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We report a case of living-related partial liver transplantation for decompensated hepatitis B without reactivation of hepatitis B in the following 30 months, and we analyze the factors that indicate a favorable prognosis for transplantation. The 42-year-old female patient received continuously administered lamivudine before transplantation, and hepatitis B virus immunoglobulin (HBIG) from the anhepatic phase to the present. Currently, she shows a normal aminotransferase level and is negative for hepatitis B surface antigen and hepatitis B virus (HBV) DNA by polymerase chain reaction amplification. Sequence analysis was performed. The entire precore/core region and part of the polymerase region of HBV were sequenced by a direct sequencing method after polymerase chain reaction. No specific mutation was found in these regions. These observations show that the key factors in the long-term successful treatment of this patient appear to be the combination therapy of lamivudine and HBIG that the patient received from around the time of the transplantation. Furthermore, the lack of specific mutations, including lamivudine resistant-mutations, is likely to represent an additional factor in the effectiveness of this treatment.

14歳時に自己免疫性肝炎様所見を呈しautoimmune hepatitis (AIH) と診断され22歳時にPrimary sclerosing cholangitis (PSC) と診断された症例を経験した. 発症時の検査成績は, T-Bil 2.1mg/dl, GPT 323KU, ALP 2, 051IU, γ-GTP 398mU/ml, γ-glob. 4.5g/dl, 抗核抗体640倍陽性 (homogeneous pattern), 抗ミトコンドリア抗体陰性, 肝生検ではpiecemeal necrosis及びbridging necrosis, 形質細胞とリンパ球の浸潤を認めAIHと診断された. プレドニン40mgより治療開始され胆管系酵素以外の生化学dataは改善した. 18歳時より通院を中止, 21歳時に食道静脈瘤破裂にて近医受診した.22歳で肝移植目的に当院へ転院, T-Bil 18.2mg/dl, GPT 39KU, ALP 897IU, γ-GTP 141 mU/ml, γ-glob. 3.1g/dl, 抗核抗体320倍陽性 (homogeneous pattern), 抗ミトコンドリア抗体陰性, perinuclear antineutrophil cytoplasmic antibody (PANCA) 陰性で, 肝組織では, 胆汁性肝硬変, fibrous obliterative cholangitis, ductopeniaを認め, ERCにてPSCの確定診断が得られた. 若年発症のPSCは, 自己免疫的要因が強く, 留意すべき病態と考え報告する.

Non-alcoholic fatty liver disease (NAFLD) is considered to be a hepatic manifestation of metabolic syndrome. NAFLD has become an important public health issue because of its high prevalence. NAFLD consists of two clinicopathological entities: simple steatosis, which generally follows a benign non-progressive clinical course, and non-alcoholic steatohepatitis (NASH), which may progress to cirrhosis and hepatocellular carcinoma. The diagnosis of NAFLD is based on the following three criteria: non-alcoholic, detection of steatosis either by imaging or by histology, and appropriate exclusion of other liver diseases. Alcoholic liver disease can occur when daily alcohol consumption exceeds 20 g in women or 30 g in men. Thus, non-alcoholic indicates lower levels of these alcohol consumptions. However, there is still no clear consensus regarding the threshold alcohol consumption for defining non-alcoholic liver disease. Then, there is the strong recommendation for a change in the nomenclature, such as use of the term metabolic fatty liver and metabolic steatohepatitis. NASH has emerged as a clinicopathological entity, and even now, a liver biopsy remains the gold standard for making a definitive diagnosis. However, liver biopsy has several drawbacks. In general practice, NAFLD is a convenient-to-use term for the diagnosis and management of these patients, and serum biomarkers that indicate the severity of fibrosis serve as clinically useful tools for the identification of NAFLD in patients with bridging fibrosis or cirrhosis. In the future, improved understanding of the pathogenesis of NASH and new technologies may contribute to the diagnostic process and provide reliable, non-invasive alternatives to liver biopsy.

The aim of this study was to elucidate the clinical and histological features, response to corticosteroid therapy and long-term outcome of primary biliary cirrhosis (PBC) with features of autoimmune hepatitis (AIH).Among 280 PBC patients under ursodeoxycholic acid administration, we identified 28 patients with AIH features fulfilling the following criteria: sustained high levels of serum aminotransferases and high immunoglobulin G levels with positivity for antinuclear antibodies or anti-smooth muscle antibodies (ASMA) and/or histological features of moderate to severe interface hepatitis or moderate to severe lobular hepatitis. We analyzed PBC patients with AIH features, focusing mainly on therapeutic responses to corticosteroids.Patients with PBC with AIH features included 26 women (93%). Their median age was 55 years, and the median follow-up period was 7.5 years. Eight of these 28 patients were not actually treated with corticosteroids due to medical conditions. Among the 20 patients receiving corticosteroid therapy, 15 were responders and five were non-responders. A high alkaline phosphatase (ALP) level, negativity for ASMA and positivity for gp210 were identified as risk factors for lack of a response to corticosteroid therapy. Among 28 PBC patients with AIH features, the responders to corticosteroids had an excellent prognosis, while those who could not be treated with corticosteroids and non-responders to corticosteroids had a poor outcome.Patients with PBC with AIH features benefit from corticosteroid therapy. Features of PBC such as high ALP level, negativity for ASMA and positivity for gp210 appear to predict a poor response to corticosteroids.

The distinctive histologic findings in acute hepatitis A, B, and C suggest that different immunological mechanisms are involved in the pathogenesis of these diseases. This study was undertaken to define the immune response in each type of acute hepatitis by identification of the intrahepatic lymphocyte subsets. Thirty paraffin-embedded liver biopsy specimens from 10 patients with acute hepatitis A, 10 patients with acute hepatitis B, and 10 patients with acute hepatitis C were evaluated. Immunohistochemical staining was performed by the indirect immunoperoxidase technique using the following monoclonal or polyclonal antibodies: CD45RO, CD20-cy, CD57, and Mac387. Inflammatory infiltrates varied from specimen to specimen. However, CD45RO+ memory T cells were the predominant infiltrating mononuclear cells in all specimens. In the portal areas, CD45RO+ memory T cells were the most prominent in AHC, followed by AHA and AHB, and the difference between AHC and AHB was statistically significant. CD20-cy+ B cells were seen mainly in the portal areas, and were significantly less common in AHB than in AHA and AHC. In addition, the ratio of CD20-cy+ B cells to CD45RO+ memory was significantly lower in AHB than in the other types of acute hepatitis. The necrotic areas in all specimens contained mainly CD45RO+ memory cells in association with a few CD57+ NK and T cells or CD20-cy+ B cells. Our study revealed differences of the intrahepatic lymphocyte subsets among the various types of acute hepatitis, but the meaning of these differences is presently unknown. Therefore, further studies are required to fully elucidate the mechanism of the immune response in acute hepatitis.

Background Biomarker for usefulness in diagnosing advanced fibrosis in nonalcoholic fatty liver disease (NAFLD) is expected. In order to discover novel biomarkers for NAFLD and its pathogenesis, we performed matabolomics screening. Methods (1) The initial cohort was 44 NAFLD patients. (2) This validation cohort was 105 NAFLD patients, 26 primary biliary cirrhosis (PBC) patients, and 48 healthy controls. Using capillary electrophoresis and liquid chromatography with mass spectrometry, we analyzed low molecular weight metabolites in these groups. Results 1. In the initial cohort, we found 28 metabolites associated with advanced fibrosis. Among them, 4 sulfated steroids showed the greatest difference. A decrease of dehydroepiandrosterone sulfate (DHEA-S) and 5α-androstan-3β ol-17-one sulfate (etiocholanolone-S) was observed with the progression of fibrosis. Furthermore, 16 hydroxydehydroepiandrosterone sulfate (16-OH-DHEA-S) increased with the progression of fibrosis. 2. In the validation cohort, the decrease of DHEA-S and etiocholanolone-S, as well as the increase of 16-OH-DHEA-S, with the progression of fibrosis was confirmed. The 16-OH-DHEA-S/DHEA-S ratio and 16-OH-DHEA-S/etiocholanolone-S ratio were even more strongly associated with the grade of fibrosis. Among PBC patients, 16-OH-DHEA-S tended to be higher in stages 3 and 4 than in stages 1 and 2. However, levels of DHEA-S, etiocholanolone-S, and the two ratios were not associated with the stage of PBC. Conclusion Several metabolic products were found to be biomarkers of fibrosis in NAFLD and could also be useful for diagnosis of this condition. Our findings suggested disturbance of hormone metabolism in NAFLD and might lead to the development of new therapy. Electronic supplementary material The online version of this article (doi:10.1007/s00535-013-0766-5) contains supplementary material, which is available to authorized users.

Background To clarify the carcinogenic factors associated with steatohepatitis, we investigated the characteristic features of hepatic and extrahepatic malignancies in patients with cirrhotic nonalcoholic steatohepatitis (NASH-LC) and cirrhotic alcoholic liver disease (ALD-LC). Methods A total of 72 patients with NASH-LC and 85 with ALD-LC (both biopsy-proven steatohepatitis without hepatocellular carcinoma [HCC]) were assessed with regard to the development of hepatic and extrahepatic malignancies. Risk factors for HCC were analyzed. Results During follow-up, 10 NASH-LC patients and 6 ALD-LC patients developed HCC. The 5-year HCC development rate was similar for these 2 groups, being 10.5% in the NASH-LC group and 12.3% in the ALD-LC group. After adjusting for age and gender, the HCC development rates were also similar. Risk factors for HCC in the NASH-LC group were older age, higher γ-GTP level, and higher Child–Pugh score as determined by Cox hazards analysis. Regarding risk factors in the ALD-LC group, no risk factor was found by Cox hazards analysis, although diabetes mellitus led to a significantly higher HCC rate by log-rank test (p = 0.013). Regarding extrahepatic cancer, only 1 NASH-LC patient (1.4%) developed endometrial cancer. In contrast, 7 ALD-LC patients (8.2%) had other cancers (p = 0.052). Conclusions Comparison between NASH-LC and ALD-LC revealed similar HCC development curves. However, the risk factors for HCC and extrahepatic malignancies differed between the 2 diseases. In ALD-LC, the incidences of HCC and extrahepatic cancer are similar. When treating LC patients with NASH or ALD, the risk factors and extrahepatic malignancies associated with ALD-LC should be assessed.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer-related mortality (World Health Organization. Mortality database, 2010). According to the most recent nationwide Japanese registration data, primary liver cancer ranks fourth for men and sixth for women as cause of death from malignant neoplasm (Journal of Health and Welfare Statistics. Health and Welfare Statistics Association, 2009).

原発性胆汁性肝硬変(PBC)と診断後,5年の生存期間中に腹部超音波,経皮経肝門脈造影(PTP)及び肝組織所見を経過観察し得た1症例を報告する.症例は62歳の女性で無症候性PBCと診断後臨床経過中に腹水や静脈瘤などの門脈圧亢進症状をきたし肝硬変への移行が疑われた.肝組織所見は門脈域の線維化が主たる所見であり,死亡時Scheuer分類のIII期であった.PTPでは肝内門脈末梢枝にIPHに類似した異常を認め,2回のPTPの比較で門脈病変の進行が明らかであった.本症にて得られた組織およびPTP所見は本症の門脈圧亢進の発生機序を考察する上で興味がもたれた.

To elucidate gender differences and the influence of obesity and/or metabolic syndrome-related fatty liver on alcoholic liver disease (ALD), we analyzed characteristic features of ALD. We investigated 266 ALD patients (224 males and 42 females) without hepatocellular carcinoma stratified by gender and the presence of cirrhosis. Male and female patients matched for age and total ethanol intake were also analyzed. A diagnosis of ALD was based on alcohol intake (70 g daily for more than 5 years), clinical features, and exclusion of other liver diseases. The prevalence of obesity, lifestyle-related diseases, and psychological disorders were assessed. The prevalence of psychological disorders showed a significant gender difference among all ALD patients (12% in males versus 43% in females, P 0.001), as well as in patients matched for age and total ethanol intake. There were 156 cirrhotic patients. Absence of dyslipidemia, presence of diabetes, and high total ethanol intake were selected as independent predictors of cirrhosis in males by multivariate analysis after excluding laboratory data of liver function tests. The prevalence of obesity was significantly lower in cirrhotic male patients than in non-cirrhotic male patients (34% vs. 20%, P = 0.023). Among females, there were no significant predictors of cirrhosis on multivariate analysis after eliminating liver function tests. The prevalence of obesity and diabetes was similar in non-cirrhotic and cirrhotic female patients. The prevalence of psychological disorders was 47% in cirrhotic females with ALD. Obesity was not common in cirrhotic ALD. Psychological disorders seem to be important for female ALD.

Background. In hepatocellular carcinoma (HCC), a high prevalence of hepatitis C virus antibody (anti-HCV) has been reported, indicating that it may be an important etiologic factor in the pathogenesis of HCC. In this study, the authors investigated the prevalence of anti-HCV in HCC patients, as well as the same prevalence in patients with cholangiocarcinoma (CC) and combined hepatocellular–cholangiocarcinoma (combined HCC-CC), to study the clinicopathologic features of anti-HCV–positive cases. Methods. The authors examined 141 patients with primary liver cancer who were pathologically diagnosed as having HCC (121 cases), CC (13 cases), or combined HCC-CC (7 cases). Hepatitis B surface antigen (HBsAg) and anti-HCV were measured in these patients. Results. Of 121 HCC cases, 85 (70.3%) were found to be anti-HCV positive, 16 (13.2%) were HBsAg positive, and 5 (4.1%) were both anti-HCV and HBsAg positive. In 13 cases with CC and in 7 with combined HCC-CC examined, 4 (30.8%) and 5 (71.4%), respectively, were anti-HCV positive. Conclusions. The anti-HCV–positive rate was high in combined HCC-CC as well as in HCC. These three types of primary liver cancer, which were anti-HCV positive, shared two common features: male dominance and high incidences of complication with liver cirrhosis.

An association between primary sclerosing cholangitis (PSC) and chronic ulcerative colitis (CUC) is well known in Western countries, but there have been no reports on this association in Japan. We reviewed 163 consecutive CUC patients (91 males and 72 females) diagnosed from 1984 to 1990 at Tokyo Women's Medical College. Abnormal liver function tests were found in 42 patients with CUC (25.8%), but chronic liver disease was only diagnosed in seven patients (4.3%). Among these seven patients, there were four with PSC, one with small-duct PSC, one with transfusion-associated chronic hepatitis and one with Type B liver cirrhosis. No relationship was found between the documented colonic manifestations of CUC and the presence of PSC. The four PSC patients did not have a longer history of CUC at the time of diagnosis of PSC than CUC patients without PSC. At the time of PSC diagnosis, two patients were asymptomatic, one presented with right upper quadrant pain, and the other had fatigue. Three patients were diagnosed as having CUC before the onset of PSC (range 2-13 years), and the other patient had both diseases simultaneously. All four had a good prognosis. Thus PSC was the most common chronic liver disease associated with CUC in our series, and it was present in all our CUC patients with alkaline phosphatase levels exceeding twice the upper limit of normal and mild transaminase elevation.

症例は59歳男性.職業は溶接業.トロトラスト,塩化ビニールモノマー(VCM),素などの直接の汚染歴なし.昭和56年より肝障害と食道静脈瘤を指摘され,昭和59年,腹部超音波検査にて,肝内に占拠性病変を認めた為,精査目的で入院.入院時,肝脾腫,及び食道,胃静脈瘤,血小板減少を認め,各種画像診断上,肝には左葉内側区域と右葉にそれぞれ約7×10cm, 5×7cm程度の塊状の腫瘍が認められた.入院3ヵ月後に肝不全,呼吸不全にて死亡.剖検所見では,腫瘍は肉眼的には血管腫様で,総織学的には紡錘形の腫瘍細胞の充実性増生と,部分的には血管腫様の管腔形成を認め,肝血管肉鍾と診断.他臓器(肺及び骨髄)の転移巣も同様の所見であった.また,非腫瘍部の一部には,Edmondson III型の肝細胞癌も顕微鏡的に観察された.非腫瘍部には肝硬変はなく,門脈枝の硬化,異常血行路を認め,特発性門脈圧亢進症(IPH)に類似した組織像であった.

特発性門脈圧亢進症(IPH)として経過を観察されていたが,再度の肝生検で先天性肝線維症(CHF)と診断された48歳女性の1例を経験したので報告する.1990年全身倦怠感を主訴に受診.CHFを疑わせる家族歴はなく,脾腫軽度,貧血はないが,血小板は8.6万と減少していた.門脈圧亢進症としては,巨大脾腎シャントを認めた.原因となるべき肝疾患を認めず,腹腔鏡,腹部エコー,CTにて肝硬変は否定された.肝生検組織は細断されており診断は困難であったが,炎症性変化のない門脈域の線維化を認めた.以上より,IPHと診断した.1991年シャント閉鎖術施行目的で再度肝精査し,この時の肝組織で,胆管増生,門脈の低形成を認め,CHFと診断した.CHFは,本邦において今日までに約70例の報告があり,20歳以下では吐下血で発症することが多く,30歳以上では,健診などで偶然に発見されることが多く,CHFによる死亡は認めなかった.中高年に至ったCHFの中には本例のように診断の困難な例もあり,腹腔鏡・肝生検による慎重な診断が必要である.

Aim: To clarify the usefulness of colestimide in patients with nonalcoholic steatohepatitis (NASH) with hyperlipidemia. Methods: In an open-label randomized controlled trial, 17 NASH patients with hyperlipidemia received colestimide (3 g/day) for 24 weeks. There were 21 control patients. All patients received lifestyle modification therapy. Efficacy was assessed based on metabolic profile, insulin resistance, transaminases, serum hepatic fibrosis markers, adipokine levels, visceral fat on computed tomography (CT), and the fatty liver grade on CT. NASH patients with moderate to severe steatosis by histology were also evaluated separately. Results: Baseline clinical characteristics of the two groups were similar. Both groups experienced a significant decrease of BMI with no difference between them. However, visceral fat decreased significantly more in the colestimide group (P = 0.046). Aspartate aminotransferase (AST) showed a significantly greater decrease in the colestimide group compared with the control group (P = 0.042). In patients with moderate to severe histological steatosis, there were significant differences between the two groups regard to HbA1c, transaminases, and hyaluronic acid (P = 0.018 for HbA1c, P = 0.003 for AST, P = 0.042 for alanine aminotransferase, and P = 0.042 for hyaluronic acid). Steatosis significantly improved in patients in the colestimide group who had fatty liver on CT (P = 0.049). In the colestimide group, abdominal distension and/or constipation were seen, but mostly tolerable, no other clinical or laboratory adverse events associated with the use of this medicine were not observed. Conclusions: Colestimide seems to increase the efficacy of lifestyle modification in NASH patients with hyperlipidemia. Its beneficial effects were more prominent in NASH patients with moderate to severe histological steatosis.

Single nucleotide polymorphisms (SNPs) of the adiponectin gene have been reported to be associated with insulin resistance and the prevalence of type-2 diabetes. We investigated the SNPs of adiponectin in non-alcoholic fatty liver disease (NAFLD) patients. One hundred nineteen patients histologically diagnosed as having NAFLD and 115 control subjects were examined. Adiponectin SNP sites were investigated at +45 of exon 2 and at +276 of intron 2; these sites have been thought to be associated with diabetes or insulin resistance. Regarding the +276 SNP, the frequency of G/G tended to be higher in NAFLD patients than in controls, but not significantly. Among females only, however, the G/G frequency was significantly higher in NAFLD patients. As for the +45 SNP, in the severe fibrosis group, the frequency of G/G homozygotes was significantly higher than that in the mild fibrosis group, and G/G homozygotes of the +45 SNP proved by multivariate analysis to be an independent factor in severe fibrosis. In NAFLD patients with adiponectin +45 G/G, homeostasis model assessment-insulin resistance was significantly higher than in NAFLD patients without adiponectin +45 G/G. Adiponectin SNPs were found to be associated with the progression of liver fibrosis and insulin resistance, suggesting that adiponectin SNPs might play roles in the occurrence and progression of NAFLD.

Aim Because the procedure of balloon-occluded retrograde transvenous obliteration (B-RTO) causes extensive thrombosis of the major shunt that connects the spleen and gastric/renal venous systems, an increase in portal pressure is unavoidable. The aim of the present study was to assess the long-term outcome of B-RTO, including changes in esophageal varices. Methods B-RTO was conducted in 22 patients with gastric varices, who were divided according to the severity of esophageal varices at baseline; there were no esophageal varices (n = 7), F(1) varices (n = 11), and F(2) varices (n = 4). The outcome measures included the development/worsening of esophageal varices after B-RTO and survival rates. Results The cumulative bleeding-free probability for all 22 patients at 3 years after B-RTO was 100%. The overall 3-year survival was 94.4%. Seven patients who had no esophageal varices prior to B-RTO did not develop any after the procedure. Seven (63.6%) of the 11 patients with stage F(1) esophageal varices prior to B-RTO showed no changes in the varices after B-RTO, while two patients progressed to F(2) varices and two developed F(3) varices. The cumulative treatment-free probability of the esophageal varices at 24 months after B-RTO was 100% for patients without esophageal varices at baseline, 80.8% for patients with pre-existing F(1) varices, and 75% for those with pre-existing F(2) varices. Conclusion Although the B-RTO procedure is considered useful for the treatment of gastric varices, changes in hemodynamics due to obliteration of this major shunt must be taken into account and observed closely.

Aim: We described the recurrence of primary biliary cirrhosis (PBC) after living donor liver transplantation (LDLT) (Liver Transplantation, 7, 2001: 588). However, since the follow-up period in that study was insufficiently long (median 35.5 months), we performed a long-term study to further characterize recurrence of PBC after LDLT. Patients: From 1991 to 2006, 15 patients with end-stage PBC underwent LDLT at Tokyo Women's Medical University. Of these patients, we studied 8 PBC patients (age 29 to 51 years, all females) who survived LDLT for more than 5 years. The follow-up period for these patients ranged form 68 to 120 months. Immunosuppression was maintained with tacrolimus and prednisone. Laboratory examinations performed in every patient and donor before LDLT included routine biochemical studies, antimitochondrial antibody (AMA) by immunofluorescence (IF), anti-M2 by enzyme-linked immunosorbent assay as well as antinuclear antibody (ANA) by IF, and immunoglobulin. After LDLT, the same laboratory examinations were performed in patients every 6 months. Liver biopsy was performed when patients exhibited clinical or biochemical signs of graft dysfunction. In addition, protocol biopsy was performed every 1 to 2 years after LDLT. Results: At the time of LDLT, all patients had end-stage cholestatic liver failure. Seven patients were positive for AMAand anti-M2 while 1 patient was negative for these markers but strongly positive for ANA. Donors were blood relatives in 6 cases, and 2 donors who were not blood relatives still exhibited multiple HLA matches with the recipients. At the end of the study in May 2006, all patients were doing well. On laboratory examination, mild abnormal liver function test results were found in 4 patients: 3 were probably due to recurrence of PBC, 1 resulted from nonalcoholic steatohepatitis. Comparison of the AMA titer between before LDLT and the most recent follow-up visit showed an increase in three patients, a decrease in two patients and no change in three patients. In contrast, the ANA titer increased in five patients. Histologically, strong evidence of recurrent PBC was found in 4 patients, and findings compatible with PBC were present in 2 additional patients. Conclusions: Although the number of our patients is small, our findings confirm that PBC can recur at high frequency after LDLT. However, this complication has not developed to advanced stages and has not caused appreciable symptoms in our patients, all of whom have a good quality of life.

Aims: To evaluate the efficacy and mechanism of combined pantethine and probucol therapy in Japanese patients with non-alcoholic steatohepatitis (NASH), liver function, serum cytokines, serum adiponectin and liver biopsy findings were investigated. Methods: Sixteen patients with NASH and hyperlipidemia were treated with pantethine (600 mg/day) and probucol (500 mg/day) for 48 weeks. Results: The mean pretreatment aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were 66 IU/L and 113 IU/L, respectively, which showed a significant decrease with treatment to 33 IU/L and 51 IU/L (P

Aim: Transforming growth factor (TGF)-beta is a cytokine which induces apoptosis of hepatocellular carcinoma (HCC) cells. However, some HCC cells show resistance to the induction of apoptosis by TGF-beta. The analyses of the molecular mechanism underlying these phenomena may be related with the mechanisms of acquirement of drug-resistance. Thus, we examined molecularly the apoptosis-resistant HCC cells which we previously established, especially emphasizing on the analyses of the stem-cell marker expression in this study. Methods: Previously established HuH7R cells, which acquired a resistance to TGF-beta-induced apoptosis after a long-term culture under small amount of TGF-beta, and control parental HuH7 cells, were used. Apoptosis rate and cell cycle state of these cells after TGF-beta treatment were determined by a flow-cytometry. The expression levels of stem-cell surface markers, such as epithelial cell adhesion molecule (EpCAM) and CD133, and those of transcriptional factors: Oct4, HNF3alfa, HNF4beta, and C/EBPbeta, were analyzed by fluorescent labeling using specific antibodies, at 24hrs and 48hrs after TGF-beta treatment. Results: 1) HuH7 cells showed apoptosis after 48hrs of TGF-beta treatment, whereas HuH7R did not show apoptosis but entered cell-cycle arrest. 2) The expression levels of CD133 were not affected at both 24hrs and 48hrs after TGF-beta treatment, while EpCAM expression levels increased from 24hrs, in both HuH7 and HuH7R cells. 3) The expression levels of Oct4, HNF3alfa, HNF4beta, and C/EBPbeta were also increased in HCC cells. However, those expression profiles were almost comparable in between HuH7 and HuH7R cells. Conclusions: The acquisition of the resistance to TGF-beta-induced apoptosis in HCCs were not related with the expression changes of stem-cell markers. The acquisition of drug-resistance in HCC cells was considered through mechanisms other than the changes of the cellular differentiation status of HCC cells. Citation Format: Tomomi Kogiso, Etsuko Hashimoto, Kazuhisa Kodama, Yuichi Ikarashi, Nobuyuki Torii, Makiko Taniai, Katsutoshi Tokushige, Keiko Shiratori. Resistance to TGF-beta-induced apoptosis is not associated with the de-differentiation in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 354. doi:10.1158/1538-7445.AM2015-354

In this study, it was investigated whether previous hepatitis B virus (HBV) infection might be associated with carcinogenesis of hepatocellular carcinoma (HCC) with alcoholic liver disease (ALD). (Method) We compared two HCC groups with ALD, one with previous HBV infection and the other without. In addition, ALD with HCC and without were compared. (Results) The two HCC groups did not differ in terms of age, sex, liver function and liver histology. About 90% of both HCC groups had septal fibrosis or liver cirrhosis. Regarding the comparison of ALD with and without HCC, age and cumulative alcohol were significantly higher in ALD with HCC. The frequency of HBc antibody tended to be higher in ALD with HCC, but the difference was not significant. (Conclusion) There was no difference in HCC with ALD between with and without previous HBV infection. In ALD with severe fibrosis, old age and heavy cumulative alcohol intake, it was important to be on the lookout for the occurrence of HCC.

Background: Soluble tumor necrosis factor receptor (sTNFR) is reported to be a good indicator of TNF production and severity in inflammatory diseases. To investigate the clinical significance of sTNFR in non-alcoholic steatohepatitis (NASH), we measured the serum levels of soluble TNF receptor-1 and -2. Patients and Methods: We analyzed 82 NASH patients and 15 healthy subjects (control). The grades of fibrosis, inflammation and fatty deposit were evaluated on liver biopsy. The serum levels of sTNFR-1 and were measured by ELISA. Results: There was significant correlation between platelet count and sTNFRs. The titers of sTNFR-2 in NASH patients with diabetes mellitus (DM) were higher than in those without DM. The serum levels of sTNFRs in NASH patients with advanced fibrosis were increased compared with those of control and NASH patients with low-grade fibrosis. Soluble TNFRs in patients with moderate inflammation were increased compared to those of control and patients with mild inflammation. There was no significant association between fatty deposit grade and sTNFRs. Conclusion: Soluble TNFRs have a sienificant correlation with fibrosis in NASH.

Summary Aims The aim of our study was to elucidate the characteristics and natural history of Japanese nonalcoholic fatty liver disease (NAFLD). Patients and methods Two hundred and forty-seven patients were diagnosed as having biopsy-proven NAFLD at Tokyo Women's Medical University or an affiliated hospital from 1990 to June 2004. Biopsies were scored for the severity of steatosis, necro-inflammation, and fibrosis according to modified Brunt criteria. We assessed the clinicopathological features and natural history of NAFLD in patients stratified by the stage of their fibrosis. Univariate and multivariate logistic analyses were performed, and the diagnostic ability was assessed by the area under the receiver operating characteristic curve. Results Clinicopathological features : The median age of the patients was 53 years, with a range from 10 to 89 years. There were 130 males and 117 females. Histologically, 46 patients were classified as F3 (bridging fibrosis), and 43 patients had F4 (cirrhosis). Females and older patients were more common in the F3–4 patients. Most of the F3–4 patients showed mild elevation of transaminases with significant deterioration of liver function tests compared with F0–2 patients. Ten patients were simultaneously diagnosed as having cirrhotic NASH and hepatocellular carcinoma (HCC). Natural history : During follow-up (median 44 months) of the F3–4 patients, 10 patients developed liver-related morbidity and five patients developed HCC. In the F3–4 patients, the 5-year cumulative incidence of HCC was 20%. Eight patients died (two of liver failure, four of HCC and two of other carcinomas). Serum markers for detecting F3 – 4 : Serum hyaluronic acid levels can accurately evaluate NAFLD patients with F3–4. Conclusions The most important consequence of NAFLD patients with advanced fibrosis was HCC. Regular screening for this complication is extremely important.

We assessed the clinicopathological features of nonalcoholic fatty liver disease (NAFLD) in patients stratified by the stage of fibrosis. One-hundred and ninety-three patients were diagnosed as having NAFLD at Tokyo Women’s Medical University or an affiliated hospital, from 1990 to September 2003, and their clinical data were collected prospectively. The diagnosis of NAFLD was based on the following criteria: (1) the presence of steatosis (affecting >30% of hepatocytes) or steatohepatitis, with steatohepatitis being defined by detection of steatosis (affecting >10% of hepatocytes), inflammatory infiltrates, and ballooning degeneration with or without Mallory bodies, pericellular fibrosis, and perivenular fibrosis; (2) intake of less than 20g of ethanol per week; and (3) appropriate exclusion of other liver diseases. We assessed the clinicopathological features of NAFLD in patients stratified by the stage of fibrosis. The median age of the patients was 53 years, with a range of 10 to 83 years. There were 96 males and 97 females. Eighty-five percent of the patients had at least one of the features of the metabolic syndrome. Histologically, 29 patients had established cirrhosis. In regard to severe fibrosis, females and older patients were more common. There were correlations between serum hyaluronic acid level, type IV collagen level, or platelet count and the stage of fibrosis. Eight patients developed hepatocellular carcinoma. Except for 1 F3 patient, they all had cirrhosis. Liver biopsy is invasive, expensive, and not suitable for all NALFD patients. Because we found correlations between serum fibrosis markers, or platelet count and the stage of fibrosis, these markers may be useful to identify NASH with severe fibrosis among NAFLD patients.

Alcoholic hepatitis and nonalcoholic steatohepatitis (NASH) show different clinical features with similar liver histology, but both disorders may progress to cirrhosis and hepatocellular carcinoma (HCC). HCC arising in alcoholic liver disease (ALD) or NASH, without hepatitis B or C virus infection, has been a rare observation, and there are no studies comparing the characteristics of ALD and NASH patients with HCC. Therefore, we compared the characteristics of ALD and NASH patients with HCC.A total of 1202 patients received a diagnosis of HCC at Tokyo Women's Medical University from 1989 to 2003, and their clinical data were collected prospectively. A clinical diagnosis was made to diagnose ALD, and clinical and histological changes were required to diagnose NASH. Of these patients, 88 received a diagnosis of HCC arising from ALD. Among them, a biopsy specimen was obtained in 50 patients (ALD-HCC group). We compared the clinical and histological characteristics of 50 ALD and 8 NASH patients (NASH-HCC group) associated with HCC. They all were negative for hepatitis virus infection by serological methods.The most significant difference between these groups was sex. Women were significantly more common in the NASH-HCC group (6% vs. 63%; p0.0001). The median age was 65 years in the ALD-HCC group and 68 years in the NASH-HCC group. The risk factors for NASH all were high in the NASH-HCC group. However, liver function tests were similar in these groups. In the ALD-HCC group, 46 (92%) patients showed severe fibrosis; 2 had septal fibrosis and 44 had cirrhosis. All patients in the NASH-HCC group showed severe fibrosis, and seven had cirrhosis.Severe fibrosis might be an important risk factor for HCC. Patients who have ALD or NASH with cirrhosis may develop HCC. This seems to occur in a sufficient number of cases to warrant regular screening for this complication.

Cholangiocarcinomas are usually fatal neoplasms originating from bile duct epithelia. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy, including cholangiocarcinoma. However, many cholangiocarcinoma cells are resistant to TRAIL-mediated apoptosis. Thus, our aim was to examine the intracellular mechanisms responsible for TRAIL resistance in human cholangiocarcinoma cell lines. Three TRAIL-resistant human cholangiocarcinoma cell lines were identified. All of the cell lines expressed TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) and TRAIL-R2/DR5. Expression of TRAIL decoy receptors and the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) was inconsistent across the cell lines. Of the antiapoptotic Bcl-2 family of proteins profiled (Bcl-2, Bcl-xL, and Mcl-1), Mcl-1 was uniquely overexpressed by the cell lines. When small-interfering-RNA (siRNA) technology was used to knock down expression of Bcl-2, Bcl-xL, and Mcl-1, only the Mcl-1-siRNA sensitized the cells to TRAIL-mediated apoptosis. In a cell line stably transfected with Mcl-1-small-hairpin-RNA (Mcl-1-shRNA), Mcl-1 depletion sensitized cells to TRAIL-mediated apoptosis despite Bcl-2 expression. TRAIL-mediated apoptosis in the stably transfected cells was associated with mitochondrial depolarization, Bax activation, cytochrome c release from mitochondria, and caspase activation. Finally, flavopiridol, an anticancer drug that rapidly down-regulates Mcl-1, also sensitized cells to TRAIL cytotoxicity. In conclusion, these studies not only demonstrate that Mcl-1 mediates TRAIL resistance in cholangiocarcinoma cells by blocking the mitochondrial pathway of cell death but also identify two strategies for circumventing this resistance.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a potential chemotherapeutic agent for cancer, is not thought to be hepatotoxic. We have recently demonstrated, however, that bile acids increase TRAIL-R2/DR5 expression in a human liver cell line and render these cells susceptible to TRAIL-mediated apoptosis. These data suggest TRAIL may be hepatotoxic in cholestasis. The aim of this study was to directly assess TRAIL hepatotoxicity in bile duct-ligated mice, a model of extrahepatic cholestasis. Bile duct-ligated mice (3 days) were used for these studies. TRAIL-R2/DR5 expression was assessed by real-time and immunoblot analysis. The TRAIL death-inducing signaling complex (DISC) was evaluated by immunoprecipitation and immunoblot techniques. Bile duct ligation increased both liver TRAIL-R2/DR5 mRNA and protein expression (10-fold). Following TRAIL administration (60 microg/mouse, i.v.) to bile duct ligation (BDL) mice, terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive hepatocytes, liver tissue caspase 3-like activity, and serum alanine aminotransferase values increased significantly compared with vehicle-treated BDL mice. The effect of TRAIL on the liver was direct, as the TRAIL DISC (Fas-associated death domain and procaspase 8 protein) was detected in liver tissue. TRAIL-mediated hepatocyte apoptosis in bile duct-ligated mice was associated with significant hepatotoxicity, as assessed by histopathology, although there was no animal mortality. In conclusion, these data define conditions under which TRAIL is hepatotoxic.

Background & Aims: Primary sclerosing cholangitis (PSC) predisposes individuals to cholangiocarcinoma; however, the molecular mechanisms involved in the carcinogenesis process remain unclear. Because p16 INK4a inactivation has been implicated in cholangiocarcinoma, our aims were to examine PSC cholangiocytes for p16 INK4a gene mutations. Methods: We studied 4 patient groups: PSC patients without cholangiocarcinoma (n = 10), patients with PSC-associated cholangiocarcinoma (n = 10), non-PSC controls (n = 10), and disease controls with primary biliary cirrhosis (n = 10). Cholangiocytes and hepatocytes were isolated from tissue sections using laser capture microdissection. Genomic DNA was extracted, and the promoter region and the 3 exons for p16 INK4a were amplified by PCR and directly sequenced. Results: In the promoter region, 8-point mutations in 5 PSC cases and 14 mutations in 8 cholangiocarcinoma cases were observed. In exon 1, 1 PSC patient and 3 cholangiocarcinoma patients had point mutations. In contrast, no case had a mutation in exon 2 or 3. Mutations were not detected in cholangiocytes from control patients or primary biliary cirrhosis patients nor in hepatocytes from any of the groups; these data indicate that the observed base changes were disease specific and not genetic polymorphisms. Several of the promoter mutations (4 of 8) dramatically decreased promoter activity (>50% reduction in luciferase activity) in a reporter gene assay. Conclusions: The results show that functional point mutations in the p16 INK4a promoter region likely contribute to the initiation/progression of cholangiocarcinoma in PSC. Promoter mutations in CpG islands may function as a methylation equivalent phenomenon resulting in gene inactivation. GASTROENTEROLOGY 2002;123:1090-1098

Background & Aims: Hepatocyte apoptosis and fibrosis are both features of liver injury. However, the potential mechanistic link between these 2 processes remains obscure. Our aim was to ascertain if Fas-mediated hepatocyte apoptosis promotes liver fibrogenesis during extrahepatic cholestasis. Methods: Wild-type and Fas-deficient lymphoproliferation (lpr) mice underwent bile duct ligation. Liver injury was assessed by quantitating hepatocyte apoptosis with the terminal deoxynucleotide transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay and determining serum ALT values. mRNA expression was quantitated using real-time polymerase chain reaction technology. Liver fibrosis was assessed by digital image analysis of Sirius red–stained sections. Results: In 3-day bile duct ligated (BDL) animals, TUNEL-positive hepatocytes and serum ALT values were reduced in lpr versus wild-type animals. Likewise, hepatic mRNA transcripts for α-smooth muscle actin and platelet-derived growth factor receptor-β (initiation phase of stellate cell activation) and transforming growth factor β1 mRNA, collagen 1α, and tissue inhibitor of matrix metalloproteinases (perpetuation phase of stellate cell activation) were also reduced in 3-day BDL wild-type mice compared with lpr mice. Finally, in 3-week BDL mice, immunoreactivity for α-smooth muscle actin and Sirius red staining for collagen were significantly less in lpr compared with wild-type animals. Conclusion: Fas-mediated hepatocyte injury is mechanistically linked to liver fibrogenesis. These observations suggest that inhibition of Fas-mediated apoptosis may be a therapeutic antifibrogenic strategy in cholestatic liver diseases. GASTROENTEROLOGY 2002;123:1323-1330

We describe six patients with non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). From 1990 to 2001, we treated 82 patients with NASH and observed six patients (three men and three women, aged 56-72 years) in this group who were referred with HCC or developed the complication during follow-up. In five of these six patients, NASH was associated with obesity (cases 3, 4 and 5), hyperlipidemia (case 5), or diabetes mellitus (cases 1, 3 and 6). We confirmed the presence of HCC by ultrasonography-guided tumor biopsy or surgery except in case 3 where we diagnosed the tumor by ultrasonography, computed tomography and selective hepatic arteriography. The carcinomas measured 1.5-6.0 cm in diameter and three were well differentiated. When HCC was diagnosed, cirrhosis was present in all instances. Four of the six tumor patients also had esophageal varices but only one patient had a history of variceal bleeding and ascites. Treatment of HCC consisted of surgery (cases 1 and 5), transcatheter arterial embolization or infusion and/or percutaneous ethanol injection (cases 2, 3, 4, and 6). In patients with NASH cirrhosis, the development of treatable HCC is sufficiently common to warrant regular screening for this grave complication.

Aim: Some pluripotent genes and epithelial cell adhesion molecule (EpCAM) have been reported as cancer stem cell markers in hepatocellular carcinoma (HCC). Nanog was recently reported to regulate self-renewal of cancer stem cells though the insulin-like growth factor pathway in HCC. However, the expression status of the pluripotent stem cell genes and their clinicopathological significance in HCC from different etiologies remain unexplored. Here, we compared the expression status of stem cell markers in HCCs derived from hepatitis C virus (HCV)-related liver disease and from nonalcoholic steatohepatitis (NASH), and also determined the clinical characteristics of Nanog-positive HCCs in NASH patients. Methods: Resected tissues from 21 NASH-related HCC (NASH-HCC) patients with histologically proven steatohepatitis and 24 HCV-related HCC (HCV-HCC)CV-HCC)HCV patients were analyzed with all patients' written informed consent. The NASH group was defined as patients who consumed less than 20 g/day of alcohol. The expression status of Nanog, Oct4, Sox9, and EpCAM were determined by immunohistochemistry. The clinicopathological features of Nanog-positive NASH-HCC patients were analyzed. Results: 1) Nanog was strongly expressed in 14.3/8.3% of NASH-HCC/HCV-HCC patients, and in 66.7/25% of noncancerous tissues of NASH/HCV (p Conclusions: The expression status of stem cell markers differed between NASH- and HCV-related liver diseases. The expression of Nanog and EpCAM in HCC were mutually exclusive and their expression in noncancerous liver tissues may be an independent risk factor in NASH- and HCV-related HCCs, respectively. Citation Format: Tomomi Kogiso, Etsuko Hashimoto, Kazuhisa Kodama, Maki Tobari, Noriko Matsushita, Nobuyuki Torii, Makiko Taniai, Katsutoshi Tokushige, Keiko Shiratori. Differential expression of cancer stem cell markers and their clinicopathological features in hepatocellular carcinoma patients with different etiologies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3878. doi:10.1158/1538-7445.AM2014-3878

We describe the recurrence of primary biliary cirrhosis (PBC) in recipients of living liver transplants. We are not aware of similar previous reports. Because most donors for living liver transplantation (LLT) are blood relatives with close HLA matches, the recurrence of PBC in transplant recipients might offer additional insights in the pathogenesis of the condition. We studied 6 women (age, 29 to 61 years) with PBC who survived LLT for at least 1 year. Tests for antimitochondrial autoantibody (AMA), antipyruvate dehydrogenase complex-E2, immunoglobulin G (IgG) anti-M2, and IgM anti-M2 had confirmed the diagnosis. Donors were blood relatives in 5 instances, and one donor who was not a blood relative still had multiple HLA matches with the recipient. After LLT, we observed a decrease in AMA titers, but within 1 year, these titers increased again in 5 of the 6 patients to pre-LLT levels or greater. Immunoblotting analysis of the anti-M2 protein profile failed to show loss of bands and showed new bands in 3 of 6 patients. Histologically, strong evidence of recurrent PBC was found in 2 patients, and findings compatible with PBC were present in 1 additional patient. All 6 patients are doing well, without symptoms of recurrent PBC (median time post-LLT, 35.5 months; range, 12 to 50 months).

Proliferating cell nuclear antigen (PCNA), also known as cyclin, is an auxiliary protein of DNA polymerase-delta and is found only in the nuclei of proliferating cells in the late G1 and S phases. The proliferation of hepatocellular carcinoma (HCC) by immunohistochemical staining for PCNA using paraffin sections of 20 surgically resected HCC specimens was analysed. The mean percentage of PCNA-positive nuclei in the HCC tissue was 10.3% in grade I of Edmondson and Steiner's classification, 25.5% in grade II, 28.4% in grade III and 41.5% in grade IV. In early HCC, we observed only a few PCNA-positive tumour cells. However, PCNA-positive nuclei were numerous in the tumour thrombi found in portal vein branches, in regions of extracapsular tumour growth, and in the inner nodules of tumours with a nodule-in-nodule formation. Proliferating cell nuclear antigen positivity was correlated with an increase of the nucleocytoplasmic ratio of tumour cells as determined by image analysis. Our findings showed that PCNA positivity was correlated with the histological grade and invasiveness of HCC, suggesting that this antigen may be used as an indicator to predict tumour invasion in patients with HCC.

Hepatocyte apoptosis and liver fibrosis are key teamres of cholestatic liver injury. Emerging data implicate a lysosolnal, cathepsin B-dependent pathway of apoptosis in bepatocytes. In this pathway, catbepsin B is released from lysosomes and tuggers mitochondrial dyshmction l%wever, the contribution of this pathway to hver injury and the relationship ot apoptosis to fibrosis remains unclear. Our Aim was to ascertain if inactivation of cathepsin B attenuates hvcr injury and fibrogenesis dunng extrahepatic cholestasis METHODS: Wild type (catB *s ~), cathepsin B-deticient (knockout) (catB~); and R 3032 (a catbepsin B inhibitor) treated catB */* mice undement bile duct ligation, Liver injury was examined by quantitating hepatocyte apopmsis with the TUNEL assay and determining serum ALT values, mRNA expression iur markers of stellate cell activation, a4MA, and fibrogenic actiwty, TGF-b and collagen I, was quantitated using real time PCR technolo~'. Liver fibrosis was assessed by digital image analysis ot Sirras red stained sections RESULTS: TUNEL positive hepamcytes and serum Allvalues were significandy reduced in cattY: and R 3032-treated catB +j+ 3-day BDL mice vs, catB +~ BDL mice, Consistent with these observations, mitochondrial cytochn)me c release, a marker for the mitochondrial pathway of apoptosis, was reduced in catB e and in R 3032-treated catB++ compared to catB ++ BDL mice, Stellate cell activation, as assessed by a-SMA was 14.0-fold greater in catB j+ vs catB v and 3-fold greater than in R 3032treated cazB +j+ BDL mice. Collagen al(I) was 102-told greater in catB ~+ vs. catB / BDL mice and 76-told greater in catB ~ ~ vs R 3032-treated ca~B +~ BDL mice. Serum bile acid and bilimbin values were mrtually identical in nil three experimental groups indicating that the above diliFrences could not be ascribed to difterences in cholestasis. Finally, in 2 week BDL mice, Sirius red staining for hepatic collagen deposition was significantly reduced in catB ~ mice compared to wdd type animals In CONCLUSION these findings not only support a prminnent tom tor the lysosomal pathway of apoptosis in cholestatic liver injury, but also link hepatocyte apoptosis to liver fibrogenesis These observations suggest cathepsin B inactivation or inhibition may be a therapeutic antifibrogenic strategy" in cholestatic liver diseases.