Effect of halofuginone on vaginal infection.

<p>Mice, under pseudo-estrus conditions, were twice infected with 10⁷<i>Candida albicans</i> in vagina. Two days before and every two days after infection, mice were injected intraperitoneally with 5 µg/100 µl or 10 µg/100 µl of halofuginone solution or diluent of halofuginone and, in selected experiments, were treated intravaginally with 10 pg of mouse rIL-17. (A) Evaluation of IL-17 concentration by ELISA in supernatants of vaginal fluids obtained at different days after vaginal <i>Candida</i> infection and halofuginone treatment. Results are expressed as mean±SD (n = 9 mice, 3 mice for each of three separate experiments). The statistical analysis was performed using Mann-Whitney U test. * <i>p</i><0.05, ** <i>p</i><0.01 (infected halofuginone treated mice vs infected diluent treated mice). At day 4, 14 and 25 after infection, mice were treated intravaginally with 10 µg of coelenterazine and imaged in the IVIS-200™ imaging system under anesthesia using 2.5% isoflurane and the vaginal lumen was washed with 150 µl of saline. (B) In vivo imaging of mice vaginally infected with <i>Candida albicans</i> cells (gLUC) and treated with halofuginone or diluent. Images are representative of 5 out of 10 mice in two different experiments. (C) Dot plot of total photon emission from the infected regions and dot plot of CFU in vaginal washes of mice (n = 10) treated with halofuginone or diluent. The statistical analysis was performed using non-parametric Mann-Whitney U test. The median is indicated by a straight line. Data are representative of one out of two independent experiments with similar results. * <i>p</i><0.05, ** <i>p</i><0.01 (infected halofuginone treated mice vs infected diluent treated mice).</p

IL-17 and IL-23 concentration in murine vaginal washes of mice infected with <i>Candida albicans</i>.

<p>Evaluation of IL-17 (A–C) and IL-23 (D) concentration by ELISA test on supernatants of vaginal fluids obtained at different times after vaginal infection with different doses of <i>Candida albicans</i> gLUC59 (A–B) or CA1399 (D). Results are expressed as mean±SD (n = 12 mice, 4 mice for each of three separate experiments). * <i>p</i><0.05, (infected mice vs non infected mice).</p

Effect of halofuginone treatment on vaginal β defensin production.

<p>Mice, under pseudo-estrus conditions, were twice infected with 10⁷<i>Candida albicans</i> in vagina. Two days before and every two days after infection, mice were injected intraperitoneally with 5 µg/100 µl of halofuginone solution or diluent of halofuginone and were treated intravaginally with 10 pg of mouse rIL-17. At day 4, 8 and 14 after infection, mice were treated intravaginally with 10 µg of coelenterazine and imaged in the IVIS-200™ imaging system under anesthesia using 2.5% isoflurane. (A) Dot plot of total photon emission from the infected regions and dot plot of CFU in vaginal washes. The statistical analysis was performed using non-parametric Mann-Whitney U test. The median is indicated by a straight line. Data are representative of one out of two independent experiments with similar results. After 4, 8, 14, 21, 28 days from challenge, the vaginal lumen was washed with 150 µl of saline and vaginal cells were recovered for β-defensin analysis. (B) Mean of fluorescence MIF of β-defensin 1, β-defensin 2 and β-defensin 3 cells evaluated by FACS analysis. The vaginal cells recovered by vaginal washes were stained with rabbit anti-mouse BD1, goat anti-mouse BD2 or goat anti-mouse BD3 and goat anti-rabbit TRIC conjugate or rabbit anti-goat PE conjugate respectively. (C) Percentage of epithelial cells producing d β-defensin 2. Data are the mean±SD (n = 8 mice, 4 mice for each of two separate experiments). Cells of vaginal washes were stained with FITC anti mouse pan-cytokeratin and goat anti-mouse BD2 and rabbit anti-goat PE conjugate. Data are representative of one out of two independent experiments (total 8 mice). The statistical analysis was performed using Mann-Whitney U test. * <i>p</i><0.05, (infected halofuginone+IL-17 treated mice vs infected halofuginone treated mice).</p

Effect of <i>Candida albicans</i> infection on draining lumbar lymph nodes.

<p>Lymph nodes, at different times after <i>Candida</i> infection, were aseptically recovered and mechanically homogenized. Cells were cultivated untreated or in presence of heat inactivated <i>C. albicans</i> for 72 hours. In the supernatant fluids of lymph node cell culture IL-17 (A) and IL-23 (B) were analyzed by ELISA. Results are expressed as mean±SD (n = 16 mice, 4 mice for each of four separate experiments). The statistical analysis was performed using Mann-Whitney U test. * <i>p</i><0.05, (Lymphocytes from infected mice vs Lymphocytes from non infected mice). # <i>p</i><0.05, ## <i>p</i><0.01, (Lymphocytes re-stimulated from infected mice vs Lymphocytes re-stimulated from non infected mice).</p

Model of murine vaginal infection and monitoring.

<p>(A) Timeline of vaginal infection model. CD1 mice are resistant to <i>Candida</i> vaginal infection unless they are treated with estradiol valerate. CD1 mice were treated subcutaneously with estradiol valerate and infected for two consecutive days with 10 µl of 10⁹/ml suspension of <i>Candida albicans</i> cells (gLUC) into vaginal lumen. Two days before and every two days after challenge mice were treated intraperitoneally with halofuginone or diluent of halofuginone and, in selected experiments, intravaginally with 10 pg of recombinant mouse IL-17. After 4, 8, 12, 14, 18, 20, 25, 30 days post infection, mice were treated intravaginally with 10 µg of coelenterazine and imaged in the IVIS-200™ imaging system under anaesthesia using 2.5% isoflurane and the vaginal lumen was washed with 150 µl of saline using mechanical pipette. The fungal burden of vaginal fluids was evaluated by colony forming units (CFU) assay. (B) In vivo imaging of mice vaginally infected with <i>Candida albicans</i> cells (gLUC). Images are representative of 5 out of 10 mice for each experiment. C) Dot plots of total photon emission from the infected vaginal regions and corresponding CFU in vaginal washes of infected mice (n = 10). The statistical analysis was performed by non-parametric Mann-Whitney U test. The median is indicated by a straight line. Data are representative of one of two independent experiments with similar results. D) The correlation between the Total Photons emitted and CFU count in the vaginal wash was assessed using the Pearson's correlation statistics, and the correlation coefficients are shown for each time point. * <i>p</i><0.05, ** <i>p</i><0.01, (Log Photons Total Flux or Log CFU/ml of mice infected after 8, 12,14,18,20,25,30 days vs Log Photons Total Flux or Log CFU/ml of mice infected after 4 days).</p

Pharmacophore-Based Repositioning of Approved Drugs as Novel <i>Staphylococcus aureus</i> NorA Efflux Pump Inhibitors

An intriguing opportunity to address antimicrobial resistance is represented by the inhibition of efflux pumps. Focusing on NorA, the most important efflux pump of <i>Staphylococcus aureus</i>, an efflux pump inhibitors (EPIs) library was used for ligand-based pharmacophore modeling studies. By exploitation of the obtained models, an in silico drug repositioning approach allowed for the identification of novel and potent NorA EPIs