Infection with Helicobacter pylori Affects All Major Secretory Cell Populations in the Human Antrum

We have investigated how gastric H. pylori infection affects antrum secretory cell types by studying the expression of secretory proteins in antrum epithelium. Antrum biopsy specimens were prospectively collected from 102 individuals (49 H. pylori-infected). Immunohistochemistry was performed for secretory mucins (MUC5AC, MUC5B, MUC6), Trefoil factor family (TFF)-peptides (TFF1, TFF2), endocrine peptides (gastrin, chromogranin A), and proliferating cells (Ki-67). Protein expression was quantified morphometrically. H. pylori infection was significantly correlated to mucosal inflammation and to epithelial atrophy and proliferation. In H. pylori-infected patients the number of proliferating cells increased significantly, and the zone of proliferating cells shifted toward the surface epithelium of the antral glands. Infection was correlated with decreased MUC5AC, TFF1, and TFF2 expression and increased MUC6 and MUC5B expression. Endocrine cells expressing chromagranin A and gastrin shifted toward the surface epithelium of the antral glands in H. pylori-infected patients. H. pylori infection and concomitant inflammation induced increased epithelial proliferation and triggered coordinate deregulation of secretory cell populations in the antrum. In particular, infection led to a coordinated increase in cells expressing MUC6 and MUC5B at the expense of MUC5AC-producing cells.     

Helicobacter pylori infection induces a reversible expression of the CDX2 transcription factor protein in human gastric epithelium

Objective. The homeobox gene CDX2 is implicated in the appearance of intestinal metaplasia in Helicobacter pylori gastritis. The aim of this study was to investigate whether CDX2 expression in gastric mucosa occurs before the appearance of overt intestinal metaplasia in H. pylori gastritis, and whether or not this expression is reversible. Material and methods. CDX2 was studied by immunohistochemistry in a cohort of 38 patients with H. pylori gastritis before and after eradication (mean follow-up 6.3 years) of H. pylori. A cohort of 49 individuals with healthy stomachs was analysed as a control. Results. In the control group no immunostaining of CDX2 in the epithelial cells of the gastric body was found, while in 57% of the cases a mild, aberrant nuclear immunostaining of CDX2 in the non-metaplastic epithelial cells in antrum, designated as “positive staining of single cells” (PSSC), was found. In H. pylori gastritis, the PSSC was seen in antrum and corpus in 100% and 26% of the cases, respectively. The prevalence of antral PSSC was significantly increased (on average by 4-fold) in H. pylori gastritis as compared with controls. After eradication of H. pylori, the prevalence of PSSC decreased significantly in antrum but not in corpus. Conclusions. Expression of CDX2 at low intensity is common in the epithelium of normal antrum, and this expression is enhanced in H. pylori gastritis. Expression of CDX2 is reversible at least in antrum after eradication of H. pylori infection.     

Nucleotide Binding Oligomerization Domain 1 Is an Essential Signal Transducer in Human Epithelial Cells Infected with Helicobacter pylori That Induces the Transepithelial Migration of Neutrophils

Background/Aims

The cytosolic host protein nucleotide binding oligomerization domain 1 (Nod1) has emerged as a key pathogen recognition molecule for innate immune responses in epithelial cells. The purpose of the study was to elucidate the mechanism by which Helicobacter pylori infection leads to transepithelial neutrophil migration in a Nod1-mediated manner.

Methods

Human epithelial cell lines AGS and Caco-2 were grown and infected with H. pylori. Interleukin (IL)-8 mRNA expression and IL-8 secretion were assessed, and nuclear factor κB (NF-κB) activation was determined. Stable transfections of AGS and Caco-2 cells with dominant negative Nod1 were generated. Neutrophil migration across the monolayer was quantified.

Results

Cytotoxin-associated gene pathogenicity island (cagPAI)(+) H. pylori infection upregulated IL-8 mRNA expression and IL-8 secretion in AGS and Caco-2 cells compared with controls. NF-κB activation, IL-8 mRNA expression and IL-8 secretion by cagPAI knockdown strains were reduced compared with those infected with the wild-type strain. NF-κB activation, IL-8 mRNA expression and IL-8 secretion in dominant-negative (DN)-Nod1 stably transfected cells were reduced compared with the controls. The transepithelial migration of neutrophils in DN-Nod1 stably transfected cells was reduced compared with that in controls.

Conclusions

Signaling through Nod1 plays an essential role in neutrophil migration induced by the upregulated NF-κB activation and IL-8 expression in H. pylori-infected human epithelial cells.     

Relevance of high virulence Helicobacter pylori strains and futility of CDX2 expression for predicting intestinal metaplasia after eradication of infection

Abstract

Objective. Different Helicobacter pylori genotypes are associated with distinct inflammatory responses and consequent development of pre-neoplastic lesions, namely intestinal metaplasia (IM), which is dependent on the expression of CDX2. We aimed to evaluate IM progression/regression in the context of H. pylori eradication, bringing into play the effect of the virulence of infecting H. pylori strains and the hypothesis that CDX2 expression might be a marker for later development of IM. Material and methods. Sixty-five male volunteers were evaluated by endoscopy before H. pylori eradication and after a median six-year follow-up. Histological diagnosis was performed at baseline and follow-up, and baseline H. pylori genotypes and CDX2 expression in non-metaplastic foci were also assessed. Results. Fifty-one individuals remained free from infection at follow-up. Six out of 27 who had no metaplastic lesions at baseline developed IM. CDX2 nuclear expression was observed in 15 of the 21 cases (71.4%) showing no progression to IM, and in three out of six cases (50%) with progression to IM (p = 0.367). Six of the 24 cases with IM at baseline showed regression to less severe outcomes, which was less frequent in those infected with high-virulence strains (7.7% vs. 50%, p = 0.047). In the latter there is a significant persistence of lymphoid follicles. Conclusions. Our results support that under infection with high virulence H. pylori strains, IM is a point of difficult return in the gastric carcinogenic pathway. The appearance of CDX-expressing cells in non-metaplastic foci was not associated with the development of IM during the six-year follow-up.     

Helicobacter pylori Inhibits Activity of cdc2 Kinase and Delays G 2 /M to G 1 Progression in Gastric Adenocarcinoma Cell

Background: Helicobacter pylori is a bacterial pathogen strongly associated with ulcer diseases and gastric cancer. The bacterial-induced alteration of cell-cycle control in host cells may play a role in the pathogenetic mechanisms. The aims of this study were to define the effect of H. pylori on the G ₂ /M to G ₁ transition in a gastric cell line. Methods: Cultured gastric cells, AGS, were synchronized in the S/early G2 phase and treated with intact H. pylori. The cell-cycle distribution of AGS cells was determined by flow cytometry. The activity of cdc2 kinase, as well as of some parameters that affect the kinase activity, was also examined. Results: H. pylori delays cell-cycle progression at the G ₂ /M phase in AGS cells. The G ₂ /M delay was associated with reduced activity of cdc2 kinase. Both down-regulation of cell-cycle regulators (p34 ^cdc2 , cyclin B1 and cdc25C) and decreased association between p34 ^cdc2 and cyclin B1 were found to be associated with the activity of cdc2 kinase abated after the H. pylori infection. In addition, the H. pylori-induced G ₂ /M delay required direct contact between the bacteria and host cells. Conclusions: H. pylori inhibits G ₂ /M to G ₁ progression and causes a reduction of cell division in gastric epithelial cells.     

Upregulated Cyclooxygenase-2 Inhibits Apoptosis of Human Gastric Epithelial Cells Infected with Helicobacter pylori

Helicobacter pylori induces apoptosis and alters the proliferation of gastric mucosal epithelial cells. Cyclooxygenase-2 (COX-2), the inducible form of prostaglandin (PG) synthesis, is known to cause alteration in epithelial cell growth. The goal of this study was to determine whether COX-2 gene expression by H. pylori infection could influence gastric epithelial cell apoptosis. Expression of COX-2 mRNA and proteins was up-regulated in Hs746T gastric epithelial cell lines infected with H. pylori, when assessed by quantitative RT-PCR and western blot. Inhibition of COX-2 expression using NS-398, a specific COX-2 inhibitor, showed a significant increase of gastric epithelial cell apoptosis and caspase-3 activation in Hs746T cells infected with H. pylori. Moreover, the effect of NS-398 on H. pylori-induced apoptosis was reversed by the addition of PGE₂. These results suggest that up-regulated COX-2 expression by H. pylori infection can inhibit apoptosis of gastric epithelial cells.     

Trefoil factor 2 in gland mucous cell mucin in the mucous gel covering normal or damaged gastric mucosa using the Mongolian gerbil model

Objective. Trefoil factor 2 (TFF2) is localized in gastric gland mucous cells. The purpose of the study was to determine whether TFF2 and gastric mucin are localized in mucous cells and in the surface mucous gel layer (SMGL) of the normal gastric mucosa or in the mucoid cap adherent to gastric mucosal lesions in Mongolian gerbils. Material and methods. Gastric mucosal lesions were induced in Mongolian gerbils using oral administration of Helicobacter pylori (H. pylori), subcutaneous administration of indomethacin, or oral administration of 30% ethanol. Tissue samples were fixed in Carnoy's solution for preservation of the SMGL, dehydrated, and embedded in paraffin. Histochemical staining for gastric mucins and immunostaining for TFF2 were performed. Results. It was found that surface mucous cell mucin and gland mucous cell mucin were segregated in the SMGL covering the normal gastric mucosa, and the mucin of the mucoid cap covering the mucosal lesions was primarily gland mucous cell mucin. There was a co-localization of TFF2 in gland mucous cell mucin in gland mucous cells, the SMGL, and the mucoid cap. Conclusions. The co-localization of TFF2 in gland mucous cells and in the adherent mucus suggests a physical interaction between TFF2 and gland mucous cell mucin, and the participation of TFF2 trapped in the adherent mucus functions in mucosal defense, healing, and repair.     

αPix interacts with Helicobacter pylori CagA to induce IL-8 expression in gastric epithelial cells

Objective.Helicobacter pylori CagA, translocated into gastric epithelial cells, induces IL-8 expression through the signalling pathways, including extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB). We previously demonstrated that CagA interacts with host αPix. The present study was purposed to determine the role of the interaction of αPix with CagA on the signalling pathways for IL-8 expression in H. pylori-infected gastric epithelial cells. Material and methods.H. pylori HP99 strain (CagA+, VacA + ) was infected to gastric epithelial AGS cells transfected with non-targeting (NT) or αPix- targeting siRNA. Activation of signalling molecules including p21-activated kinase (PAK), ERK and NF-κB, and expression of IL-8 in the cells were assessed. Results:H. pylori CagA was delivered into AGS cells and then interacted with αPix at 4 h following H. pylori infection. PAK1, ERK and NF-κB were activated in the cells containing NT and αPix siRNA at 1–2 h following H. pylori infection. However, after 4 h, the time when CagA was delivered into the cells, the activations of PAK1, ERK and NF-κB were inhibited by down-regulation of αPix using siRNA but not by NT siRNA. The results indicate that αPix is required for H. pylori-mediated signalling of PAK1, ERK and NF-κB. Additionally, αPix siRNA suppressed IL-8 induction after translocation of CagA into the cells, indicating that interaction of CagA with αPix is critical for CagA-mediating signalling for IL-8 expression. Conclusions. The interaction of αPix with CagA activates PAK1, ERK and NF-κB, which induces IL-8 expression in H. pylori-infected gastric epithelial cells.     

Helicobacter pylori-Stimulated Interleukin-8 (IL-8) Promotes Cell Proliferation Through Transactivation of Epidermal Growth Factor Receptor (EGFR) by Disintegrin and Metalloproteinase (ADAM) Activation

Helicobacter pylori infection increases the risk of hyperplastic polyps and gastric cancer, but the mechanisms remain to be elucidated. H. pylori was recently shown to transactivate epidermal growth factor receptor (EGFR) through metalloprotease stimulation. The present study was designed to investigate the effect of interleukin-8 (IL-8) induced by H. pylori infection on EGFR transactivation and epithelial cell growth. H. pylori Sydney strain 1 (SS1) having wild-type cag⁺A was used. Phospho-EGFR assay was performed by immunoprecipitation using anti-human EGFR and anti-phosphotyrosine antibodies. DNA synthesis was evaluated by [³H]thymidine uptake using the human gastric cancer cell line, KATO III. H. pylori induced EGFR phosphorylation, and a disintegrin and metalloproteinase (ADAM) inhibitor, KB-R7785, completely suppressed EGFR phosphorylation. IL-8 also induced EGFR phosphorylation, while anti-IL-8 and anti-IL-8 receptor (CXCR1) neutralizing antibodies suppressed EGFR phosphorylation. [³H]Thymidine uptake analysis demonstrated that H. pylori increased DNA synthesis in gastric epithelial cells, and tyrosine kinase inhibitor, MEK inhibitor, and ADAM inhibitor suppressed the DNA synthesis induced by H. pylori. H. pylori-stimulated IL-8 accelerates processing of EGFR ligands through ADAM activation, and cleaved EGFR ligands bind and stimulate EGFR in paracrine and autocrine manners to induce cell proliferation. This may be one of the mechanisms of hyperplastic polyp and gastric cancer development in H. pylori-infected gastric mucosa.     

Effect of an Aluminum Hydroxide-Magnesium Hydroxide Combination Drug on Adhesion,IL-8 Inducibility,and Expression of HSP60 by Helicobacter pylori

Background: Co-magaldrox (Maalox®) is used world-wide as an antacid and as a cytoprotective agent for gastritis and peptic ulcer diseases. We examined the effects of co-magaldrox on Helicobacter pylori. Methods: Adhesion of H. pylori to human gastric epithelial cells (MKN45) was evaluated by flow cytometry. Morphologic changes in H. pylori caused by co-magaldrox were determined by scanning electron microscopy. Induction of interleukin-8 (IL-8) from MKN45 cells was examined with enzyme-linked immunosorbent assay, and the intracellular and extracellular expression of heat-shock protein 60 (HSP60) was analyzed with sodium dodecyl sulphate-polyacrylamide gel electrophoresis and flow cytometry. Results: Adhesion of H. pylori to MKN 45 cells was significantly inhibited by 1.25%-5% co-magaldrox. H. pylori aggregated with co-magaldrox according to an electron microscopic examination. IL-8 secretion from MKN45 cells after H. pylori infection was also inhibited by co-magaldrox. Extracellular expression of HSP60 on the surface of H. pylori was decreased after treatment with co-magaldrox, whereas the intracellular synthesis of HSP60 was not. HSP60-induced IL-8 secretion was significantly inhibited by co-magaldrox in a dose-dependent manner. Conclusions: These results show that co-magaldrox suppressed the expression of the following virulence factors: adhesion, IL-8 inducibility, and expression of extracellular HSP60. Therefore, co-magaldrox is a potent anti-H. pylori and cytoprotective drug.     

Alterations of Gastric Homeoprotein Expression in Helicobacter pylori Infection, Incisural Antralisation, and Intestinal Metaplasia

Purpose This study was to determine whether gastric expression of homeoproteins is altered in Helicobacter pylori infection, incisural antralisation, and intestinal metaplasia (IM). Methods Gastric biopsy specimens were taken from 98 patients with non-ulcer dyspepsia for the detection of H. pylori infection; histological examinations; immunohistochemical staining of CDX2, PDX1, PAX6, and NKX6.1. Results Of the patients, 38 were positive for H. pylori infection, 44 had antral-type mucosa at the incisura, and 22 had IM in the stomach. At the incisura, the expression of PDX1, NKX6.1, and PAX6 in cytoplasm compartment was down-regulated in antral-type mucosa compared with that in the transitional- or body-type mucosa (all P < 0.01). The expression of PDX1, PAX6, and NKX6.1 in cytoplasm at the incisura was down-regulated in H. pylori-infected patients compared with that in those without H. pylori infection (all P < 0.01). CDX2 expression in whole stomach was up-regulated, but PDX1 expression at the incisura was down-regulated in patients with IM compared with that in those without IM (all P < 0.01). Conclusions Gastric expression of PDX1, PAX 6, and NKX6.1 is down-regulated in H. pylori infection and incisural antralisation. CDX2 is up-regulated but PDX1 is down-regulated in the presence of IM.     

Expression of homeodomain protein CDX2 in gallbladder carcinomas

Purpose Caudal-related homeobox protein CDX2 plays an important role in the regulation of cell proliferation and differentiation of the intestinal epithelium. CDX2 is associated with intestinal metaplasia and carcinomas of the stomach, but the role of CDX2 in gallbladder carcinogenesis remains unknown.Methods We analyzed the expression of CDX2 and intestinal apomucin MUC2 in gallbladder cancer cell lines at the mRNA level by the RT-PCR method. We also investigated the expression of CDX2 and MUC2 in 68 primary gallbladder carcinomas by the immunohistochemical staining method and compared the expression of CDX2 with the clinicopathological factors in the gallbladder carcinoma cases.Results Expression of CDX2 and MUC2 was found in three of four gallbladder cancer cell lines at the mRNA level. In addition, we found that CDX2 was absent in the normal gallbladder epithelium, but the CDX2 protein was expressed in 25 of the 68 (36.8%) gallbladder carcinomas. Interestingly, in the tubular type gallbladder carcinomas, the frequency of CDX2 expression was much higher in the well-differentiated type than the moderately and poorly differentiated types, the difference being statistically significant (P<0.01). CDX2 expression showed a relationship with expression of MUC2 (P<0.04) in the gallbladder carcinomas. CDX2 was expressed in intestinal metaplasia and dysplasia, which are hypothesized to be premalignant conditions.Conclusion These results imply that CDX2 plays an important role in gallbladder carcinogenesis with intestinal differentiation.