Liposome-entrapped antibiotic stability assayed by microbiological assay.

<p>The stability of the liposomal formulations were examined at 37°C in an 18 h period in the presence of PBS, CAMH broth, supernatant of biofilm forming <i>P. aeruginosa</i>, a combination of DNA, F-actin, LPS, and LTA, and diluted intact or autoclaved sputum.</p

Bactericidal activity and inhibition of antibiotics by DNA, F-actin, LPS and LTA.

<p>A) Bactericidal concentrations of free tobramycin (F-TOB) and liposomal tobramycin (L-TOB) were incubated in presence of LPS/LTA (1 to 1000 mg/L). B) Bactericidal concentrations of free polymyxin B (F-PMB) and liposomal polymyxin B (L-PMB) were incubated in presence of DNA/F-actin/LPS/LTA (125 to 1000 mg/L). Growth controls are represented at 0 h (empty bar), and 3 h (dark bar). Comparisons between free and liposomal formulations were made by ANOVA one-way post <i>t</i>-test, and <i>P</i>-values were considered significant when (**) <i>p</i><0.01, (***) <i>p</i><0.001.</p

Bactericidal activity and inhibition of antibiotics by LPS and LTA.

<p>A) Bactericidal concentrations of free tobramycin (F-TOB) and liposomal tobramycin (L-TOB) were incubated in presence of LPS/LTA (1 to 1000 mg/L). B) Bactericidal concentrations of free polymyxin B (F-PMB) and liposomal polymyxin B (L-PMB) were incubated in presence of LPS/LTA (1 to 1000 mg/L). Growth controls are represented at 0 h (empty bar), and 3 h (dark bar). Comparisons between free and liposomal formulations were made by ANOVA one-way post <i>t</i>-test, and <i>P</i>-values were considered significant when (***) <i>p</i><0.001.</p

CF Sputum treatment with various antibiotic formulations.

<p>CFU counts were made after incubation of diluted CF sputum (1∶10 w/v) in PBS with two-fold dilutions of free tobramycin at 512 mg/L (F-TOB), liposomal tobramycin at 128 mg/L (L-TOB), free polymyxin B at 32 mg/L (F-PMB), and liposomal polymyxin B at 8 mg/L (L-PMB). Growth controls are represented at 0 h (empty bar), and 18 h (dark bar).</p

Minimum Bactericidal Concentrations.

<p>Bactericidal activity of free and liposomal formulations against susceptible <i>P. aeruginosa</i> ATCC 27853 strain was carried out in broth alone or in presence of DNA/F-actin/LPS/LTA at a final concentration of 1000 mg/L.</p

Bactericidal activity and inhibition of tobramycin by DNA and F-actin.

<p>Bactericidal concentrations of free tobramycin (F-TOB), and liposomal tobramycin (L-TOB) at 2 mg/L were incubated with <i>P. aeruginosa</i> (ATCC 27853), or in presence of DNA/F-actin (125 to 1000 mg/L). Growth controls are represented at 0 h (empty bar), and 3 h (dark bar). Comparisons between free and liposomal tobramycin was made by ANOVA one-way post <i>t</i>-test, and <i>P</i>-values were considered significant when (***) <i>p</i><0.001.</p

IL-1β treatments modulate the systemic cytokine response.

<p>IL-6 (A), IL-12p70 (B), TNF-α (C), IL-1β (D), KC/gro (E), and IFN-γ (F) were measured using ELISA-based assays. Generally, secretion of pro-inflammatory cytokines was dissipated in <i>Nlrp3^−/−</i> mice at 10 DPI compared to WT mice. One asterisk: <i>P</i><0.05, two asterisks: <i>P</i><0.01, three asterisks: <i>P</i><0.001; ND, not detected.</p

IL-1β treatments augment macrophage colonic infiltration.

<p>Macrophage infiltration was assessed by staining cryosections of distal colons. An increase in the number of macrophages (red; indicated by arrowhead in higher magnification panels on the left) in close proximity of <i>C. rodentium</i> (green) at 10 DPI in <i>Nlrp3^−/−</i> mice is noted. With IL-1β treatments, the macrophages in crypts and mucosal lining appeared increased and bacterial infiltration into the crypts reduced. Bar 50 µm.</p

IL-1β treatments reduce intestinal colonization and dissemination of <i>C. rodentium</i> in <i>Nlrp3^−/−</i> mice.

<p>Infected mice were given repeated IL-1β treatments on 0, 2, and 4 DPI. <i>C. rodentium</i> colonization of the (A) cecum, (B) colon, and (C) dissemination to MLN were assessed on 6 and 10 DPI by plating homogenates on MacConkey agar plates in serial dilutions. IL-1β treatments of <i>Nlrp3^−/−</i> mice significantly lowered cecum colonization on 6 DPI, and colon colonization and dissemination to MLN on 10 DPI. Values were Log₁₀ transformed and data presented as mean ± SE. One asterisk: <i>P</i><0.05, two asterisks: <i>P</i><0.01.</p

IL-1β treatments modulate <i>C. rodentium</i> penetration and epithelial integrity.

<p>Biotin was administered into the lumen of the distal colon at 6 or 10 DPI. Cryosections were stained and viewed under fluorescent microscopy for <i>C. rodentium</i> infiltration (in red; indicated by arrowheads in the enlarged image of the inset highlighted by boxes in the left panels) and biotin penetration (green; indicated by asterisks). Uninfected IL-1β-treated mice retained biotin to the apical surface. In non-treated mice at 6 DPI, the bacterial infiltration and biotin penetration was increased. IL-1β treatments at 6 DPI did not seem to have a major effect on infiltration or epithelial integrity. In non-treated mice at 10 DPI, the bacterial infiltration and epithelial barrier disruption were lowered in WT and elevated in <i>Nlrp3^−/−</i> mice, relative to 6 DPI. In IL-1β-treated mice at 10 DPI, the bacterial infiltration and barrier disruption were elevated in WT and lowered in <i>Nlrp3^−/−</i> mice. Bacterial colonization followed the same trend as biotin penetration. Magnification X400, bar 50 µm.</p