[Ethical insolvency?]. / Ethische Insolvenz?

The current DRG system has not only changed the economic structure of clinical medicine but also has profound effects on its original mission. In the present paper we discuss the fundamental limits of the DRG system and make a case for placing more emphasis on time and communication in health care.

Primary mouse hepatocytes for systems biology approaches: a standardized in vitro system for modelling of signal transduction pathways.

Complex cellular networks regulate regeneration, detoxification and differentiation of hepatocytes. By combining experimental data with mathematical modelling, systems biology holds great promises to elucidate the key regulatory mechanisms involved and predict targets for efficient intervention. For the generation of high-quality quantitative data suitable for mathematical modelling a standardised in vitro system is essential. Therefore the authors developed standard operating procedures for the preparation and cultivation of primary mouse hepatocytes. To reliably monitor the dynamic induction of signalling pathways, the authors established starvation conditions and evaluated the extent of starvation-associated stress by quantifying several metabolic functions of cultured primary hepatocytes, namely activities of glutathione-S-transferase, glutamine synthetase, CYP3A as well as secretion of lactate and urea into the culture medium. Establishment of constant metabolic activities after an initial decrease compared with freshly isolated hepatocytes showed that the cultured hepatocytes achieve a new equilibrium state that was not affected by our starving conditions. To verify the highly reproducible dynamic activation of signalling pathways in the in vitro system, the authors examined the JAK-STAT, SMAD, PI3 kinase, MAP kinase, NF-kappaB and Wnt/beta-catenin signalling pathways. For the induction of gp130, JAK1 and STAT3 phosphorylation IL6 was used, whereas TGFbeta was applied to activate the phosphorylation of SMAD1, SMAD2 and SMAD3. Both Akt/PKB and ERK1/2 phosphorylation were stimulated by the addition of hepatocyte growth factor. The time-dependent induction of a pool of signalling competent beta-catenin was monitored in response to the inhibition of GSK3beta. To analyse whether phosphorylation is actually leading to transcriptional responses, luciferase reporter gene constructs driven by multiple copies of TGFbeta-responsive motives were applied, demonstrating a dose-dependent increase in luciferase activity. Moreover, the induction of apoptosis by the TNF-like cytokine Fas ligand was studied in the in vitro system. Thus, the mouse hepatocyte in vitro system provides an important basis for the generation of high-quality quantitative data under standardised cell culture conditions that is essential to elucidate critical hepatocellular functions by the systems biology approach.

[Management of chronic hepatitis B]. / Therapie der chronischen Hepatitis B.

As a leading cause of liver cirrhosis and hepatocellular carcinoma, chronic hepatitis B poses a major health care problem. Currently approved therapeutic options include interferon-alpha, pegylated interferon-alpha, lamivudine and adefovir. Interferon-alpha can induce long-term suppression of viral replication in a proportion of patients. However, treatment is associated with considerable side effects. Lamivudine and adefovir effectively suppress viral replication and induce histological improvement in the majority of patients. However, recurrence rates are high after cessation of treatment. During long-term treatment about 20% lamivudine-resistant mutants emerge annually, while currently available studies suggest that this number is about 3% for adefovir after two years of therapy. Efficacy of the respective antivirals is affected by virological and clinical parameters, thus requiring individual treatment strategies.

[Best supportive care of hepatocellular carcinoma]. / Hepatozelluläres Karzinom.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in some areas of the world. The major etiologic risk factors include toxins (alcohol, aflatoxin B(1)), hepatitis B and C virus infection as well as various inherited metabolic disorders. The prognosis of HCC patients is generally very poor with a 5-year survival rate of less than 5%. The diagnosis is based on biochemical and imaging tests as well as histology. Therapeutic strategies include surgery (resection or liver transplantation) and non-surgical interventions, such as percutaneous ethanol injection or radiofrequency thermal ablation as well as transarterial embolization or chemoembolization. Radio- or chemotherapy are mostly ineffective. Therefore, the development and evaluation of novel HCC treatment strategies as well as the implementation of existing and the development of new measures to prevent HCC are of utmost importance. The better understanding of the clinical and molecular pathogenesis of HCC should lead to improved diagnostic, therapeutic and preventive strategies with the aim to reduce the incidence of HCC, one of the most devastating malignancies worldwide.

[The role of the virus specific T-cell response in acute and chronic HBV and HCV infection]. / Die Rolle der virusspezifischen T-Zellantwort bei der akuten und chronischen Hepatitis-B- und -C-Virusinfektion.

Infections with hepatitis B (HBV) and hepatitis C virus (HCV) are worldwide one of the most frequent causes for chronic liver disease, liver cirrhosis and hepatocellular carcinoma. The mechanisms responsible for the elimination or the persistence of the virus are not well understood. The immunopathogenesis of HBV and HCV infection is primarily mediated by virus specific CD4+- and CD8+-T-cells. During acute infection a strong and multispecific T-cell response against different viral epitopes can be detected and is associated with the clearance of the virus. In case of viral persistence virus specific T-cells contribute to liver inflammation. In this article we summarize the current concepts about the role of the virus specific T-cell response in acute and chronic HBV and HCV infection.

[Systemic treatment for hepatocellular carcinoma]. / Medikamentöse Therapie des hepatozellulären Karzinoms.

Hepatocellular carcinoma (HCC) is one of the most frequent malignancies worldwide. A variety of pharmacological strategies has been evaluated in the treatment of HCC: classical chemotherapy, tamoxifen, octreotide, thymostimulin, pravastatin, (131)I-lipiodol as well as transarterial chemoperfusion (TAC) and chemoembolisation (TACE). TACE monotherapy or TACE combined with pravastatin resulted in a survival benefit of selected HCC patients. New strategies such as immunotherapy, antiangiogenic agents or cyclooxygenase inhibitors are under clinical investigation and might play a role in future therapies for HCC. Efficient strategies for the primary prevention of HCC are available and promising concepts in the secondary prevention have been reported.

[Prevention of hepatocellular carcinoma]. / Prävention des hepatozellulären Karzinoms.

Prevention is an increasingly important area of modern medicine. One of the foremost goals in the field of hepatology is prevention of hepatocellular carcinoma since this tumor has a poor prognosis and current therapeutic options are limited. Most hepatocellular carcinomas arise from chronic liver disease and liver cirrhosis which is considered a major risk factor for tumor development. The most common etiologic factors causing liver cirrhosis include chronic viral hepatitis, alcohol consumption, toxins and a few metabolic disorders. This review discusses current therapeutic concepts for prevention and treatment of chronic liver disease and cirrhosis.

Inhibition of duck hepatitis B virus replication by intrahepatic expression of an antiviral resistance gene.

Resistance genes coding for inhibitors of hepadnaviral replication, such as ribozymes, antisense RNA, and dominant negative mutants have been shown to be effective in transfected hepatoma cells. In vivo studies, however, are not available to date. Here we expanded the use of the duck hepatitis B virus (DHBV) model for studying antiviral resistance genes in vivo. Animals were experimentally infected by intravenous injection of DHBV-positive serum in ovo. The use of recombinant human adenovirus type 5 and avian adenovirus CELO for gene transfer was evaluated. Adenovirus type 5 transduced more than 95% and CELO less than 1% of embryonic hepatocytes in vivo. Adenovirus type 5 interfered with DHBV replication (viral cross-talk), but this effect was moderate and did not preclude analysis of specific antiviral effects. Thus adenoviral transfer of a dominant negative mutant prior to DHBV infection (intracellular immunization) yielded 100-fold suppression of viral replication compared to the green fluorescent protein marker gene. Neither gene was toxic. These data demonstrate that a prototype anthepadnaviral resistance gene is functional in vivo. Duck embryos represent a useful model for evaluating gene therapeutic strategies in vivo without the need for large scale preparations of gene delivery vehicles.

Generation of stable mRNA fragments and translation of N-truncated proteins induced by antisense oligodeoxynucleotides.

Binding of phosphorothioate-modified antisense oligodeoxynucleotides (AS ODNs) to target mRNAs is commonly thought to mediate RNA degradation or block of translation. Here we demonstrate cleavage of target mRNAs within the AS ODN binding region with subsequent degradation of the 5' but not the 3' cleavage product. Some, if not all, 3' mRNA fragments lacked a 5' cap structure, whereas their poly(A) tail length remained unchanged. Furthermore, they were efficiently translated into N-terminally truncated proteins as demonstrated in three settings: production of shortened hepadnaviral surface proteins, alteration of the subcellular localization of a fluorescent protein, and shift of the transcription factor C/EBPalpha isoform expression levels. Thus, AS treatment may result in the synthesis of N-truncated proteins with biologically relevant effects.

Immunotherapy directed against alpha-fetoprotein results in autoimmune liver disease during liver regeneration in mice.

BACKGROUND & AIMS: Priming immune responses against alpha-fetoprotein (AFP) highly expressed in the majority of hepatocellular carcinomas results in significant antitumoral T-cell responses. Liver regeneration in humans and mice, however, is also associated with increased AFP expression. Therefore, we evaluated the risk of AFP-directed immunotherapeutic approaches to induce autoimmunity against the regenerating liver. METHODS: Mice were immunized with DNA encoding mouse AFP. For induction of liver regeneration, partial hepatectomy was performed and mice were monitored by serial histopathologic examinations and measurements of serum ALT activities (U/L), and by determination of the kinetics of AFP-specific T-cell responses. RESULTS: Livers of AFP immune mice without partial hepatectomy were characterized by minor lymphocytic infiltrations without transaminase elevations. By contrast, a significant hepatocyte damage was observed in regenerating liver that correlated well with the number of AFP-specific CD8(+) T cells, the activity of liver regeneration, and the level of AFP synthesis. Autoimmune liver damage was mediated by CD4(+) T cell-dependent CD8(+) cytotoxic T lymphocytes. CONCLUSIONS: These results show that priming of T-cell responses against shared tumor-specific self antigens may be accompanied by induction of autoimmunity dependent on the level of expression of the self antigen and have important implications for the development of antitumoral vaccines targeted against antigens that are not strictly tumor-specific.

Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection.

Cytotoxic T lymphocytes (CTL) play a major role in the recovery from primary viral infections and the accompanying tissue injuries. However, it is unclear to what extent the two main cytolytic pathways, perforin-granzyme A and B exocytosis and Fas ligand (FasL)-Fas interaction, contribute to these processes. Here we have employed mouse strains with either spontaneous mutations or targeted gene defects in one or more components of either of the two cytolytic pathways to analyze the molecular basis of viral clearance and induction of hepatitis during lymphocytic choriomeningitis virus infection. Our results reveal that viral clearance is solely dependent on perforin but that virus-induced liver damage only occurs when both the FasL/Fas and the perforin pathways, including granzymes A and B, are simultaneously activated. The finding that development of hepatitis but not viral clearance is dependent on the concomitant activation of FasL-Fas and perforin-granzymes may be helpful in designing novel strategies to prevent hepatic failures during viral infections.

Efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis B virus.

The Asian tree shrew, Tupaia belangeri, has been proposed as a novel animal model for studying hepatitis B virus (HBV) infection. Here, we describe a protocol for efficient and reproducible infection of primary tupaia hepatocytes with HBV. We report that human serum interferes with HBV binding to the hepatocytes, thus limiting the maximum multiplicity of infection. Purification of HBV virions by gradient sedimentation greatly enhances virus binding and infectivity. Covalently closed circular DNA was clearly detectable by Southern blot analysis and newly synthesized single-stranded HBV DNA was visible 2 weeks postinoculation. Primary tupaia hepatocytes are also susceptible to infection with the recently discovered woolly monkey hepatitis B virus (WMHBV) but not to woodchuck hepatitis virus infection. Compared to HBV, WMHBV replicated at a higher rate with single-stranded DNA detectable within the first week postinoculation. Primary tupaia hepatocytes should represent a useful system for studying early steps of HBV and WMHBV infection.

Trypsinogen gene mutations in patients with chronic or recurrent acute pancreatitis.

Three-point mutations (R117H, N211, A16V) within the cationic trypsinogen gene have been identified in patients with hereditary pancreatitis (HP). A genetic background has also been discussed for idiopathic juvenile chronic pancreatitis (IJCP), which closely mimicks the clinical pattern of HP, and alcoholic chronic pancreatitis because only a small number of heavy drinkers develop pancreatitis. This prompted us to screen 104 patients in our well-defined pancreatitis cohort for the currently known cationic trypsinogen gene mutations. The R117H mutation was detected in seven patients (six patients of two clinically classified HP families, one patient with clinically classified IJCP) and the A16V mutation in one IJCP patient. No cationic trypsinogen gene mutations were found in the remaining 96 patients with chronic and recurrent acute pancreatitis of various etiologies. Our results demonstrate the need for genetic testing to exclude HP, particularly in the presence of an atypical or unknown family history. In addition, cationic trypsinogen gene mutations are no predisposing factor in patients with chronic and recurrent acute pancreatitis of different etiologies.

[Current status of cell-based therapies in liver diseases]. / Zelltherapie in der Hepatologie.

Major strides have been made during the past 10 years in the fields of liver cell transplantation and liver-directed gene therapy. Pre-clinical studies in animals have shown that primary hepatocytes transplanted into the liver as well as intravenously transfused bone marrow stem cells can generate new liver tissue. Such cell transplantation studies have contributed to our understanding of organogenesis and hepatocyte biology. Furthermore, transplantation of xenogenic hepatocytes has led to the development of new small animal models for studying viral hepatitis. In the clinical setting, liver cell transplantation offers a wide range of potential therapeutic applications, especially in metabolic diseases. In particular, the case of a patient with Crigler-Najjar Syndrome Type I clearly demonstrated the long-term viability of transplanted hepatocytes with stable metabolic function. Further studies are warranted to assess the full potential of cell- based therapies and their clinical application.

[Cell transplantation in hepatology]. / Zelltransplantation in der Hepatologie.

Preclinical studies have shown that hepatocytes transplanted into the liver function normally on a long term basis. The procedure is well tolerated and offers a wide range of potential clinical applications. Clinical case studies have been performed in the setting of acute and chronic liver failure as well as metabolic diseases. In particular, the case of a patient with Crigler-Najjar Syndrome Type I clearly demonstrated the long term viability of transplanted hepatocytes with stable metabolic function. Further studies are warranted to assess the full therapeutic potential of hepatocyte transplantation.

Ligand-mediated retargeting of recombinant adenovirus for gene transfer in vivo.

The development of efficient and safe methods for in vivo gene transfer is central to the success of gene therapy. Recombinant adenoviral vectors, although highly efficient, are limited by the host immune response, potential safety hazards due to obligatory cotransfer of viral proteins, and their broad tissue tropism. Here, we demonstrate in an animal model that host range and tissue tropism of a recombinant adenovirus from a distant species can be modified by complexing adenovirus with a cell-specific ligand. Thus, a replication-deficient lacZ recombinant human adenovirus, which naturally does not infect avian cells, allowed highly efficient and specific gene transfer to the liver of ducks in vivo when complexed with N-acetylglucosamine, a ligand for the chicken hepatic lectin. This combination of ligand-mediated receptor targeting with adenoviral uptake and intracellular processing of a given gene represents a novel approach to gene therapy of inherited and acquired liver diseases.

Dominant negative mutants of the duck hepatitis B virus core protein interfere with RNA pregenome packaging and viral DNA synthesis.

Dominant negative (DN) mutants of the hepadnaviral core protein are potent inhibitors of viral replication. We have previously shown that fusion of sequences derived from the duck hepatitis B virus (DHBV) polymerase (Pol), DHBV small surface protein (S), bacterial beta-galactosidase (lacZ), or green fluorescent protein (GFP) to the carboxy terminus of the DHBV core protein yields DN mutants that inhibit viral replication at the posttranslational level. To elucidate the mechanism(s) of their antiviral action, we analyzed the effect of the DN mutants on RNA pregenome packaging and nucleocapsid assembly. Core-Pol and core-S, but not core-lacZ or core-GFP, markedly interfered with RNA pregenome packaging. Nucleocapsid formation was not affected by any of the mutants. The DN core-GFP fusion protein formed mixed particles with wild-type core protein in the cytoplasm of cotransfected cells and interfered with reverse transcription of the viral pregenome. A subpopulation of chimeric nucleocapsids, however, was shown to overcome the block in DNA synthesis and produce mature viral DNA. Thus, at least 2 steps within the viral life cycle can be targeted by DN DHBV core proteins: 1) packaging of the viral pregenome; and 2) reverse transcription within mixed particles. The fact that some mixed particles retain replication competence demonstrates a high structural flexibility of nucleocapsids and indicates a possible mechanism of viral escape.