Increased numbers of immature plasma cells in peripheral blood specifically overexpress chemokine receptor CXCR3 and CXCR4 in patients with ulcerative colitis.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease featuring infiltration by plasma cells producing immunoglobulins. We have reported previously the specific and significant proliferation of immature plasma cells in the inflamed colonic and pouch mucosa of UC patients. The aim of this study was to characterize peripheral blood immature plasma cells and the migration mechanisms of such immature plasma cells to inflamed sites in UC. The characteristics of peripheral blood immature plasma cells and chemokine receptor expression were examined by flow cytometry. Expression of mucosal chemokine was quantified using real-time reverse transcription-polymerase chain reaction and immunohistochemistry. The number of peripheral blood immature plasma cells was significantly higher in patients with active UC and active Crohn's disease (CD) than in healthy controls. The proportion of immature plasma cells was correlated positively with clinical activities of UC and CD. Many peripheral blood immature plasma cells were positive for CXCR3, CXCR4, CCR9 and CCR10. Expression of CXCR3 and CXCR4 in UC patients was significantly higher than in controls. CXCL9, CXCL10 and CXCL11 mRNA levels in colonic mucosa of inflamed IBD were higher than in controls. Immunofluorescence study also showed abundant CXCR3-positive immature plasma cells in the inflamed colonic mucosa of UC. Increased numbers of immature plasma cells may migrate towards inflammatory sites of UC via the CXCR3 axis, and may participate in UC pathogenesis.

Interleukin-10 gene transfer to peritoneal mesothelial cells suppresses peritoneal dissemination of gastric cancer cells due to a persistently high concentration in the peritoneal cavity.

Interleukin (IL)-10 has potent biological properties including an inhibitory action on the proliferation and metastasis of various cancer cells. However, it is difficult to maintain a high concentration of this cytokine as it has a short half life. In this study, we evaluated whether peritoneal mesothelial cells (PMCs) could be suitable for maintaining a high concentration of IL-10 using adenoviral gene transfer. We also evaluated the therapeutic effects of an intraperitoneal injection with adenoviral vector containing mouse IL-10 gene (Ad-mIL-10) using a mouse peritoneal dissemination model of MKN45 gastric cancer cells. We demonstrated that in vitro transfection efficiency of a recombinant adenovirus containing the bacterial beta-galactosidase gene (Ad-LacZ) was approximately 10-fold higher for primarily isolated PMCs than MKN45. The entire peritoneum was transfected until 3 weeks after an intraperitoneal Ad-LacZ injection. Ad-mIL-10 treatment increased intraperitoneal IL-10 levels until 3 weeks after treatment, and then significantly inhibited peritoneal cancer growth by inhibiting angiogenesis. This treatment also improved cachexia and prolonged mice survival. We thus concluded that IL-10 gene transfer in PMCs could be a new strategy for the prevention of peritoneal dissemination of gastric cancer due to the resulting persistently high IL-10 concentration in the peritoneal cavity.

Non-steroidal anti-inflammatory drug-induced small intestinal damage is Toll-like receptor 4 dependent.

BACKGROUND: Enterobacteria and cytokines both play roles in the pathophysiology of NSAID-induced enteropathy. Toll-like receptor (TLR) 4 recognises lipopolysaccharide (LPS), resulting in activation of an inflammatory cascade via the accessory protein MyD88. AIMS: To investigate role of TLR4 in inflammatory responses in indomethacin-induced enteropathy. METHODS: Indomethacin was administered p.o. to non-fasting rats and mice to induce small intestinal damage. The extent of such damage was evaluated by measuring the injured area stained dark blue with Evans blue. Rats were given antibiotics (ampicillin, aztreonam or vancomycin) p.o., or intraperitoneal LPS (a TLR4 ligand) or neutralising antibodies against neutrophils, tumour necrosis factor (TNF)-alpha, or monocyte chemotactic protein (MCP)-1. Furthermore, the intestinal ulcerogenicity of indomethacin was examined in TLR4-mutant, TLR4(-/-), and MyD88(-/-) mice. RESULTS: Indomethacin induced small intestinal damage with an increase in expression of TNF-alpha and MCP-1 in both rats and mice. Antibodies against neutrophils, TNF-alpha and MCP-1 inhibited the damage by 83%, 67% and 63%, respectively, in rats. Ampicillin and aztreonam also inhibited this damage, and decreased the number of Gram-negative bacteria in the small intestinal contents of the rat. However, vancomycin, which exhibited no activity against Gram-negative bacteria, had no preventive effect against this damage. Administration of LPS 1 h after indomethacin aggravated the damage, whereas LPS pretreatment inhibited it with reduction of expression of TLR4 and cytokines. In TLR4-mutant mice, the damage and cytokine expression were markedly inhibited. TLR4(-/-) and MyD88(-/-) mice were also resistant to the damage. CONCLUSIONS: Indomethacin may injure the small intestine through a TLR4/MyD88-dependent pathway.

Roles of cyclooxygenase-2 and prostaglandin E receptors in gastric mucosal defense in Helicobacter pylori-infected mice.

BACKGROUND/AIM: Helicobacter pylori (H. pylori) induces cyclooxygenase-2 (COX-2) expression. The aim of this study was to assess the roles of COX-2 and PGE2 receptors (EPs) in gastric defense in H. pylori-infected mice. METHODS: Gastric lesions were induced by oral administration of 0.15N HCl in 60% ethanol (HCl/EtOH) to mice infected with H. pylori, and macroscopically evaluated 30 min later. Mice were administered NS-398 (COX-2 selective inhibitor) concomitantly with selective EP agonists 4 hours before HCl/EtOH challenge. RESULTS: H. pylori infection prevented the gastric damage induced by HCl/ EtOH, and this protective effect was abolished by NS-398. Selective agonists of EP1, EP2, and EP4, but not the EP3 agonist, reversed the inhibitory effect of NS-398 on prevention of damage by H. pylori infection. The EP4 agonist and EP2/EP4 agonists inhibited the increase in TNF-alpha mRNA expression and neutrophilic infiltration caused by NS-398, respectively. CONCLUSION: COX-2-derived PGE2 may play an important role in resistance to HCl/EtOH damage in H. pylori-infected mice by activating EP1, EP2, and EP4.

Lafutidine can improve the quality of gastric ulcer healing in humans: a randomized, controlled, multicenter trial.

Improving the quality of ulcer healing (QOUH) is one of the valid methods of prevention of relapse of gastric ulcers. We investigated the effect of lafutidine on the QOUH of gastric ulcer compared with famotidine in a randomized, multi-centre controlled trial. Consecutive 80 patients with a gastric ulcer were randomly assigned to receive twice daily either lafutidine (10 mg) or famotidine (20 mg) for 12 weeks. Esophagogastroduodenoscopy was performed to examine the ulcer healing rate and rate of flat type ulcer scars using dye-contrast. The gastric ulcer healing rate was 92.1% in the lafutidine group (35/38) and 94.7% in the famotidine group (36/38). The rate of flat-type ulcer scars was significantly higher in the lafutidine group (68.4%, 26/38) than in the famotidine group (42.1%, 16/38) (P = 0.021). In conclusion, the present study demonstrated that lafutidine, as compared to famotidine, yields a significantly superior QOUH in patients with gastric ulcers in the clinical setting.

Roles of cyclooxygenase 2 and microsomal prostaglandin E synthase 1 in rat acid reflux oesophagitis.

BACKGROUND: Although prostaglandin E2 (PGE2), cyclooxygenase 2 (COX-2), and microsomal prostaglandin E synthase 1 (mPGES-1) are known to play a role in various inflammatory events, their roles in the pathogenesis of gastro-oesophageal reflux disease are not known. AIMS: We examined the dynamics of COX-1, COX-2, mPGES-1, mPGES-2, cytosolic PGES (cPGES), and PGE2 synthetic activity in rat acid reflux oesophagitis and the effects of COX-2 inhibitors on the severity of oesophagitis. METHODS: Acid reflux oesophagitis was induced by ligating the transitional region between the forestomach and the glandular portion and wrapping the duodenum near the pylorus. Rats were killed on day 3 (acute phase) or day 21 (chronic phase) after induction of oesophagitis. RESULTS: Expression of COX-2 and mPGES-1 was markedly increased in oesophagitis while modest changes in COX-1, cPGES, and mPGES-2 expression were observed. COX-2 and mPGES-1 were colocalised in epithelial cells of the basal layer, as well as inflammatory and mesenchymal cells in the lamina propria and submucosa. COX-2 inhibitors significantly reduced the severity of chronic oesophagitis but did not affect acute oesophageal lesions. COX-2 inhibitors significantly inhibited the increase in PGE2 synthesis in oesophageal lesions on both days 3 and 21. Epithelial proliferation was significantly increased in the basal layer on day 21. Inflammatory cells and epithelial cells of the basal layer exhibited reactions for EP4 in oesophagitis. CONCLUSION: PGE2 derived from COX-2 and mPGES-1 plays a significant role in the pathogenesis of chronic acid reflux oesophagitis, and possibly in basal hyperplasia and persistent inflammatory cell infiltration.

Augmented expression of secondary lymphoid tissue chemokine and EBI1 ligand chemokine in Crohn's disease.

BACKGROUND: A dominant T helper type 1 (Th1) immune response is thought to be involved in Crohn's disease (CD). SLC/CCL21 and ELC/CCL19, chemokines that regulate T cell homing and promote recirculating T and dendritic cell (DC) interactions, help control antigen specific T cell responses. AIMS: To investigate the Th1 response and SLC and ELC in CD pathogenesis. METHODS: Surgically resected intestine and mesenteric lymph nodes (MLNs) from controls and patients with CD and ulcerative colitis (UC) were investigated. CD3, CD83, HECA452, VEGFR3, SLC, ELC, and CCR7 expression was studied immunohistochemically. CCR7 mRNA was quantified using real time RT-PCR. RESULTS: ELC was almost undetectable in intestinal samples. SLC was found sporadically in lymphoid follicles, lymphoid aggregate venules, and lymphatic vessels. In MLNs, SLC was highly expressed in high endothelial venules (HEVs), lymphatic vessels, and stromal DCs, predominantly in T cell areas. ELC was highly expressed in mature DCs. There were significantly more SLC positive HEVs and ELC positive mature DCs, important components of T cell areas, in CD. SLC, ELC, and CCR7 mRNA was significantly higher in CD MLNs compared with UC. CD MLNs had increased expression of SLC and ELC, mainly in HEVs, mature DCs, and lymphatic vessels, inducing T cell hyperplasia. CCR7 mRNA was increased in T cell areas. CONCLUSION: The dominant Th1 immune response is facilitated by interaction of SLC positive HEVs/lymphatic vessels, ELC positive mature DCs, and CCR7 positive T cells in hyperplastic T cell areas. In CD, memory T cells and mature DCs may home to MLN.

Accumulation of mitochondrial DNA mutation with colorectal carcinogenesis in ulcerative colitis.

We recently reported that oxidative stress elicited by chronic inflammation increases the mutation of mitochondrial DNA (mtDNA) and possibly correlates with precancerous status. Since severe oxidative stress is elicited in the colorectal mucosa of individuals with ulcerative colitis (UC), the possible occurrence of an mtDNA mutation in the inflammatory colorectal mucosa and colitic cancer was investigated. Colorectal mucosal specimens were obtained from individuals with UC with and without colitic cancer and from control subjects. The frequency of mtDNA mutations was higher in colorectal mucosal specimens from patients with UC than that from control subjects. The levels of 8-hydroxy-2'-deoxyguanosine, a DNA adduct by reactive oxygen species, were significantly higher in UC than in control. Specimens from patients with colitic cancer contained a significantly higher number of mtDNA mutations. The present observations suggest that the injury followed by the regeneration of colorectal mucosal cells associated with chronic inflammation causes accumulation of mtDNA mutations. The increased instability of genes, including those on the mtDNA, is consistent with the high and multicentric incidence of colorectal cancer in individuals with UC. Thus, analysis of mtDNA could provide a new criterion for the therapeutic evaluation, and may be useful for the prediction of risk of carcinogenesis.

Prevalence of symptomatic gastro-oesophageal reflux disease in Japanese patients with peptic ulcer disease after eradication of Helicobacter pylori infection.

BACKGROUND: The association between cure of Helicobacter pylori infection and the development of gastro-oesophageal reflux disease is controversial. AIM: To examine the prevalence of symptomatic GERD (sGERD) in Japanese patients with peptic ulcer disease after successful eradication and identify associated factors affecting sGERD development. METHODS: We retrospectively examined 72 patients (40 gastric ulcer and 32 duodenal ulcer) with successful eradication. Associated factors such as age, gender, drinking and smoking habits, body mass index, presence of gastric atrophy and hiatal hernia were analysed. RESULTS: Seven (9.7%) of 72 peptic ulcer patients newly developed sGERD. There were no differences in mean age, gender, smoking habit, drinking habit, body mass index, or presence of gastric atrophy and hiatal hernia between the sGERD and non-sGERD groups, while the proportion of subjects aged over 70 was significantly higher in the sGERD than the non-sGERD group. Six of 40 patients with gastric ulcer newly developed sGERD while only one of 32 patients with duodenal ulcer developed it. CONCLUSION: Approximately 10% of Japanese patients with peptic ulcer disease newly developed sGERD after cure of H. pylori infection. Age > 70 years was associated with development of sGERD. Eradication in patients in this age group should be carefully determined.

Correlation of MAP kinases with COX-2 induction differs between MKN45 and HT29 cells.

BACKGROUND: Mitogen-activated protein (MAP) kinases, including extracellular signal-regulated kinases (ERK),c-Jun NH2-terminal kinases (JNK) and p38 MAP kinase (p38 MAPK) are important intermediates of the signal-transduction pathway from the cell surface to the nucleus. Expression of cyclooxygenase (COX)-2, associated with proliferation, apoptosis or both of gastrointestinal cancer cells, is mediated through MAP kinase families. However, the correlation between respective MAP kinase signals and COX-2 in the proliferation of gastric and colon cancer cells has not been well elucidated. AIM: We examined the effect of selective inhibitors of MAP kinases and COX-2 on serum-induced proliferation of gastric (MKN45) and colon (HT29) cancer cells. METHODS: After 24-h serum starvation, cancer cells were stimulated with 2% serum and COX-2 inhibitors (NS398 10 micromol/L, or etodolac 100 micromol/L) or 1 h after preincubation with inhibitors for ERK (PD98059 20 micromol/L) or p38 MAPK (SB203580 10 micromol/L). Phosphorylated MAP kinases and COX-2 protein were evaluated by Western blotting, and the proliferation of cancer cells was estimated by 3H-thymidine incorporation. Transcription factors nuclear factor-kappaB and CREB were assayed by an electorophoretic mobility shift assay. RESULTS: Serum increased the proliferation of MKN45 and HT29 cells by 280% and 200%, respectively, compared with the control levels (100%). In both cancer cells, phosphorylated MAP kinases were increased within 30 min after stimulation. PD98059 and SB203580 inhibited the serum-induced proliferation of MKN45 by 21% and 51% and of HT29 by 81% and 69%, respectively. NS398 and etodolac inhibited the proliferation of HT29 by 21% and 41%, respectively, but not that of MKN45. PD98059 and SB203580 also suppressed serum-induced expression of COX-2 protein in HT29 cells. In addition to the activation of MAP kinases and COX-2, activities of nuclear factor-kappaB and CREB were also increased during HT29 cell proliferation. CONCLUSIONS: These results suggest that the correlation of MAP kinases with COX-2 induction for cell proliferation differs between MKN45 and HT29 cells.

Comparison of the effects of rebamipide with those of cimetidine on chronic gastritis associated with Helicobacter pylori in Mongolian gerbils.

BACKGROUND AND AIMS: The effects of rebamipide on chronic gastritis associated with Helicobacter pylori have not been well-defined. We compared these effects of rebamipide with those of cimetidine in Mongolian gerbils infected with H. pylori. METHODS: Mongolian gerbils with or without H. pylori were divided into 10 groups 6 weeks after inoculation and fed diets containing a drug (rebamipide or cimetidine) or control diet. All animals were sacrificed 4 weeks after grouping. Their stomachs were examined for histology, colonization by H. pylori, myeloperoxidase activity (myeloperoxidase), production of neutrophil chemokine (CINC/KC) and tumour necrosis factor-alpha (TNF-alpha), and serum gastrin levels. RESULTS: H. pylori colonized all of the inoculated animals. Neither rebamipide nor cimetidine decreased myeloperoxidase activity, but each reduced wet stomach weight in H. pylori-infected animals. The amount of increase in CINC/KC and TNF-alpha in gastric tissue caused by H. pylori infection was decreased by treatment with rebamipide or cimetidine. H. pylori infection increased serum gastrin levels, and this increase was significantly enhanced by cimetidine but not rebamipide. CONCLUSIONS: Rebamipide may improve H. pylori-infected chronic gastritis by preventing the production of inflammatory cytokines and chemokines, as does cimetidine, but may be preferable to cimetidine for long-term administration for treatment of H. pylori-infected chronic gastritis due to its effect on serum gastrin levels.

Cigarette smoking and alcohol consumption associated with gastro-oesophageal reflux disease in Japanese men.

BACKGROUND: Associations between lifestyle factors and gastro-oesophageal reflux disease (GORD) have been conflicting. We aimed to examine these associations in Japanese men. METHODS: We performed a cross-sectional study of Japanese male workers who visit a clinic for a routine health check-up and asked them to fill out a self-report questionnaire. Logistic regression was used to calculate odds ratios (OR) with 95% confidence intervals (CI) for GORD, defined as heartburn and/or acid regurgitation at least twice weekly. RESULTS: Of the 4095 eligible subjects, 276 (6.7%) were diagnosed as having GORD. Current smoking was significantly associated with CORD compared with non-smoking (OR = 1.35, 95% CI, 1.01-1.82). Moderate drinking (16-37 mL/day) and heavy drinking (> or = 38 mL/day) were also associated with GORD, while age and body mass index were not. After adjustment for age, daily alcohol consumption and body mass index, an increase in number of pack-years of cigarette smoking was significantly associated with an increased OR of GORD (P for trend = 0.034), and the OR for persons whose number of pack-years of cigarette smoking was more than 20.1 was 1.45 (CI 1.04-2.04) compared with non-smokers. CONCLUSION: Cigarette smoking and alcohol consumption are associated with an increased odds ratio for GORD in Japanese men.

Validity of simple mucosal biopsy criteria combined with endoscopy predicting patients with ulcerative colitis ultimately requiring surgery: a multicenter study.

BACKGROUND: Recent mucosal biopsy criteria combined with endoscopy effectively differentiate patients with ulcerative colitis ultimately requiring surgery (UC-S) from those receiving medication alone (UC-M). However, the criteria were inconvenient in practical use because of the need for complicated calculations, and the validity has not been verified in other institutes where the indications for surgery may differ. The aims of this multicenter study were to propose simple criteria in which calculation can be performed by mental arithmetic and to measure their validity. METHODS: Based on the above original criteria, we constructed simple criteria in which coefficients and constant were simplified to integral numbers. The criteria consisted of the diagnostic categories, highest-risk, high-risk, unpredictable, low-risk, and lowest-risk of surgery. The validity of these proposed criteria was evaluated in 121 patients with UC-S and 186 with UC-M from 11 institutes. RESULTS: The categories of high-risk and low-risk had sensitivities exceeding 86.0% and specificities exceeding 95.2%, and the validities were maintained at high levels in most individual institutes. There was little difference in validity between the proposed and original criteria when testing using the same patients. CONCLUSIONS: Despite simplified coefficients and constant, the proposed criteria reliably predicted the eventual clinical outcome of patients with ulcerative colitis and would be helpful in determining the necessity of surgery.

Increased expression of epidermal growth factor receptors in basal cell hyperplasia of the oesophagus after acid reflux oesophagitis in rats.

BACKGROUND: Epidermal growth factor (EGF), which binds to EGF receptors (EGF-R), stimulates oesophageal epithelial cell proliferation, enabling rapid repair after mucosal injury. In the normal human oesophageal epithelium, EGF-R expression is present and confined to the basal layer. AIM: To examine histological changes in and dynamics of EGF-R expression during healing after acid reflux oesophagitis in a rat model. METHODS: Gastric acid reflux oesophagitis was induced in Wistar rats by ligation of the pylorus and the transitional region between the forestomach and the grandular portion for 5 h, followed by release of both ligations. Rats were killed 7 and 14 days after production of oesophagitis to examine macroscopic and histological changes as well as dynamics of EGF-R expression. Epithelial cell proliferation was assessed by bromodeoxyuridine (BrdU) uptake, and expression of EGF-R mRNA and protein by RT-PCR and Western blotting or immunohistochemistry. RESULTS: Gastric acid reflux induced erosive and ulcerative mucosal lesions in the lower and middle part of the oesophagus. These lesions were healed by 14 days and histologically showed thickening of the oesophageal epithelium from 41.11 +/- 3.09 microm in controls to 142.73 +/- 11.59 microm (P < 0.001) in ligated rats, as well as elongation of papillae and basal cell hyperplasia. The number of BrdU-positive cells among basal cells on day 14 was significantly increased from 7.1 +/- 0.8/field in controls to 30.9 +/- 3.0/field in ligated rats. Expression of EGF-R mRNA and protein was significantly increased on day 14 and most basal cells were immunohistochemically positive in both BrdU and EGF-R staining. CONCLUSION: Acid reflux-induced oesophageal injury caused basal cell hyperplasia with an increase in cell proliferation and EGF-R expression. Activation of EGF-R gene and protein in response to acid reflux-induced injury may facilitate mucosal healing. These results suggest that epidermal growth factor receptors play a crucial role in healing after acid reflux oesophagitis in rats.

High molecular protein of Helicobacter pylori responsible for inhibition of ornithine decarboxylase activity of human gastric cultured cells.

BACKGROUND: Ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis, mediates epithelial cell proliferation and plays a critical role in the optimal repair of gastric mucosal damage. Several studies have shown that Helicobacter pylori inhibits the growth and proliferation of gastric cells in vitro. AIM: To test whether H. pylori extract affects ODC mRNA expression and its enzyme activity in gastric cells and to examine the partial characterization of the molecule responsible for this effect. METHODS: Human gastric cells (MKN-45) were used. Bacterial extracts from various E. coli or H. pylori strains, namely (1) cagA+, vacA+, CagA+, VacA+; (2) cagA+, vacA+, CagA+ VacA-; or (3) cagA-, vacA+, CagA-, VacA- were added to the cells. Cell proliferation was assessed by [3H]-thymidine incorporation, viability by MTT assay and LDH release test, ODC enzyme activity by 14CO2 counts from L-[1(14)C]ornithine, and ODC mRNA by Northern blotting. RESULTS: H. pylori and E. coli extract did not affect viability of gastric cells. H. pylori extract, especially extracts containing a protein greater than 50 kDa, significantly inhibited proliferation and ODC activity of gastric cells while E. coli extract had no effect. Inhibition of ODC activity was found in extracts of all H. pylori strains, irrespective of CagA and VacA protein expression. Serum stimulation induces an increase in ODC mRNA while H. pylori extract did not affect ODC mRNA expression. CONCLUSION: High molecular weight (greater than 50 kDa) proteins of H. pylori extract without CagA or VacA protein inhibited proliferation and ODC activity of human gastric cells, but did not affect ODC mRNA expression, suggesting that inhibition of ODC activity is regulated at the post-transcriptional level.

IgG subclasses of anti Saccharomyces cerevisiae antibody in inflammatory bowel disease.

Elevation of serum anti Saccharomyces cerevisiae antibody (ASCA) has been reported in patients with Crohn's disease. We analysed the subclasses of Immunoglobulin (Ig) G reaction in ASCA in sera from patients with inflammatory bowel disease, healthy controls, and patients with intestinal Behçet's disease. Serum samples were obtained from 29 patients with Crohn's disease, 30 patients with ulcerative colitis, 7 patients with intestinal Behçet's disease, and 12 healthy controls. Serum IgG subclasses IgG1, IgG2, IgG3, and IgG4 of ASCA were analysed using ELISA. IgG4 ASCA was significantly increased in patients with inflammatory bowel disease. In patients with intestinal Behçet's disease, IgG1, IgG3, and IgG4 ASCA were increased. Differential responses, in terms of subclasses in ASCA, were found in patients with inflammatory bowel disease and patients with intestinal Behçet's disease, which may represent different pathophysiologies of these intestinal inflammatory diseases.