Additional file 1 of Sustaining training effects through physical activity coaching (STEP): a randomized controlled trial

Additional file 1. Methods detailed information and Figure AF1 (Algorithm for the calculation of the PA weekly goal in steps/day)

Effect of 1,25(OH)₂D on the <i>ex vivo</i> inflammatory response of alveolar macrophages from non-smoking and smoking subjects.

<p>Alveolar macrophages from non-smokers (n = 5) or smokers (n = 5) were stimulated for 48h with 10 nM 1,25(OH)₂D or vehicle, followed by an additional stimulation for 24h with LPS/IFN-γ. Levels of (A) IL-8, (B) TNF-α, (C) MCP-1 and (D) IL-6 were measured in supernatants of alveolar macrophage cultures. mean±SEM. *p<0.05, ***p<0.001.</p

Effect of CSE on vitamin D metabolism in THP-1 macrophages.

<p>THP-1 macrophages were stimulated for 16h with 10% CSE or vehicle, followed by an additional stimulation for 24h with 10 nM 1,25(OH)₂D or vehicle. mRNA expression levels of (A) VDR, (B) CYP27B1 and (C) CYP24A1 were determined in cell lysates. mean±SEM. *p<0.05, ***p<0.001</p

Primers used for quantitative PCR analysis.

<p>Primers used for quantitative PCR analysis.</p

Effect of 1,25(OH)₂D on <i>ex vivo</i> antibacterial response of alveolar macrophages from non-smoking and smoking subjects.

<p>Alveolar macrophages from non-smokers or smokers were stimulated for 48h with 10 nM 1,25(OH)₂D or vehicle. (A) Phagocytosis and (B) oxidative burst by alveolar macrophages from non-smokers (n = 7) and smokers (n = 11) following bacterial challenge. Graphs show the percentage of alveolar macrophages that have internalized <i>E</i>. <i>coli</i> bacteria (A) or have produced reactive oxygen species (B). (C) Cathelicidin levels were measured in supernatants of alveolar macrophage cultures from non-smokers (n = 5) and smokers (n = 5). mean±SEM. *p<0.05, **p<0.01.</p

Effect of 1,25(OH)₂D on the inflammatory response in CSE-treated THP-1 macrophages.

<p>THP-1 macrophages were stimulated for 16h with 10% CSE or vehicle, followed by an additional stimulation for 24h with 10 nM 1,25(OH)₂D or vehicle. mRNA and protein levels of (A) IL-8, (B) TNF-α and (C) MCP-1 were determined in cell lysates and culture supernatants, respectively. Independent experiments were performed in triplicate. mean±SEM. *p<0.05, **p<0.01, ****p<0.0001.</p

1,25-Dihydroxyvitamin D Modulates Antibacterial and Inflammatory Response in Human Cigarette Smoke-Exposed Macrophages

<div><p>Cigarette smoking is associated with increased inflammation and defective antibacterial responses in the airways. Interestingly, vitamin D has been shown to suppress inflammation and to improve antibacterial defense. However, it is currently unknown whether vitamin D may modulate inflammation and antibacterial defects in human cigarette smoke (CS)-exposed airways. To explore these unresolved issues, alveolar macrophages obtained from non-smoking and smoking subjects as well as human cigarette smoke extract (CSE)-treated THP-1 macrophages were stimulated with 1,25-dihydroxyvitamin D (1,25(OH)₂D) to address inflammatory and antibacterial responses. Although basal levels of inflammatory cytokines and chemokines did not differ between non-smoking and smoking subjects, 1,25(OH)₂D did reduce levels of IL-6, TNF-α and MCP-1 in alveolar macrophages in response to LPS/IFN-γ, although not statistically significant for TNF-α and IL-6 in smokers. CSE did not significantly alter vitamin D metabolism (expression levels of CYP24A1 or CYP27B1) in THP-1 macrophages. Furthermore, stimulation with 1,25(OH)₂D reduced mRNA expression levels and/or protein levels of IL-8, TNF-α and MCP-1 in CSE-treated THP-1 macrophages. 1,25(OH)₂D did not improve defects in phagocytosis of <i>E</i>. <i>coli</i> bacteria or the oxidative burst response in CSE-treated THP-1 macrophages or alveolar macrophages from smokers. However, 1,25(OH)₂D significantly enhanced mRNA expression and/or protein levels of the antimicrobial peptide cathelicidin in alveolar macrophages and THP-1 macrophages, independently of CS exposure. In conclusion, our results provide the first evidence that vitamin D could be a new strategy for attenuating airway inflammation and improving antibacterial defense in CS-exposed airways.</p></div

Effect of 1,25(OH)₂D on antibacterial response in CSE-treated THP-1 macrophages.

<p>THP-1 macrophages were stimulated for 16h with 10% or 25% CSE or vehicle, followed by an additional stimulation for 24h with 10 nM 1,25(OH)₂D or vehicle. (A) Phagocytosis and (B) oxidative burst by THP-1 macrophages following bacterial challenge. Graphs show the percentage of alveolar macrophages that have internalized <i>E</i>. <i>coli</i> bacteria (A) or have produced reactive oxygen species (B). (C) mRNA and protein levels of cathelicidin were determined in cell lysates and culture supernatants, respectively. Independent experiments were performed in triplicate. mean±SEM. **p<0.01, ***p<0.001, ****p<0.0001.</p

Additional file 1: of End-stage cystic fibrosis lung disease is characterised by a diverse inflammatory pattern: an immunohistochemical analysis

Table S1. Detailed overview of the used primary and secondary antibodies, and chromogens. Superscript numbers (1–5) above the catalogue number indicate which primary antibody is combined with which secondary antibody and chromogen. Abbreviation: RTU = ready to use. Table S2. Inter- and intra-observer variability in the counting of the myeloid and lymphoid cells, expressed by means of the Spearman’s rank correlation coefficient. Significant correlations are indicated with *, with *** = p < 0.001. Table S3. Overview of the number of analysed follicles in each compartment for every included tissue block. CF11-20 represents the female CF patients. Table S4. Quantification of the myeloid cell types and lymphoid follicles according to localization (subdivided in the three compartments: airways, parenchyma and perivascular). The p-values in the right-hand column are the result of Kruskal-Wallis 1-way ANOVA testing. Significant differences with airways are indicated with *, with * = p < 0.05, ** = p < 0.01 and *** = p < 0.001. These values are the results of Dunn’s post hoc testing. Figure S1. Histological section of formalin-fixed paraffin-embedded CF lung tissue. Section was stained for mast cells (tryptase). Representative image showing an airway surrounded by circular fibrosis suggestive of constrictive bronchiolitis. The bronchiole is accompanied by its blood vessel. Abbreviations: AW = airway, BV = blood vessel. Scale bar = 100 μm. Figure S2. Serial histological sections of formalin-fixed paraffin-embedded CF lung tissue. Both images show the same lymphoid follicles located in the proximity of an airway. Panel A shows a CD20 staining of all B cells lying organised in germinal centres. These are surrounded by a paracortex staining positively for CD3 T cells (panel B). High endothelial venules (green arrowhead) allowing extravasation of naïve B and T cells into the lymphoid follicle are located in the T cell area. AW = airway. Scale bar = 50 μm. (DOCX 19517 kb

Additional file 1: of The aging lung: tissue telomere shortening in health and disease

Supplementary materials, methods and results. (DOCX 21 kb