Tomoyuki Ohno

Antimicrobial Activity of Essential Oils against Helicobacter pylori

Background. Helicobacter pylori is an important pathogen responsible for gastroduodenal diseases in humans. Although the eradication of H. pylori using antibiotics often improves gastroduodenal diseases, resistance to the antibiotics is emerging.

Cytokine Expression and Production by Purified Helicobacter pylori Urease in Human Gastric Epithelial Cells

ABSTRACT Cytokines have been proposed to play an important role inHelicobacter pylori-associated gastroduodenal diseases, but the exact mechanism of the cytokine induction remains unclear. H. pylori urease, a major component of the soluble proteins extracted from bacterial cells, is considered to be one of the virulence factors for the inflammation in the gastric mucosa that is produced in H. pylori infection. However, the response of human gastric epithelial cells to the stimulation of urease has not been investigated. In the present study, we used human gastric epithelial cells in a primary culture system and examined whetherH. pylori urease stimulates the gastric epithelial cells to induce proinflammatory cytokines by reverse transcription-PCR and enzyme-linked immunosorbent assay. First, by using peripheral blood mononuclear cells (PBMC) and a gastric cancer cell line (MKN-45 cells), we confirmed the ability of purified H. pylori urease to induce the production of proinflammatory cytokines. Furthermore, we demonstrated that the human gastric epithelial cells produced interleukin-6 (IL-6) and tumor necrosis factor alpha, but not IL-8, following stimulation with purified urease. The patterns of cytokine induction differed among human PBMC, MKN-45 cells, and human gastric epithelial cells. These results suggest that the human gastric epithelial cells contribute to the induction of proinflammatory cytokines by the stimulation of H. pylori urease, indicating that the epithelial cells were involved in the mucosal inflammation that accompanied H. pylori infection.

Role of Deletion Located between the Intermediate and Middle Regions of the Helicobacter pylori vacA Gene in Cases of Gastroduodenal Diseases

ABSTRACT The vacuolating cytotoxin gene of Helicobacter pylori, vacA, induces cytoplasmic vacuolation in gastric epithelial cells. Recently, the vacA intermediate (i) region, which is located between the signal (s) and middle (m) regions, was identified as a third polymorphic determinant of vacA activity. In vacA, there are approximately 81-bp deletions between the vacA i and m regions (denoted the d region). The aim was to clarify the roles of the vacA d region in relation to H. pylori-related diseases and histopathological gastric mucosal changes. We assessed the vacA signal s-, m-, i-, and d-region genotypes and cagA status in H. pylori isolates recovered from Western countries (n = 266) and East Asian countries (n = 244) by PCR. In East Asian countries, there were no relationships between the vacA genotypes and the clinical outcomes and histopathological changes. In Western countries, strains with the vacA s1, m1, i1, or d1 (no deletion) genotype significantly increased the risk for the development of gastric cancer compared with the risk from strains with the s2, m2, i2, or d2 genotype (adjusted odd ratios, 3.17 [95% confidence interval {CI}, 1.07 to 9.45] for s1, 10.65 [95% CI, 3.36 to 31.35] for m1, 8.57 [95% CI, 2.85 to 25.81] for i1, and 8.04 [95% CI, 2.67 to 24.16] for d1). The highly virulent vacA genotypes significantly enhanced neutrophil infiltration and gastric atrophy in univariant analysis, whereas only the vacA d-region genotype was significantly associated with neutrophil infiltration and gastric atrophy in both the antrum and the corpus by multiple linear regression analysis. The presence of the vacA d1 genotype in H. pylori strains could be an improved predictor of histological inflammation and the potential for atrophy compared with the presence of the vacA s-, m-, and i-region genotypes in Western countries.

Gastric mucosal interleukin-17 and -18 mRNA expression in Helicobacter pylori-induced Mongolian gerbils

Helicobacter pylori infection causes characteristic mucosal infiltration of inflammatory cells, resulting in the development of peptic ulcers and gastric cancer in approximately 10% of cases. Different clinical expressions of the infection may reflect different patterns of cytokine expression. Interleukin (IL)‐1ß, tumor necrosis factor (TNF)‐α, IL‐17, and IL‐18 have been reported to be involved in H. pylori‐induced gastric mucosal inflammation, but the details and relation to different patterns of inflammation remain unclear. Moreover, the proinflammatory virulence factor outer inflammatory protein (OipA) was reported to be associated with gastric mucosal inflammatory cytokine levels. To clarify these findings, Mongolian gerbils were infected for up to 12 months with wild‐type H. pylori 7.13 or with isogenic oipA mutants for 3 months, and mucosal cytokines (IL‐1ß, IL‐17, IL‐18, and TNF‐α) mRNA levels were then assessed using real‐time RT‐PCR. Antral mucosal IL‐1β and IL‐18 mRNA levels peaked 1 month after infection, whereas the peak of TNF‐α mRNA was at 6–12 months; IL‐17 levels peaked at 12 months. The inflammatory cell infiltration and mRNA levels of all cytokines studied were significantly lower in oipA mutants than in wild‐type‐infected gerbils. Mucosal IL‐1ß, IL‐17, and TNF‐α expression, but not that of IL‐18, were significantly associated with the grade of inflammatory cell infiltration. The pattern of increased inflammatory cytokines differed relative to the phase of the infection and pattern of inflammation. OipA appears to play a role in IL‐1ß, IL‐17, and TNF‐α expression and the resulting inflammation. (Cancer Sci 2009)

Functional and Intracellular Signaling Differences Associated with the Helicobacter pylori AlpAB Adhesin from Western and East Asian Strains

Following adhesion of Helicobacter pylori to gastric epithelial cells, intracellular signaling leads to cytokine production, which causes H. pylori-related gastric injury. Two adjacent homologous genes (alpA and alpB), which encode H. pylori outer membrane proteins, are thought to be associated with adhesion and cytokine induction. We co-cultured gastric epithelial cells with wild type H. pylori strains and their corresponding alpA/alpB-deleted mutants (ΔalpAB). Results were confirmed by complementation. Flow cytometry confirmed that AlpAB was involved in cellular adhesion. Deletion of alpAB reduced interleukin (IL)-6 induction in gastric epithelial cells. Deletion of alpAB reduced IL-8 induction with East Asian but not with Western strains. All AlpAB-positive strains tested activated the extracellular signal-regulated kinase, c-Fos, and cAMP-responsive element-binding protein. Activation of the Jun-N-terminal kinase, c-Jun, and NF-κB was exclusive to AlpAB from East Asian strains. ΔalpAB mutants poorly colonized the stomachs of C57BL/6 mice and were associated with lower mucosal levels of KC and IL-6. Our results suggest that AlpAB may induce gastric injury by mediating adherence to gastric epithelial cells and by modulating proinflammatory intracellular signaling cascades. Known geographical differences in H. pylori-related clinical outcomes may relate to differential effects of East Asian and Western types of AlpAB on NF-κB-related proinflammatory signaling pathways.

Effects of Blood Group Antigen–Binding Adhesin Expression during Helicobacter pylori Infection of Mongolian Gerbils

Helicobacter pylori outer membrane proteins, such as the blood group antigen-binding adhesin (BabA), are associated with severe pathological outcomes. However, the in vivo role of BabA during long-term infection is not clear. In this study, Mongolian gerbils were infected with H. pylori and necropsied continuously during 18 months. Bacterial clones were recovered and analyzed for BabA expression, Leb-binding activity, and adhesion to gastric mucosa. BabA expression was completely absent by 6 months post-infection. Loss of BabA expression was attributable to nucleotide changes within the babA gene that resulted in a truncated BabA. In response to the infection, changes in the epithelial glycosylation pattern were observed that were similar to responses observed in humans and monkeys. Furthermore, infections with BabA-expressing and BabA-nonexpressing H. pylori showed no differences in colonization, but infection with the BabA-expressing strain exhibited histological changes and increased inflammatory cell infiltration. This suggests that BabA expression contributes to severe mucosal injury.

Role of Tumor Necrosis Factor-Alpha and Interferon-Gamma in Helicobacter pylori Infection

Immune responses to Helicobacter pylori infection play important roles in gastroduodenal diseases. The contributions of tumor necrosis factor‐α (TNF‐α) and interferon‐7 (IFN‐γ) to the induction of gastric inflammation and to the protection from H. pylori infection were investigated using TNF‐α gene‐knockout (TNF‐α−/−) mice and IFN‐γ gene‐knockout (IFN‐γ−/−) mice. We first examined the colonizing ability of H. pylori strain CPY2052 in the stomach of C57BL/6 wild‐type and knockout mice. The number of H. pylori colonized in the stomach of IFN‐γ−/− and TNF‐α−/− mice was higher than that of wild‐type mice. These findings suggest that TNF‐α and IFN‐γ may play a protective role in H pylori infection. Furthermore, we examined the contribution of TNF‐α and IFN‐γ to gastric inflammation. The CPY2052‐infected TNF‐α−/− mice showed a moderate infiltration of mononuclear cells in the lamina propria and erosions in the gastric epithelium as did wild‐type mice, whereas the CPY2052‐infected IFN‐γ−/− mice showed no inflammatory findings even 6 months after infection. These results demonstrate that IFN‐γ may play an important role in gastric inflammation induced by H. pylori infection, whereas TNF‐α may not participate in the development of inflammatory response.

Helicobacter pylori outer membrane proteins on gastric mucosal interleukin 6 and 11 expression in Mongolian gerbils

Background and Aim:  The levels of interleukin (IL)‐6 and IL‐11 in the gastric mucosa are related to mucosal inflammation; however, the chronological changes in cytokine expression during different phases of Helicobacter pylori infection and the effects of H. pylori virulence factors, particularly those of outer membrane proteins, remain obscure. The aim of this study was to clarify the chronological changes in cytokine levels in relation to several H. pylori outer membrane proteins.

Antibacterial Effect of Kampo Herbal Formulation Hochu-Ekki-To (Bu-Zhong-Yi-Qi-Tang) on Helicobacter pylori Infection in Mice

Because Helicobacter pylori (H. pylori) infection is a major cause of gastroduodenal diseases in humans, the eradication of H. pylori using antibiotics is very effective for the treatment of gastroduodenal diseases. However, it has recently been reported that resistance to these antibiotics is developing. In the present study, the antibacterial effect of a Kampo (traditional Japanese medicine) herbal formulation, Hochu‐ekki‐to (HET; Formula repletionis animalis et supletionis medii), against H. pylori was examined in vitro and in vivo. HET inhibited the growth of antibiotic‐resistant strains of H. pylori as well as antibiotic‐sensitive strains at a dose of 2.5 mg/ml in vitro. When 1,000 mg/kg of HET was administered orally to C57BL/6 mice for 7 days before or after inoculation with H. pylori, H. pylori in the stomach was significantly reduced in the HET‐pre‐treatment group compared with the control group. Furthermore, HET in combination with antibiotics completely eradicated the bacteria in mice. The expression of interferon (IFN)‐γ was induced in the gastric mucosa of the mice pre‐treated with HET. There were no significant differences between the colonization of H. pylori in the control and HET treatment groups in IFN‐γ gene‐deficient mice. These results suggest that the antibacterial effect of HET may be partly due to IFN‐γ induction, and that HET may be clinically useful for treatment of H. pylori infection.

Relationship between Helicobacter pylori hopQ genotype and clinical outcome in Asian and Western populations.

Background and Aims:  Outer membrane proteins of Helicobacter pylori mediate important pathogen–host interactions such as colonization, adhesion and the inflammatory response. hopQ genotypes have been suggested to be associated with increased risk of peptic ulcer. The aim of this study was to test the relation of hopQ genotype to H. pylori‐related disease and histological changes in Asian and Western countries.