40- to 100-kD protein(s) of Helicobacter pylori stimulate DNA synthesis in epithelial cell lines without affecting apoptosis.

BACKGROUND: Previous in vitro studies have demonstrated that water extracts and sonicates of Helicobacter pylori increase DNA synthesis in a small intestinal epithelial cell line. The aim of this study was to identify mitogenic factor(s) in a water extract of a H. pylori strain and to examine their effects on DNA synthesis and apoptosis in vitro. METHODS: IEC-6 and FHs 74 cells were incubated for 24 h with different dilutions of a water extract of H. pylori (cytotoxic strain 88-23) or with 6 protein fractions obtained by gel filtration. Cells were labeled with tritiated thymidine and processed for autoradiography. DNA synthesis was evaluated by the labeling index (LI%). The proportion of IEC-6 cells undergoing apoptosis and/or necrosis was evaluated by flow cytometry using fluorescein isothiocyanate (FITC)-labeled annexin-V and propidium iodide. In vitro caspase activity was also determined as an alternative method for detection of apoptosis. RESULTS: The water extract of H. pylori 88-23 markedly increased DNA synthesis in both epithelial cell lines (p < 0.01). A marked stimulation of DNA synthesis was also observed in IEC-6 cells incubated with fraction II- containing proteins of a molecular weight ranging between 40 and 100 kD (p < 0.01). A lesser stimulation of DNA synthesis was observed in cells incubated with higher concentrations of the other protein fractions (p < 0.01). Neither the water extract of H. pylori 88-23 nor the protein fraction II (40-100 kD) induced apoptosis in IEC-6 cells. CONCLUSION: A water extract of H. pylori 88-23 and a protein fraction containing proteins with molecular weights of 40-100 kD stimulate DNA synthesis in a rat and human small intestinal cell line. Apoptosis was unaffected by the water extract and by protein fraction II, which indicate that the H. pylori-derived mitogen(s) have the capacity to directly enhance epithelial cell proliferation in vitro.

In vitro aging of Helicobacter pylori: changes in morphology, intracellular composition and surface properties.

BACKGROUND: During the conversion from the bacillary into the coccoid form, Helicobacter pylori organisms are known to change extensively. The aim of this study was to determine some of the changes that occur regarding morphology, intracellular composition and surface properties during the aging of bacteria in vitro. MATERIALS AND METHODS: H. pylori from agar plate cultures of different ages was used in this study. The intracellular composition of the two morphological forms of the bacteria was tested by density centrifugation, DNA extraction and quantitative OD, mRNA and ATP measurements. Immunoblotting was used to observe changes in secreted/superficial protein patterns, and hydrophobicity measurements were used to observe changes in surface properties. RESULTS: All bacillary H. pylori organisms changed morphology gradually over 10 days of culture. Rods had a higher density than cocci; bacteria stored in PBS had the highest density and bacteria stored in water had the lowest. The quantitative DNA, RNA and ATP content were reduced in the aging bacteria. Fewer immunogenic proteins were expressed, and an increased surface hydrophobicity was observed in the older cultures. CONCLUSION: This study highlights several aspects of H. pylori aging in vitro and shows some of the differences that exist between bacillary and coccoid forms. This information is important for understanding the transmission and survival of H. pylori outside the human host, as the degradative changes in the intracellular composition and the surface properties shown here point to dead bacteria, and not to a viable but nonculturable form.

[Development of resistant Helicobacter pylori in Sweden. Tendency toward increasing resistance to clarithromycin]. / Resistensutveckling hos Helicobacter pylori i Sverige. Tendens till ökning av resistens mot klaritromycin.

This study was designed to investigate the development of antibiotic resistance in Swedish H pylori isolated 1990-96. A total of 415 isolates collected from 10 clinical microbiology laboratories were examined. Three different methods of susceptibility testing were compared: agar dilution, the E-test and disc diffusion. None of the isolates was resistant to ampicillin or tetracycline, but approximately 30 per cent were resistant to metronidazole. An increase in resistance to clarithromycin was noted during 1996 (9% resistant strains), though the significance of this finding needs further investigation. The comparison of susceptibility tests suggested disc diffusion to be of doubtful value, but the E-test to be appropriate for testing H pylori. However, the number of isolates resistant to metronidazole was higher with the E-test than with agar dilution. The value of testing susceptibility to metronidazole is discussed.