P. J. Sterk

Relationship between the inflammatory infiltrate in bronchial biopsy specimens and clinical severity of asthma in patients treated with inhaled steroids.

BACKGROUND: Current guidelines on the management of asthma advocate the use of anti-inflammatory treatment in all but mild disease. They define disease control in terms of clinical criteria such as lung function and symptoms. However, the relationship between the clinical control of the disease and inflammation of the airways is not clear. A cross sectional study was therefore undertaken to investigate the relationship between airways inflammation and measures of clinical control and bronchial hyperresponsiveness in asthmatic patients treated with inhaled steroids. METHODS: Twenty six atopic adults (19-45 years) with mild to moderate asthma (baseline forced expiratory volume in one second (FEV1) > or = 50% predicted, concentration of histamine causing a 20% fall in FEV1 (PC20) 0.02-7.6 mg/ml) on regular treatment with inhaled steroids entered the study. Diary card recordings during the two weeks before a methacholine challenge test and bronchoscopic examination were used to determine peak flow variability, symptom scores, and use of beta 2 agonists. Biopsy specimens were taken by fibreoptic bronchoscopy from the carina of the right lower and middle lobes, and from the main carina. Immunohistochemical staining was performed on cryostat sections with monoclonal antibodies against: eosinophil cationic protein (EG1, EG2), mast cell tryptase (AA1), CD45, CD22, CD3, CD4, CD8, CD25, and CD45RO. The number of positively stained cells in the lamina propria was counted twice by using an interactive display system. RESULTS: There were no differences in cell numbers between the three sites from which biopsy specimens were taken. The PC20 for methacholine was inversely related to the average number of total leucocytes, EG1+, and EG2+ cells, mast cells, CD8+, and CD45RO+ cells in the lamina propria. These relationships were similar for each of the biopsy sites. Symptom scores, beta 2 agonist usage, FEV1, and peak flow variability were not related to any of the cell counts. CONCLUSIONS: Infiltration of inflammatory cells in the lamina propria of the airways seems to persist in asthmatic outpatients despite regular treatment with inhaled steroids. The number of infiltrating leucocytes such as mast cells, (activated) eosinophils, CD8+, and CD45RO+ cells in bronchial biopsy specimens from these patients appears to be reflected by airway hyperresponsiveness to methacholine, but not by symptoms or lung function. These findings may have implications for the adjustment of anti-inflammatory treatment of patients with asthma.

Standardised methodology of sputum induction and processing

Research into the pathogenetic mechanisms and clinical monitoring of inammatory diseases of any system is complete only if it involves the study of both the underlying pathological features and the physio- logical consequences that result from what are always complex inammatory processes. Respiratory dis- eases, including those of the airways, are no exception in this respect. It is, therefore, not surprising that the development over the last 25 years of techniques enabling the study of inammatory processes in the airways has revolutionised understanding of the commonest pulmonary diseases, asthma and chronic obstructive pulmonary disease (COPD). Airways inammation is now an established feature and a central consideration of any treatment strategy for both of these conditions. Most of the initial observations made in asthma, documenting the involvement of eosinophils, mast cells, T-cells and more recently structural cells, ® broblasts, endothelial cells and epithelial cells, were made in studies using ® breoptic bronchoscopy in

Diagnosis and definition of severe refractory asthma: an international consensus statement from the Innovative Medicine Initiative (IMI)

Patients with severe refractory asthma pose a major healthcare problem. Over the last decade it has become increasingly clear that, for the development of new targeted therapies, there is an urgent need for further characterisation and classification of these patients. The Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) consortium is a pan-European public-private collaboration funded by the European Commission Innovative Medicines Initiative of the European Union. U-BIOPRED aims to subphenotype patients with severe refractory asthma by using an innovative systems biology approach. This paper presents the U-BIOPRED international consensus on the definition and diagnosis of severe asthma, aligning the latest concepts in adults as well as in children. The consensus is based on existing recommendations up to 2010 and will be used for the selection of patients for the upcoming U-BIOPRED study. It includes the differentiation between ‘problematic’, ‘difficult’ and ‘severe refractory’ asthma, and provides a systematic algorithmic approach to the evaluation of patients presenting with chronic severe asthma symptoms for use in clinical research and specialised care.

Recurrent Exacerbations in Severe Asthma Are Associated with Enhanced Airway Closure During Stable Episodes

Excessive airway narrowing is a cardinal feature of asthma, and results in closure of airways. Therefore, asthmatic patients in whom airway closure occurs relatively early during expiration might be prone to severe asthma attacks. To test this hypothesis, we compared closing volume (CV) and closing capacity (CC) in a group of asthmatic patients with recurrent exacerbations (more than two exacerbations in the previous year; difficult-to-control asthma), consisting of 11 males and two females, aged 20 to 51 yr, with those in a group of equally severely asthmatic controls without recurrent exacerbations (stable asthma) consisting of 13 males and two females aged 18 to 52 yr. Both groups used equivalent doses of inhaled corticosteroids and were matched for sex, age, atopy, postbronchodilator FEV(1), and provocative concentration of methacholine causing a 20% decrease in FEV(1). They were studied during a clinically stable period of their disease. The patients inhaled 400 microg salbutamol via a spacer device, after which TLC and RV were measured by multibreath helium equilibration, together with the slope of Phase 3 (dN(2)), CV, and CC, by single-breath nitrogen washout. CV and CC were expressed as ratios of VC and TLC, respectively, and all data are presented as % predicted (mean +/- SEM). There was no difference in TLC in patients with difficult-to-control asthma and those with stable asthma (106.7 +/- 4.0% predicted versus 101.7 +/- 4.3% predicted, p = 0.40), RV (113.1 +/- 7.8% predicted versus 100.9 +/- 7.1% predicted, p = 0.26), or dN(2) (142.7 +/- 16.3% predicted versus 116.0 +/- 20.2% predicted, p = 0.23). In contrast, CV and CC were increased in the patients with difficult-to-control asthma as compared with the group with stable asthma (CV: 159.5 +/- 26.8% predicted versus 98.8 +/- 12.5% predicted, p = 0.024; CC: 114.0 +/- 6.4% predicted versus 99.9 +/- 3. 6% predicted, p = 0.030). These findings show that asthmatic individuals with recurrent exacerbations have increased CV and CC as compared with equally severely asthmatic but stable controls, even after bronchodilation during well-controlled episodes. The findings imply that airway closure at relatively high lung volumes under clinically stable conditions might be a risk factor for severe exacerbations in asthmatic patients.

Repeatability of cellular and soluble markers of inflammation in induced sputum from patients with asthma

Sputum induced by inhalation of nebulized hypertonic saline is increasingly used to monitor airways inflammation in asthma. The aim of this study was to assess the repeatability of measuring cellular and soluble markers of inflammation in whole sputum samples as obtained by sputum induction in patients both with mild and moderate-to-severe asthma. Twelve patients with mild, atopic asthma without inhaled steroid treatment and nine patients with moderate-to-severe, atopic asthma treated with inhaled steroids were studied on two separate days at least 2 days apart. Whole sputum samples, induced by inhalation of hypertonic (4.5%) saline, were homogenized, and analysed for differential cell counts and for concentrations of albumin, fibrinogen, interleukin-8 (IL-8), and eosinophil cationic protein (ECP). Repeatability was expressed as intraclass correlation coefficient (Ri), and as coefficient of repeatability (CR) in percentage cells or in doubling concentration. Samples from two patients with mild asthma contained more than 80% squamous cells and were excluded from analysis. The repeatability for cell differential counts in both groups combined was: for neutrophils, Ri = 0.57 and CR = 31.0; for eosinophils, Ri = 0.85 and CR = 12.4; and for lymphocytes, Ri = 0.76 and CR = 6.9. The repeatability of the fluid phase measurements was: for albumin, Ri = 0.71 and CR = 3.2; for fibrinogen, Ri = 0.88 and CR = 2.8; for IL-8, Ri = 0.66 and CR = 2.2; and for ECP, Ri = 0.82 and CR = 1.1. We conclude that the repeatability of cellular and soluble markers of inflammation in induced sputum from patients with mild and moderate-to-severe asthma is satisfactory. Hence, induced sputum, processed by using the whole expectorated sample, seems to be a valuable method to monitor airway inflammation in asthma.

Relationship between exhaled nitric oxide and airway hyperresponsiveness following experimental rhinovirus infection in asthmatic subjects

Exhaled nitric oxide (NO) is elevated in asthmatics, and varies with disease severity. We postulated that a respiratory virus infection increases exhaled NO levels in asthma, and examined the relationship between the virus-induced changes in exhaled NO and in airway hyperresponsiveness to histamine. In a parallel study, seven patients underwent experimental rhinovirus 16 (RV16) inoculation at days 0 and 1, whilst seven patients received placebo. Exhaled NO was measured at baseline (day 0) and at days 1, 2 and 3 after inoculation. Histamine challenges were performed prior to (day -7) and after inoculation (day 3), and were expressed as provocative concentration causing a 20% fall in forced expiratory volume in one second (FEV1) (PC20). Following RV16 infection there was a significant increase in NO at days 2 and 3 as compared to baseline (median change (range): 4.2 (7.5) parts per billion (ppb), p=0.03, and 3.0 (10.1) ppb, p=0.02, respectively). Furthermore, PC20 decreased significantly following RV16 infection (mean+/-SD change in doubling dose: -0.65+/-0.54, p=0.02), whereas PC20 did not change in the placebo group (p=0.1). There was a significant correlation between the RV16-induced changes in exhaled NO levels at day 2 and the accompanying changes in PC20 at day 3 (rank correlation coefficient (rs): 0.86, p=0.01). Hence, the greater the increase in exhaled NO, the smaller the decrease in PC20. We conclude that rhinovirus infection increases exhaled nitric oxide levels in asthmatics, and that this increase is inversely associated with worsening of airway hyperresponsiveness to histamine. These results suggest that viral induction of nitric oxide synthase within the airways may play a protective role in exacerbations of asthma.

Comparison of inflammatory cell counts in asthma: induced sputum vs bronchoalveolar lavage and bronchial biopsies

Background Induced sputum potentially allows monitoring of airway inflammation in patients with asthma in a non‐invasive way. However, the relationship between the cellular content in sputum and airway tissue has not been fully clarified.

Enhanced bronchial expression of vascular endothelial growth factor and receptors (Flk-1 and Flt-1) in patients with chronic obstructive pulmonary disease

Background: Ongoing inflammatory processes resulting in airway and vascular remodelling characterise chronic obstructive pulmonary disease (COPD). Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) could play a role in tissue remodelling and angiogenesis in COPD. Methods: The cellular expression pattern of VEGF, Flt-1, and KDR/Flk-1 was examined by immunohistochemistry in central and peripheral lung tissues obtained from ex-smokers with COPD (forced expiratory volume in 1 second (FEV1) <75% predicted; n = 14) or without COPD (FEV1 >85% predicted; n = 14). The immunohistochemical staining of each molecule was quantified using a visual scoring method with grades ranging from 0 (no) to 3 (intense). Results: VEGF, Flt-1, and KDR/Flk-1 immunostaining was localised in vascular and airway smooth muscle (VSM and ASM) cells, bronchial, bronchiolar and alveolar epithelium, and macrophages. Pulmonary endothelial cells expressed Flt-1 and KDR/Flk-1 abundantly but not VEGF. Bronchial VEGF expression was higher in microvascular VSM cells and ASM cells of patients with COPD than in patients without COPD (1.7 and 1.6-fold, p<0.01, respectively). VEGF expression in intimal and medial VSM (1.7 and 1.3-fold, p<0.05) of peripheral pulmonary arteries associated with the bronchiolar airways was more intense in COPD, as was VEGF expression in the small pulmonary vessels in the alveolar region (1.5 and 1.7-fold, p<0.02). In patients with COPD, KDR/Flk-1 expression was enhanced in endothelial cells and in intimal and medial VSM (1.3, 1.9 and 1.5-fold, p<0.02) while endothelial Flt-1 expression was 1.7 times higher (p<0.03). VEGF expression was significantly increased in bronchiolar and alveolar epithelium as well as in bronchiolar macrophages (1.5-fold, p<0.001). The expression of VEGF in bronchial VSM and mucosal microvessels as well as bronchiolar epithelium was inversely correlated with FEV1 (r<−0.45; p<0.01). Conclusions: VEGF and its receptors Flt-1 and KDR/Flk-1 may be involved in peripheral vascular and airway remodelling processes in an autocrine and/or paracrine manner. This system may also be associated with epithelial cell viability during airway wall remodelling in COPD.

The protective effect of a beta 2 agonist against excessive airway narrowing in response to bronchoconstrictor stimuli in asthma and chronic obstructive lung disease.

Beta 2 agonists reduce airway hypersensitivity to bronchoconstrictor stimuli acutely in patients with asthma and chronic obstructive lung disease. To determine whether these drugs also protect against excessive airway narrowing, the effect of inhaled salbutamol on the position and shape of the dose-response curves for histamine or methacholine was investigated in 12 patients with asthma and 11 with chronic obstructive lung disease. After pretreatment with salbutamol (200 or 400 micrograms) or placebo in a double blind manner dose-response curves for inhaled histamine and methacholine were obtained by a standard method on six days in random order. Airway sensitivity was defined as the concentration of histamine or methacholine causing a 20% fall in FEV1 (PC20). A maximal response plateau on the log dose-response curve was considered to be present if two or more data points for FEV1 fell within a 5% response range. In the absence of a plateau, the test was continued until a predetermined level of severe bronchoconstriction was reached. Salbutamol caused an acute increase in FEV1 (mean increase 11.5% predicted in asthma, 7.2% in chronic obstructive lung disease), and increase in PC20 (mean 15 fold in asthma, fivefold in chronic obstructive lung disease), and an increase in the slope of the dose-response curves in both groups. In subjects in whom a plateau of FEV1 response could be measured salbutamol did not change the level of the plateau. In subjects without a plateau salbutamol did not lead to the development of a plateau, despite achieving a median FEV1 of 44% predicted in asthma and 39% in chronic obstructive lung disease. These results show that, although beta 2 agonists acutely reduce the airway response to a given strength of bronchoconstrictor stimulus, they do not protect against excessive airflow obstruction if there is exposure to relatively strong stimuli. This, together with the steepening of the dose-response curve, could be a disadvantage of beta 2 agonists in the treatment of moderate and severe asthma or chronic obstructive lung disease.

Three phenotypes of adult-onset asthma.

Adult‐onset asthma differs from childhood‐onset asthma in many respects. It is more heterogeneous, often severe and frequently associated with loss of lung function. To identify underlying mechanisms of adult‐onset asthma and to capture predictors of disease progression, detailed characterization and phenotyping is necessary.