Effects of PGE₂ on HIV-1 release and viral infectivity.

<p>A) HEK 293T cell line was transfected with pNL4.3-Luc-R-E- and CXCR4-tropic env glycoprotein vector, and treated with PGE₂ (0.1 µM) 2d. Agp24 levels in supernatants were quantified by ELISA. B) To study the infectivity of new HIV virus the TZM-bl cells were incubated with the supernatants from PBL infected and treated or not with PGE₂ (0.1 µM) as detailed in Materials and Methods. After 16 h, the cells were lysed and luciferase activity was determined. Results are shown as percentage of non treated. C) PBL or purified primary CD4 T cells were infected with HIV-1_NL4-3 isolate and cultured during 2 d. Bellow cells were treated with PGE₂ 1 d more, and then co-cultured with the CMSF labeled target cells in presence or not of PGE₂. Effectors and target cells were seeded at a 2∶1 ratio in mixed co-culture. Anti-CD4 antibody Leu3a (0.25 µg/ml) was used as positive control. Virus transfer was assessed by flow cytometry for intracellular Gag CA p24 in target cells at 6 h after the start of co-culture. Results represent the means of 3 individual experiments. Error bars indicate standard error values. Statistical differences in comparison to control values (A, B) or shown as a percentage of positive cells for HIV-1 transfer ± SEM, *:p<0.05.</p

Prostaglandin E₂ Reduces the Release and Infectivity of New Cell-Free Virions and Cell-To-Cell HIV-1 Transfer

<div><p>Background</p><p>The course of human immunodeficiency virus type-1 (HIV-1) infection is influenced by a complex interplay between viral and host factors. HIV infection stimulates several proinflammatory genes, such as cyclooxigense-2 (COX-2), which leads to an increase in prostaglandin (PG) levels in the plasma of HIV-1-infected patients. These genes play an indeterminate role in HIV replication and pathogenesis. The effect of prostaglandin E₂ (PGE₂) on HIV infection is quite controversial and even contradictory, so we sought to determine the role of PGE₂ and the signal transduction pathways involved in HIV infection to elucidate possible new targets for antiretrovirals.</p><p>Results</p><p>Our results suggest that PGE₂ post-infection treatment acts in the late stages of the viral cycle to reduce HIV replication. Interestingly, viral protein synthesis was not affected, but a loss of progeny virus production was observed. No modulation of CD4 CXCR4 and CCR5 receptor expression, cell proliferation, or activation after PGE₂ treatment was detected. Moreover, PGE₂ induced an increase in intracellular cAMP (cyclic AMP) levels through the EP2/EP4 receptors. PGE₂ effects were mimicked by dbcAMP and by a specific Epac (exchange protein directly activated by cyclic AMP) agonist, 8-Cpt-cAMP. Treatment with PGE₂ increased Rap1 activity, decreased RhoA activity and subsequently reduced the polymerization of actin by approximately 30% compared with untreated cells. In connection with this finding, polarized viral assembly platforms enriched in Gag were disrupted, altering HIV cell-to-cell transfer and the infectivity of new virions.</p><p>Conclusions</p><p>Our results demonstrate that PGE₂, through Epac and Rap activation, alters the transport of newly synthesized HIV-1 components to the assembly site, reducing the release and infectivity of new cell-free virions and cell-to-cell HIV-1 transfer.</p></div

Inhibition of HIV-1 replication in human PBL by post-treatment with PGE₂.

<p>A) Human PBL were infected with X4 strain HIV-1_NL4.3 (20 ng HIV-1_NL4.3 p24Gag/10⁶ cells) for 2 h and treated with PGE₂ (0.1 and 1 µM) each 2 d. AZT (0.5 µM) was used as a positive control of inhibition. The culture supernatants were removed each 2 days and soluble p24 viral protein was monitored by ELISA at the indicted times. Results are shown as means ± SEM and expressed as a percentage of the value of the utreated control cultures. B) Representative WB for intracellular p24 protein determination in uninfected PBL or infected untreated and treated with PGE₂ (0.1, 1 µM) or AZT (0.5 µM) at the indicated times. Antibody directed against α-tubulin was used as a protein loading control. Bottom, graph depicting the results obtained after performing a densitometer analysis of the blots. For quantification, the pixel intensity of each band was normalized to the amount of tubulin. Statistical differences in comparison to HIV-1-infected cells *:p<0.05.; **:p<0.01.</p

Role of different EP receptors and cAMP on HIV-1 repression.

<p>A) Infected PBL were treated with different agonists: butaprost (EP2) (10 µM), sulprostone (EP1/EP3) (10 µM) or misoprostol (EP4, EP3>EP2) (35 µM), or. Agp24 levels were quantified by ELISA 3 d later. B) PGE₂ interaction with EP2/EP4 receptors induces cAMP liberation. PBL were treated with PGE₂ (0.1 µM) for the indicated times, and intracellular cAMP was quantified as described under “Material and Methods”. C) Effect of dbcAMP (200 µM) on Agp24 production in human PBL after 3 days of infection. Statistical differences in comparison to HIV-1-infected (A,C) or control cells (B) *:p<0.05.; **:p<0.01.</p

PGE₂ does not affect initial phases of HIV-1 viral cycle.

<p>PBL were treated with PGE₂ (0.1, 1 µM) for 16 hours, then cells were infected with HIV_NL4.3 insolate (15 ng/10⁶ cells) for 2 h and immediately afterwards cells were lysated and p24 protein was quantified by ELISA A) and western blot B). Data represents p24 concentration relative to infected and non-treated cells (mean ± SEM) of three experiments performed in triplicate in each case. T20 (5 µM) was used as a positive control of entry inhibition. To convert band intensity into a quantitative measurement, the Western blot was analyzed densitometrically. Data present the fold induction relative to control cells C) PGE₂ effect in post-entry steps were analyzed by infection with one-cycle viral particles VSV-pseudotyped-pNL4-3.Luc (100 ng HIV-1_NL4.3 p24^Gag/10⁶ cells). Data are shown as percentage of non treated cells. Results are from three independent experiments performed in triplicate. D) To study PGE₂ effect in LTR-transactivation, 8E5/LAV were used. Cells were activated or not with PMA (15 ng/ml) and treated with PGE₂ for 3 days. The culture supernatants were removed and p24 viral protein was monitored by ELISA. All data are shown as percentage of non treated cells.</p

Effects of PGE₂ treatment on actin poymerization and Gag distribution in T cell.

<p>Actin polymerization triggered by PGE₂ (0.1 µM) for the indicated times in HIV-1 infected cells. Data are from three independent experiments, presented as mean ±SEM. D) Actin polimerization and Gag distribution in a T cell line treated or not with PGE₂. Infected CEM-T cells treated or not with PGE₂ (0.1 µM) for 16 h, were stained with FITC-labeled Phalloidin, and RD1-labeled mouse anti-CA p24 monoclonal antibody, in order to visualize actin organization and Gag distribution, respectively. Scale bar: 5 µm.</p

PGE₂ regulates T cell actin polymerization throughout Epac/Rap1 and RhoA signaling.

<p>A) PBL were infected and cultured in the presence of different concentrations of 8-CPT-2-OMe-cAMP (1, 10, 100 µM) or 6-Bnz-cAMP (1, 10, 100 µM) for 3 d. Supernatants were collected and Agp24 levels were determined by ELISA. B) Effect of PKA inhibitors (H89 5 µM, KT5720 200 nM) on PBL treated with PGE₂ 3d post-infection. C) Pull-down assay of active Rap on infected CEM-T cells for the indicated times. Western blots were probed for Rap1. D) Effect of PI3k and ERK1 inhibitors, wortmanin (100 nM) and PD98059 (10 µM) respectively, on HIV-1 replication in PGE₂-stimulated PBL. E) Immunoblot analysis of pull-down assay for Rho-GTP. Rho-A antibody was used to detect activated Rho-GTP from a pull-down assay using beads covalently bound to Rhotekin and also total Rho-A in cell lysates. Error bars indicate standard error values. Statistical differences in comparison to non-treated HIV-1-infected cells *:p<0.05.</p

Risk factors for first-line HAART discontinuation among antiretroviral-naive paediatric patients.

<p>Legend: HR, hazard ratio; CI, confidence intervals; PMTCT, antiretroviral prophylaxis of HIV mother-to-child transmission; NNRTI, non-nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; n.a., not available; ^aper 100 cells/µL; ^bexcludes one patient who was treated with a triple NNRTI.</p

Kaplan–Meier survival curves for time spent on first vs. second vs. third HAART (A) and for time spent on first HAART by adherence to antiretrovirals (B).

<p>Kaplan–Meier survival curves for time spent on first vs. second vs. third HAART (A) and for time spent on first HAART by adherence to antiretrovirals (B).</p

Campanula sp.

<p>SD, standard deviation; ART, antiretroviral therapy; PI, protease inhibitors; NRTI, nucleoside reverse transcriptase inhibitors; NNRTI, non-NRTI; FI, fusion inhibitors, InI; integrase inhibitors.</p>*<p>Transferred to adult units with available resistance genotyping profile.</p>**<p>Statistical differences (p<0.05) have been found between transferred and non-transferred patients for these features.</p