Effect of Nitric Oxide Donors on Anthrax Lethal Toxin Inactivation in Rat Model

炭疽感染在今天仍然是一种生物恐怖分子的健康威胁，能够在相对较短的时间内影响大量个人。确定有效但廉价的治疗方法，可以长期储存并迅速大规模实施，将对美国公众在炭疽爆发时具有重要价值。 炭疽致死毒素(LeTx)的产生是炭疽致病的核心。这是一种二元型毒素，由A(致死因子，LF)和B组分(保护性抗原，PA)组成。PA是细胞摄取有毒成分Lf所必需的。致死因子是一种锌依赖的蛋白水解酶，除一种丝裂原激活的蛋白激酶(MPKK1到4和6)外，所有的酶都失活。这些MPKK参与细胞生存所必需的信号通路。我们和其他实验室已经证明，LeTx可以产生类似于活细菌感染时观察到的休克和致命性。重要的是，用选择性的PA抗体抑制细胞对LeTx的摄取在细菌挑战的炭疽模型中是保护的，强调了这种毒素对炭疽致死性的重要贡献。然而，尽管证明了抗体制剂的有效性，但它们的价格昂贵，而且可能难以大规模管理。此外，能够产生对这种抗体制剂的影响不敏感的毒素的生物工程炭疽菌株可以用相对较少的资源来制造。开发能够抑制炭疽毒素的替代药物是有必要的。 在之前的体内工作中，我们发现LeTx是一种有效的内源性一氧化氮(NO)产生的抑制因子，可以抑制致死性脂多糖和大肠杆菌攻击大鼠的内源性一氧化氮(NO)的产生。虽然在炭疽感染过程中的这种抑制本身可能通过抑制宿主的先天性免疫反应来促进细菌的生长，但它也可能在其他方面帮助微生物。与此一致的是，在大鼠实验室的活体实验中，我们观察到能够迅速增加NO水平的非致命性剂量的内毒素实际上对致命的LeTx攻击具有保护作用。这些活体研究结果表明，在炭疽感染期间，给予NO供体制剂可能有治疗效果。根据类型的不同，没有捐赠剂可以廉价地生产、大量储存和可以口服生物利用。在炭疽病暴发期间，这种药物可以相对有效地大规模使用。 本方案旨在研究NO供体制剂治疗LeTx相关性休克的潜力。作为原则调查的证据，它将重点放在可以静脉注射以确保分娩的药物上。这项研究将包括两个部分。在第一部分(研究1)中，三种不同的NO供体硝普钠(SNP)、S-亚硝基-L-谷胱甘肽(GSNO)和3-吗啉酮-1(SIN-1)在平均动脉压(MBP)下降15%的情况下，产生可测(对照的15%)并持续24小时的循环NO水平的能力将在大鼠导管模型中被测试。这些数据被用来开发针对LeTx的SIN-1治疗方案。 方案的第二部分(研究2)测试了SIN-1处理抑制LT致死效应的能力。在最初的实验中，我们发现用SIN-1预处理LT确实能抑制LTS的体内致死效应。目前正在进行实验，以确定在LT挑战期间静脉注射SIN-1是否也具有保护作用。研究2的数据在2012年美国胸科学会国际会议上公布。

Anthrax infection remains a bioterrorist health threat today capable of affecting a large number of individuals in a relatively brief period. Defining effective but inexpensive therapies that can be stock-piled for prolonged periods and quickly administered on a large scale would be of great value for the US public in the event of an anthrax outbreak. Production of anthrax lethal toxin (LeTx) is central to the pathogenesis of anthrax. This is a binary type toxin consisting of an A (lethal factor, LF) and B component (protective antigen, PA). PA is necessary for cellular uptake of the toxic moiety LF. Lethal factor is a zinc dependent protease which inactivates all but one mitogen activated protein kinase kinase (MPKK 1 to 4 and 6). These MPKKs participate in signaling pathways necessary for cell survival. We and other labs have shown that LeTx can produce shock and lethality similar to that observed during live bacterial infection. Importantly, inhibition of the cellular uptake of LeTx with selective antibodies to PA is protective in bacteria challenged anthrax models, emphasizing the important contribution this toxin makes to the lethality of anthrax. Despite their demonstrated effectiveness however, antibody preparations are expensive and may be difficult to administer on a large scale. Furthermore, bioengineered strains of anthrax capable of producing toxin insensitive to the effects of such antibody preparations can be manufactured with relatively few resources. Developing alternative agents capable of inhibiting anthrax toxin is warranted. In prior in vivo work we found that LeTx was a potent inhibitor of endogenous nitric oxide (NO) production in lethal lipopolysaccharide (LPS) and E. coli challenged rats. While such inhibition during anthrax infection itself may promote bacterial growth by suppressing host innate immune responses, it may aid the microbe in other ways as well. Consistent with this, in in vivo experiments in the rat lab, we observed that nonlethal doses of LPS capable of rapidly increasing NO levels were actually protective with lethal LeTx challenge. These in vivo findings suggest that administration of NO donor agents may be of therapeutic benefit during anthrax infection. Depending on type, NO donor agents can be produced inexpensively, stored in large quantities, and can be orally bioavailable. Such agents could be administered relatively efficiently on a large scale during an anthrax outbreak. The present protocol is designed to investigate the potential of NO donor agents for the treatment of LeTx associated shock. As a proof of principle investigation, it will focus on agents that can be administered intravenously to ensure delivery. The study will consist of two parts. In the first part (Study 1), the ability of three different NO donors sodium nitroprusside (SNP), S-nitroso-L-glutathione (GSNO) and 3-morpholinosdnonime (SIN-1) to produce increases in circulating NO levels that are measurable (>15% of control) and sustainable over 24 hours with <15% decreases in mean arterial blood pressure (MBP) will be tested in a catheterized rat model. This data was employed to develop a regimen of SIN-1 treatment directed against LeTx. The second part of the protocol (Study 2), has tested the ability of SIN-1 treatment to inhibit the lethal effects of LT. In initial experiments we showed that pretreatment of LT with SIN-1 did inhibit LTs in vivo lethal effects. Experiments are now underway to determine whether administration of SIN-1 intravenously during LT challenge will also have protective effects. Data from Study 2 was presented at the International Conference of the American Thoracic Society 2012.