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ABSTRACT: 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 77 days 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.
Journal of Viral Hepatitis 04/2001; 8(2):132-8. · 4.09 Impact Factor
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ABSTRACT: Six 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (the present cholesterol-lowering drugs known as statins), lovastatin (L), simvastatin (S), pravastatin (P), fluvastatin (F), atorvastatin (A) and cerivastatin (C) are shown to be potent inhibitors of cholesterol synthesis in human hepatocytes, the target tissue for these drugs in man. All six inhibited in the nM range (IC(50) values: 0.2-8.0 nM). As daily used cholesterol-lowering drugs they are likely coadministered with other drugs. While several cytochrome P450 (CYP) enzymes are involved in drug metabolism in the liver and thus play an important role in drug-drug interaction it was investigated which of these enzymes are influenced by the active forms of the six statins. These enzyme activities were studied in human liver microsomal preparations, and in simian and human hepatocytes in primary culture. The following CYP reactions were used: nifedipine aromatization (CYP3A4), testosterone 6beta-hydroxylation (CYP3A4), tolbutamide methylhydroxylation (CYP2C9), S-mephenytoin 4-hydroxylation (CYP2C19), bufuralol 1'-hydroxylation (CYP2D6), aniline 4-hydroxylation (CYP2E1), coumarin 7-hydroxylation (CYP2A6) and 7-ethoxyresorufin O-dealkylation (CYP1A1/2). In the human liver microsomes the statins (concentrations up to 400 microM) did not influence the CYP1A1/2 activity and hardly the CYP2A6 and CYP2E1 activities. Except P, the other five statins were stronger inhibitors of the CYP2C19 activity with IC(50) values around 200 microM and the same holds for the effect of A, C and F on the CYP2D6 activity. L and S were weaker inhibitors of the latter enzyme activity, whereas P did not influence both activities. About the same was observed for the statin effect on CYP2C9 activity, except that F was a strong inhibitor of this activity (IC(50) value: 4 microM). Using the assay of testosterone 6beta-hydroxylation the CYP3A4 activity was decreased by L, S and F with IC(50) values of about 200 microM and a little more by C and A (IC(50) around 100 microM). P had hardly an effect on this activity. To a somewhat less extent the same trend was seen when CYP3A4 activity was measured using nifedipine as substrate. The inhibitory effects observed in microsomes were verified in suspension culture of freshly isolated hepatocytes from Cynomolgus monkey (as a readily available model) and of human hepatocytes. In general the same trends were seen as in the human microsomes, except that in some cases the inhibition of the CYP activity was less, possibly by the induction of the particular CYP enzyme by incubation of the cells with a particular statin. F remained a strong inhibitor of CYP2C9 activity in human and monkey hepatocytes. A induced the CYP2C9 in monkey hepatocytes but was an inhibitor of the CYP2C9 in human hepatocytes. A, S, L and C were moderate inhibitors in both cellular systems of CYP3A4. P was not affecting any of the CYP activities in the three systems studied. It is concluded that different CYP enzymes interact with different statins and therefore differences in between these drugs are to be expected when drug-drug interaction is considered.
Biopharmaceutics & Drug Disposition 01/2001; 21(9):353-64. · 2.07 Impact Factor
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ABSTRACT: Six 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (the present cholesterol-lowering drugs known as statins), lovastatin (L), simvastatin (S), pravastatin (P), fluvastatin (F), atorvastatin (A) and cerivastatin (C) are shown to be potent inhibitors of cholesterol synthesis in human hepatocytes, the target tissue for these drugs in man. All six inhibited in the nM range (IC50 values: 0.2–8.0 nM). As daily used cholesterol-lowering drugs they are likely coadministered with other drugs. While several cytochrome P450 (CYP) enzymes are involved in drug metabolism in the liver and thus play an important role in drug–drug interaction it was investigated which of these enzymes are influenced by the active forms of the six statins. These enzyme activities were studied in human liver microsomal preparations, and in simian and human hepatocytes in primary culture. The following CYP reactions were used: nifedipine aromatization (CYP3A4), testosterone 6β-hydroxylation (CYP3A4), tolbutamide methylhydroxylation (CYP2C9), S-mephenytoin 4-hydroxylation (CYP2C19), bufuralol 1′-hydroxylation (CYP2D6), aniline 4-hydroxylation (CYP2E1), coumarin 7-hydroxylation (CYP2A6) and 7-ethoxyresorufin O-dealkylation (CYP1A1/2). In the human liver microsomes the statins (concentrations up to 400 µM) did not influence the CYP1A1/2 activity and hardly the CYP2A6 and CYP2E1 activities. Except P, the other five statins were stronger inhibitors of the CYP2C19 activity with IC50 values around 200 µM and the same holds for the effect of A, C and F on the CYP2D6 activity. L and S were weaker inhibitors of the latter enzyme activity, whereas P did not influence both activities. About the same was observed for the statin effect on CYP2C9 activity, except that F was a strong inhibitor of this activity (IC50 value: 4 µM). Using the assay of testosterone 6β-hydroxylation the CYP3A4 activity was decreased by L, S and F with IC50 values of about 200 µM and a little more by C and A (IC50 around 100 µM). P had hardly an effect on this activity. To a somewhat less extent the same trend was seen when CYP3A4 activity was measured using nifedipine as substrate. The inhibitory effects observed in microsomes were verified in suspension culture of freshly isolated hepatocytes from Cynomolgus monkey (as a readily available model) and of human hepatocytes. In general the same trends were seen as in the human microsomes, except that in some cases the inhibition of the CYP activity was less, possibly by the induction of the particular CYP enzyme by incubation of the cells with a particular statin. F remained a strong inhibitor of CYP2C9 activity in human and monkey hepatocytes. A induced the CYP2C9 in monkey hepatocytes but was an inhibitor of the CYP2C9 in human hepatocytes. A, S, L and C were moderate inhibitors in both cellular systems of CYP3A4. P was not affecting any of the CYP activities in the three systems studied. It is concluded that different CYP enzymes interact with different statins and therefore differences in between these drugs are to be expected when drug–drug interaction is considered. Copyright © 2000 John Wiley & Sons, Ltd.
Biopharmaceutics & Drug Disposition 11/2000; 21(9):353 - 364. · 2.07 Impact Factor
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ABSTRACT: Previously, we have determined that human annexin V (hAV), a Ca2+-dependent phospholipid-binding protein, and not rat AV, binds specifically to small hepatitis B surface antigen (SHBsAg), and that transfection of a rat hepatoma cell line with a construct containing the hAV gene led to hAV expression and conferred susceptibility to hepatitis B virus (HBV) infection. In this work, we have examined the effect of administration of hAV on in vitro binding of SHBsAg to human and to rat hepatocytes and on in vitro HBV infection. The results showed that hAV could bind to human as well as to rat hepatocytes. Because of this property, excess hAV was unable to prevent HBV infection in primary cultures of human hepatocytes. On the other hand, it enabled rat hepatocytes to specifically bind SHBsAg and conferred susceptibility to HBV infection. After infection of primary cultures of rat hepatocytes in the presence of hAV, HBV mRNA, covalently closed circular (ccc) DNA, replicative intermediates, hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg) and secreted HBV DNA were detected. After infection in the absence of hAV, no markers of HBV replication were detected. Hence, from the present study we conclude that hAV is involved in facilitating HBV entry, leading to successful HBV infection in primary cultures of rat hepatocytes, while it is not effective in preventing HBV infection in primary cultures of human hepatocytes.
Journal of Viral Hepatitis 04/2000; 7(2):104-14. · 4.09 Impact Factor
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ABSTRACT: 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 cell's 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.
American Journal Of Pathology 01/2000; 155(6):1831-9. · 4.89 Impact Factor
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ABSTRACT: We have previously demonstrated that human liver Annexin V (hAV), a Ca2+-dependent phospholipid binding protein, binds specifically to small HBsAg (SHBsAg). Because of the propensity of AV to bind phospholipids, we here examine the role of phospholipids, as component of the HBV envelope, in binding to hAV and in HBV infection.
The influence of phospholipids (phosphatidylserine and phosphatidylcholine) on the binding of hAV to SHBsAg or to anti-hAV monoclonals was determined by ELISA. Their influence on HBV infection was investigated using an in vitro HBV infection assay.
Two monoclonals, specific against hAV, were able to block the binding of hAV to SHBsAg and recognized different epitopes of hAV. The binding of one of these monoclonals to hAV could be inhibited by phosphatidylserine, but not by phosphatidylcholine. Further experiments revealed that phosphatidylserine could also inhibit the binding of hAV to SHBsAg and could even prevent HBV infection in vitro. Phosphatidylcholine had no effect on the binding of hAV to SHBsAg and could not prevent HBV infection in vitro. However, since phosphatidylserine was not able to abolish the binding of the other blocking monoclonal to hAV, a non-phospholipid component of the HBV envelope must also be involved in hAV binding.
These results indicate that phosphatidylserine and a non-phospholipid component of the HBV envelope are involved in hAV binding and in HBV infection.
Journal of Hepatology 12/1999; 31(5):783-90. · 9.26 Impact Factor
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ABSTRACT: 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.
Journal of Virological Methods 09/1999; 81(1-2):155-8. · 2.01 Impact Factor
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ABSTRACT: Previously, we have shown that small hepatitis B surface antigen (SHBsAg) binds specifically to human annexin V (hAV) and that hAV plays a key role in the initial steps of hepatitis B virus (HBV) infection. We have also demonstrated the spontaneous development of anti-idiotypic antibodies (antibodies to HBsAg Ab2) in rabbits immunized with hAV. As Ab2 is able to inhibit the binding of hAV to SHBsAg, Ab2 might contain epitope(s) mimicking a region of hAV for binding to SHBsAg. Identification of this epitope will therefore reveal a SHBsAg sequence involved in hAV binding. Using a panel of synthetic peptides covering the region of SHBsAg located on the outer surface of the virus, binding studies showed that the region incorporating amino acids (aa) 125-131 of SHBsAg is important for binding to Ab2 and consequently also for binding to hAV. Further experiments revealed that not only this region, but also the region incorporating aa 158-169, is involved in the binding of SHBsAg to hAV. As these regions are located in the structural vicinity according to the topological model of HBsAg proposed by Chen et al., our findings suggest that these regions are parts of a conformational epitope of SHBsAg for binding to hAV. Because of the crucial role of hAV in HBV infection, further studies on the HBsAg epitopes for hAV binding may lead to the development of a new generation of vaccines or molecules for prevention and for treatment of patients with chronic hepatitis B.
Journal of Viral Hepatitis 07/1999; 6(4):277-85. · 4.09 Impact Factor
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ABSTRACT: 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 microg 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 microg 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.
Journal of Viral Hepatitis 05/1999; 6(3):229-36. · 4.09 Impact Factor
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ABSTRACT: Previously, we have found that human liver annexin V (hA-V; in earlier reports referred as Endonexin II) is a specific hepatitis B surface antigen (HBsAg) binding protein. In this study, we demonstrate that transfection of rat hepatoma FTO 2B cells, a cell line that is not infectable by hepatitis B virus (HBV) and does not express hA-V, with a construct containing the hA-V gene, resulted in hA-V expressing cells susceptible to HBV infection. After in vitro infection, transfected FTO cells (assigned as FTO 9.1 cells) expressing hA-V in cultures were shown to contain HBV-precore/core, X mRNAs, and covalently closed circular (ccc) DNA as detected by polymerase chain reaction (PCR). The presence of HBV ccc and replicative intermediate DNA was also demonstrated by Southern blot hybridization assay. HBV DNA secreted in the culture medium was also evident as determined by quantitative branched DNA (bDNA) assay. HBsAg and hepatitis B core antigen (HBcAg) could also be detected by an immunocytochemical method in 10% to 15% of the cells at day 3 and day 5 after infection. Infectivity of in vitro-propagated HBV was demonstrated by infection of the naive FTO 9.1 cells with the culture supernatant from HBV-carrier cultures. In contrast to primary cultures of human hepatocytes and FTO 9.1 cells, primary rat and mouse hepatocytes, as well as rat hepatoma cell lines that do not express hA-V, are not susceptible to HBV infection. These findings suggest that hA-V plays a key role in the initial step of HBV infection and that the species-specific susceptibility to HBV infection and replication in hepatocytes is associated with the expression of hA-V.
Hepatology 03/1999; 29(2):576-84. · 11.66 Impact Factor
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ABSTRACT: 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 in vitro 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 12,000-14,000) 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.
Journal of Viral Hepatitis 12/1998; 5(6):377-87. · 4.09 Impact Factor
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L Sheng,
A Soumillion,
N Beckers,
C G Wu,
C Verslype,
F Nevens,
J Pirenne,
R Aerts,
H Kosala,
J Fevery, S H Yap
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ABSTRACT: Hepatitis G virus (HGV) is a recently discovered RNA virus, which belongs to the Flaviviridae family. Although HGV infection is usually not associated with elevated serum transaminases, some recent studies have reported that HGV infection is found in a significant number of patients with fulminant hepatitis and may play a role in its etiopathogenesis. In this study the prevalence of HGV infection was determined in 500 healthy blood donors and in 24 patients admitted to hospital because of acute liver failure caused by fulminant hepatitis. The presence of HGV RNA was tested in sera, obtained at admission and before any transfusion was given, by a sensitive seminested reverse transcriptase-polymerase chain reaction (RT-PCR) assay specific for detection of the non-structural (NS)5 region. Nine of the 500 blood donors (1.8%) and two of the 24 patients (8.3%) were found to be HGV RNA positive. One patient was co-infected with HCV and was known to be an intravenous (i.v.) drug user. After intensive supporting treatment, this patient recovered completely. The second patient had no serological markers of known viral hepatitis infection, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV), Epstein-Barr virus (EBV) and herpes simplex virus (HSV). This patient was successfully transplanted. From both patients, from HGV RNA-positive healthy blood donors and from other patients coinfected with HCV, a part of the HGV NS3 region (nucleotides 4191-4345, EMBL entry U45966) was cloned and sequenced. Sequence comparison revealed that the NS3 region of HGV in patients with fulminant hepatitis contained three nucleotide substitutions as part of the six substitutions described in previous work. These nucleotide substitutions were not found in the tested blood donors or in patients with HCV co-infection. Our findings therefore support the concept of the association of fulminant hepatitis with infection of a specific HGV strain.
Journal of Viral Hepatitis 10/1998; 5(5):301-6. · 4.09 Impact Factor
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L Sheng,
A Soumillion,
K Peerlinck,
C Verslype,
R Schelstraete,
F Gyselinck,
M P Emonds,
G Hess,
J Vermylen,
J Desmyter, S H Yap
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ABSTRACT: In a previous study, we have determined the prevalence of serum HGV-RNA in patients with congenital clotting disorders. Twenty-six (15%) of 175 patients investigated were serum HGV-RNA positive. In addition, HGV-RNA was detectable in peripheral blood mononuclear cells (PBMC) in ten percent of the cases, three of these patients were serum HGV-RNA negative. In the present study, we have determined the prevalence of anti-HGV-E2 antibodies in the same patient population. Anti-HGV-E2 as determined by ELISA was detected in 45 patients (25.7%). Forty of these patients were serum HGV-RNA negative. Ninety-two percent of the 26 HGV viremic patients and all but one patient (44 patients) with detectable anti-HGV-E2 had coinfection with the hepatitis C virus (HCV). Of these coinfected patients, 62.5% of HGV viremic patients and 53% of anti-HGV-E2 positive patients showed elevated serum ALT levels. Anti-HGV-E2 seroconversion is thus not associated with HCV infection. Two patients who were solely infected with HGV had normal serum ALT levels. In a retrospective longitudinal study, we have observed in 15 patients that serum HGV-RNA persisted during one to 19 years of follow-up, while anti-HGV-E2 was repeatedly negative. Five additional patients who were anti-HGV-E2 positive with concomitant detectable HGV-RNA (4 patients in serum and 1 patient in PBMC) became HGV-RNA negative during follow-up, ranging from 1 to 8 years after the first detection of anti-HGV-E2 antibodies. Two patients had lost anti-HGV-E2 antibodies 3 to 6 years after the seroconversion without the re-appearance of serum HGV-RNA. From these findings, it is clear that the prevalence rate of HGV infection in patients with clotting disorders as determined by PCR assay for HGV-RNA and anti-HGV-E2 by ELISA is actually higher than the prevalence of HGV viremia. Although HGV viremia may persist for longer than 19 years, most of the patients infected with HGV may clear the viremia spontaneously. The clearance of viremia is usually associated with seroconversion to anti-HGV-E2. In addition, anti-HGV-E2 may be lost during years of follow-up without the reappearance of the HGV-RNA. Although HGV infection does not seem to influence the fate of HCV infection and does not induce increased levels of serum ALT, the clinical significance of long-term infection remains to be established.
Thrombosis and Haemostasis 05/1998; 79(4):752-5. · 5.04 Impact Factor
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L Sheng,
A Widyastuti,
H Kosala,
J Donck,
Y Vanrenterghem,
E Setijoso,
A Soumillion,
C Verslype,
R Schelstraete,
M P Emonds,
G Hess, S H Yap
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ABSTRACT: A recently discovered non-A-E hepatitis virus has been designated as hepatitis G virus (HGV) and identified as a new member of the Flaviviridae family. Infection by this virus is thought to be associated with blood-borne hepatitis and usually in the presence of hepatitis C or hepatitis B virus (HBV) infection. In this study, the presence of HGV-RNA in serum or plasma and the prevalence of antibodies against an HGV envelope protein (E2) were investigated in patients undergoing chronic hemodialysis using a sensitive reverse-transcriptase polymerase chain reaction and an enzyme-linked immunosorbent assay, respectively. HGV-RNA was detected in 19 of 112 patients investigated (17%) and anti-E2 antibodies were detected in 15 of 106 patients studied (14.2%). With the exception of two patients, the appearance of anti-E2 is associated with the clearance of serum HGV-RNA. The total prevalence of current (HGV-RNA positivity) and/or past (anti-E2 positivity) HGV infection in this patient population is thus 28.6% (32 of 112 patients were positive for serum HGV-RNA and/or anti-E2 antibodies). In apparently healthy blood donors, serum HGV-RNA was detected in four of 358 individuals (1.12%) and anti-E2 was not detected in 50 individuals investigated. From the 19 patients with serum HGV-RNA positivity, nine were coinfected with other hepatitis viruses (seven with HBV; one with HBV, hepatitis C virus [HCV], and hepatitis D virus; and one with HBV and cytomegalovirus). Thirteen of 15 patients with anti-E2 positivity (10 were positive for only anti-E2 and three were also positive for anti-HBc) had no detectable HGV-RNA. In two patients, both HGV-RNA and anti-E2 antibodies were concomitantly present (both patients were coinfected with HCV or HBV). Of the HGV-infected patients, only three who were coinfected with HBV showed elevated serum alanine aminotransferase levels. The serum HCV-RNA and/or anti-HCV were detected in five (4.5%) of 112 patients. From these findings, we conclude that there is a high prevalence of HGV infection (28.6%) compared with HCV (4.5%) in patients undergoing hemodialysis in our hospital. However, approximately 50% of patients had spontaneously lost the viremia and developed anti-HGV-E2 antibodies. We confirm that HGV infection alone is not associated with elevated serum transaminases, and the appearance of anti-HGV-E2 is usually accompanied with clearance of serum HGV-RNA. In contrast to the results of our previous study, the majority of patients infected with HGV are not coinfected with HCV, indicating that HGV is capable of independent transmission. It is likely that there is a preferential HGV acquisition in the hemodialysis unit. The clinical significance of long-term infection with HGV remains to be established.
American Journal of Kidney Diseases 03/1998; 31(2):218-23. · 5.43 Impact Factor
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ABSTRACT: In a previous study, we have demonstrated that primary human hepatocytes in culture are susceptible for Plasmodium falciparum sporozoite invasion and for development of parasites into exo-erythrocytic forms. In a separate study we demonstrated the involvement of two human liver plasma membrane proteins (55 kD and 20 kD) in the invasion of P. falciparum sporozoites in vitro. In this study, we have unravelled the nature of the 55 kD protein.
For the identification of this protein, a 53-58 kD membrane protein fraction from human liver was isolated, radioactively labelled, incubated with sporozoites and cross-linked. After reduction of the cross-linker, the released proteins were mixed with unlabelled 53-58 kD protein fraction and separated on two-dimensional SDS-PAGE. Autoradiography showed a single spot corresponding to a protein of 55 kD and pI of 5.7-5.8.
Amino acid sequencing revealed the 55 kD protein as carboxylesterase. The biological activity of purified human liver carboxylesterase and of an antiserum against carboxylesterase on sporozoite invasion in vitro was evaluated. Human carboxylesterase as well as a rabbit antiserum against carboxylesterase inhibited the invasion of P. falciparum sporozoites into primary human hepatocytes in culture. A number of carboxylesterase cDNA clones were isolated from a human liver cDNA library. Sequence analysis revealed two different iso-types. Immunoaffinity purified recombinant human carboxylesterase was shown also to inhibit the invasion of sporozoites into primary human hepatocytes. Immunocytochemical analysis of the localisation of carboxylesterase in primary cultures of human hepatocytes using specific antibodies, showed its presence inside the hepatocytes and on the membrane.
Carboxylesterase plays a role in the invasion process of P. falciparum sporozoites into human hepatocytes in vitro. The implications of these findings are further discussed.
Journal of Hepatology 11/1997; 27(4):688-98. · 9.26 Impact Factor
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ABSTRACT: The hepatitis G virus (HGV) has recently been identified as a new member of the Flaviviridae family. Infection by this virus is thought to be associated with blood borne hepatitis. In this study, the presence of HCV- and HGV-RNAs in serum or plasma (175 patients) and in peripheral blood mononuclear cells (PBMC) (133 patients) was investigated in patients with clotting disorders using a sensitive reverse transcriptase polymerase chain reaction (RT-PCR). HGV-RNA was detected in serum of 26 patients (14.8%). In apparently healthy blood donors, serum HGV-RNA was detected in 4 of 358 individuals investigated (1.12%). Ninety two percent of the 26 serum HGV-RNA positive patients had coinfection with the hepatitis C virus (HCV), especially with HCV genotype 1b, the most common genotype in Belgium. Of these coinfected patients, 15 (62.5%) showed elevated serum ALT levels. Two patients who were solely infected with HGV had normal serum ALT.HGV-RNA in PBMC was found in 18 patients, of whom 3 were negative for serum HGV-RNA. As in case of HCV, HGV-RNA in PBMC is preferentially sensitive to interferon treatment. Nevertheless, rapid reappearance of HGV-RNA in PBMC was observed after cessation of treatment. In one patient, persistent serum ALT elevation seems to be associated with continued HGV viremia, despite the disappearance of serum HCV-RNA.
Thrombosis and Haemostasis 06/1997; 77(5):868-72. · 5.04 Impact Factor
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ABSTRACT: Hepatitis B virus (HBV) infection is still a major public health problem worldwide. Although much information about the molecular biology of HBV has been gained in the last decades, little is known about the mechanism of attachment and penetration of the HBV particle into human hepatocytes. The HBV envelope proteins are important for the interaction between the HBV particle and the hepatocyte plasma membrane. Although initially it was suggested that the preS2 domain could act, via polymerized human serum albumin, as an attachment site to human hepatocytes, in recent years other observations showed that the preS1 domain is probably the most important attachment site to human hepatocytes. However, controversial findings on cellular proteins for binding to the preS1 domain has been described, namely the IgA-, the IL6-, the asialoglycoprotein receptor and GAPD. Although the preS1 attachment site may be important, apo H has been shown to bind specifically to small HBsAg. Recently, we have identified human liver Annexin V as a specific small HBsAg-binding protein. In a preliminary report, the direct involvement of human Annexin V in the initial step of HBV infection has been demonstrated. A rat hepatoma cell line, which does not express human Annexin V and which is not infectable by HBV, gained the ability to become infected by HBV after transfection with human Annexin V. This result may facilitate the progress of HBV receptor research and elucidate the molecular mechanism of the initial step of HBV infection.
Journal of Viral Hepatitis 06/1997; 4(3):145-53. · 4.09 Impact Factor
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ABSTRACT: Following the discovery in 1989 of the hepatitis C virus (HCV), sensitive and specific diagnostic assays have shown the involvement of this RNA virus in a myriad of liver disease and extrahepatic pathology. The genetic heterogeneity of HCV is an exciting challenge in the field of epidemiology, diagnosis, treatment and vaccine development. Although viral eradication has not been achieved in the majority of patients by using interferon as single agent, the combination with ribavirin seems to have a better outlook.
Acta clinica Belgica 02/1997; 52(1):36-48. · 0.59 Impact Factor
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ABSTRACT: We perfused livers from fed rats with a balanced salt solution containing 1 mmol/L glucose. Under these conditions a low steady rate of glycogenolysis was observed (approximately 1.7 micromol glucose equivalents/g/min; 20% of the maximal glycogenolytic activity). Nitric oxide (NO) transiently stimulated hepatic glucose production. A maximal response (on average doubling basal glucose output) was observed with 34 micromol/L NO. The same concentration of nitrite (NO2-) was ineffective. Half-maximal effects were seen at 8 to 10 micromol/L NO, irrespective of the flow direction (portocaval or retrograde). This glycogenolytic response to NO corresponded to a partial activation of phosphorylase. The NO effect was not additive to maximal stimulation of glycogenolysis (7.7 +/- 0.2 micromol hexose equivalents/g/min; n = 4) by 100 micromol/L dibutyryl cyclic adenosine monophosphate (Bt2cAMP). The requirement for activation of phosphorylase was also evidenced by the ineffectiveness of NO in phosphorylase-kinase-deficient livers of gsd/gsd rats. The NO effect was blocked by co-administration of cyclooxygenase inhibitors (50 micromol/L ibuprofen, 50 micromol/L indomethacin, or 2 mmol/L aspirin), suggesting a mediatory role of prostanoids from nonparenchymal cells. This conclusion was confirmed by the fact that NO did not activate phosphorylase in isolated hepatocytes. Moreover, NO was no longer glycogenolytic in livers perfused with Ca2+-free medium, in agreement with the known mediatory role of Ca2+ in prostanoid-mediated responses. Surprisingly, in Ca2+-free medium NO inhibited the basal glucose production. This coincided with an increased elution of cyclic guanosine monophosphate (cGMP). Inhibition of glycogenolysis by NO under these conditions was blocked by 1 mmol/L theophylline, suggestive for involvement of cGMP-stimulated cAMP phosphodiesterase. However, we could not confirm that an increase in cGMP resulted in a drop in cAMP. In conclusion, NO recruits opposing mechanisms with respect to modulation of basal hepatic glycogenolysis. In the presence of Ca2+, activation of phosphorylase with stimulation of glycogenolysis dominates. Cyclooxygenase inhibitors abolish this effect. Activation by NO of the cyclooxygenase in nonparenchymal cells is a distinct possibility. In the absence of Ca2+, inhibition of basal glycogenolysis becomes observable. It remains to be established whether this results from cGMP-mediated stimulation of hydrolysis of cAMP.
Hepatology 07/1996; 23(6):1564-71. · 11.66 Impact Factor
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ABSTRACT: Octreotide has been proposed for the treatment of variceal bleeding. The effects on portal pressure, however, have been variable in published studies. As bleeding is more directly related to pressure in the varices, this study investigated the effect on variceal pressure of octreotide and terlipressin, a vasoactive drug with a well established effect. Variceal pressure was measured during four to eight minutes by a continuous non-invasive endoscopic registration method. Thirty patients in whom a stable variceal pressure recording had been obtained during at least one minute, were randomised to receive either 2 mg terlipressin, 50 micrograms octreotide or an identical volume of saline, as a single intravenous injection given over 60 seconds. For the final analysis three patients had to be excluded because of lack of a satisfactory recording. There were no significant clinical differences between the three groups of patients. Placebo administration did not induce significant changes, but a mean decrease in variceal pressure of -27% was noted with terlipressin, starting from two minutes onwards. Variceal pressure changes after injection of octreotide were variable and the mean change in pressure did not reach statistical significance. Seven of 10 patients showed a temporary increase in variceal pressure. In conclusion, terlipressin induces a significant and progressive decrease in variceal pressure but inconsistent variations of variceal pressure changes were seen after octreotide administration. This is probably related to its effect on central venous pressure. This study also shows that continuous variceal pressure recording with the non-invasive endoscopic registration technique detects in an accurate way the effect of vasoactive drugs on variceal pressure, because placebo injection did not produce significant changes.
Gut 02/1996; 38(1):129-34. · 10.11 Impact Factor
