Comparison of Anthrax Edema and Lethal Toxins in a Rat M

吸入性炭疽芽孢杆菌(B.anthracis)作为生物恐怖主义制剂的威胁是当今美国的一个重要问题。肺部吸入后的系统性炭疽杆菌会导致休克和肺水肿，尽管有有效的抗生素和其他支持，但在近50%的病例中是致命的。炭疽杆菌产生2种毒素：由致死因子(LF)和保护性抗原(PA)组成的致死毒素(LeTx)和由水肿因子(EF)和保护性抗原(PA)组成的水肿性毒素(ETx)。大量数据支持LeTx在炭疽杆菌发病机制中的核心作用。致死因子是一种锌蛋白酶，可使丝裂原活化蛋白激酶(MAPKK或MEKS)家族成员失活，从而抑制下游丝裂原活化蛋白激酶(MAP)的磷酸化和激活，如细胞外信号调节激酶(ERK)、c-Jun氨基末端/应激激活蛋白激酶(JNK/SAPK)和p38。缺乏LeTx的突变株的毒力要低得多，在许多动物模型中，直接注射LeTx是致命的。重组形式的蛋白质的出现为LeTx的研究提供了便利。 然而，尽管LeTx在炭疽杆菌的发病机制中可能很重要，但在动物模型中使用LeTx虽然会产生休克和致命性，但并不会导致在死于这种感染的患者中发现的典型的出血组织损伤。这表明，除了LeTx之外，毒力因子，如ETX，可能参与了这种微生物的致病作用。尽管缺乏ETX的炭疽杆菌突变体已被报道保持毒力，但研究支持这种蛋白在疾病发病机制中的作用。ETX是一种腺苷环化酶，能将ATP转化为cAMP，并能改变宿主的免疫和其他反应。过去在一些动物模型中使用这种毒素的早期制剂的研究表明，ETX会增加LeTx的作用。然而，由于缺乏重组制剂，ETX的研究受到阻碍。这样的准备现在已经可以使用了。目前，为了更好地确定炭疽杆菌的发病机制及其治疗，一个关键的问题是了解ETX单独和与LeTx联合作用的方式。 在本实验中，对最近可用的重组ETX和LeTx的制备进行了研究。我们首先比较了单独增加LeTx和ETX浓度的影响。在这些实验中，ETX的剂量低于LeTx的对数会导致低血压和类似LeTx的死亡。因此，ETX本身似乎具有潜在的致死作用，其浓度与LeTx的致死剂量相当。然后我们测试了ETX和LeTx的联合作用，发现它们的作用是相加的，而不是协同作用。在最近完成的实验中，我们描述了相似致死剂量的ETX和LeTx单独或联合使用对心肺和组织学变化以及宿主炎症和防御反应的影响。我们还发现保护性抗原导向抗体对ETX和LeTx的结合具有保护作用。这些研究最近已被接受出版。 在该方案的最后一组实验中，我们具体观察了ETX和LeTx单独和联合使用对心肌功能和心肌细胞结构的影响，通过电子显微镜进行评估。

The threat of inhaled Bacillus anthracis (B. anthracis) as an agent of bioterrorism is an important problem in the United States today. Systemic B. anthracis following pulmonary inhalation results in shock and pulmonary edema and is fatal in almost 50% of cases despite effective antibiotic and other support. B. anthracis produces 2 toxins; lethal toxin (LeTx) comprised of lethal factor (LF) and protective antigen (PA), and edema toxin (ETx) comprised of edema factor (EF) and PA. Much data supports a central role of LeTx in the pathogenesis of B. anthracis. Lethal factor is a zinc protease that could inactivate members of the mitogen-activated protein kinases (MAPKKs or MEKs) family and subsequently inhibits phosphorylation and activation of downstream mitogen-activated protein kinases (MAP kinases) such as extracellular signal regulated kinase (ERK), c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38. Mutant strains lacking LeTx are much less virulent and the direct administration of LeTx is lethal in many animal models. Work with LeTx has been facilitated by the availability of recombinant forms of the protein. Despite its likely importance in the pathogenesis of B. anthracis however, administration of LeTx in animal models, while producing shock and lethality, does not result in the characteristic hemorrhagic tissue injury found in patients dying with this infection. This has suggested that virulence factors in addition to LeTx, such as ETx, may contribute to the pathogenesis of this microbe. Although B. anthracis mutants lacking ETx have been reported to retain virulence, studies support a role for this protein in the pathogenesis of the disease. ETx is an adenyl cyclase that transforms ATP to cAMP and can alter immune and other responses by the host. Past studies in some animal models with early preparations of this toxin had suggested that ETx would add to the effects of LeTx. However, studies with ETx were hampered by lack of recombinant preparations. Such preparations are now becoming available. A critical question at this time, both for better defining the pathogenesis of B. anthracis as well as its treatment is to understand how ETx functions alone and in combination with LeTx. In the present experiments, recently available preparations of recombinant ETx and LeTx were investigated. We first compared the effects of increasing concentrations of LeTx and ETx alone. In these experiments, ETx in doses less than a log less than LeTx resulted in hypotension and lethality similar to LeTx. Thus, ETx alone appears to have potentially lethal effects at concentration comparable to lethal doses of LeTx. We then tested the effects of ETx and LeTx in combination and found that their effects are additive but not synergistic. In recent experiments now completed, we described the effects of similarly lethal doses of ETx and LeTx either alone or in combination on cardiopulmonary and histology changes and the host inflammatory and defense responses. We also showed that protective antigen directed antibody was protective against the combination of ETx and LeTx. These studies have recently been accepted for publication. In one final set of experiments from this protocol we are specifically looking at the effects of ETx and LeTx alone and in combination on myocardial function and myocardial cellular structure as assessed by electrom microscopy.