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Lower exhaled nitric oxide in infants with Cystic Fibrosis compared to healthy controls

  • Insa Korten
    Affiliations
    Paediatric Respiratory Medicine, Department of Pediatrics, Inselspital, Bern University Hospital, University of Bern, Switzerland

    Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland

    University Children's Hospital (UKBB), Basel, Switzerland
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  • Margot Liechti
    Affiliations
    Paediatric Respiratory Medicine, Department of Pediatrics, Inselspital, Bern University Hospital, University of Bern, Switzerland
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  • Florian Singer
    Affiliations
    Paediatric Respiratory Medicine, Department of Pediatrics, Inselspital, Bern University Hospital, University of Bern, Switzerland

    Division of Respiratory Medicine, University Children's Hospital Zurich, Switzerland
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  • Gaudenz Hafen
    Affiliations
    Department of Paediatrics, Respiratory Unit, Lausanne University Hospital, Lausanne, Switzerland
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  • Isabelle Rochat
    Affiliations
    Department of Paediatrics, Respiratory Unit, Lausanne University Hospital, Lausanne, Switzerland
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  • Pinelopi Anagnostopoulou
    Affiliations
    Paediatric Respiratory Medicine, Department of Pediatrics, Inselspital, Bern University Hospital, University of Bern, Switzerland
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  • Dominik Müller-Suter
    Affiliations
    Division of Respiratory Medicine, Children's Hospital Aarau, Switzerland
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  • Jakob Usemann
    Affiliations
    Paediatric Respiratory Medicine, Department of Pediatrics, Inselspital, Bern University Hospital, University of Bern, Switzerland

    University Children's Hospital (UKBB), Basel, Switzerland
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  • Alexander Moeller
    Affiliations
    Division of Respiratory Medicine, University Children's Hospital Zurich, Switzerland
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  • Urs Frey
    Affiliations
    University Children's Hospital (UKBB), Basel, Switzerland
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  • Philipp Latzin
    Affiliations
    Paediatric Respiratory Medicine, Department of Pediatrics, Inselspital, Bern University Hospital, University of Bern, Switzerland

    University Children's Hospital (UKBB), Basel, Switzerland
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  • Carmen Casaulta
    Correspondence
    Corresponding author at: Division of Respiratory Medicine, Department of Pediatrics, Inselspital and University of Bern, Freiburgstrasse 31, 3010 Bern, Switzerland.
    Affiliations
    Paediatric Respiratory Medicine, Department of Pediatrics, Inselspital, Bern University Hospital, University of Bern, Switzerland
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  • for the SCILD and BILD study group
    Author Footnotes
    1 Swiss Cystic Fibrosis Infant Lug Development (SCILD) cohort, current study group: Jürg Barben, MD, St. Gallen; Carmen Casaulta, MD, Bern; Gaudenz Hafen, MD, Lausanne; Elisabeth Kieninger, MD, PhD, Bern; Insa Korten, MD, Bern; Philipp Latzin, MD, PhD, Bern; Alexander Moeller, MD, Zürich; Anne Mornand, MD, Geneva; Dominik Müller-Suter, MD, Aarau; Nicolas Regamey, MD, PhD, Lucerne; Isabelle Rochat MD, Lausanne; Florian Singer, MD, PhD, Zürich; Daniel Trachsel, MD, Basel; Sophie Yammine, MD, PhD, Bern; Maura Zanolari, MD, Bellinzona.
    ,
    Author Footnotes
    2 Basel Bern Infant Lung Development (BILD) cohort, current study group: Pinelopi Anagnostopoulou, MD, Bern; Urs Frey, MD, PhD, Basel; Oliver Fuchs, MD, PhD, Bern; Olga Gorlanova, MD, Basel; Philipp Latzin, MD, PhD, Bern; Ines Mack, MD, Basel; Elena Proietti, MD, PhD, Zurich; Anne Schmidt, MD, PhD, London; Jakob Usemann, MD, Basel.
  • Author Footnotes
    1 Swiss Cystic Fibrosis Infant Lug Development (SCILD) cohort, current study group: Jürg Barben, MD, St. Gallen; Carmen Casaulta, MD, Bern; Gaudenz Hafen, MD, Lausanne; Elisabeth Kieninger, MD, PhD, Bern; Insa Korten, MD, Bern; Philipp Latzin, MD, PhD, Bern; Alexander Moeller, MD, Zürich; Anne Mornand, MD, Geneva; Dominik Müller-Suter, MD, Aarau; Nicolas Regamey, MD, PhD, Lucerne; Isabelle Rochat MD, Lausanne; Florian Singer, MD, PhD, Zürich; Daniel Trachsel, MD, Basel; Sophie Yammine, MD, PhD, Bern; Maura Zanolari, MD, Bellinzona.
    2 Basel Bern Infant Lung Development (BILD) cohort, current study group: Pinelopi Anagnostopoulou, MD, Bern; Urs Frey, MD, PhD, Basel; Oliver Fuchs, MD, PhD, Bern; Olga Gorlanova, MD, Basel; Philipp Latzin, MD, PhD, Bern; Ines Mack, MD, Basel; Elena Proietti, MD, PhD, Zurich; Anne Schmidt, MD, PhD, London; Jakob Usemann, MD, Basel.
Open ArchivePublished:July 14, 2017DOI:https://doi.org/10.1016/j.jcf.2017.05.005

      Abstract

      Exhaled nitric oxide (FENO) is a well-known, non-invasive airway biomarker. In patients with Cystic Fibrosis (CF) FENO is decreased. To understand if reduced FENO is primary related to Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) dysfunction or an epiphenomenon of chronic inflammation, we measured FENO in 34 infants with CF prior to clinical symptoms and in 68 healthy controls. FENO was lower in CF compared to controls (p = 0.0006) and the effect was more pronounced in CF infants without residual CFTR function (p < 0.0001). This suggests that FENO is reduced in CF early in life, possibly associated with underlying CFTR dysfunction.

      1. Introduction

      The fractional concentration of exhaled nitric oxide (FENO) is a well-known biomarker for airway inflammation and elevated in a number of inflammatory disorders of the lung [
      • Barnes P.J.
      • Dweik R.A.
      • Gelb A.F.
      • Gibson P.G.
      • George S.C.
      • Grasemann H.
      • et al.
      Exhaled nitric oxide in pulmonary diseases: a comprehensive review.
      ]. In patients with Cystic Fibrosis (CF) and predominantly neutrophilic airway inflammation, FENO is decreased [
      • de Winter-de Groot K.M.
      • van der Ent C.K.
      Nitric oxide in cystic fibrosis.
      ,
      • Robroeks C.M.
      • Rosias P.P.
      • van Vliet D.
      • Jobsis Q.
      • Yntema J.B.
      • Brackel H.J.
      • et al.
      Biomarkers in exhaled breath condensate indicate presence and severity of cystic fibrosis in children.
      ,
      • Keen C.
      • Gustafsson P.
      • Lindblad A.
      • Wennergren G.
      • Olin A.C.
      Low levels of exhaled nitric oxide are associated with impaired lung function in cystic fibrosis.
      ]. The following underlying causes have been discussed: (i) reduced NO synthase isoenzyme (NOS) expression, (ii) lack of NOS substrates, (iii) reduced NOS function through endogenous inhibitors (e.g. methylated arginine derivatives and polyamines), (iv) NO decomposition by bacterial reductases or neutrophilic myeloperoxidase, or (v) impaired NO diffusion through viscous mucus [
      • Barnes P.J.
      • Dweik R.A.
      • Gelb A.F.
      • Gibson P.G.
      • George S.C.
      • Grasemann H.
      • et al.
      Exhaled nitric oxide in pulmonary diseases: a comprehensive review.
      ,
      • Grasemann H.
      • Schwiertz R.
      • Grasemann C.
      • Vester U.
      • Racke K.
      • Ratjen F.
      Decreased systemic bioavailability of L-arginine in patients with cystic fibrosis.
      ,
      • Malinovschi A.
      • Ludviksdottir D.
      • Tufvesson E.
      • Rolla G.
      • Bjermer L.
      • Alving K.
      • et al.
      Application of nitric oxide measurements in clinical conditions beyond asthma.
      ,
      • Chapman A.L.
      • Morrissey B.M.
      • Vasu V.T.
      • Juarez M.M.
      • Houghton J.S.
      • Li C.S.
      • et al.
      Myeloperoxidase-dependent oxidative metabolism of nitric oxide in the cystic fibrosis airway.
      ,
      • Grasemann H.
      • Al-Saleh S.
      • Scott J.A.
      • Shehnaz D.
      • Mehl A.
      • Amin R.
      • et al.
      Asymmetric dimethylarginine contributes to airway nitric oxide deficiency in patients with cystic fibrosis.
      ,
      • Grasemann H.
      • Shehnaz D.
      • Enomoto M.
      • Leadley M.
      • Belik J.
      • Ratjen F.
      L-ornithine derived polyamines in cystic fibrosis airways.
      ]. Reduced levels of NO or NOS have been related to a number of adverse effects, such as increased airway narrowing, reduced ciliary motility and susceptibility to infections [
      • de Winter-de Groot K.M.
      • van der Ent C.K.
      Nitric oxide in cystic fibrosis.
      ,
      • Jiao J.
      • Wang H.
      • Lou W.
      • Jin S.
      • Fan E.
      • Li Y.
      • et al.
      Regulation of ciliary beat frequency by the nitric oxide signaling pathway in mouse nasal and tracheal epithelial cells.
      ].
      Absent or residual function of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein in CF patients results in insufficient NOS induction, which however appears reversible [
      • Steagall W.K.
      • Elmer H.L.
      • Brady K.G.
      • Kelley T.J.
      Cystic fibrosis transmembrane conductance regulator-dependent regulation of epithelial inducible nitric oxide synthase expression.
      ]. Reduced FENO normalizes in patients with CF after treatment with Ivacaftor, one of the first approved CFTR-targeting drugs for certain gating mutations [
      • Grasemann H.
      • Gonska T.
      • Avolio J.
      • Klingel M.
      • Tullis E.
      • Ratjen F.
      Effect of ivacaftor therapy on exhaled nitric oxide in patients with cystic fibrosis.
      ]. This suggests that decreased FENO in CF is not an epiphenomenon of chronic inflammation or infection, but might be reduced in CF airways early in life, possibly associated with the defect in CFTR.
      In order to understand whether reduced FENO in CF airways can be detected early in life, FENO measurements in a very young age and before the onset of first apparent infections need to be explored. We thus measured FENO in infants with CF and healthy controls at five to twelve weeks of age.

      2. Methods

      We enrolled infants with CF diagnosed by new-born screening and contemporary healthy infants aged five to twelve weeks, matched 1:2 based on season of birth and sex, from two ongoing birth cohort studies, the Swiss Cystic Fibrosis Infant Lung Development (SCILD) cohort [
      • Mika M.
      • Korten I.
      • Qi W.
      • Regamey N.
      • Frey U.
      • Casaulta C.
      • et al.
      The nasal microbiota in infants with cystic fibrosis in the first year of life: a prospective cohort study.
      ] and the Basel Bern Infant Lung development (BILD) cohort [
      • Fuchs O.
      • Latzin P.
      • Kuehni C.E.
      • Frey U.
      Cohort profile: the Bern infant lung development cohort.
      ] born between 2011 and 2015. Exclusion criterion was a history of respiratory symptoms suggesting upper- or lower respiratory tract infection prior to the study. Clinical examination was normal in all infants. Infants with asymptomatic bacterial colonization were not excluded (n = 5). All lung function measurements were performed in the study centres Basel (n = 1) and Bern, and infants were between five and twelve weeks of age. FENO measurements were performed between 9:30 a.m. and 3.30 p.m. during tidal breathing, in regular, quiet sleep as previously described [
      • American Thoracic S
      • European Respiratory S
      ATS/ERS recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide, 2005.
      ,
      • Frey U.
      • Stocks J.
      • Sly P.
      • Bates J.
      Specifications for signal processing and data handling used for infant pulmonary function testing. European Respiratory Society/American Thoracic Society.
      ,
      • Latzin P.
      • Kuehni C.E.
      • Baldwin D.N.
      • Roiha H.L.
      • Casaulta C.
      • Frey U.
      Elevated exhaled nitric oxide in newborns of atopic mothers precedes respiratory symptoms.
      ,
      • Hall G.L.
      • Reinmann B.
      • Wildhaber J.H.
      • Frey U.
      Tidal exhaled nitric oxide in healthy, unsedated newborn infants with prenatal tobacco exposure.
      ]. In brief, FENO measurements were obtained online using a tight fitting face mask (covering nostrils and mouth) with a rapid response chemiluminescence analyser, a previously validated device (CLD 77; Eco Medics AG, Duernten, Switzerland; analysis software: WBreath 3.28, ndd, Zurich, Switzerland). To avoid contamination by ambient NO, we used NO-free air for inspiration. FENO was sampled breath-by-breath during the third quartile of expiration and averaged across 100 consecutive breaths. This was validated to be the measurement set up with the least variability of FENO [
      • Hall G.L.
      • Reinmann B.
      • Wildhaber J.H.
      • Frey U.
      Tidal exhaled nitric oxide in healthy, unsedated newborn infants with prenatal tobacco exposure.
      ]. As FENO is flow dependent, correcting for expiratory flow was performed as described previously [
      • Latzin P.
      • Kuehni C.E.
      • Baldwin D.N.
      • Roiha H.L.
      • Casaulta C.
      • Frey U.
      Elevated exhaled nitric oxide in newborns of atopic mothers precedes respiratory symptoms.
      ,
      • Hall G.L.
      • Reinmann B.
      • Wildhaber J.H.
      • Frey U.
      Tidal exhaled nitric oxide in healthy, unsedated newborn infants with prenatal tobacco exposure.
      ]. Results are thus presented for both FENO and V′NO (NO output = FENO multiplied with corresponding expiratory flow), which were the primary outcomes. Additionally, flow and respiratory rate were secondary outcomes. We compared NO levels between healthy controls and infants with CF. Subsequently, patients were stratified into CFTR groups with (i) no residual function (two copies of class I and/or class II mutations), (ii) residual function (infants with at least one copy of a class III-VI mutation, and (iii) unclassified mutations. As FENO data were not perfectly normally distributed, we illustrate average estimates in both mean and median. We performed both Wilcoxon-Man-Whitney test and linear regression after log transformation of variables and adjusted for possible confounders (age, sex, maternal atopy, smoking during pregnancy and time of day of measurement). The study was performed in Basel and Bern and approved by the Ethics committees Basel and Bern, Switzerland. Informed consent was obtained from the parents.

      3. Results

      We analysed 102 measurements in 34 infants with CF and 68 healthy controls. For anthropometric data and results of measurements, see Table 1.
      Table 1Study population and FENO measurements.
      Healthy infants (n = 68)Infants with CF (n = 34)
      Gender, female33 (49%)12 (35%)
      Age at study date, wk5 (+/−1.2)8 (+/−2.5)
      Gestational age, wk39.5 (+/−1.4)38.7(+/−1.7)
      Length at birth, cm50 (+/−2.3)50 (2.8)
      Weight at birth, g3400 (+/−567)3310 (+/−569)
      Weight at study date, g4430 (+/−485)4970 (+/−1413)
      CF mutation
      CF infants were grouped in 1: two known copies of class I and/or II mutations 2: at least one copy of a class III-VI mutation 3. >=1 mutation not classified or unknown mutation. All children in group #3 however had two copies of disease causing mutations.
      :
       No residual CFTR function (class I and/or II)19 (56%)
       Residual CFTR function (class I/II and III-VI)7 (21%)
       Unknown CFTR function8 (23%)
       FENO ppb17.0 (+/−5.0)13.7 (+/−5.3)
       FENO ppb median (IQR)16.4 (13.8–18.7)12.2 (10.2–16.3)
       V′NO nl/min46.3 (+/−11.5)38.5 (+/−15.7)
       Flow exp. ml/s47.8 (+/−11.0)48.2 (+/−11.1)
       Respiratory Rate (1/min)43.1 (+/−8.5)43.0 (+/−8.5)
      Results are displayed in number (%) and mean (+/− SD) if not stated otherwise.
      wk = weeks.
      a CF infants were grouped in 1: two known copies of class I and/or II mutations 2: at least one copy of a class III-VI mutation 3. > = 1 mutation not classified or unknown mutation. All children in group #3 however had two copies of disease causing mutations.
      Significantly lower FENO and V′NO values were found in infants with CF compared to healthy controls (median difference for FENO 4.25 ppb, p = 0.0006 and for V′NO 12.15 nl/min, p = 0.002). Flow and respiratory rate were comparable between groups, thus lower FENO values were not due to differences in breathing patterns. The stratified analysis based on CFTR function revealed that in infants without residual CFTR function, FENO levels were even lower compared to healthy controls (median difference 4.4 ppb, p < 0.0001; V′NO 13.25 nl/min, p = 0.0001), see Fig. 1.
      Fig. 1
      Fig. 1(A) FENO and (B) V′NO measurements in healthy infants (n = 68) and infant with CF with (I) no CFTR residual function (n = 19), (II) with CFTR residual function (n = 7) and (III) unknown CFTR function (n = 8). Each dot symbolizes one infant. Lines indicate mean +/−SD.
      FENO levels between infants with and without residual CFTR function also differed significantly (p = 0.005), however numbers were low. All findings were confirmed when analysed in linear regression and after adjustment for confounders, assuring robustness of our results (see Table 2A, Table 2B).
      Table 2AComparison of lung function measurements between healthy infants and CF infants.
      Unadjusted modelAdjusted model
      Coef95% CIp-valueCoef95% CIp-value
      FENO (log)0.24[−0.37, −0.11]<0.00010.27[−0.47, −0.07]0.01
      V′NO (log)0.23[−0.35, −0.10]0.0010.24[−0.44, −0.05]0.014
      Flow (log)0.01[−0.09, 0.11]0.850.03[−0.13, 0.19]0.7
      Respiratory rate (log)0[−0.08, 0.08]0.970.03[−0.10, 0.16]0.6
      Linear regression model comparing measurements between healthy infants (n = 68) and infants with CF (n = 34), after log transformation of variables to obtain normal distribution. Baseline are healthy infants, adjusted model: adjusted for gender (reference category = female), age at measurement, maternal atopy (reference category = no atopy), smoking in pregnancy (reference category = non-smokers), and time of day of measurement (reference category = before 11:00 a.m.; 1 = 11:00–12.00 a.m.; 2 = 12:00 a.m.–3.30 p.m.), Coef = beta Coefficient; 95% CI = 95% Confidence interval.
      Table 2BComparison of lung function measurements between healthy infants and CF infants with class I/II mutation.
      Unadjusted modelAdjusted model
      Coef95% CIp-valueCoef95% CIp-value
      FENO (log)0.37[−0.52, −0.22]<0.00010.32[−0.54, −0.11]0.004
      V′NO (log)0.34[−0.48, −0.20]<0.00010.08[−0.39, −0.03]0.047
      Flow (log)0.02[−0.10, 0.15]0.730.13[−0.05, 0.31]0.2
      Respiratory rate (log)0.02[−0.07, 0.12]0.630.10[−0.04, 0.24]0.2
      Linear regression model comparing measurements between healthy infants (n = 68) and infants with CF and class I/II mutations (n = 19), after log transformation of variables to obtain normal distribution. Baseline are healthy infants, adjusted model: adjusted for gender (reference category = female), age at measurement, maternal atopy (reference category = no atopy), smoking in pregnancy (reference category = non-smokers), and time of day of measurement (reference category = before 11:00 a.m.; 1 = 11:00–12.00 a.m.; 2 = 12:00 a.m. – 3.30 p.m.), Coef = beta Coefficient; 95% CI = 95% Confidence interval.
      Five infants received antibiotic therapy prior or during measurements, indication was asymptomatic bacterial colonization (throat swab), but there was no association between therapy and FENO levels (results not shown).

      4. Discussion

      Exhaled FENO is reduced in young infants with CF and this effect is more pronounced in infants without residual CFTR function. Importantly, FENO measurements were performed prior to first respiratory symptoms, suggesting absence of significant lower airway disease in the majority of children. Data were also robust after adjusting for other known confounders such as atopy. Our results are in line with a previous preliminary study (five infants with CF) [
      • Elphick H.E.
      • Demoncheaux E.A.
      • Ritson S.
      • Higenbottam T.W.
      • Everard M.L.
      Exhaled nitric oxide is reduced in infants with cystic fibrosis.
      ] and support our hypothesis that FENO is reduced early in CF life and possibly associated with the underlying CFTR dysfunction. Low NOS expression seems related to CFTR mutation and residual function. Increase in FENO values following treatment with Ivacaftor in patients with a class III mutation [
      • Grasemann H.
      • Gonska T.
      • Avolio J.
      • Klingel M.
      • Tullis E.
      • Ratjen F.
      Effect of ivacaftor therapy on exhaled nitric oxide in patients with cystic fibrosis.
      ,
      • Kotha K.
      • Szczesniak R.D.
      • Naren A.P.
      • Fenchel M.C.
      • Duan L.L.
      • McPhail G.L.
      • et al.
      Concentration of fractional excretion of nitric oxide (FENO): a potential airway biomarker of restored CFTR function.
      ] further supports the hypothesis that FENO is a proxy of CFTR function. It may be an important player in progression of CF lung disease, as reduced FENO is associated with reduced neutrophil sequestration, bacteriostatic properties, and mucociliary transport [
      • de Winter-de Groot K.M.
      • van der Ent C.K.
      Nitric oxide in cystic fibrosis.
      ].
      Thus, FENO levels in early life might be of prognostic value for disease development. Furthermore, treatment response in FENO after CFTR modifier therapy might be a promising biomarker as the measurement is fast and easy [
      • Grasemann H.
      • Gonska T.
      • Avolio J.
      • Klingel M.
      • Tullis E.
      • Ratjen F.
      Effect of ivacaftor therapy on exhaled nitric oxide in patients with cystic fibrosis.
      ,
      • Gaston B.
      • Ratjen F.
      • Vaughan J.W.
      • Malhotra N.R.
      • Canady R.G.
      • Snyder A.H.
      • et al.
      Nitrogen redox balance in the cystic fibrosis airway: effects of antipseudomonal therapy.
      ]. It has been evaluated as appropriate measurement method in infancy in numerous studies [
      • American Thoracic S
      • European Respiratory S
      ATS/ERS recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide, 2005.
      ,
      • Frey U.
      • Stocks J.
      • Sly P.
      • Bates J.
      Specifications for signal processing and data handling used for infant pulmonary function testing. European Respiratory Society/American Thoracic Society.
      ,
      • Latzin P.
      • Kuehni C.E.
      • Baldwin D.N.
      • Roiha H.L.
      • Casaulta C.
      • Frey U.
      Elevated exhaled nitric oxide in newborns of atopic mothers precedes respiratory symptoms.
      ,
      • Hall G.L.
      • Reinmann B.
      • Wildhaber J.H.
      • Frey U.
      Tidal exhaled nitric oxide in healthy, unsedated newborn infants with prenatal tobacco exposure.
      ]. However, possible “contamination” of FENO with nasal NO should be considered. We assume this contribution negligible as nasal cavities are not fully developed in young infants [
      • Szolar D.
      • Preidler K.
      • Ranner G.
      • Braun H.
      • Kugler C.
      • Wolf G.
      • et al.
      The sphenoid sinus during childhood: establishment of normal developmental standards by MRI.
      ] and the end-expiratory portion of tidal breaths where FENO is measured, represents mainly alveolar gas [
      • Hall G.L.
      • Reinmann B.
      • Wildhaber J.H.
      • Frey U.
      Tidal exhaled nitric oxide in healthy, unsedated newborn infants with prenatal tobacco exposure.
      ].
      To summarize, our preliminary results of lower FENO in CF airways early after birth, and the association with underlying CFTR dysfunction, might open up a new chapter in the field of early FENO measurements in patients with CF.

      Disclosures

      No conflicts of interest, financial or otherwise, are declared by the author(s).

      Funding

      The study was funded by the Swiss National Science Foundation (SNF 320030_163311).
      The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

      Acknowledgements

      We appreciate the contribution of S. Lüscher, S. Schmid, G. Wirz, M. Graf and L. Beul-Beguin (Division of Respiratory Medicine, Department of Paediatrics, Inselspital and University of Bern, Bern, Switzerland) for data collection.

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