Gisela Margarit

Airway inflammation in the elite athlete and type of sport

Background: The prevalence of asthma and bronchial hyper-responsiveness is greater in elite athletes than in the general population, and its association with mild airway inflammation has recently been reported. Objective: To study the relationship between the type of sport practised at the highest levels of competition (on land or in water) and sputum induction cell counts in a group of healthy people and people with asthma. Material and methods: In total, 50 athletes were enrolled. Medical history, results of methacholine challenge tests and sputum induced by hypertonic saline were analysed Results: Full results were available for 43 athletes, who were classified by asthma diagnosis and type of sport (land or water sports). Nineteen were healthy (10 land and 9 water athletes) and 24 had asthma (13 land and 11 water athletes). Although the eosinophil counts of healthy people and people with asthma were significantly different (mean difference 3.1%, 95% CI 0.4 to 6.2, p = 0.008), analysis of variance showed no effect on eosinophil count for either diagnosis of asthma or type of sport. However, an effect was found for neutrophil counts (analysis of variance: F = 2.87, p = 0.04). There was also a significant correlation between neutrophil counts and both duration of training and bronchial hyper-responsiveness among athletes exposed to water (Spearman’s rank correlations, 0.36 and 0.47, p = 0.04 and 0.04, respectively). Conclusions: Elite athletes who practice water sports have mild neutrophilic inflammation, whether or not asthma is present, related to the degree of bronchial hyper-reactivity and the duration of training in pool water.

Inflamación bronquial, clínica respiratoria y función pulmonar en el síndrome de Sjögren primario

INTRODUCTION There is no information available regarding the relationship between the respiratory symptoms or lung function and bronchial inflammation, measured by induced sputum. OBJECTIVES Description of the clinical characteristics, radiographic images and lung function of patients suffering from Primary Sjögren Syndrome (PSS), and to assess the relationship with the inflammatory airway profile. METHODS We analysed clinical, radiology, lung function tests, bronchial hyperresponsiveness and inflammatory data in the induced sputum from 36 consecutive patients with PSS. RESULTS A total of 58% of patients had hoarseness and 42% had cough and dispnea. No lung dysfunction was observed, although 46% (n=16) had a positive bronchial response. Lymphocytosis >2.6% in induced sputum was observed in 69% of all sputa. There was chronic cough in 29% of patients with lymphocytosis (n=24), whereas 73% were normal (n=11) (P=.02). The duration time of cough was less for the former (P=.02). On the contrary a positive bronchial response was associated with lymphocytosis >2.6% (P=.02). Lipophages were present in 55% of pathological sputa (n=22) (index >15) versus 18% of the non-pathological ones (n=11) (P=.05). CONCLUSION Hoarseness, cough and dyspnea are frequent respiratory symptoms in PSS, although there is a wide variation in the relationship with bronchial responsiveness and airway inflammation. Lymphocytosis in the airways is another site of the infiltrative process in PSS, and the induced sputum is a complementary tool in the identification of active inflammatory process.

Markers of Airway Remodeling in Induced Sputum From Healthy Smokers

OBJECTIVE Airway remodeling in chronic obstructive pulmonary disease (COPD) has been linked to the equilibrium between matrix metalloproteinase (MMP) 9 and its inhibitor, tissue inhibitor of metalloproteinase (TIMP) 1. However, that equilibrium has not been analyzed in healthy smokers. The aim of this study was to assess the equilibrium between MMP-9 and TIMP-1 in induced sputum from healthy smokers, healthy nonsmokers (controls), and patients with COPD. PATIENTS AND METHODS Samples of induced sputum were obtained from 35 individuals: 12 healthy smokers, 12 controls, and 11 patients with COPD. In each sample, a differential cell count was performed and enzyme-linked immunosorbent assays were used to analyze the concentrations of MMP-9 (total and active fraction) and TIMP-1. RESULTS Compared with controls, healthy smokers were found to have a higher mean (SD) concentration of total MMP-9 (273 [277] ng/mL vs 128 [146] ng/mL) and a higher ratio of total MMP-9 to TIMP-1 (0.16 [0.14] vs 0.08 [0.06]). However, the ratio of active MMP-9 to TIMP-1 was similar in the 2 groups. Samples from patients with COPD had the highest concentrations of total MMP-9 (477 [262] ng/mL) and active MMP-9 (178 [126] ng/mL) and the lowest concentrations of TIMP-1 (1.044 [1.036] microg/mL). When all groups were considered together, there was an inverse relationship between the MMP-9/TIMP-1 ratio and the forced expiratory volume in the first second (FEV1). The relationship between the active MMP-9/TIMP-1 ratio and FEV1 was even stronger, and the relation of both ratios with FEV1 became stronger still when smoking was considered. CONCLUSIONS Healthy smokers had a higher concentration of total MMP-9 and that concentration was correlated with their exposure to tobacco smoke. Maintenance of the active MMP-9/TIMP-1 ratio in healthy smokers may explain the absence of progressive airway obstruction. Measurement of active MMP-9 concentration could be useful for assessment of airway remodeling.

Método para amplificar cultivos primarios de células epiteliales bronquiales

Objetivo Los cultivos celulares son un buen modelo para el estudio de las enfermedades pulmonares, pero son dificiles de reproducir y producen un numero limitado de celulas. El objetivo de este estudio ha sido desarrollar un metodo que incrementase la produccion de celulas epiteliales bronquiales (CEB) humanas en cultivos primarios. Material y metodos Se proceso un total de 12 muestras (9 procedentes de muestras quirurgicas y 3 de biopsias endoscopicas) en placas recubiertas de colageno tipo I con medio suplementado para CEB. Al iniciarse la proliferacion celular a su alrededor, los explantes se extrajeron y subcultivaron sucesivamente. Las celulas restantes se dejaron proliferar y se tripsinizaron tras alcanzar mas del 50% de confluencia. Se valoraron el numero de celulas obtenidas, la viabilidad y la citoqueratina 7. Resultados El numero total de celulas obtenidas con este metodo supero en una media de 3 veces el numero de CEB humanas obtenidas en cultivos primarios simples. El numero maximo de subcultivos fue de 5, la viabilidad media (± desviacion estandar) fue de 91,9 ± 11,7% y el porcentaje de celulas positivas para la citoqueratina 7 del 30,71 ± 10,68%. Conclusiones El metodo descrito para amplificar cultivos primarios de CEB permite incrementar la produccion de celulas obtenidas.

Bronchial Exudate of Serum Proteins During Asthma Attack

OBJECTIVE Although altered vascular permeability and edema of the bronchial mucosa are associated with asthma attack, their influence on its severity remains unknown. We address this issue by comparing relative indices for the concentration of albumin (RIAlb) and alpha2-macroglobulin (RIalpha2M) in induced sputum and peripheral blood from patients with exacerbated asthma, patients with stable asthma, and control subjects. PATIENTS AND METHODS Forty-six volunteers participated in the study: 14 with exacerbated asthma (forced expiratory volume in the first second [FEV1] 74.3% [SD, 20.8%] of reference), 23 with stable asthma (FEV1 93.6% [7.5%]), and 9 controls (FEV1 101.1% [9.9%]). The concentrations of albumin and alpha2-macroglobulin were quantified by immunoturbidimetry and immunonephelometry, respectively. The relative index was then calculated by dividing the concentration in sputum supernatant by the concentration in peripheral blood. RESULTS The mean RIAlb was 1.2 (1.1) in the control group, 2.9 (3.1) in the stable asthma group, and 6.0 (6.7) in the exacerbated asthma group. The RIalpha2M values were 11.7 (10.9), 11.9 (14.7), and 3.2 (3.8) for the control group and stable and exacerbated asthma groups, respectively. The increases in the RIAlb values between all groups, and the decrease in the RIalpha2M value between the exacerbated asthma and control groups were statistically significant (P<.05). The percentage of neutrophils, but not of eosinophils, in sputum was correlated with the RIAlb (r=0.39; P=.008) but not the RIalpha2M (r=-0.035; P=.82). FEV1 displayed an inverse relationship with the RIAlb (r=-0.43; P=.009) but not with the RIalpha2M (r=-0.206; P=.24). No correlation was found between oxyhemoglobin saturation and either the RIAlb (r=-0.33; P=.19) or the RIalpha2M (r=-0.12; P=.84). CONCLUSIONS Vascular permeability is altered during asthma exacerbations and appears to be correlated with the presence of neutrophils and the degree of bronchial obstruction.

Bronchial Plasma Exudation After Adenosine Monophosphate or Methacholine Challenge

Nonspecific hyperresponsiveness to adenosine monophosphate is better related to airway inflammation than methacholine. Adenosine induces mast cells and other cells to release inflammatory mediators that produce bronchoconstriction and perhaps other inflammatory effects, such as plasma exudation, which have not been well studied. We compared the plasma exudation effect, as measured in induced sputum, between adenosine and methacholine challenge in healthy and asthmatic subjects. In a cross-over design, 42 subjects were randomly challenged with adenosine or methacholine. After recovery, induced sputum was collected on 2 separate days, 48 to 72 hours apart. In the control group, an additional challenge with saline was performed. Differential cell counts and albumin and alpha2-macroglobulin levels were determined. The sputum volume obtained was sufficient to measure proteins in only 34 subjects: 10 healthy individuals and 24 mild asthmatics. There was a significant difference between adenosine and methacholine in sputum albumin (mean differences: 68[73.4] μg/L in controls, p = 0.039 and 48.0[162.9] μg/L in asthmatics) and cell counts, but only a tendency in alpha2-macroglobulin. PC20 adenosine was better related to eosinophil counts than methacholine (r = −0.44, p = 0.014). Albumin or alpha2-macroglubulin levels were not significantly correlated with baseline FEV1, PC20, or eosinophil counts. Adenosine, but not methacholine challenge, produces a mild airway plasma exudation that does not seem to be relevant to bronchoconstriction. However, this could be relevant, to some supernatant measurements after adenosine challenge.

Expansion of Primary Bronchial Epithelial Cell Cultures

OBJECTIVE Cell cultures provide a good model for studying lung diseases but they are difficult to reproduce and the number of cells obtained is limited. The aim of this study was to develop a way to increase the production of human bronchial epithelial cells (BEC) in primary cultures. MATERIAL AND METHODS A total of 12 samples (9 from surgical specimens and 3 from endoscopic biopsies) were processed on plates coated with type I collagen with growth medium supplemented for BEC. When cell proliferation started, the explants were removed for successive subculturing. The remaining cells were left to proliferate and were trypsinized after 50% confluence. We recorded the number of cells obtained, cell viability, and the percentage positive for cytokeratin 7. RESULTS The total number of cells obtained by this method was 3-fold the number of human BEC obtained with simple primary cultures. The maximum number of subcultures was 5, mean (SD) cell viability was 91.9% (11.7%), and the percentage of cells positive for cytokeratin 7 was 30.71% (10.68%). CONCLUSIONS The described method for expanding primary BEC cultures increases cell production.