From single molecule to microfluidic 3D tissue platforms: novel multiscale tools to investigate hyper-stimulated immune cells in the circulation

项目摘要 多发性硬化症（MS）发病的确切机制，这种疾病影响超过200万人 全世界约有40万人，美国约有40万人，尽管该领域的大多数专家都认为， MS涉及针对身体中枢神经系统（CNS）的异常免疫介导的应答。 具体来说，在中枢神经系统中，免疫系统的成分攻击髓鞘，髓鞘是一种基于蛋白质的物质， 包围着神经纤维这种对髓鞘的攻击导致多发性瘢痕病变（因此，多发性硬化症）， 导致疾病症状。在免疫系统中，越来越多的证据表明，T细胞是免疫系统中最重要的细胞。 负责神经纤维脱髓鞘的血流成分。虽然不清楚是什么 触发T细胞攻击髓磷脂，越来越清楚的是，在体内，细胞外ATP可能是一种 是控制T细胞功能及其从血流到中枢神经系统的主要决定因素， 参与髓鞘的损伤。需要创新的分析工具来研究T- 在组织，细胞， 和分子尺度。为满足这一需要，设立了一个由多名调查员组成的调查小组， 在流体平台，纳米生物传感器和生物样品方面的专业知识，提出了一系列具体的目标 这将证明MS中的T细胞活化是由于异常的葡萄糖加工和ATP产生， 由MS红细胞释放。我们建议开发一种创新的微流体平台， 电纺纤维将在受控的体外平台上创建独特的3D环境，以改善监测 T细胞活化/粘附和外渗穿过培养到膜上的组织。接下来，我们将采用 经典的细胞测定方法，以确定MS红细胞具有独特的葡萄糖加工能力 源于红细胞中葡萄糖转运蛋白（GLUT 1）的过度表达。在aim 3中， 将开发通道改性的纳米移液管，用于对葡萄糖进行定量的纳米级测定 和单个红细胞水平的ATP转运。这些纳米级传感器将证实， 目标2中的“全球”发现（葡萄糖转运蛋白增加和ATP过度产生）确实影响了 葡萄糖摄取，并且摄取与ATP释放有关，从而提供前所未有的代谢见解 炎症反应的起源在提案的最终目标中，我们将联合收割机与 目标1-3的发现，对从MS患者获得的红细胞进行药物操作， 对照以确定MS红细胞中异常的葡萄糖转运是否是细胞外ATP的来源 T细胞活化和粘附的产生和失调。这些目标的成功实现将 不仅提供了对影响MS脱髓鞘的因素的深入了解，而且还提供了平台技术， 多领域的细胞分析。

PROJECT SUMMARY The exact mechanism underlying the onset of Multiple Sclerosis (MS), a disease that affects over 2 million people worldwide and ~ 400,000 in the United States, is unknown although most experts in the field agree that MS involves an abnormal immune-mediated response against the body’s central nervous system (CNS). Specifically, in the CNS, components of the immune system attack myelin, the protein-based substance that surrounds nerve fiber. This attack on myelin results in multiple scar lesions (hence, Multiple Sclerosis) that lead to disease symptoms. Within the immune system, evidence continues to mount that T-cells are the bloodstream components responsible for the demyelination of the nerve fiber. While it is not clear what triggers the T-cells to attack myelin, it is becoming increasingly clear that in vivo, extracellular ATP may be a major determinant in controlling T-cell function and their passage from the bloodstream to the CNS where they participate in the damage to myelin. Innovative analytical tools are needed to investigate the mechanism of T- cell activation, adhesion, and transfer (extravasation) from the bloodstream to the CNS, at the tissue, cellular, and molecular scales. To meet this need, an investigative team consisting of multiple investigators with expertise in fluidic platforms, nanoscale biosensors, and biological samples, proposes a set of specific aims that will prove that the T-cell activation in MS is due to abnormal glucose processing and ATP production and release by the MS red blood cell. We propose that the development of an innovative microfluidic platform with electrospun fibers will create a unique 3D-environment on a controlled in vitro platform for improved monitoring of T-cell activation/adhesion and extravasation across a tissue cultured to a membrane. Next, we will employ classical cell assay methods to establish that the MS red blood cell has unique glucose processing capabilities stemming from an overexpression of the glucose transporter found in the red blood cell (GLUT1). In aim 3, ion channel modified nanopipettes will be developed to perform quantitative, nanoscale determinations on glucose and ATP transport at the single red blood cell level. These nanoscale sensors will confirm that the somewhat “global” findings in aim 2 (increased glucose transporter and overproduction of ATP) are indeed affecting glucose uptake, and that the uptake is linked to ATP release, thus providing unprecedented metabolic insight on the genesis of inflammatory response. In the final aim of the proposal, we will combine our tools and discoveries from aims 1-3 with pharmaceutical manipulation of red blood cells obtained from MS patients and controls to determine if abnormal glucose transport in the MS red blood cell is the origin of extracellular ATP production and dysregulation of T-cell activation and adhesion. The successful completion of these aims will not only provide insight into factors affecting demyelination in MS, but also provide platform technologies for cellular analyses across multiple fields.