Antistaphylococcal effects of GML and DDG in a rabbit Wiffle ball infection model.

<p>Rabbits (n = 5 in each group) were infected with 3×10⁸ CFU/ml <i>S. aureus</i> MN8, and compounds (final concentration 1 mg/ml) were instilled into the Wiffle balls every-other-day and rabbits monitored up to 7 days. Survival of the rabbits (A), bacterial counts (B), TSST-1 production (C), and TNF-α levels (D) in the Wiffle balls. TSST-1 presented as percent of day 2 TSST-1 concentrations of the control rabbits (GML, close bars; DDG, open bars). Error bars are SEM. Symbols: ○, control; ▪, GML; ▴, DDG; *, p<0.05.</p

Stability of the compounds to <i>Staphylococcus aureus</i> (MN8) lipase.

<p>(A) Glycerol monolaurate (GML). (B) Dodecylglycerol (DDG). Clear zone indicates that the compound was degraded. Arrow denotes of the radius of the clear zone on the slide.</p

Cytotoxicity of GML and DDG to Human Vaginal Epithelial Cells (HVECs).

<p>HVECs were exposed to GML (▪) and DDG (▴) for 6 h. Cytotoxicity was accessed by measuring the release of LDH. Error bars are SEM. The dashed line indicates median cell survival (LD₅₀). Symbols:▪, GML; ▴, DDG.</p

Dodecylglycerol (DDG) inhibition of <i>Staphylococcus aureus</i>.

<p>DDG concentrations 25, 50, and 100 µg/ml were tested versus <i>S. aureus</i> isolates from different PFGE types, USA400 MRSA (A), USA400 MSSA (B), USA300 MRSA (C), USA200 MRSA (D), USA200 MSSA (E), atopic dermatitis strains (F), vaginal strains from healthy women (G), for 18 h at 37°C with shaking. The dashed line indicates the starting inocula. Each triangle (▴) indicates one isolate. The bars represent the mean±SEM of bacterial density in the group.</p

Glycerol monolaurate (GML) inhibition of <i>Staphylococcus aureus</i>.

<p>GML concentrations 50, 100, and 500 µg/ml were tested versus <i>S. aureus</i> isolates from different PFGE types, USA400 MRSA (A), USA400 MSSA (B), USA300 MRSA (C), USA200 MRSA (D), USA200 MSSA (E), atopic dermatitis strains (F), vaginal strains from healthy women (G), for 18 h at 37°C with shaking. The dashed line indicates the starting inocula. Each square (▪) indicates one isolate. The bars represent the mean±SEM of bacterial density in the group.</p

Effects of GML and DDG on <i>Staphylococcus aureus</i> Toxic Shock Syndrome Toxin-1 (TSST-1) production.

<p>(A) <i>S. aureus</i> MN8 was exposed to GML 0, 25 and 50 µg/ml for 6 and 24 h, and bacterial densities at 6 and 24 h were determined by plate counts. (B) The corresponding concentrations of TSST-1 of the above GML experiment. (C) <i>S. aureus</i> MN8 was exposed to DDG 0, 5, 15, and 25 µg/ml for 6 and 24 h. (D) The corresponding concentrations of TSST-1 from above DDG experiments. TSST-1 concentrations are presented as percent of the TSST-1 concentrations in 24 h control samples. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007499#s2" target="_blank">Results</a> are mean±SEM. The dashed line indicates the starting inocula. *, p<0.05.</p

TSST-1 induces IL-8 and activates NF-κB from HVEC with minimal toxicity.

<p>HVEC were exposed to TSST-1 (10–500 µg/ml) for 6 h, then cell viability (A) and IL-8 production (B) were determined. Viability was determined using an MTT assay. IL-8 was measured from culture supernatants by ELISA and expressed as fold increase in IL-8 production compared to media only control (TSST-1 0 µg/ml). Data presented are representative of three independent experiments done in triplicate, mean ± SD. * denotes p<0.05 compared to media only control.</p

TSST-1 induces cytokines from immortalized HVEC.

<p>HVECs were exposed to TSST-1 (50 and 100 µg/ml) or media only controls (0 µg/ml) for 6 h, then RNA was harvested and supernates collected. (A) changes in expression of proinflammatory genes were measured by real time RT-PCR, and (B–E) secreted cytokines were measured via multiplex cytokine array. Data presented in 2A are representative of three independent experiments conducted in triplicate, data presented in 2B–E are from one experiment conducted in triplicate, mean ± SD. * denotes p<0.05 compared to media only control.</p

Curcumin inhibits <i>S. aureus</i> exoprotein-induced IL-8.

<p>(A) Porcine vaginal explants (5 mm) were treated with curcumin in 5 µl of 100% DMSO and incubated for 18 h. Tissue viability was measured using a MTT assay and normalized to tissue left untreated (media only: no DMSO or curcumin). * denotes p<0.05 compared to vehicle control (0 nmol curcumin in 100% DMSO) by ANOVA. Curcumin inhibits IL-8 production. Porcine vaginal explants were left unstimulated (B) or stimulated with filtered <i>S. aureus</i> culture supernates for 1 h (C) and then exposed to curcumin (5 µl/explant in 10% DMSO) and incubated for an additional 6 h. Tissue was disrupted and IL-8 was measured by ELISA. * denotes p<0.05 compared to vehicle control (0 nmol curcumin in 10% DMSO) by ANOVA. Data presented are representative of three independent experiments done in triplicate, mean ± SD.</p

Primers used in reverse transcription polymerase chain reaction.

<p>Primers used in reverse transcription polymerase chain reaction.</p