Analysis of Complement Activation and Regulation by Mass Cytometry

通过质谱流式细胞术分析补体激活和调节

• 批准号: 9235239
• 负责人: DENNIS EMIL HOURCADE
• 金额: $ 20.51万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-15 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9235239
• 关键词: Address; Adjuvant; Age related macular degeneration; Alpha Cell; Anaphylatoxins; Antibodies; Antibody Formation; Antigen-Antibody Complex; Autoimmunity; B-Cell Activation; Binding Proteins; CD4 Positive T Lymphocytes; Cell Death; Cell Differentiation process; Cell Line; Cell Survival; Cells; Clinical; Collection; Complement; Complement 3 Convertase; Complement Activation; Complement Membrane Attack Complex; Computer software; Cytolysis; Cytometry; Dangerousness; Development; Diagnostic; Disease; Disease Progression; Effector Cell; Emerging Technologies; Epithelial Cells; Failure; Feedback; Flow Cytometry; Generations; Genomics; Goals; Heavy Metals; Hemolytic-Uremic Syndrome; Human; Immune system; Individual; Infectious Agent; Inflammatory; Inflammatory Response; Injury; Instruction; Investigation; Isotope Labeling; Isotopes; Lupus; Mass Spectrum Analysis; Measures; Mediating; Membrane; Methods; Nature; Onset of illness; Opsonin; Pathway interactions; Phenotype; Play; Proteins; Reagent; Recruitment Activity; Regulation; Reporter; Resistance; Role; Signal Transduction; Site; Structure of retinal pigment epithelium; Surface; T-Lymphocyte; Therapeutic Agents; Therapeutic Intervention; Time; Tissues; Work; antibody conjugate; base; cell type; design; human disease; immune clearance; inflammatory milieu; membrane assembly; new technology; novel; novel diagnostics; novel therapeutic intervention; pathogen; public health relevance; response; tool

 DESCRIPTION (provided by applicant): Analysis of complement activation and regulation by mass cytometry SUMMARY Complement (C) is a collection of 50-60 soluble and membrane-bound proteins that constitutes the first line of defense of the intravascular space. Complement marks infectious agents for immune clearance or lysis, promotes the local inflammatory response, and facilitates B cell activation and antibody production (i.e. nature's adjuvant) as wel as T cell effector responses. Complement is also a principal cause of tissue damage: Complement-related disease and injury states can be traced to inappropriate complement activation (humoral autoimmunity), inadequate complement regulation (PNH, atypical hemolytic uremic syndrome, age-related macular degeneration), or a failure to effectively clear immune-complexes and/or cell debris (lupus). Therapeutic agents have begun to emerge in the clinical setting to inhibit systemic complement activity. The three complement activation pathways converge with the assembly of C3 convertases on a target surface. C3 convertase generates opsonins that promote target clearance, release anaphylatoxins that activate and recruit inflammatory cells, initiate the assembly of the membrane attack complex (MAC, C5b-9), and, importantly, engage a positive feedback mechanism that amplifies all of these activities. The MAC forms a pore that compromises membrane integrity and commonly leads to pathogen lysis. Host cells are protected from complement activity by surface regulators that inhibit convertase formation, compromise convertase stability and obstruct MAC assembly and function. Nucleated cells respond to complement attack by engaging intracellular pathways that further determine cell survival, cell differentiation or cell death. Mass cytometry (MC) is an emerging technology that provides the unique ability to measure >40 proteins on a single cell basis. We propose to use MC to profile the complement activators, regulators, and intracellular responses as a cell undergoes complement attack. We have selected two vastly different cell types of translational significance for hypothesis driven analyses, retinal pigment epithelial (RPE) cells and T cells, and propose the following Specific Aims: 1. To construct and validate a panel of complement-specific heavy isotope-labeled reporter antibodies; 2. To profile by mass cytometry the responses of a human RPE cell line to sublytic C attack, and compare to those findings to the responses of other epithelial cell types; 3. To profile by mass cytometry the responses of CD4+ T cells undergoing C activation. We anticipate the proposed work will generate a set of novel, unique and critical tools to delineate in unprecedented detail the activation and regulation of complement at the membrane and intracellular levels and to define the impact that intracellular responses play on comlement-dependent disease. Further, the reagents/methods we propose to generate/establish will facilitate the development of a new generation of C-based diagnostics in subsequent investigations and likely drive a paradigm shift in C-based therapeutic intervention.

 描述（通过应用程序提供）：通过质量细胞术摘要补体（C）对完成激活和调节的分析是50-60固体和膜结合蛋白的集合，构成了血管内空间的第一道防线。补体标志着免疫清除或裂解的传染性剂，促进局部炎症反应，并促进B细胞激活和抗体产生（即自然的调整），如T细胞效应子的反应。 Complement is also a principal cause of tissue damage: Complement-related disease and injury states can be traced to inappropriate completion activation (humoral autoimmunity), inadequate completion regulation (PNH, atypical hemolytic uremic syndrome, age-related macular degeneration), or a failure to effectively clear immunocomplexes and/or cell debris (lupus).治疗剂已经开始在临床环境中抑制全身完成活动。三个完成激活途径与C3转换酶在目标表面的组装相聚。 C3转化酶会产生促进靶向清除率，释放激活和募集炎症细胞的过敏毒素，启动膜攻击复合物的组装（MAC，C5B-9）的组装，并具有积极反馈机制，以放大所有这些活动。 MAC形成损害膜完整性并通常导致病原体裂解的孔。宿主细胞受到抑制转化酶形成，损害转换稳定性以及阻塞MAC组装和功能的表面调节剂的填充活性。成核细胞通过参与细胞内途径来反应补体攻击，从而进一步确定细胞存活，细胞分化或细胞死亡。质量细胞仪（MC）是一种新兴技术，可提供独特的能力，以单个细胞为基础测量> 40个蛋白质。我们建议使用MC来介绍补体激活剂，调节剂和细胞内反应，因为细胞会经历补体攻击。我们已经选择了两种截然不同的翻译意义细胞类型，用于假设驱动的分析，视网膜色素上皮（RPE）细胞和T细胞，并提出以下特定目的：1。构建和验证一组补体特异性同位素标记的报告基因抗体； 2。通过质量细胞仪概况人类RPE细胞系对Sublytic C攻击的响应，并将这些发现与其他上皮细胞类型的响应进行比较； 3。通过质量细胞术谱图CD4+ T细胞经历C激活的反应。我们预计拟议的工作将产生一系列新颖，独特和关键的工具，以史无前例的细节描绘出膜和细胞内水平的完成的激活和调节，并确定细胞内反应对注释依赖性疾病的影响。此外，我们建议生成/建立的试剂/方法将支持新一代基于C的诊断，并可能在后续的投资中发展，并可能推动基于C的基于C的治疗干预范围的范式转变。