INTRODUCTION

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Keywords: asthma; bronchial hyperreactivity; children; FEV₁

Nonspecific bronchial hyperreactivity (BHR) is a common characteristic of asthma and is shown to be a risk factor in the development and outcome of asthma (1). On the other hand, the cross-sectional correspondence between BHR and asthma, although very close, is less than perfect (4). Therefore, several factors are likely to have an impact on the degree of BHR.

Cross-sectional studies have related nonspecific BHR to high concentrations of specific IgE antibodies or positive skin tests against common inhaled allergens (5), severity of asthma (5, 8), levels of serum total IgE (8, 10), and lung function (6, 8, 10, 11). Furthermore, results of longitudinal studies demonstrated that lower lung function and the presence of atopy at enrollment predicted a higher degree of BHR at the end of the follow-up (6, 11).

In this study, the aim was to investigate the impact of some risk factors on the severity of current BHR, such as initial severity and duration of disease, age at onset of disease, presence of allergy to specific allergens, and level of lung function and serum total IgE at referral. We also aimed to determine the cross-sectional factors associated with severity of BHR in childhood asthma.

	METHODS

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Subjects

Children with asthma regularly followed between 1992 and 2000 by the outpatient clinic of Marmara University Hospital's Pediatric Allergy and Immunology Division were selected according to the following inclusion criteria: (1) to be able to perform a lung function test (LFT) properly; (2) to have been followed by regular visits (i.e, every 2 to 3 mo) by our program for at least 1 yr until the time of study. Diagnosis of asthma is defined according to criteria established in the International Consensus Report on Diagnosis and Management of Asthma (14). The study was approved by the ethics committee of the hospital, and parents of all participants signed informed consent forms.

Study Design

According to the asthma follow-up protocol of our division, a detailed entry questionnaire (including data on age at onset of disease, duration of symptoms at referral, frequency of asthma symptoms, seasonal effects, family history, triggers for symptoms, severity of disease, presence of other atopic diseases, serum total IgE, skin-prick test [SPT], LFT results, mode and dose of prescribed treatment, etc.) is filled out for each patient on the first visit. Follow-up visits are scheduled every 2 to 3 mo, and a follow-up form (including data on the interim, prescribed treatment, and results of tests performed on that visit) is filled out at each visit. Data obtained on those visits have been entered in a database program during each visit by the secretary of our division.

According to our database program, 142 subjects met the inclusion criteria and were invited for a study visit. Of those 142 subjects, 102 individuals responded and 98 of them [mean (± SD) age = 11.0 ± 3.4 yr, male/female = 50/48] were entered into the study. The computer records of these 98 subjects were reviewed, and data at referral were collected, including sex, age at onset of disease, family history of atopy, serum total IgE level, results of LFTs and SPTs, and severity and duration of disease at referral. An end-of-study visit was performed on all of the subjects included. Patients were invited specifically for lung function, skin-prick, and bronchial provocation testing, and determination of serum total IgE level on this visit. Data on current age, number of acute asthma attacks experienced during the past 12 mo, and the period without asthmatic symptoms before the time of the last visit were recorded.

Follow-up and Therapy

According to the asthma treatment protocol at Marmara University Pediatric Allergy Division, patients with intermittent asthma receive only inhaled ₂-agonists on an "as needed" basis. Patients with persistent asthma who are assigned for an anti-inflammatory therapy receive inhaled budesonide for a daily total dose of 800 µg for a month. At the end of 1 mo if an improvement is achieved, the dose is decreased to a total daily dose of 400 µg. During the follow-up period, the dose of medication needed for control of respiratory symptoms is adjusted to find the minimal dose. In addition, all patients receive inhaled ₂-agonists on an as needed basis. For the sake of clarity on assessment of anti-inflammatory treatments in patients, we had chosen a standardized treatment protocol and had chosen to use inhaled budesonide as the only anti-inflammatory medication since 1989.

All the patients followed at our Outpatient Clinic are asked to maintain symptom and medication score diaries supplied by us and to bring them on each visit. Patients keep a diary recording use of the prescribed anti-inflammatory treatment; daily symptoms, including cough, shortness of breath, sleep disturbance; and need for additional ₂-agonist treatment. Some patients also record daily peak flow measurements in their diaries. Patient evaluation includes LFTs, daily symptom scores, as well as the use of both prophylactic and need-based medication and peak flow meter results on follow-up visits paid every 2 to 3 mo, and more frequently if necessary.

Assessment of disease severity. For statistical purposes, patients were scored as 1, 2, 3, and 4, depending on frequency of asthma attacks at referral, as follows: 1 =  3 to 4 episodes a year; 2 = 1 to 2 episodes a month; 3 = 1 to 2 episodes a week; and 4 = more frequent.

Lung Function

Forced expiratory volume in one second (FEV₁) and forced expiratory flow between 25% and 75% of vital capacity (FEF_25-75%) were measured according to standardized guidelines with a spirometer (Puritan-Bennett, Wilmington, Ireland) when the subjects were asymptomatic. Three measurements were made, and the highest value obtained was recorded. Normal lung function test standards of Polgar and Promadhat (15) were used to generate the predicted values. The data are presented as percentage of predicted value and adjusted for sex, age, and height. Lung functions measured at referral (or soon after referral) and at the end-of-study visit were used in the analyses.

SPT and Serum Total IgE Level

Serum total IgE level was measured with the immulite method, both at referral and at the final visit, by the laboratory of the University Hospital. SPT was done by employing a panel of 24 allergen extracts (ALK Allergologisk; Laboratorium A/S, Copenhagen, Denmark) in addition to positive and negative controls (histamine dihydrochloride and saline) on the volar surface of the forearm. The same panel of allergen skin tests was applied at referral, and then again at the final visit, by the same two technicians of our laboratory. A wheal size equal or larger than that obtained with histamine was judged as positive. A positive and negative skin test was defined as 1 and 0, respectively.

Bronchial Provocation Test

Nonspecific bronchial reactivity to methacholine was measured with the tidal breathing method (16). Oral or inhaled ₂-agonists and antihistamines were withheld 24 h and 4 d before the test day, respectively. The aerosol was generated by a jet nebulizer (DeVilbiss PulmoAide, Chicago, IL) calibrated to give a constant output of 0.13 to 0.15 ml/min. The mist was inhaled through a face mask for 2 min during tidal breathing. FEV₁ was measured before the start of the procedure and 30 to 90 s after each inhalation. FEV₁ obtained before the start of the procedure was used as baseline. After inhalation of isotonic saline, methacholine chloride (Sigma Chemical Co., St. Louis, MO) was inhaled in doubling concentrations from 0.0625 mg/ml to 32 mg/ml. The results were expressed as the logarithm of the provocative concentration of methacholine causing a 20% reduction of FEV₁ (log PC₂₀), which was obtained from the log dose-response curve by linear interpolation of the last two points.

Statistical Analysis

All analyses were carried out by means of the SPSS statistical package program (Release 5.0.1; SPSS Inc., Cary, NC). Changes during the follow-up were tested for significance with the Wilcoxon signed rank test, paired t test, and McNemar's chi-square test for paired samples.

To examine which covariables at referral are associated with level of BHR at the end of follow-up, a multiple linear regression analysis was carried out on the log PC₂₀ at the final visit. Covariables included in this model were as follows: age, age at onset of disease, sex, family history of atopy, duration and severity of disease at referral, initial FEV₁ %, SPT result, and log of serum total IgE level. We transformed all serum IgE values to logarithmic function to have normally skewed data during the multiple regression analyses.

A cross-sectional multiple linear regression analysis was carried out to examine which covariables are associated with the level of BHR, including only covariables of the final visit. These covariables were as follows: age, sex, period without asthmatic symptoms before the time of the last visit, number of asthma attacks experienced during the past 12 mo, FEV₁ %, FEF_25-75%, log serum IgE level, and results of SPT.

To determine whether various measures at referral and at the end of study are independently associated with current BHR, another multiple linear regression analysis was carried out with the inclusion of a combination of some initial and final covariables. The covariables included in this analysis were age, age at onset of disease, sex, family history of atopy, level of lung function (FEV₁%), severity and duration of disease at referral, number of asthma attacks experienced during the past 12 mo, period without asthmatic symptoms before the time of the last visit, log serum IgE, and results of LFTs and SPTs obtained at the final visit. A p value less than 0.05 was considered significant.

	RESULTS

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Ninety-eight asthmatic children followed by the Pediatric Allergy and Immunology Division of Marmara University Hospital regularly for a duration of 3.2 ± 1.9 yr (mean ± SD) with an age range of 5.3 to 19 yr [mean (± SD) age = 11.0 ± 3.4 yr, male/female = 50/48] were included. The characteristics of the study group are summarized in Table 1.

The mean serum total IgE levels and percentage of prick test positivity did not change significantly between the two surveys. FEV₁ values showed statistically significant increase during follow-up, whereas FEF_25-75% did not change significantly between the two surveys (p = 0.03 and p = 0.556, respectively). Inhaled budesonide therapy was withdrawn in 17 of the 79 (22%) patients during the follow-up (p = 0.004, McNemar's chi-square test).

Cross-sectional Analysis for the Severity of BHR

Number of asthma attacks experienced during the past 12 mo, period without asthmatic symptoms before the time of the last visit, level of lung function, log IgE level, and sensitivity to allergens evaluated at the time of bronchial provocation testing were found not to be associated with the severity of BHR (Table 2).

Risk Factors at Referral for the Severity of Final BHR

A lower level of FEV₁ % at referral was found to be an important predictor of more severe BHR at the end of follow-up (p = 0.012). None of the other risk factors evaluated in this analysis predicted the severity of final BHR (Table 3).

Multiple Regression Analysis, Including a Combination of Initial and Final Risk Factors for Severity of BHR

To determine whether various measures at referral and at the end of study are independently associated with BHR, the analyses were repeated with the inclusion of a combination of some initial and final risk factors. A lower FEV₁ % at referral was found to be significantly associated with a more severe current BHR (p = 0.012). On the other hand, none of the other risk factors evaluated in this analysis predicted the severity of final BHR (Table 4).

	DISCUSSION

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The present study demonstrates that the level of FEV₁ % at referral has significant impact on the degree of BHR at the end of a mean of a 3-yr follow-up in children with asthma. In addition, the results of our study suggest that, although the appropriate anti-inflammatory therapy could optimize the level of FEV₁, it did not have much effect on serum total IgE level and sensitivity to inhalant allergens.

Several studies demonstrated the association of decreased lung function in childhood to severe BHR in adulthood (6, 11). In a longitudinal study, in a cohort of 892 schoolchildren reevaluated after a 3.5-yr interval, the strongest determinants of the decline in BHR were found to be baseline lung function and degree of atopic status (13).

In another longitudinal study, Martinez and colleagues investigated the factors affecting wheezing at 6 yr of age. They showed that children who started wheezing in early life and continued to wheeze were more likely than the children who never wheezed to have mothers with a history of asthma, to have elevated serum IgE levels and normal lung function in the first year of life. On the other hand, those children were shown to have diminished lung function at 6 yr of age. They concluded that such children already had substantial deficits in lung function by the age of 6 yr, which may reflect the effects of the chronic disease process on the bronchi (17).

In the current study, FEV₁ % significantly improved within a mean of 3 yr of treatment and follow-up. One of the factors for this improvement could be the regular use of anti-inflammatory therapy. This underscores the importance of early treatment to optimize lung function in children with asthma.

Many studies have related BHR to allergic skin test reactivity (5, 12, 13); other studies have shown relation to serum total IgE levels (8, 11, 12) and severity of disease (11, 18). Present data indicate that the presence of a high IgE concentration or of skin test reaction to relevant allergens not only relates to BHR measured simultaneously, but also appears to encourage the retention of BHR (6, 12, 13). In contrast to the findings of most other studies (5, 11), we observed no relationship between high serum total IgE levels, allergic skin test reactivity, and BHR in our study group.

In our country, factors that increase serum IgE other than allergy, such as parasitic infections, are more prevalent (47.9%) (19). One of the reasons why we could not demonstrate any significant relationship between BHR and higher concentrations of IgE could be this geographical factor in our study population.

Another explanation for the absence of association between BHR and higher IgE concentrations in our study may imply the distinct genetic regulation of these factors. The importance of IgE in the development of BHR has been studied in different murine models. These data indicate that IgE is not required for the development of eosinophilic airway inflammation and BHR in mice (20). Equivalent degrees of eosinophilic infiltration of the bronchial mucosa and BHR are elicited by means of allergen inhalation in wild-type and IgE-deficient mice generated by gene targeting (21).

In this study, inhaled anti-inflammatory therapy was successfully discontinued in 22% of our patients during the follow-up period. This finding could partly be explained by the use of appropriate anti-inflammatory therapy that resulted in an increase in the value of FEV₁ and also by the close follow-up of patients. One of the factors that strengthens the value of our study is the use of only one anti-inflammatory medication, namely budesonide, in all patients. This standardized treatment protocol enabled us to avoid the complexity that may arise from using different modes of anti-inflammatory therapy.

We acknowledge the limitation of our study, which was partly retrospective in design. On the other hand, data at referral were collected not from patient notes, but from our database program which had been set up for this purpose. The heterogenous age range of our study population could be another limitation, but the majority of the subjects (75%) were between 7 and 14 yr of age.

In summary, this study showed that a decreased FEV₁ % at referral is associated with a more severe BHR, whereas no such association was shown with allergic skin test reactivity and serum IgE level at the end of a 3-yr follow-up in children with asthma.