PATHOGENESIS AND PREVENTION OF IDDM

IDDM 的发病机制和预防

• 批准号: 2385626
• 负责人: HUGH O MCDEVITT
• 金额: $ 78.83万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-15 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2385626
• 关键词: NOD mouse; disease /disorder etiology; disease /disorder prevention /control; genetically modified animals; insulin dependent diabetes mellitus

The purpose of this research program is to bring together five investigators in the Departments of Microbiology and Immunology, and Pathology to develop a series of collaborative, research projects. These research projects are designed to apply the techniques of molecular immunology to understanding the development and specificity of the CD4+ T responses to islet cell antigens in type I diabetes in the NOD mouse, in NOD transgenic models expressing human IDDM susceptibility genes, and to develop novel methods of altering the immune responses to prevent IDDM. Drs. Yueh-Hsiu Chien will develop specific T cell staining reagents by producing soluble biotinylated I-A/g7 molecules with a covalently bound peptide coupled to fluorescent streptavidin. The peptide epitopes of glutamic acid decarboxylase and pre pro-insulin will be identified in Dr. McDevitt's laboratory (under the support of DK-51667). Dr. McDevitt will utilize NOD mice transgenic for HLA-DQ8, 7, and 6 to identify the immunodeficient peptide epitopes of human GAD 65 and human PPI presented by IDDM susceptible (DQ8), 7, and 6 to identify the immunodominant peptide epitopes of human GAD 65 and human PPI presented by IDDM susceptible (DQ8) and resistant (DQ7,6) alleles in man. The same T cell receptor staining reagents will be developed for detection of GAD and PPI specific, DQ8 restricted T cells in pre-diabetic and recent onset diabetic patients. In a collaboration between Dr. Chien and Dr. Mark Davis, the polymerase chain reaction (PCR) will be used to characterize and sequence T cell receptor V alpha and V beta sequences from single T cells isolated from the islets of 14-18 day old NOD mice. These T cell receptor V beta and V alpha sequences have detectable shared CDR 3 sequence motifs, suggesting an antigen specific T cell response. T cell receptors from single cells will be expressed in transfected T cell lines and transgenic mice to permit a determination of their function. Transgenic T cells will be tested for their antigenic specificity using a panel of recombinant murine islet cell antigens produced in Dr. McDevitt's laboratory. Dr. Crabtree will produce transgenic mice expressing a mutant cyclophlin able to bind and inhibit calcineurin only when complexed to a modified cyclosporin A. Modified cyclosporin A will be used to inhibit signaling in T cells, to inhibit T cell activation and T cell development, and to induce tolerance to transplanted tissues and islet cell antigens. In collaboration with Dr. McDevitt, Dr. Crabtree will introduce the modified cyclophilin gene into the NOD mouse strain. Modified cyclosporin A will be used to block T cell signaling at various time points following birth to induce tolerance to islet cell antigens. Dr. Weissman and Dr. Judith Shizuru will isolate of hematopoietic stem cells to analyze the effects of whole bone marrow (WBM) versus hematopoietic stem cell (HSC) transplantation in replacing the endogenous lymphoid compartment. H2 matched or syngeneic bone marrow will be used. These experiments will explore the potential for utilizing bone marrow transplantation to prevent the development of type I diabetes in susceptible individuals. Dr. Weissman will also use targeted recombination to analyze the development of the T cell receptor repertoire in NOD mice with endogenous and transplanted bone marrow. These studies will rely on the same techniques used by Drs. Chien and Davis to trace the development of islet cell specific T cells in the NOD mouse.

该研究计划的目的是将五个 微生物学和免疫学部门的研究人员，以及 开发一系列协作，研究项目的病理学。这些 研究项目旨在应用分子技术 了解CD4+ T的发展和特异性的免疫学 对I型糖尿病的胰岛细胞抗原的反应 点头表达人IDDM敏感性基因的转基因模型，并 开发改变免疫反应以防止IDDM的新方法。 博士。 Yueh-Hsiu Chien将通过 与共价结合的可溶性生物素化的I-A/G7分子 肽与荧光链霉亲和素偶联。肽表位 谷氨酸脱羧酶和前胰岛素将在Dr. 麦克德维特的实验室（在DK-51667的支持下）。麦克德维特博士 利用HLA-DQ8、7和6的NOD小鼠转基因来识别 呈现的人GAD 65和人PPI的免疫缺陷肽表位 IDDM易感（DQ8），7和6识别免疫肽 IDDM易感性提出的人GAD 65和人类PPI的表位（DQ8） 和人类中的抗性（DQ7,6）等位基因。相同的T细胞受体染色 将开发试剂以检测GAD和PPI特异性DQ8 糖尿病前和最近发作的糖尿病患者的T细胞受到限制。在 Chien博士和聚合酶链Mark Davis博士之间的合作 反应（PCR）将用于表征和序列T细胞受体 从胰岛分离的单个T细胞中的V alpha和Vβ序列 14-18天大的点头小鼠。这些T细胞受体Vβ和V alpha 序列具有可检测的共享CDR 3序列基序，这表明 抗原特异性T细胞反应。来自单细胞的T细胞受体将 在转染的T细胞系和转基因小鼠中表达以允许A 确定其功能。转基因T细胞将进行测试 使用一组重组鼠胰岛细胞的抗原特异性 麦克德维特博士实验室生产的抗原。 Crabtree博士将生产 表达突变体环链林的转基因小鼠能够结合并抑制 钙调蛋白仅在复合到修饰的环孢菌素A时。 环孢菌素A将用于抑制T细胞中的信号传导，以抑制T 细胞激活和T细胞发育，并诱导耐受性 移植组织和胰岛细胞抗原。与博士合作 McDevitt，Crabtree博士将将改良的环磷脂基因引入 点头小鼠应变。修饰的环孢菌素A将用于阻断T细胞 出生后各个时间点发出信号，以诱发宽容 胰岛细胞抗原。魏斯曼博士和朱迪思·史祖鲁博士将隔离 造血干细胞分析整个骨髓的作用（WBM） 与造血干细胞（HSC）移植相对于替代 内源性淋巴室。 H2匹配或合成性骨髓将 被使用。这些实验将探索利用骨骼的潜力 骨髓移植以防止I型糖尿病的发展 敏感的人。魏斯曼博士还将使用有针对性的重组 分析NOD小鼠中T细胞受体库的发展 内源性和移植的骨髓。这些研究将依靠 DRS使用的相同技术。 Chien和Davis追踪发展 NOD小鼠中的胰岛细胞特异性T细胞。