MCP 1 neutralizing antibody was added to PDGF BB treated conditioned media prior to HBMEC exposure for currently 24 h. Monocytes were obtained from HIV 1, HIV 2 and hepa titis B seronegative donor leukopacks, and separated by countercurrent centrifugal elutriation as previously described. Monocytes were washed with PBS and fluorescently labeled with 10 uM Cell tracker green for 10 minutes at room temperature. Labeled cells were added to the upper Inhibitors,Modulators,Libraries compart ments of transwell inserts and allowed to transmigrate at 37 C in a humid atmosphere of 5% CO2 for 24 h. Trans migrated monocytes were quantified using florescent plate reader. Statistical analysis Statistical analysis was performed using one way analysis of variance with a post hoc Students t test. Results were judged statistically significant if P 0.
Inhibitors,Modulators,Libraries 05 by analysis of variance. Results HIV 1 mediated Inhibitors,Modulators,Libraries upregulation of PDGF B and MCP 1 in astrocytes Since astrocytes in the CNS are exposed to HIV 1, we first sought to examine the modulation of PDGF B and MCP 1 by HIV 1. Purified HIV 1 LAI virus obtained by high speed ultracentrifugation and resuspended in astro cyte serum free media was used for these experiments. Serum starved astrocytes were exposed to purified virus at a MOI of 0. 1 for 6 h followed by assessment of RNA levels by real time RT PCR. The MOI of HIV 1 LAI used was based upon our previous study. As shown in Figure 2A, HIV 1 LAI significantly upregulated both PDGF B and MCP 1 mRNA levels. To confirm whether increased mRNA levels of PDGF B translated into increased protein, a western blot analysis was performed on lysates of astrocytes exposed to HIV 1 LAI for 24 h.
As shown in Figure 2B exposure to HIV 1 LAI also induced upregulation of PDGF BB protein. Likewise, supernatants from Inhibitors,Modulators,Libraries A172 cells treated with HIV LAI were analyzed for MCP 1 levels via ELISA. As shown in Figure 2D, HIV 1 LAI exposure also resulted in increased MCP 1 levels. To determine whether HIV mediated induction of MCP 1 could, in part, be explained due to increased Inhibitors,Modulators,Libraries PDGF BB levels, astrocytes were treated with the PDGF receptor blocker STI 571 for 1 h prior to HIV 1 exposure and assessed for MCP 1 expression. Blocking the PDGF R significantly reduced HIV mediated induction of MCP 1 RNA and protein. PDGF BB mediated upregulation of MCP 1 in astrocytes All experiments involving the treatment of cells with ex ogenous PDGF BB protein were conducted under serum free conditions since PDGF promoter is known to have serum response elements. this website To investigate the role of PDGF on MCP 1 expression, A172 cells were treated with recombinant PDGF BB for the indicated times and mRNA levels were assessed by RT PCR and real time RT PCR. The concentration of PDGF BB used was based on previous studies.