Regional Variability of Lung Inflammation in Cystic Fibrosis. KEITH C. MEYER, ANUJA SHARMA, with the Technical Assistance of NANCY S. ROSENTHAL,.
Regional Variability of Lung Inflammation in Cystic Fibrosis KEITH C. MEYER, ANUJA SHARMA, with the Technical Assistance of NANCY S. ROSENTHAL, KIMBERLY PETERSON, and LYNETTE BRENNAN Department of Medicine, Section of Pulmonary and Critical Care Medicine, University of Wisconsin Medical School, Madison, Wisconsin
Chest radiography in patients with cystic fibrosis (CF) frequently shows more severe changes in the upper lobes. We performed bronchoalveolar lavage (BAL) on 12 clinically stable, young adult patients with CF to determine whether inflammation varies significantly among geographically distinct areas of the lung. We found that absolute numbers of neutrophils were generally greater in BAL fluid from the upper lobe (25.7 6 7.9 3 105 neutrophils/ml [mean 6 SEM]) of the right lung than that obtained from the right lower lobe (6.8 6 2.8 3 105 neutrophils/ml; p , 0.01). The mean value of unopposed neutrophil elastase activity in upper-lobe BAL fluid (227 6 91 nmol peptide hydrolyzed/ml/min) was also significantly greater than that in lower-lobe BAL fluid (84 6 43 nmol/peptide hydrolyzed/ml/ min; p , 0.01), and similar differences were found for myeloperoxidase activity and DNA content. Neutrophil influx and unopposed neutrophil elastase for a given region correlated inversely with lung function or percentage of ideal body weight, and upper- versus lower-lobe differences were more pronounced in subjects with better preservation of lung function. Our findings suggest that regional variation in inflammation must be considered when utilizing BAL to study lower respiratory tract inflammation in CF or to monitor responses to therapeutic interventions that can potentially diminish lung inflammation. Our findings may also have implications for the study of the natural history of lung inflammation and infection in neonates, infants, and young children with CF. Meyer KC, Sharma A, Rosenthal NS, Peterson K, Brennan L. Regional variability of lung inflammation in AM J RESPIR CRIT CARE MED 1997;156:1536–1540. cystic fibrosis.
Mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which encodes a transmembrane chloride channel in epithelial cells (1), cause abnormal exocrine gland secretions, leading to chronic endobronchial infection and lung destruction in patients with cystic fibrosis (CF). Despite exuberant expression of CFTR in the developing normal lung in utero (2), the CF lung is essentially normal in utero and shortly after birth. However, lung involvement is the major cause of morbidity and accounts for more than 90% of the mortality in CF (3). The clinical course of lung disease in CF varies but is eventually characterized by intermittent exacerbations of endobronchial infection that typically become more apparent in the second decade of life, although lower respiratory tract inflammation and infection can be detected at bronchoscopy in many infants and young children (4–6), including many who are asymptomatic.
(Received in original form January 27, 1997 and in revised form June 27, 1997) Supported by a Teaching and Research Scholar Award from the American College of Physicians. Dr. Meyer is the recipient of an American College of Physicians Teaching and Research Scholar Award. Dr. Sharma is the recipient of a third-year-fellow award from the Cystic Fibrosis Foundation. Correspondence and requests for reprints should be addressed to Dr. Keith C. Meyer, Section of Pulmonary and Critical Care Medicine, Department of Medicine, H6/380 Clinical Sciences Center, 600 Highland Ave., Madison, WI 53792. Am J Respir Crit Care Med Vol 156. pp 1536–1540, 1997
It is increasingly recognized that neutrophil-dominated lower respiratory tract inflammation plays a key role in progression of lung disease and decline of lung function in CF (7–9). Neutrophil-derived proteases such as neutrophil elastase (NE) can overwhelm antielastase defenses which predominantly consist of a1-antiprotease and secretory leukoprotease inhibitor. Unopposed NE in bronchoalveolar lavage (BAL) fluid has been found in young adult patients with respiratory exacerbations of their disease (9), in stable patients with clinically mild lung disease (10), and in infants (4, 5) or young children (6). Active unopposed elastase prevents clearance of bacteria by cleaving various opsonins or opsonin receptors (11, 12), directly damages the lung by disrupting lung matrix proteins (13), promotes airway obstruction by stimulating mucus secretion (14), and degrades a1-antiprotease (15). Another neutrophil-derived mediator, myeloperoxidase, may contribute to lung damage by catalyzing the formation of hypochlorous acid, a potent oxidant that can inactivate a1-antiprotease (16, 17) or cause cross-linking of tyrosine residues in various proteins (18). Neutrophilderived oxyradical species and proteolytic enzymes in the CF lung may synergistically damage various glycoproteins, including a1-antiprotease (19). Radiographically, early evidence of lung disease in CF is most apparent in the upper lung fields. Changes of bronchiectasis detected by computed tomographic scanning tend to be most extensive in the upper robes (20–22). We therefore hypothesized that neutrophil-dominated inflammation would be more intense in respiratory secretions obtained via BAL from an upper lobe than from those obtained from a lower lobe. To test this hypothesis, we performed BAL sequentially
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in lower-, middle-, and upper-lobe segments of the right lung in 12 clinically stable, volunteer subjects with CF. We quantitated neutrophils in BAL and measured unopposed NE activity, myeloperoxidase activity, concentration of a1-antiprotease complexed with NE, total DNA, density of aerobic bacteria in BAL fluid via quantitative culture, and lipopolysaccharide (LPS) concentrations in BAL fluid.
METHODS Study Population The study population consisted of 12 outpatients with CF. This protocol was approved by the institutional Human Subjects Committee, and written informed consent was obtained from all volunteers. Ten of these 12 patients had sequential BAL performed in the right lower lobe, middle lobe, and upper lobe to compare differences among different lung regions. The middle lobe was not sampled in two additional patients. BAL fluid from the right middle lobe was obtained from 20 never-smoking, age-matched volunteers studied for comparative purposes.
Bronchoscopy and BAL Bronchoscopy and BAL were performed in the morning after an overnight fast in all subjects as previously described (9). The distal bronchoscope was wedged in a segmental bronchus and four 40-ml aliquots of sterile, nonpyrogenic, isotonic sodium chloride solution were sequentially instilled through the bronchoscope and immediately recovered by gentle suction via a hand-held syringe. The anterior basilar segment of the right lower lobe was lavaged first, the medial segment of the right middle lobe next, and the anterior segment of the right upper lobe last.
Processing of BAL Aliquots of BAL fluid from each segment were pooled, filtered through one layer of a loose, sterile gauze to remove mucus plugs, and centrifuged at 400 3 g for 10 min at 228 C. Aliquots of the resulting BAL supernatant fluid were immediately frozen at 2708 C until assayed. Some aliquots had phenylmethenesulfonyl fluoride (PMSF; Sigma Chemical Co., St. Louis, MO) added prior to freezing to inhibit serine protease activity. A hemocytometer and cytocentrifuge preparations of cell suspensions stained with Diff-Quik (Baxter, Miami, FL) were used to obtain total and differential cell counts.
Measurement of NE and Myeloperoxidase NE activity was determined spectrophotometrically at 410 nm via an initial velocity assay using a specific substrate, MeO-Suc-Ala-AlaPro-Val-pNA, as previously described (23). Myeloperoxidase enzyme activity, presumably released by activated or injured polymorphonuclear leukocytes, was quantified in samples of cell-free BAL fluid supernatants using a continuous, initial velocity spectrophotometric assay employing 3,39,5,59-tetramethyl benzidine (TMB) as the oxidizable substrate as previously described (23). An immunoassay kit and protocol available from Merck (Darmstadt, Germany) were used to determine levels of NE complexed with a1-antiprotease complex (NE/ a1-antiprotease complex) in BAL as reported previously (9).
DNA Content of BAL DNA sodium salt from salmon testes (Sigma) was used for standards and controls. The 2 mg/ml stock solution was diluted to 800 mg/ml with assay diluent and stored in aliquots at 2708 C. Twofold serial dilutions were done from the 800 mg/ml aliquot to obtain a standard curve. Controls were prepared to fall within the low and high areas of the standard curve and stored at 2708 C. Fifty microliters of BAL fluid, standards, and controls were added to appropriate wells in microtiter plates (Immulon 2 flat-bottom plates; Dynatech Laboratories, Chantilly, VA). Samples, standards, and controls were all run in triplicate. Fifty microliters of 20% 3,5-diaminobenzoic acid hydrochloride (DABA) substrate (Aldrich Chemical Co., Milwaukee, WI) was then added to each well, and the plates were sealed tightly with plate sealers (Fisher Scientific, Itasca, IL). The plates were thoroughly mixed on a titerplate shaker (Lab-Line Instruments, Melrose Park, IL) until
a homogeneous mixture was obtained and were incubated for 1 h at 608 C. Fifty microliters of 5 N HCl was added to each well to stop the reaction. The plates were read on a fluorometer plate reader (Cambridge Technology Inc., Watertown, MA) using a 400 nm filter for excitation and a 530 nm filter for emission.
Microbiology and BAL LPS Concentrations An aliquot of uncentrifuged BAL was treated with N-acetyl cysteine (0.125 g per 50-ml lavagate) to disperse mucus in the specimen as previously described (23). Dilutions of the suspension were then plated on media to obtain quantitative colony counts of aerobic bacteria. Blood agar, chocolate agar, eosin methylene blue, mannitol salt, and special media for P. cepacia and Hemophilus influenzae were used as culture media to isolate these pathogens if present. The Limulus amoebocyte lysate assay (BioWhittaker, Walkersville, MD) was used according to the manufacturer’s protocol to measure endotoxin concentrations in BAL fluid.
Statistical Analysis Individual assays were performed in triplicate. Mean values were used in subsequent data analysis. All data were analyzed preliminarily on electronic spreadsheets (SuperCalc4; Computer Associates, San Jose, CA). Subsequent analysis including independent t tests, paired t tests. Wilcoxon signed-rank tests, and multivariate analysis and multiple regression analysis was performed using a database-statistics package for microcomputers (Abstat 4.1; Anderson-Bell, Parker, CO).
RESULTS Clinical Data
Characteristics of the 12 patients who underwent regional BAL are given in Table 1. All patients were chronically colonized with Pseudomonas aeruginosa except for one who repeatedly had only Staphylacoccus aureus in sputum or BAL. Subjects with CF ranged in age from 18 to 44 yr. All except one were pancreatic insufficient. All subjects were clinically stable and afebrile; none had been hospitalized or given intravenous antibiotics during a period > 1 mo prior to bronchoscopy. No patients had been treated with recombinant human DNase prior to the time of study. The FEV1% predicted ranged from 40 to 100% for patients with CF, and we separated the patients into two subsets on the basis of severity of lung disease for the purpose of additional statistical analysis. The patient with only S. aureus isolated from both BAL and sputum (never colonized with P. aeruginosa) and the pancreatic-sufficient patient were in the group with FEV1 > 65% of predicted. Cell Profiles and BAL DNA Content
A marked influx of neutrophils was observed in BAL from all CF patients which generally was greater for BAL from upper lobes than that from the lower lobes (Table 2). Neutrophil
TABLE 1 CLINICAL FEATURES OF STUDY PATIENTS
n Age, yr Sex, male/female % Ideal body weight* Pulmonary function* FEV1 % FVC % FEV1/FVC FEF25–75%
All Patients
Patients with FEV1% > 65
Patients with FEV1% < 50
12 24 6 2 10/2 91 6 4
8 24 6 3 6/2 96 6 5
4 23 6 2 4/0 80 6 3†
69 6 6 82 6 5 66 6 3 39 6 8
81 6 5 91 6 3 71 6 3 52 6 9
44 6 2† 64 6 2† 56 6 3† 15 6 1†
* Data represent mean values 6 SEM. † p , 0.05 versus patients with FEV1 % > 65.
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BAL CHARACTERISTICS* Patients with Cystic Fibrosis (n 5 12) Upper Lobe
Lower Lobe
3.4 6 1.1 55 6 3
1.1 6 0.4† 61 6 2
25.2 6 5.4 2.5 6 0.8 1.2 6 0.5 71.1 6 5.6 2.6 6 0.8 41 6 13 630 6 236 50 6 18 190 6 97 227 6 91 196 6 34 400 6 179
1997
per milliliter (r 5 0.92, p , 0.0001). Values for middle-lobe BAL were generally similar to those for lower-lobe BAL (data not shown).
TABLE 2
Cells/ml % Return Cell differential % Macrophages % Lymphocytes % Eosinophils % Neutrophils Neutrophils/ml DNA, mg/ml Total protein, mg/ml CFU 3 105/ml LPS, U/ml NE activity, nmol/min/ml NE/a1AP complex, ng/ml MPO activity, nmol/min/ml
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48.0 6 7.2† 4.9 6 1.2 0.7 6 0.3 46.6 6 7.3† 0.7 6 0.3† 14 6 3† 295 6 79† 50 6 31 30 6 13 84 6 43† 407 6 127 148 6 71†
Normal Subjects (n 5 20) 0.10 6 0.009 68 6 2 88.8 6 1.4 9.1 6 1.2 0.5 6 0.2 1.6 6 0.4 0.002 6 0.0007 12 6 1 76 6 6 ND ND ND 1.0 6 0.1 ND
Definition of abbreviations: Cells/ml 5 cells/ml BAL fluid 3 106; % Return 5 percentage of lavage fluid retrieved of 160 ml instilled; Neutrophils/ml 5 neutrophils/ml BAL fluid 3 106; CFU 5 colony-forming units; LPS 5 lipopolysaccharide; NE 5 neutrophil elastase; a1AP 5 a1-antiprotease; MPO 5 myeloperoxidase; ND 5 none detected. * Data expressed as mean 6 SEM. † p , 0.01 versus upper-lobe values.
BAL Neutrophil Mediators and NE/a1-Antiprotease Complex
Active, unopposed NE was present in upper-lobe BAL from all CF patients except for one who had . 105 P. aeruginosa cultured from all three areas (right lower lobe [RLL], right middle lobe [RML], and right upper lobe [RUL] but had relatively low BAL neutrophil counts, no NE activity in BAL, and well-preserved lung function (FEV1% 5 92%). The patient who had never been colonized with P. aeruginosa had active NE in BAL from the RUL (and . 105 cfu/ml of S. aureus) but not from the RML or RLL, which also had no bacterial growth. Two other patients with relatively well-preserved lung function had essentially no unopposed NE activity in BAL from the RLL. NE activity was greater in upper-lobe BAL for nine of the 12 subjects (Figure 1). Concentrations of a1-antiprotease bound to NE tended to be greater for lower-lobe samples as compared with upper-lobe samples, and levels of this complex were virtually the same for BAL aliquots treated with PMSF versus those not treated. Myeloperoxidase activity was present in all BAL. NE activity correlated well with myeloperoxidase activity (r 5 0.99), and both myeloperoxidase and NE activities correlated well with absolute numbers of neutrophils in BAL. Aerobic Bacteria and BAL Endotoxin Concentrations
concentration in upper-lobe BAL was greater than that for lower-lobe BAL for every subject with CF. Differences between upper and lower lobes were more prominent for patients with better-preserved lung function (Table 3). DNA concentrations in BAL fluid from CF patients were significantly elevated above values for BAL from normal subjects. Additionally, DNA content in BAL was greater for BAL from upper lobe versus lower lobe in every subject with CF. DNA concentration in BAL fluid correlated well with neutrophils
All CF patients had . 105 cfu/ml of aerobic bacteria cultured from RUL BAL, with one exception as noted above. This patient, who had very well preserved lung function (FEV1% 5 100%), had no bacterial growth from the RLL, RML, or RUL; cytospins were negative for bacteria on gram stain but P. aeruginosa grew from culture of expectorated sputum. Another patient had only S. aureus at . 105 cfu/ml isolated from the RUL with negative RML and RLL culture, and no LPS was detected in BAL from any lung region. Culture of RML BAL showed > 105 cfu/ml for six of the 10 patients who un-
TABLE 3 DEGREE OF LUNG FUNCTION IMPAIRMENT AND BAL CHARACTERISTICS Patients with FEV1 % > 65 (n 5 8)
Cells/ml % Return Cell differential % Macrophages % Lymphocytes % Eosinophils % Neutrophils Neutrophils/ml NE activity, nmol/min/min NE/a1AP complex, ng/ml MPO activity, nmol/min/ml Total protein, mg/ml
Patients with FEV1 % < 50 (n 5 4)
Upper Lobe
Lower Lobe
Upper Lobe
Lower Lobe
1.7 6 0.4 59 6 3
0.4 6 0.08† 64 6 3
6.8 6 2.6 51 6 8
2.3 6 0.8 59 6 6
29.6 6 7.7 1.9 6 0.8 0.7 6 0.3 67.9 6 8.1 1.4 6 0.4 106 6 37
57.9 6 8.5† 4.6 6 1.6 0.3 6 0.2 37.5 6 9.0† 0.2 6 0.08† 15 6 8†
16.5 6 2.1 3.7 6 1.8 2.3 6 1.3 77.6 6 3.5 5.0 6 1.8 470 6 235
28.3 6 6.5 5.5 6 2.2 1.5 6 0.7 64.9 6 5.9 1.6 6 0.6 224 6 101
238 6 37
507 6 177
170 6 56
207 6 101
192 6 67
46 6 11†
816 6 495
354 6 186
313 6 73
177 6 38†
1263 6 621
533 6 181
Definition of abbreviations: Cells/ml 5 cells/ml BAL fluid 3 106; % Return 5 percentage of lavage fluid retrieved of 160 ml instilled; Neutrophils/ml 5 neutrophils/ml BAL fluid 3 106; NE 5 neutrophil elastase; a1AP 5 a1-antiprotease; MPO 5 myeloperoxidase. * Data expressed as mean 6 SEM. † p , 0.05 versus upper lobe for patients with FEV1 % > 65.
Figure 1. Paired values for neutrophil elastase activity from BAL sampling of upper- and lower-lobe lung segments of patients with CF. One patient (not shown) had no detectable unopposed elastase activity in the upper or lower lobe.
Meyer and Sharma: Regional Lung Inflammation in Cystic Fibrosis
derwent RML BAL, and BAL from the RLL showed > 105 cfu/ml for six of 12 specimens. Two of the 11 patients harboring P. aeruginosa in their secretions had no bacterial growth from RLL BAL. P. aeruginosa was the predominant pathogen (ratio > 1,000:1) in all quantitative BAL cultures with the exception of the subject with only S. aureus isolated from respiratory secretions. Colony counts (colony-forming units per milliliter) for P. aeruginosa correlated with LPS concentration (r 5 0.54) in BAL fluid from the various lung segments lavaged. However, aerobic bacterial colony counts did not correlate significantly with any parameter of inflammation or pulmonary function. In contrast, LPS concentrations in BAL correlated significantly with unopposed NE activity (r 5 0.67, p , 0.0001) and myeloperoxidase activity (r 5 0.66, p , 0.0001). Data Analysis and Correlations
Activities of NE and myeloperoxidase in supernatant BAL fluid correlated well with total neutrophils in BAL (r 5 0.82 and r 5 0.80, respectively). Unopposed elastase activity correlated significantly with FEV1% for RUL (r 5 20.57) or RLL (r 5 20.58) BAL. Neutrophils per milliliter also correlated significantly with FEV1% for RUL (r 5 20.68) and RLL (r 5 20.63). Similarly, percentage of ideal body weight correlated inversely with neutrophils per milliliter in BAL for RUL (r 5 20.60) or RLL (r 5 20.55) and unopposed elastase in BAL for RUL (r 5 20.43) or RLL (r 5 20.50).
DISCUSSION We found that neutrophil-dominated lower respiratory tract inflammation in this group of stable outpatients with CF varied according to geographic lung region. Inflammation was significantly greater in BAL obtained from the right upper lobe versus than from the right lower lobe (or the right middle lobe; data not shown). Differences in the degree of neutrophil influx and activities of unopposed NE and myeloperoxidase between the upper and lower lobes were greatest for the group of patients with better-preserved lung function. Those with worse lung function had relatively greater amounts of neutrophils and neutrophil mediators present in BAL fluid from the lower lobe than those with better lung function and generally had a smaller degree of difference in mediator levels between upper and lower lobes. Although a considerable amount of NE/a1-antiprotease complex was present, unopposed NE activity was detected in nearly all BAL specimens, particularly in BAL from the upper lobe. Our findings also demonstrate that young adult patients who appear to be clinically stable (including some with FEV1% and other pulmonary function testing within the range of normal and who have little or no sputum expectoration and have seldom or never required hospitalization for treatment of pulmonary exacerbations) harbor significant amounts of bacteria (> 105 cfu/ml) in at least one lung segment and have a marked degree of neutrophil-dominated inflammation. These findings complement those of Konstan and associates (10), who found significant infection and neutrophil-dominated inflammation in BAL from adolescent and young adult CF patients with stable, clinically mild lung disease. The inverse correlation of total neutrophils and unopposed elastase activity with tests of pulmonary function such as FEV1% suggest that the magnitude of such ongoing inflammation is linked to deleterious effects on lung function, although such associations in cross-sectional studies must be interpreted with care. Interestingly, colony counts of aerobic bacteria did not correlate well with total neutrophils or neutrophil mediators, a
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finding similar to that previously observed for a different group of patients with CF (23). However, the concentration of LPS in BAL fluid did correlate well with NE and myeloperoxidase activity for BAL from a given geographic lung segment. Also, a considerable amount of inflammation was present in the areas from which no bacteria were cultured or seen on gramsmear of cytospins. Although we cannot explain the discordance between LPS concentrations and colony-forming units on quantitative bacterial culture, the ability of phagocytic cells to ingest and kill bacteria may be better preserved in lung segments with relatively low colony-forming units but higher LPS concentrations, but of residual LPS may persist in epithelial lining fluid. The right upper lobe (and possibly the apical-posterior and anterior segments of the left upper lobe to a similar degree) may be involved more severely in the early stages of lung disease and may serve as a reservoir of aerobic bacteria that cannot be cleared from the lung because of relatively inefficient clearance of secretions from upper lung regions via cough. Although studies in infants and toddlers (4, 5) have found the presence of inflammation in some subjects when bacteria or bacterial products cannot be isolated, BAL in at least one of these studies (4) was usually performed in the lingula of the left lung, a typically less severely involved lung region than the right upper lobe by computed tomography (20). Although bacteria were often not detected in BAL from the lingula despite the presence of inflammation in this area, foci of infection and inflammation may still have been present in the nonlavaged right upper lobe. Some of the subjects in our study had 105 to 108 cfu/ml of aerobic bacteria in BAL from the right upper lobe versus no bacterial growth in culture nor identification of bacteria on stained cytospins from BAL obtained from the right lower or middle lobe, demonstrating that large numbers of bacteria may be recovered from one lung segment but not from another segment in a different lobe. The underlying reasons for regional variation in bronchial inflammation and destruction are unclear. The upper lobes, particularly the right upper lobe, appear to be most susceptible to atelectasis and aspiration pneumonitis in the newborn infant (24). This may, in part, account for the finding by Maffessanti and colleagues (20) that the most severe bronchiectatic changes are found in the upper lobes and particularly in the right upper lobe. Furthermore, aspiration is a relatively common event during sleep (25) or with eating (26) in normal subjects; infants and young children with CF have a high incidence of gastroesophageal reflux (27–29); and recumbent positioning for postural drainage and percussion can induce gastroesophageal reflux (30). Because bicarbonate ion is transported via CFTR (31), and cAMP–stimulated bicarbonate secretion is depresssd across CF airway epithela (31, 32), airway epithelial cells may not be capable of rapidly secreting bicarbonate to buffer acidic secretions in the airspaces. We raise the possibility that aspiration, particularly if combined with some degree of gastroesophageal reflux and aspiration of relatively acidic secretions, may damage airspaces of the upper lobes (especially the right upper lobe) in infants with CF and promote the establishment of infection and inflammation in these areas, initially with relative sparing of other lung regions. In summary, our findings of significant regional variation in lung inflammation and bacterial burden in CF have implications for investigations that attempt to better understand the natural history of the evolving inflammatory lung lesion of CF and for the use of BAL to study the effect of treatments designed to diminish lower respiratory tract inflammation. Investigations that employ bronchoscopy to assess lower respiratory tract inflammation, particularly longitudinal studies,
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must take into consideration regional variability in inflammation. Bronchoscopy should be standardized and a specific site or sites chosen for investigation in cross-sectional or longitudinal studies. Studies utilizing BAL to assess lung inflammation and infection in infants and toddlers should attempt to sample secretions simultaneously from multiple lung regions, including the right upper lobe, to better determine if inflammation can be present without coexistent aerobic bacterial infection. Such studies may improve our knowledge of the natural history of the inflammatory lung lesion and the role of bacterial infection in the evolution of lung disease in infants and young children with CF. Acknowledgment : The writers thank Lorna Will for her assistance in enrolling patients in this study.
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