Lydiane Pichard-Garcia

Serum-Derived Hepatitis C Virus Infection of Primary Human Hepatocytes Is Tetraspanin CD81 Dependent

ABSTRACT 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. In conclusion, CD81 is involved in HCVser infection of human hepatocytes, and comparative studies of HCVser versus JFH1/HCVcc infection of human hepatocytes and Huh-7.5 cells revealed that the cell-virion combination is determinant of the entry process.

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. The more recently identified type III IFNs (3 IFN λ or IL-28/IL-29) bind an unrelated cell-type restricted receptor. Downstream of these two receptor complexes is a shared Jak/Stat pathway. Several mechanisms that contribute to the shut down of the IFN-induced signaling have been described at the molecular level. In particular, it has long been known that type I IFN induces the establishment of a desensitized state. In this work we asked how the IFN-induced desensitization integrates into the network built by the multiple type I IFN subtypes and type III IFNs. We show that priming of cells with either type I IFN or type III IFN interferes with the cells ability to further respond to all IFN α subtypes. Importantly, primed cells are differentially desensitized in that they retain sensitivity to IFN β. We show that USP18 is necessary and sufficient to induce differential desensitization, by impairing the formation of functional binding sites for IFN α2. Our data highlight a new type of differential between IFNs α and IFN β and underline a cross-talk between type I and type III IFN. This cross-talk could shed light on the reported genetic variation in the IFN λ loci, which has been associated with persistence of hepatitis C virus and patients response to IFN α2 therapy.

Use of long-term cultures of human hepatocytes to study cytochrome P450 gene expression

Publisher Summary This chapter discusses the cellular machinery necessary for cytochrome P450 (CYP)1-3 gene induction is functional in human hepatocytes in long-term primary cultures (LTPC). First, thyrosine aminotransferase (TAT), a glucocorticoid receptor (GR)-driven gene, appears to be expressed constitutively in these cultures, suggesting a constitutive expression of GR. As this receptor controls the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) expression, it is not surprising that these two nuclear receptors are significantly expressed and functional, and this is finally consistent with CYP2B-2C and CYP3A gene induction. The constitutive expression of CYP2C9, a primary glucocorticoid responsive gene, is consistent with the constitutive expression of GR. LTPC provides an invaluable means to investigate the inducible expression and functional activity of human CYPs in their natural cellular environment. Indeed, it is remarkable that the protein and mRNA levels, as well as the enzyme activity of human CYP genes, including CYP1A2, CYP2C9, and CYP3A4, are maintained for at least 1 month in these cultures. However, further medium modifications are currently being tested to increase the expression of other CYP forms, including CYP2C19, CYP2D6, and CYP2E1.

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. The data show that ketoconazole inhibits GR transcriptional activity and competes with dexamethasone for hGR binding. In primary human hepatocytes, ketoconazole inhibits the expression of 1) GR-responsive genes tyrosine aminotransferase and both PXR and CAR; 2) CAR and PXR target genes, including cytochromes P450 (P450) CYP2B6, CYP2C9, and CYP3A4; UDP-glucuronosyltransferase 1A1, glutathione S-transferases A1 and A2; and transporter proteins (phase III) solute carrier family 21 form A6 and multidrug resistance protein 2. In parallel experiments, ketoconazole affected neither the expression of GR, the expression of glyceraldehyde-3-phosphate dehydrogenase, nor the inducible expression of CYP1A1 and 1A2. Miconazole behaved like ketoconazole, whereas fluconazole had no effect. We conclude that, in addition to their well known inhibitory effect on P450 enzyme activities, ketoconazole and miconazole are antagonists of hGR. These results provide a novel molecular mechanism by which these compounds may exert adverse and toxic effects on drug metabolism and other functions in human.

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

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. Such functional interferences provide new insight, first for understanding how xenobiotics might exert adverse effects, and second how physiopathological stimuli affect xenobiotic metabolism.

Contribution of human cytochrome P‐450 isoforms to the metabolism of the simplest phenothiazine neuroleptic promazine

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. The experiments were performed in the following in vitro models: (A) a study of promazine metabolism in liver microsomes—(a) correlations between the rate of promazine metabolism and the level and activity of CYPs; (b) the effect of specific inhibitors on the rate of promazine metabolism (inhibitors: CYP1A2—furafylline, CYP2D6—quinidine, CYP2A6+CYP2E1—diethyldithiocarbamic acid, CYP2C9—sulfaphenazole, CYP2C19—ticlopidine, CYP3A4—ketoconazole); (B) promazine biotransformation by cDNA‐expressed human CYPs (Supersomes 1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2E1, 3A4); (C) promazine metabolism in a primary culture of human hepatocytes treated with specific inducers (rifampicin—CYP3A4, CYP2B6 and CYP2C inducer, 2,3,7,8‐tetrachlordibenzeno‐p‐dioxin (TCDD)—CYP1A1/1A2 inducer). In human liver microsomes, the formation of promazine 5‐sulphoxide and N‐desmethylpromazine was significantly correlated with the level of CYP1A2 and ethoxyresorufin O‐deethylase and acetanilide 4‐hydroxylase activities, as well as with the level of CYP3A4 and cyclosporin A oxidase activity. Moreover, the formation of N‐desmethylpromazine was correlated well with S‐mephenytoin 4′‐hydroxylation. Furafylline (a CYP1A2 inhibitor) and ketoconazole (a CYP3A4 inhibitor) significantly decreased the rate of promazine 5‐sulphoxidation, while furafylline and ticlopidine (a CYP2C19 inhibitor) significantly decreased the rate of promazine N‐demethylation in human liver microsomes. The cDNA‐expressed human CYPs generated different amounts of promazine metabolites, but the rates of CYP isoforms to catalyse promazine metabolism at therapeutic concentration (10 μM) was as follows: 1A1>2B6>1A2>2C9>3A4>2E1>2A6>2D6>2C19 for 5‐sulphoxidation and 2C19>2B6>1A1>1A2>2D6>3A4>2C9>2E1>2A6 for N‐demethylation. The highest intrinsic clearance (Vmax/Km) was found for CYP1A subfamily, CYP3A4 and CYP2B6 in the case of 5– sulphoxidation, and for CYP2C19, CYP1A subfamily and CYP2B6 in the case of N‐demethylation. In a primary culture of human hepatocytes, TCDD (a CYP1A subfamily inducer), as well as rifampicin (mainly a CYP3A4 inducer) induced the formation of promazine 5‐sulphoxide and N‐desmethylpromazine. Regarding the relative expression of various CYPs in human liver, the obtained results indicate that CYP1A2 and CYP3A4 are the main isoforms responsible for 5‐sulphoxidation, while CYP1A2 and CYP2C19 are the basic isoforms that catalyse N‐demethylation of promazine in human liver. Of the other isoforms studied, CYP2C9 and CYP3A4 contribute to a lesser degree to promazine 5‐sulphoxidation and N‐demethylation, respectively. The role of CYP2A6, CYP2B6, CYP2D6 and CYP2E1 in the investigated metabolic pathways of promazine seems negligible.

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. This review focuses on these different cellular models, their advantages and disadvantages at the biological and experimental levels, and their respective use for evaluating the effect of antiviral molecules on different steps of HCV biology including virus entry, replication, particles generation and excretion, as well as on the modulation by the virus of the host cell response to infection.

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 clotting activity of each factor correlated well with antigen level. In addition, fibrinogen and fibrin were detected in the fibrillar material following incubation of the culture medium with thromboplastin. Moreover, adding vitamin K 1 to the culture medium resulted in a significant increase of factors II and VII and a reciprocal decrease of the PIVKA‐II, and adding von Willebrand factor resulted in a drastic increase of the level of FVIII:Ag. We conclude that, in our culture system, normal adult human hepatocytes retain their capacity to secrete haemostasis proteins for at least 30 days.

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. EIN was less toxic than COL based on both parameters within the concentration range 1-100 microM. 10 microM concentration of EIN did not induce the expression of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 isoforms, which were evaluated at the levels of mRNAs, proteins and specific activities in culture. EIN in concentrations up to 200 microM had no effect on marker activities of CYP1A2, 2C9, 2E1 and 3A4 in human liver microsomes. It was concluded that EIN in concentrations up to 10 microM is not cytotoxic in primary human hepatocytes as revealed by albumin secretion and LDH leakage. Possible drug-drug interactions of EIN due to effects on cytochromes P4501A2, 2C9, 2E1 and 3A4 isoforms are unlikely because inhibition/induction studies show any lack of such effects. As EIN was shown to have better antifibrotic properties than COL (European Journal of Clinical Investigation 1997, 2, 77), it can be used as a COL substitute with anticipated fewer side-effects.

Expression and DNA-binding activity of C/EBPα and C/EBPβ in human liver and differentiated primary hepatocytes

Abstract 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/EBP α and C/EBP β 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. Results : C/EBP mRNAs, isoforms and DNA-binding activities were low/undetectable in lobectomies. In contrast, several C/EBP α (47, 45, 35 and 33 kDa) and C/EBP β isoforms (47, 43, 40, 35 and 21 kDa) were observed in needle biopsies. In cultured hepatocytes, the C/EBP expression pattern dramatically changed with time. C/EBP α mRNA and the 45 kDa isoform increased in parallel, reaching a maximum after 3–4 weeks coincident with weak DNA-binding activity. C/EBP β mRNA and isoform expression increased rapidly reaching a plateau within 1–2 weeks; all C/EBP β isoforms were phosphorylated. C/EBP β exhibited greater DNA-binding activity than C/EBP α , and this activity paralleled C/EBP β isoform expression. Conclusions : C/EBP isoforms exhibit markedly different expression patterns in lobectomies, needle biopsies and cultured hepatocytes. Stress stimuli during and/or after surgery for lobectomy resections may account for this difference. The pattern of C/EBP isoform expression in long-term highly differentiated cultured hepatocytes is close to that observed in needle biopsies.