CHRONIC MYOCARDITIS IN A COXSACKIEVIRUS MURINE MODEL

柯萨奇病毒鼠模型中的慢性心肌炎

基本信息

批准号：
3365093
负责人：
Charles J Gauntt
金额：
$ 20.46万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-01-01 至 1995-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3365093
关键词：
Coxsackievirus autoantibody cell type chronic disease /disorder clone cells disease /disorder model fibroblasts genome immunopathology in situ hybridization laboratory mouse monoclonal antibody myocarditis myocardium disorder polymerase chain reaction

项目摘要

The goals of this proposal are to identify mechanisms by which a small nonenveloped RNA genome virus induces severe focal inflammation in heart tissues of mice, as a model of human disease myocarditis. Most human patients with chronic myocarditis produce autoantibodies to antigens on cardiac tissue. In a murine model of coxsackievirus B3 (CVB3)-induced chronic myocarditis, autoantibodies are also produced against cardiac cell antigens. Administration of murine sera containing CVB3-neutralizing antibodies into CVB3-inoculated mice at 3 days post-inoculation exacerbates myocarditis, suggesting the presence of pathologic antibodies. Preliminary studies of neutralizing monoclonal antibodies (mAbs) against CVB3 demonstrate shared epitopes between CVB3 particles and cultured murine cardiac fibroblasts, as assessed by antibody binding studies, complement- mediated lysis of fibroblasts and stimulation of synthesis of a macrophage chemoattractant by fibroblasts. One mAb induces myocarditis in normal mice. Mechanisms by which eight mAbs could contribute to pathogenesis of myocarditis in mice will be identified. Induction of myocarditis by mAB will be confirmed. The capacity of mAbs for participating in complement- or killer cell-mediated lysis of murine cardiac fibroblasts will be quantitatively assessed. mAb-enhanced production of soluble factors from cardiac fibroblasts will be quantified. The viral polypeptide(s) to which the eight mAbs bind will be identified. Several laboratories have shown that enteroviral genomes are present in heart tissues of 15-25% of patients with myocarditis when infectious virus cannot be detected. CVB3 murine models of chronic myocarditis will be used to determine whether viral genomes persist in heart tissues, using in situ transcription and polymerase chain reaction. To understand the molecular basis of CVB3- induced myocarditis, the genomes of highly myocarditic (CVB3m) and amyocarditic (CVB3o) variants will be cloned. Reciprocal recombinant viruses will be generated by construction of hybrid genomes from infectious cDNA molecules of each genome and assessed for myocarditis-inducing ability in adolescent mice. Infectious hybrid cDNA molecules will be constructed with progressively smaller nucleotide regions associated with myocarditis to generate recombinant viruses. The nucleotide regions associated with myocarditis will be sequenced. CVB3 infection of some murine strains induces T cells which lyse uninfected target cells. Experiments will determine whether T lymphocytes from CVB3m-inoculated mice with chronic myocarditis will proliferate in vitro in response to soluble antigens extracted from normal murine hearts/cultured cardiac fibroblasts and whether extent of proliferation is predictive of severity of myocarditis.

该提案的目标是确定小规模的机制无包膜RNA基因组病毒引起心脏严重局灶性炎症小鼠组织，作为人类疾病心肌炎的模型。最人性化慢性心肌炎患者会产生针对抗原的自身抗体心脏组织。在柯萨奇病毒 B3 (CVB3) 诱导的小鼠模型中慢性心肌炎也会产生针对心肌细胞的自身抗体抗原。施用含有 CVB3 中和剂的鼠血清接种 CVB3 的小鼠体内抗体在接种后 3 天会加剧心肌炎，提示存在病理抗体。初步的针对 CVB3 的中和单克隆抗体 (mAb) 的研究证明 CVB3 颗粒和培养小鼠之间共有表位通过抗体结合研究评估，心脏成纤维细胞，补体介导成纤维细胞裂解和刺激巨噬细胞合成成纤维细胞的化学引诱剂。一种单克隆抗体在正常情况下会诱发心肌炎老鼠。八种单克隆抗体可能导致疾病发病的机制将鉴定小鼠心肌炎。 mAB诱导心肌炎将被确认。 mAb 参与补体的能力或杀伤细胞介导的小鼠心脏成纤维细胞裂解进行了定量评估。 mAb 增强可溶性因子的产生心脏成纤维细胞将被量化。病毒多肽八种单克隆抗体结合将被识别。多个实验室已表明 15-25% 患者的心脏组织中存在肠道病毒基因组当无法检测到传染性病毒时患有心肌炎。 CVB3 小鼠慢性心肌炎模型将用于确定病毒是否基因组持续存在于心脏组织中，利用原位转录和聚合酶链式反应。了解 CVB3- 的分子基础诱发心肌炎，高度心肌炎（CVB3m）和将克隆心肌病 (CVB3o) 变体。互惠重组病毒将通过从传染性病毒中构建混合基因组来产生每个基因组的 cDNA 分子并评估其诱发心肌炎的能力在青春期小鼠中。将构建感染性杂交 cDNA 分子与心肌炎相关的核苷酸区域逐渐变小产生重组病毒。相关的核苷酸区域心肌炎将进行测序。某些小鼠品系的 CVB3 感染诱导 T 细胞裂解未感染的靶细胞。实验将确定接种 CVB3m 的小鼠的 T 淋巴细胞是否患有慢性心肌炎会响应可溶性抗原而在体外增殖从正常小鼠心脏/培养的心脏成纤维细胞中提取，增殖程度是否可以预测心肌炎的严重程度。