Neutrophil responses in the proximal (bronchial wash) and distal (bronchoalveolar lavage) airway lumen, as well as the epithelium and submucosa of the bronchial airway in healthy subjects exposed to air and ozone.

<p>Data are given as means with standard deviation and comparisons of the post air and ozone neutrophil numbers at each time point were performed using a paired t-test. </p

Peripheral blood total leukocyte and neutrophil numbers at 1.5, 6 and 18 hours post air and ozone challenge.

<p>Data are illustrated as means with standard deviations. Comparisons of cell numbers post air and ozone challenge were performed using a paired t-test.</p

Cartoon of the exposure protocol employed in the current study, with three separate groups undergoing blood and bronchoscopy-based sampling at various times post a standardised two hour filtered air and ozone exposure.

<p>Cartoon of the exposure protocol employed in the current study, with three separate groups undergoing blood and bronchoscopy-based sampling at various times post a standardised two hour filtered air and ozone exposure.</p

Significant associations (Pearson correlation) between the observed systemic neutrophil response at 1.5, 6 and 18 hour post-exposure (parameter post ozone minus that post air) with that observed in the airway lumen (sampled by BW), the bronchial airway epithelium and submucosa at the equivalent sampling time point.

<p>The Pearson correlation coefficient (r) and the 2-tailed significance (P) for each association is illustrated together with a linear regression through the data.</p

Percentage (mean ± SD) of blood vessels immunostaining for P-selectin and ICAM in the submucosa of bronchial biopsies obtained 1.5, 6 and 18 hours after the end of a 2 hour exposure to air and 0.2 ppm ozone.

<p>Comparison of the extent of immunoreactivity was performed using a Students paired t-test.</p

Particulate Oxidative Burden Associated with Firework Activity

Firework events are capable of inducing particulate matter (PM) episodes that lead to exceedances of regulatory limit values. As short-term peaks in ambient PM concentration have been associated with negative impacts on respiratory and cardiovascular health, we performed a detailed study of the consequences of firework events in London on ambient air quality and PM composition. These changes were further related to the oxidative activity of daily PM samples by assessing their capacity to drive the oxidation of physiologically important lung antioxidants including ascorbate, glutathione and urate (oxidative potential, OP). Twenty-four hour ambient PM samples were collected at the Marylebone Road sampling site in Central London over a three week period, including two major festivals celebrated with pyrotechnic events: Guy Fawkes Night and Diwali. Pyrotechnic combustion events were characterized by increased gas phase pollutants levels (NOx and SO2), elevated PM mass concentrations, and trace metal concentrations (specifically Sr, Mg, K, Ba, and Pb). Relationships between NOx, benzene, and PM10 were used to apportion firework and traffic source fractions. A positive significant relationship was found between PM oxidative burden and individual trace metals associated with each of these apportioned source fractions. The level of exposure to each source fraction was significantly associated with the total OP. The firework contribution to PM total OP, on a unit mass basis, was greater than that associated with traffic sources: a 1 μg elevation in firework and traffic PM fraction concentration was associated with a 6.5 ± 1.5 OPT μg−1 and 5.2 ± 1.4 OPT μg−1 increase, respectively. In the case of glutathione depletion, firework particulate OP (3.5 ± 0.8 OPGSH μg−1) considerably exceeded that due to traffic particles (2.2 ± 0.8 OPGSH μg−1). Therefore, in light of the elevated PM concentrations caused by firework activity and the increased oxidative activity of this PM source, there is value in examining if firework derived PM is related to acute respiratory outcomes

Effect of 25(OH)D3 on antioxidant responses in primary HBEC cultures.

(A) Ratio of reduced (GSH) to oxidised (GSSG) glutathione in 24 hour cultures of HBECs treated with/without 100nM 25(OH)D3; n = 6. (B) Fold-increase in 8-isoprostane levels in culture supernatants from primary HBECs cultured with 50μg/ml NIST with/without 100nM 25(OH)D3 for 24 hours, compared to VC control cultures; ratio paired t-test, n = 8. Statistical significance as follows: *, p ≤ 0.05.</p

Transcription microarray of 24 hour primary human bronchial epithelial cell (HBEC) cultures stimulated with NIST in the presence/absence of vitamin D.

(A) Volcano plot of the 510 genes with ≥ 1.4 fold differential expression comparing 50μg/ml NIST stimulated to unstimulated 24 hour cultures (n = 4), showing fold-change in gene expression in NIST stimulated cultures in the presence vs absence of 100nM 1,25(OH)2D3 (horizontal axis) plotted again probability of statistical significance for that fold-change (vertical axis). (B) Plot showing microarray results for fold-change in expression of all cytokine genes upon stimulation of HBECs with 50μg/ml NIST (horizontal axis) and fold-change in gene expression upon addition of 100nM 1,25(OH)2D3 to NIST-stimulated cultures (vertical axis).</p

Comparison of expression of vitamin D axis genes between HBECs from healthy and asthmatic donors.

(A) Induction of CYP24A1 by NIST with 100nM 1,25(OH)2D3, relative to the unstimulated control condition, in 4 hour and 24 hour HBEC cultures. H, Healthy donor HBECs; A, Asthmatic donor HBEC cultures. 4hr healthy, n = 5; 4hr asthmatic, n = 5; 24hr healthy, n = 7; 24hr asthmatic, n = 6.(B) Expression of CYP24A1 as measured by qPCR relative to 18S in 4 hour and 24 hour HBEC cultures stimulated with 50μg/ml NIST and 100nM 1,25(OH)2D3. 4hr healthy, n = 5; 4hr asthmatic, n = 5; 24hr healthy, n = 7–8; 24hr asthmatic, n = 6. (C) Induction of CAMP by NIST with 100nM 1,25(OH)2D3, relative to the unstimulated control condition, in 4 hour and 24 hour HBEC cultures. 4hr healthy, n = 5; 4hr asthmatic, n = 5; 24hr healthy, n = 5; 24hr asthmatic, n = 6. (D) Expression of CAMP as measured by qPCR relative to 18S in 4 hour and 24 hour HBEC cultures stimulated with 50μg/ml NIST and 100nM 1,25(OH)2D3. 4hr healthy, n = 5; 4hr asthmatic, n = 5; 24hr healthy, n = 7; 24hr asthmatic, n = 6.</p

Effects of 1,25(OH)₂D3 on production of IL-6, CXCL8 and GM-CSF by NIST stimulated HBEC cultures.

(A) Addition of 100nM 1,25(OH)2D3 reduced production of IL-6 by primary HBEC cultures stimulated for 24 hours with 50μg/ml NIST, but not CXCL8 or GM-CSF. VC: NIST vehicle control. n = 14–17. (B) Fold-increase in production of IL-6, CXCL8, and GM-CSF above that in unstimulated cultures upon stimulation with 50μg/ml NIST in the presence/absence of 1,25(OH)2D3, by disease status. Two-way ANOVAs with Bonferroni-corrected post-tests. n = 8–9 healthy, n = 7–8 asthmatic. (C) Percentage suppression by 1,25(OH)2D3 of cytokine production in PM-stimulated cultures of HBECs from healthy donors compared to asthmatic donors. Un-paired t-tests. n = 8–9 healthy, n = 7–8 asthmatic. Statistical significance as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001, ****, p ≤ 0.0001.</p