Masakazu Yasumaru

Increased frequency of HLA DR13 in hepatitis C virus carriers with persistently normal ALT levels

The aim of this study was to clarify the relationship between human leukocyte antigen DR allele distribution and the degree of liver cell injury of hepatitis C virus (HCV) carriers in Japan. The subjects, 68 HCV carriers, were divided into two groups according to the laboratory data and liver histology. Those in the asymptomatic carrier group (n = 19) had normal ALT levels persistently for 8–153 months (mean 25.7 months) and were diagnosed histologically as normal liver, nonspecific reactive hepatitis or chronic persistent hepatitis. Those in the chronic active hepatitis group (n = 49) had elevated ALT levels and were diagnosed histologically with chronic active hepatitis. The human leukocyte antigen DR alleles of all subjects were defined using the polymerase chain reaction restriction fragment length polymorphism method. The expression of human leukocyte antigen class I antigen and intercellular adhesion molecule 1 on the hepatocyte membrane were also examined in 14 patients from each group using an indirect immunohistochemical method. The frequency of DR13 (42.1%) in the asymptomatic carrier group was significantly higher (Pc < 0.003) than that of the chronic active hepatitis group (4.1%). There were no significant differences for the other DR alleles. The frequencies of expression of human leukocyte antigen class I antigen and intercellular adhesion molecule 1 on the hepatocyte membrane of the asymptomatic carrier group were significantly less than those of the chronic hepatitis group (64% vs. 100% P < 0.05, 29%; vs. 71% P < 0.05, respectively), although there was no significant difference in the serum HCV‐RNA titer between the two groups (106.4±1.1 vs. 106.5±0.7 copies/mL). These results demonstrate that the cellular immune response of the asymptomatic carrier group is less activated than the response of the chronic active hepatitis group and that HLA DR13 may be closely associated with this low activity of hepatitis among HCV carriers.

Involvement of bone marrow-derived cells in healing of experimental colitis in rats

Bone marrow is reported to contain hematopoietic stem cells and other adult somatic stem cells that have phenotypes of cells composing tissues other than bone marrow. To explore the implication of bone marrow‐derived cells in the treatment of inflammatory bowel diseases, experimental colitis was induced in wild‐type rats after transplantation of bone marrow from transgenic rats expressing green fluorescence protein (GFP). Chronic colitis was induced 21 days later using 30 mg 2,4,6‐trinitrobenzenesulfonic acid (TNBS). Control rats received saline. At 28, 56, and 224 days after TNBS administration, rats were euthanized, and tissues were removed and processed for paraffin‐embedded sections. Cells derived from bone marrow were identified by immunohistochemistry using anti‐GFP antibody. To identify the phenotypes of the cells expressing GFP, we conducted serial‐section analysis and double‐staining analysis using antibodies against cytokeratin (epithelial cells) or vimentin (interstitial cells). In the present study, GFP‐positive, bone marrow‐derived cells occupied 37.6% and 4.25% of the colonic epithelium at 28 days and 56 days after the induction of TNBS‐colitis, respectively. Also, significant amounts of mucosal and submucosal interstitial cells were derived from the bone marrow. These findings showed that a large amount of bone marrow‐derived cells were involved in regeneration of the colon after experimental colitis in rats.

IgG Oligosaccharide Alterations Are a Novel Diagnostic Marker for Disease Activity and the Clinical Course of Inflammatory Bowel Disease

BACKGROUND AND AIMS:Patients with inflammatory bowel disease (IBD) share several immunologic similarities with rheumatoid arthritis (RA). Patients with RA have significantly increased levels of serum agalactosyl immunoglobulin G (IgG). Our aim was to investigate the clinical significance of analyzing the oligosaccharide structure of serum IgG in patients with IBD.METHODS:Serum IgG oligosaccharide structures were analyzed using high-performance liquid chromatography in 60 patients with Crohns disease (CD), 58 patients with ulcerative colitis (UC), 27 healthy volunteers (HV), and 15 disease controls (DC). The activity and mRNA level of beta-1,4-galactosyltransferase (Beta4GalT) in antibody-secreting cells were investigated in these subjects.RESULTS:The agalactosyl fraction of the fucosylated IgG oligosaccharides (G0F/G2F) in CD and UC was significantly greater than that in HV and DC (P < 0.001). The percentage of subjects with a high G0F/G2F in CD, UC, HV, and DC was 72%, 33%, 0%, and 0%, respectively. G0F/G2F, which is significantly correlated with disease severity in both CD and UC, had higher sensitivity to diagnose IBD compared with anti-Saccharomyces cerevisiae antibody. Moreover, G0F/G2F was significantly correlated with the prognosis of UC patients: patients with a high G0F/G2F did not maintain long-term remission. The activity and mRNA level of Beta4GalT were significantly elevated in UC but not in CD.CONCLUSIONS:G0F/G2F is a potentially effective diagnostic marker of disease activity in both CD and UC, and of the clinical course in UC. A pathophysiologic difference between CD and UC was also demonstrated.

Efficiency of bone marrow-derived cells in regeneration of the stomach after induction of ethanol-induced ulcers in rats

BackgroundBone marrow contains hematopoietic stem cells, nonhematopoietic mesenchymal stem cells, and several precursor cells for osteoblasts, chondrocytes, adipocytes, myocytes, hepatocytes, and even neural cells. Research findings indicate that multipotent stem cells in the adult body may be used to recover the lost functions of damaged tissues. This study examined the involvement of bone marrow-derived cells in the regeneration of the stomach after experimental gastric ulcers were produced in rats.MethodsWe transplanted the bone marrow of transgenic rats that expressed green fluorescence protein (GFP) throughout the body. Twenty-one days after the bone marrow transplantation (BMT), gastric ulceration was induced, using absolute ethanol. Control animals received saline. After various observation periods, rats harboring GFP-positive bone marrow-derived cells were killed, and the tissues were removed and processed to prepare paraffin-embedded sections. Cells expressing GFP were identified by conventional immunohistochemistry, using anti-GFP antibody. To identify whether cells expressing GFP were epithelial cells or interstitial cells such as fibroblasts, serial sections were examined with anti-cytokeratin antibody or anti-vimentin antibody, respectively. Furthermore, to confirm that cells expressing GFP were epithelial cells or interstitial cells, we used double-staining analysis with anti-GFP antibody or anti-cytokeratin antibody, respectively.ResultsGFP-positive, bone marrow-derived cells were found in the cytokeratin-positive gastrointestinal epithelium, as well as among vimentin-positive interstitial cells. Interestingly, the proportions of GFP-positive, cytokeratin-positive epithelial cells and vimentin-positive interstitial cells were significantly greater in the ethanol-treated damaged stomachs than in the saline-treated controls.ConclusionsThe present study clearly demonstrates that bone marrow-derived cells are involved in the regeneration of the stomach after ethanol-induced ulcers in rats.

Cyclo-oxygenase-2 inhibitors suppress epithelial cell kinetics and delay gastric wound healing in rats

Background and Aims : The present study examined the effects of NS‐398, a specific cyclo‐oxygenase‐2 inhibitor, on gastric mucosal cell kinetics and gastric wound healing following acid‐induced injury.

Synergistic antitumor effects of celecoxib with 5-fluorouracil depend on IFN-γ

Cyclooxygenase‐2 (COX‐2) inhibitors are effective chemopreventive agents against colorectal cancers. For treatment of advanced cancers, combination of COX‐2 inhibitors and chemotherapy has been attempted, but the results are still controversial. In the present study, the effects of the COX‐2 inhibitor celecoxib on the anticancer potential of chemotherapy, and its mechanisms of action were investigated in point of the angiogenesis, using an advanced cancer model in mice. BALB/c mice were inoculated with colon 26 cells. After the allograft grew up to 5 mm in diameter, the animals received celecoxib, 5FU, or a combination of 5FU and celecoxib (5FU/celecoxib). After 21‐days of the treatment, 5FU/celecoxib significantly inhibited the tumor growth and the tumor vessel density compared with the other groups. Celecoxib, 5FU or 5FU/celecoxib significantly suppressed the VEGF content in tumor tissues. 5FU/celecoxib also enhanced IFN‐γ levels in tumor tissue, which could be involved in the potent antitumor effects of 5FU/celecoxib. This hypothesis was proven, because in IFN‐γ knockout (IFN‐γ−/−) mice, the inhibitory effects of 5FU/celecoxib on angiogenesis and tumor growth were significantly impaired compared with that in wild type mice. These results suggest that celecoxib enhances the antitumor effect of 5FU against an advanced colon cancer model by suppressing angiogenesis. In addition to VEGF, IFN‐γ has pivotal roles in tumor suppression induced by celecoxib.

A New Prognostic System for Hepatocellular Carcinoma Including Recurrent Cases : A Study of 861 Patients in a Single Institution

Objective To manage hepatocellular carcinoma (HCC) patients surviving for a long term, the treatment strategy for recurrent cancer is as important as that for the initial treatment. However, no prognostic scoring system has been available for patients with HCC recurrence. The purpose of this study was to develop a new staging system for deciding the treatment strategy not only for first-time diagnosed patients but also for recurrent patients. Methods A total of 861 cases diagnosed at our single institution from 1993 to 2003 were included. Overall survival was the only end point. The Cox model was used for multivariate analyses. Results As of August 2004, 344 cases (59%) had died. Overall median survival time was 41 months. For multivariate Cox regression analysis, independent predictive factors of survival were the number of recurrences, the Child-Pugh score, 3 nodules less than 3 cm and none of vascular invasion, and the α-fetoprotein level. A simple scoring system was thus developed, assigning scores (0/1) to the 4 covariates of the final model. Compared with the other scoring systems, the new scoring system has a greater discriminant ability. Conclusions We concluded that our scoring system can serve as a new prognostic system that reflects the spread of HCC, treatment response, and liver function. It should be very useful as the only method which can be applied for patients with recurrence.

Cultured bone marrow cell local implantation accelerates healing of ulcers in mice

BackgroundThe therapeutic potential of bone marrow (BM)-derived cells in ulcers is not known. This study aimed to clarify (1) cell types that are derived from the BM which infiltrate ulcers; (2) whether BM-derived cells or gastric myofibroblasts can be used for cell transplantation to treat ulcers; and (3) the phenotypes of such transplantable cells.Methods(1) Wild-type mice were transplanted with BM cells of green fluorescent protein (GFP)-transgenic mice. Acetic acid-induced gastric ulcers were produced in mice after BM transplantation. (2) BM cells and gastric myofibroblasts were isolated from GFP-transgenic mice. Bone marrow cells attached to plastic dishes were selected for expansion. Gastric ulcers were induced, and BM-derived cells, myofibroblasts, or phosphate-buffered saline were injected around ulcers. The ulcer healing process was examined macroscopically and histologically. (3) Expression of growth factors and cytokines in transplantable cells was examined by reverse transcriptase-polymerase chain reaction.Results(1) GFP-positive cells with interstitial phenotypes were observed at the ulcerated area. (2) Ulcer healing was significantly promoted by the injection of BM-derived cells compared to controls on day 7, but not on day 3. The BM-derived cells were observed in the tissue surrounding the ulcer. However, myofibroblasts were not found. (3) The BM-derived cells expressed hepatocyte growth factor, transforming growth factor-β1, and other stromal factors before transplantation, and had mesenchymal phenotypes after transplantation.ConclusionsBM-derived cells are involved in the ulcer healing. BM-derived cells, but not myofibroblasts, are locally implantable to ulcers. Thus, BM-derived cells can be transplanted to accelerate ulcer healing.

Upregulation of GRAIL is associated with remission of ulcerative colitis

Abrogating tolerance against unidentified antigens is a critical step in the pathogenesis of ulcerative colitis (UC). T cell anergy, one of the main mechanisms of tolerance, has been shown to be induced by E3 ubiquitin ligases, such as gene related to anergy in lymphocytes (GRAIL), Itch, and c-Cbl in mice. However, it is not well known whether these E3 ligases play roles in human diseases. The pathophysiological role of the E3 ligases in patients with UC was investigated. At first, the expression of GRAIL, Itch, and c-Cbl in human anergic T cells was analyzed by quantitative RT-PCR and Western immunoblotting. Next, the mRNA expression of the E3 ligases was analyzed in peripheral CD4+ T cells of 20 patients with UC and 10 healthy volunteers (HV). mRNA expression was analyzed in patients with active UC before and after treatment with prednisolone and leukocytapheresis. Anergic human CD4+ T cells expressed significantly higher levels of GRAIL, Itch, and c-Cbl than nonanergic cells. GRAIL expression was significantly higher in patients with UC in remission than in patients with active disease and in HV (P < 0.01). The level of GRAIL expression was also significantly increased in patients with active disease whose clinical activity index scores improved after treatment (P < 0.05). There were no significant differences in Itch and c-Cbl expression among patients with active UC, patients with UC in remission, and HV. These data suggest that GRAIL plays an important role in maintaining remission in patients with UC.

Ameboma mimicking colon cancer

A 45-year-old man was referred to our endoscopy center because of hematochezia and abdominal pain; fecal occult blood test results were positive. He had no personal or family history of inflammatory bowel disease or colon cancer, but he was homosexual. Laboratory data, including antiHIV antibody and tumor markers, did not demonstrate any abnormal findings. Bacterial culture showed normal bacterial flora. Colonoscopy with indigo carmine dye spraying showed a large ulcerative lesion in the cecum with elevated margins suggesting colonic cancer with submucosal invasion (A, B). Histology demonstrated surface exudates showing trophozoites of Entamoeba histolytica with scattered ingested erythrocytes; no malignant cells were evident (C, periodic acid-Schiff, orig. mag. 200). A serum anti-amoebic antibody was positive. A diagnosis of ame-