Isocapnic Hyperventilation Of Cold Air Research Articles

Profile of MUC5AC and MUC5B mucins expression in asthma patients under cold exposure

Introduction. Cold airway hyperresponsiveness (CAH) is a common condition in patients with asthma, which worsens the clinical course of the disease and the patients’ quality of life. MUC5AC and MUC5B are the main secreted mucins in the respiratory tract, which are involved in normal mucociliary clearance, but also capable of provoking the development of pathological changes in case of dysregulation of their balanced production.Aim. The aim of this study was to determine the dynamics of MUC5AC and MUC5B expression during experimental cooling in patients with asthma depending on the status of CAH.Materials and methods. The study enrolled 98 subjects including 26 patients with chronic non-obstructive bronchitis without exacerbation (control group) and 72 patients with asthma. The expression of MUC5AC, MUC5B and TRPM8 was determined in the upper respiratory tract by quantitative reverse transcription PCR. The production of MUC5AC and MUC5B was also measured in sputum by ELISA. All patients underwent a bronchoprovocation test with isocapnic cold air hyperventilation to detect CAH, and a similar nasal challenge was performed to assess the effect of cooling on the expression of the studied genes.Results. Patients with asthma had 4.22-fold increase in the expression of MUC5AC (p=0.02) in the nasal epithelium as compared with the control group. CAH was associated with an initial 7.33-fold upregulation of MUC5AC (p=0.008) as well as with further increase in MUC5AC expression but a decrease in MUC5B in response to cooling, which was not observed in asthma patients without CAH. Basal TRPM8 expression was associated with baseline level of MUC5AC (ρ=0.41, p=0.04), MUC5B (ρ=0.55, p<0.001) and amount of sputum produced after the cold bronchoprovocation.Conclusion. Asthma patients with CAH demonstrate a more pronounced imbalance in the production of mucins, which is aggravated by cold exposure. This, in turn, can lead to a number of pathological disorders associated with a more severe course of the disease.

Efficiency of using a short-acting β2-agonist for the relief of acute cold bronchospasm in asthma patients with cold airway hyperresponsiveness

Excessive sensitivity of the respiratory tract to physical and chemical environmental triggers can vary for many reasons, reducing the therapy effectiveness in a patient with asthma. Aim of the study was to investigate the effectiveness of a short-acting bronchodilator in patients with asthma for the relief of acute cold bronchospasm after a test of isocapnic cold air hyperventilation (ICHV). Material and methods. In 281 (161 women; 120 men, p > 0.05) asthma patients with cold airway hyperresponsiveness (CAHR), the change in airway patency (FEV1) and the efficacy of short-acting β2-agonists (SABA) after a 3-minute isocapnic hyperventilation with cold (–20 ºС) air. Results. According to clinical data, patients had persistent asthma, mean age 35 (26; 44) years, 49 % of patients smoked, ACT 16 (12; 20) points, FEV1 90.0 ± 1.0 % predicted, FEV1/VC 71.4 ± 0.6 %, the increase in FEV1 after inhalation of SABA (∆FEV1β) was 11.1 (5.1; 20.5) %. The change in FEV1 for the ICHV varied within –16 (–22.0; –12.0) %. The use of SABA after the IHCV showed different efficacy for relief of an attack of cold bronchospasm. The median value of ∆FEV1β after ICHV was 21.1 (11.6; 33.3) %, with a range of –48.6 to 108.2 %. In the general group, there was a direct relationship between the level of asthma control in terms of ACT points and the baseline FEV1 (Rs = 0.17; p = 0.007), MEF25-75 (Rs = 0.18; p = 0.008), ∆FEV1β in response to SABA (Rs = –0.17; p = 0.0104), as well as the severity of the bronchial response to the ICHV (Rs = 0.15; p = 0.014). The latter, in turn, correlated with ∆FEV1β after ICHV (Rs = –0.28; p < 0.0001) after HCI. Conclusions. There is a differentiated airway response to SABA after acute cold bronchoprovocation. The results obtained can serve as an important tool for phenotyping of asthma patients with CAHR for the medication correction of сold bronchospasm.

Nasal mucosa secretion exudation response to cold air in bronchial asthma patients

Background. Combined airway hyper responsiveness to cold and hypoosmotic stimuli in asthma patients results in impairment of lung respiration function and poor disease control compared to patients with isolated airway hyper responsiveness to only one of the stimuli or without such responsiveness that can be connected with edema or mucus hypersecretion. Aim. The purpose of the study is the estimation of the processes of mucin secretion, plasma exudation and oxidative stress in response to cold air in asthma patients with combined airway responsiveness to cold and hypoosmotic stimuli using nasal mucosa as a model. Materials and methods. 23 patients with asthma participated in the study. For the nasal lavage procedure, a nasal cavity was pre-washed at least three times in 5-min intervals with 5 ml saline solution (~36 °C). A control nasal lavage was done 5 min after the last washing with a dwelling time of 1 min in the nasal cavity. Directly after the control lavage, a cold air nasal challenge was done: a participant was asked to breathe deeply at the pace of a metronome to ensure hyperventilation inhaling cold air (–20 °C) through the nose and exhaling through the mouth for 5 min. Nasal lavages were taken at 1 min, 15, and 30 min after the challenge. Mucin secretion was estimated on the basis of total protein (TP) content, total carbohydrates (TC), and water-soluble forms of mucins MUC5AC and MUC5B in the lavage fluids. For the estimation of plasma exudation, the concentration of α2-macroglobulin (α2-MG) was measured. Oxidative stress was estimated by the content of thiobarbituric acid-reactive substances (TBARS) in lavage fluid. Lung function and airway responsiveness were studied by the forced expiration spirometry method and the bronchial challenge tests with isocapnic cold air hyperventilation (CAHV) and distilled water inhalation (DWI). Results. According to the bronchial challenge tests, the patients were divided into groups: 1) without airway responsiveness to the cold and osmotic stimuli (n = 6); 2) combined airway responsiveness to both stimuli (drop in FEV1 by 10% or more after CAHV and DWI) (n = 11); 3) isolated airway responsiveness to only one of the stimuli (n = 6). In the total group of asthma patients, the mean content of TP, TC, α2-MG, and TBARS increased by 63%, 109, 47, and 68%, respectively, after the cold air nasal challenge, whereas MUC5AC and MUC5B decreased by 15 and 20%, respectively. Secretion and exudation in the nasal mucosa were more pronounced in asthma patients of group 2 in comparison with other groups. Oxidative stress was lower in group 1. There were two interesting correlations between bronchi responsiveness to CAHV and DWI and changes in the content of the biomarkers after the cold air nasal challenge in group 2: 1) ∆FEV1 after CAHV and TC level at 15 min (r = –0,65; р = 0,0401) and at 30 min (r = –0,82; р = 0,0034); 2) ∆FEV1 after DWI and the change of α2-MG at 1 min after the cold air nasal challenge (r = –0,67; р = 0,0242). Conclusion. In accordance with the unified airway model, the found correlations may indicate that prolonged mucin secretion after cold air breathing is a negative factor for the bronchi response to cold air, whereas enhanced plasma exudation determines the bronchi responsiveness to a hypoosmotic stimulus. Nasal mucosa is a promising model for the simultaneous investigation of molecular processes of airway secretion, exudation and oxidative response in asthma patients.

Correlation of brain electrical activity and motivation in healthy people

Motivation dominates in the structure of the personality and is one of the basic notions which explains the dynamics of the behavior. The literature has little data about neurophysiology of motivation. The aim of the research was to study the correlation between the motivational sphere and electrical activity of the brain at the influence of different provocations. 24 healthy people at the age of 26-36 years were examined. The results of motivation tests turned out to be uniform (the motivation to success was of a moderate or high level, there were mean values of readiness to risk and low motivation to achievement and approval). Multiple correlations between different types of motivation and electrical activity of the brain at rest, at hyperventilation with room temperature air and at isocapnic cold air hyperventilation were revealed.

Birth weight and adult lung function: a within-pair analysis of twins followed up from birth

The aim of the study was to evaluate whether there is any association between intrauterine growth and later lung function or bronchial reactivity in early adulthood in line with Barker's hypothesis. Nineteen twin pairs with disproportionate intrauterine growth pattern were followed up from birth: either one of the pairs had intrauterine growth retardation (birth weight <2 SD) or the within-pair birth weight difference was >1.3 SD. Flow-volume spirometry, followed by isocapnic hyperventilation of cold air, was performed at the ages of 8-16 and 14-22 years in 1993 and 1999. Wilcoxon's matched-pairs analysis was used to compare smaller and larger twin pairs. In 1993, there were no significant differences between the groups in either spirometry or cold air challenge. In 1999, such a difference was found in forced expiratory volume % (FEV%) and forced expiratory flow (FEF) at 25%-75%, the smaller twin pairs having lower values. In 1993, nine subjects reacted to cold air (>9% decrease in FEV in 1 second). In 1999, only four subjects reacted to cold air, and they all belonged to the group of smaller twins (P=0.04). Lung function evaluated by FEV% and FEF25-75 was lower and responses to cold air were more common at the median age of 16 years in twins with impaired intrauterine growth.

Bronchial hyper‐responsiveness predicts the development of mild clinical asthma within 2 yr in school children with hay‐fever

In children with mild asthma, symptoms are not always apparent. Therefore, results of tests play an important role for the diagnosis. First, to investigate whether children with bronchial hyper-responsiveness (BHR) but no symptoms of asthma in 1992 had developed clinical asthma at follow up in 1994. The second aim was to find out the diagnostic properties of tests for asthma/allergic inflammation, using either doctor diagnosed asthma (DDA), self-assessed symptoms of asthma or iso-capnic hyperventilation of cold air (IHCA), as the standard, to diagnose asthma in a group of children with hay fever. Twenty-eight children with pollinosis, 12 of them with a history of asthma for the first time during the season 1992, were studied during the birch pollen season and in the autumn of 1994. During both periods, the bronchial hyper-reactivity was estimated by methacholine bronchial provocation tests (MBPT), bronchial variability by peak expiratory flow rate variability, subjective symptoms of asthma by visual analogue scale (VAS) and bronchial inflammation by serum and urine levels of inflammatory mediators. In 1994 IHCA was added during both seasons. Eight of 16 children with BHR but without clinical asthma in 1992 had developed asthma in 1994, 14 of 16 reacted to IHCA and 13 to MBPT. All 12 children with DDA in 1992 had still asthma in 1994 and 14 children with BHR in 1992 had persistent BHR in 1994. Of 23 children with BHR in 1992, 17 had DDA in 1994 and all maintained their BHR. Furthermore, 20 of them reacted to IHCA in 1994. In 1994, 24 of 28 hay-fever children had a positive IHCA tests and 24 had positive MBPT. In relation to VAS, the sensitivity of IHCA and MBPT to predict present asthma was high, but the specificity low, whereas the specificity of most other tests was high, but based on few individuals. In relation to DDA both the IHCA test (65-80%) and the MBPT test (79-85%) had a high sensitivity and it was three to six times more likely to find a positive test among asthmatics than in non-asthmatics. Children with hay fever without clinical asthma have a high risk of developing asthma within 2 yr. In relation to DDA, inhalation of cold air and the MBPT showed a high sensitivity.

Single-isomer R-salbutamol is not superior to racemate regarding protection for bronchial hyperresponsiveness

Bronchial hyper-reactivity (BHR) has been suggested to follow cessation of regular medication with racemic salbutamol. This study aimed at investigating the effects from medication with R,S- and R-salbutamol on bronchial response to provocation with isocapnic hyperventilation of cold air (IHCA).Twenty-six patients with mild to moderate asthma were enrolled in a double-blind, randomised, cross-over study. Bronchial response to provocation was measured before and after 1 week's medication. Doses of 0.63mg R-salbutamol or 1.25mg R/S-salbutamol were inhaled three times daily during medication-weeks and a wash-out week intervened. Tests were performed 6h after the last dose of test drug. Impulse oscillometry and forced expiratory volume during one second were methods used to identify bronchial response to provocation. Two patients withdrew from the investigation due to side-effects, one from R- the other from R,S-salbutamol.Comparable resting bronchial conditions were indicated by differences in baseline lung function values of <2% between study days. No statistically significant medication-dependent differences in BHR could be demonstrated between treatment groups. However, 15 patients exhibited higher (P=0.03) post-treatment BHR after pure R-salbutamol than after R,S-salbutamol. Furthermore, plasma concentrations of R-salbutamol tended to be lower (P=0.08) after medication with R- than after R,S-salbutamol despite equal doses of R-salbutamol given during the two separate treatment periods. We also found that considerable amounts of S-salbutamol were retrieved in plasma after medication with pure R-salbutamol.We conclude that we were unable to demonstrate favourable effects of R-salbutamol over R,S-salbutamol regarding response to provocation with IHCA after regular medication of 1 week's duration.

The Diagnostic Value of Isocapnic Hyperventilation of Cold Air in Adults with Suspected Asthma

Background Asthmatic patients frequently suffer cold-weather-associated respiratory symptoms. The sensitivity, specificity, accuracy and diagnostic value of isocapnic hyperventilation of cold air(IHCA) using a multistep method was investigated in patients suspected to have asthma. Method One hundred and 29 adult patients who had an IHCA performed between july 1999 and December 2000, had an methacholine bronchoprovocation test because of a clinical suspicion of asthma. Results According to strict criteria, 50 were defined as asthmatics and 79 as symptomatic nonashmatics. There were no differences in age, sex and smoking state between the asthmatic and symptomatic nonasthmatic groups. There was a significant decrease in the percentage reduction in the forced expiratory volume in 1 second(FEV1) after the IHCA between the asthmatics(-10.0±6.8%) and the symptomatic nonasthmatics(-2.3±2.5%). The factors associated with a reactivity to IHCA were FEV1/FVC, FEF25-75/FVC and FEV1(% of predicted). The accuracy was highest using a 7% fall in FEV1; the sensitivity was 76% and the specificity 96%. Conclusion IHCA is a specific, although not a sensitive, test for diagnosing asthma in adult patients. Furthermore, the diagnostic cut-off value of the different methods of IHCA need to be determined.

Analysis of T-cell activation after bronchial allergen challenge in patients with atopic asthma

Background: T helper cells are a heterogeneous group of cells that have phenotypic and functional differences. Activated T helper cells have been found in peripheral blood after allergen challenge of subjects with atopic asthma, but the phenotypes of specific T helper subpopulation involved remains to be identified. Objective: To characterize the T cell activation markers that may be regulated by allergens, we analyzed peripheral blood lymphocytes obtained before and after allergen challenge from subjects with atopic asthma. Methods: We analyzed the distribution of the cell surface activation markers, interleukin 2 receptor (IL-2R) and major histocompatibility complex class II antigens (MHC II) among T helper subpopulations classified as naive (CD45RA) or memory (CD45RO) phenotypes. Nine adult subjects with atopic asthma underwent bronchoprovacative allergen inhalation and isocapnic cold air hyperventilation (ISH) challenge followed by serial spirometry. Peripheral blood mononuclear cells (PBMC) were isolated at baseline and 2 and 24 hours after challenge. Four-color flow cytometry was used to analyze the expression and distribution in vivo of IL-2R and MHC II activation markers on naive and memory T cell subsets after challenge. Results: At 2 and 24 hours after allergen challenge, there was a significant increase in the CD45RO+IL-2R+ T helper cells compared with baseline (mea n ± SE, baseline, 12.5% ± 1% versus 2 hours, 18.1% ± 1% and 24 hours, 17.8% ± 2%, p < 0.025). MHC II expression was not significantly increased after challenge on naive and memory T helper cells and coexpression of IL-2R and MHC II was only found in a small proportion of CD45RO+ T helper cells (2.7% ± 1%). No changes of IL-2R or MHC II expression on T helper subsets were observed after ISH challenge in the same patients. We also found that 31% to 46% of T helper cells coexpress CD45RA and CD45RO simultaneously, and upregulation of IL-2-R and MHC II expression occurs only on those T helper cells that express CD45RO. Conclusions: We have found that T helper cells express both CD45RA and CD45RO isoforms, which suggests the existence of a transitional phenotype among naive and memory T helper cells in peripheral blood. In subjects with atopic asthma, our in vivo analysis characterizes two populations of activated memory T helper cells based on the expression of IL-2R or MHC II surface molecules after allergen challenge.(J Allergy Clin Immunol 1998;101:699-708.)

The diagnostic value of cold air hyperventilation in adults with suspected asthma

The diagnostic value of isocapnic hyperventilation of cold air (IHCA) is not fully established. All 342 adult patients in whom IHCA had been performed because of a clinical suspicion of asthma between 1992 and 1994 were analysed retrospectively in the authors' hospital. In addition, 26 healthy subjects were recruited. According to strict criteria, the patients were divided into asthmatics and symptomatic non-asthmatics. For the calculations of sensitivity, specificity and accuracy, the symptomatic non-asthmatic group served as a control. The post-test probability of asthma after IHCA was determined for all the possible pre-test probabilities by applying Bayes' theorem. A linear regression model was used to investigate the factors associated with the reactivity to IHCA. A single 4-min IHCA and skin prick tests were performed in the healthy subjects. Of the 287 patients in the final analysis, 113 were defined as asthmatics and 174 as symptomatic non-asthmatics. The accuracy was highest using a 9·0% fall in forced expiratory volume in 1 s (FEV 1) as a cut-off value; the specificity was then 86·8% and the sensitivity 31·9%. The authors found IHCA to be a useful diagnostic test only if the pre-test probability of asthma is between 0·30 and 0·56. The positive final diagnostic gain of IHCA is 22% at its best, but the negative gain is negligible for all possible pre-test probabilities. Factors associated with reactivity to IHCA were young age and, to a lesser extent, a history of cold-weather-associated respiratory symptoms and pre-challenge bronchial obstruction. If a rigid cut-off value for a positive response is used in all age groups, the specificity of IHCA is good but the sensitivity is unacceptably low in adults. The diagnostic value of IHCA might increase if age is taken into account when defining the cut-off value.

Respiratory impedance measurements in a dose-response study of isocapnic hyperventilation with cold air.

Spirometry and impedance of the respiratory system were compared after increasing rates of isocapnic cold air hyperventilation (IHCA) in 10 asthmatics and 11 healthy nonsmokers. In the control group IHCA at 60% of indirect maximum breathing capacity (IMBC) resulted in a significant, equal increase in resistance at lower and higher frequencies, suggesting central airway constriction. In the asthmatics negative frequency dependence of resistance and a significant increase in resonant frequency and decrease in the reactance at 8 Hz were found at 40 and 60% IMBC. These changes suggest an increase in the resistance of the peripheral airways. The frequency dependence of resistance and resonant frequency were found to be the most sensitive measures to differentiate between normals and asthmatics. It is concluded that impedance measurements provide a suited method to assess the response in cold air provocation tests.

Comparison of Responses to Methacholine and Cold Air in Patients Suspected of Having Asthma

In 140 adult patients with FEV1 greater than or equal to 60 percent predicted referred because of suspected asthma, we compared responses to methacholine and isocapnic cold-air hyperventilation. Most challenges were accomplished on the same day, cold air always being done first. The cold-air test employed a single episode of hyperventilation (target, 30 times the FEV1), and subsequent FEV1 changes were noted, a decrease of 10 percent being defined as a positive test result. For methacholine, subjects inhaled aerosols of increasing concentrations and the dose associated with a 20 percent decline in FEV1 (PC20) was noted; positive tests were defined as a 20 percent decrease at concentrations less than or equal to 8 mg/ml. Of the 140 patients, 65 had negative results on both challenges. Twelve patients had positive results on cold-air testing but negative responses to methacholine, and 17 had the opposite result. Among patients with positive results to either test, there was a significant correlation (p less than 0.001) between change in FEV1 with cold air and log PC20, but there was considerable scatter, the results of one test accounting for 25 percent of the variability in the other. Some scatter may have been related to the methods we used, but much was probably due to the patients themselves. Neither test should be used on an exclusive basis to make the diagnosis of asthma.

Role of cooling and drying in hyperventilation induced asthma.

Respiratory heat loss has been proposed as a mechanism of exercise induced asthma. Whether the predominant stimulus is airway drying or cooling remains unclear. We have measured changes in FEV1 after isocapnic cold air hyperventilation (CAH) (-23.4 degrees (SD 0.43 degrees) C) and dry ambient air hyperventilation (AAH) (18.7 degrees (0.52 degrees)C) in seven asthmatic patients (mean age 31 (SD 9) years and baseline FEV1 3.2(0.9)1) and in seven normal subjects (age 28(6) years and FEV1 3.6(0.7)1). The inspired water content in both cases was 0.3 mg/l air. The rate of respiratory heat exchange per breath was calculated in watts (W) with microcomputer based equipment. Cold air hyperventilation caused a fall in FEV1 almost twice that of ambient air hyperventilation at each level of ventilation: CAH v AAH (% fall) 8.0 (5.1) v 3.9 (4.0) at 15 l/min, 11.6 (7.8) v 7.0 (4.4) at 30 l/min, and 20.7 (10.9) v 12.4 (6.3) at 60 l/min. Identical latent heat loss (evaporative drying) was imposed on the airway during the two challenges. Sensible heat loss (convective cooling) in cold air hyperventilation was 41 W at 15 l/min, 63 W at 30 l/min, and 114 W at 60 l/min; whereas in ambient air hyperventilation the loss was 6, 13, and 23 W respectively. It is concluded that the rate of cooling of the upper airway is the predominant stimulus in hyperventilation induced asthma.

Effect of terfenadine on the response to exercise and cold air in asthma.

To assess the role of histamine as a mediator in the response to exercise and isocapnic hyperventilation of cold air (IHCA) in asthma, we studied nine asthmatic subjects, age 13 to 25 years. All had exercise induced asthma (EIA) and positive responses to IHCA. Baseline lung function was measured before standardized challenges with histamine, exercise and IHCA. On separate days, these tests were repeated 3 h after a single oral dose of 120 mg terfenadine (TF). Histamine responsiveness decreased significantly, with a provocative concentration, producing a greater than or equal to 20% fall in FEV1 (PC20), of 1.1 +/- 0.8 mg/ml (mean +/- SEM) before and 12.0 +/- 4.9 mg/ml after the antihistamine. EIA was significantly less after TF, with 53 +/- 5% mean maximal falls in FEV1 from baseline before, and 29 +/- 9% after treatment (P less than 0.01, paired t-test). In contrast, the effect of TF on the response to IHCA was insignificant, with mean maximal falls of 45 +/- 7% in FEV1 before, and 41 +/- 7% after treatment. There was a correlation between PC20 and lowest FEV1 (% predicted) for EIA (r = 0.56, P less than 0.05), but not for IHCA (r = 0.34, NS). This study indicates a role of histamine as a mediator in EIA but not in IHCA, supporting different mechanisms for both stimuli.

Asymptomatic bronchial hyperresponsiveness in rhinitis

Methacholine inhalation tests are used to help in the diagnosis of asthma when spirometry is normal. However, the significance of increased methacholine responsiveness in patients with rhinitis and no symptoms of asthma is not known. One possibility is that it is a false positive result; another possibility is that it indicates subclinical asthma. We investigated these possibilities in 25 patients with rhinitis, whose attending physician had not made a diagnosis of asthma, by comparing responsiveness to methacholine expressed as the provocation concentration to cause a fall in FEV 1 of 20% (PC 20) with responsiveness to the natural stimulus of isocapnic hyperventilation of cold air and the diurnal variation of peak flow rate. Asthma was recognized objectively by variable airflow obstruction documented by one of the latter two tests. The PC 20 ranged between 4 and >64 mg/ml. In 10 patients the PC 20 was <16 mg1ml. Five of these patients had bronchoconstriction in response to hyperventilation, and a further two patients demonstrated increased variability of peak flow rates. Thus, in seven of 10 patients, increased bronchial responsiveness was confirmed by the use of two different methods, although they were asymptomatic, and the increased response to methacholine was not a false positive result. In the remaining three patients the PC 20 was borderline increased (8 to 16 mg/ml). The results indicate that methacholine responsiveness in the asthmatic range in patients with rhinitis is associated with variable airflow obstruction and subclinical asthma.

Bronchial responsiveness to histamine or methacholine in asthma: measurement and clinical significance

Bronchial responsiveness is the term used to describe the tendency of the airways to bronchoconstrict to specific stimuli such as allergens and isocyanates, which select a limited population of apparently sensitized subjects, and to nonspecific (nonallergic) stimuli, which affect most asthmatic persons. Specific bronchial responsiveness to allergic stimuli is difficult to quantitate because commonly available allergen extracts are not well standardized for the number and concentration of components. Nonspecific responsiveness can be quantitated by inhalation tests with histamine or methacholine, by exercise, or by isocapnic hyperventilation of cold air, and may be increased in asthma and other conditions such as chronic obstructive bronchitis and cystic fibrosis. In this article we will discuss the measurement of nonspecific bronchial responsiveness by inhalation tests with histamine or methacholine and its relationship to the occurrence and severity of asthma.