The Effects of B. anthracis Cell Wall in a Small Animal

炭疽杆菌（B.炭疽病）是当今美国的一个主要生物恐怖主义威胁。在2001年的生物恐怖袭击中，所有需要血管加压药支持的患者的死亡率为100%。这与更常见的细菌性脓毒症形成对比，其中需要类似治疗的患者的死亡率通常接近60%。尽管致死毒素和水肿毒素（分别为LeTx和ETx）的产生有助于与B相关的休克。在炭疽病中，单独施用这些毒素不会导致活细菌感染所注意到的全谱组织损伤。这表明，其他毒力因子有助于这种生物的休克。B细胞壁。炭疽菌就是这样一种候选菌。 革兰氏阳性菌的细胞壁由几种结构大分子组成，包括肽聚糖（PG）和磷壁酸（TA）。肽聚糖层是由两种交替氨基糖（N-乙酰葡糖胺（GlcNAc）和N-乙酰胞壁酸（MurNAc））的线性链形成的晶格结构。该层在革兰氏阳性细菌中（20至80 nm）比在革兰氏阴性细菌中（7至8 nm）厚得多，并且占前者细菌干重的约90%，但仅占后者的10%。磷壁酸是通过磷酸二酯键连接的甘油或核糖醇的聚合物。在革兰氏阴性菌中不存在。它们可以与肽聚糖层的N-乙酰胞壁酸共价键合，或与细胞质膜中发现的质膜脂质连接，形成脂磷壁酸（LTA）。这些组分在细菌细胞壁中起结构作用，赋予细胞壁形状和结构强度，以及抵消细胞质的渗透压。然而，除了它们的结构功能外，这些分子，就像革兰氏阴性细菌的脂多糖（LPS）一样，也可以作为先天免疫系统的强大刺激剂。肽聚糖与某些革兰氏阳性菌表面的PG识别蛋白（PGRP）、Toll样受体2（TLR-2）、核苷酸结合寡聚化结构域（NOD）蛋白（NOD 1、NOD 2或cryopyrin）的相互作用导致Caspase-1、NF-κ B和MAP激酶的活化以及随后多种促炎细胞因子和趋化因子的表达和释放。来自某些细菌的脂儿茶酸也可以与CD 14、CD 36、TLR-2和可能的LBP相互作用以诱导细胞因子和趋化因子释放。这些细胞壁成分激活炎症反应可进而产生与脓毒症和脓毒性休克相关的血流动力学不稳定和组织损伤。 革兰氏阳性菌细胞壁肽聚糖和脂磷酸的特殊结构和免疫刺激作用各不相同。一些细菌的细胞壁，如高毒力的S。金黄色葡萄球菌是一种强烈的炎症刺激物，并且在体内给药时具有高毒性。然而，其他类型细菌的细胞壁，如无毒的B。枯草杆菌，诱导很少的炎症反应。现在从体外研究中获得的数据表明，B。炭疽菌细胞壁具有相当大的促炎作用。在一系列的实验中，Popov等人已经证明了B。炭疽菌细胞壁强烈刺激外周血单核细胞产生炎性细胞因子TNF β，IL-1？O和IL-6（17）。然而，这些刺激作用是否导致体内可观察到的损伤仍有待检验。同样重要的是要注意，致死性感染B。动物和人类中的炭疽病与细菌负荷有关，这些细菌负荷比通常观察到的毒性细菌大得多。因此，细胞壁的数量可能有助于损伤与B。炭疽杆菌的感染率可能比其他细菌类型高得多。 在一系列的实验和研究中，我们已经确定了高纯度形式的LeTx和ETx，单独和一起对大鼠心肺功能，炎症细胞因子和趋化因子的产生，一氧化氮的释放和组织学变化的影响。在我们开发的模型中，毒素在24小时内输注，以更好地模拟活细菌感染期间发生的模式。 本研究采用大鼠模型研究B的作用。炭疽菌细胞壁在迄今为止的实验中，我们已经注意到，尽管细胞壁的输注与循环白色血细胞和血小板的剂量依赖性减少相关，与炎症反应一致，但血压没有降低，并且尽管使用相当高的细胞壁剂量，致死率是最小的。细胞壁作为推注给药对循环细胞和血小板产生类似的作用，但确实产生一些血液动力学不稳定性和更大的致死性。然而，总体而言，这些研究支持B.炭疽菌细胞壁必须大量存在才能在B期间引起休克和致死。炭疽感染。正在编写描述本方案早期工作的摘要，其他研究正在进行中。

Bacillus anthracis (B. anthracis) is a major bioterrorism threat in the United States (US) today. In the 2001 bioterrorism attack, the mortality rate in all patients who required vasopressor support was 100%. This contrasts with more commonly encountered types of bacterial sepsis in which mortality rates in patients requiring similar treatment is typically close to 60%. Although production of both lethal and edema toxins (LeTx and ETx respectively) contribute to the shock associated with B. anthracis, administration of these toxins alone do not result in the full spectrum of tissue injury noted with live bacterial infection. This suggests that other virulence factors contribute to shock with this organism. The cell wall of B. anthracis is one such candidate. The cell wall of gram positive bacteria is composed of several structural macromolecules, including peptoglycans (PG) and teichoic acids (TA). The peptidoglycan layer is a crystal lattice structure formed from linear chains of two alternating amino sugars, N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc). This layer is substantially thicker in Gram-positive bacteria (20 to 80 nm) than in Gram-negative bacteria (7 to 8 nm) and comprises around 90% of the dry weight of the former bacteria but only 10% of the latter. Teichoic acids are polymers of glycerol or ribitol linked via phosphodiester bonds. They are not found in gram-negative bacteria. They can be either covalently bonded to N-acetylmuramic acid of the peptidoglycan layer or linked to the plasma membrane lipids found in the cytoplasmic membrane forming lipoteichoic acids (LTA). These components serve a structural role in the bacterial cell wall, giving the wall shape and structural strength, as well as counteracting the osmotic pressure of the cytoplasm. In addition to their structural functions however, these molecules, just like lipopolysaccharide (LPS) from gram-negative bacteria, can also serve as powerful stimulants of the innate immune system. Interaction of peptidoglycan with PG recognition proteins (PGRPs), toll-like receptor 2 (TLR-2), nucleotide-binding oligomerization domain (NOD) proteins (NOD1, NOD2 or cryopyrin) on the surface of some gram-positive bacteria results in the activation of Caspase-1, NF-kB and MAP kinases and the subsequent expression and release of a variety of pro-inflammatory cytokines and chemokines. Lipotechoic acid from some bacteria can also interact with CD14, CD36, TLR-2 and possibly LBP to induce cytokine and chemokine release. Activation of the inflammatory response by these cell wall components can in turn produce the hemodynamic instability and tissue injury associated with sepsis and septic shock. The specific structure and immunostimulatory effects of cell wall peptidoglycan and lipotechoic acid varies among gram-positive bacteria. The cell wall of some, such as highly virulent S. aureus, is a strong inflammatory stimulant and is highly toxic when administered in vivo. The cell wall from other bacteria types however, like nonvirulent B. subtilis, induces little inflammatory response. Data now available from in vitro studies indicates that B. anthracis cell wall has considerable pro-inflammatory effects. In a series of experiments, Popov et al. has shown that B. anthracis cell wall strongly stimulates peripheral blood monocyte production of the inflammatory cytokines TNF??, IL-1?O, and IL-6 (17). It remains to be tested however, whether these stimulatory effects result in observable injury in vivo. It is also important to note that lethal infection with B. anthracis both in animals and man is associated with bacterial loads that are much greater than is typically observed with virulent bacteria. Thus, the amount of cell wall that may contribute to injury with B. anthracis, may be much greater than with other bacteria types. In a series of experiments and studies, we have defined the effects of highly purified forms of LeTx and ETx, both alone and together on cardiopulmonary function, inflammatory cytokine and chemokine production, nitric oxide release and histological changes in rats. In the model we developed, toxin is infused over 24 h to better simulate the pattern that occurs during live bacterial infection. The present study is employing the rat model to study the effects of B. anthracis cell wall. In experiments to date we have noted that although infusion of cell wall is associated with dose dependent reductions in circulating white blood cells and platelet, consistent with an inflammatory response, blood pressure is not decreased and lethality, despite using fairly high cell wall doses, is minimal. Administration of cell wall as a bolus creates similar effects on circulating cells and platelets, but does produce some hemodynamic instability and more lethality. Overall however these studies support the possibility that B. anthracis cell wall must be present in very amounts to contribute to shock and lethality during B. anthracis infections. Abstracts are being prepared describing early work in this protocol and other studies are ongoing.