Bile Acids in Sputum and Increased Airway Inflammation in Patients With Cystic Fibrosis

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1 CHEST Bile Acids in Sputum and Increased Airway Inflammation in Patients With Cystic Fibrosis Original Research Ans Pauwels, MSc ; Ann Decraene, PhD ; Kathleen Blondeau, PhD ; Veerle Mertens, PhD ; Ricard Farre, PhD ; Marijke Proesmans, MD, PhD ; Pascal Van Bleyenbergh, MD ; Daniel Sifrim, MD, PhD ; and Lieven J. Dupont, MD, PhD CYSTIC FIBROSIS Background: Up to 80% of patients with cystic fibrosis (CF) may have increased gastroesophageal reflux and aspiration of duodenogastric contents into the lungs. We aimed to assess aspiration in patients with CF by measuring duodenogastric components in induced sputum and to investigate whether the presence of bile acids (BAs) in sputum was correlated with disease severity and markers of inflammation. Methods: In 41 patients with CF, 15 healthy volunteers, 29 patients with asthma, and 28 patients with chronic cough, sputum was obtained after inhalation of hypertonic saline. Sputum supernatant was tested for BA and neutrophil elastase. Spirometry and BMI were assessed on the day of sputum collection. Results: Two of 15 healthy patients (13%), eight of 29 patients (28%) with asthma, four of 28 patients (14%) with chronic cough, and 23 of 41 patients (56%) with CF had BA in sputum. BA concentrations were similar in patients who are positive for BA with genotype F508del homozygote, F508del heterozygote, and other CF mutations and were not related with BMI and age. Patients with CF with BA in sputum had a higher concentration of neutrophil elastase compared with patients without BA in sputum (31.25 [ ] mg/ml vs [ ] mg/ml, P,.05). There was a significant correlation between BA concentrations and dynamic lung volumes (FEV 1 % predicted [r520.53, P,.01], FVC% [ r520.59, P,.01]) as well as with number of days of antibiotic IV treatment ( r5 0.58, P,.01). Conclusions: BAs are present in the sputum of more than one-half of patients with CF, suggesting aspiration of duodenogastric contents. Aspiration of BA was associated with increased airway inflammation. In patients with BA aspiration, the levels of BA were clearly associated with the degree of lung function impairment as well as the need for IV antibiotic treatment. CHEST 2012; 141(6): Abbreviations: AZM 5 azithromycin; BA 5 bile acid; BALF 5 BAL fluid; CF 5 cystic fibrosis; GER 5 gastroesophageal reflux; NADH 5nicotinamide adenine dinucleotide hydrogen; NSE 5neutrophil elastase; PPI 5 proton pump inhibitor Gastroesophageal reflux (GER) in cystic fibrosis (CF) was first reported in and has a prevalence between 35% and 81%. 2-4 Several factors can favor GER in CF, including delayed gastric emptying, low basal lower esophageal sphincter pressure, and an increased number of transient lower esophageal sphincter relaxations. 5,6 Recently, 24-h impedance-ph monitoring, which makes it possible to measure both acid and non-acid reflux, has become the gold standard for GER detec- tion. With this technique, Blondeau et al 7 studied 33 adults with CF and found increased acid reflux in % and increased weakly acidic reflux in 21%. This group studied 24 children with CF with the same technique and found increased GER in 67%, with GER events being mainly acidic. 8 The highest concern about increased GER in CF is the alleged occurrence of aspiration of (duodeno)- gastric contents into the lungs, which may result in an exaggerated bronchial inflammatory reaction. Ledson et al 9 described tracheal acidification in four of 11 patients with CF, and recently bile acids (BAs) were found in the saliva of almost one-half of adults with CF and in 35% of children with CF. 8 Original Research

2 T he impact of increased GER/aspiration on lung function impairment in CF evolution remains unclear. Data from the European Epidemiologic Registry of CF show that patients with CF with GER have lower pulmonary function than those without GER. 10 Limiting factors to assess the relevance of GER and aspiration in CF are the invasiveness of reflux moni- toring and lack of sensitive and specific markers to screen for aspiration. Detection of duodenogastric markers in BAL fluid (BALF) is specific but not practical because bronchoscopy is invasive and not routinely performed for patients with CF. Measuring the presence of duodenogastric components in saliva is simple, but, if positive, only indicates regurgitation of gastric contents and suggests a higher risk for aspiration. In contrast, detection of duodenogastric components in sputum is noninvasive and can confirm aspiration into the airways. The aims of our study were to assess the extent of aspiration in CF by measuring BA in sputum and to correlate aspiration with markers of airway inflammation and disease severity. Study Design and Population Materials and Methods T his was a cross-sectional study comparing patients with CF to healthy control subjects, patients with asthma, and patients with chronic cough. Healthy volunteers were recruited from the hospital personnel; subjects with prior history of any respiratory disease, typical reflux symptoms, current smoking, and abnormal spirometry were excluded. Patients with asthma and patients with chronic cough were recruited at the outpatient clinic of the University Hospital Gasthuisberg. Patients with CF were recruited at the CF outpatient clinic of the University Hospital Gasthuisberg. Patients colonized with Burkholderia or with a current or recent (, 4 weeks) acute exacerbation, as defined by Fuchs et al, 11 or who underwent lung transplantation, were excluded. Clinical evaluation and sputum induction were performed consecutively on the same day, between 2:00 pm and 4:00 pm. Past and current medical history of the patients with CF was Manuscript received July 4, 2011; revision accepted November 1, Affiliations: fi From the Translational Research Center for Gastrointestinal Disorders (Ms Pauwels and Drs Blondeau, Mertens, Farre, and Sifrim), and Laboratory of Pneumology (Drs Decraene and Dupont), University of Leuven, Leuven, Belgium; Department of Pediatrics (Dr Proesmans), and Adult CF Center, Department of Respiratory Medicine (Drs Van Bleyenbergh and Dupont), University Hospital Gasthuisberg, Leuven, Belgium; and Barts and The London School of Medicine and Dentistry, Queen Mary (Dr Sifrim), University of London, London, England. Funding/Support: This work is supported by a grant from FWO Vlaanderen (Scientific Research Fund, Flanders, Belgium). Correspondence to: Lieven J. Dupont, MD, PhD, Department of Respiratory Medicine, 49 Herestraat, B-3000 Leuven, Belgium; lieven.dupont@uzleuven.be. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details. DOI: /chest obtained from hospital records, and patients were asked to fill in a reflux questionnaire specifi cally designed for the CF popula- tion. Spirometry was performed according to American Thoracic Society (ATS) criteria. 12 The Ethics Committee of the University Hospital Gasthuisberg approved the study and informed consent was obtained from all subjects or parents (CME UZ Leuven, S51060-B ). Sputum Induction and Processing In subjects with CF and healthy control subjects, sputum was induced with 5% NaCl (7-min inhalation via DeVilbiss nebulizer). 13 Before and after inhalation, subjects were asked to rinse their mouth thoroughly and cough deeply, expectorating a sputum sample. Sputum plugs were selected and separated from saliva and processed using an adapted protocol from Pizzichini et al. 14 Sputum Differential Cell Count Cytospins of sputum cells were obtained using a Shandon cytocentrifuge and were stained with May-Grünwald Giemsa (Diff- Quick stain kit; Medion Diagnostics). Cell differential count was performed on the total number of leukocytes without considering epithelial cells. Sputum Supernatant Analysis S putum supernatant was analyzed for BA using a commercially available enzymatic assay (Bioquant). In the presence of thionicotinamide adenine dinucleotide, the enzyme 3-a -hydroxysteroid dehydrogenase converts BA to 3-keto steroids and thio-nicotinamide adenine dinucleotide hydrogen (NADH). The reaction is reversible and 3- a-hydroxysteroid dehydrogenase can convert 3-keto steroids and thio-nadh to BA and thio-nicotinamide adenine dinucleotide. In the presence of excess NADH, the enzyme cycling occurs efficiently and the rate of formation of thio-nadh is determined by measuring specific change of absorbance at 405 nm. The lowest level of detection was 0.2 m mol/l. Neutrophil elastase (NSE) was measured in sputum supernatant with an enzymatic assay. As standard, commercially available human leukocyte elastase was used and the substrate was N-methoxysuccinyl-ALA-ALA-PRO-VAL-p-nitroanilide (Sigma). Absorbance was measured at 405 nm and lowest level of detection was 0.5 pg/ml. Statistical Analysis Data are presented as median (25th-75th percentile), unless stated otherwise. Deviations from Gaussian distribution were tested using the Kolmogorov-Smirnov test. Comparison between groups was done using the nonparametric Mann-Whitney U test. The Fisher exact test was used to compare categorical variables. Correlations were made using the Pearson test or the Spearman test, as appropriate. Statistical significance was considered when P,.05. Subjects Results Fifteen healthy volunteers (two men) with mean age of 27 years (range, 21-44), 29 patients with chronic cough (13 men) with mean age of 52 years (range, 14-79), 28 patients with asthma (11 men) with mean age of 54 (range, 16-75), and 41 patients with CF CHEST / 141 / 6 / JUNE,

3 (24 men) with a mean age of 24 years (range, 11-43) were recruited (Table 1 ). Thirty-three of 41 patients returned the reflux questionnaire; 12 (36%) had typical reflux symptoms. BAs in Sputum BAs were found in sputum of two of 15 healthy control subjects (13%), in four of 28 patients (14%) with chronic cough, in eight of 29 patients (28%) with asthma, and in 23 of 41 patients (56%) with CF ( Fig 1 ). Median concentration of BA in the 23 patients with CF was 2.1 ( ) mmol/l. BAs were found in sputum of three of eight children (38%) with CF compared with 20 of 33 adults (61%) with CF (P 5.2) (Table 2 ). In patients who are BA-positive with CF, BA concentrations did not differ between children and adults. Compared with adults, children had better lung function (FEV 1 % 89 [77-102] vs 65 [47-82], P5.002; FVC % 97 [88-105] vs 87 [72-92], P 5.04), lower NSE concentrations (8.01 [ ] m g/ml vs [ ] mg/ml, P5.005), and less sputum neutrophilia ( [ ] vs [ ], P 5.008). BA concentrations were similar in patients who were positive for BA with genotype F508del homozygote, F508del heterozygote, and other CF mutations (2.2 [ ] vs 2.0 [ ] vs 1.7 [ ], P 5 NS). BAs were present in sputum of five of 12 patients with, and in 14 of 21 patients without, typical reflux symptoms ( P5.3); the median concentration of BA did not differ between both groups. Fifteen patients (36%) were receiving azithromycin (AZM), 10 patients (24%) were receiving proton pump inhibitor (PPI) Table 1 Patient Characteristics Characteristics N 5 41 Mean age, y (range) 24 (11-43) Sex, male (female) 24 (17) Pancreatic insufficiency fi (sufficiency) 34 (7) Genotype F508del homozygous 19 F508del heterozygous 15 Other 7 Colonization with Pseudomonas aeruginosa 19 Staphylococcus aureus 21 No. of patients With CFRDM 7 Treated with PPI 10 Treated with AZM 15 Treated with oral bile salts 9 Median BMI, kg/m 2 (25%-75%) ( ) Median FEV 1 % predicted (25%-75%) 69 (50-88) Median FVC % predicted (25%-75%) 72 (50-87) AZM 5 azithromycin; CFRDM 5 cystic fibrosis-related diabetes mellitus; PPI 5 proton pump inhibitor. Figure 1. The concentration of BA in the sputum of patients with CF (n 5 41) was significantly higher compared with healthy control subjects (n 515) and patients with CC (1.5 [0-2.2] mmol/l vs 0 [0-0] m mol/l vs 0 [0-0] mmol/l) ( P,.01). Concentrations of BA were not significantly different between patients with CF and patients with asthma (1.5 [0-2.2] mmol/l vs 0 [0-2.0] mmol/l) (P5.1). BA 5 bile acid; CC 5 chronic cough; CF 5 cystic fibrosis. therapy, and nine patients (22%) were given oral bile salts. There were no differences in BA detection rate/concentration with respect to treatment with AZM, PPI, or oral BA supplements. Relation Between Presence of BAs and Disease Severity Comparison of patient characteristics, pulmonary function, cell count, and sputum NSE levels between patients with and without BA in sputum is depicted in Table 3. There was no correlation between BA concentrations in sputum and BMI or age. Median concentrations of NSE and sputum neutrophilia were significantly higher in subjects with CF compared with healthy control subjects (NSE [ ] mg/ml vs 1.41 [ ] mg/ml, P,.0001; neutrophilia [ ] vs [ ], P,.0001). We could not find any differences in sputum neutrophilia between patients with CF with BA and patients with CF without BA ( [ ] vs [ ], P 5.8). Patients with CF with BA in sputum had significantly higher levels of NSE compared with those without BA (P 5.005) (Table 3, Fig 2 ). There was a significant correlation between BA concentrations and NSE levels in patients with CF with detectable BA in sputum (r50.60, P 5.002) (Fig 3 ). Lung function at time of sputum sampling and the number of days of IV antibiotic therapy in 2 years preceding sputum collection were not significantly different between patients with and without BA in sputum (Table 3 ). However, there was a significant negative correlation between lung function and 1570 Original Research

4 Table 2 Characteristics of Adults With CF and Children With CF Characteristics Patients 18 y (n 5 33) Patients, 18 y (n 5 8) P Value Mean age, y (range) 27 (18-43) 13 (11-17)... Sex, male (female) 18 (15) 6 (2).4 Pancreatic insufficiency fi (suffifi ciency) 26 (7) 6 (0).6 Genotype F508del homozygous F508del heterozygous Other No. of patients treated with (%) PPI 8 (24) 2 (25)... AZM 14 (42) 1 (12)... Oral bile salts 6 (18) 3 (37)... Median BA, mmol/l (25%-75%) 2.2 ( ) 1.5 ( ).2 Median NSE, mg/ml (25%-75%) ( ) 8.01 ( ) a.005 Median sputum neutrophilia (25%-75%) ( ) b ( ) a,c.008 Median FEV 1 % predicted (25%-75%) 65 (47-82) 89 (77-102) a.002 Median FVC% predicted (25%-75%) 87 (72-92) 97 (88-105) a.04 BA 5 bile acid; CF 5 cystic fibrosis; NSE 5 neutrophil elastase. See Table 1 legend for expansion of other abbreviations. a P,.05. b n 528. c n 57. BA concentrations in patients with detectable BA in sputum (FEV 1 % r520.53, P,.01; FVC % r , P,.01) ( Fig 4 ). We also found a significant correla- tion in patients who were BA-positive between BA concentration and the number of days of IV antibiotic ther apy in 2 years preceding sputum collection ( r5 0.58, P5.009). Discussion It is well established that patients with CF have a high prevalence of acid GER. 2-4,15 A major concern in CF is the risk of aspirating duodenogastric contents into the lungs, which could result in an increased bronchial inflammatory reaction. Measuring specific markers of aspiration may help to establish the Table 3 Comparison of Patients Who Are Positive for BA and Patients Who Are Negative for BA Characteristics Patients Who Are BA-Positive (n 5 23) Patients Who Are BA-Negative (n 5 18) P Value Mean age, y (range) 25 (11-43) 24 (11-42) Sex, male (female) 15 (8) 9 (9).36 Pancreatic insufficiency fi (suffifi ciency) 20 (3) 15 (3) Genotype... F508del homozygous 12 7 F508del heterozygous 8 7 Other 3 4 Median BMI, kg/m 2 (25%-75%) ( ) ( ).75 No. of patients With (without) CFRDM 2 (21) 5 (13).2 With (without) AZM 9 (14) 6 (12).75 With (without) PPI 8 (15) 2 (16).14 With (without) oral bile salts 6 (17) 3 (15).71 Median NSE, mg/ml (25%-75%) ( ) ( ) a.005 Median sputum neutrophilia (25%-75%) ( ) b ( ) c.75 Median sputum neutrophilia, % (25%-75%) 94 (83-95) b 94 (88-97) c.31 Median FEV 1 % predicted (25%-75%) 68 (48-87) 70 (49-91).71 Median FVC % predicted (25%-75%) 88 (70-93) 88 (75-100).34 Median No. of days of antibiotic IV treatments in the past 2 y (25%-75%) 14 (0-48) b 15 (0-48) d.72 See Table 1 and 2 legends for expansion of abbreviations. a P,.05. b n c n d n CHEST / 141 / 6 / JUNE,

5 Figure 2. There is a significant difference in NSE concentration in patients with CF with and without BA in sputum (31.25 [ ] m g/ml vs [ ] mg/ml) (P,.01). NSE 5 neutrophil elastase. See Figure 1 legend for expansion of other abbreviations. potential role of GER and aspiration in CF. The gold standard in detecting aspiration would be measuring (duodeno)-gastric contents in BALF; however, this is invasive and not routinely performed in CF. The present study aimed to assess aspiration by measuring BA in sputum of patients with CF and to correlate it with markers of inflammation and other indices of CF lung disease. The main findings of the study were: (1) 56% of patients with CF have detectable BA in sputum, suggestive of aspiration of duodenogastric contents into the lungs; (2) levels of sputum NSE, an inflammatory marker in CF airways, were signifi- cantly higher in patients with BA in sputum compared with those without detectable BA; (3) when present, BA concentrations in sputum were signif- Figure 3. There is a significant correlation between BA concen- trations and NSE levels in patients with CF with detectable BA in sputum (r5 0.60, P 5.002). 5 patients who are BA-negative; 5 patients who are BA-positive. See Figure 1 and 2 legends for expansion of abbreviations. icantly and inversely correlated with spirometry (FEV 1 % and FVC%); and (4) in patients with BA in sputum, there was a significant correlation between BA concentrations and the number of days of IV antibiotic therapy. Although objective evidence is scarce, it has been suggested that patients with CF are at risk for aspiration of duodenogastric contents. Several methods have been used to detect gastric aspiration into the lungs, like pulmonary scintigraphy, 16 ambulatory 24-h laryngeal or pharyngeal ph measurements, 9 and lipidladen macrophages or duodenogastric components in BALF However, pulmonary scintigraphy has low sensitivity; laryngeal or pharyngeal ph measurements can only detect proximal reflux of acidic components, and bronchoscopy with BALF is invasive and not routinely performed in CF. In the current study, we did not assess the mechanisms for BA aspiration. The presence of BA in sputum is most likely related to the occurrence of duodenogastroesophageal reflux. Hallberg et al 21 found higher levels of bilirubin in the stomach of patients with CF compared with healthy control subjects, suggesting increased duodenogastric reflux and a higher risk for duodenogastroesophageal reflux in CF. Detection of BA in sputum is specific of aspiration of duodenogastric components and not for GER per se, meaning that patients with only GER might be missed. Several studies demonstrated that patients with CF might have increased reflux without having typical symptoms, that is, silent GER. Button et al 22 described silent GER in 60% of adults with CF, 22 and our group previously reported silent GER in 57% of adult patients with CF. In the current study, we found a poor correlation between typical reflux symptoms and aspiration. A possible explanation for the high prevalence of silent reflux and aspiration could be the occurrence of nocturnal reflux associated with impaired protective mechanisms, either chemical (in saliva) or mechanical (upper esophageal sphincter reflexes, lung clearance). Alternatively, patients with CF might be hyposensitive to acid reflux. Both hypotheses require further investigation. The link between aspiration and increased bronchial inflammation is under current investigation. We found that sputum NSE levels were significantly higher in patients with BA in sputum compared with those without BA. We could not find a difference in NSE levels between the group of patients with CF treated with AZM compared with those not treated with AZM, which confirms fi ndings by Equi et al. 23 A previous article by our group suggested that BA con centrations in BALF from lung transplant recipients lowered after treatment with AZM. 24 In this study, we found no differences in BA concentrations between patients treated or not treated with AZM Original Research

6 Figure 4. There is a significant correlation between FEV % predicted and BA concentration in patients with CF with detect- 1 able BA in sputum ( r , P,.01). 5 patients who are BA-negative; 5 patients who are BA-positive. See Figure 1 legend for expansion of abbreviations. This might be due to the small cohort of patients studied, and to the fact that patients are not studied before and after treatment. Aspiration of BA in CF appeared to be associated with more airway inflammation and the degree of aspiration appears to be related to the extent of airway inflammation. These findings are in line with previous investigations. Aspiration can lead to chemical injury, which can provoke airway constriction and edema followed by an inflammatory response. Several groups found evidence of neutrophilic inflammation of the airways in patients with GER. Carpagnano et al 25 and Jatakanon et al 26 found increased sputum neutrophilia in GER, and Sacco et al 27 reported an increased proportion of neutrophils in BALF of children with GER and asthma-like symptoms. D Ovidio et al 28 and Vos et al 29 found a correlation between BA levels and IL-8 concentrations in BALF of lung transplant recipients, suggesting the link between aspiration and airway inflammation may be attributed to a BA-induced expression of IL-8, a potent activator of neutrophils. A recent study by McNally et al 30 showed that high concentrations of pepsin in BALF from children with CF were associated with high levels of IL-8. Wu et al 31 demonstrated that exposure of airway epithelial cells to chenodeoxycholic acid, a major component of BA, leads to an increased production of IL-8 in vitro. We found a negative correlation between BA levels in sputum and lung function as well as with the number of days of IV antibiotic treatment in 2 years preceding sputum induction. This is in agreement with previous studies showing that GER/aspiration may affect disease severity in CF. 10 Coexistence of increased GER and poor lung function does not directly imply a causal relationship and may be the combined result of a more severe CF disease expression. Furthermore, impairment of lung function in CF is considered to be multifactorial. The aims of this study were to assess the extent of aspiration in CF by measuring BA in sputum, and to correlate aspiration with markers of airway inflamma- tion and disease severity. Although this is a prospective, cross-sectional study, there are some weaknesses in this study. The main weakness is the lack of comparison with a gold standard for aspiration, namely bronchoscopy with collection of BALF during the same period. Unfortunately, we were unable to perform 24-h impedance-ph monitoring as an objective test for reflux measurements, and we were not able to motivate all of the patients and/or parents to return the reflux questionnaire. In conclusion, determining BA in sputum could be an alternative tool in the assessment of aspiration. It can also be used to identify the relationship between aspiration and lung function impairment. Outcome therapeutic studies, combining PPI therapy with prokinetics or even antireflux surgery, are required to further establish the exact role of aspiration on bronchial inflammation and progression of lung disease in CF. Acknowledgments Author contributions: Ms Pauwels: collected the data, performed experiments, performed statistical analysis, and wrote the manuscript. Dr Decraene:contributed to data collection, performed experiments, provided technical support, and approved the manuscript. Dr Blondeau: contributed to the general discussion and writing and review of the manuscript and provided technical support. Dr Mertens:contributed to data collection, provided technical support, and approved the manuscript. Dr Farre: contributed to the general discussion and writing and review of the manuscript and provided technical support. Dr Proesmans: helped recruit the children with CF and contributed to the general discussion, writing, and review of the manuscript. Dr Van Bleyenbergh: helped recruit the adults with CF and contributed to the general discussion, writing, and review of the manuscript. Dr Sifrim: specialized in GER, supervised the project, and contributed to the writing, review, and revision of the manuscript. Dr Dupont: supervised the project and contributed to the writing, review, and revision of the manuscript. 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. Role of sponsors: The sponsor had no role in the design of the study, the collection and analysis of the data, or in the preparation of the manuscript. References 1. Feigelson J, Sauvegrain J. Letter: gastro-esophageal reflux in mucoviscidosis [in French]. Nouv Presse Med ;4(38): Brodzicki J, Trawińska-Bartnicka M, Korzon M. Frequency, consequences and pharmacological treatment of gastroesophageal CHEST / 141 / 6 / JUNE,

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