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: 9689765
• 负责人: EMIL Raphael UNANUE
• 金额: $ 40.47万
• 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/9689765
• 关键词: Alleles; Antigens; 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; Lymphoid Tissue; Mass Spectrum Analysis; Measures; Mouse Strains; Mus; Non obese; Organ; Pathogenesis; Peptides; Peripheral; Phagocytes; Population; Prediabetes syndrome; Process; Proteins; Reaction; Sampling; Seminal; Site; Source; Spleen; 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

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.

项目摘要 我们提出了一个合作项目，涉及有经验的免疫学家在自身免疫性 糖尿病专家、质谱专家和内分泌专家-糖尿病专家。我们的共同努力 为了特异性地鉴定来自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， 糖尿病易感小鼠，这应该导致有针对性的评估人类样本。最终 目的是鉴定形成自身反应性MHC-II应答底物的肽， 启动并延续这一过程。重要的是要确定相关的致糖尿病抗原， 从糖尿病前期到初始进展的自身免疫反应的不同阶段：它将 包括其特征、β细胞来源、参与疾病过程和部位 的介绍。 目的1包括鉴定来源于人的β细胞颗粒的新肽， 老鼠和人类都是。我们研究了从β细胞分离的囊泡，主要关注 毛体在不同的阶段，将分离出核小体和胰岛素致密核心颗粒 糖尿病的发展，他们的肽将被分离和检查复杂的质量 使用最先进的仪器和技术进行光谱分析。我们将重点放在 肽中独特的结构或翻译后变化，以及它们是否 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， 来自最近患病个体的人胰岛的胞吐产物。 目标2将是通过质量鉴定的新肽的免疫学验证 光谱法免疫学和生物化学测定将用于验证β 细胞衍生肽结合I-Ag 7并在体外和体内活化T细胞。 目标3将是在胰岛中鉴定和验证MHC-II结合肽组， 次级淋巴器官我们计划检测从MHC-II洗脱的肽组 从胰岛、外周淋巴结和脾脏获得的吞噬细胞表达的分子 NOD小鼠，并按照目标1/2对其进行表征/验证。这是一个开创性的测试， 证明一种免疫原性肽，也就是说，它在体内与MHC-II结合是阳性鉴定 等位基因。