TH-17 Axis Plays a Crucial Role in the Generation of GPI-induced Arthritis

S156 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 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, 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, Huazhong University of Science and Technology, Wuhan, 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/cEts 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

Abstracts

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, ENA78, 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 Comprehensive Human Science, University of Tsukuba, Tsukuba, Japan, Satoshi Ito, Instructor, Graduate School of Comprehensive Human Science, University of Tsukuba, 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 (P b 0.01), 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 (P b 0.05). 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 (BCLL) 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

S157 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