Protective Effects of Anti-C5a in Sepsis

描述（由申请人提供）：我们将继续研究，以确定与盲肠结扎和穿刺（CLP）后啮齿动物发生脓毒性心肌病相关的分子机制，重点是C5 a过敏毒素及其受体C5 aR和C5 L2的作用。目的1将定义C3，C5，C5 a受体（C5 aR，C5 L2）和IL-17从假手术和CLP心肌细胞（CM）自发释放心脏抑制细胞因子（IL-12，TNF-1，IL-6）的要求。将这些数据与CLP小鼠心脏匀浆和血浆中细胞因子的存在进行比较。目的2将确定CLP后CM上C5 a受体和心脏抑制细胞因子（IL-12、TNF 1和IL-6）上调的时间过程。目的3将评估从假手术和CLP啮齿动物获得的CM中的电生理反应（动作电位、Ca 2+瞬变、K+和Ca 2+电流）以及功能反应（通过测量细胞内钙瞬变和肌细胞收缩），作为CLP后时间的函数。此外，将研究假手术和CLP CM在体外暴露于C5 a后的电生理和收缩反应变化，以确定C5 a导致CM收缩功能障碍的分子机制。目的4将使用添加了不同浓度的C5 a或心脏抑制细胞因子或两者的假手术和CLP CM，评估C5 a诱导CM变化的能力（如目的3所述）。我们将确定C5 a和心脏抑制细胞因子的组合在电生理功能障碍的发展中是否存在协同作用。目的5将采用非侵入性超声心动图方法来评估CLP小鼠与假手术小鼠相比的收缩和舒张功能以及全身血管阻力以及其他参数。我们将尝试确定C3，C5，C5 a和IL-17受体在CM功能障碍中的作用。这些研究还将采用C5 a或IL-17中和抗体治疗的CLP小鼠。C5 aR基因敲除小鼠模型也将用于确定C5 a信号通路在脓毒性心肌病发病机制中的作用。总的来说，这些研究应该确定C5 a及其受体以及心脏抑制细胞因子在脓毒性心肌病发展中的作用，以及如何避免或治疗这种并发症。 公共卫生相关性：脓毒症仍然是一个艰巨的问题，在人类与单一的FDA批准的干预。根据位置和临床细节，死亡率可能高达50- 60%，鉴于北美每年有60万或更多的脓毒症患者，这是一个特别令人担忧的统计数据。很明显，我们对脓毒症的认识不完全。在通过了解脓毒症的分子决定因素来解决这一障碍之前，治疗将是支持性的，并且非常昂贵。

DESCRIPTION (provided by applicant): We will continue our studies to define molecular mechanisms related to development of septic cardiomyopathy in rodents after cecal ligation and puncture (CLP), with an emphasis on the roles of C5a anaphylatoxin and its receptors, C5aR and C5L2. Aim 1 will define the requirements for C3, C5, C5a receptors (C5aR, C5L2) and IL-17 for spontaneous release of cardiosuppressive cytokines (IL-12, TNF1, IL-6) from sham and CLP cardiomyocytes (CMs). These data will be compared to the presence of cytokines in heart homogenates and plasma from CLP mice. Aim 2 will determine the time course after CLP for upregulation of receptors for C5a and the cardiosuppressive cytokines (IL-12, TNF1 and IL-6) on CMs. Aim 3 will assess electrophysiological responses (action potentials, Ca2+ transients, and K+ and Ca2+ currents) as well as functional responses (by measuring intracellular calcium transients and myocyte contraction) in CMs obtained from sham and CLP rodents as a function of time after CLP. In addition, sham and CLP CMs will be studied for changes in electrophysiological and contractile responses after in vitro exposure to C5a in an effort to define molecular mechanisms by which C5a causes contractile dysfunction in CMs. Aim 4 will assess the ability of C5a to induce changes in CMs (as described in Aim 3), using sham and CLP CMs to which varying concentrations of C5a or cardiosuppressive cytokines, or both, have been added. We will determine if there is a synergy in development of electrophysiological dysfunction with combination of C5a and cardiosuppressive cytokines. Aim 5 will employ non-invasive echocardiography approaches to assess systolic and diastolic function and systemic vascular resistance, as well as other parameters, in CLP mice as compared to sham mice. We will attempt to define the roles of C3, C5, C5a and IL-17 receptors in CM dysfunction. These studies will also feature CLP mice that have been treated with neutralizing antibodies to C5a or IL-17. A C5aR knockout mouse model will also be utilized to define the role of the C5a signaling pathways in the pathogenesis of septic cardiomyopathy. Collectively, these studies should define the roles of C5a and its receptors as well as cardiosuppressive cytokines in the development of septic cardiomyopathy and how this complication can be averted or treated. PUBLIC HEALTH RELEVANCE: Sepsis continues to be a daunting problem in humans with a single FDA approved intervention. Depending on location and clinical details, the mortality rate may be as high as 50-60%, an especially alarming statistic in view of 600,000 or more patients with sepsis in North America each year. It is clear that we have incomplete understanding of sepsis. Until this obstacle is resolved through an understanding of the molecular determinants of sepsis, treatment will be supportive and extremely costly.