AA, but not IFN-α, induces cell death in HAM/TSP PBMCs.

<p>HAM/TSP PBMCs (n = 6) were treated for 72 hours in the absence or presence of low-dose AA (10 µg/ml), high-dose AA (100 µg/ml) or IFN-α2A (1000 IU/ml). (A) The percentage of cell death was quantified by trypan blue dye exclusion and is shown in the <i>y</i>-axis, whereas the treatment conditions are shown in the <i>x</i>-axis. The one-sample t-test p-values are indicated by asterisks (^*<0.05, ^**<0.01 and ^***<0.001). (B) Fluorescence microscopy images of Hoechst 33342-stained PBMCs are shown for untreated (NT) and high-dose AA-treated PBMCs of one representative HAM/TSP patient (n = 3). Nuclear condensation and DNA fragmentation (f) was observed for AA-treated PBMCs.</p

Overview of the top 20 of the most significant down- and up-regulated genes by high-dose AA (100 µg/ml) in MT-2 cells.

<p>Overview of the top 20 of the most significant down- and up-regulated genes by high-dose AA (100 µg/ml) in MT-2 cells.</p

AA treatment dose-dependently induces cell death in HTLV-1-infected cell lines.

<p>HTLV-1-infected CD4+ T-cell lines were cultured for 48 hours with no treatment (NT), low-, intermediate-, high-dose AA (10, 50, 100 µg/ml) or IFN-α (1000 IU/ml). Flow cytometric quantification of cells with DNA degradation (Hoechst 33342-positive subdiploid cells) and proliferating cell nuclear antigen (PCNA)-positive cells are shown in the <i>y</i>-axis for both (A) MT-4 (n = 4) as well as (B) MT-2 cells (n = 3), whereas the treatment conditions are shown in the <i>x</i>-axis. ANOVA with Bonferroni post-test for multiple testing was used and the p-values are indicated by asterisks (^*<0.05, ^**<0.01 and ^***<0.001). (C) Confocal microscopy images of Hoechst 33342-stained MT-2 cells are shown for untreated and high-dose AA-treated MT-2 cells. Nuclear condensation (c) was observed for the AA-treated cells, whereas normal nuclei and mitosis (m) were observed for the untreated cells.</p

High-dose AA, but not IFN-α, exerts strong antiproliferative effects in HAM/TSP PBMCs.

<p>(A) Lymphoproliferation of normal donors (NDs) PBMCs (n = 4) and HAM/TSP PBMCs (n = 3) was quantified by [³H]thymidine incorporation (cpm), performed in triplicate. NDs and HAM/TSP PBMCs were cultured for 5 days with no treatment (NT), high-dose AA (100 µg/ml) or IFN-α (1000 IU/ml). Thymidine incorporation is shown in the <i>y</i>-axis, whereas the treatment conditions are shown in the <i>x</i>-axis. ANOVA with Bonferroni post-test for multiple testing was used and the p-values are indicated by asterisks (^*<0.05, ^**<0.01 and ^***<0.001). (B) NDs PBMCs (n = 3) and HAM/TSP PBMCs (n = 6) were cultured in the absence or presence of low-dose AA (10 µg/ml), high-dose AA (100 µg/ml) or IFN-α2A (1000 IU/ml) for 72 hours. Direct cell counts of viable cells were quantified by trypan blue dye exclusion. ANOVA with post-test for linear trend was used and the p-values are indicated by asterisks (^*<0.05, ^**<0.01 and ^***<0.001).</p

Overview of the significant down-regulated genes and the top 20 of the most significant up-regulated genes by IFN-α in MT-2 cells.

<p>Overview of the significant down-regulated genes and the top 20 of the most significant up-regulated genes by IFN-α in MT-2 cells.</p

Cell death is the principal high-dose AA-modulated molecular network in MT-2 cells.

<p>The networks were generated through the use of IPA (Ingenuity® Systems, <a href="http://www.ingenuity.com" target="_blank">www.ingenuity.com</a>), based on genes regulated by high-dose AA treatment in MT-2 cells (after 48 hours of culture). The principal molecular network is graphically displayed, identified as cell death. The red and green colour indicate up- or down-regulation, respectively, with the intensity indicating the degree of the gene transcription change. The log₂ fold-change values and p-values are indicated below each molecule. Network shapes are represented in the legend. Full lines represent direct interactions, while dashed lines indirect interactions.</p

Schematic overview of normal donors, asymptomatic carriers and HAM/TSP patients.

<p>EDSS = expanded disability status scale, “0” reflecting normal neurological examination and “10” death; PVL = proviral load expressed as HTLV-1 copies per 10⁴ PBMCs; ND = not determined; predn = prednisone; AA = high-dose ascorbic acid; 3TC = lamivudine; baclo = baclofen; AZT = zidovudine; F = female; M = male.</p

High-dose AA treatment decreases IL-6 and HTLV-1 p19 levels in HTLV-1-infected cell line supernatant.

<p>HTLV-1-infected CD4+ T-cell lines were cultured for 48 hours with no treatment (NT), low-, intermediate-, high-dose AA (10, 50, 100 µg/ml) or IFN-α (n = 4). (A) IL-6 levels were quantified in cell-free supernatant of MT-2 cells and shown in the <i>y</i>-axis. HTLV-1 p19 concentration (pg/ml) was determined in cell-free supernatant of (B) MT-2 and (C) MT-4 cells and shown in the <i>y</i>-axis. The treatment conditions are shown in the <i>x</i>-axis. ANOVA with Bonferroni post-test for multiple testing was used and the p-values are indicated by asterisks (^*<0.05, ^**<0.01 and ^***<0.001).</p