CYTOKINE PRODUCTION IN RA SYNOVIAL TISSUE AFTER MUCOSAL TOLERIZATION

在初步实验中，我们已经证明：i)细菌来源的肽（大肠杆菌dnaJ热休克蛋白）是类风湿关节炎（RA）患者促炎T细胞反应的靶标。dnaJ与HLA DRBI*0401具有相同的RA易感性序列（共享表位）。基于这些初步结果，我们提出dnaJ可能是导致RA慢性自身免疫性炎症的抗原之一；ii)使用我们开发的一种新的技术方法（T细胞捕获：TCC），可以鉴定、分离和表征RA患者血液中与共享表位发生反应的T细胞；iii)我们已经确定了一种dnaj衍生的肽（dnaJP1），该肽已在一项旨在调节RA中这些促炎反应的临床试验中成功进行了I期测试。该建议与一项实验性II期，双盲，安慰剂对照试验有关dnaJPl对RA炎症反应的调节。在该研究中，我们将通过临床和免疫学结果测量来评估免疫调节治疗的疗效。我们将扩展这一分析，以研究治疗对抗原特异性T细胞的影响，以确定疾病活动性和治疗效果的替代标记物。试验预定在2000年2月开始。上述项目虽然全面分析了免疫调节治疗在RA患者中引起的免疫变化，但在评估炎症滑膜部位治疗引起的表型和功能变化方面仍存在不足。在这里，我们建议从UCSD的试验中招募的患者队列中获得滑膜活检样本，纵向分析这种变化。将对样品进行细胞因子生产，并将其与二期项目获得的免疫学和临床数据相关联。该项目的具体目标是：(1)在基线和治疗结束时（持续6个月）收集II期试验患者的滑膜活检；（二）建立适合分子生物学、免疫学和组织学研究的样品库；(3)通过real-time PCR （TaqMan）提取评估促炎和耐受性细胞因子的产生和趋化因子受体的表达。

In preliminary experiments that prompted this grant application, we have shown that: i) peptides of bacterial origin (E. coli dnaJ heat shock protein) are a target of pro-inflammatory T cell responses in rheumatoid arthritis (RA) patients. dnaJ shares with HLA DRBI*0401 the susceptibility sequence to RA (shared epitope). Based upon these initial results, we have proposed dnaJ as one of the possible antigens, which may contribute to the generation of chronic autoimmune inflammation in RA; ii) T cells in the blood of RA patients that react with the shared epitope can be identified, isolated and characterized, using a novel technical approach (T cell capture: TCC), which we have developed; iii) We have identified a dnaJ-derived peptide (dnaJP1) which has been successfully tested in Phase I of a clinical trial aimed at modulating these pro-inflammatory responses in RA. This proposal is connected to a pilot Phase II, double blind, placebo controlled trial of modulation of inflammatory responses to dnaJPl in RA. We will evaluate in that study efficacy of immunomodulatory treatment by clinical and immunological outcome measurements. We will extend this analysis to study the effect of the treatment on antigen-specific T cells, in order also to identify surrogate markers for disease activity and treatment efficacy. The trial is due to start in February 2000. The above mentioned project, while providing a comprehensive analysis of immune changes induced in RA patients by an immunomodulatory treatment, still falls short in evaluating phenotypical and functional changes induced by the treatment at synovial sites of inflammation. Here, we propose to analyze such changes longitudinally in synovial biopsies samples obtained from the cohort of patients enrolled in the trial at the UCSD site. Cytokine production of the samples will be performed, and correlated with immunological and clinical data obtained from the Phase II project. The specific aims for this project are: (1) To collect synovial biopsies from patients enrolled in the Phase II trial at baseline and end-treatment (six months duration) points; (2) to establish a library of samples suitable for molecular biology, immunology and histology studies; and (3) to extract evaluate production of pro-inflammatory and tolerogenic cytokines and chemokine receptors expression by real-time PCR (TaqMan)