Naoki Sawai

Induction of various cytokines and development of severe mucosal inflammation by cagA gene positive Helicobacter pylori strains

Background —Helicobacter pyloristrains possessing the cagA gene are thought to induce interleukin 8 (IL-8) in gastric mucosa. However, it is still unclear whether a relation exists between the cagA gene and the expression patterns of cytokines other than IL-8. Aims —To investigate the relation between the cagA gene and the production of various cytokine proteins using an enzyme linked immunosorbent assay (ELISA). Patients and methods —In 184 patients, the cagA gene was detected by polymerase chain reaction (PCR), and levels of production of IL-1β, IL-6, IL-7, IL-8, IL-10, and tumour necrosis factor α (TNF-α) in antral biopsy specimens were measured by ELISA. Results—Mucosal levels of IL-1β, IL-6, IL-8, and TNF-α were significantly higher in H pyloripositive than in H pylori negative patients. Furthermore, the mucosal levels of IL-1β and IL-8 were significantly higher in specimens infected with cagApositive strains than in those infected with cagAnegative strains. In H pylori positive patients, the mucosal level of IL-8 was closely correlated with that of IL-1β (p<0.0001), and the mucosal level of IL-6 was closely correlated with that of TNF-α (p<0.0001). Conclusion—These findings suggest that the ability to induce cytokines differs among the strains;cagA + strains induce various kinds of cytokines and may cause severe inflammation, whereascagA − strains induce IL-8 and IL-1β only weakly and may cause only mild inflammation. However, as most patients infected with the cagA + strains have gastritis, these strains may not be equivalent to ulcerogenic strains.

Chemokines in the gastric mucosa in Helicobacter pylori infection

Background—Although chemokines have been suggested to play an important role in Helicobacter pyloriassociated gastritis, few studies have investigated the role of chemokines other than interleukin 8 (IL-8) in gastric mucosa. Aims—To investigate the expression and production patterns of various chemokines using gastric biopsy specimens. Methods—In 192 patients, expression patterns of C-X-C chemokines (IL-8 and growth regulated α (GROα)) and C-C chemokines (regulated on activation, normal T cell expressed and presumably secreted (RANTES), monocyte chemotactic and activating factor (MCAF), macrophage inflammatory protein 1α (MIP-1α), and MIP-1β) were examined using reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA).cagA gene was identified using PCR. Results—H pylori infection was associated with increased rates of expression of mRNA for IL-8, GROα, RANTES, and MIP-1α and with increased levels of mucosal IL-8 and GROα. IL-8 and GROα levels correlated with the density of H pylori in both the antrum and corpus. The levels of these chemokines correlated with cellular infiltration in the antrum but not the corpus. cagA gene positive H pyloriinfection was associated with increased rates of expression of mRNA for IL-8 and GROα and with increased levels of these chemokines. Conclusion—H pylori infection is associated with increased expression rates and production of C-X-C chemokines (IL-8 and GROα), but not with increased production of C-C chemokines. Although H pylori infection is associated with increased C-X-C chemokines in the antrum and corpus, there is a difference in the inflammatory response between these two areas of the stomach.

Expression of Cytokine mRNA in Gastric Mucosa with Helicobacter pylori Infection

BACKGROUND We have studied the cytokine production patterns in gastric mucosal biopsy specimens with and without the Helicobacter pylori infection, using a reverse transcription-polymerase chain reaction (RT-PCR) method capable of detecting low levels of specific mRNA. METHODS Total RNA was prepared from biopsy specimens with the acid guanidinium thiocyanate-phenol-chloroform method. cDNA was synthesized by M-MLV RTase and amplified using the oligonucleotide primers specific for interleukin-1 beta (IL-1beta), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, tumor necrosis factor-alpha (TNF-alpha), interferon-alpha (IFN-alpha), IFN-beta, and IFN-gamma by PCR methods. RESULTS Although IL-1 beta and IFN-gamma mRNA were detected in most specimens, IL-2, IL-3, IL-4, IL-5, IL-9, TNF-alpha, and IFN-beta mRNA were not detected at all. The expressions of IL-7 and IL-8 mRNA were significantly higher in H. pylori-positive gastritis than in H. pylori-negative normal controls. There was a significant correlation between the expression of IL-8 mRNA and the severity of gastritis both in the antrum and in the corpus. On the other hand, there was a significant correlation between the expression of IL-7 mRNA and the severity of gastritis only in the corpus. CONCLUSIONS These findings suggest that some cytokines, especially IL-7 and IL-8, play some roles in H. pylori-associated gastritis.

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.

Levels of Interleukin-18 Are Markedly Increased in Helicobacter pylori–Infected Gastric Mucosa among Patients with Specific IL18 Genotypes

BACKGROUND The cellular immune response in gastric mucosa infected with Helicobacter pylori is proposed to be predominantly of the T helper cell type 1 type. METHODS Interleukin (IL)-18, IL-12, and interferon (IFN)-gamma levels were measured in gastric mucosal biopsy specimens by reverse-transcription polymerase chain reaction (PCR) and by enzyme-linked immunosorbent assay; IL18 polymorphisms were determined by PCR. RESULTS Biopsy specimens from 128 patients (56 with nonulcer dyspepsia, 28 with gastric ulcers, 28 with duodenal ulcers, and 16 with gastric cancer) were examined; 96 patients had H. pylori infection. IL-18 levels were markedly up-regulated in mucosa infected with H. pylori (P < .001), whereas IL-12 and IFN-gamma levels were independent of H. pylori status. IL-18 levels correlated with IFN-gamma levels only in infected patients (R = 0.31 to R = 0.51). IL-18 levels were the determining factor for monocyte infiltration in H. pylori-infected mucosa (P < .001). H. pylori-infected patients displaying IL18 -607C/C and -137G/G had higher IL-18 levels than did those with other genotypes and were more likely to experience treatment failure. CONCLUSION H. pylori infection induces IL-18 in the gastric mucosa. H. pylori-infected patients with IL18 -607C/C and -137G/G have higher IL-18 levels, which causes severe gastric inflammation. IL18 genotype might be a marker for predicting the effects of eradication therapy.

Analysis of the 13C‐urea breath test for detection of Helicobacter pylori infection based on the kinetics of Δ‐13CO2 using laser spectroscopy

We have previously reported on laser spectroscopy as a simple alternative to mass spectrometry. To validate a simplified 13C‐urea breath test (UBT) with laser spectroscopy for the detection of Helicobacter pylori in clinical use, we evaluated the optimal time of breath sample collection. The 13C‐UBT was carried out on each of 102 infected and 70 non‐infected subjects (32 without eradication and 38 after eradication therapy). Breath samples were taken at five time points within 60 min followed by 100 mg of 13C‐urea administration. The ratio of 13CO2 to 12CO2 was measured using laser spectroscopy and the recovery of tracer in the exhaled breath was calculated. Results were compared with histological and culture examinations of gastric biopsies to establish the infection status. For statistical evaluation of 13C‐UBT, the optimal timing of breath sample collection was examined on the basis of the kinetics of Δ‐13CO2. In 32 H. pylori‐negative patients (without therapy), the mean ± 2SD of Δ‐13CO2 was at its minimum 20 min after urea ingestion whereas in H. pylori‐positive patients, the mean ± SD Δ‐13CO2 was maximum at 20 min. In addition, receiver operating characteristic (ROC) curve analysis showed that the cut‐off value was estimated between 2.5–3.0 per mil (‰) at 20 min before therapy. Based on the histology and culture results, the sensitivity, specificity and positive and negative predictive values were 98.0%, 100%, 100% and 94.1%, respectively. In conclusion, 13C‐UBT with laser spectroscopy is a non‐invasive, simple, sensitive and specific test to determine H. pylori status. Our findings suggest that in clinical use, measurements made at 20 min after substrate administration could be recommended for most sensitive and specific 13C‐UBT results.

REGRESSION OF ATYPICAL LYMPHOID HYPERPLASIA AFTER ERADICATION OF HELICOBACTER PYLORI

A rare case of endescopic and histological regression of a gastric lymphoid mucosal lesion after eradication ofHelicobacter pylori is reported. A 72-year-old man was suspected of having a low-grade B-cell gastric mucosa-associated lymphoid tissue (MALT) lymphoma by endoscopic and histological findings. Histology of biopsy specimens showed massive infiltration of atypical lymphocytes and lymphoepithelial lesions. Immunohistochemical staining revealed kappa light chain expression in the infiltrated atypical lymphocytes to be twofold that of lambda light chain. The above diagnosis was thus highly suspected but not confirmed. Antibiotic therapy was given on the basis of evidence ofH. pylori infection. Successful eradication ofH. pylori resulted in remarkable improvement of endoscopic and histological findings. Follow-up studies were carried out 8 months after eradication, with no evidence of relapse. The eradication ofH. pylori appears to be an effective alternative therapy for B-cell lymphoproliferative disease, although longer follow-up and further studies are needed before this treatment can be establisted.