A multiplexed micro scale assay for real time analysis of pediatric immune cell function

用于实时分析儿科免疫细胞功能的多重微量测定

基本信息

批准号：
10132990
负责人：
David J Beebe
金额：
$ 36.31万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10132990
关键词：
Adoption Allergic Allergic Disease Asthma Biological Biological Assay Biological Markers Birth Blood Blood Volume Blood flow Blood specimen CD28 gene CD3 Antigens CXCL12 gene Cell Separation Cell physiology Cells Cellular Assay Cellular Metabolic Process Chemotaxis Child Childhood Clinical Research Cohort Studies Collection Cytolysis Data Development Devices Disease Environmental Exposure Environmental Risk Factor Enzyme-Linked Immunosorbent Assay Equipment Extrinsic asthma Farming environment Funding Gene Expression Gene Expression Profiling Genetic Risk Health Heel Heterogeneity Hypersensitivity Immune Immune system Infant Interleukin-13 Interleukin-4 Interleukin-5 Label Life Measurement Measures Metabolic Methods Microfluidics Microscopy Modeling Molecular Optics P-Selectin Pain Painless Pediatric Research Phagocytosis Phenotype Population Population Density Production RNA Reactive Oxygen Species Reagent Risk Running Sampling Specificity Surface System T cell differentiation T-Lymphocyte Technology Testing Time United States National Institutes of Health Validation Venipunctures Whole Blood Wisconsin assay development base cellular imaging cohort cytokine experience extracellular immune function immunological status innovation magnetic beads metabolic imaging microfluidic technology milliliter miniaturize multiple omics multiplex assay neutrophil next generation sequencing novel pediatric patients response sample collection

项目摘要

Abstract Restricted amounts of biological samples that can be collected from pediatric patients for the study of immune function are often insufﬁcient for conventional assays. Development of novel, multi-parameter sample sparing assays is necessary for advancing the fundamental understanding of the immune system. In this proposal, we outline three aims which detail the addition and integration of new biological readouts to an existing microﬂuidic technology and the use of this expanded platform to investigate the mechanisms for how allergies and asthma develop in children. The kit-on-a-lid- assay (KOALA) is an easy to use self-contained microﬂuidic platform which is capable of isolating neutrophils from whole blood, and assessing their chemotactic abilities. In Aim 1 we propose to adapt and validate assays for measuring neutrophil ROS production, NETosis, phagocytosis, cytokine production, and gene expression (RT- qPCR) into KOALA. We also plan to modify the cell capture speciﬁcity of KOALA to allow us to isolate T cells from whole blood and assess their cytokine production and gene expression (RT-qPCR). The last component of Aim 1 is to test and compare a new, less invasive blood draw method called Hemolink™ against traditional blood draw methods in terms of immune cell phenotype. In Aim 2 we propose to use a single-cell real-time non-invasive label-free microscopy method called optical metabolic imaging (OMI) to identify single-cell heterogeneity in activation within T cell and neutrophil populations. OMI is compatible with the other endpoints and can be run simultaneously. In Aim 3 will apply our multiplexed KOALA assays to blood samples from a cohort of children with allergic disease and matched healthy controls from the WISC cohort (an ongoing study at UW that includes farm and non-farm children). We will compare our neutrophil and T cell function and phenotype results from these children with the extensive measurements being done as part of the WISC cohort study. In Aim 3 we will also extend these analyses to a group of infants from the CREW study (a new study at UW that aims to identify early life genetic and environmental risk factors for allergic diseases and asthma) using the HemoLink™ device to draw blood with minimal discomfort and proﬁle type 2 T cell function (T cells differentiated to secrete IL-4, IL-5, IL-13 and other type 2 cytokines) using KOALA. If successful in our aims, we will have created a miniaturized, highly multiplexed, self-contained microﬂuidic platform that is capable of measuring biological markers related to immune status and function. This system will have then been validated in pediatric subjects and ready for transition into clinical research.

抽象的可以从儿科患者身上采集的生物样本数量有限对于免疫功能的研究来说，常规测定通常是不够的。开发新颖的多参数样品保留测定方法对于在这项提案中，我们增进了对免疫系统的基本了解。概述了三个目标，详细说明了新生物读数的添加和整合现有的微流体技术以及使用这个扩展平台进行研究儿童过敏和哮喘的发生机制。分析（KOALA）是一个易于使用的独立微流控平台，能够从全血中分离中性粒细胞并评估其趋化能力。目标 1 我们建议调整和验证中性粒细胞 ROS 测量方法产生、NETosis、吞噬作用、细胞因子产生和基因表达（RT- qPCR）到 KOALA 中，我们还计划将 KOALA 的细胞捕获特异性修改为允许我们从全血中分离 T 细胞并评估其细胞因子的产生和目标 1 的最后一个组成部分是测试和比较基因表达。与传统抽血相比，称为 Hemolink™ 的新型微创抽血方法在目标 2 中，我们建议使用单细胞。称为光学代谢成像的实时非侵入性无标记显微镜方法 (OMI) 识别 T 细胞和中性粒细胞内激活的单细胞异质性 OMI 与其他端点兼容并且可以同时运行。在目标 3 中，我们将采用我们的多重 KOALA 检测方法来检测来自一组患者的血液样本。患有过敏性疾病的儿童和来自 WISC 队列的匹配健康对照（华盛顿大学正在进行的研究，包括农场和非农场儿童）。这些儿童的中性粒细胞和 T 细胞功能和表型结果作为 WISC 队列研究的一部分，我们将进行广泛的测量。还将这些分析扩展到 CREW 研究中的一组婴儿（一项新研究华盛顿大学旨在确定生命早期遗传和环境过敏风险因素疾病和哮喘）使用 HemoLink™ 设备以最少的速度抽血不适和 2 型 T 细胞功能（分化为分泌 IL-4、IL-5、 IL-13 和其他 2 型细胞因子）如果使用 KOALA 成功实现我们的目标，我们将拥有创建了一个小型化、高度复用、独立的微流体平台能够测量与免疫状态和功能相关的生物标志物。然后系统将在儿科科目中得到验证，并准备好过渡到临床研究。