Yuta Wakui

Transient elastography for measurement of liver stiffness measurement can detect early significant hepatic fibrosis in Japanese patients with viral and nonviral liver diseases

BackgroundMany studies have reported the efficiency of transient elastography, a noninvasive, reproducible, and reliable method for predicting liver fibrosis, in patients with chronic hepatitis C (CHC) and B (CHB), but there are few reports about nonviral chronic liver disease (CLD) such as primary biliary cirrhosis (PBC), nonalcoholic steatohepatitis (NAFLD), and autoimmune hepatitis (AIH). We therefore compared the efficiency of transient elastography between CHC and nonviral CLD.MethodsWe assessed the accuracy of liver stiffness measurement (LSM) using Fibroscan, and compared these values with those of hyaluronic acid, type 4 collagen, platelet count, prothrombin index, and AST/platelet ratio index (APRI) as indices for the diagnosis of liver fibrosis in 114 patients with a variety of chronic liver diseases: CHC (n = 51), CHB (n = 11), NAFLD (n = 17), PBC (n = 20), and AIH (n = 15). The histology was assessed according to the METAVIR score by two pathologists.ResultsThe number of fibrosis stage (F0/1/2/3/4) with CHC was 9/15/12/6/10, and that with nonviral CLD was 10/21/11/4/6, respectively. The ability, assessed by area under receiver operating characteristic (AUROC) curve, to predict liver fibrosis F ≥ 2 for LSM, HA, type 4 collagen, platelet count, prothrombin index, and APRI, was 0.92, 0.81, 0.87, 0.85, 0.85, and 0.92 in CHC patients, respectively; and 0.88, 0.72, 0.81, 0.67, 0.81, and 0.77 in nonviral CLD patients, respectively.ConclusionsIn patients with nonviral CLD, LSM was most helpful in predicting significant fibrosis (F ≥ 2). Transient elastography is a reliable method for predicting significant liver fibrosis, not only in CHC patients but also in nonviral CLD patients.

Possible involvement and the mechanisms of excess trans-fatty acid consumption in severe NAFLD in mice

BACKGROUND & AIMS Excessive trans-fatty acids (TFA) consumption has been thought to be a risk factor mainly for coronary artery diseases while less attention has been paid to liver disease. We aimed to clarify the impact of TFA-rich oil consumption on the hepatic pathophysiology compared to natural oil. METHODS Mice were fed either a low-fat (LF) or high-fat (HF) diet made of either natural oil as control (LF-C or HF-C) or partially hydrogenated oil, TFA-rich oil (LF-T or HF-T) for 24 weeks. We evaluated the liver and body weight, serological features, liver lipid content and composition, liver histology and hepatic lipid metabolism-related gene expression profile. In addition, primary cultures of mice Kupffer cells (KCs) were evaluated for cytokine secretion and phagocytotic ability after incubation in cis- or trans-fatty acid-containing medium. RESULTS The HF-T-fed mice showed significant increases of the liver and body weights, plasma alanine-aminotransferase, free fatty acid and hepatic triglyceride content compared to the HF-C group, whereas the LF-T group did not differ from the LF-C group. HF-T-fed mice developed severe steatosis, along with increased lipogenic gene expression and hepatic TFA accumulation. KCs showed increased tumor necrosis factor secretion and attenuated phagocytotic ability in the TFA-containing medium compared to its cis-isomer. CONCLUSIONS Excessive consumption of the TFA-rich oil up-regulated the lipogenic gene expression along with marked hepatic lipid accumulation. TFA might be pathogenic through causing severe steatosis and modulating the function of KCs. The quantity and composition of dietary lipids could be responsible for the pathogenesis of non-alcoholic steatohepatitis.

Four‐year study of lamivudine and adefovir combination therapy in lamivudine‐resistant hepatitis B patients: influence of hepatitis B virus genotype and resistance mutation pattern

Summary.  To investigate the efficacy of long‐term lamivudine (3TC) and adefovir dipivoxil (ADV) combination therapy in 3TC‐resistant chronic hepatitis B virus (HBV) infected patients, we analysed 28 3TC‐resistant patients treated with the combination therapy during 47 months (range, 9–75). At 12, 24, 36, and 48 months, the rates of virological response with undetectable HBV DNA (≤2.6 log copies/mL) were 56, 80, 86, and 92%, respectively. Among 17 hepatitis B e antigen (HBeAg)‐positive patients, HBeAg disappeared in 24% at 12 months, 25% at 24 months, 62% at 36 months, and 88% at 48 months. When HBV genotypes were compared, patients with genotype B achieved virological response significantly more rapidly than those with genotype C (P = 0.0496). One patient developed virological breakthrough after 54 months, and sequence analysis of HBV obtained from the patient was performed. An rtA200V mutation was present in the majority of HBV clones, in addition to the 3TC‐resistant mutations of rtL180M+M204V. The rtN236T ADV‐resistant mutation was observed in only 25% clones. In vitro analysis showed that the rtA200V mutation recovered the impaired replication capacity of the clone with the rtL180M+M204V mutations and induced resistance to ADV. Moreover, rtT184S and rtS202C, which are known entecavir‐resistant mutations, emerged in some rtL180M+M204V clones without rtA200V or rtN236T. In conclusion, 3TC+ADV combination therapy was effective for most 3TC‐resistant patients, especially with genotype B HBV, but the risk of emergence of multiple drug‐resistant strains with long‐term therapy should be considered. The mutation rtA200V with rtL180M+M204V may be sufficient for failure of 3TC+ADV therapy.

The Nutritional Index ‘CONUT’ Is Useful for Predicting Long-Term Prognosis of Patients with End-Stage Liver Diseases

Organ allocation in Japan remains difficult due to the shortage of deceased-donor livers. The screening tool for controlling nutritional status (CONUT) has been considered to be an established assessment model for evaluating nutritional aspects in surgical patients. However, the application of this CONUT for evaluating the prognosis of patients with end-stage liver diseases has not been evaluated. We assessed the predictability of the prognoses of 58 patients with end-stage liver disease using various prognostic models. The patients registered at the transplantation center of Tohoku University Hospital for the waiting list of Japan Organ Transplant Network for liver transplantation were retrospectively analyzed. The prognoses of the patients were evaluated using the following 5 models: CONUT, the model for ELD with incorporation of sodium (MELD-Na), Child-Turcotte-Pugh score (CTP), prognostic nutritional indices (Onodera: PNI-O), and the Japan Medical Urgency criteria of the liver (JMU). Coxs proportional hazard model, log-rank test and concordance(c)-static were used for the statistics. The indices were 17.74 ± 5.80 for MELD-Na, 9.21 ± 2.19 for CTP, 33.92 ± 11.16 for PNI-O, and 7.57 ± 3.09 for CONUT. Univariate analysis revealed the significance of CONUT (p = 0.017, Odds: 1.325) but not MELD-Na, CTP, JMU or PNI-O for prediction. The cumulative survival rate was clearly discriminated at CONUT point 7. The c-static was 0.081 for the 6-month (M) survival rate, 0.172 for 12M, 0.517 for 36M, 0.821 for 48M, and 0.938 for 60M for CONUT. In conclusion, CONUT shows best predictability for the distant prognoses of patients with ELD.

Hepatitis B Virus Replication Could Enhance Regulatory T Cell Activity by Producing Soluble Heat Shock Protein 60 From Hepatocytes

BACKGROUND HBcAg-specific regulatory T (T(reg)) cells play an important role in the pathogenesis of chronic hepatitis B. Soluble heat shock proteins, especially soluble heat shock protein 60 (sHSP60), could affect the function of T(reg) cells via Toll-like receptor. METHODS We analyzed the relationship between soluble heat shock protein production and hepatitis B virus (HBV) replication with both clinical samples from HBeAg-positive patients with chronic hepatitis B (n= 24) and HBeAb-positive patients with chronic hepatitis B (n= 24) and in vitro HBV-replicating hepatocytes. Thereafter, we examined the biological effects of sHSP60 with isolated T(reg) cells. RESULTS The serum levels of sHSP60 in patients with chronic hepatitis B were statistically significantly higher than those in patients with chronic hepatitis C (P<.01), and the levels of sHSP60 were correlated with the HBV DNA levels (r = 0.532; P<.001) but not with the alanine aminotransferase levels. Moreover, the levels of sHSP60 in HBV-replicating HepG2 cells were statistically significantly higher than those in control HepG2 cells. Preincubation of CD4(+) CD25(+) cells with recombinant HSP60 (1 ng/mL) statistically significantly increased the frequency of HBcAg-specific interleukin 10-secreting T(reg) cells. The frequency of IL7R(-)CD4(+)CD25(+) cells, the expression of Toll-like receptor 2, and the suppressive function of T(reg) cells had declined during entecavir treatment. CONCLUSION The function of HBcAg-specific T(reg) cells was enhanced by sHSP60 produced from HBV-infected hepatocytes. Entecavir treatment suppressed the frequency and function of T(reg) cells; this might contribute to the persistence of HBV infection.

Enhanced intracellular retention of a hepatitis B virus strain associated with fulminant hepatitis.

A plasmid carrying 1.3-fold HBV genome was constructed from a HBV strain that caused five consecutive cases of fulminant hepatitis (pBFH2), and HepG2 cells were transfected with pBFH2 or its variants. The pBFH2 construct with A1762T/G1764A, G1862T, and G1896A showed the largest amount of core particle-associated intracellular HBV DNA, but no significant increase of extracellular HBV DNA in comparison with the wild construct, suggesting that these mutations might work together for retention of the replicative intermediates in the cells. The retention might relate to the localization of hepatitis B core antigen (HBcAg) in the nucleus of HepG2, which was observed by confocal fluorescence microscopy. HBcAg immunohistochemical examination of liver tissue samples obtained from the consecutive fulminant hepatitis patients showed stronger staining in the nucleus than acute hepatitis patients. In conclusion, the fulminant HBV strain caused retention of the core particles and the core particle-associated HBV DNA in the cells.

Enhanced Replication of Hepatitis B Virus With Frameshift in the Precore Region Found in Fulminant Hepatitis Patients

BACKGROUND The genotype B of hepatitis B virus (HBV) was reported to associate with fulminant hepatitis (FH). We aimed to clarify the characteristics of HBV obtained from FH patients in an area of Japan where genotype B HBV is prevalent. METHODS Using serum samples of 16 HBV-associated FH patients, partial HBV sequences were determined. The effects of HBV mutation/insertion/deletion were evaluated using an in vitro HBV replication system. RESULTS Of the 16 HBV isolates, 31% belonged to subgenotype B1/Bj, 38% were subgenotype B2/Ba, and 31% were subgenotype C2/Ce. Notably, the single nucleotide insertion/deletion that resulted in a frameshift of the precore protein was found exclusively in 60% of B1/Bj strains. An in vitro study showed that all of the frameshift mutants had significantly higher amounts of HBV DNA than did the wild type. One of the isolates had a novel insertion of A between nucleotides 1900 and 1901, which resulted in a 3-nucleotide change within the Kozak sequence of the core protein and enhanced the core protein expression in vitro. CONCLUSIONS The frameshift insertion/deletion in the precore region enhanced HBV replication and might be associated with the development of FH by the subgenotype B1/Bj HBV.

Inhibitory effect on hepatitis B virus in vitro by a peroxisome proliferator-activated receptor-γ ligand, rosiglitazone

Although chronic infection of hepatitis B virus (HBV) is currently managed with nucleot(s)ide analogues or interferon-alpha, the control of HBV infection still remains a clinical challenge. Peroxisome proliferator-activated receptor (PPAR) is a ligand-activated transcription factor, that plays a role in glucose and lipid metabolism, immune reactions, and inflammation. In this study, the suppressive effect of PPAR ligands on HBV replication was examined in vitro using a PPARalpha ligand, bezafibrate, and a PPARgamma ligand, rosiglitazone. The effects were examined in HepG2 cells transfected with a plasmid containing 1.3-fold HBV genome. Whereas bezafibrate showed no effect against HBV replication, rosiglitazone reduced the amount of HBV DNA, hepatitis B surface antigen, and hepatitis B e antigen in the culture supernatant. Southern blot analysis showed that the replicative intermediates of HBV in the cells were also inhibited. It was confirmed that GW9662, an antagonist of PPARgamma, reduced the suppressive effect of rosiglitazone on HBV. Moreover, rosiglitazone showed a synergistic effect on HBV replication with lamivudine or interferon-alpha-2b. In conclusion, this study showed that rosiglitazone inhibited the replication of HBV in vitro, and suggested that the combination therapy of rosiglitazone and nucleot(s)ide analogues or interferon could be a therapeutic option for chronic HBV infection.

Plasma L-cystine/L-glutamate imbalance increases tumor necrosis factor-alpha from CD14+ circulating monocytes in patients with advanced cirrhosis.

Background and Aims The innate immune cells can not normally respond to the pathogen in patients with decompensated cirrhosis. Previous studies reported that antigen-presenting cells take up L-Cystine (L-Cys) and secrete substantial amounts of L-Glutamate (L-Glu) via the transport system Xc- (4F2hc+xCT), and that this exchange influences the immune responses. The aim of this study is to investigate the influence of the plasma L-Cys/L-Glu imbalance observed in patients with advanced cirrhosis on the function of circulating monocytes. Methods We used a serum-free culture medium consistent with the average concentrations of plasma amino acids from patients with advanced cirrhosis (ACM), and examined the function of CD14+ monocytes or THP-1 under ACM that contained 0–300 nmol/mL L-Cys with LPS. In patients with advanced cirrhosis, we actually determined the TNF-alpha and xCT mRNA of monocytes, and evaluated the correlation between the plasma L-Cys/L-Glu ratio and TNF-alpha. Results The addition of L-Cys significantly increased the production of TNF alpha from monocytes under ACM. Monocytes with LPS and THP-1 expressed xCT and a high level of extracellular L-Cys enhanced L-Cys/L-Glu antiport, and the intracellular GSH/GSSG ratio was decreased. The L-Cys transport was inhibited by excess L-Glu. In patients with advanced cirrhosis (n = 19), the TNF-alpha and xCT mRNA of monocytes were increased according to the Child-Pugh grade. The TNF-alpha mRNA of monocytes was significantly higher in the high L-Cys/L-Glu ratio group than in the low ratio group, and the plasma TNF-alpha was significantly correlated with the L-Cys/L-Glu ratio. Conclusions A plasma L-Cys/L-Glu imbalance, which appears in patients with advanced cirrhosis, increased the TNF-alpha from circulating monocytes via increasing the intracellular oxidative stress. These results may reflect the immune abnormality that appears in patients with decompensated cirrhosis.

Rapid reduction of hepatitis C virus‐Core protein in the peripheral blood improve the immunological response in chronic hepatitis C patients

Aim:  The extracellular hepatitis C virus (HCV)‐antigen, including HCV‐Core protein, can suppress immune cells. Recently, the efficacy of double filtration plasmapheresis (DFPP) for chronic hepatitis C (CHC) was reported. However, the mechanism of efficacy of DFPP might not be only the reduction of HCV but also the effect of immune cells via direct and/or indirect mechanisms. The aim of this study is to analyze the virological and immunological parameters of difficult‐to‐treat HCV patients treated with DFPP combined with Peg‐interferon and RBV (DFPP/Peg‐IFN/RBV) therapy.