Maintained Smoking Cessation for 6 Months Equilibrates the Percentage of Sputum CD8+ Lymphocyte Cells with That of Nonsmokers

Little is known about the longitudinal effects of smoking cessation on sputum inflammatory cells. We aimed to investigate the changes in sputum inflammatory cells and T-lymphocyte subpopulations after 6 and 12 months smoking cessation. Induced sputum was obtained from 68 healthy smokers before and after 6 months (n = 21) and 1 year (n = 14) smoking cessation and from ten healthy never-smokers. Inflammatory cells were identified by morphology and T-lymphocyte subpopulations by flow cytometry. Sputum macrophages were decreased after 12 months of smoking cessation in comparison to baseline, while neutrophils increased. Moreover, CD8+ T-cells were decreased in smokers before smoking cessation compared to never-smokers and increased in smokers after 6 months of smoking cessation in comparison to baseline; result that was maintained after 1 year of smoking cessation. These novel findings indicate that smoking cessation can equilibrate certain inflammatory cells of smokers with those of nonsmokers, within 6 months of smoking cessation

Detection of Pgp 170 by Western Blotting in the 10 <i>Leishmania</i> isolates.

<p>The Pgp 170 molecules (130-200 kDa) were evident in the “resistant” (high Rhod-123 efflux) but not in the “susceptible” (low Rhod-123 efflux) isolates. The “resistant” human isolate (H2) presented lower signal compared to the dog “resistant” isolates. For the evaluation of the Pgp expression in each isolate, the C219 monoclonal antibody and exponential phase promastigotes, at a concentration of 10⁷ parasites, were used.</p

Treatment with Glucantime resulted in an increase in the number of Pgp molecules.

<p>Mean Fluorescent Intensity (MFI) of the 10 <i>Leishmania</i> isolates, measured by Flow Cytometry, showed an increase in the number of Pgp molecules, in all isolates, if they had previously been treated with the drug Glucantime compared to the untreated controls. This increase was not linear (r₂ = 0.27).</p

Detection of Pgp 170 molecules, by Transmission Electron Microscopy, after immunogold labeling.

<p><i>Leishmania</i> amastigote (inside a THP-1 infected cell): cytoplasm of the THP-1 cell (a); <i>Leishmania infantum</i> body (b). Black spots (as indicated by white arrow) show Pgp 170 molecules (gold granules after immunogold labeling using the C219 monoclonal antibody).</p

Survival of <i>Leishmania</i> promastigotes in different Amphotericin B concentrations, in time.

<p>Survival of the “resistant” dog isolate D5 (a) and of the “susceptible” dog isolate D1 (b), in different drug concentrations for 6 days.</p

Location of Pgp molecules in the body of <i>Leishmania</i> observed under Confocal Microscopy.

<p><i>Leishmania</i> amastigotes (inside THP-1 infected cells) marked with double immunofluorescence: positive dog serum and FITC (green signal) (a); Pgp 170 molecules marked with the C219 monoclonal antibody and the infrared Cy3 anti-mouse IgG secondary antibody (red signal) (b); overlay of image a and b (c). Image c confirms the location of Pgp 170 molecules on the membrane of the parasite body.</p

Mean Fluorescent Intensity (MFI) before and after treatment with Glucantime.

<p>In all 10 isolates, irrespective of the rate of Rhod-123 efflux (slope “α”), treatment with Glucantime resulted in an increase of MFI, estimated by Confocal Microscopy (CM), indicating an increase in the number of Pgp 170 molecules.</p

Number of Pgp molecules in a ''resistant'' and a ''susceptible'' isolate before/after exposure to Glucantime.

<p>The number of Pgp 170 molecules was found in higher numbers in the “resistant” compared to the “susceptible” isolate. This number increased, in both isolates, after exposure to Glucantime.</p

Rate of efflux of Rhod-123, in the 10 <i>Leishmania</i> isolates, estimated by Flow Cytometry.

<p>Efflux, both in the absence (slope “α”) and presence (slope “β”) of Verapamil hydrochloride was found to be correlated to the influx (the amount of drug that entered the parasite body: Mean Fluorescent Intensity on baseline (MFI) (c = 0.63 and c = 0.58, respectively). Efflux was blocked by Verapamil hydrochloride in all isolates except in isolate D2, with the lowest slope “α”, for which slope “β” had the same value (0.16)*.</p

Rhod-123 efflux in <i>Leishmania</i> promastigotes observed by Flow Cytometry.

<p>High Rhod-123 efflux in the “resistant” dog isolate D5 (a). Low Rhod-123 efflux in the “susceptible” dog isolate D1 (b). Measurements were taken every 30 minutes, for two hours.</p