P GINGIVALIS MEMBRANE BLEBS-MODULATORS OF PMN FUNCTION

厌氧菌通常是从临床感染中恢复的 遍及全身。类杆菌物种数量的增加，作为 一种多微生物区系，最常与 慢性化脓性感染。类杆菌的诱导能力 对混合感染的慢性化已得到证实，但尚不清楚 尽管有人认为它们的影响可能是在宿主防御上 细胞，主要是中性粒细胞(PMN)。渐进式 破坏性牙周病变含有数量增加的卟啉单胞菌 牙龈杆菌(前身为牙龈类杆菌)，我们假设这是 微生物通过影响中性粒细胞影响疾病进展 在牙周环境中发挥作用。我们已经证明了一些 牙龈假单胞菌的亲本菌株会影响中性粒细胞的功能，而其他菌株则不会。 PMN调节活性位于外膜，可能不是 暴露在被严密包裹的菌株中。然而，菌株释放了大量的 膜泡进入细胞外环境及我们的初步研究 研究表明，以前的“PMN-非反应性”亲本菌株会释放“PMN- “反应性”膜泡。 因为在生理应激时，滤过泡释放会增加 与牙周爆裂相吻合，并且是牙周爆裂的重要指标 销毁，这项建议的具体目标是针对(1) 对人体生理生化影响的表征 外周血中性粒细胞、牙龈假单胞菌和外膜蛋白，(2) 确定气泡和外周是否存在PMN调节活性 先前被描述为“PMN-非反应”菌株的膜，以及(3) 沙门氏菌主要外膜成分的分离和鉴定 牙龈假单胞菌。我们将表征来自初始膜的PMN响应 通过动员细胞内第二信使的去极化，以 激活呼吸爆发、脱颗粒和中性粒细胞运动。 这些检测将在对趋化性和非趋化性 趋化刺激与膜对反应的调节 将对气泡和外膜蛋白进行评估。PMN调制 将检查和比较滤过泡和外膜的活性。 各种先前被描述为“PMN-反应性”和 “PMN-无反应”亲本菌株。PMN调节分子将被分离 使用Zwitterant洗涤剂提取细菌表面成分 然后进行层析和电泳法分离纯化 用PMN检测作为生物活性的检测。

Anaerobic bacteria are frequently recovered from clinical infections throughout the body. Increased numbers of Bacteroides species, as part of a polymicrobial flora, are most often associated with the presence of chronic, purulent infections. The ability of Bacteroides species to induce chronicity to mixed infection has been demonstrated but not understood although it has been suggested that their effect may be on host defense cells, primarily the polymorphonuclear leukocyte (PMN). Progressive destructive periodontal lesions contain increased numbers of Porphyromonas gingivalis (formerly Bacteroides gingivalis) and we hypothesize that this microorganism exerts its effect on disease progression by affecting PMN function in the periodontal environment. We have demonstrated that some parent strains of P. gingivalis affect PMN function whereas others do not. The PMN-modulating activity located in the outer membrane and may not be exposed in heavily encapsulated strains. However, strains release numerous membrane blebs into the extracellular environment and our initial studies have shown that previously "PMN-unreactive" parent strains release "PMN- reactive" membrane blebs. Since bleb release is increased at times of physiologic stress and may coincide with, and be a important indicator of, bursts of periodontal destruction, the specific aims of this proposal are directed towards (1) characterizing the physiological and biochemical effects on human peripheral blood PMN P. gingivalis blebs and outer membrane proteins, (2) identifying the presence of PMN-modulating activity in the blebs and outer membranes of previously characterized "PMN-unreactive" strains, and (3) isolating and characterizing the responsible outer membrane components of P. gingivalis. We will characterize the PMN response from initial membrane depolarization through mobilization of intracellular second messengers, to activation of the respiratory burst, degranulation and PMN locomotion. These assays will be performed in response to chemotactic and non- chemotactic stimulation and the modulation of the responses by membrane blebs and outer membrane proteins will be evaluated. PMN-modulating activity will be examined and compared for blebs and outer membrane components of a variety of previously characterized "pmn-reactive" and "PMN-unreactive" parent strains. PMN-modulating molecules will be isolated using Zwittergent detergent extraction of the bacterial surface components followed by chromatographic and electrophoretic separation and purification using PMN assays as a test for biological activity.