INTRODUCTION

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Wheezing in young children is very common (15 to 35% of children) and worldwide there has been a significant increase in its reported prevalence. Despite improved understanding of pathology and immunology of asthma in childhood, little information is available about the airways immunopathology of infants and toddlers who recurrently wheeze (1). Wheezy infants are a heterogeneous group and many patterns of wheezing disorders seem to exist (2). Indeed some children wheeze with infection only during the first years of life and do not subsequently develop asthma (3) and some may have early childhood asthma. Among the large number of salient questions concerning the pathogenesis of asthma in early life, one is whether wheezing attacks are modulated by an inflammatory response of the airways (1). This unresolved question has a direct impact on the therapy of wheeze exacerbation in children less than 4 yr of age (4, 5). Much physiological knowledge has been gained by pulmonary function tests that have demonstrated that wheezing illness in early infancy may be associated with a reduced airway caliber (3, 6, 7). However, lung function tests are not directly related to the presence of an underlying inflammation of the airway and cannot give clear information in this sense.

In the last few years there has been an increased interest in developing noninvasive parameters to assess the extent of underlying inflammation of the airway and to evaluate the effect of treatment. Recent studies have suggested that measurement of exhaled nitric oxide (ENO) may be a valuable noninvasive biomarker of disease activity in asthmatic patients (8- 10). The concentration of ENO is increased in patients with asthma: this could be consistent with induction of inducible nitric oxide synthase (iNOS), an enzyme responsible for the synthesis of NO, in association with activation of inflammatory cells of the airways (10). While it is known that ENO is increased in schoolchildren and adults with acute asthma (8, 13- 15), to our knowledge no studies have investigated if this phenomenon also occurs in young children with recurrent wheeze exacerbation. The aim of this study is therefore to evaluate if ENO is increased also in infants with an acute episode of wheezing and its variations during therapy with systemic corticosteroids.

	METHODS

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Study Population

We included in the study the following groups of infants and young children (Table 1):

Group 1: Acute wheeze group with recurrent exacerbation. This study group included 15 infants and young children from 7 to 33 mo of age who came to the Department of Pediatrics of Padova, Italy, with wheeze exacerbation. Patients were considered for enrollment if they had had at least three prior episodes of wheezing, had been previously treated with ₂-agonist by inhalation, and presented a moderate exacerbation as defined by clinical criteria. The children included in this group were treated with the following therapy: oral prednisone (1 mg/ kg in one dose for 5 d) (Deltacortene; Lepetit, Lainate, Italy) and albuterol (Broncovaleas; Valeas, Milano, Italy) by nebulization (0.15 mg/kg/dose in 3 ml of normal saline 3-4 times a day) as recommended by international guidelines (4). Exhaled NO in these children was measured on two occasions: once during the acute wheeze presentation and once again after 5 d of prednisone treatment. Each patient was evaluated by the same physician (E.B.) using a clinical asthma score. The parameters included were: wheezing, accessory respiratory muscle use and dyspnea, pulse rate, and respiratory rate. All had wheezing documented by auscultation. During the course of treatment parents of children were asked to keep a record at home of coughing, wheezing, and night sleep. Excluded from the study were children who developed fever or bronchopneumonia, who presented with heart disease or other chronic lung disease, or who had taken a course of systemic steroids within 3 wk of their visit to the hospital.

Group 2: Control group. Nine healthy children with similar anthropometric characteristics of wheezy children were recruited. They had no history of asthma, eczema, or other allergic disease and they had no history of upper respiratory tract infection in the 3 wk previous to the study. Two of them undergoing elective surgery for urologic malformations were measured for ENO immediately before and after intubation.

Group 3: First-time viral wheeze group. This group included 6 children from 9 to 14 mo of age evaluated during the first episode of wheeze associated with upper respiratory tract infection. Exclusion criteria were the same as in Group 1. In addition, children with family history of asthma and allergy were excluded.

Exhaled Air Collection and ENO Measurement

Mixed ENO air was measured by a tidal breathing method using a chemiluminescence analyzer (CLD 700 Al-Med; Ecophysics, Durnten, Switzerland). Infants were seated on the legs of the mother with a mask held over the mouth. The nostrils were closed. The mask was connected to a 2-way valve that allows inspiration of NO-free air from a collapsible reservoir to ensure no contamination by ambient NO (16) and expiration into a 2-L polyvinylchloride (PVC) collection bag (Braun, Milano, Italy). The collection bag was attached to the expiratory side of the valve only after 10 breaths of NO-free air in order to permit a wash-out of the lungs. The collection bag was filled with exhaled air of the subject and the sample was immediately analyzed by chemiluminescence. The apparatus provides an expiratory resistance of 2 cm H₂O at the mouth. We have previously shown that the PVC collection bags were impermeable to NO and stability was found for at least 2 h using air containing different concentrations of NO. Immediately before the test, NO-free air was obtained by passing ambient air through a purafill converter (Maihak, Milano, Italy) and was stored in a collapsible reservoir. Before the test it was checked for NO content using the chemiluminescence analyzer.

Two children of the control group undergoing elective surgery for urologic malformations were measured for ENO before and after endotracheal intubation. Before intubation measurement was done as previously described. The patients were then intubated and stabilized. A two-way valve was attached to the end of the endotracheal tube. The child was ventilated with an anesthetic bag connected to a reservoir containing NO-free air and, after 10 manual ventilations to wash out the lungs, a 2-L reservoir was connected to the expiratory side of the valve and filled with the exhaled air of the subject.

Before each study, the chemiluminescence analyzer was calibrated with a certified calibration mixture (300 parts per billion [ppb]) of NO in nitrogen (SIAD, Bergamo, Italy) with guaranteed stability (13). Ethical committee approval was obtained for the study and all parents gave informed consent.

Statistical Analysis

ENO concentrations are reported in parts per billion. The results are shown as mean ± SEM. Nonparametric analyses were employed. In wheezing children ENO measurements before and after steroid treatment were compared by Wilcoxon matched-paired test. For the comparison of ENO values between wheezing children and healthy children, a Mann-Whitney U test was used. The relation between ENO concentrations and the age and weight of patients was evaluated by linear regression. All tests were performed with two tails. Statistical analysis was performed using a computer package (Statistica; Microsoft Corp., Redmond, WA). A p value of less than 0.05 was considered significant.

	RESULTS

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The subjects' characteristics are summarized in Table 1. Fifteen children with an acute episode of wheeze (Group 1) were recruited for the study and 13 completed the protocol: one was excluded for technical problems with the analyzer and the other one because of poor compliance with the therapy. We found that after 5 d of treatment, all children showed a clinical improvement as demonstrated by the clinical scores evaluated by the physician and by the parents (data not shown). All showed resolution of auscultatory abnormalities by physical examination. There was no significant difference in mean age between Groups 1 and 2 (p = NS) although the age range of controls was greater. In Group 1, family history of asthma and allergic diseases was positive in seven of 13 children and two of them presented mild eczema.

ENO Determinations

During the acute episode of wheezing of children with recurrent exacerbation (Group 1), the mean (± SEM) value of ENO was 14.1 ± 1.8 ppb, significantly higher than in healthy control children (5.6 ± 0.5 ppb, p < 0.001). After 5 d of therapy with prednisone, ENO concentrations were significantly (p < 0.01) reduced by 52% to a mean value of 5.9 ± 0.7 ppb, compared with baseline (Figure 1). Within this group, the mean value of ENO was slightly higher in the subgroup with family history of asthma and allergy compared with the remainder of subjects (15.1 ± 2.8 versus 12.8 ± 2.5 ppb), but the difference was not significant (p = 0.3). There was no significant correlation (p = NS) between values of ENO and the age or weight of the patients. Measurement of ENO was easily accomplished in all subjects and no complications were observed during measurements. No significant relationship was found between the changes in ENO and the changes in symptom scores (r = 0.3, p = NS).

View larger version (15K): [in this window] [in a new window]   Figure 1.   Exhaled NO concentrations in wheezy children with recurrent exacerbation (Group 1) before and after corticosteroid treatment. Group mean (± SEM) and individual values (dark circles) with the corresponding ENO values after treatment are shown. Gray circles represent individual values of healthy control subjects (Group 2). Two of them were measured immediately before and after tracheal intubation for elective surgery.

The children with first-time wheeze (Group 3) showed a mean value of ENO of 8.3 ± 1.3 ppb, significantly lower (p < 0.05) than the value of children with recurrent wheeze during exacerbation (Group 1) and similar (p = NS) to that of control children (Group 2). In two children measurement of ENO was done before and after tracheal intubation. While breathing NO-free air, the preintubation ENO concentration was 6 ppb during mask breathing and 5 ppb after intubation in one child and 7 ppb before intubation and 6 ppb after intubation in the other one.

	DISCUSSION

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Measurement of ENO has been proposed as a noninvasive approach for assessing disease activity in asthmatic patients (8- 10, 13-15). In this study we describe a method for measuring ENO in young children and, for the first time, provide evidence that also very young children with recurrent wheeze exacerbation have increased values of ENO that, after corticosteroid treatment, rapidly decrease to levels similar to those of healthy control children (Figure 1). This suggests that, in infants with recurrent wheeze exacerbation, airway inflammation could be present at a very early age.

The present data extend our previous findings (13) and those of others in schoolchildren and adults (8, 14, 15) showing the presence of raised levels of ENO during asthma exacerbation. The mechanisms that lead to an increased production of ENO are still unclear. This increase is thought to reflect the expression of the iNOS enzyme in superficial airways cells and macrophages stimulated by a variety of proinflammatory cytokines, and the decrease after steroid therapy seems due to the effects of these drugs on the iNOS gene (11). This hypothesis is supported by the fact that airway epithelial cells taken from asthmatic patients immunostain strongly for iNOS (12). On the contrary, consistent with their lack of anti-inflammatory effect, ₂-agonists do not influence ENO in asthmatic patients (13, 17).

A number of studies have provided evidence that oral steroids are beneficial for the treatment of asthma exacerbation in asthmatic schoolchildren (18). On the contrary, the utility of systemic steroids in the treatment of young children with acute episodes of wheeze is still debated (5), probably because of a lack of simple pulmonary function tests and reliable noninvasive markers of airway inflammation to judge the effects of treatment. The present study did not attempt to find out definitively if systemic steroids are effective in the treatment of acute wheeze in young children. For this purpose a placebo-controlled design would have been necessary.

We did not investigate if the wheezing attacks were precipitated by viral infections that, in this age range, are known to be the most frequent triggers for the exacerbation of asthma (19). In this case a hypothesis to explain the increased values of ENO is that viral infections could be a priming event in inducing an inflammatory response of the airways in predisposed subjects by causing the release of inflammatory mediators (19, 20). In keeping with this possibility de Gouw and coworkers (21) have recently shown that experimental rhinovirus 16 infection increases ENO levels in asthmatic subjects in vivo.

Probably more patterns of wheezing disorder exist in infancy and airway obstruction results from a number of different pathophysiological processes with different causes (2, 3). It is unknown whether these different patterns seen clinically are associated with different patterns of airway inflammation. In our study children with recurrent wheeze (Group 1) represent a select subset of wheezers with an increased predisposition to asthma. They showed raised values of ENO suggesting the presence of underlying airway inflammation. On the other hand, the children at first-time wheezy episode (Group 3) presented values of ENO similar to those of control subjects. Similar findings have been reported by Stevenson and coworkers (22) who demonstrated that atopic children with recurrent wheeze, as young as 3 yr of age, present ongoing airway inflammation characterized by eosinophils and mast cell recruitment in lavage studies, clearly differentiating children with virus-induced wheeze in whom the neutrophil dominates. Even if direct data from BAL or lung biopsy (22, 23) are vital for a better understanding of the processes that lead to recurrent wheezing in young children, they cannot routinely be justified in studying minors. There is therefore an increasing need of reliable noninvasive markers to assess airway inflammation and to evaluate the effect of treatment. If ENO is confirmed to represent a reliable marker of airway inflammation, it might help to distinguish children with early-onset asthma from transient wheezy syndromes of infancy and perhaps to identify specific risk groups for appropriate early intervention.

Measurement of ENO is influenced by several technical factors in cooperative subjects (24) but only few preliminary studies have been published in children who are not able to cooperate (25). It has been demonstrated that nasal production of NO could affect measurement at the mouth (24). To address the question of whether the levels of ENO collected in the reservoir are a reflection of NO produced in the airway, ENO measurement was performed in two children before and after tracheal intubation for elective surgery and showed similar values. This suggests that ENO measured with this method derived from the lower airways. However, more work is necessary to standardize the techniques and methods of analysis in young children.

In conclusion, these data show that infants with recurrent wheezing have elevated levels of ENO during exacerbation that rapidly decrease after steroid treatment. This suggests that airway inflammation could be present at a very early age in young children with recurrent episodes of wheeze. Further work is needed to confirm these preliminary observations, and comparison between measurement of ENO and direct measures of airway inflammation is necessary to evaluate whether this noninvasive marker will be helpful to better characterize the different wheezing phenotypes of infancy.