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Aims: The multisociety consensus nomenclature has introduced steatotic liver disease (SLD) with diverse subclassifications, which are metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction and alcohol-associated steatotic liver disease (MetALD), alcohol-associated liver disease (ALD), specific etiology, and cryptogenic. We investigated their prevalence, as per the new definition, in individuals undergoing health check-ups. Additionally, we analyzed the distribution of Fibrosis-4 (FIB-4) index and vibration-controlled transient elastography (VCTE)-derived liver stiffness measurement (LSM) for MASLD., Methods: In this cross-sectional study, 6530 subjects undergoing a health check-up in Japan were included. Conventional B-mode ultrasound was carried out on all 6530 subjects, and those with MASLD underwent VCTE., Results: The prevalence of SLD was 39.5%, comprising MASLD 28.7%, MetALD 8.6%, ALD 1.2%, specific etiology SLD 0.3%, and cryptogenic SLD 0.7%. Subjects with VCTE-derived LSM ≥8 kPa constituted 2.1% of MASLD. FIB-4 ≥1.3 showed that the sensitivity, specificity, positive predictive value (PPV), and negative predictive value for diagnosing VCTE-derived LSM ≥8 kPa were 60.6%, 77.0%, 5.3%, and 98.9%, respectively. The referral rate to specialists was 23.8% using FIB-4 ≥1.30. "FIB-4 ≥1.3 in subjects <65 years and FIB-4 ≥2.0 in subjects ≥65 years" showed higher PPV (6.7%) and lower referral rate (17.1%) compared with FIB-4 ≥1.3, but the sensitivity (54.5%) did not show adequate diagnostic capability as a noninvasive test for diagnosing VCTE-derived LSM ≥8 kPa., Conclusions: Acknowledging the selection bias in hepatology centers, we undertook this prospective health check-up study. Although the FIB-4 index proves to be a convenient marker, it might not perform well as a primary screening tool for liver fibrosis in the general population (UMIN Clinical Trials Registry No. UMIN000035188)., (© 2024 Japan Society of Hepatology.)

Background and Aims: Because the accuracy of the Fibrosis-4 (FIB-4) index for predicting liver fibrosis changes with age, the need for different cut-offs in various age groups has frequently been discussed. We developed the age-independent score, the Fibrosis-3 (FIB-3) index, and have shown its usefulness in patients with metabolic dysfunction-associated steatotic liver disease (MASLD). This study aimed to validate the diagnostic ability of the FIB-3 index to predict fibrosis progression using a large new patient cohort., Methods: The ability of the FIB-3 index to predict liver fibrosis was analyzed by comparing it with that of the FIB-4 index using data from 1398 patients with MASLD enrolled in the Asia-based clinical outcome NAFLD study., Results: The areas under the receiver operating characteristic curves for predicting fibrosis stage F3 or higher were not different between the FIB-3 and FIB-4 indices in the entire cohort. Using the single ideal cut-offs of the indices (3.41 for FIB-3 index and 2.01 for FIB-4 index), the predictive accuracy of the FIB-3 index was not significantly different from that of the FIB-4 index among patients aged <60 years; however, the accuracy of the FIB-3 index was significantly higher than that of the FIB-4 index in those aged ≥60 years (0.645 and 0.529, respectively; p < 0.0001)., Conclusion: The high ability of the FIB-3 index with a single cut-off to predict liver fibrosis in patients with MASLD was confirmed. The FIB-3 index could serve as a useful tool for assessing liver fibrosis regardless of age., (© 2024 Japan Society of Hepatology.)

Aim: This study aims to evaluate the efficacy and safety of lenvatinib radiofrequency ablation (RFA) sequential therapy for certain hepatocellular carcinoma (HCC) patients., Methods: One hundred and nineteen patients with unresectable HCC in the intermediate stage with Child-Pugh A were retrospectively recruited in a multicenter setting. Those in the lenvatinib RFA sequential therapy group received lenvatinib initially, followed by RFA and the retreatment with lenvatinib. The study compared overall survival (OS), progression-free survival (PFS), tumor response, and adverse events (AEs) between patients undergoing sequential therapy and lenvatinib monotherapy., Results: After propensity score matching, 25 patients on sequential therapy and 50 on monotherapy were evaluated. Independent factors influencing OS were identified as sequential therapy, modified albumin-bilirubin (mALBI) grade, and relative dose intensity (%) with hazard ratios (HRs) of 0.381 (95% confidence interval [CI], 0.186-0.782), 2.220 (95% CI, 1.410-3.493), and 0.982 (95% CI, 0.966-0.999), respectively. Stratified analysis based on mALBI grades confirmed the independent influence of treatment strategy across all mALBI grades for OS (HR, 0.376; 95% CI, 0.176-0.804). Furthermore, sequential therapy was identified as an independent factor of PFS (HR, 0.382; 95% CI, 0.215-0.678). Sequential therapy significantly outperformed monotherapy on survival benefits (OS: 38.27 vs. 18.96 months for sequential therapy and monotherapy, respectively, p = 0.004; PFS: 13.80 vs. 5.32 months for sequential therapy and monotherapy, respectively, p < 0.001). Sequential therapy was significantly associated with complete response by modified Response Evaluation Criteria in Solid Tumors (odds ratio, 63.089). Ten of 119 patients experienced grade 3 AEs, with no AE beyond grade 3 observed., Conclusion: Lenvatinib RFA sequential therapy might offer favorable tolerability and potential prognostic improvement compared to lenvatinib monotherapy., (© 2024 Japan Society of Hepatology.)

Aim: The noninvasive tests (NITs) Agile 3+ and Agile 4 effectively identify patients with nonalcoholic fatty liver disease (NAFLD) complicated with advanced fibrosis (F3-4) and cirrhosis (F4), respectively. Little information is available on associations between Agile scores and intra-/extrahepatic events. The aim of this study was to determine the predictive performance of Agile scores for intra-/extrahepatic events in Asian patients with biopsy-proven NAFLD., Methods: We undertook a retrospective multicenter cohort study to investigate associations between intra-/extrahepatic events and two Agile scores, Agile 3+ and Agile 4. The scores were obtained by combining clinical parameters and liver stiffness measurement using transient elastography., Results: Among 403 enrolled patients, 11 had liver-related events (LREs), including seven with hepatocellular carcinoma (HCC). The incidence of LREs and HCC showed a stepwise increase in the advanced fibrosis group (F3-4), Agile 3+ rule-in (F3-4, highly suspected), and Agile 4 rule-in (F4, highly suspected) groups, compared to their counterparts. Hazard ratios for LREs in the advanced fibrosis group, Agile 3+ rule-in, and Agile 4 rule-in groups were 4.05 (p = 0.03), 23.5 (p = 0.003), and 45.5 (p < 0.001), respectively. The predictive performance results for Agile 3+ and Agile 4 were 0.780 and 0.866, respectively, which were higher than for fibrosis (0.595). Unlike for LREs, Agile scores failed to identify patients with extrahepatic events, including cardiovascular events and extrahepatic cancer., Conclusions: Agile 3+ and Agile 4 scores are excellent NITs for predicting LREs in patients with NAFLD, possibly without histological assessment., (© 2023 Japan Society of Hepatology.)

Background: Clinical trials enroll patients with active fibrotic nonalcoholic steatohepatitis (NASH) (nonalcoholic fatty liver disease [NAFLD] activity score ≥ 4) and significant fibrosis (F ≥ 2); however, screening failure rates are high following biopsy. We developed new scores to identify active fibrotic NASH using FibroScan and magnetic resonance imaging (MRI)., Methods: We undertook prospective primary (n = 176), retrospective validation (n = 169), and University of California San Diego (UCSD; n = 234) studies of liver biopsy-proven NAFLD. Liver stiffness measurement (LSM) using FibroScan or magnetic resonance elastography (MRE), controlled attenuation parameter (CAP), or proton density fat fraction (PDFF), and aspartate aminotransferase (AST) were combined to develop a two-step strategy-FibroScan-based LSM followed by CAP with AST (F-CAST) and MRE-based LSM followed by PDFF with AST (M-PAST)-and compared with FibroScan-AST (FAST) and MRI-AST (MAST) for diagnosing active fibrotic NASH. Each model was categorized using rule-in and rule-out criteria., Results: Areas under receiver operating characteristic curves (AUROCs) of F-CAST (0.826) and M-PAST (0.832) were significantly higher than those of FAST (0.744, p = 0.004) and MAST (0.710, p < 0.001). Following the rule-in criteria, positive predictive values of F-CAST (81.8%) and M-PAST (81.8%) were higher than those of FAST (73.5%) and MAST (70.0%). Following the rule-out criteria, negative predictive values of F-CAST (90.5%) and M-PAST (90.9%) were higher than those of FAST (84.0%) and MAST (73.9%). In the validation and UCSD cohorts, AUROCs did not differ significantly between F-CAST and FAST, but M-PAST had a higher diagnostic performance than MAST., Conclusions: The two-step strategy, especially M-PAST, showed reliability of rule-in/-out for active fibrotic NASH, with better predictive performance compared with MAST. This study is registered with ClinicalTrials.gov (number, UMIN000012757)., (© 2023 Japan Society of Hepatology.)

Aim: Agile 3+ and Agile 4 scores, based on liver stiffness measurement (LSM) by transient elastography and clinical parameters, were recently reported to be effective in identifying advanced fibrosis and cirrhosis in nonalcoholic fatty liver disease (NAFLD). This study aimed to validate the utility of these scores in Japanese patients with NAFLD., Methods: Six hundred forty-one patients with biopsy-proven NAFLD were analyzed. The severity of liver fibrosis was pathologically evaluated by one expert pathologist. The LSM, age, sex, diabetes status, platelet count, and aspartate aminotransferase and alanine aminotransferase levels were used to calculate Agile 3+ scores, and the parameters above excluding age were used for Agile 4 scores. The diagnostic performance of the two scores was evaluated using receiver operating characteristic (ROC) curve analysis. Sensitivity, specificity, and predictive values of the original low cut-off (for rule-out) value and high cut-off (for rule-in) value were tested., Results: For diagnosis of fibrosis stage ≥3, the area under the ROC (AUROC) was 0.886, and the sensitivity of the low cut-off value and the specificity of the high cut-off value were 95.3% and 73.4%, respectively. For diagnosis of fibrosis stage 4, AUROC, the sensitivity of the low cut-off value, and the specificity of the high cut-off value were 0.930, 100%, and 86.5%, respectively. Both scores had higher diagnostic performance than the FIB-4 index and the enhanced liver fibrosis score., Conclusions: Agile 3+ and Agile 4 are reliable noninvasive tests to identify advanced fibrosis and cirrhosis in Japanese NAFLD patients with adequate diagnostic performance., (© 2023 Japan Society of Hepatology.)

Aim: Impacts of platelet counts at the time of liver biopsy on hepatocellular carcinoma (HCC) development in patients with nonalcoholic fatty liver disease (NAFLD) remain unknown. The aim of this study was to investigate the prognostic value of platelet counts in patients with biopsy-confirmed NAFLD using data from a multicenter study., Methods: One thousand three hundred ninety-eight patients were included in this subanalysis of the CLIONE (Clinical Outcome Nonalcoholic Fatty Liver Disease) in Asia study. Liver biopsy specimens were pathologically diagnosed, and histologically scored using the NASH Clinical Research Network system. Demographic, clinical, laboratory, and pathological data were collected., Results: During a median follow-up period of 4.6 years (range, 0.3-21.6 years), which corresponds to 8874 person-years, 37 patients developed HCC. Using a cut-off baseline platelet count of 192 × 109/L, the lower platelet group had a higher HCC rate than the higher platelet group (6.7% vs. 0.4%; p < 0.001). This cut-off value significantly stratified the event-free rate for HCC. Lower platelet counts were associated with an increased risk of HCC development. Relative to patients with platelet counts of 192 × 109/L, patients with platelet counts of 100 × 109/L had an unadjusted hazard ratio (HR) for HCC development of 7.37 (95% confidence interval [CI], 3.81-14.2) and an adjusted HR of 11.2 (95% CI, 3.81-32.7; p < 0.001), adjusting for age, sex, NASH, and diabetes., Conclusions: Baseline platelet counts of 192 × 109/L and lower are associated with a higher risk of developing HCC in patients with biopsy-confirmed NAFLD and require active surveillance., (© 2023 Japan Society of Hepatology.)

Aim: The enhanced liver fibrosis (ELF) test is a noninvasive method for diagnosing hepatic fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). This multicenter cohort study aimed to evaluate the accuracy of the ELF test and compare it with other noninvasive tests in Japan., Methods: We analyzed 371 Japanese patients with biopsy-proven NAFLD. We constructed area under the receiver operator characteristic curves (AUROC) to determine the diagnostic accuracies of the ELF test, the Mac-2-binding protein glycosylation isomer (M2BPGi), the Fibrosis-4 (FIB-4) index, and combinations of these indices., Results: In patients with F0/F1/F2/F3/F4 fibrosis, the median values of the ELF test were 8.98/9.56/10.39/10.92/11.41, respectively. The AUROCs of the ELF test for patients with F0 versus F1-4, F0-1 versus F2-4, F0-2 versus F3-4, and F0-3 versus F4 fibrosis were 0.825/0.817/0.802/0.812, respectively. The AUROCs of the ELF test were greater than those of the FIB-4 index and M2BPGi at each fibrosis stage. Respective low and high cut-off values yielded sensitivities and specificities for predicting advanced fibrosis (≥F3) of 91.1% and 50.8%, and 38.5% and 92.8%, respectively. For F3 or F4 fibrosis, the combined values from the ELF test and FIB-4 index showed a sensitivity of 98.5%, and the combined values from the ELF test and M2BPGi assay showed a specificity of 97.5%., Conclusions: In Japan, the ELF test predicts NAFLD-related fibrosis from its early stages. The diagnostic ability of the ELF test was not inferior to that of other indices, and the combined values of ELF plus other indices were more accurate., (© 2022 The Authors. Hepatology Research published by John Wiley & Sons Australia, Ltd on behalf of Japan Society of Hepatology.)

Aim: We aimed to evaluate the diagnostic accuracy of the measurement of serum type IV collagen 7S (T4C7S) concentration for the staging of liver fibrosis in patients with nonalcoholic fatty liver disease (NAFLD)., Methods: A systematic search or published works was carried out using the PubMed, Cochrane Library, and Web of Science Core Collection databases for studies of the accuracy of serum T4C7S concentration for the staging of fibrosis using Fibrosis stage (F)0-4 in patients with NAFLD diagnosed by liver biopsy., Results: Nine articles describing 1475 participants with NAFLD were included. For fibrosis ≥F1, with n = 849, summary estimates of sensitivity of 0.79, specificity of 0.69, and area under the curve (AUC) of 0.80 were obtained using a median T7C4S cut-off value of 4.6 ng/ml. For fibrosis ≥F2, with n = 1,090, summary estimates of sensitivity of 0.78, specificity of 0.78, and AUC of 0.84 were obtained using a median cut-off value of 4.9 ng/ml. For fibrosis ≥F3, with n = 1311 participants and a median cut-off value of 5.4 ng/ml, a pooled sensitivity of 0.82, specificity of 0.81, and AUC of 0.83 were obtained. For fibrosis ≥F4, with n = 753 and a median cut-off value of 6.6 ng/ml, a pooled sensitivity of 0.85, specificity of 0.81, and AUC of 0.85 were obtained., Conclusions: Serum T4C7S concentration was found to be an accurate method of staging liver fibrosis in patients with NAFLD., (© 2022 Japan Society of Hepatology.)

Nonalcoholic fatty liver disease (NAFLD) has become a serious public health issue not only in Western countries but also in Japan. Within the wide spectrum of NAFLD, nonalcoholic steatohepatitis (NASH) is a progressive form of disease that often develops into liver cirrhosis and increases the risk of hepatocellular carcinoma (HCC). While a definite diagnosis of NASH requires liver biopsy to confirm the presence of hepatocyte ballooning, hepatic fibrosis is the most important prognostic factor in NAFLD. With so many NAFLD patients, it is essential to have an effective screening method for NAFLD with hepatic fibrosis. As HCC with non-viral liver disease has increased markedly in Japan, effective screening and surveillance of HCC are also urgently needed. The most common death etiology in NAFLD patients is cardiovascular disease event. Gastroenterologists must, therefore, pay close attention to CVD when examining NAFLD patients. In the updated guidelines, we propose screening and follow-up methods for hepatic fibrosis, HCC, and CVD in NAFLD patients. Several drug trials are ongoing for NAFLD/NASH therapy, however, there is currently no specific drug therapy for NAFLD/NASH. In addition to vitamin E and thiazolidinedione derivatives, recent trials have focused on sodium glucose co-transporter 2 (SGLT2) inhibitors and glucagon-like peptide-1 (GLP-1) analogues, and effective therapies are expected to be developed. These practical guidelines for NAFLD/NASH were established by the Japanese Society of Gastroenterology in conjunction with the Japan Society of Hepatology. Clinical evidence reported internationally between 1983 and October 2018 was collected, and each clinical and background question was evaluated using the Grades of Recommendation Assessment, Development and Evaluation (GRADE) system. This English summary pro- vides the core essentials of these clinical practice guidelines, which include the definition and concept, screening systems for hepatic fibrosis, HCC and CVD, and current therapies for NAFLD/NASH in Japan., (This article has been co-published with permission in Hepatology Research and Journal of Gastroenterology. © 2021 The Authors. The articles are identical except for minor stylistic and spelling differences in keeping with each journal’s style. Either citation can be used when citing this article.)

Aim: To develop a novel noninvasive test using an artificial intelligence (AI)/neural network (NN) system (named Fibro-Scope) to determine the fibrosis stage in nonalcoholic steatohepatitis (NASH)., Methods: Three hundred twenty-four and 110 patients with histologically diagnosed nonalcoholic fatty liver disease (NAFLD) were enrolled for training and validation studies, respectively. Two independent pathologists histologically diagnosed patients with NAFLD for the validation study. Fibro-Scope was undertaken using 12 items: age, sex, height, weight, waist circumference, aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase, cholesterol, triglyceride, platelet count, and type 4 collagen 7s., Results: Differentiation of F0 versus F1-4 using the Fibro-Scope revealed 99.5% sensitivity, 90.9% specificity, 97.4% positive predictive value, and 98.0% negative predictive value in a training study with gray zone analysis, which was also effective in the analysis without gray zone. Discrimination was also excellent when comparing F0-1 versus F2-4 and F0-2 versus F3-4. In a validation study with gray zone analysis, differentiation of F0 from F1-4 using Fibro-Scope was also excellent. The discrimination of F0-1 from F2-4 using Fibro-Scope with gray zone analysis showed over 80% sensitivity and specificity in the histological diagnosis of both pathologists, but was lower without the gray zone analysis. The discrimination of F0-2 from F3-4 was effective in the analysis with gray zone; however, their sensitivity and specificity were slightly inferior in the analysis without gray zone., Conclusions: Artificial intelligence/neural network algorithms termed Fibro-Scope are easy to use and can accurately differentially diagnose minimal, moderate, and advanced fibrosis. Fibro-Scope will promote rapid NASH diagnosis and facilitate diagnosing the fibrosis stage in NASH., (© 2021 The Authors. Hepatology Research published by John Wiley & Sons Australia, Ltd on behalf of Japan Society of Hepatology.)

Aim: We aimed to develop a novel noninvasive test using an artificial intelligence (AI)/neural network (NN) system (named nonalcoholic steatohepatitis [NASH]-Scope) to screen nonalcoholic fatty liver disease (NAFLD) and NASH., Methods: We enrolled 324 and 74 patients histologically diagnosed with NAFLD for training and validation studies, respectively. Two independent pathologists histologically diagnosed patients with NAFLD for validation study. Additionally, 48 subjects who underwent a medical health checkup and did not show fatty liver ultrasonographically and had normal serum aminotransferase levels were categorized as the non-NAFLD group. NASH-Scope was based on 11 clinical values: age, sex, height, weight, waist circumference, aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase, cholesterol, triglyceride, and platelet count., Results: The sensitivity, specificity, positive predictive value, negative predictive value, and area under the receiver operator characteristic curve of NASH-Scope for distinguishing NAFLD from non-NAFLD in the training study and validation study were 99.7% versus 97.2%, 97.8% versus 97.8%, 99.7% versus 98.6%, 97.8% versus 95.7%, and 0.999 versus 0.950, respectively. Those for distinguishing NASH with fibrosis from NAFLD without fibrosis were 99.5% versus 90.7%, 84.3% versus 93.3%, 94.2% versus 98.0%, 98.6% versus 73.7%, and 0.960 versus 0.950. These results were excellent, even when the output data were divided into two categories without any gray zone., Conclusions: The AI/NN system, termed as NASH-Scope, is practical and can accurately differentially diagnose between NAFLD and non-NAFLD and between NAFLD without fibrosis and NASH with fibrosis. Thus, NASH-Scope is useful for screening nonalcoholic fatty liver and NASH., (© 2021 The Authors. Hepatology Research published by John Wiley & Sons Australia, Ltd on behalf of Japan Society of Hepatology.)

Aim: Hepatitis B virus (HBV) relies on glycosylation for crucial functions, such as entry into host cells, proteolytic processing and protein trafficking. The aim of this study was to identify candidate molecules for the development of novel antiviral agents against HBV using an siRNA screening system targeting the host glycosylation pathway., Methods: HepG2.2.15.7 cells that consistently produce HBV were employed for our in vitro study. We investigated the effects of siRNAs that target 88 different host glycogenes on hepatitis B surface antigen (HBsAg) and HBV DNA secretion using the siRNA screening system., Results: We identified four glycogenes that reduced HBsAg and/or HBV DNA secretion; however, the observed results for two of them may be due to siRNA off-target effects. Knocking down ST8SIA3, a member of the sialyltransferase family, significantly reduced both HBsAg and HBV DNA secretion. Knocking down GALNT7, which transfers N-acetylgalactosamine to initiate O-linked glycosylation in the Golgi apparatus, also significantly reduced both HBsAg and HBV DNA levels., Conclusions: These results showed that knocking down the ST8SIA3 and GALNT7 glycogenes inhibited HBsAg and HBV DNA secretion in HepG2.2.15.7 cells, indicating that the host glycosylation pathway is important for the HBV life cycle and could be a potential target for the development of novel anti-HBV agents., (© 2020 The Japan Society of Hepatology.)

Aim: Liver biopsy is still required for the diagnosis of hepatocellular ballooning and inflammation, which are important histological features of non-alcoholic steatohepatitis. We undertook this multicenter, cross-sectional study to identify novel blood markers for the diagnosis of hepatocellular ballooning., Methods: We enrolled 176 patients, of whom 132 were proven by liver biopsy as having non-alcoholic fatty liver disease (NAFLD) and classified as non-ballooning (ballooning grade 0) (n = 83) or ballooning (ballooning grade 1 and 2) (n = 49) by a central pathology review. We carried out gas chromatography-mass spectrometry, hydrophilic interaction liquid chromatography tandem mass spectrometry, and lipidomics with plasma., Results: As correlates of hepatocellular ballooning, among the clinical parameters, serum type IV collagen 7S correlated most significantly with the ballooning grade (correlation coefficient [CC] = 0.463; P < 0.001). Among the metabolic/lipidomic markers, phosphatidylcholine (PC) (aa-44:8) correlated most significantly with the ballooning grade (CC = 0.394; P < 0.001). The area under the receiver operating characteristic curve of type IV collagen 7S, choline, and lysophosphatidylethanolamine (LPE) (e-18:2), was 0.846 (95% confidence interval, 0.772-0.919)., Conclusions: Plasma levels of PC were positively correlated, and those of lysophosphatidylcholine and LPE were negatively correlated with hepatocellular ballooning in NAFLD patients. These non-invasive metabolic/lipidomic-based plasma tests might be useful to distinguish between cases of NAFLD with and without hepatocellular ballooning., (© 2020 The Japan Society of Hepatology.)

Aim: Recently, FibroScan-AST (FAST) score was reported to be effective for identifying non-alcoholic steatohepatitis (NASH) with significant activity and fibrosis in non-alcoholic fatty liver disease (NAFLD). The aim of this study was to confirm the diagnostic accuracy of FAST score of Japanese patients and compare the cut-off values and diagnostic accuracy between the FibroScan M and XL probes., Methods: Eighty-two and 84 patients were included the verification and validation sets, respectively. All patients were diagnosed with NAFLD by biopsy by two central expert pathologists. Liver stiffness measurements and controlled attenuation parameter were carried out, and diagnostic performance was evaluated using receiver operating characteristic (ROC) curve analysis., Results: No significant difference existed in FAST score between the M and XL probes (0.489 vs. 0.483, P = 0.187). No significant difference existed in the area under the ROC between the two probes (M, 0.7598; XL, 0.7614; P = 0.958). According to the Youden index, the cut-off value using the M probe was 0.57 with 68.2% sensitivity and 78.3% specificity. For the XL probe, the cut-off value was 0.56 with 68.2% sensitivity and 73.3% specificity. To obtain sensitivity and specificity values higher than 90%, cut-off values of 0.35 and 0.66 were chosen for the M probe and 0.32 and 0.63 were chosen for the XL probe., Conclusions: There was no significant difference in diagnostic accuracy of FAST score between the FibroScan M and XL probes. The FAST score can be used to identify NASH with significant risk in Japanese patients regardless of probe selection., (©2020 The Japan Society of Hepatology.)

Aim: The Enhanced Liver Fibrosis (ELF) test comprises a logarithmic algorithm combining three serum markers of hepatic extracellular matrix metabolism. We aimed to evaluate the performance of ELF for the diagnosis of liver fibrosis and to compare it with that of liver stiffness measurement (LSM) by FibroScan in non-alcoholic fatty liver disease., Methods: ELF cut-off values for the diagnosis of advanced fibrosis were obtained using receiver operating characteristic analysis in patients with biopsy-confirmed non-alcoholic fatty liver disease (training set; n = 200). Diagnostic performance was analyzed in the training set and in a validation set (n = 166), and compared with that of LSM in the FibroScan cohort (n = 224)., Results: The area under receiver operating characteristic curve was 0.81 for the diagnosis of advanced fibrosis, and the ELF cut-off values were 9.34 with 90.4% sensitivity and 10.83 with 90.6% specificity in the training set, and 89.8% sensitivity and 85.5% specificity in the validation set. There was no significant difference in the area under the receiver operating characteristic curve between ELF and LSM (0.812 and 0.839). A combination of ELF (cut-off 10.83) and LSM (cut-off 11.45) increased the specificity to 97.9% and the positive predictive value, versus ELF alone. Sequential use of the Fibrosis-4 index (cut-off 2.67) and ELF (cut-off 9.34) increased the sensitivity to 95.9%., Conclusions: ELF can identify advanced liver fibrosis in non-alcoholic fatty liver disease, and its diagnostic accuracy is comparable to that of FibroScan. According to the clinical setting, combinations or sequential procedures using other non-invasive tests complement the diagnostic performance of ELF for the identification of advanced fibrosis., (© 2020 The Japan Society of Hepatology.)

Aim: We investigated the characteristics of serum autotaxin (ATX) and its diagnostic performance for liver fibrosis in a large cohort of patients with non-alcoholic fatty liver disease (NAFLD)., Methods: We compared the usefulness of ATX and other fibrosis markers in 307 biopsy-confirmed NAFLD patients. In addition, in 145 participants with NAFLD, we compared the diagnostic performance of ATX with that of non-invasive imaging methods (vibration-controlled transient elastography and magnetic resonance elastography [MRE])., Results: Serum ATX concentration was significantly correlated with fibrosis stage in male and female NAFLD patients. In male patients, the area under the receiver operating characteristic (AUROC) curve values of ATX for the diagnosis of ≥stage 1, ≥stage 2, ≥stage 3, and ≥stage 4 fibrosis were 0.65, 0.75, 0.81, and 0.95, respectively. In female NAFLD participants, the AUROC values were all >0.81. The sensitivity of ATX was highest for the diagnosis of ≥stage 2 and ≥stage 3 fibrosis in both men and women with NAFLD. In the comparison between ATX and non-invasive imaging methods, the AUROC for MRE was the highest at every stage of fibrosis., Conclusions: Serum ATX concentration is significantly correlated with fibrosis stage in NAFLD patients. The diagnostic accuracy of ATX for liver fibrosis is lower than that of MRE, but the sensitivities of ATX for the diagnosis of ≥stage 2 and ≥stage 3 were highest. We conclude that ATX is useful for the selection of patients requiring further evaluation for liver fibrosis., (© 2019 The Japan Society of Hepatology.)

Aim: Although liver biopsy is the gold standard for the diagnosis and staging of non-alcoholic fatty liver disease (NAFLD), repeated assessment of patients' liver tissue conditions are impractical. We assessed the 10-year changes in liver stiffness measurements (LSM) utilizing vibration-controlled transient elastography in NAFLD patients., Methods: From January 2006 to September 2007, LSM was carried out for 97 biopsy-proven NAFLD patients. Of these, 34 patients underwent 10-year LSM reassessments (14 of them with paired biopsies)., Results: We evaluated the changes in the fibrosis stage as estimated using LSM (FS-LSM). Over a 10-year period, 32.4% had FS-LSM progression, 50% had static disease, and 17.6% had FS-LSM improvement. From among the initially diagnosed non-alcoholic steatohepatitis patients, 18% had progressed to considerable stage 4 (cirrhosis) 10 years later. In this cohort, none of the patients who had been initially diagnosed as FS-LSM stage 0 had progressed to cirrhosis 10 years later. The changes in LSM were correlated with the change in the histological fibrosis stage, the NAFLD activity score, and the change in the sum of the steatosis, activity, and fibrosis score. Improving more than 1 body mass index (kg/m 2 ) and having a higher initial aspartate aminotransferase, alanine aminotransferase (ALT), or ALT responder (>30% improvement or reduction to less than 40 IU/L) were factors contributing to LSM improvements (≥2 kPa)., Conclusions: Vibration-controlled transient elastography is likely to become a more clinically important tool for the long-term monitoring of NAFLD patients., (© 2019 The Japan Society of Hepatology.)

Aims: No pharmacological therapies are approved for non-alcoholic fatty liver disease (NAFLD). Luseogliflozin, a sodium glucose cotransporter 2 inhibitor, has been developed for the treatment of adults with type 2 diabetes (T2DM). The aim of this prospective, single-arm study is to evaluate the efficacy of luseogliflozin on hepatic fat content and glycated hemoglobin (HbA1c) in T2DM patients with NAFLD., Methods: Forty T2DM patients with NAFLD were treated with luseogliflozin 2.5 mg/day for 24 weeks. Primary end-points were changes in HbA1c and hepatic steatosis evaluated by magnetic resonance imaging-hepatic fat fraction from baseline. Secondary end-points were changes in metabolic and hepatic function-related parameters, including hepatic fibrosis markers (Fibrosis-4 index, NAFLD fibrosis score, type IV collagen 7S. and Wisteria floribunda agglutinin-positive Mac-2 binding protein)., Results: Not only HbA1c and transaminase activities but also hepatic fat content were significantly decreased after 24 weeks of therapy with luseogliflozin. The reduction of hepatic fat content was significantly correlated with the reduction of alanine aminotransferase. Although hepatic fibrosis markers were unchanged, serum ferritin levels reduced and serum albumin significantly increased after the treatment., Conclusion: Luseogliflozin can be a novel promising agent for the treatment of T2DM patients with NAFLD. Prospective randomized controlled trials are warranted to confirm this impact of luseogliflozin onT2DM with NAFLD., (© 2018 The Japan Society of Hepatology.)

Aim: In order to know the present status of drug-induced liver injury (DILI) in Japan, we present the data of prospectively collected DILI cases between 2010 and 2018 from 27 hospitals., Methods: Drug-induced liver injury cases diagnosed by DILI experts from 27 hospitals all over Japan have been prospectively collected since 2010. Alanine aminotransferase level ≥150 U/L and/or alkaline phosphatase ≥2× upper limit of normal were inclusion criteria., Results: In total, data of 307 cases (125 male and 182 female individuals) aged between 17 and 86 years old were collected. The types of liver injury were as follows: 64% hepatocellular type, 20% mixed type, and 16% cholestatic type. A drug-induced lymphocyte stimulation test was carried out in 59% of cases, and was positive in 48% and semipositive in 3% of cases. Eosinophilia ≥6% was observed in 27% of cases. Fifty-three percent of DILI cases occurred within 30 days and 79% of DILI cases occurred within 90 days after starting drug administration. By the diagnostic scale of the Digestive Disease Week (DDW)-Japan 2004 workshop, 93.8% of cases were diagnosed as "highly probable", and 5.9% as "possible"., Conclusions: Japanese DILI patients are somewhat different from those of Europe and North America. The diagnostic scale of the DDW-Japan 2004 workshop has been used in Japan. However, there are many issues to improve the causality assessment of DILI that we must investigate in the future. It is critical to elucidate the mechanisms of drug metabolism and the pathophysiology of liver injury by various drugs to prevent DILI., (© 2018 The Japan Society of Hepatology.)

Aim: Pruritus is a common comorbidity in chronic liver disease. The aim of this study was to clarify the prevalence of pruritus and its characteristics in patients with chronic liver disease., Methods: A total of 1631 patients with chronic liver disease who attended one of nine joint-research facilities from January to June 2016 were enrolled. We investigated the prevalence of pruritus, itch location, itch duration, daily itch fluctuation, seasonal itch exacerbation, treatment drugs, and therapeutic effects using a medical interview questionnaire., Results: The median age was 66 years and 890 (54.6%) patients were women. The prevalence of pruritus was 40.3% (658/1631), and it differed according to the underlying liver disease. The most frequent body location for pruritus was on the back (63.1%). Pruritus duration was more than 6 months in 252 (38.3%) patients. The severity of pruritus, assessed using a visual analog scale, was higher during the day than at night (median, 4 vs. 3, P < 0.001). Seasonal exacerbation was observed in 296 (45.0%) patients. Although 301 (45.7%) patients were treated with antipruritic agents, 57.8% (174/301) patients reported an insufficient effect. Active hepatitis B virus infection (odds ratio [OR], 2.51; P = 0.043), primary biliary cholangitis (OR, 3.69; P = 0.018), diabetes (OR, 1.57; P = 0.010), and aspartate aminotransferase ≥60 U/L (OR, 2.06; P = 0.011) were independent factors associated with pruritus., Conclusion: The prevalence of pruritus varies according to the chronic liver disease etiology. Underlying liver disease, aspartate aminotransferase ≥60 U/L, and comorbid diabetes are factors associated with pruritus in patients with chronic liver disease., (© 2017 The Japan Society of Hepatology.)

Aim: The progression of non-alcoholic fatty liver disease (NAFLD) is affected by epigenetics. We undertook co-methylation and differentially methylated region (DMR) analyses to identify the genomic region that is under epigenetic regulation during NAFLD progression., Methods: We collected liver biopsy specimens from 60 Japanese patients with NAFLD and classified these into mild (fibrosis stages 0-2) or advanced (fibrosis stages 3-4) NAFLD. We carried out a genome-wide DNA methylation analysis and identified the differentially methylated CpGs between mild and advanced NAFLD. Differentially methylated regions with multiple consecutive differentially methylated CpGs between mild and advanced NAFLD were extracted., Results: Co-methylation analysis showed that individual differentially methylated CpG sites were clustered into three modules. The CpG sites clustered in one module were hypomethylated in advanced NAFLD and their annotated genes were enriched for "immune system" function. The CpG sites in another module were hypermethylated and their annotated genes were enriched for "mitochondria" or "lipid particle", and "lipid metabolism" or "oxidoreductase activity". Hypomethylated DMRs included tumorigenesis-related genes (FGFR2, PTGFRN, and ZBTB38), the expressions of which are upregulated in advanced NAFLD. Tumor suppressor MGMT had two DMRs and was downregulated. Conversely, FBLIM1 and CYR61, encoding proteins that reduce cell proliferation, showed hypomethylated DMRs and were upregulated. Expression of the antioxidant gene NQO1 was upregulated, with a hypomethylated DMR. The DMR containing cancer-related MIR21 was hypomethylated in advanced NAFLD., Conclusions: Co-methylation and DMR analyses suggest that the NAFLD liver undergoes mitochondrial dysfunction, decreased lipid metabolism, and impaired oxidoreductase activity, and acquires tumorigenic potential at the epigenetic level., (© 2017 The Japan Society of Hepatology.)

Aim: Several studies on the efficacy of ezetimibe, a potent inhibitor of cholesterol absorption, in treating non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) have been published; however, the results are inconsistent. We undertook a meta-analysis to evaluate the efficacy of ezetimibe in treating NAFLD and NASH., Methods: PubMed, Medline, and Cochrane Library Full Text Database were searched until June 2016. The main inclusion criteria included original studies investigating the use of ezetimibe for the treatment of NAFLD and NASH. Identification of published work and data extraction were carried out by two reviewers based on the inclusion and exclusion criteria. All analyses were carried out using Comprehensive Meta-Analysis version 3 software., Results: An initial search identified 103 peer-reviewed articles and abstracts. Six studies (two randomized controlled and four single-arm trials) involving 273 participants with NAFLD and NASH were identified. Ezetimibe significantly reduced serum aspartate aminotransferase, alanine aminotransferase, and γ-glutamyl transpeptidase levels, and hepatic steatosis and hepatocyte ballooning. However, hepatic inflammation and fibrosis did not improve by ezetimibe treatment in patients with NAFLD and NASH. In randomized controlled trials, only hepatocyte ballooning improved with ezetimibe treatment., Conclusions: Although ezetimibe attenuated serum liver enzymes and hepatic steatosis and ballooning in six studies, it improved only hepatocyte ballooning in randomized controlled trials. Larger studies and more randomized placebo-controlled trials are necessary to determine the effects of ezetimibe on NAFLD and NASH., (© 2017 The Japan Society of Hepatology.)

Aim: Non-alcoholic fatty liver disease (NAFLD) progresses because of the interaction between numerous genes. Thus, we carried out a weighted gene coexpression network analysis to identify core gene networks and key genes associated with NAFLD progression., Methods: We enrolled 39 patients with mild NAFLD (fibrosis stages 0-2) and 21 with advanced NAFLD (fibrosis stages 3-4). Total RNA was extracted from frozen liver biopsies, and sequenced to capture a large dynamic range of expression levels., Results: A total of 1777 genes differentially expressed between mild and advanced NAFLD (q-value <0.05) clustered into four modules. One module was enriched for genes that encode cell surface or extracellular matrix proteins, and are involved in cell adhesion, proliferation, and signaling. This module formed a scale-free network containing four hub genes (PAPLN, LBH, DPYSL3, and JAG1) overexpressed in advanced NAFLD. PAPLN is a component of the extracellular matrix, LBH and DPYSL3 are reported to be tumor suppressors, and JAG1 is tumorigenic. Another module formed a random network, and was enriched for genes that accumulate in the mitochondria. These genes were downregulated in advanced NAFLD, reflecting impaired mitochondrial function. However, the other two modules did not form unambiguous networks. KEGG analysis indicated that 71 differentially expressed genes were involved in "pathways in cancer". Strikingly, expression of half of all differentially expressed genes was inversely correlated with methylation of CpG sites (q-value <0.05). Among clinical parameters, serum type IV collagen 7 s was most strongly associated with the epigenetic status in NAFLD., Conclusions: Newly identified core gene networks suggest that the NAFLD liver undergoes mitochondrial dysfunction and fibrosis, and acquires tumorigenic potential epigenetically. Our data provide novel insights into the pathology and etiology of NAFLD progression, and identify potential targets for diagnosis and treatment., (© 2017 The Japan Society of Hepatology.)

Liver-related diseases are the leading causes of death in patients with type 2 diabetes mellitus (T2DM) in Japan. Type 2 diabetes mellitus is closely associated with non-alcoholic fatty liver disease (NAFLD), which is the most prevalent chronic liver disease worldwide. Non-alcoholic steatohepatitis (NASH), a severe form of NAFLD, can lead to hepatocellular carcinoma and hepatic failure. Non-alcoholic steatohepatitis can be called "diabetic hepatopathy". There are no established pharmacotherapies for NAFLD/NASH patients with T2DM. Although metformin is established as the first-line therapy for T2DM, given its relative safety and beneficial effects on glycosylated hemoglobin, weight, and cardiovascular mortality, this agent is not recommended as specific therapy for NASH/NAFLD due to lack of clinical evidence. The effects of pioglitazone on NASH histology with T2DM have been extensively proved, but several concerns exist, such as body weight gain, fluid retention, cancer incidence, and bone fracture. In recent years, novel antidiabetic medications have been approved for T2DM, such as glucagon-like peptide 1 receptor agonists, dipeptidyl peptidase 4 inhibitors, and sodium/glucose cotransporter 2 inhibitors. A key clinical question for hepatologists is what kinds of antidiabetic medications are the most appropriate for the treatment of NAFLD accompanied by T2DM, to prevent progression of hepatic fibrosis resulting in HCC/liver-related mortality without increased risk of cardiovascular events. This review focuses on novel antidiabetic agents and future perspectives on the treatment of NAFLD/NASH with T2DM., (© 2017 The Japan Society of Hepatology.)

Aim: There are a considerable number of patients with non-obese non-alcoholic fatty liver disease (NAFLD). However, the clinical characteristics of non-obese NAFLD is not fully understood. We investigated genetic and other clinical parameters in non-obese and obese NAFLD., Methods: The single nucleotide polymorphism rs738409 in the patatin-like phospholipase 3 gene (PNPLA3) was genotyped by the Invader assay in 540 NAFLD patients (134 non-obese and 406 obese) and 1012 control subjects (782 non-obese and 230 obese). All NAFLD patients underwent liver biopsy. Odds ratios were calculated by multiple logistic regression analysis using age, sex, body mass index (BMI), type 2 diabetes mellitus (T2DM) and rs738409 genotype as explanatory variables., Results: Non-obese NAFLD subjects had a higher rs738409 GG genotype than obese NAFLD. Multiple logistic regression analysis indicated that the odds ratios of T2DM and rs738409 GG genotype for NAFLD were higher in non-obese than in obese groups. In non-obese NAFLD, rs738409 GG genotype was associated with lobular inflammation, hepatocyte ballooning and NAFLD activity score. In obese NAFLD, BMI and T2DM but not rs738409 GG genotype were associated with severity of histology., Conclusion: We demonstrated that the risk factors for the development and progression of NAFLD were different between non-obese and obese patients and that PNPLA3 rs738409 was strongly associated with the development and progression of non-obese NAFLD., (© 2016 The Japan Society of Hepatology.)

Nonalcoholic fatty liver disease (NAFLD) is currently the most common cause of chronic liver disease in industrialized countries worldwide, and has become a serious public health issue not only in Western countries but also in many Asian countries including Japan. Within the wide spectrum of NAFLD, non-alcoholic steatohepatitis (NASH) is a progressive form of disease, which often develops into liver cirrhosis and increases the risk of hepatocellular carcinoma. In turn, a large proportion of NAFLD/NASH is the liver manifestation of metabolic syndrome, suggesting that NAFLD/NASH plays a key role in the pathogenesis of systemic atherosclerotic diseases. Currently, a definite diagnosis of NASH requires liver biopsy, though various non-invasive measures are under development. The mainstays of prevention and treatment of NAFLD/NASH include dietary restriction and exercise; however, pharmacological approaches are often necessary. Currently, vitamin E and thiazolidinedione derivatives are the most evidence-based therapeutic options, although the clinical evidence for long-term efficacy and safety is limited. This practice guideline for NAFLD/NASH, established by the Japanese Society of Gastroenterology in cooperation with The Japan Society of Hepatology, covers lines of clinical evidence reported internationally in the period starting from 1983 through January 2012, and each clinical question was evaluated using the GRADE system. Based on the primary release of the full version in Japanese, this English summary provides the core essentials of this clinical practice guideline comprising the definition, diagnosis, and current therapeutic recommendations for NAFLD/NASH in Japan., (© 2015 The Japan Society of Hepatology.)

Glycogen storage disease (GSD) type Ia is caused by a deficiency in glucose-6-phosphatase. Long-term complications, including renal disease, gout, osteoporosis and pulmonary hypertension, develop in patients with GSD type Ia. In the second or third decade, 22-75% of GSD type Ia patients develop hepatocellular adenoma (HCA). In some of these patients, the HCA evolves into hepatocellular carcinoma. However, little is known about GSD type Ia patients with HCA who develop cholangiocellular carcinoma (CCC). Here, we report for the first time, a patient with GSD type Ia with HCA, in whom intrahepatic CCC was developed., (© 2014 The Japan Society of Hepatology.)

Aim: The spleen is not believed to contribute to hematopoiesis in healthy adults. However, several reports have demonstrated that the spleen in adults contains a large number of hematopoietic stem/progenitor cells (HSC). Although splenectomy increases platelet and leukocyte counts, the effects of splenectomy on circulating HSC have not been elucidated. In this study, we evaluated the association between the number of circulating HSC and splenectomy in patients with hepatitis C virus (HCV)-associated liver cirrhosis (LC)., Methods: In 48 patients with various stages of HCV-associated chronic liver disease and seven patients with LC who underwent splenectomy, and 10 healthy volunteers, we determined the numbers of circulating CD34 + cells and colony-forming unit culture by flow cytometry and methylcellulose culture, respectively. Plasma stromal cell-derived factor-1α (SDF-1α) concentrations were measured using an enzyme-linked immunosorbent assay., Results: The numbers of circulating CD34 + cells and colony-forming unit culture decreased but the plasma SDF-1α concentration increased with the progression of liver disease. There was an inverse correlation between the number of circulating HSC and the plasma SDF-1α concentration. The numbers of circulating HSC and platelets were determined before and after splenectomy in seven patients with LC. In these patients, the numbers of circulating HSC and platelets increased significantly after splenectomy and the enhancing effect persisted for a long time., Conclusion: Our data suggest that the spleen plays an important role in modulating HSC dynamics in patients with HCV-associated chronic liver disease. Our results also imply that splenectomy may improve liver function in patients with LC., (© 2014 The Japan Society of Hepatology.)

Serum ferritin was recently reported to have low diagnostic accuracy for the detection of advanced fibrosis in patients with non-alcoholic fatty liver disease (NAFLD). To corroborate these findings, we investigated the diagnostic accuracy of serum ferritin levels for detecting liver fibrosis in NAFLD patients utilizing a large Japanese cohort database. A total 1201 biopsy-proven NAFLD patients, seen between 2001 and 2013, were enrolled into the Japan Study Group of NAFLD. Analysis was performed on data from this cohort comparing between serum ferritin levels and hepatic histology. Serum ferritin increased with increasing histological grade of steatosis, lobular inflammation and ballooning. Multivariate analyses revealed that sex differences, steatotic grade and fibrotic stage were independently associated with serum ferritin levels (P < 0.0001, <0.0001, 0.0248, respectively). However, statistical analyses performed using serum ferritin levels demonstrated that the area under the receiver-operator curve for detecting fibrosis was not adequate for rigorous prediction. Several factors including sex differences, steatosis and fibrosis were found to correlate with serum ferritin levels. Therefore, serum ferritin may have low diagnostic accuracy for specifically detecting liver fibrosis in NAFLD patients due to the involvement of multiple hepatocellular processes., (© 2014 The Japan Society of Hepatology.)

Aim: Recently, several studies have shown the existence of associations between lipoprotein profiles and hepatitis C virus (HCV), although only a limited amount of information is available about the mechanisms underlying the changes in the lipoprotein profiles associated with HCV. In this study, we investigated the association between lipoprotein profile, classified according to the particle size, and lipoprotein metabolism., Methods: We used four kinds of cells for this experiment; full-length genome HCV RNA replicon cells (OR6), sub-genomic HCV RNA replicon cells (sO), and OR6c cells and sOc cells, which were the same cell lines treated with interferon-α. The triglyceride (TG) levels in the lipoprotein subclasses of the culture medium were measured by high-performance liquid chromatography. The mRNA expression levels of several molecules associated with lipoprotein metabolism were measured in the OR6, OR6c, sO and sOc cells. To confirm some of the results obtained using the in vitro system, liver biopsy samples obtained from the patients were also examined., Results: The content of TG in the large low-density lipoprotein (LDL) and medium LDL in the culture medium was increased only in the OR6 cells. The hepatic triglyceride lipase (HTGL) mRNA expression levels were lower in the OR6 cells than in the OR6c cells (P < 0.01). Examination of the HTGL expression levels in the patients' livers revealed a decrease in HTGL expression in the chronic hepatitis C liver as compared with that in the chronic hepatitis B or non-alcoholic steatohepatitis liver (P < 0.01)., Conclusion: We showed that HCV inhibits HTGL production in hepatocytes, inducing a change of the lipoprotein profile., (© 2013 The Japan Society of Hepatology.)

Objective: Absence of a late-night cortisol nadir is a consistent biochemical abnormality in patients with cortisol-producing adenoma. We evaluated the abnormality of late-night urinary free cortisol to creatinine ratio (late-night UFCCR) in patients with subclinical Cushing's syndrome (SCS)., Methods: Fifty-eight patients with incidentally detected adrenocortical adenomas [SCS: 9; nonfunctioning adenoma (NF): 49] were enrolled as subjects. Values measured in all patients were urinary free cortisol accumulated between 9:00 p.m. and 11:00 p.m. (late-night UFCCR), serum cortisol at 11:00 p.m. (midnight serum cortisol: MSC), serum cortisol after 1-mg overnight dexamethasone suppression test (1 mg-DST) and 24-h urinary free cortisol (UFC)., Results: Median late-night UFCCR value in SCS was significantly higher than that in NF (P < 0·001). Significant correlations were observed between late-night UFCCR and each of serum cortisol after 1 mg-DST and MSC (r = 0·537, P < 0·001 and r = 0·556, P < 0·001, respectively). There was no significant correlation between serum cortisol after 1 mg-DST and 24-h UFC (r = 0·211, P = 0·112). In receiver operating characteristic analysis for diagnosis of SCS, the areas under the curves of late-night UFCCR and 24-h UFC were 0·937 (95% confidence interval 0·865-1·008) and 0·726 (0·874-0·999), respectively. Late-night UFCCR cut-off value of 4·9 nmol/μmol Cre showed a sensitivity of 100% and a specificity of 76·6%., Conclusion: Patients with SCS showed higher late-night UFCCR values than those with NF. Late-night UFCCR was significantly correlated with autonomous cortisol production findings. Diagnostic performance of late-night UFCCR was superior to 24-h UFC. These results suggest that late-night UFCCR might represent one of the simple and reliable tests for SCS diagnosis., (© 2013 John Wiley & Sons Ltd.)

Aim: There have been some reports on the use of paired biopsies for monitoring disease progression in non-alcoholic fatty liver disease (NAFLD) patients. Recently, transient elastography has been developed as a non-invasive method for predicting the severity of liver fibrosis. We investigated 4-year disease progression in NAFLD patients by evaluating liver stiffness measurements (LSM) obtained using transient elastography., Methods: Of 97 biopsy-proven NAFLD patients whose LSM were obtained 4 years prior, we revaluated 36 who were available for follow up and compared their current stage of fibrosis with that at initial assessment. Fibrosis stage was diagnosed according to the cut-off values previously reported by our institution. We also investigated correlations between changes in LSM and changes in non-invasive fibrosis markers obtained by performing biochemical tests and clinical data., Results: A total of nine (25%) patients had fibrosis progression, 17 (47.2%) had static disease and 10 (27.8%) had fibrosis remission. Among patients with stage 0 fibrosis as per initial LSM (n = 16), 11 had static disease and five had fibrosis progression according to LSM obtained 4 years later. Changes in LSM correlated with changes in the FIB4 index (R = 0.519, P = 0.0011) and aspartate aminotransferase-to-platelet ratio index (R = 0.346, P = 0.0412), but not with changes in other non-invasive markers., Conclusion: Transient elastography is non-invasive and easy to use; therefore, it can be a useful tool for monitoring the severity of hepatic fibrosis in NAFLD patients., (© 2013 The Japan Society of Hepatology.)

Aim: A rapid and non-invasive method of detecting fibrosis in patients with chronic liver diseases is of major clinical interest. The purpose of this study was to comparatively investigate the effectiveness of the Liver Fibrosis Index (LF Index) calculated using real-time tissue elastography (RTE) in patients with non-alcoholic fatty liver disease (NAFLD) and patients with chronic hepatitis C (CHC)., Methods: Twenty-seven patients with biopsy-proven NAFLD and 93 patients with biopsy-proven CHC were included. They underwent transient elastography (TE), serum liver fibrosis marker testing and RTE to calculate the LF Index., Results: The LF Index showed a stepwise increase with increasing histological severity of fibrosis in CHC patients (P = 0.0102), whereas no significant correlation of the LF Index with the histological severity of liver fibrosis in NAFLD patients (P = 0.852). There was a significant correlation between the LF Index and liver stiffness measured by TE in CHC patients (r = 0.319, P = 0.0009). On the other hand, no such correlation was observed in NAFLD patients. While in CHC patients, the LF Index was correlated with the FIB-4 index, no such correlation was observed in NAFLD patients., Conclusion: The LF Index calculated by RTE is effective for assessment of liver fibrosis in patients with CHC. On the other hand, it is not useful in patients with NAFLD. This is the first study to compare the clinical usefulness of RTE as non-invasive assessment of liver fibrosis between CHC and NAFLD. Further investigations are required to refine statistical assessment of RTE., (© 2012 The Japan Society of Hepatology.)

Aim:   Choline is a dietary component that is crucial for normal cellular function. Choline is predominantly absorbed from the small intestine and completely metabolized in the liver. We recently demonstrated that free choline (fCh) levels in blood reflect the level of phosphatidylcholine synthesis in the liver and is correlated with the onset of non-alcoholic steatohepatitis (NASH). Our aim here was to validate the utility of this biomarker for NASH diagnosis., Methods:   Our cohort consisted of 110 patients with biopsy proven non-alcoholic fatty liver disease (NAFLD) from four centers across Japan and 25 age-matched healthy controls. Plasma fCh levels were measured using high-performance liquid chromatography., Results:   Patients with diagnosed or borderline NASH had significantly increased plasma fCh levels when compared with control subjects, or patients not diagnosed with NASH. Interestingly, an association between plasma fCh levels and expression of microsomal triglyceride transfer protein, which catalyzes the transfer of triglyceride, was reflected in the markedly negative correlation between these two variables in patients with NAFLD. Moreover, the grade of liver steatosis and fibrosis stage increased with increasing plasma fCh levels (P < 0.05). The area under the receiver-operating characteristic (ROC) curves for NASH, including borderline diagnosis, was 0.811. Additionally, the areas under the ROC for fibrosis stage were 0.816 for >stage 1, 0.805 for >stage 2, 0.809 for >stage 3 and 0.818 for >stage 4., Conclusion:   Plasma fCh levels are closely related to the grade of liver steatosis and fibrosis, and predict NASH severity. Plasma fCh levels are therefore a potential diagnostic marker for early-stage NASH in clinical practice., (© 2012 The Japan Society of Hepatology.)

Liver fibrosis has been gaining noticeable attention because it may lead to end-stage liver cirrhosis and ultimately to hepatocellular carcinoma. Thus, a precise estimation of the degree of liver fibrosis is crucial for predicting prognosis and deciding management of patients with chronic liver diseases. Many non-invasive approaches for the evaluation of liver fibrosis have been developed. Among these procedures, transient elastography has recently drawn great attention. Transient elastography has been reported to be well correlated with the degree of liver fibrosis by many investigators and various institutions. Since the degree of liver fibrosis is considered as a strong predictor of risk for hepatocellular carcinoma development, several trials have been performed to verify the usefulness of measurement of liver stiffness to predict the emergence of hepatocellular carcinoma. From these studies, transient elastography seems to be a promising procedure to predict the risk of hepatocellular carcinoma; however, further cohorts with long-term monitoring of liver stiffness are needed to confirm the usefulness of this method., (© 2011 The Japan Society of Hepatology.)

Liver abscess is recognized as a life-threatening disease. However, even in recent years, approximately 50% of liver abscess cases are considered to be cryptogenic. Here, we report a case of liver abscess associated with periodontal bacterial infection by Fusobacterium necrophorum, which is commonly found in the oropharyngeal flora. A 36-year-old man presented with fever and contrast-enhanced abdominal computed tomography revealed multiple liver abscesses. F.necrophorum was isolated from oral smears, liver aspirates and blood samples. Liver abscesses caused by periodontal bacterial infection are rare, however, the incidence is expected to increase in the future, as periodontitis is extremely common and is on the rise as one of the most common chronic infections in the world. A systemic survey including periodontitis may be required for the exact diagnosis of the source of infection., (© 2011 The Japan Society of Hepatology.)

Aim: Although non-alcoholic fatty liver disease (NAFLD) is now a common cause of chronic liver disease, discriminating between simple steatosis and non-alcoholic steatohepatitis (NASH), especially early-stage NASH, remains difficult. We investigated the clinical usefulness of measuring the spleen volume as a marker of early-stage NASH., Methods: We evaluated computed tomography (CT) images obtained in 84 patients with histologically diagnosed NAFLD (22 with simple steatosis, 62 with NASH with mild fibrosis [stages 1-2]). We defined the data obtained by the following formula as a spleen-body index (SBI): SBI = maximal CT axial section area of the spleen (cm(2))/body surface area (BSA) (cm(2)) x 10(4). We compared the SBI between patients with simple steatosis and those with NASH with mild fibrosis., Results: The mean SBI of the simple steatosis group was 15.8 +/- 3.9, while that of the NASH with mild fibrosis group was 18.7 +/- 5.7. This difference between the two groups was significant (P = 0.0314). A multiple logistic regression analysis showed that the SBI was significantly correlated with the discrimination of simple steatosis and NASH with mild fibrosis. The area under the receiver-operator curve was 0.661 for distinguishing between simple steatosis and NASH with mild fibrosis (P = 0.026, 95% confidence interval = 0.532-0.789)., Conclusion: Spleen enlargement may be a distinct feature of NASH, especially early-stage NASH. SBI might be a non-invasive and simple method of differentiating NASH and simple steatosis.

Aim: Non-alcoholic steatohepatitis (NASH) is considered a hepatic manifestation of metabolic syndrome. However, effective drug therapy for NASH has not been established yet. In the present study, we evaluated the efficacy of 6 months of ezetimibe treatment for NASH patients with dyslipidemia for the comparison of improvement of the clinical parameters and histological alterations., Methods: We prospectively evaluated 10 consecutive NASH patients with dyslipidemia who agreed to participate in this study. The patients were given ezetimibe (10 mg/day) for 6 months, and clinical parameters and histological alterations were comparatively evaluated before and after treatment. All the patients were given standard calorie diet (30 kcal/kg per day, carbohydrate 50-60%, fat 20-30%, protein 15-20%) and exercise counseling from 3 months before the ezetimibe treatment., Results: The serum aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase, low-density lipoprotein cholesterol, high-sensitivity C-reactive protein and type IV collagen 7 s levels were significantly improved by the treatment with ezetimibe for 6 months. In histological observations, follow-up liver biopsies revealed that the NAS score and steatosis grade were also significantly improved. The fibrosis stage did not change significantly, but six of the 10 patients exhibited an improvement in their fibrosis stage., Conclusion: Major clinical parameters and histological observations were significantly improved by the treatment with ezetimibe. Our pilot study demonstrated the efficacy of ezetimibe for drug therapy of NASH and may lead to a large-scale clinical trial in the future.

Aim: Congenital extrahepatic portosystemic shunt (CEPS) is a rare anomaly in which the enteric blood bypasses the liver and drains into the systemic veins through various venous shunts. Patients with CEPS often have liver tumors and complications such as cardiac or other anomalies, but portosystemic encephalopathy and gastrointestinal bleeding occur only occasionally. The clinical problems differ for each individual with CEPS, and establishing a prognosis can be very difficult., Methods: We reviewed the clinical features of 136 reported cases of CEPS and classified these cases according to their portosystemic shunts., Results: We classified portal blood flow directly into the inferior vena cava (IVC) as type A (88 cases), portal blood flow into the renal vein as type B (36 cases), and portal blood flow into the iliac vein via an inferior mesenteric vein as type C (12 cases). Type A patients were complicated with cardiac anomalies at a higher rate than other types. Type C patients had lower prevalences of cardiac anomalies and portosystemic encephalopathy than the other types, but the prevalence of gastrointestinal bleeding was significantly higher (P < 0.0001). The prognosis of CEPS has improved, and only six deaths have been previously reported, all of which occurred in type A patients., Conclusions: We reviewed the previously reported cases of CEPS. Classification according to the portosystemic shunt system might be useful for investigating the clinical features of CEPS.

Aim: Genetic factors as well as environmental factors play an important role in the development of non-alcoholic fatty liver disease (NAFLD). Recently, inducible nitric oxide synthase (iNOS) was significantly higher in the severest form of non-alcoholic steatohepatitis (NASH), and nitric oxide (NO) has been determined to play an important role in the process of fibrosis in NASH. In this study, we investigated iNOS gene polymorphisms for associations with NAFLD., Methods: A total of 115 NAFLD patients, consisting of 65 patients with NASH and 50 patients with simple steatosis, in whom a positive diagnosis had been made by liver biopsy, and 435 healthy control subjects, were recruited into this study., Results: We investigated 10 single nucleotide polymorphisms (SNP) of the iNOS gene, one of which, rs1060822, had the lowest P-value in the allele frequency model (P = 0.00078) with an odds ratio (95% confidence interval) of 0.49 (0.32-0.75). Four SNP, rs2297510, rs2297511, rs2797512 and rs1060822, were significantly associated with NAFLD, even when the most conservative Bonferroni's correction was applied. Linkage disequilibrium analysis revealed that SNP rs1060822 and three other SNP, rs2297510, rs2297511 and rs2797512, were in the same block. We also investigated associations between rs1060822 genotypes and the fibrosis index, and the results of the analysis revealed an additive increase in the fibrosis index and intrahepatic iNOS mRNA expression in the patients with the T allele of rs1060822., Conclusion: This is the first study to identify genetic variations in iNOS that may influence the risk of NAFLD and liver fibrosis in NAFLD.

This study examined the spawning season, spawning frequency and batch fecundity of yellow sea bream Dentex hypselosomus in the East China Sea to reassess the previously reported reproductive characteristics of the species. Time-course sampling showed that this species had a diurnal ovarian maturation rhythm. Late tertiary yolk-stage oocytes appeared 2 days before spawning, starting the process of germinal vesicle movement and breakdown. On the day of spawning, ovulation and subsequent spawning occurred in the early morning (0400-0800 hours). Postovulatory follicles disappeared from the ovaries within c. 24 h of ovulation. Seasonal changes in the ovarian conditions indicated that this species spawned more or less throughout the year, with the peak ranging from spring to autumn. The compositions of the developing oocytes and degenerating postovulatory follicles in the ovaries suggested that most females spawned repeatedly over 2 to 3 consecutive days during the peak of the spawning season. Somatic body condition did not have a significant effect on batch fecundity, but there was a significant relationship between batch fecundity and fork length according to spawning status. Females spawning on consecutive days were more fecund than those spawning every other day. The findings show that this species has much greater reproductive potential than previously estimated.

Aim: Hepatocellular carcinoma (HCC) is one of the most commonly occurring malignances worldwide. Curative therapies such as resection, percutaneous ethanol injection (PEI) and radiofrequency ablation (RFA) have been applied to patients with early-stage HCC. Patients with more advanced cancers require local or systemic therapies. We present the results of our retrospective review conducted to evaluate whether transarterial chemoembolization (TACE) alone and combined TACE with percutaneous ablation for HCC exhibited superior efficacy to palliative treatment., Methods: The effects of TACE and of the combined therapies (TACE + PEI or TACE + RFA) on the long-term survival rates were evaluated in 268 untreated HCC patients by various statistical analyses., Results: The cumulative survival rates in the TACE alone group were significantly superior to those in the palliative treatment group. Further, the cumulative survival rates in the combined TACE + PEI/RFA group were significantly superior to those in the TACE alone group. When the comparison among the groups was restricted to patients with two or three tumors fulfilling the Milan criteria, significantly greater prolongation of survival was observed in the combined TACE + PEI/RFA group than in the PEI/RFA alone group., Conclusions: The aforementioned treatment modalities yielded greater improvements of the survival rate and survival duration as compared to palliative treatment in HCC patients. Furthermore, in terms of the effect on the survival period, combined TACE + PEI/RFA therapy was more effective than TACE monotherapy, and also more effective than PEI or RFA monotherapy in cases with multiple tumors fulfilling the Milan criteria.

Aim: Human hepatocytes are known to express an array of inflammatory cytokines and chemokines. In this study, we examined the potential roles of hepatocytes in regulating immune responses in the liver, by assessing the induction of Th1- or Th2-specific chemokines in HepG2 cells after various inflammatory stimulations., Methods: HepG2 cells were stimulated with IL-1alpha, IFN-gamma, IL-4, IL-10, and/or CCL2, harvested at several time points, and served for the analyses of cytokine/chemokine mRNA expressions by semi-quantitative RT-PCR., Results: (i) IL-1alpha up-regulated mRNA levels of CXCL8, CXCL10, and CCL2. IFN-gamma increased those of CXCL9, CXCL10, and CCL5, while IL-4 or IL-10 had no effect. (ii) Addition of IL-4 to the culture of IFN-gamma-stimulated cells, down-regulated CXCL9 and CXCL10 mRNA levels. (iii) Addition of IFN-gamma to the culture of IL-1alpha-stimulated cells, further up-regulated CXCL9 and CXCL10 mRNA levels. Addition of IL-4 decreased CXCL8 and CXCL10 levels, and increased CCL2 level in IL-1alpha-stimulated cells. (iv) CCL2 induced IL-4 mRNA expression., Conclusions: IFN-gamma augmented mRNA expression of Th1-specific chemokines (CXCL9 and CXCL10) in HepG2 cells. IL-4 had no effect on those of Th2-spesific chemokines (CCL17 and CCL22); however, it was supposed to augment Th2 response indirectly through the induction of CCL2 under the inflammatory condition. The findings suggest that hepatocytes have ability to promote immune responses in the liver toward the direction, initially determined by the cytokine balances in the local inflammatory region.

Aim: Non-alcoholic steatohepatitis (NASH) is a subset of non-alcoholic fatty liver disease (NAFLD) and sometimes progresses to cirrhosis and liver failure. In this study we analyzed the expression profile of genes and biological pathways involved in NASH in comparison with non-NASH by gene set enrichment analysis (GSEA) employing a DNA microarray technique., Methods: mRNA from liver biopsy specimens was collected from a group of NASH patients and a group of non-NASH patients. We analyzed the relative abundance of mRNA using high-density oligonucleotide microarrays containing probes for 54 675 known genes, and investigated the pathogenetic mechanisms of NASH by means of a powerful technique for analyzing molecular profiling data, GSEA., Results: The results showed that the level of expression of 27 gene sets was significantly higher and the level of expression of 25 gene sets was significantly lower in the NASH samples than in the non-NASH samples. Based on these results we created an online, publicly available, searchable database containing the data for the gene expression profiles of the NASH patients (http://www2.genome.rcast.u-tokyo.ac.jp/&#95;&#95;&#95;/NASH/NASH&#95;GSEA2/)., Conclusion: Our data revealed differences in expression of many gene sets that are involved in the pathogenesis of NASH.

The intravenous injection of concanavalin A (Con A) activates T cells and induces cytokine dependent liver injury in mice. However, the effect of repeated administrations of Con A has not been fully investigated. Female BALB/c mice were intravenously injected with Con A (20mg/kg) or saline once a week for six times. Mice were rechallenged with Con A 17 days after repeated administrations of Con A. Repeated Con A administrations elicited a sustained inhibition of rechallenged-Con A-induced liver injury. Plasma TNF-alpha and IFN-gamma levels after rechallenge of Con A were decreased compared with that of repeated saline treatments. By contrast, plasma IL-4 and IL-10 levels after rechallenge of Con A were increased. In spleen cells prepared from repeated Con A treated mice, the production of TNF-alpha and IFN-gamma 24h after co-incubation with Con A decreased, and that of IL-4 and IL-10 increased. In naive mice, plasma ALT level after Con A injection was decreased by the transfer of spleen cells prepared from the repeated Con A treated mice. The repeated administrations of Con A elicited Th1 to Th2 cytokine shift and the tolerant state against the Con A-induced liver injury in mice.

Fucoidan is a complex of sulfated polysaccharides derived from non-mammalian origin such as marine brown algae and induces cytokine expression. We investigated the effect of fucoidan on concanavalin A (Con A)-induced liver injury in mice. Liver injury was induced by an intravenous injection of Con A (18.5mg/kg). Various doses of fucoidan (1-30mg/kg) were intravenously administered 30min before Con A injection. The plasma alanine aminotransferase (ALT) and several cytokines levels were determined, and hepatic histological changes were also assessed. The effect of fucoidan administration by itself on induction of interleukin (IL)-10 in plasma and liver tissue was investigated. Con A administration induced an elevation of plasma ALT level, and fucoidan administration dose-dependently prevented the Con A-induced elevation of plasma ALT. Con A administration increased plasma TNF-alpha and IFN-gamma levels, and fucoidan pretreatment significantly inhibited these alterations and increased plasma IL-10 level. The inhibitory effect of fucoidan on Con A-induced liver injury and production of proinflammatory cytokines were reversed by anti-mouse IL-10 antibody pretreatment. Fucoidan induced the IL-10 production in plasma and liver tissue. These findings suggest that fucoidan prevents Con A-induced liver injury by mediating the endogenous IL-10 production and the inhibition of proinflammatory cytokine in mice.