Respiratory Diseases in Infants and Children (European Respiratory Monograph)

Respiratory Diseases in Infants and Children Preface During the mid 1900s, there was a growing interest for respirator...

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Respiratory Diseases in Infants and Children

Preface During the mid 1900s, there was a growing interest for respiratory medicine in infants and children and, as such, diagnostic tools and techniques for assessment of lung function were developed. Paediatric pulmonology was further elaborated in Europe during the second part of the 20th century. The Paediatric Assembly within the European Respiratory Society (ERS) was established in 1993, 3 years after the Society was founded. Since then, the Paediatric Assembly has grown and is now the second largest Assembly within the ERS. There may be a number of factors which have influenced this increasing attention for paediatric pulmonology. The identification of specific disease entities such as cystic fibrosis, increasing incidence and prevalence of asthma, access to new and efficacious anti-asthma drugs, and the development of novel diagnostic techniques are all factors that are most likely to have contributed to the growing interest. Under these circumstances, it is a pleasure to announce the present issue of the European Respiratory Monograph, which is focused on respiratory diseases in infants and children. The Guest Editors have not attempted to cover the whole field, but rather to create an update of the important areas, as well as focusing on recent developments and future needs. As respiratory problems in childhood gradually transform into respiratory problems in adults, this Monograph is not addressed to paediatricians in particular. It concerns all who are interested in respiratory function and disorders. K. Larsson Editor in Chief

Eur Respir Mon, 2006, 37, viii. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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INTRODUCTION

U. Frey*, J. Gerritsen# *Paediatric Respiratory Medicine, Dept of Paediatrics, University of Berne, 3010 Inselspital, Bern, Switzerland. Fax: 41 316329484; E-mail: [email protected]. #Beatrix Children’s Hospital, University Medical Centre Groningen, Groningen, The Netherlands. Fax: 31 503614235; E-mail: [email protected]

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

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CHAPTER 1

Epidemiology of respiratory diseases in infants and children E. von Mutius* *Correspondence: E. von Mutius, Dr von Haunersches Kinderspital, Ludwig-Maximilian-Universita¨t Mu¨nchen, Lindwurmstrasse 4, D-80337 Mu¨nchen, Germany. Fax: 49 8951604452; E-mail: Erika.Von. [email protected]

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

Transient wheezing in infancy Wheeze is a very prevalent symptom during the first to third years of life [1]. Prospective studies in general population samples have estimated that no less than a third Eur Respir Mon, 2006, 37, 1–7. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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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 67–74 yrs. Males, in particular, showed significant reductions in forced expiratory volume in one second (FEV1) and FEV1/forced vital capacity ratio. Such deficits were more frequently found in subjects who were diagnosed with pneumonia before the age of 2 yrs than among nonaffected individuals. Likewise, pneumonia in the first year of life was confirmed as a risk factor for adult lung function in another longitudinal UK study [7]. In these subjects, followed from birth up to the age of 35 yrs, pneumonia before the age of 7 yrs was associated with reduced ventilatory function, independent of the development of asthma and wheeze. Nevertheless, it remains unclear whether childhood pneumonia causes the loss of lung function until adulthood, or whether it is merely a marker and indicator of children who already exhibit poor lung function before the commencement of disease. Such children may have had the transient form of wheeze early in life, but data to support or refute this notion are lacking.

Wheeze and asthma at school age Children who have developed asthma by school age are, to a large extent, subjects with some form of atopic sensitisation. There are, however, a significant number of children 2

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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 subject’s 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 [14–16]. 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 Children’s 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 hen’s eggs was 3

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found to be the best predictor for the subsequent development of asthma in the European birth cohort [20]. Since there is a strong association between allergic sensitisation and asthma at school age, the level of allergen exposure has been fiercely discussed as a potential determinant of the incidence of asthma. There is evidence to suggest that a higher level of allergen exposure is a risk factor for the development of atopic sensitisation, specifically to the allergen in question. For example, in the MAS birth cohort, levels of house dust mite and cat allergens in the first years of life were related to sensitisation to house dust mite and cats, respectively, at the ages of 3 and 6 yrs [21]. In contrast, the level of allergen exposure at an early age and later was not related, either in subjects with or without a family history of asthma and allergies, to the development of asthma at school age in the same European cohort [22]. These findings are corroborated by the results of the longitudinal prospective Prevention and Incidence of Asthma and Mite Allergy birth cohort in the Netherlands, in which no clear effect of early-life allergen exposure and development of recurrent wheezing was seen [23]. Finally, interventional trials significantly reducing the amount of house dust mite allergen indoor exposure, such as the Manchester study, have not demonstrated a protective effect against the development of asthma and wheeze in the active group [24]. On the contrary, an increased risk of atopy was found to be associated with these avoidance measures. Therefore, recent evidence does not support the notion that allergen exposure is a risk factor for the incidence of asthma and wheeze in childhood. However, allergen exposure, via the alleviation of sensitisation and the augmentation of allergic airway inflammation in these sensitised subjects, may contribute to the severity and chronicity of the asthmatic condition.

Asthma progression in adolescence and adulthood Still too little is known about the progression of asthma and wheeze from childhood to adolescence. Prospective studies have shown that the great majority of asthmatics lose their symptoms during puberty. A cohort study of Australian schoolchildren studied at the age of 8–10 yrs and again at the age of 12–14 yrs showed that the persistence of bronchial hyperresponsiveness into adolescence was related to its severity at school age, the atopic status of the child and the occurrence of asthma in its parents [25]. The majority of children showing a light or mild degree of airway hyperresponsiveness lost their increased response at age 12–14 yrs, whereas only 15.4% with severe or moderate hyperresponsiveness at school age were normoreactive at adolescence. Recent findings also suggest that the decline in asthma prevalence during puberty may be attributable to the vanishing disease expression of the nonatopic wheezing phenotype associated with viral infections. Indeed, the observations from the Tucson birth cohort showing that lower respiratory tract illnesses due to RSV and other viruses early in life were associated with a diminishing risk of recurrent wheeze during school years support this notion [26]. Therefore, virally associated wheezing may have a better prognosis than atopy-related asthma. The British National Childhood Development Study, a longitudinal survey of all people in England, Scotland and Wales born during 1 week in 1958, is of great interest when investigating asthma incidence throughout childhood and early adult life. Between the ages of 7 and 33 yrs, only 5% of symptomatic subjects showed persisting wheezing at all times [27]. There was, however, much remittance and recurrence of symptoms. More than half of the subjects who wheezed before the age of 7 yrs and reported wheezing in the previous year at the age of 33 yrs had been free of attacks between the ages of 16 and 23 yrs. Subjects can also completely lose their symptoms. No less than 35% of subjects wheezing at the age of 7 yrs showed complete remission after adolescence. It is 4

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noteworthy that the incidence of wheeze and asthma after adolescence was strongly associated with taking up active smoking.

Outlook and future questions One of the emerging challenges in the epidemiology of respiratory diseases in children is a better characterisation of the different asthma and wheeze phenotypes. Such improvement may be achieved through refined analysis of existing prospective data from various birth cohorts around the world. Furthermore, the identification of genetic factors underlying various mechanisms in these conditions is promising. It is already becoming apparent that, as would be expected, genetic factors are differentially related to either atopy or asthma in various populations. The rapidly increasing literature relating various genetic factors to asthma and wheeze will add to the complexity of the syndrome. However, these analyses will eventually help in the distinction between various disease phenotypes by linking the identified underlying pathways to clinical and epidemiological observations. Likewise, other basic science tools investigating immune responses, airway inflammation and remodelling, and other aspects of the pathophysiology will help to further elucidate the complex nature of this syndrome. Improved classification will also advance the ability to identify the relevant environmental determinants of the various phenotypes. Indeed, there is increasing evidence showing that the effect of a certain environmental exposure depends upon the exposed phenotype, the timing of the exposure and the underlying genetic disposition. For example, viral infections have been clearly shown to be a strong determinant of transient early wheeze in the first 1–3 yrs of life, but may exert protective effects against subsequent asthma development into school age and adolescence. Neglecting to distinguish between these phenotypes results in a blurred perception of the relevant environmental exposure. In this case, the direction of the association will, in such a scenario, depend upon the number of study subjects with either transient wheeze or ongoing asthma in that population rather than on the true relationship between exposure and disease incidence. The addition of genetic factors to the puzzle will not only help in identifying the underlying mechanisms, as discussed earlier, but, by considering both factors, namely genetic alterations and environmental exposures, and studying the importance of their interaction for the development of specified phenotypes, the true relevance of the environmental exposure will become apparent for that specific phenotype. For example, a large meta-analysis of passive smoke exposure has revealed that baseline lung function, as assessed by FEV1, is mildly, but significantly affected. The summary estimate showed a reduction in FEV1 of 1.4% of the predicted value in exposed subjects. In contrast, consideration of genetic susceptibility reveals much stronger effects of passive smoke exposure. Susceptibility to second-hand tobacco smoke exposure can, for example, be assessed by genotyping the glutathione S-transferase gene, which encodes an important enzyme in detoxification pathways. Certain polymorphisms result in a null variant with low levels of enzyme production. In subjects in whom such low levels have been identified through genetic analyses, the impact of environmental tobacco smoke exposure is much stronger. In a German cross-sectional survey, the FEV1 was reduced by w5% pred in exposed subjects and measures of small airways by i15% pred. Future directions in the investigation of asthma and wheeze during childhood years should aim at a better classification of affected subjects and a thorough analysis of the associated genetic factors and environmental exposures. The impact of their interaction on the incidence of a specific phenotype should be assessed, as well as taking the timing of the exposure into account. 5

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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 2–3 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 1. 2. 3. 4. 5. 6.

7. 8. 9.

Martinez FD, Wright AL, Taussig LM, Holberg CJ, Halonen M, Morgan WJ. Asthma and wheezing in the first six years of life. N Engl J Med 1995; 332: 133–138. Johnston SL. Overview of virus-induced airway disease. Proc Am Thorac Soc 2005; 2: 150–156. Lemanske RF Jr, Jackson DJ, Gangnon RE, et al. Rhinovirus illnesses during infancy predict subsequent childhood wheezing. J Allergy Clin Immunol 2005; 116: 571–577. Tager I, Ngo L, Hanrahan J. Maternal smoking during pregnancy. Effects on lung function during the first 18 months of life. Am J Respir Crit Care Med 1995; 152: 977–983. Hanrahan J, Tager IB, Segal MR, et al. The effect of maternal smoking during pregnancy on early infant lung function. Am Rev Respir Dis 1992; 145: 1129–1135. Shaheen SO, Barker DJ, Shiell AW, Crocker FJ, Wield GA, Holgate ST. The relationship between pneumonia in early childhood and impaired lung function in late adult life. Am J Respir Crit Care Med 1994; 149: 616–619. Johnston ID, Strachan DP, Anderson HR. Effect of pneumonia and whooping cough in childhood on adult lung function. N Engl J Med 1998; 338: 581–587. Williams HMK. Prevalence, natural history, and relationship of wheezy bronchitis and asthma in children. An epidemiological study. BMJ 1969; 4: 321–325. Phelan P, Robertson C, Olinsky A. The Melbourne Asthma Study: 1964–1999. J Allergy Clin Immunol 2002; 109: 189–194.

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10. 11. 12. 13. 14. 15.

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Sears MR, Greene JM, Willan AR. A longitudinal, population-based, cohort study of childhood asthma followed to adulthood. N Engl J Med 2003; 349: 1414–1422. Kurukulaaratchy RJ, Matthews S, Arshad SH. Defining childhood atopic phenotypes to investigate the association of atopic sensitization with allergic disease. Allergy 2005; 60: 1280–1286. Gern JE, Brooks GD, Meyer P, et al. Bidirectional interactions between viral respiratory illnesses and cytokine responses in the first year of life. J Allergy Clin Immunol 2006; 117: 72–78. Copenhaver CC, Gern JE, Liz Z, et al. Cytokine response patterns, exposure to viruses, and respiratory infections in the first year of life. Am J Respir Crit Care Med 2004; 170: 175–180. Celedon JC, Wright RJ, Litonjua AA, et al. Day care attendance in early life, maternal history of asthma, and asthma at the age of 6 years. Am J Respir Crit Care Med 2003; 167: 1239–1243. 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; 343: 538–543. Kramer U, Heinrich J, Wjst M, Wichmann HE. Age of entry to day nursery and allergy in later childhood. Lancet 1999; 353: 450–454. Illi S, von Mutius E, Lau S, et al. Early childhood infectious diseases and the development of asthma up to school age: a birth cohort study. BMJ 2001; 322: 390–395. 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: 1356–1361. Illi S, von Mutius E, Lau S, et al. The pattern of atopic sensitization is associated with the development of asthma in childhood. J Allergy Clin Immunol 2001; 108: 709–714. Wahn U, Bergmann R, Kulig M, Forster J, Bauer CP. The natural course of sensitisation and atopic disease in infancy and childhood. Pediatr Allergy Immunol 1997; 8: Suppl. 10, 16–20. Lau S, Falkenhorst G, Weber A, et al. High mite-allergen exposure increases the risk of sensitization in atopic children and young adults. J Allergy Clin Immunol 1989; 84: 718–725. Lau S, Illi S, Sommerfeld C, et al. Early exposure to house dust mite and cat allergens and the development of childhood asthma. Lancet 2000; 356: 1392–1397. Brussee JE, Smit HA, van Strien RT, et al. Allergen exposure in infancy and the development of sensitization, wheeze, and asthma at 4 years. J Allergy Clin Immunol 2005; 115: 946–952. Woodcock A, Lowe LA, Murray CS, et al. Early life environmental control: effect on symptoms, sensitization, and lung function at age 3 years. Am J Respir Crit Care Med 2004; 170: 433–439. Peat JK, Salome CM, Sedgwick CS, Kerrebijn J, Woolcock AJ. A prospective study of bronchial hyperresponsiveness and respiratory symptoms in a population of Australian schoolchildren. Clin Exp Allergy 1989; 19: 299–306. Stein R, Sherrill D, Morgan WJ, et al. Respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years. Lancet 1999; 354: 541–545. Strachan DP, Butland BK, Anderson HR. Incidence and prognosis of asthma and wheezing illness from early childhood to age 33 in a national British cohort. BMJ 1996; 312: 1195–1199.

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CHAPTER 2

Lung development from infancy to adulthood P.J.F.M. Merkus*, A.A. Hislop# *Division of Respiratory Medicine, Dept of Paediatrics, Erasmus Medical Centre, Sophia Children’s Hospital, Rotterdam, the Netherlands. #Institute of Child Health, University College London, London, UK. Correspondence: P.J.F.M. Merkus, Division of Respiratory Medicine, Dept of Paediatrics, Erasmus Medical Centre, Sophia Children’s Hospital, P.O. Box 2060, 3000 CB, Rotterdam, the Netherlands. Fax: 31 104636801; E-mail: [email protected]

The growth and development of the respiratory system has been the topic of several extensive reviews and chapters in textbooks [1–9]. Therefore, this chapter only briefly summarises established knowledge about the normal growth and development of the human respiratory system, and refers to review articles for further reading. For the effects of asthma or allergy, infections, environment, mechanical ventilation and respiratory infections on the development of the respiratory system, the reader is referred to corresponding chapters in the present Monograph. Special attention is given to the tools available for longitudinal assessment of growth, recent insight into the interaction between airway and vascular growth, and the impact of premature birth on the development of the respiratory system.

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

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

Lung function measurements Lung function measurements seem the most suitable tool for studying the development of the respiratory system longitudinally, especially because of the noninvasive nature of the techniques. However, lung function studies usually focus on assessment of airway function or patency, and are notoriously insensitive to peripheral airway function [10] and cannot detect parenchymal abnormalities (see below). Furthermore, they may be hard to interpret because they are the net result of numerous independent factors that Eur Respir Mon, 2006, 37, 8–21. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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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 7–8 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 2–4 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 [16–18] and scoring systems [19] have been developed for high-resolution computed tomographic scans, which make them attractive and interesting tools for assessing pulmonary changes during growth [14, 20]. Although providing only structural information (at relatively high lung volumes), valuable information is obtained about the peripheral structure of the lung, which can easily be missed when using lung function testing alone [21, 22]. However, because the radiation burden cannot be ignored, computed tomography is currently unsuitable for the assessment of prospective developmental studies in healthy subjects or in those with minor respiratory disease [23]. Possibly, magnetic resonance imaging studies will prove to be a suitable research tool not involving radiation.

Anatomical development of the respiratory system The formation of the human respiratory system, with its pulmonary and bronchial circulation, is the net result of a complex interaction between growth factors, hormones (sex hormones, thyroid hormones and corticosteroids), genetic factors (sex and race), nutrition (quality and quantity), exposures to insults such as environmental tobacco smoke and drugs, and physical stimuli, such as stretch, foetal breathing movements 9

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

Genetics (sex and race)

Nutrition (quantity and quality) Treatment (drugs, artificial ventilation) Pre-natal and post-natal respiratory system growth

Prematurity, SGA (IUGR)

Physical forces (stretch, foetal breathing, intra- and extrathoracic space, fluid)

Exposure to hormones (testosterone, corticosteroids, thyroid hormone) and insult (drugs, passive smoking) Fig. 1. – Schematic representation of pre-natal and post-natal factors influencing lung growth and development. SGA: small for gestational age; IUGR: intra-uterine growth retardation.

(FBMs) and amniotic fluid volume (fig. 1). Much of the integration of positive and negative signalling pathways is still unknown. Lung development has been divided into four stages: embryonic, pseudoglandular, canalicular and alveolar. The age of transition from stage to stage varies between individuals and species, and, in some species (e.g. rat and mouse), the alveolar stage can be entirely post-natal. The stages can be summarised as follows.

Embryonic stage During the embryonic stage (up to 7 weeks of gestation), the lung bud appears as a ventral diverticulum of the foregut and divides within the surrounding mesenchyme. Whether or not foregut endoderm cells transform to form the lung bud is critically dependent upon the transcription factor hepatocyte nuclear factor-3. Several reviews have addressed the involvement of this and other transcription factors [24–26]. 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

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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 (5–17 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 8–10 weeks of gestation [30–33]. 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 (16–27 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 20–22 weeks of gestation. Type II cells develop identifiable lamellar bodies at y24 weeks of gestation. However, surfactant is only detected in the amniotic fluid 4–5 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 blood–gas barrier as thin as that of the adult (y0.6 mm). This is sufficient to sustain life in extremely premature infants. 11

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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 200–600 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 2–4 yrs, after which growth occurs mainly by dimensional expansion [37].

Airway and blood vessel interaction during lung development Based on studies since the mid-1990s, there is currently a much better understanding of the interaction between the growth of the vasculature and the formation of the bronchial tree and acinus [6, 41–43]. During early foetal development, the airways act as a template for pulmonary blood vessel development in that the vessels form by vasculogenesis around the branching airways. During this phase, the epithelial cells induce angiogenesis and vasculogenesis, in which VEGF plays a crucial role [6]. Later in gestation, however, the capillary bed is essential for alveolar formation. From the canalicular stage onwards, the capillaries seem to cause epithelial cells to differentiate into type I and II pneumocytes and lead to further development of the alveoli [44]. In rodents, it was demonstrated that the absence of VEGF is associated with retarded alveolar multiplication and a reduction in capillary number [45]. In addition, when antiangiogenic factors were given to rats, they reduced the number of small arteries and retarded alveolar growth [46]. Furthermore, counteracting stimuli, such as endothelialmonocyte-activating polypeptide II, which is able to modulate the angiogenesis, are also active [47]. The disrupted capillary bed in bronchopulmonary dysplasia is associated with a decrease in levels of VEGF and its receptor [48]. A summary of the factors and pathways involved has been published previously [49]. It is important to remember that normal pre-natal lung development occurs in the hypoxic environment of the uterus. In vitro cultures of mouse lung buds demonstrated a reduction in branching rate when exposed to 20% instead of 3% oxygen [41]. Indeed, in vitro studies demonstrate that the low oxygen intra-uterine environment enhances branching of both distal lung epithelium and vascular tissue, and that pulmonary vascular development appears to be ratelimiting for epithelial branching morphogenesis [41]. Hence, abnormalities in foetal lung development affect both airways and blood vessels [43]. A typical example is congenital diaphragmatic hernia, in which there is a reduction in the numbers of arteries and airways along the main pathway, with a subsequent reduction in alveolar number. Similar abnormalities were found in renal agenesis, thoracic dystrophy and idiopathic pulmonary hypoplasia [43]. These and other data indicate that the capillary bed is 12

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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 [54–57]. With the current knowledge that concentrations of oxygen of i21% negatively affect further outgrowth of the vascular bed, and that alveolar and airway development depend on vascular development, it can be explained why premature lungs arrest their development at birth, and may have a strikingly simplified architecture [52]. Further studies are needed to assess whether this also implies that these lungs have fewer peripheral airway generations (alveolar ducts), and to what extent the alveolar surface is diminished.

Factors affecting lung growth Programming The hypothesis of programming was launched in 1991 by Barker et al. [58] to explain the associations found between early-life respiratory disease and increased respiratory morbidity and mortality in the elderly [59]. The concept of programming implies that the structure and function of organs and tissues are permanently altered in their design (programmed) by factors operating during sensitive periods of foetal or early post-natal life [59, 60]. Since then, numerous epidemiological studies have published supportive evidence for this theory. Factors that affect programming include: genetics (including factors determining sex or race); quality and quantity of nutrition; placental characteristics; maternal drugs and hormones; exposures to toxins (such as nicotine and other compounds in cigarette smoke); mechanical factors (such as amniotic fluid volume, diaphragmatic integrity, stretch and FBMs); and duration of gestation (see above). Hence, it is assumed that the growth and development of the respiratory system are largely programmed in utero [60], and there are reasons to assume that, once the basic structure of the respiratory system has been realised during this critical phase, the development of lung function and anatomy follows a more or less fixed course, and exhibits tracking well into adolescence in healthy subjects [61, 62] and those with respiratory disease [63–65]. One of the best-known and -studied determinants playing a role in the growth and development of the airways and lung parenchyma is the combined effects of space and mechanical forces, or stretch [66]. Studies of such mechanical determinants of lung growth have been reviewed in several articles [4, 5]. Foetal lung fluid volume and its maintenance are essential to normal lung growth and development. It is the balance between production by the distal airways and drainage through swallowing or release into the amniotic space which appears to be important in normal lung development [4]. 13

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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

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occurring thereafter) [82]. However, there is now evidence to suggest that the lung has more potential to recover, with reparative growth after insults to the lung and compensatory growth after volume loss, even after lung growth is supposedly complete [7]. Several factors that have been implicated in playing a role in post-natal lung growth have been used in experimental studies in an attempt to induce such growth [7]. These include all compounds or stimuli that play a role in the interaction between vasculogenesis/angiogenesis and the formation of airways and maturation of the acinus: increased oxygen demand due to various causes, mechanical strain (from movements, ventilation or surgical interventions), hypoxia, hormones (growth hormone and corticosteroids) and several growth factors (including platelet-derived growth factor, retinoic acid, VEGF and nitric oxide). Studies to date have demonstrated that enhancement of lung growth varies among species, and it is currently unclear whether enhancement of post-natal lung growth will become a realistic treatment modality in humans [7].

Effect of sex on lung and airway development At birth, male infants possess more alveoli [37] and probably exhibit a smaller airway calibre for a given body size than do females. For the same lung size, females have larger airways, resulting in higher forced expiratory flows and lower airways resistance. This has been demonstrated for infants, children and adolescents, as reviewed previously [2]; in adults, however, the opposite has been described [77, 78]. This can be explained by increased airway growth relative to volume in adolescent males compared with females during puberty [61]. These and many other studies demonstrate that the growth and development of the airways and airspaces differs according to sex and depends on age [2, 83–85]. This has significant implications for several functional characteristics, and is likely to influence the epidemiology of various respiratory disorders, respiratory morbidity, the natural course of respiratory diseases, hospital admission rate and mortality [86].

Effects of medication on the structural and functional development of the respiratory system Glucocorticosteroids administered to foetuses and (premature) infants may have beneficial as well as detrimental effects on foetal lung development [87, 88]. Most histological or structural evidence is derived from animal and/or in vitro studies. Previous studies on developing rats have shown that injection of steroids into both the mother before birth and the offspring after birth leads to attenuation of alveolar septation [89, 90]. This is due to precocious thinning of the matrix and maturation of the epithelial cells and microvasculature. Confirmation of this effect has come from studies on sheep, which have shown abnormal lung function as well as a reduction in the number of alveoli. The number of doses of steroid and the time of gestation at which they are given does not affect the outcome, but the effect is less in females [91]. Glucocorticoids are known to inhibit lysyl oxidase activity, resulting in diminished cross-linking of the collagen and elastin fibre network, and thus altering the structural integrity of the lung. This could have functional consequences for the tethering of the airways in the lung parenchyma, but studies addressing this are lacking to date. A recent study has compared the effect of inhaled and injected steroids in rabbits aged 1–5 weeks. In this species, there is still alveolar development occurring, but, as in humans, some of 15

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the alveoli have developed before birth. Rabbits received aerosolised budesonide or injected dexamethasone. The injected steroid had a more deleterious effect on body weight, lung volume, alveolar number, surface area and function than inhaled steroid. Treatment with inhaled steroid caused specific growth retardation of the lung, but was not sufficient to affect lung function. Alveolar size and number and elastin content, when related to lung volume, were not affected, suggesting normal structural development but inhibition of total growth. However, small peripheral airway walls were thinner and had fewer alveolar attachment points, with a greater distance between attachments [92]. Thus, developing lungs are sensitive to inhaled glucocorticoids, the use of glucocorticoids in young infants and children should be monitored, and only the lowest doses that yield a significant clinical improvement should be used. Although b2-agonists have been used therapeutically since the 1960s, little is known about how they affect the growing lung. Salbutamol has been shown to inhibit multiplication of human adult smooth muscle cells, and repeated or prolonged exposures inhibit DNA synthesis without evidence of desensitisation [93]. This may be an advantage where there is an excess of bronchial smooth muscle, but may be deleterious during the normal growth period, particularly during the first few months after birth. Since the 1990s, the use of anticholinergic drugs has been added, although there is no proof that it improves the response of airways in wheezy infants. Experimental evidence has shown that, in guinea pigs with increased bronchial smooth muscle, tiotropium bromide (a muscarinic receptor antagonist) reduces contractility and contractile protein expression [94]. Acetylcholine is known to increase cell multiplication, and thus muscarinic receptor antagonists may prevent this. In rats, there are a greater number of muscarinic receptors in the lungs during foetal life than later in life [95], which may be important for the development of the bronchial smooth muscle. The effect of these agents requires further investigation.

Recommendations for the future Inhaled and systemic drugs are being prescribed to large numbers of infants and children with respiratory disorders. Studies into their long-term effects on alveolar and capillary formation and the mechanics of airways and airspaces in such humans are lacking. There is a need for improved or new techniques to monitor the airway and gas exchange function of the lung tissue longitudinally, and, in particular, for new approaches to assessing the inhomogeneity of diffusion capacity and ventilation. The architecture of the peripheral lung, including the alveoli, determines airway function, and may be damaged or disrupted following pre-natal smoking, prematurity, congenital cardiac malformations, the use of drugs or various respiratory diseases. Therefore, it is necessary to improve or develop imaging techniques suitable for monitoring the development of the peripheral airways, vasculature and lung parenchyma noninvasively and with minimal radiation use. In addition, there remains a need for standardised histological morphometric studies of paediatric lungs throughout childhood. By combining all of the functional, histological, biochemical and genetic studies, progress will be made in understanding the mechanisms of lung growth and their relative importance in contributing to the function of the respiratory system.

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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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Dezateux C, Stocks J. Lung development and early origins of childhood respiratory illness. Br Med Bull 1997; 53: 40–57. Merkus PJ, Borsboom GJ, Van Pelt W, et al. Growth of airways and air spaces in teenagers is related to sex but not to symptoms. J Appl Physiol 1993; 75: 2045–2053. Hibbert ME, Hudson IL, Lanigan A, Landau LI, Phelan PD. Tracking of lung function in healthy children and adolescents. Pediatr Pulmonol 1990; 8: 172–177. Horak E, Lanigan A, Robets M, et al. Longitudinal study of childhood wheezy bronchitis and asthma: outcome at age 42. BMJ 2003; 326: 422–423. Filipone M, Sartor M, Zacchello F, Baraldi E. Flow limitation in infants with bronchopulmonary dysplasia and respiratory function at school age. Lancet 2003; 361: 753–754. Morgan WJ, Stern DA, Sherrill DL, et al. Outcome of asthma and wheezing in the first six years of life: follow-up through adolescence. Am J Respir Crit Care Med 2005; 172: 1253–1258. Smith PG, Janiga KE, Bruce MC. Strain increases airway smooth muscle cell proliferation. Am J Respir Cell Mol Biol 1994; 10: 85–90. Harding R. Fetal breathing movements. In: Crystal RG, West JB, eds. The Lung: Scientific Foundations. New York, NY, Raven Press, 1991; pp. 1655–1663. Hallak M, Moise K, Lira N, Smith EO, Cotton DB. The effect of tocolytic agents (indomethacin and terbutaline) on fetal breathing and body movements: a prospective, randomized, double-blind, placebo-controlled clinical trial. Am J Obstet Gynecol 1992; 167: 1059–1063. Mariotti V, Marconi AM, Pardi G. Undesired effects of steroids during pregnancy. J Matern Fetal Neonatal Med 2004; 16: Suppl. 2, 5–7. Maritz GS, Morley CJ, Harding R. Early developmental origins of impaired lung structure and function. Early Hum Dev 2005; 81: 763–771. Elliot JG, Carroll NG, James AL, Robinson PJ. Airway alveolar attachment points and exposure to cigarette smoke in utero. Am J Respir Crit Care Med 2003; 167: 45–49. Hofhuis W, de Jongste JC, Merkus PJFM. Adverse health effects of prenatal and postnatal tobacco smoke exposure on children. Arch Dis Child 2003; 88: 1086–1090. Stocks J, Dezateux C. The effect of parental smoking on lung function and development during infancy. Respirology 2003; 8: 266–285. Lawlor DA, Ebrahim S, Davey Smith G. Association of birth weight with adult lung function: findings from the British Women’s Heart and Health Study and a meta-analysis. Thorax 2005; 60: 851–858. Chan KN, Noble-Jamieson CN, Elliman A, Bryan EM, Silverman M. Lung function in children of low birth weight. Arch Dis Child 1989; 64: 1284–1293. Green M, Mead J, Turner JM. Variability of maximal expiratory flow–volume curves. J Appl Physiol 1974; 37: 67–74. Mead J. Dysanapsis in normal lungs assessed by the relationship between maximal flow, static recoil, and vital capacity. Am Rev Respir Dis 1980; 121: 339–342. Martin TR, Castile RG, Fredberg JJ, Whol ME, Mead J. Airway size is related to sex but not lung size in normal adults. J Appl Physiol 1987; 63: 2042–2047. Martin TR, Feldman HA, Fredberg JJ, Castile RG, Mead J, Whol ME. Relationship between maximal expiratory flows and lung volumes in growing humans. J Appl Physiol 1988; 65: 822–828. Parker AL, Abu-Hijleh M, McCool FD. Ratio between forced expiratory flow between 25% and 75% of vital capacity and FVC is a determinant of airway reactivity and sensitivity to methacholine. Chest 2003; 124: 63–69. Munakata M, Ohe M, Homma Y, Kawakami Y. Pulmonary dysanapsis, methacholine airway responsiveness and sensitization to airborne antigen. Respirology 1997; 2: 113–118. Cagle PT, Thurlbeck WM. Postpneumonectomy compensatory lung growth. Am Rev Respir Dis 1988; 138: 1314–1326. Becklake MR. Gender differences in airway behaviour (physiology) over the human lifespan. In: Buist S, Mapp CE, Rossi A, eds. Respiratory Diseases in Women. Eur Respir Mon 2003; 25: 8–25.

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84. 85. 86. 87. 88.

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Baraldo S, Saetta M. Sex differences in airway anatomy over human lifespan. In: Buist S, Mapp CE, Rossi A, eds. Respiratory Diseases in Women. Eur Respir Mon 2003; 25: 1–7. Hibbert M, Lannigan A, Ravven J, Landau L, Phelan P. Gender differences in lung growth. Ped Pulmonol 1995; 19: 129–134. Boezen HM, Jansen DF, Postma DS. Sex and gender differences in lung development and their clinical significance. Clin Chest Med 2004; 25: 237–245. Bolt RJ, van Weissenbruch MM, Lafeber HN, Delemarre-van de Waal HA. Glucocorticoids and lung development in the fetus and preterm infant. Pediatr Pulmonol 2001; 32: 76–91. Garbrecht MR, Klein JM, Schmidt TJ, Snyder JM. Glucocorticoid metabolism in the human fetal lung: implications for lung development and the pulmonary surfactant system. Biol Neonate 2006; 89: 109–119. Schittny JC, Djonov V, Fine A, Burri PH. Programmed cell death contributes to postnatal lung development. Am J Respir Cell Mol Biol 1998; 18: 786–793. Vyas J, Kotecha S. Effects of antenatal and postnatal corticosteroids on the preterm lung. Arch Dis Child 1997; 77: 147–150. Jobe AH. Glucocorticoids, inflammation and the perinatal lung. Semin Neonatol 2001; 6: 331–342. Kovar J, Willet KE, Hislop A, Sly PD. Impact of postnatal glucocorticoids on early lung development. J Appl Physiol 2005; 98: 881–888. Stewart AG, Tomlinson PR, Wilson JW. b2-adrenoceptor agonist-mediated inhibition of human airway smooth muscle cell proliferation: importance of the duration of b2-adrenoceptor stimulation. Br J Pharmacol 1997; 121: 361–368. Gosens R, Zaagsma J, Grootte Bromhaar M, Nelemans A, Meurs H. Acetylcholine: a novel regulator of airway smooth muscle remodelling? Eur J Pharmacol 2004; 500: 193–201. Pulera N, Bernard P, Carrara M, Bencini C, Pacifici GM. Muscarinic cholinergic receptors in lung of developing rats. Dev Pharmacol Ther 1988; 11: 142–146.

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CHAPTER 3

Assessing lung growth and function in infants and young children M. Gappa*, J. Stocks#, U. Frey} *Paediatric Pulmonology and Neonatology, Hanover Medical School, Hanover, Germany. #Portex Respiratory Unit, Institute of Child Health, London, UK. }Paediatric Respiratory Medicine, University of Berne, Inselspital Bernese University Hospital, Berne, Switzerland. Correspondence: M. Gappa, Paediatric Pulmonology and Neonatology, Medizinische Hochschule Hannover, Carl-Neuberg-Str.1, D-30625 Hannover, Germany. Fax: 49 5115329125; E-mail: gappa.monika@ mh-hannover.de

During the first years of life, the lung undergoes major structural and functional changes [1, 2], together with rapid growth of all structures involved, making this period of life particularly susceptible to adverse influences of environmental as well as diseaserelated factors. Respiratory morbidity remains a major challenge not only for the child and their family but also for the paediatric pulmonologist. It is unlikely that appropriate preventive measures or therapeutic interventions can be developed unless there is a firm understanding about the basic structure and function of the respiratory system, and how these change with age. Direct study of its structural development is obviously always difficult when human subjects, especially infants and children, are concerned. This implies that it is necessary to mainly rely on indirect measures, particularly lung function assessments. Structural changes in the growing lung include alveolar growth and multiplication, growth and maturation of the lung parenchyma, vascular development, growth of the airways and maturation of the airway wall structures, all of which are influenced by the simultaneous growth of the thoracic cage (fig. 1) [1, 3, 4]. Ideally, assessment of lung function would serve to describe the phenotypic consequences of developmental processes in healthy individuals, and the consequences of both intra-uterine and postnatal insults. True longitudinal assessment of respiratory function from birth through childhood, during which changes in lung volume and mechanics secondary to disease can be distinguished from those occurring with the physiological growth and development of the structures involved, would facilitate understanding of the progression and natural history of early lung disease, and the ability to monitor early changes and evaluate the effect of treatment. In order to better understand the impact of respiratory disease, as well as therapeutic interventions, on lung function at this young age, its potentially long-lasting impact with respect to its effect on lung growth and development must always be considered. Several observations during recent years have supported these concepts of early programming and tracking of lung function in health and disease, with correlations between respiratory morbidity in early infancy and adult life being clearly demonstrated (see below) [5–8]. 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 [10–12], infants born small for gestational age [13, 14] and those whose mothers smoked during pregnancy [14–16], which is not made up during later Eur Respir Mon, 2006, 37, 22–40. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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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. – Structure–function relationships in the developing lung. In addition to the structural components mentioned in the diagram, developmental changes to the chest wall and control of breathing have to be considered, which may influence all functional parameters. Disturbed development of vascular structures, alveoli and airways, as well as regional ventilation inhomogeneity, may also lead to ventilation/perfusion mismatching. Pa,O2: arterial oxygen tension; Pa,CO2: arterial carbon dioxide tension.

infancy, support this hypothesis. Early exposure to environmental insults, such as air pollution, has also been shown to alter pulmonary function into school age [17]. Furthermore, there is increasing evidence that early lung function may be predictive of lung function, and thus respiratory morbidity, in later life (the concept of tracking) [5, 8, 10, 18], and that children with cystic fibrosis (CF) may have early impairments in lung function even before there is clinically apparent lung disease [19, 20]. These studies emphasise the need to assess function during this vulnerable early period as a measure of the growth and development of the airways and lungs, in both health and disease, as a basis for understanding not only the early determinants of airway function in health but also the pathophysiology in different diseases in order to develop appropriate preventive and treatment strategies. The aims of this chapter are to: 1) provide a brief overview of the lung function techniques that are currently used to assess lung function in infants and young children, with special emphasis on the most recent developments; 2) discuss models and concepts of (patho)physiological mechanisms in the growing lung, early childhood lung diseases and appropriate treatment strategies; 3) discuss important future questions in this field; and 4) discuss what is required to find answers to these questions.

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

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breathers, since nasal resistance comprisesy50% of total airway resistance. Furthermore, the upper airways are known to play an important role in modulating expiratory flow, and thus the end-expiratory lung volume. This is necessary because the chest wall is highly compliant in infants, with minimal outward elastic recoil, such that, under passive conditions, the lungs recoil to a much lower lung volume in relation to total lung capacity (TLC), with a tendency towards airway closure at low lung volumes. The physiological mechanisms preventing the lungs from collapsing, which include a high respiratory frequency, short expiratory time, post-inspiratory braking and laryngeal modulation of expiratory airflow, often result in a variable end-expiratory level. This may impede assessment not only of lung volume but also of mechanics and forced expiratory flows, which are highly volume-dependent and hence need to be related to the lung volume at which they were measured. In addition, lung volume measured throughout the period in life during which the end-expiratory level, and thus functional residual capacity (FRC), is actively maintained will never be directly comparable to measures of resting lung volume later in life. Although the time between the second and sixth birthday used to be regarded as a black box as there were no suitable methods for testing young children during the preschool years, most tests of lung function routinely used in older children and adults have recently been successfully adapted for this age group. Although some of the issues discussed above are less relevant to this age group, rapid growth of all pulmonary structures has to be considered when measuring and interpreting lung function in both infants and very young children. From birth, growth in lung volume occurs by multiplication of alveoli until the age of y18 months [21, 22]. When multiplication is complete, further growth in lung volume occurs via the alveoli increasing in diameter and surface area. In contrast, at birth, the conducting airways are complete in number, with a subsequent two- to three-fold symmetrical increase in length and diameter through to adulthood [23]. These disproportionate developmental patterns of lung volume and airway growth are reflected in the concept of dysanapsis [24–26], and influence the rate of lung emptying in relation to lung volume with growth [23]. It must be remembered that accurate interpretation of lung function tests in both infants and preschool children, in whom pulmonary and somatic growth are so rapid, is highly dependent upon accurate recordings of height and weight at each test occasion.

Overview of current techniques As summarised in recent reviews on the assessment of lung function and the application of such tests in infants and preschool children [2, 27–36], assessment of lung function during the first years of life remains a challenge. In infants, tests require highly specialised equipment with regard to frequency response, safety, minimisation of dead space and resistance; usually have to be performed by two experienced investigators; are time-consuming and frequently require sedation. These factors, together with constraints due to parental work patterns, limit acceptability, study duration and the frequency with which tests can be repeated [37, 38]. In addition, interpretation of test results may be limited because of the lack of appropriate reference values and the difficulty in recruiting healthy control groups due to ethical constraints in many centres. Until recently, assessment of lung function in infants has, indeed, been restricted to a few specialised and research-orientated centres throughout the world. However, an international task force, with input from both the European Respiratory Society (ERS) and American Thoracic Society (ATS), has responded to increasing interest in this field by producing a series of manuscripts summarising the state of the art in infant lung function testing and 24

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proposing standards for equipment requirements and the most commonly used techniques for infant studies [37–42]. 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 y3–15 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

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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 [51–54]. 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, 54–57]. 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 7–10 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, 60–62]. 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

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been shown that spirometry can be successfully performed by the majority of preschool children [63–65]. 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 [67–69], 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, 71–77]. 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 [74–76], 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

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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 [85–87] or laser monitoring [88]. Such measurements have been performed during spontaneous sleep, as well as following challenges with gas mixtures known to influence control of breathing. Several factors influence tidal breathing pattern and waveform in infants and young children; the pattern of tidal breathing changes rapidly during early post-natal life, and is strongly influenced by equipment configuration and sleep state. Recently, newer analytical methods have been proposed which focus on the long-range fluctuations in tidal breathing signals containing information on breathing regulation [89, 90]. Such methods are promising since they consider breathing control using a more comprehensive system-dynamic approach. Of similar interest are new methods for studying the interaction between control of breathing and airway mechanics, which have currently been investigated only in older children [91, 92]. Further promising research in the field has been carried out by observing fluctuation in tidal breathing following spontaneous sighs. Sighs not only influence airway mechanics but also alter control of breathing on a short time scale [93].

Models and concepts of (patho)physiological mechanisms in the growing lung and early lung disease and treatment strategies Programming The long-observed association between childhood lower respiratory tract illness and subsequent development of adult chronic respiratory disease has been confirmed in numerous recent epidemiological studies [94–98]. The nature of this link, the biological mechanisms which mediate it, and the genetic, developmental and environmental factors which influence its expression have been the focus of considerable research effort in recent years. One concept evoked to explain this association is that of programming, the permanent alteration of the structure and function of organs and tissues by factors operating during sensitive periods in foetal or early post-natal life [95]. Factors implicated in the programming of the respiratory system that have been demonstrated via lung function measurements in infants include foetal nutrition [14, 99, 100], foetal 28

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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, 104–106]. 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 Children’s 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, 111–113]. The most recent follow-up data from the Tuscon study 29

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demonstrate that the group with the lowest lung function during infancy retained this low level throughout childhood and puberty [8]. This concept of tracking, i.e. that early lung function predicts subsequent development of function, has also been demonstrated on an individual level [5, 10, 67, 99]. Although there are some discrepancies in the literature with respect to pre-existing respiratory dysfunction and the course of subsequent clinical disease, these epidemiological studies provide evidence that assessment of respiratory function can be used to describe the phenotypic appearance of structural changes, resulting from a huge number of potential pre- and post-natal factors. The use of such tests to identify individual infants at risk of subsequent disease is, however, not currently feasible due to the marked intersubject variability.

Early lung disease Much has been learnt about early pulmonary disease in CF [114]. Although spirometry has been shown to be an insensitive marker of early airway disease in preschool and school-aged children with CF [31, 52, 55, 115], forced expiratory flows from raised lung volume appear to be very discriminatory during infancy [60, 61]. Marked structural changes have been demonstrated using computed tomography (CT) in children with entirely normal spirometric results, with progression of these changes not being detected by repeat spirometry [116–118]. A combination of the raised-volume technique and highresolution CT (HRCT) may prove a powerful diagnostic tool if concerns regarding ionising radiation can be addressed [119, 120]. The greater sensitivity of forced expiratory manoeuvres in young children compared to older subjects may be explained by differences in airway wall stability, different proportions of airway diameter and length in relation to lung volume, and differences in chest wall compliance. Multiple-breath washout appears to be a more sensitive method of identifying children aged w3 yrs with early pulmonary changes [51–53]; however, this remains to be proven in infants and very young children. From studies using multiple-breath inert-gas-washout techniques, it appears likely that pulmonary disease starts within the more peripheral airways in CF, resulting in the observed ventilation inhomogeneity. Parameters of ventilation inhomogeneity, such as the lung clearance index, have the advantage of being relatively constant throughout life [55], thereby negating the need for age- or height-dependent reference equations, at least beyond the first 6–12 months of life. There is also recent evidence that parameters of ventilation inhomogeneity reflect progression of disease more sensitively than conventional tests such as spirometry [121].

Future questions Clinical relevance The increasing recognition that early lung growth and development are important to long-term respiratory health is reflected by the expanding role of infant lung function testing in both clinical and research studies. Following years of study of molecular biology and gene polymorphisms, the importance of using lung function testing as a noninvasive tool for describing the phenotypic consequences has now been accepted [122]. Although there is increasing evidence elucidating the functional development of the lung, which demonstrates the importance of early programmers and the tracking of lung function from the first months of life in both health and disease, there is still little evidence as to whether early lung function tests are sensitive enough to detect clinically relevant early changes in lung function in the individual patient. Nevertheless, continuing 30

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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

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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

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there should be a move away from the traditional practice of expressing results as a percentage of the predicted value, which gives no indication of what the normal range might be, instead reporting results with sds or z-scores. The latter would not only indicate the magnitude of any changes in relation to normal between-subject variability for a particular test but also facilitate longitudinal follow-up and comparison of results from different tests [27].

Development of the respiratory system Although there have been considerable advances in the tracking of airway function during lung growth and development, there is growing evidence that prematurity, early lung disease and/or environmental toxic influences disturb not only airway and vascular development but also alveolar development. There are currently no means of directly assessing alveolar number, size and surface. The use of HRCT has provided insight into structural development [119, 120], but it is unlikely to be accepted as a routine tool, especially in the absence of overt disease, because of the relatively large amount of radiation exposure associated with this technique [129]. In addition, the resolution of HRCT remains insufficient to assess structure down to the acinar level. Micro-CT is being investigated in animal studies, but currently has no place in paediatric respiratory medicine [130]. Magnetic resonance imaging of the lungs using hyperpolarised helium is being discussed as a means of combining structural and functional assessment of the lung, but, again, this approach currently remains strictly within the research arena, and there is limited experience in children [131–133]. More realistically, techniques that permit partitioning of mechanical properties into airway and tissue components might reach the level at which they could be used more widely [80]. This may be of particular interest for assessing the effects of prematurity and monitoring infants in the intensive care unit, where parenchymal disease is a major component of both acute and chronic respiratory illness. Further insight into airway wall characteristics appears essential to clarifying the role of developmental changes versus inflammation in wheezing disorders and bronchial hyperresponsiveness [30, 80]. Accurate assessment of airway wall properties could also help differentiate wheezing associated with reduced airway size from that due to altered airway wall mechanics, as in congenital tracheobronchomalacia or secondary to inflammatory processes. Attempts to describe airway wall properties in infants are sparse, but both the FOTs and a high-speed interrupter technique may provide further insight. Assessment of vascular development may be particularly interesting in pre-term infants. However, there is currently no technique available that has been evaluated for assessing pulmonary blood flow noninvasively. Only techniques that are repeatable, noninvasive and applicable in all age groups are likely to be successful when outcome measures for clinical studies are sought. Both multiple-breath washout and the LFOT appear promising. With multiple-breath washout, the current gold standard for measuring tracer gas concentrations is the use of a mass spectrometer [31, 55]. However, there is no commercially available equipment with appropriate software. Alternatively, an ultrasonic flow-head may be used [45, 54, 56, 134].

Dynamic behaviour of the developing respiratory system Traditionally, the respiratory system has been considered a steady-state mechanical structure; however, recent data show that the system behaves in a more dynamic fluctuating manner. Analysis of the variability and correlation properties of these fluctuations in lung function provides interesting information on the developmental and 33

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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 structure–function 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.

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Shaheen SO, Barker DJ, Shiell AW, Crocker FJ, Wield GA, Holgate ST. The relationship between pneumonia in early childhood and impaired lung function in late adult life. Am J Respir Crit Care Med 1994; 149: 616–619. Dezateux C, Stocks J. Lung development and early origins of childhood respiratory illness. Br Med Bull 1997; 53: 40–57. Barker DJ, Godfrey KM, Fall C, Osmond C, Winter PD, Shaheen SO. Relation of birth weight and childhood respiratory infection to adult lung function and death from chronic obstructive airways disease. BMJ 1991; 303: 671–675. Le Souef PN. Pediatric origins of adult lung diseases. 4. Tobacco related lung diseases begin in childhood. Thorax 2000; 55: 1063–1067. Hoo AF, Stocks J, Lum S, et al. Development of lung function in early life: influence of birth weight in infants of nonsmokers. Am J Respir Crit Care Med 2004; 170: 527–533. Lucas JS, Inskip HM, Godfrey KM, et al. Small size at birth and greater postnatal weight gain: relationships to diminished infant lung function. Am J Respir Crit Care Med 2004; 170: 534–540. Le Souef P. Infant lung function, bronchial responsiveness and the development of asthma. Pediatr Allergy Immunol 2000; 11: Suppl. 13, 15–18. Warner JO. The early life origins of asthma and related allergic disorders. Arch Dis Child 2004; 89: 97–102. Frey U, Kuehni C, Roiha H, et al. Maternal atopic disease modifies effects of prenatal risk factors on exhaled nitric oxide in infants. Am J Respir Crit Care Med 2004; 170: 260–265. Gappa M, Stocks J, Merkus P. Lung growth and development after preterm birth: further evidence. Am J Respir Crit Care Med 2003; 168: 399–400. Hofhuis W, Huysman MW, van der Wiel EC, et al. Worsening of V’maxFRC in infants with chronic lung disease in the first year of life: a more favorable outcome after high-frequency oscillation ventilation. Am J Respir Crit Care Med 2002; 166: 1539–1543. Jobe AH. An unknown: lung growth and development after very preterm birth. Am J Respir Crit Care Med 2002; 166: 1529–1530. Hoo AF, Dezateux C, Hanrahan JP, Cole TJ, Tepper RS, Stocks J. Sex-specific prediction equations for Vmax(FRC) in infancy: a multicenter collaborative study. Am J Respir Crit Care Med 2002; 165: 1084–1092. Stocks J, Gappa M, Rabbette PS, Hoo AF, Mukhtar Z, Costeloe KL. A comparison of respiratory function in Afro-Caribbean and Caucasian infants. Eur Respir J 1994; 7: 11–16. Stocks J, Henschen M, Hoo AF, Costeloe K, Dezateux C. Influence of ethnicity and gender on airway function in preterm infants. Am J Respir Crit Care Med 1997; 156: 1855–1862. Stein RT, Martinez FD. Asthma phenotypes in childhood: lessons from an epidemiological approach. Paediatr Respir Rev 2004; 5: 155–161. Turner SW, Palmer LJ, Rye PJ, et al. The relationship between infant airway function, childhood airway responsiveness, and asthma. Am J Respir Crit Care Med 2004; 169: 921–927. Turner SW, Palmer LJ, Rye PJ, et al. Infants with flow limitation at 4 weeks: outcome at 6 and 11 years. Am J Respir Crit Care Med 2002; 165: 1294–1298. Murray CS, Pipis SD, McArdle EC, Lowe LA, Custovic A, Woodcock A. Lung function at one month of age as a risk factor for infant respiratory symptoms in a high risk population. Thorax 2002; 57: 388–392. Gappa M, Ranganathan SC, Stocks J. Lung function testing in infants with cystic fibrosis: lessons from the past and future directions. Pediatr Pulmonol 2001; 32: 228–245. Aurora P. Multiple breath washout in preschool children – FRC and ventilation inhomogeneity. Paediatr Respir Rev 2006; 7: Suppl. 1, S14–S16. de Jong PA, Nakano Y, Lequin MH, et al. Progressive damage on high resolution computed tomography despite stable lung function in cystic fibrosis. Eur Respir J 2004; 23: 93–97. Tiddens HA. Detecting early structural lung damage in cystic fibrosis. Pediatr Pulmonol 2002; 34: 228–231. Brody AS, Klein JS, Molina PL, Quan J, Bean JA, Wilmott RW. High-resolution computed

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tomography in young patients with cystic fibrosis: distribution of abnormalities and correlation with pulmonary function tests. J Pediatr 2004; 145: 32–38. Long FR. High-resolution CT of the lungs in infants and young children. J Thorac Imaging 2001; 16: 251–258. Long FR, Williams RS, Castile RG. Structural airway abnormalities in infants and young children with cystic fibrosis. J Pediatr 2004; 144: 154–161. Kraemer R, Blum A, Schibler A, Ammann RA, Gallati S. Ventilation inhomogeneities in relation to standard lung function in patients with cystic fibrosis. Am J Respir Crit Care Med 2005; 171: 371–378. Coates AL. Classical respiratory physiology – gone the way of the dinosaurs? Do we need a Jurassic park? Pediatr Pulmonol 2000; 30: 1–2. Bacharier LB, Strunk RC, Mauger D, White D, Lemanske RF. Jr, Sorkness CA. Classifying asthma severity in children: mismatch between symptoms, medication use, and lung function. Am J Respir Crit Care Med 2004; 170: 426–434. Edwards LJ. Modern statistical techniques for the analysis of longitudinal data in biomedical research. Pediatr Pulmonol 2000; 30: 330–344. Frey U. Clinical applications of infant lung function testing: does it contribute to clinical decision making? Paediatr Respir Rev 2001; 2: 126–130. Godfrey S, Bar-Yishay E, Avital A, Springer C. What is the role of tests of lung function in the management of infants with lung disease? Pediatr Pulmonol 2003; 36: 1–9. Jones M, Castile R, Davis S, et al. Forced expiratory flows and volumes in infants. Normative data and lung growth. Am J Respir Crit Care Med 2000; 161: 353–359. Stocks J, Sly P, Tepper, RS, Morgan W. Infant Respiratory Function Testing. New York, NY, Wiley-Liss, 1996. de Jong PA, Mayo JR, Golmohammadi K, et al. Estimation of cancer mortality associated with repetitive computed tomography scanning. Am J Respir Crit Care Med 2006; 173: 199–203. Chon D, Simon BA, Beck KC, et al. Differences in regional wash-in and wash-out time constants for xenon-CT ventilation studies. Respir Physiol Neurobiol 2005; 148: 65–83. Koumellis P, van Beek EJ, Woodhouse N, et al. Quantitative analysis of regional airways obstruction using dynamic hyperpolarized 3He MRI – preliminary results in children with cystic fibrosis. J Magn Reson Imaging 2005; 22: 420–426. Altes TA, Rehm PK, Harrell F, Salerno M, Daniel TM, De Lange EE. Ventilation imaging of the lung: comparison of hyperpolarized helium-3 MR imaging with Xe-133 scintigraphy. Acad Radiol 2004; 11: 729–734. Schreiber WG, Morbach SE, Stavngaard T, et al. Assessment of lung microstructure with magnetic resonance imaging of hyperpolarized helium-3. Respir Physiol Neurobiol 2005; 148: 23– 42. Fuchs S, Buess C, Gappa M. Improved system for ultrasonic measurement of functional residual capacity and ventilation inhomogeneity. Eur Respir J 2005; 26: 38s. Frey U, Maksym GN, Silverman M, Suki B. New approaches to the understanding of complex chronic lung diseases. In: Frey U, Gerritsen J, eds. Respiratory Diseases in Infants and Children. Eur Respir Mon 2006; 37: 345–360.

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CHAPTER 4

Remodelling in paediatric respiratory disease and impact on growth and development D.N. Payne, S. Saglani, A. Bush Dept of Paediatric Respiratory Medicine, Royal Brompton Hospital and Airways Diseases Section, National Heart and Lung Institute, Imperial College, London, UK. Correspondence: A. Bush, Dept of Paediatric Respiratory Medicine, 4th Floor, Chelsea Wing, Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK. Fax: 44 2073518763; E-mail: a.bush@ rbht.nhs.uk

Remodelling is the collective term used to describe the structural changes seen in the lungs of patients with respiratory disease. These structural alterations involve residential airway cells and, possibly, bone marrow-derived pleotropic cells recruited from the circulation. Structural changes have been reported in a number of respiratory diseases, although they are most commonly described in the airways of patients with asthma [1]. The features of airway wall remodelling in asthma are shown in table 1. Until recently, the focus has largely been on studies involving adults, due to practical and ethical constraints limiting access to tissue from infants and children. However, in the last few years, a number of groups have begun to study the paediatric airway [2–9], 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, 41–59. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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Table 1. – Features of airway wall remodelling in asthma Goblet cell hyperplasia Thickening of the epithelial reticular basement membrane Increased number of submucous glands Increase in blood vessel number and area Smooth muscle hypertrophy and hyperplasia Increase in airway wall collagen

characteristic feature of asthma in adults, in both school-age and preschool children, leading some investigators to suggest that remodelling occurs early and may develop in parallel with, but separate from, airway inflammation [2, 4, 9]. That structural changes may precede, or even be a prerequisite for, inflammation, has also been proposed [11, 12]. In contrast to studies involving adults, in whom asthma is always established at the time of investigation, the ability to investigate airway changes in infants and young children [2, 4, 13, 14], at a time when the airways are still developing and diseases such as asthma are just beginning to manifest themselves, provides an opportunity to address some of the fundamental questions regarding remodelling. These include the following. 1) When do the changes begin? 2) What initiates the early changes? Is it inflammation or some other factor? 3) What modulates them? Is it inflammation or some other factor? 4) Do they

SM Blood vessel

SM

RBM

Epi

Fig. 1. – Low-power view of an endobronchial biopsy, stained with haematoxylin and eosin, from a child with asthma. RBM: reticular basement membrane; Epi: epithelium; SM: smooth muscle.

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RBM

RBM

Epi

Fig. 2. – High-power view of an endobronchial biopsy, stained with haematoxylin and eosin. RBM: reticular basement membrane; Epi: epithelium.

matter? Are they harmful or protective? 5) What features are unique to asthma, and what are common to other diseases? The importance of the fact that structural changes in childhood occur with the background of an airway or lung that is still developing cannot be overstated. Thus, in order to interpret correctly the changes seen in children, it is essential to understand the process of normal airway development (see Chapter 2). A priority for future research must therefore be to study normal human airway development antenatally and in the first few years of life, in order to understand the mechanisms and significance of structural changes in children with respiratory disease.

Current limitations of knowledge Airway remodelling clearly encompasses virtually every airway wall component (table 1). In the paediatric field, studies are limited mainly to measurements of the RBM. Some work has been reported on what has been termed the epithelial–mesenchymal trophic unit (EMTU; see below), although the concept of the EMTU must still be considered hypothetical, albeit a helpful exploratory model. RBM thickening is a characteristic pathological feature of asthma in adults and is relatively easy to measure and quantify, so it is an important as well as convenient measurement to make. However, the paucity of data on other structural elements means that many of the conclusions of this chapter have to be based on incomplete evidence. Furthermore, almost all studies in the paediatric literature are in the context of wheeze and asthma, and little is known about remodelling in other paediatric respiratory diseases. The second major problem is the lack of true control data. Unlike in adults, it is unethical to perform bronchoscopy in children solely for research purposes [15]. 43

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However, it is safe and therefore legitimate to perform endobronchial biopsy at the time of a clinically indicated bronchoscopy, with the approval of the Institutional Ethics Committee, the consent of the family and the age-appropriate assent of the child [16]. There are detailed guidelines for the processing of endobronchial biopsy [15]. Most of the available control data are obtained from children without asthma undergoing bronchoscopy for investigation of respiratory symptoms, such as stridor, haemoptysis, recurrent infection or chronic cough, or from tissue obtained post mortem [2, 7– 9]. However, this is not the same as data on normal children. The third issue relates to the association between structural changes and physiology. The two main issues are the relationship (if any) between structural changes and fixed airflow obstruction, and between structural changes and airway hyperresponsiveness. Most papers on the subject of remodelling state that "structural changes may lead to irreversible airways obstruction". While there is some evidence to support this statement, the available data are limited. Kasahara et al. [17] demonstrated an association between RBM thickness and forced expiratory volume in one second (FEV1; the thicker the RBM, the lower the FEV1) in adults with asthma, following treatment with systemic corticosteroids and inhaled b2-agonists. Benayoun et al. [18] studied airway smooth muscle in airway biopsy and showed an increase in smooth muscle in those patients with the most severe impairment of lung function. Interestingly, a disease control group, consisting of adults with chronic obstructive pulmonary disease (COPD), had a similar deficit in lung function, but without any increase in airway smooth muscle. The relationship between structural airway changes and airway hyperresponsiveness (AHR) also needs to be explored further. While structural changes are generally considered to contribute to AHR associated with asthma [19], it has also been suggested that one of the characteristic structural changes, RBM thickening, may actually be protective against bronchospasm [20]. If this is the case, then attempts to reduce RBM thickness may actually be misguided. Clearly, understanding the significance of airway remodelling is crucial and must be a focus of future research. One of the difficulties in assessing the relationship between airway structure and function is in determining what constitutes true, fixed airflow obstruction. The traditional method is to measure the acute response to bronchodilator. This is convenient but unlikely to be valid in many contexts. In severe asthma, neither acute bronchodilator administration nor even a 2-week course of prednisolone is necessarily predictive of best lung function in the following year [21]. No one method is likely to be truly predictive of best lung function; the choice lies between the acute response to bronchodilator and the response to a prolonged course of steroids in some form, which may be combined with acute bronchodilator administration [7, 17]. The choice of route of administration includes a period of inhaled corticosteroids, oral prednisolone or even intramuscular triamcinolone, the latter having the merit of ensuring that nonadherence is not an issue [22]. The chosen method will probably be a compromise between what is practical and ethical, and what is desirable for true scientific rigour. However, the investigator will have to acknowledge the imperfections of whatever method is chosen.

What have been the most recent fundamental developments in the field? RBM thickening: a consequence or cause of asthma? The relationship between remodelling and inflammation in asthma is unclear. It should be noted that in both asthma and other diseases, it may not be valid to consider 44

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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.

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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 1–3 yrs of age, increasing to school age, a concept which fits with the lung function findings in different cohorts. The weaknesses of the pathological data must be acknowledged. First, they are not longitudinal; they are not even serial cross-sections of the same population. Secondly, until these two (infant and preschool) cohorts have been followed up into mid-childhood, it can only be speculated, based on predictive factors established by others [31, 32], as to which of the children will be the true asthmatics. Third, although RBM thickening has been shown to be present, it is not known whether this is important or merely a marker for some other, as-yet undetermined change in the airway wall. Nonetheless, the pathological and epidemiological data strongly suggest that the real changes of remodelling are a very early event in asthma [2, 4, 29], which may be preceded by symptoms [14]. However, in order to interpret accurately the significance of the changes seen in symptomatic children, better data from genuine healthy controls are needed, as too little is currently known about the normal developmental structural airway changes in infancy and early childhood.

RBM thickening occurs early and is nonprogressive in children with asthma The paradigm that repeated cycles of acute inflammation or unremitting chronic inflammation eventually lead to remodelling would logically predict a relationship between duration of asthma and the severity of structural changes. Furthermore, it would also predict that anti-inflammatory therapy is all that is needed to prevent airway remodelling. Recent paediatric data in older children, as well as the studies in preschool children, referred to above, have challenged this model. RBM thickness in biopsies obtained from a group of children (aged 6–16 yrs) with difficult asthma was compared with data from the following four other groups: 1) adults with mild asthma; 2) adults who had been ventilated for asthma; 3) healthy adult controls; and 4) paediatric controls, as described above [8]. RBM thickness was the same in all three asthma groups and was significantly higher than in both control groups. In the group of children with difficult asthma, there was no relationship between RBM thickness and duration of symptoms, level of 46

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treatment, or any marker of inflammation studied [7, 8]. Although these data are crosssectional, it is difficult to reconcile them with any mechanism postulating progressive and ongoing activity in at least this aspect of remodelling. Set against these data are the "acute remodelling" studies. Segmental allergen challenge involves the endobronchial instillation of an appropriate allergen, with bronchoscopy, bronchoalveolar lavage (BAL) and endobronchial biopsy performed before and after allergen challenge, to study the inflammatory and other changes. In one study, nine adults with mild asthma underwent endobronchial challenge to an allergen, to which they were sensitive on skin-prick testing [33]. Twenty-four hours after challenge, there was evidence of both epithelial cell and fibroblast activation, with a significant increase in the deposition of the matrix protein tenascin within the RBM. For obvious practical reasons, the resolution of these changes (if any) could not be followed by serial bronchoscopies. The significance of acute airway challenges is difficult to assess. In real life, sensitised subjects are likely to undergo repeated allergen airway challenges. If, with each challenge, there is deposition of matrix tenascin, with no mechanism of resolution, then over the years the airway would be obliterated altogether. Is there tolerance to challenge over time? Do acute changes, such as those described above, resolve completely? What is the relationship with chronic remodelling? These questions need further work if they are to be answered, but a study of the mechanisms of resolution of acute remodelling might allow us to understand and modulate chronic airway wall changes (see below).

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

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unscreened population of infants, newly diagnosed with CF, there is evidence of RBM thickening, although to a lesser degree than in asthmatics [37]. The present authors hypothesise that some factor(s) possibly, but not necessarily, related to RBM thickening and probably, but not necessarily, related to infection and inflammation, irretrievably impair lung function early in the course of CF. The later destructive processes may be separate from these early phenomena. Modulation of early changes may allow patients to attain better lung function after diagnosis and hence prolong survival.

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

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

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The epithelial–mesenchymal trophic unit. A complementary viewpoint is that the changes seen in remodelling may represent the consequences of an abnormal repair mechanism within the airway, following epithelial injury [54]. This model proposes that the epithelium and its response to injury is the primary abnormality in asthma. It also suggests a key role for the interaction between the epithelium and the associated subepithelial tissue, which may represent a reactivation of the processes responsible for embryonic lung development. Airway development in utero depends on interaction between the developing epithelium (endoderm) and the surrounding mesenchymal tissue, with branching of the primitive endoderm failing to occur in the absence of the surrounding mesenchyme. This has led to the use of the term EMTU to describe the anatomical and functional relationship between these two layers. Some of the proteins which are intimately involved in embryonic airway development, such as tenascin, fibronectin and collagens, are also associated with remodelling changes in asthma. This, along with data supporting the presence of signalling between epithelial and mesenchymal (e.g. fibroblasts) cells, has led to the suggestion that the structural airway changes occurring in the asthmatic airway represent reactivation of the EMTU. A role for epithelial–mesenchymal interaction in RBM thickening in childhood asthma has been suggested by Fedorov et al. [9], based on data examining the expression of epidermal growth factor receptor (EGFR), which is increased in response to epithelial injury within the epithelium of airway tissue from children with and without asthma. Immunostaining for EGFR expression was most intense in children with severe asthma, with a significant positive correlation between EGFR expression and RBM thickness. The suggestion is that epithelial injury results in the release, by epithelial cells, of an array of growth factors, including fibroblast growth factor (FGF), platelet-derived growth factor and TGF-b. These stimulate increased fibroblast proliferation, which in turn leads to an increase in RBM thickness. However, these data cannot determine whether or not the proposed exaggerated repair process is a cause or consequence of asthma and thus whether or not it reflects the inception or the progression of the disease. It should further be noted that the validity of the concept of the EMTU awaits further data.

Animal models. Cross-sectional studies cannot be anything other than descriptive and hypothesis-generating. For ethical reasons, intervention studies designed to test a specific hypothesis, with biopsies obtained before and after intervention, cannot be performed in children. Such studies can, however, be performed in animals, provided appropriate models exist and that their limitations are acknowledged. Animal studies are expensive and the use of animals close to humans, such as primates, is even more costly. The advantage of animal models is that they provide an opportunity to explore potentially relevant mechanisms of airway remodelling. However, a key difference between animal models of asthma and the human form of the disease relates to the concepts of heredity and risk factors for asthma. Most animal models rely on post-natal sensitisation to induce atopy. Although a model using inhalational sensitisation has been developed [55], the majority use intraperitoneal sensitising injections and it is difficult to see how these are relevant to human asthma. There is no role in animal models for the effects of parental atopy, a major influence on the development of asthma in children [31], or the circumstances of the mother’s 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

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relationship between inflammation and remodelling. However, there are significant differences between the murine and the human airway. For example, the normal murine airway does not possess a RBM, which develops only after allergen challenge. Studies involving mice, and probably all animals, can therefore only ever be hypothesisgenerating. A more appropriate model (albeit very expensive) for studying both normal airway development and remodelling is a primate model [56–61]. Not only does the airway wall in the primate closely resemble that of the human airway, but the use of infant monkeys also provides an opportunity to investigate the mechanisms of airway remodelling in the developing airway (table 2). In addition, by examining the whole airway, rather than endobronchial biopsy, the assessment of airway structural changes is not limited to the RBM but includes other features such as airway smooth muscle. The available data from primate models demonstrate that RBM development occurs post-natally and that the growth factor, FGF-2, appears to play an important role, initially being stored within basal epithelial cells before accumulating in the RBM [57, 58]. Allergen challenge leads to an increase in RBM thickness, associated with an increase in certain components of the RBM, including collagens, the proteoglycan perlecan and FGF-2. The authors of these studies have suggested that the thickened RBM may act as a source of growth factors and proteins that are necessary to allow infiltrating subepithelial inflammatory cells to move across the RBM into the epithelium and airway lumen [58]. The same investigators have also studied smooth muscle within the airway, showing an increase in airway smooth muscle following allergen challenge [60]. As well as being able to describe normal airway development and the effects of allergen challenge, this primate model provides the opportunity to investigate the progression and/or resolution of remodelling changes and to examine the effects of interventions that may inhibit remodelling. RBM changes persist in sensitised monkeys that continue to be exposed to allergen every month [59]. Interestingly, it is not known whether the changes resolve in the absence of any further allergen challenge. However, of potential therapeutic interest is the finding that resolution of remodelling changes has been demonstrated in young (3–5-yr-old) rhesus monkeys following repeated inhalation of immunostimulatory DNA sequences (ISS) [61]. These sequences contain a CpG dinuncleotide (CpG motif) that is characteristic of bacterial DNA but relatively rare in vertebrate DNA. Thus, these data provide the potential to shed light on the factors that regulate remodelling and to increase understanding of the functional significance of the structural changes. The relevance to humans of these findings in a primate model needs Table 2. – Comparison between the development of the reticular basement membrane in infant rhesus monkeys and humans Rhesus monkey

Human

RBM develops after birth and full adult thickness is reached by 6 months of age

RBM is present in infants with respiratory symptoms. Unclear when RBM first appears or when maximal thickness is reached Constituents include collagens (I, III, V), tenascin, fibronectin

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

Increase in RBM thickness in preschool children with severe wheeze

RBM: reticular basement membrane; FGF: fibroblast growth factor. 50

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

The related diseases RBM thickening cannot be said to be pathognomic or diagnostic of asthma, given that it has also been described in a number of other diseases [20, 34–37]. This suggests that this particular structural change may represent a normal response to injury. It is possible that the control mechanisms, which regulate the extent of RBM thickening, may be set at a different level in diseases such as asthma, in which the highest values of RBM thickness are usually seen. The histological data on other components of the airway wall in children are limited, so little can be said about other aspects of remodelling in other diseases. However, it is clear that structural airway changes are found in other paediatric respiratory diseases, such as CF, primary ciliary dyskinesia, other causes of bronchiectasis, obliterative bronchiolitis and gastro-oesophageal reflux disease. The extent to which these changes are reversible is a particular area of interest and debate [62].

The treatment strategies. There is little data on the effect of treatment on structural airway changes. What evidence there is comes from studies of adult asthma and again focuses on measurements of RBM thickness. Two studies have documented a reduction in RBM thickness following prolonged treatment with inhaled corticosteroids [63, 64]. Ward et al. [63] performed a double-blind, randomised, placebo-controlled, parallel group study of high-dose inhaled fluticasone (1.5 mg?day-1) involving 35 steroid-naı¨ve 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 [65–67]. 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

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beneficial effects of montelukast may have been due to a reduction in the degree of airway fibrosis, rather than an effect on airway inflammation. However, in the absence of inflammatory or histological data, this remains speculative. One aspect of airway remodelling described in adult asthma, which has not been studied in children, is the increase in blood vessel number and area. Macrolides, another class of drug that has been used to treat asthma, may have a potential role to play in the modulation of this particular structural change. Fourteen-membered macrolides appear to reduce tumour angiogenesis by an unknown mechanism and therefore it is possible that bronchial neovascularisation could be reduced [72]. Roxithromycin inhibits tumour necrosis factor-a-induced vascular endothelial growth factor production [73]. Angiogenesis may also be inhibited indirectly via effects on IL-8, which seems to be angiogenic as well as pro-inflammatory. A rapamycin analogue inhibits epidermal growth factorinduced proliferation in a murine model of lung inflammation and remodelling [74]. If this effect were the same in the human lower airway, this could have profound implications for prevention of airway remodelling associated with angiogenesis. The development of novel treatments targeting remodelling changes will gain greater impetus if it can be demonstrated that resolution of remodelling has an impact on symptoms and lung function. In this context, animal models will have an important role to play. In particular, monkey work demonstrating that inhalation of ISS can lead to resolution of remodelling changes provides an exciting stimulus for further therapeutic research [61].

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

What is needed to find answers on these questions? Noninvasive ways of serially measuring remodelling It is unlikely that serial bronchial biopsies will be performed in large cohorts of children, so there is an urgent need to validate noninvasive tests which could be used 52

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prospectively and longitudinally. Potential candidates include imaging techniques, lung function, and measurement of mediators in urine, blood, induced sputum or exhaled breath condensate (EBC). There are important general principles in the understanding of noninvasive markers; the first is to determine the purpose of measuring them. The requirements are different for mechanistic studies, as opposed to monitoring or guiding treatment. Group differences in mediators, with overlap between groups, are very useful for pointing towards potentially interesting mechanisms of disease, but may not be useful in an individual child. The ideal marker must be stable, reproducible, able to discriminate between normal and disease, and sensitive to changes in clinical state over time, whether due to disease or treatment. No such marker exists at this time, but the questions are important and the long-term aims must be remembered, particularly when considering cross-sectional studies, which comprise the bulk of what is available. In adults, quantitative measurement of airway wall thickness with high-resolution CT has been used to study airway wall changes, as discussed earlier [17]. Airway wall area and thickness were measured using quantitative CT, after pre-treatment with oral steroids and bronchodilators, to eliminate as far as possible any reversible changes. These indices were higher in asthmatics than normal subjects. There were strong correlations between both wall area and wall thickness and RBM thickness in endobronchial biopsy, with strong, inverse correlations between FEV1 and all three airway wall parameters. In another study, wall thickness ratio and area was compared in four groups of adults, as follows: 1) near fatal, 2) moderate, 3) mild asthma, and 4) normal controls [75]. All asthmatics had greater airway wall thickness than controls, with thicker walls present in the more severe asthmatics. Another study attempted to correlate biomarkers of remodelling with radiological measurements. In stable adult asthmatics, sputum matrix metalloproteinase (MMP)-9 was inversely correlated with wall area [76]. Tissue inhibitor of metalloproteinase (TIMP)-1 was positively correlated with wall area and thickness and the molar ratio of MMP-9 to TIMP-1 was positively correlated with post-bronchodilator spirometry. Taken together, in adults, quantitative CT shows promise as a noninvasive marker of airway remodelling. By contrast, the current authors were unable to correlate RBM thickness with CT measurements of bronchial wall thickness in children with severe asthma [77]. There are several possible reasons for this. The children may have had more movement artefact due to difficulty in breath-holding, or more prominent cardiac pulsation, making the measurement less precise. The children may have had nonspecific reasons for airway wall thickening, such as gastro-oesophageal reflux or recurrent viral infections. RBM thickening may correlate poorly with other more prominent features of airway wall thickening due to remodelling in children. Even if the measurements were to be refined, it is difficult to see how radiological imaging could do more than measure the sum of all the changes (reversible and irreversible), rather than the individual components (see introduction). It is possible that, in the future, magnetic resonance imaging (MRI) techniques could allow spectroscopic analysis of the changes in airway wall components. It might be possible to measure bronchial blood flow with MRI angiography, but it might be difficult to distinguish dilatation of normal bronchial arteries secondary to inflammation from airway neovascularisation as part of the remodelling process. An alternative to imaging would be to assess the activity of the remodelling process by direct measurement of modulators of the process. Superficially attractive options are EBC and induced sputum. However, these techniques may not be sensitive to processes deep in the airway wall and therefore blood or urine may be more useful. Urine is an attractive source of biomarkers. Desmosine (DES) and isodesmosine (IDES) are amino acids derived exclusively from cross-linked elastin. Hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) are amino acids derived exclusively from cross-linked collagen. All 53

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have been measured in urine, but there are no data correlating them with endobronchial biopsy in children. CF adults had raised urinary levels of all these markers [78, 79]; elevations have been also been described in emphysema [80], exacerbations of COPD and other inflammatory lung diseases [81, 82]. There were higher levels of DES, but not hydroxyproline, in the urine of patients with a more rapid decline in lung function with age [83]. A study published in abstract form in CF children reported a correlation between urinary desmosines and BAL neutrophil elastase [84]. Taken together, these data are very suggestive that DES, IDES, HP and LP in the urine are potentially useful markers of tissue destruction. They are not specific to the lung, but in the context of isolated respiratory disease, an elevation in levels is most likely attributable to events within the lungs. However, it is important to emphasise that their relationship to remodelling, as opposed to tissue destruction, is unclear. Longitudinal studies, comparing urinary markers with findings on endobronchial biopsy, are required to address this. With regard to the use of EBC, preliminary data are available showing significant correlations between RBM thickness on endobronchial biopsy and cys-LT levels in EBC in a group of children with asthma [85]. Further work is needed to evaluate whether cysLTs will turn out to be useful clinically in the assessment of remodelling.

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

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

Application of techniques to assess structures other than RBM There are a large number of established techniques to study remodelling, including immunohistochemistry, in situ hybridisation, genomics, proteomics and metabolomics, 54

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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 "let’s 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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CHAPTER 5

Immunology and defence mechanism of the developing lung B. Schaub*, R. Lauener #, S.L. Prescott},z *Pediatric Pulmonary Division, University Children’s Hospital Munich, LMU Munich, Munich, Germany. # Zurich University, Children’s Hospital, Center for Allergy Research, Zurich, Switzerland. }School of Paediatrics and Child Health, University of Western Australia, and zPrincess Margaret Hospital, Perth, Western Australia, Australia. Correspondence: B. Schaub, Pediatric Pulmonary Division, University Children’s Hospital Munich, LMU Munich, Lindwurmstr. 4, 80337 Munich, Germany. Fax: 49 8951604764; E-mail: Bianca.Schaub@med. uni-muenchen.de

Complex systems have evolved to protect the host from potentially noxious environmental agents. This is most critical in mucosal and epithelial surfaces that are in direct environmental contact. Local events in these tissues are critical for programming all systemic and local defence systems, culminating in a highly adaptive surveillance network, which is environmentally relevant. Environmental exposures at mucosal and epithelial surfaces have a number of critical effects. First, in the post-natal period, environmental exposure plays a key role in driving global immune maturation, which appears to be dependent on exogenous factors (namely microbial exposure) to develop normally. Secondly, the pattern of environmental antigen exposure determines the specificity of responses required for host defence. Finally, environmental and endogenous conditions during antigen processing in local tissues appear to influence the patterns of immune maturation and resulting immune responses. These concepts are discussed more fully below with respect to immune development in the respiratory tract. Although mucosal events arguably play the most pivotal role in immune development, these events are still poorly understood in humans because of logistical and ethical limitations of studies in this area, particularly in young children. Much current understanding is extrapolated from studies of systemic immune function, animal models or indirect measures of mucosal immune function. This chapter discusses how environmental influences and local endogenous factors (such as collectins, neuropeptides, and oxidative stress) contribute to the developing immunity in the lung.

Immune development of the healthy lung Antenatal events and influences on immune maturation As with most other systems, immune programming in the antenatal period is highly developmentally regulated; however, there is evidence that programmed development can be influenced by environmental exposures in this period, including infection [1], maternal diet [2, 3] and smoking [3, 4], which can modify immune responses detected in the neonatal period (fig. 1). Foetal responses are clearly sensitive to the ambient cytokine environment of pregnancy, and the first cellular responses (in foetal life) universally reflect the "normal T-helper (Th)2-skew" of pregnancy [5]. This, together with mounting Eur Respir Mon, 2006, 37, 60–78. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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Environmental factors, allergens, vaccines, antibiotics, infections, pathogens

Defence mechanism Mannosebinding lectin Oxidative stress

Antimicrobial substances Surfactant Microbial stimulation proteins Neuropeptides

Nasal mucosa Intestinal mucosa Bone marrow Lymph node Thymus Th1 Peripheral blood Naive HSC T-cell TLR T-reg TLR

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Th2 DC

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Fig. 1. – Schematic overview of immunological and defence mechanisms of the developing lung. HSC: haematopoietic stem cell; TLR: toll-like receptor; DC: dendritic cell; Th: T-helper cell; T-reg: regulatory T-cells.

evidence of pre-symptomatic differences in the immune function of newborns who later develop allergic disease [6], has generated more intense interest in the role of antenatal events in immune programming and disease pathogenesis. In addition to environmental effects in pregnancy, one of the authors has recently explored the role of direct materno– foetal interactions on foetal immune programming [7]. The present authors have noted that maternal reactivity to foetal allo-antigens is related to the pattern of developing foetal immune responses, as well as the subsequent development of allergic disease [7]. Together with previous observations that maternal atopy may have a stronger influence on neonatal immune responses than paternal atopy [8], these observations suggest that materno–foetal interactions could be an important determinant of immune reactivity in the early post-natal period, and this needs to be investigated further. As the immunologically active interface between the foetus and the mother, and being the major source of cytokines and other immune mediators detected in the foetus [9, 10], the placenta has enormous potential to influence foetal immune development. Low-grade inflammation is characteristic of all pregnancies and complex pathways have evolved to minimise this. The present authors speculate that variation in the propensity for inflammatory responses and/or the capacity to regulate these in the placenta is of key importance in establishing early patterns of foetal immune responsiveness. Placental cells are also likely to be sensitive to adverse environmental exposures that may affect pathways which underpin the dramatic increase in immune disease in very early life. As yet, this has not been investigated in this context, but is an important area for future research. Thus, while genetics provide the blueprint for immune development, environmental factors (including both maternal exposures and direct maternal influences in utero) play a critical role in determining how genes are expressed [11]. These effects appear to be mediated by direct chemical effects on the DNA or associated proteins (with resulting DNA methylation or histone modification) [11]. This "epigenetic model" supports the observations that while patterns of neonatal cord blood immune responses are associated 61

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with atopic heredity ([12] and others), these can also be altered by environmental modification [1–4]. Thus, as a result of the uterine environment, genes may be differentially expressed or silenced during critical stages of development and dictate future patterns of disease susceptibility. Less is known about local mucosal immune development in the human foetus. In the mature airway, dendritic cell (DC) networks play a central role in processing antigens and programming T-cell responses [13]. These are poorly developed in neonatal animals [14, 15], and there is some evidence that DCs are rarely seen in humans airways, even in the first year of life in the absence of respiratory infection [16], as discussed further below. The development of these networks and associated lymphoid tissues appears to occur largely in the post-natal period and is driven by environmental exposures. The largest source of antigenic load in the post-natal period occurs through the gut, and cells critical to the mucosal associated lymphoid system begin to appear in the foetal gut early in the second trimester [17], including macrophages (14 weeks), T- and B-cells (14 weeks) and DCs (16 weeks) [17]. The role of these cells in the antenatal period is not clear, but there has been interest in the potential effects of cytokines, environmental proteins (including allergens [18]) and other factors (such as sCD14, the soluble form of CD14 [19]) that have been detected in amniotic fluid [17]. Potentially, variations in the content of amniotic fluid, which bathes the respiratory and gastrointestinal mucosa, could modify the local milieu and patterns of maturation. To date, only one study has shown a relationship between amniotic fluid content (sCD14) and the risk of subsequent allergic disease [19], and this needs to be explored further.

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

The effects of environmental factors directly encountered in the respiratory tract. Local encounter with noxious environmental factors, including irritants (such as cigarette smoke), and respiratory pathogens is likely to influence the development of immune networks in the airways. Airways antigen-presenting cell (APC; namely DC) populations appear to have a major role in the late-phase inflammatory response [13, 20] and are therefore likely to contribute to the development of airway damage in inflammatory airway disease. These cells play a critical role in programming T-cell responses following their migration-induced maturation in regional nodes [13]. Age-related immaturity in DC function [21] is associated with reduced capacity for these cells to respond to inflammatory stimuli. Local airway DC networks are less developed in infant animals, and additionally these DC populations display markedly attenuated responses to inflammatory triggers [14, 15]. Similarly, during the first year of life, human infants do not typically show DCs in the airways in the absence of inflammation [16]. However, despite this immaturity, mature DCs do appear in association with severe respiratory infection [16]. This suggests that local tissue events, such as infection, in infancy can influence the maturation of DCs and modify subsequent downstream T-cell programming in early life. 62

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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 Th1–interferon (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 [30–34]. 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 [35–37], 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

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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 [43–46]. 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

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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 [59–62]. 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, 63–67]. 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 [65–67] 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

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risk. It has already been noted that other dietary interventions (using n-3 polyunsaturated fatty acids) can also modify maturation of circulating bone marrow precursors with associated clinical effects [73]. Although this is likely to be mediated through different pathways, it illustrates that the bone marrow is readily influenced by environmental changes. Together, these findings support observations that precursor populations in the bone marrow can be influenced by mucosal events in a bi-directional manner [68], although the mechanisms are not clear. Thus, despite the lack of direct passage through the gut, less mature forms of these cells, which undergo initial maturation in the bone marrow, appear to be influenced by remote events at mucosal surfaces (namely the gut). In summary, collectively this literature provides a strong theoretical basis for future studies to investigate the effects of intestinal microflora and other mucosal exposures on the key cells involved in the development and regulation of allergen-specific response, including direct effects on populations that traverse the gut before homing to effector sites (T- and B-cells), as well as indirect (but independent) effects on bone marrowderived population (DCs and myeloid precursors) before they reach their effector sites.

Defence mechanism in respiratory disease While exogenous/environmental factors may influence the susceptibility to respiratory disease, endogenous factors play a key role in the modulation and interaction of innate and adaptive immune responses in the respiratory tract (fig. 1). These involve a range of local mechanisms, including ancient host defence mechanisms of cell-mediated immunity, such as microbial stimulation and the induction of antimicrobial substances expressed in the respiratory tract in response to pathogens. The collectin family, namely surfactant protein (SP)-D and SP-A, as well as mannose-binding proteins, exert important protective functions in pulmonary host defence, but may also be important in pulmonary disease states, such as allergic inflammation [74–79]. There is also growing recognition that locally produced neuropeptides are involved in immune development. Finally, the generation and control of the oxidative species may be important not only for local defence but also in shaping subsequent patterns of response and disease susceptibility.

Significance of microbial stimulation and antimicrobial substances Epidemiological and murine studies have suggested a role for microbial stimulation in the development or, potentially, prevention of allergic pulmonary disease [80–85]. Endotoxin levels (endotoxin being representative for TLR4 ligands) in child mattresses in rural areas of Austria, Switzerland and Germany were inversely correlated with the occurrence of atopic diseases [81, 86]. In murine studies, administration of TLR4 and TLR2 ligands before allergic sensitisation could reduce allergic parameters in a model of allergic asthma [87]. As allergic immune responses develop early in childhood, exposure to endotoxin early in life may shift the child’s immune response more towards a Th1 phenotype, as found following local LPS administration on the nasal mucosa of nonatopic children [80]. The interaction of T regs with DCs may explain the protective role of endotoxin. An increased gene expression of CD14 and TLR2 on leukocytes in the previously mentioned rural population may be a potential biological marker for microbial exposure earlier in life [88]. Antimicrobial peptides are expressed in the respiratory tract acting as effectors of the innate immune system. Antimicrobial components of the airway secretion include 66

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

Role of surfactant proteins in respiratory diseases Host defence. Proteins produced locally in the respiratory tract, such as the surfactant proteins, are crucial for host defence. While pulmonary surfactant comprises the two hydrophobic proteins, SP-B and SP-C, relevant for adsorption and distribution of surfactant at the air–liquid interface, the collectins SP-D and SP-A have important functions during the immune development in the interaction with adaptive immune responses. One member of the collagenous C-type lectin family, SP-D, is primarily produced in the lung by alveolar type II cells and by nonciliated Clara cells, a subset of bronchiolar epithelial cells [91]. Pulmonary collectins may contribute to protection against local pulmonary inflammation, including cytokine production [74, 92, 93] elicited by gram-positive and gram-negative bacteria [75, 94–99].

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 [100–102]. 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 innate–innate 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

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early stages of allergen challenge in the absence of SP-D [94]. Whether TLR4 expression is SP-D-dependent or co-acting in allergen-induced immune responses requires further investigation. Several studies have highlighted the potential of collectins (including recombinant fragments and protein-free synthetic phospholipid-based surfactant) as therapeutic molecules [93, 107, 109, 110]. SP-D, or molecules derived from these collectins, may be good candidates for prevention or treatment of lung infection, due to their ability to interact with various microorganisms and to regulate the inflammatory response. Interestingly, SP-D is also expressed in extrapulmonary sites such as the GIT, which is, as discussed earlier, crucial in the development of mucosal immunity. Interaction of SPD with other proteins of the innate mucosal immune system in the GIT could potentially contribute to immune modulatory mechanisms. This may have implications in, for example, dietary modulations of the innate intestinal immune system [111], leading to speculation about a more global role of surfactant proteins in local innate host defence. Taken together, it is suggested that pulmonary collectins, such as SP-D or SP-A, participate in the modulation of innate and adaptive immune responses and can influence lung diseases such as infections or allergic inflammation. Whether they might have a therapeutic role in humans has to be further elucidated.

Relevance of mannose-binding lectins in respiratory disease Mannose-binding lectin (MBL) also belongs to the collectin family and represents a pattern recognition receptor of the innate immune system. MBL interacts with a wide range of bacteria, viruses, fungi and protozoa by binding to a selection of sugars such as mannose, N-acetyl-d-glucosamine or mannosamine, fucose and glucose. It promotes phagocytosis by activation of the complement system, as well as through complementindependent direct cell-surface receptor pathways. As a key factor involved in first-line defence, MBL is important for protection against respiratory tract infections [112–114] before the onset of antibody production [115]. Accordingly, MBL deficiency has been associated with increased susceptibility to acute respiratory tract infections, particularly during early childhood [116]. Incomplete activation of the MBL-MASP (MBLassociated serine protease) pathway may also contribute to an increased risk of infectious disease [117, 118]. Although originally identified as a functional opsonic defect [119], MBL may also play an immune-modulatory role potentially through the modulation of cytokine release [120], as seen in other infectious diseases or disease states complicated by infections such as cystic fibrosis, HIV, hepatitis C or autoimmune disease. Although therapeutic applications are still limited, fresh-frozen plasma has previously shown clinical benefit in children with opsonic defects [121]. More recently, purified plasma-derived MBL has become available and is now planned for clinical trials [122] to assess efficacy and to determine appropriate therapeutic indications.

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

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

Importance of oxidative stress in lung diseases While oxidant generation is part of the normal metabolism of most cells, cells involved in first-line host defence can also produce larger amounts of specialised oxidants with bactericidal properties. In the airways, oxidants are produced by activated eosinophils, neutrophils, monocytes and macrophages, as well as resident bronchial epithelial cells. This includes the production of myeloperoxidase (MPO) by neutrophils, monocytes and macrophages or eosinophil peroxidase (EPO) from eosinophils. Common inflammatory conditions, such as, asthma are frequently associated with increased EPO and MPO, as well as other markers of oxidation, such as hydrogen peroxide and nitric oxide, which can be measured in exhaled breath condensates. To counter the potentially noxious properties of oxidants, the lung has a well-adapted antioxidant system. Imbalances between these pathways can lead to excessive oxidative damage (as seen in inflammatory disease states) or increased susceptibility to infection (as seen in congenital disorders of oxidative metabolism). Endogenous antioxidant systems can be either impaired or activated in association with chronic inflammation, illustrating the complexity of these still poorly understood regulatory systems. For example, Cu,Zn-superoxide dismutase (SOD) activity [130] and peroxynitrite inhibitory activity [131] may be decreased in chronic inflammation, whereas other antioxidant systems are increased, including cyclin-dependent kinase inhibitor p21 and extracellular glutathione peroxidase [132, 133]. Studies examining the effects of different levels of exogenous (dietary) antioxidants are also confusing and inconsistent, including the effects of selenium, vitamin C and vitamin E [27, 134–136]. Other promising new antioxidants are planned for clinical trials, such as nitrones, which are radical-trapping antioxidants that inhibit the formation of 69

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intracellular oxidants by forming stable compounds or non-peptidyl SOD analogues [137, 138]. Further research is needed to understand the complex interactions that influence oxidative balance in the local tissues, particularly the therapeutic potential of different antioxidant products.

New perspectives For several pulmonary diseases, targeting the innate immune system could provide therapeutic benefits. For example, for asthma, with a huge prevalence rising over the last 10 years, no new effective regimens have reached the clinics apart from anti-IL-5 and anti-IgE. While corticosteroids are effective in symptom control and blocking inflammatory cells, they do not specifically alter the prevailing Th2 cell response. In this regard, longer-lasting and more effective treatments are needed, and several options are currently being developed. First, immunotherapy could provide therapeutic as well as potentially preventive tools. Recently, efforts are being directed towards using TLR ligation. Central to this approach would be to change the Th2-dominant inflammation, as seen in allergic inflammation to a Th1 response. So far, sustained efforts are on the increase for oligonucleotides containing nonmethylated CpG motifs to shift the balance of Th2-mediated diseases to a Th1-type response [139, 140]. Other potential therapeutic targets, emphasising the potential value of the TLR family as a target for a new generation of immunopotentiating compounds, include other modulators of Th1 responses, such as the TLR7 ligand imiquimod [141], and the TLR4 ligand l-carrageenan, a polygalactan [142]. Stimulation of the immune system through parasites also has to be considered [143]. Other potential targets for therapeutic regimens are DCs and T-regulatory cells, which are pivotal for maintaining the Th1/Th2 balance and immunomodulation. However, the effective use of immunotherapy to date is limited because of the complexity of the immune system, and in particular because of the early development and lack of understanding of the essential mechanisms of immunotherapy, as well as its potential side-effects. Secondly, adjuvants to vaccines are potentially beneficial as they target the innate immune system, and are crucial for prevention and management of infectious diseases. Again, several types of TLR ligands, such as TLR7 agonists (imiquimod or resiquimod), TLR9 agonists (CpG ODNs) and, most compelling, the TLR4 agonists (lipid A analogues) [144], have been shown efficacious as vaccine adjuvants [141]. Thirdly, in acute pulmonary infections, receptor antagonists could be used to induce or enhance host resistance against viral and bacterial infection by activation through TLRs or NOD (nucleotide-binding oligomerisation domain) 1/2 [145]. Fourthly, targeting intracellular pathways may be a strategy for prevention or treatment of several inflammatory diseases, not only regarding the lung. Promising candidates would be the NF-kB signalling pathway [146], including downstream elements such as involved kinases, the MyD88 adaptor family in TLR signalling, the NOD-protein family members and TIR domains [147] and other adaptor proteins.

Conclusion The interaction between the environment and the host has shaped the immune system during evolution; similarly, the ontogenetic development of the immune system is the result of an interaction between the host’s genetic background and its environment. It is proposed that an individual organism’s 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

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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 host’s age, e.g. the same exposure to microbial compounds modulates airway responsiveness differently at different ages [84]. A host’s immune response is therefore not only modulated by gene–environment interactions, but rather by a gene– environment–time interaction, i.e. an interaction between the host’s genetic background, environmental factors and the host’s 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 host’s genetic background and its environment. It is proposed that an individual organism’s 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 host’s immune response is not only modulated by gene–environment interactions, but rather by interaction between the host’s genetic background, environmental factors and the host’s 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

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CHAPTER 6

Allergy and the paediatric lung during development A. Custovic*, K.C. Lødrup Carlsen #, K. Ha˚kon Carlsen} *North West Lung Centre, Wythenshawe Hospital, University of Manchester, Manchester, UK. #Ulleva˚l University Hospital, Dept of Paediatrics, and }Voksentoppen BKL, Rikshospitalet (National Hospital), Oslo, Norway. Correspondence: K.C. Lødrup Carlsen, Dept of Paediatrics, Ulleva˚l University Hospital, NO-0407 Oslo, Norway. Fax: 47 22118663; E-mail: [email protected] and [email protected]

Development of allergic diseases involves many aspects, often mixed, sometimes confused and at other times clarified into discussions of clinical disease, lung function effects or objective findings of allergic sensitisation. The following chapter will focus upon allergic disease phenotypes (wheezy lower respiratory tract disease, asthma and/or allergic rhinitis), allergic sensitisation (the presence of specific immunoglobulin (Ig)E antibodies to allergens and/or positive skin sensitisation) and markers of allergic inflammation in relation to the developing lung in early life. However, understanding lung development and the underlying mechanisms of asthma requires assessment of lung function combined with a thorough knowledge of lung physiology and its relationship with allergic sensitisation and environmental exposures in infants and preschool children. Thus, the present chapter may to some extent overlap with some of the previous and following chapters, which discuss each of these features separately. Risk factors for allergic disease entities such as asthma may not be identical to those for allergic sensitisation. This is exemplified by the increased risk of asthma but not allergic sensitisation, in childhood by exposure to tobacco-smoke products [1]. However, reduced lung function has been convincingly demonstrated in children born to, or living with, smoking mothers [2–7]. Furthermore, the classical triad of atopic eczema, asthma and allergic sensitisation (with allergic rhinitis as the most common manifestation) most frequently appear in that order during early childhood, but any one may develop in the absence of the other manifestations. Thus, deciding whether the clinical entity appearing first is a risk factor for, or a first clinical manifestation preceding, the other allergic diseases as part of an "atopic phenotype" is more than a semantic exercise. It will have implications for understanding of the underlying pathological mechanisms of how and when "allergy" may affect the developing lung. Thus, in the present chapter, an attempt will be made to shed some light on the risk factors for allergic diseases and/or allergic sensitisation versus risk factors for altered lung function development in infants and preschool children. Furthermore, possible interactions between allergic disease and lung development and growth, and when these interactions may occur, will be discussed.

Asthma, wheeze and lung function Most asthma cases begin in early childhood [8], often in association with reduced lung function [9, 10] and/or increased airway responsiveness in infancy [9–11]. Two major Eur Respir Mon, 2006, 37, 79–92. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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studies have recently confirmed that subjects with reduced lung function and persistent asthma in childhood have impairments in lung function that continue into adulthood [12, 13]. In the Melbourne Asthma Study, the magnitude of difference in lung function between different asthma severity groups observed in childhood (aged 7–10 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 9–26 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 2–5 yrs, children are generally too young to cooperate, and the majority are not able to perform adequate forced breathing manoeuvres [14]. Consequently, most of the current research on asthma and allergic diseases in early childhood is based on questionnaires, with no objective measures of lung function. This is less than ideal, and several studies have reported that parents often confuse wheeze with other respiratory sounds, which may lead to under- or overestimation of the true prevalence of wheeze [15, 16]. Furthermore, in a recent large study in which lung function was measured in preschool children and compared between those with parentally reported wheeze, which was either confirmed or not by a physician, children with parentally reported and physician-confirmed wheeze had markedly reduced lung function compared with those with unconfirmed wheeze [17]. However, there was no difference in lung function between children with unconfirmed wheeze (y30% of all parentally reported wheeze) and those who have never wheezed. These findings add further weight to the argument that many parents have little understanding of what medical professionals mean by the term "wheeze" and indicate that the epidemiological studies based only on questionnaires must be interpreted with caution. This emphasises the importance of using objective measures of lung function wherever possible, both in research studies and in clinical practice.

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

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Lung function in early life Development of techniques for use both in naturally sleeping or awake infants [34–36], as well as in sedated infants [37–40], has led to successful assessments of lung function in newborns and infants up to y18 months of age. These techniques have enabled elucidation of factors that influence lung function very early in life (e.g. maternal smoking, maternal asthma, sex) [41–43]. Several studies have suggested that dynamic lung volumes can be reliably measured in preschool children using conventional lung function testing [44–47]. However, whilst all of these studies recruited subjects aged 3– 6 yrs, very few children were at the younger end of the age range (e.g. in one study of 112 children, only nine were 3–4 yrs of age) [45]. When refusal rate is taken into account, the success rate of spirometry among preschool children appears to be only 38.4% [42]. In a recent study of 355 patients, v10% of the 3–4-yr-old children were able to perform three acceptable manoeuvres [46]. Thus, standard spirometry is a difficult, time-consuming and often an unreliable procedure to perform in preschool children. There is a growing interest in developing objective measures of lung function which can be applied in young preschool children to elucidate the end-organ factors involved in asthma development and to allow more accurate identification of children who are likely to be at risk of persistent symptoms. Specific airways resistance (sRaw), which is a measure of airway calibre corrected for lung size, can be measured during normal tidal breathing using a single-step procedure which obviates the need for panting manoeuvres against a closed shutter [48]. sRaw can also be measured with the child accompanied by an adult inside the body plethysmograph [49–51], which makes it a potentially useful respiratory measurement in very young children. Other lung function techniques potentially suitable for use in pre-school children include forced and impulse oscillation and the interrupter technique. However, both the oscillation and interrupter technique have been shown to be less sensitive than sRaw in detecting changes in airway resistance after bronchodilation or airway challenge [52, 53]. The value of tidal flow–volume loops are still debated, even though repeated studies have found reduced time to peak flow6total expiratory time-1 (tPTEF/tE) in newborn babies and infants born to smoking mothers [2, 54, 55], and that reduced tPTEF/tE during infancy was associated with later wheezing respiratory illness [9, 56–59].

Early life lung function and wheeze phenotypes The present understanding of the nature of childhood wheezing illness has been augmented to some extent by the characterisation of distinct wheeze phenotypes in childhood (never-wheezers, transient early wheezers, late-onset wheezers and persistent wheezers) [9, 60]. In a recent study from France, reduced maximal flow at functional residual capacity at age 17 months was associated with persistent but not transient wheeze [61] and, in a Norwegian study, compliance of the respiratory system was reduced at birth among children with asthma persisting from before 2 yrs to 10 yrs of life [57]. However, in the Tucson study, persistent wheezers had significantly reduced lung function only at 6 yrs of age and not during the first year of life, compared with children who had never wheezed [60]. Transient early wheezers tended to have reduced lung function both in infancy and at 6 yrs of age. In contrast, a recent Australian study suggested that transient early wheezers have normal lung function at age 1 month and that reduced lung function in infancy was associated with persistent wheeze by 11 yrs of age [62]. Similarly, among 614 Norwegian children, reduced tPTEF/tE, as well as reduced compliance (but not resistance) of the respiratory system at birth, was associated with a 81

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three-fold increased risk of ongoing asthma at 10 yrs [57]. Although definitions of wheeze phenotypes were not identical, it is still difficult to explain the differences between these studies, as the techniques used to measure infant lung function appear similar. Among 1– 2-yr-old children, reduced tPTEF/tE has been found in asymptomatic children with recurrent wheeze [63] or asthma [64], and has been associated with bronchial obstruction after provocation with methacholine [65]. Recent data on lung function in early preschool age (which was largely unavailable previously) filled the gap in the young preschool age. Among 4-yr-old children enrolled in the Dutch Prevention and Incidence of Asthma and Mite Allergy (PIAMA) study, resistance measured by the interrupter technique was higher in children with persistent wheeze than in children who had never wheezed and those with transient early wheeze [66]. In the Manchester Asthma and Allergy study (MAAS), specific airway resistance at 3 and 5 yrs was reduced in children with persistent wheeze compared with transient early wheezers and non-wheezy children, with transient wheezers falling between children who have never wheezed and persistent wheezers [67]. These data suggest that among young preschool children, both transient and persistent wheezers have reduced lung function compared with non-wheezy children, and the deficit appears to be greater in persistent wheezers.

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

Sensitisation and lung function Recent cohort studies utilising lung function measurement in preschool age have confirmed that lung function is reduced in children with a history of wheeze [68]. However, a striking finding was observed among healthy children at age 3 yrs: lung function was reduced among those sensitised to common inhalant allergens, even in the absence of any respiratory symptoms [68]. Furthermore, parental sensitisation status affected a child’s lung function, but there was no interaction with a child’s atopy. However, whilst there was no interaction between a child’s sensitisation and sensitisation in parents, a significant interaction was observed between maternal asthma and a child’s sensitisation status; if the child was sensitised, there was a significant reduction in lung function in the offspring of asthmatic mothers [68]. This may result from a shared 82

a)

1.0

Predicted probability for persistent wheeze

ALLERGY AND THE PAEDIATRIC LUNG

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1

b)

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Predicted probability for late-onset wheeze

0.0

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.55

0.67

0.82 1.00 1.22 1.49 sRaw at age 3 yrs kPa·s-1

1.82

Fig. 1. – Fitted predicted probability curve for a) persistent wheezing and b) late-onset wheezing by age 5 yrs in relation to specific airway resistance (sRaw) at 3 yrs of age. Taken from [67], with permission.

environment, either antenatally or post-natally. It is worth emphasising that in children who have never wheezed, the size of the difference in lung function between those who were atopic and had a mother with asthma in comparison with all others was greater than the recently reported difference between children with confirmed asthma and healthy controls in a study using the same methodology [53]. Association between atopic sensitisation and impaired lung function have previously been demonstrated in older children. Ulrik and Backer [69] found that sensitisation to dust mite had a negative impact on FEV1 in non-asthmatic children aged 7–17 yrs, with no evidence of airway hyperreactivity. In most studies investigating the relationship between allergy and respiratory disease, sensitisation is considered only as a dichotomous variable, i.e. individuals are assigned as either sensitised or not [70]. Furthermore, various cut-off values have been used to define sensitisation (e.g. w0.35, w0.7 or w1 kUA?L-1 of allergen-specific IgE (where UA is the number of units of allergen-specific IgE) or a skin test reactionw0, 1, 2 or 3 mm) [71, 72]. 83

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1.5

sRaw kPa·s-1

1.4

1.3

1.2

1.1

0.1

0.3

1

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 child’s allergic sensitisation was further extended by a recent study demonstrating that the absolute specific IgE antibody levels offer more information than just the presence of specific IgE [73]. Increasing specific IgE antibody levels to common inhalant allergens (dust mite, cat and dog) or increasing size of the wheal on skin-prick testing were associated with reduced lung function in preschool children (fig. 2) [73]. Total IgE was found to be a poorer predictor of lung function than the sum of specific IgEs. This suggests that labelling subjects as sensitised or not based on arbitrary cut-offs for either specific IgE levels or the size of skin-test wheal is an oversimplification of a trait that may not be dichotomous in its relationship to the paediatric lung.

Allergen exposure, pet ownership and early life lung function The relationship between allergen exposure, sensitisation and the development of asthma is complex. Whilst a dose–response relationship has been demonstrated between mite allergen exposure and specific sensitisation, this exposure does not appear to be related to the development of asthma [74]. The effect of cat and dog ownership and exposure to respective allergens on the development of sensitisation and asthma is even less clear. For example, based on the available evidence, any association between pet ownership, sensitisation and asthma can be supported, i.e. risk, protection or no effect [74]. However, there is little information on the effect of allergen exposure or pet ownership on early life lung function. Recent data from a prospective birth cohort study suggested that children aged 3 yrs who were both sensitised and currently exposed to high levels of sensitising allergen (mite, cat and/or dog) had significantly worse lung function compared with those who were either not sensitised or were sensitised but not currently exposed (table 1) [75]. Therefore, sensitisation per se may have little effect on lung function in preschool children in the absence of exposure to sensitising allergen but has a major effect within the context of specific exposure. Taken together with previously 84

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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 (1.04–1.24) 1.10 (1.05–1.15) 1.21 (1.08–1.37) 1.38 (1.26–1.51)

Taken from [75], with permission.

mentioned data on the quantitative relationship between specific IgE levels and lung function, these data may indicate that the level of IgE (or the size of wheal on skin testing) to a certain degree reflects personal allergen exposure and offers more valuable information about the nature of the relationship between allergy and lung compared with a simple dichotomised atopy parameter. Cat and dog ownership, either at birth or at 3 yrs of age, had no effect on lung function [75]. Furthermore, after adjusting for the history of wheeze, lung function was substantially reduced in children who were sensitised and highly exposed to allergen and had both parents with asthma, compared with those with none or any one of these features. This indicates that there is a genetic component which interacts with environmental exposures affecting early life lung function [75]. A recent study provided the first evidence for the genetic component of the early life lung function, demonstrating the association of ADAM33 polymorphisms with reduced lung function at both 3 and 5 yrs of age [76]. Recent data from the intervention arm of the UK MAAS study raise questions about the nature of the relationship between allergic sensitisation and lung function in early childhood [77]. Stringent environmental control during pregnancy and early life resulted in increased sensitisation to dust mite but better lung function in children at high risk of allergic disease at age 3 yrs, i.e. there was a disconnection between sensitisation and lung function consequent to intervention. The absence of allergen exposure in sensitised children could not explain the observed effect, since lung function was markedly better in the intervention group both among sensitised and nonsensitised children. In children with longitudinal lung function data, there was no difference in lung function between the groups in infancy, but there was a marked difference at age 3 yrs, i.e. the difference between the groups is likely to have arisen after 4 weeks but before 3 yrs of age due to some factor(s) affected by environmental control (fig. 3) [77].

Other environmental influences on lung function Several environmental factors, in addition to those mentioned above, have been shown to reduce lung function or enhance the natural decline in lung function including air pollution in adults [78, 79] and children [80–82], and viral infections in children [83] and adults [84]. One important environmental factor which may have an impact upon lung function development and growth is treatment prescribed for asthma and allergic diseases. Whereas antibiotic treatment in early life has been shown to be related to increased allergic sensitisation [85], no such relationship to lung function has been demonstrated. Conversely, early respiratory tract infections have been shown to have an impact upon lung function in infancy [86], as well as later lung function in childhood [87], adolescence [88] and adulthood [84, 89]. It has been suggested that respiratory tract infections in early childhood may cause chronic obstructive lung disease in adulthood [90]. Respiratory syncytial virus infections, which have been related to later reductions in lung function [88], 85

a)

1.0

In V 'max FRC, GM and 95% CI

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0.8 0.6 0.4 0.2 0.0

In sRaw, GM and 95% CI

b)

HRC HRA Lung function (V 'max FRC) at age 4 weeks

0.4 0.3 0.2 0.1 0.0 -0.1

HRC HRA Lung function (s Raw) at age 3 yrs

Fig. 3. – Prospective data on lung function in the intervention (HRA; n=14) and control (HRC; n=18) groups in the Manchester Asthma and Allergy study in infancy and at 3 yrs of age. V9maxFRC: maximum expiratory flow at functional residual capacity; GM: geometric mean; CI: confidence interval; sRaw: specific airway resistance. Taken from [77], with permission.

have also been related to development of allergic diseases and allergic sensitisation [91], although this remains controversial. In adults, the importance of early anti-inflammatory treatment with inhaled steroids has been demonstrated as being related to airways remodelling [92]. An observational study [93] also indicated this importance with respect to lung function growth in schoolchildren, and a recent report from the Netherlands supported this view [94]; however, another study could not confirm this [95], and its importance is even more highly debatable in younger children. A recent report from a birth cohort study showed that children with recurrent episodes of bronchial obstruction had reduced lung function, as assessed by tidal breathing measurements before treatment was started, and children who later started with inhaled steroids, albeit before the age of 2 yrs, had reduced lung 86

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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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Impact of genetic factors on lung development in health and disease P. LeSoue¨f *, M. Kabesch# *School of Paediatrics and Child Health, University of Western Australia, Perth, Australia. #University Children’s Hospital, Ludwig Maximilian’s University Munich, Munich, Germany. Correspondence: M. Kabesch, University Children’s Hospital, Ludwig Maximilians University Munich, Lindwurmstrasse 4, D-80337 Munich, Germany. Fax: 49 8951604764; E-mail: Michael.Kabesch@ med.uni-muenchen.de

Within the last few decades, genetics has succeeded in identifying the causes of a number of monogenetic inherited diseases caused by defined mutations in single genes. In this process, the genetic causes for a number of rare, and not so rare, lung diseases were established. Foremost, the gene for cystic fibrosis (CF) has been identified [1] andw1,000 disease-associated mutations have been found in this gene, the CF transmembrane regulator (CFTR) gene. However, early studies established that correlations between CFTR genotype and CF phenotype were not straightforward. As in many other so-called monogenetic diseases, what initially looked like a classic monogenetic disease evolved into a complex picture of mutations in major and minor genes [2]. For the CFTR gene, specific CF phenotypes cannot be assigned to given CFTR alterations. Rather, modifier genes seem to be involved in directing a proportion of the clinical expression of the disease [2]. Thus, it became obvious that lung development, breathing itself and lung immunology are the product of a sophisticated network of factors, some of which are under genetic control. Furthermore, common genetic alterations leading to genetic variation within a population will seldom be apparent or cause disease. In general, these modifier genes in respiratory diseases may belong to two different groups of genes: genes that modify lung structure and genes that modulate respiratory as well as general immunity.

Patchwork genetics: small effects add up From a population point of view, and in the light of evolution, genetic variability in these factors makes sense, as a wide spectrum of similar but not identical individuals increase the chance that a species will survive encounters with different and variable environmental challenges. However, in some cases, disease may occur at the edges of this distribution due to excessive variation in these modifying factors, and this may occur for a number of reasons, as follows: 1) when a fundamental lung protein is severely disturbed by a major genetic alteration, as in CF; 2) under certain environmental conditions where the limits of the genetically determined adaptation are exceeded (e.g. chemical exposure at the workplace); and 3) in a situation where the system is overwhelmed, i.e. small genetic changes add up and/or are combined with minor environmental effects which, by themselves, would not be sufficient to lead to disease. This is the case in so-called complex diseases, such as asthma and chronic obstructive pulmonary disease (COPD), where a Eur Respir Mon, 2006, 37, 93–107. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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strong genetic background is present, but the expression of symptoms is dependent on environmental factors; this is more obvious in one disease than the other.

What is normal? Based on this paradigm, with the exception of some monogenetic disorders, genetic susceptibility to disease depends on many genetic factors. Genetic variation is a crucial principle in evolution and is ensured by recombination of maternal and paternal DNA in sexual reproduction. Furthermore, genetic change is achieved by the constant, spontaneous mutation rate of the genome. Even though such an event is relatively rare on an individual level, the effect on a population level is substantial over a long period of time. Thus, more than a million loci in the human genome are currently known to be polymorphic, which means that, on average, one polymorphic locus (base exchange) is present at least every 5,000–10,000 bases. At every single polymorphic site, i3% of individuals differ in the respective allele from the rest of the population. While random mutation is the default setting driving evolution, conservation of the DNA sequence is an active process. Highly relevant genome areas are protected from random changes within the members of a species and are also conserved throughout evolution. Certain structures have remained unchanged between mouse and humans or even between plants and humans. These conserved areas have either critical regulatory functions in the genome, e.g. controlling gene expression, or serve as exons, DNA templates used for transcription and translation into proteins.

Genetic change as a driving force in the interaction with environment Some genes involved in lung development or respiratory immunity have turned out to be highly variable, showing a mutation rate of w1 per 5,000 bases, while others are remarkably conserved. Few known mutations and polymorphisms will alter the function of a gene. Most polymorphisms are functionally silent and evolutionarily neutral. Definite functional changes can only be assigned to a minority of the polymorphisms, so far identified in candidate genes for respiratory diseases. In addition, these functional changes that can lead to increased or decreased gene transcription or translation, a diminished or exaggerated function of the protein, or a change in the structure of the protein, may be subtle rather than substantial. For example, a single base change in the CD14 promoter has been shown to alter the binding affinity of a transcription factor at that position. By itself, this is not a major change, as promoter activity is only decreased by y20% in vitro [3]. Compared with artificially introduced mutations in other parts of the promoter (which do not occur in nature), where a change in function by 10-fold or more can be achieved in an in vitro system, the effect of the CD14 promoter polymorphism seems at first to be unimpressive. However, this is the size of effect that would be expected from a useful genetic variation in nature, as it would alter but not destroy the function of a gene. Common genetic changes do not usually lead to disease by themselves, they simply modestly alter a single gene function, making these polymorphisms valuable for evolution on a population level by increasing variance within the population. However, what is advantageous for a species may be very inconvenient for an individual, as the burden of changes may, by chance, accumulate in certain individuals and then lead to disease. However, other than in many well-defined monogenetic metabolic diseases, 94

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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 individual’s genetically determined susceptibility to develop the condition and the personal history of previous exposure to environmental triggers. However, when histamine is administered in a sufficiently high dose, almost every individual, irrespective of their genetic makeup, will develop bronchial constriction. Overall, very few respiratory diseases may be due to monogenetic disorders, where the function of a single crucial gene is so severely altered that the natural development or function of the lung is fundamentally disturbed. CF is the best known and studied genetic lung disease, and has already been mentioned. Cilial dyskinesia may turn out to be due to a series of related major gene defects caused by a limited number of alterations in ciliarelated genes [4]. In addition, some other monogenetic disorders may affect the lung as part of a multi-organ disease [5]. However, these most severe but rare diseases only make up a small proportion of the vast number of patients seen in respiratory clinics all over the world. Other genes, so-called modifier genes, which may also determine and direct the development and function of the lung irrespective of disease, may have more impact on common diseases such as asthma and COPD.

Genetics of structural genes may influence the modelling and remodelling of the lung in health and disease As genetic research is driven primarily by the aim to understand and finally resolve disease rather than to investigate functional variance in healthy individuals, it is not surprising that most knowledge about the normal function of genes derives from genetic studies in diseases such as asthma, which has been a hotspot for genetics for some time now. In asthma, at least four genes, previously not known to relate to any kind of lung disease, have been identified by a purely genetic tool, so-called positional cloning [6–9]. As it turns out, some of these genes may be involved in basic pulmonary and immunological function. The ADAM33 (a disintegrin and metalloproteinase domain 33) gene is one of these genes and is suspected to be important in lung development and remodelling. Located at chromosome 20p13, it was initially identified by linkage analysis and positional cloning in a joint British and American project [7]. More than 100 common polymorphisms in and around the gene have been discovered and numerous replication studies have been conducted to clarify which ADAM33 polymorphisms contribute to asthma in different populations around the world. These studies have led to controversial results. Replication studies mostly focused on those 19 polymorphisms associated with asthma in at least one of the original populations. While all except three published studies reported some associations between ADAM33 and asthma susceptibility (which may in part be due to a positive publication bias), the amount of 95

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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 [10–16]. 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

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mechanisms of lung development and function, which, in the case of ADAM33, is just beginning.

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

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various respiratory diseases and may also be able to exert nonspecific effects on disease. In addition to effects of structural genes, such as ADAM33 and detoxification genes, genes involved in inflammatory processes may also contribute to lung health and disease. Recent studies have also suggested that structural cells in the lung, such as epithelial and smooth muscle cells, may exert immunological functions.

Lung genes may influence immunity in different ways One of the genes of pulmonary origin that has been initially linked to asthma in genetic studies, but that may turn out to be involved in a much wider range of respiratory diseases, is Clara cell protein 16 (CC16; also referred to as uteroglobulin and Clara cell secretory protein 10). CC16, secreted in large amounts in airways by the nonciliated bronchiolar Clara cells, is a potent immunosuppressive agent, inhibiting the activity of phospholipase A2 [26], interferon (IFN)-c [27], and neutrophil and monocyte migration in the lung [28]. The CC16 gene is located on chromosome 11q12–13, and an adenine/ guanine polymorphism 38 base pairs (A38G) downstream from the transcriptional start site has been identified [29]. Gene expression studies have shown that the 38A allele has a 25% lower transcription level than the 38G allele, and this difference in expression levels could therefore also decrease the CC16-associated anti-inflammatory protection of the lung in carriers of the 38A allele. Several genetic studies (but not all) have associated the CC16 polymorphism with asthma [30, 31] and, in a study of German children, asthmatics with the 38AA genotype showed increased airway responsiveness to histamine or exercise [32]. In addition, the presence of CC16 has also been associated with protection from various forms of pulmonary disorders, such as acute respiratory distress [33] and oxidative stress reactions [34]. Recently, studies have investigated whether these asthmaindependent effects are also influenced by genetic alterations. In a German population of 117 cases of acute respiratory distress syndrome and 373 controls, the same A38G polymorphism was found to alter neither the susceptibility nor the outcome of the disease [35].

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

Genetics and the development of enhanced allergic responses Since the late 1990s, a large number of studies have examined genetic variations in common immunological pathways to determine their potential effect on the development of allergy and atopy. Most of these studies have reported associations between particular polymorphisms and atopic or respiratory phenotypes. Many of these observations have been replicated in more than one population. Some are backed by positive linkage data and sound functional data that reasonably establish the causal nature of these relationships. A smaller number of studies have also included measurement of tissue levels of the output of the gene in question. The combination of replicated associations 98

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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 8–25 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

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Fig. 1. – Number of positive skin prick tests (SPTs) with genotype CD14 C-159T from age 8–25 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.

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examples of such relationships. For interleukin (IL)-4 receptor (IL-4R), several lines of evidence suggest that IL-4R polymorphisms contribute to the development of asthma in children. IL-4 is a Th2 cytokine and, together with its receptor, is involved in the generation of atopic inflammation. Several polymorphisms have been found in IL-4 and functional studies have demonstrated in vivo differences between alleles for C-589T [46, 47]. In a recent study, IL-4 -589T was more frequent in children with asthma compared with controls, and IL-4R 576Q was more frequent in children with atopic asthma [48]. The importance of haplotype analyses in such studies was demonstrated by the association between the IL-4 -34T/-589T haplotype and asthma and between the IL-4R I50A/576Q haplotype and atopic asthma [48]. A further study showed that there were also interrelationships between IL-4R and IL-13 polymorphisms [49]. Several other significant relationships between alleles in Th1 and Th2 pathway genes and asthma phenotypes in children have been demonstrated [50].

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

Other consequences of the early impairment of the immune system in atopic children The main focus of researchers in examining the early maturation of the immune system has been to understand the way in which Th2 responses evolve. This focus on the pathway to IgE production has provided a great deal of useful knowledge. However, as noted above, recent research has uncovered co-existing problems with Th1 responses. These problems are well illustrated by Rowe et al. [53], who studied cytokine responses to vaccination with diphtheria and tetanus toxins. They noted that, after vaccination, the ratio of the Th1 cytokine IFN-c to either of the two Th2 cytokines (IL-5 or IL-13) was significantly lower in those with versus those without a family history of atopy (fig. 2), 100

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5

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Fig. 2. – Mean ratios of interferon (IFN)-c to interleukin (IL)-5 from in vitro stimulation of peripheral blood mononuclear cells with tetanus toxoid in children with or without an atopic family history (AFH). F: AFH -ive; &: AFH zive. Reproduced from [53] with permission.

but that when present at 6 and 12 months of age, this situation had resolved itself by 18 months of age [53]. This impairment in Th1 responses appears to be associated with an impaired ability to resist more the serious consequences of respiratory syncytial virus infection [54].

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

Genetics of impaired antibody responses The important point of these studies with respect to the influence of genetics on the development of the immune system and the consequences for the respiratory system is that genetic mechanisms can be expected to play a major role in causing these problems. Recent genetic studies have begun to demonstrate where the problems might lie. For example, in a study of responses to the seven-valent pneumococcal vaccine, for each of 101

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the seven antigens, children with the CD14 -159C allele had lower specific IgG responses than those who had at least one -159T allele [58]. CD14 -159C was also associated with lower serum levels of CD14 and increased numbers of episodes of otitis media compared with the CD14 -159T allele. Given the consistent associations of the -159C allele with atopy, this study provides strong evidence linking the CD14 -159C allele with atopy and impaired vaccine responses. Further work on the same cohort has shown similar findings for IL-4 C-589T, IL-4R G2979T and IL-4Ralpha Gln551Arg, and, in each case, the allele associated with increased IgE responses was associated with decreased specific IgG responses to each of seven pneumococcal antigens [58]. Hence, genotypes associated with increased IgE responses in children are also associated with decreased specific IgG responses to foreign proteins.

Influence of maternal smoking on the relationship between genetics and immune responses in early life Passive smoke exposure, whether via the placental circulation in utero or via the air post-natally, has been shown in a large number of studies to be associated with decreases in lung function [59] and increases in airway responsiveness measured soon after birth [60, 61] and also later in childhood [62]. In addition to effects directly on lung tissue modified by lung genes and detoxification enzymes, smoke exposure has also been implicated for increases in atopic responses [63, 64] and respiratory infections [65]. An important study that provides evidence of a possible mechanism for the effect of smoking was recently published [40]. In this study, which was a genome-wide screen for linkage related to asthma, no evidence of linkage was found for the cytokine-rich area of chromosome 5q.31. However, when only children of females who smoked during pregnancy were examined, a highly significant result was obtained [40] (fig. 3). These data point to maternal smoking exerting a specific and potent environmental effect on particular genes in an area of the human genome where a number of immunity genes are 3 .0

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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.

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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 infant’s ability to produce specific antibodies is impaired if they have specific variations in certain genes associated with atopy and that this problem is greatly increased by exposure to parental smoking.

Hypothesis arising from data: wheeze in early life is related to a relative state of immunodeficiency For many years, researchers have recognised that the prevalence of wheeze is greatest in infants and that maternal smoking is a major environmental risk factor for infant wheeze. Research efforts have focused on attempting to unravel the exaggerated IgE responses that are associated with allergy and asthma. Some of these studies have produced data on specific IgG and cellular responses and have shown that these responses are often impaired in atopic children. More recent studies have demonstrated that maternal smoking makes a major environmental contribution to further impairing these responses. In the last year or two, genetic studies have begun to dissect these impaired responses by being able to show related allele-specific data in genes in the Th2 inflammatory pathway. These data can be used to generate the hypothesis that wheeze in early life is a consequence of a relative state of immunodeficiency that co-exists in those with a predisposition to atopy and that this state of immunodeficiency is further impaired by maternal smoking or other genetic changes in lung structure. When the immune system matures during the first few years of life, the immunodeficiency largely resolves. In other words, the reason wheeze is so common in early life may be that there are large numbers of infants who are poorly equipped to fight respiratory viral infections and the infants with the greatest problem in this area are those with delayed post-natal maturation of the immune system whose mothers smoked during pregnancy. Clearly, much more work is needed to substantiate this hypothesis and to determine whether it offers new avenues of therapeutic intervention.

Conclusion Taken together, a complex picture of interaction between genetic factors and lung development in health and disease evolves. Genetic variation affects lung structure and lung development, as well as general and respiratory immunity. The sum of these variations represents an individual’s 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.

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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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with delayed maturation of the antibody response to pneumococcal vaccine. Clin Exp Immunol 2000; 122: 16–19. Prescott SL, Sly PD, Holt PG. Raised serum IgE associated with reduced responsiveness to DPT vaccination during infancy. Lancet 1998; 351: 1489. Holt PG, Rowe J, Loh R, Sly PD. Developmental factors associated with risk for atopic disease: implications for vaccine strategies in early childhood. Vaccine 2003; 21: 3432–3435. Wiertsema S. Innate and adaptive immunity in otitis media. PhD Thesis. University of Utrecht, Utrecht, The Netherlands, 2005. Young S, Sherrill DL, Arnott J, Diepeveen D, LeSouef PN, Landau LI. Parental factors affecting respiratory function during the first year of life. Pediatr Pulmonol 2000; 29: 331–340. Goldstein AB, Castile RG, Davis SD, et al. Bronchodilator responsiveness in normal infants and young children. Am J Respir Crit Care Med 2001; 164: 447–454. Adler A, Ngo L, Tosta P, Tager IB. Association of tobacco smoke exposure and respiratory syncitial virus infection with airways reactivity in early childhood. Pediatr Pulmonol 2001; 32: 418– 427. Turner SW, Palmer LJ, Rye PJ, et al. Determinants of airway responsiveness to histamine in children. Eur Respir J 2005; 25: 462–467. Noakes PS, Holt PG, Prescott SL. Maternal smoking in pregnancy alters neonatal cytokine responses. Allergy 2003; 58: 1053–1058. Weiss ST, Tager IB, Munoz A, Speizer FE. The relationship of respiratory infections in early childhood to the occurrence of increased levels of bronchial responsiveness and atopy. Am Rev Respir Dis 1985; 131: 573–578. Peat JK, Keena V, Harakeh Z, Marks G. Parental smoking and respiratory tract infections in children. Paediatr Respir Rev 2001; 2: 207–213. Baynam GS, Kusel M, Khoo SK, et al. Association between IL-4 C-589T and total specific IgG to diphtheria toxoid and parental smoking in children at risk of atopy. Am J Respir Crit Care Med 2005; 2: A693.

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Gene–environment interaction and respiratory disease in children J. Gerritsen*, N.E. Reijmerink*,#, M. Kerkhof }, D.S. Postma# Depts of *Paediatrics, #Internal Medicine and Pulmonology, and }Epidemiology and Statistics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. Correspondence: J. Gerritsen, University Medical Center Groningen, Beatrix Children’s Hospital, P.O. Box 30.001, 9700 RB Groningen, The Netherlands. Fax: 31 503614235; E-mail: j.gerritsen@med. umcg.nl

Determining the exact genetic aetiology of complex lung diseases, such as asthma, remains a significant problem. The main reasons for this are that asthma is polygenic, interaction between genetic (host) and environmental factors is involved, and there is a wide heterogeneity of asthma phenotypes [1]. A genetic basis for asthma has been demonstrated in numerous family studies. The findings were consistent, irrespective of whether the study was performed in twins, trios or by segregation analysis of extended pedigrees [2]. Many investigators have found evidence of linkage between genetic markers and asthma, as well as its associated phenotypes, and to date seven genes have been found by positional cloning [1]. For many years, the role of environmental exposures to viruses, nonspecific stimuli and allergens in the daily morbidity of asthma and atopy has been recognised. An example of the direct relationship between exposure and morbidity is the early and late asthmatic reaction that occurs after exposure to house dust (mite), and the subsequent increase in response to nonspecific stimuli [3]. Thus, nowadays it is accepted that next to genetic basis, the environment also plays an important role in asthma development. More specific genes, as well as their interactions, have been recognised as important and as crucial factors in the development of asthma and atopy [4–7]. 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 [6–9].

Gene–gene interaction Gene–gene 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, 108–119. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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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 [14–18]. 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 gene–gene 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. Gene–gene 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 gene–gene 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 gene–gene interaction in complex diseases, since this may elucidate pathways that play a role in disease development, severity and progression.

Gene–environment 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. Gene–environment interactions can be assessed in case–control 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 60–80%, leaving a remaining 20–40% for environmental 109

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contributions [28–30]. Until recently, genetic and environmental susceptibility were studied separately, or the potential interaction between the two sources was evaluated by stratifying the effect of exposures by family history [30]. Significant interactions, demonstrated in both linkage and association studies, indicate that many early life exposures influence the risk for asthma and its related phenotypes in a genotype-specific manner. These early life exposures include exposure to endotoxins [31–34], viruses [5, 35], pets [36, 37], day care environment [5] and environmental tobacco smoke [38–41]. Several models of gene–environment interactions in asthma and atopy have been suggested by Vercelli [42], and modified by Martinez [6] and Ober and Thompson [7]. For example, the CD14 gene is considered as a potentially critical player in the gene– environment interactions leading to asthma and atopy. Several studies have shown an association with variations in the CD14 gene and atopic phenotypes, such as IgE [9, 42, 43]. The importance of the gene is also confirmed by genetic linkage studies, which suggest that one or more loci on chromosome 5q controls for the levels of serum IgE [43– 45]. Considering gene–environment interactions, theoretically, several models can be postulated as already published [6, 7]. The first model is that the phenotype is expressed when the genotype is present; environmental factor does not have an influence on the onset of disease (fig. 1). Thus, the strength of the expression of the phenotypes depends on the genotype and is not influenced by environmental exposure. In a real-life situation it is not easy to find a good example. A previously mentioned example is CF, in which the severity of the disease is predominantly determined by the genetic effect and the gene–gene interaction [10]. Nevertheless, also in CF, infections as an exogenous factor and pancreatic insufficiency as an endogenous factor contribute to the expression of the severity and prognosis of disease. In the second model (fig. 2), the environmental influence is the same for all genotypes, regardless of the level of exposure. The influence of the genotypes on the strength of the phenotype is always identical. The consequence of this is that in all environments the estimated heritability of a disease is always similar. If the studies are well designed and well performed the results will be highly reproducible irrespective of the populations, with the exception that, as in CF, gene–gene interactions are not crucial in the expression of the phenotype. A well-known example is phenyketonuria (PKU), a recessive disorder

Phenotype value

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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: ? ? ? ?.

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Fig. 2. – The association between a phenotype and environmental exposure is plotted for different genotypes. There is no gene–environmental interaction; therefore, the curves for the three genotypes are parallel. Genotype A: –––; genotype B: - - -; genotype C: ? ? ? ?.

of metabolism in which phenyalanine cannot be converted to tyrosine. The gene for phenylalanine hydrolase has been cloned and mapped to chromosome 12q24.1 [46]. More than 240 mutations have been defined in this disease. There is little variation in the presentation of the disease between the different genotypes in the presence of phenylalanine in the diet. In all patients with PKU, restriction of dietary intake of phenylalanine can completely prevent disease development in all genotypes. The third model (fig. 3) presents the extent genotype influence on the phenotypes can vary in different environments. Clear examples include the high exposure to microbes in the farming environment and the low exposure to house dust mite in the high-mountain environment. Both environments decrease the expression of the asthma phenotype, the former by high exposure to microbes and the latter by strong reduction of house dust

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Fig. 3. – The association between a phenotype and environmental exposure is plotted for different genotypes. Model of reaction of a case in which gene–environment interaction is present. The variation is explained by gene–environment interaction. Genotype A: –––; genotype B: - - -; genotype C: ? ? ? ?.

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

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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: ? ? ? ?.

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Figures 1–4 provide an impression of possible relationships between genes and environment. Asthma is a complex disease in which many different genes and multiple environmental factors (endotoxins, air pollution, viral infections, bacterial infections, food, pesticides, heavy metals, environmental tobacco smoke exposure, etc.) play a role. Thus, given the myriad possible interactions between these genes and environmental factors, it is still a simplification of one reality. Many clinicians feel that investigating these relationships is like searching for a needle in a haystack or looking at a mathematical model with an infinite number of variables. The best defined phenotypes for asthma and atopy are BHR, lung function and IgE. Figure 5 demonstrates the possible role of genes in the expression of phenotypes and the interaction with the environment. Gene A has a direct effect on BHR without any influence from the environment, while the environment affects the influence of gene B on BHR, leading to increased BHR. Gene C has no effect on BHR and is also not influenced by the environment, but is directly related to lung function. Gene D and E only initiate an elevated IgE when both genotypes are present and are influenced by the environment. A wide variety exists in phenotypes of genetically manipulated plants and of invertebrates in which the genetic traits and environment can be fully controlled. This effect is ascribed to phenotypic plasticity, which is the development of different phenotypes for the same genotype in the same environments [52]. Studies in humans into the relationships of asthma, allergies and environmental factors have investigated an immense number of variables, making these studies very complex and, generally, meaning they offer multi-interpretable results. Phenotypic plasticity is likely to add to this complexity, especially in males.

In utero environment It has long been assumed that the safest place for the child is the womb, since it protects the genes from any environmental influence. However, it is becoming more and more clear that many intra-uterine factors can play a role in the development of the respiratory system and the evolution of the immune system. The target of a toxic insult to the lungs during its development is likely to involve the disruption and/or alteration of a specific molecular signal or transcription factor but, to date, little information is available as to the precise effect of such exposures. An important aspect is timing of exposure during development, which appears to be critical to its effects. For example, Gene B

Gene C

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Environmental factors

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Fig. 5. – The possible role of genes in the expression of phenotypes and the interaction with the environment. BHR: bronchial hyperreactivity; Ig: immunoglobulin.

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maternal malnutrition during gestation may significantly retard foetal growth and the development of the lungs, leading to compromised lung function throughout life [53]. In contrast, exposure to environmental toxins, such as passive cigarette smoke, may actually accelerate the maturation of specific cell types in the foetal lung [54, 55]. The results of such an effect on overall lung-function changes from the newborn to the adult age are unknown. In general, very little is known regarding the precise effects of maternal personal exposure, such as vitamin intake, smoking and nutritional factors, air pollution, viral infections, etc. on the foetus. It is likely that the exposure affects growth changes of the respiratory system which may continue after birth. A limitation in the research of prenatal effects on the development of the respiratory system is that, for example, exact lung function measurements can only be reliable and performed on a large scale from y2– 6 yrs of age, depending on the method of lung-function measurement used. Consequently, pre-natal and post-natal effects are difficult to disentangle. Nevertheless, it has been suggested that changes of the respiratory system later in life are already measurable shortly after birth [56]. Children from mothers with asthma have a greater risk of asthma compared with children from fathers with asthma, which refers to the importance of the pre-natal environment on subsequent risks. This "parent-of-origin" effect, in which an allele is associated with asthma or atopy only when it is inherited from the mother, has been confirmed in several studies [57, 58]. A study carried out in 200 Dutch families showed that the influence of susceptibility genes for asthma might become apparent with exposure to cigarette smoke only in utero and early childhood [59]. The studies mentioned previously provide strong circumstantial, though not physiological, evidence that in utero and early childhood exposure may contribute to disease development early or later in life in interaction with genetic factors.

Sex as an endogeneous factor The influences of maternal and paternal history of atopy and asthma on asthma in the offspring differ, as already stated. In addition, studies on cord-blood IgE show that the influence of maternal history of atopy or asthma is stronger in young males than in young females, suggesting that hormonal factors in the offspring may modify the effects of maternal or paternal inheritance [60]. During childhood and adolescence, young males are nearly twice as likely as young females to develop asthma and this continues until the age ofy14 yrs [61, 62]. A change to female predominance occurs during late adolescence in females, which exists throughout adulthood, and asthma tends to be more severe in female adults [63–65]. The exact mechanisms of these differences on the molecular and genetic levels are unravelled. Before puberty, no differences are observed in the production of sex hormones; changes during puberty lead to the typical differences between males and females. From the onset, the airways of females are smaller than males. From this perspective it might be expected that young females have more respiratory symptoms than young males. However, this is in contrast with what has previously been found in young males and females. Therefore, it is evident that the differences in respiratory symptoms, allergy and asthma between young males and females cannot be accredited to anatomical differences in the respiratory system. An explanation for this shift might be the presence of important genes on the x-chromosome, which are switched off during childhood and switched on during puberty and adulthood. During the switch-off phase in childhood, the influence of the genes is minimal and makes the immune system behave similarly in young males and females. During puberty and adulthood, the genes on the extra x-chromosome are switched on, which may induce a predominant influence on the disease expression and severity of asthma. Whether these 114

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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 Xp22.3–Xp22.2, 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 Xpter–Xqter and Xq13.1–Xq28, 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 [72–74]. Another important gene located on chromosome Xq13.2–21.1 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 (gene–gene interaction) and whether other genes on the x-chromosome play a role in the sex-related differences in disease expression have still to be elucidated.

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

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time, thus allowing a genetical–genomics approach towards identification of genes in asthma and its related phenotypes [77]. It can be expected that with the development of these techniques and the possibilities of advanced analysis, the links between asthma and allergy genes, environmental factors and the development of asthma will be unravelled in the future.

Summary The exact genetic aetiology of asthma is complex. The reasons are that in asthma more than one gene is involved, there is interaction between genetic (host) and environmental factors, and there is wide heterogeneity of asthma phenotypes. The present chapter discusses gene–gene interaction, gene–environment 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, gene–environment interaction, gene–gene interaction.

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CHAPTER 9

Clinically relevant early functional and diagnostic markers of lung disease in children J.C. de Jongste*, E. Baraldi #, E. Lombardi } Depts of Paediatrics *Erasmus University Medical Center, Sophia Children’s Hospital, Rotterdam, The Netherlands. #University of Padova, Padova, and }Anna Meyer Children’s University Hospital, Florence, Italy. Correspondence: J.C. de Jongste, Dept of Paediatrics, Erasmus University Medical Center, Sophia Children’s Hospital, PO Box 2060, 3000 CB Rotterdam, The Netherlands. Fax: 31 104636811; E-mail: [email protected]

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

Fractional concentration of nitric oxide in exhaled air Since the 1990s, the exhaled nitric oxide fraction (Fe,NO) has been extensively studied and validated as a noninvasive marker of airway inflammation in asthma, and has been standardised for use in clinical practice [1]. There are now detailed guidelines for Fe,NO measurement in children and adults [1, 2]. Normative values have been published for children (fig. 1) [3]. It is now well established that Fe,NO is the first bedside test that reflects eosinophilic airway inflammation in the bronchial mucosa. In older children, the preferred measurement technique requires a single breath, constant low-flow exhalation against a resistance in order to avoid contamination with nasal air. The airflow is sampled and fed directly into the analyser. Results are immediately available. The chemiluminescence analysers for nitric oxide (NO) measurement are expensive and technically complicated, and are mainly used in academic research centres. Recently, compact hand-held analysers have been developed which are much less expensive and will facilitate more widespread introduction of the method. To date, the main utility for Fe,NO is in clinical asthma management. Allergic asthmatics have high Fe,NO that shows a rapid, dose-dependent response to corticosteroids. Treatment decisions in asthma have traditionally been made on the Eur Respir Mon, 2006, 37, 120–141. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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40.00 35.00

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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

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l

1000 900 800 700 600 500 400

s s

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300 200

100

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Start

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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

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80 s

60 40 20 0

s s l l

-2

s s l l

s l l

l l

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Fig. 3. – Individual follow-up of exhaled nitric oxide fraction (Fe,NO) in asthmatic children after discontinuation of inhaled steroids at t=0 because of clinical remission. Those children who developed a relapse show a steep increase in Fe,NO ahead of symptoms; stable children had nitric oxide values that remained low. +: relapse after 36 days; ': relapse after 35 days; #: individual who remained asymptomatic; $: individual who remained asymptomatic. Individual nitric oxide values of four children are shown, two without relapse ($ and #) and two with relapse (+ and '). Data were obtained from [6].

Fe,NO in young children Wheezing in infants is common. Some young children wheeze with infection only during the first years of life and do not subsequently develop asthma; however, some may have early childhood asthma. Clinically, it is difficult and often impossible to identify those infants who are more likely to become asthmatics. For these reasons the need for 122

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developing practical, noninvasive markers to reflect asthmatic airway inflammation is especially important in young children who wheeze, in whom other objective diagnostic tools, such as spirometry or bronchial challenges, cannot be easily applied in clinical practice [2]. There are only a limited number of reports on the use of Fe,NO in infants and preschool children with asthma [7–15]. Recently, Malmberg et al. [11] demonstrated that Fe,NO is superior to lung function and bronchodilator responsiveness in identifying preschool children with asthma. In agreement with this study, Avital et al. [9] showed that Fe,NO can differentiate young children with asthma from nonasthmatic children with chronic cough. In children with recurrent wheeze, raised Fe,NO values suggest the presence of eosinophilic airway inflammation and these patients may, therefore, be most likely to respond to treatment with inhaled corticosteroids (ICS). Recently, Moeller et al. [16] have demonstrated that moderate doses of ICS reduce levels of Fe,NO in the absence of significant changes in lung function and symptoms. Similar findings with a reduction of Fe,NO have been reported in infants after therapy with montelukast [14]. In a large epidemiological survey, Brussee et al. [12] found that 4-yr-old children with symptoms of asthma and sensitisation had a higher Fe,NO than healthy children, but the difference was much smaller than in older children, which would limit the applicability in individual children. Moreover, high-risk children from allergic parents had similar Fe,NO values to children from nonallergic parents, suggesting that Fe,NO values may have a limited potential as a predictor of sensitisation in 4-yr-old children.

Methods for measuring Fe,NO in infants and preschool children In 2002, a joint European Respiratory Society (ERS)/American Thoracic Society (ATS) task force on exhaled NO measurement in children published a statement providing recommendations and suggestions for the measurement of Fe,NO in young children [2]. On-line measurement of Fe,NO during spontaneous breathing has been applied in children aged 2–5 yrs [7]. Fe,NO is measured on-line during spontaneous breathing and the exhalation flow is manually adjusted at 50 mL?s-1 by changing the exhalation resistance. The method still requires passive cooperation inasmuch as the child needs to breathe slowly and regularly through a mouthpiece. Measurements during tidal breathing with uncontrolled flow are technically easier and therefore attractive. The tidal breathing method in infants is potentially simple and noninvasive and both on- and off-line techniques have been applied without the use of sedatives [8–9, 17, 18]. Currently, there is no standardised tidal breathing method to recommend for use in infants and young children and more research is needed to solve some methodological issues [19]. As Fe,NO is flow-dependent, a scatter of data due to variation in expiratory flows is possible. The disadvantage of mixed expiratory air is that it may be contaminated with ambient NO and NO from the upper airways. Whilst the inspiratory NO contamination can be limited by inhalation of NO-free air, the use of a two-compartment face mask can limit nasal contamination [20, 21]. There is limited experience with single-breath methods for measuring Fe,NO in infants. A modification of the raised-volume rapid thoracoabdominal compression technique has been used to measure Fe,NO during a single, slow forced exhalation [10]. NO levels are measured on-line; the plateau of NO achieved during constant expiratory flow is then determined [10, 21]. With this technique, it is important to use a two-compartment face mask to separate nasal and oral compartment [21]. This method is limited in that sedation, specialised equipment and skilled operators are needed. 123

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

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

Exhaled breath condensate In the past years there has been increasing interest in measuring exhaled breath condensate (EBC) compounds in subjects with pulmonary diseases [26–28]. Exhaled breath obtained through the cooling of exhaled air contains water vapour and microdroplets whose composition appears to reflect airway lining fluid [27]. This fluid contains various nonvolatile and over 200 volatile substances. EBC consists of w99% of water generated by the respiratory tract. A much smaller fraction is derived from respiratory droplets released from the airway surfaces and subsequently incorporated in the water deposited in the condenser. The condensate does not, however, contain inflammatory cells of the airways. EBC collection is totally noninvasive and is therefore particularly easy to perform in children, including those with severe disease [29]. Unfortunately limited information is available concerning its use in preschool children [30]. Recently, an ATS/ERS task force has developed guidelines on standardisation and analysis of EBC [31]. The principle of sampling the airways by EBC is that mediators from airways are released from the airway lining fluid, carried up by exhaled breath and subsequently collected by condensation of the exhalate [26–28, 32]. In order to collect EBC, children are asked to breathe tidally for 10–15 min. The use of noseclips is controversial because of the possibility of nasal contamination. A saliva trap is recommended because many relevant mediators may be present in large amounts in the saliva. By checking amylase activity of EBC, saliva contamination can be excluded [31]. EBC can be collected from children as young as 3–4 yrs of age using the same technique as used in adults. It is possible to use a face mask, and EBC has been successfully collected from babies in this 124

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way. Collection by continuous aspiration through a nasal cannula or from a mechanical ventilator circuit can also be considered [30, 33]. Disadvantages of EBC analysis are that most of the measurements are not on real time and concentrations of mediators in EBC are close to the detection limit of current available assays which were developed for use in other media (blood, urine) with higher mediator concentrations than condensate. To date the majority of exhaled markers have been measured by immunoassay. More sensitive techniques, such as high-performance liquid chromatography and gas chromatography/ mass spectrometry (GC/MS), should therefore be explored to validate or replace commercially available immunoassay (table 1). Potentially important variables, which may influence the composition of EBC, include minute ventilation, humidity of inspired air, collection temperature, and nasal and salivary contamination [32]. Several markers of inflammation and lipid peroxidation have been detected in EBC of asthmatic adult and children (table 2). A large number of mediators have been measured in EBC, including hydrogen peroxide, isoprostanes, prostaglandins, leukotrienes, nitrogen oxides, aldehydes, cytokines, etc., and new molecules continue to be added to this list. In addition, the acidity of EBC can be measured [27].

Hydrogen peroxide Oxidative stress contributes to the pathogenesis of several inflammatory lung diseases. Hydrogen peroxide (H2O2) is a marker of oxidative stress and it is one of the more extensively studied markers in asthma. H2O2 in EBC can be measured by colorimetric or fluorimetric methods. Jo¨bsis et al. [34] have defined reference values in healthy children. H2O2 levels are related to the eosinophil differential counts in induced sputum and to airway responsiveness [35].

Leukotrienes Cysteinyl leukotrienes (cys-LTs) are inflammatory metabolites derived from arachidonic acid through the 5-lipoxygenase pathway. They are potent airway constrictors and pro-inflammatory mediators. LTs can be measured by enzyme-linked immunoassay (EIA) and GC/MS in EBC [36]. Increased values of EBC cys-LTs have been found in allergic asthmatic children despite corticosteroid treatment [37]. Interestingly, normal values of EBC cys-LTs were found in atopic nonasthmatic children, suggesting that eicosanoids are involved in the pathogenesis of asthma [38]. Reduced cys-LTs values were reported after 3 months of house dust mite avoidance in allergic asthmatic children [39].

Table 1. – Exhaled breath condensate assays Colorimetric or fluorimetric Immunoassays ELISA Radioimmunoassay Analytical techniques Gas chromatography/mass spectrometry High-performance/liquid chromatography Ion chromatographic method Liquid chromatography/tandem mass spectrometry Future Metabonomics, proteomics, infrared laser spectrometry

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Table 2. – List of potential inflammatory markers in exhaled breath condensate Compound class

Examples

Eicosanoids

8-Isoprostane Cys-leukotrienes Leukotriene B4 Prostaglandins PGE2 PGF2a Thromboxane

Hydrogen peroxide Lipid peroxides

Malondialdehyde a,b-Unsaturated aldehydes Saturated aldehydes

Glutathione Ammonia NO products

Nitrites Nitrates Nitrotyrosine Nitrosothiols Cytokines IL-1b IL-2 IL-6 IL-8 Tumour necrosis factor

Proteins

PGE2: prostaglandin E2; PGF2a: prostaglandin F2a; NO: nitric oxide; IL: interleukin.

Cytokines Cytokines in EBC are usually quantified by EIA/ELISA kits. Several different cytokines have been identified in EBC, although at very low levels, close to the lower limit of detection. Increased level of interleukin (IL)-4 and decreased level of interferon (IFN)-c were described in EBCs of asthmatic children [40].

Isoprostanes Isoprostanes are mediators of oxidative stress [41]. They are relatively stable and specific for lipid peroxidation, which makes them potentially reliable biomarkers. Isoprostanes can be measured by EIA kits and GC/MS. Increased levels of 8-isoprostane have been found in asthmatics despite treatment with ICS, suggesting that these drugs may not be fully effective in reducing oxidative stress [37, 42]. 8-Isoprostane concentration is also elevated in patients with COPD, interstitial lung disease and CF. Aldehydes are products of lipid peroxidation found in EBC that seem to reflect oxidant-induced damage of the airways. Elevated levels of malondialdehyde measured by liquid chromatography-tandem mass spectrometry were recently detected in children with asthma exacerbation [43]. Nitrotyrosine is a stable compound expressing involvement of NO-derived oxidants in the lung. It can be measured with EIA, is increased in the EBC of asthmatic subjects and is associated with worsening of asthma symptoms [44]. Glutathione is a protective antioxidant in the lung. Glutathione levels in condensate have been measured by liquid chromatography with fluorescence detection. Reduced 126

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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 acid–base status in the airway of asthmatic patients [27]. Similar results have been found in children with stable asthma [45]. Furthermore, EBC pH levels correlate with inflammatory cells in induced sputum, suggesting that EBC pH may reflect ongoing inflammation [46]. Measurement of EBC pH is highly reproducible.

Condensate The evidence suggests a potential role of EBC in the monitoring of airway inflammation and oxidative stress. However, the lack of standardisation of EBC collection and analysis is currently the primary limitation of this technique and is likely to explain most of the variability of the results reported in the literature. In addition, longterm prospective studies correlating EBC findings with measures of disease control and established measures of lung pathology (bronchoalveolar lavage (BAL) analysis, biopsy histology) are necessary to demonstrate and validate the clinical relevance of EBCderived markers.

Markers in blood and urine Several markers in blood and urine have been evaluated for prediction and diagnosis of asthma, and for monitoring asthma and CF; studies on other respiratory diseases are still lacking.

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

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Eosinophils and their products Eosinophils and their products play an important role in allergic inflammation and asthma. An eosinophil blood count i4% at 1 yr of age has been shown to be a risk factor for persisting asthma at 13 yrs of age in wheezing children v6 yrs of age, and has been included in a clinical index to define the risk of asthma in young children with recurrent wheezing [55]. Eosinophil granule proteins, mainly eosinophil cationic protein (ECP) and eosinophil protein X (EPX), have been measured in serum as indirect parameters of eosinophil activity. In a recent study of 968 children aged 6 yrs, serum ECP levels were found to be higher in children with current asthma and severe atopy, suggesting that serum ECP assessment might be helpful in detecting persistent asthma [56]. However, sensitivity and specificity are too low for diagnostic use in individual patients [57, 58], and there is no additive value in detecting asthma compared with a family history of atopy [58, 59]. Determination of serum eosinophil granule proteins may reflect the effects of anti-inflammatory treatments on eosinophil activity [58]. The clinical use of these measurements for assessment of asthma severity has not been validated [58, 60]. Since the results depend on sampling procedures and are affected by circadian and seasonal variations, they should be performed under standardised conditions [58, 60]. EPX, a toxic protein present in eosinophil granules, is released by activated eosinophils. It is the only basic eosinophil protein that can be measured accurately in urine (uEPX) [61]. uEPX can be regarded as a marker of eosinophil degranulation in vivo [62]. uEPX levels in allergic asthmatic children were found to be significantly higher than in healthy controls [63–66]. uEPX levels were increased in symptomatic compared with asymptomatic children with asthma, and were significantly elevated during acute asthma exacerbations [67–68]. Treatment with inhaled steroids reduced uEPX [66]. As would be expected for an inflammation marker, the association between uEPX/c, where c is [creatinine], and pulmonary function tests is either weak or absent [67, 69]. Others found no correlation between uEPX/c and BAL cell counts in asthmatic patients [62]. Measuring uEPX in urine is a simple and attractive test that should be further explored for monitoring eosinophilic airway inflammation in children.

Cytokines Cytokines, such as IL-1, IL-4, IL-5, IL-6, IL-8, IFN-c, granulocyte-macrophage colony-stimulating factor and tumour necrosis factor-a, have been measured in peripheral blood in patients with asthma, CF and other respiratory disorders, as well as in healthy controls [70]. IL-10 production by peripheral blood monocytes has been shown to be reduced in subjects with atopic asthma [71] and increased in the convalescent phase of respiratory syncytial virus infection in infants with subsequent recurrent wheezing [72]. It has been proposed to be helpful in distinguishing atopic asthma from other nonatopic wheezing conditions [73]. However, cytokine measurements for clinical purposes are strongly limited by their poor sensitivity and the difficulty in interpreting the results [74].

Pulmonary function tests Pulmonary function tests (PFTs) play an important role in the diagnosis and monitoring of paediatric lung disease [75]. Although many children with lung disease may present with normal PFTs, the evidence of impaired ventilatory function and a bronchodilator response may be very helpful for the diagnosis and severity assessment of paediatric lung disease [76, 77]. A thorough description of paediatric PFTs has recently 128

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been published in this series [78]. This section will focus on the clinically relevant aspects of PFTs and bronchial challenge tests in diagnosing and managing lung disease in children.

Pulmonary function tests in school children In the "cooperative" child (i6 yrs old) spirometry is the most used PFT. Spirometry is relatively simple to perform and is repeatable. In asthmatic children, spirometric parameters detect airflow limitation and help to diagnose and monitor the disease [79]. Asthma guidelines thus recommend the performance of spirometry at the time of diagnosis, after the treatment has been started (to document an improvement in lung function), every time the treatment is changed and at regular intervals depending on the severity of the disease [79, 80]. The forced expiratory volume in one second (FEV1) has been shown to be the most reliable spirometry parameter and may be used to classify the severity of airway disease [80]. However, in conditions with intermittent airway obstruction, such as asthma, a normal spirometry does not exclude disease and a bronchial challenge test can be considered. A recent study has demonstrated that, in 5– 18-yr-old asthmatic children, FEV1 is generally normal and does not correlate inversely with asthma severity classified by symptoms frequency and medication usage, whereas the ratio of FEV1 to forced vital capacity (FEV1/FVC) declines as asthma severity increases [81]. The mean forced expiratory flow between 25 and 75% of FVC (FEF25–75%) is more sensitive for peripheral airway obstruction than the other spirometric parameters [82]. However, the high variability of FEF25–75% in the general population limits its use to detect airflow limitation in routine clinical practice [83]. Peak expiratory flow (PEF) has been used to monitor lung function and help adjust asthma treatment. However, it has been shown that daily PEF measurements in children are able to detect only about one-third of the clinically important episodes of deterioration, while children may also report false positive episodes of PEF decrease [84]. The measurement of PEF variability in children is also difficult to interpret in clinical practice due to the high diurnal variability in a high percentage of normal subjects [85]. For these reasons, the use of portable home spirometers (capable of measuring FEV1, FVC, FEV1/FVC and FEF25–75%) has been proposed [82, 86]. Nevertheless, the use of PEF meters is still recommended, along with that of (mini)spirometers, in asthma guidelines as an objective measure of lung function [75, 79–80]. Several studies have shown a relationship between the level of pulmonary function in childhood and subsequent lung function and respiratory symptoms in adulthood. FEV1 as percentage of predicted (FEV1 % pred) in children has been found to predict adult FEV1 [87]. In a recent study, a higher FEV1 in childhood and more improvement in FEV1 from the ages of 5–14 yrs to 21–33 yrs were associated with both complete and clinical asthma remission at ages 32–42 yrs [88]. The same study showed that 57% of subjects in clinical remission had bronchial hyperresponsiveness and/ or a low lung function at age 32–42 yrs, supporting the view that defining remission only on the basis of symptoms and need for treatment will overlook subjects with subclinically active disease and possibly associated airway remodelling [88].

Pulmonary function tests in preschool children The poor cooperation of children v6 yrs of age in performing standard PFTs has limited lung function evaluation, especially in preschool children (3–6 yrs old). Children in this age group are too old to be sedated for infant PFTs (see below) and too young to perform the manoeuvres required in the PFTs for school-age children. Recently, several techniques that only require passive cooperation have become commercially available. 129

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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 [91–98]. Most of the reference values were collected in the field, showing that the interrupter technique is suitable for epidemiological studies [13]. Studies evaluating Rint changes in response to bronchodilator treatment have shown that Rint is able to detect changes in airway calibre after bronchodilator in preschool children [99, 100]. However, the definition of a cut-off value for a clinically significant decrease in Rint in response to bronchodilator inhalation and the role of Rint in challenge tests remain to be established. The good repeatability and feasibility of Rint measurements, as well as the agreement with other PFTs [101, 102], and the applicability over a wide age range make the Rint attractive for the assessment of lung function in preschool children both in research and clinical practice [103, 104].

Fig. 4. – Measurement conditions of the interrupter technique in preschool children.

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Forced oscillation technique The forced oscillation technique (FOT) is another method to assess respiratory mechanics. Like the interrupter technique, FOT is performed at tidal breathing. Recommendations for its use have recently been published [105]. The principle of FOT is that an external pressure (forced oscillations) applied at the upper airways will cause a mechanical response of the respiratory system (changes in airflow and pressure) that can be measured to determine respiratory impedance with its two components, resistance (Rrs) and reactance (Xrs). The frequencies of flow oscillations, generated by a loudspeaker, usually vary between 4 and 32 Hz. In children, Rrs is frequency dependent, with higher Rrs at lower frequencies [105, 106]. From clinical studies, it appears that Rrs at low frequencies (6–8 Hz) allows the best discrimination between healthy subjects and various obstructive conditions [107]. The feasibility of FOT Rrs in children ranges between 79 and 95% [91, 108]; the reproducibility in children and adults is similar. Rrs and Xrs are also useful indices in establishing positive reactions to bronchial challenge tests [107, 109]. Normative data on reference values and bronchodilator response in healthy and asthmatic subjects were reported for preschool children [91, 95, 108, 110–112].

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

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

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results reported so far suggest that MBW is promising for detecting early lung disease in preschool children.

Spirometry Spirometry has also been performed in preschool children. Several studies show that, under specific conditions, its feasibility in preschool children ranges between 47 and 92% [120, 121] and can be improved by the use of incentive software [120]. Recommendations for spirometry in preschool children have recently been published [122]. Furthermore, reference values have been reported for spirometry in preschool children [123, 124] and clinical data on the usefulness of spirometry in preschool children with CF have been published [125].

Bronchial challenge tests Since bronchial hyperreactivity (BHR) is one of the characteristic features of asthma [80], its presence is often helpful in the diagnostic process. The main pathological factors that underlie BHR are presumably airway inflammation and bronchial remodelling [126]. However, the relationship between BHR and asthma is complex in children. While asthmatic children have BHR, BHR is not the same as asthma [127]. BHR has been reported in patients with CF or allergic rhinitis and in 7–33% of asymptomatic children [128]. In addition, there is no close correlation between airway inflammation markers in induced sputum and methacholine BHR in asymptomatic children [129]. However, most children with recurrent wheezing have BHR [127, 128]. These conflicting findings can be partially explained by the kind of stimulus used to assess BHR. Bronchial stimuli are generally described as either "direct" or "indirect". Direct stimuli include methacholine, carbachol, histamine and arachidonic acid metabolites; they cause bronchoconstriction by directly activating contraction of bronchial smooth muscle cells after binding to their relevant receptors [125]. Indirect stimuli include exercise, adenosine, cold air, hypertonic solutions and ultrasonically nebulised distilled water. Their effect is considered to be mainly due to the release of mediators from intermediary cells (mainly mast cells) [130]. Since the 1990s, an increasing body of evidence has shown that indirect stimuli have a better correlation with airway inflammation than direct stimuli [130, 131]. This helps improve current understanding of why indirect stimuli are reported to be more specific and less sensitive for a diagnosis of asthma, while direct stimuli have proven to be more sensitive and less specific. Bronchial challenge tests are not necessary to diagnose asthma when PFTs show reversibility after bronchodilator inhalation or when the clinical picture is highly suggestive for asthma. Demonstrating or ruling out BHR may be important in difficult cases. The choice of bronchial stimulus to be used depends, as always, on the question to be answered. A direct challenge will be very helpful in ruling out asthma (when negative), while it will not be able to confirm it (when positive) [126]. Conversely, an indirect challenge will be more helpful in confirming a diagnosis of asthma (when positive) than ruling it out (when negative) [126]. It has been proposed to perform serial BHR measurements to monitor anti-inflammatory therapy in adult asthmatics [132]. However, the benefits of this strategy, fewer exacerbations, were at the cost of a higher steroid dose. Guidelines for methacholine and exercise challenge tests [133] and for indirect challenge tests [130] have been published. Studies on whether BHR can be a predictor of asthma in asymptomatic children has so far produced conflicting answers [134, 135]. Cold air challenge at 6 yrs of age was a significant predictor of a low level of lung function at 16 yrs of age [136]. Studies 132

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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 1–13yr-old children with isolated chronic cough is a strong risk factor for the development of asthma 10 yrs later [138].

Infant pulmonary function tests The lung function techniques that can be used for infants have recently been reviewed [78]. In general, sedation is required for infant pulmonary function tests (IPFTs), as the infants cannot actively cooperate. The various IPFT techniques are limited to specialised paediatric lung function laboratories in academic clinics, as they require complicated and expensive, often custom-built equipment and dedicated, trained personnel to obtain reliable results. A growing number of clinical studies employing IPFTs have been published, and results of IPFTs have proven useful in understanding the epidemiology of infant lung disease, to document the nature and extent of lung involvement in various diseases, to assess treatment effects and to follow normal and pathological lung development. In all of these fields, the impact on diagnosis and management on the individual level has remained limited. Tests that can be applied to infants include plethysmography, flow-volume measurements during tidal breathing and forced expiration (rapid thoraco-abdominal compression or "squeeze"), with or without prior inflation of the lungs towards near-total lung capacity, interrupter resistance and compliance, and gas-mixing techniques to assess ventilation homogeneity, as described above. Also, exhaled air can be analysed for inflammatory markers, including exhaled nitric oxide, although the methodology has not been standardised [2]. To date, IPFTs have been important as clinical and epidemiological research tools. Their value as diagnostic or monitoring tests in routine clinical practise is limited by the demanding methodology and need for sedation, which preclude frequent routine clinical use of current IPFT techniques.

Imaging techniques Lung function tests are relatively insensitive to detect localised damage to the lungs and airways [139]. Imaging by means of computed tomography (CT) is superior to lung function for the assessment of progression of lung disease in CF [140, 141]. Standardised CT scores have been developed and routine monitoring of CF lung disease by means of CT scanning has become clinical routine in several CF centres. The radiation dose associated with regular CT scanning is still a concern, and in a worst case scenario may cause a small increased risk of cancer, depending on the assumptions [141]. Development of new magnetic resonance imaging (MRI) techniques to replace CT scanning would solve the problem of radiation dose, and MRI is a promising alternative for the future. However, the quality of MRI images of the lung is still far inferior to that of CT, but this could be improved by using specific techniques such as the use of inhaled gases to enhance contrast. Until now, imaging techniques have not played an important part in diagnosis and monitoring of prevalent lung diseases. No specific radiological abnormalities have been found in asthma, although airway wall thickness has been shown to be increased on CT, and was to some extent associated with disease severity [142]. 133

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Important future questions The study results of Fe,NO in diagnosing and monitoring of asthma as described above critically depend on the cut-off levels of Fe,NO and symptom scores, and it is unclear what the effect of other cut-off levels or alternative dosing schedules would have been. Other ways of dealing with Fe,NO are still unexplored, for instance the use of personal-best values, or the effect of more frequent monitoring. Fe,NO reflects not only inflammation, but also the direct effects of steroids and viral infections. This raises the question as to whether steroid downtitration is the proper response to a reduction of Fe,NO. Doubling the steroid dose has only limited effect on elevated Fe,NO in asthmatic children with elevated Fe,NO, despite conventional doses of inhaled steroids, and the mechanism behind this observation is not clear [5]. It is possible that monitoring of Fe,NO may be more useful for tapering than for stepping up steroids. Individual Fe,NO data may be puzzling and seem to suggest heterogeneity in the Fe,NO response to steroids. This may be due to faulty inhaler techniques, but there may also be genetic heterogeneity. Other exhaled markers of airway disease, including all those in breath condensate, are still in a very preliminary stage; clearly, standardised methodology, issues of reproducibility and biological validity and prospective evaluation is needed for condensate markers of interest. New techniques to assess condensate components, including mass spectrometry and gas chromatography, hold promise for the future development of this interesting area. The exact role of the various PFTs in the clinical management of children with lung disease remains to be determined, both in infants [143] and in older children [144, 145]. In addition, there is a need for more feasible tests of infant lung function that can be applied without the need for sedation. Clearly, reliable prediction of asthma is an important issue with potentially great implications. Combinations of lung function tests and inflammation markers, together with genetic information and knowledge of exposures may well turn out to be reliable predictors of future chronic illness and need to be explored. Not covered in this chapter are genetic studies, which hold great promise for the identification of children at risk for certain lung diseases, and have already been shown to be capable of diagnosing asthma with high accuracy based on patterns of gene activation, as shown by gene array chips. Several questions regarding the role of bronchial challenge tests need to be answered. The merits of BHR in young children in whom cut-off values are unclear need to be established. Cut-off values in adults (for methacholine challenge, usually a provocative concentration causing a 20% fall in FEV1 of 8.0 mg?mL-1) have also been used in children without any dose adjustment [95]. It has been pointed out that this practice is likely to be inappropriate in young children [95], since smaller children would receive a higher dose relative to their lung size, thus helping to explain the reported higher BHR in younger children. Indeed, lower doses and different levels of response were found to be more appropriate for young children [138]. Several studies have assessed bronchial reactivity in preschool children [13, 34, 81, 101, 121, 134, 146], however, due to the lack of data on the bronchial response to inhaled stimuli in healthy preschool children, the use of bronchial challenge tests in this age group remains at present a research tool. Imaging techniques should be further developed and explored for their potential as diagnostic, prognostic and monitoring tools in paediatric lung disease. It may be possible to overcome the present limitations of MRI for this purpose in order to limit the radiation dose associated with more sophisticated radiographic techniques, such as highresolution CT and volumetric CT.

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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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Clinically relevant early functional and diagnostic markers of lung disease in the paediatric intensive care unit A. Schibler *, J.J. Pillow #,} *Queensland Paediatric Intensive Care Service, Brisbane, Queensland, #Telethon Institute for Child Health Research and Centre for Child Health Research, and }School of Women’s and Infants’ Health, University of Western Australia, Perth, Australia. Correspondence: A. Schibler, Queensland Paediatric Intensive Care Service, Paediatric Intensive Care Unit, Mater Children’s Hospital, South Brisbane 41010 QLD, Australia. Fax: 41 738401642; E-mail: [email protected]

Respiratory care in paediatric and neonatal intensive care has undergone significant changes in recent years. New ventilatory strategies, such as lung-protective ventilation using permissive hypercapnia or high-frequency oscillatory ventilation (HFOV), have been introduced, whilst various forms of synchronised and noninvasive ventilation are increasingly utilised. In extremely pre-term babies with respiratory distress syndrome, there is a growing trend away from prolonged mechanical ventilation to prophylactic surfactant administration and early extubation to nasal continuous positive airway pressure. Many of these new concepts of respiratory support originate from studies in adults and there is a relative paucity of objective evidence regarding their efficacy in paediatric patients. There is a growing awareness of the clinical importance of functional and diagnostic markers to predict the outcome (defined as risk of death or risk to develop chronic lung disease) of children suffering from acute respiratory distress syndrome (ARDS) and bronchiolitis. Currently, markers are not applied in a standardised fashion, with little integration of markers between physiological, immunological, genetic or structural markers of disease severity and outcome. This is further complicated by rapid and ongoing change in treatment strategies, and understanding of pathophysiology. Nonetheless, an overview of current knowledge and thinking, and outlining potential future approaches is potentially worthwhile to promote discussion and the development of new study hypotheses.

Current concepts of acute respiratory failure in paediatric intensive care units Acute respiratory failure or ARDS comprises the severe end of the spectrum of acute lung injury (ALI), which is a frequent presenting feature or complication of critical illness. ARDS is diagnosed if arterial oxygen tension (Pa,O2)/inspiratory oxygen fraction (FI,O2) is v200 mmHg (26.6 kPa) and bilateral chest infiltrate without cardiogenic cause is present. It is a heterogeneous lung disease, which may have a pulmonary or extrapulmonary cause [1], the presence or absence of pre-existing lung disease prior to ARDS, or the presence or absence of immune compromise in the patient. Representing a major health problem to critical care physicians, the heterogeneity of the disease and lack Eur Respir Mon, 2006, 37, 142–152. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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

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

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

Current concept of lung recruitment Patients with ARDS are characterised by a reduction in the range of pulmonary volume excursions, because of the reduction in ventilated units (collapsed or fluid-filled alveoli), and a smaller change in volume per unit of change in pressure (decreased tissue compliance due to oedema). ARDS patients require additional PEEP to keep the lung open at end-expiration and increased peak inspiratory pressures to deliver adequate tidal volume for gas exchange. The pressure–volume (PV) curve is used as an orientation for setting optimal PEEP in patients with ALI or ARDS [21]. The initial part of the PV curve (fig. 1) represents the amount of pressure required to open collapsed peripheral alveoli, followed by a portion of the curve with impaired elastic properties (lower inflection point). With increasing pressure, a steep, almost linear, section of the PV curve is observed. Recent mathematical models suggest that the steep part of the PV curve is not characterised by optimal stretching of alveolar tissue, but by the "popping open" of individual lung units and increases in the volume of the lung unit from zero to that appropriate for its trans-alveolar pressure [22]. The pattern of alveolar recruitment follows a power law and is often referred to as an "avalanche" [23, 24]. Each increment in pressure causes an increase in volume of newly recruited alveoli that is much greater than that of alveoli which are already inflated. As the rate of recruitment diminishes and finally stops, the PV curve flattens at higher applied pressures and overdistension of alveolar structures occurs (upper inflection point). During deflation, the PV curve exhibits a similar sigmoid shape but with a significant hysteresis compared with the inflation. The slope of the initial part of the deflation limb shows the total compliance of all alveoli that were inflated at end-inspiration. When pressure during deflation decreases

Volume L

UIP

LIP Pressure cmH2O Fig. 1. – An example of a pressure–volume curve. LIP: lower inflation point; UIP: upper inflation point.

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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 pressure–impedance curves (equivalent to PV curves) from a healthy subject in the right lateral position. The pressure–impedance curve of the left (nondependent) lung is less steep and reaches a plateau at lower pressures compared with the right (dependent) lung. Thus, even in healthy lungs, "local" PV curves are significantly different if measured in the dependent and nondependent lung. As clinical PV curves are indicative of global rather than regional lung recruitment, they are unable to describe the heterogeneous disease character of injured and healthy zones. The rate of recruitment and de-recruitment at a clinically chosen pressure level may be optimal for one lung region but not so for another. If the pressure is too low, no opening of collapsed alveoli occurs; if it is too high, potentially harmful overdistension and VILI may occur in less-diseased areas of the lung.

Techniques to measure pressure–volume curves PV curves may be obtained using a super-syringe technique [27, 28]. The lungs are inflated stepwise with fixed volume steps until an airway pressure of 45 cmH2O is achieved. Alternatively, a rapid airway occlusion technique can be used at different points in the respiratory cycle during mechanical ventilation. Newer ventilators now produce a PV curve of the patient using a constant inspiratory flow or constant increase in airway pressure. These methods require paralysis and sedation and are currently restricted to relatively few commercially available ventilators. 0.25 0.20 Impedance change

0.15 0.10 0.05 0.00 -0.05 -0.10 -0.15 -0.20

3 5

10

15

20

25 30 Pressure

35

40

45

50

Fig. 2. – The impedance change of the global (——), and right (– – –) and left (-----) lung are displayed against pressure change for a healthy subject obtained in right lateral position, where impedance is measured relative to baseline tidal volume breathing. The graph shows that impedance change is relatively higher in the right lung than in the left lung. The left lung reaches a plateau earlier than the right lung. The left lung in right lateral position is already well expanded at end-expiratory level and experiences less inflation during the recruitment manoeuvre than the right lung.

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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 2–3 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 (PA–a,O2). In neonates, the PA–a,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

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pressure w40 cmH2O and a PA–a,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

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recent animal evidence in an infant rat model suggests that infantile lungs are more tolerant to high tidal volumes than adult rat lungs [39]. In the study by Kornecki et al. [39], infant and adult rats were exposed to large tidal volumes either proportional to body weight or total lung capacity. The visco-elastic properties, inflammatory markers and lung histology were significantly more deranged after ventilation in the adult rats than infant rats. In infants without pre-existing lung pathology, Plotz et al. [40] showed that a remarkable, predominately pro-inflammatory immune response could be observed after 2 h of mechanical ventilation, if they are ventilated with tidal volumes of 10 mL?kg-1.

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

Clinical markers predicting outcome Flori et al. [33] recently reported mortality rates in 320 patients with ALI; they found that 54% of patients with near drowning, 39% of patients with associated cardiac disease and 31% of patients with sepsis died. Mortality rate was greater in patients presenting with two or more nonpulmonary organ system failures. Interestingly, children with CNS dysfunction had the greatest mortality risk. They also investigated whether outcome could be predicted from the ventilatory settings required for adequate ventilation and they found that Pa,O2/FI,O2 ratio and OI predict poor outcome (unlike in adults). If the patient at admission presents with a Pa,O2/FI,O2 ratio v100 the mortality rate is 34%, whereas if the Pa,O2/FI,O2 ratio is 200–300 at admission then the mortality rate is only 13%. Traber et al. [35] identified additional clinically important information that can be obtained. Patients with ARDS after bone marrow transplant and immune suppression have the highest mortality rate (63 and 41%, respectively). Ex-premature infants with chronic lung disease have a mortality risk of 33%. Not surprisingly, both studies found that immune-compromised patients are more likely to succumb to the immense inflammatory burst in ARDS, and it is common knowledge that ARDS patients often die because of multiple-organ failure secondary to lung injury (as well as harmful mechanical ventilation). In ex-premature infants with chronic lung disease, additional lung injury may reduce the functional capacity of the lung to below the level that is compatible with life. The more restricted the functional capacity of the lung, the more likely an unfavourable outcome. High supplemental oxygen and high prior Pa,CO2 are poor prognostic factors. These children die either due to chronic respiratory failure or because 148

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of intercurrent respiratory infection/aspiration causing a severe ARDS. In summary, the higher the PEEP, mean airway pressure and the initial oxygen requirement, the more likely it is that the patient will die.

Genetic markers. Respiratory syncytial virus (RSV) bronchiolitis is the most common, severe lower-respiratory-tract infection leading to PICU admission. It is well established that RSV infection in infancy is associated with recurrent wheezing and asthma during the first years of life [44]. Furthermore, it is well recognised that infants with certain preconditions are more likely to present with severe RSV infection, including expremature infants with chronic lung disease, infants with cystic fibrosis and infants with congenital heart defects [45]. Of these infants, y40–50% 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 [49–51]. 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 [52–54]. 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.

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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 15–20 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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Wheezing disorders in young children: one disease or several phenotypes? J. Grigg*, M. Silverman# *Academic Division of Paediatrics, Queen Mary’s School of Medicine and Dentistry, London, #Division of Child Health and Institute for Lung Health, University of Leicester, Leicester, UK. Correspondence: J. Grigg, Academic Division of Paediatrics, Queen Mary’s School of Medicine and Dentistry, 4 Newark Street, London, E1 2AT, UK. Fax: 44 1162523282; E-mail: [email protected]

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, 153–169. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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Wheeze is generated at sites of flow limitation

Airway calibre

Velocity of airflow

Mucus or other obstruction downstream

Properties of the airway wall

Fig. 1. – Factors that affect the volume and pitch of wheeze from a vibrating flow-limiting segment. Indirectly, wheeze is also affected by the compliance of the lungs and the degree to which expiration is forceful rather than passive.

due to misunderstandings result in underestimation of the true prevalence of wheeze in high-risk groups [4]. Most reported wheeze in preschool children occurs in acute, short-lived episodes, in association with viral (upper) respiratory infection and in the absence of interval symptoms (fig. 2) [7]. This contrasts with the situation in later childhood asthma. The distinction has lead to some inconsistency of nomenclature. Some use the umbrella term "asthma" for all common preschool wheezing disorders (i.e. those with no specific diagnosis), assuming them to represent "variable, widespread intrathoracic airway narrowing" and therefore to conform to the basic definition of asthma. Others reserve the term asthma for (mainly allergic) children with both virus-induced wheezy episodes (or "attacks") and chronic (interval) symptoms. This debate itself encapsulates the question:

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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].

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is preschool wheeze a single disorder or are there several distinct phenotypes of wheezing disease?

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

Evidence supporting different phenotypes of preschool wheeze Preschool wheezing disorders in general (and childhood asthma in particular) represent complex interactions of many processes, both endogenous (genotype or physiology) and exogenous (environment, including intra-uterine environment; therapeutic agents) in the developing child (undergoing alveolisation and lung growth). It would be surprising if the symptoms of variable wheezing, representing variable airway obstruction, were not the common end-point of a number of discrete disease processes, each with its distinctive pattern, pathophysiology and therapeutic response.

Clinical and epidemiological evidence. Wheeze is reported by w30% of young children during the first 3 yrs of life. The prevalence falls to about half in older children. There is much evidence that this nonprogressive or "transient" wheeze [5] is a separate phenotype, distinguished from "persistent" or "late-onset" wheeze in several respects (table 1). The research literature is bedevilled by inconsistent nomenclature, itself evidence that more than one phenotype exists (table 2). The most common clinical phenotype in children v3 yrs of age is exclusive viral wheeze, a disorder characterised by acute episodes of wheeze, cough and breathlessness in association with viral respiratory tract infections, with few or no interval symptoms (fig. 2). This phenotype accounts for about two-thirds of wheezers v3 yrs of age. Episodes may be severe enough to warrant hospital admission, but the illness is generally mild and resolves with age. It is sometime referred to as "post-bronchiolitic wheeze" (although acute bronchiolitis is a relatively rare precursor of exclusive viral wheeze) or, in the USA, "recurrent bronchiolitis". In cross-sectional [8] and longitudinal [9] studies, the prevalence of viral wheeze falls with age and has similar features to transient wheeze. Whether exclusive viral wheeze and early transient wheeze are identical conditions remains to be proved.

Genotype

Environment

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Fig. 3. – The concept of phenotypes. "Therapy" is an environmental variable, introducing a feedback mechanism.

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Table 1. – Features common to both "transient" wheeze and exclusive viral wheeze in early childhood, which distinguish them from "persistent", "late-onset" childhood wheeze and classical atopic asthma Childhood prognosis Lung function Allergy Maternal smoking Nursery attendance Long-term prognosis Ethnicity Inflammatory basis Parental influence

Better (by definition, in the case of transient wheeze) Developmental airway dysfunction prior to onset# No more common than in reference population Significantly increases risk Significantly increases risk More rapid decline in FEV1(?); marker for COPD(?) Less frequently reported in preschool children of South Asian origin No eosinophilic airway inflammation between episodes Maternal w paternal influence

FEV1: forced expiratory volume in one second; COPD: chronic obstructive pulmonary disease. #: This has not been confirmed by all research groups. Table 2. – Some commonly used labels for phenotypes of preschool wheezing disorders Label Retrospective labels Transient wheeze Persistent wheeze Late onset wheeze Clinical labels Viral wheeze Exclusive viral wheeze# Asthma} Post-bronchiolitic wheeze

Definition Wheeze early in life (usually v3 yrs) which remits Wheeze early in life which persists (usually to school age) Wheeze which develops only after early life (usually w3 yrs) A discrete episode (or "attack") of wheeze in association with (viral) RTI Wheezing only in association with viral RTI, without interval symptoms Wheezing both with and between viral RTI Wheezing episodes that follow acute infantile bronchiolitis for a variable period of time

RTI: respiratory tract infection. #: Synonym: recurrent bronchiolitis (USA); }: synonyms: chronic wheeze, viral wheeze with interval symptoms, or "multiple-trigger" wheeze.

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

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

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bronchoalveolar lavage found no evidence of airway eosinophilia in preschool children and infants with severe attacks of wheeze, some of whom had persistent symptoms [15, 16]. The discrepancy may, in part, be due to the lack of consistency in defining the phenotypes of wheeze. In interpreting data on airway inflammation during acute attacks of preschool viral wheeze, it is important to consider the setting where the sampling has taken place. In a primary-care setting, the majority of children presenting with an acute asthma attack will have the "exclusive" viral wheeze phenotype. In a secondary care setting, it is possible (but as yet unproven) that there will be a shift towards children at risk of, or with, atopic asthma and interval symptoms. In children hospitalised for viral wheeze, systemic eosinophil activation, systemic neutrophil activation and increased cysteinyl leukotriene production have been reported [17–21]. To date, none of these indirect markers have been validated using direct airway sampling. However, in an adult model of exclusive viral wheeze, in which healthy adults who wheezed only with colds were experimentally infected with coronavirus, McKean and co-workers [22, 23] found that acute wheeze is characterised by neutrophilic, but not eosinophilic, inflammation in the lower respiratory tract. In summary, there is good evidence to show that children who wheeze exclusively with colds do not have chronic eosinophilic airway inflammation, and that during an acute viral-triggered attack, neutrophilic inflammation predominates. There is, however, a minority of preschool wheezers who have eosinophilic airways inflammation between viral-triggered attacks. Many uncertainties remain. The point of reference for inflammation in asthma, i.e. classical atopic asthma, has itself undergone recent re-evaluation. For example, viraltriggered attacks of classical atopic asthma are associated with neutrophilic, and not eosinophilic, airways inflammation [24, 25]. It is therefore possible that exclusive viral wheeze is atopic asthma without a propensity to develop chronic inflammation. Future research should be directed at noninvasive sampling during acute wheeze episodes. Indeed one study suggests that induced sputum can be collected in symptomatic infants and during asymptomatic interval periods [26]. The key issue related to inflammation during the interval periods between viral-triggered attacks is how to accurately identify chronic eosinophilic inflammation in uncooperative toddlers. Indirect markers of eosinophilic inflammation, such as urinary eosinophil protein-X [27], are, at best, nonspecific predictors of eosinophilic activation in the airways. Similarly, the association between exhaled nitric oxide and airway eosinophilia is not as strong as first hoped, at least in adults [28]. Analysis of exhaled breath condensate holds promise as a means of exploring differences in the underlying pathophysiology [29]. For children with severe symptoms, it may be ethically acceptable to perform more invasive tests, such as bronchoscopy and bronchial biopsy, before embarking on long-term, high-dose inhaled corticosteroid therapy.

Physiological evidence. There is little information on pulmonary function during acute wheezy episodes in young children. Whether the pattern of physiological disturbance during episodes could distinguish between different phenotypes seems unlikely. However, between episodes of viral wheeze, in older children [30] and in adults [22], there is little evidence of either persistent airway obstruction or of bronchial hyperresponsiveness (BHR), in contrast to the usual situation in classical atopic asthma. Conversely, the little data that exist for recurrently wheezy infants and young children suggest that between episodes there may be both airway obstruction and abnormal airway wall compliance [31]. There are inconsistencies in the data on BHR. While BHR during symptom-free intervals in wheezy infants is no different from nonwheezy control subjects [32], BHR measured in the first weeks of post-natal life predicts both infant wheeze [33] and 157

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subsequent asthma [34]. However, BHR is clearly associated with classical atopic asthma in schoolchildren. To explain these apparent contradictions, it must be proposed that the "partial phenotype" of BHR is not a single, uniform condition, but that the mechanisms of BHR in infancy (BHR of developmental origin) differ from those in atopic asthma (BHR of inflammatory origin). The issue may be clarified by systematic measurement of BHR using direct challenge (e.g. with methacholine) and indirect, inflammation-sensitive challenge (e.g. with adenosine) in early childhood, and relating the results to other phenotypic features and outcomes.

Response to treatment. It is tempting to argue that differences in responsiveness to antiinflammatory treatments in preschool children with different patterns of wheezing strengthen the case for different phenotypes. However, interpreting data from studies that have not been specifically designed to compare the responsiveness of different phenotypes to treatment, must be treated with great caution. For example, the ineffectiveness of inhaled corticosteroids in some randomised controlled trials in preschool asthmatics could, on the one hand, be consentient with a nonatopic wheeze phenotype. On the other hand, these data could be consistent with the lack of effectiveness of inhaled steroids in preventing viral-triggered attacks of wheeze reported in a study of school-age children, some of whom had clear evidence of atopic asthma [35]. As previously mentioned, the best therapeutic trial to justify phenotypes, is one that: 1) draws on epidemiological and inflammatory marker data to develop a set of mutually exclusive phenotypes; 2) recruits all wheezy children; and 3) stratifies children a priori into these "best guess" groupings. Only by using this type of design, will the question of whether differences in responsiveness to therapy between phenotypes really do exist, and of whether any differences are of clinical relevance, be answered. Some issues relevant to the delineation of phenotypes are discussed in the Treatment section.

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

Cluster analysis. Cluster analysis encompasses a wide range of techniques designed to detect clusters among a sample of individuals. All these methods rely on observations 158

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Factor analyses to reduce number of features

Cluster analysis Clusters Statistical properties of clusters

Refine model

Utility of clusters in research

Utility of clusters in clinical practice

Fig. 4. – Steps in cluster analysis.

made on a given set of features. If this set consists of only one dichotomous feature, say the presence or absence of a certain symptom such as "wheeze" or "cough", an obvious clustering of the subjects would be to place them into two groups, one consisting of individuals with the symptom and the other consisting of those without the symptom. When multiple features are observed, finding similar response patterns across all variables becomes much more complicated and requires multivariate techniques. The task may be further complicated by including data of different modes (e.g. categorical, continuous, count data, etc.). Clustering methods can be placed into two broad categories: probabilistic clustering and nonprobabilistic clustering. Nonprobabilistic clustering is concerned only with the task of identifying groups of individuals and does not make any assumptions about how the data may have been generated. These methods usually follow one of two approaches. The first, partitional clustering, begins with an initial partition, meaning that each individual is placed into one of a specified number of groups, say, k. The subjects are then iteratively reallocated to groups while optimising a given criterion. In k-means clustering, for instance, optimality is achieved when the variation of the observed feature is minimised within clusters. The second approach, hierarchical clustering, does not require the number of groups to be specified in advance. Instead, the groups are hierarchically constructed by joining subjects, or subgroups of subjects, into ever larger groups based on certain measurements of distance or similarity. Here, the subjects are interpreted as points within a space in which distance can be measured. For instance three features observed on two individuals can be interpreted as x, y, z coordinates of two points representing the two individuals in three-dimensional space. The Euclidean distance between the points is the length of the straight line connecting the two points. Such concepts of distance can be generalised to spaces with any number of dimensions, each dimension representing a measurable feature. Typically, the output of a hierarchical clustering algorithm is a dendrogram (fig. 5). This is a tree diagram showing the 159

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1

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Fig. 5. – Example of the output from hierarchical cluster analysis: a dendrogram.

hierarchical merging structure. The subjects are ordered along the abscissa. Each horizontal line represents a merger while the vertical location of the line along the ordinate represents the distance between the subjects or subgroups being merged. Probabilistic clustering attempts to actually model the process that gave rise to the data. These models are generally specified in terms of a probability distribution, consisting of a mixture of component distributions, each of which represents a cluster to be identified. Assuming that the observed data are a realisation of the specified mixture, the unknown parameters of the component distributions can be estimated by maximum likelihood or Bayesian methods. Once these parameters have been estimated, each individual can be assigned to the group from which its observed features would most likely have originated. Probabilistic clustering methods are typically more computationally intensive and require careful thought in specifying the model. However, they are also more flexible in accommodating various types of data, and allow statistical testing of certain hypotheses. Cluster analysis techniques have been applied to other diseases and have begun to be explored in relation to airway disease in general and wheezing disorders in childhood [38, 39]. In summary, there is no answer to the question: how many phenotypes are there? But simply raising the question may focus research endeavour in the fields of epidemiology, physiology and therapeutics to try to provide solutions. At the moment, it can confidently be stated that the care of young wheezy children is hampered by placing them all into one single diagnostic lump.

Treatment Few trials in preschool children have addressed the issue of asthma phenotypes, and some anti-asthma therapies are not licensed for the whole preschool age range. In spite of this, the UK Sign/British Thoracic Society Guidelines [40] (fig. 6) recommend regular inhaled corticosteroids (ICS) at up to 400 mg beclomethasone dipropionate or equivalent per day for anything but trivial preschool asthma. Inhaled salbutamol or terbutaline are the mainstay for treating acute symptoms. 160

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Step 4: Persistent poor control Refer to respiratory paediatrician

Step 3: Add-on therapy In children aged 25 yrs, consider trial of leukotriene receptor agonist In children <2 yrs, consider proceeding to step 4

Step 2: Regular preventer therapy Add inhaled steroid 200400 µg·day-1#,¶ or leukotriene receptor agonist if inhaled steroid cannot be used Start at dose of inhaled steroid appropriate to severity of disease

Step 1: Mild intermittent asthma Inhaled short-acting b2-agonist as required Fig. 6. – British Thoracic Society/UK Sign recommendation for nonacute treatment of preschool asthma [40]. # : beclomethasone diproprionate or equivalent; }: higher nominal doses may be required if drug delivery is difficult. Taken with permission from [34].

Regular inhaled corticosteroids. Consensus amongst experts is needed for developing guidelines, especially where the evidence base is sparse. The recommendation for ICSs in preschool asthma smoothes over some of the discrepancies in published data. For example, in many of the trials of regular ICSs in preschool children, either the asthma phenotype recruited is unclear or the primary outcome variable is not given, or a combination of both. There are indeed randomised controlled trials of regular ICSs that show a reduction in symptoms [41–44], but there are also trials where no beneficial effect has been detected [45, 46]. The study by Wilson et al. [47] provides the key to understanding this inconsistency in the published data. This randomised controlled trial recruited preschool children with the exclusive viral wheeze phenotype and found no clinical benefit from 4 months’ daily treatment with budesonide 400 mg per day, results that can now be explained by the absence of chronic airway inflammation in this phenotype (see above). Thus, studies of regular inhaled steroids that have recruited a high proportion of exclusive viral wheezers will tend to be negative. Conversely, trials that have recruited children at increased risk of atopic asthma are more likely to be positive. Indeed, the recent Prevention of Early Asthma in Kids trial, which recruited preschool children with asthma with risk factors for atopic asthma, found, as a secondary outcome analysis, 161

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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

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use. To date, the beneficial effect of intermittent use of montelukast in viral wheeze has been published as an abstract [61].

Treatment of acute attacks. For clinically severe attacks of wheeze in preschool children, current therapies are based on studies of proven efficacy in older children and adults. For home treatment, inhaled b2-agonist (four to six puffs of salbutamol as required, maximum 4 hourly) by a metered-dose inhaler and spacer, with a mask for children v3 yrs of age is recommended [40]. It remains unclear whether inhaled shortacting b2-agonists are effective in the youngest preschool children (v2 yrs) [15], but since they are safe, they should be used. If the variable benefit of inhaled therapy is due to relative resistance of bronchial smooth muscle, or impaired delivery in youngest preschool children, parenteral therapy may be more effective. Indeed, a recent review of a singledose intravenous salbutamol bolus (15 mg?kg-1 administered over 10 min) for the initial treatment of children, concluded that it had the potential to shorten the duration of severe attacks and reduce overall requirements for inhaled salbutamol maintenance [62]. There are no data on whether ipratropium bromide provides additional benefit to short-acting b2-agonists for home-based treatment, as it does when given in hospital [63]. The efficacy of systemic steroids in acute attacks of preschool asthma is unclear. The two recent randomised controlled trials have specifically recruited preschool children with viral-triggered attacks. First, preschool children who had previously been hospitalised with an attack of viral-triggered wheeze were randomised to receive a short course of oral steroids or placebo during their next attack [19]. Secondly, children presenting to an admission unit were randomised to oral prednisolone or placebo [64]. On the one hand, parent-initiated oral steroid therapy improved neither the symptom scores nor the number of salbutamol actuations required per day [19]. On the other hand, issuing steroids to children at the time of presentation to hospital reduced the need for "additional" asthma medication in children who were subsequently hospitalised [64]. Until further data appear, it is reasonable to reserve systemic steroids for preschool children admitted to hospital with severe viral-triggered wheeze and not to routinely use parent-initiated therapy.

Future trial design. To better target therapy to the heterogeneous mix of asthma phenotypes in the preschool period, it is important that trials do not overlook potentially responsive subgroups (e.g. children with chronic atopic asthma) but are not so restrictive that the results are generalisable only to specialist secondary care. An ideal randomised controlled trial would be one that recruits all wheezy preschool children and is sufficiently powered to be able to stratify by phenotype. Indeed, response to therapy may itself be an input variable in defining phenotype. It may well be that preschool asthma is the ideal condition for an "individualised" treatment approach, i.e. using statistical analysis of the risk factors for each child to predict the potential benefits of chronic anti-inflammatory therapy or assessing individual responsiveness by prescribing placebo and active drugs in a crossover (n=1 trial) design. If it proves to be feasible to sample lower airway inflammation using a modified induced sputum technique or by analysis of exhaled breath, it may also be possible to target and adjust inhaled steroids to suppressing sputum eosinophilia, a method of proven value in adults [65]. The key research questions are as follows. 1) How can we identify those children with chronic airway or lung tissue inflammation, and thus avoid using ICSs in those with no airway inflammation between attacks? 2) What is the optimal intermittent therapy for exclusive viral wheeze (high-dose inhaled steroids or montelukast, or some combination)? 3) Is there a role for LABAs as add-on therapy for young children at an increased risk of atopic asthma? 4) Could flexible dosing that is licensed for the budesonide/ 163

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

The natural history of preschool wheeze Most of the present knowledge of the natural history of preschool wheeze has been derived from retrospective and cross-sectional studies, often based on clinical populations. Recently, the first prospective longitudinal (cohort) studies of random samples of the whole population have begun to be reported. The most notable, the Tucson study [5], which has provided a benchmark, has been followed by more focussed projects. Some of these attempt to combine an experimental intervention, such as house dust mite avoidance, with an observational arm, potentially leading to bias in population recruitment and unintended effects on the outcomes of interest. A number of useful lessons have been learned from these studies.

The prognosis of preschool wheeze. Algorithms for prediction of outcome have been determined by several groups. In general, they predict the likelihood of persistence of wheeze (or asthma) at school age of those with preschool symptoms [66]. The Tucson algorithm has been successfully used to identify high-risk groups of preschool wheezers for therapeutic trials [67]. To some extent, prediction algorithms derived in one setting (the desert environment of Arizona, USA) seem to be robust enough to apply in another (temperate Western Europe). The precision of prediction algorithms is likely to be improved by collecting data specific to the local environment, by taking into account ethnic factors and by refining phenotype definitions, as previously discussed.

Is preschool wheeze a precursor of later disease? There is clearly evidence for a link between early wheezing illness and asthma in schoolchildren; however, the interpretation is conflicting, perhaps because the issue comprises several different questions. If there are several distinct preschool wheeze phenotypes (atopic asthma, exclusive viral wheeze, etc.), then the relationship with later disease needs to be considered separately for each; this is obviously very complex, and figure 7 provides a simple model. For each phenotype, if there is an association with later asthma. 1) The two may be identical, and thus simply a continuation, as is the case for chronic atopic asthma. Early onset may be the result of chance environmental exposures or greater inherent (genetic) risk. 2) The two may be identical, but the result of a common set of causal factors. Some risk factors for adult-onset atopic asthma are similar to those of childhood-onset disease [68], and many of the usual features of the illness persist in remission [69, 70]. The factors that switch on the asthma phenotype in high-risk individuals and, conversely, which later permit remission, would merit further detailed investigation. 3) Developmental "damage" as a result of preschool illness, such as remodelling, programming or disrupted development of the lungs, may lead to later airway disease, 164

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Environment

Preschool wheeze phenotype 1

(ii)

Late onset of wheeze phenotype 1

(i)

Wheeze phenotype 1 (continued)

(iii) "Damage"

New phenotype of wheeze 2

Fig. 7. – Possible scenarios for an association between wheeze phenotypes in preschool and older children. The same model can be used to explore a possible relationship between preschool wheeze and chronic obstructive pulmonary disease in adults. The three outcomes labelled (i), (ii) and (iii) are described in detail in the Is preschool wheeze a precursor of later disease? section.

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

Implications for the future There are several important conclusions from this chapter, which may inform the direction of research in preschool wheezing disease. These are as follows. 1) There are probably several distinct phenotypes to be delineated. 2) The natural history should be described for locations other than the USA desert environment (as in the Tucson study). 3) Appropriate therapeutic trials await better phenotype definition and better understanding of pathophysiology. 4) The impact of early disease on the developing lung, a potential risk factor for chronic obstructive pulmonary disease, should be determined. 165

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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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Frey U. Why are infants prone to wheeze? Physiological aspects of wheezing disorders in infants. Swiss Med Wkly 2001; 131: 400–406. Elphick HE, Ritson S, Rodgers H, Everard ML. When a "wheeze" is not a wheeze: acoustic analysis of breath sounds in infants. Eur Respir J 2000; 16: 593–597. Cane RS, Ranganathan SC, McKenzie SA. What do parents of wheezy children understand by "wheeze"? Arch Dis Child 2000; 82: 327–332. Chauliac ES, Silverman M, Zwahlen M, Strippoli M-PF, Brooke AM, Kuehni CE. The therapy of preschool wheeze: appropriate and fair? Pediatr Pulmonol 2006; 41: 829–838. Taussig LM, Wright AL, Holberg CJ, Halonen M, Morgan WJ, Martinez FD. Tucson Children’s Respiratory Study: 1980 to present. J Allergy Clin Immunol 2003; 111: 661–675. Lowe L, Murray CS, Martin L, et al. Reported versus confirmed wheeze and lung function in early life. Arch Dis Child 2004; 89: 540–543. Silverman M, Wang M, Hunter G, Taub N. Episodic viral wheeze in preschool children: effect of topical nasal corticosteroid prophylaxis. Thorax 2003; 58: 431–434. Dodge RR, Burrows B. The prevalence and incidence of asthma and asthma-like symptoms in a general population sample. Am Rev Respir Dis 1980; 122: 567–575. Kuehni C, Strippoli M, Low N, Brooke AM, Silverman M. Asthma phenotypes in white and south Asian preschool children in the UK. Eur Respir J 2006 (In press). Vignola AM, Chanez P, Campbell AM, et al. Airway inflammation in mild intermittent and in persistent asthma. Am J Respir Crit Care Med 1998; 157: 403–409. Stevenson EC, Turner G, Heaney LG, et al. Bronchoalveolar lavage findings suggest two different forms of childhood asthma. Clin Exp Allergy 1997; 27: 1027–1035. Maclennan C, Hutchinson P, Holdsworth S, Bardin PG, Freezer NJ. Airway inflammation in asymptomatic children with episodic wheeze. Pediatr Pulmonol 2006; 41: 577–583. Saglani S, Nicholson AG, Scallan M, et al. Investigation of young children with severe recurrent wheeze: any clinical benefit? Eur Respir J 2006; 27: 29–35. Ward C, Pais M, Bish R, et al. Airway inflammation, basement membrane thickening and bronchial hyperresponsiveness in asthma. Thorax 2002; 57: 309–316. Le Bourgeois M, Goncalves M, Le Clainche L, et al. Bronchoalveolar cells in childrenv3 years old with severe recurrent wheezing. Chest 2002; 122: 791–797.

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Bronchiolitis in infants and children J.L.L. Kimpen*, J. Hammer# *Wilhelmina Children’s Hospital, University Medical Center Utrecht, Utrecht, The Netherlands. #Abteilung fu¨r Intensivmedizin und Pneumologie, Universita¨ts-Kinderklinik beider Basel, Basel, Switzerland. Correspondence: J.L.L. Kimpen, Wilhelmina Children’s Hospital, University Medical Center Utrecht, POB 85090, 3508 AB Utrecht, The Netherlands. Fax: 31 302505346; E-mail: [email protected]

Bronchiolitis is a common lower respiratory tract disease of infants and young children. Most episodes are caused by respiratory syncytial virus (RSV), although recently an increasing number of respiratory viruses have been incriminated in causing the same clinical syndrome. All children have been infected with RSV at least once before their second birthday. The majority develop simple upper respiratory tract symptoms undistinguishable from a common cold, while y40% have signs of lower airway inflammation. Only 1–2% of all children need hospitalisation. However, because of the almost universal infection during the first year of life, the absolute number of patients admitted during yearly RSV epidemics is dramatic, causing logistical problems, especially on paediatric intensive care units (5–10% of the hospitalised patients need mechanical ventilation), and having a serious effect on healthcare expenditures. Moreover, nearly half of the patients admitted for RSV-related lower respiratory tract disease develop recurrent episodes of wheezing up to the age of 11–13 yrs, which impacts considerably on their quality of life. A number of comprehensive reviews have been published recently on several aspects of RSV-related disease, both from the clinical point of view and from the basic science and translational research arena [1–3]. The present chapter will focus on the most important recent developments in epidemiology, pathogenesis of acute bronchiolitis and recurrent wheezing, and prevention and treatment strategies.

Virology and epidemiology RSV is a single-stranded RNA virus belonging to the genus pneumovirus of the family Paramyxoviridae (fig. 1). Two RSV strains (A and B) and a large number of serotypes and genotypes have been identified as human pathogens. It is unlikely that a difference in virulence between these strains influences disease outcome. Zambon et al. [4] performed a phylogenetic analysis of clinical RSV isolates from three consecutive seasons in a large community-based surveillance study. They showed that different viral strains circulated in the community at the same time, infecting both adults and paediatric patients alike, and causing the complete spectrum of disease from upper respiratory infection, which can be treated in the outpatient clinic, to severe disease necessitating admission to the hospital. This confirmed earlier findings of Kneyber et al. [5], who showed that there was no difference in solid outcome parameters, such as length of hospital stay, intensive care admission and supplemental oxygen requirement, when comparing hospitalised bronchiolitis patients infected with type A and type B RSV. Finally, Smyth et al. [6] did not find differences in requirement for supplemental oxygen or mechanical Eur Respir Mon, 2006, 37, 170–190. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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Fig. 1. – Respiratory syncytial virus (electron microscopy; courtesy of M. Kneyber, Free University Medical Center, Amsterdam, The Netherlands).

ventilation in relation to RSV virus genotypes, according to variations in the small hydrophobic or nucleoprotein of the virus. RSV bronchiolitis has, in general, been considered a disease at the extreme ends of life, i.e. the very young infant and the elderly. It has recently been shown that RSV can infect people of all age groups and is responsible for more or less severe community-acquired lower respiratory tract disease. Zambon et al. [4] showed that RSV is responsible for 10– 25% of episodes of flu-like illness in 5–64-yr-olds during an observational, communitybased study. If flu-like illness, characterised by fever, cough and respiratory tract symptoms, was used to select eligible patients, disease caused by RSV could not be differentiated from infections by influenza virus. Hall et al. [7] prospectively studied viral respiratory infections in 2,960 healthy adults from 1975 to 1995, during the months that RSV was active in the community. Of the subjects studies, 7% (211 patients) acquired acute RSV infection, which was symptomatic in the majority of patients. Although most had upper respiratory tract symptoms, 26% manifested tracheobronchitis, bronchiolitis and wheezing, with a mean duration of 9.5 days and a high rate of absence from work. Although the clinical characteristics of RSV- and influenza-infected patients differed significantly (with more respiratory symptoms in the RSV group and more generalised symptoms in the influenza group), there was a considerable overlap, making a clinical aetiological diagnosis difficult. Falsey et al. [8] prospectively evaluated all respiratory illnesses in a cohort of healthy elderly patients and adults with chronic heart or lung conditions, over four consecutive seasons. RSV infection was responsible for 3–7% of respiratory illness episodes in the healthy elderly subjects and for 4–10% in the high-risk adult subjects. Again, 89% of RSV infections were symptomatic. These studies suggest that RSV needs to be considered in the differential diagnosis of lower respiratory tract disease in adults during the winter season, especially if the individual has an underlying chronic cardiorespiratory condition. The majority of bronchiolitis episodes are caused by RSV, with estimates ranging 50– 90%. However, other viruses (influenza virus, parainfluenza virus, rhinovirus and adenovirus) rarely cause the same clinical picture in young children. Recently, two new 171

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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 4–12% 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

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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

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Pathogenesis of disease It is becoming increasingly clear that local and systemic immune mechanisms initiated by RSV infection of the respiratory epithelial cells are mainly responsible for the pathogenesis of disease. RSV has a direct cytopathic effect, but infection by itself is insufficient to cause severe disease [23]. The inflammatory infiltrate in the lungs, devoted to eliminating the virus from the body, causes most of the pathology. Several arguments support this statement. First, during immunisation trials with a formalin-inactivated virus, it was noted that children became more severely ill than controls after subsequent natural infection [24]. Secondly, although immunodeficient laboratory animals challenged with RSV shed the virus for prolonged periods of time, they do not develop signs and symptoms unless they are reconstituted with cellular components of the specific immune system [25]. Thirdly, although RSV infection of severely immunocompromised children occurs and can occasionally be very severe, a much higher incidence of RSVrelated morbidity and mortality would be expected against the background of the ubiquitous presence of RSV in the community during yearly winter epidemics. Fourthly, RSV-related symptoms are at their worst at a point in the course of infection when viral replication is already decreasing and the immune response has reached its maximum. Finally, antiviral therapy with ribavirin does not influence the course of the disease considerably, although RSV is highly sensitive to the titres of drug that can be obtained in the lungs. The immunological mechanisms underlying disease pathogenesis after infection with RSV have been the subject of extensive research since the vaccine trials in the 1960s. Although a number of possible pathogenetic mechanisms have been proposed, no one single hypothesis provides all the answers. Probably the development of bronchiolitis after primary infection with RSV, the later development of recurrent wheezing, and the relative protection for severe illness after second and later infections, are the result of a complex interplay of a broad array of different factors. The most important recent developments are summarised in the following paragraphs.

Role of T-cells and antigen-presenting cells A specific antiviral immune response is a prerequisite to clear the virus from the lungs. RSV-specific CD4z and CD8z T-cells have a pivotal role. In the absence of specific Tcells, the virus is shed from the upper and lower airways for much longer periods than in the presence of an intact immune system. However, when laboratory animals are reconstituted with selected populations of RSV-specific T-cells, they not only clear the virus, but also develop an inflammatory infiltrate in the lungs, leading to further damage of infected epithelium and bronchial hyperreactivity, similar to bronchiolitis in infants. These experiments prove that, although necessary for recovery and protection, RSVspecific T-cells contribute to the pathogenesis of disease. It has been shown in the mouse model that CD4z T-cells directed to the fusion (F) protein of RSV elicit a protective, beneficial immune response, characterised by the induction of an effective cytotoxic CD8zT-cell response, a strong antiviral antibody response and T-helper cell (Th) type 1 cytokine production, e.g. interferon (IFN)-c. Conversely, CD4z T-cells directed to the attachment (G) protein of the virus result in a Th2 response, e.g. interleukin (IL)-4, IL-5 and IL-10, and a strong eosinophil influx into the airways [2, 26, 27]. However, this clear difference under controlled circumstances in the mouse model is more controversial in humans. Although a Th2-skewed immune response was found by some investigators, it could not be shown by others [28, 29]. Moreover, if RSV bronchiolitis was a Th2-type disease, a strong correlation with allergy would be expected, both as a risk factor for 174

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severe disease or as an outcome parameter after acute RSV bronchiolitis. However, most prospective, long-term follow-up studies do not show a relationship between RSV disease and allergy [30, 31]. Only the study by Sigurs et al. [32] suggests that RSV might be a risk factor for sensitisation against aeroallergens and subsequent allergic symptoms. Although it is evident that T-cells play an important role in both the recovery from and the pathogenesis of RSV-related disease, their action is not necessarily following the Th1/Th2 paradigm. The role of innate immunity has recently received attention. Eosinophils, neutrophils and antigen-presenting cells are all contributing to the pathogenesis of disease. Neutrophilic granulocytes and their secretory products can readily be detected in the airways of infected children and correlate with disease severity. McNamara et al. [33] measured high concentrations of IL-9 in the airways of children with severe RSV disease and proved neutrophils as the source of this regulatory pro-inflammatory cytokine. Garofalo et al. [34] correlated high levels of eosinophil cationic protein with disease severity in children with RSV infection. RSV inhibits apoptosis of these cells in vitro [35]. Monocytes and dendritic cells are mobilised to the mucosa during RSV infection. These cells could play a role in enhancing the immune response, contributing to the development of airway inflammation and airway hyperreactivity [36, 37]. Alternatively, they could contribute to a delayed activation of T-cells, possibly delaying an effective antiviral response [38].

Dual infections It has been suggested that concurrent infections with other microorganisms might contribute to the severity of disease due to RSV. Hament et al. [39] have shown in vitro that pneumococcal binding to respiratory epithelial cells is enhanced by pre-infection of the cells with RSV (fig. 4). Also, concomitant infection resulted in a more pronounced attachment of pneumococci to human epithelial cells. The effect was dose-dependent and was present to a different degree in different pneumococcal strains. These in vitro data were supported by clinical data from South Africa suggesting that pneumococci have a major role in the development of pneumonia associated with RSV [40]. Moreover, in a large study, Semple et al. [41] showed that co-infection with RSV and hMPV resulted in more severe disease. The rate of dual infection was 72% in a population of bronchiolitis cases admitted to the paediatric intensive care versus 10% of cases admitted to the medium care ward.

Genetics of the host RSV bronchiolitis is the result of a complex interplay between the virus and the host immune response. There is increasing evidence that the genetic make-up of an individual, mirrored by the presence of polymorphisms in genes important for the immune response, determines to a significant extent the outcome of infection. This has been shown for several infectious diseases, e.g. tuberculosis and otitis media. Recently, polymorphisms in genes playing a role in the innate and specific antiviral response have been associated with severity of disease after infection with RSV (fig. 5). Lo¨fgren et al. [42] showed that polymorphisms in genes coding for surfactant protein (SP)-A and -D are associated with a variable outcome after infection with RSV [42, 43]. One particular SP-A haplotype, 6A2/1A3c, was associated with more severe RSV disease, while another haplotype, 6A/1Ab, seemed to be protective. In the SP-D gene, the variant with a methionine at position 11 was associated with more severe disease. In addition, polymorphisms in innate immunity genes can modify the immune response upon infection with RSV. The findings of Venter et al. [44] and van Bleek et al. [45] 175

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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

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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

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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.

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Hull et al. [48] demonstrated that the IL-8-251A polymorphism in the gene encoding for the chemokine IL-8 was significantly more prevalent in children with severe RSV disease versus controls. They suggested that this polymorphism was associated with higher IL-8 production after in vitro stimulation with either endotoxin or RSV. Moreover, this polymorphism seems to be associated with long-term consequences of RSV bronchiolitis. Hoebee et al. [49] reported on polymorphisms in the promoter region of IL-4, the IL-4 receptor and IL-10. In a large group of patients, using RSV bronchiolitis and controls, they showed that the IL-4-590T polymorphism causes a cytosine change to thymidine at position 590, and that this was associated with hospitalisation during RSV infection. This association was stronger in children w6 months of age. The IL-4Ra-R551 polymorphism was similarly associated with severe RSV disease in children w6 months. These results were confirmed by Choi et al. [50] in a Korean population. In addition, Hoebee et al. [51] showed that children homozygous for IL-10-592C or IL-10592A were more prone to severe RSV disease than heterozygotes. Wilson et al. [52] showed the association of two single nucleotide polymorphisms in the IL-10 gene locus to be associated with the need for mechanical ventilation. This unique example of homozygous disadvantage had not previously been described for RSV. Finally, Hull et al. [53] described the association of two polymorphisms in the chemokine receptor 5 with severe disease. This receptor is recognised by RANTES (regulated on activation, normal T-cell expressed and secreted) and macrophage inflammatory protein-1a, and both chemokines are suggested to play an important role in the pathogenesis of RSV-related disease [54].

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

RSV bronchiolitis and childhood asthma Epidemiology Several studies have confirmed a strong link between RSV bronchiolitis in early life and the subsequent development of recurrent wheezing and asthma. Wheezing episodes 178

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following RSV bronchiolitis occur in 42–71% of infants, usually within 12–24 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 18–20 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

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sensitisation after RSV infection. These factors include: timing of RSV infection with respect to allergen exposure, prior allergic sensitisation, environmental conditions, exposure to endotoxin and the genetic background of the animal [66]. It seems that despite some similarities, the mechanisms leading to bronchial hyperresponsiveness induced by RSV are different from those that follow allergen sensitisation and challenge [67].

Pathogenesis of bronchial hyperresponsiveness after bronchiolitis It has been suggested that the immune response in children who develop recurrent wheezing after RSV bronchiolitis is characterised by the release of Th2-type cytokines [68]. However this could not be confirmed by other studies [29]. Besides the possible role of variations in the RSV-specific immune mechanisms, there is evidence from animal studies and recently one human study that the prototypic neurotrophin growth factor (NGF), its receptors and the brain-derived neurotrophic factor are upregulated during acute RSV infection [69–72]. NGF is not only a major determinant of the acute neurogenic inflammation, but may also be responsible for long-term effects on airway reactivity. Piedimonte et al. [71] have recently shown that RSV causes a persistent increase in the vascular permeability in response to sensory nerve stimulation, which is still present after the acute phase of the infection has resolved. Piedimonte et al. [71] proposed that sensory innervation in the airways undergoes remodelling after an acute RSV infection, resulting in increased supply and/or responsiveness of the neural network. This could explain the development of airway hyperresponsiveness observed in a large proportion of children with a history of RSV bronchiolitis. Animal studies have also found that RSV can persist in the lungs, even in the presence of adequate T-cell immunity. Such latent RSV infection could increase susceptibility to viral or nonviral antigens and facilitate the development of atopy [73]. Nevertheless, it currently remains unclear how to explain the link between RSV bronchiolitis during infancy and later asthma. Is it that viral infections directly damage the infant respiratory system and promote the development of later asthma, or does bronchiolitis unmask an inherent susceptibility or pre-existing lung function abnormalities? Both explanations are not mutually exclusive.

Prevention of post-bronchiolitic wheeze At present no study has convincingly identified any treatment strategy that can reliably prevent post-bronchiolitic wheezing. Several placebo-controlled trials studied the benefit of corticosteroid treatment on the development of subsequent wheezing. The majority of these studies showed that administration of nebulised budesonide or oral prednisolone during the acute episode do not prevent post-bronchiolitic wheezing or asthma in later life [74–77]. It is unclear at present whether preventing or delaying RSV infection could reduce the number of children who suffer from recurrent wheeze during childhood. Results in animal studies suggest that palivizumab might protect against the development of later asthma. Investigators have demonstrated a protective role for palivizumab on RSVinduced neurogenic inflammation and measured a significant decrease in bronchial hyperresponsiveness until 9 weeks after RSV infection in palivizumab-treated animals [78, 79]. Human studies examining the long-term benefits of passive immunisation with palivizumab are currently underway. 180

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Prevention and treatment Prevention Prevention of RSV bronchiolitis in high-risk infants has evolved from standard intravenous immunoglobulin to RSV-hyperimmune, polyclonal intravenous globuline (RSV-IGIV; Respigam1; MedImmune, Gaithersburg, MD, USA) to monthly passive immunisations with an intramuscular, humanised murine monoclonal antibody palivizumab (Synagis1; Abbott Laboratories, Abbott Park, Illinois, IL, USA). RSVIGIV has never received much popularity in Europe due to the long duration of intravenous administration, the theoretical risk of infection, the possible interference with life-attenuated vaccinations and its high costs. In two double-blind, placebocontrolled trials, monthly palivizumab at a dose of 15 mg?kg-1 i.m. for 5 months significantly reduced RSV-related hospitalisations (by 55% in 1,502 infants with prematurity and/or bronchopulmonary dysplasia/chronic lung disease and by 45% in 1,287 infants with haemodynamically significant congenital heart disease) [80, 81]. Apart from the reduction in hospital admissions, palivizumab and RSV-IGIV provided no benefit for more serious outcomes such as mortality. Due to its ease of administration, safety and effectiveness, palivizumab has become the drug of choice for RSV prophylaxis of high-risk infants. The official recommendations of the American Academy of Paediatrics for RSV prophylaxis [82] have not received widespread acceptance in Europe. The astronomical costs of palivizumab and the local variability in RSV severity have led to a debate about how and when it should be used [83, 84]. Most infants requiring hospitalisation for severe RSV bronchiolitis are previously healthy infants born at term and do not qualify for RSV prophylaxis. None of the cost studies has shown economic benefit for palivizumab prophylaxis, although it is difficult to generalise the conclusions of such studies [85–90]. The risk of serious RSV disease will be low for many infants included in the approved indications and the cost may outweigh the potential benefits. Hence, many countries have published their own, much more restrictive recommendations limiting palivizumab mostly to pre-term infants v1 yr of age who require active treatment of chronic lung disease at the time of the RSV season [91–94]. Palivizumab has not been formally assessed in other high-risk infants, such as those with immunodeficiency or cystic fibrosis, who might potentially benefit from passive immunisation. On the horizon is a new monoclonal antibody that has proven to be superior to palivizumab in microneutralisation studies. This compound is currently being investigated in two phase 3 clinical studies that began in November 2004 [95, 96]. Despite an immense amount of research having taken place during the last decades, the highly complex immunopathology of RSV disease has so far hindered the development of a safe and effective vaccine. Research was held up greatly by the experience with a crude formalin-inactivated, alum-precipitated RSV vaccine that potentiated RSV disease in clinical trials [97]. Several strategies for the development of vaccine candidates are currently followed, including live attenuated vaccines, subunit vaccines, recombinant vectors expressing the protective antigens of RSV and DNA vaccines [96, 98]. In addition, drugs that block RSV replication based on nucleic acid strategies have emerged as potentially viable drug-development options [99].

Treatment It is well recognised that supportive therapy remains the mainstay for the treatment of bronchiolitis and that there is little justification for most of the pharmacological agents 181

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traditionally administered to bronchiolitic children. The new paradigm of evidence-based medicine, together with increased cost-awareness, has driven many efforts to educate physicians and hospitals in the treatment of RSV bronchiolitis at home and in the hospital, since it has been repeatedly demonstrated that inappropriate or unnecessary medications are administered to infants with bronchiolitis [100–103]. Most children with bronchiolitis can be managed at home by their parents with careful attention to feeding and respiratory status. The decision to admit infants with bronchiolitis to the hospital is based on their age, the presence of risk factors for severe disease, the severity of respiratory distress, the ability to take oral fluids, and the social and local circumstances. Treatment of hospitalised infants includes humidified oxygen to maintain saturations above 92%, moderate fluid supply and minimal handling. Chest physiotherapy plays no role in the treatment of bronchiolitic infants and is not without potential risks [104]. Many pharmacological agents commonly used in the treatment of asthma are also widely used in the management of bronchiolitis. The rationale for the treatment of bronchiolitis with bronchodilators is weak. Airway obstruction is mainly a result of mucus plugs and cellular debris from bronchial inflammation and epithelial necrosis. The contribution of airway smooth muscle constriction (which is treatable) to the airway obstruction is minor in most cases. Furthermore, the role of inhaled b-selective bronchodilators has been debated for many years, and it is now well established that they play a very minor role in the treatment of bronchiolitis. They produce only modest, short-term improvements in clinical scores, but do not decrease the rate or duration of hospitalisation. The inclusion of recurrent wheezers may have even biased these results in favour of bronchodilators [105, 106]; however, this small benefit must be weighed against the costs of these agents. Inhaled epinephrine is thought to be the better agent with which to treat airway obstruction in acute bronchiolitis, because epinephrine stimulates the a-adrenoreceptor causing arteriolar vasoconstriction in the airway mucosa and subsequent reduction of bronchial mucosal oedema [107]. Many studies reported some improvements in shortterm outcomes, such as clinical scores or respiratory mechanics. A systematic review of eight randomised controlled trials on the use of epinephrine in bronchiolitis, however, came to the conclusion that there is no significant outcome benefit that would justify its use, although it is superior to salbutamol and placebo for its short-term benefits [108]. For treatment with corticosteroids, a recent meta-analysis found no benefit in either length of hospital stay or clinical score for infants and young children with uncomplicated bronchiolitis compared with placebo [109]. Specific data on the harm of corticosteroid therapy in this patient population are lacking, and evidence available today suggests that corticosteroids should not be used in bronchiolitis. This is true, independent of the route of medication delivery or the dose, for both infants presenting in the emergency room and those requiring hospitalisation. It remains unclear whether steroids have a potential benefit in infants with chronic lung disease. Infants progressing into respiratory failure from bronchiolitis are more likely to have underlying risk factors and are characterised by immaturity of their cellular immune system [110]. This particular group of infants might benefit from therapies that have shown no effect in mild bronchiolitis. However, no medical therapy has yet satisfied such expectations for critically ill children. Unfortunately, the majority of studies on virusinduced respiratory failure have not stratified their patients for the underlying pathophysiology, obstructive (bronchiolitis) versus restrictive (pneumonia, acute respiratory distress syndrome) disease [111, 112] (fig. 7). The most promising intervention is the use of surfactant, which is justified on the presence of a dysfunctional surfactant system in severe bronchiolitis due to increased surfactant inactivation [113]. The three previous randomised-controlled surfactant trials were small and difficult to 182

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compare because of the different ventilator strategies [114–116]. A meta-analysis of the three studies demonstrated a nonsignificant reduction in the duration of mechanical ventilation by 2.6 days [117]. No clear benefit has been found for the use of steroids in three small trials performed in critically ill children with RSV-induced respiratory failure. Larger studies are required to substantiate the weak evidence that corticosteroids may accelerate clinical recovery in those children with obstructive disease [118]. Ribavirin is a nucleoside analogue resembling guanosine that interferes with viral protein synthesis and is the only therapeutic agent available to treat the established infection. The agent is now little used because of its high cost and the disappointing results from placebo-controlled trials [119–122]. It remains unclear if certain patients, particularly the immunocompromised infants, may benefit from ribavirin therapy. Other treatment modalities that have been tried and remain without benefit include inhaled nitric oxide [123–125], heliox [126], immune globulin [127], palivizumab [128] and erythropoietin [129]. The low mortality of bronchiolitis suggests the use of expensive therapy with questionable efficacy should be reserved for selected clinical situations. Physicians must recognise that no treatment of bronchiolitis will reduce the length of stay in the hospital. The implementation of evidence-based clinical practice guidelines can result in significant changes in practice and may be cost saving [130].

Summary Bronchiolitis is a common clinical syndrome in infancy. It is caused most often by respiratory syncytial virus, although recently other causative respiratory viruses have been identified. Bronchiolitis is followed in a considerable number of cases by recurrent episodes of wheezing in the first years of life, but probably has no relation to the development of atopy later in childhood. Although there is no effective treatment for bronchiolitis, disease progression after infection can be attenuated by prophylactic administration of humanised monoclonal antibodies. The pathogenesis of viral bronchiolitis is the subject of intensive research because it is clear that the immune response initiated by the viral infection is partially responsible for the damage of the airways. The cells that have been implicated in this process are virus-specific cytotoxic and T-helper cells, as well as antigen-presenting cells, such as dendritic cells. Keywords: Corticosteroids, genetic polymorphism, palivizumab, respiratory syncytial virus, wheezing.

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Simoes EAF. Respiratory syncytial virus infection. Lancet 1999; 354: 847–852. Openshaw PJM, Tregoning JS. Immune responses and disease enhancement during respiratory syncytial virus infection. Clin Microbiol Rev 2005; 18: 541–555. Hall CB. Respiratory syncytial virus and parainfluenza virus. N Engl J Med 2001; 25: 1928. Zambon MC, Stockton JD, Clewley JP, Fleming DM. Contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study. Lancet 2001; 358: 1410–1416. Kneyber MC, Brandenburg AH, Rothbarth PH, De Groot R, Ott A, Van Steensel-Moll HA. Relationship between clinical severity of respiratory syncytial virus infection and subtype. Arch Dis Child 1996; 75: 137–140.

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6. 7. 8. 9. 10. 11. 12.

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Asthma in school-aged children and adolescents J.H. Wildhaber *, F.H. Sennhauser *, P.L.P. Brand # *University Children’s Hospital Zurich, Zurich, Switzerland. klinieken, Zwolle, The Netherlands.

#

Princess Amalia Children’s Clinic, Isala

Correspondence: J.H. Wildhaber, University Children’s Hospital Zu¨rich, Steinwiesstrasse 75, 8032 Zu¨rich, Switzerland. E-mail: [email protected]

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

The most recent fundamental developments in the field Discrepancy between goals set in guidelines and goals achieved in daily practice According to the guidelines, the goals of asthma treatment are: optimal asthma control with little or no symptoms; undisturbed sleep; no severe exacerbations; no emergency visits; normal lung function; and no limitations to daily activities such as school and sports [1, 2]. Surveys in adults and children have shown consistently that these goals are usually not met [3–7]. For instance, in a study of 572 Swiss children with asthma [7], 49% of patients had troublesome symptoms, with disturbed sleep, restricted activities and school absence (fig. 1). Compliance with ICS was poor: one in three patients took their ICS v3 months per year [7]. In an American study, many physicians chose not to prescribe ICS to children with asthma because they did not believe the medication would be helpful [8]. Many patients with asthma use their inhalers incorrectly and adherence to self-management plans is usually poor [9, 10]. Preventive measures are also often not established. In the Swiss survey, 36% of children reporting symptoms on exposure to pets kept their pets nonetheless [7]. In an American study, half of the environmental measures taken by parents to reduce exposure to harmful stimuli in their asthmatic children were unlikely to be beneficial [11]. These findings indicate undertreatment of childhood asthma by both patients and physicians [3, 8, 12]. However, surveys in the USA and Finland found that forced expiratory volume in one second (FEV1) was normal in most children diagnosed with asthma, even when their Eur Respir Mon, 2006, 37, 191–216. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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90 80 70 60 50 40 30 20 10 0

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79 1012 Age yrs

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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

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Fig. 2. – Changes in a) forced expiratory volume in one second (FEV1) after bronchodilator and b) dose of histamine causing a 20% fall in FEV1 (PC20), in three groups of asthmatic children, treated for up to 4 yrs with budesonide (???????), nedocromil (– – –) or placebo (––––), in the Childhood Asthma Management Program study. Reproduced from [20] with permission.

rates, oral steroid courses and symptom-free days. Differences in post-bronchodilator FEV1 between the two groups were surprisingly small, however (fig. 3) [22]. In a study from New Zealand [23], following up a large group of children from the age of 9 yrs until the age of 26 yrs, lung function appeared to "track" throughout childhood and into adulthood (fig. 4), suggesting that loss of lung function in asthma occurs early in the course of the disease (i.e. in early childhood) [24]. These results may be viewed as evidence that childhood asthma can be controlled by ICS therapy, but that the natural history of asthma is one of chronicity and persistent

100 l

l l

l l

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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 ƒ2 yrs at study entry. Data taken from the Steroid Therapy as Regular Treatment trial [22].

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Fig. 4. – Changes in forced expiratory volume in one second (FEV1)/forced vital capacity (FVC) from school age to early adulthood in a) males and b) females from a birth cohort study of 613 subjects in New Zealand. A clear "tracking" pattern is obvious, irrespective of the severity of asthma. %: no wheezing ever; $: transient wheezing; &: intermittent wheezing; ,: remission; +: relapse; ): persistent wheezing. Reproduced from [23] with permission.

lung function abnormalities [25]. Such an interpretation could fuel carelessness in treating childhood asthma: if long-term outcome is not influenced by ICS therapy, why bother taking the treatment daily for years on end? The present authors believe, however, that alternative explanations are likely for the data reviewed above. The patients in the CAMP study and in the Dutch study had had asthma for an average of 5 yrs before ICS therapy was instituted [20, 21]. In the START study, patients had asthma that was diagnosed ƒ2 yrs ago. In the START study, however, adding ICS therapy to study treatment was allowed at the discretion of the physician. Being almost twice as common in the placebo group as in the budesonide group, this effect reduced the apparent effect of the study drug (budesonide) on lung function considerably. Finally, the New Zealand cohort study was recruited in the 1960s, well before the introduction of ICS treatment. The reasonable conclusion from these data, therefore, would be that chronic persistent asthma that has not been treated with ICS for years on end is associated with persistent lung function abnormalities. In most cases of childhood asthma, however, lung function normalises during ICS therapy [14, 22]. At present, it is unclear whether ICS therapy started early in the course of the disease alters its natural history. Even if this is not the case, the clinically relevant improvement in symptoms, lung function, daily activity levels and quality of life consistently observed in long-term studies of ICS therapy in childhood asthma justify its ongoing use and emphasise the need for methods to improve long-term adherence to treatment. The relevance of this was demonstrated recently in a study from the Netherlands [26]. Close follow-up focused on improving inhalation technique and adherence to therapy was associated with improvements in airways hyperresponsiveness and quality of life, whilst at the same time the dose of ICS could be reduced by 25% (fig. 5). Conversely, it has been shown that poor adherence to treatment is associated with an increased risk of asthma exacerbations in children [27]. Based on the evidence reviewed above and by others, it is fair to conclude that most children with apparently troublesome or difficult asthma have either poorly treated asthma (owing to poor adherence or poor inhalation technique) or no asthma at all [28]. 194

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Paediatric QoL score

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Fig. 5. – a) Changes in paediatric asthma quality-of-life (QoL) scores in patients newly referred to a paediatric asthma clinic for chronic persistent asthma, followed up by paediatrician (&) or asthma nurse (&). Improvements in QoL scores were both statistically significant (pv0.01) and clinically relevant (an increase of w0.5 is considered to be clinically meaningful). b) This improvement was obtained despite a reduction in inhaled steroid dose in both groups (%: paediatrician; +: asthma nurse) by, on average, 26%. The authors concluded that improvements in inhalation technique were the most likely explanation for these findings [26].

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

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Using this definition, 23% of patients responded to fluticasone, 5% to montelukast and 17% to both [30]. Montelukast-responders were more likely to be younger and to have asthma of shorter duration, while fluticasone-responders were more likely to be atopic, to have high exhaled nitric oxide (NO) levels, and to be hyperresponsive [30]. Interestingly, more than half of the patients appeared to respond to neither therapy. Clearly, such results are dependent on the definition of "response". Although a 7.5% improvement in FEV1 seems a logical definition of response, it should be noted that this was a short-term study (each treatment period lasted 8 weeks), and that many patients had normal lung function at the start of the study (mean FEV1 in the whole study population was 90% of predicted), so there was little room for improvement [30]. The same problem appeared in the CAMP study (fig. 2) [20], in which post-bronchodilator FEV1, the primary outcome variable, was normal or near-normal in the majority of patients at the start of the trial. If this variable alone had been examined, CAMP would have been viewed as a negative study. This suggests that defining response in terms of a single outcome parameter may be tricky. Moreover, in many clinical trials, changes in the primary end-point follow a normal distribution between patients. While the mean response may be relatively small, a certain subgroup of patients may show dramatic improvements. It is tempting to explore this phenomenon after the data have been obtained, but such a post hoc analysis is scientifically invalid for purposes other than to generate new hypotheses, which must then be tested in separate studies [31]. Another factor compounding the issue of treatment response is that responses to treatment may vary over time in individual patients. While differences in response to therapy are apparent, studying such differences in treatment response is difficult and depends, among other factors, on the choice of the primary end-point and the effect size of the primary end-point considered to reflect a clinically meaningful response (i.e. the definition of treatment response). The art and science of defining treatment response in childhood asthma is in its infancy, but will undoubtedly become a major issue in future studies. It is highly likely that "blanket advice" (as is the rule now in guidelines) will be replaced by individualised advice in future guidelines on asthma therapy but also in guidelines on the prevention of asthma [32].

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

Current concepts and strategies Pathophysiological mechanisms Airway inflammation is widely accepted as the key pathophysiological mechanism in asthma, both in adults and in school-aged children and adolescents. An excellent review 196

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on asthmatic airways inflammation is available elsewhere [35]. It should be stressed, however, that most data on inflammation in asthma are obtained in otherwise healthy, young adults with atopic asthma. Although studies on the pathophysiology of childhood asthma are on the increase, the evidence on inflammation and repair mechanisms in childhood asthma is still very scanty indeed. The results of published studies are summarised in tables 1 and 2. The data in these studies are inconsistent and often not in accordance with studies in adults. For instance, although evidence of eosinophilic inflammation has been found in many studies of childhood asthma [36, 39, 41, 43, 48–50, 53, 55, 56, 59], a number of other studies could not confirm this [38, 40, 42, 51, 52, 54]. Neutrophil counts in bronchial biopsies and bronchoalaveolar lavage (BAL) fluid have sometimes been found to be elevated [43, 52, 53, 55], and in one study they even appeared to relate to the persistence of the disease [55]. However, in many other studies they were not elevated [38, 40, 42, 49, 50, 51, 56]. The only really consistent finding is thickening of Table 1. – Results of mucosal biopsy studies in childhood asthma First author [Ref.] VAN DEN TOORN [36]

TSCHERNIG [37]

Subjects

Main biopsy findings

Comments

Young adults (18–25 yrs old) with a) asthma since childhood (n=19), b) childhood asthma in remission (n=18), and c) controls (n=17) Trachea samples of infants with SIDS (n=29)

RBM thickening, eosinophils, mast cells and T-cells

Biopsy findings comparable in ongoing asthma and asthma in remission

No dendritic cells in tracheal mucosa in infants dying of SIDS RBM thickening, active fibroblasts, mast cells and lymphocytes RBM thickening, eosinophils

Dendritic cells found in tracheal mucosa in older children Eosinophils found in only one patient

COKUGRAS [38]

Children (5–14 yrs old) with moderate asthma (n=10)

PAYNE [39]

Children (6–16 yrs old) with difficult asthma (n=19)

JENKINS [40]

Children (6–17 yrs old) with difficult-to-control asthma (n=6)

RBM thickening, smooth muscle hypertrophy, lymphocytes

BARBATO [41]

Children (4–12 yrs old) with mild-to-moderate asthma (n=9) Children (6–16 yrs old) with difficult asthma (n=27)

RBM thickening, eosinophils

PAYNE [42]

DE

BLIC [43]

SAGLANI [44]

Children (4–18 yrs old) with difficult asthma (n=28)

Infants (3–26 months old) with severe wheeze or cough (n=53)

RBM thickening, CD4z lymphocyte density higher in patients with persistent airflow limitation RBM thickening, eosinophils, neutrophils; IFN-c levels higher in children with few symptoms No RBM thickening, no eosinophils

Biopsies taken after 2 weeks of oral prednisolone No eosinophils or neutrophils found; normal FEV1 possible despite significant RBM thickening Biopsy findings comparable in atopic nonasthmatic subjects Biopsies taken after 2 weeks of oral prednisolone; no differences between asthmatics and controls in number of eosinophils, neutrophils and CD4z lymphocytes Bronchial biopsies taken 4–6 weeks after a 2-week course of oral prednisolone RBM thickness lower in infants than in 6–16-yr-old children with asthma

RBM: reticular basement membrane; SIDS: sudden infant death syndrome; FEV1: forced expiratory volume in one second; IFN: interferon. 197

Infants (0–2 yrs old) with troublesome wheezing (n=13)

Children (1–15 yrs old) with atopic asthma (n=52); children (1–14 yrs old) with nonatopic asthma (n=23) Children (4–15 yrs) with asthma (n=14); children (5–46 months old) with recurrent wheezing (n=26) Children (2–10 yrs old) with acute asthma exacerbations (n=18) Children (6–36 months old) with recurrent troublesome wheezing (n=36)

Children (3–17 yrs old) with allergic asthma (n=13) Children (0–5 yrs) with troublesome wheezing (n=20) Children (4–15 yrs) with asthma (n=16); infants (0–2 yrs old) with recurrent viral wheeze (n=30) Infants (0–3 yrs old; n=21) and children (3–16 yrs old; n=58) with difficult ("unusual") asthma Children (5–10 yrs old) with atopic asthma in remission (n=25); children (3–7 yrs old) with episodic viral wheeze in remission (n=10)

AZEVEDO [47]

STEVENSON [48]

BARBATO [52]

198

Children (10–14 yrs old) with refractory asthma (n=28)

MAHUT [58]

Exhaled NO correlated to ECP levels, IFN-c/IL-4 ratio and TGF-b levels in BAL

Exhaled NO correlates with eosinophil counts

Eosinophils increased in atopic compared with nonatopic asthma; neutrophils elevated in persistent compared with episodic asthma Eosinophils increased in asthmatics in remission

Eosinophil counts not increased; neutrophil counts elevated in wheezy infants compared with controls No increase in eosinophils but neutrophils higher in asthmatics than in controls Lymphocytes, neutrophils, and eosinophils increased in wheezy children Neutrophil counts elevated in recurrent viral wheeze and only mildly elevated in asthma

Eosinophils increased

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

Main BAL findings

Neutrophils, lymphocytes, alveolar macrophages and mast cells not elevated in either group. Nonbronchoscopic technique in patients undergoing intubation for elective surgery Nonbronchoscopic technique in patients undergoing intubation for elective surgery

No increase in eosinophils, IL-8 and ECP in asthma; ECP was correlated with neutrophil count

ECP and MPO were elevated in asthmatics

No control group, but comparison with infants with acute viral bronchiolitis (neutrophils) ECP was elevated in asthmatics and correlated with neutrophil count

Also compared with children with cystic fibrosis, in whom neutrophil counts were highly elevated

Nonbronchoscopic technique in patients undergoing intubation for elective surgery

Comments

TNF: tumour necrosis factor; ECP: eosinophilic cationic protein; MPO: myeloperoxidase; IL: interleukin; NO: nitric oxide; IFN: interferon; TGF: transforming growth factor.

Children (5–14 yrs old) with asthma (n=29)

WARKE [57]

WARKE [56]

JUST [55]

MARGUET [54]

KRAWIEC [53]

AZEVEDO [51]

KIM [50]

MARGUET [49]

AZEVEDO [46]

Infants (0–2 yrs old) with troublesome recurrent or persistent wheeze (n=20) Infants (0–2 yrs old) with troublesome wheezing (n=13)

Subjects

GALOPPIN [45]

First author [Ref.]

Table 2. – Results of bronchoalveolar lavage (BAL) studies in childhood asthma

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the reticular basement membrane, which was observed in almost all biopsy studies [36, 38, 40–43], even in young children [39], but not in infants [44]. A major problem in all biopsy and BAL studies in childhood asthma is the small sample size (tables 1 and 2) and the highly selected nature of most study cohorts, which tend to consist of children with troublesome, therapy-resistant or otherwise unusual clinical phenotypes of asthma. In some studies, biopsies were taken directly after a 2week course of systemic steroids, which may have had considerable influence on the findings [39, 42]. At present, therefore, the presence and nature of airway inflammation in usual or average clinical expressions of childhood asthma is largely unknown, and it is quite likely that it differs considerably from the findings in adults. In addition, there is evidence to suggest that the inflammatory profile of episodic viral wheeze differs from that of atopic asthma [48, 49, 54, 55]. Clearly, a major research challenge is to try to examine airways inflammation in childhood asthma in more detail in the future.

Related diseases: is asthma a risk factor for chronic obstructive pulmonary disease? Recent studies show that asthma may be a lifelong disease. Althoughy50% of children with asthma go into remission during adolescence or early adulthood, evidence suggests that airway inflammation in these individuals continues to exist [36, 56]. In addition,y50% of the subjects with remission in early adulthood relapse later in life [60, 61]. A lower FEV1 in childhood is a strong predictor of asthma in adulthood [60–62]. In addition, as discussed above, FEV1 appears to track throughout life: children with lower FEV1 will continue to have lower FEV1 in adulthood (fig. 4) [23, 63, 64]. In the CAMP study, children with lower FEV1 were more likely to show a rapid decline in post-bronchodilator FEV1 over time [65], suggesting that children with more severe asthma may show more rapid deterioration in lung function over time. In most children with asthma, however, the rate of decline of FEV1 isv1% pred per year [65]. The mechanism behind this decline in lung function over time in asthmatics is not entirely clear. There is some evidence from animal studies that chronic allergic inflammation could induce the destructive changes observed in chronic obstructive pulmonary disease (COPD) [66], but comparable pathophysiological data in humans is lacking. Once chronic asthma persists into adulthood, the rate of decline of FEV1 increases to about or above 1% pred per year [67]. Interestingly, young children with episodic viral wheeze or "wheezy bronchitis" also show reduced lung function and an increased rate of decline in adulthood [68, 69]. This may also be related to the tracking phenomenon: it is by now well established that episodic viral wheeze in preschool children is associated with reduced airway calibre [70, 71]. This is in keeping with the hypothesis that risk factors operating in early life or probably even in utero are important in determining the risk of developing chronic diseases (such as COPD) in adulthood [72]. Although these data suggest that chronic asthma may be one of the risk factors for COPD, the important issue of whether effective therapy for asthma can prevent this has not been resolved. There is some evidence to suggest that treatment with ICS may reduce loss of FEV1 over time in children with asthma [21].

Current treatment strategies Long-acting b2-agonists: maintenance therapy, gaining asthma control and single inhaler therapy ICS are still the cornerstone of asthma therapy in children. As reviewed above, there are good reasons to believe that the poor level of asthma control achieved by many 199

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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 other’s 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 4–16 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 study’s inclusion criteria (FEV1 60–100% 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

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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 hypothalamic–pituitary– adrenal axis function was observed [85].

Leukotriene receptor antagonists Montelukast, a leukotriene receptor antagonist approved for once-daily oral use, has been quite extensively studied in childhood asthma. The drug is effective compared with placebo, both in school-aged and preschool children [86, 87]. Perhaps the most promising effect of the compound is its ability to reduce the number of virus-induced wheezing episodes in young children [88]. Head-on comparisons of montelukast with ICS are rare in children. In one such study, budesonide was more effective than montelukast in reducing asthmatic symptoms and airways hyperresponsiveness caused by allergen exposure [89]. In another, fluticasone was more effective than montelukast in improving FEV1 in asthmatic children, although a subgroup of children responded more favourably to montelukast than to fluticasone [30]. The present view of montelukast in asthma in school-aged children appears to be that it is an alternative to adding a long-acting b2-agonist in step 3 therapy. It is not unlikely that the indication for montelukast will expand to children with mild asthma [30] and to preschool children with episodic viral wheeze [88].

Anti-IgE To date, one study [90] has examined the effect of anti-IgE (omalizumab) on childhood asthma. In this study, children with atopic asthma receiving omalizumab showed fewer exacerbations and a larger reduction in ICS dose than children receiving placebo. Although omalizumab was well tolerated, its high cost and administration by monthly subcutaneous injection limit its use in clinical practice considerably.

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

How can adherence be improved? Looking at asthma statistics from epidemiological studies in developed countries it has become obvious that the problem of poor asthma control is one of education (both for 201

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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 child’s 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 doctor’s and the patient’s 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.

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This requires an active attitude by doctors as well as their patients, along with close follow-up. Such an approach has been shown to lead to improvements in bronchial hyperresponsiveness and quality of life without an increase in medication. This is probably the result of improved patient and doctor education, and improved inhalation technique (fig. 5) [26]. In addition, it has been shown that educating patients regarding the self-management of their asthma reduces both direct and indirect costs [96, 97]. A comprehensive treatment approach, taking into account patients’ and caregivers’ individual needs and expectations, should become the cornerstone of any asthma management plan in developed countries. In developing countries, poverty and lack of education are likely contributing factors to poor asthma control caused by poor adherence to treatment. Many patients in these countries simply do not have access to or awareness of the appropriate treatment. Poor healthcare infrastructure and/or the perception of asthma treatment as a low priority may add to the problem of undertreatment of asthma in developing countries. Improving the availability of appropriate treatment should be a major goal in improving asthma control in developing countries.

Defining relevant outcome measures? The assessment and evaluation of asthma relies on subjective markers, such as symptom scores, the need for rescue medications and quality of life, and on objective markers, such as lung function tests, including peak expiratory flow, FEV1, level of bronchial hyperresponsiveness and markers of inflammation. The frequency of rescue bronchodilator use is an excellent surrogate marker for poor asthma control. In the Salbuterol Multi-center Asthma Research Trial study, the apparent correlation of untoward outcomes associated with short-acting b2-agonist use was most noticeable in African-Americans, who were almost half as likely to be on concomitant ICS as their Caucasian counterparts [98]. The use of rescue bronchodilators is part of most symptom scores. However, if a symptom score is to serve as an instrument not only in follow-up but also in the primary evaluation, it would be better to exclude rescue bronchodilator use. In addition, a symptom score including medication use does not address possible differences in therapy strategies by various doctors and patients. The quality of life of patients with asthma may be misunderstood by healthcare professionals. Therefore, systemic assessment with standardised questionnaires [99–102] may contribute to better understanding between physicians, patients and their families [103, 104]. Differences in expectations and responsibilities are common. Whereas the patient’s view is that of a subjective satisfaction based on short-term expectations, the doctor’s responsibility is that of objective improvement based on long-term expectations (fig. 7). National and international guidelines recommend an initial evaluation and regular follow-up for assessing the severity and control of asthma [1, 2]. Pulmonary function tests, mainly spirometry, are generally considered the standard criterion for objective asessment of asthma severity during initial evaluation and follow-up, but even simple spirometry is commonly unavailable in primary care [4]. At the very least, the initial assessment of asthma severity should include an assessment of FEV1, according to the guidelines. Children with mild persistent asthma are expected to have FEV1 valuesw80% pred; children with moderate persistent asthma are thought to have values 60–80% pred; while FEV1 values v60% pred are viewed as evidence of severe persistent asthma in children [1, 2]. However, given the evidence that most asthmatic children have FEV1 values in the normal range, independent of disease severity, it may be necessary to redefine asthma severity in terms of FEV1 values for future guidelines [67]. 203

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Quality of life

a

b

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 [109–114]. 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 [115–117]. 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

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It will be crucial to define additional airway inflammatory markers that may be specific for the evaluation and monitoring of certain disease patterns or therapeutic strategies [124]. While biopsy, BAL and sputum induction may be difficult and not suitable for use in clinical practice, it should be stressed that these are highly valuable basic and clinical research tools. In summary, it seems logical that specific single diagnostic markers have to be defined for individual patient groups, as there is such heterogeneity in the disease expression; and that, if generally applied, a set of various diagnostic markers is more likely to be useful in larger populations.

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

BHR

Asthma Intrinsic asthma

Atopy

High serum IgE Fig. 8. – Asthma is a heterogenous disorder. BHR: bronchial hyperresponsiveness; IgE: immunoglobulin E.

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

Improving the definition of new therapy strategies? Based on the recognition that airway inflammation is the key feature in the pathophysiology underlying asthma, ICS have become the mainstay of maintenance therapy for childhood asthma. In certain cases, fear of steroid side-effects may limit the adherence to ICS treatment, but this problem can usually be solved by appropriate education of patients and parents. The search for new inhaled steroids with an improved safety profile would probably provide some, but not a spectacular, benefit in terms of widespread acceptance of ICS therapy. Conversely, a combination of anti-inflammatory treatment strategies might be more promising in improving outcomes in childhood asthma. Thus, the therapeutic potential in asthma of other anti-inflammatory drugs, such as statins, warrants further evaluation [132]. In addition, therapeutic strategies aimed at newly discovered pathways in the inflammatory cascade may also become increasingly important. For example, the role of toll-like receptors in the innate immune system has recently been recognised to be important in asthma [133]. Similarly, impaired innate immunity involving a major deficiency in IFN-b appears to lead to virus-induced asthma exacerbations [134]. Therefore, administration of toll-like receptor ligands or exogenous IFN-b by inhalation may provide new ways of reducing airway inflammation or preventing viral exacerbations in asthma. Over the last few years, airway remodelling has become recognised as an important pathophysiological feature of chronic persistent asthma. A combined therapy regimen may more successfully inhibit remodelling processes than a monotherapy with inhaled steroids, at least in adults [135–138]. Finding the pathophysiological mechanisms of 206

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remodelling processes may help in the development of specific and timely defined therapeutic strategies (fig. 9). Several mechanisms appear to be involved in these processes, mainly growth factors, such as vascular endothelial growth factor, basic fibroblast growth factor and angiogenin, which are involved in the angiogenesis seen in airway remodelling [139, 140]. Inhibition of such specific growth factors may, therefore, become a new therapeutic strategy in the future. Most drugs are delivered to the patient by inhalation. Despite the fact that this is clinically succesful in many patients, poor inhalation technique is a very common problem in children with asthma (and their parents) [9, 10, 141, 142]. This can be a major reason for therapy failure and poor adherence. Consequently, there is a need for improvement in drug output and deposition into the lower airways by inhaler devices used for childhood asthma and in their ease of use, in particular in the younger age group.

Is asthma a systemic disease? Data from the literature suggest a systemic link between various mucosal sites of the airways [124]. Although it is recognised that this link involves the bloodstream, bone marrow and mucosa-associated lymphoid tissue, the exact mechanisms of interaction between upper and lower airways in children are incompletely understood [124]. Both genetic predisposition and environmental factors appear to contribute to the development of specific phenotypes expressing disease activity in the upper airways, lower airways, or both. The understanding of the mechanisms underlying the relationship between allergic rhinitis, asthma and other atopic diseases may help to define strategies to influence clinical manifestations of allergic disease in the future. For the time being, it has been shown that treating allergic rhinitis in patients with both asthma and allergic rhinitis not only improves upper airway symptoms, but also improves asthma control (united airways) [143, 144]. ?

Inflammation

Bronchial disease

?

Remodelling

Clinical revevance

?

Time

?

Fig. 9. – Asthma is a heterogeneous disorder with variable expression of its underlying pathophysiological mechanisms. This may implicate that the timing and the type of treatment used should be based on individual characteristics.

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

Finding the answers to these questions It is obvious that more studies are needed to address the questions raised above. More effective educational programmes for patients and their caregivers, as well as for health providers, have to be developed and implemented in asthma care worldwide. Communication and collaboration has to be implemented as an integral part of asthma management on all levels of patient care. Studies examining tools to improve adherence with current medical therapy and to improve inhalation technique with current inhalation devices are of equal importance to studies aimed at developing new drugs and new inhalers. Studies using invasive measures, such as biopsies and BAL, are pertinent for the further understanding the mechanisms of the disease and hence, for the development of new treatment strategies. It is essential that such studies continue to be performed despite ethical constraints. Individual patients and patient groups should be better characterised in order to reduce heterogeneity and diversity of study patients, and to improve understanding, applicability, and generalisability of study results. Patient groups with specific disease characteristics regarding genetics and pathophysiology have to be defined by characteristic diagnostic markers. More studies of gene–environment interactions in asthma and of its pathophysiology are needed to improve understanding of individual responses to current as well as novel treatment strategies, in order to provide tailor-made individual treatment recommendations rather than blanket advice aimed at whole populations. In addition, long-term clinical trials and well-designed observational studies assessing the impact of treatment regimens on morbidity and mortality continue to have a high priority. Preferably, such studies should not only be performed with the support of the pharmaceutical companies manufacturing these medications, but also independently. In addition, workshops and other meetings of scientists and practitioners to enhance the exchange of ideas, data and experience, should be facilitated independent of support from pharmaceutical companies. Improved understanding of asthma as a systemic disease with a link to disease manifestation in other mucosal sites may help to define interventional strategies to influence disease progression. Well-designed long-term observational studies are needed to improve understanding of the natural course of the disease and the effects of current and novel therapeutic strategies on this natural history. 208

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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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140. Hoshino M, Aoike N, Takahashi M, Nakamura Y, Nakagawa T. Increased immunoreactivity of stromal cell-derived factor-1 and angiogenesis in asthma. Eur Respir J 2003; 21: 804–809. 141. Amirav I, Newhouse MT. Treatment failures in children with asthma due to inappropriate use of Turbuhaler. J Pediatr 2002; 140: 483. 142. Kamps AW, Brand PL, Roorda RJ. Determinants of correct inhalation technique in children attending a hospital-based asthma clinic. Acta Paediatr 2002; 91: 159–163. 143. Fuhlbrigge AL, Adams RJ. The effect of treatment of allergic rhinitis on asthma morbidity, including emergency department visits. Curr Opin Allergy Clin Immunol 2003; 3: 29–32. 144. Adams RJ, Fuhlbrigge AL, Finkelstein JA, Weiss ST. Intranasal steroids and the risk of emergency department visits for asthma. J Allergy Clin Immunol 2002; 109: 636–642.

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Bronchopulmonary dysplasia: current models and concepts A. Greenough*, S. Kotecha#, E. Vrijlandt} *Division of Asthma, Allergy and Lung Biology, King’s College London School of Medicine at Guy’s, King’s College and St Thomas’ Hospitals, London, #Dept of Child Health, Wales College of Medicine, Cardiff University, Cardiff, UK, and }Division of Paediatric Pulmonology, Beatrix Children’s Hospital, Groningen University Hospital, The Netherlands. Correspondence: A. Greenough, Regional Neonatal Intensive Care Centre supported by the WellChild Trust, 4th Floor Golden Jubilee Wing, King’s College Hospital, Denmark Hill, London SE5 9RS, UK. Fax: 44 2073468284; E-mail: [email protected]

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

Models of bronchopulmonary dysplasia Animal models have significantly improved the present understanding of the development and prevention of BPD, but positive effects in animal models do not necessarily translate into clinically meaningful outcomes in prematurely born infants. Eur Respir Mon, 2006, 37, 217–233. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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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.

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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 infant’s 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. 219

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The mechanism responsible for the excess lung fluid is not clear; possible explanations include abnormal protein permeability and increased filtration pressure in the pulmonary circulation. It is also likely that the reduced number of lung microvessels, through which the blood must flow, contribute to excessive filtration pressure and accumulation of lung fluid. These abnormalities of lung vascular development, overgrowth of vascular smooth muscle and decreased number of small blood vessels, have been described in infants with severe BPD.

Risk factors for BPD There are many risk factors for the development of BPD. These include prematurity, low birthweight and a genetic predisposition, including possibly a family history of atopy. Most attention, however, has focussed on the role of supplementary oxygen therapy and mechanical ventilation. Both are important antecedents of lung injury; in many animal models, increased reactive oxygen species and barotrauma/volutrauma result in an inflammatory response similar to that seen in the lungs of the prematurely born infant. Data from animal models suggests that the pre-term mammal is susceptible to damage from volutrauma in the first minutes after birth. Bjorklund et al. [13] found that six inflations of 60 cmH2O, each lasting 5 s, given to pre-term lambs before the administration of surfactant resulted in lower compliance for the next 4 h and worse lung histology than that seen in controls. Five sustained inflations of 8, 16 and 32 mL?kg-1 resulted in dose-dependent lung damage in pre-term lambs, but even those receiving 8 mL?kg-1, similar to the volume generated by the spontaneous breathing term infants, had worse lung mechanics than the nonintubated controls [14]. These effects are likely to be due to volutrauma rather than barotrauma, as similar experiments on adult rats and infant rabbits [15] have highlighted that lung injury can largely be eliminated by restricting chest wall expansion with either rubber bands or plaster casts. Patent ductus arteriosus (PDA) and fluid overload have been implicated in the pathogenesis of BPD. Excess fluid and the increased microvascular permeability with subsequent formation of pulmonary oedema contribute to poor lung function with exaggeration of hypoxaemia, hypercapnia and ventilator dependency. Recent attention has focused on infection, especially antenatal infection, in causing lung injury in susceptible infants. It is thought that antenatal infection initiates an inflammatory response in the foetal lung, which may prime the lung to greater lung injury when exposed post-natally to mechanical ventilation and oxygen supplementation. Spontaneous pre-term onset labour or pre-labour, pre-term rupture of membranes are thought to occur as a result of ascending infection from the vagina. Although a multitude of infections have been implicated in the initiating pre-term labour, disappointingly the results of antibiotic treatment of mothers presenting in pre-term labour with intact or ruptured membranes have been disappointing [16, 17]. U. urealyticum has been identified in the lungs of infants who develop BPD; a review of 17 studies demonstrated that the relative risk for BPD development in babies colonised with U. urealyticum was 1.72 (95% confidence interval (CI) 1.5–1.96). It is unclear, however, whether the organism is an innocent bystander or is causative of lung injury in vulnerable infants.

Preventive strategies Preventative strategies have been aimed at preventing or minimising lung injury and, more recently, promoting lung growth. Large randomised trials have been undertaken with varying results: no positive effect 220

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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

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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 0.12–3.78) or to treat a PDA (RR 1.52, 95% CI 0.83–2.81) [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 risk–benefit 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

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hydrocortisone on survival without BPD was terminated prematurely because of an excess of gastrointestinal perforations in the hydrocortisone group [35]. Inhaled steroids have fewer side-effects but also fewer positive effects, but do facilitate extubation and reduce the need for later rescue systemic steroids. Meta-analysis of randomised trials [36] has also demonstrated that vitamin A supplementation reduced death or oxygen requirement at 1 month of age and oxygen requirement at 36 weeks of PMA, but sideeffects meant that levels should be carefully monitored and further work is needed to define the optimum dosage, mode and duration of treatment. Some preventative therapies, although not shown to reduce BPD, may have other beneficial effects on prematurely born infants’ respiratory status. Prematurely born infants are relatively deficient in antioxidant enzyme systems, such as superoxide dismutase (SOD), and have low levels of antioxidants, such as vitamins C and E. They are therefore more vulnerable to oxygen toxicity. There is some evidence to suggest that improving antioxidant defences may improve the respiratory outcome of pre-term infants [37]. However, in two randomised trials, SOD administration was not associated with a reduction in BPD; in one there was a lower frequency of respiratory episodes (wheezing, asthma, pulmonary infections) severe enough to require treatment [38]. Administration of antioxidants antenatally might reduce BPD, not only by increasing antioxidant defences, but perhaps also by reducing pre-term delivery, since maternal supplementation with the antioxidant vitamins C and E has been shown to reduce the occurrence of pre-eclampsia [39]. There is also evidence from animal models that antenatal vitamin supplementation might impact favourably on lung growth and development; whether this occurs in infants is currently being investigated in a multicentre trial.

Treatment strategies Much of the treatment of infants with BPD is based on extrapolation from what is known about the pathophysiology of the condition, rather than the results of randomised intervention trials. Peak inspiratory pressures and inspired oxygen concentrations are kept to the minimum compatible with acceptable blood gases, and this includes allowing the carbon dioxide levels to rise, providing the infant does not develop a respiratory acidosis. Anecdotally, various ventilation modes have been used with success in infants with BPD, but none has been investigated systematically. To minimise further lung damage, BPD infants should be weaned from the ventilator as soon as possible, but, theoretically, this might result in greater compromise by increasing the work of breathing and interfering with adequate enteral nutrition. Excessive fluid should be avoided and clinical experience suggests BPD infants do not tolerate fluid intakes w150 mL?kg-1, or even lower levels if given enterally. Yet, BPD infants require a greater calorie intake than age-matched infants without respiratory disease, thus concentrated feeds, calorie supplements or modular component additives are frequently used. Given that excessive weight gain and crossing of centiles is associated with adverse long-term outcomes, it is essential that the appropriate nutrition of BPD infants is investigated. Supplementary oxygen is the mainstay of therapy for BPD infants, yet the amount of oxygen that should be delivered, as indicated by the target levels of oxygen saturation, remains controversial. In the Supplemental Therapeutic Oxygen for Prethreshold Retinopathy of Prematurity (STOP-ROP) Trial, premature infants were randomised to be maintained at 96–99% or 89–94% oxygen saturation levels [40]. Although, there were 223

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no significant differences regarding milestones or growth, more infants in the higher oxygen saturation group were still hospitalised at 50 weeks PMA and receiving diuretics. In addition, a greater proportion (13.2 versus 8.5%) of the higher saturation group developed pneumonia or exacerbations of BPD, perhaps indicating that the higher saturation levels were disadvantageous. The STOP-ROP Trial [40] was not designed to test the efficacy of different oxygen saturation levels with regard to pulmonary problems, but disadvantages of a higher saturation level were also reported from the Benefits Of Oxygen Saturation Targeting (BOOST) trial [41]. In the BOOST Trial, pre-term infants who were oxygen dependent at 32 weeks PMA and randomised to oxygen saturations of 95–98% rather than 91–94%, required supplementary oxygen for longer and more required supplementary oxygen at home [41]. However, these findings were expected and no significant differences were demonstrated with respect to growth and neurodevelopmental outcomes at a corrected age of 12 months. Further studies are therefore required to determine the most appropriate oxygen saturations levels, particularly in those BPD infants who have pulmonary hypertension. BPD infants with chronic oxygen dependency are often considered for "home oxygen". The criteria vary, but usually include that the baby’s only ongoing medical need is a requirement for supplementary oxygen, that they have good growth and that they are without frequent episodes of desaturation. In addition, in some centres, babies are sent home while they require tube feeding. Anecdotally, such a policy allows babies to be discharged home earlier, on average y2 weeks earlier, with financial savings and apparently no increase in subsequent admissions [42]. Families of home-oxygen babies, however, do require appropriate support, as they have been noted to be more prone to pre- as well as post-discharge anxiety, and the mothers have less vitality and more mental health problems. The true cost–benefit ratio of home-oxygen therapy requires testing. Rarely, infants with BPD may be considered for home ventilation. Criteria include maintenance of their carbon dioxide levels within safe limits on ventilatory equipment that is operable by the family at home, stable airway and medical condition and nutritional intake that is adequate to maintain expected growth and development. It is also important that the parents understand the long-term prognosis and are willing and capable of meeting the special needs of their child in the home setting, and that it is practical to provide the level of support and intervention that the child requires at home. The parents must also be able to evaluate their infant’s respiratory condition, use the equipment and be able to deal with all emergency procedures. The most common obstacles in Europe regarding home ventilation include funding for staffing and equipment, local organisational delays, unsuitable family housing or a change in the child’s medical condition, which affects the level of support required at home [43]. Most studies describe infants who showed clinical improvement and home ventilation could be stopped successfully after some months or even years, but not all children benefit from long-term ventilation. If home care is not possible due to family unwillingness, inability to cope or family desertion, then suitable alternative long-term arrangements should be sought, as it is not appropriate for a child to grow up in a hospital environment. The duration of home ventilation varies from 0.5–4 yrs. Medications should be administered to infants with BPD in the knowledge of the following: 1) many treatments have positive effects, but these may be short-lived with few or no long-term benefits demonstrated in randomised trials; 2) side-effects are common and may not be avoided by using the inhalational route; and 3) on current evidence, use of medications should be individualised and only continued while there is a demonstrable positive effect. Systemic administration of a proximal loop diuretic can acutely increase lung compliance and reduce airway resistance, facilitating a reduction in ventilatory requirements and transiently improving blood gases in both ventilated and nonventilated 224

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infants. Howver, chronic treatment has important side-effects. Hypokalaemia and metabolic acidosis can exacerbate carbon dioxide retention and hypercalciuria can lead to nephrocalcinosis (see below). Distal tubular diuretics have fewer side-effects but also have a smaller effect on lung function [44, 45]. Systematic review of the results of randomised trials has not highlighted any long-term benefits of chronic treatment with any type of diuretics, regardless of the method of administration. BPD infants have peribronchiolar smooth muscle hypertrophy and can have a positive response to bronchodilators, even while on the neonatal intensive care unit (NICU). Inhaled b2agonists and anticholinergics can temporarily improve lung function and blood gases and have synergistic effects [46]. There are, however, no randomised trials with long-term outcomes to inform whether chronic therapy on the NICU is appropriate. Results of small randomised trials conducted at follow-up suggest that inhaled bronchodilators are helpful in prematurely born infants who are symptomatic, but are of no benefit if given routinely [47]. Meta-analysis of the results of randomised trials has demonstrated that corticosteroids given to infants w3 weeks of age reduces the following: 1) oxygen dependency at 36 weeks PMA; 2) failure to extubate; 3) the need for rescue dexamethasone;and 4) the necessity for home oxygen therapy [48]. Fewer side-effects have been reported from the previously mentioned trials compared with the prophylactic trials, and this may reflect the older age of the infants or the fact that in some of the studies, "rescue" treatment was allowed. In a small randomised trial, inhaled corticosteroid administration was associated with a reduction in symptoms and bronchodilator requirement of prematurely born infants wheezy at follow-up [49]. The most appropriate duration and dosage of therapy needs investigating. BPD infants can suffer severe consequences of respiratory syncytial viral (RSV) infection, as shown by the greater need for hospital and paediatric intensive care unit admission [50]; retrospective studies have also highlighted that healthcare utilisation is increased at follow-up. Currently, there is no safe and effective vaccine against RSV. Palivizumab, a humanised monoclonal antibody, has been demonstrated to reduce the hospitalisation rate in BPD infants [51]; whether it improves the long-term outcome in BPD infants requires appropriate testing.

Related morbidity Respiratory BPD is associated with long-term respiratory morbidity. BPD infants have a high readmission rates in the first 2 yrs after birth, particularly due to respiratory infections [50]. Troublesome respiratory symptoms requiring treatment are common, even in young adults. In a recently reported follow-up study [52], 19-yr-old females who had had BPD were found to have a higher prevalence of doctor-diagnosed asthma, wheeze and shortness of breath than sex-matched controls. Long-term studies have also demonstrated lung function abnormalities, airway obstruction, and airway hyperreactivity and hyperinflation persisting into adolescence [53, 54].

Central and upper airways Endotracheal tubes may injure the upper airway causing laryngeal oedema; infants who have suffered prolonged or repeated intubations are at greatest risk. The prevalence of tracheal/bronchomalacia in pre-term infants with BPD varies from 16–45% [55–57]. 225

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The pathophysiology of tracheal/bronchomalacia in this patient group remains unclear; in particular it is unknown whether it contributed to the initial ventilatory requirement or was a consequence of long-term mechanical ventilation. Tracheobronchial abnormalities should be considered as a cause of persistent pulmonary problems in infants with BPD.

Cardiovascular system Pulmonary hypertension was often reported in infants who developed classical BPD, but is less common in new BPD cases. Whether this is due to a change in the underlying pathology in the more immature infant or to improvements in therapies, including home oxygen programmes, remains speculative. Infants with BPD who do develop pulmonary hypertension have an increased mortality rate. Increased work of breathing and hypoxic pulmonary vasoconstriction may potentiate the development of cor pulmonale. The incidence of systemic hypertension is higher in infants with BPD (13–43%) than in prematurely born infants with RDS only (1–9%) and in infants born at term (0.7–3%) [58]. Systemic hypertension may contribute to the development of left ventricular hypertrophy, which is often associated with right ventricular hypertrophy in infants with BPD. The pathogenesis of systemic hypertension and left ventricular hypertrophy overlap: metabolic effects of chronic hypoxaemia, hypercarbia and acidosis can increase cardiac output and stimulate the renin-angiotensin system, thereby elevating afterload. Chronic adrenergic stimulation from ongoing stress and neurohumoral stimulation, exogenous administration of medication (such as b-adrenergic agonists and steroids) and impaired lung clearance of norepinephrine have been implicated in the development of left ventricular hypertrophy [59]. It is unknown whether impaired metabolic function of the lung contributes to the pathophysiology of BPD by increasing circulating catecholamine levels, or if it is a marker of severe pulmonary vascular disease. It has been speculated that high catecholamine levels may lead to left ventricular hypertrophy or systemic hypertension. Other possible causes for systemic hypertension are renal damage due to nephrocalcinosis or prolonged umbilical arterial catheterisation. Prominent bronchial or other systemic-to-pulmonary collateral vessels were noted in early morphometric studies of infants with BPD. Although these collateral vessels are generally small, large collaterals may contribute to significant shunting of blood flow to the lung, causing oedema and the need for high levels of supplementary oxygen.

Growth retardation and gastrointestinal problems The causes of malnutrition and growth failure in BPD infants include decreased nutrient intake, hypoxia, concomitant dysfunction of other organ systems and increased requirements for energy [53]. In general, there is no significant impairment of nutrient absorption or digestion, unless there is concomitant bowel disease. Malnutrition can delay somatic growth and the development of new alveoli, and decreased muscle strength makes successful weaning from mechanical ventilation less likely and causes patients to be more prone to infections. Decreased glutathione levels may impair the response to oxidant-induced lung injury, and protein undernutrition may interfere with lung growth and DNA synthesis. Rib fractures, together with generalised bone demineralisation, are frequently observed in patients with BPD. This is usually secondary to dietary or parenteral deficiency of calcium or vitamin D and excessive calciuria resulting from chronic diuretic therapy. Gastro-oesophageal reflux (GER) is common (63%) in infants born prior to 32 weeks of gestational age and may contribute to malnutrition, the chronic inflammatory process and lung damage, but is poorly correlated with BPD [60]. The prevalence in prematurely 226

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born children with BPD is so high that those who have persistent respiratory difficulties not responding to therapy or with persistent vomiting or failure to thrive should undergo evaluation for GER and aspiration.

Renal problems The aetiology of nephrocalcinosis is multifactorial. Risk factors include immaturity, low glomerular filtration rate (causing low urinary flow), high intakes of calcium and phosphorus (to prevent rickets) with high excretion of calcium, low citrate excretion and use of diuretics. Prematurity-associated nephrocalcinosis resolves in months to years in most patients, but is still present in 15% at the age of 30 months [61]. While proximal tubular function is unaffected, high blood pressure and impaired glomerular and distal tubular function appear to occur more frequently than in healthy children [61]. In rare instances, this condition leads to renal calculi or renal insufficiency.

Neurological system and development Infants with BPD are at increased risk for cerebral palsy, microcephaly and neurodevelopmental delay affecting both cognitive (speech development, performance, IQ and receptive language) and motor function compared with premature control children matched for gestational age [62, 63]. Generalised hypotonia results in early gross motor delay. Risks of delay may be compounded by co-existing conditions, such as hearing loss, severe intracranial bleed and poor social environment. Long-term follow-up identified at 8 yrs of age either a dramatic improvement or long-term adverse effects on cognitive and academic achievement above and beyond the effects of very low birthweight [62, 64]. This highlights the need for continued monitoring of the learning, behaviour and development of BPD children, so that intervention can be planned for those with children who are at risk of school-age problems [62]. Long-term studies suggest that infants who received prolonged steroid therapy have worse neurological outcome, including an increased incidence of cerebral palsy, than control infants [65]. Observational studies have shown an association between transiently low thyroid hormone levels in pre-term infants in the first weeks of life (transient hypothyroxinaemia) and abnormal neurodevelopmental outcome. Thyroid hormone therapy might prevent this morbidity, but review of the research to date does not support the use of thyroid hormones in pre-term infants to reduce neonatal mortality, improve neurodevelopmental outcome or to reduce the severity of respiratory distress syndrome [66].

Ophthalmological problems Infants born prematurely have incompletely vascularised retinas and a peripheral avascular zone, the area of which depends on the gestational age. In premature infants, normal retinal vascular growth that would occur in utero ceases and there is loss of some of the developed vessels. With maturation of the infant, the resulting nonvascularised retina becomes increasingly metabolically active and hypoxic. Retinal neovascularisation, is induced by hypoxia, and occurs at y32–34 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

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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 (20–50%) in BPD infants is much greater than for other high-risk premature infants [72, 73]. An auditory brainstem response test conducted at the time of hospital discharge does not accurately predict later conductive hearing problems, hence infants with BPD should have routine audiological evaluation toward the end of the first year of life.

Sudden infant death syndrome In the 1980s, a seven-fold increase in the incidence of sudden infant death syndrome (SIDS) was noted retrospectively in low-birthweight infants with BPD [74]. Studies performed in the 1990s, however, found that pre-term infants with BPD are not at increased risk from SIDS compared with pre-term infants without this condition [75]. Risk factors for SIDS among infants aged 24–32 weeks gestation appear to be associated more with sociodemographic characteristics than medical problems. This suggests that for the immediate future, the risk for SIDS among very pre-term infants will be best addressed through further modification of the environment and parent behaviour [75].

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

Important future questions The most important future questions are the following. 1) What is the most effective and safe preventative therapy? Areas meriting further exploration are the resolution of lung injury in pre-term infants by the process of neutrophil apoptosis, as this appears to be inhibited in the more immature compared with the more mature infants [76], and an adequately powered trial of treatment for antenatally acquired infection. 2) Which 228

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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

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Edwards EA, O’Toole M, Wallis C. Sending children home on tracheostomy dependent ventilation: pitfalls and outcomes. Arch Dis Child 2004; 89: 251–255. Kao LC, Durand DJ, McCrea RC, Birch M, Powers RJ, Nickerson BJ. Randomised trial of long term diuretic therapy for infants with oxygen dependent bronchopulmonary dysplasia. J Pediatr 1994; 124: 772–781. Brion LP, Primak RA, Ambrosio-Perez L. Diuretics acting on the distal renal tubule for preterm infants with (or developing) chronic lung disease. Cochrane Database Syst Rev 2002; 1: CD001817. Ng GYT, da Silva O, Ohlsson A. Bronchodilation for the prevention and treatment of chronic lung disease in preterm infants. Cochrane Database Syst Rev 2001; 23: CD003214. Yuksel B, Greenough A, Maconachie I. Effective bronchodilator therapy by a simple spacer device for wheezy premature infants in the first two years of life. Arch Dis Child 1990; 65: 782–785. Halliday H, Ehrenkranz RA, Doyle LW. Delayed (w3 weeks) postnatal corticosteroids for chronic lung disease in preterm infants. Cochrane Database Syst Rev 2003; 1: CD110045. Yuksel B, Greenough A. Randomised trial of inhaled steroids in preterm infants symptomatic at follow up. Thorax 1992; 47: 910–913. Greenough A, Alexander J, Burgess S, et al. Health care utilisation of prematurely born, preschool children related to hospitalisation for RSV infection. Arch Dis Child 2004; 89: 673–678. The IMPACT-RSV Study Group. Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in highrisk infants. Pediatrics 1998; 102: 531–537. Vrijlandt EJ, Gerritsen J, Boezen HM, Duiverman EJ. Gender differences in respiratory symptoms in 19 year old adults born preterm. Respir Res 2005; 6: 117. Allen J, Zwerdling R, Ehrenkrnaz R, et al. American Thoracic Society. Statement on the care of the child with chronic lung disease of infancy and childhood. Am J Respir Crit Care Med 2003; 168: 356–396. Northway WH Jr, Moss RB, Carlisle KB, et al. Late pulmonary sequalae of bronchopulmonary dysplasia. N Engl J Med 1990; 323: 1793–1799. Downing GJ, Kilbride HW. Evaluation of airway complications in high-risk preterm infants: application of flexible fiberoptic airway endoscopy. Pediatrics 1995; 95: 567–572. Cohn RC, Kercsmar C, Dearborn D. Safety and efficacy of flexible endoscopy in children with bronchopulmonary dysplasia. Am J Dis Child 1988; 142: 1225–1228. Miller RW, Woo P, Kellman RK, Slagle TS. Tracheobronchial abnormalities in infants with bronchopulmonary dysplasia. J Pediatr 1987; 111: 779–782. Abman SH, Warady BA, Lum GM, Koops BL. Systemic hypertension in infants with bronchopulmonary dysplasia. J Pediatr 1984; 104: 928–931. Abman SH, Schaffer MS, Wiggins J, Washington R, Manco-Johnson W, Wolfe RR. Pulmonary vascular extraction of circulating norepinephrine in infants with bronchopulmonary dysplasia. Pediatr Pulmonol 1987; 3: 386–391. Akinola E, Rosenkrantz TS, Pappagallo M, McKay K, Hussain N. Gastroesophageal reflux in infants v32 weeks gestational age at birth: lack of relationship to chronic lung disease. Am J Perinatol 2004; 21: 57–62. Schell-Feith EA, Kist-van Holthe JE, van Zwieten PH, et al. Preterm neonates with nephrocalcinosis: natural course and renal function. Pediatr Nephrol 2003; 18: 1102–1108. Short EJ, Klein NK, Lewis BA, et al. Cognitive and academic consequences of bronchopulmonary dysplasia and very low birth weight: 8-year-old outcomes. Pediatrics 2003; 112: e359. Lewis BA, Singer LT, Fulton S, et al. Speech and language outcomes of children with bronchopulmonary dysplasia. J Commun Disord 2002; 35: 393–406. Robertson CM, Etches PC, Goldson E, Kyle JM. Eight-year school performance, neurodevelopmental, and growth outcome of neonates with bronchopulmonary dysplasia: a comparative study. Pediatrics 1992; 89: 365–372. O’Shea TM, Kothadia JM, Klinepeter KL, et al. Randomized placebo-controlled trial of a 42-day tapering course of dexamethasone to reduce the duration of ventilator dependency in very low

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birth weight infants: outcome of study participants at 1-year adjusted age. Pediatrics 1999; 104: 15–21. Osborn DA. Thyroid hormones for preventing neurodevelopmental impairment in preterm infants. Cochrane Database Syst Rev 2001; 4: CD001070. Arden GB, Sidman RL, Arap W, Schlingeman RO. Spare the rod and spoil the eye. Br J Ophthalmol 2005; 89: 764–769. Smith LE. Pathogenesis of retinopathy of prematurity. Growth Horm IGF Res 2004; 14: Suppl. A, S140–S144. Flynn JT, Bancalari E, Snyder ES, et al. A cohort study of transcutaneous oxygen tension and the incidence and severity of retinopathy of prematurity. N Engl J Med 1992; 326: 1050–1054. The International Classification of Retinopathy of Prematurity revisited. Arch Ophthalmol 2005; 123: 991–999. Marsh RR, Handler SD. Hearing impairment in ventilator-dependent infants and children. Int J Pediatr Otorhinolaryngol 1990; 20: 213–217. Gray PH, Sarkar S, Young J, Rogers YM. Conductive hearing loss in preterm infants with bronchopulmonary dysplasia. J Paediatr Child Health 2001; 37: 278–282. Werthammer J, Brown ER, Neff RK, Taeusch HW Jr. Sudden infant death syndrome in infants with bronchopulmonary dysplasia. Pediatrics 1982; 69: 301–304. Gray PH, Rogers Y. Are infants with bronchopulmonary dysplasia at risk for sudden infant death syndrome? Pediatrics 1994; 93: 774–777. Malloy MH. Sudden infant death syndrome among extremely preterm infants: United States 1997–1999. J Perinatol 2004; 24: 181–187. Kotecha S, Mildner RJ, Usher LR, et al. The role of neutrophil apoptosis in the resolution of acute lung injury in newborn infants. Thorax 2003; 58: 961–967. Kotecha S. Lung growth: implications for the newborn infant. Arch Dis Child Fetal Neonatal Ed 2000; 84: F69–F74.

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Cystic fibrosis A. Bush*,#, M. Go¨tz},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: [email protected]

In 1989, cystic fibrosis transmembrane conductance regulator (CFTR), the gene for cystic fibrosis (CF), was cloned and, since then, there has been an explosion of knowledge about the molecular biology of the gene product and the basic biology of the airway surface in particular. The quest is a cure for the disease, which is as yet elusive. Furthermore, the understanding of the disease has broadened from that of a lung and pancreatic disorder to a multisystem disease in which complications as diverse as osteopaenia and urinary incontinence are important to CF patients. Such is the variety of the research approaches being taken that no one chapter can possibly hope to encompass them all. Therefore the answers to the questions posed by the editors must to some extent reflect personal choice. Interested readers are referred to a recent publication [1].

What have been the most recent fundamental developments in CF? The expanding diagnosis of CF, and therefore the new diagnostic approaches: atypical CF and CF with normal CFTR CF is the most common inherited disease of white races, with a variable prevalence throughout Europe [2]. The underlying cause has been thought to be dysfunction or absence of CFTR, with the gene for this protein being localised to the long arm of chromosome 7. Historically, the diagnosis of CF was easy, being made at the autopsy of a baby or infant by the pathologist. In the 1950s, when survival had improved, measurement of sweat electrolytes became the diagnostic gold standard, and indeed to this day w98% CF patients have a positive sweat test [3]. For most patients, a typical clinical picture will point to the need for a sweat test; the usual problem is not interpreting the result, but remembering to ask for the test. Detailed guidelines have been published [4]. The sweat test, although pivotal, will not be discussed further in the present chapter because there are no significant new conceptual advances in that field. For a small minority of cases, an approach beyond simple sweat testing is needed because, by the 1990s, it had become clear that there were patients with typical phenotypic CF and an equivocal or even normal sweat test [5, 6]. In this situation, specific testing of CFTR structure or function, or less specific tests of the downstream consequences of reduced or absent CFTR function, are needed to make a diagnosis. A recent consensus group [7] defined the diagnostic criteria for CF as the following: 1) any of a clinical phenotype, CF in a sibling, or a positive newborn screening test; plus Eur Respir Mon, 2006, 37, 234–290. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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

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

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defects in proteins involved in CFTR processing, or interacting with CFTR, will be found to account for cases of atypical CF.

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

Nonspecific testing for CF Downstream effects of reduced or absent CFTR function. Ancillary testing may clarify the diagnosis, although it is important not to over-call the significance of minor abnormalities. Furthermore, the more specific tests for CFTR dysfunction have proven to be negative, the more alternative diagnoses, such as Schwachman–Diamond syndrome or primary ciliary dyskinesia (PCD), should be considered. Pancreatic function is most conveniently assessed by measuring human faecal elastase on a spot sample of stool, and this test is generally very sensitive and specific in the context of suspected CF [15]. This test may be misleading in the first week of life [16]. It cannot distinguish pancreatic insufficiency due to CF from other causes of pancreatic insufficiency and may not be useful in short gut syndrome, ileostomy patients or acute diarrhoea [17]. It can be performed even if the patient is taking pancreatic enzyme replacement therapy. Other supportive tests would be computed tomography (CT) scanning of the sinuses (a completely normal scan would be extremely unusual in CF); CT scanning of the chest (bronchiectasis) and abdomen (macronodular cirrhosis); and bronchoalveolar lavage (BAL) revealing a neutrophilic inflammation and typical CF organisms. In male adults, azoospermia is usual in CF; at any age, bilateral absence of the vas on palpation and ultrasound would be a pointer to CF.

What is a CF diagnosis? Typical versus atypical CF. The phenotype of typical CF includes the well-known respiratory manifestations but the hallmark is pancreatic insufficiency. Atypical CF patients are pancreatically sufficient, but have one or more other organ manifestations of the disease. The present authors are not sure this is quite the right division: most would think that the pancreatically sufficient CF patient with bronchiectasis and chronic bronchopulmonary infection with mucoid Pseudomonas aeruginosa has a very typical phenotype. The difficulty is classifying and diagnosing patients with no pulmonary or pancreatic phenotype but with, for example, congenital bilateral absence of the vas deferens or sinusitis.

CFTR-related disease. A number of studies have determined that there are a higherthan-expected number of patients with conditions that may be part of the CF phenotype, such as allergic bronchopulmonary aspergillosis (ABPA), chronic sinusitis and acute pancreatitis who have CFTR mutations [18, 19]. Of course, a few are eventually 236

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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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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 1–2 yrs, and this may clarify the situation.

Conclusions. As more has been discovered about CFTR, the diagnostic criteria for CF have become more opaque. Generally, the more doubt that exists, the more likely that ever greater sophistication of testing will increase confusion, rather than clarify matters. There is no one agreed approach; a pragmatic view for the clinician and a more interrogative one for the scientist are appropriate. For the clinician, the following criteria apply. 1) Do not forget to think of CF as a diagnosis (all too often the real problem in clinical practice), in particular in a screened population, which will still contain undiagnosed CF patients, albeit with a reduced prevalence (one in 70,000 [10]). 2) If it looks like CF, treat it as such. 3) Adjust treatment intensity to the severity of disease manifestations; what is appropriate in a child with severe bronchiectasis and severe airway obstruction will not be practical or desirable in a child who is asymptomatic but has two potentially disease-producing mutations in the CFTR gene. 4) Remain vigilant for deterioration; mild CF can only truly be safely diagnosed in the geriatric age group if there are no significant symptoms: never in a child! For the scientist, study of atypical phenotypes and diseases associated with a high prevalence of single CFTR mutations may yield a rich harvest of understanding of the biology of CFTR and its interactions with other proteins, which may eventually lead to new therapeutic approaches.

Multifunctional nature of CFTR: not just a chloride channel For many years, CFTR has been thought of as a chloride channel, with a few other functions. This is in part because measurement of sweat chloride is a superb diagnostic test and also because loss of electrolytes in the sweat perfectly accounts for two common disease manifestations, namely heat exhaustion and pseudo-Bartter’s syndrome. However, the connection between other CF disease manifestations and defective chloride function has been much harder to establish. CFTR has been shown to have a number of different functions (table 1). Although some, such as tooth colour, are probably not of any pathophysiological significance, the role of, for example, glutathione transport or aquaporin regulation in the production of CF disease should not be assumed to be minimal. Alternative approaches that point to the multifunctional nature of CFTR include gene expression studies (as manifest by mRNA changes) in CF knockout mouse lung tissue [22], pharmacological manipulation of CFTR in cell lines [23], and proteomics approaches in serum [24] and BAL [25], all of which have shown that alterations in CFTR function affect multiple other genes and proteins. The present authors suggest that the following points are important considerations in the pathophysiology of the clinical CF disease. 1) Different functions of CFTR may cause disease in different organs. The relationship between ion transport and lung disease is controversial. A recent study reported that residual chloride transport function in the nasal epithelium correlated with pancreatic function, and sodium transport was more significantly correlated with lung disease [26]. In contrast, in males but not females, the residual CFTR response to low chloride/isoprenaline correlated with forced expiratory volume in one second (FEV1) at age 19 yrs [27]. A third study showed no relationship at all between nasal ion transport and lung disease [28]. The role of sodium hyperabsorption in the pathophysiology of CF lung disease is discussed in 238

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Table 1. – Putative functions# of cystic fibrosis transmembrane conductance regulator (CFTR) Putative functions of CFTR Cl-, HCO3z transport Regulation of other ion channels, especially epithelial sodium channel Control of extracellular pH Intravesicular acidification Endocytic cycling Aquaporin 3 regulation Epithelial cell apoptosis Pseudomonas aeruginosa binding and internalisation Gap junction communication Ca2z regulation Extracellular fluid layer thickness Glutathione transport Increased mucus sulphation, decreased sialylation Activity of nuclear factor-kB Chemokine production Whitens teeth (mice) # : It is unclear which functions are important in producing the cystic fibrosis clinical phenotype in different organ systems.

more detail below. However, taken together, the evidence that chloride transport bears any relationship with the pulmonary phenotype is not convincing. 2) It could be speculated that local modifying factors might mean that residual ion channel function in the nose is different to that in the lower airway. For example, neutrophil elastase, which is abundant in the lower airway, increases epithelial sodium transport [29]. 3) CFTR processing is different in different organs, and at different developmental time periods. This is true both for wildtype CFTR and the mutant protein. For example, the mutant DF508 CFTR is almost completely destroyed intracellularly in the sweat gland, with virtually none reaching the apical cell membrane, whereas more translocates to the apical membrane in the airway and colon [30]. 4) There is differential regulation of CFTR function. For example, chloride transfer requires ATP and glutathione ADP [31]. From this, it follows that different mutations could, at least in theory, have differential effects on different functions of CFTR. 5) From these points, it follows on crucially that therapeutic strategies which correct only one or a limited number of functions of CFTR may have no effect on the CF clinical phenotype, if the wrong function has been targeted. Theoretically, different genetic and environmental modifiers could affect functions of CFTR selectively, or an interaction could only be significant in patients with particular genotypes. The complexities of genotype–phenotype correlations will be returned to in a later section. In summary, the present authors suggest that the assumption that all clinical phenotypes of CF are manifestations of chloride transport dysfunction is likely to be incorrect.

Novel monitoring strategies: high-resolution computed tomography, new lung function tests, the role of bronchoscopy Why do we need new techniques to detect early lung disease? The conventional clinical evaluation of babies relies heavily on symptoms, physical signs and the occasional chest radiograph. When school age is reached, spirometry can be performed, but by this 239

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stage there may already be previously undetectable evidence of fixed airflow obstruction. Lung function studies using the raised volume, rapid thoraco-abdominal compression (RVRTC) technique have shown that at diagnosis, unscreened infants have evidence of airflow obstruction [32, 33], even if there is no clinical or microbiological evidence of airway disease. Follow-up over 6 months showed no evidence of "catch-up" growth [34]. Bronchoscopy has shown evidence of inflammation and infection even in screened infants at a few weeks of age [35]. The preschool years can thus be a "silent" time, during which some, but by no means all, infants suffer probably irretrievable lung damage. The advent of novel therapies, such as genotype-specific pharmacological manipulation of CFTR and gene and stem cell therapy, make it all the more urgent to determine which preschool children are starting to deteriorate and which are likely to do well long term on conventional therapy. These novel treatments are likely to work best at an early stage, before irreversible lung destruction has supervened. An understanding of how to detect benefit is needed; mortality rate, which is of most concern to patients and families, is thankfully far too low to be a realistic end-point in clinical trials. Novel medications are also likely to have risks, which means that, at least initially, one would be unwilling to offer them to infants doing very well on conventional therapy. This is particularly relevant at the time of rapid alveolar growth, in the first 18–24 months of life [36–38]; little is known about medication safety in this context or how to monitor any potential damaging effects. Thus, with the increasing sophistication of treatment and the recognition that there are important early changes of CF lung disease in particular, the need to detect the earliest signs of deterioration has become imperative. Bronchoscopy and BAL, high-resolution computed tomography (HRCT) and sophisticated indices of lung function, such as lung clearance index (LCI), have increasingly superseded the conventional clinical tools of upper airway culture, chest radiograph and spirometry, respectively.

Detection of early lung disease Bronchoscopy. Bronchoscopy can be used to detect occult infection and to study early onset inflammation. Currently, the latter is a research tool only, because effective and safe anti-inflammatory strategies that are applicable to asymptomatic infants are not presently available. In terms of detection of infection, conventional practice in the clinic is to perform upper airway cultures, usually using a cough swab or a nasopharyngeal aspirate. In general, a negative upper airway culture is predictive of a negative BAL, but positive cultures are poorly predictive of a positive BAL [39]. Nonetheless, most physicians would err on the side of caution and treat a positive upper airway culture. The role of routine bronchoscopy in CF, as with HRCT, is undetermined. Practice varies between those who perform annual surveillance bronchoscopies in all children who are unable to expectorate, some units who virtually never resort to the bronchoscope, and probably the majority who use bronchoscopy as part of the diagnostic work-up in children who are not doing well on conventional therapy. In comparison with the lack of an attempt to secure a real evidence base in the studies in HRCT, there is a superb, prospective, longitudinal trial in Australasia, in which a routine bronchoscopy strategy is being compared with standard treatment in nearly 200 children. The results of this trial will be very informative but will not available for several years. Currently, it is known that BAL will identify organisms that are missed by upper airway cultures. It is also known that BAL itself is not a gold standard and that different pathogens may be isolated from different lobes in the same patient [40], and also that inflammatory marker levels may differ between lobes. Thus, even BAL can hardly be said to be the gold standard for determining the presence of infection. Another approach to the problem of detecting infection, which may, unlike 240

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BAL, sample all lobes, is sputum induction, which has previously shown to be safe in CF [41, 42]. Ho et al. [43] compared the culture results in spontaneously expectorated and induced sputum in 43 patients, two of whom could not tolerate sputum induction. Ho et al. [43] used 6% saline and pre-nebulised with salbutamol. Twenty-five patients had identical results (nine positive); 12 out of 16 had at least one more pathogen isolated by sputum induction and, in one patient, spontaneously produced sputum grew an extra pathogen. This technique holds promise in the clinic but is time-consuming, and it is surprising that Ho et al. [43] did not use salbutamol via a spacer instead of a nebuliser. Also, the study by Ho et al. [43] included young children who would not have been expected to expectorate spontaneously. An alternative approach is to have the child cough directly onto a microbiology culture plate ("cough plate") [44]. Induced sputum has been used to diagnose tuberculosis in African babies [45] and perhaps should be used more readily in symptomatic patients in this age group, prior to resorting to bronchoscopy.

Imaging techniques. Conventional practice is an annual chest radiograph, which can be scored in a number of different ways. Gross clinical disease is usually very obvious but chest radiography is much less sensitive to early changes. Although sophisticated systems like the Wisconsin score have been devised with early stage disease in mind, they are time-consuming to administer and have not found widespread use. This has led to consideration of the role of HRCT scanning in routine clinical practice. Three recent papers [46–48] have addressed this topic. Brody et al. [46] performed HRCT scans on a single occasion in 60 CF children aged 6–10 yrs with "mild" lung disease (forced vital capacity (FVC) w85%, but, as might be expected, FEV1 was often considerably less than this). More than one-third of patients had bronchiectasis, nearly two-thirds had air trapping and only a quarter had a normal scan. A semiquantitative scoring system was developed; there was reasonable (w80%) agreement between the three observers and they concluded that HRCT can detect changes in children with "normal" lung function. A longitudinal study [47] confirmed the greater sensitivity of HRCT compared with spirometry ("lung function tests"!). Forty-eight children had scans 2 yrs apart. Five different semiquantitative scores were used. Independent of which scoring system was used, spirometry was shown to remain stable while HRCT findings deteriorated. A weakness of the study [47] is the authors’ assumptions about what is and is not irreversible change. This may seem a strange comment but the reader should reflect how, in another context, even dramatic pneumatoceles, for example, can regress completely with time. The authors made no attempt to determine any clinical utility of their imaging findings. A third paper did move from the rather 19th century approach of estimating degrees of greyness and dilatation and making a semi-arbitrary scoring system, to digital handling of digital material [48]. The important findings relate to 10 normal and 10 CF subjects who were scanned at total lung capacity and functional residual capacity (FRC). The changes in voxel density were calculated and displayed graphically. This was a proof of concept study: the lung function comparator was spirometry. No clinical correlations were attempted, but the study again proved that spirometry is not a sensitive technique. The children studied were old enough to be able to cooperate with the respiratory manoeuvres required, but the use of sedation and bag and mask ventilation to take over respiration, analogous to RVRTC, allows the technique to be adapted for babies [49]. The use of true quantitative scoring is an advantage but, of course, it comes at a price of increased radiation dose. Current papers have a number of problems that must be considered in any critical assessment of the role of CT in CF; these are as follows. 1) There are no studies in 241

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peer-reviewed journals comparing HRCT with tests of lung function that are much more sensitive than spirometry, e.g. LCI (see below); a study reported in abstract form showed that a normal LCI excluded significant structural damage on HRCT [50]. 2) In many cases, there is use of semiquantitative scoring on digital data, which should be mathematically handled. 3) There has been a lack of attempts to quantify clinical utility or correlation with clinical status. 4) A radiation dose is being administered to lungs, breasts and potentially thyroid [51] in a population who are possibly at greater risk of at least some epithelial cancers [52]. 5) There is a paucity of reproducibility data, which is essential when HRCT is being advocated as a trial end-point in particular [53]. Better evidence is needed of what is "irreversible" and what can be corrected with growth or treatment. What, then, is the current role of HRCT in the detection of early lung disease in CF? It is clear that if there was no issue about radiation or fiscal cost, HRCT would be a routine, annual or more frequent test. The present authors believe that continuing to study the role of HRCT in the context of proper clinical trials is fully justified, that HRCT is a fully justified clinical investigation in children not making good progress for reasons that are unclear; however, evidence that regular HRCT as a clinical tool is justified has not yet been accumulated.

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

VBE: volume of back extraction; FVC: forced vital capacity; FEVt: forced expired volume in t seconds; FET: forced expiratory time. Data refers to a summary of the data from [54]. 242

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expiratory volume in t seconds) measures the emptying of different airway generations at different ages. There is a paucity of data following CF children serially through the preschool years. Nielsen et al. [55] followed 30 children in a 4-yr prospective study. They measured specific airway resistance (sRaw) using plethysmography, resistance using the interrupter technique (Rint), and impulse oscillation. They looked at the responses to cold air and bronchodilators and performed spirometry at school age. Only sRaw was consistently abnormal in CF. Lung function parameters appeared to show tracking during the preschool years. There was no difference in bronchodilator responsiveness or the results of cold air challenge. FEV1 in school age correlated significantly with sRaw only (r2=0.80), implying that Rint and impulse oscillation were insufficiently sensitive to detect disease. The study by Nielsen et al. [55] implies that there is significant early loss of lung function in CF, probably in infancy, and that intensive therapy, even over several years, does not reverse this loss of function. The question is: what is the most sensitive physiological technique for studying early CF lung disease? Gas mixing is exquisitely sensitive to distal airway disease. The physiological background has been long established, but recently Gustafsson and coworkers [56, 57] in particular have reintroduced the technique, using the wash-in and wash-out curves of an inert marker gas, such as sulphur hexafluoride. In essence, the child breathes in a known concentration of the marker gas during quiet tidal breathing, usually while distracted by watching a video. When equilibration has been reached, the child is disconnected from the tracer and the concentration is measured as the gas is washed out of the lungs during continued tidal breathing. The LCI is calculated from the time for the tracer gas concentration to fall to 1/40th of the starting level, and the total volumes (measured as number of FRCs) expired, currently measured with a mass spectrometer. The technique has been used in babies and toddlers (from whom only passive cooperation is required), as well as older children and adults. A further attraction is that LCI appears to have the same normal range, irrespective of age or size, thus obviating the problems of suitable controls for a population like CF, in whom impaired growth and nutrition is likely. Two recent papers have addressed the use of LCI in early stage CF [58, 59], both were cross-sectional. The first compared LCI with FEV1 in CF and controls [58], and compared Swedish and British data. The principle findings were that whereas there were significant negative correlations between LCI and both FEV1 and flow at 25% of vital capacity, most children with a normal FEV1 had increased (abnormal) LCI, implying that LCI was more sensitive. The normal values for LCI were similar in both countries, implying that the technique is transferable. The second paper [59], compared plethysmography (sRaw) spirometry [54] and LCI in a cross-sectional study. More abnormalities were detected by LCI than the other two techniques, and LCI was the only parameter which differed significantly between those who were and were not infected with P. aeruginosa. Longitudinal studies are the hardest to perform but the most informative of all to read. Kraemer et al. [60] studied 142 children and adolescents with CF, diagnosed on standard grounds who had documented onset of chronic P. aeruginosa infection and who had complete documentation of clinical details and lung function between the ages of 6 and 20 yrs. Those infected only intermittently with P. aeruginosa or with Burkholderia cepacia were excluded. Reflecting their population there was a high prevalence of unusual compound heterozygotes (3905insT/DF508, 9.2%; R553X/DF508, 8.5%). They performed spirometry, and measured FRC and sRaw plethysmographically, and LCI using nitrogen wash-out. Salient features of their detailed paper were as follows. 1) LCI deteriorated first, followed by mean forced expiratory flow between 25 and 75% of FVC, followed (surprisingly) by FVC ahead of FEV1. 2) When FEV1 was normal, LCI was 243

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abnormal in 52.5%; conversely, an abnormal FEV1 was only missed by LCI in 0.5%. FEV1 stabilised at 12 yrs of age, whereas hyperinflation continued to worsen. 3) Even at age 20 yrs, the slope of progression was still best for LCI and worse for FEV1. There are some caveats concerning this impressive data collection. The population was selected for infection status, and most were relatively well at age 20 yrs (only 10 deaths), so more data are needed on the relative values of these techniques in late-stage disease. There were no data from the preschool years. Nonetheless, these data would confirm cross-sectional data [48] that LCI is the most sensitive measure of deterioration in lung function. Most technique-based papers have not attempted correlation with other relevant indices, a point which will be returned to. One recent paper looked at the correlations between low frequency, forced expired oscillation [61], a lung function test previously shown to be relatively insensitive [55]. In this cross-sectional study, the children were undergoing bronchoscopy as part of their routine work-up, and the authors confirmed that infection, not symptoms, were correlated with inflammation, as judged by BAL neutrophilia, interleukin (IL)-8, neutrophil elastase and leukotriene (LT)B4. There were some correlations between inflammatory markers and lung function parameters, but no consistent picture emerged. This paper perhaps mainly confirms the lack of sensitivity of the forced oscillation techniques in early CF lung disease. What, then, is the role of LCI in monitoring early CF lung disease? It would seem likely that it is the most sensitive of current techniques and needs to be used as a comparator with, for example, CT scanning (see below). More information is needed about within-subject reproducibility before it can be recommended as a treatment outcome. It is also conceivable that it may be too sensitive and might detect abnormalities of no practical significance. More longitudinal data, in particular in more severely affected patients, are required to settle this question. For later-stage disease, LCI may be too sensitive, and spirometry will remain the preferred measure, pending the development of better tools. Finally, LCI measurements need to be adapted for robust, cheap equipment, which can be made widely available, if it is to become a routine clinical tool.

The role of macrolides Macrolide antibiotics are conventionally thought of as being derived from Streptomyces spp. They have a common macrolytic lactone ring, to which sugar moieties are attached. The first antibiotic to be discovered was erythromycin (a 14membered ring) in 1952 [62]. Clarithromycin (14-membered ring) and azithromycin (15membered ring) are more popular in clinical practice because of fewer side-effects (principally gastrointestinal) and longer dosing intervals. Other 14-membered ring compounds include roxithromycin and troleandomycin. Spiramycin, josamycin and midecamycin are 16-membered ring compounds. The latest group to be synthesised, the ketolides, are characterised by a keto group instead of a sugar moiety at position 3 of the lactone ring [63]. Macrolides are ubiquitous in nature; more than 2,000 different compounds are known, from sources as diverse as algae, lichens, insects and invertebrates. These compounds have recently been reviewed in detail [64–69], and a summary of their nonantibacterial actions is been given in table 3. It should be noted that different classes of macrolides may have different effects and extrapolation of effects between different classes is unwise. Macrolides, in particular erythromycin, have been used to treat community-acquired gram-positive respiratory infections, particularly otitis media, acute tonsillitis and mild cases of lower respiratory tract infection. More recently, the newer macrolides have been found to be useful in the treatment of resistant Mycobacterium tuberculosis and also 244

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Table 3. – Putative actions of macrolide antibiotics Effects described Downregulation of pro-inflammatory cytokines IL-1b, IL-6, IL-8, TNF-a, granulocyte-macrophage colony-stimulating factor Reduced neutrophil migration to IL-8 stimulation Reduced P. aeruginosa adherence Impaired P. aeruginosa viability and protein synthesis Upregulation of other members of the ABC cassette, e.g. MDR, MRP Effects on airway remodelling: inhibition of ET-1 and ET-1-induced VEGF production Suppression of iNOS production Binding to nuclear transcription factors AP-1 and NF-kB Reduced elastase production and oxidant burst Diminution of conversion of P. aeruginosa to the mucoid phenotype Reduced P. aeruginosa flagellin Reduction of mucus secretion and viscosity Reduction of acetylcholine release from cholinergic nerve terminals Inhibition of membrane-derived, toxic phospholipids Reduction of neutrophil adherence to endothelium and epithelium via ICAM-1, VCAM-1 and LFA-3; reduction of CD11b/CD18 Acceleration of neutrophil apoptosis Inhibition of P. aeruginosa quorum sensing; reduction in biofilm formation Synergy with other antibiotics, e.g. tobramycin Reduction in bronchial hyperreactivity Inhibition of fibroblast proliferation Treatment of occult S. aureus, H. influenzae or atypical mycobacterial infection

Note that different classes of macrolides may have differing properties [64–69]. 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, mean¡sd age 27.9¡6.5 yrs, 245

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Table 4. – Summary of published clinical trials of azithromycin (AZM)

Trial design AZM dose mg Subjects n Subject age yrs Treatment period months FEV1 drug versus placebo mean relative difference Other clinical outcomes Inflammatory markers Adverse effects

Australia [75]

UK [76]

USA [77]

Two-centres, parallel

Single-centre, crossover 250 or 500 daily 41 children w8 yrs 6 Median relative difference z5.4% Decrease in oral antibiotics

Multicentre, parallel

250 daily 60 adults 3 Mean relative difference z3.6% Decrease in intravenous antibiotics, increase in quality of life Decrease in CRP None

No difference in sputum IL-8, neutrophil elastase None

250 or 500, three times per week 185 children and adults w6 yrs 6 Mean relative difference z6.2% Decrease nonquinolone antibiotics, decrease in exacerbation, increase in weight Modest decrease in sputum elastase,no change in IL-8 Nausea, diarrhoea, wheezing

FEV1: forced expiratory volume in one second; CRP: C-reactive protein; IL: interleukin. Adapted from [1] with permission.

mean¡sd FEV1 56.6¡22.3%, 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 3.62¡1.78% (p=0.047) and -5.73¡1.66% (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 (8.1–18.6) and median (range) FEV1 and FVC were 61% (33–80) and 80% (53–99), respectively. The primary end-point was FEV1, which improved by 5.4% (95% confidence interval (CI) 0.8–10.5%) 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 5.3–16.5), whereas those who also inhaled rhDNase had a change of -3.6% (95% CI -22–3.9). 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 child’s 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

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A subsequent culture positive for non-tuberculous mycobacteria resulted in the patient being withdrawn from the study; however, they were included in the analysis on an intention-to-treat basis. The results showed a treatment benefit of 0.093 L or 6.21% for FEV1, and 4.95% for FVC (all highly statistically significant). There was marked variation in individual response;y12% increased FEV1 by i15% on azithromycin (none on placebo), but some actually deteriorated. Any benefit was lost within 28 days of discontinuing therapy. There was a 40% reduction in infective exacerbations defined as the use either of intravenous antibiotics or quinolones. The azithromycin group gained 800 g in weight compared with placebo. Physical functioning on the quality-of-life score improved significantly on azithromycin. There was no emergence of resistant microorganisms; there were more new isolates of S. aureus in the placebo group. Nausea, diarrhoea and, despite improved lung function, wheeze were more common in the azithromycin group. These trials have confirmed that for many, but not all, individuals with CF, azithromycin therapy improves pulmonary function. No one can predict which patients will benefit from treatment and the mechanism of action is unknown. Unlike in diffuse panbronchiolitis, there does not seem to be an effect on airway neutrophilia, but as with diffuse panbronchiolitis, chronic P. aeruginosa infection is not a prerequisite for benefit. The optimal dose and dosing frequency is not known. There are worldwide differences in how macrolides are used in CF. Some clinics use them as part of their routine treatment of chronic infection with P. aeruginosa. The present authors are more cautious, having taken account of the lack of knowledge of possible long-term side-effects in children; a 4– 6-month therapeutic trial of daily azithromycin is carried out in children who are not doing well on conventional therapy, irrespective of their sputum bacteriology, and the medication is discontinued if there is no benefit. This is clearly an area in which more work is needed, but also one in which clear-cut benefit for patients has been established. That at least some CF patients benefit from macrolides is indubitable, and has been confirmed by a Cochrane review [79]. The mechanisms of benefit have not been determined. The potentially beneficial effects of macrolides have been reviewed [64–69], and are summarised in table 3. Some are disease-specific (e.g. effect on the multi-drug resistant protein for CF), some are infection-specific (quorum sensing in P. aeruginosa infection) and others are generic. It should not be assumed that the same mechanisms are operative in all diseases in which macrolides are therapeutic, nor that a single mechanism is necessarily operative in a given disease.

Recognition of new multisystem complications When CF was first recognised as a separate entity in 1938, virtually all patients died in infancy from malnutrition and chronic respiratory infection. The concept of CF as a multisystem disease has developed as patients survive into adult life. The implications are: first, that these complications need to be detected early and managed effectively; and secondly, that their development paediatric practice needs to be confirmed, so that preventive strategies can be developed. The classical example of this would be CF bone disease (see below).

Insulin deficiency. Although there has been controversy about whether hyperglycaemia relates to peripheral insulin resistance or reduced pancreatic insulin production, current understanding favours the latter [80]. Approximately one-third of adult CF patients will eventually develop diabetes. The onset of diabetes is preceded by a "pre-diabetic" phase in which nutrition and lung function deteriorates [81, 82]. In females, but not males, diabetes is associated with increased mortality and, indeed, accounts for most of the excess 247

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mortality in CF females [83, 84]. Progressive pancreatic b-cell dysfunction, which is already subnormal in CF patients with oral glucose tolerance test (OGTT)-defined normal glucose tolerance status deteriorates further with consequential worsening of glycaemic status. This suggests that insulinopenia plays a prominent role in the pathogenesis of glucose intolerance and subsequent development of CF-related diabetes mellitus (CFRDM). Recent evidence casts doubt on the role of the OGTT as a gold standard for establishing CFRDM, as some children with a deteriorating clinical state may show normal OGTT responses and yet benefit from additional small doses of insulin [85]. The role of annual OGTTs is controversial. While many recommend that this test be part of the annual assessment from the age of 10 yrs [86], it may lull the clinician into a false sense of security (as described above), and it is arguable whether a child who has normal lung function and nutrition would accept a new invasive treatment on the basis of a blood test. In a child who is not doing well, an OGTT must be performed, but, if this is normal, additional intermittent blood glucose assessments may well reveal transient asymptomatic hyperglycaemia. Many recommend that all OGTTs be performed in the extended 180-min variant and that serum insulin levels be monitored concomitantly as impaired insulin secretion is common (65%), even in children with normal glucose tolerance [87]. Standardisation of the diagnosis of CFRDM and glucose intolerance is important, but until this is agreed, standard diagnostic procedures will be different in different centres. Early introduction of small doses of insulin in seemingly non-CFRDM with intermittent hyperglycaemia may dramatically improve the patient’s well-being. It remains to be seen whether insulin therapy generally should be considered in completely well CF children before the onset of overt CFRDM. They may be very vulnerable to the effects of hyperglycaemia. It may be that inhaled insulin may have a role [88]. Although oral hypoglycaemics were popular and are still the subject of therapeutic trials, logically the treatment of the deficiency state is a direct replacement of what is deficient, rather than trying to force the production of more insulin, which is unlikely to occur.

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

CF bone disease. By definition, osteopenia is a bone density between 1 and 2.5 sd below the bone density of a normal young adult. Osteoporosis is defined as i2.5 sd below that 248

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reference point. Recent data show an incidence of osteopenia in up to two-thirds of adult patients with CF and osteoporosis in a quarter [93]. Pathological fractures are common, and can include vertebral collapse, rib fractures and kyphosis [94], which may impair cough and sputum clearance and further reduce the capacity for physical exercise. There are multiple possible reasons for reduced bone mineral density in CF. These include inadequate physical activity, chronic airway inflammation with spillover of cytokines, such as IL-1, IL-6 and tumour necrosis factor (TNF)-a, into the systemic circulation, decreased calcium and vitamin uptake due to anorexia or pancreatic insufficiency leading to low vitamin D and K levels, delayed puberty and hypogonadism, and the use of oral and high-dose inhaled corticosteroids. In the transplant patient, immunosuppression and glucocorticoid use may worsen the problem. Independent risk factors are homo- and heterozygosity for DF508, male sex, greater severity of lung disease and malnutrition [95]. The CFTR null mouse model indicates that even in the absence of obvious nutritional and therapeutic differences, the CFTR mutation is associated with severe osteopenia [96]. The bone disease of CF has been studied in a few histomorphometry reports. It is heterogeneous, with reduced bone formation, but, surprisingly, also decreased bone resorption in some cases. There has been evidence of a generalised mineralisation defect but true osteomalacia is rare. Bone marker studies suggest that there is a background of low bone formation, especially during puberty, with periods of increased bone resorption [97]. Many CF clinics now recommend routine assessment starting in adolescence or young adulthood using dual energy X-ray absorption (DEXA) scans. These are performed every 1–2 yrs, depending on the level of risk. Age-related standard normal values must be taken into account, which may be a particular problem in paediatrics [98]. The management of bone disease is, firstly, prevention. Efforts to increase bone mineral density are particularly important during key areas of bone accretion in childhood and adolescence. There are no trial data upon which to base recommendations, but from studies in other conditions, the following criteria are recommended. 1) Maximise weight-bearing exercise. Even relatively simple programmes have been shown to increase bone mineral density [99]. 2) Maximise the intake of milk. In a study of asthmatic adolescent girls, increasing milk intake by delivering an extra pint to the house resulted in a significant increase in bone mineral density, still detectable 3.5–5 yrs after the intervention. Calcium supplements are not as effective as calcium in dairy products [100, 101]. 3) Ensure vitamin D levels are adequate by measuring them at least annually and supplementing as appropriate. However, in a large group of children cared for in a specialist centre, calcium homeostasis was normal with no abnormality of 25 hydroxyvitamin D. Levels were lower in adolescents and there was no association with pancreatic insufficiency or liver disease [102]. 4) Vitamin K has a key role in bone metabolism. Measurement of levels in serum is expensive and measurement of prothrombin ratio is insensitive, so many clinics recommend that vitamin K should be given empirically. 5) Pubertal stage should be monitored and any delay in onset of puberty treated. If bone disease is established, treatment is with oral antiresorptive agents such as biphosphonates. Pamidronic acid, etidronic acid and alendronic acid inhibit recruitment and function of osteoclasts, thereby reducing bone resorption. Alendronate, 10 mg?day-1 orally for a year [94], resulted in a significant gain in bone density. These are promising agents for long-term prevention and management, but many unanswered questions remain, not least at what stage they should be started. A future treatment option may be anabolic agents. Teriparatide is a Food and Drug Administration-approved anabolic bone growth agent, but data in CF are lacking. GH treatment for 1 yr improved height, weight, lean tissue mass and bone mineral content in prepubertal children aged 7.5–12.75 yrs [103]. 249

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Stress incontinence. Stress urinary incontinence occurs when the pressure within the abdomen is higher than the urethral resistance. This can happen while coughing, laughing, sneezing, huffing, bending, lifting a heavy object, doing physiotherapy, participating in vigorous sports activities, such as trampoline jumping and during sexual intercourse. While already common in non-CF females (30%), stress urinary incontinence occurs in close to 70% of CF females agedy30 yrs. The dominant precipitants in CF are coughing (84%) and laughing (68%). The problem has been addressed and assessed only in recent years and embarrassment was the main reason for not seeking medical advice. Social life may be affected, as reported by one-third of respondents of an Australian survey [104]. The differential diagnosis of unwillingness to perform physiotherapy includes stress incontinence during treatment. A sympathetic enquiry should always be part of the assessment of such children. It is not only adult females who are affected; an Australian study showed a median age of onset of incontinence of 13 yrs [105]. No relationship was seen with age, lung function, body mass index or menstrual status. There is little evidence base for treatment. CF females are prone to vaginal candidasis, which may worsen stress incontinence, and this should be treated [106], as should urinary tract infection. Pelvic floor muscle exercises are helpful in establishing improved muscle strength, leading to reduced leakage [107]. Biofeedback for training pelvic floor musculature may be more acceptable but there are no data for CF. Tension-free vaginal tape should be considered for females with urinary stress incontinence for whom conservative (nonsurgical) treatments have failed to provide a solution after 3–6 months. The current surgical gold standard is colposuspension; the help of a gynaecological expert should be sought when advising CF females. In the future, duloxetine, a selective serotonin and noradrenaline re-uptake inhibitor, may be useful [108].

Chronic pain. Assessment of pain in patients of all age groups has not generally been part of CF care concepts until relatively recently. Disease-related pain is often in the chest, and headache is also common. Other considerations include back pain, abdominal pain and musculoskeletal pain. Joint pain was found in 17% of an Australian paediatric CF population [109]. Recent work [110] on acute and chronic pain in 46 patients aged 8– 17 yrs showed pain predominantly in the abdominal/pelvic region (50%), followed by chest pain (37%) and head/neck pain (33%). Pain management consisted of rest, medication, heat or cold, the support of family and friends, and distraction therapy. No patient resorted to opioids. Children with chest pain reported significantly greater perceived functional limitations due to pain than patients without chest pain. Assessment and treatment of pain should routinely be included in the overall management plan. The negative effect of pain on pulmonary function and on quality of life should be taken into account.

Advances in sexuality, reproduction issues and management of the pregnant female with CF Sexuality. In both sexes, the subject of sexuality and reproduction should be addressed in early adolescence in order to avoid misleading information from noncompetent sources. Obstructive azoospermia in CF males is almost universal and is an early cause of concern in parents of patients with CF or in male patients once they reach adolescence. Male fertility should be discussed specifically, and before puberty. Males should be reassured that pubertal changes will be normal and sexual intercourse unaffected [111]. They need to differentiate between potency, which is unaffected, and fertility, which may be achieved by new microsurgical techniques outlined below. They should also 250

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understand that infertility is not a protection against sexually transmitted diseases and barrier methods are as essential for them as any other males. Post-pubertal males should be offered semen analysis. The usual outcome is absence of sperm and low volume of ejaculate. Occasionally congenital bilateral absence of the vas deferens may be found with none of the usual clinical CF organ manifestations. There is a higher than expected prevalence of mild CF mutations in this group [112]. Some undoubtedly have a pure genital (nonclassical) form of CF (two CF-producing mutations found); others have a single mutation and no other manifestation of CF. Female sexuality is hardly affected and sexual life is normal. However, in both sexes, sexual enjoyment may be affected in cases of severe physical distress (weakness, shortness of breath, copious expectorate). This can sometimes be alleviated by planning sexual intercourse in the morning, when the patient is not so tired. Females with CF need to consider contraception when they are sexually active. Contraception using barrier methods is no more prone to failure than in non-CF persons. Oral contraceptives have been found to be safe despite worries concerning liver function being affected by the progestogen component of contraceptives. There are some concerns that very low-dose oestrogen contraceptives may be less effective in CF females.

Reproduction issues. Female fertility in CF appears to be reduced, although no recent work has been devoted to the subject. Whether reduced water content of cervical mucus and/or increased viscosity are of major importance is unanswered. Primary (10%) or secondary amenorrhoea may be a problem, with advanced lung disease or poor nutritional status. As many as 50% of females with CF have anovulatory cycles [113]. Male reproductive choices have improved with percutaneous or microsurgical epididymal or testicular sperm aspiration and subsequent in vitro fertilisation. Intracytoplasmic sperm injection has become the preferred approach for azoospermia [114]. Older alternative methods, such as donor semen fertilisation or adoption, may have a role in selected cases. Genetic counselling is an important aspect for both CF males and females. The child of a CF parent is at best an obligate carrier; if the partner is an unsuspected carrier, or even has mild, undiagnosed CF, then the chances of producing a CF baby are 50 and 100%, respectively.

Management of pregnant females with CF. Pregnancies in females with CF are increasing concurrently with increased life expectancy. A US Cystic Fibrosis Foundation registry report covering 1985–1997, with data on 680 pregnancies inw8,000 females with CF, assessed the impact of pregnancy on survival [115]. Those entering pregnancy had a better initial health state (lung function, weight) and their 10-yr survival was higher than in those who did not become pregnant. This is presumably an example of reverse causation (the less-healthy females opted not to become pregnant). In a separate analysis, possibly confounding factors, such as chronic infection with P. aeruginosa and pancreatic function, were taken into account, but similar results were obtained. Using Cox proportional hazard modelling to adjust for baseline age, FEV1 % pred, weight, height and pulmonary exacerbation rate?yr-1, pregnancy was not associated with an increased risk of death. Pregnancy did not add an additional adverse prognostic effect in any subgroup, including patients with FEV1 v40% pred or diabetes mellitus. Thus, females with CF who became pregnant were initially healthier and had better 10-yr survival rates than females with CF who did not become pregnant. After adjustment for the initial severity of illness, females who became pregnant did not have a significantly shortened survival. Similar results were obtained in 75 pregnancies in French CF mothers [116]. There was almost a 30% incidence of low-weight newborns. 251

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Although earlier reports of pregnancy in females with CF had shown alarming rates of maternal complications, these recent data support a much more optimistic outlook. The pre-pregnancy severity of CF seems to be the decisive determinant of the subsequent course of the mother. On a cautionary note, it should be mentioned that pregnancy in CF is associated with decreased insulin sensitivity and high hepatic glucose production, in addition to inherent decreased insulin secretion. Pregnancy in CF is also associated with increased protein turnover and less response to the anticatabolic effect of insulin. These changes may predispose the pregnant CF females to early development of diabetes and poor weight gain [117]. Close collaboration between the CF team and the obstetrician is essential in all CF pregnancies. Undoubtedly, females with CF should and will make their own reproductive decisions. Couples do need to bear in mind that an already sick females with CF who becomes pregnant may deliver very prematurely and die soon afterwards, leaving a bereaved partner with a pre-term, neurodevelopmentally handicapped child.

The possibility of genotype-specific therapy The molecular abnormalities of CFTR processing have been divided into five classes [118]. Class I mutations are characterised by premature cessation of mRNA synthesis, for example, due to a mutation causing a premature stop codon (e.g. G542X). This is a particularly important category in some Jewish populations (see below). The commonest mutation in white races, DF508, is the exemplar of class II mutations. CFTR mRNA is produced and transcribed, but the protein is misfolded and, instead of tracking to the apical membrane, is ubiquitinated and marked for destruction in the endoplasmic reticulum. Class III mutations, G551D, for example, are characterised by abnormal CFTR reaching the cell surface but the regulation of the protein is abnormal, leading to abnormal function. Class IV mutations reach the cell surface but have abnormal channel function (e.g. R117H). Class V mutations lead to reduced amounts of CFTR at the apical cell membrane. These include missense mutations (A455E) and alternate splicing mutants (3849z10kbCRT), which lead to reduced synthesis of CFTR. There are also rarer variants, where CFTR fails to track selectively to the apical membrane but instead is present nonselectively all over the cell surface, and variants where the half-life of CFTR at the apical membrane is greatly reduced. This classification has use as a prognostic tool, at least for groups of patients [119]. Those homozygous for severe mutations (classes I and II, usually pancreatic insufficient) have been shown to have a worse survival than those with mild mutations, who are usually pancreatic sufficient. However, the possibility of therapeutic manipulation of the mutations in a class-specific manner is currently a hot topic. The first realisation that DF508 was, potentially at least, partially functional came when it was expressed in the Xenopus oocyte and was shown to function, as a chloride channel at 26uC but not at mammalian body temperature (37uC). This observation was confirmed by other in vitro stimulation studies. Amongst other compounds that had this putatively beneficial effect on DF508 was sodium phenylbutyrate, already licensed for the treatment of some urea cycle disorders. A randomised, double-blind clinical trial of this compound showed some restoration of ion channel function, measured in the nose [120]. Chloride channel function was shown by a response to low chloride and isoprenaline solution but, potentially crucially, there was no change in the response to amiloride, implying unaltered ENaC hyperfunction. Further work is proceeding with sodium phenylbutyrate, including the potential to combine it with a CFTR stimulator such as genistein [121]. Sildenafil has also been shown to have in vitro effects on DF508 trafficking [122]. 252

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It is likely that it will be easy to find at least male volunteers if sildenafil ever comes to a large clinical trial! The use of increased DF508 CFTR trafficking in cell systems in vitro has been the basis of a huge fiscal investment in high throughput, robotic screening for candidate compounds for genotype-specific pharmacotherapy. Many thousands of compounds from chemical libraries can be screened in a day for this activity. It should be noted that this approach, brilliant as it is, runs the risk of missing important compounds (which could act in different ways to increase DF508 CFTR function) and also raise false hopes if the trafficked CFTR cannot, in fact, correct the missing relevant function for the particular organ (usually the lung). At the time of writing, this approach has unfortunately not delivered any really promising compounds. A novel approach for class I mutations came with the in vitro observation that some aminoglycosides (not tobramycin) lead to premature stop codons being ignored and protein continuing to be transcribed. Gentamicin nose drops have been shown to increase chloride channel function in the nose, and molecular studies showed increased CFTR trafficking. This was shown to be a genotype-specific effect by looking at a control group of DF508 homozygotes, in whom, as predicted, no change was shown [123]. An extension of this approach, to allow lower risk of aminoglycoside toxicity, is to either coadminister polyanions, which directly reduce toxicity and prolong the effect, or look for alternative compounds with the same effects [124]. The use of genotype-specific, pharmacological manipulation of mutant CFTR remains a tantalising and exciting possibility. Proof of concept has been shown, but as yet nothing is ready to be used as a practical clinical tool. Furthermore, the concerns about correcting relevant functions, discussed elsewhere in this chapter, still remain.

Current models and concepts Pathophysiological mechanisms Airway surface liquid: low volume versus high salt models. The link between the basic molecular biology of CFTR and the typical clinical phenotype of CF lung disease has been difficult to make. The two current hypotheses have led to the "high salt" and "low volume" models, of which the latter seems more plausible. The high salt model postulated that the defect in ion transport leads to a high salt epithelial lining fluid, which in turn leads to inactivation of small polypeptides (bdefensins, lactoferrin, lysozyme), which function as primary defence mechanisms for the airway [125]. The model was bolstered by elegant in vitro work, but technically difficult measurements of the epithelial lining fluid suggested that the fluid was isotonic and not particularly high in sodium. The low volume hypothesis focuses on the height of the periciliary liquid layer [126]. Respiratory cilia are another important primary airway defence mechanism, which need a tightly regulated layer of fluid in which to function. The fluid needs to be sufficiently shallow that when the cilia are fully extended, they engage the mucus layer to propel it upwards in a gyratory fashion, and must be sufficiently deep to accommodate the ciliary recovery stroke. The total airway surface decreases dramatically from distal to proximal (bronchioles to trachea), and in normal subjects, if the proximal airways are not to be flooded with fluid produced in the bronchioles, fluid absorption must take place to tightly regulate the height of the periciliary fluid. This is the function of the ENaC channel; in CF, the model proposes that active hyperabsorption of sodium and passive hyperabsorption of water leads to a reduction in the height of the periciliary fluid layer, impairment of ciliary function and, 253

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thus, a breech in the airway defences against infection. This concept is supported by elegant studies in excised tissue and cell culture, which nicely demonstrate reduction in periciliary fluid height. It is difficult to believe that the low volume hypothesis on its own can account for CF lung disease but it does seem likely that failure to defend the periciliary layer is part of the story. However, this hypothesis leads to the assumption that impaired mucociliary clearance is pivotal in CF lung disease. This collapses as the sole explanation for the following reasons. 1) Mucociliary clearance (MCC) is virtually absent in PCD, but lung disease is milder and infection with typical CF pathogens, such as mucoid P. aeruginosa, is rarer and occurs much later [127]. 2) Other factors must be invoked to account for the narrow range of pathogens isolated in CF and the exaggerated inflammatory response to infection (see below). 3) There is some evidence that mucociliary clearance is normal at least early on in CF and that abnormal clearance is secondary to infection and inflammation [128, 129] Nonetheless, the low volume hypothesis has lead to important understanding of the CF airway.

Importance of epithelial sodium channel versus chloride transport. The relatively poor correlation between chloride transport and CF lung disease and the difficulty of constructing biologically plausible models to connect the two, together with the insights of the low volume hypothesis, have led to a focus on ENaC overactivity as the pivotal feature of CF lung disease. The CFTR knockout mouse has an intestinal phenotype but as a model for CF lung disease, has proved disappointing. For example, the establishment of infection with P. aeruginosa requires nebulisation of the microorganism with agar beads; this is quite unlike the human airway, wherein it is virtually impossible to avoid spontaneous chronic Pseudomonas infection. A much better model of CF airway disease has come from a normal mouse which overexpresses the b-subunit of ENaC [130]. There was airway surface liquid depletion, with spontaneous mucus plugging, neutrophilic inflammation and poor bacterial clearance, and the picture is very reminiscent of natural CF lung disease. Postulating that ENaC overexpression, and not failure of chloride transport as the cause of CF lung disease, would also solve the "O’Brodovich 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. Liddle’s syndrome is due to a b-ENaC gain of function mutation (R566X), which causes systemic 254

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hypertension but not any lung disease [135, 136]. The mouse with Liddle’s 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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inflammatory response in CF is not related to the underlying CFTR defect, but is a secondary response to prolonged and chronic infection. In summary, the balance of the evidence is in favour of the hypothesis that the exuberant and exaggerated inflammatory response in the CF airway is secondary to infection, and not a primary part of the CF defect, but the question has yet to be settled definitively.

Related diseases What can be learnt from similarities and differences between CF and PCD? Even with the best treatment, there is a remorseless deterioration in lung function in CF patients. By contrast, two studies have shown that, whatever the level of lung function at diagnosis of PCD, it can be stabilised using conventional treatment [127, 149]. The range of infecting organisms is not dissimilar between the two conditions, at least late in the disease, even including mucoid P. aeruginosa and atypical mycobacteria. Clearly any therapeutic strategy that could convert a deteriorating disease (CF) into a stable phenotype like PCD would be valuable. The inflammatory phenotype in PCD is little studied. Least controversially, although levels of NO are low in CF, they are much lower in PCD [150, 151], so whatever the putative beneficial host defence effects of nitric oxide (NO), it would be predicted that strategies to increase airway NO are likely to be beneficial. However, as a salutary check to over-confident predictions, despite the excellent theoretical reasons put forward above, a recent study showed that inhaled arginine both increases exhaled NO and spirometry in CF patients [152]. These results need to be confirmed but are an encouragement to humility in theoreticians. Sputum examination reveals a neutrophilic cytology [153] and similar levels of IL-8 to those in CF [154]. Little is known about the other inflammatory mediators; one study has suggested that, at least in stable PCD, LTB4 is not a significant mediator [155]. However, it is important if, as it seems, IL-8 levels are higher in PCD because, if confirmed, it would mean that using reduction in IL-8 as a surrogate end-point in CF trials is unlikely to be useful. Clearly, this is an area in which further work might be very fruitful. It may be that gene chip arrays, proteomic and metabolomic approaches may be helpful in generating hypotheses about the reasons for the differences between these diseases.

What can be learnt from similarities and differences between bronchiectasis in the developing and developed world? Non-CF bronchiectasis is an orphan disease, at least in the UK [156], despite evidence that it may be more common than previously appreciated. In other contexts, a number of studies have documented that bronchiectasis may be much more severe in indigenous peoples such as the Pacific islanders and Alaskans [157–160]. 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 256

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Guthrie and hypothyroid screening. Cut-off levels are laboratory dependent. Mutation analysis using commercial kits mostly screening for 32 mutations may be part of the protocol. Internationally, newborn screening is only patchily offered. Overall, the US Center for Disease Control and Prevention feels that newborn screening for CF is justified [162] and that benefits outweigh risks [163]. At present, the median age of a CF diagnosis with newborn screening is y0.5 months compared with14.5 months, in the USA, for a conventional diagnosis [164]. An early diagnosis of CF has a number of potential benefits and it primarily enables detection of asymptomatic infants. They must be referred to established CF programmes so early detection of complications and rapid initiation of therapy can be practiced. Hopefully, this will diminish disease-related weight loss, pulmonary infections and, possibly, deterioration of lung function. While longevity is steadily increasing, it is becoming clear that halting disease progression before damage becomes established is a paramount goal of management. Other benefits include the obviation of the frustration of the family at fruitless appointments with health professionals [165] and false reassurance before the diagnosis is made. Finally, the tragedy of a late diagnosis, preventing the family from having an antenatal diagnosis in a subsequent pregnancy, is avoided. Most of the evidence in favour of screening is from observational studies. The only randomised controlled trial was from Wisconsin, USA [166]. There is cumulative evidence that CF detected by newborn screening results in better and early nutritional treatment leading to improved growth and, in one study, better cognitive development [167]. Other benefits may include reduced hospitalisations and improved survival rates. Beneficial outcomes regarding pulmonary function are less certain. Good nutritional status early in life and good subsequent lung function are related [168, 169]. Advantages in the first year of life for CF diagnosed by newborn screening are avoidance of stunting (height v3rd percentile), wasting (weight v3rd percentile), hypoproteinaemic oedema, chronic airway infection with P. aeruginosa and hospitalisation for complications. Observational studies reviewed recently [170] provide indirect evidence that newborn screening may improve pulmonary health and survival in patients with CF. Early asymptomatic diagnosis (not by newborn screening) in children born after 1987 was shown to result in improved lung function for as long as up to 9 yrs of age [171]. Improvements in early treatment strategies may have allowed early diagnosis to lead to more aggressive therapies resulting in improved pulmonary health. However, it is unclear to what degree early nutritional status may reflect early pulmonary status, which in turn may predict pulmonary outcomes [170]. A long-term observational study [172] indicates that early treatment made possible by neonatal screening may be important in determining subsequent clinical outcomes for children with CF. Comparing an historical before-screening group with a screening-based group, it was shown that the screened CF group had a significantly better lung function with an FEV1 of 9.4% higher at age 10 yrs than in the nonscreened children. These data are, at present, not supported by the Wisconsin randomised clinical trial of CF newborn screening, as they do not show improved pulmonary outcomes in the screened group [173]. This may be because of poor infection control in one of the centres to which screened babies were referred. There may be a potential downside to newborn screening for CF. This includes psychological stress for parents during the confirmatory diagnostic process [174]. In general, however, benefit outweighs psychological stress, including that during the ruling out of CF after a false positive CF diagnosis. There may be an early introduction to hospital settings with added risks of P. aeruginosa acquisition [163]. Rigorous infection control policies therefore must be mandatory for all screened positive babies. Even considering this, the present evidence clearly underscores that newborn screening for CF makes sense and that national health authorities should cover the financial and organisational costs. 257

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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 4–16 yrs. Nonmucoid and mucoid P. aeruginosa were acquired at median ages of 1.0 and 13.0 yrs, respectively. In contrast with the short transition time from no P. aeruginosa to nonmucoid P. aeruginosa, the transition time from nonmucoid to mucoid P. aeruginosa was relatively long (median, 10.9 yrs) and could be slightly extended by antibiotic treatment [178]. Epidemic, antibiotic-resistant P. aeruginosa pose special problems [179] and may be suggestive of patient-to-patient transfer in a nosocomial setting. Cohort segregation is effective in reducing transmission risks and molecular characterisation of P. aeruginosa, although expensive and rarely performed, would be the ideal instrument to determine needs for patient isolation and infection control. There are no universally accepted guidelines for the treatment of respiratory infections. Current antimicrobial strategies include the following: 1) prevention and treatment of S. aureus; 2) eradication of early P. aeruginosa isolates; and 3) for chronic P. aeruginosa infection, continuous suppressive therapy, intermittent intravenous treatment, either planned or to treat an actual or impending pulmonary exacerbation.

Staphylococcus aureus. Whether early anti-staphylococcal prophylaxis is beneficial is unclear [180]. If this strategy is to be adopted, then narrow-spectrum antibiotics are preferable and cephalosporins avoided. Both observational data from a European registry using mainly oral cephalosporins [181] and a formal study from the US using cephalexin [182] indicate that whilst S. aureus is suppressed there is an unacceptable increase of P. aeruginosa infection. The pragmatic proposal of use of prophylactic anti-staphylococcal medication between diagnosis and ƒ3 yrs [183] should be viewed with caution as undesirable effects, such as changes in bacteriological spectrum and resistance, may merely be brought down to lower age ranges. If S. aureus is isolated from cough swab or sputum, irrespective of symptoms, then treatment with oral flucloxacillin or co-amoxyclav is indicated. In cases of communityacquired methicillin-resistant S. aureus, options are fucidic acid, rifampicin, cotrimoxazole, clindamycin and tetracyclines orally, depending on the age of the patient and the sensitivity of the strain. Linezolid is effective but should be kept as reserve antibiotic because of expense and potential toxicity, which includes hepatotoxicity and recent reports of severe optic neuropathy [184–186]. It can be used parenterally and orally. 258

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Teicoplanin is the drug of choice if intravenous treatment is indicated, which is unusual, with vancomycin as an alternative.

Pseudomonas aeruginosa. Eradication strategies for first isolation of P. aeruginosa improves the chances of avoiding or delaying chronic infection. A number of approaches exist, without the superiority of one over the other having definitively been established. These include: combined oral ciprofloxacin and inhaled colistin for periods of ƒ3 months [187]; and inhaled tobramycin (TOBI1; Chiron, Emeryville, CA, USA) [188] used for i4 weeks.Early antibiotic therapy leads toa P. aeruginosa-free periodof a median (range)of 18 (4–80) months. New acquisition with different P. aeruginosa genotypes occurs in 73% of episodes [189]. In the Danish CF Centre, early, aggressive eradication therapy has now been used for 15 yrs without giving rise to resistance to the antibiotics and without serious sideeffects. Although there is agreement that eradication regimes are mandatory and it is negligent not to treat a first isolation, the best regime is still not known. Chronic Pseudomonas aeruginosa infections. Based on the Cochrane review [190] and the recent TOBI1 trial [191], there is no doubt that long-term inhaled antibiotics should be prescribed to patients chronically infected with P. aeruginosa. The best regime is not known. Most clinicians use either or both of colistin and tobramycin or other aminoglycoside. For patients who are not responding well, the nonevidence-based approach of alternate months of TOBI1 and colistin is popular. However, more evidence from trials of longer duration is needed to determine if this benefit is maintained, as well as to determine the significance of development of antibiotic-resistant organisms. The Danish CF centre advocates regular 3-monthly intravenous antibiotic courses, arguing that this leads to better maintenance of lung function and less progress of Pseudomonas antibody production [192]. However, a 3-yr study did not demonstrate an advantage of a policy of elective antibiotic treatment over symptomatic treatment in patients with CF chronically infected with P. aeruginosa [193]. It should also be noted that as CF patients live longer and are exposed to multiple courses of antibiotics, there is an increased incidence of antibiotic allergy [194, 195], side-effects such as nephrotoxicity [196, 197] and, probably, selection of resistant organisms [198]. Acute exacerbations of pulmonary disease are treated with intravenous combination treatment with b-lactam antibiotics and aminoglycosides [199]. No major new strategies have been documented. Combinations are mandatory [200] and the recent finding of bacterial biofilm production being associated with subinhibitory aminoglycoside antibiotics supports this notion [201]. It should be noted that there is no evidence that in vitro sensitivities of the organism correlate with in vivo response to treatment [202, 203]. Optimal treatment duration is unknown but is usually i2–3 weeks. Once-daily tobramycin administration [204] has equal efficacy compared with three times-daily administration, with potentially less nephrotoxicity. There is interest in combining this with continuous intravenous ceftazidime [205, 206], rather than three times-daily boluses, as on pharmacokinetic grounds this may be more effective and is also potentially more convenient to the patient, who can carry a simple and portable infusion pump. Home antibiotic intravenous treatment is cost-effective and less disruptive to the patient, but recent data highlight the need for careful selection and closer supervision of such subjects as clinical outcome, as defined by spirometric parameters and body weight, was better after a course of treatment in hospital than after home treatment [207].

Attention to airway clearance: physiotherapy, devices, rhDNase and exercise. Whether airway clearance techniques are necessary from diagnosis in the asymptomatic child is unclear, but benefits are certainly obvious in the chronically productive patient. 259

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Responses to treatments are highly unpredictable in the individual and there is no universal best means to improve MCC in patients with CF.

Physiotherapy. Chest physiotherapy (PT) traditionally forms an integral part of virtually all CF treatment regimes. Despite this, the physiology and consequences of this intervention remain unclear and scientific evidence as to the long-term benefit of the various forms of PT is largely unresolved. It is unclear whether this treatment should be initiated in children diagnosed by screening. The effects of PT, in particular head-down drainage positions, on gastro-oesophageal reflux are controversial [208–210]. To date there are no comparative data on early PT favouring one approach over another. Caregivers should at the very minimum know how to check the child’s chest and have the expertise ready should the need for action arise. The treatment regimes should be individualised by experienced physiotherapists, since no one regime has evidence to favour it ahead of others [211]. In young adults, vibration using oscillation of the airflow was compared with a number of other manual or device-assisted methods, such as percussion or the flutter [212]. Peak expiratory flow rate was higher with vibration than with other methods, potentially indicating some evidence for the physiological rationale for the use of vibration to aid secretion clearance. In a recent Cochrane review on the benefit of positive expiratory pressure (PEP) to assist airway clearance, no clear evidence was found that PEP was a more or less effective intervention overall than other forms of physiotherapy. There was limited evidence that PEP was preferred by participants compared with other techniques, but this finding was from low-quality studies. Longterm studies had equivocal or conflicting results regarding the effect on FEV1. In all studies with an intervention period of i1 month, measures of participant preference were in favour of PEP [213]. Other outcome measures were, however, not addressed adequately. In an overview of five Cochrane systematic reviews, some evidence was obtained that PEP was at least as effective as other forms of airway clearance [214]. Physical training was addressed in particular and this was found to be beneficial. The Cochrane systematic reviews summarised in the paper provided some evidence to support the inclusion of physical therapies in the care-management plan of CF. A recent survey performed by the American College of Chest Physicians, which addressed nonpharmacological airway clearance methods and concentrated mainly on different breathing techniques including postural drainage, chest wall percussion and vibration, and the forced expiration technique (huffing), concluded that some are effective in increasing sputum production but their long-term efficacy in improving outcomes compared with unassisted cough alone is unknown [215]. It appears difficult to draw firm general conclusions concerning PT, so individual choices should be respected, with regular reviews by the physiotherapist. Well-designed, adequately powered and longterm studies are needed. Healthy children with CF pose special challenges. It remains to be seen whether there will ever be an answer to this issue both in terms of physiology or clinical effectiveness [216].

Devices. Many devices have been proposed to aid mucociliary clearance and sputum expectoration. These include the flutter and Acapella devices, high-frequency chest compression, the percussionaire intrapercussive ventilators, the Hayek oscillators, and others. The major drawback with almost all of these is the paucity of convincing data derived from properly designed studies. Only careful and long-term individual assessments will provide information on any benefits to patients. These would include improved or maintained lung function and simple surrogate markers, such as sputum volume or weight. Individual choice with assessment from a physiotherapist is suggested. 260

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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. 261

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Physical exercise should be an integral part of CF therapy. Benefits include general well-being, clearance of secretions and, for weight-bearing exercise, increased bone mineral density. The literature on respiratory muscle function is partly controversial indicating both normal to increased and also decreased values [228]. Recently the 6-min walk test was found to accurately determine the level of physical ability in children with CF [229]. Although an increased pro-inflammatory cytokine response is elicited by exercise [230], the benefits clearly outweigh any theoretical disadvantages. Although there is a very major interest in physical exercise training programmes and this kind of treatment is almost universally adopted [231], there are no standardised protocols that define exercise loads, supplementary oxygen, nutritional regimes and psychological support at any stage of the disease. As with physiotherapy, an individualised approach is advised.

Maintain good nutrition. This essential part of CF care can only be summarised very briefly here, for reasons of space. The brevity of the section does not reflect a lack of importance of the subject. More details can be found in standard texts.

Early and accurate diagnosis of pancreatic insufficiency. This is usually obvious from the history of profuse, offensive fatty stools, and is most conveniently confirmed by measuring human faecal elastase on a small stool sample; in pancreatic-insufficient CF this is undetectable, even when taking pancreatic enzyme supplementation [15]. Children who are pancreatic sufficient at diagnosis may develop pancreatic insufficiency, and any change in bowel habit in a pancreatic-sufficient patient should lead to a measurement of stool elastase. If doubt remains, which is unusual, a 3-day faecal fat measurement is made.

Institute pancreatic enzyme replacement therapy at an appropriate dose. Entericcoated pancreatic microspheres are the treatment of choice. They should be given with all meals and snacks, titrated to the fat content. The supervision of an experienced dietician is mandatory. The replacements are given prior to and with food, but may sometimes need to be given after meals if there is accelerated preferential gastric emptying of the microspheres [232]. The microspheres work most efficiently in an alkaline environment. The duodenum in CF may be more acidic than normal because of loss of the bicarbonaterich pancreatic secretion. Some children require acid-lowering strategies, such as proton pump inhibitors, to maximise enzyme efficiency.

A high-fat, high-calorie diet is essential. Arguments continue as to whether poor nutrition causes deterioration in lung function or the reverse is the sequence of events. Likewise, it is unclear whether the CF defect itself is causal of an increased calorie requirement or it is a secondary consequence of infection. Whichever is the case, maintenance of good nutrition is a pivotal part of CF care. The dietician will encourage "unhealthy" eating, including full-cream milk and fried food, and other high fat food. Fatsoluble vitamins are routinely prescribed.

Routine monitoring of nutrition. Height, weight and (in young children) head circumference should be measured at least four times a year and plotted on centile charts. Other useful adjuncts are calculation of body mass index and measurement of skin fold thickness. Any falling off from the centiles should be investigated promptly (see below). Levels of vitamin A, D, and E are measured as part of the routine annual assessment [233].

The CF child with nutritional failure. It should not be assumed that poor weight gain is due to inadequate pancreatic enzyme supplementation, and the dose unthinkingly 262

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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 child’s 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 Crohn’s or coeliac disease, both of which are commoner in CF [234, 235]? 3) Pseudo-Bartter’s syndrome should be eliminated, particularly in hot weather, by measuring urine and plasma electrolytes, including plasma bicarbonate [236]. 4) Cows’ milk allergy is another possibility [237], although the importance of dairy products in nutrition is such that this diagnosis should only be accepted if really stringently confirmed by a double-blind challenge. 5) Bowel infection with giardiasis [238], or a blind loop syndrome as a complication of neonatal surgery, is another consideration. 6) Short bowel syndrome or the effects of ileal resection, after neonatal surgery for meconium ileus.

The role of nutritional support. The first step in nutritional support is optimisation of meals and snacks, with the help of an experienced dietician. High calorie supplements are widely prescribed, but a recent trial suggested that benefit is not likely to be great [239]. Overnight feeding through a gastrostomy may lead to dramatic weight gains and may be particularly valuable if meals have become times of tension and quarrelling as a reluctant child is cajoled, pestered and bullied into trying to eat.

The CF child with abdominal pain. The usual causes of this are distal intestinal obstruction syndrome (DIOS) or simple childhood constipation. Abdominal pain should not prompt an uncritical increase in pancreatic enzyme therapy, and this approach leads to the iatrogenic disaster of fibrosing colonopathy [240, 241]. DIOS is not seen in other malabsorptive states; it is characterised by subacute bowel obstruction due to inspissated fatty faecal masses in the colon. Treatment acutely is with gastrograffin or Klean-Prep1 (Klean-Prep, Norgine Lim, Harefield, UK); a full review of diet and pancreatic enzymes to prevent recurrence is mandated. It must be distinguished from simple constipation, which is more common in CF. If there is any doubt, a 3-day faecal fat is performed. Other causes of abdominal pain in CF include the following: 1) acute appendicitis, which is frequently mistaken for DIOS [242]; 2) intussuception, which is commoner in CF than in the normal population [243]; 3) adhesions following neonatal surgery; 4) gall stones; 5) gastro-oesophageal reflux; and 6) rarely, torsion of an ovarian cyst or other non-CF causes of abdominal pain [244].

Early detection of multisystem complications. Proposed screening tests have been discussed above. Clearly, effective interventions need to be in place if early detection is worthwhile. It is also true that the "annual assessment" has become much more complex, burdensome and time-consuming for both patient and professional. Data gathering for its own sake must be resisted. Examples where early detection may be helpful and lead to a change in treatment include the following. 1) Insulin deficiency by glucose tolerance test and random home monitoring, with early institution of insulin therapy. 2) Abdominal ultrasound with early detection of liver disease, and initiating therapy with ursodeoxycholic acid (although the benefits of this treatment are untested). 3) Measurement of bone mineral density by DEXA scan, with early initiation of biphosphonates. Other tests for which there are unanswered questions include the following. 1) The role of HRCT scanning: will it result in clinical benefit, commensurate with the associated 263

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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 child’s 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 child’s 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 children’s 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]. 264

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

Is the Larsen hypothesis tenable? A radical hypothesis that has recently been proposed is that CF is a developmental disease, with all the action being antenatal, and the corollary that only curative attempts made before birth have any hope of success [253]. The proponents of this hypothesis have some compelling challenges to pose. These paradoxes are as follows. 1) In vitro CFTR protein interactions are not always mirrored in CF tissues. For example, enhanced sodium absorption is absent in sweat glands, despite the presence of ENaC. 2) The disease-specific manifestations of CF do not mirror the tissue distribution of CFTR; the heart and kidneys express CFTR, but there is no CF-specific disease of these organs. 3) As discussed above, patients can manifest a CF disease phenotype, without having any CFTR mutation. An additional piece of supportive evidence is the increasing amount of data cataloguing the multiple gene and protein consequences of CFTR function, discussed above [22–25]. Although most would agree that the weight of the evidence is against this hypothesis, nonetheless it is sufficiently challenging as to deserve consideration. There can be no question that CFTR is widely expressed in the lungs during foetal life, and that the amounts dwindle towards term. As with much lung development, the precise role(s) of CFTR are not known. Larsen and co-workers [253, 254] rightly draw attention to the fact that it is difficult to put together CFTR dysfunction(s) and the clinical phenotype of CF, and hypothesise that some disease manifestations are as a result of downstream disturbances of function in pathways which need to interact with CFTR. From this not implausible position, the group boldly hypothesised that downstream effects might occur in cells many years after their progenitors could not interact with CFTR, and as a result produce the disease CF. A number of studies have shown that transient overexpression of CFTR in the foetal airway by gene transfer experiments lead to permanent phenotypic changes, including epithelial cell proliferation, increase in 265

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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 266

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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.

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

Demonstration of restoration of CFTR function(s). This inevitably leads back to the question: what function does the CFTR gene have? A recent gene therapy trial restored ƒ25% of chloride channel function measured electrophysiologically [260], as did the use of oral 4-sodium phenylbutyrate [120]. Neither strategy corrected the overexpression of ENaC function. If, as the present authors have argued above, sodium transport is pivotal in CF lung disease, then restoration of chloride channel function (above) will not achieve the desired effect.

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

Will stem cells work? The short answer to the question is no, not on their own. The problems of gene therapy in the airway include: vector delivery to a distorted and often poorly ventilating airway; the evasion of the normal host defences against the approach of foreign molecules; the traversing of intracellular barriers; the transcription of the inserted gene; and the expression and correct deployment of the resultant protein. These could all be circumvented if pluripotent stem cells can be harvested from the bone marrow, subjected to corrective gene therapy in vitro and re-infused to repopulate the airway and correct the CF lung disease. The death blow to the idea that stem cells on their own can repopulate 267

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the lung comes from studies in CF patients who have undergone lung transplantation. There has never been any report of recurrence of CF in the transplanted lungs, no matter how many years the patient has survived after transplantation, which would surely have happened were there to be a normal trafficking of stem cells between bone marrow and airway. However, there are some possible pointers that an innovative strategy may allow tracking of stem cells to the airway. There have been reports of Y chromosome positive cells in the airways of male donor lungs transplanted into female recipients, and also in the female recipients of donor male bone marrow [262, 263]. There have also been reports that CD34z cells implicated in airway remodelling have been isolated from peripheral blood, presumably en route between the bone marrow and the airway. These studies provide no more than proof of possibility, and a cure for CF with stem cells seems a long way away.

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

When is inflammation beneficial? Why does it remain acute neutrophilic, not chronic? Clearly, the immune/inflammatory response has a biological purpose, as shown by the catastrophic infections in children with congenital or acquired immunodeficiency. It would seem likely that early on in CF, the inflammatory response is beneficial and wards off early infection with P. aeruginosa. In late-stage disease, it is clearly both ineffectual and harmful. What is not known is when the transition to harm occurs. This is important because immunosuppression with steroids, for example, while definitely beneficial (at the price of side-effects) in late disease, may not be helpful early on. Indeed, in the oral steroid trial, there were benefits only in the group chronically infected with P. aeruginosa [264]. This issue was brought into sharp focus by a recent randomised, placebocontrolled trial of an LTB4 receptor antagonist, which was halted prematurely because of increased adverse events (infective exacerbations) in the treatment group [265]. Thus, further work is needed to determine the proportions of good and bad inflammation at different stages of CF. The second challenging inflammation question is: why is there persistent acute, rather than chronic, infection in the CF airway (defining acute and chronic by the cellular pattern, not by time duration)? The neutrophil is the hallmark cell of the acute inflammatory response to bacterial infection in particular. The outcome is usually resolution (as in uncomplicated lobar pneumonia) or progression to chronicity (for example, tuberculosis), when the neutrophil has largely been replaced by chronic inflammatory cells. This clearly does not happen in CF (nor does it happen in PCD and non-CF bronchiectasis). Why? And would it be better for the organism if the severe persistent acute phenotype infection gave way to chronicity? Lipoxin production is one way the body switches off an acute inflammatory phenotype. In one study, patients with CF were found to have reduced airway lipoxin levels and administration of exogenous lipoxin attenuated the lung disease of a CF mouse model [144]. 268

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

What are the key modifier genes? Although for large groups there are correlations between genotype and at least some aspects of clinical phenotype, and between genotype and prognosis, individuals with the same genotype at the CFTR locus may have very different disease phenotypes. This is true even for full siblings brought up within the same environment; for example, one sibling may have devastating lung disease, while the other has normal lung function but hepatosplenomegaly. Thus, there must be powerful genetic modifiers of the CF phenotype within the karyotype. It should also be said that there are clearly profound environmental influences: in one study from the USA, wealthy patients with CF (never on Medicaid) had a three-fold lower death rate than impoverished patients (always on Medicaid) [271]. The importance of careful consideration of gene–environment interactions, and not just genes alone, is discussed below. The key reason for bothering with modifier genes is that if a modifier gene product can convert severe lung disease to a mild phenotype, that product may make a very promising therapeutic target. It has to be said that this goal appears to be in the distant future. 269

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How is cross-infection and the emergence of new pathogens to be prevented? Infection control has been addressed in recently published reviews in North America [272] and Europe [198]. This is a highly contentious area. It is known that living together under the same roof for several days will lead to cross-infection; it is not known how short a time is safe. Passing in the corridor at a brisk walk? On the same train for a journey? Attending a football match? Starting with the noncontroversial, the following points are known: 1) cross-infection definitely occurs with B. cepacia complex, some, but not all, strains of P. aeruginosa and methicillin-resistant S. aureus (MRSA); 2) crossinfection does not occur with some strains of P. aeruginosa, atypical mycobacteria and H. influenzae; and 3) previous sputum microbiology is not predictive of current infection status. Recommendations would therefore be as follows. 1) Basic hygiene measures, such as hand washing, are mandatory and should be rigorous [273]. 2) People with CF who are infected with a pathogen that has been shown to be associated with cross-infection should be separated from others with CF, both inside and outside the hospital, as far as possible. There is obviously no point in making impractical suggestions and complete isolation outside the hospital (for example, in schools) may be inconsistent with disability legislation in some countries and with patient confidentiality. 3) For paediatric outpatient clinics, many now adopt a system whereby the child and family go into a room and the professionals enter the room in turn, rather than the conventional model in which children wait to be seen in a communal area. The room is carefully cleaned between patients. This model results in substantially fewer patients being seen per clinic and also diminishes flexibility in seeing acutely unwell children in the clinic. 4) Adult outpatients can be segregated on microbiological status within the usual communal waiting area and be asked to sit a set distance away from each other to minimise aerosol spread. 5) In hospital, each patient should have a single room with en-suite facilities. If this is impractical, then no more than one CF patient per single bay is permitted. 6) Microbiological surveillance for the early detection of cross-infection is vital. The gold standard, molecular typing of every isolate, is probably too expensive for most clinics. Antibiotic resistance patterns are deceptive, and therefore of little value [274]. The price of cohort isolation includes loss of the psychosocial support that families offer to each other. To some extent this can be overcome by the use of Internet technology. Although it may sound harsh, patients’ self-help groups are an ideal source for pathogen transmission and as such should be viewed with great reservation. S. aureus and P. aeruginosa are the "classical" CF pathogens and are covered elsewhere in this chapter. Recently, and probably as a consequence of increased longevity but also due to intensive antibiotic treatments, a number of new pathogens have been described and their role in CF has been addressed in some detail [275]. There are no set treatment guidelines for these and relatively little is known of transmissibility. Most are of environmental origin and are characterised by innate resistance to most major antibiotics. Particular attention should be paid to the prevention of transmission to other patients. Treatment of MRSA has been discussed in a previous section.

Burkholderia cepacia complex. There are i10 variants in this family ("genomovars"). At least in Central Europe, Burkholderia cenocepacia (genomovar III) is the most likely to be associated with virulence and high transmissibility, thus carrying a poor prognosis whilst the other Burkholderia spp. may be of lesser importance. Recently, B. dolosa (genomovar VI) was found to carry a poor prognosis, with a 13% probability of dying within 18 months of established infection [276]. If B. cepacia is suspected, the identity of the isolate must be confirmed in a reference laboratory, if misdiagnosis is to be avoided. 270

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The organism requires selective culture media to be detected with accuracy. Only after 1 yr of at least three B. cepacia-negative cultures can the organisms be considered cleared. Antibiotic treatment is difficult. Even when in vitro testing suggests sensitivity, in vivo results are disappointing. Synergy testing with double or preferably triple antibiotic combinations appears to offer the best chance to establish possible treatments [277]. Meropenem combined with cotrimoxazole, piperacillin/tazobactam, doxycycline and ceftazidime are the most likely antibiotics to be useful. Recently, temocillin has been successfully used in a series of 20 B. cepacia complex patients [278]. It is crucial to cohort isolate the different genomovars separately; before this was appreciated, patients carrying a benign genomovar were grouped together with genomovar III patients, acquired genomovar III, and eventually died from it.

Stenotrophomonas maltophilia. The role of this organism remains uncertain and it is not generally treated unless the clinical situation requires intervention. Cross-infection does not appear to be a problem and clinic separation is not warranted under present circumstances. Stenotrophomonas maltophilia does not lead to a change in clinical condition in most patients, and frequently disappears spontaneously from the airway [279, 280]. If treatment is considered at all, cotrimoxazole combined with ticarcillinclavulanic acid is used. Alternatives are ciprofloxacin plus ticarcillin/clavulanic acid or piperacillin/ tazobactam.

Achromobacter (Alcaligenes) xylosoxidans. This is a rod-shaped organism of unknown pathogenicity in CF. In some patients (without CF), it has been reported to cause septicaemia, peritonitis in patients on peritoneal dialysis, pneumonia and ear infection. Transmission amongst CF patients does not seem to be a problem and there are no reports of accelerating loss of lung function. There are no figures on the incidence of the organism in large CF collectives. Treatment is not normally an issue but, in exceptional cases, ureidopenicillins or ceftazidime may be promising.

Pandoraea apista. Some of these organisms may cause chronic infection and can be transmitted between CF patients. Epidemic forms were seen in the Danish CF Centre and cohort isolation was effective in preventing further spread [281]. Treatment is difficult and there is a paucity of evidence.

Nontuberculous mycobacteria. A number of nontuberculous mycobacteria cause human disease and may be seen in CF (Mycobacterium avium, M. kansasii, M. abscessus and others). Their origin is mostly environmental, and cross-infection is not an issue. They are frequently only commensals in CF and only patients with multiple positive cultures should be considered for treatment. In general, a period of observation, with serial CT scans, is recommended [282, 283]. These organisms are frequently multiply resistant, and expert advice should be sought if treatment is contemplated.

How is evidence obtained for the treatment strategies particularly used in early stage disease? In order to obtain evidence for treatment strategies used in early stage disease, the following are needed. 1) International agreement as to the hierarchy of important questions and the order in which they should be answered. 2) An absolute commitment internationally to recruit all screened CF infants to multicentre trials, to answer these questions. 3) Funding to ensure these trials are adequately powered and are of sufficient duration, to answer questions definitively. 4) Simple and cheap end-points, which do not 271

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require expensive and sophisticated apparatus, so that many centres can be involved in trials. 5) Excellent surrogate end-points in these trials: ortality will never be satisfactory.

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

What are the best treatment strategies for liver disease? Liver disease is becoming increasingly important as CF patients live longer. Out of a total of 21,742 patients with CF summarised in the 2003 US Cystic Fibrosis Foundation Patient Registry, 5.5% were affected by some type of liver disease. Just 10 of these underwent liver transplantation. This is in contrast with figures from Canada, where CF liver disease was observed in 18, 29 and 41% at ages 2, 5 and 12 yrs and cirrhosis occurred in 19 (7.8%) patients at a median age of 10 yrs [286]. Patients with a history of meconium 272

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ileus (incidence rate ratio 5.5; 2.7–11), male sex (2.5; 1.3–4.9) or severe mutations (2.4; 1.2–4.8) 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 20–30 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. 273

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What is needed to find answers to these questions? A really good CF lung disease animal model The CF mouse has proved to be a disappointment with respect to the pulmonary phenotype. A model of chronic P. aeruginosa infection has been established in these mice by administering infected agar beads by aerosol. The difficulty in establishing this infection in the CF mouse is in stark contrast to the almost impossibility of preventing it in humans. It could be argued that the difficulty of a suitable model of the CF lung phenotype has already been achieved with the ENaC b-subunit overexpressing mouse [130]. That this is a model of a chronic infective lung disease is indisputable; that it is specific for CF is uncertain and, if it is, the implications for therapeutic targets are profound (see above). It is unlikely that the CF mouse will ever be a really good model for CF lung disease, since, quite apart from anything else, it lacks submucosal glands, the chief site of CFTR expression in humans. Alternatives, such as the ferret, would potentially be of interest. The ideal animal would have submucosal glands and similar ion transport physiology to humans, both in terms of ENaC function and the nature of non-CFTR chloride channels. Such an animal could be produced by gene knockout, RNA interference or pharmacologically, if CFTR could be completely inactivated by an otherwise nontoxic compound.

Studies that determine why PCD is a mild lung disease and CF is severe The phenotypic differences between PCD and CF have been described above. The study of the differences in the key inflammatory mediators, or possible tissue-destructive enzymes, may lead to new targets and the avoidance of irrelevant ones. There are a number of noninvasive techniques, such as spontaneous and induced sputum, exhaled breath condensate and exhaled breath, and powerful molecular techniques, which could be applied to samples (genomics, proteomics) that could be utilised to probe the similarities and differences between these conditions.

Systematic clinical trials of treatment, such that all newly screened babies are included Treatment options for newly diagnosed babies have recently been reviewed. It is very clear that the evidence base for much of what is suggested is very poor. The advent of widespread newborn screening presents the opportunity to address this, if there is the collective will to do so. The best model is that of the paediatric oncologists, who enrol almost all children with cancer into studies. The disease that is particularly studied is acute lymphoblastic leukaemia, in which the prognosis has been transformed by a series of therapeutic trials (the UK Acute Lymphoblastic Leukaemia Trials). The oncologists have the advantage that a decisive end-point (death) is either reached or quickly averted. However, there is no reason to avoid tackling the more difficult issues in CF. The requirements are as follows. 1) A will to enrol the vast majority of newly diagnosed, screened babies into a succession of focused trials. 2) A realisation that these will be medium and long term, not short term. 3) The use of simple end-points that can be applied all over Europe, using data that are already being collecting, such as cough swabs, height and weight, and (in older children) spirometry. 274

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This will only be achieved if core funding can be obtained for the data collection and analysis, and there is a collective will to make this happen, from a large number of CF clinicians and national and international interest groups.

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

Hypothesis-driven genetic modifier studies, with findings tested in a second similar population before being accepted Criteria for an adequate and informative genetic association study have recently been succinctly summarised [293] and may equally be applied to genetic modifier studies. The criteria are as follows. 1) The sample size must be adequate and a pre-study power calculation is a prerequisite. 2) The controls must be carefully chosen from the same population as the disease group; a ratio of controls to cases of 2:1 increases power. 3) The choice of polymorphisms/associations sought should be logical. 4) The phenotypes must be carefully described. 5) Issues of multiple testing must be faced and allowed for in the statistical testing, especially if the same population is used for multiple association studies. 6) Any findings should be replicated in a second, independent sample to be credible. 7) The findings should enhance knowledge of the mechanisms of disease or its treatment. The current "state of the art" for CF modifier gene studies unfortunately seems to be to collect DNA from as many patients as possible (sometimes controlling for the CF genotype, e.g. exclusively DF508/DF508), and hoping that something will fall out from either a genome-wide scan or an analysis of polymorphisms in likely candidate genes, such as ENaC or other ion channels, inflammatory cytokine genes, etc. The present authors believe this approach is, unfortunately, naı¨ve, and almost certainly doomed to failure for the following reasons. 1) Genes do not exist in isolation, but they interact with the environment; for example, polymorphisms of the glutathione S-transferase gene are thought to interact with the severity of CF lung disease [294, 295]. However, powerful interactions with environmental factors have been shown to be important in the context of childhood lung function and air pollution. For example, maternal but not paternal glutathione S-transferase genotype is an important determinant of the effects of maternal smoking during smoking on foetal airway development and later respiratory symptoms [296]. 2) Genetic influences may only operate at a particular developmental stage, and mixing of paediatric and adult studies may obscure this. 3) Genetic influences may be important only at particular stages of the disease; for example, by the time chronic infection with P. aeruginosa has been established, the effect of genes influencing the early binding of the organism to epithelial cells are likely to be irrelevant. 4) Genes and 275

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treatment policy may interact; upregulation of a putative epithelial receptor for P. aeruginosa leading to earlier infection will be more relevant to prognosis in clinics where a vigorous eradication policy is not pursued. 5) Polymorphisms may show a biphasic influence, being protective in some environments but detrimental in others. For example, the CD14/-260 CRT polymorphism in the CD14 promoter was associated with higher levels of both total and specific immunoglobulin E to aeroallergens in children in regular contact with domestic pets, but the opposite relationship, not explained by endotoxin levels, in children in contact with stable animals [297]. 6) Ethnic factors, including genetic variations, may lead to different polymorphisms being relevant in different populations. Whereas it is important to confirm a putative modifier in one study, in a second study in a different population, if the second population is too different from the first, the polymorphism may be discarded as irrelevant, whereas it is in fact relevant only in some populations. 7) It is at least conceivable that studying one genotype exclusively (e.g. DF508/DF508) may miss the effect of polymorphisms relevant in other genotypes. These are not counsels of despair of ever finding anything, merely a plea for hypothesis-driven, focused studies. These studies also have to be adequately powered. Ideally the target population has to be big enough for a "double-study" approach, in which half the population is used as a hypothesis-generating exercise to find a modifier, and the other half is used to test that the modifier is important in the generated hypothesis. Both halves must be large enough to give adequate study power. The complexities of gene–environment interactions in infant wheeze should have taught us the limitations of mere DNA collection. A clinical phenotype needs to be thought of as the product of the interactions between the internal (intra-uterine) and external environment, and the genes; neglect of any part of this "phenotype equation" will limit the ability of studies to detect important modifiers. A recent large study exactly highlights the limitations of collecting only DNA. Drumm et al. [298] studied two cohorts of patients. The first, used to generate associations, consisted of 808 patients who were homozygous for DF508. They attempted to replicate the work of others by looking for associations between disease severity. They studied 10 different genes (table 5) and validated their findings in a second cohort (n=498) with a variety of genotypes (70% DF508 homozygotes). They had no information on environmental factors. They found a rough doubling of the odds ratio for severe lung disease for the highest risk transforming growth factor-b1 genotype (codon 10 CC), their Table 5. – Putative polymorphisms in modifier genes studied by DRUMM et al. [298] Genes studied

Polymorphisms

A1-antiprotease

S allele (T2313A) Z allele (G4627A) 39 enhancer (G1237A) D or I deletion A46G C79G Null deletion A1375G G-1082A Null structural polymorphisms (B, C, D) XA/O T5220G Promotor (C-509T) Codon 10 (C29T) Codon 25 (G74C) Promotor (G-308A

Angiotensin-converting enzyme b2-Adrenergic receptor Glutathione S-transferase 1 Glutathione S-transferase 2 Interleukin 10 Mannose-binding lectin 2 Nitric oxide synthase 3 Transforming growth factor-b1 Tumour necrosis factor-a

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sole positive finding. To put this in perspective, there is a three-fold risk of dying in CF patients always relying on Medicaid compared with those never relying on Medicaid [270]. However, this impressive study has missed the boat, as the hugely discordant clinical phenotypes and prognosis in genotype-identical siblings is well known. The present authors suspect that a better way forward will be to study, in enormous detail, the differences between small groups of such discordant sibling pairs, in the hope of finding the really dramatic modifier that would provide a useful therapeutic target. Another approach would be to study, in detail, the differences between congenital bilateral absence of the vas deferens (CABVD) patients, in whom lavage studies have shown evidence of bacterial infection and a muted inflammatory response [299], but no evidence of overt lung disease in CF patients in whom infection and inflammation lead to airway destruction. The likely explanation for the mild lung phenotype in CABVD is an as yet undiscovered protective mechanism, which could offer therapeutic targets. The present study will undoubtedly guide and inform mechanistic studies, but has not generated therapeutic targets.

Summary and conclusions CF is probably the paediatric respiratory disease that has seen the most change in the last 15 yrs, both in terms of understanding of the molecular and cellular changes, and also the changing understanding of the nature of the disease. Concepts that were set in stone have been challenged. Future research is needed to determine why patients with CF get the typical lung phenotype, how to find specific treatments and know they are effective, and how to treat the multisystem disease that CF has now become.

Summary Twenty years ago, cystic fibrosis was considered a disease mainly of children, affecting the lungs and the digestive systems, diagnosed using the sweat test. Since then, the gene for CF has been discovered, leading to great increases in the knowledge about the fundamental molecular and cellular biology of the airway; the diagnostic spectrum has also been expanded to mild and atypical cases, requiring more sophisticated diagnostic testing, with mild cases presenting in adult life. Specialist care has lead to an increase in survival, so that shortly there will be more adults than children with the disease. The nature of the disease is now appreciated to affect nearly all systems in the body, including the bones and the genitourinary system. Psychosocial issues of living with a chronic disease have become increasingly important. These new complications have lead to searches for new preventative strategies in childhood and have posed novel treatment challenges in adults. The expectation for treatment has switched from the reactive and symptomatic, to curative strategies, including gene therapy and phenotype-specific treatments, such as aminoglycosides, to overcome premature stop codons. There remain many unanswered questions about basic pathophysiology and much treatment is not evidence-based. The ongoing challenge for clinicians is to establish a firm evidence base for therapy; for basic scientists, it is to understand the crucial steps leading from an absent or dysfunctional protein to the clinical disease, and to target those functions that are crucially disease-producing. Keywords: Cystic fibrosis, diabetes, gene therapy, nutrition, osteopaenia, Pseudomonas. 277

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209. Button BM, Heine RG, Catto-Smith AG, et al. Chest physiotherapy in infants with cystic fibrosis: to tip or not? A five-year study. Pediatr Pulmonol 2003; 35: 208–213. 210. Phillips GE, Pike SE, Rosenthal M, Bush A. Holding the baby: head downwards positioning for physiotherapy does not cause gastro-oesophageal reflux. Eur Respir J 1998; 12: 954–957. 211. Lannefors L, Button BM, McIlwaine M. Physiotherapy in infants and young children with cystic fibrosis: current practice and future developments. J R Soc Med 2004; 97: Suppl. 44, 8–25. 212. McCarren B, Alison JA. Physiological effects of vibration in subjects with cystic fibrosis. Eur Respir J 2006; 27: 1204–1209. 213. Elkins M, Jones A, van der Schans C. Positive expiratory pressure physiotherapy for airway clearance in people with cystic fibrosis. Cochrane Database Syst Rev 2006; 2: CD003147. 214. Bradley JM, Moran FM, Elborn JS. Evidence for physical therapies (airway clearance and physical training) in cystic fibrosis: an overview of five Cochrane systematic reviews. Respir Med 2006; 100: 191–201. 215. McCool FD, Rosen MJ. Nonpharmacologic airway clearance therapies: ACCP evidence-based clinical practice guidelines. Chest 2006; 129: Suppl. 1, 250S–259S. 216. Main E, Prasad A, Schans C. Conventional chest physiotherapy compared to other airway clearance techniques for cystic fibrosis. Cochrane Database Syst Rev 2005; 1: CD002011. 217. Suri R. The use of human deoxyribonuclease (rhDNase) in the management of cystic fibrosis. BioDrugs 2005; 19: 135–144. 218. Suri R, Grieve R, Normand C, et al. Effects of hypertonic saline, alternate day and daily rhDNase on healthcare use, costs and outcomes in children with cystic fibrosis. Thorax 2002; 57: 841–846. 219. Suri R, Metcalfe C, Lees B, et al. Comparison of hypertonic saline and alternate-day or daily recombinant human deoxyribonuclease in children with cystic fibrosis: a randomised trial. Lancet 2001; 358: 1316–1321. 220. Quan JM, Tiddens HA, Sy JP, et al. A two-year randomized, placebo-controlled trial of dornase alfa in young patients with cystic fibrosis with mild lung function abnormalities. J Pediatr 2001; 139: 813–820. 221. Hodson ME, McKenzie S, Harms HK, et al. Investigators of the Epidemiologic Registry of Cystic Fibrosis. Dornase alfa in the treatment of cystic fibrosis in Europe: a report from the Epidemiologic Registry of Cystic Fibrosis. Pediatr Pulmonol 2003; 36: 427–432. 222. Paul K, Rietschel E, Ballmann M, et al. Bronchoalveolar Lavage for the Evaluation of Antiinflammatory Treatment Study Group. Effect of treatment with dornase alpha on airway inflammation in patients with cystic fibrosis. Am J Respir Crit Care Med 2004; 169: 719–725. 223. Donaldson SH, Bennett WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. Mucus clearance and lung function in cystic fibrosis with hypertonic saline. N Engl J Med 2006; 354: 241– 250. 224. Ratjen F. Restoring airway surface liquid in cystic fibrosis. N Engl J Med 2006; 354: 291–293. 225. Elkins MR, Robinson M, Rose BR, et al. National Hypertonic Saline in Cystic Fibrosis (NHSCF) Study Group. A controlled trial of long-term inhaled hypertonic saline in patients with cystic fibrosis. N Engl J Med 2006; 354: 229–240. 226. Nixon PA, Orenstein DM, Kelsey SF, Doershuk CF. The prognostic value of exercise testing in patients with cystic fibrosis. N Engl J Med 1992; 327: 1785–1788. 227. Moorcroft AJ, Dodd ME, Morris J, Webb AK. Symptoms, lactate and exercise limitation at peak cycle ergometry in adults with cystic fibrosis. Eur Respir J 2005; 25: 1050–1056. 228. Lands LC, Heigenhauser GJ, Jones NL. Respiratory and peripheral muscle function in cystic fibrosis. Am Rev Respir Dis 1993; 147: 865–869. 229. Cunha MT, Rozov T, de Oliveira RC, Jardim JR. Six-minute walk test in children and adolescents with cystic fibrosis. Pediatr Pulmonol 2006; 41: 618–622. 230. Ionescu AA, Mickleborough TD, Bolton CE, et al. The systemic inflammatory response to exercise in adults with cystic fibrosis. J Cyst Fibros 2006; 5: 105–112. 231. Barker M, Hebestreit A, Gruber W, Hebestreit H. Exercise testing and training in German CF centers. Pediatr Pulmonol 2004; 37: 351–355.

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232. Taylor CJ, Hillel PG, Ghosal S, et al. Gastric emptying and intestinal transit of pancreatic enzyme supplements in cystic fibrosis. Arch Dis Child 1999; 80: 149–152. 233. Carr SB, Dinwiddie R. Annual review or continuous assessment? J R Soc Med 1996; 89: Suppl. 27, 3–7. 234. Dobbin CJ, Moriarty C, Bye PT. Granulomatous diseases in a patient with cystic fibrosis. J Cyst Fibros 2003; 2: 35–37. 235. Rabinowitz I. Diagnosis of cystic fibrosis and celiac disease in an adult: one patient, two diseases, and three reminders. Respir Care 2005; 50: 644–645. 236. Kennedy JD, Dinwiddie R, Daman-Willems C, Dillon MJ, Matthew DJ. Pseudo–Bartter’s syndrome in cystic fibrosis. Arch Dis Child 1990; 65: 786–787. 237. Lucarelli S, Quattrucci S, Zingoni AM, et al. Food allergy in cystic fibrosis. Minerva Pediatr 1994; 46: 543–548. 238. Walkowiak J, Krawczynski M, Herzig KH. Giardiasis aggravates malabsorption in cystic fibrosis. Scand J Gastroenterol 2004; 39: 607–608. 239. Poustie VJ, Russell JE, Watling RM, Ashby D, Smyth RL, CALICO Trial Collaborative Group. Oral protein energy supplements for children with cystic fibrosis: CALICO multicentre randomised controlled trial. BMJ 2006; 332: 632–636. 240. Smyth RL, van Velzen D, Smyth AR, Lloyd DA, Heaf DP. Strictures of ascending colon in cystic fibrosis and high-strength pancreatic enzymes. Lancet 1994; 343: 85–86. 241. MacSweeney E, Oades PJ, Buchdahl R, Rosenthal M, Bush A. Colonic strictures in cystic fibrosis. Lancet 1995; 345: 752–756. 242. Shields MD, Levison H, Reisman JJ, Durie PR, Canny GJ. Appendicitis in cystic fibrosis. Arch Dis Child 1991; 66: 307–310. 243. Hafen GM, Taylor AC, Oliver MR, et al. Intussusceptions arising from two different sites in a child with cystic fibrosis. Pediatr Pulmonol 2005; 40: 358–361. 244. Dalzell AM, Heaf DP, Carty H. Pathology mimicking distal intestinal obstruction syndrome in cystic fibrosis. Arch Dis Child 1990; 65: 540–541. 245. Go¨tz I, Go¨tz M. Cystic fibrosis: psychological issues. Paediatr Respir Rev 2000; 1: 121–127. 246. Cowen L, Mok J, Corey M, MacMillan H, Simmons R, Levison H. Psychologic adjustment of the family with a member who has cystic fibrosis. Pediatrics 1986; 77: 745–753. 247. Sawyer EH. Family functioning when children have cystic fibrosis. J Pediatr Nurs 1992; 7: 304–311. 248. Quittner AL, DiGirolamo AM, Michel M, Eigen H. Parental response to cystic fibrosis: a contextual analysis of the diagnosis phase. J Pediatr Psychol 1992; 17: 683–704. 249. Patterson JM, McCubbin HI, Warwick WJ. The impact of family functioning on health changes in children with cystic fibrosis. Soc Sci Med 1990; 31: 159–164. 250. McCubbin H, McCubbin M, Patterson J, Cauble A, Wilson L, Warwick W. CHIP: Coping Health Inventory for Parents: an assessment of parental coping patterns in the care of the chronically ill child. J Marriage Family 1983; 45: 359–370. 251. Bluebond-Langner M, Angst D, Bryan Lask B. Psychosocial Aspects of Cystic Fibrosis. London, Hodder Arnold, 2001. 252. DeLambo KE, Ievers-Landis CE, Drotar D, Quittner AL. Association of observed family relationship quality and problem-solving skills with treatment adherence in older children and adolescents with cystic fibrosis. J Pediatr Psychol 2004; 29: 343–353. 253. Larson JE, Cohen JC. Developmental paradigm for early features of cystic fibrosis. Pediatr Pulmonol 2005; 40: 371–377. 254. Larson JE, Delcarpio JB, Farberman MM, Morrow SL, Cohen JC. CFTR modulates lung secretory cell proliferation and differentiation. Am J Physiol Lung Cell Mol Physiol 2000; 279: L333–L341. 255. Morrow SL, Larsen JE, Nelson S, Sekhon HS, Ren T, Cohen JC. Modification of development by the CFTR gene in utero. Mol Genet Metab 1998; 65: 203–212. 256. Larson JE, Morrow SL, Happel L, Sharp JF, Cohen JC. Reversal of cystic fibrosis phenotype in mice by gene therapy in utero. Lancet 1997; 349: 619–620.

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257. Davis PB, Byard PJ, Konstan MW. Identifying treatments that halt progression of pulmonary disease in cystic fibrosis. Pediatr Res 1997; 41: 161–165. 258. Prentice RL. Surrogate endpoints in clinical trials: definition and operational criteria. Stat Med 1989; 8: 431–440. 259. Fleming TR, deMets DL. Surrogate endpoints in clinical trials: are we being misled? Ann Intern Med 1996; 125: 605–613. 260. Alton EW, Stern M, Farley R, et al. Cationic lipid-mediated CFTR gene transfer to the lungs and nose of patients with cystic fibrosis: a double-blind placebo-controlled trial. Lancet 1999; 353: 947–954. 261. Coates AL, Bush A. Basic science research vs. clinical research in cystic fibrosis: has the pendulum swung too far? Pediatr Pulmonol 2003; 36: 175–177. 262. Spencer H, Rampling D, Aurora P, et al. Transbronchial biopsies provide longitudinal evidence for epithelial chimerism in children following sex mismatched lung transplantation. Thorax 2005; 60: 60–62. 263. Suratt BT, Cool CD, Serls AE, et al. Human pulmonary chimerism after hematopoietic stem cell transplantation. Am J Respir Crit Care Med 2003; 168: 318–322. 264. Eigen H, Rosenstein B, Fitxsimmons S, Schidlow D. A multi-center study of alternate day prednisolone therapy in patients with cystic fibrosis. J Pediatr 1995; 126: 515–523. 265. Konstan MW, Doring G, Lands LC, et al. Results of a phase 11 clinical trial of BIIL 284 BS (an LTB4 receptor antagonist) for the treatment of CF lung disease. Pediatr Pulmonol Suppl 2005; 28: 125–126. 266. Moss RB, Mayer-Hamblett N, Wagener J, et al. Randomized, double-blind, placebo-controlled, dose-escalating study of aerosolized interferon gamma-1b in patients with mild to moderate cystic fibrosis lung disease. Pediatr Pulmonol 2005; 39: 209–218. 267. McElvaney NG, Hubbard RC, Birrer P, et al. Aerosol alpha 1-antitrypsin treatment for cystic fibrosis. Lancet 1991; 337: 392–394. 268. Martin SL, Downey D, Bilton D, et al. Safety and efficacy of recombinant alpha(1)-antitrypsin therapy in cystic fibrosis. Pediatr Pulmonol 2006; 41: 177–183. 269. McElvaney NG, Nakamura H, Birrer P, et al. Modulation of airway inflammation in cystic fibrosis: in vivo suppression of interleukin-8 levels on the respiratory epithelial surface by aerosolization of recombinant secretory leukoprotease inhibitor. J Clin Invest 1992; 90: 1296–1301. 270. Balfour-Lynn IM, Mohan U, Bush A, Rosenthal M. Intravenous immunoglobulin for cystic fibrosis lung disease: a case series of 16 children. Arch Dis Child 2004; 89: 315–319. 271. Schechter MS, Shelton BJ, Margolis PA, Fitzsimmons SC. The association of socioeconomic status with outcomes in cystic fibrosis patients in the United States. Am J Respir Crit Care Med 2001; 163: 1331–1337. 272. Saiman L, Siegel J. Cystic Fibrosis Foundation Consensus Conference on Infection Control Participants. Infection control recommendations for patients with cystic fibrosis: microbiology, important pathogens, and infection control practices to prevent patient-to-patient transmission. Am J Infect Control 2003; 31: Suppl. 3, S1–S62. 273. Geddes DM. Of isolates and isolation: Pseudomonas aeruginosa in adults with cystic fibrosis. Lancet 2001; 358: 522–523. 274. Davies G, McShane D, Davies JC, Bush A. Multiresistant Pseudomonas aeruginosa in a pediatric cystic fibrosis center: natural history and implications for segregation. Pediatr Pulmonol 2003; 35: 253–256. 275. LiPuma JJ. Burkholderia and emerging pathogens in cystic fibrosis. Semin Respir Crit Care Med 2003; 24: 681–692. 276. Kalish LA, Waltz DA, Dovey M, et al. Impact of Burkholderia dolosa on lung function and survival in cystic fibrosis. Am J Respir Crit Care Med 2006; 173: 421–425. 277. Aaron SD, Ferris W, Henry DA, Speert DP, Macdonald NE. Multiple combination bactericidal antibiotic testing for patients with cystic fibrosis infected with Burkholderia cepacia. Am J Respir Crit Care Med 2000; 161: 1206–1212.

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278. Lekkas A, Gyi KM, Hodson ME. Temocillin in the treatment of Burkholderia cepacia infection in cystic fibrosis. J Cyst Fibrosis 2006; 5: 121–124. 279. Marchac V, Equi A, Le Bihan-Benjamin C, Hodson M, Bush A. Case-control study of Stenotrophomonas maltophilia acquisition in cystic fibrosis patients. Eur Respir J 2004; 23: 98–102. 280. Goss CH, Mayer-Hamblett N, Aitken ML, Rubenfeld GD, Ramsey BW. Association between Stenotrophomonas maltophilia and lung function in cystic fibrosis. Thorax 2004; 59: 955–959. 281. Jorgensen IM, Johansen HK, Frederiksen B, et al. Epidemic spread of Pandoraea apista, a new pathogen causing severe lung disease in cystic fibrosis patients. Pediatr Pulmonol 2003; 36: 439–446. 282. Olivier KN, Weber DJ, Wallace RJ Jr, et al. Non-tuberculous mycobacteria. 1: multicenter prevalence study in cystic fibrosis. Am J Respir Crit Care Med 2003; 167: 828–834. 283. Olivier KN, Weber DJ, Lee JH, et al. Non-tuberculous mycobacteria. 11: nested-cohort study of impact on cystic fibrosis lung disease. Am J Respir Crit Care Med 2003; 167: 835–840. 284. Schuepp KG, Jauernig J, Janssens HM, et al. In vitro determination of the optimal particle size for nebulized aerosol delivery to infants. J Aerosol Med 2005; 18: 225–235. 285. Meers P, Neville M, Kurumunda R, et al. SLITTM amikacin drug release mediated by P. aeruginosa infection. Pediatr Pulmonol Suppl 2005; 28: 265–266. 286. Lamireau T, Monnereau S, Martin S, Marcotte JE, Winnock M, Alvarez F. Epidemiology of liver disease in cystic fibrosis: a longitudinal study. J Hepatol 2004; 41: 920–925. 287. Colombo C, Battezzati PM, Crosignani A, et al. Liver disease in cystic fibrosis: a prospective study on incidence, risk factors, and outcome. Hepatology 2002; 36: 1374–1382. 288. Lenaerts C, Lapierre C, Patriquin H, et al. Surveillance for cystic fibrosis-associated hepatobiliary disease: early ultrasound changes and predisposing factors. J Pediatr 2003; 143: 343–350. 289. Colombo C, Battezzati PM, Podda M, Bettinardi N, Giunta A. Ursodeoxycholic acid for liver disease associated with cystic fibrosis: a double-blind multicenter trial. The Italian Group for the Study of Ursodeoxycholic Acid in Cystic Fibrosis. Hepatology 1996; 23: 1484–1490. 290. Lindblad A, Glaumann H, Strandvik B. A two-year prospective study of the effect of ursodeoxycholic acid on urinary bile acid excretion and liver morphology in cystic fibrosisassociated liver disease. Hepatology 1998; 27: 166–174. 291. Nousia-Arvanitakis S, Fotoulaki M, Economou H, Xefteri M, Galli-Tsinopoulou A. Long-term prospective study of the effect of ursodeoxycholic acid on cystic fibrosis-related liver disease. J Clin Gastroenterol 2001; 32: 324–328. 292. Debray D, Lykavieris P, Gauthier F, et al. Outcome of cystic fibrosis-associated liver cirrhosis: management of portal hypertension. J Hepatol 1999; 31: 77–83. 293. Hall IP, Blakey JD. Genetic association studies in thorax. Thorax 2005; 60: 357–359. 294. Hull J, Thomson AH. Contribution of genetic factors other than CFTR to disease severity in cystic fibrosis. Thorax 1998; 53: 1018–1021. 295. Flamant C, Henrion-Caude A, Boelle PY, et al. Glutathione-S-transferase M1, M3, P1 and T1 polymorphisms and severity of lung disease in children with cystic fibrosis. Pharmacogenetics 2004; 14: 295–301. 296. Gilliland FD, Li YF, Dubeau L, et al. Effects of glutathione S-transferase M1, maternal smoking during pregnancy, and environmental tobacco smoke on asthma and wheezing in children. Am J Respir Crit Care Med 2002; 166: 457–463. 297. der W, Klimecki W, Yu L, et al. Opposite effects of CD 14/-260 on serum IgE levels in children raised in different environments. J Allergy Clin Immunol 2005; 116: 601–607. 298. Drumm ML, Konstan M, Schluchter MD, et al. Genetic modifiers of lung disease in cystic fibrosis. N Engl J Med 2005; 353: 1443–1453. 299. Gilljam M, Moltyaner Y, Downey GP, et al. Airway inflammation and infection in congenital bilateral absence of the vas deferens. Am J Respir Crit Care Med 2004; 169: 174–179.

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Current issues in the basic mechanisms, pathophysiology, diagnosis and management of primary ciliary dyskinesia T. Ferkol*,#, H.M. Mitchison}, C. O’Callaghanz, M. Leigh §, J. Carson §,ƒ, H. Lie**, D. Rosenbluth**, S.L. Brody ** Depts of *Paediatrics, #Cell Biology and Physiology, and **Internal Medicine, Washington University School of Medicine, St. Louis, MO, and Depts of §Pediatricsand ƒCell and Developmental Biology, University of North Carolina, Chapel Hill, NC, USA. }Dept of Paediatrics and Child Health, Royal Free and University College Medical School, University College LondonzDept of Paediatrics and Infection, Immunity and Inflammation, University of Leicester, Leicester, UK. Correspondence: S.L. Brody, Dept of Internal Medicine, Washington University School of Medicine, Box 8052, 660 South Euclid Avenue, Saint Louis, MO 63110, USA. Fax: 1 3143628987; E-mail: sbrody@im. wustl.edu

Primary ciliary dyskinesia (PCD) is a genetic disorder resulting from the dysfunction of motile cilia. Major clinical manifestations are recurrent infections of the upper and lower respiratory tract, including otitis media, sinusitis and bronchiectasis. In half of all infected individuals, randomisation of left–right 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 [1–6]. 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 Bardet–Biedl 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, 291–313. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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Recent developments in understanding cilia classes and ciliogenesis Classification of cilia Cilia are broadly classified as motile and primary cilia. Motile cilia contain dynein proteins functioning as motors to provide directed ciliary motion. Primary cilia lack motors but are specialised environmental sensors. The recognition that many diseases are related to hereditary defects in motile and primary cilia structure has led to an explosion in the study of cilia biology, particularly related to primary cilia. The area of most rapid growth has been the study of primary cilia defects in polycystic kidney disease [7–12].

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 left–right asymmetry [13]. The structure of the ciliary axoneme is reviewed briefly in this section, within the context of PCD genetics. Each normal cilium is a composed of y250 proteins organised around pairs of longitudinal microtubules of a- and b-tubulin, in a well-known pattern of nine outer-circle doublets that surround a central pair, thus creating the "nineztwo" organisation observed in electron photomicrographs of the axoneme cross-section (fig. 1a). Dynein complexes are visualised as inner or outer "arms" extending from outer doublets. Radial spoke proteins join the membrane sheath surrounding the central pair with the outer doublets and nexin proteins that form a circumferential network linking the peripheral doublets. The microtubules with their cognate proteins are anchored to a a)

b)

c)

Fig. 1. – a) Transmission electron photomicrograph of a ciliary axoneme cross-section from a normal individual demonstrating the nine surrounding and two central microtubule pairs. The outer and inner dynein arms are attached to each of the surrounding nine pairs (circled area). b) Photomicrograph of an axoneme from a primary ciliary dyskinesia (PCD) patient demonstrating absent inner and outer dynein arms (circled area). c) Computer-enhanced digital processing of the difference between normal (a) and PCD (b) images obtained from the highlighted regions. The composite confirms the absence of both arms relative to normal position noted by the green highlighted areas which, are illustrated by the red arrows. The black arrow shows the localisation of the ciliary axoneme outer membrane.

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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 400–500 kDa) and the large number of proteins associated with dyneins makes identification of mutations in genes required for creating motor complexes difficult.

Primary cilia Nearly all vertebrate cells have a single cilia transiently observed during interphase [16]. This has been termed a "primary cilium". The structure of the primary cilia retain the nine outer microtubule pairs but lack the outer arms and the central pair; hence they are termed "ninezzero" cilia. The cilium extending into the environment is hypothesised to function as a sensor to detect many types of information. The best-characterised primary cilia are specialised for vision, hearing and olfactory functions. Most recently, primary cilia in kidney-tubule epithelial cells have been shown to detect flow by bending, resulting in the transmission of a calcium-mediated signal to the cell [8]. In a similar manner, nonmotile cilia in the embryonic node are proposed to detect motile cilia flow to signal asymmetric body formation during early development (see below) [17].

Ciliogenesis, assembly, cilia transcriptomes and proteomes Many concepts of ciliogenesis and cilia assembly have been gleaned from the seminal work by Sorokin [18], which describes the ultrastructure of developing ciliated airway epithelial cells in electron micrographs. Another major observation, made more recently, was the identification of proteins transported to and from the cytoplasm into the axoneme for assembly and maintenance of the cilia, termed intraflagellar transport (IFT). IFT studies have revealed that dyneins, and other building blocks of the cilia, dock at the basal body while awaiting transport along the axonemal microtubules as cargo on kinesin motor proteins [19]. The IFT process also controls cilia length. Mutations or deficiencies in IFT transport components result in failed transport and are hypothesised to be the basis of defects in sensory cilia in some forms of polycystic kidney disease [11, 12]. In motile cilia, mutations in dyneins may also result in a "traffic jam" at the basal body or proximal axoneme, leading to failed transport of cilia motor proteins and subsequently a PCD phenotype [20]. These studies emphasise the impact of a dynein mutation and provide one mechanism for failure of dynein arm formation in PCD. Few "master genes" that regulate ciliogenesis have been identified. In developing airways, ciliated epithelial cells, as well as Clara cells, express transcription factors TTF1 and Foxa2 (HNF-3b) prior to ciliogenesis [21]. Factors that commit a cell to the ciliated 293

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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 [25–28]. In these studies, axonemes from human airway cells or Chlamydomonas were isolated and subjected to proteomic analysis, or Chlamydomonas RNA was analysed by microarrray to identify genes expressed during ciliogenesis. Taken together, these studies: 1) confirmed that a large number of human and Chlamydomonas proteins are shared; 2) identified regulatory proteins and others previously not suspected to play a role in ciliogenesis; 3) revealed that genes identified in primary cilia, and mutated in polycystic kidney disease, are present in motile cilia of Chlamydomonas; and 4) provided the basis for creation of biochemical pathways of cilia formation.

Progress in the genetic basis of PCD Recent advances in human genetics of PCD The inheritance of PCD is autosomal recessive with some rare cases of autosomal dominant or X-linked inheritance [29, 30]. The precise disease incidence is not known but is estimated at one per 25,000 live births in Caucasians [6, 31]. Since the association of Situs inversus and bronchiectasis alone is often used to make the diagnosis of PCD, this incidence is likely to be an underestimate [3]. The disease prevalence is higher in certain populations, such as those where consanguineous marriages are customary and those that are isolated for cultural or geographical reasons [32, 33]. The variation in clinical symptoms in PCD and the complexity of the cilia and sperm proteins led to early predictions of locus heterogeneity for the disorder [31]. The underlying ciliary ultrastructural defects are diverse but dominated by absent dynein motor proteins. Molecular genetic and linkage analyses in PCD families and model organisms and identification of mutations in PCD genes have confirmed extensive genetic heterogeneity, even in patients with the same ultrastructural defect [34, 35]. Efforts to map a major locus within a large PCD cohort were not successful [36]; however, the development of higher resolution, high throughput linkage mapping resources, such as single nucleotide polymorphism-based genome-wide genotyping panels, make re-visiting this approach more appropriate [37]. A major goal of PCD investigation is the determination of the molecular basis of disease by identifying and characterising the disease-causing genes. The locus heterogeneity underlying PCD has greatly impeded efforts to find the genes responsible for the disease. The results of human genetic-linkage studies of PCD and laterality defects are summarised in table 1. Successful strategies used to map the genes causing PCD genes have involved candidate gene analysis and positional cloning by homozygosity mapping in inbred families. Small, inbred populations, especially if families have a common ultrastructural defect, are more likely to share ancestral founder mutations. 294

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Table 1. – Human genetic linkage studies of primary ciliary dyskinesia (PCD) and laterality defects Disorder PCD PCD PCD PCD Situs inversus and probably PCD PCD PCD PCD PCD PCD and Usher type I syndrome PCD and cystic kidney disease PCD and retinitis pigmentosa Left–right axis abnormality Left–right axis abnormality

Chromosome location

Locus/gene

Function

Reference

6p21 Several 9p13–p21 19q13-qter 7p15

HLA-DR7

Unknown

DNAI1 CILD2 locus DNAH11

Dynein intermediate chain

[38, 39] [36] [35] [40] [41]

Dynein heavy chain

2q32 5p15 16p12 15q13–15 14q32

DNAH7 DNAH5

Dynein heavy chain Dynein heavy chain

USH1 (?EMAP)

9q31

INVS

EMAP is a microtubuleassociated protein Unknown

Xp21

RPGR

Xq26 2q21

[42] [34] [32] [32] [43, 44] [10]

ZIC3

Retinitis pigmentosa GTPase regulator gene Zinc finger transcription factor

[45]

CFC1

Extracellular signaling protein

[46]

[30]

HLA: human leukocyte antigen; DNAH: dynein axonemal heavy chain; EMAP: evoked muscle action potential; GTPase: guanosine triphosphatase.

Mutations in two genes encoding axonemal, outer dynein-arm structural components have been shown to cause recessive PCD in a proportion of patients with outer dyneinarm defects; but no mutations have yet been reported to account for other ultrastructural abnormalities in PCD patients. Mutations in genes coding for proteins that compose the outer arms are the DNAI (dynein axonemal intermediate chain)1 gene on human chromosome 9p [35, 47, 48] and the DNAH (dynein axonemal heavy chain)5 gene on chromosome 5p [49]. Identification of these two genes was aided by selective screening of candidate disease genes with homologues in Chlamydomonas [50]. The DNAI1 and DNAH5 proteins are orthologues of the 78 kDa intermediate chain (IC78) and gamma heavy chain (HCc) of the Chlamydomonas outer-arm dynein, respectively. Dysfunction of IC78/DNAI1 and HCc/DNAH5 result in ciliary dysmotility implying these proteins are essential for outer arm assembly or attachment to the outer doublet. Six DNAI1 mutations have been published, including missense, nonsense and deletion mutations. Three screens in 47 PCD families showed that 12 out of 94 disease chromosomes had a DNAI1 mutation, which is a frequency of 13% [34, 35, 47, 48]. Of these, half the mutant alleles were the same mutation, 219z3insT, a splice site mutation arising from insertion of a single T nucleotide. This frequency suggests a founder effect for DNAI1 or a mutation hotspot in the gene. Ten mutations in the DNAH5 gene have been reported [34, 51]. This is a very large gene to screen and, to date, results from only 25 PCD families compatible for linkage to DNAH5 have been published. Mutations were found in eight families, including mostly truncation mutations predicting loss of the encoded proteins motor- and microtubule-binding sites, and two missense mutations located in evolutionarily conserved amino acids that are presumably critical for function. No mutation hotspots were found. In patients homozygous for the same DNAH5 truncation or missense mutation, no differences in the clinical phenotype were observed compared with patients with a combination of different (compound heterozygous) mutations. However, a patient homozygous for a splice site mutation (IVS74-1GwC) had a partial outer dynein arm deficiency with shortened outer dynein arms, in contrast to patients homozygous for two truncation mutations who had complete absence of outer 295

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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 left–right asymmetry to PCD. It has been noted that certain PCD patient groups have a lower or higher than predicted occurrence of Kartagener’s Syndrome (PCD with Situs inversus) [32]. Further molecular genetic studies will elucidate whether randomisation of situs only applies to a subset of cases of PCD and whether certain PCD mutations and genes may be more closely associated with laterality defects. To minimise the chance of locus heterogeneity, patients have sometimes been divided according to situs for genetic studies [36, 47]. Laterality has been found to require normal cilia function in the embryonic node (see below). Further studies to identify differences in gene expression in cilia of the embryonic node and the cilia of the respiratory epithelial cells may help to explain some of the observed frequencies in situs abnormalities and respiratory cilia defects causing PCD.

Primary cilia disorders and overlap cilia diseases Research in cilia-related disease has exploded after proteins coded for by genes mutant in polycystic kidney disease were found to be expressed in the primary cilium or basal body of renal tubule cells [8]. Patients with this disease develop multiple renal cysts and can also have cysts in the pancreas and liver, implicating defects in cells with primary cilia lining the ducts of these organs. Adult-onset polycystic kidney disease and the related paediatric syndrome of nephronophthisis are genetically heterogeneous causes of chronic renal failure. Nephronophthisis is an autosomal recessive disease in childhood due to mutations of one of five different genes (all expressed in basal bodies and/or cilia), while adult-onset autosomal recessive or dominant forms of polycystic kidney disease are due to mutations in polycystin 1 or 2 genes, also expressed in basal bodies/cilia [9–12]. Mice deficient in genes encoding these proteins develop cystic kidneys and pancreas similar to the human disease [7, 12]. Mutations in other genes cause retinitis pigmentosa with polycystic kidney disease, constituting the Senior–Loken syndrome [9]. The mechanistic relationship between defects in primary cilia proteins and the lesion of polycystic kidney disease is not precisely known. Despite rare case reports, there is no apparent clinical relationship between polycystic kidney disease and PCD. However, an infrequent patient with PCD has features that point to additional roles of disease-causing genes in sensory cilia, including retinitis pigmentosa and left–right asymmetry [10, 29, 30]. These observations suggest that proteins mutated in these individuals may have functions in motile and sensory cells of the embryonic node, motile ciliated cells of the respiratory tract, and in sensory cilia of the eye and kidney. Interestingly, genes that are mutated in polycystic kidney disease 296

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have been identified in proteomic and gene array studies of Chlamydomonas and human respiratory cilia [25–28]. BBS is a rare autosomal recessive disorder with multiple phenotypes in varied combinations including retinopathy, polycystic kidneys, central obesity, polydactyly, male hypogonadism, cognitive impairment, diabetes mellitus and congenital heart defects [59]. Mutations at eight genetic loci have been identified in BBS. Several BBS proteins are expressed in basal bodies in C. elegans and defects in intraflagellar transport have been identified in mice with deleted BBS genes [25, 60]. The observation that BBS genes have a function in microtubule anchoring and cell cycle have lead to much speculation regarding the relationship of the BBS proteins to primary cilia function in nonsensory organs, as well as motile cilia. Defects associated with motile cilia have been found in BBS families, including Situs inversus and hydrocephalus. Mice deficient in BBS4 fail to form sperm flagella, suggesting shared function between cilia types [61]. Additionally, BBS genes have also been identified in genomic and proteomic analysis of motile cilia [25, 27, 61].

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

Left–right Hydrocephalus Cilia ultra-structure Other cilia-related asymmetry defect problems

DNAHc5 (mdnah5)

Random

Yes

DNAHc1 (mdhc7)

Normal

No

DNAHc11 (lrd) Tektin2 (tektin-t)

Random Normal

No No

DPCD/DNAPolymeraseLambda Foxj1

Random

Yes

Random

Yes

Random

Yes

Tg737orpk (Ift88, Polaris,Tct10)

Absent outer arm

Immotile cilia, sinusitis Normal Decrease sperm and tracheal cilia beat frequency Normal None Absent inner arm Decreased sperm and trachea cilia motility Absent inner arm Sinusitis, immotile sperm Failure of basal Absent motile bodies to dock axonemes# Absent and Polycystic kidneys, abnormal photoreceptor primary cilia and sperm defects

#

: ciliary aplasia. 297

Survival post-natal

Reference

3–4 weeks [34, 63, 73] Normal

[65]

Normal Normal

[66] [67]

1–4 months

[5, 64]

0–4 weeks

[23, 68]

Embryonic lethal

[69, 70]

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(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 left–right asymmetry that has been fundamental for identifying genes that regulate the development of left–right 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 left–right 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 left–right asymmetry.

Strategies for cilia gene targeting in mouse models. The identification of the role of genes coding for proteins expressed in cilia can be studied in mice rendered deficient through gene targeting, However, no mouse with deletion of a gene known to be mutant in human PCD (i.e. DNAH5) is currently capable of survival for study of PCD pathology in lung infection, nitric oxide generation and for development of PCD therapies. The use of conditional deletion of PCD genes in mice may favour improved survival for these studies. This could be accomplished by the use of a regulated FOXJ1 promoter to control deletion of cilia-specific genes [71]. In addition, to study human PCD mutations where proteintrafficking defects may be responsible for disease, mice deficient in the murine orthologue of the human mutant genes could be complemented with the mutant human PCD gene using bacterial artificial chromosomes.

Future challenges in PCD genetics Genetic studies will aid in the investigation of functional differences or tissue-specific expression differences in PCD genes. Many proteins that are components of cilia are members of large gene families with diversity of function and tissue-specific expression patterns. For example, the HUGO (Human Genome Organisation) Gene Nomenclature Committee has designated gene symbols for 15 axonemal heavy chain dyneins, and it has not yet been determined which are the most important for function of cilia in different locations in the body. For example, homology and expression studies suggest that DNAH8 might substitute for DNAH5 function in the testis [72]. Based on the absence of dynein arms in ultrastructure studies, it is often assumed that the dynein proteins themselves are mutated in PCD. Further studies of the relationship between the function of other proteins important in ciliogenesis and the associated ultrastructural changes of cilia should be performed. Identification of the genetic basis of PCD will probably expand as the process of cilia assembly is better understood and as gene-sequencing costs decrease. 298

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

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

Situs variations in PCD. Although the randomisation of situs is considered secondary to abnormal nodal cilia causing abnormal nodal flow, no PCD patient with either a transposition defect or central microtubular agenesis has been reported to have Situs inversus [76, 77]. This may be explained by the observation that cilia with these defects have a circular beat pattern similar to that of nodal cilia. Thus, it could be predicted that the movement of nodal cilia would not be affected and flow across the node would be in the usual leftward direction, resulting in normal situs. Specific mutations in cilia proteins may, therefore, lead to a variety of effects on nodal structure and function, resulting in varied situs defects observed in some PCD patients. For example, although situs totalis is typically seen in PCD, asymmetry defects may involve some, but not all organs in the thorax and abdomen (situs ambiguus) or be associated with congenital heart deformities, asplenia or polysplenia [78]. The relationship of these problems to cilia function within the node is not yet understood. 299

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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 (7–8 microns) and beat at twice the frequency (37–40 Hz in rats) [79]. There is a clear link between dysfunction of ependymal cilia and hydrocephalus. For example, hydrocephalus can be induced in experimental models by metavanadate, an inhibitor of ciliary movement [80] or by damage to the ciliated ependyma by bacteria, such as Streptococcus pneumoniae [81]. As noted above, mice deficient in some cilia proteins (table 2), including DNAH5 or foxj1, developed progressive unobstructed hydrocephalus leading to death [23, 56, 82]. The Tg737orpk mouse, deficient in Polaris, an IFT protein expressed in primary cilia, also develops hydrocephalus. These mice were found to have abnormal chloride production in cerebral spinal fluid (CSF), leading to the hypothesis that primary cilia regulate ion transport in CSF [69] and indicating the need for further study of the roles of both motile and nonmotile cilia in the brain. Hydrocephalus is rare in human PCD. Slightly enlarged brain ventricles in pre-natal ultrasound examinations of embryos with Situs inversus have been noted [83]; however, progression of hydrocephalus is likely to be unusual since there are few case reports of hydrocephalus with PCD [84]. The wider aqueduct of the human compared with the mouse may explain the low incidence of hydrocephalus observed in humans in contrast to the more uniform occurrence in mice with ciliary defects [82].

Low exhaled nasal nitric oxide in PCD An interesting PCD phenotype possibly related to cilia function is low exhaled nasal nitric oxide (NO). An initial report that children with Kartagener’s syndrome produced very low levels of expired nasal NO [85] was validated by several studies demonstrating that individuals of all ages with PCD have 10–20% of normal levels [3, 86, 87]. Interestingly, nasal NO levels measured in parents of the PCD patients, who are obligate heterozygotes, were intermediate to levels of PCD and normal individuals [3]. Together, these findings suggest that a mutant protein interferes with a normal cellular process directly or indirectly related to ciliated cell functions. In normal individuals, NO recovered from the nose is several log-fold higher than from lower airways, reflecting a different regulation of NO production, metabolism and/or clearance from the two sites. Alteration in these processes may account for the low levels of nasal NO in PCD. Within the nose and respiratory tract, postulated functions for NO include regulation of ciliary motility and antimicrobial activity [88, 89]. A family of nitric oxide synthase (NOS) enzymes catalyses the formation of NO in the presence of appropriate substrates (l-arginine and oxygen) and cofactors. Three mammalian NOS isoforms have been identified: nNOS (neuronal NOS or NOS I); iNOS (inducible NOS or NOS II); and eNOS (endothelial NOS or NOS III). Both iNOS and eNOS have been localised in nasal epithelium [90, 91]. Immunolocalisation studies have shown that eNOS is expressed close to the base of cilia and may play a role in regulating ciliary beat [91]. iNOS is increased in nasal epithelial cells obtained from patients with allergic rhinitis, suggesting a role in the regulation of inflammatory processes [90]. A potential area of investigation is iNOS and eNOS expression in epithelial cells of PCD patients with and without infection, as well as the effect of NO addition on cilia cell function. 300

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

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

Underappreciated clinical features of PCD in the neonate and infant PCD may present with a variety of clinical features, mimicking respiratory diseases, such as CF or asthma, thus making prompt diagnosis a challenge and often leading to delayed diagnosis. One study reported that the mean age of PCD diagnosis was slightly w4 yrs of age despite early manifestations of PCD in infancy [96]. Accumulating information reveals that half to three-quarters of patients with PCD have significant respiratory symptoms shortly after birth [1, 3, 97]. Many reports emphasise atelectasis or pneumonia associated with significant respiratory distress in neonates with PCD. The association of neonatal respiratory distress with PCD point to the critical nature of cilia function for effective clearance of foetal lung fluid. In infancy, chronic cough and persistent rhinitis, often present since birth, are common clues to underlying PCD. As a consequence, the infant may struggle with poor feeding and failure to thrive. Rhinitis and 301

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chronic cough in infancy typically leads to chronic sinopulmonary infections and eventual PCD diagnosis later in childhood [96].

Ultrastructure analysis of cilia Afzelius [98], and other authors, characterised the first ultrastructural lesions by electron microscopy that elucidated the pathophysiological basis of PCD. These studies showed absent axonemal dynein arms of airway cilia and sperm flagella as the causative basis for ciliary immotility and resultant respiratory disease and infertility. Since these early reports of PCD demonstrating completely absent inner and outer dynein arms and absent ciliary motility, a number of phenotypic variations including missing or dysmorphic inner or outer arms, and changes in the structural organisation of auxiliary axonemal elements, such as radial spokes, have been noted [92, 93, 99]. The most common ultrastructural defects detected in a PCD population are outer-arm defects that can be related to genetic mutations in heavy chain dynein genes [3]. Electron microscopy of sperm tail flagella or of respiratory cilia has been recognised as the "gold standard" for the diagnosis of PCD. Ultrastructural analysis has limitations that may lead to confounding results. Among the problems associated with such analyses are poor sample quality, processing and procedural errors, and the inherent limitations of electron optics related to imaging of ultra-thin sections. As individuals with PCD generally present with upper respiratory symptoms, including chronic sinusitis, acquisition of a sample of epithelium containing an adequate number of ciliated cells is often problematic. Moreover, because of the chronic nature of infection and inflammation, acquired ciliary defects are often present in the sample, along with necrotic cell debris that interferes with optimal imaging. This has had the unfortunate result of erroneous reports of "new" index lesions said to confer PCD. The processing of specimens for electron microscopic examination requires exposure of the cells and tissues to fixatives intended to stabilise cell and organelle structure, exposure of the tissues to heavy metal-containing stains to impart differential electron opacity to specific chemical groups, and embedment of the specimen in an epoxy resin for ultra-thin sectioning. Under conditions of excellent quality control, electron dense precipitates, swelling and shrinking of tissues, and suboptimal section quality and thickness are limited. The accelerating voltage, operational modes of the transmission electron microscope, and the physical characteristics of the electron beam also contribute to the quality and resolution of micrographs intended for analysis. For example, a typical ultra-thin section is 60–90 nm in thickness and, for a given axoneme, such a section may reveal a wide variety of profiles. Thus, it is of paramount importance that a sufficient number of clear cross-sections are obtained to ensure a reliable interpretation. Computer-assisted programmes that enhance the electron micrograph image to improve detection of cilia defects are being evaluated (fig. 1c) [100, 101].

Emerging questions for ultrastructural imaging studies in PCD. The small dimensions, particularly thinness, of ultra-thin sections severely limit approaches to analyse the organisation of axonemal elements in three dimensions and their linear distribution along the length of axonemes. Furthermore, staining characteristics of conventional heavy metal stains used in biological electron microscopy may not be capable of generating the contrast necessary for imaging certain axonemal elements integral to ciliary function. Cilia from patients with PCD often exhibit marginal motility and ultrastructural evidence of irregular distributions of dynein arms. Thus, it is plausible to hypothesise that dynein organisation and distribution are altered along the length of 302

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the axoneme and that these differences are genotypic. Although elegant morphometric studies using computer-assisted analyses [102] and freeze-fracture methodology [103] have demonstrated the spatial organisation of axonemal elements in wildtype and mutant eukaryotic Protists, similar analyses have not been performed in axonemes from documented PCD patients. Moreover, the vast majority of ultrastructural studies of PCD have focused on the fine structure of axonemes to characterise ciliary anomalies, but have failed to analyse the "upstream" events of ciliogenesis as an initiating event in the formation of abnormal cilia in PCD. A promising area of PCD research in recent years has been the search for and identification of candidate genes, and fundamental data that relates specific mutations to cilia ultrastructure has not been realised [34, 48]. As both genetic and proteomic studies yield new information and as imaging techniques undergo further refinement, biological electron microscopy as well as other high-resolution imaging techniques will continue to play an important role in the molecular level characterisation of axonemal organisation in this syndrome.

Expired nasal NO for PCD diagnosis Several studies have demonstrated that exhaled nasal NO is very low in individuals with PCD, and the ease of test performance suggests this as a useful screening or supportive diagnostic test for PCD. However, low nasal NO levels have also been reported in disorders with overlapping clinical features including CF, panbronchiolitis and nasal polyposis [86, 104, 105]. The low NO levels in these other diseases may be due to secondary cilia dysfunction, but the shared clinical features indicate that further assessment is warranted before this test is routinely relied on for definitive diagnosis of PCD. Alternatively, the data indicate that in some individuals with specific clinical findings it is possible that the diagnosis of PCD could be supported if CF was excluded. Recent efforts have focused on standardising nasal NO measurement by a noninvasive procedure to allow comparisons between studies [106]. During the measurement, several manoeuvres may be used to close the soft palate and thereby limit "contamination" of nasal air by air exhaled by the lower airways (exhaled NO from lower airways is typically much lower than nasal NO). These techniques have been used reproducibly in children w5 yrs of age, but may be difficult in younger children who cannot cooperate with palate closure manoeuvres.

Areas for investigation of exhaled NO related to PCD. Exhaled nasal NO is now used in large PCD centres as a screening test. However, the underlying pathophysiological mechanism responsible for the result is unknown, making interpretation difficult. Studies to elucidate NO pathways in patients with PCD are required. This may be facilitated by studies of mice with known dynein arm mutations (knockout mice) or in vitro cell culture of airway epithelial cells from patients with defined dynein arm mutations. Clinical trials to assess the contribution of genetic, infectious and inflammatory factors are also necessary. Although preliminary studies have not revealed differences in low NO production relative to different PCD mutations, it may be useful to investigate exhaled NO in individuals with novel defects not involving common dynein arm genes.

Cilia beat analysis Qualitative assays for ciliary function, including measures of mucociliary clearance using the saccharin test and radioisotopic methods, have limited usefulness because they are poorly standardised, cannot be used in young children, and do not distinguish between primary and secondary causes of ciliary dyskinesia. The recent advent of 303

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high-resolution digital high-speed video imaging allowing the precise beat pattern of cilia to be viewed frame by frame in different planes has revealed that reproducible differences in ciliary beat pattern are related to specific underlying ultrastructural abnormalities [76]. These findings have led to the concept that detailed analysis of cilia beat could be used for clinical assessment and the diagnosis of PCD.

Classes of cilia beat relative to ultrastructure defect. High-resolution video imaging of cilia from nasal or lower airway sampling has shown that respiratory cilia do not beat with a classical forward power stroke and recovery stroke that sweeps to the side as previously assumed. They simply beat forward and backward in a planar motion, without a sideways recovery sweep [107]. In patients with isolated outer dynein arm or outer and inner dynein arm defects, the majority of cilia are immotile and, at best, simply flicker. Cilia from patients with an isolated inner dynein arm defect or a radial spoke defect combined with an inner dynein arm defect have a stiff forward stroke with markedly reduced amplitude. The beat frequency of these two groups differs. The inner dynein arm defect has a mean beat frequency of 8.1 Hz while the radial spoke defect frequency is slower at 6 Hz. The stiff beat pattern supports the importance of the inner dynein arm in the bending of the ciliary axoneme. The third type of beat pattern is found in patients with PCD associated with ciliary transposition and central microtubular agenesis. In the ciliary transposition, the gap left by an absent central microtubular pair is filled by a peripheral microtubule pair and associated dynein arms that transpose to the centre of the axoneme. In microtubular agenesis a proportion of cilia lack the central microtubular pair [77]. In both defects, all cilia have an oval gyrating pattern with cilia beating with a mean frequency of 10.7 Hz which is within the normal range of ciliary beat frequency (normal mean 12 Hz; range 9.7–18.7 Hz) measured using the high-speed video system [76]. The implications of these findings are significant, as ciliary beat frequency is typically measured using indirect techniques, such as the photomultiplier and modified photodiode that cannot assess ciliary beat pattern. Thus, the use of beat frequency alone as a screening test to determine those patients requiring further investigation will miss PCD due to ciliary transposition defects or central microtubular agenesis. Cilia-beat analysis requires demanding expertise and expensive instrumentation. The routine use of this methodology for the diagnosis of PCD is, therefore, currently limited to a few specialised centres. However, information from these studies can be used to characterise specific groups of patients with cilia defects, make genotype–phenotype correlations, and provide useful information for assessment of results from other diagnostic testing methods.

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

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PCD biology using electron microscopy, cilia beat analysis, immunostaining and biochemical methods.

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

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

Current concepts in the clinical management of PCD Therapeutic approach and prognosis of PCD No therapies are known that correct ciliary dysfunction and clinical studies with sufficient statistical power to prove the efficacy of specific therapies have not been performed. Similarly, few retrospective studies are of sufficient size to make strong conclusions regarding current therapy. Instead, most therapies are based on experience from centres treating large numbers of individuals with PCD or are extrapolated from treatment of diseases with similar clinical features, including CF and bronchiectasis of multiple aetiologies. The goals of current PCD therapy are: 1) maintenance of 305

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mucociliary clearance; 2) optimal management of otitis media and sinusitis; and 3) bacteria culture-directed antibiotic therapy for upper and lower airways infection. Three small longitudinal studies of PCD suggest that the onset of airway disease occurs early in childhood [110–112], stressing the need for early diagnosis, close monitoring for airway infection, and, ultimately, long-term multicentre studies. It is widely agreed that early diagnosis and antibiotic use have improved the prognosis for PCD. In contrast to CF, many individuals with PCD have a normal or near-normal lifespan. However, like CF, chronic lung disease is the most common limiting problem, leading to severe pulmonary disability and eventually to respiratory failure in some individuals.

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

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

Directed antibiotic therapy for lung disease Directed antibiotic therapy practice for PCD is based on experience in CF clinics [95]. Here, management of pulmonary disease involves regular monitoring of pulmonary function using spirometry, chest radiographs, respiratory cultures by sputum or throat swab (based on age), and treating respiratory infections with antimicrobial agents based on careful laboratory assay of sensitivities. One retrospective study of PCD at a 306

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specialised centre in the USA found that this was commonly the practice in PCD management [3]. In half the cases, antibiotics were provided as i.v. therapy. Prospective longitudinal surveys of bacterial species recovered from respiratory samples of PCD patients have not been published. The most common organism recovered from patients with PCD is Haemophilus influenzae, particularly in children [3]. Other common organisms are Pseudomonas aeruginosa, Staphylococcus aureus, and S. pneumoniae. As in CF, chronic infection with mucoid strains of P. aeruginosa can occur, but chronic infection with P. aeruginosa appears at a later age when compared with CF. Oral or systemic antibiotics should be prescribed as indicated for worsening respiratory symptoms, infection or spirometry, and should be chosen on the basis of prior sputum culture results. As in other bronchiectatic syndromes, chronic suppressive oral or inhaled antibiotics may be prescribed as maintenance therapy but could enhance the development of antimicrobial resistance.

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

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

Summary and conclusion Since the 1990s, major advances in PCD are related to discoveries of the genetic and molecular basis of this disease. Specifically, identification of dynein gene mutations that 307

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cause the syndrome of PCD in many affected individuals, and confirmation of the biological basis of disease by demonstration of a similar phenotype in mice that are rendered dynein gene deficient. In addition, a role for cilia in the establishment of left– right asymmetry and hydrocephalus has been discovered in PCD-related research. However, genetic mutations have not been identified in a significant number of patients with PCD, pointing to the need for further studies of cilia biology and genetics. Thus, there are significant problems related to easily making the diagnosis of PCD. In this regard, the use of exhaled NO appears promising and requires a better understanding of the mechanism of altered NO excretion in PCD. Finally, multicentre studies of genotype– phenotype relationships and therapeutic modalities will need to be organised.

Summary Primary ciliary dyskinesia (PCD) is a genetic disorder resulting from dysfunction of motile cilia. Epithelial cells containing motile cilia are localised in the respiratory tree, ventricles of the brain, oviduct, sperm and the embryonic node. In these epithelial cells, dysfunction accounts for the major symptoms of PCD, including otitis media, sinusitis and bronchiectasis, Situs inversus (in half of the patients) and, more rarely, infertility and hydrocephalus. While the understanding of cellular and molecular mechanisms responsible for these symptoms has recently progressed, genetic analysis has identified mutations in only two axonemal dynein genes that can account for abnormal cilia ultrastructure and beat frequency in a subpopulation of individuals. Thus, investigations are directed towards expanding understanding of the genetic basis of PCD by identification of proteins with roles in ciliogenesis, and increasing the scope of genes and populations subject to genetic analyses. To support the diagnosis of PCD, efforts are currently directed toward the optimal use of cilia ultrastructure and beat analysis, and interpretation of low levels of exhaled nasal nitric oxide. While there is no specific therapy for PCD, maintenance of mucociliary clearance and culturedirected antibiotic therapy are current cornerstones of therapy. The establishment of new methodologies for PCD diagnosis and therapies will require evaluation of relationships between specific genetic mutations, disease phenotypes and therapeutic responses to be carried out in multicentre cooperative trials. Keywords: Bronchiectasis, cilia, dynein, left–right asymmetry, mouse models, nitric oxide.

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Hossain T, Kappelman MD, Perez-Atayde AR, Young GJ, Huttner KM, Christou H. Primary ciliary dyskinesia as a cause of neonatal respiratory distress: implications for the neonatologist. J Perinatol 2003; 23: 684–687. Afzelius BA. A human syndrome caused by immotile cilia. Science 1976; 193: 317–319. Schneeberger EE, McCormack J, Issenberg HJ, Schuster SR, Gerald PS. Heterogeneity of ciliary morphology in the immotile-cilia syndrome in man. J Ultrastruct Res 1980; 73: 34–43. Carson JL, Hu SS, Collier AM. Computer-assisted analysis of radial symmetry in human airway epithelial cilia: assessment of congenital ciliary defects in primary ciliary dyskinesia. Ultrastruct Pathol 2000; 24: 169–174. Escudier E, Couprie M, Duriez B, et al. Computer-assisted analysis helps detect inner dynein arm abnormalities. Am J Respir Crit Care Med 2002; 166: 1257–1262. Mastronarde DN, O’Toole ET, McDonald KL, McIntosh JR, Porter ME. Arrangement of inner dynein arms in wild-type and mutant flagella of Chlamydomonas. J Cell Biol 1992; 118: 1145–1162. Goodenough UW, Heuser JE. Substructure of inner dynein arms, radial spokes, and the central pair/projection complex of cilia and flagella. J Cell Biol 1985; 100: 2008–2018. Nakano H, Ide H, Imada M, et al. Reduced nasal nitric oxide in diffuse panbronchiolitis. Am J Respir Crit Care Med 2000; 162: 2218–2220. Colantonio D, Brouillette L, Parikh A, Scadding GK. Paradoxical low nasal nitric oxide in nasal polyposis. Clin Exp Allergy 2002; 32: 698–701. American Thoracic Society, European Respiratory Society. ATS/ERS Recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide, 2005. Am J Respir Crit Care Med 2005; 171: 912–930. Chilvers MA, O’Callaghan C. Analysis of ciliary beat pattern and beat frequency using digital high speed imaging: comparison with the photomultiplier and photodiode methods. Thorax 2000; 55: 314–317. Jorissen M, Willems T. Success rates of respiratory epithelial cell culture techniques with ciliogenesis for diagnosing primary ciliary dyskinesia. Acta Otorhinolaryngol Belg 2000; 54: 357–365. Randell SH, Walstad L, Schwab UE, Grubb BR, Yankaskas JR. Isolation and culture of airway epithelial cells from chronically infected human lungs. In vitro Cell Dev Biol Anim 2001; 37: 480–489. Corkey CW, Levison H, Turner JA. The immotile cilia syndrome. A longitudinal survey. Am Rev Respir Dis 1981; 124: 544–548. Ellerman A, Bisgaard H. Longitudinal study of lung function in a cohort of primary ciliary dyskinesia. Eur Respir J 1997; 10: 2376–2379. Hellinckx J, Demedts M, De Boeck K. Primary ciliary dyskinesia: evolution of pulmonary function. Eur J Pediatr 1998; 157: 422–426. Hadfield PJ, Rowe-Jones JM, Bush A, Mackay IS. Treatment of otitis media with effusion in children with primary ciliary dyskinesia. Clin Otolaryngol 1997; 22: 302–306. Denoyelle F, Roger G, Ducroz V, Escudier E, Fauroux B, Garabedian EN. Results of tympanoplasty in children with primary ciliary dyskinesia. Arch Otolaryngol Head Neck Surg 1998; 124: 177–179. Noone PG, Bennett WD, Regnis JA, et al. Effect of aerosolized uridine-5’-triphosphate on airway clearance with cough in patients with primary ciliary dyskinesia. Am J Respir Crit Care Med 1999; 160: 144–149. Smit HJ, Schreurs AJ, Van den Bosch JM, Westermann CJ. Is resection of bronchiectasis beneficial in patients with primary ciliary dyskinesia? Chest 1996; 109: 1541–1544. Macchiarini P, Chapelier A, Vouhe P, et al. Double lung transplantation in situs inversus with Kartagener’s syndrome. Paris-Sud University Lung Transplant Group. J Thorac Cardiovasc Surg 1994; 108: 86–91. Rabago G, Copeland JG 3rd, Rosapepe F, et al. Heart-lung transplantation in situs inversus. Ann Thorac Surg 1996; 62: 296–298.

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Adipositas in infants and children: a new disease on the horizon K.G. Tantisira, D.R. Gold Channing Laboratory, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA. Correspondence: K.G. Tantisira, Channing Laboratory, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA. Fax: 1 6175250958; E-mail: kelan. [email protected]

Obesity is one of the most prevalent chronic disorders of childhood, affecting 22 million children worldwide [1]. In the period 1999–2002, y15% of children and adolescents in the USA met the criteria for obesity [2, 3]. Using National Health and Nutrition Examination Survey (NHANES) data, the prevalence of obesity, defined by a body mass index (BMI) i95th percentile, for children of the same age and sex doubled between 1976–1980 and 1999–2002 [4]. Similar increases have been noted in other developed countries [5–8]. For instance, the prevalence of obesity in Japanese schoolaged children doubled from 5 to 10% during 1973–1994 [9]. In the USA in 1998, the prevalence of childhood obesity was 21.5% for African-Americans, 21.8% for Hispanic Americans and 12.3% for non-Hispanic Whites [3]. Despite these ethnic disparities, increases in body mass have occurred among both sexes and across all ethnic and socioeconomic groups [2]. Childhood obesity is also increasing in developing countries, where it is not uncommon to note demographic characteristics that simultaneously include both high prevalences of obesity and malnourished children [1, 10]. Thus, obesity in childhood is clearly evolving into a truly global health issue. Although no single explanation for the increased prevalence of childhood obesity exists, multiple theories abound. The Western lifestyle hypothesis builds upon the increased frequency of obesity in developed countries compared to Third World countries. Certainly, as countries make the transition from primarily agricultural societies towards increasing industrialisation, the incidence and prevalence of obesity tend to increase. For instance, in Thailand, the per capita gross national product doubled between 1990 and 1996. The prevalence of overweight adults, defined as a BMI of i25.0 kg?m-2, rose from 7.7 to 13.2% in males and from 15.7 to 25.0% in females between 1991 and 1996 [11]. Similar trends were seen in children, with pronounced differences by socioeconomic and urban versus rural settings [11]. The hypotheses for the development of obesity in relationship to Western lifestyle have focused primarily on caloric consumption and decreased physical activity [3, 11]. Other common hypotheses to explain the "obesity epidemic" include the Barker or foetal origins hypothesis [12, 13], changes in the relative proportion of dietary fats and type of carbohydrates [1, 11], and interactions of genetic predisposition with environmental influences [12, 14, 15]. Childhood obesity is associated with a variety of both long- and shorter-term sequelae. Several studies have reported an increase in cardiovascular and all-cause mortality rate in adults who were overweight or obese as children or adolescents [16–18]. Childhood obesity may increase mortality risk in adulthood through its cumulative effects on elevated blood pressure, lipid abnormalities or type 2 diabetes mellitus [8, 19, 20]. Childhood obesity is also associated with more immediate decrements in health and Eur Respir Mon, 2006, 37, 314–344. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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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 1979–1981 to US$127 million during 1997–1999 [23]. There is rapidly evolving evidence that obesity may substantially affect respiratory as well as cardiovascular health. Respiratory complications of obesity in children are the focus of the present chapter, after discussion of definitions of childhood obesity. Data are reviewed on the relation of obesity to pulmonary function, airways responsiveness (AHR), asthma and obstructive sleep apnoea (OSA) in children. Subsequently, the potential pathophysiological mechanisms that may help to explain these associations and the requirement for further research into these mechanisms are discussed. Finally, recommendations for individual and social practice designed to reduce childhood obesity are reviewed. Although the present chapter focuses on pulmonary manifestations of obesity in children, these are supplemented with recent findings from adult human and animal studies for situations for which the literature in children is sparse or unavailable.

Body mass: the correct obesity phenotype? Most of the literature involving childhood adiposity has focused on some measure of BMI (body mass in kilograms divided by height in metres squared), including using population cut-offs of the 85th percentile to define overweight and the 95th percentile to define obesity. Indeed, the use of BMI has been proposed as the primary standard for the worldwide definition of overweight and obesity in children [24]. Nonetheless, multiple other obesity phenotypes have been described. For example, central, including truncal and abdominal, adiposity is linked with insulin resistance, whereas centrifugal or gluteal adiposity is not [25]. BMI is probably a good clinical measure for the definition of obesity in adolescents [22]. In pre-adolescents, however, BMI may be representative of increases in other growth parameters, such as muscle mass [22, 26, 27], and, therefore, should be used with caution. The best epidemiological measure of body fat in children and adolescents remains controversial [28], but may be one utilising skinfold measures (e.g. triceps skinfolds) [29, 30]. Other commonly employed field measures of adiposity in children include waist circumference, waist/hip ratio and measures of bioelectric impedance [31, 32]. Dual-energy X-ray absorptiometry (DXA) [31, 33] has become a widely accepted standard for measuring body fat in clinical studies of both children and adults, due to its relative ease of use, low radiation exposure and clinical reproducibility [34]. One advantage of DXA is the ability to assess total as well as regional body composition (i.e. trunk, arms and legs). Computed tomography and magnetic resonance imaging scans may be more accurate but their use is limited in clinical studies of children due to their cost and radiation exposure [32]. Although the criticism that BMI correlates with overall growth has been demonstrated to be true when growth is evaluated by measured height, one study noted that it remains a valid measure of obesity in 5–12-yr-old children since the degree of measured adiposity also increases with height [35]. In particular, Freedman et al. [35] found a very high correlation (r=0.85–0.90) between BMI and measured body fat in these younger children. A follow-up study by the same group evaluated 1,196 children aged 5–18 yrs, comparing BMI with DXA-measured fat mass and fat-free mass [36]. In those children with a BMI v50th percentile for age and sex, BMI strongly correlated with fat-free mass (r=0.56– 0.83). However, for a BMI i85th percentile, BMI correlated primarily with fat mass 315

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(r=0.85–0.96), supporting the continued usage of the current BMI definitions of overweight and obesity as valid measures of adiposity. Few studies are available that evaluate the relation of obesity phenotypes other than BMI to respiratory disease. Therefore, in the present chapter, the definition of obesity is assumed to be based upon BMI measurements. However, where available, data based upon other measurements of adiposity are cited.

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

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perspective, an increased ventilatory responsiveness to hypoxia and relatively decreased ventilatory responsiveness to hypercapnia have been noted in obese subjects [53]. Obesity and fat distribution pattern may have independent effects on ventilatory function. FVC, FEV1 and TLC were found to be significantly lower in 42 subjects with an upper body fat distribution (central obesity) as measured by waist/hip ratio [45]. Sex differences in fat distribution (fat is distributed more peripherally in females and more centrally in males) may also contribute to the observed greater impact of fat mass on FVC in males [54]. A large cross-sectional analysis of 1,634 adults noted that increased waist/hip ratio was associated with more pronounced decrements in FEV1 and FVC in males than in females [55]. Obesity in children may also result in little or no change in spirometric indices [56, 57]. In a descriptive study of 64 obese children (median age 12 yrs; median BMI 30.1 kg?m-2), Li et al. [58] noted overall median values within the normal range for both spirometric and lung volumes when obesity was assessed as a BMI i95th percentile. However, 30 of the 64 (46%) showed reduced FRC, consistent with the adult literature on this topic. Additionally, these authors noted a moderate decrement in diffusing capacity of the lung for carbon monoxide (DL,CO) in a third of the subjects, which has not been reported in adults. When truncal obesity was assessed by DXA, TLC and RV were also noted to be reduced, supporting the contention that DXA may be a better measure than BMI of body fat that might affect the pulmonary architecture. Decrements in DL,CO with obesity were also noted in one other study of 13 morbidly obese children [59]. The children in that study ranged 150–300% of ideal body weight. In addition to the DL,CO, the children exhibited decrements in FEV1, forced expiratory flow between 25 and 75% of vital capacity, and expiratory reserve volume. Aside from the changes in DL,CO, this series of children indicate that the pulmonary function abnormalities in the morbidly obese child generally parallel those expected of the obese adult. The effects of body mass on pulmonary function have also been evaluated in several paediatric cohorts. In a study of 1,041 childhood asthmatics [27], BMI was positively associated with increased FEV1 and FVC. However, increased BMI corresponded to decrements in the FEV1/FVC ratio. This study was limited by the lack of children at the extremes of the body mass distribution (no child was w150% of the age and sex distributions). The authors concluded that, over the range of normal to mild obesity, BMI was a proxy for growth (as with the aforementioned muscularity effect), leading to the associations of increases in spirometric volumes with increased BMI. They did, however, speculate that the decrements in FEV1/FVC with increasing BMI may play an important role in the obesity–lung function relationship. Supporting this, both the Six Cities Study longitudinal data on 9,828 children and cross-sectional data on 2,176 Italian children also noted decrements in the FEV1/FVC ratio in association with increased BMI [60, 61]. In a cohort of 8,484 Australian schoolchildren, Lazarus et al. [26] analysed the relationship of both body weight and body fat measured via skinfold thickness with spirometric lung volumes. These authors also noted marked age- and height-adjusted increases in both FEV1 and FVC in association with increased weight, again supporting the notion of increased growth in association with increased body mass. However, in the evaluation of adiposity via skinfold measurements, the highest tertile of body fat was consistently associated with lower FEV1 and FVC in this study, suggesting once again that, in paediatric studies, BMI in normal-to-mildly obese children might not be the optimal measure of adiposity since it cannot optimally distinguish lean tissue mass from fat mass. Fung et al. [62] also noted increased spirometric flows correlating with increased BMI in a population of Chinese schoolchildren. Nevertheless, overweight children (w90% of the predicted value) showed decrements in these pulmonary measures 317

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in association with increasing BMI, supporting other studies noting a classic restrictive ventilatory defect in obese children [59].

Obesity and AHR In addition to the effects that obesity has on static lung volumes and spirometric measures, obesity has also been independently linked to AHR. Total respiratory resistance has been estimated to be about twice as high in obese compared to nonobese adults [63]. The increased respiratory resistance associated with obesity has been suggested to result from the reduction in lung volumes [64]. Mechanistically, this is similar to other manoeuvres known to reduce lung volumes, such as supine position or ribcage strapping, which have also been noted to increase respiratory resistance [65, 66]. It was noted previously that FRC is reduced in obesity due to the effect of the abdominal contents on the position of the diaphragm. Obesity has also been associated with decrements in tidal volume [52], which fails to increase during times of dynamic stress, such as exercise [67]. Moreover, in morbid obesity, the majority of tidal breaths are taken at around the closing volume [68]. Decrements in FRC and low tidal volumes infer low cycling frequencies, resulting in the conversion of airway smooth muscle from rapidly cycling actin–myosin cross-bridges to slowly cycling latch-bridges [69, 70]. The attainment of the latch state has been hypothesised to be the reason that obstruction persists in asthmatic airways [69, 70]. The latch state has also been postulated to result in increased AHR [69, 70]. Furthermore, these effects may be enhanced by breathing at around the closing volume [70, 71]. The latch state may thus explain the observations that decrements in FRC, as occur in obesity, have been tightly correlated with increased airways resistance [64, 72] and responsiveness to methacholine [71]. A separate but related mechanism to the latch state results from the high frequencies but low tidal volumes at which the obese individual breathes [51, 52]. Normally, the tidal action of spontaneous breathing imposes tidal strains on airway smooth muscle; these tidal strains are extremely potent natural bronchodilatory agents [73, 74]. Since the tidal volumes of the obese individual are decreased compared to normal subjects, this bronchodilatory mechanism is attenuated and, therefore, also predisposes to increased AHR compared with the lean individual [75]. The effects of obesity on AHR may be accentuated in children for several reasons. Smaller more immature airways may be more prone to the latch state [74]. Additionally, the mechanical load of obesity may affect lung growth [76], leading to reductions in pulmonary function with the subsequent risk of AHR. Obesity may also lead to accelerated airway remodelling, leading to fixed obstruction. The remodelling is generally not reversible with subsequent weight loss [77]. Finally, inflammatory pathways may be activated in association with obesity in children, leading to an increased predisposition towards AHR; these pathways are reviewed in greater detail in the subsequent sections. The association between obesity and AHR has been evaluated in three large epidemiological studies of adults. Litonjua et al. [78] reported an association between increased BMI and incident AHR in a case–control study of 305 males. Specifically, the odds ratio (OR) of developing significant AHR was 10.0 (95% confidence interval (CI) 2.6–37.9) for the highest quintile of body mass compared with the middle quintile. Interestingly, there appeared to be a U-shaped relationship, with the lowest quintile also being associated with AHR (OR 7.0; 95% CI 1.8–27.7). The findings of increased AHR with increased BMI were also noted in two large cross-sectional studies of adults [79, 80]. In children, although AHR has recently been correlated with asthma severity [81], little is known about the relationship between BMI and AHR. In a cross-sectional populationbased study of teenagers in Taiwan, Huang et al. [82] noted a decreased prevalence of 318

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AHR in the lowest quintile of BMI in teenage females. These findings were not noted in males. In their analysis of the Childhood Asthma Management Program cohort, Tantisira et al. [27] noted an increased provocative concentration of drug causing a 20% fall in FEV1 (decreased AHR) in association with increased BMI. However, this did not persist after adjustment for baseline FEV1, suggesting that the relationship was primarily mediated by increased airways growth in association with increased BMI. Two other epidemiological studies, including a cross-sectional study of 5,993 children [83] and a longitudinal study of 757 children [84], also noted no independent effect of BMI on methacholine sensitivity. Nevertheless, the consistent association of obesity with exercise-induced bronchospasm in children [85, 86] suggests that a relationship between increased AHR and BMI may yet exist. Further study is warranted in this area.

Obesity and asthma Interestingly, the recent increase in the prevalence of obesity has been accompanied by a similar rise in the associated rates of asthma. Currently, an estimated 300 million individuals worldwide are estimated to have asthma [87]. Although asthma affects people of all ages, 50% of all asthma cases are diagnosed by the age of 3 yrs and 90% by the age of 6 yrs [88]. Since 1980, data from the National Center for Health Statistics demonstrate that asthma prevalence has increased by y80%, with the self-reported prevalence of asthma in children aged 5–14 yrs rising from 42.8 per 1,000 population in 1980 to 74.4 per 1,000 population in 1994 in the USA [89]. Worldwide, the prevalence of asthma also continues to rise in children and young adults [90, 91]. Although this increase has occurred in all ages of children and young adults, it has been most pronounced in children aged v5 yrs. Asthma is the leading cause of hospitalisation in children and the most common reason for days lost from school [92]. Currently, medications and healthcare utilisation for childhood asthma cost yUS$10 billion annually. Given the dramatic rise in prevalence of both obesity and asthma, it is not surprising that there has been an increasing body of literature relating to the association between BMI and asthma. In the paediatric population, the frequency with which obesity was listed as a secondary diagnosis for asthma-related hospitalisations increased from 5.9% in 1979–1981 to 8.1% in 1997–1999 [23], a rise of nearly 40%. Asthma was the most common principal diagnosis in hospitalisations when obesity was listed as a secondary diagnosis. Population-based subgroup analysis of asthma risk has revealed a similar story. In an analysis of populations at risk of the development of physician-diagnosed asthma, Rodriguez et al. [93] evaluated 12,388 children, aged 2 months to 16 yrs. The highestrisk subgroup identified by signal detection analysis was composed of children with a parental history of asthma or hay fever aged i10 yrs with a BMI i85th percentile (31.0% current asthma). From an epidemiological perspective, increases in BMI have been associated with raised asthma prevalence [94–99], raised asthma incidence [61, 100–102], asthma severity [103, 104] and asthma persistence [105] in children. At the extremes of BMI, a similar increase in the prevalence of asthma has been noted with overweight [93] and obesity in children [104]. Furthermore, this relationship may be influenced by sex. Although greater pre-adolescent asthma incidence [106] and severity [107, 108] have generally been associated with male children, the obesity–asthma relationship appears to be a phenomenon of female children. The risk of prevalent asthma has been demonstrated to be higher in obese female children than nonobese female or male children of any body mass in cross-sectional studies of German [109] and UK [94] children. Additionally, increases in BMI have been associated with incident wheezing, peak flow variability and bronchodilator response in school-aged females but not males [110]. Finally, although 319

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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 [111–114]. 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 BMI–asthma relationship appears stronger in nonatopic than in atopic children [99, 102]. These latter two points indicate possible mechanistic bases for the obesity–asthma relationship.

Obesity and OSA In adults, obesity is clearly the single largest risk factor for the development of OSA [116]. It is important to acknowledge the role of obesity in the development of OSA in children. In children, hypertrophy of the adenoids and tonsils is seen as the most prevalent cause of OSA [117]. However, of children diagnosed with OSA, 10–23% have Table 1. – Selected association studies of obesity–asthma in children Study [Ref.]

Country

NSHG [94]

UK

NHANES III [99]

USA

Subjects n Age yrs 14908

4–11

Design

Parameter

OR/RR (95% CI)

Cross-sectional

Asthma Persistent wheeze (all) Persistent wheeze (females) Persistent wheeze (males) Ever asthma Current asthma Recent wheeze Current asthma

1.28 (1.11–1.48)# 1.57 (1.18–2.07)#

7505

4–17

Cross-sectional

Western Australia Australia Pregnancy Cohort [101] CHS [102] USA

2860

6

Cross-sectional

3792

7–18

Prospective: 5-yr follow-up

Six Cities [61]

9828

6–14

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 (1.33–3.24)# 1.29 (0.89–1.86)# 1.77 (1.44–2.19)} 1.98 (1.54–2.53)} 1.48 (1.24–1.76)} 1.83 (1.34–2.52)z 1.86 (1.14–3.06)§ 1.60 (1.08– 2.36) 1.52 (1.14–2.03) 1.77 (1.26–2.49) 1.16 (0.63–2.15) 2.24 (1.14–4.40)ƒ 1.04 (0.60–1.82)ƒ 3.11 (1.55–6.24)## 3.81 (1.88–7.72)##

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: Children’s 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); ƒ: highest versus lowest quintile (RR); ##: highest versus third quintile (RR). Males in the lowest quintile also showed an increased RR (2.76 (95% CI 1.34–5.67)), supporting a J-shaped relationship between weight gain and asthma. 320

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a)

b)

9 8

6 5

0

n

2 1 3 4 5 Annual change in BMI z-score quintile

n

n

n

n

1

n

n

2

n

3

n

4 n

RR (95% CI)

7

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 1.59–14.15) [119]. In a retrospective review of polysomnographic data from 90 paediatric subjects, obese children showed higher apnoea/hypopnoea indices and, concomitantly, higher systolic and diastolic blood pressure measurements [120]. This suggests an interactive role for obesity with OSA in the development of hypertension. OSA in children may also be an independent risk factor, in combination with obesity, for the development of asthma [121]. In addition to classic OSA, obese children may also suffer from obesity hypoventilation syndrome, which comprises obesity, chronic daytime hypercapnia and hypoxaemia, polycythaemia, hypersomnolence and right ventricular failure [37]. Obesity hypoventilation syndrome is commonly known as Pickwickian syndrome [122], and, although this term was coined in reference to an overweight boy with excessive somnolence described by Charles Dickens in the Pickwick Papers [123], the precise incidence and prevalence of obesity hypoventilation syndrome has not been studied in children.

Physiological and epidemiological associations of obesity with respiratory disease Much can be learned about the effects of obesity on pulmonary function in children from the classic physiology studies performed in adults. In obese adolescents and morbidly obese children of any age, the primary changes noted include decrements in the FRC. FEV1, FVC and TLC decrease with increasing degrees of obesity. In turn, decrements in FRC and low tidal volumes may be associated with the latch state, leading 321

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

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

Obesity and gastro-oesphageal reflux disease Although primarily a mechanical effect in relation to obesity, gastro-oesophageal reflux (GOR) is associated with a multitude of respiratory problems in children. These 322

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include chronic cough, apparent life-threatening events, respiratory depression, interstitial lung disease, recurrent pneumonias, infantile wheezing, vocal cord dysfunction and asthma [126–132]. The estimated prevalence of GOR in asthmatic children ranges 50–60% [133]. Possible mechanisms for GOR-related asthma symptoms include acid-induced bronchoconstriction, by either direct microaspiration or vagally mediated reflex [132]. Medical or surgical therapy of GOR disease results in asthma symptom improvement in y70% of patients [134]. Obesity has been frequently cited as an independent risk factor for GOR and GOR symptoms [135–137]. A recent meta-analysis of studies focusing on the obesity–GOR relationship yielded pooled adjusted ORs for GOR symptoms of 1.43 (95% CI 1.16–1.77) for a BMI of 25–30 kg?m-2 and 1.94 (95% CI 1.47–2.57) for a BMI of w30 kg?m-2 [138]. Mechanically, this effect may be mediated via increased abdominal pressures, which increase the gastro-oesophageal pressure gradient [139, 140]. Both medical [141] and surgical [137] weight loss regimens have been associated with improvement in GOR symptoms. These findings have led to the speculation that GOR might mediate the asthma–obesity relationship [114, 142].

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

Cytokines produced by adipocytes: tumour necrosis factor-a and interleukin-6. Multiple studies have demonstrated associations between tumour necrosis factor (TNF)-a, interleukin (IL)-6, IL-1b, and C-reactive protein and the obese state [145–148]. Moreover, IL-6 and TNF-a have been demonstrated to be constitutively expressed by adipocytes and to correlate with total fat mass [149, 150]. Clearly, asthma is also a disease characterised by inflammation. Although most of the recent focus has been on IL-4 and IL-5 as the primary cytokine mediators of extrinsic (allergic) asthma, there is evidence that the obesity-regulated cytokines may influence airways disease as well. IL-1b has been associated with induction of increased levels of IL-5 from CD4z T-cells [151]. In asthma, upon exposure to allergens, the production of TNF-a increases [152]. In turn, TNF-a increases both IL-4 and IL-5 production [153, 154]. IL-6 production is increased in asthma and has been associated with histamine, IL-4, IL-5, TNF-a and IL-1 stimulation [151, 152, 155]. It has been postulated that IL-6 is responsible for the modulation of immunoglobulin (Ig)E production by IL-4 [156]. Finally, it has been demonstrated that IL-6 causes substantial subepithelial fibrosis in animal models and may be a key modulator of airway remodelling in asthma [157].

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

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Leptin and other factors upregulate adhesion molcules on endotheial cells, leading to monocyte transmigration Leptin Visfatin Adiponectin IL-6 MCP-1 Other factors

Leptin Adiponectin IL-6 MCP-1 Other factors

Increased TNF-a-inhibits adiponectin

TNF-a IL-6 MCP-1 Other cytokines and chemokines (resistin and adipsin)

TNF-a IL-6 MCP-1 Other cytokines (resistin and adipsin)

Obesity Fig. 2. – Adipose tissue: cellular components and molecules produced. Adipose tissue is composed of adipocytes and the stromovascular fraction, which includes macrophages. In the nonobese subject, adipocytes produce leptin, adiponectin, visfatin, interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1 and other factors. Macrophages produce tumour necrosis factor (TNF)-a, IL-6, MCP-1, and other cytokines and chemokines. In human subjects, the ultimate cellular source of adipsin and resistin seems to be the macrophage. In obesity, leptin and possibly other factors produced by adipocytes, macrophages or both upregulate adhesion molecule levels on endothelial cells, leading to transmigration of bone marrow-derived monocytes and, thus, an increase in numbers of white-adipose-tissue-resident macrophages, and higher levels (q) of TNF-a, IL-6 and chemokines compared with those in lean persons. At the same time, adiponectin production by adipocytes is reduced (Q), possibly through upregulated local TNF-a levels. Reproduced from [144] with permission.

[166] and impaired respiratory control, especially during sleep [167]. These findings may have direct relevance to the relationship of obesity with pulmonary function and with OSA. Indeed, a recent study noted that leptin levels are elevated in OSA patients with chronic hypercapnia compared with those who are eucapnic [168]. Although the smaller lung size in the mouse model of leptin deficiency may, at least in part, be related to the mechanical effects of obesity on total lung compliance, the overall effects of leptin on the developing respiratory system are difficult to ignore. Moreover, given the importance of lung size in the aetiology of asthma, understanding the role of leptin in foetal lung development may prove to be very important [92]. From an immunological perspective, leptin is member of the IL-6 family of cytokines [143]. In CD4z T-cells, leptin increases T-helper cell (Th) type 1 and suppresses Th2 cytokine production [169], whereas, in lipopolysaccharide-stimulated macrophages, leptin increases production of TNF-a, IL-6 and IL-12 [170]. In relating obesity to asthma, what is not currently known is whether the relative resistance that occurs in the hypothalamic leptin receptors as part of human obesity extends to the leptin receptors on Th and other cells involved in the asthmatic response. In the brain, the mechanism of leptin resistance is via upregulation of suppressor of cytokine signalling (SOCS)-3, a phosphatase that limits leptin receptor signalling [171]. Since SOCS-3 decreases 324

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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 leptin’s 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 [175–177]. 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 5.9¡3.4 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 (2.06–7.24) ng?mL-1 in the asthmatics and 2.26 (1.26–4.71) ng?mL-1 in the controls (p=0.008). A second case–control study also showed elevations of mean serum leptin level in 23 asthmatic children (19.3¡5.1 ng?mL-1) compared with 20 controls (9.8¡1.6 ng?mL-1; pv0.001) at baseline [182]. Interestingly, after 4 weeks of budesonide treatment, the leptin levels normalised (10.6¡1.6 ng?mL-1). A third case–control 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 case–control study comparing outcomes of 74 very low birthweight versus 64 normal birthweight children at 12 yrs of age, leptin levels were considerably higher in the 27 overweight than in the 111 nonoverweight children (median 18.1 versus 2.8 ng?ml-1; pv0.001) [184]. Interestingly, in the overweight children, current asthmatics showed leptin levels that were twice as high of those of children without current asthma (median 30.8 versus 14.3 ng?ml-1; p=0.14), although this was not the case in the nonoverweight children. Although this difference in overweight was not significant, this was probably due to the small numbers of overweight asthmatics in the cohort.

Other hormonal, peptide and adipokine influences on obesity and respiratory disease. Although the role of leptin has been closely evaluated with regard to obesity and lung disease, several other factors involved in fat regulation may also affect the inflammatory cascade and, subsequently, lung disease. In addition to neuropeptide Y, these include vitamin D, corticotrophin-releasing factor, adiponectin and plasminogen activator inhibitor-1. Each of these are briefly discussed in the following paragraphs. Vitamin D is a pleiotropic hormone. In its active form as 1,25-dihydroxycholecalciferol, its inflammatory effects include augmenting the differentiation of naı¨ve 325

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T-lymphocytes towards a Th2 immunophenotype [185]. Mice deficient in the vitamin D receptor also demonstrate a Th2 cytokine profile [186]. The role of vitamin D in obesity lies in its regulatory effects of increasing intracellular calcium levels. Increased intracellular calcium stimulates lipogenesis and suppresses lipolysis, resulting in increased adiposity [187]. Therefore, increasing vitamin D level could lead to both increased adiposity and increases in the Th2 immunophenotype. However, in two large epidemiological studies of adults, higher serum levels of 25-hydroxycholecalciferol were associated with lower, not higher, measured body fat [188, 189]. These studies did not measure the active form of vitamin D, however, and their serum measurements did not account for the fat solubility of this hormone. Vitamin D has been evaluated in relation to atopy and asthma. In murine models of airway inflammation, exogenous vitamin D administration is associated with significantly increased levels of ovalbumin-specific IgE as well as a shift towards a Th2 immune profile [190]. In a human cohort study evaluating 7,648 Finnish subjects from birth to age 31 yrs, regular supplementation with vitamin D was associated with increased ORs for atopy (1.46 (95% CI 1.4–2.0)) and asthma (1.35 (95% CI 0.99–1.8)) compared with irregular or no supplementation [191]. No study to date has evaluated vitamin D specifically in the obesity–asthma relationship. Corticotropin-releasing factor (CRF) is involved in numerous physiological and behavioural actions, including strong anorectic and thermogenic effects. Indeed, CRF has been proposed as a potential target for the pharmacological treatment of obesity [192, 193]. Not surprisingly, both murine and human models of obesity are associated with decrements in CRF level [194]. Although CRF has not been extensively studied as a risk factor for respiratory disease, in a murine model of asthma, Silverman et al. [195] reported that the CRF knockout mouse demonstrates marked increases in airway inflammation, lung tissue resistance, and IL-4, IL-5 and IL-13 level. The authors concluded that relative CRF deficiency states, as with obesity, might be associated with an increased propensity to develop asthma. Adiponectin is the most abundant adipokine; its primary role is the regulation of insulin sensitivity [196]. Unlike other adipokines, adiponectin levels are depressed in obesity and increase with weight loss [144]. Adiponectin shares structural homology with TNF-a and inhibits production of TNF-a, IL-6, nuclear factor-kB and the endothelial adhesion molecules, intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin [197–200], while inducing IL-10 and IL-1 receptor antagonist [201, 202]. Interestingly, adiponectin inhibits the proliferation and migration of cultured vascular smooth muscle cells [203]. Since adiponectin receptors are also present on airway smooth muscle cells, a direct role for adiponectin in AHR has been hypothesised [75]. Additionally, upregulation of endothelial adhesion molecules, as would be expected with obesity, have been associated with asthma and AHR in human studies [204–207]. Plasminogen activator inhibitor (PAI)-1 is the major endogenous inhibitor of fibrinolysis and plasmin activation. Adipocytes produce and secrete PAI-1, and levels of PAI-1 are increased in obesity [144]. Increased PAI-1 levels, through their effects on extracellular matrix turnover, may predispose towards AHR [75]. This has been demonstrated in murine models of asthma, in which PAI-1 is required for the AHR induced by lipopolysaccharide and for the collagen and fibrin deposition associated with airway remodelling [208, 209]. In summary, it is now clear that obesity is an inflammatory condition and that the adipocyte is active in the cellular production of multiple pro-inflammatory agents, including leptin. Many of these inflammatory agents, in turn, have been associated with providing the appropriate milieu for alterations in lung development, predisposition towards AHR, dysregulation of respiratory control or susceptibility to asthma. 326

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Sex-specific effects of obesity As noted previously, although studies of pre-pubertal children have not noted a consistent sex-related effect, the association between obesity and asthma has been particularly strong in post-pubertal females. In a study of 16,862 children aged 9–14 yrs, BMI correlated with prevalence of asthma in both males and females. Interestingly, asthma risk was inversely related to Tanner stage in males (relative risk (RR) of 0.3 for stage V compared to stage I) but positively related to Tanner stage in females (RR of 1.6 for stage V compared with stage I) [100]. In a longitudinal study of 1,246 children, females, but not males, who had begun puberty and became overweight or obese between the ages of 6 and 11 yrs were 6.8 times (95% CI 2.4–19.4) more likely to develop new wheezing compared to the nonobese females [110]. This was further supported by increases in peak flow variability and salbutamol-responsiveness in the obese females. The sex differences noted in obese asthmatics may simply be a reflection of the increased incidence [210] and prevalence [89, 211, 212] of asthma in adult females of any size. This has been postulated as a primary airway size effect [210]. However, females also appear to have a higher prevalence of AHR than males [213, 214], which persists despite adjustment for airway size. Although the mechanisms behind these associations have yet to be clarified, obesity may amplify these associations via the mechanical effects noted above. Similarly, leptin levels [215–217] are elevated in females compared with their male counterparts and may portend to an enhanced inflammatory state. One other potential reason for the sex difference noted is the sex hormone oestrogen. Post-menopausal hormone replacement therapy (HRT) has been associated with a significantly increased RR of incident asthma in females (1.49 for ever using HRT versus never using HRT) [218]. In adolescent females, elevated oestrogen levels accompany pubertal onset; in turn, pubertal onset in females may be linked to increased BMI [219, 220]. In obesity, although androgen levels are elevated, peripheral aromatisation of androstenedione to oestrone and testosterone to oestrogen occurs within the stroma of adipose tissue [222]. Combined with the decreased sex-hormone-binding globulin levels found in obesity, this results in an oestrogen amplification effect on sensitive tissues [221]. During the menstrual cycle, peak oestrogen levels have been associated with increased symptoms and decreased pulmonary function in asthmatic females [222]. From an immunological perspective, oestrogen administration results in a shift in the immunological reaction from a Th1 to a Th2 type [223], increases IL-4 and IL-13 production from blood monocytes [224], and increases eosinophil recruitment [225] and degranulation [226]. These changes exemplify those typically found in asthma.

Genetic effects of obesity It has long been known that both asthma and obesity are genetic diseases that run in families. The heritability (proportion of phenotypic variance that can be attibutable to genetic factors) of BMI, fat mass and percentage fat mass have been reported to be as high as 90, 65 and 80%, respectively. Similarly, the heritability of asthma diagnosis, AHR and FEV1 have been reported to be as high as 75, 66 and 39%, respectively. Extensive genetic epidemiological studies individually focusing on asthma and lung function phenotypes, as well as on obesity and its related phenotypes, have been performed in recent years. Reviews of the genetic epidemiology of these complex genetic traits are readily available [14, 15, 227–229]. There are several ways in which obesity genes might influence respiratory function and disease pathogenesis. First, genetic studies performed in each of these individual disease states have revealed several candidate genes that have been associated with both obesity and asthma. Secondly, other obesity candidate genes 327

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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 5q31–q32, 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 [234–236]. 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 [246–248]. Increased arm fat in small-for-gestational-age babies has been noted as early as 2–5 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

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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 [256–259]. 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 obesity–asthma association have noted the expected inverse relation and close correlation between physical activity and BMI [266, 267]. Several authors have speculated that the obesity–asthma 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 obesity–asthma 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 [277–279], 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

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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, 279–290]. Although vitamins A and E, carotene, riboflavin and pyridoxine have been associated with reduced lung function and asthma, their role remains controversial [291–293]. 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 [297–299] and increased AHR [285]. Supplementation with vitamin C has been demonstrated to decrease asthma severity and frequency [300], exercise-induced bronchospasm [301] and AHR to methacholine [302].

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

Conclusion: evolving policies addressing the global obesity epidemic Throughout the present chapter, the increasing evidence that obesity in children is associated with the development of respiratory disease through a variety of potential pathophysiological mechanisms has been presented. Recommendations regarding further research in this field have been published [92]. Clearly, adequate prevention and treatment of respiratory disorders in children warrants discussion related to global treatment of the obesity epidemic. Furthermore, since lung function decrements, AHR and asthma in adults may have a primary aetiological basis in childhood, early intervention in the obese child may prevent subsequent morbidity as adults. The World Health Organization has recognised obesity in general, and childhood obesity in particular, as a substantial health problem. Key elements of their recently published "WHO global strategy on diet, physical activity and health" [303] include: 1) achieving energy balance and a healthy weight; 2) limiting salt, sugar and total fat intake and shifting consumption towards polyunsaturated fats; 3) increased consumption of fruits, vegetables, legumes, and whole grains and nuts; 4) engaging in daily moderate physical activity for i30 min; 4) creating supportive policies that promote education and availability and accessibility of low-fat high-fibre foods and that provide opportunities for physical activity; and 5) addressing prevalent obesity through clinical programmes and staff training. Other national panels have made similar recommendations, with an emphasis on early recognition and prevention of obesity, given the recognised difficulties in treating established obesity [304]. Many of these panels emphasise the importance of identifying 330

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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 obesity–respiratory 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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New approaches to the understanding of complex chronic lung diseases U. Frey*, G.N. Maksym#, M. Silverman}, B. Sukiz *Paediatric Respiratory Medicine, Dept of Paediatrics, University Hospital of Bern, Bern, Switzerland. # School of Biomedical Engineering, Dalhousie University, Halifax, NS, Canada. }Institute for Lung Health, Leicester University, Leicester, UK. zDept of Biomedical Engineering, Boston University, Boston, MA, USA. Correspondence: U. Frey, Paediatric Respiratory Medicine, Dept of Paediatrics, University Hospital of Bern, Inselspital, 3010 Bern, Switzerland. Fax: 41 316329484; E-mail: [email protected]

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, 345–360. Printed in UK - all rights reserved. Copyright ERS Journals Ltd 2006; European Respiratory Monograph; ISSN 1025-448x.

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a) Trigger

Inflammation

Bronchial hyperactivity

Airway obstruction

Symptoms

b) Long-term impact

Genetic background Growth and development Information storage

Time-varying noisy environmental stimuli (allergens, infections, drugs, pollutants, etc.) Short-term impact

Complex nonlinear dynamic respiratory system

Fluctuating symptoms of disease

Functional modules Metabolic pathways

Short term

Memory effects Long term

Inflammatory Immunological Mechanical Neurorespiratory control

Fig. 1. – a) An analytical reductionist approach and b) a system approach to the complex behaviour of asthma. The classical paradigm of asthma is based on the concept of an inducer or trigger causing airway inflammation, leading to bronchial hyperactivity and airway obstruction and resulting in symptoms (a). Although this analytical approach provides insight into the behaviour of mechanisms involved in cascades of reaction, it cannot explain all of the phenomena in a complex chronic disease such as asthma. Systems biology attempts to understand the behaviour of complete biological systems based on network considerations (b) [4]. This alternative systems approach to asthma is presented as an example of a fluctuating chronic disease with multiple components. It is proposed that, in such a complex system, the fluctuating output or symptoms are the result of noisy environmental inputs (such as allergen exposure, infection, air pollution, etc.), but modified by many interacting components of various subsystems (inflammatory system, immune system, lung mechanics, neurorespiratory control, etc.), each having internal feedback loop mechanisms. Some of these feedback mechanisms may act slowly over medium or long timescales of hours, weeks or months (e.g. the immune system), and some over short timescales of seconds or minutes (e.g. neurorespiratory control). It is important to note that such systems store information (memory) and can behave in a highly nonlinear manner. Thus, small perturbations may lead to exaggerated peak responses (e.g. in fatal asthma). In this model, even genetic adaptation to environmental pressure (i.e. evolutionary process) can be seen as a long-term effect [4]. Individual components of such systems cannot be described in detail, but statistical techniques are available for describing their overall properties.

e.g. in fatal asthma [1], why bronchial hyperactivity may persist for months after a single allergen exposure [2] or why people with occupational asthma may have persistent symptoms long after they have ceased to be exposed to the relevant environment [3]. Such behaviour cannot be accounted for by the classical paradigm of asthma being caused by a series of events consisting of a trigger leading to inflammation leading to bronchial hyperactivity, airway obstruction and finally symptoms (fig. 1a). 346

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The present authors propose that this and other clinical examples suggest that, during the course of asthma, and possibly also under healthy conditions, the respiratory system, with its multitude of components and interactions, exhibits stochastic behaviour coupled with complex nonlinear dynamics, leading to substantial fluctuations in physiological and clinical variables. The dynamic properties of this system are strikingly analogous to other networks in physics and biology. The current state of the system is not independent of its history, implying that the temporal patterns of such systems exhibit memory effects. Previous events often have a long-lasting effect, as evidenced by long-range correlation [5, 6]. Understanding the functioning of such systems and predicting certain events, such as an asthma attack, are not possible within the realm of reductionism and require a probabilistic systems approach, which is the main topic of the present chapter.

State of the art In the present chapter, examples of recent research based on a comprehensive systembased approach are presented. Specifically, an overview of the behaviour of the lung in the framework of network phenomena is provided, and it is demonstrated that its fluctuating temporal physiological behaviour can be an expression of a homeokinetically regulated dynamic nonlinear system.

Lung structure Fractal airway network structure: implications for disease progression. The threedimensional structure of the airways is a well-known example of a complex fractal structure. Fractals are self-similar structures, in which magnified subparts resemble the entire structure (fig. 2a) [7]. With regard to the airway tree, the self-similarity is manifest in a branching pattern that repeats itself over multiple length scales [8–10]. 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 [11–14]. 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 347

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Time Fig. 2. – Fractal properties of a) a structure, the bronchial tree and b) a time series, a fluctuating physiological signal. The bronchial tree is typical of a fractal structure in which the geometry is similar over different length scales or generations. The mean diameter as a function of length scale follows a power law (fig. 3). The diameter ratio between successive generations is constant (scale invariance). In an ideal fractal time series, the fluctuations at different timescales are similar. In biological time series, this similarity can only be described on a statistical level.

feature of a power-law distribution is that its tail is very long (note the logarithmic scale in fig. 3), compared with familiar distributions such as the Gaussian distribution. Since the tail of a power law can be orders of magnitude larger than the tail of a Gaussian distribution, the probability of a large or rare event occurring in a power-law model is also orders of magnitude higher. Most distributions have a characteristic value (or scale), such as the value corresponding to the peak of the Gaussian distribution. Power-law 348

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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

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Fig. 5. – Emphysematous clusters in chronic obstructive pulmonary disease: computed tomographic image of the lung of an emphysematous patient. Colour clusters represent contiguous areas of low attenuation, where gas exchange is already compromised. Note the significant spatial heterogeneity and considerable size variability of the colour clusters. The size distribution of these clusters also follows a power law, and the exponent is sensitive to emphysema progression. Reproduced from [29] with permission.

is merely the superposition of their respective separate outputs. The situation is drastically different in nonlinear systems since even a small input acting on a nonlinear system in the presence of another input can induce dramatic and unexpected changes in the system, with large subsequent fluctuations in its output variables [32]. This is perhaps one of the reasons why it is so difficult to describe the general properties of complex nonlinear systems [33]. Furthermore, in a spatially extended nonlinear system, such as a complex network, there can be multiple inputs acting at different sites and scales. Often one or more of the external perturbations are not known. They too may fluctuate widely. An example is the effect of day-to-day or seasonal variations in pollen count, pollution levels or infections when coupled with immune mechanisms, inflammation, and the neurological control and mechanics of breathing, which can have unpredictable effects on the clinical status and well-being of asthmatics. The results are significant short- and long-term fluctuations in the output variables of the system (airway obstruction, wheeze and dyspnoea, etc.). Even if all of the inputs are known, since physiological systems are rarely 351

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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.

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

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

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thought that measurements of short-term variations in airway resistance may be a useful additional diagnostic parameter in asthma [40]. Airway resistance can be measured quickly and without disturbing the respiratory system by using the forced oscillation technique [40, 46]. With this technique, airway resistance can be obtained over several breaths, and the magnitude of the variation can be assessed using the sd or logarithm of sd of several resistance measurements. Variation is elevated in asthma in measurements obtained at frequencies of 6 Hz in adults [40] and 10 Hz in children [47]. Furthermore, preliminary data show that administration of the bronchodilator salbutamol to asthmatic children aged 6–13 yrs decreases the variation in resistance in children at 4– 14 Hz (unpublished data). Variation in resistance was the only measure that changed significantly, since forced expiratory volume in one second, forced mid-expiratory flow and mean airway resistance were unaltered. This implies that the variation in resistance reflects changes in airway smooth muscle activity. Conversely, Que et al. [40] showed that methacholine challenge led to increased variation in resistance in healthy adults. Variation in activation of airway smooth muscle could be due to many factors, including changes in agonist deposition, changes in relaxation following constriction and even changes in the load acting on the muscle [48], all of which can vary throughout the airway tree. Indeed, spatial heterogeneity of airway constriction is a well-established feature of asthma, measured by both forced oscillation and imaging techniques [25, 47, 49]. Thus, short-term variation in airway resistance is likely to be the result of time-varying spatial heterogeneity of airway constriction. However, variation in airway resistance can also occur due to other factors, such as changes in glottis aperture and changes in the number of open airways during a breath, as well as changes in lung volume with breathing. Indeed, lung volume is a potent determinant of airway resistance, and thus its variation must also strongly affect variations in resistance [50, 51]. Que et al. [40] showed that changing from the upright to supine position resulted in a significant increase in variation in airway resistance, which was associated with a decrease in lung volume and airway diameter. Changing lung volume can have two effects on variations in airway resistance. A decrease in volume can lead to unloading of airway smooth muscle, which allows any variation in airway smooth muscle activation to translate into larger changes in airway diameter, or a decrease in volume with no change in smooth muscle activation leads to an increased variation in airway resistance due to a geometric effect caused by the inverse fourth power dependence of resistance on diameter. The preliminary data mentioned above and obtained in children show that both probably occur. The principal advantage of measurement of resistance variation using the forced oscillation technique is that it is simple for children to perform, requiring only a mouthpiece, and, in principle, could be adapted to a face mask. If a 3-min series of measurements of variation in airway resistance is related to severity of disease, then this could be a powerful clinical tool for determining adequate therapy in asthma. However, before studies are undertaken to assess the use of short-term variability in improving clinical outcomes, the role of lung volume in airway diameter variation, as well as the effect of control of breathing on airway diameter, must first be established. Nevertheless, although the direct mechanisms may be different and the subsequent fluctuations unrelated, variation in airway function from short to long timescales appears to be ubiquitous in asthma. Similar to physical systems, the transition from short- to long-term variability occurs smoothly, and it is likely that both result from the interactions between environmental fluctuations and memory effects of the dynamic nonlinear system.

Long-term fluctuations and variability in respiratory airway function over days and weeks. Asthma is a chronic disease of the airways with multiple long-acting influences, such as inflammation and immunological and mechanical (remodelling) mechanisms. The 353

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complex interactions between endogenous and environmental factors result in a highly variable pattern of airway obstruction over time. Fluctuations in airway calibre result in episodic symptoms of wheeze, dyspnoea or cough. Long-term fluctuations in asthma can, for example, be seen in series of daily symptoms scores or daily lung function measurements. Understanding and predicting such fluctuations is difficult not only because environmental stimuli are not always recognisable and simple to quantify, but also because the correlation between stimuli and symptoms is poor [1, 52, 53]. The lack of a deterministic relationship between a stimulus and its outcome is an essential feature of complex dynamic systems. In many complex systems, these fluctuations exhibit longrange correlations, which are typically scale-invariant and thus show similar statistical properties over different timescales (fig. 2b). In the time domain, processes with scaleinvariant correlation properties are called fractal processes. It has recently been shown that the day-to-day lung function fluctuations in chronic asthma exhibit fractal properties characterised by long-range correlations [54]. Often fractal processes arise when signals travel through structures which themselves have spatial fractal scaling. An example is the His–Purkinje system in the heart [55]. In this regard, it is important to note that fractal properties are found in both the structure and function of the respiratory system. An example of a twice daily peak expiratory flow (PEF) series [54] of 6 months’ duration is shown in figure 6a. It can be seen that PEF behaves in a highly variable fashion but not randomly over time. In order to analyse such PEF series, the temporal pattern was investigated in 80 asthmatic subjects [54] who had taken part in a long-term clinical trial [56]. In particular, it was examined whether the statistical and correlation properties of the time series of PEF recordings could be used to predict the risk of a subsequent episode of asthma. It was found that the fluctuations in a series of daily PEF measurements exhibited fractal-type long-range correlations. These correlation properties of the signal can be characterised by so-called detrended fluctuation analysis [5, 57], which measures the extent to which mean variations in the past affect variation in the present. Often the correlation function follows a power law, which allows the complex behaviour to be characterised with a simple number, a, the exponent of the power law (fig. 6b). An a of 0.5 represents a random uncorrelated process; the higher a is, the more tightly correlated the time series and the greater the degree of internal memory. It was found that the a of the PEF series correlated with asthma severity [54]. What are the consequences of these findings? In a temporal process with long-range correlations, the amplitudes of fluctuations that have occurred in the past influence current values. However, the influence of previous fluctuations decreases as the time interval increases. The value of a, and hence the strength of these long-range correlations, is influenced by the integrative contribution of inflammation and immunological and mechanical (remodelling) mechanisms. Thus, asthma, as a chronic disease, behaves as a complex nonlinear system with internal memory properties related to disease severity. The clinical surrogate for this memory of asthma, embodied in the immune system, chronic airway inflammation and structural remodelling, may be manifest as persistent bronchial hyperactivity after a single allergen challenge [2], persistent symptoms following stimulus removal in occupational asthma [2, 3, 58] or persistent remodelling, and inflammation after clinical remission of asthma [52, 53]. Although it is impossible to disentangle all of the individual components of the system and its memory properties, it is possible to describe the overall dynamic behaviour of the disease in a comprehensive integrative manner using a systems approach. Internal long-range correlations are also important to the stability of the system. By knowing both past PEFs, embodied in the memory (a and coefficient of variation of the PEF series), it is possible to calculate the probability or risk (more precisely, the conditional probability) that, given the current PEF, a severe episode of asthma (for example, with a PEF v60% of the predicted value) will occur within a given time period 354

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(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.

Current models and concepts Alternative approaches to the respiratory system and chronic respiratory diseases have been presented, considering the respiratory system as a complex dynamic system with 355

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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.

Important future questions Although the systems approach may be seen as a strategy complementary to the conventional analytical reductionist approach, it naturally opens up new possibilities and questions. 1) Is the reductionist asthma paradigm of "trigger plus bronchial hyperresponsiveness leads to bronchoconstriction" still valid? The systems approach adds two new dimensions: the effects of past events memorised in the system and the nonlinear reactions between past and current stimuli. Such considerations might also aid better understanding of the mechanisms of symptom relapse after disease remission [52, 53]. 2) Could the concept of programming [59–61] of lung function in disease be better understood within the framework of the systems approach? Could key programmers, for example, maternal smoking during pregnancy, which affect lung growth and development in early life, not just be understood as very long-acting mechanical memory effects? Could immune deviation early in life not just be understood as very long-acting immunological memory effects in the development of allergy. 3) Is it sufficient to design research studies with two-point observation based on initial conditions and outcomes? It would be better to design studies with long-term serial observations in order to better understand the dynamics of the disease process, which might permit statistical prediction of exacerbations, as demonstrated in [54]. 4) Could different phenotypes of a chronic disease (e.g. brittle asthma and episodic or chronic asthma) be better defined using the systems approach? By describing long-range correlations, stability and the predictability of future events, the overall properties of the system can be used to classify subjects. The hypothesis that these clinical phenotypes were important could then be tested by determining their natural history, epidemiology and response to treatment. 5) Would parameters related to the systems approach be useful additional outcome measures in drug trials? Outcomes could be determined more rapidly, and hence more cheaply, as the result of intensive observations if the short-term analysis [52] predicts longer-term responses. 6) Can the systems approach help to provide hypotheses for experimental (reductionist) science and vice versa?

What is needed to answer these questions? Application of the systems approach to medicine is still in its infancy, and a series of studies are required to establish its clinical significance. However, it is important to learn 356

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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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