Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma.

The molecular events that drive hepatitis B virus (HBV)-mediated transformation and tumorigenesis have remained largely unclear, due to the absence of a relevant primary model system. Here we propose the use of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. We first describe a primary ex vivo HBV-infection model derived from healthy donor liver organoids after challenge with recombinant virus or HBV-infected patient serum. HBV-infected organoids produced covalently closed circular DNA (cccDNA) and HBV early antigen (HBeAg), expressed intracellular HBV RNA and proteins, and produced infectious HBV. This ex vivo HBV-infected primary differentiated hepatocyte organoid platform was amenable to drug screening for both anti-HBV activity and drug-induced toxicity. We also studied HBV replication in transgenically modified organoids; liver organoids exogenously overexpressing the HBV receptor sodium taurocholate co-transporting polypeptide (NTCP) after lentiviral transduction were not more susceptible to HBV, suggesting the necessity for additional host factors for efficient infection. We also generated transgenic organoids harboring integrated HBV, representing a long-term culture system also suitable for viral production and the study of HBV transcription. Finally, we generated HBV-infected patient-derived liver organoids from non-tumor cirrhotic tissue of explants from liver transplant patients. Interestingly, transcriptomic analysis of patient-derived liver organoids indicated the presence of an aberrant early cancer gene signature, which clustered with the hepatocellular carcinoma (HCC) cohort on The Cancer Genome Atlas Liver Hepatocellular Carcinoma dataset and away from healthy liver tissue, and may provide invaluable novel biomarkers for the development of HCC and surveillance in HBV-infected patients.

Development of C-TILDA: A modified TILDA method for reservoir quantification in long term treated patients infected with subtype C HIV-1.

A better characterization of the HIV reservoir is pivotal for the development of effective eradication strategies. Accurate quantification of the latent reservoir remains challenging. Starting from a regular blood draw, the Tat/Rev induced limiting dilution assay (TILDA) combines serial dilution of CD4+ T cells with a PCR-based detection of HIV-1 spliced mRNA produced upon cell stimulation. Here we adapted the original protocol for HIV-1 subtype B to detect tat/rev mRNAs transcribed from reactivated latently infected cells in long term suppressed patients infected with HIV-1 subtype C. Given the heterogeneity of global HIV epidemiology, it is pivotal to develop assays with optimal performances also in patients infected with non-B subtypes. We observed that, in these patients infected with subtype C virus, the HIV reservoir quantified by TILDA correlates with both the time of virological suppression and CD4/CD8 ratio.

Follicular helper T cells serve as the major CD4 T cell compartment for HIV-1 infection, replication, and production.

In the present study, we have investigated the distribution of HIV-specific and HIV-infected CD4 T cells within different populations of memory CD4 T cells isolated from lymph nodes of viremic HIV-infected subjects. Four memory CD4 T cell populations were identified on the basis of the expression of CXCR5, PD-1, and Bcl-6: CXCR5(-)PD-1(-)Bcl-6(-), CXCR5(+)PD-1(-)Bcl-6(-), CXCR5(-)PD-1(+)Bcl-6(-), and CXCR5(+)PD-1(+)Bcl-6(+). On the basis of Bcl-6 expression and functional properties (IL-21 production and B cell help), the CXCR5(+)PD-1(+)Bcl-6(+) cell population was considered to correspond to the T follicular helper (Tfh) cell population. We show that Tfh and CXCR5(-)PD-1(+) cell populations are enriched in HIV-specific CD4 T cells, and these populations are significantly increased in viremic HIV-infected subjects as compared with healthy subjects. The Tfh cell population contained the highest percentage of CD4 T cells harboring HIV DNA and was the most efficient in supporting productive infection in vitro. Replication competent HIV was also readily isolated from Tfh cells in subjects with nonprogressive infection and low viremia (<1,000 HIV RNA copies). However, only the percentage of Tfh cells correlated with the levels of plasma viremia. These results demonstrate that Tfh cells serve as the major CD4 T cell compartment for HIV infection, replication, and production.

Incomplete IgG response to HIV-1 proteins and low avidity levels in recently converted HIV patients treated with early antiretroviral therapy.

OBJECTIVES: To evaluate the evolution of antibody avidity and Western blot reactivity in recently infected HIV-1 subjects and to study the impact of highly active antiretroviral therapy (HAART) on avidity maturation of HIV-1-specific immunoglobulin G (IgG) in patients with recent HIV-1 infection. METHODS: Thirty-six HIV-1 seroconverters were enrolled in this study and followed longitudinally over 24 months to evaluate if the administration of antiretroviral therapy during primary infection affects Western blot reactivity and the evolution of antibody avidity. The patients were divided into two groups; group A consisted of 19 HIV-1-untreated patients who did not receive any drug treatment during our follow-up period; group B consisted of 17 subjects who were treated early with an association of two nucleoside reverse transcriptase inhibitors (NRTI) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) within 3 months after seroconversion. RESULTS: At diagnosis, Western blot analysis and avidity index (mean value) were exactly matched in untreated and treated patients; subsequently, however, a significantly lower reactivity to HIV-1 pol and gag proteins and a lower avidity index (mean values) were observed in HAART-treated patients up until the end of the follow-up period. CONCLUSIONS: The impaired production and maturation of the humoral immunological response in antiretroviral-treated patients might be related to a rapid suppression of HIV replication, driven by HAART. These results could have important implications in understanding the complex mechanism of the immune response during HIV infection.

HIV-1 Tat protein enhances RANKL/M-CSF-mediated osteoclast differentiation.

Impaired osteoblast/osteoclast cross-talk and bone structure homeostasis resulting in osteopenia/osteoporosis are often observed in HIV seropositive patients but the causal mechanisms remain unsettled. This study analyzed the biological effects of Tat on peripheral blood monocyte-derived osteoclast differentiation. Tat enhances osteoclast differentiation and activity induced by RANKL plus M-CSF treatment increasing both the mRNA expression of specific osteoclast differentiation markers, such as cathepsin K and calcitonin receptor, and TRAP expression and activity. These Tat-related biological effects may be related, at least in part, to the induction of c-fos expression and AP-1 activity. c-fos up-regulation was triggered by Tat when cell cultures were co-treated with RANKL/M-CSF and an analysis of c-fos promoter with c-fos deletion mutant constructs disclosed specific c-fos promoter domains targeted by Tat. Together, these results show that Tat may be considered a viral factor positively modulating the osteoclastogenesis and then bone resorption activity suggesting a pathogenetic role of this viral protein in the HIV-related osteopenia/osteoporosis.

Analysis of the effects of specific protease inhibitors on OPG/RANKL regulation in an osteoblast-like cell line.

Bone mass loss with the subsequent development of osteopenia and osteoporosis is related to HIV infection and antiretroviral treatment, even though the mechanisms involved have not yet been elucidated. In this report analyzes the early effects of some specific protease inhibitors on OPG/RANKL yielding and cell survival in osteoblast-like HOBIT cell line. None of the compounds, tested at scalar concentrations (C1, C2, C3), affected cell survival except for tipranavir that elicited a reliable induction of apoptosis at the highest concentration (C3). Atazanavir, saquinavir and indinavir did not affect OPG/RANKL in the cell surnatant in our experimental conditions. By contrast, at optimal concentration (C2), fosamprenavir induced a significant increase in OPG associated with a RANKL decrease whereas tipranavir down-regulated both OPG and RANKL (at C2 and C3) and darunavir increased RANKL only at C3 concentration. Together these data (coupled with the analysis of OPG/RANKL ratio) indicate that at early times and at optimal concentrations the PIs did not impair the OPG/RANKL system with the exception of fosamprenavir that showed a relative positive OPG/RANKL ratio regulation. Instead, cell cultures treated by the highest concentrations of tipranavir or darunavir showed a change in cell survival or an increase in RANKL, with a negative effect on the OPG/RANKL balance.

HIV-1 triggers apoptosis in primary osteoblasts and HOBIT cells through TNFalpha activation.

Several HIV-1 infected patients show bone loss and osteopenia/osteoporosis during the course of disease. The mechanisms underlying this degenerative process are largely unsettled and it has not been determined yet whether bone dysfunction is linked to HIV-1-mediated direct and/or indirect effects on osteoblasts/osteoclasts cross-talk regulation. This study investigated the effects of HIV-1(IIIb) and HIV-1(ADA) strains on osteoblasts using the osteoblast-derived cell line (HOBIT) and primary human osteoblasts as cellular models. The challenge of these cell cultures by both HIV-1 strains triggered a significant apoptosis activation unrelated to viral infection, since proviral HIV-1 DNA and supernatant HIV-1 RNA were not detected by real time PCR or b-DNA assays respectively. Under the experimental conditions, even heat-inactivated HIV-1 or cross-linked recombinant gp120 treatment of HOBIT and osteoblasts induced programmed cell death, suggesting that apoptosis is regulated by the interaction between HIV-1 gp120 and cell membrane. The analysis of cell culture supernatants showed a significant up-regulation of TNFalpha, a pleiotropic protein considered an apoptosis inducer in the osteoblast model. In fact, pretreatment of HOBIT and osteoblast cell cultures with anti-TNFalpha polyclonal antibody tackled effectively HIV-1 related induction of cell apoptosis. As a whole, these results indicate that HIV-1 may impair bone mass structure homeostasis by TNFalpha regulated osteoblast apoptosis.

RANKL/OPG/TRAIL plasma levels and bone mass loss evaluation in antiretroviral naive HIV-1-positive men.

Osteopenia and osteoporosis are common in HIV-1-infected individuals and represent a challenge in clinical and therapeutic management. This report investigated osteopenia/osteoporosis in a group of 31 antiretroviral naive HIV-1-positive men and the role of specific molecules belonging to TNF and the TNF-receptor family in HIV-1-related bone mass loss. Osteoprotegerin (OPG), the receptor activator of NF-kappab-ligand (RANKL), and the TNF-related apoptosis-inducing ligand (TRAIL) were significantly increased in the plasma of antiretroviral naive HIV-1-positive patients compared to a control group of healthy blood donors. In addition, TRAIL and RANKL plasma concentrations were positively correlated to HIV-1-RNA viral load. Measurement of bone mineral density in 20 out of 31 HIV-1-positive subjects disclosed osteopenia/osteoporosis in 40% of these patients. The antiretroviral naive HIV-1-positive subjects with low bone mineral density had a decreased plasma OPG/RANKL ratio and a plasma RANKL concentration >500 pg/ml. Together, these data indicate that plasma concentrations of specific factors involved in bone homeostasis were increased during HIV-1 infection and that RANKL and OPG/RANKL ratio deregulation may be involved in osteopenia/osteoporosis occurring in antiretroviral naive HIV-1 individuals.

HIV-1 negatively affects the survival/maturation of cord blood CD34(+) hematopoietic progenitor cells differentiated towards megakaryocytic lineage by HIV-1 gp120/CD4 membrane interaction.

To investigate the mechanisms involved in the human immunodeficiency virus type 1 (HIV-1)-related thrombocytopenia (TP), human umbilical cord blood (UCB) CD34(+) hematopoietic progenitor cells (HPCs) were challenged with HIV-1(IIIb) and then differentiated by thrombopoietin (TPO) towards megakaryocytic lineage. This study showed that HIV-1, heat-inactivated HIV-1, and HIV-1 recombinant gp120 (rgp120) activated apoptotic process of megakaryocyte (MK) progenitors/precursors and decreased higher ploidy MK cell fraction. All these inhibitory effects on MK survival/maturation and platelets formation were elicited by the interaction between gp120 and CD4 receptor on the cell membrane in the absence of HIV-1 productive infection. In fact, in our experimental conditions, HPCs were resistant to HIV-1 infection and no detectable productive infection was observed. We also evaluated whether the expression of specific cytokines, such as TGF-beta1 and APRIL, involved in the regulation of HPCs and MKs proliferation, was modulated by HIV-1. The specific protein and mRNA detection analysis, during TPO-induced differentiation, demonstrated that HIV-1 upregulates TGF-beta1 and downregulates APRIL expression through the CD4 engagement by gp120. Altogether, these data suggest that survival/differentiation of HPCs committed to MK lineage is negatively affected by HIV-1 gp120/CD4 interaction. This long-term inhibitory effect is also correlated to specific cytokines regulation and it may represent an additional mechanism to explain the TP occurring in HIV-1 patients.