HRF, an NFkB antagonist targeting multiple pathogenes

HRF，一种针对多种病原体的 NFkB 拮抗剂

• 批准号: 6818642
• 负责人: Malgorzata Simm
• 金额: $ 32.78万
• 依托单位: ST. LUKE'S-ROOSEVELT INST FOR HLTH SCIS
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6818642
• 关键词: Campylobacter; HeLa cells; Salmonella typhimurium; Shigella; antiinflammatory agents; biotechnology; clinical research; cooperative study; dengue; gel mobility shift assay; genetic transcription; host organism interaction; human tissue; immune response; immunoaffinity chromatography; immunomodulators; immunoregulation; immunotherapy; inflammation; mixed tissue /cell culture; molecular cloning; neoplastic cell culture for noncancer research; nuclear factor kappa beta; nucleic acid sequence; polymerase chain reaction; recombinant proteins; secretory protein; site directed mutagenesis

DESCRIPTION (provided by applicant): This application for funds is submitted in response to RFA AI-03-017 and proposes characterization and cloning of a human gene product as a broad spectrum immunotherapeutic for the treatment of infection with multiple Category A and B priority pathogens. Infection by or exposure to some viruses or Gram-negative bacteria induces inflammatory responses that contribute significantly to disease pathogenesis. Common to many such responses is the activation of nuclear factor kB (NFkB) - directed transcription. In our ongoing investigation of HRF, an anti-HIV-1 factor secreted by a transformed human T cell line, we found that it acts through interference with NFkB binding to kB on chromosomal DNA. In recent studies we confirmed that HRF blocks NFkB induction by lipopolysaccharides of several pathogenic bacteria and NFkB activation of inflammatory genes in human cells. Recent HRF peptide sequence findings enabled us to identify a homologous coding sequence, facilitating molecular cloning. This application is directed toward characterization of HRF as an antagonist of NFkB-directed inflammatory responses to several high priority pathogens and to completion of our ongoing program of cloning HRF. We propose to: 1) determine the activity of HRF in human cells in culture in NFkB-directed responses to Shigella, Salmonella, Campylobacter, and Dengue; 2) clone and express recombinant HRF and evaluate its activity in culture for treatment of common inflammatory response to different pathogens; 3) determine the molecular mechanism of action through which HRF antagonizes NFkB-directed transcription. These studies will employ several prokaryotic-eukaryotic coculture systems, molecular cloning, PCR site-directed mutagenesis, and electrophoretic mobility shift assays of protein-protein and protein-DNA complexes. This program will result in the development of a new broad spectrum immunotherapeutic based upon a human protein for the control of NFkB-directed acute inflammatory responses to multiple pathogens.

描述（由申请人提供）：本资金申请是为了回应 RFA AI-03-017 而提交的，并提议对人类基因产品进行表征和克隆，作为治疗多种 A 类和 B 类优先病原体感染的广谱免疫疗法。感染或接触某些病毒或革兰氏阴性细菌会诱发炎症反应，从而对疾病发病机制产生重大影响。许多此类反应的共同点是核因子 kB (NFkB) 定向转录的激活。在我们对 HRF（一种由转化的人类 T 细胞系分泌的抗 HIV-1 因子）进行的持续研究中，我们发现它通过干扰 NFkB 与染色体 DNA 上 kB 的结合发挥作用。在最近的研究中，我们证实 HRF 可以阻断几种病原菌脂多糖诱导的 NFkB 诱导以及人类细胞中炎症基因的 NFkB 激活。最近的 HRF 肽序列发现使我们能够鉴定同源编码序列，从而促进分子克隆。该应用旨在表征 HRF 作为 NFkB 介导的针对几种高优先级病原体的炎症反应的拮抗剂，并完成我们正在进行的克隆 HRF 计划。我们建议：1）确定培养的人类细胞中 HRF 的活性，以 NFkB 为导向，对志贺氏菌、沙门氏菌、弯曲杆菌和登革热作出反应； 2) 克隆和表达重组HRF并评估其在培养物中治疗不同病原体常见炎症反应的活性； 3)确定HRF拮抗NFkB介导的转录的分子作用机制。这些研究将采用多种原核-真核共培养系统、分子克隆、PCR 定点诱变以及蛋白质-蛋白质和蛋白质-DNA 复合物的电泳迁移率变化分析。该计划将开发出一种基于人类蛋白质的新型广谱免疫疗法，用于控制 NFkB 介导的针对多种病原体的急性炎症反应。