INFLAMMATORY NEUROLOGICAL DISEASES

描述：这是一项研究IL12在多种疾病中的作用的新建议 神经紊乱模型和各种系统的研究。他的 研究将分为三个具体目标。在第一个目标中，他 建议研究IL12在体内多种细胞中的表达和诱导 使用鼠标的系统。对于这些系统中的每一个，他将包括 用敏感株和耐药株来确定IL12是否是关键 可能与易感性或抗药性有关的显著特征。 要检查的系统有：内毒素血症、MHV感染、李斯特氏菌 感染、伯氏疟原虫感染和流行性出血热。在这些系统中，他 将检查疾病发展过程中的多个时间点， 在高峰期、恢复期或慢性期。他的“朗读”将是 用核糖核酸酶保护实验分析p35和嵌合脑的基因表达。他 详细介绍了所有这些系统和研究的方法。最后，他会 还要检查中枢神经系统组织及其提取物中的各种 通过聚合酶链式反应和原位杂交检测其他促炎和抗炎细胞因子。 第二个具体目标是分析星形胶质细胞的组织培养和 小胶质细胞(他已经证明了这两种细胞在内毒素上都能产生IL-12 刺激)和腹膜巨噬细胞。他将定义时间进程 脂多糖诱导IL-12分泌减少的机制 通过用环己胺阻断蛋白质组装来进行新的合成，并尝试 阐明内毒素的诱导是否是通过该物质对 基因转录。本文使用的检测方法包括p35和p40的分析。 RNA诱导、抗IL12的酶联免疫吸附试验系统和使用 人类淋巴母细胞系是从自己或同伙身上产生的！ 第三个目标是创造一个稳定的转基因IL12生产菌 转基因小鼠。他将使用他已经成功使用的GFAP启动子系统 用于过去。目前，他确实拥有转基因创始人和 是培育它们以建立稳定的双基因转基因的过程 具有一致的IL12表达。他将使用插槽印迹或聚合酶链式反应进行跟踪 后代体内的转基因。IL-12一度稳定表达的细胞系 转基因技术已经建立，他将把它们广泛应用于 定义IL12是否以及如何 影响通常适用于特定模型的标准更改。 首先，他将分析转基因患者的临床表型和行为 VS法线。他将描述发育和解剖分布 IL12在中枢神经系统的表达、胶质细胞增生程度、小胶质细胞密度和 髓鞘和少突胶质细胞改变(如果有)。详细解剖检查 神经元的分布和它的改变将使用 计算机辅助图像分析辅助的各种神经元标记物。 还将进行超微结构研究，以检查中枢神经系统的精细结构 对于在组织逻辑上看不到的任何细胞成分的变化 光学显微镜。中枢神经系统的性质、分布、发病和类型 炎症将被定义。他已经争取到了两个人的努力 神经病理学家在这方面的帮助，并包括一个表格列出了 需要检查的参数有很多。自创始人以来，老鼠已经展示了中枢神经系统 自发发炎时，他会做详细的免疫组织化学检查 分析脑内皮细胞以确定哪种粘连 分子就在那里。在第三个目标的第三部分，他将尝试 定义参与或偏离正常神经免疫的能力 反应。这些措施将包括EAE诱导，将IL12转基因 SCID背景或

DESCRIPTION: This is a new proposal to study the role of IL12 in a variety of models of neurological disorders and in a variety of systems. His studies are to be divided into three specific aims. In the first aim he proposes to study IL12 expression and induction in a variety of in vivo systems using mice. For each of these systems he will include members of both susceptible and resistant strains to define if IL12 is a critical distinguishing feature that might be linked to susceptibility or resistance. The systems to be examined are: LPS endotoxemia, MHV infection, Listeria infection, Plasmodium berghii infestation, and EAE. In these systems he will examine a number of time points during the development of the illness, at it's peak and during recovery or chronic phases. His "read out" will be analysis by RNase protection assay of mRNa for p35 and embedded brains. He details the methods for all of these systems and studies. Finally, he will also examine the CNS tissues and extracts from them for a wide variety of other pro- and anti-inflammatory cytokines by PCR and in situ hybridization. The second specific aim is to analyze tissue cultures of astrocytes and microglial cells (both of which he has demonstrated to make IL12 on LPS stimulation) and peritoneal macrophages. He will define the time course of IL12 induction by LPS, attempt to define the mechanism of secretion being de novo synthesis by blocking protein assembly with cyclohexamide, and try to elucidate if the LPS induction is via a direct effect of the substance on gene transcription. Assays to be used here include analysis of p35 and p40 RNA induction, an anti-IL12 ELISA system, and a bioassay for IL12 using a human lymphoblastoid cell line generated from himself or an associate! The third aim is the creation of a stable transgenic IL12 producing transgenic mouse. He will use the GFAP promoter system he has successfully used in the past. AT this time he does possess the transgenic founders and is the process of breading them to establish a stable bigenic transgenic with consistent IL12 expression. He will use slot blots or PCR to follow the transgenes in the offspring. Once stable lines of IL12 expressing transgenics are established, he will use them in a wide number of experimental systems and manipulations to define whether and how IL12 may influence the standard changes typically attending the particular model. First he will analyze the clinical phenotype and behavior of the transgenics vs normals. He will describe the developmental and anatomic distribution of IL12 expression in the CNS, the degree of gliosis, microglial density and myelin and oligodendroglial changes (if any). Detailed anatomic examination of the neuronal distribution and its alterations will be defined using a variety of neuronal markers assisted by computer assisted image analysis. Ultrastructural studies will also be done to examine the CNS fine structure for changes in any cellular constituent not histo-logically seen with the light microscope. The nature, distribution, onset and type of CNS inflammation will be defined. He has enlisted the effort of two neuropathologists to help in this effort, and includes a table listing the numerous parameters to be examined. Since the founder mice have shown CNS inflammation spontaneously, he will do a detailed immunohistochemical analysis of the endothelium of the brain to determine which adhesion molecules are there. In the third part of the third aim he will attempt to define ability to participate in or deviate from normal neuroimmunological reactions. These will include EAE induction, putting the IL12 transgene on the SCID background or