TRAUMA & ORGAN FAILURE--ROLE OF LPS BINDING PROTEIN

脓毒症和脓毒性休克仍然是发病率和死亡率的主要原因 外科重症患者中。 在革兰氏阴性脓毒症中， 内毒素（LPS）与单核细胞上的CD 14受体结合， 炎症介质的释放可导致组织和器官损伤 失败 LPS结合蛋白（LBP）增强LPS与CD 14的结合 复杂. 当与LBP结合时，低许多倍浓度的LPS可以 激活宿主细胞。 我们最近首次报道LBP 通过损伤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 局部产生的LBP调节对LPS的局部免疫应答， 革兰氏阴性感染，用于定位感染因子， 将主机防御集中在本地站点。 然而，如果局部免疫 激活太大，并溢出到循环中，或者如果 激活持续时间过长，肝外诱导LBP可能 易患多器官功能衰竭 我们建议研究控制 肝细胞以外的细胞产生LBP，并将其与 由肝细胞自身产生。 在准备阶段，我们克隆了老鼠 LBP cDNA，纯化的大鼠LBP蛋白，产生的抗LBP抗体，定义于 LBP调节的体外和体内模型， 对大鼠LBP启动子进行测序，表达重组大鼠LBP。 AIM I 将定义脂多糖结合蛋白的调节因子 在体外由肺、肾和肝外细胞产生。 研究 将在非肝细胞以及肝细胞中进行，并与 更好地定义LBP的组织特异性调节背后的机制。 我们开始通过定义调节LBP生产的细胞因子。 核 将进行连续测定以确认和定量转录 感应 我们将描述启动子DNA元件和核 控制LBP基因转录的因子。 最后我们将 确定LBP的产生是否受mRNA的改变控制 稳定性或翻译效率作为其mRNA的3 '-UTR中的元件 结果显示 AIM II将定义脂多糖结合的调节 体内肝外组织的蛋白质产生。 使用两种模型， 损伤（后肢血栓注射和失血性休克），我们将 确定在体外调节LBP产生的相同细胞因子是否 还调节体内LBP的产生。 进一步表征 产生LBP的细胞也将完成。 AIM III将决定 局部产生的（肺）脂多糖结合的功能作用 蛋白质对宿主对LPS的反应。 利用出血、受伤或 腺病毒载体含有LBP cDNA，我们将诱导增加局部 肺生产的lBP，并确定在何种程度上增加局部 LBP水平增强肺内免疫细胞活化和组织 全身性或创伤性LPS损伤。 在我们完成学业后， 我们将确定LBP是如何在肝外组织中诱导的， 损伤以及这种局部LBP的病理生理意义 生产 这些信息将提供重要的和以前 对革兰氏阴性脓毒症创伤后反应的见解不可用 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.