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International audience; BACKGROUNDLiver fibrosis can result in end-stage liver failure and death.AIMTo examine human liver fibrogenesis and anti-fibrotic therapies, we evaluated the three dimensional ex vivo liver slice (LS) model.METHODSFibrotic liver samples (F0 to F4 fibrosis stage according to the METAVIR score) were collected from patients after liver resection. Human liver slices (HLS) were cultivated for up to 21 days. Hepatitis C virus (HCV) infection, alcohol (ethanol stimulation) and steatosis (palmitate stimulation) were examined in fibrotic (F2 to F4) liver slices infected (or not) with HCV. F0-F1 HLS were used as controls. At day 0, either ursodeoxycholic acid (choleretic and hepatoprotective properties) and/or α-tocopherol (antioxidant properties) were added to standard of care on HLS and fibrotic liver slices, infected (or not) with HCV. Expression of the biomarkers of fibrosis and the triglyceride production were checked by quantitative reverse transcription polymerase chain reaction and/or enzyme-linked immunosorbent assay.RESULTSThe cultures were viable in vitro for 21 days allowing to study fibrosis inducers and to estimate the effect of anti-fibrotic drugs. Expression of the biomarkers of fibrosis and the progression to steatosis (estimated by triglycerides production) was increased with the addition of HCV and /or ethanol or palmitate. From day 15 of the follow-up studies, a significant decrease of both transforming growth factor β-1 and Procol1A1 expression and triglycerides production was observed when a combined anti-fibrotic treatment was applied on HCV infected F2-F4 LS cultures.CONCLUSIONThese results show that the human three dimensional ex vivo model effectively reflects the in vivo processes in damaged human liver (viral, alcoholic, nonalcoholic steatohepatitis liver diseases) and provides the proof of concept that the LS examined model permits a rapid evaluation of new anti-fibrotic therapies when used alone or in combination

Induction of broadly neutralizing antibodies targeting ectodomain of the transmembrane (TM) glycoprotein gp41 HIV-1 provides a basis for the development of a universal anti-viral vaccine. The HeLa cell-derived TZM-bl reporter cell line is widely used for the estimation of lentiviruses neutralization by immune sera. The cell line is highly permissive to infection by most strains of HIV, SIV, and SHIV. Here we demonstrated that TZM-bl cells express a 48 kDa non-glycosylated protein (p48) recognized by broadly neutralizing monoclonal antibody (mAb) 2F5 targeting the ELDKWA (aa 669–674) epitope of gp41TM of HIV-1. A significant amount of p48 was found in the cell supernatant. The protein was identified as human kynureninase (KYNU), which has the ELDKWA epitope. The protein is further called “p48 KYNU”. The HIV-1 neutralization by mAb 2F5 and 4E10 in the presence of p48KYNU was tested on Jurkat and TZM-bl cells. It was demonstrated that p48KYNU reduces neutralization by 2F5-like antibodies, but it has almost no effect on mAb 4E10. Therefore, p48KYNU can attenuate HIV-1 neutralization by 2F5-like antibodies and hence create false-negative results. Thus, previously tested immune sera that recognized the ELDKWA-epitope and demonstrated a “weak neutralization” of HIV-1 in TZM-bl assay should be reevaluated.

The liver is the central metabolic organ in the human body, and also plays an essential role in innate and adaptive immunity. While mouse models offer significant insights into immune-inflammatory liver disease, human immunology differs in important respects. It is not easy to address those differences experimentally. Therefore, to improve the understanding of human liver immunobiology and pathology, we have established precision-cut human liver slices to study innate immunity in human tissue. Human liver slices collected from resected livers could be maintained in ex vivo culture over a two-week period. Although an acute inflammatory response accompanied by signs of tissue repair was observed in liver tissue following slicing, the expression of many immune genes stabilized after day 4 and remained stable until day 15. Remarkably, histological evidence of pre-existing liver diseases was preserved in the slices for up to 7 days. Following 7 days of culture, exposure of liver slices to the toll-like receptor (TLR) ligands, TLR3 ligand Poly-I:C and TLR4 ligand LPS, resulted in a robust activation of acute inflammation and cytokine genes. Moreover, Poly-I:C treatment induced a marked antiviral response including increases of interferons IFNB, IL-28B and a group of interferon-stimulated genes. Therefore, precision-cut liver slices emerge as a valuable tool to study human innate immunity.

The infection with the hepatitis C virus (HCV) is an example of the translational research success. The reciprocal interactions between clinicians and scientists have allowed in 30 years the initiation of empirical treatments by interferon, the discovery of the virus, the development of serological and virological tools for diagnosis but also for prognosis (the non-invasive biochemical or morphological fibrosis tests, the predictors of the specific immune response including genetic IL28B polymorphisms). Finally, well-tolerated and effective treatments with oral antivirals inhibiting HCV non-structural viral proteins involved in viral replication have been marketed this last decade, allowing the cure of all infected subjects. HCV chronic infection, which is a public health issue, is a hepatic disease, which may lead to a cirrhosis and an hepatocellular carcinoma (HCC) but also a systemic disease with extra-hepatic manifestations either associated with a cryoglobulinemic vasculitis or chronic inflammation. The HCV infection is the only chronic viral infection, which may be cured: the so-called sustained virologic response, defined by undetectable HCV RNA 12 weeks after the end of the treatment, significantly reduces the risk of morbidity and mortality associated with hepatic and extra-hepatic manifestations, which are mainly reversible. The history of HCV ends with the pangenotypic efficacy of the multiple combinations, easy to use for 8-12 weeks with one to three pills per day and little problems of tolerance. This explains the short 30 years from the virus discovery to the viral hepatitis elimination policy proposed by the World Health Organization (WHO) in 2016.

Hepatitis C virus (HCV) is a major cause of liver diseases including liver cirrhosis and hepatocellular carcinoma. Approximately 3% of the world population is infected with HCV. Thus, HCV infection is considered a public healthy challenge. It is worth mentioning, that the HCV prevalence is dependent on the countries with infection rates around 20% in high endemic countries. The review summarizes recent data on HCV molecular biology, the physiopathology of infection (immune-mediated liver damage, liver fibrosis and lipid metabolism), virus diagnostic and treatment. In addition, currently available in vitro, ex vivo and animal models to study the virus life cycle, virus pathogenesis and therapy are described. Understanding of both host and viral factors may in the future lead to creation of new approaches in generation of an efficient therapeutic vaccine.

International audience; BACKGROUND & AIMS:Chronic infection with HCV remains a public health problem with approximately 150 million people infected worldwide. HCV intersects with lipid metabolism for replication and entry; and plasma concentrations of apolipoproteins have been identified as predictors for response to therapy. Herein, we conducted a screen of plasma proteins, including all apolipoproteins, to identify correlates of response to pegylated-interferon/ribavirin (PR) and HCV non-structural protein 3 (NS3) inhibitors (i.e., telaprevir/boceprevir) therapy in treatment-experienced cirrhotic patients from the ANRS CUPIC cohort.METHODS:We analysed 220 baseline plasma protein concentrations in 189 patients using Luminex technology and analyzed results.RESULTS:We identified baseline levels of apolipoprotein H (apoH) as a surrogate marker for sustained virological response (SVR). Notably, increased plasma concentration of apoH, used in combination with known clinical parameters, established a robust model with improved classification of patients as likely to achieve SVR (AUC = 0.77, Se = 66%, Sp = 72%, NRI = 39%). Moreover, we provide mechanistic information that indicates a previously unidentified role for apoH during viral entry. Using a human liver slices HCV infection model, we demonstrate that apoH limits replication.CONCLUSION:These data support testing of new biomarker strategies for the management of cirrhotic HCV patients and expand our understanding of how apoH may intersect with HCV infection

HCV is one of the major causes of liver disease worldwide and has a high risk of progress in cirrhosis and hepatocellular carcinoma, the incidence of which is increasing. Currently, the licensed standard treatment for HCV infection is pegylated IFN-α and ribavirin; in HCV genotype 1-infected patients, the first-generation HCV NS3/4 protease inhibitors telaprevir and boceprevir may be added to the standard of care with higher antiviral potency. Although the sustained viral response rate of treatment has improved over the years, this therapy is not effective in all patients. In addition, several toxic side effects, complication and high cost limits patient compliance and the efficacy of the treatment. There is no easy model of HCV infection, and it is necessary to develop useful in vitro and in vivo models to study the pathobiology of HCV infection, including early events of acute infection (viral entry, immunological mechanisms and genetic predictors), as well as to evaluate the potency of the HCV antiviral drugs and vaccines. This article summarizes the in vitro and in vivo models that are currently used for the study of HCV infection, elucidating the early events of acute infection. This is a major issue that will probably allow us to reduce the risk of chronicity and aid the development of prophylactic and therapeutic vaccines.

The development of human cultured hepatitis C virus (HCV) replication-permissive hepatocarcinoma cell lines has provided important new virological tools to study the mechanisms of HCV infection; however, this experimental model remains distantly related to physiological and pathological conditions. Here, we report the development of a new ex vivo model using human adult liver slices culture, demonstrating, for the first time, the ability of primary isolates to undergo de novo viral replication with the production of high-titer infectious virus as well as Japanese fulminant hepatitis type 1, H77/C3, and Con1/C3. This experimental model was employed to demonstrate HCV neutralization or HCV inhibition, in a dose-dependent manner, either by cluster of differentiation 81 or envelope protein 2–specific antibodies or convalescent serum from a recovered HCV patient or by antiviral drugs. Conclusion: This new ex vivo model represents a powerful tool for studying the viral life cycle and dynamics of virus spread in native tissue and also allows one to evaluate the efficacy of new antiviral drugs. (HEPATOLOGY 2012;56:861–872)

Jaagsiekte retrovirus is an exogenous (exJSRV) beta-retrovirus with a simple genome. It causes lower airway epithelial cell tumors in small ruminants. Endogenous (enJSRV) counterparts of exJSRV are present in different copy numbers in numerous Bovidae family members.This work has focused on enJSRV in Simmental (Germany) and Limousine (France) beef breeds of domestic cattle and domestic goat. Of the enJSRV sequences in cattle, the orf-x sequences were about 99% identical, the LTR sequences were about 97% identical and the env sequences were nearly 95% identical to the corresponding endogenous sequences in sheep. A significant polymorphism of the proviral sequences between the cattle breeds was noted. Clonal analyses of the amplicons suggest two enJSRV proviruses in cattle genome. The endogenous sequences revealed in goat were closer to enzootic nasal tumor virus (ENTV) from goat rather than to enJSRV from sheep.The expression of enJSRV in cattle was partial (env only) and detected exclusively in bone marrow.

Jaagsiekte retrovirus (JSRV) causes ovine pulmonary adenomatosis (OPA) that resembles bronchioloalveolar carcinoma (BAC) in humans. To test the possible role of JSRV in human diseases, DNA specimens from 103 individuals either healthy or suffering from lung carcinomas were analyzed for JSRV sequences. orf-x sequences were detected in 19 of 64 samples and gag-prt sequences in 4 of 38 samples, predominantly in individuals from Africa. Sequences obtained from orf-x amplimers varied in-between each other and differed from control endogenous ovine JSRV sequence. No association with lung cancer was found. This is the first report of JSRV-like sequences detected in humans.

International audience; Hepatitis C virus (HCV) establishes infection using host lipid metabolism pathways that are thus considered potential targets for indirect anti-HCV strategies. HCV enters the cell via clathrin-dependent endocytosis, interacting with several receptors, and virus-cell fusion, which depends on acidic pH and the integrity of cholesterol-rich domains of the hepatocyte membrane. The ATP-binding Cassette Transporter A1 (ABCA1) mediates cholesterol efflux from hepatocytes to extracellular Apolipoprotein A1 and moves cholesterol within cell membranes. Furthermore, it generates high-density lipoprotein (HDL) particles. HDL protects against arteriosclerosis and cardiovascular disease. We show that the up-regulation of ABCA1 gene expression and its cholesterol efflux function in Huh7.5 hepatoma cells, using the liver X receptor (LXR) agonist GW3965, impairs HCV infection and decreases levels of virus produced. ABCA1-stimulation inhibited HCV cell entry, acting on virus-host cell fusion, but had no impact on virus attachment, replication, or assembly/secretion. It did not affect infectivity or properties of virus particles produced. Silencing of the ABCA1 gene and reduction of the specific cholesterol efflux function counteracted the inhibitory effect of the GW3965 on HCV infection, providing evidence for a key role of ABCA1 in this process. Impaired virus-cell entry correlated with the reorganisation of cholesterol-rich membrane microdomains (lipid rafts). The inhibitory effect could be reversed by an exogenous cholesterol supply, indicating that restriction of HCV infection was induced by changes of cholesterol content/distribution in membrane regions essential for virus-cell fusion. Stimulation of ABCA1 expression by GW3965 inhibited HCV infection of both human primary hepatocytes and isolated human liver slices. This study reveals that pharmacological stimulation of the ABCA1-dependent cholesterol efflux pathway disrupts membrane cholesterol homeostasis, leading to the inhibition of virus-cell fusion and thus HCV cell entry. Therefore besides other beneficial roles, ABCA1 might represent a potential target for HCV therapy.

To determine the mechanisms by which human immunodeficiency virus type 1 (HIV-1) crosses the placenta into the fetal blood, 12 matched samples of serial maternal blood, term placentas, and infant blood obtained from a cohort of pregnant women in Cameroon identified as predominantly infected by subtype A viruses were studied. HIV-1 env sequences were detected by polymerase chain reaction (PCR) in both chorionic villi and enriched trophoblastic cells of all 12 placentas but at variable rates of detection. Heteroduplex mobility assay analysis showed the presence of multiple HIV-1 env quasispecies in sequential maternal peripheral blood mononuclear cell samples, but only a small number of env variants were found in chorionic villi and enriched trophoblastic cells. These data indicate that HIV-1 env sequences are always present in term placentas of seropositive women, contrasting with the low frequency at which infection is diagnosed by PCR in neonates with tat, gag, and env primers. Maternal HIV-1 variants appear to undergo a strong negative selection by different cell populations within the placental villi.

AIM: To evaluate the antiviral potency of a new anti-hepatitis C virus (HCV) antiviral agent targeting the cellular autophagy machinery. METHODS: Non-infected liver slices, obtained from human liver resection and cut in 350 μm-thick slices (2.7 × 106 cells per slice) were infected with cell culture-grown HCV Con1b/C3 supernatant (multiplicity of infection = 0.1) cultivated for up to ten days. HCV infected slices were treated at day 4 post-infection with GNS-396 for 6 d at different concentrations. HCV replication was evaluated by strand-specific real-time quantitative reverse transcription - polymerase chain reaction. The infectivity titers of supernatants were evaluated by foci formation upon inoculation into naive Huh-7.5.1 cells. The cytotoxic effect of the drugs was evaluated by lactate dehydrogenase leakage assays. RESULTS: The antiviral efficacy of a new antiviral drug, GNS-396, an autophagy inhibitor, on HCV infection of adult human liver slices was evidenced in a dose-dependent manner. At day 6 post-treatment, GNS-396 EC50 was 158 nmol/L without cytotoxic effect (compared to hydroxychloroquine EC50 = 1.17 μmol/L). CONCLUSION: Our results demonstrated that our ex vivo model is efficient for evaluation the potency of autophagy inhibitors, in particular a new quinoline derivative GNS-396 as antiviral could inhibit HCV infection in a dose-dependent manner without cytotoxic effect.

We cloned and sequenced gp41 HIV-1 from plasma of AIDS patients under HAART and T-20 (enfuvirtide, Fuzeon) therapy and revealed several T-20 resistance-associated mutations. Two mutations, a single V38A and a double N43T-N44K were the most frequent; however, they were not found together in one clone. We anticipated that simultaneous mutations of these three residues might play a vital role in the viral life cycle. To address this problem, we introduced N43T-N44K and V38M + N43T–N44K substitutions to a cloned gp41 and introduced modified gp41 into the pNL4-3 molecular clone. HEK293T cells were transfected with the obtained vectors and released viruses were examined for reverse transcriptase (RT) activity, infectivity on reporter TZM-bl cells, and in Western blotting. Nearly equal RT activity was demonstrated in viruses with and without mutations. However, viruses with the V38M + N43T–N44K mutations were not infectious and, as shown by Western blotting, gPr160 cleavage was impaired. These data suggest that V38M + N43T–N44K mutations perturbed the natural conformation of gPr160 in a way that access of furin to the cleavage site (REKR) was blocked. Therefore, the residues V38 + N43–N44 retain the gPr160 conformation in proximity to the furin cleavage site and, as a consequence, are critical for virus infectivity. These data may explain why viruses with V38M + N43T–N44K mutations were not previously detected in the plasma of T-20-experienced patients.

International audience; Invariant natural killer T (iNKT) cells promote immune responses to various pathogens, but exactly how iNKT cells control antiviral responses is unclear. Here, we showed that iNKT cells induced tissue-specific antiviral effects in mice infected by lymphocytic choriomeningitis virus (LCMV). Indeed, iNKT cells inhibited viral replication in the pancreas and liver but not in the spleen. In the pancreas, iNKT cells expressed the OX40 molecule and promoted type I interferon (IFN) production by plasmacytoid dendritic cells (pDCs) through OX40-OX40 ligand interaction. Subsequently, this iNKT cell-pDC cooperation attenuated the antiviral adaptive immune response in the pancreas but not in the spleen. The dampening of pancreatic anti-LCMV CD8(+) T cell response prevented tissue damage in transgenic mice expressing LCMV protein in islet beta cells. Thus, this study identifies pDCs as an essential partner of iNKT cells for mounting an efficient, nondeleterious antiviral response in peripheral tissue.

Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant child's virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other diseases. The mechanisms of virus pathogenesis are still obscure. The occurrence of defective proviruses in HTLV-1-infected cell lines and the peripheral blood mononuclear cells (PBMC) of infected individuals is a frequent feature of virus infection. We detected defective proviruses with large internal deletions in PBMC from ATLL and HAM/TSP patients and in asymptomatic HTLV-1 carriers. Seventeen PCR-amplified defective proviruses were sequenced, and three types of deletions were found. Besides truncated MA and the 5′ end of the genome, truncated CA, truncated SU, and more frequently truncated TM linked to the pX region were detected. Reverse transcription-PCR analysis of PBMC from ATLL patients and asymptomatic carriers also revealed RNA transcripts with large internal deletions. Analysis of two RT-PCR cDNA clones confirmed a Gag-TM-pX structure of the transcripts. Most defective proviruses contained numerous internal stop codons, but some were capable of coding for the truncated MA linked to a variable out-of-frame peptide. Cloned defective proviruses with long open reading frames were subjected to in vitro transcription-translation followed by radioimmunoprecipitation, which showed expression of chimeric proteins between 8 and 12 kDa. Possible roles of defective proviruses and chimeric proteins are discussed, although there is no firm association with pathogenesis.

Human immunodeficiency virus, generated during contact between HIV-infected cells and the apical surface of an epithelial cell, can cross a tight epithelial barrier by transcytosis. We show that transcytosis of primary HIV isolates is blocked by dimeric IgA or IgM against HIV envelope proteins. Neutralization occurs intracellularly within the apical recycling endosome, and immune complexes are specifically recycled to the mucosal surface. One epitope involved in neutralization is a conserved sequence of the gp41 HIV envelope protein subunit. Finally, transcytosis also occurs across functional human mucosal tissue in a process inhibited by a serosal internalization of IgM against the HIV envelope protein. These results suggest that induction of mucosal immunity to HIV envelope proteins may impair the transcytotic route of HIV mucosal transmission.

Summary Sixteen matching sera and DNA samples from healthy African blood donors living in rural areas of Guinea were analysed for the presence of type D retrovirus markers. Screening for the antibodies against structural proteins of Mason-Pfizer monkey virus (M-PMV) was carried out by Western blot with a purified M-PMV as an antigen. Eight out of 16 sera samples were found to contain antibodies against at least two gag genecoded proteins, and three of these were weakly positive against env gene-coded protein. Using PCR amplification and Southern hybridization, we detected M-PMV-like gag sequences in 11 out of 16 samples and env -related sequences in 8 out of 16 samples. Six DNAs were found to contain both M-PMV gag - and env -related sequences. Restriction endonudease analysis of the PCR-amplified gag sequences from two individuals and direct DNA sequencing analysis of the amplirners confirmed their M-PMV-like origin. Detection of antibodies and M-PMV-related sequences in blood donors from Guinea, but not m French or Algerian blood donors, indicated exogenous SRV infection in humans from certain geographic areas of Western Africa.

Viruses, and more particularly retroviruses, have been postulated to play a role in the pathogenesis of autoimmune diseases. In a search for spumaretrovirus infection markers, we screened a group of 29 patients with Graves disease and a representative healthy population (23 subjects) as a control. Southern blot hybridization under stringent conditions, of patients' DNA extracted from peripheral blood lymphocytes, with a spumaretrovirus-specific genomic probe derived from the human spumaretrovirus (HSRV) prototype, gave a positive signal in 10 cases. Moreover, by PCR, HSRV-related sequences were detected in the DNA of 19 patients (66%). Positive DNA samples in Southern blots were also positive in PCR for all regions tested (gag, bel1, bel2, long terminal repeat). Amplified (gag and bel2) products were cloned and sequenced; they showed high homology with HSRV. On the other hand, all 23 control subjects were negative by both procedures. Sera from both populations were examined for the presence of antibodies reactive with antigens of the spumaretrovirus family. These sera were negative by several immunodetection techniques: ELISA, indirect immunofluorescence, serum neutralization, and Western blotting. These results strongly suggest the existence of an association between Graves disease and the presence of HSRV-related infection markers.

The sera of two patients with autoimmune disorders recognize in HeLa cell extracts two proteins with apparent molecular masses of 37,000 (p37) daltons and 32,000 daltons (p32). These proteins are non covalently associated with 5S RNA and sediment as 7-10 S particles in sucrose density gradients. Both proteins are antigenetically related to TFIIIA, a previously described protein of Xenopus laevis, which is known as a 5S RNA transcription factor and occurs in oocytes as a noncovalent complex with 5S RNA. Like TFIIIA, HeLa cell proteins p37 binds in vitro to 5S RNA and to cloned 5S RNA genes. These results suggest that protein p37 fulfils in HeLa cells a function similar to that of TFIIIA in amphibian oocytes, ie control of 5S RNA transcription.

Summary PSL(p55) is a nuclear 55kD antigen present in various mammalian cell systems, which has been first identified by use of human autoimmune antibodies (Barque et al. 1983, EMBO J. 2, 743). It has been shown to be associated with interphase chromatine and to be synthetised in during the S phase of the cell cycle. In this work, we have analysed the status of PSL in promyelocytic HL-60 human cells in exponential or stationary growth, or undergoing granulocytic differentiation in presence of Retinoic acid. By use of 2-dimensional electrophoresis, PSL was found to be composed of two acidic proteins designated p55 A and p55 B . Unexpectedly, estimated 10–20 fold higher amounts of each species were found in cells treated for 5 days with 10 −6 M Retinoic acid, than in asynchronously growing cells or resting cells. Moreover, the p55 A protein was phosphorylated during the process. On the basis of these results, PSL appears to be involved in some steps of the granulocytic differentiation process.

A 55 Kd nuclear antigen, designated PSL has been found using a human autoimmune serum. This antigen accumulates in the nucleus, during the S phase, transiently associates with chromatin and possesses a DNA-binding ability. It appears to be highly conserved among eukariotic species including mammals, birds and insects.

An S phase-related nuclear 55K antigen, also designated PSL, has been characterized in various mammalian cells, using a human serum from a patient with autoimmune disorders (Barque et al., EMBO j 2 (1983) 743) [6]. In this report, we show by immunoelectron microscopy that the p55 protein associates in situ with the chromatin of rat hepatocytes. This association is a transient one, as indirect immunofluorescence studies show that PSL does not bind to individualized metaphase chromosomes. Furthermore, immunoprecipitation tests indicate that the majority of PSL is in the non-chromosomal cell fraction. These results suggest that this nuclear antigen is directly involved in the DNA replication process.

Major progress has been made in understanding the pathophysiology of fibrosis, but despite a wide range of therapeutic targets, the gap between basic and clinical research persists due to limitations in the duration of clinical studies and invasive evaluation methods. The ex vivo culture model of human liver slices with a duration of up to 21 days was used to study variations in fibrogenesis in the human liver at different stages of F0 to F4 fibrosis, in particular the early stages of molecular liver fibrogenesis due to ethanol, HCV, or steatosis, which are the main factors in clinical liver injury. The expression of several molecules involved in the fibrosis process, such as TGF-β1, procolA1, MMP-2, MMP-9, α-SMA, HSP47, VEGF and the production of triglycerides increased significantly throughout the kinetics. As ongoing randomized clinical trials have certain limitations (based on serial liver biopsies, long duration (3 years) to generate speculative positive results, placebo-controlled studies with a single drug), our study provided proof of concept that the ex vivo model of human liver slice culture allows the rapid evaluation of the potency of new anti-fibrotic therapies alone or in combination.The human liver slice culture model can allow studies of immune components of liver disease. Basic research suggests that the development of fibrosis is subject to type 3 inflammation, guided by IL-17A and IL-22. Type 3 inflammation can be regulated by active metabolites of vitamin A, of which the liver is a storage site. In human liver samples available to us, we have shown that the concentration of IL-17A and IL-22 in liver tissue does not follow the severity of fibrosis as opposed to TGF-β1. The immune profile of the liver differs from that of circulating blood, with the preponderance of non-canonical immune populations, such as Mucosal-Associated Invariant T cells (MAIT), LTγδ, NKT cells. IL-17A secretion is mainly carried out by the Th17 population in the blood, while in the liver, the sources of IL-17A diversify, giving way to LTh17, MAIT and LTc17 cells. Stimulation of human liver slices with IL-17A revealed the fibrotic potential of IL-17A but did not reveal the dialogue with the retinoic acid (the active metabolite of vitamin A). On the other hand, the study on the energetic potential of the Huh7.5.1 hepatocarcinoma cell line using SeaHorse, revealed the effect of RA in compensating for IL-17A-induced disturbances in mitochondrial respiration. This compensation was translated by the activation of autophagy in the cells of the lineage, which was not the case in the human liver slice model.This work shows the significant need for human tissue-based models to better understand, describe and resolve existing problems in Hepatology.; Des progrès majeurs ont été réalisés dans la compréhension de la physiopathologie de la fibrose, mais malgré un large éventail de cibles thérapeutiques, le fossé entre la recherche fondamentale et la recherche clinique persiste en raison des limitations de la durée des études cliniques et des méthodes d'évaluation invasives. Le modèle ex vivo de culture de tranches de foie humain d'une durée allant jusqu'à 21 jours a permis d'étudier les variations de la fibrogenèse dans le foie humain à différents stades de la fibrose de F0 à F4, en particulier les premières étapes de la fibrogenèse hépatique moléculaire due à l'éthanol, au VHC ou à la stéatose, principaux facteurs de la lésion hépatique en clinique. L'expression de plusieurs molécules impliquées dans le processus de fibrose, telles que TGF-β1, procolA1, MMP-2, MMP-9, α-SMA, HSP47, VEGF et la production de triglycérides a augmenté de manière significative tout au long de la cinétique. Comme les essais cliniques randomisés en cours présentent certaines limites (basés sur des biopsies hépatiques en série, longue durée (3 ans) pour générer des résultats positifs spéculatifs, études placebo contrôlées avec un seul médicament), notre étude a fourni la preuve de concept que le modèle ex vivo de culture de tranches de foie humain permet l'évaluation rapide de la puissance des nouvelles thérapies anti-fibrotiques seules ou en combinaison.Le modèle de culture de tranches du foie humain peut permettre l’étude des composants immunitaires des maladies du foie. Les données de recherche fondamentale suggèrent que le développement de la fibrose est soumis à l’inflammation du type 3, guidée par IL-17A et IL-22. L’inflammation du type 3 peut être régulée par les métabolites actifs de la vitamine A, dont le foie est un lieu du stockage. Sur les échantillons de foie humain à notre disposition, nous avons montré, que la concentration d’IL-17A et d’IL-22 dans le tissu hépatique n’est pas corrélée à la gravité de fibrose par opposition au TGF-β1. Le profil immunitaire du foie se diffère de celui du sang circulant, avec la prépondérance des populations immunitaires non-canoniques, comme les MAIT (Mucosal-Associated Invariant T cells), les LTγδ, les cellules NKT. La sécrétion d’IL-17A est liée à la population Th17 dans le sang, tandis que dans le foie, les sources d’IL-17A se diversifient, laissant la place aux cellules LTh17, MAIT et LTc17. La stimulation des tranches de foie humain par IL-17A montre le potentiel fibrogène de l’IL-17A, mais n’a pas permis de voir la relation avec l’acide rétinoïque (le métabolite actif de la vitamine A). D’autre part, l’étude sur le potentiel énergétique de la lignée cellulaire d’hépatocarcinome Huh7.5.1 à l’aide de SeaHorse, a révélé l’effet de l’AR à savoir la compensation des perturbations dans la respiration mitochondriale induites par l’IL-17A. Cette compensation se traduit par l’activation de l’autophagie dans les cellules de la lignée, ce qui n’était pas le cas dans le modèle des tranches de foie humain.Ce travail montre le besoin significatif de modèles à la base du tissu humain pour mieux comprendre, décrire et résoudre des questions non résolues en Hépatologie.