Association of Helicobacter pylori and gastric autoimmunity: A population-based study

Abstract Based on clinical studies, a negative association between Helicobacter pylori and autoimmune corpus gastritis is described. In the present investigation of an unselected population of 1461 adults we can state, however, that there exists a relationship between H. pylori infection and the development of gastric corpus autoimmunity. As confirmation for the gastric autoantibody development through molecular mimicry, a high homology (72% in 25 amino acid overlap) between the beta subunit of H. pylori urease and that of H + K + ATPase, the gastric parietal cell autoantigen, was revealed.     

Helicobacter pylori induces activation of human peripheral γδ+ T lymphocytes

Helicobacter pylori is a gram-negative bacterium that causes gastric and duodenal diseases in humans. Despite a robust antibody and cellular immune response, H. pylori infection persists chronically. To understand if and how H. pylori could modulate T cell activation, in the present study we investigated in vitro the interaction between H. pylori and human T lymphocytes freshly isolated from peripheral blood of H. pylori-negative donors. A direct interaction of live, but not killed bacteria with purified CD3+ T lymphocytes was observed by microscopy and confirmed by flow cytometry. Live H. pylori activated CD3+ T lymphocytes and predominantly γδ+ T cells bearing the TCR chain Vδ2. Upon interaction with H. pylori, these cells up-regulated the activation molecule CD69 and produced cytokines (such as TNFα, IFNγ) and chemokines (such as MIP-1β, RANTES) in a non-antigen-specific manner. This activation required viable H. pylori and was not exhibited by other gram-negative bacteria. The cytotoxin-associated antigen-A (CagA), was at least partially responsible of this activation. Our results suggest that H. pylori can directly interact with T cells and modulate the response of γδ+ T cells, thereby favouring an inflammatory environment which can contribute to the chronic persistence of the bacteria and eventually to the gastric pathology.     

Effect of probiotics and triple eradication therapy on the cyclooxygenase (COX)-2 expression, apoptosis, and functional gastric mucosal impairment in Helicobacter pylori-infected Mongolian gerbils

BACKGROUND: Helicobacter pylori infection in Mongolian gerbils is an established experimental model of gastric carcinogenesis that mimics H. pylori-positive patients developing gastric ulcer and gastric cancer, but the effect of probiotic therapy on functional aspects of this infection remains unknown. METHODS: We compared the effects of intragastric inoculation of gerbils with H. pylori strain (cagA+ vacA+, 5 x 10(6) colony forming units/ml) with or without triple therapy including omeprazole, amoxicillin, and tinidazol or probiotic bacteria Lacidofil. Histology of glandular mucosa, the viable H. pylori, and density of H. pylori colonization were evaluated. The gastric blood flow was measured by H2-gas clearance method; the plasma gastrin and gastric luminal somatostatin were determined by RIA and expression of cyclooxygenase (COX)-2 and apoptotic Bax and Bcl-2 proteins were evaluated by Western blot. RESULTS: The gastric H. pylori infection was detected in all animals by histology and H. pylori culture. Basal gastric acid was significantly reduced in H. pylori-infected animals but not in those with triple therapy or Lacidofil. Early lesions were seen already 4 weeks upon H. pylori inoculation and consisted of chronic gastritis and glandular atypia associated with typical regenerative hyperplasia and increased mitotic activity and formation of apoptotic bodies. The H. pylori infection was accompanied by the fall in gastric blood flow, the marked increase in plasma gastrin, the significant fall in gastric somatostatin levels and Bcl-2 protein expression, and the rise in expression of COX-2 and Bax proteins. These mucosal changes were counteracted by the triple therapy and Lacidofil. CONCLUSIONS: H. pylori infection in gerbils, associated with regenerative hyperplasia of glandular structure, results in the suppression of gastric secretion, overexpression of COX-2, and enhancement in apoptosis and impairment of both, gastric blood flow and gastrin-somatostatin link that were reversed by anti-H. pylori triple therapy and attenuated by probiotics.     

Mucosal immune responses are related to reduction of bacterial colonization in the stomach after therapeutic Helicobacter pylori immunization in mice

The aim of this study was to investigate the capacity of oral and parenteral therapeutic immunization to reduce the bacterial colonization in the stomach after experimental Helicobacter pylori infection, and to evaluate whether any specific immune responses are related to such reduction. C57BL/6 mice were infected with H. pylori and thereafter immunized with H. pylori lysate either orally together with cholera toxin or intraperitoneally (i.p.) together with alum using immunization protocols that previously have provided prophylactic protection. The effect of the immunizations on H. pylori infection was determined by quantitative culture of H. pylori from the mouse stomach. Mucosal and systemic antibody responses were analyzed by ELISA in saponin extracted gastric tissue and serum, respectively, and mucosal CD4+ T cell responses by an antigen specific proliferation assay. Supernatants from the proliferating CD4+ T cells were analyzed for Th1 and Th2 cytokines. The oral, but not the parenteral therapeutic immunization induced significant decrease in H. pylori colonization compared to control infected mice. The oral immunization resulted in markedly elevated levels of serum IgG+M as well as gastric IgA antibodies against H. pylori antigen and also increased H. pylori specific mucosal CD4+ T cell proliferation with a Th1 cytokine profile. Although the parenteral immunization induced dramatic increases in H. pylori specific serum antibody titers, no increases in mucosal antibody or cellular immune responses were observed after the i.p. immunization compared to control infected mice. These findings suggest that H. pylori specific mucosal immune responses with a Th1 profile may provide therapeutic protection against H. pylori.     

Effect of dietary anti-urease immunoglobulin Y on Helicobacter pylori infection in Mongolian gerbils

BACKGROUND AND AIM: Helicobacter pylori is known to be a major pathogenic factor in the development of gastritis, peptic ulcer disease and gastric cancer. Recently, chicken egg yolk immunoglobulin Y (IgY) has been recognized as an inexpensive antibody source for passive immunization against gastrointestinal infections. The present study was designed to investigate the effect of anti-urease IgY on H. pylori infection in Mongolian gerbils. METHODS: H. pylori-infected Mongolian gerbils were administered a diet containing anti-urease IgY, with or without famotidine (F). After 10 weeks, bacterial culture and measurement of the gastric mucosal myeloperoxidase (MPO) activity were performed. In a second experiment, another group of gerbils was started on a diet containing F + IgY a week prior to H. pylori inoculation. After 9 weeks, these animals were examined. RESULTS: In the H. pylori-infected gerbils, there were no significant differences in the level of H. pylori colonization among the different dietary and control groups. However, the MPO activity was significantly decreased in the H. pylori group administered the F + IgY diet compared with that in the H. pylori group administered the IgY, F, or control diet. Furthermore, in the gerbils administered the F + IgY diet prior to the bacterial inoculation, inhibition of H. pylori colonization and suppression of the elevated gastric mucosal MPO activity were observed. CONCLUSIONS: Oral administration of urease-specific IgY not only inhibited H. pylori disease activity in H. pylori-infected gerbils, but also prevented H. pylori colonization in those not yet infected. These encouraging results may pave the way for a novel therapeutic and prophylactic approach in the management of H. pylori-associated gastroduodenal disease.     

Helicobacter pylori encoding the pathogenicity island activates matrix metalloproteinase 1 in gastric epithelial cells via JNK and ERK

Helicobacter pylori colonizes the human gastric epithelium and induces an inflammatory response that is a trigger for gastric carcinogenesis. Matrix metalloproteinases (MMPs) have recently been shown to be up-regulated in gastric epithelial cells infected with H. pylori and might contribute to the pathogenesis of peptic ulcer. The aim of this study was to extend the knowledge about the effect of H. pylori infection on MMP-1 expression by gastric epithelial cells, the kinetics of induction, the pathogenetic properties of the bacterium, and the intracellular signaling pathways required for MMP-1 up-regulation. Expression of MMP-1 was induced more than 10-fold by co-culture of AGS+cells with H. pylori strains carrying the pathogenicity island (PAI). H. pylori strains with mutations in the PAI and a defective type IV secretion system had no effect on MMP-1. Double immunofluorescence revealed strong MMP-1 staining in epithelial cells of gastric biopsies at sites of bacterial attachment. In vitro, MMP-1 is up-regulated by interleukin-1beta and tumor necrosis factor-alpha, but these regulatory mechanisms are not operating in H. pylori infection as shown by inhibitory antibodies. Specific inhibitors of JNK kinase and ERK1/2 kinase were found to suppress the H. pylori-induced MMP-1 expression and activity. AGS cells treated with antisense MMP-1 showed a significantly reduced potential to degrade reconstituted basement membrane. Our results suggest that in gastric epithelial cells, H. pylori up-regulates MMP-1 in a type IV secretion system-dependent manner via JNK and ERK1/2. Induction of MMP-1 is further implicated in complex processes induced by H. pylori, resulting in tissue degradation and remodeling of the gastric mucosa.     

Helicobacter pylori, T cells and cytokines: the "dangerous liaisons"

Helicobacter pylori infection is the major cause of gastroduodenal pathologies, but only a minority of infected patients develop chronic and life threatening diseases, as peptic ulcer, gastric cancer, B-cell lymphoma, or autoimmune gastritis. The type of host immune response against H. pylori is crucial for the outcome of the infection. A predominant H. pylori-specific Th1 response, characterized by high IFN-gamma, TNF-alpha, and IL-12 production associates with peptic ulcer, whereas combined secretion of both Th1 and Th2 cytokines are present in uncomplicated gastritis. Gastric T cells from MALT lymphoma exhibit abnormal help for autologous B-cell proliferation and reduced perforin- and Fas-Fas ligand-mediated killing of B cells. In H. pylori-infected patients with autoimmune gastritis cytolytic T cells infiltrating the gastric mucosa cross-recognize different epitopes of H. pylori proteins and H+K+ ATPase autoantigen. These data suggest that peptic ulcer can be regarded as a Th1-driven immunopathological response to some H. pylori antigens, whereas deregulated and exhaustive H. pylori-induced T cell-dependent B-cell activation can support the onset of low-grade B-cell lymphoma. Alternatively, H. pylori infection may lead in some individuals to gastric autoimmunity via molecular mimicry.     

Helicobacter pylori urease binds to class II MHC on gastric epithelial cells and induces their apoptosis

Infection by Helicobacter pylori leads to injury of the gastric epithelium and a cellular infiltrate that includes CD4+ T cells. H. pylori binds to class II MHC molecules on gastric epithelial cells and induces their apoptosis. Because urease is an abundant protein expressed by H. pylori, we examined whether it had the ability to bind class II MHC and induce apoptosis in class II MHC-bearing cells. Flow cytometry revealed the binding of PE-conjugated urease to class II MHC+ gastric epithelial cell lines. The binding of urease to human gastric epithelial cells was reduced by anti-class II MHC Abs and by staphylococcal enterotoxin B. The binding of urease to class II MHC was confirmed when urease bound to HLA-DR1-transfected COS-1 (1D12) cells but not to untransfected COS-1 cells. Urease also bound to a panel of B cell lines expressing various class II MHC alleles. Recombinant urease induced apoptosis in gastric epithelial cells that express class II MHC molecules, but not in class II MHC- cells. Also, Fab from anti-class II MHC and not from isotype control Abs blocked the induction of apoptosis by urease in a concentration-dependent manner. The adhesin properties of urease might point to a novel and important role of H. pylori urease in the pathogenesis of H. pylori infection.     

cag+ Helicobacter pylori induce transactivation of the epidermal growth factor receptor in AGS gastric epithelial cells

The gastric pathogen Helicobacter pylori is known to activate epithelial cell signaling pathways that regulate numerous inflammatory response genes. The aim of this study was to elucidate the pathway leading to extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in H. pylori-infected AGS gastric epithelial cells. We find that H. pylori, via activation of the epidermal growth factor (EGF) receptor activates the small GTP-binding protein Ras, which in turn, mediates ERK1/2 phosphorylation. cag+ strains of H. pylori are able to induce greater EGF receptor activation than cag- strains, and studies with isogenic mutants indicate that an intact type IV bacterial secretion system is required for this effect. Blockade of EGF receptor activation using tyrphostin AG1478 prevents H. pylori-mediated Ras activation, inhibits ERK1/2 phosphorylation, and substantially decreases interleukin-8 gene expression and protein production. Investigations into the mechanism of EGF receptor activation, using heparin, a metalloproteinase inhibitor and neutralizing antibodies reveal that H. pylori transactivates the EGF receptor via activation of the endogenous ligand heparin-binding EGF-like growth factor. Transactivation of gastric epithelial cell EGF receptors may be instrumental in regulating both proliferative and inflammatory responses induced by cag+ H. pylori infection.     

Helicobacter pylori cag pathogenicity island-positive strains induce syndecan-4 expression in gastric epithelial cells

Helicobacter pylori is recognized as the main cause of gastritis and is associated with gastric carcinogenesis. Syndecan-4 represents the major source of heparan sulfate (HS) in the gastric cells. HS proteoglycans expressed on the cell surface constitute targets for H. pylori at the early stage of infection. The aim of this study was to determine whether H. pylori induction of syndecan-4 expression is affected by the virulence characteristics of the infecting strain, namely the cytotoxic-associated gene ( cag ) pathogenicity island (PAI). We observed that individuals infected with highly pathogenic H. pylori strains express syndecan-4 in the foveolar epithelium of the gastric mucosa. The association between the cag PAI status of the infecting strain and syndecan-4 expression was further demonstrated by infection of gastric epithelial cell lines with a panel of cag PAI⁺ and cag PAI⁻ H. pylori strains, showing that expression of syndecan-4 was significantly increased in response to infection with the highly pathogenic strains. Moreover, infection of gastric cells with cag A and cag E mutant strains further confirmed that syndecan-4 induction is dependent on an intact cag PAI. The present study shows that highly pathogenic H. pylori strains induce syndecan-4 expression, both in human gastric mucosa and in gastric cell lines, in a cag PAI-dependent manner.     

Adoptive transfer of splenocytes in SCID mice implicates CD4+ T cells in apoptosis and epithelial proliferation associated with Helicobacter pylori-induced gastritis

Helicobacter pylori causes severe, rapidly progressive gastritis in severe combined immunodeficient (SCID) mouse recipients of congenic splenocytes. The H. pylori-infected and uninfected C57BL/6J and recipient SCID mice were evaluated to detect CD4+ and CD8+ T cells, B cells, apoptotic epithelial cells, and epithelial cell proliferation at postinoculation weeks 5, 6, 8, and 12. Serum was evaluated for anti-H. pylori IgG and IgM. In all H. pylori-infected mice, gastric CD4+ cell scores were increased, compared with scores for uninfected controls. Recipient mice differed, however, according to the source of the transferred CD4+ cells. The CD4+ cell scores for recipients of splenocytes from H. pylori-infected (immune) donors were indistinguishable from those for wild-type donor mice at all time points. In contrast, gastric mucosal CD4+ cell scores did not become significantly high until two weeks after transfer (postinoculation week 6) in recipients of cells from uninfected (na?ve) donors. Gastric epithelial apoptosis and the gastric epithelial proliferation zone were significantly (P < 0.05) increased in infected recipient and donor, compared with non-recipient and uninfected mice at postinoculation week 12. Results indicated that CD4 cells are sensitized in vivo and migrate to the gastric mucosa where they induce gastritis in response to H. pylori antigens. Influx of CD4 cells and gastritis are correlated with epithelial proliferation and apoptosis, and suggest that CD4-dependent H. pylori gastritis leads to epithelial damage with attendant proliferative and metaplastic responses.     

Cag pathogenicity island-independent up-regulation of matrix metalloproteinases-9 and -2 secretion and expression in mice by Helicobacter pylori infection

Helicobacter pylori cag pathogenicity island (PAI) is a major determinant of gastric injury via induction of several matrix metalloproteinases (MMPs). In the present study, we examined the influence of the cag PAI on gastric infection and MMP-9 production in mice and in cultured cells. A new mouse colonizing Indian H. pylori strain (AM1) that lacks the cag PAI was used to study the cag PAI importance in inflammation. Groups of C57BL/6 mice were inoculated separately with H. pylori strains AM1 and SS1 (cag+), gastric tissues were histologically examined, and bacterial colonization was scored by quantitative culture. Mice infected with either cag+ or cag- H. pylori strains showed gastric inflammation and elevated MMP-3 production. Significant up-regulation of pro-MMP-9 secretion and gene expression in H. pylori infected gastric tissues indicate dispensability of cag PAI for increased pro-MMP-9 secretion and synthesis in mice. In agreement, cell culture studies revealed that both AM1 and SS1 were equipotent in pro-MMP-9 induction in human gastric epithelial cells. Both strains showed moderate increase in MMP-2 activity in vivo and in vitro. In addition, increased secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 induced pro-MMP-9 secretion and synthesis in AM1 or SS1 strain-infected mice suggesting elicitation of pro-inflammatory cytokines by both cag- and cag+ genotype. Moreover, tissue inhibitors of metalloproteinase-1 expression were decreased with increase in pro-MMP-9 induction. These data show that H. pylori may act through different pathways other than cag PAI-mediated for gastric inflammation and contribute to up-regulation of MMP-9 via pro-inflammatory cytokines.     

Increased expression of the urokinase plasminogen activator system by Helicobacter pylori in gastric epithelial cells

The gastric pathogen Helicobacter pylori (H. pylori) is linked to peptic ulcer and gastric cancer, but the relevant pathophysiological mechanisms are unclear. We now report that H. pylori stimulates the expression of plasminogen activator inhibitor (PAI)-1, urokinase plasminogen activator (uPA), and its receptor (uPAR) in gastric epithelial cells and the consequences for epithelial cell proliferation. Real-time PCR of biopsies from gastric corpus, but not antrum, showed significantly increased PAI-1, uPA, and uPAR in H. pylori-positive patients. Transfection of primary human gastric epithelial cells with uPA, PAI-1, or uPAR promoters in luciferase reporter constructs revealed expression of all three in H+/K+ATPase- and vesicular monoamine transporter 2-expressing cells; uPA was also expressed in pepsinogen- and uPAR-containing trefoil peptide-1-expressing cells. In each case expression was increased in response to H. pylori and for uPA, but not PAI-1 or uPAR, required the virulence factor CagE. H. pylori also stimulated soluble and cell surface-bound uPA activity, and both were further increased by PAI-1 knockdown, consistent with PAI-1 inhibition of endogenous uPA. H. pylori stimulated epithelial cell proliferation, which was inhibited by uPA immunoneutralization and uPAR knockdown; exogenous uPA also stimulated proliferation that was further increased after PAI-1 knockdown. The proliferative effects of uPA were inhibited by immunoneutralization of the EGF receptor and of heparin-binding EGF (HB-EGF) by the mutant diphtheria toxin CRM197 and an EGF receptor tyrosine kinase inhibitor. H. pylori induction of uPA therefore leads to epithelial proliferation through activation of HB-EGF and is normally inhibited by concomitant induction of PAI-1; treatments directed at inhibition of uPA may slow the progression to gastric cancer.     

Increase of antigen-presenting cells in the gastric mucosa of Helicobacter pylori-infected children

BACKGROUND: Infection with Helicobacter pylori leads to an increase of T cells in the gastric mucosa of children. In contrast to peripheral blood, where monocytes are the most abundant antigen-presenting cells, CD14+ macrophages are very rare in infected gastric mucosa. We postulated that other types of antigen-presenting cells must be present in infected gastric mucosa. MATERIAL AND METHODS: Antral biopsies were obtained from 56 children. The cellular expression of major histocompatibility complex (MHC) class II molecules, CD1a/b, and CD23, which are involved in antigen presentation were analyzed by immunohistochemistry. In addition, T cells (CD4, CD8, CD25, and gamma/delta-TCR), B cells (anti-IgM), macrophages (CD14) and granulocytes (CD15) were quantified. RESULTS: Twenty-eight children were H. pylori-infected. Thirteen children were healthy, 15 had other gastric pathologies. T cells (p<.0001), B cells (p<.0001), CD23+ (p<.0001), and CD1a/b+ (p<.005) cells were significantly increased in the lamina propria of H. pylori-infected children, whereas macrophages were rare without significant differences among the groups. Within the epithelium, CD8+ T lymphocytes predominated clearly over CD4+ cells. H. pylori-negative children had only few MHC class II-positive cells within the gastric epithelium, whereas MHC class II antigens were strongly expressed on epithelial cells (p<.0001) of all H. pylori-infected children. CONCLUSION: Helicobacter pylori infection leads to an enhanced expression of antigen-presenting molecules together with a parallel rise of T cells in the lamina propria. This may represent an effort of the immune system to optimize local immune responses against H. pylori. We speculate that the epithelium participates in the initiation of a local immune response against H. pylori.     

Vacuolating cytotoxin A is associated with increased thrombin generation in gastric mucosa

BACKGROUND: Activation of the coagulation system is a critical response for both the repair of tissue injury and the host defense against microbial pathogens. Activation of the coagulation cascade culminates with the generation of thrombin. In vitro studies have shown that thrombin protects gastric epithelial cells from injury. The present study was undertaken to assess in vivo the relationship between gastric intramucosal generation of thrombin and Helicobacter pylori infection. MATERIALS AND METHODS: This study comprised 59 patients with gastroduodenal disorders. There were 27 patients with H. pylori infection (Hp+), 14 without it (Hp-), and 18 patients with cured H. pylori infection (Hp c). The gastric intramucosal concentrations of thrombin-antithrombin complex (TAT), epidermal growth factor (EGF), prostaglandin E2 (PGE2), and vacuolating cytotoxin A (VacA) were measured by specific immunoassays. RESULTS: The level of TAT was significantly increased in patients with Hp+ compared to Hp- and Hp c. The levels of TAT, EGF and PGE2 were higher in VacA (+) patients than in those with VacA (-). VacA induced significant expression of tissue factor in gastric epithelial cells in vitro. The gastric intramucosal level of VacA antigen was proportionally and significantly correlated with TAT, EGF and PGE2 in Hp+ patients. The level of TAT was proportionally and significantly correlated with EGF in Hp+ patients but not in Hp- and HP c patients. CONCLUSIONS: These results showed that VacA produced by H. pylori is associated with increased thrombin generation, and that thrombin may play a protective role in H. pylori-associated gastroduodenal disorders.     

Toll-Like Receptor 4 +3725 G/C Polymorphism, Helicobacter pylori Seropositivity, and the Risk of Gastric Atrophy and Gastric Cancer in Japanese

Background:  Toll-like receptor 4 (TLR4) Asp299Gly and Thr399Ile polymorphisms were reported to be a risk factor of gastric carcinoma or its precursors in Caucasian and Indian population, but these polymorphisms are absent in Japanese. We investigated the associations of TLR4 +3725 G/C polymorphism, another functional polymorphism of TLR4 , with risk of gastric cancer and gastric atrophy in Japanese.
Materials and Methods:  Study subjects were 583 histologically diagnosed gastric cancer patients and age- and sex-matched 1592 control outpatients, who visited Aichi Cancer Center Hospital from 2001 to 2005. Serum anti- H. pylori IgG antibody and pepsinogens were measured to evaluate H. pylori infection and gastric atrophy, respectively. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by a logistic model.
Results:  Among the seropositive subjects, the age- and sex-adjusted OR of gastric atrophy was 1.17 (95%CI: 0.91–1.50) for G/C , 1.20 (95%CI: 0.76–1.89) for C/C , and 1.18 (95%CI: 0.93–1.49) for G/C + C/C relative to G/G genotype. The age- and sex-adjusted OR of severe gastric atrophy among H. pylori seropositive subjects was 1.43 (95%CI: 0.99–2.06) for G/C , 1.47 (95%CI: 0.76–2.88) for C/C , and 1.43 (95%CI: 1.01–2.04) for G/C + C/C . The OR of gastric cancer compared with gastric atrophy controls was not statistically significant.
Conclusion:  Our study found that TLR4 +3725 G/C polymorphism was a risk factor of severe gastric atrophy in H. pylori seropositive Japanese. Our results underscored the significance of the variations in host innate immunity due to TLR4 polymorphism as genetic predispositions to gastric precancerous lesions in Eastern Asian populations with the same backgrounds.     

幽门螺杆菌在胃内定植的相关影响因素

胃内定植是引起幽门螺杆菌(Helicobacter pylori,H.pylori)感染的先决条件。H.pylori可穿过胃黏液层并与胃上皮细胞相互作用。这个定植过程主要受到H.pylori动力和尿素酶的影响。同时H.pylori形态、胃内pH、外膜蛋白及益生菌等也在其中扮演重要角色。该研究主要对H.pylori胃内定植过程中的相关影响因素进行综述。     

In-Cell Western analysis of Helicobacter pylori-induced phosphorylation of extracellular-signal related kinase via the transactivation of the epidermal growth factor receptor

Helicobacter pylori activates extracellular-signal related (ERK) kinases in gastric epithelial cells, via transactivation of the EGF receptor (EGFR). H. pylori activation of EGFR may be relevant to epithelial hyperproliferation and gastric carcinogenesis. The aim of this study was to develop an 'In-Cell Western' (ICW) assay for quantitative examination of H. pylori-induced epithelial signalling, to enable the role of the EGFR in H. pylori-induced phosphorylation of ERK in epithelial cells to be ascertained. H. pylori strains were co-incubated with A431 and AGS cells. pERK and total ERK were quantified in situ using ICW analysis. H. pylori strains both with, and without a cag PAI, and Helicobacter felis, significantly increased pERK levels in A431 cells. The EGFR inhibitor EKB-569 dose-dependently reduced H. pylori-induced ERK phosphorylation in A431 and AGS cells. A significantly lower reduction was observed with cag+ strains in A431 but not AGS cells. The cag PAI was not necessary for EGFR signal transactivation. These data suggest that H. pylori induces pERK in epithelial cells partly via the EGFR pathway. Additional signalling mechanisms are likely to be involved in H. pylori-induced ERK phosphorylation. ICW analysis is a rapid quantitative method for evaluating the effects of inhibitors on H. pylori-induced cell signalling pathways of relevance to gastric carcinogenesis.