Current Therapy of Chronic Viral Hepatitis B, C and D.

The majority of chronic viral hepatitis cases are induced via infection with the hepatitis B virus (HBV), hepatitis C virus (HCV), or hepatitis D virus (HDV). These patients are at increased risk for progressive liver disease leading to cirrhosis as well as hepatocellular carcinoma (HCC). HBV infection is well controlled by the currently available nucleosides as well as nucleotides, and the development of cirrhosis can be prevented. Additionally, it has been shown that HBV-induced liver fibrosis can regress during successful antiviral treatment; however, a "functional cure", i.e., loss of HBsAg, is a rare event when these drugs are used. Therefore, novel therapeutic strategies are aiming at the selective suppression of HBsAg levels in combination with immunostimulation. The development of directly acting antivirals (DAAs) has revolutionized HCV therapy, as almost all patients can be cured via this treatment. Additionally, DAA therapy has few, if any, side effects, and is generally well tolerated by patients. HDV remains the most challenging type of chronic viral hepatitis. Although novel therapeutic options have recently been approved, response rates are still less favorable compared to HBV and HCV. This review discusses current and future options for the treatment of chronic HBV, HCV, and HDV infection.

[Cryoglobulinemic vasculitis]. / Kryoglobulinämische Vaskulitis.

Cryoglobulinemic vasculitis (CV) is a rare immune complex disease of small vessels (capillaries, venules or arterioles) with detection of cryoglobulins (CG). These are serum proteins that precipitate at temperatures below the normal body temperature. The laboratory diagnostics are logistically challenging because the temperature of the blood sample must be maintained continuously at 37 °C until arrival in the laboratory to prevent early precipitation of the proteins with adsorption to corpuscular blood components. Cryoglobulins can be divided into three classes (types I-III), with each class associated with specific underlying diseases and symptom complexes. Cryoglobulinemia can be caused by hematological, virological or autoimmune diseases and mixed forms also occur. The most common cause to date is a hepatitis C infection. Treatment of the underlying disease is obligatory, with antiviral treatment of hepatitis C offering the rare possibility of causal treatment. Depending on the severity of cryoglobulinemia, immunosuppressive therapy is indicated to prevent permanent damage caused by the inflammation.

Tissue-based quantitative proteome analysis of human hepatocellular carcinoma using tandem mass tags.

CONTEXT AND OBJECTIVE: Human hepatocellular carcinoma (HCC) is a severe malignant disease, and accurate and reliable diagnostic markers are still needed. This study was aimed for the discovery of novel marker candidates by quantitative proteomics. METHODS AND RESULTS: Proteomic differences between HCC and nontumorous liver tissue were studied by mass spectrometry. Among several significantly upregulated proteins, translocator protein 18 (TSPO) and Ras-related protein Rab-1A (RAB1A) were selected for verification by immunohistochemistry in an independent cohort. For RAB1A, a high accuracy for the discrimination of HCC and nontumorous liver tissue was observed. CONCLUSION: RAB1A was verified to be a potent biomarker candidate for HCC.

One Sample, One Shot - Evaluation of sample preparation protocols for the mass spectrometric proteome analysis of human bile fluid without extensive fractionation.

The proteome analysis of bile fluid represents a promising strategy to identify biomarker candidates for various diseases of the hepatobiliary system. However, to obtain substantive results in biomarker discovery studies large patient cohorts necessarily need to be analyzed. Consequently, this would lead to an unmanageable number of samples to be analyzed if sample preparation protocols with extensive fractionation methods are applied. Hence, the performance of simple workflows allowing for "one sample, one shot" experiments have been evaluated in this study. In detail, sixteen different protocols implying modifications at the stages of desalting, delipidation, deglycosylation and tryptic digestion have been examined. Each method has been individually evaluated regarding various performance criteria and comparative analyses have been conducted to uncover possible complementarities. Here, the best performance in terms of proteome coverage has been assessed for a combination of acetone precipitation with in-gel digestion. Finally, a mapping of all obtained protein identifications with putative biomarkers for hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) revealed several proteins easily detectable in bile fluid. These results can build the basis for future studies with large and well-defined patient cohorts in a more disease-related context. BIOLOGICAL SIGNIFICANCE: Human bile fluid is a proximal body fluid and supposed to be a potential source of disease markers. However, due to its biochemical composition, the proteome analysis of bile fluid still represents a challenging task and is therefore mostly conducted using extensive fractionation procedures. This in turn leads to a high number of mass spectrometric measurements for one biological sample. Considering the fact that in order to overcome the biological variability a high number of biological samples needs to be analyzed in biomarker discovery studies, this leads to the dilemma of an unmanageable number of necessary MS-based analyses. Hence, easy sample preparation protocols are demanded representing a compromise between proteome coverage and simplicity. In the presented study, such protocols have been evaluated regarding various technical criteria (e.g. identification rates, missed cleavages, chromatographic separation) uncovering the strengths and weaknesses of various methods. Furthermore, a cumulative bile proteome list has been generated that extends the current bile proteome catalog by 248 proteins. Finally, a mapping with putative biomarkers for hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) derived from tissue-based studies, revealed several of these proteins being easily and reproducibly detectable in human bile. Therefore, the presented technical work represents a solid base for future disease-related studies.

Effectiveness and Safety of Tenofovir Disoproxil Fumarate in Chronic Hepatitis B: A 3-Year Prospective Field Practice Study in Germany.

BACKGROUND AND AIMS: Multiple clinical trials have demonstrated the efficacy and safety of tenofovir disoproxil fumarate (TDF) in chronic hepatitis B (CHB). However, long-term efficacy and safety data for TDF in real-life clinical practice are limited. METHODS: Prospective German field practice study in CHB-mono-infected patients. Patients were TDF-naïve but could have been treated previously with other HBV antivirals. RESULTS: Efficacy analysis included 400 patients; 301 (75 %) completed 36 months of TDF treatment. Both treatment-naïve and treatment-experienced patients showed a rapid decline in HBV DNA within 3 months of TDF initiation. After 36 months, HBV DNA < 69 IU/mL was achieved by 91 % of treatment-naïve patients (90 and 92 % in hepatitis B "e" antigen [HBeAg]-positive and [HBeAg]-negative, respectively) and 96 % of treatment-experienced patients (93 and 97 %, respectively). Three patients experienced virologic breakthrough, all with reported non-compliance. Overall, 5.7 % HBeAg-positive and 2.2 % HBeAg-negative patients lost hepatitis B surface antigen. Safety data were consistent with the known TDF safety profile; the most commonly reported adverse events possibly related to TDF were fatigue (2.0 %) and headache (2.0 %). Few patients (1.3 %) experienced renal-related adverse reactions. Creatinine clearance remained relatively stable over time; patients responded favorably where TDF was dose adjusted per label for decreased creatinine clearance. CONCLUSIONS: TDF showed a favorable tolerability profile and induced rapid and sustained suppression of HBV DNA in patients with CHB treated for up to 3 years in routine clinical practice, irrespective of treatment history. Efficacy and safety in this heterogeneous patient population were consistent with data from clinical trials.

Quantitative Tissue Proteomics Analysis Reveals Versican as Potential Biomarker for Early-Stage Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most aggressive tumors, and the treatment outcome of this disease is improved when the cancer is diagnosed at an early stage. This requires biomarkers allowing an accurate and early tumor diagnosis. To identify potential markers for such applications, we analyzed a patient cohort consisting of 50 patients (50 HCC and 50 adjacent nontumorous tissue samples as controls) using two independent proteomics approaches. We performed label-free discovery analysis on 19 HCC and corresponding tissue samples. The data were analyzed considering events known to take place in early events of HCC development, such as abnormal regulation of Wnt/b-catenin and activation of receptor tyrosine kinases (RTKs). 31 proteins were selected for verification experiments. For this analysis, the second set of the patient cohort (31 HCC and corresponding tissue samples) was analyzed using selected (multiple) reaction monitoring (SRM/MRM). We present the overexpression of ATP-dependent RNA helicase (DDX39), Fibulin-5 (FBLN5), myristoylated alanine-rich C-kinase substrate (MARCKS), and Serpin H1 (SERPINH1) in HCC for the first time. We demonstrate Versican core protein (VCAN) to be significantly associated with well differentiated and low-stage HCC. We revealed for the first time the evidence of VCAN as a potential biomarker for early-HCC diagnosis.

Analysis of disease-associated protein expression using quantitative proteomics­fibulin-5 is expressed in association with hepatic fibrosis.

Hepatic fibrosis and cirrhosis are major health problems worldwide. Until now, highly invasive biopsy remains the diagnostic gold standard despite many disadvantages. To develop noninvasive diagnostic assays for the assessment of liver fibrosis, it is urgently necessary to identify molecules that are robustly expressed in association with the disease. We analyzed biopsied tissue samples from 95 patients with HBV/HCV-associated hepatic fibrosis using three different quantification methods. We performed a label-free proteomics discovery study to identify novel disease-associated proteins using a subset of the cohort (n = 27). Subsequently, gene expression data from all available clinical samples were analyzed (n = 77). Finally, we performed a targeted proteomics approach, multiple reaction monitoring (MRM), to verify the disease-associated expression in samples independent from the discovery approach (n = 68). We identified fibulin-5 (FBLN5) as a novel protein expressed in relation to hepatic fibrosis. Furthermore, we confirmed the altered expression of microfibril-associated glycoprotein 4 (MFAP4), lumican (LUM), and collagen alpha-1(XIV) chain (COL14A1) in association to hepatic fibrosis. To our knowledge, no tissue-based quantitative proteomics study for hepatic fibrosis has been performed using a cohort of comparable size. By this means, we add substantial evidence for the disease-related expression of the proteins examined in this study.

A structured proteomic approach identifies 14-3-3Sigma as a novel and reliable protein biomarker in panel based differential diagnostics of liver tumors.

Hepatocellular carcinoma (HCC) is a major lethal cancer worldwide. Despite sophisticated diagnostic algorithms, the differential diagnosis of small liver nodules still is difficult. While imaging techniques have advanced, adjuvant protein-biomarkers as glypican3 (GPC3), glutamine-synthetase (GS) and heat-shock protein 70 (HSP70) have enhanced diagnostic accuracy. The aim was to further detect useful protein-biomarkers of HCC with a structured systematic approach using differential proteome techniques, bring the results to practical application and compare the diagnostic accuracy of the candidates with the established biomarkers. After label-free and gel-based proteomics (n=18 HCC/corresponding non-tumorous liver tissue (NTLT)) biomarker candidates were tested for diagnostic accuracy in immunohistochemical analyses (n=14 HCC/NTLT). Suitable candidates were further tested for consistency in comparison to known protein-biomarkers in HCC (n=78), hepatocellular adenoma (n=25; HCA), focal nodular hyperplasia (n=28; FNH) and cirrhosis (n=28). Of all protein-biomarkers, 14-3-3Sigma (14-3-3S) exhibited the most pronounced up-regulation (58.8×) in proteomics and superior diagnostic accuracy (73.0%) in the differentiation of HCC from non-tumorous hepatocytes also compared to established biomarkers as GPC3 (64.7%) and GS (45.4%). 14-3-3S was part of the best diagnostic three-biomarker panel (GPC3, HSP70, 14-3-3S) for the differentiation of HCC and HCA which is of most important significance. Exclusion of GS and inclusion of 14-3-3S in the panel (>1 marker positive) resulted in a profound increase in specificity (+44.0%) and accuracy (+11.0%) while sensitivity remained stable (96.0%). 14-3-3S is an interesting protein biomarker with the potential to further improve the accuracy of differential diagnostic process of hepatocellular tumors. This article is part of a Special Issue entitled: Medical Proteomics.

Identification of novel biomarker candidates for the immunohistochemical diagnosis of cholangiocellular carcinoma.

The aim of this study was the identification of novel biomarker candidates for the diagnosis of cholangiocellular carcinoma (CCC) and its immunohistochemical differentiation from benign liver and bile duct cells. CCC is a primary cancer that arises from the epithelial cells of bile ducts and is characterized by high mortality rates due to its late clinical presentation and limited treatment options. Tumorous tissue and adjacent non-tumorous liver tissue from eight CCC patients were analyzed by means of two-dimensional differential in-gel electrophoresis and mass-spectrometry-based label-free proteomics. After data analysis and statistical evaluation of the proteins found to be differentially regulated between the two experimental groups (fold change ≥ 1.5; p value ≤ 0.05), 14 candidate proteins were chosen for determination of the cell-type-specific expression profile via immunohistochemistry in a cohort of 14 patients. This confirmed the significant up-regulation of serpin H1, 14-3-3 protein sigma, and stress-induced phosphoprotein 1 in tumorous cholangiocytes relative to normal hepatocytes and non-tumorous cholangiocytes, whereas some proteins were detectable specifically in hepatocytes. Because stress-induced phosphoprotein 1 exhibited both sensitivity and specificity of 100%, an immunohistochemical verification examining tissue sections of 60 CCC patients was performed. This resulted in a specificity of 98% and a sensitivity of 64%. We therefore conclude that this protein should be considered as a potential diagnostic biomarker for CCC in an immunohistochemical application, possibly in combination with other candidates from this study in the form of a biomarker panel. This could improve the differential diagnosis of CCC and benign bile duct diseases, as well as metastatic malignancies in the liver.

KIR2DL3⁺NKG2A⁻ natural killer cells are associated with protection from productive hepatitis C virus infection in people who inject drugs.

BACKGROUND & AIMS: Despite continuous high-risk behavior, a subgroup among people who inject drugs (PWID) remains seronegative for hepatitis C virus (HCV) suggesting that a state of "natural resistance" to HCV Infection may exist. Homozygosity for KIR2DL3 and its ligand HLA-C1 group alleles has been associated with control of HCV infection, however, the mechanism mediating this protective effect remained unclear. METHODS: Peripheral NK cells from PWID (n=104) were phenotypically and functionally characterized by multicolor flow cytometry. Expression levels of the NK cell receptor ligands were analysed in liver biopsies and primary human hepatocytes. RESULTS: HCV seronegative PWID (n=34) had increased levels of KIR2DL3(+)NKG2A(-) NK cells compared to healthy controls (n=10; p<0.001) and PWID with chronic (n=38; p<0.001) or resolved infection (n=37; p<0.001). There was an inverse correlation between the frequency of KIR2DL3(+) and NKG2A(+) NK cells (r=-0.53; p<0.0001). Importantly, expression of HLA-E, the ligand for NKG2A, was significantly upregulated in liver biopsies of HCV infected patients (n=51) compared to HBV infected patients (n=22; p<0.01) and correlated with HCV viral load (r=0.32; p<0.0029). In functional analyses KIR2DL3(-)NKG2A(+) NK cells but not KIR2DL3(+)NKG2A(-) NK cells were significantly inhibited by HLA-E ligation. Accordingly, interferon gamma secretion of NK cells from PWID with chronic infection but not from HCV seronegative PWID was significantly suppressed in the presence of HLA-E. CONCLUSIONS: KIR2DL3(+)NKG2A(-) NK cells are not sensitive to HLA-E-mediated inhibition and may thereby control early HCV infection prior to seroconversion and result in an apparent state of "natural resistance" to HCV in PWID.

Mini-laparoscopy is superior in detecting liver cirrhosis and metastases in liver cancer: an over 10-year experience in 1,788 cases.

BACKGROUND/AIMS: Mini-laparoscopy has, since its first description in 1998, proven to be a valuable diagnostic method in liver diseases. We re-evaluated the significance of mini-laparoscopy for diagnosis and staging of liver disease and primary liver and bile duct cancer. PATIENTS AND METHODS: 1,788 consecutive patients who received a diagnostic mini-laparoscopy between 10/1998 and 06/2011 were included in this retrospective cohort study. RESULTS: In chronic liver disease, cirrhosis was detected by mini-laparoscopy in 27% of cases. A comparison of microscopic versus macroscopic diagnosis of cirrhosis revealed a sampling error for histology alone of 21%. Macroscopic inspection of the liver surface contributed to the diagnosis of unknown liver diseases in approximately 38%. In patients with bile duct or liver cancer, mini-laparoscopy led to upstaging of the disease in 33 and 23%, respectively. Major complications (bowel perforation and delayed bleeding) occurred in 0.39% of cases. CONCLUSIONS: Mini-laparoscopy is a valuable procedure with significant diagnostic impact in known and unknown inflammatory and malignant liver diseases. It can be safely performed even in patients with acute liver failure and severe coagulopathy and the diagnostic value does not differ from diagnostic laparoscopy performed with standard instruments.

Analysis of U2 small nuclear RNA fragments in the bile differentiates cholangiocarcinoma from primary sclerosing cholangitis and other benign biliary disorders.

BACKGROUND AND OBJECTIVES: Up to now the diagnosis of early stage cholangiocarcinoma (CC) has remained difficult, with low sensitivities reported for current diagnostic methods. Based on recent promising findings about circulating U2 small nuclear RNA fragments (RNU2-1f) as novel blood-based biomarkers for pancreatic and colorectal adenocarcinoma, we studied the utility of RNU2-1f as a diagnostic marker of CC in bile fluid. METHODS: Bile fluid was collected from patients with CC (n = 12), controls (patients with choledocholithiasis) (n = 11) and with primary sclerosing cholangitis (PSC; n = 11). RNU2-1f levels were measured by real-time polymerase chain reaction normalized to cel-54. RESULTS: Measurement of RNU2-1f levels in bile fluids enabled the differentiation of patients with CC from controls in all cases. Furthermore, RNU2-1f levels in bile fluids of patients with CC were significantly higher than in patients with PSC, resulting in a receiver-operating characteristic curve area of 0.856, with sensitivity of 67 % and specificity of 91 %. CONCLUSIONS: Our data suggest that the measurement of RNU2-1 fragments detected in the bile fluid can be used as a diagnostic marker for CC and should be included in future prospective diagnostic studies for this disease entity.

A practical data processing workflow for multi-OMICS projects.

Multi-OMICS approaches aim on the integration of quantitative data obtained for different biological molecules in order to understand their interrelation and the functioning of larger systems. This paper deals with several data integration and data processing issues that frequently occur within this context. To this end, the data processing workflow within the PROFILE project is presented, a multi-OMICS project that aims on identification of novel biomarkers and the development of new therapeutic targets for seven important liver diseases. Furthermore, a software called CrossPlatformCommander is sketched, which facilitates several steps of the proposed workflow in a semi-automatic manner. Application of the software is presented for the detection of novel biomarkers, their ranking and annotation with existing knowledge using the example of corresponding Transcriptomics and Proteomics data sets obtained from patients suffering from hepatocellular carcinoma. Additionally, a linear regression analysis of Transcriptomics vs. Proteomics data is presented and its performance assessed. It was shown, that for capturing profound relations between Transcriptomics and Proteomics data, a simple linear regression analysis is not sufficient and implementation and evaluation of alternative statistical approaches are needed. Additionally, the integration of multivariate variable selection and classification approaches is intended for further development of the software. Although this paper focuses only on the combination of data obtained from quantitative Proteomics and Transcriptomics experiments, several approaches and data integration steps are also applicable for other OMICS technologies. Keeping specific restrictions in mind the suggested workflow (or at least parts of it) may be used as a template for similar projects that make use of different high throughput techniques. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.

Proteomic differences between hepatocellular carcinoma and nontumorous liver tissue investigated by a combined gel-based and label-free quantitative proteomics study.

Proteomics-based clinical studies have been shown to be promising strategies for the discovery of novel biomarkers of a particular disease. Here, we present a study of hepatocellular carcinoma (HCC) that combines complementary two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography (LC-MS)-based approaches of quantitative proteomics. In our proteomic experiments, we analyzed a set of 14 samples (7 × HCC versus 7 × nontumorous liver tissue) with both techniques. Thereby we identified 573 proteins that were differentially expressed between the experimental groups. Among these, only 51 differentially expressed proteins were identified irrespective of the applied approach. Using Western blotting and immunohistochemical analysis the regulation patterns of six selected proteins from the study overlap (inorganic pyrophosphatase 1 (PPA1), tumor necrosis factor type 1 receptor-associated protein 1 (TRAP1), betaine-homocysteine S-methyltransferase 1 (BHMT)) were successfully verified within the same sample set. In addition, the up-regulations of selected proteins from the complements of both approaches (major vault protein (MVP), gelsolin (GSN), chloride intracellular channel protein 1 (CLIC1)) were also reproducible. Within a second independent verification set (n = 33) the altered protein expression levels of major vault protein and betaine-homocysteine S-methyltransferase were further confirmed by Western blots quantitatively analyzed via densitometry. For the other candidates slight but nonsignificant trends were detectable in this independent cohort. Based on these results we assume that major vault protein and betaine-homocysteine S-methyltransferase have the potential to act as diagnostic HCC biomarker candidates that are worth to be followed in further validation studies.

No beneficial effect of all-trans retinoic acid in previous non-responder patients with chronic hepatitis C: the ATRACTION study, a phase II randomised trial.

BACKGROUND: Preclinical data suggested all-trans retinoic acid (tretinoin) as a potential antiviral agent against chronic hepatitis C infection. AIMS: To assess efficacy, safety, and tolerability of tretinoin in combination with peg-interferon and ribavirin in genotype-1 infected patients with prior non-response. METHOD: We performed an open-label multicentre clinical trial. Patients were randomised to either receive additional tretinoin (45mg/m(2)/day) for 12 weeks (arm A), or peg-interferon and ribavirin alone (arm B). Primary endpoint was the slope of the third phase of viral decline (Mδ) as determined in an established kinetic model known to correlate with treatment outcome. Secondary endpoints were additional kinetic parameters, viral response rates, safety, and tolerability. RESULTS: 27 patients in arm A and 30 patients in arm B were treated per protocol until week 12. Viral kinetic parameters did not differ. Rates of early virological response (>2log10 drop at week 12) were similar (10/27 versus 11/30 patients). In arm A, patients experienced a higher rate and intensity of adverse events, most commonly skin and mucosal dryness, and headache. CONCLUSION: Addition of tretinoin was safe and acceptably well tolerated. However, it did not influence viral kinetics and thus cannot be further considered as a treatment option.

Selective internal radiotherapy (SIRT) of hepatic tumors: how to deal with the cystic artery.

PURPOSE: Selective internal radiotherapy (SIRT) with the beta emitter yttrium-90 (Y90) is a rapidly developing therapy option for unresectable liver malignancies. Nontarget irradiation of the gallbladder is a complication of SIRT. Thus, we aimed to assess different strategies to avoid infusion of Y90 into the cystic artery (CA). METHODS: After hepatic digital subtraction angiography and administration of technetium-99m-labeled human serum albumin ((99)mTc-HSA), 295 patients with primary or secondary liver tumors underwent single-photon emission computed tomography/computed tomography (SPECT/CT). Different measures were taken before repeated Y90 mapping and SIRT to avoid unintended influx into the CA where necessary. Clinical symptoms, including pain, fever, or a positive Murphy sign, were assessed during patient follow-up. RESULTS: A significant (99)mTc-HSA accumulation in the gallbladder wall (higher (99)mTc-HSA uptake than in normal liver tissue) was seen in 20 patients. The following measures were taken to avoid unintended influx into the CA: temporary/permanent occlusion of the CA with gelfoam (n = 5)/microcoil (n = 1), induction of vasospasm with a microwire (n = 4), or altering catheter position (n = 10). Clinical signs of cholecystitis were observed in only one patient after temporary CA occlusion with gelfoam and were successfully treated by antibiotics. Cholecystectomy was not required for any patient. CONCLUSION: It is important to identify possible nontarget irradiation of the gallbladder. The risk for radiation-induced cholecystitis can be easily minimized by temporary or permanent CA embolization, vasospasm induction, or altering the catheter position.

Selective internal radiation therapy of hepatocellular carcinoma: potential hepatopulmonary shunt reduction after sorafenib administration.

Sorafenib, a protein kinase inhibitor, is a systemic drug that has been licensed for the treatment of hepatocellular carcinoma (HCC). This retrospective study assessed whether the administration of sorafenib can result in a reduction of the hepatopulmonary shunt (HPS) before selective internal radiation therapy (SIRT). After exclusion from SIRT because of high HPS, computed tomography scan indicated a shunt reduction in seven patients with HCC receiving sorafenib. Repeated measurements revealed HPS reduction (from 26.5% to 7.5% on average), and subsequent SIRT became possible. In conclusion, sorafenib may reduce HPS in patients with advanced HCC in some cases.

[Toll-like receptor dependent innate immune responses by primary mouse hepatocytes and its control of HBV replication].

OBJECTIVE: This report aims to investigate the Toll-like receptor (TLR) signaling pathways and induced antiviral activity in hepatocytes. METHODS: We isolated primary hepatocytes from wild-type C57BL/6 mice and examined the expression of TLR by realtime RT-PCR. Hepatocytes were stimulated with TLR 1-9 agonists and the supernatants were harvested. The secretion of cytokines were tested by ELISA. The antiviral effectors in supernatants were assayed via virus protection assay (in EMCV system) and the control of HBV replication were assessed via Southern blotting (in HBV system). RESULTS: We demonstrated that hepatocytes expressed TLR1-9. In accordance with these TLR expression profiles, hepatocytes responded to all TLR ligands by producing inflammatory cytokines (TNF-α or IL-6), to TLR -1,-3,-7 and -9 ligands by producing type I IFN (IFN-α or IFN-ß). Only TLR 3 and TLR 7 agonists could stimulate the production of high amounts of antiviral mediators by hepatocytes in virus protection assay. By contrast, supernatants from TLR1, -3 and -4 directly stimulated hepatocytes and TLR 3, -7 and -9 transfected hepatocytes were able to potently suppress HBV replication. CONCLUSION: Primary hepatocytes display a unique TLR signaling pathway and can control HBV replication after stimulation by TLR agonists in mice. It may be helpful for the development of TLR-based therapeutic approaches against hepatotropic virus.

Non-alcoholic fatty liver disease progresses to hepatocellular carcinoma in the absence of apparent cirrhosis.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in developed countries, and accumulating evidence suggests it as the hepatic manifestation of the metabolic syndrome (MS). Although the published prevalence of hepatocellular carcinoma (HCC) is low in NAFLD/NASH patients, most of these data have been derived from areas endemic for viral hepatitis. We recruited 162 adults with HCC between February 2007 and March 2008, investigated the underlying etiologies and determined the prevalence of the MS and related features within each group. Patients with NAFLD/NASH-associated HCC exhibited a higher prevalence of metabolic features (Type 2 diabetes mellitus, hypertension, dyslipidemia, coronary artery disease) compared to non-NAFLD/NASH-HCC. Intriguingly, a significant number (41.7%; p < 0.005) of individuals with NAFLD/NASH-HCC had no evidence of cirrhosis. Patients with alcohol-induced liver disease also displayed many features (14/19, 73.7%) of the MS, although, in contrast to NAFLD/NASH-HCC, alcohol-associated HCC was highly associated with cirrhosis (95.0%; p = 0.064). NAFLD/NASH as the hepatic entity of the MS may itself pose a risk factor for HCC, even in the absence of cirrhosis. The MS may also promote development of HCC among those with alcoholic liver disease. Increased awareness of liver manifestations in the MS may instigate early interventions against developing HCC.