Biomedical subjects
F E Hargreave
Publications and source records attributed to F E Hargreave.
Stable COPD: predicting benefit from high-dose inhaled corticosteroid treatment.
The role of inhaled corticosteroids in the management of chronic obstructive pulmonary disease (COPD) remains controversial. The purpose of this study was to evaluate whether sputum eosinophilia (defined as eosinophils > or = 3%) predicts clinical benefit from inhaled corticosteroid treatment in patients with smoking-related clinically stable moderate-to-severe COPD. Forty consecutive patients with effort dyspnoea (mean age 67 yrs; 52 pack-yr smoking history; post-bronchodilator forced expiratory volume in one second (FEV1) <60% predicted, consistent with moderate-to-severe smoking-related chronic airflow limitation) were enrolled. Subjects were treated with inhaled placebo followed by inhaled budesonide (Pulmicort Turbuhaler 1,600 microg.day(-1)), each given for 4 weeks. While the treatment was single-blind (subject level), sputum cell counts before and after treatment interventions were double-blind, thus removing bias. Outcome variables included spirometry, quality-of-life assessment and 6-min walk test. Sputum eosinophilia was present in 38% of subjects. In these, budesonide treatment normalised the eosinophil counts and, in comparison to placebo treatment, resulted in clinically significant improvement in the dyspnoea domain of the disease-specific chronic respiratory questionnaire (0.8 versus 0.3) and a small but statistically significant improvement in post-bronchodilator spirometry (FEV1 100 mL versus 0 mL; p<0.05). In conclusion, sputum eosinophilia predicts short-term clinical benefit from high-dose inhaled corticosteroid treatment in patients with stable moderate-to-severe chronic obstructive pulmonary disease.
Problems with using exhaled nitric oxide to guide asthma treatment.
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Asthma, COPD and bronchitis are just components of airway disease.
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Determining asthma treatment by monitoring sputum cell counts: effect on exacerbations.
One important goal of asthma treatment is to reduce exacerbations. The current authors investigated if the use of sputum cell counts to guide treatment would achieve this goal. A total of 117 adults with asthma were entered into a multicentre, randomised, parallel group-effectiveness study for two treatment strategies over a 2-yr period. In one strategy (the clinical strategy: CS) treatment was based on symptoms and spirometry. In the other (the sputum strategy: SS) sputum cell counts were used to guide corticosteroid therapy to keep eosinophils 250 microg, and was due to fewer eosinophilic exacerbations. The cumulative dose of corticosteroid during the trial was similar in both groups. Monitoring sputum cell counts was found to benefit patients with moderate-to-severe asthma by reducing the number of eosinophilic exacerbations and by reducing the severity of both eosinophilic and noneosinophilic exacerbations without increasing the total corticosteroid dose. It had no influence on the frequency of noneosinophilic exacerbations, which were the most common exacerbations.
Steroid naive eosinophilic asthma: anti-inflammatory effects of fluticasone and montelukast.
BACKGROUND: Inhaled corticosteroids and leukotriene receptor antagonists reduce airway eosinophilia and have been used as first line anti-inflammatory therapy for mild persistent asthma. METHODS: A multicentre, randomised, placebo controlled, parallel group study was performed to compare the anti-inflammatory effects of fluticasone propionate and montelukast as measured by sputum eosinophils in 50 adults with symptomatic steroid naive asthma and sputum eosinophilia of > or =3.5%. RESULTS: Eighteen patients received low dose fluticasone (250 mug/day), 19 received montelukast (10 mg/day), and 13 were given placebo for 8 weeks. Fluticasone treatment resulted in a greater reduction in sputum eosinophils (geometric mean (SD) 11.9 (2.3)% to 1.7 (5.1)%) than montelukast (10.7 (2.3)% to 6.9 (3.8)%; p = 0.04) or placebo (15.4 (2.4)% to 7.8 (4.2)%; p = 0.002), and improvement in FEV(1) (mean (SD) 2.6 (0.9) l to 3.0 (0.9) l) than montelukast (2.8 (0.7) l to 2.8 (0.9) l; p = 0.02) or placebo (2.4 (0.8) l to 2.4 (0.9) l; p = 0.01). Treatment with fluticasone suppressed sputum eosinophilia within a week while montelukast only attenuated it. The effect of montelukast was maximal at 1 week and was maintained over 4 weeks. The effect of fluticasone was maintained over 8 weeks while that of montelukast was not. CONCLUSIONS: Montelukast is not as effective as low dose fluticasone in reducing or maintaining an anti-inflammatory effect in steroid naive eosinophilic asthma.
Failure of montelukast to reduce sputum eosinophilia in high-dose corticosteroid-dependent asthma.
Sputum eosinophilia is a sensitive predictor of benefit from corticosteroid treatment. Montelukast is a cysteinyl leukotriene antagonist, which also reduces sputum and blood eosinophils. The present study examined the possibility that montelukast has an added eosinophil-lowering effect in subjects with asthma who are corticosteroid responsive but relatively corticosteroid resistant. A total of 14 clinically stable adults with asthma requiring minimum treatment with a high-dose inhaled steroid or prednisone, with baseline sputum eosinophilia (> or =5%), were randomised to receive 4 weeks of 10 mg montelukast or placebo daily in a double-blind crossover trial. The primary outcome was the effect of treatment on the percentage of sputum eosinophils. Secondary outcomes were changes in the blood eosinophil count, symptoms, forced expiratory volume in one second, peak expiratory flow and the need for salbutamol. The median (interquartile range, i.e. 75th-25th centile) for sputum eosinophils at baseline was 15.7% (22). The effect of adding montelukast was not significantly different from that of placebo, sputum eosinophils being 9.3% (18.9) after montelukast and 11.3% (22.8) after placebo. No difference was detected on secondary outcomes. No crossover interactions were observed. In conclusion, the addition of montelukast to existing high-dose corticosteroid therapy in subjects with asthma with elevated sputum eosinophils does not provide additional attenuation of airway eosinophilia.
Effect of freezing of sputum samples on flow cytometric analysis of lymphocyte subsets.
Sputum samples should be processed shortly after induction to prevent cell degradation. For intermediate storage, freezing of homogenised samples or immediate fixation have been shown to be suitable for cytospins. The aim of this study was to investigate whether freezing or immediate fixation of sputum affect the analysis of lymphocyte subsets by flow cytometry. Selected plugs from 24 sputum samples were homogenised. One aliquot was processed immediately and analysed by flow cytometry. A second aliquot was homogenised, frozen at -20 C after addition of dimethylsulfoxide and stored for a median time of 6 days. In six samples a third aliquot was fixed in formalin after induction and stored for up to 72 h before further processing. Compared to immediate processing, percentages of total lymphocytes and T-suppressor cells were elevated after being frozen, with a minor decrease in the T4/T8 ratio. Proportions of total lymphocytes, T-helper and T-suppressor cells correlated between native and frozen samples, intra-class correlation coefficients being 0.74, 0.85 and 0.70, respectively. The formalin-fixed aliquots could not be analysed with the antibodies used. In conclusion, freezing seems to be a suitable technique to store sputum samples for flow cytometry of CD3, CD4 and CD8 lymphocyte subsets. Its effects were minor compared to the variation between subjects.
Indirect airway challenges.
Indirect challenges act by causing the release of endogenous mediators that cause the airway smooth muscle to contract. This is in contrast to the direct challenges where agonists such as methacholine or histamine cause airflow limitation predominantly via a direct effect on airway smooth muscle. Direct airway challenges have been used widely and are well standardised. They are highly sensitive, but not specific to asthma and can be used to exclude current asthma in a clinic population. Indirect bronchial stimuli, in particular exercise, hyperventilation, hypertonic aerosols, as well as adenosine, may reflect more directly the ongoing airway inflammation and are therefore more specific to identify active asthma. They are increasingly used to evaluate the prevalence of bronchial hyperresponsiveness and to assess specific problems in patients with known asthma, e.g. exercise-induced bronchoconstriction, evaluation before scuba diving. Direct bronchial responsiveness is only slowly and to a modest extent, influenced by repeated administration of inhaled steroids. Indirect challenges may reflect more closely acute changes in airway inflammation and a change in responsiveness to an indirect stimulus may be a clinically relevant marker to assess the clinical course of asthma. Moreover, some of the indirect challenges, e.g. hypertonic saline and mannitol, can be combined with the assessment of inflammatory cells by induction of sputum.
A method to preserve sputum for delayed examination.
Examination of sputum cell counts is limited by the need to process samples within hours of expectoration. The validity and repeatability of a method to preserve sputum for delayed processing and examination were investigated in this study. Portions of selected sputum from 39 subjects were dispersed with dithiothreitol (routine method), or were fixed in a dithiothreitol-formaldehyde mixture for 48-72 h before dispersal with trypsin (preservation method). Total and differential cell counts using the two methods were compared; within-method repeatability of the preservation method was also examined. The intraclass correlation coefficient (ICC) for total cell counts and percentage of eosinophils, neutrophils and macrophages in sputum processed by the two methods was 0.89, 0.86, 0.91 and 0.90, respectively. Within-method repeatability (ICC) of the preservation method for the same cellular indices was 0.99, 0.94, 0.97 and 0.97, respectively. The interobserver repeatability for eosinophils, neutrophils and macrophages was 0.96, 0.97 and 0.97 using the preservation method, and 0.96, 0.99 and 0.99 using the routine method, respectively. This method of sputum preservation and dispersal is valid, reliable and convenient, and may be used for delayed processing and examination.
Airway inflammation in COPD: physiological outcome measures and induced sputum.
Chronic obstructive pulmonary disease (COPD) is a result of airway inflammation, and the best predictor of COPD is the early detection of airflow limitation by spirometry. The Global Initiative for Obstructive Lung Disease Workshop Report defines airflow limitation using simple spirometric indices. Available guidelines categorise the severity of COPD using forced expiratory volume in one second (FEV1) and forced vital capacity (FVC), with symptoms playing a minor role in the assessment. Current standards define COPD by progressive loss of FEV1, and thus longitudinal decline in FEV1 will be the primary outcome variable for intervention studies aimed at preventing or reducing the loss of pulmonary function. There is evidence, however, that the variable FEV1/FVC and FEV1 are often not measured properly in all settings. This article will discuss the roles of physiological measurements in diagnosing COPD and physiological outcome measures for COPD. It does not formally compare physiological measures with other outcome measures, such as symptoms or quality of life. Additionally, improved treatment of established disease requires a better understanding of the inflammatory process and its clinical effects and treatment. The inflammatory process, and how drugs affect it, can be studied noninvasively or relatively noninvasively by using refined methods of examining spontaneous or induced sputum. Enhanced understanding of the use of induced sputum will assist in predicting patients' responses to short- and long-term inhaled corticosteroid treatment, and the methods of sputum examination need to be simplified so that they can be applied more easily to clinical practice.
Standardised methodology of sputum induction and processing.
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Clinical applications of assessment of airway inflammation using induced sputum.
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The use of induced sputum in clinical trials.
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Induced sputum: time from expectoration to processing.
One of the limitations in the use of induced sputum to measure indices of airway inflammation is the perceived need to process the sample within 2 h. Therefore, the authors investigated whether the processing of induced sputum could be delayed. Induced sputum samples obtained from asthmatic subjects (n=30) were examined. Each sample was stored at 4 degrees C. A portion was selected and processed within 2 h and the remaining expectorate (sputum plus saliva) was refrigerated. Later an equal amount was selected and processed at either 9 (n=15) or 18 (n=15) h. The sputum was examined for cell counts and viability, fluid-phase eosinophil cationic protein (ECP), interleukin-8 (IL-8) and fibrinogen. Repeatability of measurements was assessed by the interclass correlation coefficient (ICC). Measurements obtained at 9 h did not differ from those made at 2 h and the repeatability was excellent (ICC 0.88-0.99). However, by 18 h the median cell viability was reduced from 65.0% to 43.0% and the ICC was generally lower: 0.10 for total cell count, 0.24 for viability, 0.60 for neutrophils, 0.90 for eosinophils, 0.56 for macrophages, 0.76 for ECP, 0.82 for IL-8 and 0.84 for fibrinogen. The results indicate that when induced sputum from subjects with asthma is kept at 4 degrees C, examination of cell counts can be delayed for < or = 9 h and for the fluid-phase indices measured for < or = 18 h. Further investigation of this issue is required for spontaneous sputum, other airway diseases and other inflammatory markers.
Standardised methodology of sputum induction and processing. Future directions.
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Eosinophilic bronchitis.
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The use of sputum cell counts to evaluate asthma medications.
Total and differential cell counts from hypertonic-induced, dithiothreitol-dispersed sputum provide reproducible measurements of airway inflammatory cell counts, which are responsive to treatment with anti-inflammatory drugs. They have helped to understand the kinetics of inflammatory cell changes in asthma after the reduction of corticosteroids and the subsequent re-introduction of treatment. They have identified that the presence of sputum eosinophilia in asthma, chronic cough and chronic airflow limitation is a predictor of steroid-responsiveness and of lack of 'asthma control'. They can be used to study the dose-response effect of inhaled corticosteroids and may be useful to establish the relative potency of different corticosteroid formulations and delivery devices. Sputum cell counts are also useful to study the potential anti-inflammatory effects of drugs like theophylline, long-acting beta-adrenoceptor agonists, leukotriene antagonists and newer drugs in development. They may be helpful to select add-on therapy to corticosteroids in 'difficult-to-control' asthma.