INTESTINAL MECHANISMS OF C DIFFICILE TOXINS

本提案的总体目标是阐明 C.艰难梭菌毒素损害肠道。 净化后的毒素 使用顺序离子交换从肉汤培养物上清液制备 色谱法、高效液相色谱法（HPLC），和 用单克隆抗体进行免疫亲和层析。 的氨基酸 纯化毒素的组成和序列将与其他毒素进行比较。 已知的细菌肠毒素和细胞毒素， 使用去污剂将毒素转化为结合和催化亚单位， 试剂和尿素。 毒素A和B的机制将在体外使用成纤维细胞进行研究。 和肠细胞系。 因为毒素B会引起 成纤维细胞和平滑肌细胞的微丝，我们将进一步 探索毒素B对其他细胞骨架和基质蛋白的影响， 成纤维细胞以及暴露于毒素B的肠上皮细胞。 将通过免疫荧光法测定细胞骨架和基质蛋白 显微镜和SDS-PAGE。 毒素对细胞内钙离子的影响 将探索靶细胞中的释放。 我们最近展示了 毒素A可引起人体细胞内游离Ca ~（++）浓度急剧升高， 中性粒细胞 我们将测定细胞内钙离子浓度， 其他靶细胞包括肠上皮细胞。 这些结果将 与磷脂酶C激活的测量相关， 靶细胞中细胞蛋白的磷酸化。 这些研究应 阐明毒素A和B激活的细胞机制。 将在家兔回肠和结肠中研究毒素的体内效应 循环 我们将确定纯化毒素A和B的影响，或 两者的混合物，对肠上皮细胞形态，细胞骨架蛋白，流体 分泌、渗透性和大分子合成。 我们会研究 中性粒细胞和肥大细胞对肠炎的作用 环氧合酶和脂氧合酶途径的药理学阻断剂，氧 自由基清除剂和抗组胺剂。 最后，我们将研究受体与放射性标记毒素A和B的结合， 靶细胞和动物及人类大小刷状缘膜 肠子 由于感染C. difficile似乎不会导致 疾病的新生儿和婴儿，我们建议测量毒素结合， 肠上皮细胞在不同阶段的出生后发展，以确定是否 生命早期缺乏毒素结合与缺乏肠道反应相关。

The overall goals of this proposal are to elucidate the mechanisms by which C. difficile toxins damage the intestinal tract. Purified toxins will be prepared from broth culture supernatants using sequential ion exchange chromatography, high performance liquid chromatography (HPLC), and immunoaffinity chromatography with monoclonal antibodies. The amino acid composition and sequence of purified toxins will be compared with other known bacterial enterotoxins and cytotoxins and will attempt to dissociate toxins into binding and catalytic sub-units using detergents, reducing agents and urea. The mechanisms of toxins A and B will be studied in vitro using fibroblast and intestinal cell lines. Since toxin B causes profound changes in microfilaments of fibroblasts and smooth muscle cells, we will further explore the effects of toxin B on other cytoskeletal and matrix proteins in fibroblasts as well as intestinal epithelial cells exposed to toxin B. Cytoskeletal and matrix proteins will be assayed by immunofluorescent microscopy and SDS-PAGE. The effects of toxins on intracellular calcium release in target cells will be explored. We have recently demonstrated that toxin A causes a dramatic rise in intracellular ionized Ca++ in human neutrophils. We will determine intracellular calcium concentration in other target cells including the enterocyte. These results will be correlated with measurements of phospholipase C activation and phosphorylation of cellular proteins in target cells. These studies should elucidate the cellular mechanisms activated by toxins A and B. The in vivo effects of toxins will be studied in rabbit ileal and colonic loops. We will determine the effects of purified toxins A and B, or mixtures of the two, on enterocyte morphology, cytoskeletal proteins, fluid secretion, permeability and macro-molecular synthesis. We will study the contributions of neutrophils and mast cells to the enteritis by using pharmacologic blockers of cyclooxygenase and lipoxygenase pathways, oxygen radical scavengers and antihistamines. Finally, we will study receptor binding to radiolabelled toxins A and B to target cells and brush border membranes of animal and human small and large intestine. Because infection with C. difficile appears not to cause disease in newborns and infants, we propose to measure toxin binding to enterocytes at various stages of post-natal development to determine if lack of toxin binding in early life correlates with lack of gut response.