local myeloid cell population is unable to 

secrete cysteine, unlike circulating monocytes from peripheral 

blood. As a consequence, mucosal T lymphocytes contain 

significantly lower GSH concentrations than blood T lymphocytes. 

Enhancement of GSH levels in mucosal T cells by 

providing cysteine either directly or by adding blood 

monocytes restores their antigen receptor reactivity. This 

mechanism appears to be relevant for their behaviour in 

inflammatory bowel diseases where GSH levels in mucosal T 

cells from inflammatory sites are at least as high as in 

circulating blood T cells. Here, myeloid cells with the capacity 

to secrete cysteine are frequently detected. Importantly, 

this is not only due to their influx from blood but, at least in 

part, to a functional and phenotypic change under altered 

microenvironmental conditions. Supported by a grant from 

DFG/SFB405/B6. 

doi:10.1016/j.clim.2007.03.130 

Su.92 Whole Genome Association Identifies Novel 

Susceptibility Genes for Crohn's Disease and 

Implicates a Crucial Role for Autophagy 

John Rioux, Associate Professor, Universite de Montreal, 

Department of Medicine, Montreal, QC, Canada, Ramnik 

Xavier, Associate Professor, Massachusetts General Hospital, 

Harvard Medical School, Boston, MA, Kent Taylor, Associate 

Professor, Cedars Sinai Medical Center, Los Angeles, CA, 

Philippe Goyette, Scientist, Montreal Heart Institute, 

Montreal, QC, Canada, Mark Silverberg, Associate Professor, 

Mount Sinai Hospital, Toronto, ON, Canada, Alan Huett, 

Postdoctoral Fellow, Massachusetts General Hospital, 

Harvard Medical School, Boston, MA, Todd Green, Project 

Manager, Broad Institute of MIT and Harvard, Cambridge, 

MA, Petric Kuballa, Postdoctoral Fellow, Massachusetts 

General Hospital, Harvard Medical School, Boston, MA, 

Michael Barmada, Associate Professor, University of 

Pittsburgh, Pittsburgh, PA, Lisa Datta, John Hopkins 

University School of Medicine, Baltimore, MD, Yin Yao 

Shugart, Associate Professor, John Hopkins University, 

Bloomberg School of Public Health, Baltimore, MD, Edmond 

Jean Bernard, Assistant Professor, Universite de Montreal, 

Montreal, QC, Canada, Ling Mei, Cedars Sinai Medical 

Center, Los Angeles, CA, Dan Nicolae, Associate Professor, 

University of Chicago, Departments of Medicine and 

Statistics, Chicago, IL, Hillary Steinhart, Associate 

Professor, Mount Sinai Hospital and University of Toronto, 

Toronto, ON, Canada, Jerome Rotter, Professor, Cedars Sinai 

Medical Center, Los Angeles, CA, Judy Cho, Yale University, 

New Haven, CT, Mark Daly, The Broad Institute of MIT and 

Harvard, Cambridge, MA, Miguel Regueiro, Associate 

Professor of Medicine, University of Pittsburgh, Division 

of Gastroenterology, Hepatology and Nutrition, Pittsburgh, 

PA, Philip Schumm, University of Chicago, Department 

of Health Studies, Chicago, IL, Richard Duerr, Associate 

Professor, University of Pittsburgh, School of Medicine, 

Pittsburgh, PA, Steven Brant, Meyerhoff Inflammatory 

Bowel Disease Center, John Hopkins University School 

of Medicine, Baltimore, MD 

In 2002, the NIDDK IBD Genetics Consortium was founded 

by six Genetic Research Centers located in Canada and the 

US. Our first stage genome-wide association (GWA) study of 

308,332 autosomal single nucleotide polymorphisms (SNPs) 

in 567 ileal CD samples and 571 controls identified genetic 

variants within the IL23R gene that influence an individual's 

risk for developing IBD (Duerr et al. Science 2006). In the 

current study, we typed an independent set 401 patients with 

ileal CD and 433 controls for the exact same set of SNPs. We 

have since performed a combined analysis of all cases and 

controls as well as an independent replication study that 

establish five regions of unambiguously confirmed association 

of genome-wide significance. Specifically, in addition to 

the established CARD15 and IL23R associations, we report 

three novel confirmed associations with variation in the 

PHOX2B and ATG16L1 genes and in an intergenic region on 

10q21.1. Moreover, we also identify a statistically significant 

excess of additional regions showing nominal replication of 

association that likely contain additional gene(s). We further 

demonstrate that the ATG16L1 gene is expressed in intestinal 

epithelial cells and that functional knock down of this gene 

abrogates autophagy of Salmonella typhimurium. Together 

these findings implicate that autophagy and other host 

responses to intra-cellular microbes are involved in the pathogenesis 

of CD. 

doi:10.1016/j.clim.2007.03.131 

Su.93 A High Density Association Study of the 

Extended Major Histocompatibility Locus in 

Ulcerative Colitis 

Philippe Goyette, Research Associate, Montreal Heart 

Institute, Montreal, QC, Canada, Todd Green, The Broad 

Institute of MIT & Harvard, Cambridge, MA, Paul de Bakker, 

The Broad Institute of MIT & Harvard, Cambridge, MA, 

Daniel Mirel, The Broad Institute of MIT & Harvard, 

Cambridge, MA, Christine Stevens, The Board Institute 

of MIT & Harvard, Cambridge, MA, Anna Latiano, IRCCS 

Casa Sollievo della Sofferenza, Italy, Jorge Oksenberg, 

University of California, San Francisco, San Francisco, CA, 

S. Hauser, University of California, San Francisco, San 

Francisco, CA, Vitto Annese, Medical School and University 

of Foggia, Italy, John Rioux, Montreal, QC, Canada, 

Mark Daly, The Broad Institute of MIT & Harvard, 

Cambridge, MA 

Ulcerative colitis (UC) and Crohn's disease (CD) are chronic 

inflammatory diseases of the gastrointestinal tract. While 

the study of genetic risk factors in CD has led to the identification 

of multiple risk factors, few have been convincingly 

identified in UC. Several independent genome-wide 

linkage scans, however, have suggested that the major 

histocompatibility complex (MHC) region plays a role in the 

genetic susceptibility to UC. While several putative association 

results have been reported for UC in this region, findings

Abstracts 

have not been consistent due to the limited number of alleles 

tested, to small sample sizes and to extensive LD in the 

region. We have recently reported the creation of a high 

density genetic map, including 7500 SNPs and DIPs, as well as 

the classical HLA genes. In the current study, we have 

selected 1500 of the most informative SNPs in order to 

capture the common variability in this region. Using this 

panel, we performed an association study in a cohort of 643 

UC cases and 1056 controls. Single marker association testing 

identified 35 SNPs with significant association (b4.5×10 − 5 ) 

to UC. The peak of association, supported by multiple SNPs, 

is localized in the HLA-DRA and HLA-DQA gene regions. HLA 

tagging SNPs also identify this region as containing a UC 

susceptibility locus. Our initial analyses suggest that a 

common variant (∼30% frequency) in this region can explain 

the observed association. Higher density association mapping 

of this region and replication in an independent cohort 

will be necessary to identify causal allele(s). 

doi:10.1016/j.clim.2007.03.132 

Su.94 TRAM-34, a Blocker of the Calcium-activated 

Potassium Channel KCa3.1, Prevents Acute 

Transplant Rejection 

Heike Wulff, Assistant Professor, University of California, 

Davis, Davis, CA, Yi-Je Chen, Graduate Student, University 

of California, Davis, Surgical and Radiological Sciences, 

Davis, CA, Girija Raman, Postdoctoral Researcher, 

University of California, Davis, Medical Pharmacology, 

Davis, CA, Clare Gregory, Professor, University of California, 

Davis, Surgical and Radiological Sciences, Davis, CA 

Rejection of transplanted organs is caused by an immune 

response of recipient T cells against the donor MHC. One 

emerging new target for the suppression of T cell activation 

is the calcium-activated potassium channel KCa3.1. In 

addition to T and B cells, KCa3.1 is also expressed in 

macrophages, proliferating vascular smooth muscle and 

endothelial cells and has been shown to play an important 

role in the proliferation and migration of these cells. In 

2000, we designed a selective small molecule KCa3.1 

blocker called TRAM-34 (EC50=20 nM) and showed that it 

suppressed T cell proliferation and synergized with cyclosporine 

(PNAS 2000, 97: 8151). TRAM-34 has a half-life of 

1.8 hours in rats and of 2.1 hours in rhesus macaques, 

prevents restenosis in rats and exhibits no signs of acute of 

long-term toxicity. Encouraged by these results we are here 

testing TRAM-34 alone or in combination with cyclosporine 

in a model of acute transplant rejection. Hearts are 

heterotopically transplanted from male Brown Norway to 

male Lewis rats. While transplanted hearts in vehicle 

treated animals failed within 9 days (n=6), transplanted 

hearts in TRAM-34 treated rats (10 mg/kg twice daily) 

remained functional for an average of 25 days (n=6). In 

comparison, treatment with low dose cyclosporine (2 mg/kg 

Neoral®) resulted in a graft survival time of 18 days (n=6). 

Combinations of TRAM-34 with low dose cyclosporine are 

currently being tested. Supported by NIH and UC Davis. 

doi:10.1016/j.clim.2007.03.133 

Su.95 Low Doses IVIG with Plasmapheresis and 

Rituximab in Positive Cross Match Patients 

Miguel Rodriguez, Immunoallergist, Hospital 

Especialidades, IMSS, Guadalajara, Mexico, Luis Vales- 

Alberto, MD, Hospital Especialidades IMSS, Departmento de 

Nefrologia, Guadalajara, Mexico, Felicia Castro-Ramos, 

Biologist, Hospital Especialidades IMSS, Nephrology Unit, 

Guadalajara, Mexico, Francisco Monteon-Ramos, 

Nephrologist, Hospital de Especialidades IMSS, Nephrology 

Unit, Guadalajara, Efrain Montano-Gonzales, 

Immunoallergologist, Hospital Especialidades UMAE, IMSS 

Department of Immunology and Allergy, Guadalajara, 

Mexico, Alejandro Bautista-Vazquez, Nephrologist, Hospital 

de Especialidades, CMENGLANDUMAE, IMSS, Unit of 

Nephrology, Guadalajara, Mexico, Salvador Chavez, MD, 

Hospital Especialidades IMSS, Departamento de Nefrologia, 

Guadalajara, Mexico 

Introduction: Renal Transplant (RT) is the best option for 

End Stage Renal Disease (ESRD) Positive Cross Match (+XM) is 

formal contraindication to RT. Protocols have been developed 

to overcome this problem. Methods and Materials: Simple 

not ramdomized sample. Patients ABO compatible with 

+XM (up to 30%) by FlowCytometry (FC) to living related 

donor. Patients scheme was: PP after that IVIG 100 mg/kg 3 

times every other day. If XM becomes negative we administrated 

Rituximab and go to RT. After transplant 3 more 

sessions of PP and IVIG were given. Results: We treated 6 

patients, 3 in 2nd RT. One persisted positive after scheme 

and was not transplanted. Average PRA Class I 91.83% (SD 

6.1) Class II 24.14 (SD 31.7. Five transplanted patients with 

satisfactory evolution after 7.6 Mo (SD 3.9 Mo) with excellent 

renal function: creatinine 0.9 (SD 0.07 mg/dl) and creatinine 

depuration 84 (SD 10 ml/min/1.73 m2. One case presented 

cellular rejection (percutaneous renal biopsy with negative 

c4d) 10 weeks after RT that respond to IV steroids. We do not 

detect any infectious complication. Discussion: It is possible 

to desensitize positive cross match patients with this 

schedule of very low IVIG doses, diminishing costs and 

without side effects. Until now results are very satisfactory, 

we need more cases to obtain experience. 

doi:10.1016/j.clim.2007.03.135 

S173 

Su.96 Semi-mature Bone Marrow Derived DC Expand 

Foxp3+CD4+CD25+CD127- Regulatory T Cells with 

Suppressive Activity in the Macaque 

Aureli Moreau, PhD student, INSERM U643- ITERT/U649, 

Nantes, France, Gaelle Beriou, PhD, Center of Neurologic 

Diseases, Boston, MA, Jack-Yves Deschamps, DVM, ENVN, 

Nantes, France, Joanna Ashton-Chess, PhD, INSERM 

U643- ITERT, Nantes, France, Heslan Michele, Engineer, 

INSERM U643- ITERT, Nantes, France, Elise Chiffoleau, CR2 

INSERM, INSERM U643- ITERT, Nantes, France, Regis Josien, 

CR1, INSERM U643- ITERT, Nantes, France, Philippe Moullier, 

DR2 INSERM, U649, Nantes, France, Brigitte Alliot-Licht, 

PhD, INSERM U643- ITERT, Nantes, France, Maria-Cristina 

Cuturi, DR2, INSERM U643- ITERT, Nantes, France 

Over the past 10 years, several types of tolerogenic dendritic 

cells (DC) and T regulatory cells (Treg) have been


identified that play a pivotal role in the control of autoimmunity 

and transplantation tolerance in rodents. Recent 

studies have shown that immature DC and ex vivo expanded 

Treg could be potential reagents to promote antigen-specific 

tolerance in vivo. To date, these works have been carried out 

mostly in rodents and will need to be validated in primates 

before being applied in the clinic. In order to better 

characterise tolerogenic DC and Treg in primates we first 

generated and characterised different types of DC from 

macaque bone marrow (BMDC). Among these cells, the most 

immature BMDC (adherent cells resulting from culture in the 

presence of GM-CSF alone) could be good candidates for in 

vivo testing in protocols of tolerance induction. We were also 

able to demonstrate that in the presence of IL-2, semimature 

BMDC (cultured with GM-CSF and IL-4) have the 

ability to expand CD4+CD25+CD127-Foxp3+ macaque Treg 

cells (25 fold in 7 days) and additionally increase their suppressive 

activity. These results represent an important step 

towards the potential use of tolerogenic DC and/or Treg as an 

adoptive cell therapy to induce antigen-specific tolerance in 

pre-clinical, non-human primate models. 

doi:10.1016/j.clim.2007.03.136 

Su.98 Low Doses of Intravenous Immunoglobulin in 

Renal Transplant Patients with High Levels of 

Anti-HLA Panel Reactive Antibodies (PRA) 

Miguel Rodriguez, Immunologist, Hospital Especialidades, 

IMSS Department Inmunologia, Guadalajara, Mexico, 

Alejandro Bautista, Nephrologist, Hospital Especialidades 

IMSS, Department Nephrology, Guadalajara, Mexico, 

Francisco Monteon, MD, Hospital Especialidades Division of 

Transplantes, Guadalajara, Mexico, Felicia Castro, 

Pharmacobiology Chemist, Hospital Especialidades, Division 

of Transplantes, Guadalajara, Mexico, Efrain Montaño, MD, 

Hospital Especialidades IMSS, Department Inmunologia 

Clínica, Guadalajara, Mexico, Antonio Flores, MD, Hospital 

Especialidades, IMSS Department Nefrologia, Guadalajara, 

Mexico 

Introduction: The renal transplant (RT) is the best 

treatment for the patients with End Stage Renal Disease 

(ESRD). Patient with anti-HLA antibodies has a short graft life. 

Different studies shown that intravenous immunoglobulin 

(IVIG) diminishes anti-HLA antibodies. Methods and materials: 

An open, not randomized clinical trial. We used 100 mg/kg 

of IVIG in patient with PRA higher than 305 with negative 

crossmatch to donor (XM−) by flow cytometry (FC). We 

administered 3 doses of IVIG 100 mg/kg every other day. One 

week after the PRA was repeated. Patients with PRA lower 

than 30%, were considered desensitized and proceeded to RT, 

monitoring the occurrence of rejections and graft survival. 

Results: We reported 8 patients (3M/5F), age of 22.8 SD 

2.4 years. Four patients with second RT (50%). PRA previous 

88.65% (SD 5.65%) Class I and 50.09% (SD 40.32%) Class II. After 

treatment 7.66% (SD 12.32%) Class I and 11.39% (SD25.37%) 

Class II. PRA diminished in 7 patients and in 1 patient 

remained elevated. Five received a RT from living related 

donor, ABO compatible (71.4%) and two of them from cadaver 

(28.6). The monitoring of this group is 46+–25.7 weeks. One 

patient (from cadaver) presented two rejection crises both 

resolved favorably. Creatinine being 0.95 mg/dl (SD 0.4). 

Creatinine depuration (MDRD)88.6 SD 22.05 ml/min/1.73m2. 

We did not document any infectious process within the group. 

Discussion: IVIG in low doses confer a protective effect, in 

patients with high PRA. We need more monitoring to evaluate 

the impact in the long term. 

doi:10.1016/j.clim.2007.03.137 

Su.99 Efficient Ex Vivo Priming of Naïve Precursors 

into EBV-specific Type-1 CD8+ T Cell Responses from 

EBV Seronegative Pediatric HTx Patients Requires 

IL-12 and IL-27 

Diana Metes, Assistant Professor, University of Pittsburgh, 

Transplantation Surgery, Pittsburgh, PA, Camila Macedo, 

Postdoctoral Associate, University of Pittsburgh, 

Transplantation Surgery, Pittsburgh, PA, Walter Storkus, 

Professor, University of Pittsburgh, Dermatology, 

Pittsburgh, PA, Iulia Popescu, Research Instructor, 

University of Pittsburgh, Transplantation Surgery, 

Pittsburgh, PA, Steve Webber, Professor, University of 

Pittsburgh, Pediatrics, Pittsburgh, PA 

The risk of post-transplant lymphoproliferative disorders 

(PTLD) is increased in pediatric recipients, and cellular 

immunotherapy with EBV specific cytotoxic T lymphocytes 

(CTLs) represents a promising therapeutic strategy to restore 

cellular immunity in PTLD patients. Although we were 

effective at priming strong EBV-specific CTL responses from 

EBV-seronegative healthy adults using DC-based protocols, 

similar attempts were unsuccessful for EBV-seronegative 

pediatric Tx patients. Peripheral blood lymphocytes were 

obtained from 5 EBV seronegative HTx patients and were 

stimulated ex vivo for 10 days with autologous mature DC 

pulsed with a cocktail of MHC class I-restricted EBV peptides 

as follows: (i)DC/peptide only (ii)DC/peptide +IL-12 (an 

adjuvant cytokine for promoting Type-1 immunity-IFNgama 

secretion) (iii)DC/peptide +IL-27 (a potent early cytokine 

regulator of Type-1 commitment) (IV)DC/peptide+IL-12+IL-27. 

EBV-specific CD8+ T cell responses were screened by flow 

cytometry and IFNgama ELISPOT assays. The DC/peptide + 

IL-12 +IL-27 approach yielded the best yields (1.7X), the 

optimal EBV-specific CD8+ T cell expansion (from undetectable 

to 1.5% +0.5 EBV-tetramer+ CD8 T cells), and optimal 

EBV specificity (from 3+2 spots/105 to 45+15 spots/105 

CD8+ T cells in ELISPOT assays). DC/peptide (9+2 spots/105) 

or the DC/peptide+IL-12 (12 +4 spots/105) protocols were 

unsuccessful in this setting. These results suggest that 

priming of functional EBV-specific CD8+ Tcells from pediatric 

EBV seronegative HTx patients is feasible, but EBV-Ag presentation 

by DC may require IL-27 in addition to IL-12 to 

promote Tc1 cells from naive CD8+ T cell precursors ex vivo. 

doi:10.1016/j.clim.2007.03.138 

Su.100 Pharmacodynamic Controlled Tapering of 

Cyclosporine A: A New Step Towards Individualized 

Immunosuppressive Therapy in Transplanted Patients 

Thomas Giese, Group Leader, Institute of Immunology, 

Heidelberg, Germany, Claudia Sommerer, Physician,

Abstracts 

Department of Nephrology, Heidelberg, Germany, Martin 

Zeier, Medical Director, Department of Nephrology, 

Heidelberg, Germany, Stefan Meuer, Director, Institute of 


Background: At present it is unclear which dose of Cyclosporine 

A (CsA) is optimal with respect to immunosuppressive 

efficacy and drug specific side effects at the level of the 

individual patient. Recently we proposed the quantitative 

assessment of NFAT regulated gene expression in peripheral 

lymphocytes as a new tool to measure the individual degree 

of immunosuppression by CsA and demonstrated that a 

significant correlation exists between suppression of NFATregulated 

genes and clinical complications such as infections 

and malignancies. The reduction of CsA dosage under close 

pharmacodynamic monitoring may lead to reduced drug 

specific side effects while maintaining optimal graft protection. 

Methods: Eight stable renal transplant recipients were 

enrolled and longitudinally followed for 27 (12–44) months. 

Median CsA dose was 1.83 mg/kg/day at the time of 

enrolment and was tapered to 1.19 mg/kg/day. NFATregulated 

gene expression was measured in peripheral 

blood lymphocytes stimulated with PMA and ionomycin ex 

vivo. All patients had an ultrasound guided allograft biopsy. 

Results: The stepwise reduction of CsA was inversely 

correlated to the residual NFAT-regulated gene expression. 

In seven patients there were no signs of acute rejection in 

the whole study period. One patient had a BANFF Ia 

rejection. In this patient the mean residual NFAT-regulated 

gene expression had increased up to 47% in 6 months prior to 

rejection. All patients without rejection had a median NFATregulated 

gene expression of 25%. Conclusion: The measurement 

of residual NFAT-related gene expression is a helpful 

and reliable tool in individualizing the immunosuppressive 

therapy in long-term allograft recipients. 

doi:10.1016/j.clim.2007.03.139 

Su.101 A Non-allogeneic Stimulus Triggers the 

Production of De Novo HLA Antibodies in Healthy 

Adults 

Jose Crispin, PhD Student, Instituto Nacional de Ciencias 

Medicas y Nutricion SZ, Mexico City, Mexico, Luis Morales 

Buenrostro, Investigator, Department of Nephrology, 


Mexico City, Mexico, Maria Ines Vargas Rojas, PhD Student, 



Mexico, Lluvia Marino Vazquez, Undergraduate Student, 

Department of Nephrology, Instituto Nacional de Ciencias 

Medicas y Nutricion SZ, Mexico City, Mexico, Claudia de Leo, 

Chemist, Department of Transplantation, Instituto Nacional 

de Ciencias Medicas y Nutricion SZ, Mexico City, Mexico, 

Josefina Alberu, Head of Department, Department of 

Transplantation, Instituto Nacional de Ciencias Medicas y 

Nutricion SZ, Mexico City, Mexico 

Transplant recipients develop antibodies against a wide 

array of HLA specificities, and not only against the antigens 

to which they have been exposed. The aim of the present 

study was to establish if HLA-unrelated immune stimulation 

is capable of triggering the production of HLA antibodies. We 

determined the presence of HLA antibodies in 20 healthy 

adults before and after the administration of an immune 

stimulus (hepatitis B vaccine plus tuberculin skin test). HLA 

antibodies were assessed with a flow-cytometry based 

system. At baseline, HBsAg antibodies were detected in 

protective titers in 75% of the subjects; HLA antibodies were 

negative in all but one. One week after the antigenic 

stimulus, HBsAg antibody titers increased significantly, 

without any detectable changes in HLA antibodies. Surprisingly, 

HLA antibodies developed in 8 participants 1 month 

after the application of the stimulus. One additional subject 

developed HLA antibodies 1 month later. Therefore 9/20 

subjects became PRA positive during the observation period. 

Antibody specificities were identified by single-antigen 

assay. The alloantibody response elicited by the immune 

stimulus was not consistent with memory cell stimulation. 

The findings of this study demonstrate that a non-HLA 

antigenic stimulus is capable of eliciting the development of 

HLA antibodies in healthy adults. 

doi:10.1016/j.clim.2007.03.140 

S175 

Su.102 Activation of Transcription Factor 

PPARγ In Vivo Protects Against Alloreactive Vascular 

Remodeling of Human Artery 

Ivana Kawikova, Associate Research Scientist, Yale School of 

Medicine, New Haven, CT, Yi Tai, Associate Research 

Scientist, Transplantation Surgery, New Haven, CT, Marc 

Lorber, Professor, Transplantation Surgery, Yale School of 

Medicine, New Haven, CT, George Tellides, Associate 

Professor, Cardiothoracic Surgery, New Haven, CT, Jordan 

Pober, Professor, Immunobiology, Yale School of Medicine, 

New Haven, CT, Alfred Bothwell, Professor, Immunobiology, 

New Haven, CT 

PPARγ is a ligand-activated transcription factor that 

controls lipogenesis and plays a role in regulation of immune 

responses. PPARγ ligands inhibited inflammation in several 

animal disease models. Here we tested effects of PPARγ 

ligands in the in vivo model of human graft arteriosclerosis 

(GA) in humanized (SCID)/beige mice. Human artery was 

inserted into mouse abdominal aorta, allowed to heal and 

then the mice were injected with alloreactive PBMC (3×108 

ip/mouse). This led to the development of GA in human 

artery within 4 weeks, while mouse vessels remained intact. 

Treatment with endogenous PPARγ ligands, 15-deoxy-prostaglandin 

J2(15-d-PGJ2; 50 μg/kg ip for 4 weeks) or 

pharmacological PPARγ ligand, ciglitazone (2 mg/kg for 4 

weeks ip.) prevents the vascular remodeling. PPARN=antagonist, 

GW9662 (300 μg/kg ip for 4 weeks), significantly 

reduced the protective effects of PPARγ ligands suggesting 

that the protection is mainly mediated via PPARγ activation. 

Since T cells infiltrating GA lesion produce IFNγ that can on 

its own induce GA of human artery, we tested the effects of 

15-d-PGJ2(50 μg/kg ip. for 4 weeks) also in this model and 

found that the treatment inhibited also IFNγ-induced GA. In 

summary, activation of PPARN=protects human artery 

against alloreactive GA and the mechanisms involve effects 

on pathways downstream of IFNγ. These studies may open 

new possibilities for the treatment of human graft rejection


since glitazons are PPARγ ligands that are already FDA 

approved drugs used for treatment of diabetes type 2. 

doi:10.1016/j.clim.2007.03.141 

Su.103 Potential Role of Donor-derived Regulatory 

T Cells in the Suppression of Alloimmune Responses 

Fleur Samantha Benghiat, MD, Institute for Medical 

Immunology, Gosselies, Belgium 

Naturally occurring CD4+CD25+FoxP3+ regulatory cells 

(Tregs) are essential for the regulation of allogeneic responses. 

In view of the small numbers of circulating Treg cells, 

many protocols nowadays attempt to expand recipient Tregs 

ex vivo in order to achieve allograft tolerance by infusing 

them back to the recipient. But these are tedious procedures. 

We instead hypothesized that Tregs coming from the 

same donor as the allograft could be easily extracted in large 

numbers and could target the graft by recognizing selfantigens 

on the graft. For this purpose, in vitro experiments 

were performed with C57BL/6 CD4+CD25neg cells as 

responders and bm12 bone marrow derived immature 

dendritic cells as stimulators. The efficacy of syngeneic 

(bm12) versus allogeneic (C57BL/6) Tregs in regulating Th1 

and Th2 alloresponses were compared. Our results show that 

syngeneic Tregs are as efficient as allogeneic Tregs to 

suppress IL-2, IFN-g and IL-13 in a dose dependent manner. 

Nevertheless, the more Tregs were added, the more the 

proliferation – tested by thymidine incorporation – was 

enhanced. Using CFSE staining and Thy1.1–Thy1.2 congenic 

markers, it was confirmed that proliferating cells were 

Foxp3+ Tregs originally coming from naturally occurring Tregs 

added to the culture. Treg proliferation was still observed 

when CD4+CD25neg alloreactive cells were substituted by 

recombinant IL-2. Similar results were observed with human 

cells. In conclusion, allogeneic and syngeneic Tregs are 

equally efficient in regulating Th1 and Th2 alloresponses in 

vitro and proliferate under the cover of IL-2. Ongoing experiments 

are investigating the role of donor Tregs in allograft 

acceptance. 

doi:10.1016/j.clim.2007.03.142 

Su.104 HMGB1 and IL-1 are Endogenous Mediators 

Linking Cell Injury to the Adaptive Immune 

Response 

Deepak A. Rao, MD/PhD Student, Immunobiology, Yale 

University School of Medicine, New Haven, CT, Jordan S. 

Pober, Professor and Vice-Chair, Immunobiology, Yale 

University School of Medicine, New Haven, CT 

Pre- or peri-operative allograft injury predisposes to 

acute and chronic allograft rejection; however, the mediators 

released by injured tissues that promote rejection 

remain unclear. We and others have demonstrated that IFNgamma-producing 

T cells play a key role in the development 

of chronic vascular rejection. Here, we report that freezethaw 

lysates of human umbilical vein endothelial cells (EC) 

increase the amount of IFN-gamma produced by T cells when 

added to co-cultures of CD4+ T cells with HLA-DR+ allogeneic 

EC. Immunoadsorption of HMGB1, a mediator released by 

necrotic cells, partly removes activity from the lysates. 

Recombinant HMGB1 has no effect on EC but increases IFNgamma 

production by CD4+ Tcells activated by allogeneic EC 

or by anti-CD3 and anti-CD28 mAbs. However, HMGB1enhanced, 

but not basal, IFN-gamma production is markedly 

reduced in co-cultures thoroughly depleted of monocytes. 

HMGB1 preparations rigorously depleted of endotoxin act on 

monocytes via TLR4 and CD14 to induce IL-1beta release, 

and neutralization of IL-1beta significantly reduces the 

effect of HMGB1 on EC-T cell co-cultures containing 

monocytes. EC lysates enhance IFN-gamma production in 

the absence of monocytes, and neutralization of IL-1alpha 

blocks almost all of this activity. Memory T cells express IL- 

1R1, and both IL-1alpha and IL-1beta increase IFN-gamma 

production from memory CD4+ T cells activated by allogeneic 

EC or anti-CD3 and anti-CD28 mAbs. We conclude that 

damaged EC may release HMGB1 and IL-1alpha, both of 

which directly co-stimulate T cell production of IFN-gamma. 

In addition, HMGB1 induces monocyte secretion of IL-1beta, 

further promoting IFN-gamma production. Supported by the 

NIH. 

doi:10.1016/j.clim.2007.03.144 

Su.105 Alloantigen-specific Treg: Repertoire 

Selection, TH17 Cell Differentiation and 

Prolongation of Organ Allograft Survival 

Giorgio Raimondi, Postdoctoral Scholar, Starzl 

Transplantation Institute, University of Pittsburgh, 

Department of Surgery, Pittsburgh, PA, Tokita Daisuke, 

Postdoctoral Associate, Starzl Transplantation Institute, 

University of Pittsburgh, Department of Surgery, 

Pittsburgh, PA, Zhiliang Wang, Research Assistant Professor, 

Starzl Transplantation Institute, University of Pittsburgh, 

Department of Surgery, Pittsburgh, PA, Tina Supter, 

Postdoctoral Associate, Starzl Transplantation Institute, 

University of Pittsburgh, Department of Surgery, 

Pittsburgh, PA, Angus Thomson, Professor of Surgery, Starzl 

Transplantation Institute, University of Pittsburgh, 

Department of Surgery, Pittsburgh, PA 

Little information exists regarding the selection and 

functional evaluation of CD4+CD25+Foxp3+ naturally-arising 

regulatory T cells (Treg) for promotion of transplant 

tolerance. We have investigated/optimized procedures for 

the selection of murine alloantigen (Ag)-specific Treg 

(AAsTreg), and tested their function in a MHC-mismatched 

heart allograft model. When naïve Treg were stimulated with 

mature (LPS-stimulated), allogeneic DC, with a high IL-2 

concentration (500 U/ml), significant numbers of Foxp3cells 

quickly accumulated. Intracellular flow analysis identified 

these Foxp3- cells as IL-17 producers (TH17). The 

expansion of Treg during an MLR supported then the use of 

immature allogeneic DC as stimulators. However, determination 

of Treg responder frequency (based on CFSE dilution 

analysis) during the MLR (immature DC:CD4+ T cells), 

revealed that an unexpected high frequency of Treg was 

induced to proliferate (approximately 27% of Foxp3+ cells, 

compared with the anticipated 5–8% of non-Treg). The high 

levels of IL-2 accounted for most of what we determined to

Abstracts 

be an ‘Ag-non-specific’ stimulation, causing the high 

precursor frequency measured. Based on these results, we 

used immature allogeneic DC and a mixture of stimulatory 

cytokines (low concentration) to select AAsTreg that proved 

extremely powerful Ag-specific inhibitors of alloreactive T 

cells proliferation (several-fold superior to freshly-isolated 

Treg). Moreover, 50% of heart transplants in normal animals 

given a short course of sub-therapeutic rapamycin, and 

injected with AAsTreg on day 7 post-transplant, are currently 

beating N70 days. Accounting for of all these observations 

and further optimization of the AAsTreg adoptive transfer 

strategy will be key to successful implementation of this 

clinically relevant regimen. 

doi:10.1016/j.clim.2007.03.145 

Su.107 Polyclonal Anti-thymocyte Globulin Exhibits 

Consistent Immunosuppressive Capabilities Beyond 

Cell Depletion 

Gina LaCorcia, Principal Research Associate, Genzyme Corp. 

IMD Biology and Transplant Research, Framingham, MA, 

Mark Swistak, Senior Research Associate, Genzyme Corp. 

IMD Biology and Transplant Research, Framingham, MA, 

Carla Lawendowski, Principal Research Associate, Genzyme 

Corp. IMD Biology and Transplant Research, Framingham, 

MA, Tim Weeden, Senior Research Associate, Genzyme Corp. 

IMD Biology and Transplant Research, Framingham, MA, Su 

Duan, Principal Research Associate, Genzyme Corp. IMD 

Biology and Transplant Research, Framingham, MA, Sharon 

Nahill, Scientific Director, Genzyme Corp. IMD Biology and 

Transplant Research, Framingham, MA, John Williams, Vice 

President, Genzyme Corp. IMD Biology and Transplant 

Research, Framingham, MA, John Dzuris, Senior Scientist, 

Genzyme Corp. IMD Biology and Transplant Research, 

Framingham, MA 

Thymoglobulin® [Anti-thymocyte Globulin (Rabbit)] is a 

purified, gamma globulin produced by immunization of 

rabbits with human thymocytes. It is an FDA approved therapy 

for acute organ transplant rejection. Although the 

mechanism of action of Thymoglobulin® is not fully understood, 

its efficacy is thought to be the result of lymphocyte 

depletion via cytotoxic antibodies directed against antigens 

expressed on human T lymphocytes. Thymoglobulin® also 

contains antigen reactivities that may account for downmodulation 

of T cell activation and lymphocyte trafficking. 

The in vitro studies presented here demonstrate some of the 

immunosuppressive effects of different manufactured 

batches of Thymoglobulin®. We show that culture of 

human PBMC with Thymoglobulin® results in the emergence 

of an increased population of Foxp3+ regulatory T cells (T 

reg) which are functional in that they potently suppress in 

vitro T cell activation. All 14 retained batches of manufactured 

Thymoglobulin® tested induced T reg that were 

immunosuppressive. Thymoglobulin® also interacts with 

many different cell surface receptors in a consistent and 

reproducible manner. Flow cytometric analysis demonstrated 

that all batches of Thymoglobulin® tested were 

comparable in binding ability to several cell surface 

receptors. One receptor to which Thymoglobulin® has 

reactivity is the chemokine receptor CXCR4. We demonstrate 

that 14 manufactured batches of Thymoglobulin® tested 

inhibit chemotaxis of the Jurkat Tcell line in response to SDF-1. 

These studies provide additional insights into the mechanisms 

by which Thymoglobulin® contributes to prevention and 

treatment of transplant rejection episodes and underscores 

the overall consistency of the immunosuppressive capability 

among different manufactured batches of Thymoglobulin®. 

doi:10.1016/j.clim.2007.03.146 

Su.108 KIR Genotyping of the Venezuelan Mestizo 

Population by PCR-SSP 

Angela Conesa, Bioanalist, Institute of Immunology, 

Caracas, Venezuela, Felix Toro, Biologist, Institute of 

Immunology, Caracas, Venezuela, Nubia Silva, Biologist, 

Institute of Immunology, Caracas, Venezuela, Maria-Elena 

Marquez, Biologist, Institute of Immunology, Caracas, 

Venezuela, Paolo Tassinari, MD, Institute of Immunology, 

Caracas, Venezuela, Isaac Blanca, Biologist, Institute of 

Immunology, Caracas, Venezuela 

Killer-cell-Immunoglobulin-like Receptors (KIR) belong to a 

family of NK cell receptors that recognize MHC-I molecules. 17 

KIR genes have been identified and clustered in 2 major 

haplotypes (A and B). These haplotypes show variations in 

geographical distribution among different human populations. 

Goals: a) To standardize the PCR technique for KIR genotyping 

in DNA samples, b) To determine KIR gene frequency in the 

Venezuelan mestizo population. Methods: Genomic DNA was 

isolated from Peripheral Blood Leucocytes. KIR genotyping was 

determined by PCR-SSP technique (1). Different experimental 

conditions were assessed: primers and DNA sample concentration, 

time and temperature of annealing and buffer composition. 

A commercial kit for KIR genotyping (Invitrogen) was used 

as reference assay. Results: We accomplished gene amplification 

of 14 KIR genes and the pseudogenes 2DP1 and 3DP1A/B. 

From 40 DNA samples analyzed, 100% (f=1) were positive for 

KIR2DL1, 2DL4, 2DS4, 3DL1 and pseudogenes 2DP1 and 3DP1. 

Other KIR showed a gene frequency b1: 2DL2(.28), 2DL3(.78), 

2DL5(.5), 2DS1(.39), 2DS2(.28), 2DS3(.18), 2DS5(.29), 

3DL2(.78), 3DL3(.73) and 3DS1(.33). Conclusions: a) Our 

preliminary results showed that frequency of inhibitory KIR 

genes was N0,73, except for KIR2DL2 and 2DL5, whereas 

frequency of the activating KIR2DS4 gene was equal to 1, 

showing an A haplotype similar to the Caucasian population, 

b) The PCR-SSP technique represents an efficient and low 

cost method for KIR genotyping. Key words, KIR, ADN, PCR- 

SSP. Supported by FONACIT G-2005000395. (1)Tissue Antigen 

2002; 59:184–193. 

doi:10.1016/j.clim.2007.03.147 

S177 

Su.109 Immunological Properties of Human 

Embryonic Stem Cell-derived Oligodendrocyte 

Progenitor Cells 

Ross Okamura, Scientist, Geron Corporation, Cell Biology, 

Menlo Park, CA, Melinda Au, Senior Research Associate, 

Geron Corporation, Cell Biology, Menlo Park, CA, Catherine 

Priest, Associate Director, Geron Corporation, Cell Biology, 

Menlo Park, CA, Anish Majumdar, Senior Director, Geron


Corporation, Cell Biology, Menlo Park, CA, Jerrod Denham, 

Senior Manager, Geron Corporation, Product Development, 

Menlo Park, CA 

A major concern in the use of allotransplantation of human 

embryonic stem cell (hESC)-derived cell therapies is the 

possibility of rejection by the host's immune system. We have 

previously reported that the undifferentiated hESC lines H1, 

H7 and H9 possess unique immunological properties which 

may make them less susceptible to rejection. To determine if 

differentiation alters the immunogenicity of hESCs, we chose 

to study hESC-derived oligodendrocyte progenitor cells (OPC) 

that have the potential for clinical application to treat 

patients with spinal cord injury. Undifferentiated H1 and H1derived 

OPCs express low to moderate levels of surface HLA-A, 

B,C antigens and lack surface expression of HLA-DR,DP,DQ 

antigens. In an in vitro mixed lymphocyte reaction (MLR) 

assay, both hESCs and OPCs stimulated weak T cell proliferation 

compared to allogeneic dendritic cells. Both undifferentiated 

hESCs (uhESCs) and their differentiated OPC 

derivatives were largely resistant to NK cell-mediated lysis. 

We have also investigated whether hESC-derived OPCs are 

susceptible to the presence of anti-Neu5Gc antibodies in 

normal human sera. We observed that of ten sera from normal 

healthy individuals representing all four blood groups, uhESCs 

showed no susceptibility to lysis and eight sera did not show 

measurable cytotoxicity against OPCs. The same sera when 

tested against murine embryonic fibroblasts (MEFs) demonstrated 

significant complement-dependent lysis. Taken 

together, these data show that the hESC-derived OPCs retain 

some of the unique immunological properties of the parental 

cell line from which they were differentiated. 

doi:10.1016/j.clim.2007.03.148 

Su.110 Adhesion Molecules in Pancreatitisassociated 

Lung Injury 

Roman Vatseba, Medical University, Lviv, Ukraine, Serge 

Chooklin, Medical University, Lviv, Ukraine, Myroslav Lyba, 

Medical University, Lviv, Ukraine 

Respiratory failure is typical for acute necrotizing pancreatitis. 

Unfortunately, the pathogenesis of these changes is 

fully unknown. The role of the endothelium in the human 

acute pancreatitis is still unclear. Applying the ELISA 

technique, plasma levels of soluble E-selectin (sE-selectin) 

and sICAM-1 were studied in 51 patients with acute 

pancreatitis. Twenty-five of them had respiratory complications. 

In this study enrolled patients who admitted to clinic 

within 48 hours after disease onset. The control group 

compiled 10 healthy volunteers. Already after admission the 

increased levels of adhesion molecules were noted in all 

patients. By that, the highest levels were observed in 

patients with lung injury. During first 7 days plasma levels 

of sE-selectin and sICAM-1 practically did not change in 

patients with necrotizing pancreatitis, while in patients with 

the pancreatitis-associated lung injury its levels gradually 

increased starting from the third day. There was a clear 

correlation between sE-selectin, sICAM-1 and severity of 

hemodynamic compromise was established. The intensity of 

endothelial activation, measured by adhesion molecules 

concentration, considered to be key features of systemic 

inflammation in severe pancreatitis. These data pointed on 

the role of adhesion molecules in the pathogenesis of 

pancreatitis-associated lung injury. Blockade of ICAM-1 and 

E-selectin synthesis and its receptors may be a novel target 

in the management of pancreatitis-associated lung injury. 

doi:10.1016/j.clim.2007.03.149 

Su.111 Aire Deficient Mice—A Model for 

Autoimmune Lung Disease 

Anthony Shum, Clinical Fellow, University of California, San 

Francisco Department of Medicine, San Francisco, CA, Jason 

DeVoss, Postdoctoral Fellow, University of California, San 

Francisco Diabetes Center, San Francisco, CA, Mark 

Anderson, Assistant Professor, University of California, San 

Francisco Diabetes Center and Department of Medicine, San 

Francisco, CA 

Aire deficient mice have a mutation for the gene encoding 

AIRE, a transcription factor that drives the ectopic expression 

of organ-specific antigens in the thymus. A deficiency in AIRE 

results in a breakdown of central tolerance toward selfantigens 

resulting in organ-specific autoimmune disease. The 

AIRE knockout mouse is a model for Autoimmune Polyglandular 

Syndrome Type 1, a disorder characterized by polyglandular 

autoimmune disease. Pulmonary manifestations, 

while rare, have been reported, including lymphocytic 

interstitial pneumonitis and bronchiolitis obliterans organizing 

pneumonia. Our goals are to 1) understand the mechanism 

of autoimmune-mediated attack and its relationship to the 

thymus 2) identify lung specific auto-antigens. Methods: AIRE 

knockout mice were examined for lung histology and 

immunohistochemistry. Serum from knockout mice was used 

for immunoblotting and indirect immunofluorescence. Flow 

cytometry and intracellular cytokine staining was performed 

on lymphocytes isolated from AIRE knockout lungs. Results: 

Lung histology reveals perivascular and peribronchiolar 

mononuclear inflammation. Immunoblots reveal a predominant 

antigen at 84 kilodaltons. The presence of autoantibody 

to this antigen correlates with the presence of histologic 

infiltrates. Indirect immunofluorescence on lung sections 

from unaffected mice show staining of bronchiolar epithelium. 

Immune cell subtypes visualized on sections of knockout 

mice reveal a predominance of CD4+ cells. Flow cytometric 

analysis of lymphocytes from lungs of knockout mice confirm 

the predominance of CD4+ T cells. Intracellular cytokine 

staining of lymphocytes reveal the predominance of the TH1 

cytokine IFN-γ. Conclusions: AIRE deficient mice are a 

promising model of spontaneous autoimmune lung disease 

offering the potential to identify lung auto-antigens. 

doi:10.1016/j.clim.2007.03.150 

Su.112 The XX Sex Chromosome Complement, as 

compared to the XY, Confers Greater Susceptibility to 

Experimental Autoimmune Diseases — EAE and SLE 

Deborah Smith, Doctoral Candidate, University of California, 

Los Angeles Neuroscience, Los Angeles, CA, Sienmi Du, 

Research Assistant, University of California, Los Angeles

Abstracts 

Neurology, Los Angeles, CA, Seema Tiwari-Woodruff, 

Associate Professor, University of California, Los Angeles 

Neurology, Los Angeles, CA, Arthur P. Arnold, Chair, 

Distinguished Professor, University of California, Los Angeles 

Physiological Science, Los Angeles, CA, Jennifer K. King, 

Post-Residency Fellow, University of California, Los Angeles, 

Rheumatology, Los Angeles, CA, Ram Raj Singh, Professor 

of Medicine, University of California, Los Angeles Medicine- 

Rheumatology, Los Angeles, CA, Rhonda R. Voskuhl, 

Professor in Residence, University of California, Los 

Angeles Neurology, Los Angeles, CA 

The majority of autoimmune diseases are more common 

in women as compared to men. This may be attributed to 

differences in sex hormones, sex chromosomes or both. To 

determine the contribution of sex chromosomes to sex 

differences in susceptibility to autoimmune disease, we used 

animal models characterized by a known female preponderance, 

experimental autoimmune encephalomyelitis (EAE) 

and pristane-induced lupus, each in SJL mice. Mice which 

have Sry, the gene that encodes for testicular development, 

deleted from the Y chromosome were backcrossed onto the 

SJL strain, resulting in EAE and lupus-susceptible mice which 

differ in their complement of sex chromosomes, while having 

the same gonadal type, thereby permitting assessment of 

sex chromosome effects not confounded by differences in 

sex hormones (ovary bearing XX vs. XY- mice). When 

comparing EAE in ovariectomized XX vs. XY- SJL mice, 

disease was more severe and there was more inflammation in 

the spinal cords of mice with the XX sex chromosome 

complement. Also, when lupus was induced in ovariectomized 

XX vs. XY- SJL mice, mortality, nephritis and autoantibody 

levels were each greater in mice with the XX sex 

chromosome complement. The differences in susceptibility 

to EAE and lupus also occurred when comparing castrated XX 

Sry versus XY- Sry mice (testis bearing mice). This is the first 

evidence that XX sex chromosome complement, as compared 

to XY, confers greater susceptibility to two immunopathologically 

distinct diseases. This may be relevant to the 

increased susceptibility of women to a variety of distinct 

autoimmune diseases. 

doi:10.1016/j.clim.2007.03.151 

Su.113 Expression of the Renal Antigen Neprhin by 

Human Medullary Thymic Epithelial Cells 

Michael Tasch, Senior Fellow, University of Washington, 

Department of Medicine, Seattle, WA, Kimberly Muczynski, 

Associate Professor, University of Washington, Department 

of Medicine, Seattle, WA, Anne Stevens, Assistant Professor, 

University of Washington, Department of Pediatrics, 

Seattle, WA, J. Lee Nelson, Professor, Fred Hutchinson 

Cancer Research Center, Division of Clinical Research, 

Seattle, WA 

Idiopathic nephrotic syndrome, a set of glomerular pathologies 

often leading to end-stage renal disease, is associated 

with disturbances in Tcell function. This has led to the 

view that these diseases are a consequence of T cell 

mediated autoimmunity, though the relevant autoantigen(s) 

remain unidentified. Nephrin is a possible target for 

autoimmunity in the kidney. The protein product of the 

NPHS1 gene and an essential component of the glomerular 

filtration barrier, nephrin is expressed proximal to autoimmune 

renal lesions and conforms to the definition of a 

tissue restricted antigen (TRA). Since intrathymic expression 

of other TRAs has been implicated in the establishment of 

central tolerance and protection from organ-specific autoimmunity, 

we hypothesized that nephrin is expressed by cells 

of the human thymus. RT-PCR analysis showed that NPHS1 

mRNA is present in all human thymi (N=12) tested. Using 

primer sets flanking sequences encoding immunoglobulinlike 

domains, we showed that the thymic-specific transcript 

appears identical to that from kidney. FACS-purified CD326+, 

HLA-DR+ medullary thymic epithelial cells (mTEC) exhibited 

the highest concentration of NPHS1 transcripts. Immunofluorescence 

analysis showed that nephrin protein is 

expressed in thymus sections. Nephrin exhibited a predominantly 

medullary pattern of expression, co-localizing with 

HLA-DR and cytokeratin, consistent with mTEC, although 

infrequent nephrin-positive cells were seen in the cortex and 

capsule also. Nephrin staining also showed distinct intracellular 

patterns including surface, intracellular granules and 

punctate structures. These data provide a strong rationale 

for exploring nephrin's role in negative selection and the role 

of nephrin-specific T cells in human nephrotic syndrome. 

doi:10.1016/j.clim.2007.03.153 

S179 

Su.114 Low-dose of Local Radiation Treatment Can 

Inhibit the Progression of Experimental 

Autoimmune Nephritis by Promoting Apoptosis 

M. S. Razzaque, Research Scientist, Department of 

Pathology, Nagasaki University, Nagasaki, Japan, L. Diange, 

Research Scientist, Department of Pathology, Nagasaki 

University, Nagasaki, Japan, A. Nazneen, Research 

Scientist, Department of Pathology, Nagasaki University, 

Nagasaki, Japan, T. Taguchi, Professor, Department of 

Pathology, Nagasaki University, Nagasaki, Japan 

Autoimmune crescentic glomerulonephritis (CrGN) is a 

rapidly progressive form of nephritis and usually resistant to 

therapeutic intervention. Apoptosis plays important role in 

resolution of CrGN. We investigated the effects of local 

radiation treatment in the progression of CrGN in rats. Three 

experimental groups were studied; Group-I: sham-operated 

control (n=12); Group-II: intravenous injection of rabbit 

anti-rat GBM antibody (nephrotoxic serum, NTS) (n=23); and 

Group-III: a single low-dose of 0.5 Gy X-ray radiation 

treatment to local bilateral kidneys at 6, 13, 20, and 

27 days after NTS injection (n=55). In Group-III, the level 

of BUN and serum creatinine was significantly decreased 

after radiation treatment compared with age-matched 

Group-II nephritic rats (pN0.05 or 0.001). Glomerular 

hypercellularity, crescents, global sclerosis and tubulointerstitial 

damage gradually developed throughout the time 

course in Group-II rats, were significantly less (PN 0.05 or 

0.001) after radiation treatment in Group-III. The expression 

of active caspases-3 and -7 was increased for irradiated 

kidneys, compared with their corresponding Group-II rats. 

Western blot analysis showed that 17 kDa active caspase-3 

and 35 kDa active caspase-7 expressions markedly increased


in Group-III kidneys, compared with the Group-II. Increased 

expression of p53 protein was also observed in radiation 

treated kidneys compared with groups Group-II. Low-dose of 

local radiation to the kidneys, can delay the progression of 

CrGN in rats by p53-dependent radiation-induced apoptosis. 

Caspases-3 and -7 are the key mediators in such apoptosis. 

This study shows that radiation treatment is effective in 

preventing acute glomerular inflammation and crescent 

formation in the rat model of autoimmune CrGN. 

doi:10.1016/j.clim.2007.03.154 

Su.116 IL-1beta, MMP-1, and MMP-3 Transcript 

Levels are Associated with CD3delta Expression in 

the Osteoarthritic Synovial Membrane 

Carla Scanzello, Rheumatology Fellow, Department of 

Rheumatology, Hospital for Special Surgery, New York, NY, 

Andrew Pearle, Assistant Attending Orthopedic Surgeon, 

Department of Orthopedics, Hospital for Special Surgery, 

New York, NY, Mary Crow, Attending Physician, Department 

of Rheumatology, Hospital for Special Surgery, New York, 

NY, Edward DiCarlo, Associate Attending Pathologist, 

Department of Laboratory Medicine, Hospital for Special 

Surgery, New York, NY 

Many patients with osteoarthritis (OA) develop inflammatory 

infiltrates containing T lymphocytes within the 

synovial membrane (SM). These infiltrates have the 

potential to augment cytokine (i.e.IL-1beta and TNFalpha) 

and degradative enzyme (i.e. MMPs) production by 

macrophages or synovial fibroblasts. Purpose: The aim of 

this study was to determine if lymphocytic synovial 

infiltration in patients with OA is associated with increased 

cytokines and enzyme expression implicated in cartilage 

catabolism. Methods: SM specimens from 37 OA patients 

undergoing total hip or knee arthroplasty (n=31) or 

arthroscopy (n=6) were obtained. Total RNA was extracted 

and cDNA synthesized. Expression levels of CD3delta, IL- 

1beta, TNF-alpha, IL-15, IL-6, IL-10, TGF-beta, MMP-1, and 

MMP-3, were measured by real-time PCR. Synovial inflammation 

was graded in H&E sections on a four-point scale. 

Correlation coefficients were calculated to determine if 

cytokine and enzyme mRNA levels varied with CD3delta 

expression or histologic score. Results: CD3delta transcript 

levels correlated most significantly with IL-1beta (r=0.677, 

pb0.0001), but were also associated with TNF-alpha 

(r=0.487, p=0.003), IL-15 (r=0.524, p=0.001), IL-10 

(r=0.570, p=0.0004), and TGF-beta (r=0.381, p=0.022). 

CD3delta levels were also associated with MMP-1 

(r=0.432, p=0.008) and MMP-3 (r=0.506, r=0.0014) transcripts. 

In addition, MMP-1 expression was significantly 

associated with increasing histologic inflammatory score 

(r=0.412, p=0.011). Conclusions: mRNA levels of IL-1beta, 

MMP-1 and MMP-3 correlated with levels of CD3delta 

transcripts within the SM of patients with both knee and 

hip OA.These relationships suggest that synovial infiltrating 

T cells may contribute to disease pathogenesis in OA by 

promoting synthesis of these mediators of joint damage. 

doi:10.1016/j.clim.2007.03.155 

Su.117 An In Silico Model of Regulatory T Cell 

Function 

Paul Taylor, Postdoctoral Researcher, La Jolla Institute for 

Allergy and Immunology, San Diego, CA, Matthias von 

Herrath, Primary Investigator, La Jolla Institute for Allergy 

and Immunology, San Diego, CA 

Regulatory T (Treg) cells participate in response to 

infection reported so far involve chronic or persistent viral 

infections. The findings that Treg cells influence the functional 

immunity during viral infections however, might 

indicate that, in some cases, virus-specific Treg cells not 

only influence immune pathology or prevent pathogen elimination 

but also can promote a generalized state of 

immuno-suppression in vivo such that the host is more 

susceptible to secondary infections with other pathogens or 

has reduced tumour resistance. The regulatory mechanism by 

which Tregs act has yet to be definitively characterized. 

There is opposing evidence to suggest either cell-to-cell 

contact being required with the cell being suppressed or a 

cytokine mediated mechanism, either through immunosuppressive 

cytokines or by acting as a growth factor ‘sink’. We 

have developed a cellular automaton that is an in silico representation 

of the cells of the immune system. The 

automaton aims to faithfully simulate these cells and models 

their interaction with cognate rule sets derived from in vitro 

and in vivo experimentation. The automaton has been 

developed to provide a stochastic model of a Treg suppression 

assay which can be used to investigate the possible mechanisms 

behind their immuno-suppressive effect. The model can 

rapidly simulate a large number of assays under a wide range 

of parameters and the detailed results allow for the analysis 

of the parameters at a detail level currently impossible in 

vitro. The insights obtained by using the automaton provide a 

powerful tool for directing in vitro research into Treg 

function. 

doi:10.1016/j.clim.2007.03.156 

Su.118 Data Analysis Protocols for Identifying 

Cytokine Signatures in Breast Cancer and Type 1 

Diabetes 

Janet Siebert, Data Architect, CytoAnalytics, Denver, CO, 

Margaret Inokuma, Scientist, BD Biosciences, San Jose, CA, 

Nathan Pennock, Professional Research Assistant, Webb- 

Waring Institute, The University of Colorado Denver and 

Health Sciences Center, Denver, CO, Dan Waid, Researcher, 

Webb-Waring Institute, Denver, CO, Corazon dela Rosa, 

Research Scientist, Medicine/Oncology/Tumor Vaccine 

Group, University of Washington, Seattle, WA, Mary Disis, 

Professor, Medicine/Oncology/Tumor Vaccine Group, 

University of Washington, Seattle, WA, Jack Dunne, 

Research Scientist, BD Biosciences, San Jose, CA, David 

Wagner, Assistant Professor, Webb-Waring Institute, The 

University of Colorado Denver and Health Sciences Center, 

Denver, CO, Holden Maecker, Research Scientist, BD 

Biosciences, San Jose, CA 

Large clinical studies of cytokine expression generate 

complex data sets which can be difficult to analyze. With the 

identification of cytokine signatures and biomarkers a goal of

Abstracts 

much immunodiagnostic research, fast and effective methods 

for extracting meaning from the data are needed. This 

interdisciplinary study presents several data analysis protocols 

for identfying cytokine signatures. Two different data 

sets are considered. The first examines cytokine signatures 

of T cell responses to tumor antigens in breast cancer 

patients (IFNγ, IL-2, and TNFα as expressed by CD4+ and 

CD8+ T cells). The second explores cytokine signatures of 

Type 1 diabetes patients (IFNγ, IL-1b, IL-2, IL-4, IL-5, IL-6, IL- 

8, IL-10, TNFα and TNFβ; on T cells). In the breast cancer 

study, a successful response to a foreign antigen (CMV 

antigen in CMV seropositive donors) was compared to an 

unsuccessful response to a self antigen (CEA, Her2/neu, and 

MAGE-3). The differing magnitude of the cytokine responses 

required data normalization techniques to support visual and 

statistical comparison of the cytokine responses. In the Type 

1 diabetes study, measurements were collected for 10 

cytokines and 4 activation environments, for a total of 40 

different measurements per donor. High-throughput data 

analysis techniques allowed quick identification of significant 

differences between healthy and diseased cohorts, and 

the exploration of differing donor-level signature patterns. 

Taken together, these protocols demonstrate effective 

methods for identifying signature patterns of multiple 

cytokines in complex data sets. 

doi:10.1016/j.clim.2007.03.157 

Su.119 Carbon Monoxide Generated by Heme 

Oxygenase-1 Activity Confers Tolerogenic Capacity 

to Dendritic Cells 

Christine Chauveau, Postdoctoral Fellow, ITERT-INSERM 

U643, Nantes, France, Régis Brion, Technician, ITERT-INSERM 

U643, New York, AK, Séverine Remy, Engineer, ITERT-INSERM 

U643, Nantes, France, Marcelo Hill, Postdoctoral Fellow, 

ITERT-INSERM U643, Nantes, France, Pierre-Joseph Royer, 

Postdoctoral Fellow, INSERM U601, Nantes, France, Séverine 

Tanguy-Royer, PhD, ITERT-INSERM U643, Nantes, France, 

Laurent Tesson, Engineer, ITERT-INSERM U643, Nantes, 

France, Roberto Motterlini, GroupLeader, Northwick Park 

Hospital, Harrow, Roberta Foresti, Group Leader, Northwick 

Park Hospital, Harrow, Ignacio Anegon, Group Leader, 

ITERT-INSERM U643, Nantes, France 

Heme oxygenase -1 (HO-1) is the inducible heme oxygenase 

that catabolizes the degradation of heme into 

biliverdin Fe 2+ and carbon monoxide (CO). Biliverdin is 

subsequently reduced into bilirubin by biliverdin reductase, 

and Fe2+ induces the expression of ferritin. We have 

recently described that immature DCs express HO-1 and 

that the expression of HO-1 confers tolerogenic capacity to 

DCs. We now demonstrate that treatment of human 

monocyte-derived DCs by exogenous CO blocks LPS-induced 

increases in MHC II, CD83, CD80, CD86 expression and proinflammatory 

IL-12 cytokine production associated with DC 

maturation, while preserving the expression of the antiinflammatory 

cytokine IL-10. CO treated DCs also display a 

reduced capacity to induce T cell allogeneic proliferation in 

vitro. In contrast, treatment of DCs with bilirubin, biliverdin 

and deferoxamine (which mimics the action of ferritin) had 

no effect on DC phenotype or cytokine production. These 

observations indicate that the effect of HO-1 on DC function 

is mediated through CO. HO-1 induced the expression of IL- 

10 and the inhibition of DC maturation by HO-1 was partially 

prevented by anti-IL-10 antibodies. Preliminary results show 

that HO-1 expression by DCs inhibits autoimmune diabetes 

in a transgenic model. In conclusion, we show the capacity 

of CO generated by HO-1 activity to block DC maturation 

and to inhibit pro-inflammatory and allogeneic immune 

responses while preserving IL-10 production. This novel 

immune function for CO may be of interest for the inhibition 

of immune responses in autoimmune diseases, transplantation, 

and other conditions involving activation of the 

immune system. 

doi:10.1016/j.clim.2007.03.158 

Su.120 Real-time Characterization of Antibodies 

and Biomarker Detection in the Study of 

Immune-based Disease 

Jean-Francois Houle, Director, Axela Biosensors, Toronto, 

ON, Canada 

We describe a versatile platform for more informed decisions 

in assay development for biomarker detection and 

characterization of the etiological agents of some immune 

disorders and monitoring responses to therapeutic interventions. 

Diffraction-based sensing is a simple, sensitive technique 

for detecting real-time biomolecular interactions in 

complex media. Briefly, capture molecules or substrates are 

immobilized on a flat surface in a specific optimized grating 

that produces a strong diffraction pattern when illuminated. 

Binding of biomolecules to the patterned capture molecules 

causes a change in the diffractive properties of the pattern, 

which increases the diffracted signal intensity. Conversely, 

release of the substrate or interacting species leads to a 

measurable decrease in signal. We have used this ability to 

continuously observe antibody–antigen interactions as a 

means to significantly accelerate immunoassay development. 

Representative examples include antibody characterization, 

activity measurements, antibody mapping and 

pairing studies, buffer optimization for assays as well as 

quantitative analyte measurements. Furthermore, we have 

demonstrated simultaneous measurement of biomarkers 

from high micromolar through single-digit picomolar levels 

in a single sample. 

doi:10.1016/j.clim.2007.03.159 

S181 

Su.121 Inhibition of CXCR2 Expression in HL60 Cells 

by CXCR2 Antisense RNA 

Lingjie Liao, Postdoctoral Researcher, Department of 

Pediatrics, Tongji Hospital, Huazhong University of Science 

and Technology, Wuhan, China, Wei Wang, Postgraduate 

Student, Department of Pediatrics, Tongji Hospital, 




and Technology, Wuhan, China, Xiaoping Luo, Chief 

Physician, Department of Pediatrics, Tongji Hospital, 

Huazhong University of Science and Technology, Wuhan,


China, Guanxin Shen, Professor, Department of Immunology, 

Tongji Medical College, Huazhong University of Science and 

Technology, Wuhan, China 

Summary: CXC chemokine receptor 2 (CXCR2) is the 

common receptor of several potent chemokines, which plays 

an important role in the neutrophil-mediated inflammation. 

In this study, we investigated the effect of CXCR2 antisense 

RNA on the gene expression and chemotactic activity in 

promyelocytic HL60 cells. A human CXCR2 antisense RNA 

recombinant vector, pcDNA-ASCXCR2 complementary to nt 

−21∼ nt 1080 of CXCR2 was constructed and transfected into 

HL60 cells by electroporation. The stable transfected clones 

were screened by G418. The expression of CXCR2 gene was 

measured by RT-PCR and immunohistochemistry. Chemotactic 

response to IL-8 was determined using Boyden chamber 

procedure. CXCR2 expression in pcDNA-ASCXCR2 transfected 

HL60 cells was significantly decreased at mRNA and protein 

levels, as compared with those of untransfected cells. 

Furthermore, chemotactic indexes of HL60 cells were 

markedly decreased by pcDNA-ASCXCR2 transfection. In 

conclusion, CXCR2 antisense RNA can suppress chemotactic 

activity through depleting CXCR2 expression on plasma 

membrane of targeted cells, which would be a new 

therapeutic strategy for inflammatory diseases. This work 

was supported by the NSFC National Science Fund 

£¨No.30571643£No.30672380£©, and National Key Basic 

Research Program of China (2005CB522901, 2005CB522507) 

and Clinical Subject Key Project from the Ministry of Health. 

doi:10.1016/j.clim.2007.03.160 

Su.122 Functional Polymorphisms of MICA and MICB 

in NK Cell Activation 

Amonrat Jumnainsong, PhD Candidate, Faculty of Medical 

Technology, Mahidol University, Bangkok, Thailand, Khon 

Kaen, Thailand, Hugh Reyburn, Department of Pathology, 

University of Cambridge, Cambridge, MA, Mar Vales-Gomez, 

Department of Pathology, University of Cambridge, 

Cambridge, MA, Chanvit Leelayuwat, Assistant Professor, 

Department of Clinical Immunology, Khon Kaen University, 

Khon Kaen, Thailand 

MICA and MICB are ligands of NKG2D that expressed on the 

cell surface of many immune cells. The MIC expressions are 

increased in response to cellular stress including tumor 

transformation and viral infection. Although MICA and MICB 

genes are highly polymorphic and associated with some 

diseases, the evidence demonstrating different capability of 

diverse alleles to activate NK cells is rare. In this study, we 

investigated the effect of various MICA and MICB alleles in NK 

cell activation. C1R stable transfected cells expressing 

different MICA or MICB alleles were tested for NK cell 

activation by chromium releasing assay and IFN-f× production. 

MICA*004 transfectants expressing MICA at a higher level 

could induce NK cells less than transfectants expressing 

MICA*01801 and 045. It is known that MICA amino acid at 

position 129 affects NKG2D binding. The methionine at this 

position can bind to NKG2D stronger than that of valine. 

MICA*004 carries valine whereas MICA*01801 and 045 carry 

methionine at this position. Thus, it is conceivable that this 

position may also affect NK cell activation. MICB*002 and 

MICB*004 transfectants could express MICB at the same level 

but MICB*002 expressing cells could activate NK cells 

stronger than those of MICB*004. From crystal structure of 

NKG2D-MIC binding, position 52 in the alpha 1 domain is 

located near the NKG2D binding site. Thus, the alteration of 

charge between aspartic acid and asparagine (in MICB*004) 

may affect NK cell activation. These results suggested that 

the MICA/B polymorphisms affecting NK cell activation 

could be relevant to diseases and transplantations. 

doi:10.1016/j.clim.2007.03.161 

Su.123 The Role of Regulatory T Cells in the 

Pathogenesis of Experimental Biliary Atresia 

Alexander Miethke, Clinical Fellow, Cincinnati Children’s 

Hospital Medical Center, Pediatric Gastroenterology, 

Cincinnati, OH, Pranavkumar Shivakumar, Research 

Associate, Cincinnati Children’s Hospital Medical Center, 

Division of Pediatric Gastroenterology, Cincinnati, OH, 

Jorge Bezerra, Professor of Pediatrics, Cincinnati Children’s 

Hospital Medical Center, Division of Pediatric 

Gastroenterology, Cincinnati, OH 

In rotavirus induced experimental biliary atresia (BA) the 

extraheaptic bile ducts of newborn mice undergo a Th1regulated 

fibroinflammatory obstruction similar to BA in 

children. Here we investigate the role of regulatory T (Treg) 

cells in the pathogenesis of experimental BA. Using 3 color 

flow cytometry identifying CD4+CD25+Foxp3+, CD4+CD25+ 

CD103+ and CD4+CD25+CTLA-4+ populations, we found that 

Treg cells had low abundance in livers of 3-day-old mice as 

compared to adult mice. Infection of neonatal mice with 

rotavirus did not induce an emergence of Treg cells in the liver 

within 3 days post infection. However, 7 days after infection, 

at a time when neonatal mice develop jaundice, acholic 

stools and bilirubinuria, CD4+CD25+ lymphocytes co-staining 

for Foxp3+, CD103+ and CTLA-4+ increased by 3-, 10- and 43fold, 

respectively in the liver when compared to age-matched 

uninfected controls. Real-time PCR analysis of purified 

lymphocyte subsets revealed a 970 fold increase of IL-10 

mRNA expression in CD4+CD25+ Treg cells from livers of RRV 

infected mice compared to saline controls; however, similar 

increase in TGF β1 expression was not found. In conclusion, 

postnatal viral challenge did not induce a rise in Treg cells 

within 3 days of life. While there was an increased expression 

of functional markers (CTLA-4 and IL-10), activated Treg cells 

failed to surge TGF β1 mRNA expression at the time of duct 

obstruction. We speculate that a deficit in number and 

selective function of neonatal Treg cells may be responsible 

for the susceptibility of neonates to biliary atresia. 

doi:10.1016/j.clim.2007.03.162 

Su.124 Association of HLA DRB1*04 and HLA 

DQB1*06 with Severe Early Childhood Caries 

Hossein Neamatollahi, Dentist, Pediatric Department, 

School of Dentistry, Mashhad, University of Medical 

Sciences, Mashhad, Iran, Jalil Tavakkol-Afshari, Vice 

President for Research, Head, Department of

Abstracts 

Immunogenetics and Cell Culture, Bu-Ali Research Institute 

(BARI), Department of Immunogenetics and Cell Culture, 

Mashhad, Iran, Alireza Sarraf, Dentist, Pediatric 

Department, School of Dentistry, Mashhad University of 

Medical Sciences, Mashhad, Iran, Ali Bagherian, Dentist, 

School of Dentistry, Mashhad University of Medical 

Sciences, Mashhad, Iran, Habibollah Esmaili, 

Epidemiologist, Epidemiology and Statistics Department, 

Ghaem Medical Center, Mashhad University of Medical 

Sciences, Mashhad, Iran, Nasrin Moheghi, Researcher, Bu- 

Ali Research Institute (BARI), Department of 

Immunogenetics, Mashhad, Iran 

Severe early childhood caries (SECC) is one of the most 

common diseases in childhood. Etiology of SECC is multifactorial 

and both genetic and environmental factors play 

important roles in the pathogenesis of disease. Genetic 

variation of the host may contribute to the different risks of 

dental caries. Genetic factors such as the Human Leukocyte 

Antigen (HLA) have been recently introduced as predisposing 

factors. The aim of this study was looking for an association 

between HLA-DRB1*04 and HLADQB1*06 with SECC for early 

diagnosis as well as prevention of the disease. In this study 

we extracted the genomic DNAs from the whole blood 

samples of 44 patients with SECC and 35 caries-free children 

(control group) by salting out method. We amplified the 

genomic DNA by PCR Sequence Specific Primer (PCR-SSP) and 

HLA-typing was performed for both alleles. The results 

revealed a significant increase in the frequency of 

HLADRB1*04 in the patient group (P-Value =0.019). The 

odds ratio for this allele was detected to be 10. Frequency 

of HLA-DQB1*06 allele was not significantly different 

between two groups (P-Value=0.37). 

The above results suggest that HLA-DRB1*04 is associated 

with the susceptibility to SECC. Therefore, the detection of 

HLA-DRB1*04 allele as a molecular marker for early diagnosis 

of SECC is recommended. 

doi:10.1016/j.clim.2007.03.163 

Su.125 The Genetic Relationship Among Eleven 

Persian Ethnic Groups: An Anthropological View 

Based on HLA Class II Gene Polymorphism 

Shirin Farjadian, Assistant Professor, Shiraz University 

Medical Sciences, Immunology Department, Shiraz, Iran, 

Abbas Ghaderi, Full Professor, Immunology Department, 

Shiraz, Iran 

Besides being considered by immunologists for its pivotal 

role in the immune response, highly polymorphic HLA genes 

are still regarded as useful markers by anthropologists for 

determination of genetic relationship and interaction among 

different populations. Knowledge of the HLA allele distributions 

in various populations is also critical for establishing 

bone marrow donor registries; study of HLA associated 

disease and designing of peptide vaccines against infections, 

tumors, or autoimmune diseases. In this study, HLA class II 

profiles were determined by molecular methods in 816 DNA 

samples from eleven ethnic groups of Persia and the genetic 

relationship of Persians to Asians and Europeans were 

investigated. DRB1*1103=04, DQA1*0501, and DQB1*0301 

were the most frequent alleles and DRB1*1103=04- 

DQA1*0501-DQB1*0301 was the most common haplotype in 

Persia. Six samples typed as DRB1*1605 by direct sequencing 

of exon 2 and all of them were heterozygote for DRB1 locus 

and belonged to DRB1*1605-DQA1*0101/2-DQB1*0502 haplotype. 

The results of neighbor-joining and correspondence 

analysis revealed a close genetic relationship among Persian 

subpopulations that were well separated from other Asians 

and European populations. The results of AMOVA also 

revealed that the main variation component (96.9%) was 

contributed to by the within-population level and genetic 

differentiation (FST) was 0.03 when all Persian subpopulations 

considered as one group. 

doi:10.1016/j.clim.2007.03.164 

Su.126 IFN-g Increases Neurogenesis in Aging and 

Alzheimer's-like Disease 

Shai Abutbul, PhD Student, The National Institute of 

Biotechnology in the Negev, Department of Microbiology 

and Immunology, Beer-Sheva, Israel, Rona Baron, MSc, The 

National Institute of Biotechnology in the Negev, 

Department of Microbiology and Immunology, Beer Sheva, 

Israel, Anna Nemirovsky, MSc, The National Institute of 


and Immunology, Beer sheva, Alon Monsonego, PhD, The 

National Institute of Biotechnology in the Negev, 

Department of Microbiology and Immunology, Beer Sheva, 

Israel, Yair Fisher, BSc, The National Institute of 


and Immunology, Beer Sheva, Israel 

The birth of new neurons is maintained in the adult central 

nervous system (CNS) within restricted areas in the brain, 

primarily the dentate gyrus (DG) and the subventricular zone 

(SVZ). Neurogenesis significantly declines with aging, possibly 

to a greater extent with the progression of Alzheimer's disease 

(AD). The primary cause of this decline is unclear, but brainendogenous 

and peripheral immune mechanisms associated 

with aging and the progression of AD appear to affect stem cell 

proliferation and differentiation in the brain. In contrast to 

the suppression of neurogenesis by proinflammatory cytokines 

(such as IL-1b, TNF-a, and IL-6) associated with chronic glial 

activation and neurotoxicity in the aging or AD brain, our 

studies in IFNg-transgenic mice with AD-like pathology show 

that the T cell-derived cytokine IFN-g, expressed under the 

myelin basic protein promoter, induces an approximately 

twofold increase in the quantity of neuronal progenitor cells in 

the DG of aged, but not young, mice. Limited expression of 

IFN-g in the periphery also resulted in a significant decrease in 

the portion of CD4+CD25+ and CD4+Foxp3+ regulatory T cells 

among the CD4+ T cell population in the spleens of the 

transgenic mice (vs. control). IFN-g has often been implicated 

in brain inflammation and neuronal damage, although recent 

studies suggest that it plays a neuroprotective role. Our 

findings also demonstrate a novel role for IFN-g in neuronal 

recovery by acting directly on brain endogenous cells and/or 

indirectly by increasing peripheral immunity. 

doi:10.1016/j.clim.2007.03.165 

S183


Su.127 Astrocyte-Mediated Regulation of Human 

CNS Inflammation 

Alex Kostianovsky, MD, Center for Neurologic Diseases, 

Harvard Medical School, Boston, MA, David E. Anderson, 

PhD, Center for Neurologic Diseases, Harvard Medical 


Microglial activation, or a lack thereof, is associated with 

numerous central nervous system (CNS) neurodegenerative 

and inflammatory diseases, including Alzheimer's disease, 

multiple sclerosis, and CNS tumors. We are interested in 

better understanding the extent to which astrocytes modulate 

microglial and T cell activation within the CNS. Using 

primary human astrocytes co-cultured with ex vivo human 

monocytes or primary human microglia, we report that 

astrocytes potently inhibit monocyte/microglial activation 

induced with pathogen-derived (TLR agonists) but not T cellderived 

(CD40L) inflammatory signals. More specifically, 

human astrocytes inhibit TNF-± secretion and induce IL-10 

secretion. Comparisons between primary human astrocytes 

and primary human malignant glioma cell lines (transformed 

astrocytes) have demonstrated that gliomas inhibit TNF-± 

and induce IL-10 to greater extents than do primary astrocytes; 

moreover, they can inhibit monocyte activation 

induced with both TLR and CD40L signals. Trans-well assays 

demonstrate that inhibition of TNF-± secretion is mediated 

by both soluble and contact-dependent mechanisms, whereas 

induction of IL-10 is contact-dependent. Candidate molecules 

such as TGF- 2 , IL-10, COX2, and CD200 do not appear to 

be responsible for the astrocyte-associated suppression of 

TNF-±. Preliminary data suggest indicate that up-regulation 

of members of the STAT gene family in transformed 

astrocytes is associated with the immunosuppressive phenotype. 

Collectively, these results indicate that astrocytes 

serve an immunoregulatory role within the CNS, which may 

be amplified or suppressed in different CNS diseases. In the 

context of malignant gliomas, amplification of the normal 

immunoregulatory function of astrocytes may explain microglial 

defects and impaired T cell immunity associated with 

these tumors. 

doi:10.1016/j.clim.2007.03.166 

Su.128 Peripheral Markers of Encephalitis on 

Monocytes from SIV-infected Rhesus Monkeys 

Maria Cecilia Marcondes, Staff Scientist, The Scripps 

Research Institute, Molecular and Integrative Neurosciences 

Department, La Jolla, CA, Tricia Burdo, Research Associate, 

The Scripps Research Institute, Molecular and Integrative 

Neurosciences Department, La Jolla, CA, Caroline Lanigan, 

Scientific Associate, The Scripps Research Institute, 

Molecular and Integrative Neurosciences Department, La 

Jolla, CA, Howard Fox, Associate Professor, The Scripps 

Research Institute, Molecular and Integrative Neurosciences 

Department, La Jolla, CA, Debbie Watry, Research 

Assistant, The Scripps Research Institute, Molecular and 

Integrative Neurosciences Department, La Jolla, CA 

In HIV-1 infected people, the accumulation of macrophages 

(MØs) in the brain correlates with dementia and 

encephalitis. We hypothesized that a pattern of monocyte 

surface marker expression may serve as a peripheral marker 

for CNS disease. Using the SIV/rhesus monkey model, we first 

analyzed functionally relevant surface markers on monocytes 

and macrophages from blood and organs upon 

termination in groups of animals that later did (SIVE) or did 

not (SIVnoE) develop encephalitis. Cluster analysis revealed 

that the pattern of staining of cells from the brain was 

closest to that found in blood, compared to spleen, liver, 

bone marrow and bronchioalveolar lavage. In the blood, the 

predominant (CD16low) population of monocytes from SIVE 

animals had higher levels of both CD44v6 and CCR5 compared 

to SIVnoE animals. Furthermore the inflammatory CD16+ 

subpopulation of blood monocytes from SIVE animals 

expressed high levels of CD44v6, but not CCR5, compared 

to those from SIVnoE animals. In the brain, only CCR5 

expression on CD16+ macrophages correlated with encephalitis. 

A longitudinal analysis of blood monocyte markers 

revealed that, beginning at 3 weeks prior to termination, 

both CD16low and CD16+ monocytes indeed upregulated 

CD44v6. This provides a potential biomarker to determine 

individuals who may develop the central nervous system 

complications of HIV infection. Furthermore, given its role in 

cellular adhesion and as a receptor for osteopontin, CD44v6 

upregulation on monocytes offers functional clues to the 

pathogenesis of such complications, and provides a target for 

preventative and therapeutic measures. (NIH Grant 1R01 

NS045534-04). 

doi:10.1016/j.clim.2007.03.169 

Su.129 Polymerized-Type I Collagen a Biodrug for 

the Treatment of Patients with Knee Osteoarthritis 

Janette Furuzawa-Carballeda, Chemistry, Instituto Nacional 

de Ciencias Medicas y Nutricion SZ, Department of 

Immunology and Rheumatology, Mexico, Olga Muñoz- 

Chable, Physician, Instituto Nacional de Ciencias Medicas y 

Nutricion SZ, Department of Immunology and 

Rheumatology, Mexico, Israel Macias-Hernandez, Physician, 


Department of Immunology and Rheumatology, Mexico, 

Andres Agualimpia-Janning, Physician, Instituto Nacional de 

Ciencias Medicas y Nutricion SZ, Department of Immunology 

and Rheumatology, Mexico 

Polymerized-Type I Collagen (Collagen-PVP) is an antiinflammatory 

and a tissue regenerator biodrug. In vivo 

study: Patients (n=53) were treated with 12 intraarticular 

injections of 2 ml of collagen-PVP (16.6 mg of collagen) 

(n=27) or 2 ml of placebo (n=26) during 6 months. The 

follow up was done during the next 6 months. The primary 

endpoints included WOMAC, Lequesne index and pain 

intensity on a VAS. Secondary outcomes were patient and 

investigator global score and biodrug evaluation. Clinical 

improvement was determined if the decrease in pain 

exceeds 20 mm on VAS and patients achieved at least 

20% of improvement in primary endpoints from baseline to 

week 24. In vitro study: Cartilage and synovial tissue cocultures 

from 5 patients with OA were performed. Tissues 

were cultured during 7 days with/without 1% Collagen-PVP 

and they were stained with Alcian Blue technique. IL-1β, 

IL-8, IL-10, IL-12, TNF-α and IFN-γ were measured in

Abstracts 

supernatants by ELISA. COMP, type II collagen, TNF-α, IL-10 

and Ki-67 expression was determined by immunohistochemistry. 

Patients had a statistically significant improvement 

(pb0.05) in Collagen-PVP-treated versus baseline and versus 

placebo in Lequesne Index, WOMAC, VAS, patient global score 

and physician drug evaluation. The histological analysis 

showed that Collagen-PVP increased 3 to 6-fold proteoglycans, 

Ki-67, COMP and type II collagen. IL-1β and TNF-α 

production was inhibited; meanwhile IL-10 levels were upregulated 

treated versus untreated-cultures. Collagen-PVP 

was safe and more effective than placebo for the treatment 

of knee OA, probably due to the induction of tissue 

regeneration and the down-regulation of inflammation. 

doi:10.1016/j.clim.2007.03.170 

Su.132 Antibody-oligo Arrays for Multiplex Surface 

Marker Profiling of Immune Cells Subsets 

Michael Kattah, MSTP Student, Stanford University 

Immunology and Rheumatology, Stanford, CA, John Coller, 

Research and Development Engineer, School of Medicine, 

MDRP’S, Stanford Functional Genomics Facility, Stanford, 

CA, Golnaz Alemi, Medical Student, Drexel University, 

Stanford, CA, Neekaan Oshidary, Undergraduate Student, 

Stanford University Immunology and Rheumatology, 

Stanford, CA, Paul J. Utz, Associate Professor of Medicine 

(Immunology and Rheumatology), Medicine, Immunology 

and Rheumatology and Center for Clinical Immunology at 

Stanford, Stanford, CA 

While the field of proteomics holds great promise for the 

study of immune-related disorders, methods for analyzing 

specific proteins in a high-throughput manner are still needed. 

In an effort to develop a multi-analyte proteomics platform 

using monoclonal antibodies, we are developing a new assay 

termed antibody-oligo arrays (AOAs). This method employs a 

panel of monoclonal antibodies, each tagged with a unique 

oligonucleotide sequence identifier. After staining a sample, 

the complex mixture of tags is amplified and hybridized to a 

custom DNA microarray, providing an indirect measure of the 

relative levels of each analyte in the original sample. In an 

effort to identify cell-surface markers that are either up- or 

down-regulated in a given cell population, we have developed 

a cell-surface AOA to profile 1–2×106 cells with a panel of 90 

surface markers and 4 isotype controls in two 48-plex 

reactions. Although there are many potential applications for 

this technology, we have performed pilot experiments using 

both cancer cell lines and primary human lymphocytes. 

Preliminary results have identified changes on the surface of 

primary human naïve CD4+CD45RA+CD45RO-CD25- Tcells upon 

polyclonal activation in vitro with anti-CD3 and anti-CD28 

coated beads. The markers identified in this screen agree with 

previously described activation markers and were all validated 

by conventional flow cytometry. In the future we hope to 

profile cell-surface markers on Tregulatory and IL-17 secreting 

Th17 cells with our newly developed cell-surface AOA in order 

to identify targets for future functional studies and for 

potential therapeutic interventions. 

doi:10.1016/j.clim.2007.03.171 

Su.133 Industrial Science: Biomarker Discovery 

from Plasma: Maximizing Proteomic Breath and 

Depth Using Protein Partitioning and Fractionation 

Followed by Shotgun Mass Spectrometry 

Shijun Sheng, Senior Research Technologist, Johns Hopkins 

Bayview NHLBI Proteomics Center, Baltimore, MD, Qin Fu, 

Research Associate, Johns Hopkins Bayview NHLBI 

Proteomics Center, Baltimore, MD, Jeff Chapman, Director, 

Beckman Coulter, Biomarker Discovery and Automation 

Business Center, Fullerton, CA, Jennifer Van Eyk, Associate 

Professor Medicine, Johns Hopkins University-Bayview 

Campus, Baltimore, MD, Jerald Feitelson, Manager 

Strategic Marketing, Beckman Coulter, Biomarker Discovery 

and Automation Business Center, Fullerton, CA 

The potential of proteomics to identify new diagnostic 

markers was widely touted but has met with limited success 

due to technical issues related to reproducibility, sample 

requirements, availability of well characterized clinical 

cohorts for discovery and validation, and ability to obtain 

sufficient proteome depth and coverage. To discover novel 

biomarkers while overcoming previous limitations in proteomic 

methodology, we have developed a robust biomarker 

discovery pipeline that allows the detection and quantification 

of proteins below 100 pg/ml. The proteomic pipeline 

involves two key sample preparation steps at a large scale: 

(a) partitioning (removal) of the top 12 most abundant 

plasma proteins using immunoaffinity chromatography, and 

(b) subsequent two-dimensional liquid chromatography that 

fractionates intact proteins based on two of their key 

intrinsic properties, pI and hydrophobicity. Fractions are 

split allowing the third intrinsic protein parameter, intact 

mass, to be obtained using MALDI-TOF mass spectrometry. 

When combined with analysis of proteome maps based on 

aligned second dimension chromatograms, we can identify 

fractions that differ between individual samples in large data 

sets. To push the proteomics envelope, we carried out 

multiple analyses on each individual sample prior to pooling 

and quantitative shotgun LC-MS/MS to identify and characterize 

proteins. This biomarker discovery pipeline is being 

applied to unprecedented broad and deep analyses of 100 

individuals attempting to obtain quantitative protein characterization 

(isoform and PTM) data. We have shown that this 

scale of discovery proteomics (1 mL plasma per patient, 100 

patients and controls) was able to identify candidate 

biomarkers of a crucially important disease. 

doi:10.1016/j.clim.2007.03.173 

S185 

Su.134 Human C-Reactive Protein Augments the Gut 

Ischemia/Reperfusion Injury in Mice 

Xinyue Lu, Research Fellow, Department Cellular Injury, 

Walter Reed Army Institute of Research, Silver Spring, MD, 

Russell Peckham, Principle Investigator, Corresponding 

Author, Department Cellular Injury, Walter Reed Army 

Institute of Research, Silver Spring, MD, Michael Falabella, 

Research Assistant, Department Cellular Injury, Walter Reed 

Army Institute of Research, Silver Spring, MD, George C. 

Tsokos, Principle Investigator, Professor, Department 

Cellular Injury, Walter Reed Army Institute of Research, 

Silver Spring, MD


C-reactive protein (CRP) has been shown to increase 

ischemia/reperfusion (IR) injury in models of acute myocardial 

infarction and stroke. The role of CRP on intestinal IR which 

results in serious local and systemic inflammatory reactions is 

unknown. We adapted a murine superior mesenteric artery IR 

model which we used to study the effect of CRP. Human 

purified CRP (250, 500, and 1000 μg per mouse) was 

administered prior to the ischemia period of 30 min and tissue 

injury was evaluated after 2 hours of reperfusion. Intestinal 

injury damage, estimated using an established score, was 

statistically significantly higher in all CRP-treated groups 

compared to non-treated animals. The increased injury score 

was associated with increased intestinal myeloperoxidase 

activity only in the CRP-treated animals. In addition, the levels 

of the proinflammatory cytokines IL-6, IL-1α,TNF-α mRNA, as 

determined by real-time RT-PCR, were increased in the 

intestines of CRP-treated animals compared to the nontreated 

animals. Immunofluorescent staining revealed that 

human CRP was deposited in the intestinal tissues and that it 

co-localized with complement C3, factor H, MBL-A and C5b-9. 

Our results indicate CRP enhances intestinal IR injury probably 

by recruiting and activating the complement pathway. 

doi:10.1016/j.clim.2007.03.174 

Su.136 Expression, Histological Localization of hfgl2 

and Its Gene Regulation in Cancer 

Kai Su, Postgraduate Student, Department of Infectious 


and Technology, Wuhan, China, Fang Chen, Resident, 



China, Ping Zhang, Graduate Student, Department of 


Science and Technology, Wuhan, China, Xiaomin Qin, Doctor 

in Charge, Department of Infectious Disease, Tongji 


Wuhan, China, Meifang Han, Doctor in Charge, Department 

of Infectious Disease, Tongji Hospital, Huazhong University 

of Science and Technology, Wuhan, China, Weiming Yan, 

Assistant Researcher, Department of Infectious Disease, 

Tongji Hospital, Huazhong University of Science and 

Technology, Wuhan, China, Bin Pi, Assistant Researcher, 



China, Dong Xi, Assistant Research, Department of 


Science and Technology, Wuhan, China, Xiaoping Luo, Chief 

Physician, Department of Pediatrics of Tongji Hospital, 




and Technology, Wuhan, China 

To study the role of fibrinogen-like protein 2/fibroleukin 

(fgl2) in tumor development, human malignant tumor tissues 

from 133 patients with various kinds of cancers and the 

animal tumor tissues from human hepatocellular carcinoma 

(HCC) model on nude mice established by using different HCC 

cell lines were used to detect the fgl2 expression through an 

immunohistological method. Antibodies against different 

cellular markers (CD8, CD57, CD68 and vWF) were used to 

determine the cellular types in which the fgl2 was expressed. 

Fgl2 was detected in 127 out of 133 patients' samples and 

human HCC nude mice model. Fibrin (nogen) co-localization 

with fgl2 expression was determined by double staining. 

Particularly, it was evidenced that fgl2 was highly expressed 

not just in cancer cells, but also in interstitial infiltrated cells 

including macrophages, NK cells, CD8+T lymphocytes and 

vascular endothelial cells. In situ hybridization shows fgl2 

mRNA stained in associated location. Furthermore, IL-2, IFN-γ 

and TNF-a can dramatically up-regulate fgl2 mRNA (increased 

within the range of 10–100 fold) and protein expression in 

both THP-1 and HUVEC cell lines. One-stage clotting assay 

demonstrated that the significantly increased procoagulant 

activity after cytokines stimulation of both cell lines. 

The present study shows that fg12 contribute to the 

hypercoagulability in cancer, and thus in turn induces tumor 

angiogenesis and metastasis. This work was supported by 

Natural Science Foundation of China (NSFC 30225040, 

30571643, 30672380), National Key Basic Research Program 

of China (2005CB522901, 2005CB522507) and Clinical Subject 

Key Project from the Ministry of Health. 

doi:10.1016/j.clim.2007.03.175 

Su.137 CpG ODN Reduced Amyloid-β 

Accumulation Through Up-Regulated F-Spondin on 

Mouse BV2 Microglial Cell 

Yung Chih Cheng, PhD Student, Institute of Biochemical 

Science, National Taiwan University, Taipei, Taiwan, Cheng- 

Chin Kuo, Postdoctoral Fellowtor, Agricultural 


Taiwan, Shu-Mei Liang, Professor, Agricultural 


Taiwan 

Alzheimer's disease, the most common senile dementia, is 

characterized by amyloid plaques, vascular amyloid, neurofibrillary 

tangles, and progressive neurodegeneration. Amyloid 

plaques are mainly composed of amyloid-beta (A-β) 

peptides, which are derived from processing of the betaamyloid 

precursor protein (APP) by secretases. In this study, 

we found that BV2 microglial cells stimulated with unmethylated 

CpG-ODN not only increased the level of F-spondin but 

also resistant to A-β formation. Similar results were observed 

in CpG ODN stimulated RAW264.7 cells. Results from both real 

time PCR and western blotting demonstrated that CpG ODN 

regulated F-spondin expression at both transcriptional and 

translational level. Inhibition of TLR9 and MyD88 by inhibitors 

or dominant negative mutants indicated that CpG ODN upregulated 

F-spondin was TLR9/MyD88 dependent. In addition, 

we also found that a downstream kinase of TLR9, PI3K, 

was essential for CpG ODN-induced F-spondin expression. 

Depletion of F-spondin by small interfering RNA attenuated 

the prevention of CpG ODN on APP degradation by secretases, 

thereby increasing the level of A-β and soluble APP in culture 

medium. Collectively, these findings suggest that CpG ODN 

increases F-spondin via a TLR9/MyD88/PI3K pathway. The 

enhancement of F-spondin plays a critical role in the 

reduction of A-β formation mediated by CpG ODN, thereby 

reducing Alzheimer disease occurrence. These results may

Abstracts 

provide an insight for developing a potential drug for the 

treatment of Alzheimer's disease. 

doi:10.1016/j.clim.2007.03.176 

Su.138 Loss of Th1 and Effector T Cell Function in 

Chronic Versus Subacute Hypersensitivity 

Pneumonitis 

Lourdes Barrera, Biological Sciences Doctor, National 

Institute of Respiratory Diseases, Mexico City, Mexico, 

Felipe Mendoza, MsC, National Institute of Respiratory 

Diseases, Mexico City, Mexico, Moises Selman, National 

Insitute of Respiratory Diseases, Mexico City, Mexico 

Hypersensitivity pneumonitis (HP) represents an immunologically-mediated 

inflammation caused by a variety of 

antigens. 21 patients with Sub-HP, 20 with Chr-HP and 8 

healthy subjects were studied. T cells from bronchoalveolar 

lavage (BAL) were analyzed by flow cytometry. Cytokine 

expression and memory functional profile were evaluated in 

cell cultures. Chr-HP patients showed lower BAL lymphocytosis, 

higher CD4+/CD8+ ratio (3.5–3.3 versus 1.2–0.4 and 

1.7–1.5 from controls and Sub-HP; pb0.05), and a decrease 

of f× fÔT cells (2.5+0.9 versus 13.3+6.3 and 10.4+5.6 from 

controls and Sub-HP; pb0.01). Chr-HP had an increase in the 

terminally differentiated memory CD4+ and CD8+ T cells 

subsets compared with the subacute group (p b0.05). 

Memory cells from Chr-HP showed lower IFN-f× production, 

and decreased cytotoxicity of CD8+ T-lymphocytes. Chr-HP 

displayed a Th2-like phenotype with decreased CXCR3/CCR4 

ratio on T cells (2.5–1.8 versus 14.8–8.8; pb0.01) and lower 

levels of the CXCR3 ligands, IP-10/CXCL10 and MIG/CXCL9 in 

BAL supernatants. BAL lymphocytes from Chr-HP, stimulated 

with the specific antigen expressed lower levels of Th1 

cytokines and higher levels of Th2 cytokines (IFN-f× /IL-4 

ratio: 0.7–0.3% versus 2.1–0.6% from Sub-HP; pb0.01). 

Differences between chronic and subacute patients include 

an increase in CD4+/CD8+ ratio, a decrease of f× fÔT cells, a 

skewing towards Th2 as opposed to Th1 and exhaustion of 

effector CD8+ T cells. These findings may provide insight in 

the development of new therapeutic strategies e.g. the 

inhibition of GATA-3 transcription factor to modify the Th1/ 

Th2 cell balance and the blocking of PD-1 receptor to restore 

the Tcell function may be useful in the treatment for chronic HP. 

doi:10.1016/j.clim.2007.03.558 

Su.139 Analysis of Natural Killer Cells Isolated from 

Human Decidua: Evidence that 2B4 (CD244) 

Functions as an Inhibitory Receptor and Blocks NK 

Cell Function 

Gabriella Pietra, PhD, DIMES-University of Genoa, 

Genova, Italy, Paola Vacca, PhD Student, DIMES-University 

of Genoa, Genova, Italy, Lorenzo Moretta, MD, G Gaslini 

Institute, Genova, Italy, Federico Prefumo, MD, G 

Gaslini Institute, Genova, Italy, Maria Cristina Mingari, 

PhD, National Cancer Institute, Genova, Italy 

During the first trimester of pregnancy, CD56bright NK 

cells constitute more than 50% of the lymphocytes present 

in the decidua, a tissue in which B and T lymphocytes are 

rare. The role they have at this site is still unclear. It is 

currently believed that in the early stages of gestation, 

decidual NK (dNK) cells possess the ability to regulate 

trophoblastic differentiation and invasion. In this study we 

analyzed dNK cells for their phenotype and functional 

capability. We show that dNK cells express normal surface 

levels of certain activating receptors, including NKp46, 

NKG2D and 2B4, as well as of killer-cell immunoglobulin-like 

receptors (KIRs) and CD94/NKG2A receptor. In addition, 

high levels of cytoplasmic granules despite their CD56bright 

CD16- surface phenotype characterize them. Moreover, we 

provide evidence that in dNK cells, activating NK receptors 

display normal triggering capability while 2B4 functions as 

an inhibitory receptor. Thus, crosslinking of 2B4 resulted in 

inhibition of both cytolytic activity and interferon-gamma 

production. Clonal analysis revealed that, in the majority of 

dNK cell clones, the 2B4 inhibitory function is related to the 

deficient expression of SLAM-associated protein (SAP) 

mRNA. Moreover biochemical analysis revealed low levels 

of SAP protein in dNK polyclonal population. Thus, in the 

absence of SAP, SHP-1 phosphatase can bind to 2B4 and 

mediate inhibition of the dNK-mediated cytolysis CD48+ 

target cells (eg EBV+ B cells). This suggests that dNK cells, 

although potentially capable of killing, are inhibited in their 

function when interacting with cells expressing CD48 (the 

2B4 ligand). 

doi:10.1016/j.clim.2007.03.555 

S187 

Su.140 Population of T, T Reg, NK Cells and Their 

Activation and Inhibitory Receptors in Peripheral 

Blood of Healthy Women During the Menstrual 

Cycle 

Erika Aurora Martínez García, Master in Science 

(Immunology), University of Guadalajara, Guadalajara, 

Mexico, Victor Ermilo Arana Argaez, Master in Science 

(Immunology), University of Guadalajara, Guadalajara, 

Mexico, Beatriz Teresita Martín Márquez, Master in 

Science (Immunology), University of Guadalajara, 

Guadalajara, Mexico, Pedro Ernesto Sánchez Hernández, 

PhD, University of Guadalajara, Guadalajara, Mexico, Luz 

Maria Adriana Balderas Peña, PhD, Unidad de 

Investigación Medica en Epidemiología Clínica, UMAE, HE, 

CMNO Instituto Mexicano del Segu, Guadalajara, Mexico, 

Trinidad García Iglesias, PhD, University of Guadalajara, 

Guadalajara, Mexico, Alicia Del Toro Arreola, Master in 

Science, University of Guadalajara, Guadalajara, Mexico, 

Oscar Javier López León Murguía, MD, University of 

Guadalajara, Guadalajara, Mexico, José Francisco Muñoz 

Valle, PhD, University of Guadalajara, Guadalajara, 

Mexico, Adrián Daneri Navarro, PhD, University of 

Guadalajara, Guadalajara, Mexico, Mónica Vázquez Del 

Mercado Espin, PhD, University of Guadalajara, 

Guadalajara, Mexico 

There is evidence that the maternal immune system is 

influence by changes in the hormonal levels during the 

menstrual cycle. So far, the information related to the levels 

of T, T reg, NK cells and their activation and inhibitory 

receptors is scarce. Aim: To analyze the populations of T, T


reg, NK cells and their activation and inhibitory receptors of 

peripheral blood of healthy women during the menstrual 

cycle. Material and Methods: We studied 10 women not using 

hormonal contraceptives in the day 5 of the follicular phase 

and 21st of the luteal phase of the menstrual cycle. 

Peripheral blood lymphocyte subsets and their receptors 

were determined by flow cytometry. Results: We do not 

observe changes in the percentages of CD4+ and CD8+ Tcells. 

With respect to ILT-2 and CD94/NKG2 receptors expressed in 

CD4+ T cells and at the levels of T reg cells (CD4+CD25+ 

Foxp3+) we noted an increased in the follicular phase. On 

the other hand, the levels of NK cells remain constant in the 

two menstrual phases as well as their receptors NKG2D and 

ILT-2. According to the receptors NKp30 and NKp46 we 

identified major fluctuations in the follicular phase and the 

opposite for CD94/NKG2. Conclusions: We observed an 

increase in day 5 and decay in day 21 of the menstrual 

cycle in the majority of the cellular populations and their 

receptors. Perhaps these results are product of the necessary 

mechanisms in menstrual cycle to prepare the 

processes of implantation in early pregnancy. 

doi:10.1016/j.clim.2007.03.556


FOCIS 2007 Abstract Index by Author 

Abbas, Alexander R. Sa.149 

Abel, Lucia Cristina Jamli F.64 

Abello, Paula Sa.65 

Aberle, Teresa Sa.27 

Abhinandan, Raghavan Su.87 

Abolhassani, Mohsen Sa.17 

Abouljoud, Marwan F.71 

Abrams, Elaine F.94 

Abramson, Tzvia F.69 

Abril, Annette Sa.26 

Abutbul, Shai Su.126 

Adams, Jason OR.59 

Adams, Katherine Sa.98 

Adib, Minoo F.125 

Adler, Adam OR.81 

Adriani, Marsilio OR.29 

Agarwal, Rajeev K. OR.38 

Aghasizadeh, Navid Su.62 

Agualimpia-Janning, Andres Su.129 

Aguiar, Paloma Sa.134 

Aguilar-Lemarrroy, Adriana C. Su.55 

Aguilar-Velazquez, Gustavo Su.76 

Aguilera, Raquel Sa.133 

Aguillon, Juan C. Sa.65 

Ahearn, Joseph M. Sa.31, Sa.35 

Aherns, Richard F.124 

Ahlmen, Jarl F.129 

Ailes, Elizabeth F.44 

Aksentijevich, Ivona Sa.49, Sa.50 

Alam, Munir F.72 

Alami Chentoufi, Aziz F.53 

Albani, Salvatore F.20, Sa.66, Sa.67, Sa.72, 

Sa.73 

Alberu, Josefina Su.101 

Alcocer Varela, Jorge F.93, Sa.81 

Alemi, Golnaz 

Alesahebfosoul, Fereshteh F.111, F.125 

Alkan, Sefik Su.57 

Allaire, Normand OR.55 

Allauzen, Sophie F.136 

Allen, Hamish Sa.40 

Allende, Gloria F.18 

Allie, Rameeza Sa.145 

Alliot-Licht, Brigitte Su.96 

doi:10.1016/j.clim.2007.03.541 



Allison, James E. Sa.152 

Almeida, Alexandre F.67, F.98 

Alonso, Liv an Sa.147 

Alper, Chester Su.59, Su.60 

Althshuler, David OR.80 

Altshuler, David Su.35 

Alves, Venancio F.64 

Aly, Theresa A. OR.78 

Amato, Anthony Su.31 

Amox, Diane Sa.66 

Anderson, Amy F.57 

Anderson, David E. OR.95, OR.101, Sa.114, 

Su.127, Su.38 

Anderson, David F.15, OR.83, Sa.131 

Anderson, Mark F.12, OR.60, Su.111 

Anderson, Richard C.E. OR.58 

Anderson, Richard Sa.120 

Anderson, Stacie M. OR.29 

Anegon, Ignacio OR.71, Su.119 

Annels, Nicola F.103 

Annese, Vitto Su.93 

Ansaldo, Gianluca Sa.112 

Antel, Jack Su.10, Su.16 

Anthony, Donald F.71 

Aoki, Joseph OR.29 

Aono, Shelly F.61 

Aparicio, Carlos F.129 

Arab, Hamid Reza Su.50 

Arana Argaez, Victor Ermilo Su.140, Sa.44 

Aranami, Toshimasa OR.24, Su.12 

Aravena, Octavio Sa.65 

Arbour, Nathalie OR.33, OR.41, Su.16, 

Su.29 

Arechiga, Adrian F.28, Sa.148 

Arend, William P. Sa.71 

Aricò, Maurizio F.81 

Arif, Sefina OR.18 

Armengol, Maria Pilar F.26 

Armstrong, Don OR.79 

Arnold, Arthur P. Su.134 

Arnold, Dilani F.80 

Arouche Camara Lopes, 

Luiz Heraldo 

Sa.123 

Arshi, Saba Sa.7

S190 FOCIS 2007 Abstract Index by Author 

Ashayeri, Hassan Sa.7 

Ashfield, Rebecca OR.9 

Ashley, Charles OR.77, Sa.120 

Ashman, Robert F. OR.44 

Ashton-Chess, Joanna Su.113 

Ashutosh, Mangalam OR.61 

Aspsater, Mahsa F.129 

Atkinson, Mark OR.15, OR.2, OR.56 

Au, Liemin OR.49 

Au, Melinda Su.109 

Aud, Dee Su.40 

Autschbach, Frank Su.90, Su.91 

Avanesyan, Armine OR.97 

Awasthi, Amit Su.32 

Azadi, Gholomreza Sa.7 

Azmi, Hooman F.134 

Azuma, Miyuki F.33 

Azzari, Chiara F.91 

B. Elkon, Keith Sa.22 

Babu, Sunanda R. OR.78, F.12 

Bacchetta, Rosa F.91 

Badolato, Raffaele F.12, F.91 

Badr El-Din, Nariman Sa.124 

Badur, Selim Sa.109 

Baecher-Allan, Clare OR.77, Sa.120 

Baeten, Dominique Sa.57, Sa.58, Sa.59 

Baez, Ineavely F.113 

Bagely, Jessamyn Sa.11 

Baghani, Zahra 

Bagherian, Ali: 

Su.50 

Bailey, Samantha OR.34 

Baker, Harold F.104 

Baker, Mark Su.101 

Baker, Rocky F.25 

Balboni, Imelda Sa.46 

Balderas Peña, Luz Maria Su.140 

Adriana 

Balmer, Paul F.126, F.65 

Banda, Nirmal Sa.63 

Bankey, Paul F.70 

Baranyi, Ulrike Sa.11 

Baranzini, Sergio Su.15 

Barba-Barajas, Martha Su.55 

Barbour, Gene OR.3 

Barcellos, Lisa Sa.152 

Baric, Ralph F.46 

Barker, Jennifer F.12 

Barker, Jonathan OR.39 

Barmada, Michael Su.92 

Baron, Rona Su.126 

Bar-Or, Amit OR.41, OR.86 

Barrera, Lourdes Su.138 

Barros, Noac Chuffi F.96 

Barsky, Lora F.113 

Bartholomew, Richard OR.13 

Bartunkova, Jirina F.116, Sa.117 

Baschal, Erin E. OR.78 

Bason, Caterina F.65, OR.23, 1202 

Basso, Alexandre S. Su.28 

Bastos, Fabiana Su.19 

Batliwalla, Franak OR.55, Sa.55, Sa.61 

Bautista, Alejandro Su.98 

Bautista-Vazquez, Alejandro Su.95 

Bautz, David Sa.136 

Bayer, Monika Sa.126 

Begovich, Ann Sa.150 

Behrens, Marshall Sa.154 

Behrens, Timothy W. OR.80 

Bein, Gregor Su.41 

Belaunzaran, Francisco F.97 

Belmares, Michael Sa.132 

Belouski, Shelley F.137 

Benard, Gil F.52 

Bender, James Sa.106 

Benghiat, Fleur Samantha Su.103, Su.130 

BenMohamed, Lbachir F.53 

Benson, Charles OR.28, Sa.125 

Benson, Jacqueline OR.40 

Benvenuti, Luiz A. Su.124 

Benvenuto, Federica Sa.130 

Berer, Kerstin OR.20 

Berg, Ulla F.122 

Bergan, Lindsay Sa.97 

Berghöfer, Beate Su.41 

Bergholdt, Regine F.22 

Bergmann, Christoph OR.73 

Beriou, Gaelle Su.96 

Bernstein, Allan L. Su.2 

Bethunaickan, Ramalingam Sa.45 

Bettahi, Ilham F.53 

Bettelli, Estelle Su.42 

Bezerra, Jorge Su.123 

Bhairo, Michelle Sa.113 

Bhan, M.K. F.68 

Bhat, Roopa OR.96 

Bhatnagar, Shinjini F.68 

Bianchi, Lucia F.91 

Bianco, Nicolás Sa.85 

Bianco, Nicole OR.89 

Bidar, Maryam Su.62 

Biedermann, Tilo Sa.153 

Bienkowska, Jadwiga OR.55, Sa.55 

Bilton, Diana F.67 

Binder, Steven Sa.79 

Birmelin, Jennifer F.89 

Bishop, Gail A. Sa.68 

Blanca, Isaac Sa.85, Su.108 

Blanchard, Carine F.124 

Blankenhorn, Elizabeth OR.82 

Bloomfield, Tiffany Sa.96 

Bluestone, Jeffrey A. OR.16, OR.59, F.33, F.34 

Boardman, Cynthia Su.48 

Bohmova, Kristyna F.19 

Bollyky, Paul Sa.128 

Bolton, Wade F.129 

Bonadkar, Sasha Su.45 

Borgonovo, Giacomo OR.62 

Borrow, Ray F.126, F.65 

Bossi, Giovanna OR.9, Sa.98 

Bothwell, Alfred Su.102 

Bottini, Nunzio Sa.153 

Boudakov, Ivo F.38, Sa.127 

Bourcier, Kasia Sa.151, Su.37


Bourdette, Dennis OR.13 

Bour-Jordan, Helene F.33 

Bowman, Edward Su.91 

Boyer, Scott Su.63 

Boyle, John F.76 

Bradley, Brenda OR.3 

Bradshaw, Elizabeth Su.31 

Braemswig, Kira OR.15 

Branch, Rodshawn F.120 

Branigan, Patrick OR.40 

Braud, Cristophe Sa.57 

Braun, Jonathan OR.100, Su.82, Su.89 

Braun, Michel Sa.89 

Braun, Werner OR.22 

Braunstein, Jutta Su.90 

Bravo-Cuéllar, Alejandro Su.55 

Bresson, Damien OR.14, F.7 

Brewer, Joanna Sa.107 

Brion, Régis Su.119 

Brito, Cyro Alves Sa.16 

Brizuela, J.A. Su.32 

Brod, Staley Su.20 

Brodsky, Nina N. OR.52 

Brook, Azam Sa.105, Su.62 

Brooks-Worrell, Barbara OR.49 

Brorsson, Caroline F.22 

Brouard, Sophie Sa.57 

Brown, Chris F.132 

Brown, Margaret R. OR.52 

Bruce, Jeffrey N. Sa.114 

Bruce, Jeffrey Sa.120 

Bruner, Ben OR.89 

Bruner, Gail R. Sa.24 

Brunner, Tali Su.10 

Brusko, Todd OR.2 

Bryant, Shaughn Sa.32 

Buckle, Guy Su.27 

Buckner, Jane OR.19, OR.7, OR.76, F.28, 

Sa.128 

Bucknum, Amanda Sa.96 

Budinsky, Vit F.109 

Bupp, Melanie F.33 

Burdo, Tricia Su.128 

Burge, Matthew Sa.88 

Burke, George W. F.18 

Burrows, Gregory OR.10 

Butcher, Eugene C. OR.37 

Butte, Atul OR.53, F.9 

Buus, Søren F.53 

Cai, Chun Sa.134 

Cai, Qing Sa.150 

Calabresi, Peter Sa.145 

Calder, Claudia OR.86 

Callewaert, Nico Sa.38 

Camara-Lopes, Luiz F.131, Sa.121 

Cameron, Brian OR.9 

Campagnolo, Denise Su.9 

Canavez, Flavio Sa.121 

Candotti, Fabio OR.29 

Cannon, Jennifer Sa.116 

Cantaert, Tineke Sa.57, Sa.58, Sa.59 

Cantu-Brito, Carlos F.93 

Capocasale, Renold OR.40 

Capon, Francesca OR.39 

Carmans, Sofie OR.94 

Carmignani, Giorgio Sa.112 

Carmody, Francis F.13 

Carneiro-Sampaio, Magda Maria F.82 

Carter, Robert Sa.75 

Carulli, John OR.55, Sa.55 

Casanova, Jean-Laurent F.96 

Caspi, Rachel R. OR.38, OR.35 

Castro-Ramos, Feliciano Su.95 

Catanzarite, Tatiana Sa.132 

Cayrol, Romain OR.33, OR.88 

Chaim, Jacob OR.79 

Chan, Chi-Chao OR.35, OR.38 

Chang, Monica Sa.150 

Chang, Shun-Chiao Sa.53 

Chang, Vicky Su.82 

Chao, Nelson Sa.86 

Chapel, Helen F.80, F.83 

Chapman, Jeff Su.133 

Chau, Sandra F.27 

Chauveau, Christine Su.119 

Chavali, Arvind Su.84 

Chavez, Raul Su.93 

Chavez, Salvador Su.95 

Cheeran, Daniel OR.82 

Chella, David OR.61 

Chen, Benny Sa.86 

Chen, Cheng-Hsu OR.31 

Chen, Dong OR.42, OR.47 

Chen, Fang Su.136 

Chen, Genhui Sa.143 

Chen, James Sa.135 

Chen, Jing F.94 

Chen, Li-Chen F.88 

Chen, Ling Su.82 

Chen, Qian F.134 

Chen, Yi-Je Su.94 

Chen, Zoe OR.35 

Cheney, Richard Sa.111 

Cheng, Mickie OR.60, F.12 

Cheng, Yung Chih Su.137 

Chiffoleau, Elise OR.71, Su.96 

Chin, Hyunsook Sa.152 

Chini, Loredana F.93 

Chinn, Ivan F.84 

Chirinos, Perla Sa.90 

Cho, Judy Su.92 

Choi, Bong-Kum Sa.90, Sa.91 

Choi, Jae Eun Su.49 

Choi, James Su.62 

Chong, Mark Su.3 

Chooklin, Serge Su.110 

Choudhury, Arpita Sa.41 

Chow, Anthony W. Sa.93 

Choy, Edwin Su.35 

Christen, Selina Sa.126 

Christen, Urs 2201, OR.62, Sa.126 

Chruscinski, Andrzej J. Sa.30 

Chu, Alvina D. Sa.30 

S191


Chung, Denise OR.19 

Chung, James Su.69 

Ciancio, Gaetano F.18 

Citron, John Sa.152 

Ciullini Mannurita, Sara F.91 

Clarke, Grace Su.94 

Clayton, David OR.85 

Coccia, Marco Sa.137 

Cody, Virginia OR.43 

Cohen, Irun F.20 

Cohen, Philip L. OR.58 

Cohen, Philip Sa.41, Sa.50 

Cohen, Smadar Su.10 

Cohen, Stanley Sa.62 

Coito, Ana OR.97 

Cole, Derek Sa.142 

Collanieri, Anna Cristina F.67 

Coller, John Su.132 

Collins, Mary Sa.142, Su.9 

Concannon, Patric Sa.148 

Condamine, Thomas OR.71 

Conesa, Angela Su.108 

Constabtino-Silva, Rosemeire F.99, F.96, Su.75 

Navickas 

Contreras, Carmen F.51 

Contreras-Levicoy, Juan Sa.65 

Cooke, Lawrence Su.46 

Cooper, Leslie Sa.154 

Coppieters, Ken Sa.38, Sa.95, Sa.23 

Coram, Marc OR.53 

Cornelius, Jennine Su.84 

Corneta, Elaine Cristina Sa.123 

Corneta, Elaine F.131 

Correa, Alexandre OR.100, F.100 

Correa, MRF Su.75 

Cort, Laura OR.82 

Costa, Cristiana F.103 

Costantino, Cristina F.115 

Costenbader, Karen H. Sa.53 

Courtenay-Luck, Nigel Sa.107 

Cowan, Morton OR.25 

Creusot, Remi J. F.5 

Crispín, José Carlos Sa.81 

Crispin, Jose F.93, Su.101, Sa.20 

Croen, Lisa Sa.152 

Croft, Michael F.10 

Cronk, Katharine Sa.114 

Crow, Mary Sa.47, Sa.92, Su.116 

Cruzado-Park, Ingrid D. F.72 

Cruz-Robles, David Sa.64 

Cua, Daniel OR.35 

Cuchacovich, Miguel Sa.65 

Cunningham, Matthew Su.96 

Cunninghame Graham, Deborah OR.80 

Cutter, Gary Sa.66 

Cuturi, Maria-Cristina OR.71, Su.96 

Czelusta, Adam Sa.12 

da Silva Duarte, Alberto Jose F.67, F.97, F.98 

Dai, Yang F.27, F.30 

Daikh, David Sa.82 

Dailey, Michael E. Sa.138, Su.5 

Daisuke, Goto Sa.40 

Daisuke, Tokita Su.105 

Daly, Mark Su.35, Su.93 

Damle, Aarti OR.55, Sa.63 

Dan, Nicolae Su.92 

Dandekar, Abhya Sa.17 

Daneri Navarro, Adrián Su.140 

Dang, Demi F.5, F.9, OR.16 

Danilenko, Dimitry OR.37 

Dannecker, Guenther E. F.135 

Dardalhon, Valerie OR.34 

Darden, Janice F.133 

Das, Shampa Su.59, Su.60 

Dasgupta, Gargi F.53 

Datta, Lisa Su.92 

Dave, Amy F.10 

Davey, Michael OR.84, Su.79 

David, Chella Sa.154, Sa.78 

Davidson, Anne Sa.45 

Davies, Robert F.80 

Davis, Ian D. Su.54 

D’Costa, Sybil F.136, F.72 

De, Asit F.72 

De, Mita F.70 

de Almeida, Alexandre F.97 

de Bakker, Paul Su.93 

de Beaucoudrey, Ludovic F.92 

De Jager, Philip OR.83, Su.27, Su.35, 

Su.36, Sa.139 

de Jager, Wilco F.20, Sa.75 

De Keyser, Filip Sa.59 

de Krijger, Ronald F.103 

de Leo, Claudia Su.101 

De Rycke, Leen Sa.58, Sa.59 

De Sanctis, Juan Sa.1, OR.81 

De Vos, Martine Sa.23 

de Vries, Niek Sa.57 

Degauque, Nicolas F.3 

Deisenhammer, Florian Su.8 

Dekan, Gerhard OR.64 

Del Toro Arreola, Alicia Su.140 

dela Rosa, Corazon Sa.101, Su.118 

Dellanegra, Marinella F.96 

Delovitch, Terry L. OR.30, F.4, F.23 

Demartino, Julie Su.58 

Denham, Jerrod Su.109 

Dennis, Rebecca OR.9 

Derfanoux, Nadine A. Sa.52 

Derfuss, Tobias 3202 

Desaire, Heather F.72 

Deschamps, Jack-Yves Su.96 

Deshmukh, Umesh Sa.78 

Deshpande, Chetan Sa.62 

Devlin, Blythe F.84 

DeVoss, Jason Su.111 

Di Meglio, Paola OR.39 

Di Mitri, Diletta Su.43 

Diamantopoulos, Stavros F.18 

Diange, L. Su.114 

Diarra, Danielle Sa.51 

DiCarlo, Edward Su.116 

Diehl, Lauri Su.84


Dinarello, Charles F.130 

Ding, Li F.66 

Dinnall, Joudy-Ann Sa.42 

Dishaw, Larry J. F.86 

Disis, Mary Sa.101, Su.118 

Dissanayake, Samudra Sa.115 

Dittmer, Dirk Su.39 

Dodelet-Devillers, Aurore OR.33, OR.88 

Doi, Yoshimitsu Su.13 

Dolcino, Marzia OR.23 

Dombrovskiy, Viktor Su.74 

Domingues Ferreira, Mauricio F.67, F.97, F.98 

Dominguez-Rodriguez, Su.55 

Jorge R. 

Dong, Jiaxin Su.47 

Dorsey, Morna J. F.86 

Dotta, Francesco F.24 

Douhan III, John Sa.142 

Downes, Kate OR.85 

D'Souza, Patricia F.133 

Du, Jianguang OR.72 

Du, Sienmi Su.112 

Du, Xiaoyan Sa.71 

Duan, Su Su.107 

Duarte, Alberto J.S. Sa.49, F.52, F.86, Sa.50, 

F.96, F.99, Su.75, Sa.16, 

F.92 

Dubey, Devendra Su.59, Su.60 

Dubrava, Tatyana Sa.92 

Duchateau, Jean 

Dugast, Anne-Sophie 

F.127 

Dumont, Debora OR.87 

Dunger, David OR.85 

Dunn, Shannon OR.69 

Dunne, Jack Su.118 

Dunne, John Sa.101 

Dunussi-Joannopoulos, Sa.142 

Kyriaki 

Durinovic-Bello, Ivana F.32 

Dusilova Sulkova, Sylva F.109 

Dutt, Suparna OR.68 

Dutz, Jan P. F.21 

Dzuris, John Su.107 

Eagar, Todd F.33 

Easley, Kirk F.94 

Eastham-Anderson, Jeffrey Su.55 

Ebeling, Cory OR.65 

Edberg, Jeffrey Sa.84 

Edgerton, Colin Sa.79 

Edmond, Jean Su.92 

Edwards, Hannah OR.6 

Egeler, Maarten F.103 

Eibel, Hermann OR.32 

Eisenbarth, George S. OR.78, F.12, F.17, F.7, 

OR.1, OR.21 

Eisenberg, Robert A. OR.58, Sa.41, Sa.42 

El Baz, Mimouna F.127 

El Khoury, Joseph OR.90 

Elewaut, Dirk Sa.23, Sa.38 

Elliott, John OR.21 

Ellis, Richard OR.18 

Elloumi, Houda F.66 

Elmets, Craig Su.89 

Elyaman, Wassim Su.22 

Emanuel, Katy Sa.103 

Engelmann, Peter F.31 

Epstein, Michelle OR.64 

Eriksson, Anna Sa.135 

Esmaili, Habibollah Su.124 

Estevam, Jose Sa.151, Su.37 

Estrada-Garcia, Iris Su.76 

Estrada-Parra, Sergio Su.76 

Evanko, Stephen Sa.128 

Exley, Andrew F.126, F.65 

Ezekowitz, Alan Sa.63 

Fafutis, Mary F.53 

Fahmidekar, Mohammad Ali Su.78 

Falabella, Michael Su.134 

Falk, Ronald Sa.136 

Fanslow, William 3201 

Farber, Donna OR.46 

Farid-Hosseini, Reza Sa.9, Su.66 

Fariss, Robert Su.81 

Farjadian, Shirin Su.125 

Farkas, Klara F.31 

Farshad, Shohreh F.46 

Fathman, C. Garrison F.5, F.9, OR.16 

Faure, Olivier Sa.106 

Fedynyshyn, Joe F.136 

Feingersh, Diane Sa.137 

Feitelson, Jerald Su.133 

Felton, Jamie OR.49 

Fenoglio, Daniela Sa.112 

Feo, Lourdes Sa.32 

Ferbas, John F.137, Su.86 

Fergus, Gleeson F.80 

Fernandez, Marco Antonio F.26 

Fernando, Michelle Sa.74 

Ferrada, Carlos Sa.122 

Ferrao, Maria do Socorro F.96 

Ferraz Neto, Ben-Hur F.64 

Ferrera, Francesca OR.12 

Ferrone, Soldano Sa.111 

Ferry, Berne F.83 

Field, Elizabeth H. Sa.138 

Field, Leigh F.22 

Fife, Brian T. F.33 

Filaci, Gilberto Sa.112 

Filippi, Christophe 2201, F.7 

Finegood, Diane F.21 

Fiorillo, Edoardo Sa.153 

Fisher, Yair Su.126 

Flanagan, Ken Su.84 

Flavell, Richard Su.79 

Fleisher, Thomas 3201 

Flores, Antonio Su.98 

Fohner, Alison Sa.10 

Forbes, Elizabeth F.124 

Foresti, Roberta Su.119 

Forman, Daron F.114 

Forman, Stephen Sa.87 

Forsyth, Ramses F.105 

S193


Fossati, Gianluca Su.102, Su.85 

Foulkes, Roly Su.86 

Fousteri, Georgia F.10 

Fox, Edward Su.21 

Fransson, Moa Sa.100 

Fraser, Patricia Sa.53 

Fravega, Marco Sa.112 

Frederiksen, 

Su.54 

Klaus Stensgaard 

Frenkel, Dan Su.28 

Friend, Samantha Sa.66 

Frieri, Marianne Sa.8 

Frulloni, Luca 1202 

Fu, Qin Su.133 

Fujimoto, Manabu F.108, Sa.25, Su.63, 

Su.67, Su.68, Su.68 

Fujio, Keishi Sa.22 

Fujita, Mayumi Su.14 

Fujita, Tomoyuki Sa.25 

Fujiwara, Daisuke OR.100, Su.89 

Fulcher, Jennifer Sa.135 

Fung, Erik F.11 

Fung, John J. OR.31, Su.121 

Funke, Benjamin Su.90 

Furuzawa-Carballeda, Janette Sa.64, Su.129 

Fusaro, Ana Elisa Sa.16 

Gadkar, Kapil OR.59 

Gailey, Ryan Sa.104 

Gaines, Elizabeth Su.65 

Gallucci, Stefania Sa.42 

Gambhir, Sam S. F.5 

Gambineri, Eleonora F.91 

Ganjali, Rashin Sa.48, Su.66 

Gao, Sui F.50 

García Iglesias, Trinidad Su.140 

Garcia, Bertha OR.47, 1201 

Garcia, Michael Sa.71 

Garmendia, Jenny Sa.1 

Garvik, Barbara Sa.97 

Gattorno, Marco Sa.130 

Geba, Gregory Sa.5 

Gelli, Anna Maria Grazia 

Geng, Xuan F.21 

George, Tracy OR.68 

George, Heavner OR.40 

Gailey, Ryan Sa.104 

Ghaderi, Abbas Su.125 

Ghaffari, Javad Sa.109 

Ghahramani, Negar Sa.72, Sa.73 

Ghanekar, Smita Sa.133 

Ghasemi Hashtroodi, Leila F.1 

Ghio, Massimo Sa.33 

Ghoneum, Alia Sa.124 

Ghoneum, Mamdooh Sa.124 

Ghorayeb, Christine OR.86 

Giese, Thomas OR.46, Su.100, Su.107, 

Su.15, Su.90 

Gilbert, Leona OR.63 

Ginesta, Alexandra Sa.122 

Giuliani, Fabrizio Su.14 

Gluhcheva, Yordanka Georgieva Sa.108 

Gneiss, Claudia Su.8 

Golbin, Jason Sa.6 

Gold, Ralf Su.5 

Goldblum, Randall OR.22 

Goldoni, Adriana Leticia OR.48 

Goldrath, Ananda Su.61 

Goltsev, Anatoliy Sa.92 

Gomez-Contreras, Piedad C. Su.55 

Gonzalez, Carlos Sa.73 

Goodwin, Kelley Su.79 

Gorczynski, Reg Sa.127 

Gorczynski, Reginald F.38 

Gordon, John OR.65 

Gorelik, Elieser Sa.123 

Gosch, Courtney Sa.66 

Gostick, Emma Sa.98 

Goto, Daisuke Su.45 

Gottlieb, Alice Su.74 

Goverman, Joan OR.36 

Govindarajan, Rajagopalan OR.61 

Goyette, Philippe Su.108, Su.93 

Gozin, Michael Su.28 

Gozzard, Neil Su.86 

Graham, Kareem L. OR.91 

Graham, Robert R. OR.80 

Green, Todd Su.92, Su.93 

Greenbaum, Carla F.28 

Greenfield, Edward A. Sa.146 

Greer, Allison F.15 

Gregersen, Peter OR.55, Sa.55, Sa.61 

Gregory, Clare Su.94 

Greidinger, Eric L. Sa.28 

Greiner, Dale OR.56 

Griffiths, Gillian F.81 

Grimbacher, Bodo F.89 

Gros, Philippe F.27 

Gross, Jennifer Sa.97 

Grumach, Anete Sevciovic F.96, F.99, Su.75, F.92 

Gubbels Bupp, Melanie Sa.129 

Guberski, Dennis OR.82 

Guillaume, Marie Paule F.90, F.134 

Guillen, Cecilia F.51 

Guillermo, Roy Sa.120 

Gulati, Reema F.13, F.56 

Guleria, Indira F.33 

Gunderson, Erica Sa.152 

Gupta, Santosh F.68 

Gürses, Atilla Su.98 

Gutenberger, Sylvia F.75 

Haaland, Perry Sa.101 

Habiby Kermany, 

Sa.134 

Mohammad 

Hackstein, Holger Su.41 

Haensch, Gertrud Maria OR.98 

Hafler, David A. OR.77, OR.83, OR.94, 

OR.95, F.15, F.115, 

Sa.114, Sa.120, Sa.139, 

Sa.146, Sa.151, Su.27, 

Su.35, Su.36, Su.37, 

Su.38, Su.41 

Hahn, Bevra OR.4, OR.12, Sa.36, Sa.76


Hahn-Zoric, Mirjana F.122 

Hale, Matthew OR.92 

Hallam, Robert F.65, F.126 

Hamada, Takashi OR.97 

Hamm, Stefanie OR.32 

Han, David Su.34 

Han, May H. Su.34 

Han, Meifang Su.136, Su.43 

Hanak, Viktor Sa.6 

Hang, Howard F.115 

Hanlon, Douglas OR.43 

Hansen, Anna M. OR.38 

Hansen, Lasse Tengbjerg Su.54 

Hanson, Imelda C. Sa.12 

Hanson, Lars Åke F.122 

Hansson, Sverker F.122 

Hao, Qian-Lin F.113 

Haqqani, Arsalan OR.2 

Harley, John B. Sa.24, Sa.79 

Harley, John Sa.47, OR.81 

Hartnett, Mark F.9 

Hartt-Meyers, Jennifer Sa.146 

Hasegawa, Minoru Sa.25, Su.63, Su.67 

Haskins, Kathryn F.25, OR.3 

Hastings, William F.15 

Haug, Markus F.135 

Hauser, S. Su.93 

Haworth, Charles F.65 

Haynes, Barton OR.25 

Hazzan, Marc Sa.89 

Headley, Mark OR.57 

Hegde, Ganapati Sa.102, Sa.103 

Heidtmann, Antje Su.90 

Heinlen, Latisha Sa.79 

Hellings, Niels OR.87, Su.18, Su.23 

Hendriks, Jerome JJA OR.87 

Hennon, Teresa OR.89 

Henry, Alistair Su.85 

Hensen, Karen Su.23 

Heppert, Volkmar F.49 

Hering, Bernhard F.24 

Hernandez-Flores, Georgina Su.55 

Herold, Kevan OR.56 

Herrera-Gonzàlez, Norma Sa.110 

Herrinton, Lisa Sa.152 

Heslan, Michele OR.71, Su.96 

Hewitt, Kyle Sa.115 

Hickman, Scott F.127 

Hickman, Suzanne OR.90 

Hiestand, Peter Su.3 

Highton, John Sa.88 

Hill, Marcello OR.71 

Hillary, Steinhart Su.92 

Hintermann, Edith OR.62, Sa.126 

Hirasawa, Masao Su.53 

Hirsch, J. Su.19 

Hladikova, Zuzana F.19 

Ho, Anthony Sa.113 

Ho, I-Cheng Su.40 

Ho, Peggy P. Sa.54, Sa.71, Sa.88 

Hoefferer, Liane Su.7 

Hoffman, Robert W. Sa.26, Sa.28 

Hogan, Simon F.124 

Hogan, Susan Sa.136 

Hogendoorn, Pancras F.103 

3202 

Hojaili, Bernard OR.9 

Holdener, Martin OR.62 

Holers, V Michael Sa.63 

Holland, Steven M. OR.52, F.66 

Hollenberg, Morley OR.65 

Holness, Claire F.9 

Holzer, Ursula OR.65 

Hood, Zach Su.20 

Hoogeboom, Manja F.103 

Horai, Reiko OR.29 

Horn, Julia F.89 

Horwitz, David A. OR.75, Sa.140 

Hosseini-Farahabadi, Sara Sa.9, Su.66 

Hosseinkhani, Ayda F.46, F.62 

Hou, Stephen F.130 

Houle, Jean-Francois Su.120 

Hrotekova, Zuzana F.19, Sa.95 

Hsieh, Meng-Ying F.88 

Hu, Lina Sa.145 

Hu, Paul OR.64 

Hua, Jing Sa.47 

Huang, Jing-Long F.88 

Huber, Janine OR.40 

Hudson, Michael Sa.116 

Hueber, Wolfgang Sa.54, Sa.61 

Huegel, Alyssa OR.2 

Huett, Alan Su.92 

Huibregtse, Inge F.116 

Husain, Zaheed Su.59, Su.60 

Hussain, Shabbir F.23 

Huttenlocher, Anna OR.5 

Hwang, Hank Su.47 

Hwang, Sunil Su.34 

Iacomini, John Sa.18 

Ifergan, Igal OR.33, OR.88, Su.29 

Ikeda, Osamu F.58 

Iking-Konert, Christof F.49 

Iliev, Iliyan D. OR.70 

Im, Suhn-young Su.51 

Imitola, Jaime Su.22 

Indiveri, Francesco OR.12, Sa.112, Sa.33 

Inman, Robert Sa.47, 1201 

Inokuma, Margaret Sa.101, Su.118 

Inoue, Asuka Su.45 

Ishida, Masato Su.52 

Ishiura, Nobuko F.108 

Ismail, Heba OR.49 

Israel, David OR.74 

Itano, Andrea Su.69 

Ito, Satoshi Sa.48, Su.45 

Iwai, Hideyuki F.9, OR.16 

Iwakura, Yoichiro OR.35 

Iwanami, Keiichi Su.45 

Jaberi, Yahya F.1 

Jachimowicz, Edward F.129 

Jacob, Chaim O. Sa.94 

S195


Jacob, Cristina Miuki Abe F.82 

Jacob, Noam OR.93 

Jacobi, Christian Su.15 

Jacobson, Robert OR.26, F.63 

Jacobson, Stefan H. F.122 

Jacques, Peggy Sa.23 

Jahromi, Mohamed M. OR.78 

Jaimes, Kimberly Sa.26 

Jaimes, Maria F.133 

Jain, Ashish 3201, OR.27 

Jaing, Tang-Her F.88 

Jakobsen, Bent OR.9, Sa.98 

Jalahej, Heyam F.31 

James, Judith OR.81, Sa.79 

Jamshidzadeh, Akram F.48, F.62 

Jann, Monica OR.71 

Jasinski, Jean F.17, F.7, OR.1, OR.21 

Jave-Suarez, Luis F. Su.55 

Jawa, Vibha Su.42 

Jeffrey, Lisa F.120 

Jennette, J. Charles Sa.136 

Jensen-Jarolim, Erika OR.15 

Jerath, Maya Su.58 

Jerome, Rotter Su.92 

Jesus, Adriana Sa.49, Sa.50 

Ji, Shaoquan OR.39, Su.47 

Jiang, Guoping OR.74 

Jill, Giles OR.40 

Jimenez-Martinez, Maria Su.64, Su.76 

Carmen 

Joe, Selby Sa.152 

Johannes, Kellsey OR.60 

John, Eigth Author Su.107 

Johnson, Jonas OR.73 

Johnson, Kelly OR.1, OR.21 

Joo, Sung-Yeon Sa.90, Sa.91 

Jose Francisco, 

Sa.52 

Muñoz-Valle 

Joshi, Avadhut Sa.103 

Joshi, Shantaram Sa.103, Sa.116 

Josien, Regis OR.71, Su.96 

Juan, Gloria Su.69 

Juan, Manel F.26 

Juang, Yuang-Taung OR.48 

Jumnainsong, Amonrat Su.122 

Kagoda, Mercy F.113 

Kaieda, Shinjiro Su.14 

Kallas, Esper F.64 

Kalyanasundaram, 

F.73 

Ramaswamy 

Kamphuis, Sylvia F.20 

Kandeel, Fouad Sa.87 

Kang, Mi Jin Sa.90, Sa.91 

Kao, Amy H. Sa.31 

Karabeg, Adela OR.64 

Karagiannis, Panos Sa.112 

Karbach, Julia OR.9 

Karlson, Elizabeth W. Sa.53 

Karmali, Rafik F.127 

Karow, Margaret F.131 

Karter, Andrew Sa.152 

Kasman, Ian OR.37, Su.84 

Kassam, Nasim Sa.146, Su.38 

Kastelein, Robert Su.79 

Kataoka, Kazuo Su.24 

Kattah, Michael Su.132 

Katz, Jonathan F.8 

Kaufman, Kenneth OR.81 

Kawaciuk, Ivan Sa.117 

Kawikova, Ivana Su.102 

Kebir, Hania OR.33, OR.88, Su.29 

Kedem, Hassya F.69 

Keller, Baerbel F.75 

Kellermann, Sirid-Aimee Sa.52 

Kelley, Keith F.137 

Kelly, Jennifer OR.81 

Kennedy, Jeff Su.85 

Kent, Sally F.15, F.24 

Kenyon, Norma F.24 

Keogh, Elissa Sa.66, Sa.72, Sa.73 

Kern, Marlena OR.55 

Khaje Daluei, Mohammad Sa.16 

Khalili, Houman OR.55 

Khatri, Ismat F.38 

Khoury, Samia Su.27, Su.35 

Kianifard, Farid Sa.5 

Kiany, Simin F.45, F.46, F.48, F.62 

Kilpatrick, Jeff OR.81 

Kim, Han-A Su.51 

Kim, Il Hwan Su.64 

Kim, Junwoo Sa.134 

Kim, Patrick Sa.134 

Kim, Seon-Hee OR.89 

Kim, Sung-Joo Sa.100 

Kimberly, Robert Sa.84 

Kimmie, Crystal OR.49 

King, Jennifer K. Su.112 

King, Marie OR.56 

Kiran, Bayram Sa.109 

Kirby, Martha OR.29 

Kis, Janos F.31 

Kitaichi, Nobuyoshi Sa.21 

Kivisakk, Pia Su.22 

Kivovich, Violetta OR.63 

Klareskog, Lars Sa.61 

Klein, Mark F.20 

Klinkhammer, Thomas Su.46 

Knight, Seth Sa.75 

Knittelfelder, Regina OR.15 

Ko, Hon-Sum Sa.19 

Ko, Hyun-Mi Su.51 

Kobayashi, Koichi Su.79 

Kobayashi, Masakazu F.17 

Kodama, Keichi OR.16, F.5, F.9 

Koffeman, Eva Sa.66 

Kohlmoos, Cassidy OR.93 

Kohm, Adam Su.13 

Komura, Kazuhiro Su.63 

Komuves, Laszlo Su.84 

Korchynska, Olga F.41 

Korinets, Yaroslav F.41 

Korman, Alan Sa.137 

Korn, Thomas OR.19, Su.32


Koscielna, Katarzyna Sa.8 

Koss, Michael OR.93 

Kostianovsky, Alex Su.127 

Kostov, Georgi Stefanov Sa.108 

Kovacs, Jospeh 3201 

Kovats, Susan Sa.132 

Kozlova, Yulia Sa.101 

Kramer, Ivan F.39, F.74 

Krensky, Alan M. Sa.10 

Kretowski, Adam OR.78 

Kretsos, Kosmas Sa.52 

Kreuwel, Huub OR.59 

Krienke, Stefan Sa.47, 1201 

Krueger, Gerald Sa.150 

Kruithof, Elli Sa.59 

Krutsick, Amy Sa.104 

Krutzik, Peter OR.92 

Kuballa, Petric Su.92 

Kuchroo, Juhi OR.101, Su.38 

Kuchroo, Vijay K. OR.19, OR.95, F.15, 

Sa.146, Su.32 

Kudrycki, Katherine Sa.59 

Kudva, Yogish OR.61 

Kühne, Ronald Sa.129 

Kuis, Wietse F.20, Sa.67 

Kumar, Krishan Sa.8 

Kumar, Vijay F.110 

Kulhankova, Katarina Sa.138 

Kuo, Cheng-Chin F.121, Su.137 

Kuo, Liang-Mou OR.31, OR.74 

Kuo, Ming-Ling F.88 

Kusunoki, Susumu Su.24 

Kwon, Sung(Steve) F.73 

Kye, Young Chul Su.49 

Kyjovska, Drahomira Sa.95 

Kyttaris, Vasileios C. OR.48 

La Cava, Antonio OR.12, OR.4, Sa.36, 

Sa.76, Su.9 

LaCorcia, Gina Su.107 

Lage, Agustin Sa.147 

Lai, Chen-Yen F.121 

Lam, Queenie Lai Kwan OR.98 

Lam, Tong OR.65 

Lamb, Roberta OR.40 

Lamberth, Kasper F.53 

Lanchbury, Jerry OR.39 

Land, Michael F.100 

Landa, Rosa Sa.88 

Lang, Jiena F.34 

Lanigan, Caroline Su.128 

Larocca, Nancy Sa.1 

LaRosa, David F.57 

Laroy, Wouter Sa.38 

Lasitschka, Felix Su.105, Su.91 

Lathey, Janet F.120 

Latiano, A. Su.93 

Lau, Chak-Sing Sa.80 

Law, Adam F.12 

Lawendowski, Carla Su.107 

Lawson, Alastair Su.87 

Lawson, Jenifer OR.6 

Lederman, Michael F.73 

Le, Ngocdiep Sa.86 

Lee, Annette Sa.55 

Lee, Benhur Sa.151 

Lee, Chung Sa.36 

Lee, Hai-Chon Sa.13 

Lee, Hern-Ku Su.51 

Lee, Jin Su.49 

Lee, JungHwa Su.49 

Lee, Kwang Chul Su.49 

Lee, Li-Fen Sa.88 

Lee, Michael R. F.34 

Lee, Sun min Sa.106 

Lee, Tzielan Sa.70 

Lee, Wen-I F.88 

Lee-Chan, Edwin F.6 

Leelayuwat, Chanvit Sa.125, Su.122 

Lehman, Thomas Sa.47 

Leif, Jean OR.97 

Leite, Katia Sa.132 

Leiva, Lily E. F.101 

Lemar, Hadia Sa.137 

Lenert, Petar OR.44 

Leon, Juan F.44 

Leon-Murguia, Oscar Sa.44 

Leotlela, Poloko Sa.115 

Leppert, Mark Sa.150 

Lerma-Diaz, Jose M. Su.70 

Leung, Hin-Tak OR.85 

Leung, Lawrence Sa.71 

Levi, Dina F.14 

Levinson, Arnold F.57 

Levinson, Ralph Su.82 

Levisetti, Matteo OR.17 

Levy, Gary Su.59 

Li, Bin Sa.143 

Li, De-Kun Sa.152 

Li, Haowei Sa.93 

Li, Jian OR.40 

Li, Marcella F.17 

Li, Mu OR.12, OR.47, 1201 

Li, Quan-Zhen OR.81 

Li, Sharon F.13 

Li, Wentian Sa.25, Sa.55 

Li, Xinrui Sa.84 

Li, Zhuqing Su.78, Su.81 

Liang, Chi-Ming F.121 

Liang, Shu-Mei F.121, Su.137 

Liao, Hua-Xin F.72 

Liao, Lingjie Su.121 

Liddy, Nathaniel Sa.114 

Lierl, Michelle B. Su.131 

Liggitt, Denny OR.36 

Lightwood, Daniel Su.86 

Lim, Wee Kiak Su.78 

Limb, Cindy Sa.46 

Limón-Camacho, Leonardo Sa.20, Sa.64 

Lin, Chia-Lei Sa.87 

Lin, Erina F.100 

Lin, Jean Sa.31, Sa.35 

Lin, Syh-Jae F.92 

Linares, Marisela Su.76 

S197


Lindesmith, Lisa F.44 

Ling, Eleanor F.16 

Ling, Mei Su.92 

Linhart, Birgit Sa.11 

Linington, Christopher OR.20, 3202 

Link, Jason OR.10 

Lipets, Irina Su.74 

Littman, Dan R. OR.72 

Liu, Baoying Su.78, Su.81 

Liu, Chau-Ching Sa.31, Sa.35 

Liu, Chunyu Sa.55 

Liu, Edwin F.17, OR.21 

Liu, Mingfeng Su.43 

Liu, Peng Su.58 

Liu, Quanhai OR.41 

Liu, Ruolan Su.9, Su.9 

Liu, Shuying F.87, 3201 

Liu, Wenxia Sa.134 

Liu, Yingge Sa.153 

Lock, Christopher OR.96 

Logani, Jyoti F.68 

Long, S. Alice OR.7, OR.76 

Loo, Ray Mun Sa.8 

Lopes, Jared E. OR.72 

Lopez-Granados, Eduardo F.83 

López León Murguía, 

Su.140 

Oscar Javier 

López, Mercedes N. Sa.122 

Lorber, Marc Su.102 

Lord, James Sa.128 

Lories, Rik Sa.23 

Loskog, Angelica Sa.100 

Louvet, Cedric F.34 

Lu, Lina OR.31, OR.74 

Lu, Liwei OR.98 

Lu, Wen OR.60 

Lu, Xinyue Su.134 

Luckner, Claudia Sa.113 

Luger, Dror OR.35, Su.54 

Lühder, Fred Su.5, Su.5 

Luna-Baca, Gabriel Andres Su.64, Su.76 

Lunardi, Claudio 1202, OR.23 

Lundsgaard, Dorthe Su.54 

Luo, Jinquan Sa.141 

Luo, Xiaoping OR.86, F.50, Su.43, 

Su.136 

Luster, Andrew OR.90 

Lutsenko, Elena Sa.92 

Ly, Dalam F.4, F.23 

Lyba, Myroslav Su.110 

Lyle, Michael Sa.143 

Lyons, Kathryn Su.58 

Ma, Chi OR.27 

Ma, Hak-Ling Su.72 

Macaubas, Claudia Sa.62 

Macedo, Camila Su.99 

Macias-Hernandez, Israel Su.129 

Macip-Rodríguez, Perla Sa.64 

Macpherson, Michael OR.100 

Maecker, Holden F.133, Sa.101, Su.118 

Magliocco, Melissa Su.74 

Mahadevan, Daruka Su.46 

Mahesh, Sankara Su.81 

Mahesh, Sankaranarayana Su.78 

Mahon, Tara OR.9, Sa.98 

Maier, Lisa OR.83 

Maino, Vernon Sa.101 

Majumdar, Anish Su.127 

Malik, Sanober Sa.24 

Malter, James OR.99 

Mamura, Mizuko Sa.40, Su.61 

Manak, Mark F.120 

Manku, Harinder OR.80 

Manns, Michael OR.62 

Mansour, Eli F.99 

Manzi, Susan Sa.35 

Marcenaro, Stefania F.81 

Marchetti, Piero F.24 

Marcondes, Maria Cecilia Su.128 

Marietta, Eric F.116 

Marinkovich, Vincent A. F.128, F.132 

Marino Vazquez, Lluvia Su.118 

Marisis, Tatiane F.131 

Mark, Daly Su.92 

Markert, Louise F.84 

Markovic, Svetomir Sa.119 

Marquez, Maria Elena Sa.85, Su.126 

Marrero, Idania F.30 

Marti, Luciana F.64 

Martín Márquez, Beatriz Teresita Sa.44, Su.140 

Martin, Andrew Su.87 

Martin, Tammy Su.79 

Martínez García, Erika Su.140, Sa.44 

Martini, Alberto Sa.130, Su.137 

Martini, Stefania F.85 

Martinic, Marianne 2201, F.7 

Maryam, Tadayon F.106 

Masewicz, Susan A. F.32 

Massanari, Marc Sa.5 

Massoco, Cristina F.131, Sa.121 

Matarese, Giuseppe OR.4 

Matejkova, Eva Sa.95 

Mather, Ian OR.20 

Mathey, Emily 3202 

Mathieu, Suzanne Sa.32 

Matozan, Katja Su.7 

Matsevitaya, Irina Sa.92 

Matsuda, Tadashi F.58, Su.52 

Matsumoto, Isao Sa.40, Su.45 

Matsumoto, Mitsuru OR.24, Sa.141 

Matthews, Kate OR.64 

Maykut, Robert Sa.5 

Mazhani, Maryam Sa.48 

McArdel, Shannon Sa.114, Su.31 

McArthur, Grant Su.54 

McClain, Micah Sa.79 

McCrae, Ellie OR.41 

McGrail, Kieran OR.2 

McNeal, Erin-Joi F.44 

McPherson, Michael Su.89 

Means, Terry OR.90 

Meinl, Edgar 3202 

Mellins, Elizabeth Sa.148, Sa.62


Mendonça, Marcelo F.52 

Mendoza, Felipe Su.138 

Meraz-Rios, Marco Antonio Sa.110 

Merrill, Joan OR.81 

Mesirov, Jill Su.46 

Metes, Diana Su.99 

Meuer, Stefan Sa.113, Su.15, Su.90, 

Su.91, Su.100 

Meuwissen, Pieter Su.36 

Miao, Dongmei OR.21, F.17 

Michalek, Jaroslav F.19, Sa.104 

Michaud, Gregory Sa.127 

Midoro-Horiuti, Terumi OR.22 

Mielants, Herman Sa.23 

Miescher, Sylvia Su.22 

Miethke, Alexander Su.123 

Miettunen, Paivi Sa.69 

Miguel, Regueiro Su.92 

Mikita, Allison F.3 

Miklos, David B. OR.43 

Mileti, Erika OR.70 

Milkovich, Kimberly F.71 

Miller, Stephen OR.19, OR.34 

Miller-Graziano, Carol F.70 

Millonig, Alban Su.8 

Milosevic, Ivan F.105 

Min, Wei-Ping OR.42, OR.47, 1201 

Minarik, Ivo Sa.117 

Mingari, Maria Cristina Su.139 

Minnig, Kathrin Su.7 

Minor, Dana F.101 

Mi Qing, Sheng F.23 

Mirel, Daniel Su.109 

Misawa, Tamako Su.6 

Misbah, Siraj F.80 

Misiara, Antonio Sa.123, F.131 

Misiura, Angela F.41 

Mistry, Jehangir Su.47 

Miyake, Sachiko Su.12, Su.13, Su.14 

Miyamoto, Katsuichi Su.24 

Miyashiro, Joy Sa.142 

Miyashita, Minoru Su.53 

Mizuno, Miho Su.14 

Mo, Lian Su.99 

Modrusan, Zora Su.84 

Moe, Christine F.44 

Moheghi, Nasrin Su.50, Su.124 

Molloy, Peter Sa.98 

Monjure, Hanh F.101 

Monsonego, Alon OR.38, Su.10 

Montaño, Efrain Su.98 

Montano-Gonzales, Efrain Su.95 

Monteon, Francisco Su.98 

Monteon-Ramos, Francisco Su.112 

Montero, Enrique Sa.147 

Montgomery, Mitzi Su.21 

Montoya, Margarita F.51 

Moonka, Dilip F.71 

Moore, Adrian Su.86 

Moore, Carolina OR.97 

Moore, Marcus Su.52 

Mor, Guillermo Sa.116 

Moraes Vasconcelos, Dewton F.67, F.92, F.96, F.97, 

F.98, F.103, F.131, Sa.121, 

Sa.123, Su.75 

Morales Buenrostro, Luis Su.101 

Mordes, John OR.56, OR.82 

Moreau, Aurelie Su.96 

Moreira-Filho, Carlos F.66 

Moreno, Dolores Sa.1 

Moreno-Fierros, Leticia Su.56 

Moretta, Lorenzo F.81, Sa.130, Su.139 

Morrow, Matthew R. F.86 

Moshkoff, Dmitry F.120 

Moskowitz, Keith F.120 

Motterlini, Roberto Su.119 

Moullier, Philippe Su.96 

Moulton, Vaishali R. OR.48 

Mouri, Yasuhiro Sa.141 

Mouritzen, Ulrik Su.54 

Moviglia, Gustavo A. Su.19 

Moviglia, M.T. Su.19 

Moya, R. Su.19 

Muczynski, Kimberly Su.113 

Mueller, Isabelle OR.76 

Munder, Markus Sa.113 

Munger, Corey Sa.102, Sa.103 

Munirathinam, Gnanasekar F.73 

Muñoz Valle, José Francisco Su.140 

Muñoz-Chable, Olga Su.129 

Munroe, Melissa E. Sa.68 

Muromoto, Ryuta Su.52 

Murphy, Andrew F.124 

Murray, Joseph F.116 

Murtaza, Anwar Sa.32 

Myles, Timothy Sa.78 

Nadeau, Kari C. Sa.10 

Nahill, Sharon OR.19, Su.107 

Nakajima, Toshihiro Sa.56 

Nakayama, Maki OR.1, OR.21, F.17 

Nance, Christina OR.26 

Nasr, Mohamed Sa.138 

Navratil, Jeannine S. Sa.31 

Nazneen, A. Su.114 

Ndejembi, Modesta OR.46 

Neamatollahi, Hossein Su.124 

Neethling, Francisca OR.54, Sa.106 

Negi, Surendra OR.22 

Negri Santi, Tatiana F.69, F.97 

Neil, Risch Sa.152 

Nejad Shahrokhabadi, Khadijeh Sa.105 

Nelson, David 3201 

Nelson, Edward F.130 

Nelson, J. Lee Su.113 

Nemirovsky, Anna Su.126 

Nepom, Gerald OR.51, F.32, Sa.128 

Nesbitt, Andrew Su.85, Su.86 

Nesheim, Steven F.94 

Nesspor, Tom OR.40 

Nestle, Frank OR.39 

Nevin, Barry Sa.113 

Newell, Karen OR.41 

Newsom, Brian Su.21 

S199


Ng, Gordon Su.69 

Nguyen, Alex F.53 

Nguyen, Khoa Sa.10, Sa.62 

Nguyen, Tffany OR.54, Sa.106 

Nickoloff, Brian Sa.115 

Nico, Marcelo Mentha Su.75 

Niesters, Marieke F.103 

Niewold, Timothy Sa.47 

Nikoopour, Enayat F.6 

Ning, Bo OR.22 

Ning, Qin F.50, Su.43, Su.121, 

Su.136 

Nishimoto, Kevin F.130 

Nishimura, Toshihiko Sa.71 

Nishioka, Kusuki Sa.56 

Nishiya, Ana F.92 

Noaman, Eman Sa.124 

Nolan, Garry OR.92 

Nolan, GP OR.50 

Norowski, Elaine OR.82 

Notarangelo, Luigi F.91 

Noval Rivas, Magali Sa.89 

Nussenblatt, Robert Su.78, Su.81 

O’Brien, Peter Sa.104 

Ochs, Hans F.78, F.79, 3201 

O’Conner, Kevin Sa.114, Su.31, Su.47 

Offner, Halina OR.10, OR.13 

Ogawa, Masafumi Su.12 

Ohno, Shigeaki Sa.21 

Okamoto, Akiko Sa.22 

Okamura, Ross Su.109 

Oki, Shinji Su.13, Su.14 

Oksenberg, Jorge OR.69, Su.93 

Oldham, Elizabeth OR.39 

Oldham, Janine 2201 

Oliveira, Joao B. F.82, F.92, Sa.49, F.96, 

Su.75, Sa.50 

Olson, Julie Su.26 

Ondr, Jennifer F.8 

O’Neil, William M. Sa.43 

Onengut-Gumuscu, Suna Sa.148 

Ootsuka, Takao Su.14 

Opoka, Robert F.8 

O’Quinn, Darrell 3203 

Orange, Jordan F.76 

Orban, Tihamer F.31 

Oreizi, Farzad F.125 

Orihuela, Ana Su.31 

Orii, Noemia F.96 

Oritani, Kenji F.58, Su.52 

Orlovsky, Yevgeniya OR.40 

Orru, Valeria Sa.153 

Ortiz-Lazareno, Pablo C. Su.55 

O’Shea, Adam F.13, F.56 

Oshidary, Neekaan Su.132 

Ostankov, Maxim Sa.92 

Oukka, Mohamed OR.19, Su.32 

Ousman, Shalina OR.69, OR.94 

Out, Theo Sa.58 

Ouyang, Wenjun OR.37 

Ovsyannikova, Inna F.63 

Packwood, Kerri F.83 

Padyukov, Leonid F.122 

Page, Matt Su.87 

Paglia, Michael F.13, F.56 

Pahwa, Savita F.94 

Pai, Sung-Yun Su.40 

Palframan, Roger Su.86 

Palian, Beth Sa.140 

Palleros, C.A. Su.19 

Palmer, Jeanne Sa.86 

Palmer, Jerry OR.49 

Palumbo, Michael F.14 

Palumbo, Paul F.94 

Pan, Kuang-Hung Sa.62 

Pandey, Niranjan Su.57 

Pantanelli, Seth Su.81 

Pareigis, Elizabeth R. OR.44 

Paris, Kenneth F.101 

Parker, Alex OR.8 

Parrish, Yasmin F.113 

Pasalar, Mehdi F.114 

Passerini, Laura F.91 

Paston, Samantha Sa.98 

Pastorino, Antonio Carlos F.82 

Patel, Dhavalkumar Su.39, Su.58 

Paterson, Blake Su.57 

Pathoulakis, Charalabos F.61 

Patil, Tanvi F.38 

Payne, Kimberly F.113 

Peakman, Mark OR.6, OR.18 

Pearle, Andrew Su.116 

Pearson, Todd OR.56 

Pease, Larry Sa.119 

Peckham, Russell Su.134 

Pegram, Mark Sa.107 

Penaranda, Cristina OR.59 

Pende, Daniela F.81 

Peng, Stanford Sa.129, Su.57 

Peng, Ruoqi Su.58 

Penny, Richard Sa.18 

Pereda, Cristian Sa.122 

Perez, OD OR.50 

Perez, Rolando Sa.147 

Perez-Tapia, Mayra Su.76 

Peritt, David OR.40 

Perkins, Izabella F.44 

Perrin, Steve OR.55 

Perroni, Lucia Beatrice F.91 

Pesce, Barbara Sa.65 

Pessoa, Mário F.64 

Peter, Hans Hartmut OR.32, F.75, F.89 

Peter, Lipsky OR.81 

Peterlana, Dimitri OR.23, 1202 

Petrovic, Aleksandra F.86 

Petrovic-Stojkovic, Sanja Su.28 

Petry, Douglas OR.101 

Pi, Bin Su.136 

Pieri, Patrícia de Campos F.82 

Pietra, Gabriella Su.139 

Pihoker, Catherine F.28 

Pilat, Nina Sa.11 

Pillai, Asha OR.68


Pinsky, Norman F.63 

Pinto, Jorge Andrade F.99 

Pires Correa, Alexandre F.99 

Pitashny, Milena Sa.27 

Pixley, John S. Sa.43 

Plagnol, Vincent OR.85 

Planck, Steve Su.79 

Planck, Steven OR.84 

Ploegh, Hidde F.115 

Pober, Jordan Su.102, Su.104 

Pociot, Flemming F.22 

Poland, Gregory F.63 

Polychronakos, Constantin F.14 

Ponath, Paul F.58 

Ponte, Joe F.56 

Poole, Brian D. OR.63 

Popescu, Iulia Su.99 

Popov, Igor Sa.47, 1201 

Popplewell, Andrew Su.87 

Porcelli, A. F.23 

Prakken, Berent F.20, Sa.67 

Prasad, Shashi OR.43 

Prat, Alexandre OR.33, OR.88, Su.29 

Pratt, Nicol F.32 

Prefumo, Federico Su.139 

Pressler, Barrrak Sa.136 

Prestigiacomo, Tony Sa.79 

Preston, Ben Sa.137 

Preston, Gloria Sa.136 

Pricop, Luminita OR.93 

Priest, Catherine Su.109 

Prokoptchuk, Natalia F.41 

Puccetti, Antonio OR.23, 1202 

Puckett, Lindsay Su.28 

Pugliese, Alberto F.18, F.24 

Pujol Borrell, Ricardo F.26 

Punwani, Divya F.83 

Purbhoo, Marco OR.9 

Purcell, Shaun F.22 

Putterman, Chaim OR.93 

Pyne, Saumyadipta Su.36 

Qian, Shiguang OR.31, OR.74 

Rabellino, Enrique F.72, F.129 

Rachitskaya, Aleksandra V. OR.38 

Rafatpanah, Houshang Sa.9, Su.66 

Ragheb, Jack F.134 

Raimondi, Giorgio Su.105 

Rajagopolan, Govind Sa.78 

Rakhmanov, Mirzokhid F.75 

Rakhshandeh, Hassan Sa.105 

Ram, Aarthi OR.26 

Raman, Girija Su.94 

Ramanujam, Meera Sa.45 

Ramos Moreira Leite, Katia Sa.123 

Rao, Deepak A. Su.104 

Rapacki, Kristoffer F.22 

Rasband, Matthew Su.26, 3202 

Rashtak, Shadi F.116 

Rasouli, Manoochehr F.45, F.46, F.48, F.64 

Rassassi, Khadir Sa.151, Su.37 

Ratnayake, Chitra K. F.72 

Ravetti, Jean Louis Sa.112 

Ray, Pratima F.70 

Razzaque, M.S. Su.114 

Read, Jennifer F.94 

Reddy, Jay Su.32 

Reich, David Sa.139 

Reichardt, Holger Su.20 

Reid, Jessica Sa.96 

Reiff, Andreas OR.79 

Relman, David F.69 

Remy, Séverine Su.119 

Rescigno, Maria OR.70 

Reséndiz Albor, Aldo Arturo Su.56 

Rewers, Marian J. OR.78 

Reyburn, Hugh Su.122 

Reyes, Candice F.136 

Reyes, Diego Sa.122 

Richard, Julie OR.19 

Richards, William Sa.51 

Ricordi, Camillo F.24 

Rider, Beverley J. OR.23, F.6 

Rieben, Robert Su.7 

Rieck, Mary F.28, OR.76, Sa.148 

Riedl, Marc F.100 

Riemer, Angelika B. OR.15 

Rigato, Paula Ordonhez Sa.16 

Riggs, James Sa.96 

Rihal, Pardeep S. Sa.12 

Riker, Adam Sa.115 

Riley, Christopher Su.46 

Rinderknecht, Cornelia Sa.132 

Rioux, John Sa.74, Su.92, Su.93 

Rita, Bottino OR.56 

Rivitti, Evandro F.99 

Rizzi, Marta OR.12, OR.32, Sa.33 

Robbins, Paul Sa.91 

Roberto, Eigth Author F.24 

Roberts, Robert OR.42, F.87 

Robinson, William OR.94, Sa.54, Sa.61, 

Sa.71, Su.34 

Robotham, Jason M. Sa.18 

Roby, Keith Su.48 

Rodrigo, Evelyn 2201 

Rodrigues, Denise F.64 

Rodriguez Manzanet, Roselynn Sa.146 

Rodriguez, Benigno F.71 

Rodriguez, Miguel Su.95, Su.98 

Roep, Bart F.2, OR.11, OR.6 

Rogerio, Jaqueline F.120 

Rojas, Mauricio Su.58 

Romeo, Elisa 

Römisch, Jürgen Su.15 

Roncarolo, Maria Grazia F.91 

Ronchese, Franca Sa.6 

Roord, Sarah Sa.67 

Rosenbaum, James OR.84, Su.79 

Rosengren, Sanna Sa.70 

Rosenthal, Devin Sa.115 

Rosenzweig, Holly OR.84, Su.79 

Rosenzweig, Michael F.114, F.56, F.13 

Rossin, Elizabeth Su.27, Su.35, Su.36 

S201


Rossini, Aldo OR.56 

Rothenberg, Marc F.124 

Rottembourg, Diane OR.14 

Rottiers, Pieter F.116, Sa.46 

Rouse, Todd Sa.138 

Roux, Kenneth H. Sa.17, Sa.18 

Roy, Debasmita Su.39 

Roy, Dolly Su.34 

Royer, Pierre-Joseph Su.119 

Rubertone, Mark Sa.79 

Rubinstein, Mark Su.61 

Ruitenberg, Joyce Sa.133 

Rummens, Jean-Luc Su.23 

Ruzek, Melanie OR.19 

Ryan, Jenna F.63 

Ryu, Jay Sa.6 

S. Grumach, Anete F.67, F.98, F.101 

Sabater, Lidia F.26 

Sabeti, Pardis Sa.74 

Saikali, Philippe Su.16 

Saito, Yuki Su.67 

Salazar, Lorena Sa.65 

Salazar-Onfray, Flavio OR.45, Sa.65, Sa.122 

Salek Moghddam, Alireza Sa.7 

Salman, Andac Sa.109 

Saltzman, Mark Sa.110 

Salzer, Ulrich OR.32, F.89 

Sampaio, Elizabeth F.66 

Samstag, Yvonne 

Sánchez Hernández, 

Pedro Ernesto 

Su.90 

Sanda, Srinath F.2 

Sandborg, Christy Sa.62 

Santacruz-Valdes, 

Su.64, Su.76 

Concepcion 

Santamaria, Pere F.21 

Santoro, Alessandra F.81 

Saoudi, Abdelhadi Su.111 

Sarraf, Alireza Su.124 

Sathe, Shridhar Sa.17, Sa.18 

Sato, Jun-ichi Su.6 

Sato, Maria Notomi Sa.16 

Sato, Shinichi Sa.33, Su.63 

Sawalha, Amr H. OR.81, Sa.24 

Sayegh, Mohamed Su.22 

Scalapino, Kenneth Sa.82 

Scallon, Bernie OR.40 

Scanzello, Carla Su.116 

Schaefer, Catherine Sa.152 

Schartner, Jill OR.5 

Schatz, Desmond OR.2 

Schein, Catherine Sa.5 

Schenk, Erin Sa.119 

Schett, Georg Sa.51 

Schlesier, Michael F.89 

Schnell, Christian Su.3 

Schoeb, Trenton 3203 

Scholler, Nathalie Sa.97 

Schout, Denise F.52 

Schreiner, Bettina OR.34 

Schrodi, Steven OR.69 

Schroeder, Andreas Su.90 

Schroeder-Braunstein, Jutta Su.91 

Schwartzberg, Pamela L. OR.29 

Schweitzer, Barry Sa.116 

Scofield, R Hal Sa.24 

Scott, Benjamin Sa.42 

Seamon, Vanessa Sa.17 

Seddiki, Nabila OR.30 

Seeborg, Filiz Sa.12 

Seeuws, Sylvie Sa.23 

Segal, Gabriela Sa.122 

Segura, Jorge F.51 

Seidu, Luqman F.124 

Sekine, Yuichi F.58, Su.67 

Selby, Mark Sa.137 

Selman, Moises Su.138 

Sercarz, Eli F.27, F.30 

Seroogy, Christine F.110 

Servais, Genevieve F.127 

Seshasayee, Dhaya Sa.149 

Sessarego, Marta Sa.33 

Sette, Jr., Hoel F.64 

Setty Balakrishnan, Anand F.73 

Sewell, Andy Sa.98 

Seyeddi, Ronak Sa.85 

Seyfer, Sarah OR.44 

Seyfert-Margolis, Vicki Su.1, Su.50 

Shampain, Kimberly L. Sa.114 

Shao, Wenhai OR.58 

Sharif, Shadi Sa.71 

Sharifi, Faranak F.1 

Sharma, Meera F.79 

Sharp, Veronika OR.50 

Shea, Yvonne F.68 

Shealy, David OR.40 

Shearer, William OR.26, F.94 

Shen, Guanxin Su.121 

Shen, Jijia F.134 

Shen, Ling Sa.152 

Shen, Zhong-Jian OR.99 

Sheng, Shijun Su.133 

Shi, Fu-Dong Su.9 

Shi, Jishu F.61 

Shirdel, Babak F.27 

Shivakumar, Pranavkumar Su.123 

Shizuru, Judith Sa.88 

Shoda, Lisl OR.59 

Shohreh, Farshad F.62 

Shoor, Stanford Sa.152 

Shugart, Yin Yao Su.92 

Shultz, Leonard OR.56 

Shum, Anthony Su.111 

Shum, Tony F.12 

Sido, Bernd Su.90, Su.91 

Siebert, Janet Sa.101, Su.118 

Siegel, Richard M. OR.29 

Sierra, Juan F.93 

Silberman, Daniel Sa.96 

Silva, Clovis Sa.56, Sa.57 

Silva, Léia C. Rodrigues F.52 

Silva, Nubia Su.108 

Silver, Phyllis OR.35, OR.38


Silverberg, Mark Su.92 

Sim, Davis Sa.78 

Simonian, Michael H. F.72 

Simonson, William OR.5 

Sims, Martin Sa.42 

Sin, Sang-Hoon Su.39 

Singer, Josef OR.15 

Singh, Bhagirath F.6 

Singh, Harvir Sa.30 

Singh, Manisha F.55 

Singh, Ram Raj OR.66, Sa.76, Sa.94, 

Su.112, Sa.135 

Sirota, Marina OR.53 

Siwak, Edward OR.26 

Skak, Kresten Su.54 

Skowera, Anna OR.18 

Skrumsager, Birte K. Su.69 

Slaets, Leen OR.87 

Slavik, Jacqueline Sa.146 

Slavonic, Michael F.13 

Sleasman, John W. F.90 

Smit, Helga Su.114 

Smith, Deborah Su.112 

Smith, Michael Sa.116 

Smitz, John Sa.136 

Smogorzewska, Monika F.113 

Smyth, Deborah OR.85 

Snowden, James Su.87 

Snyder, James F.134 

Snyder, Michael Sa.116 

Sobel, Raymond A. Sa.146, OR.69, OR.91, 

OR.94, Sa.97, Su.32, 

Su.34 

Sochorova, Klara F.109 

Sohn, Jeongwon Su.49 

Solbrig, Camille OR.43 

Soler-Ferran, Dulce Su.27 

Somers, Veerle Su.18, Su.23 

Somerville, John Sa.106 

Sommerer, Claudia Su.100 

Son, Hye-Youn Sa.90, Sa.91 

Son, Sang Wook Su.49 

Song, Jason Sa.71 

Soper, David OR.8 

Sorensen, Ricardo U. OR.5 

Soto-Abraham, Virginia Sa.72 

Soulillou, Jean-Paul Sa.65 

Sousa-Canavez, Juliana F.131, Sa.121, Sa.123 

Spier, Catherine Su.46 

Sprent, Jonathan Su.61 

Spycher, Martin O. Su.7 

Sripa, Banchob Sa.111 

Standifer, Nathan OR.51 

Stanimirovic, Danica OR.88 

Staquet, Kim OR.40 

Stechova, Katerina F.19 

Steinman, Lawrence OR.69, OR.94, OR.96, 

Su.34 

Stephens, Tracey A. F.6 

Sternjak, Alexander OR.10 

Stevens, Anne Su.113 

Stevens, Christine Sa.74, Su.93 

Stevens, Helen OR.85 

Still, Travis F.24 

Stinissen, Piet OR.87, Su.18, Su.23, 

Su.31 

Stolina, Marina Sa.51 

Storch, Maria OR.20, 3202 

Stourac, Petr F.62 

Straub, Irena F.135 

Strauss, Laura OR.73 

Strober, Samuel OR.68 

Strober, Warren 3201 

Strobl, Frank Sa.104 

Strom, Terry B. Sa.146 

Stromnes, Ingunn OR.36 

Struthers, Mary Su.58 

Su, Kai Su.136 

Su, Kaihong Sa.84 

Su, Maureen F.12 

Suen, Yu Su.48 

Suez, Daniel F.78, F.79 

Suggs, Sid F.137 

Sugiyama, Kenji Su.52 

Sumida, Takayuki Sa.40, Su.45 

Sun, Hongtao 1201 

Sun, Lulu F.102 

Sun, Xiaocui OR.72 

Supter, Tina Su.105 

Suresh, Lakshmanan F.110 

Sutton, Deborah OR.9, Sa.98 

Suzuki, Motohiko OR.42, OR.47, 1201 

Swanson, Steven J. Su.42 

Swerkersson, Svante F.122 

Swiergala, Elzbieta F.22 

Swistak, Mark Su.107 

Szalai, Alexander Sa.84 

Szereday, Laszlo F.31 

Szot, Gregory L. F.34 

Szuhai, Karoly F.103 

Tabunoki, Hiroko Su.6 

Tachdjian, Raffi F.100 

Tagawa, Asako OR.24 

Taguchi, T. Su.114 

Tahmasebifar, Neda F.122 

Tai, Yi Su.102 

Tak, Paul-Peter Sa.57, Sa.58, Sa.59 

Takahashi, Kazue Sa.63 

Takehara, Kazuhiko Sa.25, Su.67 

Tálamo, Carlos Sa.1 

Tamaki, Kunihiko F.108, Su.68 

Tamayo, Pablo Su.36 

Tamiz, Amir Su.57 

Taneja, Veena Sa.154 

Tang, Anita OR.46 

Tang, Liren Sa.143 

Tang, MengXiang Sa.101 

Tang, Qizhi OR.59, F.33 

Tanguy-Royer, Séverine Su.119 

Tanji, Maury M. F.52 

Tasch, Michael Su.113 

Tassinari, Paolo Su.108 

Tati, Abdellatif F.103 

S203


Taub, Dennis Sa.115 

Tavakkol Afshari, Jalil Sa.9, Sa.48, Sa.105, 

Su.50, Su.62, Su.66, 

Su.124 

Tavares, R.C. F.104 

Tawde, Pallavi Sa.17, Sa.18 

Taylor, Kent Su.92 

Taylor, Paul Su.117 

Tedder, Thomas Su.85 

Teklenburg, Gijs F.20 

Tellides, George Su.119, Su.129 

Temmerman, Stephane OR.27 

Tenenbaum, Jessica Sa.46 

Terrett, Jon OR.72 

Tesson, Laurent Su.119 

Teuber, Suzanne OR.50, OR.63 

Thebault, Pamela OR.71 

Thewissen, Marielle Su.18, Su.23 

Thiel, Jens F.85, F.95 

Thomas, John F.137 

Thomson, Angus Su.105 

Thornton, Angela M. OR.29 

Thurlings, Rogier Sa.58 

Tian, Yan F.3 

Tibshirani, Robert Sa.69 

Timmel, Amanda OR.5 

Timmer, Trieneke Sa.58 

Tirabasi, Rebecca OR.82 

Tisch, Roland Sa.136 

Tiwari, Ruby OR.22 

Tiwari-Woodruff, Seema Su.112 

Tobiasova, Zuzana F.109, Sa.117 

Todd, John A. F.11 

Todd, John OR.85 

Todorov, Ivan Sa.87 

Toft, Michelle OR.90 

Togher, Lisa OR.14, 2201, F.7, F.16 

Tomi, Chiharu Su.28 

Tommasini, Alberto F.95 

Tomooka, Beren OR.94, Sa.61 

Tonel, Giulia OR.39 

Tonkin, Daniel OR.3 

Toro, Felix Su.108 

Torre, Giancarlo Sa.112 

Torres-Lozano, Carlos F.77, Su.70 

Tötterman, Thomas Sa.100 

Touil, Tarik OR.41 

Traggiai, Elisabetta Sa.130 

Traverso, Paolo Sa.112 

Tree, Timothy OR.6, OR.18 

Trembath, Richard OR.39 

Tremoulet, Adriana Sa.74 

Treszl, Andras F.31 

Tripp, Catherine Sa.32 

Trucco, Massimo OR.56 

Trueblood, Esther Su.69 

Tsokos, George C. OR.48, Su.134 

Tsoukas, Christos M. F.91 

Tsuchihashi, Sei-ichiro Su.128 

Tsutsumi, Akito Sa.40, Su.45 

Turna, Akif Sa.109 

Tyson, Kerry Su.87 

Uccelli, Antonio Sa.130 

Ueda, Masumi OR.28, Sa.125 

Untersmayr, Eva OR.15 

Uratsu, Sandra Sa.17 

Urban, Nicole Sa.97 

Utz, Paul J. OR.50, OR.75, Sa.30, 

Su.132 

Uzel, Gulbu OR.52 

Vacca, Paola Su.139 

Vaickus, Lou F.114 

Vaitaitis, Gisela M. OR.67 

Valdes-Ferrer, Sergio Ivan F.93 

Valdez, Patricia OR.37 

Valenta, Rudolf Sa.11 

Valenzuela, David F.124 

Vales-Alberto, Luis Su.95 

Vales-Gomez, Mar Su.122 

Valle, Sollange F.99 

Van Belle, Tom F.16 

Van Beneden, Katrien Sa.23, Sa.38 

van de Ven, Annick Sa.72, Sa.73 

van den Brandt, Jens Su.5 

van der Pouw-Kraan, Tineke Sa.58 

van Deventer, Sander 

Van Eyk, Jennifer 

F.116 

van Kooten, Cees F.122 

Van Landeghen, Megan OR.7 

Van Noort, Johannes OR.94 

van Rooijen, Nico Sa.22 

van Wijk, Femke Sa.67 

Vandenbark, Arthur OR.10, OR.13 

Vanderlocht, Joris OR.87 

Vandooren, Bernard Sa.58, Sa.59 

Varela, Gabriela Su.19 

Varela, Jorge Alcocer Sa.20 

Vargas Rojas, Maria Ines F.93, Sa.20, Sa.64, Sa.81, 

Su.101 

Vasquez, Ismael Sa.110 

Vatseba, Roman Su.110 

Vázquez Del Mercado Espin, Sa.44, Su.140 

Mónica 

Venken, Koen Su.18, Su.23 

Verbruggen, Gust Sa.23, Sa.38 

Verrijn Stuart, Annemarie F.20 

Veys, Eric M. Sa.59 

Victor, Jeferson Russo Sa.16 

Vidlakova, Petra Sa.95 

Vierkant, Robert F.63 

Vieths, Stefan Sa.17 

Viglietta, Vissia Su.27 

Vilela, Maria Marluce F.99 

Villaggio, Barbara Sa.112 

Villanueva, Cleva Sa.110 

Vladau, Costin F.124, OR.42, OR.47, 1201 

Vliagoftis, Harissios Sa.7 

Vollmer, Timothy Su.9 

von Gynz Rekowski, Kathrin Su.21 

von Herrath, Matthias OR.14, F.2, 2201, F.7, F.10, 

F.16, Sa.137, Su.117 

Vose, Julie Sa.102, Sa.103 

Voskuhl, Rhonda R. Su.112


Vrabelova, Zuzana F.19 

Vugler, Alex Su.86 

Vyse, Timothy J. OR.80, Sa.74 

Wade, Michael OR.7 

Wadia, Persis P. OR.53 

Wagner, Christof F.49 

Wagner, David OR.67, F.25, Sa.131, 

Su.118 

Waid, Dan Su.118 

Wakeland, Edward OR.81 

Waldmann, Herman F.13 

Waliszewska, Alicja Sa.139 

Walker, Mindi OR.76 

Walker, Neil OR.85 

Walsh, Emily Sa.74 

Wandstrat, Amy OR.81 

Wang, Fang Sa.17 

Wang, James Sa.94 

Wang, Julie OR.75, Sa.140 

Wang, Kevin YehSheng F.87 

Wang, Shen-Wu F.137 

Wang, Wei Su.121 

Wang, Zhiliang Su.123 

Wang, Zhimo F.50 

Warnatz, Klaus OR.32, F.75, F.89 

Wasserfall, Clive OR.2, OR.56 

Watanabe, Rei Su.68 

Watanabe, Yoko Su.45 

Watry, Debbie Su.128 

Wawrousek, Eric OR.94 

Weaver, Casey 3203 

Webber, Steve Su.99 

Webster, John Sa.143 

Weeraratna, Ashani Sa.115 

Wei, Bo OR.100, Su.89 

Wei, Nathan Sa.70 

Weidanz, Jon A. OR.54, Sa.106, Sa.108 

Weiner, Howard Su.27, Su.28 

Weiner, Mira Su.27 

Weisenburger, Dennis Sa.102 

Wekerle, Thomas Sa.11 

Wellcome Trust Case Contr OR.85 

Wen, Xiangshu OR.66, Sa.135 

Wentzensen, Andreas F.49 

Wernet, Dorothee F.135 

Westall, Frederick Su.1, Su.2 

Westerberg, Per-Anton F.122 

Westerfield, Susan OR.26 

Whartenby, Katharine Sa.145 

Whitehead, Terry Su.47 

Whiteside, Theresa L. OR.73 

Whiting, Chan C. OR.59, Sa.52 

Wicker, Linda F.11, F.27, OR.83 

Wight, Thomas Sa.128 

Wijayawardana, Sameera F.98 

Wijbrandts, Carla Sa.66, Sa.67 

Wilkinson, Julie F.129, Sa.2 

Williams, Jim Su.34 

Williams, John Su.107 

Williams-Weese, Courtney Sa.24 

Willis, Van OR.52 

Willison, LeAnna N. Sa.18 

Wishart, Neil Sa.32 

Witte, Alison Sa.137 

Wolf, Bryan J. Sa.43 

Wolslegel, Kristen Sa.149 

2201 

Wong, Maida Sa.80 

Wongsena, Wachanan Sa.111 

Wood, Allyson Sa.63 

Woodburn, Tito OR.54 

Wotring, Michael OR.49 

Wu, Jianfeng OR.37 

Wu, Michele F.53 

Wu, Paul OR.13 

Wucherpfennig, Kai Su.32 

Wulff, Heike Su.94 

Wullner, Danika Su.42 

Xavier, Ramnik Su.92 

Xi, Dong F.50, Su.43, Su.136 

Yaghoubi, Shahriar F.5 

Yagishita, Naoko Sa.56 

Yagita, Hideo F.33 

Yamamoto, Kazuhiko Sa.30 

Yamamoto, Nobuto OR.28, Sa.125 

Yamamura, Takashi OR.24, Su.12, Su.13, 

Su.14 

Yamasaki, Satoshi Sa.56 

Yan, Weiming F.50, Su.43, Su.136 

Yancopoulos, George F.124 

Yang, Jiajin Sa.136 

Yang, Jun-Qi OR.66 

Yang, Li OR.95 

Yang, Lihu Su.58 

Yang, Sherry Sa.115 

Yang, Wei F.3 

Yano, Masashi Sa.141 

Yao, Qingping Sa.94 

Yaremtchuk, Tatiana F.41 

Yasui, Teruhito F.58 

Ye, Shuang OR.75, Sa.140 

Yelensky, Roman Su.35 

Yener, Alper Sa.109 

Yeung, Joseph Sa.80 

Yin, Zhenyu OR.31, OR.74 

Yong, Pierre F.76 

Yoo, Tai June Sa.134 

Yoshiga, Yohei Sa.40 

Yoshimura, Akihiko F.58 

Young, Daniel OR.59, Sa.52 

Young, Debbie OR.87 

Youssef, Sawsan OR.69 

Yu, Liping OR.1, F.17 

Yue, Erica Su.42 

Z, Stephen Su.69 

Zabaleta-Lanz, Mercedes Sa.85 

Zabel, Brian A. OR.91 

Zamir, Ehud F.12 

Zang, Yun Sa.28 

Zarabi, Mohammad Su.62 

S205


Zarkeshfard, Bahareh F.125 

Zeckcer, Israel Su.75 

Zehnder, Roland Su.7 

Zeier, Martin Su.100 

Zeiseniss, Peter Sa.66 

Zelazny, Adrian F.66 

Zeldin, Robert Sa.12 

Zeng, Defu Sa.87 

Zhang, Chunyan Sa.87 

Zhang, Dong F.3 

Zhang, Li F.12 

Zhang, Ping Su.136 

Zhang, Xiao Sa.66 

Zhang, Xiuli F.53 

Zhang, Xusheng OR.47, F.109, Sa.54 

Zhang, Yiqun F.21 

Zhang, Zili OR.84 

Zhao, Hong Sa.75 

Zhao, Lei Sa.153 

Zhao, Xiaoyan Sa.54 

Zheng, Chengjie OR.101 

Zheng, Song Guo OR.75, Sa.140 

Zheng, Xin Xiao F.3 

Zheng, Xiufen OR.42, OR.47, 1201 

Zheng, Yan OR.37 

Zheng, Yanan OR.59 

Zhou, Baohua OR.57 

Zhou, Bin Sa.134 

Zhou, Jing OR.63 

Zhou, Liang OR.72 

Zhou, Tong OR.47, Sa.75, Sa.84 

Zhou, Xiaochuan Sa.142 

Zhou, Yixuan Sa.134 

Zhu, Chuanlong F.51 

Zhu, Xiaoming F.55, OR.57 

Zidovetzki, Raphael OR.79 

Ziegler, Steven F. OR.72, Sa.13 

Zielinska, Ewa F.113 

Zollner, Ricardo F.99 

Zonneveld-Huijssoon, Evelien Sa.67 

Zucchiatti, Andrea F.70 

Zuniga, Luis OR.91, OR.69 

Zvaifler, Nathan Sa.70 

Zvetkova, Elissaveta Borissova Sa.108


Abstract Index by Category 

Diabetes and other autoimmune endocrine diseases 

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, 

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, 

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 

Immunity and infection 

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, 

F.62, F.63; F.64, F.65, F.66, F.67, F.68, F.69, F.70, F.71, F.72, F.73 

Immunodeficiency: primary or acquired 

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, 

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 

Laboratory immunology 

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, 

F.128, F.129, F.130, F.131, F.132, F.133, F.134, F.135, F.136, F.137 

Allergy/Asthma 

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 

Autoimmune rheumatologic diseases 

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, 

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, 



Sa.82, Sa.84, Sa.85, Sa.86 

Bone marrow or stem cell transplantation 

OR.53, OR.68, Sa.87, Sa.88, Sa.89, Sa.90, Sa.91, Sa.92, Sa.93, Sa.94, Sa.95 

Immuno-oncology 

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, 

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, 

Sa.121, Sa.122, Sa.123, Sa.124, Sa.125, Su.83 

General autoimmunity 

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, 

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, 

Sa.151, Sa.152, Sa.153, Sa.154, Sa.155 

doi:10.1016/j.clim.2007.03.542 


www.elsevier.com/locate/yclim

S208 Abstract Index by Category 

Autoimmune neurologic diseases 

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, 

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, 

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 

Cytokines/Chemokines 

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, 

Su.51, Su.52, Su.53, Su.54, Su.55, Su.56, Su.57, Su.58, Su.59, Su.60, Su.61, Su.62 

Immuno-dermatology 

OR.39, Su.63, Su.64, Su.65, Su.66, Su.67, Su.68, Su.69, Su.70, Su.72, Su.74, Su.75 

Immunology of the eye 

OR.35, Su.76, Su.78, Su.79, Su.81, Su.82, Su.83 

Inflammatory bowel disease 

OR.70, OR.100, Su.84, Su.85, Su.86, Su.87, Su.89, Su.90, Su.91, Su.92, Su.93 

Organ transplantation 

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, 

Su.108, Su.109 

Other 

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, 

Su.126, Su.127, Su.128, Su.129, Su.132, Su.133, Su.134, Su.136, Su.137, Su.138 

Reproductive immunology 

OR.60, Su.139, Su.140

Oral Presentations - Federation of Clinical Immunology Societies

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