Beneficial Effect of Vitamin D on Non-Alcoholic Fatty Liver Disease (NAFLD) Progression in the Zebrafish Model.

A major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma, non-alcoholic fatty liver disease (NAFLD) results from excessive liver fat accumulation. Vitamin D (VitD) plays multiple important roles in diverse physiologic processes. Here, we describe the role of VitD in the complex pathogenesis of NAFLD and explore the possible therapeutic role of VitD supplementation in NAFLD therapy. To compare the effect of VitD to other interventions such as low-calorie diet, we induced NAFLD in young adult zebrafish (Danio rerio, AB strain) and monitored the effects of VitD supplementation on the disease course. The zebrafish administered with high-dose VitD (1.25 µg) had significantly reduced liver fat compared to those that received low-dose VitD (0.049 µg) or caloric restriction. Gene expression analysis revealed that VitD downregulated several pathways that may play a role in NAFLD etiology, which affected fatty acid metabolism, vitamins and their cofactors, ethanol oxidation, and glycolysis. The pathway analysis revealed that the cholesterol biosynthesis pathway and the isoprenoid biosynthetic process pathway were significantly upregulated whereas the small molecule catabolic process pathway significantly downregulated following the exposure of NAFLD zebrafish model to high VitD dose. Therefore, our findings suggest the association of novel biochemical pathways with NAFLD and highlight the potential of VitD supplementation to reverse the severity of NAFLD, especially in younger people.

Longitudinal kinetics of RBD+ antibodies in COVID-19 recovered patients over 14 months.

We describe the longitudinal kinetics of the serological response in COVID-19 recovered patients over a period of 14 months. The antibody kinetics in a cohort of 192 recovered patients, including 66 patients for whom follow-up serum samples were obtained at two to four clinic visits, revealed that RBD-specific antibodies decayed over the 14 months following the onset of symptoms. The decay rate was associated with the robustness of the response in that antibody levels that were initially highly elevated after the onset of symptoms subsequently decayed more rapidly. An exploration of the differences in the longitudinal kinetics between recovered patients and naïve vaccinees who had received two doses of the BNT162b2 vaccine showed a significantly faster decay in the naïve vaccinees, indicating that serological memory following natural infection is more robust than that following to vaccination. Our data highlighting the differences between serological memory induced by natural infection vs. vaccination contributed to the decision-making process in Israel regarding the necessity for a third vaccination dose.

25-Hydroxyvitamin D Inhibits Hepatitis C Virus Production in Hepatocellular Carcinoma Cell Line by a Vitamin D Receptor-Independent Mechanism.

Previously, we have reported that the active vitamin D metabolite, calcitriol and vitamin D3 (cholecalciferol), both remarkably inhibit hepatitis C virus production. The mechanism by which vitamin D3 exerts its effect is puzzling due to the low levels of calcitriol produced in vitamin D3-treated Huh7.5 cells. In this study, we aimed to explore the mechanism of vitamin D3 anti-hepatitis C virus effect. We show that vitamin D3 activity is not mediated by its metabolic conversion to calcitriol, but may be due to its primary metabolic product 25(OH)D3. This is inferred from the findings that 25(OH)D3 could inhibit hepatitis C virus production in our system, and that adequate concentrations needed to exert this effect are produced in Huh7.5 cells treated with vitamin D3. Using the CRISPR-Cas9 editing technology to knockout the vitamin D receptor, we found that the antiviral activity of vitamin D3 and 25(OH)D3 was not impaired in the vitamin D receptor knockout cells. This result indicates that 25(OH)D3 anti-hepatitis C virus effect is exerted by a vitamin D receptor-independent mode of action. The possibility that vitamin D3 and 25(OH)D3, being 3ß-hydroxysteroids, affect hepatitis C virus production by direct inhibition of the Hedgehog pathway in a vitamin D receptor-independent manner was ruled out. Taken together, this study proposes a novel mode of action for the anti-hepatitis C virus activity of vitamin D3 that is mediated by 25(OH)D3 in a vitamin D receptor-independent mechanism.

Correction: Modeling suggests that microliter volumes of contaminated blood caused an outbreak of hepatitis C during computerized tomography.

[This corrects the article DOI: 10.1371/journal.pone.0210173.].

Modeling suggests that microliter volumes of contaminated blood caused an outbreak of hepatitis C during computerized tomography.

BACKGROUND & AIMS: Acute hepatitis C (AHC) is not frequently identified because patients are usually asymptomatic, although may be recognized after iatrogenic exposures such as needle stick injuries, medical injection, and acupuncture. We describe an outbreak of AHC among 12 patients who received IV saline flush from a single multi-dose vial after intravenous contrast administration for a computerized tomography (CT) scan. The last patient to receive IV contrast with saline flush from a multi-dose vial at the clinic on the previous day was known to have chronic HCV genotype 1b (termed potential source, PS). Here we sought to confirm (via genetic analysis) the source of infection and to predict the minimal contaminating level of IV saline flush needed to transmit infectious virus to all patients. METHODS: In order to confirm the source of infection, we sequenced the HCV E1E2 region in 7 CT patients, in PS, and in 2 control samples from unrelated patients also infected with HCV genotype 1b. A transmission probabilistic model was developed to predict the contamination volume of blood that would have been sufficient to transmit infectious virus to all patients. RESULTS: Viral sequencing showed close clustering of the cases with the PS. The transmission probabilistic model predicted that contamination of the multi-dose saline vial with 0.6-8.7 microliters of blood would have been sufficient to transmit infectious virus to all patients. CONCLUSION: Analysis of this unique cohort provides a new understanding of HCV transmission with respect to contaminating volumes and viral titers.

Transplantation of Livers from Old Donors: Pushing the Envelope Beyond the Seventh Decade.

BACKGROUND: The lack of organs for liver transplantation has prompted transplant professionals to study potential solutions, such as the use of livers from donors older than 70 years. This strategy is not widely accepted because potential risks of vascular and biliary complications and recurrence of hepatitis C. OBJECTIVES: To examine the efficacy and safety of liver grafts from older donors for transplantation. METHODS: A retrospective analysis of data on 310 adults who underwent deceased donor liver transplantation between 2005 and 2015 was conducted. We compared graft and recipient survival, as well as major complications, of transplants performed with grafts from donors younger than 70 years (n=265, control group) and those older than 70 years (n=45, older-donor group), followed by multivariate analysis, to identify risk factors. RESULTS: There was no significant difference between the control and older-donor group at 1, 5, and 10 years of recipient survival (79.5% vs. 73.3%, 68.3% vs. 73.3%, 59.2% vs. 66.7%, respectively) or graft survival (74.0% vs. 71.0%, 62.7% vs. 71.0%, 54.8% vs. 64.5%, respectively). The rate of biliary and vascular complications was similar in both groups. Significant risk factors for graft failure were hepatitis C (hazard ratio [HR] = 1.92, 95% confidence interval [95%CI] 1.16-2.63), older donor age (HR = 1.02, 95%CI 1.007-1.031), and male gender of the recipient (HR = 1.65, 95%CI 1.06-2.55). CONCLUSIONS: Donor age affects liver graft survival. However, grafts from donors older than 70 years may be equally safe if cold ischemia is maintained for less than 8 hours.

The utility of magnetic resonance cholangiopancreatography for the assessment of patients with elevated liver enzyme levels.

PURPOSE: To evaluate the usefulness of magnetic resonance cholangiopancreatography (MRCP) in patients referred for assessment of the cause of elevated liver enzymes. METHODS: The MRCP scans of 170 patients between 2009 and 2011 with elevated liver enzyme levels were blindly and independently rereviewed by two experienced radiologists. Biochemical data were collected from the medical records. Receiver operating characteristics curve analyses were carried out. Cutoff levels of the enzymes to predict pathological MRCPs were determined by the area under the curve (AUC). Sensitivity, specificity, negative predictive value, and positive predictive value were calculated. RESULTS: MRCP scans of 134 patients were identically diagnosed by the two reviewers as nonpathological and 22 as pathological and 14 as uncertain. The agreement between the two reviewers was κ=0.62, indicating good agreement. The percentage of pathological MRCP performed because of elevated liver enzymes was only 14%. For patients with inflammatory bowel disease, the frequency was 36%. On receiver operating characteristics curve analysis, Az values were high for alkaline phosphatase 160 U/l (AUC=0.725) and γ-glutamyl transferase 270 U/l (AUC=0.617). Alkaline phosphatase and γ-glutamyl transferase had a high negative, but low positive predictive value for distinguishing pathological from normal scans. CONCLUSION: MRCP does not contribute markedly toward the evaluation of the cause of elevated enzyme levels, except in patients with inflammatory bowel disease. These findings can provide practical guidelines for evaluation of patients with abnormal liver enzymes and for alleviation of the financial burden from health providers.

The use of epitope arrays in immunodiagnosis of infectious disease: hepatitis C virus, a case study.

Serodiagnosis of infectious disease is often based on the detection of pathogen-specific antibodies in a patient's blood. For this, mixtures of pathogen-related antigens are used as bait to capture corresponding antibodies in solid phase immunoassays such as enzyme immunoassay (EIA). Western blots provide improved diagnostic power as compared with EIA due to the fact that the mixture of markers in the EIA well is resolved and tested as individual antigens on the Western blot. Hence, confirmation of EIA results is accomplished using the antigen arrays of Western blots. Here we took this approach one step further and tested the attributes of using epitope arrays in a diagnostic platform coined "combinatorial diagnostics." As a case in point, we tested a panel of phage-displayed epitope-based markers in the serodiagnosis of hepatitis C virus (HCV). The repertoire of HCV antigens was deconvoluted into panels of distinct linear and conformational epitopes and tested individually by quantitative EIA. Combinatorial diagnostics proved to be effective for the discrimination between positive and negative sera as well as serotyping of HCV.

Extended peginterferon plus ribavirin treatment for 72 weeks versus standard peginterferon plus ribavirin treatment for 48 weeks in chronic hepatitis C genotype 1 infected slow-responder adult patients.

BACKGROUND: The standard length of peginterferon plus ribavirin treatment for chronic hepatitis C virus (HCV) genotype 1 infected patients is 48 weeks. However, the number of patients demonstrating a sustained virological response is not high. In order to improve sustained virological response, extending the length of the treatment period has been suggested. OBJECTIVES: To study the benefits and harms of extended 72-week treatment in comparison with 48-week treatment with peginterferon plus ribavirin in patients with chronic HCV genotype 1 infection who have shown a slow antiviral response. SEARCH METHODS: We searched the Cochrane Hepato-Biliary Group Controlled Trials Register, Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library, MEDLINE, EMBASE, Science Citation Index Expanded, and LILACS until November 2011. We identified further trials by reviewing reference lists and contacting principal authors. SELECTION CRITERIA: Trials were eligible for this review if they included patients infected with hepatitis C virus genotype 1 who had a slow antiviral response, and if those patients were randomised to completing 72 weeks versus 48 weeks of treatment with pegylated interferon and ribavirin. DATA COLLECTION AND ANALYSIS: Two authors independently assessed the trials for risk of bias, and extracted the data. The primary outcomes were overall mortality, liver-related mortality, and liver-related morbidity. We extracted data separately according to two definitions of slow responders: 1) patients with ≥ 2 log viral reduction but still detectable HCV RNA after 12 weeks of treatment and undetectable HCV RNA after 24 weeks of treatment; 2) patients with detectable HCV RNA after four weeks of treatment. We calculated risk ratios from individual trials as well as in the meta-analyses of trials. MAIN RESULTS: We included seven trials with 1369 participants. All trials had high risk of bias. Five trials used our first definition of slow responders, and three other trials (including one that used both definitions) used the second definition. None of the included trials mentioned our primary outcomes. However, regarding the secondary outcomes, extension of the treatment period to 72 weeks increased the sustained virological response according to both definitions (71/217 (32.7%) versus 52/194 (26.8%); risk ratio (RR) 1.43, 95% confidence interval (CI) 1.07 to 1.92, P = 0.02, I(2) = 8%; and 265/499 (53.1%) versus 207/496 (41.7%); RR 1.27, 95% CI 1.07 to 1.50, P = 0.006, I(2) = 38%), with a risk difference of 0.11 and calculated number needed to treat of nine. The end of treatment response was not significantly different between the two treatment groups. The number of participants who relapsed virologically was found to be lower in the groups that had been treated for 72 weeks using both definitions (27/84 (32.1%) versus 46/91 (50.5%); RR 0.59, 95% CI 0.40 to 0.86, P = 0.007, I(2) = 18%, 3 trials; and 85/350 (24.3%) versus 146/353 (41.4%); RR 0.59, 95% CI 0.47, 0.73, P < 0.000001, I(2) = 0%, 3 trials). The length of treatment did not significantly affect the adherence (247/279 (88.5%) versus 252/274 (92.0%); RR 0.95, 95% CI 0.84 to 1.07, P = 0.42, I(2) = 69%, 3 trials). In the single trial that reported adverse events, no significant difference was seen between the two treatment groups. AUTHORS' CONCLUSIONS: This review demonstrates higher a proportion of sustained virological response after extension of treatment from 48 weeks to 72 weeks in HCV genotype 1 infected patients in whom HCV RNA was still detectable but decreased by ≥ 2 log after 12 weeks and became negative after 24 weeks of treatment, and in patients with detectable HCV RNA after four weeks of treatment with peginterferon plus ribavirin. The observed intervention effects can be caused by both systematic error (bias) and random errors (play of chance). There was no reporting on mortality and the reporting of clinical outcomes and adverse events was insufficient. More data are needed in order to recommend or reject the policy of extending the treatment period for slow responders.

Removal of hepatitis C virus-infected cells by a zymogenized bacterial toxin.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and has become a global health threat. No HCV vaccine is currently available and treatment with antiviral therapy is associated with adverse side effects. Moreover, there is no preventive therapy for recurrent hepatitis C post liver transplantation. The NS3 serine protease is necessary for HCV replication and represents a prime target for developing anti HCV therapies. Recently we described a therapeutic approach for eradication of HCV infected cells that is based on protein delivery of two NS3 protease-activatable recombinant toxins we named "zymoxins". These toxins were inactivated by fusion to rationally designed inhibitory peptides via NS3-cleavable linkers. Once delivered to cells where NS3 protease is present, the inhibitory peptide is removed resulting in re-activation of cytotoxic activity. The zymoxins we described suffered from two limitations: they required high levels of protease for activation and had basal activities in the un-activated form that resulted in a narrow potential therapeutic window. Here, we present a solution that overcame the major limitations of the "first generation zymoxins" by converting MazF ribonuclease, the toxic component of the E. coli chromosomal MazEF toxin-antitoxin system, into an NS3-activated zymoxin that is introduced to cells by means of gene delivery. We constructed an expression cassette that encodes for a single polypeptide that incorporates both the toxin and a fragment of its potent natural antidote, MazE, linked via an NS3-cleavable linker. While covalently paired to its inhibitor, the ribonuclease is well tolerated when expressed in naïve, healthy cells. In contrast, activating proteolysis that is induced by even low levels of NS3, results in an eradication of NS3 expressing model cells and HCV infected cells. Zymoxins may thus become a valuable tool in eradicating cells infected by intracellular pathogens that express intracellular proteases.

Vitamin D: an innate antiviral agent suppressing hepatitis C virus in human hepatocytes.

UNLABELLED: Vitamin D supplementation was reported to improve the probability of achieving a sustained virological response when combined with antiviral treatment against hepatitis C virus (HCV). Our aim was to determine the in vitro potential of vitamin D to inhibit HCV infectious virus production and explore the mechanism(s) of inhibition. Here we show that vitamin D(3) remarkably inhibits HCV production in Huh7.5 hepatoma cells. These cells express CYP27B1, the gene encoding for the enzyme responsible for the synthesis of the vitamin D hormonally active metabolite, calcitriol. Treatment with vitamin D(3) resulted in calcitriol production and induction of calcitriol target gene CYP24A1, indicating that these cells contain the full machinery for vitamin D metabolism and activity. Notably, treatment with calcitriol resulted in HCV inhibition. Collectively, these findings suggest that vitamin D(3) has an antiviral activity which is mediated by its active metabolite. This antiviral activity involves the induction of the interferon signaling pathway, resulting in expression of interferon-ß and the interferon-stimulated gene, MxA. Intriguingly, HCV infection increased calcitriol production by inhibiting CYP24A1 induction, the enzyme responsible for the first step in calcitriol catabolism. Importantly, the combination of vitamin D(3) or calcitriol and interferon-α synergistically inhibited viral production. CONCLUSION: This study demonstrates for the first time a direct antiviral effect of vitamin D in an in vitro infectious virus production system. It proposes an interplay between the hepatic vitamin D endocrine system and HCV, suggesting that vitamin D has a role as a natural antiviral mediator. Importantly, our study implies that vitamin D might have an interferon-sparing effect, thus improving antiviral treatment of HCV-infected patients.

The role of oncogenic viruses in the pathogenesis of hepatocellular carcinoma.

HBV and HCV have major roles in hepatocarcinogenesis. More than 500 million people are infected with hepatitis viruses and, therefore, HCC is highly prevalent, especially in those countries endemic for HBV and HCV. Viral and host factors contribute to the development of HCC. The main viral factors include the circulating load of HBV DNA or HCV RNA and specific genotypes. Various mechanisms are involved in the host-viral interactions that lead to HCC development, among which are genetic instability, self-sufficiency in growth signals, insensitivity to antigrowth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasiveness. Prevention of HBV by vaccination, as well as antiviral therapy against HBV and for HCV seem able to inhibit the development of HCC.

Engineered toxins "zymoxins" are activated by the HCV NS3 protease by removal of an inhibitory protein domain.

The synthesis of inactive enzyme precursors, also known as "zymogens," serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated "zymogenized" chimeric toxins (which we denote "zymoxins"). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the "zymoxin" approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.

Inhibition of protease-inhibitor-resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies.

Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons resistant to small-molecule inhibitors of the NS3/4A protease, and replicons derived from different HCV genotypes. The combination of intrabodies and interferon-α had an additive inhibitory effect on RNA replication in the replicon model. Intrabody expression also inhibited production of infectious HCV in a cell culture system. The NS3 protease activity was inhibited by the intrabodies in NS3-expressing cells. In contrast, cell-free synthesis of HCV RNA by preformed replicase complexes was not inhibited by intrabodies, suggesting that the major mode of inhibition of viral replication is inhibition of NS3/4A protease activity and subsequent suppression of viral polyprotein processing.

Lamivudine or adefovir dipivoxil alone or combined with immunoglobulin for preventing hepatitis B recurrence after liver transplantation.

BACKGROUND: Recurrence of hepatitis B virus (HBV) infection in the liver graft is a grave complication following liver transplantation for HBV cirrhosis. Hepatitis B immunoglobulin (HBIg) seems effective in increasing survival after liver transplantation. HBIg and anti-viral drugs are given alone or in combination for its prevention. OBJECTIVES: To assess the benefits and harms of different regimens for preventing HBV reactivation following liver transplantation. SEARCH STRATEGY: We searched The Cochrane Hepato-Biliary Group Controlled Trials Register, The Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library, MEDLINE, EMBASE, and Science Citation Index Expanded until February 2010. We attempted to identify further trials by reviewing the reference lists and contacting the principal authors of identified trials. SELECTION CRITERIA: Randomised clinical trials addressing benefits and harms of lamivudine or adefovir dipivoxil alone or in combination with hepatitis B immunoglobulins (HBIg) for preventing recurrent HBV infection in patients who are liver transplanted due to HBV infection with or without hepatocellular carcinoma. DATA COLLECTION AND ANALYSIS: Two authors independently assessed the trials for risk of bias and extracted data. We contacted study authors whenever information was lacking. We collected information on adverse events. The primary outcomes were all-cause mortality and reappearance of hepatitis B surface antigen in serum after liver transplantation. Relative risks were calculated from individual trials. MAIN RESULTS: Four trials, recruiting 136 participants, were included. Two trials compared lamivudine alone versus HBIg alone. Randomisation was performed one week after transplantation in one of the trials and after six months after transplantation in another; from transplantation until randomisation, HBIg alone was given to all patients in the two trials. A third trial compared combination treatment with lamivudine and HBIg versus lamivudine alone after one month of combination treatment, and a fourth trial compared the combination of lamivudine and HBIg versus a combination of lamivudine and adefovir dipivoxil after at least 12-month of lamivudine and HBIg combination treatment. Statistically significant differences were not detected in any of the comparisons and outcomes. All trials were open-labelled, and none of the trials were adequately powered to show a difference in HBV recurrence. No meta-analyses were performed since the identified trials assessed different comparisons. AUTHORS' CONCLUSIONS: This review could not derive clear evidence from randomised clinical trials for the treatment of patients with chronic HBV following liver transplantation for preventing recurrence of HBV infection. Large randomised clinical trials comparing long-term combination treatment to each of the monotherapy alone, including the newer antiviral drugs, are needed.

Bezafibrate treatment of primary biliary cirrhosis following incomplete response to ursodeoxycholic acid.

BACKGROUND AND GOALS: Ursodeoxycholic acid (UDCA) is the only current pharmacologic treatment for primary biliary cirrhosis (PBC). However, some patients show persistent liver biochemical abnormalities even after 6 to 12 months treatment. Bezafibrate retard is a commonly used medication for hyperlipidemia. In Japanese studies, it was found to lower liver enzyme levels, apparently through its action on multiple drug resistance gene 3, a transport element of the ATP-dependent bile secretion system, and on peroxisome proliferator-activated receptor-alpha. The aim of this study was to evaluate the effect of adding bezafibrate to the treatment regimen in patients with PBC and a partial response to UDCA. STUDY: The study group included 8 White patients, 7 women and 1 man, aged 52 to 76 years with PBC who had been treated at our Liver Institute with UDCA (900 mg/d to 1500 mg/d) for 2 to 11 years (mean, 5.7 y) with only a partial response (19% to 56% reduction in alkaline phosphatase level). Bezafibrate (400 mg/d) was added to UDCA and the patients were followed for 4 to 12 months. RESULTS: Alkaline phosphatase levels (normal range, 35 to 104 U/L) decreased in all patients, from 140 to 360 U/L (mean, 201.2) to 68 to 158 U/L (mean, 98.4), and normalized in 6 patients. In addition, levels of gamma-glutamyl transferase (normal range, 6 to 42 U/L) decreased from 70 to 192 U/L (mean, 130) to 41 to 122 U/L (mean, 71.8). These findings were maintained throughout follow-up. CONCLUSIONS: Combination therapy with bezafibrate and UDCA improves the biochemical profile of patients with PBC who respond only partially to UDCA. A larger controlled study is needed to evaluate the clinical implications of these findings.

A comparison of transjugular and plugged-percutaneous liver biopsy in patients with contraindications to ordinary percutaneous liver biopsy and an "in-house" protocol for selecting the procedure of choice.

The purpose of this study was to evaluate the effectiveness and safety of transjugular liver biopsy (TJLB) and plugged-percutaneous liver biopsy (PB) in consecutive patients with severe liver disease associated with impaired coagulation, ascites, or both and to verify the in-house protocol used to select the appropriate procedure. In 2000-2006, 329 patients (208 male [62.8%] and 121 female [37.2%]), aged 1 month to 81 years (mean, 46.8 years), underwent 150 TJLBs (39.1%) or 233 PBs (60.9%) procedures at a major tertiary center, as determined by an in-house protocol. The groups were compared for specimen characteristics, technical success, and complications. Technical success rates were 97.4% for TJLB (146/150) and 99.1% for PB (231/233). TJLB was associated with a lower average core length (1.29 vs. 1.43 cm) and lower average number of specimens obtained (2.44 vs. 2.8), but both methods yielded sufficient tissue for a definitive diagnosis. There were no major complications in either group. TJLB and PB can be safely and effectively performed for the diagnosis of hepatic disease in patients with contraindications for standard percutaneous liver biopsy. When both are technically available, we suggest PB as the procedure of choice, especially in transplanted livers.

Expression of liver-specific markers in naïve adipose-derived mesenchymal stem cells.

BACKGROUND: Increasing evidence suggests that adipose tissue contains mesenchymal stem cells (MSC) that possess the ability to transdifferentiate into other cell types including hepatocytes, similar to bone marrow-derived stem cells. The existence of precommitted cells in the MSC population may explain transdifferentiation. AIMS: Our aim was to identify a population of putative hepatocyte-like precursor cells in human adipose tissue. METHODS: We analysed the 'basal' hepatic potential of undifferentiated, naïve human adipose-derived mesenchymal stem cells (hADMSC). hADMSC were isolated from human adipose tissue and characterized for cell surface markers and for liver-specific gene expression. RESULTS: The isolated undifferentiated naïve hADMSCs expressed MSC surface markers. They also expressed alpha-fetoprotein, CK18, CK19 and HNF4, which are known as early liver expressing genes. Interestingly, the undifferentiated naïve hADMSC were also positive for albumin, G-6-P and alpha-1-antitrypsin (AAT), which are all known to be predominantly expressed in adult liver cells. These cells acquired a hepatocyte-specific phenotype and function upon treatment with a differentiation medium, resulting in the upregulation of albumin, G-6-P and AAT. Moreover, urea production, glycogen storage ability and cellular uptake of indocyanine green, which were absent in the basal state, were evident in the treated cells. CONCLUSIONS: Our findings suggest the presence of cells with hepatocyte-like properties that are isolated from human adipose tissue and that can readily acquire hepatocyte-like functions. Adipose tissue could thus be an exciting alternative means for repopulating the liver after various injuries, and might serve as a source for the transplantation of liver cells.

Hepatitis C virus targets over-expression of arginase I in hepatocarcinogenesis.

Hepatitis C virus (HCV) infection is often associated with chronic liver disease, which is a major risk factor for the development of hepatocellular carcinoma (HCC). To study the HCV host-cell relationship on the molecular level, HepG2 and Huh7 cells were stably transfected with an infectious cDNA clone of HCV or with empty vector. Evidence for HCV replication was obtained in both culture systems. HCV also stimulated growth in vitro. To identify genes whose altered expression by HCV are important to the pathogenesis of infection, RNAs were isolated from HepG2-HCV and HepG2-vector cells and subjected to microarray analysis. The results showed that arginase 1 mRNA and protein were elevated about threefold in HCV positive compared with negative cells (p < 0.01). Arginase 1 expression was elevated in more than 75% of HCV infected liver samples compared with paired HCC from the same patients (>33% positive) and to uninfected liver tissues (0% positive). Arginase 1 specific siRNA inhibited the ability of HCV to stimulate hepatocellular growth in culture by >70%, suggesting that the metabolism of arginine to ornithine may contribute to HCV mediated stimulation of hepatocellular growth. Introduction of arginase specific siRNA also resulted in increased nitric oxide synthase (iNOS) (>1.2-fold), nitric oxide (NO) production (>3-fold) and increased cell death (>2.5-fold) in HCV positive compared with negative cells, suggesting that these molecules potentially contribute to hepatocellular damage. Hence, an important part of the mechanism whereby HCV regulates hepatocellular growth and survival may be through altering arginine metabolism.

Postshunt hepatic encephalopathy in liver transplant recipients.

BACKGROUND: Preexisting spontaneous portosystemic shunts increase the risk of posttransplantation portal vein thrombosis. Portosystemic shunts may also be placed surgically to manage posttransplant portal vein stenosis/thrombosis. Both types may be complicated by hepatic encephalopathy. METHODS: The database of a major tertiary medical center from 1999 to 2006 was searched for liver transplant recipients with hepatic encephalopathy and stable liver function. The medical and imaging files were reviewed for risk factors, management, and outcome. RESULTS: Of the 244 patients who underwent liver transplantation during the study period, four (1.6%) met the inclusion criteria. Median age at transplantation was 49 years (range 39-54); median time to the first episode of hepatic encephalopathy after transplantation was 23 months (range 2-40). In two patients, a distal splenorenal shunt placed at 1 and 7 months after transplantation to treat portal vein thrombosis led to hepatic encephalopathy at 1 and 33 months later. Both responded to medical therapy. The other two patients had spontaneous splenorenal shunts, and hepatic encephalopathy appeared 33 months and 12 months after transplantation. Treatment consisted of transhepatic percutaneous portal vein dilatation with stent insertion in the first patient and interposition of a venous graft between the superior mesenteric and left intrahepatic portal veins to reroute splanchnic flow in the second patient. CONCLUSIONS: Portosystemic shunts in liver transplant recipients with stable graft function may be associated with hepatic encephalopathy. Pretransplant assessment to detect unknown spontaneous shunts is important. Restoration of portal flow is the preferred procedure in this setting.