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
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