Binding of the Helicobacter pylori vacuolating cytotoxin to target cells

The vacuolating cytotoxin of Helicobacter pylori, VacA, enters the cytoplasm of target cells and causes vacuolar degeneration by interfering with late stages of endocytosis. By using indirect immunofluorescence and flow cytometry, we have demonstrated that VacA binds to specific high-affinity cell surface receptors and that this interaction is necessary for cell intoxication.     

Effects of Helicobacter pylori vacuolating cytotoxin on primary cultures of human gastric epithelial cells

BACKGROUND: Many Helicobacter pylori strains produce a cytotoxin that induces cytoplasmic vacuolation in various types of eukaryotic cells. In contrast with the marked cell vacuolation that occurs in vitro in response to this cytotoxin, comparatively little epithelial vacuolation has been observed in the gastric mucosa of H pylori infected persons. AIMS: Experiments were performed to determine the susceptibility of human gastric epithelial cells in vitro to H pylori vacuolating cytotoxin activity. METHODS: Human gastric epithelial cells, harvested from upper gastrointestinal endoscopic biopsy specimens, were incubated overnight with broth culture supernatants from either a wild type cytotoxin producing (tox+) H pylori strain or an isogenic mutant strain that lacks cytotoxin activity. RESULTS: Prominent cytoplasmic vacuolation occurred in response to tox+ supernatant, but not supernatant from the isogenic mutant strain. Primary human gastric epithelial cells were significantly more sensitive to H pylori vacuolating cytotoxin activity than were either HeLa or AGS cells. Exposure of human gastric epithelial cells to high concentrations of tox+ supernatant for 48 hours caused lethal cell injury. CONCLUSIONS: These studies indicate that primary human gastric epithelial cells are highly sensitive to H pylori vacuolating cytotoxin activity.     

The m2 form of the Helicobacter pylori cytotoxin has cell type-specific vacuolating activity

The Helicobacter pylori toxin VacA causes vacuolar degeneration in mammalian cell lines in vitro and plays a key role in peptic ulcer disease. Two alleles, m1 and m2, of the mid-region of the vacA gene have been described, and the m2 cytotoxin always has been described as inactive in the in vitro HeLa cell assay. However, the m2 allele is associated with peptic ulcer and is prevalent in populations in which peptic ulcer and gastric cancer have high incidence. In this paper, we show that, despite the absence of toxicity on HeLa cells, the m2 cytotoxin is able to induce vacuolization in primary gastric cells and in other cell lines such as RK-13. The absence of Hela cell activity is due to an inability to interact with the cell surface, suggesting a receptor-mediated interaction. This result is consistent with the observation that the m2 allele is found in a population that has a high prevalence of peptic ulcer disease and gastric cancer. VacA is the first bacterial toxin described for which the same active subunit can be delivered by different receptor binding domains.     

Enterotoxic effect of the vacuolating toxin produced by Helicobacter pylori in Caco-2 cells

Preliminary clinical evidence suggests that Helicobacter pylori may be associated with diarrhea through its vacuolating toxin (VacA). To establish whether VacA induces intestinal secretion, epithelial damage, or both, purified pH-activated VacA was added to Caco-2 cell monolayers mounted in Ussing chambers, and electrical parameters were monitored. Mucosal addition of VacA induced an increase in short circuit current, consistent with enterotoxic effect. The effect was time- and dose-dependent and saturable. It was not found if the toxin was not pH-activated, added to the serosal side, or preheated. In cells preloaded with the Ca2+ buffering compound BAPTA/AM or with the Cl- channel inhibitor 5-nitro-2-3-(3-phenylpropylamino)benzoic acid, short circuit current did not change, indicating that VacA induces activation of Ca2+-dependent Cl- channels. VacA did not show cytopathic effects, as judged by tissue resistance. These results support the hypothesis that H. pylori may be associated with diarrhea through production of VacA.     

In vitro activity of omeprazole in combination with several antimicrobial agents against clinical isolates of Helicobacter pylori

An agar dilution checkerboard method was used to evaluate the in vitro activity of omeprazole combined with clarithromycin, amoxicillin, and ceftibuten, respectively, against clinical isolates of Helicobacter pylori. Mueller-Hinton agar plus 5% horse blood, an inoculum of 10(6) cfu/ml, and incubation of 72 h in a CO2 atmosphere were used. With the omeprazole and clarithromycin combination, synergism was observed in 51.5% and partial synergism in 39.3% of 33 strains; with omeprazole and amoxicillin, synergism was observed in 3% and partial synergism in 60.6% of 33 strains; and with omeprazole and ceftibuten, synergism was observed in 33.3% and partial synergism in 50% of 24 strains. Antagonism between omeprazole and each antibiotic was exhibited in 0%, 6.1%, and 4.1% of these groups of strains, respectively. Of the antibiotic combinations tested, omeprazole plus clarithromycin exhibited synergism (partial or total) in the highest percentage of strains.     

Diversity among clinical isolates of Helicobacter pylori in Korea

Eleven strains of Helicobacter pylori were isolated from biopsy specimens obtained at the A-San Medical Center from December, 1995 to February, 1996. Every H. pylori positive patient was diagnosed to have chronic, erosive, or mild superficial gastritis. To determine the diversity in clinical isolates, the following were studied: total protein profile, plasmid profile, presence of cagA, and variation in DNA sequence. Protein profiles of nine isolates were similar to each other while two isolates had a 35 kDa protein which did not appear in others. The presence of cagA was detected with PCR in seven isolates (63%). Among eleven isolates, seven (63%) carried plasmids. Each isolate showed a big diversity with a PCR-based randomly amplified polymorphic DNA method. Even though all H. pylori isolates used in this study were isolated from gastritis patients at the same hospital, their molecular and biological characteristics were quite different from another showing a big diversity in H. pylori.     

Lack of correlation between vacuolating cytotoxin activity, cagA gene in Helicobacter pylori, and peptic ulcer disease in children

To determine the prevalence of the cagA gene and vacuolating cytotoxin in Helicobacter pylori isolates obtained from children and to characterize the relationship between cagA, cytotoxin production, and ulcerogenesis, pediatric Helicobacter pylori isolates were tested for cagA by the polymerase chain reaction and for vacuolating cytotoxin by a cell culture assay. Helicobacter pylori isolates were obtained from 33 children referred for upper gastrointestinal endoscopy. Twenty-six of these isolates were tested for cagA by the polymerase chain reaction; all 26 (100%) were positive. Of the 26 children from whom these isolates were obtained, 26 (100%) had chronic gastritis and 12 (46%) had duodenal ulcers. Nine (30%) of 30 isolates tested showed expression of vacuolating cytotoxin, only three of which came from patients with duodenal ulceration (odds ratio 0.81, 95% confidence interval 0.1-5.3). Of the 23 cagA-positive isolates tested for cytotoxin, only nine (39%) were positive. There was no association between vacuolating cytotoxin and clinical symptoms, nor was cytotoxicity associated with ulcerogenesis. In summary, the findings suggest that cagA is not a marker of duodenal ulceration or of vacuolating cytotoxin production in children referred for endoscopy.     

Prevalence of antimicrobial resistance in eighty clinical isolates of Helicobacter pylori

The actions of different adrenoceptor antagonists on gastric potential difference (PD), electrical current (I) and resistance (R) were studied, using the voltage clamp technique. In an isolated gastric mucosal tissue, 5% ethanol was able to reduce the PD and I across the gastric mucosa. Direct incubation with propranolol 10(-4) mol/l either from the mucosal or submucosal sides attenuated such effects. Intraperitoneal administration of propranolol (2.5-10 mg/kg), a nonselective beta-adrenoceptor blocker with significant membrane-stabilizing activity, given 30 min before the preparation of the gastric tissue, not only alleviated the fall in PD and I across the gastric mucosa, but also increased the R of the stomach tissue. Butoxamine, a selective beta2-antagonist, produced the similar but less significant effects in the same experimental setting. Metroprolol, a beta1-adrenoceptor blocker, given by the similar doses did not produce significant effects. Nonselective beta-adrenoceptor blocker, nadolol but not the beta- and alpha-adrenoceptor blocker, labetalol, also significantly preserved the decrease of PD induced by ethanol, but to a lesser extent. These findings suggest that blockade of the beta2-receptors in the gastric mucosa together with membrane-stabilizing activity could improve the integrity of the gastric mucosa, and these effects are probably acting through its direct action on the tissue.     

Transport and storage of Helicobacter pylori from gastric mucosal biopsies and clinical isolates

Presence of multivessel coronary artery disease (MVD) identifies a high risk subgroup after acute myocardial infarction (AMI). Dobutamine stress echocardiography (DSE) has recently emerged as a promising non invasive test to detect the presence and extent of coronary artery disease. Forty six consecutive patients (38 males, 8 females; mean age 48.6 +/- 10.4 years) of Q-wave acute myocardial infarction were subjected to submaximal treadmill test (TMT) and dobutamine stress echocardiography to see their ability to predict multivessel coronary artery disease as detected by coronary angiography before hospital discharge. Dobutamine infusion was started at 5 micrograms/kg/min to a maximum of 40 micrograms/kg/min, to achieve 70 percent of the age predicted heart rate. Appearance of new regional wall motion abnormality was interpreted as positive DSE for MVD. Mean peak infusion dose of dobutamine used in the study was 28.56 +/- 5.67 micrograms/kg/min. In none of the patients, the test had to be terminated due to side effects. The sensitivity and specificity of DSE to predict MVD was 80 percent and 93.7 percent, respectively as compared to 45 percent and 86 percent for submaximal TMT. Thus, DSE in patients of AMI before hospital discharge is a safe procedure with fairly accurate prediction of multivessel coronary artery disease.     

Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori using short oligonucleotide probes containing repetitive sequences

The ability of oligonucleotide probes containing short repetitive sequence motifs to differentiate between isolates of Helicobacter pylori was investigated. Genomic DNA preparations from H. pylori were digested with the restriction enzyme HindIII, electrophoresed in agarose gels and transferred to nylon filters. Five separate oligonucleotide probes were tested for hybridization sequentially to fingerprint the digested DNA from a panel of 29 clinical isolates and one type strain of H. pylori, and their relative discriminatory abilities were assessed. Four probes, (GACA)4, (GT)8, (GTG)5 and (GGAT)4, were each shown to yield highly informative hybridization band profiles allowing differentiation of H. pylori isolates. The DNA fingerprints of individual isolates obtained with each probe were distinct and reproducible. Direct comparison with ribotyping revealed that oligonucleotide fingerprinting had far superior discriminatory power. Computer-assisted similarity analysis of (GGAT)4-generated hybridization profiles of pairwise combinations of H. pylori isolates revealed that there was no correlation between ribotype and oligonucleotide fingerprint patterns. The results of this study demonstrate that oligonucleotide probes containing microsatellite sequences provide a new and powerful tool for isolate discrimination of H. pylori.     

Cytotoxic effect of ammonia produced by Helicobacter pylori urease on the cultural cells

We investigated the action of the ammonia produced by Helicobacter pylori urease on the cultured cells. The urease was purified from supernatant fluid of sonicated cell of H. pylori cultured on blood agar for 2 days at 37 degrees C under microaerophilic condition. Purification was carried out by DEAE-Sepharose chromatography, Phenyl-Sepharose chromatography, Sephacryl S-200 SF chromatography and fast protein liquid chromatography on Mono-Q. Vero, HeLa and Intestin 407 cells with or without the addition of 30 mM urea were exposed to the purified urease. Those cells showed cytotoxic effects within 80 minutes after addition of purified urease in the presence of urea. The ammonia production was observed on tissue culture medium within 10 minutes, and the ammonia concentration ranged from 5.56 mg/ml to 7.3 mg/ml and pH in the medium was over pH 9.0. No such effect was observed on the cells exposed to urease without urea. Ammonia water added to Vero cells showed the same cytotoxic effect within 70 minutes on the production of ammonia and raised the pH. However, when the cells were exposed to the ammonia water pre-neutralized to a given pH 7-8 using 1 N HCl cytotoxic effect was not observed. It was concluded that the cytotoxic effect of H. pylori urease was dependent on ammonia generated by hydrolysis of urea.     

In vitro susceptibility of clinical yeast isolates to fluconazole and terconazole

OBJECTIVE: Fifty clinical yeast isolates, representing equally Candida albicans, Candida krusei, Candida parapsilosis, Candida tropicalis, and Torulopsis glabrata, were tested in vitro for their susceptibility to terconazole and fluconazole. STUDY DESIGN: The minimal inhibitory concentrations of terconazole and fluconazole were determined by use of a proposed standardized broth macrodilution assay. Also, the response of selected yeast isolates to 25 micrograms of either drug was measured by agarose disk diffusion experiments. RESULTS: For all species the minimum inhibitory concentrations for terconazole were significantly lower than those for fluconazole (p < 0.05). In fact, for each individual isolate the minimum inhibitory concentration of terconazole was consistently lower than that of fluconazole. Differences in the geometric mean of terconazole and fluconazole minimum inhibitory concentrations were largest among C. krusei and T. glabrata, followed by C. parapsilosis, C. tropicalis, and C. albicans, in order of decreasing difference. Disk diffusion experiments suggested that terconazole is a more effective fungistatic agent than fluconazole is. CONCLUSION: Terconazole may be more effective than fluconazole against yeast species other than C. albicans.     

Granulocyte activation by Helicobacter pylori

Helicobacter pylori-associated gastritis (HAG) is characterized by granulocytic and mononuclear cell infiltrates within infected gastric mucosa. Since the bacterium does not invade the epithelial layer, it must be assumed that components or products of the pathogen which permeate the epithelial barrier may initiate chemotaxis and activation of neutrophils. The aim of this study was to evaluate the effect of H. pylori water soluble protein (WSP) components on the induction of granulocyte adherence and activation. The results show that H. pylori WSP led to enhanced expression of the beta 2-integrin CD11b/CD18 on the granulocyte surface. Following upregulation of this adhesion molecule, activated granulocytes demonstrated increased adhesion to human endothelial cells (HUVEC) in culture. These observations support the hypothesis that in vivo neutrophil activation may be a direct result of H. pylori constituents promoting transendothelial migration into the lamina propria of infected gastric mucosa.     

Beneficial effects of Helicobacter pylori eradication on migraine

BACKGROUND/AIMS: Migraine is a commonly unilateral throbbing headache, which has been associated with disorders of the vascular tone. Helicobacter pylori, the most relevant cause of gastritis and peptic ulcer, has been recently associated with a typical functional vascular disorder such as primary Raynaud phenomenon. The aim of this study was to assess the prevalence of H. pylori for patients affected by migraine and the effects of H. pylori eradication on migraine symptoms. METHODOLOGY: Two-hundred and twenty-five patients were consecutively enrolled between October 1996 and January 1997. H. pylori was assessed by 13C-urea breath test. Infected subjects were eradicated of the bacterium; frequency, intensity and duration of attacks of migraine were assessed during a 6 month follow-up period. RESULTS: H. pylori was detected in 40% of the patients. Eighty-three percent of the patients who underwent therapy were eradicated. Intensity, duration and frequency of attacks of migraine were significantly reduced in all eradicated patients. CONCLUSIONS: H. pylori is common in subjects with migraine. Bacterium eradication causes a significant decrease in attacks of migraine. The reduction of vasoactive substances produced during infection may be the pathogenetic mechanism underlying the phenomenon.     

Helicobacter mustelae and Helicobacter pylori bind to common lipid receptors in vitro

Helicobacter pylori is a recently recognized human pathogen causing chronic-active gastritis in association with duodenal ulcers and gastric cancer. Helicobacter mustelae is a closely related bacterium with similar biochemical and morphologic characteristics. H. mustelae infection of antral and fundic mucosa in adult ferrets causes chronic gastritis. An essential virulence property of both Helicobacter species is bacterial adhesion to mucosal surfaces. The aim of this study was to determine whether H. mustelae binds to the same lipids shown previously to be receptors for H. pylori adhesion in vitro. By using thin-layer chromatography overlay and a receptor-based enzyme-linked immunosorbent assay, H. mustelae was found to bind the same receptor lipids as H. pylori, namely, phosphatidylethanolamine and gangliotetraosylceramide. In addition, both H. pylori and H. mustelae bound to a deacylplasmalogen phosphatidylethanolamine. In contrast to H. pylori, H. mustelae binding to receptors was unaffected by motility or viability. Murine monoclonal and bovine polyclonal antibodies against exoenzyme S, and exoenzyme S itself (from Pseudomonas aeruginosa), inhibited binding of H. mustelae to phosphatidylethanolamine and gangliotetraosylceramide. These findings show that H. mustelae binds in vitro to the same lipid receptors as H. pylori and suggest that the adhesion of H. mustelae to such species is mediated by preformed, surface-exposed adhesins which include an exoenzyme S-like protein.     

Production of chemoattractant by Helicobacter pylori

Helicobacter pylori is present in the antral region of the stomach in a majority of patients with gastritis type B. The specific mechanism whereby the organism participates in the development of disease remains uncertain. Since the organism is not invasive, we postulate that H. pylori produces a chemoattractant that recruits inflammatory cells to the antral region of the stomach. H. pylori was grown under microaerophilic conditions at 37 degrees C for 72 hr in Brucella broth containing 1% fetal bovine serum. Culture supernates were harvested after removal of organisms by centrifugation and filtration. The putative chemoattractant in culture supernates as well as that which might be present endogenously in the growth medium (negative control) was assayed against human neutrophils (PMN) in modified Boyden blind-well chambers using 3.0-microns membranes. We found that H. pylori supernates are chemotactic and showed up to 130% activity when compared to the positive chemoattractant control (zymosan-activated serum, a source of C5a). Minimal activity was observed with virgin growth medium. The chemoattractant activity is proportional to the number of colony forming units (CFU) of H. pylori. Preliminary characterization of the activity shows that the chemoattractant is stable in a boiling water bath for 15 min, activity is lost within 1 hr in acid or alkali, and the chemotactic factor has an approximate molecular weight of 8500 daltons. The factor has no amino-sugar and is negative for the lipid A portion of lipopolysaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro cytotoxic effect of difluoromethylomithine increased nonspecifically by peptide coupling

Difluoromethylomithine (DFMO)-peptide conjugates were synthesized as prodrugs to improve the cytotoxic efficacy of DFMO. All conjugates inhibited cell growth in different cell lines more effectively than DFMO itself. The best cytotoxic effect was achieved in all cell lines by DFMO-Glu-His-Phe-Arg-Trp-Gly-OMe, where the carrier peptide is a melanotropin hormone fragment. Although this conjugate is capable of displacing labeled melanotropin from its receptor, its cytotoxic effect on the receptor-positive human melanoma cell line has not been proven to be receptor-mediated. The differences in the cytotoxicities of the congeners seem to be influenced, at least in part, by the nature of the carrier molecule.     

Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori

Helicobacter pylori, one of the most common bacterial pathogens of humans, colonizes the gastric mucosa, where it appears to persist throughout the host's life unless the patient is treated. Colonization induces chronic gastric inflammation which can progress to a variety of diseases, ranging in severity from superficial gastritis and peptic ulcer to gastric cancer and mucosal-associated lymphoma. Strain-specific genetic diversity has been proposed to be involved in the organism's ability to cause different diseases or even be beneficial to the infected host and to participate in the lifelong chronicity of infection. Here we compare the complete genomic sequences of two unrelated H. pylori isolates. This is, to our knowledge, the first such genomic comparison. H. pylori was believed to exhibit a large degree of genomic and allelic diversity, but we find that the overall genomic organization, gene order and predicted proteomes (sets of proteins encoded by the genomes) of the two strains are quite similar. Between 6 to 7% of the genes are specific to each strain, with almost half of these genes being clustered in a single hypervariable region.