GAD ANTIBODY MEDIATED CONTROL OF EPITOPE SPECIFIC ANTIGEN PRESENTATION

在临床发病前和发病时，胰岛素依赖型糖尿病 (IDDM)与胰岛自身免疫密切相关，特别是与 抗GAD65-MR 65,000亚型自身抗体的存在 谷氨酸脱羧酶。GAD65抗体具有较高的诊断价值 对IDDM的敏感性、特异性和预测价值。The Now 我们研制成功的标准化GAD65Ab放射结合测定法 用我们的GAD65基因克隆人胰岛GAD65后制备 放射性GAD65通过体外转录和翻译相结合的方法 用于证明表位特异性GAD65Ab具有更高的诊断能力 对IDDM的敏感性高于非表位分析的GAD65Ab。我们的目标是 探讨GAD65抗体相关自身免疫的机制 IDDM有两个具体目标。1)检验表位特异性假设 GAD65Ab在中间表型中决定进展为IDDM。 具体地说，我们将从以下方面构建组合抗体库：1) 新发IDDM患者(项目3)；2)GAD65Ab阳性健康人 未进展为IDDM的个人(项目4)；3)GAD65Ab阳性 对2型糖尿病(NIDDM)患者进行分类和治疗 (项目3)为了分离与IDDM相关的GAD65反应的Fab B.确定重链和轻链抗-HBs的cDNAs序列 用GAD65表位特异性淘选法筛选出GAD65Ab。2)测试 GAD65抗体介导的GAD65摄取偏离人类白细胞抗原-65的假设 克隆性T细胞反应受限。具体地说，我们将：A.转化 人B细胞表位特异性GAD65Ab的摄取、加工 和介绍；B.探索抗体的精细特异性在 GAD65多肽和人类白细胞抗原对GAD65表位呈递的影响 与项目1合作进行的限制性T细胞克隆。 项目2将与项目1合作，通过以下方式定义机制 B淋巴细胞膜上结合的GAD65Ab可能偏离抗原- 介绍MHC II类抗原和随后的T细胞反应。 来自项目1的GAD65多肽特异性和DRB1或DQB1限制性T细胞 将用于确定偏差响应。与您的合作 项目3是研究新发的IDDM患者和GAD65Ab阳性患者 对2型糖尿病(NIDDM)患者进行分类和治疗。这些 患者将与非进展期标志物阳性的健康人进行比较 项目4中的受试者。

Before and at the clinical onset, insulin-dependent diabetes mellitus (IDDM) is closely associated with islet auto-immunity, in particular with the presence of auto-antibodies (Ab) against GAD65 - the Mr 65,000 isoform of glutamic acid decarboxylase. GAD65Ab have a high diagnostic sensitivity, specificity and predictive value for IDDM. The now standardized GAD65Ab radiobinding assay that we successfully developed after cloning human islet GAD65 using our GAD65 cDNA plasmid to prepare radioactive GAD65 by coupled in vitro transcription and translation was used to demonstrate that epitope-specific GAD65Ab have a higher diagnostic sensitivity for IDDM than non-epitope analyzed GAD65Ab. The objective is to determine the mechanisms of GAD65 antibody-associated auto-immunity in IDDM in two Specific Aims. 1) To test the hypothesis that epitope-specific GAD65Ab determine progression to IDDM in intermediary phenotypes. Specifically, we will construct combinatorial antibody libraries from: 1) new onset IDDM patients (Project 3); 2) GAD65Ab- positive healthy individuals not progressing to IDDM (Project 4); 3) GAD65Ab-positive patients who are classified and treated for type 2 (NIDDM) diabetes (Project 3) in order to isolate Fab reactive with IDDM-associated GAD65 epitopes; b. determine the cDNA sequences of heavy and light chain anti- GAD65Ab selected by GAD65 epitope-specific panning. 2) To test the hypothesis that GAD65 antibody-mediated uptake of GAD65 deviate HLA- restricted clonal T cell responses. Specifically we will: a. transfect epitope-specific GAD65Ab to human B cells to determine uptake, processing and presentation; b. explore the role of antibody fine specificity in influencing GAD65 epitope presentation using GAD65 peptide and HLA restricted T cell clones in collaboration with Project 1. Project 2 will collaborate with project 1 to define the mechanisms by which membrane-bound GAD65Ab on B lymphocytes may deviate antigen- presentation on MHC Class II antigens and subsequent T-cell responses. GAD65-peptide specific and DRB1 or DQB1-restricted T cells from project 1 will be used to determine deviation responses. The collaboration with Project 3 is to study new onset IDDM patients and GAD65Ab-positive patients who are classified and treated for type 2 (NIDDM) diabetes. These patients will be compared with non-progressing marker-positive healthy subjects individuals from Project 4.