Background: To study the first steps of liver molecular fibrogenesis in our ex vivo model of human liver slices culture. Methods: Human fibrotic liver samples (F0 to F4 fibrosis stage according to the METAVIR score) were collected after liver resection. Human liver slices of 350 µm (2.7x106cells per slice) were cultivated for up to 21 days. Fibrotic liver slices F2 to F4 (FLS) were infected (or not) with HCVcc supernatant infection [Con1/C3 (genotype1b)] (MOI = 0.1) (INF FLS). F0-F1 HLS (HLS) were used as controls. At day 0, either ursodeoxycholic acid (UDCA, only choleretic and hepatoprotective properties) and/or atocopherol (Toco, anti-oxydant properties which could reduce fibrosis progression) were added to standard of care concentrations on HLS and FLS. The following fibrosis markers expression were assayed in HLS, in FLS and in INF FLS, [tumor growth factor b (TGFβ), Heat shock protein 47 (Hsp47), Alpha smooth muscle actin (α-Sma), Procollagen1 A1 (Procl1A1), Matrix metalloproteinases 2, 9 (MMP-2, 9), Vascular Endothelial Growth Factor (VEGF)] and checked by RT-qPCR and the triglyceride production by ELISA assays. Results: The TGFβ, Hsp47,α-Sma, Procl1A1, MMP-2, MMP-9, VEGF expression and the triglyceride production increased significantly in both HLS and FLS but to a higher extent in FLS as compared to HLS, with a triglyceride production higher [~x 3.2] in FLS. HCV infection increased significantly the fibrotic markers expression and triglyceride production in non-infected (NI) FLS and INF FLS to a higher extent in INF FLS as compared to controls. The induced molecular fibrogenesis significantly increased, particularly true for the advanced stage of fibrosis, including cirrhosis. Under a-tocopherol exposure, a significant reduction of TGF-β RNA expression was observed in NINF and INF FLS but only for the stage F4. UDCA did not impact TGFβ RNA expression in NINF F0 to F4 liver slices, but significantly reduces its expression in F2F3F4 infected liver slices. Combination of UDCA and Toco treatments did not result in an additive effect and only reduced the TGFβ RNA expression in F2-F4 INF HLS but not in F2-F4 NI HLS. Conclusion: Our ex vivo model of human liver slices culture, supporting hepatocyte-specific gene expression for 21 days, provides an easy tool for studying human molecular liver fibrogenesis which could accelerate the evaluation of the potency of new anti-fibrotic therapies, alone or in combination.

BACKGROUND AND AIMS: Interferon-gamma (IFN-c) is a dimerized soluble cytokine which is critical to the defense against HCV infection as it can inhibit HCV replication in the replicon system.It has been proven that polymorphisms in some genes may influence the persistence of HCV infection, clinical outcome, HCV replication, and liver damage.METHODS: Family-based association designs offer a compromise between traditional linkage studies and case-control association studies.In these studies, association is assessed within families, and hence the confounding due to population heterogeneity can be eliminated.In our study 177 families have been recruited from The Upper, Middle, and Lower Egypt governments.These families include 323HCV patients as well as 347 non infected individuals.All were subjected to Routine clinical & laboratory investigations in addition to PCR HCV, HCV genotyping.Polymorphism of IFN-c (+874T/A, rs 62559044) was genotyped using ARM-PCR and confirmed using direct sequencing specific primers.RESULTS: Groups of our study were in Hardy-Weinberg equilibrium for IFN-c (+874T/A, rs 62559044).The T of IFN-c (+874T) allele was associated with 2 folds increased risk for HCV patients as compared to non-infected exposed person (OR = 1.9597, 95% CI 1.5269-2.5152,z statistic 5.284, p < 0.0001).The distribution of INF-c polymorphism was 10.81% AA, 51.39% AT &37.77%TT in HCV infected patients while in HCV-negative patients the distribution was 35.73% AA, 52.17% AT& 12.1%TT CONCLUSIONS: The T allele of rs 62559044 SNP of +IFNc gene polymorphism may have a crucial role of susceptibility to HCV infection in Egyptian families.

expression in liver biopsies and serum specimens obtained sequentially from the acute phase of HCV infection in chimpanzees to determine the kinetics of miR-122 and their relationship to HCV replication, interferon (IFN) type I response and liver pathology.Material and methods: Liver biopsies and sera were obtained from three chimpanzees infected with genotype 1a HCV and observed over a period up to 201 days after inoculation (DAI).Chimpanzees started to have HCV RNA detected in serum from DAI 4 to 7. Two chimpanzees (CH6413 and CH256) remained HCV RNA-positive throughout the study period, one (CH1541) cleared the infection by DAI 131.Hepatic and serum levels of miR-122 and hepatic levels of IFN-a, IFN-b and 2 5 oligoadenylate synthetase-2 (2,5-OAS-2) mRNAs were measured by real-time PCR.Results: Hepatic levels of miR-122 decreased to approximately 50% of preinoculation values at the peak of HCV replication in series of 5 to 12 consecutive weekly biopsies and returned to initial levels during the period when HCV RNA titers were declining (DAI 75 to 201 in CH256, DAI 50 to 201 in CH6413, DAI 61 to 131 in CH1541).This inverse correlation was statistically significant in two chimpanzees (r = -0.681;p = 0.015 in CH256; r = -0.098,p = 0.003 in CH1541).In all 3 chimpanzees, the decrease in hepatic miR-122 was associated with rise in levels of hepatic IFN-b and INF-a and 2,5-OAS-2 mRNA.The profile of miR-122 in serum correlated positively with serum ALT activity values (r = 0.75 to 0.9; p ≤ 0.002).Conclusions: In the chimpanzee model, negative correlations between miR-122 expression in the liver and HCV replication together with evidence of IFN type I induction contribute to molecular pathogenesis of the acute phase on HCV infection.Changes of miR-122 in serum positively correlated with profile of ALT activity and suggested that serum miR-122 may be a biomarker of HCV-associated liver injury.

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.

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.

ABSTRACT 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.

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.

For several decades, African green monkeys (AGMs) and macaques have been used as sources of cells and blood for vaccine trials. The health of animals is assessed visually and by serology for infections. However, even seronegative animals, especially from the wild, could be infected with latent viruses and retroviruses, so their biological material may be a source of infections.

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.

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 gene-coded 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 endonuclease analysis of the PCR-amplified gag sequences from two individuals and direct DNA sequencing analysis of the amplimers confirmed their M-PMV-like origin. Detection of antibodies and M-PMV-related sequences in blood donors from Guinea, but not in French or Algerian blood donors, indicated exogenous SRV infection in humans from certain geographic areas of Western Africa.