Effects of D,L-Buthionine- (S,R) sulphoximine (BSO) and desferrioxamine in combination with ellagic acid on the FcM29 strain.

<p>Values are the means of the FIC₅₀ (which is an interaction coefficient indicating whether the combined effect of the drugs is synergistic, additive, or antagonistic) and standard deviations for assays run in triplicate on different days.</p><p>The combination was considered to be synergistic if the FIC₅₀ was <1, additive if the FIC₅₀ was equal to 1 and antagonistic if the FIC₅₀ was >1.</p

ROS generation in <i>Plasmodium falciparum</i> was detected by DCFDA probe.

<p>All parasitized erythrocytes were labeled with DCFDA for 30 minutes. After washing, they were incubated for 3 hours with or without ellagic acid, ascorbic acid and NAC, alone and in combination. The level of ROS in the parasites was detected by DCF-dependent measurements. The data represent the mean ±SEM of four different experiments with triplicate sets in each assay. In the first column (<b>A</b>), the fluorescence intensity in parasites without ellagic acid is represented. The black band (control value) corresponded to the fluorescence intensity in untreated parasites incubated 3 hours. The grey and white bands corresponded to the fluorescence intensity in parasites only treated with NAC (857.10⁻⁶ M) and ascorbic acid (113.10⁻⁶ M) respectively. In the next four columns, the black bands are the fluorescence of parasites after treatment with various concentrations (between 6.61×10⁻⁹ and 6610×10⁻⁹ M) ellagic acid alone. The grey and white bands correspond to the ellagic acid treatment with NAC and ascorbic acid respectively with the same concentrations as in the first column. **p<0.01 indicates a significant difference compared with the untreated parasites. The quantity amount of ROS was compared according to the increased of EA concentration, ## p<0.05 indicates the significant difference.</p

Inhibition of ellagic acid antiplasmodial activity by antioxidants (ascorbic acid, N-acetyl-L- cysteine and glutathione ethyl-ester) on the FcM29 strain of <i>Plasmodium falciparum</i>.

<p>Ascorbic acid (113.55 µM) and N-acetyl-L-cysteine (857 µM) had no effect on <i>in vitro Plasmodium</i> growth. Ascorbic acid and NAC significantly decreased (p<0.05) the ellagic acid antiplasmodial efficacy since the IC₅₀ obtained were increased 2.4 fold and 1.5 fold respectively. Data are expressed as mean ±SEM for three independent experiments with triplicate sets in each assay.</p>**<p>p<0.05 indicates a significant difference compared with ellagic acid mean IC_50.</p

<i>Ex-vivo</i> experiments: short-term culture of <i>P. yoelii</i> with ellagic acid.

<p>Data are expressed as mean ±SEM for three independent experiments with triplicate sets in each assay.</p><p>The radioactive micromethod was used after 24 h of incubation at 37°C, to determine the IC₅₀ of ellagic acid in the “<i>P.yoelii high GSH</i>” strain <i>versus</i> the controls corresponding to the wild type <i>P yoelii</i>.</p

Cell-bound galectin-3 does not potentiate PLM-A-induced TNF-α production.

<p>(<b>A</b>). The levels of endogenous galectin-3 bound to the J774 cell surface were determined by immunofluorescent staining using anti-galectin-3 antibody and FITC-conjugated secondary antibody. Cells were then fixed and analyzed by fluorescence microscopy. (<b>B</b>). Alternatively, cells were incubated with 10 µg/ml of exogenous rGal-3 for 1h at 20°C before staining and analysis performed as described in (A). Insert shows high magnification of labeled cells. (<b>C</b> and <b>D</b>) Cells were preincubated for 1h with (■) or not (□) 10 µg/ml of exogenous rGal-3 prior to the addition of 10 µg/ml of PLM-A or PLM-BMT6∆ (<b>C</b>) 10 µg/ml of curdlan or 500 ng/ml of Pam3CSK4 (<b>D</b>). Cells were cultivated for additional 4h. Then, supernatants were collected and TNF-α concentration was determined by ELISA. Results are presented as the mean ± standard deviation from 3 independent experiments. *<i>p<0</i>.<i>05; **p<0.01; ***p<0.0005</i>.</p

Long chain oligomannosides are required for PLM-induced TNF-α production.

<p>(<b>A</b>). J774 macrophages were incubated with increasing concentration of PLM-A (●), PLM-B (■) or PLM-BMT6∆ (○) and cultivated for 5h at 37°C. Cell culture supernatants were harvested and TNF-α production was evaluated by ELISA. (<b>B</b>). Alternatively, cells were stimulated with PLM-A (50 µg/ml) or with curdlan (100 µg/ml) and Pam3CSK4 (500 ng/ml) as positive controls. Data represents the mean ± standard deviation from 3 independent experiments, each one performed in triplicates. (<b>C</b> and <b>D</b>). J774 macrophages were pretreated (■) or not (□) with Latrunculin A for 30 min prior to PLMs addition at the indicated concentrations (<b>C</b>) or live <i>C. albicans</i> serotype A and B yeast cells at a 5 yeast : 1 cell ratio (<b>D</b>). Curdlan and Pam3CSK4 were used as positive controls. TNF-α concentration in cell-free supernatants was determined by ELISA after 4h incubation. Data are representative of two independent experiments performed in triplicates. *p<0.05<i>; **p<0.01; ***p<0.0005</i>.</p

Structural comparison between glycan chains of PLM-A, PLM-B and PLM-BMT6Δ.

<p>(<b>A</b>). General structure of PLMs used in the present study, showing the differences in glycan chain length linked to the lipid backbone. (<b>B</b>). PLM-A was isolated from SC5314, a WT <i>C. albicans</i> serotype A strain; PLM-B, isolated from the NIHB strain, a serotype B strain and; PLM-BMT6∆, isolated from the <i>bmt6</i>∆ mutant. Following, PLMs were submitted to hydrolysis and analyzed by Fluorophore-assisted carbohydrate electrophoresis (FACE) method. The analysis shows the different mannosidic chains released after the hydrolysis protocol. Isolated mannosides (M2, M3, M4, M8, M11-13) were used as control. </p

Soluble galectin-3 potentiates PLM-A-induced TNF-α production.

<p>Exogenous rGal-3 (10 µg/ml) was preincubated (■) or not (□) with increasing concentrations of PLM-A (A), PLM-B (B) or PLM-BMT6∆ (C) for 1h. The mixtures were then added to J774 macrophages and incubated for an additional 4h. TNF-α concentration in cell-free supernatants was determined by ELISA. (D) Exogenous rGal-3 (10 µg/ml) was preincubated (■) or not (□) with 10 µg/ml of curdlan or 500 ng/ml of Pam3CSK4. Results are represented as the mean ± standard deviation from 3 independent experiments. P values refer to the statistical differences between the effect exerted by galectin-3 alone and in association with PLMs. *<i>p<0</i>.<i>05; **p<0.01</i>.</p

PLMs induce caspase-1 activation independent on ROS production.

<p>(<b>A</b>-<b>C</b>). J774 macrophages were primed with LPS (100 ng/ml) for 2h. Then, indicated stimuli were added and cells were cultivated for additional 4h. (<b>A</b>). Caspase-1 activation was revealed by addition of fluorescent FAM-YVAD-FMK and examined by fluorescent microscopy. Adenosine triphosphate (ATP) and Aluminum hydroxide (Alum) were used as positive control for caspase-1 activation. (<b>B</b>). Total pro-caspase-1 and cleaved caspase-1 levels were revealed by western blot analysis. (<b>C</b>). Production of IL-1β after stimulation with PLMs of LPS-primed cells was measured after 12- and 24h-incubation by ELISA. (<b>D</b>). Macrophages were pretreated or not with Latrunculin-A and then stimulated with indicated PLMs. Total pro-caspase-1 and cleaved caspase-1 levels were revealed by western blot analysis. Data are representative of 4 independent experiments. (<b>E</b> and <b>F</b>). Macrophages were pretreated or not with Latrunculin A for 30min. Live yeast cells at 5 yeast: 1 cell ratio (<b>E</b>) or PLMs (50µg/ml) (<b>F</b>) were then added. Chemiluminescence was monitored for 2h in the presence of 66 µM of 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol). 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 µM) was used as a positive control. Results are representative of 2 independent experiments.</p

Regulation of CD36 mRNA expression by tumor necrosis factor (TNF)α and adalimumab

<p><b>Copyright information:</b></p><p>Taken from "Tumor necrosis factor alpha and adalimumab differentially regulate CD36 expression in human monocytes"</p><p>http://arthritis-research.com/content/9/2/R22</p><p>Arthritis Research & Therapy 2007;9(2):R22-R22.</p><p>Published online 2 Mar 2007</p><p>PMCID:PMC1906797.</p><p></p> TNFα decreases CD36 mRNA expression and adalimumab increases CD36 mRNA expression. Human monocytes were incubated with macrophage-serum-free medium (M-SFM) alone (control), or with M-SFM containing TNFα (10 ng/ml) or adalimumab (Ada; 1 μg/ml) for 4 h. CD36 mRNA expression was quantified using RT-PCR and normalized to β-actin. Data represent the mean ± standard error of the relative quantification of CD36 mRNA expression measured in three experiments. *Significantly different from control (< 0.05)