CHLAMYDIAL PNEUMONITIS IN HSP 60-IMMUNE MANIPULATED MICE

HSP 60 免疫小鼠中的衣原体肺炎

• 批准号: 2376425
• 负责人: BERNHARD KALTENBOECK
• 金额: $ 9.06万
• 依托单位: AUBURN UNIVERSITY AT AUBURN
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2376425
• 关键词: Chlamydiaceae; bacterial vaccines; chlamydial disease; delayed hypersensitivity; disease /disorder model; genetic strain; genetically modified animals; helper T lymphocyte; homozygote; humoral immunity; immune tolerance /unresponsiveness; immunization; immunocytochemistry; immunologic assay /test; inbreeding; interleukin 10; interleukin 12; laboratory mouse; polymerase chain reaction; recombinant proteins; respiratory disorder; stress proteins; western blottings

The intracellular bacterium, Chlamydia (C.) pneumoniae, has emerged over the last decade as a major human respiratory pathogen. C. pneumoniae infection also has been associated with atheromatous plaques in coronary heart disease. Much of the tissue injury in chlamydial disease presumably results from a deleterious immune response to the chlamydial protein hsp60 of the 60 kDa family of heat shock proteins. We propose to define the pathogenetic role of chlamydial hsp60 in a mouse model of C. pneumoniae pneumonitis. We will generate mice immunotolerant to hsp60 with two novel approaches, and expect these animals to be, in part, resistant to immunopathology induced by infection with whole organisms. We also will prime mice to T-Helper-cell 1 (TH1)- or TH2- dominated immunity against hsp60 by DNA-vaccination with plasmid vectors simultaneously expressing chlamydial hsp60 and murine interleukin-12 (IL- 12) or IL- 10, and anticipate differentially modulated immunopathology. These experiments will help to understand the regulation of immunopathological versus protective responses to chlamydial infections, and thus aid in the design of effective chlamydial vaccines, and of strategies to ameliorate the sequelae of these infections. The specific aims of the proposed studies are: (1) Generate a homozygous, inbred mouse line immunotolerant to C. pneumoniae hsp60 through transgenic thymic expression of hsp6O during embryonic development (hsp60+ mouse). (2) Induce peripheral immunological tolerance or hyporesponsiveness to C. pneumoniae hsp60 in mice through oral administration of hsp60 covalently conjugated to cholera toxin B subunit (hsp60-orally tolerized mouse). (3) Induce immunological hyperresponsiveness to C. pneumoniae hsp60 in mice through intradermal injection of free plasmid DNA capable of coexpressing hsp60 with murine IL-12 or IL-10 in epithelial cells (IL-12 or IL-10 hsp60 DNA-vaccinated mouse). (4) Characterize the immune response to C. pneumoniae hsp60 and (5) analyze and compare disease pathogenesis and immune responses to experimental C. pneumoniae pneumonitis in unmanipulated mice, in hsp60+ mice, in hsp60-orally tolerized mice, and in IL-12 and IL- 10 hsp6O DNA-vaccinated mice. Humoral immunity will be assessed by enzyme immunoassays and immunoblot assays, and cell mediated immune responses will be analyzed by delayed type hypersensitivity and lymphoproliferation tests. Tissue sections of lung will be scored for seventy of lesions. Distribution of chlamydiae will be evaluated by cell culture isolation and in situ polymerase chain reaction (PCR). Inflammatory cell types, and TH1, TH2, and fibrogenic cytokine expression will be determined by immunohistochemical staining and reverse transcriptase in situ PCR.

胞内细菌衣原体（Chlamydia（C.）肺炎，已经出现在 在过去十年中是人类呼吸道的主要病原体。C.肺炎 感染也与冠状动脉粥样硬化斑块有关， 心脏病 衣原体病中的大部分组织损伤 由对衣原体蛋白HSP 60的有害免疫应答引起 60 kDa热休克蛋白家族的成员。 我们建议确定衣原体热休克蛋白60在小鼠中的致病作用， 模型C.肺炎我们将产生小鼠免疫耐受 用两种新的方法，并期望这些动物， 部分，抵抗由整体感染引起的免疫病理学 有机体我们还将使小鼠接种T辅助细胞1（TH 1）-或TH 2-。 质粒DNA疫苗诱导的HSP 60免疫应答 同时表达衣原体hsp 60和鼠白细胞介素-12（IL-12）， 12)或IL- 10，并预测差异调节的免疫病理学。 这些实验将有助于理解 对衣原体感染的免疫病理学反应与保护性反应， 从而有助于设计有效的衣原体疫苗， 改善这些感染后遗症的策略。 所提出的研究的具体目标是：（1）产生纯合的， 免疫耐受C. pneumoniae hsp 60通过转基因 胚胎发育过程中hsp 60的胸腺表达（hsp 60+小鼠）。 (2)诱导对C. 肺炎热休克蛋白60在小鼠体内的表达 与霍乱毒素B亚单位偶联（hsp 60-口服耐受小鼠）。 （三） 诱导对C.小鼠肺炎热休克蛋白60 通过皮内注射能够共表达 hsp 60与上皮细胞中的鼠IL-12或IL-10（IL-12或IL-10 hsp 60 DNA接种的小鼠）。(4)描述对C. 肺炎热休克蛋白60;（5）分析比较疾病的发病机制， 实验C的免疫应答肺炎 未操作小鼠、hsp 60+小鼠、hsp 60-口服耐受小鼠和 IL-12和IL- 10 hsp 60 DNA接种的小鼠。体液免疫将是 通过酶免疫测定和免疫印迹测定以及细胞介导的 通过迟发型超敏反应分析免疫应答， 淋巴细胞增殖试验。肺组织切片将进行评分 70例病变。将按细胞评价衣原体分布 培养分离和原位聚合酶链反应（PCR）。 炎性细胞类型和TH 1、TH 2和纤维化细胞因子表达 将通过免疫组织化学染色和反向 转录酶原位PCR