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

基本信息

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
项目类别：
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

项目摘要

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，浸润胰岛的细胞外囊泡 (EV) 是直径为 30 – 的双层膜结构。 1000 nm 由全身细胞释放到血液中，浓度约为每毫升 1010 nm。它们的蛋白质、dsDNA 寡聚体、microRNA (miRNA)、mRNA 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功能作用，还可以提供有关电动汽车的诊断报告因此，EV 甚至可以作为 T1D 生物标志物的潜在来源。提供对胰岛微环境的遗传和功能特征的见解。我们提出了一项从胰岛来源的囊泡中寻找 T1D 生物标志物的计划，该计划将我们的尖端技术结合起来，在 AIM 1 中，我们将微流控纳米颗粒富集技术转化为一套多参数分析方法。利用一套独特的批量和单 EV (sEV) 转录组学和蛋白质组学方法来全面研究来自健康捐献者和 T1D 患者的胰岛细胞的分子含量以及衍生的 EV 从这些培养的细胞中，这将产生一组可以独特地识别来自 β 细胞的 EV 的蛋白质和 RNA，以及对胰岛 EV 异质性的见解在这里我们也开始解决分子问题。 T1D 发病机制特有的特征对胰岛浸润性 CD8+ T 细胞的研究常常受到阻碍。胰岛浸润 CD8+ T 细胞将 EV 释放到血液中的事实有限。流提供了一个独特的机会来监测胰岛浸润 T 细胞的活动在 AIM 2 中，我们提出了一种纳米颗粒技术来捕获带有 EV 的 T 细胞受体 (TCR)。我们计划对来自健康和 T1D 血清的这些 EV 进行表征，以确定发生这种情况的 EV 群体。监测这些 EV 群体可以揭示关键的局部细胞。正如我们预期的那样，胰腺中的事件如 T 细胞激活和细胞毒性。胰岛衍生的 EV，在 AIM 3 中，我们提供了一种支持微流体的解决方案，以丰富胰岛衍生的 EV 这种集成的样品处理工作流程允许从人血清中提取 β 细胞和浸润性 CD8+ T 细胞。从有限体积的血清中自动分离胰岛来源的 EV，只需最少的人工时间分析。储存的血清样本将产生一组与 T1D 发展相关的候选生物标志物，这将重要的是，拟议的研究不仅会提供 EV- 基于 T1D 生物标志物，还生成对设计和开发具有高价值的数据集 T1D 的抗原特异性免疫疗法。我们的多学科团队由在 EV 生物学、T1D 领域具有专业知识的科学家和研究员组成发病机制、免疫学、多组学疾病诊断、计算生物学和检测开发。