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journal.publications.chestnet.

org 1013
[ Original Research Signs and Symptoms of Chest Diseases ]
Manuscript received January 15, 2014; revision accepted April 11, 2014;
originally published Online First May 29, 2014.
ABBREVIATIONS: CXCL 5 C-X-C motif ligand; CXCR 5 C-X-C motif
receptor; IL-1RA 5 IL-1 receptor antagonist; IRAK 5 IL-1 receptor-
associated kinase; NF- k B 5 nuclear factor- k B; PBB 5 protracted bacte-
rial bronchitis; PELI1 5 pellino-1; Q 5 quartile; TLR 5 Toll-like receptor;
TNF 5 tumor necrosis factor
AFFILIATIONS: From the Priority Research Centre for Asthma and
Respiratory Diseases (Drs Baines, Simpson, and Gibson), Te University
of Newcastle, Callaghan, NSW; Department of Respiratory and Sleep
Medicine (Drs Baines, Simpson, and Gibson), Hunter Medical Research
Institute, John Hunter Hospital, New Lambton Heights, NSW; School of
Medicine (Drs Upham, Yerkovich, and Marchant and Ms Carroll), Te
University of Queensland, Brisbane, QLD; Qld Lung Transplant Service
(Dr Yerkovich), Te Prince Charles Hospital, Brisbane, QLD; Depart-
ment of Respiratory Medicine (Drs Chang and Marchant), Queensland
Childrens Medical Research Institute, Royal Childrens Hospital,
Brisbane, QLD; and Child Health Division (Dr Chang), Menzies School
of Health Research, Darwin, NT, Australia.
FUNDING/SUPPORT: Drs Gibson , Upham, and Chang are supported by
National Health and Medical Research Council (NHMRC; Common-
wealth of Australia) fellowships [Grants 569240, 511019, and 545216,
respectively]. Dr Baines is supported by a research fellowship from Te
University of Newcastle. Te study was funded by the Financial Markets
Foundation for Children [Grant 2010-005], NHMRC [Grant 1042601],
and NHMRC Centre of Research Excellence (CRE) in Lung Health of
Aboriginal and Torres Strait Islander Children [Grant 1040830].
CORRESPONDENCE TO: Katherine J. Baines, PhD, Level 2 West, HMRI
Bldg, Lot 1 Kookaburra Circuit, New Lambton Heights, NSW 2305,
Australia; e-mail: katherine.baines@newcastle.edu.au
2014 AMERICAN COLLEGE OF CHEST PHYSICIANS. Reproduction of
this article is prohibited without written permission from the American
College of Chest Physicians. See online for more details.
DOI: 10.1378/chest.14-0131
Mediators of Neutrophil Function in Children With
Protracted Bacterial Bronchitis
Katherine J. Baines , PhD ; John W. Upham , PhD ; Stephanie T. Yerkovich , PhD ; Anne B. Chang , PhD ;
Julie M. Marchant , PhD ; Melanie Carroll , BSc ( Hons ); Jodie L. Simpson , PhD ; and Peter G. Gibson , MBBS
BACKGROUND: Protracted bacterial bronchitis (PBB) is a common and treatable cause of
chronic wet cough in children in which the mechanisms are not understood. Tis study inves-
tigates the IL-1 pathway and a neutrophil gene expression signature in PBB.
METHODS: BAL was collected from children in an experimental cohort (n 5 21, PBB; n 5 33,
control subjects), and a second validation cohort (n 5 36, PBB; n 5 11, control subjects). IL-1 b ,
IL-1 receptor antagonist (IL-1RA), and a -defensins 1-3 were assayed by enzyme-linked immu-
nosorbent assay, western blot, and quantitative real-time polymerase chain reaction, together
with selected IL-1 pathway members and neutrophil-related molecules.
RESULTS: In the experimental cohort, children with symptomatic PBB had signifcantly higher
levels of IL-1 b and a -defensin gene and protein expression. Expression of the neutrophil che-
mokine receptor C-X-C motif receptor 2 was also higher in PBB. IL-1RA protein was higher,
however, the IL-1RA:IL-1 b ratio was lower in children with PBB than control subjects. In the
validation cohort, protein and gene expression of IL-1 b and a -defensins 1-3 were confrmed
higher, as was gene expression of IL-1 pathway members and C-X-C motif receptor 2. IL-1 b
and a -defensin 1-3 levels lowered when PBB was treated and resolved. In children with recurrent
PBB, gene expression of the IL-1 b signaling molecules pellino-1 and IL-1 receptor-associated
kinase 2 was signifcantly higher. IL-1 b protein levels correlated with BAL neutrophilia and
the duration and severity of cough symptoms. IL-1 b and a -defensin 1-3 levels were highly
correlated.
CONCLUSIONS: PBB is characterized by increased IL-1 b pathway activation. IL-1 b and related
mediators were associated with BAL neutrophils, cough symptoms, and disease recurrence,
providing insight into PBB pathogenesis. CHEST 2014; 146(4):1013- 1020
Downloaded From: http://journal.publications.chestnet.org/ by M Darwich on 10/12/2014
1014 Original Research [ 1 4 6 # 4 CHEST OCTOBER 2 0 1 4 ]
Protracted bacterial bronchitis (PBB) is an important
and common cause of chronic wet cough in children.
1

Once correctly diagnosed, the childs cough resolves
with prolonged antibiotic therapy.
2
PBB is now interna-
tionally accepted as a diagnostic entity
3
and has been
incorporated into national
4
and international
5
pediatric
guidelines for cough management. However, despite
this increased clinical recognition, the underlying mech-
anisms of PBB remain to be elucidated.
Prior studies have shown that bacterial colonization and
airway neutrophilia are present in children with PBB.
2,6

Tis was associated with upregulation of the Toll-like
receptors (TLRs) TLR2 and TLR4 in the BAL of chil-
dren with PBB.
6
Tis implicates persistent neutrophilic
infammation in the pathogenesis of PBB and suggests
that neutrophil pathway mediators such as IL-1 b may
play an important role in pathogenesis. In adults with
neutrophilic asthma, using gene expression profling we
have implicated the IL-1 and tumor necrosis factor
(TNF)- a /nuclear factor- k B (NF- k B) pathways in
sputum
7
and a blood gene expression signature
involving neutrophil defensins and proteases.
8
Since
neutrophils play a role in both PBB and neutrophilic
asthma, there may be common mechanisms involved.
Terefore, this study evaluated these pathways and
mediators in two cohorts of PBB and control children.
We hypothesized that IL-1 b and the neutrophil gene
expression signature would be elevated in PBB and
related to symptoms and recurrence.
Materials and Methods
Subject Recruitment and Sampling
Infammatory mediators were evaluated in two cohorts. Te experi-
mental cohort (n 5 54) comprised subjects and BAL samples collected
in previous studies.
6,9
Te validation cohort (n 5 47) was obtained
from a second cohort with purposive matching of control subjects.
2

Te selection of samples for analysis was based on the availability of
specimens and clinical diagnosis; details of enrollment of children is
described previously.
2,6,9
A clinical history was obtained on the day of
the bronchoscopy, and parents were provided with a cough diary card
10

used to document response to antibiotics, defned as absence of cough
or . 75% reduction in score (for 3 days) within 2 weeks of antibi-
otic use (amoxycillin-clauvanate 45 mg/kg/d in two doses for 14 days
2
)
postbronchoscopy. In the validation cohort, children with PBB were
contacted at monthly intervals to document recurrence.
PBB was defned as the presence of a history of chronic ( . 4 weeks)
wet cough and a response to antibiotic treatment with resolution of the
cough within 2 weeks in the absence of signs and symptoms of other
diseases. Symptomatic PBB was defned as children with PBB who
were coughing when bronchoscopy was undertaken. Resolved PBB
was defned as children who previously had a chronic wet cough that
responded to 2 weeks of antibiotics and who were cough-free at the
time when bronchoscopy was undertaken. Recurrent PBB was defned
prospectively as more than three episodes of wet cough responding to
antibiotic treatment within 12 months following the initial diagnosis,
and nonrecurrent PBB as those with fewer than three episodes in the
same timeframe.
Experimental cohort control was a convenient sample of children
undergoing gastroscopy, whereas in the validation cohort control sub-
jects were age-matched and obtained opportunistically from children
undergoing bronchoscopy for assessment of the airways (eg, stridor)
with no history of chronic cough and no respiratory infection in the pre-
ceding 2 weeks. Informed consent was obtained, and the studies were
approved by the Ethics Committees of the Royal Childrens Hospital
and University of Queensland (HREC/03/QRCH/17).
Target Selection and Gene Expression
Inflammatory gene expression was determined in RNA extracted
from BAL cell pellets using real-time quantitative polymerase chain
reaction and standardized TaqMan methods as described in detail
in e-Appendix 1 . Genes tested include those previously identifed as
increased in sputum in neutrophilic asthma and include IL-1 b ( IL1B ),
IL-1 receptor 2 ( IL1R2 ), IL-1 receptor antagonist ( IL1RN ), pellino-1
( PELI1 ), and IL-1 receptor-associated kinase 2 ( IRAK2 ); TNF- a /NF- k B
pathway members TNF receptor superfamily member 1B ( TNFRSF1B )
and NF- k light polypeptide gene enhancer in B cells 2 ( NFKB2 ); and
the chemoattractant receptor C-X-C motif receptor 2 ( CXCR2 ).
7
Also
tested was a blood neutrophil gene expression signature including the
a -defensins ( DEFA1-3 and DEFA4 ), protease elastase ( ELANE ), and
cathepsin G ( CTSG ).
8

Protein Measurements
IL-1 b (undiluted) and IL-1 receptor antagonist (IL-1RA) (one-ffh
dilution) protein levels were measured in BAL supernatant using the
DuoSet enzyme-linked immunosorbent assay as per the manufacturers
instructions (R&D Systems, Inc). a -Defensins 1-3 (also known as the
human neutrophil peptides 1-3) were measured in BAL supernatant
(undiluted) using the Human HNP1-3 enzyme-linked immunosorbent
assay kit as per the manufacturers instructions (HK317; Hycult Biotech).
Western blot was performed on undiluted BAL from a subset of subjects
in the experimental cohort as described in e-Appendix 1 .
Statistical Analysis
Data were analyzed using Stata 11 (StataCorp LP) and reported as
median (quartile [Q]1, Q3). Statistical comparisons were performed
using the two-sample Wilcoxon rank sum (Mann-Whitney) test for
nonparametric data with P , .05 considered signifcant. Spearman rank
correlations were used to test relationships.
Results
Clinical Characteristics
Te children with PBB in both the experimental (n 5 21)
( Table 1 ) and validation (n 5 36) ( Table 1 ) cohorts com-
prised mainly infants and young children with similar
profles of moderate cough severity and a mean symp-
tom duration of . 20 weeks. Lung infamma tion was
present with increased BAL cellularity including neutro-
phils. Te validation cohort tended to have more males
and less intense airway neutrophilia than the experi-
mental cohort. Te control subjects were older in the
Downloaded From: http://journal.publications.chestnet.org/ by M Darwich on 10/12/2014
journal.publications.chestnet.org 1015
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1016 Original Research [ 1 4 6 # 4 CHEST OCTOBER 2 0 1 4 ]
experimental cohort, but ages were similar in the valida-
tion cohort.
Gene expression was measured in the entire experimental
cohort, and protein was assessed in 30 control subjects
and 21 subjects with PBB. In the validation cohort,
IL-1 b and IL-1RA protein were measured in 10 control
subjects and 29 PBB subjects, a -defensin 1-3 protein
was measured in fve control subjects and 24 subjects
with PBB, and gene expression was assessed in 10 con-
trol subjects and 36 subjects with PBB, except for C-X-C
motif receptor (CXCR) 2 (eight control subjects and
25 subjects with PBB) due to insuf cient remaining
samples.
Mediators in the Experimental Cohort
Tere was altered expression of IL-1 pathway members
IL1B , IL1R2 , IL1RN , PELI1 , and IRAK2 and the neutro-
phil chemokine receptor CXCR2 in PBB ( Table 1 ) but
not TNF- a /NF- k B pathway members TNFRSF1B
[control subjects: 1.03 (0.77, 1.35); PBB: 1.09 (0.94, 1.49);
P 5 .183] and NFKB2 [control subjects: 1.06 (0.63, 1.53);
PBB: 1.16 (0.84, 1.48); P 5 .419]. Gene expression of
DEFA1-3 was higher in PBB, but ELANE [control sub-
jects: 1.21 (0.49, 2.04); PBB: 1.45 (0.92, 2.06); P 5 .370]
and CTSG [control subjects: 0.91 (0.72, 1.29); PBB: 0.84
(0.57, 1.35); P 5 .299] were not changed, and DEFA4
was not detected.
Tere were higher levels of a -defensins 1-3 ( Fig 1A ,
Table 1 ), IL-1 b ( Fig 1B , Table 1 ), and IL-1RA protein
levels ( Table 1 ) in PBB, and a lower IL-1RA to IL-1 b
ratio ( Fig 1C , Table 1 ). Western blot confrmed the
presence of active IL-1 b ( Fig 1D ). Protein and gene
expression for IL-1 b were signifcantly correlated in
PBB (Spearman r 5 0.65, P 5 .001).
Mediator in the Validation Cohort
Signifcant diferences in the experimental cohort were
evaluated in a clinical validation cohort to relate medi-
ator levels to symptom status and disease recurrence.
Tere was signifcantly higher gene expression of IL1B ,
IL1R2 , IL1RN , DEFA1-3 , and CXCR2 in PBB ( Table 1 ).
IL-1 b protein was also signifcantly higher ( Table 1 ),
corresponding to a decreased IL-1RA to IL-1 b ratio in
PBB ( Table 1 ). However, gene expression of PELI1 and
IRAK2 did not reach signifcance between patients with
PBB and control subjects. In PBB, the level of gene and
protein expression of a -defensins 1-3, IL-1 b , and IL-1RA
were signifcantly correlated ( a -defensins 1-3: Spearman
r 5 0.46, P 5 .023; IL-1 b : Spearman r 5 0.68, P , .001;
IL-1RA: Spearman r 5 0.56, P 5 .002).
Clinical Signicance of Mediator Levels
Te relationship between mediators and clinical features
was explored by comparing symptomatic PBB (n 5 26)
with treated and resolved PBB (n 5 10), recurrent
(n 5 29) with nonrecurrent PBB (n 5 6), and investi-
gating correlations. Compared with symptomatic PBB,
resolved PBB showed much lower BAL neutrophils
[symptomatic PBB: 47% (12, 66); resolved PBB: 5% (2, 7)
neutrophils; P , .001], IL-1 b protein ( Fig 2A ), and
a -defensin 1-3 protein ( Fig 2B ), but little diference in
gene expression of IL-1 pathway or neutrophil-related
mediators (data not shown).
When children with recurrent PBB were evaluated at
baseline and compared with those with nonrecurrent
PBB, there were no signifcant diferences in a -defensins
1-3, IL-1 b , IL-1RA, or CXCR2 expression. However,
downstream IL-1 pathway signaling molecules PELI1
( Fig 2C ) and IRAK2 ( Fig 2D ) were signifcantly higher
at baseline in those with recurrent vs nonrecurrent PBB,
suggesting that activation of IL-1 signaling predicts PBB
recurrence. Similar trends were seen whether the partic-
ipants with recurrent and nonrecurrent PBB were
symptomatic or resolved at the time of sampling (for
resolved nonrecurrent PBB, n 5 3; resolved recurrent
PBB, n 5 7; and for symptomatic nonrecurrent PBB,
n 5 3; symptomatic recurrent PBB, n 5 22). PELI1 gene
Figure 1 IL-1 b expression in BAL samples from the experimental
cohort including 30 control subjects and 21 subjects with PBB. A-D,
(A) a -defensins 1-3 and (B) IL-1 b protein levels are higher in PBB,
(C) the IL-1RA to IL-1 b ratio is decreased in PBB with results being
presented as the median and the error bar as the upper interquartile range,
and (D) Pro-IL-1 b (17-kDa form) is present in the BAL supernatant of
subjects with PBB but not control subjects, demonstrated by western blot.
CTRL 5 control BAL sample; IL-1RA 5 IL-1 receptor antagonist;
PBB 5 protracted bacterial bronchitis BAL sample; STD 5 western
blot standard.
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journal.publications.chestnet.org 1017
Figure 3 A-C, Levels of IL-1 b protein detected in the BAL of children with PBB are correlated with (A) BAL neutrophilia, (B) cough duration, and
(C) severity of cough symptoms (C, n 5 8 score of 1; n 5 10 score of 2; and n 5 26 score of 3 or more, results are presented as mean and the error bar as
SEM). **Dunn post hoc P , .01 vs children with PBB and a cough score of 1. D, IL-1 b protein was signifcantly correlated with a -defensin 1-3 protein in
the BAL of children with PBB. See Figure 1 legend for expansion of abbreviations.
expression remained signifcantly higher in symptom-
atic recurrent PBB ( P 5 .03) or resolved recurrent PBB
( P 5 .04). IRAK2 gene expression was still higher in
symptomatic recurrent PBB ( P 5 .13) or resolved recur-
rent PBB ( P 5 .09), however, we lost signifcance with
the reduced sample size.
IL-1 b was signifcantly associated with the BAL neutro-
philia ( r 5 0.75, P , .0001) (Fig 3A) and duration of
cough in weeks ( r 5 0.29, P 5 .046) (Fig 3B). IL-1 b levels
were signifcantly higher in children with PBB and a
cough score of three or more ( P 5 .006) (Fig 3C). IL-1 b
and a -defensin 1-3 levels were signifcantly correlated
( Fig 3D ), and a -defensin 1-3 levels were also associated
with BAL neutrophils [Spearman r 5 0.48; P 5 .001].
Discussion
Tis study has identifed increased expression of
neutrophil-related mediators in PBB, including IL-1 path-
way members, neutrophil a -defensins, and the chemokine
receptor CXCR2. Elevated IL-1 b in PBB was con-
frmed in two clinical cohorts and was associated with
Figure 2 A-D, In symptomatic PBB, the levels of (A) a -defensins 1-3 and (B) IL-1 b protein in BAL are higher. IL-1 pathway signaling is associated
with recurrence of PBB shown by elevated gene expression of (C) PELI1 and (D) IRAK2. Results are presented as median and the error bar as the upper
interquartile range. IRAK 5 IL-1 receptor-associated kinase; PELI1 5 pellino-1. See Figure 1 legend for expansion of other abbreviations.
Downloaded From: http://journal.publications.chestnet.org/ by M Darwich on 10/12/2014
1018 Original Research [ 1 4 6 # 4 CHEST OCTOBER 2 0 1 4 ]
symptomatic PBB. Importantly, IL-1 b protein levels
were correlated with BAL neutrophilia, as well as the
duration and severity of cough symptoms. Additionally,
baseline expression (time of bronchoscopy) of IL-1
pathway signaling members IL-1 receptor associated
kinase (IRAK) 2 and pellino-1 (PELI1) was higher in
those children who were more likely to experience
disease recurrence (more than three episodes of wet
cough in the year following baseline bronchoscopy). Te
expression of a -defensins 1-3 was increased in PBB and
was signifcantly correlated with IL-1 b .
IL-1 b is an important mediator of the infammatory
response and host defense, however, dysregulated and
persistent IL-1 b release can harm the host and has been
linked to the pathogenesis of several diseases such as
rheumatoid arthritis, type 2 diabetes mellitus, and
atherosclerosis, as well as certain specifc autophagocytic
conditions.
11
IL-1 b is secreted as the inactive pro mole-
cule, proIL-1 b , and is then processed enzymatically to
activated IL-1 b . Typically this occurs via caspase-1 and
infammasome activation.
12
However, activation of
IL-1 b when released from neutrophils is not exclusively
dependent on caspase-1, as enzymes including neutro-
phil elastase are able to cleave IL-1 b into its active
form.
13
Released and active IL-1 b binds to its receptor
IL-1R1 and initiates a signaling cascade through MyD88
and IRAK1/IRAK4, assisted by IRAK2 and PELI1.
Efects of IL-1 b are blocked through its decoy receptor
IL-1 receptor 2 and receptor antagonist IL-1RA.
14
Tis
study shows that IL-1 b is associated with increased BAL
neutrophilia, suggesting that either neutrophils are the
source of this cytokine production, or alternatively that
IL-1 b induces neutrophil infltration into the lung. Either
way, our previous reports
6
detail increases in neutrophil
proteases including matrix metalloproteinase-9, which
may lead to activation of IL-1 b -mediated neutrophil infux.
IL-1 b has been observed to be elevated in other airway
diseases characterized by airway neutrophilia and/or
infection, such as neutrophilic asthma,
7
COPD,
15
cystic
fbrosis,
16
and non-cystic fbrosis bronchiectasis.
17
IL-1 b
is increased in stable COPD as well as acute exacerba-
tions, where it is associated with bacterial infection.
18

Bacterial infection is frequently detected in PBB and the
relationship between bacteria and IL-1 b activation in
PBB needs further research. In a number of models of
Pseudomonas pulmonary infection, IL-1 b production
occurs in response to bacterial infection
19
and is a deter-
minant of neutrophil infux likely through C-X-C motif
ligand (CXCL) 8. IL-1 plays a key role in coordinating
chemokine responses that lead to neutrophil infltration
in the lung.
20
Elevated IL-1 b in PBB is, therefore, consis-
tent with known host responses in neutrophilic airway
diseases, and suggests bacterial infection and neutrophil
infux are key features leading to ongoing IL-1 b release
and PBB symptoms.
Although PBB responds well to prolonged antibiotic
therapy, it can recur. In another infective disease, viral
encephalitis, levels of IL-1 b and IL-1RA were related to
prognosis.
21
We found that IL-1 b levels were highest in
symptomatic PBB and subjects with PBB who had
higher cough scores. IL-1 signaling molecules IRAK2
and PELI1 had signifcantly increased gene expression
at baseline in children who went on to develop recur-
rent PBB. Tis suggests that IL-1 b pathway activation
may determine PBB recurrence. PELI1 is important in
regulating the innate immune response of the epithe-
lium to rhinovirus infection, including CXCL8 produc-
tion and neutrophil recruitment.
22
IRAK2 is critical in
sustaining late-phase infammatory responses afer TLR
stimulation, which leads to increased cytokine produc-
tion.
23
We have previously reported the upregulation of
PELI1 and IRAK2 in response to rhinovirus infection of
human primary bronchial epithelial cells in COPD.
24

Tis evidence collectively suggests that IRAK2 and
PELI1 promote neutrophilic airway infammation trig-
gered by infection and IL-1 b and that this response is
dysregulated in PBB and contributes to disease
recurrence.
Tis study also reports increased expression of the CXCL8
high-af nity G-protein-coupled receptor CXCR2 and
the neutrophil a -defensins 1-3 in PBB. CXCR2 is
thought to be involved in uncontrolled neutrophil infux
into the airways in acute lung injury.
25
Defensins are
small arginine-rich cationic peptides that have antimi-
crobial activity against a broad range of pathogens and
exert their antimicrobial efect through membrane per-
meabilization. Te level of neutrophil a -defensins 1-3
was higher in symptomatic PBB and was signifcantly
correlated with IL-1 b , indicating that these molecules
may interact and infuence PBB pathogenesis. Indeed,
recent evidence implicates a -defensins in the release of
IL-1 b from lipopolysaccharide-primed macrophages
through the P2X7 receptor.
26
Intratracheal instillation of
a -defensins in mice leads to acute lung infammation
and dysfunction involving neutrophil infux and elas-
tase release.
27
a -defensins have a cytotoxic efect,
induce IL-8 and IL-1 b gene expression, IL-8 protein
production, and NF- k B binding activity in human
bronchial epithelial cells.
28
Expression of TNF/NF- k B
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journal.publications.chestnet.org 1019
Acknowledgments
Author contributions: K. J. B., J. W. U., A. B. C.,
and P. G. G. are guarantors of this paper and
take responsibility for the integrity of the
work as a whole, from inception to published
article, and conceived and designed the
study. K. J. B., A. B. C., J. M. M., and P. G. G.
contributed to data collection and interpreta-
tion; K. J. B. wrote the frst draf of the manu-
script and performed data analysis; K. J. B.
and P. G. G. contributed to the writing of the
manuscript; and K. J. B., J. W. U., S. T. Y.,
A. B. C., J. M. M., M. C., J. L. S., and P. G. G.
contributed to the editing, revising, and
reviewing of the manuscript.
Financial/nonfnancial disclosures: Te
authors have reported to CHEST the
following conficts: Dr Upham has been the
recipient of peer-reviewed research fund-
ingfrom the National Health and Medical
Research Council (Commonwealth of
Australia), received speaking fees from
AstraZeneca, Boehringer Ingelheim GmbH,
and Novartis Corp, and sits on the medical
advisory boards for Boehringer Ingelheim
GmbH, Te Menarini Group, and Novartis
Corp. Drs Baines, Yerkovich, Chang, Marchant,
Simpson, and Gibson and Ms Carroll have
reported that no potential conficts of interest
exist with any companies/organizations whose
products or services may be discussed in this
article.
Role of sponsors: Te study sponsors had no
role in study design; the collection, analysis,
and interpretation of data; the writing of the
report; or the decision to submit the paper
for publication. No form of payment was
given to anyone to produce the manuscript.
Other contributions: We are grateful to all of
the parents and children who participated in
this study. We also thank Brent Masters, Helen
Buntain, Paul Francis, Nigel Dore, and Alan
Isles for allowing us to recruit their patients
into the study; Carol Willis for maintaining
the database; and Sophie Anderson-James and
Helen Petsky for collecting the specimens and
clinical data. We also thank Naomi Fibbens,
Melinda Tooze, and Kellie Fakes for their tech-
nical assistance with laboratory measurements.
Additional information: Te e-Appendix
can be found in the Supplemental Materials
section of the online article.
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