S Weston

Reticular basement membrane fragmentation and potential epithelial mesenchymal transition is exaggerated in the airways of smokers with chronic obstructive pulmonary disease

Background and objective:  In COPD, the airways are chronically inflamed, and we have now observed fragmentation of the reticular basement membrane (Rbm). This appears to be a hallmark of the process known as epithelial mesenchymal transition (EMT), in which epithelial cells migrate through the Rbm and differentiate into fibroblasts. The aim of this study was to confirm the extent and relevance of Rbm fragmentation in smokers and patients with COPD, and to undertake a preliminary analysis of some classical markers of EMT.

Olfactory ensheathing cells promote collateral axonal branching in the injured adult rat spinal cord

In recent years, injection of olfactory ensheathing cells (ECs) into the spinal cord has been used as an experimental strategy to promote regeneration of injured axons. In this study, we have compared the effects of transplanting encapsulated ECs with those injected directly into the spinal cord. The dorsal columns of adult rats were cut at T(8-9) and rats in experimental groups received either EC-filled porous polymer capsules or culture medium (CM)-filled capsules with ECs injected at the injury site. Control rats were in three groups: (1) uninjured, (2) lesion with transplantation of CM-filled capsules and (3) lesion with transplantation of CM-filled capsules and injections of CM. Three weeks after injury, Fluororuby was injected into the hindlimb motor and somatosensory cortex to label corticospinal neurons. Observations indicated that there were a few regenerating fibres, up to 10, in the EC-treated groups. In rats that received encapsulated ECs, regenerating fibres were present in close association with the capsule. Rats that received EC injections demonstrated a significant increase in the number of collateral branches from the intact ventral corticospinal tract (vCST) compared with the corresponding control, CM-injected group (P=0.003), while a trend for increased collateral branches was observed in rats that received encapsulated ECs (P=0.07).

Increased vascular permeability precedes cellular inflammation as asthma control deteriorates

Background Airway microcirculation is abnormal in asthma but the role of vascular changes in asthma deteriorations remains poorly defined. We prospectively assessed the vascular changes accompanying worsening of asthma control by using an inhaled corticosteroid (ICS) dose‐reduction model.

Changes in airway histone deacetylase2 in smokers and COPD with inhaled corticosteroids: a randomized controlled trial.

The expression of HDAC2 is reported as reduced in chronic obstructive pulmonary disease (COPD). We assessed HDAC2 expression within the airways of smokers and subjects with COPD and effects of inhaled corticosteroids (ICS), using immuno-histology to contrast with previous molecular methodology. Endobronchial biopsies (ebb) from current smokers with COPD (COPD-CS; n = 15), ex-smokers with COPD (COPD-ES; n = 17), smokers with normal lung function (NS; n = 16) and normal controls (NC; n = 9) were immunostained for HDAC2. A double-blinded, randomized, placebo-controlled 6 months intervention study assessed effects of ICS on HDAC2 in 34 COPD subjects. There was no difference in epithelial HDAC2 staining in all groups. There was a significant reduction in total cell numbers in the lamina propria (LP) in COPD-CS and NS (p<0.05). LP cellularity correlated inversely with smoking history in COPD-CS (R = −0.8, p<0.003). HDAC2 expression increased markedly in NS (p<0.001); in contrast COPD-CS was associated with suppressed signal (p<0.03), while normal in COPD-ES. ICS did not affect HDAC2 cell staining. Our findings suggest that airway HDAC2 expression is increased in the LP by smoking itself, but is reduced in COPD. Ex-smokers have normalised HDAC2 cell expression, but ICS had no effect. The paper emphasise the pit-falls of relying on molecular data alone to define airway changes. Clinical Trial Registration Information: Name of registry The Australian New Zealand Clinical Trials Registry (ANZCTR) Registry number ACTRN12612001111864

Nerve growth factor promotes olfactory axonal elongation.

&NA; An explant culture system was used to test the effect of nerve growth factor (NGF) on olfactory axonal elongation. Statistical analysis showed that exogenously applied NGF (50 ng/ml) significantly enhanced olfactory neurite elongation from E14 rat olfactory epithelial explants (p = 0.025). Immunostaining showed that the neurites expressed active TrkA receptors and that S‐100‐positive ensheathing cells were also present. In a separate experiment, immunoassay confirmed that following a growth period of 72 h, E14 presumptive olfactory bulb expressed and secreted NGF into the culture medium. The results indicate that during ontogeny, the olfactory bulb secretes NGF which binds to olfactory axons and facilitates their elongation.

Mast cells in COPD airways: relationship to bronchodilator responsiveness and angiogenesis

We have investigated whether mast cells are associated with bronchodilator responsiveness and airway vascular changes in chronic obstructive pulmonary disease (COPD) airways. We have previously shown that the reticular basement membrane is hypervascular and the lamina propria is hypovascular in COPD. Bronchial biopsies from 32 COPD subjects, 15 smokers with normal lung function and 17 controls, were immunostained for factor VIII, mast cell tryptase and chymase antibodies. Mast cells in the airway smooth muscle, the reticular basement membrane and the underlying lamina propria were quantitated. 41% of COPD subjects had significant bronchodilator responsiveness, but this was not related to smooth muscle mast cell numbers. The reticular basement membrane had greater mast cell density in all groups compared with controls (p<0.01). In this compartment, perivascular mast cell density was related to hypervascularity. Lamina propria mast cell density was increased only in COPD (p<0.05). Perivascular mast cell density in the lamina propria was not related to its decreased vessel density. Bronchodilator responsiveness in COPD is not related to large airway smooth muscle mast cells of either type; both reticular basement membrane and lamina propria mast cells are increased in COPD patients, and perivascular mast cells may be involved in increased angiogenesis in the reticular basement membrane.

Profiling cellular and inflammatory changes in the airway wall of mild to moderate COPD

The objective of this study was to enumerate total cells and the number of inflammatory cell differentials in large airways (LAs) versus small airways (SAs) of mild‐moderate COPD, and against appropriate controls.

Substance P in the hypoglossal nucleus of the rat

The distribution of substance P (SP)-containing synaptic terminals in the hypoglossal nucleus (XII) of adult rats was examined by retrograde peroxidase labelling and immunocytochemistry. From the location of peroxidase injections into the tongue and of labelled neurones in the ventral lamina of XII, motor neurones that supply intrinsic vertical, longitudinal and transverse fibres as well as the extrinsic muscle genioglossus appear to have been labelled. SP-containing terminals were found making contact, and sometimes dual synapses, with unlabelled neuronal dendrites but not with retrogradely labelled somata or dendrites. These findings suggest that SP terminals may contact dendrites of interneurones or of neurones supplying other extrinsic muscles located in the anterior part of the tongue. Dual SP-containing synapses between XII motor neurones may be the means by which tongue muscle fibres are recruited and their function synchronized.

The main rhinovirus respiratory tract adhesion site (ICAM-1) is upregulated in smokers and patients with chronic airflow limitation (CAL)

BackgroundICAM-1 is a major receptor for ~60% of human rhinoviruses, and non-typeable Haemophilus influenzae, two major pathogens in COPD. Increased cell-surface expression of ICAM-1 in response to tobacco smoke exposure has been suggested. We have investigated epithelial ICAM-1 expression in both the large and small airways, and lung parenchyma in smoking-related chronic airflow limitation (CAL) patients.MethodsWe evaluated epithelial ICAM-1 expression in resected lung tissue: 8 smokers with normal spirometry (NLFS); 29 CAL patients (10 small-airway disease; 9 COPD-smokers; 10 COPD ex-smokers); Controls (NC): 15 normal airway/lung tissues. Immunostaining with anti-ICAM-1 monoclonal antibody was quantified with computerized image analysis. The percent and type of cells expressing ICAM-1 in large and small airway epithelium and parenchyma were enumerated, plus percentage of epithelial goblet and submucosal glands positive for ICAM- 1.ResultsA major increase in ICAM-1 expression in epithelial cells was found in both large (p < 0.006) and small airways (p < 0.004) of CAL subjects compared to NC, with NLFS being intermediate. In the CAL group, both basal and luminal areas stained heavily for ICAM-1, so did goblet cells and sub-mucosal glands, however in either NC or NLFS subjects, only epithelial cell luminal surfaces stained. ICAM-1 expression on alveolar pneumocytes (mainly type II) was slightly increased in CAL and NLFS (p < 0.01). Pack-years of smoking correlated with ICAM-1 expression (r = 0.49; p < 0.03).ConclusionAirway ICAM-1 expression is markedly upregulated in CAL group, which could be crucial in rhinoviral and NTHi infections. The parenchymal ICAM-1 is affected by smoking, with no further enhancement in CAL subjects.

S89 Epithelial Mesenchymal Transition (EMT) in Chronic Obstructive Pulmonary Disease (COPD) Airways is Attenuated by Inhaled Corticosteroids (ICS)

Introduction and Objectives We recently reported1, 2 that EMT is active in the airways of COPD patients; in this process epithelial cells change shape and become motile, then digest through the reticular basement membrane (Rbm) which becomes fragmented and transition to a mesenchymal fibroblast-like cell. We also demonstrated that the Rbm is hyper-vascular, a combined picture specifically suggesting active EMT-type-III, which is a dangerous, pre-malignant condition. This may well be the link between COPD and lung cancer. In this study, we have assessed the effects of ICS on markers of EMT in endobronchial biopsies (ebb) in COPD. Methods A double-blinded, randomised, placebo-controlled study assessed the effects of inhaled fluticasone propionate (FP: 500µg twice daily) on EMT in 34 COPD patients. Ebb were assessed for EMT related Rbm fragmentation (core structural marker) and immunostained for the EMT signatures S100A4 (a fibroblast epitope), matrix-metalloproteinase-9 (MMP-9) and the epithelial activation marker, epidermal growth factor receptor (EGFR). Results Table 1. Abstract S89 Table 1 Comparison of different indices at baseline and after treatment between the two groups. Data expressed as medians and ranges Markers Placebo Fluticasone Propionate Comparison Before After Before After % Rbm fragmentation 24(6–100)* 26(2–48) 19(0.2–42) 2(0–88) †! S100A4 positive cells in basal epithelium (BE) per mm of Rbm 19(2–31)* 17(10–35) 25(2–55) 12(0.6–24) †! S100A4 positive cells in Rbm per mm of Rbm 23(14–82)* 29(3–48) 44(15–92) 20(2–60) †! MMP-9 positive cells in Rbm clefts per mm of Rbm 11(0–41)* 13(0–27) 5(0–43) 0(0–6)† EGFR % in the epithelium 14(3–38)* 10(1–39) 34(14–59) 5(2–43) †! * No significant difference at baseline between placebo/ICS groups, but all significantly different to normal controls at baseline (p<0.007). Significant differences: † Change within ICS group (p<0.003); !changes over time with ICS compared to those on placebo for Rbm fragmentation (p<0.04); S100A4 in BE (p<0.009) and Rbm (p<0.002); EGFR (p<0.02).  No significant difference after the treatment compared to normal controls. Conclusions This is the first study to report the positive effects of ICS on EMT markers in COPD. This may be the mechanistic link between ICS and its reported preventive action against smoking-related lung cancer in COPD. References Sohal SS, et al. Respirology 2010, 15(6):930–938. Sohal SS, et al. Respir Res 2011, 12(1):130.