Respiratory Diseases in Infants and Children

Respiratory Diseases in Infants and
Children
Preface
During the mid 1900s, there was a growing interest for respiratory medicine in infants
and children and, as such, diagnostic tools and techniques for assessment of lung
function were developed. Paediatric pulmonology was further elaborated in Europe
during the second part of the 20th century.
The Paediatric Assembly within the European Respiratory Society (ERS) was
established in 1993, 3 years after the Society was founded. Since then, the Paediatric
Assembly has grown and is now the second largest Assembly within the ERS.
There may be a number of factors which have influenced this increasing attention for
paediatric pulmonology. The identification of specific disease entities such as cystic
fibrosis, increasing incidence and prevalence of asthma, access to new and efficacious
anti-asthma drugs, and the development of novel diagnostic techniques are all factors
that are most likely to have contributed to the growing interest.
Under these circumstances, it is a pleasure to announce the present issue of the
European Respiratory Monograph, which is focused on respiratory diseases in infants and
children. The Guest Editors have not attempted to cover the whole field, but rather to
create an update of the important areas, as well as focusing on recent developments and
future needs. As respiratory problems in childhood gradually transform into respiratory
problems in adults, this Monograph is not addressed to paediatricians in particular. It
concerns all who are interested in respiratory function and disorders.
K. Larsson
Editor in Chief
Eur Respir Mon, 2006, 37, viii. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph;
ISSN 1025-448x.
viii
INTRODUCTION
U. Frey*, J. Gerritsen#
*Paediatric Respiratory Medicine, Dept of Paediatrics, University of Berne, 3010 Inselspital, Bern,
Switzerland. Fax: 41 316329484; E-mail: urs.frey@insel.ch. #Beatrix Childrens Hospital, University Medical
Centre Groningen, Groningen, The Netherlands. Fax: 31 503614235; E-mail: j.gerritsen@med.umcg.nl
Paediatric respiratory medicine is a growing and continuously changing field with an

increasing impact for many adult respiratory diseases. The field is so large that it is
impossible to cover all aspects in one issue of this series. Therefore, the current
Monograph focuses on a few important topics.
With increasing pollution and a particular susceptibility of children to these
environmental toxins, as well as changing lifestyles in the Western world, lung problems
in children have become a major health issue. Many of these environmental factors will not
only have an immediate effect on childrens health, but may also have a long-lasting impact
on their growth and development. Whether this will result in a change in respiratory
morbidity in adults in the future is still not very well known. For these reasons, the first few
chapters of this Monograph focus on the developmental aspects of paediatric respiratory
diseases. The impact of genetic and environmental factors, and their interaction with growth
and structural development of the lung and with immune and allergy development in
children, is discussed. This includes the impact of remodelling on lung growth.
In the following chapters, new diagnostic ideas are provided for the paediatric
respiratory clinician. Over the past few years, there has been a growing body of literature
presenting new diagnostic techniques to assess lung function, inflammatory and allergic
markers of lung disease in the outpatient clinic and in paediatric intensive care units.
However, it is getting increasingly difficult to decide whether these techniques are clinically
relevant and how they should be used and interpreted. This is followed by a discussion of
the major disease groups in paediatric respiratory medicine, including the increasingly
important problem of childhood adipositas and its impact on the respiratory system.
The final chapter of the Monograph provides a summary on new approaches of how to
examine and perhaps better understand complex respiratory disease with its multitude of
influencing factors, interactions and impact on growth and development. Such new
concepts have been frequently used in statistical physics and have begun to find their way
into natural sciences.
A large amount of summaries and reviews on each of these particular fields and many
up-to-date textbooks have been published recently. It was decided that there is no need
for another "ordinary" textbook, which can never be complete and cover all aspects of a
particular field. Many authors have supported the idea that the current Monograph should
be more conceptual and visionary. Thus, the authors were asked to focus on the four main
following questions: 1) What have been the most recent fundamental developments in your
field?; 2) What are the current models and concepts of the pathophysiological mechanisms,
the related diseases and the treatment strategies?; 3) What are the important future
questions?; and 4) What is needed to find answers on these questions?
The Guest Editors have aimed to form author teams of experienced and innovative
people in the field who can come up with new concepts and ideas. These concepts should
inspire clinicians to critically re-evaluate their strategies, but should also stimulate young
researchers. The Guest Editors hope that you will like the concept of this Monograph, and
think that this issue is similarly interesting for adult respiratory specialists who should be
increasingly interested in the early paediatric origins of respiratory disease.
Eur Respir Mon, 2006, 37, ix. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph;
ISSN 1025-448x.
ix
CHAPTER 1
Epidemiology of respiratory diseases in

infants and children
E. von Mutius*
*Correspondence: E. von Mutius, Dr von Haunersches Kinderspital, Ludwig-Maximilian-Universitat
Munchen, Lindwurmstrasse 4, D-80337 Munchen, Germany. Fax: 49 8951604452; E-mail: Erika.Von.
Mutius@med.uni-muenchen.de
The bulk of the epidemiological literature on childhood respiratory diseases is on

asthma, (recurrent) wheeze and bronchitis, the latter mostly being very ill defined.
Therefore, the present chapter focuses on childhood wheeze and asthma. Over the last
few decades, there has been increasing interest in the clinical and research question as to
whether childhood asthma is more likely to be a syndrome than a single disease entity.
Clinically, all physicians have seen children with mild forms of wheeze whose symptoms
disappear at school age or during puberty. In other patients, symptoms become manifest
during the early years of life, and persist and progress throughout childhood and
adolescence, with significant morbidity, need for long-term therapy and frequent use of
healthcare resources. Some of these patients continue to be symptomatic during puberty
and into adult age, whereas many other patients, at least transiently, lose their symptoms
during puberty, in some to relapse in young adult age. This diversity of clinical
manifestation may be the expression of a spectrum of severity within a single illness.
Alternatively, asthma may be heterogeneous in nature. There has been increasing
recognition over recent years that asthma is, in fact, not a single entity, and that similar
clinical disease manifestations, such as wheeze and cough, may be the expression of
different underlying mechanisms. Therefore, different phenotypes of asthma are likely to
exist. This notion is supported by findings from epidemiological studies investigating the
natural course of wheeze from birth to school age, adolescence and adulthood.
It remains unknown how many different disease mechanisms are implicated in the
development of wheeze and asthma in infants and children. In the following paragraphs,
phenotypes that have been characterised in some detail during recent years are presented.
It must, however, be realised that these proposed phenotypes may not be definitive, since
progress, particularly in the field of genetics, may increase future understanding.
Therefore, phenotypic definitions of wheeze and the asthmatic syndrome may be
reshaped in the future. Studies of the genetics, immunology and cellular biology of
asthma and wheeze currently under way will help to identify forms of the disease in which
certain underlying mechanisms are predominant. This differentiation is not purely of
theoretical and scientific interest, but the goal of these activities is eventually to identify
therapies that are specific to certain asthma phenotypes, and will be able to control
symptoms, as well as to pre-empt and control the implied mechanisms.
Transient wheezing in infancy

Wheeze is a very prevalent symptom during the first to third years of life [1].
Prospective studies in general population samples have estimated that no less than a third
Eur Respir Mon, 2006, 37, 17. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph;
ISSN 1025-448x.
E. VON MUTIUS
of all children experience at least one episode of bronchial obstruction during the first
3 yrs of life. The peak incidence, therefore, occurs early in life and then decreases
markedly. The great majority of these incident cases are related to infections with
respiratory viruses, particularly respiratory syncytial virus (RSV), parainfluenza virus
and rhinovirus [2, 3]. A large proportion of these infants and young children with one or
more episodes of wheezing during the first 3 yrs of life have a good prognosis, since 60%
of them will have stopped wheezing by the early school years [1]. This form of transient
early wheeze is not related to allergic conditions, such as atopic dermatitis, increased
levels of total or specific immunoglobulin (Ig) E in the serum, and the development of
hay fever in later years. Furthermore, these children are not more likely to be an
offspring of parents affected by asthma or other allergic conditions.
Epidemiological studies have identified a number of factors that are associated with
this transient form of wheezing. There are two main risk factors, maternal smoking
during pregnancy and lower levels of lung function measured in the first 2 weeks of life
before any lower respiratory tract illness has occurred. Exposure to maternal smoking
during pregnancy has, interestingly, been linked to lower values of various lung function
parameters in infancy [4, 5], thereby suggesting the possibility that exposure to tobacco
smoke products during pregnancy may alter the development of the lung in utero. The
relationship between reduced levels of lung function and transient early wheezing is,
however, also observed in children not exposed to tobacco smoke. Genetic factors may
determine these lower levels of lung function. It seems reasonable to assume that many genes
are involved in the regulation of airway size and tone, as well as lung size, including among
infants with transient early wheeze. However, data to support these notions are still lacking.
Little is known about the fate of infants with transient early wheeze beyond childhood.
Will these children develop symptoms and signs of other respiratory diseases, such as
chronic obstructive pulmonary disease, particularly after taking up smoking later in life?
There are no data for such long-term follow-ups, which is not surprising given the
difficulty inherent in such long-term prospective studies. In retrospective surveys, early
childhood infections were identified as predictors of adult pulmonary function. Whether
these acute respiratory illnesses were indeed infectious processes or a noninfectious
exacerbation of pre-existing obstructive airway disease remains unclear, since most of
these studies made retrospective assessments of exposure. Shaheen et al. [6] assessed the
relationship of several childhood respiratory illnesses as documented in health visit
records to lung function at age 6774 yrs. Males, in particular, showed significant
reductions in forced expiratory volume in one second (FEV1) and FEV1/forced vital
capacity ratio. Such deficits were more frequently found in subjects who were diagnosed
with pneumonia before the age of 2 yrs than among nonaffected individuals. Likewise,
pneumonia in the first year of life was confirmed as a risk factor for adult lung function in
another longitudinal UK study [7]. In these subjects, followed from birth up to the age of
35 yrs, pneumonia before the age of 7 yrs was associated with reduced ventilatory function,
independent of the development of asthma and wheeze. Nevertheless, it remains unclear
whether childhood pneumonia causes the loss of lung function until adulthood, or whether it
is merely a marker and indicator of children who already exhibit poor lung function before
the commencement of disease. Such children may have had the transient form of wheeze
early in life, but data to support or refute this notion are lacking.
Wheeze and asthma at school age

Children who have developed asthma by school age are, to a large extent, subjects with
some form of atopic sensitisation. There are, however, a significant number of children
2
CHILDHOOD RESPIRATORY DISEASE EPIDEMIOLOGY
who have frequent wheeze until school age, but who have no detectable IgE in their
serum. In UK literature, the term "wheezy bronchitis" has been coined as distinct from
the label "asthma" [8]. In children with wheezy bronchitis, attacks are thought to be
triggered by viral infections alone, whereas, in asthmatic subjects, other factors, such as
exercise, allergen exposure, irritants and stress, can also induce symptoms. To date, it is
not clear whether the wheezy bronchitis phenotype is the same as the nonatopic wheezing
phenotype. However, both entities seem to share a number of characteristics, amongst
others a relatively good prognosis. Children with wheezy bronchitis have been shown to
retain normal lung function in long-term follow-up studies in Australia and New Zealand
[9, 10]. In the Isle of White study, nonatopic wheezers also showed normal lung function
at school age and the beginning of adolescence [11].
The role of viral infections in exacerbating wheeze and/or inducing wheeze and
asthma-like symptoms has been debated at great length. There is increasing evidence to
suggest that host characteristics play a role in determining a subjects response to a viral
infection. One of the best-studied exposures is infection with RSV. A number of
observations have suggested that children who had been infected with RSV and
developed airway obstruction were more likely to experience continued and ongoing
wheeze episodes years after the original RSV infection than those who had not [2].
Recent observations suggest that the configuration of the immune response prior to any
viral infection determines the risk of virally induced wheeze. In a cohort of infants
with a positive family history of asthma and allergy, immune responses were
measured prospectively, and specific viral respiratory infections were identified in
early infancy [12]. The results demonstrated that mitogen- and cytokine-induced
responses were immature at birth in these high-risk children, and that the quality of these
responses was related to the risk of subsequent wheezing. In particular, vigorous
interleukin-13 and interferon gamma responses were associated with a reduced risk of
developing wheezing.
Exposure to other children early in life increased the risk of symptomatic infections
with rhinovirus and RSV [13]. Although the rate of rhinovirus-associated wheeze was
greatly increased by exposure to day care and/or siblings, there was relatively little effect
on RSV-associated wheezing. Interestingly, the authors also showed a small but
measurable effect of frequent infections being associated with a smaller decline in
interferon gamma responses during the first year of life, in accordance with the hygiene
hypothesis. The epidemiological literature supports this observation. A number of
studies have clearly shown a protective effect of day care early in life on the subsequent
development of frequent wheeze and asthma [1416]. Likewise, frequent episodes of a
runny nose in the first year of life were associated with a lower risk of asthma at school
age in the prospective Multicenter Allergy Study (MAS) birth cohort [17].
Allergic asthma
The most persistent and usually more severe form of recurrent wheeze is associated
with evidence of IgE-mediated immune responses to food and aeroallergens. A number
of studies have shown that most cases of allergic asthma show their first symptoms
during early life [1]. In the large prospective Childrens Respiratory Study (Tucson, AZ,
USA), children sensitised to Alternaria, which was the main local aeroallergen associated
with asthma, started wheezing during the second and third years of life [18]. A European
birth cohort study has shown that children who will eventually develop asthma by the age
of 7 yrs will not only start wheezing but also develop atopic sensitisation early in life [19].
Most allergies, at this age, are directed towards foods. Sensitisation to hens eggs was
3
E. VON MUTIUS
found to be the best predictor for the subsequent development of asthma in the European
birth cohort [20].
Since there is a strong association between allergic sensitisation and asthma at school
age, the level of allergen exposure has been fiercely discussed as a potential determinant
of the incidence of asthma. There is evidence to suggest that a higher level of allergen
exposure is a risk factor for the development of atopic sensitisation, specifically to the
allergen in question. For example, in the MAS birth cohort, levels of house dust mite and
cat allergens in the first years of life were related to sensitisation to house dust mite and
cats, respectively, at the ages of 3 and 6 yrs [21]. In contrast, the level of allergen exposure
at an early age and later was not related, either in subjects with or without a family
history of asthma and allergies, to the development of asthma at school age in the same
European cohort [22]. These findings are corroborated by the results of the longitudinal
prospective Prevention and Incidence of Asthma and Mite Allergy birth cohort in the
Netherlands, in which no clear effect of early-life allergen exposure and development of
recurrent wheezing was seen [23]. Finally, interventional trials significantly reducing the
amount of house dust mite allergen indoor exposure, such as the Manchester study, have
not demonstrated a protective effect against the development of asthma and wheeze in
the active group [24]. On the contrary, an increased risk of atopy was found to be
associated with these avoidance measures. Therefore, recent evidence does not support
the notion that allergen exposure is a risk factor for the incidence of asthma and wheeze
in childhood. However, allergen exposure, via the alleviation of sensitisation and the
augmentation of allergic airway inflammation in these sensitised subjects, may contribute
to the severity and chronicity of the asthmatic condition.
Asthma progression in adolescence and adulthood

Still too little is known about the progression of asthma and wheeze from childhood to
adolescence. Prospective studies have shown that the great majority of asthmatics lose
their symptoms during puberty. A cohort study of Australian schoolchildren studied at
the age of 810 yrs and again at the age of 1214 yrs showed that the persistence of
bronchial hyperresponsiveness into adolescence was related to its severity at school age,
the atopic status of the child and the occurrence of asthma in its parents [25]. The
majority of children showing a light or mild degree of airway hyperresponsiveness lost
their increased response at age 1214 yrs, whereas only 15.4% with severe or moderate
hyperresponsiveness at school age were normoreactive at adolescence. Recent findings
also suggest that the decline in asthma prevalence during puberty may be attributable to
the vanishing disease expression of the nonatopic wheezing phenotype associated with
viral infections. Indeed, the observations from the Tucson birth cohort showing that
lower respiratory tract illnesses due to RSV and other viruses early in life were associated
with a diminishing risk of recurrent wheeze during school years support this notion [26].
Therefore, virally associated wheezing may have a better prognosis than atopy-related
asthma.
The British National Childhood Development Study, a longitudinal survey of all
people in England, Scotland and Wales born during 1 week in 1958, is of great interest
when investigating asthma incidence throughout childhood and early adult life. Between
the ages of 7 and 33 yrs, only 5% of symptomatic subjects showed persisting wheezing at
all times [27]. There was, however, much remittance and recurrence of symptoms. More
than half of the subjects who wheezed before the age of 7 yrs and reported wheezing in
the previous year at the age of 33 yrs had been free of attacks between the ages of 16 and
23 yrs. Subjects can also completely lose their symptoms. No less than 35% of subjects
wheezing at the age of 7 yrs showed complete remission after adolescence. It is
4
noteworthy that the incidence of wheeze and asthma after adolescence was strongly
associated with taking up active smoking.
Outlook and future questions

One of the emerging challenges in the epidemiology of respiratory diseases in children
is a better characterisation of the different asthma and wheeze phenotypes. Such
improvement may be achieved through refined analysis of existing prospective data from
various birth cohorts around the world. Furthermore, the identification of genetic factors
underlying various mechanisms in these conditions is promising. It is already becoming
apparent that, as would be expected, genetic factors are differentially related to either
atopy or asthma in various populations. The rapidly increasing literature relating various
genetic factors to asthma and wheeze will add to the complexity of the syndrome.
However, these analyses will eventually help in the distinction between various disease
phenotypes by linking the identified underlying pathways to clinical and epidemiological
observations. Likewise, other basic science tools investigating immune responses, airway
inflammation and remodelling, and other aspects of the pathophysiology will help to
further elucidate the complex nature of this syndrome.
Improved classification will also advance the ability to identify the relevant
environmental determinants of the various phenotypes. Indeed, there is increasing
evidence showing that the effect of a certain environmental exposure depends upon the
exposed phenotype, the timing of the exposure and the underlying genetic disposition.
For example, viral infections have been clearly shown to be a strong determinant of
transient early wheeze in the first 13 yrs of life, but may exert protective effects against
subsequent asthma development into school age and adolescence. Neglecting to distinguish
between these phenotypes results in a blurred perception of the relevant environmental
exposure. In this case, the direction of the association will, in such a scenario, depend upon
the number of study subjects with either transient wheeze or ongoing asthma in that
population rather than on the true relationship between exposure and disease incidence.
The addition of genetic factors to the puzzle will not only help in identifying the
underlying mechanisms, as discussed earlier, but, by considering both factors, namely
genetic alterations and environmental exposures, and studying the importance of their
interaction for the development of specified phenotypes, the true relevance of the
environmental exposure will become apparent for that specific phenotype. For example,
a large meta-analysis of passive smoke exposure has revealed that baseline lung function,
as assessed by FEV1, is mildly, but significantly affected. The summary estimate showed
a reduction in FEV1 of 1.4% of the predicted value in exposed subjects. In contrast,
consideration of genetic susceptibility reveals much stronger effects of passive smoke
exposure. Susceptibility to second-hand tobacco smoke exposure can, for example, be
assessed by genotyping the glutathione S-transferase gene, which encodes an important
enzyme in detoxification pathways. Certain polymorphisms result in a null variant with
low levels of enzyme production. In subjects in whom such low levels have been identified
through genetic analyses, the impact of environmental tobacco smoke exposure is much
stronger. In a German cross-sectional survey, the FEV1 was reduced by w5% pred in
exposed subjects and measures of small airways by i15% pred.
Future directions in the investigation of asthma and wheeze during childhood years
should aim at a better classification of affected subjects and a thorough analysis of the
associated genetic factors and environmental exposures. The impact of their interaction
on the incidence of a specific phenotype should be assessed, as well as taking the timing of
the exposure into account.
5
E. VON MUTIUS
Summary
The application of epidemiological methods in the investigation of paediatric
respiratory disease has greatly contributed to the understanding of these illnesses. In
childhood asthma and allergies, results of longitudinal cohort studies have pointed
towards the developmental aspect of paediatric diseases which arise, become manifest
and disappear at various ages. Several wheezing phenotypes have been confirmed in a
number of studies.
First, transient wheeze in infancy must be regarded as a separate condition being
associated with risk factors, such as maternal smoking, premature birth and low birth
weight. There is good evidence to suggest that reduced lung function after birth, before
any wheezing illness has occurred, contributes to the underlying mechanisms. Viral
infections are potent triggers of symptoms among children with this phenotype. The
prognosis is good as children outgrow their symptoms between 23 yrs of age.
Secondly, nonatopic wheezing after toddler and school age has been documented. This
phenotype is characterised by the lack of detectable immunoglobulin E antibodies,
allergic comorbidities, and often by the absence of airway hyperresponsiveness.
Children with nonatopic wheeze are likely to lose their symptoms around school age
and retain normal lung function. In contrast, children with the atopic wheezing
phenotype are most likely to develop a chronic course of the illness with significant
impairment in lung function and the development of airway hyperresponsiveness.
Over adolescence, a significant proportion of these children lose their symptoms, but
new onset of illness, particularly among females, is also seen at that age. Risk factors
for the persistence of asthma and wheeze during puberty include the severity of atopy
and airway hyperresponsiveness.
Keywords: Asthma, atopy, epidemiology, viral infections, wheeze.
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CHAPTER 2
Lung development from infancy to

adulthood
P.J.F.M. Merkus*, A.A. Hislop#
*Division of Respiratory Medicine, Dept of Paediatrics, Erasmus Medical Centre, Sophia Childrens
Hospital, Rotterdam, the Netherlands. #Institute of Child Health, University College London, London, UK.
Correspondence: P.J.F.M. Merkus, Division of Respiratory Medicine, Dept of Paediatrics, Erasmus
Medical Centre, Sophia Childrens Hospital, P.O. Box 2060, 3000 CB, Rotterdam, the Netherlands. Fax:
31 104636801; E-mail: p.j.f.m.merkus@erasmusmc.nl
The growth and development of the respiratory system has been the topic of several
extensive reviews and chapters in textbooks [19]. Therefore, this chapter only briefly
summarises established knowledge about the normal growth and development of the
human respiratory system, and refers to review articles for further reading. For the
effects of asthma or allergy, infections, environment, mechanical ventilation and
respiratory infections on the development of the respiratory system, the reader is referred
to corresponding chapters in the present Monograph. Special attention is given to the
tools available for longitudinal assessment of growth, recent insight into the interaction
between airway and vascular growth, and the impact of premature birth on the
development of the respiratory system.
Tools for assessing respiratory system development and growth

Traditionally, studies into the growth and development of the respiratory system have
been direct and precise but cross-sectional, or indirect and less precise but longitudinal.
Anatomical and histological studies

Obviously, anatomical and histological studies have provided information about the
dimensions, architecture and composition of lung tissue, but not about its functional
characteristics nor about prospective changes. Other disadvantages include the lack of
numbers of autopsy procedures, which makes it hard to determine differences between
groups of subjects, such as those due to age or sex.
Lung function measurements

Lung function measurements seem the most suitable tool for studying the development
of the respiratory system longitudinally, especially because of the noninvasive nature of
the techniques. However, lung function studies usually focus on assessment of airway
function or patency, and are notoriously insensitive to peripheral airway function [10]
and cannot detect parenchymal abnormalities (see below). Furthermore, they may be
hard to interpret because they are the net result of numerous independent factors that
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LUNG DEVELOPMENT INTO ADULTHOOD
may even counteract each other [11]. Functional studies on the pulmonary and bronchial
circulation are scarce and are not discussed here.
Developmental physiology is largely covered in the chapter of Gappa et al. [12], and
will not be detailed here. However, changes in respiratory physiology affect the degree to
which lung function data can be interpreted and compared between children of various
ages.
It is essential to realise that infants and toddlers have to ventilate with a flaccid ribcage,
which results in less effective or even paradoxical ventilation, a diaphragm that operates
less effectively, and lung parenchyma that is less compliant than in later life, resulting in a
lowered functional residual capacity. The increases in thoracic stiffness and lung
compliance in the first years of life result in a gradual increase in end-expiratory volume
with age. This process continues during childhood, and is responsible for the relative
underdistension of the lung below the age of 78 yrs and for its relative overdistension in
children above this age [13]. This was confirmed by a recent imaging study [14].
This phenomenon puts older studies in a different perspective. The observation that
the relative resistance of peripheral airways in infants and young children is high
compared to that in older subjects [15] may partly reflect the additional increased airways
resistance due to underdistension of the lung. After the age of 24 yrs, alveolar growth
occurs mainly by enlargement, as occurs in the airways from the start. Hence, in healthy
infants and preschool children, lungs are relatively underdistended and airway closure
may occur at the end of a normal tidal expiration.
Obviously, the consequences of increased lung distension in later childhood are
improvements in gas exchange [3] and a lower risk of microatelectasis [13]. With respect
to the interpretation of lung growth, increasing volumes with age do not necessarily
reflect alveolar multiplication or dimensional growth, but can also reflect enlargement
due to higher inflation levels. Decreased resistance measurements during growth may
partly be due to higher inflation levels rather than dimensional growth of the bronchial
tree. These aspects are difficult to correct for when trying to assess growth from lung
function data.
Imaging techniques
Promising new applications [1618] and scoring systems [19] have been developed for
high-resolution computed tomographic scans, which make them attractive and
interesting tools for assessing pulmonary changes during growth [14, 20].
Although providing only structural information (at relatively high lung volumes),
valuable information is obtained about the peripheral structure of the lung, which can
easily be missed when using lung function testing alone [21, 22]. However, because the
radiation burden cannot be ignored, computed tomography is currently unsuitable for
the assessment of prospective developmental studies in healthy subjects or in those with
minor respiratory disease [23]. Possibly, magnetic resonance imaging studies will prove to
be a suitable research tool not involving radiation.
Anatomical development of the respiratory system

The formation of the human respiratory system, with its pulmonary and bronchial
circulation, is the net result of a complex interaction between growth factors, hormones
(sex hormones, thyroid hormones and corticosteroids), genetic factors (sex and race),
nutrition (quality and quantity), exposures to insults such as environmental tobacco
smoke and drugs, and physical stimuli, such as stretch, foetal breathing movements
9
P.J.F.M. MERKUS, A.A. HISLOP
Genetics
(sex and race)
Nutrition
(quantity and quality)
Treatment
(drugs, artificial ventilation)
Pre-natal and post-natal
respiratory system growth
Prematurity,
SGA (IUGR)
Physical forces
(stretch, foetal breathing,
intra- and extrathoracic
space, fluid)
Exposure to hormones (testosterone,

corticosteroids, thyroid hormone)
and insult (drugs, passive smoking)
Fig. 1. Schematic representation of pre-natal and post-natal factors influencing lung growth and development.
SGA: small for gestational age; IUGR: intra-uterine growth retardation.
(FBMs) and amniotic fluid volume (fig. 1). Much of the integration of positive and
negative signalling pathways is still unknown.
Lung development has been divided into four stages: embryonic, pseudoglandular,
canalicular and alveolar. The age of transition from stage to stage varies between
individuals and species, and, in some species (e.g. rat and mouse), the alveolar stage can
be entirely post-natal. The stages can be summarised as follows.
Embryonic stage
During the embryonic stage (up to 7 weeks of gestation), the lung bud appears as a
ventral diverticulum of the foregut and divides within the surrounding mesenchyme.
Whether or not foregut endoderm cells transform to form the lung bud is critically
dependent upon the transcription factor hepatocyte nuclear factor-3. Several reviews
have addressed the involvement of this and other transcription factors [2426].
Interaction with the surrounding mesenchyme determines the initiation and
complexity of the branching pattern. An airway continues to increase in length when
stripped of its surrounding mesenchyme, but does not branch, whereas mesenchyme
transplanted from an area of active branching stimulates an otherwise dormant epithelial
tube to divide [27]. Numerous factors are implicated in the regulation of branching, as
recently reviewed [28], with one of the most important being vitamin A, or retinoic acid,
which is able to influence the transcription of multiple genes, affecting the development
and homeostasis of various organs, including the lung. Maternal overdoses of retinoic
acid, as well as vitamin A deprivation, are known to cause dose- and time-dependent
defects in primary and secondary branching, leading to lobar, unilateral or bilateral lung
agenesis or hypoplasia [28].
By 6 weeks of gestation, the two lungs are separated from the foregut and there are
two or three generations of airways lined with endoderm, which give rise to the
specialised epithelial cells of the lung, whereas all other elements of the lung originate
from the mesenchyme. The pulmonary arteries are thought to be derived from the sixth
aortic arches and are found alongside the developing airways [4]. As early as 34 days of
10
gestation, each lung bud is supplied by a pulmonary artery extending from the aortic sac.
This is connected via a capillary plexus in the mesenchyme around the single lung bud to
a pulmonary vein connected to the prospective left atrium, suggesting that a continuous
circulation is already present [29].
Pseudoglandular stage
The airway buds continue to divide into the mesenchyme during the pseudoglandular
stage (517 weeks of gestation), and all pre-acinar airways are present by 17 weeks of
gestation. As the airways increase in size, the walls differentiate and smooth muscle,
cartilage, submucosal glands and connective tissue appear. From 11 weeks of gestation,
the epithelium differentiates into ciliated, goblet and basal cells (stem cells), with Clara
cells in the peripheral airways. By 24 weeks of gestation, the airways have the same wall
structure as they have in the adult. Smooth muscle cells are present in the human trachea
and lobar bronchi, and are innervated by 810 weeks of gestation [3033]. First-trimester
human tracheal smooth muscle cells exhibit a fluctuating resting membrane potential
that is associated with the spontaneous development of tone and peristalsis-like
contractions of the airway, which help move the liquid within the airway lumen [31, 32].
As each new airway bud forms by division peripherally, a halo of endothelial cells
forming capillary tubules surrounds them, probably as a result of the action of vascular
endothelial growth factor (VEGF) produced by the endoderm cells. These tubules
coalesce alongside the penultimate airway to form the pulmonary arteries and veins.
Thus, new vessels are formed by vasculogenesis within the mesenchyme. The airway acts
as a template for the pulmonary vessels, which become progressively longer by the
sustained addition of the newly formed tubules to the existing vessels. Thus, the preacinar branching of both arteries and veins is also complete by 17 weeks of gestation [30,
34]. The airways also influence the structure of the arterial wall in that the first layer of
smooth muscle cells found around the newly formed arteries appears to derive from the
bronchial smooth muscle cells of the adjacent airway. Later, putative muscle cells are
recruited from the mesenchyme and lay down elastic laminae and collagen [30].
Innervation of the blood vessels follows muscularisation. The bronchial arteries form
independently of the pulmonary arteries from 8 weeks of gestation, and grow from the
descending aorta and enter the lung at the hilum. They extend down the intrapulmonary
airway as the cartilage plates differentiate and form a subepithelial and an adventitial
plexus. By birth, they extend to the end of the bronchioli. The peripheral bronchial veins
drain into the pulmonary veins.
Canalicular stage
During the canalicular stage (1627 weeks of gestation), the pre-acinar airways
continue to increase in size and differentiate, but there is still division at the periphery to
form the prospective respiratory bronchioli (two to three generations) and beyond this
the prospective alveolar ducts, which are at this time saccular in shape. Type I and II
alveolar epithelial cells can be identified lining saccular air spaces by 2022 weeks of
gestation. Type II cells develop identifiable lamellar bodies at y24 weeks of gestation.
However, surfactant is only detected in the amniotic fluid 45 weeks later. The thinning
of the epithelium to form the type I cells is led by capillaries which come to lie under the
epithelium, and this leads to the formation of a bloodgas barrier as thin as that of the
adult (y0.6 mm). This is sufficient to sustain life in extremely premature infants.
11
Alveolar stage
At the beginning of the alveolar stage (27 weeks to term), the walls of the saccules
contain discrete bundles of elastin and muscle beneath the epithelium, which form small
crests subdividing the walls [35]. These crests elongate to produce primitive alveolar
walls, which, at this time, have a double capillary supply below the epithelium on each
side of the wall, with mesenchymal tissue between the two. The two layers of capillaries
then coalesce to form a single sheet and the mature cup-shaped alveoli line elongated
saccules, now defined as alveolar ducts, and part of the wall of the respiratory bronchioli
[36]. The number of alveoli increases with gestational age, and, by term, between a third
and a half of the adult number is present [35]. The increase in lung volume seen during
late gestation is caused mainly by the increase in alveolar number. Alveolar surface area
increases and shows a linear relationship with age and body weight. The number of
alveoli both at birth and in the adult has been variously reported; however, it is agreed
that male children and adults possess more alveoli than female children and adults [37,
38], with estimated adult alveolar numbers ranging 200600 million [39, 40]. Initial postnatal lung growth occurs mainly by increase in alveolar number, and numbers increase
little or not at all after the age of 24 yrs, after which growth occurs mainly by
dimensional expansion [37].
Airway and blood vessel interaction during lung development

Based on studies since the mid-1990s, there is currently a much better understanding of
the interaction between the growth of the vasculature and the formation of the bronchial
tree and acinus [6, 4143]. During early foetal development, the airways act as a template
for pulmonary blood vessel development in that the vessels form by vasculogenesis
around the branching airways. During this phase, the epithelial cells induce angiogenesis
and vasculogenesis, in which VEGF plays a crucial role [6]. Later in gestation, however,
the capillary bed is essential for alveolar formation. From the canalicular stage onwards,
the capillaries seem to cause epithelial cells to differentiate into type I and II pneumocytes
and lead to further development of the alveoli [44].
In rodents, it was demonstrated that the absence of VEGF is associated with retarded
alveolar multiplication and a reduction in capillary number [45]. In addition, when antiangiogenic factors were given to rats, they reduced the number of small arteries and
retarded alveolar growth [46]. Furthermore, counteracting stimuli, such as endothelialmonocyte-activating polypeptide II, which is able to modulate the angiogenesis, are also
active [47]. The disrupted capillary bed in bronchopulmonary dysplasia is associated with
a decrease in levels of VEGF and its receptor [48]. A summary of the factors and
pathways involved has been published previously [49]. It is important to remember that
normal pre-natal lung development occurs in the hypoxic environment of the uterus. In
vitro cultures of mouse lung buds demonstrated a reduction in branching rate when
exposed to 20% instead of 3% oxygen [41]. Indeed, in vitro studies demonstrate that the
low oxygen intra-uterine environment enhances branching of both distal lung epithelium
and vascular tissue, and that pulmonary vascular development appears to be ratelimiting for epithelial branching morphogenesis [41]. Hence, abnormalities in foetal lung
development affect both airways and blood vessels [43]. A typical example is congenital
diaphragmatic hernia, in which there is a reduction in the numbers of arteries and
airways along the main pathway, with a subsequent reduction in alveolar number.
Similar abnormalities were found in renal agenesis, thoracic dystrophy and idiopathic
pulmonary hypoplasia [43]. These and other data indicate that the capillary bed is
12
essential for alveolar formation in the last trimester of gestation, and this has important
clinical implications for pre-term infants.
It has long been recognised that lung function in adolescents born prematurely can be
diminished due to prematurity per se rather than due to neonatal lung disease [50].
Although the lungs of premature infants may be damaged by infant respiratory distress
syndrome, infections, hyperoxia and mechanical ventilation, with the risk of developing
bronchopulmonary dysplasia, there is now convincing evidence that prematurity alone
may also result in permanent alterations to the way in which the lungs, vessels and
bronchial tree develop [51, 52]. Infants born at a gestational age of 24 weeks (canalicular
stage) are about to begin forming the distal saccules of the lung in parallel with
development of the alveolar capillary bed, and this anatomical development seems to be
arrested by premature birth [53]. This is also compatible with the reported diminished
growth of airway function in the first year of life [5457].
With the current knowledge that concentrations of oxygen of i21% negatively affect
further outgrowth of the vascular bed, and that alveolar and airway development depend
on vascular development, it can be explained why premature lungs arrest their
development at birth, and may have a strikingly simplified architecture [52]. Further
studies are needed to assess whether this also implies that these lungs have fewer
peripheral airway generations (alveolar ducts), and to what extent the alveolar surface is
diminished.
Factors affecting lung growth

Programming
The hypothesis of programming was launched in 1991 by Barker et al. [58] to explain
the associations found between early-life respiratory disease and increased respiratory
morbidity and mortality in the elderly [59]. The concept of programming implies that the
structure and function of organs and tissues are permanently altered in their design
(programmed) by factors operating during sensitive periods of foetal or early post-natal
life [59, 60]. Since then, numerous epidemiological studies have published supportive
evidence for this theory.
Factors that affect programming include: genetics (including factors determining sex
or race); quality and quantity of nutrition; placental characteristics; maternal drugs and
hormones; exposures to toxins (such as nicotine and other compounds in cigarette
smoke); mechanical factors (such as amniotic fluid volume, diaphragmatic integrity,
stretch and FBMs); and duration of gestation (see above). Hence, it is assumed that the
growth and development of the respiratory system are largely programmed in utero [60],
and there are reasons to assume that, once the basic structure of the respiratory system
has been realised during this critical phase, the development of lung function and
anatomy follows a more or less fixed course, and exhibits tracking well into adolescence
in healthy subjects [61, 62] and those with respiratory disease [6365].
One of the best-known and -studied determinants playing a role in the growth and
development of the airways and lung parenchyma is the combined effects of space and
mechanical forces, or stretch [66].
Studies of such mechanical determinants of lung growth have been reviewed in several
articles [4, 5]. Foetal lung fluid volume and its maintenance are essential to normal lung
growth and development. It is the balance between production by the distal airways and
drainage through swallowing or release into the amniotic space which appears to be
important in normal lung development [4].
13
Since stretch is known to induce the release of mitogenic growth factors [66], it
constitutes a stimulus for pre-natal and post-natal lung growth. It is for this reason that
FBMs are highly important for the proper development of the lung and respiratory
muscles [67].
A number of compounds and conditions can modify foetal breathing frequency and
amplitude. Hypercapnia, hyperglycaemia, acidosis, fever, caffeine and theophylline,
terbutaline and indomethacin can increase FBMs [4, 68], whereas nicotine, alcohol,
several sedative and narcotic drugs, corticosteroids, hypoxia, hypoglycaemia, prostaglandin E2 and infections inhibit FBMs [4, 67, 69]. The effects due to maternal smoking,
in particular, have been extensively studied, since it is a common toxic exposure, usually
with long-term exposure during pregnancy. Passive pre-natal smoking is associated with
irreversible alterations in lung growth [70]. Histological alterations have been shown in
experimental studies [8] and in humans [71], as well as reduced lung function [72, 73].
Little is known about the effects of maternal disease or stress in general on FBMs.
Since FBMs are of such importance, it is warranted that any negative influence on FBMs
should be minimised, and research is needed to assess the therapeutic advantage of
stimulated FBMs in pathological pregnancies, such as those with intra-uterine growth
retardation or diminished FBMs.
Another recognised covariable is birthweight, which may reflect, to some extent, the
quality and quantity of nutrition, placental function and/or genetic factors. Birthweight
was found to show a modest positive association with adult lung function, which
indicates that intra-uterine factors might play a role in lung development [74]. This was
also found in premature children; childhood lung function was found to be strongly
associated with birthweight, much more so than neonatal illness and/or subsequent
treatment [75].
Dysanapsis
Knowledge about the growth patterns of the airways and airspaces is based on crosssectional anatomical studies and longitudinal and cross-sectional lung function
assessment; all have their limitations. The human bronchial tree is formed during the
first trimester of pregnancy and its branching is complete by the end of gestation. Alveoli
only begin to appear around week 29 of pregnancy, and there is an enormous increase in
alveolar number during the first 2 yrs of life; thus the growth patterns of airways and
alveoli differ in their timing. Also, in later life, there are phases during which growth of
the airways and alveoli cannot be described as isotropic [61]. This phenomenon of
unequal growth has been coined dysanapsis [76, 77], and partly explains the occasionally
large between-subject differences in forced expiratory flows and other measures of airway
patency, since the degree and timing of the phenomenon may differ between subjects.
Solid longitudinal studies into the issue of dysanapsis are scarce [61, 78], but there are
indications that dysanapsis originates in early childhood [79], suggesting that it may very
well be a consequence of pre-natal programming (see above).
Although dysanapsis may constitute a completely normal variation in anatomy, it
seems to partly determine deposition patterns of inhaled substances [80, 81], and may
constitute a risk factor for the development of respiratory symptoms [7] and have
prognostic consequences. For example, it can partly explain the differences in prevalence
and severity of respiratory disease and hospital admission rate between male and female
children (see below).
It has traditionally been assumed that structural changes are irreversible after the
completion of normal alveolar development (i.e. with final alveolar numbers almost
attained at the age of 2 yrs, and before the age of 8 yrs, with dimensional growth
14
occurring thereafter) [82]. However, there is now evidence to suggest that the lung has
more potential to recover, with reparative growth after insults to the lung and
compensatory growth after volume loss, even after lung growth is supposedly complete
[7]. Several factors that have been implicated in playing a role in post-natal lung growth
have been used in experimental studies in an attempt to induce such growth [7]. These
include all compounds or stimuli that play a role in the interaction between
vasculogenesis/angiogenesis and the formation of airways and maturation of the
acinus: increased oxygen demand due to various causes, mechanical strain (from
movements, ventilation or surgical interventions), hypoxia, hormones (growth hormone
and corticosteroids) and several growth factors (including platelet-derived growth factor,
retinoic acid, VEGF and nitric oxide). Studies to date have demonstrated that
enhancement of lung growth varies among species, and it is currently unclear whether
enhancement of post-natal lung growth will become a realistic treatment modality in
humans [7].
Effect of sex on lung and airway development

At birth, male infants possess more alveoli [37] and probably exhibit a smaller airway
calibre for a given body size than do females. For the same lung size, females have larger
airways, resulting in higher forced expiratory flows and lower airways resistance. This
has been demonstrated for infants, children and adolescents, as reviewed previously [2];
in adults, however, the opposite has been described [77, 78]. This can be explained by
increased airway growth relative to volume in adolescent males compared with females
during puberty [61]. These and many other studies demonstrate that the growth and
development of the airways and airspaces differs according to sex and depends on age [2,
8385]. This has significant implications for several functional characteristics, and is
likely to influence the epidemiology of various respiratory disorders, respiratory
morbidity, the natural course of respiratory diseases, hospital admission rate and
mortality [86].
Effects of medication on the structural and functional

development of the respiratory system
Glucocorticosteroids administered to foetuses and (premature) infants may have
beneficial as well as detrimental effects on foetal lung development [87, 88]. Most
histological or structural evidence is derived from animal and/or in vitro studies. Previous
studies on developing rats have shown that injection of steroids into both the mother
before birth and the offspring after birth leads to attenuation of alveolar septation [89,
90]. This is due to precocious thinning of the matrix and maturation of the epithelial cells
and microvasculature. Confirmation of this effect has come from studies on sheep, which
have shown abnormal lung function as well as a reduction in the number of alveoli. The
number of doses of steroid and the time of gestation at which they are given does not
affect the outcome, but the effect is less in females [91].
Glucocorticoids are known to inhibit lysyl oxidase activity, resulting in diminished
cross-linking of the collagen and elastin fibre network, and thus altering the structural
integrity of the lung. This could have functional consequences for the tethering of the
airways in the lung parenchyma, but studies addressing this are lacking to date. A recent
study has compared the effect of inhaled and injected steroids in rabbits aged 15 weeks.
In this species, there is still alveolar development occurring, but, as in humans, some of
15
the alveoli have developed before birth. Rabbits received aerosolised budesonide or
injected dexamethasone. The injected steroid had a more deleterious effect on body
weight, lung volume, alveolar number, surface area and function than inhaled steroid.
Treatment with inhaled steroid caused specific growth retardation of the lung, but was
not sufficient to affect lung function. Alveolar size and number and elastin content, when
related to lung volume, were not affected, suggesting normal structural development but
inhibition of total growth. However, small peripheral airway walls were thinner and had
fewer alveolar attachment points, with a greater distance between attachments [92].
Thus, developing lungs are sensitive to inhaled glucocorticoids, the use of
glucocorticoids in young infants and children should be monitored, and only the
lowest doses that yield a significant clinical improvement should be used.
Although b2-agonists have been used therapeutically since the 1960s, little is known
about how they affect the growing lung. Salbutamol has been shown to inhibit
multiplication of human adult smooth muscle cells, and repeated or prolonged exposures
inhibit DNA synthesis without evidence of desensitisation [93]. This may be an
advantage where there is an excess of bronchial smooth muscle, but may be deleterious
during the normal growth period, particularly during the first few months after birth.
Since the 1990s, the use of anticholinergic drugs has been added, although there is no
proof that it improves the response of airways in wheezy infants. Experimental evidence
has shown that, in guinea pigs with increased bronchial smooth muscle, tiotropium
bromide (a muscarinic receptor antagonist) reduces contractility and contractile protein
expression [94]. Acetylcholine is known to increase cell multiplication, and thus
muscarinic receptor antagonists may prevent this. In rats, there are a greater number of
muscarinic receptors in the lungs during foetal life than later in life [95], which may be
important for the development of the bronchial smooth muscle. The effect of these agents
requires further investigation.
Recommendations for the future

Inhaled and systemic drugs are being prescribed to large numbers of infants and
children with respiratory disorders. Studies into their long-term effects on alveolar and
capillary formation and the mechanics of airways and airspaces in such humans are
lacking.
There is a need for improved or new techniques to monitor the airway and gas
exchange function of the lung tissue longitudinally, and, in particular, for new
approaches to assessing the inhomogeneity of diffusion capacity and ventilation.
The architecture of the peripheral lung, including the alveoli, determines airway
function, and may be damaged or disrupted following pre-natal smoking, prematurity,
congenital cardiac malformations, the use of drugs or various respiratory diseases.
Therefore, it is necessary to improve or develop imaging techniques suitable for
monitoring the development of the peripheral airways, vasculature and lung parenchyma
noninvasively and with minimal radiation use.
In addition, there remains a need for standardised histological morphometric studies
of paediatric lungs throughout childhood. By combining all of the functional,
histological, biochemical and genetic studies, progress will be made in understanding
the mechanisms of lung growth and their relative importance in contributing to the
function of the respiratory system.
16
Summary
Since the 1980s, it has become increasingly clear that conditions during foetal life and
early childhood are of paramount importance for optimal growth and development of
the respiratory system. The development of the pulmonary vasculature interacts with
that of the bronchial tree, and this has important clinical consequences for premature
infants and children with congenital cardiovascular abnormalities. Therapeutic
options for preventing abnormal development have been lacking until now. The
anatomical and functional development of the lung appears especially vulnerable to a
whole range of insults during gestation and the first few years of life, and a significant
proportion of adult lung disease probably has its origin in utero or in early infancy.
Many conditions and treatment modalities may affect lung maturation and growth,
including the drugs administered during early life. The magnitude of these effects in
humans needs to be studied further.
Promoting or facilitating optimal lung growth in foetuses and infants and reducing the
incidence of respiratory tract illness in infancy may reduce the incidence of chronic
adult lung disease in future generations.
There is a need for improved or new imaging techniques suitable for monitoring the
development of the peripheral airways, vasculature and lung parenchyma noninvasively and without radiation, and there remains a need for standardised histological
morphometric studies of paediatric lungs throughout childhood. By combining
functional, histological, biochemical and genetic studies, progress will be made in
understanding mechanisms of lung growth and their relative importance in
contributing to the function of the respiratory system.
Keywords: Airways, dysanapsis, imaging, lung growth, prematurity, pulmonary
vasculature.
Support statement: A.A. Hislop was supported, in part, by Actelion Pharmaceuticals
(London, UK).
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21
CHAPTER 3
Assessing lung growth and function in

infants and young children
M. Gappa*, J. Stocks#, U. Frey}
*Paediatric Pulmonology and Neonatology, Hanover Medical School, Hanover, Germany. #Portex
Respiratory Unit, Institute of Child Health, London, UK. }Paediatric Respiratory Medicine, University of
Berne, Inselspital Bernese University Hospital, Berne, Switzerland.
Correspondence: M. Gappa, Paediatric Pulmonology and Neonatology, Medizinische Hochschule Hannover, Carl-Neuberg-Str.1, D-30625 Hannover, Germany. Fax: 49 5115329125; E-mail: gappa.monika@
mh-hannover.de
During the first years of life, the lung undergoes major structural and functional
changes [1, 2], together with rapid growth of all structures involved, making this period
of life particularly susceptible to adverse influences of environmental as well as diseaserelated factors. Respiratory morbidity remains a major challenge not only for the child
and their family but also for the paediatric pulmonologist. It is unlikely that appropriate
preventive measures or therapeutic interventions can be developed unless there is a firm
understanding about the basic structure and function of the respiratory system, and how
these change with age. Direct study of its structural development is obviously always
difficult when human subjects, especially infants and children, are concerned. This
implies that it is necessary to mainly rely on indirect measures, particularly lung function
assessments.
Structural changes in the growing lung include alveolar growth and multiplication,
growth and maturation of the lung parenchyma, vascular development, growth of the
airways and maturation of the airway wall structures, all of which are influenced by the
simultaneous growth of the thoracic cage (fig. 1) [1, 3, 4]. Ideally, assessment of lung
function would serve to describe the phenotypic consequences of developmental
processes in healthy individuals, and the consequences of both intra-uterine and postnatal insults. True longitudinal assessment of respiratory function from birth through
childhood, during which changes in lung volume and mechanics secondary to disease can
be distinguished from those occurring with the physiological growth and development of
the structures involved, would facilitate understanding of the progression and natural
history of early lung disease, and the ability to monitor early changes and evaluate the
effect of treatment.
In order to better understand the impact of respiratory disease, as well as therapeutic
interventions, on lung function at this young age, its potentially long-lasting impact with
respect to its effect on lung growth and development must always be considered. Several
observations during recent years have supported these concepts of early programming
and tracking of lung function in health and disease, with correlations between respiratory
morbidity in early infancy and adult life being clearly demonstrated (see below) [58].
The foetal origins hypothesis states that programming of organ function, due to
stimuli or insults during critical periods in early foetal life, may have life-long
consequences [9]. Recent data showing a reduction in lung function shortly after birth in
healthy pre-term infants [1012], infants born small for gestational age [13, 14] and those
whose mothers smoked during pregnancy [1416], which is not made up during later
ISSN 1025-448x.
22
LUNG GROWTH AND FUNCTION
Tissue
Pa,O2, PPa,CO2
Pa,O2,
a,CO2
Oxygen uptake
Alveolar size
Alveolar number
Lung volume
Ventilation inhomogeneities
Convection/diffusion disturbance
Vasculature
Airways
Size
Number
Localisation
Compliance
Inertance
Compliance
Resistance
Respiratory
Airway
Ventilation inhomogeneity
Bronchial responsiveness
Airway diameter
Tube length
Airway wall stability
Fig. 1. Structurefunction relationships in the developing lung. In addition to the structural components
mentioned in the diagram, developmental changes to the chest wall and control of breathing have to be
considered, which may influence all functional parameters. Disturbed development of vascular structures, alveoli
and airways, as well as regional ventilation inhomogeneity, may also lead to ventilation/perfusion mismatching.
Pa,O2: arterial oxygen tension; Pa,CO2: arterial carbon dioxide tension.
infancy, support this hypothesis. Early exposure to environmental insults, such as air
pollution, has also been shown to alter pulmonary function into school age [17].
Furthermore, there is increasing evidence that early lung function may be predictive of
lung function, and thus respiratory morbidity, in later life (the concept of tracking) [5, 8,
10, 18], and that children with cystic fibrosis (CF) may have early impairments in lung
function even before there is clinically apparent lung disease [19, 20]. These studies
emphasise the need to assess function during this vulnerable early period as a measure of
the growth and development of the airways and lungs, in both health and disease, as a
basis for understanding not only the early determinants of airway function in health but
also the pathophysiology in different diseases in order to develop appropriate preventive
and treatment strategies.
The aims of this chapter are to: 1) provide a brief overview of the lung function
techniques that are currently used to assess lung function in infants and young children,
with special emphasis on the most recent developments; 2) discuss models and concepts
of (patho)physiological mechanisms in the growing lung, early childhood lung diseases
and appropriate treatment strategies; 3) discuss important future questions in this field;
and 4) discuss what is required to find answers to these questions.
Developmental aspects of assessment of lung function during

the first years of life
The major difference in assessing lung function during the first 2 yrs of life, compared
with measures of pulmonary function from approximately the third birthday onwards,
relates to the need to perform measurements while the infants are sleeping, most
commonly following sedation with chloral hydrate. In contrast to older subjects,
measurements are generally performed in the supine position, using a face mask.
Developmental aspects that have to be considered include the fact that infants are nose
23
M. GAPPA ET AL.
breathers, since nasal resistance comprisesy50% of total airway resistance. Furthermore,

the upper airways are known to play an important role in modulating expiratory flow,
and thus the end-expiratory lung volume. This is necessary because the chest wall is
highly compliant in infants, with minimal outward elastic recoil, such that, under passive
conditions, the lungs recoil to a much lower lung volume in relation to total lung capacity
(TLC), with a tendency towards airway closure at low lung volumes. The physiological
mechanisms preventing the lungs from collapsing, which include a high respiratory
frequency, short expiratory time, post-inspiratory braking and laryngeal modulation of
expiratory airflow, often result in a variable end-expiratory level. This may impede
assessment not only of lung volume but also of mechanics and forced expiratory flows,
which are highly volume-dependent and hence need to be related to the lung volume at
which they were measured. In addition, lung volume measured throughout the period in
life during which the end-expiratory level, and thus functional residual capacity (FRC), is
actively maintained will never be directly comparable to measures of resting lung volume
later in life.
Although the time between the second and sixth birthday used to be regarded as a
black box as there were no suitable methods for testing young children during the
preschool years, most tests of lung function routinely used in older children and adults
have recently been successfully adapted for this age group. Although some of the issues
discussed above are less relevant to this age group, rapid growth of all pulmonary
structures has to be considered when measuring and interpreting lung function in both
infants and very young children. From birth, growth in lung volume occurs by
multiplication of alveoli until the age of y18 months [21, 22]. When multiplication is
complete, further growth in lung volume occurs via the alveoli increasing in diameter and
surface area. In contrast, at birth, the conducting airways are complete in number, with a
subsequent two- to three-fold symmetrical increase in length and diameter through to
adulthood [23]. These disproportionate developmental patterns of lung volume and
airway growth are reflected in the concept of dysanapsis [2426], and influence the rate of
lung emptying in relation to lung volume with growth [23]. It must be remembered that
accurate interpretation of lung function tests in both infants and preschool children, in
whom pulmonary and somatic growth are so rapid, is highly dependent upon accurate
recordings of height and weight at each test occasion.
Overview of current techniques

As summarised in recent reviews on the assessment of lung function and the
application of such tests in infants and preschool children [2, 2736], assessment of lung
function during the first years of life remains a challenge. In infants, tests require highly
specialised equipment with regard to frequency response, safety, minimisation of dead
space and resistance; usually have to be performed by two experienced investigators; are
time-consuming and frequently require sedation. These factors, together with constraints
due to parental work patterns, limit acceptability, study duration and the frequency with
which tests can be repeated [37, 38]. In addition, interpretation of test results may be
limited because of the lack of appropriate reference values and the difficulty in recruiting
healthy control groups due to ethical constraints in many centres. Until recently,
assessment of lung function in infants has, indeed, been restricted to a few specialised and
research-orientated centres throughout the world. However, an international task force,
with input from both the European Respiratory Society (ERS) and American Thoracic
Society (ATS), has responded to increasing interest in this field by producing a series of
manuscripts summarising the state of the art in infant lung function testing and
24
proposing standards for equipment requirements and the most commonly used
techniques for infant studies [3742]. These processes, together with the gradual
development of appropriate commercial equipment that meets these specifications, have
now opened the possibility of reliably assessing the usefulness of infant lung function
testing in clinical practice.
In preschool children, the challenge is to adapt equipment, methods and measurement
conditions from routine application in older children and adults to the special
requirements of this age group. Relevant factors for improving the feasibility of assessing
lung function in preschool children include a playful environment, adaptation to the
short attention span at this young age and considerable patience of all involved in
performing the measurements. These efforts have been met with remarkable success in
that, with specially trained operators and a suitable environment, many pulmonary
function tests now appear to be feasible in i50% of 3-yr-olds and the majority of
children aged w4 yrs. Techniques that have been recently adapted for the preschool age
group include spirometry [28], the forced oscillation technique (FOT) [30], the interrupter
technique, plethysmographic assessments of specific airway resistance [43], and measures
of FRC and gas mixing efficiency using gas dilution and washout techniques [31]. A joint
ATS/ERS Task Force is working to produce recommendations for the use of these tests
in preschool children, which will highlight the current state of knowledge and indicate
which further data are required before definitive guidelines can be developed.
An increasing variety of methods are available for assessing lung volume, respiratory
mechanics and control of breathing. The most commonly used methods, including
whole-body plethysmography for assessment of lung volumes and airway resistance [42],
multiple-breath nitrogen-washout assessments of lung volume [40], forced expiratory
manoeuvres within the tidal volume range [41] and passive respiratory mechanics [39],
have been summarised in detail before [44]. More recent developments include
application of multiple-breath inert-gas-washout measurement for measuring both
lung volume and ventilation inhomogeneity [31, 45], forced expiratory manoeuvres over
an extended volume range [46] and assessment of partitioned respiratory mechanics using
FOTs [30, 47]. For assessing lung growth and function, there is no single technique that
can accurately describe the complex maturational changes of the lung and airways in
healthy children, or changes secondary to potential intra-uterine and post-natal insults in
disease, a combination of carefully selected techniques applied longitudinally over the
period of interest being essential to achieving this aim. The advantages and limitations of
some of the most commonly used techniques are briefly summarised below.
Lung volume
The FRC is the only lung volume that can be readily assessed in infants and very young
children, using either whole-body plethysmography or gas-dilution. The use of the raised
volume technique, in combination with plethysmography, which potentially permits
assessment of partitioned lung volumes over the full volume range in infants, is still
restricted to a few specialised centres [46, 48, 49]. Plethysmography permits assessment of
the FRC, including the volume trapped behind obstructed airways. In infants,
plethysmography usually requires sedation. Commercial equipment, which fulfils the
equipment requirements proposed by the international task force, is now available for
subjects who weigh y315 kg. Plethysmography is not a mobile technique, thus
precluding assessment in the intensive care unit as a bedside tool. Although a collation of
international reference data has been published [42], data obtained using the new
generation of body plethysmographs yield lower values than previously reported [50].
Although plethysmography is not suitable for assessment of lung volume in awake
25
M. GAPPA ET AL.
children agedv6 yrs, it may still be feasible to assess specific airways resistance in this age
group [43].
Gas-dilution techniques, such as helium-dilution or multiple-breath nitrogen- or
sulphur-hexafluoride-washout, are more time-consuming, at least in the presence of
airway disease. However, as these techniques only require quiet tidal breathing, without
any airway occlusion, they may be applied without sedation in the youngest infants and
are applicable at the bedside. With appropriate adaptation of the size of the equipment
and the bypass flow, such a technique may be applied in all age groups, including
preschool children, too old for sedation and too young to cooperate for standard wholebody plethysmography [5154]. In contrast to plethysmography, gas-dilution techniques
only measure gas readily communicating with the large airways. Principally, multiplebreath washout tests can be performed using a nitrogen-washout technique or by adding
an inert tracer gas to the bypass flow during the wash-in period. Although there have
been numerous studies using the nitrogen-washout technique in the past, there is
currently no commercially available equipment utilising this technique. Instead, there has
been increased interest multiple-breath inert-gas-washout measurement, primarily using
helium or sulphur hexafluoride as the tracer gas [31, 45, 51, 52, 5457]. In addition to
assessment of FRC, multiple-breath washout data can be used to calculate indices of
ventilation inhomogeneity, which may be very sensitive in the detection of peripheral
airways disease (see below). As stated above, interpretation of longitudinal data on lung
volume from infancy to childhood has to take changes in breathing pattern, determinants
of resting lung volume, sleep state, posture and relative dead spaces (including that
arising from the use of a mask versus a mouthpiece) into account. Most importantly,
however, measurement of lung volume is only ever a rough estimate of lung growth, since
none of the techniques described above can reflect the number or size of the alveoli.
Airway function
Spirometry remains the most commonly used test of lung function in older children.
However, it should be remembered that forced expiratory manoeuvres only describe the
function of the conducting airways. Depending on the age and size of the child, the
function of airways beyond generations 710 are unlikely to be reflected in these
measurements [58]. In infants and very young children, the rapid thoracoabdominal
compression (RTC) technique has been standardised, with appropriate commercial
equipment being available. However, the maximal forced expired flow at FRC, the
parameter most commonly reported from the RTC technique, is heavily dependent upon
the end-expiratory level, partially accounting for the high observed inter-individual
variability [59]. In addition, airway function is likely to be influenced by both the size of
the conducting airways and the stability of the airway wall, the effects of which cannot be
differentiated between using this technique. A raised-volume (RV) RTC following lung
inflation to near TLC is now increasingly being used, and may be more sensitive to early
pulmonary changes [49, 6062]. However, this technique remains rather invasive, and
differences in equipment and measurement protocol make interpretation of results and
comparison between different centres difficult [46]. With regard to longitudinal
assessment of airway function beyond infancy, preliminary data from London are
encouraging in suggesting that data on forced expiratory volume in time (FEVt) and
maximum expiratory flow when x% of the vital capacity remains to be exhaled (MEFx)
obtained using the RVRTC technique tie in with conventional spirometric results in
young preschool children [44]. As stated above, with adaptation of conventional
spirometry to the younger age group, including playful training, the use of selected
computer incentives and the development of appropriate quality control measures, it has
26
been shown that spirometry can be successfully performed by the majority of preschool
children [6365]. The relative usefulness of spirometric indices compared to other
parameters of lung function for assessing pulmonary changes in diseases such as CF
remains the subject of further research [52, 66].
Although, in the past, plethysmographic assessment of airway resistance greatly
increased the understanding of lung growth and development [6769], its use in infants is
currently limited by the lack of validated commercially available equipment [70].
Respiratory mechanics
The occlusion techniques for assessing passive respiratory mechanics have been
standardised by the ERS/ATS task force [39], are quick and easy to apply, and can be
used in spontaneously breathing, as well as mechanically ventilated, infants [36].
However, both resistance and compliance are dependent upon the lung volume at which
they are measured. In addition, the calculated results do not allow separation of the
different components of respiratory mechanics into the lung parenchyma and airways,
which becomes increasingly important as more is understood about developmental
processes and influencing factors. Partitioning of mechanics has been demonstrated in
both infants and young children using the interrupter technique, the low-frequency input
impedance FOT (LFOT) and the transfer impedance technique [47, 7177].
The interrupter technique has been most commonly used in preschool children [30, 78,
79], but its feasibility in unsedated young infants has also been demonstrated [77]. A
major potential problem of both input impedance measurements and the interrupter
technique is upper airway shunt compliance. This poses a particular problem in the
presence of a gas-filled face mask, which should be replaced by a putty-filled firm silicone
mask in infants. Although, for preschool children, standards for the measurement
procedure and analysis of the interrupter technique have recently been developed by a
joint ERS/ATS working group [44], opening the field for clinical studies, these issues,
together with assessment of the potential clinical validity of this bedside technique,
remain to be evaluated in infants.
Other potentially interesting techniques, which currently remain within the research
arena, are the LFOT and transfer impedance technique for measuring impedance. Both
have helped improve understanding about the contribution of tissue mechanics to
asthma and wheezing disorders in infants, particularly during bronchial challenge tests.
There is new evidence from such measurements that bronchoconstrictor agents may
increase not only airway but also parenchymal impedance [75]. Although current data
modelling has some limitations with respect to separating the effects of ventilation
inhomogeneities due to inter-regional flows or tissue damping, as well as separating the
influence of airway resistance [7476], these findings are nevertheless highly interesting,
and show the importance of tissue properties during induced bronchoconstriction. Highfrequency input impedance measurements have recently shown that airway wall
mechanical properties in infants with wheezing disorders are relevant to the phenomenon
of flow limitation. Such measurements might help in elucidating the role of changes in
airway wall mechanics following remodelling early in life and during development [80
82]. In infants, the equilibrium between tissue properties, lung volume, airway wall
compliance and airway diameter is highly complex and dynamically interacting. Such
lung function techniques may be particularly useful in future studies of respiratory
disease in neonatal intensive care, during which the underlying pathophysiology
frequently includes both the airways and parenchyma [83]. It has recently been
demonstrated that a brief respiratory pause may be sufficient to apply LFOT in infants,
including unsedated neonates, encouraging further work towards a clinically relevant
measurement technique [83].
27
M. GAPPA ET AL.
Control of breathing
Measurement of airflow during tidal breathing is one of the most commonly and easily
performed lung function tests in the newborn infant [84]. Tidal breathing measurements
change with alterations in lung mechanics, and have been used extensively, in the past, to
evaluate the effect of different treatments and monitor functional progress over time.
However, tidal breathing reflects not only the mechanical properties of the respiratory
system but also alterations in control of breathing. These two factors are not easily
separated. Tidal breathing measurements have also been used to assess parameters of
control of breathing in sleep-related breathing disorders (SRBDs). SRBDs can occur in
infants delivered prematurely or those with chronic lung disease of infancy (CLDI [84]),
upper airway problems or tracheomalacia, or impaired central respiratory drive
(congenital central hypoventilation syndrome). SRBDs in CLDI are often due to
immature control of breathing in combination with impaired lung mechanics, and result
in clinical signs such as sleep fragmentation, apnoea, hypoxaemia or even bradycardia.
SRBDs predominantly occur during the early neonatal developmental period, which is
marked by maturation of cardiorespiratory control, lung growth and sleep organisation.
Control of breathing analysis has mostly been derived from tidal flow measurements,
measured either directly via a flow meter or indirectly via observation of chest and
abdominal movements using respiratory inductance plethysmography [8587] or laser
monitoring [88]. Such measurements have been performed during spontaneous sleep, as
well as following challenges with gas mixtures known to influence control of breathing.
Several factors influence tidal breathing pattern and waveform in infants and young
children; the pattern of tidal breathing changes rapidly during early post-natal life, and is
strongly influenced by equipment configuration and sleep state. Recently, newer
analytical methods have been proposed which focus on the long-range fluctuations in
tidal breathing signals containing information on breathing regulation [89, 90]. Such
methods are promising since they consider breathing control using a more
comprehensive system-dynamic approach. Of similar interest are new methods for
studying the interaction between control of breathing and airway mechanics, which have
currently been investigated only in older children [91, 92]. Further promising research in
the field has been carried out by observing fluctuation in tidal breathing following
spontaneous sighs. Sighs not only influence airway mechanics but also alter control of
breathing on a short time scale [93].
Models and concepts of (patho)physiological mechanisms in the

growing lung and early lung disease and treatment strategies
Programming
The long-observed association between childhood lower respiratory tract illness and
subsequent development of adult chronic respiratory disease has been confirmed in
numerous recent epidemiological studies [9498]. The nature of this link, the biological
mechanisms which mediate it, and the genetic, developmental and environmental factors
which influence its expression have been the focus of considerable research effort in
recent years. One concept evoked to explain this association is that of programming, the
permanent alteration of the structure and function of organs and tissues by factors
operating during sensitive periods in foetal or early post-natal life [95]. Factors
implicated in the programming of the respiratory system that have been demonstrated via
lung function measurements in infants include foetal nutrition [14, 99, 100], foetal
28
exposure to maternal smoking during pregnancy [16, 62], pre-term delivery [10, 11] and
exposure to environmental allergens or viral respiratory infections during infancy [101,
102]. Small-for-gestational-age infants have been found to exhibit diminished airway
function when measured using the RVRTC technique, with the effect persisting
throughout the first year of life [14, 99]. Similarly, there is now overwhelming evidence
that parental smoking has an adverse effect on airway function in both otherwise healthy
infants and infants with lung disease. A family history of atopy, particularly in the
mother, has been shown to influence respiratory function [67], e.g. including production
of nitric oxide [103]. All of these studies highlight the fact that multiple complex
interactions influence early respiratory function, and that the effect of single exposures or
risk factors should never be considered in isolation when interpreting lung function data
in either health or disease. For example, little is known about the impact of pre-term
delivery on airway development, although it has been shown that this may result in a
relative increase in the amount of bronchial smooth muscle and the number of goblet
cells, particularly among those who require mechanical ventilatory support [23]. Recent
publications have suggested that pre-term delivery, even in the absence of any neonatal
respiratory disease or ventilatory support, may have an adverse effect on subsequent lung
growth and development, which persists and may even worsen throughout the first years
of life [10, 11, 104106]. These studies have shown that lung volume may be smaller,
ventilation homogeneity impaired and compliance reduced during the neonatal period
[32, 33, 36]. Although most parameters tend to improve during the first year of life,
relative airway function, as reflected by forced expiratory flows, may further deteriorate
[10, 34]. These data have revolutionised the picture of CLDI or bronchopulmonary
dysplasia, since the target group for potential therapeutic interventions can no longer be
defined simply as pre-term infants with prolonged oxygen dependency after birth.
Unfortunately, many of the supposed structural changes in pre-term lungs, such as
alterations in the number and size of alveoli, cannot be differentiated between using
commonly applied techniques. In addition, forced expiratory flows result from a complex
interaction between airway size, the surrounding lung tissue and airway wall mechanics.
At present, there are few data regarding airway wall mechanics in infants, but it is likely
that altered airway wall development contributes significantly to the observed functional
changes. Some of the newer techniques for assessing partitioned mechanics may help to
clarify these issues [47].
An intrinsic factor that has consistently been shown to have a marked effect on
respiratory function is sex, as reflected in the increased prevalence of wheezing illnesses
and reduced forced expiratory flows in male compared with female children, especially
during the first years of life. This has necessitated the development of sex-specific
reference equations [107], which are important if significant changes are not to be missed
in females, or, conversely, overestimated in males. Similarly, differences in breathing
pattern and lower nasal and total airway resistance observed in Afro-Caribbean
compared to Caucasian infants [108, 109] point to intrinsic/genetic factors influencing
lung growth and development independent of intra-uterine factors or insults during early
post-natal life.
Tracking
One of the first large epidemiological studies that prospectively assessed development
of respiratory function in relation to clinical course was the Tucson Childrens
Respiratory Study [8, 110]. The observation that a pre-morbid reduction in respiratory
function is a risk factor for subsequent wheezing illness has been confirmed by later
studies [67, 111113]. The most recent follow-up data from the Tuscon study
29
M. GAPPA ET AL.
demonstrate that the group with the lowest lung function during infancy retained this
low level throughout childhood and puberty [8]. This concept of tracking, i.e. that early
lung function predicts subsequent development of function, has also been demonstrated
on an individual level [5, 10, 67, 99]. Although there are some discrepancies in the
literature with respect to pre-existing respiratory dysfunction and the course of
subsequent clinical disease, these epidemiological studies provide evidence that
assessment of respiratory function can be used to describe the phenotypic appearance
of structural changes, resulting from a huge number of potential pre- and post-natal
factors. The use of such tests to identify individual infants at risk of subsequent disease is,
however, not currently feasible due to the marked intersubject variability.
Early lung disease

Much has been learnt about early pulmonary disease in CF [114]. Although spirometry
has been shown to be an insensitive marker of early airway disease in preschool and
school-aged children with CF [31, 52, 55, 115], forced expiratory flows from raised lung
volume appear to be very discriminatory during infancy [60, 61]. Marked structural
changes have been demonstrated using computed tomography (CT) in children with
entirely normal spirometric results, with progression of these changes not being detected
by repeat spirometry [116118]. A combination of the raised-volume technique and highresolution CT (HRCT) may prove a powerful diagnostic tool if concerns regarding
ionising radiation can be addressed [119, 120]. The greater sensitivity of forced expiratory
manoeuvres in young children compared to older subjects may be explained by
differences in airway wall stability, different proportions of airway diameter and length
in relation to lung volume, and differences in chest wall compliance. Multiple-breath
washout appears to be a more sensitive method of identifying children aged w3 yrs with
early pulmonary changes [5153]; however, this remains to be proven in infants and very
young children. From studies using multiple-breath inert-gas-washout techniques, it
appears likely that pulmonary disease starts within the more peripheral airways in CF,
resulting in the observed ventilation inhomogeneity. Parameters of ventilation
inhomogeneity, such as the lung clearance index, have the advantage of being relatively
constant throughout life [55], thereby negating the need for age- or height-dependent
reference equations, at least beyond the first 612 months of life. There is also recent
evidence that parameters of ventilation inhomogeneity reflect progression of disease
more sensitively than conventional tests such as spirometry [121].
Future questions
Clinical relevance
The increasing recognition that early lung growth and development are important to
long-term respiratory health is reflected by the expanding role of infant lung function
testing in both clinical and research studies. Following years of study of molecular
biology and gene polymorphisms, the importance of using lung function testing as a
noninvasive tool for describing the phenotypic consequences has now been accepted
[122]. Although there is increasing evidence elucidating the functional development of the
lung, which demonstrates the importance of early programmers and the tracking of lung
function from the first months of life in both health and disease, there is still little
evidence as to whether early lung function tests are sensitive enough to detect clinically
relevant early changes in lung function in the individual patient. Nevertheless, continuing
30
efforts to standardise tests of infant and preschool lung function and develop reliable
commercially available equipment will hopefully permit relevant clinical questions, such
as those posed below, to be addressed in the future.
Can infant lung function tests be used for early diagnosis and recognition of disease
before clinical symptoms occur? From preliminary evidence, clinical conditions such as
CF and bronchial asthma might profit from such a functional diagnostic approach.
Identifying at-risk children might be helpful for preventive therapeutic strategies. Further
research is required to show whether early recognition and subsequent preventive
treatment are clinically useful target strategies.
Can infant lung function tests be used to monitor disease severity and progression?
There is evidence, in both school- and preschool-aged children, that disease progression in
CF and severity in bronchial asthma are reflected in lung function test results [52, 53, 121
123]. Assessment of lung growth and development requires serial measurements in a
longitudinal manner. This is important as repeat cross-sectional studies may not reflect
growth within a given population [124]. With regard to infancy and early childhood, this is
challenged by the need to sedate infants for most lung function tests, and by the lack of
appropriate longitudinal reference data for interpretation of the results (see below).
Recruiting and measuring suitable control groups are likely to require a multicentric
approach, which has been facilitated by recent standardisation of the most commonly
used techniques. Similar efforts should be undertaken for the most promising newer
techniques, requiring close collaboration between centres and manufacturers of potential
equipment. However, even if these challenges are met, other problems arise concerning the
longitudinal assessment of lung and airway growth. As discussed above, measurements of
lung volume in infants are never directly comparable to those in older children because of
the dynamic elevation of the end-expiratory level during the first year of life. When
measuring forced expiratory volumes such as FEV0.5, which is feasible across all age
groups, changes in measurement conditions should be considered, as discussed above. In
addition, during the preschool years, FEV0.5 may reflect the central airways more than
when the same parameter is measured during infancy, due to the reduced rate of lung
emptying with growth. Factors determining forced expiratory volumes are complex, and
it is unlikely that FEV0.5 measured during infancy and early childhood will provide similar
information to that obtained when measuring FEV1 in older subjects. Furthermore, even
interpretation of repeat measurement of FEV0.5 within a subject is difficult because it is
unlikely to provide information about the same airway generations with ongoing growth
[44]. Knowledge of within-subject between-occasion repeatability in health will also be
essential to the meaningful interpretation of serial measurements in disease, and
evaluation of whether such assessments are useful in the clinical management of
individual infants.
Can infant lung function tests be used to assess bronchial responsiveness? Assessing
bronchodilator response is probably one of the most important clinical applications of
lung function testing in older children and adults. Similarly, assessing the response to
bronchoconstrictors may be useful in excluding a diagnosis of asthma. However, although
there have been numerous articles reporting the assessment of bronchial responsiveness in
infants and preschoolers using a variety of lung function techniques, the role of these tests
in infants and young children has not yet been clearly defined [125, 126]. Although there is
evidence that the airways are fully innervated at birth and that bronchial responsiveness
may be a risk factor for developing asthma later in life, the discriminatory power of such
tests has been debated by some. Forced expiratory manoeuvres using the RTC technique
31
M. GAPPA ET AL.
have been the commonest method of assessing response to both bronchodilators and
bronchoprovocation; however, the concomitant changes in FRC may mask changes in
airway function. In infants, the situation is complex since heightened responsiveness may
result from a range of factors, including anatomically small airways, increased smooth
muscle tone, relatively thick airway walls, decreased chest wall recoil and increased chest
wall compliance. In addition, the equivocal findings in the literature can, at least partly, be
explained by the fact that there is currently no consensus as to which techniques may be
most useful for assessing changes in airway function, which agent should be used, the
dosage and delivery efficacy of the aerosol, how to quantify the airway response or the
potential clinical utility of the information obtained [2, 44]. It may, therefore, prove to be
impossible to interpret age-related changes in bronchial responsiveness during these first
years of life. Nevertheless, there is an urgent need for further studies to systematically
address questions regarding how bronchial reactivity is best assessed in this age group and
whether such investigations can contribute to better disease management.
Can infant lung function tests be used to predict long-term outcome? Some
knowledge of the expected long-term outcome might be particularly useful in guiding
practitioners and advising parents of children with CLDI or CF and other chronic
respiratory problems. As mentioned above, this is currently only possible at the
population level, and further work is required before it can be directly related to the
individual infant. As with all diagnostic tests, if it is to be used in this way, the results of
infant lung function tests would need to be interpreted with respect to all other relevant
clinical and background information
What is normal?
Reference equations are essential for expressing pulmonary function in relation to that
which would be expected for healthy children of similar age, sex, body size and ethnic
group; characterising and monitoring disease severity; expanding knowledge regarding
growth and development; and studying mechanisms of normal and abnormal function
and the natural history of the disease. The use of control groups is often the preferred
option in research studies, but any attempt to use infant lung function tests to determine
the nature or severity of lung disease in an individual will be thwarted unless appropriate
reference data are available. Unfortunately this overriding requirement is challenged by
the difficulty of undertaking such measurements in a sufficient number of healthy infants
using identical equipment, measuring conditions and methods. Moreover, the reference
population needs to cover the entire age and body size range likely to be encountered
clinically and to be matched for ethnic group, socioeconomic factors and environmental
exposures, such as pre- and post-natal tobacco smoke exposure. Since the most
meaningful results from clinical studies are likely to be gained from serial studies,
interpretation should ideally be with respect to longitudinal data from healthy infants,
although such data are currently very rare. Although some reference equations have been
published for various infant lung function tests [107, 127, 128], many are based on
relatively small numbers and may not be appropriate for use with the current
commercially available equipment [50]. Given the time-consuming nature of studying
infants, and the limited number of healthy subjects likely to be studied in most
institutions, there is an overwhelming need for prospective multicentric initiatives to
collate the data collected using a standardised protocol and equipment for both the well
established and recently developed infant lung function tests. Having done this, there is a
need for appropriate modelling in order to take age, sex and body size and ethnic group
into account, as well as relevant exposures, such as maternal smoking. Furthermore,
32
there should be a move away from the traditional practice of expressing results as a
percentage of the predicted value, which gives no indication of what the normal range
might be, instead reporting results with sds or z-scores. The latter would not only
indicate the magnitude of any changes in relation to normal between-subject variability
for a particular test but also facilitate longitudinal follow-up and comparison of results
from different tests [27].
Development of the respiratory system

Although there have been considerable advances in the tracking of airway function
during lung growth and development, there is growing evidence that prematurity, early
lung disease and/or environmental toxic influences disturb not only airway and vascular
development but also alveolar development. There are currently no means of directly
assessing alveolar number, size and surface. The use of HRCT has provided insight into
structural development [119, 120], but it is unlikely to be accepted as a routine tool,
especially in the absence of overt disease, because of the relatively large amount of
radiation exposure associated with this technique [129]. In addition, the resolution of
HRCT remains insufficient to assess structure down to the acinar level. Micro-CT is
being investigated in animal studies, but currently has no place in paediatric respiratory
medicine [130]. Magnetic resonance imaging of the lungs using hyperpolarised helium is
being discussed as a means of combining structural and functional assessment of the
lung, but, again, this approach currently remains strictly within the research arena, and
there is limited experience in children [131133]. More realistically, techniques that
permit partitioning of mechanical properties into airway and tissue components might
reach the level at which they could be used more widely [80]. This may be of particular
interest for assessing the effects of prematurity and monitoring infants in the intensive
care unit, where parenchymal disease is a major component of both acute and chronic
respiratory illness. Further insight into airway wall characteristics appears essential to
clarifying the role of developmental changes versus inflammation in wheezing disorders
and bronchial hyperresponsiveness [30, 80]. Accurate assessment of airway wall
properties could also help differentiate wheezing associated with reduced airway size
from that due to altered airway wall mechanics, as in congenital tracheobronchomalacia
or secondary to inflammatory processes. Attempts to describe airway wall properties in
infants are sparse, but both the FOTs and a high-speed interrupter technique may
provide further insight. Assessment of vascular development may be particularly
interesting in pre-term infants. However, there is currently no technique available that
has been evaluated for assessing pulmonary blood flow noninvasively.
Only techniques that are repeatable, noninvasive and applicable in all age groups are
likely to be successful when outcome measures for clinical studies are sought. Both
multiple-breath washout and the LFOT appear promising. With multiple-breath
washout, the current gold standard for measuring tracer gas concentrations is the use
of a mass spectrometer [31, 55]. However, there is no commercially available equipment
with appropriate software. Alternatively, an ultrasonic flow-head may be used [45, 54,
56, 134].
Dynamic behaviour of the developing respiratory system

Traditionally, the respiratory system has been considered a steady-state mechanical
structure; however, recent data show that the system behaves in a more dynamic
fluctuating manner. Analysis of the variability and correlation properties of these
fluctuations in lung function provides interesting information on the developmental and
33
M. GAPPA ET AL.
disease properties of the respiratory system. Future research should focus increasingly on
the dynamic properties of the respiratory system (see [135]).
Conclusions
In conclusion, assessment of lung function during the first years of life has provided
important insight into early growth and development and appears to support current
pathophysiological concepts such as programming and tracking. Although measurement
of lung function is now feasible at almost any age, true longitudinal assessments have
only rarely been performed. Current techniques may be used to provide outcome
measures in clinical trials, but their role in the clinical management of the individual
infant remains doubtful. More sophisticated techniques need to be developed further in
order to describe the complex aspects of the developing lung more adequately. It is likely
that the development of noninvasive imaging techniques, as well as methods for assessing
both alveolarisation and the pulmonary vasculature, will be required to fully understand
the structurefunction relationships of the lung during early life. One major task for the
future is to assess lung growth and development serially in healthy infants, with the aim
of not only understanding the influence of genetic and environmental factors, and their
interactions, on respiratory health but also providing essential reference data, with which
to detect subtle differences in pulmonary function early during the course of a disease,
before irreversible changes have occurred.
Summary
During the first years of life, the lung undergoes a period of most rapid growth and
development of all structures involved, making this period of life particularly
susceptible to adverse environmental and disease-related factors. Lung function
testing allows indirect noninvasive assessment of the functional consequences
reflecting this developmental process. Measurements of respiratory function can
now be carried out at most ages, with methodological guidelines being available for
most infant lung function techniques. Published studies incorporating functional
assessment of the lung and the airways appear to support current pathophysiological
concepts, such as early programming of lung function or tracking. However, direct
assessment of alveolar and vascular development is currently not feasible using
conventional methods. In addition, the complex interaction between airway
dimensions, airway wall characteristics, chest wall and tissue mechanics, all influencing
airway function, are not yet fully understood. The dynamic behaviour of the
respiratory system has only recently received attention with regard to the paediatric
population. A variety of newer techniques are being explored in order to clarify these
issues, including low-frequency forced oscillation and new imaging techniques.
Keywords: Infant, lung development, respiratory function, toddler.
34
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40
CHAPTER 4
Remodelling in paediatric respiratory

disease and impact on growth and
development
D.N. Payne, S. Saglani, A. Bush
Dept of Paediatric Respiratory Medicine, Royal Brompton Hospital and Airways Diseases Section,
National Heart and Lung Institute, Imperial College, London, UK.
Correspondence: A. Bush, Dept of Paediatric Respiratory Medicine, 4th Floor, Chelsea Wing, Royal
Brompton Hospital, Sydney Street, London SW3 6NP, UK. Fax: 44 2073518763; E-mail: a.bush@
rbht.nhs.uk
Remodelling is the collective term used to describe the structural changes seen in the
lungs of patients with respiratory disease. These structural alterations involve residential
airway cells and, possibly, bone marrow-derived pleotropic cells recruited from the
circulation. Structural changes have been reported in a number of respiratory diseases,
although they are most commonly described in the airways of patients with asthma [1].
The features of airway wall remodelling in asthma are shown in table 1. Until recently,
the focus has largely been on studies involving adults, due to practical and ethical
constraints limiting access to tissue from infants and children. However, in the last few
years, a number of groups have begun to study the paediatric airway [29], with changes
described in childhood asthma similar to those seen in adults (figs 1 and 2). As a result,
these new findings have begun to challenge the previously held assumptions about the
mechanisms and significance of remodelling.
Despite the similarities in the structural changes reported in both children and adults,
there remains a fundamental difference in the remodelling process between these two age
groups. In adult-onset disease, changes occur in airways that are already fully developed.
This contrasts significantly with the situation in infants and children in whom airway
development is still ongoing. Issues peculiar to infancy and childhood include the
following: 1) the physiological changes in airway calibre and length as normal growth
proceeds; 2) the developmental changes in the immune system, including the plasticity of
T-helper (Th) cell type 1 and Th2 responses, at least in the early months of life; 3)
exposure to a range of pathogens, viruses in particular, for the first time; and 4)
paediatric airway issues, such as gastro-oesophageal reflux and aspiration.
The mechanisms involved and the functional significance of structural airway changes
may therefore differ considerably between children and adults. Specifically, interference
with normal airway growth at crucial time periods may have particularly long-term
effects, by analogy with the critical period for alveolar development, which largely ends
by the age of 3 yrs, with little evidence of catch-up growth thereafter. In support of this
hypothesis is the finding that children with any wheezing phenotype presenting before the
age of 6 yrs had evidence of airway obstruction at age 16 yrs, whereas those in whom
wheezing commenced after the age of 6 yrs had normal spirometry at 16 yrs [10].
The most popular hypothesis is that structural airway changes in asthma develop
secondary to repeated episodes of airway inflammation. However, recent reports have
described thickening of the epithelial reticular basement membrane (RBM), a
ISSN 1025-448x.
41
D.N. PAYNE ET AL.
Table 1. Features of airway wall remodelling in asthma

Goblet cell hyperplasia
Thickening of the epithelial reticular basement membrane
Increased number of submucous glands
Increase in blood vessel number and area
Smooth muscle hypertrophy and hyperplasia
Increase in airway wall collagen
characteristic feature of asthma in adults, in both school-age and preschool children,

leading some investigators to suggest that remodelling occurs early and may develop in
parallel with, but separate from, airway inflammation [2, 4, 9]. That structural changes
may precede, or even be a prerequisite for, inflammation, has also been proposed [11, 12].
In contrast to studies involving adults, in whom asthma is always established at the time
of investigation, the ability to investigate airway changes in infants and young children
[2, 4, 13, 14], at a time when the airways are still developing and diseases such as asthma
are just beginning to manifest themselves, provides an opportunity to address some of the
fundamental questions regarding remodelling. These include the following. 1) When do
the changes begin? 2) What initiates the early changes? Is it inflammation or some other
factor? 3) What modulates them? Is it inflammation or some other factor? 4) Do they
SM
Blood
vessel
SM
RBM
Epi
Fig. 1. Low-power view of an endobronchial biopsy, stained with haematoxylin and eosin, from a child with
asthma. RBM: reticular basement membrane; Epi: epithelium; SM: smooth muscle.
42
REMODELLING IN INFANTS AND CHILDREN
RBM
RBM
Epi
Fig. 2. High-power view of an endobronchial biopsy, stained with haematoxylin and eosin. RBM: reticular
basement membrane; Epi: epithelium.
matter? Are they harmful or protective? 5) What features are unique to asthma, and what
are common to other diseases?
The importance of the fact that structural changes in childhood occur with the
background of an airway or lung that is still developing cannot be overstated. Thus, in
order to interpret correctly the changes seen in children, it is essential to understand the
process of normal airway development (see Chapter 2). A priority for future research
must therefore be to study normal human airway development antenatally and in the first
few years of life, in order to understand the mechanisms and significance of structural
changes in children with respiratory disease.
Current limitations of knowledge

Airway remodelling clearly encompasses virtually every airway wall component
(table 1). In the paediatric field, studies are limited mainly to measurements of the RBM.
Some work has been reported on what has been termed the epithelialmesenchymal
trophic unit (EMTU; see below), although the concept of the EMTU must still be
considered hypothetical, albeit a helpful exploratory model. RBM thickening is a
characteristic pathological feature of asthma in adults and is relatively easy to measure
and quantify, so it is an important as well as convenient measurement to make. However,
the paucity of data on other structural elements means that many of the conclusions of
this chapter have to be based on incomplete evidence. Furthermore, almost all studies in
the paediatric literature are in the context of wheeze and asthma, and little is known
about remodelling in other paediatric respiratory diseases.
The second major problem is the lack of true control data. Unlike in adults, it is
unethical to perform bronchoscopy in children solely for research purposes [15].
43
D.N. PAYNE ET AL.
However, it is safe and therefore legitimate to perform endobronchial biopsy at the time
of a clinically indicated bronchoscopy, with the approval of the Institutional Ethics
Committee, the consent of the family and the age-appropriate assent of the child [16].
There are detailed guidelines for the processing of endobronchial biopsy [15]. Most of the
available control data are obtained from children without asthma undergoing
bronchoscopy for investigation of respiratory symptoms, such as stridor, haemoptysis,
recurrent infection or chronic cough, or from tissue obtained post mortem [2, 7 9].
However, this is not the same as data on normal children.
The third issue relates to the association between structural changes and physiology.
The two main issues are the relationship (if any) between structural changes and fixed
airflow obstruction, and between structural changes and airway hyperresponsiveness.
Most papers on the subject of remodelling state that "structural changes may lead to
irreversible airways obstruction". While there is some evidence to support this statement,
the available data are limited. Kasahara et al. [17] demonstrated an association between
RBM thickness and forced expiratory volume in one second (FEV1; the thicker the
RBM, the lower the FEV1) in adults with asthma, following treatment with systemic
corticosteroids and inhaled b2-agonists. Benayoun et al. [18] studied airway smooth
muscle in airway biopsy and showed an increase in smooth muscle in those patients with
the most severe impairment of lung function. Interestingly, a disease control group,
consisting of adults with chronic obstructive pulmonary disease (COPD), had a similar
deficit in lung function, but without any increase in airway smooth muscle.
The relationship between structural airway changes and airway hyperresponsiveness
(AHR) also needs to be explored further. While structural changes are generally
considered to contribute to AHR associated with asthma [19], it has also been suggested
that one of the characteristic structural changes, RBM thickening, may actually be
protective against bronchospasm [20]. If this is the case, then attempts to reduce RBM
thickness may actually be misguided. Clearly, understanding the significance of airway
remodelling is crucial and must be a focus of future research.
One of the difficulties in assessing the relationship between airway structure and
function is in determining what constitutes true, fixed airflow obstruction. The
traditional method is to measure the acute response to bronchodilator. This is
convenient but unlikely to be valid in many contexts. In severe asthma, neither acute
bronchodilator administration nor even a 2-week course of prednisolone is necessarily
predictive of best lung function in the following year [21]. No one method is likely to be
truly predictive of best lung function; the choice lies between the acute response to
bronchodilator and the response to a prolonged course of steroids in some form, which
may be combined with acute bronchodilator administration [7, 17]. The choice of route
of administration includes a period of inhaled corticosteroids, oral prednisolone or even
intramuscular triamcinolone, the latter having the merit of ensuring that nonadherence is
not an issue [22]. The chosen method will probably be a compromise between what is
practical and ethical, and what is desirable for true scientific rigour. However, the
investigator will have to acknowledge the imperfections of whatever method is chosen.
What have been the most recent fundamental developments in

the field?
RBM thickening: a consequence or cause of asthma?
The relationship between remodelling and inflammation in asthma is unclear. It should
be noted that in both asthma and other diseases, it may not be valid to consider
44
Lung function
remodelling as a single entity; different components may have different relationships with
inflammation. Possible relationships include the following: 1) remodelling is a direct
consequence of ongoing airway inflammation; 2) an underlying factor causes both
inflammation and remodelling as separate processes, in parallel but at different rates; and
3) the primary defect in asthma is an abnormality of airway structure, including airway
matrix components, and this is a prerequisite for the development of airway
inflammation.
Currently, the validity of these concepts is not known. One important source of
information is those cohort studies in which longitudinal lung function measurements
have been made. These epidemiological data are discussed in detail elsewhere, in Chapter
1 of this Monograph. No cohort has been followed through from before birth to old age,
so conclusions have to be based on a composite of cohort studies and other
epidemiological evidence. The physiological findings of the prospective, longitudinal
cohort studies in childhood asthma can be summarised as follows. 1) Babies with
transient wheeze (predominantly associated with viral colds, stopping before 3 yrs of age)
are born with airflow obstruction and continue to have lung function impairment at
16 yrs of age [10, 23]. 2) Babies with persistent (usually atopic) wheeze are born with
normal lung function, but by the age of 6 yrs have airflow obstruction, which persists
into adolescence [10, 23]. 3) Children whose first episode of wheeze occurs after the age of
6 yrs show no evidence of airflow obstruction at 16 yrs of age [10]. 4) From the age of
7 yrs until at least the mid-40s, lung function in transient wheezers and atopic asthmatics
follows exactly parallel tracks, with the atopic asthmatics having a lower starting point
[24, 25]. 5) Lung aging, manifest by worsening airflow obstruction, is accelerated by
active smoking, and also accelerated in asthmatics and children who previously had
transient wheeze [26, 27]. However, lung aging is a late phenomenon. 6) A major
determinant of chronic obstructive airways disease in the elderly is early life events,
decades previously [28].
The most logical conclusion from these data is that the atopic infants who wheeze
before the age of 6 yrs suffer structural damage before age 6 yrs, and that thereafter the
process "burns out" and is stable (fig. 3). The nature of this "hit" is currently unknown. It
is unlikely that there will ever be prospective, serial airway biopsy studies commencing in
Early hit#
Age yrs
Fig. 3. Hypothesised relationship between development of structural airway changes and lung function
impairment in childhood asthma (see text for discussion). #: The development of structural airway changes and
lung function impairment, with subsequent tracking of lung function over time.
45
D.N. PAYNE ET AL.
childhood and weaker evidence will still need to be relied on, either serial noninvasive
measurements (see below) or cross-sectional studies in different groups of children at
different ages. Recent cross-sectional data have suggested a pathological mechanism that
is compatible with physiological data. In one study, symptomatic infants (median age
12 months) undergoing bronchoscopy as part of their diagnostic work-up were
investigated [14]. Subjects were assigned to one of the following three groups, based
on plethysmographic data: 1) infants with increased airways resistance, which was
acutely reversible to bronchodilator; 2) infants with increased airways resistance, which
did not reverse with bronchodilator; and 3) infants who had normal lung function.
Airway biopsies taken from the main carina showed no difference in RBM thickness
between the three groups and, interestingly, no evidence of airway inflammation.
Comparison with biopsies obtained from healthy adults and paediatric "controls"
showed that RBM thickness was similar in the infants and the older control groups.
In a second cross-sectional study in preschool children (median age 3 yrs) [29], RBM
thickness was measured in biopsies from subjects with true wheeze, identified from a
video questionnaire [30], and compared with data from two other groups; subjects with
reported, but unconfirmed, wheeze and a "normal control" group. RBM thickness was
greater in the confirmed wheeze group compared with controls. However, the absolute
values of RBM thickness were less than those reported in older schoolchildren with
difficult asthma [6, 7]. These pathological data therefore imply that RBM thickening may
begin within a window of 13 yrs of age, increasing to school age, a concept which fits
with the lung function findings in different cohorts.
The weaknesses of the pathological data must be acknowledged. First, they are not
longitudinal; they are not even serial cross-sections of the same population. Secondly,
until these two (infant and preschool) cohorts have been followed up into mid-childhood,
it can only be speculated, based on predictive factors established by others [31, 32], as to
which of the children will be the true asthmatics. Third, although RBM thickening has
been shown to be present, it is not known whether this is important or merely a marker
for some other, as-yet undetermined change in the airway wall. Nonetheless, the
pathological and epidemiological data strongly suggest that the real changes of
remodelling are a very early event in asthma [2, 4, 29], which may be preceded by
symptoms [14]. However, in order to interpret accurately the significance of the changes
seen in symptomatic children, better data from genuine healthy controls are needed, as
too little is currently known about the normal developmental structural airway changes
in infancy and early childhood.
RBM thickening occurs early and is nonprogressive in children with asthma

The paradigm that repeated cycles of acute inflammation or unremitting chronic
inflammation eventually lead to remodelling would logically predict a relationship
between duration of asthma and the severity of structural changes. Furthermore, it
would also predict that anti-inflammatory therapy is all that is needed to prevent airway
remodelling.
Recent paediatric data in older children, as well as the studies in preschool children,
referred to above, have challenged this model. RBM thickness in biopsies obtained from
a group of children (aged 616 yrs) with difficult asthma was compared with data from
the following four other groups: 1) adults with mild asthma; 2) adults who had been
ventilated for asthma; 3) healthy adult controls; and 4) paediatric controls, as described
above [8]. RBM thickness was the same in all three asthma groups and was significantly
higher than in both control groups. In the group of children with difficult asthma, there
was no relationship between RBM thickness and duration of symptoms, level of
46
treatment, or any marker of inflammation studied [7, 8]. Although these data are crosssectional, it is difficult to reconcile them with any mechanism postulating progressive and
ongoing activity in at least this aspect of remodelling.
Set against these data are the "acute remodelling" studies. Segmental allergen challenge
involves the endobronchial instillation of an appropriate allergen, with bronchoscopy,
bronchoalveolar lavage (BAL) and endobronchial biopsy performed before and after
allergen challenge, to study the inflammatory and other changes. In one study, nine
adults with mild asthma underwent endobronchial challenge to an allergen, to which they
were sensitive on skin-prick testing [33]. Twenty-four hours after challenge, there was
evidence of both epithelial cell and fibroblast activation, with a significant increase in the
deposition of the matrix protein tenascin within the RBM. For obvious practical reasons,
the resolution of these changes (if any) could not be followed by serial bronchoscopies.
The significance of acute airway challenges is difficult to assess. In real life, sensitised
subjects are likely to undergo repeated allergen airway challenges. If, with each challenge,
there is deposition of matrix tenascin, with no mechanism of resolution, then over the
years the airway would be obliterated altogether. Is there tolerance to challenge over
time? Do acute changes, such as those described above, resolve completely? What is the
relationship with chronic remodelling? These questions need further work if they are to
be answered, but a study of the mechanisms of resolution of acute remodelling might
allow us to understand and modulate chronic airway wall changes (see below).
RBM thickening is also seen early in the course of cystic fibrosis

Although RBM thickening is a characteristic feature of asthma, it also occurs in other
diseases. Adult studies have described RBM thickening in eosinophilic bronchitis [34],
allergic rhinitis [20], post-lung transplant and even diabetes [35, 36]. What is not known is
whether or not the mechanism and nature of RBM thickening is the same in all disease.
Thickening has also been reported in children with cystic fibrosis (CF) [37]. It is not in
dispute that babies with CF are born with essentially structurally normal lungs or that
when death from respiratory failure ensues, the airways are structurally very abnormal.
Furthermore, from an early age, there is evidence of chronic infection and an exuberant
inflammatory response. The relationship between infection and inflammation is reviewed
in Chapter 15. Still less is known about the relationship between infection, inflammation
and airway wall damage. Possible models, which are not mutually exclusive, are as
follows: 1) infection and inflammation lead to cycles of airway damage and repair,
eventually causing airway destruction; 2) airway destruction proceeds in parallel with
infection and inflammation, possibly as a result of the basic defect in CF transmembrane
conductance regulator; and 3) different components of the airway wall changes may be
modulated by different processes.
The differentiation between these models is of practical as well as theoretical
importance, as with asthma. The airway destruction is so much greater in CF than
asthma that, if anything, the question is even more important in CF. Is control of
infection with antibiotics, and possibly with supplementary anti-inflammatory therapy,
all that is needed to prevent the airways disintegrating in CF? Or should we be looking to
discover and modulate a separate pathway of airway wall changes in this disease?
The serial lung function data are more scanty than in asthma, but are also suggestive of
an "early hit" model. Infants diagnosed with CF following a clinical presentation (i.e. an
unscreened population) have evidence of airflow obstruction at diagnosis, even in the
absence of any apparent respiratory symptoms [38, 39]. Furthermore, two studies have
shown tracking of lung function in the preschool years, i.e. there was no "catch-up"
growth in lung function despite treatment in specialist centres [40, 41]. Even in an
47
D.N. PAYNE ET AL.
unscreened population of infants, newly diagnosed with CF, there is evidence of RBM
thickening, although to a lesser degree than in asthmatics [37]. The present authors
hypothesise that some factor(s) possibly, but not necessarily, related to RBM thickening
and probably, but not necessarily, related to infection and inflammation, irretrievably
impair lung function early in the course of CF. The later destructive processes may be
separate from these early phenomena. Modulation of early changes may allow patients to
attain better lung function after diagnosis and hence prolong survival.
Invasive techniques are becoming more acceptable in children

There are currently no satisfactory noninvasive markers of remodelling in children (see
below) and, in any case, such a marker would need to be validated against invasive
studies. Endobronchial biopsy has long been an acceptable diagnostic and research
procedure in adults and is used diagnostically in children. BAL and non-bronchoscopic
lavage have been used in children for research purposes [42, 43], despite the fact that fever
is common after the procedure. Although the concept of research endobronchial biopsy
at the time of a clinically indicated procedure has been questioned, the current authors
and others have published work establishing its safety and usefulness in children [2, 9, 13,
44, 45]. Harvesting epithelial cells by brushing has also been used safely for research [46,
47]. If sound ethical principles are followed, there is no reason not to use the
opportunities afforded by clinically indicated invasive procedures, such as bronchoscopy,
to carry out research.
Current models and concepts

The pathophysiological mechanisms
Structural changes occurring secondary to airway inflammation. As discussed above,
the most popular hypothesis is that structural airway changes develop as a consequence of
repeated bouts of airway inflammation. The data from allergen challenge studies support
this hypothesis, with further evidence from research, both in humans and in mice,
investigating the effects of treatment with antibody to interleukin (IL)-5. Using a mouse
model, Humbles et al. [48] demonstrated that eosinophil-deficient mice were protected
from the peribronchiolar collagen deposition and increase in airway smooth muscle
associated with allergen challenge. In humans, treatment with anti-IL-5 (three infusions
given at 4-week intervals) reduces airway eosinophil numbers, along with a reduction in
the expression of tenascin, as well as other components of the RBM (lumican and
procollagen III) [49]. Anti-IL-5 treatment is also associated with a significant reduction in
the numbers and percentage of airway eosinophils expressing mRNA for transforming
growth factor (TGF)-b1 and the concentration of TGF-b1 in BAL fluid. Together, these
data suggest that eosinophils may contribute to remodelling of the RBM in asthma by
regulating the deposition of extracellular matrix proteins. Related studies using mouse
models of asthma have also demonstrated an association between eosinophil-derived
TGF-b and airway fibrosis, following allergen challenge [50, 51], with anti-IL-5 reducing
the fibrotic changes within the airway [50, 52]. In addition, therapeutic treatment of mice
with anti-TGF-b antibody has been shown to reduce peribronchiolar extracellular matrix
deposition, airway smooth muscle cell proliferation, and mucus production in the lung
without affecting established airway inflammation and Th2 cytokine production [53].
These data therefore suggest that it might be possible to uncouple airway inflammation
and remodelling during prolonged allergen challenge.
48
The epithelialmesenchymal trophic unit. A complementary viewpoint is that the

changes seen in remodelling may represent the consequences of an abnormal repair
mechanism within the airway, following epithelial injury [54]. This model proposes that
the epithelium and its response to injury is the primary abnormality in asthma. It also
suggests a key role for the interaction between the epithelium and the associated
subepithelial tissue, which may represent a reactivation of the processes responsible for
embryonic lung development. Airway development in utero depends on interaction
between the developing epithelium (endoderm) and the surrounding mesenchymal tissue,
with branching of the primitive endoderm failing to occur in the absence of the
surrounding mesenchyme. This has led to the use of the term EMTU to describe the
anatomical and functional relationship between these two layers. Some of the proteins
which are intimately involved in embryonic airway development, such as tenascin,
fibronectin and collagens, are also associated with remodelling changes in asthma. This,
along with data supporting the presence of signalling between epithelial and mesenchymal
(e.g. fibroblasts) cells, has led to the suggestion that the structural airway changes
occurring in the asthmatic airway represent reactivation of the EMTU.
A role for epithelialmesenchymal interaction in RBM thickening in childhood asthma
has been suggested by Fedorov et al. [9], based on data examining the expression of
epidermal growth factor receptor (EGFR), which is increased in response to epithelial
injury within the epithelium of airway tissue from children with and without asthma.
Immunostaining for EGFR expression was most intense in children with severe asthma,
with a significant positive correlation between EGFR expression and RBM thickness.
The suggestion is that epithelial injury results in the release, by epithelial cells, of an array
of growth factors, including fibroblast growth factor (FGF), platelet-derived growth
factor and TGF-b. These stimulate increased fibroblast proliferation, which in turn leads
to an increase in RBM thickness. However, these data cannot determine whether or not
the proposed exaggerated repair process is a cause or consequence of asthma and thus
whether or not it reflects the inception or the progression of the disease. It should further
be noted that the validity of the concept of the EMTU awaits further data.
Animal models. Cross-sectional studies cannot be anything other than descriptive and
hypothesis-generating. For ethical reasons, intervention studies designed to test a specific
hypothesis, with biopsies obtained before and after intervention, cannot be performed in
children. Such studies can, however, be performed in animals, provided appropriate
models exist and that their limitations are acknowledged. Animal studies are expensive
and the use of animals close to humans, such as primates, is even more costly. The
advantage of animal models is that they provide an opportunity to explore potentially
relevant mechanisms of airway remodelling. However, a key difference between animal
models of asthma and the human form of the disease relates to the concepts of heredity
and risk factors for asthma. Most animal models rely on post-natal sensitisation to induce
atopy. Although a model using inhalational sensitisation has been developed [55], the
majority use intraperitoneal sensitising injections and it is difficult to see how these are
relevant to human asthma. There is no role in animal models for the effects of parental
atopy, a major influence on the development of asthma in children [31], or the
circumstances of the mothers pregnancy (e.g. diet, smoking). Viral infection is another
key feature of asthma in children, and one which is not addressed by allergen challenge
models in animals.
A number of different models have been developed and these include the use of rodents
(mice, rats) and mammals, both primates (monkeys) and nonprimates (sheep, cat). The
use of mouse models is widespread and provides the opportunity to investigate in great
detail mechanisms and pathways of potential importance, particularly regarding the
49
D.N. PAYNE ET AL.
relationship between inflammation and remodelling. However, there are significant

differences between the murine and the human airway. For example, the normal murine
airway does not possess a RBM, which develops only after allergen challenge. Studies
involving mice, and probably all animals, can therefore only ever be hypothesisgenerating.
A more appropriate model (albeit very expensive) for studying both normal airway
development and remodelling is a primate model [5661]. Not only does the airway wall
in the primate closely resemble that of the human airway, but the use of infant monkeys
also provides an opportunity to investigate the mechanisms of airway remodelling in the
developing airway (table 2). In addition, by examining the whole airway, rather than
endobronchial biopsy, the assessment of airway structural changes is not limited to the
RBM but includes other features such as airway smooth muscle.
The available data from primate models demonstrate that RBM development occurs
post-natally and that the growth factor, FGF-2, appears to play an important role,
initially being stored within basal epithelial cells before accumulating in the RBM [57,
58]. Allergen challenge leads to an increase in RBM thickness, associated with an increase
in certain components of the RBM, including collagens, the proteoglycan perlecan and
FGF-2. The authors of these studies have suggested that the thickened RBM may act as a
source of growth factors and proteins that are necessary to allow infiltrating subepithelial
inflammatory cells to move across the RBM into the epithelium and airway lumen [58].
The same investigators have also studied smooth muscle within the airway, showing an
increase in airway smooth muscle following allergen challenge [60].
As well as being able to describe normal airway development and the effects of allergen
challenge, this primate model provides the opportunity to investigate the progression
and/or resolution of remodelling changes and to examine the effects of interventions that
may inhibit remodelling. RBM changes persist in sensitised monkeys that continue to be
exposed to allergen every month [59]. Interestingly, it is not known whether the changes
resolve in the absence of any further allergen challenge. However, of potential
therapeutic interest is the finding that resolution of remodelling changes has been
demonstrated in young (35-yr-old) rhesus monkeys following repeated inhalation of
immunostimulatory DNA sequences (ISS) [61]. These sequences contain a CpG
dinuncleotide (CpG motif) that is characteristic of bacterial DNA but relatively rare in
vertebrate DNA. Thus, these data provide the potential to shed light on the factors that
regulate remodelling and to increase understanding of the functional significance of the
structural changes. The relevance to humans of these findings in a primate model needs
Table 2. Comparison between the development of the reticular basement membrane in infant rhesus
monkeys and humans
Rhesus monkey
Human
RBM develops after birth and full adult

thickness is reached by 6 months of age
RBM is present in infants with respiratory

symptoms. Unclear when RBM first appears or when
maximal thickness is reached
Constituents include collagens (I, III, V),
tenascin, fibronectin
Constituents of RBM include collagens,

proteoglycans (perlecan, lumican) and FGF-2
(initially stored in the basal epithelium)
Effect of allergen challenge on sensitised infants
Increase in RBM thickness, with increase in collagens,
proteoglycans (perlecan, lumican) and FGF-2
Focal areas of RBM thinning, in association with
trafficking of inflammatory cells across the RBM
Increase in RBM thickness in preschool

children with severe wheeze
RBM: reticular basement membrane; FGF: fibroblast growth factor.

50
to be investigated. To begin with, it should be possible to compare the immunohistochemical features of the developing monkey and human airway, given the increased
availability of airway tissue obtained from infants and children.
The related diseases

RBM thickening cannot be said to be pathognomic or diagnostic of asthma, given that it
has also been described in a number of other diseases [20, 3437]. This suggests that this
particular structural change may represent a normal response to injury. It is possible that
the control mechanisms, which regulate the extent of RBM thickening, may be set at a
different level in diseases such as asthma, in which the highest values of RBM thickness are
usually seen. The histological data on other components of the airway wall in children are
limited, so little can be said about other aspects of remodelling in other diseases. However,
it is clear that structural airway changes are found in other paediatric respiratory diseases,
such as CF, primary ciliary dyskinesia, other causes of bronchiectasis, obliterative
bronchiolitis and gastro-oesophageal reflux disease. The extent to which these changes are
reversible is a particular area of interest and debate [62].
The treatment strategies. There is little data on the effect of treatment on structural
airway changes. What evidence there is comes from studies of adult asthma and again
focuses on measurements of RBM thickness. Two studies have documented a reduction in
RBM thickness following prolonged treatment with inhaled corticosteroids [63, 64].
Ward et al. [63] performed a double-blind, randomised, placebo-controlled, parallel
group study of high-dose inhaled fluticasone (1.5 mg?day-1) involving 35 steroid-nave
adults with asthma. BAL and airway biopsy studies were carried out at baseline and after
3 and 12 months of treatment. A significant reduction in RBM thickness was noted only
after 12 months of treatment. In the same study, a significant reduction in eosinophils (%)
and mast cells (%) in BAL was demonstrated after 3 months treatment, with no further
effect after a year. This study perhaps supports the hypothesis of dissociation between
inflammation and remodelling. In an earlier study, Sont et al. [64] measured RBM
thickness at the beginning and the end of a 2-yr study designed to test the effect of
measuring AHR to methacholine as an aid to clinical management in adults with asthma.
Adjustments in treatment were made according to a standardised protocol. Patients
treated according to the AHR strategy had a lower incidence of mild exacerbations of
asthma compared with the reference group and received a higher daily dose of inhaled
steroids (median difference of 400 mg?day-1). A significant reduction in RBM thickness
was demonstrated in the AHR group, but not in the reference group. From these two
studies, it is clear that prolonged high doses of inhaled corticosteroids are needed if they
are to be used to modulate remodelling. The potential dangers of this approach (in
particular, hypoglycaemia due to adrenal suppression) are well known [6567].
Treatment with anti-leukotrienes may also have a role to play in the modulation of
remodelling in asthma. In vitro work demonstrates that leukotriene D4 can enhance
collagen production by activated myofibroblasts, in the presence of TGF-b [68].
Leukotriene D4, in the presence of IL-5, can also increase the production of eosinophilderived TGF-b [69]. There is evidence from murine models of asthma that the cysteinyl
leukotriene-1 receptor (Cys-LT1) antagonist montelukast can significantly reduce airway
eosinophil infiltration, mucus plugging, smooth muscle hyperplasia and subepithelial
fibrosis in ovalbumin-sensitised/challenged mice [70]. In an open study of seven children
with asthma, treatment with montelukast was associated with a reduction in gas trapping
on computed tomography (CT) scan and an improvement in lung function (reduction in
residual volume) in six children [71]. The authors of this study [71] suggested that the
51
D.N. PAYNE ET AL.
beneficial effects of montelukast may have been due to a reduction in the degree of
airway fibrosis, rather than an effect on airway inflammation. However, in the absence of
inflammatory or histological data, this remains speculative.
One aspect of airway remodelling described in adult asthma, which has not been
studied in children, is the increase in blood vessel number and area. Macrolides, another
class of drug that has been used to treat asthma, may have a potential role to play in the
modulation of this particular structural change. Fourteen-membered macrolides appear
to reduce tumour angiogenesis by an unknown mechanism and therefore it is possible
that bronchial neovascularisation could be reduced [72]. Roxithromycin inhibits tumour
necrosis factor-a-induced vascular endothelial growth factor production [73]. Angiogenesis may also be inhibited indirectly via effects on IL-8, which seems to be angiogenic as
well as pro-inflammatory. A rapamycin analogue inhibits epidermal growth factorinduced proliferation in a murine model of lung inflammation and remodelling [74]. If
this effect were the same in the human lower airway, this could have profound
implications for prevention of airway remodelling associated with angiogenesis.
The development of novel treatments targeting remodelling changes will gain greater
impetus if it can be demonstrated that resolution of remodelling has an impact on symptoms
and lung function. In this context, animal models will have an important role to play. In
particular, monkey work demonstrating that inhalation of ISS can lead to resolution of
remodelling changes provides an exciting stimulus for further therapeutic research [61].
What are the important future questions?

The present knowledge of remodelling in paediatric respiratory disease is limited and
there are many more questions than answers. A priority must be to improve current
understanding of the mechanisms involved in normal airway development and the
response to injury. Only if the normal can be understood can we really make sense of the
changes seen in disease. Questions regarding normal development include the following.
1) What is the sequence of changes within the normal developing airway? 2) What controls
this process and at what age is development (as compared with growth in size) complete?
3) Are the responses to epithelial injury the same as those that regulate early development?
(An answer to this question is fundamental to the credibility of the concept of the EMTU).
A better understanding of normal development will then allow specific questions to be
asked regarding the remodelling process, as follows. 1) When do remodelling changes begin
and what drives them? 2) Is the repair process in asthma intrinsically abnormal or just an
exaggeration of the normal response? 3) How can the "acute" RBM thickening seen in
segmental airway challenge in adults be reconciled with the evidence that RBM thickening
does not seem to be progressive, i.e. what modulates the remodelling process? 4) What (if
anything) leads to resolution of acute remodelling? And is there any relationship at all
between acute remodelling changes and chronic structural changes in the airway? 5) What
is the significance of chronic structural changes in the airway wall, i.e. their effect on
physiology, inflammation? 6) Can these changes be reversed? And if so, how?
What is needed to find answers on these questions?

Noninvasive ways of serially measuring remodelling
It is unlikely that serial bronchial biopsies will be performed in large cohorts of
children, so there is an urgent need to validate noninvasive tests which could be used
52
prospectively and longitudinally. Potential candidates include imaging techniques, lung

function, and measurement of mediators in urine, blood, induced sputum or exhaled
breath condensate (EBC).
There are important general principles in the understanding of noninvasive markers;
the first is to determine the purpose of measuring them. The requirements are different
for mechanistic studies, as opposed to monitoring or guiding treatment. Group
differences in mediators, with overlap between groups, are very useful for pointing
towards potentially interesting mechanisms of disease, but may not be useful in an
individual child. The ideal marker must be stable, reproducible, able to discriminate
between normal and disease, and sensitive to changes in clinical state over time, whether
due to disease or treatment. No such marker exists at this time, but the questions are
important and the long-term aims must be remembered, particularly when considering
cross-sectional studies, which comprise the bulk of what is available.
In adults, quantitative measurement of airway wall thickness with high-resolution CT
has been used to study airway wall changes, as discussed earlier [17]. Airway wall area
and thickness were measured using quantitative CT, after pre-treatment with oral
steroids and bronchodilators, to eliminate as far as possible any reversible changes. These
indices were higher in asthmatics than normal subjects. There were strong correlations
between both wall area and wall thickness and RBM thickness in endobronchial biopsy,
with strong, inverse correlations between FEV1 and all three airway wall parameters. In
another study, wall thickness ratio and area was compared in four groups of adults, as
follows: 1) near fatal, 2) moderate, 3) mild asthma, and 4) normal controls [75]. All
asthmatics had greater airway wall thickness than controls, with thicker walls present in
the more severe asthmatics. Another study attempted to correlate biomarkers of
remodelling with radiological measurements. In stable adult asthmatics, sputum matrix
metalloproteinase (MMP)-9 was inversely correlated with wall area [76]. Tissue inhibitor
of metalloproteinase (TIMP)-1 was positively correlated with wall area and thickness and
the molar ratio of MMP-9 to TIMP-1 was positively correlated with post-bronchodilator
spirometry. Taken together, in adults, quantitative CT shows promise as a noninvasive
marker of airway remodelling.
By contrast, the current authors were unable to correlate RBM thickness with CT
measurements of bronchial wall thickness in children with severe asthma [77]. There are
several possible reasons for this. The children may have had more movement artefact due
to difficulty in breath-holding, or more prominent cardiac pulsation, making the
measurement less precise. The children may have had nonspecific reasons for airway wall
thickening, such as gastro-oesophageal reflux or recurrent viral infections. RBM
thickening may correlate poorly with other more prominent features of airway wall
thickening due to remodelling in children. Even if the measurements were to be refined, it
is difficult to see how radiological imaging could do more than measure the sum of all the
changes (reversible and irreversible), rather than the individual components (see
introduction). It is possible that, in the future, magnetic resonance imaging (MRI)
techniques could allow spectroscopic analysis of the changes in airway wall components.
It might be possible to measure bronchial blood flow with MRI angiography, but it
might be difficult to distinguish dilatation of normal bronchial arteries secondary to
inflammation from airway neovascularisation as part of the remodelling process.
An alternative to imaging would be to assess the activity of the remodelling process by
direct measurement of modulators of the process. Superficially attractive options are
EBC and induced sputum. However, these techniques may not be sensitive to processes
deep in the airway wall and therefore blood or urine may be more useful. Urine is an
attractive source of biomarkers. Desmosine (DES) and isodesmosine (IDES) are amino
acids derived exclusively from cross-linked elastin. Hydroxylysylpyridinoline (HP) and
lysylpyridinoline (LP) are amino acids derived exclusively from cross-linked collagen. All
53
D.N. PAYNE ET AL.
have been measured in urine, but there are no data correlating them with endobronchial
biopsy in children. CF adults had raised urinary levels of all these markers [78, 79];
elevations have been also been described in emphysema [80], exacerbations of COPD and
other inflammatory lung diseases [81, 82]. There were higher levels of DES, but not
hydroxyproline, in the urine of patients with a more rapid decline in lung function with age
[83]. A study published in abstract form in CF children reported a correlation between
urinary desmosines and BAL neutrophil elastase [84]. Taken together, these data are very
suggestive that DES, IDES, HP and LP in the urine are potentially useful markers of tissue
destruction. They are not specific to the lung, but in the context of isolated respiratory
disease, an elevation in levels is most likely attributable to events within the lungs.
However, it is important to emphasise that their relationship to remodelling, as opposed to
tissue destruction, is unclear. Longitudinal studies, comparing urinary markers with
findings on endobronchial biopsy, are required to address this.
With regard to the use of EBC, preliminary data are available showing significant
correlations between RBM thickness on endobronchial biopsy and cys-LT levels in EBC
in a group of children with asthma [85]. Further work is needed to evaluate whether cysLTs will turn out to be useful clinically in the assessment of remodelling.
Good animal models

The advantages and disadvantages of the available animal models have been discussed
earlier. It is likely that mouse models will continue to be used to explore potential
mechanisms and generate hypotheses, with primate models providing the bridge between
murine and human studies. The ability to study inflammation and remodelling in the
developing airway of infant monkeys constitutes a major attraction of the primate model.
Better use of the opportunities of anaesthesia or bronchoscopy to obtain

bronchial tissue
Paediatric studies are less far advanced than adults and, in particular, the opportunities to
examine tissue are far more limited. The problem has been made worse by recent scandals at
Alder Hey and Bristol Childrens Hospitals in Liverpool and Bristol, respectively, in the
UK. An urgent priority is to maximise the opportunities currently available. Perhaps a point
is being reached when the concepts of nonbronchoscopic BAL and brush biopsy, as well as
endobronchial biopsy at the time of a clinically-indicated bronchoscopy, can be extended to
performing bronchoscopy, with airway and brush biopsy, at the time of general anaesthesia
for another procedure. Another possible source might be surgical lobectomy specimens of,
for example, a small distal congenital thoracic malformation, or at the time of organ
harvesting for lung transplant, using for scientific studies a cuff from the proximal bronchus
which would otherwise have been discarded at the time of anastomosis to the recipient.
Obviously, all such proposals should be scrutinised by an ethics committee, be open and
transparent, and be subject to fully informed consent by all relevant parties. These proposals
might be thought of as rather extreme but unless ways of ethically harvesting more tissue are
found, it is unlikely that progress will be made. It is particularly urgent to obtain tissue both
from children who do not have any airway disease and from children with mild asthma.
Application of techniques to assess structures other than RBM

There are a large number of established techniques to study remodelling, including
immunohistochemistry, in situ hybridisation, genomics, proteomics and metabolomics,
54
which have been used in adult studies. The aim should be to apply these techniques in a
focussed, hypothesis-driven manner, rather than in a "lets measure everything we can
think of on every bit of tissue we can find" way. This is particularly important when
considering the tiny fragments of tissue from infant biopsies. These are so precious that it
is likely that the best approach will be to perform hypothesis-generating studies in older
children, in whom bigger biopsies can be obtained, and test the hypotheses in infants.
Conclusions
The following conclusions can be drawn.
1) There are limited data regarding remodelling in children.
2) Prioritising this area of research will be beneficial as the available data suggest that
remodelling occurs early, with significant long-term consequences.
3) This is not an easy area, in view of ethical and practical constraints.
4) Focus needs to be on maximising opportunities for obtaining airway tissue from
controls and subjects with disease.
5) Understanding normal airway development is essential to interpreting the changes
in disease accurately.
6) Animals are not humans but models such as transgenic mice offer a potentially
powerful, hypothesis-generating tool to stimulate human studies.
7) Animal models also suggest that resolution of remodelling is possible; this raises the
potential for disease-modifying treatments in humans if remodelling is really harmful
rather than protective.
Summary
Remodelling is the collective term used to describe the structural changes seen in the
lungs of patients with respiratory disease. Structural changes have been reported in a
number of conditions, although they are most commonly described in the airways of
patients with asthma, with changes recently described in children similar to those seen
in adults. As a result, these findings in children have led investigators to challenge the
previously held assumptions that remodelling develops as a result of persistent airway
inflammation and that structural changes are associated with progressive impairment
of lung function. Prioritising this area of research will be beneficial as the limited data
available suggest that remodelling occurs early, with significant long-term consequences. However, this is not an easy area to research, in view of ethical and
practical constraints. Efforts therefore need to be made to maximise the opportunities
for obtaining airway tissue from controls and subjects with disease. In addition, a
better understanding of normal airway development is essential in order to interpret
the changes in disease accurately.
Keywords: Airway inflammation, airway remodelling, asthma, endobronchial biopsy.
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59
CHAPTER 5
Immunology and defence mechanism of the

developing lung
B. Schaub*, R. Lauener #, S.L. Prescott},z
*Pediatric Pulmonary Division, University Childrens Hospital Munich, LMU Munich, Munich, Germany.
#
Zurich University, Childrens Hospital, Center for Allergy Research, Zurich, Switzerland. }School of
Paediatrics and Child Health, University of Western Australia, and zPrincess Margaret Hospital, Perth,
Western Australia, Australia.
Correspondence: B. Schaub, Pediatric Pulmonary Division, University Childrens Hospital Munich, LMU
Munich, Lindwurmstr. 4, 80337 Munich, Germany. Fax: 49 8951604764; E-mail: Bianca.Schaub@med.
uni-muenchen.de
Complex systems have evolved to protect the host from potentially noxious
environmental agents. This is most critical in mucosal and epithelial surfaces that are
in direct environmental contact. Local events in these tissues are critical for
programming all systemic and local defence systems, culminating in a highly adaptive
surveillance network, which is environmentally relevant. Environmental exposures at
mucosal and epithelial surfaces have a number of critical effects. First, in the post-natal
period, environmental exposure plays a key role in driving global immune maturation,
which appears to be dependent on exogenous factors (namely microbial exposure) to
develop normally. Secondly, the pattern of environmental antigen exposure determines
the specificity of responses required for host defence. Finally, environmental and
endogenous conditions during antigen processing in local tissues appear to influence the
patterns of immune maturation and resulting immune responses. These concepts are
discussed more fully below with respect to immune development in the respiratory tract.
Although mucosal events arguably play the most pivotal role in immune development,
these events are still poorly understood in humans because of logistical and ethical
limitations of studies in this area, particularly in young children. Much current
understanding is extrapolated from studies of systemic immune function, animal models
or indirect measures of mucosal immune function. This chapter discusses how
environmental influences and local endogenous factors (such as collectins, neuropeptides, and oxidative stress) contribute to the developing immunity in the lung.
Immune development of the healthy lung

Antenatal events and influences on immune maturation
As with most other systems, immune programming in the antenatal period is highly
developmentally regulated; however, there is evidence that programmed development
can be influenced by environmental exposures in this period, including infection [1],
maternal diet [2, 3] and smoking [3, 4], which can modify immune responses detected in
the neonatal period (fig. 1). Foetal responses are clearly sensitive to the ambient cytokine
environment of pregnancy, and the first cellular responses (in foetal life) universally
reflect the "normal T-helper (Th)2-skew" of pregnancy [5]. This, together with mounting
ISSN 1025-448x.
60
IMMUNOLOGY AND DEFENCE IN LUNG DEVELOPMENT
Environmental factors,
allergens, vaccines,
antibiotics, infections,
pathogens
Defence mechanism
Mannosebinding
lectin
Oxidative stress
Antimicrobial substances Surfactant

Microbial stimulation
proteins
Neuropeptides
Nasal mucosa
Intestinal
mucosa
Bone marrow
Lymph node
Thymus
Th1
Peripheral blood
Naive
HSC
T-cell
TLR T-reg
TLR
Mucosal inflammation
Lung disease
Th2
DC
Genetic factors
Fig. 1. Schematic overview of immunological and defence mechanisms of the developing lung. HSC:
haematopoietic stem cell; TLR: toll-like receptor; DC: dendritic cell; Th: T-helper cell; T-reg: regulatory T-cells.
evidence of pre-symptomatic differences in the immune function of newborns who later

develop allergic disease [6], has generated more intense interest in the role of antenatal
events in immune programming and disease pathogenesis. In addition to environmental
effects in pregnancy, one of the authors has recently explored the role of direct materno
foetal interactions on foetal immune programming [7]. The present authors have noted
that maternal reactivity to foetal allo-antigens is related to the pattern of developing
foetal immune responses, as well as the subsequent development of allergic disease [7].
Together with previous observations that maternal atopy may have a stronger influence
on neonatal immune responses than paternal atopy [8], these observations suggest that
maternofoetal interactions could be an important determinant of immune reactivity in
the early post-natal period, and this needs to be investigated further.
As the immunologically active interface between the foetus and the mother, and being
the major source of cytokines and other immune mediators detected in the foetus [9, 10],
the placenta has enormous potential to influence foetal immune development. Low-grade
inflammation is characteristic of all pregnancies and complex pathways have evolved to
minimise this. The present authors speculate that variation in the propensity for
inflammatory responses and/or the capacity to regulate these in the placenta is of key
importance in establishing early patterns of foetal immune responsiveness. Placental cells are
also likely to be sensitive to adverse environmental exposures that may affect pathways
which underpin the dramatic increase in immune disease in very early life. As yet, this has not
been investigated in this context, but is an important area for future research.
Thus, while genetics provide the blueprint for immune development, environmental
factors (including both maternal exposures and direct maternal influences in utero) play a
critical role in determining how genes are expressed [11]. These effects appear to be
mediated by direct chemical effects on the DNA or associated proteins (with resulting
DNA methylation or histone modification) [11]. This "epigenetic model" supports the
observations that while patterns of neonatal cord blood immune responses are associated
61
B. SCHAUB ET AL.
with atopic heredity ([12] and others), these can also be altered by environmental
modification [14]. Thus, as a result of the uterine environment, genes may be
differentially expressed or silenced during critical stages of development and dictate
future patterns of disease susceptibility.
Less is known about local mucosal immune development in the human foetus. In the
mature airway, dendritic cell (DC) networks play a central role in processing antigens
and programming T-cell responses [13]. These are poorly developed in neonatal animals
[14, 15], and there is some evidence that DCs are rarely seen in humans airways, even in
the first year of life in the absence of respiratory infection [16], as discussed further below.
The development of these networks and associated lymphoid tissues appears to occur
largely in the post-natal period and is driven by environmental exposures. The largest
source of antigenic load in the post-natal period occurs through the gut, and cells critical
to the mucosal associated lymphoid system begin to appear in the foetal gut early in the
second trimester [17], including macrophages (14 weeks), T- and B-cells (14 weeks) and
DCs (16 weeks) [17]. The role of these cells in the antenatal period is not clear, but there
has been interest in the potential effects of cytokines, environmental proteins (including
allergens [18]) and other factors (such as sCD14, the soluble form of CD14 [19]) that have
been detected in amniotic fluid [17]. Potentially, variations in the content of amniotic
fluid, which bathes the respiratory and gastrointestinal mucosa, could modify the local
milieu and patterns of maturation. To date, only one study has shown a relationship
between amniotic fluid content (sCD14) and the risk of subsequent allergic disease [19],
and this needs to be explored further.
Central role of mucosal events in post-natal immune maturation

In the post-natal period, maturation of both the innate and adaptive immune systems
is driven by environmental exposures, which largely occur at mucosal surfaces,
particularly through the gut and the upper respiratory tract. Logically, there must be
functional pathways that "translate" mucosal events into appropriate peripheral immune
responses in tissues such as the lung, although these are not well defined. These pathways
must fundamentally underpin all aspects of environmentally driven post-natal immune
maturation.
The effects of environmental factors directly encountered in the respiratory tract.

Local encounter with noxious environmental factors, including irritants (such as cigarette
smoke), and respiratory pathogens is likely to influence the development of immune
networks in the airways. Airways antigen-presenting cell (APC; namely DC) populations
appear to have a major role in the late-phase inflammatory response [13, 20] and are
therefore likely to contribute to the development of airway damage in inflammatory
airway disease. These cells play a critical role in programming T-cell responses following
their migration-induced maturation in regional nodes [13].
Age-related immaturity in DC function [21] is associated with reduced capacity for
these cells to respond to inflammatory stimuli. Local airway DC networks are less
developed in infant animals, and additionally these DC populations display markedly
attenuated responses to inflammatory triggers [14, 15]. Similarly, during the first year of
life, human infants do not typically show DCs in the airways in the absence of
inflammation [16]. However, despite this immaturity, mature DCs do appear in
association with severe respiratory infection [16]. This suggests that local tissue events,
such as infection, in infancy can influence the maturation of DCs and modify subsequent
downstream T-cell programming in early life.
62
In animals, resting DCs stimulate Th2 development unless they receive obligatory
Th1-trophic signals during antigen processing [22]. These signals may typically occur
under conditions of infection or other local stress [23, 24]. Thus, variations in DC
maturation (as a result of both environmental and endogenous factors) could have a key
role in determining the subsequent pattern of local T-cell responses.
Despite this, the relationship between early respiratory tract infections and chronic
airway inflammation (and allergic airway disease) has been confusing. These infectious
agents have been clearly identified as asthma triggers in children with established disease,
and, in addition, early respiratory syncytial virus (RSV) infection in infancy has also been
long regarded as a risk factor for subsequent asthma, at least in the first 6 yrs of life [25].
This may be partly because of the Th2-trophic properties of this and other respiratory
viruses [26], but it may also be an indirect consequence of the delayed capacity to mount
Th1interferon (IFN)-c responses in the early post-natal period [27]. Predisposition to
wheezing lower respiratory infection in the first year of life is a strong risk factor for
asthma at 6 yrs of age in both nonatopic and atopic children [28]. This strongly suggests
that significant infection-induced airway inflammation during the early period of postnatal lung growth and development can have profound long-term effects that appear to
be more marked than inflammation occurring at later ages [29]. However, the notion that
infection can serve only as a priming factor for subsequent allergic inflammation is at
odds with other observations that under some circumstances infections appear to protect
from allergic disease [3034].
Together, these observations suggest that early encounter with infectious agents has
the potential to accelerate the maturation of local immune networks (including DC
networks), producing Th1 defence responses, which may override the Th2 default response
in immunologically immature infants. The complexity of these relationships needs to be
further dissected. In particular, variations in the consequences of infections on allergic
propensity may involve differences in the timing of exposure, the nature of the infectious
agent and the location of the infection (upper or lower airway), in addition to genetic factors.
Links between the enteric flora and the lung. Allergic predisposition is associated with
immaturity of a number of aspects of early immune function, in particular Th1 function
[3537], but potentially also underlying APC signalling [38, 39] and immature precursor
populations [40], as previously shown. Given that microbial exposure through the
gastrointestinal tract (GIT) [41] is arguably the strongest driving influence for immune
maturation [42], the mechanisms by which events in the gut influence the development of
these key effector cell populations will provide the key as to how the immune system
"translates" environmental exposures (predominantly through the gut) into adaptive
peripheral immune responses in other tissues. This discussion will explore two pathways
by which enteric microflora are likely to influence the respiratory tract and other aspects
of the peripheral immune system: 1) direct influences on lymphocyte populations (B-, Tand regulatory cells), which recirculate through the gut mucosa during their normal
maturation; and 2) indirect effects on precursor populations within the bone marrow
(including immature APC), which are affected by microflora without direct passage
through gut tissues.
Direct effects on lymphocytes that transit the gut during their development
The present authors hypothesise that intestinal flora influence the maturation of a
large pool of immature precursor cells that circulate through the gut and subsequently
home to tissues throughout the body, particularly to other mucosal surfaces (namely the
respiratory tract), where they develop their mature functional attributes. These
63
B. SCHAUB ET AL.
precursors can develop into a diverse range of lymphocytes (including regulatory cells),
depending on ambient maturational signals, and this could logically explain the
apparently diverse effects of intestinal macrobiotics. It could also explain how events in
the gut mucosa can influence local immune development in remote tissues. Alteration in
microflora or events that lead to inflammation in the gut could logically modify the local
milieu, and the rate and pattern of precursor maturation. This is supported by
observations that infants who develop allergic disease (manifest in other tissues) have
differences in very early colonisation patterns [4346].
For many years the "common mucosal immune system" has been recognised as a
functional entity [47]. Although separate, the mucosal immune system is functionally
integrated with the peripheral ("systemic") immune system [48, 49]. The gut appears to be
an early extra-thymic reservoir for T- and B-cell precursors [50, 51] that mature and
eventually migrate to the periphery according to the local immunological needs of the
host [49]. It is highly likely that early mucosal events influence the rate and pattern of
maturation of precursor cells in the mesenteric lymphoid tissues. Although maturation
into tissue-homing immunoglobulin (Ig)A-bearing B- and T-cells is well described [47,
49, 52], it is likely that these include subpopulations of maturing regulatory cells
(including CD4zCD25zT-cells), which play a key role in controlling peripheral immune
responses. An effect on functional maturation of thymic-derived precursors is supported
by other recent observations that probiotics induce functional CD4z regulatory cells
(bearing transforming growth factor-b), which are associated with clinical benefits (an
amelioration of colitis) in an animal model [53]. Thus, it is highly plausible that intestinal
flora may influence the maturation of a large pool of immature precursor cells that
circulate through the gut and subsequently home to tissues throughout the body,
particularly to other mucosal surfaces where they have diverse effects. These cells
ultimately seed to other mucosal sites (namely the respiratory tract), where they play a
major role in local defence through the production of secretory IgA (B-cells). This also
provides an explanation for previous observations that probiotic species in the gut
influence (salivary) IgA production in distal sites [54].
Finally, there is also a very strong case for investigating the effects of intestinal flora
and other environmental exposures on CD4zCD25z T-regulatory cells, which are
emerging as important candidates in the pathogenesis of allergic disease and logical
targets for therapy [55]. Already there is evidence that the therapeutic effects of
immunotherapy are at least in part mediated through these cells [56]. Although activated
by antigen, CD4zCD25z regulatory cells have antigen-nonspecific suppressive effects.
These cells are also activated by microbial signals, mainly via toll-like receptor (TLR)4
and TLR9 [57], which provide a logical pathway by which enteric flora (probiotics) can
nonspecifically modulate bystander cell function. However, in adaptive responses, the
expression of regulatory activity is dependent on the level of danger to the host. In the
presence of pathogen-associated inflammation, microbial encounter (and a high level of
interleukin (IL)-6 production) can also block the suppressive effect of CD4zCD25z
regulatory cells, allowing activation of pathogen-specific adaptive immune responses
[58]. Thus, the activation and expression of T-regulatory cell function depends on the
context of bacterial encounter, and it is speculated that intestinal flora (which do not
typically induce strong inflammatory signals) are more likely to promote regulatory
function than to suppress it. This is supported by animal models in which probiotic
intestinal flora appear to induce regulatory T-cell populations [53]. While environmental
microbes are proposed to exert their effect through modulation of DC-function-guiding
regulatory T-cells (T-regs), microbes can affect the innate immune system itself in the
sense of activating mechanisms such as lipopolysaccharide (LPS) tolerance.
This mechanism presumably works by upregulating negative inhibitory feedback
mechanisms.
64
Indirect effects on bone marrow precursors that develop into APC and other
tissue-homing effector cells which do not directly passage the gut
It has been hypothesised that the maturation of bone marrow-derived APC
populations is dependent on microbial signals from the environment [5962]. Variations
in level and function of APC populations and less mature bone marrow precursor cells
are evident in peripheral blood before they reach the tissues and undergo final
maturation events. These variations are associated with allergic disease susceptibility [38,
6367]. This strongly implies that there are indirect signals from the gut to developing
precursors in the bone marrow, just as there is evidence of signalling from other tissues to
the bone marrow during inflammatory events [68]. This is supported by recent studies
showing that changes in gut flora are associated with direct effects on bone marrow
precursor populations entering the circulation [61]. Together, these observations suggest
that intestinal microflora could also inhibit allergic inflammation by influencing
developing DC and precursor cells in the bone marrow before they home to local tissues.
The maturation and function of APC are strongly dictated by environmental microbial
exposure, which occurs predominantly through the gut in early life. These cells
(particularly tissue DCs) provide critical regulatory signals during T-cell activation in
regional nodes and play a fundamental role in programming subsequent effector
responses. Unless they receive obligatory Th1-trophic signals during maturation and
antigen processing, DC preferentially stimulate Th2 development [22]. Logically, these
cells are of fundamental interest in mediating the apparent "Th2 inhibitory" effects of
microbiotics.
There has been much work demonstrating how probiotic intestinal microflora directly
enhance the activity of DC populations that reside within the human gut [69, 70].
However, these studies do not address the more fundamental question of how intestinal
microflora affect DC populations in other tissues where allergic inflammation is manifest
(such as the skin and the respiratory tract). These DC and other APC (monocytes)
develop in the bone marrow and are measurable in peripheral blood before they seed to
distal tissue sites. Although they do not transit the gut, these APC appear equally
dependent on environmental microbial exposure as the major stimulus for normal
maturation [60]. Delays in systemic maturation of the APC compartment have been
implicated as one of the most likely mechanisms for the increasing propensity for allergic
disease [71], as a presumed result of reduced microbial burden in infancy [71]. In support
of this, circulating monocytes in infant animals mature at significantly different rates
depending on enteric microflora exposure [62]; this occurs with two-fold lower function
in germ-free animals [60]. Although DCs derived from murine bone marrow cultures are
activated directly in vitro by probiotics to produce strong IL-12 and tumour necrosis
factor (TNF)-a responses [72], this is unlikely to be relevant in vivo, except for DCs that
ultimately home to the gut. This suggests other indirect influences between events in the
gut and developing bone marrow populations. Preliminary studies in humans suggest
that this effect could be directly on the bone marrow [61]. The study in question found
that oral probiotic supplementation was associated with significant changes in the
numbers of circulating (CD34z) bone marrow precursor cells in peripheral blood [61].
Although this needs to be examined further, it is highly relevant in the context of allergic
disease, as it has previously been shown that variation in the function of these marrow
precursor populations is associated with both established allergic disease [6567] and
high risk for allergic disease in early infancy [40]. More pronounced immaturity of APC
(major histocompatibility complex class II expression) [38] and precursor populations
[40] have been associated with a higher allergic risk, suggesting that factors that can
enhance maturation of bone marrow-derived cells may have a role in modifying disease
65
B. SCHAUB ET AL.
risk. It has already been noted that other dietary interventions (using n-3
polyunsaturated fatty acids) can also modify maturation of circulating bone marrow
precursors with associated clinical effects [73]. Although this is likely to be mediated
through different pathways, it illustrates that the bone marrow is readily influenced by
environmental changes.
Together, these findings support observations that precursor populations in the bone
marrow can be influenced by mucosal events in a bi-directional manner [68], although the
mechanisms are not clear. Thus, despite the lack of direct passage through the gut, less
mature forms of these cells, which undergo initial maturation in the bone marrow,
appear to be influenced by remote events at mucosal surfaces (namely the gut).
In summary, collectively this literature provides a strong theoretical basis for future
studies to investigate the effects of intestinal microflora and other mucosal exposures on
the key cells involved in the development and regulation of allergen-specific response,
including direct effects on populations that traverse the gut before homing to effector
sites (T- and B-cells), as well as indirect (but independent) effects on bone marrowderived population (DCs and myeloid precursors) before they reach their effector sites.
Defence mechanism in respiratory disease

While exogenous/environmental factors may influence the susceptibility to respiratory
disease, endogenous factors play a key role in the modulation and interaction of innate
and adaptive immune responses in the respiratory tract (fig. 1). These involve a range of
local mechanisms, including ancient host defence mechanisms of cell-mediated
immunity, such as microbial stimulation and the induction of antimicrobial substances
expressed in the respiratory tract in response to pathogens. The collectin family, namely
surfactant protein (SP)-D and SP-A, as well as mannose-binding proteins, exert
important protective functions in pulmonary host defence, but may also be important in
pulmonary disease states, such as allergic inflammation [7479]. There is also growing
recognition that locally produced neuropeptides are involved in immune development.
Finally, the generation and control of the oxidative species may be important not only
for local defence but also in shaping subsequent patterns of response and disease
susceptibility.
Significance of microbial stimulation and antimicrobial substances

Epidemiological and murine studies have suggested a role for microbial stimulation in
the development or, potentially, prevention of allergic pulmonary disease [8085].
Endotoxin levels (endotoxin being representative for TLR4 ligands) in child mattresses in
rural areas of Austria, Switzerland and Germany were inversely correlated with the
occurrence of atopic diseases [81, 86]. In murine studies, administration of TLR4 and
TLR2 ligands before allergic sensitisation could reduce allergic parameters in a model of
allergic asthma [87]. As allergic immune responses develop early in childhood, exposure
to endotoxin early in life may shift the childs immune response more towards a Th1
phenotype, as found following local LPS administration on the nasal mucosa of nonatopic children [80]. The interaction of T regs with DCs may explain the protective role of
endotoxin. An increased gene expression of CD14 and TLR2 on leukocytes in the
previously mentioned rural population may be a potential biological marker for
microbial exposure earlier in life [88].
Antimicrobial peptides are expressed in the respiratory tract acting as effectors of the
innate immune system. Antimicrobial components of the airway secretion include
66
lysozymes, lactoferrin, secretory leukoprotease inhibitor, cathelicidins and defensins,

which are the most widely studied family of peptides present in airway fluid. Betadefensins originate from epithelial cells, macrophages and lymphocytes, while alphadefensins are found in neutrophils. As an example, human beta-defensin (HBD)-2 is
upregulated either in response to bacterial infection or by endogenous inflammatory
cytokines. HBD-2 has in vitro antimicrobial activity against yeast and gram-positive as
well as gram-negative bacteria. Its chemotactic properties towards immature T-cells are
an additional feature contributing to airway and gut mucosal defence. One of the present
authors has shown that human TLR2 mediates cellular activation in response to
bacterial lipoproteins, resulting in an adapted innate immune response [89]. In addition,
in human tracheobronchial epithelium, LPS has been shown to induce HBD-2 expression
[90]. In summary, TLR-mediated recognition of microbial structures and the ensuing
cellular activation reflect the afferent arm of the innate immune system, whereas
induction of antimicrobial peptides and other mechanisms may be regarded as the
efferent, effector arm of the pulmonary innate immune system conferring protection
against foreign invasions.
Role of surfactant proteins in respiratory diseases

Host defence. Proteins produced locally in the respiratory tract, such as the surfactant
proteins, are crucial for host defence. While pulmonary surfactant comprises the two
hydrophobic proteins, SP-B and SP-C, relevant for adsorption and distribution of
surfactant at the airliquid interface, the collectins SP-D and SP-A have important
functions during the immune development in the interaction with adaptive immune
responses. One member of the collagenous C-type lectin family, SP-D, is primarily
produced in the lung by alveolar type II cells and by nonciliated Clara cells, a subset of
bronchiolar epithelial cells [91]. Pulmonary collectins may contribute to protection
against local pulmonary inflammation, including cytokine production [74, 92, 93] elicited
by gram-positive and gram-negative bacteria [75, 9499].
Allergic pulmonary inflammation. More recently, it has been proposed that SP-D can
diminish allergic inflammation potentially by immune modulation via communication
between B- and T-lymphocytes [100102]. One of the present authors has shown in a
murine model of asthma that pulmonary allergic inflammation is increased in SP-D
deficiency [94], and that SP-D deficiency resulted in persistent T-cell activation in another
murine model [102]. In vitro human studies reveal that SP-D suppresses allergen-induced
lymphocyte proliferation and IL-2-dependent T-cell proliferation [100]. Furthermore, SPD inhibits allergen-induced histamine release and proliferation of peripheral blood
mononuclear cells from asthmatic and nonasthmatic children [103]. Higher levels of SP-D
are present in the bronchoalveolar lavage (BAL) fluid of asthmatic adults [104] and this is
also the case in murine models of allergic airways inflammation [105, 106]. Application of
endogenous SP-D can suppress allergic airway inflammation [107]; however, at maximal
allergen exposure, SP-D may not be sufficient to reduce allergic inflammation [94]. These
data add to the emerging role of SP-D in modulating cellular immune responses after
allergen challenge [108].
Potential interactions of SP-D with another innate receptor, TLR4, may offer new
possibilities of innateinnate interactions in host defence mechanisms. Previous reports
indicate that an intact TLR4 complex is necessary for SP-A-induced activation of the
transcription factor nuclear factor (NF)-kB and of several cytokines, such as TNF-a and
IL-10 [101]. The present authors have shown that TLR4 expression is increased in
wildtype mice in vivo after allergen challenge, while TLR4 expression was diminished at
67
B. SCHAUB ET AL.
early stages of allergen challenge in the absence of SP-D [94]. Whether TLR4 expression
is SP-D-dependent or co-acting in allergen-induced immune responses requires further
investigation.
Several studies have highlighted the potential of collectins (including recombinant
fragments and protein-free synthetic phospholipid-based surfactant) as therapeutic
molecules [93, 107, 109, 110]. SP-D, or molecules derived from these collectins, may be
good candidates for prevention or treatment of lung infection, due to their ability to
interact with various microorganisms and to regulate the inflammatory response.
Interestingly, SP-D is also expressed in extrapulmonary sites such as the GIT, which is,
as discussed earlier, crucial in the development of mucosal immunity. Interaction of SPD with other proteins of the innate mucosal immune system in the GIT could potentially
contribute to immune modulatory mechanisms. This may have implications in, for
example, dietary modulations of the innate intestinal immune system [111], leading to
speculation about a more global role of surfactant proteins in local innate host defence.
Taken together, it is suggested that pulmonary collectins, such as SP-D or SP-A,
participate in the modulation of innate and adaptive immune responses and can influence
lung diseases such as infections or allergic inflammation. Whether they might have a
therapeutic role in humans has to be further elucidated.
Relevance of mannose-binding lectins in respiratory disease

Mannose-binding lectin (MBL) also belongs to the collectin family and represents a
pattern recognition receptor of the innate immune system. MBL interacts with a wide
range of bacteria, viruses, fungi and protozoa by binding to a selection of sugars such as
mannose, N-acetyl-d-glucosamine or mannosamine, fucose and glucose. It promotes
phagocytosis by activation of the complement system, as well as through complementindependent direct cell-surface receptor pathways. As a key factor involved in first-line
defence, MBL is important for protection against respiratory tract infections [112114]
before the onset of antibody production [115]. Accordingly, MBL deficiency has been
associated with increased susceptibility to acute respiratory tract infections, particularly
during early childhood [116]. Incomplete activation of the MBL-MASP (MBLassociated serine protease) pathway may also contribute to an increased risk of infectious
disease [117, 118]. Although originally identified as a functional opsonic defect [119], MBL
may also play an immune-modulatory role potentially through the modulation of cytokine
release [120], as seen in other infectious diseases or disease states complicated by infections
such as cystic fibrosis, HIV, hepatitis C or autoimmune disease.
Although therapeutic applications are still limited, fresh-frozen plasma has previously
shown clinical benefit in children with opsonic defects [121]. More recently, purified
plasma-derived MBL has become available and is now planned for clinical trials [122] to
assess efficacy and to determine appropriate therapeutic indications.
Impact of neuropeptides in respiratory disease

It is increasingly recognised that the nervous system is closely interconnected with the
immune development to optimise defence mechanisms within the respiratory tract [123].
For example, several neuropeptides, such as vasoactive peptide, somatostatin, substance
P and calcitonin gene related-protein, are involved in T-cell activation. While
neuropeptides are known to be released from nerve endings, inflammatory immune
cells, such as monocytes, DCs, eosinophils and mast cells, can also release these
substances. On release, they can exert direct stimulatory and inhibitory effects on T-cell
68
activation and indirect effects through modulation of recruitment and activation of

professional APCs such as DCs.
Somatostatin is a typical example. It inhibits hormone release in the anterior pituitary
gland and the GIT system, and is found in sympathetic and sensory neurons in the
peripheral nervous system. It is found in lymphoid organs, and receptors for
somatostatin are located in lymphoid follicle germinal centres [124], as well as on
lymphocytes and monoctes. Somatostatin generally inhibits T-cell proliferation [125] and
suppresses IFN-c production [126]; however, the distinct mechanism of the immunomodulatory role of somatostatin in DC activation remains to be determined.
While most research on immune function has focused on lymphocytes, neuroendocrine
interactions with macrophages, particularly airway macrophages, may be important for
the maintenance of lung homeostasis in the first line of defence to inhaled particles.
Airway macrophages, most studied in pulmonary macrophage populations, are
originally derived from monocyte precursors in the bone marrow [127]. In inflammatory
diseases such as asthma, they may be derived from precursors in the airway interstitium,
or through proliferation. One postulate is that paracrine and autocrine interactions may
sustain the suppressor effect of airway macrophages within the microenvironment of the
airway. In summary, the immune system is intricately related to the nervous and
endocrine system acting via bidirectional communication and provides homeostasis for
the host during different "stress conditions".
Future in vivo studies could enhance understanding of the role of neuropeptides in
migration of lymphocytes, modulation of Th cell differentiation or induction of tolerance
[128, 129]. Furthermore, use of pharmacological antagonists as well as knockout mouse
models lacking specific neuropeptides or their receptors would facilitate this matter.
Ultimately, clinical studies as interventional trials can prove the contribution of
neuropeptides to human T-cell-mediated diseases of the lung, such as allergic inflammation.
Importance of oxidative stress in lung diseases

While oxidant generation is part of the normal metabolism of most cells, cells involved
in first-line host defence can also produce larger amounts of specialised oxidants with
bactericidal properties. In the airways, oxidants are produced by activated eosinophils,
neutrophils, monocytes and macrophages, as well as resident bronchial epithelial cells.
This includes the production of myeloperoxidase (MPO) by neutrophils, monocytes and
macrophages or eosinophil peroxidase (EPO) from eosinophils. Common inflammatory
conditions, such as, asthma are frequently associated with increased EPO and MPO, as
well as other markers of oxidation, such as hydrogen peroxide and nitric oxide, which
can be measured in exhaled breath condensates. To counter the potentially noxious
properties of oxidants, the lung has a well-adapted antioxidant system. Imbalances
between these pathways can lead to excessive oxidative damage (as seen in inflammatory
disease states) or increased susceptibility to infection (as seen in congenital disorders of
oxidative metabolism).
Endogenous antioxidant systems can be either impaired or activated in association
with chronic inflammation, illustrating the complexity of these still poorly understood
regulatory systems. For example, Cu,Zn-superoxide dismutase (SOD) activity [130] and
peroxynitrite inhibitory activity [131] may be decreased in chronic inflammation, whereas
other antioxidant systems are increased, including cyclin-dependent kinase inhibitor p21
and extracellular glutathione peroxidase [132, 133].
Studies examining the effects of different levels of exogenous (dietary) antioxidants are
also confusing and inconsistent, including the effects of selenium, vitamin C and vitamin
E [27, 134136]. Other promising new antioxidants are planned for clinical trials, such as
nitrones, which are radical-trapping antioxidants that inhibit the formation of
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B. SCHAUB ET AL.
intracellular oxidants by forming stable compounds or non-peptidyl SOD analogues

[137, 138]. Further research is needed to understand the complex interactions that
influence oxidative balance in the local tissues, particularly the therapeutic potential of
different antioxidant products.
New perspectives
For several pulmonary diseases, targeting the innate immune system could provide
therapeutic benefits. For example, for asthma, with a huge prevalence rising over the last
10 years, no new effective regimens have reached the clinics apart from anti-IL-5 and
anti-IgE. While corticosteroids are effective in symptom control and blocking
inflammatory cells, they do not specifically alter the prevailing Th2 cell response. In
this regard, longer-lasting and more effective treatments are needed, and several options
are currently being developed.
First, immunotherapy could provide therapeutic as well as potentially preventive tools.
Recently, efforts are being directed towards using TLR ligation. Central to this approach
would be to change the Th2-dominant inflammation, as seen in allergic inflammation to a
Th1 response. So far, sustained efforts are on the increase for oligonucleotides containing
nonmethylated CpG motifs to shift the balance of Th2-mediated diseases to a Th1-type
response [139, 140]. Other potential therapeutic targets, emphasising the potential value
of the TLR family as a target for a new generation of immunopotentiating compounds,
include other modulators of Th1 responses, such as the TLR7 ligand imiquimod [141],
and the TLR4 ligand l-carrageenan, a polygalactan [142]. Stimulation of the immune
system through parasites also has to be considered [143]. Other potential targets for
therapeutic regimens are DCs and T-regulatory cells, which are pivotal for maintaining
the Th1/Th2 balance and immunomodulation. However, the effective use of
immunotherapy to date is limited because of the complexity of the immune system,
and in particular because of the early development and lack of understanding of the
essential mechanisms of immunotherapy, as well as its potential side-effects.
Secondly, adjuvants to vaccines are potentially beneficial as they target the innate
immune system, and are crucial for prevention and management of infectious diseases.
Again, several types of TLR ligands, such as TLR7 agonists (imiquimod or resiquimod),
TLR9 agonists (CpG ODNs) and, most compelling, the TLR4 agonists (lipid A
analogues) [144], have been shown efficacious as vaccine adjuvants [141].
Thirdly, in acute pulmonary infections, receptor antagonists could be used to induce or
enhance host resistance against viral and bacterial infection by activation through TLRs
or NOD (nucleotide-binding oligomerisation domain) 1/2 [145].
Fourthly, targeting intracellular pathways may be a strategy for prevention or
treatment of several inflammatory diseases, not only regarding the lung. Promising
candidates would be the NF-kB signalling pathway [146], including downstream
elements such as involved kinases, the MyD88 adaptor family in TLR signalling, the
NOD-protein family members and TIR domains [147] and other adaptor proteins.
Conclusion
The interaction between the environment and the host has shaped the immune system
during evolution; similarly, the ontogenetic development of the immune system is the
result of an interaction between the hosts genetic background and its environment. It is
proposed that an individual organisms immune system is shaped by the interaction
between its genetic background and environmental influences. With regard to the
immune system, microbes are the most relevant constituent of the environment. The
70
innate immune response is not only the first response to microbial molecules, but it also
modulates any subsequent antigen-specific adaptive immune response. The effect of such
stimulation of the innate immune system by microbial compounds may depend on the
hosts age, e.g. the same exposure to microbial compounds modulates airway
responsiveness differently at different ages [84]. A hosts immune response is therefore
not only modulated by geneenvironment interactions, but rather by a gene
environmenttime interaction, i.e. an interaction between the hosts genetic background,
environmental factors and the hosts age. Effects early in life have the potential to set the
course for modulation of the immune response with long-lasting effects, such as a
propensity for allergic diseases. Mucosal and epithelial surfaces of the body are the sites
of the first contact between microbes and the host, and the place where initial immune
responses take place. The gut occupies a particularly important role, given the exposure
of the gut-associated immune system to microbes. The immunological effects of exposure
to microbes, however, are not limited to the site of exposure, but rather may manifest at
distant sites, as suggested, for example, by data showing an association between gut
microflora and the development of atopic airway diseases in children. More detailed
insights into the mechanisms governing the modulation of immune responses by
exposure of the immune system to microbes may lead to novel approaches both for
therapy as well as for prevention of immunologically mediated diseases of the lung. The
art of putting into practice such new approaches will be to induce the desired control
mechanisms of the immune system without suppressing antimicrobial or antitumour
defence mechanisms and without inducing inflammatory reactions.
Summary
The ontogenetic development of the immune system is the result of an interaction
between the hosts genetic background and its environment. It is proposed that an
individual organisms immune system is shaped by the interaction between its genetic
background and environmental influences. With regard to the immune system,
microbes are the most relevant constituent of the environment. The innate immune
response is not only the first response to microbial molecules, but it also modulates any
subsequent antigen-specific adaptive immune response. Thus, a hosts immune
response is not only modulated by geneenvironment interactions, but rather by
interaction between the hosts genetic background, environmental factors and the
hosts age. Mucosal and epithelial surfaces of the body are the sites of the first contact
between microbes and the host, and the place where initial immune responses take
place. The gut occupies a particularly important role, given the exposure of the gutassociated immune system to microbes. The immunological effects of exposure to
microbes, however, are not limited to the site of exposure, but rather may manifest at
distant sites, as suggested, for example, by data showing an association between gut
microflora and the development of atopic airway diseases in children. More detailed
insights into the mechanisms governing the modulation of immune responses by
exposure of the immune system to microbes may lead to novel approaches both for
therapy as well as for prevention of immunologically mediated diseases of the lung.
The art of putting into practice such new approaches will be to induce the desired
control mechanisms of the immune system without suppressing antimicrobial or
antitumour defence mechanisms and without inducing inflammatory reactions.
Keywords: Adaptive immune response, defence, gut, innate immune response, lung
development.
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B. SCHAUB ET AL.
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78
CHAPTER 6
Allergy and the paediatric lung during

development
A. Custovic*, K.C. Ldrup Carlsen #, K. Hakon Carlsen}
*North West Lung Centre, Wythenshawe Hospital, University of Manchester, Manchester, UK. #Ulleval
University Hospital, Dept of Paediatrics, and }Voksentoppen BKL, Rikshospitalet (National Hospital),
Oslo, Norway.
Correspondence: K.C. Ldrup Carlsen, Dept of Paediatrics, Ulleval University Hospital, NO-0407 Oslo,
Norway. Fax: 47 22118663; E-mail: k.c.l.carlsen@medisin.uio.no and kclo@uus.no
Development of allergic diseases involves many aspects, often mixed, sometimes

confused and at other times clarified into discussions of clinical disease, lung function
effects or objective findings of allergic sensitisation. The following chapter will focus
upon allergic disease phenotypes (wheezy lower respiratory tract disease, asthma and/or
allergic rhinitis), allergic sensitisation (the presence of specific immunoglobulin (Ig)E
antibodies to allergens and/or positive skin sensitisation) and markers of allergic
inflammation in relation to the developing lung in early life.
However, understanding lung development and the underlying mechanisms of asthma
requires assessment of lung function combined with a thorough knowledge of lung
physiology and its relationship with allergic sensitisation and environmental exposures in
infants and preschool children. Thus, the present chapter may to some extent overlap with
some of the previous and following chapters, which discuss each of these features separately.
Risk factors for allergic disease entities such as asthma may not be identical to those
for allergic sensitisation. This is exemplified by the increased risk of asthma but not
allergic sensitisation, in childhood by exposure to tobacco-smoke products [1]. However,
reduced lung function has been convincingly demonstrated in children born to, or living
with, smoking mothers [27]. Furthermore, the classical triad of atopic eczema, asthma
and allergic sensitisation (with allergic rhinitis as the most common manifestation) most
frequently appear in that order during early childhood, but any one may develop in the
absence of the other manifestations. Thus, deciding whether the clinical entity appearing
first is a risk factor for, or a first clinical manifestation preceding, the other allergic
diseases as part of an "atopic phenotype" is more than a semantic exercise. It will have
implications for understanding of the underlying pathological mechanisms of how and
when "allergy" may affect the developing lung.
Thus, in the present chapter, an attempt will be made to shed some light on the risk
factors for allergic diseases and/or allergic sensitisation versus risk factors for altered lung
function development in infants and preschool children. Furthermore, possible
interactions between allergic disease and lung development and growth, and when
these interactions may occur, will be discussed.
Asthma, wheeze and lung function

Most asthma cases begin in early childhood [8], often in association with reduced lung
function [9, 10] and/or increased airway responsiveness in infancy [911]. Two major
ISSN 1025-448x.
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studies have recently confirmed that subjects with reduced lung function and persistent
asthma in childhood have impairments in lung function that continue into adulthood [12,
13]. In the Melbourne Asthma Study, the magnitude of difference in lung function
between different asthma severity groups observed in childhood (aged 710 yrs) did not
increase over time by 42 yrs of age (i.e. childhood lung function deficits tracked into
adulthood) [12]. A study in New Zealand that followedw1,000 participants from the age
of 926 yrs demonstrated that subjects with a low post-bronchodilator forced expiratory
volume (FEV1)/forced vital capacity ratio at age 26 yrs already had reduced lung
function at the age of 9 yrs [13].
Lung function is routinely measured in adults and older children with respiratory
diseases to aid diagnosis, monitor disease progression and evaluate treatment. However,
between the ages of 25 yrs, children are generally too young to cooperate, and the
majority are not able to perform adequate forced breathing manoeuvres [14].
Consequently, most of the current research on asthma and allergic diseases in early
childhood is based on questionnaires, with no objective measures of lung function. This
is less than ideal, and several studies have reported that parents often confuse wheeze
with other respiratory sounds, which may lead to under- or overestimation of the true
prevalence of wheeze [15, 16]. Furthermore, in a recent large study in which lung function
was measured in preschool children and compared between those with parentally
reported wheeze, which was either confirmed or not by a physician, children with
parentally reported and physician-confirmed wheeze had markedly reduced lung
function compared with those with unconfirmed wheeze [17]. However, there was no
difference in lung function between children with unconfirmed wheeze (y30% of all
parentally reported wheeze) and those who have never wheezed. These findings add
further weight to the argument that many parents have little understanding of what
medical professionals mean by the term "wheeze" and indicate that the epidemiological
studies based only on questionnaires must be interpreted with caution. This emphasises
the importance of using objective measures of lung function wherever possible, both in
research studies and in clinical practice.
Risk factors for allergic diseases and allergic sensitisation in

childhood
In 1873, being "well educated" had already been identified as a risk factor for allergic
rhinitis [18] in adults. "Affluent" or "Western" society has persistently been found to
increase the risk of asthma and allergic sensitisation [19], and this has been well
documented in studies of the former East versus West Germany before and after
unification [20, 21]. However, the increased risk of asthma among children of lower-class,
inner-city families [22] points to the complex pattern of environmental risk for allergic
diseases. No specific lifestyle factors have been universally identified as main risk factors,
although the "hygiene hypothesis" [23] gained support in Europe, where less allergic
sensitisation and asthma was found among children of farmers living in close contact
with livestock [24], as well as in adult farmers [25]. Conversely, other populations have
found increased allergic sensitisation and asthma among farmers children [26, 27], and
genetic susceptibility may contribute to this uncertainty [28].
Well-recognised risk factors for both asthma and allergic sensitisation during
childhood are being male and having a positive family history of allergic disease [29
32]. Both factors have also been implied for reduced lung function in infants [5, 6, 33, 34].
80
ALLERGY AND THE PAEDIATRIC LUNG
Lung function in early life

Development of techniques for use both in naturally sleeping or awake infants [3436],
as well as in sedated infants [3740], has led to successful assessments of lung function in
newborns and infants up to y18 months of age. These techniques have enabled
elucidation of factors that influence lung function very early in life (e.g. maternal
smoking, maternal asthma, sex) [4143]. Several studies have suggested that dynamic
lung volumes can be reliably measured in preschool children using conventional lung
function testing [4447]. However, whilst all of these studies recruited subjects aged 3
6 yrs, very few children were at the younger end of the age range (e.g. in one study of 112
children, only nine were 34 yrs of age) [45]. When refusal rate is taken into account, the
success rate of spirometry among preschool children appears to be only 38.4% [42]. In a
recent study of 355 patients, v10% of the 34-yr-old children were able to perform three
acceptable manoeuvres [46]. Thus, standard spirometry is a difficult, time-consuming and
often an unreliable procedure to perform in preschool children.
There is a growing interest in developing objective measures of lung function which
can be applied in young preschool children to elucidate the end-organ factors involved in
asthma development and to allow more accurate identification of children who are likely
to be at risk of persistent symptoms. Specific airways resistance (sRaw), which is a
measure of airway calibre corrected for lung size, can be measured during normal tidal
breathing using a single-step procedure which obviates the need for panting manoeuvres
against a closed shutter [48]. sRaw can also be measured with the child accompanied by an
adult inside the body plethysmograph [4951], which makes it a potentially useful
respiratory measurement in very young children. Other lung function techniques
potentially suitable for use in pre-school children include forced and impulse oscillation
and the interrupter technique. However, both the oscillation and interrupter technique
have been shown to be less sensitive than sRaw in detecting changes in airway resistance
after bronchodilation or airway challenge [52, 53].
The value of tidal flowvolume loops are still debated, even though repeated studies
have found reduced time to peak flow6total expiratory time-1 (tPTEF/tE) in newborn
babies and infants born to smoking mothers [2, 54, 55], and that reduced tPTEF/tE during
infancy was associated with later wheezing respiratory illness [9, 5659].
Early life lung function and wheeze phenotypes

The present understanding of the nature of childhood wheezing illness has been
augmented to some extent by the characterisation of distinct wheeze phenotypes in
childhood (never-wheezers, transient early wheezers, late-onset wheezers and persistent
wheezers) [9, 60]. In a recent study from France, reduced maximal flow at functional
residual capacity at age 17 months was associated with persistent but not transient
wheeze [61] and, in a Norwegian study, compliance of the respiratory system was reduced
at birth among children with asthma persisting from before 2 yrs to 10 yrs of life [57].
However, in the Tucson study, persistent wheezers had significantly reduced lung
function only at 6 yrs of age and not during the first year of life, compared with children
who had never wheezed [60]. Transient early wheezers tended to have reduced lung
function both in infancy and at 6 yrs of age. In contrast, a recent Australian study
suggested that transient early wheezers have normal lung function at age 1 month and
that reduced lung function in infancy was associated with persistent wheeze by 11 yrs of
age [62]. Similarly, among 614 Norwegian children, reduced tPTEF/tE, as well as reduced
compliance (but not resistance) of the respiratory system at birth, was associated with a
81
A. CUSTOVIC ET AL.
three-fold increased risk of ongoing asthma at 10 yrs [57]. Although definitions of wheeze
phenotypes were not identical, it is still difficult to explain the differences between these
studies, as the techniques used to measure infant lung function appear similar. Among 1
2-yr-old children, reduced tPTEF/tE has been found in asymptomatic children with
recurrent wheeze [63] or asthma [64], and has been associated with bronchial obstruction
after provocation with methacholine [65].
Recent data on lung function in early preschool age (which was largely unavailable
previously) filled the gap in the young preschool age. Among 4-yr-old children enrolled in
the Dutch Prevention and Incidence of Asthma and Mite Allergy (PIAMA) study,
resistance measured by the interrupter technique was higher in children with persistent
wheeze than in children who had never wheezed and those with transient early wheeze
[66]. In the Manchester Asthma and Allergy study (MAAS), specific airway resistance at
3 and 5 yrs was reduced in children with persistent wheeze compared with transient early
wheezers and non-wheezy children, with transient wheezers falling between children who
have never wheezed and persistent wheezers [67]. These data suggest that among young
preschool children, both transient and persistent wheezers have reduced lung function
compared with non-wheezy children, and the deficit appears to be greater in persistent
wheezers.
Predicting wheeze phenotypes

Clinically, it has proven difficult to predict which preschool children will have only
transient early life symptoms and to distinguish them from those whose symptoms will
persist. It has recently been demonstrated that among children with a history of wheeze
within the first 3 yrs of life, lung function at age 3 yrs was reduced in those who
subsequently continued with wheezing (persistent wheezers) compared with children who
stopped wheezing (transient early wheezers) [67]. However, there were no differences in
lung function at age 3 yrs between children who had never wheezed compared with those
who developed wheeze after 3 yrs of age (i.e. late-onset wheezers). Thus, reduced lung
function at age 3 yrs predicted the persistence of symptoms in children who wheezed
within the first 3 yrs of life, but was not associated with the onset of wheeze after age
3 yrs (fig. 1) [67]. It is tempting to speculate that measuring lung function in symptomatic
young preschool children may enable the targeting of children who are most likely to
benefit from treatment interventions and monitoring, whereas classification into
wheezing phenotypes can only be done in a retrospective manner when the development
of wheezing illness is known.
Sensitisation and lung function

Recent cohort studies utilising lung function measurement in preschool age have
confirmed that lung function is reduced in children with a history of wheeze [68].
However, a striking finding was observed among healthy children at age 3 yrs: lung
function was reduced among those sensitised to common inhalant allergens, even in the
absence of any respiratory symptoms [68]. Furthermore, parental sensitisation status
affected a childs lung function, but there was no interaction with a childs atopy.
However, whilst there was no interaction between a childs sensitisation and sensitisation
in parents, a significant interaction was observed between maternal asthma and a childs
sensitisation status; if the child was sensitised, there was a significant reduction in lung
function in the offspring of asthmatic mothers [68]. This may result from a shared
82
a)
1.0
Predicted probability for persistent wheeze
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
b)
1.0
Predicted probability for late-onset wheeze
0.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.55
0.67
0.82
1.00
1.22
1.49
sRaw at age 3 yrs kPas-1
1.82
Fig. 1. Fitted predicted probability curve for a) persistent wheezing and b) late-onset wheezing by age 5 yrs in
relation to specific airway resistance (sRaw) at 3 yrs of age. Taken from [67], with permission.
environment, either antenatally or post-natally. It is worth emphasising that in children

who have never wheezed, the size of the difference in lung function between those who
were atopic and had a mother with asthma in comparison with all others was greater than
the recently reported difference between children with confirmed asthma and healthy
controls in a study using the same methodology [53].
Association between atopic sensitisation and impaired lung function have previously
been demonstrated in older children. Ulrik and Backer [69] found that sensitisation to
dust mite had a negative impact on FEV1 in non-asthmatic children aged 717 yrs, with
no evidence of airway hyperreactivity.
In most studies investigating the relationship between allergy and respiratory disease,
sensitisation is considered only as a dichotomous variable, i.e. individuals are assigned as
either sensitised or not [70]. Furthermore, various cut-off values have been used to define
sensitisation (e.g. w0.35, w0.7 or w1 kUA?L-1 of allergen-specific IgE (where UA is the
number of units of allergen-specific IgE) or a skin test reactionw0, 1, 2 or 3 mm) [71, 72].
83
A. CUSTOVIC ET AL.
1.5
sRaw kPas-1
1.4
1.3
1.2
1.1
0.1
0.3
3
10
30
100
Sum IgE to mite, cat and dog
300
1000
Fig. 2. Association between lung function at age 5 yrs and a sum of mite, cat and dog allergen-specific
immunoglobulin (Ig)E antibodies shown as a regression line () with 95% confidence intervals (------).
p=0.004. sRaw: specific airway resistance. Taken from [73], with permission.
The observation of the association between early life lung function and a childs allergic
sensitisation was further extended by a recent study demonstrating that the absolute
specific IgE antibody levels offer more information than just the presence of specific IgE
[73]. Increasing specific IgE antibody levels to common inhalant allergens (dust mite, cat
and dog) or increasing size of the wheal on skin-prick testing were associated with
reduced lung function in preschool children (fig. 2) [73]. Total IgE was found to be a
poorer predictor of lung function than the sum of specific IgEs. This suggests that
labelling subjects as sensitised or not based on arbitrary cut-offs for either specific IgE
levels or the size of skin-test wheal is an oversimplification of a trait that may not be
dichotomous in its relationship to the paediatric lung.
Allergen exposure, pet ownership and early life lung function

The relationship between allergen exposure, sensitisation and the development of
asthma is complex. Whilst a doseresponse relationship has been demonstrated between
mite allergen exposure and specific sensitisation, this exposure does not appear to be
related to the development of asthma [74]. The effect of cat and dog ownership and
exposure to respective allergens on the development of sensitisation and asthma is even
less clear. For example, based on the available evidence, any association between pet
ownership, sensitisation and asthma can be supported, i.e. risk, protection or no effect
[74]. However, there is little information on the effect of allergen exposure or pet
ownership on early life lung function. Recent data from a prospective birth cohort study
suggested that children aged 3 yrs who were both sensitised and currently exposed to
high levels of sensitising allergen (mite, cat and/or dog) had significantly worse lung
function compared with those who were either not sensitised or were sensitised but not
currently exposed (table 1) [75]. Therefore, sensitisation per se may have little effect on
lung function in preschool children in the absence of exposure to sensitising allergen but
has a major effect within the context of specific exposure. Taken together with previously
84
Table 1. Estimated marginal means of the specific airway resistance (sRaw) levels in relation to allergen
sensitisation and specific allergen exposure status
sRaw geometric mean (95% confidence interval)
Not sensitised, not exposed
Not sensitised, exposed
Sensitised, not exposed
Sensitised, exposed
1.14 (1.041.24)
1.10 (1.051.15)
1.21 (1.081.37)
1.38 (1.261.51)
Taken from [75], with permission.
mentioned data on the quantitative relationship between specific IgE levels and lung
function, these data may indicate that the level of IgE (or the size of wheal on skin
testing) to a certain degree reflects personal allergen exposure and offers more valuable
information about the nature of the relationship between allergy and lung compared with
a simple dichotomised atopy parameter. Cat and dog ownership, either at birth or at
3 yrs of age, had no effect on lung function [75].
Furthermore, after adjusting for the history of wheeze, lung function was substantially
reduced in children who were sensitised and highly exposed to allergen and had both
parents with asthma, compared with those with none or any one of these features. This
indicates that there is a genetic component which interacts with environmental exposures
affecting early life lung function [75]. A recent study provided the first evidence for the
genetic component of the early life lung function, demonstrating the association of
ADAM33 polymorphisms with reduced lung function at both 3 and 5 yrs of age [76].
Recent data from the intervention arm of the UK MAAS study raise questions about
the nature of the relationship between allergic sensitisation and lung function in early
childhood [77]. Stringent environmental control during pregnancy and early life resulted
in increased sensitisation to dust mite but better lung function in children at high risk of
allergic disease at age 3 yrs, i.e. there was a disconnection between sensitisation and lung
function consequent to intervention. The absence of allergen exposure in sensitised
children could not explain the observed effect, since lung function was markedly better in
the intervention group both among sensitised and nonsensitised children. In children
with longitudinal lung function data, there was no difference in lung function between
the groups in infancy, but there was a marked difference at age 3 yrs, i.e. the difference
between the groups is likely to have arisen after 4 weeks but before 3 yrs of age due to
some factor(s) affected by environmental control (fig. 3) [77].
Other environmental influences on lung function

Several environmental factors, in addition to those mentioned above, have been shown
to reduce lung function or enhance the natural decline in lung function including air
pollution in adults [78, 79] and children [8082], and viral infections in children [83] and
adults [84].
One important environmental factor which may have an impact upon lung function
development and growth is treatment prescribed for asthma and allergic diseases.
Whereas antibiotic treatment in early life has been shown to be related to increased
allergic sensitisation [85], no such relationship to lung function has been demonstrated.
Conversely, early respiratory tract infections have been shown to have an impact upon
lung function in infancy [86], as well as later lung function in childhood [87], adolescence
[88] and adulthood [84, 89]. It has been suggested that respiratory tract infections in early
childhood may cause chronic obstructive lung disease in adulthood [90]. Respiratory
syncytial virus infections, which have been related to later reductions in lung function [88],
85
a)
1.0
In V 'max FRC, GM and 95% CI
A. CUSTOVIC ET AL.
0.8
0.6
0.4
0.2
0.0
In sRaw, GM and 95% CI
b)
HRC
HRA
Lung function (V 'max FRC) at age 4 weeks
0.4
0.3
0.2
0.1
0.0
-0.1
HRC
HRA
Lung function (s Raw) at age 3 yrs
Fig. 3. Prospective data on lung function in the intervention (HRA; n=14) and control (HRC; n=18) groups in
the Manchester Asthma and Allergy study in infancy and at 3 yrs of age. V9maxFRC: maximum expiratory flow
at functional residual capacity; GM: geometric mean; CI: confidence interval; sRaw: specific airway resistance.
Taken from [77], with permission.
have also been related to development of allergic diseases and allergic sensitisation [91],
although this remains controversial.
In adults, the importance of early anti-inflammatory treatment with inhaled steroids
has been demonstrated as being related to airways remodelling [92]. An observational
study [93] also indicated this importance with respect to lung function growth in
schoolchildren, and a recent report from the Netherlands supported this view [94];
however, another study could not confirm this [95], and its importance is even more
highly debatable in younger children. A recent report from a birth cohort study showed
that children with recurrent episodes of bronchial obstruction had reduced lung function,
as assessed by tidal breathing measurements before treatment was started, and children
who later started with inhaled steroids, albeit before the age of 2 yrs, had reduced lung
86
function as compared with those with earlier inhaled steroid treatment. Furthermore,
lung function improved significantly in children who received inhaled steroids, and the
improvement was related to the duration of inhaled steroid treatment [96]. A
randomised, clinical, placebo-controlled trial demonstrated by forced expiratory flows
that lung function improved in infants treated with inhaled steroids as compared with
placebo-treated infants [97]. However, in very early childhood, there is concern regarding
a possible negative effect of steroids upon lung growth and development, resulting from
reports from animal studies with high doses of systemic steroids [98, 99]; the impact upon
the young human airway is not known.
Conclusions
Reduced early life lung function is associated with persistent wheezing independent of
atopic sensitisation. It is possible that in addition to being "remodelled" as a consequence
of inflammatory process, the airways could be "pre-modelled" as one of the prerequisites
for subsequent development of wheeze, with allergic sensitisation contributing to a
further reduction in lung function during the development [68, 77]. Studies support that
such pre-modelling may have effect long into adult life [90]. Children with comparatively
smaller deficits in lung function may develop only transient wheezing. In children with a
history of wheeze in early life and a deficit in lung function, early development of IgEmediated sensitisation further increases the risk of persistence of symptoms. Monitoring
of lung function and atopic sensitisation in symptomatic children and understanding
their relationship from an early age may enable identification of children at risk of
persistent disease.
Summary
The clinical entities of asthma, atopic eczema and allergic rhinitis may appear alone or
in any combination. The link between the developing and growing lung and these
clinical diseases is not clear, although several risk factors for asthma are similar to risk
factors for reduced lung function in early life. Even less is known about possible
associations between environmental exposure, allergic sensitisation and lung function
in early life, and whether patho-physiological mechanisms related to allergic
sensitisation also play a role in lung development and growth in the young child.
In recent years, the availability of equipment for measuring various aspects of lung
function from birth through infancy, preschool age into school age and adolescence
has increased greatly. This will increase the possibility of unravelling some of the
current questions in years to come.
Keywords: Allergy, asthma, lung development, lung function.
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CHAPTER 7
Impact of genetic factors on lung

development in health and disease
P. LeSouef *, M. Kabesch#
*School of Paediatrics and Child Health, University of Western Australia, Perth, Australia. #University
Childrens Hospital, Ludwig Maximilians University Munich, Munich, Germany.
Correspondence: M. Kabesch, University Childrens Hospital, Ludwig Maximilians University Munich,
Lindwurmstrasse 4, D-80337 Munich, Germany. Fax: 49 8951604764; E-mail: Michael.Kabesch@
med.uni-muenchen.de
Within the last few decades, genetics has succeeded in identifying the causes of a
number of monogenetic inherited diseases caused by defined mutations in single genes. In
this process, the genetic causes for a number of rare, and not so rare, lung diseases were
established. Foremost, the gene for cystic fibrosis (CF) has been identified [1] andw1,000
disease-associated mutations have been found in this gene, the CF transmembrane
regulator (CFTR) gene. However, early studies established that correlations between
CFTR genotype and CF phenotype were not straightforward. As in many other so-called
monogenetic diseases, what initially looked like a classic monogenetic disease evolved
into a complex picture of mutations in major and minor genes [2]. For the CFTR gene,
specific CF phenotypes cannot be assigned to given CFTR alterations. Rather, modifier
genes seem to be involved in directing a proportion of the clinical expression of the
disease [2]. Thus, it became obvious that lung development, breathing itself and lung
immunology are the product of a sophisticated network of factors, some of which are
under genetic control. Furthermore, common genetic alterations leading to genetic
variation within a population will seldom be apparent or cause disease. In general, these
modifier genes in respiratory diseases may belong to two different groups of genes: genes
that modify lung structure and genes that modulate respiratory as well as general
immunity.
Patchwork genetics: small effects add up

From a population point of view, and in the light of evolution, genetic variability in
these factors makes sense, as a wide spectrum of similar but not identical individuals
increase the chance that a species will survive encounters with different and variable
environmental challenges. However, in some cases, disease may occur at the edges of this
distribution due to excessive variation in these modifying factors, and this may occur for
a number of reasons, as follows: 1) when a fundamental lung protein is severely disturbed
by a major genetic alteration, as in CF; 2) under certain environmental conditions where
the limits of the genetically determined adaptation are exceeded (e.g. chemical exposure
at the workplace); and 3) in a situation where the system is overwhelmed, i.e. small
genetic changes add up and/or are combined with minor environmental effects which, by
themselves, would not be sufficient to lead to disease. This is the case in so-called complex
diseases, such as asthma and chronic obstructive pulmonary disease (COPD), where a
ISSN 1025-448x.
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P. LESOUEF, M. KABESCH
strong genetic background is present, but the expression of symptoms is dependent on

environmental factors; this is more obvious in one disease than the other.
What is normal?
Based on this paradigm, with the exception of some monogenetic disorders, genetic
susceptibility to disease depends on many genetic factors. Genetic variation is a crucial
principle in evolution and is ensured by recombination of maternal and paternal DNA in
sexual reproduction. Furthermore, genetic change is achieved by the constant,
spontaneous mutation rate of the genome. Even though such an event is relatively
rare on an individual level, the effect on a population level is substantial over a long
period of time. Thus, more than a million loci in the human genome are currently known
to be polymorphic, which means that, on average, one polymorphic locus (base
exchange) is present at least every 5,00010,000 bases. At every single polymorphic site,
i3% of individuals differ in the respective allele from the rest of the population. While
random mutation is the default setting driving evolution, conservation of the DNA
sequence is an active process. Highly relevant genome areas are protected from random
changes within the members of a species and are also conserved throughout evolution.
Certain structures have remained unchanged between mouse and humans or even
between plants and humans. These conserved areas have either critical regulatory
functions in the genome, e.g. controlling gene expression, or serve as exons, DNA
templates used for transcription and translation into proteins.
Genetic change as a driving force in the interaction with

environment
Some genes involved in lung development or respiratory immunity have turned out to
be highly variable, showing a mutation rate of w1 per 5,000 bases, while others are
remarkably conserved. Few known mutations and polymorphisms will alter the function
of a gene. Most polymorphisms are functionally silent and evolutionarily neutral.
Definite functional changes can only be assigned to a minority of the polymorphisms, so
far identified in candidate genes for respiratory diseases. In addition, these functional
changes that can lead to increased or decreased gene transcription or translation, a
diminished or exaggerated function of the protein, or a change in the structure of the
protein, may be subtle rather than substantial. For example, a single base change in the
CD14 promoter has been shown to alter the binding affinity of a transcription factor at
that position. By itself, this is not a major change, as promoter activity is only decreased
by y20% in vitro [3]. Compared with artificially introduced mutations in other parts of
the promoter (which do not occur in nature), where a change in function by 10-fold or
more can be achieved in an in vitro system, the effect of the CD14 promoter
polymorphism seems at first to be unimpressive. However, this is the size of effect that
would be expected from a useful genetic variation in nature, as it would alter but not
destroy the function of a gene.
Common genetic changes do not usually lead to disease by themselves, they simply
modestly alter a single gene function, making these polymorphisms valuable for
evolution on a population level by increasing variance within the population. However,
what is advantageous for a species may be very inconvenient for an individual, as the
burden of changes may, by chance, accumulate in certain individuals and then lead to
disease. However, other than in many well-defined monogenetic metabolic diseases,
94
GENES IN HEALTH AND DISEASE
where loss of function in any one of the many crucial bottle-neck enzymes may lead to a
halt in metabolism and thus to disaster, breathing, lung development and lung
immunology seem to rely on more redundant mechanisms. In lung diseases, such as
asthma or COPD, genetic susceptibility may only turn into disease when various
components of a developmental or functional pathway are affected by genetic changes or
when certain environmental trigger factors hit a more susceptible individual. Of course,
in many environmental exposures, dosage is critical and independent of the genetic
susceptibility, and exposure above a certain threshold (e.g. heavy smoking) usually leads
to disease independent of the genetic makeup of an individual. The occurrence of disease
in that instance would be a phenocopy rather than a genetic version of the disease, as it
would result from strong environmental influences only. An example of this can be found
in pulmonary function assessment, as follows: response to histamine in a certain dose
range is a sensitive measurement for bronchial hyperresponsiveness (BHR), which is
usually dependent on an individuals genetically determined susceptibility to develop the
condition and the personal history of previous exposure to environmental triggers.
However, when histamine is administered in a sufficiently high dose, almost every
individual, irrespective of their genetic makeup, will develop bronchial constriction.
Overall, very few respiratory diseases may be due to monogenetic disorders, where the
function of a single crucial gene is so severely altered that the natural development or
function of the lung is fundamentally disturbed. CF is the best known and studied genetic
lung disease, and has already been mentioned. Cilial dyskinesia may turn out to be due to
a series of related major gene defects caused by a limited number of alterations in ciliarelated genes [4]. In addition, some other monogenetic disorders may affect the lung as
part of a multi-organ disease [5]. However, these most severe but rare diseases only make
up a small proportion of the vast number of patients seen in respiratory clinics all over
the world. Other genes, so-called modifier genes, which may also determine and direct the
development and function of the lung irrespective of disease, may have more impact on
common diseases such as asthma and COPD.
Genetics of structural genes may influence the modelling and

remodelling of the lung in health and disease
As genetic research is driven primarily by the aim to understand and finally resolve
disease rather than to investigate functional variance in healthy individuals, it is not
surprising that most knowledge about the normal function of genes derives from genetic
studies in diseases such as asthma, which has been a hotspot for genetics for some time
now. In asthma, at least four genes, previously not known to relate to any kind of lung
disease, have been identified by a purely genetic tool, so-called positional cloning [69].
As it turns out, some of these genes may be involved in basic pulmonary and
immunological function. The ADAM33 (a disintegrin and metalloproteinase domain 33)
gene is one of these genes and is suspected to be important in lung development and
remodelling. Located at chromosome 20p13, it was initially identified by linkage analysis
and positional cloning in a joint British and American project [7]. More than 100
common polymorphisms in and around the gene have been discovered and numerous
replication studies have been conducted to clarify which ADAM33 polymorphisms
contribute to asthma in different populations around the world. These studies have led to
controversial results. Replication studies mostly focused on those 19 polymorphisms
associated with asthma in at least one of the original populations. While all except three
published studies reported some associations between ADAM33 and asthma
susceptibility (which may in part be due to a positive publication bias), the amount of
95
variation between these associations was profound. The two largest studies so far
conducted on ADAM33 [10, 11] came to the conclusion that no significant association
can be assumed between ADAM33 polymorphisms and asthma. All replication studies
tested multiple polymorphism and multiple outcome variables [1016]. Thus, caution is
also necessary in the interpretation of any study that reports positive association results.
Intriguingly, even in the positive studies, there is very little consensus on the
polymorphisms that show associations in different populations.
Genetic and environmental variability may be one possible explanation for this
diversity in results between study populations. However, other factors may also
contribute to these inconsistent results of replication. One possibility is that ADAM33
polymorphisms noted in the original report are not the true cause for the linkage signal
observed and that other polymorphisms in ADAM33 or even in other genes in linkage
with ADAM33 are responsible. By testing the published ADAM33 polymorphisms, one
may or may not concomitantly measure the effect of the "true" asthma risk gene on
chromosome 20p13, as different populations may represent different haplotype and
linkage blocks, either linking or not linking certain ADAM33 polymorphisms to the real
risk gene or risk polymorphism.
Interestingly, however, the strength of association with ADAM33 increased when
asthma with BHR was analysed as a distinct phenotype. Thus, ADAM33 may be more
involved in airway remodelling than being of general immunological importance. Indeed,
gene expression could be detected in lung fibroblasts and airway smooth muscle cells. In
Dutch studies in adults, ADAM33 polymorphisms were associated with an accelerated
decline in lung function, which may support the hypothesis that ADAM33 is involved in
airway remodelling [15, 16]. However, recent data suggests that ADAM33 is also
expressed in different isoforms, which may be genetically regulated, in embryonic lung
tissue [17]. While several ADAM33 protein isoforms also occur in adult bronchial
smooth muscle cells, ADAM33 is expressed in human embryonic bronchi and
surrounding mesenchyme, suggesting a role in smooth muscle development. The
identification of ADAM33 in embryonic mesenchymal cells may indicate that ADAM33
is not only involved in remodelling of airways in asthma later in life, but that it may
actually play a role in the initial development of the airway wall. Genetic alterations in
this early "modelling" may increase bronchial responsiveness and influence the
susceptibility for obstructive airway diseases, such as asthma, later on. The role of
these ADAM33 isoforms in these developmental processes is still poorly understood.
However, an increase in the total amount of ADAM33 mRNA is unlikely to be the
problem that leads to disease, but rather an altered expression profile of different
isoforms of the ADAM33 protein that could change the proteins role. As most
polymorphisms in the ADAM33 gene have been located in the intronic regions of the
gene, it could be speculated that these polymorphisms may influence splicing. However,
no direct link between such a polymorphism and splice regulation has been established
either in vivo or in vitro so far. Even though ADAM33 is a very attractive candidate gene
for asthma based on the model proposed for its function and expression in cells
important in the lung, there is still no firm evidence for its function in either asthma or
airway modelling or remodelling. In addition, no functional role of ADAM33
polymorphisms has yet been described.
The heterogeneity in replication results for ADAM33 may well be due to a different
weight of certain polymorphisms and genes in the development of asthma in different populations. Expressed in more general terms, various sets of genes have a different
weight in the development of common diseases in different ethnically and genetically
diverse groups. What can be learned from ADAM33 is that finding genes for a complex
genetic trait, such as asthma or other common respiratory disorders, may lead not only
to the discovery of a disease gene but may increase the understanding of underlying
96
mechanisms of lung development and function, which, in the case of ADAM33, is just
beginning.
Genetic modification of exposure to air pollutants in the lung

In addition to ADAM33, a number of other genes may be involved in lung
development. Some of these genes may be influenced by genetic alterations and become
disease relevant depending on certain environmental stimuli. The first candidates for
these effects are detoxification genes in connection with exposure to toxic substances
from the environment. A number of these genes exist, and one of the best-studied groups
of genes in this area is the glutathione S-transferases (GSTs) [18]. Four cytosolic classes
of GSTs exist (alpha (A), mu (M), pi (P) and theta (T)), and various subclasses are
defined. Located mainly in the cytosol, GST enzymes catalyse the conjugation of
electrophilic substrates to glutathione but also contribute to peroxidase and isomerase
activities [18]. GSTs facilitate responses to oxidative stress reactions and are involved in
major detoxification pathways of polycyclic aromatic hydrocarbons and detoxify
benzo[a]pyrene [19]. Common deletions of the GSTM1 and GSTT1 genes affecting 50
and 15%, respectively, of the European population result in a complete loss of the gene
and the respective enzyme function [20]. In addition, common polymorphisms in the
GSTP1 gene have been described to lead to amino acid changes, as previously revised
[21]. Genetic alterations, and in the case of GSTM1 and GSTT1 the complete loss of the
gene, may significantly alter an individuals ability to detoxify components found in air
pollution, primarily in passive and active smoke exposure.
Experimental findings and data from population genetic studies indicate that
individuals with a decreased function of GST enzymes are at a higher risk of developing
asthma and asthma symptoms in combination with in utero environmental tobacco
smoke (ETS) exposure, later passive ETS exposure or active smoking, than those
exposed children with an intact GST system. These exposures are interconnected and are
thus difficult to decipher. However, all types of tobacco exposure showed independent
effects on respiratory health in GST negative individuals and trends for dose-dependent
effects were observed. The role of the GST system, and genetic alterations within that
system, in the development of childhood asthma does not appear to be limited to the
modification of active and passive smoking effects. Recent studies indicate that
alterations in GST enzymes may also be involved in mediating negative health effects
caused by other forms of air pollution [22]. Studies from Mexico City, Mexico, showed
that GSTM1 deficiency in children with a high level of ozone exposure increased the risk
for asthma in an interactive manner [22]. In addition, in China, children homozygous for
GSTP1 Ile105 and exposed to high levels of air pollution had a higher risk of developing
asthma [23].
GST deficiency in combination with air pollution not only leads to asthma, but also
alters basic lung function and leads to the development of more general respiratory
symptoms, such as wheezing and cough [20, 24, 25]. Lung development, as inferred by
lung function measurements, seems to be diminished in GST-deficient children when
mothers smoke during pregnancy [20] and also when passive smoke exposure occurs later
in life [24, 25].
Thus, genetic changes in these detoxification enzymes appear to modify the effect of
common environmental hazards in general. If these interactions do lead to disease or
unspecific respiratory symptoms, additional factors are likely to be involved. In addition,
genetic alterations in GSTs or other similar pulmonary modifier genes, such as
a1-antitrypsin or tissue growth factor, may specifically influence the clinical expression of
97
various respiratory diseases and may also be able to exert nonspecific effects on disease.
In addition to effects of structural genes, such as ADAM33 and detoxification genes,
genes involved in inflammatory processes may also contribute to lung health and disease.
Recent studies have also suggested that structural cells in the lung, such as epithelial and
smooth muscle cells, may exert immunological functions.
Lung genes may influence immunity in different ways

One of the genes of pulmonary origin that has been initially linked to asthma in genetic
studies, but that may turn out to be involved in a much wider range of respiratory
diseases, is Clara cell protein 16 (CC16; also referred to as uteroglobulin and Clara cell
secretory protein 10). CC16, secreted in large amounts in airways by the nonciliated
bronchiolar Clara cells, is a potent immunosuppressive agent, inhibiting the activity of
phospholipase A2 [26], interferon (IFN)-c [27], and neutrophil and monocyte migration
in the lung [28]. The CC16 gene is located on chromosome 11q1213, and an adenine/
guanine polymorphism 38 base pairs (A38G) downstream from the transcriptional start
site has been identified [29]. Gene expression studies have shown that the 38A allele has a
25% lower transcription level than the 38G allele, and this difference in expression levels
could therefore also decrease the CC16-associated anti-inflammatory protection of the
lung in carriers of the 38A allele. Several genetic studies (but not all) have associated the
CC16 polymorphism with asthma [30, 31] and, in a study of German children, asthmatics
with the 38AA genotype showed increased airway responsiveness to histamine or exercise
[32]. In addition, the presence of CC16 has also been associated with protection from
various forms of pulmonary disorders, such as acute respiratory distress [33] and
oxidative stress reactions [34]. Recently, studies have investigated whether these asthmaindependent effects are also influenced by genetic alterations. In a German population of
117 cases of acute respiratory distress syndrome and 373 controls, the same A38G
polymorphism was found to alter neither the susceptibility nor the outcome of the disease
[35].
Immunogenetics and lung development

Genetics play an important role in the development of the immune system, which in
turn has important effects on the respiratory system. Environmental factors create added
complexity by strongly influencing the relationship between genetics and immune-system
development. Evidence is accumulating rapidly in this area and the available data suggest
that it will be one of considerable and increasing importance in the future.
Genetics and the development of enhanced allergic responses

Since the late 1990s, a large number of studies have examined genetic variations in
common immunological pathways to determine their potential effect on the development
of allergy and atopy. Most of these studies have reported associations between particular
polymorphisms and atopic or respiratory phenotypes. Many of these observations have
been replicated in more than one population. Some are backed by positive linkage data
and sound functional data that reasonably establish the causal nature of these
relationships. A smaller number of studies have also included measurement of tissue
levels of the output of the gene in question. The combination of replicated associations
98
between a particular allele and a specific phenotype, supporting functional data and data
on tissue levels of the protein output of the gene, provide reasonable evidence of causality
[36].
An excellent example of a polymorphism that fulfils all these criteria and one that is
perhaps the best-established genetic variant causing atopy in children is the CD14 C159T promoter polymorphism. CD14 is an important receptor for lipopolysaccharides
and components of bacterial cell walls and plays a crucial role in directing T-helper cell
(Th) type 1 and Th2 responses [37]. The CD14 C-159T promoter polymorphism is
localised on chromosome 5.31.1 [38], a region that, in genome-wide screening studies, has
shown strong linkage with atopic phenotypes in some [39, 40], weak linkage in others and
no linkage in others [41].
The C allele has been associated with reduced CD14 production in in vitro studies [3],
reduced levels of circulating CD14 (which would tend to enhance Th2 immunological
responses) [42], increased serum levels of specific immunoglobulin (Ig)E [42], increased
serum levels of total IgE [43], increases in positive skin-prick tests to common allergens in
an adult population [43] and age-specific increases in positive skin-prick tests in a
population of children followed longitudinally from 825 yrs of age [44] (fig. 1).
The potential role of the environment in producing and sustaining these associations is
still not clear. The role must be substantial, since in some places in the world, there is very
little allergy or asthma, whereas in others, strong relationships are found between the
same alleles and outcomes [45]. However, from the context of children living a "Western"
lifestyle, the environmental factors that are involved, although largely still unknown,
appear to produce a similar pattern of associations in populations living in a broad range
of societies, in varying climates and in widely separate geographical locations.
In general in Western society, there is biological plausibility for the relationships
between genetics and immunological factors. Alleles that enhance Th2 immunological
responses in vitro are usually associated with increased IgE levels in vivo and with
increases in prevalence of atopic diseases, such as dermatitis, rhinitis and asthma, as
demonstrated by the CD14 C-159T example quoted above. There are several other
Mean number positive SPTs
1.4
**
n
**
1.2
1.0
0.8
0.6
0.4
l
s
10
12
n
s
l
n
s
l
s
s
l
l
s
0.2
0.0
14
Age yrs
16
18
25
Fig. 1. Number of positive skin prick tests (SPTs) with genotype CD14 C-159T from age 825 yrs. Those with
CD14 -159CC (&) had a greater number of positive SPTs versus those with CD14-159CT ($) and CD14-159TT
(+). *: pv0.05; **: pv0.01. Reproduced from [44] with permission.
99
examples of such relationships. For interleukin (IL)-4 receptor (IL-4R), several lines of
evidence suggest that IL-4R polymorphisms contribute to the development of asthma in
children. IL-4 is a Th2 cytokine and, together with its receptor, is involved in the
generation of atopic inflammation. Several polymorphisms have been found in IL-4 and
functional studies have demonstrated in vivo differences between alleles for C-589T [46,
47]. In a recent study, IL-4 -589T was more frequent in children with asthma compared
with controls, and IL-4R 576Q was more frequent in children with atopic asthma [48].
The importance of haplotype analyses in such studies was demonstrated by the
association between the IL-4 -34T/-589T haplotype and asthma and between the IL-4R
I50A/576Q haplotype and atopic asthma [48]. A further study showed that there were
also interrelationships between IL-4R and IL-13 polymorphisms [49]. Several other
significant relationships between alleles in Th1 and Th2 pathway genes and asthma
phenotypes in children have been demonstrated [50].
Relevance of genetic studies to understanding the early

development of the immune system
These studies demonstrate that development of the immune system and its relationship
with the respiratory system is influenced by genetic variation. However, one of the most
interesting issues with respect to the early development of the immune system is the
difference in rate of development of the immune system between atopic and non-atopic
children. This area of research has received increasing attention in recent years. A
longitudinal study of the early maturation of Th1 and Th2 responses in early life
demonstrated that those destined to develop atopy had both an impairment in early Th1
responses and a delay in mounting Th2 responses, followed by the development of
augmented Th2 responses [51]. These important observations establish the disordered
function of the immune system in the first year or two of life in children with a genetic
predisposition to atopy. Investigating why atopic children have these aberrant responses
and impaired development can be approached by dissecting the genetic variations that
contribute to these responses. Although such studies are planned, very little is known
about any of the variations that play a role in this area at this stage. Indeed, a follow-up
study of the above cohort at 6 yrs demonstrated that the best predictor of outcome at this
age was family history of allergy rather than anything that had been measured soon after
birth [52]. These findings can be interpreted as further evidence that genetics is playing
the crucial role in determining both alterations in early maturation of the immune system
and in setting the level of clinical and immunological function during childhood.
Other consequences of the early impairment of the immune

system in atopic children
The main focus of researchers in examining the early maturation of the immune system
has been to understand the way in which Th2 responses evolve. This focus on the
pathway to IgE production has provided a great deal of useful knowledge. However, as
noted above, recent research has uncovered co-existing problems with Th1 responses.
These problems are well illustrated by Rowe et al. [53], who studied cytokine responses to
vaccination with diphtheria and tetanus toxins. They noted that, after vaccination, the
ratio of the Th1 cytokine IFN-c to either of the two Th2 cytokines (IL-5 or IL-13) was
significantly lower in those with versus those without a family history of atopy (fig. 2),
100
IFN-g/IL-5
4
3
2
1
0
6 months
12 months
18 months
Fig. 2. Mean ratios of interferon (IFN)-c to interleukin (IL)-5 from in vitro stimulation of peripheral blood
mononuclear cells with tetanus toxoid in children with or without an atopic family history (AFH). F: AFH -ive;
&: AFH zive. Reproduced from [53] with permission.
but that when present at 6 and 12 months of age, this situation had resolved itself by
18 months of age [53]. This impairment in Th1 responses appears to be associated with
an impaired ability to resist more the serious consequences of respiratory syncytial virus
infection [54].
Impairment of response to vaccines in atopic children

The most controlled exposure to antigens in early life is the administration of vaccines.
The tightly controlled dose and timing of exposure to vaccines provides an excellent
opportunity to examine immune system responses to foreign proteins. In general, atopic
children have a much poorer response to vaccines than non-atopic children for both
humoral and cellular responses. For example, fewer atopic children responded to
pneumococcal vaccine than non-atopic children [55]. They also demonstrated reduced
responsiveness to diphtheria, pertussis and tetanus vaccination during infancy [56].
Observations such as these led to the concern that atopic children are at risk of poor
protection from vaccines in infancy, as well as an inferior ability to resist infections in
early life [57]. However, the intriguing aspect of these studies is that the problem that
atopic subjects face with respect to their immune system related to vaccines is in the
production of specific IgG antibodies and specific T-cell responses rather than the
abnormalities of IgE production, which is the usual focus of studies in atopic subjects.
Genetics of impaired antibody responses

The important point of these studies with respect to the influence of genetics on the
development of the immune system and the consequences for the respiratory system is
that genetic mechanisms can be expected to play a major role in causing these problems.
Recent genetic studies have begun to demonstrate where the problems might lie. For
example, in a study of responses to the seven-valent pneumococcal vaccine, for each of
101
the seven antigens, children with the CD14 -159C allele had lower specific IgG responses
than those who had at least one -159T allele [58]. CD14 -159C was also associated with
lower serum levels of CD14 and increased numbers of episodes of otitis media compared
with the CD14 -159T allele. Given the consistent associations of the -159C allele with
atopy, this study provides strong evidence linking the CD14 -159C allele with atopy and
impaired vaccine responses. Further work on the same cohort has shown similar findings
for IL-4 C-589T, IL-4R G2979T and IL-4Ralpha Gln551Arg, and, in each case, the allele
associated with increased IgE responses was associated with decreased specific IgG
responses to each of seven pneumococcal antigens [58]. Hence, genotypes associated with
increased IgE responses in children are also associated with decreased specific IgG
responses to foreign proteins.
Influence of maternal smoking on the relationship between

genetics and immune responses in early life
Passive smoke exposure, whether via the placental circulation in utero or via the air
post-natally, has been shown in a large number of studies to be associated with decreases
in lung function [59] and increases in airway responsiveness measured soon after birth
[60, 61] and also later in childhood [62]. In addition to effects directly on lung tissue
modified by lung genes and detoxification enzymes, smoke exposure has also been
implicated for increases in atopic responses [63, 64] and respiratory infections [65]. An
important study that provides evidence of a possible mechanism for the effect of smoking
was recently published [40]. In this study, which was a genome-wide screen for linkage
related to asthma, no evidence of linkage was found for the cytokine-rich area of
chromosome 5q.31. However, when only children of females who smoked during
pregnancy were examined, a highly significant result was obtained [40] (fig. 3). These
data point to maternal smoking exerting a specific and potent environmental effect on
particular genes in an area of the human genome where a number of immunity genes are
3 .0
L od score
2.5
2.0
1.5
1.0
0.5
0.0
50
100
Distance cm
150
200
Fig. 3. Results from genome-wide linkage analysis of asthma in European-American families from the
Collaborative Study for the Genetics of Asthma on the basis of passive smoke exposure for chromosome 5.
: Lod scores from all asthmatic subjects; ------: exposed asthmatic subjects; ???????????: asthmatic subjects not
exposed to smoke. Reproduced from [40] with permission.
102
amassed. These observations have been extended by a subsequent study that has
examined vaccine responses in children from a cohort selected for parental atopy. In this
study, preliminary data have shown that specific anti-tetanus and anti-diphtheria IgG
levels were reduced in children exposed to parental smoking, but that this relationship
was only evident amongst those with alleles of IL-4R and IL-4 previously associated with
atopy [66]. These findings can be summarised as showing that an infants ability to
produce specific antibodies is impaired if they have specific variations in certain genes
associated with atopy and that this problem is greatly increased by exposure to parental
smoking.
Hypothesis arising from data: wheeze in early life is related to

a relative state of immunodeficiency
For many years, researchers have recognised that the prevalence of wheeze is greatest
in infants and that maternal smoking is a major environmental risk factor for infant
wheeze. Research efforts have focused on attempting to unravel the exaggerated IgE
responses that are associated with allergy and asthma. Some of these studies have
produced data on specific IgG and cellular responses and have shown that these
responses are often impaired in atopic children. More recent studies have demonstrated
that maternal smoking makes a major environmental contribution to further impairing
these responses. In the last year or two, genetic studies have begun to dissect these
impaired responses by being able to show related allele-specific data in genes in the Th2
inflammatory pathway.
These data can be used to generate the hypothesis that wheeze in early life is a
consequence of a relative state of immunodeficiency that co-exists in those with a
predisposition to atopy and that this state of immunodeficiency is further impaired by
maternal smoking or other genetic changes in lung structure. When the immune system
matures during the first few years of life, the immunodeficiency largely resolves. In other
words, the reason wheeze is so common in early life may be that there are large numbers
of infants who are poorly equipped to fight respiratory viral infections and the infants
with the greatest problem in this area are those with delayed post-natal maturation of the
immune system whose mothers smoked during pregnancy. Clearly, much more work is
needed to substantiate this hypothesis and to determine whether it offers new avenues of
therapeutic intervention.
Conclusion
Taken together, a complex picture of interaction between genetic factors and lung
development in health and disease evolves. Genetic variation affects lung structure and
lung development, as well as general and respiratory immunity. The sum of these
variations represents an individuals genetic makeup, determining their susceptibility to
developing a disease. Environmental factors influence the expression of phenotypes and,
finally, the development of clinical symptoms. Primarily, smoke exposure seems to be a
common, well-studied and utterly unnecessary hazard for the respiratory health of
children, affecting diverse pulmonary and immunological pathways. While it will not be
possible to alter the genetic makeup of patients in the near future, knowledge about the
genetic influences on pulmonary health and disease will undoubtedly increase.
103
Summary
Few lung diseases are caused by monogenetic disorders. However, respiratory health
and the development of lung diseases are strongly influenced by genes that modify
pulmonary development and the capability to react to environmental challenges.
Genetic variation, a driving force of evolution and an important guarantee of a broad
range of adaptive potential on the population level to increase survival of the species,
may turn into a burden when changes accumulate in certain individuals, thus leading
to disease. Genetic variations in genes that modify pulmonary health have now been
identified in many cases. Some of these common alterations affect genes involved in
pulmonary structure, detoxification and inflammation, but may also affect immunity
genes, which in turn may have profound effects on pulmonary health. In this context,
genetic susceptibility determines the potential of the organism to interact with the
environment and it is only recently that some of these interactions have been
identified. Smoke exposure seems to be of particular importance as it interacts with a
multitude of genetically determined mechanisms, aggravating immunological as well
as respiratory problems.
Keywords: Atopy, development, genetics, immunology, lung, smoking.
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107
CHAPTER 8
Geneenvironment interaction and

respiratory disease in children
J. Gerritsen*, N.E. Reijmerink*,#, M. Kerkhof }, D.S. Postma#
Depts of *Paediatrics, #Internal Medicine and Pulmonology, and }Epidemiology and Statistics, University
Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Correspondence: J. Gerritsen, University Medical Center Groningen, Beatrix Childrens Hospital, P.O.
Box 30.001, 9700 RB Groningen, The Netherlands. Fax: 31 503614235; E-mail: j.gerritsen@med.
umcg.nl
Determining the exact genetic aetiology of complex lung diseases, such as asthma,
remains a significant problem. The main reasons for this are that asthma is polygenic,
interaction between genetic (host) and environmental factors is involved, and there is a
wide heterogeneity of asthma phenotypes [1]. A genetic basis for asthma has been
demonstrated in numerous family studies. The findings were consistent, irrespective of
whether the study was performed in twins, trios or by segregation analysis of extended
pedigrees [2]. Many investigators have found evidence of linkage between genetic
markers and asthma, as well as its associated phenotypes, and to date seven genes have
been found by positional cloning [1].
For many years, the role of environmental exposures to viruses, nonspecific stimuli
and allergens in the daily morbidity of asthma and atopy has been recognised. An
example of the direct relationship between exposure and morbidity is the early and late
asthmatic reaction that occurs after exposure to house dust (mite), and the subsequent
increase in response to nonspecific stimuli [3]. Thus, nowadays it is accepted that next to
genetic basis, the environment also plays an important role in asthma development.
More specific genes, as well as their interactions, have been recognised as important
and as crucial factors in the development of asthma and atopy [47].
Notwithstanding the progress that has been made over recent years, it has still not been
fully elucidated which major and minor genes are responsible for the development of
asthma and atopy. It also remains to be clarified how the interaction occurs between the
genes already found and to what extent the expression of these genes is dependent on
other environmental and endogenous factors. Additionally, the mechanisms of gene
environment interaction have also been subject to different interpretations, as recently
discussed [69].
Genegene interaction
Genegene interaction in the development of lung disease has been extensively
investigated in cystic fibrosis (CF) [10]. Soon after the discovery of the DF508 mutation,
on chromosome 7q, in the CF transmembrane conductance regulator (CFTR) gene, it
became clear that there is great variability of pulmonary phenotypes and survival in CF,
even among patients homozygous for the most prevalent mutation DF508 [11]. This
variability could partly be explained by modifying environmental factors, such as severe
pulmonary infections, nutritional status, early development of liver disease and other
ISSN 1025-448x.
108
GENEENVIRONMENT INTERACTION
concomitant diseases [12]. Recently, it has been shown that additional genetic variation
(i.e. presence of "modifier" genes [13]) also contributes to the expression of the final
phenotype. This has been tested on chromosome 19q13.2 in several of the 10 genes [11].
In a large North American CF population, it has been shown that polymorphisms in the
promoter and codon 10 region of the transforming growth factor (TGF)-b1 gene, on
chromosome 19q13.2, are associated with pulmonary phenotypes predictive of the longterm outcome of patients with CF homozygote for DF508 in the CFTR gene.
Interestingly, recent association studies have also linked these TGF-b1 polymorphisms to
atopy, asthma and chronic obstructive pulmonary disease [1418]. The polymorphisms
of TGF-b1 are functional in that they are related to abnormalities of the airways, such as
induction of extracellular matrix in asthmatic airway smooth muscle and orchestration of
airway remodelling [19, 20].
Another example of genegene interaction was reported by Blumenthal et al. [21] in a
collaborative study on the genetics of asthma, in which a nonparametric gene analysis
approach was performed. When conditioning on chromosome 11q, there was increased
evidence for linkage in four other chromosomal regions, 5q, 8p, 12p and 14q, but not for
20p. Genegene interaction analysis has also been performed with candidate genes of
asthma. Interleukin (IL)-13 and IL-4RA are both key molecules in T-helper 2 signalling
[22]. Variations in the IL-13 gene have been associated with bronchial hyperreactivity
(BHR), asthma susceptibility and immunoglobulin (Ig)E. While a borderline significant
association was observed between polymorphisms in IL-4RA and BHR and asthma,
both BHR and IgE are risk factors for asthma. Thus, interaction between the genes could
be expected. Indeed, when both genes were analysed in combination, individuals with the
risk genotypes had a nearly 2.5 times greater risk of developing asthma than individuals
with either genotype alone and a five-fold risk compared with those without these
genotypes.
These findings make it likely that genegene interaction plays an important role in
asthma as in CF, although the mechanisms by which interaction between modifier genes
and the candidate asthma genes act are still to be unravelled. It also stresses the
importance of studying genegene interaction in complex diseases, since this may
elucidate pathways that play a role in disease development, severity and progression.
Geneenvironment interaction
The environment has been highlighted as one of the factors that plays a role in the
pathogenesis of asthma and atopy. Strong indicators were exacerbations of allergic
rhinitis, particularly during the pollen season, and the beneficial effect of house dust mite
avoidance in the mountains on asthma severity, BHR and medication use [23]. However,
the allergic reactions occur in patients with established disease. Therefore, this does not
prove whether the environment also contributes to disease development, severity and/or
progression. Epidemiological studies have shown that exposure to allergens is related to
the development of allergic diseases [24, 25]. Furthermore, in studies in farmers and areas
with high infection rates, the environment can also have a protective effect on the
development of asthma and allergy [26, 27]. Since, as previously mentioned, the role of
genes in asthma is also established, it is plausible that genes constitute the link between
the environment and development of atopy and asthma.
Geneenvironment interactions can be assessed in casecontrol and cohort studies as
well as in family based genetic studies. Twin studies have provided suggestive evidence
for both genetic and environmental contributions to asthma. The heritability of asthma
has been reported to vary 6080%, leaving a remaining 2040% for environmental
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J. GERRITSEN ET AL.
contributions [2830]. Until recently, genetic and environmental susceptibility were

studied separately, or the potential interaction between the two sources was evaluated by
stratifying the effect of exposures by family history [30]. Significant interactions,
demonstrated in both linkage and association studies, indicate that many early life
exposures influence the risk for asthma and its related phenotypes in a genotype-specific
manner. These early life exposures include exposure to endotoxins [3134], viruses [5, 35],
pets [36, 37], day care environment [5] and environmental tobacco smoke [3841].
Several models of geneenvironment interactions in asthma and atopy have been
suggested by Vercelli [42], and modified by Martinez [6] and Ober and Thompson [7].
For example, the CD14 gene is considered as a potentially critical player in the gene
environment interactions leading to asthma and atopy. Several studies have shown an
association with variations in the CD14 gene and atopic phenotypes, such as IgE [9, 42,
43]. The importance of the gene is also confirmed by genetic linkage studies, which
suggest that one or more loci on chromosome 5q controls for the levels of serum IgE [43
45]. Considering geneenvironment interactions, theoretically, several models can be
postulated as already published [6, 7].
The first model is that the phenotype is expressed when the genotype is present;
environmental factor does not have an influence on the onset of disease (fig. 1).
Thus, the strength of the expression of the phenotypes depends on the genotype and is
not influenced by environmental exposure. In a real-life situation it is not easy to find a
good example. A previously mentioned example is CF, in which the severity of the
disease is predominantly determined by the genetic effect and the genegene interaction
[10]. Nevertheless, also in CF, infections as an exogenous factor and pancreatic
insufficiency as an endogenous factor contribute to the expression of the severity and
prognosis of disease.
In the second model (fig. 2), the environmental influence is the same for all genotypes,
regardless of the level of exposure. The influence of the genotypes on the strength of the
phenotype is always identical. The consequence of this is that in all environments the
estimated heritability of a disease is always similar. If the studies are well designed and
well performed the results will be highly reproducible irrespective of the populations,
with the exception that, as in CF, genegene interactions are not crucial in the expression
of the phenotype. A well-known example is phenyketonuria (PKU), a recessive disorder
Phenotype value
High
Low
High
Low
Environmental exposure
Fig. 1. The association between a phenotype and environmental exposure is plotted for different genotypes.
There is no environmental interaction for any genotype. Genotype A: ; genotype B: - - -; genotype C: ? ? ? ?.
110
Phenotype value
High
Low
Low
High
There is no geneenvironmental interaction; therefore, the curves for the three genotypes are parallel. Genotype
A: ; genotype B: - - -; genotype C: ? ? ? ?.
of metabolism in which phenyalanine cannot be converted to tyrosine. The gene for

phenylalanine hydrolase has been cloned and mapped to chromosome 12q24.1 [46].
More than 240 mutations have been defined in this disease. There is little variation in the
presentation of the disease between the different genotypes in the presence of
phenylalanine in the diet. In all patients with PKU, restriction of dietary intake of
phenylalanine can completely prevent disease development in all genotypes.
The third model (fig. 3) presents the extent genotype influence on the phenotypes can
vary in different environments. Clear examples include the high exposure to microbes in
the farming environment and the low exposure to house dust mite in the high-mountain
environment. Both environments decrease the expression of the asthma phenotype, the
former by high exposure to microbes and the latter by strong reduction of house dust
Phenotype value
High
Low
Low
High
Model of reaction of a case in which geneenvironment interaction is present. The variation is explained by
geneenvironment interaction. Genotype A: ; genotype B: - - -; genotype C: ? ? ? ?.
111
J. GERRITSEN ET AL.
mite exposure. Immunologically, this can be explained by maternal exposure to

environments rich in microbes; these exposures eventually determine the priming of the
unborn childs immune response. For example, research has shown that these pre-natal
exposures affect the expression of the toll-like receptors 2 and 4 and CD14 in school-age
children [47]; these receptors are important in the development and maintenance of the
immune system.
The effects of the genotypes are different and cannot be predicted at the different levels
of exposure. It is more likely that individuals with genotype C have the phenotype
studied when exposed at a low level, whereas individuals with genotype A are protected.
However, at high exposure, individuals with genotype A are at an increased risk to
express the phenotype and individuals with genotype C are protected. The final
interaction model is presented in figure 4.
In the paper by Ober and Thompson [7], a list is presented of the known asthma and
atopy genes found by positional cloning following linkage studies and candidate-gene
studies. Despite the large number of successful studies there is no single gene that has
been replicated in all studies and the information of the interaction between the
environment and these genes is limited and not always consistent.
An important example of geneenvironment interaction is the CD14 genotype,
endotoxin exposure and asthma i.e. the functional promoter polymorphism, -159C/T, in
the gene encoding the monocyte receptor for endotoxin i.e. CD14. Children with the TT
genotype had reduced serum levels of circulating soluble CD14 levels and IgE [48]. The
association with the T allele and reduced risk for atopy was replicated in some, but not
all, subsequent studies [49]. However, studies carried out in a farming population
revealed an association between the T allele and an increased risk for atopy [50]. This
leads to the suggestion that the CD14 variant, CD14-159C/T, interacts with
environmental levels of endotoxin to determine whether an individual is at risk or
even protected from asthma and atopy [51]. This has recently been confirmed in children
from Barbados, in whom the TT genotype was protective against asthma in
environments with low house-dust endotoxin levels, but associated with risk for
asthma in children from homes with high levels [33]. Further studies are underway,
especially in children of farmers and children living in a Steiner-lifestyle environment.
Phenotype value
High
Low
Low
High
The allele is associated with increased expression of the phenotype which will depend on the degree of exposure.
At low levels of exposure, expression of the phenotype is higher for genotype C (- - -). At lower levels of
exposure, expression of the phenotype is higher for genotype A (). Genotype B: ? ? ? ?.
112
Figures 14 provide an impression of possible relationships between genes and

environment. Asthma is a complex disease in which many different genes and multiple
environmental factors (endotoxins, air pollution, viral infections, bacterial infections,
food, pesticides, heavy metals, environmental tobacco smoke exposure, etc.) play a role.
Thus, given the myriad possible interactions between these genes and environmental
factors, it is still a simplification of one reality. Many clinicians feel that investigating
these relationships is like searching for a needle in a haystack or looking at a
mathematical model with an infinite number of variables.
The best defined phenotypes for asthma and atopy are BHR, lung function and IgE.
Figure 5 demonstrates the possible role of genes in the expression of phenotypes and the
interaction with the environment. Gene A has a direct effect on BHR without any
influence from the environment, while the environment affects the influence of gene B on
BHR, leading to increased BHR. Gene C has no effect on BHR and is also not influenced
by the environment, but is directly related to lung function. Gene D and E only initiate an
elevated IgE when both genotypes are present and are influenced by the environment.
A wide variety exists in phenotypes of genetically manipulated plants and of
invertebrates in which the genetic traits and environment can be fully controlled. This
effect is ascribed to phenotypic plasticity, which is the development of different
phenotypes for the same genotype in the same environments [52]. Studies in humans into
the relationships of asthma, allergies and environmental factors have investigated an
immense number of variables, making these studies very complex and, generally,
meaning they offer multi-interpretable results. Phenotypic plasticity is likely to add to
this complexity, especially in males.
In utero environment
It has long been assumed that the safest place for the child is the womb, since it
protects the genes from any environmental influence. However, it is becoming more and
more clear that many intra-uterine factors can play a role in the development of the
respiratory system and the evolution of the immune system. The target of a toxic insult to
the lungs during its development is likely to involve the disruption and/or alteration of a
specific molecular signal or transcription factor but, to date, little information is
available as to the precise effect of such exposures. An important aspect is timing of
exposure during development, which appears to be critical to its effects. For example,
Gene B
Gene C
Gene D
Gene E
Environmental
factors
Gene A
Phenotypes
BHR
Lung function
IgE
Normal IgE
Fig. 5. The possible role of genes in the expression of phenotypes and the interaction with the environment.
BHR: bronchial hyperreactivity; Ig: immunoglobulin.
113
J. GERRITSEN ET AL.
maternal malnutrition during gestation may significantly retard foetal growth and the
development of the lungs, leading to compromised lung function throughout life [53]. In
contrast, exposure to environmental toxins, such as passive cigarette smoke, may actually
accelerate the maturation of specific cell types in the foetal lung [54, 55]. The results of
such an effect on overall lung-function changes from the newborn to the adult age are
unknown. In general, very little is known regarding the precise effects of maternal
personal exposure, such as vitamin intake, smoking and nutritional factors, air pollution,
viral infections, etc. on the foetus. It is likely that the exposure affects growth changes of
the respiratory system which may continue after birth. A limitation in the research of prenatal effects on the development of the respiratory system is that, for example, exact lung
function measurements can only be reliable and performed on a large scale from y2
6 yrs of age, depending on the method of lung-function measurement used.
Consequently, pre-natal and post-natal effects are difficult to disentangle. Nevertheless,
it has been suggested that changes of the respiratory system later in life are already
measurable shortly after birth [56].
Children from mothers with asthma have a greater risk of asthma compared with
children from fathers with asthma, which refers to the importance of the pre-natal
environment on subsequent risks. This "parent-of-origin" effect, in which an allele is
associated with asthma or atopy only when it is inherited from the mother, has been
confirmed in several studies [57, 58]. A study carried out in 200 Dutch families showed
that the influence of susceptibility genes for asthma might become apparent with
exposure to cigarette smoke only in utero and early childhood [59].
The studies mentioned previously provide strong circumstantial, though not
physiological, evidence that in utero and early childhood exposure may contribute to
disease development early or later in life in interaction with genetic factors.
Sex as an endogeneous factor

The influences of maternal and paternal history of atopy and asthma on asthma in the
offspring differ, as already stated. In addition, studies on cord-blood IgE show that the
influence of maternal history of atopy or asthma is stronger in young males than in
young females, suggesting that hormonal factors in the offspring may modify the effects
of maternal or paternal inheritance [60]. During childhood and adolescence, young males
are nearly twice as likely as young females to develop asthma and this continues until the
age ofy14 yrs [61, 62]. A change to female predominance occurs during late adolescence
in females, which exists throughout adulthood, and asthma tends to be more severe in
female adults [6365]. The exact mechanisms of these differences on the molecular and
genetic levels are unravelled. Before puberty, no differences are observed in the
production of sex hormones; changes during puberty lead to the typical differences
between males and females. From the onset, the airways of females are smaller than
males. From this perspective it might be expected that young females have more
respiratory symptoms than young males. However, this is in contrast with what has
previously been found in young males and females. Therefore, it is evident that the
differences in respiratory symptoms, allergy and asthma between young males and
females cannot be accredited to anatomical differences in the respiratory system. An
explanation for this shift might be the presence of important genes on the x-chromosome,
which are switched off during childhood and switched on during puberty and adulthood.
During the switch-off phase in childhood, the influence of the genes is minimal and
makes the immune system behave similarly in young males and females. During puberty
and adulthood, the genes on the extra x-chromosome are switched on, which may induce
a predominant influence on the disease expression and severity of asthma. Whether these
114
genes on the x-chromosome are directly responsible for this sex change or whether they
act as modifier genes in combination with switched on mechanisms of the hormone
chromosomes is not clear.
Given the two alleles of the x-chromosome in young females and one in young males, it
is interesting to investigate whether specific single nucleotide polymorphisms in genes
which are located on the x-chromosome are associated with asthma development and/or
severity in young females or adult females. Candidate genes on the x-chromosome with a
possible linkage to asthma and atopy are toll-like receptor (TLR)-7 and TLR-8, both of
which are located at Xp22.3Xp22.2, playing a role in both innate and adaptive
responses. TLR-7 receptors are mainly expressed in the lung and placenta, whereas TLR8 receptors are mainly expressed in the lung and peripheral leukocytes [66]. Viral
products may activate TLR-7 or may generate a ligand that interacts with TLR-7,
besides T-regulatory cells which express TLR-7 and -8 [67]. Other genes are IL-13
receptor a1 (IL-13a1) and IL-13a2, which are located at XpterXqter and Xq13.1Xq28,
respectively. The receptor for IL-13 is composed of IL-13a1 and one of the forms of the
IL-4 receptor on chromosome 5q [68, 69]. IL-13 is secreted from CD4z T-cells, mast
cells, basophils and eosinophils. It is a central mediator of allergen-induced airway
hyperresponsiveness and is associated with elevated serum IgE levels [70, 71]. A
noncoding variant of IL-13R-a1 is associated with high IgE levels, particularly in males,
suggesting an x-linked inheritance of high IgE levels [7274]. Another important gene
located on chromosome Xq13.221.1 is cysteinyl-leukotriene receptor 1 (cysLT1), which,
via leukotriene (LT)C4, LTD4 and LTE4, plays a role in mediating human asthma and
activating of at least the two receptors cysLT1 and cysLT2. Activation of these receptors
induces many of the relevant biological effects in the pathophysiology of asthma [75, 76].
These genes on the x-chromosome have a direct relationship with asthma and allergy.
The exact functions of these genes and how they interplay with asthma genes (genegene
interaction) and whether other genes on the x-chromosome play a role in the sex-related
differences in disease expression have still to be elucidated.
Conclusions and future perspectives

It is evident that the environment plays a pivotal role in the development and severity
of asthma and allergy. The exact role of the environment in disease development and
progression still has to be unravelled. This is relevant since it offers opportunities for
early and life-long intervention. Furthermore, knowledge about the genetics of these
diseases and the interplay with the environment is essential. However, since the genetics
of asthma and allergy as polygenetic diseases is extremely complex, the discovered genes
only partly shed light on the risks of development and progression of diseases. Thus,
research in geneenvironment interaction may still be a matter of trying to find exits
within this labyrinth. Genome-wide screens are very expensive and the signals, although
interesting, so far do not provide the solution to solving the puzzle as to why some
individuals develop allergy and asthma and others do not. Other more costly and
intensive ways to approach this issue are fine-mapping strategies of chromosomal
regions, leading to genome-wide association mapping. These require large cohorts due to
the multiple genetic and environmental factors involved. Recent collaborative studies of
different genetic centres involved in large (birth) cohorts, such as in GABRIEL,
extensively increases the power. The expectations are that this will provide new and
important information. Another approach is to perform comparative studies translating
findings in animals, for example mice, to humans. Furthermore, the introduction of
micro-arrays or DNA-chip technology offers the opportunity to carry out highthroughput analysis of biological systems to investigate a high number of genes at one
115
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time, thus allowing a geneticalgenomics approach towards identification of genes in

asthma and its related phenotypes [77].
It can be expected that with the development of these techniques and the possibilities
of advanced analysis, the links between asthma and allergy genes, environmental factors
and the development of asthma will be unravelled in the future.
Summary
The exact genetic aetiology of asthma is complex. The reasons are that in asthma more
than one gene is involved, there is interaction between genetic (host) and
environmental factors, and there is wide heterogeneity of asthma phenotypes. The
present chapter discusses genegene interaction, geneenvironment interaction, the
influence of the in utero environment, and the role of sex. The overall conclusion is that
the environment plays a pivotal role in the development and severity of asthma and
allergy, although the role of the environment in disease development and progression
is still to be unravelled. Collaborative studies of different genetic centres with a large
number of subjects are needed to extensively increase the power. However, new
techniques offer the opportunity to identify genes in asthma and the related
phenotypes. With this approach it can be expected that the links between asthma, and
allergy genes, environmental factors will be uncovered.
Keywords: Allergy, asthma, geneenvironment interaction, genegene interaction.
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119
CHAPTER 9
Clinically relevant early functional and

diagnostic markers of lung disease in
children
J.C. de Jongste*, E. Baraldi #, E. Lombardi }
Depts of Paediatrics *Erasmus University Medical Center, Sophia Childrens Hospital, Rotterdam, The
Netherlands. #University of Padova, Padova, and }Anna Meyer Childrens University Hospital, Florence,
Italy.
Correspondence: J.C. de Jongste, Dept of Paediatrics, Erasmus University Medical Center, Sophia
Childrens Hospital, PO Box 2060, 3000 CB Rotterdam, The Netherlands. Fax: 31 104636811; E-mail:
j.c.dejongste@erasmusmc.nl
Respiratory symptoms are extremely common in young children. Most children

presenting with cough, wheeze, shortness of breath or other symptoms have benign
conditions, including recurrent viral infections and mild asthma, while others have more
serious underlying disorders. To identify the children whose symptoms are due to an
underlying disease and to separate these from the large group of children with benign,
self-limiting symptoms, there is a need for diagnostic tests that can be applied in clinical
practise over a wide age range, and give results that are relevant to the individual child. In
the present chapter, the authors present a brief overview of tests that are relevant to the
detection of lung disease in children, including conventional lung function tests,
techniques to study markers in exhaled air, disease markers in blood or urine and new
imaging techniques.
Fractional concentration of nitric oxide in exhaled air

Since the 1990s, the exhaled nitric oxide fraction (Fe,NO) has been extensively studied
and validated as a noninvasive marker of airway inflammation in asthma, and has been
standardised for use in clinical practice [1]. There are now detailed guidelines for Fe,NO
measurement in children and adults [1, 2]. Normative values have been published for
children (fig. 1) [3]. It is now well established that Fe,NO is the first bedside test that
reflects eosinophilic airway inflammation in the bronchial mucosa.
In older children, the preferred measurement technique requires a single breath,
constant low-flow exhalation against a resistance in order to avoid contamination with
nasal air. The airflow is sampled and fed directly into the analyser. Results are
immediately available. The chemiluminescence analysers for nitric oxide (NO)
measurement are expensive and technically complicated, and are mainly used in
academic research centres. Recently, compact hand-held analysers have been developed
which are much less expensive and will facilitate more widespread introduction of the
method.
To date, the main utility for Fe,NO is in clinical asthma management. Allergic
asthmatics have high Fe,NO that shows a rapid, dose-dependent response to
corticosteroids. Treatment decisions in asthma have traditionally been made on the
ISSN 1025-448x.
120
MARKERS OF LUNG DISEASE IN CHILDREN
40.00
35.00
Fe,NO ppb
30.00
25.00
20.00
15.00
10.00
5.00
0.00
10
Age yrs
15
20
Fig. 1. Normal values for exhaled nitric oxide fraction (Fe,NO) in children, measured with the on-line single
breath method and a flow of 50 mL?s-1. : mean and upper 95% Fe,NO level (n=405); : mean and
upper 95% Fe,NO level without outliers (n=389); -------: mean and upper 95% Fe,NO level without outliers and
"atopics" (n=332). Reproduced from [3] with permission.
basis of symptoms, either with or without a measure of airway patency such as peak flow.
However, within an asthma population, both symptoms and airway obstruction do not
accurately reflect the presence and severity of airway inflammation. Fe,NO inflammometry can be used to identify a patient with eosinophilic airway inflammation.
Possible applications of Fe,NO include diagnosis of asthma, prediction of steroid
response, monitoring of steroid treatment and treatment compliance, steroid dose
titration, prediction of exacerbation or relapse, and screening for asthma.
A number of recent studies have indicated that Fe,NO is indeed useful in asthma
management. Smith et al. [4] performed a controlled study where Fe,NO was used to
downtitrate steroids in adult asthmatics. The results showed that at the end, the Fe,NO
group used a significant 45% lower steroid dose than the control group, but, nevertheless,
had at least the same level of asthma control by all other end-points. Pijnenburg et al. [5]
performed a paediatric study, where Fe,NO guided the steroid dosing. In this study, the
Fe,NO group showed a significant improvement of bronchial hyperresponsiveness (fig. 2),
and less severe exacerbations than the control group, without the need for more steroids.
Recent studies have described that Fe,NO predicts loss of asthma control or relapse after
tapering the dose, or after stopping steroids (fig. 3) [6]. Another study found Fe,NO levels
to be a good predictor of a clinical response to inhaled steroids in steroid-naive adults
and children with chronic respiratory symptoms not typical for asthma [4]. These studies
show the feasibility of Fe,NO measurement in paediatric and adult clinical practise, and
are suggestive of a significant benefit of monitoring of Fe,NO in asthmatic subjects.
A diagnostic application for Fe,NO in clinical practise is primary ciliary dyskinesia
(PCD). In this rare syndrome, chronic airway infection and bronchiectasis develop as a
consequence of a genetic defect leading to reduced ciliary function and impaired
mucociliary clearance. Children with PCD have abnormally low Fe,NO values, with
minimal overlap with healthy controls. Low Fe,NO in children with chronic respiratory
infection should therefore alert for possible PCD and prompt for specific studies of
ciliary function. Nasal NO is even more discriminative and is now recommended as the
screening tool of choice for PCD.
121
J.C. DE JONGSTE ET AL.
1000
900
800
700
600
500
400
s
s
Mean PD20 methacholine g
2000
300
200
100
Start
12 months
Start
Visit
12 months
Fig. 2. Response to steroids in children with exhaled nitric oxide fraction management ($) and control group
(() at start of study and 12 months later. PD20: provocative dose of methacholine causing a 20% fall in forced
expiratory volume in one second. Reproduced from [5] with permission.
120
s
s
100
Fe,NO ppb
80
s
60
40
20
0
s
s
l
l
-2
s
s
l
l
s
l
l
l
l
l
l
12
Time weeks
l
l
24
Fig. 3. Individual follow-up of exhaled nitric oxide fraction (Fe,NO) in asthmatic children after discontinuation
of inhaled steroids at t=0 because of clinical remission. Those children who developed a relapse show a steep
increase in Fe,NO ahead of symptoms; stable children had nitric oxide values that remained low. +: relapse after
36 days; ': relapse after 35 days; #: individual who remained asymptomatic; $: individual who remained
asymptomatic. Individual nitric oxide values of four children are shown, two without relapse ($ and #) and
two with relapse (+ and '). Data were obtained from [6].
Fe,NO in young children

Wheezing in infants is common. Some young children wheeze with infection only
during the first years of life and do not subsequently develop asthma; however, some may
have early childhood asthma. Clinically, it is difficult and often impossible to identify
those infants who are more likely to become asthmatics. For these reasons the need for
122
developing practical, noninvasive markers to reflect asthmatic airway inflammation is

especially important in young children who wheeze, in whom other objective diagnostic
tools, such as spirometry or bronchial challenges, cannot be easily applied in clinical
practice [2]. There are only a limited number of reports on the use of Fe,NO in infants and
preschool children with asthma [715]. Recently, Malmberg et al. [11] demonstrated that
Fe,NO is superior to lung function and bronchodilator responsiveness in identifying
preschool children with asthma. In agreement with this study, Avital et al. [9] showed
that Fe,NO can differentiate young children with asthma from nonasthmatic children with
chronic cough. In children with recurrent wheeze, raised Fe,NO values suggest the
presence of eosinophilic airway inflammation and these patients may, therefore, be most
likely to respond to treatment with inhaled corticosteroids (ICS). Recently, Moeller
et al. [16] have demonstrated that moderate doses of ICS reduce levels of Fe,NO in the
absence of significant changes in lung function and symptoms. Similar findings with a
reduction of Fe,NO have been reported in infants after therapy with montelukast [14]. In a
large epidemiological survey, Brussee et al. [12] found that 4-yr-old children with
symptoms of asthma and sensitisation had a higher Fe,NO than healthy children, but the
difference was much smaller than in older children, which would limit the applicability in
individual children. Moreover, high-risk children from allergic parents had similar Fe,NO
values to children from nonallergic parents, suggesting that Fe,NO values may have a
limited potential as a predictor of sensitisation in 4-yr-old children.
Methods for measuring Fe,NO in infants and preschool children

In 2002, a joint European Respiratory Society (ERS)/American Thoracic Society
(ATS) task force on exhaled NO measurement in children published a statement
providing recommendations and suggestions for the measurement of Fe,NO in young
children [2].
On-line measurement of Fe,NO during spontaneous breathing has been applied in
children aged 25 yrs [7]. Fe,NO is measured on-line during spontaneous breathing and
the exhalation flow is manually adjusted at 50 mL?s-1 by changing the exhalation
resistance. The method still requires passive cooperation inasmuch as the child needs to
breathe slowly and regularly through a mouthpiece.
Measurements during tidal breathing with uncontrolled flow are technically easier and
therefore attractive. The tidal breathing method in infants is potentially simple and
noninvasive and both on- and off-line techniques have been applied without the use of
sedatives [89, 17, 18]. Currently, there is no standardised tidal breathing method to
recommend for use in infants and young children and more research is needed to solve
some methodological issues [19].
As Fe,NO is flow-dependent, a scatter of data due to variation in expiratory flows is
possible. The disadvantage of mixed expiratory air is that it may be contaminated with
ambient NO and NO from the upper airways. Whilst the inspiratory NO contamination
can be limited by inhalation of NO-free air, the use of a two-compartment face mask can
limit nasal contamination [20, 21]. There is limited experience with single-breath methods
for measuring Fe,NO in infants. A modification of the raised-volume rapid thoracoabdominal compression technique has been used to measure Fe,NO during a single, slow
forced exhalation [10]. NO levels are measured on-line; the plateau of NO achieved
during constant expiratory flow is then determined [10, 21]. With this technique, it is
important to use a two-compartment face mask to separate nasal and oral compartment
[21]. This method is limited in that sedation, specialised equipment and skilled operators
are needed.
123
Nasal nitric oxide

NO is present in the nasal cavity in much higher concentrations compared with the
lower airways, and nasal NO (nNO) is affected by inflammation of the upper airways.
The possible use of nNO measurements in the diagnosis and treatment of upper airway
disease still needs to be further evaluated because of the variable and inconsistent
findings until now, with two exceptions: 1) PCD, and 2) cystic fibrosis (CF). It is well
known that the nNO levels in these diseases are reduced, independent of measurement
method, and nNO is now recommended as a first-line screening tool for PCD [22, 23].
Markers in exhaled air: other substances

Apart from NO, an increasing number of gas phase compounds have been identified in
exhaled air, including carbon monoxide and ethane. These may reflect oxidative stress
and lipid metabolism, and there is some evidence that they relate to airway inflammation
in adults with asthma, chronic obstructive pulmonary disease (COPD) and CF [24]. The
detection of components in exhaled air has been facilitated by the use of mass
spectrometry, by which minute amounts of volatile molecules can be identified in
relatively small samples. Using mass spectrometry, large numbers of different volatiles
have been identified and quantified in exhaled air samples [25]. It seems possible that such
analysis may enable simultaneous analysis of a spectrum of markers that may
differentiate between different types of airway inflammation. However, no meaningful
results have as yet been published for paediatric populations. Future studies will have to
address the reproducibility and biological validity of any new gas phase marker, and its
possible use in relation to airways diseases.
Exhaled breath condensate

In the past years there has been increasing interest in measuring exhaled breath
condensate (EBC) compounds in subjects with pulmonary diseases [2628]. Exhaled
breath obtained through the cooling of exhaled air contains water vapour and microdroplets whose composition appears to reflect airway lining fluid [27]. This fluid contains
various nonvolatile and over 200 volatile substances. EBC consists of w99% of water
generated by the respiratory tract. A much smaller fraction is derived from respiratory
droplets released from the airway surfaces and subsequently incorporated in the water
deposited in the condenser. The condensate does not, however, contain inflammatory
cells of the airways. EBC collection is totally noninvasive and is therefore particularly
easy to perform in children, including those with severe disease [29]. Unfortunately
limited information is available concerning its use in preschool children [30].
Recently, an ATS/ERS task force has developed guidelines on standardisation and
analysis of EBC [31]. The principle of sampling the airways by EBC is that mediators from
airways are released from the airway lining fluid, carried up by exhaled breath and
subsequently collected by condensation of the exhalate [2628, 32]. In order to collect EBC,
children are asked to breathe tidally for 1015 min. The use of noseclips is controversial
because of the possibility of nasal contamination. A saliva trap is recommended because
many relevant mediators may be present in large amounts in the saliva. By checking
amylase activity of EBC, saliva contamination can be excluded [31]. EBC can be collected
from children as young as 34 yrs of age using the same technique as used in adults. It is
possible to use a face mask, and EBC has been successfully collected from babies in this
124
way. Collection by continuous aspiration through a nasal cannula or from a mechanical

ventilator circuit can also be considered [30, 33]. Disadvantages of EBC analysis are that
most of the measurements are not on real time and concentrations of mediators in EBC are
close to the detection limit of current available assays which were developed for use in other
media (blood, urine) with higher mediator concentrations than condensate. To date the
majority of exhaled markers have been measured by immunoassay. More sensitive
techniques, such as high-performance liquid chromatography and gas chromatography/
mass spectrometry (GC/MS), should therefore be explored to validate or replace
commercially available immunoassay (table 1). Potentially important variables, which
may influence the composition of EBC, include minute ventilation, humidity of inspired
air, collection temperature, and nasal and salivary contamination [32].
Several markers of inflammation and lipid peroxidation have been detected in EBC of
asthmatic adult and children (table 2). A large number of mediators have been measured
in EBC, including hydrogen peroxide, isoprostanes, prostaglandins, leukotrienes,
nitrogen oxides, aldehydes, cytokines, etc., and new molecules continue to be added
to this list. In addition, the acidity of EBC can be measured [27].
Hydrogen peroxide
Oxidative stress contributes to the pathogenesis of several inflammatory lung diseases.
Hydrogen peroxide (H2O2) is a marker of oxidative stress and it is one of the more
extensively studied markers in asthma. H2O2 in EBC can be measured by colorimetric or
fluorimetric methods. Jobsis et al. [34] have defined reference values in healthy children.
H2O2 levels are related to the eosinophil differential counts in induced sputum and to
airway responsiveness [35].
Leukotrienes
Cysteinyl leukotrienes (cys-LTs) are inflammatory metabolites derived from
arachidonic acid through the 5-lipoxygenase pathway. They are potent airway
constrictors and pro-inflammatory mediators. LTs can be measured by enzyme-linked
immunoassay (EIA) and GC/MS in EBC [36]. Increased values of EBC cys-LTs have
been found in allergic asthmatic children despite corticosteroid treatment [37].
Interestingly, normal values of EBC cys-LTs were found in atopic nonasthmatic
children, suggesting that eicosanoids are involved in the pathogenesis of asthma [38].
Reduced cys-LTs values were reported after 3 months of house dust mite avoidance in
allergic asthmatic children [39].
Table 1. Exhaled breath condensate assays

Colorimetric or fluorimetric
Immunoassays
ELISA
Radioimmunoassay
Analytical techniques
Gas chromatography/mass spectrometry
High-performance/liquid chromatography
Ion chromatographic method
Liquid chromatography/tandem mass spectrometry
Future
Metabonomics, proteomics, infrared laser spectrometry
125
Table 2. List of potential inflammatory markers in exhaled breath

condensate
Compound class
Examples
Eicosanoids
8-Isoprostane
Cys-leukotrienes
Leukotriene B4
Prostaglandins
PGE2
PGF2a
Thromboxane
Hydrogen peroxide
Lipid peroxides
Malondialdehyde
a,b-Unsaturated aldehydes
Saturated aldehydes
Glutathione
Ammonia
NO products
Nitrites
Nitrates
Nitrotyrosine
Nitrosothiols
Cytokines
IL-1b
IL-2
IL-6
IL-8
Tumour necrosis factor
Proteins
PGE2: prostaglandin E2; PGF2a: prostaglandin F2a; NO: nitric oxide;

IL: interleukin.
Cytokines
Cytokines in EBC are usually quantified by EIA/ELISA kits. Several different
cytokines have been identified in EBC, although at very low levels, close to the lower limit
of detection. Increased level of interleukin (IL)-4 and decreased level of interferon (IFN)-c
were described in EBCs of asthmatic children [40].
Isoprostanes
Isoprostanes are mediators of oxidative stress [41]. They are relatively stable and
specific for lipid peroxidation, which makes them potentially reliable biomarkers.
Isoprostanes can be measured by EIA kits and GC/MS. Increased levels of 8-isoprostane
have been found in asthmatics despite treatment with ICS, suggesting that these drugs
may not be fully effective in reducing oxidative stress [37, 42]. 8-Isoprostane
concentration is also elevated in patients with COPD, interstitial lung disease and CF.
Aldehydes are products of lipid peroxidation found in EBC that seem to reflect
oxidant-induced damage of the airways. Elevated levels of malondialdehyde measured by
liquid chromatography-tandem mass spectrometry were recently detected in children
with asthma exacerbation [43].
Nitrotyrosine is a stable compound expressing involvement of NO-derived oxidants in
the lung. It can be measured with EIA, is increased in the EBC of asthmatic subjects and
is associated with worsening of asthma symptoms [44].
Glutathione is a protective antioxidant in the lung. Glutathione levels in condensate
have been measured by liquid chromatography with fluorescence detection. Reduced
126
concentrations have been found in children with acute asthma with respect to healthy
controls suggesting a deficiency of antioxidant capacity in asthma [43].
Acidity
Airway pH homeostasis is maintained by a balance of different buffer systems and the
production and release of acids and bases in the airways. Up to three log order decreases
in EBC pH have been described in acute asthma, suggesting that the simple measurement of
EBC pH could be used to study acidbase status in the airway of asthmatic patients [27].
Similar results have been found in children with stable asthma [45]. Furthermore, EBC pH
levels correlate with inflammatory cells in induced sputum, suggesting that EBC pH may
reflect ongoing inflammation [46]. Measurement of EBC pH is highly reproducible.
Condensate
The evidence suggests a potential role of EBC in the monitoring of airway
inflammation and oxidative stress. However, the lack of standardisation of EBC
collection and analysis is currently the primary limitation of this technique and is likely to
explain most of the variability of the results reported in the literature. In addition, longterm prospective studies correlating EBC findings with measures of disease control and
established measures of lung pathology (bronchoalveolar lavage (BAL) analysis, biopsy
histology) are necessary to demonstrate and validate the clinical relevance of EBCderived markers.
Markers in blood and urine

Several markers in blood and urine have been evaluated for prediction and diagnosis
of asthma, and for monitoring asthma and CF; studies on other respiratory diseases are
still lacking.
Immunoglobulin E
An association between total serum immunoglobulin E (IgE) during the first year of
life and subsequent allergic disease by the age of 2 yrs was first reported in 1975 [47].
Subsequent studies assessing cord serum IgE as a predictor for allergic disease and
asthma later in life, mostly up to 5 yrs of age, have shown conflicting results [48, 49]. A
recent study has shown that cord serum total IgE levels i0.5 kU?L-1 were significantly
associated with asthma 10 yrs of age, but not at 4 yrs of age, suggesting that high cord
serum IgE levels are predictive of late-onset asthma [50]. Since this association was also
present in children with no positive skin prick tests, these data suggest that the
correlation between cord serum IgE and subsequent asthma at 10 yrs of age is not
necessarily mediated by allergic sensitisation [50]. Several studies have shown that serum
IgE may predict allergic airway disease; however, wheezy infants and young children
come into remission more often if they are not sensitised than if they are [51, 52].
Furthermore, sensitisation to aero-allergens in asthmatic children is a risk factor for
increased disease severity [53]. A recent study in 4-yr-old children has shown that
increased IgE levels were significantly more prevalent among those with allergic disease
[54]. The sensitivity of the test could be increased by using the sum of specific IgE levels in
combination with the number of positive allergens [54].
127
Eosinophils and their products

Eosinophils and their products play an important role in allergic inflammation and
asthma. An eosinophil blood count i4% at 1 yr of age has been shown to be a risk factor
for persisting asthma at 13 yrs of age in wheezing children v6 yrs of age, and has been
included in a clinical index to define the risk of asthma in young children with recurrent
wheezing [55]. Eosinophil granule proteins, mainly eosinophil cationic protein (ECP) and
eosinophil protein X (EPX), have been measured in serum as indirect parameters of
eosinophil activity. In a recent study of 968 children aged 6 yrs, serum ECP levels were
found to be higher in children with current asthma and severe atopy, suggesting that
serum ECP assessment might be helpful in detecting persistent asthma [56]. However,
sensitivity and specificity are too low for diagnostic use in individual patients [57, 58], and
there is no additive value in detecting asthma compared with a family history of atopy
[58, 59]. Determination of serum eosinophil granule proteins may reflect the effects of
anti-inflammatory treatments on eosinophil activity [58]. The clinical use of these
measurements for assessment of asthma severity has not been validated [58, 60]. Since the
results depend on sampling procedures and are affected by circadian and seasonal
variations, they should be performed under standardised conditions [58, 60].
EPX, a toxic protein present in eosinophil granules, is released by activated
eosinophils. It is the only basic eosinophil protein that can be measured accurately in
urine (uEPX) [61]. uEPX can be regarded as a marker of eosinophil degranulation in vivo
[62]. uEPX levels in allergic asthmatic children were found to be significantly higher than
in healthy controls [6366]. uEPX levels were increased in symptomatic compared with
asymptomatic children with asthma, and were significantly elevated during acute asthma
exacerbations [6768]. Treatment with inhaled steroids reduced uEPX [66]. As would be
expected for an inflammation marker, the association between uEPX/c, where c is
[creatinine], and pulmonary function tests is either weak or absent [67, 69]. Others found
no correlation between uEPX/c and BAL cell counts in asthmatic patients [62].
Measuring uEPX in urine is a simple and attractive test that should be further explored
for monitoring eosinophilic airway inflammation in children.
Cytokines
Cytokines, such as IL-1, IL-4, IL-5, IL-6, IL-8, IFN-c, granulocyte-macrophage
colony-stimulating factor and tumour necrosis factor-a, have been measured in
peripheral blood in patients with asthma, CF and other respiratory disorders, as well
as in healthy controls [70]. IL-10 production by peripheral blood monocytes has been
shown to be reduced in subjects with atopic asthma [71] and increased in the convalescent
phase of respiratory syncytial virus infection in infants with subsequent recurrent
wheezing [72]. It has been proposed to be helpful in distinguishing atopic asthma from
other nonatopic wheezing conditions [73]. However, cytokine measurements for clinical
purposes are strongly limited by their poor sensitivity and the difficulty in interpreting
the results [74].
Pulmonary function tests

Pulmonary function tests (PFTs) play an important role in the diagnosis and
monitoring of paediatric lung disease [75]. Although many children with lung disease
may present with normal PFTs, the evidence of impaired ventilatory function and a
bronchodilator response may be very helpful for the diagnosis and severity assessment of
paediatric lung disease [76, 77]. A thorough description of paediatric PFTs has recently
128
been published in this series [78]. This section will focus on the clinically relevant aspects
of PFTs and bronchial challenge tests in diagnosing and managing lung disease in
children.
Pulmonary function tests in school children

In the "cooperative" child (i6 yrs old) spirometry is the most used PFT. Spirometry is
relatively simple to perform and is repeatable. In asthmatic children, spirometric
parameters detect airflow limitation and help to diagnose and monitor the disease [79].
Asthma guidelines thus recommend the performance of spirometry at the time of
diagnosis, after the treatment has been started (to document an improvement in lung
function), every time the treatment is changed and at regular intervals depending on the
severity of the disease [79, 80]. The forced expiratory volume in one second (FEV1) has
been shown to be the most reliable spirometry parameter and may be used to classify the
severity of airway disease [80]. However, in conditions with intermittent airway
obstruction, such as asthma, a normal spirometry does not exclude disease and a
bronchial challenge test can be considered. A recent study has demonstrated that, in 5
18-yr-old asthmatic children, FEV1 is generally normal and does not correlate inversely
with asthma severity classified by symptoms frequency and medication usage, whereas
the ratio of FEV1 to forced vital capacity (FEV1/FVC) declines as asthma severity
increases [81]. The mean forced expiratory flow between 25 and 75% of FVC (FEF2575%)
is more sensitive for peripheral airway obstruction than the other spirometric parameters
[82]. However, the high variability of FEF2575% in the general population limits its use
to detect airflow limitation in routine clinical practice [83]. Peak expiratory flow (PEF)
has been used to monitor lung function and help adjust asthma treatment. However, it
has been shown that daily PEF measurements in children are able to detect only about
one-third of the clinically important episodes of deterioration, while children may also
report false positive episodes of PEF decrease [84]. The measurement of PEF variability
in children is also difficult to interpret in clinical practice due to the high diurnal
variability in a high percentage of normal subjects [85]. For these reasons, the use of
portable home spirometers (capable of measuring FEV1, FVC, FEV1/FVC and FEF2575%)
has been proposed [82, 86]. Nevertheless, the use of PEF meters is still recommended, along
with that of (mini)spirometers, in asthma guidelines as an objective measure of lung
function [75, 7980]. Several studies have shown a relationship between the level of
pulmonary function in childhood and subsequent lung function and respiratory
symptoms in adulthood. FEV1 as percentage of predicted (FEV1 % pred) in children has
been found to predict adult FEV1 [87]. In a recent study, a higher FEV1 in childhood and
more improvement in FEV1 from the ages of 514 yrs to 2133 yrs were associated with
both complete and clinical asthma remission at ages 3242 yrs [88]. The same study
showed that 57% of subjects in clinical remission had bronchial hyperresponsiveness and/
or a low lung function at age 3242 yrs, supporting the view that defining remission only
on the basis of symptoms and need for treatment will overlook subjects with subclinically
active disease and possibly associated airway remodelling [88].
Pulmonary function tests in preschool children

The poor cooperation of children v6 yrs of age in performing standard PFTs has
limited lung function evaluation, especially in preschool children (36 yrs old). Children
in this age group are too old to be sedated for infant PFTs (see below) and too young to
perform the manoeuvres required in the PFTs for school-age children. Recently, several
techniques that only require passive cooperation have become commercially available.
129
These techniques are particularly suitable for lung function assessment in preschool
awake children. Unfortunately, there is still a lack of standardisation for most of these
techniques. The ERS/ATS Joint Group for Pulmonary Function Testing in Infants and
Young Children is currently working to produce international recommendations for
most of the techniques applicable in preschool children. Also, the relative role of each
available pulmonary function technique in the clinical management of lung disease in
preschool children still remains to be established.
Interrupter resistance
The interrupter resistance (Rint) is a noninvasive method for measurement of airflow
resistance during tidal breathing; it uses an interrupter system to measure flow and
pressure at the mouth (fig. 4). Its main assumption is that, during a sudden and transient
interruption of the tidal airflow, alveolar pressure and mouth pressure equilibrate within
a few milliseconds [89]. If flow is measured immediately before interruption, the ratio of
flow to pressure changes gives the Rint [90]. The feasibility of the interrupter technique in
preschool children ranges between 79 and 98% [91, 92]. The short- and long-term
repeatability of Rint is known, and reference values have been published for the
interrupter technique in preschool children [9198]. Most of the reference values were
collected in the field, showing that the interrupter technique is suitable for
epidemiological studies [13]. Studies evaluating Rint changes in response to bronchodilator treatment have shown that Rint is able to detect changes in airway calibre
after bronchodilator in preschool children [99, 100]. However, the definition of a cut-off
value for a clinically significant decrease in Rint in response to bronchodilator
inhalation and the role of Rint in challenge tests remain to be established. The good
repeatability and feasibility of Rint measurements, as well as the agreement with other
PFTs [101, 102], and the applicability over a wide age range make the Rint attractive for
the assessment of lung function in preschool children both in research and clinical
practice [103, 104].
Fig. 4. Measurement conditions of the interrupter technique in preschool children.
130
Forced oscillation technique

The forced oscillation technique (FOT) is another method to assess respiratory
mechanics. Like the interrupter technique, FOT is performed at tidal breathing.
Recommendations for its use have recently been published [105]. The principle of FOT is
that an external pressure (forced oscillations) applied at the upper airways will cause a
mechanical response of the respiratory system (changes in airflow and pressure) that can
be measured to determine respiratory impedance with its two components, resistance
(Rrs) and reactance (Xrs). The frequencies of flow oscillations, generated by a
loudspeaker, usually vary between 4 and 32 Hz. In children, Rrs is frequency dependent,
with higher Rrs at lower frequencies [105, 106]. From clinical studies, it appears that Rrs
at low frequencies (68 Hz) allows the best discrimination between healthy subjects and
various obstructive conditions [107]. The feasibility of FOT Rrs in children ranges
between 79 and 95% [91, 108]; the reproducibility in children and adults is similar. Rrs and Xrs
are also useful indices in establishing positive reactions to bronchial challenge tests [107,
109]. Normative data on reference values and bronchodilator response in healthy and
asthmatic subjects were reported for preschool children [91, 95, 108, 110112].
Whole body plethysmography

Whole body plethysmography used to be an unsuitable technique for most preschool
children. Dab and Alexander [113] proposed a simplified, one-step method to measure
specific airway resistance (sRaw) using body plethysmography. This method has the
advantage of not requiring thoracic gas volume measurements, thus avoiding the need to
breathe against a closed shutter. Recently, further adaptations were made to the
technique, making it more acceptable for preschool children [91, 114]. Although
the measurement of plethysmographic sRaw has not yet been standardised, and the
equipment is expensive and cumbersome, clinical studies [114, 115], as well as the
availability of reference values [91], illustrate its potential usefulness as a clinical and
research tool.
Multiple breath washout

Multiple breath washout (MBW) was described in 1953 for assessing lung volume and
measuring overall ventilation inhomogeneity during tidal breathing [116]. The technique
used in the first description was nitrogen washout using 100% oxygen. In the subsequent
years, inert nonresident gases were introduced (helium and sulphur hexafluoride) and
reference values have been reported for functional residual capacity using helium dilution
in preschool children [117]. In relatively recent years, the analysis of ventilation
inhomogeneities has been improved and several indices reflecting overall ventilation
inhomogeneity, and hence peripheral airway disease, have been described. The most
commonly used are the lung clearance index (LCI, the number of lung volumes required
to complete the washout) and the mixing ratio (MR, the ratio between the actual and the
ideal number of breaths needed to complete the washout) [118]. Two recent papers have
compared spirometry and plethysmography findings with LCI and MR using MBW with
4% helium and 4% sulphur hexafluoride in preschool and school children with CF and in
healthy subjects [118, 119]. Abnormal ventilation distribution was present in the majority
of children with CF, including young children with normal spirometry or plethysmography measurements. These results suggest that MBW is more sensitive than other PFTs
in detecting early lung disease in children with CF. Although much more work needs to
be done before this technique can be implemented for routine use in clinical practice, the
131
results reported so far suggest that MBW is promising for detecting early lung disease in
preschool children.
Spirometry
Spirometry has also been performed in preschool children. Several studies show that,
under specific conditions, its feasibility in preschool children ranges between 47 and 92%
[120, 121] and can be improved by the use of incentive software [120]. Recommendations
for spirometry in preschool children have recently been published [122]. Furthermore,
reference values have been reported for spirometry in preschool children [123, 124] and
clinical data on the usefulness of spirometry in preschool children with CF have been
published [125].
Bronchial challenge tests

Since bronchial hyperreactivity (BHR) is one of the characteristic features of asthma
[80], its presence is often helpful in the diagnostic process. The main pathological factors
that underlie BHR are presumably airway inflammation and bronchial remodelling [126].
However, the relationship between BHR and asthma is complex in children. While
asthmatic children have BHR, BHR is not the same as asthma [127]. BHR has been
reported in patients with CF or allergic rhinitis and in 733% of asymptomatic children
[128]. In addition, there is no close correlation between airway inflammation markers in
induced sputum and methacholine BHR in asymptomatic children [129]. However, most
children with recurrent wheezing have BHR [127, 128].
These conflicting findings can be partially explained by the kind of stimulus used to
assess BHR. Bronchial stimuli are generally described as either "direct" or "indirect".
Direct stimuli include methacholine, carbachol, histamine and arachidonic acid
metabolites; they cause bronchoconstriction by directly activating contraction of
bronchial smooth muscle cells after binding to their relevant receptors [125]. Indirect
stimuli include exercise, adenosine, cold air, hypertonic solutions and ultrasonically
nebulised distilled water. Their effect is considered to be mainly due to the release of
mediators from intermediary cells (mainly mast cells) [130]. Since the 1990s, an increasing
body of evidence has shown that indirect stimuli have a better correlation with airway
inflammation than direct stimuli [130, 131]. This helps improve current understanding of
why indirect stimuli are reported to be more specific and less sensitive for a diagnosis of
asthma, while direct stimuli have proven to be more sensitive and less specific. Bronchial
challenge tests are not necessary to diagnose asthma when PFTs show reversibility after
bronchodilator inhalation or when the clinical picture is highly suggestive for asthma.
Demonstrating or ruling out BHR may be important in difficult cases. The choice of
bronchial stimulus to be used depends, as always, on the question to be answered. A
direct challenge will be very helpful in ruling out asthma (when negative), while it will not
be able to confirm it (when positive) [126]. Conversely, an indirect challenge will be more
helpful in confirming a diagnosis of asthma (when positive) than ruling it out (when
negative) [126]. It has been proposed to perform serial BHR measurements to monitor
anti-inflammatory therapy in adult asthmatics [132]. However, the benefits of this
strategy, fewer exacerbations, were at the cost of a higher steroid dose. Guidelines for
methacholine and exercise challenge tests [133] and for indirect challenge tests [130] have
been published.
Studies on whether BHR can be a predictor of asthma in asymptomatic children has so
far produced conflicting answers [134, 135]. Cold air challenge at 6 yrs of age was a
significant predictor of a low level of lung function at 16 yrs of age [136]. Studies
132
attempting to investigate whether BHR in children with isolated cough is associated with
subsequent asthma have reported conflicting results. A positive methacholine challenge
test in 74 children and young adults with cough was not able to predict subsequent
asthma [137]. A more recent study, however, concludes that methacholine BHR in 113yr-old children with isolated chronic cough is a strong risk factor for the development of
asthma 10 yrs later [138].
Infant pulmonary function tests

The lung function techniques that can be used for infants have recently been reviewed
[78]. In general, sedation is required for infant pulmonary function tests (IPFTs), as the
infants cannot actively cooperate. The various IPFT techniques are limited to specialised
paediatric lung function laboratories in academic clinics, as they require complicated and
expensive, often custom-built equipment and dedicated, trained personnel to obtain
reliable results. A growing number of clinical studies employing IPFTs have been
published, and results of IPFTs have proven useful in understanding the epidemiology of
infant lung disease, to document the nature and extent of lung involvement in various
diseases, to assess treatment effects and to follow normal and pathological lung
development. In all of these fields, the impact on diagnosis and management on the
individual level has remained limited. Tests that can be applied to infants include
plethysmography, flow-volume measurements during tidal breathing and forced
expiration (rapid thoraco-abdominal compression or "squeeze"), with or without prior
inflation of the lungs towards near-total lung capacity, interrupter resistance and
compliance, and gas-mixing techniques to assess ventilation homogeneity, as described
above. Also, exhaled air can be analysed for inflammatory markers, including exhaled
nitric oxide, although the methodology has not been standardised [2].
To date, IPFTs have been important as clinical and epidemiological research tools.
Their value as diagnostic or monitoring tests in routine clinical practise is limited by the
demanding methodology and need for sedation, which preclude frequent routine clinical
use of current IPFT techniques.
Imaging techniques
Lung function tests are relatively insensitive to detect localised damage to the lungs
and airways [139]. Imaging by means of computed tomography (CT) is superior to lung
function for the assessment of progression of lung disease in CF [140, 141]. Standardised
CT scores have been developed and routine monitoring of CF lung disease by means of
CT scanning has become clinical routine in several CF centres. The radiation dose
associated with regular CT scanning is still a concern, and in a worst case scenario may
cause a small increased risk of cancer, depending on the assumptions [141]. Development
of new magnetic resonance imaging (MRI) techniques to replace CT scanning would
solve the problem of radiation dose, and MRI is a promising alternative for the future.
However, the quality of MRI images of the lung is still far inferior to that of CT, but this
could be improved by using specific techniques such as the use of inhaled gases to
enhance contrast. Until now, imaging techniques have not played an important part in
diagnosis and monitoring of prevalent lung diseases. No specific radiological
abnormalities have been found in asthma, although airway wall thickness has been
shown to be increased on CT, and was to some extent associated with disease severity
[142].
133
Important future questions

The study results of Fe,NO in diagnosing and monitoring of asthma as described above
critically depend on the cut-off levels of Fe,NO and symptom scores, and it is unclear what
the effect of other cut-off levels or alternative dosing schedules would have been. Other
ways of dealing with Fe,NO are still unexplored, for instance the use of personal-best
values, or the effect of more frequent monitoring. Fe,NO reflects not only inflammation,
but also the direct effects of steroids and viral infections. This raises the question as to
whether steroid downtitration is the proper response to a reduction of Fe,NO. Doubling
the steroid dose has only limited effect on elevated Fe,NO in asthmatic children with
elevated Fe,NO, despite conventional doses of inhaled steroids, and the mechanism
behind this observation is not clear [5]. It is possible that monitoring of Fe,NO may be
more useful for tapering than for stepping up steroids. Individual Fe,NO data may
be puzzling and seem to suggest heterogeneity in the Fe,NO response to steroids. This may
be due to faulty inhaler techniques, but there may also be genetic heterogeneity.
Other exhaled markers of airway disease, including all those in breath condensate, are
still in a very preliminary stage; clearly, standardised methodology, issues of
reproducibility and biological validity and prospective evaluation is needed for
condensate markers of interest. New techniques to assess condensate components,
including mass spectrometry and gas chromatography, hold promise for the future
development of this interesting area.
The exact role of the various PFTs in the clinical management of children with lung
disease remains to be determined, both in infants [143] and in older children [144, 145]. In
addition, there is a need for more feasible tests of infant lung function that can be applied
without the need for sedation.
Clearly, reliable prediction of asthma is an important issue with potentially great
implications. Combinations of lung function tests and inflammation markers, together
with genetic information and knowledge of exposures may well turn out to be reliable
predictors of future chronic illness and need to be explored. Not covered in this chapter
are genetic studies, which hold great promise for the identification of children at risk for
certain lung diseases, and have already been shown to be capable of diagnosing asthma
with high accuracy based on patterns of gene activation, as shown by gene array chips.
Several questions regarding the role of bronchial challenge tests need to be answered.
The merits of BHR in young children in whom cut-off values are unclear need to be
established. Cut-off values in adults (for methacholine challenge, usually a provocative
concentration causing a 20% fall in FEV1 of 8.0 mg?mL-1) have also been used in
children without any dose adjustment [95]. It has been pointed out that this practice is
likely to be inappropriate in young children [95], since smaller children would receive a
higher dose relative to their lung size, thus helping to explain the reported higher BHR in
younger children. Indeed, lower doses and different levels of response were found to be
more appropriate for young children [138]. Several studies have assessed bronchial
reactivity in preschool children [13, 34, 81, 101, 121, 134, 146], however, due to the lack of
data on the bronchial response to inhaled stimuli in healthy preschool children, the use of
bronchial challenge tests in this age group remains at present a research tool.
Imaging techniques should be further developed and explored for their potential as
diagnostic, prognostic and monitoring tools in paediatric lung disease. It may be possible
to overcome the present limitations of MRI for this purpose in order to limit the
radiation dose associated with more sophisticated radiographic techniques, such as highresolution CT and volumetric CT.
134
Summary
A brief overview of tests that are relevant to the detection of lung disease in children is
presented in this chapter. Exhaled nitric oxide (NO) is a noninvasive and well-validated
marker of eosinophilic airway inflammation and is useful in asthma diagnosis and
management. Elevated exhaled NO fraction is characteristic for atopic asthma
and responds dose-dependently to steroid treatment. Nasal NO is a highly specific
and sensitive screening test for primary ciliary dyskinesia. Exhaled breath condensate
(EBC) may in part reflect the composition of airway lining fluid. The lack of
standardisation of EBC collection and analysis is currently the primary limitation of this
technique and is likely to explain most of the variability of the results. Eosinophils and
their products play an important role in allergic inflammation and asthma. However,
serum or urinary eosinophil cationic protein and eosinophil protein X are too variable
for diagnostic use in individual patients. Pulmonary function tests play an important
role in the diagnosis and monitoring of paediatric lung disease. Bronchial challenge tests
with spasmogens (methacholine) may be helpful in ruling out asthma when negative, but
are not diagnostic if positive. The value of infant lung function tests as diagnostic or
monitoring tools in routine clinical practice is limited by the demanding methodology
and need for sedation. Computed tomography (CT) is superior to lung function for the
assessment of progression of lung disease in cystic fibrosis. Development of new
magnetic resonance imaging techniques to replace CT scanning would solve the problem
of radiation dose, and is a promising alternative for the future.
Keywords: Asthma, cystic fibrosis, eosinophilic inflammation, exhaled breath condensate, exhaled nitric oxide, markers of inflammation.
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141
CHAPTER 10
Clinically relevant early functional and

diagnostic markers of lung disease in the
paediatric intensive care unit
A. Schibler *, J.J. Pillow #,}
*Queensland Paediatric Intensive Care Service, Brisbane, Queensland, #Telethon Institute for Child
Health Research and Centre for Child Health Research, and }School of Womens and Infants Health,
University of Western Australia, Perth, Australia.
Correspondence: A. Schibler, Queensland Paediatric Intensive Care Service, Paediatric Intensive Care
Unit, Mater Childrens Hospital, South Brisbane 41010 QLD, Australia. Fax: 41 738401642; E-mail:
andreas_schibler@mater.org.au
Respiratory care in paediatric and neonatal intensive care has undergone significant
changes in recent years. New ventilatory strategies, such as lung-protective ventilation
using permissive hypercapnia or high-frequency oscillatory ventilation (HFOV), have
been introduced, whilst various forms of synchronised and noninvasive ventilation are
increasingly utilised. In extremely pre-term babies with respiratory distress syndrome,
there is a growing trend away from prolonged mechanical ventilation to prophylactic
surfactant administration and early extubation to nasal continuous positive airway
pressure. Many of these new concepts of respiratory support originate from studies in
adults and there is a relative paucity of objective evidence regarding their efficacy in
paediatric patients. There is a growing awareness of the clinical importance of functional
and diagnostic markers to predict the outcome (defined as risk of death or risk to develop
chronic lung disease) of children suffering from acute respiratory distress syndrome
(ARDS) and bronchiolitis. Currently, markers are not applied in a standardised fashion,
with little integration of markers between physiological, immunological, genetic or
structural markers of disease severity and outcome. This is further complicated by rapid
and ongoing change in treatment strategies, and understanding of pathophysiology.
Nonetheless, an overview of current knowledge and thinking, and outlining potential
future approaches is potentially worthwhile to promote discussion and the development
of new study hypotheses.
Current concepts of acute respiratory failure in paediatric

intensive care units
Acute respiratory failure or ARDS comprises the severe end of the spectrum of acute
lung injury (ALI), which is a frequent presenting feature or complication of critical
illness. ARDS is diagnosed if arterial oxygen tension (Pa,O2)/inspiratory oxygen fraction
(FI,O2) is v200 mmHg (26.6 kPa) and bilateral chest infiltrate without cardiogenic cause
is present. It is a heterogeneous lung disease, which may have a pulmonary or
extrapulmonary cause [1], the presence or absence of pre-existing lung disease prior to
ARDS, or the presence or absence of immune compromise in the patient. Representing a
major health problem to critical care physicians, the heterogeneity of the disease and lack
ISSN 1025-448x.
142
MARKERS OF LUNG DISEASE IN THE PICU
of consistency in defining diagnostic criteria has complicated the design and

interpretation of clinical trials, as well as the development and acceptance of markers
of disease.
As with any therapy, mechanical ventilation has side-effects and may lead to
secondary ventilator-induced lung injury (VILI) [2]. Overdistension of alveoli
(volutrauma), repeated opening and closing of alveoli (atelectrauma) and oxygen
toxicity contribute to VILI. Many experimental and human studies have demonstrated
that VILI is mediated by localised inflammation and the systemic release of inflammatory
cytokines [3]. The inflammatory response is clinically relevant as it aggravates the
underlying lung disease and the lung-borne inflammatory process evolves into a systemic
inflammatory response leading to multiple organ failure [4]. For the clinician, knowledge
of markers that assess the severity and nature of the primary lung disease, and which can
be used to monitor progression of lung disease and accompanying complications of VILI
would provide useful information for outcome prediction, the likelihood of development
of chronic lung disease, and which may guide therapeutic interventions.
The severity of primary lung disease is assessed most commonly with physiological
parameters, such as FI,O2, Pa,O2, arterial oxygen saturation (Sa,O2) and ventilator settings
(pressures) giving information on gas exchange and mechanical properties of the lung. It
is not possible to differentiate VILI from primary lung disease using these parameters. A
more promising approach suggests that the measurement of inflammatory markers
may quantify the degree of VILI [5]. An understanding of current concepts of
ventilatory strategies is essential to appreciate the value and utility of some prognostic
markers.
Latest fundamental developments in ventilation strategies

Whilst ARDS represents a major health problem for critical care physicians, the only
effective treatment strategy for decreasing mortality in a large randomised, multi-centre
trial [6] has been the recent application of lung protective ventilation strategy [7]. Several
different concepts of lung protection have synergistically improved outcome in ARDS.
The success of high-frequency oscillation in the ventilated neonates is critically
dependent on the utilisation of initial high mean airway pressures [8]. Translation of
this concept to ventilation at conventional frequencies in the early 1990s highlighted the
importance of the application of positive end-expiratory pressure (PEEP) to achieve
improved oxygenation [9]. In the mid-to-late 1990s, lung-protective ventilation using low
tidal volume and allowing high carbon dioxide levels (permissive hypercapnia) was
shown to improve outcome in adults with ARDS [10, 11]. A ventilation strategy using
low tidal volume in combination with high PEEP is commonly acknowledged as
standard treatment in paediatric intensive care units (PICUs), despite remaining
controversial [12, 13]. More recently, the concept of the "baby lung" has been evolved
[14], which argues that in acute respiratory failure, ventilation is directed into
functionally "normal" compartments of the lung. The available volume of such
compartments is substantially less than the volume of a healthy lung. Strategies aiming
for low tidal volume ventilation will protect these compartments from VILI [2]. An
important consideration of the baby lung approach is that tidal volumes need to increase
with recruitment of previously nonaerated lung units and resolution of lung disease. The
recent concept of ventilator-induced diaphragmatic dysfunction as a potential cause of
weaning failure in ARDS has highlighted the potential importance of assisted ventilation
modalities in maintaining neural activation and mechanical activity of the diaphragm
[15, 16]. A further major focus of recent approaches to lung protective ventilation has
143
A. SCHIBLER, J.J. PILLOW
been the use of lung recruitment manoeuvres to prevent atelectotrauma or to re-open

previously collapsed areas of the lung to participate in gas exchange [6, 17, 18]. During
HFOV, lung recruitment is normally performed with stepwise increases in mean airway
pressure, until oxygenation is optimised with subsequent reduction of pressure to the
lowest distending pressure that maintains an "open lung" [8]. HFOV has been used
successfully in paediatric and adult patients with ARDS [19], although it has not been
subjected to a rigorous controlled trial. In conventional ventilation, sigh manoeuvres are
used to recruit lung volume. Incorporation of sigh into lung-protective ventilation
improves recruitment and oxygenation [20].
Current concept of lung recruitment

Patients with ARDS are characterised by a reduction in the range of pulmonary
volume excursions, because of the reduction in ventilated units (collapsed or fluid-filled
alveoli), and a smaller change in volume per unit of change in pressure (decreased tissue
compliance due to oedema). ARDS patients require additional PEEP to keep the lung
open at end-expiration and increased peak inspiratory pressures to deliver adequate tidal
volume for gas exchange. The pressurevolume (PV) curve is used as an orientation for
setting optimal PEEP in patients with ALI or ARDS [21]. The initial part of the PV curve
(fig. 1) represents the amount of pressure required to open collapsed peripheral alveoli,
followed by a portion of the curve with impaired elastic properties (lower inflection
point). With increasing pressure, a steep, almost linear, section of the PV curve is
observed. Recent mathematical models suggest that the steep part of the PV curve is not
characterised by optimal stretching of alveolar tissue, but by the "popping open" of
individual lung units and increases in the volume of the lung unit from zero to that
appropriate for its trans-alveolar pressure [22]. The pattern of alveolar recruitment
follows a power law and is often referred to as an "avalanche" [23, 24]. Each increment in
pressure causes an increase in volume of newly recruited alveoli that is much greater than
that of alveoli which are already inflated. As the rate of recruitment diminishes and
finally stops, the PV curve flattens at higher applied pressures and overdistension of
alveolar structures occurs (upper inflection point). During deflation, the PV curve
exhibits a similar sigmoid shape but with a significant hysteresis compared with the
inflation. The slope of the initial part of the deflation limb shows the total compliance of
all alveoli that were inflated at end-inspiration. When pressure during deflation decreases
Volume L
UIP
LIP
Pressure cmH2O
Fig. 1. An example of a pressurevolume curve. LIP: lower inflation point; UIP: upper inflation point.
144
and falls below the highest alveolar opening pressure, lung units start to collapse. The
point of maximal curvature on the deflation limb may indicate optimal lung volume [8,
25]. The last part of the deflation curve is characterised by minimal change in volume,
indicating that most of the collapsible lung has become atelectatic.
The measurement of PV curves has some important limitations [26]. Lung
compartments heavily affected by the disease process may never open with a PV
manoeuvre, even when high pressures are used. The PV curve predominately reflects the
recruitment characteristics of the "good" lung. Figure 2 shows pressureimpedance
curves (equivalent to PV curves) from a healthy subject in the right lateral position. The
pressureimpedance curve of the left (nondependent) lung is less steep and reaches a
plateau at lower pressures compared with the right (dependent) lung. Thus, even in
healthy lungs, "local" PV curves are significantly different if measured in the dependent
and nondependent lung. As clinical PV curves are indicative of global rather than
regional lung recruitment, they are unable to describe the heterogeneous disease
character of injured and healthy zones. The rate of recruitment and de-recruitment at a
clinically chosen pressure level may be optimal for one lung region but not so for another.
If the pressure is too low, no opening of collapsed alveoli occurs; if it is too high,
potentially harmful overdistension and VILI may occur in less-diseased areas of the lung.
Techniques to measure pressurevolume curves

PV curves may be obtained using a super-syringe technique [27, 28]. The lungs are
inflated stepwise with fixed volume steps until an airway pressure of 45 cmH2O is
achieved. Alternatively, a rapid airway occlusion technique can be used at different
points in the respiratory cycle during mechanical ventilation. Newer ventilators now
produce a PV curve of the patient using a constant inspiratory flow or constant increase
in airway pressure. These methods require paralysis and sedation and are currently
restricted to relatively few commercially available ventilators.
0.25
0.20
Impedance change
0.15
0.10
0.05
0.00
-0.05
-0.10
-0.15
-0.20
3 5
10
15
20
25 30
Pressure
35
40
45
50
Fig. 2. The impedance change of the global (), and right ( ) and left (-----) lung are displayed against
pressure change for a healthy subject obtained in right lateral position, where impedance is measured relative to
baseline tidal volume breathing. The graph shows that impedance change is relatively higher in the right lung
than in the left lung. The left lung reaches a plateau earlier than the right lung. The left lung in right lateral
position is already well expanded at end-expiratory level and experiences less inflation during the recruitment
manoeuvre than the right lung.
145
Outcome prediction
Functional markers
Physiological parameters. In clinical practice, it is tempting to correlate the mechanical
impairment of the respiratory system with the severity of the disease and to set the
ventilatory parameters accordingly. The benefit of adjusting ventilatory strategy
according to respiratory mechanics remains uncertain. Two randomised trials in an
adult population showed that protective ventilatory strategy individually tailored to the
PV curve minimised pulmonary and systemic inflammation [3] and decreased mortality
[6]. Ranieri et al. [3] investigated 44 patients suffering from severe ARDS. In the group
receiving lung protective ventilation strategy, a PV curve was performed and PEEP set to
23 cmH2O above the lower inflexion point. The concentration of inflammatory markers
(tumour necrosis factor-a and interleukin (IL)-6) 36 h post-randomisation was
significantly lower in the lung-protective group than in the control group. Amato et al.
[6] randomly allocated patients with ARDS into a control group (tidal volume 12 mL?kg-1
and PEEP adjusted to lowest possible level for adequate oxygenation) and into a lungprotective group (6 mL?kg-1). In the lung-protective group, the optimal PEEP was
adjusted to above the lower inflection point of the PV curve. The mortality rate in the
lung-protective strategy group was 37% lower than in the control group (29 versus 66%).
One of the welcome side-effects of performing a PV curve is that the lungs are recruited
during the manoeuvre. Furthermore, the lungs are more efficiently ventilated if
ventilation is continued on the deflation than on the inflation limb of a PV (recruitment)
manoeuvre [29]. Rimensberger and coworkers [8, 29] demonstrated that animals
ventilated on the deflation limb after a sustained inflation of the lung had reduced lung
injury compared with controls ventilated at the same PEEP levels. To implement these
results, the clinician needs first to perform a diagnostic PV curve and then determine the
optimal distending pressures on the deflation limb. A second PV curve must be
performed afterwards, and ventilation should be continued at the previously defined
optimal pressure on the deflation limb.
Scoring systems. An alternative method to assess the severity of lung disease and relate it
to outcome is to utilise a scoring system. Only a few studies have investigated outcome
prediction in children with ARDS based on measures of oxygenation, ventilation, lung
compliance, mean airway pressure, the Pa,O2/FI,O2 ratio and alveolar-to-arterial oxygen
tension difference (PAa,O2). In neonates, the PAa,O2 was widely accepted as a predictor of
death in severe respiratory failure [30] and has been used as a criterion for extracorporeal
membrane oxygenation [31]. The importance of this predictor decreased with the
introduction of surfactant therapy in the early 1990s. Timmons et al. [32] estimated
mortality risk for 470 paediatric patients with acute respiratory failure (PEEP i6 cmH2O
and FI,O2 i0.5) using the Pediatric Respiratory Failure (PeRF) score, which includes age,
operative status, Pediatric Risk of Mortality score, FI,O2, respiratory rate, peak
inspiratory pressure, PEEP, Pa,O2 and carbon dioxide arterial pressure (Pa,CO2). The area
under the receiver-operating characteristic curve for PeRF score was 0.77, indicating a
high predictive power. The subgroup with the highest mortality was patients after bone
marrow transplantation (mortality rate of 91%). More recently, Flori et al. [33] reported
risk factors associated with mortality in paediatric ALI. They described 328 patients with
ALI with an overall mortality rate of 22%. A decreased Pa,O2/FI,O2 ratio (v300) at
presentation to the PICU, the presence of nonpulmonary and non-central nervous system
(CNS) organ dysfunction and isolated CNS dysfunction were associated with high
mortality. Rivera et al. [31] found that a combination of maximum peak inspiratory
146
pressure w40 cmH2O and a PAa,O2 w580 mmHg (77.14 kPa) was associated with a
mortality of 81%. Paret et al. [34] found that a ventilation index (VI=Pa,CO26peak
inspiratory pressure6respiratory rate/1,000) w65 predicted death with a power of w90%.
Arnold et al. [19] demonstrated in children treated with HFOV that an oxygenation
index (OI=(mean airway pressure6FI,O26100)/Pa,O2) of i28 discriminates survivors from
nonsurvivors with a w70% probability. Traber et al. [35] recently reported a study, in
which they prospectively followed 131 paediatric patients with acute hypoxic respiratory
failure. They reported that a high OI in the first 12 h of mechanical ventilation is
associated with poor outcome, but an absolute threshold could not be defined.
Surprisingly, they used rather low PEEP values in their study. The usefulness of these data
may be limited because calculated indices based on physiological parameters and
ventilatory settings in a patient supported by mechanical ventilation is a physiciandirected variable, i.e. at a given degree of lung disease, a high PEEP would probably result
in improvement in Pa,O2 at any FI,O2. It is therefore important to be aware of the
ventilation strategy used when comparing outcome studies.
Post-PICU functional outcome. There is only one study measuring functional outcome
of children surviving ARDS. Fanconi et al. [36] followed nine children surviving 0.9
4.2 yrs after ARDS with pulmonary function and found that all patients had abnormal
ventilation distribution measured with a multi-breath nitrogen washout. There was a
significant correlation between length of FI,O2 w0.5 and peak inspiratory pressure with
measured lung function abnormalities in the post-PICU period.
Inflammatory markers. One of the great limitations in ALI research has been the lack of
valid markers of lung injury or systemic inflammation that can be used to predict the
severity, outcome or response to therapy. It is widely accepted that the outcomes of ALI
and ARDS are related to the magnitude and duration of the pulmonary inflammatory
response [3]. Cytokines are an important component of the pathophysiology of the
inflammatory response associated with ARDS. Recently, Parsons et al. [5] reported the
results of plasma measurements of key pro- and anti-inflammatory cytokines in adult
patients with ARDS enrolled in a randomised controlled trial, either ventilated with
6 mL?kg-1 or 12 mL?kg-1 tidal volume. The primary hypothesis was that at the onset of
ALI, patients with more severe systemic inflammation would have a worse prognosis.
They found a strong association in both treatment groups between outcome and plasma
IL-6 and IL-8 levels measured at study onset. Furthermore, they observed that low tidal
volume ventilation (6 mL?kg-1) was associated with a more rapid attenuation of the
inflammatory response. In a paediatric study, Flori et al. [37] analysed soluble
intercellular adhesion molecule-1 (sICAM) in paediatric lung injury and compared the
levels in mechanically ventilated children without lung pathology. sICAM was higher in
the ALI group but, in addition, there was an association between sICAM level and
outcome. A sICAM level w1,000 ng?mL-1 had high specificity for identifying
nonsurvivors and prolonged mechanical ventilation. These studies are an important
step in better separation of primary and secondary lung disease.
Understanding the interrelationship between ventilatory strategy and inflammatory
response is essential to appreciating the relevance of the inflammatory response.
Cytokines can leak from the inflammatory sites in the lungs. Alternatively, cytokines may
be produced in response to, for example, bacterial products, such as endotoxin, that leak
from the lung into the circulation. It follows that inflammation and mechanical injury are
linked pathophysiologically in the lungs and lead synergistically to a more pronounced
inflammatory response [38]. These observations may explain why mechanical ventilation
with larger tidal volumes is not as harmful in noninflamed normal lungs. Interestingly,
147
recent animal evidence in an infant rat model suggests that infantile lungs are more
tolerant to high tidal volumes than adult rat lungs [39]. In the study by Kornecki et al.
[39], infant and adult rats were exposed to large tidal volumes either proportional to body
weight or total lung capacity. The visco-elastic properties, inflammatory markers and
lung histology were significantly more deranged after ventilation in the adult rats than
infant rats. In infants without pre-existing lung pathology, Plotz et al. [40] showed that a
remarkable, predominately pro-inflammatory immune response could be observed after
2 h of mechanical ventilation, if they are ventilated with tidal volumes of 10 mL?kg-1.
Imaging techniques. Monitoring of optimal recruitment can be carried out by computed

tomography (CT) scans of the lung, demonstrating that most areas of the lung are aerated
after a recruitment manoeuvre. CT scans are rarely used clinically for titration of
ventilatory settings. A promising alternative technique is electrical impedance
tomography (EIT) [41, 42]. A transectional image of one plane of the lung is obtained
by sending a small current through 16 electrodes placed circumferentially around the
chest. A representative image of local impedance change of that plane of the lung can be
obtained by analysing the back projection of the electrical signal with a complex
mathematical algorithm. Measured local impedance change correlates with local tidal
volume change and an estimate of local ventilation distribution is obtained. This
technique allows demonstration of changes in local ventilation and end-expiratory level.
EIT not only provides images of ventilation distribution but also enables the measurement
of impedance time-course analysis. During a PV or pressureimpedance manoeuvre, the
local mechanical characteristics of the lung can be measured. Kunst et al. [43] showed
that the posterior lung in the supine position has a significantly different pressure
impedance curve than the anterior lung. EIT therefore enables the clinician to identify
lung areas with different elastic properties and help to prevent potentially harmful
overdistension of the lung.
Clinical markers predicting outcome

Flori et al. [33] recently reported mortality rates in 320 patients with ALI; they found
that 54% of patients with near drowning, 39% of patients with associated cardiac disease
and 31% of patients with sepsis died. Mortality rate was greater in patients presenting
with two or more nonpulmonary organ system failures. Interestingly, children with CNS
dysfunction had the greatest mortality risk. They also investigated whether outcome
could be predicted from the ventilatory settings required for adequate ventilation and
they found that Pa,O2/FI,O2 ratio and OI predict poor outcome (unlike in adults). If the
patient at admission presents with a Pa,O2/FI,O2 ratio v100 the mortality rate is 34%,
whereas if the Pa,O2/FI,O2 ratio is 200300 at admission then the mortality rate is only
13%. Traber et al. [35] identified additional clinically important information that can be
obtained. Patients with ARDS after bone marrow transplant and immune suppression
have the highest mortality rate (63 and 41%, respectively). Ex-premature infants with
chronic lung disease have a mortality risk of 33%. Not surprisingly, both studies found
that immune-compromised patients are more likely to succumb to the immense
inflammatory burst in ARDS, and it is common knowledge that ARDS patients often die
because of multiple-organ failure secondary to lung injury (as well as harmful mechanical
ventilation). In ex-premature infants with chronic lung disease, additional lung injury
may reduce the functional capacity of the lung to below the level that is compatible with
life. The more restricted the functional capacity of the lung, the more likely an
unfavourable outcome. High supplemental oxygen and high prior Pa,CO2 are poor
prognostic factors. These children die either due to chronic respiratory failure or because
148
of intercurrent respiratory infection/aspiration causing a severe ARDS. In summary, the

higher the PEEP, mean airway pressure and the initial oxygen requirement, the more
likely it is that the patient will die.
Genetic markers. Respiratory syncytial virus (RSV) bronchiolitis is the most common,
severe lower-respiratory-tract infection leading to PICU admission. It is well established
that RSV infection in infancy is associated with recurrent wheezing and asthma during the
first years of life [44]. Furthermore, it is well recognised that infants with certain
preconditions are more likely to present with severe RSV infection, including expremature infants with chronic lung disease, infants with cystic fibrosis and infants with
congenital heart defects [45]. Of these infants, y4050% require invasive mechanical
ventilation [46].
The intense airway inflammatory response associated with RSV infection may be an
important determinant in the severity of the disease. Wilson et al. [47] recently described
an association of the need for mechanical ventilation with genetic variability at the IL-10
gene locus. It has been estimated in the past thaty70% of the inter-individual variation in
the production of IL-10 is genetically determined [48]. These findings support the motion
that genetic predisposition is an important early marker for lung disease in PICU [4951].
Genetic polymorphisms associated with pulmonary surfactant collectin protein (SP)-A,
SP-B and SP-D genes are associated with genetic susceptibility to ARDS and severe RSV
infections [5254]. Other polymorphisms in genes coding for cytokines and pulmonary
renin-angiotensin activity have also been linked to the susceptibility to and severity of
ARDS [55, 56].
Future questions
The identification of early markers of disease severity is standard in other areas of
adult medicine. In contrast, ALI and ARDS are clinical syndromes that are diagnosed
when a patient develops critical hypoxaemia and bilateral pulmonary in the absence of
cardiac failure. There is a need for development of diagnostic tests to assess severity and
underlying cause for acute lung injury that encompass functional, structural,
inflammatory and genetic aspects of ARDS. The evolving nature of mechanical
ventilatory modalities suggests that emphasis needs to be placed on markers that are
equally applicable across a range of ventilatory strategies, and not on those that can only
be performed in the paralysed subject. Studies that assess long-term outcome of
paediatric survivors of ARDS over several years into adulthood will further inform
clinical treatments and decision-making in the intensive care unit. Such a project can only
be accomplished through close collaboration between paediatric intensive care and
paediatric respiratory units.
149
Summary
Predicting outcome and potential lung disease for any child suffering from severe
respiratory failure in the paediatric intensive care unit is difficult. The causes of the
acute respiratory failure are diverse and still not well understood. In simple terms, it
may be concluded that high pressures and supplementary oxygen in the phase of acute
respiratory failure increase the likelihood that complications and subsequent death
may occur. As a simple rule of thumb, if the oxygenation index after intubation
exceeds 1520 and there follows prolonged mechanical ventilation, a poor outcome is
likely.
Keywords: Acute respiratory distress syndrome, chronic lung disease, hyaline
membrane disease, lung recrutiment, outcome, oxygenation index.
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152
CHAPTER 11
Wheezing disorders in young children: one

disease or several phenotypes?
J. Grigg*, M. Silverman#
*Academic Division of Paediatrics, Queen Marys School of Medicine and Dentistry, London, #Division of
Child Health and Institute for Lung Health, University of Leicester, Leicester, UK.
Correspondence: J. Grigg, Academic Division of Paediatrics, Queen Marys School of Medicine and
Dentistry, 4 Newark Street, London, E1 2AT, UK. Fax: 44 1162523282; E-mail: j.grigg@qmul.ac.uk
The problems of preschool wheezing disorders derive mainly from their dependence on
the single poorly characterised symptom "wheeze" to encapsulate a complex set of
asthma-like disorders. In this respect, they differ from other well-established chronic
diseases, such as Type I diabetes or chronic inflammatory bowel disease, or indeed
asthma in older children and young adults, which are characterised by a constellation of
their clinical features and underlying pathophysiology. As a preface to this chapter, the
current understanding of wheeze in preschool children and why the symptom has
hampered progress will be explored.
Multiphonic wheeze is a high-pitched sighing or whistling sound from the
intrathoracic airways and is heard mainly during expiration. It is the audible
manifestation of airway oscillations at points of flow limitation (fig. 1). Flow limitation
is the result of the complex interaction between the elastic properties of the lungs and of
the airways, as well as the airway calibre [1]. It is not usually possible to determine the
main sites of airway narrowing from the properties of the sound, since flow limitation
may develop some way downstream of the main problem. Other clinical features may
help in localisation; for example, hyperinflation suggests widespread peripheral
narrowing.
Wheeze rarely occurs in isolation in young children. Cough and breathlessness are
common accompaniments. In infants, particularly during common viral respiratory tract
infections, rattly sounds (colloquially called "ruttles" in the East Midlands of the UK),
probably caused by mucus in large airways, may mask wheeze or be mislabelled by
parents as wheeze. The two sounds produce distinct phonographic patterns and should
be easily distinguished by clinicians [2]. Whether or not this confusion is important in
clinical practice has yet to be determined, but recent research on parental understanding
of wheeze suggests that parents (and even inexperienced doctors) may have some
difficulty [3].
Parental misclassification of noisy breathing in young children is widespread, but more
common in socioeconomically deprived families and in ethnic minority groups in the
UK, possibly contributing to the poorer health outcome for their children [4]. Moreover,
much of the epidemiology of preschool wheezing disorders is derived from parentcompleted questionnaires, and therefore prone to error from misclassification. As a
counsel of perfection, parental reports of preschool wheeze should be confirmed by
auscultation by an experienced doctor or at a home visit by a nurse [5, 6]. Inevitably, this
will reduce the size of studies (and therefore their statistical power) and lead to different
types of bias, compared with questionnaire-based research. However, in spite of the high
prevalence of reported wheeze, the present authors research suggests that most errors
ISSN 1025-448x.
153
J. GRIGG, M. SILVERMAN
Wheeze is generated at
sites of flow limitation
Airway calibre
Velocity of airflow
Mucus or other
obstruction downstream
Properties of the airway wall
Fig. 1. Factors that affect the volume and pitch of wheeze from a vibrating flow-limiting segment. Indirectly,
wheeze is also affected by the compliance of the lungs and the degree to which expiration is forceful rather than
passive.
due to misunderstandings result in underestimation of the true prevalence of wheeze in

high-risk groups [4].
Most reported wheeze in preschool children occurs in acute, short-lived episodes, in
association with viral (upper) respiratory infection and in the absence of interval
symptoms (fig. 2) [7]. This contrasts with the situation in later childhood asthma. The
distinction has lead to some inconsistency of nomenclature. Some use the umbrella term
"asthma" for all common preschool wheezing disorders (i.e. those with no specific
diagnosis), assuming them to represent "variable, widespread intrathoracic airway
narrowing" and therefore to conform to the basic definition of asthma. Others reserve the
term asthma for (mainly allergic) children with both virus-induced wheezy episodes (or
"attacks") and chronic (interval) symptoms. This debate itself encapsulates the question:
4.0
Score
3.0
l l
l
2.0
1.0
0.0
l
l
l
l
l l l l l l l
-7
l l l l l
0
7
Time relative to the beginning of an episode days
14
Fig. 2. Mean daily symptom scores (95% confidence intervals of mean) recorded from 7 days before until 14
days after the onset of acute viral episodes in preschool children with histories of exclusive viral wheeze. There
is a very low level of interval symptoms between these brief, self-limiting attacks. Reproduced with permission
from [7].
154
WHEEZING DISORDERS IN YOUNG CHILDREN
is preschool wheeze a single disorder or are there several distinct phenotypes of wheezing
disease?
Are there several phenotypes of preschool wheeze?

The concept of phenotype
The concept of the phenotype, as applied here, relates not to the individual but to the
group. It implies a group of individuals with clinical features sufficiently distinct from
other groups to represent a useful clinical entity (or distinct "disease"; fig. 3). Scientific
medicine (for instance, evidence-based therapy) is very largely dependent on placing
patients as closely as possible into disease pigeon-holes. Clearly, neither one extreme, in
which every wheezy child has a unique phenotype, nor the other, placing all wheezy
children in the same disease entity "asthma", provides much guidance for prevention,
prognosis or treatment. A utilitarian position falls between these two extremes.
Evidence supporting different phenotypes of preschool wheeze

Preschool wheezing disorders in general (and childhood asthma in particular)
represent complex interactions of many processes, both endogenous (genotype or
physiology) and exogenous (environment, including intra-uterine environment; therapeutic agents) in the developing child (undergoing alveolisation and lung growth). It
would be surprising if the symptoms of variable wheezing, representing variable airway
obstruction, were not the common end-point of a number of discrete disease processes,
each with its distinctive pattern, pathophysiology and therapeutic response.
Clinical and epidemiological evidence. Wheeze is reported by w30% of young children

during the first 3 yrs of life. The prevalence falls to about half in older children. There is
much evidence that this nonprogressive or "transient" wheeze [5] is a separate phenotype,
distinguished from "persistent" or "late-onset" wheeze in several respects (table 1). The
research literature is bedevilled by inconsistent nomenclature, itself evidence that more
than one phenotype exists (table 2).
The most common clinical phenotype in children v3 yrs of age is exclusive viral
wheeze, a disorder characterised by acute episodes of wheeze, cough and breathlessness
in association with viral respiratory tract infections, with few or no interval symptoms
(fig. 2). This phenotype accounts for about two-thirds of wheezers v3 yrs of age.
Episodes may be severe enough to warrant hospital admission, but the illness is generally
mild and resolves with age. It is sometime referred to as "post-bronchiolitic wheeze"
(although acute bronchiolitis is a relatively rare precursor of exclusive viral wheeze) or, in
the USA, "recurrent bronchiolitis". In cross-sectional [8] and longitudinal [9] studies, the
prevalence of viral wheeze falls with age and has similar features to transient wheeze.
Whether exclusive viral wheeze and early transient wheeze are identical conditions
remains to be proved.
Genotype
Environment
Clinical
phenotype
Therapy
Outcome
Fig. 3. The concept of phenotypes. "Therapy" is an environmental variable, introducing a feedback mechanism.
155
Table 1. Features common to both "transient" wheeze and exclusive viral wheeze in early childhood, which
distinguish them from "persistent", "late-onset" childhood wheeze and classical atopic asthma
Childhood prognosis
Lung function
Allergy
Maternal smoking
Nursery attendance
Long-term prognosis
Ethnicity
Inflammatory basis
Parental influence
Better (by definition, in the case of transient wheeze)

Developmental airway dysfunction prior to onset#
No more common than in reference population
Significantly increases risk
Significantly increases risk
More rapid decline in FEV1(?); marker for COPD(?)
Less frequently reported in preschool children of South Asian origin
No eosinophilic airway inflammation between episodes
Maternal w paternal influence
FEV1: forced expiratory volume in one second; COPD: chronic obstructive pulmonary disease. #: This has not
been confirmed by all research groups.
Table 2. Some commonly used labels for phenotypes of preschool wheezing disorders
Label
Retrospective labels
Transient wheeze
Persistent wheeze
Late onset wheeze
Clinical labels
Viral wheeze
Exclusive viral wheeze#
Asthma}
Post-bronchiolitic wheeze
Definition
Wheeze early in life (usually v3 yrs) which remits
Wheeze early in life which persists (usually to school age)
Wheeze which develops only after early life (usually w3 yrs)
A discrete episode (or "attack") of wheeze in association with (viral) RTI
Wheezing only in association with viral RTI, without interval symptoms
Wheezing both with and between viral RTI
Wheezing episodes that follow acute infantile bronchiolitis for a variable period of time
RTI: respiratory tract infection. #: Synonym: recurrent bronchiolitis (USA); }: synonyms: chronic wheeze, viral
wheeze with interval symptoms, or "multiple-trigger" wheeze.
Other phenotypes of preschool wheeze include an illness resembling classical asthma,

with chronic, interval symptoms between episodes and often with an atopic personal and
family history; an episodic bronchitic illness in which a rattly (presumed mucous or
productive) cough is the principal feature. Rarer specific disorders, such as congenital
airway disorders (tracheo-bronchomalacia), chronic lung disease of prematurity and
obliterative bronchiolitis (either post-adenoviral or associated with recurrent microaspiration) may have a wheezy component. Overlap or the coincidental occurrence of
two independent disorders in individual children creates a wide potential spectrum. At
the same time, it creates the sort of uncertainty that makes clinical paediatric medicine
such a challenge.
Inflammatory basis. The point of reference for understanding lung inflammation in

preschool asthma is adult atopic asthma. Atopic adult asthmatics have a tendency to
develop persistent eosinophilia in their bronchial submucosa and airway lumen [10].
Thus, inhaled corticosteroid therapy needs to be given continuously, otherwise chronic
inflammation and symptoms return. However, as described above, most preschool
children with asthma have exclusive viral-triggered wheeze (table 2). Direct sampling of
the lower airway between viral-triggered attacks in this group has found no evidence of
chronic airway eosinophilia or abnormal T-cell activation [11, 12]. At the other end of the
preschool asthma spectrum are the minority of children who almost certainly have
classical atopic asthma. In a recent study, Saglani et al. [13] found evidence of tissue
eosinophilia and thickened reticular basement membrane, both hallmarks of atopic
asthma [14], in children aged between 3 months and 5 yrs referred to a tertiary clinic with
severe recurrent wheeze. In contrast, two other studies using bronchoscopy and
156
bronchoalveolar lavage found no evidence of airway eosinophilia in preschool children

and infants with severe attacks of wheeze, some of whom had persistent symptoms [15,
16]. The discrepancy may, in part, be due to the lack of consistency in defining the
phenotypes of wheeze.
In interpreting data on airway inflammation during acute attacks of preschool viral
wheeze, it is important to consider the setting where the sampling has taken place. In a
primary-care setting, the majority of children presenting with an acute asthma attack will
have the "exclusive" viral wheeze phenotype. In a secondary care setting, it is possible
(but as yet unproven) that there will be a shift towards children at risk of, or with, atopic
asthma and interval symptoms. In children hospitalised for viral wheeze, systemic
eosinophil activation, systemic neutrophil activation and increased cysteinyl leukotriene
production have been reported [1721]. To date, none of these indirect markers have
been validated using direct airway sampling. However, in an adult model of exclusive
viral wheeze, in which healthy adults who wheezed only with colds were experimentally
infected with coronavirus, McKean and co-workers [22, 23] found that acute wheeze is
characterised by neutrophilic, but not eosinophilic, inflammation in the lower respiratory
tract. In summary, there is good evidence to show that children who wheeze exclusively
with colds do not have chronic eosinophilic airway inflammation, and that during an
acute viral-triggered attack, neutrophilic inflammation predominates. There is, however,
a minority of preschool wheezers who have eosinophilic airways inflammation between
viral-triggered attacks.
Many uncertainties remain. The point of reference for inflammation in asthma, i.e.
classical atopic asthma, has itself undergone recent re-evaluation. For example, viraltriggered attacks of classical atopic asthma are associated with neutrophilic, and not
eosinophilic, airways inflammation [24, 25]. It is therefore possible that exclusive viral
wheeze is atopic asthma without a propensity to develop chronic inflammation. Future
research should be directed at noninvasive sampling during acute wheeze episodes.
Indeed one study suggests that induced sputum can be collected in symptomatic infants
and during asymptomatic interval periods [26]. The key issue related to inflammation
during the interval periods between viral-triggered attacks is how to accurately identify
chronic eosinophilic inflammation in uncooperative toddlers. Indirect markers of
eosinophilic inflammation, such as urinary eosinophil protein-X [27], are, at best,
nonspecific predictors of eosinophilic activation in the airways. Similarly, the association
between exhaled nitric oxide and airway eosinophilia is not as strong as first hoped, at
least in adults [28]. Analysis of exhaled breath condensate holds promise as a means of
exploring differences in the underlying pathophysiology [29]. For children with severe
symptoms, it may be ethically acceptable to perform more invasive tests, such as
bronchoscopy and bronchial biopsy, before embarking on long-term, high-dose inhaled
corticosteroid therapy.
Physiological evidence. There is little information on pulmonary function during acute

wheezy episodes in young children. Whether the pattern of physiological disturbance
during episodes could distinguish between different phenotypes seems unlikely. However,
between episodes of viral wheeze, in older children [30] and in adults [22], there is little
evidence of either persistent airway obstruction or of bronchial hyperresponsiveness
(BHR), in contrast to the usual situation in classical atopic asthma. Conversely, the little
data that exist for recurrently wheezy infants and young children suggest that between
episodes there may be both airway obstruction and abnormal airway wall compliance [31].
There are inconsistencies in the data on BHR. While BHR during symptom-free
intervals in wheezy infants is no different from nonwheezy control subjects [32], BHR
measured in the first weeks of post-natal life predicts both infant wheeze [33] and
157
subsequent asthma [34]. However, BHR is clearly associated with classical atopic asthma
in schoolchildren. To explain these apparent contradictions, it must be proposed that the
"partial phenotype" of BHR is not a single, uniform condition, but that the mechanisms
of BHR in infancy (BHR of developmental origin) differ from those in atopic asthma
(BHR of inflammatory origin). The issue may be clarified by systematic measurement of
BHR using direct challenge (e.g. with methacholine) and indirect, inflammation-sensitive
challenge (e.g. with adenosine) in early childhood, and relating the results to other
phenotypic features and outcomes.
Response to treatment. It is tempting to argue that differences in responsiveness to antiinflammatory treatments in preschool children with different patterns of wheezing
strengthen the case for different phenotypes. However, interpreting data from studies that
have not been specifically designed to compare the responsiveness of different phenotypes
to treatment, must be treated with great caution. For example, the ineffectiveness of
inhaled corticosteroids in some randomised controlled trials in preschool asthmatics
could, on the one hand, be consentient with a nonatopic wheeze phenotype. On the other
hand, these data could be consistent with the lack of effectiveness of inhaled steroids in
preventing viral-triggered attacks of wheeze reported in a study of school-age children,
some of whom had clear evidence of atopic asthma [35]. As previously mentioned, the best
therapeutic trial to justify phenotypes, is one that: 1) draws on epidemiological and
inflammatory marker data to develop a set of mutually exclusive phenotypes; 2) recruits
all wheezy children; and 3) stratifies children a priori into these "best guess" groupings.
Only by using this type of design, will the question of whether differences in
responsiveness to therapy between phenotypes really do exist, and of whether any
differences are of clinical relevance, be answered. Some issues relevant to the delineation
of phenotypes are discussed in the Treatment section.
How many phenotypes are there?

The techniques needed to answer this question are common to many types of
taxonomy. But in relation to human disease, the answer is rarely black and white, as may
appear to be the case as in animal or plant taxonomy. The answer for human phenotypes
is more often utilitarian, and in this respect, resembles the answers to questions about the
relative values of particular diagnostic tests (such as "Which test has the best ratio of
sensitivity to specificity?"). Indeed, the taxonomy of early childhood wheeze may differ,
depending on the purpose of the classification: genetics, prognosis or response to treatment.
Asthma and airway disorders are multidimensional. Describing disease phenotypes in
a single dimension, such as clinical severity, sputum eosinophil count or BHR, cannot be
sufficient. However, choosing which dimensions to combine in order to characterise a
disease is not straightforward. The "dimensions" of preschool wheeze may include:
clinical features, immunology, physiological features, inflammatory markers, pathology,
prognosis, response to therapy and genotype. Some features will be closely correlated
with each other, such as eosinophilia and allergy, while some require repeated
observations, such as prognosis. These add statistical complexity to the overall
complexity of the search for phenotypic diversity in wheezing disease. Factor analysis, as
a first step, can help to minimise duplication by grouping the clinical features into
correlated sets or factors [36, 37]. One general approach to the search for useful
subgroups (or phenotypes) is termed "cluster analysis" (fig. 4).
Cluster analysis. Cluster analysis encompasses a wide range of techniques designed to

detect clusters among a sample of individuals. All these methods rely on observations
158
Factor analyses to
reduce number of features
Cluster analysis
Clusters
Statistical properties
of clusters
Refine
model
Utility of clusters in
research
Utility of clusters in
clinical practice
Fig. 4. Steps in cluster analysis.
made on a given set of features. If this set consists of only one dichotomous feature, say the
presence or absence of a certain symptom such as "wheeze" or "cough", an obvious
clustering of the subjects would be to place them into two groups, one consisting of
individuals with the symptom and the other consisting of those without the symptom.
When multiple features are observed, finding similar response patterns across all variables
becomes much more complicated and requires multivariate techniques. The task may be
further complicated by including data of different modes (e.g. categorical, continuous,
count data, etc.).
Clustering methods can be placed into two broad categories: probabilistic clustering
and nonprobabilistic clustering. Nonprobabilistic clustering is concerned only with the
task of identifying groups of individuals and does not make any assumptions about how
the data may have been generated. These methods usually follow one of two approaches.
The first, partitional clustering, begins with an initial partition, meaning that each
individual is placed into one of a specified number of groups, say, k. The subjects are then
iteratively reallocated to groups while optimising a given criterion. In k-means clustering,
for instance, optimality is achieved when the variation of the observed feature is
minimised within clusters. The second approach, hierarchical clustering, does not require
the number of groups to be specified in advance. Instead, the groups are hierarchically
constructed by joining subjects, or subgroups of subjects, into ever larger groups based
on certain measurements of distance or similarity. Here, the subjects are interpreted as
points within a space in which distance can be measured. For instance three features
observed on two individuals can be interpreted as x, y, z coordinates of two points
representing the two individuals in three-dimensional space. The Euclidean distance
between the points is the length of the straight line connecting the two points. Such
concepts of distance can be generalised to spaces with any number of dimensions, each
dimension representing a measurable feature. Typically, the output of a hierarchical
clustering algorithm is a dendrogram (fig. 5). This is a tree diagram showing the
159
3
Subjects
Fig. 5. Example of the output from hierarchical cluster analysis: a dendrogram.
hierarchical merging structure. The subjects are ordered along the abscissa. Each
horizontal line represents a merger while the vertical location of the line along the
ordinate represents the distance between the subjects or subgroups being merged.
Probabilistic clustering attempts to actually model the process that gave rise to the
data. These models are generally specified in terms of a probability distribution,
consisting of a mixture of component distributions, each of which represents a cluster to
be identified. Assuming that the observed data are a realisation of the specified mixture,
the unknown parameters of the component distributions can be estimated by maximum
likelihood or Bayesian methods. Once these parameters have been estimated, each
individual can be assigned to the group from which its observed features would most
likely have originated. Probabilistic clustering methods are typically more computationally intensive and require careful thought in specifying the model. However, they are
also more flexible in accommodating various types of data, and allow statistical testing of
certain hypotheses. Cluster analysis techniques have been applied to other diseases and
have begun to be explored in relation to airway disease in general and wheezing disorders
in childhood [38, 39].
In summary, there is no answer to the question: how many phenotypes are there? But
simply raising the question may focus research endeavour in the fields of epidemiology,
physiology and therapeutics to try to provide solutions. At the moment, it can
confidently be stated that the care of young wheezy children is hampered by placing them
all into one single diagnostic lump.
Treatment
Few trials in preschool children have addressed the issue of asthma phenotypes, and
some anti-asthma therapies are not licensed for the whole preschool age range. In spite of
this, the UK Sign/British Thoracic Society Guidelines [40] (fig. 6) recommend regular
inhaled corticosteroids (ICS) at up to 400 mg beclomethasone dipropionate or equivalent
per day for anything but trivial preschool asthma. Inhaled salbutamol or terbutaline are
the mainstay for treating acute symptoms.
160
Step 4: Persistent poor control

Refer to respiratory paediatrician
Step 3: Add-on therapy

In children aged 25 yrs, consider trial of
leukotriene receptor agonist
In children <2 yrs, consider proceeding to step 4
Step 2: Regular preventer therapy

Add inhaled steroid 200400 gday-1#,
or leukotriene receptor agonist
if inhaled steroid cannot be used
Start at dose of inhaled steroid appropriate
to severity of disease
Step 1: Mild intermittent asthma

Inhaled short-acting b2-agonist as required
Fig. 6. British Thoracic Society/UK Sign recommendation for nonacute treatment of preschool asthma [40].
#
: beclomethasone diproprionate or equivalent; }: higher nominal doses may be required if drug delivery is
difficult. Taken with permission from [34].
Regular inhaled corticosteroids. Consensus amongst experts is needed for developing

guidelines, especially where the evidence base is sparse. The recommendation for ICSs in
preschool asthma smoothes over some of the discrepancies in published data. For
example, in many of the trials of regular ICSs in preschool children, either the asthma
phenotype recruited is unclear or the primary outcome variable is not given, or a
combination of both. There are indeed randomised controlled trials of regular ICSs that
show a reduction in symptoms [4144], but there are also trials where no beneficial effect
has been detected [45, 46]. The study by Wilson et al. [47] provides the key to
understanding this inconsistency in the published data. This randomised controlled trial
recruited preschool children with the exclusive viral wheeze phenotype and found no
clinical benefit from 4 months daily treatment with budesonide 400 mg per day, results
that can now be explained by the absence of chronic airway inflammation in this
phenotype (see above). Thus, studies of regular inhaled steroids that have recruited a high
proportion of exclusive viral wheezers will tend to be negative. Conversely, trials that have
recruited children at increased risk of atopic asthma are more likely to be positive. Indeed,
the recent Prevention of Early Asthma in Kids trial, which recruited preschool children
with asthma with risk factors for atopic asthma, found, as a secondary outcome analysis,
161
that 350 mg beclomethasone dipropionate equivalent per day (88 mg fluticasone twice a
day) during the 2-yr treatment period, resulted in fewer exacerbations requiring oral
steroids (57 versus 89%) but not days of bronchodilator use or unscheduled physician
visits [48]. Given this modest potential benefit, and until there is a systematic review that
includes unpublished negative trials (e.g. Glaxo trial FMS30058 [49]), the view of a recent
editorial that prolonged ICS treatment for toddlers with asthma should be "highly
selective" [50] seems sensible. In general, the threshold for starting chronic inhaled steroid
therapy should fall with increased age, since the risk that interval (persistent) symptoms
are due to atopic asthma is higher in older preschool children. If inhaled steroids were
without side-effects, the issue of steroid responsiveness would not be such an important
issue. However, the systemic bioavailability of inhaled steroids is significantly increased in
the noninflamed lung [51], and children with exclusive viral wheeze may therefore be at
increased risk of side-effects if inhaled steroids are given between attacks. These sideeffects are not necessarily trivial and may include acute life-threatening adrenal
suppression [52]. Taken as whole, the evidence of trials of ICS strongly suggests the
presence of multiple phenotypes of preschool asthma.
"Add-on" therapy. In school-age children and adults, when used in conjunction with
inhaled steroids, long-acting b2-agonists (LABAs) result in a 19% relative reduction in the
risk of patients experiencing one or more exacerbations requiring systemic corticosteroids
[53]. To date, LABAs are not recommended for preschool children [40]. However,
Primhak et al. [54] have shown that inhaled LABA Serevent1 (salmeterol xinafoate;
Allen and Hanburys, Uxbridge, UK) protects against methacholine-induced wheeze in
preschool children with a history of recurrent asthma. To date, Serevent1 is licensed in
the UK only for children aged i4 yrs. This therapeutic gap leaves the cysteinyl
leukotriene receptor antagonist, montelukast (licensed over the whole preschool age
range), as the major "add-on" option when control is suboptimal on ICSs.
Although there is evidence that montelukast is efficacious in preschool asthma, the
trial data are difficult to interpret because of the following points: 1) the primary
outcome is related to safety and not efficacy; 2) some trials did not stratify for use of
ICSs; and 3) there was phenotypic heterogeneity of recruited children. For example, a
large randomised controlled trial of montelukast reported that 4 mg montelukast per day
improved asthma symptoms in preschool children [55], but the primary outcome was
related to safety, and a significant minority of children were receiving ICSs (thus in the
majority, montelukast was used as a monotherapy). In school-age children, a Cochrane
systematic review reported that inhaled steroids at a dose of 400 mg?day-1 of
beclomethasone or equivalent are more effective than anti-leukotriene agents given in
the usual licensed doses [56]. Monotherapy with montelukast cannot therefore be
recommended for preschool asthma, if an inhaled steroid is an option [40].
Intermittent therapy. If regular ICSs are not effective in exclusive viral wheeze, are there
any ways of preventing attacks? Three studies showed only minimal benefit from the use
of high-dose (w1,500 mg per day) ICSs started at the first sign of a cold or lower
respiratory tract symptoms, and continued for a short period of time (up to 10 days) [57
59]. Bisgaard [60] reported that regular daily montelukast therapy in children aged 2
5 yrs, with a history of "intermittent" asthma associated with common cold and minimal
or no symptoms between the episodes, reduced the rate of asthma exacerbations, the time
to first exacerbations, the rate of ICS use and b-agonist usage. Thus, monotherapy with
montelukast could be justified for this phenotype, since regular ICSs are likely to be
ineffective (see above). As cysteinyl receptor blockade with montelukast lasts only 24 h
with no evidence of a long-term, carry-over effect, it may be better suited to intermittent
162
use. To date, the beneficial effect of intermittent use of montelukast in viral wheeze has
been published as an abstract [61].
Treatment of acute attacks. For clinically severe attacks of wheeze in preschool

children, current therapies are based on studies of proven efficacy in older children and
adults. For home treatment, inhaled b2-agonist (four to six puffs of salbutamol as
required, maximum 4 hourly) by a metered-dose inhaler and spacer, with a mask for
children v3 yrs of age is recommended [40]. It remains unclear whether inhaled shortacting b2-agonists are effective in the youngest preschool children (v2 yrs) [15], but since
they are safe, they should be used. If the variable benefit of inhaled therapy is due to
relative resistance of bronchial smooth muscle, or impaired delivery in youngest preschool
children, parenteral therapy may be more effective. Indeed, a recent review of a singledose intravenous salbutamol bolus (15 mg?kg-1 administered over 10 min) for the initial
treatment of children, concluded that it had the potential to shorten the duration of severe
attacks and reduce overall requirements for inhaled salbutamol maintenance [62]. There
are no data on whether ipratropium bromide provides additional benefit to short-acting
b2-agonists for home-based treatment, as it does when given in hospital [63].
The efficacy of systemic steroids in acute attacks of preschool asthma is unclear. The
two recent randomised controlled trials have specifically recruited preschool children
with viral-triggered attacks. First, preschool children who had previously been
hospitalised with an attack of viral-triggered wheeze were randomised to receive a
short course of oral steroids or placebo during their next attack [19]. Secondly, children
presenting to an admission unit were randomised to oral prednisolone or placebo [64].
On the one hand, parent-initiated oral steroid therapy improved neither the symptom
scores nor the number of salbutamol actuations required per day [19]. On the other hand,
issuing steroids to children at the time of presentation to hospital reduced the need for
"additional" asthma medication in children who were subsequently hospitalised [64].
Until further data appear, it is reasonable to reserve systemic steroids for preschool
children admitted to hospital with severe viral-triggered wheeze and not to routinely use
parent-initiated therapy.
Future trial design. To better target therapy to the heterogeneous mix of asthma
phenotypes in the preschool period, it is important that trials do not overlook potentially
responsive subgroups (e.g. children with chronic atopic asthma) but are not so restrictive
that the results are generalisable only to specialist secondary care. An ideal randomised
controlled trial would be one that recruits all wheezy preschool children and is sufficiently
powered to be able to stratify by phenotype. Indeed, response to therapy may itself be an
input variable in defining phenotype. It may well be that preschool asthma is the ideal
condition for an "individualised" treatment approach, i.e. using statistical analysis of the
risk factors for each child to predict the potential benefits of chronic anti-inflammatory
therapy or assessing individual responsiveness by prescribing placebo and active drugs in
a crossover (n=1 trial) design. If it proves to be feasible to sample lower airway
inflammation using a modified induced sputum technique or by analysis of exhaled
breath, it may also be possible to target and adjust inhaled steroids to suppressing sputum
eosinophilia, a method of proven value in adults [65].
The key research questions are as follows. 1) How can we identify those children with
chronic airway or lung tissue inflammation, and thus avoid using ICSs in those with no
airway inflammation between attacks? 2) What is the optimal intermittent therapy for
exclusive viral wheeze (high-dose inhaled steroids or montelukast, or some combination)? 3) Is there a role for LABAs as add-on therapy for young children at an increased
risk of atopic asthma? 4) Could flexible dosing that is licensed for the budesonide/
163
formoterol combination inhaler in older children be applied to preschool children with a

combination of chronic interval symptoms and episodic viral-triggered attacks? 5) Are
systemic steroids effective in treating viral-triggered attacks? 6) Do any therapies
currently available alter the natural history of early childhood wheeze?
Answering these questions, and thereby improving current understanding of how the
underlying inflammatory substrate corresponds with the symptom pattern, may help in
devising the best way of stratifying children in clinical trials. However, the most
important resource is the clinician who understands the complexity of preschool asthma
and can develop large, adequately powered, independent randomised controlled trials
with clear primary outcomes, and who can critically appraise the resulting data and
follow up subjects long-term.
The natural history of preschool wheeze

Most of the present knowledge of the natural history of preschool wheeze has been
derived from retrospective and cross-sectional studies, often based on clinical
populations. Recently, the first prospective longitudinal (cohort) studies of random
samples of the whole population have begun to be reported. The most notable, the
Tucson study [5], which has provided a benchmark, has been followed by more focussed
projects. Some of these attempt to combine an experimental intervention, such as house
dust mite avoidance, with an observational arm, potentially leading to bias in population
recruitment and unintended effects on the outcomes of interest. A number of useful
lessons have been learned from these studies.
The prognosis of preschool wheeze. Algorithms for prediction of outcome have been
determined by several groups. In general, they predict the likelihood of persistence of
wheeze (or asthma) at school age of those with preschool symptoms [66]. The Tucson
algorithm has been successfully used to identify high-risk groups of preschool wheezers
for therapeutic trials [67]. To some extent, prediction algorithms derived in one setting
(the desert environment of Arizona, USA) seem to be robust enough to apply in another
(temperate Western Europe). The precision of prediction algorithms is likely to be
improved by collecting data specific to the local environment, by taking into account
ethnic factors and by refining phenotype definitions, as previously discussed.
Is preschool wheeze a precursor of later disease? There is clearly evidence for a link
between early wheezing illness and asthma in schoolchildren; however, the interpretation
is conflicting, perhaps because the issue comprises several different questions. If there are
several distinct preschool wheeze phenotypes (atopic asthma, exclusive viral wheeze, etc.),
then the relationship with later disease needs to be considered separately for each; this is
obviously very complex, and figure 7 provides a simple model. For each phenotype, if
there is an association with later asthma.
1) The two may be identical, and thus simply a continuation, as is the case for chronic
atopic asthma. Early onset may be the result of chance environmental exposures or
greater inherent (genetic) risk.
2) The two may be identical, but the result of a common set of causal factors. Some
risk factors for adult-onset atopic asthma are similar to those of childhood-onset disease
[68], and many of the usual features of the illness persist in remission [69, 70]. The factors
that switch on the asthma phenotype in high-risk individuals and, conversely, which later
permit remission, would merit further detailed investigation.
3) Developmental "damage" as a result of preschool illness, such as remodelling,
programming or disrupted development of the lungs, may lead to later airway disease,
164
Genetics
Environment
Preschool wheeze
phenotype 1
(ii)
Late onset of wheeze

phenotype 1
(i)
Wheeze phenotype 1
(continued)
(iii)
"Damage"
New phenotype of
wheeze 2
Fig. 7. Possible scenarios for an association between wheeze phenotypes in preschool and older children. The
same model can be used to explore a possible relationship between preschool wheeze and chronic obstructive
pulmonary disease in adults. The three outcomes labelled (i), (ii) and (iii) are described in detail in the Is
preschool wheeze a precursor of later disease? section.
possibly of a different phenotype. For instance, repeated viral inflammatory episodes

might affect airway structure, function or alveolisation, leading to small airway disease
or to bronchial remodelling, thus increasing the risk of later wheezing or even leading to a
phenotype with characteristics different from classical atopic asthma. For example,
asthma in nonatopic schoolchildren has yet to be explained.
There is increasing evidence for a contribution of early life events to the risk of chronic
obstructive pulmonary disease in late adult life [71]; this has been suspected for many
years [72]. Among these, exclusive viral wheeze may be a candidate. The only (weak)
evidence is that despite normal spirometric lung function as young adults, the later
decline in forced expiratory volume in one second was greater for those with a history of
exclusive viral wheeze in childhood (albeit not confined to the preschool years) than in
those with childhood asthma and those without childhood respiratory symptoms [73].
Potential explanations are similar to those mentioned above for asthma (fig. 7). There are
no truly longitudinal studies.
To explore a developmental link between early and late airway disease, the
longitudinal cohorts should be studied in hypothesis-testing research. For example,
the technology is now available to undertake longitudinal studies of alveolar structural
[74] and functional development [75] to test the hypothesis that a (putative) link between
early viral wheeze in the first 2 yrs of life (at a time of active alveolisation) and later
chronic obstructive pulmonary disease could be explained by alveolar mal-development.
This is an exciting prospect, as "alveolar therapy" becomes a realistic possibility.
Implications for the future

There are several important conclusions from this chapter, which may inform the
direction of research in preschool wheezing disease. These are as follows. 1) There are
probably several distinct phenotypes to be delineated. 2) The natural history should be
described for locations other than the USA desert environment (as in the Tucson study). 3)
Appropriate therapeutic trials await better phenotype definition and better understanding of
pathophysiology. 4) The impact of early disease on the developing lung, a potential risk
factor for chronic obstructive pulmonary disease, should be determined.
165
Summary
Wheeze is a symptom with a complex physiological basis. This leads to the possibility
that, within the spectrum of wheezing disorders in young children, several different
disorders or phenotypes exist, each with its own aetiology, pathophysiology and
prognosis. If so, this has important therapeutic implications. Evidence for at least two
clearly distinct phenotypes, exclusive viral wheeze, an illness resembling classical
chronic asthma, is strong. Clinical and epidemiological observations supported by
studies of airway inflammation, pulmonary physiology and randomised controlled
trials support the existence of these two phenotypes. However, new statistical
techniques, falling broadly within clustering methodology, may lead to more subtle
classifications. The prognosis of early wheezing disorders, and in particular their role
in chronic obstructive pulmonary disease in adult life, remains to be studied in detail.
Keywords: Cluster analysis, phenotype, preschool, treatment, wheeze.
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169
CHAPTER 12
Bronchiolitis in infants and children

J.L.L. Kimpen*, J. Hammer#
*Wilhelmina Childrens Hospital, University Medical Center Utrecht, Utrecht, The Netherlands. #Abteilung
fur Intensivmedizin und Pneumologie, Universitats-Kinderklinik beider Basel, Basel, Switzerland.
Correspondence: J.L.L. Kimpen, Wilhelmina Childrens Hospital, University Medical Center Utrecht, POB
85090, 3508 AB Utrecht, The Netherlands. Fax: 31 302505346; E-mail: j.kimpen@umcutrecht.nl
Bronchiolitis is a common lower respiratory tract disease of infants and young

children. Most episodes are caused by respiratory syncytial virus (RSV), although
recently an increasing number of respiratory viruses have been incriminated in causing
the same clinical syndrome. All children have been infected with RSV at least once before
their second birthday. The majority develop simple upper respiratory tract symptoms
undistinguishable from a common cold, while y40% have signs of lower airway
inflammation. Only 12% of all children need hospitalisation. However, because of the
almost universal infection during the first year of life, the absolute number of patients
admitted during yearly RSV epidemics is dramatic, causing logistical problems,
especially on paediatric intensive care units (510% of the hospitalised patients need
mechanical ventilation), and having a serious effect on healthcare expenditures.
Moreover, nearly half of the patients admitted for RSV-related lower respiratory tract
disease develop recurrent episodes of wheezing up to the age of 1113 yrs, which impacts
considerably on their quality of life.
A number of comprehensive reviews have been published recently on several aspects of
RSV-related disease, both from the clinical point of view and from the basic science and
translational research arena [13]. The present chapter will focus on the most important
recent developments in epidemiology, pathogenesis of acute bronchiolitis and recurrent
wheezing, and prevention and treatment strategies.
Virology and epidemiology

RSV is a single-stranded RNA virus belonging to the genus pneumovirus of the family
Paramyxoviridae (fig. 1). Two RSV strains (A and B) and a large number of serotypes
and genotypes have been identified as human pathogens. It is unlikely that a difference in
virulence between these strains influences disease outcome. Zambon et al. [4] performed a
phylogenetic analysis of clinical RSV isolates from three consecutive seasons in a large
community-based surveillance study. They showed that different viral strains circulated
in the community at the same time, infecting both adults and paediatric patients alike,
and causing the complete spectrum of disease from upper respiratory infection, which
can be treated in the outpatient clinic, to severe disease necessitating admission to the
hospital. This confirmed earlier findings of Kneyber et al. [5], who showed that there was
no difference in solid outcome parameters, such as length of hospital stay, intensive care
admission and supplemental oxygen requirement, when comparing hospitalised
bronchiolitis patients infected with type A and type B RSV. Finally, Smyth et al. [6]
did not find differences in requirement for supplemental oxygen or mechanical
ISSN 1025-448x.
170
BRONCHIOLITIS IN INFANTS AND CHILDREN
Fig. 1. Respiratory syncytial virus (electron microscopy; courtesy of M. Kneyber, Free University Medical
Center, Amsterdam, The Netherlands).
ventilation in relation to RSV virus genotypes, according to variations in the small

hydrophobic or nucleoprotein of the virus.
RSV bronchiolitis has, in general, been considered a disease at the extreme ends of life,
i.e. the very young infant and the elderly. It has recently been shown that RSV can infect
people of all age groups and is responsible for more or less severe community-acquired
lower respiratory tract disease. Zambon et al. [4] showed that RSV is responsible for 10
25% of episodes of flu-like illness in 564-yr-olds during an observational, communitybased study. If flu-like illness, characterised by fever, cough and respiratory tract
symptoms, was used to select eligible patients, disease caused by RSV could not be
differentiated from infections by influenza virus. Hall et al. [7] prospectively studied
viral respiratory infections in 2,960 healthy adults from 1975 to 1995, during the months
that RSV was active in the community. Of the subjects studies, 7% (211 patients)
acquired acute RSV infection, which was symptomatic in the majority of patients.
Although most had upper respiratory tract symptoms, 26% manifested tracheobronchitis, bronchiolitis and wheezing, with a mean duration of 9.5 days and a high rate of
absence from work. Although the clinical characteristics of RSV- and influenza-infected
patients differed significantly (with more respiratory symptoms in the RSV group and
more generalised symptoms in the influenza group), there was a considerable overlap,
making a clinical aetiological diagnosis difficult. Falsey et al. [8] prospectively evaluated
all respiratory illnesses in a cohort of healthy elderly patients and adults with chronic
heart or lung conditions, over four consecutive seasons. RSV infection was responsible
for 37% of respiratory illness episodes in the healthy elderly subjects and for 410% in
the high-risk adult subjects. Again, 89% of RSV infections were symptomatic. These
studies suggest that RSV needs to be considered in the differential diagnosis of lower
respiratory tract disease in adults during the winter season, especially if the individual has
an underlying chronic cardiorespiratory condition.
The majority of bronchiolitis episodes are caused by RSV, with estimates ranging 50
90%. However, other viruses (influenza virus, parainfluenza virus, rhinovirus and
adenovirus) rarely cause the same clinical picture in young children. Recently, two new
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J.L.L. KIMPEN, J. HAMMER
viruses have been identified in the Netherlands, and have subsequently been isolated
elsewhere, that are responsible for a minority of cases of bronchiolitis in infants: human
metapneumovirus (hMPV) and human corona virus (HCoV-NL63) [9, 10]. hMPV is
thought to cause 412% of bronchiolitis episodes worldwide, although its prevalence
differs from country to country. The prevalence of HCoV-NL63 is not known.
hMPV is a single-stranded RNA virus belonging to the genus metapneumovirus in the
family Paramyxoviridae. It is more closely related to avian metapneumoviruses than to
RSV. Genetic differences between hMPV and RSV are the lack of both nonstructural
proteins (NS1 and NS2) in hMPV, and a different gene order on the genome [11]. hMPV
has been shown to cause productive infection of ciliated respiratory epithelial cells and
discrete clinical symptoms in primates, comparable with the effects of RSV in humans
[12]. Clinical disease caused by hMPV is very similar to the illness caused by RSV,
although hMPV patients tend to be slightly older and less dyspnoeic [13, 14]. As with
RSV, most people are infected at an early age. The effect of hMPV on recurrent wheezing
in childhood and, later, asthma has not been reported.
In the Netherlands, HCoV-NL63 was isolated from a 7-month-old child with
bronchiolitis and conjunctivitis [10]. The virus was subsequently identified in more
clinical specimens, suggesting it to be widespread within the population. HCoV-NL63
was found in 19 out of 525 clinical specimens in Canada [15]. Patients belonged to all age
groups and had symptoms in the upper and lower respiratory tract, conjunctivitis and
fever. In Connecticut, USA, a closely related coronavirus (HCoV-NH) was isolated from
paediatric patients with respiratory symptoms [16].
Additional viruses or virus strains will probably be identified in the future, until most if
not all bronchiolitis episodes are accounted for aetiologically.
RSV infects all children before the age of 2 yrs. Half of the patients develop lower
airway disease and a minority need admission to the hospital, and eventually intensive
care treatment. The illness starts with a few days of watery rhinitis, followed by
tachypnoea, dyspnoea, retractions and often wheezing on auscultation. Percutaneous
oxygen saturation shows hypoxia, and radiography of the chest shows bilateral patchy
infiltrates and hyperinflation (fig. 2). Viral antigen can be readily detected in exfoliated
nasal epithelial cells, using a direct immunofluorescence test (fig. 3). Risk factors for
severe disease are prematurity, chronic lung disease of the neonate and congenital heart
disease, especially if accompanied by pulmonary hypertension. When more than one risk
factor is involved, the prognosis deteriorates accordingly [17]. Most children admitted
with acute RSV bronchiolitis do not belong to a high-risk group; however, most high-risk
patients do not develop serious RSV disease. This suggests that nonmedical or
environmental risk factors play a disease-modifying role. These factors have been studied
in several large cohorts of healthy children and neonates belonging to a well-defined risk
group, usually with a history of prematurity.
Carbonell-Estrany et al. [18] studied a nationwide cohort of premature infants born
at v32 weeks of gestation and determined additional risk factors during hospitalisation
for RSV infection. Independent prognostic factors for RSV-related hospitalisation were
as follows: lower gestational age, a chronological age of v3 months at the onset of the
RSV season, living with school-age siblings and exposure to tobacco smoke. The group
extended their analysis to the group of premature infants born at a gestational age of 33
35 weeks and confirmed the findings from the first study [19]. In addition, living with
more than four persons in the home, breastfeeding forv2 months and a family history of
wheezing were associated with a higher risk for hospitalisation due to RSV infection in
the earlier study.
Law et al. [20] analysed a similar cohort in Canada (Pediatric Investigators
Collaborative Network on Infections in Canada; the PICNIC study) and reported the
same risk factors. In addition, day-care attendance and male sex were significantly
172
Fig. 2. Radiograph of the thorax of a patient with respiratory syncytial virus bronchiolitis, showing
hyperinflation and patchy bilateral infiltrates.
Fig. 3. Direct immunofluorescence test showing viral protein in exfoliated nasopharyngeal epithelial cells.
associated with a high hospitalisation risk. Surprisingly, a family history of eczema was
protective. These data were confirmed by Liese et al. [21] in the Munich RSV Study Group.
It is clear from these studies and others that many environmental and demographic
risk factors contribute to the risk of hospitalisation after initial RSV infection in early
childhood [22].
173
Pathogenesis of disease
It is becoming increasingly clear that local and systemic immune mechanisms initiated
by RSV infection of the respiratory epithelial cells are mainly responsible for the
pathogenesis of disease. RSV has a direct cytopathic effect, but infection by itself is
insufficient to cause severe disease [23]. The inflammatory infiltrate in the lungs, devoted
to eliminating the virus from the body, causes most of the pathology. Several arguments
support this statement. First, during immunisation trials with a formalin-inactivated
virus, it was noted that children became more severely ill than controls after subsequent
natural infection [24]. Secondly, although immunodeficient laboratory animals
challenged with RSV shed the virus for prolonged periods of time, they do not develop
signs and symptoms unless they are reconstituted with cellular components of the specific
immune system [25]. Thirdly, although RSV infection of severely immunocompromised
children occurs and can occasionally be very severe, a much higher incidence of RSVrelated morbidity and mortality would be expected against the background of the
ubiquitous presence of RSV in the community during yearly winter epidemics. Fourthly,
RSV-related symptoms are at their worst at a point in the course of infection when viral
replication is already decreasing and the immune response has reached its maximum.
Finally, antiviral therapy with ribavirin does not influence the course of the disease
considerably, although RSV is highly sensitive to the titres of drug that can be obtained
in the lungs.
The immunological mechanisms underlying disease pathogenesis after infection with
RSV have been the subject of extensive research since the vaccine trials in the 1960s.
Although a number of possible pathogenetic mechanisms have been proposed, no one
single hypothesis provides all the answers. Probably the development of bronchiolitis
after primary infection with RSV, the later development of recurrent wheezing, and the
relative protection for severe illness after second and later infections, are the result of a
complex interplay of a broad array of different factors. The most important recent
developments are summarised in the following paragraphs.
Role of T-cells and antigen-presenting cells

A specific antiviral immune response is a prerequisite to clear the virus from the lungs.
RSV-specific CD4z and CD8z T-cells have a pivotal role. In the absence of specific Tcells, the virus is shed from the upper and lower airways for much longer periods than in
the presence of an intact immune system. However, when laboratory animals are
reconstituted with selected populations of RSV-specific T-cells, they not only clear the
virus, but also develop an inflammatory infiltrate in the lungs, leading to further damage
of infected epithelium and bronchial hyperreactivity, similar to bronchiolitis in infants.
These experiments prove that, although necessary for recovery and protection, RSVspecific T-cells contribute to the pathogenesis of disease. It has been shown in the mouse
model that CD4z T-cells directed to the fusion (F) protein of RSV elicit a protective,
beneficial immune response, characterised by the induction of an effective cytotoxic
CD8zT-cell response, a strong antiviral antibody response and T-helper cell (Th) type 1
cytokine production, e.g. interferon (IFN)-c. Conversely, CD4z T-cells directed to the
attachment (G) protein of the virus result in a Th2 response, e.g. interleukin (IL)-4, IL-5
and IL-10, and a strong eosinophil influx into the airways [2, 26, 27]. However, this clear
difference under controlled circumstances in the mouse model is more controversial in
humans. Although a Th2-skewed immune response was found by some investigators, it
could not be shown by others [28, 29]. Moreover, if RSV bronchiolitis was a Th2-type
disease, a strong correlation with allergy would be expected, both as a risk factor for
174
severe disease or as an outcome parameter after acute RSV bronchiolitis. However, most
prospective, long-term follow-up studies do not show a relationship between RSV disease
and allergy [30, 31]. Only the study by Sigurs et al. [32] suggests that RSV might be a risk
factor for sensitisation against aeroallergens and subsequent allergic symptoms.
Although it is evident that T-cells play an important role in both the recovery from
and the pathogenesis of RSV-related disease, their action is not necessarily following the
Th1/Th2 paradigm.
The role of innate immunity has recently received attention. Eosinophils, neutrophils
and antigen-presenting cells are all contributing to the pathogenesis of disease.
Neutrophilic granulocytes and their secretory products can readily be detected in the
airways of infected children and correlate with disease severity. McNamara et al. [33]
measured high concentrations of IL-9 in the airways of children with severe RSV disease
and proved neutrophils as the source of this regulatory pro-inflammatory cytokine.
Garofalo et al. [34] correlated high levels of eosinophil cationic protein with disease
severity in children with RSV infection. RSV inhibits apoptosis of these cells in vitro [35].
Monocytes and dendritic cells are mobilised to the mucosa during RSV infection.
These cells could play a role in enhancing the immune response, contributing to the
development of airway inflammation and airway hyperreactivity [36, 37]. Alternatively,
they could contribute to a delayed activation of T-cells, possibly delaying an effective
antiviral response [38].
Dual infections
It has been suggested that concurrent infections with other microorganisms might
contribute to the severity of disease due to RSV. Hament et al. [39] have shown in vitro that
pneumococcal binding to respiratory epithelial cells is enhanced by pre-infection of the cells
with RSV (fig. 4). Also, concomitant infection resulted in a more pronounced attachment
of pneumococci to human epithelial cells. The effect was dose-dependent and was present
to a different degree in different pneumococcal strains. These in vitro data were supported
by clinical data from South Africa suggesting that pneumococci have a major role in the
development of pneumonia associated with RSV [40]. Moreover, in a large study, Semple
et al. [41] showed that co-infection with RSV and hMPV resulted in more severe disease.
The rate of dual infection was 72% in a population of bronchiolitis cases admitted to the
paediatric intensive care versus 10% of cases admitted to the medium care ward.
Genetics of the host

RSV bronchiolitis is the result of a complex interplay between the virus and the host
immune response. There is increasing evidence that the genetic make-up of an individual,
mirrored by the presence of polymorphisms in genes important for the immune response,
determines to a significant extent the outcome of infection. This has been shown for
several infectious diseases, e.g. tuberculosis and otitis media. Recently, polymorphisms in
genes playing a role in the innate and specific antiviral response have been associated
with severity of disease after infection with RSV (fig. 5).
Lofgren et al. [42] showed that polymorphisms in genes coding for surfactant protein
(SP)-A and -D are associated with a variable outcome after infection with RSV [42, 43].
One particular SP-A haplotype, 6A2/1A3c, was associated with more severe RSV disease,
while another haplotype, 6A/1Ab, seemed to be protective. In the SP-D gene, the variant
with a methionine at position 11 was associated with more severe disease.
In addition, polymorphisms in innate immunity genes can modify the immune response
upon infection with RSV. The findings of Venter et al. [44] and van Bleek et al. [45]
175
b)
a)
c)
d)
Fig. 4. Respiratory syncytial virus (RSV) infection of human epithelial cell line enhances adherence of
pneumococci. Adherence of pneumococci to a) uninfected and b) RSV-infected monolayers of Hep2-cells,
demonstrated by immunofluorescence with pneumococcus-specific antibodies. Scanning electron micrographs of
c) uninfected and d) RSV-infected Hep2-cells, showing adhering pneumococci. Scale bars=20 mm.
show that individual variation exists in reaction to T-cell epitopes on the N, F and G
proteins of RSV, even if these epitopes are well conserved in wildtype strains of RSV.
Venter et al. [44] showed that out of 37 adult volunteers, 21 reacted with a vigorous IFNc response upon challenge of their cells with overlapping peptides of the N protein of
RSV. Using peptide mapping, they showed a human leucocyte antigen (HLA)-B*08restricted epitope that induced a strong cytolytic response. This epitope also induced lysis
in HLA-A*02-restricted cells. van Bleek et al. [45] used a similar IFN-c Elispot
technique to study individual variation in reaction upon challenge with major
histocompatibility complex class II epitopes derived from the F protein of RSV. A set
of 31 overlapping epitopes was studied in the context of the most prevalent HLA
haplotypes. Although the cells of all individuals reacted to a number of peptides with a
176
Nonspecific handling
of viral particles
Surfactant proteins A and D
Innate immune response

and antigen presentation
MHC-restricted epitope
recognition
Specific antiviral
immune response
IL-4, IL-4R, IL-8, CCR5, IL-10

(TNF-a, TGF-b, IFN-g, IL-6)
Fig. 5. Polymorphisms in genes of different phases of the immune response determining disease outcome.
MHC: major histocompatibility complex; IL: interleukin; CCR5: chemokine receptor 5; TNF: tumour necrosis
factor; TGF: transforming growth factor; IFN: interferon.
measurable IFN-c response, there was a striking inter-individual variation in the extent
to which epitopes were recognised by different individuals. The same research group
extended their research to epitopes of the G protein and found similar results [46] (fig. 6).
Tal et al. [47] showed that the Asp299Gly and Thr399Ile polymorphisms in the tolllike receptor-4 were associated with more severe RSV disease, either each of the
mutations alone or in co-segregation. Severe RSV cases were defined as children in need
of admission and eventual supportive care. They were compared with a group of mild,
outpatient RSV cases and a group of healthy adult controls.
56
200
120
40
50
Spots106 PBMC
30
10
14 15
20
19
13
50
38 39
28
1
72
57
58
84
2
39
33
45
57
43
57
30
56
60
40
20
40
20
66
56
80
60
40
20
80
1415
51
39
10 15
84
3
91
4
14
37
23
Pool-1 Pool-2
50 51
40
Pool-3
Pool-5 Pool-6
79
77
Pool-7
Pool-8
87
95
95
77
Pool-9 Pool-10 Pool-11 Pool-12 Pool-13 Pool-14 Pool-15
Fig. 6. Determination of antigenic peptides within positive peptide pools. Direct interferon-c ELISPOT assays
were performed with single 18-mer F peptides at 20 mM. Only peptides of pools that were positive in previous
assays were tested. The values given are the number of spots per 10-6 peripheral blood mononuclear cells
(PBMC) minus the number of spots in unstimulated PBMC. Rows from top to bottom represent the following:
donor VB-5, human leukocyte antigen (HLA)-A2, B35, 62, C4 DRBI *0401, DRBI*0403, DQBI*03; donor CE3, HLA-A2, B40, DRBI*03, *0401, DQBI*02, 03; donor VB-2, HLA-A1, 29, B44, 57, DRBI*0701, DRBI*1101,
DQBI*03; donor CH-1, HLA-A2, B27, 60, C2, 3, DRBI*15, *16, DQBI*0602; and donor CE-4, HLA-A3, 28,
B7, 35, DRBI*01, DQBI*05. Reproduced from [45] with permission.
177
Hull et al. [48] demonstrated that the IL-8-251A polymorphism in the gene encoding
for the chemokine IL-8 was significantly more prevalent in children with severe RSV
disease versus controls. They suggested that this polymorphism was associated with
higher IL-8 production after in vitro stimulation with either endotoxin or RSV.
Moreover, this polymorphism seems to be associated with long-term consequences of
RSV bronchiolitis.
Hoebee et al. [49] reported on polymorphisms in the promoter region of IL-4, the IL-4
receptor and IL-10. In a large group of patients, using RSV bronchiolitis and controls,
they showed that the IL-4-590T polymorphism causes a cytosine change to thymidine at
position 590, and that this was associated with hospitalisation during RSV infection.
This association was stronger in children w6 months of age. The IL-4Ra-R551
polymorphism was similarly associated with severe RSV disease in children w6
months. These results were confirmed by Choi et al. [50] in a Korean population. In
addition, Hoebee et al. [51] showed that children homozygous for IL-10-592C or IL-10592A were more prone to severe RSV disease than heterozygotes. Wilson et al. [52]
showed the association of two single nucleotide polymorphisms in the IL-10 gene locus to
be associated with the need for mechanical ventilation. This unique example of
homozygous disadvantage had not previously been described for RSV.
Finally, Hull et al. [53] described the association of two polymorphisms in the
chemokine receptor 5 with severe disease. This receptor is recognised by RANTES
(regulated on activation, normal T-cell expressed and secreted) and macrophage
inflammatory protein-1a, and both chemokines are suggested to play an important role
in the pathogenesis of RSV-related disease [54].
The influence of age

RSV bronchiolitis runs its most severe course in the very young child, i.e. v6 months
old. Bont et al. [55] showed that nearly all children admitted to the intensive care unit for
mechanical ventilation had a post-conceptional age of v44 weeks. It is plausible that
small infants with a less mature immune response are unable to mount a strong antiviral
IFN-c response, making them most vulnerable to severe disease [56]. This is supported by
an elegant study by Culley et al. [57], who infected mice at different ages with RSV and
challenged the animals 2 weeks later with a second infection. Mice infected for the first
time at a very early age developed more severe symptoms (such as weight loss) than mice
infected later in life, where a weak IFN-c response but a strong IL-4 response was
mounted, and developed lung eosinophilia. All of this suggests a disadvantageous
immune response against the virus. Conversely, mice infected at a later stage did not
loose weight and mounted a strong IFN-c and only a very weak IL-4 response, leading to
neutrophil and not eosinophil influx in the airways. These findings suggest that the ability
to develop an efficient antiviral, i.e. Th1, response increases with age, which could be
reflected in the way RSV is handled in early life. These findings fit well with the
theoretical maturation model in the airways, which was proposed by Holt et al. [58], but
whether these findings in laboratory animals can be readily extrapolated to the human is
controversial.
RSV bronchiolitis and childhood asthma

Epidemiology
Several studies have confirmed a strong link between RSV bronchiolitis in early life
and the subsequent development of recurrent wheezing and asthma. Wheezing episodes
178
following RSV bronchiolitis occur in 4271% of infants, usually within 1224 months [59].
Current findings are in favour of the hypothesis that recurrent wheezing after bronchiolitis is
a mainly nonallergic condition that resolves with age, but that this may be different in
atopic and nonatopic children [60]. There is a debate as to whether RSV bronchiolitis is
also a risk factor for allergic sensitisation and early onset of immunoglobulin (Ig)Eassociated asthma.
The first prospective study to report a relationship between RSV bronchiolitis in
early life and the subsequent development of wheezing and atopy in childhood was
performed by Stein et al. [30]. They found that children suffering from RSV
bronchiolitis in early life have an increased risk of subsequent wheezing until 10 yrs of
age. In most children, post-bronchiolitic wheezing resolved by 13 yrs of age. This large
cohort of children with mild RSV bronchiolitis not requiring hospital admission had no
increased risk for the development of atopic asthma in later life. This suggests that RSV
bronchiolitis and atopic status are independent risk factors for the development of
asthma. This hypothesis is supported by a study by Korppi et al. [61], who found no
increased rate of asthma or allergic sensitisation after 1820 yrs in subjects requiring
hospitalisation for RSV bronchiolitis in infancy when compared with control subjects
from nonatopic families. Mild lung function abnormalities, such as decreased forced
expiratory volume in one second and mid-expiratory flow, were common in adults with
a history of RSV hospitalisation in infancy. It was concluded that bronchiolitis does
not directly predispose to the development of asthma and atopy, but is more likely to
serve as a marker for the presence of immunological abnormalities that are common to
both conditions. A second prospective study was described by Bont et al. [31]. They
followed a large cohort of children hospitalised for RSV bronchiolitis and observed
recurrent wheezing in approximately half of the patients during the first year of followup. This proportion decreased over time; however, the decrease was not linear, but
showed a seasonal pattern. The same group found that increased production of
monocyte-derived IL-10 is associated with the development of recurrent wheezing. This
indicates that virus-induced changes in monocyte cytokine responses can lead to late
asthmatic symptoms and that an allergen-driven Th2 cytokine profile is not necessary
for such sequelae [62].
Allergic sensitisation
Only the third prospective study linked RSV bronchiolitis with allergic sensitisation
and the development of allergic asthma in later life [32]. Sigurs et al. [32] found that RSV
bronchiolitis severe enough to require hospitalisation is a strong risk factor for the
development of allergic asthma in early adolescence. They followed a cohort of 47
children until they were 13 yrs of age. Acute bronchiolitis was the most important factor
predisposing for asthma. Sensitisation to common inhaled allergens assessed by skinprick tests or serum IgE antibodies was nearly twice as frequent as in the control group.
The finding that severe RSV infection predisposes to allergic sensitisation has yet to be
confirmed by other human studies.
It has been shown in guinea pigs that prior allergic sensitisation potentiates the
physiological and structural changes induced by acute RSV bronchiolitis [63]. Hence,
atopic status may increase the severity of RSV infections in children. Inversely, studies
using animal models have attempted to explain how early exposure to RSV can promote
an environment that contributes to the development of subsequent allergic airway
disease. It has been demonstrated in mice that RSV infection increases airway
hyperresponsiveness and peribronchial inflammation in response to allergen challenge
[64, 65]. Animal models have suggested many factors that may contribute to allergic
179
sensitisation after RSV infection. These factors include: timing of RSV infection with
respect to allergen exposure, prior allergic sensitisation, environmental conditions,
exposure to endotoxin and the genetic background of the animal [66]. It seems that despite
some similarities, the mechanisms leading to bronchial hyperresponsiveness induced by
RSV are different from those that follow allergen sensitisation and challenge [67].
Pathogenesis of bronchial hyperresponsiveness after bronchiolitis

It has been suggested that the immune response in children who develop recurrent
wheezing after RSV bronchiolitis is characterised by the release of Th2-type cytokines
[68]. However this could not be confirmed by other studies [29]. Besides the possible role
of variations in the RSV-specific immune mechanisms, there is evidence from animal
studies and recently one human study that the prototypic neurotrophin growth factor
(NGF), its receptors and the brain-derived neurotrophic factor are upregulated during
acute RSV infection [6972]. NGF is not only a major determinant of the acute
neurogenic inflammation, but may also be responsible for long-term effects on airway
reactivity. Piedimonte et al. [71] have recently shown that RSV causes a persistent
increase in the vascular permeability in response to sensory nerve stimulation, which is
still present after the acute phase of the infection has resolved. Piedimonte et al. [71]
proposed that sensory innervation in the airways undergoes remodelling after an acute
RSV infection, resulting in increased supply and/or responsiveness of the neural network.
This could explain the development of airway hyperresponsiveness observed in a large
proportion of children with a history of RSV bronchiolitis. Animal studies have also
found that RSV can persist in the lungs, even in the presence of adequate T-cell
immunity. Such latent RSV infection could increase susceptibility to viral or nonviral
antigens and facilitate the development of atopy [73]. Nevertheless, it currently remains
unclear how to explain the link between RSV bronchiolitis during infancy and later
asthma. Is it that viral infections directly damage the infant respiratory system and
promote the development of later asthma, or does bronchiolitis unmask an inherent
susceptibility or pre-existing lung function abnormalities? Both explanations are not
mutually exclusive.
Prevention of post-bronchiolitic wheeze

At present no study has convincingly identified any treatment strategy that can reliably
prevent post-bronchiolitic wheezing. Several placebo-controlled trials studied the benefit
of corticosteroid treatment on the development of subsequent wheezing. The majority of
these studies showed that administration of nebulised budesonide or oral prednisolone
during the acute episode do not prevent post-bronchiolitic wheezing or asthma in later
life [7477].
It is unclear at present whether preventing or delaying RSV infection could reduce the
number of children who suffer from recurrent wheeze during childhood. Results in
animal studies suggest that palivizumab might protect against the development of later
asthma. Investigators have demonstrated a protective role for palivizumab on RSVinduced neurogenic inflammation and measured a significant decrease in bronchial
hyperresponsiveness until 9 weeks after RSV infection in palivizumab-treated animals
[78, 79]. Human studies examining the long-term benefits of passive immunisation with
palivizumab are currently underway.
180
Prevention and treatment

Prevention
Prevention of RSV bronchiolitis in high-risk infants has evolved from standard
intravenous immunoglobulin to RSV-hyperimmune, polyclonal intravenous globuline
(RSV-IGIV; Respigam1; MedImmune, Gaithersburg, MD, USA) to monthly passive
immunisations with an intramuscular, humanised murine monoclonal antibody
palivizumab (Synagis1; Abbott Laboratories, Abbott Park, Illinois, IL, USA). RSVIGIV has never received much popularity in Europe due to the long duration of
intravenous administration, the theoretical risk of infection, the possible interference
with life-attenuated vaccinations and its high costs. In two double-blind, placebocontrolled trials, monthly palivizumab at a dose of 15 mg?kg-1 i.m. for 5 months
significantly reduced RSV-related hospitalisations (by 55% in 1,502 infants with
prematurity and/or bronchopulmonary dysplasia/chronic lung disease and by 45% in
1,287 infants with haemodynamically significant congenital heart disease) [80, 81]. Apart
from the reduction in hospital admissions, palivizumab and RSV-IGIV provided no
benefit for more serious outcomes such as mortality.
Due to its ease of administration, safety and effectiveness, palivizumab has become the
drug of choice for RSV prophylaxis of high-risk infants. The official recommendations of
the American Academy of Paediatrics for RSV prophylaxis [82] have not received
widespread acceptance in Europe. The astronomical costs of palivizumab and the local
variability in RSV severity have led to a debate about how and when it should be used
[83, 84]. Most infants requiring hospitalisation for severe RSV bronchiolitis are
previously healthy infants born at term and do not qualify for RSV prophylaxis. None of
the cost studies has shown economic benefit for palivizumab prophylaxis, although it is
difficult to generalise the conclusions of such studies [8590]. The risk of serious RSV
disease will be low for many infants included in the approved indications and the cost
may outweigh the potential benefits. Hence, many countries have published their own,
much more restrictive recommendations limiting palivizumab mostly to pre-term infants
v1 yr of age who require active treatment of chronic lung disease at the time of the RSV
season [9194].
Palivizumab has not been formally assessed in other high-risk infants, such as those
with immunodeficiency or cystic fibrosis, who might potentially benefit from passive
immunisation. On the horizon is a new monoclonal antibody that has proven to be
superior to palivizumab in microneutralisation studies. This compound is currently being
investigated in two phase 3 clinical studies that began in November 2004 [95, 96].
Despite an immense amount of research having taken place during the last decades, the
highly complex immunopathology of RSV disease has so far hindered the development
of a safe and effective vaccine. Research was held up greatly by the experience with a
crude formalin-inactivated, alum-precipitated RSV vaccine that potentiated RSV disease
in clinical trials [97]. Several strategies for the development of vaccine candidates are
currently followed, including live attenuated vaccines, subunit vaccines, recombinant
vectors expressing the protective antigens of RSV and DNA vaccines [96, 98]. In
addition, drugs that block RSV replication based on nucleic acid strategies have emerged
as potentially viable drug-development options [99].
Treatment
It is well recognised that supportive therapy remains the mainstay for the treatment of
bronchiolitis and that there is little justification for most of the pharmacological agents
181
traditionally administered to bronchiolitic children. The new paradigm of evidence-based

medicine, together with increased cost-awareness, has driven many efforts to educate
physicians and hospitals in the treatment of RSV bronchiolitis at home and in the
hospital, since it has been repeatedly demonstrated that inappropriate or unnecessary
medications are administered to infants with bronchiolitis [100103].
Most children with bronchiolitis can be managed at home by their parents with careful
attention to feeding and respiratory status. The decision to admit infants with
bronchiolitis to the hospital is based on their age, the presence of risk factors for severe
disease, the severity of respiratory distress, the ability to take oral fluids, and the social
and local circumstances. Treatment of hospitalised infants includes humidified oxygen to
maintain saturations above 92%, moderate fluid supply and minimal handling. Chest
physiotherapy plays no role in the treatment of bronchiolitic infants and is not without
potential risks [104].
Many pharmacological agents commonly used in the treatment of asthma are also
widely used in the management of bronchiolitis. The rationale for the treatment of
bronchiolitis with bronchodilators is weak. Airway obstruction is mainly a result of
mucus plugs and cellular debris from bronchial inflammation and epithelial necrosis. The
contribution of airway smooth muscle constriction (which is treatable) to the airway
obstruction is minor in most cases. Furthermore, the role of inhaled b-selective
bronchodilators has been debated for many years, and it is now well established that they
play a very minor role in the treatment of bronchiolitis. They produce only modest,
short-term improvements in clinical scores, but do not decrease the rate or duration of
hospitalisation. The inclusion of recurrent wheezers may have even biased these results in
favour of bronchodilators [105, 106]; however, this small benefit must be weighed against
the costs of these agents.
Inhaled epinephrine is thought to be the better agent with which to treat airway
obstruction in acute bronchiolitis, because epinephrine stimulates the a-adrenoreceptor
causing arteriolar vasoconstriction in the airway mucosa and subsequent reduction of
bronchial mucosal oedema [107]. Many studies reported some improvements in shortterm outcomes, such as clinical scores or respiratory mechanics. A systematic review of
eight randomised controlled trials on the use of epinephrine in bronchiolitis, however,
came to the conclusion that there is no significant outcome benefit that would justify its
use, although it is superior to salbutamol and placebo for its short-term benefits [108].
For treatment with corticosteroids, a recent meta-analysis found no benefit in either
length of hospital stay or clinical score for infants and young children with
uncomplicated bronchiolitis compared with placebo [109]. Specific data on the harm
of corticosteroid therapy in this patient population are lacking, and evidence available
today suggests that corticosteroids should not be used in bronchiolitis. This is true,
independent of the route of medication delivery or the dose, for both infants presenting
in the emergency room and those requiring hospitalisation. It remains unclear whether
steroids have a potential benefit in infants with chronic lung disease.
Infants progressing into respiratory failure from bronchiolitis are more likely to have
underlying risk factors and are characterised by immaturity of their cellular immune
system [110]. This particular group of infants might benefit from therapies that have
shown no effect in mild bronchiolitis. However, no medical therapy has yet satisfied such
expectations for critically ill children. Unfortunately, the majority of studies on virusinduced respiratory failure have not stratified their patients for the underlying
pathophysiology, obstructive (bronchiolitis) versus restrictive (pneumonia, acute
respiratory distress syndrome) disease [111, 112] (fig. 7). The most promising
intervention is the use of surfactant, which is justified on the presence of a dysfunctional
surfactant system in severe bronchiolitis due to increased surfactant inactivation [113].
The three previous randomised-controlled surfactant trials were small and difficult to
182
a)
Flow mLs-1
b)
d)
Flow mLs-1
210
140
140
70
70
0
c)
e)
210
21
42
63
84
400
f)
350
0
Volume mL
350
200
21
42
63
84
700
200
200
0
Volume mL
175
Fig. 7. Two distinct patterns of respiratory syncytial virus-induced respiratory failure. 1) Obstructive disease.
Bronchiolitis is characterised a) radiographically by pulmonary hyperinflation and a concave (obstructive) flow
volume curve during b) passive and c) forced expiration. 2) Restrictive disease. Pneumonia or acute respiratory
distress syndrome is characterised d) radiographically by alveolar infiltrates and by linear (restrictive) flow
volume curves during e) passive and f) forced expiration. Adapted from [111].
183
compare because of the different ventilator strategies [114116]. A meta-analysis of the

three studies demonstrated a nonsignificant reduction in the duration of mechanical
ventilation by 2.6 days [117]. No clear benefit has been found for the use of steroids in
three small trials performed in critically ill children with RSV-induced respiratory failure.
Larger studies are required to substantiate the weak evidence that corticosteroids may
accelerate clinical recovery in those children with obstructive disease [118].
Ribavirin is a nucleoside analogue resembling guanosine that interferes with viral
protein synthesis and is the only therapeutic agent available to treat the established
infection. The agent is now little used because of its high cost and the disappointing
results from placebo-controlled trials [119122]. It remains unclear if certain patients,
particularly the immunocompromised infants, may benefit from ribavirin therapy.
Other treatment modalities that have been tried and remain without benefit include
inhaled nitric oxide [123125], heliox [126], immune globulin [127], palivizumab [128] and
erythropoietin [129]. The low mortality of bronchiolitis suggests the use of expensive
therapy with questionable efficacy should be reserved for selected clinical situations.
Physicians must recognise that no treatment of bronchiolitis will reduce the length of
stay in the hospital. The implementation of evidence-based clinical practice guidelines
can result in significant changes in practice and may be cost saving [130].
Summary
Bronchiolitis is a common clinical syndrome in infancy. It is caused most often by
respiratory syncytial virus, although recently other causative respiratory viruses have
been identified. Bronchiolitis is followed in a considerable number of cases by
recurrent episodes of wheezing in the first years of life, but probably has no relation to
the development of atopy later in childhood. Although there is no effective treatment
for bronchiolitis, disease progression after infection can be attenuated by prophylactic
administration of humanised monoclonal antibodies. The pathogenesis of viral
bronchiolitis is the subject of intensive research because it is clear that the immune
response initiated by the viral infection is partially responsible for the damage of the
airways. The cells that have been implicated in this process are virus-specific cytotoxic
and T-helper cells, as well as antigen-presenting cells, such as dendritic cells.
Keywords: Corticosteroids, genetic polymorphism, palivizumab, respiratory syncytial
virus, wheezing.
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190
CHAPTER 13
Asthma in school-aged children and

adolescents
J.H. Wildhaber *, F.H. Sennhauser *, P.L.P. Brand #
*University Childrens Hospital Zurich, Zurich, Switzerland.
klinieken, Zwolle, The Netherlands.
Princess Amalia Childrens Clinic, Isala
Correspondence: J.H. Wildhaber, University Childrens Hospital Zurich, Steinwiesstrasse 75, 8032
Zurich, Switzerland. E-mail: johannes.wildhaber@kispi.unizh.ch
Based on the viewpoint that airway inflammation is the key pathophysiological

mechanism in childhood asthma, inhaled corticosteroids (ICS) have become the
cornerstone of its maintenance therapy. International guidelines have been revised and
updated, using an evidence-based approach rather than one based on expert opinion and
consensus. It would be assumed that such up-to-date evidence-based guidelines would be
widely accepted and used in clinical practice. Recent surveys, however, indicate that
guidelines are poorly adhered to, both by physicians and by patients. It is also becoming
increasingly acknowledged that while ICS effectively reduce symptoms of asthma, they
do not appear to alter the natural history of the disorder. This chapter will explore
explanations for these apparently disappointing findings, and will suggest strategies for
improvement.
The most recent fundamental developments in the field

Discrepancy between goals set in guidelines and goals achieved in daily practice
According to the guidelines, the goals of asthma treatment are: optimal asthma control
with little or no symptoms; undisturbed sleep; no severe exacerbations; no emergency
visits; normal lung function; and no limitations to daily activities such as school and
sports [1, 2]. Surveys in adults and children have shown consistently that these goals are
usually not met [37]. For instance, in a study of 572 Swiss children with asthma [7], 49%
of patients had troublesome symptoms, with disturbed sleep, restricted activities and
school absence (fig. 1). Compliance with ICS was poor: one in three patients took their
ICS v3 months per year [7]. In an American study, many physicians chose not to
prescribe ICS to children with asthma because they did not believe the medication would
be helpful [8]. Many patients with asthma use their inhalers incorrectly and adherence to
self-management plans is usually poor [9, 10]. Preventive measures are also often not
established. In the Swiss survey, 36% of children reporting symptoms on exposure to pets
kept their pets nonetheless [7]. In an American study, half of the environmental measures
taken by parents to reduce exposure to harmful stimuli in their asthmatic children were
unlikely to be beneficial [11]. These findings indicate undertreatment of childhood
asthma by both patients and physicians [3, 8, 12].
However, surveys in the USA and Finland found that forced expiratory volume in one
second (FEV1) was normal in most children diagnosed with asthma, even when their
ISSN 1025-448x.
191
J.H. WILDHABER ET AL.
100
Control of asthma %
90
80
70
60
50
40
30
20
10
0
46
79
1012
Age yrs
1316
Fig. 1. Control of asthma in children of different age groups. &: good control; h: satisfactory control; &:
unsatisfactory control; &: poor control. Control of asthma was significantly better in older children (p=0.0001).
Reproduced from [8] with permission.
symptoms and medication usage indicated poor control [13, 14]. This suggests that
children may also be treated with anti-asthma drugs for nonspecific respiratory
symptoms [15].
Clearly, goals defined in asthma guidelines reflect an "ideal" scenario, which is different
from what is happening in real life.
Maintenance therapy does not seem to alter the natural history of asthma
When ICS were first found to reduce asthma symptoms and improve lung function and
airways hyperresponsiveness [16, 17], it was hoped that starting ICS soon after
establishing a diagnosis of asthma in children could induce a long-lasting remission and
alter the natural persistence of the disorder. At that time, it had already been established
that asthma, once it has become chronic and persistent, tends to relapse after the
withdrawal of long-term maintenance treatment with ICS [18]. In an observational study,
improvements in lung function were more pronounced in children who started
budesonide therapy early in the course of their disease than in patients in whom
asthma had been present for some years before the start of ICS therapy [19].
Recent data appear to suggest, however, that ICS maintenance therapy does not alter
the natural history of childhood asthma. In the Childhood Asthma Management
Program (CAMP) study, budesonide maintenance therapy was clearly superior to
nedocromil or placebo treatment in terms of exacerbation rates, symptom-free days and
bronchial hyperresponsiveness. Unexpectedly, however, post-bronchodilator FEV1
improved to a similar degree during the study in all three treatment arms (fig. 2) [20].
Comparable findings were reported in a long-term study of budesonide treatment in
children from the Netherlands [21].
In the Steroid Therapy as Regular Treatment (START) study, children with recently
(v2 yrs) diagnosed asthma were recruited. They were treated with budesonide or placebo
for 3 yrs. Again, budesonide was clearly superior to placebo, with respect to exacerbation
192
ASTHMA IN SCHOOLCHILDREN AND ADOLESCENTS
105
3
PC20 mgmL-1
b) 4
FEV1 % pred
a) 110
100
95
90
2
Time yr
2
Time yr
Fig. 2. Changes in a) forced expiratory volume in one second (FEV1) after bronchodilator and b) dose of
histamine causing a 20% fall in FEV1 (PC20), in three groups of asthmatic children, treated for up to 4 yrs with
budesonide (???????), nedocromil ( ) or placebo (), in the Childhood Asthma Management Program
study. Reproduced from [20] with permission.
rates, oral steroid courses and symptom-free days. Differences in post-bronchodilator

FEV1 between the two groups were surprisingly small, however (fig. 3) [22].
In a study from New Zealand [23], following up a large group of children from the age
of 9 yrs until the age of 26 yrs, lung function appeared to "track" throughout childhood
and into adulthood (fig. 4), suggesting that loss of lung function in asthma occurs early in
the course of the disease (i.e. in early childhood) [24].
These results may be viewed as evidence that childhood asthma can be controlled by
ICS therapy, but that the natural history of asthma is one of chronicity and persistent
100
l
l
l
l
l
Baseline
1 yr
3 yr
FEV1 % pred
90
80
70
60
50
Fig. 3. Post-bronchodilator forced expiratory volume in one second (FEV1) in 7,421 children and adults
treated for 3 yrs with budesonide () or placebo ( ) who had had asthma for 2 yrs at study entry.
Data taken from the Steroid Therapy as Regular Treatment trial [22].
193
b)
a) 95
90
FEV1/FVC %
l
t
n
85
u
l
t
n
u
t
l
u
l
t
n
u
t
l
n
s
u
u
80
l
u
n
s
t
l
u
n
t
s
l
u
n
t
s
s
u
l
u
n
s
t
u
u
u
t
l
n
n
s
u
u
l
n
t
s
u
l
n
s
t
u
u
t
l
n
s
u
u
l
t
n
s
u
s
u
s
75
70
11 13 15
18
Age yrs
21
26
11 13 15
18
Age yrs
21
26
Fig. 4. Changes in forced expiratory volume in one second (FEV1)/forced vital capacity (FVC) from school
age to early adulthood in a) males and b) females from a birth cohort study of 613 subjects in New Zealand. A
clear "tracking" pattern is obvious, irrespective of the severity of asthma. %: no wheezing ever; $: transient
wheezing; &: intermittent wheezing; ,: remission; +: relapse; ): persistent wheezing. Reproduced from [23]
with permission.
lung function abnormalities [25]. Such an interpretation could fuel carelessness in

treating childhood asthma: if long-term outcome is not influenced by ICS therapy, why
bother taking the treatment daily for years on end?
The present authors believe, however, that alternative explanations are likely for the
data reviewed above. The patients in the CAMP study and in the Dutch study had had
asthma for an average of 5 yrs before ICS therapy was instituted [20, 21]. In the START
study, patients had asthma that was diagnosed 2 yrs ago. In the START study,
however, adding ICS therapy to study treatment was allowed at the discretion of the
physician. Being almost twice as common in the placebo group as in the budesonide
group, this effect reduced the apparent effect of the study drug (budesonide) on lung
function considerably. Finally, the New Zealand cohort study was recruited in the 1960s,
well before the introduction of ICS treatment. The reasonable conclusion from these
data, therefore, would be that chronic persistent asthma that has not been treated with
ICS for years on end is associated with persistent lung function abnormalities. In most
cases of childhood asthma, however, lung function normalises during ICS therapy [14,
22]. At present, it is unclear whether ICS therapy started early in the course of the disease
alters its natural history. Even if this is not the case, the clinically relevant improvement
in symptoms, lung function, daily activity levels and quality of life consistently observed
in long-term studies of ICS therapy in childhood asthma justify its ongoing use and
emphasise the need for methods to improve long-term adherence to treatment. The
relevance of this was demonstrated recently in a study from the Netherlands [26]. Close
follow-up focused on improving inhalation technique and adherence to therapy was
associated with improvements in airways hyperresponsiveness and quality of life, whilst
at the same time the dose of ICS could be reduced by 25% (fig. 5). Conversely, it has been
shown that poor adherence to treatment is associated with an increased risk of asthma
exacerbations in children [27].
Based on the evidence reviewed above and by others, it is fair to conclude that most
children with apparently troublesome or difficult asthma have either poorly treated
asthma (owing to poor adherence or poor inhalation technique) or no asthma at all [28].
194
Paediatric QoL score
a)
4
b) 300
Baseline
6 months
u
s
u
s
250
u
s
mgday-1
12 months
s
u
200
150
100
Baseline
3
Follow-up months
12
Fig. 5. a) Changes in paediatric asthma quality-of-life (QoL) scores in patients newly referred to a paediatric
asthma clinic for chronic persistent asthma, followed up by paediatrician (&) or asthma nurse (&).
Improvements in QoL scores were both statistically significant (pv0.01) and clinically relevant (an increase of
w0.5 is considered to be clinically meaningful). b) This improvement was obtained despite a reduction in inhaled
steroid dose in both groups (%: paediatrician; +: asthma nurse) by, on average, 26%. The authors concluded
that improvements in inhalation technique were the most likely explanation for these findings [26].
Individual response to therapy

In clinical trials and meta-analyses, mean responses to therapy are compared between
treatment groups. Based on differences in these mean treatment responses, judgements
are made regarding the relative effectiveness of different treatments in asthma. Although
variability in responses to therapy has always been acknowledged, the systematic study
of differences between individuals in treatment response has only just begun. Responses
to inhaled b2-agonists have been found to be dependent on genetic polymorphisms of the
b2-adrenergic receptor [29]. Patients with the arginine (Arg)/Arg genotype, which is more
common in African-Americans, responded poorly to albuterol (salbutamol), while
patients with the glycine (Gly)/Gly genotype showed much more favourable responses to
the same drug. In a crossover study of 126 children with asthma [30], responses to
montelukast and fluticasone were compared not only at group level but also between
individuals. "Treatment response" was operationalised as a 7.5% improvement in FEV1.
195
Using this definition, 23% of patients responded to fluticasone, 5% to montelukast and

17% to both [30]. Montelukast-responders were more likely to be younger and to have
asthma of shorter duration, while fluticasone-responders were more likely to be atopic, to
have high exhaled nitric oxide (NO) levels, and to be hyperresponsive [30]. Interestingly,
more than half of the patients appeared to respond to neither therapy. Clearly, such results
are dependent on the definition of "response". Although a 7.5% improvement in FEV1
seems a logical definition of response, it should be noted that this was a short-term study
(each treatment period lasted 8 weeks), and that many patients had normal lung function at
the start of the study (mean FEV1 in the whole study population was 90% of predicted), so
there was little room for improvement [30]. The same problem appeared in the CAMP
study (fig. 2) [20], in which post-bronchodilator FEV1, the primary outcome variable, was
normal or near-normal in the majority of patients at the start of the trial. If this variable
alone had been examined, CAMP would have been viewed as a negative study. This
suggests that defining response in terms of a single outcome parameter may be tricky.
Moreover, in many clinical trials, changes in the primary end-point follow a normal
distribution between patients. While the mean response may be relatively small, a certain
subgroup of patients may show dramatic improvements. It is tempting to explore this
phenomenon after the data have been obtained, but such a post hoc analysis is scientifically
invalid for purposes other than to generate new hypotheses, which must then be tested in
separate studies [31]. Another factor compounding the issue of treatment response is that
responses to treatment may vary over time in individual patients.
While differences in response to therapy are apparent, studying such differences in
treatment response is difficult and depends, among other factors, on the choice of the
primary end-point and the effect size of the primary end-point considered to reflect a
clinically meaningful response (i.e. the definition of treatment response). The art and
science of defining treatment response in childhood asthma is in its infancy, but will
undoubtedly become a major issue in future studies. It is highly likely that "blanket advice"
(as is the rule now in guidelines) will be replaced by individualised advice in future
guidelines on asthma therapy but also in guidelines on the prevention of asthma [32].
The gap between patients and doctors beliefs

Although the approach of basing guidelines on evidence from clinical trials is laudable,
clinical practice is quite different from clinical trials. First, adherence to medication is
usually higher in trials than in practice. When improvements are large irrespective of
treatment group, this may obscure effects of the treatment being studied [33]. In addition,
as discussed above, the choice of inclusion and exclusion criteria influences the
generalisability of study results: they can only be applied to patients with similar
characteristics, which excludes many patients in clinical practice. Finally, outcome
measures in clinical trials are usually chosen because they can be measured easily or
because they make sense to doctors, not because they are meaningful to patients
(surrogate end-points) [34]. Most children with asthma dont really care what their FEV1
is, as long as they dont experience symptoms or limitations in daily life.
Current concepts and strategies

Pathophysiological mechanisms
Airway inflammation is widely accepted as the key pathophysiological mechanism in
asthma, both in adults and in school-aged children and adolescents. An excellent review
196
on asthmatic airways inflammation is available elsewhere [35]. It should be stressed,

however, that most data on inflammation in asthma are obtained in otherwise healthy,
young adults with atopic asthma. Although studies on the pathophysiology of childhood
asthma are on the increase, the evidence on inflammation and repair mechanisms in
childhood asthma is still very scanty indeed. The results of published studies are
summarised in tables 1 and 2. The data in these studies are inconsistent and often not in
accordance with studies in adults. For instance, although evidence of eosinophilic
inflammation has been found in many studies of childhood asthma [36, 39, 41, 43, 4850,
53, 55, 56, 59], a number of other studies could not confirm this [38, 40, 42, 51, 52, 54].
Neutrophil counts in bronchial biopsies and bronchoalaveolar lavage (BAL) fluid have
sometimes been found to be elevated [43, 52, 53, 55], and in one study they even appeared
to relate to the persistence of the disease [55]. However, in many other studies they were
not elevated [38, 40, 42, 49, 50, 51, 56]. The only really consistent finding is thickening of
Table 1. Results of mucosal biopsy studies in childhood asthma
First author [Ref.]
VAN DEN TOORN [36]
TSCHERNIG [37]
Subjects
Main biopsy findings
Comments
Young adults (1825 yrs old)

with a) asthma since
childhood (n=19),
b) childhood asthma in
remission (n=18),
and c) controls (n=17)
Trachea samples of infants
with SIDS (n=29)
RBM thickening,
eosinophils, mast
cells and T-cells
Biopsy findings comparable

in ongoing asthma and
asthma in remission
No dendritic cells in
tracheal mucosa in
infants dying of SIDS
RBM thickening, active
fibroblasts, mast cells
and lymphocytes
RBM thickening,
eosinophils
Dendritic cells found in

tracheal mucosa in
older children
Eosinophils found in
only one patient
COKUGRAS [38]
Children (514 yrs old) with

moderate asthma (n=10)
PAYNE [39]

difficult asthma (n=19)
JENKINS [40]

difficult-to-control
asthma (n=6)
RBM thickening,
smooth muscle
hypertrophy,
lymphocytes
BARBATO [41]
Children (412 yrs old)

with mild-to-moderate
asthma (n=9)
RBM thickening,
eosinophils
PAYNE [42]
DE
BLIC [43]
SAGLANI [44]

Infants (326 months old)

with severe wheeze or
cough (n=53)
RBM thickening,
CD4z lymphocyte
density higher in
patients with persistent
airflow limitation
RBM thickening,
eosinophils, neutrophils;
IFN-c levels
higher in children
with few symptoms
No RBM thickening,
no eosinophils
Biopsies taken after

2 weeks of oral
prednisolone
No eosinophils or
neutrophils found;
normal FEV1 possible
despite significant
RBM thickening
Biopsy findings
comparable in atopic
nonasthmatic subjects
Biopsies taken after
2 weeks of oral prednisolone;
no differences between
asthmatics and controls
in number of eosinophils,
neutrophils and CD4z
lymphocytes
Bronchial biopsies taken
46 weeks after a 2-week
course of oral prednisolone
RBM thickness lower
in infants than in
616-yr-old children
with asthma
RBM: reticular basement membrane; SIDS: sudden infant death syndrome; FEV1: forced expiratory volume in
one second; IFN: interferon.
197
Infants (02 yrs old) with troublesome

wheezing (n=13)
Children (115 yrs old) with atopic

asthma (n=52); children (114 yrs old)
with nonatopic asthma (n=23)
Children (415 yrs) with asthma (n=14);
children (546 months old) with
recurrent wheezing (n=26)
Children (210 yrs old) with acute
asthma exacerbations (n=18)
Children (636 months old) with recurrent
troublesome wheezing (n=36)
Children (317 yrs old) with allergic

asthma (n=13)
Children (05 yrs) with troublesome
wheezing (n=20)
Children (415 yrs) with asthma (n=16);
infants (02 yrs old) with recurrent viral
wheeze (n=30)
Infants (03 yrs old; n=21) and children
(316 yrs old; n=58) with difficult
("unusual") asthma
Children (510 yrs old) with atopic asthma
in remission (n=25); children (37 yrs old)
with episodic viral wheeze in remission (n=10)
AZEVEDO [47]
STEVENSON [48]
BARBATO [52]
198
Children (1014 yrs old) with refractory

asthma (n=28)
MAHUT [58]
Exhaled NO correlated to ECP levels,

IFN-c/IL-4 ratio and TGF-b levels in BAL
Exhaled NO correlates with eosinophil counts
Eosinophils increased in atopic compared with

nonatopic asthma; neutrophils elevated in
persistent compared with episodic asthma
Eosinophils increased in asthmatics in remission
Eosinophil counts not increased; neutrophil

counts elevated in wheezy infants compared
with controls
No increase in eosinophils but neutrophils
higher in asthmatics than in controls
Lymphocytes, neutrophils, and eosinophils
increased in wheezy children
Neutrophil counts elevated in recurrent viral
wheeze and only mildly elevated in asthma
Eosinophils increased
Alveolar macrophages show less adenyl

cyclase uptake in wheezers than in controls
Alveolar macrophages in wheezers show
increased release of eicasanoids compared
with controls
Alveolar macrophages in wheezers show
increased production of TNF-a compared
with controls
Atopic asthma: eosinophils and mast cells
increased; nonatopic asthma: total cell
count increased
Eosinophils and CD4/CD8 ratio increased
in asthma, but not in recurrent wheeze
Main BAL findings
Neutrophils, lymphocytes, alveolar macrophages

and mast cells not elevated in either group.
Nonbronchoscopic technique in patients
undergoing intubation for elective surgery
No increase in eosinophils, IL-8

and ECP in asthma; ECP was correlated
with neutrophil count
ECP and MPO were elevated in asthmatics
No control group, but comparison with infants

with acute viral bronchiolitis (neutrophils)
ECP was elevated in asthmatics and
correlated with neutrophil count
Also compared with children with cystic fibrosis,

in whom neutrophil counts were highly elevated

Comments
TNF: tumour necrosis factor; ECP: eosinophilic cationic protein; MPO: myeloperoxidase; IL: interleukin; NO: nitric oxide; IFN: interferon; TGF: transforming growth factor.
Children (514 yrs old) with asthma (n=29)
WARKE [57]
WARKE [56]
JUST [55]
MARGUET [54]
KRAWIEC [53]
AZEVEDO [51]
KIM [50]
MARGUET [49]
AZEVEDO [46]

recurrent or persistent wheeze (n=20)
wheezing (n=13)
Subjects
GALOPPIN [45]
First author [Ref.]
Table 2. Results of bronchoalveolar lavage (BAL) studies in childhood asthma
the reticular basement membrane, which was observed in almost all biopsy studies [36,
38, 4043], even in young children [39], but not in infants [44].
A major problem in all biopsy and BAL studies in childhood asthma is the small
sample size (tables 1 and 2) and the highly selected nature of most study cohorts, which
tend to consist of children with troublesome, therapy-resistant or otherwise unusual
clinical phenotypes of asthma. In some studies, biopsies were taken directly after a 2week course of systemic steroids, which may have had considerable influence on the
findings [39, 42]. At present, therefore, the presence and nature of airway inflammation in
usual or average clinical expressions of childhood asthma is largely unknown, and it is
quite likely that it differs considerably from the findings in adults. In addition, there is
evidence to suggest that the inflammatory profile of episodic viral wheeze differs from
that of atopic asthma [48, 49, 54, 55]. Clearly, a major research challenge is to try to
examine airways inflammation in childhood asthma in more detail in the future.
Related diseases: is asthma a risk factor for chronic obstructive pulmonary

disease?
Recent studies show that asthma may be a lifelong disease. Althoughy50% of children
with asthma go into remission during adolescence or early adulthood, evidence suggests
that airway inflammation in these individuals continues to exist [36, 56]. In addition,y50%
of the subjects with remission in early adulthood relapse later in life [60, 61]. A lower FEV1
in childhood is a strong predictor of asthma in adulthood [6062]. In addition, as discussed
above, FEV1 appears to track throughout life: children with lower FEV1 will continue to
have lower FEV1 in adulthood (fig. 4) [23, 63, 64]. In the CAMP study, children with lower
FEV1 were more likely to show a rapid decline in post-bronchodilator FEV1 over time [65],
suggesting that children with more severe asthma may show more rapid deterioration in
lung function over time. In most children with asthma, however, the rate of decline of FEV1
isv1% pred per year [65]. The mechanism behind this decline in lung function over time in
asthmatics is not entirely clear. There is some evidence from animal studies that chronic
allergic inflammation could induce the destructive changes observed in chronic obstructive
pulmonary disease (COPD) [66], but comparable pathophysiological data in humans is
lacking. Once chronic asthma persists into adulthood, the rate of decline of FEV1 increases
to about or above 1% pred per year [67].
Interestingly, young children with episodic viral wheeze or "wheezy bronchitis" also show
reduced lung function and an increased rate of decline in adulthood [68, 69]. This may also be
related to the tracking phenomenon: it is by now well established that episodic viral wheeze in
preschool children is associated with reduced airway calibre [70, 71]. This is in keeping with
the hypothesis that risk factors operating in early life or probably even in utero are important
in determining the risk of developing chronic diseases (such as COPD) in adulthood [72].
Although these data suggest that chronic asthma may be one of the risk factors for
COPD, the important issue of whether effective therapy for asthma can prevent this has
not been resolved. There is some evidence to suggest that treatment with ICS may reduce
loss of FEV1 over time in children with asthma [21].
Current treatment strategies

Long-acting b2-agonists: maintenance therapy, gaining asthma control and single
inhaler therapy
ICS are still the cornerstone of asthma therapy in children. As reviewed above, there
are good reasons to believe that the poor level of asthma control achieved by many
199
children with asthma in clinical practice [3, 4, 7] is primarily caused by poor adherence to
treatment by physicians, patients and parents.
In contrast to the situation in adults, where adding long-acting b2-agonists to ICS has
clearly been shown to be more effective than increasing the dose of ICS in patients with
asthma uncontrolled on ICS alone [1, 2], studies have failed to show similar effects in
children. In fact, the only study comparing a doubling in ICS dose with the addition of
long-acting b2-agonists to ICS and with an unchanged dose of ICS in children
uncontrolled on ICS alone showed no differences between the three groups [33].
Interestingly, all three groups showed an improvement in FEV1 (even the group in which
ICS dose remained unchanged), suggesting an important role for improved adherence to
treatment during the study. Although beneficial effects on a range of surrogate endpoints of adding long-acting b2-agonists to ICS have been reported in clinical trials [73
77], a systematic review showed that exacerbation rates (one of the most important endpoints for patients) was not reduced by adding long-acting b2-agonists [78]. Evidently,
although the addition of a long-acting b2-agonist has been recommended as the first
choice for "step 3" treatment in evidence-based guidelines [1, 2], the evidence to support
this in children has been lacking.
Recent studies, however, have shed some interesting new light on this issue. Not only
do ICS and long-acting b2-agonists show additive effects on inflammatory pathways,
they also enhance each others beneficial properties in vitro [79]. In a clinical trial in which
control of asthma was sought quite agressively in adolescents and adults, asthma was
totally or well controlled more frequently and with a lower ICS dose with a combination
product of fluticasone and salmeterol than with fluticasone alone [80]. Perhaps even more
impressive were the results of a study in which a combination of budesonide and
formoterol was used both as maintenance and as reliever medication ("single inhaler
therapy") in adults and in children 416 yrs of age, and compared to budesonide/
formoterol and to budesonide maintanance therapy with terbutaline as rescue
medication [81]. The cumulative severe exacerbation rate in the single inhaler therapy
group (in which the daily dose of budesonide/formoterol in children was only 100/6 mg
once daily) was y15%, compared to 30% in the two other groups [81]. Although these
results appear promising, the studys inclusion criteria (FEV1 60100% pred, with w12%
improvement in FEV1 after bronchodilator) limit its generalisability to the majority of
children with asthma, who have FEV1 levels in the normal range [67].
Other relatively novel approaches to asthma therapy in children include extrafine ICS
aerosols targeted at small airways, the addition of leukotriene receptor antagonists and
anti-immunoglobulin (Ig)E antibodies.
Extrafine ICS
Small airways are increasingly recognised as an important site of asthmatic airways
inflammation [82]. ICS aerosols based on extrafine solutions can be expected to penetrate
better into peripheral airways than than the regular ICS suspension aerosols. Extrafine
beclomethasone solution aerosol has been shown to be safe and effective in childhood
asthma compared with placebo [83], and the clinical effects of regular beclomethasone
aerosol and extrafine beclomethasone aerosol at half the daily dose were comparable [84].
However, the hypothesis that targeting small airways in this fashion is superior to using
traditional ICS formulations has not been substantiated in clinical trials in children to
date. It should also be kept in mind that a higher proportion of peripheral deposition
might lead to higher systemic absorption and hence to potential systemic side-effects,
which may be more pronounced in children than in adults. However, in a recent study in
24 children with asthma, such systemic side-effects of ciclesonide, a novel ICS in an
200
extrafine aerosol solution, were not found. During 2-week treatment with daily dosages
up to 160 mg?day-1 (which are the dosages intended for clinical use in children), no
evidence of systemic side-effects on lower-leg growth rate or hypothalamicpituitary
adrenal axis function was observed [85].
Leukotriene receptor antagonists

Montelukast, a leukotriene receptor antagonist approved for once-daily oral use, has
been quite extensively studied in childhood asthma. The drug is effective compared with
placebo, both in school-aged and preschool children [86, 87]. Perhaps the most promising
effect of the compound is its ability to reduce the number of virus-induced wheezing
episodes in young children [88]. Head-on comparisons of montelukast with ICS are rare
in children. In one such study, budesonide was more effective than montelukast in
reducing asthmatic symptoms and airways hyperresponsiveness caused by allergen
exposure [89]. In another, fluticasone was more effective than montelukast in improving
FEV1 in asthmatic children, although a subgroup of children responded more favourably
to montelukast than to fluticasone [30].
The present view of montelukast in asthma in school-aged children appears to be that
it is an alternative to adding a long-acting b2-agonist in step 3 therapy. It is not unlikely
that the indication for montelukast will expand to children with mild asthma [30] and to
preschool children with episodic viral wheeze [88].
Anti-IgE
To date, one study [90] has examined the effect of anti-IgE (omalizumab) on childhood
asthma. In this study, children with atopic asthma receiving omalizumab showed fewer
exacerbations and a larger reduction in ICS dose than children receiving placebo.
Although omalizumab was well tolerated, its high cost and administration by monthly
subcutaneous injection limit its use in clinical practice considerably.

As discussed above, the main reason for poor asthma control and hence for insufficient
asthma therapy is poor adherence. Therefore, improving adherence by any means seems
to be the most urgent need in asthma. Other problems may be the lack of diagnostic
markers for asthma in general and for individual treatment responses in particular.
Understanding the origin of the disease in more detail, especially the variety of gene
environment interactions as well as the variety of underlying pathophysiological
mechanisms, may help to define diagnostic tools as well as new therapeutic strategies.
The understanding of asthma as part of a systemic disease including all mucosal surfaces
(united airways disease) may greatly improve treatment outcome. As the long-term
effects of anti-inflammatory therapy on the natural course of asthma are largely
unknown, more research is needed in this area.
How can adherence be improved?

Looking at asthma statistics from epidemiological studies in developed countries it has
become obvious that the problem of poor asthma control is one of education (both for
201
patients and providers) and communication (two-way between providers and patients)
[4, 7].
Many medical records do not include the basic clinical information required to assess
asthma management [91]. Clinicians should not assume that they can predict which
patient (or family) is more or less compliant with management plans [11]. Multiple family
parameters, such as emotional characteristics, asthma management behaviours and
physiological factors influence asthma outcomes [92]. Parents reports of wheeze (they
use other words for wheeze and label other sounds as wheeze) and clinicians findings
differ [93, 94]. Airway inflammation as the underlying cause of asthma and symptoms is
often paid insufficient attention by patients and doctors. Although highly effective antiinflammatory medications are available, these are not taken or prescribed regularly in
many patients [4, 7]. Patients perceptions of asthma control and the prevalence of
symptoms are often mismatched (fig. 6).
At present, this mismatch can only be solved by the use of good communication skills,
experience and common sense by the practitioner. The logical approach is to try to define
shared treatment goals and management strategies between children with asthma, their
parents and the medical team. A recent study in atopic dermatitis showed that parents of
patients preferred sharing decisions with their doctor, but were forced to take on a more
active role because they felt that their childs condition was not taken sufficiently
seriously [95]. This may also be the case in asthma; it is an issue worth exploring.
Nonadherence
Patients
expectation
Doctors
view
Concordance
Fig. 6. Viewpoints and compliance. The circles represent the doctors and the patients beliefs. Best adherence
to treatment is achieved when the two circles are congruent, whereas the worst adherence is expected when the
circles do not overlap.
202
This requires an active attitude by doctors as well as their patients, along with close
follow-up. Such an approach has been shown to lead to improvements in bronchial
hyperresponsiveness and quality of life without an increase in medication. This is
probably the result of improved patient and doctor education, and improved inhalation
technique (fig. 5) [26]. In addition, it has been shown that educating patients regarding
the self-management of their asthma reduces both direct and indirect costs [96, 97].
A comprehensive treatment approach, taking into account patients and caregivers
individual needs and expectations, should become the cornerstone of any asthma
management plan in developed countries.
In developing countries, poverty and lack of education are likely contributing factors
to poor asthma control caused by poor adherence to treatment. Many patients in these
countries simply do not have access to or awareness of the appropriate treatment. Poor
healthcare infrastructure and/or the perception of asthma treatment as a low priority
may add to the problem of undertreatment of asthma in developing countries. Improving
the availability of appropriate treatment should be a major goal in improving asthma
control in developing countries.
Defining relevant outcome measures?

The assessment and evaluation of asthma relies on subjective markers, such as
symptom scores, the need for rescue medications and quality of life, and on objective
markers, such as lung function tests, including peak expiratory flow, FEV1, level of
bronchial hyperresponsiveness and markers of inflammation.
The frequency of rescue bronchodilator use is an excellent surrogate marker for poor
asthma control. In the Salbuterol Multi-center Asthma Research Trial study, the
apparent correlation of untoward outcomes associated with short-acting b2-agonist use
was most noticeable in African-Americans, who were almost half as likely to be on
concomitant ICS as their Caucasian counterparts [98]. The use of rescue bronchodilators
is part of most symptom scores. However, if a symptom score is to serve as an instrument
not only in follow-up but also in the primary evaluation, it would be better to exclude
rescue bronchodilator use. In addition, a symptom score including medication use does
not address possible differences in therapy strategies by various doctors and patients.
The quality of life of patients with asthma may be misunderstood by healthcare
professionals. Therefore, systemic assessment with standardised questionnaires [99102]
may contribute to better understanding between physicians, patients and their families
[103, 104]. Differences in expectations and responsibilities are common. Whereas the
patients view is that of a subjective satisfaction based on short-term expectations, the
doctors responsibility is that of objective improvement based on long-term expectations
(fig. 7).
National and international guidelines recommend an initial evaluation and regular
follow-up for assessing the severity and control of asthma [1, 2]. Pulmonary function
tests, mainly spirometry, are generally considered the standard criterion for objective
asessment of asthma severity during initial evaluation and follow-up, but even simple
spirometry is commonly unavailable in primary care [4]. At the very least, the initial
assessment of asthma severity should include an assessment of FEV1, according to the
guidelines. Children with mild persistent asthma are expected to have FEV1 valuesw80%
pred; children with moderate persistent asthma are thought to have values 6080% pred;
while FEV1 values v60% pred are viewed as evidence of severe persistent asthma in
children [1, 2]. However, given the evidence that most asthmatic children have FEV1
values in the normal range, independent of disease severity, it may be necessary to
redefine asthma severity in terms of FEV1 values for future guidelines [67].
203
Quality of life
c
d
a1 b1
c1
Objective outcome measure
d1
Fig. 7. The relationship between objective outcome measure and subjectively perceived quality of life is not
linear. For example, a huge improvement (from d1 to c1) or deterioration (from c1 to d1) in lung function does
not necessarily mean a huge improvement or loss in quality of life (d to c or c to d, respectively). However, a
small improvement (from b1 to a1) or deterioration (from a1 to b1) can have a huge impact on quality of life
(b to a and a to b, respectively).
The rationale for regular monitoring of lung function comes from two observations.
First, it has been shown that lower FEV1 values are a poor prognostic factor for the
outcome of asthma, both in the following year [105], and later in life as an adult [23, 60
62]. In addition, lung-function monitoring helps to identify "poor perceivers" who fail to
report wheeze or dyspnoea when their airways are obstructed [106, 107]. Despite these
apparently logical reasons for lung-function monitoring, there is no firm evidence from
randomised controlled trials to support monitoring of FEV1 in childhood asthma [67,
108]. Home monitoring of peak expiratory flow (PEF) has been advocated in guidelines
as an objective measure of asthma severity and to aid in self-management. It has been
shown, however, that the correlations between PEF and individual symptom scores,
spirometry and bronchial hyperresponsiveness in asthmatic children are weak [109114].
Moreover, the information provided in a PEF diary by apparently well-motivated
children with asthma and their families is unreliable [113]. In summary, there is no
evidence to support the routine use of home PEF monitoring in asthma management in
childhood [108]. Although novel portable electronic devices measuring PEF and FEV1
may allow a more accurate and reliable objective measure for home monitoring in
asthmatic children, they have been shown not to be of additional benefit in the
monitoring and follow-up of asthma [115117].
Whereas guidelines recommend that the monitoring of asthma should be based on
symptoms and lung function tests, only additional measurements such as airway
hyperresponsiveness and sputum eosinophils have been shown to lead to improved
outcomes, at least in adults [118, 119]. Although these results support the hypothesis
that such surrogate markers of airway inflammation are helpful in disease evaluation
and monitoring, their measurements have been time-consuming and difficult to
perform. Conversely, measurement of NO in exhaled breath (eNO) is an easily
obtained, noninvasive surrogate marker of airway inflammation in children. Recent
studies have shown that eNO monitoring is helpful in anti-inflammatory dose
adjustment in adults [120], and in prediction of exacerbations [121], control of
adherence [122] and monitoring of disease in children [123]. It is likely that this will be
implemented in future guidelines.
204
It will be crucial to define additional airway inflammatory markers that may be specific
for the evaluation and monitoring of certain disease patterns or therapeutic strategies [124].
While biopsy, BAL and sputum induction may be difficult and not suitable for use in clinical
practice, it should be stressed that these are highly valuable basic and clinical research tools.
In summary, it seems logical that specific single diagnostic markers have to be defined
for individual patient groups, as there is such heterogeneity in the disease expression; and
that, if generally applied, a set of various diagnostic markers is more likely to be useful in
larger populations.
How can we better understand individual responses to treatment?

It is now widely accepted that asthma is not a homogeneous but a heterogeneous
disorder. Several types of asthma in school-aged children are recognised, mainly allergic
(extrinsic) asthma and nonallergic (intrinsic) asthma. The heterogeneity of these different
types is highlighted by different pathophysiological patterns underlying various clinical
disease expressions [125]. The main pathophysiological mechanisms, such as airway
inflammation, airway remodelling and bronchial hyperresponsiveness are differently
expressed in different disease phenotypes (fig. 8).
It has been well accepted that T-helper (Th) cell type 2 cells play a pivotal role in
driving airway inflammation, both in atopic and in nonatopic asthma. Surprisingly,
however, treatments therapeutically targeting the Th2 pathway (either in general or at
specific steps of the pathway) have shown only modest benefits [126]. This may be
explained by recent data showing heterogeneity in the immune response patterns of
asthmatic children [127]. Atopy to inhalant allergens in children appears to be associated
with a mixed Th1/Th2 immune response profile. The contribution of individual Th1- and
Th2-associated effector mechanisms to this mixed response profile is highly heterogeneous. The immunologically hyperresponsive phenotype, consisting of high levels of
phytohaemagglutinin-induced interleukin-10, tumour necrosis factor-a and interferon
(IFN)-c, appears to be restricted to nonatopics with bronchial hyperresponsiveness. The
Th1 response not only has a protective role but also seems to have a driving role in
airway inflammation, as it both antagonises and synergises Th2 response [128]. These
findings may explain the disappointing results of trials employing Th2 antagonists and
may help develop understanding of individual responses to treatment observed in
practice.
Immune response to aeroallergens
BHR
Asthma
Intrinsic asthma
Atopy
High serum IgE

Fig. 8. Asthma is a heterogenous disorder. BHR: bronchial hyperresponsiveness; IgE: immunoglobulin E.
205
If the underlying pathophysiological mechanisms of asthma are heterogeneous, it

might be expected that the aetiology of these mechanisms, in particular the gene
environment interaction, is heterogeneous as well, and should, therefore, be assessed at
an individual level [32]. Many research groups are trying to identify genetic
polymorphisms associated with an increased risk of asthma [129]. However, a genetic
polymorphism may only be a risk for asthma when associated with an exposure to a
specific environmental risk factor. In addition, not only the exposure per se may be
important, but also the level and the timing of exposure. As a result, a specific exposure
may have both protective and causative roles in disease development and progression
(such as exposure to cats and dogs, which is described in more detail in Chapter 2).
Improved understanding of the variation in geneenvironment interaction, as well as
in pathophysiological mechanisms, should help to define novel individual primary,
secondary and tertiary therapy strategies. This should help to tailor individualised
asthma therapy and prevention, replacing the strategies aimed at the whole population
that are the current paradigm. Given the complexity of the issue itself and of the
necessary studies to explore it, it is unlikely that such individualised treatment will
become available in the next 10 years.
As pointed out earlier in this chapter, studying differences in response to treatment in
asthmatic children is hindered by the lack of a uniformly accepted definition of treatment
response in asthma. This issue has been elegantly addressed in a recent editorial [130],
which stated: "A limitation in the interpretation of the asthma literature is the
inconsistency in the definition of response. The heterogeneity and variability of asthma
make it difficult to achieve consensus for this term. This is compounded by the lack of
standardised means to assess asthma control, a measure of response."
Despite these difficulties, the importance of addressing individual therapy responses,
as well as the limitations of meta-analyses in this respect, is increasingly acknowledged
[131].
Improving the definition of new therapy strategies?

Based on the recognition that airway inflammation is the key feature in the
pathophysiology underlying asthma, ICS have become the mainstay of maintenance
therapy for childhood asthma. In certain cases, fear of steroid side-effects may limit the
adherence to ICS treatment, but this problem can usually be solved by appropriate
education of patients and parents. The search for new inhaled steroids with an improved
safety profile would probably provide some, but not a spectacular, benefit in terms of
widespread acceptance of ICS therapy. Conversely, a combination of anti-inflammatory
treatment strategies might be more promising in improving outcomes in childhood
asthma. Thus, the therapeutic potential in asthma of other anti-inflammatory drugs, such
as statins, warrants further evaluation [132]. In addition, therapeutic strategies aimed at
newly discovered pathways in the inflammatory cascade may also become increasingly
important. For example, the role of toll-like receptors in the innate immune system has
recently been recognised to be important in asthma [133]. Similarly, impaired innate
immunity involving a major deficiency in IFN-b appears to lead to virus-induced asthma
exacerbations [134]. Therefore, administration of toll-like receptor ligands or exogenous
IFN-b by inhalation may provide new ways of reducing airway inflammation or
preventing viral exacerbations in asthma.
Over the last few years, airway remodelling has become recognised as an important
pathophysiological feature of chronic persistent asthma. A combined therapy regimen
may more successfully inhibit remodelling processes than a monotherapy with inhaled
steroids, at least in adults [135138]. Finding the pathophysiological mechanisms of
206
remodelling processes may help in the development of specific and timely defined
therapeutic strategies (fig. 9).
Several mechanisms appear to be involved in these processes, mainly growth factors,
such as vascular endothelial growth factor, basic fibroblast growth factor and
angiogenin, which are involved in the angiogenesis seen in airway remodelling [139,
140]. Inhibition of such specific growth factors may, therefore, become a new therapeutic
strategy in the future.
Most drugs are delivered to the patient by inhalation. Despite the fact that this is
clinically succesful in many patients, poor inhalation technique is a very common
problem in children with asthma (and their parents) [9, 10, 141, 142]. This can be a major
reason for therapy failure and poor adherence. Consequently, there is a need for
improvement in drug output and deposition into the lower airways by inhaler devices
used for childhood asthma and in their ease of use, in particular in the younger age
group.
Is asthma a systemic disease?

Data from the literature suggest a systemic link between various mucosal sites of the
airways [124]. Although it is recognised that this link involves the bloodstream, bone
marrow and mucosa-associated lymphoid tissue, the exact mechanisms of interaction
between upper and lower airways in children are incompletely understood [124]. Both
genetic predisposition and environmental factors appear to contribute to the
development of specific phenotypes expressing disease activity in the upper airways,
lower airways, or both. The understanding of the mechanisms underlying the
relationship between allergic rhinitis, asthma and other atopic diseases may help to
define strategies to influence clinical manifestations of allergic disease in the future. For
the time being, it has been shown that treating allergic rhinitis in patients with both
asthma and allergic rhinitis not only improves upper airway symptoms, but also
improves asthma control (united airways) [143, 144].
?
Inflammation
Bronchial disease
Remodelling
Clinical
revevance
Time
Fig. 9. Asthma is a heterogeneous disorder with variable expression of its underlying pathophysiological
mechanisms. This may implicate that the timing and the type of treatment used should be based on individual
characteristics.
207
How can we influence the natural course of the disease?

As pointed out previously, it is unclear whether ICS therapy influences the natural
course of childhood asthma. Obviously, an answer to this question is needed urgently.
Due to the complexity of study design and the ethical issues involved, randomised
controlled clinical trials are not likely to provide the answer. Observational cohort
studies, therefore, are needed to assess the effect of current asthma treatment on the
natural course of disease and to tease out the point in the disease course at which it would
be best to intervene. The present authors believe that early intervention with ICS,
commencing shortly after the establishment of the diagnosis of asthma in children, and
continuing for prolonged periods, along with extensive education and close follow-up to
ensure proper adherence to treatment and correct inhalation technique, will provide the
best strategy to influence the natural course of asthma in the near future. In addition, it is
likely that combined or novel treatment approaches will be particularly helpful to those
patients with poor or limited response to ICS treatment.
Finding the answers to these questions

It is obvious that more studies are needed to address the questions raised above. More
effective educational programmes for patients and their caregivers, as well as for health
providers, have to be developed and implemented in asthma care worldwide.
Communication and collaboration has to be implemented as an integral part of
asthma management on all levels of patient care. Studies examining tools to improve
adherence with current medical therapy and to improve inhalation technique with
current inhalation devices are of equal importance to studies aimed at developing new
drugs and new inhalers.
Studies using invasive measures, such as biopsies and BAL, are pertinent for the
further understanding the mechanisms of the disease and hence, for the development of
new treatment strategies. It is essential that such studies continue to be performed despite
ethical constraints. Individual patients and patient groups should be better characterised
in order to reduce heterogeneity and diversity of study patients, and to improve
understanding, applicability, and generalisability of study results.
Patient groups with specific disease characteristics regarding genetics and pathophysiology have to be defined by characteristic diagnostic markers. More studies of
geneenvironment interactions in asthma and of its pathophysiology are needed to
improve understanding of individual responses to current as well as novel treatment
strategies, in order to provide tailor-made individual treatment recommendations
rather than blanket advice aimed at whole populations. In addition, long-term clinical
trials and well-designed observational studies assessing the impact of treatment
regimens on morbidity and mortality continue to have a high priority. Preferably, such
studies should not only be performed with the support of the pharmaceutical
companies manufacturing these medications, but also independently. In addition,
workshops and other meetings of scientists and practitioners to enhance the exchange
of ideas, data and experience, should be facilitated independent of support from
pharmaceutical companies.
Improved understanding of asthma as a systemic disease with a link to disease
manifestation in other mucosal sites may help to define interventional strategies to
influence disease progression. Well-designed long-term observational studies are needed
to improve understanding of the natural course of the disease and the effects of current
and novel therapeutic strategies on this natural history.
208
Until such new data become available, clinicians will need to focus on working with
children with asthma and their families, developing shared treatment goals and
expectations. Only through extensive education, close collaboration and follow-up can
adherence to treatment be improved, and undesirably poor asthma outcomes be
prevented.
Key messages
1) It is unclear whether the natural course of the disease can be influenced by antiinflammatory therapy and, if so, how early such a treatment has to be established.
2) Despite highly effective anti-inflammatory drugs (inhaled corticosteroids) being
available, they are not taken as prescribed in many cases. Goals defined in asthma
guidelines reflect an "ideal" scenario, which differs from what is happening in real life.
3) Improved understanding of individual response to treatment, and underlying
mechanisms, will change treatment strategies for asthma in the future, from blanket
advice given to all asthmatic patients to more tailor-made, individualised, treatment
plans.
4) Improved education of and collaboration with patients and parents is likely to be a
key factor in improving short- and long-term outcomes in children with asthma.
Summary
Although inhaled corticosteroids have become the key aspect of maintenance therapy
in childhood asthma, it is not clear at what point such treatment should begin and
indeed whether anti-inflammatory therapy can influence the natural course of the
disease.
A further complication is the fact that, in many cases, anti-inflammatory drugs are not
taken as prescribed. Thus, idealised asthma guidelines do not reflect the reality of
asthma management.
As understanding of the underlying mechanisms of, and individual responses to, antiinflammatory treatment improves, it is likely that treatment strategies will evolve
towards more individualised, bespoke plans based upon this new information.
With this in mind, clinicians need to work closely with children with asthma, as well as
with their families, to develop an understanding of individual cases and to educate
patients about their treatment. In this way, short- and long-term outcomes can be
improved.
Keywords: Adherence, asthma, inhaled corticosteroids, remodelling, self-management.
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216
CHAPTER 14
Bronchopulmonary dysplasia: current

models and concepts
A. Greenough*, S. Kotecha#, E. Vrijlandt}
*Division of Asthma, Allergy and Lung Biology, Kings College London School of Medicine at Guys,
Kings College and St Thomas Hospitals, London, #Dept of Child Health, Wales College of Medicine,
Cardiff University, Cardiff, UK, and }Division of Paediatric Pulmonology, Beatrix Childrens Hospital,
Groningen University Hospital, The Netherlands.
Correspondence: A. Greenough, Regional Neonatal Intensive Care Centre supported by the WellChild
Trust, 4th Floor Golden Jubilee Wing, Kings College Hospital, Denmark Hill, London SE5 9RS, UK.
Fax: 44 2073468284; E-mail: anne.greenough@kcl.ac.uk
Bronchopulmonary dysplasia (BPD) represents a spectrum of disease, whereby infants

remain oxygen dependent for prolonged periods and have abnormal chest radiological
findings. A variety of names, including chronic lung disease (CLD) of prematurity, have
been given to this condition. The consensus at a National Institute of Child Health and
Human Development (NICHD)-sponsored workshop was to use the term BPD to
describe all prolonged oxygen-dependent infants, as BPD rather than CLD better
distinguishes the neonatal lung process from chronic lung illnesses seen in later life [1].
The first report of BPD was by Northway et al. [2], who described four stages of BPD
according to a sequence of chest radiograph changes. Since that report, the spectrum of
disease has changed, with the introduction of new modes of mechanical ventilation with
gentler delivery of pressure and volume, routine administration of treatments, such as
surfactant, and the survival of extremely prematurely born infants. The incidence of BPD
in very low birthweight infants has been reported to vary from 15 to 50%, the differences
relate to the proportions of very immature infants included in the populations studied
and the definition of BPD used. The incidence of BPD is inversely related to gestational
age [3]. Various criteria have been used to diagnose BPD, including oxygen dependency
at 28 days of age or 36 weeks post-menstrual age (PMA) and the chest radiograph
appearance. A consensus regarding definition would permit comparisons between centres
and with historical data. At the NICHD-sponsored workshop, it was proposed that
babies should be considered to have BPD if they had been oxygen dependent for
i28 days, then be classified as suffering from mild, moderate or severe BPD according
to their respiratory support requirements at a later date [1].
In this chapter, the long-term morbidity associated with BPD, which emphasises the
need for successful preventative strategies, is summarised. Models currently used to
investigate the pathogenesis and efficacy of therapies and the quality of evidence
supporting current prophylactic therapies and BPD treatments are described.
Furthermore, the important future research questions are highlighted.
Models of bronchopulmonary dysplasia

Animal models have significantly improved the present understanding of the
development and prevention of BPD, but positive effects in animal models do not
necessarily translate into clinically meaningful outcomes in prematurely born infants.
ISSN 1025-448x.
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A. GREENOUGH ET AL.
Using prematurely delivered baboons and lambs, the responses to injuries inflicted
antenatally (e.g. amniotic injections of bacterial toxin or microbes such as Ureaplasma
urealyticum) or post-natally (oxygen supplementation or mechanical ventilation) have
been investigated. The baboon model, although very expensive, is the closest model to
the human premature infant. Rodent and rabbit models of BPD have also been used;
these are easier to handle. There are, however, limitations to animal models, and these
include differences in lung growth and development compared with the human infant. In
addition, animal models are delivered at predetermined times, whereas human infants
frequently deliver following pre-term labour and factors that lead to pre-term labour may
also prime the foetus to lung injury.
Pathology of BPD
The pathology of infants with BPD has changed over the last four decades from socalled "classical" to "new" BPD, reflecting differences in the patients and the therapies
used.
When Northway et al. [2] described classical BPD, his population was relatively
mature and they responded to the risk factors for BPD with fibrosis and smooth muscle
augmentation of medium-sized airways, resulting in airway obstruction. The present
population of BPD infants are often born very prematurely and lung fibrosis is replaced
by abnormalities of lung growth, with less smooth muscle encircling larger airways, but
markedly decreased numbers of alveoli [4, 5]; this is often termed new BPD. As a
consequence, the chest radiograph appearance has changed from one that demonstrated
cystic abnormalities and interstitial fibrosis (fig. 1) to often one showing only smallvolume hazy lung fields. The differing responses to the risk factors for BPD may reflect
that the more mature BPD infants described by Northway et al. [2] were delivered at a
relatively late stage in the development of the lung with alveolarisation having
commenced, whereas the more prematurely born infant may be delivered in the saccular
stage of development.
Fig. 1. Chest radiograph of an infant with severe bronchopulmonary dysplasia. Note the gross widespread
interstitial changes. The infant also has osteopenia.
218
PREVENTION AND MANAGEMENT OF BPD
Pathogenesis
Lung inflammation is important in the pathogenesis of BPD. The neutrophil is central
in mediating this inflammation and many pro-inflammatory cytokines, such as
interleukin (IL)-1b, IL-6 and the neutrophil chemotactic factor IL-8, are increased in
babies who develop BPD [6, 7]. Furthermore, products from activated neutrophils, such
as proteases and reactive oxygen species, have been described in infants who develop
BPD. The inflammatory phase may commence antenatally, with infection being the most
likely initiator. Yoon et al. [8] described increased pro-inflammatory cytokines, including
IL-1, IL-6 and tumour necrosis factor-a, in the amniotic fluid of females who
subsequently delivered prematurely and whose infants progressed to develop BPD.
Resolution of the acute lung injury appears to be mediated by alveolar macrophages, the
numbers increase during the second week after birth in infants who develop BPD.
Macrophages synthesise and release growth factors, which lead to repair and remodelling of
the injured lung. The same growth factors are involved in normal lung growth; thus, it is
likely that they are responsible for the dysregulated lung growth that is observed in infants
who develop BPD.
Any of the risk factors for BPD described below can lead to the inflammatory
pulmonary response seen in animal models. The challenge is to understand why the very
immature infant responds to similar insults with greater pulmonary injury than the
relatively mature infant. Is it that the extremely pre-term infants lungs are simply fragile
and are severely injured despite use of relatively low ventilatory pressures, or is it because
their enzymatic systems (antioxidant, protease, neutrophil apoptosis) are too immature
to cope with the inflammatory insult? An alternative theory for the dysregulated lung
growth seen in infants who develop BPD is that their vascular development may be
abnormal, which leads to abnormalities of lung growth. In a series of experiments in
rodents, Kasahara et al. [9] demonstrated that chronic inhibition of vascular endothelial
growth factor (VEGF) receptors led to pulmonary hypertension, as well as abnormal
lung growth. The angiogenic VEGF is a potent endothelial cell growth and permeability
factor, and is highly expressed in the lung. Expression of different VEGF isoforms and
their receptors (Flt-1 and Flk-1) appears to be developmentally regulated, with increased
expression toward term coincident with the phase of active microvascular angiogenesis.
VEGF and its receptors are significantly decreased in BPD, possibly leading to failure to
expand the capillary network. Interestingly, addition of nitric oxide to the rodent model
led to improved alveolarisation [10]. It is likely that injury to either epithelial cells or
endothelial cells will disrupt the normal pattern of lung development and maturation.
BPD infants can develop pulmonary hypertension. The exact mechanisms are
incompletely understood, but are related to interactions between the disruption of lung
vascular growth and development by premature birth, acute injury and an inability to
achieve normal post-natal adaptation of the lung circulation after birth. In a baboon
model of BPD, disruption of lung vascular growth was evidenced by abnormalities in
microvascular development, angiogenic growth factors and endothelial cell receptors,
which resulted in dysmorphic capillaries [11]. Structural changes in the lung vasculature
contribute to high pulmonary resistance through narrowing of the vessel diameter and
decreased vascular compliance [12]. In addition to these structural changes, the
pulmonary circulation is further characterised by abnormal vasoreactivity and decreased
angiogenesis. The development of pulmonary hypertension may also relate to an inability
to achieve normal post-natal adaptation of the lung circulation. This was evaluated in a
post mortem study of distal lung specimens from chronically ventilated pre-term and
control lambs. Prolonged mechanical ventilation was associated with inhibition of the
normal post-natal decrease in pulmonary vascular resistance and led to lung oedema.
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A. GREENOUGH ET AL.
The mechanism responsible for the excess lung fluid is not clear; possible explanations
include abnormal protein permeability and increased filtration pressure in the pulmonary
circulation. It is also likely that the reduced number of lung microvessels, through which
the blood must flow, contribute to excessive filtration pressure and accumulation of lung
fluid. These abnormalities of lung vascular development, overgrowth of vascular smooth
muscle and decreased number of small blood vessels, have been described in infants with
severe BPD.
Risk factors for BPD

There are many risk factors for the development of BPD. These include prematurity,
low birthweight and a genetic predisposition, including possibly a family history of
atopy. Most attention, however, has focussed on the role of supplementary oxygen
therapy and mechanical ventilation. Both are important antecedents of lung injury; in
many animal models, increased reactive oxygen species and barotrauma/volutrauma
result in an inflammatory response similar to that seen in the lungs of the prematurely
born infant. Data from animal models suggests that the pre-term mammal is susceptible
to damage from volutrauma in the first minutes after birth. Bjorklund et al. [13] found
that six inflations of 60 cmH2O, each lasting 5 s, given to pre-term lambs before the
administration of surfactant resulted in lower compliance for the next 4 h and worse lung
histology than that seen in controls. Five sustained inflations of 8, 16 and 32 mL?kg-1
resulted in dose-dependent lung damage in pre-term lambs, but even those receiving
8 mL?kg-1, similar to the volume generated by the spontaneous breathing term infants,
had worse lung mechanics than the nonintubated controls [14]. These effects are likely to
be due to volutrauma rather than barotrauma, as similar experiments on adult rats and
infant rabbits [15] have highlighted that lung injury can largely be eliminated by
restricting chest wall expansion with either rubber bands or plaster casts. Patent ductus
arteriosus (PDA) and fluid overload have been implicated in the pathogenesis of BPD.
Excess fluid and the increased microvascular permeability with subsequent formation of
pulmonary oedema contribute to poor lung function with exaggeration of hypoxaemia,
hypercapnia and ventilator dependency.
Recent attention has focused on infection, especially antenatal infection, in causing
lung injury in susceptible infants. It is thought that antenatal infection initiates an
inflammatory response in the foetal lung, which may prime the lung to greater lung injury
when exposed post-natally to mechanical ventilation and oxygen supplementation.
Spontaneous pre-term onset labour or pre-labour, pre-term rupture of membranes are
thought to occur as a result of ascending infection from the vagina. Although a multitude
of infections have been implicated in the initiating pre-term labour, disappointingly the
results of antibiotic treatment of mothers presenting in pre-term labour with intact or
ruptured membranes have been disappointing [16, 17]. U. urealyticum has been identified
in the lungs of infants who develop BPD; a review of 17 studies demonstrated that the
relative risk for BPD development in babies colonised with U. urealyticum was 1.72 (95%
confidence interval (CI) 1.51.96). It is unclear, however, whether the organism is an
innocent bystander or is causative of lung injury in vulnerable infants.
Preventive strategies
Preventative strategies have been aimed at preventing or minimising lung injury and,
more recently, promoting lung growth.
Large randomised trials have been undertaken with varying results: no positive effect
220
on BPD, but other important clinical benefits; a positive impact on BPD, but serious
side-effects; and no positive effects. The results of these trials need to be interpreted
bearing in mind factors such as the adequacy of the controls and whether the results are
limited to particular populations.
Other strategies have been tested only in physiological studies, small randomised or
nonrandomised trials with short-term outcomes or their results compared with those of
historical controls. Experience with patient-triggered ventilation and high-frequency
ventilation highlights the fact that such evidence does not necessarily translate into longterm benefits in randomised trials.
When considering the efficacy of preventative strategies, it is important to consider
that BPD may not be the correct outcome and respiratory status at follow-up should be
determined.
Systematic reviews of many randomised trials have demonstrated that both antenatal
administration of corticosteroids and post-natal surfactant significantly reduce the
incidence of neonatal death and respiratory distress syndrome (RDS), but do not favourably
impact on the incidence of BPD. Arguably, this is because both therapies improve the
survival of very immature infants, who are at greatest risk of BPD. Whether the new
generation of surfactants will be more efficacious with regard to BPD is not known.
Many ventilation modes have been shown to have positive effects in studies with
physiological end-points and even in some randomised trials. The inappropriateness of
using the results of a single randomised trial to inform routine clinical practise is
demonstrated by the experience with high-frequency jet ventilation (HFJV). In one
study, HFJV was associated with a reduction in the incidence of BPD at 36 weeks and a
need for home oxygen [18], but a second trial [19] was halted for safety reasons, as infants
exposed to HFJV as opposed to conventional ventilation had higher rates of severe
intracranial haemorrhage (41 versus 22%) and periventricular leukomalacia (31 versus
6%). Certain ventilation modes have been investigated in a number of randomised trials,
but to date systematic review of such trials has failed to identify a mode with a substantial
impact on BPD. For example, patient-triggered compared with conventional ventilation
was not shown to reduce BPD and the only positive effect was, if it was started in the
recovery phase of RDS, it shortened weaning from mechanical ventilation [20]. It is
possible that the limited efficacy of patient-triggered ventilation may reflect deficiencies
in the ventilators and/or triggering systems used in the trials. Results from physiological
studies suggest that adequate gas exchange is achieved at lower pressures and with less
asynchrony using the newer triggered modes; whether this translates into less lung injury
and better long-term respiratory outcomes is not known. Interpretation of ventilation
trials is complicated by differences in the way in which the ventilation modes have been
used, as this can influence their efficacy. For example, high-frequency oscillation
ventilation (HFOV) can be used either with a low-volume strategy, in which pressures are
minimised with the hope of preventing damage due to baro-/volutrauma, or a highvolume strategy, in which mean airway pressure is elevated to promote optimum alveolar
expansion. Results from a surfactant-deficient rabbit model demonstrated that the highvolume strategy was associated with less damage to the lungs [21] and this is in keeping
with more favourable results being found in the trials using the high-volume strategy.
Meta-analysis of the results of 11 trials in which infants were randomised to receive
HFOV or intermittent positive-pressure ventilation in the first 24 h after birth [22]
demonstrated that HFOV was associated with a modest reduction in BPD in survivors at
term. Certain trials, however, differed in their results and the HFOV strategy used, but
also in the comparator groups. Courtney et al. [23] reported that HFOV reduced the
combined outcome of BPD and death in comparison with that experienced by infants
supported by synchronous intermittent mandatory ventilation (SIMV). That result,
however, may not reflect that HFOV was beneficial, but rather that SIMV was
221
A. GREENOUGH ET AL.
disadvantageous, because if a low SIMV rate (v20 bpm) is used, the work of breathing is
increased [24]. In another trial, no short-term [3] or longer-term [25] benefits or
disadvantages of HFOV were noted; the control group were supported by "conventional"
ventilator modes, including synchronised ventilation. Other respiratory support
strategies have not been exposed to rigorous testing. Many practitioners have adopted
a policy of CPAP rather than intubation and ventilation. In a baboon model, such a
policy was associated with less injury to the immature lung, but evidence that such a
strategy reduces BPD in prematurely born infants is either from comparison between
centres or to historical controls. To date, the few randomised trials that have been
performed have been too small to address clinically meaningful outcomes.
Infection, particularly if temporarily associated with a PDA, has been associated with
an increased risk of BPD. Whether aggressive therapy of infection reduces BPD has not
been adequately tested. Only two studies with a total colonisation rate ofv40 infants [26]
could be included in a recent Cochrane review investigating whether antibiotic treatment
of U. urealyticum decreased mortality and BPD. Fluid overload worsens lung function
but the converse does not improve long-term respiratory outcome and may impair
nutritional intake. The impacts of PDA treatment and fluid restriction have been
disappointing. A Cochrane review reported no statistically significant difference in the
development of BPD when ibuprofen was given to prevent a PDA (relative risk (RR)
0.67, 95% CI 0.123.78) or to treat a PDA (RR 1.52, 95% CI 0.832.81) [27]. In addition,
treatment of asymptomatic PDA with indomethacin in three randomised trials, although
significantly reducing the incidence of symptomatic PDA, resulted in only a small
decrease in the duration of requirement for supplementary oxygen [28]. Fluid restriction
in early trials reduced PDA, but has not subsequently been shown to reduce BPD [29].
Evidence from animal models may not always be replicated in humans. For example, a
protective effect of polyunsaturated fatty acids against lung injury was reported in
experimental rats [30] but no protection against BPD in pre-term infants has been
demonstrated in several randomised trials [31]. The efficacy of a strategy, however, may
vary according to the population studied. Nitric oxide has been shown to be lung
protective in premature lamb models with RDS and associated with increased alveolar
growth in lung-injured neonatal rats, yet early randomised trials in prematurely born
infants demonstrated no or little benefit of inhaled nitric oxide (iNO). Those studies,
however, included infants with severe respiratory failure. More recently, in a singlecentre study, iNO administration was associated with a decrease in the combined
outcome of death and BPD and intracranial haemorrhage in infants with mild
respiratory failure [32]. Clearly it is important to confirm or refute those observations in a
larger population.
Other therapies have been demonstrated to have positive effects on BPD, but they
have limited use because of side-effects. Meta-analysis demonstrated that corticosteroids
systemically administered in the first 96 h after birth reduce oxygen dependency at
28 days and 36 weeks [33], but they have acute side-effects and, more importantly, longterm adverse effects on neurodevelopmental outcomes and lung structure. Given at a
critical period of lung growth of between 4 and 14 days after birth, corticosteroids
resulted in rats of normal body size, but with increased lung volumes and enlarged
airspaces and decreased alveolar surface area. The outgrowth of new alveolar septa was
partly suppressed and after drug withdrawal the lungs remained emphysematous with
larger and fewer airspaces [34]. In view of those adverse effects, there has been interest in
assessing lower doses of systemically administered steroids, using alternative corticosteroids to dexamethasone and the inhalation route. Anecdotally, one-tenth of the
previously used dexamethasone dose has short-term positive effects, but the riskbenefit
ratio of such a regime requires careful investigation. Unfortunately, other steroid
preparations may also have side-effects; a randomised study investigating the effect of
222
hydrocortisone on survival without BPD was terminated prematurely because of an

excess of gastrointestinal perforations in the hydrocortisone group [35]. Inhaled steroids
have fewer side-effects but also fewer positive effects, but do facilitate extubation and
reduce the need for later rescue systemic steroids. Meta-analysis of randomised trials [36]
has also demonstrated that vitamin A supplementation reduced death or oxygen
requirement at 1 month of age and oxygen requirement at 36 weeks of PMA, but sideeffects meant that levels should be carefully monitored and further work is needed to
define the optimum dosage, mode and duration of treatment.
Some preventative therapies, although not shown to reduce BPD, may have other
beneficial effects on prematurely born infants respiratory status. Prematurely born
infants are relatively deficient in antioxidant enzyme systems, such as superoxide
dismutase (SOD), and have low levels of antioxidants, such as vitamins C and E. They
are therefore more vulnerable to oxygen toxicity. There is some evidence to suggest that
improving antioxidant defences may improve the respiratory outcome of pre-term
infants [37]. However, in two randomised trials, SOD administration was not associated
with a reduction in BPD; in one there was a lower frequency of respiratory episodes
(wheezing, asthma, pulmonary infections) severe enough to require treatment [38].
Administration of antioxidants antenatally might reduce BPD, not only by increasing
antioxidant defences, but perhaps also by reducing pre-term delivery, since maternal
supplementation with the antioxidant vitamins C and E has been shown to reduce the
occurrence of pre-eclampsia [39]. There is also evidence from animal models that
antenatal vitamin supplementation might impact favourably on lung growth and
development; whether this occurs in infants is currently being investigated in a
multicentre trial.
Treatment strategies
Much of the treatment of infants with BPD is based on extrapolation from what is
known about the pathophysiology of the condition, rather than the results of randomised
intervention trials.
Peak inspiratory pressures and inspired oxygen concentrations are kept to the
minimum compatible with acceptable blood gases, and this includes allowing the carbon
dioxide levels to rise, providing the infant does not develop a respiratory acidosis.
Anecdotally, various ventilation modes have been used with success in infants with BPD,
but none has been investigated systematically. To minimise further lung damage, BPD
infants should be weaned from the ventilator as soon as possible, but, theoretically, this
might result in greater compromise by increasing the work of breathing and interfering
with adequate enteral nutrition.
Excessive fluid should be avoided and clinical experience suggests BPD infants do not
tolerate fluid intakes w150 mL?kg-1, or even lower levels if given enterally. Yet, BPD
infants require a greater calorie intake than age-matched infants without respiratory
disease, thus concentrated feeds, calorie supplements or modular component additives
are frequently used. Given that excessive weight gain and crossing of centiles is associated
with adverse long-term outcomes, it is essential that the appropriate nutrition of BPD
infants is investigated.
Supplementary oxygen is the mainstay of therapy for BPD infants, yet the amount of
oxygen that should be delivered, as indicated by the target levels of oxygen saturation,
remains controversial. In the Supplemental Therapeutic Oxygen for Prethreshold
Retinopathy of Prematurity (STOP-ROP) Trial, premature infants were randomised to
be maintained at 9699% or 8994% oxygen saturation levels [40]. Although, there were
223
A. GREENOUGH ET AL.
no significant differences regarding milestones or growth, more infants in the higher

oxygen saturation group were still hospitalised at 50 weeks PMA and receiving diuretics.
In addition, a greater proportion (13.2 versus 8.5%) of the higher saturation group
developed pneumonia or exacerbations of BPD, perhaps indicating that the higher
saturation levels were disadvantageous. The STOP-ROP Trial [40] was not designed to
test the efficacy of different oxygen saturation levels with regard to pulmonary problems,
but disadvantages of a higher saturation level were also reported from the Benefits Of
Oxygen Saturation Targeting (BOOST) trial [41]. In the BOOST Trial, pre-term infants
who were oxygen dependent at 32 weeks PMA and randomised to oxygen saturations of
9598% rather than 9194%, required supplementary oxygen for longer and more
required supplementary oxygen at home [41]. However, these findings were expected and
no significant differences were demonstrated with respect to growth and neurodevelopmental outcomes at a corrected age of 12 months. Further studies are therefore required
to determine the most appropriate oxygen saturations levels, particularly in those BPD
infants who have pulmonary hypertension.
BPD infants with chronic oxygen dependency are often considered for "home oxygen".
The criteria vary, but usually include that the babys only ongoing medical need is a
requirement for supplementary oxygen, that they have good growth and that they are
without frequent episodes of desaturation. In addition, in some centres, babies are sent
home while they require tube feeding. Anecdotally, such a policy allows babies to be
discharged home earlier, on average y2 weeks earlier, with financial savings and
apparently no increase in subsequent admissions [42]. Families of home-oxygen babies,
however, do require appropriate support, as they have been noted to be more prone to
pre- as well as post-discharge anxiety, and the mothers have less vitality and more mental
health problems. The true costbenefit ratio of home-oxygen therapy requires testing.
Rarely, infants with BPD may be considered for home ventilation. Criteria include
maintenance of their carbon dioxide levels within safe limits on ventilatory equipment
that is operable by the family at home, stable airway and medical condition and
nutritional intake that is adequate to maintain expected growth and development. It is
also important that the parents understand the long-term prognosis and are willing and
capable of meeting the special needs of their child in the home setting, and that it is
practical to provide the level of support and intervention that the child requires at home.
The parents must also be able to evaluate their infants respiratory condition, use the
equipment and be able to deal with all emergency procedures. The most common
obstacles in Europe regarding home ventilation include funding for staffing and
equipment, local organisational delays, unsuitable family housing or a change in the
childs medical condition, which affects the level of support required at home [43]. Most
studies describe infants who showed clinical improvement and home ventilation could be
stopped successfully after some months or even years, but not all children benefit from
long-term ventilation. If home care is not possible due to family unwillingness, inability
to cope or family desertion, then suitable alternative long-term arrangements should be
sought, as it is not appropriate for a child to grow up in a hospital environment. The
duration of home ventilation varies from 0.54 yrs.
Medications should be administered to infants with BPD in the knowledge of the
following: 1) many treatments have positive effects, but these may be short-lived with few
or no long-term benefits demonstrated in randomised trials; 2) side-effects are common
and may not be avoided by using the inhalational route; and 3) on current evidence, use
of medications should be individualised and only continued while there is a demonstrable
positive effect.
Systemic administration of a proximal loop diuretic can acutely increase lung
compliance and reduce airway resistance, facilitating a reduction in ventilatory
requirements and transiently improving blood gases in both ventilated and nonventilated
224
infants. Howver, chronic treatment has important side-effects. Hypokalaemia and

metabolic acidosis can exacerbate carbon dioxide retention and hypercalciuria can lead
to nephrocalcinosis (see below). Distal tubular diuretics have fewer side-effects but also
have a smaller effect on lung function [44, 45]. Systematic review of the results of
randomised trials has not highlighted any long-term benefits of chronic treatment with
any type of diuretics, regardless of the method of administration. BPD infants have
peribronchiolar smooth muscle hypertrophy and can have a positive response to
bronchodilators, even while on the neonatal intensive care unit (NICU). Inhaled b2agonists and anticholinergics can temporarily improve lung function and blood gases and
have synergistic effects [46]. There are, however, no randomised trials with long-term
outcomes to inform whether chronic therapy on the NICU is appropriate. Results of
small randomised trials conducted at follow-up suggest that inhaled bronchodilators are
helpful in prematurely born infants who are symptomatic, but are of no benefit if given
routinely [47].
Meta-analysis of the results of randomised trials has demonstrated that corticosteroids
given to infants w3 weeks of age reduces the following: 1) oxygen dependency at
36 weeks PMA; 2) failure to extubate; 3) the need for rescue dexamethasone;and 4) the
necessity for home oxygen therapy [48]. Fewer side-effects have been reported from the
previously mentioned trials compared with the prophylactic trials, and this may reflect
the older age of the infants or the fact that in some of the studies, "rescue" treatment was
allowed. In a small randomised trial, inhaled corticosteroid administration was
associated with a reduction in symptoms and bronchodilator requirement of prematurely
born infants wheezy at follow-up [49]. The most appropriate duration and dosage of
therapy needs investigating.
BPD infants can suffer severe consequences of respiratory syncytial viral (RSV)
infection, as shown by the greater need for hospital and paediatric intensive care unit
admission [50]; retrospective studies have also highlighted that healthcare utilisation is
increased at follow-up. Currently, there is no safe and effective vaccine against RSV.
Palivizumab, a humanised monoclonal antibody, has been demonstrated to reduce the
hospitalisation rate in BPD infants [51]; whether it improves the long-term outcome in
BPD infants requires appropriate testing.
Related morbidity
Respiratory
BPD is associated with long-term respiratory morbidity. BPD infants have a high
readmission rates in the first 2 yrs after birth, particularly due to respiratory infections
[50]. Troublesome respiratory symptoms requiring treatment are common, even in young
adults. In a recently reported follow-up study [52], 19-yr-old females who had had BPD
were found to have a higher prevalence of doctor-diagnosed asthma, wheeze and
shortness of breath than sex-matched controls. Long-term studies have also demonstrated lung function abnormalities, airway obstruction, and airway hyperreactivity and
hyperinflation persisting into adolescence [53, 54].
Central and upper airways

Endotracheal tubes may injure the upper airway causing laryngeal oedema; infants
who have suffered prolonged or repeated intubations are at greatest risk. The prevalence
of tracheal/bronchomalacia in pre-term infants with BPD varies from 1645% [5557].
225
A. GREENOUGH ET AL.
The pathophysiology of tracheal/bronchomalacia in this patient group remains unclear;

in particular it is unknown whether it contributed to the initial ventilatory requirement or
was a consequence of long-term mechanical ventilation. Tracheobronchial abnormalities
should be considered as a cause of persistent pulmonary problems in infants with BPD.
Cardiovascular system
Pulmonary hypertension was often reported in infants who developed classical BPD,
but is less common in new BPD cases. Whether this is due to a change in the underlying
pathology in the more immature infant or to improvements in therapies, including home
oxygen programmes, remains speculative. Infants with BPD who do develop pulmonary
hypertension have an increased mortality rate. Increased work of breathing and hypoxic
pulmonary vasoconstriction may potentiate the development of cor pulmonale.
The incidence of systemic hypertension is higher in infants with BPD (1343%) than in
prematurely born infants with RDS only (19%) and in infants born at term (0.73%)
[58]. Systemic hypertension may contribute to the development of left ventricular
hypertrophy, which is often associated with right ventricular hypertrophy in infants with
BPD. The pathogenesis of systemic hypertension and left ventricular hypertrophy
overlap: metabolic effects of chronic hypoxaemia, hypercarbia and acidosis can increase
cardiac output and stimulate the renin-angiotensin system, thereby elevating afterload.
Chronic adrenergic stimulation from ongoing stress and neurohumoral stimulation,
exogenous administration of medication (such as b-adrenergic agonists and steroids) and
impaired lung clearance of norepinephrine have been implicated in the development of
left ventricular hypertrophy [59]. It is unknown whether impaired metabolic function of
the lung contributes to the pathophysiology of BPD by increasing circulating
catecholamine levels, or if it is a marker of severe pulmonary vascular disease. It has
been speculated that high catecholamine levels may lead to left ventricular hypertrophy
or systemic hypertension. Other possible causes for systemic hypertension are renal
damage due to nephrocalcinosis or prolonged umbilical arterial catheterisation.
Prominent bronchial or other systemic-to-pulmonary collateral vessels were noted in
early morphometric studies of infants with BPD. Although these collateral vessels are
generally small, large collaterals may contribute to significant shunting of blood flow to
the lung, causing oedema and the need for high levels of supplementary oxygen.
Growth retardation and gastrointestinal problems

The causes of malnutrition and growth failure in BPD infants include decreased
nutrient intake, hypoxia, concomitant dysfunction of other organ systems and increased
requirements for energy [53]. In general, there is no significant impairment of nutrient
absorption or digestion, unless there is concomitant bowel disease. Malnutrition can
delay somatic growth and the development of new alveoli, and decreased muscle strength
makes successful weaning from mechanical ventilation less likely and causes patients to
be more prone to infections. Decreased glutathione levels may impair the response to
oxidant-induced lung injury, and protein undernutrition may interfere with lung growth
and DNA synthesis. Rib fractures, together with generalised bone demineralisation, are
frequently observed in patients with BPD. This is usually secondary to dietary or
parenteral deficiency of calcium or vitamin D and excessive calciuria resulting from
chronic diuretic therapy.
Gastro-oesophageal reflux (GER) is common (63%) in infants born prior to 32 weeks
of gestational age and may contribute to malnutrition, the chronic inflammatory process
and lung damage, but is poorly correlated with BPD [60]. The prevalence in prematurely
226
born children with BPD is so high that those who have persistent respiratory difficulties
not responding to therapy or with persistent vomiting or failure to thrive should undergo
evaluation for GER and aspiration.
Renal problems
The aetiology of nephrocalcinosis is multifactorial. Risk factors include immaturity,
low glomerular filtration rate (causing low urinary flow), high intakes of calcium and
phosphorus (to prevent rickets) with high excretion of calcium, low citrate excretion and
use of diuretics. Prematurity-associated nephrocalcinosis resolves in months to years in
most patients, but is still present in 15% at the age of 30 months [61]. While proximal
tubular function is unaffected, high blood pressure and impaired glomerular and distal
tubular function appear to occur more frequently than in healthy children [61]. In rare
instances, this condition leads to renal calculi or renal insufficiency.
Neurological system and development

Infants with BPD are at increased risk for cerebral palsy, microcephaly and
neurodevelopmental delay affecting both cognitive (speech development, performance,
IQ and receptive language) and motor function compared with premature control
children matched for gestational age [62, 63]. Generalised hypotonia results in early gross
motor delay. Risks of delay may be compounded by co-existing conditions, such as
hearing loss, severe intracranial bleed and poor social environment. Long-term follow-up
identified at 8 yrs of age either a dramatic improvement or long-term adverse effects on
cognitive and academic achievement above and beyond the effects of very low
birthweight [62, 64]. This highlights the need for continued monitoring of the learning,
behaviour and development of BPD children, so that intervention can be planned for
those with children who are at risk of school-age problems [62]. Long-term studies
suggest that infants who received prolonged steroid therapy have worse neurological
outcome, including an increased incidence of cerebral palsy, than control infants [65].
Observational studies have shown an association between transiently low thyroid
hormone levels in pre-term infants in the first weeks of life (transient hypothyroxinaemia)
and abnormal neurodevelopmental outcome. Thyroid hormone therapy might prevent
this morbidity, but review of the research to date does not support the use of thyroid
hormones in pre-term infants to reduce neonatal mortality, improve neurodevelopmental
outcome or to reduce the severity of respiratory distress syndrome [66].
Ophthalmological problems
Infants born prematurely have incompletely vascularised retinas and a peripheral
avascular zone, the area of which depends on the gestational age. In premature infants,
normal retinal vascular growth that would occur in utero ceases and there is loss of some
of the developed vessels. With maturation of the infant, the resulting nonvascularised
retina becomes increasingly metabolically active and hypoxic. Retinal neovascularisation, is induced by hypoxia, and occurs at y3234 weeks PMA. In retinopathy of
prematurity (ROP), angiogenesis has a predominant role [67]. Angiogenesis is controlled
by many factors, including the expression of VEGF, which in turn is regulated by
absolute and relative lack of oxygen, and insulin-like growth factor (IGF)-1. In
premature infants, the absence of IGF-1 (normally provided by the placenta and the
amniotic fluid) stops blood vessel growth [68]. Oxygen saturations w95% and arterial
227
A. GREENOUGH ET AL.
oxygen tension w80 mmHg (10.64 kPa) are associated with higher incidences of ROP
[69]. Experimental studies have focussed on the role of oxygen, but there have been many
recorded instances of ROP in premature infants not exposed to elevated oxygen
concentrations and other factors, such as sepsis, hypercarbia or hypocapnia, vitamin E
deficiency, lactic acidosis and anaemia, have been implicated in the pathophysiology of
ROP. Extreme prematurity is the most significant risk factor.
Vision loss from ROP is a consequence of excessive overgrowth of new vessels in the
retina and vitreous cavity. There are five stages of the abnormal vascular response at the
junction of the vascularised and avascular retina [70]. The more posterior the disease and
the greater the amount of involved retinal vascular tissue, the more serious the disease.
Although ablation treatment, laser photocoagulation or cryotherapy of the retina
reduces the incidence of blindness by 25%, the visual outcomes after treatment are often
poor in those who reach late-stage disease.
Auditory deficits
Pre-term infants with BPD are at high risk of persistent conductive hearing loss.
Although conductive impairment is implicated in many cases, the degree of impairment is
greater than that usually seen with middle ear effusion [71]. The incidence of hearing
impairment (2050%) in BPD infants is much greater than for other high-risk premature
infants [72, 73]. An auditory brainstem response test conducted at the time of hospital
discharge does not accurately predict later conductive hearing problems, hence infants
with BPD should have routine audiological evaluation toward the end of the first year of
life.
Sudden infant death syndrome

In the 1980s, a seven-fold increase in the incidence of sudden infant death syndrome
(SIDS) was noted retrospectively in low-birthweight infants with BPD [74]. Studies
performed in the 1990s, however, found that pre-term infants with BPD are not at
increased risk from SIDS compared with pre-term infants without this condition [75].
Risk factors for SIDS among infants aged 2432 weeks gestation appear to be associated
more with sociodemographic characteristics than medical problems. This suggests that
for the immediate future, the risk for SIDS among very pre-term infants will be best
addressed through further modification of the environment and parent behaviour [75].
Most recent fundamental developments

The most recent fundamental developments concern the following: 1) the
inflammatory nature of BPD; and 2) the roles of VEGF and nitric oxide in lung growth.

The most important future questions are the following. 1) What is the most effective
and safe preventative therapy? Areas meriting further exploration are the resolution of
lung injury in pre-term infants by the process of neutrophil apoptosis, as this appears to
be inhibited in the more immature compared with the more mature infants [76], and an
adequately powered trial of treatment for antenatally acquired infection. 2) Which
228
infants are at highest risk of developing BPD and what is the most sensitive method of
identifying such infants? 3) What is the pathogenesis of pulmonary hypertension in
children with BPD? Is it possible to prevent pulmonary hypertension and, if not, what is
the best way to monitor and treat the condition? 4) What is the best way to stimulate
somatic growth and what is the relationship between somatic growth and lung growth? 5)
Who are eligible candidates for long-term ventilation and will long-term mechanical
ventilation be an option to promote growth by decreasing the caloric loss due to work of
breathing? Will some children inevitably "grow out of their lungs"? 6) What are the longterm consequences of new BPD? A major concern for infants with BPD is the
abnormalities of lung growth that have been described either in those who have died [77]
or from animal models. There is a concern that these infants may develop chronic
obstructive pulmonary disease prematurely in young adulthood. 7) What is the
additional risk of neurodevelopmental delay and persistent conductive hearing loss in
prematurely born children who have had BPD? What is the effectiveness of special
education?
In order to answer these questions, the following are needed: 1) a multidisciplinary
approach between geneticists, obstetricians, physiologists, paediatricians, educational
psychologists, etc.; 2) longitudinal studies; and 3) international collaboration and
randomised trials adequately powered to detect differences in clinically meaningful longterm outcomes.
Summary
Bronchopulmonary dysplasia (BPD) is a common adverse outcome of very premature
birth. BPD infants suffer prolonged oxygen dependency, troublesome respiratory
symptoms, lung function abnormalities at follow-up and related problems, including
pulmonary and systemic hypertension, neurodevelopmental delay and conductive
hearing loss. There are many risk factors for BPD development, including oxygen
toxicity, volutrauma and infection, as well as prematurity. Studies in animal models
have demonstrated that these factors lead to the inflammatory pulmonary response
seen in infants with BPD. In addition, it has been highlighted that abnormal vascular
development may lead to impaired lung growth. Nowadays, infants are described as
having "new" BPD, with abnormalities of lung growth being more prominent than the
fibrosis and smooth muscle augmentation of the airways seen previously in severe or
classical BPD. Preventative strategies have largely been aimed at preventing or
minimising lung injury and have had limited success. Despite many randomised trials,
the optimum ventilation mode with regard to preventing BPD has not been identified,
and, although systemically administered corticosteroids in the first 96 h after birth are
efficacious, concerns regarding serious adverse effects preclude their use. Supplementary oxygen is the mainstay of treatment for BPD infants, but further work is
necessary to identify the optimum oxygen saturation level, particularly in infants with
pulmonary hypertension. On current evidence, the use of medications in BPD infants
should be individualised and only continued whilst there is evidence of a clinically
important response. Research areas regarding prevention of BPD that merit further
investigation are antioxidant supplementation, resolution of lung injury by neutrophil
apoptosis, treatment of antenatally acquired infection and prophylactic administration of nitric oxide to promote angiogenesis and alveolarisation.
Keywords: Angiogenesis, antioxidants, inflammation, lung growth, volutrauma.
229
A. GREENOUGH ET AL.
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Abman SH, Warady BA, Lum GM, Koops BL. Systemic hypertension in infants with
bronchopulmonary dysplasia. J Pediatr 1984; 104: 928931.
Abman SH, Schaffer MS, Wiggins J, Washington R, Manco-Johnson W, Wolfe RR. Pulmonary
vascular extraction of circulating norepinephrine in infants with bronchopulmonary dysplasia.
Akinola E, Rosenkrantz TS, Pappagallo M, McKay K, Hussain N. Gastroesophageal reflux in
infants v32 weeks gestational age at birth: lack of relationship to chronic lung disease. Am J
Perinatol 2004; 21: 5762.
Schell-Feith EA, Kist-van Holthe JE, van Zwieten PH, et al. Preterm neonates with
nephrocalcinosis: natural course and renal function. Pediatr Nephrol 2003; 18: 11021108.
Short EJ, Klein NK, Lewis BA, et al. Cognitive and academic consequences of bronchopulmonary
dysplasia and very low birth weight: 8-year-old outcomes. Pediatrics 2003; 112: e359.
Lewis BA, Singer LT, Fulton S, et al. Speech and language outcomes of children with
bronchopulmonary dysplasia. J Commun Disord 2002; 35: 393406.
Robertson CM, Etches PC, Goldson E, Kyle JM. Eight-year school performance,
neurodevelopmental, and growth outcome of neonates with bronchopulmonary dysplasia: a
comparative study. Pediatrics 1992; 89: 365372.
OShea TM, Kothadia JM, Klinepeter KL, et al. Randomized placebo-controlled trial of a 42-day
tapering course of dexamethasone to reduce the duration of ventilator dependency in very low
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infants. Cochrane Database Syst Rev 2001; 4: CD001070.
Arden GB, Sidman RL, Arap W, Schlingeman RO. Spare the rod and spoil the eye. Br J
Ophthalmol 2005; 89: 764769.
Smith LE. Pathogenesis of retinopathy of prematurity. Growth Horm IGF Res 2004; 14: Suppl. A,
S140S144.
Flynn JT, Bancalari E, Snyder ES, et al. A cohort study of transcutaneous oxygen tension and the
incidence and severity of retinopathy of prematurity. N Engl J Med 1992; 326: 10501054.
The International Classification of Retinopathy of Prematurity revisited. Arch Ophthalmol 2005;
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Marsh RR, Handler SD. Hearing impairment in ventilator-dependent infants and children. Int J
Pediatr Otorhinolaryngol 1990; 20: 213217.
Gray PH, Sarkar S, Young J, Rogers YM. Conductive hearing loss in preterm infants with
bronchopulmonary dysplasia. J Paediatr Child Health 2001; 37: 278282.
Werthammer J, Brown ER, Neff RK, Taeusch HW Jr. Sudden infant death syndrome in infants
with bronchopulmonary dysplasia. Pediatrics 1982; 69: 301304.
Gray PH, Rogers Y. Are infants with bronchopulmonary dysplasia at risk for sudden infant death
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Kotecha S. Lung growth: implications for the newborn infant. Arch Dis Child Fetal Neonatal Ed
2000; 84: F69F74.
233
CHAPTER 15
Cystic fibrosis
A. Bush*,#, M. Gotz},z
*Imperial School of Medicine at National Heart and Lung Institute, and #Royal Brompton Hospital,
London, UK. }Medical University of Vienna, and zDept of Paediatrics and Adolescent Medicine,
Respiratory and Infectious Diseases, Vienna, Austria.
Correspondence: A. Bush, Dept of Paediatric Respiratory Medicine, Royal Brompton Hospital, Sydney
Street, London SW3 6NP, UK. Fax: 44 2073518763; E-mail: a.bush@rbht.nhs.uk
In 1989, cystic fibrosis transmembrane conductance regulator (CFTR), the gene for
cystic fibrosis (CF), was cloned and, since then, there has been an explosion of knowledge
about the molecular biology of the gene product and the basic biology of the airway
surface in particular. The quest is a cure for the disease, which is as yet elusive.
Furthermore, the understanding of the disease has broadened from that of a lung and
pancreatic disorder to a multisystem disease in which complications as diverse as
osteopaenia and urinary incontinence are important to CF patients. Such is the variety of
the research approaches being taken that no one chapter can possibly hope to encompass
them all. Therefore the answers to the questions posed by the editors must to some extent
reflect personal choice. Interested readers are referred to a recent publication [1].
What have been the most recent fundamental developments in

CF?
The expanding diagnosis of CF, and therefore the new diagnostic approaches:
atypical CF and CF with normal CFTR
CF is the most common inherited disease of white races, with a variable prevalence
throughout Europe [2]. The underlying cause has been thought to be dysfunction or
absence of CFTR, with the gene for this protein being localised to the long arm of
chromosome 7. Historically, the diagnosis of CF was easy, being made at the autopsy of
a baby or infant by the pathologist. In the 1950s, when survival had improved,
measurement of sweat electrolytes became the diagnostic gold standard, and indeed to
this day w98% CF patients have a positive sweat test [3]. For most patients, a typical
clinical picture will point to the need for a sweat test; the usual problem is not
interpreting the result, but remembering to ask for the test. Detailed guidelines have been
published [4]. The sweat test, although pivotal, will not be discussed further in the present
chapter because there are no significant new conceptual advances in that field. For a
small minority of cases, an approach beyond simple sweat testing is needed because, by
the 1990s, it had become clear that there were patients with typical phenotypic CF and an
equivocal or even normal sweat test [5, 6]. In this situation, specific testing of CFTR
structure or function, or less specific tests of the downstream consequences of reduced or
absent CFTR function, are needed to make a diagnosis.
A recent consensus group [7] defined the diagnostic criteria for CF as the following:
1) any of a clinical phenotype, CF in a sibling, or a positive newborn screening test; plus
ISSN 1025-448x.
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CYSTIC FIBROSIS
2) evidence of CFTR dysfunction, such as raised sweat chloride, positive genotype or

abnormal nasal potential difference (PD).
This section will discuss how well these criteria hold up in the light of new information.
Specific testing for CF will be described first, followed by the role of testing for
downstream effects of CFTR dysfunction. The concepts of typical and atypical CF, preCF and clinical scenarios where the diagnosis is doubtful will then be discussed. It should
be noted that as newborn screening becomes more widely introduced, most of the
obvious cases (95% in one series [8]) will be diagnosed, and the challenge for the future
will increasingly be: 1) to remember that screening does not preclude the need to consider
CF in the differential diagnosis of paediatric and adult disease in many systems; and 2) to
deal with an increasingly greater proportion of atypical and "difficult" cases, for which
the sweat test is not an adequate diagnostic tool.
Specific testing for CF

Genotype. The CF gene is localised on the long arm of chromosome 7. Genetic testing has
shown that some patients have two known CF disease-producing mutations, confirming
the diagnosis. To date i1,300 alterations in the DNA composition of CFTR have been
detected [9]. These comprise disease-producing mutations and harmless polymorphisms.
Thus, in assessing the significance of a genetic test result, the following must be
remembered: 1) changes in DNA per se do not define a disease (a point that is returned to
below); 2) two known CF disease-producing mutations (e.g. DF508), in the setting of an
appropriate disease phenotype, establish the diagnosis of CF; 3) failure to find two CF
disease-producing mutations cannot exclude the diagnosis of CF; and 4) the effects of a
known CF disease-producing mutation may be abrogated by a second mutation in the
same gene.
A paper purporting to describe a group of patients with a completely normal CFTR
gene sequence and a CF phenotype has further extended the complexities of genetic
testing [10]. A total of 74 patients with suspected but unconfirmed CF were referred
from 34 centres and underwent mutation analysis and complete gene sequencing.
Twenty-nine patients were found to have two mutations (genetic diagnosis of CF), 14
had one mutation and 30 had no mutations. There were no phenotypic differences
between the groups. Bolstering the claim that an atypical CF phenotype could exist
with normal CFTR was the finding of siblings with atypical CF phenotypes who were
discordant at the CFTR locus and who had evidence of some CFTR function on nasal
PD testing.
One obvious solution for the apparent paradox of CF with normal CFTR gene
sequences is that there is a genetic defect in a protein involved in the complex intracellular
processing of CFTR, or one of the many at the cell surface with which CFTR interacts.
At a conservative estimate,w20 proteins are required to enable CFTR to traverse the cell
to the apical membrane and, on arrival, CFTR interacts with i10 different proteins [11].
Although some of these proteins are likely to be essential to so many intracellular
processes that a mutation would be lethal at the embryo stage, it is surely not too fanciful
to suggest the possibility that CFTR dysfunction may be secondary to failure of normal
CFTR to interact with a mutated protein, either within the cell or at the cell surface.
There is precedent for this: deficiency of surfactant protein (SP)-B or -C may result in
neonatal interstitial lung disease with a histological picture of pulmonary alveolar
proteinosis. Investigation of babies with this picture revealed some with completely
normal SP gene sequences, but a defect in ABCA3, a lipid transporter involved with the
coplex post-translational processing of SPs [12]. The present authors predict that genetic
235
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A. BUSH, M. GO
defects in proteins involved in CFTR processing, or interacting with CFTR, will be

found to account for cases of atypical CF.
Electrical potential differences. In some cases, the diagnosis was still obscure; for these
patients, measurement of electrical PDs using an exploring catheter in the nose and a
subcutaneous reference electrode is helpful. Features seen in CF patients which differ
from the norm are as follows: 1) a lower (more negative) PD; 2) a bigger positive deflection
when the nasal mucosal surface is perfused with amiloride, which blocks the epithelial
sodium channel (ENaC); and 3) an absence of a negative deflection when CFTR is
stimulated by mucosal perfusion with a low chloride, isoprenaline-containing solution
[13]. In some centres, equivalent measurements are made on rectal biopsies in an Ussing
chamber in vitro [14]. Hence, specific testing of CFTR structure and function by genotype
analysis and PD measurements may confirm the diagnosis of CF.
Nonspecific testing for CF

Downstream effects of reduced or absent CFTR function. Ancillary testing may clarify
the diagnosis, although it is important not to over-call the significance of minor
abnormalities. Furthermore, the more specific tests for CFTR dysfunction have proven to
be negative, the more alternative diagnoses, such as SchwachmanDiamond syndrome or
primary ciliary dyskinesia (PCD), should be considered. Pancreatic function is most
conveniently assessed by measuring human faecal elastase on a spot sample of stool, and
this test is generally very sensitive and specific in the context of suspected CF [15]. This test
may be misleading in the first week of life [16]. It cannot distinguish pancreatic
insufficiency due to CF from other causes of pancreatic insufficiency and may not be
useful in short gut syndrome, ileostomy patients or acute diarrhoea [17]. It can be
performed even if the patient is taking pancreatic enzyme replacement therapy. Other
supportive tests would be computed tomography (CT) scanning of the sinuses (a
completely normal scan would be extremely unusual in CF); CT scanning of the chest
(bronchiectasis) and abdomen (macronodular cirrhosis); and bronchoalveolar lavage
(BAL) revealing a neutrophilic inflammation and typical CF organisms. In male adults,
azoospermia is usual in CF; at any age, bilateral absence of the vas on palpation and
ultrasound would be a pointer to CF.
What is a CF diagnosis?
Typical versus atypical CF. The phenotype of typical CF includes the well-known
respiratory manifestations but the hallmark is pancreatic insufficiency. Atypical CF
patients are pancreatically sufficient, but have one or more other organ manifestations of
the disease. The present authors are not sure this is quite the right division: most would
think that the pancreatically sufficient CF patient with bronchiectasis and chronic
bronchopulmonary infection with mucoid Pseudomonas aeruginosa has a very typical
phenotype. The difficulty is classifying and diagnosing patients with no pulmonary or
pancreatic phenotype but with, for example, congenital bilateral absence of the vas
deferens or sinusitis.
CFTR-related disease. A number of studies have determined that there are a higherthan-expected number of patients with conditions that may be part of the CF phenotype,
such as allergic bronchopulmonary aspergillosis (ABPA), chronic sinusitis and acute
pancreatitis who have CFTR mutations [18, 19]. Of course, a few are eventually
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CYSTIC FIBROSIS
determined to have late-diagnosed CF. However, it would seem that the presence of one
CFTR mutation, if there are other risk factors (either genetic or environmental), may
increase the risk of other diseases, even though CFTR function is 50% normal. In the
patient with a single CFTR mutation and ABPA, a sweat test and if possible nasal PD
measurements should be performed, but if these are normal, CF can be excluded with a
high degree of certainty. However, CF is a diagnosis that can never be excluded with
complete assurance.
Pre-CF and subclinical CF. These diagnostic difficulties have lead to the proposal that a
long-established oncological concept may be useful in this context also. Pre-malignant
diseases, which may never progress to cancer, and which do not require immediate
treatment, have long been recognised; the present authors proposed that this concept
should be extended to CF [20]. A pre-CF state may be chemical (abnormal sweat test, no
disease), electrical (abnormal nasal potential) or genetic (two abnormalities in the CFTR
locus). Subclinical CF is characterised by subtle but definite evidence of end organ
dysfunction. The disease CF is just that: an actual clinical disease of one or more organs.
Although the disease manifestations of CF may be mild, the present authors believe that
there is an important distinction between mild disease and subtle abnormalities, of no
clinical importance to the patient and only detectable by sophisticated investigation.
These subtle abnormalities are not considered to qualify for the label of the disease CF,
although they may be a marker that clinical vigilance to detect progression to the disease is
warranted. There is no clear-cut boundary between pre-CF and subclinical CF, just as
there is none between subclinical CF and the clinical disease; diagnostic systems are no
substitute for clinical judgment.
The normal child who tests positive for CFTR dysfunction. One clinical conundrum is
the management of the child who is entirely well but, usually as a result of a diagnosis in a
family member, has been found to have a positive sweat test or two CF disease-producing
mutations, but in whom no actual manifestations of the disease have been found. The
special problems of newborn screening are discussed later in this chapter. An older child
with positive tests but no disease would be assigned the label chemical or genetic pre-CF,
depending on which test result(s) were abnormal, and an open discussion about follow-up
and treatment options would follow with the whole family. It would be made very clear as
to what evidence is and is not available regarding the rate of progression in groups of
patients; ignorance as to how individuals will behave would also be acknowledged. It is
quite clear that such a child should be evaluated carefully for subclinical problems and
followed up carefully. It is unlikely that the child would do two 30-min sessions of
physiotherapy a day (even if appropriate) but checking that the chest was clear with a few
huffs or similar technique once a day would be reasonable. Pre-CF may progress to CF
and vigilance for the first sign of deterioration is imperative, at which point therapy must
be intensified; however, it is also possible that the child may remain disease-free for many
years.
The child with a CF phenotype and negative tests for CFTR dysfunction. A CF
diagnosis presents a more straightforward scenario. Diagnostic testing and therapeutic
endeavour must be kept separate. Whatever the underlying diagnosis and the results of
any diagnostic testing, clinical manifestations of a disease must be treated. Failure to do so
may lead to considerable morbidity [21]. Thus bronchopulmonary infection is treated
with appropriate antibiotics, depending on the micro-organism. This includes nebulae
antibiotics for P. aeruginosa. Airway secretions are cleared by physiotherapy and
pancreatic enzymes are given for pancreatic insufficiency. The diagnostic label is less
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A. BUSH, M. GO
important than ensuring that the child receives adequate treatment. Of course it is
important to exclude conditions for which a specific therapy is available, such as
agammaglobulinaemia. When this has been carried out, the final label becomes a matter
of clinical judgement. It is appropriate to diagnose CF on purely clinical grounds [3].
Diagnostic testing may be repeated after 12 yrs, and this may clarify the situation.
Conclusions. As more has been discovered about CFTR, the diagnostic criteria for CF
have become more opaque. Generally, the more doubt that exists, the more likely that
ever greater sophistication of testing will increase confusion, rather than clarify matters.
There is no one agreed approach; a pragmatic view for the clinician and a more
interrogative one for the scientist are appropriate. For the clinician, the following criteria
apply. 1) Do not forget to think of CF as a diagnosis (all too often the real problem in
clinical practice), in particular in a screened population, which will still contain
undiagnosed CF patients, albeit with a reduced prevalence (one in 70,000 [10]). 2) If it
looks like CF, treat it as such. 3) Adjust treatment intensity to the severity of disease
manifestations; what is appropriate in a child with severe bronchiectasis and severe airway
obstruction will not be practical or desirable in a child who is asymptomatic but has two
potentially disease-producing mutations in the CFTR gene. 4) Remain vigilant for
deterioration; mild CF can only truly be safely diagnosed in the geriatric age group if there
are no significant symptoms: never in a child!
For the scientist, study of atypical phenotypes and diseases associated with a high
prevalence of single CFTR mutations may yield a rich harvest of understanding of the
biology of CFTR and its interactions with other proteins, which may eventually lead to
new therapeutic approaches.
Multifunctional nature of CFTR: not just a chloride channel

For many years, CFTR has been thought of as a chloride channel, with a few other
functions. This is in part because measurement of sweat chloride is a superb diagnostic
test and also because loss of electrolytes in the sweat perfectly accounts for two common
disease manifestations, namely heat exhaustion and pseudo-Bartters syndrome.
However, the connection between other CF disease manifestations and defective
chloride function has been much harder to establish. CFTR has been shown to have a
number of different functions (table 1). Although some, such as tooth colour, are
probably not of any pathophysiological significance, the role of, for example, glutathione
transport or aquaporin regulation in the production of CF disease should not be
assumed to be minimal. Alternative approaches that point to the multifunctional nature
of CFTR include gene expression studies (as manifest by mRNA changes) in CF
knockout mouse lung tissue [22], pharmacological manipulation of CFTR in cell lines
[23], and proteomics approaches in serum [24] and BAL [25], all of which have shown
that alterations in CFTR function affect multiple other genes and proteins. The present
authors suggest that the following points are important considerations in the
pathophysiology of the clinical CF disease.
1) Different functions of CFTR may cause disease in different organs. The relationship
between ion transport and lung disease is controversial. A recent study reported that
residual chloride transport function in the nasal epithelium correlated with pancreatic
function, and sodium transport was more significantly correlated with lung disease [26]. In
contrast, in males but not females, the residual CFTR response to low chloride/isoprenaline
correlated with forced expiratory volume in one second (FEV1) at age 19 yrs [27]. A third
study showed no relationship at all between nasal ion transport and lung disease [28]. The
role of sodium hyperabsorption in the pathophysiology of CF lung disease is discussed in
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CYSTIC FIBROSIS
Table 1. Putative functions# of cystic fibrosis transmembrane

conductance regulator (CFTR)
Putative functions of CFTR
Cl-, HCO3z transport
Regulation of other ion channels, especially epithelial sodium channel
Control of extracellular pH
Intravesicular acidification
Endocytic cycling
Aquaporin 3 regulation
Epithelial cell apoptosis
Pseudomonas aeruginosa binding and internalisation
Gap junction communication
Ca2z regulation
Extracellular fluid layer thickness
Glutathione transport
Increased mucus sulphation, decreased sialylation
Activity of nuclear factor-kB
Chemokine production
Whitens teeth (mice)
#
: It is unclear which functions are important in producing the cystic
fibrosis clinical phenotype in different organ systems.
more detail below. However, taken together, the evidence that chloride transport bears any
relationship with the pulmonary phenotype is not convincing.
2) It could be speculated that local modifying factors might mean that residual ion
channel function in the nose is different to that in the lower airway. For example,
neutrophil elastase, which is abundant in the lower airway, increases epithelial sodium
transport [29].
3) CFTR processing is different in different organs, and at different developmental
time periods. This is true both for wildtype CFTR and the mutant protein. For example,
the mutant DF508 CFTR is almost completely destroyed intracellularly in the sweat
gland, with virtually none reaching the apical cell membrane, whereas more translocates
to the apical membrane in the airway and colon [30].
4) There is differential regulation of CFTR function. For example, chloride transfer
requires ATP and glutathione ADP [31]. From this, it follows that different mutations
could, at least in theory, have differential effects on different functions of CFTR.
5) From these points, it follows on crucially that therapeutic strategies which correct
only one or a limited number of functions of CFTR may have no effect on the CF clinical
phenotype, if the wrong function has been targeted.
Theoretically, different genetic and environmental modifiers could affect functions of
CFTR selectively, or an interaction could only be significant in patients with particular
genotypes. The complexities of genotypephenotype correlations will be returned to in a
later section. In summary, the present authors suggest that the assumption that all
clinical phenotypes of CF are manifestations of chloride transport dysfunction is likely to
be incorrect.
Novel monitoring strategies: high-resolution computed tomography, new lung

function tests, the role of bronchoscopy
Why do we need new techniques to detect early lung disease? The conventional
clinical evaluation of babies relies heavily on symptoms, physical signs and the occasional
chest radiograph. When school age is reached, spirometry can be performed, but by this
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stage there may already be previously undetectable evidence of fixed airflow obstruction.
Lung function studies using the raised volume, rapid thoraco-abdominal compression
(RVRTC) technique have shown that at diagnosis, unscreened infants have evidence of
airflow obstruction [32, 33], even if there is no clinical or microbiological evidence of
airway disease. Follow-up over 6 months showed no evidence of "catch-up" growth [34].
Bronchoscopy has shown evidence of inflammation and infection even in screened infants
at a few weeks of age [35]. The preschool years can thus be a "silent" time, during which
some, but by no means all, infants suffer probably irretrievable lung damage.
The advent of novel therapies, such as genotype-specific pharmacological manipulation of CFTR and gene and stem cell therapy, make it all the more urgent to determine
which preschool children are starting to deteriorate and which are likely to do well long
term on conventional therapy. These novel treatments are likely to work best at an early
stage, before irreversible lung destruction has supervened. An understanding of how to
detect benefit is needed; mortality rate, which is of most concern to patients and families,
is thankfully far too low to be a realistic end-point in clinical trials. Novel medications
are also likely to have risks, which means that, at least initially, one would be unwilling to
offer them to infants doing very well on conventional therapy. This is particularly
relevant at the time of rapid alveolar growth, in the first 1824 months of life [3638];
little is known about medication safety in this context or how to monitor any potential
damaging effects.
Thus, with the increasing sophistication of treatment and the recognition that there are
important early changes of CF lung disease in particular, the need to detect the earliest
signs of deterioration has become imperative. Bronchoscopy and BAL, high-resolution
computed tomography (HRCT) and sophisticated indices of lung function, such as lung
clearance index (LCI), have increasingly superseded the conventional clinical tools of
upper airway culture, chest radiograph and spirometry, respectively.
Detection of early lung disease

Bronchoscopy. Bronchoscopy can be used to detect occult infection and to study early
onset inflammation. Currently, the latter is a research tool only, because effective and safe
anti-inflammatory strategies that are applicable to asymptomatic infants are not presently
available. In terms of detection of infection, conventional practice in the clinic is to
perform upper airway cultures, usually using a cough swab or a nasopharyngeal aspirate.
In general, a negative upper airway culture is predictive of a negative BAL, but positive
cultures are poorly predictive of a positive BAL [39]. Nonetheless, most physicians would
err on the side of caution and treat a positive upper airway culture. The role of routine
bronchoscopy in CF, as with HRCT, is undetermined. Practice varies between those who
perform annual surveillance bronchoscopies in all children who are unable to expectorate,
some units who virtually never resort to the bronchoscope, and probably the majority
who use bronchoscopy as part of the diagnostic work-up in children who are not doing
well on conventional therapy. In comparison with the lack of an attempt to secure a real
evidence base in the studies in HRCT, there is a superb, prospective, longitudinal trial in
Australasia, in which a routine bronchoscopy strategy is being compared with standard
treatment in nearly 200 children. The results of this trial will be very informative but will
not available for several years. Currently, it is known that BAL will identify organisms
that are missed by upper airway cultures. It is also known that BAL itself is not a gold
standard and that different pathogens may be isolated from different lobes in the same
patient [40], and also that inflammatory marker levels may differ between lobes. Thus,
even BAL can hardly be said to be the gold standard for determining the presence of
infection. Another approach to the problem of detecting infection, which may, unlike
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CYSTIC FIBROSIS
BAL, sample all lobes, is sputum induction, which has previously shown to be safe in CF
[41, 42]. Ho et al. [43] compared the culture results in spontaneously expectorated and
induced sputum in 43 patients, two of whom could not tolerate sputum induction. Ho et
al. [43] used 6% saline and pre-nebulised with salbutamol. Twenty-five patients had
identical results (nine positive); 12 out of 16 had at least one more pathogen isolated by
sputum induction and, in one patient, spontaneously produced sputum grew an extra
pathogen. This technique holds promise in the clinic but is time-consuming, and it is
surprising that Ho et al. [43] did not use salbutamol via a spacer instead of a nebuliser.
Also, the study by Ho et al. [43] included young children who would not have been
expected to expectorate spontaneously. An alternative approach is to have the child cough
directly onto a microbiology culture plate ("cough plate") [44]. Induced sputum has been
used to diagnose tuberculosis in African babies [45] and perhaps should be used more
readily in symptomatic patients in this age group, prior to resorting to bronchoscopy.
Imaging techniques. Conventional practice is an annual chest radiograph, which can

be scored in a number of different ways. Gross clinical disease is usually very obvious
but chest radiography is much less sensitive to early changes. Although sophisticated
systems like the Wisconsin score have been devised with early stage disease in mind,
they are time-consuming to administer and have not found widespread use. This has
led to consideration of the role of HRCT scanning in routine clinical practice. Three
recent papers [4648] have addressed this topic. Brody et al. [46] performed HRCT
scans on a single occasion in 60 CF children aged 610 yrs with "mild" lung disease
(forced vital capacity (FVC) w85%, but, as might be expected, FEV1 was often
considerably less than this). More than one-third of patients had bronchiectasis, nearly
two-thirds had air trapping and only a quarter had a normal scan. A semiquantitative
scoring system was developed; there was reasonable (w80%) agreement between the
three observers and they concluded that HRCT can detect changes in children with
"normal" lung function.
A longitudinal study [47] confirmed the greater sensitivity of HRCT compared with
spirometry ("lung function tests"!). Forty-eight children had scans 2 yrs apart. Five
different semiquantitative scores were used. Independent of which scoring system was
used, spirometry was shown to remain stable while HRCT findings deteriorated. A
weakness of the study [47] is the authors assumptions about what is and is not
irreversible change. This may seem a strange comment but the reader should reflect how,
in another context, even dramatic pneumatoceles, for example, can regress completely
with time. The authors made no attempt to determine any clinical utility of their imaging
findings.
A third paper did move from the rather 19th century approach of estimating degrees of
greyness and dilatation and making a semi-arbitrary scoring system, to digital handling
of digital material [48]. The important findings relate to 10 normal and 10 CF subjects
who were scanned at total lung capacity and functional residual capacity (FRC). The
changes in voxel density were calculated and displayed graphically. This was a proof of
concept study: the lung function comparator was spirometry. No clinical correlations
were attempted, but the study again proved that spirometry is not a sensitive technique.
The children studied were old enough to be able to cooperate with the respiratory
manoeuvres required, but the use of sedation and bag and mask ventilation to take over
respiration, analogous to RVRTC, allows the technique to be adapted for babies [49].
The use of true quantitative scoring is an advantage but, of course, it comes at a price of
increased radiation dose.
Current papers have a number of problems that must be considered in any critical
assessment of the role of CT in CF; these are as follows. 1) There are no studies in
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peer-reviewed journals comparing HRCT with tests of lung function that are much more
sensitive than spirometry, e.g. LCI (see below); a study reported in abstract form showed
that a normal LCI excluded significant structural damage on HRCT [50]. 2) In many
cases, there is use of semiquantitative scoring on digital data, which should be
mathematically handled. 3) There has been a lack of attempts to quantify clinical utility
or correlation with clinical status. 4) A radiation dose is being administered to lungs,
breasts and potentially thyroid [51] in a population who are possibly at greater risk of at
least some epithelial cancers [52]. 5) There is a paucity of reproducibility data, which is
essential when HRCT is being advocated as a trial end-point in particular [53]. Better
evidence is needed of what is "irreversible" and what can be corrected with growth or
treatment.
What, then, is the current role of HRCT in the detection of early lung disease in CF? It
is clear that if there was no issue about radiation or fiscal cost, HRCT would be a routine,
annual or more frequent test. The present authors believe that continuing to study the
role of HRCT in the context of proper clinical trials is fully justified, that HRCT is a fully
justified clinical investigation in children not making good progress for reasons that are
unclear; however, evidence that regular HRCT as a clinical tool is justified has not yet
been accumulated.
Physiological techniques. Spirometry is a standard clinical procedure in school-age

children, and babies can be sedated for pulmonary function testing, such as RVRTC and
plethysmography. The advantage of RVRTC, in which the abdominal compression is
applied after passive inflation to total lung capacity, is that the flowvolume curve
produced is very similar to that of conventional spirometry. This is unlike the
conventional squeeze technique, where the compression is applied at the end of
inspiration and flow at FRC is measured. The London Collaborative CF group used the
RVRTC technique to study CF infants 6 months apart, and compared them with controls
[31]. This report demonstrated that even despite intensive therapy in specialised centres,
there was no catch-up growth in airway function. The need for sedation, trained personnel
and complex apparatus means that even in babies, RVRTC cannot be used at every clinic
visit and the technique cannot be used at all in toddlers. However, the same group has
looked at means of performing spirometry in preschool children (both normal subjects
and those with CF), and, most importantly, developing adequate quality control (table 2)
[54]. The authors used incentive spirometry and achieved success rates of 64% in the 2
v4 yr age bracket. It should be noted that up to 25 manoeuvres were recorded, and in
conditions such as asthma, which are characterised by airway instability, this could lead to
bronchoconstriction. Nonetheless, the RVRTC technique, preschool spirometry and
conventional clinic spirometry allow essentially the same measurements to be made across
the entire age span. However, it should be noted that interpretation is far from
straightforward; lung emptying is progressively less efficient with age, so FEVt (the forced
Table 2. Quality control criteria for spirometry in the preschool years
All curves must be visually inspected
Start of test can be quantitatively expressed as in adults, but VBE w80 mL or VBE/FVC w12.5% should prompt
re-inspection of the data, rather than automatic rejection
FEVt should only be reported if FET for that expiration is wt
FEV0.5, FEV0.75 and FEV1 should be reported when available (see above)
Repeatability criteria of 100 mL and 10% of best effort may be more appropriate for DFVC and DFEVt
VBE: volume of back extraction; FVC: forced vital capacity; FEVt: forced expired volume in t seconds; FET: forced
expiratory time. Data refers to a summary of the data from [54].
242
CYSTIC FIBROSIS
expiratory volume in t seconds) measures the emptying of different airway generations at

different ages.
There is a paucity of data following CF children serially through the preschool years.
Nielsen et al. [55] followed 30 children in a 4-yr prospective study. They measured
specific airway resistance (sRaw) using plethysmography, resistance using the interrupter
technique (Rint), and impulse oscillation. They looked at the responses to cold air and
bronchodilators and performed spirometry at school age. Only sRaw was consistently
abnormal in CF. Lung function parameters appeared to show tracking during the
preschool years. There was no difference in bronchodilator responsiveness or the results
of cold air challenge. FEV1 in school age correlated significantly with sRaw only
(r2=0.80), implying that Rint and impulse oscillation were insufficiently sensitive to detect
disease. The study by Nielsen et al. [55] implies that there is significant early loss of lung
function in CF, probably in infancy, and that intensive therapy, even over several years,
does not reverse this loss of function.
The question is: what is the most sensitive physiological technique for studying early
CF lung disease? Gas mixing is exquisitely sensitive to distal airway disease. The
physiological background has been long established, but recently Gustafsson and coworkers [56, 57] in particular have reintroduced the technique, using the wash-in and
wash-out curves of an inert marker gas, such as sulphur hexafluoride. In essence, the
child breathes in a known concentration of the marker gas during quiet tidal breathing,
usually while distracted by watching a video. When equilibration has been reached, the
child is disconnected from the tracer and the concentration is measured as the gas is
washed out of the lungs during continued tidal breathing. The LCI is calculated from the
time for the tracer gas concentration to fall to 1/40th of the starting level, and the total
volumes (measured as number of FRCs) expired, currently measured with a mass
spectrometer. The technique has been used in babies and toddlers (from whom only
passive cooperation is required), as well as older children and adults. A further attraction
is that LCI appears to have the same normal range, irrespective of age or size, thus
obviating the problems of suitable controls for a population like CF, in whom impaired
growth and nutrition is likely.
Two recent papers have addressed the use of LCI in early stage CF [58, 59], both were
cross-sectional. The first compared LCI with FEV1 in CF and controls [58], and
compared Swedish and British data. The principle findings were that whereas there were
significant negative correlations between LCI and both FEV1 and flow at 25% of vital
capacity, most children with a normal FEV1 had increased (abnormal) LCI, implying
that LCI was more sensitive. The normal values for LCI were similar in both countries,
implying that the technique is transferable. The second paper [59], compared
plethysmography (sRaw) spirometry [54] and LCI in a cross-sectional study. More
abnormalities were detected by LCI than the other two techniques, and LCI was the only
parameter which differed significantly between those who were and were not infected
with P. aeruginosa.
Longitudinal studies are the hardest to perform but the most informative of all to read.
Kraemer et al. [60] studied 142 children and adolescents with CF, diagnosed on standard
grounds who had documented onset of chronic P. aeruginosa infection and who had
complete documentation of clinical details and lung function between the ages of 6 and
20 yrs. Those infected only intermittently with P. aeruginosa or with Burkholderia cepacia
were excluded. Reflecting their population there was a high prevalence of unusual
compound heterozygotes (3905insT/DF508, 9.2%; R553X/DF508, 8.5%). They performed spirometry, and measured FRC and sRaw plethysmographically, and LCI using
nitrogen wash-out. Salient features of their detailed paper were as follows. 1) LCI
deteriorated first, followed by mean forced expiratory flow between 25 and 75% of FVC,
followed (surprisingly) by FVC ahead of FEV1. 2) When FEV1 was normal, LCI was
243
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A. BUSH, M. GO
abnormal in 52.5%; conversely, an abnormal FEV1 was only missed by LCI in 0.5%.
FEV1 stabilised at 12 yrs of age, whereas hyperinflation continued to worsen. 3) Even at
age 20 yrs, the slope of progression was still best for LCI and worse for FEV1. There are
some caveats concerning this impressive data collection. The population was selected for
infection status, and most were relatively well at age 20 yrs (only 10 deaths), so more data
are needed on the relative values of these techniques in late-stage disease. There were no
data from the preschool years. Nonetheless, these data would confirm cross-sectional
data [48] that LCI is the most sensitive measure of deterioration in lung function.
Most technique-based papers have not attempted correlation with other relevant
indices, a point which will be returned to. One recent paper looked at the correlations
between low frequency, forced expired oscillation [61], a lung function test previously
shown to be relatively insensitive [55]. In this cross-sectional study, the children were
undergoing bronchoscopy as part of their routine work-up, and the authors confirmed
that infection, not symptoms, were correlated with inflammation, as judged by BAL
neutrophilia, interleukin (IL)-8, neutrophil elastase and leukotriene (LT)B4. There were
some correlations between inflammatory markers and lung function parameters, but no
consistent picture emerged. This paper perhaps mainly confirms the lack of sensitivity of
the forced oscillation techniques in early CF lung disease.
What, then, is the role of LCI in monitoring early CF lung disease? It would seem
likely that it is the most sensitive of current techniques and needs to be used as a
comparator with, for example, CT scanning (see below). More information is needed
about within-subject reproducibility before it can be recommended as a treatment
outcome. It is also conceivable that it may be too sensitive and might detect
abnormalities of no practical significance. More longitudinal data, in particular in
more severely affected patients, are required to settle this question. For later-stage
disease, LCI may be too sensitive, and spirometry will remain the preferred measure,
pending the development of better tools. Finally, LCI measurements need to be adapted
for robust, cheap equipment, which can be made widely available, if it is to become a
routine clinical tool.
The role of macrolides

Macrolide antibiotics are conventionally thought of as being derived from
Streptomyces spp. They have a common macrolytic lactone ring, to which sugar
moieties are attached. The first antibiotic to be discovered was erythromycin (a 14membered ring) in 1952 [62]. Clarithromycin (14-membered ring) and azithromycin (15membered ring) are more popular in clinical practice because of fewer side-effects
(principally gastrointestinal) and longer dosing intervals. Other 14-membered ring
compounds include roxithromycin and troleandomycin. Spiramycin, josamycin and
midecamycin are 16-membered ring compounds. The latest group to be synthesised, the
ketolides, are characterised by a keto group instead of a sugar moiety at position 3 of the
lactone ring [63]. Macrolides are ubiquitous in nature; more than 2,000 different
compounds are known, from sources as diverse as algae, lichens, insects and
invertebrates. These compounds have recently been reviewed in detail [6469], and a
summary of their nonantibacterial actions is been given in table 3. It should be noted that
different classes of macrolides may have different effects and extrapolation of effects
between different classes is unwise.
Macrolides, in particular erythromycin, have been used to treat community-acquired
gram-positive respiratory infections, particularly otitis media, acute tonsillitis and mild
cases of lower respiratory tract infection. More recently, the newer macrolides have been
found to be useful in the treatment of resistant Mycobacterium tuberculosis and also
244
CYSTIC FIBROSIS
Table 3. Putative actions of macrolide antibiotics

Effects described
Downregulation of pro-inflammatory cytokines IL-1b, IL-6, IL-8, TNF-a, granulocyte-macrophage
colony-stimulating factor
Reduced neutrophil migration to IL-8 stimulation
Reduced P. aeruginosa adherence
Impaired P. aeruginosa viability and protein synthesis
Upregulation of other members of the ABC cassette, e.g. MDR, MRP
Effects on airway remodelling: inhibition of ET-1 and ET-1-induced VEGF production
Suppression of iNOS production
Binding to nuclear transcription factors AP-1 and NF-kB
Reduced elastase production and oxidant burst
Diminution of conversion of P. aeruginosa to the mucoid phenotype
Reduced P. aeruginosa flagellin
Reduction of mucus secretion and viscosity
Reduction of acetylcholine release from cholinergic nerve terminals
Inhibition of membrane-derived, toxic phospholipids
Reduction of neutrophil adherence to endothelium and epithelium via ICAM-1, VCAM-1 and LFA-3;
reduction of CD11b/CD18
Acceleration of neutrophil apoptosis
Inhibition of P. aeruginosa quorum sensing; reduction in biofilm formation
Synergy with other antibiotics, e.g. tobramycin
Reduction in bronchial hyperreactivity
Inhibition of fibroblast proliferation
Treatment of occult S. aureus, H. influenzae or atypical mycobacterial infection
Note that different classes of macrolides may have differing properties [6469]. IL: interleukin; TNF: tumour
necrosis factor; P. aeruginosa: Pseudomonas aeruginosa; MDR: multidrug resistance protein; MRP: multidrug
resistance-associated protein; ET: endothelin; VEGF: vascular endothelial growth factor; iNOS: inducible nitric
oxide synthase; AP: activator protein; NK-kB: nuclear factor-kB; ICAM: intercellular adhesion molecule; VCAM:
vascular cell adhesion molecule; LFA: leukocyte function-associated antigen; S. aureus: Staphylococcus aureus;
H. influenzae: Haemophilus influenzae.
atypical mycobacterial infection. They are very safe and generally well tolerated.
Macrolide resistance in the community is related to prescribing frequency. They are not,
however, conventionally thought to be active against the gram-negative bacteria that are
a particular problem to the CF airway.
Interest in their nonantibacterial, immunomodulatory effects first arose from the
dramatic benefits seen in patients with diffuse panbronchiolitis. This is a disease of
middle-aged people in the Far East and is characterised by many of the phenotypic
features of CF [70]. Presentation is with cough, chronic sputum production and
breathlessness, with coarse crackles heard on auscultation. There is a mixed obstructive
and restrictive pattern physiologically. HRCT scanning reveals bronchiectasis. Sputum
cultures are positive for Haemophilus influenzae, Staphylococcus aureus and, most
strikingly, mucoid strains of P. aeruginosa. As a result of chance observations, it became
clear that long-term, low-dose erythromycin dramatically improved prognosis, changing
10-yr survival fromv20% tow90% [71]. A series of elegant studies established that diffuse
panbronchiolitis is characterised by a neutrophilic BAL and that macrolide treatment
therapy reduced lavage neutrophil chemo-attractant activity and neutrophil counts [72,
73]. The response did not depend on the patient being chronically infected with mucoid
P. aeruginosa [74]. Treatment with erythromycin or, if this fails, clarithromycin, is
essentially curative of a once fatal condition. This extraordinary result led to interest in
the use of macrolides in CF.
Three different double-blind, randomised, placebo-controlled trials have established a
role for macrolides in some patients with CF (table 4). The first was a double-blind,
parallel group study [75] in which 60 adults with stable CF, meansd age 27.96.5 yrs,
245
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A. BUSH, M. GO
Table 4. Summary of published clinical trials of azithromycin (AZM)
Trial design
AZM dose mg
Subjects n
Subject age yrs
Treatment period months
FEV1 drug versus placebo
mean relative difference
Other clinical outcomes
Inflammatory markers
Adverse effects
Australia [75]
UK [76]
USA [77]
Two-centres, parallel
Single-centre,
crossover
250 or 500 daily
41 children
w8 yrs
6
Median relative
difference z5.4%
Decrease in oral
antibiotics
Multicentre, parallel
250 daily
60 adults
3
Mean relative
difference z3.6%
Decrease in intravenous
antibiotics, increase in
quality of life
Decrease in CRP
None
No difference in
sputum IL-8,
neutrophil elastase
None
250 or 500, three times per week

185 children and adults
w6 yrs
6
Mean relative difference z6.2%
Decrease nonquinolone
antibiotics, decrease in
exacerbation, increase in weight
Modest decrease in sputum
elastase,no change in IL-8
Nausea, diarrhoea, wheezing
FEV1: forced expiratory volume in one second; CRP: C-reactive protein; IL: interleukin. Adapted from [1] with
permission.
meansd FEV1 56.622.3%, were given azithromycin (250 mg) once daily or placebo for
3 months. In the azithromycin group, FEV1 and FVC remained stable, but in the placebo
group there was a deterioration of 3.621.78% (p=0.047) and -5.731.66% (p=0.001),
respectively. This is quite a rapid deterioration, equating to y15 and 22% annual rate of
decline, respectively. The azithromycin group used fewer courses of intravenous
antibiotics (0.37 versus 1.13, p=0.016) and quality of life improved as opposed to
remaining stable. C-reactive protein declined from 10.4 to 5.4 ng?mL-1 in the treatment
group and remained stable in the placebo group. There was no change in sputum
bacteriology. The present authors performed a crossover study in children, who were
given daily azithromycin or matched placebo in random order, in a dose of 500 mg if
their weight was i40 kg and 250 mg if it was v40 kg [76]. Forty-one children were
recruited and provided evaluable data. Median (range) age at recruitment was 14.2 yrs
(8.118.6) and median (range) FEV1 and FVC were 61% (3380) and 80% (5399),
respectively. The primary end-point was FEV1, which improved by 5.4% (95%
confidence interval (CI) 0.810.5%) with azithromycin compared with placebo. Half
had an improvement in FEV1 of i10%. There were no side-effects. A post hoc
comparison of the results in patients who were and were not also inhaling human
deoxyribonuclease (rhDNase), which an in vitro study suggested might be inhibited by
azithromycin [78], showed that the patients not also inhaling rhDNase (n=26) had an
increase in FEV1 of 11.5% (95% CI 5.316.5), whereas those who also inhaled rhDNase
had a change of -3.6% (95% CI -223.9). However, the study was not powered for
subgroup analyses; this was a post hoc comparison and the latter finding has not been
confirmed in the third study [77]. The third study [77] was a parallel group study, with a
2-week run-in period, 168-day treatment period and a 28-day wash-out period. The dose
used was 500 mg if the childs weight was i40 kg and 250 mg if it was v40 kg, and was
given 3 days in the week, with provision for stepdown if it was not tolerated. A total of
185 patients were randomised, of whom 87 received azithromycin and 98 placebo. Only
one patient (in the placebo group) was lost to follow-up. The groups were well matched.
The mean age was 20 yrs, just over one-half were male, mean height was 162 cm, mean
weight just over 50 kg, and starting FEV1 was y70% predicted. Around 40% were
homozygous for DF508. All had chronic infection with P. aeruginosa and smear
positivity for non-tuberculous mycobacteria at entry was an exclusion criteria.
246
CYSTIC FIBROSIS
A subsequent culture positive for non-tuberculous mycobacteria resulted in the patient

being withdrawn from the study; however, they were included in the analysis on an
intention-to-treat basis. The results showed a treatment benefit of 0.093 L or 6.21% for
FEV1, and 4.95% for FVC (all highly statistically significant). There was marked
variation in individual response;y12% increased FEV1 by i15% on azithromycin (none
on placebo), but some actually deteriorated. Any benefit was lost within 28 days of
discontinuing therapy. There was a 40% reduction in infective exacerbations defined as
the use either of intravenous antibiotics or quinolones. The azithromycin group gained
800 g in weight compared with placebo. Physical functioning on the quality-of-life score
improved significantly on azithromycin. There was no emergence of resistant microorganisms; there were more new isolates of S. aureus in the placebo group. Nausea,
diarrhoea and, despite improved lung function, wheeze were more common in the
azithromycin group.
These trials have confirmed that for many, but not all, individuals with CF,
azithromycin therapy improves pulmonary function. No one can predict which patients
will benefit from treatment and the mechanism of action is unknown. Unlike in diffuse
panbronchiolitis, there does not seem to be an effect on airway neutrophilia, but as with
diffuse panbronchiolitis, chronic P. aeruginosa infection is not a prerequisite for benefit.
The optimal dose and dosing frequency is not known. There are worldwide differences in
how macrolides are used in CF. Some clinics use them as part of their routine treatment
of chronic infection with P. aeruginosa. The present authors are more cautious, having
taken account of the lack of knowledge of possible long-term side-effects in children; a 4
6-month therapeutic trial of daily azithromycin is carried out in children who are not
doing well on conventional therapy, irrespective of their sputum bacteriology, and the
medication is discontinued if there is no benefit. This is clearly an area in which more
work is needed, but also one in which clear-cut benefit for patients has been established.
That at least some CF patients benefit from macrolides is indubitable, and has been
confirmed by a Cochrane review [79]. The mechanisms of benefit have not been
determined. The potentially beneficial effects of macrolides have been reviewed [6469],
and are summarised in table 3. Some are disease-specific (e.g. effect on the multi-drug
resistant protein for CF), some are infection-specific (quorum sensing in P. aeruginosa
infection) and others are generic. It should not be assumed that the same mechanisms are
operative in all diseases in which macrolides are therapeutic, nor that a single mechanism
is necessarily operative in a given disease.
Recognition of new multisystem complications

When CF was first recognised as a separate entity in 1938, virtually all patients died in
infancy from malnutrition and chronic respiratory infection. The concept of CF as a
multisystem disease has developed as patients survive into adult life. The implications
are: first, that these complications need to be detected early and managed effectively; and
secondly, that their development paediatric practice needs to be confirmed, so that
preventive strategies can be developed. The classical example of this would be CF bone
disease (see below).
Insulin deficiency. Although there has been controversy about whether hyperglycaemia
relates to peripheral insulin resistance or reduced pancreatic insulin production, current
understanding favours the latter [80]. Approximately one-third of adult CF patients will
eventually develop diabetes. The onset of diabetes is preceded by a "pre-diabetic" phase in
which nutrition and lung function deteriorates [81, 82]. In females, but not males, diabetes
is associated with increased mortality and, indeed, accounts for most of the excess
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mortality in CF females [83, 84]. Progressive pancreatic b-cell dysfunction, which is

already subnormal in CF patients with oral glucose tolerance test (OGTT)-defined normal
glucose tolerance status deteriorates further with consequential worsening of glycaemic
status. This suggests that insulinopenia plays a prominent role in the pathogenesis of
glucose intolerance and subsequent development of CF-related diabetes mellitus
(CFRDM). Recent evidence casts doubt on the role of the OGTT as a gold standard
for establishing CFRDM, as some children with a deteriorating clinical state may show
normal OGTT responses and yet benefit from additional small doses of insulin [85]. The
role of annual OGTTs is controversial. While many recommend that this test be part of
the annual assessment from the age of 10 yrs [86], it may lull the clinician into a false sense
of security (as described above), and it is arguable whether a child who has normal lung
function and nutrition would accept a new invasive treatment on the basis of a blood test.
In a child who is not doing well, an OGTT must be performed, but, if this is normal,
additional intermittent blood glucose assessments may well reveal transient asymptomatic
hyperglycaemia. Many recommend that all OGTTs be performed in the extended 180-min
variant and that serum insulin levels be monitored concomitantly as impaired insulin
secretion is common (65%), even in children with normal glucose tolerance [87].
Standardisation of the diagnosis of CFRDM and glucose intolerance is important, but
until this is agreed, standard diagnostic procedures will be different in different centres.
Early introduction of small doses of insulin in seemingly non-CFRDM with intermittent
hyperglycaemia may dramatically improve the patients well-being. It remains to be seen
whether insulin therapy generally should be considered in completely well CF children
before the onset of overt CFRDM. They may be very vulnerable to the effects of
hyperglycaemia. It may be that inhaled insulin may have a role [88]. Although oral
hypoglycaemics were popular and are still the subject of therapeutic trials, logically the
treatment of the deficiency state is a direct replacement of what is deficient, rather than
trying to force the production of more insulin, which is unlikely to occur.
Anabolic hormones in CF. As CF is a hypercatabolic condition, either as a possible

consequence of the underlying disorder or, which is more likely, as a secondary
consequence of the burden of infection and inflammation, the use of anabolic hormones
may seem logical. There have been a few small studies of the use of growth hormone [89
91]. Insulin-like growth factor (IGF)-I, formerly known as somatomedin C, mediates
some but not all of the metabolic actions of growth hormone (GH) and has both GH-like
and insulin-like actions in vivo. GH and IGF-I both have a net anabolic effect in humans,
enhancing whole body protein synthesis over a period of weeks and perhaps months [92].
Both hormones favourably improve body composition in GH-deficient subjects, with an
increase in lean body mass and decreased adiposity. A potent glucose-lowering effect is
typically observed after IGF-I administration, with improved insulin sensitivity and
marked lowering of circulating insulin concentrations. This observation means IGF-I is a
potentially more convenient anabolic agent to use in conditions in which carbohydrate
metabolism is more likely to be impaired, such as in CF. IGF-I increases lipid oxidation
only when given chronically, usually in states of chronic insulinopenia. The role of IGF-1
(and GH) in a CF patient remains to be established. It appears conceivable that in the
future, a kind of calculated "doping" will be introduced in patients with CF, but careful
determination of which patients might improve most without untoward side-effects
remains an unsettled issue. It is not yet necessary to ask the "drug cheats" from athletics to
bring their methods to the CF clinic.
CF bone disease. By definition, osteopenia is a bone density between 1 and 2.5 sd below
the bone density of a normal young adult. Osteoporosis is defined as i2.5 sd below that
248
CYSTIC FIBROSIS
reference point. Recent data show an incidence of osteopenia in up to two-thirds of adult

patients with CF and osteoporosis in a quarter [93]. Pathological fractures are common,
and can include vertebral collapse, rib fractures and kyphosis [94], which may impair
cough and sputum clearance and further reduce the capacity for physical exercise. There
are multiple possible reasons for reduced bone mineral density in CF. These include
inadequate physical activity, chronic airway inflammation with spillover of cytokines,
such as IL-1, IL-6 and tumour necrosis factor (TNF)-a, into the systemic circulation,
decreased calcium and vitamin uptake due to anorexia or pancreatic insufficiency leading
to low vitamin D and K levels, delayed puberty and hypogonadism, and the use of oral
and high-dose inhaled corticosteroids. In the transplant patient, immunosuppression and
glucocorticoid use may worsen the problem. Independent risk factors are homo- and
heterozygosity for DF508, male sex, greater severity of lung disease and malnutrition [95].
The CFTR null mouse model indicates that even in the absence of obvious nutritional and
therapeutic differences, the CFTR mutation is associated with severe osteopenia [96].
The bone disease of CF has been studied in a few histomorphometry reports. It is
heterogeneous, with reduced bone formation, but, surprisingly, also decreased bone
resorption in some cases. There has been evidence of a generalised mineralisation defect
but true osteomalacia is rare. Bone marker studies suggest that there is a background of
low bone formation, especially during puberty, with periods of increased bone resorption
[97].
Many CF clinics now recommend routine assessment starting in adolescence or young
adulthood using dual energy X-ray absorption (DEXA) scans. These are performed
every 12 yrs, depending on the level of risk. Age-related standard normal values must be
taken into account, which may be a particular problem in paediatrics [98].
The management of bone disease is, firstly, prevention. Efforts to increase bone
mineral density are particularly important during key areas of bone accretion in
childhood and adolescence. There are no trial data upon which to base recommendations, but from studies in other conditions, the following criteria are recommended.
1) Maximise weight-bearing exercise. Even relatively simple programmes have been
shown to increase bone mineral density [99]. 2) Maximise the intake of milk. In a study of
asthmatic adolescent girls, increasing milk intake by delivering an extra pint to the house
resulted in a significant increase in bone mineral density, still detectable 3.55 yrs after
the intervention. Calcium supplements are not as effective as calcium in dairy products
[100, 101]. 3) Ensure vitamin D levels are adequate by measuring them at least annually
and supplementing as appropriate. However, in a large group of children cared for in a
specialist centre, calcium homeostasis was normal with no abnormality of 25
hydroxyvitamin D. Levels were lower in adolescents and there was no association
with pancreatic insufficiency or liver disease [102]. 4) Vitamin K has a key role in bone
metabolism. Measurement of levels in serum is expensive and measurement of
prothrombin ratio is insensitive, so many clinics recommend that vitamin K should
be given empirically. 5) Pubertal stage should be monitored and any delay in onset of
puberty treated.
If bone disease is established, treatment is with oral antiresorptive agents such as
biphosphonates. Pamidronic acid, etidronic acid and alendronic acid inhibit recruitment
and function of osteoclasts, thereby reducing bone resorption. Alendronate, 10 mg?day-1
orally for a year [94], resulted in a significant gain in bone density. These are promising
agents for long-term prevention and management, but many unanswered questions
remain, not least at what stage they should be started.
A future treatment option may be anabolic agents. Teriparatide is a Food and Drug
Administration-approved anabolic bone growth agent, but data in CF are lacking. GH
treatment for 1 yr improved height, weight, lean tissue mass and bone mineral content in
prepubertal children aged 7.512.75 yrs [103].
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Stress incontinence. Stress urinary incontinence occurs when the pressure within the
abdomen is higher than the urethral resistance. This can happen while coughing, laughing,
sneezing, huffing, bending, lifting a heavy object, doing physiotherapy, participating in
vigorous sports activities, such as trampoline jumping and during sexual intercourse.
While already common in non-CF females (30%), stress urinary incontinence occurs in
close to 70% of CF females agedy30 yrs. The dominant precipitants in CF are coughing
(84%) and laughing (68%). The problem has been addressed and assessed only in recent
years and embarrassment was the main reason for not seeking medical advice. Social life
may be affected, as reported by one-third of respondents of an Australian survey [104].
The differential diagnosis of unwillingness to perform physiotherapy includes stress
incontinence during treatment. A sympathetic enquiry should always be part of the
assessment of such children. It is not only adult females who are affected; an Australian
study showed a median age of onset of incontinence of 13 yrs [105]. No relationship was
seen with age, lung function, body mass index or menstrual status.
There is little evidence base for treatment. CF females are prone to vaginal candidasis,
which may worsen stress incontinence, and this should be treated [106], as should urinary
tract infection. Pelvic floor muscle exercises are helpful in establishing improved muscle
strength, leading to reduced leakage [107]. Biofeedback for training pelvic floor
musculature may be more acceptable but there are no data for CF. Tension-free vaginal
tape should be considered for females with urinary stress incontinence for whom
conservative (nonsurgical) treatments have failed to provide a solution after 36 months.
The current surgical gold standard is colposuspension; the help of a gynaecological
expert should be sought when advising CF females. In the future, duloxetine, a selective
serotonin and noradrenaline re-uptake inhibitor, may be useful [108].
Chronic pain. Assessment of pain in patients of all age groups has not generally been part
of CF care concepts until relatively recently. Disease-related pain is often in the chest, and
headache is also common. Other considerations include back pain, abdominal pain and
musculoskeletal pain. Joint pain was found in 17% of an Australian paediatric CF
population [109]. Recent work [110] on acute and chronic pain in 46 patients aged 8
17 yrs showed pain predominantly in the abdominal/pelvic region (50%), followed by
chest pain (37%) and head/neck pain (33%). Pain management consisted of rest,
medication, heat or cold, the support of family and friends, and distraction therapy. No
patient resorted to opioids. Children with chest pain reported significantly greater
perceived functional limitations due to pain than patients without chest pain. Assessment
and treatment of pain should routinely be included in the overall management plan. The
negative effect of pain on pulmonary function and on quality of life should be taken into
account.
Advances in sexuality, reproduction issues and management of the pregnant

female with CF
Sexuality. In both sexes, the subject of sexuality and reproduction should be addressed in
early adolescence in order to avoid misleading information from noncompetent sources.
Obstructive azoospermia in CF males is almost universal and is an early cause of
concern in parents of patients with CF or in male patients once they reach adolescence.
Male fertility should be discussed specifically, and before puberty. Males should be
reassured that pubertal changes will be normal and sexual intercourse unaffected [111].
They need to differentiate between potency, which is unaffected, and fertility, which may
be achieved by new microsurgical techniques outlined below. They should also
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understand that infertility is not a protection against sexually transmitted diseases and
barrier methods are as essential for them as any other males.
Post-pubertal males should be offered semen analysis. The usual outcome is absence of
sperm and low volume of ejaculate. Occasionally congenital bilateral absence of the vas
deferens may be found with none of the usual clinical CF organ manifestations. There is
a higher than expected prevalence of mild CF mutations in this group [112]. Some
undoubtedly have a pure genital (nonclassical) form of CF (two CF-producing mutations
found); others have a single mutation and no other manifestation of CF.
Female sexuality is hardly affected and sexual life is normal. However, in both sexes,
sexual enjoyment may be affected in cases of severe physical distress (weakness, shortness
of breath, copious expectorate). This can sometimes be alleviated by planning sexual
intercourse in the morning, when the patient is not so tired.
Females with CF need to consider contraception when they are sexually active.
Contraception using barrier methods is no more prone to failure than in non-CF persons.
Oral contraceptives have been found to be safe despite worries concerning liver function
being affected by the progestogen component of contraceptives. There are some concerns
that very low-dose oestrogen contraceptives may be less effective in CF females.
Reproduction issues. Female fertility in CF appears to be reduced, although no recent

work has been devoted to the subject. Whether reduced water content of cervical mucus
and/or increased viscosity are of major importance is unanswered. Primary (10%) or
secondary amenorrhoea may be a problem, with advanced lung disease or poor
nutritional status. As many as 50% of females with CF have anovulatory cycles [113].
Male reproductive choices have improved with percutaneous or microsurgical
epididymal or testicular sperm aspiration and subsequent in vitro fertilisation.
Intracytoplasmic sperm injection has become the preferred approach for azoospermia
[114]. Older alternative methods, such as donor semen fertilisation or adoption, may
have a role in selected cases.
Genetic counselling is an important aspect for both CF males and females. The child of
a CF parent is at best an obligate carrier; if the partner is an unsuspected carrier, or even
has mild, undiagnosed CF, then the chances of producing a CF baby are 50 and 100%,
respectively.
Management of pregnant females with CF. Pregnancies in females with CF are

increasing concurrently with increased life expectancy. A US Cystic Fibrosis Foundation
registry report covering 19851997, with data on 680 pregnancies inw8,000 females with
CF, assessed the impact of pregnancy on survival [115]. Those entering pregnancy had a
better initial health state (lung function, weight) and their 10-yr survival was higher than
in those who did not become pregnant. This is presumably an example of reverse
causation (the less-healthy females opted not to become pregnant). In a separate analysis,
possibly confounding factors, such as chronic infection with P. aeruginosa and pancreatic
function, were taken into account, but similar results were obtained. Using Cox
proportional hazard modelling to adjust for baseline age, FEV1 % pred, weight, height
and pulmonary exacerbation rate?yr-1, pregnancy was not associated with an increased
risk of death. Pregnancy did not add an additional adverse prognostic effect in any
subgroup, including patients with FEV1 v40% pred or diabetes mellitus. Thus, females
with CF who became pregnant were initially healthier and had better 10-yr survival rates
than females with CF who did not become pregnant. After adjustment for the initial
severity of illness, females who became pregnant did not have a significantly shortened
survival. Similar results were obtained in 75 pregnancies in French CF mothers [116].
There was almost a 30% incidence of low-weight newborns.
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Although earlier reports of pregnancy in females with CF had shown alarming rates of
maternal complications, these recent data support a much more optimistic outlook. The
pre-pregnancy severity of CF seems to be the decisive determinant of the subsequent
course of the mother. On a cautionary note, it should be mentioned that pregnancy in CF
is associated with decreased insulin sensitivity and high hepatic glucose production, in
addition to inherent decreased insulin secretion. Pregnancy in CF is also associated with
increased protein turnover and less response to the anticatabolic effect of insulin. These
changes may predispose the pregnant CF females to early development of diabetes and
poor weight gain [117]. Close collaboration between the CF team and the obstetrician is
essential in all CF pregnancies.
Undoubtedly, females with CF should and will make their own reproductive decisions.
Couples do need to bear in mind that an already sick females with CF who becomes
pregnant may deliver very prematurely and die soon afterwards, leaving a bereaved
partner with a pre-term, neurodevelopmentally handicapped child.
The possibility of genotype-specific therapy

The molecular abnormalities of CFTR processing have been divided into five classes
[118]. Class I mutations are characterised by premature cessation of mRNA synthesis, for
example, due to a mutation causing a premature stop codon (e.g. G542X). This is a
particularly important category in some Jewish populations (see below). The commonest
mutation in white races, DF508, is the exemplar of class II mutations. CFTR mRNA is
produced and transcribed, but the protein is misfolded and, instead of tracking to the
apical membrane, is ubiquitinated and marked for destruction in the endoplasmic
reticulum. Class III mutations, G551D, for example, are characterised by abnormal
CFTR reaching the cell surface but the regulation of the protein is abnormal, leading to
abnormal function. Class IV mutations reach the cell surface but have abnormal channel
function (e.g. R117H). Class V mutations lead to reduced amounts of CFTR at the apical
cell membrane. These include missense mutations (A455E) and alternate splicing
mutants (3849z10kbCRT), which lead to reduced synthesis of CFTR. There are also
rarer variants, where CFTR fails to track selectively to the apical membrane but instead
is present nonselectively all over the cell surface, and variants where the half-life of
CFTR at the apical membrane is greatly reduced.
This classification has use as a prognostic tool, at least for groups of patients [119].
Those homozygous for severe mutations (classes I and II, usually pancreatic insufficient)
have been shown to have a worse survival than those with mild mutations, who are
usually pancreatic sufficient. However, the possibility of therapeutic manipulation of the
mutations in a class-specific manner is currently a hot topic. The first realisation that
DF508 was, potentially at least, partially functional came when it was expressed in the
Xenopus oocyte and was shown to function, as a chloride channel at 26uC but not at
mammalian body temperature (37uC). This observation was confirmed by other in vitro
stimulation studies. Amongst other compounds that had this putatively beneficial effect
on DF508 was sodium phenylbutyrate, already licensed for the treatment of some urea
cycle disorders. A randomised, double-blind clinical trial of this compound showed some
restoration of ion channel function, measured in the nose [120]. Chloride channel
function was shown by a response to low chloride and isoprenaline solution but,
potentially crucially, there was no change in the response to amiloride, implying
unaltered ENaC hyperfunction. Further work is proceeding with sodium phenylbutyrate, including the potential to combine it with a CFTR stimulator such as genistein
[121]. Sildenafil has also been shown to have in vitro effects on DF508 trafficking [122].
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It is likely that it will be easy to find at least male volunteers if sildenafil ever comes to a
large clinical trial!
The use of increased DF508 CFTR trafficking in cell systems in vitro has been the basis
of a huge fiscal investment in high throughput, robotic screening for candidate
compounds for genotype-specific pharmacotherapy. Many thousands of compounds
from chemical libraries can be screened in a day for this activity. It should be noted that
this approach, brilliant as it is, runs the risk of missing important compounds (which
could act in different ways to increase DF508 CFTR function) and also raise false hopes
if the trafficked CFTR cannot, in fact, correct the missing relevant function for the
particular organ (usually the lung). At the time of writing, this approach has
unfortunately not delivered any really promising compounds.
A novel approach for class I mutations came with the in vitro observation that some
aminoglycosides (not tobramycin) lead to premature stop codons being ignored and
protein continuing to be transcribed. Gentamicin nose drops have been shown to
increase chloride channel function in the nose, and molecular studies showed increased
CFTR trafficking. This was shown to be a genotype-specific effect by looking at a control
group of DF508 homozygotes, in whom, as predicted, no change was shown [123]. An
extension of this approach, to allow lower risk of aminoglycoside toxicity, is to either coadminister polyanions, which directly reduce toxicity and prolong the effect, or look for
alternative compounds with the same effects [124].
The use of genotype-specific, pharmacological manipulation of mutant CFTR remains
a tantalising and exciting possibility. Proof of concept has been shown, but as yet nothing
is ready to be used as a practical clinical tool. Furthermore, the concerns about correcting
relevant functions, discussed elsewhere in this chapter, still remain.

Pathophysiological mechanisms
Airway surface liquid: low volume versus high salt models. The link between the basic
molecular biology of CFTR and the typical clinical phenotype of CF lung disease has been
difficult to make. The two current hypotheses have led to the "high salt" and "low volume"
models, of which the latter seems more plausible.
The high salt model postulated that the defect in ion transport leads to a high salt
epithelial lining fluid, which in turn leads to inactivation of small polypeptides (bdefensins, lactoferrin, lysozyme), which function as primary defence mechanisms for the
airway [125]. The model was bolstered by elegant in vitro work, but technically difficult
measurements of the epithelial lining fluid suggested that the fluid was isotonic and not
particularly high in sodium. The low volume hypothesis focuses on the height of the
periciliary liquid layer [126]. Respiratory cilia are another important primary airway
defence mechanism, which need a tightly regulated layer of fluid in which to function.
The fluid needs to be sufficiently shallow that when the cilia are fully extended, they
engage the mucus layer to propel it upwards in a gyratory fashion, and must be
sufficiently deep to accommodate the ciliary recovery stroke. The total airway surface
decreases dramatically from distal to proximal (bronchioles to trachea), and in normal
subjects, if the proximal airways are not to be flooded with fluid produced in the
bronchioles, fluid absorption must take place to tightly regulate the height of the
periciliary fluid. This is the function of the ENaC channel; in CF, the model proposes
that active hyperabsorption of sodium and passive hyperabsorption of water leads to a
reduction in the height of the periciliary fluid layer, impairment of ciliary function and,
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thus, a breech in the airway defences against infection. This concept is supported by
elegant studies in excised tissue and cell culture, which nicely demonstrate reduction in
periciliary fluid height.
It is difficult to believe that the low volume hypothesis on its own can account for CF
lung disease but it does seem likely that failure to defend the periciliary layer is part of the
story. However, this hypothesis leads to the assumption that impaired mucociliary
clearance is pivotal in CF lung disease. This collapses as the sole explanation for the
following reasons. 1) Mucociliary clearance (MCC) is virtually absent in PCD, but lung
disease is milder and infection with typical CF pathogens, such as mucoid P. aeruginosa,
is rarer and occurs much later [127]. 2) Other factors must be invoked to account for the
narrow range of pathogens isolated in CF and the exaggerated inflammatory response to
infection (see below). 3) There is some evidence that mucociliary clearance is normal at
least early on in CF and that abnormal clearance is secondary to infection and
inflammation [128, 129] Nonetheless, the low volume hypothesis has lead to important
understanding of the CF airway.
Importance of epithelial sodium channel versus chloride transport. The relatively poor
correlation between chloride transport and CF lung disease and the difficulty of
constructing biologically plausible models to connect the two, together with the insights of
the low volume hypothesis, have led to a focus on ENaC overactivity as the pivotal feature
of CF lung disease. The CFTR knockout mouse has an intestinal phenotype but as a
model for CF lung disease, has proved disappointing. For example, the establishment of
infection with P. aeruginosa requires nebulisation of the microorganism with agar beads;
this is quite unlike the human airway, wherein it is virtually impossible to avoid
spontaneous chronic Pseudomonas infection. A much better model of CF airway disease
has come from a normal mouse which overexpresses the b-subunit of ENaC [130]. There
was airway surface liquid depletion, with spontaneous mucus plugging, neutrophilic
inflammation and poor bacterial clearance, and the picture is very reminiscent of natural
CF lung disease.
Postulating that ENaC overexpression, and not failure of chloride transport as the
cause of CF lung disease, would also solve the "OBrodovich paradox" [131]. This can be
approached in two ways. First, CFTR is normally widely distributed in the foetal lung
and yet there is no foetal CF lung phenotype. This is because the foetal lung is a sodium
excreting, not absorbing, organ. Secondly, CF lung disease begins distally, despite CFTR
being much more abundant in the proximal airway, because ENaC is distributed much
more peripherally and the low volume hypothesis predicts maximal sodium reabsorption
distally in order not to flood the proximal airways. These postulations have some support
from a clinical study of patients with systemic pseudohypoaldosteronism, a disease
caused by loss of function mutations in ENaC [132]. This group demonstrated that
airway surface fluid volume was more than twice the normal level, as predicted by the low
volume hypothesis. Interestingly, in three adults, mucociliary clearance was increased. At
first sight this seems surprising; it would be expected that failure of ciliary function could
result not just from reduced airway surface liquid height, with crushing down of the cilia
by the mucus layer and failure of the recovery stroke, but also too great a surface height,
leading to failure of the tips of the cilia to engage the mucus layer. In systemic
pseudohypoaldosteronism, the periciliary fluid height is normal and the extra water is in
the mucus layer. These concepts are summarised in a recent review [133]. It should also be
noted that ENaC mutations with functional consequences may cause a CF-like disease
[134].
Further data have supported the role of CFTR as the controller of ENaC. Liddles
syndrome is due to a b-ENaC gain of function mutation (R566X), which causes systemic
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hypertension but not any lung disease [135, 136]. The mouse with Liddles syndrome has
a normal airway surface liquid height, despite ENaC gain of function. However, the
double mutant (Liddle and CFTR -/-) has the reduced airway surface liquid height, as
predicted from the model that hypothesises that CFTR suppresses, or at least controls,
ENaC function in order to keep the airway surface appropriately hydrated [137].
Against this hypothesis is the finding that there was no difference in nasal PD
measurements between patients with mild and severe mutations [138]. However, it has
previously been argued that this might be misleading, since nasal PDs may not
necessarily reflect bronchial PD due to local interactions; direct endobronchial PD
measurements can be made [139] and it is these that need to be correlated with disease
manifestations in the lower airway.
Taken together, the balance of the evidence is that correcting the failure of chloride
absorption alone may not correct the CF pulmonary phenotype. This has implications
for the assessment of new therapies.
Inflammation and infection: which comes first? There is no dispute that the two main
components of CF airway disease are chronic infection with a relatively narrow range of
pathogens (S. aureus and gram-negative rods, especially P. aeruginosa) and an exuberant
host inflammatory response. Neutrophils undergo necrosis within the airway lumen
because sheer numbers are thought to overwhelm the physiological apoptotic
mechanisms. This results in the release of tissue-damaging mediators, such as
neutrophil elastase. What is not clear is the relationship between infection and
inflammation. There are four broad current hypotheses describing the relationship, not all
necessarily mutually exclusive, as follows. 1) The CF airway is itself pro-inflammatory in
the absence of infection; this is based in part on studies of explanted human CF foetal
tracheas in the flanks of immunosuppressed mice [140]. 2) The CF airway has an excessive
inflammatory response to infection [141], evidence includes a greater number of airway
neutrophils per bacterium in CF compared with other diseases characterised by chronic
bronchial sepsis. The serial data on bronchoscopy and BAL in screened CF infants from
Australia would suggest that infection is a prerequisite for inflammation [142]. 3) Failure
of resolution of inflammation, due to IL-10 deficiency [143], or failure of the lipoxin
pathway (see below) [144] is the main problem. 4) One group has suggested that if
allowance is made for increased epithelial binding of bacteria, particularly P. aeruginosa
[145], then the inflammatory response per bound bacterium is normal [146].
New data implicating IL-10 in early CF lung disease have recently been reported [147].
The effect of intratracheal lipopolysaccharide (LPS) in CF knockout mice was compared
with an IL-10 knockout animal. The use of LPS was intended to eliminate any possible
confounding effects of differing epithelial bacterial adherence between the two models.
Compared with wildtype, both knockout mice exhibited a greater neutrophilic
inflammation, a more prolonged consumption of I-kB (the nuclear factor (NF)-kB
inhibitor protein) and production of NF-kB, and more intense production of the
cytokines TNF-a, IL-1b and macrophage inflammatory protein (MIP)-2. These changes
were abrogated in the CF mice by IL-10 treatment, suggesting that this may be a useful
target in the human disease.
Recently, the effects of different classes of CFTR mutations on the inflammatory
response in mice have been reported [148]. Four mutations were studied (R117H, S489X,
Y122X, DF508) and infected with P. aeruginosa containing agarose beads. The BAL
levels of the cytokines TNF-a, IL-1b, IL-6, MIP-2, and keratinocyte chemo-attractant,
and the eicosanoids prostaglandin E2 and LTB4 were found to be essentially no different
between the strains. This lack of difference is strongly suggestive that the dysregulated
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inflammatory response in CF is not related to the underlying CFTR defect, but is a

secondary response to prolonged and chronic infection.
In summary, the balance of the evidence is in favour of the hypothesis that the
exuberant and exaggerated inflammatory response in the CF airway is secondary to
infection, and not a primary part of the CF defect, but the question has yet to be settled
definitively.
Related diseases
What can be learnt from similarities and differences between CF and PCD? Even
with the best treatment, there is a remorseless deterioration in lung function in CF
patients. By contrast, two studies have shown that, whatever the level of lung function at
diagnosis of PCD, it can be stabilised using conventional treatment [127, 149]. The range
of infecting organisms is not dissimilar between the two conditions, at least late in the
disease, even including mucoid P. aeruginosa and atypical mycobacteria. Clearly any
therapeutic strategy that could convert a deteriorating disease (CF) into a stable
phenotype like PCD would be valuable. The inflammatory phenotype in PCD is little
studied. Least controversially, although levels of NO are low in CF, they are much lower
in PCD [150, 151], so whatever the putative beneficial host defence effects of nitric oxide
(NO), it would be predicted that strategies to increase airway NO are likely to be
beneficial. However, as a salutary check to over-confident predictions, despite the
excellent theoretical reasons put forward above, a recent study showed that inhaled
arginine both increases exhaled NO and spirometry in CF patients [152]. These results
need to be confirmed but are an encouragement to humility in theoreticians.
Sputum examination reveals a neutrophilic cytology [153] and similar levels of IL-8 to
those in CF [154]. Little is known about the other inflammatory mediators; one study has
suggested that, at least in stable PCD, LTB4 is not a significant mediator [155]. However,
it is important if, as it seems, IL-8 levels are higher in PCD because, if confirmed, it would
mean that using reduction in IL-8 as a surrogate end-point in CF trials is unlikely to be
useful. Clearly, this is an area in which further work might be very fruitful. It may be that
gene chip arrays, proteomic and metabolomic approaches may be helpful in generating
hypotheses about the reasons for the differences between these diseases.
What can be learnt from similarities and differences between bronchiectasis in the
developing and developed world? Non-CF bronchiectasis is an orphan disease, at least
in the UK [156], despite evidence that it may be more common than previously
appreciated. In other contexts, a number of studies have documented that bronchiectasis
may be much more severe in indigenous peoples such as the Pacific islanders and Alaskans
[157160]. This difference in severity may merely reflect poverty, poor access to
healthcare, and an unhealthy environment. However, it might be worth speculating that
there are host genetic or other factors that are responsible for the severe clinical picture in
these patients [161]. Differences between mild and severe bronchiectatics might be a useful
clue as to what to search for that makes CF lung disease so severe.
Treatment strategies
Make the diagnosis early, preferably by screening. Although politicians debate the
merits of screening, it is virtually impossible to find a CF family, or a CF Healthcare
Professional, who is not in favour of it. Neonatal screening programmes generally rely on
detection of increased levels of immunoreactive trypsin in bloodspots, collected as part of
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Guthrie and hypothyroid screening. Cut-off levels are laboratory dependent. Mutation
analysis using commercial kits mostly screening for 32 mutations may be part of the
protocol. Internationally, newborn screening is only patchily offered. Overall, the US
Center for Disease Control and Prevention feels that newborn screening for CF is justified
[162] and that benefits outweigh risks [163]. At present, the median age of a CF diagnosis
with newborn screening is y0.5 months compared with14.5 months, in the USA, for a
conventional diagnosis [164]. An early diagnosis of CF has a number of potential benefits
and it primarily enables detection of asymptomatic infants. They must be referred to
established CF programmes so early detection of complications and rapid initiation of
therapy can be practiced. Hopefully, this will diminish disease-related weight loss,
pulmonary infections and, possibly, deterioration of lung function. While longevity is
steadily increasing, it is becoming clear that halting disease progression before damage
becomes established is a paramount goal of management. Other benefits include the
obviation of the frustration of the family at fruitless appointments with health
professionals [165] and false reassurance before the diagnosis is made. Finally, the tragedy
of a late diagnosis, preventing the family from having an antenatal diagnosis in a
subsequent pregnancy, is avoided.
Most of the evidence in favour of screening is from observational studies. The only
randomised controlled trial was from Wisconsin, USA [166]. There is cumulative
evidence that CF detected by newborn screening results in better and early nutritional
treatment leading to improved growth and, in one study, better cognitive development
[167]. Other benefits may include reduced hospitalisations and improved survival rates.
Beneficial outcomes regarding pulmonary function are less certain. Good nutritional
status early in life and good subsequent lung function are related [168, 169]. Advantages
in the first year of life for CF diagnosed by newborn screening are avoidance of stunting
(height v3rd percentile), wasting (weight v3rd percentile), hypoproteinaemic oedema,
chronic airway infection with P. aeruginosa and hospitalisation for complications.
Observational studies reviewed recently [170] provide indirect evidence that newborn
screening may improve pulmonary health and survival in patients with CF. Early
asymptomatic diagnosis (not by newborn screening) in children born after 1987 was
shown to result in improved lung function for as long as up to 9 yrs of age [171].
Improvements in early treatment strategies may have allowed early diagnosis to lead to
more aggressive therapies resulting in improved pulmonary health. However, it is unclear
to what degree early nutritional status may reflect early pulmonary status, which in turn
may predict pulmonary outcomes [170]. A long-term observational study [172] indicates
that early treatment made possible by neonatal screening may be important in
determining subsequent clinical outcomes for children with CF. Comparing an historical
before-screening group with a screening-based group, it was shown that the screened CF
group had a significantly better lung function with an FEV1 of 9.4% higher at age 10 yrs
than in the nonscreened children. These data are, at present, not supported by the
Wisconsin randomised clinical trial of CF newborn screening, as they do not show
improved pulmonary outcomes in the screened group [173]. This may be because of poor
infection control in one of the centres to which screened babies were referred.
There may be a potential downside to newborn screening for CF. This includes
psychological stress for parents during the confirmatory diagnostic process [174]. In
general, however, benefit outweighs psychological stress, including that during the ruling
out of CF after a false positive CF diagnosis. There may be an early introduction to
hospital settings with added risks of P. aeruginosa acquisition [163]. Rigorous infection
control policies therefore must be mandatory for all screened positive babies. Even
considering this, the present evidence clearly underscores that newborn screening for CF
makes sense and that national health authorities should cover the financial and
organisational costs.
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Treat infection. Lower respiratory infections are almost universal in CF patients. BAL in
Australian infants of mean age 2.6 months mainly diagnosed on newborn screening
showed the presence of S. aureus in almost 40%, of whom more than one-third were
symptom free [175]. Infected infants had more macrophages, neutrophils and IL-8 in their
BAL fluid. Despite normal lung structure at birth, early lower respiratory infection
represents a significant early feature of CF.
Although S. aureus is generally the first major pathogen demonstrated in BAL fluid or
deep throat cultures, P. aeruginosa is clearly the dominant player regarding lung
pathology and prognosis. Acquisition of P. aeruginosa is usually of the environmental
phenotype initially [176] and is nonmucoid and antibiotic sensitive. During the first 3 yrs
of life 72% of patients with CF isolate P. aeruginosa on at least one culture, and this
increases to w97% when culture and serology results are combined. Once mucoid P.
aeruginosa has become established, the prognosis becomes worse [177]. Triggers for
change from nonmucoid to mucoid strains are poorly understood but formation of
biofilms and microcolonies represents an advanced stage of infection with limited
treatment response. Early detection of P. aeruginosa offers a chance for intervention and
a window of opportunity for suppression and possible eradication (by aggressive antipseudomonal treatment) of the initially nonmucoid P. aeruginosa [178]. The Wisconsin
group [178] prospectively investigating the epidemiology of P. aeruginosa showed that in
their screened population, the age-specific prevalence of mucoid P. aeruginosa increased
markedly from age 416 yrs. Nonmucoid and mucoid P. aeruginosa were acquired at
median ages of 1.0 and 13.0 yrs, respectively. In contrast with the short transition time
from no P. aeruginosa to nonmucoid P. aeruginosa, the transition time from nonmucoid
to mucoid P. aeruginosa was relatively long (median, 10.9 yrs) and could be slightly
extended by antibiotic treatment [178]. Epidemic, antibiotic-resistant P. aeruginosa pose
special problems [179] and may be suggestive of patient-to-patient transfer in a
nosocomial setting. Cohort segregation is effective in reducing transmission risks and
molecular characterisation of P. aeruginosa, although expensive and rarely performed,
would be the ideal instrument to determine needs for patient isolation and infection
control. There are no universally accepted guidelines for the treatment of respiratory
infections. Current antimicrobial strategies include the following: 1) prevention and
treatment of S. aureus; 2) eradication of early P. aeruginosa isolates; and 3) for chronic
P. aeruginosa infection, continuous suppressive therapy, intermittent intravenous treatment, either planned or to treat an actual or impending pulmonary exacerbation.
Staphylococcus aureus. Whether early anti-staphylococcal prophylaxis is beneficial is

unclear [180]. If this strategy is to be adopted, then narrow-spectrum antibiotics are
preferable and cephalosporins avoided. Both observational data from a European registry
using mainly oral cephalosporins [181] and a formal study from the US using cephalexin
[182] indicate that whilst S. aureus is suppressed there is an unacceptable increase of P.
aeruginosa infection. The pragmatic proposal of use of prophylactic anti-staphylococcal
medication between diagnosis and 3 yrs [183] should be viewed with caution as
undesirable effects, such as changes in bacteriological spectrum and resistance, may
merely be brought down to lower age ranges.
If S. aureus is isolated from cough swab or sputum, irrespective of symptoms, then
treatment with oral flucloxacillin or co-amoxyclav is indicated. In cases of communityacquired methicillin-resistant S. aureus, options are fucidic acid, rifampicin, cotrimoxazole, clindamycin and tetracyclines orally, depending on the age of the patient and the
sensitivity of the strain. Linezolid is effective but should be kept as reserve antibiotic
because of expense and potential toxicity, which includes hepatotoxicity and recent
reports of severe optic neuropathy [184186]. It can be used parenterally and orally.
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Teicoplanin is the drug of choice if intravenous treatment is indicated, which is unusual,

with vancomycin as an alternative.
Pseudomonas aeruginosa. Eradication strategies for first isolation of P. aeruginosa

improves the chances of avoiding or delaying chronic infection. A number of approaches
exist, without the superiority of one over the other having definitively been established.
These include: combined oral ciprofloxacin and inhaled colistin for periods of 3 months
[187]; and inhaled tobramycin (TOBI1; Chiron, Emeryville, CA, USA) [188] used for
i4 weeks.Early antibiotic therapy leads toa P. aeruginosa-free periodof a median (range)of
18 (480) months. New acquisition with different P. aeruginosa genotypes occurs in 73% of
episodes [189]. In the Danish CF Centre, early, aggressive eradication therapy has now been
used for 15 yrs without giving rise to resistance to the antibiotics and without serious sideeffects. Although there is agreement that eradication regimes are mandatory and it is
negligent not to treat a first isolation, the best regime is still not known.
Chronic Pseudomonas aeruginosa infections. Based on the Cochrane review [190] and
the recent TOBI1 trial [191], there is no doubt that long-term inhaled antibiotics should
be prescribed to patients chronically infected with P. aeruginosa. The best regime is not
known. Most clinicians use either or both of colistin and tobramycin or other
aminoglycoside. For patients who are not responding well, the nonevidence-based
approach of alternate months of TOBI1 and colistin is popular. However, more evidence
from trials of longer duration is needed to determine if this benefit is maintained, as well as
to determine the significance of development of antibiotic-resistant organisms. The
Danish CF centre advocates regular 3-monthly intravenous antibiotic courses, arguing
that this leads to better maintenance of lung function and less progress of Pseudomonas
antibody production [192]. However, a 3-yr study did not demonstrate an advantage of a
policy of elective antibiotic treatment over symptomatic treatment in patients with CF
chronically infected with P. aeruginosa [193]. It should also be noted that as CF patients
live longer and are exposed to multiple courses of antibiotics, there is an increased
incidence of antibiotic allergy [194, 195], side-effects such as nephrotoxicity [196, 197] and,
probably, selection of resistant organisms [198].
Acute exacerbations of pulmonary disease are treated with intravenous combination
treatment with b-lactam antibiotics and aminoglycosides [199]. No major new strategies
have been documented. Combinations are mandatory [200] and the recent finding of
bacterial biofilm production being associated with subinhibitory aminoglycoside
antibiotics supports this notion [201]. It should be noted that there is no evidence
that in vitro sensitivities of the organism correlate with in vivo response to treatment [202,
203]. Optimal treatment duration is unknown but is usually i23 weeks. Once-daily
tobramycin administration [204] has equal efficacy compared with three times-daily
administration, with potentially less nephrotoxicity. There is interest in combining this
with continuous intravenous ceftazidime [205, 206], rather than three times-daily boluses,
as on pharmacokinetic grounds this may be more effective and is also potentially more
convenient to the patient, who can carry a simple and portable infusion pump. Home
antibiotic intravenous treatment is cost-effective and less disruptive to the patient, but
recent data highlight the need for careful selection and closer supervision of such subjects
as clinical outcome, as defined by spirometric parameters and body weight, was better
after a course of treatment in hospital than after home treatment [207].
Attention to airway clearance: physiotherapy, devices, rhDNase and exercise.

Whether airway clearance techniques are necessary from diagnosis in the asymptomatic
child is unclear, but benefits are certainly obvious in the chronically productive patient.
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Responses to treatments are highly unpredictable in the individual and there is no

universal best means to improve MCC in patients with CF.
Physiotherapy. Chest physiotherapy (PT) traditionally forms an integral part of virtually

all CF treatment regimes. Despite this, the physiology and consequences of this
intervention remain unclear and scientific evidence as to the long-term benefit of the
various forms of PT is largely unresolved. It is unclear whether this treatment should be
initiated in children diagnosed by screening. The effects of PT, in particular head-down
drainage positions, on gastro-oesophageal reflux are controversial [208210]. To date
there are no comparative data on early PT favouring one approach over another.
Caregivers should at the very minimum know how to check the childs chest and have the
expertise ready should the need for action arise. The treatment regimes should be
individualised by experienced physiotherapists, since no one regime has evidence to
favour it ahead of others [211].
In young adults, vibration using oscillation of the airflow was compared with a
number of other manual or device-assisted methods, such as percussion or the flutter
[212]. Peak expiratory flow rate was higher with vibration than with other methods,
potentially indicating some evidence for the physiological rationale for the use of
vibration to aid secretion clearance. In a recent Cochrane review on the benefit of
positive expiratory pressure (PEP) to assist airway clearance, no clear evidence was found
that PEP was a more or less effective intervention overall than other forms of
physiotherapy. There was limited evidence that PEP was preferred by participants
compared with other techniques, but this finding was from low-quality studies. Longterm studies had equivocal or conflicting results regarding the effect on FEV1. In all
studies with an intervention period of i1 month, measures of participant preference
were in favour of PEP [213]. Other outcome measures were, however, not addressed
adequately. In an overview of five Cochrane systematic reviews, some evidence was
obtained that PEP was at least as effective as other forms of airway clearance [214].
Physical training was addressed in particular and this was found to be beneficial. The
Cochrane systematic reviews summarised in the paper provided some evidence to
support the inclusion of physical therapies in the care-management plan of CF. A recent
survey performed by the American College of Chest Physicians, which addressed
nonpharmacological airway clearance methods and concentrated mainly on different
breathing techniques including postural drainage, chest wall percussion and vibration,
and the forced expiration technique (huffing), concluded that some are effective in
increasing sputum production but their long-term efficacy in improving outcomes
compared with unassisted cough alone is unknown [215]. It appears difficult to draw firm
general conclusions concerning PT, so individual choices should be respected, with
regular reviews by the physiotherapist. Well-designed, adequately powered and longterm studies are needed. Healthy children with CF pose special challenges. It remains to
be seen whether there will ever be an answer to this issue both in terms of physiology or
clinical effectiveness [216].
Devices. Many devices have been proposed to aid mucociliary clearance and sputum
expectoration. These include the flutter and Acapella devices, high-frequency chest
compression, the percussionaire intrapercussive ventilators, the Hayek oscillators, and
others.
The major drawback with almost all of these is the paucity of convincing data derived
from properly designed studies. Only careful and long-term individual assessments will
provide information on any benefits to patients. These would include improved or
maintained lung function and simple surrogate markers, such as sputum volume or
weight. Individual choice with assessment from a physiotherapist is suggested.
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rhDNase. The benefits of rhDNase have recently been reviewed [217]. Once-daily
application is the most widely used mucoactive therapy in patients with CF. rhDNase
reduces the viscoelasticity of sputum from patients with CF and enhances the clearance of
secretions. Clinical trials have shown rhDNase to be a well-tolerated treatment that
improves lung function and reduces exacerbation rate in CF patients with moderate and
severe lung disease. However, the response to treatment is heterogeneous and only a
proportion of patients with CF actually benefit. At present, it is impossible to predict
which patients will benefit from rhDNase. Many CF centres have developed formal n-of-1
trials of treatment to find out who benefits. rhDNase is an expensive therapy and is mainly
used in patientsw5 yrs of age with moderate-to-severe lung disease. Recently, it has been
shown that giving rhDNase on an alternate-day basis [218], rather than daily, is equally
effective, potentially reducing costs and treatment time. Comparisons with another
mucoactive drug, hypertonic saline (HS) [219], have shown rhDNase to be more effective,
although some patients who did not respond to rhDNase had a beneficial response to HS.
One randomised study [220] and observational data from the European Epidemiologic
Registry of Cystic Fibrosis (ERCF) [221] suggested that younger patients were likely to
benefit more from treatment. However, the extra treatment time may be difficult for a well
CF patient to accept.
rhDNase was initially considered to be pro-inflammatory, but recent data failed to
detect any adverse effects on airway inflammation. The Bronchoalveolar Lavage in the
Evaluation of Anti-Inflammatory Treatment (BEAT) Study is a multicentre open study
intended to evaluate the evolution of inflammation in CF patients with early lung disease
and its modulation by rhDNase treatment [222] and has suggested some antiinflammatory benefits. In this study, a total of 105 patients with CF (i5 yrs of age)
having normal spirometry, were randomised to receive rhDNase (2.5 mg?day-1) or no
rhDNase. A significant increase in neutrophils was observed over the 3-yr study period in
both untreated patients and control subjects, whereas neutrophils remained unchanged
in patients treated with rhDNase. Neutrophil elastase and IL-8 concentrations also
increased in untreated patients and remained stable in patients on rhDNase. It was
concluded that in patients with CF, an increase in neutrophilic airway inflammation is
found that is positively influenced by rhDNase treatment.
Hypertonic saline. Restoring airway surface liquid in CF by inhalation of HS results
in improved MCC for many hours [223]; whether this is due to increased volumes of
airway surface liquid, induction of cough or a combination of both remains open [224].
An Australian study of HS (7%) showed statistically significant but clinically trivial
improvements in spirometry, but a remarkable decrease in infective exacerbations
[225]. Therefore, HS may be considered an inexpensive, safe and effective additional
therapy, providing unpleasant taste, irritant potential and extra treatment time are
acceptable.
Exercise. Physical fitness represents a prognostic marker; patients with higher levels of
aerobic fitness are more than three times as likely to survive than patients with lower levels
of fitness [226]. In contrast, CF patients with severe lung disease (FEV1 v40% pred) have
significantly higher breathlessness, lower muscle effort scores, lower peak lactate, lower
peak heart rate and a mean ventilation exceeding the predicted, thus confirming that
ventilation is the major factor limiting exercise [227]. Progressive hyperinflation adds
strain to the inspiratory muscles and decreased chest wall compliance (hyperinflation
leading to tidal breathing being on the plateau of the normal compliance curve) increases
oxygen consumption. Low activity and physical deconditioning, as well as poor
nutritional state, are additional factors contributing to poor performance.
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Physical exercise should be an integral part of CF therapy. Benefits include general

well-being, clearance of secretions and, for weight-bearing exercise, increased bone
mineral density. The literature on respiratory muscle function is partly controversial
indicating both normal to increased and also decreased values [228]. Recently the 6-min
walk test was found to accurately determine the level of physical ability in children with
CF [229]. Although an increased pro-inflammatory cytokine response is elicited by
exercise [230], the benefits clearly outweigh any theoretical disadvantages. Although
there is a very major interest in physical exercise training programmes and this kind of
treatment is almost universally adopted [231], there are no standardised protocols that
define exercise loads, supplementary oxygen, nutritional regimes and psychological
support at any stage of the disease. As with physiotherapy, an individualised approach is
advised.
Maintain good nutrition. This essential part of CF care can only be summarised very
briefly here, for reasons of space. The brevity of the section does not reflect a lack of
importance of the subject. More details can be found in standard texts.
Early and accurate diagnosis of pancreatic insufficiency. This is usually obvious from
the history of profuse, offensive fatty stools, and is most conveniently confirmed by
measuring human faecal elastase on a small stool sample; in pancreatic-insufficient CF
this is undetectable, even when taking pancreatic enzyme supplementation [15]. Children
who are pancreatic sufficient at diagnosis may develop pancreatic insufficiency, and any
change in bowel habit in a pancreatic-sufficient patient should lead to a measurement of
stool elastase. If doubt remains, which is unusual, a 3-day faecal fat measurement is made.
Institute pancreatic enzyme replacement therapy at an appropriate dose. Entericcoated pancreatic microspheres are the treatment of choice. They should be given with all
meals and snacks, titrated to the fat content. The supervision of an experienced dietician is
mandatory. The replacements are given prior to and with food, but may sometimes need
to be given after meals if there is accelerated preferential gastric emptying of the
microspheres [232]. The microspheres work most efficiently in an alkaline environment.
The duodenum in CF may be more acidic than normal because of loss of the bicarbonaterich pancreatic secretion. Some children require acid-lowering strategies, such as proton
pump inhibitors, to maximise enzyme efficiency.
A high-fat, high-calorie diet is essential. Arguments continue as to whether poor

nutrition causes deterioration in lung function or the reverse is the sequence of events.
Likewise, it is unclear whether the CF defect itself is causal of an increased calorie
requirement or it is a secondary consequence of infection. Whichever is the case,
maintenance of good nutrition is a pivotal part of CF care. The dietician will encourage
"unhealthy" eating, including full-cream milk and fried food, and other high fat food. Fatsoluble vitamins are routinely prescribed.
Routine monitoring of nutrition. Height, weight and (in young children) head
circumference should be measured at least four times a year and plotted on centile charts.
Other useful adjuncts are calculation of body mass index and measurement of skin fold
thickness. Any falling off from the centiles should be investigated promptly (see below).
Levels of vitamin A, D, and E are measured as part of the routine annual assessment [233].
The CF child with nutritional failure. It should not be assumed that poor weight gain is
due to inadequate pancreatic enzyme supplementation, and the dose unthinkingly
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increased. If the child is takingw10,000 lipase units?kg-1?day-1, and certainly if the dose is
w15,000 lipase units?kg-1?day-1, then investigation is needed. Key questions include the
following. 1) Is the childs intake of calories adequate? 2) Is the child taking their enzymes
in an appropriate manner? Is compliance an issue? 3) Is the child malabsorbing? A 3-day
faecal fat should be performed.
Consideration should also be given to alternative diagnoses, which include the following.
1) Has the child developed glucose intolerance (see above)? 2) Could the child have Crohns
or coeliac disease, both of which are commoner in CF [234, 235]? 3) Pseudo-Bartters
syndrome should be eliminated, particularly in hot weather, by measuring urine and
plasma electrolytes, including plasma bicarbonate [236]. 4) Cows milk allergy is another
possibility [237], although the importance of dairy products in nutrition is such that this
diagnosis should only be accepted if really stringently confirmed by a double-blind
challenge. 5) Bowel infection with giardiasis [238], or a blind loop syndrome as a
complication of neonatal surgery, is another consideration. 6) Short bowel syndrome or the
effects of ileal resection, after neonatal surgery for meconium ileus.
The role of nutritional support. The first step in nutritional support is optimisation of
meals and snacks, with the help of an experienced dietician. High calorie supplements are
widely prescribed, but a recent trial suggested that benefit is not likely to be great [239].
Overnight feeding through a gastrostomy may lead to dramatic weight gains and may be
particularly valuable if meals have become times of tension and quarrelling as a reluctant
child is cajoled, pestered and bullied into trying to eat.
The CF child with abdominal pain. The usual causes of this are distal intestinal
obstruction syndrome (DIOS) or simple childhood constipation. Abdominal pain should
not prompt an uncritical increase in pancreatic enzyme therapy, and this approach leads
to the iatrogenic disaster of fibrosing colonopathy [240, 241]. DIOS is not seen in other
malabsorptive states; it is characterised by subacute bowel obstruction due to inspissated
fatty faecal masses in the colon. Treatment acutely is with gastrograffin or Klean-Prep1
(Klean-Prep, Norgine Lim, Harefield, UK); a full review of diet and pancreatic enzymes
to prevent recurrence is mandated. It must be distinguished from simple constipation,
which is more common in CF. If there is any doubt, a 3-day faecal fat is performed. Other
causes of abdominal pain in CF include the following: 1) acute appendicitis, which is
frequently mistaken for DIOS [242]; 2) intussuception, which is commoner in CF than in
the normal population [243]; 3) adhesions following neonatal surgery; 4) gall stones; 5)
gastro-oesophageal reflux; and 6) rarely, torsion of an ovarian cyst or other non-CF
causes of abdominal pain [244].
Early detection of multisystem complications. Proposed screening tests have been

discussed above. Clearly, effective interventions need to be in place if early detection is
worthwhile. It is also true that the "annual assessment" has become much more complex,
burdensome and time-consuming for both patient and professional. Data gathering for its
own sake must be resisted. Examples where early detection may be helpful and lead to a
change in treatment include the following. 1) Insulin deficiency by glucose tolerance
test and random home monitoring, with early institution of insulin therapy.
2) Abdominal ultrasound with early detection of liver disease, and initiating therapy
with ursodeoxycholic acid (although the benefits of this treatment are untested).
3) Measurement of bone mineral density by DEXA scan, with early initiation of
biphosphonates.
Other tests for which there are unanswered questions include the following. 1) The role
of HRCT scanning: will it result in clinical benefit, commensurate with the associated
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radiation risks, and how often and with what protocol should it be performed?
2) Regular surveillance bronchoscopy: the results of the Australasian trial are awaited.
Holistic care of the patient and family at all stages of the disease. CF affects the
individual and the whole family, and the presence of biopsychosocial stressors may add to
the burden caused by the disease. CF does not necessarily cause long-term serious family
dysfunction, but it changes family structures and often taxes the family system beyond its
strength [245]. Thus, the care of the individual is seen as only part of the picture and family
centred care is essential. Taking biopsychosocial circumstances into account will permit a
more personalised approach and support of patients, families and dependants. In
addition, a spiritual (not necessarily religious) dimension may be important for patient
and caregiver systems and should be respected by the CF support team. Additional
resources may thus be mobilised and contribute to strength. This systems thinking
requires knowledge of the background of the CF individual and his or her attitudes,
beliefs and motives. Stage and severity of the illness may be indicators of the care needed.
One way of judging family function is the Family Assessment Measure (FAM).
Encouragingly, in 299 families with preschool children with CF, FAM scores were in the
normal range [246]. In a group of 32 families with children aged 3 months to 4 yrs with
CF, defective family functioning was not detected using the Feetham Family Functioning
Scale (FFFS) [247].
Family dysfunction tends to increase with progressive illness, indicating family
vulnerability and possibly marital dissatisfaction. Conversely, the childs illness may
draw parents closer together and may lead to stronger bonding within family systems
[248]. Intrafamilial stress and strains have a negative effect on physical health variables of
CF children [249]. Associations between parental coping and the childs health status
were documented in a study of 100 families with a child with CF. Fathers played an
equally important role as mothers in maintaining both psychological stability and
pulmonary function [250].
While issues of patient and family interactions have been explored in detail in
childhood CF, there is a paucity of data in adults with CF. Problem-solving
approaches based on individual orientation and formation of coping strategies may be
more effective than family therapy, as utilised in younger childrens families. These
appear to be the logical age-dependent instruments with which to offer comprehensive
guidance. Family strengths and capabilities will have to be appraised in all contexts to
establish coping and problem-solving communication. Older children and adolescents
who come from families experiencing unhappy and conflicted relationships may be at
greater risk for poor adherence to treatments; thus, family relationships are
appropriate targets for interventions aimed at improving adherence [251]. McCubbin
et al. [250] defined the following three coping patterns for families managing a child
with CF: 1) maintaining family integration, cooperation and an optimistic definition
of the situation; 2) maintaining social support, self-esteem and psychological stability;
and 3) understanding the healthcare situation through communication with other
parents (which may be difficult in modern segregated clinics) and the entire healthcare
team.
This means that caregivers should invest in themselves as individuals, should meet the
needs of individual family members (for example, normal siblings) and that couples
should aim at maintaining their relationship as partners. Free communication within the
family and expression of feelings leads to better physical and emotional outcomes than
suppression of these issues. These important subjects are discussed further in a recent
publication [252].
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What are the important future questions?

What property of CFTR is important for disease in which organ?
The multiple properties of CFTR have been discussed in detail previously. The
importance of this question can be illustrated by an example: whereas a number of gene
therapy trials in the nose and lung, using various vectors, have been able to demonstrate
some restoration of (presumed CFTR-mediated) chloride transport, none has
demonstrated any change in ENaC function. Assuming that the tools of gene therapy
are perfected or, for that matter, perfect phenotype-specific pharmacotherapy is
discovered, and chloride channel function is totally restored while ENaC hyperfunction
continues unabated, will CF lung disease have been cured? In favour of the proposition
that it will be are two observations. The first is that the CF mouse is a poor lung disease
model, and this may be due to expression of alternative chloride channels in the lung. The
second is that gene therapy in vitro and in vivo lead to a reduction of P. aeruginosa
binding to epithelial cells back to normal levels [143]. From the databases, the prognosis
of never-infected patients is much better than those with chronic P. aeruginosa infection,
so abrogation of the abnormal adherence of this organism could potentially convert
severe CF lung disease to mild. Against the importance of CFTR chloride channel
function in the pathogenesis of CF lung disease is the excellence of the CF-like airway
phenotype in the ENaC b-subunit overexpressing mouse, and the plausible pathophysiological concepts to which ENaC hyperfunction leads [130]. At this stage the question is
unanswered, but all working with CF must be aware of its potential importance.
Is the Larsen hypothesis tenable?

A radical hypothesis that has recently been proposed is that CF is a developmental
disease, with all the action being antenatal, and the corollary that only curative attempts
made before birth have any hope of success [253]. The proponents of this hypothesis have
some compelling challenges to pose. These paradoxes are as follows. 1) In vitro CFTR
protein interactions are not always mirrored in CF tissues. For example, enhanced
sodium absorption is absent in sweat glands, despite the presence of ENaC. 2) The
disease-specific manifestations of CF do not mirror the tissue distribution of CFTR; the
heart and kidneys express CFTR, but there is no CF-specific disease of these organs. 3)
As discussed above, patients can manifest a CF disease phenotype, without having any
CFTR mutation.
An additional piece of supportive evidence is the increasing amount of data
cataloguing the multiple gene and protein consequences of CFTR function, discussed
above [2225]. Although most would agree that the weight of the evidence is against this
hypothesis, nonetheless it is sufficiently challenging as to deserve consideration.
There can be no question that CFTR is widely expressed in the lungs during foetal life,
and that the amounts dwindle towards term. As with much lung development, the precise
role(s) of CFTR are not known. Larsen and co-workers [253, 254] rightly draw attention
to the fact that it is difficult to put together CFTR dysfunction(s) and the clinical
phenotype of CF, and hypothesise that some disease manifestations are as a result of
downstream disturbances of function in pathways which need to interact with CFTR.
From this not implausible position, the group boldly hypothesised that downstream
effects might occur in cells many years after their progenitors could not interact with
CFTR, and as a result produce the disease CF. A number of studies have shown that
transient overexpression of CFTR in the foetal airway by gene transfer experiments lead
to permanent phenotypic changes, including epithelial cell proliferation, increase in
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lamellar body formation and enhanced resistance to bacterial infection in later life [254,
255]. However, although mouse CFTR knockout animals have disrupted lung
development, this does not appear to be the case in humans. That there are changes
early on in infancy is indisputable, but at birth, there is very little if any change from
normal.
The real challenge to the CF community comes from controversial data that
temporary application of gene therapy in utero in mice results in permanent cure of the
CF phenotype [256]. It is difficult to see how this could happen and others have had
difficulty in reproducing the results. Nonetheless, the possibility that in utero gene
therapy is the only hope of cure must not be dismissed out of hand, depressing as this
thought may be, in that the approach of antenatal manipulation of the karyotype seems
unlikely to command support.
How will we know if we find a cure and what are the best surrogate end-points?
At first sight this seems to be a curious question; how could we not know when we have
cured CF? It is not as easy as one might think at first, and we could even be sitting on the
cure now and not know about it. The present section is very speculative but is intended to
stimulate lateral thinking.
What will a cure do? This is speculative but it seems likely that a cure will not reverse
gross saccular bronchiectasis, and it seems not unlikely that the cycles of inflammation,
infection and tissue destruction reach a stage which is self-perpetuating, such that even
were complete restoration of all CFTR functions possible in the airway, the patient would
still die. Thus, the cure must be tested in patients who have milder disease. However, if
their disease is so mild, is it feasible to detect an improved prognosis?
One thing is clear; mortality is not a feasible end-point. The mortality curves for CF
are now happily so flat that it would take decades and thousands of patients to show
improved survival. The rate of change of lung function, at least as measured by
spirometry, is also flat [257]. Huge numbers of patients will be required to be followed for
prolonged periods to detect a change, unless the "cure" is so dramatic that there is a
restoration of lung function to normal in relatively mild patients. This too seems
unlikely; reversal of established structural airway wall changes. Thus, it is concluded that
surrogate end-points are needed. The reasons for using surrogates can be summarised as
follows. 1) Reduced sample size, since surrogate outcomes may occur more frequently
(reduced infective exacerbations versus mortality, for example). 2) Need for shorter
follow-up time and smaller sample size. 3) Ability to bring medications into the clinical
arena faster.
The requirements of a good surrogate have been summarised [258]. These include the
following. 1) The surrogate must correlate with the desired outcome. 2) Interventions will
have the same effect on the surrogate as the desired outcome, i.e. will lead to the same
efficacy conclusions. 3) They will be simple to measure. 4) They will have a short latency
(i.e. gives a relevant signal quickly, both to natural history and treatment effects).
The most common reason for failure of surrogates to predict clinical effect is when the
surrogate is unrelated to disease pathophysiology, i.e. a mere epiphenomemnon like the
colour of the teeth of CF mice [259].
Potential surrogate end-points. There are three potential approaches, each with
potential problems. These are as follows: 1) direct demonstration of the CFTR synthesis
(mRNA, protein); 2) demonstration of restoration of CFTR function(s); and 3)
demonstration of reversal of clinical manifestations of disease. Each has its problems and
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CYSTIC FIBROSIS
will be considered in turn. Although most of the focus of this work has come from gene
therapy, the concepts apply equally to other specific therapeutic strategies.
Direct demonstration of the CFTR synthesis (mRNA, protein). Superficially this

would seem attractive; CFTR was not being made and now it is. However, in a protein
which undergoes such complex post-translational processing as CFTR, the mere presence
of mRNA is an insufficient guarantee of functional protein, although of course failure to
detect CFTR mRNA would be a big blow to any trial of specific therapy. Likewise, CFTR
may reach the apical cell membrane, but if it is prevented from functioning properly, the
disease may not be affected. Remembering the sophisticated coordination of ion channels
at the apical cell membrane required of CFTR, it should be acknowledged that the mere
presence of CFTR no more guarantees function than the presence of the conductor of the
La Scala orchestra somewhere in Northern Italy guarantees that the orchestra will play
the overture of the opera Rigoletto correctly and at the scheduled start of the opera!
Demonstration of restoration of CFTR function(s). This inevitably leads back to the

question: what function does the CFTR gene have? A recent gene therapy trial restored
25% of chloride channel function measured electrophysiologically [260], as did the use
of oral 4-sodium phenylbutyrate [120]. Neither strategy corrected the overexpression of
ENaC function. If, as the present authors have argued above, sodium transport is pivotal
in CF lung disease, then restoration of chloride channel function (above) will not achieve
the desired effect.
Demonstration of reversal of clinical manifestations of disease. This is the obvious

aim of therapy but the hardest to demonstrate. The difficulty of showing a decline in lung
function has been discussed above. Reduction of the increased adherence of P. aeruginosa
to epithelial cells has its attractions; reduction of the intense inflammatory response is also
attractive. However, the present authors would urge caution in putting too much weight
on a single pro-inflammatory cytokine, such as IL-8, until it is known if it is pivotal to the
abnormal process. Clearance of a hitherto chronic infection would be clear-cut evidence
of a specific benefit but is an unlikely goal in the chronically infected CF patient.
Thus, two tremendous questions remain. How can we cure CF? And, how do we know
if we have done it? If we get our end-points wrong, we may focus our therapeutic drive
against mere epiphenomena or discard promising compounds because their actions do
not conform to our prejudices of the moment. In the end, a pragmatic approach is likely
to have to be taken and studies started using the best likely novel therapy; academic
theorising has its limits and may be wrong (see the arginine/CF data above). Ultimately,
theorising has to stop or all action will be paralysed. If a trial fails, there will always be
plenty of people to point out after the event the reasons why this should have been
obvious from the outset [261].
Will stem cells work?

The short answer to the question is no, not on their own. The problems of gene therapy
in the airway include: vector delivery to a distorted and often poorly ventilating airway;
the evasion of the normal host defences against the approach of foreign molecules; the
traversing of intracellular barriers; the transcription of the inserted gene; and the
expression and correct deployment of the resultant protein. These could all be
circumvented if pluripotent stem cells can be harvested from the bone marrow, subjected
to corrective gene therapy in vitro and re-infused to repopulate the airway and correct the
CF lung disease. The death blow to the idea that stem cells on their own can repopulate
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the lung comes from studies in CF patients who have undergone lung transplantation.
There has never been any report of recurrence of CF in the transplanted lungs, no matter
how many years the patient has survived after transplantation, which would surely have
happened were there to be a normal trafficking of stem cells between bone marrow and
airway. However, there are some possible pointers that an innovative strategy may allow
tracking of stem cells to the airway. There have been reports of Y chromosome positive
cells in the airways of male donor lungs transplanted into female recipients, and also in
the female recipients of donor male bone marrow [262, 263]. There have also been reports
that CD34z cells implicated in airway remodelling have been isolated from peripheral
blood, presumably en route between the bone marrow and the airway. These studies
provide no more than proof of possibility, and a cure for CF with stem cells seems a long
way away.
Can designer macrolides be found?

The intriguing properties of the macrolides have been reviewed (see above and table 3).
If their property or properties that accounted for the benefit in CF was known, then the
many hundreds could be screened to find the one that maximally expressed that property
or one could even be synthesised. Ideally it would not have any antibacterial property or,
at least, did not induce macrolide resistance, a real problem when macrolides are widely
prescribed.
When is inflammation beneficial? Why does it remain acute neutrophilic, not

chronic?
Clearly, the immune/inflammatory response has a biological purpose, as shown by the
catastrophic infections in children with congenital or acquired immunodeficiency. It
would seem likely that early on in CF, the inflammatory response is beneficial and wards
off early infection with P. aeruginosa. In late-stage disease, it is clearly both ineffectual
and harmful. What is not known is when the transition to harm occurs. This is important
because immunosuppression with steroids, for example, while definitely beneficial (at the
price of side-effects) in late disease, may not be helpful early on. Indeed, in the oral
steroid trial, there were benefits only in the group chronically infected with P. aeruginosa
[264]. This issue was brought into sharp focus by a recent randomised, placebocontrolled trial of an LTB4 receptor antagonist, which was halted prematurely because of
increased adverse events (infective exacerbations) in the treatment group [265]. Thus,
further work is needed to determine the proportions of good and bad inflammation at
different stages of CF.
The second challenging inflammation question is: why is there persistent acute, rather
than chronic, infection in the CF airway (defining acute and chronic by the cellular
pattern, not by time duration)? The neutrophil is the hallmark cell of the acute
inflammatory response to bacterial infection in particular. The outcome is usually
resolution (as in uncomplicated lobar pneumonia) or progression to chronicity (for
example, tuberculosis), when the neutrophil has largely been replaced by chronic
inflammatory cells. This clearly does not happen in CF (nor does it happen in PCD and
non-CF bronchiectasis). Why? And would it be better for the organism if the severe
persistent acute phenotype infection gave way to chronicity? Lipoxin production is one
way the body switches off an acute inflammatory phenotype. In one study, patients with
CF were found to have reduced airway lipoxin levels and administration of exogenous
lipoxin attenuated the lung disease of a CF mouse model [144].
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Can an effective anti-inflammatory strategy be found?

Modulating the immune response should be carried out with caution. Oral steroids in
high doses are beneficial but at the unacceptable cost of side-effects. The effect of lowdose steroids has never been trialled (and probably never will be). Inhaled corticosteroids
are largely ineffective and certainly over-prescribed. There are some novel strategies that
show promise, and the key question is: which agent, and at which stage of the disease, is
most likely to benefit the patient? A number of possibilities have been trialled. These
include the following.
1) Aerosolised interferon-c1b. However, this was ineffective in an adult trial [266].
2) Nebulised a1-antitrypsin (a1-AT). Neutrophil elastase (NE) is one of the main end
products of chronic inflammation, resulting from major neutrophil recruitment and
subsequent necrosis. It is normally neutralised by endogenous a1-AT. In CF, this
mechanism is overwhelmed by excessive NE and becomes ineffective. Following early
reports of the use of nebulised a1-AT in CF [267], a recent pilot study showed no
statistical difference for sputum-free NE activity between a1-AT and placebo, although a
trend toward reduction of NE activity in treated patients was observed [268]. In addition,
lung function, sputum bacteriology and sputum IL-8 were not statistically different
among the two groups. The drug was found to be safe and well tolerated. It remains to be
seen whether new, improved drug delivery systems might beneficially modify these
results. Another biologically active agent, secretory leukoprotease inhibitor, showed
some beneficial effects in a pilot study [269], but there seems to be no recent interest in
taking this forward.
3) Monthly intravenous immunoglobulin G was investigated retrospectively in 16
children with severe obstructive CF lung disease, leading to some lung function
improvement and reduction of inhaled or oral corticosteroid medication [270].
4) Anti-inflammatory but nonanti-infective agents, such as colchicine, methotrexate,
cyclosporine A, montelukast, pentoxifylline, nutritional supplements (fish oil and other
antioxidants), may have a role in individual management, on an experimental basis.
Thus, tantalisingly, some potentially beneficial agents are available but, as yet, there is
insufficient data available to determine how best to deploy them, in which patients and at
what stage of the illness.
What are the key modifier genes?

Although for large groups there are correlations between genotype and at least some
aspects of clinical phenotype, and between genotype and prognosis, individuals with the
same genotype at the CFTR locus may have very different disease phenotypes. This is
true even for full siblings brought up within the same environment; for example, one
sibling may have devastating lung disease, while the other has normal lung function but
hepatosplenomegaly. Thus, there must be powerful genetic modifiers of the CF
phenotype within the karyotype. It should also be said that there are clearly profound
environmental influences: in one study from the USA, wealthy patients with CF (never
on Medicaid) had a three-fold lower death rate than impoverished patients (always on
Medicaid) [271]. The importance of careful consideration of geneenvironment
interactions, and not just genes alone, is discussed below.
The key reason for bothering with modifier genes is that if a modifier gene product
can convert severe lung disease to a mild phenotype, that product may make a very
promising therapeutic target. It has to be said that this goal appears to be in the distant
future.
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How is cross-infection and the emergence of new pathogens to be prevented?

Infection control has been addressed in recently published reviews in North America
[272] and Europe [198]. This is a highly contentious area. It is known that living together
under the same roof for several days will lead to cross-infection; it is not known how
short a time is safe. Passing in the corridor at a brisk walk? On the same train for a
journey? Attending a football match? Starting with the noncontroversial, the following
points are known: 1) cross-infection definitely occurs with B. cepacia complex, some,
but not all, strains of P. aeruginosa and methicillin-resistant S. aureus (MRSA); 2) crossinfection does not occur with some strains of P. aeruginosa, atypical mycobacteria and H.
influenzae; and 3) previous sputum microbiology is not predictive of current infection
status.
Recommendations would therefore be as follows. 1) Basic hygiene measures, such as
hand washing, are mandatory and should be rigorous [273]. 2) People with CF who are
infected with a pathogen that has been shown to be associated with cross-infection
should be separated from others with CF, both inside and outside the hospital, as far as
possible. There is obviously no point in making impractical suggestions and complete
isolation outside the hospital (for example, in schools) may be inconsistent with disability
legislation in some countries and with patient confidentiality. 3) For paediatric outpatient clinics, many now adopt a system whereby the child and family go into a room
and the professionals enter the room in turn, rather than the conventional model in
which children wait to be seen in a communal area. The room is carefully cleaned
between patients. This model results in substantially fewer patients being seen per clinic
and also diminishes flexibility in seeing acutely unwell children in the clinic. 4) Adult outpatients can be segregated on microbiological status within the usual communal waiting
area and be asked to sit a set distance away from each other to minimise aerosol spread.
5) In hospital, each patient should have a single room with en-suite facilities. If this is
impractical, then no more than one CF patient per single bay is permitted.
6) Microbiological surveillance for the early detection of cross-infection is vital. The
gold standard, molecular typing of every isolate, is probably too expensive for most
clinics. Antibiotic resistance patterns are deceptive, and therefore of little value [274].
The price of cohort isolation includes loss of the psychosocial support that families
offer to each other. To some extent this can be overcome by the use of Internet
technology. Although it may sound harsh, patients self-help groups are an ideal source
for pathogen transmission and as such should be viewed with great reservation.
S. aureus and P. aeruginosa are the "classical" CF pathogens and are covered elsewhere
in this chapter. Recently, and probably as a consequence of increased longevity but also
due to intensive antibiotic treatments, a number of new pathogens have been described
and their role in CF has been addressed in some detail [275]. There are no set treatment
guidelines for these and relatively little is known of transmissibility. Most are of
environmental origin and are characterised by innate resistance to most major
antibiotics. Particular attention should be paid to the prevention of transmission to
other patients. Treatment of MRSA has been discussed in a previous section.
Burkholderia cepacia complex. There are i10 variants in this family ("genomovars").
At least in Central Europe, Burkholderia cenocepacia (genomovar III) is the most likely to
be associated with virulence and high transmissibility, thus carrying a poor prognosis
whilst the other Burkholderia spp. may be of lesser importance. Recently, B. dolosa
(genomovar VI) was found to carry a poor prognosis, with a 13% probability of dying
within 18 months of established infection [276]. If B. cepacia is suspected, the identity of
the isolate must be confirmed in a reference laboratory, if misdiagnosis is to be avoided.
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The organism requires selective culture media to be detected with accuracy. Only after
1 yr of at least three B. cepacia-negative cultures can the organisms be considered cleared.
Antibiotic treatment is difficult. Even when in vitro testing suggests sensitivity, in vivo
results are disappointing. Synergy testing with double or preferably triple antibiotic
combinations appears to offer the best chance to establish possible treatments [277].
Meropenem combined with cotrimoxazole, piperacillin/tazobactam, doxycycline and
ceftazidime are the most likely antibiotics to be useful. Recently, temocillin has been
successfully used in a series of 20 B. cepacia complex patients [278]. It is crucial to cohort
isolate the different genomovars separately; before this was appreciated, patients
carrying a benign genomovar were grouped together with genomovar III patients,
acquired genomovar III, and eventually died from it.
Stenotrophomonas maltophilia. The role of this organism remains uncertain and it is not
generally treated unless the clinical situation requires intervention. Cross-infection does
not appear to be a problem and clinic separation is not warranted under present
circumstances. Stenotrophomonas maltophilia does not lead to a change in clinical
condition in most patients, and frequently disappears spontaneously from the airway
[279, 280]. If treatment is considered at all, cotrimoxazole combined with ticarcillinclavulanic acid is used. Alternatives are ciprofloxacin plus ticarcillin/clavulanic acid or
piperacillin/ tazobactam.
Achromobacter (Alcaligenes) xylosoxidans. This is a rod-shaped organism of

unknown pathogenicity in CF. In some patients (without CF), it has been reported to
cause septicaemia, peritonitis in patients on peritoneal dialysis, pneumonia and ear
infection. Transmission amongst CF patients does not seem to be a problem and there are
no reports of accelerating loss of lung function. There are no figures on the incidence of
the organism in large CF collectives. Treatment is not normally an issue but, in
exceptional cases, ureidopenicillins or ceftazidime may be promising.
Pandoraea apista. Some of these organisms may cause chronic infection and can be
transmitted between CF patients. Epidemic forms were seen in the Danish CF Centre and
cohort isolation was effective in preventing further spread [281]. Treatment is difficult and
there is a paucity of evidence.
Nontuberculous mycobacteria. A number of nontuberculous mycobacteria cause

human disease and may be seen in CF (Mycobacterium avium, M. kansasii, M. abscessus and
others). Their origin is mostly environmental, and cross-infection is not an issue. They are
frequently only commensals in CF and only patients with multiple positive cultures should be
considered for treatment. In general, a period of observation, with serial CT scans, is
recommended [282, 283]. These organisms are frequently multiply resistant, and expert advice
should be sought if treatment is contemplated.
How is evidence obtained for the treatment strategies particularly used in early
stage disease?
In order to obtain evidence for treatment strategies used in early stage disease, the
following are needed. 1) International agreement as to the hierarchy of important
questions and the order in which they should be answered. 2) An absolute commitment
internationally to recruit all screened CF infants to multicentre trials, to answer these
questions. 3) Funding to ensure these trials are adequately powered and are of sufficient
duration, to answer questions definitively. 4) Simple and cheap end-points, which do not
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require expensive and sophisticated apparatus, so that many centres can be involved
in trials. 5) Excellent surrogate end-points in these trials: ortality will never be
satisfactory.
How can treatment be made less burdensome?

The time required for daily treatment and the variable emotional burden are the two
most dominant sources of stress for those suffering from CF, and the latter frequently
depends on the former. Despite hope of future research results beneficial for the patient,
most parents and some children feel that there is no light at the end of the tunnel. The
inevitable downhill course, however slow, puts treatment adherence at risk and may at
times lead to a happy-go-lucky attitude. Furthermore, many patients are well and
(rightly) see themselves as a fit person who has CF, not a CF patient. Treatment may be
made more manageable by adopting the following techniques. 1) Flexibility with
physiotherapy regimes; a device which can be used in the car on the way to school may be
physiologically less ideal than postural drainage (although this is contentious), but a
device used is better than postural drainage not done. 2) No medication should be given
by nebuliser if there is an alternative drug delivery system. 3) The new fast, intelligent
nebulisers should be used to minimise treatment time for those medications that must be
nebulised, for example, the eFlowTM (PARI GmbH, Starnberg, Germany) perforated
vibrating nebuliser [284]. 4) The number of nebulised treatments must be minimised. The
present authors doubt that anyone will regularly have two antibiotic, two HS and one
rhDNase nebulisations, five in all, every day. Choices must be made. 5) The frequency of
nebulised therapy should be minimised; rhDNase can be given alternate days and the
new liposomal formulations of nebulised antibiotics may only need to be given twice a
week [285]. 6) Part of the regular clinic review must be to go through the list of
medications with the patient to see if any can be stopped. 7) If adherence is a real
problem, try to agree with the child which medications are essential (pancreatic enzyme
replacement) and which, although desirable, may be omitted for periods of time (vitamin
supplements). 8) Remember that if a new treatment is added, then the patient will almost
certainly drop another one off the list.
The support of clinical psychology, a functional CF team and education towards
self-care independence may, and should, be able to reduce the burden of it all. In
particular, psychological interventions with a broad range of modalities are able to
beneficially alter disease perception. However hard patients and families try, they
cannot get rid of CF. Knowing the biopsychosocial background of the family may help
the identification and interpretation of problems presented within the complexity of
CF, some of which are wrongly attributed to CF [245]. By better understanding of CF
achieved by information and demonstration as well as by reinforcement (praise, reward
systems), compromises may be reached. This will usually be far from acceptance but
may beneficially influence treatment adherence and reduce perception of CF to be an
everlasting burden.
What are the best treatment strategies for liver disease?

Liver disease is becoming increasingly important as CF patients live longer. Out of a
total of 21,742 patients with CF summarised in the 2003 US Cystic Fibrosis Foundation
Patient Registry, 5.5% were affected by some type of liver disease. Just 10 of these
underwent liver transplantation. This is in contrast with figures from Canada, where CF
liver disease was observed in 18, 29 and 41% at ages 2, 5 and 12 yrs and cirrhosis occurred
in 19 (7.8%) patients at a median age of 10 yrs [286]. Patients with a history of meconium
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ileus (incidence rate ratio 5.5; 2.711), male sex (2.5; 1.34.9) or severe mutations (2.4;
1.24.8) at multivariate analysis appear to have a higher risk developing liver disease
[287]. The evidence base for treatment is scant and the difficulties in studying CF liver
disease include the following. 1) Difficulty in detection: CF liver disease may be present
with normal liver function tests and no hepatomegaly. 2) Difficulty in agreeing diagnostic
end-points: liver biopsy is not useful because the disease is patchy. 3) Liver function tests
do not correlate with presence or progress of CF liver disease. 4) Difficulty in agreeing
surrogate end-points in clinical trials: mortality is not suitable but the significance of
other potential end-points and the relationship to mortality is unclear.
Focal biliary cirrhosis or multilobular cirrhosis continue to be the dominant
manifestations of CF liver involvement. Other manifestations are hepatomegaly,
abnormality of liver function tests, hepatic fibrosis, steatosis and biliary tract
involvement (gallstones, microgallbladder, cholangitis, bile duct obstruction at the
level of the head of the pancreas and, rarely, sclerosing cholangitis and malignancies).
Although the development of cirrhosis is only gradually progressive, the significant
increase of life expectancy makes liver disease a more important consideration and, in
fact, is now the second most frequent cause of death in CF.
Detection is difficult and the yearly assessment of liver function is not particularly
helpful. Ultrasound of the abdomen as a routine at least every 2 yrs may help detection.
It should also be performed if any liver function test is elevated beyond twice the upper
limit of normal, if the liver or spleen is palpable, if there is evidence of hypersplenism or if
the patient has had a gastro-intestinal haemorrhage. The sensitivity of detection of
hepatic fibrosis is variable and operator dependant, but major liver or bile duct
pathology will be demonstrated readily [288]. Magnetic resonance imaging or CT
scanning should be considered.
What are the treatment strategies that can be offered today and what is the evidence
that they work? Ursodeoxycholic acid (UDCA) is the drug of choice to attempt slow
progression of CF liver disease. UDCA is a hydrophilic bile acid that improves secretion
of bile acids, may improve bile flow and has immunomodulatory properties that may
reduce immune-mediated liver damage. While the evidence of effectiveness in non-CF
patients is reasonably convincing, much controversy remains regarding its role in CF. In
an Italian prospective, multicentre, double-blind study, UDCA administration for 1 yr
improved clinical and biochemical parameters in CF patients with liver disease, but there
was no assessment of liver histology [289]. In a smaller study involving 10 patients treated
with UDCA for 2 yrs, liver morphology improved and liver disease-associated
inflammation decreased [290]. In an open study involving 70 CF patients who were
given UDCA (20 mg?kg-1) over a period of 10 yrs [291], the progression of nodular
biliary cirrhosis ultrasound changes was arrested, hepatic function was preserved and no
variceal bleeding was observed. No case of focal biliary cirrhosis progressed to nodular
biliary cirrhosis. Furthermore, the multifocal, multilobular changes suggestive of focal
biliary cirrhosis on ultrasound scan were reversed to normal. UDCA is safe, with
virtually no toxicity, and it is therefore recommended that the drug be commenced in a
dosage of 2030 mg?kg-1 once signs of liver disease become demonstrable. Adequate fatsoluble vitamins should be given, including vitamin K if not already prescribed.
Oesophageal varices indicating portal hypertension are treated by variceal ligation and
sclerotherapy, and this is usually very effective in controlling bleeding. Portosystemic
shunting may prove successful in stabilising patients and may stave off liver
transplantation for many years [292]. Generally, hepatocyte function is preserved for
many years. Liver transplantation is used for end-stage liver disease and portal
hypertension should be controlled by measures short of transplantation. There is a lack
of large published series and outcomes appear to vary.
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What is needed to find answers to these questions?

A really good CF lung disease animal model
The CF mouse has proved to be a disappointment with respect to the pulmonary
phenotype. A model of chronic P. aeruginosa infection has been established in these mice
by administering infected agar beads by aerosol. The difficulty in establishing this
infection in the CF mouse is in stark contrast to the almost impossibility of preventing it
in humans. It could be argued that the difficulty of a suitable model of the CF lung
phenotype has already been achieved with the ENaC b-subunit overexpressing mouse
[130]. That this is a model of a chronic infective lung disease is indisputable; that it is
specific for CF is uncertain and, if it is, the implications for therapeutic targets are
profound (see above). It is unlikely that the CF mouse will ever be a really good model
for CF lung disease, since, quite apart from anything else, it lacks submucosal glands, the
chief site of CFTR expression in humans. Alternatives, such as the ferret, would
potentially be of interest. The ideal animal would have submucosal glands and similar
ion transport physiology to humans, both in terms of ENaC function and the nature of
non-CFTR chloride channels. Such an animal could be produced by gene knockout,
RNA interference or pharmacologically, if CFTR could be completely inactivated by an
otherwise nontoxic compound.
Studies that determine why PCD is a mild lung disease and CF is severe
The phenotypic differences between PCD and CF have been described above. The
study of the differences in the key inflammatory mediators, or possible tissue-destructive
enzymes, may lead to new targets and the avoidance of irrelevant ones. There are a
number of noninvasive techniques, such as spontaneous and induced sputum, exhaled
breath condensate and exhaled breath, and powerful molecular techniques, which could
be applied to samples (genomics, proteomics) that could be utilised to probe the
similarities and differences between these conditions.
Systematic clinical trials of treatment, such that all newly screened babies are
included
Treatment options for newly diagnosed babies have recently been reviewed. It is
very clear that the evidence base for much of what is suggested is very poor. The
advent of widespread newborn screening presents the opportunity to address this, if
there is the collective will to do so. The best model is that of the paediatric
oncologists, who enrol almost all children with cancer into studies. The disease that is
particularly studied is acute lymphoblastic leukaemia, in which the prognosis has been
transformed by a series of therapeutic trials (the UK Acute Lymphoblastic Leukaemia
Trials). The oncologists have the advantage that a decisive end-point (death) is either
reached or quickly averted. However, there is no reason to avoid tackling the more
difficult issues in CF. The requirements are as follows. 1) A will to enrol the vast
majority of newly diagnosed, screened babies into a succession of focused trials. 2) A
realisation that these will be medium and long term, not short term. 3) The use of
simple end-points that can be applied all over Europe, using data that are already
being collecting, such as cough swabs, height and weight, and (in older children)
spirometry.
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This will only be achieved if core funding can be obtained for the data collection and
analysis, and there is a collective will to make this happen, from a large number of CF
clinicians and national and international interest groups.
Really good end-points in infancy and the preschool years

There is substantial physiological and some pathological evidence that early events in
the CF airway have far-reaching consequences, and therefore early intervention is
necessary to prevent the CF child being programmed for suboptimal lung function. The
requirements for the ideal end-point for CF trials in preschool children are as follows:
1) no safety issues; 2) requires no active or passive cooperation; 3) rapid and easy to
perform, and expensive apparatus and highly trained personnel not required; 4) can be
used repeatedly in the same child; 5) good discriminator between health and disease,
reproducible over time, sensitive to changes in clinical sate over time and with treatment;
and 6) good prognostic indicator.
As discussed above, there are a number of potential end-points, none of which meet
these stringent requirements, including HRCT, LCI and bronchoscopy. Perhaps the two
most promising are HRCT (provided the radiation dose can be reduced to very low
levels) and LCI (provided really cheap gas analysers can be produced).
Hypothesis-driven genetic modifier studies, with findings tested in a second

similar population before being accepted
Criteria for an adequate and informative genetic association study have recently been
succinctly summarised [293] and may equally be applied to genetic modifier studies. The
criteria are as follows. 1) The sample size must be adequate and a pre-study power
calculation is a prerequisite. 2) The controls must be carefully chosen from the same
population as the disease group; a ratio of controls to cases of 2:1 increases power. 3) The
choice of polymorphisms/associations sought should be logical. 4) The phenotypes must be
carefully described. 5) Issues of multiple testing must be faced and allowed for in the
statistical testing, especially if the same population is used for multiple association studies.
6) Any findings should be replicated in a second, independent sample to be credible. 7) The
findings should enhance knowledge of the mechanisms of disease or its treatment.
The current "state of the art" for CF modifier gene studies unfortunately seems to be to
collect DNA from as many patients as possible (sometimes controlling for the CF
genotype, e.g. exclusively DF508/DF508), and hoping that something will fall out from
either a genome-wide scan or an analysis of polymorphisms in likely candidate genes,
such as ENaC or other ion channels, inflammatory cytokine genes, etc. The present
authors believe this approach is, unfortunately, nave, and almost certainly doomed to
failure for the following reasons. 1) Genes do not exist in isolation, but they interact with
the environment; for example, polymorphisms of the glutathione S-transferase gene are
thought to interact with the severity of CF lung disease [294, 295]. However, powerful
interactions with environmental factors have been shown to be important in the context
of childhood lung function and air pollution. For example, maternal but not paternal
glutathione S-transferase genotype is an important determinant of the effects of maternal
smoking during smoking on foetal airway development and later respiratory symptoms
[296]. 2) Genetic influences may only operate at a particular developmental stage, and
mixing of paediatric and adult studies may obscure this. 3) Genetic influences may be
important only at particular stages of the disease; for example, by the time chronic
infection with P. aeruginosa has been established, the effect of genes influencing the early
binding of the organism to epithelial cells are likely to be irrelevant. 4) Genes and
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treatment policy may interact; upregulation of a putative epithelial receptor for P.

aeruginosa leading to earlier infection will be more relevant to prognosis in clinics where a
vigorous eradication policy is not pursued. 5) Polymorphisms may show a biphasic
influence, being protective in some environments but detrimental in others. For example,
the CD14/-260 CRT polymorphism in the CD14 promoter was associated with higher
levels of both total and specific immunoglobulin E to aeroallergens in children in regular
contact with domestic pets, but the opposite relationship, not explained by endotoxin
levels, in children in contact with stable animals [297]. 6) Ethnic factors, including genetic
variations, may lead to different polymorphisms being relevant in different populations.
Whereas it is important to confirm a putative modifier in one study, in a second study in a
different population, if the second population is too different from the first, the
polymorphism may be discarded as irrelevant, whereas it is in fact relevant only in some
populations. 7) It is at least conceivable that studying one genotype exclusively (e.g.
DF508/DF508) may miss the effect of polymorphisms relevant in other genotypes.
These are not counsels of despair of ever finding anything, merely a plea for
hypothesis-driven, focused studies. These studies also have to be adequately powered.
Ideally the target population has to be big enough for a "double-study" approach, in
which half the population is used as a hypothesis-generating exercise to find a modifier,
and the other half is used to test that the modifier is important in the generated
hypothesis. Both halves must be large enough to give adequate study power. The
complexities of geneenvironment interactions in infant wheeze should have taught us
the limitations of mere DNA collection. A clinical phenotype needs to be thought of as
the product of the interactions between the internal (intra-uterine) and external
environment, and the genes; neglect of any part of this "phenotype equation" will limit
the ability of studies to detect important modifiers.
A recent large study exactly highlights the limitations of collecting only DNA. Drumm
et al. [298] studied two cohorts of patients. The first, used to generate associations,
consisted of 808 patients who were homozygous for DF508. They attempted to replicate the
work of others by looking for associations between disease severity. They studied 10
different genes (table 5) and validated their findings in a second cohort (n=498) with a
variety of genotypes (70% DF508 homozygotes). They had no information on
environmental factors. They found a rough doubling of the odds ratio for severe lung
disease for the highest risk transforming growth factor-b1 genotype (codon 10 CC), their
Table 5. Putative polymorphisms in modifier genes studied by DRUMM et al. [298]
Genes studied
Polymorphisms
A1-antiprotease
S allele (T2313A)
Z allele (G4627A)
39 enhancer (G1237A)
D or I deletion
A46G
C79G
Null deletion
A1375G
G-1082A
Null structural polymorphisms (B, C, D)
XA/O
T5220G
Promotor (C-509T)
Codon 10 (C29T)
Codon 25 (G74C)
Promotor (G-308A
Angiotensin-converting enzyme
b2-Adrenergic receptor
Glutathione S-transferase 1
Glutathione S-transferase 2
Interleukin 10
Mannose-binding lectin 2
Nitric oxide synthase 3
Transforming growth factor-b1
Tumour necrosis factor-a
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sole positive finding. To put this in perspective, there is a three-fold risk of dying in CF
patients always relying on Medicaid compared with those never relying on Medicaid [270].
However, this impressive study has missed the boat, as the hugely discordant clinical
phenotypes and prognosis in genotype-identical siblings is well known. The present authors
suspect that a better way forward will be to study, in enormous detail, the differences
between small groups of such discordant sibling pairs, in the hope of finding the really
dramatic modifier that would provide a useful therapeutic target. Another approach would
be to study, in detail, the differences between congenital bilateral absence of the vas
deferens (CABVD) patients, in whom lavage studies have shown evidence of bacterial
infection and a muted inflammatory response [299], but no evidence of overt lung disease in
CF patients in whom infection and inflammation lead to airway destruction. The likely
explanation for the mild lung phenotype in CABVD is an as yet undiscovered protective
mechanism, which could offer therapeutic targets. The present study will undoubtedly
guide and inform mechanistic studies, but has not generated therapeutic targets.
Summary and conclusions

CF is probably the paediatric respiratory disease that has seen the most change in the last
15 yrs, both in terms of understanding of the molecular and cellular changes, and also the
changing understanding of the nature of the disease. Concepts that were set in stone have
been challenged. Future research is needed to determine why patients with CF get the typical
lung phenotype, how to find specific treatments and know they are effective, and how to
treat the multisystem disease that CF has now become.
Summary
Twenty years ago, cystic fibrosis was considered a disease mainly of children, affecting
the lungs and the digestive systems, diagnosed using the sweat test. Since then, the gene
for CF has been discovered, leading to great increases in the knowledge about the
fundamental molecular and cellular biology of the airway; the diagnostic spectrum has
also been expanded to mild and atypical cases, requiring more sophisticated diagnostic
testing, with mild cases presenting in adult life. Specialist care has lead to an increase in
survival, so that shortly there will be more adults than children with the disease. The
nature of the disease is now appreciated to affect nearly all systems in the body,
including the bones and the genitourinary system. Psychosocial issues of living with a
chronic disease have become increasingly important. These new complications have
lead to searches for new preventative strategies in childhood and have posed novel
treatment challenges in adults. The expectation for treatment has switched from the
reactive and symptomatic, to curative strategies, including gene therapy and
phenotype-specific treatments, such as aminoglycosides, to overcome premature
stop codons. There remain many unanswered questions about basic pathophysiology
and much treatment is not evidence-based. The ongoing challenge for clinicians is to
establish a firm evidence base for therapy; for basic scientists, it is to understand the
crucial steps leading from an absent or dysfunctional protein to the clinical disease,
and to target those functions that are crucially disease-producing.
Keywords: Cystic fibrosis, diabetes, gene therapy, nutrition, osteopaenia, Pseudomonas.
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290
CHAPTER 16
Current issues in the basic mechanisms,

pathophysiology, diagnosis and
management of primary ciliary dyskinesia
T. Ferkol*,#, H.M. Mitchison}, C. OCallaghanz, M. Leigh , J. Carson ,, H. Lie**, D. Rosenbluth**,
S.L. Brody **
Depts of *Paediatrics, #Cell Biology and Physiology, and **Internal Medicine, Washington University
School of Medicine, St. Louis, MO, and Depts of Pediatricsand Cell and Developmental Biology,
University of North Carolina, Chapel Hill, NC, USA. }Dept of Paediatrics and Child Health, Royal Free
and University College Medical School, University College LondonzDept of Paediatrics and Infection,
Immunity and Inflammation, University of Leicester, Leicester, UK.
Correspondence: S.L. Brody, Dept of Internal Medicine, Washington University School of Medicine, Box
8052, 660 South Euclid Avenue, Saint Louis, MO 63110, USA. Fax: 1 3143628987; E-mail: sbrody@im.
wustl.edu
Primary ciliary dyskinesia (PCD) is a genetic disorder resulting from the dysfunction
of motile cilia. Major clinical manifestations are recurrent infections of the upper and
lower respiratory tract, including otitis media, sinusitis and bronchiectasis. In half of
all infected individuals, randomisation of leftright symmetry results in Situs inversus
or complex cardiac situs defects. Less appreciated features of PCD are newborn
respiratory distress, failure to thrive during childhood and infertility in adults.
Comprehensive reviews summarising the common clinical aspects of PCD have
recently been published [16]. This chapter summarises the status of investigative areas
relevant to PCD with a focus on genetics, pathophysiology, diagnostic testing and
therapy.
Important findings in the past 5 yrs that have advanced the understanding of PCD are
particularly related to the molecular basis of disease. The field of cilia biology has rapidly
expanded with the discovery that genetic defects in cilia proteins are also responsible for
polycystic kidney disease, neurosensory hearing and vision loss, and the multi-system
BardetBiedl Syndrome (BBS). These findings, and the completed sequencing of the
human and Chlamydomonas genomes, have made the identification of new proteins with
roles in cilia function possible. Although several genes that code for dynein proteins have
been proposed as PCD-causing candidates, only a handful of mutations have been
identified. Thus, there is a need for advances in genetics to discover other mutations
responsible for PCD.
The greatest current challenge remains the care of individual patients with PCD. To
avoid delayed diagnosis in the newborn infant or child, there is a need to develop a
defined set of clinical and genetic studies that are sensitive, specific and cost-effective.
Many of these tests will be surrogates for formal genetic testing as it evolves.
Identification of the ideal diagnostic studies is intrinsically linked to understanding the
biology and pathophysiology of PCD. To achieve these goals, organised, multicentre
studies for diagnosis and care of this relatively rare disease will be required to allow
meaningful analysis of genetic features, which can be linked to clinical and biological
traits. Similarly, this approach can be used to determine the efficacy of current and new
PCD therapies.
ISSN 1025-448x.
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T. FERKOL ET AL.
Recent developments in understanding cilia classes and

ciliogenesis
Classification of cilia
Cilia are broadly classified as motile and primary cilia. Motile cilia contain dynein
proteins functioning as motors to provide directed ciliary motion. Primary cilia lack
motors but are specialised environmental sensors. The recognition that many diseases are
related to hereditary defects in motile and primary cilia structure has led to an explosion
in the study of cilia biology, particularly related to primary cilia. The area of most rapid
growth has been the study of primary cilia defects in polycystic kidney disease [712].
Motile cilia
Motile cilia in humans are located on the apical surface of epithelial cells within: 1) the
respiratory tract composing the upper and lower airways; 2) the central nervous system
within the choroid plexus, ependymal cells of the ventricles and the spinal column; and
3) reproductive organs, including the oviduct and the testes as sperm flagellae. A
population of cells containing motile cilium was also identified in the embryonic node, a
structure transiently present in the midline of the embryo during the somite development
stage and responsible for establishing leftright asymmetry [13].
The structure of the ciliary axoneme is reviewed briefly in this section, within the
context of PCD genetics. Each normal cilium is a composed of y250 proteins organised
around pairs of longitudinal microtubules of a- and b-tubulin, in a well-known pattern of
nine outer-circle doublets that surround a central pair, thus creating the "nineztwo"
organisation observed in electron photomicrographs of the axoneme cross-section
(fig. 1a). Dynein complexes are visualised as inner or outer "arms" extending from outer
doublets. Radial spoke proteins join the membrane sheath surrounding the central pair
with the outer doublets and nexin proteins that form a circumferential network linking
the peripheral doublets. The microtubules with their cognate proteins are anchored to a
a)
b)
c)
Fig. 1. a) Transmission electron photomicrograph of a ciliary axoneme cross-section from a normal individual
demonstrating the nine surrounding and two central microtubule pairs. The outer and inner dynein arms are
attached to each of the surrounding nine pairs (circled area). b) Photomicrograph of an axoneme from a
primary ciliary dyskinesia (PCD) patient demonstrating absent inner and outer dynein arms (circled area). c)
Computer-enhanced digital processing of the difference between normal (a) and PCD (b) images obtained from
the highlighted regions. The composite confirms the absence of both arms relative to normal position noted by
the green highlighted areas which, are illustrated by the red arrows. The black arrow shows the localisation of
the ciliary axoneme outer membrane.
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PRIMARY CILIARY DYSKINESIA
basal body, held within the apical domain of the cell cytoplasm by a macromolecular
complex.
Dynein genes, the leading candidate of most PCD cases, code for proteins that provide
cilia. Dyneins are members of a large family composed of axonemal and cytoplasmic
dyneins, which are classed according to size as heavy, intermediate, light-intermediate
and light chains. Dyneins are highly conserved across phyla, including in the ciliated
green alga, Chlamydomonas reinhardtii. This organism has served as an important tool
for understanding the structure and function of human cilia [14, 15]. Motor function of
the dynein is generated through adenosine triphosphate hydrolytic activity sites, which
are conserved within nucleotide binding motifs called P-loops [14]. The N-terminal
region of heavy-chain dynein binds to other dynein molecules, creating complexes of
microtubule arms of multiple polypeptides of different dynein classes as with other
proteins. In PCD, the most prevalent ultrastructural defects are shortened or absent
dynein arms (inner, outer or both; fig. 1b), attributed to mutation(s) in one dynein gene.
However, the genetic basis of arm defects remains undetermined in most patients. The
large size of the dynein proteins (e.g. heavy chains 400500 kDa) and the large number of
proteins associated with dyneins makes identification of mutations in genes required for
creating motor complexes difficult.
Primary cilia
Nearly all vertebrate cells have a single cilia transiently observed during interphase
[16]. This has been termed a "primary cilium". The structure of the primary cilia retain
the nine outer microtubule pairs but lack the outer arms and the central pair; hence they
are termed "ninezzero" cilia. The cilium extending into the environment is hypothesised
to function as a sensor to detect many types of information. The best-characterised
primary cilia are specialised for vision, hearing and olfactory functions. Most recently,
primary cilia in kidney-tubule epithelial cells have been shown to detect flow by bending,
resulting in the transmission of a calcium-mediated signal to the cell [8]. In a similar
manner, nonmotile cilia in the embryonic node are proposed to detect motile cilia flow to
signal asymmetric body formation during early development (see below) [17].
Ciliogenesis, assembly, cilia transcriptomes and proteomes

Many concepts of ciliogenesis and cilia assembly have been gleaned from the seminal
work by Sorokin [18], which describes the ultrastructure of developing ciliated airway
epithelial cells in electron micrographs. Another major observation, made more recently,
was the identification of proteins transported to and from the cytoplasm into the
axoneme for assembly and maintenance of the cilia, termed intraflagellar transport
(IFT). IFT studies have revealed that dyneins, and other building blocks of the cilia, dock
at the basal body while awaiting transport along the axonemal microtubules as cargo on
kinesin motor proteins [19]. The IFT process also controls cilia length. Mutations or
deficiencies in IFT transport components result in failed transport and are hypothesised
to be the basis of defects in sensory cilia in some forms of polycystic kidney disease [11,
12]. In motile cilia, mutations in dyneins may also result in a "traffic jam" at the basal
body or proximal axoneme, leading to failed transport of cilia motor proteins and
subsequently a PCD phenotype [20]. These studies emphasise the impact of a dynein
mutation and provide one mechanism for failure of dynein arm formation in PCD.
Few "master genes" that regulate ciliogenesis have been identified. In developing
airways, ciliated epithelial cells, as well as Clara cells, express transcription factors TTF1
and Foxa2 (HNF-3b) prior to ciliogenesis [21]. Factors that commit a cell to the ciliated
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T. FERKOL ET AL.
cell phenotype or that regulate the initiation of ciliogenesis are unknown. Centriole
(basal body) precursors, as detected by transmission electron microscopy, are the earliest
known marker of a cell committed to ciliogenesis. Studies in Chlamydomonas indicated
that c-tubulin is essential for basal body production [22]. Also, transcription factor Foxj1
is required for docking basal bodies at the apical membrane and subsequent axoneme
production [23, 24].
Strategies for identification of proteins that are dysfunctional in PCD and related
cilia diseases. Any defect in cilia assembly could potentially result in PCD. To identify the
large number of proteins with a role in the structure, function and assembly of the cilia,
genomic and proteomic studies of human and Chlamydomonas cilia have been performed
[2528]. In these studies, axonemes from human airway cells or Chlamydomonas were
isolated and subjected to proteomic analysis, or Chlamydomonas RNA was analysed by
microarrray to identify genes expressed during ciliogenesis. Taken together, these studies:
1) confirmed that a large number of human and Chlamydomonas proteins are shared;
2) identified regulatory proteins and others previously not suspected to play a role in
ciliogenesis; 3) revealed that genes identified in primary cilia, and mutated in polycystic
kidney disease, are present in motile cilia of Chlamydomonas; and 4) provided the basis
for creation of biochemical pathways of cilia formation.
Progress in the genetic basis of PCD

Recent advances in human genetics of PCD
The inheritance of PCD is autosomal recessive with some rare cases of autosomal
dominant or X-linked inheritance [29, 30]. The precise disease incidence is not known but
is estimated at one per 25,000 live births in Caucasians [6, 31]. Since the association of
Situs inversus and bronchiectasis alone is often used to make the diagnosis of PCD, this
incidence is likely to be an underestimate [3]. The disease prevalence is higher in certain
populations, such as those where consanguineous marriages are customary and those
that are isolated for cultural or geographical reasons [32, 33]. The variation in clinical
symptoms in PCD and the complexity of the cilia and sperm proteins led to early
predictions of locus heterogeneity for the disorder [31]. The underlying ciliary
ultrastructural defects are diverse but dominated by absent dynein motor proteins.
Molecular genetic and linkage analyses in PCD families and model organisms and
identification of mutations in PCD genes have confirmed extensive genetic heterogeneity,
even in patients with the same ultrastructural defect [34, 35]. Efforts to map a major locus
within a large PCD cohort were not successful [36]; however, the development of higher
resolution, high throughput linkage mapping resources, such as single nucleotide
polymorphism-based genome-wide genotyping panels, make re-visiting this approach
more appropriate [37].
A major goal of PCD investigation is the determination of the molecular basis of
disease by identifying and characterising the disease-causing genes. The locus
heterogeneity underlying PCD has greatly impeded efforts to find the genes responsible
for the disease. The results of human genetic-linkage studies of PCD and laterality
defects are summarised in table 1. Successful strategies used to map the genes causing
PCD genes have involved candidate gene analysis and positional cloning by
homozygosity mapping in inbred families. Small, inbred populations, especially if
families have a common ultrastructural defect, are more likely to share ancestral founder
mutations.
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Table 1. Human genetic linkage studies of primary ciliary dyskinesia (PCD) and laterality defects
Disorder
PCD
PCD
PCD
PCD
Situs inversus and
probably PCD
PCD
PCD
PCD
PCD
PCD and Usher
type I syndrome
PCD and cystic
kidney disease
PCD and retinitis
pigmentosa
Leftright axis
abnormality
Leftright axis
abnormality
Chromosome location
Locus/gene
Function
Reference
6p21
Several
9p13p21
19q13-qter
7p15
HLA-DR7
Unknown
DNAI1
CILD2 locus
DNAH11
Dynein intermediate chain
[38, 39]
[36]
[35]
[40]
[41]
Dynein heavy chain
2q32
5p15
16p12
15q1315
14q32
DNAH7
DNAH5
Dynein heavy chain

Dynein heavy chain
USH1 (?EMAP)
9q31
INVS
EMAP is a microtubuleassociated protein

Unknown
Xp21
RPGR
Xq26
2q21
[42]
[34]
[32]
[32]
[43, 44]
[10]
ZIC3
Retinitis pigmentosa GTPase

regulator gene
Zinc finger transcription factor
[45]
CFC1
Extracellular signaling protein
[46]
[30]
HLA: human leukocyte antigen; DNAH: dynein axonemal heavy chain; EMAP: evoked muscle action potential;
GTPase: guanosine triphosphatase.
Mutations in two genes encoding axonemal, outer dynein-arm structural components

have been shown to cause recessive PCD in a proportion of patients with outer dyneinarm defects; but no mutations have yet been reported to account for other ultrastructural
abnormalities in PCD patients. Mutations in genes coding for proteins that compose the
outer arms are the DNAI (dynein axonemal intermediate chain)1 gene on human
chromosome 9p [35, 47, 48] and the DNAH (dynein axonemal heavy chain)5 gene on
chromosome 5p [49]. Identification of these two genes was aided by selective screening of
candidate disease genes with homologues in Chlamydomonas [50]. The DNAI1 and
DNAH5 proteins are orthologues of the 78 kDa intermediate chain (IC78) and gamma
heavy chain (HCc) of the Chlamydomonas outer-arm dynein, respectively. Dysfunction
of IC78/DNAI1 and HCc/DNAH5 result in ciliary dysmotility implying these proteins
are essential for outer arm assembly or attachment to the outer doublet.
Six DNAI1 mutations have been published, including missense, nonsense and deletion
mutations. Three screens in 47 PCD families showed that 12 out of 94 disease
chromosomes had a DNAI1 mutation, which is a frequency of 13% [34, 35, 47, 48]. Of
these, half the mutant alleles were the same mutation, 219z3insT, a splice site mutation
arising from insertion of a single T nucleotide. This frequency suggests a founder effect
for DNAI1 or a mutation hotspot in the gene. Ten mutations in the DNAH5 gene have
been reported [34, 51]. This is a very large gene to screen and, to date, results from only
25 PCD families compatible for linkage to DNAH5 have been published. Mutations were
found in eight families, including mostly truncation mutations predicting loss of the
encoded proteins motor- and microtubule-binding sites, and two missense mutations
located in evolutionarily conserved amino acids that are presumably critical for function.
No mutation hotspots were found. In patients homozygous for the same DNAH5
truncation or missense mutation, no differences in the clinical phenotype were observed
compared with patients with a combination of different (compound heterozygous)
mutations. However, a patient homozygous for a splice site mutation (IVS74-1GwC) had
a partial outer dynein arm deficiency with shortened outer dynein arms, in contrast to
patients homozygous for two truncation mutations who had complete absence of outer
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T. FERKOL ET AL.
dynein arms [42]. The total contribution to PCD of DNAH5 cannot be accurately
estimated, although one estimate suggests that DNAI1 and DNAH5 together account for
24% of PCD patients [52].
In addition, two other dyneins are likely to have a role in PCD. DNAH11 is a dynein
heavy chain gene located on chromosome 7p, which is the human homologue of the left
right dynein gene (lrd), also defective in the inversus viscerum (iv) mouse model of Situs
inversus. Mutations in DNAH11 are associated with Situs inversus and probably a
minority of cases of PCD [41]. The DNAH7 protein was shown to be absent in the cilia
from PCD patient cells that lacked inner dynein arms [42]. However, since mutations
were not detected in the gene sequence it is possible that the underlying defect was in an
associated gene. Candidate genes unsuccessfully screened for mutations in selected
populations of PCD patients include: DNAI2 [53]; HFH4/FOXJ1 [54]; DNAH9 [55];
TCTE3 [56]; hPF20 [57]; DPCD [5]; and DNAL1 [58].
Future genetic studies of the relationship of leftright asymmetry to PCD. It has been
noted that certain PCD patient groups have a lower or higher than predicted occurrence
of Kartageners Syndrome (PCD with Situs inversus) [32]. Further molecular genetic
studies will elucidate whether randomisation of situs only applies to a subset of cases of
PCD and whether certain PCD mutations and genes may be more closely associated with
laterality defects. To minimise the chance of locus heterogeneity, patients have sometimes
been divided according to situs for genetic studies [36, 47]. Laterality has been found to
require normal cilia function in the embryonic node (see below). Further studies to
identify differences in gene expression in cilia of the embryonic node and the cilia of the
respiratory epithelial cells may help to explain some of the observed frequencies in situs
abnormalities and respiratory cilia defects causing PCD.
Primary cilia disorders and overlap cilia diseases

Research in cilia-related disease has exploded after proteins coded for by genes mutant
in polycystic kidney disease were found to be expressed in the primary cilium or basal
body of renal tubule cells [8]. Patients with this disease develop multiple renal cysts and
can also have cysts in the pancreas and liver, implicating defects in cells with primary cilia
lining the ducts of these organs. Adult-onset polycystic kidney disease and the related
paediatric syndrome of nephronophthisis are genetically heterogeneous causes of chronic
renal failure. Nephronophthisis is an autosomal recessive disease in childhood due to
mutations of one of five different genes (all expressed in basal bodies and/or cilia), while
adult-onset autosomal recessive or dominant forms of polycystic kidney disease are due
to mutations in polycystin 1 or 2 genes, also expressed in basal bodies/cilia [912]. Mice
deficient in genes encoding these proteins develop cystic kidneys and pancreas similar to
the human disease [7, 12]. Mutations in other genes cause retinitis pigmentosa with
polycystic kidney disease, constituting the SeniorLoken syndrome [9]. The mechanistic
relationship between defects in primary cilia proteins and the lesion of polycystic kidney
disease is not precisely known.
Despite rare case reports, there is no apparent clinical relationship between polycystic
kidney disease and PCD. However, an infrequent patient with PCD has features that
point to additional roles of disease-causing genes in sensory cilia, including retinitis
pigmentosa and leftright asymmetry [10, 29, 30]. These observations suggest that
proteins mutated in these individuals may have functions in motile and sensory cells of
the embryonic node, motile ciliated cells of the respiratory tract, and in sensory cilia of
the eye and kidney. Interestingly, genes that are mutated in polycystic kidney disease
296
have been identified in proteomic and gene array studies of Chlamydomonas and human
respiratory cilia [2528].
BBS is a rare autosomal recessive disorder with multiple phenotypes in varied
combinations including retinopathy, polycystic kidneys, central obesity, polydactyly,
male hypogonadism, cognitive impairment, diabetes mellitus and congenital heart
defects [59]. Mutations at eight genetic loci have been identified in BBS. Several BBS
proteins are expressed in basal bodies in C. elegans and defects in intraflagellar transport
have been identified in mice with deleted BBS genes [25, 60]. The observation that BBS
genes have a function in microtubule anchoring and cell cycle have lead to much
speculation regarding the relationship of the BBS proteins to primary cilia function in
nonsensory organs, as well as motile cilia. Defects associated with motile cilia have been
found in BBS families, including Situs inversus and hydrocephalus. Mice deficient in
BBS4 fail to form sperm flagella, suggesting shared function between cilia types [61].
Additionally, BBS genes have also been identified in genomic and proteomic analysis of
motile cilia [25, 27, 61].
Animal models of PCD

Mutations of Chlamydomonas [62], and especially mice, have been useful for
understanding PCD genetics and pathophysiology. Deletion of PCD candidate genes in
mice have provided biological proof-of-concept for a genetic relationship between dynein
function, cilia dysfunction, leftright asymmetry, hydrocephalus and other features of
human disease (table 2). For example, outer arm component DNAH5 that is mutated in
human disease was an ideal target for deletion in mice. Dnahc5 (the mouse orthologue of
human DNAH5) deficient mice demonstrated phenotypes similar to PCD patients with
DNAH5 mutations, but also displayed marked hydrocephalus, a rare finding in PCD
[63]. Other knockout mice have revealed a potential role for novel cilia genes in PCD. A
mouse designed to be deficient for the DNA polymerase lambda (Poll), surprisingly
resulted in absent inner dynein arms and a PCD phenotype [64]. Careful genomic
analysis of the gene deletion strategy used to generate this mouse suggested that a gene on
the opposite DNA strand was also likely to be deleted. This gene, named Deleted in PCD
Table 2. Mouse models of cilia defects
Gene deleted
Leftright Hydrocephalus Cilia ultra-structure Other cilia-related

asymmetry
defect
problems
DNAHc5 (mdnah5)
Random
Yes
DNAHc1 (mdhc7)
Normal
No
DNAHc11 (lrd)
Tektin2 (tektin-t)
Random
Normal
No
No
DPCD/DNAPolymeraseLambda
Foxj1
Random
Yes
Random
Yes
Random
Yes
Tg737orpk (Ift88,
Polaris,Tct10)
Absent outer arm
Immotile cilia,
sinusitis
Normal
Decrease sperm
and tracheal cilia
beat frequency
Normal
None
Absent inner arm Decreased sperm
and trachea cilia
motility
Absent inner arm Sinusitis, immotile
sperm
Failure of basal
Absent motile
bodies to dock
axonemes#
Absent and
Polycystic kidneys,
abnormal
photoreceptor
primary cilia
and sperm defects
: ciliary aplasia.
297
Survival
post-natal
Reference
34 weeks [34, 63, 73]

Normal
[65]
Normal
Normal
[66]
[67]
14 months
[5, 64]
04 weeks
[23, 68]
Embryonic
lethal
[69, 70]
T. FERKOL ET AL.
(Dpcd), was found to code for a novel protein likely to be expressed during ciliogenesis
[5].
Mice with disruption of other genes coding for cilia components have had less severe
PCD syndromes, indicating that additional proteins are capable of compensatory roles
affecting the phenotype in these strains. Deletion of the mouse inner arm dynein heavy
chain (mdhc)-7 (renamed Dnahc1) resulted in impaired flagellar and ciliary motility in
mice [65]. Deletion of the basal body protein tektin2 (tektin-t), a protein expressed during
basal body formation and in the axoneme, resulted in defective inner arm structure but
only male infertility [67]. These targeted deletions of cilia proteins support the notion that
human mutations in cilia genes may result in a spectrum of clinical presentations,
including milder phenotypes.
Deletions of other genes have provided important information for the role of cilia in
development and insight into the biological basis of PCD. As noted above, Dnahc11 is
the gene interrupted in the spontaneous mutant iv, a mouse with randomised leftright
asymmetry that has been fundamental for identifying genes that regulate the
development of leftright axis [66]. This mouse has an embryonic node defect, without
a known defect in motile or sensory cilia in extra-nodal tissues. In contrast, deletion of
the forkhead transcription factor, foxj1, also resulted in randomised leftright axis, but
with absent formation of motile cilia [23, 68]. This deletion also results in lethal
hydrocephalus but provides further biological evidence of the relationship between cilia
function and leftright asymmetry.
Strategies for cilia gene targeting in mouse models. The identification of the role of
genes coding for proteins expressed in cilia can be studied in mice rendered deficient
through gene targeting, However, no mouse with deletion of a gene known to be mutant in
human PCD (i.e. DNAH5) is currently capable of survival for study of PCD pathology in
lung infection, nitric oxide generation and for development of PCD therapies. The use of
conditional deletion of PCD genes in mice may favour improved survival for these studies.
This could be accomplished by the use of a regulated FOXJ1 promoter to control deletion
of cilia-specific genes [71]. In addition, to study human PCD mutations where proteintrafficking defects may be responsible for disease, mice deficient in the murine orthologue
of the human mutant genes could be complemented with the mutant human PCD gene
using bacterial artificial chromosomes.
Future challenges in PCD genetics

Genetic studies will aid in the investigation of functional differences or tissue-specific
expression differences in PCD genes. Many proteins that are components of cilia are
members of large gene families with diversity of function and tissue-specific expression
patterns. For example, the HUGO (Human Genome Organisation) Gene Nomenclature
Committee has designated gene symbols for 15 axonemal heavy chain dyneins, and it has
not yet been determined which are the most important for function of cilia in different
locations in the body. For example, homology and expression studies suggest that
DNAH8 might substitute for DNAH5 function in the testis [72].
Based on the absence of dynein arms in ultrastructure studies, it is often assumed that
the dynein proteins themselves are mutated in PCD. Further studies of the relationship
between the function of other proteins important in ciliogenesis and the associated
ultrastructural changes of cilia should be performed. Identification of the genetic basis of
PCD will probably expand as the process of cilia assembly is better understood and as
gene-sequencing costs decrease.
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New concepts in the cellular and molecular basis of PCD

pathophysiology
The role of cilia in leftright asymmetry
A relationship between recurrent sinopulmonary infections and leftright reversal of
thoracic and abdominal organs (Situs inversus) was the basis of Kartageners syndrome.
Consistently, half of all PCD patients have Situs inversus, indicating that this is a random
process. One of the recent, most exciting and important observations in developmental
biology has been the discovery of a cilia-dependent mechanism for determination of left
right asymmetry. Asymmetry is determined by the embryonic node, a dish-like structure
containing motile cilia that is transiently present along the midline of the embryonic
notochord prior to the development of leftright axis. This structure is highly conserved
in vertebrates. The cilia of the node have a rotational beat pattern that results in a
leftward nodal flow of extracellular fluid, thought to determine situs [13]. Mice with Situs
inversus have been shown to have absent or immotile nodal cilia and lack of nodal flow,
resulting in random determination of situs [13, 17, 73]. Evidence that directional flow was
generated by cilia was further supported by the creation of an experimental model where
nodal flow could be controlled [74]. If nodal flow was in the rightward direction, most of
the embryos exhibited a reversal of situs. Artificial leftward or rightward flow in an iv
mutant mouse embryo resulted in normal and reversed leftright patterning, respectively.
Controversies in cilia function in leftright asymmetry. An area of active research is

how flow is detected within the node to activate downstream patterning genes required for
asymmetric organ positioning. One mechanism proposes that the flow generated by
motile cilia within the node signals laterality by bending nonmotile sensory cilia that are
also within the node [17]. In this paradigm, nonmotile sensory cilia activate calciumdependent signals on the right or left edge of the node to trigger downstream programmes
[17]. More recently, it has shown that fibroblast growth factor can trigger the release of
"nodal vesicular packets" containing sonic hedgehog and retinoic acid that engage
receptors at the edge of the node to trigger laterality programmes [75]. Thus, the precise
role of the motile and sensory cilia within the node is undefined. Further generation of
mouse models with proteins that can be imaged (e.g. by targeting the green fluorescent
protein gene) may be useful for studying asymmetry mechanisms. In addition, future
studies will also need to determine the proteins that compose node cilia, compared with
respiratory cilia and sperm flagellae.
Situs variations in PCD. Although the randomisation of situs is considered secondary to

abnormal nodal cilia causing abnormal nodal flow, no PCD patient with either a
transposition defect or central microtubular agenesis has been reported to have Situs
inversus [76, 77]. This may be explained by the observation that cilia with these defects
have a circular beat pattern similar to that of nodal cilia. Thus, it could be predicted that
the movement of nodal cilia would not be affected and flow across the node would be in
the usual leftward direction, resulting in normal situs. Specific mutations in cilia proteins
may, therefore, lead to a variety of effects on nodal structure and function, resulting in
varied situs defects observed in some PCD patients. For example, although situs totalis is
typically seen in PCD, asymmetry defects may involve some, but not all organs in the
thorax and abdomen (situs ambiguus) or be associated with congenital heart deformities,
asplenia or polysplenia [78]. The relationship of these problems to cilia function within the
node is not yet understood.
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T. FERKOL ET AL.
Hydrocephalus in PCD
The precise function of the cilia that line the cerebral ventricles and aqueducts are
unknown; however, orientation and distribution suggest a role in cerebral spinal fluid
flow. Compared with respiratory epithelia, the ependymal cells have fewer cilia (y40 per
cell), are of longer length (78 microns) and beat at twice the frequency (3740 Hz in
rats) [79]. There is a clear link between dysfunction of ependymal cilia and
hydrocephalus. For example, hydrocephalus can be induced in experimental models
by metavanadate, an inhibitor of ciliary movement [80] or by damage to the ciliated
ependyma by bacteria, such as Streptococcus pneumoniae [81]. As noted above, mice
deficient in some cilia proteins (table 2), including DNAH5 or foxj1, developed
progressive unobstructed hydrocephalus leading to death [23, 56, 82]. The Tg737orpk
mouse, deficient in Polaris, an IFT protein expressed in primary cilia, also develops
hydrocephalus. These mice were found to have abnormal chloride production in cerebral
spinal fluid (CSF), leading to the hypothesis that primary cilia regulate ion transport in
CSF [69] and indicating the need for further study of the roles of both motile and
nonmotile cilia in the brain.
Hydrocephalus is rare in human PCD. Slightly enlarged brain ventricles in pre-natal
ultrasound examinations of embryos with Situs inversus have been noted [83]; however,
progression of hydrocephalus is likely to be unusual since there are few case reports of
hydrocephalus with PCD [84]. The wider aqueduct of the human compared with the
mouse may explain the low incidence of hydrocephalus observed in humans in contrast
to the more uniform occurrence in mice with ciliary defects [82].
Low exhaled nasal nitric oxide in PCD

An interesting PCD phenotype possibly related to cilia function is low exhaled nasal
nitric oxide (NO). An initial report that children with Kartageners syndrome produced
very low levels of expired nasal NO [85] was validated by several studies demonstrating
that individuals of all ages with PCD have 1020% of normal levels [3, 86, 87].
Interestingly, nasal NO levels measured in parents of the PCD patients, who are obligate
heterozygotes, were intermediate to levels of PCD and normal individuals [3]. Together,
these findings suggest that a mutant protein interferes with a normal cellular process
directly or indirectly related to ciliated cell functions.
In normal individuals, NO recovered from the nose is several log-fold higher than
from lower airways, reflecting a different regulation of NO production, metabolism
and/or clearance from the two sites. Alteration in these processes may account for the
low levels of nasal NO in PCD. Within the nose and respiratory tract, postulated
functions for NO include regulation of ciliary motility and antimicrobial activity [88,
89]. A family of nitric oxide synthase (NOS) enzymes catalyses the formation of NO in
the presence of appropriate substrates (l-arginine and oxygen) and cofactors. Three
mammalian NOS isoforms have been identified: nNOS (neuronal NOS or NOS I);
iNOS (inducible NOS or NOS II); and eNOS (endothelial NOS or NOS III). Both
iNOS and eNOS have been localised in nasal epithelium [90, 91]. Immunolocalisation
studies have shown that eNOS is expressed close to the base of cilia and may play a
role in regulating ciliary beat [91]. iNOS is increased in nasal epithelial cells obtained
from patients with allergic rhinitis, suggesting a role in the regulation of inflammatory
processes [90]. A potential area of investigation is iNOS and eNOS expression in
epithelial cells of PCD patients with and without infection, as well as the effect of NO
addition on cilia cell function.
300
Chronic sinusitis and bronchiectasis

The leading cause of morbidity and mortality in PCD is recurrent respiratory tract
infection leading to chronic airways infection and bronchiectasis. In PCD, the aetiology
of airways infection is considered to be the result of defective mucociliary clearance in
epithelium in contact with the environment [92, 93]. The major effect is an increased
burden of bacteria in the middle ear, paranasal sinuses and airway [3]. A paradigm
developed for chronic bronchitis and cystic fibrosis (CF) that has been applied to PCD, is
that repeated infection and inflammation leads to injury, tissue remodeling and persistent
infection in the form of biofilms [94, 95]. Little information is available to indicate that
these processes are fundamentally different in PCD compared with other diseases with
abnormal clearance. The availability of animal models of PCD and large multicentre
trials will be useful to elucidate mechanisms of chronic infection and inflammation in
PCD.
Current and emerging strategies for the clinical diagnosis of

PCD
Perspective on studies required for the diagnosis of PCD
Until genetic mutations are better understood and definitive genetic methods are
developed, there is general agreement among practitioners that the diagnosis of PCD
should be based on the presence of the typical clinical phenotype plus specific
ultrastructural defects of cilia identified by transmission electron microscopy [1, 3]. Since
the clinical features of PCD, particularly in the newborn and young child, are shared with
a variety of diseases, clinicians must maintain an awareness of the disease to ensure a
timely and accurate diagnosis. Cilia ultrastructure analysis is a relatively expensive test
and a broad differential diagnosis should be considered before this test is pursued.
Furthermore, it must be stressed that defects in both cilia beat frequency and cilia
ultrastructure are often the result of infection, allergy, or other inflammatory disease,
rather than a genetic defect in cilia structure. Also, once embarking on making the
diagnosis of PCD, serious consideration should be given to the expertise required to
obtain appropriate samples and perform ultrastructural analysis of cilia, as noted below.
Of all the strategies emerging to aid in the PCD diagnosis, exhaled nasal NO is perhaps
the most promising.
Underappreciated clinical features of PCD in the neonate and infant

PCD may present with a variety of clinical features, mimicking respiratory diseases,
such as CF or asthma, thus making prompt diagnosis a challenge and often leading to
delayed diagnosis. One study reported that the mean age of PCD diagnosis was slightly
w4 yrs of age despite early manifestations of PCD in infancy [96]. Accumulating
information reveals that half to three-quarters of patients with PCD have significant
respiratory symptoms shortly after birth [1, 3, 97]. Many reports emphasise atelectasis or
pneumonia associated with significant respiratory distress in neonates with PCD. The
association of neonatal respiratory distress with PCD point to the critical nature of cilia
function for effective clearance of foetal lung fluid. In infancy, chronic cough and
persistent rhinitis, often present since birth, are common clues to underlying PCD. As a
consequence, the infant may struggle with poor feeding and failure to thrive. Rhinitis and
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chronic cough in infancy typically leads to chronic sinopulmonary infections and

eventual PCD diagnosis later in childhood [96].
Ultrastructure analysis of cilia

Afzelius [98], and other authors, characterised the first ultrastructural lesions by
electron microscopy that elucidated the pathophysiological basis of PCD. These studies
showed absent axonemal dynein arms of airway cilia and sperm flagella as the causative
basis for ciliary immotility and resultant respiratory disease and infertility. Since these
early reports of PCD demonstrating completely absent inner and outer dynein arms and
absent ciliary motility, a number of phenotypic variations including missing or
dysmorphic inner or outer arms, and changes in the structural organisation of auxiliary
axonemal elements, such as radial spokes, have been noted [92, 93, 99]. The most
common ultrastructural defects detected in a PCD population are outer-arm defects that
can be related to genetic mutations in heavy chain dynein genes [3]. Electron microscopy
of sperm tail flagella or of respiratory cilia has been recognised as the "gold standard" for
the diagnosis of PCD.
Ultrastructural analysis has limitations that may lead to confounding results. Among
the problems associated with such analyses are poor sample quality, processing and
procedural errors, and the inherent limitations of electron optics related to imaging of
ultra-thin sections. As individuals with PCD generally present with upper respiratory
symptoms, including chronic sinusitis, acquisition of a sample of epithelium containing
an adequate number of ciliated cells is often problematic. Moreover, because of the
chronic nature of infection and inflammation, acquired ciliary defects are often present in
the sample, along with necrotic cell debris that interferes with optimal imaging. This has
had the unfortunate result of erroneous reports of "new" index lesions said to confer
PCD.
The processing of specimens for electron microscopic examination requires exposure
of the cells and tissues to fixatives intended to stabilise cell and organelle structure,
exposure of the tissues to heavy metal-containing stains to impart differential electron
opacity to specific chemical groups, and embedment of the specimen in an epoxy resin for
ultra-thin sectioning. Under conditions of excellent quality control, electron dense
precipitates, swelling and shrinking of tissues, and suboptimal section quality and
thickness are limited. The accelerating voltage, operational modes of the transmission
electron microscope, and the physical characteristics of the electron beam also contribute
to the quality and resolution of micrographs intended for analysis. For example, a typical
ultra-thin section is 6090 nm in thickness and, for a given axoneme, such a section may
reveal a wide variety of profiles. Thus, it is of paramount importance that a sufficient
number of clear cross-sections are obtained to ensure a reliable interpretation.
Computer-assisted programmes that enhance the electron micrograph image to improve
detection of cilia defects are being evaluated (fig. 1c) [100, 101].
Emerging questions for ultrastructural imaging studies in PCD. The small

dimensions, particularly thinness, of ultra-thin sections severely limit approaches to
analyse the organisation of axonemal elements in three dimensions and their linear
distribution along the length of axonemes. Furthermore, staining characteristics of
conventional heavy metal stains used in biological electron microscopy may not be
capable of generating the contrast necessary for imaging certain axonemal elements
integral to ciliary function. Cilia from patients with PCD often exhibit marginal motility
and ultrastructural evidence of irregular distributions of dynein arms. Thus, it is plausible
to hypothesise that dynein organisation and distribution are altered along the length of
302
the axoneme and that these differences are genotypic. Although elegant morphometric
studies using computer-assisted analyses [102] and freeze-fracture methodology [103] have
demonstrated the spatial organisation of axonemal elements in wildtype and mutant
eukaryotic Protists, similar analyses have not been performed in axonemes from
documented PCD patients. Moreover, the vast majority of ultrastructural studies of PCD
have focused on the fine structure of axonemes to characterise ciliary anomalies, but have
failed to analyse the "upstream" events of ciliogenesis as an initiating event in the
formation of abnormal cilia in PCD. A promising area of PCD research in recent years
has been the search for and identification of candidate genes, and fundamental data that
relates specific mutations to cilia ultrastructure has not been realised [34, 48]. As both
genetic and proteomic studies yield new information and as imaging techniques undergo
further refinement, biological electron microscopy as well as other high-resolution
imaging techniques will continue to play an important role in the molecular level
characterisation of axonemal organisation in this syndrome.
Expired nasal NO for PCD diagnosis

Several studies have demonstrated that exhaled nasal NO is very low in individuals
with PCD, and the ease of test performance suggests this as a useful screening or
supportive diagnostic test for PCD. However, low nasal NO levels have also been
reported in disorders with overlapping clinical features including CF, panbronchiolitis
and nasal polyposis [86, 104, 105]. The low NO levels in these other diseases may be due
to secondary cilia dysfunction, but the shared clinical features indicate that further
assessment is warranted before this test is routinely relied on for definitive diagnosis of
PCD. Alternatively, the data indicate that in some individuals with specific clinical
findings it is possible that the diagnosis of PCD could be supported if CF was excluded.
Recent efforts have focused on standardising nasal NO measurement by a noninvasive
procedure to allow comparisons between studies [106]. During the measurement, several
manoeuvres may be used to close the soft palate and thereby limit "contamination" of
nasal air by air exhaled by the lower airways (exhaled NO from lower airways is typically
much lower than nasal NO). These techniques have been used reproducibly in children
w5 yrs of age, but may be difficult in younger children who cannot cooperate with palate
closure manoeuvres.
Areas for investigation of exhaled NO related to PCD. Exhaled nasal NO is now used
in large PCD centres as a screening test. However, the underlying pathophysiological
mechanism responsible for the result is unknown, making interpretation difficult. Studies
to elucidate NO pathways in patients with PCD are required. This may be facilitated by
studies of mice with known dynein arm mutations (knockout mice) or in vitro cell culture
of airway epithelial cells from patients with defined dynein arm mutations. Clinical trials
to assess the contribution of genetic, infectious and inflammatory factors are also
necessary. Although preliminary studies have not revealed differences in low NO
production relative to different PCD mutations, it may be useful to investigate exhaled
NO in individuals with novel defects not involving common dynein arm genes.
Cilia beat analysis

Qualitative assays for ciliary function, including measures of mucociliary clearance
using the saccharin test and radioisotopic methods, have limited usefulness because
they are poorly standardised, cannot be used in young children, and do not distinguish
between primary and secondary causes of ciliary dyskinesia. The recent advent of
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high-resolution digital high-speed video imaging allowing the precise beat pattern of cilia
to be viewed frame by frame in different planes has revealed that reproducible differences
in ciliary beat pattern are related to specific underlying ultrastructural abnormalities [76].
These findings have led to the concept that detailed analysis of cilia beat could be used for
clinical assessment and the diagnosis of PCD.
Classes of cilia beat relative to ultrastructure defect. High-resolution video imaging of

cilia from nasal or lower airway sampling has shown that respiratory cilia do not beat with
a classical forward power stroke and recovery stroke that sweeps to the side as previously
assumed. They simply beat forward and backward in a planar motion, without a sideways
recovery sweep [107]. In patients with isolated outer dynein arm or outer and inner dynein
arm defects, the majority of cilia are immotile and, at best, simply flicker. Cilia from
patients with an isolated inner dynein arm defect or a radial spoke defect combined with
an inner dynein arm defect have a stiff forward stroke with markedly reduced amplitude.
The beat frequency of these two groups differs. The inner dynein arm defect has a mean
beat frequency of 8.1 Hz while the radial spoke defect frequency is slower at 6 Hz. The
stiff beat pattern supports the importance of the inner dynein arm in the bending of the
ciliary axoneme. The third type of beat pattern is found in patients with PCD associated
with ciliary transposition and central microtubular agenesis. In the ciliary transposition,
the gap left by an absent central microtubular pair is filled by a peripheral microtubule
pair and associated dynein arms that transpose to the centre of the axoneme. In
microtubular agenesis a proportion of cilia lack the central microtubular pair [77]. In both
defects, all cilia have an oval gyrating pattern with cilia beating with a mean frequency of
10.7 Hz which is within the normal range of ciliary beat frequency (normal mean 12 Hz;
range 9.718.7 Hz) measured using the high-speed video system [76]. The implications of
these findings are significant, as ciliary beat frequency is typically measured using indirect
techniques, such as the photomultiplier and modified photodiode that cannot assess
ciliary beat pattern. Thus, the use of beat frequency alone as a screening test to determine
those patients requiring further investigation will miss PCD due to ciliary transposition
defects or central microtubular agenesis.
Cilia-beat analysis requires demanding expertise and expensive instrumentation. The
routine use of this methodology for the diagnosis of PCD is, therefore, currently limited
to a few specialised centres. However, information from these studies can be used to
characterise specific groups of patients with cilia defects, make genotypephenotype
correlations, and provide useful information for assessment of results from other
diagnostic testing methods.
Other methodologies for diagnosis of PCD

Primary airway epithelial cell culture to exclude secondary damage. It must be
stressed that defects in both cilia beat frequency and cilia ultrastructure are often the result
of infection, allergy or inflammatory disease, rather than PCD. In this regard, culture of
human airway cells offers a strategy for obtaining samples in a controlled environment.
Culture of primary human respiratory epithelial cells from nasal sinus or lower airway
biopsies or brushing can be performed and cells differentiated using airliquid interface
conditions [108]. These cultures require specialised knowledge and experience, but are
now routinely carried out in many research laboratories. Bacterial contamination with
resistant organisms in the samples must be considered when culturing these cells but can
be prevented by the use of specific antibiotics [109]. High-quality samples generated using
these methods contain abundant ciliated cells that can be a powerful reagent for studies of
304
PCD biology using electron microscopy, cilia beat analysis, immunostaining and
biochemical methods.
Analysis of dynein protein localisation. Detection and intracellular localisation of

DNAH5 by immunofluorescent microscopy may also aid clinical diagnosis of PCD. In a
recent study, antibodies to DNAH5 and DNAH9 were used for intracellular localisation
of dyneins in respiratory tract epithelial cells or sperm [20]. The results showed that
individuals with PCD due to known DNAH5 mutations had either absent staining or
abnormal accumulation of the dyneins within the region of the basal bodies. Absent
staining was associated with cilia being immotile, whereas cells with abnormal
intracellular localisation had minimally motile cilia. The development of a panel of
robust antibodies directed toward multiple cilia proteins may enable screening respiratory
epithelial cell or sperm samples if used in conjunction with other testing such as exhaled
NO, and careful exclusion of secondary causes of cilia damage. Similar approaches can
also be used to uncover mechanisms of cilia assembly and the effect of specific cilia gene
mutations on protein trafficking and cilia function.
Challenges for the diagnosis of PCD

New methods for PCD diagnosis must be carefully evaluated in prospective studies. In
general, the most sensitive, specific, and cost-efficient adjuncts to clinical diagnosis
should be determined. Currently, the gold standard is ultrastructural analysis; however,
this is expensive and requires specialised facilities. These problems can be somewhat
overcome by centralisation of sample analysis at dedicated laboratories. Ultimately,
genetic testing must be used. Some simple genetic testing could be developed, such as a
specific test for the 219z3insT mutation, to detect an estimated 50% of DNAI1 mutations
in outer dynein arm defects. For the future, further PCD gene identification will be
necessary to facilitate development of less invasive diagnostic methods.
As diagnostic tools are developed and standardised for PCD, it must be determined
how various diagnostic results vary with specific genotypes and considered that there are
unusual or mild phenotypes that have cilia defects. In addition, a broad range of
individuals with diseases that mimic PCD must be included in testing. PCD is likely to
exist as a continuum; milder phenotypes certainly exist that would be manifested by
subtle or no apparent structural defects and ciliary dysfunction. Thus, new tools must be
studied within the context of existing diagnostic standards in a broad range of patients.
To accomplish this, specialised PCD research centres should have available methods, as
noted above, to facilitate advances in diagnosis.
Current concepts in the clinical management of PCD

Therapeutic approach and prognosis of PCD
No therapies are known that correct ciliary dysfunction and clinical studies with
sufficient statistical power to prove the efficacy of specific therapies have not been
performed. Similarly, few retrospective studies are of sufficient size to make strong
conclusions regarding current therapy. Instead, most therapies are based on experience
from centres treating large numbers of individuals with PCD or are extrapolated from
treatment of diseases with similar clinical features, including CF and bronchiectasis of
multiple aetiologies. The goals of current PCD therapy are: 1) maintenance of
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mucociliary clearance; 2) optimal management of otitis media and sinusitis; and 3)

bacteria culture-directed antibiotic therapy for upper and lower airways infection.
Three small longitudinal studies of PCD suggest that the onset of airway disease occurs
early in childhood [110112], stressing the need for early diagnosis, close monitoring for
airway infection, and, ultimately, long-term multicentre studies. It is widely agreed that
early diagnosis and antibiotic use have improved the prognosis for PCD. In contrast to
CF, many individuals with PCD have a normal or near-normal lifespan. However, like
CF, chronic lung disease is the most common limiting problem, leading to severe
pulmonary disability and eventually to respiratory failure in some individuals.
Management of otological and sinus disease

Chronic otitis media with effusion is a near universal manifestation of PCD, indicating
that patients should be routinely screened for hearing deficits. Aggressive antibiotic use
has been the practice for managing otitis media; however, overuse may lead to resistant
organisms. The use of myringotomy tubes in children is common in the PCD population
[3, 93]. The practice remains controversial as tubes may lead to chronic mucoid
discharge, temporary or persistent perforation, and tympanosclerosis, while offering no
clear advantage in improving hearing thresholds when compared with a strategy of
watchful waiting as hearing generally improves with time [113]. If needed, tympanoplasty
has been employed to successfully improve hearing in children [114]. Hearing aids should
be provided to children and adults with conductive hearing impairment.
Like otitis media, chronic rhinitis and sinusitis affect almost all individuals with PCD
[1, 3, 93]. Chronic sinusitis has been managed symptomatically with nasal steroids, sinus
lavage and intermittent courses of systemic antibiotics. Functional endoscopic sinus
surgery may help promote drainage and local delivery of medications in those refractory
to medical therapy [1].
Enhancing mucociliary clearance

Experience from the management of CF and bronchiectasis from other causes has
shown that enhancing mucociliary clearance should be the cornerstone of daily therapy
for most individuals with PCD. Cough is the major route for mucociliary clearance and
should be encouraged (not suppressed). Routine airway clearance using any of the
available physiotherapy techniques from conventional postural drainage and percussion
to percussive or oscillary vests, along with routine use of inhaled b2-agonist
bronchodilators, which may increase ciliary beat frequency, and exercise should be
employed in all patients. A brief trial of aerosolised uridine-5-triphosphate (UTP) has
been shown to increase airway clearance in PCD patients [115], but insufficient evidence
is available to recommend the routine use of this agent, as well as DNAse or antiinflammatory agents used in CF.
Directed antibiotic therapy for lung disease

Directed antibiotic therapy practice for PCD is based on experience in CF clinics [95].
Here, management of pulmonary disease involves regular monitoring of pulmonary
function using spirometry, chest radiographs, respiratory cultures by sputum or throat
swab (based on age), and treating respiratory infections with antimicrobial agents based
on careful laboratory assay of sensitivities. One retrospective study of PCD at a
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specialised centre in the USA found that this was commonly the practice in PCD
management [3]. In half the cases, antibiotics were provided as i.v. therapy.
Prospective longitudinal surveys of bacterial species recovered from respiratory
samples of PCD patients have not been published. The most common organism
recovered from patients with PCD is Haemophilus influenzae, particularly in children [3].
Other common organisms are Pseudomonas aeruginosa, Staphylococcus aureus, and S.
pneumoniae. As in CF, chronic infection with mucoid strains of P. aeruginosa can occur,
but chronic infection with P. aeruginosa appears at a later age when compared with CF.
Oral or systemic antibiotics should be prescribed as indicated for worsening respiratory
symptoms, infection or spirometry, and should be chosen on the basis of prior sputum
culture results. As in other bronchiectatic syndromes, chronic suppressive oral or inhaled
antibiotics may be prescribed as maintenance therapy but could enhance the
development of antimicrobial resistance.
Chronic lung disease, pulmonary resection and lung transplantation

Complications of bronchiectasis become more prominent with increasing age. The
decline in lung function as measured by the forced expiratory volume in one second (FEV1)
appears to be significantly less than that seen in CF; thus, patients have better long-term
prognosis and survival when compared with CF patients [3]. Surgical resection of
bronchiectasis has been performed in patients with PCD and may be considered for patients
with localised disease refractory to medical management [116]. Since there has never been a
prospective evaluation of surgical resection in PCD, careful consideration should be given
prior to performing a resection, and referral to a tertiary-care centre with experience in the
surgical management of patients with bronchiectasis should be entertained.
While preservation of native lung function should be the goal of management, patients
with end-stage lung disease secondary to PCD-related bronchiectasis with or without
Situs inversus have successfully undergone heart-lung, double lung or living donor lobar
lung transplants [117, 118]. Any of these procedures is technically more difficult in
patients with Situs inversus due to the challenges presented by anatomic considerations
at the anastamotic sites, but assuming these are overcome, then long-term survival
should be similar to other transplant recipients.
Future directions for studies of PCD management

As noted previously, the relatively low incidence of PCD mandates that evaluation of
therapies be carried out using multicentre strategies. Large, prospective clinical trials in
management of otitis, sinusitis and bronchiectasis are lacking. Questions as to the benefit
of directed antibiotic therapies, the use of agents to enhance cilia beat frequency, or
measurement of NO levels are potential areas for study. Multicentre studies have been
organised in the USA, UK and Europe. Further collaboration can be achieved through
foundations supporting patients with PCD and their families, and lung disease Councils,
many of which are accessible via the Internet. These organisations are invaluable for
informing patients of clinical studies and encouraging financial support from
government, charitable organisations and pharmaceutical companies.
Summary and conclusion

Since the 1990s, major advances in PCD are related to discoveries of the genetic and
molecular basis of this disease. Specifically, identification of dynein gene mutations that
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cause the syndrome of PCD in many affected individuals, and confirmation of the
biological basis of disease by demonstration of a similar phenotype in mice that are
rendered dynein gene deficient. In addition, a role for cilia in the establishment of left
right asymmetry and hydrocephalus has been discovered in PCD-related research.
However, genetic mutations have not been identified in a significant number of patients
with PCD, pointing to the need for further studies of cilia biology and genetics. Thus,
there are significant problems related to easily making the diagnosis of PCD. In this
regard, the use of exhaled NO appears promising and requires a better understanding of
the mechanism of altered NO excretion in PCD. Finally, multicentre studies of genotype
phenotype relationships and therapeutic modalities will need to be organised.
Summary
Primary ciliary dyskinesia (PCD) is a genetic disorder resulting from dysfunction of
motile cilia. Epithelial cells containing motile cilia are localised in the respiratory tree,
ventricles of the brain, oviduct, sperm and the embryonic node. In these epithelial
cells, dysfunction accounts for the major symptoms of PCD, including otitis media,
sinusitis and bronchiectasis, Situs inversus (in half of the patients) and, more rarely,
infertility and hydrocephalus. While the understanding of cellular and molecular
mechanisms responsible for these symptoms has recently progressed, genetic analysis
has identified mutations in only two axonemal dynein genes that can account for
abnormal cilia ultrastructure and beat frequency in a subpopulation of individuals.
Thus, investigations are directed towards expanding understanding of the genetic basis
of PCD by identification of proteins with roles in ciliogenesis, and increasing the scope
of genes and populations subject to genetic analyses. To support the diagnosis of PCD,
efforts are currently directed toward the optimal use of cilia ultrastructure and beat
analysis, and interpretation of low levels of exhaled nasal nitric oxide. While there is
no specific therapy for PCD, maintenance of mucociliary clearance and culturedirected antibiotic therapy are current cornerstones of therapy. The establishment of
new methodologies for PCD diagnosis and therapies will require evaluation of
relationships between specific genetic mutations, disease phenotypes and therapeutic
responses to be carried out in multicentre cooperative trials.
Keywords: Bronchiectasis, cilia, dynein, leftright asymmetry, mouse models, nitric
oxide.
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313
CHAPTER 17
Adipositas in infants and children: a new

disease on the horizon
K.G. Tantisira, D.R. Gold
Channing Laboratory, Brigham and Womens Hospital and Harvard Medical School, Boston, MA, USA.
Correspondence: K.G. Tantisira, Channing Laboratory, Brigham and Womens Hospital and Harvard
Medical School, 181 Longwood Avenue, Boston, MA 02115, USA. Fax: 1 6175250958; E-mail: kelan.
tantisira@channing.harvard.edu
Obesity is one of the most prevalent chronic disorders of childhood, affecting

22 million children worldwide [1]. In the period 19992002, y15% of children and
adolescents in the USA met the criteria for obesity [2, 3]. Using National Health and
Nutrition Examination Survey (NHANES) data, the prevalence of obesity, defined by a
body mass index (BMI) i95th percentile, for children of the same age and sex doubled
between 19761980 and 19992002 [4]. Similar increases have been noted in other
developed countries [58]. For instance, the prevalence of obesity in Japanese schoolaged children doubled from 5 to 10% during 19731994 [9]. In the USA in 1998, the
prevalence of childhood obesity was 21.5% for African-Americans, 21.8% for Hispanic
Americans and 12.3% for non-Hispanic Whites [3]. Despite these ethnic disparities,
increases in body mass have occurred among both sexes and across all ethnic and
socioeconomic groups [2]. Childhood obesity is also increasing in developing countries,
where it is not uncommon to note demographic characteristics that simultaneously
include both high prevalences of obesity and malnourished children [1, 10]. Thus, obesity
in childhood is clearly evolving into a truly global health issue.
Although no single explanation for the increased prevalence of childhood obesity
exists, multiple theories abound. The Western lifestyle hypothesis builds upon the
increased frequency of obesity in developed countries compared to Third World
countries. Certainly, as countries make the transition from primarily agricultural
societies towards increasing industrialisation, the incidence and prevalence of obesity
tend to increase. For instance, in Thailand, the per capita gross national product doubled
between 1990 and 1996. The prevalence of overweight adults, defined as a BMI of
i25.0 kg?m-2, rose from 7.7 to 13.2% in males and from 15.7 to 25.0% in females
between 1991 and 1996 [11]. Similar trends were seen in children, with pronounced
differences by socioeconomic and urban versus rural settings [11]. The hypotheses for the
development of obesity in relationship to Western lifestyle have focused primarily on
caloric consumption and decreased physical activity [3, 11]. Other common hypotheses to
explain the "obesity epidemic" include the Barker or foetal origins hypothesis [12, 13],
changes in the relative proportion of dietary fats and type of carbohydrates [1, 11], and
interactions of genetic predisposition with environmental influences [12, 14, 15].
Childhood obesity is associated with a variety of both long- and shorter-term sequelae.
Several studies have reported an increase in cardiovascular and all-cause mortality rate in
adults who were overweight or obese as children or adolescents [1618]. Childhood
obesity may increase mortality risk in adulthood through its cumulative effects on
elevated blood pressure, lipid abnormalities or type 2 diabetes mellitus [8, 19, 20].
Childhood obesity is also associated with more immediate decrements in health and
ISSN 1025-448x.
314
OBESITY AND RESPIRATORY DISEASE
quality of life in childhood [21]. Childhood obesity substantially increases the risk of later
morbidity, regardless of whether or not the obesity persists into adulthood [22].The
economic burden of obesity in children continues to climb, with recent obesity-associated
annual hospital costs (based on 2001 constant USA dollar value) in the USA increasing
more than three-fold, from US$35 million during 19791981 to US$127 million during
19971999 [23].
There is rapidly evolving evidence that obesity may substantially affect respiratory as
well as cardiovascular health. Respiratory complications of obesity in children are the
focus of the present chapter, after discussion of definitions of childhood obesity. Data are
reviewed on the relation of obesity to pulmonary function, airways responsiveness
(AHR), asthma and obstructive sleep apnoea (OSA) in children. Subsequently, the
potential pathophysiological mechanisms that may help to explain these associations and
the requirement for further research into these mechanisms are discussed. Finally,
recommendations for individual and social practice designed to reduce childhood obesity
are reviewed. Although the present chapter focuses on pulmonary manifestations of
obesity in children, these are supplemented with recent findings from adult human and
animal studies for situations for which the literature in children is sparse or unavailable.
Body mass: the correct obesity phenotype?

Most of the literature involving childhood adiposity has focused on some measure of
BMI (body mass in kilograms divided by height in metres squared), including using
population cut-offs of the 85th percentile to define overweight and the 95th percentile to
define obesity. Indeed, the use of BMI has been proposed as the primary standard for the
worldwide definition of overweight and obesity in children [24]. Nonetheless, multiple
other obesity phenotypes have been described. For example, central, including truncal
and abdominal, adiposity is linked with insulin resistance, whereas centrifugal or gluteal
adiposity is not [25]. BMI is probably a good clinical measure for the definition of obesity
in adolescents [22]. In pre-adolescents, however, BMI may be representative of increases
in other growth parameters, such as muscle mass [22, 26, 27], and, therefore, should be
used with caution. The best epidemiological measure of body fat in children and
adolescents remains controversial [28], but may be one utilising skinfold measures (e.g.
triceps skinfolds) [29, 30]. Other commonly employed field measures of adiposity in
children include waist circumference, waist/hip ratio and measures of bioelectric
impedance [31, 32]. Dual-energy X-ray absorptiometry (DXA) [31, 33] has become a
widely accepted standard for measuring body fat in clinical studies of both children and
adults, due to its relative ease of use, low radiation exposure and clinical reproducibility
[34]. One advantage of DXA is the ability to assess total as well as regional body
composition (i.e. trunk, arms and legs). Computed tomography and magnetic resonance
imaging scans may be more accurate but their use is limited in clinical studies of children
due to their cost and radiation exposure [32].
Although the criticism that BMI correlates with overall growth has been demonstrated
to be true when growth is evaluated by measured height, one study noted that it remains
a valid measure of obesity in 512-yr-old children since the degree of measured adiposity
also increases with height [35]. In particular, Freedman et al. [35] found a very high
correlation (r=0.850.90) between BMI and measured body fat in these younger children.
A follow-up study by the same group evaluated 1,196 children aged 518 yrs, comparing
BMI with DXA-measured fat mass and fat-free mass [36]. In those children with a BMI
v50th percentile for age and sex, BMI strongly correlated with fat-free mass (r=0.56
0.83). However, for a BMI i85th percentile, BMI correlated primarily with fat mass
315
K.G. TANTISIRA, D.R. GOLD
(r=0.850.96), supporting the continued usage of the current BMI definitions of

overweight and obesity as valid measures of adiposity. Few studies are available that
evaluate the relation of obesity phenotypes other than BMI to respiratory disease.
Therefore, in the present chapter, the definition of obesity is assumed to be based upon
BMI measurements. However, where available, data based upon other measurements of
adiposity are cited.
Associations of obesity with respiratory function and disease in

children
Obesity and pulmonary function
Obesity alone, in the absence of other known causes, can be associated with the
sensation of dyspnoea at rest. The prevalence of symptoms increases with increasing BMI
or waist circumference [37]. Traditionally, obesity has been one of the factors cited to be
associated with classic extrapulmonary restrictive physiology, with spirometric evidence
of decreased forced expiratory volume in one second (FEV1), decreased forced vital
capacity (FVC) and preserved FEV1/FVC ratio [38]. The mechanism behind this cited
restrictive defect has generally been decreases in pulmonary compliance associated with
the accumulation of fat in and around the chest wall, diaphragm and abdomen [37, 39,
40]. Lung compliance is commonly reduced by i25% [40]. However, this classic
restrictive decrement applies primarily to morbid obesity [41, 42]. In studies directed at
evaluating contributions of body weight to pulmonary function, the additional variance
explained by BMI in linear regression models has been found to be modest in children
[26]. Part of the poor predictive value of body mass in relation to FEV1 may be related to
the use of BMI as a linear term. Indeed, in both adults and children, it has been noted
that initial increases in level of body mass are associated with increased FEV1
(muscularity effect) [26, 27, 43]. However, further increases in body mass are
subsequently associated with decrements in FEV1 (obesity effect) [43]. This is further
supported by the fact that increases in measured fat mass in lieu of BMI have consistently
been associated with decrements in lung function [26, 44, 45].
Obesity also contributes significantly to other physiological changes in pulmonary
function. The most common abnormality in pulmonary function of obese subjects is a
reduction in expiratory reserve volume and functional residual capacity (FRC) [37, 42,
46]. The mass loading effect of obesity decreases the FRC but does not decrease residual
volume (RV); therefore, expiratory reserve volume declines. RV related to total lung
capacity (TLC) may even be higher than normal in morbid obesity, but TLC, maximum
voluntary ventilation and vital capacity may be reduced [41].
In addition to changes in spirometric measurments and lung volumes, oxygenation and
ventilation may also be affected in obesity. Normoxia or mild hypoxaemia is usually
present in the upright position, although many obese patients develop further
hypoxaemia when supine [47]. Gas exchange abnormalities may be the result of
ventilation/perfusion mismatch, with preserved perfusion but diminished ventilation to
the lung bases due to atelectasis. In obesity, rates of total oxygen consumption and
carbon dioxide production are increased even at rest [48, 49]. Obese patients also dedicate
a disproportionately high percentage of total oxygen consumption to performing
respiratory work even during quiet breathing [50]. These patients generally exhibit an
elevated resting respiratory frequency with a tidal volume that is normal overall but
reduced when adjusted for lean body mass [51, 52]. The net effect is one of increased
minute ventilation compared with normal subjects. Finally, from a gas exchange
316
perspective, an increased ventilatory responsiveness to hypoxia and relatively decreased

ventilatory responsiveness to hypercapnia have been noted in obese subjects [53].
Obesity and fat distribution pattern may have independent effects on ventilatory
function. FVC, FEV1 and TLC were found to be significantly lower in 42 subjects with
an upper body fat distribution (central obesity) as measured by waist/hip ratio [45]. Sex
differences in fat distribution (fat is distributed more peripherally in females and more
centrally in males) may also contribute to the observed greater impact of fat mass on
FVC in males [54]. A large cross-sectional analysis of 1,634 adults noted that increased
waist/hip ratio was associated with more pronounced decrements in FEV1 and FVC in
males than in females [55].
Obesity in children may also result in little or no change in spirometric indices [56, 57].
In a descriptive study of 64 obese children (median age 12 yrs; median BMI 30.1 kg?m-2),
Li et al. [58] noted overall median values within the normal range for both spirometric
and lung volumes when obesity was assessed as a BMI i95th percentile. However, 30 of
the 64 (46%) showed reduced FRC, consistent with the adult literature on this topic.
Additionally, these authors noted a moderate decrement in diffusing capacity of the lung
for carbon monoxide (DL,CO) in a third of the subjects, which has not been reported in
adults. When truncal obesity was assessed by DXA, TLC and RV were also noted to be
reduced, supporting the contention that DXA may be a better measure than BMI of
body fat that might affect the pulmonary architecture.
Decrements in DL,CO with obesity were also noted in one other study of 13 morbidly
obese children [59]. The children in that study ranged 150300% of ideal body weight. In
addition to the DL,CO, the children exhibited decrements in FEV1, forced expiratory flow
between 25 and 75% of vital capacity, and expiratory reserve volume. Aside from the
changes in DL,CO, this series of children indicate that the pulmonary function
abnormalities in the morbidly obese child generally parallel those expected of the
obese adult.
The effects of body mass on pulmonary function have also been evaluated in several
paediatric cohorts. In a study of 1,041 childhood asthmatics [27], BMI was positively
associated with increased FEV1 and FVC. However, increased BMI corresponded to
decrements in the FEV1/FVC ratio. This study was limited by the lack of children at the
extremes of the body mass distribution (no child was w150% of the age and sex
distributions). The authors concluded that, over the range of normal to mild obesity,
BMI was a proxy for growth (as with the aforementioned muscularity effect), leading to
the associations of increases in spirometric volumes with increased BMI. They did,
however, speculate that the decrements in FEV1/FVC with increasing BMI may play an
important role in the obesitylung function relationship. Supporting this, both the Six
Cities Study longitudinal data on 9,828 children and cross-sectional data on 2,176 Italian
children also noted decrements in the FEV1/FVC ratio in association with increased BMI
[60, 61].
In a cohort of 8,484 Australian schoolchildren, Lazarus et al. [26] analysed the
relationship of both body weight and body fat measured via skinfold thickness with
spirometric lung volumes. These authors also noted marked age- and height-adjusted
increases in both FEV1 and FVC in association with increased weight, again supporting
the notion of increased growth in association with increased body mass. However, in the
evaluation of adiposity via skinfold measurements, the highest tertile of body fat was
consistently associated with lower FEV1 and FVC in this study, suggesting once again
that, in paediatric studies, BMI in normal-to-mildly obese children might not be the
optimal measure of adiposity since it cannot optimally distinguish lean tissue mass from
fat mass. Fung et al. [62] also noted increased spirometric flows correlating with
increased BMI in a population of Chinese schoolchildren. Nevertheless, overweight
children (w90% of the predicted value) showed decrements in these pulmonary measures
317
in association with increasing BMI, supporting other studies noting a classic restrictive
ventilatory defect in obese children [59].
Obesity and AHR

In addition to the effects that obesity has on static lung volumes and spirometric
measures, obesity has also been independently linked to AHR. Total respiratory
resistance has been estimated to be about twice as high in obese compared to nonobese
adults [63]. The increased respiratory resistance associated with obesity has been
suggested to result from the reduction in lung volumes [64]. Mechanistically, this is
similar to other manoeuvres known to reduce lung volumes, such as supine position or
ribcage strapping, which have also been noted to increase respiratory resistance [65, 66].
It was noted previously that FRC is reduced in obesity due to the effect of the abdominal
contents on the position of the diaphragm. Obesity has also been associated with
decrements in tidal volume [52], which fails to increase during times of dynamic stress,
such as exercise [67]. Moreover, in morbid obesity, the majority of tidal breaths are taken
at around the closing volume [68]. Decrements in FRC and low tidal volumes infer low
cycling frequencies, resulting in the conversion of airway smooth muscle from rapidly
cycling actinmyosin cross-bridges to slowly cycling latch-bridges [69, 70]. The
attainment of the latch state has been hypothesised to be the reason that obstruction
persists in asthmatic airways [69, 70]. The latch state has also been postulated to result in
increased AHR [69, 70]. Furthermore, these effects may be enhanced by breathing at
around the closing volume [70, 71]. The latch state may thus explain the observations that
decrements in FRC, as occur in obesity, have been tightly correlated with increased
airways resistance [64, 72] and responsiveness to methacholine [71].
A separate but related mechanism to the latch state results from the high frequencies
but low tidal volumes at which the obese individual breathes [51, 52]. Normally, the tidal
action of spontaneous breathing imposes tidal strains on airway smooth muscle; these
tidal strains are extremely potent natural bronchodilatory agents [73, 74]. Since the tidal
volumes of the obese individual are decreased compared to normal subjects, this
bronchodilatory mechanism is attenuated and, therefore, also predisposes to increased
AHR compared with the lean individual [75].
The effects of obesity on AHR may be accentuated in children for several reasons.
Smaller more immature airways may be more prone to the latch state [74]. Additionally,
the mechanical load of obesity may affect lung growth [76], leading to reductions in
pulmonary function with the subsequent risk of AHR. Obesity may also lead to
accelerated airway remodelling, leading to fixed obstruction. The remodelling is generally
not reversible with subsequent weight loss [77]. Finally, inflammatory pathways may be
activated in association with obesity in children, leading to an increased predisposition
towards AHR; these pathways are reviewed in greater detail in the subsequent sections.
The association between obesity and AHR has been evaluated in three large
epidemiological studies of adults. Litonjua et al. [78] reported an association between
increased BMI and incident AHR in a casecontrol study of 305 males. Specifically, the
odds ratio (OR) of developing significant AHR was 10.0 (95% confidence interval (CI)
2.637.9) for the highest quintile of body mass compared with the middle quintile.
Interestingly, there appeared to be a U-shaped relationship, with the lowest quintile also
being associated with AHR (OR 7.0; 95% CI 1.827.7). The findings of increased AHR
with increased BMI were also noted in two large cross-sectional studies of adults [79, 80].
In children, although AHR has recently been correlated with asthma severity [81], little is
known about the relationship between BMI and AHR. In a cross-sectional populationbased study of teenagers in Taiwan, Huang et al. [82] noted a decreased prevalence of
318
AHR in the lowest quintile of BMI in teenage females. These findings were not noted in
males. In their analysis of the Childhood Asthma Management Program cohort,
Tantisira et al. [27] noted an increased provocative concentration of drug causing a 20%
fall in FEV1 (decreased AHR) in association with increased BMI. However, this did not
persist after adjustment for baseline FEV1, suggesting that the relationship was primarily
mediated by increased airways growth in association with increased BMI. Two other
epidemiological studies, including a cross-sectional study of 5,993 children [83] and a
longitudinal study of 757 children [84], also noted no independent effect of BMI on
methacholine sensitivity. Nevertheless, the consistent association of obesity with
exercise-induced bronchospasm in children [85, 86] suggests that a relationship between
increased AHR and BMI may yet exist. Further study is warranted in this area.
Obesity and asthma

Interestingly, the recent increase in the prevalence of obesity has been accompanied by
a similar rise in the associated rates of asthma. Currently, an estimated 300 million
individuals worldwide are estimated to have asthma [87]. Although asthma affects people
of all ages, 50% of all asthma cases are diagnosed by the age of 3 yrs and 90% by the age
of 6 yrs [88]. Since 1980, data from the National Center for Health Statistics demonstrate
that asthma prevalence has increased by y80%, with the self-reported prevalence of
asthma in children aged 514 yrs rising from 42.8 per 1,000 population in 1980 to 74.4
per 1,000 population in 1994 in the USA [89]. Worldwide, the prevalence of asthma also
continues to rise in children and young adults [90, 91]. Although this increase has
occurred in all ages of children and young adults, it has been most pronounced in
children aged v5 yrs. Asthma is the leading cause of hospitalisation in children and the
most common reason for days lost from school [92]. Currently, medications and
healthcare utilisation for childhood asthma cost yUS$10 billion annually.
Given the dramatic rise in prevalence of both obesity and asthma, it is not surprising
that there has been an increasing body of literature relating to the association between
BMI and asthma. In the paediatric population, the frequency with which obesity was
listed as a secondary diagnosis for asthma-related hospitalisations increased from 5.9% in
19791981 to 8.1% in 19971999 [23], a rise of nearly 40%. Asthma was the most common
principal diagnosis in hospitalisations when obesity was listed as a secondary diagnosis.
Population-based subgroup analysis of asthma risk has revealed a similar story. In an
analysis of populations at risk of the development of physician-diagnosed asthma,
Rodriguez et al. [93] evaluated 12,388 children, aged 2 months to 16 yrs. The highestrisk subgroup identified by signal detection analysis was composed of children with a
parental history of asthma or hay fever aged i10 yrs with a BMI i85th percentile
(31.0% current asthma).
From an epidemiological perspective, increases in BMI have been associated with
raised asthma prevalence [9499], raised asthma incidence [61, 100102], asthma severity
[103, 104] and asthma persistence [105] in children. At the extremes of BMI, a similar
increase in the prevalence of asthma has been noted with overweight [93] and obesity in
children [104]. Furthermore, this relationship may be influenced by sex. Although greater
pre-adolescent asthma incidence [106] and severity [107, 108] have generally been
associated with male children, the obesityasthma relationship appears to be a
phenomenon of female children. The risk of prevalent asthma has been demonstrated
to be higher in obese female children than nonobese female or male children of any body
mass in cross-sectional studies of German [109] and UK [94] children. Additionally,
increases in BMI have been associated with incident wheezing, peak flow variability and
bronchodilator response in school-aged females but not males [110]. Finally, although
319
not well studied in children, in adult asthmatics, both medical and surgical weight loss
have been consistently associated with dramatic improvements in lung function, asthma
symptoms and medication usage [111114].
Five large epidemiological studies relating obesity to asthma are summarised in
table 1. Although a few studies have failed to demonstrate a relationship between BMI
and asthma [83, 115], in general, significant correlations between increased BMI and
asthma have been reported. Three interesting points have been noted in these studies.
First, one study demonstrating a significant relationship between BMI and asthma did
not note any associations between body fat, as measured by skinfold thickness, and
asthma. [94]. Secondly, two studies reported a stronger association between BMI and
asthma in female than in male children [61, 94], with one reporting a possible J-shaped
relationship between change in BMI and asthma in male children that may help to
explain the discrepancy between the sexes (fig. 1). Finally, two studies have noted that the
BMIasthma relationship appears stronger in nonatopic than in atopic children [99, 102].
These latter two points indicate possible mechanistic bases for the obesityasthma
relationship.
Obesity and OSA

In adults, obesity is clearly the single largest risk factor for the development of OSA
[116]. It is important to acknowledge the role of obesity in the development of OSA in
children. In children, hypertrophy of the adenoids and tonsils is seen as the most
prevalent cause of OSA [117]. However, of children diagnosed with OSA, 1023% have
Table 1. Selected association studies of obesityasthma in children
Study [Ref.]
Country
NSHG [94]
UK
NHANES III [99]
USA
Subjects n Age yrs

14908
411
Design
Parameter
OR/RR (95% CI)
Cross-sectional
Asthma
Persistent
wheeze (all)
Persistent wheeze
(females)
Persistent
wheeze (males)
Ever asthma
Current asthma
Recent wheeze
Current asthma
1.28 (1.111.48)#
1.57 (1.182.07)#
7505
417
Cross-sectional
Western Australia Australia

Pregnancy
Cohort [101]
CHS [102]
USA
2860
Cross-sectional
3792
718
Prospective:
5-yr follow-up
Six Cities [61]
9828
614
Prospective:
5-yr follow-up
USA
Obese
Overweight
Overweight nonatopic
Overweight atopic
Highest BMI (females)
Highest BMI (males)
Weight gain (females)
Weight gain (males)
2.07 (1.333.24)#
1.29 (0.891.86)#
1.77 (1.442.19)}
1.98 (1.542.53)}
1.48 (1.241.76)}
1.83 (1.342.52)z
1.86 (1.143.06)
1.60 (1.08 2.36)
1.52 (1.142.03)
1.77 (1.262.49)
1.16 (0.632.15)
2.24 (1.144.40)
1.04 (0.601.82)
3.11 (1.556.24)##
3.81 (1.887.72)##
OR: odds ratio (for asthma/wheeze groups shown); RR: relative risk (of physician-diagnosed asthma in obese/
overweight subjects); CI: confidence interval; NSHG: National Study of Health and Growth; NHANES: National
Health and Nutrition Examination Survey; CHS: Childrens Health Study; BMI: body mass index. #: BMI at or
above 90th percentile versus BMI at or below 10th percentile (OR); }: BMI at or above 75th percentile versus BMI
at or below 25th percentile (OR); z: overweight versus normal (OR); : obese versus overweight (OR); : highest
versus lowest quintile (RR); ##: highest versus third quintile (RR). Males in the lowest quintile also showed an
increased RR (2.76 (95% CI 1.345.67)), supporting a J-shaped relationship between weight gain and asthma.
320
a)
b)
9
8
6
5
2
1
3
4
5
Annual change in BMI z-score quintile
4
n
RR (95% CI)
2
1
3
4
5
Annual change in BMI z-score quintile
Fig. 1. Relative risk (RR) of incident asthma with persistent wheeze by quintile of annual change in
body mass index (BMI) z-score in a) male and b) female children. The reference group is the third quintile.
Although change in BMI is associated with a J-shaped effect on the RR of asthma in males, there appears to
be more of a traditional threshold effect on the RR in females. Reproduced from [61] with permission.
obesity as a primary risk factor [118]. Population-based studies of OSA generally indicate
that snoring and other symptoms of sleep-disordered obstructive breathing are two to
three times more common in obese than nonobese children [116]. In a study evaluating
risk factors for polysomnographically diagnosed OSA in paediatric family members of
known probands with OSA versus family members of neighbourhood controls, Redline
et al. [119] reported that a significantly higher proportion of children of the adult
probands with OSA were obese (11.7 versus 4.8%; p=0.03). Both BMI as a linear term and
presence of obesity were strongly associated with risk of OSA. The adjusted OR for risk
of OSA given obesity in this paediatric cohort was 4.69 (95% CI 1.5914.15) [119]. In a
retrospective review of polysomnographic data from 90 paediatric subjects, obese
children showed higher apnoea/hypopnoea indices and, concomitantly, higher systolic
and diastolic blood pressure measurements [120]. This suggests an interactive role for
obesity with OSA in the development of hypertension. OSA in children may also be an
independent risk factor, in combination with obesity, for the development of asthma
[121]. In addition to classic OSA, obese children may also suffer from obesity
hypoventilation syndrome, which comprises obesity, chronic daytime hypercapnia and
hypoxaemia, polycythaemia, hypersomnolence and right ventricular failure [37]. Obesity
hypoventilation syndrome is commonly known as Pickwickian syndrome [122], and,
although this term was coined in reference to an overweight boy with excessive somnolence
described by Charles Dickens in the Pickwick Papers [123], the precise incidence and
prevalence of obesity hypoventilation syndrome has not been studied in children.
Physiological and epidemiological associations of obesity with respiratory disease

Much can be learned about the effects of obesity on pulmonary function in children
from the classic physiology studies performed in adults. In obese adolescents and
morbidly obese children of any age, the primary changes noted include decrements in
the FRC. FEV1, FVC and TLC decrease with increasing degrees of obesity. In turn,
decrements in FRC and low tidal volumes may be associated with the latch state, leading
321
to increased AHR. Although it can be assumed that the changes due to increased work
of breathing and ventilation/perfusion mismatch should be similar to those of adults,
decrements in DL,CO accompanying childhood obesity have yet to be explained and
differ from those in adults. Although BMI is a reasonable proxy for adiposity (but not
for fat distribution) in adolescents and adults, increasing BMI has been consistently
associated with increasing spirometric volumes in younger children, indicating
that BMI may be more of a proxy for somatic growth. Other measures of adiposity
should be considered when evaluating potential effects of obesity on lung function in
pre-adolescent children.
There is evolving epidemiological evidence in children that obesity is associated with
AHR, asthma and OSA. However, there have been criticisms of the epidemiological
studies performed to date, including the relative paucity of studies evaluating AHR and
OSA, use of self-reported diagnosis of asthma, use of self-reported anthropometric
measures (such as weight and height), directionality of causation (with cross-sectional
data), diagnostic misclassification, inadequate adjustment for potential confounders
(such as diet or physical activity) and publication bias [124, 125]. Even in prospective
studies of overweight and asthma in early childhood, although overweight often precedes
asthma diagnoses, studies are less certain as to whether overweight precedes or tracks
with the precursors to asthma diagnosis (airway inflammation, airway reactivity or
wheeze). Nevertheless, the consistency of the associations noted, the results from
prospective studies, the reversibility with weight loss (in adults), other supporting
literature from adult studies and the identification of sound mechanistic bases for a
pathophysiological association (discussed below) lend support to the validity of an
association between obesity and AHR, asthma and obesity. The relative contribution of
obesity-related airway inflammation to obesity-associated decrements in lung function is
not well understood. Additional clarification via further research is also needed [92, 124]
in order to: 1) better define whether or not there truly is a sex-specific relationship in
children; 2) clarify the role of weight gain versus static weight measurements;
3) determine the best obesity phenotype to evaluate; and 4) gain better insights into
the role of other influences of obesity on the respiratory system.
Pathophysiological basis for the association of obesity with

respiratory dysfunction
Traditionally, the relationship between obesity and respiratory disease has been viewed
from a mechanical perspective. That is, how the effects of obesity on respiratory
compliance, FRC, closing volume and latch might affect an individuals susceptibility to
respiratory symptoms and disease. Although the mechanical effects of obesity remain
important, the adipocyte and other aspects of obesity have the potential to influence
respiratory disease through a diverse array of mechanisms. Obesity is an inflammatory
state. It may also influence sex hormone production, helping to explain any sex-specific
associations. Factors may also exist that influence both obesity and respiratory disease,
including in utero programming, genetics, diet and physical activity. Each of these
mechanisms are reviewed briefly in the following paragraphs. Although most of the
literature on this topic has evolved with primary reference to the obesityasthma
relationship, these mechanisms may influence other respiratory disorders as well.
Obesity and gastro-oesphageal reflux disease

Although primarily a mechanical effect in relation to obesity, gastro-oesophageal
reflux (GOR) is associated with a multitude of respiratory problems in children. These
322
include chronic cough, apparent life-threatening events, respiratory depression,

interstitial lung disease, recurrent pneumonias, infantile wheezing, vocal cord dysfunction and asthma [126132]. The estimated prevalence of GOR in asthmatic children
ranges 5060% [133]. Possible mechanisms for GOR-related asthma symptoms include
acid-induced bronchoconstriction, by either direct microaspiration or vagally mediated
reflex [132]. Medical or surgical therapy of GOR disease results in asthma symptom
improvement in y70% of patients [134]. Obesity has been frequently cited as an
independent risk factor for GOR and GOR symptoms [135137]. A recent meta-analysis
of studies focusing on the obesityGOR relationship yielded pooled adjusted ORs for
GOR symptoms of 1.43 (95% CI 1.161.77) for a BMI of 2530 kg?m-2 and 1.94 (95% CI
1.472.57) for a BMI of w30 kg?m-2 [138]. Mechanically, this effect may be mediated via
increased abdominal pressures, which increase the gastro-oesophageal pressure gradient
[139, 140]. Both medical [141] and surgical [137] weight loss regimens have been
associated with improvement in GOR symptoms. These findings have led to the
speculation that GOR might mediate the asthmaobesity relationship [114, 142].
Immune modification by obesity

In addition to its primary role in energy storage, adipose tissue is increasingly being
recognised as having diverse roles in the regulation of physiological and pathological
processes, including modulating inflammatory and immune responses. The role of
adipose tissue in inflammation has recently been reviewed (fig. 2) [143, 144]; salient
features with regard to respiratory disease will now be discussed.
Cytokines produced by adipocytes: tumour necrosis factor-a and interleukin-6.

Multiple studies have demonstrated associations between tumour necrosis factor (TNF)-a,
interleukin (IL)-6, IL-1b, and C-reactive protein and the obese state [145148]. Moreover,
IL-6 and TNF-a have been demonstrated to be constitutively expressed by adipocytes and
to correlate with total fat mass [149, 150].
Clearly, asthma is also a disease characterised by inflammation. Although most of the
recent focus has been on IL-4 and IL-5 as the primary cytokine mediators of extrinsic
(allergic) asthma, there is evidence that the obesity-regulated cytokines may influence
airways disease as well. IL-1b has been associated with induction of increased levels of
IL-5 from CD4z T-cells [151]. In asthma, upon exposure to allergens, the production of
TNF-a increases [152]. In turn, TNF-a increases both IL-4 and IL-5 production [153,
154]. IL-6 production is increased in asthma and has been associated with histamine,
IL-4, IL-5, TNF-a and IL-1 stimulation [151, 152, 155]. It has been postulated that IL-6
is responsible for the modulation of immunoglobulin (Ig)E production by IL-4 [156].
Finally, it has been demonstrated that IL-6 causes substantial subepithelial fibrosis in
animal models and may be a key modulator of airway remodelling in asthma [157].
Leptin. Leptin is a hormone produced by adipocytes that acts in the hypothalamus to

signal satiety and to increase metabolism. As the protein product of the putative ob gene
[158, 159], leptin deficiency is a rare cause of congenital obesity [160]. However, in the vast
majority of obese children and adults, the leptin level is elevated and correlates well with
BMI, probably due to insensitivity to endogenous leptin [161, 162]. The role of leptin in
the relationship between obesity and asthma is unknown; however, it may be related to
either leptins effect on foetal lung development, its immunological properties, its
relationship to AHR or its links with sympathetic nervous system tone. In the developing
lung, leptin stimulates surfactant synthesis in foetal lung cells [163, 164] and proliferation
of tracheal epithelial cells [165]. Mice that lack leptin exhibit markedly reduced lung size
323
Leptin and other factors upregulate

adhesion molcules on endotheial cells,
leading to monocyte transmigration
Leptin
Visfatin
Adiponectin
IL-6
MCP-1
Other factors
Leptin
Adiponectin
IL-6
MCP-1
Other factors
Increased
TNF-a-inhibits
adiponectin
TNF-a
IL-6
MCP-1
Other cytokines and chemokines
(resistin and adipsin)
TNF-a
IL-6
MCP-1
Other cytokines
(resistin and adipsin)
Obesity
Fig. 2. Adipose tissue: cellular components and molecules produced. Adipose tissue is composed of adipocytes
and the stromovascular fraction, which includes macrophages. In the nonobese subject, adipocytes produce
leptin, adiponectin, visfatin, interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1 and other factors.
Macrophages produce tumour necrosis factor (TNF)-a, IL-6, MCP-1, and other cytokines and chemokines. In
human subjects, the ultimate cellular source of adipsin and resistin seems to be the macrophage. In obesity,
leptin and possibly other factors produced by adipocytes, macrophages or both upregulate adhesion molecule
levels on endothelial cells, leading to transmigration of bone marrow-derived monocytes and, thus, an increase
in numbers of white-adipose-tissue-resident macrophages, and higher levels (q) of TNF-a, IL-6 and chemokines
compared with those in lean persons. At the same time, adiponectin production by adipocytes is reduced (Q),
possibly through upregulated local TNF-a levels. Reproduced from [144] with permission.
[166] and impaired respiratory control, especially during sleep [167]. These findings may
have direct relevance to the relationship of obesity with pulmonary function and with
OSA. Indeed, a recent study noted that leptin levels are elevated in OSA patients with
chronic hypercapnia compared with those who are eucapnic [168]. Although the smaller
lung size in the mouse model of leptin deficiency may, at least in part, be related to the
mechanical effects of obesity on total lung compliance, the overall effects of leptin on the
developing respiratory system are difficult to ignore. Moreover, given the importance of
lung size in the aetiology of asthma, understanding the role of leptin in foetal lung
development may prove to be very important [92].
From an immunological perspective, leptin is member of the IL-6 family of cytokines
[143]. In CD4z T-cells, leptin increases T-helper cell (Th) type 1 and suppresses Th2
cytokine production [169], whereas, in lipopolysaccharide-stimulated macrophages,
leptin increases production of TNF-a, IL-6 and IL-12 [170]. In relating obesity to
asthma, what is not currently known is whether the relative resistance that occurs in the
hypothalamic leptin receptors as part of human obesity extends to the leptin receptors on
Th and other cells involved in the asthmatic response. In the brain, the mechanism of
leptin resistance is via upregulation of suppressor of cytokine signalling (SOCS)-3, a
phosphatase that limits leptin receptor signalling [171]. Since SOCS-3 decreases
324
interferon (IFN)-c signalling [172], a relative resistance to leptin in T-cells would result in
the leptin immunological profile switching from Th1 toward Th2.
Leptin may also modulate AHR. Ozone-induced AHR was investigated in normal and
obese knockout mice. Obese mice demonstrated enhanced AHR to ozone (a nonspecific
trigger of AHR), with concomitant increases in ozone-induced neutrophil influx and
eotaxin release into bronchoalveolar lavage fluid [173]. Exogenous leptin administration
did not attenuate the inflammation in the knockout mice, but increased airway
inflammation in the wildtype mice. A follow-up study demonstrated a marked increase in
ovalbumin-sensitised methacholine responsiveness in leptin-infused mice compared with
saline-infused mice [76]. Serum IgE levels were also markedly increased in the leptin/
ovalbumin group compared with either the leptin/nonsensitised group or the saline/
ovalbumin group. Thus, elevations of serum leptin levels may help to explain the
relationship between obesity and AHR.
A final effect of leptin that could have important implications for asthma is its ability
to activate the sympathetic nervous system, an effect that may be related to leptins close
interaction with neuropeptide Y in the hypothalamus. The neuropeptide Y system
strongly stimulates feeding behaviour and has strong effects on energy storage in adipose
tissue [174, 175]. In obesity, elevations of leptin level are associated with increases in both
peripheral sympathetic nervous system activity and neuropeptide Y levels [175177].
Although increased catecholamine release would be expected to have an impact on lung
function, the role of neuropeptide Y in asthma is also intriguing. Serum levels of
neuropeptide Y may be increased during exacerbations of asthma [178]. Immunologically, neuropeptide Y specifically suppresses differentiated Th1 cells in their production of
IFN-c and stimulates the production of IL-4 by Th2 cells [179, 180].
Although few paediatric asthma studies have measured leptin, in a study of 102
asthmatic children (mean age 5.93.4 yrs) and 33 healthy paediatric controls, Guler et
al. [181] reported that asthmatic children exhibited significantly higher leptin levels, with
median (interquartile range) levels of 3.53 (2.067.24) ng?mL-1 in the asthmatics and 2.26
(1.264.71) ng?mL-1 in the controls (p=0.008). A second casecontrol study also showed
elevations of mean serum leptin level in 23 asthmatic children (19.35.1 ng?mL-1)
compared with 20 controls (9.81.6 ng?mL-1; pv0.001) at baseline [182]. Interestingly,
after 4 weeks of budesonide treatment, the leptin levels normalised (10.61.6 ng?mL-1).
A third casecontrol study did not note any relationship between leptin and mild asthma
in children [183]. Leptin has also been investigated in obese asthmatic children. In a
nested casecontrol study comparing outcomes of 74 very low birthweight versus 64
normal birthweight children at 12 yrs of age, leptin levels were considerably higher in the
27 overweight than in the 111 nonoverweight children (median 18.1 versus 2.8 ng?ml-1;
pv0.001) [184]. Interestingly, in the overweight children, current asthmatics showed
leptin levels that were twice as high of those of children without current asthma (median
30.8 versus 14.3 ng?ml-1; p=0.14), although this was not the case in the nonoverweight
children. Although this difference in overweight was not significant, this was probably
due to the small numbers of overweight asthmatics in the cohort.
Other hormonal, peptide and adipokine influences on obesity and respiratory

disease. Although the role of leptin has been closely evaluated with regard to obesity and
lung disease, several other factors involved in fat regulation may also affect the
inflammatory cascade and, subsequently, lung disease. In addition to neuropeptide Y,
these include vitamin D, corticotrophin-releasing factor, adiponectin and plasminogen
activator inhibitor-1. Each of these are briefly discussed in the following paragraphs.
Vitamin D is a pleiotropic hormone. In its active form as 1,25-dihydroxycholecalciferol, its inflammatory effects include augmenting the differentiation of nave
325
T-lymphocytes towards a Th2 immunophenotype [185]. Mice deficient in the vitamin D

receptor also demonstrate a Th2 cytokine profile [186]. The role of vitamin D in obesity
lies in its regulatory effects of increasing intracellular calcium levels. Increased
intracellular calcium stimulates lipogenesis and suppresses lipolysis, resulting in
increased adiposity [187]. Therefore, increasing vitamin D level could lead to both
increased adiposity and increases in the Th2 immunophenotype. However, in two large
epidemiological studies of adults, higher serum levels of 25-hydroxycholecalciferol were
associated with lower, not higher, measured body fat [188, 189]. These studies did not
measure the active form of vitamin D, however, and their serum measurements did not
account for the fat solubility of this hormone. Vitamin D has been evaluated in relation
to atopy and asthma. In murine models of airway inflammation, exogenous vitamin D
administration is associated with significantly increased levels of ovalbumin-specific IgE
as well as a shift towards a Th2 immune profile [190]. In a human cohort study evaluating
7,648 Finnish subjects from birth to age 31 yrs, regular supplementation with vitamin D
was associated with increased ORs for atopy (1.46 (95% CI 1.42.0)) and asthma (1.35
(95% CI 0.991.8)) compared with irregular or no supplementation [191]. No study to
date has evaluated vitamin D specifically in the obesityasthma relationship.
Corticotropin-releasing factor (CRF) is involved in numerous physiological and
behavioural actions, including strong anorectic and thermogenic effects. Indeed, CRF
has been proposed as a potential target for the pharmacological treatment of obesity
[192, 193]. Not surprisingly, both murine and human models of obesity are associated
with decrements in CRF level [194]. Although CRF has not been extensively studied as a
risk factor for respiratory disease, in a murine model of asthma, Silverman et al. [195]
reported that the CRF knockout mouse demonstrates marked increases in airway
inflammation, lung tissue resistance, and IL-4, IL-5 and IL-13 level. The authors
concluded that relative CRF deficiency states, as with obesity, might be associated with
an increased propensity to develop asthma.
Adiponectin is the most abundant adipokine; its primary role is the regulation of
insulin sensitivity [196]. Unlike other adipokines, adiponectin levels are depressed in
obesity and increase with weight loss [144]. Adiponectin shares structural homology with
TNF-a and inhibits production of TNF-a, IL-6, nuclear factor-kB and the endothelial
adhesion molecules, intercellular adhesion molecule-1, vascular cell adhesion molecule-1
and E-selectin [197200], while inducing IL-10 and IL-1 receptor antagonist [201, 202].
Interestingly, adiponectin inhibits the proliferation and migration of cultured
vascular smooth muscle cells [203]. Since adiponectin receptors are also present on
airway smooth muscle cells, a direct role for adiponectin in AHR has been hypothesised
[75]. Additionally, upregulation of endothelial adhesion molecules, as would be
expected with obesity, have been associated with asthma and AHR in human studies
[204207].
Plasminogen activator inhibitor (PAI)-1 is the major endogenous inhibitor of
fibrinolysis and plasmin activation. Adipocytes produce and secrete PAI-1, and levels
of PAI-1 are increased in obesity [144]. Increased PAI-1 levels, through their effects on
extracellular matrix turnover, may predispose towards AHR [75]. This has been
demonstrated in murine models of asthma, in which PAI-1 is required for the AHR
induced by lipopolysaccharide and for the collagen and fibrin deposition associated with
airway remodelling [208, 209].
In summary, it is now clear that obesity is an inflammatory condition and that the
adipocyte is active in the cellular production of multiple pro-inflammatory agents,
including leptin. Many of these inflammatory agents, in turn, have been associated with
providing the appropriate milieu for alterations in lung development, predisposition
towards AHR, dysregulation of respiratory control or susceptibility to asthma.
326
Sex-specific effects of obesity

As noted previously, although studies of pre-pubertal children have not noted a
consistent sex-related effect, the association between obesity and asthma has been
particularly strong in post-pubertal females. In a study of 16,862 children aged 914 yrs,
BMI correlated with prevalence of asthma in both males and females. Interestingly,
asthma risk was inversely related to Tanner stage in males (relative risk (RR) of 0.3 for
stage V compared to stage I) but positively related to Tanner stage in females (RR of 1.6
for stage V compared with stage I) [100]. In a longitudinal study of 1,246 children,
females, but not males, who had begun puberty and became overweight or obese between
the ages of 6 and 11 yrs were 6.8 times (95% CI 2.419.4) more likely to develop new
wheezing compared to the nonobese females [110]. This was further supported by
increases in peak flow variability and salbutamol-responsiveness in the obese females.
The sex differences noted in obese asthmatics may simply be a reflection of the
increased incidence [210] and prevalence [89, 211, 212] of asthma in adult females of any
size. This has been postulated as a primary airway size effect [210]. However, females also
appear to have a higher prevalence of AHR than males [213, 214], which persists despite
adjustment for airway size. Although the mechanisms behind these associations have yet
to be clarified, obesity may amplify these associations via the mechanical effects noted
above. Similarly, leptin levels [215217] are elevated in females compared with their male
counterparts and may portend to an enhanced inflammatory state.
One other potential reason for the sex difference noted is the sex hormone oestrogen.
Post-menopausal hormone replacement therapy (HRT) has been associated with a
significantly increased RR of incident asthma in females (1.49 for ever using HRT versus
never using HRT) [218]. In adolescent females, elevated oestrogen levels accompany
pubertal onset; in turn, pubertal onset in females may be linked to increased BMI [219,
220]. In obesity, although androgen levels are elevated, peripheral aromatisation of
androstenedione to oestrone and testosterone to oestrogen occurs within the stroma of
adipose tissue [222]. Combined with the decreased sex-hormone-binding globulin levels
found in obesity, this results in an oestrogen amplification effect on sensitive tissues [221].
During the menstrual cycle, peak oestrogen levels have been associated with increased
symptoms and decreased pulmonary function in asthmatic females [222]. From an
immunological perspective, oestrogen administration results in a shift in the
immunological reaction from a Th1 to a Th2 type [223], increases IL-4 and IL-13
production from blood monocytes [224], and increases eosinophil recruitment [225] and
degranulation [226]. These changes exemplify those typically found in asthma.
Genetic effects of obesity

It has long been known that both asthma and obesity are genetic diseases that run in
families. The heritability (proportion of phenotypic variance that can be attibutable to
genetic factors) of BMI, fat mass and percentage fat mass have been reported to be as
high as 90, 65 and 80%, respectively. Similarly, the heritability of asthma diagnosis, AHR
and FEV1 have been reported to be as high as 75, 66 and 39%, respectively. Extensive
genetic epidemiological studies individually focusing on asthma and lung function
phenotypes, as well as on obesity and its related phenotypes, have been performed in
recent years. Reviews of the genetic epidemiology of these complex genetic traits are
readily available [14, 15, 227229]. There are several ways in which obesity genes might
influence respiratory function and disease pathogenesis. First, genetic studies performed
in each of these individual disease states have revealed several candidate genes that have
been associated with both obesity and asthma. Secondly, other obesity candidate genes
327
are clustered in chromosomal regions that have also been linked to asthma and other
respiratory traits. Their close proximity may indicate increased potential for inheritance
of these two traits simultaneously. Finally, candidate genes for obesity may encode
protein products that may directly influence the asthma state, such as the adipokines and
cytokines noted in the previous section.
There are two genes for which strong associations have been found with both the
obesity and asthma disease phenotypes. These singular candidate genes encode the b2adrenergic receptor and TNF-a. The gene encoding the b2-adrenergic receptor is located
on chromosome 5q31q32, a region linked to both asthma and obesity. Polymorphisms
of the b2-adrenergic receptor are thought to be associated with specific asthma
phenotypes and responses to treatment. For instance, in asthma, the Gln27Glu
polymorphism has been found to be associated with elevated serum IgE levels [230] and a
protective effect against methacholine challenge [231]. In obesity, the Gln27Glu
polymorphism has been demonstrated to be significantly associated with overall obesity
[232, 233].
The TNF-a gene complex is located on chromosome 6p21.3, another region linked to
both asthma and obesity. The TNF-a-308 and lymphotoxin A NcoI polymorphisms, as
well as the lymphotoxin A NcoI/TNF-308*2 extended haplotype, have been associated
with asthma [234236]. The latter haplotype [237], as well as the isolated TNF-a-308*2
polymorphism [238], have also been associated with AHR. Concurrently, TNF-a-308*2
is associated with BMI [239] and obesity [240].
Genome-wide scans of asthma to date have noted several consensus regions of linkage
[241]. These regions include, in addition to 5q and 6p, portions of chromosomal areas 2p,
11q and 12q. Comparative analysis of these five chromosomal asthma linkage peaks with
those of candidate obesity genes shows considerable overlap. This further supports the
hypothesis that the underlying genetic susceptibility to asthma may be shared with that
for obesity.
In utero programming
Asthma is primarily a disease of early childhood, with 90% of all cases diagnosed by
the age of 6 yrs. There is increasing evidence that pre-natal events affect the subsequent
development of asthma [242, 243]. The idea that foetal programming can affect the
subsequent development of chronic disease was popularised by Barker and Martyn
[244], and is often referred to as the Barker hypothesis. This foetal-origins hypothesis
proposes that these diseases originate through adaptations that the foetus makes when it
is undernourished. Such diseases may be consequences of programming, whereby a
stimulus or insult at a critical sensitive period of early life results in long-term changes in
physiology or metabolism [245].
Foetal programming and birthweight have been correlated with the subsequent
development of obesity. Studies have noted that low birthweight is associated with an
increased percentage of body fat and central fat distribution in children [246248].
Increased arm fat in small-for-gestational-age babies has been noted as early as
25 months of age compared with average babies [246]. Low birthweight has also been
associated with centripetal obesity in adolescents [249]. At the other extreme of
birthweight, foetal macrosomia has also been associated with the subsequent
accumulation of excess subcutaneous fat in childhood [250] and the development of
obesity as adults [251]. One plausible biochemical link between these apparently
disparate associations is leptin. Umbilical cord leptin levels are elevated in both large-forgestational-age neonates [252, 253] and in intra-uterinely growth retarded humans [252]
and animals [254]. Thus, infants exposed to poor nutrition during the early trimesters
328
may be programmed for enhanced leptin production and subsequent adipose tissue
deposition, whereas those overweight infants exposed to high nutrition, especially late in
pregnancy, exemplify increased leptin concentrations typical of the obese adult.
Low birthweight has also been associated with decrements in pulmonary function and
asthma risk. Barker et al. [255] noted that decreasing birthweight was associated with
lower lung function and risk of death from obstructive airways disease in adults. Since
then, consistent reports of associations between an increased risk of asthma and low
birthweight have appeared [256259]. The mechanism behind this relationship may be a
compromised development of the lungs and, therefore, pulmonary function [255, 260].
The prototypical example of the relationship of foetal development to both asthma
and obesity is the Dutch winter famine of 1944/1945. Females exposed during early and
mid-pregnancy to the severe nutritional limitations imposed by the famine had offspring
of reduced birth size [261, 262]. The risk of obstructive airways disease was also increased
in those exposed to famine in early and mid-gestation, but not in late gestation [263].
Interestingly, in separate follow-up studies, obesity prevalence was higher in males
exposed to famine during early-to-mid-gestation and lower in the last trimester [264] and
in females exposed early in gestation [265].
Physical activity
Studies of the obesityasthma association have noted the expected inverse relation and
close correlation between physical activity and BMI [266, 267]. Several authors have
speculated that the obesityasthma relationship may simply be a reflection of a sedentary
lifestyle [95, 268]. The lack of full lung expansion associated with exercise may lead to
increased AHR [269, 270]. In recent studies, increased physical fitness has been
associated with decreases in the RR of incident asthma in schoolchildren [84] and in twins
discordant for the diagnosis of asthma [271]. In the study of schoolchildren, decreased
physical fitness was also significantly correlated with the subsequent development of
AHR to methacholine [84]. However, although physical activity may explain a portion of
the obesityasthma relationship, energy expenditure in leisure-time physical activities
was shown to be comparable-to-increased in asthmatics versus nonasthmatics in a large
Canadian cohort [272], suggesting that physical activity alone cannot explain the entirety
of the relationship.
Diet
The relationship between diet and obesity is an obvious one. Interestingly, obese
subjects may consume no more calories than lean controls [273]. Indeed, analysis of the
NHANES I data based on 24-h food recalls found a negative correlation between
overeating and overweight [274]. However, the type of food consumed by obese
individuals tends to be of poor nutritive value [273] and rich in total fat [275, 276]. Levels
of vitamins A, C and E, carotenes, riboflavin, pyridoxine, zinc, magnesium and n-3 fatty
acids have been noted to correlate inversely with body fat [277279], whereas levels of n-6
fatty acids correlated positively with body fat [279, 280]. Paradigms for the treatment of
obesity include decreasing total fat intake and ensuring adequate intake of vitamins and
minerals [281].
The dietary factors mentioned above may affect asthma and pulmonary function as
well, although relatively few longitudinal cohort and interventional studies have been
performed [282]. Total fat intake has been associated with the diagnosis of asthma [283,
284]. Zinc and magnesium deficiencies have been associated with asthma symptoms and
bronchial reactivity [285, 286]. Zinc deficiency may also lead to an enhanced Th2 immune
329
response [287]. Oxidant stress may enhance the inflammatory response of the respiratory
tract; therefore, much attention has been devoted to the relationship between dietary
antioxidants and asthma. Increased dietary n-3 fatty acid intake (primarily focusing on
fish oils) has been generally associated with protection from asthma, whereas n-6 fatty
acids may increase asthma risk; however, this remains controversial [282, 279290].
Although vitamins A and E, carotene, riboflavin and pyridoxine have been associated
with reduced lung function and asthma, their role remains controversial [291293]. The
role of vitamin C is more compelling. Lower vitamin C levels have been associated with
high asthma prevalence in adults [294] and children [295], increased respiratory
symptoms [296], reduced pulmonary function [297299] and increased AHR [285].
Supplementation with vitamin C has been demonstrated to decrease asthma severity and
frequency [300], exercise-induced bronchospasm [301] and AHR to methacholine [302].
Obesity mechanisms modulating respiratory disease

Although the mechanistic effects of obesity on the respiratory system remain
important, especially for morbid obesity, it is becoming increasingly clear that adipose
tissue is a vibrant system that can modulate both inflammatory processes and enhance
sex-specific effects through increased oestrogen production. The adipokine leptin has
been carefully studied, and has pleiotropic effects on lung development, respiratory
control, cytokine production, AHR and sympathetic activation. Each of these effects
may influence the pathogenesis of respiratory disease. Other adipokines may have similar
effects. Additionally, other risk factors for the development of obesity, including
genetics, in utero programming, physical activity and diet, may also directly influence
susceptibility to respiratory disease.
Conclusion: evolving policies addressing the global obesity

epidemic
Throughout the present chapter, the increasing evidence that obesity in children is
associated with the development of respiratory disease through a variety of potential
pathophysiological mechanisms has been presented. Recommendations regarding
further research in this field have been published [92]. Clearly, adequate prevention
and treatment of respiratory disorders in children warrants discussion related to global
treatment of the obesity epidemic. Furthermore, since lung function decrements, AHR
and asthma in adults may have a primary aetiological basis in childhood, early
intervention in the obese child may prevent subsequent morbidity as adults. The World
Health Organization has recognised obesity in general, and childhood obesity in
particular, as a substantial health problem. Key elements of their recently published
"WHO global strategy on diet, physical activity and health" [303] include: 1) achieving
energy balance and a healthy weight; 2) limiting salt, sugar and total fat intake and
shifting consumption towards polyunsaturated fats; 3) increased consumption of fruits,
vegetables, legumes, and whole grains and nuts; 4) engaging in daily moderate physical
activity for i30 min; 4) creating supportive policies that promote education and
availability and accessibility of low-fat high-fibre foods and that provide opportunities
for physical activity; and 5) addressing prevalent obesity through clinical programmes
and staff training.
Other national panels have made similar recommendations, with an emphasis on early
recognition and prevention of obesity, given the recognised difficulties in treating
established obesity [304]. Many of these panels emphasise the importance of identifying
330
at-risk children who might benefit most from early intervention. Strategies for reducing
chronic respiratory diseases by reducing childhood obesity will require culturally relevant
social as well as individualised approaches. The relative success of public health
community-based approaches in reducing obesity or persistence of wheeze needs to be
formally evaluated, so that the lessons learned from these efforts can be applied to future
public health programmes. Through these individual, school, community and globally
based efforts, the hope of ameliorating obesity-related morbidity in children, including
respiratory disorders, may become a reality.
Summary
The incidence and prevalence of obesity are increasing rapidly in children throughout
many parts of the world. Among the many sequelae of childhood obesity, respiratory
complications have been largely underappreciated. Body mass index (BMI) remains
the primary means of evaluating obesity in children and adults, although some
criticisms have been directed at this measure. Epidemiological and human
physiological studies have noted associations between childhood obesity and lung
function. Although morbid obesity is associated with the traditional restrictive defect,
increases in BMI have been associated with decrements in the forced expiratory
volume in one second/forced vital capacity ratio. Obese children also show decrements
in functional residual capacity and the diffusing capacity of the lung for carbon
monoxide, this latter observation has not been noted in obese adults. Obesity has been
associated with incident and prevalent asthma. Although obesity has also been
associated with increased airways responsiveness in adults, studies in children have
yielded conflicting results. Obesity remains a major risk factor for obstructive sleep
apnoea in adults. Although mechanical effects have been traditionally thought of as
the pathophysiological basis for the obesityrespiratory disease link, there is
increasing evidence that obesity may influence the lung via inflammatory, genetic,
sex-specific, developmental and dietary mechanisms. Of greatest current interest to the
research community is the diverse array of inflammatory processes that accompany
increased adiposity. Given the evidence from both human association studies and the
literature surrounding the mechanisms behind these associations, it is becoming
evident that adequate prevention and treatment of respiratory disorders in children
will include community, national and global strategies for addressing the obesity
epidemic.
Keywords: Asthma, body mass index, children, inflammation, obesity, respiratory.
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344
CHAPTER 18
New approaches to the understanding of

complex chronic lung diseases
U. Frey*, G.N. Maksym#, M. Silverman}, B. Sukiz
*Paediatric Respiratory Medicine, Dept of Paediatrics, University Hospital of Bern, Bern, Switzerland.
#
School of Biomedical Engineering, Dalhousie University, Halifax, NS, Canada. }Institute for Lung
Health, Leicester University, Leicester, UK. zDept of Biomedical Engineering, Boston University, Boston,
MA, USA.
Correspondence: U. Frey, Paediatric Respiratory Medicine, Dept of Paediatrics, University Hospital of
Bern, Inselspital, 3010 Bern, Switzerland. Fax: 41 316329484; E-mail: urs.frey@insel.ch
The core physiological functions of the cardiopulmonary system are to facilitate gas
exchange and to supply the tissues of the body with oxygenated blood. Exchange of
oxygen and carbon dioxide occurs in the lungs at the level of the alveoli. Fresh air flows
into the lung through the three-dimensional branching structure of the airways and
diffuses across the thin walls of the alveolar membrane into the blood. Breathing itself as
well as airway function is influenced by neuroregulatory control. The structure as well as
the function of the cardiopulmonary system is complex, and includes subsystems such as
those involved in host defence, immune mechanisms and inflammation, which are
themselves inhomogeneous and irregular. Even more important, all of these subsystems
do not function independently of each other but are highly interlinked, constituting a
network at various scales ranging from molecular through cellular to organ level. Further
interactions occur within the organism (for instance at neurohumoral level) and with the
external environment.
Most studies that have led to the immense advances in the understanding of the
pathophysiology of the respiratory system have followed a reductionist or analytical
approach, trying to isolate and identify specific mechanisms within this network
responsible for certain processes or disease states. This analytical approach consists of
breaking the whole into its components, in order to eventually understand the whole
through the totality of its parts. This mechanistic or reductionist philosophy began with
Galileo Galilei, Rene Descartes and Sir Isaac Newton. Despite the advances that have
resulted, the mechanistic scientific approach has significant limitations since it assumes a
certain level of predictability between any given stimulus and its resulting reaction. In
other words, the overall assumption is that, by knowing the isolated properties of every
component part, the response of the entire system to any stimulus can be predicted.
A number of phenomena in biology and medicine, however, cannot be explained by
such direct deterministic relationships. Since most biological systems are complex and
built up of a large number of components and interactions, the stimulation of a single
component may affect a large number of other components in the network structure in a
highly nonlinear manner. In such network constructs, it may even be that information
from previous stimuli is stored such that cumulative or delayed system effects may occur.
The complex chronic disease asthma provides well-known examples. Although the
important mechanisms of asthma (fig. 1a) are known, including allergic inflammation,
bronchial hyperactivity and airway obstruction, it is still poorly understood why there is
such a weak relationship between the strength of adverse exposure (trigger) and outcome,
ISSN 1025-448x.
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a)
Trigger
Inflammation
Bronchial
hyperactivity
Airway
obstruction
Symptoms
b)
Long-term
impact
Genetic background
Growth and development
Information storage
Time-varying
noisy environmental
stimuli (allergens,
infections, drugs,
pollutants, etc.)
Short-term
impact
Complex nonlinear
dynamic respiratory system
Fluctuating symptoms
of disease
Functional modules
Metabolic pathways
Short term
Memory effects
Long term
Inflammatory
Immunological
Mechanical
Neurorespiratory
control
Fig. 1. a) An analytical reductionist approach and b) a system approach to the complex behaviour of asthma.
The classical paradigm of asthma is based on the concept of an inducer or trigger causing airway inflammation,
leading to bronchial hyperactivity and airway obstruction and resulting in symptoms (a). Although this
analytical approach provides insight into the behaviour of mechanisms involved in cascades of reaction, it
cannot explain all of the phenomena in a complex chronic disease such as asthma. Systems biology attempts to
understand the behaviour of complete biological systems based on network considerations (b) [4]. This
alternative systems approach to asthma is presented as an example of a fluctuating chronic disease with multiple
components. It is proposed that, in such a complex system, the fluctuating output or symptoms are the result of
noisy environmental inputs (such as allergen exposure, infection, air pollution, etc.), but modified by many
interacting components of various subsystems (inflammatory system, immune system, lung mechanics,
neurorespiratory control, etc.), each having internal feedback loop mechanisms. Some of these feedback mechanisms
may act slowly over medium or long timescales of hours, weeks or months (e.g. the immune system), and some over
short timescales of seconds or minutes (e.g. neurorespiratory control). It is important to note that such systems store
information (memory) and can behave in a highly nonlinear manner. Thus, small perturbations may lead to
exaggerated peak responses (e.g. in fatal asthma). In this model, even genetic adaptation to environmental pressure (i.e.
evolutionary process) can be seen as a long-term effect [4]. Individual components of such systems cannot be described
in detail, but statistical techniques are available for describing their overall properties.
e.g. in fatal asthma [1], why bronchial hyperactivity may persist for months after a single
allergen exposure [2] or why people with occupational asthma may have persistent
symptoms long after they have ceased to be exposed to the relevant environment [3]. Such
behaviour cannot be accounted for by the classical paradigm of asthma being caused by a
series of events consisting of a trigger leading to inflammation leading to bronchial
hyperactivity, airway obstruction and finally symptoms (fig. 1a).
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UNDERSTANDING COMPLEX CHRONIC LUNG DISEASES
The present authors propose that this and other clinical examples suggest that, during
the course of asthma, and possibly also under healthy conditions, the respiratory system,
with its multitude of components and interactions, exhibits stochastic behaviour coupled
with complex nonlinear dynamics, leading to substantial fluctuations in physiological
and clinical variables. The dynamic properties of this system are strikingly analogous to
other networks in physics and biology. The current state of the system is not independent of
its history, implying that the temporal patterns of such systems exhibit memory effects.
Previous events often have a long-lasting effect, as evidenced by long-range correlation [5, 6].
Understanding the functioning of such systems and predicting certain events, such as
an asthma attack, are not possible within the realm of reductionism and require a
probabilistic systems approach, which is the main topic of the present chapter.
State of the art

In the present chapter, examples of recent research based on a comprehensive systembased approach are presented. Specifically, an overview of the behaviour of the lung in
the framework of network phenomena is provided, and it is demonstrated that its
fluctuating temporal physiological behaviour can be an expression of a homeokinetically
regulated dynamic nonlinear system.
Lung structure
Fractal airway network structure: implications for disease progression. The threedimensional structure of the airways is a well-known example of a complex fractal
structure. Fractals are self-similar structures, in which magnified subparts resemble the
entire structure (fig. 2a) [7]. With regard to the airway tree, the self-similarity is manifest in
a branching pattern that repeats itself over multiple length scales [810]. In such a fractal
branching network, there is a constant scaling relationship at each generation
characterised by a single number, the fractal dimension. Self-similar structures such as
the bronchial tree exhibit so-called power-law properties (fig. 3). Such scale-invariant
power-law statistical properties are typical of many network structures [1114].
Many organs exhibit a fractal-type structure, since the distribution of blood flow [9,
15] and airflow [10] are optimised and coupled [16] in the lung. Although fractality is
optimal for lung ventilation [10], it also shows some unexpected peculiarities in diseases.
For example, it has recently been shown that the fractal dimension of the airway tree
decreases significantly in fatal asthma compared with the nonasthmatic state, implying
reduced complexity of the airway structure due to long-term remodelling of the lung [17].
The structural organisation of this fractal network has important consequences for
physiology and medicine [18]. In the normal lung and under natural breathing
conditions, all airways are open, providing only airflow resistance to breathing. The
airways and alveoli are coated with a thin liquid layer stabilised by surfactant. When the
outward elastic tethering forces become smaller than the inward surface-tensiongenerated forces, segments in the airway tree close by either developing a liquid bridge or
compliant collapse [19, 20]. Thus airway segments can develop closure when the lung
volume is reduced below the so-called closing volume. In diseases such as asthma, airway
closure may develop during normal breathing [21], due to the additional force generated
by the contractile apparatus of airway smooth muscle cells. If the closed segments do not
reopen during inspiration, then ventilation and gas exchange may be impaired, leading to
potentially severe consequences. The reopening of closed airway segments during
inspiration occurs in avalanches (fig. 4), and the size distribution of these avalanches
follows a distinct distribution function, a power law (fig. 3) [22]. The characteristic
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U. FREY ET AL.
a)
Decreasing timescale
b)
Time
Fig. 2. Fractal properties of a) a structure, the bronchial tree and b) a time series, a fluctuating physiological
signal. The bronchial tree is typical of a fractal structure in which the geometry is similar over different length
scales or generations. The mean diameter as a function of length scale follows a power law (fig. 3). The diameter ratio
between successive generations is constant (scale invariance). In an ideal fractal time series, the fluctuations at different
timescales are similar. In biological time series, this similarity can only be described on a statistical level.
feature of a power-law distribution is that its tail is very long (note the logarithmic scale
in fig. 3), compared with familiar distributions such as the Gaussian distribution. Since
the tail of a power law can be orders of magnitude larger than the tail of a Gaussian
distribution, the probability of a large or rare event occurring in a power-law model is
also orders of magnitude higher. Most distributions have a characteristic value (or scale),
such as the value corresponding to the peak of the Gaussian distribution. Power-law
348
Log probability
l
l
l
l
l
100
101
102
103
Log category scale
104
105
Fig. 3. Power-law distribution. Power-law statistical distributions are fundamentally different from normal
(Gaussian) distributions, since they do not have a characteristic value (or scale) that is largely preferred over
others. A power-law distribution can be described by a single exponent (slope b), which is similar at all scales.
Examples include bronchial generation and properties of a times series; scales include amplitude of signal, interevent interval and airway diameter of a lung structure etc.
a)
b)
Fig. 4. Patchy distribution of airway closure. During the slow inflation of a collapsed lung, airway segments
do not open steadily but in discrete steps (white: open segments; red: closed segments). Entire segments fill with
air in an avalanche-type manner as soon as a critical opening pressure is reached. The size distribution of the
avalanches follows a distinct distribution function, a power law. The behaviour can be mimicked by a threedimensional model of the airway tree a) before and b) after an avalanche suddenly opens many segments.
Reproduced from [23] with permission.
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U. FREY ET AL.
distributions, however, do not have a characteristic value that is largely preferred over
others and are said to be scale-free [18]. An important feature of power-law distributions
is thus the similar statistical properties at different scales.
The bronchial tree structure also has an enormous effect on other phenomena closely
related to asthma. For example, not only airway opening but also the process of closure
can occur in avalanches. In a lung with small ventilation inhomogeneities, bronchoconstriction does not take place homogeneously in all airway segments. Although, it has
been known since the mid-1970s that the distribution of ventilatory inhomogeneities in
asthma is nonhomogeneous [24], Venegas and co-workers [25, 26] recently demonstrated, using positron emission tomographic imaging, that, once smooth muscle
activation reaches a critical level, localised clusters of poorly ventilated airways can
develop abruptly in discrete steps. These steps are called catastrophic shifts or avalanches
and lead to a new stable condition. As a consequence of the fractal network structure,
with its elastic interactions through the parenchyma, initial small ventilation
inhomogeneities lead to self-organised patches of poorly ventilated lung during
bronchoconstriction. Thus, an airway cannot close or open without influencing
neighbouring airways, and the constriction of a single airway has consequent effects
on other airways. This study is fascinating, since it offers a new understanding of asthma
attacks on a structural level. In asthma, the airways in the lung are likely to be close to
the local critical closing threshold pressure, which means that a small additional stimulus
can cause a catastrophic avalanche with severe impairment of lung function. Such
threshold-based mechanisms are highly nonlinear in nature, which may offer an
explanation for the poor relationship between trigger and outcome in asthma in general
and fatal asthma attacks in particular, as noted previously.
Lung tissue network structure: implications for disease progression. In the lung, it is
not only the airway tree that forms a network. The elastic structure of the lung
parenchyma can also be understood as a network, and the development of disease
progression in emphysema can be better understood using network considerations.
Emphysema, one type of chronic obstructive pulmonary disease (COPD), is a disease of
the elastic fibre network of the lung tissue, which is slowly destroyed over a period of years
[27, 28]. High-resolution computed tomography (HRCT) is a sensitive method for
examining lung structure and its alterations in COPD [29]. Zones of lung emphysema,
represented on HRCT as areas of low attenuation, do not develop homogeneously, but in
clusters of widely different sizes (fig. 5). The statistical distribution of the cluster sizes
again follows a power law, which may imply that the stress distribution within the fibre
network of the lung might also follow a power law. Mishima et al. [29] demonstrated that
disease progression in emphysema is consistent with the breakdown of a network
comprised of elastic springs, and Suki et al. [30] showed that, for this process to be
consistent with the computed tomography images, the breakdown has to be governed by
mechanical forces. Following the failure of an elastic element, stresses are redistributed
within the network. Consequently, neighbouring elements can become overloaded and
fail, leading to an avalanche of failure. Indeed, the condition of COPD subjects can
suddenly and irreversibly deteriorate following an exacerbation episode [31]. Thus, it
becomes obvious that disease progression is not a linear sequential process but can
happen in discrete steps or sudden deteriorations without any obvious trigger, which then
result in a sudden rearrangement of the forces within the fibre network.
Lung function
Temporal fluctuations in lung function variables in disease. The most important
feature of a linear system is that the overall effect of the superposition or sum of two inputs
350
Fig. 5. Emphysematous clusters in chronic obstructive pulmonary disease: computed tomographic image of the
lung of an emphysematous patient. Colour clusters represent contiguous areas of low attenuation, where gas
exchange is already compromised. Note the significant spatial heterogeneity and considerable size variability of
the colour clusters. The size distribution of these clusters also follows a power law, and the exponent is sensitive
to emphysema progression. Reproduced from [29] with permission.
is merely the superposition of their respective separate outputs. The situation is drastically
different in nonlinear systems since even a small input acting on a nonlinear system in the
presence of another input can induce dramatic and unexpected changes in the system, with
large subsequent fluctuations in its output variables [32]. This is perhaps one of the
reasons why it is so difficult to describe the general properties of complex nonlinear
systems [33]. Furthermore, in a spatially extended nonlinear system, such as a complex
network, there can be multiple inputs acting at different sites and scales. Often one or
more of the external perturbations are not known. They too may fluctuate widely. An
example is the effect of day-to-day or seasonal variations in pollen count, pollution levels
or infections when coupled with immune mechanisms, inflammation, and the neurological
control and mechanics of breathing, which can have unpredictable effects on the clinical
status and well-being of asthmatics. The results are significant short- and long-term
fluctuations in the output variables of the system (airway obstruction, wheeze and
dyspnoea, etc.). Even if all of the inputs are known, since physiological systems are rarely
351
U. FREY ET AL.
deterministic, repeated observations always lead to variability, even under the most
carefully controlled experimental conditions. In order to characterise these fluctuations, it
is therefore necessary to use statistical methods.
The use of statistical methods simply to reduce output measurement noise is a narrow
objective. Many studies have demonstrated that the most important information
characterising complex nonlinear systems is embedded in their output fluctuations [5, 22,
34]. Indeed, when certain physiological variables are measured continuously, it has been
found that the healthy state is marked by high degrees of variability, and disease states
are characterised by a reduction in variability, as, for example, first described for cardiac
frequency in adults [33, 36] or during foetal distress [37]. The nature of the information
hidden behind short-term variability and long-term fluctuations in airway function is
explored below.
The origin of fluctuations in airway function. If short-term variability in airway

function (over a timescale of seconds) and long-term variability (over days and weeks)
were proportional, this could imply a single, but complex, overall mechanism acting
similarly on all timescales. This has not been investigated systematically other than some
preliminary evidence which does not support a proportional relationship [38]. Thus, it is
more likely that there are multiple processes underlying airway function variation. For
example, if it is assumed that the fluctuations are the expression of a nonlinear dynamic
network with memory, the effects of memory may occur due to a number of parallel
feedback processes, with different response times keeping the process within limits. Since
chemoreceptors and stretch receptors have very short response times (seconds) with
respect to airway function, whereas immunological, inflammatory and remodelling
processes can have response times over minutes, hours, days or years (fig. 1b), the overall
fluctuations may be influenced by integrative inputs of all of these feedbacks and external
triggers superimposed on a given genetic background. Such a system is bound to be highly
adaptive to external perturbations and new conditions which keep the system within
limits. Both a lack of variation and excessive variation can be abnormal. For the healthy
physiological system, the term homeokinesis has been proposed to describe such
behaviour [39]. In summary, the present authors propose the model shown in figure 1b as
a starting point for considering chronic diseases, such as asthma, based on a systems
biology approach, and illustrate how information regarding disease severity can be
extracted by analysing the statistical fluctuations and memory effects of system variables.
Short-term fluctuations and variability in respiratory airway function. Control of

breathing and the structural interactions between lung volume and airway patency are
likely to dominate the fluctuations in airway function over very short timescales of
seconds to minutes. Based on such considerations, Que et al. [40] proposed an interesting
model. They postulated that not only airway calibre but also many other controlled
systems show nonrandom, nonperiodic systematic variations with time. Systematic
variations in airway calibre mean that there are systematic variations in dead space. This,
combined with variations in tidal volume, results in breath-by-breath variations in carbon
dioxide elimination and oxygen uptake. These are propagated into systematic variations
in oxygen pulse and acidbase balance and so on. Fluctuations in one parameter lead to
similar fluctuations in others. Indeed, variability analysis related to respiratory
physiology has been performed based on various signals, such as tidal breathing [41],
end-tidal oxygen and carbon dioxide signals [41], inter-breath intervals [42] and foetal
breathing [43, 44]. In tidal breathing, fluctuations have been used to quantify control of
breathing in immature and young infants [42, 45].
How can information from such short-term fluctuations be useful in understanding
airway disease? Variability in airway function occurs over short periods of time, and it is
352
thought that measurements of short-term variations in airway resistance may be a useful

additional diagnostic parameter in asthma [40]. Airway resistance can be measured
quickly and without disturbing the respiratory system by using the forced oscillation
technique [40, 46]. With this technique, airway resistance can be obtained over several
breaths, and the magnitude of the variation can be assessed using the sd or logarithm of
sd of several resistance measurements. Variation is elevated in asthma in measurements
obtained at frequencies of 6 Hz in adults [40] and 10 Hz in children [47]. Furthermore,
preliminary data show that administration of the bronchodilator salbutamol to
asthmatic children aged 613 yrs decreases the variation in resistance in children at 4
14 Hz (unpublished data). Variation in resistance was the only measure that changed
significantly, since forced expiratory volume in one second, forced mid-expiratory flow
and mean airway resistance were unaltered. This implies that the variation in resistance
reflects changes in airway smooth muscle activity. Conversely, Que et al. [40] showed that
methacholine challenge led to increased variation in resistance in healthy adults.
Variation in activation of airway smooth muscle could be due to many factors, including
changes in agonist deposition, changes in relaxation following constriction and even
changes in the load acting on the muscle [48], all of which can vary throughout the airway
tree. Indeed, spatial heterogeneity of airway constriction is a well-established feature of
asthma, measured by both forced oscillation and imaging techniques [25, 47, 49]. Thus,
short-term variation in airway resistance is likely to be the result of time-varying spatial
heterogeneity of airway constriction.
However, variation in airway resistance can also occur due to other factors, such as
changes in glottis aperture and changes in the number of open airways during a breath, as
well as changes in lung volume with breathing. Indeed, lung volume is a potent
determinant of airway resistance, and thus its variation must also strongly affect
variations in resistance [50, 51]. Que et al. [40] showed that changing from the upright to
supine position resulted in a significant increase in variation in airway resistance, which
was associated with a decrease in lung volume and airway diameter. Changing lung
volume can have two effects on variations in airway resistance. A decrease in volume can
lead to unloading of airway smooth muscle, which allows any variation in airway smooth
muscle activation to translate into larger changes in airway diameter, or a decrease in
volume with no change in smooth muscle activation leads to an increased variation in
airway resistance due to a geometric effect caused by the inverse fourth power
dependence of resistance on diameter. The preliminary data mentioned above and
obtained in children show that both probably occur. The principal advantage of
measurement of resistance variation using the forced oscillation technique is that it is
simple for children to perform, requiring only a mouthpiece, and, in principle, could be
adapted to a face mask. If a 3-min series of measurements of variation in airway
resistance is related to severity of disease, then this could be a powerful clinical tool for
determining adequate therapy in asthma. However, before studies are undertaken to
assess the use of short-term variability in improving clinical outcomes, the role of lung
volume in airway diameter variation, as well as the effect of control of breathing on
airway diameter, must first be established. Nevertheless, although the direct mechanisms
may be different and the subsequent fluctuations unrelated, variation in airway function
from short to long timescales appears to be ubiquitous in asthma. Similar to physical
systems, the transition from short- to long-term variability occurs smoothly, and it is
likely that both result from the interactions between environmental fluctuations and
memory effects of the dynamic nonlinear system.
Long-term fluctuations and variability in respiratory airway function over days and
weeks. Asthma is a chronic disease of the airways with multiple long-acting influences,
such as inflammation and immunological and mechanical (remodelling) mechanisms. The
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U. FREY ET AL.
complex interactions between endogenous and environmental factors result in a highly

variable pattern of airway obstruction over time. Fluctuations in airway calibre result in
episodic symptoms of wheeze, dyspnoea or cough. Long-term fluctuations in asthma can,
for example, be seen in series of daily symptoms scores or daily lung function
measurements. Understanding and predicting such fluctuations is difficult not only
because environmental stimuli are not always recognisable and simple to quantify, but
also because the correlation between stimuli and symptoms is poor [1, 52, 53]. The lack of
a deterministic relationship between a stimulus and its outcome is an essential feature of
complex dynamic systems. In many complex systems, these fluctuations exhibit longrange correlations, which are typically scale-invariant and thus show similar statistical
properties over different timescales (fig. 2b). In the time domain, processes with scaleinvariant correlation properties are called fractal processes. It has recently been shown
that the day-to-day lung function fluctuations in chronic asthma exhibit fractal properties
characterised by long-range correlations [54]. Often fractal processes arise when signals
travel through structures which themselves have spatial fractal scaling. An example is the
HisPurkinje system in the heart [55]. In this regard, it is important to note that fractal
properties are found in both the structure and function of the respiratory system.
An example of a twice daily peak expiratory flow (PEF) series [54] of 6 months
duration is shown in figure 6a. It can be seen that PEF behaves in a highly variable
fashion but not randomly over time. In order to analyse such PEF series, the temporal
pattern was investigated in 80 asthmatic subjects [54] who had taken part in a long-term
clinical trial [56]. In particular, it was examined whether the statistical and correlation
properties of the time series of PEF recordings could be used to predict the risk of a
subsequent episode of asthma. It was found that the fluctuations in a series of daily PEF
measurements exhibited fractal-type long-range correlations. These correlation properties of the signal can be characterised by so-called detrended fluctuation analysis [5, 57],
which measures the extent to which mean variations in the past affect variation in the
present. Often the correlation function follows a power law, which allows the complex
behaviour to be characterised with a simple number, a, the exponent of the power law
(fig. 6b). An a of 0.5 represents a random uncorrelated process; the higher a is, the more
tightly correlated the time series and the greater the degree of internal memory. It was
found that the a of the PEF series correlated with asthma severity [54].
What are the consequences of these findings? In a temporal process with long-range
correlations, the amplitudes of fluctuations that have occurred in the past influence
current values. However, the influence of previous fluctuations decreases as the time
interval increases. The value of a, and hence the strength of these long-range correlations,
is influenced by the integrative contribution of inflammation and immunological and
mechanical (remodelling) mechanisms. Thus, asthma, as a chronic disease, behaves as a
complex nonlinear system with internal memory properties related to disease severity.
The clinical surrogate for this memory of asthma, embodied in the immune system,
chronic airway inflammation and structural remodelling, may be manifest as persistent
bronchial hyperactivity after a single allergen challenge [2], persistent symptoms
following stimulus removal in occupational asthma [2, 3, 58] or persistent remodelling,
and inflammation after clinical remission of asthma [52, 53]. Although it is impossible to
disentangle all of the individual components of the system and its memory properties, it is
possible to describe the overall dynamic behaviour of the disease in a comprehensive
integrative manner using a systems approach.
Internal long-range correlations are also important to the stability of the system. By
knowing both past PEFs, embodied in the memory (a and coefficient of variation of the
PEF series), it is possible to calculate the probability or risk (more precisely, the
conditional probability) that, given the current PEF, a severe episode of asthma (for
example, with a PEF v60% of the predicted value) will occur within a given time period
354
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Fig. 6. a) Representative time series of twice-daily peak expiratory flow (PEF) measurements observed over a
6-month period, showing fluctuations. Despite appearances, the fluctuations are not random but ordered,
meaning that any particular value is dependent upon previous values. Data from [54]. b) Corresponding
schematic representation of the detrended fluctuation function F(n) used to detect long-range correlation
properties. In order to calculate F(n), the time series was integrated and divided into equally sized nonoverlapping windows of length n. A linear regression line was fitted through the data in each window and the
time series were locally detrended by subtracting the regression line from the data. This removes the effects of
general trends, which could lead to spurious correlations. The F(n) was computed for each window of length as
the root mean square of the detrended and integrated time series. This calculation was repeated for different
values of n of increasing window length, and a was obtained as the slope of a straight line fit to F(n) on a
double logarithmic plot (a=0.78). A time series with no correlations has an a of 0.5. The higher the a the more
highly correlated the time series. Fractal time series are often seen in processes with biological feedback
mechanisms, in which a characterises the feedback or memory property of the whole system.
(say, the next month). Using such mathematical analysis, the impact of treatment has
been tested. Clinical and physiological data from the long-term clinical trial [56] were
obtained during three 6-month cross-over treatment periods: regular short-acting b2agonist, regular long-acting b2-agonist (salmeterol 50 mg twice daily), and matching
placebo. Interestingly, this internal memory was not altered by long-acting b2-agonists.
However, repetitive short-acting b2-agonists altered the correlated nature of the PEF
time series such that the time series became similar to a random process. Since a random
process is less predictable, this finding has important consequences for the risk of future
severe obstructive episodes. Although long-acting b2-mimetics significantly decreased
this risk, regular short-acting salbutamol tended to increase this risk. Although shortacting b2-mimetics are the first-line drug for on-demand relief of bronchial obstruction, it
appears that, when given regularly, they drive the internal regulation of airway tone
towards a random process and make the system less stable and hence an exacerbation
event significantly more likely. What is the potential general message of this latter
finding? The present authors speculate that short-acting regularly given pulsatile
treatment might disturb the internal homeokinesis of normal airway function. It also
reveals that, in future drug trials, it may be necessary to consider the timing effects of
regularly given drugs on the system dynamics of a chronic disease.

Alternative approaches to the respiratory system and chronic respiratory diseases have
been presented, considering the respiratory system as a complex dynamic system with
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U. FREY ET AL.
multiple network-type links and interactions and long-term memory. Such an approach
offers the possibility of better understanding the complex temporal behaviour in a more
comprehensive manner. Simple descriptors, such as the correlation exponent (a)
characterising the memory of a fluctuating disease process, may be the basis of newly
defined temporal phenotypes of chronic diseases. With regard to the stability of a system
and the predictability of the associated disease process, acute asthma attacks with severe
bronchoconstriction may be seen as an avalanche-type redistribution of energy in a
network within the lung, with unexpectedly severe consequences for gas exchange as a
result of the catastrophic closure of entire airway clusters. Minuscule triggers may cause
large nonlinear effects when the system is close to a critical condition. Multiple
immunological, inflammatory and mechanical factors acting on different timescales
contribute to the long-term memory effects in the respiratory system, which ultimately
lead to the build up of such critical conditions. Environmental exposures or endogenous
noise may then lead to unpredictable effects.

Although the systems approach may be seen as a strategy complementary to the
conventional analytical reductionist approach, it naturally opens up new possibilities and
questions.
1) Is the reductionist asthma paradigm of "trigger plus bronchial hyperresponsiveness
leads to bronchoconstriction" still valid? The systems approach adds two new
dimensions: the effects of past events memorised in the system and the nonlinear
reactions between past and current stimuli. Such considerations might also aid better
understanding of the mechanisms of symptom relapse after disease remission [52, 53].
2) Could the concept of programming [5961] of lung function in disease be better
understood within the framework of the systems approach? Could key programmers, for
example, maternal smoking during pregnancy, which affect lung growth and
development in early life, not just be understood as very long-acting mechanical
memory effects? Could immune deviation early in life not just be understood as very
long-acting immunological memory effects in the development of allergy.
3) Is it sufficient to design research studies with two-point observation based on initial
conditions and outcomes? It would be better to design studies with long-term serial
observations in order to better understand the dynamics of the disease process, which
might permit statistical prediction of exacerbations, as demonstrated in [54].
4) Could different phenotypes of a chronic disease (e.g. brittle asthma and episodic or
chronic asthma) be better defined using the systems approach? By describing long-range
correlations, stability and the predictability of future events, the overall properties of the
system can be used to classify subjects. The hypothesis that these clinical phenotypes
were important could then be tested by determining their natural history, epidemiology
and response to treatment.
5) Would parameters related to the systems approach be useful additional outcome
measures in drug trials? Outcomes could be determined more rapidly, and hence more
cheaply, as the result of intensive observations if the short-term analysis [52] predicts
longer-term responses.
6) Can the systems approach help to provide hypotheses for experimental
(reductionist) science and vice versa?
What is needed to answer these questions?

Application of the systems approach to medicine is still in its infancy, and a series of
studies are required to establish its clinical significance. However, it is important to learn
356
from other fields of research. In physics, the analysis of complex systems has been
underway since the 1960s. Interestingly enough, physicists teach that everyday clinical
experience may be more important than had been anticipated. Goldenfeld and
Kadanoff [62] write, in their review on complexity: "As science turns to complexity, one
must realise that complexity demands attitudes quite different from those heretofore
common in physics. Up to now, physicists looked for fundamental laws true for all times
and all places. But each complex system is different; apparently there are no general laws
for complexity. Instead, one must reach for "lessons" that might, with insight and
understanding, be learned in one system and applied to another. Maybe physics studies
will become more like human experience."
Perhaps the same is true for the medical and biological sciences. Even if this is not the
case for the scientific method in medicine, the approach has been used in everyday
medicine. Indeed, it is likely that experienced general practitioners would intuitively
agree with this, since every patient is treated on an individual basis. Nevertheless, this
field is open to new studies investigating old diseases within the general framework of the
systems approach.
Summary
Despite recent advances in the understanding of chronic respiratory diseases, such as
bronchial asthma, the complexity of such diseases has made it difficult to obtain a
comprehensive picture of the multiple mechanisms involved. With regard to asthma, a
large variety of genetic factors, environmental triggers, interactions between
inflammatory and repair mechanisms, structural alterations of the airways and
changes in the immune system are involved in the disease process. Several strategies
have been used to target problems related to asthma. They are mainly based on
phenomenologically characterising subgroups of patients with similarities in disease
conditions, or based on mechanisms linking genetic or pathophysiological abnormalities to clinical phenotypes. This reductionist approach can help in the
understanding of individual components or the interaction of single components of
the disease system; however, this approach often fails to cope with the overall
complexity and temporal pattern of the disease. There are still a number of
unexplained phenomena in asthma, such as unexpected fatal attacks, the persistence of
symptoms after removal of the trigger in occupational asthma or the persistence of
bronchial hyperactivity following a bronchial challenge with an allergen or viral
infections. These phenomena, including the lack of a well-defined relationship between
trigger and symptoms (attacks), point to the existence of a nonlinear relationship
among the subcomponents of the system. Additionally, bronchial hyperactivity,
allergic sensitisation or remodelling phenomena are consistent with memory effects in
the system. The analysis of nonlinear dynamic systems with memory effects has been a
challenge for several decades. This chapter demonstrates how the techniques borrowed
from statistical physics can be applied to fluctuations in physiological variables in
order to better understand asthma. It is proposed that the next steps in understanding
and treating chronic diseases in general need to utilise tools from complex systems
analysis, which should find their way into life sciences and medicine.
Keywords: Complexity, fluctuation, lung function, respiratory system, variability.
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Respiratory Diseases in Infants and Children

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Respiratory Diseases in Infants and

Paediatric respiratory medicine is a growing and continuously changing field with an

Epidemiology of respiratory diseases in

The bulk of the epidemiological literature on childhood respiratory diseases is on

Transient wheezing in infancy

Wheeze and asthma at school age

CHILDHOOD RESPIRATORY DISEASE EPIDEMIOLOGY

Asthma progression in adolescence and adulthood

CHILDHOOD RESPIRATORY DISEASE EPIDEMIOLOGY

Outlook and future questions

CHILDHOOD RESPIRATORY DISEASE EPIDEMIOLOGY

Lung development from infancy to

Tools for assessing respiratory system development and growth

Anatomical and histological studies

Lung function measurements

LUNG DEVELOPMENT INTO ADULTHOOD

Anatomical development of the respiratory system

P.J.F.M. MERKUS, A.A. HISLOP

Exposure to hormones (testosterone,

LUNG DEVELOPMENT INTO ADULTHOOD

P.J.F.M. MERKUS, A.A. HISLOP

Airway and blood vessel interaction during lung development

LUNG DEVELOPMENT INTO ADULTHOOD

Factors affecting lung growth

P.J.F.M. MERKUS, A.A. HISLOP

LUNG DEVELOPMENT INTO ADULTHOOD

Effect of sex on lung and airway development

Effects of medication on the structural and functional

P.J.F.M. MERKUS, A.A. HISLOP

Recommendations for the future

LUNG DEVELOPMENT INTO ADULTHOOD

P.J.F.M. MERKUS, A.A. HISLOP

LUNG DEVELOPMENT INTO ADULTHOOD

P.J.F.M. MERKUS, A.A. HISLOP

LUNG DEVELOPMENT INTO ADULTHOOD

Assessing lung growth and function in

LUNG GROWTH AND FUNCTION

Developmental aspects of assessment of lung function during

breathers, since nasal resistance comprisesy50% of total airway resistance. Furthermore,

Overview of current techniques

LUNG GROWTH AND FUNCTION

LUNG GROWTH AND FUNCTION

Models and concepts of (patho)physiological mechanisms in the

LUNG GROWTH AND FUNCTION

Early lung disease

LUNG GROWTH AND FUNCTION

LUNG GROWTH AND FUNCTION

Development of the respiratory system

Dynamic behaviour of the developing respiratory system

LUNG GROWTH AND FUNCTION

LUNG GROWTH AND FUNCTION

LUNG GROWTH AND FUNCTION

Remodelling in paediatric respiratory

D.N. PAYNE ET AL.

Table 1. Features of airway wall remodelling in asthma

characteristic feature of asthma in adults, in both school-age and preschool children,

REMODELLING IN INFANTS AND CHILDREN

Current limitations of knowledge

D.N. PAYNE ET AL.

What have been the most recent fundamental developments in

REMODELLING IN INFANTS AND CHILDREN

D.N. PAYNE ET AL.

RBM thickening occurs early and is nonprogressive in children with asthma

REMODELLING IN INFANTS AND CHILDREN