Patients
The study was a prospective study based on 80 asthmatic children, aged 7 – 10, who were diagnosed with bronchial asthma <5 years of age based on mAPI (modified API) (Asthma Predicted Index) criteria (11,12). Children were registered, diagnosed and followed up in our outpatient allergy outpatient clinic. Patients with other chronic diseases such as congenital or acquired heart and lung diseases, rheumatologic diseases and immunodeficiency were excluded from the study.
Parents were requested to attend the clinic with their children by phone call. They were interviewed personally. At the first visit they were informed about the purpose of the study, how to score allergy and asthma symptoms and underwent skin testing. Demographic data regarding sex, type of residence, exposure to molds, animals at home, exposure to tobacco, perinatal history (type of delivery, APGAR score, birth weight) as well as family and medical history were taken, patients were classified as atopic based on history and positive skin prick testing. In patients unable to undergo skin testing, a serum specific IgE for a specific allergen was employed. At this visit children were divided into two groups: remission group - children in whom asthma treatment was discontinued at the last visit to the allergy clinic due to the absence of clinical symptoms of the disease and persistent group - children who remained on anti-asthmatic therapy due to asthma symptoms. Patients were recruited to each study group until the number of 40 children in each group was reached. The second visit took place six months after the first one, and the third visit took place 12 months from the first visit to confirm asthma status. During each visit lung function and exhaled nitric oxide concentration were measured and the patients’ diaries were evaluated. Additionally, at the third visit, blood samples were obtained from all patients to assess immunological parameters.
Asthma diagnosis in early childhood
The childhood asthma was defined as positive by using the modified Asthma Predicted Index (mAPI) which requires 4 or more wheezing episodes in the last year, in addition to one of three major criteria: physician-diagnosed parental asthma, physician-diagnosed child eczema, sensitization to ≥ 1 aeroallergen or two out of three minor criteria: wheezing apart from colds, blood eosinophils ≥ 4%, sensitization to ≥ 1 food allergen (11, 12).
Asthma remission
Clinical remission of asthma in our patient was defined as the absence of asthma symptoms for at least 12 months without the use of inhaled corticosteroids and short acting beta2 agonists. In the cases with clinical remission, the spirometry and reversibility test confirmed complete remission (13).
Allergen sensitization
Skin prick testing to standard allergen extracts was performed using Allergopharma, Reinbek, Germany. Reaction >3mm in diameter above the negative control recorded at 15 min were considered positive.
The measurements of serum specific IgE was applied, if necessary. Allergen sensitization was defined as specific IgE of ≥0.35 KU/L for at least one of tested allergens (chemiluminescence method (CLIA), Immulite 2000, XPI, Siemens, Germany). For the purpose of the study we defined allergy as the presence of serum IgE of ≥0.35 KU/L specific for allergens: i.e. dust mites, moulds, cat and dog dander, grasses, wild grasses, and tree pollen.
Symptom-medication score
The combined symptom-medication score (SMS) was calculated as a sum of symptom score (SS) and the medication score (MS) recorded by the patients on a daily basis between the visits (14). SS was recorded with the use of a point score system for the most common symptoms of rhinitis (rhinorrhea, sneezing, itching, and nasal congestion), conjunctivitis (ocular pruritus, watery eyes, itching), and respiratory disorders (cough, wheeze, and dyspnea). The results of the score helped to document asthma remission/asthma symptoms as well as allergic rhinitis (AR).
Lung function, body plethysmography (sRtot, Rocc)
All pulmonary function tests were performed with a Master Screen unit (Erich Jaeger Gmbh-Hochberg, Germany), as described elsewhere, in accordance with the ATS/ERS guidelines (15,16). The highest of 3 successful measurements of spirometry was taken and analysed. The results were expressed as the percentage of a predicted value.
Nitric oxide (NO) measurement
Fractional exhaled nitric oxide (FeNO) was measured with a chemiluminescence analyser (Sievers NOA 280i, CO, USA). Exhalations were performed in accordance with the ATS/ERS guidelines (17). Children had to maintain a constant expiratory flow of 50 mL/s for at least 3 seconds and exhalations were repeated until three measurements were within 5% of the mean.
Immunological assessment
The following panel of antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin-chloro-phyll-protein (Per-CP), or allophycocyanin (APC), was used for the following assays: PPARG-FITC, CD11c-PE, CD 25 FITC, CD 4 PerCP, CD 71 PE, CD 73, PerCP, Anti- GARP APC, FOXP3 PE, (all antibodies from Becton Dickinson, San Diego, CA, USA) and SOCS3 (from Abbexa, Cambridge, UK). All procedures were carried out according to the manufacturer's instructions. The detailed description of the immunological tests and cells preparation is included in an appendix.
The study was approved by the Medical Ethics Committee of the Medical University of Lodz. All parents or legal guardians gave their oral and written consent for the evaluation of data from the medical documentation of their children.
Statistical methods
Statistical analysis was conducted in three steps. The first and second step comprised the logistic regression analysis in univariate followed by the multivariate model. Significant (p<0,1) predictors of asthma remission defined in univariate models were included into the final multivariate model. During the third step, immunological parameters were compared in relation to the presence or absence of previously defined clinical predictors of asthma remission. The significance threshold of p-level was set at 0.05. The statistical analysis was performed with the STATISTICA 13.1 (StatSoft Poland, Kraków).
Results
Eighty patients were included into the analysis. Clinical characteristics of study groups are presented in Table 1.
Clinical predictors of asthma remission
In the first step all available clinical data as independent variables were included into the univariate model of logistic regression analysis together with asthma remission as a dependent variable. The results are shown in Table 2. The presence of mAPI criteria such as inhalant allergy, wheezing apart from colds, an increased eosinophil blood count, as well as the presence of house dust mite (HDM)-allergy and allergic rhinitis, were associated with a reduced prevalence of asthma remission.
Inflammatory/immunoregulatory predictors of asthma remission
In the second step inflammatory/immunoregulatory data (categorized according to lower and upper quartiles) as independent variables were included into the univariate model of logistic regression analysis together with asthma remission as dependent variable. Only the FeNO level was associated with asthma remission (Table 3). The second versus the first quartile of FeNO increased the prevalence of asthma remission, but the third and fourth did not affect it.
Finally, all statistical predictors were included into the one model of multivariate logistic regression analysis. The results showed that only the current allergic rhinitis independently decreased the probability of asthma remission (OR=0,15; 95%CI:0,039 to 0,56).
Associations between clinical and inflammatory/immunoregulatory predictors of asthma remission
The presence of increased eosinophil blood count in API (Asthma Predictive Index) criteria was associated with a lower expression of CD25 positive cells (Table 4). Current HDM-allergy was associated with a higher FeNO level and a higher expression of CD25CD71 positive cells. Allergic rhinitis was associated with a higher expression of PPAR and CD25CD71 positive cells; in the same group of patients a lower expression of GARP positive cells were observed.
Allergy to house dust mite allergens and the presence of allergic rhinitis symptoms determine the expression of CD25 + CD71 + cells in patients with/without asthma. Figure 1.