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.

本建议的目标是确定一个小的 无包膜RNA基因组病毒引起严重的心脏局灶性炎症 小鼠组织，作为人类疾病心肌炎的模型。 大多数人类 慢性心肌炎患者产生自身抗体， 心脏组织 在柯萨奇病毒B3（CVB 3）诱导的小鼠模型中， 慢性心肌炎，自身抗体也产生对心脏细胞 抗原 施用含有CVB 3中和的鼠血清 在接种后3天将抗体注入接种CVB 3的小鼠， 心肌炎，表明存在病理性抗体。 初步 抗CVB 3中和性单克隆抗体的研究 证实了CVB 3颗粒和培养的小鼠之间的共有表位 心脏成纤维细胞，如通过抗体结合研究评估的，补体- 介导的成纤维细胞溶解和巨噬细胞合成的刺激 成纤维细胞的化学引诱物。 一种mAb诱导正常人心肌炎 小鼠 八种单克隆抗体可能有助于发病机制的机制 将鉴定小鼠的心肌炎。 单克隆抗体诱发心肌炎 将得到证实。 mAb参与补体的能力- 或杀伤细胞介导的鼠心脏成纤维细胞裂解将被 定量评估。 mAb增强的可溶性因子的产生来自 将对心脏成纤维细胞进行定量。 所述病毒多肽， 将鉴定8种mAb结合。 几个实验室已经证明 肠道病毒基因组存在于15-25%的患者的心脏组织中， 当感染性病毒不能被检测到时， CVB 3鼠 慢性心肌炎模型将用于确定是否存在病毒性心肌炎。 基因组在心脏组织中持续存在，使用原位转录， 聚合酶链反应 为了了解CVB 3的分子基础， 诱导的心肌炎，高度心肌炎（CVB 3 m）和 将克隆心肌肌萎缩（CVB 30）变体。 相互重组 病毒将通过从感染性病毒中构建杂交基因组来产生。 每个基因组的cDNA分子并评估心肌炎诱导能力 在青春期的老鼠身上。 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 来产生重组病毒。 相关的核苷酸区域 将对心肌炎进行测序。 几种小鼠株的CVB 3感染 诱导T细胞裂解未感染的靶细胞。 实验将 检测CVB 3 m接种小鼠的T淋巴细胞是否 心肌炎会在体外对可溶性抗原作出反应而增殖 提取自正常鼠心脏/培养的心脏成纤维细胞， 增殖程度是否是心肌炎严重程度的预测指标。