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CHLAMYDIAL PNEUMONITIS IN HSP 60-IMMUNE MANIPULATED MICE

HSP 60 免疫小鼠中的衣原体肺炎

基本信息

批准号：
6163704
负责人：
BERNHARD KALTENBOECK
金额：
$ 10.71万
依托单位：
AUBURN UNIVERSITY AT AUBURN
依托单位国家：
美国
项目类别：
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

项目摘要

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，已经出现了在过去的十年里，它是人类呼吸道的主要病原体。肺炎衣原体感染也与冠状动脉中的动脉粥样斑块有关心脏病。衣原体病的大部分组织损伤可能是对衣原体蛋白Hsp60的有害免疫反应所致属于60 kDa的热休克蛋白家族。我们建议确定衣原体Hsp60在小鼠中的致病作用肺炎衣原体肺炎模型。我们将产生免疫耐受小鼠有两种新的方法，预计这些动物将在部分，对整体感染诱导的免疫病理产生抵抗有机体。我们还将小鼠准备到T辅助细胞1(TH1)-或TH2- 质粒载体DNA疫苗对HSP60的优势免疫衣原体Hsp60和小鼠白介素12的同时表达 12)或IL-10，并预测差异调节的免疫病理。这些实验将有助于理解生物多样性的调节。对衣原体感染的免疫病理和保护性反应，从而帮助设计有效的衣原体疫苗，并改善这些感染的后遗症的策略。拟议研究的具体目标是：(1)产生纯合子，转基因近交系小鼠对肺炎链球菌Hsp60的免疫耐受 HSP60+小鼠胚胎发育过程中胸腺hsp60的表达 (2)诱导外周免疫耐受或对C. 小鼠口服Hsp60共价感染肺炎热休克蛋白60的研究结合霍乱毒素B亚单位(HSP60口服耐受小鼠)。(3) 诱导小鼠对肺炎链球菌Hsp60的免疫高反应性通过皮内注射可共表达的游离质粒DNA HSP60与小鼠IL-12或IL-10在上皮细胞(IL-12或IL-10)的结合 DNA疫苗接种小鼠)。(4)对C. 肺炎热休克蛋白60和(5)病机分析比较实验性肺炎衣原体肺炎的免疫应答未处理的小鼠，Hsp60+小鼠，口服Hsp60耐受小鼠，以及 IL-12和IL-10 hsp60DNA疫苗免疫小鼠。体液免疫将是用酶免疫测定法和免疫印迹测定法以及细胞介导法进行评估免疫反应将通过迟发型超敏反应和淋巴增殖试验。肺组织切片将被计分为 70%的病变。衣原体的分布将由细胞进行评估培养分离和原位聚合酶链式反应(PCR)。炎性细胞类型与TH1、TH2和纤维化细胞因子的表达将通过免疫组织化学染色和逆转转录酶原位聚合酶链式反应。