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Rationale: Markers of inflammatory activity are important for assessment and management of many respiratory diseases. Markers that are currently unrecognized may be more valuable than those presently believed to be useful.Objectives: To identify potential biomarkers of suppurative and inflammatory lung disease in induced sputum samples.Methods: Induced sputum was collected from 20 healthy control subjects, 24 patients with asthma, 24 with chronic obstructive pulmonary disease, 28 with cystic fibrosis (CF), and 19 with bronchiectasis. Twelve patients with CF had sputum sampled before and after antibiotic therapy for an infective exacerbation. The fluid phase of induced sputum was analyzed by surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy on three protein array surfaces. Some protein markers were selected for identification, and relevant ELISA assays sought. For 12 patients with CF, both SELDI-TOF and ELISA monitored changes in inflammatory responses during infective exacerbations.Measurements and Main Results: SELDI-TOF identified potential biomarkers that differentiated each of the disease groups from healthy control subjects: at a significance of P < 0.01, there were 105 for asthma, 113 for chronic obstructive pulmonary disease, 381 for CF, and 377 for bronchiectasis. Peaks selected for protein identification yielded calgranulin A, calgranulin B, calgranulin C, Clara cell secretory protein, lysosyme c, proline rich salivary peptide, cystatin s, and hemoglobin alpha. On treatment of an infective CF exacerbation, SELDI-TOF determined falls in levels of calgranulin A and calgranulin B that were mirrored by ELISA-measured falls in calprotectin (heterodimer of calgranulins A and B).Conclusions: Proteomic screening of sputum yields potential biomarkers of inflammation. The early development of a clinically relevant assay from such data is demonstrated. [ABSTRACT FROM AUTHOR]

Inhaled corticosteroid/long-acting β2-agonist (ICS/LABA) fixed-dose combinations are recommended regular maintenance options for asthma. ICS/LABAs containing formoterol may also be indicated for single maintenance and reliever therapy (SMART). This analysis evaluated the frequency of SMART dosing of budesonide/formoterol fixed-dose combination (BFC) in the United Kingdom. Secondary objectives were to assess adherence and use of short-acting ß2-agonists (SABAs). This was a descriptive analysis of treatment patterns using the UK Clinical Practice Research Datalink-GP OnLine Database data (2009-2013). SMART dosing was determined when prescription instructions contained guidance for daily dosing plus 'and when required'. Treatment and prescription refill patterns of BFC and SABA were described in the year following the index date to identify adherence and SMART dosing instructions versus other dosing regimens. Of 14,818 patients identified, 173 (1.2%) had evidence of prescriptions for SMART dosing at their index BFC prescription. Despite being prescribed SMART dosing, 91 of 173 patients (53%) were additionally dispensed SABA in the year following the index date. The mean number of BFC inhalers used was less than required for daily treatment for SMART and non-SMART dosing groups (4.7 and 4.8, respectively).This analysis suggests that SMART dosing is infrequent when examining dosing instructions. Therefore, results of randomised clinical trials using SMART dosing may not translate to clinical practice in the United Kingdom because of the low level of SMART prescription, concurrent use of SABA, and inadequate refill persistence observed. Further research is needed to understand SMART dosing in real-world clinical practice.

Background: Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTR gene therapy in patients with cystic fibrosis., Methods: We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Patients (aged ≥12 years) with a forced expiratory volume in 1 s (FEV1) of 50-90% predicted and any combination of CFTR mutations, were randomly assigned, via a computer-based randomisation system, to receive 5 mL of either nebulised pGM169/GL67A gene-liposome complex or 0.9% saline (placebo) every 28 days (plus or minus 5 days) for 1 year. Randomisation was stratified by % predicted FEV1 (<70 vs ≥70%), age (<18 vs ≥18 years), inclusion in the mechanistic substudy, and dosing site (London or Edinburgh). Participants and investigators were masked to treatment allocation. The primary endpoint was the relative change in % predicted FEV1. The primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01621867., Findings: Between June 12, 2012, and June 24, 2013, we randomly assigned 140 patients to receive placebo (n=62) or pGM169/GL67A (n=78), of whom 116 (83%) patients comprised the per-protocol population. We noted a significant, albeit modest, treatment effect in the pGM169/GL67A group versus placebo at 12 months' follow-up (3.7%, 95% CI 0.1-7.3; p=0.046). This outcome was associated with a stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group. We recorded no significant difference in treatment-attributable adverse events between groups., Interpretation: Monthly application of the pGM169/GL67A gene therapy formulation was associated with a significant, albeit modest, benefit in FEV1 compared with placebo at 1 year, indicating a stabilisation of lung function in the treatment group. Further improvements in efficacy and consistency of response to the current formulation are needed before gene therapy is suitable for clinical care; however, our findings should also encourage the rapid introduction of more potent gene transfer vectors into early phase trials., Funding: Medical Research Council/National Institute for Health Research Efficacy and Mechanism Evaluation Programme., (Copyright © 2015 Alton et al. Open Access article distributed under the terms of CC BY. Published by Elsevier Ltd.. All rights reserved.)

Background: Clinical trials in cystic fibrosis (CF) have been hindered by the paucity of well characterised and clinically relevant outcome measures., Aim: To evaluate a range of conventional and novel biomarkers of CF lung disease in a multicentre setting as a contributing study in selecting outcome assays for a clinical trial of CFTR gene therapy., Methods: A multicentre observational study of adult and paediatric patients with CF (>10 years) treated for a physician-defined exacerbation of CF pulmonary symptoms. Measurements were performed at commencement and immediately after a course of intravenous antibiotics. Disease activity was assessed using 46 assays across five key domains: symptoms, lung physiology, structural changes on CT, pulmonary and systemic inflammatory markers., Results: Statistically significant improvements were seen in forced expiratory volume in 1 s (p<0.001, n=32), lung clearance index (p<0.01, n=32), symptoms (p<0.0001, n=37), CT scores for airway wall thickness (p<0.01, n=31), air trapping (p<0.01, n=30) and large mucus plugs (p=0.0001, n=31), serum C-reactive protein (p<0.0001, n=34), serum interleukin-6 (p<0.0001, n=33) and serum calprotectin (p<0.0001, n=31)., Discussion: We identify the key biomarkers of inflammation, imaging and physiology that alter alongside symptomatic improvement following treatment of an acute CF exacerbation. These data, in parallel with our study of biomarkers in patients with stable CF, provide important guidance in choosing optimal biomarkers for novel therapies. Further, they highlight that such acute therapy predominantly improves large airway parameters and systemic inflammation, but has less effect on airway inflammation.

Rationale: Bronchiectasis is a chronic debilitating disease with few evidence-based long-term treatments., Objectives: A randomized controlled trial assessing the efficacy of nebulized gentamicin therapy over 1 year in patients with non-cystic fibrosis bronchiectasis., Methods: Sixty-five patients were randomized to either twice-daily nebulized gentamicin, 80 mg, or nebulized 0.9% saline, for 12 months. All were reviewed at three-monthly intervals during treatment and at 3 months' follow-up., Measurements and Main Results: At each review the following were assessed: quantitative and qualitative sputum bacteriology; sputum purulence and 24-hour volume; FEV(1), FVC, and forced expiratory flow, midexpiratory phase; exercise capacity; Leicester Cough Questionnaire and St. George's Respiratory Questionnaire; and exacerbation frequency. Fifty-seven patients completed the study. At the end of 12 months' treatment, compared with the saline group, in the gentamicin group there was reduced sputum bacterial density with 30.8% eradication in those infected with Pseudomonas aeruginosa and 92.8% eradication in those infected with other pathogens; less sputum purulence (8.7% vs. 38.5%; P < 0.0001); greater exercise capacity (510 [350-690] m vs. 415 [267.5-530] m; P = 0.03); and fewer exacerbations (0 [0-1] vs. 1.5 [1-2]; P < 0.0001) with increased time to first exacerbation (120 [87-161.5] d vs. 61.5 [20.7-122.7] d; P = 0.02). The gentamicin group had greater improvements in Leicester Cough Questionnaire (81.4% vs. 20%; P < 0.01) and St. George's Respiratory Questionnaire (87.5% vs. 19.2%; P < 0.004) score. No differences were seen in 24-hour sputum volume, FEV(1), FVC, or forced expiratory flow, midexpiratory phase. No P. aeruginosa isolates developed resistance to gentamicin. At follow-up, all outcome measures were similar to baseline., Conclusions: Regular, long-term nebulized gentamicin is of significant benefit in non-cystic fibrosis bronchiectasis but treatment needs to be continuous for its ongoing efficacy. Clinical trial registered with www.clinicaltrials.gov (NCT 00749866).

The use of a combination inhaler containing budesonide and formoterol as both maintenance and quick relief therapy (SMART) has been recommended as an improved method of using inhaled corticosteroid/long-acting beta agonist (ICS/LABA) therapy. Published double-blind trials show that budesonide/formoterol therapy delivered in SMART fashion achieves better asthma outcomes than budesonide monotherapy or lower doses of budesonide/formoterol therapy delivered in constant dosage. Attempts to compare budesonide/formoterol SMART therapy with regular combination ICS/LABA dosing using other compounds have been confounded by a lack of blinding and unspecified dose adjustment strategies. The asthma control outcomes in SMART-treated patients are poor; it has been reported that only 17.1% of SMART-treated patients are controlled. In seven trials of 6-12 months duration, patients using SMART have used quick reliever daily (weighted average 0.92 inhalations/day), have awakened with asthma symptoms once every 7-10 days (weighted average 11.5% of nights), have suffered asthma symptoms more than half of days (weighted average 54.0% of days) and have had a severe exacerbation rate of one in five patients per year (weighted average 0.22 severe exacerbations/patient/year). These poor outcomes may reflect the recruitment of a skewed patient population. Although improvement from baseline has been attributed to these patients receiving additional ICS therapy at pivotal times, electronic monitoring has not been used to test this hypothesis nor the equally plausible hypothesis that patients who are non-compliant with maintenance medication have used budesonide/formoterol as needed for self-treatment of exacerbations. Although the long-term consequences of SMART therapy have not been studied, its use over 1 year has been associated with significant increases in sputum and biopsy eosinophilia. At present, there is no evidence that better asthma treatment outcomes can be obtained by moment-to-moment symptom-driven use of ICS/LABA therapy than conventional physician-monitored and adjusted ICS/LABA therapy.

Background: Adequate monitoring of cystic fibrosis lung disease is difficult. CF exacerbation offers a unique setting to test the utility of biomarkers in the assessment of changing airways inflammation. We hypothesised that levels of calprotectin in sputum (and serum) would change informatively following treatment of an exacerbation., Methods: 27 patients with CF were recruited at onset of pulmonary exacerbation. Sputum and serum were collected at the start and end of anti-biotic therapy. Sputum calprotectin, interleukin-8 (IL8), and myeloperoxidase (MPO) were measured, as were serum calprotectin, CRP and vascular endothelial growth factor (VEGF)., Results: Sputum calprotectin decreased following treatment of an exacerbation (p<0.05), and was superior to other sputum markers. Serum calprotectin, CRP, and VEGF also decreased significantly (p=0.002, p=0.002, p=0.013 respectively). Serum calprotectin level following treatment had predictive value for time to next exacerbation (p=0.032)., Conclusions: This study demonstrates the superiority of calprotectin (in sputum and serum) as a biomarker of CF exacerbation over better-established markers.

Background: Induced sputum cytology and protein biomarkers can be used to assess airways inflammation. Increases in sputum iron have been described in inflammatory lung disease. We hypothesized that other sputum metals may be affected by airways inflammation and investigated their potential value as biomarkers., Methods: Sputum was obtained from 20 healthy control subjects and from patients with inflammatory pulmonary diseases (23 with cystic fibrosis [CF], 16 with bronchiectasis, 17 with asthma, and 23 with COPD), and iron, zinc, manganese, and copper were measured. Fourteen patients with CF were also studied through an exacerbation cycle., Results: Sputum zinc and iron were elevated in CF and non-CF bronchiectasis vs controls (P < .001, zinc; P < .01 iron). Manganese was elevated in asthma (P < .01) and bronchiectasis (P < .05) vs controls. Copper was elevated in CF vs controls (P < .05). Zinc decreased (P < .01) following treatment of CF exacerbation. In subjects with CF zinc levels correlated with other biomarkers., Conclusions: These results suggest a relationship of high concentrations of total zinc and iron with airways inflammation in CF and non-CF bronchiectasis, with longitudinal changes being observed in CF. Further work is required to elucidate potential inflammatory mechanisms related to these observations.

Background: In adults with asthma, ventilation heterogeneity, independent of inflammation, has been hypothesised to be associated with airway remodelling. Bronchial biopsy in preschool children with wheeze demonstrates early structural changes. Ventilation heterogeneity is sensitive to airway disease in other paediatric respiratory conditions such as cystic fibrosis, so may be sensitive to early airway disease in asthma. An observational study was performed in which it was hypothesised that ventilation heterogeneity (lung clearance index (LCI) and phase III slope indices (S(cond) and S(acin))) were more sensitive than conventional measurements (forced expiratory volume in 1 s (FEV(1)) and exhaled nitric oxide (Feno)) for detecting residual airways disease in children with well controlled asthma., Methods: In 31 children with asthma of mean age 10.6 years (range 5-15), FEV(1), LCI, S(cond) and S(acin) were measured at two separate visits, before and after blinded salbutamol or placebo, with Feno measured once. 29 healthy volunteers of mean age 11.2 years (range 5-16) completed measurements at one visit only., Results: Baseline mean (SD) LCI was significantly higher in children with asthma than in controls (6.69 (0.91) vs 6.24 (0.47), p = 0.02). There were no significant differences in FEV(1) or median Feno. Following salbutamol there was a small significant change in mean (SD) FEV(1) (from -1.26 (1.25) to -0.93 (0.23), p = 0.03) but not in LCI, S(cond) or S(acin). Importantly, LCI remained significantly higher after bronchodilator in children with asthma than in controls (6.64 (0.69), p = 0.01)., Conclusion: This study identifies the presence of residual ventilation heterogeneity in children with well controlled asthma and normal FEV(1). The role of LCI in measuring early airway disease in children with asthma requires further exploration, possibly as a surrogate of structural remodelling.

Background: For cystic fibrosis (CF) patients there is a lack of good assays of disease activity and response to new therapeutic interventions, including gene therapy. Current measures of airways inflammation severity are insensitive or non-specific., Methods: Bronchoalveolar lavage fluid from 39 CF children and 38 respiratory disease controls was obtained at bronchoscopy and analysed by surface enhanced laser desorption ionisation time of flight (SELDI-TOF) mass spectrometry. Recognized proteins were assessed for CF disease specificity. Individual protein identification of specific peaks was performed., Results: 1277 proteins/peptides, >4 kDa, were detected using 12 different surfaces and binding conditions. 202 proteins/peptides were differentially expressed in the CF samples (p<0.001), 167 up-regulated and 35 down-regulated. The most discriminatory biomarker had a mass of 5.163 kDa. The most abundant, with a mass of 10.6 kDa, was identified as s100 A8 (calgranulin A)., Conclusions: The application of SELDI-TOF mass spectrometry allows evaluation of proteins in BAL fluid avoiding the limitations of only analysing predetermined proteins and potentially identifying proteins not previously appreciated as biomarkers. Its application to cystic fibrosis should enable appropriate evaluation of evolving illness, of gene therapy and other new therapies.

S(cond) and S(acin) are derived from analysis of concentration-normalized phase III slopes (Sn(III)) of a multiple breath inert gas washout. Studies in healthy and COPD subjects suggest these reflect ventilation heterogeneity in conducting and acinar airway zones respectively, but similar studies in cystic fibrosis (CF) are lacking. S(cond), S(acin) and lung clearance index (LCI, a measure of overall gas mixing efficiency) were measured in 22 adults and 18 children with CF and 17 adult and 29 child controls. Plethysmography and gas transfer measurements were performed in adults, and spirometry in all subjects. S(cond) was elevated in almost all CF patients, including children with mild disease and normal LCI. However, S(cond) did not correlate with other measurements and appeared to reach a maximum; further increase in ventilation heterogeneity being restricted to S(acin). The nature and/or severity of CF lung disease may invalidate assumptions underlying the ability to separate phase III slope analysis of ventilation heterogeneity into proximal and peripheral components, and LCI may be a better indicator of gas mixing in this population.

Background: Lung clearance index (LCI) is a sensitive marker of early lung disease in children but has not been assessed in adults. Measurement is hindered by the complexity of the equipment required. The aims of this study were to assess performance of a novel gas analyser (Innocor) and to use it as a clinical tool for the measurement of LCI in cystic fibrosis (CF)., Methods: LCI was measured in 48 healthy adults, 12 healthy school-age children and 33 adults with CF by performing an inert gas washout from 0.2% sulfur hexafluoride (SF6). SF6 signal:noise ratio and 10-90% rise time of Innocor were compared with a mass spectrometer used in similar studies in children., Results: Compared with the mass spectrometer, Innocor had a superior signal:noise ratio but a slower rise time (150 ms vs 60 ms) which may limit its use in very young children. Mean (SD) LCI in healthy adults was significantly different from that in patients with CF: 6.7 (0.4) vs 13.1 (3.8), p<0.001. Ten of the patients with CF had forced expiratory volume in 1 s > or = 80% predicted but only one had a normal LCI. LCI repeats were reproducible in all three groups of subjects (mean intra-visit coefficient of variation ranged from 3.6% to 5.4%)., Conclusions: Innocor can be adapted to measure LCI and affords a simpler alternative to a mass spectrometer. LCI is raised in adults with CF with normal spirometry, and may prove to be a more sensitive marker of the effects of treatment in this group.

Background: Treatment of acute asthma is based on rapid reversal of bronchospasm and airway inflammation. Magnesium sulphate (MgSO(4)) is known to have a bronchodilator effect on smooth muscle but studies have shown conflicting results on its efficacy in acute asthma, although its use is recommended in national and international guidelines., Aims: To determine if intravenous MgSO(4), when used as an adjunct to standard therapy, improves the outcome in acute asthma., Methods: A double blind, randomised placebo controlled trial comparing 1.2g MgSO(4) with standard therapy in adult patients with acute asthma. Patients had a PEF

Increased mucosal vascularity is a hallmark of airway inflammation in asthma. It was hypothesised that this would lead to a detectable increase in respiratory heat and moisture loss (RHML), which would reflect the degree of airway inflammation present. A total of 23 subjects with asthma and 18 healthy controls had RHML measured in a cross-sectional study. The measurements were made using a device that combines temperature and humidity measurement during inspiration and expiration and allows precise control over inspirate conditions and ventilatory pattern. The subjects with asthma underwent parallel measurements of exhaled nitric oxide, sputum eosinophil percentage and exhaled breath condensate pH. Mean+/-SD RHML was elevated in patients with asthma (98.1+/-7.3 J.L(-1)) compared with control subjects (91.9+/-4.5 J.L(-1)). RHML measurement in asthma correlated with sputum eosinophil percentage. This novel correlation between thermal and cellular measurements in asthma suggests that both of these noninvasive indices are sensitive to the degree of underlying chronic airway inflammation.

Exhaled breath condensate pH and ammonium reflect asthmatic status and acute exacerbations in adults. The aim of this study was to assess whether pH and ammonium could reflect asthma and its severity in children. The current study comprised two parts: 1) a cross-section of 74 children with asthma (median age 10.5 yrs) compared with 47 healthy controls (median age 10 yrs); and 2) longitudinal assessment of eight children (mean age 8.5 yrs) admitted with asthma exacerbation. Condensate pH and ammonium were compared with clinical observations. In the cross-sectional part of the study, lower per cent forced expiratory volume in one second was associated with more symptoms and treatment. There was no significant difference between median pH in children with stable asthma (6.05) compared with controls (5.90). Ammonium was significantly lower in children with asthma (median 258 microM) compared with controls (median 428 microM). No association was found between ammonium or pH and lung function or symptom-free days. In the longitudinal study, significant improvements in oxygen saturation and respiratory rate with treatment of an acute exacerbation were not reflected by changes in pH or ammonium. In conclusion, pH does not appear to reflect disease or severity in children with asthma. Ammonium was significantly lower in children with asthma when compared with controls.

Background: The effects of breathing pattern and inspired air conditions on the volume and content of exhaled breath condensate (EBC) were investigated., Methods: Total exhaled water (TEW), EBC volume, pH, nitrite and protein concentrations were measured in three groups of 10 healthy subjects breathing into a condenser at different target minute ventilations (Vm), tidal volumes (Vt), and inspired air conditions., Results: The volumes of both TEW and EBC increased significantly with Vm. For Vm 7.5, 15 and 22.5 l/min, mean (SD) EBC was 627 (258) microl, 1019 (313) microl, and 1358 (364) microl, respectively (p<0.001) and TEW was 1879 (378) microl, 2986 (496) microl, and 4679 (700) microl, respectively (p<0.001). TEW was significantly higher than EBC, reflecting a condenser efficiency of 40% at a target Vm of 7.5 l/min which reduced to 29% at Vm 22.5 l/min. Lower Vt gave less TEW than higher Vt (26.6 v 30.7 microl/l, mean difference 4.1 (95% CI 2.6 to 5.6), p<0.001) and a smaller EBC volume (4.3 v 7.6 microl/l, mean difference 3.4 (95% CI 2.3 to 4.5), p<0.001). Cooler and drier inspired air yielded less water vapour and less breath condensate than standard conditions (p<0.05). Changes in the breathing pattern had no effect on EBC protein and nitrite concentrations and pH., Conclusion: These results show that condensate volume can be increased by using high Vt and increased Vm without compromising the dilution of the sample.

Objectives: To assess the effect of montelukast versus salmeterol added to inhaled fluticasone propionate on asthma exacerbation in patients whose symptoms are inadequately controlled with fluticasone alone. Design and setting A 52 week, two period, double blind, multicentre trial during which patients whose symptoms remained uncontrolled by inhaled corticosteroids were randomised to add montelukast or salmeterol., Participants: Patients (15-72 years; n = 1490) had a clinical history of chronic asthma for > or = 1 year, a baseline forced expiratory volume in one second (FEV1) value 50-90% predicted, and a beta agonist improvement of > or = 12% in FEV1., Main Outcome Measures: The primary end point was the percentage of patients with at least one asthma exacerbation., Results: 20.1% of the patients in the group receiving montelukast and fluticasone had an asthma exacerbation compared with 19.1% in the group receiving salmeterol and fluticasone; the difference was 1% (95% confidence interval -3.1% to 5.0%). With a risk ratio (montelukast-fluticasone/salmeterol-fluticasone) of 1.05 (0.86 to 1.29), treatment with montelukast and fluticasone was shown to be non-inferior to treatment with salmeterol and fluticasone. Salmeterol and fluticasone significantly increased FEV1 before a beta agonist was used and morning peak expiratory flow compared with montelukast and fluticasone (P < or = 0.001), whereas FEV1 after a beta agonist was used and improvements in asthma specific quality of life and nocturnal awakenings were similar between the groups. Montelukast and fluticasone significantly (P = 0.011) reduced peripheral blood eosinophil counts compared with salmeterol and fluticasone. Both treatments were generally well tolerated., Conclusion: The addition of montelukast in patients whose symptoms remain uncontrolled by inhaled fluticasone could provide equivalent clinical control to salmeterol.

Background: The loss of cystic fibrosis transmembrane conductance regulator (CFTR) mediated chloride conductance does not fully explain the diverse pathologies evident in patients with cystic fibrosis (CF). Bicarbonate (HCO(3)(-)) secretion is also impaired in CFTR expressing tissues and CFTR is thought to regulate HCO(3)(-) secretion at the apical membrane of epithelial cells. We hypothesised that the epithelial lining fluid (ELF) of patients with CF would be acidified and that this may be worsened during an infective exacerbation due to the increased inflammatory burden., Methods: pH and nitrite levels in exhaled breath condensate (EBC) from 12 healthy non-smoking controls and 30 patients with CF (11 of whom were in an infective exacerbation) were measured. A further nine patients were studied before and after intravenous antibiotic treatment for an exacerbation of CF., Results: The pH of EBC was significantly lower in patients with stable CF than in controls (5.88 (0.32) v 6.15 (0.16), p=0.017), and was further reduced in CF patients with an exacerbation (5.32 (0.38), p=0.001) compared with stable CF patients. EBC pH increased significantly following antibiotic treatment from 5.27 (0.42) to 5.71 (0.42), p=0.049). Nitrite levels in EBC were increased in CF patients with an exacerbation compared with control subjects (4.4 (4.0) micro m v 1.6 (1.6) micro m p=0.047). No correlation was found between EBC pH and nitrite levels., Conclusions: These findings support the hypothesis that airway acidification occurs in CF. This acidity is in part a function of inflammation as the pH of the EBC of patients increased significantly with treatment of an exacerbation, although not to control levels. Acidic pH of the ELF may play a role in the pathophysiology of CF lung disease and requires further investigation.

Neutrophil apoptosis represents a major mechanism involved in the resolution of acute inflammation. In contrast to the effect of hypoxia observed in many other cell types, oxygen deprivation, as we have shown, causes a profound but reversible delay in the rate of constitutive apoptosis in human neutrophils when aged in vitro. This effect was mimicked by exposing cells to 2 structurally unrelated iron-chelating agents, desferrioxamine (DFO) and hydroxypyridines (CP-94), and it appeared specific for hypoxia in that no modulation of apoptosis was observed with mitochondrial electron transport inhibitors, glucose deprivation, or heat shock. The involvement of chelatable iron in the oxygen-sensing mechanism was confirmed by the abolition of the DFO and CP-94 survival effect by Fe(2+) ions. Although hypoxia inducible factor-1alpha (HIF-1alpha) mRNA was identified in freshly isolated neutrophils, HIF-1alpha protein was only detected in neutrophils incubated under hypoxic conditions or in the presence of DFO. Moreover, studies with cyclohexamide demonstrated that the survival effect of hypoxia was fully dependent on continuing protein synthesis. These results indicate that the neutrophil has a ferroprotein oxygen-sensing mechanism identical to that for erythropoietin regulation and results in HIF-1alpha up-regulation and profound but reversible inhibition of neutrophil apoptosis. This finding may have important implications for the resolution of granulocytic inflammation at sites of low-oxygen tension.

Study Objectives: To investigate whether differing airway interleukin (IL)-18 levels may be implicated in the pathogenesis of asthma and sarcoidosis., Setting: University teaching hospital., Patients and Methods: IL-18 levels were measured in BAL fluid and in the supernatant of lipopolysaccharide (LPS)-stimulated alveolar macrophages obtained by BAL from 15 patients with sarcoidosis, 11 patients with asthma, and 13 healthy subjects. We also examined the relationship between IL-18 levels and macrophage and lymphocyte concentrations in BAL fluid. IL-18 was measured using an in-house enzyme-linked immunosorbent assay., Results: IL-18 levels were significantly lower in BAL fluid from patients with asthma (median, 0.0 pg/mL; interquartile range, 0.0 to 0.0 pg/mL) compared to patients with sarcoidosis (median, 222.0 pg/10(6); interquartile range, 110 to 340 pg/mL; p = 0.009, Mann Whitney rank-sum test) and healthy control subjects (median, 162 pg/mL; interquartile range, 38 to 203 pg/mL; p = 0.025, Mann Whitney rank-sum test). Individual analyses comparing IL-18 levels with BAL macrophage counts, and IL-18 with lymphocyte counts in the three groups showed no correlation between these indexes. The mean levels of IL-18 in unstimulated macrophage supernatants were 410 pg/10(6) cells for patients with asthma, 723.4 pg/10(6) cells for patients with sarcoidosis, and 734.8 pg/10(6) cells for healthy control subjects (p > 0.05). Stimulated macrophages from patients with sarcoidosis responded with increasing amounts of IL-18 at lower doses of LPS than macrophages from healthy control subjects or patients with asthma., Conclusion: Our findings suggest that inherently low levels of IL-18 may be associated with the pathogenesis of asthmatic airway inflammation.

Background: The success of haematopoietic (bone marrow or peripheral blood) stem cell transplantation (SCT) is compromised by pulmonary complications. We hypothesised that a proinflammatory alveolar microenvironment, reflected in alveolar macrophage (AM) cytokine production, would predispose to such complications., Methods: AM were isolated from adult SCT recipients by bronchoalveolar lavage before SCT (n=32) and during post-transplant pancytopenia (n=23). Concentrations of tumour necrosis factor (TNF)alpha, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin (IL)-1 beta, IL-6, and IL-8 in 24 hour AM culture medium were measured by enzyme linked immunosorbent assay and compared with both the occurrence of post-SCT lung disease and with subjects' previous respiratory histories., Results: Eleven subjects developed lung disease within 6 months of SCT. These subjects had higher median pre-transplant AM TNFalpha (8 (IQR 1-8) v 2 (1-5) ng/10(6)AM, p=0.01, median difference (D) = 3, 95% CI 0.1 to 7), GM-CSF (5 (0.7-8) v 0.2 (0.1-0.8), p=0.006, D = 4, 95% CI 0.5 to 7), and IL-6 (0.5 (0.1-1) v 0.1 (0.02-0.3), p=0.049, D = 0.3, 95% CI 0.0002 to 1) production than remaining subjects; IL-1 beta and IL-8 did not differ. During pancytopenia high AM GM-CSF production again predicted later lung disease (1 (0.7-9) v 0.1 (0.06-0.3), p=0.01, D = 1, 95% CI 0.1 to 6). A history of recent chest disease was associated with high AM TNFalpha and GM-CSF production and with post-SCT lung disease. Pre-SCT lung function was unrelated to post-SCT lung disease., Conclusions: Recent respiratory disease and persistent proinflammatory AM behaviour detectable before transplantation are associated with lung disease following SCT. These associations may prove useful in pre-transplant risk assessment.

Study Objective: To examine whether atopy influences exhaled nitric oxide (NO) levels in adults with established asthma., Setting: Specialist respiratory unit in a university teaching hospital., Patients: Twenty-eight asthmatics (mean FEV(1), 85.7%) receiving short-acting inhaled bronchodilators and a range of inhaled steroids (0 to 4,000 microg/d)., Interventions: Subjects were studied on two occasions, 5 to 7 days apart, between September and March., Measurements and Results: On the first day, FEV(1), exhaled NO, and histamine challenge were performed. On the second day, exhaled NO, total IgE, and skin-prick testing to six common allergens were conducted. Exhaled NO was measured with the single exhalation method. We found exhaled NO levels to correlate positively with total IgE (r = 0.43, p = 0.02) and number of positive skin-prick tests (p = 0. 002). By contrast, there was no significant correlation between exhaled NO and FEV(1) or the provocative concentration causing a 20% fall in FEV(1). Subanalyses of steroid-treated and steroid-naive patients in this group revealed the same findings., Conclusion: Exhaled NO levels in asthmatics correlate more closely with atopy than with bronchial hyperreactivity and lung function.

The authors have observed that some patients with acute exacerbations of asthma do not have substantially higher levels of exhaled nitric oxide (NO). The study examined whether this could be explained by the effect of airway calibre on exhaled NO. Exhaled NO, height and forced expiratory volume in one second (FEV1) were measured in 12 steroid-naive asthmatics and 17 normal subjects. For comparison, another group of patients with airways disease (34 cystic fibrosis patients) were also studied. In 20 asthmatics (on various doses of inhaled steroids, 0-3,200 microg x day-1), exhaled NO was measured before and after histamine challenge (immediately after reaching the provocative concentration causing a 20% fall in FEV1) and in 12 of these patients, also after nebulized salbutamol to restore FEV1 to baseline. Studies were also conducted to examine possible confounding effects of repeated spirometry (as would occur in histamine challenge) and nebulized salbutamol alone in exhaled NO levels. Exhaled NO was measured using a single exhalation method with a chemiluminescence analyser at a constant flow rate and mouth pressure. There was a significant correlation between FEV1 and exhaled NO in steroid naive asthmatics (r=0.9, p<0.001) and cystic fibrosis patients (r=-0.48, p<0.05) but not in normal subjects (r=-0.13, p=0.61). Exhaled NO decreased significantly after histamine challenge and returned to baseline after bronchodilation by nebulized salbutamol (mean+/-SEM: 23.6+/-3.6 parts per billion (ppb) (prehistamine), 18.2+/-2.7 ppb (posthistamine) and 23.6+/-3.8 ppb (postsalbutamol) p=0.001). Repeated spirometry and nebulized salbutamol did not affect exhaled NO measurements significantly. Exhaled nitric oxide levels appear to be lower in circumstances of smaller airway diameter. Hence, within a subject nitric oxide levels may be artefactually decreased during bronchoconstriction. This may be caused by increased airflow velocity in constricted airways when the exhalation rate is kept constant.

Identifying noninvasive markers of pulmonary inflammation would be useful in assessing new therapies in children. Breath condensate is a simple and potentially acceptable sample medium even in small children. The technique has previously been used in adults, but not children with cystic fibrosis. The technique was assessed in 36 children with cystic fibrosis (mean age 10.4 yrs) and 17 control subjects, analysing samples for nitrite, interleukin(IL)-8 and salivary and nasal contamination. Correlations were made between levels of the inflammatory markers and forced expiratory volume in one second/forced vital capacity, chest radiograph score and use of inhaled steroids. On samples without significant contamination (<10 u x L(-1) amylase) nitrite was detected in 93% of samples at a median concentration of 3.0 microM compared with 50% of control samples at a median of 0.5 microM. Condensate amylase levels did not correlate with the nitrite value obtained (r=0.31). IL-8 was detected in 33% of CF samples. Breath condensate is an acceptable method of sample collection in children. Nitrite was raised in breath condensate from patients with cystic fibrosis when compared with control subjects.

Background: Dysphonia is a known local adverse effect of inhaled corticosteroids. This symptom was investigated by laryngoscopy and assessment in a voice laboratory. The effects of changing the treatment of patients with dysphonia, reported whilst using the pMDI, to pMDI plus Nebuhaler or Tubuhaler was also assessed., Methods: Seventy-two patients reporting dysphonia and taking inhaled steroids from a pMDI entered a 12-week, open, parallel group study. Fifty-one completed the study per protocol; 26 in the Nebuhaler group [21 female, mean age 57 years (22-77)] and 25 in the Turbuhaler group [18 female, mean age 58 years (21-81)]. A dysphonia diary card was completed weekly. Voice laboratory assessments and laryngoscopy were performed on entry and at 12 weeks., Results: There were no differences in voice laboratory data, laryngoscopic evidence of disordered glottic closure and diary data between the two groups at 12 weeks. At study entry laryngoscopic appearances were normal in almost half the patients. Vocal cord bowing was rarely seen. Glottic closure changed in nine patients during the study period, but there was no correlation with voice symptoms. The trend of symptomatic improvement of voice status in the Turbuhaler group did not correlate with voice laboratory assessments and laryngoscopic evidence of disordered glottic closure. After 4 weeks, 40% of patients using Turbuhaler and 8% in the Nebuhaler group scored their voice status as better (P < 0.02) but there was no significant difference between the two groups at 12 weeks (Turbuhaler 52%, Nebuhaler 23%, P=0.08)., Conclusion: This study does not support the view that dysphonia in asthmatics inhaling corticosteroids is usually caused by myopathic bowing of the vocal cord muscles.

Background: Little is known about the management of acute asthma prior to hospital admission. Pre-hospital treatment of patients referred to hospital with acute asthma was therefore studied in 150 patients divided into three groups: those in the Edinburgh Emergency Asthma Admission Service (EEAAS) who can contact an ambulance and present directly to respiratory services when symptoms arise (n = 38), those under continuing supervision at a hospital respiratory outpatient clinic (n = 54), and those managed solely in primary care (n = 58)., Methods: Standardised admission forms detailing aspects of pre-hospital management, case records, GP referral letters, and ambulance patient transport forms were analysed., Results: In each group airflow obstruction had improved upon arrival at hospital, the effect being most marked in patients transported by ambulance (p<0. 001) and in those receiving nebulised beta(2) agonists prior to admission (p<0.005). However, 25% of patients arrived without having nebulised beta(2) agonists and 37% without having glucocorticoids. EEAAS patients were least likely to receive nebulised beta(2) agonists before arrival at hospital (p<0.05). This observation was attributable to a tendency for these patients to travel to hospital by car rather than by ambulance., Conclusions: There is an important shortfall in administration of bronchodilators and glucocorticoids for acute asthma before arrival at hospital. Ambulances equipped with nebulised bronchodilators provide the optimal mode of transport to hospital for patients with acute asthma. In Edinburgh ambulances are not being used by a significant proportion of the population with asthma, possibly because of the mistaken belief that personal transport arrangements reduce journey time to hospital.

Nebulized recombinant human deoxyribonuclease (DNase) reduces sputum viscosity and improves lung function in some cystic fibrosis patients, but individual responses are unpredictable. The aim of this study was to investigate how DNase can be targeted to those cystic fibrosis patients who would benefit most. The Scottish Cystic Fibrosis Group agreed on a randomized, double-blind, placebo-controlled n-of-1 assessment protocol. Patients underwent a maximum of three 4-week assessment periods (2 weeks saline, 2 weeks DNase each). Measurements performed at hospital (exercise, oximetry and spirometry) and home (symptom scores) were used to derive a scoring system to discriminate maximally between DNase and placebo effects. The data on 89 4-week assessments in 52 patients were reported. Twenty-four patients have completed the assessment process (12 responders and 12 nonresponders) to date. Forced expiratory volume in one second (FEVI) was the best discriminator of response, rising by >200 mL after DNase in 33 of 89 (37%) assessments compared with 3 of 89 (3%) after saline. N-of-1 trials, while laborious, permitted genuine treatment effects to be quantified within individuals with confidence, permitting appropriate treatment targeting. This provides a model of how other new expensive therapies may be introduced to maximize patient benefit.

Stimulated inflammatory cells release large amounts of hydrogen peroxide (H2O2). Breath condensate H2O2 has been shown to be elevated in stable asthmatic children, chronic obstructive pulmonary disease and intubated adult respiratory distress syndrome. In cystic fibrosis airways, where neutrophilic inflammation dominates, it is postulated that H2O2 in breath condensate would be elevated and may be used as a marker of airways inflammation. Expired breath condensate was collected from 16 clinically stable cystic fibrosis (CF) patients (mean age 25.3 yrs, mean forced expiratory volume in one second (FEV1) 50.2%) and 14 normal subjects (mean age 29.9 yrs). Total plasma leukocyte, neutrophil, monocyte and eosinophil counts and lung function were also measured on the day of collection. A method of breath condensate collection excluding the confounding factors of nasal air and saliva contamination was validated and used and H2O2 measured fluorometrically using an optimized assay. The median level of H2O2 concentration in breath condensate of CF patients was lower than that in normal subjects (0.064 versus 0.089 microM), but this did not reach statistical significance (p = 0.20, Mann-Whitney rank sum test). Within the CF group, there was no correlation between H2O2 concentration and lung function. Expired breath condensate H2O2 is not elevated in patients with cystic fibrosis, and is thus not a suitable marker of airways inflammation in these patients. Possible explanations include physical barriers to its detection caused by viscous airways secretions, reaction with other reactive species or increased antioxidant activity caused by trapping of positively charged antioxidants in negatively charged airways secretions.

Background: Nitric oxide (NO) is released by activated macrophages, neutrophils, and stimulated bronchial epithelial cells. Exhaled NO has been shown to be increased in patients with asthma and has been put forward as a marker of airways inflammation. However, we have found that exhaled NO is not raised in patients with cystic fibrosis, even during infective pulmonary exacerbation. One reason for this may be that excess airway secretions may prevent diffusion of gaseous NO into the airway lumen. We hypothesised that exhaled NO may not reflect total NO production in chronically suppurative airways and investigated nitrite as another marker of NO production., Methods: Breath condensate nitrite concentration and exhaled NO levels were measured in 21 clinically stable patients with cystic fibrosis of mean age 26 years and mean FEV1 57% and 12 healthy normal volunteers of mean age 31 years. Breath condensate was collected with a validated method which excluded saliva and nasal air contamination and nitrite levels were measured using the Griess reaction. Exhaled NO was measured using a sensitive chemiluminescence analyser (LR2000) at an exhalation rate of 250 ml/s. Fourteen patients with cystic fibrosis had circulating plasma leucocyte levels and differential analysis performed on the day of breath collection., Results: Nitrite levels were significantly higher in patients with cystic fibrosis than in normal subjects (median 1.93 microM compared with 0.33 microM). This correlated positively with circulating plasma leucocytes and neutrophils (r = 0.6). In contrast, exhaled NO values were not significantly different from the normal range (median 3.8 ppb vs 4.4 ppb). There was no correlation between breath condensate nitrite and lung function and between breath condensate nitrite and exhaled NO., Conclusions: Nitrite levels in breath condensate were raised in stable patients with cystic fibrosis in contrast to exhaled NO. This suggests that nitrite levels may be a more useful measure of NO production and possibly airways inflammation in suppurative airways and that exhaled NO may not reflect total NO production.

Macrophage migration inhibitory factor (MIF) is a potent proinflammatory mediator that has been shown to potentiate lethal endotoxemia and to play a potentially important regulatory role in human acute respiratory distress syndrome (ARDS). We have investigated whether eosinophils are an important source of MIF and whether MIF may be involved in the pathophysiology of asthma. Unstimulated human circulating eosinophils were found to contain preformed MIF. Stimulation of human eosinophils with phorbol myristate acetate in vitro yielded significant release of MIF protein. For example, eosinophils stimulated with phorbol myristate acetate (100 nM, 8 h, 37 degreesC) released 1,539+/-435 pg/10(6) cells of MIF, whereas unstimulated cells released barely detectable levels (< 142 pg/10(6) cells, mean+/-SEM, n = 8). This stimulated release was shown to be (a) concentration- and time-dependent, (b) partially blocked by the protein synthesis inhibitor cycloheximide, and (c) significantly inhibited by the protein kinase C inhibitor Ro-31,8220. In addition, we show that the physiological stimuli C5a and IL-5 also cause significant MIF release. Furthermore, bronchoalveolar lavage fluid obtained from asthmatic patients contains significantly elevated levels of MIF as compared to nonatopic normal volunteers (asthmatic, 797.5+/-92 pg/ml; controls, 274+/-91 pg/ml). These results highlight the potential importance of MIF in asthma and other eosinophil-dependent inflammatory disorders.