THEILERS DEMYELINATION--ROLE OF M0S AND OLIGODENDROCYTES

这项拟议研究的目的是阐明在泰勒氏小鼠脑脊髓炎病毒(TMEV)诱导的脱髓鞘疾病中病毒持续存在的细胞位置和分子基础。TMEV诱导的脱髓鞘疾病是多发性硬化症(MS)的一个高度相关的模型系统，流行病学证据强烈支持环境因素的参与，最有可能的是病毒感染。Theler模型的优点是病毒是小鼠的自然病原体(因此该模型不是人为设计的)，中枢神经系统疾病的慢性化，以及许多临床、病理、免疫学和遗传上与MS相似的地方。在这种感染中，髓鞘破坏似乎是由免疫介导的，而不是由于病毒对少突胶质细胞的细胞溶解作用；然而，我们现在认为这两种机制都有助于病理。研究表明，TMEV的持久性对于维持病理过程是必要的，并且巨噬细胞(Mphi)中存在主要的病毒抗原(Ag)负载。因此，Mphis似乎是病毒持续存在的优先部位(靶点)。TMEV在Mphis中的增殖受到限制的事实与RNA病毒只有在病毒生长受限的条件下才能持续存在的想法是一致的。相反，一些少突胶质细胞也被感染(在持续期间)，但感染的程度存在争议。由于病毒的持久性，病毒特异性的CD4+T细胞对病毒表位的反应维持在高水平。主要组织相容类(MHC)II限制性的CD4+Th1 T细胞(与病毒免疫相关)介导的TMEV特异性迟发型超敏反应(DTH)在脱髓鞘中起免疫病理作用。(1)通过竞争性逆转录-聚合酶链式反应(CRT-PCR)和实时定量RT-PCR方法，测定小鼠中枢神经系统(急性期和持续期)合成的TMEV RNA分子(基因组)的数量，以及正负病毒RNA链的比例。(2)感染中心、病毒抗原和病毒基因组阳性小鼠中枢神经系统Percoll梯度分离的Mphis、星形胶质细胞和少突胶质细胞的百分率。(3)病毒清除改变对免疫缺陷小鼠耐药株TMEV持久性细胞部位的影响。(4)菜豆病毒在三种小鼠少突胶质细胞系和原代培养的小鼠星形胶质细胞中的生长动力学。

The goal of the proposed research is to elucidate the cellular sites and molecular basis of viral persistence in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. TMEV-induced demyelinating disease is a highly relevant model system for multiple sclerosis (MS) in which epidemiologic evidence strongly supports involvement of an environmental factor, most likely a virus infection. The strengths of the Theiler's model are the facts that the virus is a natural pathogen of mice (therefore the model is not contrived), the chronicity of the CNS disease, and the many clinical, pathological, immunological and genetic parallels with MS. In this infection, myelin breakdown appears to be immune-mediated rather than due to a cytolytic effect of the virus on oligodendrocytes; however, we now believe that both mechanisms contribute to the pathology. It has been shown that TMEV persistence is necessary to sustain the pathologic process, and that a predominant viral antigen (Ag) load resides in macrophages (Mphis). Mphis therefore appear to be the preferential site (target) for viral persistence. The fact that TMEV multiplication in Mphis is restricted is consistent with the idea that RNA viruses persist only under restricted conditions of virus growth. In contrast, some oligodendrocytes are also infected (during persistence), but the extent of the infection is disputed. As a result of viral persistence, virus-specific CD4+ T cell responses to viral epitopes are sustained at high levels. TMEV-specific delayed-type hypersensitivity (DTH) mediated by major histocompatibility class (MHC) II-restricted CD4+ Th1 T cells (which are associated with immunity to viruses) plays an immunopathologic role in demyelination. The following studies will determine: (1) The number of TMEV RNA molecules (genomes) synthesized in the CNS of mice over time (acute and persistent phases) by competitive reverse transcription-polymerase chain reaction (cRT-PCR) and real-time quantitative RT-PCR, and the ratio of positive to negative viral RNA strands. (2) The percent of infected Mphis, astrocytes and oligodendrocytes isolated on Percoll gradients from the CNS of mice inoculated ic with BeAn virus positive for infectious centers, viral Ag and viral genomes. (3) The effect of altered viral clearance on the cellular sites of TMEV persistence in resistant strains of immunodeficient mice. (4) The kinetics of BeAn virus growth in three murine oligodendrocyte cell lines and primary murine astrocyte cultures.