Renal grafts from anti-hepatitis B core-positive donors: a quantitative review of the literature.

BACKGROUNDS AND AIMS: Organ shortage is a major problem in transplantation. The use of organs from hepatitis B surface antigen (HBsAg)-negative and hepatitis B core antibody (HBcAb)-positive donors could significantly increase the donor pool. However, little information is available about the impact of HBcAb status of renal donors on viral transmission to recipients. To address this issue, the present quantitative review of relevant studies has been performed. MATERIALS AND METHODS: Electronic databases including Medline, EMBASE, ISI, and Scopus were systematically searched for studies that evaluated risk of hepatitis B virus (HBV) transmission through renal transplantation from HBsAg-/HBcAb+ donors. Eligible studies were identified according to predefined criteria. The final outcome was one of HBV markers seroconversion defined as HBsAg, hepatitis B surface antibody (HBsAb), or HBcAb detection in previously seronegative end-stage renal disease (ESRD) patients after transplantation, and without other identified major sources of infection. RESULTS: Nine studies with 1385 eligible kidney recipients were included. In total, 45 subjects showed seroconversion of HBV markers as follows: HBsAg (n = 4) (0.28%; 95% confidence interval [CI] 0.006; 0.57), HBcAb (n = 32), HBsAb (n = 5), and either HBcAb or HBsAb (n = 4). The total rate of seroconversion after renal transplantation was calculated to be 3.24% (95% CI: 2.31-4.18). CONCLUSION: Our review indicates that the risk of HBV transmission from HBcAb-positive kidney donors is extremely low. Therefore, kidneys from these donors can be transplanted safely into ESRD patients.

Weight loss and severe jaundice in a patient with hyperthyroidism.

BACKGROUND: Thyrotoxicosis may significantly alter hepatic function and is associated with autoimmune disorders of the liver. CASE REPORT: We report the case of a thyrotoxic patient with Graves' disease and histologically established cholestatic hepatitis. Medical treatment of hyperthyroidism normalized liver function tests. CONCLUSIONS: In patients with elevated liver function parameters and jaundice of unknown origin, thyroid function should generally be tested. Moreover, medical treatment of hyperthyroidism with thyrostatics may cause severe hepatitis whereas untreated hyperthyroid patients are at risk of developing chronic liver failure.

New aspects of an anti-tumour drug: sorafenib efficiently inhibits HCV replication.

BACKGROUND AND AIMS: Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and is associated with significant morbidity and mortality. Since there is evidence for an interaction of NS5A with c-Raf we studied whether the c-Raf inhibitor sorafenib affects HCV replication. METHODS: HCV replicating HuH7.5 cells were treated with sorafenib and examined for HCV RNA titres by northern blotting or real time polymerase chain reaction (PCR), for core, NS3 and NS5A expression by immunostaining, and for replication by luciferase reporter assays. RESULTS: Here we demonstrate that in cells replicating infectious HCV particles, NS5A recruits c-Raf to the replicon complex resulting in the activation of c-Raf. Therefore, we studied the effect of inhibition of c-Raf on HCV replication using the anti-tumour drug sorafenib that is known to inhibit c-Raf with high specificity. Sorafenib efficiently blocks HCV replication and viral gene expression. In addition, in HCV-replicating cells sorafenib decreased the hyperphosphorylated form of NS5A and resulted in the formation of additional hypophosphorylated forms. Further, sorafenib caused a rapid dissociation of lipid droplets. We provide evidence that the antiviral effect of sorafenib indeed is caused by inhibition of c-Raf. By contrast, inhibition of targets downstream of c-Raf or inhibition of tyrosine kinases by sunitinib did not affect HCV replication. CONCLUSION: Our data demonstrate that the well-characterised anti-tumour drug sorafenib efficiently blocks HCV replication in vitro. This novel effect of sorafenib should be further explored as an antiviral strategy for patients with chronic HCV infection.

CpG-island methylation of the ER promoter in colorectal cancer: analysis of micrometastases in lymph nodes from UICC stage I and II patients.

Patients with UICC stage II colorectal cancer (CRC) have a risk of approximately 20% to develop disease recurrence after tumour resection. The presence and significance of micrometastases for locoregional recurrence in these patients lacking histopathological lymph node involvement on routine stained HE sections is undefined. Oestrogen receptor (ER) promoter methylation has earlier been identified in CRC. Therefore, we evaluated the methylation status of the ER promoter in lymph nodes from 49 patients with CRC UICC stage I and II as a molecular marker of micrometastases and predictor of local recurrence. DNA from 574 paraffin-embedded lymph nodes was isolated and treated with bisulphite. For the detection of methylated ER promoter sequences, quantitative real-time methylation-specific PCR was used. Of the 49 patients tested, 15 (31%) had ER methylation-positive lymph nodes. Thirteen of those (86%) remained disease free and two (14%) developed local recurrence. In the resected lymph nodes of 34 of the 49 patients (69%), no ER promoter methylation could be detected and none of these patients experienced a local relapse. The methylation status of the ER promoter in lymph nodes of UICC stage I and II CRC patients may be a useful marker for the identification of patients at a high risk for local recurrence.

An error model for protein quantification.

MOTIVATION: Quantitative experimental data is the critical bottleneck in the modeling of dynamic cellular processes in systems biology. Here, we present statistical approaches improving reproducibility of protein quantification by immunoprecipitation and immunoblotting. RESULTS: Based on a large data set with more than 3600 data points, we unravel that the main sources of biological variability and experimental noise are multiplicative and log-normally distributed. Therefore, we suggest a log-transformation of the data to obtain additive normally distributed noise. After this transformation, common statistical procedures can be applied to analyze the data. An error model is introduced to account for technical as well as biological variability. Elimination of these systematic errors decrease variability of measurements and allow for a more precise estimation of underlying dynamics of protein concentrations in cellular signaling. The proposed error model is relevant for simulation studies, parameter estimation and model selection, basic tools of systems biology. AVAILABILITY: Matlab and R code is available from the authors on request. The data can be downloaded from our website www.fdm.uni-freiburg.de/~ckreutz/data.

The molecular biology of hepatitis B virus.

Infection with the hepatitis B virus (HBV) or related hepadnaviruses is associated with a wide spectrum of liver diseases, including hepatocellular carcinoma (HCC). In this article we review the current state of knowledge about the structure, genetic organization and life cycle of HBV. The mechanisms of viral pathogenesis and HCC development remain poorly understood, but new approaches may soon begin to shed light on these areas.

Cyclin D1 overexpression and p53 inactivation immortalize primary oral keratinocytes by a telomerase-independent mechanism.

The immortalization of human cells is a critical step in multistep carcinogenesis. Oral-esophageal carcinomas, a model system to investigate molecular mechanisms underlying squamous carcinogenesis, frequently involve cyclin D1 overexpression and inactivation of the p53 tumor suppressor. Therefore, our goal was to establish the functional role of cyclin D1 overexpression and p53 inactivation in the immortalization of primary human oral squamous epithelial cells (keratinocytes) as an important step toward malignant transformation. Cyclin D1 overexpression alone was found to induce extension of the replicative life span of normal oral keratinocytes, whereas the combination of cyclin D1 overexpression and p53 inactivation led to their immortalization. This study also demonstrates that immortalization of oral keratinocytes can be independent of telomerase activation, involving an alternative pathway of telomere maintenance (ALT).

Reversal of drug resistance of hepatocellular carcinoma cells by adenoviral delivery of anti-ABCC2 antisense constructs.

Human cancers are characterized by a high degree of drug resistance. The multidrug resistance transporters MDR1-P-glycoprotein (ABCB1) and ABCC2 (MRP2) are expressed in a variety of human cancers, including hepatocellular carcinoma (HCC). The ABCC2 gene encodes a membrane protein involved in the ATP-dependent transport of conjugates of lipophilic substances. In this study we analyzed the effect of an ABCC2 antisense construct on the chemosensitization of HepG2 cells. Adenoviral vectors were constructed to allow an efficient expression of anti-ABCC2 antisense constructs. The effective target sequence comprised nucleotides 2543-2942 of the human ABCC2 cDNA. Adenoviral delivery of the ABCC2 antisense construct resulted in a reduced IC(50) for doxorubicin (12-fold), vincristine (50-fold), cisplatin (25-fold) and etoposide (VP-16) (25-fold). The adenoviral delivery of the ABCC2 antisense construct was so efficient that chemosensitization of HepG2 cells could even be demonstrated in mass cell cultures without a selection of transduced cells for single ABCC2 antisense-expressing HCC cell clones. After transfection of the ABCC2 antisense-expressing construct, HepG2 cells had significantly reduced ABCC2 mRNA and ABCC2 protein levels. Transduction of the ABCC2 antisense-expressing construct into HepG2 cells resulted in the accumulation of the high-affinity ABCC2 substrate Fluo-3. HepG2 tumors stably transfected with an anti-ABCC2 antisense construct regressed significantly in nude mice upon vincristine treatment. In addition, significant tumor regression was also observed when adenovirus-expressing anti-ABCC2 antisense construct was directly injected into HepG2 tumors in nude mice. Our study demonstrates the specific reversal of ABCC2-related drug resistance in adenovirus-transduced HepG2 cells and in HepG2 tumors in nude mice expressing this ABCC2 antisense construct.

CD117 (c-kit) expression in human hepatocellular carcinoma.

AIMS: Although various methods of treatment have been tried, treatment options for advanced hepatocellular carcinoma (HCC) remain limited. Expression of the platelet-derived growth factor has been shown in HCC, which may derive from hepatic stem cells that express the c-kit proto-oncogene. Because of the promising results of imatinib and the key role played by c-kit in gastrointestinal stromal tumours and other solid tumours, the aim of this study was to determine the prevalence of c-kit (CD117) overexpression in patients with HCC. MATERIALS AND METHODS: A retrospective study of 258 archival specimens of subjects with histologically confirmed HCC was carried out. Expression of the c-kit proto-oncogene was evaluated by immunohistochemistry using rabbit anti-CD117 antibody A4502. RESULTS: The overall percentage of positive immunohistochemical staining of HCCs was 2.3% (6/258). CONCLUSIONS: Our results suggest that CD117 is not significantly overexpressed in HCC and there seems to be no role for the use of imatinib.

Effect of vardenafil, an inhibitor of phosphodiesterase-5, on portal haemodynamics in normal and cirrhotic liver -- results of a pilot study.

BACKGROUND: Dysregulation of the cyclic guanosine 3',5' monophosphate-nitric oxide system is in part responsible for portal hypertension in cirrhosis. AIM: To test the effects of inhibitors of phosphodiesterase-5 on portal haemodynamics. METHODS: To 18 healthy subjects and 18 patients with Child A liver cirrhosis, 10 mg of vardenafil, an inhibitor of phosphodiesterase-5, were administered orally. Doppler sonographic measurements of hepatic and splanchnic blood flow, systemic blood pressure and heart rate were recorded before, 1 h after, and 48 h after the application. Vardenafil plasma levels were determined after 1 h. In five patients, invasive registration of free and wedged hepatic vein pressure was performed. RESULTS: Portal venous flow increased in patients from 0.82 +/- 0.30 L/min (mean +/- s.d.) by 26% (CI: 16-37%, P = 0.0004) and in healthy subjects from 0.75 +/- 0.20 L/min (mean +/- s.d.) by 19% (CI: 9-28%; P = 0.0010). Celiac and hepatic artery resistivity indices rose significantly. Systemic blood pressure decreased slightly in patients. The wedged hepatic venous pressure gradient decreased in four of five patients with liver cirrhosis. Vardenafil plasma levels were higher in patients (14 +/- 10 microg/L) than in healthy subjects (9 +/- 6 microg/L; n.s.). CONCLUSIONS: Inhibition of phosphodiesterase-5 increases portal flow and lowers portal pressure by a decrease in sinusoidal resistance and may be a novel therapeutic strategy for portal hypertension.

Growth suppression of the hepatocellular carcinoma cell line Hepa1-6 by an activatable interferon regulatory factor-1 in mice.

Hepatocellular carcinoma (HCC) is a highly malignant tumor with a poor prognosis and few therapeutic options. The aim of the study was to evaluate the potential of IFN regulatory factor-1 (IRF-1) for cytokine gene therapy of HCC using an IRF-1/human estrogen receptor fusion protein (IRF-1hER), which is reversibly activatable by beta-estradiol (E2). IRF-1hER stably expressing murine Hepa1-6 HCC cells (HepaIRF-1hER) were characterized by lowMHC 1, highCD54, and lack of MHC II, CD80, and CD86 expression. Activation of HepaIRF-1hER cells induced a highMHC I, lowMHC II, and highCD54 phenotype. Furthermore, they were characterized by IFN-beta secretion, decreased anchorage-independent growth in a soft agar assay, and diminished cell growth. Tumor growth in E2-treated syngeneic C57L/J mice, but not in E2-untreated mice, was suppressed. These E2-treated mice were protected against rechallenge with HepaIRF-1hER and wild-type Hepa1-6 tumors even in the absence of E2, suggesting induction of tumor specific immunity. In fact, significant CTL activity against Hepa1-6 tumors and the endogenously expressed HCC-specific self antigen alpha-fetoprotein was observed. Antitumoral effects, however, were only partially dependent on both CD4+ and CD8+ T cells. IRF-1 treatment of mice bearing HepaIRF-1hER tumors resulted in growth arrest of tumors, and a significant survival benefit was observed in comparison to E2-untreated mice. In conclusion, our data demonstrate that IRF-1 suppresses HCC growth through both a direct antitumor growth effect and enhanced immune cell recognition of the tumor and is a promising candidate for gene therapy of HCC.

The transcription factor c-JUN/AP-1 promotes HBV-related liver tumorigenesis in mice.

Hepatocellular carcinoma (HCC) develops as a consequence of chronic inflammatory liver diseases such as chronic hepatitis B virus (HBV) infection. The transcription factor c-Jun/activator protein 1 (AP-1) is strongly expressed in response to inflammatory stimuli, promotes hepatocyte survival during acute hepatitis and acts as an oncogene during chemically induced liver carcinogenesis in mice. Here, we therefore aimed to characterize the functions of c-Jun during HBV-related liver tumorigenesis. To this end, transgenic mice expressing all HBV envelope proteins (HBV(+)), an established model of HBV-related HCC, were crossed with knockout mice lacking c-Jun specifically in hepatocytes and tumorigenesis was analyzed. Hepatic expression of c-Jun was strongly induced at several time points during tumorigenesis in HBV(+) mice, whereas expression of other AP-1 components remained unchanged. Importantly, formation of premalignant foci and tumors was strongly reduced in HBV(+) mice lacking c-Jun. This phenotype correlated with impaired hepatocyte proliferation and increased expression of the cell cycle inhibitor p21, whereas hepatocyte survival was not affected. Progression and prognosis of HBV-related HCC correlates with the expression of the cytokine osteopontin (Opn), an established AP-1 target gene. Opn expression was strongly reduced in HBV(+) livers and primary mouse hepatocytes lacking c-Jun, demonstrating that c-Jun regulates hepatic Opn expression in a cell-autonomous manner. These findings indicate that c-Jun has important functions during HBV-associated tumorigenesis by promoting hepatocyte proliferation as well as progression of dysplasia. Therefore, targeting c-Jun may be a useful strategy to prevent hepatitis-associated tumorigenesis.

T cell response in hepatitis C virus infection.

Hepatitis C virus (HCV) is a hepatotropic RNA virus that causes acute and chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. It is widely accepted that cellular immune responses play an important role in viral clearance and disease pathogenesis. However, HCV often evades effective immune recognition and has a propensity to persist in the majority of acutely infected individuals (ca. 80%). The immunological and virological basis for the inefficiency of the cellular immune response to clear or control the virus is not known. Recent studies, however, have provided new insights into the mechanisms of viral clearance and persistence that will be discussed in detail.

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.

Genetic variants of hepatitis B virus and their clinical relevance.

Infection with hepatitis B virus (HBV) leads to a wide spectrum of clinical presentations ranging from an asymptomatic carrier state to self-limited acute or fulminant hepatitis to chronic hepatitis with progression to cirrhosis and hepatocellular carcinoma (HCC). Infection with HBV is one of the most common viral diseases affecting man. Both viral factors as well as the host immune response have been implicated in the pathogenesis and clinical outcome of HBV infection. Evidence has been accumulating that HBV mutants are associated with certain clinical disease manifestations, may affect the natural course of the infection and confer resistance to antivirals. Naturally occurring mutations have been identified in the structural and non-structural genes as well as regulatory elements of the virus. The best characterized mutants comprise the pre-core (pre-C) stop codon mutation resulting in a loss of hepatitis B e antigen (HBeAg), defined clusters of mutations in the core promotor resulting in enhanced viral replication and mutations in the hepatitis B core and surface antigens (HBcAg and HBsAg) altering the antigenicity of the virus. More recently, several mutations in the reverse transcriptase/polymerase gene have been identified conferring resistance to antivirals used for the treatment of chronic hepatitis B. In this review, we will focus on the biological phenotype of HBV genetic variants and discuss their clinical relevance for the pathogenesis of HBV-induced liver disease.

Cationic liposome-mediated gene delivery to the liver and to hepatocellular carcinomas in mice.

The potential of cationic liposomes as nonviral vectors for in vivo gene delivery to the liver and to intrahepatic hepatocellular carcinoma (HCC) was investigated. Mice were injected via the tail vein or portal vein with a cationic lipid complexed to plasmid DNA (pDNA) encoding the chloramphenicol acetyltransferase (CAT) reporter gene at various cationic lipid:pDNA molar ratios to analyze the efficiency of gene delivery after intravenous administration. Tail vein injection resulted in high CAT expression levels in lung and spleen and low levels in the liver. Portal vein injection, by comparison, significantly enhanced hepatic reporter gene expression but also resulted in pronounced hepatic toxicity. Gene delivery to intrahepatic tumors produced by intrahepatic injection of human HCC cells was analyzed in nude mice. Tail vein injection as well as portal vein injection resulted in low levels of gene expression in intrahepatic tumors. By comparison, high levels of gene expression were achieved by direct, intratumoral injection of liposome-pDNA complexes, with only minimal expression in the surrounding normal liver. Therefore, direct liposome-pDNA complex injection appears far superior to systemic or portal intravenous administration for gene therapy of localized intrahepatic tumors, and may be a useful adjunct in the treatment of human HCCs.

Antiviral activities of penciclovir and famciclovir on duck hepatitis B virus in vitro and in vivo.

Chronic hepatitis B virus (HBV) infection is a major health problem worldwide. Antiviral strategies available at present, including interferon-alpha, have only limited efficacy, leading us to analyse the antiviral effects of penciclovir and famciclovir in the duck hepatitis B virus (DHBV) model of HBV infection in vitro and in vivo. In DHBV-infected duck hepatocytes, penciclovir effectively inhibited viral replication, with a concentration giving half-maximal inhibition of 0.25 microM. Furthermore, in vivo, penciclovir and its orally administered prodrug famciclovir strongly inhibited DHBV replication. These data demonstrate that penciclovir and famciclovir both have strong antiviral activities, and suggest that these agents might be useful for treating HBV infection in humans.

Colorectal cancer: future population screening for early colorectal cancer.

Colorectal cancer (CRC) is one of the most frequent cancers in Western countries. The identification of individuals at risk and the early diagnosis of CRC are of critical importance since a large proportion can be prevented or cured by surgical removal before metastasis has occurred. With increasing understanding of the genetic basis of hereditary and sporadic (non-hereditary) CRC, it becomes feasible to detect genetic alterations by molecular techniques. Familial adenomatous polyposis (FAP), hereditary nonpolyposis colorectal cancer (HNPCC), as well as early stages of spontaneous CRC, can be diagnosed by molecular characterisation of the adenomatous polyposis coli (APC) gene, the RAS oncogene and other genes in DNA from peripheral blood, stool or intestinal biopsies. With a better understanding of the genetic events leading to malignant transformation, molecular population screening should allow us to identify individuals at risk as well as patients with an early and potentially curable CRC. At present, careful patient and family history, physical examination and testing for occult blood as well as colonoscopy are still the key elements for clinical patient management. Molecular diagnosis will hopefully soon complement these analyses and should result in a reduction of morbidity and mortality from CRC.

Hepatitis B virus replication and viral antigen synthesis in hepatocyte lines derived from normal human liver.

Transient transfection and in vitro infection experiments were performed to characterize replication and antigen synthesis of the hepatitis B virus (HBV) in human hepatocyte lines HH29 and HHY41, derived from normal liver tissue. These liver cell lines are capable of supporting HBV replication and gene expression at levels similar to the human hepatoma cell line HuH-7. Strikingly, a very tight adhesion of HBV to the outer cell membrane of HH29 and HHY41 was observed under conditions that removed HBV to undetectable levels from HuH-7 hepatoma cells. However, no productive HBV infection could be established in these cells as determined by the absence of viral transcripts and de novo antigen synthesis. In conclusion, the human hepatocyte cell lines HH29 and HHY41 may be useful to study important aspects of late steps in the replication of HBV, but appear to lack certain cellular components that play a pivotal role during early steps of the viral life cycle.

Hepatocellular carcinoma and hepatitis B virus infection: molecular evidence for monoclonal origin and expansion of malignantly transformed hepatocytes.

The clonality of tumor cells was studied in a patient with metastasizing hepatocellular carcinoma (HCC). Using hepatitis B virus (HBV) DNA as a genetic marker, the pattern of integration of viral DNA into the tumor cell genome was determined by Southern blot analyses of DNAs extracted from different HCC lesions in the liver and both lungs. All tumor tissues examined were found to have viral DNA integrated into the same site(s) of the cellular genome. This finding provides direct molecular evidence for a monoclonal origin and expansion of malignantly transformed hepatocytes during tumor growth and metastasis. This characteristic is similar to other human cancers associated with viral infections, such as adult T-cell leukemia, Burkitt's lymphoma, or cervical cancer, and is important for our understanding of viral oncogenesis in man.