USP18-based negative feedback control is induced by type I and type III interferons and specifically inactivates interferon α response.

Type I interferons (IFN) are cytokines that are rapidly secreted upon microbial infections and regulate all aspects of the immune response. In humans 15 type I IFN subtypes exist, of which IFN α2 and IFN β are used in the clinic for treatment of different pathologies. IFN α2 and IFN β are non redundant in their expression and in their potency to exert specific bioactivities.

Use of human hepatocytes to investigate HCV infection.

Investigations on the biology of hepatitis C virus (HCV) have been hampered by the lack of small animal models. Efforts have therefore been directed to designing practical and robust cellular models of human origin able to support HCV replication and production in a reproducible and physiologically pertinent manner. Different systems have been constructed based on hepatoma or other cell lines, sub-genomic and genomic replicons, productive replicons, and immortalized hepatocytes.

Use of human hepatocytes to investigate blood coagulation factor.

Coagulation is the complex process by which activation of plasmatic haemostasis proteins ends up with the generation of fibrin. Most of the plasma coagulation proteins are synthesized in hepatocytes. The aim of this chapter is to describe experimental procedures allowing to measure the secretion by primary human hepatocytes and functional activity (including production of fibrillar material from extracellular medium) of haemostasis proteins including factors II, V, VII, VIII, PIVKA-II (protein induced by vitK 1 absence or antagonist II), antithrombin and protein S.

Cellular models for the screening and development of anti-hepatitis C virus agents.

Investigations on the biology of hepatitis C virus (HCV) have been hampered by the lack of small animal models. Efforts have therefore been directed to designing practical and robust cellular models of human origin able to support HCV replication and production in a reproducible, reliable and consistent manner. Many different models based on different forms of virions and hepatoma or other cell types have been described including virus-like particles, pseudotyped particles, subgenomic and full length replicons, virion productive replicons, immortalised hepatocytes, fetal and adult primary human hepatocytes.

Serum-derived hepatitis C virus infection of primary human hepatocytes is tetraspanin CD81 dependent.

Hepatitis C virus-positive serum (HCVser, genotypes 1a to 3a) or HCV cell culture (JFH1/HCVcc) infection of primary normal human hepatocytes was assessed by measuring intracellular HCV RNA strands. Anti-CD81 antibodies and siRNA-CD81 silencing markedly inhibited (>90%) HCVser infection irrespective of HCV genotype, viral load, or liver donor, while hCD81-large intracellular loop (LEL) had no effect. However, JFH1/HCVcc infection of hepatocytes was modestly inhibited (40 to 60%) by both hCD81-LEL and anti-CD81 antibodies.

The low-density lipoprotein receptor plays a role in the infection of primary human hepatocytes by hepatitis C virus.

Background/aims: The direct implication of low-density lipoprotein receptor (LDLR) in hepatitis C virus (HCV) infection of human hepatocyte has not been demonstrated. Normal primary human hepatocytes infected by serum HCV were used to document this point.

Methods: Expression and activity of LDLR were assessed by RT-PCR and LDL entry, in the absence or presence of squalestatin or 25-hydroxycholesterol that up- or down-regulates LDLR expression, respectively.

Ketoconazole and miconazole are antagonists of the human glucocorticoid receptor: consequences on the expression and function of the constitutive androstane receptor and the pregnane X receptor.

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) play a major part in the control of drug metabolism and transport. We have previously shown that PXR and CAR expression is controlled by the glucocorticoid receptor (GR) and proposed the existence of a signal transmission cascade GR-(PXR/CAR)-drug metabolizing and transporter systems. In the current study, we investigated the effect of ketoconazole and other azole-derived drugs, miconazole and fluconazole, on the transcriptional activity of the human GR (hGR) in HeLa and HepG2 cells, and in primary human hepatocytes.

Role of CYP3A4 in the regulation of the aryl hydrocarbon receptor by omeprazole sulphide.

Cross-talk between nuclear receptors involved in the control of drug metabolism is being increasingly recognised as a source of drug side effects. Omeprazole is a well known activator of the aryl hydrocarbon receptor (AhR). We investigated the regulation of AhR by omeprazole-sulphide, a degradation metabolite of omeprazole, using CYP1A mRNA induction, reporter gene assay, receptor DNA binding, ligand binding, nuclear translocation, trypsin digests, and drug metabolism analysis in mouse Hepa-1c1c7, human HepG2 cells and primary human hepatocytes.

Cross-talk between xenobiotic detoxication and other signalling pathways: clinical and toxicological consequences.

1. Recent investigations on nuclear receptors and other transcription factors involved in the regulation of genes encoding xenobiotic metabolizing and transport systems reveal that xenobiotic-dependent signalling pathways are embedded in, and establish functional interactions with, a tangle of regulatory networks involving the glucocorticoid and oestrogen receptors, the hypoxia-inducible factor, the vitamin D receptor and other transcription factors/nuclear receptors controlling cholesterol/bile salt homeostasis and liver differentiation. 2.

Secretion of functional plasma haemostasis proteins in long-term primary cultures of human hepatocytes.

This study was designed to investigate the ability of long-term primary cultures of adult human hepatocytes to secrete the main haemostasis proteins. Factors II, V, VII, VIII, PIVKA-II (protein induced by vitamin K 1 absence or antagonist II), fibrinogen and antithrombin were quantified in culture medium by immunological methods and by measuring the coagulant activity of factors II, V and VII. All the haemostasis protein antigens except the factor VIII antigen (FVIII:Ag) were found in the culture medium throughout the culture period.

The metabolism of the piperazine-type phenothiazine neuroleptic perazine by the human cytochrome P-450 isoenzymes.

Identification of cytochrome P-450 isoenzymes (CYPs) involved in perazine 5-sulphoxidation and N-demethylation was carried out using human liver microsomes and cDNA-expressed human CYPs (Supersomes). In human liver microsomes, the formation of perazine metabolites correlated significantly with the level of CYP1A2 and ethoxyrezorufin O-deethylase activity, as well as with the level of CYP3A4 and cyclosporin A oxidase activity. Moreover, the formation of N-desmethylperazine also correlated well with S-mephenytoin 4'-hydroxylase activity (CYP2C19).

The olivacine derivative s 16020 (9-hydroxy-5,6-dimethyl-N-[2-(dimethylamino)ethyl)-6H-pyrido(4,3-B)-carbazole-1-carboxamide) induces CYP1A and its own metabolism in human hepatocytes in primary culture.

The olivacine derivative 9-hydroxy-5,6-dimethyl-N-[2-(dimethylamino)ethyl)-6H-pyrido(4,3-b)-carbazole-1-carboxamide (S 16020) exhibits a potent antitumor activity. However, when administered in cancer patients, its blood clearance increases after repeated administrations, whereas the volume of distribution remains constant, suggesting that the drug is able to induce its own metabolism. The aim of this work was to identify the enzymes involved in S 16020 metabolism and determine whether this molecule is an enzyme inducer in human hepatocytes in primary cultures.

Effects of cytochrome P-450 inducers on the perazine metabolism in a primary culture of human hepatocytes.

The metabolism of perazine in a primary culture of human hepatocytes after treatment of cells with TCDD (a CYP1A1/2 inducer) or rifampicin (mainly a CYP3A4 inducer) were studied in vitro. The concentrations of perazine and its main metabolites (perazine 5-sulfoxide, N-desmethylperazine) formed in hepatocytes were assayed in the extracellular medium using the HPLC method. TCDD and rifampicin induced the formation of perazine 5-sulfoxide, however, such an effect was not observed in the case of N-desmethylperazine.

Colchicine down-regulates cytochrome P450 2B6, 2C8, 2C9, and 3A4 in human hepatocytes by affecting their glucocorticoid receptor-mediated regulation.

The xenobiotic-mediated induction of three major human liver cytochrome P450 genes, CYP2B6, CYP2C9, and CYP3A4, is known to be regulated by the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). CAR and PXR are regulated, at least in part, by the glucocorticoid receptor (GR) and the hypothesis of a signal transduction cascade GR-[CAR/PXR]-P450 has been proposed. This study was aimed at testing this hypothesis in primary human hepatocytes by using the tubulin network disrupting agent colchicine.

Contribution of human cytochrome p-450 isoforms to the metabolism of the simplest phenothiazine neuroleptic promazine.

1. The aim of the present study was to identify human cytochrome p-450 isoforms (CYPs) involved in 5-sulphoxidation and N-demethylation of the simplest phenothiazine neuroleptic promazine in human liver. 2.

Chiral aspects of metabolism of antiinflammatory drug flobufen in human hepatocytes.

The metabolism of the nonsteroidal antiinflammatory drug flobufen, 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid, was studied in primary cultures of human hepatocytes prepared by two-step collagenase perfusion of livers from four donors. Racemic flobufen or its individual enantiomers, R-(+)- and S-(-)-flobufen were used as substrates. Aliquots of culture medium were collected during 24-h incubation.

Perazine as a potent inhibitor of human CYP1A2 but not CYP3A4.

The effects of perazine on the activities of CYP1A2 and CYP3A4 in a primary culture of human hepatocytes of one patient were studied in vitro. The CYPs activities were assessed by measuring the rate of acetanilide 4-hydroxylation (CYP1A2) and cyclosporine A oxidation (CYP3A4) after treatment with TCDD (a CYP1A subfamily inducer) or rifampicin (mainly a CYP3A4 inducer). The amounts of the metabolites formed in hepatocytes were assayed in the extracellular medium using the HPLC method.

Carbonyl reduction of the potential cytostatic drugs benfluron and 3,9-dimethoxybenfluron in human in vitro.

Benfluron (B, [5-(2-N-oxo-2-N',N"-dimethylaminoethoxy)-7-oxo-7H-benzo[c]fluorene]) is a potential benzo[c]fluorene antineoplastic agent with high activity against a broad spectrum of experimental tumors in vitro and in vivo. The structure of B has been modified to repress its rapid deactivation through carbonyl reduction on C7. 3,9-Dimethoxybenfluron (D, [3,9-dimethoxy-5-(2-N-oxo-2-N',N"-dimethylaminoethoxy)-7-oxo-7H-benzo[c]fluorene]) is one of the B derivatives developed.

Comparative effect of colchicine and colchiceine on cytotoxicity and CYP gene expression in primary human hepatocytes.

The aims of the present study were (1) to determine the cytotoxicity of colchiceine (EIN) in comparison with that of colchicine (COL); (2) to evaluate the effect of EIN on cytochrome P450 (CYP) expression and activity. Primary human hepatocytes were the model of choice for cytotoxicity and CYP expression experiments. LDH leakage and albumin secretion served as cytotoxicity parameters.

Transcriptional regulation of CYP2C9 gene. Role of glucocorticoid receptor and constitutive androstane receptor.

Although cytochrome P450 2C9 (CYP2C9) is a major CYP expressed in the adult human liver, its mechanism of regulation is poorly known. In previous work, we have shown that CYP2C9 is inducible in primary human hepatocytes by xenobiotics including dexamethasone, rifampicin, and phenobarbital. The aim of this work was to investigate the molecular mechanism(s) controlling the inducible expression of CYP2C9.

Expression and DNA-binding activity of C/EBPalpha and C/EBPbeta in human liver and differentiated primary hepatocytes.

Background/aims: Limited information is available on the expression and role of C/EBP factors in human liver and hepatocytes. We investigated the expression and DNA-binding activity of C/EBPalpha and C/EBPbeta in human liver needle biopsies, surgical lobectomies and differentiated cultured hepatocytes derived from lobectomies.

Methods: RNA and protein extracts were analyzed by RNAse protection, immunoblot and gel shift assays.

Induction of CYP2C genes in human hepatocytes in primary culture.

The expression and inducibility of four CYP2C genes, including CYP2C8, -2C9, -2C18, and -2C19, was investigated in primary cultures of human hepatocytes. By the use of RNase protection assay and specific antibodies, each CYP2C mRNA and protein were quantified unequivocally. The four CYP2C mRNAs were expressed in human livers and cultured primary hepatocytes, but only the CYP2C18 protein was not detected.

Interleukin-6 negatively regulates the expression of pregnane X receptor and constitutively activated receptor in primary human hepatocytes.

The marked impairment of hepatic drug metabolism during inflammation and infections has been known for many years and shown to result from down-regulation of cytochrome P450s (CYP) by cytokines. However, the mechanism of this repression is unknown. Using primary cultures of human hepatocytes, we show here that interleukin-6 (IL-6) rapidly and markedly decreases the expression of PXR (pregnane X receptor) and CAR (constitutively activated receptor) mRNAs, but does not affect the levels of dioxin receptor and glucocorticoid receptor mRNA.

Human hepatocytes in primary culture predict lack of cytochrome P-450 3A4 induction by eletriptan in vivo.

Eletriptan (Relpax) is a novel 5-hydroxytryptamine (serotonin)(1D/1B) agonist currently in development for the acute treatment of migraine. The aim of this work was to evaluate the relative induction potency of eletriptan in vitro compared with well characterized cytochrome P-450 (CYP) inducers with primary cultures of human hepatocytes and to relate this to the situation in vivo. Eletriptan was a weak inducer of CYP3A4 protein and cyclosporin A oxidation in four of the six cultures used, whereas rifampicin was a potent inducer in all cultures.