SIALIC ACID MODULATION REGULATES NEUTROPHIL DIAPEDESIS

描述（改编自申请人的摘要）：唾液酸是 作为糖缀合物存在于哺乳动物细胞上的氨基糖， 赋予这些细胞负表面电荷。唾液酸的损失 发生在骨髓成熟和激活期间，并伴随 通过增强运动性、变形性和收缩性。唾液酸酶是 从糖缀合物中快速除去唾液酸残基的酶 并且在中性粒细胞活化时迅速移动到质膜。 相反，通过将唾液酸残基加回糖缀合物上， 唾液酸转移酶（ST）可以将糖缀合物恢复到预活化的 状态因此，两者上唾液酸残基的量和分布都是相同的。 中性粒细胞和内皮细胞（EC）可能代表了一种快速的 中性粒细胞对局部炎症反应的适应性机制 刺激并离开循环。在具体目标I中，PI将检查 唾液酸对中性粒细胞各阶段的调节作用 腹膜透析：滚动粘连、牢固粘连和跨内皮粘连 迁移（TEM）。将添加静息或活化的人中性粒细胞 到EC单层，无论是休息或激活短效 （组胺）或长效（TNF）刺激或用 去甲肾上腺素酶将测量中性粒细胞粘附和TEM， 对IL-8或缓冲液的应答（自发）。PMN的作用 唾液酸酶将使用以下药物抑制剂进行评估： 唾液酸酶和多克隆抗唾液酸酶抗体先前显示， 抑制中性粒细胞唾液酸酶。中性粒细胞ST的表达 将测定唾液酸酶。在具体目标II中，申请人将 确定唾液酸调节EC单层的作用， 中性粒细胞粘附和TEM。内源性EC的初步证据 唾液酸酶将通过酶活性和免疫活性两者来延伸。 侦测在《特定目的III》中，他将确定同源性的程度， 中性粒细胞唾液酸酶与最近描述的人唾液酸酶克隆 从淋巴母细胞的MHC区域，并可能发挥作用， 在T细胞活化中。将纯化中性粒细胞唾液酸酶， 从结合到抗唾液酸酶抗体的溶胞产物洗脱的溶胞产物测序， 固体基质这些序列将用于构建引物， 从人HL-60细胞文库中克隆唾液酸酶。内皮 细胞唾液酸酶将被分离、克隆并与嗜中性粒细胞进行比较 Aim III中的唾液酸酶。这些研究将确定一种新的机制， 快速适应，时间限制的局部反应， 炎症该提案将提供新的目标和额外的 治疗炎症的方法。

DESCRIPTION (Adapted from the applicant's abstract): Sialic acids are amino sugars that are present on mammalian cells as glycoconjugates and impart to these cells a negative surface charge. The loss of sialic acid occurs during both myeloid maturation and activation and is accompanied by enhanced motility, deformability and adhesiveness. Sialidases are enzymes that rapidly remove sialic acid residues from glycoconjugates and on neutrophil activation are rapidly mobilized to plasma membrane. In contrast, by adding sialic acid residues back onto glycoconjugates, sialyltransferases (ST) may restore glycoconjugates to the preactivated state. Thus, the amount and distribution of sialic acid residues on both neutrophils and perhaps endothelial cells (EC) may represent a rapidly adaptable mechanism by which neutrophils respond to local inflammatory stimuli and leave the circulation. In Specific Aim I the PI will examine the effect of sialic acid modulation at each stage of neutrophil diapedeis: rolling adhesion, firm adhesion and transendothelial migration (TEM). Resting or activated human neutrophils will be added to EC monolayers that are either resting or activated with short-acting (histamine) or long acting (TNF) stimuli or treated with neutroaminidase. Neutrophil adherence and TEM will be measured in response to either IL-8 or to buffer (spontaneous). The role of PMN sialidase will be assessed with the use of pharmacologic inhibitors of sialidase and a polyclonal antisialidase antibody previously shown to inhibit neutrophil sialidase. The expression of PMN ST relative to sialidase will be determined. In Specific Aim II the applicant will determine the role of sialic acid modulation of EC monolayers in neutrophil adherence and TEM. Preliminary evidence for an endogenous EC sialidase will be extended by both enzymatic activity and immune detection. in Specific AIM III he will determine the degree of homology of neutrophil sialidase with a recently described human sialidase cloned from the MHC region of lymphoblastoid cells and which may play a role in T cell activation. Neutrophil sialidase will be purified and sequenced from lysates eluded from antisialidase antibody bound to a solid matrix. These sequences will be used to construct primers from cloning of the sialidase from a human HL-60 cell library. Endothelial cell sialidase will be isolated, cloned and compared to neutrophil sialidase in Aim III. These studies will define a new mechanism for a rapidly adaptable, temporally- restricted local response to inflammation. This proposal will provide new targets and additional approaches to the treatment of inflammation.