Effects of Growth and Development on Lung Function
Models for Study of Childhood Asthma

WILLIAM W. BUSSE, SUSAN P. BANKS-SCHLEGEL, and GARY L. LARSEN

Division of Allergy and Clinical Immunology, Department of Medicine, University of Wisconsin, Madison, Wisconsin; Division of Lung Diseases, National Heart, Lung and Blood Institute, Bethesda, Maryland; and National Jewish Medical and Research Center and Section of Pediatric Pulmonary Medicine, University of Colorado School of Medicine, Denver, Colorado

	INTRODUCTION

TOP INTRODUCTION REFERENCES

A previous NHLBI workshop, Childhood- versus Adult-Onset Asthma (1), raised the possibility that important differences exist between asthma in children and adults. Furthermore, it was suggested that investigation into childhood asthma would provide unique insights into asthma on a mechanistic and therapeutic level. Moreover, a concept has begun to emerge to indicate that the development of asthma in childhood is influenced by intrauterine as well as environmental and genetic factors that express themselves early in life and ultimately determine not only the onset of asthma but also its persistence and severity. Therefore, childhood asthma may be the critical window of research opportunity to establish the pathogenesis of asthma and its development.

Major advances toward the pathogenesis of asthma have come from study of adult asthma and have formed the basis of the hypothesis that airway inflammation is an essential component of asthma and a critical factor in its pathogenesis. Although the clinical features of asthma are similar in adults and childhood, studies to establish the features of airway inflammation in childhood asthma are lacking and, thus, an important clue to mechanism of disease in the child is missing.

A knowledge of the normal growth and development of the lung as well as other systems of the body that influence lung function is important to fully understand certain disease processes. In this respect, many pulmonary diseases that primarily involve the airways begin early in life and may have part of their pathogenesis tied to a disruption of normal developmental processes. For example, asthma commonly presents within the first few months to years of life (2), involves both large and small airways, and is felt to be modulated by immune/inflammatory responses within the host. Scientific knowledge is incomplete regarding the ontogeny of airway function (including airway responsiveness), immune responses, and inflammatory reactions to both immunologic and nonimmunologic stimuli. In this respect, a better understanding of the normal effects of growth factors with age, greater knowledge about developmental aspects of immune responses, and more information about changes in neural control mechanisms within airways with growth may be central to understanding this dynamic process (3). Because children are the primary "model" for studies of this nature, clinical investigations addressing pathogenesis at the onset of the disease have been limited in number and difficult to conduct. In addition, information on the pathology of asthma in children of various ages is limited in terms of the numbers of observations available for review (4). To establish the scope of this problem, define the primary needs in terms of where more scientific knowledge is needed, and gather information on the instruments that are available for studies of this type, a workshop was convened. The central focus of this meeting was to review developmental processes of lung growth that are of potential relevance to asthma. In addition to a primary objective of helping to define research priorities, the goal of the workshop was to facilitate communication and future collaborations among laboratories that are interested in the developmental aspects of airway disease.

	OVERVIEW OF LUNG GROWTH AND DEVELOPMENT

Lung development is under the influence of many factors, including transcriptional molecules to control growth and cell differentiation. Since many of these influences occur early in utero, the development of lung growth is likely well established by the time the child is born. From studies of lung development, it has been noted that a central feature in normal airway function is the influence of factors that reduce surface tension in the conducting airways and maintain their patency. Abnormalities in surfactant activity early in life are an example where changes in lung function can be predicted by knowledge and function of this surface tension reducing factor. Furthermore, "knock-out" and transgenic animal models have provided unique opportunities to determine the influence of various genes on lung growth, development, and ultimate function. Because abnormalities in the regulation of lung growth and development have had important influence on lung function, it is logical to assume that asthma will be similarly affected, and understanding these interrelationships will prove to be critical to improved understanding of asthma.

	MATURATION OF MECHANICAL DETERMINANTS OF AIRWAY RESPONSIVENESS

Airway hyperresponsiveness is a defining characteristic of asthma, although the mechanisms for the heightened responsiveness remain unclear. Furthermore, recurrent wheezing may not be a manifestation of airway hyperresponsiveness or asthma in infancy. Therefore, to evaluate the relationships between airway hyperresponsiveness and asthma in infants, it is important to understand the mechanisms for changes in airway responsiveness that occur between infancy and adulthood. Various studies suggest that airway responsiveness declines between infancy and adulthood in normal subjects (5). These findings strongly suggest that there are maturational differences in airway responsiveness, and differences in lung size and inhaled dose of agonist do not necessarily account for the observed maturational differences in airway reactivity among infants, children, and adults. Yet it remains unclear whether alterations in lung mechanics early in life, either congenital or acquired, contribute to the heightened airway responsiveness early in life and the persistence of airway hyperresponsiveness throughout childhood and adulthood.

	RISK FACTORS IN THE DEVELOPMENT OF ASTHMA

New evidence has emerged during the last few years that strongly suggests that events occurring during fetal and early postnatal life play a decisive role as risk factors for the development of asthma. It has been recently shown that sensitization to aeroallergens may occur in utero. There is also strong evidence suggesting that maternal asthma and maternal allergic rhinitis are more strongly associated with the risk of developing sensitization to aeroallergens and asthma in children than are paternal asthma and paternal allergic rhinitis. It is thus possible that interactions occur between the mother's immune system and the child's immune system prenatally, and that this interaction may predispose the infant to the development of allergic disease and asthma. Furthermore, it has been shown that, even among subjects who do not have asthmatic symptoms in early life, serum IgE levels and eosinophil counts measured before the age of 1 yr predict the development of asthma later in life. Particularly striking in this regard are differences in eosinophilic responses to viruses during infancy among subjects who subsequently develop asthma and those who do not. Early onset asthma is also associated with significant deficits in lung function, which are not observed for these same children before they start having asthma symptoms. Thus, the hypothesis has been proposed that asthma is a disease in which the patterns of maturation of the lungs and the immune system are altered early in life. The relationship of lung growth and development to this process is not clearly established. These alterations may trigger the first asthmatic attacks, and these attacks, by establishing the inflammatory phenotype that is characteristics of asthma, may predispose for changes in lung structure and function that are the basis for the self-perpetuating chronic nature of the disease.

	ROLE OF RESPIRATORY INFECTIONS ON LUNG GROWTH AND DEVELOPMENT

In children, allergen exposure and viral infections are two of the most common factors capable of producing changes in lung function and are responsible for much of the morbidity attributed to asthmatic exacerbations (8). A common manifestation by which both allergen exposure and viral infections could induce airway obstruction is through the augmentation of cytokine production, which results in upregulation of resident or inflammatory cell functions, which then contributes to alteration in airway physiological responses. In a rat model for virus-induced airway dysfunction, structural and functional changes can be produced that vary depending upon the age at the time of infection and the strain of animal being infected (11, 12). Furthermore, serial measurements of lung function and evaluation of lung histopathology reveal that viral respiratory infections in rats can produce an asthma-like syndrome that is characterized by increased airway inflammation (increased mast cells and eosinophils), alterations in airway resistance and dynamic compliances, and enhanced airway responsiveness (13, 14). Based upon observations such as these, it has been proposed that the susceptibility to virus-induced chronic airway dysfunction is controlled at the level of cytokine response to the infecting virus, i.e., high gene expression of IL-4 and IL-5 in airways and low gene expression of IL-2 and interferon-. Furthermore, these observations suggest that the development of respiratory viral infections, in a genetically susceptible individual, may be critical in determining the eventual outcome of the infection and also the likelihood for the development of persistent asthma.

	IMPORTANCE OF NEUROREGULATION IN GROWTH AND DEVELOPMENT

Control of airway function may vary significantly as a function of age. For example, it has been shown that postnatal maturation in the piglet enhances contractile responses of airway smooth muscle exposed to cholinergic stimulation (15) or tachykinins, such as substance P (SP) and neurokinin A (NKA). Maturational changes as well as alterations with age in receptor affinity for an agonist and G protein coupling may all contribute to these physiologic changes (16). Furthermore, the role of various modulators of cholinergically mediated airway contractile responses and the location of these responses may vary with development. In this respect, histamine has a differential distribution of contractile responses in the developing piglet in that the dominant effect is in the lung parenchyma during early postnatal life but involves both airways and lung parenchyma at later states of development (17). Insults to the airway early in postnatal life may also influence the response to factors that modulate airway function. For example, preliminary studies suggest that when maturing rat pups are exposed to hyperoxic stress, there is a significant increase in preprotachykinin gene expression in the lungs (18). Consequent production of SP can lead to enhanced cholinergic sensitivity of tracheal smooth muscle in this model. Relaxant responses within the lung may also vary as a function of age and the insults to which the airways are subject (19).

	GROWTH AND DEVELOPMENT OF AIRWAY SMOOTH MUSCLE

Mechanisms that potentially contribute to age-dependent differences in airway smooth muscle (ASM) function have been identified: (1) airway acetylcholinesterase activity is reduced in immature swine compared with older animals; thus, the relative intensity of acetycholinergic stimulation may be enhanced in younger animals; (2) the myosin content of airways throughout the lung has been reported to increase (20) or decrease (21) with age in pigs, though both studies concluded that the ratio of SM2 to SM1 isoforms of smooth muscle myosin heavy chain increases with aging; (3) cell surface membrane receptors and channels may change with age; for example, the affinity of muscarinic receptors for muscarinic agonists may decrease with age (22); (4) signal transduction may change with age, as immature rabbit tracheal smooth muscle exhibits greater basal and carbachol-stimulated inositol (1,4,5)-triphosphate levels (23); and (5) calcium sensitivity of the contractile apparatus of tracheal smooth muscle from fetal and suckling pigs exceeds that of tissue from older animals by several-fold (20).

Given the complex and apparently disparate way in which development can alter these aspects of isolated airway function in animal models, it is imperative to determine the physiologic relevance of findings in animals for human airway function by direct study of human tissues. This is especially true in light of the substantial heterogeneity of function recently identified among individual ASM cells isolated from individual airways of animals. Once identified, it may then be challenging to determine how these structural and functional changes with development influence in vivo bronchial reactivity.

	REGULATION OF IgE SYNTHESIS IN MAN AND ROLE OF IGE IN ASTHMA

In addition to respiratory infections, the development of IgE sensitivity is also critical to the pathogenesis of asthma. Studies of the regulation of IgE synthesis indicate that the B-cell antigen CD40 is unique in its capacity to deliver a signal, which, in conjunction with cytokines, induces isotype switching, and this process involves a recombination between repetitive switch sequences located upstream of the genes encoding for the immunoglobulin heavy chains, in this case, IgE. At the present, the molecular mechanisms for CD40 signaling that lead to an isotype switch remain unclear. Nonetheless, tyrosine kinases and other associated proteins appear to be critical in this whole process. Because this event occurs early in life and is an important risk factor for the development of asthma, the interaction and regulation of IgE synthesis is a key factor in the development of allergic disease and asthma. The influence of age and interrelationship with other environmental factors in this process is, at present, poorly understood.

	CELLULAR IMMUNITY AND THE REGULATION OF AIRWAY FUNCTION

T cells expressing activation markers have been identified in the peribronchial tissues and bronchoalveolar lavage fluid (BALF) in asthma (24). The mechanisms by which these lymphocytes initiate, contribute to, and/or sustain the immune/ inflammatory responses seen in this disease are not entirely clear. However, T cells are essential for immunologic memory, cytokines from T cells such as IL-4 (and IL-13) are essential for IgE synthesis, and T lymphocytes also contribute to the development of the inflammatory response through the elaboration of a number of cytokines, among which IL-5 may be pivotal in terms of the eosinophil response in the lungs of the sensitized host. Whether cytokine production by other cell types (e.g., mast cells and basophils) is sufficient to replace T cells in this function is not known. Although evidence indicates the importance of T cells to development and regulation of airway inflammation, it is essential to establish the effects of host age and maturation on their function and possible cytokine pattern (25).

	GENETIC INFLUENCE IN THE DEVELOPMENT OF AIRWAY DISEASE

A genetic component in asthma is suggested by familial aggregation and in twin concordance rates (28). Although asthma is difficult to characterize, several features closely associated with this disorder are easily measured and objectively defined, including bronchial hyperresponsiveness and the presence of atopy. In pursuit of this question, various chromosomal regions have been studied that have candidate genes which are relevant to asthma and allergy (29). Linkages to chromosome 5q, which contains numerous candidate loci including the IL-4 cytokine group, and 12q, where there are genes for interferon- and nitric oxide synthase, are important areas for investigation. Evidence has been shown that genetic loci that determine a significant portion of the observed biological variability in bronchial responsiveness and serum IgE were co-inherited, and these traits have been mapped to chromosome 5q 31-q33.16; furthermore, preliminary data suggest that a major gene for susceptibility to asthma maps to this same region. Thus, significant progress has been made toward identifying genes important in susceptibility to asthma, with the delineation of several chromosomal regions showing linkages to asthma and associated intermediate phenotypes. This information not only shows a genetic basis characteristic of asthma but also may be important in determining the factors important in the regulation and eventual expression of clinical asthma.

	INTERACTIONS OF ALLERGEN SENSITIZATION AND NEUROREGULATION

Studies in experimental models of airway disease have demonstrated several alterations of the airways' neural control in response to allergen sensitization. Prominent among these changes is an alteration in prejunctional mechanisms leading to an imbalance in neural regulation of airway smooth muscle. Employing well-defined models of airway inflammation (allergen-induced asthmatic responses in mice and rabbits), various changes in cholinergic as well as nonadrenergic, noncholinergic (NANC) responses have been defined. These include increased in vitro airway responsiveness to electrical field stimulation of nerves (25, 33), enhanced release of acetylcholine from cholinergic nerves (34), functional loss of M₂ muscarinic autoreceptors (34, 35), loss of the NANC inhibitory system (33), and increased airway smooth muscle contractile responses to SP (36). Under these conditions, an imbalance between excitatory and inhibitory mechanisms is produced that may contribute to the development of altered airway caliber and/or responsiveness. It is interesting to note that observations in asthma as well as other mammalian models of human disease have also implicated similar alterations in the airways' mechanisms of neural control (35, 37).

Despite the potential importance of neural mechanisms in the pathogenesis of airway diseases, limited data exist on the distribution and functional status of autonomic nerves in developing airways. Furthermore, little is known about the effects of environmental stimuli on mechanisms of neural control in young animals. Recent evidence suggests the existence of significant age-related changes in autonomic regulation of functions within the airways. In addition, it appears that events occurring early in life may disrupt normal maturational processes and lead to abnormalities that persist in fully grown animals (38).

	THE USE OF TRANSGENIC ANIMALS TO STUDY THE PATHOGENESIS OF ASTHMA

The use of overexpression transgenic technology to characterize the effects on airway pathology and physiology of cytokines implicated in the asthmatic diathesis has provided invaluable insights. For example, IL-11 is a cationic IL-6-type cytokine produced by epithelial cells, airway smooth muscle cells, and fibroblasts in response to other cytokines (IL-1 and TGF-), histamine, major basic protein, and respiratory tropic viruses (respiratory syncytial virus [RSV], rhinovirus [RV], and parainfluenza virus [PIV-3]). It induces airways hyperresponsiveness (AHR) in vivo and can be found in exaggerated quantities in the nasal secretions of children with viral upper respiratory tract infections. To characterize its effects when chronically expressed, the clara cell 10 kD protein (CC10) promoter was used to overexpress IL-11 in the murine airway. The airways of the transgene animals manifest a number of abnormalities including: (1) peribronchiolar mononuclear infiltrates composed largely of B cells and CD4⁺ and CD8⁺ T cells; and (2) bronchial remodeling with subepithelial fibrosis with enhanced deposition of type III and, to a lesser extent, type I collagen and the enhanced accumulation of fibroblasts, myofibroblasts, and myocytes beneath normal basement membranes. Physiologic evaluation demonstrated elevated levels of baseline airways resistance and AHR to methacholine. These animals have features that are reminiscent of aspects of human asthma and animal models of virus-induced inflammation and AHR.

The role that a particular protein plays in the pathogenesis of airway inflammation and AHR can be readily evaluated via the generation of animals with targeted gene disruptions. A large number of animals have already been produced that are relevant to the asthmatic diathesis. Limitations of this approach include the frequent occurrence of embryonic lethality and the possible induction of cellular pathways of redundancy that may or may not be physiologically relevant. To resolve these issues, approaches such as the cre-loxP system are being developed that will hopefully produce organ-specific, temporally regulatable, targeted gene disruptions.

	THE IMPORTANCE OF GENETIC ABNORMALITIES IN ADRENERGIC FUNCTION TO THE PATHOGENESIS OF ASTHMA

The structure of the ₂-adrenergic receptor (₂AR) varies within the human population due to polymorphisms at four loci within the coding block (39, 40). The significance of this finding has been pursued along three lines of investigation: determination of the relevance of polymorphisms to receptor function, delineation of the mechanism by which these structural changes alter receptor function, and assessment of relationships between polymorphic variants and asthma or its treatment. The ₂AR polymorphisms consist of single amino acid variations at residues 16 (Arg or Gly), 27 (Gln or Glu), 34 (Val or Met), and 164 (Thr or Ile), with the first residue in each pair representing the "wild-type." The latter two polymorphisms are in transmembrane spanning regions I and IV, respectively, while the former two are in the extracellular amino-terminus. No polymorphism is uniquely associated with asthma, with each occurring with similar frequencies in normal subjects and patients with asthma.

With a clearer understanding of the receptor phenotypes, studies have been performed to assess their role in adult asthma. The possibility that they may dictate certain asthmatic phenotypes, severity of disease, bronchial hyperreactivity, or response to -agonist therapy has been considered. In addition, tachyphylaxis to chronic -agonists, which is an area of concern since it may be associated with a loss of control and poor outcome, may be related to ₂AR genotype, given that two of the polymorphisms alter agonist-promoted downregulation. In studies to date (39, 41), these notions appear to be true. In pediatric asthma, much less is known about ₂AR responsiveness and chronic -agonist use. No studies assessing the physiologic or clinical relevance of ₂AR polymorphisms in pediatric asthma have been carried out nor is information available on the developmental aspects of this regulatory process.

	INSIGHTS IN AIRWAY INJURY FROM STUDIES OF CYSTIC FIBROSIS

Cystic fibrosis (CF) is characterized by airway infection and an intense neutrophilic inflammation. A series of studies involving invasive procedures in older patients with mild disease and in very young patients has revealed marked abnormalities at a very early stage of disease with respect to neutrophil-dominated inflammation and infection. A study of patients greater then 12 yr of age who had normal pulmonary function and did not produce sputum revealed infection with traditional CF pathogens in each case (44). In addition, patients also had increased neutrophils in the airway as assessed by bronchoalveolar lavage (57% in patients with CF versus 3% in control subjects). This study demonstrated that clinically stable patients with normal lung function and no sputum production still have marked abnormalities within the lung's microenvironment. A study in younger children confirmed the presence of neutrophilia as well as proinflammatory cytokines in toddlers and school-age children (45). Thus, it appears that the airway microenvironment in infants and young children with CF is similar to that in older patients as determined by sputum as well as bronchoalveolar lavage. These findings demonstrate that even in the absence of clinical signs of disease, abnormalities are still present within the microenvironment of the lung. Moreover, the suppurative lung disease of later life is initiated in infancy, possibly in the absence of infection. Studies of this nature may provide surrogate markers for future interventions in early infancy. Proposed new mechanisms for pathogenesis will require testing in infants and young children before many of the secondary effects of CF airways disease take place.

	STUDIES OF CHILDHOOD ASTHMA

Various techniques might be used to address the immunopathogenesis of childhood asthma, including, but not limited to, bronchoscopy with lavage and/or endobronchial biopsy. Thus far, bronchoalveolar lavage in children with asthma is limited and has been applied primarily to older children with this disease (46, 47). However, recent experience utilizing this invasive procedure to study other airway diseases in infants and preschool children, including CF (48) as well as bronchipulmonary dysplasia (49) and recurrent infantile wheezing (50), suggests this might be accomplished in a safe manner and yield important information regarding disease pathogenesis closer to the onset of disease.

Recent experience has been reported in terms of characterizing the cellular environment of the lungs of normal infants and small children using lavage obtained by nonbronchoscopic techniques at the time of endotracheal intubation for elective surgery (51). In addition, preliminary work employing the same technique has been done on atopic children with asthma and children with virus-associated wheeze (52). Children with asthma appear to have significant increases in eosinophils and mast cells recovered by lavage when compared with both normal subjects and those with virus-associated wheeze.

	RECOMMENDATIONS FOR FUTURE DIRECTIONS

Based upon the presentations and discussions of the topics reviewed above, several recommendations were made in terms of future directions. They include the following:

1. Better insight into the natural history of asthma is needed. For example, longitudinal studies that assess the importance of the age at which allergen sensitization occurs in relation to the persistence of the syndrome could shed light on asthma pathogenesis and persistence into adult life. Emphasis might be placed on enrolling families where both parents have asthma, and thus, a high percentage of their offspring may develop the disease.
2. The influence of growth and development on the response to insults and repair to the airways early in life should also receive emphasis. Clinically, the most important insults currently identified appear to be viral respiratory infections, allergens, and the environmental pollutant cigarette smoke. Of these insults early in life, the potential for interaction between viral respiratory infections and allergens appears especially important, as well as host factors. While observations in infants and children are necessary, mammalian models of disease will again be important in addressing mechanistic questions. Given the potential vulnerability of developing airways to insults in early life, emphasis should be placed on defining the sequelae of insults at an early age in terms of their potential to produce prolonged airways dysfunction. The elucidation of these mechanisms will improve our knowledge of factors capable of influencing the structure and function of airways after early insults. In children at risk for airways disease, potential insults commonly occur together. Therefore, more attention should be focused on the interactions of insults in terms of their ability to act synergistically to alter airway function and, as noted above, interactions of viral respiratory infections with potential allergens need to be addressed in more detail in terms of the potential for an infection to enhance allergic sensitization as a function of postnatal development.
3. The importance of intrauterine exposure to potential allergens in terms of the consequences to the child should be defined. While observations in humans can be made, the ability to address questions in a mechanistic manner and to manipulate this system in terms of maneuvers to increase or decrease in utero exposure is limited. In this respect, the use and continued development of mammalian models alongside clinical studies will be critical.
4. Asthma often starts early in life. Yet studies to establish features of airway disease in childhood asthma are lacking. Little in the way of research bronchoscopies are performed in the pediatric population. A workshop is needed to develop guidelines and establish a policy statement that relates to the use of invasive techniques to study childhood asthma. In particular, guidelines and experience in bronchoscopy/ BAL/biopsy in normal children, those with stable asthma, and those with asthmatic flares need to be defined. A similar effort to assess the safety of these procedures in adults with asthma laid the initial groundwork for safely applying these techniques to study the pathogenesis of asthma in adults (53). As part of the deliberations, the formidable problems of providing informed consent as well as the need to include normal groups of subjects must be addressed.
5. Studies are also needed to identify early markers of airway inflammation that may reflect disease activity in blood and/ or urine. Ideally, when working with children, this marker should be easily assessed and correlate with disease activity as defined by more invasive procedures or with tests of lung function. In infants and young children, a more accurate assessment of the natural history of asthma will rely on reliable markers of disease activity. Moreover, there is a need for standardization of the measurements obtained in studies involving infants and children. This applies to physiologic assessments as well as the invasive procedures that may be applied in clinical studies to address pathogenesis.
6. Pathophysiology of airways obstruction needs to be explored. There are different mechanisms of airway obstruction. Thus, the physiological and biochemical basis for the airways obstruction needs to be defined, as well as its location, extent of reversibility, and whether the mechanisms differ between children and adults.
7. Approaches to primary prevention in childhood asthma, such as environmental interventions versus immune modulation, need to be investigated.
8. A major issue in childhood asthma relates to the role that viral infection in early years plays in the asthmatic diathesis. Available data show a prominent association between pediatric viral infection and the subsequent development of asthma. It is not known, however, if this association involves cause and effect. Cellular and molecular mechanisms by which viruses, individually and in combination with allergen, regulate airway inflammation and airway wall remodeling and alter the process of immune sensitization in atopic and nonatopic individuals and appropriate animal models need to be determined. The functional consequences of virus-induced alterations in airways inflammation and airway wall remodeling also need to be characterized.

	Footnotes

Correspondence and requests for reprints should be addressed to Susan P. Banks-Schlegel, Ph.D., Senior Scientific Advisor, Airway Biology and Disease Program, Division of Lung Diseases, National Heart, Lung and Blood Institute, Two Rockledge Centre, Suite 10018, 6701 Rockledge Drive, MSC 7952, Bethesda, MD 20892-7952.

(Received in original form December 30, 1996 and in revised form March 11, 1997).

	References

TOP INTRODUCTION REFERENCES

1. Busse, W., S. Banks-Schlegel, and G. L. Larsen. 1995. Childhood- versus adult-onset asthma. Am. J. Respir. Crit. Care Med. 151: 1635-1639 [Medline].

2. Yunginger, J. W., C. E. Reed, E. J. O'Connell, L. J. Melton III, W. M. O'Fallon, and M. D. Silverstein. 1992. A community-based study of the epidemiology of asthma: incidence rates, 1964-1983. Am. Rev. Respir. Dis. 146: 888-894 [Medline].

3. Hislop, A. A., J. Wharton, K. M. Allen, J. M. Polak, and S. G. Haworth. 1990. Immunochemical localization of peptide-containing nerves in human airways: age-related changes. Am. J. Respir. Cell Mol. Biol. 3: 191-198 .

4. Richards, W., and J. R. Patrick. 1965. Death from asthma in children. Am. J. Dis. Child. 110: 4-23 .

5. Montgomery, G. L., and R. S. Tepper. 1990. Changes in airway reactivity with age in normal infants and young children. Am. Rev. Respir. Dis. 142: 1372-1376 [Medline].

6. Tepper, R. S.. 1987. Airway reactivity in infants: a positive response to methacholine and metaproterenol. J. Appl. Physiol. 62: 1155-1159 [Abstract/Free Full Text].

7. Tepper, R. S., J. Stevens, and H. Eigen. 1994. Heightened airway responsiveness in normal female children compared with adults. Am. J. Respir. Crit. Care Med. 149: 678-681 [Abstract].

8. Castleman, W. L., R. L. Sorkness, R. F. Lemanske Jr., and P. K. McAllister. 1990. Viral bronchiolitis during early life induces increased numbers of bronchiolar mast cells and airway hyperresponsiveness. Am. J. Pathol. 137: 821-831 [Abstract].

9. Cypcar, D., J. Stark, and R. F. Lemanske, Jr. 1992. The impact of respiratory infections on asthma. In D. A. Stempel and S. J. Szefler, editors. The Pediatric Clinics of North America. W. B. Saunders Company, Philadelphia. 1259-1276.

10. Duff, A. L., E. S. Pomeranz, L. E. Gelber, H. W. Price, H. Farris, F. G. Hayden, T. A. E. Platts-Mills, and P. W. Heymann. 1993. Risk factors for acute wheezing in infants and children: viruses, passive smoke, and IgE antibodies to inhalant allergens. Pediatrics 92: 535-540 [Abstract/Free Full Text].

11. Sheth, K. D., R. L. Sorkness, J. J. Clough, P. K. McAllister, W. L. Castleman, and R. F. Lemanske Jr.. 1994. Reversal of persistent postbronchiolitis abnormalities with dexamethasone in rats. J. Appl. Physiol. 76: 333-338 [Abstract/Free Full Text].

12. Sorden, S. D., and W. L. Castleman. 1995. Virus-induced increases in airway mast cells in Brown Norway rats are associated with enhanced pulmonary viral replication and persisting lymphocytic infiltration. Exp. Lung Res. 21: 197-213 [Medline].

13. Sorkness, R., R. F. Lemanske Jr., and W. L. Castleman. 1991. Persistent airway hyperresponsiveness after neonatal viral bronchiolitis in rats. J. Appl. Physiol. 70: 375-383 [Abstract/Free Full Text].

14. Sorkness, R., J. J. Clough, W. L. Castleman, and R. F. Lemanske Jr.. 1994. Virus-induced airway obstruction and parasympathetic hyperresponsiveness in adult rats. Am. J. Respir. Crit. Care Med. 150: 28-34 [Abstract].

15. Rodriguez, R. J., I. A. Dreshaj, G. Kumar, M. J. Miller, and R. J. Martin. 1994. Maturation of the cholinergic response of tracheal smooth muscle in the piglet. Pediatr. Pulmonol. 18: 28-33 [Medline].

16. Haxhiu-Poskurica, B., P. Ernsberger, M. A. Haxhiu, M. J. Miller, L. Cattarossi, and R. J. Martin. 1993. Development of cholinergic innervation and muscarinic receptor subtypes in piglet trachea. Am. J. Physiol. 264(Lung Cell. Mol. Physiol. 8):L606-L614.

17. Dreshaj, I. A., M. A. Haxhiu, C. F. Potter, F. H. Agani, and R. J. Martin. 1996. Maturational changes in responses of tissue and airway resistance to histamine. J. Appl. Physiol. 81: 1785-1791 [Abstract/Free Full Text].

18. Agani, F. H., R. J. Martin, C. F. Farver, I. A. Dreshaj, J. E. Krause, M. A. Haxhiu, and C. H. Chang. 1995. Increased preprotachykinin gene expression in lungs of newborn rats exposed to hyperoxia. Pediatr. Res. 37: 323A .

19. Jakupaj, M., R. J. Martin, I. A. Dreshaj, C. F. Potter, and M. A. Haxhiu. 1996. Nitric oxide (NO) modulates contractile responses of airway smooth muscle during early postnatal life (abstract). Pediatr. Res. 39: 335A .

20. Sparrow, M. P., and H. W. Mitchell. 1990. Contraction of smooth muscle of pig airway tissues from before birth to maturity. J. Appl. Physiol. 68: 468-477 [Abstract/Free Full Text].

21. Murphy, T. M., R. W. Mitchell, A. Halayko, J. Roach, L. Roy, E. A. Kelly, N. M. Munoz, N. L. Stephens, and A. R. Leff. 1991. Effect of maturational changes in myosin content and morphometry on airway smooth muscle contraction. Am. J. Physiol. 260(Lung Cell. Mol. Physiol. 4):L471-L480.

22. Rothberg, K. G., P. L. Morris, and J. S. Douglas. 1987. Characterization of cholinergic muscarinic receptors in cow tracheal muscle membranes: effect of maturation. Biochem. Pharmacol. 36: 1687-1695 [Medline].

23. Rosenberg, S. M., G. T. Berry, J. R. Yandrasitz, and M. M. Grunstein. 1991. Maturational regulation of inositol 1,4,5-triphosphate metabolism in rabbit airway smooth muscle. J. Clin. Invest. 88: 2032-2038 .

24. Gelfand, E. W., and G. L. Larsen. 1996. Airway hyperresponsiveness: induction by T cells. In S. B. Liggett and D. A. Meyers, editors. The Genetics of Asthma. Marcel Dekker, Inc., New York. 17-37.

25. Larsen, G. L., H. Renz, J. E. Loader, K. L. Bradley, and E. W. Gelfand. 1992. Airway response to electrical field stimulation in sensitized inbred mice: passive transfer of increased responsiveness with peribronchial lymph nodes. J. Clin. Invest. 89: 747-752 .

26. Garssen, G., F. P. Nijkamp, H. V. D. Vliet, and H. V. Loveren. 1991. T-cell-mediated induction of airway hyperreactivity in mice. Am. Rev. Respir. Dis. 144: 931-938 [Medline].

27. Renz, H., K. Bradley, J. Saloga, J. Loader, G. L. Larsen, and E. W. Gelfand. 1993. T cells expressing specific V elements regulate IgE production and airways responsiveness in vivo. J. Exp. Med. 177: 1175-1180 [Abstract/Free Full Text].

28. Lander, E. S., and N. J. Schork. 1994. Genetic dissection of complex traits. Science 265: 2037-2048 [Abstract/Free Full Text].

29. Marsh, D. G., J. D. Neely, D. R. Breazeale, B. Ghosh, L. R. Freidhoff, E. Ehrlich-Kautzky, C. Schou, G. Krishnaswamy, and T. H. Beaty. 1994. Linkage analysis of IL-4 and other chromosome 5q 31.1 markers and total serum immunoglobulin E concentrations. Science 264: 1152-1156 [Abstract/Free Full Text].

30. Meyers, D. A., D. S. Postma, C. I. M. Panhuysen, J. Xu, P. J. Amelung, R. C. Levitt, and E. R. Bleecker. 1994. Evidence for a locus regulating total serum IgE levels mapping to chromosome 5.  Genomics 23: 464-470 [Medline].

31. Shirakawa, T., A. Li, M. Dubowitz, J. W. Dekker, A. E. Shaw, J. A. Faux, C. Ra, W. O. C. M. Cookson, and J. M. Hopkin. 1994. Association between atopy and variants of the  subunit of the high-affinity immunoglobulin E receptor. Nature Genet. 7: 125-130 [Medline].

32. Postma, D. S., E. R. Bleecker, P. J. Amelung, K. J. Holroyd, C. I. M. Panhuysen, D. A. Meyers, and R. C. Levitt. 1995. Genetic susceptibility to asthma: bronchial hyperresponsiveness coinherited with a major gene for atopy. N. Engl. J. Med. 333: 894-900 [Abstract/Free Full Text].

33. Fame, T. M., G. N. Colasurdo, J. E. Loader, J. P. Graves, and G. L. Larsen. 1994. Decrease in the airways' nonadrenergic noncholinergic inhibitory system in allergen sensitized rabbits. Pediatr. Pulmonol. 17: 296-303 [Medline].

34. Larsen, G. L., T. M. Fame, H. Renz, J. E. Loader, J. Graves, M. Hill, and E. W. Gelfand. 1994. Increased acetylcholine release in tracheas from allergen-exposed IgE-immune mice. Am. J. Physiol. 266(Lung Cell. Mol. Physiol. 10):L263-L270.

35. Fryer, A. D., and M. Wills-Karp. 1991. Dysfunction of M₂-muscarinic receptors in pulmonary parasympathetic nerves after antigen challenge. J. Appl. Physiol. 71: 2255-2261 [Abstract/Free Full Text].

36. Colasurdo, G. N., J. E. Loader, J. P. Graves, and G. L. Larsen. 1995. Substance P-induced contraction of airway smooth muscle in normal and allergen-sensitized rabbits: mechanisms of action. J. Appl. Physiol. 78: 428-432 [Abstract/Free Full Text].

37. Minette, P. A., J. W. Lammers, C. M. Dixon, M. T. McCusker, and P. J. Barnes. 1989. A muscarinic agonist inhibits reflex bronchoconstriction in normal but not in asthmatic subjects. J. Appl. Physiol. 67: 2461-2465 [Abstract/Free Full Text].

38. Colasurdo, G. N., J. E. Loader, J. P. Graves, and G. L. Larsen. 1994. Maturation of nonadrenergic noncholinergic inhibitory system in normal and allergen-sensitized rabbits. Am. J. Physiol. 267(Lung Cell. Mol. Physiol. 11):L739-L744.

39. Reihsaus, E., M. Innis, N. MacIntyre, and S. B. Liggett. 1993. Mutations in the gene encoding for the ₂-adrenergic receptor in normal and asthmatic subjects. Am. J. Respir. Cell Mol. Biol. 8: 334-339 .

40. Liggett, S. B. 1996. The genetics of ₂-adrenergic receptor polymorphisms: relevance to receptor function and asthmatic phenotypes. In S. B. Liggett and D. A. Meyers, editors. The Genetics of Asthma. Marcel Dekker, Inc., New York. 455-478.

41. Turki, J., J. Pak, S. A. Green, R. J. Martin, and S. B. Liggett. 1995. Genetic polymorphisms of the ₂-adrenergic receptor in nocturnal asthma: evidence that Gly16 correlates with the nocturnal phenotype. J. Clin. Invest. 95: 1635-1641 .

42. Hall, I. P., A. Wheatley, P. Wilding, and S. B. Liggett. 1995. Association of Glu27 ₂-adrenoceptor polymorphism with lower airway reactivity in asthmatic subjects. Lancet 345: 1213-1214 [Medline].

43. Ohe, M., M. Munakata, N. Hizawa, A. Itoh, I. Doi, E. Yamaguchi, Y. Homma, and Y. Kawakami. 1995. ₂-adrenergic receptor gene restriction fragment length polymorphism and bronchial asthma. Thorax 50: 353-359 [Abstract/Free Full Text].

44. Konstan, M. W., K. A. Hilliard, T. M. Norvell, and M. Berger. 1994. Bronchoalveolar lavage findings in cystic fibrosis patients with stable, clinically mild lung disease suggest ongoing infection and inflammation. Am. J. Respir. Crit. Care Med. 150: 448-454 [Abstract].

45. Wilmott, R. W., J. T. Kassab, P. L. Killian, W. R. Benjamin, S. D. Douglas, and R. E. Wood. 1990. Increased levels of interleukin-1 in bronchoalveolar washings from children with bacterial pulmonary infections. Am. Rev. Respir. Dis. 142: 365-368 [Medline].

46. Ferguson, A. C., and F. W. M. Wong. 1989. Bronchial hyperresponsiveness in asthmatic children: correlation with macrophages and eosinophils in broncholavage fluid. Chest 96: 988-991 [Abstract/Free Full Text].

47. Ferguson, A. C., M. Whitelaw, and H. Brown. 1992. Correlation of bronchial eosinophil and mast cell activation with bronchial hyperresponsiveness in children with asthma. J. Allergy Clin. Immunol. 90: 609-613 [Medline].

48. Khan, T. Z., J. S. Wagener, T. Bost, J. Martinez, F. J. Accurso, and D. W. H. Riches. 1995. Early pulmonary inflammation in infants with cystic fibrosis. Am. J. Respir. Crit. Care Med. 151: 1075-1082 [Abstract].

49. Yoder, M. D., R. Chua, and R. Tepper. 1991. Effect of dexamethasone on pulmonary inflammation and pulmonary function of ventilator-dependent infants with bronchopulmonary dysplasia. Am. Rev. Respir. Dis. 143: 1044-1048 [Medline].

50. Galoppin, L., J. de Blic, I. Azevedo, P. Scheinmann, B. B. Vargaftig, and M. Bachelet. 1994. Nonspecific refractoriness to adenylyl cyclase stimulation in alveolar macrophages from infants with recurrent bronchiolitis. J. Allergy Clin. Immunol. 93: 885-890 [Medline].

51. Heaney, L. G., E. C. Stevenson, G. Turner, I. S. Cadden, R. Taylor, M. D. Shields, and M. Ennis. 1996. Investigating paediatric airways by non-bronchoscopic lavage: normal cellular data. Clin. Exp. Allergy 26: 799-806 [Medline].

52. Heaney, L. G., M. D. Shields, I. S. Cadden, E. C. Stevenson, R. Taylor, and M. Ennis. 1994. Non-bronchoscopic lavage of paediatric airways: evidence for ongoing inflammation in childhood asthma. Clin. Exp. Allergy 244: 991 .

53. Goldstein, R. A., and S. S. Hurd. 1985. Summary and recommendations of a workshop on the investigative use of fiberoptic bronchoscopy and bronchoalveolar lavage in asthmatics. Am. Rev. Respir. Dis. 132: 180-182 [Medline].