Cytochemical staining for GGT enzyme activity in sputum samples.

<p>(A) Neutrophils with different levels of GGT activity in sputum films of patients with cystic fibrosis. (B) GGT-negative epithelial cell surrounded by GGT-positive neutrophils is also shown. Magnification 100×.</p

Total and fractional GGT activity in CF sputum.

<p>CF sputum samples were solubilised, centrifuged at 10,000×g and the supernatants analyzed by high-performance gel filtration chromatography. The table reports the whole GGT activity of each solubilised sputum and the activities corresponding to the two different GGT fractions identified by gel filtration chromatography. Data were are expressed as U/L.</p

GGT release by neutrophils.

<p>Neutrophils isolated from fresh buffy coats were incubated in (A) RPMI-1640 alone, (B) in the presence of ionomycin (0.5 µM; 15 min) or (C) fMLP (1 µM; 120 min). GGT activity was measured in the 10,000×g centrifuged supernatants. Results are means ± SD of three separate determinations. Data were analyzed by Student's t test; (*) p<0.0001.</p

Relationship between GGT activity and MPO levels in CF sputum samples.

<p>MPO levels were detected by western blot analysis in samples of solubilised sputum pellets and correlated with GGT activity of solubilised sputum supernatants. A) Lane 1–7, CF samples; lane 8, control (neutrophils). B) Data reported are expressed as a <i>ratio</i> of MPO against GADPH band densities, while GGT values are normalized on protein content. R² = 0.683; p = 0.02.</p

High-performance gel filtration chromatography of soluble fraction of CF sputum.

<p>Supernatants obtained from sputum solubilisation and centrifugation at 10,000×g were ultracentrifuged again at 100,000×g before analysis. A) 100,000×g supernatant; B) 100,000×g pellet. Data represent one representative separation out of three and are expressed as arbitrary units (a.u.) of fluorescence.</p

GGT activity in neutrophils fractions obtained on Percoll gradients.

<p>A high GGT activity was found in fractions γ, β₁ and in the fractions between β₂ and β₁ (β₂β₁). Data reported were obtained from neutrophils isolated from three different healthy donors. Cy = cytosol; γ = secretory vesicles and plasma membranes; β₂ = gelatinase granules; β₁ = specific granules; α = azurophilic granules; γβ₂, β₂β₁ and β₁α are the fractions recovered among the main bands.</p

Relationship between sputum GGT activity and FEV1 values of CF patients.

<p>R² = 0,717, p<0.02.</p

Inhibition of <i>Pseudomonas aeruginosa</i> secreted virulence factors reduces lung inflammation in CF mice

<p><b>Background</b>: Cystic fibrosis (CF) lung infection is a complex condition where opportunistic pathogens and defective immune system cooperate in developing a constant cycle of infection and inflammation. The major pathogen, <i>Pseudomonas aeruginosa</i>, secretes a multitude of virulence factors involved in host immune response and lung tissue damage. In this study, we examined the possible anti-inflammatory effects of molecules inhibiting <i>P. aeruginosa</i> virulence factors.</p> <p><b>Methods</b>: Pyocyanin, pyoverdine and proteases were measured in bacterial culture supernatant from different <i>P. aeruginosa</i> strains. Inhibition of virulence factors by sub-inhibitory concentrations of clarithromycin and by protease inhibitors was evaluated. Lung inflammatory response was monitored by in vivo bioluminescence imaging in wild-type and CFTR-knockout mice expressing a luciferase gene under the control of a bovine IL-8 promoter.</p> <p><b>Results</b>: The amount of proteases, pyocyanin and pyoverdine secreted by P. aeruginosa strains was reduced after growth in the presence of a sub-inhibitory dose of clarithromycin. Intratracheal challenge with culture supernatant containing bacteria-released products induced a strong IL-8-mediated response in mouse lungs while lack of virulence factors corresponded to a reduction in bioluminescence emission. Particularly, sole inactivation of proteases by inhibitors Ilomastat and Marimastat also resulted in decreased lung inflammation.</p> <p><b>Conclusions</b>: Our data support the assumption that virulence factors are involved in <i>P. aeruginosa</i> pro-inflammatory action in CF lungs; particularly, proteases seem to play an important role. Inhibition of virulence factors production and activity resulted in decreased lung inflammation; thus, clarithromycin and protease inhibitors potentially represent additional therapeutic therapies for <i>P. aeruginosa</i>-infected patients.</p

Relationship between GGT activity and cysteinyl-glycine (CysGly) levels in whole sputum.

<p>Data were obtained from seven different samples of CF sputum. (A) Free and (B) protein bound CysGly. A) R² = 0,811, p<0.01; B) R² = 0,917, p<0.001.</p

Microbiological characterization of CF sputum samples.

<p>Microbiological characterization of CF sputum samples.</p