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 存在抗GAD 65-Mr 65，000同种型的自身抗体（Ab） 谷氨酸脱羧酶GAD 65抗体具有较高的诊断价值， 敏感性、特异性和预测价值。当下 我们成功开发的标准化GAD 65 Ab放射结合试验 用我们的GAD 65 cDNA质粒克隆人胰岛GAD 65后， 放射性GAD 65通过体外转录和翻译偶联， 用于证明表位特异性GAD 65 Ab具有更高的诊断特异性， 与非表位分析的GAD 65 Ab相比，目标是 确定GAD 65抗体相关自身免疫的机制， 两个具体目标的胰岛素依赖型糖尿病。1)为了验证表位特异性 GAD 65 Ab决定中间表型向IDDM的进展。 具体地，我们将从以下构建组合抗体文库：1） 新发IDDM患者（项目3）; 2）GAD 65 Ab阳性的健康人 未进展为IDDM的个体（项目4）; 3）GAD 65 Ab阳性 被分类为2型（NIDDM）糖尿病并接受治疗的患者 （项目3）为了分离与IDDM相关的GAD 65反应的Fab 表位; B.确定重链和轻链抗- 通过GAD 65表位特异性淘选选择GAD 65 Ab。2)测试 假设GAD 65抗体介导GAD 65摄取偏离HLA- 限制性克隆T细胞应答。具体而言，我们将：a.转染 表位特异性GAD 65 Ab至人B细胞，以确定摄取、加工 和呈现; B.探讨抗体精细特异性在 使用GAD 65肽和HLA影响GAD 65表位呈递 限制性T细胞克隆与项目1合作。 项目2将与项目1合作，通过以下方式确定机制： B淋巴细胞上的膜结合GAD 65 Ab可能偏离抗原- MHC II类抗原的呈递和随后的T细胞应答。 来自项目1的GAD 65肽特异性和DRB 1或DQB 1限制性T细胞 将用于确定偏差响应。合作与 项目3是研究GAD 65 Ab阳性的新发IDDM患者， 2型糖尿病（NIDDM）的分类和治疗患者。这些 将患者与非进展性标志物阳性的健康受试者进行比较。 项目4中的个人。

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.