1. health and fitness
2. disease
3. asthma

Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 1107
IgE sensitization against food
allergens - Natural history, relation
to airway inflammation and asthma
ANTONIOS PATELIS
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2015
ISSN 1651-6206
ISBN 978-91-554-9256-4
urn:nbn:se:uu:diva-251396
Dissertation presented at Uppsala University to be publicly examined in Hedstrandssalen, Ing
70, Akademiska Sjukhuset, Uppsala, Tuesday, 2 June 2015 at 13:15 for the degree of Doctor
of Philosophy (Faculty of Medicine). The examination will be conducted in Swedish. Faculty
examiner: Professor Eva Rönmark.
Abstract
Patelis, A. 2015. IgE sensitization against food allergens. Natural history, relation to airway
inflammation and asthma. Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 1107. 66 pp. Uppsala: Acta Universitatis Upsaliensis.
ISBN 978-91-554-9256-4.
Background: According to recent studies in children, IgE sensitization not only against
perennial allergens, but also against food allergens, is related to asthma risk and increased airway
inflammation. During the last decade, a new technique for IgE determination based on allergen
components has become available, but its use in epidemiological studies has been limited.
Aims: To investigate the relationship between the pattern of IgE sensitization to allergen
components and the prevalence of asthma, airway inflammation and hyperresponsiveness in a
population-based setting. To examine the relationship of IgE sensitization to allergen extract,
and airway inflammation, airway hyperresponsiveness and blood eosinophilia in asthmatics. To
examine the natural history of IgE sensitization to food allergens in adults. To compare extractbased and component-based IgE measurements in relation with new-onset respiratory disease
and airway inflammation and hyperresponsiveness.
Methods:The present thesis is based on cross-sectional and longitudinal analyses of the
adult, the population-based study ECRHS (European Community Health Survey) and a crosssectional, observational study of young subjects with asthma. IgE sensitization was examined
by means of both extract-based and component-based tests. Airway inflammation was assessed
by exhaled NO and airway hyperresponsiveness with methacholine test.
Results:IgE sensitization to food allergens independently related to increased airway
inflammation in both a population-based study and a study of asthmatics. Furthermore, a relation
was found with increased blood eosinophils in asthmatics. The decrease in prevalence of IgE
sensitization against food allergens during a 9-year follow-up was larger than the decrease
of aeroallergens. Subjects with IgE sensitization to both cat extract and components showed
more frequent airway inflammation, greater bronchial responsiveness and higher likelihood of
developing asthma and rhinitis than subjects with IgE sensitization only to cat extract.
Conclusions: The presence of IgE antibodies against food allergens was independently
associated with airway and systemic inflammation. Both aeroallergens and food allergens
should be examined in order to understand the signaling of local and systemic inflammation in
asthma. Prevalence of IgE sensitization to food decreased in adults to a larger extent than IgE
sensitization against aeroallergens. Measurement of IgE sensitization to cat allergen components
appears to have a higher clinical value than extract-based measurement
Keywords: allergen components, extract-based, exhaled NO, airway hyperresponsiveness,
blood eosinophil count, IgE sensitization, food hypersensitivity, food allergens, perennial
allergens, natural history, markers of systemic inflammation, markers of local inflammation
Antonios Patelis, Department of Medical Sciences, Akademiska sjukhuset, Uppsala
University, SE-75185 Uppsala, Sweden.
© Antonios Patelis 2015
ISSN 1651-6206
ISBN 978-91-554-9256-4
urn:nbn:se:uu:diva-251396 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-251396)
When you feel that you want to quit
after a bad result think that nobody
managed to walk without falling,
nobody managed to run without
stopping to get some breaths.
List of papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I
Patelis, A., Gunnbjörnsdottir, M., Malinovschi, A., Matsson, P.,
Onell, A., Högmann, M., Alving, K., Janson, C., (2012), Population-based study of multiplexed IgE sensitization in relation to
asthma, exhaled nitric oxide and bronchial responsiveness.
JACI 130(2):397-402 e 392
II
Patelis, A., Janson, C., Borres, M.P., Nordvall, L., Alving, K.,
Malinovschi, A., (2014), Aeroallergen and food sensitization
and local and systemic inflammation in asthma. Allergy
69(3):380-7
III
Patelis, A., Gunnbjörnsdottir, M., Borres, M.P., Burney, P.,
Gisslasson, T., Torén, K., Forsberg, B., Alving, K., Malinovschi, A., Janson, C., (2014), Natural history of perceived
food hypersensitivity and IgE sensitization to food allergens in
a cohort of adults. PLoS One 10; 9(1):e85333
IV
Patelis, A., Gunnbjörnsdottir, M., Alving, K., Borres, M.P.,
Högmann, M., Janson, C., Malinovschi, A., (2015), Allergen
extract vs. component sensitization and airway inflammation,
responsiveness and new-onset respiratory disease (Submitted)
Reprints were made with permission from the respective publishers.
Contents
Introduction ................................................................................................... 11 IgE sensitization and asthma .................................................................... 11 IgE sensitization and airway inflammation .............................................. 11 IgE sensitization and airway hyperresponsiveness................................... 12 IgE sensitization and blood eosinophil count ........................................... 12 Importance of type of IgE sensitization ................................................... 13 IgE sensitization against aeroallergens and respiratory disease ............. 13 IgE sensitization against food allergens and adverse food reactions ..... 14 IgE sensitization against food allergens and respiratory disease ........... 14 Methods for examining IgE sensitization ................................................. 15 Extract-based tests ............................................................................... 15 Component-based tests ........................................................................ 15 Extract-based test vs. component-based tests ...................................... 16 Aims .............................................................................................................. 18 Material and methods .................................................................................... 19 Population................................................................................................. 19 Questionnaires .......................................................................................... 21 Definition of asthma ................................................................................. 22 Definition of rhinitis ................................................................................. 22 Definition of food hypersensitivity .......................................................... 22 Presence and degree of IgE sensitization ................................................. 22 Blood eosinophil count............................................................................. 28 Measurements of exhaled NO .................................................................. 28 Smoking history ....................................................................................... 28 Lung function ........................................................................................... 28 Methacholine challenge test ..................................................................... 29 Statistical methods ........................................................................................ 30 Ethics ............................................................................................................ 32 Results ........................................................................................................... 33 Paper I ...................................................................................................... 33 Paper II ..................................................................................................... 35 Paper III .................................................................................................... 36 Paper IV ................................................................................................... 39 Discussion ..................................................................................................... 43 Asthma, degree of inflammation and airway hyperresponsiveness in
relation to pattern of IgE sensitization in a general population and in a
cohort of asthmatics ................................................................................. 43 IgE sensitization against food allergens and exhaled NO and blood
eosinophil levels ....................................................................................... 44 Methodological issues and clinical utility of multiplex, componentbased vs. limited panel of extract-based IgE testing ................................ 45 Cat and timothy components’ predictive value for the risk of higher
airway inflammation and hyperresponsiveness and development of
new-onset respiratory disease................................................................... 46 Change in the prevalence of IgE sensitization to food allergens.............. 47 Reported food hypersensitivity symptoms after food intake .................... 48 Study limitations ........................................................................................... 49 Future research .............................................................................................. 50 Conclusions ................................................................................................... 51 Clinical implications ..................................................................................... 53 Aknowledgements......................................................................................... 54 References ..................................................................................................... 56 Abbreviations
B-Eos
BMI
ECRHS
FeNO
FEV1
FVC
IgE
IL
iNOS
ISAC
ISU
MIDAS
PD20 FEV1
sIgE
Blood eosinophil count
Body mass index
European Community Respiratory Health Study
Fraction of exhaled nitric oxide
Forced expiratory volume in one second
Forced vital capacity
Immunoglobulin E
interleukin
inducible NO synthase
Immuno-solid-phase allergen chip
ISAC standardized units
Minimally-invasive diagnostics for asthma allergic
diseases
Cumulative dose of methacholine causing a 20 %
reduction in FEV1
specific IgE antibodies in serum
9
Introduction
IgE sensitization and asthma
Asthma is a significant health problem affecting 300 million people of all
ages worldwide (1). The prevalence of asthma is increasing in most countries, especially among children. Consequences of asthma can be heavy for
the individual due to limitation of daily activities and poorer quality of life,
but also for society due to healthcare costs. In some cases, uncontrolled
asthma without proper treatment can be fatal (1). According to a UK study,
deaths because of anaphylaxis affected mostly patients treated for asthma,
and in most of these individuals, asthma was poorly controlled (2). Our understanding of asthma has progressed greatly in the last decades and modern
treatment can be effective, allowing asthmatics to lead normal lives (3).
However, asthma control – the goal of asthma treatment – is not achieved in
more than 50 % of patients (4, 5).
The prevalence of allergic diseases is also increasing in many countries
and it is estimated that 25 % of the population worldwide suffers from allergies (6). Allergic sensitization is a known risk factor for asthma (7) and sensitization to multiple allergens is associated with a high prevalence (8) and
severity of asthma (9). Synthesis of IgE against allergens is a prerequisite for
developing allergic diseases such as allergic rhinitis and asthma (10), although many sensitized individuals do not develop symptoms (11). Moreover, there are subjects who have non-allergic asthma (3). The prevalence of
atopy in asthmatics approaches 80 % in a national survey in the United
States and could be the causative factor in over 50 % of asthma cases (7).
The European Community Respiratory Health Study (ECRHS) found a
prevalence of atopy of 70 % in asthmatics (12) and an overall attributable
fraction of asthma symptoms caused by atopy of 30 % (13).
IgE sensitization and airway inflammation
One of the key features in asthma is chronic airway inflammation, in which
many cells and cellular elements play a role (1). This chronic inflammation
in the airway mucosa accounts to a large extent for the symptoms that an
asthmatic patient experiences, as the accompanying airway hyperresponsiveness results in repeated attacks of wheezing, breathlessness, chest tight11
ness and coughing, particularly at night (14). Exhaled nitric oxide (NO) originates from the bronchial epithelium and can be used as a marker of airway
inflammation (15). A marked upregulation of inducible NO synthase (iNOS)
in the epithelium of the airways of asthmatics through the signaling pathway
of interleukins IL-4 and IL-13 seems to be the main cause for increased NO
levels (16-18). By contrast, patients with non-allergic asthma have lower
levels of exhaled NO (19, 20) often within normal range. However, these
levels appear to be higher than in their non-allergic healthy counterparts
(21). Increased exhaled NO levels have been associated with allergic sensitization and the degree of IgE sensitization is related to exhaled NO (22, 23).
Exhaled NO is not a marker of atopy per se (24), but rather a result of the
subclinical airway inflammation caused by allergen exposure through either
environmental allergens (25) or under-controlled challenge (18). Finally,
having increased NO levels is a prognostic factor for the development of
respiratory symptoms in the future, in both children (26) and adults (27).
IgE sensitization and airway hyperresponsiveness
Airway hyperresponsiveness to specific and non-specific stimuli (1) is a
consistent and defining clinical feature of asthma. Airway hyperresponsiveness consists of a variable and a persistent component (28, 29) and can be
measured as the responsiveness to direct (histamine, methacholine) and indirect (mannitol, adenosine monophosphate, exercise, cold air, hyperventilation) stimuli (30, 31). According to previous studies (28) the persistent component is observed frequently, but not always, and is related to the chronicity
of the disease. Chronic effects of airway inflammation lead to airway remodeling which includes increased contractility of smooth muscles, increased wall thickness and reduced airway caliber (28). Furthermore, other
studies (32, 33) indicate that the variable component reflects airway inflammation and is related to allergic sensitization. There is persuasive evidence
(28) that allergen-induced reactions in the airways, such as airway hyperresponsiveness and eosinophilic inflammation, are IgE-dependent. Additionally, the monoclonal anti-(Fc)-IgE omalizumab inhibits these asthmatic responses as well as allergen-induced airway hyperresponsiveness and eosinophilia (34, 35). Airway hyperresponsiveness is associated not only with the
presence, but also with the degree of atopic sensitization (36).
IgE sensitization and blood eosinophil count
Peripheral blood eosinophilia is also recognized as a feature of allergic sensitization (37-39) and asthma (40). Regardless of the clinical history of respiratory symptoms, exposure to allergens is sufficient for increased eosinophil
12
counts in sensitized subjects (37). Blood eosinophilia is primarily triggered
by IL-5 (41), but can be also driven by other mechanisms, such as rhinovirus
infections (42) or chronic rhinosinusitis (43). Eosinophils are major effector
cells in asthma (40), although some patients, especially those with nonallergic asthma, have a neutrophilic or paucigranulocytic profile (3). The
previous theory, that systemic eosinophilic inflammation originates in a
spillover from the inflamed airways, is challenged by a new one suggesting
that they are two independent inflammatory components (24). This notion is
supported by the finding that airway inflammation and blood eosinophilia
correlate only moderately (44). Furthermore, local anti-inflammatory drugs,
such as inhaled steroids, do not affect peripheral eosinophilia (45) as much
as systemic anti-inflammatory drugs like antileukotrienes (46, 47). The systemic eosinophilic inflammation, measured by blood eosinophil count (BEos), is of importance for asthma, as the number of eosinophils in peripheral
blood and in bronchial lavage from subjects with asthma is associated with
disease severity (48) and asthma-related visits to emergency departments
(49).
Importance of type of IgE sensitization
IgE sensitization against aeroallergens and respiratory disease
IgE sensitization is associated with developing asthma and other atopic diseases, but not in an absolute manner. Rather, certain patterns (50) and degrees (51) of IgE sensitization are linked to a higher probability of new-onset
allergic disease. Sensitization against perennial aeroallergens is the type of
IgE sensitization that seems to have the higher clinical impact on asthma and
its clinical features. Specifically, asthma is strongly associated with elevated
IgE and sensitization to indoor perennial allergens, notably those derived
from cat (52) and mite (53). Sensitization to perennial but not seasonal
aeroallergens is reported to be the main determinant of high exhaled NO
levels (22). Sensitization to indoor allergens (mite or cat) has the strongest
association with airway hyperresponsiveness in ECRHS (54) and other surveys (53-56). Having IgE antibodies against perennial allergens is related to
new-onset asthma (57, 58), whereas IgE sensitization against seasonal (pollen) aeroallergens has been shown to be more related to rhinitis than asthma
in both cross-sectional (59) and longitudinal studies (60). There are also
geographical differences concerning which allergic profile is most clinically
relevant for asthma and airway inflammation. IgE sensitization against mite
allergens is more important in Southern Europe (61, 62) while IgE sensitization against furry animal (cat and dog) is more important in Northern Europe
(8, 63).
13
IgE sensitization against food allergens and adverse food
reactions
It is important to point out the difference between IgE sensitization towards
food allergens, IgE- mediated food allergy and food hypersensitivity. The
majority of subjects sensitized to food allergens do not present clinical IgEmediated food allergy; only about one tenth of those sensitized to a food
allergen develop symptoms of food allergy (64). It is also a fact that most
adverse reactions after food ingestion are not IgE-mediated (65). Therefore,
they cannot be classified as IgE-mediated food allergy, but should rather be
characterized as food hypersensitivity (66). This term covers all non-toxic
food reactions which are further divided in immune-mediated (IgE- and nonIgE-mediated food allergy) and non-immune-mediated (10). Food hypersensitivity is also common. Previous population studies in the 1990s have estimated the prevalence of self-reported adverse reactions after food consumption to 12-20 % in adults (67-69). Food hypersensitivity reactions are usually
non-fatal, but have important impact on quality of life even if they are just
perceived reactions and not physician-diagnosed (70). Further studies in
adults are needed as there are limited data on the prevalence and distribution
of IgE sensitization to food allergens in relation to food hypersensitivity.
In one study it was found that 15 % to 20 % of the population has IgE antibodies against at least one food allergenic source (64). In the same crosssectional study (64), prevalence of IgE sensitization towards food allergens
was highest in children, at 28 %, and decreased with age to 13 % in adults
older than 60 years old. The majority of allergic reactions to food allergens
begin in childhood and few treatment alternatives can influence their natural
history (71). Existing longitudinal studies (72, 73) about the natural course
of IgE sensitization to food are few and most cover only childhood, where
prevalence remains stable during the first 5-6 years of life and then begins to
decline. Unfortunately, data about sensitization in adults are scarce. The
prevalence of food allergy has been found to be 6 % in children (74) and 4 %
in adults (75). Childhood food allergies are usually outgrown (76-79), with
the exception of peanut allergies, which usually persist (80, 81).
IgE sensitization against food allergens and respiratory disease
It is important to map IgE sensitization against food allergens. According to
recent studies in children (82, 83), children sensitized to food allergens have
increased levels of FeNO and have an increased risk for asthma. Moreover,
IgE sensitization to food is highly prevalent in adults (39) as well as children
(84) with asthma, and is associated with increased use of healthcare and
asthma medication (84). Furthermore, IgE-mediated food allergy can act as a
trigger for asthma symptoms (3) and may be an underestimated risk factor
for severe asthma (85). In patients with diagnosed food-induced anaphylaxis,
14
the severe allergic reaction may include a life-threatening asthma attack (86).
It is important to define predisposing factors associated with worse asthma
control, as asthma is often not optimally controlled, despite existing therapies (4, 87, 88). Moreover, recognizing risk factors for new-onset asthma
and modifying them may reduce the burden of disease in the future (89).
Methods for examining IgE sensitization
Extract-based tests
The most important step in the diagnostic algorithm of allergic diseases is a
thorough clinical case history. Traditionally IgE sensitization has been examined with skin prick tests (SPT) or in vitro specific tests (sIgE), when
patient history has suggested sensitization against an allergenic source (90).
These IgE antibody tests are based on crude extracts consisting of allergenic
and non-allergenic molecules from that source (91). While the quality of the
extracts has varied in the past (90, 92, 93), advances in characterization and
standardization have improved quality (94, 95). Skin prick test is a cheaper
method with high sensitivity that allows immediate reading, while blood
tests constitute an automatic quantitative method appropriate for high sensitivity monitoring of sensitization (91).
Component-based tests
To supplement the traditional extract-based test, component-resolved IgE
antibody determination, based on microarray techniques, was recently introduced into the field of clinical Allergology (96). The utilization of advances
in DNA technology allowed the characterization and cloning of various allergenic molecules in order to determine important components of various
allergic diseases (97-100). The availability of allergenic components in the
last decades exploded and created a need for systematic allergen nomenclature. The World Health Organization and International Union of Immunological Species Allergen Nomenclature Subcommittee were charged with
establishing and maintaining the systematic nomenclature and a detailed
database of characterized allergenic proteins. Allergenic molecules are
named using their Latin family name (genus and species). For example, the
abbreviation Fel d is used for allergenic components from Felis domesticus
(cat). A number is added to the name to distinguish the various components
from the same species (e.g., Fel d 1, Fel d 2, etc.). The numbers are given to
allergens in order of identification.
The various allergenic components are grouped into protein families,
based on their structure and biological function (101). Some protein families,
such as storage proteins in peanuts (e.g., Ara h 2) and hazelnuts (e.g., Cor a
15
9), more frequently trigger severe allergic reactions such as anaphylaxis,
while other allergenic molecules are considered more innocent as they associate to sensitization without severe clinical symptoms. Components belonging to the same family have similar antibody binding sites (epitopes) and the
same IgE antibody can bind allergens from different allergen sources. For
example, Bet v 1, the major birch pollen allergen, shows high homology
with other members of pathogenesis-related (PR)-10 protein family such as
Ara h 8, a peanut allergen, or Cor a 1, a hazelnut allergen. IgE antibodies
against birch cross-react with peanut and hazelnut causing allergic symptoms
usually in form of oral allergy syndrome. These cross-reactive allergen components can explain many sensitizations and clinical symptoms from related
allergenic sources (102-105).
In contrast to cross-reactive components, there are also components specific for a certain allergenic source. This is an important utility of component-based diagnostics as it identifies markers of primary sensitization and
increases the accuracy of diagnosis (91). Component-based analysis facilitates a better understanding of primary sensitizations (106, 107) and eases a
risk assessment for future severe allergic reactions (102, 108). This can improve the selection of patients appropriate for immunotherapy and the effect
of targeted allergen avoidance or the need to proceed with food challenges
(102). However, the geographical differences in distribution and prevalence
of various sensitization patterns and disease expression should be taken in
consideration in the evaluation of the information obtained with this method.
Component-resolved IgE antibody determination is a new semiquantitative technique, still under validation and it is considered to be a third
line tool after clinical history and extract-based tests (91). This new technology can also be useful as a tool for evaluation of the epidemiology of IgE
sensitization in the future (109).
Extract-based test vs. component-based tests
Several child cohort studies (110-112) have examined sensitization using
both extract-based tests and allergen components, though none of these studies included a direct comparison. IgE testing against peanut components had
a high diagnostic accuracy in children with peanut allergy verified through
food challenges. Only a few cross-sectional surveys (113-115) have examined the performance of IgE determination using allergen components in
relation to IgE determination using extracts, or examined the relation of IgE
determination using allergen components to clinical symptoms (116). Reports generally indicate a high degree of agreement between single and multiplex measurements of IgE to allergen components (113), as well as between measurements using allergen extract and the respective components
(117-122). However, differences have been reported for some allergens
(among them cat, timothy and Dermatophagoides pteronyssinus) (114, 115),
16
with a fraction of subjects showing sensitization in extract-based tests, but
not in tests using allergen components. This results in a high negative percent agreement and a lower positive percent agreement, despite the overall
high concordance between the two types of IgE measurements. The clinical
importance of this discordance has not been studied in relation to present
airway inflammation and responsiveness or in relation to later development
of allergic airway disease.
17
Aims
1) to investigate the relationship between the pattern of IgE sensitization, obtained using the microarray technique, and the prevalence of
asthma, levels of exhaled NO and airway hyperresponsiveness in a
population-based setting.
2) to investigate the IgE sensitization profile in asthmatics and examine
the association between this profile and exhaled nitric oxide, blood
eosinophil count and airway responsiveness to methacholine.
3) to examine the natural course of food hypersensitivity symptoms and
IgE sensitization to food allergens in adults and to investigate the
risk factors for new-onset food hypersensitivity and sensitization.
4) to compare the performance of extract-based and component-based
measurements of IgE sensitization to cat, timothy and birch, in relation to current and new-onset rhinitis and asthma, as well as in relation to airway inflammation and hyperresponsiveness.
18
Material and methods
Population
The populations of Papers I, III and IV were based on the European Community Respiratory Health Survey (ECRHS) I (1991-92), II (2000-01) and
III (2010-2012) (123), an international multicenter study of asthma and allergy (ECRHS) and its follow-ups after ~10 and ~20 years respectively. The
details of the designs of ECRHS I, II and III have been published (123).
Each participant was sent a brief questionnaire (Stage 1) and among those
who responded, a random sample was invited to undergo a more detailed
clinical examination (Stage 2). A ”symptomatic” sample consisting of additional subjects who in Stage 1 reported symptoms of asthma attacks, using
asthma medication or waking with shortness of breath was also studied. In
ECRHS II, subjects who had participated in Stage 2 were invited to participate in a follow-up study. Subjects answered a standardized questionnaire
administered by trained interviewers and underwent lung function tests and
blood tests. In the Uppsala study center, 679 subjects were reinvestigated in
ECRHS II. ECRHS III encompassed 302 subjects who had participated in
ECRHS II. Rhinitis and asthma symptoms were questionnaire-assessed again
in ECRHS III.
Paper I included 467 people from the Uppsala study center in ECRHS II,
who were examined with ImmunoCAP ISAC. Of these people, 96 were diagnosed with symptomatic asthma. Methacholine challenge test results were
available from 362 subjects and exhaled NO from 288.
Paper III comprised 2,307 individuals from Sweden and Iceland who answered questions about food hypersensitivity symptoms in both ECRHS I
and II. In a subgroup of these ECRHS participants from the random sample
(n = 807), for whom there were measurements of IgE to food allergens at
ECRHS I (39) (124), these measurements were performed again in the ECRHS II. (Figure 1)
19
Figure 1. Flow chart explaining the studied population of Paper II.
Paper IV was based on 451 subjects from Uppsala center, who participated
in the ECRHS II, which was a 9-year follow-up of the first ECRHS (125)
and 302 subjects from the ECRHS III, which was a 10-year follow-up of
ECRHS II.
This study focused on 302 people who were examined with both allergen
extracts (ImmunoCAP IgE) and allergen components (ImmunoCAP ISAC)
at baseline and followed up 10 years later with a questionnaire about respiratory symptoms and disease in the ECRHS III (Figure 2).
20
ECRHS II
ECRHS III
Questionnaire
about
respiratory
symptoms and
disease
(N=451)
Measurements of FeNO
and
methacholine challenge
test
IgE measurements
with
extract-based
(ImmunoCAP sIgE)
and component-based tests
(ImmunoCap ISAC)
Questionnaire
about
respiratory
symptoms and
disease
(N=302)
Figure 2. Flow chart explaining the studied population of Paper IV.
Paper II was based on subjects who participated in a project run within the
framework of an industry-academy collaboration on Minimally-Invasive
Diagnostics for Asthma and allergic diseases (MIDAS). Subjects were recruited from both primary and specialist care in Uppsala, Sweden. A total of
408 children and young adults (10-34 years) with physician-diagnosed asthma (according to their medical records) and daily treatment with inhaled
corticosteroids and/or oral antileukotrienes during at least three months of
the year before the study were included in Paper II. Further, 118 nonasthmatic controls were randomly chosen from the population registry and
also included in the paper.
Questionnaires
The main questionnaire used in ECRHS I, II and III (http://www.ecrhs.org
(125)) gathered information about respiratory symptoms, respiratory disease
(asthma and rhinitis), food hypersensitivity and smoking history.
21
Definition of asthma
In Papers I, III and IV, a person was recorded as having asthma if he/she had
ever been diagnosed with asthma and had an asthma attack or one of the
following symptoms during the last 12 months: nocturnal chest tightness,
attack of shortness of breath, chest wheezing or whistling (126).
In Paper II, subjects participated in detailed interviews containing questions regarding symptoms of asthma, rhinitis and sinusitis as well as medication use.
The degree of asthma control was assessed with the Asthma Control Test
(ACT) (127), which is a questionnaire consisting of 5 questions, each with a
5-point scale. The total ACT score is between 5 and 25, with a lower score
indicating poorer control of asthma. An ACT score ≥ 20 reflects wellcontrolled asthma (127).
Definition of rhinitis
A person was considered to have allergic rhinitis if he/she gave a positive
answer to the question ‘‘Do you have any nasal allergies, including hay fever?’’ (128).
Definition of food hypersensitivity
An individual was considered to have food hypersensitivity (10) if he/she
answered yes to the following questions “Have you ever had an illness or
trouble caused by eating a particular food or foods?” and if so, “Have you
nearly always had the same illness or trouble after eating this type of food?”
Respondents were then asked to list the food(s) and symptoms (67).
Presence and degree of IgE sensitization
In Paper I, the presence of IgE antibodies was examined using microarray
chip technology (ImmunoCAP ISAC; Phadia/Thermo Fischer Scientific,
Uppsala, Sweden) (129) (130) (Table 1). Sensitization is given in ISU units
as a semi-quantitative estimate of specific IgE antibody titer. Subjects were
considered non-IgE-sensitized if the signal was non-measurable or very low
(< 0.3 ISU). The IgE antibody levels were added together (sum IgE) (22) in
order to get an assessment of the overall levels of IgE sensitization in four
allergen categories: food allergens, seasonal allergens (pollen), perennial
allergens (animal, mite, mold), and other allergens.
22
Table 1. List of available allergen components examined with ISAC chip used in
papers I and IV.
Component
Foods from plant origin
Any
Brazil nut
rBer e 1
Sesame
nSes i 1
Peanut
nAra h 1
nAra h 2
nAra h 3
rAra h 8
Hazelnut
rCor a 1,0401
Apple
rMal d 1
Peach
rPru p 1
nPru p 3
Soybean
rGly m 4
Celery
rApi g 1
Carrot
rDau c 1
Kiwi
Act d 1
Act d 2
Act d 5
Act d 8
Cashew
Ana o 2
Hazelnut
Cor a 8
Cor a 9
Soy
Gly m 5
Gly m 6
Wheat
Gliadin
Tri a 19
Tri a 18
Tri a aA_TI
Foods from animals
Any
Egg white
nGal d 2
Milk
nBos d 4
nBos d 5
nBos d 8
nBos d lactoferrin
Carp
rCyp c 1
Cod
rGad c 1
23
Shrimp
rPen a 1
nPen i 1
nPen m 1
Egg white
nGal d 1
nGal d 3
Egg yolk/chicken
Grass pollen
nGal d 5
Any
Bermuda
Timothy
nCyn d 1
rPhl p 1
rPhl p 2
nPhl p 4
rPhl p 5
rPhl p 6
rPhl p 7
rPhl p 11
rPhl p 12
Tree pollen
Any
Olive
nOle e 1
nOle e 2
Plane tree
rPla a 1
rPla a 2
Cypress
Birch
nCup a 1
rBet v 1
rBet v 2
rBet v 4
Weed pollen
Alder
rAln g 1
Hazel
rCor a 1,0101
Japanese cedar
Cry j 1
Any
Ragweed
nAmb a 1
Mugwort
nArt v 1
Wall pellitory
Par j 2
Saltwort
Sal k 1
nArt v 3
Animal
Any
Cat
rFel d 1
nFel d 2
rFel d 4
Dog
rCan f 1
rCan f 2
24
nCan f 3
Mite
Mouse
nMus m 1
Cow
nBos d 6
Horse
Equ c 3
Any
House dust mite
nDer p 1
nDer p 2
rDer p 10
nDer f 1
nDer f 2
Storage mite
Mold
r Eur m 2
Any
Alternaria
rAlt a 1
Aspergillus
rAsp f 1
rAlt a 6
rAsp f 2
rAsp f 3
rAsp f 6
rAsp f 4
Cladosporium
Cockroach
rCla h 8
Any
rBla g 1
rBla g 2
nBla g 7
nBla g 4
nBla g 5
Latex
Any
rHev b 6
rHev b 8
rHev b 1
rHev b 3
rHev b 5
Other
Any
Mercury
rMer a 1
Bromelin
nAna c 2
Bee
nApi m 1
Anisakis
rAni s 3
nApi m 4
rAni s 1
25
In Paper II, IgE against a mix of food allergens (fx5), hazelnut, shrimp,
peach, a mix of aeroallergens (Phadiatop) and Alternaria alternata were
measured in all subjects. Subjects with titers of ≥ 0.35 kU/L against the
aeroallergen or food mix were further characterized regarding IgE-antibody
levels against each individual allergen in the respective panels: egg white,
milk, cod fish, wheat, peanut, and soybean for the food allergen panel and
cat, dog, horse, Dermatophagoides pteronyssinus, Dermatophagoides farinae, Cladosporium herbarum, birch, timothy grass, and mugwort for the
aeroallergen panel. All measurements of IgE were done with the ImmunoCAP system (Immunodiagnostics, Thermo Fisher Scientific, Uppsala,
Sweden). The results are presented in kU/L and values of IgE ≥ 0.35 kU/L
for an individual allergen defined a subject as being sensitized to that allergen (131). Subjects with IgE-antibody levels < 0.35 kU/L were given an
arbitrary value of 0.17 kU/L. The IgE antibody levels were added together
(sum IgE) (22), to get an assessment of the overall levels of IgE sensitization
in five different allergen categories: Mite (D. pteronyssinus, D. farinae),
Mold (Cladosporium herbarum, Alternaria alternata/Alternaria tenuis),
Furry animal (cat, horse, dog), Pollen (timothy, birch, mugwort) and Food
(wheat, peanut, soy, shrimp, egg, milk, fish) (Table 2).
Table 2. List of allergens examined with ImmunoCAP in Papers II, III and IV
(*only in Paper II).
Aeroallergens
Mite
Mold
Furry animal
Pollen
Food
Plant origin
Animal origin
D. pteronyssinus
D. farinae
Cladosporium herbarum
Alternaria alternata/Alternaria tenuis
Cat
Horse
Dog
Timothy
Birch
Mugwort
Wheat
Peanut
Soy
Hazelnut *
Peach *
Shrimp *
Egg
Milk
Fish
We chose not to include hazelnut and peach when evaluating the results in
the group of food allergens due to the large extent of cross-reactivity in
26
birch-sensitized subjects with hazelnut (94 %) and peach (73 %). In order to
adjust for a potential cross-reactivity between horse and cat IgE (132), we
have performed additional analyses after removing horse-sensitized subjects
from the group sensitized to furry animals.
In Paper III, venous blood samples were drawn and frozen at -20 ºC for
measurement of total serum IgE (total IgE) and serum IgE antibodies to cat,
D. pteronyssinus, grass, birch, and Cladosporium, as part of the ECRHS I
and II protocol (123). Measurement of IgE antibodies against birch was performed in all centers in the ECRHS I and in the Uppsala center in ECRHS II.
In addition, IgE antibodies to a mix of food allergens (containing egg white,
milk, cod fish, wheat, peanut, and soybean) were measured using the same
technique and reagents in ECRHS I and II (Pharmacia CAP System, MultiCap fx5, Thermo Fisher Scientific, Uppsala, Sweden). Serum with a positive
reaction to the food panel (fx5) was further analyzed for the single allergens
in the panel. The results are presented in kU/L, and values of 0.35 kU/L and
above were regarded as positive sensitization (131). A titer below the detection level (< 0.35 kU/L) was arbitrarily given the value 0.17 kU/L. All samples from ECRHS I from Reykjavík, Uppsala, Gothenburg and Umeå were
analyzed in Uppsala, as previously described (39), while samples from the
random sample of ECRHS II (depending on availability of residual samples)
were analyzed within the frame of EuroPREVALL in Manchester, as described elsewhere (133).
In Paper IV, the presence of IgE antibodies was examined using both an
allergen extract-based method (22) for birch, timothy, Dermatophagoides
pteronyssinus, Cladosporium herbarum and cat (ImmunoCAP system: Immunodiagnostics, Thermo Fisher Scientific, Uppsala, Sweden) and chip
technology (ImmunoCAP ISAC 103; Thermo Fischer Scientific) (129).
Dermatophagoides pteronyssinus and Cladosporium herbarum are not presented in the paper due to the small number of sensitized subjects to the respective component allergens (3.2 % and 0.2 %, as previously reported
(134)). The sub-analyses focusing on the groups with negative sensitization
to allergen components but positive to extract were done only for timothy
and cat, as this group was very small for birch. A subject was defined as IgEsensitized to an allergen extract in case of IgE values ≥ 0.35 kU/L for this
allergen (131). Subjects with IgE antibody levels < 0.35 kU/L (the lower
limit of detection) were given an arbitrary value of 0.17 kU/L. Regarding
component sensitization, subjects were considered non-IgE-sensitized if the
measurement was lower than 0.3 ISU. The IgE antibody levels were added
together (Sum IgE) (22) in order to get an assessment of the overall levels of
IgE sensitization to components of timothy, birch and cat, respectively. Subjects with non-detectable IgE antibody levels to any of the components of an
allergen were given an arbitrary value of 0.05 ISU for Sum IgE of each respective allergen.
27
Blood eosinophil count
Blood eosinophil count (B-Eos) was measured with a clinical routine method
(Cell-Dyn Sapphire, Abbott, Illinois, USA).
Measurements of exhaled NO
In Papers I, III and IV, FeNO measurements were performed in accordance
with the American Thoracic Society/European Respiratory Society recommendations (135) using an exhalation flow rate of 50 ml/s, with the exception that no initial vital capacity maneuver was performed, as a deep breath
with slow inhalation was found to be sufficient (136). The system used for
NO measurements was a computer-based single-breath NO system from
Nitrograf (Hässelby, Sweden) with a chemiluminescence analyzer (Sievers
NOA 280; Sievers, Boulder, Colorado, USA) in Papers I, III and IV, and
NIOX® Flex; Aerocrine AB, Solna, Sweden in Paper II. Measurements were
performed in duplicate and if the difference between individual measurements exceeded 10 %, additional measurements were done.
Smoking history
Smoking history was assessed by questionnaire. In ECRHS subjects who
have smoked at least one pack-year and were still smoking were defined as
current smokers. In MIDAS study current smokers were defined as individuals reporting current smoking of at least one cigarette/day during the last 6
months prior to the study.
Lung function
In Papers I, III and IV, forced expiratory volume in one second (FEV1) was
measured using a dry rolling-seal spirometer (Model 2130; Sensor Medics,
Anaheim, California, USA). Up to five technically acceptable maneuvers
were measured. In Paper II, flow-volume curves were performed using a
Master Scope spirometer (Erich Jaeger, Wurzburg, Germany). Measurements were performed in accordance with the ATS/ERS recommendations
(137).
28
Methacholine challenge test
Details of the methacholine challenge test have been described previously
(12). In Papers I and IV, the level of airway hyperresponsiveness was expressed as a dose-response slope (55), where a lower value indicates a higher
degree of airway hyperresponsiveness.
In Paper II, bronchial provocation tests were performed with Aerosol
Provocation System (Viasys Healthcare GmbH) using a simplified protocol
(138). Two minutes after each inhalation, FEV1 was measured and PD20
FEV1 (the cumulative dose of methacholine causing a 20 % reduction in
FEV1) was calculated by logarithmic interpolations using an integrated program.
29
Statistical methods
Statistical analyses were performed using STATA 11.0 software (Stata
Corp., 2001, Texas, USA) in Paper I and STATA 12.1 software (Stata Corp.,
College Station, Texas, USA) in Papers II, III and IV.
In Paper I, logistic regression was used to analyze the correlation between
the type of sensitization and asthma. Linear regression was used when analyzing correlation between the type of sensitization and exhaled NO and
correlation between sensitization and airway hyperresponsiveness. These
correlations were further tested in models that included age, sex, body mass
index and smoking history.
In Paper II, we used percentage differences of FeNO, PD20 and B-Eos as a
measure of effect. Percentage differences of FeNO, PD20 and B-Eos were
obtained from linear regression models, where the log-transformed value
was the outcome variable. The regression coefficient for the predictor variable of interest (e.g., food sensitization) was back-transformed, as described
in previous studies (21), in order to obtain fold differences of FeNO, PD20
and B-Eos (e.g., food sensitized vs. non-food-sensitized), and these were in
turn converted to percentage differences by multiplying the obtained fold
differences by 100 and subtracting 100 %. We estimated percentage differences of FeNO, PD20 and B-Eos for every 1 % increase of grade of IgE sensitization by back-transforming the regression coefficient from the model
where both outcome and predictor were log-transformed (139).
In Paper III, multiple logistic regression models were used when examining predictors for new-onset food hypersensitivity, and persistent food sensitization. These models always included age, sex, body mass index, rhinitis,
current asthma, total IgE, IgE against food allergens, sample and IgE against
aeroallergens.
In Paper IV, linear regression was performed to assess correlation between log-transformed titers of IgE for allergen extract and sum of IgE for
different components for the corresponding allergen using multiplex technique. To evaluate the agreement between allergen extract and component
IgE testing, calculations were made of the positive percent agreement (PPA),
i.e., the percentage of subjects positive to allergen components (> 0.3 ISU)
out of all subjects who were positive using the extract-based method at either
a cut-off of > 0.35 kUA/L or an arbitrarily chosen (114, 115) higher cut-off
of > 1 kUA/L, and of the negative percent agreement (NPA), i.e., the percentage of subjects who were negative to allergen components (< 0.3 ISU)
30
out of all subjects who were negative to the extract. A p–value of < 0.05 was
considered statistically significant.
31
Ethics
All subjects gave their written permission for the utilization of personal data
for the purpose of these studies. Studies within Papers I, II, III and IV were
approved by the Ethics Committee at the Medical Faculty at Uppsala University. The study in Paper III was also approved by the Ethics Committee at
University of Iceland.
32
Results
Paper I
Prevalence of IgE sensitization to various allergens
Of the 103 allergen components on the ISAC chip, 78 were detected in at
least one of the tested individuals. IgE antibodies against at least one component were detected in 141 (38.0 %) of the non-asthmatic and 70 (72.9 %)
of the asthmatic subjects (p < 0.0001). The asthmatic subjects had a significantly higher prevalence of IgE antibodies against foods of plant origin,
grass, tree and weed pollen, furry animals, mold, latex and other allergens
(p < 0.05).
Independent association of IgE sensitization to various groups of allergens
with asthma, FeNO and airway hyperresponsiveness
Multiple variable analyses were performed with the allergen components
merged into four groups, as presented in Table 3. Asthma was independently
related to having IgE against pollen and perennial allergens, increased FeNO
was independently related to having IgE against food allergens and perennial
allergens, while increased airway responsiveness was independently associated only with IgE against perennial allergens. These results were consistent
in a similar model where sum ISU titers for allergens within the respective
categories were calculated for each allergy category and inserted in the multiple linear regression models as predictors.
33
Table 3. Independent association between having specific IgE against groups of
allergen components and asthma, exhaled NO and airway hyperresponsiveness,
expressed as dose-response slope to methacholine challenge, where a negative value
indicates greater responsiveness.
Asthma
(OR (95 % CI)#
FeNO
(% difference
(95 % CI))#
20.2 (2.3,44.5)*
(n = 70)
Slope
(Beta (95 % CI))#
Food
1.20 (0.69-2.32)
-0.32 (-0.86, 0.23)
(foods of plant and animal (n = 118) §
(n = 81)
origin)
Pollen
2.21 (1.20-4.07)*
12.2 (-4.5, 34.9)
-0.20 (-0.75, 0.34)
(grass, tree and weed)
(n = 143)
(n = 82)
(n = 108)
Perennial
5.59 (3.10-10.1)*
47.9 (25.9, 77.8)* -1.43 (-2.03, -0.83)*
(animal, mite, mold)
(n = 87)
(n = 57)
(n = 65)
Other
2.02 (0.76-5.35)
9.6 (-18.7,47.9)
-0.80 (-1.76, 0.17)
(cockroach, latex, other)
(n = 25)
(n = 14)
(n = 19)
#adjusted for the variables in the table and age, sex, body mass index and smoking history.
§ number of subjects sensitive to respective allergen among subjects with recorded asthma
status (n = 467), FeNO (n = 288) or slope (n = 362).
*p < 0.05
The combined effect of IgE sensitization to different groups of allergens was
analyzed by combining sensitization to the different allergen groups
(Figure 3). A combination that included IgE antibodies against perennial
allergens was independently associated with asthma. Sensitization to food
allergens was related to asthma and increased FeNO in subjects who were
also sensitized to pollen and/or perennial allergens. The highest association
to asthma, FeNO and airway hyperresponsiveness was found in subjects who
had IgE antibodies against components from all three of these groups
(Table 4).
34
Figure 3. Asthma prevalence (percentage, left panel), FeNO levels (geometrical
mean (95 % CI) on a log-scale, middle panel) and airway hyperresponsiveness
(mean +/- SEM, right panel) in relation to sensitization to food, pollen, and perennial
allergen components. * p < 0.05 compared with the group without any allergic sensitization
Table 4. Asthma, exhaled NO and airway hyperresponsiveness expressed as doseresponse slope to methacholine challenge, where a negative value indicates greater
responsiveness in relation to sensitization (mean (95 % CI)).
Food
§
Pollen Perennial
$
£
N
Asthma
(OR (95 % CI))#
FeNO
(% difference
(95 % CI))#
Slope
(Beta (95 % CI))#
No
No
No
259
Yes
No
No
38 0.49 (0.16-2.02) 2.3 (-18.7, 31.8) -0.01 (-0.82, 0.79)
No
Yes
No
46 1.67 (0.68-4.10) 2.3 (-20.6, 28.8) -0.23 (-0.91, 0.46)
No
No
Yes
21 8.18 (3.00-22.3)* 62.2 (23.0,118)* -1.78 (-2.81, -0.76)*
Yes
Yes
No
37 4.43 (1.95-10.1)* 73.8 (34.9,123)* -0.82 (-1.68, 0.04)
No
Yes
Yes
23 8.69 (3.32-22.8)* 66.0 (23.0,123)* -1.18 (-2.13, -0.23)*
Yes
No
Yes
6
2.22 (0.24-20.8) 118 (17.5, 307)* -0.35 (-2.60, 1.90)
Yes
Yes
Yes
37 18.3 (7.83-42.5)* 90.5 (47.9, 139)* -2.41 (-3.23, -1.60)*
# adjusted for age, sex, body mass index and smoking history, *p < 0.05. § both plant and
animal origin, $ grass, tree and weed, £ animal, mite, mold.
Paper II
Prevalence of IgE sensitization to various allergens
The prevalence of sensitization to all allergens was higher among asthmatics
than among controls (p < 0.05). Sensitizations to cat, dog or timothy were
the most common among asthmatics, with prevalence figures between 50
and 60 %, whereas hazelnut, birch and timothy, with prevalence figures of
up to 20 %, were the most common sensitizations among controls.
35
Association between IgE sensitization and FeNO, airway hyperresponsiveness and B-Eos in asthmatics
FeNO, airway responsiveness and B-Eos were higher in sensitized asthmatics compared with non- sensitized asthmatics for all groups of allergens (all
p-values < 0.05). FeNO was independently associated with the presence of
IgE (≥ 0.35 kU/L) against mold, furry animals and food, after adjustment for
type of sensitization, age, sex, height, smoking history and medication. Similarly, airway hyperresponsiveness was independently associated with having
IgE against mold and furry animals, whereas B-Eos was independently associated with the presence of IgE against mold, furry animals and food
(Figure 4). Furthermore, FeNO was independently associated with the degree (sum IgE) of furry animal and food IgE sensitization; airway hyperresponsiveness was independently associated with the degree of mold and
furry animal IgE sensitization; and B-Eos was independently associated with
the degree of mold, furry animal and food IgE sensitization.
Fe NO
Mi te
••
B Eos
Mi te
Mol d
Mol d
••
•
Mol d
Pollen
Pollen
Furry
Furry
Furry
Food
Food
Food
Pollen
~!;;)
Percentage difference (95% Cl)*
•
Mi te
l
Percentage difference (95% Cl)*
•
!;;)
~!;;)
Percentage difference (95% Cl)*
Figure 4. Independent association (expressed as % difference (95 % CI))* in FeNO,
airway hyperresponsiveness (PD20) (expressed as dose-response slope to methacholine challenge, where a lower value indicates greater responsiveness) and B-Eos
between subjects sensitized and non-sensitized to specific allergen groups (significant difference (p < 0.05) means that the line of error bar does not cross the zeropercentage difference line).
*Adjusted for the variables in the figure and age, sex, height, smoking history, use of inhalant
corticosteroids and use of antileukotrienes.
§ No. of subjects sensitive to the respective allergens among asthmatics with FeNO (n = 402),
PD20 (n = 352) or B-Eos(n = 402).
Paper III
Natural history of food hypersensitivity symptoms
Around 21 % of the interviewed subjects reported food hypersensitivity
symptoms in both ECRHS I and II (Table 5). The most common foods relat36
ed to these symptoms were fruits, followed by nuts and then vegetables. The
prevalence of food hypersensitivity in relation to ingestion of fish (including
seafood and shellfish) increased significantly from ECRHS I to II and this
became the fourth most common cause of food hypersensitivity, alongside
vegetables. Food hypersensitivity symptoms due to dairy products, wheat
products (gluten, cereal, and wheat) or spices (herbs, chili, and garlic) also
increased significantly from ECRHS I to II (Table 5).
Table 5. Prevalence (n (%)) of type of food reported as cause of food hypersensitivity (n = 2,358).
ECRHS I
1991-92
ECRHS II
2000-01
p-value
Any food
498 (21.1)
526 (22.3)
0.58
Fruits
209 (8.9)
225 (9.5)
0.31
Nuts
163 (5.3)
147 (6.2)
0.6
Vegetables
102 (3.4)
106 (4.5)
0.13
Fish, seafood, shellfish
75 (2.5)
106 (4.5)
<0.001
Chocolate
38 (1.3)
23 (1)
0.31
Egg
33 (1.1)
20 (0.9)
0.34
Milk and dairy products
31 (1)
60 (2.5)
<0.001
Meat
11 (0.4)
19 (0.8)
0.13
Gluten, cereal, wheat products
8 (0.3)
14 (0.6)
0.049
Herbs, chili, garlic
9 (0.3)
20 (0.9)
0.004
37
Natural history of prevalence of IgE sensitization to food and aeroallergens
Prevalence of IgE sensitization to at least one food allergen decreased by
56 % during the study (Figure 5A). Peanut IgE sensitization had the highest
prevalence among the food allergens; it decreased by 67 %. The prevalence
of soy IgE sensitization decreased significantly between ECRHS I and II,
whereas similar prevalence figures for IgE sensitization to wheat, milk, egg
and fish were found at both time points (Figure 5B). No significant change
of prevalence of cat, birch and mite IgE sensitization was found, whereas
prevalence of timothy grass IgE sensitization decreased by 15 % (prevalence
decrease from 17 % to 15 %) (Figure 5A).
Figure 5. Prevalence of sensitization to food (n = 807) and aeroallergens (n = 804)
(panel A) and prevalence of IgE sensitization to individual food allergens (panel B)
in ECRHS I and II. Birch pollen was measured only in Uppsala (n = 138).
38
Incidence of food hypersensitivity
The incidence of food hypersensitivity was 11.7 % during this 9-year study.
The incidence in the symptomatic sample was higher than in the random
sample (20.6 % vs. 9.9 %, p < 0.001). Female sex OR: 1.8 (1.3, 2.6), rhinitis
OR: 1.8 (1.2, 2.6), eczema OR: 1.8 (1.3, 2.5), and presence of IgE sensitization to aeroallergens OR: 1.9 (1.3, 2.8) were associated with new-onset food
hypersensitivity. Odds ratios were adjusted for the variables in the model
and age, body mass index, current smoking, sample and total IgE.
Paper IV
IgE sensitization assessed by analysis using allergen extracts and components
Generally, more subjects were sensitized to allergen extracts than to allergen
components, for all investigated allergens (Figure 6). A total of 48 % of subjects who were sensitized to cat extract were not sensitized to any investigated cat allergen component. The 56 individuals who were positive for the cat
extract but negative for the cat allergen components had mostly low IgE
levels to cat, with a geometrical mean of 1.3 (1.0, 1.7) kU/L, versus 5.6 (4.3,
7.3) kU/L in subjects IgE-sensitized to both cat extract and components.
After choosing a higher cut-off for being sensitized to allergen extract
(1 kU/L), 34 % of individuals positive to cat extract (according to the higher
cut off) were still not sensitized to any investigated cat component. This high
percentage of individuals not sensitized to cat components decreased to 23 %
for cut-off > 2 kU/L IgE against cat extract and to 16 % for cut-off > 3 kU/L
IgE against cat extract. For timothy, 21 % of the subjects who were sensitized to timothy extract were not sensitized to timothy components. The
highest agreement was seen for birch, for which only 14 % of the extractsensitized subjects were not sensitized to birch components. The agreement
between component and extract IgE sensitization was lower for cat (kappa:
0.61 ± 0.04) than timothy (kappa: 0.81 ± 0.05) (p = 0.002) or birch (kappa:
0.89 ± 0.05) (p < 0.001).
Strong correlations were found between IgE antibody concentrations for
allergen extracts and the sum of IgE for different components of separate
allergens: r = 0.95 for birch, r = 0.96 for timothy, and r = 0.83 for cat
(Figure 6).
39
Figure 6. Contingency tables and scatter plots of IgE levels against extract (Y-axis)
vs. sum of components (X-axis) (both axes log-scales). The number of sensitized
subjects for each allergen according to each method is presented in the table in each
panel.
40
IgE sensitization patterns for cat and timothy in relation to new-onset rhinitis and asthma
In multiple logistic regression models, new-onset rhinitis was associated
with sensitization to cat components but not with sensitization to the extract
(Figure 7A). This association remained significant when analyzing the additive value of sensitization to cat components in a model containing age, sex,
BMI, smoking and asthma at baseline (Pseudo-R2 increased from 13 % to
20 %, p = 0.007), while no additive value of sensitization to cat extract was
found (p = 0.15). New-onset asthma was associated with sensitization to
both cat extract and cat components (Figure 7B). Similarly, there was a consistent additive explanatory value of IgE sensitization to cat components for
new-onset asthma (Pseudo-R2 increased from 7 % to 22 %, p = 0.005), but
this was not found for the extract (p = 0.15). Sensitization to timothy allergen components was associated with new-onset rhinitis (OR 4.0 (1.03,
15.7)), but this was not found for timothy extract (OR 2.4 (0.6, 9.6)). No
association between new-onset of asthma and sensitization to timothy extract
was found (OR 0.6 (0.1, 5.7)), and no subjects with sensitization to timothy
components had new-onset asthma in the present study.
Figure 7. Odds ratios for new-onset rhinitis# and new-onset asthma* for subjects
sensitized to cat allergens (Panels A & B). #Adjusted for age, BMI, sex, smoking,
asthma at baseline, *Adjusted for age, BMI, sex, smoking, rhinitis at baseline.
41
Relative importance of different cat and timothy components for airway inflammation and responsiveness and new-onset respiratory disease
An independent association was found between sensitization to Fel d 1 or
Fel d 2 and FeNO (p = 0.002 and p < 0.001) and airway hyperresponsiveness
(p = 0.001 and p = 0.02) when analyzing independent effects of IgE sensitization to cat components in the same model (Table 6). Regarding new-onset
respiratory disease, only a trend towards an association between IgE sensitization to Fel d 1 and new-onset rhinitis (p = 0.083) was found (Table 6).
Regarding timothy components, only IgE sensitization to Phl p1 was independently associated with higher present increase in FeNO, [7.2 % (0.7,
14.9), p = 0.03] and airway hyperresponsiveness [regression coefficient: -1.1
(-1.9,-0.3), p = 0.009]. Furthermore, IgE sensitization to Phl p1 was associated with new-onset rhinitis [OR: 8.2 (1.2, 55.7), p = 0.03].
Table 6. Independent association between FeNO, slope (where lower values indicate
more airway hyperresponsiveness) new-onset rhinitis# and new-onset asthma* and
cat components in the same model.
Components
rFel d 1
nFel d 2
rFel d 4
% difference
(95 % CI)
% difference
(95 % CI)
% difference
(95 % CI)
14.9 (7.2, 23.1)
23.1 (7.2, 41.4)
1.4 (-6.7, 7.2)
Beta (95 % CI)
Beta (95 % CI)
Beta (95 % CI)
-1.5 (-2.4, -0.7)
-1.5 (-2.8, -0.2)
-0.5 (-1.5, 0.6)
OR (95 % CI)
OR (95 % CI)
OR (95 % CI)
New-onset rhinitis # 7.0 (0.8, 63.2)
Omitted
1.9 (0.1, 53.2)
New-onset asthma*
7.6 (0.5, 110)
7.8 (0.6, 98.1)
FeNO§
Slope§
3.4 (0.3, 25.3)
#Adjusted for age, BMI, sex, smoking, asthma at baseline.
*Adjusted for age, BMI, sex, smoking, rhinitis at baseline.
§ Adjusted for age, BMI, sex, smoking.
42
Discussion
Asthma, degree of inflammation and airway
hyperresponsiveness in relation to pattern of IgE
sensitization in a general population and in a cohort of
asthmatics
Having IgE antibodies against perennial aeroallergens was the type of sensitization that had the strongest relation to asthma, exhaled NO and airway
hyperresponsiveness in a population-based study (Paper I). This result is in
line with other analyses from the ECRHS cohort (22, 54, 55) and other studies (53, 58, 140). Sensitization to pollen was also an independent risk factor
for asthma but not for elevated FeNO or increased airway responsiveness.
The latter could be due to the fact that most subjects (80 %) in this population were examined outside the pollen season (141). Other studies have
found that sensitization to pollen is less related to asthma and airway hyperresponsiveness compared with sensitization to perennial allergens (142). An
increasing prevalence of asthma and higher exhaled NO levels and airway
hyperresponsiveness could be found with increasing numbers of sensitizations, when grouped into perennial, pollen and food allergen sensitizations.
This is in line with previous studies where multiple sensitization related to
higher risk of asthma (8) and bronchial responsiveness (36).
In Paper II, we observed an association between IgE sensitization to
aeroallergens and local and systemic inflammation in asthmatics. The markers of local inflammation, FeNO and airway responsiveness were independently associated with the presence of IgE antibodies against mold and
furry animals. FeNO has previously been reported to be increased in asthmatic children sensitized to furry animals (63) and is also associated with the
degree of IgE sensitization to furry animals in asthmatic children (63) and
adults (23). Mold sensitization was related to high FeNO levels in a population-based study (22). The levels of FeNO were significantly higher in patients with asthma who were both sensitized and exposed to a relevant
aeroallergen (mite, cat or dog) than in those who were sensitized but not
exposed (143). However, in the present study, mite sensitization was not
related to increased FeNO, which probably reflects the low exposure to mite
allergens in Sweden (144).
43
In Paper I, we found an independent association of perennial (furry animals, mite and mold) IgE sensitization with airway hyperresponsiveness
(134). Mold sensitization was the type of sensitization associated with the
largest increase in airway hyperresponsiveness and previous studies have
shown that mold sensitization is associated with more severe asthma in
young adults (145) and more frequent admission to intensive care (146).
IgE sensitization against food allergens and exhaled NO
and blood eosinophil levels
In Paper I, sensitization to food allergen components was independently
associated with increased FeNO. In subjects dually sensitized to pollen and
food allergens, an increased risk of asthma and elevated FeNO was seen,
whereas no such association was found in subjects who were only sensitized
to pollen. Previous studies have shown a significant association between
food allergy and asthma, which was even stronger in children with multiple
food allergies or severe food allergy. These findings suggest that food allergy could be an under-recognized risk factor for problematic asthma (85).
In Paper II, we could confirm the finding from the population-based study
(Paper I) that food IgE sensitization is associated with increased airway inflammation. Although we have no data on food allergen exposure in Paper
II, it is reasonable to assume that only a minority of the subjects with IgE
antibodies towards a food allergen have symptoms of food allergy (64) and
therefore avoid the food in question.
The marker of systemic eosinophil inflammation used in the present
study, i.e., the number of blood eosinophils, was independently associated
with the presence of IgE sensitization to mold, furry animals and food, and
was also independently associated with the degree of mold, furry animal and
food IgE sensitization. Development of eosinophilia is common in asthmatics with mold sensitization (147). Current exposure to furry animals also
associates with blood eosinophilia (148), and multisensitization to furry animal-derived components associates with increased numbers of blood eosinophils in patients with severe asthma (149). The relation between food allergen sensitization and eosinophilia has not been studied previously in asthmatics, although such a relation has been reported in a population-based
study of adults (39) and in children with food allergy (38) or atopic dermatitis (150). The systemic eosinophilic inflammatory component is of importance for asthma, as the number of eosinophils in peripheral blood and in
bronchial lavage from subjects with asthma is associated with disease severity (48). We have recently reported that both blood eosinophil counts and
FeNO are independently associated with wheeze and asthma events in a
large American population survey (49).
44
Mechanistically, exposure of the gastrointestinal tract to food allergens
may cause subclinical inflammation in the gastrointestinal tract with activation of Th-2 lymphocytes and production of Th-2 cytokines, such as IL-5,
IL-4 and IL-13. These cytokines can drive inflammation in the primed tissue
of the airway mucosa in subjects sensitized to both aeroallergens and food
allergens through IL-4/IL-13 and upregulation of iNOS and blood eosinophilia via the IL-5 inflammatory pathway (151) (Figure 8). Additionally,
some of the food allergens may be inhaled and exposure to aerosolized food
has been reported to result in asthma symptoms in asthmatic children with
food allergies (152).
Figure 8. Proposed mechanism.
Methodological issues and clinical utility of multiplex,
component-based vs. limited panel of extract-based IgE
testing
About 70 % of asthmatic subjects and 40 % of non-asthmatic subjects were
IgE-sensitized. Previous analyses of IgE sensitization in the ECRHS II population have only included four individual allergens: mite, cat, timothy and
Cladosporium (22). By using the microarray technique, we identified IgE
antibodies against almost 80 components in this population. This increased
the prevalence of IgE sensitization from 28 % to 38 % in the non-asthmatic
group, but had a relatively limited effect in the asthmatic group, where the
prevalence of IgE sensitization only increased from 70 % to 73 %. A possi45
ble explanation is that the four allergens tested previously are the most
common allergens causing mono-sensitization. Therefore, co-sensitization to
other allergen components tested in the microarray does not increase the
prevalence of atopy to a large extent. This is supported by a previous study
from ECRHS (12). The results in the asthma group suggest that there is a
proportion of 20-30 % of asthmatics who are not IgE-sensitized even when
extended IgE characterization is performed. Additional information in terms
of the number of allergen components a subject is sensitized to is obtained
using the microarray, and this has been reported to be related to the risk of
developing allergic disease (153).
The high overall correlation between IgE sensitization to allergen extract
and the corresponding components in Paper IV is in agreement with previous
studies on measurements of IgE towards single allergen components, which
have shown strong agreement with extracts of birch (117), timothy (118,
119), and cat (120, 122). Further, high concordance has been shown between
the use of timothy extract and allergen components measured with multiplex
technique (114, 115). However, in our study, large variations in the positive
percent agreement between the methods were noted, ranging between 52 %
and 85 %, with the lowest PPA for cat. The PPAs for timothy and cat were
lower than those presented by Bonini et al (114) and Melioli et al (115).
Given that these two studies and our study use the same version of the ISAC
microarray (with 103 allergen spots), a possible explanation for this difference could be that our study is population-based while the other two studies
examined multi-sensitized subjects, who probably had higher IgE antibody
concentrations overall. In the present study, the greatest discordance was
seen for comparisons between allergen extract and allergen components for
cat. The subjects who were not sensitized to cat components on the ISAC
chip generally had lower levels of IgE to cat extract. However, there was an
overlap in IgE levels to cat between subjects sensitized and subjects not sensitized to cat components and we still had discordant groups when raising
the threshold for IgE sensitization to cat extract to levels higher than 1, 2 or
3 kU/L.
Cat and timothy components’ predictive value for the
risk of higher airway inflammation and
hyperresponsiveness and development of new-onset
respiratory disease
Assessment with cat components had a higher predictive value for the risk of
developing asthma and rhinitis than analyses with extracts. The reason for
this is unclear, but it may relate to the fact that allergen extracts contain other
allergen components not tested for, and that these components are of less
46
clinical importance. Sensitization to certain allergen components might be
more clinically relevant, as timothy component Phl p 1 was independently
associated with new-onset rhinitis, higher FeNO and airway hyperresponsiveness, while cat components Fel d 1 and Fel d 2 were associated with
higher airway hyperresponsiveness and FeNO. A previous study has also
shown the association of Phl p 1 with new-onset rhinitis (154), but no other
study examined the association between timothy or cat components and
new-onset rhinitis, FeNO or airway hyperresponsiveness. Another explanation may be that analysis of allergen components by multiplex technique is
less sensitive (115), meaning that we will only detect individuals with higher
IgE antibody concentrations, with a concurrent increased risk of developing
airway inflammation and respiratory symptoms. However, this theory fails to
explain why we could still find a significant proportion of subjects without
sensitization to cat allergen components even when looking at subjects with
IgE levels > 1 kU/L towards the extract.
IgE sensitization to cat and timothy allergen components had added predictive value for new-onset asthma and rhinitis compared with a confirmed
sensitization to the corresponding extracts. Earlier studies on the predictive
value of extract-based IgE measurements for the onset of allergic diseases in
adults have shown that IgE to grass and birch pollen allergens were related
to new-onset rhinitis (60, 155), and IgE to cat was related to new-onset
asthma (57). To our knowledge, only one study has examined the prognostic
value of IgE sensitization to allergen components. This study was performed
in children and found a relation between timothy components and new-onset
rhinitis (154).
Change in the prevalence of IgE sensitization to food
allergens
A novel finding of Paper III was the marked reduction of prevalence of IgE
sensitization to food allergens over time in adults. The most prominent decrease was noticed for peanut and soy, foods which sensitized subjects can
probably avoid more easily than wheat, milk and fish. The natural history of
prevalence of IgE sensitization to food allergens has previously been studied
only in children (72). For aeroallergens, the natural history of prevalence of
IgE sensitization has been examined in longitudinal studies in adults (156162), but the results are contradictory and show the prevalence decreasing
(159, 162), remaining stable (156, 163) or increasing (157, 158, 160, 161)
over time. The decrease in prevalence of IgE sensitization to food allergens
does not appear to be related to the initial age of the subjects, which tends to
preclude the possibility that the effect is related to a specific birth cohort.
Reductions of similar size could be observed in both younger and older sub47
jects. This could indicate that the decreasing prevalence is related to aging.
Previous longitudinal studies (156, 158, 160, 161) on the prevalence of IgE
sensitization to aeroallergens in relation to age have reported mutually inconsistent results.
Reported food hypersensitivity symptoms after food
intake
To our knowledge, no previous studies have analyzed longitudinal changes
of self-reported perceived food hypersensitivity in adults. The prevalence of
food hypersensitivity reported was in line with reports from previous crosssectional studies (67-69, 164). Our results, showing an unchanged prevalence of food hypersensitivity over time, are contrary to what is known about
the natural history of food hypersensitivity in children, where major remission of symptoms has been reported (165-167).
48
Study limitations
In Paper I, enrichment of the randomly selected study group with a symptomatic sample (22) increased the group of asthmatics and allowed the analysis
of relationship to asthma, but may have made the results less generalizable to
general populations. The measurements of exhaled NO were not available in
all subjects and therefore some of the analyses with regard to airway inflammation might have been underpowered.
In Paper II, the lack of data on exposure to allergens, especially food allergens, was a study limitation. The use of component-resolved diagnosis
could have given more information regarding cross-reactivities. Simply
summing up IgE titers against different allergens does not take into account
primary sensitization versus cross-reactivities. Furthermore, componentresolved diagnosis would have allowed us to analyze if some allergen components for the same allergen were more important than others in relation to
local and systemic inflammation.
A weakness of Paper III is that we were not able to assess the persistence/remission of food hypersensitivity as the questions referred to ever
having had food hypersensitivity. A limitation is the fairly limited number of
participants for whom we had data from repeated measurements of IgE
against food allergens. Another limitation is that several of the most common food allergens in adulthood, such as hazelnut, shrimp, peach and apple
(133), were not analyzed at both time points.
A weakness of Paper IV may be that the definition of allergic disease was
questionnaire-based. Further, caution should be exercised in making inferences about how results based on multiplex technique can be applied to
analyses of IgE to single components.
49
Future research
Molecular-based analysis of IgE sensitization can enhance the determination
of IgE through extract-based tests. Large-scale multicenter studies will help
identify which groups of patients are appropriate for this type of IgE testing.
Furthermore, the value of specific allergen components to longitudinally
predict new-onset/persistent respiratory symptoms remains to be examined
further and in greater detail among asthmatics and general populations.
Some allergen components from the same allergenic source can be more
clinically important than others in relation to local and systemic inflammation. Such components must be spotted in order to identify atopic subjects
who are going to develop allergic disease, use more healthcare and require
more intense therapy in the future.
50
Conclusions
1. The type and degree of IgE sensitization to aeroallergens were important
predictors of asthma, airway inflammation and reactivity.
2. Microarray chips marginally increased the information about prevalence
(yes/no) of IgE sensitization compared with a standard panel of a limited
number of allergens.
3. The known relationship of IgE sensitization against perennial allergens
with airway inflammation, airway hyperresponsiveness and blood eosinophilic count was confirmed in children and young adults with asthma
(Figure 9A).
4. IgE sensitization to food allergens is associated with increased local
inflammation in a population-based material and to increased local and
systemic inflammation in an asthmatic population (Figure 9B).
5. The prevalence of IgE sensitization to food allergens in adults decreased
over a 9-year period, while the prevalence of food hypersensitivity remained unchanged.
6. Measurement of IgE sensitization to cat allergen components appeared
to have a higher clinical value than extract-based measurement, as it related better to airway inflammation and responsiveness and had a higher
prognostic value for the development of asthma and rhinitis over a 12year period.
51
Figure 9. Graphical summary of the main findings. We confirmed previous findings
that IgE sensitization against perennial allergens relates to increased airway inflammation, airway hyperresponsiveness and blood eosinophilic count (Panel A). Our
data support the new finding that IgE sensitization against food allergens also relates
to increased airway inflammation and blood eosinophilic count (Panel B).
52
Clinical implications
Studies of the association between IgE sensitization profiles and local and
systemic inflammation may enable personalized, biomarker-based treatment
of patients with asthma in the future. Different allergy profiles can associate
to differing extents with the various features of respiratory disease, suggesting that they also have differing therapeutic needs. Identifying these needs
can be the first step towards individually tailored treatments.
Multiplex component analysis is of clinical use in identifying true food allergies. Its epidemiological use remains to be established, and in Paper I we
have used only the information regarding perennial, pollen and food sensitization, not the full information regarding the degree of sensitization to all
allergen components. By taking advantage of information about primary
sensitization and cross-reactivity as well, it would be possible to get a detailed allergic profile of a population and the relation of these sensitizations
with ongoing respiratory disease, airway inflammation and responsiveness.
In Paper IV, we identified allergen components that were associated with
new-onset respiratory symptoms and airway inflammation and responsiveness. Such information can enable physicians to detect patients who would
be affected more severely and require more healthcare in the future. That in
turn would help choosing appropriate candidates for immunotherapy and
other therapies, improving their efficacy.
53
Aknowledgements
The work of this PhD thesis was performed at the Departments of Medical
Sciences: Clinical Physiology and Medical Sciences: Respiratory,Allergy
and Sleep Research.
The financial support needed to perform these studies was obtained from:
Swedish Agency for Innovation Systems, Swedish Heart and Lung Foundation, Vårdal Foundation for Health Care Science and Allergy Research,
Swedish Association against Asthma and Allergy, Thermo Fisher Scientific,
Sweden (formerly Phadia), Kronoberg Region, Södra Sjukvårdsregionen,
Agnes and Mac Rudberg’s Foundation, Bror Hjerpstedt Foundation.
I would like to express my sincere gratitude to the following persons:
Andrei Malinovschi, my main supervisor, for his patience for my endless
questions. His answers helped understand better the new enviroment of both
research and the new country and enable me to evolve as a researcher and a
person.
Christer Janson, my co-supervisor for correcting my mistakes and showing a paternal patience he led our research through his experience to successful paths.
Maria Gunnbjörnsdottir, my co-supervisor, for being my ‘mother’ in research and in my first years in Uppsala. Even if she was later in Iceland I
knew that there was a person that could turn to.
Kjell Alving for his expertise by giving advice not only about nitric oxide
but explaining me also the difficult pathways of interleukins and other allergic mechanisms.
Magnus P. Borres for his contribution and important advice about components and other practical tips.
Marianne Högman for her valuable tips about nitric oxide and her overall
contribution.
My co-authors Lennart Nordvall, Per Mattsson, Annica Önell, Peter Burney, Thorarinn Gislason, Kjell Torén, Bertil Forsberg for a successful collaboration.
Mary Kämpe for her support and her good words and advice when I
needed them mostly.
54
Gunnis and Gunilla for their smiling faces and good mood. Of course the
rest of the research group at Lung and Allergy Clinic in Uppsala.
My colleagues and head of the clinic in Växjö for their understanding and
their help during the time that I had to research.
My wife for her love and her understanding. Without her it would have
been impossible to cope with the difficulties the last year. I am grateful that
you followed me everywhere in that journey and made this journey more
pleasant.
My daughter whose smile make me forget all difficulties despite she fell
asleep during all my presentations home.
My family in Greece. Αφιερωμένο επισης στην οικογένεια μου που δεν
τους έχω ευχαριστήσει για όσα έχουν κάνει και κάνουν για μένα.
My friends in Lidköping Nancy (Poulen), in Göteborg Kostas (Pitsal),
Menelis, Zoe, in Uppsala Giorgos (Paoktsis saved me the last minute),
Giorgos (Tsakonas), Katerina, Poppi , Stauros, Phillip, Anna (the dancing
crocks), Spiros(Nepal), Dimitris (teamate), Betty, Paulos (hotel and sports
bar), Kostas(Unicorn), Stergios (Geitonas), Hanna, in Växjö Petros, Natasa,
Kleri, Nikos, Dimitris, Poli, Anna, Dimitris,in Greece Thanos (Karamitsos),
Donna, Ntina, Kostas (Zouri), Maria, Stauros(best man), Aigli (best judge),
Dimitris, Margarita, Basilis, Maria, Panos, Dimitra, Spiros(treme), Michael
(bro), Christos (bro), Dimos (italian bro), the members of the holy
quadruplet (Giorgos G, Giorgos Chatz, Christos) and many others.
To those who I forget to mention I apologize but the list is getting longer
and the deadline for printing the thesis is getting closer quickly.
55
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66
Acta Universitatis Upsaliensis
Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 1107
Editor: The Dean of the Faculty of Medicine
A doctoral dissertation from the Faculty of Medicine, Uppsala
University, is usually a summary of a number of papers. A few
copies of the complete dissertation are kept at major Swedish
research libraries, while the summary alone is distributed
internationally through the series Digital Comprehensive
Summaries of Uppsala Dissertations from the Faculty of
Medicine. (Prior to January, 2005, the series was published
under the title “Comprehensive Summaries of Uppsala
Dissertations from the Faculty of Medicine”.)
Distribution: publications.uu.se
urn:nbn:se:uu:diva-251396
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2015