Kazuhito Sugimura

p16INK4 is inactivated by extensive CpG methylation in human hepatocellular carcinoma

Abstract Background & Aims: The molecular status of the p16 INK4 tumor-suppressor gene has not been fully elucidated in hepatocellular carcinoma. The aim of this study was to clarify the mechanism that gives rise to inactivation of p16 INK4 in hepatocellular carcinoma. Methods: The status of p16 INK4 was evaluated in 60 hepatocellular carcinomas by immunohistochemical staining, differential polymerase chain reaction, single-strand conformational polymorphism, methylation-specific polymerase chain reaction, and methylation-sensitive single nucleotide primer extension. Results: Immunohistochemical staining showed that 29 of the 60 tumors exhibited complete loss of p16 INK4 expression. High levels of DNA methylation were detected in 24 of 29 cases of hepatocellular carcinoma with negative p16 INK4 expression, with methylation of 60%–85% of the CpG islands. In contrast, the level of methylation was p16 INK4 staining, and no methylation was detected in tumors with positive immunostaining. Intragenic alteration of p16 INK4 was detected in 4 cases. Conclusions: A strong correlation was found between the extent of methylation and the degree of expression of p16 INK4 in tumor tissues, indicating that epigenetic change due to extensive CpG methylation is the main cause of inactivation of p16 INK4 in hepatocellular carcinoma. GASTROENTEROLOGY 1999;116:394-400

Spectrum of cytokine gene expression in intestinal mucosal lesions of Crohn's disease and ulcerative colitis

Background/Aims: We investigated the mRNA expression of spectrum of cytokines in the colonic mucosa in inflammatory bowel disease (IBD). Methods: The expression of cytokine gene was evaluated by using the reverse transcription-polymerase chain reaction and the radioactivity of amplified cDNA standardized by coamplified β-actin cDNA. Results: Crohn’s disease and ulcerative colitis showed significantly increased expression of IL-1β, IL-6, IL-8 and TNF-α mRNA as compared with controls (p < 0.05). Conclusion: Proinflammatory cytokines such as IL-1β, IL-6, IL-8 and TNF-α are closely involved in the immune abnormalities of inflammatory mucosal lesions in IBD.

Selective depletion of neutrophils by a monoclonal antibody, RP-3, suppresses dextran sulphate sodium-induced colitis in rats

Administration of dextran sulphate sodium to animals induces acute colitis characterized by infiltration of large numbers of neutrophils into the colonic mucosa, which histologically resembles human active ulcerative colitis. It has been reported that neutrophils and the reactive oxygen metabolites produced by them are involved in the progress of ulcerative colitis. This study was intended to clarify their roles by using this animal model. First, possible sources and species of reactive oxygen metabolites were determined using luminol‐dependent chemiluminescence with addition of enzyme inhibitors and reactive oxygen metabolite scavengers. Next, to examine whether neutrophils and hypochlorous acid derived from them contribute to tissue injury, we administered RP‐3, a monoclonal antibody capable of selectively depleting neutrophils, and taurine, a hypochlorous acid scavenger, to rats treated with dextran sulphate sodium. Addition of azide, taurine, catalase, superoxide dismutase and dimethyl sulphoxide into colonic mucosal scrapings significantly inhibited chemiluminescence production, but allopurinol and indomethacin had no effects. These results suggest that excessive hypochlorous acid, hydrogen peroxide, superoxide anion and hydroxyl radical are generated by the inflamed colonic mucosa. Intraperitoneal injections of RP‐3 significantly suppressed bleeding, tissue myeloperoxidase activity, chemiluminescence production and erosion formation. On the other hand, administration of taurine tended to inhibit bleeding and erosion formation to some extent, although it could not significantly suppress them. These data suggest that neutrophils play an important role in the development of this colitis and that hypochlorous acid might be one of the causes of tissue injury induced by neutrophils.

Analysis of genes within the HLA region affecting susceptibility to ulcerative colitis

To clarify the molecular relationship between HLA loci and ulcerative colitis (UC) in Japanese patients, we performed HLA-DP genotyping by the PCR-RFLP method and studied tumor necrosis factor beta-chain genetic polymorphism by Southern hybridization, in addition to conventional serologic typing. Significant increase was observed in Bw52, DPw9 (DPB1*0901), and DR2 (DRB1*1502) in Japanese patients with UC. Linkage analysis indicated that A24-Bw52-DR2-DPw9 alleles constitute a common haplotype in Japanese UC patients. Among the patients not carrying Bw52, B13 was significantly increased and B44 was relatively increased. These Bw52, B13, and B44 alleles share the unique amino acids, serine and aspartic acid at positions 67 and 77, respectively. These positions are in the second hypervariable region of the alpha 1-domain of the HLA-B13, B44, Bw52, and B49 antigens (B49 is quite rare in the Japanese population). The inflammatory region in UC patients was found to vary depending on their HLA-B alleles. These results suggest that the HLA-B locus itself plays an important role in the susceptibility to Japanese UC.

Influence of Interleukin-10 on the Interleukin-1 Receptor Antagonist/Interleukin-1β Ratio in the Colonic Mucosa of Ulcerative Colitis

A decrease in the ratio of IL-1ra/IL-1β produced regionally by the colonic mucosa of patients with ulcerative colitis (UC) is believed to play a role in the pathogenesis of UC. To investigate factors influencing intramucosal IL-1ra/IL-1β ratios, we evaluated polymorphism of the IL-1ra gene and the production of mucosal cytokines in Japanese patients with UC. Colonic biopsy specimens of mucosal tissue were placed in organ cultures for 24 h. Then, the supernatant concentrations of IL-1β, IL-1ra, IL-8, IL-4, IL-10, and TGF-β were assayed by ELISA. Genomic DNA was extracted from patient peripheral blood samples, then IL-1ra gene polymorphism was determined using PCR amplification. The mucosa from patients with active stage UC showed a tendency toward a decreased IL-1ra/IL-1β ratio. In the resolving stage, IL-1ra/IL-1β ratios increased with increasing IL-10 and TGF-β concentrations. The addition of human recombinant IL-10 to the culture supernatants produced concentration-dependent inhibition of IL-1β. In Japanese patients with UC, the IL-1ra allele gene 2 phenotype had no effect on the IL-1ra/IL-1β ratio. Our findings suggest that a relative deficiency of IL-10 in patients with UC may contribute to persistent inflammatory changes.

Gastric carcinoid tumors without autoimmune gastritis in Japan: A relationship with Helicobacter pylori infection

In Japan, most cases of gastric carcinoid tumor (GCT) are unassociated with either autoimmune gastritis (AIG) showing type-A chronic atrophic gastritis (CAG-A) or Zollinger-Ellison syndrome (ZES). However, the pathogenesis of this tumor remains unknown. Recent studies have determined that Helicobacter pylori infection induces gastric carcinoid in Mongolian gerbils and that H. pylori lipopolysaccharide exerts a mitogenic effect on ECL cells. We examined five patients with histologically diagnosed GCT, 40 patients with H. pylori-positive gastric ulcer (Hp+GU), 24 patients with H. pylori-positive duodenal ulcer (Hp+DU), and 12 patients with AIG showing CAG-A topographically. We compared the prevalence of H. pylori infection, and the levels of gastrin and pepsinogen (PG) in the serum of patients with GCT with those of patients with Hp+GU, or Hp+DU, and AIG. We also investigated the histological characteristics of the tumor and the gastric corpus mucosa in the GCT patients. The levels of serum gastrin and PG I and II were measured using an RIA kit. In all five (100%) patients with GCT, H. pylori infection was present, without any evidence of AIG or ZES. The serum levels of gastrin in the GCT patients were higher than those in either Hp+GU or Hp+DU patients and lower than those in the AIG patients. In contrast, serum PG I levels and the PG I/II ratio were lower in the GCT group than in the Hp+GU or Hp+DU groups. Histologically, all GCTs were ECL cell tumors and peritumoral corporal mucosal atrophy was observed in four of the five patients with GCT. In conclusions, H. pylori infection and hypergastrinemia were found in the patients with GCT without AIG. This finding suggests that H. pylori infection may induce corporal mucosal atrophy and hypergastrinemia that can produce a GCT with time.

Leukocytapheresis is effective in inducing but not in maintaining remission in ulcerative colitis.

Goals and Background: Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by dense infiltration of lymphocytes, plasma cells, neutrophils, and monocyte-macrophages into the colonic mucosa. Leukocytapheresis is a procedure for selectively removing white blood cells from withdrawn blood. It is used for the treatment of several autoimmune diseases. This study was performed to evaluate the effectiveness of leukocytapheresis for inducing and maintaining remission in corticosteroid-resistant UC, as compared with corticosteroid-responsive UC. Study: Forty-five patients with active UC who were treated with a dose of 1 mg/kg per day or more of prednisolone given systemically for at least 2 weeks were evaluated. Twenty patients (6 males, 14 females) in whom improvement was induced only by high doses of prednisolone were allocated as the corticosteroid-responsive group. The other 25 patients (11 males, 14 females) who did not respond to the above-mentioned dose of prednisolone therapy were allocated as the corticosteroid-resistant group and received leukocytapheresis therapy once a week for 5 weeks. Of patients who had a remission, the corticosteroid-responsive group continued to have the conventional therapy and the corticosteroid-resistant group were given leukocytapheresis once every 4 weeks for at least 2 years as maintenance therapy. Results: Remission was induced by 5 weeks of leukocytapheresis in 23 of the 25 (92%) patients with corticosteroid-resistant active UC. The number of days required to achieve remission of UC was fewer in patients who received leukocytapheresis than in those who did not. Follow-up study of the patients who had remission showed similar relapse rates at 2 years in the patients who received leukocytapheresis and those given high doses of prednisolone alone. Conclusions: Leukocytapheresis is an effective treatment of acute corticosteroid-resistant UC but does not prevent the recurrence of UC.

The Relationship Between Iron Deficiency in Patients with Helicobacter Pylori‐Infected Nodular Gastritis and the Serum Prohepcidin Level

Helicobacter pylori (H. pylori) is recognized as a causative agent for unexplained iron‐deficiency anemia (IDA). We evaluated many background factors influencing an iron‐deficiency state in adult patients with various H. pylori‐infected upper gastrointestinal tract diseases.

Simultaneous Combined Balloon-occluded Retrograde Transvenous Obliteration and Partial Splenic Embolization for Portosystemic Shunts

PURPOSE To evaluate the efficacy and safety of simultaneous combined balloon-occluded retrograde transvenous obliteration (B-RTO) and partial splenic embolization (PSE) for gastric varices and/or hepatic encephalopathy. MATERIALS AND METHODS B-RTO was performed in 19 consecutive patients with gastric varices and/or hepatic encephalopathy, of whom 10 received simultaneous combined B-RTO and PSE (group 1) and nine received B-RTO monotherapy (group 2). To evaluate the safety of these techniques, we analyzed 20 patients who received PSE monotherapy during the same period as a control group (group 3). Outcomes were retrospectively assessed. RESULTS No significant differences were observed in baseline characteristics among the three groups except for significantly lower platelet counts and larger spleen volumes in group 3. In all cases in groups 1 and 2, gastric varices disappeared and hepatic encephalopathy improved after treatment. Procedure times were not significantly different between groups 1 and 2 (P = .7435). In group 1, the volume of sclerosing agent required for B-RTO was significantly lower (P = .0355) and exacerbation of esophageal varices was significantly less frequent (P = .0146) than in group 2. Few serious complications occurred in patients who received combined therapy. CONCLUSIONS This study indicates that concomitant PSE may help diminish the increase in portal venous pressure after B-RTO for portosystemic shunts, and may allow a reduction in the volume of hazardous sclerosing agent used. It is worth evaluating the efficacy of simultaneous B-RTO and PSE in a prospective study.

Correlation between serum phospholipase A2 IIA levels and histological activity in patients with ulcerative colitis

HeadingAbstract Background and aims. Phospholipase A2 (PLA2) participates in the regulation of phospholipid metabolism and biosynthesis of eicosanoids, serum levels of PLA2 are suggested to reflect the disease activity in patients with ulcerative colitis (UC). We examined the relationship between histological disease activity and serum levels of PLA2 IIA and also clarified mucosal production sites of PLA2 IIA by immunohistochemistry. Patients and methods. Serum samples from 44 patients with UC, 125 with Crohns diseases (CD), and 68 controls were studied. Biopsy specimens of colonic mucosa obtained from 23 patients with UC were used for assessment of histological activity. The histological score was determined active (1) or inactive (0), and the sum of each histological score from ten segments of the large intestine was assessed as disease activity. The levels of PLA2 IIA in sera were measured by a radioimmunoassay kit using a specific monoclonal antibody; immunohistochemical study was performed using the same monoclonal antibody. Results. The serum PLA2 IIA levels in patients with UC and CD were significantly higher than those of controls. Serum PLA2 IIA levels in UC were closely correlated with histological disease activity. Immunohistochemical study showed the production of PLA2 IIA by the polymorphonuclear cells, macrophages, and colonic epithelial cells. Conclusion. Serum PLA2 IIA is a good candidate for assessing disease activity in UC as one of clinical laboratory tests.