Vaccination prevents Helicobacter pylori-induced alterations of the gastric flora in mice

Molecular analysis of the gastric microflora in mice revealed that Helicobacter pylori infection causes an increase in microbial diversity. The stomachs of H. pylori-infected animals were colonized by bacteria which are naturally restricted to the lower intestinal tract. Clostridia, Bacteroides/Prevotella spp., Eubacterium spp., Ruminococcus spp., streptococci and Escherichia coli were detected exclusively in the stomachs of infected animals, whereas lactobacilli dominated the gastric flora in noninfected mice. The H. pylori-induced shifts in the gastric microbiota were independent from histological pathology and from changes in the gastric pH but were prevented by immunization of mice with live Salmonella expressing H. pylori urease. Immunized mice displayed reduced H. pylori levels in the gastric epithelium and developed a normal gastric microflora, indicating that vaccination may be protective against H. pylori-induced changes in the gastric flora.     

5-LOX inhibitor modulates the inflammatory responses provoked by Helicobacter pylori infection

BACKGROUND: Arachidonic acid metabolites have been considered as pivotal mediators in Helicobacter pylori-induced inflammatory response, which are mainly metabolized by two distinct enzymes: cyclooxygenase (COX) and lipoxygenase (LOX). While COX has become well known to play a key role in either carcinogenesis or inflammation related to H. pylori infection, little is known regarding the implication of LOX in H. pylori infection. In this study, we evaluated the roles of 5-LOX and its metabolites in H. pylori-induced host responses and further a potential beneficial action of specific LOX inhibitors against H. pylori infection. MATERIALS AND METHODS: Expressions of cytosolic phospholipase A(2) (cPLA(2)), COX-2, and 5-LOX after H. pylori infection were evaluated by immunofluorescence staining and Western blotting. Synthesis of LOX metabolites was measured with reversed-phase high-performance liquid chromatography. For analyzing the influence of 5-LOX inhibitors, nordihydroguaiaretic acid (NDGA) and geraniin, on H. pylori-induced inflammatory responses, RNase protection assay and RT-PCR were performed. RESULTS: H. pylori stimulated the translocation of cPLA(2) from cytoplasm to nucleus and increased the biosynthesis of hydroxyeicosatetraenoic acids (HETEs) as a predominant form of 5S-HETE in gastric epithelium. NDGA exerted a strong suppression activity of H. pylori-induced 5-LOX signaling. The administration of LOX inhibitors was related with down-expression of proinflammatory mediators such as interleukin-8 and tumor necrosis factor-alpha in both H. pylori-infected gastric epithelial cells and macrophage cells. CONCLUSION: LOX modulation with its specific inhibitors could impose significant anti-inflammatory responses after H. pylori infection, based on the fact that H. pylori infection provoked gastric inflammation through metabolizing arachidonic acid by the 5-LOX pathway.     

Inducible nitric oxide synthase expression before and after eradication of Helicobacter pylori in different forms of gastritis

An increased expression of inducible nitric oxide synthase (iNOS) has been observed in the inflamed human gastric mucosa as well as in some tumors. This observation suggests a pathobiological role of elevated NO production. The purpose of this study was to compare the immunohistochemical iNOS expression in the different kinds of gastritis before and after the eradication of Helicobacter pylori. We performed iNOS and H. pylori immunohistochemical staining and counted iNOS positive cells. We detected elevated expression of iNOS around sites infected with H. pylori. iNOS expression in chemical gastritis was strongly elevated in mucosal glands. After treatment, we found a significant difference in iNOS expression in patients with classical H. pylori-induced antrum predominant gastritis and in patients with active autoimmune gastritis. In the special case of progressed gastritis with intestinal metaplasia we found persistence of intestinal metaplasia, and we could not find a significant difference in the number of positive iNOS cells before and after treatment. The persistence of IM as a possibly precancerous lesion is probably at least in the antrum a source of continuous iNOS induction even after H. pylori eradication.     

Apoptosis in different gastric lesions and gastric cancer: relationship with Helicobacter pylori, overexpression of p53 and aneuploidy

Apoptosis has an essential function in maintaining the integrity of the gastrointestinal mucosa. Its deregulation is associated with the occurrence of lesions such as in atrophic gastritis, peptic ulcers, intestinal metaplasia, and stomach tumorigenesis. Thus, the aim of the present study was to investigate the frequency of apoptotic cells (apoptotic index, AI) by using two different immunohistochemical techniques, TUNEL and anti-activated caspase-3 antibody (CPP32), in gastric dyspepsia [chronic gastritis (CG, N = 34), chronic atrophic gastritis (CAG, N = 11), gastric ulcer (GU, N = 17), and intestinal metaplasia (IM, N = 15)], normal gastric mucosae (NM, N = 8), and gastric adenocarcinoma (GC, N = 12). The relationship was investigated between the AI and Helicobacter pylori infection, diagnosed by PCR, overexpression of p53 protein determined by immunohistochemistry, and aneuploidy by fluorescence in situ hybridization, as performed by our laboratory in previous studies. No significant differences were observed in AI between the different groups, whether by the TUNEL technique (F = 1.60; p = 0.1670) or by CPP32 antibody (F = 1.70; p = 0.1420). Nonetheless, CAG and CG groups had AI statistically higher than those of normal mucosae. These two groups (CAG and CG) also showed a higher frequency of apoptosis-positive cases (TUNEL+ or CPP32+). Generally, there was no correlation between the AI detected by the TUNEL and CPP32 techniques in the groups studied, except in the GC group (r = 0.70). Moreover, there was no significant association between apoptosis and H. pylori infection, overexpression of p53 protein and aneuploidy, but the H. pylori-positive cases only of GU (p = 0.0233) and IM (p = 0.0253) groups displayed a statistically higher AI compared to H. pylori-negative NM, when the CPP32 antibody technique was used. Thus, CG and CAG have increased apoptosis, which may occur independent of an association with H. pylori infection, aneuploidy and overexpression of p53 protein.     

Activation of peroxisome proliferator-activated receptor gamma suppresses nuclear factor kappa B-mediated apoptosis induced by Helicobacter pylori in gastric epithelial cells

Helicobacter pylori colonization leads to epithelial cell hyperproliferation within inflamed mucosa, but levels of apoptosis vary, suggesting that imbalances between rates of cell production and loss may contribute to differences in gastric cancer risk among infected populations. Peroxisome proliferator-activated receptor gamma (PPARgamma) regulates inflammatory and growth responses of intestinal epithelial cells. We determined whether activation of PPARgamma modified H. pylori-induced apoptosis in gastric epithelial cells. PPARgamma was expressed and functionally active in gastric epithelial cell lines sensitive to H. pylori-induced apoptosis. PPARgamma ligands 15d-PGJ(2) and BRL-49653 significantly attenuated H. pylomicronri-induced apoptosis, effects that could be reversed by co-treatment with a specific PPARgamma antagonist. Cyclopentanone prostaglandins that do not bind and activate PPARgamma had no effects on H. pylori-induced apoptosis. The ability of H. pylori to activate nuclear factor (NF)-kappaB and increase levels of the NF-kappaB target IL-8 was blocked by co-treatment with PPARgamma agonists, and direct inhibition of NF-kappaB also abolished H. pylori-stimulated apoptosis. These results suggest that activation of the PPARgamma pathway attenuates the ability of H. pylori to induce NF-kappaB-mediated apoptosis in gastric epithelial cells. Because PPARgamma regulates a multitude of host responses, activation of this receptor may contribute to varying levels of cellular turnover as well as the diverse pathologic outcomes associated with chronic H. pylori colonization.     

Host Single Nucleotide Polymorphisms of MMP-9 −1562/TIMP-1 372 Have Gender Differences in the Risk of Gastric Intestinal Metaplasia After Helicobacter pylori Infection

Background:  Helicobacter pylori infection causes chronic gastric inflammation and intestinal metaplasia (IM), related with deregulation of Wnt pathway and over-expressions of COX-2, matrix metalloproteinase (MMP), and tissue inhibitors of matrix metalloproteinase (TIMP). We thus test the host genomic predispositions related to the risk of IM after H. pylori infection.
Methods:  We enrolled 296 H. pylori -infected patients to provide gastric biopsies for histology and genomic DNA for genotypes of single nucleotide polymorphisms (SNPs), including APC , COX-2 , IL-1B , IL-1RN , IL-10 , MMP-2 , MMP-9 , TIMP-1 , and TIMP-2 determined by sequence specific oligonucleotide probe, sequence specific primers, restriction fragment length polymorphism, or real-time polymerase chain reaction.
Results:  There was no association between the presence of IM and SNPs in APC , COX-2 , IL-1B , IL-1RN , IL-10 , MMP-2 , and TIMP-2 . The risk of IM was increased up to 2.29-folds in males with TIMP-1 372 C, and 3.03-fold in females with T carrier ( p  < .05). The combination genotype of MMP-9 − 1562/ TIMP-1 372 as CC/C and CT/T in males had a 4.5-fold increased risk of IM, as compared to CC/T ( p  < .05). Females with such combination genotype as CC/T-carrier had a 3-fold risk of IM than males with CC/T ( p  < .05). In contrast, males' combination genotype as CC/C had a 3-fold risk of IM than females with CC/CC ( p =  .05).
Conclusions:  The host MMP-9 − 1562/ TIMP-1 372 SNPs had gender differences in the risk of IM after H. pylori infection, and could possibly serve as a host factor to identify the risk group harboring gastric precancerous changes after H. pylori infection.     

Causal relationship between the loss of RUNX3 expression and gastric cancer

Runx3/Pebp2alphaC null mouse gastric mucosa exhibits hyperplasias due to stimulated proliferation and suppressed apoptosis in epithelial cells, and the cells are resistant to growth-inhibitory and apoptosis-inducing action of TGF-beta, indicating that Runx3 is a major growth regulator of gastric epithelial cells. Between 45% and 60% of human gastric cancer cells do not significantly express RUNX3 due to hemizygous deletion and hypermethylation of the RUNX3 promoter region. Tumorigenicity of human gastric cancer cell lines in nude mice was inversely related to their level of RUNX3 expression, and a mutation (R122C) occurring within the conserved Runt domain abolished the tumor-suppressive effect of RUNX3, suggesting that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer.     

Urokinase plasminogen activator receptor is upregulated by Helicobacter pylori in human gastric cancer AGS cells via ERK, JNK, and AP-1

The gastric pathogen Helicobacter pylori (H. pylori) is suggested to be associated with gastric cancer progression. In this study, we investigated the effect of H. pylori on urokinase plasminogen activator receptor (uPAR) expression which has been known to correlate closely with gastric cancer invasion. H. pylori induced the uPAR expression in a time- and concentration-dependent manner. Specific inhibitors and inactive mutants of MEK-1 and JNK were found to suppress the H. pylori-induced uPAR expression and the uPAR promoter activity. Electrophoretic mobility shift assay and transient transfection study using an AP-1 decoy oligonucleotide confirmed that the activation of AP-1 is involved in the H. pylori-induced uPAR upregulation. The AGS cells treated with H. pylori showed a remarkably enhanced invasiveness, and this effect was partially abrogated by uPAR-neutralizing antibodies. These results suggest that H. pylori induces uPAR expression via Erk-1/2, JNK, and AP-1 signaling pathways and, in turn, stimulates the cell invasiveness in human gastric cancer AGS cells.     

Helicobacter pylori activates NF-kappaB via the alternative pathway in B lymphocytes

Helicobacter pylori causes various gastroduodenal diseases including gastric MALT lymphoma, but the mechanism underlying H. pylori-induced carcinogenesis is not known. The alternative pathway for NF-kappaB activation, which involves the processing of NF-kappaB2/p100 to p52, has been implicated in lymphocyte survival, attenuated apoptosis, and secondary lymphoid tissue development. In this study, we investigated H. pylori-induced activation of NF-kappaB through the alternative pathway in B lymphocytes. In immunoblot and EMSA, H. pylori induced NF-kappaB2/p100 processing to p52 and subsequent nuclear accumulation in IM-9 (human B cell line) cells and human peripheral blood B cells, but not in AGS (human gastric cancer cell line) cells. The activation of the alternative pathway was LPS-dependent but not cag pathogenicity island-dependent. Alternative pathway activation by H. pylori was associated with attenuated apoptosis. The expression levels of B lymphocyte chemoattractant, EBI-1 ligand chemokine, and stromal cell-derived factor-1alpha mRNAs were up-regulated in cocultured human B cells and in infected human gastric mucosa. In the infected mucosa, NF-kappaB2/p100 and p52 were detected immunohistochemically in the cytoplasm and nuclear compartments of lymphocytes, but not in epithelial cells. In summary, H. pylori activates the alternative NF-kappaB pathway in B lymphocytes. The effects on chemokine production and antiapoptosis mediated by H. pylori-induced processing of NF-kappaB2/p100 to p52 may drive lymphocytes to acquire malignant potential.     

Upregulation of progranulin by Helicobacter pylori in human gastric epithelial cells via p38MAPK and MEK1/2 signaling pathway: role in epithelial cell proliferation and migration

Helicobacter pylori is a major human pathogen associated with gastric diseases such as chronic active gastritis, peptic ulcer, and gastric carcinoma. The growth factor progranulin (PGRN) is a secreted glycoprotein that functions as an important regulator of cell growth, migration, and transformation. We aimed to determine the molecular mechanisms by which H. pylori upregulates the expression of PGRN and the relationship between H. pylori infection and production of PGRN in controlling cell proliferation and migration. Levels of PGRN were examined in gastric tissues from patients and in vitro in gastric epithelial cells. Cell proliferation was measured by colony formation assay. Cell migration was monitored by wound healing migration assay. PGRN protein levels were increased in patients with gastritis and gastric cancer tissue. Infection of gastric epithelial cells with H. pylori significantly increased PGRN expression in a time-dependent manner. Blockade of the p38 and MEK1/2 pathway by inhibitor inhibited H. pylori-mediated PGRN upregulation. Activation of p38 and MEK1/2 pathway by H. pylori was also identified. Knockdown of PGRN attenuated the H. pylori-induced proliferative activity and migration of cancer cells. These findings suggest that the upregulation of PGRN in H. pylori-infected gastric epithelial cells may contribute to the carcinogenic process.     

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.