NEW ASSAY FOR TCR DIVERSITY IN TMJ RHEUMATOID ARTHRITIS

活化的T细胞似乎是致病机制中的中心元素。 关节(TMJ)类风湿性关节炎(RA)。其作用机制 负责T细胞激活的原因尚不清楚。如果T 细胞根据传统的机制被激活，然后特定 免疫原(S)应通过T细胞抗原受体识别 (TCR)。无论是免疫原(S)，还是涉及的TcR都没有定义 在分子水平上。我们假设TCR的一种主要形式 在体内激活的TMJ T中可以发现可变序列 淋巴细胞。这项建议的主要目标是分析 IN表达的TCRα\Beta V区核苷酸序列多样性 活体激活的T细胞，并表征任何 优势受体。为此，我们将选择六个合适的TMJ RA 我们可以从患者身上获得体内激活的滑膜T细胞。我们 将开发一种新的检测方法，将“连接锚定”的聚合酶链式反应(LA-PCR) 变性梯度凝胶电泳(DGGE)定量 估计TCR V区序列的多样性。这些细胞将是 用LA-PCR+DGGE分析。已鉴定的优势受体物种 将被克隆和测序。这种化验方法的发展将会 允许解决克隆问题，并且在将来可能需要 确定患者特定的致病T细胞克隆。作为未来的努力， 这些主要受体的独特区域将在一个 重组系统，以及针对它们的单抗将是 已生成。优势T细胞克隆的鉴定及其研究进展 单抗可能直接导致重要的医学和科学 申请。针对主要TCR的单抗可能 事实证明，作为诊断和监测疾病的新方法是有用的 活性，并有可能用于特定的免疫治疗。

The activated T-cells appear to be a central element in the pathogenesis of temporomandibular joint (TMJ) rheumatoid arthritis (RA). The mechanisms responsible for the activation of the T cells are not known. If the T cells are activated according to conventional mechanisms, then specific immunogen(s) should be recognized through the T-cell antigen receptor (TCR). Neither the immunogen(s), nor the TCR's involved have been defined at the molecular level. We hypothesize that a predominant form of TCR variable sequence can be found among the in vivo activated TMJ T lymphocytes. The primary objective of this proposal is to analyze the diversity TCR alpha\Beta V-region nucleotide sequences expressed by the in vivo activated T cells, and characterize the nucleotide sequences of any predominant receptors. To this end, we will select six suitable TMJ RA patients from which we can obtain in vivo activated synovial T-cells. We will develop a novel assay which combines "linker-anchored" PCR (LA-PCR) and Denaturing Gradient Gel Electrophoresis (DGGE) to quantitatively estimate the diversity of TCR V-region sequences. These cells will be analyzed by the LA-PCR plus DGGE. Predominant receptor species identified on the gel will be cloned and sequenced. Development of this assay would allow resolution of the clonality, and might in the future be needed to identify patient-specific pathogenic T cell clones. As a future effort, unique regions of these predominant receptors will be expressed in a recombinant system, and monoclonal antibodies specific for them will be generated. Identification of predominant T cell clones, and development of monoclonal antibody could lead directly to important medical and scientific applications. Monoclonal antibodies specific for predominant TCR might prove useful as new assays for diagnosis and for monitoring disease activity, and potentially in specific immunotherapy.