[Re-expression of cytokeratin-19 in human hepatocyte primary culture].
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Cells, Cultured↗
Biomedical subjects
S H Yap
Publications and source records attributed to S H Yap.
Identification of human liver carboxylesterase as one of the proteins involved in Plasmodium falciparum malaria sporozoite invasion in primary cultures of human hepatocytes.
BACKGROUND/AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
Amino Acid Sequence↗
Organ and species specificity of hepatitis B virus (HBV) infection: a review of literature with a special reference to preferential attachment of HBV to human hepatocytes.
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.
Animals↗
Hepatitis C: seven years after the discovery of the causative agent.
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.
Antiviral Agents↗
Hepatitis G viral RNA in serum and in peripheral blood mononuclear cells and its relation to HCV-RNA in patients with clotting disorders.
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.
Adolescent↗
An exceptional high concentration of serum CA 19.9 in a patient with alcoholic liver disease.
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Biomarkers, Tumor↗
Modulation of basal hepatic glycogenolysis by nitric oxide.
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.
Animals↗
Assessment of variceal pressure by continuous non-invasive endoscopic registration: a placebo controlled evaluation of the effect of terlipressin and octreotide.
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.
Adult↗
Different effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors on sterol synthesis in various human cell types.
The three vastatins examined, lovastatin, simvastatin and pravastatin, are equally strong inhibitors of the sterol synthesis in human hepatocytes in culture with IC50-values of 4.1, 8.0 and 2.0 nM, respectively. However, in the human extrahepatic cells: umbilical vascular endothelial cells, retinal pigment epithelial cells, cornea fibroblasts and granulosa cells, pravastatin was much less inhibiting the sterol synthesis than lovastatin or simvastatin. It was observed as well that longer incubation with the vastatins resulted in higher IC50-values. In order to show that the feedback regulation mechanism for 3-hydroxy-3-methylglutaryl-coenzyme A reductase was involved in this phenomena mRNA levels were measured in human vascular endothelial cells after incubation with the vastatins for 3.5 h and for 20 h. Indeed, lovastatin and simvastatin gave rise to higher levels of HMG-CoA reductase mRNA after 20 h than after 3.5 h of incubation. The differences observed in different human cell types can be explained by supposing that pravastatin is transported into the human hepatocyte via a liver-specific transporter. This was supported by the results of uptake experiments with 14C-labelled pravastatin and 14C-labelled simvastatin into human hepatocytes compared to that into human umbilical endothelial cells (as an example of an extrahepatic cell type). [14C]-Simvastatin was associated with both cell types, whereas [14C]-pravastatin was hardly associated with human endothelial cells, but to a similar extent as [14C]-simvastatin with human hepatocytes.
Blotting, Northern↗
Hepatitis C virus genotypes in Belgian hemophiliacs.
Hemophiliacs who have been exposed to unheated and/or dry heated pooled clotting factor concentrates are at a high risk of chronic hepatitis C. Serum HCV-RNA was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) technique in 58 hemophiliacs positive for anti-HCV antibodies, HCV-RNA was detected in 55 patients. Based on the results of PCR positivity, HCV genotyping was carried out using genotype specific probes and the dot blot hybridization assay: 2 patients were found to be infected with type 1a (3.6%), 31 patients with type 1b (56.4%), 8 patients with type 2 (14.6%), 6 patients with type 3 (10.9%), 1 patient with type 4, and 7 patients with double infection (12.7%). As compared to a control group of 105 nonhemophilia patients with chronic hepatitis C, HCV genotype 1b was found predominant in both groups. However, double infection with two HCV types as well as a relatively high prevalence of infection with type 2 and type 3 were found in hemophiliacs.
Adolescent↗
Hepatitis C virus RNA detection and HCV genotype in patients with chronic non-A, non-B hepatitis in Jakarta.
Antibody response in HCV infection may be variable and the variability of the serological response could be due to the differences in HCV strains. Since the distribution of hepatitis C virus genotype has been found to be geographically dependent, it is important to determine the distribution of HCV genotype in various countries with high prevalence of chronic non-A, non-B hepatitis. In this study, serum HCV RNA was examined in 53 patients suspected of chronic non-A, non-B hepatitis with an anti-HCV test as determined by currently available assay. HCV viremia was detected in 48 patients (90.6%). These patients had elevated serum ALT level at the time of HCV RNA determination. Using specific genotype probes, all isolates were classified into three different genotypes. Double and triple infections were also noted. HCV genotype 1b is the predominant genotype found in chronic hepatitis C patients in Jakarta.
Adult↗
Genotype, serum level of HCV-RNA and response to interferon-alpha treatment in patients with chronic hepatitis C.
OBJECTIVE: Since only a small proportion of patients with chronic hepatitis C benefit from interferon therapy, we assessed the viral factors such as HCV genotype and the level of viraemia for prediction of interferon response. METHODS: HCV genotype and serum levels of HCV-RNA were determined in 24 patients before, during and after interferon treatment. RESULTS: Pre-treatment serum levels of HCV-RNA were significantly lower in patients with normalization of serum ALT and disappearance of serum HCV-RNA upon therapy than in patients with persistent abnormal ALT and/or serum HCV-RNA positivity. Sole infection with HCV genotype 1b was associated with a significantly higher pre-treatment serum HCV-RNA level than infection with genotype other than 1b or mixed types. CONCLUSIONS: HCV genotype and pre-treatment serum HCV-RNA levels are indeed important predictors for response to interferon treatment in patients with chronic hepatitis C.
Adolescent↗
Desmin expressing nonhematopoietic liver cells during rat liver development: an immunohistochemical and morphometric study.
The expression and cellular distribution of desmin, alpha-smooth muscle actin (A-SMA) and cytokeratin no. 8 (CK-8) and no. 18 (CK-18) in normal adult, neonatal and fetal rat liver were examined immunohistochemically on cryostat sections. At days 14 and 15 of gestation, nonhematopoietic cells in embryonic liver were strongly desmin-positive, and some of the cells, mainly located in the periphery, were also stained with anti-A-SMA. Desmin immunoreactivity gradually decreased from day 16 of gestation. A close association of desmin-positive cell processes with hematopoietic cells was observed during fetal and early neonatal development. From day 16 of gestation the pre-hepatocytes became desmin-negative, remained CK-8 and CK-18 positive. Desmin-expressing cells were numerous in the liver from the embryonic period to the neonatal age. However, their absolute number per unit area, as well as their number relative to hepatocytes, decreased with age. We suggest that desmin-positive cells in embryonic liver may act as stromal cells in the hepatic hematopoietic microenvironment and support hepatocyte development.
Actins↗
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.
Animals↗
Role of hepatitis C virus in chronic liver disease occurring after orthotopic liver transplantation.
Paediatric orthotopic liver transplant recipients may develop chronic hepatitis after surgery. To investigate the role of hepatitis C virus in this pathology a cohort of 249 paediatric orthotopic liver transplant recipients was studied. Sixteen children (6.4%) were found to have chronic hepatitis C virus hepatitis after orthotopic liver transplantation. All but one of them had serum transaminase values which were persistently raised two to eight times the upper limit of normal. Thirteen were positive for both serology and serum hepatitis C virus RNA. Serum hepatitis C virus RNA detection occurred five to 33 months before hepatitis C virus antibodies. Liver tissue hepatitis C virus RNA and hepatitis C virus core antigen were detected in five. In one patient, tissue hepatitis C virus core antigen was detected when other tests for hepatitis C were negative. Two patients had positive human cytomegalovirus serum antibodies and RNA before transplantation. Although serum hepatitis C virus RNA was not detected after transplantation, serum enzyme immunosorbent assay and tissue core antigen were still detectable in both patients. In another child, serum hepatitis C virus RNA was positive and hepatitis C virus core antigen was found on a liver biopsy specimen but antihepatitis C virus antibodies were negative as well as liver hepatitis C virus RNA. No patient developed severe liver disease or cirrhosis during a follow up of up to 72 months. It is concluded that hepatitis C virus is a significant cause of morbidity after paediatric orthotopic liver transplantation. Diagnosis cannot rely on serological testing only. The patients remained stable on follow up, but longer prospective histological studies remain necessary to establish prognosis.
Antigens, Viral↗
Hepatitis C virus-RNAs in plasma and in peripheral blood mononuclear cells of hemophiliacs with chronic hepatitis C: evidence for viral replication in peripheral blood mononuclear cells.
Hemophiliacs who have been exposed to unheated and/or dry heated pooled clotting factor concentrates are at high risk of chronic hepatitis C. Although the mechanism and site of hepatitis C virus (HCV) replication are not yet known, HCV is thought to replicate through a complementary negative RNA strand, as has been shown for flaviviruses. The detection of negative RNA strands has therefore been regarded as a marker of replication. We investigated the prevalence of HCV-RNA and of negative HCV-RNA strands in peripheral blood mononuclear cells (PBMC) and plasma of hemophiliacs. Forty-three of 47 patients studied (91%) had anti-HCV antibodies and in 36 patients HCV-RNA was detectable in PBMC. In one group of 20 patients negative HCV-RNA strands were present in PBMC and 10 of these patients also had negative HCV-RNA strands in plasma. In another group of nine patients HCV-RNA was detected in PBMC, although cDNA synthesis was carried out in the absence of primers. Only in two of these nine patients negative and positive HCV-RNA strands were demonstrated specifically in PBMC using a modified reverse transcription step. If the presence of negative HCV-RNA strands can be considered as marker of viral replication, the findings indicate that HCV can replicate in PBMC. Furthermore, in certain patients it is impossible to use the currently available technique to detect selectively positive or negative HCV-RNA strands.
Adolescent↗
In vitro binding properties of the hepatitis delta antigens to the hepatitis B virus envelope proteins: potential significance for the formation of delta particles.
To investigate the possible existence of (a) reactive binding site(s) on the hepatitis B surface antigen (HBsAg) for the hepatitis delta antigen (delta Ag) in the hepatitis delta virus (HDV), we performed binding studies using recombinant (rec)Small, recMiddle, recLarge HBsAg and recombinant small (S) and large (L) hepatitis delta antigen (recS delta Ag, recL delta Ag). Rec delta Ag was immobilized onto microtiter plates and incubated with recSmall, recMiddle and recLarge HBsAg. Of the three HBsAg proteins only the recMiddle HBsAg was found to bind to recS delta Ag. This binding was inhibited by the addition of synthetic PreS2 peptide but not by small HBsAg, indicating that the S delta Ag exhibits a PreS2 binding site. RecL delta Ag bound to all three forms of HBsAg. The binding of the HBsAg to recL delta Ag was saturable and could be blocked with an excess of HBsAg, but not with BSA. The region of the additional 19 amino acids of the L delta Ag is therefore responsible for the creation of the small HBsAg binding site on the L delta Ag. We therefore suggest that all HBsAg proteins but particularly the small HBsAg in the HDV coat seem to be involved in the interaction with the HDV core particle and that the PreS2 region of the middle HBsAg plays a crucial role in binding to small delta Ag during HDV particle formation, probably to increase the stability of the HDV particle.
Antigens, Viral↗
Detection of hepatitis C virus antigen by immuno-histochemical staining: a histological marker of hepatitis C virus infection.
Hepatitis C virus has been recognized as a major cause of non-A, non-B viral hepatitis. Although serologic tests have been commercialized, no specific histological or immuno-histochemical markers for hepatitis C virus infection are available for routine use. In an effort to detect hepatitis C virus antigen in liver tissue we investigated the immuno-reactivity to monoclonal antibodies on frozen liver tissue from a chimpanzee and patients with chronic non A, non B hepatitis. Monoclonal antibodies were developed in mice immunized with a synthetic peptide derived from hepatitis C virus core antigen. One monoclonal antibody was reactive and showed typical cytoplasmic granules in chimpanzee hepatocytes. Using this monoclonal antibody a similar staining pattern was found in the liver biopsies of 21 out of 28 chronic non-A, non-B hepatitis patients, positive for hepatitis C virus-RNA and anti-HCV. The granular immuno-reactivity was abolished after pre-incubation of this monoclonal antibody with infected chimpanzee liver or with hepatitis C virus synthetic peptide but not with normal chimpanzee or human liver tissue. There was no reactivity in four patients with hepatitis C virus-RNA-negative, anti-HCV-positive chronic non-A, non-B hepatitis, in 11 patients with chronic type B hepatitis or in 12 hepatitis C virus-RNA-negative, anti-HCV-negative patients with various liver diseases. However, staining was found in three out of four additional chronic type B hepatitis patients suspected of co-infection with non-A, non-B agents.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗