P. gingivalis lipid A species modulation of endothelial cell gene activation prog

描述（由申请人提供）：最近，我们发现了卟啉单胞菌。牙龈炎是一种牙周病病原体，改变其LPS脂质A组成，以响应生长培养基中的氯化血红素浓度。我们已经获得了富含在低和高氯化血红素浓度下发现的不同脂质A质量离子的牙龈卟啉单胞菌LPS制剂，并且已经表明这两种牙龈卟啉单胞菌LPS制剂都与TLR 4相互作用。然而，它们对来自人内皮细胞的E选择素表达具有相反的作用。在此R21探索性提议中，我们提议进一步探索这两种不同牙龈卟啉单胞菌LPS物种的可能差异基因调节程序。三种不同的LPS制剂，Pgips，含有所有已知的脂质A种类，Pg^s/uso，在高氯高铁血红素浓度下富含脂质A种类，和iego，在低氯高铁血红素浓度下富含脂质A种类，|加入到人内皮细胞中并进行微阵列分析。此外，将检查两种不同的大肠杆菌LPS制剂，一种野生型（ECwt），另一种从msbB突变体（EcmSbB）获得的效力显著降低的形式，并将其作为对照。将使用包含代表所有人类基因的47，000多个转录本的Affyestival全基因组芯片。这种方法将使我们能够测试我们的假设：牙龈卟啉单胞菌选择性地修改其脂质A组成，以改变内皮细胞功能时，它在血液丰富的环境。内皮细胞功能的改变可能导致牙周袋中的脉管系统溃疡和/或可能促进生物体的全身传播。与华盛顿大学的表达阵列中心合作，将阐明这些LPS制剂中每一种的内皮细胞基因调节程序。在一式三份分析和统计测定后，将利用GoMiner、GenMAPP和外部数据库的组合分析功能和途径组的基因表达谱，以通过聚类分析对LPS制剂之间的差异和相似性进行功能分类。基于这些参数，将通过真实的时间PCR确认选择的基因。此外，将鉴定选择基因对TLR 4活化的依赖性。该信息将确定不同的LPS种类是否引起不同的内皮细胞活化程序，并将形成数据集以检验关于牙龈卟啉单胞菌与TLR 4或其他新型LPS受体相互作用的假设。此外，微阵列分析结果将通过存放在欧洲生物信息学研究所Array Express数据库中公开。

DESCRIPTION (provided by applicant): Recently, we have discovered that Porphyromonas. gingivalis, a periopathogen, alters its LPS lipid A composition in response to the hemin concentration in the growth medium. We have obtained P. gingivalis LPS preparations that are enriched in the different lipid A mass ions found at low and high hemin concentrations and have shown that both of these P. gingivalis LPS preparations interact with TLR4. However, they have opposing effects on E selectin expression from human endothelial cells. In this R21 exploratory proposal we propose to further explore the possible differential gene modulation programs by these two different P. gingivalis LPS species. Three different LPS preparations, Pgips, containing all the known lipid A species, Pg^s/uso, enriched in the lipid A species found at high hemin concentrations, and iego, enriched in the lipid A species found at low hemin concentrations wil| be added to human endothelial cells and a microarray analysis will be performed. In addition, two different preparations of Escherichia coli LPS, one wild type (ECwt)and the other a significantly less potent form obtained from an msbB mutant (EcmSbB) will be examined and serve as controls. Affymetrix full genome chips which contain more than 47,000 transcripts representing all human genes will be utilized. This approach will allow us to test our hypothesis that: P. gingivalis selectively modifies its lipid A composition to alter endothelial cell function when it's in a blood enriched environment. Alterations in endothelial cell function may contribute to vasculature ulceration in periodontal pockets and/or may facilitate systemic dissemination of the organism. In collaboration with the Center for Expression Arrays at the University of Washington the endothelial cell gene modulation programs will be elucidated for each of these LPS preparations. After triplicate analysis and statistical determinations, gene expression profiles will be analyzed for functional and pathway groups utilizing a combination of GoMiner, GenMAPP and external databases to functionally categorize the differences and similarities among the LPS preparations by cluster analysis. Based on these parameters select genes will be confirmed by Real Time PCR. In addition, select genes will be identified for their dependence upon TLR4 for activation. This information will determine if different LPS species elicit different endothelial cell activation programs and will form a data set to test hypothesis concerning P. gingivalis interactions with TLR4 or other novel LPS receptors. Furthermore, the microarray analysis results will be made public by deposition at the European Bioinformatics Institute Array Express database.