Mutsumi Tsuchishima

Usefulness of a Combined Evaluation of the Serum Adiponectin Level, HOMA-IR, and Serum Type IV Collagen 7S Level to Predict the Early Stage of Nonalcoholic Steatohepatitis

OBJECTIVE:Since nonalcoholic steatohepatitis (NASH) may progress to cirrhosis, it is important to differentiate NASH from simple steatosis, especially in its early stages. However, a liver biopsy cannot be performed in all patients with nonalcoholic fatty liver disease (NAFLD). We herein investigated whether serum biochemical markers are useful for predicting early-stage NASH.METHOD:Nineteen patients with simple steatosis and 66 patients with early-stage NASH (stage 1–2 in Brunts criteria) were studied. The area under the receiver operating characteristic curve (AUC) was used to illustrate the diagnostic ability of serum biochemical parameters to distinguish between simple steatosis and early-stage NASH.RESULTS: The serum adiponectin level was found to be significantly lower with early-stage NASH group (3.6 μg/mL) than in the simple steatosis group (6.0 μg/mL) (P < 0.001). The AUC was high (0.765) in the early-stage NASH group, and it was also the highest among all other markers. The sensitivity of the serum adiponectin level in the diagnosis of early-stage NASH was 68%, which was higher than for any other factors, while its specificity was 79%. The corresponding sensitivity and specificity of HOMA-IR were 51% and 95%, respectively. For type IV collagen 7S, sensitivity was 41% and specificity 95%. The sensitivity of the combination of three markers was 94%, with a specificity of 74%.CONCLUSION: Approximately 90% of the patients with early-stage NASH can be predicted by a combined evaluation of the serum adiponectin level, HOMA-IR, and serum type IV collagen 7S level.

Association between functional promoter polymorphisms of macrophage migration inhibitory factor (MIF) gene and ulcerative colitis in Japan.

Macrophage migration inhibitory factor (MIF) is a key proinflammatory mediator. Two functional polymorphisms were identified in the promoter region of MIF gene. We attempted to clarify the associations between these polymorphisms and ulcerative colitis (UC). The study was performed in 111 patients with UC and 209 subjects without UC. We employed the PCR-SSCP method to detect gene polymorphisms. Overall, 5/5-CATT genotype was a decreased risk for the development of UC (OR, 0.51; 95% CI, 0.26-0.99). In addition, 7/7-CATT genotype was significantly associated with chronic continuous phenotype and distal colitis phenotype (OR, 5.49; 95% CI, 1.19-25.3, and OR, 6.10; 95% CI, 1.32-28.2, respectively), whereas 5/5-CATT genotype had an inhibitory effect on the development of UC after 20years of age (OR, 0.33; 95% CI, 0.14-0.82). On the other hand, G-173C polymorphism did not affect the susceptibility to and the phenotypes of UC. Our results suggested that tetranucleotide CATT repeat of MIF gene promoter may be associated with the development of UC and the severity of inflammation in patients with UC.

Serum Osteopontin Predicts Degree of Hepatic Fibrosis and Serves as a Biomarker in Patients with Hepatitis C Virus Infection

Background & Aims Osteopontin (OPN) is a matricellular protein that upregulates during pathogenesis of hepatic fibrosis. The present study was aimed to evaluate whether serum OPN could be used as a biomarker to assess the degree of hepatic fibrosis in patients with hepatitis C virus (HCV) infection. Methods Needle biopsy was performed on HCV patients and scored as zero fibrosis (F0), mild fibrosis (F1), moderate fibrosis (F2), severe fibrosis (F3) and liver cirrhosis (F4) based on Masson’s trichrome and α-smooth muscle actin (α-SMA) staining. Serum OPN levels were measured using ELISA and correlated with the degree of fibrosis. Furthermore, the OPN values were correlated and evaluated with platelets count, serum hyaluronic acid (HA), and collagen type IV and subjected to receiver operating characteristic (ROC) curve analysis. Results Serum OPN levels were remarkably increased from F0 through F4 in a progressive manner and the differences were significant (P < 0.001) between each group. The data were highly correlated with the degree of hepatic fibrosis. The ROC curve analysis depicted that serum OPN is an independent risk factor and an excellent biomarker and a prognostic index in HCV patients. Conclusions The results of the present study indicate that serum OPN levels reflect the degree of hepatic fibrosis and could be used as a biomarker to assess the stage of fibrosis in HCV patients which would help to reduce the number of liver biopsies. Furthermore, serum OPN serves as a prognostic index towards the progression of hepatic fibrosis to cirrhosis and hepatocellular carcinoma.

Binge alcohol consumption aggravates oxidative stress and promotes pathogenesis of NASH from obesity-induced simple steatosis.

The pathogenesis of nonalcoholic steatohepatitis (NASH) is a two-stage process in which steatosis is the “first hit” and an unknown “second hit.” We hypothesized that “a binge” could be a “second hit” to develop NASH from obesity-induced simple steatosis. Thirty-week-old male Otsuka Long-Evans Tokushima fatty (OLETF) rats were administered 10 mL of 10% ethanol orally for 5, 3, 2, and 1 d/wk for 3 consecutive weeks. As control, male Otsuka Long-Evans Tokushima (OLET) rats were administered the same amount of alcohol. Various biochemical parameters of obesity, steatosis and NASH were monitored in serum and liver specimens in untreated and ethanol-treated rats. The liver sections were evaluated for histopathological alterations of NASH and stained for cytochrome P-4502E1 (CYP2E1) and 4-hydroxy-nonenal (4-HNE). Simple steatosis, hyperinsulinemia, hyperglycemia, insulin resistance, hypertriglycemia and marked increases in hepatic CYP2E1 and 4-HNE were present in 30-wk-old untreated OLETF rats. Massive steatohepatitis with hepatocyte ballooning was observed in the livers of all OLETF rats treated with ethanol. Serum and hepatic triglyceride levels as well as tumor necrosis factor (TNF)-α mRNA were markedly increased in all ethanol-treated OLETF rats. Staining for CYP2E1 and 4-NHE demonstrated marked increases in the hepatic tissue of all the groups of OLETF rats treated with ethanol compared with OLET rats. Our data demonstrated that “a binge” serves as a “second hit” for development of NASH from obesity-induced simple steatosis through aggravation of oxidative stress. The enhanced levels of CYP2E1 and increased oxidative stress in obesity play a significant role in this process.

Predictors of Malignancy in Branch Duct Intraductal Papillary Mucinous Neoplasm of the Pancreas

OBJECTIVE Indication of surgery for branch duct intraductal papillary mucinous neoplasm (BD-IPMN) proposed by the consensus guidelines revised in 2012 was too complex to refer to in clinical practice. This study aimed to identify simple predictors of malignancy in BD-IPMN. METHODS Consecutive 202 patients with BD-IPMNs were enrolled. They comprised 35 patients that underwent surgery and 167 that were followed up without surgery by being regarded as benign neoplasms. Cutoff values of cyst size, main pancreatic duct (MPD) diameter, and mural nodule size were determined by receiver operator characteristic (ROC) curve. Factors that may discriminate benign from malignant BD-IPMNs were analyzed by multivariate logistic regression model. RESULTS Cutoff values of cyst size, MPD diameter, and mural nodule size were determined to be 30 mm, 6 mm, and 10 mm, respectively. Multivariate analysis demonstrated that mural nodule ≥10 mm (OR 198, 95% CI 23.1-1690, P<0.0001) and positive cytology (OR 634, 95% CI 49.1-8,190, P<0.0001) were predictors of malignancy in BD-IPMN. When BD-IPMNs with mural nodules ≥10 mm or positive cytology were diagnosed as malignant, sensitivity, specificity, and overall accuracy were 88%, 98%, and 97%, respectively. CONCLUSIONS Mural nodule ≥10 mm and positive cytology were demonstrated to be simple predictors of malignancy in BD-IPMN.

Correlation between adenosine triphosphate content and apoptosis in liver of rats treated with alcohol.

BACKGROUND Chronic alcohol consumption depresses adenosine triphosphate (ATP) synthesis and induces mitochondrial DNA (Mt-DNA) deletion. ATP content in cells may play a critical role in inducing cell death, apoptosis, or necrosis. However, it is unknown which type of cell death occurs in alcoholic liver disease. In this study, the deletions of hepatic Mt-DNA, hepatic ATP content, and the number of single-stranded DNA (ss-DNA) of hepatocytes in rats treated with ethanol were determined to elucidate the relationship among Mt-DNA deletion, ATP synthesis, and/or hepatic apoptosis. METHODS Sixteen male Wistar rats were fed with a liquid diet containing 36% ethanol (E group) or liquid diet without ethanol (C group) for 5 weeks. Hepatic ATP content was measured and the deletions of Mt-DNA encoding complexes I, IV, and V were determined in fresh liver tissue, and ss-DNA was stained in paraffin sections. RESULTS Fatty change was observed in the E group, but not in the C group. Hepatic ATP content in the E group was 0.44 micromol/g of liver, which was significantly lower than that in the C group (0.84 micromol/g of liver). However, no deletion of Mt-DNA encoding complexes I, IV, and V was detected in either the E or the C group. ss-DNA staining was clearly observed in the nuclei of hepatocytes in both groups. The number of ss-DNA-positive hepatocytes in the E group was 5.6 +/- 1.8/10,000 hepatocytes, which was significantly less than that in the C group: 20.6 +/- 4.8/10,000 hepatocytes. There was a positive correlation between hepatic ATP contents and the number of ss-DNA-positive cells. CONCLUSIONS The results suggest that mitochondrial function, at least in part ATP synthesis, was depressed before the damage of Mt-DNA by chronic ethanol consumption. Chronic ethanol consumption may not be responsible for the apoptosis of hepatocytes.

Imaging morphological changes of intraductal papillary mucinous neoplasm of the pancreas was associated with its malignant transformation but not with development of pancreatic ductal adenocarcinoma

BACKGROUND/OBJECTIVE A considerable number of branch duct intraductal papillary mucinous neoplasm (BD-IPMN) developed not infrequently pancreatic malignancy, either as part of IPMN (malignant IPMN) or as concomitant pancreatic ductal adenocarcinoma (PDAC). To date, imaging morphological changes predicting occurrence of malignancy in BD-IPMN are not well-investigated. This study aimed to evaluate the relationships between occurrence of malignancy in BD-IPMN and imaging morphological changes of the tumors observed during follow-up. METHODS 515 BD-IPMN patients with mural nodule <10 mm and negative cytology were included. 19 patients developed malignant IPMN and 8 patients developed concomitant PDAC during mean follow-up of 4.7 years. The following imaging morphological features were assessed: cyst/main pancreatic duct (MPD) diameter, occurrence of additional cyst/mural nodule. RESULTS Growth rate of cyst/MPD diameter were significantly larger in patients who developed malignant IPMN compared to those in patients whose IPMN remained benign (p = 0.013, p = 0.01). Occurrence of additional cyst/mural nodule were more frequently observed in patients who developed malignant IPMN (p = 0.009, p = 0.04). In contrast, none of the factors associated with imaging morphological changes of IPMN were shown to be significantly different between patients who developed concomitant PDAC and patients whose IPMN remained benign. Growth rate of MPD diameter and occurrence of additional cyst were independent factors associated with development of malignant IPMN (odds ratio 21.5, and 5.62, respectively). CONCLUSIONS Imaging morphological changes of IPMN, such as growth rate of MPD diameter and occurrence of additional cyst, could be indicators for development of malignant IPMN, but not for development of concomitant PDAC.

Histochemical Study of Hyaluronate in Alcoholic Liver Disease

Recently, it has been reported that serum hyaluronate (hyaluronic acid; HA) concentrations increase in various liver diseases, especially in alcoholic liver disease (ALD), and serum HA concentration has been used as a marker for hepatic fibrosis. However, it is unknown whether hepatic HA contents in ALD increase by alcohol or not. In this study, we histochemically stained HA in liver biopsy specimens obtained from ALD patients while actively drinking and after abstinence to clarify the effects of alcohol on hepatic HA contents. Liver biopsy specimens were obtained from 13 patients with ALD and 10 patients with non-ALD. In ALD patients, liver biopsy was performed twice within 3 days, and 4 to 8 weeks after abstinence when serum levels of AST and ALT normalized. HA in biopsy specimens was stained histochemically with biotinylated HA binding protein. Staining intensity of HA in liver tissue was also determined by computer-assisted imaging analyzer. HA staining was clearly observed in sinusoidal wall and fibrous regions around the portal tract and central vein in liver diseases. HA staining intensities in patients actively drinking with ALD increased markedly, compared with those in patients with non-ALD, and these intensities decreased with abstinence. These results clearly suggest that hepatic HA contents in ALD may be increased by alcohol in addition to hepatic fibrosis, and, therefore, increased HA deposition in the liver may be reversible by abstinence of alcohol.

Clinicopathological study of chronic hepatitis induced by alcohol with or without hepatitis G virus

The concept of chronic hepatitis induced by alcohol (AL-CH) has not been widely accepted, because AL-CH may be due to non-A-E hepatitis virus in heavy drinkers. Recently, hepatitis G virus (HGV) was identified as a positive-strand RNA virus related to members of the Flaviviridae family. In this study, we determined serum HGV in patients with AL-CH and analyzed the clinicopathological changes after abstinence to evaluate whether AL-CH is caused by alcohol or not. Serum samples were obtained from 16 patients with AL-CH who had neither hepatitis B nor C virus. The diagnosis was confirmed histologically. In eight patients, liver biopsy was performed twice, within 3 days and 4 to 8 weeks after abstinence. The NS3 region of the HGV genome was detected using an reverse transcriptase-polymerase chain reaction method. Serum levels of AST, ALT and -γ-glutamyltranspeptidase were measured once a week sequentially after admission. Serum HGV-RNA was detected in only one patient with AL-CH (6.3%). In all patients, including one patient with HGV, serum levels of AST, ALT and γ-glutamyltranspeptidase clearly decreased to normal levels after abstinence. Inflammatory activity in the periportal area of patients with actively drinking decreased or disappeared after abstinence for 4 to 8 weeks. These results suggest that HGV may not play an important role for development of AL-CH, and that AL-CH may be caused by alcohol itself, although a more larger number of patients with AL-CH are needed to obtain definitive conclusions.

MMP‐13 deletion decreases profibrogenic molecules and attenuates N‐nitrosodimethylamine‐induced liver injury and fibrosis in mice

Connective tissue growth factor (CTGF) is involved in inflammation, pathogenesis and progression of liver fibrosis. Matrix metalloproteinase‐13 (MMP‐13) cleaves CTGF and releases several fragments, which are more potent than the parent molecule to induce fibrosis. The current study was aimed to elucidate the significance of MMP‐13 and CTGF and their downstream effects in liver injury and fibrosis. Hepatic fibrosis was induced using intraperitoneal injections of N‐nitrosodimethylamine (NDMA) in doses of 10 μg/g body weight on three consecutive days of each week over a period of 4 weeks in both wild‐type (WT) and MMP‐13 knockout mice. Administration of NDMA resulted in marked elevation of AST, ALT, TGF‐β1 and hyaluronic acid in the serum and activation of stellate cells, massive necrosis, deposition of collagen fibres and increase in total collagen in the liver of WT mice with a significant decrease in MMP‐13 knockout mice. Protein and mRNA levels of CTGF, TGF‐β1, α‐SMA and type I collagen and the levels of MMP‐2, MMP‐9 and cleaved products of CTGF were markedly increased in NDMA‐treated WT mice compared to the MMP‐13 knockout mice. Blocking of MMP‐13 with CL‐82198 in hepatic stellate cell cultures resulted in marked decrease of the staining intensity of CTGF as well as protein levels of full‐length CTGF and its C‐terminal fragments and active TGF‐β1. The data demonstrate that MMP‐13 and CTGF play a crucial role in modulation of fibrogenic mediators and promote hepatic fibrogenesis. Furthermore, the study suggests that blocking of MMP‐13 and CTGF has potential therapeutic implications to arrest liver fibrosis.