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: 8503594
• 负责人: Daniel Ricklin
• 金额: $ 22.56万
• 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 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8503594
• 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; 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; screening; 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. ! !

描述（由申请人提供）：越来越多的研究提供了证据，表明传统上仅归因于先天免疫防御中的作用的人类补体系统也参与了从体内平衡到细胞开发的关键生理过程。在这种多功能性的基础上是动态的生化和细胞过程，这些过程经过精心调整以达到所需的函数。同时，外国表面，微生物入侵者或分子/细胞功能障碍会促进补体介导的炎症事件，并且临床条件与补体的参与列表正在迅速增长。因此，对定义补体反应过程的分子和细胞事件的深入了解在基础研究和临床研究中都是必不可少的。不幸的是，我们用于监视此类关键过程的工具相当钝，因为它们通常不提供必要的动态或时空分辨率。尽管长期以来已经认识到这个关键差距，但直到最近才出现适当的分析系统。在其中，基于光子晶体表面的增强的无标记生物传感器显得特别有前途，因为它们不仅允许对生物分子相互作用进行实时测量，而且还可以在单​​细胞分辨率下实时地实时地在实时的细胞分辨率下，能够检测细胞结合甚至均匀的细胞活化事件（例如GPCR介导的信号传导）；这允许使用低细胞数量和筛选原始细胞，并可以动态监测趋化性和细胞迁移。单个平台的多功能性使得这项技术非常适合分析复合物 到目前为止，生理网络，但仅描述了很少的应用。最近，我们获得了该工具（SRU BIND（R）扫描仪）的高级访问权限，并旨在探索，建立和利用这项新兴技术来开发新颖和创新的测定法，以进行补充和免疫研究。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激活事件的能力使该方法非常有趣，可以揭示过敏毒素受体的信号传导模式，在该领域中存在深刻的争议。在AIM 1中，我们将研究集中在原发性（免疫）细胞上，并通过过敏毒素C3A和C5A监测结合，激活和趋化性。在第二个目标中，我们使用该平台将外国表面的时空补体激活模式阐明，重点是生物材料应用。级联启动和扩增的特定机制以及抑制作用将使用临床研究 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的相互作用的测定，并研究其对生化和细胞水平上各种补体逃避分子的影响。这些研究将使用随时可用的试剂和细胞进行，不仅会导致新颖 与先天免疫中的关键问题相关的补充测定法，但很可能可以转移到从药物发现到生物材料工程等领域中使用扫描仪技术。呢呢