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Hepatitis B virus (HBV) infection is a prominent cause of hepatocellular carcinoma (HCC) but the underlying molecular mechanisms are complex and multiple pathways have been proposed such as the activation of the Wnt-/β-catenin-signalling and dysregulation of E-cadherin/β-catenin adherens junctions. This study aimed to identify mechanisms of how HBV infection and replication as well as HBV X protein (HBx) gene expression in the context of an HBV genome influence Wnt-/β-catenin-signalling and formation of adherens junctions and to which extent HBx contributes to this. Regulation of E-cadherin/β-catenin junctions and β-catenin-signalling as well as the role of HBx were investigated using constructs transiently or stably inducing replication of HBV+/-HBx in hepatoma cell lines. In addition, HCC and adjacent non-tumorous tissue samples from HBV-infected HCC patients and drug interference in HBV-infected cells were studied. Although HBV did not alter overall expression levels of E-cadherin or β-catenin, it diminished their cell surface localization resulting in nuclear translocation of β-catenin and activation of its target genes. In addition, HBV gene expression increased the amount of phosphorylated c-Src kinase. Treatment with Src kinase inhibitor Dasatinib reduced HBV replication, prevented adherens junction disassembly and reduced β-catenin-signalling, while Sorafenib only did so in cells with mutated β-catenin. Interestingly, none of the HBV induced alterations required HBx. Thus, HBV stimulated β-catenin-signalling and induced disassembly of adherens junctions independently of HBx through Src kinase activation. These pathways may contribute to hepatocellular carcinogenesis and seem to be more efficiently inhibited by Dasatinib than by Sorafenib.

Background The 5′-noncoding region (5′NCR) of the HCV-genome comprises an internal ribosome entry site essential for HCV-translation/replication. Phosphorothioate oligodeoxynucleotides (tS-ODN) complementary to this region can inhibit HCV-translation in vitro. In this study, bile acid conjugated tS-ODN were generated to increase cell-selective inhibition of 5′NCR-dependent HCV-translation. Methods Different bile acid conjugated tS-ODN complementary to the HCV5′NCR were selected for their inhibitory potential in an in vitro transcription/translation assay. To analyze OATP (organic anion transporting polypeptides)-selective uptake of bile acid conjugated ODN, different hepatoma cells were stably transfected with the OATP1B1-transporter and primary human hepatocytes were used. An adenovirus encoding the HCV5′NCR fused to the luciferase gene (Ad-GFP-NCRluc) was generated to quantify 5′NCR-dependent HCV gene expression in OATP-overexpressing hepatoma cells and in vivo. Results A 17mer phosphorothioate modified ODN (tS-ODN4_13) complementary to HCV5′NCR was able to inhibit 5′NCR-dependent HCV-translation in an in vitro transcription/translation test system by more than 90% and it was also effective in Huh7-cells containing the HCV subgenomic replicon. Conjugation to taurocholate (tS-ODN4_13T) significantly increased selective ODN uptake by primary human hepatocytes and by OATP1B1-expressing HepG2-cells compared to parental HepG2-cells. Correspondingly, tS-ODN4_13T significantly inhibited HCV gene expression in liver-derived OATP1B1-expressing HepG2- or CCL13-cells up to 70% compared to unconjugated tS-ODN and compared to mismatch taurocholate coupled tS-ODN. In vivo, tS-ODN4_13T showed also a trend to block 5′NCR-dependent HCV gene expression. Conclusions The tested taurocholate conjugated 17mer antisense ODN complementary to HCV5′NCV showed an increased and selective uptake by hepatocytes and liver-derived cells through OATP-mediated transport resulting in enhanced specific inhibition of HCV gene expression in vitro and in vivo. Thus, this novel approach may represent a promising strategy to improve antisense approaches with ODN in the control of hepatitis C infection.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease leading to cirrhosis and cholangiocellular carcinoma. Inhibitors of the renin-angiotensin system or the sympathetic nervous system delay liver fibrogenesis in animal models.We investigated the antifibrotic potential of telmisartan, an angiotensin II type 1 receptor antagonist, and the β-adrenoceptor blocker propranolol in the PSC-like Abcb4 knockout mouse model.Sixty-five Abcb4 (-/-) mice were treated with telmisartan for 3 or 5 months (T) and with telmisartan plus propranolol for 3, 5, or 8 months (TP), or for 2 or 5 months starting with a delay of 3 months (TP delayed). Liver hydroxyproline content, inflammation, fibrosis, and bile duct proliferation were assessed; fibrosis-related molecules were analyzed by real-time polymerase chain reaction and Western blotting.Compared to controls, telmisartan monotherapy had no significant influence on hydroxyproline; however, telmisartan plus propranolol reduced hydroxyproline (TP 3 months, p = 0.008), fibrosis score (TP 3 months and TP 8 months, p = 0.043 and p = 0.008, respectively; TP delayed 8 months, p0.0005), bile duct proliferation (TP 8 months and TP delayed 8 months, p = 0.006 and p0.0005, respectively), and procollagen α1(I), endothelin-1, TIMP-1 and MMP3 mRNA as well as α-SMA, CK-19, and TIMP-1 protein.Telmisartan plus propranolol reduces liver fibrosis and bile duct proliferation in the PSC-like Abcb4 (-/-) mouse model, even when started at late stages of fibrosis, and may thus represent a novel therapeutic option for cholestatic liver diseases such as PSC.

Hepatitis B virus (HBV) is tightly controlled by a number of noncytotoxic mechanisms. This control occurs within the host hepatocyte at different steps of the HBV replication cycle. HBV persists by establishing a nuclear minichromosome, HBV cccDNA, serving as a transcription template for the viral pregenome and viral mRNAs. Nucleoside/nucleotide analogues widely used for antiviral therapy as well as most antiviral cytokines act at steps after transcription of HBV RNAs and thus can control virus replication but do not directly affect its gene expression. Control of HBV at the level of transcription in contrast is able to restrict both, HBV replication and gene expression. In the review, we focus on how HBV is controlled at the level of transcription. We discuss how the composition of transcription factors determines HBV gene expression and replication and how this may be influenced by antivirally active substances, e.g. the cytokine IL-6 or helioxanthin analogues, or by the differentiation state of the hepatocyte.

With about 350 million virus carriers, hepatitis B virus (HBV) infection remains a major health problem. HBV is a noncytopathic virus causing persistent infection, but it is still unknown whether host recognition of HBV may activate an innate immune response. We describe that upon infection of primary human liver cells, HBV is recognized by nonparenchymal cells of the liver, mainly by liver macrophages (Kupffer cells), although they are not infected. Within 3 hours, this recognition leads to the activation of nuclear factor kappa B (NF-κB) and subsequently to the release of interleukin-6 (IL-6) and other proinflammatory cytokines (IL-8, TNF-α, IL-1β), but does not induce an interferon response. The activation of proinflammatory cytokines, however, is transient, and even inhibits responsiveness toward a subsequent challenge. IL-6 released by Kupffer cells after activation of NF-κB controls HBV gene expression and replication in hepatocytes at the level of transcription shortly after infection. Upon binding to its receptor complex, IL-6 activates the mitogen-activated protein kinases exogenous signal-regulated kinase 1/2, and c-jun N-terminal kinase, which inhibit expression of hepatocyte nuclear factor (HNF) 1α and HNF 4α, two transcription factors essential for HBV gene expression and replication. Conclusion: Our results demonstrate recognition of HBV patterns by nonparenchymal liver cells, which results in IL-6-mediated control of HBV infection at the transcriptional level. Thus, IL-6 ensures early control of the virus, limiting activation of the adaptive immune response and preventing death of the HBV-infected hepatocyte. This pattern recognition may be essential for a virus, which infects a new host with only a few virions. Our data also indicate that therapeutic neutralization of IL-6 for treatment of certain diseases may represent a risk if the patient is HBV-infected. (HEPATOLOGY 2009:50:1773–1782.)

The purpose of the present study was to characterize histopathological lesions in primary biliary cirrhosis (PBC) and to assess the relationship between histopathological lesions and biochemistry. Liver biopsies of 252 patients with PBC were investigated. A laboratory database was established. Histopathological characterization was performed. Relationships between detailed histopathological features and biochemistry were calculated statistically. Combining the data, a PBC group, consisting of an anti-mitochondrial antibody (AMA)-positive and -negative subgroup, and an overlap group were defined, with a female preponderance of >90% and higher activity of aspartate aminotransferase (AST) in the overlap group. Histopathological changes were characteristic in >80%. Periductal concentric fibrosis, lobular granuloma formation and steatosis were frequently remarkable. Correlations were found between alanine aminotransferase activity and modified hepatitis activity index in the overlap group and the AMA-positive group. A positive significant relationship was demonstrated between mean AST activity and portal fibrosis for the AMA-positive group. A highly significant positive link was seen between mean concentration of bilirubin and stage of fibrosis. Biochemistry reflects only in part the degree of severity of histopathological lesions in PBC. Histopathology indicates comorbidity in a high percentage of patients.

International audience; Hepatitis B virus (HBV) is an important human pathogen, which targets the liver extremely efficient, gaining access to hepatocytes by a so far unknown receptor and replicating in a hepatocyte-specific fashion. Cell differentiation seems to determine HBV replication. We here show that the level of hepatocyte differentiation, as indicated by hepatocyte polarization and metabolic activity, is closely correlated to the transcription of the HBV RNA pregenome. Pregenome transcription determined the level of HBV replication in various cell lines of hepatocellular origin and in primary human hepatocytes. A variety of hepatocyte-enriched nuclear factors have been described to regulate transcription of the pregenome, but it remained unknown which factors link HBV replication to hepatocyte differentiation. We determined that high expression levels of HNF4alpha but not its potential cofactors or other hepatocyte-enriched transcription factors were essential for efficient HBV replication, and link it to hepatocyte differentiation. HNF1alpha contributed to the control of HBV replication because it regulated the expression of HNF4alpha. Thus, a concerted action of HNF4alpha and HNF1alpha, which also determines morphological and functional differentiation of hepatocytes, links HBV replication to hepatocyte differentiation.

Background & Aims: Induction of heme oxygenase-1 (HO-1) has been shown to be beneficial in immune-mediated liver damage. We now investigate the effects of HO-1 induction in models of human hepatitis B virus (HBV) infection. Methods: Adenoviral transfer of an HBV 1.3 genome into wild-type mice was used as a model for acute hepatitis B. HBV transgenic animals were used as a model for chronic HBV infection. HBV replication was assessed by HBV viremia, antigenemia, and Southern blotting, liver damage was assessed by serum alanine aminotransferase activities and histopathology of liver sections. To investigate HO-1 effects on HBV replication at a molecular level, stably HBV-transfected hepatoma cells were used. HBV gene expression, protein stability, transcription, and replication were determined. HO-1 was induced by either cobalt-protoporphyrin-IX or over expressed by adenoviral gene transfer. Results: In the acute hepatitis B model, liver injury was reduced significantly after HO-1 induction. In addition, HO-1 showed a pronounced antiviral effect, which was confirmed in stably HBV-transfected hepatoma cells and in persistently HBV replicating transgenic mice. We showed that HO-1 induction repressed HBV replication directly in hepatocytes at a posttranscriptional step by reducing stability of HBV core protein and thus blocking refill of nuclear HBV covalently closed circular (ccc)DNA. Small interfering RNA directed against HO-1 proved that this effect depended on the expression level of HO-1. Conclusions: Besides its hepatoprotective effect, HO-1 showed a pronounced antiviral activity in HBV infection. Therefore, induction of HO-1 might be a novel therapeutic option for inflammatory flares of hepatitis B.

Dendritic cells (DC) of hepatitis B virus (HBV) carriers have been reported to exhibit functional impairment. Possible explanations for this phenomenon are infection of HBV by DC or alteration of DC function by HBV. We therefore analyzed whether DC support the different steps of HBV infection and replication: uptake, deposition of the HBV genome in the nucleus, antigen expression, and progeny virus release. When HBV genomes were artificially introduced into monocyte-derived DC by adenoviral vectors, low-level expression of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) but no HBV replication was detected. When monocyte-derived DC were subjected to wild-type HBV or a recombinant HBV expressing Renilla luciferase under a non-liver-specific promoter, intracellular HBV DNA was detected in a low percentage of cells. However, neither nuclear cccDNA was formed nor luciferase activity was detected, indicating that either uncoating or nucleocytoplasmic transport were blocked. To verify our observation in the in vivo situation, myeloid and plasmacytoid DC were isolated from blood of high viremic HBV carriers, and analyzed by quantitative polymerase chain reaction (PCR) and electron microscopy. Although circulating DC had in vivo been exposed to more than 10(4) HBV virions per cell, HBV genomic DNA was hardly detected, and no nuclear cccDNA was detected at all. By using electron microscopy, subviral particles were found in endocytic vesicles, but virions were undetectable as were viral capsids in the cytoplasm. In conclusion, circulating DC may take up HBV antigens, but neither support nucleocytoplasmic transport nor replication of HBV.

Despite encouraging progress, antiviral treatment is far from achieving eradication of hepatitis C virus (HCV) infection. Therefore, a large body of new patents concerning novel therapeutic concepts and agents is claimed every year. This review comprises information revealed about granted patents on experimental therapy for HCV infection from 1999 to 2002 but does not intend to be comprehensive. A great part of the inventions described represent peptidomimetic inhibitors of NS3/4–protease, but chemicals, nucleoside analogues or oligodeoxynucleotides are also discussed. The efficacy, specificity and toxicity for most of the claimed drugs are tested mainly in vitro and clinical trial data are rare. Most compounds are intended for combination therapy with well-known antivirals. Other claimed agents, such as recombinant adenoviruses or fusion proteins, are used to improve drug delivery into the cell. A new developing direction of research is the use of immunotherapeutic approaches for HCV therapy.

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease ranging from simple fatty liver to steatohepatitis, fibrosis, and cirrhosis. We aimed to analyze the diagnostic performance and clinical utility of simple noninvasive tests alone or in combination for the detection of advanced fibrosis in patients with NAFLD.Data from 323 patients with biopsy-proven NAFLD/NASH who presented to the Clinic for Gastroenterology and Hepatology, University Hospital of Cologne between July 1998 and November 2009, were analyzed retrospectively. Sensitivity, specificity, positive predictive values, and negative predictive values were determined along with the area under receiver operating characteristic curves (AUROC) using published formulas for NAFLD, FIB-4, and BARD fibrosis scores.The area under receiver operating characteristic curves were as follows: NAFLD fibrosis score 0.96 [95% confidence interval (CI), 0.92-0.99], FIB-4 0.95 (95% CI, 0.91-1.00), BARD 0.82 (95% CI, 0.71-0.92) with negative predictive values for advanced fibrosis of 96%, 98%, and 96%, respectively. When applying the NAFLD, FIB-4, or BARD scoring systems 25%, 15%, or 26% of cases with advanced fibrosis would have been missed. Combining FIB-4 and BARD in a stepwise fashion, patients would have been correctly classified without biopsy in 67% of cases without missing a single case of advanced fibrosis.The FIB-4 and NAFLD fibrosis scores perform better than the BARD scoring system. Liver biopsy can securely be replaced only with a stepwise combination of simple noninvasive tests, otherwise the assessment of risk due to advanced fibrosis may be misleading in a clinically meaningful proportion of patients.

Hepatocellular carcinoma (HCC) is the 5th common malignancy worldwide, but established markers fail to detect up to one third of HCC. We have recently identified Neighbor of Punc E11 (Nope) as a surface marker for murine fetal liver stem cells. Similar to commonly used HCC markers such as α-Fetoprotein (Afp) and Glypican-3 (Gpc-3), we here establish Nope as an oncofetal marker of murine and human HCC and investigate its specific expression in hepatoma cell lines and primary HCC. Murine and human hepatoma cell lines and Cre-inducible SV40 T-antigen transgenic mice (Alb-SV40TAg(ind)) were analyzed for Nope expression in comparison to common HCC markers by quantitative RT-PCR, Western blot analyses and immunohistochemistry. Nope expression in primary human HCC was investigated using Oncomine Microarray database. Nope expression was elevated in 8 of 10 investigated murine and human hepatoma cell lines and in all tumors of our oncogenic mouse model but remained undetectable in normal liver and at preneoplastic stages of murine hepatocarcinogenesis. Furthermore, a significant induction of Nope was detected in primary human cancers compared to corresponding normal or cirrhotic tissue. Nope expression in tumor specimens and murine cell lines correlated closely with expression levels of Gpc-3, whereas expression levels of Afp showed high variations. In conclusion, we identified Nope as a novel oncofetal surface marker for murine and human HCC. Nope is specifically expressed by epithelial tumor cells but not in preneoplastic stages and is a promising marker for clinical application because of its high detection rate in Afp-positive and Afp-negative tumors.

Background & Aims In this study, adenoviral vectors encoding an antisense RNA complementary to the 5′ non-coding region (5′NCR) of the HCV-genome were generated to inhibit HCV-RNA gene expression in cell culture and in vivo . Methods First and second-generation (with E4-deletion) adenoviruses encoding the HCV5′NCR in antisense direction (Ad-NCRas and Ad-E4del-NCRas) were generated. Inhibition of HCV gene expression was analyzed in hepatoma cells stably transfected with the HCV5′NCR cDNA fused to the firefly luciferase gene (NCRluc), as well as in the HCV subgenomic replicon (genotypes 1b and 2a) and the fully infectious HCV JFH-1 cell culture systems. For in vivo experiments, an adenovirus encoding the NCRluc-gene was injected intravenously to achieve a NCR-dependent luciferase-expression in the liver of C3H/HeNcrl-mice. Results Forty eight hours after transduction with GFP-encoding adenoviruses, >85% of HepG2-, CCL13-and Huh7-cells expressed GFP. Surprisingly, GFP-expression of E4-deleted adenoviruses was considerably reduced at the same MOI. Using antisense first-generation adenoviruses (Ad-NCRas), a significant inhibition of the 5′NCR-dependent HCV-gene expression (54±19% in HepG2-cells and 66.2±15% in Huh7-cells) was achieved 48h after transduction. In Huh7-cells containing the HCV subgenomic replicons and in infectious HCV JFH-1 cell cultures, adenovirus-mediated transcription of antisense 5′NCR significantly blocked HCV-replication (40% and 76%, respectively). Corresponding to low transgene expression, the maximal inhibition reached with Ad-delE4-NCRas was 30%. In vivo , antisense adenoviral vectors also showed a significant inhibition (40%) of NCR-dependent luciferase expression compared to control adenoviruses ( p Conclusions The data indicate that HCV gene expression can be inhibited by antisense RNA encoding adenoviruses in the tested settings.

Aims: MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs through translational repression or RNA degradation. Many fundamental biological processes are modulated by miRNAs and an important role for miRNAs in carcinogenesis is emerging. We have shown by previous screening of dysregulated miRNA, that miRNA-198 (miR-198) is highly downregulated in HCV-positive hepatocellular carcinoma (1). In the present approach we studied the function of miR-198 in hepatocellular carcinogenesis. Methods: miR-198 level was analyzed in Huh7, Hep3B, HepG2, and Pop10 hepatoma cells and in hepatocellular carcinoma of different entities by real-time PCR of reverse-transcribed total RNA extracts (2). An agoMir-198, mimicking miR-198, was constructed and transfected into Huh7 and Pop10 hepatoma cells. The agoMir-198 function was shown by reporter assays using a luciferase miR-198 fusion sequence. Cell growth, cell proliferation, and apoptosis were determined in agoMir-treated hepatoma cells by means of the "Aqueous One Solution Cell Proliferation Assay" (Promega), the Tunnel Assay (Roche) or by BRDU incorporation, respectively. Putative targets of miR198 were searched using the algorithms of Miranda, Targetscan and Pictar. Results: miR-198 expression level was repressed in all hepatoma cells tested, but was lowest in highly dedifferentiated Pop10 cells showing reduced level of liver-specific transcription factors. AgoMir-198 treatment of Huh-7 and Pop-10 cells resulted in repression of the miR-198-reporter construct and in 50% or 75% reduced cell growth of Huh7 or Pop10 cells, respectively. Screening of different databases identified the met receptor pathway, transducing HGF involved mitogenic activation, as a putative target of miR-198 repression, but also anti-apoptotic mediators have to be considered. Conclusion: miR-198, mediating reduced hepatocellular cell growth, is suggested to act as tumor suppressor miRNA in hepatocarcinogenesis. Literatur: (1) Varnholt H., U. Drebber, F. Schulze, I. Wedemeyer, P. Schirmacher, H.P. Dienes, and M. Odenthal, MicroRNA Gene Expression Profile of HCV-Associated Hepatocellular Carcinoma. Hepatology, 47(4): p. 1223-32; 2008.(2) Shi R and VL Chiang, Facile means for quantifying microRNA expression by real-time PCR. Biotechniques. 39(4): p. 519-25; 2005.

Das hepatozellulare Karzinom (HCC) zahlt weltweit zu den haufigsten malignen Tumorerkrankungen. Die Sensitivitat der Fruherkennung mit Sonografie und α-Fetoprotein (AFP) ist eingeschrankt. Daher ist die Etablierung weiterer Tumormarker essentiell. Im Mausmodell konnten wir zeigen, dass Neighbor of Punc E11 (Nope) im Rahmen der Leberentwicklung (1) und der Hepatokarzinogenese (2) induziert wird und als muriner onco-fetaler Marker eingesetzt werden kann. In dieser Arbeit soll NOPE als potentieller Marker humaner HCCs analysiert werden. Methoden: Humane Hepatoma-Zellinien (HuH7, HepG2, Hep3B) sowie Kryo-Lebergewebe von 24 Patienten mit HCC bei verschiedenen Lebererkrankungen wurden analysiert. Mittels quantitativer RT-PCR und im Western Blot (WB) wurden die Expression von NOPE und AFP im Vergleich zu normaler adulter Leber quantifiziert. Zusatzlich wurden immunhistochemische Farbungen fur NOPE in Kombination mit AFP und E-cadherin durchgefuhrt. Ergebnisse: In allen drei humanen Zellinien konnten wir eine signifikante Expression von NOPE auf RNA- und Protein-Ebene nachweisen, wahrend Proben aus humaner adulter Leber negativ waren. Nur 2 der 3 NOPE-positiven Zellinien exprimierten auch AFP (HepG2, HuH7). In der quantitativen RT-PCR zeigten ebenfalls 5 von 24 HCC-Proben eine signifikante Expression von NOPE. Es bestand keine Korrelation mit der Expression von AFP oder der zugrundeliegenden Lebererkrankung. Immmunhistochemisch zeigte sich eine uberwiegende Lokalisation von NOPE im Golgi-Apparat. Zusammenfassung: Sowohl in humanen Hepatoma-Zellinien wie auch in HCCs bei verschiedenen Lebererkrankungen konnten wir eine signifikante Expression von NOPE nachweisen, wahrend NOPE in der normalen adulten Leber und bei allen getesteten Lebergrunderkrankungen nicht exprimiert wurde. Unsere Ergebnisse zeigen, dass NOPE als neuer Marker fur humane HCCs eingesetzt werden kann. Literatur: 1) Nierhoff D, LeVoci L, Schulte S, Goeser T, Rogler LE and Shafritz DA. New cell surface markers for murine fetal liver stem cells identified through high density cDNA microarrays. Hepatology 2007, 46(2):535-47. 2) Nierhoff D*, Marquardt J*, Quasdorff M, Holz G, Arnold B, Protzer U, Goeser T Neighbor of Punc E11 (Nope): a novel marker for murine hepatocellular carcinoma Hepatology 2007, 46 (4S): 525A.

Background/Aims Epstein–Barr virus has a seroprevalence of more than 80% world wide and is known to be associated with hepatitis. However, little is known about the underlying pathogenesis and immunmechanisms and no standard diagnostic criteria to diagnose EBV-hepatitis are available. Methods We collected liver biopsies ( n =21) with the tentative diagnosis of EBV induced hepatitis according to pathological changes and traceable EBV genome by PCR. Correlation with serological data revealed acute in seven cases, convalescent in two cases, past EBV infection in six cases. Viral RNA was visualised by in situ hybridisation within nuclei of lymphocytes. Results In seven of 68 liver biopsies with the diagnosis ‘liver disease of unknown aetiology' EBV genome in the tissue was demonstrated indicating a possible role for EBV in the induction of hepatitis or a trapping of infected lymphocytes within the liver. In a control group of 20 EBV-seropositive patients with steatohepatitis EBV–DNA PCR of the liver tissue was negative. Immunohistochemistry identified CD3 and CD8 positive T-lymphocytes as the main lymphocytic infiltrate in EBV hepatitis. Conclusions EBV hepatitis should be taken into consideration in case of typical histopathological changes and a positive DNA PCR of liver biopsy. Serological confirmation of the diagnosis is inevitable.

U. Protzer’ , S. Seyfried2, M. QuasdortT’ , G. Sass2, F. Bohne’ , M. Hosel’ , P. Schirmacher3, G. Tiegs2. ‘Molecular Infgctiology at the Center,fbr Molecular Medicine, Institute ,fbr Medical Microbiology, lntntunology, and Hygiene, University of‘ Cologne, Cologne; 21nstitute of Experintental and Clinical Pharmacology and Toxicology, University of‘ ErlangenNiiremberg, Erlangen; ”Institute of Pathology, linioersiiy of Heidelberg, linioersiiy Hospital, Heidelberg E-mail: tiegs@pharmakologie.uni-erlangen.de

A new probe drug cocktail containing substrates of important drug transporters was tested for mutual interactions in a clinical trial. The cocktail consisted of (predominant transporter; primary phenotyping metric): 10 mg adefovir-dipivoxil (OAT1; renal clearance (CLR )), 100 mg sitagliptin (OAT3; CLR ), 500 mg metformin (several renal transporters; CLR ), 2 mg pitavastatin (OATP1B1; clearance/F), and 0.5 mg digoxin (intestinal P-gp, renal P-gp, and OATP4C1; peak plasma concentration (Cmax ) and CLR ). Using a randomized six-period, open change-over design, single oral doses were administrated either concomitantly or separately to 24 healthy male and female volunteers. Phenotyping metrics were evaluated by noncompartmental analysis and compared between periods by the standard average bioequivalence approach (boundaries for ratios 0.80-1.25). Primary metrics supported the absence of relevant interactions, whereas secondary metrics suggested that mainly adefovir was a victim of minor drug-drug interactions (DDIs). All drugs were well tolerated. This cocktail may be another useful tool to assess transporter-based DDIs in vivo.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths, worldwide. MicroRNAs, inhibiting gene expression by targeting various transcripts, are involved in genomic dysregulation during hepatocellular tumorigenesis. In previous studies, microRNA-198 (miR-198) was shown to be significantly downregulated in HCV-positive hepatocellular carcinoma (HCC). Herein, the function of miR-198 in hepatocellular carcinoma cell growth and gene expression was studied. In hepatoma cell-types with low levels of liver-specific transcription factor HNF1α indicating a low differentiation grade, miR-198 expression was most downregulated. However, miR-198 treatment did not restore the expression of the liver-specific transcription factors HNF1α or HNF4α. Importantly, overexpression of miR-198 in Pop10 hepatoma cells markedly reduced cell growth. In agreement, comprehensive gene expression profiling by microarray hybridisation and real-time quantification revealed that central signal transducers of proliferation pathways were downregulated by miR-198. In contrast, genes mediating cellular adherence were highly upregulated by miR-198. Thus, the low expression of E-cadherin and claudin-1, involved in cell adhesion and cell-cell contacts, was abolished in hepatoma cells after miR-198 overexpression. This definite induction of both proteins by miR-198 was shown to be accompanied by a significantly impaired migration activity of hepatoma Pop10 cells. In conclusion, miR-198 acts as a tumor suppressor by repression of mitogenic and motogenic pathways diminishing cell growth and migration.