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

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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. ! !