DIMETHYLNITROSAMINE EFFECTS ON CELLULAR IMMUNITY

在这次竞争性更新中概述的实验旨在 确定组织对二甲基亚硝胺（DMN）诱导的 肝毒性导致免疫功能障碍。我们以前的工作 表明DMN暴露导致骨髓改变， 分化、诱导血清携带的细胞因子和组织 巨噬细胞前列腺素合成增加， 呼吸爆发活性与肿瘤坏死因子α基因 表情本建议中概述的研究将继续侧重于 巨噬细胞的作用，因为这种细胞参与了这两个 炎症反应组织损伤以及调节细胞- 介导免疫（CMI）。先前开发的分子方法学 研究将用于阐明DMN的机制， 暴露会影响免疫系统。假设将在 这一建议是，缺乏足够的下调， 化学诱导的炎症反应导致不适当的 细胞因子的表达，从而影响免疫调节 反应因此，特定细胞因子的过度产生导致 免疫功能紊乱为了解决这个假设，我们将执行 定性和定量研究，以描述归纳和 DMN诱导的炎症反应的消退。这些研究将 监测细胞内流，诱导急性期蛋白， 粘附分子和细胞因子表达的变化， 免疫检测、原位杂交、酶联免疫吸附和 生物测定以及溶液杂交和聚合酶链 反应（PCR）基因扩增。其次，实验将确定 血清细胞因子活性升高的机制和 诱导"引发的"巨噬细胞群体，并改变免疫 反应（体外）。这些努力将利用"分子 表型分析"和定量PCR程序，我们已经开发， 专注于我们对巨噬细胞生成的持续兴趣 异质性以及为什么DMN诱导的分化变化导致 巨噬细胞对调节信号的反应改变。具体 将被解决的问题包括：是特定的"分子 与组织和炎性巨噬细胞相关的"表型 在DMN暴露之后？变化之间的相关性是什么 巨噬细胞标志物的功能？T细胞调节信号是否缺失 DMN引起的炎症这些问题的答案将用于 我们的第三个目标是确定细胞因子谱是否 与DMN暴露相关的生物标志物直接负责 免疫毒性，并可用于预测改变的宿主抗性（在 体内）。因此，这些研究将确定原因和影响 鉴定的生物标志物和DMN诱导的 免疫毒性这些努力的目标是确定是否 给予细胞因子可以逆转DMN诱导的免疫毒性， 对细胞因子表达的监测可以用来预测 体内免疫反应。因此，这种方法将使我们能够 建立DMN暴露后风险评估模型。 最后，将进行实验以确定是否存在 DMN引起的变化的遗传成分。

Experiments outlined in this competitive renewal are aimed at determining how the tissue response to dimethylnitrosamine (DMN)-induced hepatotoxicity leads to immune dysfunction. Our previous work demonstrated that DMN exposure resulted in altered bone marrow differentiation, induction of serum borne cytokines, and tissue macrophages with increased prostaglandin synthesis, and enhanced respiratory burst activity and tumor necrosis factor alpha gene expression. Studies outlined in this proposal will continue to focus on the role of the macrophage since this cell is involved in both the inflammatory response to tissue injury as well as regulating cell- mediated immunity (CMI). Molecular methodologies developed in prior studies will be utilized to elucidate the mechanism(s) by which DMN exposure affects the immune system. The hypothesis to be addressed in this proposal is that the lack of adequate down-regulation of the chemically-induced inflammatory response results in inappropriate cytokine expression, thus, affecting the regulation of the immune response. Consequently, the overproduction of specific cytokines leads to immune dysfunction. To address this hypothesis we will perform qualitative and quantitative studies to characterize the induction and resolution of the DMN-induced inflammatory response. These studies will monitor the cellular influx, induction of acute phase proteins and adhesion molecules, and changes in cytokine expression by utilizing immunodetection, in situ hybridization, enzyme-linked immunosorbent- and bio-assays as well as solution hybridization and polymerase chain reaction (PCR) gene amplification. Secondly, experiments will determine the mechanisms responsible for elevated serum cytokine activity and the induction of "primed" macrophage populations, and altered immune responses (in vitro). These efforts will utilize the "molecular phenotyping" and quantitative PCR procedures which we have developed to focus on our continued interest in the generation of macrophage heterogeneity and why DMN-induced changes in differentiation results in macrophages with altered responses to regulatory signals. Specific questions which will be addresses include: Are specific "molecular phenotypes" associated with tissue and inflammatory macrophages following DMN exposure? What is the correlation between changes in macrophage markers with functions? Are T cell regulatory signals absent in DMN-induced inflammation? Answers to these questions will be used in our third objective to determine whether the cytokine profiles (biomarkers) associated with DMN exposure are directly responsible for immunotoxicity and can be used to predict altered host resistance (in vivo). Hence, these studies will determine the cause and effect relationships between identified biomarkers and DMN-induced immunotoxicity. The objectives of these efforts are to determine whether administration of cytokines can reverse DMN-induced immunotoxicity or that monitoring of cytokine expression can be used to predict altered immune responses in vivo. Thus, this approach will permit us to establish a model for making risk assessments following DMN exposure. Finally, experiments will be conducted to determine whether there is a genetic component to DMN-induced changes.