Identification of relevant peptides involved in the initiation and progression of autoimmune diabetes

鉴定参与自身免疫性糖尿病发生和进展的相关肽

• 批准号: 10246429
• 负责人: EMIL Raphael UNANUE
• 金额: $ 43.48万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-20 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10246429
• 关键词: Alleles; Antigens; Autoimmune; Autoimmune Diabetes; Autoimmune Process; Beta Cell; Binding; Biochemical; Biological Assay; Blood; CD4 Positive T Lymphocytes; Cells; Cellular biology; Cytoplasmic Granules; Dendritic Cells; Development; Diabetes Mellitus; Disease; Endocrinologist; Evaluation; Glucose; Goals; Human; Hybridomas; Immunologics; Immunologist; In Vitro; Inbred NOD Mice; Individual; Insulin; Insulin-Dependent Diabetes Mellitus; Investigation; Islets of Langerhans; Lead; Lymphoid Tissue; Mass Spectrum Analysis; Measures; Mouse Strains; Mus; Non obese; Pathogenesis; Peptides; Peripheral; Phagocytes; Population; Prediabetes syndrome; Process; Proteins; Reaction; Sampling; Seminal; Site; Source; Spleen; Structure; T-Lymphocyte; Techniques; Technology; Testing; Thymus Gland; Tissues; Urine; Validation; Vesicle; autoreactivity; diabetic; diabetogenic; experience; experimental study; granule cell; immunogenic; in vivo; instrumentation; islet; lymph nodes; macrophage; non-diabetic; novel; response; secondary lymphoid organ

Project Summary We propose a collaborative project involving immunologists experienced in autoimmune diabetes, mass-spectrometrists, and endocrinologists-diabetologists. Our combined efforts are to specifically identify relevant peptides from beta cell proteins involved in the immunopathogenesis of type 1 diabetes. The project combines experimental studies in diabetes-prone mice that should lead to targeted evaluation of human samples. The ultimate goal is to identify peptides that form the substrate for the autoreactive MHC-II response that initiates and perpetuates the process. Important is to identify relevant diabetogenic antigens at different stages of the autoimmune reaction from pre-diabetes to the initial progression: it will include their characterization, source in beta cells, involvement in the disease process, and sites of presentation. Aim 1 consists of an identification of novel peptides derived from beta cell granules of both mice and humans. We examine vesicles isolated from beta cells with a major focus on crinosomes. Crinosomes and insulin dense core granules will be isolated during different stages of diabetes development, their peptides will be isolated and examined by sophisticated mass spectrometry with state of the art instrumentation and technology. We will place emphasis in unique structural or post translational changes in peptides and whether the panoply of them changes as the beta cell progresses into active diabetes. We also will examine vesicles and exocytosed products from human islets from recently diseased individuals. Aim 2 will be an immunological validation of novel peptides identified by mass spectrometry. Immunological and biochemical assays will be used to validate the ability of beta cell derived peptides to bind to I-Ag7 and activate T cells in vitro and in vivo. Aim 3 will be the Identification and validation of the MHC-II-bound peptidome in islets and secondary lymphoid organs. We plan to examine the peptidome eluted from MHC-II molecules expressed by phagocytes obtained from islets, peripheral lymph nodes, and spleens of the NOD mouse and to characterize/validate them as in Aims 1/2. This is the seminal test to prove an immunogenic peptide, that is, it's a positive identification bound in vivo to MHC-II alleles.

项目概要 我们提出了一个由在自身免疫方面经验丰富的免疫学家参与的合作项目 糖尿病学家、质谱学家和内分泌学家-糖尿病学家。我们的共同努力是 专门鉴定参与β细胞蛋白的相关肽 1 型糖尿病的免疫发病机制。该项目结合了实验研究 易患糖尿病的小鼠，这应该会导致对人类样本进行有针对性的评估。终极 目标是鉴定形成自身反应性 MHC-II 反应底物的肽， 启动并延续该过程。重要的是识别相关的糖尿病抗原 从糖尿病前期到初始进展的自身免疫反应的不同阶段： 包括它们的特征、β细胞来源、参与疾病过程和位点 的演示文稿。 目标 1 包括鉴定源自 β 细胞颗粒的新型肽 老鼠和人类。我们检查从 β 细胞中分离的囊泡，主要关注 克里诺体。 cinosomes和胰岛素致密核心颗粒将在不同阶段被分离 糖尿病发展过程中，他们的肽将被分离并通过复杂的质量检查 采用最先进的仪器和技术进行光谱测定。我们将重点 肽的独特结构或翻译后变化以及它们的整体是否 随着β细胞进展为活动性糖尿病而发生变化。我们还将检查囊泡和 来自最近患病个体的人类胰岛的胞吐产物。 目标 2 将是对通过质量鉴定的新型肽进行免疫学验证 光谱测定法。免疫学和生化检测将用于验证β的能力 细胞衍生的肽与 I-Ag7 结合并在体外和体内激活 T 细胞。 目标 3 将是胰岛和胰岛中 MHC-II 结合肽组的鉴定和验证 次级淋巴器官。我们计划检查从 MHC-II 中洗脱的肽组 由胰岛、周围淋巴结和脾脏获得的吞噬细胞表达的分子 的 NOD 鼠标并按照目标 1/2 描述/验证它们。这是开创性的测试 证明是一种免疫原性肽，即它是体内与MHC-II结合的阳性识别肽 等位基因。