CYTOKINES AND AIDS DEMENTIA COMPLEX

这项建议的重点是细胞因子产生 通过系统地渗透免疫细胞或常驻脑细胞， 在艾滋病毒感染过程中导致中枢神经系统损伤。为了检验这一假设， 采用了一种定义良好的转基因方法，其中表达了 在细胞因子中，IL-6和IL-3通过胶质细胞靶向星形胶质细胞 纤维酸性蛋白(GFAP)-融合基因构建。这有 为我们研究神经致病提供了独特而有力的模型 星形胶质细胞结构性产生细胞因子的后果 在完整的中枢神经系统中。GFAP-IL6和GFAP-IL3的初步表征 转基因小鼠已经揭开了广泛的分子、细胞和 中枢神经系统的功能改变-其中许多与 那些在艾滋病毒脑病中看到的。值得注意的是，这些研究直接 在HIV的发生中起因果作用的细胞因子 脑病和其他神经退行性疾病。在此，我们建议 建立表达细胞因子肿瘤坏死因子-α的转基因小鼠 目标是中南欧。在这一新的研究中详细的神经病理评估 模型和现有的GFAP-细胞因子小鼠都将使用 建立了一系列测试，以检查分子中中枢神经系统的变化 和细胞水平，包括核糖核酸酶保护分析，原位 杂交、Northern印迹杂交、蛋白质免疫印迹试验、 免疫标记脑的常规光镜和激光共聚焦显微镜 切片和电子显微镜。GFAP的中枢神经系统功能改变- 小鼠将在行为和行为上测定细胞因子 电生理和水平，并在可能的情况下与特定的 分子和细胞的改变。初级阶段的认定 与大脑相关的致病和功能里程碑 各种细胞因子的表达将由以下因素决定：i)详细 不同类型GFAP的发展研究与比较分析 细胞因子模型，以及ii)分析由 产生细胞因子的转基因星形胶质细胞在正常小鼠体内的移植 大脑。其他致病因素对神经系统的影响将 被评估：i)在杂交实验中培育生物小鼠 表达细胞因子的组合(即IL-6+IL-3)，以及II)通过背部- GFAP-细胞因子小鼠与SCID小鼠杂交发育的研究 免疫缺陷GFAP-细胞因子转基因动物。这些研究将 开发模型，概括多因素致病因素和 免疫缺陷的环境被认为是艾滋病毒脑病的基础。 最后，特征良好的GFAP转基因小鼠将被用于 在体内识别和评估针对有害的药物的疗效 单个细胞因子与中枢神经系统的相互作用。这项研究提供了一种独特的 一种强有力的方法来阐明细胞和分子基础 中枢神经系统细胞因子的体内病理生物学和可望推动我们的 了解艾滋病毒相关的神经系统疾病，帮助识别 治疗干预的关键目标，并促进 治疗策略的临床前评估。

This proposal focuses on the hypothesis that cytokines produced systemically and by infiltrating immune cells or resident brain cells, contribute to CNS injury during HIV-infection. To test this hypothesis, a well defined transgenic approach was employed in which the expression of the cytokines IL-6 and IL-3 was targeted to astrocytes using glial fibrillary acidic protein (GFAP)-fusion gene constructs. This has provided us with unique and powerful models to study the neuropathogenic consequences of the constitutive production of cytokines from astrocytes in the intact CNS. Initial characterization of GFAP-IL6 and GFAP-IL3 transgenic mice has unveiled wide-ranging molecular, cellular and functional alterations of the CNS-many of which share similarities to those seen in HIV encephalopathy. Significantly, these studies directly implicate cytokines in having a causal role in the genesis of HIV encephalopathy and other neurodegenerative diseases. Here we propose to develop transgenic mice with expression of the cytokine TNF-alpha targeted to the CNS. Detailed neuropathological assessment in this new model as well as in existing GFAP-cytokine mice will employ an established battery of tests to examine CNS alterations at the molecular and cellular levels, including RNase protection assays, in situ hybridization, northern blot hybridization, protein immunoblot assay, conventional light and laser confocal microscopy of immunolabeled brain sections and electron microscopy. Functional CNS alterations in the GFAP- cytokine mice will be determined at the behavioral and electrophysiological and levels and where possible be linked to specific molecular and cellular alterations. The identification of primary pathogenetic and functional milestones associated with the cerebral expression of the various cytokines will be determined by: i) detailed developmental studies and comparative analysis of the different GFAP- cytokine models, and ii) analyzing the CNS alterations resulting from the grafting of cytokine producing transgenic astrocytes in the normal mouse brain. The neurological impact of additional pathogenetic factors will be assessed: i) in cross-breeding experiments to develop biogenic mice expressing combinations of cytokines (i.e. IL-6+IL-3), and ii) by back- cross breeding GFAP-cytokine mice with SCID mice to develop immunodeficient GFAP-cytokine transgenic animals. These studies will develop models that recapitulate the multi-factorial pathogenetic and immunodeficient environments thought to underlie HIV encephalopathy. Finally, the well characterized GFAP-transgenic mice will be used to identify and assess in vivo the efficacy of drugs targeted at harmful individual cytokine-CNS interactions. This study provides a unique and powerful approach to elucidate the molecular and cellular basis for the CNS pathobiology of cytokines in vivo and can be expected to advance our understanding of HIV-associated neurological disease, help identify critical targets for therapeutic interventions and facilitate the preclinical evaluation of therapeutic strategies.