Molecular mechanisms of anthrax toxin-mediated edema

这个由Marge职业发展奖支持的项目的目标是：1) 确定ET启动和维持介质释放的细胞类型，如组胺， 前列腺素和神经激动素，参与ET介导的水肿和；2)决定这些是否相同 介体参与/负责内毒素诱导的小鼠死亡。尽管我们已经证明了ET S能够诱导COX-2的表达，使用HEK细胞，我们没有观察到COX-2mRNA的增加 基础，在人类单核细胞来源的单核细胞中。另一方面，ET引起PGE2的小幅增加 RAW264.7巨噬细胞合成及其与内毒素诱导前列腺素的协同作用 在这些细胞中产生。为了解决由ET启动的介体级联可能开始的假设 有或包括直接刺激神经细胞、背根节释放P物质(SP) 分离大鼠和兔背根神经节(DRG)细胞，加入ET进行体外培养。到目前为止，我们没有检测到 在统计学意义上，这两种类型的细胞都会释放SP。 基于我们先前的工作显示了组胺(H1)拮抗剂(吡胺胺)和环氧合酶-2的抑制剂 (塞来昔布)可通过皮内ET减轻兔皮肤的水肿，我们已对其进行了测试 药物对内毒素致BALB/CJ小鼠死亡的影响。虽然两种药物都没有显著的效果 单独而言，这种结合导致了死亡率的降低和死亡时间的延长。在这些研究中，我们 然而，值得注意的是，只有由大肠杆菌衍生的EF组成的ET才有这种作用，并且EF产生于 炭疽杆菌没有造成任何死亡。为了解决这一差异，我们通过 NIAID/BEI合同，我们正在对这两个来源的ET进行直接比较 酶和体外毒素活性，以及在小鼠和兔体内的作用。这项工作正在进行中，但 毒素效应(体外和体内)似乎有显著的和可重复的差异。 这两种制剂之间的EF。 在这个职业发展奖的第二年，我们将继续研究ET对COX-2的影响 不同细胞类型的诱导，测量肥大细胞(小鼠和人)的组胺释放 暴露于ET处理的DRG细胞的条件培养液中，表征药理作用 拮抗剂对ET相关临床体征、实验室异常、病理改变和死亡的影响 并利用COX-2和前速激肽原A基因敲除小鼠和肥大细胞缺陷小鼠研究 前列腺素、P物质和肥大细胞在内毒素诱导的小鼠病理/死亡中的作用

The objectives of this project, which is supported by a MARGE Career Development Award, are: 1) to dentify the cell types through which ET initiates and sustains the release of mediators, such as histamine, prostanoids and neurokinins, that are involved in ET-mediated edema and; 2) determine if these same mediators are involved in/responsible for ET-induced death in mice. Although we have demonstrated that ET s able to elicit COX-2 expression, using HEK cells, we have not observed an increase in COX-2 mRNA over basal, in human monocyte-derived monocytes. On the other hand, ET elicits a small increase in PGE2 synthesis from RAW264.7 macrophages and the effect is synergistic with LPS-induced prostaglandin production in these cells. To address the hypothesis that the mediator cascade initiated by ET might begin with or include direct stimulation of Substance P (SP) release from neuronal cells, dorsal root ganglion (DRG) cells were isolated from rats and rabbits and exposed to ET in vitro. Thus far, we have not detected a statistically significant release of SP from either of these cells types. Based on our earlier work showing a histamine (H1) antagonist (pyrilamine) and an inhibitor of COX-2 (celecoxib) cause a reduction in edema produced in rabbit skin by intradermal ET, we have tested these to drugs for their effect on ET-induced mortality in of BALB/cJ mice. While neither drug had a significant effect alone, the combination resulted in a reduced mortality and prolonged time to death. In these studies, we noted however, that only ET composed of E. coli-derived EF had this effect and the EF produced in B.anthracis did not cause any mortality. In order to address that discrepancy, we obtained funding through an NIAID/BEI contract, for which we are doing a direct comparison of the ET from these two sources for enzymatic and in vitro toxin activities, as well as in vivo effects in mice and rabbits. This work is ongoing, but there appears to be a significant and reproducible difference in the toxin effects (both in vitro and in vivo) between these two preparations of EF. In the second year of this career development award, we will continue to study the effect of ET on COX-2 induction in different cell types, measure histamine release from mast cells (murine and human) that are exposed to the conditioned medium of ET-treated DRG cells, characterize the effects of pharmacological antagonists on ET-associated clinical signs, laboratory abnormalities, pathological changes, and death in mice and utilize COX-2 and preprotachykinin A-knockout mice, and mast cell-deficient mice to investigate the roles of prostanoids, substance P and mast cells in ET-induced murine pathology/mortality.