Hepatitis B virus DNA in liver tissue and risk for hepatocarcinogenesis in patients with hepatitis C virus-related chronic liver disease. A prospective study.

AIMS: To prospectively study whether occult hepatitis B virus (HBV) infection can promote the development of hepatocellular carcinoma (HCC) in patients with hepatitis C virus (HCV)-related chronic liver disease. In addition, to evaluate the difference among HBV DNA-negative patients and patients with high and low HBV copy numbers. METHODS: A total of 167 patients with HCV-related chronic liver disease without HBV surface antigen (HBsAg) were studied. HBV DNA in liver tissue was determined using polymerase chain reaction (PCR). RESULTS: HBV DNA was detected in 9 of 167 patients (5.4%) by single PCR and in 25 patients (15.0%) by nested PCR. HCC developed in 12 of 167 patients (7.2%). Ten of 142 HBV DNA-negative patients (7.0%) and 2 of 9 patients with a high HBV copy number (22.2%) developed HCC, whereas none of 16 patients with a low HBV copy number developed HCC. The incidence rate of HCC in patients with a high HBV copy number was significantly higher than in HBV DNA-negative patients and patients with low HBV copy number. CONCLUSION: A high amount of HBV DNA in liver tissue of HBsAg-negative patients with HCV-related liver disease might be associated with HCC development.

Oral administration of taurine improves experimental pancreatic fibrosis.

BACKGROUND AND AIM: The mechanism of pancreatic fibrosis is unclear. Taurine is used in the clinical treatment of a wide variety of diseases, but its effect on improving pancreatic fibrosis is unknown. We examined whether a diet with added taurine improves pancreatic fibrosis induced by dibutyltin dichloride (DBTC) in an experimental chronic pancreatitis rat model. In addition, we examined the influence of taurine on pancreatic stellate cells. METHODS: Pancreatic fibrosis was induced by DBTC. Rats were fed a taurine-containing diet or a normal diet and were killed at 4 weeks. Pancreatic stellate cells were isolated from male Wistar rats. Cultured pancreatic stellate cells were incubated with or without taurine chloramine. Type I collagen and transforming growth factor-beta1 secretion was evaluated by ELISA, and matrix metalloproteinase activity was assessed by gelatin zymography. Interleukin-6, interleukin-2, and transforming growth factor-beta1 levels in the supernatants of pancreatic tissue homogenates were measured. RESULTS: Pancreatic fibrosis induced by DBTC was improved remarkably by the oral administration of the taurine-containing diet. Taurine chloramine decreased type I collagen, transforming growth factor-beta1, and matrix metalloproteinases 2 of the pancreatic stellate cell culture supernatant. Increased interleukin-6 and decreased interleukin-2 were found in the supernatants of the pancreatic tissue homogenates of DBTC-induced pancreatitis rats compared with other groups. CONCLUSION: The oral administration of taurine improves pancreatic fibrosis. Taurine chloramine inhibits transforming growth factor-beta1 produced from activated pancreatic stellate cells and improves pancreatic fibrosis.

Hepatitis C virus genotype distribution in Myanmar: Predominance of genotype 6 and existence of new genotype 6 subtype.

AIM: This study was performed to determine the prevalence and distribution of hepatitis C virus (HCV) genotypes in Myanmar. METHODS: A total of 1333 peripheral blood samples were collected from four different border cities of Myanmar. The anti-HCV antibody-positive serum samples were identified. HCV was genotyped by reverse transcriptase polymerase chain reaction, direct DNA sequencing and phylogenetic analysis on the partial core genome. RESULTS: The overall prevalence of HCV infection was 11.6% (154/1333). Regionally, it was 13.5% (47/349) in the north-eastern city, 12.8% (64/501) in the north-western city, 4.2% (16/380) in the southern city and 26.2% (27/103) in the western city. HCV was genotyped in 145/154 (94.2%) samples. Genotype 6 was the most prevalent genotype in this study (71/145, 49%), followed by genotype 3 (57/145, 39.3%), genotype 1 (16/145, 11%), and genotype 2 (1/145, 0.7%). Genotype 6 was mostly found in the northern cities and genotype 3 in the southern and western cities of Myanmar. Multiple HCV genotypes/subtypes were successfully characterized as 1a, 1b, 2a, 3a, 3b, 6m, 6n, and a new 6 subtype. Among them, subtype 6n was the most predominant subtype (38.6%), followed by subtype 3b (29.7%), 3a (9.6%), 6m (9%), 1b (6.9%), 1a (4.1%), new 6 subtype (1.4%) and 2a (0.7%). Subtype 6n was more widely distributed in the northern cities whereas subtype 3b was more common in the western city. The newly discovered genotype 6 subtype was from the northern cities. CONCLUSIONS: The results indicate there are regional differences of HCV genotype distribution in Myanmar. There is a distinct geographic variation from other South-East Asian countries in terms of the existence of the new genotype 6 subtype.

Three type 6 hepatitis C virus subgroups among blood donors in the Yangon area of Myanmar are identified as subtypes 6m and 6n, and a novel subtype by sequence analysis of the core region.

Previously, using phylogenetic analysis of NS5b sequences, we found that three type 6 variant subgroups (M6-1, M6-2 and M6-3) exist in Myanmar. According to the new nomenclature of hepatitis C, M6-1 and M6-2 belong to subtypes 6m and 6n, respectively, but M6-3 is unassigned. In this study, we sequenced and phylogenetically analyzed the core region of these type 6 variant subgroups. Serum samples assigned as 6m or 6n by NS5b sequence were also identified as 6 m or 6n by core region analysis. The M6-3 (sample name MYAN-3E-3) remained unassigned to a subgroup based on its core region analysis. The findings of this study suggest that either the core region or the NS5b region can be analyzed for HCV subtype classification.

Functional promoter upstream p53 regulatory sequence of IGFBP3 that is silenced by tumor specific methylation.

BACKGROUND: Insulin-like growth factor binding protein (IGFBP)-3 functions as a carrier of insulin-like growth factors (IGFs) in circulation and a mediator of the growth suppression signal in cells. There are two reported p53 regulatory regions in the IGFBP3 gene; one upstream of the promoter and one intronic. We previously reported a hot spot of promoter hypermethylation of IGFBP-3 in human hepatocellular carcinomas and derivative cell lines. As the hot spot locates at the putative upstream p53 consensus sequences, these p53 consensus sequences are really functional is a question to be answered. METHODS: In this study, we examined the p53 consensus sequences upstream of the IGFBP-3 promoter for the p53 induced expression of IGFBP-3. Deletion, mutagenesis, and methylation constructs of IGFBP-3 promoter were assessed in the human hepatoblastoma cell line HepG2 for promoter activity. RESULTS: Deletions and mutations of these sequences completely abolished the expression of IGFBP-3 in the presence of p53 overexpression. In vitro methylation of these p53 consensus sequences also suppressed IGFBP-3 expression. In contrast, the expression of IGFBP-3 was not affected in the absence of p53 overexpression. Further, we observed by electrophoresis mobility shift assay that p53 binding to the promoter region was diminished when methylated. CONCLUSION: From these observations, we conclude that four out of eleven p53 consensus sequences upstream of the IGFBP-3 promoter are essential for the p53 induced expression of IGFBP-3, and hypermethylation of these sequences selectively suppresses p53 induced IGFBP-3 expression in HepG2 cells.

Treatment of elderly patients with early gastric cancer by endoscopic submucosal dissection using an insulated-tip diathermic knife.

OBJECTIVE: In recent years, the number of elderly patients with early gastric cancer (EGC) has steadily been increasing. In our institute, endoscopic submucosal dissection (ESD) involving the use of an insulated-tip diathermic knife (IT-ESD) was introduced for the treatment of mucosal gastric carcinoma in 1996. The purpose of this study was to evaluate the effectiveness of IT-ESD for the treatment of elderly patients with EGC. MATERIALS AND METHODS: A total of 144 patients with EGC were treated at Shikoku Cancer Center in the 5-year period from January 2000 to December 2004, including 53 patients over 75 years old. The performance status (PS) for all patients was less than 2. We compared patient's backgrounds, the one-piece resection rate, complete resection (CR) rate, operation time, bleeding rate, perforation rate, blood pressure, and peripheral oxygen saturation (SpO(2)) between patients over 75 years of age (elderly group) and the remaining 91 younger patients (non-elderly group). RESULTS: The rate of having underlying disease was significantly higher for the elderly group (p<0.05) and we found no significant difference for the one-piece resection rate, CR rate, operation time, bleeding rate, and perforation rate between the 2 groups. There were also no significant differences for the frequency of the use of oxygen, pressor and depressor between the 2 groups. CONCLUSION: There was no significant difference in the outcome resulting from ESD between the 2 groups. Our study proves that ESD is a feasible treatment for elderly patients with EGC PS of less than 2.

Matrix metalloproteinase-2 in pancreatic juice for diagnosis of pancreatic cancer.

INTRODUCTION: Matrix metalloproteinase-2 (MMP-2) has an activity to degrade type IV collagen and is associated with invasion angiogenesis of malignant tumor. AIM: A diagnostic value of MMP-2 in pancreatic juice was studied in the diagnosis of pancreatic cancer. METHODOLOGY: Using gelatin zymography, active MMP-2 and proMMP-2 were determined in pancreatic juice obtained endoscopically from 12 patients with pancreatic cancer, 11 with chronic pancreatitis, and 7 control subjects. RESULTS: ProMMP-2 was detected in 12 of 12 patients (100%) with pancreatic cancer, 6 of 11 (54.5%) with chronic pancreatitis, and 3 of 7 (42.9%) controls. Active MMP-2 was detected in 11 patients (91.6%) with pancreatic cancer, 2 (18.2%) with chronic pancreatitis, and none of the control subjects. An activation ratio of MMP-2 (active MMP-2/total MMP-2) in pancreatic juice is significantly higher in pancreatic cancer (23.4 +/- 4.4%, mean +/- SE) than in chronic pancreatitis (2.1 +/- 1.7%) and controls (0%) (p < 0.01). Active MMP-2 was also detected in pancreatic juice from three cases of small pancreatic cancer (tumor <2 cm in diameter). CONCLUSION: Our observation suggests that detection of active MMP-2 in pancreatic juice using gelatin zymography may be useful for the diagnosis of pancreatic cancer.

Genome aberrations observed by restriction landmark genome scanning analysis of chromosomal DNA in various types of primary hepatocellular carcinoma.

BACKGROUND/AIMS: The genes responsible for hepatocellular carcinoma have not been identified. To identify the relevant genes of hepatocellular carcinoma, detailed and comprehensive information of genomic aberrations must be obtained. To reveal the chromosomal aberrations in hepatocellular carcinoma, we carried out a restriction landmark genome scanning analysis of various types of hepatocellular carcinoma. METHODOLOGY: Samples of various types of hepatocellular carcinoma, including two with multinodular-hepatocellular carcinomas, one hepatocellular carcinoma showing nodules in a nodule pattern, one hepatocellular carcinoma metastasized to different tissues, three small (< 2.0 cm) hepatocellular carcinomas and four large (> 5.0 cm) hepatocellular carcinomas were examined by the restriction landmark genome scanning method with corresponding non-hepatocellular carcinoma tissues. Restriction enzyme Not I was used as a landmark enzyme, Eco RV was used as a fragmentation enzyme, and Hin fI was used as a digestion enzyme in the gel for two-dimensional electrophoresis. RESULTS: We observed spot aberrations with different origins. Frequently observed spot aberrations originated from the change in the methylation status of repetitive sequences. No clear correlation between the pathological grade and the number or type of spot aberrations was observed. CONCLUSIONS: We demonstrated that major aberrations of restriction landmark genome scanning spots originated from the change of methylation status in hepatocellular carcinoma.

Efficacy of clinical pathway for the management of mucosal gastric carcinoma treated with endoscopic submucosal dissection using an insulated-tip diathermic knife.

OBJECTIVE: Recently, the cases of early gastric carcinomas which can be treated by endoscopic submucosal dissection (ESD) method have been increasing in our institute. Simple and precise guidelines for treating mucosal gastric carcinoma are necessary for improving the treatment outcome of this disease. In our institute, ESD using an insulated-tip diathermic knife (IT-ESD) was introduced for the treatment of mucosal gastric carcinoma in 1996. The purpose of this study was to evaluate the impact of a clinical pathway and standardize the treatment for mucosal gastric carcinoma treated with IT-ESD. MATERIALS AND METHODS: The Clinical Pathway and standardized of treatment for mucosal gastric carcinoma treated with IT-ESD were introduced at our institute in January 2002. We compared the length of hospitalization, total costs, hospital costs, operation time and bleeding rate during the 18 months before and after January 2002. RESULTS: There was no significant difference in the clinical characteristics of the 20 patients in the control group and the 23 patients in the pathway group. There were 9 and 13 bleeding cases in the respective groups. The mean length of hospitalization, total costs and hospital costs were significantly less for patients in the pathway group. There was no significant difference in the operation time or bleeding rate among the two groups. CONCLUSION: Our clinical pathway and the standardization of treatment for mucosal gastric carcinoma treated with IT-ESD proved effective for treating patients with mucosal gastric carcinoma and for minimizing the length of hospitalization without compromising patient care.

Cytoskeletal inhibitors, anti-adhesion molecule antibodies, and lectins inhibit hepatocyte spheroid formation.

We investigated the role of cytoskeletons, adhesion molecules, membrane-glycosylations, and proteoglycans in forming the shape of adult rat hepatocyte spheroids. Isolated hepatocytes were cultured on dishes coated with chondroitin sulfate phosphatidyl ethanolamine (CS-PE). Spheroid-forming ability was observed after adding cytoskeletal inhibitors (cytochalasin D, colchicine, okadaic acid, mycalolide B), anti-adhesion molecule antibodies (anti-E-cadherin, anti-connexin 32, anti-zo-1), a glycosphingolipid synthetic inhibitor (N-butyldeoxynojirimycin), a proteoglycan synthetic inhibitor (p-nitrophenyl-beta-D-xylopyranoside), and several lectins. Localization of actin was studied using confocal microscopy after rhodamine-phalloidin staining. Adding cytoskeletal inhibitors on the initial day resulted in weakly clustered cell aggregates rather than smoothly formed spheroids. These effects disappeared at lower reagent concentrations. When reagents were added on day 3, after the formation of spheroids, only mycalolide B was associated with an irregular spheroid surface; the others had no effect. Adding the anti-E-cadherin, anti-connexin 32 on the initial day showed inhibition of spheroid formation, but anti-zo-1 and proteoglycan synthetic inhibitor had no effects. Among the several lectins, only Wheat Germ Agglutinin (WGA), Ricinus communis Agglutinin I (RCA-I), and Concanavalin A (ConA) showed inhibition. These results suggest that cytoskeletal conformation and some adhesion molecules are necessary to form spheroids. Based on the interactions between lectins and hepatocytes in the present study, hepatocytes appear to contain an N-linked complex or N-linked hybrid glycosylated chains.

Establishment of a novel collagenase perfusion method to isolate rat pancreatic stellate cells and investigation of their gene expression of TGF-beta1, type I collagen, and CTGF in primary culture or freshly isolated cells.

In studies of the pathogenesis of pancreatic fibrosis, pancreatic stellate cells (PSCs) have recently gained attention. In the present study, we established a new collagenase perfusion method through thoracic aorta cannulation to isolate PSCs, and we studied gene expression of TGF-beta1, type I collagen, and connective tissue growth factor using primary cultured PSCs. Our method facilitated PSC isolation, and by our new method, 4.3 +/- 1.2 x 10(6) PSCs were obtained from a rat. In comparing the expression of these genes with that of hepatic stellate cells (HSCs), we observed a similar pattern, although PSCs expressed type I collagen gene earlier than did HSCs. These results suggest that PSCs may play an important role in fibrosis of the pancreas, as HSCs do in liver fibrosis; in addition, PSCs may exist in a preactivated state or may be more easily activated than are HSCs. We also isolated the PSCs from a WBN/Kob rat, the spontaneous pancreatitis rat, and compared the gene expression with that from a normal rat.

Analysis of HCV genotypes from blood donors shows three new HCV type 6 subgroups exist in Myanmar.

The prevalence of hepatitis C virus (HCV) genotypes in Myanmar in comparison with the rest of Southeast Asia is not well known. Serum samples were obtained from 201 HCV antibody-positive volunteer blood donors in and around the Myanmar city of Yangon. Of these, the antibody titers of 101 samples were checked by serial dilution using HCV antibody PA test II and Terasaki microplate as a low-cost method. To compare antibody titers by this method and RNA identification, we also checked HCV-RNA using the Amplicor 2.0 test. Most high-titer groups were positive for HCV-RNA. Of the 201 samples, 110 were successfully polymerase chain reaction (PCR) amplified. Among them, 35 (31.8%) were of genotype 1, 52 (47.3%) were of genotype 3, and 23 (20.9%) were of type 6 variants, and phylogenetic analysis of these type 6 variants revealed that 3 new type 6 subgroups exist in Myanmar. We named the subgroups M6-1, M6-2, and M6-3. M6-1 and M6-2 were relatively close to types 8 and 9, respectively. M6-3, though only found in one sample, was a brand-new subgroup. These subtypes were not seen in Vietnam, where type 6 group variants are widely spread. These findings may be useful for analyzing how and when these subgroups were formed.

Xanthine oxidase-derived free radicals directly activate rat pancreatic stellate cells.

BACKGROUND AND AIM: Free radicals are reported to be associated with fibrosis in the pancreas. It is generally accepted that pancreatic stellate cells (PSC) play an important role in pancreatic fibrosis. However, the exact role of free radicals in activation of PSC has not been fully elucidated. In the present study, using a superoxide dismutase (SOD) inhibitor, diethyldithiocarbamate (DDC) with cultured PSC, we investigated how free radicals act on the activation of PSC. METHODS: PSC were isolated from male Wister rats. Cultured rat PSC were incubated with DDC for 48 h. Intracellular SOD activity and lipid peroxidation were examined in DDC-treated PSC. Activation of PSC was examined by determining the expression of alpha-smooth muscle actin (alpha-SMA) by immunocytochemistry. The number of PSC using a hemocytometer, type I collagen secretion with ELISA and matrix metalloproteinases (MMP) activities with gelatin zymography were also examined. Secretion of transforming growth factor-beta1 (TGF-beta1) was evaluated by ELISA. The effects of the allopurinol, a xanthine oxidase (XOD) inhibitor, on PSC were also examined. RESULTS: DDC decreased SOD activity and increased lipid peroxidation products in PSC. DDC activated PSC, increasing the number of alpha-SMA positive cells, enhancing secretion of type I collagen and MMP, inhibiting PSC proliferation. Secretion of TGF-beta1, which is known to activate PSC, was increased by DDC treatment. These alterations were prevented by allopurinol. CONCLUSION: These results suggest that free radicals generated by XOD might directly activate PSC.

Camostat, an oral trypsin inhibitor, reduces pancreatic fibrosis induced by repeated administration of a superoxide dismutase inhibitor in rats.

BACKGROUND AND AIM: An oral trypsin inhibitor, camostat (CM), has a beneficial effect on chronic pancreatitis, but its mechanism is not yet fully understood. Recently, pancreatic stellate cells (PSC) have been reported to play an essential role in pancreatic fibrosis. An experimental model of pancreatic fibrosis induced by a superoxide dismutase (SOD) inhibitor (diethyldithiocarbamate [DDC]) was developed in rats. Thus, the effect of an oral trypsin inhibitor on pancreatic fibrosis and PSC was investigated. METHODS: Pancreatic fibrosis was induced in rats using DDC (DDC rats). DDC + CM rats were administered DDC, and subsequently were fed a diet containing CM. Immunohistochemistry of the pancreas was performed with monoclonal anti-alpha-smooth muscle actin (alpha-SMA) antibody and anti-desmin antibody. RESULTS: The DDC rats showed a significant increase in alpha-SMA-positive cells or desmin-positive cells compared with control rats. These significant increases in the fibrotic area improved after treatment with CM. The level of prolyl hydroxylase in the pancreas, which significantly increased as a result of DDC, decreased after treatment with CM. CONCLUSION: Camostat has a beneficial effect on pancreatic fibrosis induced by the administration of a SOD inhibitor, which inhibits the proliferation and activation of PSC.

Hepatitis B virus gene in liver tissue promotes hepatocellular carcinoma development in chronic hepatitis C patients.

The hepatitis B virus (HBV) gene has been detected in hepatocellular carcinoma (HCC) tissue negative for the hepatitis B surface antigen and positive for the hepatitis C virus (HCV) antibody, but the precise role of the HBV gene in hepatocarcinogenesis has yet to be clarified. We studied the HBV gene in liver tissue several years before the emergence of HCC. Eleven patients diagnosed with HCV-positive chronic liver disease and who developed HCC were assigned to group A. HBV DNA was detected in 8 of the 11 patients (73%). Twenty-five patients, who did not develop HCC, were selected as group B. Six of the group B patients were classified as DNA-positive (24%). The HBV DNA in liver tissue was found to be significantly related to HCC development (P < 0.01). Thus, the presence of the HBV gene in patients with chronic HCV associated-liver injury appears to promote hepatocarcinogenesis, although prospective studies are needed to confirm this result.