Immunoglobulin-like transcript 2 as an impaired anti-tumor cytotoxicity marker of natural killer cells in patients with hepatocellular carcinoma.

Introduction: Natural killer (NK) cells play a pivotal role in immune surveillance in the liver. We aimed to identify potential targets for NK cell-mediated immune intervention by revealing the functional molecules on NK cells in HCC patients. Methods: To evaluate the impact of aging on NK cell phenotypes, we examined NK cells from healthy volunteers (HVs) of various ages. Because ILT2 expression on CD56dim NK cells increased with increasing age, we enrolled age-matched HCC patients and HVs. We determined the NK cell phenotypes in blood mononuclear cells (PBMCs) and intrahepatic lymphocytes (IHLs) from cancerous and non-cancerous tissues. We evaluated cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) of NK cells in vitro. Results: ILT2-positive CD56dim NK cells in PBMCs were increased in HCC patients compared with HVs. In HCC patients, ILT2-positive CD56dim NK cells were increased in cancerous IHLs compared with non-cancerous IHLs and PBMCs. We examined the impact of macrophage migration inhibitory factor (MIF) on ILT2 expression in co-cultures of HCC cells and NK cells. The enhanced expression of ILT2 on CD56dim NK cells from HCC patients was inhibited by masking antibodies against MIF and CXCR4. ILT2-positive CD56dim NK cells exhibited lower capacities for cytotoxicity and ADCC than ILT2-negative cells, which were partially restored by ILT2 blockade. Conclusions: In HCC patients, ILT2 is a signature molecule for cancerous CD56dim NK cells with impaired cytolytic capacity. The MIF-CXCR4 interaction is associated with ILT2 induction on CD56dim NK cells and ILT2 serves as a target for functional NK cell restoration.

Differences in branched-chain amino acid to tyrosine ratio (BTR) among etiologies of chronic liver disease progression compared to healthy adults.

BACKGROUND: The branched-chain amino acids (BCAAs) to tyrosine (Tyr) ratio (BTR) test is used to evaluate the progression of chronic liver disease (CLD). However, the differences across sex, age, body mass index (BMI) and etiologies are still unclear. METHODS: We retrospectively reviewed data from 2,529 CLD cases with free amino acids (FAAs) in peripheral blood from four hospitals and 16,421 general adults with FAAs data from a biobank database. In total, 1,326 patients with CLD (covering seven etiologies) and 8,086 healthy controls (HCs) were analyzed after exclusion criteria. We investigated the change of BTR in HCs by sex, age and BMI and then compared these to patients divided by modified ALBI (mALBI) grade after propensity score matching. RESULTS: BTR is significantly higher in males than females regardless of age or BMI and decreases with aging in HCs. In 20 types of FAAs, 7 FAAs including BCAAs were significantly decreased, and 11 FAAs including Tyr were significantly increased by mALBI grade in total CLD. The decreasing timings of BTR were at mALBI grade 2b in all CLD etiologies compared to HCs, however in chronic hepatitis C (CHC), chronic hepatitis B (CHB) and alcoholic liver disease (ALD), BTR started to decrease at 2a. There was a positive correlation between BCAAs and albumin among parameters in BTR and mALBI. The correlation coefficients in PBC, ALD and MASLD were higher than those of other etiologies. CONCLUSIONS: BTR varies by sex and age even among healthy adults, and decreasing process and timing of BTR during disease progression is different among CLD etiologies.

Posttreatment liver function, but not baseline liver function stratifies patient survival after direct-acting antiviral treatment in decompensated cirrhosis with hepatitis C virus.

BACKGROUND: The prognosis of cirrhosis is clearly stratified by liver function. Although direct-acting antiviral (DAA) has recently been used to eliminate hepatitis C virus (HCV), it is not clear whether liver function stratifies the prognosis of decompensated cirrhotic patients treated with DAA. METHODS: A total of 206 HCV-associated decompensated cirrhotic patients who started DAA from February 2019 to December 2021 at 31 Japanese hospitals were prospectively registered. RESULTS: The median age was 68, and the proportions of patients with Child-Pugh class A (CP-A), CP-B and CP-C were 10% (20/206), 76% (156/206) and 15% (30/206), respectively. Twenty-six patients died, and two patients underwent liver transplantation (LT); the 2- and 3-year LT-free survival rates were 90.0% and 83.2%, respectively. We examined factors associated with LT-free survival using 2 models including either CP class (Model 1) or MELD score (Model 2). In multivariate Cox proportional hazard analysis, CP class at 12 weeks after the end of treatment (EOT) in Model 1 and MELD score at 12 weeks after the EOT in Model 2 were significant factors, while baseline CP class or MELD score was not. Two-year LT-free survival rates were 100%, 91.6% and 60.4% for patients with CP-A, CP-B and CP-C at 12 weeks after the EOT and 95.2% and 69.6% for patients with MELD < 15 and MELD ≥ 15 at 12 weeks after the EOT, respectively. CONCLUSIONS: The prognosis of decompensated cirrhotic patients receiving DAA was stratified by liver function at 12 weeks after the EOT, not by baseline liver function.

Exercise changes the intrahepatic immune cell profile and inhibits the progression of nonalcoholic steatohepatitis in a mouse model.

BACKGROUND: NASH is an increasingly common cause of chronic liver disease and can progress to cirrhosis and HCC. Although exercise suppresses inflammation during acute hepatitis, its impact on the progression of chronic liver disease remains unclear. Here, we investigated the effects of exercise on disease progression and intrahepatic immune cell composition in a mouse model of NASH. METHOD: Mice were assigned to 4 groups: 2 control groups (normal diet) and 2 NASH groups (western diet and low-dose carbon tetrachloride injection). One of each group remained sedentary and one was exercised on a treadmill for 12 weeks (60 min/d, 5 times/wk). All mice were then analyzed for liver histomorphology, steatosis, inflammation, and fibrosis; liver, adipose tissue, and skeletal muscle expression of genes related to metabolism and inflammation; and intrahepatic immune cell composition. RESULT: Compared with the normal diet mice, NASH mice exhibited enhanced liver steatosis, inflammation, and fibrosis; upregulated expression of liver lipogenesis-related and inflammation-related genes; and increased frequencies of intrahepatic F4/80 int CD11b hi bone marrow-derived macrophages and programmed death receptor-1 (PD-1) + CD8 + T cells. Expression of inflammatory cytokines and the frequencies of bone marrow-derived macrophages and PD-1 + CD8 + T cells correlated positively with liver steatosis, inflammation, and fibrosis. Exercise was shown to reduce NASH-induced hepatic steatosis, liver inflammation, and fibrosis; induce alterations in metabolism-related genes and inflammatory cytokines in the liver; and suppress accumulation of liver bone marrow-derived macrophages and PD-1 + CD8 + T cells. In addition, we showed that exercise induced increased expression of IL-15 in muscle and its deficiency exacerbated the pathology of NASH. CONCLUSIONS: Exercise alters the intrahepatic immune cell profile and protects against disease progression in a mouse model of NASH.

Gene-regulation modules in nonalcoholic fatty liver disease revealed by single-nucleus ATAC-seq.

We investigated the progression of nonalcoholic fatty liver disease from fatty liver to steatohepatitis using single-nucleus and bulk ATAC-seq on the livers of rats fed a high-fat diet (HFD). Rats fed HFD for 4 wk developed fatty liver, and those fed HFD for 8 wk further progressed to steatohepatitis. We observed an increase in the proportion of inflammatory macrophages, consistent with the pathological progression. Utilizing machine learning, we divided global gene regulation into modules, wherein transcription factors within a module could regulate genes within the same module, reaffirming known regulatory relationships between transcription factors and biological processes. We identified core genes-central to co-expression and protein-protein interaction-for the biological processes discovered. Notably, a large part of the core genes overlapped with genes previously implicated in nonalcoholic fatty liver disease. Single-nucleus ATAC-seq, combined with data-driven statistical analysis, offers insight into in vivo global gene regulation as a combination of modules and assists in identifying core genes of relevant biological processes.

Effects of sodium glucose cotransporter 2 inhibitors and pioglitazone on FIB-4 index in metabolic-associated fatty liver disease.

BACKGROUND: The antidiabetic drugs sodium glucose cotransporter 2 inhibitors (SGLT2is) and thiazolidinediones have beneficial effects on the liver dysfunction of patients with nonalcoholic fatty liver disease and type 2 diabetes mellitus (T2DM). We aimed to determine the efficacy of these drugs for the treatment of liver disease in patients with metabolic dysfunction-associated fatty liver disease (MAFLD) and T2DM. METHODS: We undertook a retrospective study of 568 patients with MAFLD and T2DM. Of these, 210 were treating their T2DM with SGLT2is (n = 95), 86 with pioglitazone (PIO), and 29 with both. The primary outcome was the change in Fibrosis-4 (FIB-4) index between baseline and 96 weeks. RESULTS: At 96 weeks, the mean FIB-4 index had significantly decreased (from 1.79 ± 1.10-1.56 ± 0.75) in the SGLT2i group, but not in the PIO group. The aspartate aminotransferase to platelet ratio index, serum aspartate and alanine aminotransferase (ALT), hemoglobin A1c, and fasting blood sugar significantly decreased in both groups (ALT: SGLT2i group, -17 ± 3 IU/L; PIO group, -14 ± 3 IU/L). The bodyweight of the SGLT2i group decreased, but that of the PIO group increased (-3.2 kg and +1.7 kg, respectively). When the participants were allocated to two groups according to their baseline ALT (>30 IU/L), FIB-4 index significantly decreased in both groups. In patients taking pioglitazone, the addition of SGLT2i improved liver enzymes but not FIB-4 index for 96 weeks. CONCLUSIONS: Treatment with SGLT2i causes a larger improvement in FIB-4 index than PIO in patients with MAFLD over 96 weeks.

Serum osteopontin predicts the response to atezolizumab plus bevacizumab in patients with hepatocellular carcinoma.

BACKGROUND: Combination therapy with anti-programmed death-ligand 1 and anti-vascular endothelial growth factor (VEGF) antibodies has become the standard treatment for un-resectable hepatocellular carcinoma (uHCC). We aimed to identify predictive circulating biomarkers for the outcome/response of the combination therapy in uHCC patients. METHODS: This prospective multicenter study enrolled 70 patients with uHCC who received atezolizumab and bevacizumab (Atez/Bev). We evaluated 47 circulating proteins in sera before and after 1 and 6 weeks of Atez/Bev therapy by multiplex bead-based immunoassay and ELISA. As controls, we analyzed the sera from 62 uHCC patients before treatment of lenvatinib (LEN) and healthy volunteers (HVs). RESULTS: The disease control rate was 77.1%. Median progression-free survival (PFS) was 5.7 months (95% confidence interval [CI] = 3.8-9.5). The pretreatment levels of osteopontin (OPN), angiopoietin-2, VEGF, S100-calcium-binding protein A8/S100-calcium-binding protein A9, soluble programmed cell death-1, soluble CD163, and 14 cytokines/chemokines were higher in patients with uHCC than in HVs. Among these, pretreatment OPN levels were higher in PD group than in non-PD group for Atez/Bev. The PD rate was higher in high OPN group than in low OPN group. Multivariate analysis identified high pretreatment OPN and high α-fetoprotein levels as independent predictors of PD. In the sub-analysis of Child-Pugh class A patients, PFS was also shorter in the high OPN group than in the low OPN group. Pretreatment OPN level was not associated with treatment response for LEN. CONCLUSION: High serum OPN levels were associated with poor response to Atez/Bev in patients with uHCC.

Preoperative serum brain-derived neurotrophic factor as a predictive biomarker for sepsis after living-donor liver transplantation.

AIM: Although the survival rate after living-donor liver transplantation (LDLT) is improving, sepsis still limits the prognosis. Immune dysfunction and sarcopenia are often observed in LDLT patients, and increase susceptibility to infection. Brain-derived neurotrophic factor (BDNF) is a myokine produced by immune cells and skeletal muscle. We aimed to determine whether serum BDNF could be a feasible biomarker for sepsis of LDLT patients. METHODS: We measured serum samples from 124 patients who underwent LDLT and 9 healthy volunteers for BDNF. We examined its correlation with incidence rate of sepsis. To clarify the source of BDNF, we examined its expression in lymphocytes, skeletal muscle cells, and hepatocytes. RESULTS: Patients who experienced sepsis showed worse short-term survival. Preoperative serum BDNF was lower in LDLT patients compared with healthy volunteers, and was also lower in Child-Pugh C compared with Child-Pugh A or B. Serum BDNF was inversely correlated with Model for End-Stage Liver Disease and controlling nutritional status (CONUT) scores, but had a weak positive correlation with skeletal muscle mass index (SMI). Multivariate analysis revealed that serum BDNF was independently associated with sepsis. Preoperative serum BDNF was a better predictor of sepsis in LDLT patients than CONUT score or SMI. Serum BDNF was positively correlated with lymphocyte counts, especially T cells. In vitro, T cells and skeletal muscle cells produced BDNF. CONCLUSIONS: Preoperative serum BDNF could be a predictive biomarker for sepsis after LDLT, by reflecting the systemic condition including hepatic function, nutritional status, and immune status.

Reactivation of hepatitis C virus with severe hepatitis flare during steroid administration for interstitial pneumonia.

Hepatitis C virus reactivation (HCVr) was defined previously as an increase in HCV RNA level of ≥ 1 log10 IU/mL from baseline HCV RNA level after chemotherapies or immunosuppressive therapies, but HCVr during a steroid monotherapy has rarely been reported. Here we report a 75-year-old Japanese female with chronic hepatitis C (genotype 2a) who developed HCVr after the administration of prednisolone for interstitial pneumonia. She experienced alanine aminotransferase (ALT) flare with icterus, but after the tapering of prednisolone and a liver supporting therapy, levels of HCV RNA and ALT were gradually decreased. Then, she received an anti-viral therapy with sofosbuvir/ledipasvir. Although HCV relapsed 4 weeks after the therapy, a second therapy with glecaprevir/pibrentasvir was successful. This case suggests that HCVr with hepatitis flare can occur even after a steroid monotherapy, and we should pay attention to HCVr when we administer prednisolone for patients with HCV chronic infection.

Plasma free amino acids are associated with sarcopenia in the course of hepatocellular carcinoma recurrence.

OBJECTIVE: Sarcopenia worsens the prognosis of patients with hepatocellular carcinoma (HCC). The aim of this study was to elucidate the plasma free amino acids (PFAAs) associated with sarcopenia or myosteatosis in the course of HCC recurrence. METHODS: In this cross-sectional study, 187 patients were enrolled retrospectively. All patients experienced more than one hospitalization (mean times, 2.65) owing to HCC recurrence. The skeletal muscle index (SMI) and muscle attenuation (MA) were measured by a transverse computed tomography (CT) scan image at the third lumbar vertebra (L3). The changes in the concentration of 24 PFAAs, SMI, and MA in the same patient between recurrences were defined as Δ. The associations between sarcopenia, myosteatosis, and PFAAs were evaluated by a logistic regression model. The ΔSMI and ΔMA were compared between the patients who received branched-chain amino acids (BCAAs) formulation and those who did not. RESULTS: Patients with sarcopenia showed lower survival rate; the 1-, 3-, and 5-y survival rates were 85%, 42%, and 9%, respectively. Multivariate analysis revealed that the level of total BCAAs was significantly associated with sarcopenia. The correlation coefficient value between the change of leucine (ΔLeu) and ΔSMI was highest (R = 0.256; P < 0.001) among the PFAAs. In the Child-Pugh grade B or C, the decrease of SMI was significantly more suppressed in the patients with the BCAAs formulation than in those without BCAAs formulation (ΔSMI: mean change -0.98 versus -3.45 cm²/m²; P = 0.038). CONCLUSION: Among the PFAAs, the level of BCAAs was associated with sarcopenia in the course of HCC recurrence.

Hepatitis B Virus Reactivation with Discontinuation of Nucleoside Analogue in Patients Who Received Allogeneic Hematopoietic Stem Cell Transplantation.

Reactivation of hepatitis B virus (HBV) is known to occur frequently after hematopoietic stem cell transplantation (HSCT). The reactivation can be prevented by nucleos(t)ide analogue (NA), but it is unclear how long NA should be continued. Here, we report 3 cases of HBV reactivation with discontinuation of NA following the discontinuation of immunosuppressive therapies after HSCT. Three male patients aged 34, 59, and 54 years received allogeneic HSCT (allo-HSCT) for chronic myeloid leukemia, mixed phenotype acute leukemia, and myelodysplastic syndrome, respectively. Before HSCT, 2 patients were positive for hepatitis B surface antigen (HBsAg) and 1 patient was negative for HBsAg and positive for antibodies to hepatitis B core antigen. NA (lamivudine or entecavir) was started at the same time as HSCT and stopped after the discontinuation of immunosuppressive therapies. In all patients, the serum HBV DNA levels were increased after the discontinuation of NAs. Two of the three patients developed severe hepatitis with high levels of HBV DNA (7.5 and 7.4 log IU/mL, respectively). A patient without hepatitis was re-administered NA soon after the HBV DNA started to increase (3.3 log IU/mL). Interestingly, the 2 patients who developed hepatitis cleared HBsAg promptly after the recovery from hepatitis and they could stop NAs without the reversion of HBsAg. It was speculated that transplanted immune cells, which were naïve for HBV, react strongly with HBV antigens that were increased after the NA discontinuation. The discontinuation of NA after allo-HSCT is not recommended generally because strong hepatitis might be induced even after several years.

Steatotic Hepatocytes Release Mature VLDL Through Methionine and Tyrosine Metabolism in a Keap1-Nrf2-Dependent Manner.

BACKGROUND AND AIMS: NAFLD is a lipotoxic disease wherein hepatic steatosis and oxidative stress are key pathogenic features. However, whether free amino acids (FAAs) are associated with the oxidative stress response against lipotoxicity has yet to be determined. We hypothesized that an imbalance of FAAs aggravates hepatic steatosis by interfering with the oxidative stress sensor. APPROACH AND RESULTS: C57BL/6 mouse immortalized hepatocytes, primary hepatocytes, and organoids were employed. Steatotic hepatocytes treated with oleic acid (OA) were cultured under FAA-modifying media based on the concentrations of FAAs in the hepatic portal blood of wild-type (WT) mice. As in vivo experiments, WT hepatocyte-specific Kelch-like ECH-associated protein 1 (Keap1) knockout mice (Keap1∆hepa ) and Cre- control mice (Keap1fx/fx ) were fed high-fat (HF) diets with modified amino acid content. The correlations were analyzed between the areas of lipid droplets (LDs) around central vein and plasma OA/FAA ratio in 61 patients with NAFLD. Mice fed an HF, Met-restricted, and tyrosine (Tyr)-deficient diet showed the NAFLD-like phenotype in which the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), triglyceride-rich VLDL, and fumarate were decreased in liver, but Keap1∆hepa ameliorated these phenomena. Reactive oxygen species and LDs induced by the deprivation of Met and Tyr were prevented in hepatic organoids generated from Keap1∆hepa . Dimethyl fumarate, an Nrf2 inducer, ameliorated the steatosis and increased the hepatic fumarate reduced by the deprivation of Met and Tyr in vitro. OA/Met or Tyr ratio in peripheral blood was associated with the hepatic steatosis in patients with NAFLD. CONCLUSIONS: An imbalance between free fatty acids and Met and Tyr induces hepatic steatosis by disturbing the VLDL assembling through the Keap1-Nrf2 system.

Switching to tenofovir disoproxil fumarate in entecavir-treated chronic hepatitis B patients: A pilot randomized controlled study.

Although hepatitis B surface antigen (HBsAg) removal is considered the goal of chronic hepatitis B treatment, it can rarely be achieved with nucleos(t)ide analogues (NAs). It has been reported that tenofovir disoproxil fumarate (TDF) is superior in reducing HBsAg compared with entecavir (ETV) in treatment-naïve patients; however, the effect of TDF in patients who have received NAs is still unclear. The aim of the present study was to evaluate the efficacy of switching from ETV to TDF in patients who were already receiving ETV. A pilot randomized controlled study for 2 years in patients who had been treated with ETV for >1 year and did not exhibit drug resistance was performed (Clinical trial registration: UMIN000021948, UMIN-CTR, May 1, 2016). A total of 20 patients were enrolled and 19 patients were randomized into 2 groups, a TDF-switching group (n=12) or an ETV-continuing group (n=7). The mean change in HBsAg levels after 2 years was greater in the TDF group compared with the ETV group, but the difference was not significant (-0.25 vs. -0.06 log IU/ml). In the TDF group, hepatitis B e antigen (HBeAg)-positive patients at baseline showed significantly greater changes in HBsAg (-0.63 vs. -0.03 log IU/ml; P=0.030). In contrast, no difference between HBeAg-positive and HBeAg-negative patients was observed in the ETV group. No significant differences of estimated glomerular filtration rate and inorganic phosphorus changes were observed among the TDF and ETV groups. In conclusion, a significant HBsAg decrease was not achieved after switching from ETV to TDF in the overall analysis, but HBeAg-positive patients showed a larger HBsAg decrease after switching treatment.

Rapid enlargement of a hepatic hilar cyst leading to bile duct obstruction after acute exacerbation of chronic hepatitis B.

A hepatic cyst is usually asymptomatic but, in some cases, can be associated with various complications. Here we report a rare case with rapid enlargement of a hepatic hilar cyst that induced bile duct obstruction after an acute exacerbation of chronic hepatitis B. The case is a 60-year old female who discontinued entecavir by herself. Hyperbilirubinemia was prolonged along with bile duct obstruction due to an enlarged cyst. After the administration of entecavir and steroid pulse therapy, biliary drainage and punctuation of the cyst were performed. There was no evidence of malignancy in the cyst. The therapies were not effective enough, probably due to the prior liver damage, and she died of acute on chronic liver failure. This case suggests that a hepatic hilar cyst in a patient with acute hepatitis or an acute exacerbation of chronic hepatitis can become enlarged and may affect the clinical course of hepatitis. In such a case, the size of the cyst should be monitored frequently and bile duct obstruction should be treated early if it occurs.

Methotrexate-associated Lymphoproliferative Disorder in the Liver Resembling Hepatocellular Carcinoma Treated with Transarterial Chemoembolization.

Methotrexate-related lymphoproliferative disorder (MTX-LPD) is known to be a side effect of MTX, but its involvement in the liver has been rarely reported. We herein report a 70-year-old woman with autoimmune hepatitis and rheumatoid arthritis who developed multiple liver tumors. We initially considered that she had developed rapid-growing hepatocellular carcinoma (HCC) in the cirrhotic liver based on imaging tests. A tumor biopsy and transcatheter arterial chemoembolization were thus performed. The tumors were then diagnosed as diffuse large B-cell lymphoma pathologically and considered to be MTX-LPD. This case indicates that MTX-LPD should be considered even in cirrhotic patients with liver tumors resembling HCC.

Spontaneous reactivation of hepatitis B virus with S gene mutations in an elderly patient with diabetic nephropathy.

Generally, reactivation of hepatitis B virus (HBV) infection is induced by the administration of immunosuppressants or anticancer agents, but reactivation without such drugs has rarely been reported. Here we report an elder case with spontaneous reactivation of HBV replication accompanied by hepatitis B surface antigen (HBsAg) mutations. A 69-year-old man with a history of diabetes mellitus and chronic kidney disease (CKD) was found to be positive for HBsAg (0.072 IU/ml) in June 2018. In May 2019, marked hepatic dysfunction and increased HBsAg (2533.2 IU/ml) were observed when he visited the hospital due to diarrhea and worsening of CKD. At that time, hepatitis B surface antibody (HBsAb) was positive (268.9 mIU/ml) and HBV DNA was 6.0 log IU/ml. After treatment with entecavir, HBV DNA and HBsAg rapidly decreased. Full-genome HBV sequence analysis revealed that the patient was infected with HBV of subgenotype B1 and it had an "a" determinant mutation M133L in the S gene coding HBsAg. Notably, both HBsAg and HBsAb were positive at the time of HBV reactivation, suggesting that the HBV with these mutations escaped from neutralization by HBsAb. This case suggests that immune escape mutations could play an important role in spontaneous HBV reactivation.

Effect of viral DNA methylation on expression of hepatitis B virus proteins depends on the virus genotype.

The frequency of HBV genomic methylation in the liver was reported to vary among patients, but the detailed mechanism is still unknown. In this study, the effects of HBV genome methylation on HBV replication were investigated in vitro. A total of 6 plasmids containing 1.24-fold the HBV genome of different genotypes (subgenotypes A1, A2, B1, and C2) were purified after in vitro methylation with CpG methyltransferase (M.SssI) and transfected into HepG2 cells. In genotype B and C strains, methylation markedly decreased the amount of hepatitis B e antigen (HBeAg) in the culture supernatant. A reduction of hepatitis B surface antigen (HBsAg) was found in some HBV strains but the reduction was smaller than that of HBeAg. There was no significant difference in particle-associated HBV DNA in the culture supernatant. These findings suggest that HBV genomic methylation might be involved in the HBeAg decline in genotype B and C, in part, and that the reduction of HBsAg was less than that of HBeAg. In conclusion, this study showed that the effect of HBV genomic methylation differs among HBV genotypes, suggesting a potential explanation for the different clinical outcomes of genotypes A, B, and C.

Comparison of hepatitis B virus genotypes B and C among chronically hepatitis B virus-infected patients who received nucleos(t)ide analogs: A multicenter retrospective study.

AIM: Hepatitis B virus genotype B (HBV/B) has been reported to have less risk of liver cirrhosis and hepatocellular carcinoma (HCC), but long-term observation has rarely been reported. We aimed to clarify the characteristics of HBV/B in nucleos(t)ide analog-treated patients in an area where HBV/B is more prevalent than in other areas of Japan. METHODS: A total of 498 chronically HBV-infected patients treated with nucleos(t)ide analog (lamivudine, entecavir, tenofovir disoproxil fumarate, or tenofovir alafenamide fumarate) for >6 months (mean 70.6 months) were included from nine hospitals in northeast Japan. The frequencies of hepatitis B surface antigen loss and HCC occurrence were analyzed. RESULTS: Among 427 patients whose genotype could be determined, 34.0% and 64.4% were infected with HBV/B and genotype C (HBV/C), respectively. The age of patients with HBV/B was significantly older than those with HBV/C (57.7 vs. 48.1). The cumulative rate of hepatitis B surface antigen loss was significantly higher in HBV/B than in HBV/C (3.6% vs. 0.7% at 10 years). Among 480 patients without HCC history, HCC occurrence was found in 40 patients (13.4% at 10 years). There was no cumulative rate difference of HCC occurrence among the genotypes, but after propensity score matching for age/sex, it was significantly lower in HBV/B than in HBV/C (5.3% vs. 18.5% at 10 years). CONCLUSIONS: Although a lower rate of HCC occurrence in HBV/B was shown by an age/sex-matched analysis than that in HBV/C, patients with HBV/B were significantly older and had a comparative risk of HCC occurrence in nucleos(t)ide analog-treated patients.

Small Interfering RNA Screening for the Small GTPase Rab Proteins Identifies Rab5B as a Major Regulator of Hepatitis B Virus Production.

Viruses are considered to use vesicular trafficking in infected cells, but the details of assembly/release pathways of hepatitis B virus (HBV) are still unknown. To identify key regulators of HBV production, we performed short interfering RNA (siRNA) screening for Rab proteins, which are considered to act as molecular switches in vesicular trafficking using HepG2.2.15 cells. Among 62 Rab proteins, the suppression of Rab5B most significantly increased HBV DNA in the culture supernatant. Surprisingly, 5 days after the transfection of Rab5B siRNA, HBV DNA in the supernatant was increased more than 30-fold, reflecting the increase of infectious HBV particles. Northern blotting showed that transcription of 2.4/2.1-kb mRNA coding envelope proteins containing large hepatitis B surface protein (LHBs) was increased. Analysis of hepatocyte nuclear factors (HNFs) showed that transcription of HNF4α, which is known to enhance 2.4-kb mRNA transcription, was regulated by Rab5B. Also, it was revealed that LHBs had accumulated in the endoplasmic reticulum (ER) after Rab5B depletion but not in the multivesicular body (MVB), which is thought to be an organelle utilized for HBV envelope formation. Therefore, it was considered that Rab5B is required for the transport of LHBs from the ER to MVB. Immunofluorescent microscopy showed that HBs proteins, including LHBs, colocalized with HBc in the ER of Rab5B-depleted cells, suggesting that HBV envelopment occurs not only in the MVB but also in the ER. In conclusion, Rab5B is a key regulator of HBV production and could be a target of antiviral therapy.IMPORTANCE HBV infection is a worldwide health problem, but the mechanisms of how HBV utilizes cellular machinery for its life cycle are poorly understood. In particular, it has been unclear how the viral components and virions are transported among the organelles. The HBV budding site has been reported to be the ER or MVB, but it has not been clearly determined. In this study, siRNA-based screening of Rab proteins using HBV-expressing cells showed that Rab5B, one of the Rab5 isoforms, has important roles in late steps of the HBV life cycle. Although Rab5 is known to work on early endosomes, this study showed that Rab5B plays a role in the transport of LHBs between the ER and MVB. Furthermore, it affects the transcription of LHBs. This is the first report on the mechanisms of HBV envelope protein transport among the organelles, and the results provide important insights into the therapeutic control of HBV infection.

Branched chain amino acids are associated with the heterogeneity of the area of lipid droplets in hepatocytes of patients with non-alcoholic fatty liver disease.

AIM: Macrovesicular steatosis around the central vein (zone 3) is one of the pathological features of non-alcoholic fatty liver disease or steatohepatitis (NAFLD/NASH). The aim of this study is to elucidate precisely the association between the area of lipid droplets (LDs) and the plasma metabolic parameters in patients with NAFLD/NASH. METHODS: Eighty patients with NAFLD/NASH diagnosed by needle biopsy were enrolled. The LDs around zone 3 were counted automatically by image processing software, the total area of LDs (TLDs), the maximum area of LDs (MAXLDs), the average area of LDs (AVELDs) and the heterogeneity by the coefficient of variation (CV [%]) were quantified. The correlations between these values and plasma metabolic parameters were analyzed. We evaluated the association between branched chain amino acids (BCAAs) and the heterogeneity of LDs in hepatocytes in vitro and in vivo. RESULTS: The MAXLDs was significantly correlated with more metabolic parameters than AVELDs and TLDs. The level of BCAAs was independently associated with the CV among the metabolic parameters. In early stage NAFLD, aspartate and alanine aminotransferase were significantly higher in the high CV group than in the low CV group. The high concentration of BCAAs increased the CV of LDs in hepatocytes accompanied by the expression of phosphor-p70 S6 kinase and sterol regulatory element-binding protein 1 in vitro. A high BCAA diet induced high heterogeneity of LDs around zone 3 in ob/ob mice. CONCLUSIONS: The levels of BCAAs were associated with the LD heterogeneity of hepatocytes around zone 3 in patients with NAFLD/NASH.