C. M. Habibullah

The cag Pathogenicity Island of Helicobacter pylori Is Disrupted in the Majority of Patient Isolates from Different Human Populations

ABSTRACT The cag pathogenicity island (cag-PAI) is one of the major virulence determinants of Helicobacter pylori. The chromosomal integrity of this island or the lack thereof is speculated to play an important role in the progress of the gastroduodenal pathology caused by H. pylori. We determined the integrity of the cag-PAI by using specific flanking and internally anchored PCR primers to know the biogeographical distribution of strains carrying fully integral cag-PAI with proinflammatory behavior in vivo. Genotypes based on eight selected loci were studied in 335 isolates obtained from eight different geographic regions. The cag-PAI appeared to be disrupted in the majority of patient isolates throughout the world. Conservation of cag-PAI was highest in Japanese isolates (57.1%). However, only 18.6% of the Peruvian and 12% of the Indian isolates carried an intact cag-PAI. The integrity of cag-PAI in European and African strains was minimal. All 10 strains from Costa Rica had rearrangements. Overall, a majority of the strains of East Asian ancestry were found to have intact cag-PAI compared to strains of other descent. We also found that the cagE and cagT genes were less often rearranged (18%) than the cagA gene (27%). We attempted to relate cag-PAI rearrangement patterns to disease outcome. Deletion frequencies of cagA, cagE, and cagT genes were higher in benign cases than in isolates from severe ulcers and gastric cancer. Conversely, the cagA promoter and the left end of the cag-PAI were frequently rearranged or deleted in isolates linked to severe pathology. Analysis of the cag-PAI genotypes with a different biogeoclimatic history will contribute to our understanding of the pathogen-host interaction in health and disease.

Studies on Urea Cycle Enzyme Levels in the Human Fetal Liver at Different Gestational Ages

Urea cycle enzymes involved in the detoxification of ammonia were studied in liver tissues of 57 male and 49 female fetuses of different age groups ranging from 13 to 36 wk of gestation. Surgical wedge biopsies of liver from 18 male and 12 female adults were used as controls. Significant enzyme activity was found to be present as early as the 13th wk of gestation. As gestational age advanced, enzyme activity gradually increased, reaching about 90% of the adult activity by the 36th wk of gestation.

In Vitro Insulin Production and Analysis of Pancreatic Transcription Factors in Induced Human Hepatic Progenitor Cells

BACKGROUND beta-Cell destruction and/or insufficient insulin production are the hallmarks of diabetes mellitus (type 1 diabetes). A hepatic progenitor from developing liver is sought to be one of the surrogate sources of insulin production as the pancreas and the liver share a common precursor and signals from the cardiac mesoderm. Production of insulin is possible by transfecting pancreatic transcription factors that play important roles in development of the pancreatic beta-cell. But, there is always the fear of using genetically manipulated cells for therapeutics. Hence, the present study was designed to analyze the feasibility of using primary human fetal hepatic progenitors as a potential source for insulin production. METHODS Human fetal hepatic progenitors were enriched using CD-326 magnetic cell sorting. The sorted cells were cultured with different concentrations of glucose (5-30 mM) in Dulbeccos modified Eagles medium. The amount of insulin production was estimated in the cultured cells by the chemiluminescence method. Total RNA isolated from sorted epithelial cell adhesion molecule (EpCAM)-positive cells was reverse-transcribed, and the expression of different beta-cell-producing transcriptions factors was analyzed by polymerase chain reaction (PCR). Immunocytochemical analysis was performed in cultured cells using specific insulin antibodies. RESULTS The viability of the total liver cells isolated was found to be 95%. The average number of EpCAM-positive cells in the total liver was found to be approximately 15%. An insulin kinetics study using glucose induction with different concentrations showed increased insulin secretion in response to glucose concentrations up to 20 mM. Furthermore, results of immunocytochemical analysis demonstrated intense insulin expression in EpCAM-positive cultured cells. Expression studies of the cultured EpCAM-positive cells using reverse transcription-PCR showed positive expression of the pancreatic transcription factors essential for insulin production. CONCLUSIONS The present study demonstrates that in vitro differentiation of induced human hepatic progenitors into insulin-producing cells without genetic manipulations may promote strategies for the treatment of type 1 diabetes.

OL-030 Synergistic interplay between Helicobacter pylori virulence genes and host COX-2 and iNOS enhances the risk of premalignant and malignant lesions

Background and Aim: H. pylori (Hp) strains vary in their carcinogenic potential. Both host factors and bacterial factors have been postulated to contribute to the variable outcome. Over expression of host COX-2 and iNOS has been implicated in the development of gastric carcinoma. Furthermore, the link between genotypes in relation to COX-2 and iNOS expression and disease status needs to be determined. Therefore the present study addressed to identify Hp-bearing hosts who are at greatest risk of developing precancerous lesions. Methods: A total of 240 subjects with various gastric disorders were screened. Genotyping based on cagA, cagE, cagT, vacA signal region and hrgA genes of Hp was performed using DNA from gastric biopsies. Expression of COX-2 and iNOS was assessed by RT-PCR and immunoblotting. Histological scoring of antral and corpus biopsies for the presence precancerous lesions was done. Results: The genotype cagA+/cagE+/cagT+/hrgA+/vacAs1 showed high prevalence 177 (73.7%). Among which 81.1% had overt gastric disorders whereas 46% subjects had less severe gastric disease. Histology revealed presence of atrophy in 52% vs 18%, IM in 32% vs 9% and dysplasia in 20% vs 4% respectively (Statistically significant at p < 0.01). RT-PCR and immunoblotting data showed high expression patterns of COX-2 and iNOS in overt gastric disorders than with less severe gastrointestinal disorders. Conclusion: Genotype cagT+ve/hrgA+ve/cagA+ve/cagE+ve/ vacAs1+ve and heightened expression levels of COX-2 and iNOS have higher differentiating and predictive value for the development of severe disease manifestations. This suggests that Hp induced gastric inflammatory reaction to be influenced by multiple factors, and probably results from the synergistic effect of bacterial virulence and host factors, which work together in a complex way causing various diseases in the host.