Novel Lipid Mediators in LAP and Resolution

该提案的目标是确定某些脂质第二信使分子的确切作用[即，二酰基甘油（DAG）]和内源性介质[消退素、二十二碳三烯（DT）、脂氧素]在牙周炎症中的作用，并继续开发新的分子策略来阻断炎症级联反应。我们将专注于局部侵袭性牙周炎（局限性，以前称为局限性青少年牙周炎，LJP），它提出了一个经典的中性粒细胞的“致敏”表型，即超氧化物产生在细胞刺激过程中的高反应性。中性粒细胞（PMN）从外周血分离的COPD患者表现出三倍的细胞甘油二酯和膜相关蛋白激酶（PKC）活性增加两到三倍。α-异构体的显著减少 在这些细胞中发生DAG激酶（DGK α）活性的降低，这可能是导致DAG水平升高和引发现象的原因。我们小组的工作（见项目0001）揭示了新的内源性脂质介质（LM），消退素和二十二碳三烯（DT），它们是各种动物模型（包括牙周破坏的兔模型）中有效的天然炎症抑制剂。这些化合物阻断超氧化物的产生和趋化性与某些激动剂刺激的中性粒细胞。为了实现我们的目标，我们将在遗传和生物化学水平上评估负责DGK α活性急剧下降的分子机制，确定由于DAG增加而在PMN-DRG中升高的膜结合PKC的亚型，并确定DAG/膜结合PKC的这些升高如何“引发”NADPH氧化酶复合物在PMN-DRG中产生更多的超氧化物/过氧化物。最后，我们将评估resolvins和docosatrienes对牙周炎症原位的有益作用，并将我们的结果与脂氧素及其稳定类似物的观察结果进行比较。 这些研究将使用分子生物学、生物化学和动物病理生理学技术。

The goal of this proposal is to determine the exact roles of certain lipid second messenger molecules [i.e., diacylglycerols (DAG's)] and endogenous mediators [resolvins, docosatrienes (DT), lipoxins] in periodontal inflammation, and to continue to develop novel molecular strategies to pharmacologically interrupt the inflammatory cascade. We will focus on Localized Aggressive Periodontitis (LAP, formerly known as localized juvenile periodontitis, LJP), which presents neutrophils with a classically "primed" phenotype, i.e. hyper-responsiveness in superoxide production during cell stimulation. Neutrophils (PMN) isolated from peripheral blood of LAP patients exhibit a three fold elevation in cellular diacylglycerol and a two to three fold increase in membrane associated protein kinase (PKC) activity. A marked decrease in the alpha-isoform of DAG kinase (DGKalpha) activity occurs in these cells, which is likely to be responsible for the elevated levels of DAG and the priming phenomena. Work by our group (see Project 0001) has revealed new classes endogenous lipid mediators (LM), the resolvins and docosatrienes (DT), which are potent natural inhibitors of inflammation in a wide variety of animal models including the rabbit model of periodontal destruction. These compounds block superoxide production and chemotaxis in stimulated PMN with certain agonists. To accomplish our goals, we will evaluate the molecular mechanisms responsible for the dramatic decrease in DGKalpha activity in LAP PMN at the genetic and biochemical level, identify the isoforms of membrane-bound PKC that are elevated in LAP PMN due to increased DAG, and determine how these elevations in DAG/membrane bound PKC "prime" the NADPH-oxidase complex to produce more superoxide/peroxide in LAP PMN. Finally, we will evaluate the beneficial effect of resolvins and docosatrienes on periodontal inflammation in situ and compare our results to those observed for lipoxins and their stable analogs. Techniques of molecular biology, biochemistry, and animal pathophysiology will be used in these studies.