Yoshito Nishi

Impact of Helicobacter pylori infection on gastric and plasma ghrelin dynamics in humans.

OBJECTIVES:There are contradictory reports on the relationship between Helicobacter pylori and circulating ghrelin. We sought to clarify the influence of H. pylori infection on gastric and plasma ghrelin dynamics in humans.METHODS:Using endoscopic biopsies from the corpus of 56 H. pylori-infected patients and 25 uninfected subjects, ghrelin mRNA expression levels and gastric ghrelin peptide contents were measured by real-time polymerase chain reaction and radioimmunoassay, respectively. We also measured plasma ghrelin concentrations and analyzed the numbers of ghrelin immunoreactive cells in the fundic gland area. Fifty-one patients with H. pylori infection were treated with a 7-day triple therapy consisting of lansoprazole, clarithromycin, and amoxicillin.RESULTS:The gastric ghrelin mRNA expression level of H. pylori-positive patients (1.64 ± 1.27 in arbitrary units) was significantly lower than in H. pylori-negative subjects (4.87 ± 4.1, p < 0.0001). A similar trend was noted for ghrelin peptide contents (31.2 ± 27.5 vs 81.2 ± 64.1 ng/mg protein, respectively, p < 0.0001). There was no significant difference in the number of ghrelin immunoreactive cells/mm2 in terms of H. plyori status. Plasma ghrelin concentrations in H. pylori-infected patients (144.6 ± 7.8.8 fmol/ml) were significantly lower than in uninfected subjects (196.1 ± 97.2, p < 0.05) and increased following cure of the infection. Plasma ghrelin levels correlated positively with the expression levels of ghrelin mRNA (r = 0.583, p < 0.0001) and peptide products (r = 0.574, p < 0.0001). There was a significant stepwise decrease in gastric ghrelin mRNA expression (p < 0.05), peptide contents (p < 0.01) and density of ghrelin immunoreactive cells (p < 0.05) with progression of histological severity of glandular atrophy in the corpus. The histological severity of chronic inflammation also negatively influenced the ghrelin mRNA expression (p < 0.001) and peptide production (p < 0.005).CONCLUSIONS:H. pylori infection has a negative impact on gastric and plasma ghrelin dynamics. Chronic inflammatory and atrophic changes associated with the infection may affect gastric ghrelin biosynthesis and contribute to the low circulating levels.

Enhanced Expression of Interleukin-8 and Activation of Nuclear Factor Kappa-B in Endoscopy-negative Gastroesophageal Reflux Disease

OBJECTIVE:Interleukin-8 (IL-8) mediates neutrophil trafficking via its receptors. Recent studies have shown that IL-8 is likely involved in the development and progression of erosive reflux esophagitis (RE), yet little is known about its implication in endoscopy-negative gastroesophageal reflux disease (GERD). The purpose of this study was to determine IL-8 messenger ribonucleic acid (mRNA) expression levels in endoscopy-negative GERD, along with assessment of nuclear factor kappaB (NF-κB) activation, which upregulates IL-8 expression.METHODS:We studied 31 patients with endoscopy-negative GERD, 15 patients with erosive RE, and 15 asymptomatic controls. Paired biopsy samples were taken from the esophagus 3 cm above the gastroesophageal junction; one biopsy was snap-frozen for measurement of IL-8 mRNA levels by real-time quantitative polymerase chain reaction, and another was formalin-fixed for histopathological evaluation. In nine endoscopy-negative GERD patients, the IL-8 mRNA expression levels were measured before and 8 wk after treatment with lansoprazole. We also sampled additional specimens for NF-κB-DNA binding assay and immunohistochemical analyses of NF-κB p65 and p50 subunits, IL-8 and specific IL-8 receptor, CXCR-1.RESULTS:The relative IL-8 mRNA expression levels were significantly higher in esophageal mucosa of patients with endoscopy-negative GERD than those of the controls. The presence of basal zone hyperplasia and intraepithelial neutrophils, histopathological hallmarks of GERD, were associated with higher levels of IL-8 mRNA. Lansoprazole treatment significantly reduced the IL-8 mRNA expression levels. The esophageal epithelium of patients with GERD showed intense immunoreactivity for IL-8, and expressed CXCR-1 antigen. We found NF-κB activation in esophageal mucosa in GERD patients and the NF-κB subunits were localized predominantly in the nuclei of IL-8-expressing cells.CONCLUSIONS:Our results demonstrate enhanced mucosal expression of IL-8 in incipient GERD even without mucosal breaks. NF-κB activation may be implicated in the pathogenesis in GERD.

Circulating ghrelin levels in patients with various upper gastrointestinal diseases.

The stomach is the main source of circulating ghrelin. Plasma concentrations of this hormone in patients with various upper gastrointestinal diseases remain undetermined. Thus we measured plasma ghrelin levels by radioimmunoassay in 225 subjects, including 134 Helicobacter pylori-infected and 91 uninfected subjects. They included 67 patients with chronic gastritis (CG), 26 with benign gastric polyp (BGP), 24 with gastric ulcer (GU), 24 with reflux esophagitis (RE), 18 with duodenal ulcer (DU), 28 with acute gastritis (AG), 23 with gastric cancer (GC), and 39 who had normal mucosa on upper endoscopy (N). Plasma pepsinogen I and II levels were also measured. The extent of gastritis was assessed endoscopically. Ghrelin levels differed significantly among the different disease groups. Plasma ghrelin concentrations were lowest in the CG group, followed by the GU group, and highest in the AG patients. There was a significant difference in the levels between differentiated and undifferentiated GC. Ghrelin concentrations in BGP, RE, and DU patients were comparable to those in the N group. Ghrelin circulating levels were lower in H. pylori-positive than –negative individuals, but the significant differences among disease groups were still observed in H. pylori-infected and uninfected populations. Ghrelin concentrations correlated positively with plasma pepsinogen I levels and I/II ratios and inversely with the extent of H. pylori-related gastritis. Plasma ghrelin levels varied widely in diverse conditions of the upper digestive tract, reflecting the inflammatory and atrophic events of the background gastric mucosa. Further investigation is warranted to unravel the mechanisms of the high circulating ghrelin levels in certain upper gastrointestinal diseases.

Clustering of Helicobacter pylori VacA in Lipid Rafts, Mediated by Its Receptor, Receptor-Like Protein Tyrosine Phosphatase β, Is Required for Intoxication in AZ-521 Cells

ABSTRACT Helicobacter pylori vacuolating cytotoxin, VacA, induces multiple effects on epithelial cells through different cellular events: one involves pore formation, leading to vacuolation, mitochondrial damage, and apoptosis, and the second involves cell signaling, resulting in stimulation of proinflammatory responses and cell detachment. Our recent data demonstrated that VacA uses receptor-like protein tyrosine phosphatase β (RPTPβ) as a receptor, of which five residues (QTTQP) at positions 747 to 751 are involved in binding. In AZ-521 cells, which mainly express RPTPβ, VacA, after binding to RPTPβ in non-lipid raft microdomains on the cell surface, is localized with RPTPβ in lipid rafts in a temperature- and VacA concentration-dependent process. Methyl-β-cyclodextrin (MCD) did not block binding to RPTPβ but inhibited translocation of VacA with RPTPβ to lipid rafts and all subsequent events. On the other hand, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), which disrupts anion channels, did not inhibit translocation of VacA to lipid rafts or VacA-induced activation of p38 mitogen-activated protein (MAP) kinase, but inhibited VacA internalization followed by vacuolation. Thus, p38 MAP kinase activation did not appear to be required for internalization. In contrast, phosphatidylinositol-specific phospholipase C (PI-PLC) inhibited translocation, as well as p38 MAP kinase/ATF-2 activation, internalization, and VacA-induced vacuolation. Neither NPPB nor PI-PLC affected VacA binding to cells and to its receptor, RPTPβ. Thus, receptor-dependent translocation of VacA to lipid rafts is critical for signaling pathways leading to p38 MAP kinase/ATF-2 activation and vacuolation.

Gangliosides Act as Co-receptors for Salmonella enteritidis FliC and Promote FliC Induction of Human β-Defensin-2 Expression in Caco-2 Cells

Antimicrobial peptides such as defensins are crucial for host defense at mucosal surfaces. We reported previously that Salmonella enteritidis flagellin (FliC) induced human β-defensin-2 (hBD-2) mRNA expression in Caco-2 cells via NF-κB activation (Ogushi, K., Wada, A., Niidome, T., Mori, N., Oishi, K., Nagatake, T., Takahashi, A., Asakura, H., Makino, S., Hojo, H., Nakahara, Y., Ohsaki, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Moss, J., and Hirayama, T. (2001) J. Biol. Chem. 276, 30521–30526). In this study, we examined the role of ganglioside as co-receptors with Toll-like receptor 5 (TLR5) on FliC induction of hBD-2 expression in Caco-2 cells. Exogenous gangliosides suppressed FliC induction of hBD-2 promoter activity and binding of FliC to Caco-2 cells. Incorporation of exogenous ganglioside GD1a into Caco-2 cell membranes increased the effect of FliC on hBD-2 promoter activity. In support of a role for endogenous gangliosides, incubation of Caco-2 cells with dl-threo-2-hexadecanoylamino-3-morpholino-1-phenylpropanol, a glucosylceramide synthase inhibitor, reduced FliC induction of hBD-2 promoter activity. GD1a-loaded CHO-K1-expressing TLR5 cells had a higher potential for hBD-2 induction following FliC stimulation than GD1a-loaded CHO-K1 cells not expressing TLR5. FliC increased phosphorylation of mitogen-activated protein kinase, p38, and ERK1/2. Exogenous gangliosides GD1a, GD1b, and GT1b each suppressed FliC induction of p38 and ERK1/2 phosphorylation. Furthermore, FliC did not enhance luciferase activity in Caco-2 cells transfected with a plasmid containing a mutated activator protein 1-binding site. These results suggest that gangliosides act as co-receptors with TLR5 for FliC and promote hBD-2 expression via mitogen-activated protein kinase.

Pleiotropic Effects of Proton Pump Inhibitors Guest Editor: Yuji Naito Immune and Inflammatory Responses in GERD and Lansoprazole

The exact pathophysiological mechanisms responsible for gastroesophageal reflux disease (GERD) remain unclear. Recent studies have shown that mucosal immune and inflammatory responses, characterized by specific cytokine and chemokine profiles, may underlie the diverse esophageal phenotypes of GERD. Interleukin 8 (IL-8), a representative chemokine, mediates neutrophil trafficking via its receptors, mainly CXCR-1. The IL-8 mRNA and protein levels are increased in the esophageal mucosa, not only in reflux esophagitis (RE), but also in endoscopy-negative GERD (NERD), through activation of nuclear factor-κB (NF-κB), which is a pivotal transcription factor. Mucosal IL-8 concentrations have been found to parallel the endoscopic severity of RE, implying that this cytokine is a key player in the development of GERD. The mucosal levels of the C-C chemokines, macrophage chemoattractant protein 1 (MCP-1) and regulated on activation normal T-cell-expressed and presumably secreted (RANTES), which primarily attract monocytes and lymphocytes to the site of inflammation, respectively, are also elevated in RE. The secreted levels of IL-8 and IL-1β, a prototype of proinflammatory cytokine, are maximal at the proximal segment within Barrett esophagus (BE) tissue. The expression of the two pleiotrophic proinflammatory cytokines, IL-6 and tumor necrosis factor α, is enhanced in the intestinal epithelium of BE, which places this epithelium at a higher risk for developing malignancy. BE is characterized by a distinct Th-2 predominant cytokine profile (IL-4 and -10), compared to the proinflammatory nature of RE (interferone-γ). Treatment with a proton pump inhibitor, lansoprazole reduces the mucosal levels of IL-8 mRNA and protein in GERD, including RE and NERD. This may occur in part through an anti-inflammatory action of proton pump inhibitors beyond gastric acid inhibition.

Essential domain of receptor tyrosine phosphatase β (RPTPβ) for interaction with Helicobacter pylori vacuolating cytotoxin

Helicobacter pylori produces a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Although VacA was able to interact with two receptor-like protein tyrosine phosphatases, RPTPβ and RPTPα, RPTPβ was found to be responsible for gastric damage caused by VacA. To define the region of RPTPβ involved in VacA binding, we made mutants of human cDNA RPTPβ-B, a short receptor form of RPTPβ. Immunoprecipitation experiments to assess VacA binding to RPTPβ-B mutants indicated that five residues (QTTQP) at positions 747–751 of the extracellular domain of RPTPβ-B (which is commonly retained in RPTPβ-A, a long form of RPTPβ) play a crucial role in its interaction with VacA, resulting in vacuolation as well as Git-1 phosphorylation. Transfected cells expressing deletion mutant Δ752, which lacks QTTQP, or the double point mutant Δ747 (T748A,T749A) had diminished vacuolation in response to VacA. Treatment of RPTPβ-B and Δ747 (which have QTTQP at 747–751) with neuraminidase and O-glycosidase diminished their VacA binding, whereas chondroitinase ABC did not have an effect. No inhibitory effect of pleiotrophin, a natural RPTPβ ligand, on VacA binding to RPTPβ-B or Δ747 was observed, supporting the conclusion that the extracellular region of RPTPβ-B responsible for VacA binding is different from that involved in binding pleiotrophin. These data define the region in the RPTPβ extracellular domain critical for VacA binding, in particular the sequence QTTQP at positions 747–751 with crucial threonines at positions 748 and 749 and are consistent with a role for terminal sialic acids possibly because of threonine glycosylation.

Helicobacter pylori VacA Reduces the Cellular Expression of STAT3 and Pro-survival Bcl-2 Family Proteins, Bcl-2 and Bcl-XL, Leading to Apoptosis in Gastric Epithelial Cells

BackgroundHelicobacter pylori vacuolating cytotoxin, VacA, stimulates apoptosis via a mitochondria-dependent pathway. VacA induces apoptosis via activation of the pro-apoptotic B-cell lymphoma (Bcl)-2 family proteins, Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), while the implication of such pro-survival Bcl-2 family members as Bcl-2 and Bcl-XL in the VacA-induced apoptosis remains unknown. Signal transduction and activator of transcription 3 (STAT3) is a pivotal transcription factor that upregulates Bcl-2 and Bcl-XL.AimsThis study was conducted to elicit the implication of STAT3 and pro-survival Bcl-2 and Bcl-XL in the intrinsic apoptosis.MethodsImmunoblot and reverse transcriptase real-time polymerase chain reaction (RT-PCR) were employed to assess the cellular expression of STAT3, Bcl-2, and Bcl-XL in response to purified VacA in gastric adenocarcinoma cell lines. VacA-induced apoptosis was quantitated morphologically following knockdown by each specific small interfering RNA (siRNA) or in the presence of pharmacological inhibitors.ResultsVacA reduced STAT3, Bcl-2, and Bcl-XL expression in a dose-dependent manner. Knockdown of STAT3, Bcl-2, and Bcl-XL by siRNA induced apoptosis to a similar extent in the case of sufficient VacA inoculation. The VacA-mediated reduction of STAT3 expression was independent of cellular vacuolization, since a vacuolar-type ATPase inhibitor, bafilomycin A1, did not inhibit VacA-induced reduction of STAT3, Bcl-2, and Bcl-XL expression. Instead, a c-JUN NH2-terminal kinase (JNK) inhibitor, SP600125, restored the VacA-induced reduction of STAT3 expression to the basal level.ConclusionsVacA-induced apoptosis may be, in part, implicated in the reduction of STAT3 linking to the downregulation of Bcl-2 and Bcl-XL, in association with JNK activity.

Elevated Concentrations of |[alpha]|-Defensins in Gastric Juice of Patients with Helicobacter pylori Infection

OBJECTIVE:Defensins (α- and β-defensins) are endogenous antimicrobial peptides. Little is known about α-defensins during Helicobacter pylori infection.METHODS:The concentrations of human neutrophil peptides (HNP-1, -2, and -3), the major components of neutrophils-derived α-defensins, were measured by radioimmunoassay (RIA) in plasma and gastric juice of 61 H. pylori-infected and 33 uninfected subjects, and before and after anti-H. pylori treatment in 12 patients with H. pylori-associated gastritis. Interleukin (IL)-8 concentrations in gastric juice were measured by enzyme-linked immunosorbent assay. Histological grades of gastritis and neutrophil counts (/mm2) infiltrating in the gastric mucosa were determined using two biopsy specimens taken from the antrum and corpus. Immunohistochemistry and reverse-phase high performance liquid chromatography (RP-HPLC) were used to identify HNPs 1–3.RESULTS:HNP 1–3 concentrations in gastric juice were significantly higher in H. pylori-positive than in H. pylori-negative patients and significantly decreased after cure. HNP 1–3 concentrations in gastric juice correlated with IL-8 levels and neutrophil densities in the gastric mucosa and were associated with histological degree of gastritis, especially the grades of activity. Intense immunoreactivity for anti-HNPs 1–3 antiserum was noted in infiltrating neutrophils in H. pylori-infected mucosa. In RP-HPLC analysis, all of the HNP 1–3 molecules were identified as their mature forms. Plasma HNP 1–3 concentrations were similar in H. pylori-infected and non-infected groups and showed no correlations with other parameters.CONCLUSIONS:We demonstrated significantly elevated levels of HNPs 1–3 in gastric juice during H. pylori infection. The elevation of HNPs is presumably secondary to H.pylori-associated gastric inflammation involving neutrophil infiltration.

Mucosal concentrations of proinflammatory cytokines and chemokines at gastric cardia: implication of Helicobacter pylori infection and gastroesophageal reflux.

OBJECTIVES:The pathogenesis of carditis remains unclear, although gastroesophageal reflux disease (GERD) and Helicobacter pylori infection have been proposed. Little is known about the profile of proinflammatory cytokines and chemokines in the pathogenesis of carditis.METHODS:We studied 28 patients with GERD and 40 controls. Two biopsy specimens were taken endoscopically from the cardiac mucosa within 5 mm from the squamocolumnar junction; one was snap frozen for measurement of mucosal levels of interleukin 1β (IL-1β), tumor necrosis factor-α, IL-6, IL-8, monocyte chemoattractant protein 1 (MCP-1), regulated on activation normal T-cell expressed and presumably secreted (RANTES) by enzyme-linked immunosorbent assays, while another was processed for histopathology. H. pylori infection was assessed by serology, rapid urease test, and histology with Giemsa staining. Samples were taken from the cardia of 18 H. pylori-positive patients, before and after eradication treatment.RESULTS:Carditis was significantly associated with H. pylori infection, but not GERD. IL-8, MCP-1, and RANTES levels were significantly higher in cardiac mucosa of patients with carditis than in those without it and in patients with than without H. pylori infection. IL-8 concentrations were significantly associated with the degree of neutrophil infiltration within the cardiac mucosa and decreased after cure of the infection. Mucosal MCP-1 and RANTES levels correlated positively with the grades of mononuclear cell infiltration and IL-1β concentrations.CONCLUSION:Our results indicate that chemokines produced locally in the cardiac mucosa may be involved in the development of H. pylori-associated carditis.