MOLECULAR GENETICS OF AUTOANTIBODIES IN HUMANS

这项研究的长期目标是描绘出分子和遗传 持续产生潜在致病自身抗体的基础 自身免疫性疾病。这些“疾病特异性”自身抗体水平很高。 免疫球蛋白M和免疫球蛋白G亚型的亲和性自身抗体，如免疫球蛋白类风湿 类风湿关节炎(RA)相关因子(RF)与血清中抗DNA抗体的关系 系统性红斑狼疮(SLE)。在上一个资助期内，我们的 对类风湿关节炎患者的免疫球蛋白RFS的分析表明， 患者是克隆性相关的，由自然自身抗体引起。 最近对免疫耐受的研究表明，自身反应性B细胞 要么被细胞凋亡删除，要么功能失活(无能)。 然而，某些自身反应性B细胞在自身免疫MRL中是不能容忍的。 LPR/LPR小鼠，存在Fas介导的细胞凋亡途径缺陷。它是 已知，在选择压力下，具有生存能力的变异细胞 优势可以以克隆的方式扩展。我们假设某些疾病 类风湿关节炎和系统性红斑狼疮中分泌自身抗体的特异性B细胞可能逃脱耐受 调控原因：1)与细胞凋亡相关的功能缺失突变 或凋亡抑制基因的功能获得突变(如 Bcl2)在克隆性B细胞水平，导致I耐受性 变异；或2)刺激相关基因的功能获得突变 (如B7)，导致结构性激活的无能变体。至 仔细研究这些假设，具体目标是： 1)疾病特异性自身抗体的产生和特性- 分泌B细胞系和同型特异性对照免疫球蛋白分泌细胞系 来自类风湿性关节炎和系统性红斑狼疮患者。 2)Fas介导的细胞凋亡途径的比较分析 细胞系。将对每个患者的细胞系进行Fas分析 细胞表面表达、可溶性Fas分泌与细胞凋亡 分别由Fas配体(FasL)或细胞毒性抗Fas诱导 抗体。 3)比较分析不同细胞系中Bcl2的表达。 4)Fas非依赖、表面Ig介导的比较分析 这些细胞系中的细胞凋亡途径； 5)B7共刺激分子在这些细胞中表达的比较分析 台词。 6)确定上述研究中发现的缺陷是否也 存在于新分离的自身反应性B细胞中 病人。

The long term goal of this study is to delineate the molecular and genetic basis for sustained production of potentially pathogenic autoantibodies in autoimmune diseases. These "disease specific" autoantibodies are high affinity autoantibodies of IgM and IgG isotypes, such as IgG rheumatoid factors (RFs) in rheumatoid arthritis (RA) and IgG anti-DNA antibodies in systemic lupus erythematosus (SLE). During the last funding period, our analysis of IgG RFs from RA patients has revealed that the RFs in each patient are clonally related, and arise from natural autoantibodies. Recent studies of immunological tolerance show that self-reactive B cells either are deleted by apoptosis or are functionally inactivated (anergy). However, some self-reactive B cells are not tolerized in autoimmune MRL- lpr/lpr mice, which have a defective Fas-mediated apoptosis pathway. It is known that, under selection pressure, a variant cell with a survival advantage can expand in a clonal manner. We hypothesize that some disease specific autoantibody-secreting B cells in RA and SLE may escape tolerance regulation because of: 1) loss-of-function mutations in apoptosis-related genes or gain-of-function mutations in apoptosis suppressor genes (such as Bcl-2) at the clonal B cell level, resulting i tolerance-resistant variants; or 2) gain-of-function mutations in stimulation-related genes (such as B7), resulting in constitutively activated anergic variants. To examine these hypotheses, the specific aims are: 1) Generation and characterization of disease specific autoantibody- secreting B cell lines and isotype-specific control Ig-secreting cell lines from RA and SLE patients. 2) Comparative analysis of the Fas-mediated apoptosis pathway in these cell lines. Cell lines from each patient will be analyzed for Fas expression on the cell surface, secretion of soluble Fas, and apoptosis induced separately by either Fas ligand (FasL) or cytotoxic anti-Fas antibodies. 3) Comparative analysis of Bcl-2 expression in these cell lines. 4) Comparative analysis of the Fas-independent, surface Ig-mediated apoptosis pathway in these cell lines; 5) Comparative analysis of the B7 costimulator expression in these cell lines. 6) Determination if the defects identified in the above studies are also present in freshly isolated autoreactive B cells from corresponding patients.