Helicobacter pylori HP0876 is Dispensable for Heme–Iron Acquisition but Attenuates Bacterial Adherence to Gastric Epithelial Cells

Helicobacter pylori can efficiently capture iron either from free heme or heme-containing compounds in the iron-limited gastric mucosa. However, the heme iron utilization systems of H. pylori have not been fully described to date. To investigate the contribution of genes involved in heme-iron utilization, a gene homologous to frpB, encode by hp0876 in H. pylori ATCC 26695, was inactivated by homologous recombination. Δhp0876 showed no demonstrable growth defects in the presence of the various concentrations of free iron. Moreover, when hemoglobin or heme was supplied as the sole iron sources, Δhp0876 had growth curves similar to the wild-type strain. The growth competition experiments in vitro also showed that Δhp0876 retained the ability for iron acquisition. Furthermore, IL-8 production in human gastric epithelial cells co-cultured with Δhp0876 and wild-type strain was compared, and our results indicated that lack of HP0876 affected the IL-8 release. And Δhp0876 exhibited significantly increased adherence to gastric epithelial cells. Together, our data suggests that HP0876 is dispensable for H. pylori heme-iron uptake, but it may attenuate H. pylori adherence to gastric epithelial cells, which induced decreased production of H. pylori-induced IL-8 production in gastric epithelial cells.     

Helicobacter pylori infection and CagA protein translocation in human primary gastric epithelial cell culture

BACKGROUND: Increasing evidence has shown that Helicobacter pylori CagA protein translocation into gastric epithelial cells plays an important role in the development of gastric inflammation and malignancy. Translocated CagA undergoes tyrosine phosphorylation in gastric adenocarcinoma cell line cells, and CagA involves disruption of cellular apical-junction complex in Madin-Darby canine kidney cells. METHODS: To elucidate whether these events take place in normal human gastric epithelium, we infected human primary gastric epithelial cells with H. pylori. RESULTS: Our results demonstrate that CagA protein was translocated into primary gastric epithelial cells and tyrosine phosphorylated. The translocated CagA induces cytoskeletal rearrangement and the disruption of tight junctions in primary gastric epithelial cells. CONCLUSIONS: This study provides direct evidence of the modulation of gastric epithelial cells by CagA protein translocation, and advances our understanding of the pathogenesis of H. pylori infection.     

Molecular mechanisms of epithelial-barrier disruption by Helicobacter pylori

Intact intercellular junctions and cell-matrix contacts are important structures in the formation and maintenance of epithelial-barrier functions against microbes. The human gastric pathogen Helicobacter pylori developed a remarkable network of strategies to alter these epithelial cell-cell and cell-matrix adhesions, which are implicated in inflammation, proliferation, cell migration and invasive growth. This review focuses on recent findings on H. pylori-induced host-cell signaling. We propose a stepwise model for how H. pylori interacts with components of focal adhesions and intercellular tight and adherens junctions to disrupt the epithelial layer, providing novel insights into the pathogenesis of H. pylori.     

A novel in vitro infection model of Helicobacter pylori using mucin-producing murine gastric surface mucous cells

BACKGROUND: Helicobacter pylori is found within the gastric surface mucous gel layer and in the epithelial surface. Gastric cancer cells have been used in experimental H. pylori infection in vitro, although cancer cells have some abnormalities in cellular properties. The aim of this study was to develop an in vitro H. pylori infection model using normal gastric surface cells that produce gastric mucin. MATERIALS AND METHODS: Normal murine gastric surface mucous cells (GSM06) were cultured by the liquid interface method using a serum-free medium and a collagen gel containing a fibroblast cell line (L929) and infected with H. pylori. Infection by H. pylori was assessed by enumerating the colony-forming units (CFU) of H. pylori adhered to GSM06 cells and by transmission electron microscopy. The production of mucin was determined by a lectin binding assay, sugar analysis, and MUC5AC gene expression. RESULTS: GSM06 cells cultured under these conditions produced mucin containing N-acetylgalactosamine and MUC5AC as the core protein. Significantly higher numbers of H. pylori adhered to GSM06 cells under mucin-producing conditions than under nonproducing conditions. Microscopic observation showed a filamentous structure resembling a type IV secretion system apparatus formed between the surface of GSM06 cells and H. pylori. CONCLUSIONS: This study demonstrates a novel in vitro H. pylori infection model using mucin-producing murine GSM06 cells for early stages of infection.     

The effect of ascorbic acid on Helicobacter pylori induced cyclooxygenase 2 expression and prostaglandin E2 production by gastric epithelial cells in vitro

BACKGROUND: Cyclooxygenase 2 (COX-2) is induced by the presence of Helicobacter pylori (H. pylori) on the gastric mucosa as part of the inflammatory response; this results in the synthesis of prostaglandins that amplify the local inflammatory response. The presence of H. pylori inhibits the secretion of ascorbate into the gastric lumen. Interestingly, ascorbate inhibits the growth of H. pylori and low dietary levels are associated with an increased risk of gastric adenocarcinoma. We therefore investigated the effect of ascorbate on H. pylori mediated COX-2 induction and prostaglandin production in vitro. METHODS: H. pylori was cocultured with gastric epithelial cells in the presence of ascorbate at physiological concentrations. The expression of COX-2 was assessed by Western blotting and prostaglandin E(2) (PGE(2)) was assessed by ELISA. RESULTS: Ascorbate inhibited gastric cell PGE(2) synthesis but not in COX-2 expression in response to H. pylori. In the absence of the organism, ascorbate also reduced PGE(2) expression in cells that constitutively express COX-2, again with no reduction of COX-2 protein expression. CONCLUSIONS: Physiological concentrations of ascorbate inhibit PGE(2) but not COX-2 expression in response to H. pylori in gastric epithelial cells.     

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.     

Bovine colostrum supports the serum-free proliferation of epithelial cells but not of fibroblasts in long-term culture

Medium lacking serum but supplemented with milk will support the growth of sparse cells in culture. Milk obtained within 8 h after the birth of a calf (day 1 colostrum) is the most effective in supporting proliferation. In mixed cultures of early-passage bovine embryonic kidney (BEK) or early-passage calf kidney (CK) cells, both epithelial cells and fibroblasts grow in Dulbecco’s modified eagle’s medium (DMEM) supplemented with serum. However, only cells that appear to be epithelial-like grow in DMEM supplemented with colostrum. Sparse cultures of early-passage human and rat fibroblasts that grow readily in DMEM supplemented with serum do not grow in DMEM supplemented with colostrum. Canine kidney epithelial cells (MDCK), when plated sparsely, grow exponentially in DMEM supplemented with day 1 bovine colostrum. The generation time is 26 h, the same growth rate as in DMEM supplemented with calf serum. The MDCK cells can be subcultured and regrown to confluence repeatedly in colostrum-supplemented DMEM. Growth in DMEM supplemented with colostrum does not alter the morphological characteristics of the MDCK cells, which are polygonal, contain microvilli at the apical surface, and are connected by tight junctions and desmosomes. MDCK cells do not proliferate in DMEM supplemented with milk obtained 1 wk after the birth of a calf.     

L-lactic acid secreted from gastric mucosal cells enhances growth of Helicobacter pylori

BACKGROUND: Helicobacter pylori mainly inhabit the mucus layer in the gastric mucosa. However, mechanisms involving H. pylori colonization and proliferation in gastric mucosa are not well established. This study focuses on elucidating the role of gastric mucosal cells on growth of H. pylori. MATERIALS AND METHODS: H. pylori was co-cultured with the murine gastric surface mucosal cells (GSM06), and the growth of H. pylori on the cells was assessed by enumerating the colony-forming units (CFU). The H. pylori growth factor in the culture media conditioned by GSM06 cell was purified by HPLC, and the chemical structure of the growth factor was identified by analyses of (1)H- and (13)C-NMR spectra. RESULTS: A marked increase in the number of CFU of H. pylori was observed in the GSM06 cells. The enhanced H. pylori growth was also observed when indirectly incubated with GSM06 cells through semi-permeable membrane. In addition, culture media conditioned by GSM06 cell stimulated H. pylori growth approximately one thousand-fold. By bioassay-guided purification, the H. pylori growth factor was isolated from the conditioned medium of GSM06 cells and identified as L-lactic acid. The H. pylori growth-enhancing activity under microaerobic condition was well correlated with L-lactic acid concentrations in the conditioned media. CONCLUSIONS: This study demonstrates that L-lactic acid secreted by gastric mucosal cells enhances the growth of H. pylori, and this L-lactic acid-dependent growth of H. pylori may be important to the long-term colonization of H. pylori in the stomach.     

Helicobacter pylori induces an increase in inositol phosphates in cultured epithelial cells

Abstract Helicobacter pylori is a bacterial pathogen of humans that infects the gastric mucosa. This infection has been associated with gastritis, peptic ulcers, and gastric carcinomas. Diverse in vitro studies have described efficient adherence of H. pylori to different types of epithelial cells. Because of its varied effects on host cells, we have analysed signal transduction events in H. pyfori -infected epithelial cells. Our results show that H. pylori induces an increase in inositol phosphates in all cultured epithelial cells used, including HeLa, Henle 407, Hep-2, and the human gastric adenocarcinoma cell line AGS. Bacterial growth medium supernatants induce a similar response in the host cell. The increase in inositol phosphates is not related to redistribution of cytoskeletal proteins such as actin or α-actinin nor tyrosine-phosphorylation of host cell proteins. The inositol phosphate increase is also observed in cells infected with low or non-adherent H. pylori mutants or mutants defective in the vacuolating toxin or urease holoenzyme. These results indicate that inositol phosphate release in H. pytori -infected cells is not dependent on bacterial adherence, and that a soluble bacterial factor, but not the vacuolating toxin or urease holoenzyme, mediates such an effect.     

mRNA expression of cytokines and chemokines in the normal gastric surface mucous epithelial cell line GSM06 during bacterial infection with Helicobacter felis.

BACKGROUND AND AIM: A group of the proinflammatory and chemotactic cytokines (chemokines) has been considered as an important factor in the pathomechanism of different bacterial diseases, among them the common Helicobacter pylori infection. Experimental results obtained with gastric biopsy samples of H. pylori positive patients, and with H. pylori infected tumor originated gastric cell lines indicated that these cytokines have essential roles in the development and maintenance of the immune response and inflammation of the gastric mucosa during H. pylori infection. Although the mRNA expression was shown in these biopsy samples and cell lines, it is not yet proved that the normal gastric mucosal epithelial cells themselves express these cytokines. The establishment of a gastric surface mucous cell line with non-tumor origin (GSM06), and the usage of Helicobacter felis as a model of the classic H. pylori infection gave us the possibility to check this question. MATERIALS AND METHODS: in this study GSM06 cells were infected with different numbers (10(5), 10(6), 10(7), 10(8), 10(9) bacterium/ml medium) of H. felis for two different time periods (2, 4 h). Cells treated with medium only were used as control. Then the mRNA expression of the following cytokines was measured by RT-PCR method in the GSM06 cells: proinflammatory cytokine IL1-beta, and chemokine RANTES, eotaxin, MCP-1, MIP1-alpha and MIP1-beta. RESULTS: we found that neither mRNA of the investigated cytokines was expressed constitutively, however the GSM06 cells expressed the mRNA of each cytokine during H. felis infection. CONCLUSION: our results prove that normal gastric surface mucous epithelial cells express immunologically active peptides during H. felis infection. We may suppose that the epithelial cells of the gastric mucosa contribute to the immune response and inflammation by expressing proinflammatory (IL1-beta) and chemotactic (RANTES, eotaxin, MCP-1, MIP1-alpha and beta) cytokines during H. pylori infection in human.     

Effects of Helicobacter pylori on biological characteristics of gastric epithelial cells

Infection with Helicobacter pylori (H. pylori) strains is linked to an increased risk of inflammation and gastric cancer. To investigate the effects of H. pylori on biological characteristics of gastric epithelial cells SGC-7901, derived from human adenocarcinoma, morphological appearances of both the pathogen and these cells, as well as features of attachment and internalization were observed by using transmission electron microscopy (TEM). We also investigated cell junctions and invasion by TEM and Transwell Invasion Assay. Cell proliferation and apoptosis were assessed by using chromogenic methylthiazol tetrazolium bromide (MTT) dye and flow cytometry. Three types of H. pylori were observed around, attaching to, or invading tumor cells. Cellular damage was characterized by vacuolar degeneration, dilated endoplasmic reticulum (ER), and reduction of organelles. Cell junctions and cell microvilli reduced or disappeared. H. pylori inhibited cell proliferation, whereas it had no effect on apoptosis. It also promoted gastric carcinoma cell invasion. H. pylori damages cell construction, destroys cell junctions, inhibits cell proliferation, promotes cell invasive ability, and, therefore, might accelerate the malignant progress and metastasis of gastric cancer.     

The role of phosphorylation in development of tight junctions in cultured renal epithelial (MDCK) cells.

We have explored the effect of the protein kinase inhibitor H7 on tight junction formation in a MDCK cell model for the development of cell-cell contact, tight junctions and epithelial polarity: the "Ca++ switch" model. In this developmental model, which is thought to mimic processes during the early morphogenesis of epithelial tissues, the protein kinase inhibitor H7 markedly inhibits the development of transepithelial resistance of confluent MDCK cells during the "switch" from low (1-5 microM) to normal (1.8 mM) Ca++ media compared with control MDCK cells. Moreover, indirect immunofluorescence using specific antisera against two tight junctional proteins, ZO1 and cingulin, revealed that H7 inhibits the sorting of these proteins from an intracellular site to the lateral surfaces of MDCK cells when the Ca++ in the medium is raised. These data suggest protein kinase mediation in sorting events that lead to the assembly of tight junctions.     

胚胎鼠胃粘膜上皮细胞的原代培养

采用胰酶冷消化法对胚胎鼠胃粘膜上皮细胞进行了培养,细胞培养于含２０％的胎牛血清的ＤＭＥＭ／Ｆ１２的培养液中,种植２４ｈ后开始生长,３ｄ长成片状,相差显微镜下观察９０％的细胞具有上皮细胞特征。免疫细胞化学显示：（ａ）９０％的细胞上皮角蛋白染色阳性;（ｂ）９０％的上皮细胞ＰＡＳ染色阳性;（ｃ）２０％的上皮细胞琥珀酸脱氢酶（ＳＤＨ）染色阳性。透射电镜可见微绒毛、连接复合物（紧密连接,桥粒）、丰富的糖原、线粒体。放射自显影结果表明：上皮细胞具有合成ＤＮＡ的能力,而且在培养２ｄ时增殖能力最强。本实验为研究胃粘膜的功能提供了较好的方法。     

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

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

Effect of H. pylori on the expression of TRAIL, FasL and their receptor subtypes in human gastric epithelial cells and their role in apoptosis

BACKGROUND AND AIMS: In the human stomach expression of TNF-related apoptosis inducing ligand (TRAIL) and its receptors and the modulatory role of Helicobacter pylori are not well described. Therefore, we investigated the effect of H. pylori on the expression of TRAIL, FasL and their receptors (TRAIL-R1-R4, Fas) in gastric epithelial cells and examined their role in apoptosis. MATERIALS AND METHODS: mRNA and protein expression of TRAIL, FasL and their receptors were analyzed in human gastric epithelial cells using RT-PCR, Western blot, and immunohistochemistry. Gastric epithelial cells were incubated with FasL, TRAIL and/or H. pylori, and effects on expression, cell viability and epithelial apoptosis were monitored. Apoptosis was analyzed by histone ELISA, DAPI staining and immunohistochemistry. RESULTS: TRAIL, FasL and their receptor subtypes were expressed in human gastric mucosa, gastric epithelial cell primary cultures and gastric cancer cells. TRAIL, FasL and H. pylori caused a time- and concentration-dependent induction of DNA fragmentation in gastric cancer cells with synergistic effects. In addition, H. pylori caused a selective up-regulation of TRAIL, TRAIL-R1 and Fas mRNA and protein expression in gastric cancer cells. CONCLUSIONS: Next to FasL and Fas, TRAIL and all of its receptor subtypes are expressed in the human stomach and differentially modulated by H. pylori. TRAIL, FasL and H. pylori show complex interaction mediating apoptosis in human gastric epithelial cells. These findings might be important for the understanding of gastric epithelial cell kinetics in patients with H. pylori infection.     

Helicobacter pylori inhibits gastric cell cycle progression

Helicobacter pylori infection of the gastric mucosa is associated with changes in gastric epithelial cell proliferation. In vitro studies have shown that exposure to H. pylori inhibits proliferation of gastric cells. This study sought to investigate the cell cycle progression of gastric epithelial cell lines in the presence and absence of H. pylori. Unsynchronized and synchronized gastric epithelial cell lines AGS and KatoIII were exposed to H. pylori over a 24-h period. Cell cycle progression was determined by flow cytometry using propidium iodide (PI), and by analysis of cyclin E, p21, and p53 protein expression using Western blots. In the absence of H. pylori 40, 45, and 15% of unsynchronized AGS cells were in G(0)-G(1), S, and G(2)-M phases, respectively, by flow cytometry analysis. When AGS cells were cultured in the presence of H. pylori, the S phase decreased 10% and the G(0)-G(1) phase increased 17% after 24 h compared with the controls. KatoIII cells, which have a deleted p53 gene, showed little or no response to H. pylori. When G1/S synchronized AGS cells were incubated with media containing H. pylori, the G(1) phase increased significantly (25%, P < 0.05) compared with controls after 24 h. In contrast, the control cells were able to pass through S phase. The inhibitory effects of H. pylori on the cell cycle of AGS cells were associated with a significant increase in p53 and p21 expression after 24 h. The expression of cyclin E was downregulated in AGS cells following exposure of AGS cells to H. pylori for 24 h. This study shows that H. pylori-induced growth inhibition in vitro is predominantly at the G(0)-G(1) checkpoint. Our results suggest that p53 may be important in H. pylori-induced cell cycle arrest. These results support a role for cyclin-dependent kinase inhibitors in the G(1) cell cycle arrest exerted by H. pylori and its involvement in changing the regulatory proteins, p53, p21, and cyclin E in the cell cycle.