Original article
Bronchial inflammation in seasonal allergic rhinitis with or without asthma in relation to natural exposure to pollen allergens
P. Panznera,
, I. Malkusováa, M. Vachováa, M. Li¿kaa, P. Brodskáb, O. R¿¿i¿kovác, M. Malýd
Corresponding author
a Department of Immunology and Allergology, Faculty of Medicine in Pilsen, Charles University Prague, Czech Republic
b Department of Dermatology, Faculty of Medicine in Pilsen, Charles University Prague, Czech Republic
c Department of Respiratory Medicine, Faculty of Medicine in Pilsen, Charles University Prague, Czech Republic
d National Institute of Public Health, Prague, Czech Republic
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Panzner, I. Malkusová, M. Vachová, M. Li¿ka, P. Brodská, O. R¿¿i¿ková, M. Malý" "autores" => array:7 [ 0 => array:4 [ "nombre" => "P." "apellidos" => "Panzner" "email" => array:1 [ 0 => "panzner@fnplzen.cz" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">*</span>" "identificador" => "cor0005" ] ] ] 1 => array:3 [ "nombre" => "I." "apellidos" => "Malkusová" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 2 => array:3 [ "nombre" => "M." "apellidos" => "Vachová" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 3 => array:3 [ "nombre" => "M." "apellidos" => "Li¿ka" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 4 => array:3 [ "nombre" => "P." "apellidos" => "Brodská" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">b</span>" "identificador" => "aff0010" ] ] ] 5 => array:3 [ "nombre" => "O." "apellidos" => "R¿¿i¿ková" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">c</span>" "identificador" => "aff0015" ] ] ] 6 => array:3 [ "nombre" => "M." "apellidos" => "Malý" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">d</span>" "identificador" => "aff0020" ] ] ] ] "afiliaciones" => array:4 [ 0 => array:3 [ "entidad" => "Department of Immunology and Allergology, Faculty of Medicine in Pilsen, Charles University Prague, Czech Republic" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Department of Dermatology, Faculty of Medicine in Pilsen, Charles University Prague, Czech Republic" "etiqueta" => "b" "identificador" => "aff0010" ] 2 => array:3 [ "entidad" => "Department of Respiratory Medicine, Faculty of Medicine in Pilsen, Charles University Prague, Czech Republic" "etiqueta" => "c" "identificador" => "aff0015" ] 3 => array:3 [ "entidad" => "National Institute of Public Health, Prague, Czech Republic" "etiqueta" => "d" "identificador" => "aff0020" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1052 "Ancho" => 1669 "Tamanyo" => 124172 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Sputum eosinophils (%) – classical staining, arithmetic means and 95% confidence intervals.</p>" ] ] ] "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Background</span><p id="par0005" class="elsevierStylePara elsevierViewall">Seasonal allergic rhinitis is one of the most common allergic diseases and its prevalence is steadily increasing.<a class="elsevierStyleCrossRef" href="#bib0005"><span class="elsevierStyleSup">1</span></a> Epidemiological and pathophysiological studies suggest that allergic rhinitis and asthma are closely related diseases. They often occur together and allergic rhinitis increases the risk of asthma development.<a class="elsevierStyleCrossRefs" href="#bib0010"><span class="elsevierStyleSup">2,3</span></a> The inflammatory response in both conditions is orchestrated by Th2 lymphocytes, while other cell types may play a role, eosinophils being the most important. Asthma and allergic rhinitis are two manifestations of one common allergic respiratory syndrome. The relationship between asthma and allergic rhinitis is complex and upper and lower airways interact with each other.<a class="elsevierStyleCrossRef" href="#bib0020"><span class="elsevierStyleSup">4</span></a></p><p id="par0010" class="elsevierStylePara elsevierViewall">Although the relationship between allergic rhinitis and asthma has been well established, direct links between nasal and bronchial inflammation in seasonal allergic rhinitis without asthma remain to be exactly investigated. Several authors have focused on the effect of natural allergen exposure or nasal challenge on lower airway,<a class="elsevierStyleCrossRef" href="#bib0025"><span class="elsevierStyleSup">5</span></a> but the dynamics of bronchial inflammation in patients with pollen allergic rhinitis during and out of the pollen season are not completely clear yet, as well as the exact consequences of this inflammation.</p><p id="par0015" class="elsevierStylePara elsevierViewall">The presence of bronchial inflammation in non-asthmatic patients with seasonal allergic rhinitis has previously been reported.<a class="elsevierStyleCrossRefs" href="#bib0030"><span class="elsevierStyleSup">6,7</span></a> Natural exposure to pollen allergen induces inflammatory cell recruitment leading to bronchial eosinophilic inflammation in these patients.<a class="elsevierStyleCrossRef" href="#bib0040"><span class="elsevierStyleSup">8</span></a> On the other hand, increased inflammatory markers in nasal mucosa from non-asthmatic patients with seasonal allergic rhinitis after segmental bronchial allergen provocation were found, suggesting a systemic cross-talk between upper and lower airways. High incidence of airway hyperresponsiveness in non-asthmatic patients with seasonal allergic rhinitis is also well known. Both natural exposure to pollen and nasal allergen challenge induce an increase in airway responsiveness to methacholin in non-asthmatic patients with seasonal allergic rhinitis.<a class="elsevierStyleCrossRef" href="#bib0025"><span class="elsevierStyleSup">5</span></a> In studies focusing on the relationship between sputum eosinophils and airway responsiveness in general population, no correlations were found, although there was a weak correlation found when atopic subjects were considered alone.<a class="elsevierStyleCrossRef" href="#bib0045"><span class="elsevierStyleSup">9</span></a> No significant differences were found between healthy subjects and subjects with rhinitis. Sputum eosinophil counts did not correlate with methacholine PC20 or blood eosinophils.<a class="elsevierStyleCrossRef" href="#bib0050"><span class="elsevierStyleSup">10</span></a> Some studies describe changes in sputum ECP level after nasal allergen challenge. This parameter seems also to reflect well the inflammatory changes in bronchi.<a class="elsevierStyleCrossRef" href="#bib0055"><span class="elsevierStyleSup">11</span></a></p><p id="par0020" class="elsevierStylePara elsevierViewall">Seasonal allergic rhinitis could predispose to the development of chronic bronchial inflammation as observed in asthma. Direct links between nasal and bronchial inflammation and airway responsiveness in patients with seasonal allergic rhinitis without asthma are not fully understood yet.<a class="elsevierStyleCrossRef" href="#bib0060"><span class="elsevierStyleSup">12</span></a></p><p id="par0025" class="elsevierStylePara elsevierViewall">The aim of this study was to confirm the presence of bronchial inflammation in pollen allergic subjects with seasonal allergic rhinitis with or without asthma, to compare the intensity of the inflammation during the pollen season (natural allergen exposure) with the periods out of the pollen season (before and after the season) and to compare the intensity of the inflammation in patients with allergic rhinitis with asthma vs. patients without asthma and vs. healthy controls.</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Patients</span><p id="par0030" class="elsevierStylePara elsevierViewall">38 patients with seasonal allergic rhinitis and seasonal intermittent bronchial asthma (BA), 52 patients with seasonal allergic rhinitis without asthma (SAR) and 23 healthy controls (C) were recruited consecutively from patients treated in the outpatient allergology service and from healthy volunteers. The diagnosis of asthma was based on clinical history of recurrent episodes of wheezing, breathlessness and/or cough associated with reversible airway obstruction. Historical positivity of reversibility test was required (improvement in FEV<span class="elsevierStyleInf">1</span> of at least 12% 30<span class="elsevierStyleHsp" style=""></span>min after inhalation of 400<span class="elsevierStyleHsp" style=""></span>¿g of salbutamol). 55.8% of patients in the SAR group and 55.3% of patients in the BA group used antihistamines for symptom relief during the followed pollen season. Demographic characteristics of the subjects are described in <a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>.</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><p id="par0035" class="elsevierStylePara elsevierViewall">Seasonal allergy was defined as occurrence of seasonal symptoms during spring and/or summer and at least one positive skin prick test (mean wheal diameter ≥3<span class="elsevierStyleHsp" style=""></span>mm) to a pollen allergen (trees, grass, weeds) and a positive specific serum IgE result (≥0.7<span class="elsevierStyleHsp" style=""></span>IU/ml) to this pollen allergen.</p><p id="par0040" class="elsevierStylePara elsevierViewall">All patients had a clinical history of allergic rhinitis during the previous two or more pollen seasons; all patients with asthma had a clinical history of seasonal asthma for at least the pollen season prior to the study. Asthma patients were treated only by short-acting ¿<span class="elsevierStyleInf">2</span>-agonists administered as required; no treatment with systemic, inhaled or nasal corticosteroids was used before or during the study. Healthy controls did not use any form of medication influencing allergic inflammation, they had no symptoms of rhinitis, no history of asthma or other allergic diseases and they had no positivity in skin prick testing to common aeroallergens.</p><p id="par0045" class="elsevierStylePara elsevierViewall">All subjects had no upper respiratory tract infection in the last four weeks prior to each sputum induction. Non-smoking was a prerequisite for selection. Informed consent for this study was signed by all subjects.</p><p id="par0050" class="elsevierStylePara elsevierViewall">All included subjects were examined 6–8 weeks before the expected start of their seasonal symptoms, during the period of symptoms and 6–8 weeks after the end of symptoms. Treatment with antihistamines and cromones during the period of sampling was not restricted, as well as allergen immunotherapy in maintenance phase. The comparative analysis (results not shown) proved that none of these treatments had any influence on the followed inflammatory parameters.</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Lung function measurement</span><p id="par0055" class="elsevierStylePara elsevierViewall">Measurement of basic lung function was performed by means of a computerised system MasterScope (Jaeger, Wuerzburg, Germany) according to standard guidelines. Subjects did not use short or long acting bronchodilators 12<span class="elsevierStyleHsp" style=""></span>h prior to the measurement and did not drink tea or coffee in the morning before the measurement and sputum induction.</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Sputum induction and processing</span><p id="par0060" class="elsevierStylePara elsevierViewall">Sputum was induced by the method described by Pizzichini.<a class="elsevierStyleCrossRef" href="#bib0065"><span class="elsevierStyleSup">13</span></a> Briefly: patients inhaled increasing concentrations of hypertonic saline (3%, 4% and 5% NaCl) each for 7<span class="elsevierStyleHsp" style=""></span>min through a mouthpiece with a nose clip, 15<span class="elsevierStyleHsp" style=""></span>min after premedication with 200<span class="elsevierStyleHsp" style=""></span>¿g of inhaled salbutamol. Aerosol was generated by an ultrasonic nebuliser DeVilbiss 2000 (DeVilbiss, Somerset, USA) with the output of 1<span class="elsevierStyleHsp" style=""></span>ml/min. Lung function tests were performed before sputum induction and after each period of inhalation. Subjects were encouraged to cough deeply after each period and in any other time they felt the need. Samples were collected in a sterile container and kept at 4<span class="elsevierStyleHsp" style=""></span>°C until processing.</p><p id="par0065" class="elsevierStylePara elsevierViewall">Sputum was processed according to the method described by Pin and Gibson<a class="elsevierStyleCrossRefs" href="#bib0070"><span class="elsevierStyleSup">14,15</span></a> within 2<span class="elsevierStyleHsp" style=""></span>h after collection. Sputum plugs were selected and treated with four times their weight of freshly prepared 0.1% dithiothreitol (Sputolysin, Calbiochem, San Diego, USA) at room temperature for 15<span class="elsevierStyleHsp" style=""></span>min. The suspension was four times its volume diluted with phosphate buffered saline. Dispersed samples were filtered through a 48<span class="elsevierStyleHsp" style=""></span>¿m nylon mesh to remove cell debris and mucus. The proportion of salivary squamous cells was noted and viability was determined using the trypan blue exclusion method. Only samples with cell viability ≥70% and squamous cell contamination ≤20% were considered for evaluation. Slides were prepared by centrifuging 100<span class="elsevierStyleHsp" style=""></span>¿l of the cell suspension at 450<span class="elsevierStyleHsp" style=""></span>rpm for 6<span class="elsevierStyleHsp" style=""></span>min (Cytospin IV, Shandon, England). The remaining sample volume was centrifuged at 1500<span class="elsevierStyleHsp" style=""></span>rpm for 5<span class="elsevierStyleHsp" style=""></span>min and supernatant was stored at −70<span class="elsevierStyleHsp" style=""></span>°C for later analysis.</p><p id="par0070" class="elsevierStylePara elsevierViewall">Slides for classical staining were air-dried, fixed and stained with Hemacolor Rapid (Merck, Darmstadt, Germany). Slides for immunocytochemistry staining were fixed in acetone–methanol and stored at −20<span class="elsevierStyleHsp" style=""></span>°C until use. Immunocytochemistry was performed using DAKO (Glostrup, Denmark) monoclonal antibodies, by the alkaline-phosphatase/anti-alkaline-phosphatase (APAAP) staining procedure. Antibodies anti-CD68 targeted macrophages, anti-neutrophil-elastase targeted neutrophils and anti-major basic protein (MBP) targeted eosinophils. Dilutions 1:50, 1:100, and 1:30 respectively, were used. Reading of slides obtained by either method was carried out by two investigators who had no knowledge about the clinical characteristic of the patients and 400<span class="elsevierStyleHsp" style=""></span>cells were counted. Results were expressed as percentage of the total non-squamous cell count.</p><p id="par0075" class="elsevierStylePara elsevierViewall">ECP in the sputum fluid phase was measured by chemiluminiscent immunometric assay (Immulite 2000, Siemens, Germany). The results were adjusted for the dilution factor. The detection threshold was 0.2<span class="elsevierStyleHsp" style=""></span>ng/ml.</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Statistical analysis</span><p id="par0080" class="elsevierStylePara elsevierViewall">Continuous data are presented as arithmetic mean and standard deviation values, ECP levels were log transformed and described as geometric means. Variability of the data was characterised by 95% confidence intervals. Group and time effects assessment was based on a repeated-measures analysis of variance followed by Sidak's test for multiple comparisons and orthogonal contrasts. Differences in proportions between groups were tested by Pearson's <span class="elsevierStyleItalic">¿</span><span class="elsevierStyleSup">2</span>-test. Correlations between variables were analysed using Spearman's rank correlation coefficient. <span class="elsevierStyleItalic">p</span>-Values less than or equal to 0.05 were considered statistically significant.</p></span></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Results</span><p id="par0085" class="elsevierStylePara elsevierViewall">The groups of patients and controls did not differ with respect to sex and age significantly. Sputum induction was successful in 288 (85.0%) of procedures. Technical problems in sputum processing were encountered in 77 (26.7%) of these patients’ samples (cell viability ≤70%, squamous cells contamination ≥20% or high salivary content). These samples were excluded from evaluation. Paired sputum values were available in 70 (61.9%) subjects (see <a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>). Mean weight of obtained sputum samples was 0.728<span class="elsevierStyleHsp" style=""></span>g, total non-squamous cell count 2.84<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">6</span>/ml and cell viability 73%. These parameters were not statistically different among the groups and periods of examination.</p><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Pulmonary function</span><p id="par0090" class="elsevierStylePara elsevierViewall">Mean FEV<span class="elsevierStyleInf">1</span> (% predicted) values in patients of all groups in the three timepoints are shown in <a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>. No significant differences and changes in baseline pulmonary function before, during and after the pollen season were observed in any group. No correlations between FEV<span class="elsevierStyleInf">1</span> and sputum eosinophils or ECP were found.</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Concentration of sputum ECP</span><p id="par0095" class="elsevierStylePara elsevierViewall">No significant differences in sputum ECP levels among the groups in any of the periods of examination were found. Significant rise of sputum ECP levels during the pollen season compared to ECP levels out of the season was observed only in patients with SAR (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.006). In consequence, there is an apparent although still non-significant difference in time course between the SAR group and the two other groups (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.105) (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>).</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Differential sputum cell counts</span><p id="par0100" class="elsevierStylePara elsevierViewall">Differential cell count was assessed by classical staining (Haemacolor Rapid) and by immunocytochemistry staining.</p></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Classical staining</span><p id="par0105" class="elsevierStylePara elsevierViewall">The mean eosinophil counts expressed as percentage of non-squamous cells, were on the whole significantly higher in the BA group compared to the C group (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span><<span class="elsevierStyleHsp" style=""></span>0.001) reaching significance particularly before (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.005) and during (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span><<span class="elsevierStyleHsp" style=""></span>0.001) the pollen season. The difference between the SAR and C groups is in total on the borderline of significance (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.073) but is not provable at any single time point. The difference between the BA and SAR groups was significant globally (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.007) and in particular during the pollen season (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.017). The rise of eosinophil count during the pollen season compared with values out of the pollen season was significant only in the BA group (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.014) (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>).</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Immunocytochemistry staining</span><p id="par0110" class="elsevierStylePara elsevierViewall">Similar changes as in classical staining were observed also in the detection of eosinophils by immunocytochemistry. The mean eosinophil counts expressed as percentage of the non-squamous cells were on the whole significantly higher in both the BA and SAR groups compared to the C group (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span><<span class="elsevierStyleHsp" style=""></span>0.001 and <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.003, respectively). The difference between the BA and C groups was significant in measurements before (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.046) and during (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.015) the pollen season and was on the borderline after the season (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.077). The difference between the SAR and C groups was manifested especially during the pollen season (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.016). No significant difference between the BA and SAR groups was observed in any timepoint of examination. The rise of eosinophil count during the pollen season was apparent only in the SAR group (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.073) (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>).</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia><p id="par0115" class="elsevierStylePara elsevierViewall">There were no significant differences in number of macrophages and neutrophils in sputum samples, during and out of the pollen season in any group.</p></span></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Discussion</span><p id="par0120" class="elsevierStylePara elsevierViewall">In several studies a model of artificial allergen challenge was used to study airway inflammation in asthma and rhinitis.<a class="elsevierStyleCrossRef" href="#bib0025"><span class="elsevierStyleSup">5</span></a> We decided to use the natural allergen exposure with its long-time duration to obtain results which reflect best the conditions in real disease. It was proven that bronchial and nasal allergen challenges give similar inflammatory response in the airway but less systemic inflammation than seasonal exposure.<a class="elsevierStyleCrossRef" href="#bib0055"><span class="elsevierStyleSup">11</span></a> The nasal allergen challenge was shown to elevate sputum eotaxin and sputum eosinophils.<a class="elsevierStyleCrossRef" href="#bib0080"><span class="elsevierStyleSup">16</span></a> However, other authors did not show elevation of sputum eosinophils even after subsequent multiple nasal allergen challenges which may more adequately approximate the natural exposure.<a class="elsevierStyleCrossRef" href="#bib0085"><span class="elsevierStyleSup">17</span></a> Other studies focused on soluble inflammatory mediators only<a class="elsevierStyleCrossRefs" href="#bib0090"><span class="elsevierStyleSup">18,19</span></a> and showed similar results as our study. We confirmed the findings also by following the cellular inflammatory markers in sputum and we extended the observations to two measurements out of the period of allergen exposure – both before and after the pollen season – with the aim of following the possible persistence of eosinophilic inflammation. We also aimed to minimise the possible inconsistencies in sputum cell profiles in individual patients by repeated sampling.<a class="elsevierStyleCrossRefs" href="#bib0045"><span class="elsevierStyleSup">9,20,21</span></a></p><p id="par0125" class="elsevierStylePara elsevierViewall">Induced sputum examination is a time consuming and technically not simple procedure, which limits its extension to the practice. Sometimes also safety issues of this procedure are raised. Inhalation of hypertonic saline aerosol carries the risk of bronchoconstriction in patients with bronchial hyperresponsiveness. We performed sputum induction in patients with baseline FEV<span class="elsevierStyleInf">1</span> higher than 90% predicted and we did not observe a considerable fall in FEV<span class="elsevierStyleInf">1</span> after inhalation of saline aerosol in any of the subjects. We confirmed that sputum induction is a reliable and safe alternative to obtain secretion from the lower airway, in order to study the presence and quality of cells and inflammatory mediators present in the airway and their changes during the course of the disease. Induced sputum can be used to monitor the presence and severity of lower airway inflammation both in asthma and allergic rhinitis.</p><p id="par0130" class="elsevierStylePara elsevierViewall">Sputum eosinophil count seems to be the most important marker of airway inflammation.<a class="elsevierStyleCrossRefs" href="#bib0110"><span class="elsevierStyleSup">22,23</span></a> Increased number of sputum eosinophils is associated with allergen exposure and reflects the severity of airway inflammation in asthmatic patients<a class="elsevierStyleCrossRef" href="#bib0120"><span class="elsevierStyleSup">24</span></a> and it decreases during corticosteroid therapy.<a class="elsevierStyleCrossRef" href="#bib0125"><span class="elsevierStyleSup">25</span></a> It is estimated that more than 80% patients with asthma previously not treated with corticosteroids and more than 50% of those who used inhaled corticosteroid therapy, may have elevated sputum eosinophil numbers.<a class="elsevierStyleCrossRef" href="#bib0120"><span class="elsevierStyleSup">24</span></a></p><p id="par0135" class="elsevierStylePara elsevierViewall">Results of sputum cell counts obtained by means of classical staining and by immunocytochemistry staining are comparable and 89% of samples in our study were evaluated identically in the sense of elevated eosinophil count. Differences in the percentages of eosinophils obtained by these two methods were not statistically significant and may be explained by methodology aspects: immunocytochemistry is based on the detection of cell antigenic markers (MBP) and classical differential cell count is based on morphology and cellular staining.</p><p id="par0140" class="elsevierStylePara elsevierViewall">Some studies refer the relation of airway inflammation and airway hyperresponsiveness<a class="elsevierStyleCrossRef" href="#bib0130"><span class="elsevierStyleSup">26</span></a> or report significant inverse correlation between sputum eosinophils and lung function. Other authors did not observe this correlation, or the correlation between sputum eosinophils and bronchial responsiveness. This might be due to relatively good lung function in the included asthma patients. In our study none of the enrolled patients had FEV<span class="elsevierStyleInf">1</span> lower than 90% predicted. We suppose that this is the reason why we did not observe any correlations between FEV1 and inflammatory markers.</p><p id="par0145" class="elsevierStylePara elsevierViewall">Sputum ECP levels are related to the inflammatory activity of the disease. ECP level in sputum supernatant reflects eosinophil degranulation and does not necessarily closely correlate with sputum eosinophil count,<a class="elsevierStyleCrossRef" href="#bib0135"><span class="elsevierStyleSup">27</span></a> but in general, moderate correlation between sputum ECP and eosinophil count is observed.<a class="elsevierStyleCrossRefs" href="#bib0140"><span class="elsevierStyleSup">28–30</span></a> Sputum soluble inflammatory markers may sometimes reflect better asthma activity than the number of eosinophils alone.<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">31</span></a> Elevated sputum ECP levels were also detected after single allergen challenge in patients with mild allergic asthma<a class="elsevierStyleCrossRef" href="#bib0160"><span class="elsevierStyleSup">32</span></a> and allergic rhinitis.<a class="elsevierStyleCrossRef" href="#bib0165"><span class="elsevierStyleSup">33</span></a> Our results correspond to these facts.</p><p id="par0150" class="elsevierStylePara elsevierViewall">We observed the raise of sputum ECP levels during the pollen season only in SAR patients, surprisingly not in BA patients. This may be due to slightly higher levels of ECP in BA patients present also out of the pollen season, what supports the presence of persisting allergic inflammation in the bronchi of these patients also in asymptomatic periods. On the other hand this result confirms the presence of bronchial allergic inflammation during pollen exposure in patients with no bronchial symptoms (SAR).</p><p id="par0155" class="elsevierStylePara elsevierViewall">The presence of higher numbers of eosinophils in bronchi in BA patients compared to controls confirms persisting allergic inflammation in bronchi of these patients which is present even in asymptomatic periods. This may correspond to a similar situation in asthma patients who are clinically well controlled on therapy.<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">34</span></a> It also corresponds to the fact that asthma, even when in remission, is accompanied by airway inflammation and remodelling.<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">35</span></a></p><p id="par0160" class="elsevierStylePara elsevierViewall">The difference in eosinophil counts between SAR patients and controls is only borderline in classical staining and more clearly expressed in immunocytochemistry staining. The raise in eosinophil counts in SAR patients during the pollen exposure is not pronounced in classical staining, but significantly present in immunocytochemistry staining. In BA patients, this raise is significantly present only in classical staining and not pronounced in immunocytochemistry staining probably because a certain degree of inflammation is present also in periods without allergen exposure.</p><p id="par0165" class="elsevierStylePara elsevierViewall">It seems that the immunocytochemical detection of eosinophils corresponds more closely to sputum ECP levels than the detection by means of classical staining and probably reflects better the real changes in bronchial inflammation during the followed periods.</p><p id="par0170" class="elsevierStylePara elsevierViewall">The main findings of this study indicate that sputum eosinophil counts and ECP levels are to a certain extent and with different dynamics elevated both in BA and SAR patients as a result of natural allergen exposure during pollen season. Our results support the view that subclinical inflammatory changes in the lower airway are present in patients with allergic rhinitis without asthma and in asthma patients both in symptomatic and asymptomatic periods.</p><p id="par0175" class="elsevierStylePara elsevierViewall">The natural courses of untreated or under-treated eosinophilic airway inflammation are unknown. Eosinophilic inflammation can heal spontaneously, especially if it is a result of exposure to an allergen, which ceases. Prolonged eosinophilic inflammation requires treatment with an anti-inflammatory agent. Optimal treatment of patients with SAR in the period of allergen exposure, which may stop the progression of SAR to asthma, is still under investigation. Some authors suggest that effective treatment may have to involve inhaled steroids for 2–3 months.<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">36</span></a> More, it is well known that patients with SAR without asthma symptoms may experience a sudden asthma attack.<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">37</span></a> Corticosteroid therapy was proven to be the most potent anti-inflammatory approach in allergic conditions. Anti-inflammatory effects of antihistamines are limited and their role as the first line treatment in SAR should be reconsidered from this point of view. The similar characteristics of inflammation (Th2 driven) in both BA and SAR may indicate that the therapeutic approaches should be similar as well.</p><p id="par0180" class="elsevierStylePara elsevierViewall">At present, the asthma treatment level is mostly guided solely by clinical indices such as symptoms and lung function and the current therapeutic approach is based on the assumption that there is a relationship between clinical indices of asthma severity and bronchial inflammation. However, a major drawback of these markers is their uncertain relationship with the degree of airway inflammation.<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">38</span></a> Several studies suggest that sputum eosinophils may be useful for tailoring the asthma treatment.<a class="elsevierStyleCrossRefs" href="#bib0195"><span class="elsevierStyleSup">39,40</span></a> Our study supports the idea that the treatment approach should consider the presence of allergic inflammation in the airways. This may concern both persistence of inflammation after the end of pollen exposure in patients with seasonal BA, and the presence of inflammation of bronchi in patients with SAR without asthma symptoms. It should be suitable to consider the treatment by anti-inflammatory drugs instead of antihistamines alone.</p><p id="par0185" class="elsevierStylePara elsevierViewall">In summary, the presence of allergic inflammation of lower airway was detected by means of sputum ECP levels and eosinophil counts not only in patients with seasonal asthma, but also with seasonal allergic rhinitis. This inflammation persists to certain extent even in periods without allergen exposure especially in patients with seasonal asthma. Measurement of bronchial inflammation seems essential not only to achieve proper asthma control, but also to consider the optimal treatment.</p></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Ethical disclosure</span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Protection of human and animal subjects</span><p id="par0190" class="elsevierStylePara elsevierViewall">The authors declare that the procedures followed were in accordance with the regulations of the responsible Clinical Research Ethics Committee and in accordance with those of the World Medical Association and the Helsinki Declaration.</p></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Confidentiality of data</span><p id="par0195" class="elsevierStylePara elsevierViewall">The authors declare that they have followed the protocols of their work centre on the publication of patient data and that all the patients included in the study have received sufficient information and have given their informed consent in writing to participate in that study.</p></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Right to privacy and informed consent</span><p id="par0200" class="elsevierStylePara elsevierViewall">The authors have obtained the informed consent of the patients and/or subjects mentioned in the article. The author for correspondence is in possession of this document.</p></span></span><span id="sec0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Conflict of interest</span><p id="par0205" class="elsevierStylePara elsevierViewall">All the authors declare no conflict of interest.</p></span></span>" "textoCompletoSecciones" => array:1 [ "secciones" => array:9 [ 0 => array:2 [ "identificador" => "xres414309" "titulo" => array:5 [ 0 => "Abstract" 1 => "Background" 2 => "Methods" 3 => "Results" 4 => "Conclusions" ] ] 1 => array:2 [ "identificador" => "xpalclavsec389949" "titulo" => "Keywords" ] 2 => array:2 [ "identificador" => "sec0005" "titulo" => "Background" ] 3 => array:3 [ "identificador" => "sec0010" "titulo" => "Materials and methods" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Patients" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "Lung function measurement" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "Sputum induction and processing" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "Statistical analysis" ] ] ] 4 => array:3 [ "identificador" => "sec0035" "titulo" => "Results" "secciones" => array:5 [ 0 => array:2 [ "identificador" => "sec0040" "titulo" => "Pulmonary function" ] 1 => array:2 [ "identificador" => "sec0045" "titulo" => "Concentration of sputum ECP" ] 2 => array:2 [ "identificador" => "sec0050" "titulo" => "Differential sputum cell counts" ] 3 => array:2 [ "identificador" => "sec0055" "titulo" => "Classical staining" ] 4 => array:2 [ "identificador" => "sec0060" "titulo" => "Immunocytochemistry staining" ] ] ] 5 => array:2 [ "identificador" => "sec0065" "titulo" => "Discussion" ] 6 => array:3 [ "identificador" => "sec0070" "titulo" => "Ethical disclosure" "secciones" => array:3 [ 0 => array:2 [ "identificador" => "sec0075" "titulo" => "Protection of human and animal subjects" ] 1 => array:2 [ "identificador" => "sec0080" "titulo" => "Confidentiality of data" ] 2 => array:2 [ "identificador" => "sec0085" "titulo" => "Right to privacy and informed consent" ] ] ] 7 => array:2 [ "identificador" => "sec0090" "titulo" => "Conflict of interest" ] 8 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2013-03-31" "fechaAceptado" => "2013-06-03" "PalabrasClave" => array:1 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec389949" "palabras" => array:4 [ 0 => "Asthma" 1 => "Allergic rhinitis" 2 => "Induced sputum" 3 => "Pollen allergy" ] ] ] ] "tieneResumen" => true "resumen" => array:1 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "<span class="elsevierStyleSectionTitle" id="sect0010">Background</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Nasal inflammation in allergic rhinitis enhances bronchial Th2 driven inflammation and development of asthma. We assessed bronchial inflammation induced by natural allergen exposure during pollen season in patients with pollinosis with or without asthma to show the intensity of inflammation in asthma and rhinitis and possible persistence of inflammation in periods without allergen exposure.</p> <span class="elsevierStyleSectionTitle" id="sect0015">Methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Sputum was induced in 52 patients with seasonal allergic rhinitis without asthma, 38 patients with seasonal allergic rhinitis and seasonal asthma and 23 healthy volunteers. Sampling was performed 6–8 weeks before the expected beginning of symptoms, during symptomatic period and 6–8 weeks after the end of symptoms. Sputum ECP was measured by means of chemiluminiscent immunometric assay and sputum cell counts were assessed by classical staining and immunocytochemistry.</p> <span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Sputum eosinophils were on the whole higher in both asthma and rhinitis compared to controls (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span><<span class="elsevierStyleHsp" style=""></span>0.001, <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.003). The rise of eosinophils during pollen season compared with values out of pollen season was significant in asthma (classical staining) (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.014) and slightly apparent in rhinitis (immunocytochemistry) (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.073). The seasonal rise of sputum ECP was observed only in rhinitis (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.006).</p> <span class="elsevierStyleSectionTitle" id="sect0025">Conclusions</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Inflammation of the lower airway in patients with allergic rhinitis with and without asthma has been confirmed by means of both sputum eosinophil count and sputum ECP level. Persistent inflammation of lower airway in periods without allergen exposure was proven in seasonal asthma. This may have implications for the therapy of seasonal allergic rhinitis with and without asthma in terms of promoting long-term anti-inflammatory treatment.</p>" ] ] "NotaPie" => array:1 [ 0 => array:2 [ "etiqueta" => "☆" "nota" => "<p class="elsevierStyleNotepara" id="npar0005">This research was performed with institutional support by the Faculty of Medicine in Pilsen, Charles University in Prague.</p>" ] ] "multimedia" => array:4 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1190 "Ancho" => 1670 "Tamanyo" => 138652 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Sputum ECP levels (ng/ml), geometric means and 95% confidence intervals.</p>" ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1052 "Ancho" => 1669 "Tamanyo" => 124172 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Sputum eosinophils (%) – classical staining, arithmetic means and 95% confidence intervals.</p>" ] ] 2 => array:7 [ "identificador" => "fig0015" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 1059 "Ancho" => 1669 "Tamanyo" => 125491 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Sputum eosinophils (%) – immunocytochemistry staining, arithmetic means and 95% confidence intervals.</p>" ] ] 3 => array:7 [ "identificador" => "tbl0005" "etiqueta" => "Table 1" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:2 [ "leyenda" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Age – arithmetic means<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>s.d.</p><p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">Baseline FEV1 (% predicted) – arithmetic means and 95% confidence intervals.</p>" "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head " align="" valign="top" scope="col" style="border-bottom: 2px solid black"> \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">BA \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">SAR \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">C \t\t\t\t\t\t\n \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="table-entry " align="left" valign="top">Subjects enrolled \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">38 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">52 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">23 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="table-entry " align="left" valign="top">Subjects evaluated \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">19 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">33 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">18 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="table-entry " align="left" valign="top">Sex (M/F) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">6/13 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">18/15 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">10/8 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="table-entry " align="left" valign="top">Age \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">32.3<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>8.4 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">38.8<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>10.8 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">43.3<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>11.4 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="table-entry " align="left" valign="top">Baseline FEV1 before the season \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">102.3(98.5–106.2) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">105.2(100.7–109.6) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">111.4(103–119.9) \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="table-entry " align="left" valign="top">Baseline FEV1 during the season \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">102.0(97.9–106.2) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">105.9(101.0–110.9) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">109.3(100–118.6) \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="table-entry " align="left" valign="top">Baseline FEV1 after the season \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">98.1(93.8–102.5) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">103.0(98.6–107.3) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">107.3(98.2–116.3) \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab646881.png" ] ] ] ] "descripcion" => array:1 [ "en" => "<p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Characteristics of subjects.</p>" ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0005" "bibliografiaReferencia" => array:40 [ 0 => array:3 [ "identificador" => "bib0005" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ 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| Year/Month | Html | Total | |
|---|---|---|---|
| 2024 November | 8 | 0 | 8 |
| 2024 October | 59 | 10 | 69 |
| 2024 September | 95 | 15 | 110 |
| 2024 August | 61 | 19 | 80 |
| 2024 July | 70 | 6 | 76 |
| 2024 June | 60 | 4 | 64 |
| 2024 May | 66 | 13 | 79 |
| 2024 April | 74 | 17 | 91 |
| 2024 March | 86 | 12 | 98 |
| 2024 February | 53 | 5 | 58 |
| 2024 January | 78 | 2 | 80 |
| 2023 December | 81 | 7 | 88 |
| 2023 November | 46 | 4 | 50 |
| 2023 October | 95 | 15 | 110 |
| 2023 September | 61 | 2 | 63 |
| 2023 August | 61 | 8 | 69 |
| 2023 July | 75 | 11 | 86 |
| 2023 June | 56 | 10 | 66 |
| 2023 May | 90 | 3 | 93 |
| 2023 April | 99 | 5 | 104 |
| 2023 March | 50 | 5 | 55 |
| 2023 February | 27 | 4 | 31 |
| 2023 January | 43 | 4 | 47 |
| 2022 December | 25 | 6 | 31 |
| 2022 November | 24 | 5 | 29 |
| 2022 October | 20 | 10 | 30 |
| 2022 September | 25 | 14 | 39 |
| 2022 August | 34 | 10 | 44 |
| 2022 July | 35 | 7 | 42 |
| 2022 June | 25 | 7 | 32 |
| 2022 May | 20 | 13 | 33 |
| 2022 April | 23 | 11 | 34 |
| 2022 March | 46 | 13 | 59 |
| 2022 February | 35 | 6 | 41 |
| 2022 January | 77 | 7 | 84 |
| 2021 December | 39 | 15 | 54 |
| 2021 November | 41 | 8 | 49 |
| 2021 October | 41 | 19 | 60 |
| 2021 September | 48 | 10 | 58 |
| 2021 August | 41 | 8 | 49 |
| 2021 July | 23 | 18 | 41 |
| 2021 June | 39 | 8 | 47 |
| 2021 May | 44 | 10 | 54 |
| 2021 April | 66 | 13 | 79 |
| 2021 March | 30 | 8 | 38 |
| 2021 February | 23 | 13 | 36 |
| 2021 January | 29 | 9 | 38 |
| 2020 December | 2 | 2 | 4 |
| 2020 October | 0 | 1 | 1 |
| 2020 August | 0 | 1 | 1 |
| 2020 July | 0 | 1 | 1 |
| 2020 May | 0 | 1 | 1 |
| 2020 February | 0 | 1 | 1 |
| 2019 December | 0 | 1 | 1 |
| 2019 October | 0 | 5 | 5 |
| 2019 July | 0 | 1 | 1 |
| 2019 May | 0 | 16 | 16 |
| 2018 February | 13 | 3 | 16 |
| 2018 January | 9 | 4 | 13 |
| 2017 December | 17 | 3 | 20 |
| 2017 November | 14 | 3 | 17 |
| 2017 October | 15 | 4 | 19 |
| 2017 September | 10 | 3 | 13 |
| 2017 August | 17 | 6 | 23 |
| 2017 July | 13 | 10 | 23 |
| 2017 June | 17 | 9 | 26 |
| 2017 May | 22 | 8 | 30 |
| 2017 April | 21 | 15 | 36 |
| 2017 March | 14 | 22 | 36 |
| 2017 February | 22 | 4 | 26 |
| 2017 January | 15 | 3 | 18 |
| 2016 December | 16 | 5 | 21 |
| 2016 November | 17 | 0 | 17 |
| 2016 October | 46 | 9 | 55 |
| 2016 September | 23 | 9 | 32 |
| 2016 August | 22 | 5 | 27 |
| 2016 July | 11 | 3 | 14 |
| 2016 June | 13 | 6 | 19 |
| 2016 May | 8 | 17 | 25 |
| 2016 April | 20 | 10 | 30 |
| 2016 March | 14 | 10 | 24 |
| 2016 February | 18 | 11 | 29 |
| 2016 January | 24 | 7 | 31 |
| 2015 October | 1 | 1 | 2 |
| 2015 July | 1 | 1 | 2 |
| 2015 May | 1 | 2 | 3 |
| 2015 April | 3 | 7 | 10 |
| 2015 March | 1 | 3 | 4 |
| 2015 February | 2 | 10 | 12 |
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