Primary Human Gastric Epithelial Cells Research Articles

Essential role of Helicobacter pylori apolipoprotein N-acyltransferase (Lnt) in stomach colonization.

Bacterial lipoproteins are post-translationally modified with acyl chains, anchoring these proteins to bacterial membranes. In Gram-negative bacteria, three enzymes complete the modifications. Lgt (which adds two acyl chains) and LspA (which removes the signal peptide) are essential. Lnt (which adds a third acyl chain) is not essential in certain bacteria including Francisella tularensis, Neisseria gonorrhoeae, and Acinetobacter baumannii. Deleting lnt results in mild to severe physiologic changes. We previously showed lnt is not essential for Helicobacter pylori growth in vitro. Here, the physiologic consequences of deleting lnt in H. pylori and the role of Lnt in the host response to H. pylori were examined using in vitro and in vivo models. Comparing wild-type, Δlnt, and complemented mutant H. pylori, no changes in growth rates or sensitivity to acid or antibiotics were observed. Since deleting lnt changes the number of acyl chains on lipoproteins and the number of acyl chains on lipoproteins impacts the innate immune response through Toll-like receptor 2 (TLR2) signaling, primary human gastric epithelial cells were treated with a purified lipoprotein from wild-type or lnt mutant H. pylori. Differential gene expression analysis indicated that lipoprotein from the lnt mutant induced a more robust TLR2 response. In a complementary approach, we infected wild-type and Tlr2-/- mice and found that both the wild-type and complemented mutant strains successfully colonized the animals. However, the lnt mutant strain was unable to colonize either mouse strain. These results show that lnt is essential for H. pylori colonization and identifies lipoprotein synthesis as a target for therapeutic intervention.

H. pylori infection confers resistance to apoptosis via Brd4‐dependent BIRC3 eRNA synthesis

H. pylori infection is one of the leading causes of gastric cancer and the pathogenicity of H. pylori infection is associated with its ability to induce chronic inflammation and apoptosis resistance. While H. pylori infection-induced expression of pro-inflammatory cytokines for chronic inflammation is well studied, the molecular mechanism underlying the apoptosis resistance in infected cells is not well understood. In this study, we demonstrated that H. pylori infection induced apoptosis resistance in gastric epithelial cells triggered by Raptinal, a drug that directly activates caspase-3. This resistance resulted from the induction of cIAP2 (encoded by BIRC3) since depletion of BIRC3 by siRNA or inhibition of cIAP2 via BV6 reversed H. pylori-suppressed caspase-3 activation. The induction of cIAP2 was regulated by H. pylori induced BIRC3 eRNA synthesis. Depletion of BIRC3 eRNA decreased H. pylori-induced cIAP2 and reversed H. pylori suppressed caspase-3 activation. Mechanistically, H. pylori stimulated the recruitment of bromodomain-containing factor Brd4 to the enhancer of BIRC3 and promoted BIRC3 eRNA and mRNA synthesis. Inhibition of Brd4 diminished the expression of BIRC3 eRNA and the anti-apoptotic response to H. pylori infection. Importantly, H. pylori isogenic cagA deficient mutant failed to activate the synthesis of BIRC3 eRNA and the associated apoptosis resistance. Finally, in primary human gastric epithelial cells, H. pylori also induced resistance to Raptinal-triggered caspase-3 activation by activating the Brd4-dependent BIRC3 eRNA synthesis in a CagA-dependent manner. These results identify a novel function of Brd4 in H. pylori-mediated apoptosis resistance via activating BIRC3 eRNA synthesis, suggesting that Brd4 could be a potential therapeutic target for H. pylori-induced gastric cancer.

H. pylori infection confers resistance to apoptosis via Brd4-dependent BIRC3 eRNA synthesis

H. pylori infection is one of the leading causes of gastric cancer and the pathogenicity of H. pylori infection is associated with its ability to induce chronic inflammation and apoptosis resistance. While H. pylori infection-induced expression of pro-inflammatory cytokines for chronic inflammation is well studied, the molecular mechanism underlying the apoptosis resistance in infected cells is not well understood. In this study, we demonstrated that H. pylori infection-induced apoptosis resistance in gastric epithelial cells triggered by Raptinal, a drug that directly activates caspase-3. This resistance resulted from the induction of cIAP2 (encoded by BIRC3) since depletion of BIRC3 by siRNA or inhibition of cIAP2 via BV6 reversed H. pylori-suppressed caspase-3 activation. The induction of cIAP2 was regulated by H. pylori-induced BIRC3 eRNA synthesis. Depletion of BIRC3 eRNA decreased H. pylori-induced cIAP2 and reversed H. pylori-suppressed caspase-3 activation. Mechanistically, H. pylori stimulated the recruitment of bromodomain-containing factor Brd4 to the enhancer of BIRC3 and promoted BIRC3 eRNA and mRNA synthesis. Inhibition of Brd4 diminished the expression of BIRC3 eRNA and the anti-apoptotic response to H. pylori infection. Importantly, H. pylori isogenic cagA-deficient mutant failed to activate the synthesis of BIRC3 eRNA and the associated apoptosis resistance. Finally, in primary human gastric epithelial cells, H. pylori also induced resistance to Raptinal-triggered caspase-3 activation by activating the Brd4-dependent BIRC3 eRNA synthesis in a CagA-dependent manner. These results identify a novel function of Brd4 in H. pylori-mediated apoptosis resistance via activating BIRC3 eRNA synthesis, suggesting that Brd4 could be a potential therapeutic target for H. pylori-induced gastric cancer.

Inhibition of gastric cancer cell growth by a PI3K-mTOR dual inhibitor GSK1059615

Gastric cancer (GC) is a common malignancy. Developing novel and efficient anti-GC agents is urgent. GSK1059615 is a PI3K (phosphatidylinositol 3-kinase) and mTOR (mammalian target of rapamycin) dual inhibitor. It activity in human GC cells is tested here. In AGS cells and primary human GC cells, GSK1059615 potently inhibited cell growth, survival, proliferation and cell cycle progression. Further, significant apoptosis activation was detected in GSK1059615-treated GC cells. Contrarily in the primary human gastric epithelial cells, GSK1059615 failed to induce significant cytotoxicity and apoptosis. GSK1059615 blocked PI3K-AKT-mTOR cascade activation, inducing microRNA-9 downregulation but LMX1A (LIM homeobox transcription factor 1α) upregulation in GC cells. Significantly, GSK1059615 administration (i.p., daily, at 10 or 30 mg/kg) in nude mice potently inhibited subcutaneous AGS xenograft growth. AKT-mTOR inhibition and LMX1A upregulation were detected in AGS xenograft tissues with GSK1059615 administration. Together, we conclude that GSK1059615 inhibits GC cell growth in vitro and in vivo.

AntogomiR-451 protects human gastric epithelial cells from ethanol via activating AMPK signaling

The prevention and treatment efficiency of ethanol-induced gastric epithelial injury are not satisfied. We have previously shown that AMP-activated protein kinase (AMPK) activation exerts a pro-survival function in human gastric epithelial cells (GECs). miroRNA-451 (“miR-451”)’s inhibitor, antagomiR-451, can activate AMPK signaling. In the present study, we show that forced-expression of antagomiR-451 via a lentiviral vector depleted miR-451, leading to AMPK activation in established GES-1 cells and primary human GECs. AntagomiR-451 efficiently protected GES-1 cells and primary human GECs from ethanol-induced viability reduction and apoptosis. AMPK activation is required for antagomiR-451-induced GEC protection. AMPKα1 knockdown (by targeted-shRNAs) or knockout (by CRISPR-Cas-9 KO plasmid) blocked antagomiR-451-induced AMPK activation, and GEC protection against ethanol. Further experimental results show that antagomiR-451 significantly attenuated ethanol-induced reactive oxygen species (ROS) production, lipid peroxidation and DNA damage. Collectively, antagomiR-451 protects human GECs from ethanol via activating AMPK signaling.

Characterization of Gastrospheres Using 3D Coculture System.

To understand the molecular mechanisms involved in gastric disorders and regeneration, we need an in vitro tridimensional (3D) culture model, which can mimic the in vivo gastric microenvironment. A 3D coculture system named gastrosphere is proposed herein, composed of primary human gastric epithelial and stromal cells. The primary cultures were obtained from endoscopic gastric biopsies, and after mechanical and enzymatic dispersion, epithelial (HGE3) and stromal (HGS12) cells were expanded. After extensive immunocytochemical characterization, cells were seeded onto 96-well round bottom plates previously covered with 1% agarose. Cells were cultured in KM-F12 culture medium with 10% fetal bovine serum (FBS), antibiotics, and antimycotics, in humidified air at 37°C and atmosphere containing 5% CO2 for 72h or until spheres formation. Then gastrospheres were carefully transferred to a rotary cell culture system (RCCS-4), and maintained for 07, 14, 21, and 28days. Gastrospheres were morphologically characterized by immunocytochemistry [cytokeratins (CK), vimentin, α-smooth muscle actin (α-SMA), laminin (LN), fibronectin (FN), and type IV collagen (CIV), proliferating cell nuclear antigen (PCNA)], and electron microscopy. In gastrospheres, the cytokeratin-positive epithelial cells were found in the outer layer, while vimentin-positive stromal cells were localized in the center of the gastrospheres. PCNA+ cells were mainly seen at the peripheral and in the intermediary region while nestin+ cells were also depicted in the latter zone. Scanning electron microscopy revealed groups of cohesive gastric cells at the periphery, while transmission electron microscopy demonstrated some differentiated mucous-like or zymogenic-like cells in the periphery and stromal structures located at the center of the 3D structures. Extracellular matrix was deposed between cells. Our data suggest that in vitro gastrospheres recapitulate the in vivo gastric microenvironment.

Inflammatory signaling pathways induced by Helicobacter pylori in primary human gastric epithelial cells.

Inflammatory signaling pathways induced by Helicobacter pylori remain unclear, having been studied mostly on cell-line models derived from gastric adenocarcinoma with potentially altered signaling pathways and nonfunctional receptors. Here, H. pylori-induced signaling pathways were investigated in primary human gastric epithelial cells. Inflammatory response was analyzed on chemokine mRNA expression and production after infection of gastric epithelial cells by H. pylori strains, B128 and B128Δ cagM, a cag type IV secretion system defective strain. Signaling pathway involvement was investigated using inhibitors of epidermal growth factor receptor (EGFR), MAPK, JAK and blocking Abs against TLR2 and TLR4. Inhibitors of EGFR, MAPK and JAK significantly reduced the chemokine mRNA expression and production induced by both H. pylori strains at 3 h and 24 h post-infection. JNK inhibitor reduced chemokine production at 24 h post-infection. Blocking Abs against TLR2 but not TLR4 showed significant reduction of chemokine secretion. Using primary culture of human gastric epithelial cells, our data suggest that H. pylori can be recognized by TLR2, leading to chemokine induction, and that EGFR, MAPK and the JAK/STAT signaling pathways play a key role in the H. pylori-induced CXCL1, CXCL5 and CXCL8 response in a cag pathogenicity island-independent manner.

AMP-activated protein kinase activation protects gastric epithelial cells from Helicobacter pylori-induced apoptosis

Helicobacter pylori (H pylori), infecting half of the world’s population, causes gastritis, duodenal and gastric ulcer, and gastric cancers. AMP-activated protein kinase (AMPK) is a highly conserved regulator of cellular energy and metabolism. Recent studies indicated an important role for AMPK in promoting cell survival. In this study, we discovered that H Pylori induced AMPK activation in transformed (GEC-1 line) and primary human gastric epithelial cells (GECs). Inhibition of H Pylori-stimulated AMPK kinase activity by AMPK inhibitor compound C exacerbated apoptosis in transformed and primary GECs. Meanwhile, downregulation of AMPK expression by targeted shRNAs promoted apoptosis in H pylori-infected GECs. In contrast, A-769662 and resveratrol, two known AMPK activators, or AMPKα1 over-expression, enhanced H Pylori-induced AMPK activation, and inhibited GEC apoptosis. Our data suggested that transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) could be the upstream kinase for AMPK activation by H pylori. Partial depletion of TAK1 by shRNAs not only inhibited AMPK activation, but also suppressed survival of H pylori-infected GECs. Taken together, these results suggest that TAK1-dependent AMPK activation protects GECs from H pylori-Induced apoptosis.

Gastric epithelial cells drive retinoic acid biosynthesis in human gastric dendritic cells (MUC8P.803)

Abstract Despite the high prevalence of chronic gastritis caused by H. pylori, the gastric mucosa as a unique immune environment has received little investigative attention. Here, we analyzed whether retinoic acid (RA), an important homeostatic factor in small intestine, also contributes to gastric immune regulation. Using HPLC, we detected both RA and the RA precursor molecule retinol in human gastric tissue, indicating that the gastric mucosa is an active site of RA biosynthesis. To determine the source of RA in gastric mucosa, we show that primary human gastric epithelial cells expressed RA biosynthesis genes Rdh10 and Aldh1a1, surprisingly at higher levels than small intestinal epithelial cells. Moreover, primary gastric epithelial cell cultures provided with retinol synthesized RA in vitro and induced the expression of the RA biosynthesis gene Aldh1a2 in co-cultured antigen-presenting cells through an RA-dependent mechanism, evidence that gastric epithelial cells confer the ability to generate RA to gastric DCs. Indeed, DCs purified from gastric mucosa had similar levels of aldehyde dehydrogenase activity and RA biosynthesis gene expression as small intestinal DCs, although gastric DCs lacked CD103. In summary, gastric epithelial cells have access to retinol, convert retinol to RA and drive RA biosynthesis in gastric DCs. Such RA biosynthesis capacity may enable gastric DCs to induce regulatory T cells and drive mucosal T cell homing during H. pylori infection.

Chemokines and antimicrobial peptides have a cag-dependent early response to Helicobacter pylori infection in primary human gastric epithelial cells.

Helicobacter pylori infection systematically causes chronic gastric inflammation that can persist asymptomatically or evolve toward more severe gastroduodenal pathologies, such as ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. The cag pathogenicity island (cag PAI) of H. pylori allows translocation of the virulence protein CagA and fragments of peptidoglycan into host cells, thereby inducing production of chemokines, cytokines, and antimicrobial peptides. In order to characterize the inflammatory response to H. pylori, a new experimental protocol for isolating and culturing primary human gastric epithelial cells was established using pieces of stomach from patients who had undergone sleeve gastrectomy. Isolated cells expressed markers indicating that they were mucin-secreting epithelial cells. Challenge of primary epithelial cells with H. pylori B128 underscored early dose-dependent induction of expression of mRNAs of the inflammatory mediators CXCL1 to -3, CXCL5, CXCL8, CCL20, BD2, and tumor necrosis factor alpha (TNF-α). In AGS cells, significant expression of only CXCL5 and CXCL8 was observed following infection, suggesting that these cells were less reactive than primary epithelial cells. Infection of both cellular models with H. pylori B128ΔcagM, a cag PAI mutant, resulted in weak inflammatory-mediator mRNA induction. At 24 h after infection of primary epithelial cells with H. pylori, inflammatory-mediator production was largely due to cag PAI substrate-independent virulence factors. Thus, H. pylori cag PAI substrate appears to be involved in eliciting an epithelial response during the early phases of infection. Afterwards, other virulence factors of the bacterium take over in development of the inflammatory response. Using a relevant cellular model, this study provides new information on the modulation of inflammation during H. pylori infection.

Helicobacter pylori Infection Inhibits Phagocyte Clearance of Apoptotic Gastric Epithelial Cells

Increased apoptotic death of gastric epithelial cells is a hallmark of Helicobacter pylori infection, and altered epithelial cell turnover is an important contributor to gastric carcinogenesis. To address the fate of apoptotic gastric epithelial cells and their role in H. pylori mucosal disease, we investigated phagocyte clearance of apoptotic gastric epithelial cells in H. pylori infection. Human gastric mononuclear phagocytes were analyzed for their ability to take up apoptotic epithelial cells (AECs) in vivo using immunofluorescence analysis. We then used primary human gastric epithelial cells induced to undergo apoptosis by exposure to live H. pylori to study apoptotic cell uptake by autologous monocyte-derived macrophages. We show that HLA-DR(+) mononuclear phagocytes in human gastric mucosa contain cytokeratin-positive and TUNEL-positive AEC material, indicating that gastric phagocytes are involved in AEC clearance. We further show that H. pylori both increased apoptosis in primary gastric epithelial cells and decreased phagocytosis of the AECs by autologous monocyte-derived macrophages. Reduced macrophage clearance of apoptotic cells was mediated in part by H. pylori-induced macrophage TNF-α, which was expressed at higher levels in H. pylori-infected, compared with uninfected, gastric mucosa. Importantly, we show that H. pylori-infected gastric mucosa contained significantly higher numbers of AECs and higher levels of nonphagocytosed TUNEL-positive apoptotic material, consistent with a defect in apoptotic cell clearance. Thus, as shown in other autoimmune and chronic inflammatory diseases, insufficient phagocyte clearance may contribute to the chronic and self-perpetuating inflammation in human H. pylori infection.

Increased programmed death-ligand-1 expression in human gastric epithelial cells inHelicobacter pyloriinfection

B7-H1 [programmed death-ligand-1 (PD-L1)] is a B7-family member that binds to programmed death-1 (PD-1). Recently, deficiency of PD-L1 has been demonstrated to result in accelerated gastric epithelial cell damage in gastritis, and PD-L1 is suggested to play a critical role in regulating T cell homeostasis. Here, we aimed to gain more insight into gastric PD-L1 expression, regulation and function during Helicobacter pylori infection. PD-L1 expression in human gastric epithelial cells was analysed using Western blotting, quantitative polymerase chain reaction and fluorescence activated cell sorter analysis. Furthermore, co-culture experiments of human gastric epithelial cells with primary human T cells or Jurkat T cells were conducted. PD-L1 expression in primary human gastric epithelial cells was strongly enhanced by H. pylori infection and activated T cells, and augmented markedly by further stimulation with interferon-γ or tumour necrosis factor-α. Moreover, PD-L1 expression in gastric epithelial cells significantly induced apoptosis of T cells. Our results indicate that a novel bidirectional interaction between human gastric epithelial cells and lymphocytes modulates PD-L1 expression in human gastric epithelial cells, contributing to the unique immunological properties of the stomach.

Increased expression of the urokinase plasminogen activator system by Helicobacter pylori in gastric epithelial cells

The gastric pathogen Helicobacter pylori (H. pylori) is linked to peptic ulcer and gastric cancer, but the relevant pathophysiological mechanisms are unclear. We now report that H. pylori stimulates the expression of plasminogen activator inhibitor (PAI)-1, urokinase plasminogen activator (uPA), and its receptor (uPAR) in gastric epithelial cells and the consequences for epithelial cell proliferation. Real-time PCR of biopsies from gastric corpus, but not antrum, showed significantly increased PAI-1, uPA, and uPAR in H. pylori-positive patients. Transfection of primary human gastric epithelial cells with uPA, PAI-1, or uPAR promoters in luciferase reporter constructs revealed expression of all three in H+/K+ATPase- and vesicular monoamine transporter 2-expressing cells; uPA was also expressed in pepsinogen- and uPAR-containing trefoil peptide-1-expressing cells. In each case expression was increased in response to H. pylori and for uPA, but not PAI-1 or uPAR, required the virulence factor CagE. H. pylori also stimulated soluble and cell surface-bound uPA activity, and both were further increased by PAI-1 knockdown, consistent with PAI-1 inhibition of endogenous uPA. H. pylori stimulated epithelial cell proliferation, which was inhibited by uPA immunoneutralization and uPAR knockdown; exogenous uPA also stimulated proliferation that was further increased after PAI-1 knockdown. The proliferative effects of uPA were inhibited by immunoneutralization of the EGF receptor and of heparin-binding EGF (HB-EGF) by the mutant diphtheria toxin CRM197 and an EGF receptor tyrosine kinase inhibitor. H. pylori induction of uPA therefore leads to epithelial proliferation through activation of HB-EGF and is normally inhibited by concomitant induction of PAI-1; treatments directed at inhibition of uPA may slow the progression to gastric cancer.

Helicobacter pylori alters the distribution of ZO-1 and p120ctn in primary human gastric epithelial cells

Helicobacter pylori infection is related to the development of diverse gastric pathologies, possibly by affecting epithelial junctional complexes that define cell polarity and play an essential role in transepithelial transport and cell-cell adhesion. Using primary gastric epithelial cell cultures, effects of H. pylori on the expression and localization of tight/adherence junction proteins and the resulting morphological changes and migratory capabilities were studied under in vivo-like conditions. Gastric epithelial cells were isolated from biopsies or gastrectomies and maintained in Quantum286 on collagen I-coated culture dishes or cover-slips. Cell cultures were characterized and further analyzed by western blot and immunofluorescent staining for ZO-1, p120ctn, and H. pylori CagA. Morphological changes and migratory response were monitored by time-lapse digital image microscopy. ZO-1 and p120ctn protein expression levels remain unaffected by H. pylori infection. Immunocytochemistry on H. pylori-infected primary cell monolayers focally showed disruption of intercellular ZO-1 staining and accumulation of ZO-1 in small vesicles. H. pylori infection recruited non-phosphorylated p120ctn to perinuclear vesicles. The fraction of phosphorylated p120ctn increased and could be detected in the nucleus, at the cell membrane, and at the leading edge of migrating cells. These alterations, triggered by H. pylori infection, are associated with an elongation phenotype and increased migration.

Increased gastric expression of MMP-7 in hypergastrinemia and significance for epithelial-mesenchymal signaling

Chronic hypergastrinemia is associated with enterochromaffin-like (ECL) cell hyperplasia, which may progress to gastric carcinoid tumors. The latter consists of epithelial cells and stroma, and both compartments usually regress after normalization of hypergastrinemia. We previously showed that matrix metalloproteinase (MMP)-7 in gastric epithelial cells was upregulated by Helicobacter pylori and described MMP-7-dependent reciprocal signaling between the epithelium and a key stromal cell type, the myofibroblast. Here, we describe the regulation of gastric MMP-7 by gastrin and the potential significance for recruiting and maintaining myofibroblast populations. Biopsies of the gastric corpus and ECL cell carcinoid tumors were obtained from hypergastrinemic patients. Western blot analysis, ELISA, immunohistochemistry, and promoter-luciferase (luc) reporter assays were used to study MMP-7 expression. Gastric myofibroblasts were identified by alpha-smooth muscle actin (alpha-SMA) expression, and the effects of MMP-7 on myofibroblast proliferation were investigated. In hypergastrinemic patients, there was an increased abundance of MMP-7 and alpha-SMA in gastric corpus biopsies and ECL cell carcinoid tumors. In the latter, MMP-7 was localized to ECL cells but not stromal cells, which were nevertheless well represented. Gastrin stimulated MMP-7-luc expression in both AGS-G(R) and primary human gastric epithelial cells. Conditioned medium from gastrin-treated human gastric glands stimulated myofibroblast proliferation, which was inhibited by neutralizing antibodies to MMP-7. MMP-7 increased the proliferation of myofibroblasts via the MAPK and phosphatidylinositol 3-kinase (PI3K) pathways. In conclusion, stimulation of gastric MMP-7 by elevated plasma gastrin may activate epithelial-mesenchymal signaling pathways regulating myofibroblast function via MAPK and PI3K pathways and contribute to stromal deposition in ECL cell carcinoid tumors.

Helicobacter pylori enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in human gastric epithelial cells.

To investigate the relations between tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Helicobacter pylori (H pylori) infection in apoptosis of gastric epithelial cells and to assess the expression of TRAIL on the surface of infiltrating T-cells in H pylori-infected gastric mucosa. Human gastric epithelial cell lines and primary gastric epithelial cells were co-cultured with H pylori in vitro, then recombinant TRAIL proteins were added to the culture. Apoptosis of gastric epithelial cells was determined by a specific ELISA for cell death. Infiltrating lymphocytes were isolated from H pylori-infected gastric mucosa, and expression of TRAIL in T cells was analyzed by flow cytometry. The apoptosis of gastric epithelial cell lines and primary human gastric epithelial cells was mildly increased by interaction with either TRAIL or H pylori alone. Interestingly, the apoptotic indices were markedly elevated when gastric epithelial cells were incubated with both TRAIL and H pylori (Control vs TRAIL and H pylori: 0.51+/-0.06 vs 2.29+/-0.27, P = 0.018). A soluble TRAIL receptor (DR4-Fc) could specifically block the TRAIL-mediated apoptosis. Further studies demonstrated that infiltrating T-cells in gastric mucosa expressed TRAIL on their surfaces, and the induction of TRAIL sensitivity by H pylori was dependent upon direct cell contact of viable bacteria, but not CagA and VacA of H pylori. H pylori can sensitize human gastric epithelial cells and enhance susceptibility to TRAIL-mediated apoptosis. Modulation of host cell sensitivity to apoptosis by bacterial interaction adds a new dimension to the immunopathogenesis of H pylori infection.

Adherence of isogenic flagellum-negative mutants of Helicobacter pylori and Helicobacter mustelae to human and ferret gastric epithelial cells.

Isogenic flagellum-negative mutants of Helicobacter pylori and Helicobacter mustelae were screened for their ability to adhere to primary human and ferret gastric epithelial cells, respectively. We also evaluated the adherence of an H. pylori strain with a mutation in the flbA gene, a homologue of the flbF/lcrD family of genes known to be involved in the regulation of H. pylori flagellar biosynthesis. H. pylori and H. mustelae mutants deficient in production of FlaA or FlaB and mutants deficient in the production of both FlaA and FlaB showed no reduction in adherence to primary human or ferret gastric epithelial cells compared with the wild-type parental strains. However, adherence of the H. pylori flbA mutant to human gastric cells was significantly reduced compared to the adherence of the wild-type strain. These results show that flagella do not play a direct role in promoting adherence of H. pylori or H. mustelae to gastric epithelial cells. However, genes involved in the regulation of H. pylori flagellar biosynthesis may also regulate the production of an adhesin.

[The role of heat shock protein 60 (HSP60) of Helicobacter pylori in adhesion of H. pylori to human gastric epithelial cell].

Adhesion of Helicobacter pylori to both human gastric carcinoma cell lines (MKN45, MKN28 and KATO III) and prepared primary human gastric epithelial cells were analyzed with flow-cytometry. All strains adhered to human gastric carcinoma cells. Especially, these strains strongly adhered to MKN45 cells. Adhesion of H. pylori strains to prepared primary human gastric epithelial cells was also observed. However, the adherence rates of H. pylori to these cells were different among the cells used. These results suggested that the host factor might be important for adhesion of the bacteria to human gasgric cells. In addition, H20 monoclonal antibody directed to H. pylori HSP60 inhibited the adhesion of H. pylori to both cells. These results indicate that H. pylori HSP60 might be associated with the adhesion of the bacteria to human gastric epithelial cells.

Heat-shock protein 60 homologue of Helicobacter pylori is associated with adhesion of H. pylori to human gastric epithelial cells.

A previous study reported a relationship between the expression of heat-shock protein 60 (HSP60) by Helicobacter pylori and its adhesion to human gastric carcinoma (MKN45) cells. To examine whether the HSP60 homologue of H. pylori is associated with the adhesion of H. pylori to human gastric epithelial cells, an inhibition assay of adhesion of H. pylori to MKN45 cells was performed by flow cytometric analysis with monoclonal antibody (MAb) designated as H20 recognising HSP60 of H. pylori. The rate of adhesion of H. pylori pretreated with MAbH20 to MKN45 cells was lower than that of untreated H. pylori. Primary human gastric epithelial cells from a patient with gastric cancer were also prepared for comparison in the inhibition assay with MAbH20. H. pylori adhered to the primary human gastric epithelial cells, and this adhesion was significantly inhibited by MAbH20. These results suggest that the H. pylori HSP60 homologue recognised by MAbH20 might be associated with the adhesion of H. pylori to primary human gastric epithelial cells as well as to cultured gastric cancer cells.

Effects of Helicobacter pylori vacuolating cytotoxin on primary cultures of human gastric epithelial cells.

Many Helicobacter pylori strains produce a cytotoxin that induces cytoplasmic vacuolation in various types of eukaryotic cells. In contrast with the marked cell vacuolation that occurs in vitro in response to this cytotoxin, comparatively little epithelial vacuolation has been observed in the gastric mucosa of H pylori infected persons. Experiments were performed to determine the susceptibility of human gastric epithelial cells in vitro to H pylori vacuolating cytotoxin activity. Human gastric epithelial cells, harvested from upper gastrointestinal endoscopic biopsy specimens, were incubated overnight with broth culture supernatants from either a wild type cytotoxin producing (tox+) H pylori strain or an isogenic mutant strain that lacks cytotoxin activity. Prominent cytoplasmic vacuolation occurred in response to tox+ supernatant, but not supernatant from the isogenic mutant strain. Primary human gastric epithelial cells were significantly more sensitive to H pylori vacuolating cytotoxin activity than were either HeLa or AGS cells. Exposure of human gastric epithelial cells to high concentrations of tox+ supernatant for 48 hours caused lethal cell injury. These studies indicate that primary human gastric epithelial cells are highly sensitive to H pylori vacuolating cytotoxin activity.