Hemodynamic and anti-Toxin Treatments in Anthrax Lethal Toxin Challenged Canines

尽管在2001年美国炭疽病暴发中，对常规血流动力学支持液体和血管升压剂的抗震性导致了所有5名死亡患者的死亡，但其机制和最佳处理仍不清楚。致死性(LeTx)和水肿性(ETx)毒素是炭疽病的主要致病因素。LeTx和ETx都由细胞摄取有毒物质所必需的保护性抗原(PA)、LeTx的致死因子(LF)和ETX的水肿因子(EF)组成。致死因子是一种锌金属蛋白酶，可以抑制丝裂原激活的蛋白激酶1-4、6和7，而EF是一种钙调素依赖的腺苷环化酶。最近的研究表明，LeTx和ETX产生的细胞变化不同于与更常见的细菌毒素(如内毒素)或革兰氏阳性生物的外毒素(如金黄色葡萄球菌)相关的变化。尽管有这项研究，但定义这些毒素对心血管影响的体内工作有限。此外，尽管已经做出了很大的努力来开发和测试新的毒素导向药，但很少有人注意到常规液体、血管升压剂和肌松药在炭疽毒素相关性休克中的疗效。我们实验室的大部分工作表明，这些反应可能很难预测。 在大鼠模型中，输注24小时的LeTx可产生致死性和休克，但不改变循环中的炎性细胞因子、一氧化氮或儿茶酚胺。在这个模型中，尽管PA导向的单抗(PA-mAb5H3或Raxibacumab)(HGS，Rockville，MD)是保护性的，但液体或去甲肾上腺素支持方案对脂多糖或大肠杆菌有利，不适用于LeTx(4-6)。事实上，液体不仅使LeTx的存活率变差，而且还抵消了PA-mAb的作用。 尽管提供了信息，但大鼠模型不允许持续时间(即24小时)或血流动力学监测类型(即全身和肺动脉导管测量和系列心脏成像)来充分表征来曲安奈德和ETX的心血管效应。因此，在Trans-NIH/FDA生物防御计划的支持下，建立了一种犬模型。在最初的研究中，犬的LeTx挑战产生了许多与大鼠相同的变化。LeTx还与犬的心肌抑制有关。 本研究的目的是利用目前发展起来的犬炭疽病LeTx模型，全面研究液体和血管升压剂治疗以及联合毒素导向治疗的效果。该模型的一个优势在较小的动物模型中是不可能的，那就是能够根据预负荷和灌流压力的测量来滴定血流动力学支持，就像在危重患者中所做的那样。具体地说，这项研究将解决标准方案(液体和去甲肾上腺素输注)或毒素导向疗法(即5H3-mAb或Raxibacumab)在单独或联合使用时对接受24小时LeTx注射的犬是有益的。基于对这种犬模型的早期研究，已经开发出使用预负荷措施滴定液体和去甲肾上腺素的顺序给药的治疗算法。 检查血流动力学支持和Raxibacumab与LeTx挑战的效果的研究已经完成。在血流动力学支持中加入Raxibacumab可改善血流动力学，减少血管升压剂的需求和液体滞留，并在接受LeTx挑战后增加存活率。这一数据是在2011年美国胸科学会国际会议上公布的，现已公布。(Barochia等人)保护性抗原抗体增强犬炭疽致死性毒素休克的血流动力学支持。J感染Dis.2012年；205：818 829。) 在相同的犬模型中，研究也完成了单独或与ETX挑战单独或与ETX和LeTx联合应用Raxibacumab和血流动力学支持的效果。单独使用ETX以及与ETX和LeTx一起使用时，raxibacumab还增强了血流动力学支持并改善了存活率。这些发现分别于2013年和2014年在美国胸科学会国际会议上公布。 已经提交了一份手稿供考虑出版。

Although shock resistant to conventional hemodynamic support with fluids and vasopressors contributed to death in all five nonsurvivors in the 2001 United States anthrax outbreak, its mechanisms and optimal management remain unclear. Lethal (LeTx) and edema (ETx) toxin are major contributors to the pathogenesis of anthrax. Both LeTx and ETx consist of protective antigen (PA) which is necessary for cellular uptake of the toxic moities; lethal factor (LF) for LeTx and edema factor (EF) for ETx. Lethal factor is a zinc metalloproteinase that inhibits mitogen activated protein kinase kinases (MAPKK1-4, 6 and 7) while EF is a calmodulin dependent adenyl cyclase. Recent research has shown that LeTx and ETx produce cellular changes different from those associated with more commonly encountered bacterial toxins such as lipopolysaccharide (LPS) or the exotoxins of gram-positive organisms like S. aureus. Despite this research, there has been limited in vivo work defining these toxins cardiovascular effects. Furthermore, although there have been major efforts to develop and test new toxin directed agents, little attention has been paid to the efficacy of conventional fluid, vasopressor and inotrope support during anthrax toxin associated shock. Work done largely in our laboratiory suggests these responses may be difficult to predict. In a rat model, 24 h LeTx infusion produced lethality and shock, but it did not alter circulating inflammatory cytokines, nitric oxide or catecholamines. In this model, while PA directed monoclonal antibody (PA-mAb, 5H3 or Raxibacumab) (HGS, Rockville, MD) was protective, regimens of fluid or norepinephrine support beneficial with LPS or E. coli, were not with LeTx (4 - 6). In fact fluids not only worsened survival with LeTx but also negated the effects of PA-mAb. Although informative, the rat model did not permit the duration (i.e. > 24h) or type of hemodynamic monitoring (i.e. systemic and pulmonary arterial catheter measures and serial cardiac imaging) necessary to fully characterize the cardiovascular effects of LeTx and ETx. Therefore a canine model was developed with support from the Trans-NIH/FDA Biodefense Program. In initial studies, LeTx challenge in the canine produced many of the same changes noted in the rat. LeTx was also associated with myocardial depression in the canine. The purpose of present study is to use the now developed canine anthrax LeTx model to comprehensively investigate the effects of fluid and vasopressor treatment alone and in combination with a toxin directed treatment. An advantage of this model not possible in smaller animal models is the ability to titrate hemodynamic support based on measures of preload and perfusion pressure just as it is done in critically ill patients. Specifically, this study will address whether a standard regimen (fluid and norepinephrine infusion) or a toxin directed therapy (i.e. 5H3-mAb or Raxibacumab) are beneficial when used alone or together in canines challenged with 24 h infusions of LeTx. Based on earlier studies with this canine model, treatment algorithms have been developed for the sequential administration of fluid and norepinephrine titrated using preload measures. Studies examining the effects of hemodynamic support and Raxibacumab with LeTx challenge are complete. Addition of Raxibacumab to hemodynamic support improved hemodynamics, reduced vasopressor needs and fluid retention and increased survival following LeTx challenge. This data was presented at the International Conference of the American Thoracic Society 2011 and has now been published. (Barochia et al. Protective antigen antibody augments hemodynamic support in anthrax lethal toxin shock in canines. J Infect Dis. 2012; 205: 818 829.) Studies are now also complete examining the effects of Raxibacumab and hemodynamic support alone or together with ETx challenge alone and with ETx and LeTx in combination in this same canine model. Raxibacumab also augmented hemodynamic support and improved survival with ETx alone and with ETx and LeTx together. These findings were presented at the International Conference of the American Thoracic Society in 2013 and 2014. A manuscript has been submitted for consideration of publication.