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

项目总结 多发性硬化症(MS)发病的确切机制，这种疾病影响着200多万人 世界各地的人和美国的约40万人是未知的，尽管该领域的大多数专家都同意 多发性硬化症涉及一种针对人体中枢神经系统(CNS)的异常免疫中介反应。 具体地说，在中枢神经系统中，免疫系统的组件攻击髓磷脂，这种基于蛋白质的物质 围绕着神经纤维。这种对髓鞘的攻击导致多发性疤痕病变(因此，多发性硬化症) 会导致疾病症状。在免疫系统内，不断有证据表明T细胞是 负责神经纤维脱髓鞘的血液成分。虽然还不清楚是什么 触发T细胞攻击髓鞘，越来越清楚的是，在体内，细胞外ATP可能是一种 控制T细胞功能及其从血液进入中枢神经系统的主要决定因素 参与对髓鞘的破坏。需要创新的分析工具来研究T-T的机制。 细胞的激活、黏附和从血流到中枢神经系统的转移(外渗)，在组织，细胞， 和分子尺度。为了满足这一需求，由多名调查人员组成的调查小组 在流体平台、纳米生物传感器和生物样本方面的专业知识提出了一系列具体目标 这将证明MS患者T细胞的激活是由于糖加工和ATP产生异常所致。 由多发性硬化症红细胞释放。我们建议开发一种创新的微流控平台， 电纺纤维将在受控的体外平台上创造独特的3D环境，以改进监测 T细胞的激活/黏附和渗出通过培养到膜的组织。接下来，我们将聘用 确定MS红细胞具有独特的葡萄糖处理能力的经典细胞检测方法 源于红细胞中葡萄糖转运蛋白的过度表达(GLUT1)。在目标3中，ION 将开发通道修饰的纳米管来对葡萄糖进行定量、纳米级的测定 以及在单个红细胞水平上的ATP转运。这些纳米尺度的传感器将证实某种程度上 AIM 2的“全球”发现(葡萄糖转运蛋白增加和ATP的过量生产)确实在影响 葡萄糖的摄取，并且这种摄取与ATP的释放有关，从而提供了前所未有的代谢洞察力 关于炎症反应的起源。在提案的最终目标中，我们将结合我们的工具和 AIMS 1-3对多发性硬化症患者红细胞药物处理的发现 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细胞活化和黏附的产生和失调。这些目标的成功实现将 不仅提供了对多发性硬化症脱髓鞘影响因素的洞察，而且还为 跨多个领域的细胞分析。

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.