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

Subject characteristics.

<p>Subject characteristics.</p

Panel of antibodies used.

<p>Panel of antibodies used.</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

Correlations between epithelial phenotype and measures of lung function.

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

Epithelial correlations.

<p>Epithelial correlations.</p

Viral Inhibition of Bacterial Phagocytosis by Human Macrophages: Redundant Role of CD36

<div><p>Macrophages are essential to maintaining lung homoeostasis and recent work has demonstrated that influenza-infected lung macrophages downregulate their expression of the scavenger receptor CD36. This receptor has also been shown to be involved in phagocytosis of <i>Streptococcus pneumoniae</i>, a primary agent associated with pneumonia secondary to viral infection. The aim of this study was to investigate the role of CD36 in the effects of viral infection on macrophage phagocytic function. Human monocyte-derived macrophages (MDM) were exposed to H3N2 X31 influenza virus, M37 respiratory syncytial virus (RSV) or UV-irradiated virus. No infection of MDM was seen upon exposure to UV-irradiated virus but incubation with live X31 or M37 resulted in significant levels of viral detection by flow cytometry or RT-PCR respectively. Infection resulted in significantly diminished uptake of <i>S</i>. <i>pneumoniae</i> by MDM and significantly decreased expression of CD36 at both the cell surface and mRNA level. Concurrently, there was a significant increase in IFNβ gene expression in response to infection and we observed a significant decrease in bacterial phagocytosis (p = 0.031) and CD36 gene expression (p = 0.031) by MDM cultured for 24 h in 50IU/ml IFNβ. Knockdown of CD36 by siRNA resulted in decreased phagocytosis, but this was mimicked by transfection reagent alone. When MDM were incubated with CD36 blocking antibodies no effect on phagocytic ability was observed. These data indicate that autologous IFNβ production by virally-infected cells can inhibit bacterial phagocytosis, but that decreased CD36 expression by these cells does not play a major role in this functional deficiency.</p></div

Effect of influenza infection on bacterial phagocytosis by MDM.

<p>MDM were differentiated in the presence of 2 ng/ml GM-CSF for 12 d prior to infection with H3N2 X31 influenza virus or a UV-irradiated aliquot of virus (UVX31) for 2 h. After washing, media was replaced and the cells incubated for a further 22 h before supernatants and cells were harvested for <b>(A) & (B)</b> influenza NP1 expression (% cells, n = 7) by flow cytometry. Phagocytosis of <i>S</i>. <i>pneumonia</i> was detected in <b>(C)</b> X31-infected (n = 5) MDM after a further 2 h incubation with live bacteria by culture. <b>(A)</b> Representative flow cytometry plot of MDM expressing influenza NP1. Data are expressed as means ±SE of n independent experiments and analysed using a Wilcoxon-signed rank test * p<0.05, ** p<0.01.</p

Virus-induced expression of IFNβ and effect of IFNβ on bacterial phagocytosis.

<p>MDM were differentiated in the presence of 2 ng/ml GM-CSF for 12 d prior to infection with <b>(A)</b> H3N2 X31 influenza virus or a UV-irradiated aliquot of virus (UVX31) or <b>(C)</b> M37 RSV or a UV-irradiated aliquot of virus (UV-RSV) for 2 h. After washing, media was replaced and the cells incubated for a further 22 h before supernatants and cells were harvested for IFNβ gene expression by RT-PCR (X31 n = 9, RSV n = 7). MDM were differentiated for 12 d as above before treatment without (NT) or with 50 IU/ml IFNβ for 24 h and <b>(B)</b> cells harvested for CD36 gene expression by RT-PCR (n = 5) or <b>(D)</b> phagocytosis of <i>S</i>. <i>pneumonia</i> was detected after a further 2 h incubation with live bacteria by culture (n = 5). PCR data were normalised to β2MG and are expressed as mean fold induction over the non-infected (NI) sample ± SEM. Data are expressed as means ±SE of n independent experiments and analysed using a Wilcoxon-signed rank test * p<0.05, ** p<0.01.</p

Airway Elastin is increased in severe asthma and relates to proximal wall area: histological and computed tomography findings from the U-BIOPRED severe asthma study

Background: Airway remodelling, which may include goblet cell hyperplasia / hypertrophy, changes in epithelial integrity, accumulation of extracellular matrix components, smooth muscle hypertrophy and thickening of the lamina reticularis, is a feature of severe asthma and contributes to the clinical phenotype. Objective: Within the U-BIOPRED severe asthma study, we have assessed histological elements of airway remodelling and their relationship to computed tomography (CT) measures of proximal airway dimensions. Methods: Bronchial biopsies were collected from two severe asthma groups, one non-smoker (SAn, n = 28) and one current/ex-smoker (SAs/ex, n = 13), and a mild-moderate asthma group (MMA, n = 28) classified and treated according to GINA guidelines, plus a healthy control group (HC, n = 33). Movat's pentachrome technique was used to identify mucin, elastin and total collagen in these biopsies. The number of goblet cells (mucin+) was counted as a percentage of the total number of epithelial cells and the percentage mucin epithelial area measured. The percentage area of elastic fibres and total collagen within the submucosa was also measured, and the morphology of the elastic fibres classified. Participants in the asthma groups also had a CT scan to assess large airway morphometry. Results: The submucosal tissue elastin percentage was higher in both severe asthma groups (16.1% SAn, 18.9% SAs/ex) compared with the HC (9.7%) but did not differ between asthma groups. There was a positive relationship between elastin and airway wall area measured by CT (n = 18–20, rho=0.544, p = 0.024), which also related to an increase in elastic fibres with a thickened lamellar morphological appearance. Mucin epithelial area and total collagen were not different between the four groups. Due to small numbers of suitable CT scans, it was not feasible to compare airway morphometry between the asthma groups. Conclusion: These findings identify a link between extent of elastin deposition and airway wall thickening in severe asthma