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.

摘要 1型糖尿病(T1D)中β细胞的破坏是由胰岛反应性细胞毒性T细胞介导的 渗入胰岛。细胞外小泡(EV)是一种双层膜结构，直径30- 1000纳米由全身细胞释放到血液中，浓度约为每毫升1010毫升。 它们的蛋白质、dsDNA寡聚体、microRNAs(MiRNAs)、mRNAs和其他分析物的分子含量， 可以通过电动汽车贩运发挥多种职能作用，还可以提供关于 发病部位或组织起源。因此，电动汽车可以作为T1D生物标记物的潜在来源，甚至 提供对胰岛微环境的遗传和功能特征的见解。 我们提出了一个项目，从胰岛衍生的囊泡中寻找T1D生物标记物，结合我们的尖端技术， 微流控纳米颗粒浓缩技术发展到一套多参数分析方法。在目标1中，我们 利用一套独特的大宗和单一EV(SEV)转录和蛋白质组学方法来全面 研究健康供者和T1D患者胰岛细胞及EVS来源的分子含量 从这些培养的细胞中提取。这将产生一组蛋白质和RNA，可以唯一地识别β细胞中的EV， 以及对小岛电动汽车异质性的洞察。在这里，我们也开始解析分子 T1D发病机制所特有的征象。胰岛浸润性CD8+T细胞的研究常常受到 获取捐赠者胰岛组织的途径有限。胰岛浸润性CD8+T细胞将肠道病毒送入血液的事实 STREAM为监测循环中胰岛浸润性T细胞的活动提供了独特的机会 电动汽车。在AIM 2中，我们提出了一种纳米颗粒技术来捕获携带EVS的T细胞受体(TCR) 发行量。我们计划从健康和T1D血清中鉴定这些EV，以确定EV种群 起源于胰岛浸润的CD8+T细胞。监测这些EV人群可以揭示当地的关键 胰腺中的事件，如T细胞激活和细胞毒性。因为我们预计血液丰度会很低 胰岛来源的EVS，在AIM 3中，我们提供了一种支持微流体的解决方案来丰富来自胰腺的EVS β细胞和人血清中的CD8T细胞。这一集成的样品处理工作流程允许 从有限的血清中自动分离胰岛来源的EV，只需最少的动手时间。分析 储存的血清样本将产生一组与T1D发育相关的候选生物标记物，这将 通过一个独立的队列进行验证和改进。重要的是，拟议的研究不仅将提供电动汽车- 基于T1D生物标志物，而且还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拥有EV生物学专业知识的科学家和临床医生组成，T1D 发病机制、免疫学、多组学疾病诊断学、计算生物学和化验发展。