Inhibition of gp120-induced IL-6 expression by gp120-specific siRNA.

<p>Four gp120 siRNA (A) were designed using Ambion software and commercially synthesized by Ambion Inc. Only positive strand sequences are shown in the figure. Astrocytes were transfected with 50 nmoles each of scrambled or one of the 4 different siRNA for 48 hours before gp120 transfection. IL-6 mRNA (B) and protein (C) was measured at 6 and 48 hours, post-gp120 transfection. The mRNA is presented as relative percent mRNA expression with gp120 transfected cells as positive control. The protein concentration is presented as relative percent expression. Each bar represents mean ± SE of 3 experiments with each experiment done in triplicates. The statistical significance was calculated using student's t test and ** denotes p value of ≤0.01.</p

List of the initial six barrios proposed, and *38 final community clusters selected, for the COPA project in Ponce, Puerto Rico.

List of the initial six barrios proposed, and *38 final community clusters selected, for the COPA project in Ponce, Puerto Rico.</p

Community Organizations of COPA <i>Barrios</i> mentioned in focus group discussions and in-depth interviews.

Community Organizations of COPA Barrios mentioned in focus group discussions and in-depth interviews.</p

Important problems in the communities mentioned by participants of the focus groups discussions and the in-depth interviews.

Important problems in the communities mentioned by participants of the focus groups discussions and the in-depth interviews.</p

Guide of questions for moderator to conduct interviews to assess the acceptability and feasibility of <i>Aedes aegypti</i> control strategies with community leaders from Ponce, Puerto Rico.

Guide of questions for moderator to conduct interviews to assess the acceptability and feasibility of Aedes aegypti control strategies with community leaders from Ponce, Puerto Rico.</p

Inhibition of gp120-induced IL-6 expression by chemical inhibitors and siRNA specific for the NFκB pathway.

<p>SVGA astrocytes were treated with 10 µM SC514 (IKK-2 Inhibitor) and BAY11-7082 (IKKβ inhibitor) 24 hours prior to the transfection. 1×10⁶ SVGA astrocytes were transfected with 1 µg gp120 DNA in the presence of inhibitor, which was also maintained throughout experiments. IL-6 mRNA (A) and protein (B) was measured at 6 and 48 hours, post transfection respectively. For siRNA experiments, astrocytes were transfected with 50 nmoles of either scrambled, NFκB, or RelA siRNAs for 48 hours before gp120 transfection. IL-6 mRNA (C) and protein (D) was measured at 6 and 48 hours after gp120 transfection on cells previously transfected with siRNAs. (E) SVGA astrocytes were treated with 20 nM gp120IIIB. 10 µM SC514 was added to SVGA 1 hour prior to gp120 treatment. IL-6 mRNA was measured after 1 hour of gp120IIIB treatment. The mRNA is presented as relative percent mRNA expression with gp120 transfected cells as positive control. The protein concentration is presented as relative percent expression. Each bar represents mean ± SE of 3 experiments with each experiment done in triplicates. The statistical significance was calculated using student's t test and * and ** denotes p value of ≤0.05 and ≤0.01, respectively.</p

gp120 mediated increased levels of IL-6 in primary human fetal astrocytes.

<p>1×10⁶ primary human fetal astrocytes from each of three different donors were treated with 20 nM gp120 IIIB for specific lengths of time and the cells were harvested to obtain mRNA. The cell culture supernatants were collected at the times indicated and used to quantify the level of IL-6 protein expression using a Bio-Plex assay. Levels of IL-6 mRNA expression as measured by real-time RT-PCR peaked at 1 hour (A) and IL-6 protein expressions peaked at 6 hours (B). Monoclonal antibody for gp120 was used to negate the gp120 mediated response and thus served as another control. Heat inactivated gp120 was compared with untreated control to show gp120 specific IL-6 expression (C). Different strains of gp120 (D) and monomeric gp120 SF162 and trimeric gp140 SF162 (E) increased the levels of IL-6 differentially. Primary astrocytes from different donors were transfected with gp120 plasmid in order to compare the levels of IL-6 expression after 2 hours (D). Each bar represents mean ± SE of 3 experiments with each experiment done in triplicates. The statistical significance was calculated using student's t test and * and ** denotes p value of ≤0.05 and ≤0.01, respectively.</p

Increased NF-κB-translocation in gp120-transfected astrocytes.

<p>SVGA cells were either mock-transfected or were transfected with gp120 plasmid for a period of 6 hours followed by separation of cytosolic and nuclear fractions. These proteins were electrophoresed on 10% SDS gel and transferred to PVDF membrane. A representative western blot with lane-1 (mock-transfected SVGA cytosolic fraction), lane-2 (gp120-transfected SVGA cytosolic fraction), lane-3 (Mock-transfected SVGA nuclear fraction) and lane-4 (gp120-transfected SVGA nuclear fraction) is shown. The expression levels of p50 were normalized to their respective compartmental housekeeping genes (LaminB for nucleus and β-tubulin for cytoplasm) as loading controls. The bars, shown in the chart show normalized values of the band intensities for p50 over loading controls for appropriate compartments (LaminB for nucleus and β-tubulin for cytoplasm). The bar chart represents the means and SE of the ratios of p50 to the appropriate housekeeping gene (i.e. laminB for nuclear extracts and β-tubulin for cytoplasmic extracts). The means and SE values are from 3 independent experiments. The statistical significance was calculated using student's t test and * and ** denotes p value of ≤0.05 and ≤0.01, respectively.</p

Guide of questions for moderator to conduct focus group discussions to assess the acceptability and feasibility of <i>Aedes aegypti</i> control strategies with residents from Ponce, Puerto Rico.

Guide of questions for moderator to conduct focus group discussions to assess the acceptability and feasibility of Aedes aegypti control strategies with residents from Ponce, Puerto Rico.</p

gp120-mediated increase of IL-6 expression in SVGA astrocyte cells.

<p>1×10⁶ SVGA astrocytes were transfected with 1 µg gp120 JR-FL (R5 tropic) DNA using Lipofectamine™ 2000. The cells were harvested at the times described in the text. Mock transfection was performed with transfection of equal amount of empty human vector pcDNA3.1. All times noted on the figure and listed in the text are the times at which cells and supernatants were harvested after the end of the 5 hour transfection protocol. IL-6 mRNA (A) and IL-6 protein (B) expression levels were measured using real time RT-PCR and bioplex assays respectively. In (B), open bars show empty-vector transfected mock controls and closed bars show gp120 transfected samples. (C) shows the effect of exogenous gp120 on SVGA cells. SVGA were exposed to 20 nM recombinant gp120IIIB for various lengths of time and total mRNA was isolated. IL-6 expression levels were measured using real time RT-PCR. The mRNA levels are presented as fold difference between gp120 transfected cells and control cells transfected with empty plasmid. The protein concentration is presented as ng/ml protein in supernatant. Each bar represents mean ± SE of 3 experiments with each experiment done in triplicates. The statistical significance was calculated using student's t test and * and ** denotes p value of ≤0.05 and ≤0.01, respectively.</p