S H Yap

Universitair Ziekenhuis Leuven, Leuven, VLG, Belgium

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Publications (163)610.43 Total impact

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    ABSTRACT: Abstract Aplastic anemia is a rare complication of liver transplantation (<1%). However, an increasing number of cases of aplastic anemia have been recently reported when liver transplantation is performed for non-A, non-B, non-C fulminant hepatic failure. The aim of this study is to reevaluate the importance and the incidence of aplastic anemia after liver transplantation for non-A, non-B, non-C fulminant hepatic failure, and to propose preventive measures, diagnostic and management guidelines to try to reduce the incidence, morbidity and mortality associated with this complication. In this report a case of aplastic anemia after liver transplantation for non-A, non-B, non-C fulminant hepatic failure is described. In addition, the pertinent literature on aplastic anemia after liver transplantation, since the first description of that complication in 1987, is reviewed. A 20-year-old woman developed aplastic anemia 14 weeks after liver transplantation for fulminant non-A, non-B, non-C hepatitis. After failure of G-CSF treatment, she was treated with intensive immunosuppression (FK 506, ATG, high-dose steroids). She is well 1 year post-transplantation, with normal liver tests and with bone marrow recovery. Through a Medline literature search (1988–1999), we identified 30 additional cases of aplastic anemia following liver transplantation for non-A, non-B, non-C fulminant hepatic failure. Of all liver transplantations performed for that indication at five participating centers, the mean incidence of aplastic anemia was 23.2%. Mean age was 10 years (1.2–29) and the male/female ratio was 4.6. For treating aplastic anemia, different modalities were used: ATG (n=12), ALG (n=1), OKT 3 (n=1), G-CSF (n=6), a 6-HLA-compatible bone marrow transplantation (n=3), and none (n=12). The mortality rate remains high (39%), with infections and bleeding as the two most frequent causes of death. Based on this literature review, we conclude that aplastic anemia is a relatively common complication of liver transplantation for non-A, non-B, non-C fulminant hepatic failure in children and young adults. An unknown viral agent operating through immune-mediated mechanisms is probably responsible. The myelotoxic environment inherent to transplantation (e.g. azathioprine, trimethoprim) probably has a cumulative effect. Preventive measures (e.g. not using myelotoxic drugs) should be adopted in high-risk children and young adults transplanted for non-A, non-B, non-C fulminant hepatic failure. Early detection of bone marrow depression, a low threshold for performing a bone marrow biopsy, and prompt treatment are pivotal. Intensive standards supportive care with broad-spectrum antibiotics and anti-fungal agents os essencial during phasesof pancytopenia. Although spontaneous recovery has been described under maintenance immunosuppression, increased immunosuppression, in particular with ATG, may reverse the aplatic anemia and promote bone marrow recovery. In unresponsive patients, six-HLA-identical bone marrow transplanation has been successful.
    Transplant International 03/2005; 15(2‐3):117 - 123. · 3.16 Impact Factor
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    ABSTRACT: TT viruses are single-stranded DNA viruses, suggested to be involved in non A-E hepatitis. We studied the prevalence of TTV infection in acute or chronic hepatitis in Belgium in comparison with that in blood donors and in patients regularly receiving blood products. TTV-DNA was detected by PCR using the primer set of Takahashi et al (1998) or a nested-PCR specific for genotype-2, because it had been reported that this subtype might be more pathogenic (Tagger et al. 1999). TTV-DNA was present in 49% of 128 patients with chronic hepatitis C, in 54% of 54 with chronic hepatitis B and in 54% of 24 with acute liver failure. This prevalence is similar to the 47% in 127 patients with clotting disorders, or the 64% in 103 undergoing chronic haemodialysis, but lower than the 29.7% found in 340 healthy blood donors. Significant differences in clinical or biochemical characteristics between TTV- positive or TTV-negative patients could not be substantiated. The genotype-2 subgroup comprised 3.9%, but they also did not differ from non genotype-2 patients. The prevalence of TTV infection was higher in patients than in healthy blood donors. Its clinical significance remains questionable since clinical and biochemical characteristics were not different between TTV positive and TTV negative patients. The higher prevalence of TTV in patients might be related to parenteral transmission, but the relatively high prevalence in healthy blood donors points to an additional presumably faeco-oral infection. The presence of TTV in animals suggests that infection might also originate from food. Long term follow-up will have to define whether co-infection with TTV eventually alters the natural history of chronic hepatitis.
    Acta gastro-enterologica Belgica 01/2004; 67(2):161-5. · 0.58 Impact Factor
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    ABSTRACT: A novel DNA virus, TT virus (TTV), has been proposed as a possible etiologic agent for non A-E hepatitis. The aim of the present study was to determine the prevalence of TTV infection using PCR in healthy blood donors and in patients with clotting disorders who have been investigated previously for GBV-C/HGV and HCV infection in Belgium. In this study, PCR using primers proposed by Takahashi et al. [(1998) Hepatology Research 12:233-239] proved far more sensitive than those used by Okamoto et al. [(1998) Journal of Medical Virology 56:128-132]. The sequence of the PCR products showed 87% identity to the published sequence. TTV was present in 29.7% of healthy blood donors, a figure intermediate between the low rate of infection observed in Scotland and the high rates in the Far East. TTV was detected in 46.5% of 127 patients studied with clotting disorders as compared to 79.5% for HCV and 11.8% for GBV-C/HGV infection. However, there was no impact on the level of serum transaminases. Treatment with interferon for HCV infection co-infected with TTV suppressed temporarily serum TTV DNA. Therefore, it was concluded that TTV DNA is detected frequently in serum of healthy blood donors in Belgium and more often in patients with clotting disorders. TTV does not cause liver disease or contribute to the severity of liver disease.
    Journal of Medical Virology 05/2002; 66(4):561-6. · 2.37 Impact Factor
  • Lan Lin, Johan Fevery, Sing Hiem Yap
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    ABSTRACT: Hepatitis C virus (HCV) is reported to be lymphotropic under certain circumstances. In order to evaluate viral replication in peripheral blood mononuclear cells (PBMCs), a novel strand-specific RT-PCR was developed for the determination of HCV negative-strand RNA. The detection limit of this strand-specific RT-PCR was 100 copies of HCV negative-strand RNA in the presence of 1 microg liver RNA and 10(7)-10(8) copies of positive-strand RNA. False positive PCR signals occurred only when HCV positive-strand RNA exceeded 10(9) copies. With this RT-PCR, the replicative-intermediates could be detected specifically in eight of ten liver tissues, but not in any samples of serum or plasma (0/65) of patients with chronic hepatitis C. When examining the PBMCs of 46 hepatitis C patients, including 12 patients who had undergone orthotopic liver transplantation, HCV negative-strand RNA was detected in only one patient (1/46). In addition, HCV replicative intermediates were not detected in PBMCs of patients using immunosuppressive agents. It is concluded that the replication of HCV in PBMCs is very unusual.
    Journal of Virological Methods 03/2002; 100(1-2):97-105. · 1.90 Impact Factor
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    ABSTRACT: Aplastic anemia is a rare complication of liver transplantation (<1%). However, an increasing number of cases of aplastic anemia have been recently reported when liver transplantation is performed for non-A, non-B, non-C fulminant hepatic failure. The aim of this study is to reevaluate the importance and the incidence of aplastic anemia after liver transplantation for non-A, non-B, non-C fulminant hepatic failure, and to propose preventive measures, diagnostic and management guidelines to try to reduce the incidence, morbidity and mortality associated with this complication. In this report a case of aplastic anemia after liver transplantation for non-A, non-B, non-C fulminant hepatic failure is described. In addition, the pertinent literature on aplastic anemia after liver transplantation, since the first description of that complication in 1987, is reviewed. A 20-year-old woman developed aplastic anemia 14 weeks after liver transplantation for fulminant non-A, non-B, non-C hepatitis. After failure of G-CSF treatment, she was treated with intensive immunosuppression (FK 506, ATG, high-dose steroids). She is well 1 year post-transplantation, with normal liver tests and with bone marrow recovery. Through a Medline literature search (1988-1999), we identified 30 additional cases of aplastic anemia following liver transplantation for non-A, non-B, non-C fulminant hepatic failure. Of all liver transplantations performed for that indication at five participating centers, the mean incidence of aplastic anemia was 23.2%. Mean age was 10 years (1.2-29) and the male/female ratio was 4.6. For treating aplastic anemia, different modalities were used: ATG ( n=12), ALG ( n=1), OKT 3 ( n=1), G-CSF ( n=6), a 6-HLA-compatible bone marrow transplantation ( n=3), and none ( n=12). The mortality rate remains high (39%), with infections and bleeding as the two most frequent causes of death. Based on this literature review, we conclude that aplastic anemia is a relatively common complication of liver transplantation for non-A, non-B, non-C fulminant hepatic failure in children and young adults. An unknown viral agent operating through immune-mediated mechanisms is probably responsible. The myelotoxic environment inherent to transplantation (e.g. azathioprine, trimethoprim) probably has a cumulative effect. Preventive measures (e.g. not using myelotoxic drugs) should be adopted in high-risk children and young adults transplanted for non-A, non-B, non-C fulminant hepatic failure. Early detection of bone marrow depression, a low threshold for performing a bone marrow biopsy, and prompt treatment are pivotal. Intensive standard supportive care with broad-spectrum antibiotics and anti-fungal agents is essential during phases of pancytopenia. Although spontaneous recovery has been described under maintenance immunosuppression, increased immunosuppression, in particular with ATG, may reverse the aplastic anemia and promote bone marrow recovery. In unresponsive patients, six-HLA-identical bone marrow transplantation has been successful.
    Transplant International 03/2002; 15(2-3):117-23. · 3.16 Impact Factor
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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. · 3.08 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. · 2.09 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 (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. · 2.09 Impact Factor
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    ABSTRACT: A reproducible and potentially reversible model of acute liver failure in the pig is feasible based on transient ischemia of the liver. To determine the shortest period of liver ischemia sufficient to cause 100% mortality, ischemia of the liver was induced for different lengths of time, starting with 6 hours. If the pig survived, ischemia time was prolonged for 2 hours in the next animal. In the first group, the common bile duct was not tightened. In the second group, the common bile duct was tightened. The Laboratory for Hepatopathophysiology, Catholic University, Leuven, Belgium. Female stress-negative Belgian Landrace pigs weighing 18 to 22 kg. During preparatory surgery, all ligaments around the liver and connective tissue around the liver hilum were transected and an end-to-side portacaval shunt was made. Vessel loops were placed around the branches of the hepatic artery and bile duct. Three days later, in fully awake pigs, the loops were tightened. Mortality. Development of acute liver failure was determined based on neurologic, biochemical, and pathological variables. When occluded for 10 hours, all pigs in group 2 (n = 5) [corrected] died between 12 and 17 hours after the induction of ischemia. All pigs developed typical acute liver failure. Tissue specimens showed 90% necrosis of the liver parenchyma. A highly reproducible and potentially reversible model of acute liver failure in the large animal has been established.
    Archives of Surgery 11/2000; 135(10):1183-9. · 4.10 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. · 3.08 Impact Factor
  • S De Meyer, S H Yap
    Acta gastro-enterologica Belgica 01/2000; 63(2):180-1. · 0.58 Impact Factor
  • Journal of Hepatology - J HEPATOL. 01/2000; 32:44-44.
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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. · 4.60 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. · 9.86 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. · 1.90 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. · 3.08 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. · 3.08 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. · 12.00 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. · 3.08 Impact Factor

Publication Stats

2k Citations
610.43 Total Impact Points

Institutions

  • 1990–2005
    • Universitair Ziekenhuis Leuven
      • • Department of Hepatology
      • • Department of Pathology
      Leuven, VLG, Belgium
  • 2001
    • TNO
      Delft, South Holland, Netherlands
  • 1992–2000
    • University of Leuven
      • • Department of Reproduction, Development and Regeneration
      • • Department of Clinical and Experimental Medicine
      Louvain, Flanders, Belgium
  • 1974–1990
    • Radboud University Nijmegen
      • • Department of Medical Microbiology
      • • Pharmacology and Toxicology
      Nymegen, Gelderland, Netherlands
  • 1981–1988
    • Radboud University Medical Centre (Radboudumc)
      Nymegen, Gelderland, Netherlands
  • 1986
    • Imperial Valley College
      Imperial, California, United States