LIVER/LUNG INTERACTIONS DURING GRAM NEGATIVE ENDOTOXEMIA

描述（改编自申请人摘要）：革兰氏阴性菌 细菌性脓毒症是一种肝-肺轴炎症， 器官损伤是由于炎症与抗- 炎症基因产物这种菌血症后器官功能障碍被认为是 由于氧化还原引起的继发性缺血缺氧应激可增强 （氧化还原）敏感的转录因子和随后的扩增 关键细胞因子和非细胞因子表达介导的炎症反应 基因.拟议研究的目的是检验假设， 肝和肺内菌血症后氧限制的差异 调节一组确定的氧化还原敏感性转录的激活 因子，从而改变细胞因子表达的方向相反， 器官特异性实验旨在确定以下因素的影响： 在调节中，肝与肺O2供应的继发性减少 核因子-kB（NF-κ B）激活蛋白 （AP）-1、NFIL-6和环AMP反应元件结合蛋白（CREB）。的 这些变化的生物学意义将通过检查 炎性细胞因子（TNF-α，IL-1 α， IL-1 β）、抗炎细胞因子（IL-6、IL-10）、前列腺素（PG）H 合酶-2（考克斯-2）和诱导型一氧化氮合酶（iNOS）在这些器官中表达 系统在离体器官灌注过程中的氧化还原状态。实验 也被设计来定义菌血症后蛋白质：DNA相互作用， 缺氧应激和复氧通过评估这些激活 Kupffer细胞和肺泡巨噬细胞中的转录因子。结果数据 将建立一个涉及增强型考克斯-2和CREB的自动调节环， 活性通过PGE 2依赖性抑制菌血症后细胞因子表达 以一种特定的方式。核激活的作用 蛋白质缺氧诱导因子-1协同调节内毒素诱导的细胞因子 将评估随后缺氧期间的基因表达。这些终点 还将在缺氧应激期间在清醒大鼠中进行分析， 菌血症感染后既存肝功能障碍。并行 研究中，赋予缺氧抑制性的顺式作用DNA序列， 转染的RAW 264.7细胞中内毒素诱导的细胞因子启动子活性 与TNF-α和IL-1 β报告基因构建体的表达。 这些纵向研究的结果应该提供新的见解 细胞因子和iNOS表达的转录调节， 革兰氏阴性菌血症，同时确定机制的方法， 改善肺损伤和多器官衰竭。

DESCRIPTION (Adapted from the applicant's abstract): During gram-negative bacteremic sepsis, a liver-lung axis of inflammation predisposes to critical organ injury due to an imbalanced expression of inflammatory vs. anti- inflammatory gene products. Such postbacteremic organ dysfunction is thought to be augmented by secondary ischemic-hypoxic stress owing to reduction-oxidation (redox)-sensitive transcription factors and subsequent amplification of inflammatory responses mediated by expression of key cytokine and noncytokine genes. The objective of the proposed research is to test the hypothesis that postbacteremic O2 limitation within the liver and the lungs differentially modulates the activation of a defined group of redox-sensitive transcription factors, thereby altering cytokine expression in a directionally- opposite and organ-specific manner. Experiments are designed to determine the effects of secondary reductions in the hepatic vs. pulmonary O2 supply in modulating postbacteremic transactivation of nuclear factor-kB (NF-kB), activator protein (AP)-1, NFIL-6, and the cyclic AMP response element binding protein (CREB). The biologic significance of these changes will be assessed by examining the concomitant expression of inflammatory cytokines (TNF-alpha, IL-1alpha, IL-1beta), anti-inflammatory cytokines (IL-6, IL-10), prostaglandin (PG) H synthase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in these organ systems in relation to redox status during ex situ organ perfusion. Experiments are also designed to define postbacteremic protein:DNA interactions during hypoxic stress and reoxygenation by assessing the activation of these transcription factors in Kupffer cells and alveolar macrophages. Resulting data will establish if an autoregulatory loop involving enhanced COX-2 and CREB activity suppresses postbacteremic cytokine expression by a PGE2-dependent mechanism in an organ-specific manner. The role of activation of the nuclear protein hypoxia inducible factor-1 in co-modulating endotoxin-induced-cytokine gene expression during subsequent hypoxia will be assessed. These endpoints will also be analyzed in conscious rats during hypoxic stress, with and without preexisting liver dysfunction following bacteremic infection. In parallel studies, the cis-acting DNA sequences that confer hypoxic suppressibility of endotoxin-induced cytokine promoter activity in RAW 264.7 cells transfected with TNF-alpha and IL-1beta reporter gene constructs will be identified. Results from these vertically-oriented studies should provide novel insights into the transcriptional regulation of cytokine and iNOS expression during gram-negative bacteremic sepsis while identifying mechanistic approaches to ameliorate lung injury and multiple organ failure.