INTESTINAL TOXIN RECEPTORS AND PATHOGENESIS OF DIARRHEA

本建议旨在确定 肠道对志贺氏菌毒素的反应。 我们最近发现 一个年龄依赖的，成熟的模式，在外观上的 志贺氏菌糖脂结合位点，神经酰胺三己糖（Gb 3） 毒素，并获得了初步数据 这表明毒素的分泌作用取决于 Gb 3的出现。 该提案的主要目标是 描述Gb 3的发育调控，并确认 Gb 3是小肠中的功能性受体 的 人类新生儿对临床志贺氏菌病的相对耐药性可能 与受体的发育模式有关， 志贺氏菌毒素在小肠中。 这些实验将 包括纯化和定量来自小兔子Gb 3 肠在不同年龄，以建立年龄依赖性， Gb 3的定量差异作为 肠道对志贺氏菌毒素的功能反应。 糖脂将在DEAE-Sephadex上提取和纯化 色谱和Florisil或Unisil柱，并通过 高效液相色谱法 Gb 3在近端和远端的定位和分布 远端小肠，在隐窝和绒毛细胞和亚细胞 分数，将在不同年龄进行研究。 这些实验将 描绘毒素受体的特异性膜结构域， 因此，将解释观察到的响应变化， 毒素在小肠的不同水平。 为了确认 Gb 3是参与毒素介导的 过程中，Gb 3胶束将插入到耐火材料中 新生家兔的肠道，以使它们对 毒素和Gb 3脂质体的分泌作用， 单克隆抗Gb 3抗体将用于阻断毒素- 介导结扎回肠袢中的液体反应。 监管 Gb 3的发育模式所涉及的机制将是 通过测量特定的半乳糖基转移酶活性来评价， 由于初步数据显示Gb 3水平降低， 在新生儿中有高水平的前体。 激素 还将研究Gb 3合成调节的调节。 所描述的研究将阐明发育控制 Gb 3在不同年龄家兔体内的表达和功能。 这些研究可能 为志贺氏菌毒素的发病机制提供了新的见解 介导的腹泻，一个主要的健康问题， 发达国家

This proposal is designed to identify the developmental pattern of intestinal response to shigella toxin. We have recently identified an age-dependent, maturational pattern in the appearance of the glycolipid binding site, globotriaosylceramide (Gb3), for shigella toxin in rabbit small intestine, and have obtained preliminary data which suggest that the secretory effects of the toxin depend upon the appearance of Gb3. The major goals of this proposal are to characterize the developmental regulation of Gb3 and to confirm that Gb3 is the functional receptor in the small intestine. The relative resistence of human neonates to clinical shigellosis could be related to a developmental pattern of the receptors for shigella toxin in the small intestine. These experiments will include purification and quantitation of Gb3 from rabbit small intestine at various ages in order to establish age-dependent, quantitative difference in Gb3 as the basis for variations in functional responsiveness of the intestine to shigella toxin. Glycolipids will be extracted and purified on DEAE-Sephadex chromatography and Florisil or Unisil columns and quantitated by HPLC. The localization and distribution of Gb3 in proximal and distal small intestine, in crypt and villus cells and in subcellular fractions, will be studied at various ages. These experiments will delineate the specific membrane domain of the toxin receptor and will thus explain the observed variations in the response to the toxin at various levels of the small intestine. In order to confirm that Gb3 is the functional receptor involved in the toxin mediated process, Gb3 micelles will be inserted into the refractory intestine of newborn rabbits in order to render them sensitive to the secretory effects of the toxin, and Gb3 liposomes and monoclonal anti-Gb3 antibodies will be used to block the toxin- mediated fluid response in ligated ileal loops. The regulatory mechanisms involved in the developmental patter of Gb3 will be evaluated by measuring specific galactosyltransferase activity at various ages since preliminary data reveal decreased levels of Gb3 with high levels of its precursors in neonates. Hormonal modulation of the regulation of Gb3 synthesis will also be studied. The studies described will elucidate the developmental control and function of Gb3 in rabbits at various ages. These studies may provide new insights into the pathogenesis of shigella toxin mediated diarrhea, a major health problem in both developing and developed countries.