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

评估人体补体系统动态事件的新方法

• 批准号: 8383322
• 负责人: Daniel Ricklin
• 金额: $ 20万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-03 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8383322
• 关键词: Adhesions; Agonist; Anaphylatoxins; Anti-Inflammatory Agents; Anti-inflammatory; Antibiotic Resistance; Area; Basic Science; Binding; Biochemical; Biochemical Process; Biocompatible Materials; Biological Assay; Biology; Biomaterials Research; Biosensor; Blood Platelets; Cell Count; Cell Line; Cell physiology; Cell-Cell Adhesion; Cell-Matrix Junction; Cells; Chemotaxis; Clinical; Clinical Research; Complement; Complement 3a; Complement 5a; Complement Activation; Complex; Data; Deposition; Development; Disease; Drug Delivery Systems; Dyes; Emerging Technologies; Engineering; Equilibrium; Equipment; Event; Functional disorder; G Protein-Coupled Receptor Genes; Grant; Health; Homeostasis; Human; Immune; Immune System Diseases; Immunologic Surveillance; Immunology; Implant; Individual; Infection; Inflammatory; Inflammatory Response; Label; Lead; Light; Literature; Location; Measurement; Measures; Mediating; Mediator of activation protein; Methods; Modeling; Molecular; Monitor; Morphology; Natural Immunity; Operative Surgical Procedures; Pathway interactions; Pattern; Phagocytosis; Physiological; Physiological Processes; Population; Process; Proteins; Reaction; Reagent; Regulation; Reporting; Research; Resolution; Role; Science; Screening procedure; Series; Signal Pathway; Signal Transduction; Specificity; Staphylococcus aureus; Surface; System; Technology; Therapeutic; Therapeutic Intervention; Time; Tissues; Titania; Titanium; Virulence; base; biomaterial compatibility; cell motility; clinically relevant; complement system; drug discovery; immune activation; immunoregulation; improved; inhibitor/antagonist; innovation; insight; instrument; interest; microbial; next generation; novel; novel strategies; pathogen; photonics; prevent; receptor; receptor binding; research study; response; spatiotemporal; titanium dioxide; tool

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. ! ! PUBLIC HEALTH RELEVANCE: Although the human complement system is increasingly recognized as an important mediator in physiological processes and involved in many clinical conditions, the analysis of the dynamic processes that build the base of this versatile involvement are comparatively static and limited. Here we explore a novel and innovative technological platform, which allows the assessment of dynamic biochemical and cellular processes in the context of complement biology. The development of novel assay forms is not only expected to excel immunology research but may also benefit fields ranging from drug discovery to material sciences. ! !

描述（由申请人提供）：越来越多的研究提供证据表明，传统上仅被认为在先天免疫防御中起作用的人类补体系统也参与从稳态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物化学和细胞过程，这些过程经过微调以达到所需的功能。同时，外来表面、微生物入侵者或分子/细胞功能障碍可促进补体介导的炎症事件，并且涉及补体的临床病症的列表正在迅速增长。因此，深入了解定义补体应答过程的分子和细胞事件在基础和临床研究中至关重要。不幸的是，我们用来监测这些关键过程的工具是相当钝的，因为它们往往不能提供必要的动态或时空分辨率。虽然人们早就认识到这一关键差距，但直到最近才出现适当的分析系统。其中，基于光子晶体表面的增强型无标记生物传感器似乎特别有前途，因为它们不仅允许生物分子相互作用的真实的实时测量，而且还能够检测细胞结合甚至细胞活化事件（例如，GPCR介导的信号传导）;这允许使用低细胞数和筛选原代细胞，并且能够动态监测趋化性和细胞迁移。单一平台的多功能性使该技术成为分析复杂 生理网络，但迄今为止仅描述了很少的应用。我们最近获得了这种仪器（SRU BIND（R）SCANNER）的先进使用权，旨在探索、建立和利用这种新兴技术，开发用于补体和免疫研究的新颖和创新的检测方法。在不使用染料或途径限制的情况下测量混合群体内单个细胞的激活事件的能力使得该方法对于解开过敏毒素受体的信号传导模式非常有趣，对于该方法，该领域存在深刻的争议。在目标1中，我们将研究重点放在初级（免疫）细胞上，并监测过敏毒素C3 a和C5 a的结合、活化和趋化性。在第二个目标中，我们使用该平台来阐明外来表面的时空补体激活模式，重点是生物材料应用。级联起始和扩增的具体机制及其抑制将使用临床实验研究。 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，宿主蛋白质和免疫细胞相互作用的测定，并研究其在生物化学和细胞水平上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，不仅会产生新的结果， 补体相关的分析，以回答先天免疫的关键问题，但很可能会转移到使用扫描仪技术的领域，从药物发现到生物材料工程。! ! 公共卫生关系：虽然人类补体系统越来越被认为是生理过程中的重要介质，并参与许多临床条件，建立这种多功能参与的基础的动态过程的分析是相对静态和有限的。在这里，我们探索了一个新颖的和创新的技术平台，它允许在补体生物学的背景下评估动态生化和细胞过程。新型检测形式的发展不仅有望超越免疫学研究，而且还可能使从药物发现到材料科学的领域受益。! !