INTRODUCTION

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Asthma is the most common chronic childhood disease in developed nations (1, 2). While most asthma originates in childhood (3), the natural history of asthma in childhood is poorly understood and the primary causes remain unknown. One of the most consistently reported physiologic associations of childhood asthma has been increased airway responsiveness to an inhaled agonist (4, 5), although the relationship with symptoms is inconsistent (6).

There is increasing evidence that pathophysiologic changes manifest early in life are important in the etiology of childhood asthma and other respiratory diseases (7). Very few studies have followed children from before birth to early childhood and evaluated factors related to the emergence of clinical symptoms of atopy, asthma, and lower respiratory tract illness (LRTI). None have included an assessment of airway responsiveness in infancy and in early childhood. Previous research has shown that normal infants demonstrate airway responsiveness to histamine soon after birth (8), and exhibit a range of responsiveness by 1 mo of age (9). We hypothesized that increased airway responsiveness in early life may predate symptoms resulting from airway inflammation and allergy. In this study, we report the relationships between airway responsiveness at 1 mo of age and the development of asthma, atopy, symptoms of LRTI, and decreased lung function by 6 yr of age in a prospective cohort with extensive lung function data collected in early life (9).

	METHODS

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The 253 Western Australian subjects comprising the original cohort were randomly recruited from a metropolitan hospital before birth during the years 1987 to 1991 (9). Subjects were all white, born at full term, and none had major congenital anomalies. Infants were first assessed at approximately 1 mo of age; none had previously had a LRTI or any clinically important nonrespiratory illness at this time (9).

One hundred twenty children were seen as near as was practicable to their sixth birthday. The follow-up rate was thus 47.4%, but because 100% of families able to be contacted agreed to participate in the follow-up, the relatively low follow-up rate could be attributed to the difficulty of maintaining a current contact address over 6 yr with a mobile population of young families. In each subject, all parameters were assessed during one laboratory visit at 1 mo and another at 6 yr.

Informed consent was obtained from the parents of all children at both time points. This study was conducted with the approval of the medical ethics committee of the Princess Margaret Hospital for Children, Perth, Western Australia.

Data collection at 1 mo of age have been previously described (9). Spirometry was assessed by determining the maximal flow at functional residual capacity (max_FRC) using the rapid thoracic compression technique (8). To assess response to histamine airway challenge at 1 mo, doubling concentrations of nebulized histamine from 0.125 g/ L to a maximum of 8.0 g/L were administered (9). A new concentration was delivered every 5 min during tidal breathing and respiratory function was assessed after each concentration, with a minimum of five forced expirations at each measurement. The challenge was ended when max_FRC fell by at least 40% or when the maximal concentration was reached. The provocation concentration of histamine producing a 40% decrease (PC₄₀) in max_FRC was estimated from the log dose-response curve. Individuals who had not responded by the maximal dose of histamine were assigned the value PC₄₀ = 16.0 g/L (i.e., twice the maximal dose).

At the 6-yr follow-up, spirometry was performed with a portable spirometer (Model 61000; Welch Allyn, Skaneateles Falls, NY). FEV₁ and FVC were measured according to the guidelines of the American Thoracic Society (10). Spirometric indices at age 6 are expressed both as raw values and as percent predicted values (11). Parents were requested to withhold bronchodilator or prophylactic medication from the children for appropriate periods (dependent upon the specific medication) before spirometric testing. Response to histamine challenge was assessed by the Yan rapid-inhalation technique (12) in subjects whose FEV₁ was  70% of predicted. The maximal cumulative dose given was 7.8 µmol histamine. The dose-response slope (DRS) of histamine response was calculated by the methods of O'Connor and coworkers (13).

Skin reactivity at 1 mo was measured to the following allergens: Dermatophagoides farinae, perennial ryegrass pollen, cow's milk, and egg white (Hollister-Stier, Elkhart, IN). Skin reactivity at 6 yr was measured for the same allergens plus Dermatophagoides pteronyssinus, mixed grass, cat dander, and dog dander (Hollister-Stier, Elkhart, IN). All tests were read after 15 min. A positive reaction was defined as a weal  2 mm and larger than a negative control.

Total serum immunoglobulin E (IgE) was measured with the IgE fluoroenzymeimmunoassay (FEIA) CAP system (Pharmacia Diagnostics, Uppsala, Sweden) from cord blood and from blood taken at the time of the 6-yr follow-up. Peripheral blood eosinophil counts at age 6 are expressed as absolute cell counts.

At both 1 mo and 6 yr, individual and family histories of respiratory symptoms, diagnoses, and exposures were assessed by a modified American Thoracic Society questionnaire administered to a parent (generally the mother). At 6 yr, an outcome of asthma "ever" was defined as 1 or more episodes of physician-diagnosed asthma by age 6. Current asthma was defined as the presence of current, symptomatic, physician-diagnosed asthma requiring medication. "Wheeze (ever) with colds" was defined as an affirmative answer to the question to "Has his/her chest ever sounded wheezy or whistling when they have a cold?" "Wheeze (ever) apart from colds" was defined as an affirmative answer to the question to "Has his/her chest ever sounded wheezy or whistling apart from colds?" "Usual cough" was defined as an affirmative answer to the question "Does he/she normally have a cough?" Environmental tobacco smoke exposure was defined from parental questionnaire as both current maternal smoking and maternal smoking "ever."

Statistical analysis investigated associations of PC₄₀ to histamine at 1 mo with the following outcomes at age 6: diagnosis and current symptom status with regard to asthma, wheeze and cough; total and specific serum IgE levels; blood eosinophil counts; FEV₁; FVC; and the DRS to histamine.

Total serum IgE levels, the blood eosinophil count and the DRS at 6 yr, PC₄₀, max_FRC, and at 1 mo, and cord IgE at birth were all skewed with a long right-hand tail, and were log_e transformed before analysis. A constant was added to each DRS measurement at age 6 yr to allow log_e transformation when the DRS was  0. Although PC₄₀ is technically a censored variable, only 5 of 95 (5.3%) infants did not respond to the histamine challenge by maximal dose. Consequently, PC₄₀ exhibited an approximate log-normal marginal distribution in this population and was analyzed as log_e PC₄₀.

Sex, maternal smoking at 1 mo, and family history of asthma were analyzed as binary variables. Outcomes of asthma, wheeze, and usual cough at age 6 were analyzed as binary variables. Skin reactivity to specific allergens was analyzed as a binary variable ( 1 positive reaction, 0 positive reactions), as was type of medication in current use by children with active asthma (regular bronchodilator use only, regular bronchodilator use plus prophylactic medication use). All other variables were analyzed as continuous.

Bivariate analyses were performed using unpaired Student's t tests (two-tailed, equivariance not assumed) and Pearson's correlation coefficients (14).

Generalized linear models (logistic and linear regression) (15) were constructed to investigate the relationships and predictive value of log_e PC₄₀ at 1 mo to outcomes of interest at age 6. Multiple regression models were constructed in a logical and systematic manner starting with covariates found to be significant in the bivariate analyses. Checks of goodness of fit (15) included an investigation of the need for interaction or polynomial terms, analyses of Pearson residuals, and examination of the effect of observations with high regression leverage. Definitive final models were shown to provide a valid summary of the observed data.

Analysis and data management was carried out using Minitab for Windows v12 (Minitab Inc., State College, PA) and SPlus v4.5 (Mathsoft Inc., Cambridge, MA). Formal statistical significance was defined at the conventional 5% level.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Comparison of infant data for children participating in the 6-yr follow-up with the data from the children lost to follow-up suggested no significant differences for any of the parameters of interest (Table 1). PC₄₀ testing was successfully completed on 95 of the infants followed at age 6 (79.2%). The characteristics of the study population at age 6 are given in Table 2. All of the 26 children with current, symptomatic physician-diagnosed asthma at age 6 were using prescribed "bronchodilator" medication (₂-adrenergic receptor agonists); a further 13 children were also using prescribed medications commonly defined as "prophylactic," comprising Intal (sodium cromoglycate) or inhaled steroids, or both.

Cross-sectional Analysis of Airway Responsiveness and Other Factors Assessed at 1 mo

At 1 mo, bivariate analysis indicated that log_e PC₄₀ was not significantly associated with log_e max_FRC, log_e cord total IgE level, positive skin reactivity, sex, a positive family history of asthma, or maternal smoking status.

Cross-sectional Analysis of Airway Responsiveness and Other Factors Assessed at 6 yr

At 6 yr, bivariate analysis indicated that increased log_e DRS was associated with decreased level of FEV₁ (r = 0.20, p = 0.04), with increased levels of log_e total serum IgE (r = 0.30, p = 0.01), and with increased log_e eosinophil counts (r = 0.21, p = 0.04). An increased log_e DRS was also associated with the presence of current physician-diagnosed asthma (t₂₂ = 2.72, p = 0.01), physician diagnosis of asthma ever (t₃₇ = 2.43, p = 0.02), and wheezing both with (t₃₉ = 2.18, p = 0.04) and without (t₁₅ = 2.46, p = 0.03) colds. Log_e DRS at age 6 was not significantly associated with FVC levels, usual cough, a positive family history of asthma, the use of prophylactic medication in addition to bronchodilator medication in those children with current asthma, sex, maternal smoking status (current or ever), or positive skin reactivity.

Relationship of Airway Responsiveness at 1 mo and Outcomes at 6 yr

Bivariate analyses indicated that increased airway responsiveness at 1 mo was associated with the development of physician-diagnosed asthma (ever) (t₆₉ = 2.11, p = 0.04), current physician-diagnosed asthma (t₃₉ = 2.68, p = 0.01), usual cough (t₅₀ = 2.24, p = 0.03), and wheeze apart from colds (t₁₂ = 3.00, p = 0.01) by age 6. Multiple logistic regression (Table 3) confirmed the association of log_e PC₄₀ with current asthma, asthma (ever), wheeze apart from colds and usual cough by age 6. Although bivariate analysis did not indicate a formally significant relationship of log_e PC₄₀ with wheeze with colds by age 6 (t₆₇ = 1.73, p = 0.09), multivariate analysis did indicate a significant association (Table 3). Note that an odds ratio (OR) < 1.0 in Table 3 indicates association of an outcome with decreasing log_e PC₄₀, i.e., increasing airways responsiveness. Analysis of the PC₄₀ and DRS dichotomized at their respective median values indicated that proportions of asthma were lowest in those subjects with low response to histamine challenge at both 1 mo and 6 yr and highest in the subjects with high response at both 1 mo and 6 yr (Table 4).

Bivariate analysis indicated that log_e PC₄₀ at 1 mo was associated with both FEV₁ percent predicted (r = 0.35, p = 0.001) (Figure 1) and FVC percent predicted (r = 0.31, p = 0.003), but not with the log_e DRS to histamine (r = 0.02, p = 0.82) at 6 yr. Multiple linear regression confirmed the relationship of log_e PC₄₀ at 1 mo with FEV₁ and FVC (Table 5) and the lack of relationship with the log_e DRS (data not shown). Analysis of the PC₄₀ and DRS dichotomized at their respective median values indicated that mean FEV₁ percent predicted levels were highest in those subjects with low response to histamine challenge at both 1 mo and 6 yr and lowest in the subjects with high response at both 1 mo and 6 yr (Table 4).

View larger version (12K): [in this window] [in a new window]   Figure 1.   Loge PC40 at 1 mo against FEV1 (percent predicted) at 6 yr in a cohort of Western Australian children (n = 95).

Bivariate analysis indicated that log_e PC₄₀ at 1 mo was not significantly associated with log_e eosinophil counts (r = 0.13, p = 0.24), log_e total serum IgE levels (r = 0.08, p = 0.55), or positive skin reactivity (t₈₉ = 0.22, p = 0.83) at 6 yr. Multiple linear and logistic regression models confirmed the lack of association of log_e PC₄₀ at 1 mo with any of the atopy-associated factors assessed at 6 yr (data not shown).

All reported significant associations of log_e PC₄₀ with outcomes of interest derived from multivariate modeling (Tables 3 and 5) were independent of sex, family history of asthma, log_e cord IgE, maternal smoking status at 1 mo or 6 yr, log_e max_FRC at 1 mo, skin reactivity or length 1 mo, drug therapy for asthma at 6 yr or height at 6 yr. In particular, log_e max_FRC was not a significant independent predictor of any of the outcomes investigated. A family history of asthma was closely associated with all of the outcomes investigated at age 6, with the exception of the DRS; associations of log_e PC₄₀ with outcomes of interest were unaffected if this covariate was included in the multivariate models (Tables 3 and 5). Significant interactions of PC₄₀ with other factors measured at one mo such as sex, smoking, and max_FRC were not found.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Our prospective cohort study is the first to involve both an assessment of airway responsiveness soon after birth and a follow-up in childhood. This study demonstrates that airway responsiveness to inhaled histamine in early life predicts the development of asthma, lower respiratory tract symptoms, and reduced spirometric indices by school age. Of all the variables we measured in infancy, no other variable showed such consistent associations with important clinical and physiologic outcomes at age 6.

Importantly, there were no significant differences at 1 mo of age in the parameters of interest between those children followed and not followed at 6 yr of age (Table 1), suggesting that those followed were representative of the initial study group. This was not unexpected, given that each subject for whom we had a current contact address participated in the study and it is unlikely that family mobility would be related to airway responsiveness at 1 mo. However, the finding that there were no formally significant differences between 1-mo parameters in those followed and not followed does not exclude the possibility of selection bias.

Important limitations of this study include the lack of longitudinal data during childhood up to the 6-yr follow-up in the children studied. This is particularly true with regard to physiologic measurements of lung function and atopy during the first 6 yr of life. Unfortunately, there are no practical ways of measuring lung function in children between the ages of approximately 6 mo and 5 to 6 yr; children in this age range are too developmentally mature to be sedated and studied with the "tidal volume rapid thoracic compression" technique used at 1 mo in our study and are not generally able to complete independent spirometric tests. Data regarding disease and symptom history collected from questionnaires are subject to well-known limitations regarding recall bias and recollection loss (16); this limitation may have biased our study in unknown ways, and would have particularly affected the maternal questionnaires administered at the 6-yr follow-up. Further, we did not have longitudinal data on smoke exposure or use of asthma medication during childhood. Finally, by challenging our subjects with histamine at both time points we investigated only one dimension of the "airways responsiveness" phenotype; accumulating evidence suggests that several different types of challenge may be necessary to fully elucidate this phenotype in asthma (17).

To demonstrate consistency with previous findings regarding the association of maternal smoking with spirometric measures in infants and children, the relationship of max_FRC to maternal smoking at 1 mo was investigated. While not formally significant, there was some evidence of a relationship (t₈₄ = 1.77, p = 0.08) between log_e max_FRC and maternal smoking at 1 mo. This finding and the finding that FEV₁ at 6 yr was significantly associated with maternal smoking (Table 5) were consistent with the results of previous studies (18).

Wheezing-related LRTI is very common in early childhood; approximately 30% of all children have at least one occurrence by 1 yr of age (22). Whereas most children who wheeze in early childhood stop wheezing by age 6 ("transient wheezers"), a subpopulation at high risk of developing asthma will continue to wheeze at school age ("persistent wheezers") (23). These two groups have not been easy to distinguish in infancy (23), although a longitudinal study by Martinez and colleagues indicated that decreased spirometry in infancy was present only in a group of transient wheezers (24). This was consistent with our finding that max_FRC measured at 1 mo was not a significant predictor of asthma, wheeze, or cough at age 6.

Our data (9) and those of others (26) indicate that infants exhibit a range of airway responsiveness by 1 mo of age. Previous studies have suggested that airway responsiveness in infancy does not play a fundamental role in the pathogenesis of wheezing LRTI in the first year of life (27). The evidence regarding the association between airway responsiveness measured in later childhood and the subsequent development of asthma in childhood has been inconsistent (30). Our study is the first to suggest that increased airway responsiveness in infancy identifies a subgroup of children at increased risk of asthma and wheeze by school age, and may be the earliest physiologic reflection of a fundamental predisposition to childhood asthma. Our results therefore suggest that there are important functional differences in the airways from early life.

This study provides further evidence implicating the important influence of events in early development on physiologic function and the development of asthma in later childhood (3, 25, 31). Increased airway responsiveness in early life is likely to result from complex interactions between genetic susceptibilities (35) and in utero and early exposure to environmental factors (31, 36). Studying healthy infants at such an early age, before the occurrence of any LRTI, is likely to have minimized the effect of environmental insults on airway responsiveness, particularly the effects of viral infections (37) and passive exposure to environmental tobacco smoke. Our study thus also suggests that the airway responsiveness-associated predisposition precedes exposure to viral respiratory infections, consistent with previous studies suggesting that viral LRTIs are not primary factors in the pathogenesis of childhood asthma (37). Factors such as exposure to viral infections and passive exposure to maternal tobacco smoke are likely to act as adjuvant factors that increase the risk of asthma in susceptible individuals throughout childhood.

In an earlier analysis of the first 63 subjects to be recruited into this cohort (9), maternal smoking during pregnancy and a family history of asthma in primary relatives were associated with increased airway responsiveness at 1 mo. We were unable to confirm these findings in the full cohort of 253 subjects with the statistical analyses described. The differing results found in the current study may be due both to the use of differing statistical methods and to the differing sizes of the two cohorts studied (9).

The lack of association between airway responsiveness at 1 mo and at 6 yr suggests that factors that contribute to airway responsiveness in early life may be different from those that contribute to airway responsiveness in later childhood. This is biologically plausible, as there are clear age-dependent changes in immune responses and in airway and lung geometry throughout childhood that are likely to influence nonspecific airway responsiveness (6). The differing associations of airway responsiveness at 1 mo and 6 yr with objective markers of atopy are consistent with this hypothesis; atopy appears to contribute less to increased airway responsiveness in infancy than in later childhood. This may be due to the cumulative influence of environmental factors on atopy by age 6; environmental factors may interact differently with the genetic factors determining airways responsiveness and those determining lung function by age 6. The finding that airway responsiveness measured at both 1 mo and at 6 yr were associated with asthma and asthma-associated factors at age 6 but not with each other (Table 4) is consistent with the existence of multiple, age-dependent determinants of asthma susceptibility and further suggests that these two indices may even be measuring entirely different phenomena.

The marked association between increased airway responsiveness at 1 mo and both FEV₁ and FVC at 6 yr suggests that airway responsiveness at 1 mo may be an indicator of an increase in long-term, possibly life-long, airway dysfunction. Airway responsiveness at 1 mo may therefore identify persistent wheezers with airway dysfunction, possibly owing to congenital alterations in the regulation of airway caliber or tone, who are predisposed to asthma at age 6. In contrast, airway responsiveness at 6 yr may reflect the influence of atopy-induced airway inflammation and identify atopy-related risk of emerging wheeze (38) and atopic asthma at age 6. However, whereas increased airways responsiveness at each age may mean something different in terms of asthma pathobiology, our data also suggest that children who are more responsive both in infancy and later life are at maximal risk of developing asthma and have lower lung function at age 6; conversely, children who are less responsive both in infancy and later life are at least risk of developing asthma and have higher lung function at age 6 (Table 4).

In conclusion, this study is unique in examining airway responsiveness both in early life and in early childhood, and in examining how the level of airway responsiveness in early life relates to the emergence of asthma. Further research should provide more information on the contribution of this factor to the development of asthma and assist in identifying causative genetic and environmental agents.

Increased airway responsiveness in early life is strongly predictive of the later emergence of asthma and other respiratory symptoms, and may thus be an important marker of those infants at risk of developing asthma and persistent wheezing in childhood. The identification of infants at high risk of subsequently developing asthma is important, and the current study has implications for future research on the potential for prophylactic intervention.