? The purpose of this proposal is to provide a means for the applicant, Sarah Keates, to develop as an independent scientist. She plans to carry out the work in this proposal under the guidance of her mentor Dr. Simon Robson and with the support of the gastroenterology division at BIDMC. The Career Development Plan includes course work, meetings, publishing, and mentoring goals. ? The research proposal examines the hypothesis that infection of gastric epithelial cells (GEC's) with the bacterial pathogen Helicobacter pylori causes an upregulation of Plasminogen activator inhibitor-1 (PAI-1), which contributes to gastric cancer progression.
Aim 1 examines how H. pylori upregulates PAI-1 in AGS (GEC's). Using an in vitro system the bacterial products important for the upregulation of PAI-1 and the signaling pathways that are required for PAI-1 upregulation by H. pylori will be determined. To achieve this aim GEC's will be infected with different strains of H. pylori and isogenic mutants, and overexpression of bacterial genes will be carried out. Inhibitors and dominant negative constructs will be used to elucidate signaling pathways required for H. pylori-mediated PAI-1 upregulation. Secreted PAI-1 protein will be measured by ELISA, and mRNA determined by real-time PCR and also through the use of luciferase reporters.
In Specific Aim 2, the functional effects of PAI-1 upregulation by H. pylori will be examined. It shall be determined whether PAI-1 production has an autocrine efect on epithelial cell function, and whether increased levels of PAI-1 induced by H. pylori can modulate migration and invasion of GEC's. Paracrine effects on endothelial cell function will also be examined; in order to ascertain whether PAI-1 production by H. pylori-infected GEC's contributes to angiogenesis. For this aim migration, invasion, proliferation and endothelial tube formation assays will be used.
In Specific Aim 3, it will be determined whether PAI-1 upregulation by H. pylori occurs in vivo and is strain dependent. For these studies patients infected with H. pylori or uninfected controls, will be used to determine if infection with H. pylori results in increased PAI-1 production. PAI-1 protein in serum and biopsies will be measured by ELISA, and mRNA levels in gastric biopsies determined by PCR. In those infected with H. pylori, PAI-1 levels will also be correlated with the genotype of the infecting bacterium. The source of increased PAI-1 production will also be evaluated using immunohistochemistry and laser capture microdissection. ? Results from these studies may lead to a better understanding of H. pylori pathogenesis and also the involvement of PAI-1 in the development and progression of human gastric cancer. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Scientist Development Award - Research & Training (K01)
Project #
1K01DK075942-01
Application #
7138501
Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Program Officer
Podskalny, Judith M,
Project Start
2006-08-01
Project End
2011-07-31
Budget Start
2006-08-01
Budget End
2007-07-31
Support Year
1
Fiscal Year
2006
Total Cost
$116,667
Indirect Cost
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02215
Celli, Jonathan P; Turner, Bradley S; Afdhal, Nezam H et al. (2009) Helicobacter pylori moves through mucus by reducing mucin viscoelasticity. Proc Natl Acad Sci U S A 106:14321-6
Keates, A C; Tummala, S; Peek Jr, R M et al. (2008) Helicobacter pylori infection stimulates plasminogen activator inhibitor 1 production by gastric epithelial cells. Infect Immun 76:3992-9
Katchar, Kianoosh; Kelly, Ciaran P; Keates, Sarah et al. (2007) MIP-3alpha neutralizing monoclonal antibody protects against TNBS-induced colonic injury and inflammation in mice. Am J Physiol Gastrointest Liver Physiol 292:G1263-71
Keates, Sarah; Keates, Andrew C; Katchar, Kianoosh et al. (2007) Helicobacter pylori induces up-regulation of the epidermal growth factor receptor in AGS gastric epithelial cells. J Infect Dis 196:95-103
Keates, Sarah; Han, Xinbing; Kelly, Ciaran P et al. (2007) Macrophage-inflammatory protein-3alpha mediates epidermal growth factor receptor transactivation and ERK1/2 MAPK signaling in Caco-2 colonic epithelial cells via metalloproteinase-dependent release of amphiregulin. J Immunol 178:8013-21