Comparative studies of complement responses to ICs

补体对 IC 反应的比较研究

基本信息

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
项目类别：
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

项目摘要

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（小鼠和大鼠）的补体系统在功能上表现出显着的物种差异，特别是在补体激活的经典途径方面。事实上，自 20 世纪 50 年代以来的研究已经证明，与大鼠相比，小鼠的经典途径功能显着降低。在初步数据中，我们使用血小板因子 4 (PF4) 和肝素 (KKO IC) 的单克隆抗体或多克隆兔形成的模型免疫复合物 (IC) 证实并扩展了这些发现。具体而言，我们展示了与匙孔血蓝蛋白 (DNP IC) 结合的抗二硝基苯酚 (DNP) 抗体：1) KKO 和 DNP IC 在小鼠、大鼠和人类的全血和血浆中强烈激活 C3 2) 经典途径要求。用于大鼠和人类中 KKO IC 的补体激活，但小鼠中需要替代途径 3) 将替代途径蛋白掺入小鼠的 KKO IC 中，但大鼠或人类中则不然 4)四种小鼠品系对替代途径的要求以及 5) 通过蛋白质组学在小鼠中与大鼠和人类相比替代途径蛋白的差异表达基于这些初步数据和已发表的观察结果，我们将测试损害经典途径活性的总体假设。为了检验这一假设，我们提出了以下两个目标： 1) 人类、小鼠和大鼠补体系统的比较研究在这个目标中，我们将检验遗传变异的假设。小鼠经典途径蛋白我们将应用分子方法来比较小鼠与大鼠和人类中经典途径蛋白的结构同源性。在其他研究中，我们将研究小鼠、大鼠和人类的血浆蛋白质组。蛋白质/免疫复合物相互作用、补体和补体相关蛋白质的共表达以及将补体蛋白质水平与 KKO 和 DNP IC 的功能反应相关联 2) 小鼠和人类补体系统的功能研究为此目的，我们将检验该假设。老鼠利用替代途径的 C3b 反馈循环作为对低经典途径活性的补偿反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来证明受损的经典 C3 转化酶活性降低以及 C3b 反馈循环的正常/增强活性。小鼠，检查人类和小鼠补体蛋白互换的影响，并从小鼠血清中鉴定经典途径激活的潜在鼠类抑制剂，这些研究预计将描述小鼠和人类不同补体途径反应的分子和功能基础。期待这些研究为扩展应用生成初步数据，以阐明小鼠补体反应的分子和结构差异，长期目标是开发翻译相关的动物模型。