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

炭疽感染今天仍然是一种生物恐怖主义健康威胁，能够在相对较短的时间内影响大量个人。确定有效但廉价的疗法，可以长期储存并迅速大规模管理，这对美国公众在炭疽爆发时具有巨大价值。 炭疽致死毒素（LeTx）的产生是炭疽发病机制的核心。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是细胞摄取毒性部分LF所必需的。致死因子是一种锌依赖性蛋白酶，其使除一种丝裂原活化蛋白激酶激酶（MPKK 1至4和6）之外的所有激酶失活。这些MPKKs参与细胞存活所必需的信号通路。我们和其他实验室已经表明，LeTx可以产生类似于活细菌感染期间观察到的休克和致命性。重要的是，用PA的选择性抗体抑制LeTx的细胞摄取在细菌攻击的炭疽模型中是保护性的，强调了这种毒素对炭疽致死性的重要贡献。然而，尽管它们被证明是有效的，但抗体制剂是昂贵的，并且可能难以大规模施用。此外，能够产生对这种抗体制剂的作用不敏感的毒素的炭疽的生物工程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必要的。 在先前的体内研究中，我们发现LeTx是致死性脂多糖（LPS）和大肠杆菌内源性一氧化氮（NO）产生的有效抑制剂。大肠杆菌攻击的大鼠。虽然炭疽感染过程中的这种抑制本身可能通过抑制宿主先天免疫反应来促进细菌生长，但它也可能以其他方式帮助微生物。与此一致，在大鼠实验室的体内实验中，我们观察到能够快速增加NO水平的非致死剂量的LPS实际上对致死LeTx攻击具有保护作用。这些体内研究结果表明，在炭疽感染期间，NO供体试剂的施用可能具有治疗益处。根据类型，NO供体试剂可以廉价地生产，大量储存，并且可以口服生物利用。在炭疽病爆发期间，这种药剂可以相对有效地大规模施用。 本方案旨在研究NO供体试剂用于治疗LeTx相关休克的潜力。作为原则研究的证据，它集中于可以静脉内施用以确保递送的药剂。到目前为止，研究报告由两部分组成。在第一部分（研究1）中，将在导管插入的大鼠模型中测试三种不同NO供体硝普钠（SNP）、S-亚硝基-L-谷胱甘肽（GSNO）和3-吗啉代壬肟（SIN-1）产生循环NO水平增加的能力，所述循环NO水平增加是可测量的（对照的>15%）并且持续超过24小时，平均动脉血压（MBP）降低<15%。该数据用于开发针对LeTx的SIN-1治疗方案。 该协议的第二部分（研究2），测试了SIN-1治疗抑制LT的致死作用的能力。在最初的实验中，我们表明，用SIN-1预处理LT抑制LT的体内致死作用。 随后的实验表明，SIN-1与LT的共输注（即同时通过相同的静脉导管）具有显著的保护作用，推测在输注过程中使LT失活。 最后一组实验现在已经完成，其中SIN-1和LT已经通过单独的导管输注。 这些实验的数据正在分析中，手稿正在编写中。 该研究的数据在2012年和2014年美国胸科学会国际会议上发表。

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 has focused on agents that can be administered intravenously to ensure delivery. The study has consisted of two parts thus far. 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. Subsequent experiments showed that coinfusion of SIN-1 with LT (i.e. simultaneously via the same venous catheter) was significantly protective, presumably inactivating LT during the process of infusion. A final set of experiments has now been completed in which SIN-1 and LT have been infused via separate catheters. Data from these experiments is under analysis and a manuscript is in preparation. Data from the study was presented at the International Conference of the American Thoracic Society 2012 and 2014.