S.H. Yap

Synaptophysin: A novel marker for human and rat hepatic stellate cells.

Synaptophysin is a protein involved in neurotransmitter exocytosis and is a neuroendocrine marker. We studied synaptophysin immunohistochemical expression in 35 human liver specimens (normal and different pathological conditions), in rat models of galactosamine hepatitis and carbon tetrachloride-induced cirrhosis, and in freshly isolated rat stellate cells. Synaptophysin reactivity was present in perisinusoidal stellate cells in both human and rat normal liver biopsies. The number of synaptophysin-reactive perisinusoidal cells increased in pathological conditions. Double staining for alpha-smooth muscle actin and synaptophysin, detected by confocal laser scanning microscopy, unequivocally demonstrated colocalization of both markers in lobular stellate cells. In addition, freshly isolated rat stellate cells expressed synaptophysin mRNA (detected by polymerase chain reaction) and protein. Finally, electron microscopy showed the presence of small electron translucent vesicles, comparable to the synaptophysin-reactive synaptic vesicles in neurons, in stellate cell projections. We conclude that synaptophysin is a novel marker for quiescent as well as activated hepatic stellate cells. Together with the stellate cells expression of neural cell adhesion molecule, glial fibrillary acidic protein, and nestin, this finding raises questions about its embryonic origin and its differentiation. In addition, the presence of synaptic vesicles in stellate cell processes suggests a hitherto unknown mechanism of interaction with neighboring cells.

Mycophenolic acid, an immunosuppressive agent, inhibits HBV replication in vitro

Mycophenolate mofetil (MMF), the morpholinoethyl ester of mycophenolic acid (MPA), is currently used as an immunosuppressive agent in kidney transplant recipients. After oral administration, MMF is hydrolysed to MPA, the active compound, which is a potent inhibitor of inosine monophosphate dehydrogenase (IMP‐DH). Inhibition of this enzyme results in a depletion of the intracellular GTP and dGTP pools. MPA has been shown to inhibit the replication of a number of viruses, including arena viruses (Junin and Tacaribe), yellow fever virus, reovirus‐1, parainfluenza‐3 virus, Coxsackie B4 virus, Epstein–Barr virus and human immunodeficiency virus. To examine whether MPA also has an inhibitory effect on HBV replication, experiments were performed using cultures of primary human hepatocytes and HBV‐transfected, HepG2 2.2.15 cells. After in vitro infection with HBV in human hepatocytes, HBV covalently‐closed‐circular (ccc) DNA and HBV mRNAs were detectable in the cells during the 10 days following infection. HBV DNA and hepatitis B surface antigen (HBsAg) were also secreted into the culture medium. In the presence of 10 μg ml–1 MPA (the therapeutic serum level of MPA as an immunosuppressive agent) in culture medium, HBV ccc DNA and HBV mRNAs became undetectable 5 days after treatment was started. The secretion of HBV DNA and HBsAg into the medium was also markedly reduced. No cytotoxic effect of the drug was noted during the experiments. The effect of MPA on HBV replication was abolished by the presence of guanosine (50 μg ml–1). In HepG2 2.2.15 cells (which contain an integrated tandem dimer of the HBV genome), MPA treatment had no significant inhibitory effect on the secretion of HBV DNA and HBsAg into the culture medium. HBV ccc DNA and HBV mRNAs in HepG2 2.2.15 cells were also not affected. The observed effect of MPA on HBV replication in primary human hepatocyte cultures may involve only episomal replication and may have clinical implications, especially before integration of HBV DNA into the host genome.

Hepatitis B virus infection in microcarrier‐attached immortalized human hepatocytes cultured in molecularporous membrane bags: a model for long‐term episomal replication of HBV

Studies on the pathobiology of chronic (long‐term) hepatitis B virus (HBV) infection and in vitro drug testing have been hampered by the lack of appropriate systems for culturing susceptible cells chronically infected with HBV. Most of the in vitro studies of HBV replication have been performed with HBV genome‐transduced cell lines. In this system, viral production is mainly the result of chromosomal replication. In an invitro infection system, owing to medium refreshment (which leads to the removal of infectious particles necessary for the perpetuation of infection) and to trypsinization for cell passages, it is difficult, if not impossible, to maintain chronic HBV infection, despite the use of susceptible cells. To circumvent these unfavourable factors for chronic HBV infection in vitro, we cultured microcarrier‐attached immortalized human hepatocytes, infected with HBV, in molecularporous (MW 12000–14000) membrane (dialysis) bags for a duration of 2 months. HBV covalently‐closed‐circular (ccc) DNA, HBV precore/core and X mRNAs were detected in the cells cultured in this system following infection until the end of the experiment (day 58), while in classical culture conditions (monolayer), markers of HBV replication were also detected. Production of hepatitis B surface antigen (HBsAg) and HBV DNA was detected and their levels in culture medium (collected at the end of experiments from the molecularporous membrane bags) were increased 2.86‐ and 3.28‐fold respectively. Using Southern blot analysis, HBV replicative intermediates could also be demonstrated throughout the experiments. However, integrated HBV DNA was not present. In contrast, HBV ccc DNA, HBV precore/core and X mRNAs, and replicative intermediates were not demonstrable in FTO 2B rat hepatoma cells infected in the same manner in parallel experiments. This in vitro infection system, using susceptible, immortalized human hepatocytes, therefore provides a new tool for studying the long‐term effect of HBV infection, mainly involving episomal replication in hepatocytes, and for drug testing.

Use of digoxigenin-labelled probes for the quantitation of HBV-DNA in antiviral drug evaluation.

The use of digoxigenin-labelled probes was studied for quantitation of HBV-DNA during antiviral drug evaluation. Digoxigenin (dig)-labelled probes were generated either via incorporation of dig-dUTP in a polymerase chain reaction (PCR) or a random priming reaction. Using the PCR-labelled probe (delineating a 523 bp fragment in the core gene of the HBV) as little as 1 pg of immobilized HBV-DNA could be detected following an 8 h exposure of the hybridized membrane. A close correlation (r = 0.95) was found between the amount of HBV-DNA (range 2.5-200 pg) and the signal generated by the probe hybridized to its target DNA. By using a probe that was labelled with digoxigenin via random priming, the minimal quantity of immobilized HBV plasmid DNA that could be detected following an 8 h exposure was 4 pg, whereas a 32P-labelled probe, generated in parallel by random priming, allowed the detection of 16 pg of HBV plasmid DNA following a 4-day exposure. The PCR-generated digoxigenin-labelled probe proved to be useful for antiviral drug evaluation, i.e. to detect HBV-DNA in total cellular DNA from HBV-positive hepatoma cells (HepG2.2.15) that had either been treated with reference antiviral agents or left untreated. The 50% effective concentrations (EC50) that were calculated for inhibition of HBV-DNA production by lamivudine (3TC), penciclovir (PCV), lobucavir (LBV), adefovir (PMEA) and tenofovir (PMPA) were comparable to those reported in the literature. The use of digoxigenin-labelled probes thus appears to be a simple, convenient, rapid, reliable and non-radioactive method for use for anti-HBV screening. In addition, and in contrast to 32P-labelled probes, digoxigenin-labelled probes can be stored for >1 year without loss of specific activity, which makes these probes particularly attractive for large-scale antiviral drug evaluation purposes.

Hepatitis B virus : specific binding and internalization of small HBsAg by human hepatocytes

Previously, we identified human liver endonexin II (EII) present on human hepatocyte plasma membrane as a specific hepatitis B surface antigen (HBsAg) binding protein. We also showed the spontaneous development of anti-idiotypic (anti-HBsAg) antibodies in rabbits immunized with EII and in chicken immunized with the F(ab)2 fragment of rabbit anti-EII IgG. These findings suggest the existence of a receptor-ligand relationship between EII and HBsAg. In the present study, we demonstrate that small HBsAg conjugated to 10 nm colloidal gold also binds specifically to human hepatocytes. Invagination of the coated pit region at the HBsAg binding sites on the human hepatocyte plasma membrane results in the internalization of the HBsAg-gold particles. The binding and consequently the internalization of HBsAg is inhibited by anti-EII or anti-idiotypic (anti-HBsAg) antibodies. These findings indicate that EII is directly involved in the binding and uptake of hepatitis B envelope proteins.

Hepatitis δ virus attaches to human hepatocytes via human liver endonexin II, a specific HBsAg binding protein

SUMMARY. Recently we identified human liver endonexin II (EII) as a specific hepatitis B surface antigen (HBsAg) binding protein. To investigate whether EII is also able to interact with the HBsAg envelope of the hepatitis δ virus (HδV). immunoprecipitation experiments were performed. HδV particles could be co‐precipitated by polyclonal rabbit anti‐EII, but not by rabbit anti‐glutathiontransferase (GSTφ) antibodies from an HδV‐enriched fraction containing EII or GSTφ. These findings suggest that HδV particles were co‐precipitated by anti‐EII as a consequence of the binding between HBsAg present in the HδV envelope and EII. Furthermore, binding of HδV particles to human hepatocytes could be inhibited by incubation of the liver cells with rabbit anti‐EII IgG or the HδV particles with anti‐idiotypic (anti‐HBsAg) antibodies, developed spontaneously in rabbits immunized with EII. These findings support the assumption that small HBsAg present in the HδV envelope is important for the attachment to the hepatocytes and that EII plays an important role in this process.

HVR1 quasispecies analysis from a long-term culture of hepatitis C virus in Hep G2 derived cells grown in a haemodialysis cartridge

Studies on the in vitro hepatitis C virus (HCV) infection are hampered by the lack of an appropriate system to culture permissive cells to be continuously infected with HCV. Trypsinization required for cell passage can lead to possible temporary loss of permissiveness for infection, whereas refreshment of the medium can result in loss of infectious particles necessary for perpetuation of the infection; it is therefore very difficult to maintain a continuous HCV infection in cell cultures. A new infection method was designed and evaluated in order to prevent these unfavourable circumstances. A cell line derived from the human hepatoblastoma cell line Hep G2 was grown in the extracapillary space of a haemodialysis cartridge, in the presence of a HCV‐positive inoculum, while the culture medium was recirculated through the intracapillary space, supplying the cells with nutrients and oxygen. HCV RNA could continuously be detected in the cells up to 77u2003days of culture. Sequence analysis of the HCV hypervariable region 1 (HVR1) revealed that 56% and 75%, respectively, of the clones obtained from the cells at day 20 and 40 after start of the infection were different from the clones obtained from the original inoculum and that certain nucleotide positions in this region were more susceptible to mutations, leading to an alteration in amino acid sequence. As none of these sequences were present in the clones from the inoculum, it is suggested that new HCV quasispecies have emerged as a result of viral replication in the hepatocytes in vitro. This system seems a valuable tool for the in vitro evaluation of antiviral drugs.

Concomitant findings of anti-HBc as a sole marker for hepatitis B virus infection and elevated serum transaminases are associated with a high prevalence of hepatitis C virus infection

Abstract From two hundred sixty five patients with liver disease and elevated serum transaminases who were admitted or referred to Atmajaya academic hospital in Jakarta, 37 patients were found to be anti-HBc positive as a sole marker of HBV infection. Nineteen out of these 37 patients were also anti-HCV positive, HCV-RNA was detected in 16 patients. From 1656 individuals with normal serum transaminases who underwent serological examination and consisted of 489 subjects from population based study in Kalianyar (an urban area in Jakarta), 258 students of Atmajaya Medical School, 541 women and 162 children from maternity-child clinic and 206 patients admitted to hospital without evidence of liver disease, anti-HBc as a sole marker for HBV infection was found in 213. However, HCV-RNA was detected only in 2 subjects. We conclude that concomitant findings of anti-HBc as a sole marker for HBV infection and elevated ALT are associated with a relative high prevalence of HCV infection. In a group of individuals without evidence of liver disease, HCV infection was uncommon, even when anti-HBc is present as a sole marker of HBV infection.