DIMETHYLNITROSAMINE EFFECTS ON CELLULAR IMMUNITY

在这次竞争更新中概述的实验旨在 确定组织对二甲基亚硝胺(DMN)的反应 肝毒性会导致免疫功能障碍。我们之前的工作 证明接触DMN会导致骨髓改变 血清携带的细胞因子的分化、诱导和组织 巨噬细胞前列腺素合成增加，并增强 呼吸爆发活性与肿瘤坏死因子α基因 表情。本提案中概述的研究将继续侧重于 巨噬细胞的作用，因为这个细胞参与了 对组织损伤的炎症反应以及调节细胞- 中介免疫(CMI)。分子方法论在上一期 将利用研究来阐明DMN的作用机制(S) 暴露会影响免疫系统。将在中提出的假设 这项建议是，缺乏足够的下调监管 化学诱导的炎症反应导致不适当的 细胞因子的表达，从而影响免疫的调节 回应。因此，特定细胞因子的过度生产导致 与免疫功能障碍有关。为了解决这一假设，我们将执行 定性和定量研究，以表征归纳和 消解DMN诱导的炎症反应。这些研究将 监测细胞内流、急性时相蛋白的诱导和 黏附分子与细胞因子表达的变化 免疫检测、原位杂交、酶联免疫吸附和 生物检测以及溶液杂交和聚合酶链法 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及临床意义 诱导“预置”的巨噬细胞群，改变免疫 反应(体外)。这些努力将利用“分子” 表型“和定量聚合酶链式反应程序，我们已经开发了 关注我们对巨噬细胞生成的持续兴趣 异质性和为什么DMN诱导的分化变化导致 巨噬细胞对调节信号的反应发生了变化。特定的 将解决的问题包括：是否有特定的“分子” 与组织和炎性巨噬细胞相关的表型 在接触DMN之后？这些变化之间的相关性是什么 巨噬细胞标志物有功能吗？T细胞调节信号缺失吗？ 在DMN诱导的炎症中？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 (生物标志物)与DMN接触直接相关 免疫毒性，可用于预测寄主抵抗力的改变(在 Vivo)。因此，这些研究将确定因果关系。 识别的生物标志物与DMN诱导的关系 免疫毒性。这些努力的目标是确定是否 给予细胞因子可逆转DMN诱导的免疫毒性或 对细胞因子表达的监测可以用来预测 体内的免疫反应。因此，这种方法将使我们能够 建立在接触DMN后进行风险评估的模型。 最后，将进行实验，以确定是否存在 遗传成分对DMN诱导的改变。

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.