PROCESSING OF ENDOTOXINS BY LIVER MACROPHAGES

肝脏巨噬细胞对内毒素的处理

• 批准号: 2747868
• 负责人: PETER THOMAS
• 金额: $ 14.16万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 1999-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2747868
• 关键词: Kupffer's cell; binding proteins; biological signal transduction; endotoxins; laboratory mouse; laboratory rat; lipopolysaccharides; liver metabolism; manganese; monoclonal antibody; nucleic acid probes; protein purification; protein sequence; protein structure function; tissue /cell culture; toxin metabolism; transfection

Infection due to gram negative bacteria is a major clinical problem and bacterial endotoxins in the circulation cause life threatening systemic reactions. Endotoxin is also involved in the pathology of a number of liver diseases including cirrhosis. The liver is the major site of metabolism for gut derived endotoxins. The cells responsible for endotoxin clearance from the portal blood is the liver macrophage (Kupffer cell) and co-operates with hepatocytes in detoxification. Because of continuous chronic exposure to low levels of endotoxin the Kupffer cell response to endotoxin is different to other macrophages and monocytes and the molecular details of endotoxin uptake and response in Kupffer cells in unclear. To investigate this we will: 1) Characterize the structures necessary for the binding of endotoxin to the Kupffer cells. We have identified two novel endotoxin binding proteins of 31 and 34 kD on rat Kupffer cells that are internal proteins that appear to be involved in transducing the signal for the cytokine response. Experiments are designed to purify these proteins and to determine amino acid sequences. We will produce monoclonal antibodies to both the 31 and 34 kD binding proteins using multiple antigenic peptide (MAP) technology. These antibodies will be used to study the functional significance and distribution of these binding proteins. 2) We will identify and clone and sequence the genes encoding the 31 and 34 kD endotoxin binding proteins using peptide sequences to synthesize specific oligonucleotide probes. The sequence data will be used to create a potential domain model for the binding proteins and to examine their structural relationships to other proteins. The cloned genes will also be available for transfection into eukaryotic cells and provide a model to study the biological role of these novel LPS binding proteins. 3) We will determine the role of the 31 and 34 kD binding proteins in signaling events associated with LPS interaction with Kupffer cells and in the secretion of cytokines by Kupffer cells in response to LPS. To examine the protective effects of Mn/++ and other transition metals against endotoxin and their inhibition of LPS binding to the 31 and 34 kD proteins. These studies will increase our understanding of the biological effects of endotoxins and lead to novel therapies against toxic shock.

由于革兰氏阴性细菌引起的感染是一个主要的临床问题， 流通中的细菌内毒素会导致生命威胁系统性 反应。内毒素也参与了许多 包括肝硬化在内的肝病。肝脏是 肠道衍生的内毒素的代谢。负责的单元 门毒素血液的内毒素清除率是肝巨噬细胞 （kupffer细胞）并与肝细胞合作进行排毒。 由于持续长期暴露于低水平的内毒素 库普弗细胞对内毒素的反应与其他巨噬细胞不同， 单核细胞和内毒素摄取和反应的分子细节 库普弗细胞不清楚。为了调查这一点，我们将：1）表征 内毒素与Kupffer结合所需的结构 细胞。我们已经确定了31个新型内毒素结合蛋白， 大鼠库普弗细胞上的34 kd是内部蛋白质的 参与转导细胞因子反应的信号。 实验旨在纯化这些蛋白质并确定氨基 酸序列。我们将对31和31产生单克隆抗体 34 kD结合蛋白使用多种抗原肽（MAP） 技术。这些抗体将用于研究功能 这些结合蛋白的显着性和分布。 2）我们会的 识别并克隆并测序编码31和34 kd的基因 内毒素结合蛋白使用肽序列合成 特定的寡核苷酸探针。序列数据将用于 为结合蛋白创建潜在的域模型，并检查 它们与其他蛋白质的结构关系。克隆的基因将 也可用于转染真核细胞，并提供 研究这些新型LPS结合蛋白的生物学作用的模型。 3）我们将确定31和34 kD结合蛋白在 与LPS与Kupffer细胞相互作用相关的信号事件和 在响应LP的库普弗细胞分泌细胞因子中。到 检查MN/++和其他过渡金属的保护作用 反对内毒素及其抑制LPS与31和34的结合 KD蛋白。这些研究将增加我们对 内毒素的生物学作用并导致对抗的新疗法 有毒冲击。