Micro-nanotechnologies for the analysis of islet-derived extracellular vesicles implicated in Type 1 Diabetes

用于分析与 1 型糖尿病有关的胰岛来源的细胞外囊泡的微纳米技术

• 批准号: 10706514
• 负责人: Alphonsus Ng
• 金额: $ 71.47万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-19 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10706514
• 关键词: Antigens; Attenuated; Autoantibodies; Autoantigens; Autoimmune; Back; Bar Codes; Beta Cell; Biological Markers; Biology; Blood; Blood Cells; Body Fluids; CD8-Positive T-Lymphocytes; Cell Communication; Cell Culture Techniques; Cell Line; Cells; Characteristics; Circulation; Clinic; Clinical; Collaborations; Communities; Computational Biology; Couples; Cultured Cells; Data; Data Set; Databases; Development; Diagnostic; Diameter; Disease; Disease Progression; Early Diagnosis; Event; Fingerprint; Genetic; Goals; Health; Heterogeneity; Human; Hyperglycemia; Image; Immune; Immunology; Immunotherapy; Individual; Infiltration; Insulin-Dependent Diabetes Mellitus; Islet Cell; Islets of Langerhans; Knowledge acquisition; Learning; Libraries; Light; Lipids; Measures; Mediating; Membrane; Messenger RNA; Methodology; Methods; MicroRNAs; Microfluidics; Molecular; Molecular Profiling; Monitor; Nanotechnology; Nucleic Acids; Organ; Pancreas; Pancreatic Diseases; Pathologic; Patients; Performance; Play; Population; Proliferating; Proteins; Proteome; Proteomics; Public Domains; RNA; Reporting; Research Institute; Research Personnel; Resolution; Risk; Role; Sampling; Scientist; Serum; Set protein; Site; Somatotype; Source; Specimen; Stream; Structure of beta Cell of islet; Systems Biology; T cell infiltration; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Technology; Testing; Time; Tissues; Transcript; Universities; Utah; Vesicle; Work; analytical method; assay development; autoreactive T cell; biomarker identification; candidate marker; cell type; cohort; cytotoxic CD8 T cells; cytotoxicity; design; diabetes pathogenesis; digital; disease diagnostic; ds-DNA; early detection biomarkers; extracellular vesicles; immunoregulation; innovation; insight; islet; liquid biopsy; molecular marker; multidisciplinary; multiple omics; nanoparticle; nanoscale; programs; tool; trafficking; transcriptome; transcriptomics

ABSTRACT The destruction of β cells in Type 1 Diabetes (T1D) is mediated by islet-reactive cytotoxic CD8+ T cells that infiltrate the pancreatic islet. Extracellular Vesicles (EVs) are bilayer membrane structures of diameters 30 – 1000 nm released into the blood by cells throughout the body, at concentrations on the order of 1010 per ml. Their molecular content of proteins, dsDNA oligomers, microRNAs (miRNAs), mRNAs, and other analytes, may play multiple functional roles via EV trafficking, and may also provide a diagnostic report back on the disease site or tissue of origin. As such, EVs can serve as potential sources of T1D biomarkers, perhaps even providing insights into the genetic and functional characteristics of the pancreatic islet microenvironment. We propose a program pursuing T1D biomarkers from islet-derived vesicles that couples our cutting-edge, microfluidic nanoparticle enrichment technology to a suite of multiparameter analytical methods. In AIM 1, we utilize a distinct set of bulk and single-EV (sEV) transcriptomic and proteomic methods to comprehensively investigate the molecular contents of islet cells from healthy donors and individuals with T1D, and EVs derived from those cultured cells. This will yield a set of proteins and RNAs that can uniquely identify EVs from β cells, as well as insights on the heterogeneity of the islet EVs. Here we also begin to resolve the molecular signatures that are unique to T1D pathogenesis. The study of islet-infiltrating CD8+ T cells is often impeded by the limited access to donor islet tissues. The fact that islet-infiltrating CD8+ T cells shed EVs into the blood stream provides a unique opportunity for monitoring the activities of the islet-infiltrating T cells with circulating EVs. In AIM 2, we propose a nanoparticle technology to capture T-cell receptor (TCR) bearing EVs in circulation. We plan to characterize these EVs from healthy and T1D sera to determine the EV populations that arise from pancreatic islet infiltrating CD8+ T cells. Monitoring these EV populations can reveal key local events in the pancreas such as T cell activation and cytotoxicity. As we anticipate a low blood abundance of islet-derived EVs, in AIM 3, we provide a microfluidics-enabled solution to enrich EVs derived from pancreatic β cells and infiltrating CD8+ T cells from human sera. This integrated sample processing workflow allows automatic isolation of islet-derived EVs from limited volumes of serum, with minimal hands on time. Analysis of banked serum specimens will yield a set of candidate biomarkers associated with T1D development, which will be validated and refined with an independent cohort. Importantly, the proposed study will not only provide EV- based T1D biomarkers, but also generate datasets that are of high value to the design and development of antigen-specific immunotherapies for T1D. Our multidisciplinary team is comprised of scientists and clinicians with expertise in EV biology, T1D pathogenesis, immunology, multi-omic disease diagnostics, computational biology, and assay development.

抽象的 1型糖尿病（T1D）中β细胞的破坏是由胰岛反应性细胞毒性CD8+ T细胞介导的 渗透胰岛。细胞外囊泡（EV）是直径30 - 的双层膜结构 1000 nm在整个人体中被细胞释放到血液中，浓度为每毫升1010。 它们的蛋白质，dsDNA低聚物，microRNA（miRNA），mRNA和其他分析物的分子含量， 可以通过EV运输扮演多个功能角色，还可以提供有关诊断报告 疾病部位或起源组织。因此，电动汽车可以作为T1D生物标志物的潜在来源，甚至可能 提供有关胰岛微环境的遗传和功能特征的见解。 我们提出了一个计划，从胰岛衍生的蔬菜中追求T1D生物标志物，该计划将我们的尖端融合在一起， 微流体纳米颗粒富集技术用于一组多参数分析方法。在AIM 1中，我们 利用一组不同的批量和单eV（SEV）转录组和蛋白质组学方法全面 研究来自健康供体和具有T1D的个体的胰岛细胞的分子含量，并得出EVS 来自那些培养的细胞。这将产生一组蛋白质和RNA，这些蛋白质和RNA可以从β细胞中唯一识别EV， 以及对胰岛电动汽车异质性的见解。在这里，我们也开始解决分子 T1D发病机理独有的特征。胰岛浸入CD8+ T细胞的研究通常受到 有限的供体胰岛组织的机会。胰岛浸入CD8+ T细胞将EV散发到血液中的事实 流提供了一个独特的机会，可以监视渗透胰岛渗透的T细胞的活动 电动汽车。在AIM 2中，我们提出了一种纳米颗粒技术来捕获T细胞受体（TCR）轴承EV 循环。我们计划从健康和T1D血清中表征这些电动汽车，以确定EV种群 胰岛浸润CD8+ T细胞产生。监视这些电动汽车群体可以揭示关键的本地 胰腺中的事件，例如T细胞活化和细胞毒性。正如我们预计血液丰富的 胰岛来源的电动汽车，在AIM 3中，我们提供了一个支持微芯片的解决方案，以丰富从胰腺中得出的电动汽车 β细胞和人血清浸润CD8+ T细胞。这个集成的样本处理工作流允许 从有限体积的血清中自动隔离胰岛来源的电动汽车，并且可以按时隔离。分析 银行的血清标本将产生与T1D开发相关的一组候选生物标志物，这将 通过独立队列进行验证和完善。重要的是，拟议的研究不仅会提供 基于T1D生物标志物，但也生成对设计和开发具有很高价值的数据集 T1D的抗原特异性免疫疗法。 我们的多学科团队由具有EV生物学专业知识的科学家和临床医生组成，T1D 发病机理，免疫学，多词性疾病诊断，计算生物学和测定开发。