Exhaled Nitric Oxide and Exhaled Breath Condensate ph in Severe Refractory Asthma

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1 CHEST Exhaled Nitric Oxide and Exhaled Breath Condensate ph in Severe Refractory Asthma Original Research Eleni Tseliou, MD ; Vasiliki Bessa, MD ; Georgios Hillas, MD ; Vasiliki Delimpoura, MD ; Georgia Papadaki, MD ; Charis Roussos, MD ; Spyros Papiris, MD, FCCP ; Petros Bakakos, MD ; and Stelios Loukides, MD, FCCP ASTHMA Background: Distinct inflammatory cellular phenotypes of severe refractory asthma (SRA) have been reported. Fractional exhaled nitric oxide (F e NO) primarily is related to eosinophilic inflammation. Exhaled breath condensate (EBC) ph has been suggested as a noninvasive tool in the assessment of patients with asthma. We sought to determine whether F e NO and EBC ph could identify the presence and type of the underlying cellular inflammation in patients with SRA. Methods: Twenty-nine patients with SRA, 27 patients with moderate asthma, and 17 healthy subjects underwent F e NO measurement, EBC collection for ph measurement, and sputum induction for cell count identification. Results: F e NO was significantly higher and ph significantly lower in patients with SRA than in the other groups. In SRA, F e NO levels of. 19 parts per billion were associated with a sensitivity of 0.78 and a specificity of 0.73 for sputum eosinophilia, whereas F e NO levels of, 19 parts per billion were associated with a sensitivity of 0.63 and a specificity of 0.9 for sputum neutrophilia irrespective of the presence of eosinophils. The ph failed to predict the cellular profile in SRA, but a cutoff value of, 7.37 could predict sputum eosinophilia in moderate asthma. Conclusions: In patients with SRA, different F e NO threshold values can identify those with predominant eosinophilia as well as those with neutrophilia. F e NO levels were reduced in patients with predominant neutrophilia regardless of the concomitant presence of eosinophilia. Although ph could not identify the cellular profile in SRA, it seemed to be a better index for predicting eosinophilia in moderate asthma. CHEST 2010; 138(1): Abbreviations: AUC 5 area under the curve; EBC 5 exhaled breath condensate; FeNO 5 fraction of exhaled nitric oxide; ICS 5 inhaled corticosteroids; ppb 5 parts per billion; ROC 5 receiver operating characteristic; SRA 5 severe refractory asthma Asthma is a heterogeneous, chronic inflammatory airway disease with a varying severity across its clinical spectrum. The majority of patients with asthma suffer from mild to moderate disease that can be well controlled with the regular use of low to medium doses of inhaled corticosteroids (ICS). 1 However, in a subset of patients with asthma, even Manuscript received May 28, 2009; revision accepted January 26, Affiliations: From the Second Respiratory Medicine Department (Drs Tseliou, Bessa, Delimpoura, Papiris, and Loukides), University of Athens Medical School, Attikon Hospital; First Respiratory Medicine Department (Drs Papadaki, Roussos, and Bakakos), University of Athens Medical School, Sotiria Chest Hospital; and Department of Respiratory and Critical Care Medicine (Dr Hillas), Research Unit, Sotiria Chest Hospital, Athens, Greece. Correspondence to: Stelios Loukides, MD, FCCP, Smolika , Athens, Greece; ssat@hol.gr high doses of ICS fail to control the disease, and they are considered as having severe refractory asthma (SRA). In patients with SRA, their condition remains difficult to control despite an extensive reevaluation of diagnosis, management, and observation for a period of at least 6 months by an asthma specialist. 2,3 Assessment of airway inflammation in SRA has led to the identification of patients with eosinophilic, neutrophilic, and mixed cellular profiles of the disease as well as of those with the pauciinflammatory phenotype. Identifying the cellular. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians ( site/misc/reprints.xhtml ). DOI: /chest CHEST / 138 / 1 / JULY,

2 profile in SRA may assist in understanding the pathophysiology and natural history of the disease, as well as in guiding treatment. 4 Recently, the field of noninvasive techniques for assessing airway inflammation has undergone intense development. The fraction of exhaled nitric oxide (F e NO) is recognized as an important and easily measured mediator in exhaled air. 5,6 It is primarily related to eosinophilic inflammation 7 and has been prospectively used to guide asthma treatment, with conflicting results. 8,9 Sputum induction is considered to be a semiinvasive technique that has helped clinical researchers to elucidate the inflammatory process of many airway diseases, including asthma. 10 The identification of sputum eosinophilia is a valuable parameter that predicts steroid response, can guide treatment, and can identify a subgroup of patients with SRA who experience more frequent asthma exacerbations. 11,12 The collection of exhaled breath condensate (EBC) is achieved by freezing the exhaled air with the use of special condensing devices. 13 EBC ph has been proposed as a promising noninvasive tool in the assessment of patients with asthma because it is measurable on site with a standardized methodology, is reproducible, and has normal values available. 14 There is evidence that EBC ph is significantly correlated with the number of eosinophils obtained from induced sputum in patients with moderate asthma. 15 We sought to determine whether F e NO and EBC ph differ among patients with SRA, patients with moderate asthma, and normal subjects. Additionally, we examined whether these markers can predict the cellular profile of SRA and moderate asthma in induced sputum. infection or any asthma exacerbation in the past 8 weeks prior to study admission also were excluded. The study was approved by the ethic committees of both hospitals, and all subjects provided informed consent. Study Variables FeNO Measurement: FeNO was measured with a portable nitirc oxide analyzer (NIOX MINO airway inflammation monitor; Aerocrine; Solna, Sweden). It provides measurements at 50 ml/s exhalation flow rate expressed in parts per billion (ppb). 17,18 Measurement of ph in EBC: EBC was collected and processed according to recently published recommendations, 13 using a condenser (EcoScreen; Jaeger; Wurzburg, Germany). The ph was measured with a ph meter as previously described (Consort P-903; Turnhout, Belgium). 15 Induced Sputum: Sputum was induced as previously described, 19 using all the modifications for safe measurements according to the underlying asthma severity. 20 Patients inhaled 3% saline at room temperature delivered by an ultrasonic nebulizer (DeVilbiss Co.; Heston, England) at the maximal saline output (4 ml/min) for 15 min. Sputum was processed as previously described. 21 Total cell counts were performed on a hemacytometer using trypan blue stain. Slides were prepared by using cytospin (Shandon; Runcorn, England) and May-Grunwald Giemsa staining protocols for differential cell counts. Cell counting was performed by an observer blind to the clinical characteristics of the subjects. At least 500 inflammatory cells were counted in each sample. A sample was considered adequate when the patient was able to expectorate at least 2 ml of sputum and the slides contained, 10% squamous cells on differential cell counting. Total cell count expressed as the number of cells /ml and the percentage of sputum inflammatory cells were used for our analysis. Based on reference values of induced sputum cellularity in normal volunteers proposed by previously published studies, a percentage of. 3% sputum eosinophils in differential cell counting was considered to reflect an eosinophilic profile of asthma, whereas a percentage of. 40% sputum neutrophils reflected a neutrophilic profile. 22 A percentage of. 3% eosinophils along with. 40% neutrophils was defined as a mixed granulocytic profile. Subjects Materials and Methods Lung Function: FEV1 and FVC were measured as components of flow volume using Master Screen Body (Viasys Healthcare, Jaeger; Hoechberg, Germany) according to American Thoracic Society guidelines. 25 Subjects were recruited from an open cohort of patients with asthma who were under medical follow-up care in the asthma clinics of the First and Second Respiratory Medicine University Departments in Athens, Greece. The diagnosis of asthma was established according to Global Initiative for Asthma guidelines. 1 The diagnosis of SRA was established according to American Thoracic Society criteria. 2 Twenty-nine patients with SRA were recruited. All were receiving high doses of ICS (. 1.2 mg budesonide/d or equivalent) plus long-acting b 2 -agonists. Twelve of these 29 patients also were receiving oral steroids, and three were treated with omalizumab. Twenty-seven were characterized as having moderate asthma according to the 2005 Global Initiative for Asthma classification, 16 and all were receiving low to medium doses of ICS (, m g budesonide/d or equivalent) plus longacting b 2 -agonists. Seventeen healthy, nonatopic, nonsmoking subjects composed the control group. The patients with asthma were not current-smokers or had a smoking history of, 5 pack-years. Patients with any other respiratory disease or any concomitant malignant, heart, renal, liver, or collagen disease were excluded. Patients with a respiratory tract Bronchial Hyperresponsiveness: Bronchial hyperresponsiveness was performed only in the control group and measured as the provocative dose of methacholine causing a 20% fall in FEV 1 with a commercially available system (Aerosol Provocation System; Viasys Healthcare, Jaeger) according to American Thoracic Society guidelines. 26 Atopy Status: A positive skin prick test to any of six common aeroallergens (dermatophagoides mix, cat epithelium, parietaria, olive, altenaria, cypress) was used to confirm atopy. Study Design On day 1, all subjects underwent a medical history and physical examination by an experienced pneumonologist, spirometry for measuring FEV 1, FVC before and after bronchodilation, BMI measurement, and skin prick tests. On the same day, subjects underwent F e NO measurement and EBC collection for ph measurement. The following day, sputum induction was performed only in subjects with asthma. 108 Original Research

3 Statistical Analysis Normally distributed data were presented as mean 6 SD, whereas skewed data were presented as median (interquartile range). Normality of distribution was checked with Kolmogorov- Smirnov test. Statistical comparisons within groups were estimated with one-way analysis of variance accompanied by Bonferonni post hoc test for multiple comparisons. For nonnormally distributed values, Kruskal-Wallis test was used. Statistical comparisons between SRA and moderate asthma were estimated with unpaired t tests for normally distributed values and Mann-Whitney U test for nonnormally distributed values. Correlations between both F e NO and EBC ph (dependent variables) and sputum eosinophils and neutrophils (independent variables) were performed using a linear regression analysis after proper adjustments for age, sex, BMI, atopy, and ICS (low to medium dose vs high dose) and oral steroid use. Diagnostic performance of both F e NO and EBC ph to predict sputum cellular profile was examined by constructing receiver operating characteristic (ROC) curves. The cutoff values of both F e NO and EBC ph were selected according to appropriate statistical methods in order to reflect the optimal sensitivity and specificity of the previously mentioned parameters. A P value,.05 was considered significant. Statistical analysis was performed using SPSS, version 15.0, software (SPSS Inc; Chicago, IL), MedCalc (MedCalc Software; Mariakerke, Belgium), and Graphpad Prism 3.0 (Graphpad Software; La Jolla, CA). Results ph and F e NO Values in the Three Study Groups Subjects characteristics are summarized in Table 1. The EBC ph in patients with SRA was significantly lower than in patients with moderate asthma and healthy subjects ( vs vs , respectively; P,.0001), (Fig 1A, Table 1 ). F e NO levels were significantly higher in patients with SRA than in patients with moderate asthma and healthy subjects (43 [ ] vs 25 [17-38] vs 12 [9.5-19], respectively; P,.0001) (Fig 1B, Table 1 ). Inflammatory Cells in Induced Sputum Patients with SRA had significantly higher percentages of both eosinophils and neutrophils than those with moderate asthma ( Table 1 ). Eighteen of the 29 patients with SRA had the eosinophilic profile, only two patients had the neutrophilic profile, and seven had a mixed granulocytic phenotype; the remaining two patients had neither eosinophilia nor neutrophilia. Among the 27 patients with moderate asthma, 20 had the eosinophilic profile, and none had sputum neutrophilia; the remaining seven had neither eosinophilia nor neutrophilia. Associations Between EBC ph and F eno With Cells in Induced Sputum of Patients With Asthma SRA: After the already-mentioned proper adjustments, F e NO in SRA was significantly positively associated with sputum eosinophilia (B, 1.8; P 5.01) and negatively associated with sputum neutrophilia (B, 21.3; P 5.03). No significant associations between EBC ph and any of the inflammatory cells were observed. Moderate Asthma: After similar proper adjustments, a weak significant association existed between F eno and eosinophils (B, 1.5; P 5.04). Regarding ph, a significant negative association with sputum eosinophilia was found (B, ; P 5.01). ROC Analysis SRA: An Fe NO level of. 19 ppb was associated with a sensitivity of 0.78 and a specificity of 0.73 for the identification of the eosinophilic profile, with an area under the curve (AUC) of 0.8 (95% CI, ; P ) ( Fig 2 A ). An F e NO level of, 19 ppb was associated with a sensitivity of 0.63 and a specificity of 0.9 for the identification of sputum neutrophilia (seven patients with mixed profile plus two patients with neutrophilic profile), with an AUC of 0.79 (95% CI, ; P ) ( Fig 2 B ). After subdividing patients on the basis of atopy, no significant changes of these results were observed (data not shown). A ph level of, 7.12 was associated with a sensitivity of 0.27 and a specificity of 0.9 for the identification of the eosinophilic profile, with an AUC of 0.5 (95% CI, ; P 5.9). A ph level of, 7.24 was associated with a sensitivity of 0.6 and a specificity of 0.7 for the identification of sputum neutrophilia, with an AUC of 0.7 (95% CI, ; P 5.12). Moderate Asthma: An Fe NO level of. 28 ppb was associated with a sensitivity of 0.47 and a specificity of 0.9 for the identification of the eosinophilic profile, with an AUC of 0.7 (95% CI, ; P 5.09). A ph level of, 7.37 was associated with a sensitivity of 0.8 and a specificity of 0.9 for the identification of the eosinophilic profile, with an AUC of 0.9 (95% CI, ; P ) ( Fig 3 ). Discussion In this study, different cutoff values of F eno in patients with SRA identified those with predominant sputum eosinophilia and neutrophilia. Those with sputum eosinophilia had elevated F e NO levels despite high-dose steroid treatment, whereas those with sputum neutrophilia had low F e NO levels regardless of the concomitant presence of eosinophilia. EBC ph could not identify the cellular profiles in SRA; however, it had a better prognostic performance for the eosinophilic profile in moderate asthma. Our study agrees with previous findings that showed a close relationship between increased F eno levels and eosinophilic inflammation. 7 Furthermore, CHEST / 138 / 1 / JULY,

4 Table 1 Study Subjects Characteristics Study Variable SRA (n 5 29) Moderate Asthma (n 5 27) Normal Subjects (n 5 17) P Value Age, y Sex, male (female), No. 10 (19) 10 (17) 7 (10) NS BMI, kg/m NS Atopy 10 (29) 11 (27) 0 NS FEV 1, % predicted ,.0001 FVC, % predicted F e NO, ppb 43 ( ) 25 (17-38) 12 (9.5-19),.0001 ph in EBC ,.0001 Total cell count, 10 6 cells/ml 1.7 ( ) 1.24 (1-1.6) ND.002 Eosinophils, % ND.001 Neutrophils, % ND,.0001 Macrophages, % ND.003 Lymphocytes, % 2.5 (1.25-4) 2 (1.5-5) ND NS Treatment ICS, dose a 1350 (1,100-1,600) 500 ( ) Normally distributed data are presented as mean 6 SD, whereas skewed data are presented as median (interquartile ranges), unless otherwise indicated. Bold numbers indicate significant differences, all in favor of patients with SRA compared with patients with moderate asthma and normal subjects. EBC 5 exhaled breath condensate; FeNO 5 fraction of exhaled nitric oxide; ICS 5 inhaled corticosteroids; ND 5 Not done; NS 5 nonsignificant; SRA 5 severe refractory asthma. a Dose of ICS is expressed as mg budesonide. we demonstrated that F e NO levels might be predictive of eosinophilic and neutrophilic inflammation as assessed by induced sputum in patients with SRA. The finding related to eosinophilia is in accordance with that previously reported by Silkoff et al, 27 although the biologic sample in which eosinophils were assessed was different (biopsy specimen vs induced sputum). In another study, where both biopsy specimen and sputum were used to assess airway inflammation, F e NO was increased in the eosino- philic SRA phenotype identified from the bronchial biopsy specimen but not from the induced sputum; however, the predictive value for eosinophilia was not examined. 12 The linkage between F e NO and eosinophils observed in patients with SRA might be explained by either the steroid resistance or the persistent activation of epithelial cells, which are the major site of nitric oxide synthesis and a source of eosinophilic chemoattractant factors. It is important to note that Figure 1. F e NO values in patients with SRA, patients with moderate asthma, and normal subjects ( A ). Data are expressed as median with minimum and maximum levels; P,.0001 in favor of SRA compared with the other two groups. ph values in EBC in patients with SRA, patients with moderate asthma, and normal subjects ( B ). Data are expressed as mean 6 SD. * P,.0001 in favor of SRA compared with the other two groups. ** P,.0001 between patients with moderate asthma and normal subjects. EBC 5 exhaled breath condensate; FeNO 5 fraction of exhaled nitric oxide; ppb 5 parts per billion; SR 5 severe refractory; SRA 5 severe refractory asthma. 110 Original Research

5 Figure 2. ROC curve for the evaluation of the prognostic value of F e NO for the eosinophilic profile in induced sputum in patients with SRA (n 5 29) ( A ). An F e NO level of. 19 ppb was associated with a sensitivity of 0.78 and a specificity of 0.73 for the identification of the eosinophilic profile, with an AUC of 0.8 (95% CI, ; P ). An ROC curve for the evaluation of the prognostic value of F e NO for sputum neutrophilia in induced sputum in patients with SRA (n 5 29) ( B ). An F e NO level of, 19 ppb was associated with a sensitivity of 0.63 and a specificity of 0.9 for the identification of sputum neutrophilia (seven patients with mixed profile plus two patients with neutrophilic profile), with an AUC of 0.79 (95% CI, ; P ). SRA 5 severe refractory asthma. AUC 5 area under the curve; ROC 5 receiver operating characteristic. See Figure 1 legend for expansion of other abbreviations. F e NO loses its sensitivity in relation to eosinophils when neutrophils are present along with elevated eosinophils. On the other hand, the weak association found in moderate asthma might be related to the deactivation of eosinophils by steroid treatment. 28 An interesting finding in our study was that F eno levels are negatively associated with neutrophilic inflammation in SRA, whereas low F e NO levels are predictive of neutrophilia irrespective of the presence of eosinophils. F e NO has been related primarily to eosinophilic inflammation, and thus, the crucial point is to interpret why the this relationship is abolished in the presence of neutrophilic inflammation. A possible explanation for our finding might be the conversion of nitric oxide to peroxynitrate through its reaction with high amounts of oxygen species released by neutrophils. 29 This nitric oxide modulation might overcome any effect of eosinophils on nitric oxide production. Regardless of the inflammatory profile of patients with SRA, the levels of F e NO were increased compared with patients with moderate asthma, although with a wide range of values. This finding also was documented in a recent study with selection criteria similar to ours. 30 Previous studies found no significant difference in F e NO values between SRA and moderate asthma. 12,22,27,31 These differences might be attributed to either the different selection criteria for patients with SRA used within studies 22,27,31 or the different phenotype of SRA due to patient smoking habits or exacerbation frequency. 12 EBC ph has not been studied previously in patients with SRA. Previously published data on ph evaluation support decreased ph levels in patients with moderate asthma compared with patients with mild asthma.15 In the present study, patients with SRA had lower ph levels than patients with moderate asthma and healthy subjects. This finding might be attributed to the already-reported close relationship between airway acidification and oxidative stress because the latter is further increased in SRA. 15 Nevertheless, the CHEST / 138 / 1 / JULY,

6 neutrophil counts in normal subjects as a primary outcome. 22 However, we have to address that a greater variability exists in the current literature, indicating that a higher percentage might be more proper for the definition of normal neutrophil counts in sputum. 32,33 The main clinical message derived from our results is that in patients with SRA, low F e NO values cannot exclude the presence of sputum eosinophilia. Accordingly, sputum induction might provide further information about airway inflammatory cells. In conclusion, this study suggests that F e NO is a potentially useful biomarker for the prediction of eosinophilia and neutrophilia in SRA. F e NO levels were found to be reduced in patients with sputum neutrophilia regardless of the concomitant presence of eosinophilia. Although ph could not identify the cellular profile in SRA, it seemed to be a better index for predicting eosinophilia in moderate asthma. Figure 3. ROC curve for the evaluation of the prognostic value of ph in EBC for the eosinophilic profile in induced sputum in patients with moderate asthma [n 5 27]. A ph level of, 7.37 was associated with a sensitivity of 0.8 and a specificity of 0.9 for the identification of the eosinophilic profile, with an AUC of 0.9 (95% CI, ; P ). See Figures 1 and 2 legends for expansion of abbreviations. cellular origin as well as the underlying mechanisms for airway acidification are still under investigation and might explain why ph failed to predict the cellular profile in sputum in SRA. In previous studies, ph has been related to either eosinophils (asthma) or neutrophils (COPD) but not to inflammatory infiltrations where both cells were present. 15 In our study, the significant predictive performance of ph for eosinophilia in moderate asthma may be related to the homogeneity of the cellular population that is characterized by eosinophils but not neutrophils. In addition, this finding might imply that compared with FeNO, ph is a biomarker less dependent on steroid treatment. The small number of patients with SRA included in our study is a possible limitation of the clinical validity of our findings. However, most studies of noninvasive evaluation of inflammation in SRA include a similar or even lower number of patients. 12,20,23,27 Nevertheless, we believe that the results of the present study have to be prospectively validated, and further studies are needed to elucidate the connection among F e NO, EBC ph, and sputum cellular populations. Another limitation might be the definition of normal values for sputum neutrophil counts. We chose 40% as the cutoff value to include the upper limit of SD from the study that evaluated sputum Acknowledgments Author contributions: Dr Tseliou: contributed to the performance of the F e NO and ph measurements and sputum induction and processing. Dr Bessa: contributed to the performance of the F e NO and ph measurements and sputum induction and processing. Dr. Hillas: contributed to the performance of sputum induction and processing. Dr Delimpoura: contributed to the performance of the F eno and ph measurements and sputum induction and processing. Dr Papadaki: contributed to the performance of the F eno and ph measurements and sputum induction and processing. Dr Roussos: contributed to the coordination of the whole study on behalf of the two departments. Dr Papiris: contributed to the coordination of the whole study on behalf of the two departments. Dr Bakakos: contributed to the recruitment of patients, confirmed their eligibility for entering the study, and supervised every noninvasive and semiinvasive procedure. Dr Loukides: contributed to the recruitment of patients, confirmed their eligibility for entering the study, and supervised every noninvasive and semiinvasive procedure. Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Other contributions: All work was performed at the First and Second Respiratory Medicine Departments, University of Athens Medical School, Athens, Greece. References 1. Bateman ED, Hurd SS, Barnes PJ, et al. Global strategy for asthma management and prevention: GINA executive summary. Eur Respir J ;31: American Thoracic Society. Proceedings of the ATS Workshop on Refractory Asthma. Current understanding, recommendation, and unanswered questions. Am J Respir Critical Care Med ;162: Holgate ST, Polosa R. The mechanisms, diagnosis, and management of severe asthma in adults. Lancet ;368(9537): Original Research

7 4. Chanez P, Wenzel SE, Anderson GP, et al. Severe asthma in adults: what are the important questions? J Allergy Clin Immunol ;119 (6 ): Gustafsson LE, Leone AM, Persson MG, Wiklund NP, Moncada S. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem Biophys Res Commun ;181 (2 ): Kharitonov SA, Yates D, Robbins RA, Logan-Sinclair R, Shinebourne EA, Barnes PJ. Increased nitric oxide in exhaled air of asthmatic patients. Lancet ;343 (8890 ): Kharitonov SA, Barnes PJ. Exhaled markers of pulmonary disease. Am J Respir Crit Care Med ;163 (7 ): Smith AD, Cowan JO, Brassett KP, Herbison GP, Taylor DR. Use of exhaled nitric oxide measurements to guide treatment in chronic asthma. N Engl J Med ;352 (21 ): Shaw DE, Berry MA, Thomas M, et al. The use of exhaled nitric oxide to guide asthma management: a randomized controlled trial. Am J Respir Crit Care Med ;176 (3 ): Brightling CE. Clinical applications of induced sputum. Chest ;129 (5 ): Green RH, Brightling CE, McKenna S, et al. Asthma exacerbations and sputum eosinophil counts: a randomised controlled trial. Lancet ;360 (9347 ): Lemière C, Ernst P, Olivenstein R, et al. Airway inflammation assessed by invasive and noninvasive means in severe asthma: eosinophilic and noneosinophilic phenotypes. J Allergy Clin Immunol ;118 (5 ): Horváth I, Hunt J, Barnes PJ, et al ; ATS/ERS Task Force on Exhaled Breath Condensate. Exhaled breath condensate: methodological recommendations and unresolved questions. Eur Respir J ;26 (3 ): Paget-Brown AO, Ngamtrakulpanit L, Smith A, et al. Normative data for ph for exhaled breath condensate. Chest ;126 (2 ): Kostikas K, Papatheodorou G, Ganas K, Psathakis K, Panagou P, Loukides S. ph in expired breath condensate of patients with inflammatory airway diseases. Am J Respir Crit Care Med ;165 (10 ): Global Strategy for Asthma Management and Prevention. Bethesda, MD: National Institutes of Health, National Heart, Lung, and Blood Institute; NIH publication , updated. 17. American Thoracic Society ; European Respiratory Society. ATS/ERS recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide, Am J Respir Crit Care Med ;171 (8 ): Menzies D, Nair A, Lipworth BJ. Portable exhaled nitric oxide measurement: Comparison with the gold standard technique. Chest ;131 (2 ): Kips JC, Fahy JV, Hargreave FE, Ind PW, in t Veen JC. Methods for sputum induction and analysis of induced sputum: a method for assessing airway inflammation in asthma. Eur Respir J Suppl ;26 :9S-12S. 20. Grootendorst DC, van den Bos JW, Romeijn JJ, et al. Induced sputum in adolescents with severe stable asthma. Safety and the relationship of cell counts and eosinophil cationic protein to clinical severity. Eur Respir J ;13 (3 ): Efthimiadis A, Spanevello A, Hamid Q, et al. Methods of sputum processing for cell counts, immunocytochemistry and in situ hybridisation. Eur Respir J ;20 :19S-23S. 22. Spanevello A, Confalonieri M, Sulotto F, et al. Induced sputum cellularity. Reference values and distribution in normal volunteers. Am J Respir Crit Care Med ; 162 ( 3 Pt 1 ): Jatakanon A, Uasuf C, Maziak W, Lim S, Chung KF, Barnes PJ. Neutrophilic inflammation in severe persistent asthma. Am J Respir Crit Care Med ;160 (5 Pt 1 ): Louis R, Lau LC, Bron AO, Roldaan AC, Radermecker M, Djukanović R. The relationship between airways inflammation and asthma severity. Am J Respir Crit Care Med ; 161 (1 ): American Thoracic Society. Standardization of spirometry: 1994 update. Am J Respir Crit Care Med ; 152 ( 3 ): American Thoracic Society. Guidelines for methacholine and exercise challenge testing Am J Respir Crit Care Med ;161 (1 ): Silkoff PE, Lent AM, Busacker AA, et al. Exhaled nitric oxide identifies the persistent eosinophilic phenotype in severe refractory asthma. J Allergy Clin Immunol ; 116 ( 6 ): Newton R. Molecular mechanisms of glucocorticoid action: what is important? Thorax ;55 (7 ): Jones KL, Bryan TW, Jinkins PA, et al. Superoxide released from neutrophils causes a reduction in nitric oxide gas. Am J Physiol ;275 (6 Pt 1 ): van Veen IH, Ten Brinke A, Sterk PJ, et al. Exhaled nitric oxide predicts lung function decline in difficult-to-treat asthma. Eur Respir J ;32 (2 ): Montuschi P, Corradi M, Ciabattoni G, Nightingale J, Kharitonov SA, Barnes PJ. Increased 8-isoprostane, a marker of oxidative stress, in exhaled condensate of asthma patients. Am J Respir Crit Care Med ;160 (1 ): Thomas RA, Green RH, Brightling CE, et al. The influence of age on induced sputum differential cell counts in normal subjects. Chest ;126 (6 ): Belda J, Leigh R, Parameswaran K, O Byrne PM, Sears MR, Hargreave FE. Induced sputum cell counts in healthy adults. Am J Respir Crit Care Med ;161 (2 Pt 1 ): CHEST / 138 / 1 / JULY,