Yoshiyuki Ueno

Sofosbuvir plus ribavirin in Japanese patients with chronic genotype 2 HCV infection: an open‐label, phase 3 trial

Genotype 2 hepatitis C virus (HCV) accounts for up to 30% of chronic HCV infections in Japan. The standard of care for patients with genotype 2 HCV – peginterferon and ribavirin for 24 weeks – is poorly tolerated, especially among older patients and those with advanced liver disease. We conducted a phase 3, open‐label study to assess the efficacy and safety of an all‐oral combination of the NS5B polymerase inhibitor sofosbuvir and ribavirin in patients with chronic genotype 2 HCV infection in Japan. We enrolled 90 treatment‐naïve and 63 previously treated patients at 20 sites in Japan. All patients received sofosbuvir 400 mg plus ribavirin (weight‐based dosing) for 12 weeks. The primary endpoint was sustained virologic response at 12 weeks after therapy (SVR12). Of the 153 patients enrolled and treated, 60% had HCV genotype 2a, 11% had cirrhosis, and 22% were over the aged 65 or older. Overall, 148 patients (97%) achieved SVR12. Of the 90 treatment‐naïve patients, 88 (98%) achieved SVR12, and of the 63 previously treated patients, 60 (95%) achieved SVR12. The rate of SVR12 was 94% in patients with cirrhosis and in those aged 65 and older. No patients discontinued study treatment due to adverse events. The most common adverse events were nasopharyngitis, anaemia and headache. Twelve weeks of sofosbuvir and ribavirin resulted in high rates of SVR12 in treatment‐naïve and previously treated patients with chronic genotype 2 HCV infection. The treatment was safe and well tolerated by patients, including the elderly and those with cirrhosis.

Distinct microRNAs expression profile in primary biliary cirrhosis and evaluation of miR 505-3p and miR197-3p as novel biomarkers.

Background and Aims MicroRNAs are small endogenous RNA molecules with specific expression patterns that can serve as biomarkers for numerous diseases. However, little is known about the expression profile of serum miRNAs in PBC. Methods First, we employed Illumina deep sequencing for the initial screening to indicate the read numbers of miRNA expression in 10 PBC, 5 CH-C, 5 CH-B patients and 5 healthy controls. Comparing the differentially expressed miRNAs in the 4 groups, analysis of variance was performed on the number of sequence reads to evaluate the statistical significance. Hierarchical clustering was performed using an R platform and we have found candidates for specific miRNAs in the PBC patients. Second, a quantitative reverse transcription PCR validation study was conducted in 10 samples in each group. The expression levels of the selected miRNAs were presented as fold-changes (2−ΔΔCt). Finally, computer analysis was conducted to predict target genes and biological functions with MiRror 2.0 and DAVID v6.7. Results We obtained about 12 million 32-mer short RNA reads on average per sample and the mapping rates to miRBase were 16.60% and 81.66% to hg19. In the statistical significance testing, the expression levels of 81 miRNAs were found to be differentially expressed in the 4 groups. The heat map and hierarchical clustering demonstrated that the miRNA profiles from PBC clustered with those of CH-B, CH-C and healthy controls. Additionally, the circulating levels of hsa-miR-505-3p, 197-3p, and 500a-3p were significantly decreased in PBC compared with healthy controls and the expression levels of hsa-miR-505-3p, 139-5p and 197-3p were significantly reduced compared with the viral hepatitis group. Conclusions Our results indicate that sera from patients with PBC have a unique miRNA expression profile and that the down-regulated expression of hsa-miR-505-3p and miR-197-3p can serve as clinical biomarkers of PBC.

Incidence of and risk factors for hepatocellular carcinoma in primary biliary cirrhosis: National data from Japan

Primary biliary cirrhosis (PBC) primarily affects females and is rarely complicated by hepatocellular carcinoma (HCC). Although the HCC incidence in PBC patients is low, several characteristics and risk factors associated with its development have been reported. In this study, national data concerning the current status of carcinogenesis in PBC patients in Japan are reviewed. Using data from two national questionnaire surveys, we investigated the clinicopathological findings associated with HCC in PBC patients. According to the data of all reviewed PBC patients, the HCC incidence was 2.4% (71/2946). The HCC incidence by gender was 5.1% (19/370) in males and 2.0% (52/2576) in females, and the proportion of males was 26.7%. Prognosis was significantly poorer in the PBC patients with HCC than in those without. Multivariate analysis of risk factors associated with HCC by gender revealed histological stage at the time of PBC diagnosis as an independent risk factor associated with the development of HCC in females, but not in males. Furthermore, data from another national survey of 178 PBC patients with HCC (male/female = 49/129; proportion of males 27.5%) revealed that the duration between the diagnosis of PBC and that of HCC was significantly shorter in males than in females. In addition, histological stage at the time of HCC diagnosis was an independent risk factor for HCC in females, whereas no risk factors were identified in males. Conclusion: these data indicate that males are at risk of developing HCC at any histological stage of PBC. Therefore, male PBC patients in particular should be carefully screened for HCC from the early stages of PBC. (HEPATOLOGY 2013)

Secretin Stimulates Biliary Cell Proliferation by Regulating Expression of MicroRNA 125b and MicroRNA let7a in Mice

BACKGROUND & AIMSnProliferating cholangiocytes secrete and respond to neuroendocrine hormones, including secretin. We investigated whether secretin secreted by S cells and cholangiocytes stimulates biliary proliferation in mice.nnnMETHODSnCholestasis was induced in secretin knockout (Sct(-/-)) and wild-type (control) mice by bile duct ligation (BDL). At days 3 and 7 after BDL, control and Sct(-/-) mice received tail-vein injections of morpholinos against microRNA 125b or let7a. One week later, liver tissues and cholangiocytes were collected. Immunohistochemical, immunoblot, luciferase reporter, and real-time polymerase chain reaction assays were performed. Intrahepatic bile duct mass (IBDM) and proliferation were measured. Secretin secretion was measured in conditioned media from cholangiocytes and S cells and in serum and bile.nnnRESULTSnSecretin secretion was increased in supernatants from cholangiocytes and S cells and in serum and bile after BDL in control mice. BDL Sct(-/-) mice had lower IBDM, reduced proliferation, and reduced production of vascular endothelial growth factor (VEGF) A and nerve growth factor (NGF) compared with BDL control. BDL and control mice given morpholinos against microRNA 125b or let7a had increased IBDM. Livers of mice given morpholinos against microRNA 125b had increased expression of VEGFA, and those treated with morpholinos against microRNA let7a had increased expression of NGF. Secretin regulated VEGF and NGF expression that negatively correlated with microRNA 125b and let7a levels in liver tissue.nnnCONCLUSIONSnAfter liver injury, secretin produced by cholangiocytes and S cells reduces microRNA 125b and let7a levels, resulting in up-regulation of VEGF and NGF. Modulation of cholangiocyte expression of secretin could be a therapeutic approach for biliary diseases.

Guidelines for the management of primary biliary cirrhosis: The Intractable Hepatobiliary Disease Study Group supported by the Ministry of Health, Labour and Welfare of Japan Guidelines for the management of primary biliary cirrhosis: The Intractable Hepatobiliary Disease Study Group supported by the Ministry of Health, Labour and Welfare of Japan

THE JAPANESE VERSION of the clinical practice guidelines for primary biliary cirrhosis (PBC) was developed in 2012 by the Intractable Hepatobiliary Disease Study Group, with the support of the Ministry of Health, Labour and Welfare of Japan, for the use of general physicians, gastroenterologists and hepatologists who treat patients with PBC. In preparation for developing the guidelines, the study group reviewed recent studies that provided important evidence or that were published in leading journals with a high impact factor, in addition to considering the formal consensus of experts on PBC or related subjects. Using the core keywords “primary biliary cirrhosis,” a PubMed search was conducted for English-language clinical trials, randomized clinical trials (RCTs) and meta-analyses that were published from January 1998 to December 2009 and that addressed treatment of PBC and its complications, follow-up, indication of and time of consultation for liver transplantation, or time of consultation with specialists. Medical systems and other culture-specific factors in Japan were also taken into account. Members of the task force exchanged ideas frequently during the drafting process to try and establish a consensus. The final draft was made after collecting comments from the public and all the committee members. The level of evidence (LE; Table 1) and the grade of recommendation (GR; Table 2) were based on the Medical Information Network Distribution Service in Japan (MINDS). After being modified by recent literatures published since 2010, the present English version of the guidelines was developed in order to spread our ideas and exchange opinions with physicians who are involved in the management of PBC patients overseas. *Working Subgroup (English version) for Clinical Practice Guidelines for Primary Biliary Cirrhosis (in alphabetical order): Atsumasa Komori, Clinical Research Center, National Hospital Organization Nagasaki Medical Center; Atsushi Tanaka, Department of Medicine, Teikyo University School of Medicine; Hajime Takikawa, Department of Medicine, Teikyo University School of Medicine; §Hirohito Tsubouchi, Digestive Disease and Life-style Related Disease, Kagoshima University Graduate School of Medical and Dental Sciences and Kagoshima City Hospital; †Hiromi Ishibashi, International University of Health and Welfare/Fukuoka Sanno Hospital and Clinical Research Center, National Hospital Organization Nagasaki Medical Center; Hiroto Egawa, Department of Surgery, Institute of Gastroenterology, Tokyo Women’s Medical University; Junko Hirohara, Third Department of Internal Medicine, Kansai Medical University; Ken Shirabe, Department of Surgery and Science, Kyushu University; Kenichi Harada, Department of Human Pathology, Kanazawa University Graduate School of Medicine; Makoto Nakamuta, Department of Gastroenterology, National Hospital Organization Kyushu Medical Center; Mikio Zeniya, Department of Gastroenterology, Jikei University Graduate School of Medicine; Minoru Nakamura, Clinical Research Center, National Hospital Organization Nagasaki Medical Center and Department of Hepatology, Nagasaki University Graduate School of Biomedical Sciences; Nobuyoshi Fukushima, Department of Gastroenterology, National Hospital Organization Kyushu Medical Center; Shinji Shimoda, Medicine and Biosystemic Science, Graduate School of Medical Sciences, Kyushu University; Shotaro Sakisaka, Department of Gastroenterology and Medicine, Fukuoka University Faculty of Medicine; Toshio Morizane, Japan Council for Quality Health Care; Yasuaki Takeyama, Department of Gastroenterology and Medicine, Fukuoka University Faculty of Medicine; ‡Yasuni Nakanuma, Department of Human Pathology, Kanazawa University Graduate School of Medicine; Yoshiyuki Ueno, Department of Gastroenterology, Yamagata University Faculty of Medicine (†Chairperson of the Working Group, ‡Chairperson of the PBC Subcommittee, §Chairperson of the Intractable Hepatobiliary Disease Study Group). **Correspondence: Hiromi Ishibashi, Fukuoka Sanno Hospital, International University of Health and Welfare, 3-6-45 Momochihama, Sawara-ku, Fukuoka, 814-0001, Japan. Email: hiishibashi-gi@umin.ac.jp bs_bs_banner

A Lower Level of Forced Expiratory Volume in 1 Second Is a Risk Factor for All-Cause and Cardiovascular Mortality in a Japanese Population: The Takahata Study

Chronic obstructive pulmonary disease is a known risk factor for cardiovascular death in Western countries. Because Japan has a low cardiovascular death rate, the association between a lower level of forced expiratory volume in 1 s (FEV1) and mortality in Japan’s general population is unknown. To clarify this, we conducted a community-based longitudinal study. This study included 3253 subjects, who received spirometry from 2004 to 2006 in Takahata, with a 7-year follow-up. The causes of death were assessed on the basis of the death certificate. In 338 subjects, airflow obstruction was observed by spirometry. A total of 127 subjects died. Cardiovascular death was the second highest cause of death in this population. The pulmonary functions of the deceased subjects were significantly lower than those of the subjects who were alive at the end of follow-up. The relative risk of death by all causes, respiratory failure, lung cancer, and cardiovascular disease was significantly increased with airflow obstruction. The Kaplan–Meier analysis showed that all-cause and cardiovascular mortality significantly increased with a worsening severity of airflow obstruction. After adjusting for possible factors that could influence prognosis, a Cox proportional hazard model analysis revealed that a lower level of FEV1 was an independent risk factor for all-cause and cardiovascular mortality (per 10% increase; hazard ratio [HR], 0.89; 95% confidence interval [CI], 0.82–0.98; and HR, 0.72; 95% CI, 0.61–0.86, respectively). In conclusion, airflow obstruction is an independent risk factor for all-cause and cardiovascular death in the Japanese general population. Spirometry might be a useful test to evaluate the risk of cardiovascular death and detect the risk of respiratory death by lung cancer or respiratory failure in healthy Japanese individuals.

Modulation of the biliary expression of arylalkylamine N-acetyltransferase alters the autocrine proliferative responses of cholangiocytes in rats.

Secretin stimulates ductal secretion by interacting with secretin receptor (SR) activating cyclic adenosine 3′,5′‐monophosphate/cystic fibrosis transmembrane conductance regulator/chloride bicarbonate anion exchanger 2 (cAMP⇒CFTR⇒Cl−/HCO u20093− AE2) signaling that is elevated by biliary hyperplasia. Cholangiocytes secrete several neuroendocrine factors regulating biliary functions by autocrine mechanisms. Melatonin inhibits biliary growth and secretin‐stimulated choleresis in cholestatic bile‐duct–ligated (BDL) rats by interaction with melatonin type 1 (MT1) receptor through down‐regulation of cAMP‐dependent signaling. No data exist regarding the role of melatonin synthesized locally by cholangiocytes in the autocrine regulation of biliary growth and function. In this study, we evaluated the (1) expression of arylalkylamine N‐acetyltransferase (AANAT; the rate‐limiting enzyme for melatonin synthesis from serotonin) in cholangiocytes and (2) effect of local modulation of biliary AANAT expression on the autocrine proliferative/secretory responses of cholangiocytes. In the liver, cholangiocytes (and, to a lesser extent, BDL hepatocytes) expressed AANAT. AANAT expression and melatonin secretion (1) increased in BDL, compared to normal rats and BDL rats treated with melatonin, and (2) decreased in normal and BDL rats treated with AANAT Vivo‐Morpholino, compared to controls. The decrease in AANAT expression, and subsequent lower melatonin secretion by cholangiocytes, was associated with increased biliary proliferation and increased SR, CFTR, and Cl−/HCO u20093− AE2 expression. Overexpression of AANAT in cholangiocyte cell lines decreased the basal proliferative rate and expression of SR, CFTR, and Cl−/HCO u20093− AE2 and ablated secretin‐stimulated biliary secretion in these cells. Conclusion: Local modulation of melatonin synthesis may be important for management of the balance between biliary proliferation/damage that is typical of cholangiopathies. (HEPATOLOGY 2013)

Pancreatic fat accumulation, fibrosis, and acinar cell injury in the Zucker diabetic fatty rat fed a chronic high-fat diet.

Objective The histological alteration of the exocrine pancreas in obesity has not been clarified. In the present study, we investigated biochemical and histological changes in the exocrine pancreas of obese model rats. Methods Zucker lean rats were fed a standard diet, and Zucker diabetic fatty (ZDF) rats were divided into 2 groups fed a standard diet and a high-fat diet, respectively. These experimental groups were fed each of the diets from 6 weeks until 12, 18, 24 weeks of age. We performed blood biochemical assays and histological analysis of the pancreas. Results In the ZDF rats fed a high-fat diet, the ratio of accumulated pancreatic fat area relative to exocrine gland area was increased significantly at 18 weeks of age in comparison with the other 2 groups (P < 0.05), and lipid droplets were observed in acinar cells. Subsequently, at 24 weeks of age in this group, pancreatic fibrosis and the serum exocrine pancreatic enzyme levels were increased significantly relative to the other 2 groups (P < 0.01). Conclusions In ZDF rats fed a chronic high-fat diet, fat accumulates in pancreatic acinar cells, and this fatty change seems to be related to subsequent pancreatic fibrosis and acinar cell injury.

GABA induces the differentiation of small into large cholangiocytes by activation of Ca2+/CaMK I-dependent adenylyl cyclase 8†‡§

Large, but not small, cholangiocytes (1) secrete bicarbonate by interaction with secretin receptors (SRs) through activation of cystic fibrosis transmembrane regulator (CFTR), Cl−/HCO3− (apex) anion exchanger 2 (Cl−/HCO3− AE2), and adenylyl cyclase (AC)8 (proteins regulating large biliary functions) and (2) proliferate in response to bile duct ligation (BDL) by activation of cyclic adenosine monophosphate (cAMP) signaling. Small, mitotically dormant cholangiocytes are activated during damage of large cholangiocytes by activation of D‐myo‐inositol 1,4,5‐trisphosphate/Ca2+/calmodulin‐dependent protein kinase (CaMK) I. gamma‐Aminobutyric acid (GABA) affects cell functions by modulation of Ca2+‐dependent signaling and AC. We hypothesized that GABA induces the differentiation of small into large cholangiocytes by the activation of Ca2+/CaMK I‐dependent AC8. In vivo, BDL mice were treated with GABA in the absence or presence of 1,2‐bis‐(o‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid, tetraacetoxymethyl ester (BAPTA/AM) or N‐(6‐aminohexyl)‐5‐chloro‐1‐naphtalenesulfonamide (W7) before evaluating apoptosis and intrahepatic bile ductal mass (IBDM) of small and large cholangiocytes. In vitro, control‐ or CaMK I‐silenced small cholangiocytes were treated with GABA for 3 days before evaluating apoptosis, proliferation, ultrastructural features, and the expression of CFTR, Cl−/HCO3− AE2, AC8, and secretin‐stimulated cAMP levels. In vivo administration of GABA induces the apoptosis of large, but not small, cholangiocytes and decreases large IBDM, but increased de novo small IBDM. GABA stimulation of small IBDM was blocked by BAPTA/AM and W7. Subsequent to GABA in vitro treatment, small cholangiocytes de novo proliferate and acquire ultrastructural and functional phenotypes of large cholangiocytes and respond to secretin. GABA‐induced changes were prevented by BAPTA/AM, W7, and stable knockdown of the CaMK I gene. Conclusion: GABA damages large, but not small, cholangiocytes that differentiate into large cholangiocytes. The differentiation of small into large cholangiocytes may be important in the replenishment of the biliary epithelium during damage of large, senescent cholangiocytes. (HEPATOLOGY 2013;)

Albuminuria is an independent predictor of all-cause and cardiovascular mortality in the Japanese population: the Takahata study.

BackgroundAlbuminuria is a known risk factor for cardiovascular events and premature deaths. However, the association between urinary albumin excretion and mortality is unknown in the Japanese population. To clarify this, we conducted a community-based longitudinal study.MethodsThis study included 3,445 registered Japanese subjects (mean age 62.6xa0years), with a 7-year follow-up. Albuminuria was defined as a urine albumin-creatinine ratio (ACR)xa0≥30xa0mg/g in the morning spot urine.ResultsSubjects with albuminuria (nxa0=xa0514, 14.9xa0%) were older and showed a higher prevalence of hypertension, obesity, and diabetes and lower values of estimated glomerular filtration rate (eGFR) than those without albuminuria (nxa0=xa02931, 85.1xa0%). During the follow-up, 138 subjects died. A Kaplan–Meier analysis showed that all-cause mortality significantly increased along with the increase in urine albumin excretion (log-rank test, Pxa0<xa00.001). The subjects with albuminuria showed a significantly higher mortality rate than those without albuminuria (7.4 vs. 3.4xa0%; log-rank test, Pxa0<xa00.001). A Cox proportional hazard model analysis after adjusting for possible confounders showed that albuminuria was an independent risk factor for all-cause and cardiovascular mortality (hazard ratio [HR] 1.69, 95xa0% confidence interval [CI] 1.12–2.56 and HR 2.27, 95xa0% CI 1.10–4.70, respectively) but not for noncardiovascular mortality. These associations were preserved after excluding subjects with high ACR (≥300xa0mg/g).ConclusionsAlbuminuria was a risk factor for all-cause and cardiovascular mortality in the Japanese population. To detect subjects with a high risk for premature death, measuring urinary albumin excretion might be useful.