Primary antibodies and conditions used for identification of inflammatory markers, Hsps, and pHSF-1 protein.

<p>Primary antibodies and conditions used for identification of inflammatory markers, Hsps, and pHSF-1 protein.</p

NFkB-p65 binding sites in the human <i>hsp60</i> gene.

<p>NFkB-p65 binding sites in the human <i>hsp60</i> gene.</p

Correlations between neutrophils and Hsp60.

<p>Regression analysis between number of Hsp60 positive cells (vertical axis) and number of neutrophils (horizontal axis), panels A and B, and between number of Hsp60 positive cells (vertical axis) and number of MPO positive neutrophils (horizontal axis), panels C and D, in the lamina propria of all smokers (panels A and C) and of patients with COPD alone, considered as a single group (panels B and D).</p

Immunohistochemical quantification of Hsp and HSF-1 proteins in bronchial mucosa.

<p>Abbreviations: COPD, chronic obstructive pulmonary disease; MPO, myeloperoxidase. Data expressed as median (range). Statistics: The Kruskal-Wallis test was used for multiple comparisons followed by Mann-Whitney U test for comparison between groups: #, p<0.05, significantly different from control non smokers; ε, p<0.05, significantly different from control smokers with normal lung function; The exact “p” values for comparison between groups are given in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028200#s2" target="_blank">Results</a> section.</p

Inflammatory markers in bronchial mucosa.

<p>Abbreviations: COPD, chronic obstructive pulmonary disease; MPO, myeloperoxidase; FoxP3: forkhead box P3. Data expressed as median (range). Statistics: Kruskal-Wallis test for multiple comparisons. For comparison between groups the Mann-Whitney U test was applied: #, p<0.05, significantly different from control non smokers; ε, p<0.05, significantly different from control smokers with normal lung function; ^, p<0.05, significantly different from mild/moderate COPD. The exact “p” values for comparison between groups are given in the Results section.</p

Hsps in the bronchial epithelium and lamina propria: Representative images.

<p>Photomicrographs showing frozen sections of bronchial mucosa from a control non smoker (A, C, E) and from a patient with severe stable COPD (B, D, F) immunostained to identify Hsp10 (A, B), Hsp40 (C, D), and Hsp60 (E, F). Nuclei were counterstained with haematoxylin (blue). Cells positive for Hsps are in red. Inset in F shows a cell double stained for neutrophil elastase (red) and Hsp60 (brown). Bar  = 50 microns.</p

Hsp mRNA levels, expressed as the ratio [mRNA of gene of interest] / [mRNA of housekeeping gene (GAPDH)], in the bronchial mucosa.

<p>Abbreviations: COPD, chronic obstructive pulmonary disease, Hsp, heat shock protein. Data are median (range). Relative levels of mRNAs are expressed as the ratio [mRNA gene of interest OD] / [mRNA housekeeping gene OD (GAPDH)]. Statistics: Kruskal-Wallis test was applied for multiple comparisons.</p

Quantification of mRNA in the bronchial biopsies and binding of NFkB-p65 to the human <i>hsp60</i>-gene promoter region.

<p>Panel A: Levels of HSF-1 and NFKB-p65 mRNA did not change significantly after treatment (50 or 100 µM of H₂O₂). Panel B: the binding of NFkB-p65 to the <i>hsp60</i> promoter was lower in untreated (UT) cells (lane 2) compared to the binding in cells treated with 50 or 100 µM of H₂O₂ (lanes 3 and 4, respectively). Lanes: 1, Marker; 5, DNA UT Positive control; 6, DNA UT Negative control; and 7, Input DNA.</p

Clinical characteristics of subjects studied.

<p>Patients were classified according to GOLD (<a href="http://www-goldcopd.com" target="_blank">http://www-goldcopd.com</a>) levels of severity for COPD into: mild/moderate (stages I–II) and severe/ very severe (stages III–IV).</p><p>Data are mean±SD. For COPD patients FEV₁/FVC (%) are post-bronchodilator values.</p><p>Abbreviations: n, number of subjects; M, male; F, female, FEV₁: forced expiratory volume in one second; FVC, forced vital capacity; ND, not determined; COPD, chronic obstructive pulmonary disease. Statistics. (ANOVA) #, p<0.0001, significantly different from control smokers with normal lung function and control never-smokers; ^&, p<0.0001, significantly different from mild/moderate COPD.</p

Impact of oxidative stress on the levels of Hsp60 in a human bronchial epithelial cell line.

<p>Panel A: Western blotting showing significantly increased levels of Hsp60 in 16HBE after treatment with 50 or 100 μM of H₂O₂ compared to untreated (UT) cells. The difference between 100 and 50 μM was also significant. Histograms represent the mean (SD) of the Hsp60/actin ratio. Panel B: RT-PCR showing significantly increased levels of Hsp60 only after treatment with 100 μM of H₂O₂ compared to UT treated cells. Panel C: Hsp60 increased levels in the extracellular medium (measured by ELISA tests) after treatment with 50 or 100 μM of H₂O₂ compared to untreated (UT) cells. ^: p<0.005; *: p<0.001.</p