Exhaled Nitric Oxide and Airway Hyperresponsiveness to Adenosine 5 -monophosphate and Methacholine in Children with Asthma

Transcription

1 Originl Pper Received: July 3, 2014 Accepted fter revision: Jnury 14, 2015 Published online: Mrch 13, 2015 Exhled Nitric Oxide nd Airwy Hyperresponsiveness to Adenosine 5 -monophosphte nd Methcholine in Children with Asthm Mtthew S. Perznowski Young Yoo b d Deprtment of Environmentl Helth Sciences, Milmn School of Public Helth, Columbi University, New York, N.Y., USA; b Deprtment of Peditrics, College of Medicine nd c Allergy Immunology Center, Kore University, nd d Environmentl Helth Center, Kore University Anm Hospitl, Seoul, Kore Key Words Adenosine 5 -monophosphte Airwy hyperresponsiveness Childhood sthm Exhled nitric oxide Methcholine Abstrct Bckground: There is incresing interest in the role of indirect bronchil chllenges becuse clinicl studies hve shown tht indirect irwy hyperresponsiveness (AHR) reflects underlying irwy inflmmtion better thn direct AHR. Frctionl exhled nitric oxide (FeNO) ppers to be useful clinicl tool for ssessing irwy inflmmtion noninvsively. We exmined whether FeNO is more closely relted to AHR to indirect stimuli thn AHR to direct stimuli in children with mild to moderte sthm. Methods: Fifty-nine sthmtic children ged 6 16 yers without rhinitis, underwent spirometry, FeNO mesurement nd blood tests for serum totl IgE, blood eosinophil count nd serum eosinophil ctionic protein (ECP). All subjects underwent methcholine nd denosine 5-monophosphte (AMP) chllenge tests t intervls of 3 dys. Results: In univrite liner regression nlysis, FeNO ws significntly ssocited with both PC 20 AMP ( R 2 = 0.341, p < 0.001) nd PC 20 methcholine ( R 2 = 0.188, p = 0.001). After djustment for ge, sex, serum totl IgE nd blood eosinophil count, the ssocition between FeNO nd PC 20 AMP (β = 1.98, p = 0.001) ws more robust thn tht between FeNO nd PC 20 methcholine (β = 0.87, p = 0.081). The significnt correltion between FeNO nd PC 20 AMP ws observed in the steroid-nïve group (β = 2.48, p = 0.001), but not in the steroid-treted group (β = 0.88, p = 0.463). Conclusions: FeNO levels were more closely ssocited with PC 20 AMP thn with PC 20 methcholine. This reltionship could only be seen in the steroid-nïve subjects. These results suggest tht FeNO levels in children with sthm my be more closely relted to indirect AHR thn to direct AHR S. Krger AG, Bsel Introduction Asthm is one of the most common chronic diseses in children. Airwy hyperresponsiveness (AHR) nd irwy inflmmtion re two min chrcteristics of sthm [1]. It hs been shown tht irwy inflmmtion contributes to AHR, even though the presence nd severity of AHR re relted to smooth muscle contrction. AHR is defined s n exggerted bronchoconstrictive response of the irwy to vriety of stimuli nd cn be mesured by using direct or indirect stimuli. Direct stimuli, such s with methcholine, induce irwy nrrowing minly through direct ction on the receptors of irwy smooth muscles [2], wheres indirect stimuli, such s E-Mil krger@krger.com S. Krger AG, Bsel /15/ $39.50/0 Correspondence to: Prof. Young Yoo Deprtment of Peditrics, College of Medicine, Kore University 73 Inchon-ro, Sungbuk-gu Seoul (Kore) E-Mil kore.c.kr

2 with denosine 5 -monophosphte (AMP), cuse irwy nrrowing minly vi the stimultion of primed mst-cell degrnultion nd the relese of proinflmmtory meditors [3]. Thus, irwy inflmmtion lso ppers to be mjor fctor determining the degree of AHR nd irflow obstruction. Exhled nitric oxide (eno) is noninvsive mrker of irwy inflmmtion. In recent yers, frctionl eno (FeNO) hs become vluble tool for the dignosis nd monitoring of sthm in childhood [4]. Previous studies investigting reltionships between FeNO nd AHR hve shown inconsistent results nd the correltions were generlly low [5 7]. Some studies concerning topic steroidnïve sthmtics hve demonstrted significnt ssocitions between FeNO nd AHR [5] ; others performed on steroid-treted sthmtics hve not [7, 8]. FeNO mesurement is incresingly useful for the dignosis nd mngement of sthm. However, the clinicl vlidity of FeNO in sthmtics is not well estblished or widely ccepted becuse of the significnt phenotypic heterogeneity of sthm. Moreover, FeNO levels re elevted not only in ptients with sthm but lso in ptients with rhinitis nd other upper irwy diseses s well s in topic subjects without respirtory disese [9]. Although FeNO nd AHR most likely represent different spects of inflmmtion nd structurl chnges in the irwys, further understnding of the reltionships between clinicl mrkers of irwy inflmmtion, irflow obstruction nd irwy hyperrectivity in sthmtics my hve clinicl implictions. Since AHR to indirect stimuli reflect llergic irwy inflmmtion better thn AHR to direct stimuli [10], we hypothesized tht FeNO would be more closely relted to indirect bronchil chllenge thn to direct bronchil chllenge. We thus exmined whether FeNO would be more closely ssocited with AHR to AMP thn with AHR to methcholine. Mterils nd Methods Study Popultion This study ws performed on 59 children ged 6 16 yers with mild to moderte, persistent sthm who were recruited from the Allergy Clinic of the Kore University Anm Hospitl, Seoul, Kore. Inclusion criteri were physicin-dignosed sthm nd history of episodic wheezing nd/or dyspne during the pst yer tht ws resolved with bronchodiltor. Subjects with history of ner-ftl sthm, mjor excerbtions necessitting the use of systemic corticosteroids or other respirtory diseses were excluded from the study. Seventeen subjects were tking sthm medicine regulrly prior to testing. Eleven subjects were using inhled corticosteroids (ICSs), 5 were tking leukotriene modifiers nd 1 ws tking orl theophylline s controller medicine. They were divided into steroid-treted group (n = 11) nd steroid-nïve group (n = 48). Subjects with ny history of symptoms suggestive of current llergic rhinitis, such s recurrent sneezing, rhinorrhe nd nsl stuffiness or itching in the bsence of common cold within the pst 12 months were lso excluded. The clinicl severity of sthm ws ssessed ccording to the Ntionl Asthm Eduction nd Prevention Progrm criteri [11]. Prents gve written informed consent for their children to prticipte in the study. The study protocol ws pproved by the Kore University Anm Hospitl Institutionl Review Bord (No. AN ). Pulmonry Function Tests Forced expirtory volume in 1 s (FEV 1 ) nd forced vitl cpcity (FVC) were mesured using computerized spirometer (Microspiro-HI 298, Chest, Tokyo, Jpn), in ccordnce with the recommendtions of the Americn Thorcic Society [12]. All subjects were sked to undergo spirometry in reproducible wy (i.e. coefficient of vrition of FEV 1 in 3 consecutive flow-volume curves of <5%) nd to chieve n FEV 1 of t lest 60% of the predicted vlue. The lrgest vlue of triplicte FEV 1 on ech occsion ws used for nlysis. Methcholine nd AMP Chllenges Methcholine chllenge tests were crried out using modifiction of the method described by Chi et l. [13]. AMP chllenge tests were crried out using modifiction of the method of the Europen Respirtory Society [14]. Ptients were sked to discontinue the use of inhled β2-gonists (for 24 h), orl theophylline (for 48 h), leukotriene modifiers nd ICSs (for 7 dys) t lest before testing. Ech ptient hd been free of cute respirtory trct infections for t lest 4 weeks prior to the tests. Fresh methcholine nd AMP solutions were prepred in buffered sline solution t different concentrtions (0.075, 0.15, 0.3, 0.625, 1.25, 2.5, 5, 10, 25 nd 50 mg/ml for methcholine; 3.125, 6.25, 12.5, 25, 50, 100, 200 nd 400 mg/ml for AMP). A Rosenthl- French dosimeter (Lbortory for Applied Immunology, Bltimore, Md., USA), triggered by solenoid vlve set to remin open for 0.6 s, ws used to generte n erosol from DeVilbiss 646 nebulizer (DeVilbiss Helth Cre, Somerset, P., USA) with pressurized ir t 20 psi. Ech subject inhled 5 inspirtory cpcity breths of buffered sline solution nd incresing concentrtions of methcholine or AMP, respectively, t 5-min intervls. This setup produced n output of ± ml (men ± SD) per inhltion. At ech concentrtion, FEV 1 ws mesured 90 s fter inhltion. The procedure ws terminted when FEV 1 decresed by >20% of its post-sline vlue or when the highest methcholine (50 mg/ml) or AMP (400 mg/ml) concentrtion ws reched. Percentge declines in FEV 1 from the post-sline vlue were plotted ginst the log concentrtions of inhled methcholine or AMP. PC 20 methcholine nd PC 20 AMP were clculted by interpolting between 2 djcent dt points whenever FEV 1 decresed by >20%. For sttisticl nlyses, censored vlue of 800 mg/ml for PC 20 AMP ws ssigned to subjects who did not show 20% decline in FEV 1 fter inhltion of the mximl concentrtion of AMP. A cumultive provoctive dose cusing 20% fll in FEV 1 (PD 20 AMP) ws clculted by log-liner interpoltion of the finl dt points. The dose-response rtio (DRR) ws clculted by obtining the finl percentge fll in FEV 1 nd dividing it by the cumultive dose of AMP dministered. 108 Perznowski/Yoo

3 Tble 1. Clinicl chrcteristics of the 2 study groups Prmeters Steroid-treted (n = 11) Steroid-nïve (n = 48) Totl (n = 59) Age, yers 10.0± ± ±2.7 Boys, % Positive skin-prick test, % Totl IgE, IU/ml ( ) ( ) ( ) Blood eosinophil count, /μl ( ) ( ) ( ) FeNO, ppb, b 54.1 ( ) 30.0 ( ) 33.3 ( ) Geometric men (rnge of 1 SD). b p < Mesurement of FeNO Exhled NO ws mesured ccording to Americn Thorcic Society recommendtions using n NO nlyzer (NIOX MINO nlyzer, Aerocrine, Soln, Sweden) during single-breth exhltion [15]. Subjects inhled NO-free ir to totl lung cpcity nd immeditely exhled fully ginst positive mouthpiece counter to pressure t flow rte of 50 ml/s into n on-line nlyzer to void ny nsl contmintion. Skin-Prick Tests Skin-prick tests were performed using 13 common erollergens: house dust mites ( Dermtophgoides pteronyssinus nd Dermtophgoides frine ), niml dnders (ct epithelium nd dog epithelium), pollens (mugwort, ryegrss, rgweed, hzel, lder nd ok), molds ( Aspergillus fumigtes nd Alternri lternt ) nd cockroch (Bltell germnic). The llergens were supplied by Allergophrm (Reinbek, Germny). A men whel dimeter >3 mm in the bsence of ny rection to the negtive control ws considered positive rection. Atopy ws defined s the presence of t lest 1 positive rection to these llergens greter thn the negtive control. Serum totl IgE nd Blood Eosinophil Mrkers Serum totl IgE levels were mesured using Cot-A-Count Totl IgE IRMA (Dignostic Products Co., Los Angeles, Clif., USA) ccording to the mnufcturer s instructions. The number of peripherl blood eosinophils ws counted with blood smples contining EDTA using n utomted hemtology nlyzer (Coulter Counter STKS, Beckmn Coulter, Fullerton, Clif., USA). Serum ECP levels were mesured using commercilly vilble fluoroimmunossy kit (Phrmci ECP UniCAP System FEIA, Phrmci Dignostics, Uppsl, Sweden) which hd detection limit of <2.0 μg/l. Blood smple collection, serum preprtion nd serum ECP mesurement were performed ccording to the mnufcturer s instructions. Sttisticl Anlysis A smple size of 61 ws chieved by 80% power to detect difference of 0.35 between the null hypothesis correltion of 0.00 nd the lterntive hypothesis correltion of 0.35, using 2-sided hypothesis test with significnce level of 0.05 with PASS 12 (NCSS, LLC, Kysville, Uth, USA). Dt re presented s men ± SD. The vlues for FeNO, PC 20 methcholine, PC 20 AMP, PD 20 AMP, DRR, serum totl IgE, serum ECP nd blood eosinophil counts were log trnsformed before sttisticl nlysis. The mximl percentge fll in FEV 1 from bseline fter dministrtion of AMP ws clculted. The mens or frequencies were compred between the 2 groups using the 2-tiled Student t test or the χ 2 test. Reltionships between FeNO nd serum IgE level/eosinophil count/ecp level were mesured using the Person correltion coefficients. Multivrible liner regression models were used to nlyze the ssocition between FeNO nd PC 20 methcholine/pc20 AMP, fter djustment for ge, sex, serum totl IgE level nd blood eosinophil count. The coefficient of determintion ( R 2 ) nd regression coefficient (β) were obtined from the nlyses. All sttisticl nlyses were performed using SPSS softwre (version 17; SPSS Inc., Chicgo, Ill., USA). A p vlue of <0.05 ws considered sttisticlly significnt. Results The clinicl chrcteristics of the steroid-treted nd steroid-nïve groups of children with mild to moderte, persistent sthm re shown in tble 1. There ws no significnt difference in ge, sex rtio nd blood eosinophil count between the 2 groups. The geometric men of serum totl IgE levels of the steroid-treted group [249.6 IU/ml ( )] ws not significntly different from those of the steroid-nïve group [144.0 IU/ml ( ), p = 0.290]. However, the prevlence of topy showed mrginl significnce between the 2 groups (90.9 vs. 70.8%, p = 0.050). The geometric men of FeNO levels ws significntly higher in the steroid-treted group thn in the steroid-nïve group [54.1 ppb ( ) vs ppb ( ), p = 0.006]. The pulmonry function prmeters of the 2 groups re presented in tble 2. There ws no significnt difference in bseline pulmonry function prmeters nd PC 20 methcholine between the 2 groups. The geometric men (rnge of 1 SD) of PC 20 AMP ws significntly lower in the Associtions of FeNO nd AHR to AMP 109

4 Tble 2. Pulmonry function prmeters nd AHR in steroid-treted nd steroid-nïve sthmtic children Prmeters Steroid-treted (n = 11) Steroid-nïve (n = 48) Totl (n = 59) FEV 1, % predicted 96.5± ± ±14.9 FVC, % predicted 96.1± ± ±11.9 FEV 1 /FVC, % 85.0± ± ±7.6 PC 20 methcholine, mg/ml 2.01 ( ) 4.31 ( ) 3.4 ( ) PC 20 AMP, mg/ml, b 18.5 ( ) 89.1 ( ) 66.4 ( ) PD 20 AMP, mg % fll in FEV 1 (AMP), % DRR, % fll/mg, b Geometric men (rnge of 1 SD). b p < ( ) 25.5± ( ) 63.9 (8.36±489.3) 23.7± ( ) 50.9 ( ) 24.0± ( ) 1,000 R 2 = 0.188, p = ,000 R 2 = 0.341, p = FeNO (ppb) FeNO (ppb) Methcholine PC 20 (mg/ml) b ,000 AMP PC 20 (mg/ml) Fig. 1. Assocition between FeNO nd PC 20 methcholine. b Assocition between FeNO nd PC 20 AMP. Open circles indicte steroid-nïve sthmtics nd closed circles indicte steroid-treted sthmtics. steroid-treted group thn in the steroid-nïve group [18.5 mg/ml ( ) vs mg/ml ( ), p = 0.042]. The men percent fll in FEV 1 ws not significntly different between the steroid-treted group nd the steroid-nïve group (25.5 ± 9.4 vs ± 11.8%). There were 17 subjects who did not show positive response fter inhltion of the mximl concentrtion of AMP (400 mg/ml). The DRR ws significntly higher in the steroidtreted group thn in the steroid-nïve group [1.56% fll/ mg ( ) vs. 0.30% fll/mg ( ), p = 0.046]. In the univrite nlysis, FeNO ws significntly ssocited with both PC 20 AMP ( R 2 = 0.341, p < 0.001) nd PC 20 methcholine ( R 2 = 0.188, p = 0.001; fig. 1 ). However, in the multivrible liner regression model, fter controlling for ge, sex, serum totl IgE nd blood eosinophil count, FeNO ws independently ssocited with PC 20 AMP (β = 1.98, p = 0.001), but not with PC 20 methcholine (β = 0.87, p = 0.081; tble 3 ). A significnt ssocition between FeNO nd PC 20 AMP ws found in the steroid-nïve group (β = 2.48, p = 0.001), but not in 110 Perznowski/Yoo

5 Tble 3. Multivrible models for ssocitions between FeNO nd PC 20 methcholine/pc 20 AMP in the study subjects PC 20 methcholine PC 20 AMP β 95% CI p vlue β 95% CI p vlue FeNO, ppb to to Age to to Sex to to Totl IgE, IU/ml to to Eosinophils, /μl to to Tble 4. Multivrible models for ssocitions between FeNO nd PC 20 methcholine/pc 20 AMP in the steroid-treted group PC 20 methcholine PC 20 AMP β 95% CI p vlue β 95% CI p vlue FeNO, ppb to to Age to to Sex to to Totl IgE, IU/ml to to Eosinophils, /μl to to Tble 5. Multivrible models for ssocitions between FeNO nd PC 20 methcholine/pc 20 AMP in the steroid-nïve group PC 20 methcholine PC 20 AMP β 95% CI p vlue β 95% CI p vlue FeNO, ppb to to Age to to Sex to to Totl IgE, IU/ml to to Eosinophils, /μl to to the steroid-treted group (β = 0.88, p = 0.463) ( tbles 4, 5 ). There ws positive reltionship of FeNO concentrtions to blood eosinophil mrkers ( r = 0.34, p = 0.009) nd serum ECP levels ( r = 0.29, p = 0.027). FeNO concentrtions were lso positively ssocited with serum totl IgE concentrtions ( r = 0.47, p = 0.001), but not with FEV 1 / FVC ( r = 0.11, p = 0.402). Discussion In this study, we demonstrted tht FeNO concentrtions were more closely ssocited with AHR to AMP thn with AHR to methcholine in sthmtic children. A significnt ssocition between FeNO nd AHR to AMP ws observed in the steroid-nïve group, but not in the steroid-treted group. FeNO ws positively correlted with serum totl IgE nd blood eosinophil mrkers but not with bseline pulmonry function prmeters. Airwy inflmmtion nd AHR re two min chrcteristic fetures of sthm. AHR, n exggerted response to stimuli, cn be mesured using direct (methcholine) nd indirect (AMP) stimuli. There is incresing interest in indirect testing by relesing meditors becuse AHR is n indictor of the presence of irwy inflmmtion [3]. AMP, unlike methcholine, does not ct s bronchoconstrictor gent by cting directly on irwy smooth muscle, but cts rther vi meditors re- Associtions of FeNO nd AHR to AMP 111

6 lesed from ctivted mst cells [10]. Inhled AMP cts through A2B receptors nd is thought to result in bronchoconstriction vi mst-cell degrnultion tht releses histmine nd other cytokines [16]. Inhled AMP incresed the eosinophil count in the lungs in sensitized niml model [17]. Recruited eosinophils my reflect n overll incresed inflmmtory ctivity, incresing the production of cytokines tht stimulte mst cells directly nd indirectly. Activtion of mst cells cn lso occur through the IgE-dependent pthwy, resulting in the relese of meditors tht re responsible for n influx of eosinophils in the sputum [16]. Therefore, AHR to AMP reflects underlying irwy inflmmtion more specificlly [18]. A proportion of our subjects responded to methcholine, but not to AMP. AHR to direct stimuli my be present in the bsence of AHR to indirect stimuli. This indictes tht irwy dmge or irflow limittion without irwy inflmmtion my occur in sthmtic irwys [19]. Eleven subjects were tking ICSs prior to testing. In current sthm, irwy inflmmtion is the centrl bnormlity leding to AHR, nd treting irwy inflmmtion with ICSs my hve greter effects on indirect chllenges thn on direct chllenges [8, 20]. Menwhile, irwy inflmmtion cn be noninvsively ssessed with FeNO. NO is synthesized by vrious cells through the oxidtion of L-rginine by NO synthse. In sthm, inducible NO synthse expression is upregulted by ctivting STAT-6 through interleukins 4 nd 13 produced by T helper 2 cells, mst cells nd eosinophils [4, 21]. Thus, eno directly signls the proinflmmtory cytokine mechnisms of centrl importnce in the pthophysiology of sthm. The levels of eno prllel the inflmmtory process in the sthmtic irwy, nd tretment with ICSs results in mrked reduction in the levels of eno [22]. On the other hnd, the degree of IgE sensitiztion is lso thought to be relted to eno, lthough eosinophilic inflmmtion is commonly considered n importnt contributor [21, 23]. Atopic inflmmtion is chrcterized by the recruitment nd ctivtion of eosinophils; therefore, both topy nd eosinophils might be importnt determinnts of NO production [21]. Reltionships between FeNO nd indirect AHR hve been shown in numerous studies [24 26]. Both FeNO nd indirect AHR re incresed in sthm; however, not ll sthm phenotypes re chrcterized by significnt reltionships between these 2 prmeters. Previous studies hve shown tht FeNO is significntly ssocited with indirect AHR in topic subjects, but not in nontopic subjects [24, 25]. This ssocition is, in prt, due to the differences in inflmmtory profiles or cytokine production between topic nd nontopic sthmtics. It is suggested tht both FeNO levels nd the response to AMP chllenge depend on the degree of llergic irwy inflmmtion in sthmtics [21, 24]. In this study, we demonstrted tht FeNO ws more closely ssocited with AHR to AMP thn with AHR to methcholine. Consistent with these results, Porsbjerg et l. [5] showed tht FeNO hs stronger correltion with indirect stimuli (mnnitol) thn with direct stimuli (methcholine). A previous study showed tht n improvement in PC 20 AMP but not in PC 20 methcholine fter 2 weeks of steroid therpy is ssocited with decrese in FeNO, indicting tht AHR to AMP is more closely ssocited with irwy inflmmtion [27]. When we conducted subnlyses on the steroid-treted nd steroid-nïve groups, significnt reltionships were observed only in the steroid-nïve group. Other studies hve shown no correltions between FeNO nd indirect AHR in steroid-treted subjects, supporting the notion tht the inflmmtory response in the irwys could be ttenuted by ICSs [8]. Severl points must be considered. Of the 59 subjects tht we studied, 11 were tking ICSs prior to enrollment. Treting with ICSs my ttenute inflmmtion, my possibly inhibit irwy sensitivity to indirect stimuli nd, thus, impct reltionships between the two. ICS wshout for 1 week my not hve been sufficient in our subjects. Another point to be considered is tht we ttempted to exclude current rhinitis ptients going by their symptoms. The bsence of rhinitis symptoms within the lst 12 months does not lwys exclude the possibility of llergic rhinitis. In summry, we found tht irwy inflmmtion, s ssessed by using eno, ws more closely ssocited with AMP responsiveness thn with methcholine responsiveness in steroid-nïve sthmtic children. Our results suggest tht FeNO my better reflect bronchil responsiveness when ssessed by indirect chllenge thn by direct chllenge. Acknowledgements This study ws supported by grnts from the Environmentl Helth Center for Childhood Asthm, Ministry of Environment, Kore, the Koren Helth Technology R&D Project, Ministry of Helth & Welfre, Kore (A110663) nd the Bsic Science Reserch Progrm through the Ntionl Reserch Foundtion of Kore, Ministry of Eduction, Science nd Technology, Kore (2012R1A1A ). 112 Perznowski/Yoo

7 References 1 Ntionl Hert, Lung, nd Blood Institute: Definition, pthophysiology nd pthogenesis of sthm, nd nturl history of sthm; in: Expert Pnel Report 3: Guidelines for the Dignosis nd Mngement of Asthm (NIH publiction No ). Bethesd, US Deprtment of Helth nd Humn Services, Hrgreve FE, Dolovich J, O Byrne PM, Rmsdle EH, Dniel EE: The origin of irwy hyperresponsiveness. J Allergy Clin Immunol 1986; 78: de Meer G, Mrks GB, Postm DS: Direct or indirect stimuli for bronchil chllenge testing: wht is the relevnce for sthm epidemiology? Clin Exp Allergy 2004; 34: Ludviksdottir D, Dimnt Z, Alving K, Bjermer L, Mlinovschi A: Clinicl spects of using exhled NO in sthm dignosis nd mngement. Clin Respir J 2012; 6: Porsbjerg C, Brnnn JD, Anderson SD, Bcker V: Reltionships between irwy responsiveness to methcholine nd mrkers of irwy inflmmtion, pek flow vribility nd qulity of life in sthm ptients. Clin Exp Allergy 2007; 38: Berkmn N, Avitl A, Breuer R, Brdch E, Springer C, Godfrey S: Exhled nitric oxide in the dignosis of sthm: comprison with bronchil provoction tests. Thorx 2005; 60: Vn den Berge M, Meijer RJ, Kerstjens HA, de Reus DM, Koeter GH, Kuffmn HF, Postm DS: PC 20 denosine 5-monophosphte is more closely ssocited with irwy inflmmtion in sthm thn PC 20 methcholine. Am J Respir Crit Cre Med 2001; 163: Leuppi JD, Slome CM, Jenkins CR, Koskel H, Brnnn JD, Anderson SD, Andersson M, Chn HK, Woolcock AJ: Mrkers of irwy inflmmtion nd irwy hyperresponsiveness in ptients with well-controlled sthm. Eur Respir J 2001; 18: Linhres D, Jcinto T, Pereir AM, Fonsec JA: Effects of topy nd rhinitis on exhled nitric oxide vlue systemtic review. Clin Trnsl Allergy 2011; 1: Spicuzz L, Polos R: The role of denosine s novel bronchoprovocnt in sthm. Curr Opin Allergy Clin Immunol 2003; 3: Ntionl Asthm Eduction nd Prevention Progrm: Expert Pnel Report 2: Guidelines for the Dignosis nd Mngement of Asthm (NIH publiction No ). Bethesd, US Deprtment of Helth nd Humn Services, Americn Thorcic Society: Stndrdiztion of spirometry, 1984 updte. Am J Respir Crit Cre Med 1995; 152: Chi H, Frr RS, Froehlich LA, Mthison DA, McLen JA, Rosenthl RR, Sheffer AL, Spector SL, Townley RG: Stndrdiztion of bronchil inhltion chllenge procedures. J Allergy Clin Immunol 1975; 56: Joos GF, O Connor B, Anderson SD, Chung F, Cockcroft DW, Dhlen B, DiMri G, Foresi A, Hrgreve FE, Holgte ST, Inmn M, Lotvll J, Mgnussen H, Polos R, Postm DS, Riedler J; ERS Tsk Force: Indirect irwy chllenges. Eur Respir J 2003; 21: Dweik RA, Boggs PB, Erzurum SC, Irvin CG, Leigh MW, Lundberg JO, Olin AC, Plummer AL, Tylor DR: An officil ATS clinicl prctice guideline: interprettion of exhled nitric oxide levels (FENO) for clinicl pplictions. Am J Respir Crit Cre Med 2011; 184: Vss G, Horvth I: Adenosine nd denosine receptors in the pthomechnism nd tretment of respirtory diseses. Curr Med Chem 2008; 15: Smith N, Johnson FJ: Erly- nd lte-phse bronchoconstriction, irwy hyper-rectivity nd cell influx into the lungs, fter 5 -denosine monophosphte inhltion: comprison with ovlbumin. Clin Exp Allergy 2005; 35: Anderson SD: Provoctive chllenges to help dignose nd monitor sthm: exercise, methcholine, denosine, nd mnnitol. Curr Opin Pulm Med 2008; 14: Cockcroft DW: Direct chllenge tests: irwy hyperresponsiveness in sthm: its mesurement nd clinicl significnce. Chest 2010; 138: 18S 24S. 20 O Connor BJ, Ridge SM, Brnes PJ, Fuller RW: Greter effect of inhled budesonide on denosine 5 -monophosphte-induced thn on sodium-metbisulfite-induced bronchoconstriction in sthm. Am Rev Respir Dis 1992; 146: Alving K, Mlinovschi A: Bsic spects of exhled nitric oxide. Eur Respir Mon 2010; 49: vn Rensen EL, Strthof KC, Veselic-Chrvt MA, Zwindermn AH, Bel EH, Sterk PJ: Effect of inhled steroids on irwy hyperresponsiveness, sputum eosinophils, nd exhled nitric oxide levels in ptients with sthm. Thorx 1999; 54: Jng WN, Prk IS, Choi CH, Buer S, Hrmin S, Seo SC, Choi IS, Choung JT, Yoo Y: Reltionships between exhled nitric oxide nd topy profiles in children with sthm. Allergy Asthm Immunol Res 2013; 5: Rouhos A, Ekroos H, Krjlinen J, Srn S, Sovijrvi AR: Exhled nitric oxide nd exercise-induced bronchoconstriction in young mle conscripts: ssocition only in topics. Allergy 2005; 60: Grzelewski T, Grzelewsk A, Mjk P, Stelmch W, Kowlsk A, Stelmch R, Jns A, Stelmch I: Frctionl exhled nitric oxide (FeNO) my predict exercise-induced bronchoconstriction (EIB) in schoolchildren with topic sthm. Nitric Oxide 2012; 27: Cown DC, Hewitt RS, Cown JO, Plmy R, Willimson A, Lucs SJ, Murrell CJ, Thoms KN, Tylor DR: Exercise-induced wheeze: frction of exhled nitric oxide-directed mngement. Respirology 2010; 15: vn den Berge M, Kerstjens HA, Meijer RJ, de Reus DM, Koeter GH, Kuffmn HF, Postm DS: Corticosteroid-induced improvement in the PC 20 of denosine monophosphte is more closely ssocited with reduction in irwy inflmmtion thn improvement in the PC 20 methcholine. Am J Respir Crit Cre Med 2001; 164: Associtions of FeNO nd AHR to AMP 113