Pre-diabetes is a risk factor for type 2 diabetes mellitus (DM) development. This study aimed to elucidate the impact of treatment response on sequential changes in glucose abnormalities in pre-diabetic chronic hepatitis C (CHC) patients.

Chronic Hepatitis C patients with a baseline haemoglobin A1C (A1C) range 5.7–6.4% who achieved 80/80/80 adherence were prospectively recruited. All patients received current peginterferon-based recommendations. The primary outcome measurement was their A1C level at the end of follow-up (EOF). The interaction between variants of the IL28B gene and outcomes of glucose metabolism was also measured.

A total of 181 consecutive CHC patients were enrolled. The mean A1C at EOF was 5.82 ± 0.41%, which was significantly lower than the baseline level (5.93 ± 0.21%, P < 0.001). At EOF, 63 (34.8%) patients became normoglycaemic, whereas 10 (5.5%) patients developed DM. The sustained virological response (SVR) rates of 63 normoglycaemics, 108 pre-diabetics and 10 diabetic patients at the EOF were 92.1%, 84.3% and 50% respectively (normoglycaemics vs. diabetics P = 0.003; pre-diabetics vs. diabetics P = 0.02). Achievement of an SVR was the only predictive factor associated with normoglycaemia development at EOF by multivariate logistic regression analysis (Odds ratio = 2.6, P = 0.04). The prevalence of the interleukin 28B rs8099917 TT variant in patients who developed DM (70.0%) at EOF tended to be lower than that in patients with pre-diabetics (87.0%) or normoglycaemics (92.1%).

Successful eradication of HCV improves glucose abnormalities in pre-diabetic CHC patients.

Hepatitis B immune globulin (HBIg) with or without nucleos(t)ide analogue (NA) inhibitors has been shown to prevent recurrence of hepatitis B virus (HBV) following orthotopic liver transplantation (OLT). However, the use of HBIg has many disadvantages.

The present study was performed to determine if converting patients from HBIg±NA to combination NA therapy could prevent recurrence of HBV.

Twenty-one recipients without evidence of HBV recurrence on HBIg±NA for ≥6 months were enrolled. Patients received their last injection of HBIg at the time they initiated tenofovir disoproxil fumarate/emtricitabine (TDF/FTC; Truvada^®) and were followed up for 31.1 ± 9.0 [range 15–47] months.

After 1 year, 3 patients (14%) had detectable HBsAg, one of whom was non-compliant. Two of 3 with recurrence cleared HBsAg by last follow-up on TDF/FTC; the non-compliant patient became HBV DNA-undetectable with re-institution of TDF/FTC. TDF/FTC saved $12,469/year over our standard-of-care, monthly intramuscular HBIg/lamivudine. There was no evidence of a general adverse effect of TDF/FTC on renal function. However, 3 patients developed reversible acute renal failure; on renal biopsy, 1 had possible TDF/FTC-induced acute tubular necrosis.

Substitution of TDF/FTC for HBIg prevented recurrence of HBV DNA in 100% (20/20) of patients who were compliant with the medication and led to substantial cost savings over HBIg-containing regimens.

Type 2 diabetes mellitus (T2DM) is a well-known factor risk for non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) in obese patients.

To better understand the association between T2DM and NAFLD, global changes in protein expression in diabetic and non-diabetic obese subjects were assessed by a proteomic approach.

Liver samples were obtained from diabetic and non-diabetic morbid obese subjects (BMI>40 kg/m²). Histological analysis was used to evaluate hepatic steatosis and the degree of anatomopathological alteration. Changes in protein expression were analysed by two-dimentional electrophoresis combined with MALDI-TOF mass spectrometry. Levels of glutathione, carbonyl and 4-HNE protein adducts were used to assess oxidative stress status.

Of 850 proteins analysed, 33 were differentially expressed in T2DM obese subjects. Of these, 27 were unequivocally identified by mass spectrometry. Analysis of protein sets revealed patterns of decreased abundance in mitochondrial enzymes, proteins involved in methione metabolism, and oxidative stress response. Accordingly, T2DM subjects showed decreased levels of glutathione, the antioxidant byproduct of methionine metabolism via the transsulfuration pathway, and higher levels of protein and lipid oxidative damage. Changes in detoxyfing enzymes, carbohydrate metabolism, proteasome subunits and retinoic acid synthesis were also found.

The results suggest alterations in mitochondrial function and methionine metabolism as potential contributing factors to increased oxidative stress in liver of obese diabetic patients which may be influencing the development of NAFLD and NASH.

Absence of curative treatment creates urgent need for new strategies for unresectable hepatoma. Based on former discoveries of good liver cell compatibility, safety and tumour-specific inhibition of hydroxyapatite nanoparticles (nHAP), this work tries to make nHAP serve as gene vector in the hepatoma-targeted trans-arterial embolization (TAE) gene therapy to elevate and synergize the therapeutic efficacy of TAE and target gene therapy.

Following dosage and ratio optimization, polypolex formed by surface modified nHAP and p53 expressing plasmid was applied in vitro for human hepatoma HePG2 cell, and then in vivo for rabbit hepatic VX2 tumour by injection of polypolex/lipodoil emulsion to the hepatic artery in a tumour-target manner.

In vitro, the polypolex transfected only about 5% HepG2 cells, but can elevate the inhibition of its growth and apoptosis in a much more degree while keeping safe to the normal hepatocyte line, L02. In vivo, the emulsion, with better dispersion than the polypolex and more specific tumour-target than lipiodol, mediated specific 4% p53 expression and antitumoural nanoparticle retention in the target tumour site, also significantly reduced tumour growth and prolonged the animal survival times more than the lipiodol (P < 0.05).

In all, this new treatment based on nHAP can enhance therapeutic effect of HCC safely both in vitro and in vivo.

Previous studies showed that hepatocyte nuclear factor 4α (HNF4α) may play a critical role in hepatitis B virus (HBV) replication.

This study aimed to investigate the effect of knocking down of HNF4α with RNA interference technique on HBV replication in a HBV replication mouse model.

Four HNF4α, specific short hairpin RNA (shRNA)-producing plasmids were constructed. HBV mRNA and DNA replication intermediates were analysed using Northern and Southern blot respectively. The expression of HNF4α and HBV core antigen (HBcAg) was detected using immunohistochemistry technique.

One of the HNF4α shRNAs, HNF4α shRNA1, efficiently inhibited the expression of HNF4α in HepG2 cells and mice liver. HBV RNA transcripts and DNA replication intermediates in HNF4α shRNA1 group were decreased 67.3 and 76%, respectively, in HepG2 cells, and 68.1 and 70.6% in mice liver respectively. The expression level of HBcAg in the liver was also decreased with the inhibition of HNF4α expression.

These results suggested that decreasing of HNF4α expression was associated with the reduced level of HBV replication in HepG2 cells and mice liver. These data indicated that HNF4α played a critical role in HBV replication in vivo, and HNF4α shRNA could inhibit HBV replication in vivo.

Poor cellular trafficking and suboptimal T-cell responses in liver, the hall marks of chronic hepatitis C virus (CHC) infection, might be attributed to defective antigen presentation. Controversy exists regarding role of myeloid dendritic cells (DCs) in CHC and response to antiviral treatment. This study examines functional status of DCs before and after completion of treatment with the aim to find any modulatory effect.

Frequency and functions of monocyte-derived DCs (mo-DCs) were evaluated in CHC (n = 25), before the start of therapy (CHC₀). These patients were then put on treatment with peg-interferon-α plus ribavirin for 24 or 48 weeks, and the mo-DC functions were evaluated after 6 months of completion of treatment (CHC₆) again, using multicolour flow cytometry, endocytosis assay, cytokine assay and mixed lymphocyte reaction.

Pre-treatment frequency of mo-DCs in CHC₀ was lower than that in healthy controls, which became close to normal in patients who achieved virological response (SVR+, n = 20) but not in non-responders (SVR−, n = 5). Pre-treatment levels of CD83, CD80 and CD86 on mo-DC in SVR₀+, but not SVR₀−, got upregulated after lipopolysaccharide stimulation supporting the hypothesis that DCs play deciding role in response to therapy. Post-treatment allostimulatory and phagocytosing capacity of mo-DCs in SVR+ patients indicated regain in functional capacity in these patients but not in SVR− patients.

Our results indicate that DCs in CHC patients exhibiting mature and functional phenotype prior to therapy achieve sustained virological response suggesting that functional modulation of defective DCs is directly associated with successful response to therapy.

Portal hypertension causes arterial vasodilation and sympathetic atrophy in the splanchnic area. We aimed to demonstrate a relationship between hemodynamic alterations and sympathetic atrophy by investigating a pathway from sensitive afferent signals to mesenteric sympathetic ganglia.

Experiments were conducted in sham and portal vein ligated (PVL) adult and neonatal rats treated with vehicle or capsaicin. Hemodynamic parameters, and immunohistochemistry, immunofluorescence and Western blot of different tissues were analysed.

cFos expression in the brain supraoptic nuclei was used to confirm abrogation of the afferent signal in capsaicin-treated PVL rats (effectively afferent blocked). Neonatal and adult PVL afferent blocked rats showed simultaneous prevention of hemodynamic alterations and sympathetic atrophy (measured by tyrosine hydroxylase expression in nerve structures of splanchnic vasculature). Not effectively afferent blocked rats showed none of these effects, behaving as PVL vehicle. All capsaicin treated animals presented loss of calcitonin gene-related peptide in superior mesenteric artery and ganglia, whereas neuronal nitric oxide synthase remained unaffected. Neuronal markers semaphorin-3A, nerve growth factor, its precursor and p75 neurotrophic receptor, were significantly over-expressed in the PVL sympathetic ganglia compared with sham, but not in effectively afferent blocked rats. Semaphorin-3A staining in mesenteric ganglia co-localized with vesicular acetylcholine transporter, but not with adrenergic, nitrergic and sensory axons, suggesting that semaphorin-3A might originate in preganglionic neurons.

These results indicate that the nervous system has a central role in the genesis of the circulatory abnormalities of portal hypertension, and support that mesenteric sympathetic atrophy contributes to splanchnic arterial vasodilation.

Previous studies demonstrated that the Long-Evans (LE) rats exhibited liver injury and lipid metabolic abnormalities after 8 weeks of ethanol feeding.

The goal of this study was to investigate if the LE rats develop more advanced hepatic abnormalities (e.g., fibrosis) after long-term feeding with an ethanol-containing Lieber–DeCarli diet. In addition, the contribution of early growth response-1 (EGR1) transcription factor to these pathological changes was assessed.

Long-Evans rats were fed an ethanol-containing or isocaloric control liquid diet for 18 months. Livers were processed for histological analyses, studies of fibrosis-related gene expression, cell fractionation and triglyceride measurement. Serum alanine aminotransferase (ALT) levels were assessed. DNA binding activities of p53 and the sterol regulatory element-binding protein-1c (SREBP1c) were analysed. The abundance of EGR1 and enzymes involved in fatty acid synthesis were determined. Chromatin immunoprecipitation was employed to study EGR1 binding to the SREBP1c promoter region.

Ethanol feeding generated steatosis, chicken wire fibrosis and ALT elevations in the LE rats. Fibrosis was associated with the upregulation of EGR1 and its downstream target genes. EGR1 upregulation was associated with enhanced p53 activity and an increase in the cellular p66^shc abundance. Steatosis was linked to the activation of SREBP1c. Importantly, EGR1 upregulation paralleled the expression and transcriptional activity of SREBP1c. Finally, EGR1 was shown to bind to the SREBP1c promoter region.

Long-term ethanol feeding promoted steatosis and fibrosis in LE rats via EGR1 activation. The highly abundant EGR1 bound to the SREBP1c promoter and contributed to the steatosis observed in the LE rat model.

Liver diseases are common in the United States and often require liver transplantation; however, donated organs are limited and thus alternative sources for liver cells are in high demand. Embryonic stem cells (ESC) can provide a continuous and readily available source of liver cells. ESC differentiation to liver cells is yet to be fully understood and comprehensive differentiation protocols are yet to be defined. Here, we aimed to achieve human (h)ESC differentiation into mature hepatocytes using defined recombinant differentiation factors and metabolites.

Embryonic stem cell H1 line was sub-cultured on feeder layer. We induced hESCs into endodermal differentiation succeeded by early/late hepatic specification and finally into hepatocyte maturation using step combinations of Activin A and fibroblast growth factor (FGF)-2 for 7 days; followed by FGF-4 and bone morphogenic protein 2 (BMP2) for 7 days, succeeded by FGF-10 + hepatocyte growth factor 4 + epidermal growth factor for 14 days. Specific inhibitors/stimulators were added sequentially throughout differentiation. Cells were analysed by PCR, flow cytometry, microscopy or functional assays.

Our hESC differentiation protocol resulted in viable cells with hepatocyte shape and morphology. We observed gradual changes in cell transcriptome, including up-regulation of differentiation-promoting GATA4, GATA6, POU5F1 and HNF4 transcription factors, steady levels of stemness-promoting SOX-2 and low levels of Nanog, as defined by PCR. The hESC-derived hepatocytes expressed alpha-antitrypsin, CD81, cytokeratin 8 and low density lipoprotein (LDL) receptor. The levels of alpha-fetoprotein and proliferation marker Ki-67 in hESC-derived hepatocytes remained elevated. Unlike stem cells, the hESC-derived hepatocytes performed LDL uptake, produced albumin and alanine aminotransferase and had functional alcohol dehydrogenase.

We report a novel protocol for hESC differentiation into morphological and functional yet immature hepatocytes as an alternative method for hepatocyte generation.

To validate whether the anti-cancer effect of microRNA-122 (miR-122) on hepatocellular carcinoma (HCC) is mediated through regulating Wnt/β-catenin signalling pathways.

The expression levels of miR-122 in HCC tissues and varied hepatoma cells were quantified by real-time PCR. MiR-122 agomir was transfected into HepG2, Hep3B cells to over-express miR-122. The effect of over-expression miR-122 on proliferation and apoptosis of HepG2 and Hep3B cells was evaluated using CCK-8 kit and flow cytometer respectively. The 3′-UTR segments of Wnt1 containing the miR-122 binding sites were amplified by PCR and the luciferase activity in the transfected cells was assayed. Wnt1 mRNA level was quantified using RT-PCR. Protein levels of Wnt1, β-catenin and TCF-4 were detected using Western blotting.

In comparison with the expression level of miR-122 in para-cancerous tissues or Chang liver cell, the expression level in HCC tissues or varied hepatoma cells was significantly decreased (P < 0.05). Over-expression of miR-122 significantly inhibited the proliferation (P < 0.05), and promoted the apoptosis of HepG2 and Hep3B cells. Over-expressed miR-122 down-regulated the protein levels of Wnt1, β-catenin and TCF-4 (P < 0.05). MiR-122 suppressed the luciferase activity of the pmiR-Wnt1-wt by approximately 50% compared with the negative control, while mutation or removal of the miR-122 binding site using siRNA or mir-122 inhibitor blocked the suppressive effect (P < 0.05).

MiR-122 expression is down-regulated in human HCC. Over-expression of miR-122 inhibits HCC cell growth and promotes the cell apoptosis by affecting Wnt/β-catenin-TCF signalling pathway.

Serum alanine aminotransferase (ALT) is an easily available, low-cost screening tool for detecting silent chronic liver disease. Recent studies have suggested that the currently accepted healthy ALT thresholds be lowered.

In this retrospective cross-sectional study, we determined upper thresholds for ALT values in a nationally representative healthy cohort (n = 3337) from the Fourth Korea National Health and Nutrition Examination Survey (KNHANES IV). Sensitivity and specificity of the currently used ALT threshold (40 IU/L, regardless of gender) was compared against study-derived, gender-specific ALT thresholds for detecting individuals at risk for chronic liver disease in 27 913 health check-up participants.

The 95th percentile levels for ALT in healthy weight, metabolically normal, liver disease-free KNHANES participants were 34 IU/L for men and 25 IU/L for women. The prevalence of ALT elevation among health check-up participants was 11.0% in currently used thresholds, and increased to 22.6% with study-derived, gender-specific thresholds. Of the population who were additionally defined to have elevated ALT levels under new ALT threshold, 65.7% were at risk for chronic liver disease. Sensitivity for detecting individuals at risk for chronic liver disease improved from 18 to 33% with new thresholds whereas a trade-off in specificity (from 96 to 88%) was observed.

It is recommendable to lower the current ALT thresholds to better identify individuals at risk for chronic liver disease.

Effective cytokines can drive the commitment of naive T cells to regulate immune response after antigen-mediated activation. Aims are to elucidate the clinical role of serum IL-27 and IL-6 in the different stages of naïve hepatitis B virus (HBV)-infected patients.

Samples with well-characterized clinical profiles were assessed from 395 HBV-infected patients including chronic hepatitis B (CHB) group in 291 patients, liver cirrhosis (LC) group in 57 patients, hepatocellular carcinoma (HCC) group in 47 patients. Another 139 non-HBV infected individuals were enrolled as control group (CG) including 104 with normal liver function (NF) and 35 with liver dysfunction (LD).

The HBV-infected group and separated groups presented significantly higher IL-27 and IL-6 expression than the CG or subgroups of CG. In contrast to IL-27, IL-6 showed significant differences with deteriorating liver condition compared with LC or HCC with CHB groups. Furthermore, IL-6, rather than IL-27, showed significant statistical differences in patients with advanced liver disease compared with those of mild or moderate to severe liver disease and in patients with terminal stage HCC compared with those of early to intermediate or advanced stage HCC. The data associated with liver function, including Albumin, Bilirubin, INR, Platelet and AFP levels, were significantly correlated to IL-6 expression, but had weak correlation to IL-27 expression in HBV patients.

Serum IL-27 can trigger immune response to prevent hepatic injury in different clinical-pathologic stages of HBV-infected patients earlier, but IL-6 may play an extremely important role to determine the liver progression.

The upper gastrointestinal (GI) toxicity associated with non-steroidal anti-inflammatory drugs (NSAID) use among cirrhotic patients remains unclear. The objective of this study was to evaluate the risk of upper GI adverse events associated with celecoxib and oral and parenteral non-selective NSAIDs in cirrhotic patients.

All the patients aged ≥ 20 years with a diagnosis of cirrhosis hospitalized for variceal bleeding and non-variceal upper GI adverse events (oesophageal, gastric, duodenal ulcer, bleeding; gastritis and duodenitis) in 2006 were identified using ICD-9-CM diagnosis codes from inpatient claims from the Taiwan National Health Insurance Database. In the case-crossover study design, the case period was defined as 1–30 days and the control period as 31–60 days before the date of hospitalization. The information for individual NSAID use was obtained from the outpatient pharmacy prescription database. Adjusted self-matched odds ratios (OR) and their 95% confidence intervals (CI) were estimated with a conditional logistic regression model.

A total of 4876 cirrhotic patients were included. The adjusted OR (95% CI) was 1.44 (0.89–2.31) for celecoxib, 1.87 (1.66–2.11) for oral non-selective NSAIDs and 1.90 (1.55–2.32) for parenteral NSAIDs overall. Risks were similar for variceal and non-variceal events. Concomitant use of proton pump inhibitors and histamine-2 receptor antagonists tended to decrease the upper GI toxicity associated with non-selective NSAIDs and celecoxib.

The use of nsNSAIDs but not celecoxib was associated with a two-fold increased risk of variceal and non-variceal upper GI events among cirrhotic patients.

Previous studies suggest that chronic liver disease may be related to vitamin D deficiency. It is, however, not known whether 25(OH)D levels are associated with incident hepatic decompensation and mortality in chronic liver failure.

We aimed to evaluate whether 25(OH)D serum levels are associated with Child–Pugh (CP) score, model for end-stage liver disease (MELD) score, occurrence of hepatic decompensation, and survival in patients with cirrhosis.

We enrolled 75 consecutive cirrhotic patients admitted to our outpatient liver clinic (32% females; age: 58 ± 11 years; aetiology alcohol in 61%). At baseline, 25(OH)D was determined and the degree of liver dysfunction was estimated by CP and MELD score. Thereafter patients were followed-up with respect to hepatic decompensation and mortality.

25(OH)D levels averaged 16.0 ± 9.2 ng/ml and were inversely correlated with MELD score (r = −0.34, P = 0.003) and CP score (r = −0.21, P = 0.080). Thirty-seven patients developed hepatic decompensation and 24 patients died during a median follow-up of 3.6 years. Age- and gender-adjusted relative risk (with 95% confidence interval) was 6.37 (1.75–23.2; P = 0.005) for hepatic decompensation and 4.31 (1.38–13.5; P = 0.012) for mortality within the first vs the third 25(OH)D tertile but these associations were largely attenuated towards non-significant trends after additional adjustments for CP or MELD score.

Our findings show a significant association of 25(OH)D with the degree of liver dysfunction and suggest that low 25(OH)D levels may predict hepatic decompensation and mortality in patients with chronic liver failure.

Mature hepatocytes retain the ability to regenerate the liver lobule fully in vivo following injury. Several cytokines and soluble factors (hepatocyte growth factors, epidermal growth factors, insulin and nicotinamide) are known to be important for proliferation of mature hepatocytes in vitro. However, hepatocytes monolayer-cultured on extracellular matrices have gradually lost their specific functions, particularly those in drug metabolism.

We have explored and established a new culture system for expansion of functional hepatocytes.

We evaluated two approaches for efficient expansion of mature hepatocytes: (i) Co-culture with mouse embryonic fibroblasts (MEF); (ii) Addition to culture of inhibitors of cell signals involved in liver regeneration. After expansion steps, 3-dimensional spheroid-forming culture was used to re-induce mature hepatocellular function.

The addition of inhibitors for tumour growth factor (TGF) β and glycogen synthase kinase (GSK) 3β efficiently induced in vitro expansion of mature hepatocytes. Although expression of hepatocellular functional genes decreased after expansion in monolayer culture, their expression and the activity of cytochrome P450 enzymes significantly increased with spheroid formation. Furthermore, when hepatocytes were co-cultured with MEF, addition of a MAPK/ERK kinase (MEK) inhibitor at the spheroid formation step enhanced drug-metabolism-related gene expression.

Combination of the MEF co-culture system with the addition of inhibitors of TGFβ and GSK3β induced in vitro expansion of hepatocytes. Moreover, expression of mature hepatocellular genes and the activity of drug-metabolism enzymes in expanded hepatocytes were re-induced after spheroid culture.

Mouse hepatocarcinogenesis is associated with mutations in Hras, but infrequently in Kras. The effect on carcinogenesis of developmental age at the time of ras mutation remains unknown.

We sought to compare quantitatively the effects of expressing mutant H- or Kras genes in fetal vs. adult mouse liver.

We established an inducible system of gene expression in mouse liver to define disease pathogenesis associated with activation of oncogene expression.

Diffuse expression of either oncogene in fetal or adult hepatocytes caused hepatomegaly. For mutant Hras^G12V, this phenotype was almost fully reversible and accompanied by apoptosis, indicating that maintenance of hepatomegaly requires continuous Hras^G12V expression. We also examined the effect of ras expression on growth of transplanted hepatocytes in an in vivo system that allows us to quantify hepatocyte growth effects in both permissive and restrictive hepatic growth environments. Mutant Kras^G12D had no effect on hepatocyte growth in this system. In contrast, Hras^G12V induced increased hepatocyte focus growth in quiescent liver, the hallmark of a cell autonomous growth stimulus. Hras^G12V also increased the fraction of donor hepatocyte foci that displayed extreme growth, a characteristic of preneoplastic lesions.

The primary effect of diffuse, whole-liver expression of either mutant ras gene in fetal or adult mouse liver is diffuse and progressive hepatic growth. Hras^G12V mutation influences hepatocarcinogenesis by conferring cell autonomous growth potential upon foci of expressing cells and by increasing the risk of neoplastic progression. Kras^G12D does not share these latter carcinogenic effects in mouse liver.

Hepatitis C virus (HCV) infection is associated with substantial costs to patients, their caregivers and society.

We evaluated time costs (time spent seeking healthcare) and out-of-pocket (OOP) costs for patients with HCV and their caregivers.

We measured costs for 738 HCV outpatients in a tertiary-care clinic using a patient-completed questionnaire. Time and OOP costs were compared across disease stages and sociodemographic categories. We examined the association between cost and disease stage using linear regression adjusting for age, gender, marital status, education, income and Index of Coexistent Disease (ICED) comorbidity score. Costs were expressed in 2007 Canadian dollars.

The mean annual time cost per patient was $2136 (98 h), and ranged from $281 (18 h) in individuals who had cleared the virus to $9416 in transplant recipients (420 h). Caregiver costs were reported in 10% of patients. The mean annual OOP cost per patient was $1326. Patients receiving active treatment and those with late-stage disease spent $2500–2800 per year on HCV-related healthcare, approximately 7% of their annual income. Patients who had cleared the virus had the lowest time and OOP costs. Low income and unemployed patients had higher costs.

In HCV-infected individuals, OOP and time costs represent a significant economic burden and fall disproportionately upon those least able to afford them. The lower cost burden among those who were successfully treated suggests that wider use of antiviral therapy may reduce economic burden in addition to improving health outcomes.

Acoustic radiation force impulse (ARFI) imaging is a new non-invasive, ultrasound-based method for the evaluation of liver fibrosis and cirrhosis.

To determine the diagnostic accuracy of ARFI imaging, transient elastography (TE) and Fibrotest for the evaluation of complications in patients with cirrhosis.

A total of 166 patients (109 male, mean age: 54 ± 11 years) with chronic liver disease and established cirrhosis were included in this study. ARFI-imaging of the liver and spleen, TE and Fibrotest were performed in all patients. In addition, clinical, laboratory and morphological parameters, including MELD/Child–Pugh scores, presence of oesophageal varices and hepatocellular carcinoma, history of variceal bleeding and history of hepatic encephalopathy were recorded.

Acoustic radiation force impulse liver was significantly correlated with ARFI spleen (r = 0.48, P < 0.001), TE (r = 0.75, P < 0.001) and Fibrotest (r = 0.21, P = 0.006). The diagnostic accuracy (AUROC) for the diagnosis of large oesophageal varices was 0.58 (95% CI: 0.48–0.67), 0.58 (0.49–0.67), 0.53 (0.44–0.63) and 0.50 (0.41–0.59) for ARFI liver, spleen, TE and Fibrotest respectively (P > 0.20). The AUROC for the detection of hepatocellular carcinoma (HCC) was 0.54 (0.39–0.70), 0.58 (0.44–0.73), 0.56 (0.40–0.73) and 0.72 (0.60–0.84) respectively (P > 0.20). Multiple logistic regression analysis showed that ARFI spleen better predicted the presence of large oesophageal varices and HCC compared with ARFI liver.

The diagnostic accuracy of ARFI liver and spleen was comparable to TE and Fibrotest for the detection of complications in patients with cirrhosis.

Standard-dose ribavirin is crucial for the standard-of-care treatment of chronic hepatitis C virus (HCV) infection. Equilibrative nucleoside transporter 1 (ENT1), encoded by SLC29A1 gene, is the main transporter that imports ribavirin into human hepatocytes. Aims:

To determine whether single nucleotide polymorphisms (SNPs) at the SLC29A1 gene could influence the probability of treatment response compared with other baseline and host genetic factors.

A total of 526 East Asian patients monoinfected with HCV genotype 1b who had received pegylated interferon alpha plus ribavirin therapy were enrolled in this study. They were assigned randomly to the derivation and confirmatory groups. SNPs related to the IL28B,ITPA and SLC29A1 genes were genotyped using real-time detection polymerase chain reaction. Factors associated with sustained virological response (SVR) were analysed using multiple logistic regression analysis.

Multivariate analysis for the derivation group identified six baseline variables significantly and independently associated with SVR: age [P = 0.023, odds ratio (OR) = 0.97], gender (P = 0.0047, OR = 2.25), platelet count (P = 0.00017, OR = 1.11), viral load (P = 0.00026, OR = 0.54), IL28B SNP rs12979860 (P = 1.09 × 10⁻⁷, OR = 8.68) and SLC29A1 SNP rs6932345 (P = 0.030, OR = 1.85). Using the model constructed by these independent variables, positive and negative predictive values and predictive accuracy were 73.3, 70.1 and 71.9% respectively. For the confirmatory group, they were 71.4, 84.6 and 75.3% respectively. The SLC29A1 and IL28B SNPs were also significantly associated with rapid virological response.

The SNP at the major ribavirin transporter ENT1 gene SLC29A1 was one of significantly independent factors influencing treatment response, although the impact on the prediction was small.

Cholestasis is a common disease of the liver. Chronic cholestasis eventually leads to hepatic cirrhosis and fibrosis, and rodent chronic cholestasis models are used to study aspects of fibrosis and cirrhosis. Cholestasis-induced liver injury and fibrosis are associated with increased oxidative stress and inflammation. Few pharmacological therapies exist for treatment of cholestasis or cirrhosis, but it is known that humans with better nutritional intake are less likely to develop certain types of cirrhosis. Eugenia jambolana (Jamun) is a tropical berry fruit rich in antioxidant anthocyanin compounds.

As anthocyanins decrease cellular lipid peroxidation and oxidative stress, it was hypothesized that Jamun fruit extract (JFE) administration could protect against cholestatic liver injury and inflammation in mice.

Starting 24 h after sham or bile-duct ligation (BDL) surgery, male C57Bl/6 mice were administered vehicle or JFE (100 mg/kg, po) for 10 days.

Mice that underwent BDL had elevated serum ALT levels, which were reduced to 60% by JFE treatment. Likewise, BDL caused hepatic inflammation, macrophage infiltration, fibrosis and necrosis, all of which were largely improved by JFE. Interestingly, hepatoprotection was observed in JFE-treated BDL mice, despite suppressed transporter expression and increased hepatic bile acid concentrations.

Jamun fruit phytochemicals decreased hepatic inflammation and oxidative stress, and protected against hepatocellular injury in mice. Jamun warrants further investigation as a potential antioxidant/anti-inflammatory therapy not only to treat cholestasis but also other liver diseases with an inflammatory component.

Multidrug resistance is a major reason for poor treatment results in advanced hepatoblastoma (HB). Several alternative treatment options are currently under investigation to improve the prognosis of affected patients

This study aimed to analyse the impact of sorafenib on the viability of HB cells and xenotransplanted HB tumours.

Cell viability and apoptosis were evaluated in two HB cell lines (HUH6 and HepT1) after treatment with sorafenib using MTT and Caspase 3 activation assay. Extracellular signal-regulated kinase (ERK) phosphorylation was investigated using Western blot. In addition, sorafenib (30 mg/kg) was administered orally to NMRI mice bearing subcutaneous HUH6 derived tumours. Tumour progression and viability were monitored by tumour volume and α-fetoprotein (AFP) levels, and apoptosis was assessed using TUNEL assay. Tumour angiogenesis and mean vascular density (MVD) was determined using CD31 staining, ERK phosphorylation was detected using indirect immunofluorescence.

Treatment with sorafenib led to decreased ERK phosphorylation, reduced cell viability and induction of apoptosis in HepT1 and HUH6 cells. In HB xenografts, sorafenib significantly reduced tumour growth compared with control (P < 0.05). AFP levels were lower in the sorafenib group (P = 0.07). Relative apoptotic areas detected using TUNEL assay were increased (P = 0.003). CD31 staining revealed inhibition of angiogenesis, and mean vascular density was lower in the sorafenib group (P = 0.02). ERK phosphorylation was reduced in tumours tissues after sorafenib treatment.

Treatment with sorafenib led to a potent inhibition of cell viability, tumour progression and angiogenesis. Sorafenib might therefore also be a promising treatment option for high risk or recurrent HB.

Since previous studies have investigated the population dynamics of Japan-indigenous genotype 3 hepatitis E virus (HEV) using virus sequences, more nucleotide sequences have been determined, and new techniques have been developed for such analysis.

To prevent future hepatitis E epidemic in Japan, this study aimed to elucidate the cause of past HEV expansion.

The epidemic history of Japan-indigenous genotype 3 HEV was determined using the coalescent analysis framework. Bayesian skyline plot (BSP) and Bayesian estimate of phylogeny with relaxed molecular clock models were calculated using Markov chain Monte Carlo sampling.

Japan-indigenous strains consist of New World strains (subtype 3a), Japanese strains (3b) and European strains (3e). The oldest lineage, 3b, appeared around 1929. Lineages 3a and 3e appeared around 1960. BSPs indicated similar radical population growth of the 3a and 3b lineages from 1960 to 1980.

Population dynamics of the three lineages shared some common characteristics, but had distinguishing features. The appearance of 3a and 3e lineages coincides with the increase of large-race pig importation from Europe and the USA after 1960. The epidemic phase of 3a and 3b strains from 1960 to 1980 could be related to increased opportunity for HEV infection arising from large-scale pig breeding since 1960. Our observations revealed new findings concerning the close relationship between the epidemic history of Japan-indigenous genotype 3 HEV and the improvement of the Japanese pig industry. Infection control in pig farms should be an effective method of preventing HEV infection in humans.

Although regeneration of intrahepatic bile ducts has been extensively studied and intrahepatic progenitor cells have been identified, few studies have focussed on the extrahepatic bile duct (EHBD). We hypothesized that local progenitor cells are present within the EHBD of humans. Human EHBD specimens (n = 17) were included in this study.

Specimens of normal EHBD tissue were obtained from healthy donor livers (n = 6), mildly injured EHBD from patients with cholangitis (n = 6) and severely injured EHBD from patients with ischaemic type biliary lesions (n = 5). Double immunostaining for K19 and the proliferation marker Ki-67 was performed to identify and localize proliferating cells. In addition, immunofluorescent doublestaining using antibodies against K19 and c-Kit was performed to identify and localize cholangiocytes co-expressing putative progenitor cell markers.

In normal EHBD, few Ki-67⁺ cells were detected, whereas large numbers of Ki-67⁺ were found in the diseased EHBD. In EHBD affected by cholangitis, Ki-67⁺ cells were mainly located in the basal layer of the lumen. EHBD specimens from patients with ischaemic type biliary lesions displayed histological signs of epithelial cell loss and large numbers of Ki-67⁺ cells were observed in the peribiliary glands. C-Kit expression was localized throughout the EHBD wall and immunofluorescent doublestaining identified a few K19⁺/c-Kit⁺ cells in the luminal epithelium of the EHBD as well as in the peribiliary glands.

These findings support the hypothesis that progenitor cells exist in the EHBD and that the peribiliary glands can be considered a local progenitor cell niche in the human EHBD.

While the mechanisms underlying the development of drug-induced liver injury are not clear, there is evidence to suggest that tumor necrosis factor-α (TNF-α) plays an important role in drug- or drug metabolite-induced immune responses. We hypothesized that polymorphisms in the TNF-α gene are associated with anti-tuberculosis drug (ATD)-induced hepatitis.

Patients who suffered from ATD-induced hepatitis were enrolled in the study. ATD-induced hepatitis was defined as an increase in liver transaminase levels that were more than three times the upper limit of normal. ATD-tolerant patients were used as a control. Patients were treated with first line ATD therapies including isoniazid, rifampicin, ethambutol, and pyrazinamide. We compared the genotype frequencies of the TNF-α polymorphism -308G/A in 77 patients with ATD-induced hepatitis and 229 ATD-tolerant patients.

The frequency of carrying the variant allele (AG or AA) was significantly higher in patients with ATD-induced hepatitis compared with ATD-tolerant patients [26.0% vs. 15.3%, P = 0.034, OR (95% CI) = 1.94 (1.04–3.64)] and the frequency of the A allele was significantly different between the two groups [0.143 vs. 0.079, P = 0.018, OR (95% CI) = 1.95 (1.11–3.44)].

These results reveal that the TNF-α genetic polymorphism -308G/A is significantly associated with ATD-induced hepatitis. This genetic variant may be a risk factor for ATD-induced hepatitis in individuals from Korea.

Bacterial infections are common cause of morbidity and mortality in patients with cirrhosis. The early diagnosis of these infections is rather difficult.

To assess the accuracy of acute phase proteins in the identification of bacterial infections.

Concentration of C-reactive protein (CRP), procalcitonin (PCT), lipopolysaccharide-binding protein (LBP), sCD14 and antimicrobial antibodies were measured in serum of 368 well-characterized patients with cirrhosis of whom 139 had documented infection. Clinical data was gathered by reviewing the patients’ medical charts.

Serum levels of CRP, PCT and LBP were significantly higher in patients with clinically overt infections. Among the markers, CRP – using a 10 mg/L cut-off value– proved to be the most accurate in identifying patients with infection (AUC: 0.93). The accuracy of CRP, however, decreased in advanced stage of the disease, most probably because of the significantly elevated CRP levels in non-infected patients. Combination of CRP and PCT increased the sensitivity and negative predictive value, compared with CRP on its own, by 10 and 5% respectively. During a 3-month follow-up period in patients without overt infections, Kaplan–Meier and proportional Cox-regression analyses showed that a CRP value of >10 mg/L (P = 0.035) was independently associated with a shorter duration to progress to clinically significant bacterial infections. There was no correlation between acute phase protein levels and antimicrobial seroreactivity.

C-reactive protein on its own is a sensitive screening test for the presence of bacterial infections in cirrhosis and is also a useful marker to predict the likelihood of clinically significant bacterial infections in patients without overt infections.

Biochemical tests have been recommended as endpoints for clinical trials in primary biliary cirrhosis (PBC) because the use of liver transplantation and death as endpoints in ursodeoxycholic acid (UDCA) therapeutic trials is unfeasible. The best inclusion criteria cut-off values and cut-off for demonstrating treatment success have not been defined.

Our aim was to determine the optimal biochemical values for patient inclusion and to define values for treatment success in therapeutic trials.

We performed a retrospective review of 73 patients with PBC treated with UDCA followed over 36 months. Following one year of UDCA therapy, the likelihood of developing clinical endpoints of varices, ascites, encephalopathy, death or transplantation over the ensuing two years, based on degrees of elevation of biochemical markers, was analyzed using chi-square or Fisher's exact test.

Patients with ALP≥2 X upper limit of normal (ULN) had a 2-fold greater likelihood of developing endpoints compared to patients with lower values (23% versus 11%), (p < 0.05). Patients with bilirubin > 1 mg/dL were 4 times more likely to develop endpoints compared to those with lower values (33% versus 8%), (p = 0.02). These values help identify the patient population for adjunctive therapy trials. Patients with ALP ≤1.67 X ULN and bilirubin ≤1mg/dL demonstrated the least likelihood of reaching adverse clinical endpoints and can be used to define treatment success.

Optimal ALP and Bilirubin levels can be used as appropriate biochemical criteria for patient selection and defining treatment success in future clinical trials in patients with PBC.

Cytotoxic T lymphocyte-associated factor 4 (CTLA-4) functions as a negative regulator of T cell-mediated immune response. Molecular changes associated to CTLA-4 gene polymorphisms could reduce its ability to suppress and control lymphocyte proliferation.

To evaluate the frequency of CTLA-4 gene polymorphisms in chronic hepatitis C virus (HCV) infected patients and correlate to clinical and histological findings.

We evaluated 112 HCV-infected subjects prospectively selected and 183 healthy controls. Clinical and liver histological data were analysed. −318C > T, A49G and CT60 CTLA-4 single-nucleotide polymorphisms (SNPs) were studied by PCR-RFLP and AT(n) polymorphism by DNA fragment analysis by capillary electrophoresis in automatic sequencer.

Eight AT repetitions in 3′UTR region were more frequent in HCV-infected subjects. We found a positive association of −318C and + 49G with HCV genotype 3 (P = 0.008, OR 9.13, P = 0.004, OR 2.49 respectively) and an inverse association of both alleles with HCV genotype 1 (P = 0.020, OR 0.19, P = 0.002, OR 0.38 respectively). Allele + 49G was also associated to aminotransferases quotients > 3 (qALT, P = 0.034, qAST, P = 0.041). Allele G of CT60 SNP was also associated with qAST > 3 (P = 0.012). Increased number of AT repetitions was positively associated to severe necroinflammatory activity scores in liver biopsies (P = 0.045, OR 4.62).

CTLA-4 gene polymorphisms were associated to HCV-infection. Eight AT repetitions were more prevalent in HCV-infected subjects. −318C and + 49G alleles were associated to genotypes 1 and 3 infections and increased number of AT repetitions in 3′UTR region favoured severe necroinflammatory activity scores in liver biopsies.

Hepatitis B virus surface antigen (HBsAg) quantification has been suggested to discriminate inactive carriers from hepatitis e antigen (HBeAg) negative chronic hepatitis, but it could be genotype-dependent. We studied the predictive value of HBsAg quantification in genotype C HBeAg-negative hepatitis B virus (HBV) carriers.

We recruited 104 HBeAg-negative HBV carriers with HBV DNA levels < 2,000 IU/ml and normal alanine aminotransferase (ALT) levels for at least 12 months and prospectively followed them for > 36 months. Patients were classified into two groups: inactive carriers (IC) who showed HBV DNA levels < 2,000 IU/ml and persistently ALT ≤ 40 IU/ml throughout the follow-up period and patients with HBeAg-negative chronic hepatitis (ENH).

After follow-up, 73 patients were categorized into the IC group and 31 patients into the ENH group. HBsAg levels were significantly lower in the IC group than in the ENH group. The diagnostic accuracy of single-point HBsAg levels for predicting viral activation was favourable (AUROC = 0.710, P < 0.001). Diagnostic accuracy improved when HBsAg was combined with baseline HBV DNA levels (AUROC = 0.750, P < 0.001). The combination of HBsAg levels > 850 IU/ml and HBV DNA > 850 IU/ml predicted the reactivation of HBV replication with 84.6% diagnostic accuracy.

Although it is inferior to other genotypes and to serum HBV DNA alone, single-point HBsAg level has a favourable diagnostic accuracy in genotype C HBeAg-negative HBV carriers and is expected to provide additional information for managing chronic hepatitis B.

Recent studies have focused on regulatory T cells (Tregs) in chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC) and they were also conducted independently of each other.

This study tried to characterize Tregs in blood and tumour infiltration, and to explore the correlations between Tregs and the context of chronic hepatitis B in HCC patients.

The liver-resident Tregs and CD8⁺ T cells on core biopsy were investigated using immunohistochemistry staining in individuals (n = 209) with CHB (n = 47), HCC (n = 137) or healthy controls (n = 25). Circulating Tregs were detected in the above patients with CHB (n = 27) or HCC (n = 101) by flow cytometry.

The number of tumour-infiltrating and circulating FoxP3⁺ Tregs was significantly high in patients with CHB (P < 0.001). However, there were fewer intratumoural Tregs in patients with advanced HCC than those in patients with early stage HCC (P = 0.043); In contrast, the circulating Tregs frequency increased during the progression of HCC (P = 0.024). Increased tumour-infiltrating and circulating FoxP3⁺ Tregs were associated with poor overall survival (P = 0.041, 0.002 respectively) and a shorter time to recurrence (P = 0.049, 0.002 respectively) in patients with early stage HCC. Tumour-infiltrating Foxp3 +  Tregs were related to chronic hepatitis B natural history in HCC (P = 0.012). Neither tumour-infiltrating CD8⁺ T cells nor balance of intratumoural Tregs and CD8⁺ T cells correlated with prognosis of HCC.

Increased Foxp3⁺ Tregs may represent a prognostic predictor in patients with early stage HCC. The CHB natural history influenced density of tumour-infiltrating Tregs in hepatocellular carcinoma patients with chronic hepatitis B viruses infection.

Accuracy of transient elastography (TE) in hepatitis B virus (HBV) infection has not been well established. We aimed to compare the performances of TE for the assessment of liver fibrosis in patients with chronic HBV or hepatitis C virus (HCV) infection. A secondary analysis was performed to assess whether or not alanine aminotransferase (ALT) levels would impact on the accuracy of TE.

This cross-sectional study, carried out in a single centre, included treatment-naïve patients with compensated chronic HBV or HCV infection, consecutively admitted between 2006 and 2008 for a liver biopsy and TE measurement on the same day.

A total of 202 HBV patients and 363 HCV subjects were evaluated. Overall diagnostic accuracy of TE in the HBV group was comparable to that observed in HCV patients [area under the receiver-operating characteristics (AUROCs) 0.867 ± 0.026 vs. 0.868 ± 0.019 for predicting F ≥ 2, P = 0.975; 0.902 ± 0.029 vs. 0.894 ± 0.020 for F ≥ 3, P = 0.820; and 0.935 ± 0.024 vs. 0.947 ± 0.027 for F4, P = 0.740 respectively]. TE exhibited comparable accuracies, sensitivities, specificities, predictive values and likelihood ratios in HBV and HCV groups. AUROC analysis showed no influence of ALT levels on the performance of TE in HBV individuals. ALT-specific cut-off values did not exhibit significantly higher diagnostic performances for predicting fibrosis in HBV patients with elevated ALT.

In HBV patients, TE measurement accurately predicts the absence or presence of significant fibrosis, advanced fibrosis or cirrhosis and shows similar performances as compared to HCV patients. The use of TE cut-off values adjusted to ALT level did not improve performances for estimating liver fibrosis in HBV patients.

An association between primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD) is well recognized. However, the disease course of IBD following liver transplantation (LT) for PSC remains ill-defined.

We aimed to assess the impact of IBD in patients that had undergone LT for PSC to help identify risk factors for flare and to assess the impact of IBD on graft survival.

110 patients underwent LT for PSC (Oct 1990-Aug 2009) at King's College Hospital. 74 (67%) patients had concurrent IBD and 36 had PSC alone prior to transplant. 39 patients developed IBD (flare of IBD and de-novo) post transplant. Cumulative risk for IBD at 1-, 2-, 5- and 10-years was 16%, 24%, 38% and 72% respectively. Flare of IBD occurred in 33 patients with a mean time to flare of 30 ± 28 months. De-novo IBD occurred in 6 patients (all UC). Mean time to diagnosis was 29 ± 25 months. Multivariate cox-regression analysis identified active IBD at time of LT as a significant predictor of graft failure post LT (HR 10, CI 3-39, P = 0.001) and smoking at time of transplantation and subsequent cessation predictive of recurrent IBD post transplantation (HR 17, 2-180, P = 0.02).

In conclusion, smoking at time of LT was predictive of flare of IBD and active IBD at time of transplantation had a significant effect on graft survival. Medical therapy needs to be maximised in the pre-LT period. Patients with poorly controlled IBD refractory to medical therapy should be considered for colectomy at time of transplantation.

Suppression of hepatitis B virus (HBV) DNA is more potent, and occurrence of resistant strain is rare with entecavir than lamivudine, but whether these merits result in a more favourable outcome in HBV-related decompensated cirrhosis patients is unclear.

To compare virologic response, changes in liver function, clinical course and predictive factors for early mortality after treatment between patients treated with lamivudine and those with entecavir in HBV-related decompensated cirrhosis patients.

HBV-related decompensated cirrhosis patients [Child-Turcotte-Pugh (CTP) score ≥ 7] treated with either lamivudine or entecavir were enrolled. Serum HBV DNA levels, CTP score and Model for End-stage Liver Disease (MELD) score were monitored every 3 months.

Eighty-six patients were enrolled; mean age was 54 ± 11 years, and 63 (73.3%) patients were men; 41 (47.7%) and 45 (52.3%) patients were assigned to the lamivudine group and entecavir group respectively. Although suppression of serum HBV DNA level was more potent in the entecavir group, CTP or MELD scores during the course of treatment did not differ between the two groups. Similarly, 6-month survival rates did not differ between the two groups (95.1 vs 93.2%, P = 0.684). Baseline CTP score and MELD score at 3 months of treatment were significantly associated with 6-month mortality. The 6- and 12-month mortality rates for patients with baseline CTP score ≥11 and MELD score ≥17.5 after 3 months of treatment were 42.9 and 61.9% respectively.

Although HBV DNA suppression was more potent in the entecavir group than the lamivudine group, early mortality rates did not differ between the two groups. The baseline CTP score and MELD score 3 months after initiating antiviral treatment were significant predictors of early mortality.

Detection of biliary dysplasia in PSC is essential for proper timing of liver transplantation to prevent the development of cholangiocancer, which is considered a contraindication for liver transplantation in most centres. In patients with PSC, differential diagnosis of benign, premalignant and malignant biliary strictures is difficult.

This prospective study aimed to evaluate the role of DNA analysis in combination with brush cytology, scored ERCP findings, and tumour markers to detect hepatobiliary dysplasia and malignancy.

Brush samples for cytology and for evaluation of DNA content analysed with flow cytometry came from 102 consecutive PSC patients referred for ERCP. Symptoms, serum Ca19-9 and CEA were determined at the time of index biliary examination. ERCP findings were scored for intra- and extrahepatic changes. The end-points were liver transplantation or diagnosis of malignancy or dysplasia.

Most of the patients were asymptomatic at the time of ERCP: 73% had no symptoms, and 12% had only mild symptoms. An aneuploid DNA content was evident in 20 (20%) patients, and cells suspected for malignancy in 22 (21%). Seven patients had both aneuploidity and cytology (7%) suspicious for malignancy. An end-point, diagnosis of malignancy or liver transplantation was achieved in 42 patients. Combining DNA ploidity and cytology in patients at the end-point, sensitivity was 72%, specificity 82%, positive predictive value 86% and negative predictive value 67%.

In this mostly asymptomatic PSC-patient population, 33% demonstrated abnormal brush cytology or aneuploidity. Determining DNA ploidy and brush cytology during ERCP offers a useful tool for identifying those PSC patients who are at high risk of developing cholangiocancer.

Despite a decline in cases of acute hepatitis B and the low hepatitis B virus (HBV) chronicity rates in adults, still some patients progress to HBV-related fulminant liver failure. In this review, we discuss treatment options that may prevent the progression of severe acute hepatitis B to fulminant liver failure and death. In severe acute HBV with prolonged prothrombin time and increased bilirubin, interferon failed to be effective while antiviral treatment, particularly with lamivudine, appears to improve survival (mean survival almost 80%). Outcome without antiviral therapy has remained considerably poor, whereas there is no convincing evidence of amelioration of HBV-targeted immunity. Of note, most patients who died or required transplantation despite lamivudine therapy, were started on lamivudine at advanced stages compared with those survived. This suggests that prompt and timely antiviral therapy is crucial. Owing to the abovementioned results the design of randomized placebo-control trials in the setting of severe acute hepatitis B seems unethical. On the contrary, the design of multicentre double-blind randomized trials to compare the efficacy between lamivudine and entecavir or even tenofovir in acute severe HBV cases is ideally needed, but these studies appear to be very difficult to perform considering that these cases are not frequent and therefore, it is almost impossible to have two arms adequately numerous and homogenous for statistical evaluation. Thus, in the absence of solid evidence based data, the hepatologists could treat their patients with severe acute hepatitis B with lamivudine or the most potent antivirals entecavir or tenofovir.

Serum gamma-glutamyltransferase (GGT) activity is a sensitive but non-specific marker of non-alcoholic fatty liver disease (NAFLD). Recently, four GGT fractions (big-, medium-, small-, free-GGT) were described in humans.

We aimed to investigate whether a specific GGT fraction pattern is associated with NAFLD.

Gamma-glutamyltransferase fractions were determined in patients with NAFLD (n = 90), and compared with those in control subjects (n = 70), and chronic hepatitis C (CHC, n = 45) age and gender matched.

Total GGT was elevated in NAFLD as compared to controls (median, 25°–75°percentile: 39.4, 20.0–82.0 U/L vs. 18.4, 13.2–24.9 U/L respectively, P < 0.001). All fractions were higher in NAFLD than in controls (P < 0.001). The b-GGT showed the highest diagnostic accuracy for NAFLD diagnosis [area under ROC curve (ROC-AUC): 0.85; cut-off 2.6 U/L, sensitivity 74%, specificity 81%].

Also subjects with CHC showed increased GGT (41.5, 21.9–84.5 U/L, P < 0.001 vs. controls, P = n.s. vs. NAFLD), as well as m-, s-, and f-GGT, while b-GGT did not show any significant increase (P = n.s. vs. HS, P < 0.001 vs. NAFLD). In subjects with CHC, s-GGT showed the best diagnostic value (ROC-AUC: 0.853; cut-off 14.1 U/L, sensitivity 73%, specificity 90%). Serum GGT did not show any value in the differential diagnosis between NAFLD and CHC (ROC-AUC 0.507, P = n.s.), while b-GGT/s-GGT ratio showed the highest diagnostic accuracy for distinguishing NAFLD and CHC (ROC-AUC: 0.93; cut-off value 0.16, sensitivity 82%, specificity 90%).

b-GGT increases in NAFLD, but not in CHC. GGT fraction analysis might help in improving the sensitivity and specificity of the diagnosis of NAFLD and other liver dysfunctions.

Fibrosis determines prognosis and management in patients with chronic hepatitis B and C (CHB and CHC). Transient elastography (TE) is a promising non-invasive method to assess fibrosis. We prospectively studied the performance of TE compared to histology and also whether there are differences between CHB and CHC. Only large biopsies (≥25 mm) were used.

We included 241 patients with CHB (n = 125) and CHC (n = 116), of whom we acquired 257 liver biopsies, all preceded by elastography. We correlated liver stiffness with fibrosis stage according to the METAVIR system, inflammation (Histology Activity Index), steatosis and iron. The impact of gender, age, body mass index, alcohol, alanine aminotransferase levels, platelet count, viral load and genotype on liver stiffness was evaluated.

The AUROC's for F ≥ 2 were 0.85 for CHB and 0.76 for CHC. AUROC's for F ≥ 3 were 0.91 for CHB and 0.87 for CHC and 0.90 and 0.91 for F4 for CHB and CHC respectively. For F ≥ 2 the cut-off value was 6.0 kPa for CHB and 5.0 kPa for CHC. The cut-off values for ≥F3 were 9.0 and 8.0 kPa for CHB and CHC, respectively, and 13.0 kPa for F4 in both CHB and CHC patients. Besides inflammation, all other remaining factors do not influence liver stiffness.

For the diagnosis of fibrosis stages F ≤ 2 TE is suboptimal, and inflammation may induce higher values. For stages F ≥ 3 TE performance is good and equal in both CHB and CHC patients.

Honokiol, a small active molecular compound extracted from magnolia, has recently been shown to inhibit hepatitis C virus (HCV) infection in vitro.

This study further characterized aspects of the HCV lifecycle affected by the antiviral functions of honokiol.

The influence of honokiol on HCV infection, entry, translation and replication was assessed in Huh-7.5.1 cells using cell culture-derived HCV (HCVcc), HCV pseudo-type (HCVpp) and sub-genomic replicons.

Honokiol had strong antiviral effect against HCVcc infection at non-toxic concentrations. Combined with interferon-α, its inhibitory effect on HCVcc was more profound than that of ribavirin. Honokiol inhibited the cell entry of lentiviral particles pseudo-typed with glycoproteins from HCV genotypes 1a, 1b, and 2a, but not of the vesicular stomatitis virus. It had inefficient activity on HCV internal ribosome entry site (IRES)-translation at concentrations with significant anti-HCVcc effects. The expression levels of components of replication complex, NS3, NS5A and NS5B, were down-regulated by honokiol in a dose-dependent manner. It also inhibited HCV replication dose dependently in both genotypes 1b and 2a sub-genomic replicons.

Honokiol inhibits HCV infection by targeting cell entry and replication and, only at a concentration >30 μM, IRES-mediated translation of HCV life cycle. Based on its high therapeutic index (LD₅₀/EC₉₀ = 5.4), honokiol may be a promising drug for the treatment of HCV infection.

Obesity is a common risk factor for nonalcoholic fatty liver disease (NAFLD) and chronic kidney disease (CKD). NAFLD and CKD have been associated in many epidemiological studies. We hypothesize that more severe liver disease, namely nonalcoholic steatohepatitis (NASH), is related with further renal impairment. We aimed to evaluate if changes in renal function were present in morbid obese patients with NAFLD.

Prospective and consecutive recruitment of morbid obese patients with biopsy proven NAFLD obtained during bariatric surgery. Renal function was evaluated with CKD-Epidemiology Collaboration estimated glomerular filtration rate (eGFR). Plasmatic adiponectin, leptin and active ghrelin concentrations were determined.

One hundred and forty-eight patients were included of whom 25% had NASH and 75% simple steatosis. NASH patients were older, with higher body mass index and had more frequently metabolic syndrome and lower eGFR (97 ± 22 vs 106 ± 16 ml/min/1.73², P = 0.035). NASH conferred an odds ratio (OR) 3.0 (1.3–7.0) for eGFR < 90 and OR 9.7 (1.0–96.4) for eGFR < 60 ml/min/1.73². eGFR < 90 ml/min/1.73² associated with aspartate aminotransferase [OR 2.9 (1.1–7.6)] and γ-glutamyl transpeptidase elevation [OR 3.0 (1.3–7.2)], NASH [OR 3.0 (1.3–7.0)], any lobular inflammatory activity [OR 3.0 (1.3–7.0)] and severe fibrosis [OR 3.4 (1.1–10.8)]. Neither eGFR nor liver histology was associated with adipokines levels.

In morbid obese patients, NASH, particularly lobular inflammation and advanced fibrosis, associates with mild decreases in eGFR, suggesting a common inflammatory link between liver and renal lesion.

Recently, a non-M2-related mitochondrial 60 kDa protein found to be recognized by antimitochondrial antibody (AMA) negative sera from patients with primary biliary cirrhosis (PBC) has been shown to contain parts of the five F₁-ATPase subunits α, β, γ, δ and ε. In this study, we examined whether this enzyme is, indeed, a target antigen in PBC.

Analysed were 60 AMA-positive/anti-M2-negative and 103 anti-M2-positive PBC patients, 46 patients with autoimmune hepatitis (AIH), 35 patients with primary sclerosing cholangitis (PSC), 110 patients with viral hepatitis, 40 patients with inflammatory bowel diseases (IBD), 33 patients with connective tissue diseases (systemic lupus erythematosus, mixed connective tissue disease, Sjögren disease, systemic sclerosis) and 25 blood donors. The F₁-ATPase-subunits α-δ were recombinantly expressed in Escherichia coli, purified and applied to ELISA and Western blotting.

In all, 40 of the 60 AMA-positive/anti-M2-negative (67%) and 44 (43%) of the 103 anti-M2-positive PBC-sera reacted with at least one of the F₁-subunits α-δ. The β- and γ-subunits were preferentially recognized. However, also up to 57% of patients with AIH and 34% of patients with PSC had anti-β- or γ-antibodies, while patients with viral hepatitis had these antibodies in up to 13%. Patients with IBD had anti-β and anti-γ-antibodies in up to 20 and 5% respectively. None of the patients with connective tissue diseases had antibodies to the β- and only 6% to the γ-subunit. Sera from healthy blood donors were negative.

Antibodies to the β- and γ-subunits of F₁-ATPase are further AMAs in PBC but occur also in other autoimmune liver disorders; they may be, therefore, indicators for a general autoimmune process of the liver.

The outcome of patients with hepatitis C virus (HCV) infection acquired during childhood in the absence of antiviral therapy is not clear.

The purpose of this study was to review the outcome of untreated HCV acquired in childhood. Only population-based studies were included, as referred cases would be predicted to have more severe disease.

A systematic review of the literature was completed up to October 2010 to identify studies where a population was screened for HCV infection that was presumably acquired during childhood. Demographical and clinical data were collected on infected patients who had not been treated with an antiviral. Primary outcome was development of a severe adverse outcome (cirrhosis, hepatoma, need for a liver transplant or liver-related death).

There were 25 studies reporting a total of 733 infected patients. Liver biopsy results were provided for 180 patients (25%), revealing cirrhosis in eight (1.0% of the total and 4.0% of those who had a biopsy). None of the other patients developed a severe adverse outcome. As a result of the small number of patients with a severe adverse outcome, risk factors for HCV progression could not be identified.

Although HCV can lead to liver transplantation and death during childhood, the vast majority of patients with disease acquired during childhood have slowly progressive disease. There is no clear indication for antiviral therapy in the majority of children with HCV infection.

Antiviral therapy for hepatitis B virus (HBV) infection is frequently prescribed for patients with chronic HBV infection during surveillance for hepatocellular carcinoma (HCC). In patients who subsequently develop HCC, the impact of antiviral therapy on the outcome of HCC remains unclear.

We aimed to study the impact of antiviral therapy on the survival of patients who developed HCC.

From two prospective surveillance cohorts, the use of antiviral therapy for patients with HCC was retrospectively reviewed. We compared the overall survival, liver function and tumour characteristics between patients with and without antiviral therapy during surveillance. Multivariate analysis was conducted to determine the independent prognostication of antiviral therapy.

During a median follow-up of 10.1 years of 1429 patients, 148 cases of HCC were diagnosed and followed up for a median of 5.7 years. Twenty-nine patients were given antiviral therapy during surveillance and continued treatment after diagnosis of HCC. The median survival of this group of patients was better than the rest of cohorts (hazard ratio: 0.472; 95% CI: 0.25–0.89; P = 0.0191). Use of antiviral therapy remained an independent prognostic factor after adjustment for demographic factors and tumour staging on multivariate analysis. Exploratory analysis revealed that patients who commenced antiviral therapy during surveillance had lower HBVDNA, lower serum alanine transaminase, better hepatic reserves and higher rate of local treatment at diagnosis of HCC.

This study provides evidence that commencement of antiviral therapy during the surveillance period is associated with improvement in overall survival in HBV-related HCC.

Liver-related clinical consequences of non-alcoholic fatty liver disease (NAFLD) are seen only in the minority of patients with advanced fibrosis. The aim of our study was to generate insight into a potential endocrine basis of steatohepatitis with advanced fibrosis in NAFLD.

Biopsy and blood samples were prospectively collected from patients with medically complicated obesity. Patients were categorized, according to liver histology, into: (i) normal, (ii) simple steatosis (SS), (iii) non-alcoholic steatohepatitis (NASH) with fibrosis stage (FS) 0–1 and (iv) NASH with FS ≥ 2. A broad panel of potential biomarkers included DHEA-S, growth hormone (GH), homeostasis model assessment-insulin resistance (HOMA-IR), leptin, resistin, adiponectin and cytokeratin 18 (CK-18) fragments.

We studied 160 patients (mean BMI 46.8 ± 8.2 kg/m²). Liver biopsies demonstrated normal histology in 10%, SS in 45%, NASH with FS 0–1 in 37.5% and NASH with FS ≥ 2 in 7.5%. C-reactive protein, IL-6, GH, CK-18, adiponectin, HOMA-IR and quantitative insulin sensitivity check index (QUICKI) were significantly associated with NASH in univariate analysis, but overall predictivity of these parameters was low (AUC ROC = 0.62–0.68). In contrast, all patients with NASH with FS ≥ 2 had insulin resistance, as measured by QUICKI, and GH levels <0.45 ng/ml and all but one patient with NASH FS 2–3 had low DHEA levels (<123 μg/dl).

Low serum levels of GH and DHEA are very common in patients with NASH with more advanced fibrosis. Other biomarkers, including CK-18 fragment levels, have predictivity characteristics that would be of low clinical utility for distinguishing patients with normal histology or SS from those with NASH. These findings demonstrate an endocrine profile associated with advanced fibrosis.

Vitamin D deficiency is associated with fractures, infections and death. Liver disease impairs vitamin D and vitamin D binding protein (DBP) metabolism.

We aimed to determine the impact of liver transplantation on vitamin D, particularly on DBP and free vitamin D concentrations.

Serum 25(OH)D, 1,25(OH)₂D and DBP concentrations were measured in 202 adults before liver transplantation and 3 months later in 155. Free vitamin D concentrations were estimated from these values. Risk factors for 25(OH)D deficiency (<20 ng/ml) and low 1,25(OH)₂D (<20 pg/ml) were examined with logistic regression, and changes in concentrations following transplantation with linear regression.

Pretransplant, 84% were 25(OH)D deficient, 13% had 25(OH)D concentrations <2.5 ng/ml, and 77% had low 1,25(OH)₂D. Model for end-stage liver disease score ≥20 (P < 0.005) and hypoalbuminemia (P < 0.005) were associated with low 25(OH)D and 1,25(OH)₂D concentrations. Following transplantation, 25(OH)D concentrations increased a median of 17.8 ng/ml (P < 0.001). Albumin increased from a median of 2.7 to 3.8 g/dl (P < 0.001) and DBP from 8.6 to 23.8 mg/dl (P < 0.001). Changes in total 25(OH)D were positively and independently associated with changes in DBP (P < 0.05) and albumin (P < 0.001). Free 25(OH)D concentrations rose from 6.0 to 9.7 pg/ml (P < 0.001). In contrast, total 1,25(OH)₂ Dconcentrations rose only by 4.3 pg/ml (P < 0.001) and free 1,25(OH)₂ Dconcentrations declined (P < 0.001).

Serum total and free 25(OH)D and DBP concentrations rose substantially following transplantation, while 1,25(OH)₂D concentrations showed modest changes and free 1,25(OH)₂D decreased. Studies of the effects of vitamin D status on diverse transplant complications are needed.

Nonalcoholic steatohepatitis (NASH), the most severe form of nonalcoholic fatty liver disease (NAFLD), is associated with inflammation and increased oxidative stress. The neutrophil/lymphocyte ratio (N/L) integrates information on the inflammatory milieu and physiological stress.

The aim of this study was to determine the utility of N/L ratio to predict the presence of NASH in patients with NAFLD.

Our cohort consisted of 101 consecutive patients undergoing liver biopsy for clinical suspicion of NAFLD. Patients were divided into two groups: NASH group (n = 50) and not NASH group (n = 51). The stage of fibrosis was measured using a 4-point scale. The total white cell count, neutrophil and lymphocyte counts were recorded, and the N/L ratio was calculated.

The mean age was 49.5 (±10.8) years and the mean BMI was 31.4 (±4.9) kg/m². Patients with NASH had a higher N/L ratio compared with patients with not NASH [2.5 (1.9–3.3) and 1.6 (1.2–2.0), respectively, P < 0.001]. The N/L ratio correlated with the NAFLD activity score and its individual components (steatosis, inflammation and ballooning P < 0.001). Patients with advanced fibrosis (F3-4) had an elevated N/L ratio [2.9 (2.0–3.9)] compared with patients with fibrosis stage 1–2 [1.8 (1.2–2.2)], P < 0.001. For each one-unit increase in N/L ratio, the likelihood of having NASH increased by 70% and the likelihood of having fibrosis increased by 50%.

The N/L ratio is higher in patients with NASH and advanced fibrosis. This ratio can be used as a novel noninvasive marker to predict advanced disease.

The aim of this study was to assess the patterns of lamivudine (LAM)-resistant mutations and the influence on biochemical and virological responses to adefovir (ADV) add-on LAM combination therapy in patients with LAM-resistant chronic hepatitis B (CHB).

Seventy-eight CHB patients with confirmed genotypic resistance to LAM, who initiated ADV add-on LAM combination treatment, were enrolled at our institution between April 2007 and April 2009.

The baseline tyrosine-methionine-aspartate-aspartate (YMDD) mutation patterns were as follows: rtM204I 45 (57.7%); and rtM204V + rtM204I/V 33 (42.3%). The decrease in the mean ± standard deviation (SD) serum log₁₀HBV-DNA level did not differ between the patients carrying the rtM204I vs. rtM204IV +rtM204I/V mutations at 3, 6 and 12 months after the initiation of ADV add-on LAM combination treatment. The proportion of patients who achieved ALT normalization (<40 IU/L) 12 months after the initiation of ADV add-on LAM combination treatment were significantly higher in patients with a rtM204I mutation than rtM204V+ rtM204I/V mutations (39 [86.7%] vs. 22 [66.7%], P = 0.05). The proportion of patients in whom the log₁₀HBV-DNA decreased <2 log₁₀ copies/ml, 6 months after the initiation of ADV add-on LAM combination treatment (non-responders), was significantly higher in patients with a rtM204V + rtM204I/V mutations than rtM204I mutation (7 [21.2%] vs. 2 [4.4%], P = 0.032).

Biochemical response at 12 months from baseline was better in patients with a rtM204I mutation than rtM204V+ rtM204I/V mutations. In addition, early treatment failure was more common in patients with rtM204V+ rtM204I/V mutations than a rtM204I mutation.

Somatostatin is a pleiotropic peptide, exerting a variety of effects through its receptor subtypes. Recently, somatostatin has been shown to act as a chemoattractant for haematopoietic progenitor cells and hepatic oval cells (HOC) via receptor subtype 2 and subtype 4 (SSTR4) respectively.

We investigated the in vivo effect of somatostatin/SSTR4 on HOC migration in the injured liver model of rats and the type of signalling molecules associated with the chemotactic function.

Migration assay, HOC transplantation and phosphatidylinositol-3-kinase (PI3K) signalling were assessed with or without somatostatin and an analogue of somatostatin (TT232) that specifically binds to SSTR4.

TT232 was shown to have an antimigratory action on HOC induced by somatostatin in vitro. In HOC transplantation experiments, a lower number of donor-derived cells were detected in TT232-treated animals, as compared with control animals. Activation of PI3K was observed in HOC exposed to somatostatin, and this activation was suppressed by either SSTR4 antibody or TT232-pretreatment. In addition, a PI3K inhibitor abrogated the motility of HOC.

Together, these data suggest that somatostatin stimulates the migration of HOC within injured liver through SSTR4, and this action appears to be mediated by the PI3K pathway.

Inflammation leads to transcriptional downregulation of many hepatic genes, particulary those activated by retinoid X receptor-α (RXRα) heterodimers. Inflammation-mediated reduction of nuclear RXRα levels is a main factor in reduced nuclear receptor (NR)-regulated hepatic gene expression, eventually leading to cholestasis and liver damage.

To investigate roles for RXRα in hepatic gene expression during inflammation, using two complementary mouse models: ligand activation of RXRα, and in mice expressing hepatocyte-specific expression of RXRα missing its DNA-binding domain (DBD; hs-RxrαΔex4^−/−).

To activate RXRα, mice were gavage-fed with LG268 or vehicle for 5 days. To inhibit RXRα function, hs-RxrαΔex4^−/− mice were used. All mice were injected intraperitoneally with lipopolysaccharides (LPS) or saline for 16 h prior to analysis of hepatic RNA, protein and NR–DNA binding.

LG268 treatment attenuated the LPS-mediated reductions of several RXRα-regulated genes, coinciding with maintained RXRα occupancy in both Bsep and Ostβ promoters. Lacking full hepatocyte RXRα function (hs-RxrαΔex4^−/− mice) led to enhancement of LPS-mediated changes in gene expression, but surprisingly, maintenance of RNA levels of some RXRα-regulated genes. Investigations revealed that hs-RxrαΔex4^−/− hepatocytes expressed an internally truncated, approximately 44 kDa, RXRα-form. DNA-binding capacity of NR heterodimers was equivalent in wild-type and hs-RxrαΔex4^−/− livers, but reduced by LPS in both. Chromatin immunoprecipitation quantitative PCR revealed that RXRα occupancy to the Bsep RXRα:Farnesoid X Receptor site was reduced, but not absent, in hs-RxrαΔex4^−/− livers.

There are differential regulatory roles for hepatic RXRα, both in basal and inflammatory states, suggesting new and complex multidomain roles for RXRα in regulating hepatic gene expression. Moreover, there is an unexpected non-obligate role for the DBD of RXRα.

Sphingosine kinase 1 (SphK1), which phosphorylates sphingosine to sphingosine-1-phosphate (S1P), is overexpressed in various types of cancers, and may act as an oncogene in tumorigenesis. However, little is known about the precise role of the SphK1/S1P pathway in human liver cancer, especially regarding the metastasis of hepatocellular carcinoma (HCC).

The expression of SphK1 was detected by quantitative reverse-transcription PCR. In addition, transwell cell migration and invasion assay were carried out for functional analysis. Furthermore, the level of S1P was quantified by ELISA and Rac1/Cdc42 GTPase activation was assessed by western blot analysis.

The levels of SphK1 mRNA are commonly up-regulated in HCC patients and human liver cancer cell migration and invasion can be promoted by the overexpression of SphK1. In addition, inhibition of SphK1 with either a SphK1 inhibitor or siRNA reduced human liver cancer cell migration and invasion. Furthermore, overexpression of SphK1 increased S1P levels, and the exogenous addition of S1P increased liver cell migration and invasion through the EDG1 receptor.

The results from this study provide strong evidence of a role for the SphK1/S1P/EDG1 pathway in liver metastasis, thus making it an attractive therapeutic target for the development of new anti-HCC drugs.

The hepatitis C virus (HCV) genomic database is expanding rapidly.

There is a need to provide an updated phylogenetic tree analysis based on the complete coding region of HCV.

All available HCV complete genome sequences in the HCV databases available through October 2010 were analyzed.

The assignment of all known complete sequences up-to-date confirmed the previous six major genotypes and one new sequence, which have been provisionally assigned as subtype 7a. New recombinant forms of HCV, although uncommon, have been detected and were found to have different crossover points.

This updated analysis based on the complete region of HCV confirmed the validity of the previously assigned genotypes/subtypes and provided an up-to-date reference for future basic research and clinical studies.