CHRONIC MYOCARDITIS IN A COXSACKIEVIRUS MURINE MODEL

柯萨奇病毒鼠模型中的慢性心肌炎

• 批准号: 3365094
• 负责人: Charles J Gauntt
• 金额: $ 20.33万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 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/3365094
• 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

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.

这项提案的目标是确定一种机制，通过这种机制 无包膜RNA基因组病毒在心脏引起严重的局灶性炎症 小鼠组织，作为人类疾病心肌炎的模型。最人性化 慢性心肌炎患者产生自身抗体的抗原 心脏组织。柯萨奇B3病毒(CVB3)诱导的小鼠模型 慢性心肌炎，也会产生针对心肌细胞的自身抗体 抗原。含CVB3中和小鼠血清的给药 接种CVB3的小鼠在接种后3天的抗体加剧 心肌炎，提示存在病理抗体。初步 抗CVB3中和性单抗的研究 证明CVB3颗粒与培养的小鼠具有共同的表位 通过抗体结合研究评估的心脏成纤维细胞，补体- 成纤维细胞的介导性裂解和巨噬细胞合成的刺激 成纤维细胞的趋化剂。一株单抗诱导正常大鼠心肌炎 老鼠。八种单抗参与慢性粒细胞白血病发病机制的研究 小鼠的心肌炎将被识别出来。单抗诱导心肌炎的实验研究 将会得到确认。单抗参与补体的能力 或杀伤细胞介导的小鼠心脏成纤维细胞的裂解 定量评估。单抗促进红曲霉可溶性因子的生产 心脏成纤维细胞将被量化。病毒多肽(S)对其 将确定八个mAbbs结合。几个实验室已经证明 15%-25%的患者的心脏组织中存在肠道病毒基因组 在无法检测到传染性病毒的情况下患心肌炎。CVB3小鼠 慢性心肌炎的模型将被用来确定病毒 基因组保留在心脏组织中，使用原位转录和 聚合酶链式反应。为了了解CVB3的分子基础- 诱导性心肌炎、高度心肌炎(CVB3m)和 心肌炎(CVB30)的变异体将被克隆。互换重组 病毒将通过构建具有传染性的混合基因组来产生 每个基因组的cdna分子并评估其诱发心肌炎的能力 在青春期小鼠身上。将构建具有感染性的杂交cdna分子 与心肌炎相关的核苷酸区域逐渐变小 以产生重组病毒。与之相关的核苷酸区域 心肌炎将被测序。几种小鼠的CVB3感染 诱导T细胞裂解未感染的靶细胞。实验将会 检测CVB3m接种小鼠的T淋巴细胞是否为慢性粒细胞白血病 心肌炎会在体外增殖，对可溶性抗原的反应 从正常小鼠心脏/培养的心脏成纤维细胞中提取 增殖程度是否预示心肌炎的严重程度。