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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
ix
CHAPTER 1
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.
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.
noteworthy that the incidence of wheeze and asthma after adolescence was strongly
associated with taking up active smoking.
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.
References
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Johnston SL. Overview of virus-induced airway disease. Proc Am Thorac Soc 2005; 2: 150156.
Lemanske RF Jr, Jackson DJ, Gangnon RE, et al. Rhinovirus illnesses during infancy predict
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Tager I, Ngo L, Hanrahan J. Maternal smoking during pregnancy. Effects on lung function during
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Hanrahan J, Tager IB, Segal MR, et al. The effect of maternal smoking during pregnancy on early
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Shaheen SO, Barker DJ, Shiell AW, Crocker FJ, Wield GA, Holgate ST. The relationship between
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Johnston ID, Strachan DP, Anderson HR. Effect of pneumonia and whooping cough in childhood
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Williams HMK. Prevalence, natural history, and relationship of wheezy bronchitis and asthma in
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Kurukulaaratchy RJ, Matthews S, Arshad SH. Defining childhood atopic phenotypes to
investigate the association of atopic sensitization with allergic disease. Allergy 2005; 60: 12801286.
Gern JE, Brooks GD, Meyer P, et al. Bidirectional interactions between viral respiratory illnesses
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Copenhaver CC, Gern JE, Liz Z, et al. Cytokine response patterns, exposure to viruses, and
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Celedon JC, Wright RJ, Litonjua AA, et al. Day care attendance in early life, maternal history of
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Ball TM, Castro-Rodriguez JA, Griffith KA, Holberg CJ, Martinez FD, Wright AL. Siblings, daycare attendance, and the risk of asthma and wheezing during childhood. N Engl J Med 2000;
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Kramer U, Heinrich J, Wjst M, Wichmann HE. Age of entry to day nursery and allergy in later
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Illi S, von Mutius E, Lau S, et al. Early childhood infectious diseases and the development of
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Halonen M, Stern DA, Wright AL, Taussig LM, Martinez FD. Alternaria as a major allergen for
asthma in children raised in a desert environment. Am J Respir Crit Care Med 1997; 155: 13561361.
Illi S, von Mutius E, Lau S, et al. The pattern of atopic sensitization is associated with the
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Wahn U, Bergmann R, Kulig M, Forster J, Bauer CP. The natural course of sensitisation and
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Lau S, Illi S, Sommerfeld C, et al. Early exposure to house dust mite and cat allergens and the
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CHAPTER 2
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.
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.
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)
(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].
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.
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].
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.
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
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
Eur Respir Mon, 2006, 37, 2240. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph;
ISSN 1025-448x.
22
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.
M. GAPPA ET AL.
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].
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.
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].
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 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
Eur Respir Mon, 2006, 37, 4159. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph;
ISSN 1025-448x.
41
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
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.
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.
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
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.
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).
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.
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
Human
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 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
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].
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.
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
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.
60
Environmental factors,
allergens, vaccines,
antibiotics, infections,
pathogens
Defence mechanism
Mannosebinding
lectin
Oxidative stress
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.
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.
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.
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.
B. SCHAUB ET AL.
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.
71
B. SCHAUB ET AL.
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78
CHAPTER 6
79
A. CUSTOVIC ET AL.
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.
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.
a)
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
b)
1.0
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.
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.
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)
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].
a)
1.0
A. CUSTOVIC ET AL.
0.8
0.6
0.4
0.2
0.0
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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92
CHAPTER 7
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.
93
P. LESOUEF, M. KABESCH
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.
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.
P. LESOUEF, M. KABESCH
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.
P. LESOUEF, M. KABESCH
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.
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
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
P. LESOUEF, M. KABESCH
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].
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].
P. LESOUEF, M. KABESCH
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.
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.
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
P. LESOUEF, M. KABESCH
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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CHAPTER 8
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
Eur Respir Mon, 2006, 37, 108119. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph;
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
109
J. GERRITSEN ET AL.
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
GENEENVIRONMENT INTERACTION
Phenotype value
High
Low
Low
Environmental exposure
High
Fig. 2. The association between a phenotype and environmental exposure is plotted for different genotypes.
There is no geneenvironmental interaction; therefore, the curves for the three genotypes are parallel. Genotype
A: ; genotype B: - - -; genotype C: ? ? ? ?.
Phenotype value
High
Low
Low
Environmental exposure
High
Fig. 3. The association between a phenotype and environmental exposure is plotted for different genotypes.
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.
Phenotype value
High
Low
Low
Environmental exposure
High
Fig. 4. The association between a phenotype and environmental exposure is plotted for different genotypes.
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
GENEENVIRONMENT INTERACTION
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.
GENEENVIRONMENT INTERACTION
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.
J. GERRITSEN ET AL.
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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120
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
1000
900
800
700
600
500
400
s
s
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].
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].
125
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
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.
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
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].
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.
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].
130
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].
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].
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
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
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.
142
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.
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.
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
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
Eur Respir Mon, 2006, 37, 153169. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph;
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
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.
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
is preschool wheeze a single disorder or are there several distinct phenotypes of wheezing
disease?
Genotype
Environment
Clinical
phenotype
Therapy
Outcome
Fig. 3. The concept of phenotypes. "Therapy" is an environmental variable, introducing a feedback mechanism.
155
J. GRIGG, M. SILVERMAN
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
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.
J. GRIGG, M. SILVERMAN
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.
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
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
J. GRIGG, M. SILVERMAN
3
Subjects
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
J. GRIGG, M. SILVERMAN
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].
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
J. GRIGG, M. SILVERMAN
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)
(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.
J. GRIGG, M. SILVERMAN
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
170
Fig. 1. Respiratory syncytial virus (electron microscopy; courtesy of M. Kneyber, Free University Medical
Center, Amsterdam, The Netherlands).
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.
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.
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
MHC-restricted epitope
recognition
Specific antiviral
immune response
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
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].
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].
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
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
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
Correspondence: J.H. Wildhaber, University Childrens Hospital Zurich, Steinwiesstrasse 75, 8032
Zurich, Switzerland. E-mail: johannes.wildhaber@kispi.unizh.ch
191
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
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.
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.
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].
TSCHERNIG [37]
Subjects
Comments
RBM thickening,
eosinophils, mast
cells and T-cells
No dendritic cells in
tracheal mucosa in
infants dying of SIDS
RBM thickening, active
fibroblasts, mast cells
and lymphocytes
RBM thickening,
eosinophils
COKUGRAS [38]
PAYNE [39]
JENKINS [40]
RBM thickening,
smooth muscle
hypertrophy,
lymphocytes
BARBATO [41]
RBM thickening,
eosinophils
PAYNE [42]
DE
BLIC [43]
SAGLANI [44]
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
RBM: reticular basement membrane; SIDS: sudden infant death syndrome; FEV1: forced expiratory volume in
one second; IFN: interferon.
197
AZEVEDO [47]
STEVENSON [48]
BARBATO [52]
198
MAHUT [58]
Eosinophils increased
Comments
TNF: tumour necrosis factor; ECP: eosinophilic cationic protein; MPO: myeloperoxidase; IL: interleukin; NO: nitric oxide; IFN: interferon; TGF: transforming growth factor.
WARKE [57]
WARKE [56]
JUST [55]
MARGUET [54]
KRAWIEC [53]
AZEVEDO [51]
KIM [50]
MARGUET [49]
AZEVEDO [46]
Subjects
GALOPPIN [45]
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.
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].
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.
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.
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.
BHR
Asthma
Intrinsic asthma
Atopy
205
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.
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
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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CHAPTER 14
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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
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.
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
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
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A. GREENOUGH ET AL.
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].
A. GREENOUGH ET AL.
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.
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.
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.
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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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].
234
CYSTIC FIBROSIS
TZ
A. BUSH, M. GO
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.
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
236
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
237
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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.
CYSTIC FIBROSIS
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.
TZ
A. BUSH, M. GO
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.
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.
TZ
A. BUSH, M. GO
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.
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].
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CYSTIC FIBROSIS
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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.
CYSTIC FIBROSIS
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
TZ
A. BUSH, M. GO
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
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
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
247
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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
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A. BUSH, M. GO
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.
CYSTIC FIBROSIS
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.
TZ
A. BUSH, M. GO
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.
CYSTIC FIBROSIS
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.
TZ
A. BUSH, M. GO
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
254
CYSTIC FIBROSIS
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
255
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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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CYSTIC FIBROSIS
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.
CYSTIC FIBROSIS
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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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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.
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].
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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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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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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.
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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.
CYSTIC FIBROSIS
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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.
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.
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.
CYSTIC FIBROSIS
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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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.
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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
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.
277
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A. BUSH, M. GO
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290
CHAPTER 16
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.
Eur Respir Mon, 2006, 37, 291313. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph;
ISSN 1025-448x.
291
T. FERKOL ET AL.
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.
292
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].
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.
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
[38, 39]
[36]
[35]
[40]
[41]
2q32
5p15
16p12
15q1315
14q32
DNAH7
DNAH5
USH1 (?EMAP)
9q31
INVS
Xp21
RPGR
Xq26
2q21
[42]
[34]
[32]
[32]
[43, 44]
[10]
ZIC3
[45]
CFC1
[46]
[30]
HLA: human leukocyte antigen; DNAH: dynein axonemal heavy chain; EMAP: evoked muscle action potential;
GTPase: guanosine triphosphatase.
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.
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].
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)
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
[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.
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].
T. FERKOL ET AL.
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.
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.
T. FERKOL ET AL.
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.
PCD biology using electron microscopy, cilia beat analysis, immunostaining and
biochemical methods.
T. FERKOL ET AL.
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.
T. FERKOL ET AL.
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
314
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.
in association with increasing BMI, supporting other studies noting a classic restrictive
ventilatory defect in obese children [59].
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.
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.
Country
NSHG [94]
UK
USA
411
Design
Parameter
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
2860
Cross-sectional
3792
718
Prospective:
5-yr follow-up
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.
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.
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.
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].
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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CHAPTER 18
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,
Eur Respir Mon, 2006, 37, 345360. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph;
ISSN 1025-448x.
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a)
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hyperactivity
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obstruction
Symptoms
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impact
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noisy environmental
stimuli (allergens,
infections, drugs,
pollutants, etc.)
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impact
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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).
346
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.
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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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
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104
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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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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
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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.
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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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.
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.
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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