Enhanced enzymatic production of cholesteryl 6'-acylglucoside impairs lysosomal degradation for the intracellular survival of Helicobacter pylori.

BACKGROUND: During autophagy defense against invading microbes, certain lipid types are indispensable for generating specialized membrane-bound organelles. The lipid composition of autophagosomes remains obscure, as does the issue of how specific lipids and lipid-associated enzymes participate in autophagosome formation and maturation. Helicobacter pylori is auxotrophic for cholesterol and converts cholesterol to cholesteryl glucoside derivatives, including cholesteryl 6'-O-acyl-α-D-glucoside (CAG). We investigated how CAG and its biosynthetic acyltransferase assist H. pylori to escape host-cell autophagy. METHODS: We applied a metabolite-tagging method to obtain fluorophore-containing cholesteryl glucosides that were utilized to understand their intracellular locations. H. pylori 26695 and a cholesteryl glucosyltransferase (CGT)-deletion mutant (ΔCGT) were used as the standard strain and the negative control that contains no cholesterol-derived metabolites, respectively. Bacterial internalization and several autophagy-related assays were conducted to unravel the possible mechanism that H. pylori develops to hijack the host-cell autophagy response. Subcellular fractions of H. pylori-infected AGS cells were obtained and measured for the acyltransferase activity. RESULTS: The imaging studies of fluorophore-labeled cholesteryl glucosides pinpointed their intracellular localization in AGS cells. The result indicated that CAG enhances the internalization of H. pylori in AGS cells. Particularly, CAG, instead of CG and CPG, is able to augment the autophagy response induced by H. pylori. How CAG participates in the autophagy process is multifaceted. CAG was found to intervene in the degradation of autophagosomes and reduce lysosomal biogenesis, supporting the idea that intracellular H. pylori is harbored by autophago-lysosomes in favor of the bacterial survival. Furthermore, we performed the enzyme activity assay of subcellular fractions of H. pylori-infected AGS cells. The analysis showed that the acyltransferase is mainly distributed in autophago-lysosomal compartments. CONCLUSIONS: Our results support the idea that the acyltransferase is mainly distributed in the subcellular compartment consisting of autophagosomes, late endosomes, and lysosomes, in which the acidic environment is beneficial for the maximal acyltransferase activity. The resulting elevated level of CAG can facilitate bacterial internalization, interfere with the autophagy flux, and causes reduced lysosomal biogenesis.

ß-Carotene Inhibits Expression of Matrix Metalloproteinase-10 and Invasion in Helicobacter pylori-Infected Gastric Epithelial Cells.

Matrix metalloproteinases (MMPs), key molecules of cancer invasion and metastasis, degrade the extracellular matrix and cell-cell adhesion molecules. MMP-10 plays a crucial role in Helicobacter pylori-induced cell-invasion. The mitogen-activated protein kinase (MAPK) signaling pathway, which activates activator protein-1 (AP-1), is known to mediate MMP expression. Infection with H. pylori, a Gram-negative bacterium, is associated with gastric cancer development. A toxic factor induced by H. pylori infection is reactive oxygen species (ROS), which activate MAPK signaling in gastric epithelial cells. Peroxisome proliferator-activated receptor γ (PPAR-γ) mediates the expression of antioxidant enzymes including catalase. ß-Carotene, a red-orange pigment, exerts antioxidant and anti-inflammatory properties. We aimed to investigate whether ß-carotene inhibits H. pylori-induced MMP expression and cell invasion in gastric epithelial AGS (gastric adenocarcinoma) cells. We found that H. pylori induced MMP-10 expression and increased cell invasion via the activation of MAPKs and AP-1 in gastric epithelial cells. Specific inhibitors of MAPKs suppressed H. pylori-induced MMP-10 expression, suggesting that H. pylori induces MMP-10 expression through MAPKs. ß-Carotene inhibited the H. pylori-induced activation of MAPKs and AP-1, expression of MMP-10, and cell invasion. Additionally, it promoted the expression of PPAR-γ and catalase, which reduced ROS levels in H. pylori-infected cells. In conclusion, ß-carotene exerts an inhibitory effect on MAPK-mediated MMP-10 expression and cell invasion by increasing PPAR-γ-mediated catalase expression and reducing ROS levels in H. pylori-infected gastric epithelial cells.

Helicobacter pylori infection impairs chaperone-assisted maturation of Na-K-ATPase in gastric epithelium.

Helicobacter pylori infection always induces gastritis, which may progress to ulcer disease or cancer. The mechanisms underlying mucosal injury by the bacteria are incompletely understood. Here, we identify a novel pathway for H. pylori-induced gastric injury, the impairment of maturation of the essential transport enzyme and cell adhesion molecule, Na-K-ATPase. Na-K-ATPase comprises α- and ß-subunits that assemble in the endoplasmic reticulum (ER) before trafficking to the plasma membrane. Attachment of H. pylori to gastric epithelial cells increased Na-K-ATPase ubiquitylation, decreased its surface and total levels, and impaired ion balance. H. pylori did not alter degradation of plasmalemma-resident Na-K-ATPase subunits or their mRNA levels. Infection decreased association of α- and ß-subunits with ER chaperone BiP and impaired assembly of α/ß-heterodimers, as was revealed by quantitative mass spectrometry and immunoblotting of immunoprecipitated complexes. The total level of BiP was not altered, and the decrease in interaction with BiP was not observed for other BiP client proteins. The H. pylori-induced decrease in Na-K-ATPase was prevented by BiP overexpression, stopping protein synthesis, or inhibiting proteasomal, but not lysosomal, protein degradation. The results indicate that H. pylori impairs chaperone-assisted maturation of newly made Na-K-ATPase subunits in the ER independently of a generalized ER stress and induces their ubiquitylation and proteasomal degradation. The decrease in Na-K-ATPase levels is also seen in vivo in the stomachs of gerbils and chronically infected children. Further understanding of H. pylori-induced Na-K-ATPase degradation will provide insights for protection against advanced disease.NEW & NOTEWORTHY This work provides evidence that Helicobacter pylori decreases levels of Na-K-ATPase, a vital transport enzyme, in gastric epithelia, both in acutely infected cultured cells and in chronically infected patients and animals. The bacteria interfere with BiP-assisted folding of newly-made Na-K-ATPase subunits in the endoplasmic reticulum, accelerating their ubiquitylation and proteasomal degradation and decreasing efficiency of the assembly of native enzyme. Decreased Na-K-ATPase expression contributes to H. pylori-induced gastric injury.

Helicobacter pylori inhibits GKN1 expression via the CagA/p-ERK/AUF1 pathway.

BACKGROUND: Recent studies have shown that gastrokine 1 (GKN1), an important tumor suppressor gene, is downregulated in Helicobacter pylori (H. pylori) infected gastric mucosa and gastric cancer. However, the underlying mechanism is poorly understood. Herein, we investigated the potential mechanism of H. pylori-induced GKN1 downregulation. MATERIALS AND METHODS: GKN1 and AU-rich element RNA-binding factor 1 (AUF1) expressions were assessed by quantitative real-time PCR, Western blot, or immunohistochemistry in H. pylori-infected tissues and H. pylori co-cultured cell lines. The regulation of AUF1 on GKN1 was determined by RNA pulldown assay, RNA immunoprecipitation, mRNA turnover, and luciferase activity assays. The involvement of phosphorylated extra-cellular signal-regulated kinase (p-ERK) or CagA in H. pylori-induced AUF1 expression was verified using p-ERK inhibitor or CagA knockout H. pylori. In addition, the cell proliferation and migration capacities of AUF1-knockdown cells were investigated. RESULTS: GKN1 expression progressively decreased from H. pylori-infected gastritis to gastric cancer tissues. H. pylori co-culture also induced significant GKN1 reduction in GES-1 and BGC-823 cells. Besides, the mRNA level of GKN1 and AUF1 in human gastric mucosa showed negative correlation significantly. AUF1 knockdown resulted in upregulation of GKN1 expression and promoted GKN1 mRNA decay by binding the 3' untranslated region of GKN1 mRNA H. pylori-induced AUF1 expression was associated with p-ERK activation and CagA. Furthermore, knockdown of AUF1 significantly inhibited cell viability, migration ability, and arrested fewer cells in S-phase. CONCLUSION: Our data demonstrated that H. pylori infection downregulated GKN1 expression via the CagA/p-ERK/AUF1 pathway. AUF1 promoted gastric cancer at least partly through downregulating GKN1, which presented a novel potential target for the treatment of gastric cancer.

Comparison of the Clinicopathological Characteristics and Genetic Alterations Between Patients with Gastric Cancer with or Without Helicobacter pylori Infection.

BACKGROUND: Helicobacter pylori (HP) can induce epithelial cells and intestinal metaplasia with genetic damage that makes them highly susceptible to the development of gastric cancer (GC). MATERIALS AND METHODS: Between 2005 and 2010, 356 patients with gastric cancer who received curative surgery were enrolled. Analysis of HP, Epstein-Barr virus (EBV) infection, PIK3CA amplification, and mutation analysis of 68 mutations in eight genes using a mass spectrometric single-nucleotide polymorphism genotyping technology was conducted. The clinicopathological characteristics of patients with or without HP infection were compared. RESULTS: Among the 356 patients, 185 (52.0%) had HP infection. For intestinal-type GC, patients with HP infection were more likely to be younger and had fewer PI3K/AKT pathway genetic mutations than those without HP infection. For diffuse-type GC, patients with HP infection were characterized by less male predominance, less lymphoid stroma, fewer microsatellite instability-high tumors, and fewer PI3K/AKT pathway genetic mutations than those without HP infection. Patients with HP infection had less tumor recurrence and a better 5-year overall survival (87.7% vs. 73.9%, p = .012) and disease-free survival (64.1% vs. 51.3%, p = .013) than those without HP infection, especially for intestinal-type GC. For EBV-negative GC, patients with HP infection had fewer PI3K/AKT pathway mutations and a better 5-year overall survival and disease-free survival than those without HP infection. Multivariate analysis demonstrated that HP infection was an independent prognostic factor regarding overall survival and disease-free survival. CONCLUSION: Patients with GC with HP infection were associated with fewer PI3K/AKT pathway genetic mutations and better survival than those without HP infection, especially for EBV-negative and intestinal-type GC. IMPLICATIONS FOR PRACTICE: Patients with gastric cancer with Helicobacter pylori (HP) infection had fewer PI3K/AKT pathway genetic mutations, less tumor recurrence, and better survival than those without HP infection, especially for Epstein-Barr virus (EBV)-negative and intestinal-type gastric cancer. HP infection is an independent prognostic factor regarding overall survival and disease-free survival. Future in vivo and in vitro studies of the correlation among HP infection, PI3K/AKT pathway, and EBV infection in gastric cancer are required.

Acetylation-mediated Siah2 stabilization enhances PHD3 degradation in Helicobacter pylori-infected gastric epithelial cancer cells.

Gastric epithelial cells infected with Helicobacter pylori acquire highly invasive and metastatic characteristics. The seven in absentia homolog (Siah)2, an E3 ubiquitin ligase, is one of the major proteins that induces invasiveness of infected gastric epithelial cells. We find that p300-driven acetylation of Siah2 at lysine 139 residue stabilizes the molecule in infected cells, thereby substantially increasing its efficiency to degrade prolyl hydroxylase (PHD)3 in the gastric epithelium. This enhances the accumulation of an oncogenic transcription factor hypoxia-inducible factor 1α (Hif1α) in H. pylori-infected gastric cancer cells in normoxic condition and promotes invasiveness of infected cells. Increased acetylation of Siah2, Hif1α accumulation, and the absence of PHD3 in the infected human gastric metastatic cancer biopsy samples and in invasive murine gastric cancer tissues further confirm that the acetylated Siah2 (ac-Siah2)-Hif1α axis is crucial in promoting gastric cancer invasiveness. This study establishes the importance of a previously unrecognized function of ac-Siah2 in regulating invasiveness of H. pylori-infected gastric epithelial cells.-Kokate, S. B., Dixit, P., Das, L., Rath, S., Roy, A. D., Poirah, I., Chakraborty, D., Rout, N., Singh, S. P., Bhattacharyya, A. Acetylation-mediated Siah2 stabilization enhances PHD3 degradation in Helicobacter pylori-infected gastric epithelial cancer cells.

Phytochemicals in Helicobacter pylori Infections: What Are We Doing Now?

In this critical review, plant sources used as effective antibacterial agents against Helicobacter pylori infections are carefully described. The main intrinsic bioactive molecules, responsible for the observed effects are also underlined and their corresponding modes of action specifically highlighted. In addition to traditional uses as herbal remedies, in vitro and in vivo studies focusing on plant extracts and isolated bioactive compounds with anti-H. pylori activity are also critically discussed. Lastly, special attention was also given to plant extracts with urease inhibitory effects, with emphasis on involved modes of action.

Effect of CYP2C19 Gene Polymorphisms on Proton Pump Inhibitor, Amoxicillin, and Levofloxacin Triple Therapy for Eradication of Helicobacter Pylori.

BACKGROUND The effects of PPI are variable owing to the CYP2C19 polymorphisms. However, whether the polymorphisms could affect the Hp eradication efficacy of triple therapy is still not clear. The present study aimed to assess the effects of CYP2C19 gene polymorphisms on proton pump inhibitor (PPI), amoxicillin, and levofloxacin triple therapy for Helicobacter pylori (Hp) eradication. MATERIAL AND METHODS We randomly assigned 160 Hp-positive patients with chronic gastritis to 2 groups to receive either 20 mg bid omeprazole (OAL group, n=80) or 10 mg bid rabeprazole (RAL group, n=80), combined with 1000 mg bid amoxicillin and 500 mg qd levofloxacin. The 2 groups were treated for 10 days. The CYP2C19 genotypes included wild-type, M1 mutant gene (*2, the mutation of exon 5), and M2 mutant gene (*3, the mutation of exon 4) identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFIP). According to CYP2C19 genotype combinations, the patients were divided into extensive metabolizer (EM), intermediate metabolizer (IM), and poor metabolizer (PM) subgroups. The eradication efficacy of Hp was evaluated by 14C-UBT at 28 days after treatment. RESULTS The trial was completed by 155 patients. Hp eradication rates in OAL and RAL groups were 78.2% and 88.3%, respectively, on per-protocol (PP) analysis, indicating no significant difference (P>0.05). Regarding CYP2C19 genotypes, eradication rates of 60.7%, 84.2%, and 100% were obtained for EM, IM, and PM subgroups, respectively, of the OAL group. EM group eradication rates were significantly lower than IM and PM group values (P<0.05). In the RAL group, no such difference was observed (P>0.05). Hp eradication rates were significantly lower in the EM subgroup of the OAL group compared with that of the RAL group. CONCLUSIONS Hp eradication rates were higher in the RAL group than in OAL-treated patients. Interestingly, omeprazole-based therapy was significantly affected by the CYP2C19 genotype, unlike the rabeprazole-based therapy.

Braf, Kras and Helicobacter pylori epigenetic changes-associated chronic gastritis in Egyptian patients with and without gastric cancer.

UNLABELLED: We aimed to study MLH1 and MGMT methylation status in Helicobacter pylori-associated chronic gastritis in Egyptian patients with and without gastric cancer. 39 patients were included in our study. They were divided into 2 groups; patients without (group I) and with gastric adenocarcinoma (group II). Patients were subjected to clinical examination, abdominal ultrasound and upper endoscopy for gastric biopsy. Biopsies were subjected to urease test, histological examination, and DNA purification. H. pylori, Braf, Kras, MLH1 and MGMT methylation were assessed by quantitative PCR. DNA sequencing was performed to assess Braf and Kras genes mutation. qPCR of H. pylori was significantly higher in patients with adenocarcinoma (group II) than those without adenocarcinoma (group I); with a p < 0.001 as well as in patients with age above 50 years with a p value = 0.008. By applying logistic regression analysis it was reported that the H. pylori qPCR is a significant predictor to the adenocarcinoma with OR = 1.025 (95 % CI: 1. 002-1.048), with sensitivity of 90 % and specificity of 100 %. Adenocarcinoma patients had a significantly higher mean age and levels of H. Pylori, Braf, K-ras, methylated MGMT and methylated MLH1 than those of gastritis patients. DNA sequence analysis of Braf (codon 12) and Kras (codon 600) had genes mutation in gastric adenocarcinoma versus chronic gastritis. CONCLUSION: H. pylori may cause epigenetic changes predisposing the patients to cancer stomach. Estimation of H. pylori by qPCR can be a good predictor to adenocarcinoma. Braf and Kras genes mutation were reveled in gastritis and adenocarcinoma patients.

Helicobacter pylori, cyclooxygenase-2 and evolution of gastric lesions: results from an intervention trial in China.

To investigate the role of cyclooxygenase (COX)-2/prostaglandin E2 (PGE2) in the process of Helicobacter pylori-induced gastric carcinogenesis, a prospective study based on an intervention trial was conducted in Linqu County, China. A total of 1401 subjects with histopathologic diagnosis were investigated at baseline, among those, 919 completed subsequent interventions (anti-H.pylori and/or celecoxib treatment). Expressions of COX-2 and Ki-67 were assessed by immunohistochemistry, and PGE2 levels were measured by enzyme immunoassay before and after interventions, respectively. We found a grade-response relationship between COX-2 expression level and risk of advanced gastric lesions at baseline. Stratified analysis indicated an additive interaction between COX-2 expression and H.pylori infection on the elevated risk of advanced gastric lesions. The odds ratios (ORs) for both factors combined were 9.31 [95% confidence interval (CI): 4.13-20.95] for chronic atrophic gastritis, 16.26 (95% CI: 7.29-36.24) for intestinal metaplasia and 21.13 (95% CI: 7.87-56.75) for dysplasia, respectively. After interventions, COX-2 expression and Ki-67 labeling index (LI) were decreased in anti-H.pylori group (OR: 1.65, 95% CI: 1.36-1.99 for COX-2; OR: 1.78, 95% CI: 1.49-2.12 for Ki-67) or anti-H.pylori followed by celecoxib group (OR: 1.41, 95% CI: 1.17-1.70 for COX-2; OR: 1.63, 95% CI: 1.37-1.94 for Ki-67). PGE2 levels were decreased in all treatment groups. Furthermore, the regression of gastric lesions was associated with the decrease of COX-2 expression or Ki-67 LI after interventions. Our findings indicate that H.pylori-induced COX-2/PGE2 pathways play an important role on the progression of precancerous gastric lesions in a Chinese population.

Integrin-linked kinase modulates lipopolysaccharide- and Helicobacter pylori-induced nuclear factor κB-activated tumor necrosis factor-α production via regulation of p65 serine 536 phosphorylation.

Integrin-linked kinase (ILK) is a ubiquitously expressed and highly conserved serine-threonine protein kinase that regulates cellular responses to a wide variety of extracellular stimuli. ILK is involved in cell-matrix interactions, cytoskeletal organization, and cell signaling. ILK signaling has also been implicated in oncogenesis and progression of cancers. However, its role in the innate immune system remains unknown. Here, we show that ILK mediates pro-inflammatory signaling in response to lipopolysaccharide (LPS). Pharmacological or genetic inhibition of ILK in mouse embryonic fibroblasts and macrophages selectively blocks LPS-induced production of the pro-inflammatory cytokine tumor necrosis factor α (TNF-α). ILK is required for LPS-induced activation of nuclear factor κB (NF-κB) and transcriptional induction of TNF-α. The modulation of LPS-induced TNF-α synthesis by ILK does not involve the classical NF-κB pathway, because IκB-α degradation and p65 nuclear translocation are both unaffected by ILK inhibition. Instead, ILK is involved in an alternative activation of NF-κB signaling by modulating the phosphorylation of p65 at Ser-536. Furthermore, ILK-mediated alternative NF-κB activation through p65 Ser-536 phosphorylation also occurs during Helicobacter pylori infection in macrophages and gastric cancer cells. Moreover, ILK is required for H. pylori-induced TNF-α secretion in macrophages. Although ILK-mediated phosphorylation of p65 at Ser-536 is independent of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway during LPS stimulation, upon H. pylori infection this event is dependent on the PI3K/Akt pathway. Our findings implicate ILK as a critical regulatory molecule for the NF-κB-mediated pro-inflammatory signaling pathway, which is essential for innate immune responses against pathogenic microorganisms.

Activation of EGFR and ERBB2 by Helicobacter pylori results in survival of gastric epithelial cells with DNA damage.

BACKGROUND & AIMS: The gastric cancer-causing pathogen Helicobacter pylori up-regulates spermine oxidase (SMOX) in gastric epithelial cells, causing oxidative stress-induced apoptosis and DNA damage. A subpopulation of SMOX(high) cells are resistant to apoptosis, despite their high levels of DNA damage. Because epidermal growth factor receptor (EGFR) activation can regulate apoptosis, we determined its role in SMOX-mediated effects. METHODS: SMOX, apoptosis, and DNA damage were measured in gastric epithelial cells from H. pylori-infected Egfr(wa5) mice (which have attenuated EGFR activity), Egfr wild-type mice, or in infected cells incubated with EGFR inhibitors or deficient in EGFR. A phosphoproteomic analysis was performed. Two independent tissue microarrays containing each stage of disease, from gastritis to carcinoma, and gastric biopsy specimens from Colombian and Honduran cohorts were analyzed by immunohistochemistry. RESULTS: SMOX expression and DNA damage were decreased, and apoptosis increased in H. pylori-infected Egfr(wa5) mice. H. pylori-infected cells with deletion or inhibition of EGFR had reduced levels of SMOX, DNA damage, and DNA damage(high) apoptosis(low) cells. Phosphoproteomic analysis showed increased EGFR and erythroblastic leukemia-associated viral oncogene B (ERBB)2 signaling. Immunoblot analysis showed the presence of a phosphorylated (p)EGFR-ERBB2 heterodimer and pERBB2; knockdown of ErbB2 facilitated apoptosis of DNA damage(high) apoptosis(low) cells. SMOX was increased in all stages of gastric disease, peaking in tissues with intestinal metaplasia, whereas pEGFR, pEGFR-ERBB2, and pERBB2 were increased predominantly in tissues showing gastritis or atrophic gastritis. Principal component analysis separated gastritis tissues from patients with cancer vs those without cancer. pEGFR, pEGFR-ERBB2, pERBB2, and SMOX were increased in gastric samples from patients whose disease progressed to intestinal metaplasia or dysplasia, compared with patients whose disease did not progress. CONCLUSIONS: In an analysis of gastric tissues from mice and patients, we identified a molecular signature (based on levels of pEGFR, pERBB2, and SMOX) for the initiation of gastric carcinogenesis.

Aegle marmelos fruit extract attenuates Helicobacter pylori Lipopolysaccharide induced oxidative stress in Sprague Dawley rats.

BACKGROUND: Bael (Aegle marmelos (L.) Corr.) has been widely used in indigenous systems of Indian medicine to exploit its medicinal properties including astringent, antidiarrheal, antidysenteric, demulcent, antipyretic, antiulcer, anti-inflammatory and anti cancer activities. The present study aims to evaluate the antioxidative and antiulcer effect of methanolic extract of unripe fruit of Aegle marmelos (MEAM) against Helicobacter pylori-Lipopolysaccharide (HP-LPS) induced gastric ulcer in Sprague Dawley (SD) rats. METHODS: Dose and duration of HP-LPS and MEAM were fixed based on ulcer index of gastric tissue of experimental animals. Various gastric secretory parameters such as volume of gastric juice, free and total acidity, acid output, pepsin concentration were analyzed. The activities of enzymatic antioxidants (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione transferase), non-enzymatic antioxidants (reduced glutathione, vitamin C and vitamin E) and the levels of lipid peroxidation products were measured. Histological analysis was performed to evaluate the effect of Aegle marmelos on HP-LPS induced gastric ulcer. RESULTS: Oral administration of HP-LPS (50 µg per animal) for four consecutive days resulted in induction of ulcer with the increase in gastric secretory parameters such as volume of gastric juice, free and total acidity, acid output, pepsin concentration. Oral administration of methanolic extract of Aegle marmelos fruit (MEAM) (25, 50, 100, 250 and 500 mg/kg) reduced the gastric ulcer by 2.8 %, 52.4 %, 73 %, 93 % and 93.98 %, respectively, compared to 89.2 % reduction by sucralfate (100 mg/kg). MEAM treatment significantly (p < 0.05) inhibited the increase in gastric secretory parameters in ulcerated rats, and it also prevented the reduction of enzymatic (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione transferase) and non-enzymatic antioxidants (reduced glutathione, vitamin C and vitamin E) after HP-LPS induction. In addition, lipid peroxidation was inhibited by MEAM in HP-LPS induced rats. Results of histological analysis correlated well with biochemical parameters. CONCLUSION: These observations explored the antioxidant properties of MEAM contributing to the gastroprotective effect in HP-LPS induced gastric ulcer model.

[THE EFFECTIVENESS OF ERADICATION IN PATIENTS WITH PEPTIC ULCER DISEASE ASSOCIATED WITH HELICOBACTER PYLORI, DEPENDING ON THE GENOTYPE OF THE DRUGMETABOLISM OF PROTON PUMP INHIBITORS].

One of the most likely causes of the lack of effectiveness of eradication therapy of peptic ulcer associated with Helicobacter pylori, is a feature of omeprazole metabolism by cytochrome CYP2C19. The paper work presents evidence that the rate of reduction of the clinical picture and the likelihood of scarring ulcers and eradication rates higher in patients slow metabolizers of omeprazole.

Helicobacter pylori-induced posttranscriptional regulation of H-K-ATPase α-subunit gene expression by miRNA.

Acute Helicobacter pylori infection of gastric epithelial cells induces CagA oncoprotein- and peptidoglycan (SLT)-dependent mobilization of NF-κB p50 homodimers that bind to H-K-ATPase α-subunit (HKα) promoter and repress HKα gene transcription. This process may facilitate gastric H. pylori colonization by induction of transient hypochlorhydria. We hypothesized that H. pylori also regulates HKα expression posttranscriptionally by miRNA interaction with HKα mRNA. In silico analysis of the HKα 3' untranslated region (UTR) identified miR-1289 as a highly conserved putative HKα-regulatory miRNA. H. pylori infection of AGS cells transfected with HKα 3' UTR-Luc reporter construct repressed luciferase activity by 70%, whereas ΔcagA or Δslt H. pylori infections partially abrogated repression. Transfection of AGS cells expressing HKα 3' UTR-Luc construct with an oligoribonucleotide mimetic of miR-1289 induced maximal repression (54%) of UTR activity within 30 min; UTR activity was unchanged by nontargeting siRNA transfection. Gastric biopsies from patients infected with cagA(+) H. pylori showed a significant increase in miR-1289 expression compared with uninfected patients or those infected with cagA(-) H. pylori. Finally, miR-1289 expression was necessary and sufficient to attenuate biopsy HKα protein expression in the absence of infection. Taken together, these data indicate that miR-1289 is upregulated by H. pylori in a CagA- and SLT-dependent manner and targets HKα 3' UTR, affecting HKα mRNA translation. The sensitivity of HKα mRNA 3' UTR to binding of miR-1289 identifies a novel regulatory mechanism of gastric acid secretion and offers new insights into mechanisms underlying transient H. pylori-induced hypochlorhydria.

Helicobacter pylori induces cell migration and invasion through casein kinase 2 in gastric epithelial cells.

BACKGROUND: Chronic infection with Helicobacter pylori (H. pylori) is causally linked with gastric carcinogenesis. Virulent H. pylori strains deliver bacterial CagA into gastric epithelial cells. Induction of high motility and an elongated phenotype is considered to be CagA-dependent process. Casein kinase 2 plays a critical role in carcinogenesis through signaling pathways related to the epithelial mesenchymal transition. This study was aimed to investigate the effect of H. pylori infection on the casein kinase 2-mediated migration and invasion in gastric epithelial cells. MATERIALS AND METHODS: AGS or MKN28 cells as human gastric epithelial cells and H. pylori strains Hp60190 (ATCC 49503, CagA(+)) and Hp8822 (CagA(-)) were used. Cells were infected with H. pylori at multiplicity of infection of 100 : 1 for various times. We measured in vitro kinase assay to examine casein kinase 2 activity and performed immunofluorescent staining to observe E-cadherin complex. We also examined ß-catenin transactivation through promoter assay and MMP7 expression by real-time PCR and ELISA. RESULTS: H. pylori upregulates casein kinase 2 activity and inhibition of casein kinase 2 in H. pylori-infected cells profoundly suppressed cell invasiveness and motility. We confirmed that casein kinase 2 mediates membranous α-catenin depletion through dissociation of the α-/ß-catenin complex in H. pylori-infected cells. We also found that H. pylori induces ß-catenin nuclear translocation and increases MMP7 expressions mediated through casein kinase 2. CONCLUSIONS: We show for the first time that CagA(+) H. pylori upregulates cellular invasiveness and motility through casein kinase 2. The demonstration of a mechanistic interplay between H. pylori and casein kinase 2 provides important insights into the role of CagA(+) H. pylori in the gastric cancer invasion and metastasis.

[A study of urease activity of mouth cavity microflora].

STUDY OBJECTIVE: Analysis of the prevalence of mouth cavity urealytic microflora and determination of the level of its enzymatic activity depending on concentration and amount of urea solution taken as a substrate. MATERIALS AND METHODS: 62 randomly chosen patients at the age of 5-64 took part in the study. Each of them rinsed the mouth with 50 ml of 1% urea solution. Before and after rinsing the concentration of ammonia in the mouth cavity air was measured. In patients with highest and lowest activity of mouth cavity urealytic microflora a series of tests was carried out including mouth rinsing with urea solution in various concentrations and amounts and measuring ammonia concentration before and after rinsing. Obtained results were analyzed using mathematical statistics methods. RESULTS: It was found that in 91% ± 1.8% of randomly chosen patients (p < 0.05) mouth cavity microflora showed apparent urease activity. The lowest concentration (0.0625% in 50 ml) and volume (0.5 ml of 1% solution) levels of urea solution were obtained that can exert negative influence on the results of helicobacteriosis diagnosis by means of mouth cavity air analysis. CONCLUSION: Urealytic microflora in the mouth cavity is very common and may constitute a factor that decreases the specificity of helicobacteriosis diagnosis by means of the methods based on detection of indicators of gas metabolites resulting from the enzymatic reaction in air samples taken from the mouth cavity after oral administration of urea.

Protein kinase C isozymes regulate matrix metalloproteinase-1 expression and cell invasion in Helicobacter pylori infection.

BACKGROUND: Protein kinase C (PKC) signalling is often dysregulated in gastric cancer and therefore represents a potential target in cancer therapy. The Gram-negative bacterium Helicobacter pylori, which colonises the human stomach, plays a major role in the development of gastritis, peptic ulcer and gastric adenocarcinoma. OBJECTIVE: To analyse the role of PKC isozymes as mediators of H pylori-induced pathogenesis. METHODS: PKC phosphorylation was evaluated by immunoblotting and immunohistochemistry. Gene reporter assays, RT-PCR and invasion assays were performed to assess the role of PKC in the regulation of activator protein-1 (AP-1), matrix metalloproteinase-1 (MMP-1) and the invasion of H pylori-infected epithelial cells. RESULTS: H pylori induced phosphorylation of PKC isozymes α, δ, θ in AGS cells, which was accompanied by the phosphorylation of PKC substrates, including PKCµ and myristoylated alanine-rich C kinase substrate (MARCKS), in a CagA-independent manner. Phospholipase C, phosphatidylinositol 3-kinase and Ca(2+) were crucial for PKC activation on infection; inhibition of PKC diminished AP-1 induction and, subsequently, MMP-1 expression. Invasion assays confirmed PKC involvement in H pylori-induced MMP-1 secretion. In addition, analysis of biopsies from human gastric mucosa showed increased phosphorylation of PKC in active H pylori gastritis and gastric adenocarcinoma. CONCLUSION: The targeting of certain PKC isozymes might represent a suitable strategy to interfere with the MMP-1-dependent remodelling of infected tissue and to overcome the invasive behaviour of gastric cancer cells.

Overproduction of the N-terminal anticodon-binding domain of the non-discriminating aspartyl-tRNA synthetase from Helicobacter pylori for crystallization and NMR measurements.

Aminoacyl-tRNA synthetases (aaRSs) covalently attach an amino acid to its cognate tRNA isoacceptors through an ester bond. The standard set of 20 amino acids implies 20 aaRSs for each pair of amino acid/tRNA isoacceptors. However, the genomes of all archaea and some bacteria do not encode for a complete set of 20 aaRSs. For the human pathogenic bacterium Helicobacter pylori, a gene encoding asparaginyl-tRNA synthetase (AsnRS) is absent whilst an aspartyl-tRNA synthetase (AspRS) aminoacylates both tRNA(Asp) and tRNA(Asn) with aspartate. The structural and functional basis for this non-discriminatory behavior is not well understood. Here we report the over-production of the N-terminal anticodon-binding domain of H. pylori ND-AspRS using Escherichia coli BL21(DE3) host cells. Prolonged expression of this protein resulted in a toxic phenotype, limiting the expression period to just 30min. Purified protein was monomeric in solution by gel filtration chromatography and stable up to 42°C as observed in temperature-dependent dynamic light scattering measurements. Circular dichroism indicated a mixture of α-helix and ß-sheet secondary structure at 20°C and predominantly ß-sheet at 70°C. Optimized crystallization conditions at pH 5.6 with PEG 4000 as a co-precipitant produced well-formed crystals and (1)H NMR spectrum showed a well dispersed chemical shift envelope characteristic of a folded protein.

Immune evasion by Helicobacter pylori is mediated by induction of macrophage arginase II.

Helicobacter pylori infection persists for the life of the host due to the failure of the immune response to eradicate the bacterium. Determining how H. pylori escapes the immune response in its gastric niche is clinically important. We have demonstrated in vitro that macrophage NO production can kill H. pylori, but induction of macrophage arginase II (Arg2) inhibits inducible NO synthase (iNOS) translation, causes apoptosis, and restricts bacterial killing. Using a chronic H. pylori infection model, we determined whether Arg2 impairs host defense in vivo. In C57BL/6 mice, expression of Arg2, but not arginase I, was abundant and localized to gastric macrophages. Arg2(-/-) mice had increased histologic gastritis and decreased bacterial colonization compared with wild-type (WT) mice. Increased gastritis scores correlated with decreased colonization in individual Arg2(-/-) mice but not in WT mice. When mice infected with H. pylori were compared, Arg2(-/-) mice had more gastric macrophages, more of these cells were iNOS(+), and these cells expressed higher levels of iNOS protein, as determined by flow cytometry and immunofluorescence microscopy. There was enhanced nitrotyrosine staining in infected Arg2(-/-) versus WT mice, indicating increased NO generation. Infected Arg2(-/-) mice exhibited decreased macrophage apoptosis, as well as enhanced IFN-γ, IL-17a, and IL-12p40 expression, and reduced IL-10 levels consistent with a more vigorous Th1/Th17 response. These studies demonstrate that Arg2 contributes to the immune evasion of H. pylori by limiting macrophage iNOS protein expression and NO production, mediating macrophage apoptosis, and restraining proinflammatory cytokine responses.