Induction of IL-8 but not IP-10 in response to Th2 stimulation.

<p>ppBECs were treated for 24-13 (10 ng/ml) and IL-4 (10 ng/ml) for 24 hours. Protein secretion in cell supernatant was measured by ELISA (R&D). Statistical analysis 1A n = 9 ANOVA P<0.05. 1B n = 15, Wilcoxon, p<0.05. NT-no treatment.</p

Panel of antibodies used.

<p>Panel of antibodies used.</p

Correlations between epithelial phenotype and measures of lung function.

<p>Correlations between epithelial phenotype and measures of lung function.</p

Subject characteristics.

<p>Subject characteristics.</p

Graphs showing epithelial phenotype.

<p>The percentage of epithelium with squamous metaplasia (A), that with a pseudostratified (B) and a fully squamous (C) phenotype are shown in healthy non-smokers (●), healthy smokers (■) and subjects with COPD1(▲) and COPD2(▼). Significant differences between the groups are indicated.</p

Mechanical Strain Causes Adaptive Change in Bronchial Fibroblasts Enhancing Profibrotic and Inflammatory Responses

<div><p>Asthma is characterized by periodic episodes of bronchoconstriction and reversible airway obstruction; these symptoms are attributable to a number of factors including increased mass and reactivity of bronchial smooth muscle and extracellular matrix (ECM) in asthmatic airways. Literature has suggested changes in cell responses and signaling can be elicited via modulation of mechanical stress acting upon them, potentially affecting the microenvironment of the cell. In this study, we hypothesized that mechanical strain directly affects the (myo)fibroblast phenotype in asthma. Therefore, we characterized responses of bronchial fibroblasts, from 6 normal and 11 asthmatic non-smoking volunteers, exposed to cyclical mechanical strain using flexible silastic membranes. Samples were analyzed for proteoglycans, α-smooth muscle actin (αSMA), collagens I and III, matrix metalloproteinase (MMP) 2 & 9 and interleukin-8 (IL-8) by qRT-PCR, Western blot, zymography and ELISA. Mechanical strain caused a decrease in αSMA mRNA but no change in either αSMA protein or proteoglycan expression. In contrast the inflammatory mediator IL-8, MMPs and interstitial collagens were increased at both the transcriptional and protein level. The results demonstrate an adaptive response of bronchial fibroblasts to mechanical strain, irrespective of donor. The adaptation involves cytoskeletal rearrangement, matrix remodelling and inflammatory cytokine release. These results suggest that mechanical strain could contribute to disease progression in asthma by promoting inflammation and remodelling responses.</p></div

Epithelial expression of early markers of carcinogenesis and proliferation.

<p>CEA is expressed in the luminal cells of pseudostratified epithelium (A), p53 is weakly expressed in the perinuclear region of suprabasal cells (B), nuclear expression of ki67 can also be seen (C). Positive staining is brown. No staining was observed in the negative controls sections (D). Scale bar = 20 um. The percentage of the epithelium expressing CEA (E), p53 (F) and Ki67 (G) are shown in healthy non-smokers (●), healthy smokers (■) and subjects with COPD1(▲) and COPD2(▼). Significant differences and Kruskall Wallis P values between the groups are indicated.</p

Epithelial correlations.

<p>Epithelial correlations.</p

Characterization of microfluidic culture system.

<p>Differentiated PBECs were cultured in the microfluidic culture system and after an equilibration phase of 1h apically exposed to pollen extract. a: The flow rate of pollen-challenged and control wells was determined by measuring the outlet volume per hour for a period of 12h. n = 7 independent experiments with different donors. b: The transepithelial electrical resistance (TER) of differentiated PBECs was measured before and after microfluidic culture conditions without or with pollen challenge. Cells cultured in common culture conditions without flow were used as static controls. TER was normalized to the respective value before the start of the experiment. n = 15 independent experiments using 13 different donors; *: p≤0.05.</p

Design and assembly of the microfluidic culture system.

<p>a: Schematic section of a culture well: cells are cultured on a Transwell^® inserted in the holder. A V-ring is used to seal the system. b: Schematic of the setup: each well is connected to the syringe pump. A plate is placed under the device and is moved to allow for the time-resolved fraction collection. c: Exploded view of the assembly of a single well. Five layers of dry film photoresist are laminated on top of a support layer. A holder is screwed on top of the support layer and a Transwell^® is subsequently inserted together with the V-ring. The microfluidic inlet is screwed in the holder while the outlet needle is glued on the bottom of the support layer.</p