Low cytometry (FACScan; Becton Dickinson, NJ, USA) analysis using anti-CD3 (BD Biosciences Pharmingen, CA, USA) and anti-CD68 (Southern Biotech, AL, USA) monoclonal antibodies.Results VIP and PACAP treatment inhibited HIV-1 production in macrophagesBecause activation of the receptors VPAC1 and VPAC2 has previously resulted in opposite effects during HIV-1 infection [27,28], we initially investigated whether the neuropeptides VIP and PACAP, the natural ligands of those receptors, would also T necrotic phenomena were not reported here. In the present study affect HIV-1 replication. To test this hypothesis, HIV-1-infected monocyte-derived macrophages were treated with VIP or PACAP. We first observed that both neuropeptides induced a significant reduction in virus replication (Fig. 1). VIP and PACAP were each individually able to decrease HIV-1 24195657 replication, achieving 33 and 38 of viral inhibition at 5 nM and 62 and 58 at 10 nM concentrations for VIP and PACAP, respectively. These results suggest that both neuropeptides were similarly effective in their ability to reduce HIV-1 production in macrophages. Higher concentrations of VIP or PACAP did not inhibit virus production and actually enhanced it (VIP at 100 nM), possibly due to receptor desensitization or an inverse agonist effect, as discussed later. Therefore, the next experiments were performed using the optimal inhibitory concentration of 10 nM for both neuropeptides.Macrophage production of b-chemokines and IL-Uninfected macrophages were treated with VIP or PACAP (10 nM), and concentrations of the b-chemokines CCL3 and CCL5 and of the cytokine IL-10 in the culture supernatants were measured using specific ELISA kits (R D Systems, MN, USA, and eBioscience Inc, CA, USA, respectively). The results are shown as mass/volume and also by the area under curve (AUC) transformation, which allows a global analysis of the induced production of the mediators.VIP and PACAP Inhibit HIV-1 InfectionFigure 1. VIP and PACAP inhibit HIV-1 replication. Macrophages were infected with an R5-tropic HIV-1 isolate (Ba-L) and treated once with different concentrations of the neuropeptides, as indicated. Virus replication was measured in the culture supernatants by an HIV-1 p24 ELISA 12-14 days after infection. Data represent means 6 SEM of five independent experiments for each peptide. *p#.05; ***p#.001. doi:10.1371/journal.pone.0067701.gVIP and PACAP present synergistic and additive effects on HIV-1 inhibitionAs VIP and PACAP share receptors, we analyzed whether these neuropeptides could cooperatively modulate HIV-1 replication by exposing infected macrophages to combinations of sub-optimal or optimal viral inhibitory concentrations of VIP and PACAP. Combinations of 1 nM and 5 nM significantly potentiated inhibition relative to their individual activities, while no increment of HIV-1 inhibition occurred when both peptides were combined at a concentration of 10 nM (Fig. 2). To accurately classify the nature of this Title Loaded From File finding, we calculated the interaction coefficient of VIP and PACAP at those concentrations by dividing the inhibition percentages found when the peptides were associated by the sum of the inhibition of each isolated peptide (Fig. 2D; an interaction coefficient on the order of 1 indicates an additive phenomenon, whereas values greater than 1 indicate a synergistic effect). Therefore, VIP and PACAP synergize at 1 nM and act in an additive manner on viral production at 5 nM. These results suggest that combinations of small concentrations of VIP and PACAP could re.Low cytometry (FACScan; Becton Dickinson, NJ, USA) analysis using anti-CD3 (BD Biosciences Pharmingen, CA, USA) and anti-CD68 (Southern Biotech, AL, USA) monoclonal antibodies.Results VIP and PACAP treatment inhibited HIV-1 production in macrophagesBecause activation of the receptors VPAC1 and VPAC2 has previously resulted in opposite effects during HIV-1 infection [27,28], we initially investigated whether the neuropeptides VIP and PACAP, the natural ligands of those receptors, would also affect HIV-1 replication. To test this hypothesis, HIV-1-infected monocyte-derived macrophages were treated with VIP or PACAP. We first observed that both neuropeptides induced a significant reduction in virus replication (Fig. 1). VIP and PACAP were each individually able to decrease HIV-1 24195657 replication, achieving 33 and 38 of viral inhibition at 5 nM and 62 and 58 at 10 nM concentrations for VIP and PACAP, respectively. These results suggest that both neuropeptides were similarly effective in their ability to reduce HIV-1 production in macrophages. Higher concentrations of VIP or PACAP did not inhibit virus production and actually enhanced it (VIP at 100 nM), possibly due to receptor desensitization or an inverse agonist effect, as discussed later. Therefore, the next experiments were performed using the optimal inhibitory concentration of 10 nM for both neuropeptides.Macrophage production of b-chemokines and IL-Uninfected macrophages were treated with VIP or PACAP (10 nM), and concentrations of the b-chemokines CCL3 and CCL5 and of the cytokine IL-10 in the culture supernatants were measured using specific ELISA kits (R D Systems, MN, USA, and eBioscience Inc, CA, USA, respectively). The results are shown as mass/volume and also by the area under curve (AUC) transformation, which allows a global analysis of the induced production of the mediators.VIP and PACAP Inhibit HIV-1 InfectionFigure 1. VIP and PACAP inhibit HIV-1 replication. Macrophages were infected with an R5-tropic HIV-1 isolate (Ba-L) and treated once with different concentrations of the neuropeptides, as indicated. Virus replication was measured in the culture supernatants by an HIV-1 p24 ELISA 12-14 days after infection. Data represent means 6 SEM of five independent experiments for each peptide. *p#.05; ***p#.001. doi:10.1371/journal.pone.0067701.gVIP and PACAP present synergistic and additive effects on HIV-1 inhibitionAs VIP and PACAP share receptors, we analyzed whether these neuropeptides could cooperatively modulate HIV-1 replication by exposing infected macrophages to combinations of sub-optimal or optimal viral inhibitory concentrations of VIP and PACAP. Combinations of 1 nM and 5 nM significantly potentiated inhibition relative to their individual activities, while no increment of HIV-1 inhibition occurred when both peptides were combined at a concentration of 10 nM (Fig. 2). To accurately classify the nature of this finding, we calculated the interaction coefficient of VIP and PACAP at those concentrations by dividing the inhibition percentages found when the peptides were associated by the sum of the inhibition of each isolated peptide (Fig. 2D; an interaction coefficient on the order of 1 indicates an additive phenomenon, whereas values greater than 1 indicate a synergistic effect). Therefore, VIP and PACAP synergize at 1 nM and act in an additive manner on viral production at 5 nM. These results suggest that combinations of small concentrations of VIP and PACAP could re.
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