Antonella Di Franco

Induced sputum is a reproducible method to assess airway inflammation in asthma

To evaluate the reproducibility of induced sputum analysis, and to estimate the sample size required to obtained reliable results, sputum was induced by hypertonic saline inhalation in 29 asthmatic subjects on two different days. The whole sample method was used for analysis, and inflammatory cells were counted on cytospin slides. Reproducibility, expressed by intra-class correlation coefficients, was good for macrophages (+0.80), neutrophils (+0.85), and eosinophils (+0.87), but not for lymphocytes (+0.15). Detectable differences were 5.5% for macrophages, 0.6% for lymphocytes, 5.2% for neutrophils, and 3.0% for eosinophils. We conclude that analysis of induced sputum is a reproducible method to study airway inflammation in asthma. Sample sizes greater than ours give little improvement in the detectable difference of eosinophil percentages.

Asthma Control Test (ACT): Comparison with Clinical, Functional, and Biological Markers of Asthma Control.

Background. Asthma Control Test (ACT) is a simple tool for assessing the level of asthma control in clinical practice, and it has been validated in comparison with a general clinical assessment of asthma control, including forced expiratory volume in the first second (FEV1). Objective. To evaluate the relationship between ACT score and clinical and functional findings of asthma control and biomarkers of airway inflammation. Methods. A total of 68 asthmatic patients observed in our asthma clinic (33 regularly treated with inhaled corticosteroids (ICS) and 35 ICS-naïve) filled ACT questionnaire and underwent the following measurements: (a) FEV1 before and after salbutamol; (b) exhaled nitric oxide; (c) bronchial hyperresponsiveness to methacholine; (d) sputum eosinophil count; and (e) daytime and nighttime symptoms, rescue salbutamol, and twice-daily peak expiratory flow (PEF) recording on a 4-week diary card. Results. ACT score significantly correlated with symptom score, rescue medication use, and PEF variability, but not with FEV1, FEV1 reversibility, and markers of airway inflammation, which could not distinguish controlled from uncontrolled patients according to ACT, regardless of ICS treatment. Conclusion. ACT score is a valid tool to simply assess the current level of asthma control in terms of symptoms, rescue medication use, and PEF variability. Pulmonary function and biomarkers of airway inflammation are not related to the clinical asthma control as assessed by ACT and may represent additional measurements potentially useful in asthma management.

Profiles of proinflammatory cytokines in sputum from different groups of severe asthmatic patients

BACKGROUND Severe asthma represents a heterogeneous group of patients whose characteristics of airway inflammation are poorly known. OBJECTIVE To evaluate the sputum cytokine profiles of different phenotypes of severe asthma. METHODS Severe asthmatic patients (n = 45) were divided into 3 groups: frequent exacerbations, persistent bronchoconstriction, and both features. Two other groups (9 patients with untreated mild asthma and 10 control subjects) were also studied. Selected sputum portions were assayed for differential cell count, supernatant interleukin 5 (IL-5), granulocyte-macrophage colony-stimulating factor, IL-8, and eosinophil cationic protein. RESULTS There were no statistically significant differences among the 3 severe asthma groups in terms of sputum inflammatory cell percentages, IL-8 levels, and eosinophil cationic protein levels, although IL-8 levels tended to be higher in patients with persistent bronchoconstriction. Sputum concentrations of granulocyte-macrophage colony-stimulating factor and IL-5 were significantly higher in patients with frequent exacerbations compared with the other 2 groups. Levels of IL-5 and IL-8 were higher in severe asthmatic patients compared with mild asthmatic patients and controls, whereas sputum eosinophil percentages were intermediate between those of mild asthmatic patients and controls. CONCLUSIONS Proeosinophilic cytokine levels are increased in severe asthmatic patients with frequent exacerbations but not in severe asthmatic patients with persistent bronchoconstriction, suggesting that different cytokine profiles could be associated with different phenotypes of severe asthma.

Increase in markers of airway inflammation after ozone exposure can be observed also in stable treated asthmatics with minimal functional response to ozone.

BackgroundThe discrepancy between functional and inflammatory airway response to ozone has been reported in normal subjects, but few data are available for stable asthmatics regularly treated with inhaled corticosteroids.MethodsTwenty-three well controlled, regularly treated, mild-to-moderate asthmatic patients underwent two sequential randomised exposures to either filtered air or ozone (0.3 ppm for 2 hours) in a challenge chamber. Pulmonary function (PF) was monitored, and patients with FEV1 decrease greater than 10% from pre-challenge value were considered as responders. Immediately after each exposure, exhaled breath condensate (EBC) was collected to measure malondialdehyde (MDA). Six hours after each exposure, PF and EBC collection were repeated, and sputum was induced to measure inflammatory cell counts and soluble mediators (IL-8 and neutrophil elastase). The response to ozone was also evaluated according to the presence of polymorphism in oxidative stress related NQO1 and GSTM1 genes.ResultsAfter ozone exposure, sputum neutrophils significantly increased in responders (n = 8), but not in nonresponders (n = 15). Other markers of neutrophil activation in sputum supernatant and MDA in EBC significantly increased in all patients, but only in nonresponders the increase was significant. In nonresponders, sputum eosinophils also significantly increased after ozone. There was a positive correlation between ozone-induced FEV1 fall and increase in sputum neutrophils. No difference in functional or inflammatory response to ozone was observed between subjects with or without the combination of NQO1wt- GSTM1null genotypes.ConclusionsMarkers of neutrophilic inflammation and oxidative stress increase also in asthmatic subjects not responding to ozone. A greater functional response to ozone is associated with greater neutrophil airway recruitment in asthmatic subjects.

Effects of oral prednisone on sputum eosinophils and cytokines in patients with severe refractory asthma.

BACKGROUND Severe asthma occurs in a heterogeneous group of patients in whom symptoms and airway inflammation persist despite maximal antiasthma treatment. OBJECTIVE To verify whether a short-term course of oral steroids would modify sputum inflammatory cytokine and sputum eosinophil concentrations and whether this effect is related to the presence of sputum eosinophilia. METHODS In 59 patients with severe refractory asthma, we measured pulmonary function and inflammatory markers in hypertonic saline-induced sputum before and after 2 weeks of treatment with 0.5 mg/kg of oral prednisone (n = 39) or placebo (n = 20) daily. Selected sputum portions were assayed for total and differential cell counts and supernatant interleukin (IL) 5 and IL-8 concentrations. RESULTS At baseline, no statistical differences were found among placebo- and prednisone-treated patients in terms of sputum inflammatory cell percentages and IL-5 and IL-8 concentrations. After treatment, forced expiratory volume in 1 second significantly increased and sputum eosinophil percentages and IL-5 and IL-8 concentrations significantly decreased in the prednisone group, whereas no changes were observed in the placebo group. The positive effect of prednisone treatment was observed only in patients with baseline sputum eosinophilia, whereas in noneosinophilic patients with severe asthma prednisone induced only a significant decrease of sputum IL-8. CONCLUSIONS Additional high-dose oral corticosteroids improve pulmonary function and reduce not only sputum eosinophil but also sputum proinflammatory cytokine concentrations in patients with severe refractory asthma.

Biological Markers in Induced Sputum of Patients with Different Phenotypes of Chronic Airway Obstruction

Background: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by a combination of 3 different disorders, namely chronic asthma, chronic bronchitis and pulmonary emphysema, sometimes simultaneously present in the same subject. Objectives: The aim of our study was to compare sputum inflammatory markers in patients with different phenotypes of chronic airway obstruction. Methods: Forty-five subjects (forced expiratory volume in 1 s/vital capacity, FEV1/VC: 58.8 ± 12.2%; FEV1: 49.8 ± 11.5% of predicted) were classified as chronic asthma (n = 10) or COPD patients (n = 35); the latter were further divided into patients with prevalent chronic bronchitis (n = 24) or prevalent pulmonary emphysema (n = 11) according to clinical history and functional evaluation, and underwent sputum induction and analysis of inflammatory cell and soluble mediators. Results: Patients with chronic asthma showed higher sputum eosinophil percentages and eosinophilic cationic protein levels, and lower neutrophil percentages and neutrophil elastase levels than COPD patients. Neutrophil chemotactic activity in sputum supernatant was higher than the pool of normal subjects both in chronic asthma and COPD patients. No difference in sputum cell composition and levels of soluble mediators was observed between patients with chronic bronchitis and patients with pulmonary emphysema. Conclusions: The pattern of airway inflammation in induced sputum of patients with chronic asthma is different from that of COPD patients with a similar FEV1. Among COPD patients, however, the pattern of airway inflammation shows no difference between chronic bronchitis and patients with pulmonary emphysema, suggesting that these two clinically and functionally distinct phenotypes share a common inflammatory pattern as detected by induced sputum.

Analysis of sputum cell counts during spontaneous moderate exacerbations of asthma in comparison to the stable phase.

Background. Acute airway inflammation is considered to characterize asthma exacerbations, but its specific cellular pattern has not yet been completely evaluated. Aim. To evaluate the prevalence of sputum eosinophilia during acute asthma exacerbations of moderate severity, compared with a stable phase of the disease, and to assess the concordance between changes in pulmonary function and sputum eosinophilia in the period between exacerbation and post exacerbation. Methods. We compared sputum and blood inflammatory cell counts in 29 asthmatic subjects during a spontaneous moderate exacerbation of asthma (visit 1) with sputum and blood cell counts measured 4 weeks after the resolution of asthma exacerbation (visit 2). At visit 1, all subjects required an appropriate 1 week treatment with oral corticosteroids. Results. At visit 1, all subjects were able to collect spontaneous sputum, whereas at visit 2 sputum was induced by inhalation of hypertonic saline (NaCl 3, 4, and 5%, 10 minutes each) with β2-agonist pretreatment. Asthma exacerbation was accompanied by a significant increase in sputum eosinophil percentages compared with levels after exacerbation [25%(1–78) versus 4%(0–23), p<0.05). Only four subjects showed low sputum eosinophil percentages during exacerbation, and these showed no differences in main clinical findings with respect to subjects with sputum eosinophilia. At visit 2, the stability of asthma was assessed on the basis of PEF, FEV1, symptoms, and use of rescue β2-agonist. Asthma was defined as stable in 21 out of 29 subjects. Sputum eosinophil percentages fell significantly between visit 1 and visit 2 in both stable and unstable patients, but at visit 2 sputum eosinophil percentages were still high in subjects with unstable asthma. In patients who proved to be stable at visit 2, there was a significant correlation between the changes recorded in sputum eosinophil percentages and in FEV1 between the two visits (rho: 0.723, p<0.001). Conclusion. Sputum eosinophil but not neutrophil percentages increase in most asthmatic subjects during moderate exacerbation of asthma. Changes in the degree of airway eosinophilic inflammation are related to changes in the severity of airway obstruction during asthma exacerbation.

Sputum eosinophilia is a determinant of FEV1 decline in occupational asthma: results of an observational study

Objective To evaluate the potential determinants of forced expiratory volume in 1 s (FEV1) decline in workers with occupational asthma (OA) still exposed to the causative agent. We hypothesised that sputum eosinophilia might be a predictor of poor asthma outcome after diagnosis. Setting, design and participants In a specialistic clinical centre of the University Hospital of Pisa, we studied 39 participants (28 M, 11 F) diagnosed as having OA, routinely followed up between 1990 and 2009. They were a subgroup of 94 participants diagnosed as affected by OA in that period: 9 had been removed from work at the diagnosis, 21 were excluded for having ceased occupational exposure after few months from diagnosis, and 25 were lost at the follow-up or had no acceptable sputum measurements at the diagnosis. Estimates of the decline in FEV1 were obtained by means of simple regression analysis during the period of occupational exposure after diagnosis. Logistic regression was used to analyse the effects of factors (baseline FEV1 and sputum inflammatory cells, duration and type of exposure) that may potentially influence FEV1 decline. Results At follow-up (5.7+3.7 years), most participants were still symptomatic despite inhaled corticosteroids (ICS) treatment and had their occupational exposure reduced. Participants with higher sputum eosinophils (>3%) at baseline had a significantly greater decline of FEV1 (–52.5 vs −18.6 mL/year, p=0.012). Logistic regression showed that persistent exposure and sputum eosinophilia were significantly associated with a greater decline in FEV1 (OR 11.5, 95% CI 1.8 to 71.4, p=0.009 and OR 6.7, 95% CI 1.1 to 41.7, p= 0.042, respectively). Conclusions Sputum eosinophilia at diagnosis, together with the persistence of occupational exposure during follow-up, may contribute to a greater decline in FEV1 in patients with OA still at work. Further long-term studies are required as to whether intensive ICS treatment may be beneficial for patients with OA and increase ad eosinophilic inflammation.

Magnitude of late asthmatic response to allergen in relation to baseline and allergen-induced sputum eosinophilia in mild asthmatic patients

BACKGROUND Late asthmatic response (LAR) to allergen challenge is a validated method for studying the pathogenesis of and new treatments for asthma in the laboratory. OBJECTIVE To evaluate the relationship between the magnitude of allergen-induced LAR and clinical and biological determinants, including sputum and blood eosinophil percentages and eosinophil cationic protein concentrations. METHODS Thirty-eight untreated mild asthmatic patients (mean age, 21.2 years) were selected for the presence of allergen-induced early asthmatic response (EAR) and LAR. Each patient measured methacholine responsiveness (provocation dose that caused a decrease in forced expiratory volume in 1 second of 20% [PD20FEV1]) at baseline, differential blood cell counts and eosinophil cationic protein levels in blood and induced sputum, and serum neutrophil chemotactic activity at baseline and 24 hours after allergen challenge. RESULTS A correlation was found between LAR (as area under the curve [AUC]) and sputum eosinophil percentages at baseline (r = 0.51; P = .001) and 24 hours after allergen challenge (r = 0.44; P < .007). Furthermore, we found significant correlations between AUC LAR and AUC EAR, baseline methacholine PD20FEV1, baseline blood eosinophil percentages, and baseline serum neutrophil chemotactic activity. A stepwise multiple regression analysis showed that the stronger determinants of AUC LAR were baseline sputum eosinophilia and AUC EAR. CONCLUSION Baseline sputum eosinophilia and functional findings are determinants of the magnitude of allergen-induced LAR.

Prednisone Blunts Airway Neutrophilic Inflammatory Response due to Ozone Exposure in Asthmatic Subjects

Background: The effect of corticosteroids on the ozone (O3)-induced airway inflammation is still debated. Objective: The aim of the study was to confirm the effect of a short-term treatment with oral glucocorticosteroids on O3-induced airway inflammation, detected by induced sputum analysis, and on functional response in glucocorticosteroid-naive subjects. Methods: A randomized, placebo-controlled study using oral prednisone (25 mg o.d. for 4 days) was carried out. Nine mild persistent asthmatics were exposed for 2 h, on separatedays, to 0.27 ppm O3 and to air in random order, after 4 days of treatment with prednisone (25 mg o.d.) and after 4 days of placebo.Before and after exposure, pulmonary function test was measured; 6 h afterexposure, sputum induction was done. Results: Oral glucorticosteroids did not prevent pulmonary function decrement due to O3. After placebo, the percentage of neutrophils in induced sputum was significantly higher after O3 than after air [52.1 (15.7–77.3) vs. 17.8 (1.7–58.4), p = 0.02, O3 vs. air]. This difference was lost after 4 days of treatment with prednisone [35.2% (10–96.2) vs. 30.9% (6.1–75.6), n.s., O3 vs. air]. Neutrophil elastase in sputum supernatant increased after O3 exposure in the sample obtained after placebo, but not after prednisone treatment. Conclusions: This study confirms that glucocorticosteroids reduce inflammatory airway response, but do not prevent the airway functional impairment after O3 exposure.