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Background and aims: One of the key components of inflammation is changes in vascular structure and function. This suggests that the microcirculation may be a key target of Helicobacter pylori [...]

Background and aims: The success of Helicobacter pylori eradication regimens depends on gastric pH, inflammation, and mucus thickness. Our aim was to investigate the effects of acid secretion, inflammation, and [...]

Research has been conducted on the noncytotoxic vacA type Helicobacter pylori s2 strains. Results indicate that the addition of the region that encodes 12-residue extension to type s1 vacA does not affect VacA production.

H.R. Pearce [1] N.J. Brown [1] K.D. Bardhan [2] J.C. Atherton [3] N. Kalia [1] (1.) Surgical & Anaesthetic Sciences, University of Sheffield; (2.) District General Hospital, Rotherham; (3.) Division [...]

Human and animal data have demonstrated H.pylori induced platelet aggregation in gastric vasculature. Formation of platelet aggregates may contribute to the pathogenesis of H.pylori associated gastric injury and may also [...]

Several studies have demonstrated that H. pylori induces COX-2 in gastric mucosa and suggested that this may explain the increased risk of gastric cancer with H. pylori infection. We have [...]

C.J. Hawkey [1] J.C. Atherton [1] H.C. Treichel [2] M. Ravic [3] B. Thjodleifsson [4] (1.) University Hospital, Nottingham, UK; (2.) Johanniter Krankenhaus, Genthin, Germany; (3.) Eisai Ltd, London, UK; [...]

J.R. Bebb [1] L. Leach [2] J.C. Atherton [1] (1.) Division of Gastroenterology & Institute of Infections and Immunity and (2.) School of Biomedical Sciences, University Hospital, Nottingham, UK Background: [...]

Microarrays can be used to demonstrate differences in genetic content between Helicobacter pylori strains, giving a foretaste of how research may be conducted in the near future Israel DA, Salama [...]

Background: Helicobacter pylori induces cyclooxygenase activity in the stomach, although the COX isoform and cellular source are unclear. A potential source is the vascular endothelial cell, which plays a role in regulating mucosal blood flow and inflammatory cell infiltration. Methods: We examined the effect of four strains (toxigenic and non-toxigenic) of H. pylori on COX isoform expression in vascular endothelial cells. Prostaglandin synthesis was measured by enzyme immunoassay and COX isozyme expression determined by Western blot and RT-PCR. Gene induction was examined using 5′ deletion constructs of the COX-1 and COX-2 promoters coupled with luciferase. Results: All H. pylori strains induced prostaglandin generation and expression of both COX-1 and COX-2 in HUVEC, although this was most pronounced with the highly toxigenic strain H. pylori 60190. Treatment of the cells with selective COX inhibitors demonstrated that COX-1 was predominantly responsible for the enhanced generation of prostacyclin induced by H. pylori 60190. Similar results were seen with H. pylori broth culture filtrates, suggesting that a secreted product was responsible. Induction of COX-2 reflected both enhanced gene expression and stabilization of the mRNA. Conclusions: H. pylori increased both COX-1 and COX-2 activity in vascular endothelial cells. This increased generation of endothelial cell prostacyclin may play a role in modulating mucosal blood flow, platelet function and inflammatory cell infiltration in response to H. pylori infection. The regulation of COX-1 at the transcriptional level by H. pylori described in this study is a novel finding and calls into question the traditional description of COX-1 as a purely constitutive, housekeeping gene. [ABSTRACT FROM AUTHOR]

An improved procedure is described for the dibromination of anthracene, a.ording 9,10-dibromoanthracene with yields > 95%. [ABSTRACT FROM AUTHOR]

Introduction: Levels of vacuolating cytotoxin gene (vacA) transcription vary among laboratory strains of H pylori. We aimed to study the importance of vacA transcriptional differences among wild type strains and explore whether specific genetic determinants within vacA explained these differences. Methods: H pylori single colonies were isolated from gastric biopsy specimens from 8 patients and their vacA type determined by allele-specific PCR. Total RNA was prepared for each isolate and for the control strains 60190 (Tox[sup +]; vacA s1/m1) and Tx30a (Tox; s2/m2). Level of vacA transcription was determined by densitometric analysis of RNA dot blots. Level of VacA protein production was determined by densitometric analysis of immunoblots of 24 hour broth culture supernatants. For each isolate, nucleotide sequences were determined for the region between vacA and its 5' gene, cysS (the cysS-vacA intergenic region) and For 122 nucleotides of the 5' end of vacA. Finally, the vacuolating activity of each strain was determined by cell culture assay. Results: vacA transcription varied 6 fold among the clinical isolates. VacA RNA levels correlated closely with protein levels suggesting that variation in vacA production resulted mainly from variation in transcription. Level of vacA transcription was not related to vacA genotype (genotypes of wild type isolates were: 2 s1/m1; 4 s1/m2; 1 s2/m2; 1 s2/m1) nor was it associated with disease state or specific gastric histological features. Analysis of the cysS-vacA intergenic region revealed 70% identity across all 10 strains (including control strains 60190 and Tx30a). The -10 and -35 promoter regions were well conserved. Identity within the vacA 5' untranslated region was 77%. Despite high variation within the cysSvacA intergenic region, none of the genetic differences were associated with vacA transcription level. A potential stem-loop structure at the +4 site was conserved between all strains. Conclusion: Level of vacA... [ABSTRACT FROM AUTHOR]

Background: Mastic gum is a resinous exudate obtained from the stem and leaves of Pistacia lentiscus. It is widely used in Middle Eastern and Mediterranean countries as a chewing gum and food additive. We have previously shown that it is bactericidal against H pylori in vitro and this has been independently confirmed. Since then mastic has been widely marketed both in the USA and Europe as a cure for H pylori infection and peptic ulcers. Methods and Patients: Nine H pylori positive patients were recruited from routine day case endoscopy lists. No patients had current or previous gastroduodenal ulceration or had taken antibiotics, bismuth compounds, or proton pump inhibitors for 6 weeks prior to the trial. A 13C urea breath test was performed prior to, on day 14 of and 5 weeks after treatment with mastic capsules 1 g four times a day. Results: Eight patients completed the trial protocol (one withdrew after 5 days due to nausea). All nine patients remained H pylori positive by UBT immediately after mastic treatment with unchanged UBT values (pre-treatment mean ± SEM 19.4 ± 3.2 per ml, post-treatment 18.5 ± 3.4 per ml). Eight patients attended for UBT 5 weeks after treatment finished, again with unchanged UBT values (18.2 ± 3.6 per mi). Other than the patient who withdrew, two patients reported mild adverse symptoms; one complained of fatigue and the second of constipation and bloating. Conclusion: This study shows that high dose mastic gum has no significant effect against H pylori in vivo. [ABSTRACT FROM AUTHOR]

Introduction: H pylori strains possessing the cag Pathogenicity Island (cag Pal) or expressing active vacuolating cytotoxin (vacA) are associated with increased risk of peptic ulceration and gastric adenocarcnoma. Most strains have both or neither of these virulence factors. This is unlikely to be due to genetic linkage as H pylori recombine freely and the loci are distant on the chromosome. We hypothesised that the association was due to a gain of function from possessing both factors. Methods: In two toxigenic cag[sup +] strain backgrounds, 60190 and 84-183, we constructed separate isogenic mutants null for vacA, and for each of two genes encoded on the cag Pal, cagA and cagE. These and wild type strains were passaged in parallel to avoid differential laboratory adaption. For all strains, we assayed vacA activity on cultured epithelial cells by direct counting and by neutral red uptake (NRU) assay; cag-induced pro-inflammatory activity by interleukin-8 (IL-8) ELISA on supernatant from co-cultured epithelial cells; and cagA phosphorylation in epithelial cells by immunoblot with antiphosphotyrosine antibodies. Results (mean±SD) were compared using t tests. Results: cagA[sup -] mutants had increased vacuolating activity compared with cagA[sup +] parent strains, both by direct counting and by NRU assay (60190, 0.085±0.0073 adjusted OD units v 0.131±0.017, p=0.0001). cage mutants had similar effects (60190, 0.085±0.0073 v 0.151±0.018, p=0.00005). As expected, the vacA[sup -] mutants lacked vacuolating activity. In contrast, vacA mutants were similar to vacA[sup +] parent strains in inducing 11.-8 secretion from epithelial cells and n inducing cagA phosphorylation. As expected, the cage mutant but not the cagA mutant had significantly reduced IL-8 stimulating activity (60190, 2145±140.89 pg/ml vs cagE, 662.5±90.85 p=0.O00002) and the cage mutant did not induce cagA phosphorylation in epithelial cells. Similar results... [ABSTRACT FROM AUTHOR]

Background: Clinical studies have suggested an association between cardiovascular disease and infection with H pylori. We have described strain specific H pylori-induced platelet aggregation that involves platelet glycoprotein lb and van Willebrand factor (vWf). We exam ned the role of H pylori antibodies in this aggregation. Methods and Results: Addition of pooled immunoglobin (Ig) and fibrinogen along with H pylori strain 60190 (coated in vWf) to washed platelets led to platelet aggregation (60±4% n=3). Antibodes involved were specific to H pylori as bacteria incubated in pooled g and subsequently washed could support platelet aggregation (49±7%, n=3). To confirm this, pooled Ig was incubated with H pylori or Streptococcus sanguis. Bacteria were removed by centrifugation and the depleted g used in aggregation assays. H pylori-depleted Ig showed greatly reduced platelet aggregation to H pylori (13±9%, n=3, p<0.01) while the S sanguis-depleted Ig was not affected (54±3%, n=3, p=NS). All 20 plasma donors were tested for H pylori seropositivity using a H pylori ELISA. All 4/4 of the positive volunteers consistent y aggregated in response to H pylori while only 2/15 of the H pylori negative donors aggregated consistently (p=0.039, Fisher's Exact Test). The relative risk of H pylori positivity for aggregation was 7.5 (CI 2.0-27.3). Conclusions: H pylori-induced platelet aggregation requires binding of H pylori-specific IgG. This pro-aggregatory phenotype is not unique to H pylori and has been described with S sanguis and P gingivalis. Exposure to oral bacteria such as S sanguis can lead to infective endocarditis and in the case of P gingivalis, to enhancement of atherosclerosis in animal models. Repeated exposure to proaggregatory bacteria such as certain strains of H pylori may not cause coronary artery disease but could accelerate the progression of the disease. Local platelet effects may also contribute to the pathogenesis of H... [ABSTRACT FROM AUTHOR]

Introduction: Helicobacter pylori infection is a major risk factor for distal gastric adenocarcinoma. Pathogenicity determinants include production of an active form of vacuolating cytotoxin (VacA) and possession of the cag pathogenicity island (cag Pal). Toxigenic strains cause marked epithelial cell vacuolation in vitro whilst cag-dependent effects include rearrangement of the actin cytoskeleton to form a "hummingbird" phenotype. NSAID ingestion is associated with a reduced incidence of gastric adenocarcinoma, although mechanisms are unclear We tested the hypothesis that NSAIDs modulate H. pylori-induced effects on epithelial cells, in particular effects induced by VacA and the cag Pal. Methods: AGS cells (1-2 × 10[sup 5] cells/ml) were co-cultured with H pylori strain 60190 (cag+, vacA sl/ml) or its VacA or Cage isogenic mutants at a bacteria:cell ratio of 0.02-70:1. Indomethacin (a non-selective NSAID) or vehicle control was added at a concentration of 0.01-100µM. After overnight incubation with 5% CO[sub 2] at 37°C, vacuolated and hummingbird cells were counted directly using phase contrast microscopy. Statistical analysis was by ANOVA and Student's paired t test. Results: Co-culture with H pylori strain 60190, but not its VacA mutant, increased AGS cell vacuolation from 0.8 ± 0.8 to 25.4 ± 6.5%. Indomethacin had no significant effect in the absence of H pylori, but inhibited H pylori-induced vacuolation in a dose-dependent manner (100µM: by 56 ± 14%, p=0.02; 1 µM:, by 42 ± 15%, p=0.05; 0.01µM: by 26 ± 13% p=0.26). Co-culture with H pylori strain 60190, but not its CagE mutant increased AGS hummingbirds from 0 to 6.2 ± 1.7%. Indomethacin had no effect in the absence of H pylori. In the presence of H pylori, indomethacin at 100µM surprisingly increased hummingbirds by 240 ± 98%, p=0.05. Conclusion: Indomethacin reduces H pylori-induced vacuolation, but in contrast, at... [ABSTRACT FROM AUTHOR]

Background: MMP-7 (matrilysin) is a member of the metalloproteinase family of enzymes, which are important in normal and pathological remodelling of epithelial matrix interactions, it is upreguJated in gastric cancer. We have previously shown that H pylori co-culture induces upregulation of MMP-7 in epithelial cells in vitro at both the protein and RNA level, and that this is more marked for strains with an intact cag pathogenicity island. We now examined whether this occurs in viva. Methods: Gastric biopsies were taken at endoscopy from H pylori infected (n = 17) and uninfected (n = 16) patients, and MMP-7 expression examined by immunohistochemistry (paraffin-embedded sections), ELISA (R&D systems), and Real Time PCR. H pylori strains were cultured and PCR-typed for cagA, cagE, and vacA. For immunohistochemistry, slides were examined by two blinded observers and staining intensity was graded 0-4. Results were compared by Mann Whitney U test (immunohist) and t test (ELISA, Real Time PCR). Results: Epithelial ceils from H pylori infected patients stained more intensely for MMP-7 than uninfected patients both in antrum and corpus. This was most marked superficially in the antrum (median Hp+ 2, Hp- 1, p < 0.05) and in the proliferative zone in the corpus (median Hp+ 3, Hp- 2, p < 0.05). There was also a significant increase in MMP-7 staining inflammatory cells in infected patients. ELISA confirmed these results with a threefold increase in infected patients (Hp+ 0.182 v Hp- 0.059, p = 0.009). Real Time PCR demonstrated upregulation of MMP-7 at RNA level (antrum Hp+ 0.038 v Hp0.008, p = 0.007). It was not possible to correlate changes with strain type due to small numbers of cag-negative strains. Conclusion: This study demonstrates increased expression of MMP-7 in vivo in nonmalignant gastric epithelial cells colonised by H pylori. We speculate that upregulation of MMP-7 in H pylori gastritis may play a role in H pylori induced gastric carcinogenesis. [ABSTRACT FROM AUTHOR]

Introduction: The H pylori vacuolating cytotoxin, VacA, varies between strains as a result of genetic recombination. The three main types have different toxicity (s1/m1>s1/m2>s2/m2) and this results in different disease associations. We aimed to show whether VacA can evolve to alter its toxicity within an individual stomach. Methods: Single H pylori colonies were cultured From gastric biopsies from a Mexican patient with duodenal ulcer and typed for the toxin gene, vacA, by allele specific PCR. The relationship between isolates was determined by two PCR based genomic fingerprinting methods, random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP), and for two isolates of different vacA type by partial sequencing of the housekeeping genes, A/G adenine glycosylase (mutY) and GTPase (yphC). For these two isolates, vacA was sequenced and vacuolating activity was determined on the gastric cell line, AGS. Results: Among 11 individual isolates cultured, we found 4 of vacA type sl/ml and 7 of type s1/m2. RAPD and AFLP analysis showed that all isolates were closely related. Nucleotide sequencing of mutY and yphC confirmed a clonal origin. The vacA sequence of candidate vacA s1/m1 and s1/m2 clones showed differences in two vacA segments of 439 and 378 base pairs, respectively. Outside these regions, vacA nucleotide sequences were identical. The vacA s1/m1 clone induced AGS cell vacuolation whereas the s1/m2 clone induced no vacuolation. Conclusion/Discussion: We have demonstrated evolution, by homologous recombination, of the toxin gene, vacA, and hence of toxin phenotype within an individual stomach. From our vacA sequence data, it appears most likely that the less pathogenic strain is the result of this evolution process. Evolution of VacA in vivo might represents an adaptation process For survival in the changing gastric environment. Changing H pylori virulence within an individual stomach has important implications for pathogenicity... [ABSTRACT FROM AUTHOR]

H pylori associated disease is more common in patients infected with CagA+ strains. CagA is one of 31 proteins encoded on the cag pathogenicity island (Pal). CagA itself is translocated into host cells via a syringe like structure comprising other cag encoded proteins. Here it becomes tyrosine phosphorylated and causes proliferative cell signalling and cytoskeletal changes. Interaction of the cag syringe itself with epithelial cells results in induction of proinflammatory cytokines, particularly interleukin-8 (IL-8). One or both of cagA phosphorylation and cag induced IL-8 secretion are likely responsible for the increased disease risk from CagA+ strains. Thus, we aimed to assess whether PCR detection of selected cag Pal genes could predict whether H pylori strains could induce epithelial cells to phosphorylate cagA and/or secrete IL-8. Methods/Results: H pylori strains were obtained from 26 patients from South Africa. The presence of cagA, cagE, cagM, cagT, and cag6-7 genes was determined by PCR analysis, cagA was present in all 26 strains, and 16 had all the tested cag Pal genes and so were predicted to encode an intact Pal. Possession and phosphorylation of cagA was determined by use of a cell free system and by co-culturing H pylori with AGS cells at 37° for 6 h, followed by lysis of the cells, and western blotting. Of the 26 strains, 20 induced cagA phosphorylation within AGS cells, and all of these strains induced the secretion of IL-8. Of the remaining 6 strains none induced IL-8 secretion. The degree of cagA phosphorylation induced and the amount of translocated cagA were not related to the level of lL-8 secretion induced. Six strains that possessed cagA did not produce cagA protein. Four strains predicted to lack a complete cag Pal by PCR analysis induced both cagA phosphorylation and IL-8 production, and one strain predicted to possess an intact Pal by PCR did not induce cagA phosphorylation or IL-8 production. Conclusion: PCR analysis of selected... [ABSTRACT FROM AUTHOR]

VacA, a major virulence factor of H. pylori, forms anion-selective pores within membranes, causes profound vacuolation of epithelial cells, reduces trans-epithelial resistance, and plays a role in apoptosis. Although it is clear that VacA is localised to rab7-positive vacuoles, little is known about the delivery of VacA to other compartments within epithelial cells. Methods/Results: To visualise the intracellular location of VacA, we performed direct immunofluorescence using anti-VacA antibodies on RK13 cells treated with culture supernatant from H. pylori strain 60190 (vacA s1m1) for 6 hours. As expected, most VacA localised to the vacuolar membrane, but a small proportion appeared to be free within the cytosol. To provide further evidence for the existence of cytosolic VacA, we treated RK13 cells, co-cultured for 16-24 hours with H. pylori strain 60190, with streptolysin O, which creates pores in cell plasma membranes, and showed that VacA was released. This confirmed our isolation of small quantities of VacA from crude cell cytoplasm preparations. Finally, to begin to explore how VacA may be routed deeper within cells, we treated RK13 cells co-cultured with H. pylori strain 60190 with brefeldin A, which causes disruption of the Golgi apparatus. Surprisingly, this resulted in enhanced vacuolation, implying trafficking of VacA through this compartment, beyond the vacuole. Conclusions: Most VacA delivered to epithelial cells becomes associated with vacuolar membranes but a fraction appears to be translocated into the cytosol and may be transported through the Golgi apparatus. Cytosolic VacA appears to be biologically important, as VacA has been reported to interact with the cytosolic proteins VIP54 and RACK1, and to cause apoptosis through mitochondrial damage. [ABSTRACT FROM AUTHOR]

The vacuolating cytotoxin gene, vacA, varies in its signal region (s1 or s2) and its mid region (m1 or m2). Naturally-occurring strains of H. priori are vacA s1m1 (vacuolating), s1/m2 (vacuolating RK13 cells, not HeLa/AGS) or s2m2 (non-vacuolating). We assessed the contribution of the vacA mid region to cell specific vacuolation. Methods: We constructed a vacA hybrid of toxigenic strain 60190 (s1m1) containing the m2 region (60190 s1/m2) and vacA hybrids of nontoxigenic Tx30a (s2m2) containing the m1 region (Tx30a s2m1) or both the s1 and m1 regions (Tx30a s1m1). We used natural transformation and allelic exchange of cloned vacA fragments from Tx30a and 60190. We incubated AGS, HeLa and RK13 overnight with 48hr culture supernatants. Results: Culture supernatants of the 60190 s1m1 control vacuolated all cell lines but the 60190 s1m2 hybrid vacuolated RK13 only, confirming that the m2 mid region confers cell specific vacuolating activity. Supernatants of the Tx30a s2m2 control and its s2m1 hybrid did not cause vacuolation of any cell line, showing that the m1 region alone is insufficient to confer vacuolating activity. However, the Tx30a s1m1 hybrid (A) had full vacuolating activity on all cell lines. A second independently derived Tx30a s1m1 hybrid (B), vacuolated RK13 cells, but not HeLa or AGS. Sequencing mature vacA of these hybrids, showed that hybrid B had 107 amino acids of Tx30a not present in hybrid A, and surprisingly these were within the p37 domain. Conclusion: The non-vacuolating phenotype of Tx30a is due to its vacA structure, not to other genetic elements. The m2 type of p58 binding subunit confers cell-line specificity, but such specificity can also be conferred by elements in the p37 subunit. This implies either that cell specificity of vacuolation is not due to VacA binding specificity, or that the p37 domain can influence VacA binding. [ABSTRACT FROM AUTHOR]