C1 INHIBITOR-MEDIATED PROTECTION FROM ENDOTOXIN SHOCK

描述(由申请人提供)： 输注C1抑制剂(C1INH)可保护小鼠、狗、兔和狒狒免受革兰氏阴性细菌败血症和休克的侵袭。保护被认为是通过抑制补体和/或接触系统的激活而产生的。由于C1INH是如此有效，我们推测它可能与内毒素结合，这种相互作用可能干扰内毒素激活巨噬细胞的能力。初步研究支持这一假设。特定目标1将分析C1INH-内毒素的相互作用。将使用突变的重组C1INH蛋白确定结合位置，分析其与内毒素结合的能力(酶联免疫吸附试验和表面等离子共振)，以抑制内毒素与巨噬细胞的结合和激活。C1INH对内毒素休克小鼠的保护作用将直接与其他内毒素拮抗剂相比较。具体目标2将确定C1INH在对内毒素诱导的内皮损伤的保护中的作用。为了分析C1INH对内毒素诱导的血管通透性的影响，将描述内皮细胞骨架的改变和细胞屏障功能障碍，以及C1INH、C1INH突变体和氨基末端结构域逆转内毒素介导的血管通透性增加的能力。特异靶3将表征C1INH-内毒素复合体在小鼠体内的清除和器官定位。此外，还将在体外分析不同细胞类型对C1INH-LPS复合体的内化和降解情况。C1INH对小鼠(低剂量内毒素诱导的)耐受性的影响将被确定。最后，由于脓毒症模型在生物学上与人类疾病更相关，而且一些在内毒素休克模型中具有保护作用的药物在脓毒症中可能没有保护作用(甚至可能是有害的)，因此特定目标4将分析C1INH和C1INH变体在小鼠盲肠结扎/穿孔模型中的保护作用。这些研究将有助于更好地理解C1INH在抵抗革兰氏阴性败血症和内毒素休克中所起的作用，并可能导致这些疾病的新疗法。

DESCRIPTION (provided by applicant): Infusion of C1 inhibitor (C1INH) protects mice, dogs, rabbits and baboons from gram negative bacterial sepsis and shock. Protection has been assumed to result from inhibition of activation of the complement and/or contact systems. Because C1INH is so effective, we hypothesized that it might bind to LPS, and that this interaction might interfere with the ability of LPS to activate macrophages. Preliminary studies support this hypothesis. Specific Aim 1 will analyze the C1INH-LPS interaction. The binding site will be defined using recombinant mutated C1INH proteins, which will be analyzed for the ability to bind to LPS (ELISA and surface plasmon resonance), to inhibit binding of LPS to, and activation of, macrophages. The ability of C1 INH to protect mice from endotoxin shock will be directly compared with that of other endotoxin antagonists. Specific Aim 2 will define the role of C1INH in protection from LPS-induced endothelial injury. To analyze the effect of C1INH on LPS-induced vascular permeability, endothelial cell cytoskeletal alterations and cell barrier dysfunction will be characterized, as will the ability of C1INH, C1INH mutants and the amino terminal domain to reverse LPS-mediated increased vascular permeability. Specific Aim 3 will characterize the clearance and organ localization of C1INH-LPS complexes in mice. In addition, the internalization and degradation of C1INH-LPS complexes by different cell types will be analyzed in vitro. The effect of C1INH on LPS tolerance in mice (induced by low doses of LPS) will be determined. Lastly, because sepsis models are more biologically relevant to human disease and because some agents that are protective in endotoxin shock models may not be protective (or may even be harmful) in sepsis, Specific Aim 4 will analyze the role of C1INH and C1 INH variants in protection in the cecal ligation/puncture model in mice. These studies will lead to improved understanding of the role played by C1INH in the cbrresistance to gram negative sepsis and endotoxin shock, and may lead to new therapies for these diseases.