A novel approach for assessing dynamic events in the human complement system

描述(申请人提供)：越来越多的研究提供证据表明，人类补体系统，传统上只被认为是天然免疫防御的角色，也参与了从动态平衡到细胞发育的关键生理过程。这种多功能性的基础是动态的生化和细胞过程，这些过程经过微调以达到所需的功能。与此同时，异物表面、微生物侵入或分子/细胞功能障碍可加剧补体介导的炎症事件，涉及补体的临床情况正在迅速增加。因此，深入了解决定补体反应过程的分子和细胞事件在基础和临床研究中都是必不可少的。不幸的是，我们用来监测这类关键过程的工具相当生硬，因为它们往往不能提供必要的动态或时空分辨率。尽管这一关键差距早已被认识到，但直到最近才出现了适当的分析系统。其中，基于光子晶体表面的增强型无标记生物传感器显得特别有前途，因为它们不仅可以实时测量生物分子之间的相互作用，而且能够在单细胞分辨率下实时检测细胞结合甚至细胞激活事件(例如GPCR介导的信号)；这允许使用较少的细胞数量和筛选原代细胞，并能够动态监测趋化和细胞迁移。单一平台的多功能性使这项技术成为分析复杂环境的理想选择 生理网络，但到目前为止只有很少的应用被描述。我们最近获得了这种仪器(SRU BIND(R)扫描仪)的高级访问权限，旨在探索、建立和利用这一新兴技术，为补体和免疫研究开发新颖和创新的分析方法。由于能够在不使用染料或途径限制的情况下测量混合种群中单个细胞的激活事件，因此该方法对于解开过敏毒素受体的信号模式非常有兴趣，这一点在该领域存在着深刻的争议。在目标1中，我们专注于原代(免疫)细胞的研究，并监测过敏毒素C3a和C5a的结合、激活和趋化作用。在第二个目标中，我们使用该平台来阐明由外源表面激活的时空补体模式，重点是生物材料的应用。级联启动和放大的具体机制及其抑制将在临床上进行研究 相关模型生物材料(钛)。最后，我们将探索金黄色葡萄球菌与表面、宿主蛋白和免疫细胞相互作用的方法，并从生化和细胞水平研究对其各种补体逃避分子的影响。这些研究都将使用现成的试剂和细胞进行，不仅将导致新的 补体相关分析用于回答先天免疫中的关键问题，但很可能将被转移到从药物发现到生物材料工程等领域的扫描仪技术的使用。！！

DESCRIPTION (provided by applicant): An increasing body of research provides evidence that the human complement system, which has traditionally only been attributed a role in innate immune defense, is also involved in key physiological processes ranging from homeostasis to cell development. At the base of this versatility are dynamic biochemical and cellular processes that are finely tuned to arrive at the desired function. At the same time, foreign surfaces, microbial intruders, or molecular/cellular dysfunctions can fuel complement-mediated inflammatory events, and the list of clinical conditions with involvement of complement is rapidly growing. A deep understanding of the molecular and cellular events that define the course of the complement response is therefore essential in both basic and clinical research. Unfortunately, the tools we use for monitoring such key processes are rather blunt, since they often not provide the necessary dynamics or spatiotemporal resolution. Although this critical gap has long been recognized, it is only recently that appropriate analytical systems emerge. Among those, enhanced label-free biosensors based on photonic crystal surfaces appear particularly promising, since they to not only allow real- time measurement of biomolecular interactions, but are also capable of detecting cell binding and even cellular activation events (e.g. GPCR-mediated signaling) in real-time at single cell resolution; this allows for using low cell numbers and screening of primary cells, and enables dynamic monitoring of chemotaxis and cell migration. The versatility of a single platform renders this technology ideal for analyzing complex physiological networks, yet only few application have been described so far. We were recently given advanced access to such an instrument (SRU BIND(R) SCANNER) and aim to explore, establish, and utilize this emerging technology for developing novel and innovative assays for complement and immune research. The ability to measure activation events of individual cells within mixed populations without the use of dyes or pathway restrictions makes the method highly interesting for unraveling the signaling pattern of anaphylatoxin receptors, for which profound controversy exists in the field. In aim 1, we focus our study on primary (immune) cells and monitor binding, activation and chemotaxis by anaphylatoxins C3a and C5a. In a second aim, we use the platform to shed light into the spatiotemporal complement activation pattern by foreign surfaces, with an emphasis on biomaterial applications. The specific mechanisms of cascade initiation and amplification, and the inhibition thereof will be studied using a clinically relevant model biomaterial (titanium). Finally, we will explore assays for the interaction of Staphylococcus aureus with surfaces, host proteins and immune cells, and study the effect on its various complement evasion molecules on a biochemical and cellular level. These studies, which will all be performed using readily available reagents and cells, will not only lead to novel complement-related assays for answering key questions in innate immunity, but will likely be transferrable to the use of the SCANNER technology in fields ranging from drug discovery to biomaterial engineering. ! !