PMN MOTILITY AND ADHERENCE IN NEONATES

该项目的具体目标是解决以下假设： 外周血中白细胞黏附机制的表达和/或功能 新生儿导致急性炎症缺陷并增加 对细菌感染的敏感性。这项提议的重点 将研究中性粒细胞定位的黏附机制。 在急性炎症期间的组织中，以及在组织中的特定缺陷 刚出生的。实验设计将遵循与 体外分离的分子机制和功能以确定 具体的新生儿缺陷，然后检查可能的贡献 每种缺陷对减少新生动物体内炎症的影响 模特。体外研究将建立白细胞/内皮细胞的模型 流动条件下的黏附，条件下的白细胞聚集 定义的切应力，有无内皮细胞迁移 已定义的趋化梯度和依从性机制 白细胞运动；这些研究将利用特定的探针 每种已知的粘连机制(例如，单抗，Ig-嵌合体， 转染细胞系等)。体内研究将利用新生儿 兔子作为人类新生儿的模型，我们有证据表明，新生儿 兔子表现出一些相同的主要缺陷 新生儿中性粒细胞体外培养。具体目标1.明确具体目标 新生儿中性粒细胞对内皮细胞的边际化缺陷 流动状态下的血小板单层。这涉及到调查 关于L-选择素缺乏症的分子基础，P-选择素- 和E-选择素依赖的途径，因为它们对白细胞有贡献 边集作用(滚动粘连)和β2整合素依赖机制 阻止白血球滚动。具体目标2.明确具体目标 新生儿中性粒细胞同型和异型聚集缺陷 在定义的剪切条件下。这涉及对L的调查-- 选择素和P选择素依赖的机制适用于 CD11b/CD18黏附及黏附动力学研究 去粘连。具体目标3.界定#年具体赤字 新生儿中性粒细胞的内皮迁移。这涉及到 黏附依赖的运动、迁移反应机制 细胞因子刺激的内皮细胞，以及对 趋化梯度(如IL-8)。具体目标4.定义 每种体外黏附机制缺陷对减少的贡献 兔新生儿模型中的急性炎症。这涉及到评估 急性炎症对非特异性刺激的反应，评估 利用活体显微镜观察粘附级联，并对 特定细菌(例如，B组链球菌)。

The specific aims of this project address the hypothesis that impaired expression and/or function of leukocyte adhesive mechanisms in the neonate leads to deficits in acute inflammation and increased susceptibility to bacterial infection. The focus of this proposed research will be on the adhesive mechanisms by which neutrophils localize in tissue during acute inflammation, and the specific deficits in the neonate. The experimental design will follow the pattern of working with isolated molecular mechanisms and functions in vitro to define the specific neonatal deficits, and then examining the possible contribution of each deficit to reduced inflammation in vivo in a neonatal animal model. The studies in vitro will model leukocyte/endothelial cell adhesion under conditions of flow, leukocyte aggregation under conditions of defined shear stress, transendothelial migration with and without defined chemotactic gradients, and adherence-dependent mechanisms of leukocyte motility; and these studies will utilize specific probes for each of the known adhesive mechanisms (e.g., MAbs, Ig-chimeras, transfected cell lines, etc). The studies in vivo will utilize neonatal rabbits as a model of human neonates since we have evidence that neonatal rabbits exhibit some of the same major deficits that have been seen in neonatal human neutrophils in vitro. Specific Aim 1. Define the specific deficits in margination of neonatal neutrophils on endothelial cell and platelet monolayers under conditions of flow. This involves investigation of the molecular basis for the L-selectin deficit, studies of P-selectin- and E-selectin-dependent pathways as they contribute to leukocyte margination (rolling adhesion), and beta2 integrin-dependent mechanisms for stopping rolling leukocytes. Specific Aim 2. Define the specific deficits in homotypic and heterotypic aggregation of neonatal neutrophils under defined shear conditions. This involves investigation of L- selectin-, and P-selectin-dependent mechanisms as they apply to aggregation as well as studies of the kinetics of CD11b/CD18 adhesion and de-adhesion. Specific Aim 3. Define the specific deficits in transendothelial migration of neonatal neutrophils. This involves adherence-dependent mechanisms of motility, migration in response to cytokine-stimulated endothelial cells, and migration in response to chemotactic gradients (e.g., IL-8). Specific Aim 4. Define the contribution of each in vitro deficit in adhesive mechanisms to reduced acute inflammation in a rabbit neonatal model. This involves assessment of acute inflammation in response to nonspecific stimuli, evaluation of the adhesion cascade using intravital microscopy, and responses to specific bacteria (e.g., group B Streptococcus).