Comparative studies of complement responses to ICs

补体对 IC 反应的比较研究

• 批准号: 10645672
• 负责人: Gowthami M Arepally
• 金额: $ 24.15万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10645672
• 关键词: Address; Animal Model; Animals; Antibodies; Antigen-Antibody Complex; Autoimmune; Binding; Biological; Cavia; Classical Complement Pathway; Clinical; Cold Hemagglutinin Disease; Comparative Study; Complement; Complement 3 Convertase; Complement 3b; Complement 3c; Complement Activation; Complement Inactivators; Complex; Data; Development; Dinitrophenols; Disease; Erythrocytes; Feedback; Funding; Future; Generations; Genetic; Genetic Variation; Genomics; Goals; Graft Rejection; Hemolysis; Hemolytic-Uremic Syndrome; Heparin; Human; Immune; Immunological Models; Impairment; Keyhole Limpet Hemocyanin; Laboratory Animals; Malignant Neoplasms; Mediating; Medical; Molecular; Monoclonal Antibodies; Mus; Mutation; Oryctolagus cuniculus; PF4 Gene; Pathway interactions; Physiological; Plasma; Proteins; Proteome; Proteomics; Publishing; Rattus; Serum; Study models; System; Testing; Therapeutic; Variant; Whole Blood; complement pathway; complement system; differential expression; hereditary angioneurotic edema; inhibitor; mouse model; paroxysmal nocturnal hemoglobinuria; research and development; response; species difference; success; therapeutic evaluation

The complement systems of humans and laboratory animals (mice and rats) show striking species differences in function, particularly with regards to the classical pathway of complement activation. Indeed, studies since the 1950’s have documented markedly reduced classical pathway function in mice as compared to rats and humans. In preliminary data, we confirm and extend these findings using model immune complexes (ICs) formed by monoclonal antibodies to platelet factor 4 (PF4) and heparin (KKO ICs) or polyclonal rabbit anti-dinitrophenol (DNP) antibodies to DNP conjugated with keyhole limpet hemocyanin (DNP ICs). Specifically, we show: 1) robust C3 activation by KKO and DNP ICs in whole blood and plasma from mice, rats and humans 2) classical pathway requirements for complement activation by KKO ICs in rats and human, but alternative pathway requirements in mice 3) incorporation of alternative pathway proteins into KKO ICs in mice, but not rats or humans 4) comparable requirements for the alternative pathway among four murine strains and 5) differential expression of alternative pathway proteins by proteomics in mice as compared to rats and humans. Based on these preliminary data and published observations, we will test the overall hypothesis that impaired classical pathway activity in mice is counterbalanced by the alternative pathway C3b feedback cycle. To test this hypothesis, we propose the following two aims: 1) Comparative studies of human, mouse, and rat complement systems. In this aim, we will test that hypothesis that genetic variation in murine classical pathway proteins contributes to impaired classical pathway function. We will apply molecular approaches to compare genetic sequences and perform structural homologies of classical pathway proteins in mice v rats and humans. In other studies, we will investigate the plasma proteome of mice, rats, and humans to examine protein/immune complex interactions, co-expression of complement and complement-associated proteins and correlate complement protein levels with functional responses to KKO and DNP ICs. 2) Functional studies of murine and human complement systems. In this aim, we will test the hypothesis that mice utilize the C3b feedback cycle of the alternative pathway as a compensatory response to low classical pathway activity. To test this hypothesis, we will use purified human and mouse complement proteins to demonstrate reduced impaired classical C3 convertase activity and normal/heightened activity of the C3b feedback cycle in mice, examine the effects of interchanging human and mouse complement proteins, and identify potential murine inhibitors of classical pathway activation from mouse serum. Together, these studies are expected to delineate the molecular and functional basis of divergent complement pathway responses in mice and humans. We expect these studies to generate preliminary data for an expanded application to elucidate the molecular and structural divergence of murine complement responses with the long term goal of developing translationally relevant animal models.

人类和实验动物的完成系统（小鼠和大鼠）在功能方面表现出惊人的物种差异，尤其是关于完成激活的经典途径。实际上，与大鼠和人相比，自1950年代以来的研究已经证明了小鼠的经典途径功能显着降低。在初步数据中，我们使用由单克隆抗体4（PF4）和肝素（KKO ICS）或多克隆兔抗二硝基苯酚（DNP）抗体形成的单克隆抗体（PF4）和肝素抗体（DNP）抗体形成的单克隆抗体形成的模型免疫复合物（IC）（ICS）确认并扩展了这些发现，在初步数据中，我们确认并扩展了这些发现，在初步数据中，具体而言，我们表明：1）KKO和DNP IC在全血和血浆中的强大C3激活小鼠，大鼠和人类的血浆2）KKO IC在大鼠和人类中的完成途径的经典途径要求，但在小鼠中的替代途径需求中，但在小鼠中的替代途径要求3）将替代途径的替代蛋白质中的替代蛋白纳入小鼠，但在kko中的替代途径，但不是四个人，但在kko中的替代途径，但四个人是人类的替代途径，人类是人类的4个，而人类的人类是4的4）与大鼠和人相比，蛋白质组学对替代途径蛋白的菌株和5）差异表达。基于这些初步数据和已发表的观察结果，我们将测试总体假设，即小鼠的经典途径活动受损是由替代途径C3B反馈周期抵消的。为了检验这一假设，我们提出以下两个目的：1）人，小鼠和大鼠补体系统的比较研究。在此目标中，我们将测试假说，即鼠经典途径蛋白的遗传变异有助于经典途径功能受损。我们将采用分子方法来比较小鼠和人类中经典途径蛋白的遗传序列并执行结构同源。在其他研究中，我们将研究小鼠，大鼠和人类的血浆蛋白，以检查蛋白/免疫复合物相互作用，补体和补体相关蛋白的共表达，并将补体水平与对KKO和DNP IC的功能反应相关。 2）鼠和人类完成系统的功能研究。在此目标中，我们将检验以下假设：小鼠利用替代途径的C3B反馈周期作为对低经典途径活动的补偿性响应。为了检验这一假设，我们将使用纯化的人和小鼠补体蛋白质来证明小鼠中C3B反馈周期的经典C3转化酶活性减少，并且在小鼠中的正常/增强活性，检查互换人和小鼠补体蛋白的影响，并鉴定出从小鼠血清中经典途径激活的经典途径激活的潜在鼠抑制剂。总之，这些研究有望描绘小鼠和人类的分子完成途径响应的分子和功能基础。我们希望这些研究能够生成扩展应用的初步数据，以阐明鼠完成响应的分子和结构差异，其长期目标是开发转换相关的动物模型。