Cytokines in exhaled breath condensate of children with asthma and cystic fibrosis

Department of Paediatric Pulmonology, University Hospital Maastricht, Maastricht, The Netherlands.
Annals of allergy, asthma & immunology: official publication of the American College of Allergy, Asthma, & Immunology (Impact Factor: 2.6). 03/2006; 96(2):349-55. DOI: 10.1016/S1081-1206(10)61247-1
Source: PubMed
ABSTRACT
Inflammatory mediators in exhaled breath condensate (EBC) indicate ongoing inflammation in the lungs and might differentiate between asthma and cystic fibrosis (CF).
To evaluate the presence, concentration, and short-term variability of TH1- and TH2-mediated cytokines (interferon-gamma [IFN-gamma], tumor necrosis factor alpha [TNF-alpha], interleukin 10 [IL-10], IL-5, IL-4, and IL-2) in EBC of children with asthma or CF and in controls and to analyze the discriminating ability of inflammatory markers in EBC between children with asthma or CF and controls.
Expired air was conducted through a double-jacketed glass tube cooled by circulating ice water. In 33 asthmatic children, 12 children with CF, and 35 control children, EBC was collected during tidal breathing. Cytokines were measured using flow cytometry.
Interleukin 2, IL-4, IFN-gamma, and IL-10 were detected in 16%, 16%, 11%, and 9%, respectively, of all samples in asthma and CF. Interleukin 5 and TNF-alpha were not detected in children with CF. Cytokine concentrations did not differ significantly in children with asthma vs CF. In controls, IFN-gamma, TNF-alpha, and IL-10 were detected in 9%, 14%, and 3%, respectively; IL-2, IL-4, and IL-5 were not detected in controls.
Cytokines such as IFN-gamma, TNF-alpha, IL-10, IL-5, IL-4, and IL-2 can be detected in EBC of children with asthma or CF. However, the concentrations found are close to the detection limits of the assay used. These findings emphasize the importance of developing more sensitive techniques for the analysis of EBC and of standardizing the EBC collection method.

Full-text

Available from: Philippe P R Rosias, Jan 06, 2014
Cytokines in exhaled breath condensate of
children with asthma and cystic fibrosis
Charlotte M. H. H. T. Robroeks, MD*; Quirijn Jo¨bsis, MD, PhD*; Jan G. M. C. Damoiseaux, PhD†;
Peter H. M. Heijmans, BA†; Philippe P. R. Rosias, MD*; Han J. E. Hendriks, MD, PhD*; and
Edward Dompeling, MD, PhD*
Background: Inflammatory mediators in exhaled breath condensate (EBC) indicate ongoing inflammation in the lungs and
might differentiate between asthma and cystic fibrosis (CF).
Objectives: To evaluate the presence, concentration, and short-term variability of T
H
1- and T
H
2-mediated cytokines (inter-
feron-
[IFN-
], tumor necrosis factor
[TNF-
], interleukin 10 [IL-10], IL-5, IL-4, and IL-2) in EBC of children with asthma
or CF and in controls and to analyze the discriminating ability of inflammatory markers in EBC between children with asthma
or CF and controls.
Methods: Expired air was conducted through a double-jacketed glass tube cooled by circulating ice water. In 33 asthmatic
children, 12 children with CF, and 35 control children, EBC was collected during tidal breathing. Cytokines were measured using
flow cytometry.
Results: Interleukin 2, IL-4, IFN-
, and IL-10 were detected in 16%, 16%, 11%, and 9%, respectively, of all samples in asthma
and CF. Interleukin 5 and TNF-
were not detected in children with CF. Cytokine concentrations did not differ significantly in
children with asthma vs CF. In controls, IFN-
, TNF-
, and IL-10 were detected in 9%, 14%, and 3%, respectively; IL-2, IL-4,
and IL-5 were not detected in controls.
Conclusions: Cytokines such as IFN-
, TNF-
, IL-10, IL-5, IL-4, and IL-2 can be detected in EBC of children with
asthma or CF. However, the concentrations found are close to the detection limits of the assay used. These findings
emphasize the importance of developing more sensitive techniques for the analysis of EBC and of standardizing the EBC
collection method.
Ann Allergy Asthma Immunol. 2006;96:349–355.
INTRODUCTION
Chronic lung diseases, such as asthma and cystic fibrosis
(CF), are an important source of morbidity and mortality in
children.
1,2
Airway inflammation and oxidative stress are
central features in the pathogenesis of asthma and CF.
3– 6
The
methods available for obtaining information about the pres-
ence of inflammation in the airways, such as induced sputum
or bronchoscopy with bronchoalveolar lavage and biopsies,
7
are too invasive to apply routinely or repeatedly. Monitoring
of disease activity is currently based on clinical features and
lung function test results.
7
If chronic airway inflammation is
demonstrated in a direct and specific manner, children may
be monitored more closely and, consequently, treatment may
be adjusted more appropriately to inflammation activity.
8
A
noninvasive method for measuring the nature and severity of
airway inflammation is to evaluate inflammatory mediators in
exhaled breath condensate (EBC) or in expired air.
8 –11
One of
the most developed and standardized inflammatory mediators
is exhaled nitric oxide (eNO) measurements.
12
The eNO level
is elevated in asthma, and it is related to clinical severity and
corticosteroid dosage. Recently, it was shown that eNO mea-
surement is helpful in monitoring and improving asthma
outcomes.
13
Cytokine responses are important controlling and, there-
fore, signaling elements of inflammatory and immune reac-
tions that occur in the course of a disease.
14
Allergic asthma
is considered a T
H
2-mediated disease characterized by the
production of interleukin 4 (IL-4), IL-5, and IL-6. In allergic
asthma, eosinophils become one of the major effector cells
when inflammation of the lungs is chronic. Interleukin 5 is
the primary cytokine responsible for the selective differenti-
ation of the eosinophils. In contrast, the nature of inflamma-
tion in CF is dominated by neutrophils.
15,16
The altered cyto-
kine production in patients with CF includes decreased IL-10
and increased tumor necrosis factor
(TNF-
), IL-6, and
IL-8 levels.
15
In patients with CF, the T
H
1 response, charac-
terized by the production of IL-2, interferon-
(IFN-
), and
TNF-
, predominates, as has been confirmed in patients with
chronic Pseudomonas aeruginosa lung infection.
14
Despite rapidly increasing data on inflammatory markers
in EBC, until now, only 2 research groups reported on the
detection of the cytokines IL-4,
17,18
TNF-
,
17
and IFN-
18
in
EBC of children with asthma, and 1 group reported on IL-8
in EBC of children with CF.
19
If cytokines can be detected in
* Department of Paediatric Pulmonology, University Hospital Maastricht,
Maastricht, the Netherlands.
† Department of Clinical and Experimental Immunology, University Hospi-
tal Maastricht, Maastricht, the Netherlands.
‡ Department of Paediatrics, Maasland Hospital, Sittard, the Netherlands.
Received for publication December 21, 2004.
Accepted for publication in revised form July 29, 2005.
VOLUME 96, FEBRUARY, 2006 349
Page 1
EBC, they might be important for monitoring airway inflam-
mation and for differentiating distinct diseases.
In an earlier study we did not demonstrate the presence of
IL-6, IL-8, TNF-
, or soluble p75 TNF receptor in EBC of
asthmatic children,
20
possibly owing to limitations of the
enzyme-linked immunosorbent assay, with a lower detection
limit of 20 pg/mL. These cytokines do not reflect a possible
T
H
1/T
H
2 imbalance. Therefore, the aim of the present study
was to evaluate (1) the presence, concentration, and short-
term variability of the T
H
1- and T
H
2-mediated cytokines
IFN-
, TNF-
, IL-10, IL-5, IL-4, and IL-2 in EBC of chil-
dren with asthma or CF and in controls and (2) the discrim-
inating ability of these inflammatory markers in children with
asthma or CF and in control children.
METHODS
Participants
Children aged 5 to 17 years with known asthma or CF were
recruited at the Department of Paediatric Pulmonology, Uni-
versity Hospital Maastricht. Asthma was defined as relevant
symptoms combined with the presence of reversibility with
use of a bronchodilator (increase forced expiratory volume in
1 second [FEV
1
]of8% of predicted) or bronchial hyper-
responsiveness (provocation concentration that caused a de-
crease in FEV
1
of 20% of 8 mg/mL of histamine). Cystic
fibrosis was defined as a combination of clinical features and
an abnormal sweat test result (chloride concentration 60
mmol/L). The use of antibiotics was allowed in the popula-
tion with CF. Healthy children aged 5 to 18 years were
recruited from the Outpatient Clinic of Paediatric Pulmonol-
ogy, University Hospital Maastricht. The main reason for
consultation was enuresis nocturna and constipation. The
ISAAC (International Study of Asthma and Allergies in Chil-
dren) questionnaire was used to exclude children with any
(history of) airway or allergy complaints. Exclusion criteria
for all the populations consisted of (1) diseases that might
interfere with the result of the study (eg, upper airway infec-
tion, cor vitium, anatomic anomalies of the airways, and other
chronic inflammatory diseases, such as Crohn disease and
rheumatoid arthritis), (2) mental retardation, (3) inability to
perform the EBC procedure properly, and (4) active smoking.
In all the participants, sex, age, height, weight, duration of
disease, therapy, and comorbidity were documented. Patients
with and without an exacerbation were included. Informed
consent was obtained from the parents of all the children
participating in this study, which was approved by the Maas-
tricht Ethics Committee of Maastricht University.
Lung Function Tests
Bronchodilator medication use was stopped before lung func-
tion testing: short-acting bronchodilators at least 8 hours
before and long-acting bronchodilators at least 36 hours be-
fore the test. Dynamic spirometry was performed by means of
a pneumotachograph (Masterlab; Jaeger, Wu¨rzberg, Ger-
many), with measurement of FEV
1
and forced vital capacity
(FVC) according to the standards of the European Respira-
tory Society.
21
The highest FEV
1
and FVC values of 3 forced
expiratory maneuvers were used for data analysis. The bron-
chodilating response was evaluated 15 minutes after admin-
istration of a short-acting bronchodilator and is expressed as
the increase in FEV
1
compared with the predicted value of
FEV
1
. Reversibility was defined as an increase in FEV
1
of
more than 8%. This response was not analyzed in healthy
children. Lung volumes (residual volume, intrathoracic gas
volume, and total lung capacity) were evaluated using body
plethysmography (Jaeger).
EBC Collection
Samples of EBC were obtained from children during tidal
breathing while wearing a nose clip, as described previous-
ly.
20
A 2-way nonrebreathing valve (series 1420; Hans Ru-
dolph Inc, Kansas City, KS) also served as a saliva trap. The
expired air was conducted through a 35-cm double-jacketed
glass tube cooled by circulating ice water. The collected
condensate was directly frozen in small plastic tubes by
submerging the tubes in isopentane and dry ice (80°C) and
was stored at the same temperature for a maximum of 4
weeks.
Cytokine Analysis
Cytokines were assayed using flow cytometry (CBA; BD
Biosciences, San Diego, CA).
22
Samples of EBC in a
volume of 50
L were incubated with beads of the human
T
H
1/T
H
2 cytokine CBA kit (IFN-
, TNF-
, IL-10, IL-5,
IL-4, and IL-2) in combination with phycoerythrin-conju-
gated detection antibodies. Recombinant cytokine stan-
dards were used in different concentrations (10 –10,000
pg/mL) to prepare the respective standard curves. Next,
data acquisition was performed using a flow cytometer
(FACSCalibur; BD Biosciences), and data were analyzed
using analysis software (CBA; BD Biosciences). The de-
tection limit for each cytokine was determined using the
mean fluorescence intensity of the phycoerythrine signal
of a negative control (0 pg/mL) measured at the University
Hospital Maastricht, plus 1 SD given by BD Biosciences.
These SDs were calculated after measuring the fluores-
cence of 20 replicates of a negative control. The corre-
sponding concentrations of the mean fluorescence inten-
sity of the phycoerythrine signals were calculated using the
formula of the standard curves created by us.
To determine intra-assay reproducibility, standard solu-
tions with concentrations of 2.5, 5, and 10 pg/mL were
analyzed 3 times in the same assay. The coefficients of
variation (CVs) (SD divided by the mean multiplied by
100%) at 2.5, 5, and 10 pg/mL were calculated. In addition,
short-term intrasubject reproducibility was evaluated in 30
individuals (10 patients with asthma, 10 patients with CF, and
10 controls) by analyzing the variation between 2 different
EBC samples at a 30-minute interval.
Statistical Analysis
All the data are expressed as mean SEM. Patient charac-
teristics (sometimes after logarithm transformation) were nor-
350 ANNALS OF ALLERGY, ASTHMA & IMMUNOLOGY
Page 2
mally distributed. Cytokine concentrations were not normally
distributed. Two-sided independent-samples t tests and
Mann-Whitney U tests were used for analysis. P .05 was
considered statistically significant.
RESULTS
Clinical Characteristics
Eighty children 5 years and older were included in this study:
33 asthmatic children, 12 children with CF, and 35 controls
(Table 1). Children with CF were older than the other pop-
ulations (P .002) and were taller than asthmatic children
(P .004). The healthy and asthmatic populations had com-
parable FEV
1
and FEV
1
/FVC values. In asthma, as expected,
greater reversibility while taking a bronchodilator and more
atopy were present than in children with CF (P .01 for
both). In the CF group, 3 patients were admitted to the
hospital with an exacerbation. Half of the children with CF
were colonized with P aeruginosa, and 1 child had allergic
bronchopulmonary aspergillosis.
EBC Collection
The duration of EBC collection was not significantly differ-
ent among the populations. The mean EBC collection times
in the asthma and CF populations were 19 0.4 and 20 2
minutes, respectively, and the mean volumes of collected
EBC were 1,150 100 and 1,515 180
L, respectively. In
the healthy population, the collection time and the volume of
collected EBC were 29 0.4 minutes and 2,900 219
L,
respectively. In the CF and healthy populations, more EBC
was produced per minute than in asthmatic children (P .04
and P .001, respectively). There were no adverse effects.
Exhaled Cytokines
In asthma and CF, IFN-
, IL-10, IL-4, and IL-2 were detected
(Fig 1 and Table 2). The observed concentrations were close
to the lower detection limits of the assay. In EBC of children
with asthma, all 6 measured cytokines were detected. How-
ever, IFN-
, IL-4, and IL-2 were detected in 12% to 15% of
the children, whereas TNF-
, IL-10, and IL-5 were found in
only 3% to 6%. There was no relationship between the
detection of cytokines or the cytokine levels and the use of
inhaled corticosteroids. In the CF population, IFN-
, IL-10,
IL-4, and IL-2 were found in 8% to 25% of the samples;
TNF-
and IL-5 were not detected. Cytokine concentrations
did not differ significantly in children with asthma vs CF. In
the healthy population, IFN-
, TNF-
, and IL-10 were de-
tected in 9%, 14%, and 3% of the individuals, respectively.
There was no detection of IL-5, IL-4, or IL-2 in any of these
EBC samples (Table 3). Because the controls were measured
in a separate analysis, detection limits slightly differ from
those of the diseased children.
Short-term Intrasubject and Intra-assay Reproducibility
The short-term intrasubject reproducibility of cytokines of
all the children ranged from 10% to 32% (Table 4). Most
reproducible data were obtained with measurements of
Table 1. Clinical Characteristics of the Study Population*
Characteristics
Asthma group
(n 33)
Cystic fibrosis
group (n 12)
Control group
(n 35)
Age, y 9.7 0.5† 13.3 1.0‡ 10.0 0.5
Height, cm 134.5 2.7† 151.4 5.9 141.5 3.0
Weight, kg 33.0 2.1 39.3 3.8 34.7 1.9
Sex, M/F, No. 22/11 7/5 19/16
IgE, kU/L 600 219† 126 67 NA
Exacerbation of disease, No. (%) 8 (24) 3 (25) NA
Phadiatop positive, No. (%) 25 (76)† 1 (8) NA
Active eczema, No. (%) 4 (12) NA NA
Allergic rhinitis, No. (%) 6 (18) NA NA
Inhaled corticosteroid use, No. (%) 22 (67)† 3 (25) NA
Corticosteroid dose,
g 536 89† 100 0NA
Antibiotic use, No. (%) NA 7 (58) NA
Reversibility, % predicted§ 21† 0 NA
FEV
1
, % predicted 101.5 2.9† 54.9 6.6‡ 101.3 1.5
FEV
1
/FVC 83.0 3.0† 69.8 3.8‡ 87.6 1.1
TLC, % predicted NA 95.2 4.8 NA
RV, % predicted NA 182.4 18.8 NA
ITVG, % predicted NA 114.7 7.3 NA
Abbreviations: FEV
1
, forced expiratory volume in 1 second; FVC, forced vital capacity; ITGV, intrathoracic gas volume; NA, not available; RV,
residual volume; TLC, total lung capacity.
* Data are given as mean SEM except were indicated otherwise.
P .05, asthma vs cystic fibrosis.
P .05, cystic fibrosis vs controls.
§ Reversibility is defined as the presence of a change in FEV
1
of more than 8% after use of a bronchodilator.
VOLUME 96, FEBRUARY, 2006 351
Page 3
IL-10 and IL-5 (CVs, 13% and 10%, respectively). The
largest variation was found in IFN-
data (CV, 22%–
43%). The mean (range) CVs of samples containing cyto-
kine concentrations of 2.5 vs 5 pg/mL were comparable:
31% (22%– 48%) vs 35% (19%– 46%) (Table 5). The
intra-assay reproducibility of samples containing cytokine
concentrations of 10 pg/mL was significantly better (mean
CV, 20%; range, 13%–27%; P .05). The intra-assay
reproducibility profile of the cytokines was in accordance
with the profile of in vivo intrasubject reproducibility.
DISCUSSION
The main finding of this study was the detection of the
cytokines IFN-
, TNF-
, IL-10, IL-5, IL-4, and IL-2 in EBC
originating from not only children with asthma but also
children with CF, except for TNF-
and IL-5. However,
cytokine concentrations were low and detection was possible
only in a few children. No differences in cytokine concen-
trations were found between asthma and CF. To our knowl-
edge, this is the first EBC study demonstrating IL-4 and
Figure 1. Percentage of samples in which cytokines were detected in the asthma (n 33) and cystic fibrosis (CF) (n 12) patient populations. Tumor necrosis
factor
(TNF-
) and interleukin 5 (IL-5) were detected only in exhaled breath condensate of children with asthma. IFN-
indicates interferon-
.
Table 2. Concentrations of Cytokines Detected in Exhaled Breath Condensate of Children With Asthma or Cystic Fibrosis and Percentage of
Samples With Positive Detection of Cytokines
Detection
limit,
pg/mL
Asthma group (n 33) Cystic fibrosis group (n 12) Total (n 45)
Positive
samples,
%
Concentration,
mean SEM
(range), pg/mL
Positive
samples,
%
Concentration,
mean SEM
(range), pg/mL
Positive
samples,
%
Concentration,
mean SEM
(range), pg/mL
IFN-
5.2 12 5.8 0.4 (5.2–6.7) 8 7.2 11 6.1 0.4 (5.2–7.2)
TNF-
1.3 6 1.3 0.0 (1.3–1.3) 0 NA 4 1.3 0.0 (1.3–1.3)
IL-10 2.0 3 5.6 25 2.3 0.1 (2.0–2.5) 9 3.1 0.8 (2.0–5.6)
IL-5 1.3 3 1.3 0 NA 2 1.3
IL-4 2.5 15 2.8 0.1 (2.7–3.1) 17 2.7 0.0 (2.7–2.7) 16 2.8 0.1 (2.7–3.1)
IL-2 1.4 12 3.5 1.9 (1.4–9.1) 25 1.4 0.0 (1.4–1.4) 16 2.6 1.1 (1.4–9.1)
Abbreviations: IFN-
, interferon-
; IL-10, interleukin 10; NA, not applicable; TNF-
, tumor necrosis factor
.
352 ANNALS OF ALLERGY, ASTHMA & IMMUNOLOGY
Page 4
IFN-
in EBC of children with CF and IL-2 and IL-5 in EBC
of asthmatic children. It was shown that EBC collection is a
safe and feasible method in children with asthma and CF 5
1
2
years and older, even during an exacerbation. All the patients
completed the EBC collection procedure, and no adverse
effects were reported.
One would expect different cytokine profiles in EBC orig-
inating from children with asthma vs CF, ie, an increased T
H
2
vs T
H
1 response. However, these differences were not found,
probably owing to the limited number of positive cytokine
detections. In addition, it was not possible to correlate these
findings with patient characteristics or to detect relationships
with severity of disease, the use of inhaled corticosteroids, or
the coexistence of atopy (data not shown).
Data on the variability of cytokines demonstrated accept-
able reproducibility of IL-5 and IL-10 but a much larger
variation of TNF-
and IFN-
. No other studies on the
variability of inflammatory biomarkers in EBC exist in chil-
dren. Limited data exist in adults. Reproducibility of inflam-
matory markers in EBC has been studied in few subjects.
23–27
The mean CV of eNO described was 9.5% 4.7%,
23
which
is close to the reproducibility of IL-5 in our study. It is likely
that increased standardization and the use of a more optimal
coating and condenser system will increase the detection and
reproducibility of cytokines in EBC.
Data on EBC in children are scant.
18 –20
Overall, the lack of
cytokine detection in EBC of children with asthma or CF may
be due to the following aspects. First, the inner coating of the
condenser systems affected the detection of 8-isoprostane and
albumin in EBC. The use of a silicone or glass coating
resulted in significantly better detection of albumin and 8-iso-
prostane in EBC than Teflon, aluminum, and polypropylene
coatings.
28,29
Based on these results, we used a glass con-
denser coating in this study. However, it is as yet unclear
which coating is optimal for measuring cytokines. Second,
cytokine concentrations in EBC were close to the detection
limits of the assay used. In addition, no emphasis should be
given to absolute quantitative values of cytokines in this
study. Third, the study population had well-controlled dis-
ease, when cytokines may be detectable only during an ex-
acerbation or in cases of uncontrolled disease.
Based on the results of the present study, the following
aspects should be considered when measuring cytokines in
EBC. First, more sensitive analytical methods are needed to
show differences in cytokine concentrations. Second, tech-
niques to concentrate EBC volume may increase cytokine
concentrations and, thereby, measurements may be more
reliable. We concentrated several samples 10-fold by means
of centrifugation through a 30-kDa molecular filter, but this
procedure did not really affect cytokine concentrations mea-
sured (data not shown), possibly because of the adhesion of
cytokines to the filter device. Other concentration methods,
such as freeze-drying combined with centrifugation, may be
beneficial. Third, to prevent degradation of cytokines before
analysis, samples may be protected by adding a protease
inhibitor or bovine serum albumin.
24
This study stresses the importance of further improvement
and standardization of the EBC technique, as recently re-
viewed.
8
There are indications that the currently used con-
densers are not very effective in the sampling of EBC.
28,29
A
task force of the European Respiratory Society and the Amer-
ican Thoracic Society is working on the standardization of the
EBC technique. Such recommendations on standardization of
another inflammatory marker—NO in exhaled air—already
exist and were of great value in the development and appli-
cation of this marker in clinical studies.
12
Consequently, some
important recent studies demonstrated that eNO measurement
is valuable for adjusting maintenance treatment in asthma
13
and is a predictor of asthma relapse or asthma exacerbations.
Such studies are lacking on EBC. However, eNO has limited
value for a diagnosis of asthma and probably is not very
useful in other chronic lung diseases, such as CF and chronic
obstructive pulmonary disease. After further standardization,
the noninvasive EBC technique may be a valuable additional
tool for assessing airway inflammation in various chronic
lung diseases.
Altogether, cytokines are potentially important inflamma-
tory markers for the diagnosis of inflammatory airway dis-
eases and the monitoring of chronic airway inflammation as
a result of the primary etiology of asthma or CF.
8,9,30,31
Al-
though we did not demonstrate the presence of IFN-
,
Table 3. Concentrations of Cytokines Detected in Exhaled Breath
Condensate of 35 Controls and Percentage of Samples With
Positive Detection of Cytokines
Detection
limit,
pg/mL
Positive
samples, %
Concentration,
mean SEM
(range), pg/mL
IFN-
2.7 9 2.9 0.1(2.7–3.0)
TNF-
1.1 14 1.6 0.1(1.3–2.0)
IL-10 1.6 3 1.8
IL-5 1.1 0 NA
IL-4 2.5 0 NA
IL-2 2.5 0 NA
Abbreviations: IFN-
, interferon-
; IL-10, interleukin 10; NA, not ap-
plicable; TNF-
, tumor necrosis factor
.
Table 4. Short-term Intrasubject Reproducibility
Coefficient of variation, %*
Control
group
Asthma
group
Cystic
fibrosis
group
Total
IFN-
23 43 22 32
TNF-
29 21 33 28
IL-10 13 16 10 13
IL-5 0 11 6 10
IL-4 0 27 14 21
IL-2 0 26 13 20
Abbreviations: IFN-
, interferon-
; IL-10, interleukin 10; TNF-
, tumor
necrosis factor
.
* Standard deviation divided by the mean value multiplied by 100%
VOLUME 96, FEBRUARY, 2006 353
Page 5
TNF-
, IL-10, IL-5, IL-4, and IL-2 in EBC of children with
asthma or CF, this study emphasizes the importance of opti-
mizing the EBC collection method and the development of
more sensitive assays or techniques with increased sensitivity
for T-cell cytokines.
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Table 5. Intra-assay Reproducibility at Various Cytokine Concentrations
2.5 pg/mL 5.0 pg/mL 10.0 pg/mL
Concentration,
mean, pg/mL
CV, %*
Concentration,
mean, pg/mL
CV, %*
Concentration,
mean, pg/mL
CV, %*
IFN-
3.6 48 5.6 46 8.8 23
TNF-
3.6 31 5.5 44 8.3 27
IL-10 3.7 32 5.4 30 9.6 13
IL-5 3.8 22 5.7 31 9.0 19
IL-4 3.9 33 5.1 38 7.0 20
IL-2 2.6 22 4.6 19 8.3 16
Total 3.5 31 5.3 35 8.5 20
Abbreviations: CV, coefficient of variation; IFN-
, interferon-
; IL-10, interleukin 10; TNF-
, tumor necrosis factor
.
* Standard deviation divided by the mean value multiplied by 100%
354 ANNALS OF ALLERGY, ASTHMA & IMMUNOLOGY
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Requests for reprints should be addressed to:
Charlotte M. H. H. T. Robroeks, MD
Department of Paediatric Pulmonology
University Hospital Maastricht
PO Box 5800
6202 AZ Maastricht, the Netherlands
E-mail: crob@paed.azm.nl
VOLUME 96, FEBRUARY, 2006 355
Page 7
  • Source
    • "Additionally,Colombo et al.,demonstrated IL-8 correlated with clinical biomarkers of cystic fibrosis by biochip array[31]. However, more sensitive assays, such as multiple reaction monitoring (MRM), are needed as these cytokines are often near immunological reagent dependent assay's detection limits[29]. These examples emphasize the potential for using EBC as a medium to monitor lung inflammatory mediators. "
    [Show abstract] [Hide abstract] ABSTRACT: Exhaled breath condensate (EBC) has been established as a potential source of respiratory biomarkers. Compared to the numerous small molecules identified, the protein content of EBC has remained relatively unstudied due to the methodological and technical difficulties surrounding EBC analysis. In this review, we discuss the proteins identified in EBC, by mass spectrometry, focusing on the significance of those proteins identified. We will also review the limitations surrounding mass spectral EBC protein analysis emphasizing recommendations to enhance EBC protein identifications by mass spectrometry. Finally, we will provide insight into the future directions of the EBC proteomics field.
    Full-text · Article · Sep 2014
  • Source
    • "These VOCs were not correlated with CF genotype, treatment with ICS and deoxyribonuclease, or atopic status [117]. EBC TNF-alpha, interferon-gamma, IL-2, IL-4, IL-5, and IL-10 levels can be detected in CF or asthma children [118] with reduction of EBC IL-8 levels and increasing of pH values after antibiotic treatment [119]. EBC pH values show a significant mean difference between asthmatics and nonasthmatics [120] and its variability is not influenced by clinical status changes [121]. "
    [Show abstract] [Hide abstract] ABSTRACT: Today, exhaled nitric oxide has been studied the most, and most researches have now focusd on asthma. More than a thousand different volatile organic compounds have been observed in low concentrations in normal human breath. Alkanes and methylalkanes, the majority of breath volatile organic compounds, have been increasingly used by physicians as a novel method to diagnose many diseases without discomforts of invasive procedures. None of the individual exhaled volatile organic compound alone is specific for disease. Exhaled breath analysis techniques may be available to diagnose and monitor the diseases in home setting when their sensitivity and specificity are improved in the future.
    Full-text · Article · Sep 2013
  • Source
    • "For cytokine analysis, a shift in the Th1/Th2 ratio usually accompanies with varied immune response in pathological pulmonary conditions. Examples of such approach have been reported in determining the IFN- γ(Th1)/IL-4(Th2) ratio [56, 59]. Systematic approaches, such as proteomic analysis of EBC, have been previously used and may provide a more detailed overall view about cytokine profile in the EBC. "
    [Show abstract] [Hide abstract] ABSTRACT: Exhaled breath condensate (EBC) has been increasingly studied as a noninvasive research method for sampling the alveolar and airway space and is recognized as a promising source of biomarkers of lung diseases. Substances measured in EBC include oxidative stress and inflammatory mediators, such as arachidonic acid derivatives, reactive oxygen/nitrogen species, reduced and oxidized glutathione, and inflammatory cytokines. Although EBC has great potential as a source of biomarkers in many lung diseases, the low concentrations of compounds within the EBC present challenges in sample collection and analysis. Although EBC is viewed as a noninvasive method for sampling airway lining fluid (ALF), validation is necessary to confirm that EBC truly represents the ALF. Likewise, a dilution factor for the EBC is needed in order to compare across subjects and determine changes in the ALF. The aims of this paper are to address the characteristics of EBC; strategies to standardize EBC sample collection and review available analytical techniques for EBC analysis.
    Full-text · Article · Dec 2012 · The Scientific World Journal
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