Monocyte-derived macrophages (A), monocyte-derived dendritic cells (B) and T cells (C) were incubated with R5 or X4 viruses in the presence of increasing concentrations of C14 or an unique dose of T 20 (5 μM) for 3 hours at 37°C

Cells were then washed and cultured in fresh medium for 3 days. DNA was extracted and the viral DNA was quantified by real time PCR. Means are representative of 3 independent experiments and assays were performed in triplicates. *, ≤ 0.05; **, ≤ 0.01 between untreated and treated cells.<p><b>Copyright information:</b></p><p>Taken from "Pre-clinical development as microbicide of zinc tetra-ascorbo-camphorate, a novel terpenoid derivative: Potent in vitro inhibitory activity against both R5- and X4-tropic HIV-1 strains without significant in vivo mucosal toxicity"</p><p>http://www.aidsrestherapy.com/content/5/1/10</p><p>AIDS Research and Therapy 2008;5():10-10.</p><p>Published online 3 Jun 2008</p><p>PMCID:PMC2426711.</p><p></p

Monocyte-derived macrophages, monocyte-derived dendritic cells and T cells were incubated with R5 viruses in the presence of increasing concentrations of C14 for 3 hours at 37°C

Cells were then washed and incubated with polyclonal human anti-gp160 antibodies. The staining was revealed with FITC-conjugated mouse anti-human IgG mAbs. Cells were then analysed by confocal microscopy. The experiment was performed 3 times with cells from three different donors. 30 cells were at least analyzed for each donor.<p><b>Copyright information:</b></p><p>Taken from "Pre-clinical development as microbicide of zinc tetra-ascorbo-camphorate, a novel terpenoid derivative: Potent in vitro inhibitory activity against both R5- and X4-tropic HIV-1 strains without significant in vivo mucosal toxicity"</p><p>http://www.aidsrestherapy.com/content/5/1/10</p><p>AIDS Research and Therapy 2008;5():10-10.</p><p>Published online 3 Jun 2008</p><p>PMCID:PMC2426711.</p><p></p

HIV-1 was adsorbed on poly-L-lysin pre-coated wells (Greiner Bio-One) at 4°C overnight and further incubated with C14 for 1 h

In positive and negative control wells, 1% Triton X-100 and medium were added, respectively. After four washes, activated peripheral blood lymphocytes were incubated with C14 or triton-treated or untreated HIV-1 particles. After 6 days, viral production was assessed by p24 Ag capture ELISA. Means are representative of 3 independent experiments were performed in triplicates. **, ≤ 0.01.<p><b>Copyright information:</b></p><p>Taken from "Pre-clinical development as microbicide of zinc tetra-ascorbo-camphorate, a novel terpenoid derivative: Potent in vitro inhibitory activity against both R5- and X4-tropic HIV-1 strains without significant in vivo mucosal toxicity"</p><p>http://www.aidsrestherapy.com/content/5/1/10</p><p>AIDS Research and Therapy 2008;5():10-10.</p><p>Published online 3 Jun 2008</p><p>PMCID:PMC2426711.</p><p></p

The effects of HIV and Tat treatments on the secretion of IL-18 and IL-18BP from IEC.

<p>The panels A and B show mean concentrations of IL-18 and IL-18BP, respectively, in the culture supernatants. The vertical line on each bar denotes standard deviation. The Tat used in these experiments was pre-treated with Tat-neutralizing antibodies (Neut Tat) or with control antibodies (Tat).</p

The effects of IL-18 on the expression of Tight Junction proteins in the intestinal cell monolayers.

<p>The cell monolayers were treated with IL-18 (10 ng/ml) and/or with Tat (100 ng/ml) for 24 hours, after which the cell monolayers were washed with PBS and stained with either protein specific rabbit antibodies or control antibodies (negative control), washed and stained with FITC-conjugated goat anti-rabbit antibodies. The cells were also stained with DAPI and examined under a fluorescent microscope and imaged. Staining of Caco2 monolayers for Claudin-2 (<b>A</b>) and of HT-29 monolayers for Occludin (<b>B</b>). The treatment of HT-29 monolayers also reduced expression of both Claudin-2 and Occludin when determined by Western blots (<b>C</b>).</p

IL-18 and LPS activate caspase-1 and caspase-3 in HT-29 cells.

<p>The cell monolayers were treated with IL-18 (10 ng/ml) or with LPS (10 ng/ml) for 12 hours. Thereafter, the monolayers were washed with PBS, lysed and the activation of the caspases was determined on Western blots by using antibodies specific to the activated forms of the caspases. The Figure shows results from a representative of three independent experiments.</p

IL-18 increases expression of MLCK and pMLC via ROCK, and decreases that of phosphorylated STAT-5.

<p>HT29 cell monolayers were treated with IL-18 (10 ng/ml) and/or Tat (100 ng/ml) with or without a cell permeable inhibitor of ROCK (GSK429286A, 10μM; added 30 minutes prior to the addition of the cytokine). At the indicated time points, the cells were lysed. About 25 ug of the lysate proteins were resolved on SDS-PAGE and the Western blots were performed. Panel <b>A</b> shows results of a representative of three experiments for the expression of p-MLC. Panel <b>B</b> shows expression of MLCK after treatment of the monolayers with IL-18 (10 ng per ml) and/or with Tat (100 ng/ml) after 30 minutes. Panel <b>C</b> shows expression of pSTAT-5 and STAT-5 from IL-18 (10 ng per ml) and/or with Tat (100 ng per ml)-treated cells.</p

IL-18-induced cell death is partially inhibited by inhibitors of caspase-1 and caspase-3.

<p>HT29 cell monolayers were pre-treated with cell permeable inhibitors for caspase-1 (Z-YVAD-FMK), caspase-3 (Z-DQMD-FMK) or both (50 uM each) for 30 minutes. After 24 hours, the cells were collected and apoptosis was determined by staining with FITC-annexin V and PI. Panel <b>A</b> shows dot plots of the cells stained for Annexin V and PI. Panel <b>B</b> summarizes the results from three independent experiments by showing mean %ages ± SE.</p

IL-18 decreases TEER in Caco2 monolayers.

<p>The TEER was measured during 24 hours after addition of 10 ng/ml of IL-18, 10 ng/ml IL-1β or 100 ng/ml Tat to the cell monolayers. Note dramatic decrease of the TEER occurring at hour 6 post-exposure by both IL-18 and Tat. IL-1β causes a transient increase in the TEER followed by a decrease beginning at hour 14 post-exposure. TEER was measured by the Electric Cell-substrate Impedance Sensing ECIS Zθ instrument and 8W10E+ electrode arrays were used.</p

The effects of HIV and Tat treatments on the secretion of IL-18 and IL-18BP from IEC.

<p>The panels A and B show mean concentrations of IL-18 and IL-18BP, respectively, in the culture supernatants. The vertical line on each bar denotes standard deviation. The Tat used in these experiments was pre-treated with Tat-neutralizing antibodies (Neut Tat) or with control antibodies (Tat).</p