Clinical factors associated with the therapeutic efficacy of atezolizumab plus bevacizumab in patients with unresectable hepatocellular carcinoma: A multicenter prospective observational study.

The treatment efficiency and predictors of atezolizumab plus bevacizumab therapy for unresectable hepatocellular carcinoma in real-world practice have not been established. This study aimed to assess the efficacy and safety of atezolizumab plus bevacizumab and to investigate predictors of progression-free survival and overall survival. Patients with unresectable hepatocellular carcinoma treated with atezolizumab plus bevacizumab therapy in 19 hospitals were enrolled before treatment and observed prospectively. The outcomes of 222 patients in this cohort were analyzed. The objective response rate and disease control rate were 22.0% and 70.6%, respectively, whereas the median progression-free survival was 5.7 months. Independent risk factors for shortened progression-free survival were younger age (<75 years; 3.9 months vs. 8.6 months), higher number of intrahepatic tumors (≥5; 4.0 months vs. 7.9 months), macrovascular invasion (2.3 months vs. 6.7 months), and higher neutrophil-to-lymphocyte ratio (≥3.03; 3.0 months vs. 7.8 months). The median overall survival was not reached; however, independent risk factors for shortened overall survival were absence of hyperlipidemia, higher number of intrahepatic tumors (≥5), macrovascular invasion, higher α-fetoprotein level (≥400 ng/mL), worse Child-Pugh score (≥6), and higher neutrophil-to-lymphocyte ratio (≥3.03). Severe adverse events (grade ≥3) were observed in 96 patients (36.0%), with proteinuria being the most frequent. In conclusion, patients with older age, lower number of intrahepatic tumors, absent macrovascular invasion, and lower neutrophil-to-lymphocyte ratio are expected to have better progression-free survival with atezolizumab plus bevacizumab therapy for unresectable hepatocellular carcinoma.

[An anatomical case of obstructive jaundice due to the rapid enlargement of hepatic peribiliary cysts in end-stage alcoholic liver cirrhosis].

A man in his 60s had end-stage alcoholic cirrhosis. About six months before his death, hepatic peribiliary cysts (HPBC) rapidly increased, and he developed jaundice and liver failure. The pathological autopsy performed after his death revealed that his intrahepatic bile duct was pressured due to multiple cysts caused by HPBC, which resulted in liver failure. Some cases of HPBC have been associated with alcoholic cirrhosis;however, no other cases of increased HPBC in a short period of time have been reported. Although identifying the cause of increased HPBC in a short time is difficult in this case, it may be have been caused by continuous alcohol drinking after the onset of HPBC. Most patients with HPBC have liver cirrhosis and obstructive jaundice that may promote liver failure as in this case. Therefore, patients with HPBC should not only be instructed for abstinence but also promptly consider effective treatments in the event of obstructive jaundice to prevent liver dysfunction.

Pretreatment with antibiotics is associated with reduced therapeutic response to atezolizumab plus bevacizumab in patients with hepatocellular carcinoma.

AIM: Alterations in microbial composition of gut microbiota due to antibiotics (ATB) may lead to resistance to immune checkpoint inhibitors (ICIs). This study aimed to assess the impact of ATB use on therapeutic response in patients with hepatocellular carcinoma (HCC) receiving atezolizumab plus bevacizumab. METHODS: This study retrospectively analyzed 105 patients with HCC treated with atezolizumab plus bevacizumab as a primary systemic therapy from prospectively-registered, multicenter, cohorts. Nineteen patients who received prior ATB were included in the ATB (+) group; 86 patients who did not receive prior ATB were included in the ATB (-) group. The therapeutic outcomes were compared between the two groups. RESULTS: Most of the patients' baseline characteristics were not significantly different between the two groups. The objective response rates according to the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) (30.1% vs. 11.1%; p = 0.143) and modified RECIST (mRECIST) (44.6% vs. 27.8%; p = 0.190) were not significantly different between the ATB (-) and ATB (+) groups. The disease control rates were higher in the ATB (-) group than in the ATB (+) group according to RECIST v1.1 (74.7% vs. 44.4%; p = 0.012) and mRECIST (78.3% vs. 50.0%; p = 0.020). Prior ATB use was found to be independently associated with radiological progressive disease of the first therapeutic assessment. The median progression-free survival according to RECIST v1.1 (9.1 months vs. 3.0 months; p = 0.049) and mRECIST (9.1 months vs. 3.0 months; p = 0.036), and overall survival (not reached vs. 11.4 months; p = 0.015) were longer in the ATB (-) group than in the ATB (+) group. CONCLUSIONS: Prior ATB use was associated with reduced therapeutic responses in patients with HCC receiving atezolizumab plus bevacizumab.

Two cases of delayed onset of immune-related adverse events after discontinuation of nivolumab in patients with metastatic renal cell cancer.

INTRODUCTION: Delayed onset of immune-related adverse events following immune-checkpoint inhibitor discontinuation is underrecognized because of little available evidence. CASE PRESENTATION: A 50-year-old man with metastatic renal cell carcinoma (Case 1) and 58-year-old woman with renal cell carcinoma and retroperitoneal lymph node metastasis (Case 2) underwent nivolumab therapy. Case 1: Progressive disease forced nivolumab discontinuance after 18 months, and he underwent two courses of tyrosine kinase inhibitor therapy. immune-related adverse events of pneumonitis, hepatitis, and renal dysfunction were diagnosed 142 days after nivolumab discontinuation, and he recovered with immunosuppressive treatment. Case 2: The immune-related adverse event of pneumonitis forced nivolumab discontinuance after 14 months, and two courses of tyrosine kinase inhibitor therapy were administered. The immune-related adverse event of hepatitis was diagnosed 436 days after nivolumab discontinuation, and she recovered with immunosuppressive treatment. CONCLUSION: Two patients with delayed onset of immune-related adverse events after nivolumab discontinuation were recovered with immunosuppressive treatment.

Self-expandable metallic stent placement above the papilla without endoscopic sphincterotomy in patients with distal malignant biliary obstruction.

Background and study aims The best method for endoscopic placement of self-expandable metallic stents (SEMS) for distal malignant biliary obstruction (MBO) has not yet been determined. The aim of this study was to evaluate how SEMS placement above the papilla and without endoscopic sphincterotomy (EST) impacts the time to recurrent biliary obstruction (RBO) in patients with distal MBO. Patients and methods We retrospectively reviewed data for 73 consecutive patients with unresectable distal MBO who underwent endoscopic SEMS placement for the first time at our institution between April 2014 and March 2016. We compared time to RBO of SEMS placement above the papilla (intraductal placement) with SEMS placement across the papilla (transpapillary placement). In the intraductal placement group, we also compared time to RBO of placement without EST with placement with EST. Results Endoscopic SEMS placement was performed in 30 patients with intraductal placement and in 43 patients with transpapillary placement. The median time to RBO was significantly longer with intraductal placement (307 days) than with transpapillary placement (161 days) ( P  = 0.022). Complication rates did not differ between the two groups. In both univariate and multivariate analysis, intraductal placement was an independent factor contributing to prolonged time to RBO. In intraductal placement, time to RBO was significantly longer in SEMS placement without EST than with EST (363 days vs. 227 days, respectively; P  = 0.026). Conclusions Intraductal SEMS placement, especially without EST for distal MBO contributed to longer time to RBO.

Prognostic Factors for Patients With a Large Number of Hepatocellular Carcinoma Nodules.

BACKGROUND: The prognostic factors and treatment strategies for hepatocellular carcinoma (HCC) patients with a large number of tumor nodules have not been fully elucidated. Clinical factors influencing prognosis were investigated in HCC patients with 30 or more tumor nodules. METHODS: Forty-six HCC patients with 30 or more tumor nodules participated in this study. None of them had vascular invasion and extrahepatic metastasis. Kaplan-Meier curve and Cox proportional hazard model were used for analysis. RESULTS: The median survival time of our patients was no more than 15 months, suggesting that patients with 30 or more tumor nodules may be regarded as a progressive subgroup showing poorer prognosis. In multivariate analysis, presence of between 30 and 59 tumor nodules (P = 0.002), male gender (P = 0.002), lower total bilirubin (total bilirubin < 1.0 mg/dL) (P = 0.011), transarterial chemoembolization (TACE) as an initial therapy (P = 0.027) and higher prothrombin time (P = 0.049) were significant independent factors for better overall survival. Among 39 patients who underwent TACE as an initial therapy, patients who received sorafenib therapy during follow-up showed better overall survival than those who did not (P = 0.026). Efficacy of sorafenib appeared to be more evident in patients who needed repeated transarterial treatment. CONCLUSIONS: In HCC patients with 30 or more tumor nodules, TACE as an initial therapy may be correlated with better prognosis. Sorafenib administration after the prior transarterial treatment may improve antitumor efficacy.

Development of a thrombus in the superior mesenteric artery associated with sequential therapy with tyrosine kinase inhibitors for hepatocellular carcinoma.

Tyrosine kinase inhibitors (TKIs) are widely used for systemic chemotherapy of hepatocellular carcinoma (HCC). Arterial thromboembolism (ATE) has been reported to be an adverse event associated with TKI therapy, but its incidence is rare. Here, we report a case of an HCC patient who developed a thrombus in the superior mesenteric artery (SMA) while on TKI therapy. The patient was a 78-year-old Japanese man with hepatitis C virus-associated HCC with multiple nodules. Several sessions of transarterial chemoembolization therapy caused him to become refractory to the treatment. Sorafenib and regorafenib therapy had also been previously performed, but his disease continued to progress gradually. Therefore, we started lenvatinib therapy. When a contrast-enhanced computed tomography (CT) examination was performed 2 months later, we found a thrombus in the SMA. Retrospective analysis of the CT images revealed that the thrombus formed during the sorafenib-regorafenib sequential therapy and it developed rapidly, especially during the lenvatinib therapy. An HCC patient developed a thrombus in the SMA during TKI therapy. The incidence of ATE is rare in TKI treatment; however, long-term or sequential TKI therapy may increase the frequency of ATE. Further study is needed.

Chemotherapy-Induced Sclerosing Cholangitis Caused by Systemic Chemotherapy.

A 61-year-old woman diagnosed with cervical cancer received systemic chemotherapy using paclitaxel and bevacizumab. Marked elevation of liver enzyme levels was observed. Ultrasonography and computed tomography showed wall thickening of the extrahepatic and intrahepatic bile ducts accompanied by stricture and dilatation. According to these, she was diagnosed as chemotherapy-induced sclerosing cholangitis (CISC), a form of secondary sclerosing cholangitis. Although CISC triggered by systemic chemotherapy is rare, CISC should be considered as a clinically important adverse event of chemotherapy because it causes rapid deterioration of liver function and necessitates interruption of chemotherapy.

Two-step progression of varenicline-induced autoimmune hepatitis.

We describe a rare case of drug-induced hepatitis due to the smoking cessation agent varenicline in a 46-year-old Asian woman. The liver injury progressed in two steps. First, the liver injury started in the absence of viral/autoimmune responses, and withdrawal of varenicline lowered the increase in the levels of liver enzymes immediately. Such findings suggested varenicline-induced liver injury. Second, hepatitis recurred in association with conversion of antinuclear antibody from negative to positive about 8 weeks after the initial episode. Histology upon recurrence of liver injury revealed interface hepatitis with lymphocytic and lymphoplasmacytic portal inflammatory infiltrates extending into lobules. Such findings suggested autoimmune hepatitis. Corticosteroid treatment was effective for recurrent hepatitis. The clinical course suggests that varenicline caused drug-induced liver injury and subsequent autoimmune hepatitis. Some autoimmune changes were probably involved in the mechanism of varenicline-induced liver injury.

The Effectiveness of a Liver Disease Education Class for Providing Information to Patients and Their Families.

BACKGROUND: We have been conducting liver disease education classes regularly in our hospital for the purpose of providing health information to patients and their families. METHODS: In order to evaluate the effectiveness of these classes, we conducted a questionnaire survey of patients and family members who attended the classes held three times in 2012. The cumulative total number of participants was 80 (49 patients, 26 family members, and five others). The classes focused on the following areas: 1) prevention of hepatic cancer; 2) treatment of hepatic cancer; 3) iron restriction diet for hepatitis C patients; and 4) importance of branched-chain amino acid preparations. Self-evaluation of knowledge in these areas was based on a four-point scale. RESULTS: A comparison of knowledge levels between the patients and their family members revealed no statistically significant differences. Therefore, subsequent analyses were performed by combining the patients and their families into one group. The knowledge level of the participants increased with the number of class attendances; that is, the more often they attended, the more they accumulated knowledge (Kruskal-Wallis test: P < 0.0001; P = 0.0368; P = 0.0021; and P < 0.0001). In addition, the results of the questionnaire administered immediately before and after the education class showed significant improvement in the knowledge level for each area. CONCLUSION: The results of this study indicate the liver disease education class to be effective for improving the knowledge of patients and their families. The importance of repeated information provision was also demonstrated.

CD14+ monocyte-derived galectin-9 induces natural killer cell cytotoxicity in chronic hepatitis C.

Natural killer (NK) cell activation is associated with both liver injury and persistent infection in chronic hepatitis C (CHC); however, the detailed mechanism of this activation has not yet been fully elucidated. Because galectin-9 (Gal-9) has been reported to be increased in the serum and liver tissue of CHC patients, we investigated the function of Gal-9 in NK cell activation in CHC. First, we evaluated the function of Gal-9 on NK cytotoxicity in vitro. Gal-9 treatment resulted in increased cytotoxicity of naïve NK cells, and the Gal-9-activated NK cells demonstrated cytotoxicity toward hepatoma cells and T cells. Additionally, coculturing peripheral blood mononuclear cells (PBMCs) with JFH-1/Huh7.5.1 cells increased both Gal-9 production and NK cell cytotoxicity. Next, we investigated the source of Gal-9 and the mechanism of Gal-9 production. Deletion of CD14+ monocytes from PBMCs resulted in reduced Gal-9 production in the coculture with JFH-1/Huh7.5.1 cells. Gal-9 production was driven by coculturing of PBMCs with apoptotic hepatocytes. Blocking integrin αv ß3 , a receptor for phosphatidylserine expressed on apoptotic cells, also resulted in decreased Gal-9 production. Finally, we found that serum Gal-9 levels were significantly higher in CHC patients than in healthy donors and patients who achieved sustained virologic response. Among CHC patients, serum Gal-9 levels were significantly higher in patients with elevated alanine aminotransferase (ALT) than in those with normal ALT. CONCLUSION: These results demonstrate that CD14+ monocyte-derived Gal-9 increases NK cell cytotoxicity in HCV infection, which might be associated with liver injury and persistent infection. (Hepatology 2017;65:18-31).

Quick and stable parallel puncture of hepatic tumors using a double-barreled needle direction system for ultrasound-guided bipolar radiofrequency ablation.

AIM: In bipolar radiofrequency ablation (RFA) therapy, insertion of multiple needles at appropriate points on a target is difficult. The aim of our study was to evaluate a simplified method for multi-electrode insertion using a newly developed double-barreled needle guidance system for percutaneous RFA of hepatic tumors. METHODS: RFA using two bipolar electrodes was performed in 15 consecutive patients (nine men, six women; mean age, 72.0 ± 8.2 years) with a solitary small (≤3 cm) hepatic tumor. The first five nodules were treated using the conventional puncture method with the standard attachment, then 10 nodules were ablated using the parallel puncture method with the double-barreled attachment. The times required for double-needle placement and the shapes of the ablated areas were compared between the two puncture methods. RESULTS: The parallel puncture method required a shorter time for double-needle placement than the conventional method (12 s [range, 8-24] vs 96 s [range, 50-240]; P = 0.0003), and allowed continuous observation of the tip of all needles and the size of the ablated area as it increased until completion of the ablation. The method also provided a stable ellipsoidal ablated area. The median height, width and thickness were 30 mm (range, 22-34), 30 mm (range, 21-33) and 20 mm (range, 7-25), respectively, using 20-mm electrodes, and 34 mm (range, 32-41), 36 mm (range, 35-38) and 24 mm (range, 23-24), respectively, using 30-mm electrodes. CONCLUSION: The parallel puncture method may be a feasible procedure for multi-needle RFA therapy.

S100A8 Production in CXCR2-Expressing CD11b+Gr-1high Cells Aggravates Hepatitis in Mice Fed a High-Fat and High-Cholesterol Diet.

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease with a spectrum of presentations. S100A8 has been suggested to play a pivotal role as an endogenous immune-activator in inflammatory diseases. In this study, we investigated the involvement of S100A8 in the development of NAFLD. We used a diet model of NAFLD, in which mice were fed either a high-fat and high-cholesterol diet (HFHCD) or a normal diet (ND) as a control. We also assessed liver tissues from patients with NAFLD, including patients with nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). HFHCD-fed mice, but not ND-fed mice, developed steatohepatitis. S100A8 expression was significantly elevated in the livers of HFHCD-fed mice compared with the controls. S100A8 was exclusively expressed in CXCR2-expressing CD11b(+)Gr-1(high) cells, which significantly increased in the livers of HFHCD-fed mice. These cells were F4/80 negative and did not possess a suppressor function. TNF-α expression was enhanced by S100A8 in primary liver leukocytes or a hepatocyte cell line and significantly elevated in the livers of HFHCD-fed mice. TNF-α was primarily produced from CD11b(+)F4/80(+) cells in liver leukocytes in response to S100A8. TNF-α deficiency attenuated hepatitis in HFHCD-fed mice. S100A8 was significantly more expressed in the liver tissues of patients with NASH than in those of patients with NAFL. In conclusion, these results suggest that S100A8 is primarily produced from CXCR2-expressing CD11b(+)Gr-1(high) cells, and it upregulates TNF-α production in CD11b(+)F4/80(+) cells through cellular cross-talk, which is an important mechanism in the development of NAFLD.

The Hepatitis B Virus Genotype Affects the Persistence of Viral Replication in Immunodeficient NOG Mice.

BACKGROUND & AIMS: At least eight genotypes of Hepatitis B virus (HBV) have been identified. HBV genotype C is the most common genotype in Japan, although the incidence of HBV genotype A is increasing. The reason underlying the differences in viral multiplication of the HBV genotypes is unclear, especially in vivo. The purpose of this study was to elucidate the differences in HBV load and the persistence of viremia in vivo between genotypes A and C. METHODS: Immunodeficient NOG mice were transfected by hydrodynamic injection with the HBV expression plasmids pHBA1.2 or pHBC1.2, which contain overlength (1.2-mer) copies of the genomes of HBV genotype A or C, respectively. RESULTS: One day after transfection, the number of HBcAg-positive hepatocytes and serum HBV DNA levels were similar between mice transfected with pHBA1.2 and pHBC1.2. Serum levels of HBV DNA, HBsAg and HBeAg in mice transfected with pHBA1.2 were maintained over 5 months. In contrast, those in mice with pHBC1.2 gradually decreased over time and reached undetectable levels within 3 months after transfection. HBcAg-stained hepatocytes were detected in mice transfected with pHBA1.2, but not pHBC1.2, 5 months post-transfection. Double-staining immunohistochemistry revealed that the number of cleaved caspase3-stained, HBcAg-positive hepatocytes in the pHBC1.2-transfected mice was higher than in the pHBA1.2-transfected mice 3 days post-transfection. Moreover, the plasmid DNA and covalently closed circular DNA levels were decreased in the livers of pHBC1.2-transfected mice. These results suggested that hepatocytes expressing HBV genotype C were eliminated by apoptosis in the absence of immune cells more often than in hepatocytes expressing HBV genotype A. CONCLUSIONS: Immunodeficient mice transfected with HBV genotype A develop persistent viremia, whereas those transfected with HBV genotype C exhibit transient viremia accompanied by apoptosis of HBV-expressing hepatocytes. This differences may affect the clinical courses of patients infected with HBV genotypes A and C.

Emergence of hepatitis C virus NS5A L31V plus Y93H variant upon treatment failure of daclatasvir and asunaprevir is relatively resistant to ledipasvir and NS5B polymerase nucleotide inhibitor GS-558093 in human hepatocyte chimeric mice.

BACKGROUND: Resistance-associated variants (RAVs) emerge at multiple positions spanning hepatitis C virus (HCV) NS3/4A and NS5A regions upon failure of asunaprevir/daclatasvir combination therapy. It has not been determined whether the emergence of such RAVs have an impact on re-treatment by a combination of ledipasvir and sofosbuvir, a potent regimen for HCV genotype 1 infection. METHODS: TK-NOG human hepatocyte chimeric mice were inoculated with sera from a patient with treatment failure of asunaprevir/daclatasvir therapy. RESULTS: They developed persistent HCV infection with triple variants of NS3/4A D168V, NS5A L31V plus Y93H. Administration of ledipasvir/GS-558093 (a NS5B nucleotide analog) in these mice failed to achieve end-of-treatment response or sustained virologic response, which was in sharp contrast to the results in mice with wild-type virus infection. The administration of telaprevir/GS-558093 successfully achieved it in those mice. CONCLUSIONS: Treatment failure with asunaprevir/daclatasvir may limit further treatment options. This population may represent a growing unmet medical need.

Invariant natural killer T cell deficiency leads to the development of spontaneous liver inflammation dependent on γδT cells in mice.

BACKGROUND: Immune tolerance is maintained in the liver, and perturbation of tolerance can lead to immune-mediated liver diseases such as autoimmune hepatitis (AIH). Invariant natural killer T (iNKT) cells and γδT cells have been shown to maintain immune homeostasis as regulatory cells and to play pathogenic roles in immune-mediated diseases as effector cells. We hypothesized that iNKT cells and γδT cells are involved in the maintenance of hepatic immune tolerance and immune-mediated liver disease. METHODS: We measured liver inflammation and the cytokine profiles of liver mononuclear cells in BALB/c wild-type (WT) mice and BALB/c Jα18-deficient (KO) mice lacking iNKT cells. We also examined the role of γδT cells in AIH using liver tissue from AIH patients and control subjects. RESULTS: Spontaneous liver inflammation, hepatocyte damage, and anti-nuclear-antibody production occurred in Jα18 KO mice but not in WT mice. Furthermore, liver mononuclear cells from Jα18 KO mice, but not those from WT mice, produced interleukin-17 (IL-17). γδT cells were the primary producers of the cytokine, and they were more abundant in the livers of Jα18 KO mice than in those of WT mice. In Jα18 KO mice, the administration of anti-γδT-cell-receptor antibody abolished liver inflammation, hepatocyte damage, and IL-17 production. γδT cells accumulated in the livers of AIH patients but not in those of the control subjects. CONCLUSIONS: Our results suggest a protective role for iNKT cells, a pathologic role for γδT cells, and an association between these cells in the pathogenesis of AIH.

Carbamazepine promotes liver regeneration and survival in mice.

BACKGROUND & AIMS: Carbamazepine (CBZ), a widely used anticonvulsant and mood stabilizer, activates multiple proliferative and pro-survival pathways. Here, we hypothesize that CBZ may promote hepatocellular proliferation and ameliorate liver regeneration. METHODS: C57BL6/J mice were orally administered CBZ or vehicle and underwent a 70% partial hepatectomy (PHx), 85% PHx or treatment with carbon tetrachloride (CCl4). Liver regeneration was determined by liver to body weight ratio, hepatocyte proliferation markers, and activation of intracellular signalling pathways. RESULTS: Two to 5days after the 70% PHx, the liver to body weight ratio was significantly higher in the CBZ-treated mice than in the vehicle-treated mice. CBZ treatment upregulated the number of proliferative hepatocytes following PHx or CCl4 treatment, as assessed by intrahepatic Ki-67 staining, BrdU uptake, and PCNA protein expression. PHx surgery induced the expression of several cyclins and activated Akt/mTOR signalling pathways, all of which were enhanced by CBZ treatment. The administration of the mTOR inhibitor temsirolimus abrogated the hepato-proliferative effect of CBZ. CBZ treatment significantly improved the survival rate of the mice that underwent lethal 85% massive hepatectomy. CONCLUSIONS: CBZ demonstrated a novel hepato-proliferative effect through the activation of the mTOR signalling pathway in hepatectomised mice. CBZ has the potential to be a therapeutic option for facilitating efficient liver regeneration in patients subjected to liver surgery.

Valine, the branched-chain amino acid, suppresses hepatitis C virus RNA replication but promotes infectious particle formation.

BACKGROUND & AIMS: Concentrations of the branched-chain amino acid (BCAA) in the serum of patients with liver cirrhosis correlate with their liver function. Oral administration of BCAA can ameliorate hypoalbuminemia and hepatic encephalopathy. In this study, we aim to clarify the role of BCAA in regulating the replication of the hepatitis C virus (HCV). METHODS: HCV sub-genomic replicon cells, genome-length replicon cells, and cells infected with cell culture-infectious HCV (HCVcc) were cultured in media supplemented with various concentrations of BCAA, followed by evaluation of the replicon or HCV abundance. RESULTS: BCAA was capable of suppressing the HCV replicon in a dose-dependent manner and the effect was independent of the mTOR pathway. Of the three BCAAs, valine was identified as being responsible for suppressing the HCV replicon. Surprisingly, an abundance of HJ3-5(YH/QL), an HCVcc, in Huh7 cells was augmented by BCAA supplementation. In contrast, BCAA suppressed an abundance of HJ3-5(wild), an HCVcc that cannot assemble virus particle in Huh7 cells. Internal ribosome entry site of HCV was shown to be a target of BCAA. Single-cycle virus production assays using Huh7-25 cells, which lacked CD81 expression, revealed that BCAA, especially valine, promoted infectious virus particle formation with minimal effect on virus secretion. Thus, BCAA was found to have two opposing effects on HCV production: suppression of the HCV genome RNA replication and promotion of infectious virus formation. CONCLUSIONS: BCAA accelerates HCV production through promotion of infectious virus formation in infected cells despite its suppressive effect on HCV genome replication.