The TZM-bl Reporter Cell Line Expresses Kynureninase That Can Neutralize 2F5-like Antibodies in the HIV-1 Neutralization Assay.

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.

IL-17A in Human Liver: Significant Source of Inflammation and Trigger of Liver Fibrosis Initiation.

IL-17A is considered to guide liver inflammation and fibrosis. From twenty-two human liver samples of different fibrosis stages (F0 to F4), IL-17A, IL-22, and TGFß1 protein expression in liver tissue lysates were analyzed. Ten paired samples of liver tissue (F0-F1 stage) and blood from the same patient were used to analyze intrahepatic and blood T-lymphoid IL-17A+ cells by flow cytometry. The analyses have been performed regardless of pathology, considering the stage of fibrosis. Human liver tissue was used for the primary human liver slice cultures, followed by subsequent cytokine stimulation and fibrotic markers' analysis by ELISA. IL-17A production in human liver tissue was significantly higher in the early fibrotic stage compared with the advanced stage. Th17 T cells and, to a lesser extent, MAIT cells were the main sources of IL-17A in both compartments, the liver and the blood. Moreover, the presence of liver Th17IL-17A+INFγ+ cells was detected in the liver. IL-17A stimulation of human liver slice culture increased the expression of profibrotic and pro-inflammatory markers. IL-17A, secreted by Th17 and MAIT cells in the liver, triggered fibrosis by inducing the expression of IL-6 and profibrotic markers and could be a target for antifibrotic treatment. Further amplitude studies are needed to confirm the current results.

The remarkable history of the hepatitis C virus.

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.

NKT cell-plasmacytoid dendritic cell cooperation via OX40 controls viral infection in a tissue-specific manner.

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.

Plasma apolipoprotein H limits HCV replication and associates with response to NS3 protease inhibitors-based therapy.

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.

Efficient replication of primary or culture hepatitis C virus isolates in human liver slices: a relevant ex vivo model of liver infection.

UNLABELLED: 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.

Retinoic Acid: A New Old Friend of IL-17A in the Immune Pathogeny of Liver Fibrosis.

Despite all the medical advances mortality due to cirrhosis and hepatocellular carcinoma, the end stages of fibrosis, continuously increases. Recent data suggest that liver fibrosis is guided by type 3 inflammation with IL-17A at the top of the line. The storage of vitamin A and its active metabolites, as well as genetics, can influence the development and progression of liver fibrosis and inflammation. Retinoic acid (active metabolite of vitamin A) is able to regulate the differentiation of IL-17A+/IL-22-producing cells as well as the expression of profibrotic markers. IL-17A and its pro-fibrotic role in the liver is the most studied, while the interaction and communication between IL-17A, IL-22, and vitamin A-active metabolites has not been investigated. We aim to update what is known about IL-17A, IL-22, and retinoic acid in the pathobiology of liver diseases.

Adult human liver slice cultures: Modelling of liver fibrosis and evaluation of new anti-fibrotic drugs.

BACKGROUND: Liver fibrosis can result in end-stage liver failure and death. AIM: To examine human liver fibrogenesis and anti-fibrotic therapies, we evaluated the three dimensional ex vivo liver slice (LS) model. METHODS: Fibrotic 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. RESULTS: The 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 ß-1 and Procol1A1 expression and triglycerides production was observed when a combined anti-fibrotic treatment was applied on HCV infected F2-F4 LS cultures. CONCLUSION: These 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.

The remarkable history of the hepatitis C virus.

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: Morphogenesis, infection and therapy.

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.

Anti-hepatitis C virus potency of a new autophagy inhibitor using human liver slices model.

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 × 10(6) 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.

Up-regulation of the ATP-binding cassette transporter A1 inhibits hepatitis C virus infection.

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.

Cell surface phenotype and human T lymphotropic virus type 1 antigen expression in 12 T cell lines derived from peripheral blood and cerebrospinal fluid of West Indian, Guyanese and African patients with tropical spastic paraparesis

Twelve long term cell lines were established from peripheral blood mononuclear cells (PBMC) or cerebrospinal fluid cells of patients with human T lymphotropic virus type I (HTLV-I) seropositive tropical spastic paraparesis (TSP) originating from the French West Indies, French Guyana or the Central African Republic. Most of these long term interlukin-2-dependent cell lines exhibited a pattern characteristic of CD4+ -activated T cells with high expression of CD2, CD3 and CD4 antigens, associated with a strong density of TAC and DR molecules. Nevertheless, in five cases CD8 expression was present at a significant level. HTLV-I antigens were expressed in a few cells after short-term culture and after 4 months the majority of the cells were HTLV-I positive, as demonstrated by indirect immunofluorescence (IF) using polyclonal or monoclonal anti-p19 and anti-p24 antibodies. Low and variable levels of reverse transcriptase activity were detected in supernatant fluids of these cell lines only after 4 months of culture, when at least 50 percent of the cells exhibited HTLV-I antigens by IF. However numerous type C HTLV-I-like viral particles were detected, mostly in the extracellular spaces, with rare budding particles. Similar findings were found in three T cell lines derived from West Indian and African patients with adult T-cell leukaemia/lymphoma (ATLL). Differences in high M-r polypeptides were detected by Western blot in cell lysates when comparing TSP-or ATLL-derived T cell lines. Thus a signal of 62K was easily detectable in all the TSP lines, but not in the ATLL lines. In all cell lines bands corresponding to p53, p24 and p19 viral core polypeptides were present, as was the env gene-coded protein p46. (AU)