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

描述（申请人提供）：最近，我们发现卟啉单胞菌。牙龈菌是一种周围病原体，根据生长培养基中的血红蛋白浓度改变其LPS脂质a组成。我们已经获得了富含在低和高血红素浓度下发现的不同脂质A质量离子的牙龈卟啉卟啉LPS制剂，并表明这两种牙龈卟啉卟啉LPS制剂都与TLR4相互作用。然而，它们对人内皮细胞E选择素的表达有相反的影响。在这篇R21探索性提案中，我们建议进一步探索这两种不同牙龈卟啉卟啉脂多糖可能的差异基因调控程序。三种不同的LPS制剂，Pgips，含有所有已知的脂质A种，Pg^s/uso，富集在高血红蛋白浓度下发现的脂质A种，和iego，富集在低血红蛋白浓度下发现的脂质A种，将被添加到人内皮细胞中，并进行微阵列分析。此外，两种不同的大肠杆菌LPS制剂，一种是野生型（ECwt），另一种是从msbB突变体（EcmSbB）获得的效力明显较低的形式，将被检验并作为对照。将使用Affymetrix全基因组芯片，其中包含代表所有人类基因的47,000多个转录本。这种方法将允许我们测试我们的假设：牙龈卟卟菌选择性地改变其脂质A组成，以改变内皮细胞功能，当它在血液富集的环境中。内皮细胞功能的改变可能导致牙周袋血管溃疡和/或可能促进生物体的全身传播。在与华盛顿大学表达阵列中心的合作下，内皮细胞基因调节程序将被阐明每种LPS制剂。经过三次重复分析和统计测定后，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，通过聚类分析对LPS制剂之间的异同进行功能分类。根据这些参数选择的基因将被Real Time PCR确认。此外，还将鉴定一些依赖TLR4激活的基因。这一信息将确定不同的LPS物种是否会引发不同的内皮细胞激活程序，并将形成一个数据集来检验关于牙龈假单胞菌与TLR4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沉积在欧洲生物信息学研究所阵列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.