TRAUMA & ORGAN FAILURE--ROLE OF LPS BINDING PROTEIN

败血症和感染性休克仍然是发病率和死亡率的主要原因。 在危重的外科病人中。在革兰氏阴性败血症期间， 内毒素(LPS)与单个核细胞上的CD14受体结合，导致 释放可导致组织和器官损伤的炎性介质 失败了。内毒素结合蛋白(LBP)增强内毒素与CD14的结合 很复杂。当与LBP结合时，较小浓度的LPS可以增加许多倍 激活宿主细胞。我们最近率先报道了LBP 损伤可在大鼠肝外组织中诱导产生。我们有 现在也证实了人类肝外LBP的产生。我们假设 本地生产的LBP调节本地对内毒素的免疫反应 革兰氏阴性感染，用于定位感染剂和 将主机防御集中在本地站点。然而，如果当地的免疫力 激活太大并溢出到循环中，或者如果 激活持续太长时间，肝外诱导LBP可能 为MSOF做好准备。我们建议研究控制经济增长的因素 肝细胞以外的细胞产生LBP，并与 由肝细胞本身产生。在准备过程中，我们克隆了大鼠 LBP基因，纯化的大鼠LBP蛋白，产生抗LBP抗体，定义在 LBP调节的体外和体内模型，并即将分离 并对大鼠LBP启动子进行测序，表达重组大鼠LBP。目标I 将确定调节内毒素结合蛋白的因素 在体外由肺、肾和肝外细胞产生。研究 将在非肝细胞和肝细胞中进行，并与 更好地定义LBP组织特异性调控背后的机制。 我们首先定义了调节LBP产生的细胞因子。核子 将进行连续分析以确认和量化转录 归纳法。我们将对启动子DNA元素和核进行表征 控制LBP基因转录的因素。最后，我们会 确定LBP的产生是否受mRNA的改变控制 作为其基因3‘-非编码区元件的稳定性或翻译效率 建议。AIM II将定义内毒素结合的调节 体内肝外组织的蛋白质生产。使用两种模型 受伤(后肢松节油注射和失血性休克)，我们将 确定在体外调节LBP产生的相同细胞因子 也调节体内LBP的产生。进一步刻画了 产生LBP的细胞也将完成。AIM III将决定 局部产生的(肺)内毒素结合的功能作用 宿主对内毒素的反应。使用出血、损伤或 携带LBP基因的腺病毒载体，我们会诱导局部增加 肺产生LBP，并确定局部增加到什么程度 LBP水平增强肺内免疫细胞激活和组织 全身或气管内的内毒素损伤。在我们完成学业后， 我们将定义LBP是如何在肝外组织中诱导的 局部LBP的损伤及其病理生理学意义 制作。这一信息将提供重要的和以前的 对革兰氏阴性脓毒症的创伤后反应缺乏洞察力 和内毒素血症。

Sepsis and septic shock are still major causes of morbidity and mortality among critically ill surgical patients. During gram-negative sepsis, endotoxins (LPS) binds to CD14 receptors on mononuclear cells, causing release of inflammatory mediators which can cause tissue injury and organ failure. LPS-binding protein (LBP) enhances binding of LPS to the CD14 complex. When bound to LBP, many fold smaller concentrations of LPS can activate host cells. We have recently been the first to report that LBP production is induced in extrahepatic tissues in rats by injury. We have now confirmed extrahepatic LBP production in humans also. We hypothesize that locally produced LBP modulates the local immune response to LPS and Gram-negative infection, serving to localize the infecting agent and focusing host defenses at the local site. If, however, the local immune activation is too great and spills into the circulation or if the activation persists for too long, extrahepatic induction of LBP may predispose toward MSOF. We propose to study the factors controlling the production of LBP by cells other than hepatocytes and to compare them to production by hepatocytes themselves. In preparation, we have cloned rat LBP cDNA, purified rat LBP protein, generated anti-LBP antibody, defined in vitro as well as in vivo models of LBP regulation and are about to isolate and sequence the rat LBP promoter and express recombinant rat LBP. AIM I will define the factors regulating lipopolysaccharide-binding protein production by pulmonary, renal and extrahepatic cells in vitro. Studies will be done in non-hepatocytes as well as hepatocytes and contrasted to better define the mechanism behind the tissue-specific regulation of LBP. We begin by defining the cytokines which regulate LBP production. Nuclear run-on assays will be done to confirm and quantify transcriptional induciton. We will characterize the promoter DNA elements and nuclear factors controlling transcription of the LBP gene. Finally, we will determine whether LBP production is controlled by alteractions in mRNA stability or translational efficiency as elements in the 3'-UTR of its mRNA suggest. AIM II will define the regulation of lipopolysaccharide-binding protein production by extrahepatic tissues in vivo. Using two models of injury (hindlimb turpentine injection and hemorrhagic shock), we will determine whether the same cytokines that regulate LBP production in vitro also regulate LBP production in vivo. Further characterization of the cells that produce the LBP will also be done. AIM III will determine the functional role of locally produced (pulmonary) lipopolysaccharide-binding protein on host responses to LPS. Using hemorrhage, injury, or an adenovirus vector containing the LBP cDNA, we will induce increased local pulmonary production of lBP and determine to what extent increased local levels of LBP potentiates intrapulmonary immune cell activation and tissue injury by systemic or intratracheal LPS. At the completion of our studies, we will have defined how LBP is induced in extrahepatic tissues after injury as well as the pathophysiologic significance of such local LBP production. This information will provide important and previously unavailable insights into post-traumatic responses to Gram-negative sepsis and endotoxemia.