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.