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1.
Mucosal Immunol ; 10(2): 373-384, 2017 03.
Article in English | MEDLINE | ID: mdl-27353252

ABSTRACT

p40, a Lactobacillus rhamnosus GG (LGG)-derived protein, transactivates epidermal growth factor receptor (EGFR) in intestinal epithelial cells, leading to amelioration of intestinal injury and inflammation. To elucidate mechanisms by which p40 regulates mucosal immunity to prevent inflammation, this study aimed to determine the effects and mechanisms of p40 on regulation of a proliferation-inducing ligand (APRIL) expression in intestinal epithelial cells for promoting immunoglobulin A (IgA) production. p40 upregulated April gene expression and protein production in mouse small intestine epithelial (MSIE) cells, which were inhibited by blocking EGFR expression and kinase activity. Enteroids from Egfrfl/fl, but not Egfrfl/fl-Vil-Cre mice with EGFR specifically deleted in intestinal epithelial cells, exhibited increased April gene expression by p40 treatment. p40-conditioned media from MSIE cells increased B-cell class switching to IgA+ cells and IgA production, which was suppressed by APRIL receptor-neutralizing antibodies. Treatment of B cells with p40 did not show any effects on IgA production. p40 treatment increased April gene expression and protein production in small intestinal epithelial cells, fecal IgA levels, IgA+B220+, IgA+CD19+, and IgA+ plasma cells in lamina propria of Egfrfl/fl, but not of Egfrfl/fl-Vil-Cre, mice. Thus p40 upregulates EGFR-dependent APRIL production in intestinal epithelial cells, which may contribute to promoting IgA production.


Subject(s)
Antibody Formation , B-Lymphocytes/immunology , Bacterial Proteins/metabolism , Epithelial Cells/immunology , Intestine, Small/pathology , Lacticaseibacillus rhamnosus/immunology , Tumor Necrosis Factor Ligand Superfamily Member 13/metabolism , Animals , Bacterial Proteins/immunology , Cells, Cultured , ErbB Receptors/genetics , ErbB Receptors/metabolism , Immunoglobulin A/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , RNA, Small Interfering/genetics , Transcriptional Activation , Tumor Necrosis Factor Ligand Superfamily Member 13/genetics , Up-Regulation
2.
Mucosal Immunol ; 10(1): 117-127, 2017 01.
Article in English | MEDLINE | ID: mdl-27095077

ABSTRACT

Development of the intestinal microbiota during early life serves as a key regulatory stage in establishing the host-microbial relationship. This symbiotic relationship contributes to developing host immunity and maintaining health throughout the life span. This study was to develop an approach to colonize conventionally raised mice with a model probiotic bacterium, Lactobacillus rhamnosus GG (LGG), and to determine the effects of LGG colonization on intestinal development and prevention of colitis in adulthood. LGG colonization in conventionally raised was established by administering LGG to pregnant mice starting at gestational day 18 and pups at postnatal days 1- 5. LGG colonization promoted bodyweight gain and increased diversity and richness of the colonic mucosa-associated microbiota before weaning. Intestinal epithelial cell proliferation, differentiation, tight junction formation, and mucosal IgA production were all significantly enhanced in LGG-colonized mice. Adult mice colonized with LGG showed increased IgA production and decreased susceptibility to intestinal injury and inflammation induced in the dextran sodium sulfate model of colitis. Thus, neonatal colonization of mice with LGG enhances intestinal functional maturation and IgA production and confers lifelong health consequences on protection from intestinal injury and inflammation. This strategy might be applied for benefiting health in the host.


Subject(s)
Colitis/immunology , Gastrointestinal Microbiome/immunology , Immunoglobulin A/metabolism , Intestinal Mucosa/immunology , Intestines/physiology , Lacticaseibacillus rhamnosus/immunology , Probiotics/administration & dosage , Animals , Animals, Newborn , Cell Proliferation , Cells, Cultured , Colitis/microbiology , Colitis/prevention & control , Dextran Sulfate , Disease Models, Animal , Female , Humans , Intestinal Mucosa/microbiology , Intestinal Mucosa/pathology , Intestines/microbiology , Mice , Mice, Inbred C57BL , Pregnancy , Symbiosis , Tight Junctions/pathology
3.
Oncogene ; 35(48): 6262-6269, 2016 12 01.
Article in English | MEDLINE | ID: mdl-27157617

ABSTRACT

Helicobacter pylori (H. pylori) is the strongest identified risk factor for gastric cancer, the third most common cause of cancer-related death worldwide. An H. pylori constituent that augments cancer risk is the strain-specific cag pathogenicity island, which encodes a type IV secretion system (T4SS) that translocates a pro-inflammatory and oncogenic protein, CagA, into epithelial cells. However, the majority of persons colonized with CagA+ H. pylori strains do not develop cancer, suggesting that other microbial effectors also have a role in carcinogenesis. Toll-like receptor 9 (TLR9) is an endosome bound, innate immune receptor that detects and responds to hypo-methylated CpG DNA motifs that are most commonly found in microbial genomes. High-expression tlr9 polymorphisms have been linked to the development of premalignant lesions in the stomach. We now demonstrate that levels of H. pylori-mediated TLR9 activation and expression are directly related to gastric cancer risk in human populations. Mechanistically, we show for the first time that the H. pylori cancer-associated cag T4SS is required for TLR9 activation and that H. pylori DNA is actively translocated by the cag T4SS to engage this host receptor. Activation of TLR9 occurs through a contact-dependent mechanism between pathogen and host, and involves transfer of microbial DNA that is both protected as well as exposed during transport. These results indicate that TLR9 activation via the cag island may modify the risk for malignancy within the context of H. pylori infection and provide an important framework for future studies investigating the microbial-epithelial interface in gastric carcinogenesis.


Subject(s)
Bacterial Proteins/metabolism , Helicobacter Infections/metabolism , Helicobacter Infections/microbiology , Helicobacter pylori/physiology , Toll-Like Receptor 9/metabolism , Type IV Secretion Systems , Bacterial Proteins/genetics , Biological Transport , Carcinogenesis , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Helicobacter Infections/complications , Humans , Mutation , Stomach Neoplasms/etiology
4.
Oncogene ; 34(14): 1865-71, 2015 Apr 02.
Article in English | MEDLINE | ID: mdl-24837365

ABSTRACT

Helicobacter pylori is the strongest risk factor for the development of gastric cancer. Although the specific mechanisms by which this pathogen induces carcinogenesis have not been fully elucidated, high-expression interleukin (IL)-1ß alleles are associated with increased gastric cancer risk among H. pylori-infected persons. In addition, loss of matrix metalloproteinase 7 (MMP7) increases mucosal inflammation in mouse models of epithelial injury, and we have shown that gastric inflammation is increased in H. pylori-infected MMP7(-/-) C57BL/6 mice. In this report, we define mechanisms that underpin such responses and extend these results into a genetic model of MMP7 deficiency and gastric cancer. Wild-type (WT) or MMP7(-/-) C57BL/6 mice were challenged with broth alone as an uninfected control or the H. pylori strain PMSS1. All H. pylori-challenged mice were successfully colonized. As expected, H. pylori-infected MMP7(-/-) C57BL/6 mice exhibited a significant increase in gastric inflammation compared with uninfected or infected WT C57BL/6 animals. Loss of MMP7 resulted in M1 macrophage polarization within H. pylori-infected stomachs, as assessed by Luminex technology and immunohistochemistry, and macrophages isolated from infected MMP7-deficient mice expressed significantly higher levels of the M1 macrophage marker IL-1ß compared with macrophages isolated from WT mice. To extend these findings into a model of gastric cancer, hypergastrinemic WT INS-GAS or MMP7(-/-) INS-GAS mice were challenged with H. pylori strain PMSS1. Consistent with findings in the C57BL/6 model, H. pylori-infected MMP7-deficient INS-GAS mice exhibited a significant increase in gastric inflammation compared with either uninfected or infected WT INS-GAS mice. In addition, the incidence of gastric hyperplasia and dysplasia was significantly increased in H. pylori-infected MMP7(-/-) INS-GAS mice compared with infected WT INS-GAS mice, and loss of MMP7 promoted M1 macrophage polarization. These results suggest that MMP7 exerts a restrictive role on H. pylori-induced gastric injury and the development of premalignant lesions by suppressing M1 macrophage polarization.


Subject(s)
Gastritis/microbiology , Helicobacter Infections/pathology , Macrophages/immunology , Matrix Metalloproteinase 7/genetics , Stomach , Animals , Disease Models, Animal , Gastric Mucosa/cytology , Gastric Mucosa/immunology , Gastric Mucosa/microbiology , Gastritis/immunology , Helicobacter Infections/immunology , Helicobacter pylori/immunology , Helicobacter pylori/pathogenicity , Hyperplasia/genetics , Inflammation/immunology , Inflammation/microbiology , Macrophages/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Precancerous Conditions/immunology , Precancerous Conditions/microbiology , Stomach/immunology , Stomach/microbiology , Stomach/pathology , Stomach Neoplasms/immunology , Stomach Neoplasms/microbiology
5.
Oncogene ; 34(26): 3429-40, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25174398

ABSTRACT

Helicobacter pylori infection causes gastric cancer, the third leading cause of cancer death worldwide. More than half of the world's population is infected, making universal eradication impractical. Clinical trials suggest that antibiotic treatment only reduces gastric cancer risk in patients with non-atrophic gastritis (NAG), and is ineffective once preneoplastic lesions of multifocal atrophic gastritis (MAG) and intestinal metaplasia (IM) have occurred. Therefore, additional strategies for risk stratification and chemoprevention of gastric cancer are needed. We have implicated polyamines, generated by the rate-limiting enzyme ornithine decarboxylase (ODC), in gastric carcinogenesis. During H. pylori infection, the enzyme spermine oxidase (SMOX) is induced, which generates hydrogen peroxide from the catabolism of the polyamine spermine. Herein, we assessed the role of SMOX in the increased gastric cancer risk in Colombia associated with the Andean mountain region when compared with the low-risk region on the Pacific coast. When cocultured with gastric epithelial cells, clinical strains of H. pylori from the high-risk region induced more SMOX expression and oxidative DNA damage, and less apoptosis than low-risk strains. These findings were not attributable to differences in the cytotoxin-associated gene A oncoprotein. Gastric tissues from subjects from the high-risk region exhibited greater levels of SMOX and oxidative DNA damage by immunohistochemistry and flow cytometry, and this occurred in NAG, MAG and IM. In Mongolian gerbils, a prototype colonizing strain from the high-risk region induced more SMOX, DNA damage, dysplasia and adenocarcinoma than a colonizing strain from the low-risk region. Treatment of gerbils with either α-difluoromethylornithine, an inhibitor of ODC, or MDL 72527 (N(1),N(4)-Di(buta-2,3-dien-1-yl)butane-1,4-diamine dihydrochloride), an inhibitor of SMOX, reduced gastric dysplasia and carcinoma, as well as apoptosis-resistant cells with DNA damage. These data indicate that aberrant activation of polyamine-driven oxidative stress is a marker of gastric cancer risk and a target for chemoprevention.


Subject(s)
Adenocarcinoma , Helicobacter Infections/complications , Helicobacter pylori/physiology , Oxidoreductases Acting on CH-NH Group Donors/physiology , Stomach Neoplasms , Adenocarcinoma/epidemiology , Adenocarcinoma/genetics , Adenocarcinoma/microbiology , Adult , Animals , Cells, Cultured , Colombia/epidemiology , DNA Damage/genetics , Enzyme Induction , Gerbillinae , Helicobacter Infections/genetics , Humans , Hydrogen Peroxide/metabolism , Male , Middle Aged , Oxidative Stress/genetics , Risk Factors , Stomach Neoplasms/epidemiology , Stomach Neoplasms/genetics , Stomach Neoplasms/microbiology , Polyamine Oxidase
6.
Virchows Arch ; 460(1): 19-46, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22190006

ABSTRACT

Atrophic gastritis, intestinal metaplasia, and epithelial dysplasia of the stomach are common and are associated with an increased risk for gastric cancer. In the absence of guidelines, there is wide disparity in the management of patients with these premalignant conditions. The European Society of Gastrointestinal Endoscopy, the European Helicobacter Study Group, the European Society of Pathology, and the Sociedade Portuguesa de Endoscopia Digestiva have therefore combined efforts to develop evidence-based guidelines on the management of patients with precancerous conditions and lesions of the stomach. A multidisciplinary group of 63 experts from 24 countries developed these recommendations by means of repeat online voting and a meeting in June 2011 in Porto, Portugal. The recommendations emphasize the increased cancer risk in patients with gastric atrophy and metaplasia and the need for adequate staging in the case of high-grade dysplasia, and they focus on treatment and surveillance indications and methods.


Subject(s)
Gastritis/therapy , Precancerous Conditions/pathology , Precancerous Conditions/therapy , Stomach Neoplasms/epidemiology , Stomach/pathology , Atrophy , Endoscopy, Gastrointestinal , Europe , Gastritis/diagnosis , Gastritis/pathology , Humans , Metaplasia , Portugal , Precancerous Conditions/diagnosis , Risk Factors , Societies, Medical
7.
Endoscopy ; 44(1): 74-94, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22198778

ABSTRACT

Atrophic gastritis, intestinal metaplasia, and epithelial dysplasia of the stomach are common and are associated with an increased risk for gastric cancer. In the absence of guidelines, there is wide disparity in the management of patients with these premalignant conditions. The European Society of Gastrointestinal Endoscopy (ESGE), the European Helicobacter Study Group (EHSG), the European Society of Pathology (ESP) and the Sociedade Portuguesa de Endoscopia Digestiva (SPED) have therefore combined efforts to develop evidence-based guidelines on the management of patients with precancerous conditions and lesions of the stomach (termed MAPS). A multidisciplinary group of 63 experts from 24 countries developed these recommendations by means of repeat online voting and a meeting in June 2011 in Porto, Portugal. The recommendations emphasize the increased cancer risk in patients with gastric atrophy and metaplasia, and the need for adequate staging in the case of high grade dysplasia, and they focus on treatment and surveillance indications and methods.


Subject(s)
Gastric Mucosa/pathology , Gastritis, Atrophic/pathology , Gastritis, Atrophic/therapy , Precancerous Conditions/pathology , Precancerous Conditions/therapy , Stomach Neoplasms/pathology , Biopsy , Evidence-Based Medicine , Gastritis, Atrophic/diagnosis , Gastroscopy , Helicobacter Infections/drug therapy , Helicobacter Infections/economics , Helicobacter Infections/microbiology , Helicobacter pylori , Humans , Metaplasia/pathology , Metaplasia/therapy , Pepsinogens/blood , Population Surveillance , Precancerous Conditions/diagnosis
8.
Oncogene ; 29(41): 5643-50, 2010 Oct 14.
Article in English | MEDLINE | ID: mdl-20676134

ABSTRACT

Chronic infection with cagA-positive Helicobacter pylori is the strongest risk factor for the development of gastric adenocarcinoma. The cagA gene product CagA is injected into gastric epithelial cells and disturbs cellular functions by physically interacting with and deregulating a variety of cellular signaling molecules. RUNX3 is a tumor suppressor in many tissues, and it is frequently inactivated in gastric cancer. In this study, we show that H. pylori infection inactivates the gastric tumor suppressor RUNX3 in a CagA-dependent manner. CagA directly associates with RUNX3 through a specific recognition of the PY motif of RUNX3 by a WW domain of CagA. Deletion of the WW domains of CagA or mutation of the PY motif in RUNX3 abolishes the ability of CagA to induce the ubiquitination and degradation of RUNX3, thereby extinguishing its ability to inhibit the transcriptional activation of RUNX3. Our studies identify RUNX3 as a novel cellular target of H. pylori CagA and also reveal a mechanism by which CagA functions as an oncoprotein by blocking the activity of gastric tumor suppressor RUNX3.


Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Core Binding Factor Alpha 3 Subunit/metabolism , Proteasome Endopeptidase Complex/metabolism , Amino Acid Motifs/genetics , Animals , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Binding Sites/genetics , COS Cells , Cell Line , Cell Line, Tumor , Chlorocebus aethiops , Core Binding Factor Alpha 3 Subunit/genetics , Gastric Mucosa/metabolism , Gastric Mucosa/pathology , Gastric Mucosa/virology , Helicobacter Infections/metabolism , Helicobacter Infections/virology , Helicobacter pylori/genetics , Helicobacter pylori/metabolism , Helicobacter pylori/physiology , Host-Pathogen Interactions , Humans , Immunoblotting , Immunohistochemistry , Immunoprecipitation , Mice , Mutation , Protein Binding , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , Stomach Neoplasms/virology , Transfection
9.
Clin Microbiol Infect ; 16(4): 369-78, 2010 Apr.
Article in English | MEDLINE | ID: mdl-19456839

ABSTRACT

The C-terminus of the Helicobacter pylori CagA protein is polymorphic, bearing different EPIYA sequences (EPIYA-A, B, C or D), and one or more CagA multimerization (CM) motifs. The number of EPIYA-C motifs is associated with precancerous lesions and gastric cancer (GC). The relationship between EPIYA, CM motifs and gastric lesions was examined in H. pylori-infected Colombian patients from areas of high and low risk for GC. Genomic DNA was extracted from H. pylori strains cultured from gastric biopsies from 80 adults with dyspeptic symptoms. Sixty-seven (83.8%) of 80 strains were cagA positive. The 3' region of cagA was sequenced, and EPIYA and CM motifs were identified. CagA proteins contained one (64.2%), two (34.3%) or three EPIYA-C motifs (1.5%), all with Western type CagA-specific sequences. Strains with one EPIYA-C motif were associated with less severe gastric lesions (non-atrophic and multifocal atrophic gastritis), whereas strains with multiple EPIYA-C motifs were associated with more severe lesions (intestinal metaplasia and dysplasia) (p <0.001). In 54 strains, the CM motifs were identical to those common in Western strains. Thirteen strains from the low-risk area contained two different CM motifs: one of Western type located within the EPIYA-C segment and another following the EPIYA-C segment and resembling the CM motif found in East Asian strains. These strains induced significantly shorter projections in AGS cells and an attenuated reduction in levels of CagA upon immunodepletion of SHP-2 than strains possessing Western/Western motifs. This novel finding may partially explain the difference in GC incidence in these populations.


Subject(s)
Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Helicobacter Infections/complications , Helicobacter pylori/genetics , Precancerous Conditions/microbiology , Stomach Neoplasms/microbiology , Adult , Amino Acid Sequence , Antigens, Bacterial/chemistry , Antigens, Bacterial/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Colombia , Gastric Mucosa/metabolism , Gastric Mucosa/microbiology , Helicobacter pylori/isolation & purification , Helicobacter pylori/metabolism , Humans , Male , Middle Aged , Molecular Sequence Data , Polymorphism, Genetic , Precancerous Conditions/metabolism , Precancerous Conditions/pathology , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology
10.
Infect Immun ; 76(9): 3992-9, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18519558

ABSTRACT

Chronic infection with the gastric pathogen Helicobacter pylori significantly increases the risk of developing atrophic gastritis, peptic ulcer disease, and gastric adenocarcinoma. H. pylori strains that possess the cag pathogenicity island, which translocates CagA into the host cells, augment these risks. The aim of this study was to determine the molecular mechanisms through which H. pylori upregulates the expression of plasminogen activator inhibitor 1 (PAI-1), a member of the urokinase activator system that is involved in tumor metastasis and angiogenesis. Levels of PAI-1 mRNA and protein were examined in tissues from H. pylori-infected patients and in vitro using AGS gastric epithelial cells. In vitro, cells were infected with toxigenic cag-positive or nontoxigenic cag-negative strains of H. pylori or isogenic mutants. The amount of PAI-1 secretion was measured by enzyme-linked immunosorbent assay, and mRNA levels were determined using real-time PCR. The regulation of PAI-1 was examined using the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor and small interfering RNA. Analysis of human biopsy samples revealed an increase in both PAI-1 mRNA and protein levels in patients with H. pylori gastritis compared to those of uninfected controls. Infection of AGS cells with H. pylori significantly increased PAI-1 mRNA expression and the secretion of PAI-1 protein. Moreover, PAI-1 mRNA and protein production was more pronounced when AGS cells were infected by H. pylori strains carrying a functional cag secretion system than when cells were infected by strains lacking this system. PAI-1 secretion was also reduced when cells were infected with either cagE-negative or cagA-negative mutants. The ectopic overexpression of CagA significantly increased the levels of PAI-1 mRNA and protein, whereas blockade of the ERK1/2 pathway inhibited H. pylori-mediated PAI-1 upregulation. These findings suggest that the upregulation of PAI-1 in H. pylori-infected gastric epithelial cells may contribute to the carcinogenic process.


Subject(s)
Epithelial Cells/microbiology , Gastric Mucosa/microbiology , Helicobacter pylori/physiology , Plasminogen Activator Inhibitor 1/biosynthesis , Up-Regulation , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Cell Line , Enzyme-Linked Immunosorbent Assay , Female , Gastric Mucosa/pathology , Gene Deletion , Gene Expression Profiling , Humans , Male , Middle Aged
11.
Oncogene ; 26(32): 4617-26, 2007 Jul 12.
Article in English | MEDLINE | ID: mdl-17237808

ABSTRACT

Infection with Helicobacter pylori cagA-positive strains is associated with gastric adenocarcinoma. Intestinal metaplasia is a precancerous lesion of the stomach characterized by transdifferentiation of the gastric mucosa to an intestinal phenotype. The H. pylori cagA gene product, CagA, is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation by Src family kinases. Tyrosine-phosphorylated CagA specifically binds to and activates SHP-2 phosphatase, thereby inducing cell-morphological transformation. We report here that CagA physically interacts with E-cadherin independently of CagA tyrosine phosphorylation. The CagA/E-cadherin interaction impairs the complex formation between E-cadherin and beta-catenin, causing cytoplasmic and nuclear accumulation of beta-catenin. CagA-deregulated beta-catenin then transactivates beta-catenin-dependent genes such as cdx1, which encodes intestinal specific CDX1 transcription factor. In addition to beta-catenin signal, CagA also transactivates p21(WAF1/Cip1), again, in a phosphorylation-independent manner. Consequently, CagA induces aberrant expression of an intestinal-differentiation marker, goblet-cell mucin MUC2, in gastric epithelial cells that have been arrested in G1 by p21(WAF1/Cip1). These results indicate that perturbation of the E-cadherin/beta-catenin complex by H. pylori CagA plays an important role in the development of intestinal metaplasia, a premalignant transdifferentiation of gastric epithelial cells from which intestinal-type gastric adenocarcinoma arises.


Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Cadherins/metabolism , Cell Transformation, Neoplastic/metabolism , Gastric Mucosa/metabolism , Precancerous Conditions/metabolism , Stomach Neoplasms/etiology , beta Catenin/metabolism , Adenocarcinoma/etiology , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Antigens, Bacterial/analysis , Bacterial Proteins/analysis , Cadherins/analysis , Cell Line , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cytoplasm/chemistry , Cytoplasm/metabolism , Gastric Mucosa/pathology , Gene Expression Regulation, Neoplastic , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Mucin-2 , Mucins/metabolism , Phosphorylation , Precancerous Conditions/genetics , Precancerous Conditions/pathology , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , Transcriptional Activation , Tyrosine/metabolism , beta Catenin/analysis
12.
Gut ; 54(10): 1363-9, 2005 Oct.
Article in English | MEDLINE | ID: mdl-15863471

ABSTRACT

BACKGROUND AND AIMS: Helicobacter pylori, in particular cytotoxin associated gene (cag)+ strains, have been shown to enhance gastric epithelial cell proliferation in vivo, an effect that likely contributes to gastric carcinogenesis. Early growth response gene 1 (Egr-1) is a crucial regulator of cell growth, differentiation, and survival, which is known to play a role in carcinogenesis and cancer progression. The aims of this study were to: (1) examine whether H pylori could upregulate Egr-1 in gastric epithelial cell lines; (2) determine whether there was a differential response to infection with different strains; (3) examine the role of the cag pathogenicity island in this process; and (4) elucidate the molecular mechanisms leading to Egr-1 upregulation. METHODS AND RESULTS: We found that infection of AGS cells with cag+H pylori resulted in a rapid (1-2 hours) but transient increase in Egr-1 mRNA and protein levels whereas coculture with cag- isolates did not elicit this response. Furthermore, two independent cagE- isogenic mutants of H pylori also demonstrated impaired ability to upregulate Egr-1. Upregulation of Egr-1 protein was inhibited by the extracellular regulated kinase (ERK)1/2 inhibitor PD98059 and overexpression of dominant negative MEK1 downregulated Egr-1 luciferase reporter gene activity. Treatment of AGS cells with the epidermal growth factor receptor (EGFR) kinase inhibitors PD153035 and AG1478 resulted in a reduction in H pylori mediated Egr-1 upregulation, demonstrating that EGFR transactivation plays a role in this early cellular process. CONCLUSIONS: Our findings show that cag+H pylori cause rapid induction of Egr-1 in gastric epithelial cells which may contribute to H pylori mediated pathogenesis.


Subject(s)
Antigens, Bacterial , Bacterial Proteins , ErbB Receptors/genetics , Helicobacter pylori/genetics , Stomach/physiology , Transcriptional Activation/genetics , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Cell Line , Cytotoxins/genetics , Down-Regulation/genetics , Epithelial Cells/physiology , ErbB Receptors/metabolism , Humans , Luciferases, Bacterial/genetics , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , RNA, Bacterial/analysis , RNA, Messenger/analysis , Up-Regulation/genetics
13.
J Clin Microbiol ; 40(1): 239-46, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11773122

ABSTRACT

Since the associations between Helicobacter pylori genotype and disease differ in Asia and the West, we investigated the correlation between HP0638, encoding an outer membrane protein, and potential markers of virulence (cagA, vacA, and iceA). For 109 strains from nine countries, the status of cagA, vacA, and iceA was determined by PCR and/or a line probe assay. We also studied 18 strains from 8 patients (parents and 6 daughters) from a Dutch family and paired strains collected on average 8 years apart from 11 patients. When the HP0638 signal sequences were amplified by PCR and DNA sequence determinations were performed, 89 (96%) of 93 cagA-positive strains had HP0638 in frame, versus none (0%) of 16 cagA-negative strains (P < 0.001). Among strains in which HP0638 was in frame, a six-CT dinucleotide repeat pattern was dominant in Western countries (23 of 33 strains [70%]), while a pattern of three CT repeats with another CT after four T's (3 + 1-CT-repeat pattern) was dominant in East Asia (31 of 46 strains [67%]); however, specific CT repeat patterns did not correlate with clinical outcome. HP0638 phylogenetic trees also showed geographic characters. The HP0638 frame status and CT dinucleotide repeat patterns were identical for 9 of 11 pairs of strains obtained on average 8 years apart from individuals and the 15 strains obtained from the mother and all six daughters. Thus, HP0638 frame status and cagA status are strongly correlated. The CT dinucleotide repeat pattern in the putative HP0638 signal sequence has geographic characters and appears stable in particular patients and families over a period of years. Analysis of HP0638 CT polymorphisms may serve as a new typing system to discriminate H. pylori isolates for epidemiological purposes.


Subject(s)
Antigens, Bacterial , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/metabolism , Helicobacter Infections/epidemiology , Helicobacter pylori/classification , Polymorphism, Genetic , Adult , Asia/epidemiology , Base Sequence , Colombia/epidemiology , Dinucleotide Repeats/genetics , Europe/epidemiology , Female , Helicobacter Infections/microbiology , Helicobacter pylori/genetics , Helicobacter pylori/metabolism , Helicobacter pylori/pathogenicity , Humans , Male , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA , Virulence/genetics
14.
Proc Natl Acad Sci U S A ; 98(25): 14625-30, 2001 Dec 04.
Article in English | MEDLINE | ID: mdl-11724955

ABSTRACT

Isolates of the gastric pathogen Helicobacter pylori harvested from different individuals are highly polymorphic. Strain variation also has been observed within a single host. To more fully ascertain the extent of H. pylori genetic diversity within the ecological niche of its natural host, we harvested additional isolates of the sequenced H. pylori strain J99 from its human source patient after a 6-year interval. Randomly amplified polymorphic DNA PCR and DNA sequencing of four unlinked loci indicated that these isolates were closely related to the original strain. In contrast, microarray analysis revealed differences in genetic content among all of the isolates that were not detected by randomly amplified polymorphic DNA PCR or sequence analysis. Several ORFs from loci scattered throughout the chromosome in the archival strain did not hybridize with DNA from the recent strains, including multiple ORFs within the J99 plasticity zone. In addition, DNA from the recent isolates hybridized with probes for ORFs specific for the other fully sequenced H. pylori strain 26695, including a putative traG homolog. Among the additional J99 isolates, patterns of genetic diversity were distinct both when compared with each other and to the original prototype isolate. These results indicate that within an apparently homogeneous population, as determined by macroscale comparison and nucleotide sequence analysis, remarkable genetic differences exist among single-colony isolates of H. pylori. Direct evidence that H. pylori has the capacity to lose and possibly acquire exogenous DNA is consistent with a model of continuous microevolution within its cognate host.


Subject(s)
Genetic Variation , Helicobacter pylori/genetics , Chromosomes, Bacterial/genetics , DNA, Bacterial/genetics , Drug Resistance, Bacterial/genetics , Evolution, Molecular , Helicobacter pylori/isolation & purification , Helicobacter pylori/pathogenicity , Humans , Models, Biological , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Open Reading Frames , Phylogeny , Polymorphism, Genetic , Random Amplified Polymorphic DNA Technique , Stomach/microbiology
15.
J Biol Chem ; 276(51): 48127-34, 2001 Dec 21.
Article in English | MEDLINE | ID: mdl-11604402

ABSTRACT

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.


Subject(s)
Antigens, Bacterial/genetics , Bacterial Proteins/genetics , ErbB Receptors/genetics , Gastric Mucosa/metabolism , Helicobacter pylori/physiology , Transcriptional Activation/physiology , Bacterial Proteins/physiology , Cell Line , Enzyme Activation , Epithelial Cells/metabolism , ErbB Receptors/antagonists & inhibitors , Gastric Mucosa/cytology , Gastric Mucosa/enzymology , Gene Expression Regulation, Bacterial/physiology , Helicobacter pylori/genetics , Humans , Interleukin-8/genetics , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Quinazolines , Tyrphostins/pharmacology
16.
Gut ; 49(5): 629-35, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11600464

ABSTRACT

BACKGROUND: South African Helicobacter pylori isolates are characterised by the universal presence of cagA but have differences in vacuolating cytotoxin gene (vacA) alleles which correlate with clinically significant disease. However, the candidate virulence marker gene iceA has not been investigated. AIM: To characterise the genetic organisation and heterogeneity of iceA genotypes in different South African clinical isolates. PATIENTS AND METHODS: We studied H pylori strains isolated from 86 dyspeptic patients (30 with peptic ulcer disease (PUD), 19 with distal gastric adenocarcinoma (GC), and 37 with non-erosive gastritis) for the presence of iceA1 or iceA2 genes, and for differences in the genetic organisation of iceA2 by polymerase chain reaction, Southern hybridisation analysis, and sequencing. RESULTS: Genetic analysis of iceA1 demonstrated significant homology (92-95%) with the USA type strain 26695 and probably functions as a transcriptional regulator, while a novel variant (iceA2D') of iceA2 and marked differences in predicted protein secondary structure of the iceA2 protein were defined. iceA1 was detected in 68% and iceA2 in 80% of all clinical isolates. Although approximately 40% of patients had both strains, a higher prevalence (p< 0.01) of GC patients were infected with iceA1 isolates which were invariably vacA s1/iceA1 (p< 0.005 v gastritis). Isolates from PUD patients were distinguished by the structurally altered iceA2D variant (53%; p<0.03 v gastritis) while the iceA2C variant distinguished isolates from patients with gastritis alone (67%; p< 0.005 v PUD). CONCLUSION: In this study, an association between iceA1 and GC was noted while differences in variants of iceA2 differentiated between PUD and gastritis alone. Combination analyses of iceA genotypes and vacA alleles supported these associations.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Genes, Bacterial/genetics , Helicobacter Infections/microbiology , Helicobacter pylori/genetics , Adenocarcinoma/microbiology , Animals , Bacterial Proteins/genetics , Blotting, Southern , Gastritis/microbiology , Genetic Heterogeneity , Helicobacter pylori/pathogenicity , Humans , Peptic Ulcer/microbiology , Polymerase Chain Reaction , Protein Structure, Secondary , Sequence Analysis, DNA , Stomach Neoplasms/microbiology , Virulence/genetics
17.
Aliment Pharmacol Ther ; 15(9): 1271-90, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11552897

ABSTRACT

Helicobacter pylori causes persistent inflammation in the human stomach, yet only a minority of persons harbouring this organism develop peptic ulcer disease or gastric malignancy. An important question is why such variation exists among colonized individuals. Recent evidence has demonstrated that H. pylori isolates possess substantial phenotypic and genotypic diversity, which may engender differential host inflammatory responses that influence clinical outcome. For example, H. pylori strains that possess the cag pathogenicity island induce more severe gastritis and augment the risk for developing peptic ulcer disease and distal gastric cancer. An alternative, but not exclusive, hypothesis is that enhanced inflammation and injury is a consequence of an inappropriate host immune response to the chronic presence of H. pylori within the gastric niche. Investigations that precisely delineate the mechanisms responsible for induction of gastritis will ultimately help to define which H. pylori-colonized persons bear the highest risk for subsequent development of clinical disease, and thus, enable physicians to focus eradication therapy.


Subject(s)
Adenocarcinoma/etiology , Gastritis , Helicobacter Infections/complications , Helicobacter pylori , Stomach Neoplasms/etiology , Antibody Formation , Gastritis/complications , Gastritis/microbiology , Helicobacter Infections/immunology , Helicobacter pylori/genetics , Helicobacter pylori/immunology , Helicobacter pylori/pathogenicity , Humans
19.
Semin Gastrointest Dis ; 12(3): 151-66, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11478748

ABSTRACT

Helicobacterpylori causes persistent inflammation in the human stomach, yet only a minority of persons harboring this organism develop peptic ulcer disease or gastric malignancy. H. pylori isolates possess substantial phenotypic and genotypic diversity, which may engender differential host inflammatory responses that influence clinical outcome. For example, strains that possess the cag pathogenicity island induce more severe gastritis and augment the risk for developing peptic ulcer disease and distal gastric cancer. However, important geographic differences in susceptibility to disease exist as clear-cut markers for H. pylori strains that affect certain groups of colonized individuals have little or no predictive power for other populations. Recent investigations that have more precisely delineated mechanisms of H. pylori pathogenesis will ultimately help to define which colonized persons bear the highest risk for subsequent development of clinical disease, and thus enable physicians to appropriately focus diagnostic testing and eradication therapy. Although stratification of disease risk based on H. pylori strain characteristics is unlikely to completely account for differences in clinical outcomes, it is an important first step in helping to understand the biology of long-term interactions between H. pylori and its human host.


Subject(s)
Antigens, Bacterial , Helicobacter Infections/diagnosis , Helicobacter pylori , Bacterial Proteins/genetics , Biopsy/methods , Breath Tests/methods , Gastritis/complications , Helicobacter Infections/complications , Helicobacter pylori/genetics , Helicobacter pylori/pathogenicity , Humans , Peptic Ulcer/complications , Polymerase Chain Reaction/methods , Serologic Tests/methods , Stomach Neoplasms/complications , Urea , Urease
20.
J Biol Chem ; 276(33): 31059-66, 2001 Aug 17.
Article in English | MEDLINE | ID: mdl-11397815

ABSTRACT

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.


Subject(s)
Apoptosis , Gastric Mucosa/microbiology , Helicobacter pylori/physiology , NF-kappa B/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Thiazolidinediones , Transcription Factors/physiology , Cell Line , Cyclooxygenase 2 , Humans , Isoenzymes/physiology , Membrane Proteins , Prostaglandin-Endoperoxide Synthases/physiology , Rosiglitazone , Species Specificity , Thiazoles/pharmacology
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