INFLAMMATORY NEUROLOGICAL DISEASES

描述：这是一个新的建议，研究IL 12在各种疾病中的作用。 神经系统疾病的模型和各种系统。 他 研究分为三个具体目标。 在第一个目标，他 建议研究IL 12在各种体内表达和诱导， 系统使用鼠标。 对于每一个系统，他将包括以下成员： 敏感和耐药菌株，以确定IL 12是否是关键的 可能与易感性或耐药性有关的显著特征。 待检查的系统为：LPS内毒素血症、MHV感染、李斯特菌 感染、伯氏疟原虫感染和EAE。 在这些系统中， 将检查疾病发展过程中的一些时间点， 在它的高峰期和恢复期或慢性期。 他的“宣读”将是 通过RNase保护测定法分析p35和包埋脑的mRNa。 他 详细介绍了所有这些系统和研究的方法。 最后，他会 还检查了中枢神经系统组织和提取物中的各种 其他促炎和抗炎细胞因子的PCR和原位杂交。 第二个具体目标是分析星形胶质细胞的组织培养物， 小胶质细胞（他已经证明这两种细胞都能在LPS上产生IL 12 刺激）和腹膜巨噬细胞。 他将定义 LPS诱导IL 12的分泌，试图确定IL 12的分泌机制， 通过用环己酰胺阻断蛋白质组装来进行novo合成，并试图 阐明LPS诱导是否是通过物质对 基因转录 此处使用的测定包括p35和p40的分析 RNA诱导、抗IL-12 ELISA系统和使用ELISA的IL-12生物测定 人类淋巴母细胞系是从他自己或他的同伙身上产生的！ 第三个目的是建立稳定的转基因IL 12生产细胞， 转基因小鼠 他将使用他已经成功开发的GFAP启动子系统 过去使用的。 在这个时候，他确实拥有转基因创始人， 是培育它们以建立稳定的双基因转基因 IL 12表达一致。 他将使用狭缝印迹或PCR来跟踪 后代中的转基因。 表达IL 12的一次稳定系 转基因技术已经建立，他将在许多领域使用它们， 实验系统和操作，以确定是否和如何IL 12可以 影响典型地涉及特定模型的标准变化。 首先，他将分析转基因动物的临床表型和行为 vs正常人。 他将描述的发育和解剖分布， CNS中IL 12表达、胶质增生程度、小胶质细胞密度和 髓鞘和少突胶质细胞变化（如有）。 详细解剖检查 神经元分布及其变化的定义将使用 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CNS的精细结构 对于组织学上未观察到的任何细胞成分的变化， 光镜 CNS的性质、分布、发病和类型 将定义炎症。 他召集了两个 神经病理学家，以帮助在这方面的努力，并包括一个表列出了 有很多参数需要检查。 自从创始小鼠表现出中枢神经系统 如果是自发性炎症，他会做详细的免疫组织化学 分析大脑的内皮细胞，以确定 分子在那里。 在第三个目标的第三部分，他将试图 定义参与或偏离正常神经免疫学的能力 反应. 这些将包括EAE诱导，将IL 12转基因置于 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