Representative FACS histograms demonstrating intracellular-location of TLR-4, and a reduced total TLR-4 after CSE treatment.

<p>Localisation of Toll-like receptor 4 (TLR-4). (A): Representative histogram for surface and cytoplasmic staining for IgG and TLR-4 in bronchial epithelial cells. (B): rMFI for TLR-4 in permeabilised and non-permeabilised nasal and bronchial epithelial cells. (C): Staining for TLR-4 in permeabilised bronchial epithelial cells after stimulated with 25 µg/ml <i>P aeruginosa</i> LPS, with or without 24 h pre-treatment with CSE. (D): rMFI for TLR-4 was lower in bronchial epithelial cells than for nasal epithelial cells (for control, 4 h and 24 h CSE treatment). A 24 h exposure to CSE significantly decreased rMFI for bronchial, but not for nasal, epithelial cells. Data are displayed as median (interquartile range). <b>*</b> indicates a significant difference (<i>p</i><0.05).</p

Effects of (A) 5% CSE and (B) acrolein on IL-8 release from nasal and bronchial epithelial cultures.

<p>Cells were treated 5% CSE prepared with a single cigarette for 4 h or 24 h (n = 5). In separate experiments, cells were treated with increasing concentrations of acrolein for 4 h. Supernatants were collected and assessed for IL-8 by ELISA in all cases. Data are displayed as median (interquartile range). <b>*</b> indicates a significant difference (<i>p</i><0.05).</p

Effect of LPS ±5% CSE pre-treatment on TLR-4 mRNA expression.

<p>Nasal epithelial cell cultures were pretreated with 5% CSE prepared for (A) 4 h,(B) 24 h or vehicle, and subsequently stimulated with <i>P aeruginosa</i> LPS [10–30 µg/ml]for 24 h. Experiments were repeated in bronchial epithelial cell cultures, again for (C) 4 h and (D) 24 h 5% CSE exposure.</p

Immunocytochemistry for cytokeratin 5 in nasal and bronchial epithelial cell cultures.

<p>Primary nasal (A) and bronchial (B) epithelial cell cultures were stained with a rabbit anti-human antibody against cytokeratin 5 (1∶100). The primary antibody was detected using a secondary antibody coupled to Alexafluor 568 (1∶500). Nuclei were stained blue with DAPI (×40).</p

(A) IL-8 dose response and (B) linear regression analysis from paired nasal and bronchial epithelial cells from COPD subjects after 24 h treatment with LPS.

<p>Paired nasal and bronchial epithelial cells were with various concentrations of LPS [5–25 µg/ml] for 24 h (n = 8). Supernatants were collected and assessed for IL-8 by ELISA in all cases. Data are displayed as median (interquartile range). * indicates a significant difference (<i>p</i><0.05).</p

IL-6 dose response from paired PNECs and PBECs from COPD subjects after 24 h treatment with LPS.

<p>Paired nasal and bronchial epithelial cells were with various concentrations of LPS [0–25 µg/ml] for 24 h (n = 8). Supernatants were collected and assessed for IL-6 by ELISA in all cases. Data are displayed as median (interquartile range). <b>*</b> indicates a significant difference (<i>p</i><0.05).</p

Growth curves showing total viable counts of parent MRSA and <i>P. aeruginosa</i> isolates and resistant mutants which developed following exposure to fosfomycin and tobramycin.

<p>(A) MRSA CFP13, aerobic, (B) MRSA CFP13 anaerobic, (C) <i>P. aeruginosa</i> W050 aerobic and (D) <i>P. aeruginosa</i> W050 anaerobic. Filled circle (•) Parent strain; filled square (▪) Fosfomycin resistant mutant; filled triangle (▴) Tobramycin resistant mutant.</p

Fosfomycin, tobramycin and F∶T MICs for MRSA and <i>P. aeruginosa</i> strains used in this study.

<p>Fosfomycin, tobramycin and F∶T MICs for MRSA and <i>P. aeruginosa</i> strains used in this study.</p

Fosfomycin, tobramycin and F∶T MICs for <i>P. aeruginosa</i> isolates following serial passage in sub-inhibitory antibiotic concentrations and subsequent removal of antibiotic pressure following 12 passages.

<p>(A) AY4 FOS aerobic, (B) AY4 FOS anaerobic and (C) W050 TOB aerobic. Filled circle (•) Fosfomycin MIC; filled square (▪) Tobramycin MIC; filled triangle (▴)F∶T MIC; broken line, antibiotic pressure removed.</p

Fosfomycin, tobramycin and F∶T MICs for MRSA isolate CFP8 following serial passage in sub-inhibitory antibiotic concentrations under both aerobic and anaerobic conditions.

<p>Exposure to (A) Fosfomycin aerobic, (B) Fosfomycin anaerobic, (C) Tobramycin aerobic, (D) Tobramycin anaerobic, (E) F∶T aerobic, (F) F∶T anaerobic. Filled circle (•) Fosfomycin MIC; filled square (▪) Tobramycin MIC; filled triangle (▴)F∶T MIC.</p