Shuji Iwai

In situ detection of oxidative DNA damage, 8-hydroxydeoxyguanosine, in chronic human liver disease

BACKGROUND/AIMS 8-Hydroxydeoxyguanosine (8-OHdG) is a promutagenic DNA lesion produced by oxygen radicals and is recognized as a useful marker in estimating DNA damage induced by oxidative stress. METHODS Hepatic expression of 8-OHdG was immunohistochemically investigated in control and diseased human livers. RESULTS While no positive immunolabeling for 8-OHdG was observed in control livers, 8-OHdG was widely evident in diseased livers. Nuclear expression of 8-OHdG in the hepatocytes and bile duct cells were found in various forms of chronic hepatitis. 8-OHdG-positive hepatocytes were especially abundant in the periportal area with piecemeal necrosis and prominent cell infiltration. The number of positive hepatocytes significantly increased with the progression of severity of chronic hepatitis activity (r(s)=0.68, P<0.05). In alcoholic liver disease, nuclear expression of 8-OHdG was detected in the hepatocytes in the area of alcoholic hepatitis. Regarding primary biliary cirrhosis, 8-OHdG was preferentially detected in the nuclei of injured bile ducts (11 of 12 cases, 91.7%) and occasionally (2 of 12 cases, 16.7%) in the nuclei of hepatocytes around the bile duct lesions. CONCLUSIONS These results indicate that oxidative DNA damage is common in various forms of chronic liver disease suggesting a possible link between chronic inflammation and hepatocarcinogenesis.

Usefulness of transient elastography for assessment of liver fibrosis in chronic hepatitis B: Regression of liver stiffness during entecavir therapy.

Aim:  The usefulness of transient elastography remains to be validated in chronic hepatitis B, particularly as a tool for monitoring the degree of liver fibrosis during treatment.

Physical inactivity and insufficient dietary intake are associated with the frequency of sarcopenia in patients with compensated viral liver cirrhosis.

The association between sarcopenia and nutritional status is thought to be an important problem in patients with cirrhosis. In this study, we investigated whether nutritional factors were related to sarcopenia in patients with liver cirrhosis.

Add-on combination therapy with adefovir dipivoxil induces renal impairment in patients with lamivudine-refractory hepatitis B virus.

Summary.  Combination therapy with adefovir dipivoxil (ADV) and lamivudine (LAM) is recommended for patients infected with LAM‐refractory hepatitis B virus (HBV). However, the effects of such therapy on renal function and serum phosphorus levels have not been fully evaluated. Combination therapy with ADV and LAM was given to 37 patients infected with LAM‐refractory HBV, including 17 with hepatic cirrhosis. Serum HBV DNA levels decreased to below 2.6 log10 copies/mL in 23 (62%) of 37 patients at 12 months, 25 (78%) of 32 patients at 24 months, and 16 (84%) of 19 patients at 36 months. Except for one cirrhotic patient, serum alanine aminotransferase levels were below 50 IU/L in all patients during combination therapy. Serum creatinine levels increased in 14 (38%) of 37 patients, and serum phosphate levels decreased to below 2.5 mg/mL in 6 (16%) of 37 patients during combination therapy. Patients who received combination therapy for 36 months or longer had a significantly incidence of elevated serum creatinine levels. Fanconi syndrome occurred in a 57‐year‐old woman with cirrhosis after ADV was added to LAM. Combination therapy with ADV and LAM can maintain biochemical remission in patients with LAM‐refractory HBV. However, the dosing interval of ADV should be adjusted according to renal function and serum phosphate levels in patients receiving long‐term treatment.

Noninvasive laboratory tests proposed for predicting cirrhosis in patients with chronic hepatitis C are also useful in patients with non-alcoholic steatohepatitis

BackgroundSeveral noninvasive tests have been proposed to predict cirrhosis in patients with chronic hepatitis C, but not in patients with non-alcoholic steatohepatitis (NASH). We assessed whether noninvasive laboratory tests designed to predict the risk of cirrhosis in patients with chronic hepatitis C virus (HCV) infection could be used in patients with NASH.MethodsThe subjects were 50 patients with biopsy-proved NASH and 100 age- and sex-matched patients with HCV. Aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio (AAR), age-platelet (AP) index, AST-to-platelet ratio index (APRI), cirrhosis discriminant score (CDS), and the hepatitis C antiviral long-term treatment against cirrhosis (HALT-C) model were calculated.ResultsThe areas under the receiver-operating characteristic curves of the AAR, AP index, APRI, CDS, and HALT-C model for predicting cirrhosis were respectively 0.813, 0.877, 0.786, 0.949, and 0.908 in patients with NASH and 0.555, 0.652, 0.761, 0.782, and 0.782 in patients with HCV. A CDS cutoff value of less than 5 misclassified none of the 9 patients with NASH who had cirrhosis, while a value of more than 8 misclassified none of the 41 patients with NASH without cirrhosis. With the HALT-C model, a cutoff value of less than 0.6 classified non-cirrhotic NASH, while a cutoff value of 0.97 or higher classified cirrhotic NASH. The use of CDS and HALT-C model could avoid liver biopsy for predicting cirrhosis in 60 and 48% of the patients with NASH, respectively.ConclusionsNoninvasive laboratory tests designed to predict cirrhosis in patients with HCV are also useful in patients with NASH.

Cytoglobin is expressed in hepatic stellate cells, but not in myofibroblasts, in normal and fibrotic human liver.

Cytoglobin (CYGB) is ubiquitously expressed in the cytoplasm of fibroblastic cells in many organs, including hepatic stellate cells. As yet, there is no specific marker with which to distinguish stellate cells from myofibroblasts in the human liver. To investigate whether CYGB can be utilized to distinguish hepatic stellate cells from myofibroblasts in normal and fibrotic human liver, human liver tissues damaged by infection with hepatitis C virus (HCV) and at different stages of fibrosis were obtained by liver biopsy. Immunohistochemistry was performed on histological sections of liver tissues using antibodies against CYGB, cellular retinol-binding protein-1 (CRBP-1), α-smooth muscle actin (α-SMA), thymocyte differentiation antigen 1 (Thy-1), and fibulin-2 (FBLN2). CYGB- and CRBP-1-positive cells were counted around fibrotic portal tracts in histological sections of the samples. The expression of several of the proteins listed above was examined in cultured mouse stellate cells. Quiescent stellate cells, but not portal myofibroblasts, expressed both CYGB and CRBP-1 in normal livers. In fibrotic and cirrhotic livers, stellate cells expressed both CYGB and α-SMA, whereas myofibroblasts around the portal vein expressed α-SMA, Thy-1, and FBLN2, but not CYGB. Development of the fibrotic stage was positively correlated with increases in Sirius red-stained, α-SMA-positive, and Thy-1-positive areas, whereas the number of CYGB- and CRBP-1-positive cells decreased with fibrosis development. Primary cultured mouse stellate cells expressed cytoplasmic CYGB at day 1, whereas they began to express α-SMA at the cellular margins at day 4. Thy-1 was undetectable throughout the culture period. In human liver tissues, quiescent stellate cells are CYGB positive. When activated, they also become α-SMA positive; however, they are negative for Thy-1 and FBLN2. Thus, CYGB is a useful marker with which to distinguish stellate cells from portal myofibroblasts in the damaged human liver.

Frequent detection of hepatitis B virus DNA in hepatocellular carcinoma of patients with sustained virologic response for hepatitis C virus.

Hepatocellular carcinoma (HCC) develops several years after the eradication of hepatitis C virus (HCV) by interferon therapy. Risk factors for the development of HCC are only partly understood. To elucidate the role of occult hepatitis B virus (HBV) infection in hepatocarcinogenesis in patients with sustained virologic response, the prevalences of HBV‐related makers were examined. Study group comprised 16 patients with sustained virologic response (group A) and 50 with HCV (group B). Anti‐HBc and anti‐HBs in serum were examined by enzyme‐linked immunoassay. HBV DNA in liver was examined by nested polymerase chain reaction, using primers specific for genes encoding for HBx, HBsAg, HBcAg, and HBV cccDNA. Sequence of the amplified HBV DNA for ‘a’ determinant of HBsAg was determined in HCC. Anti‐HBc was positive in 10 of 16 in group A and 25 of 50 in group B. HBV DNA in liver was detected in 12 of 16 in group A and 21 of 50 in group B (P = 0.044). In group A, HBV DNA in liver was detected frequently in patients without cirrhosis and in those with a longer period from the time of HCV eradication to the development of HCC. Mutation in ‘a’ determinant of HBsAg was found in three HCC of group A. Occult HBV infection may be one of the most important risk factors in hepatocarcinogenesis of Japanese patients with sustained virologic response. J. Med. Virol. 81:1009–1014, 2009.

Two cases of hepatocellular adenomatosis treated with transcatheter arterial embolization

Hepatocellular adenoma is a rare benign tumor of the liver. However, some complications, such as hemorrhage, rupture, and malignant transformation, have been reported previously. Surgical resection is considered to be the best choice of treatment, when adenomas are increasing in size, while resection is difficult to perform when multiple adenomas develop throughout the liver. Here, we report two cases of multiple hepatocellular adenomatosis. One patient had a history of aplastic anemia and the other had glycogen storage disease. We treated them with transcatheter arterial embolization (TAE) to prevent hemorrhage and rupture. After TAE, most parts of the adenomas showed necrotic change. These cases suggest that TAE is an effective treatment of hepatocellular adenomatosis.

Nutritional status in relation to lifestyle in patients with compensated viral cirrhosis.

AIM To assess the nourishment status and lifestyle of non-hospitalized patients with compensated cirrhosis by using noninvasive methods. METHODS The subjects for this study consisted of 27 healthy volunteers, 59 patients with chronic viral hepatitis, and 74 patients with viral cirrhosis, from urban areas. We assessed the biochemical blood tests, anthropometric parameters, diet, lifestyle and physical activity of the patients. A homeostasis model assessment-insulin resistance (HOMA-IR) value of ≥ 2.5 was considered to indicate insulin resistance. We measured height, weight, waist circumference, arm circumference, triceps skin-fold thickness, and handgrip strength, and calculated body mass index, arm muscle circumference (AMC), and arm muscle area (AMA). We interviewed the subjects about their dietary habits and lifestyle using health assessment computer software. We surveyed daily physical activity using a pedometer. Univariate and multivariate logistic regression modeling were used to identify the relevant factors for insulin resistance. RESULTS The rate of patients with HOMA-IR ≥ 2.5 (which was considered to indicate insulin resistance) was 14 (35.9%) in the chronic hepatitis and 17 (37.8%) in the cirrhotic patients. AMC (%) (control vs chronic hepatitis, 111.9% ± 10.5% vs 104.9% ± 10.7%, P = 0.021; control vs cirrhosis, 111.9% ± 10.5% vs 102.7% ± 10.8%, P = 0.001) and AMA (%) (control vs chronic hepatitis, 128.2% ± 25.1% vs 112.2% ± 22.9%, P = 0.013; control vs cirrhosis, 128.2% ± 25.1% vs 107.5% ± 22.5%, P = 0.001) in patients with chronic hepatitis and liver cirrhosis were significantly lower than in the control subjects. Handgrip strength (%) in the cirrhosis group was significantly lower than in the controls (control vs cirrhosis, 92.1% ± 16.2% vs 66.9% ± 17.6%, P < 0.001). The results might reflect a decrease in muscle mass. The total nutrition intake and amounts of carbohydrates, protein and fat were not significantly different amongst the groups. Physical activity levels (kcal/d) (control vs cirrhosis, 210 ± 113 kcal/d vs 125 ± 74 kcal/d, P = 0.001), number of steps (step/d) (control vs cirrhosis, 8070 ± 3027 step/d vs 5789 ± 3368 step/d, P = 0.011), and exercise (Ex) (Ex/wk) (control vs cirrhosis, 12.4 ± 9.3 Ex/wk vs 7.0 ± 7.7 Ex/wk, P = 0.013) in the cirrhosis group was significantly lower than the control group. The results indicate that the physical activity level of the chronic hepatitis and cirrhosis groups were low. Univariate and multivariate logistic regression modeling suggested that Ex was associated with insulin resistance (odds ratio, 6.809; 95% CI, 1.288-36.001; P = 0.024). The results seem to point towards decreased physical activity being a relevant factor for insulin resistance. CONCLUSION Non-hospitalized cirrhotic patients may need to maintain an adequate dietary intake and receive lifestyle guidance to increase their physical activity levels.

Close correlation of liver stiffness with collagen deposition and presence of myofibroblasts in non-alcoholic fatty liver disease.

Aim:  Transient elastography is known as a rapid, objective, and highly reliable technique for staging hepatic fibrosis caused by hepatitis C virus infection; however, the relationship between degree of fibrosis and the collagen deposition or the accumulation of myofibroblasts in non‐alcoholic fatty liver disease (NAFLD) remains to be further elucidated.