MICROGLIA AND SIV NEUROPATHOGENESIS

描述（改编自摘要）：该项目将测试 假设血管周围巨噬细胞是一个关键的细胞靶点 在SIV感染中。 在第一个具体目标中，首席研究员和他的同事 将确定SIV对分离的SIV亚群的体内嗜性， 脑巨噬细胞在SIV脑炎（SIVE）的进展。 这 将在回顾性和前瞻性研究中进行。 在 回顾性研究，研究人员将分析动物以前 感染了未克隆的SIVmac 251，这是一种导致更高的 发展SIVE的动物比分子克隆菌株的百分比。 这些研究将主要包括免疫组织化学分析， 使用抗许多巨噬细胞的单克隆抗体储存的组织 标记物，以及区分血管周围脑巨噬细胞和 通过与抗CD 45抗体的不同反应性， CD14。 巨噬细胞标志物的表达将与 SIV DNA和RNA的原位杂交。 对于前瞻性研究，研究人员将分离相对纯的 来自的血管周围和实质巨噬细胞/小胶质细胞群体 SIVmac 251感染的猕猴。 电阻率将通过 使用容许细胞系和通过定量PCR回收病毒。 在这些实验中，研究人员将使用在两个小时内牺牲的动物。 感染后几周-研究人员认为， 主要受感染的细胞是血管周围的--并将结果与 在稍后的时间点处死动物。 在第二个具体目标中，研究人员将确定独特的病毒 用于接种的病毒群内的基因型。 这些研究 是基于这样的观察，即特定的SIV和HIV序列是 与靶器官感染有关。 为了实现这一目标， 研究人员将使用CD 14分离脑巨噬细胞亚群， 的FACS。 来自分选细胞的病毒序列将使用 PCR检测，并分析env和nef基因型。 的选择 这些实验的基因是基于观察，这两个 基因在决定嗜神经性方面特别相关， NERPRC之前的工作中指出。 中枢神经系统嗜巨噬细胞 序列将被插入SIVmac 239骨架中， 然后分析体外系统中的复制。 最后， 这些重组病毒将在猴子身上进行试验。

DESCRIPTION (adapted from the Abstract): This project will test the hypothesis that the perivascular macrophage is a critical cellular target in SIV infection. In the first specific aim, the Principal Investigator and his associates will determine the in vivo tropism of SIV for discrete subpopulations of brain macrophages during the progression of SIV encephalitis (SIVE). This will be performed in both retrospective and prospective studies. In the retrospective studies, the researchers will analyze animals previously infected with uncloned SIVmac251, a strain that leads to a higher percentage of animals developing SIVE than molecularly cloned strains. These studies will consist primarily of immunohistochemical analyses of banked tissue using monoclonal antibodies against a number of macrophage markers, and of differentiating perivascular brain macrophage from microglia by the differential reactivity with antibodies against CD45 and CD14. Expression of macrophage markers will be correlated with expression of SIV DNA and RNA using in situ hybridization. For the prospective studies, the researchers will isolate relatively pure populations of perivascular and parenchymal macrophages/microglia from SIVmac251-infected macaques. Infectivity will be quantified by the recovery of virus using a permissive cell line, and by quantitative PCR. For these experiments, the researchers will use animals sacrificed at two weeks after infection--a time when the Investigator believes that the predominantly infected cell is perivascular--and compare the results with animals sacrificed at later time points. In the second specific aim, the researchers will identify unique viral genotypes within the swarm of viruses used for inoculation. These studies are based on the observation that specific SIV and HIV sequences are associated with infection of target organs. To accomplish this aim, the researchers will isolate subpopulations of brain macrophages using CD14 and FACS. Viral sequences from the sorted cells will be amplified using PCR, and the genotypes for env and nef will be analyzed. The choice of genes for these experiments is based on the observation that these two genes are of particular relevance in the determination of neurotropism, as indicated in previous work from the NERPRC. The CNS macrophage tropic sequences will be inserted into an SIVmac239 backbone, and the replication in an in vitro system will then be analyzed. Ultimately, these recombinant viruses will be tested in monkeys.