Julio Cortijo

Aclidinium inhibits cholinergic and tobacco smoke-induced MUC5AC in human airways

Mucus hypersecretion and mucin MUC5AC overexpression are pathological features of chronic obstructive pulmonary disease (COPD). This study examines the inhibitory effect of aclidinium, a new long-acting muscarinic antagonist, on MUC5AC expression in human airway epithelial cells. MUC5AC mRNA (RT-PCR) and protein expression (ELISA and immunohistochemistry) were studied in human bronchial tissue and differentiated human airway epithelial cells activated with carbachol (100 &mgr;M) or cigarette smoke extract in the absence or presence of aclidinium. Carbachol increased MUC5AC mRNA and protein expression in human bronchus and cultured epithelial cells. Aclidinium inhibited the carbachol-induced MUC5AC mRNA and protein expression with potency (half maximal inhibitory concentration) ∼1 nM in human bronchus and cultured airway epithelial cells. AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, inhibited carbachol-induced MUC5AC responses, indicating EGFR transactivation. Aclidinium inhibited carbachol-induced phospho-EGFR and phospho-p44/42 MAPK expression. In cultured airway epithelial cells transfected with small interfering (si)RNA against muscarinic receptor subtypes, siRNA-M3 but not siRNA-M2 blocked carbachol-induced MUC5AC expression. Cigarette smoke-induced MUC5AC upregulation in cultured airway epithelial cells was suppressed by aclidinium. In conclusion, aclidinium decreases carbachol and tobacco smoke-induced MUC5AC overexpression in human airway epithelial cells. This effect may contribute to the clinical efficacy of aclidinium in mucus hypersecretory diseases including COPD.

Effects of long‐term azithromycin therapy on airway oxidative stress markers in non‐cystic fibrosis bronchiectasis

To explore the effect of long‐term therapy with azithromycin in regards to airway oxidative stress markers in exhaled breath condensate (EBC) of adult patients with stable non‐cystic fibrosis (CF) bronchiectasis.

ISG15 Is Upregulated in Respiratory Syncytial Virus Infection and Reduces Virus Growth through Protein ISGylation

ABSTRACT Human respiratory syncytial virus (RSV), for which neither a vaccine nor an effective therapeutic treatment is currently available, is the leading cause of severe lower respiratory tract infections in children. Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that is highly increased during viral infections and has been reported to have an antiviral or a proviral activity, depending on the virus. Previous studies from our laboratory demonstrated strong ISG15 upregulation during RSV infection in vitro. In this study, an in-depth analysis of the role of ISG15 in RSV infection is presented. ISG15 overexpression and small interfering RNA (siRNA)-silencing experiments, along with ISG15 knockout (ISG15−/−) cells, revealed an anti-RSV effect of the molecule. Conjugation inhibition assays demonstrated that ISG15 exerts its antiviral activity via protein ISGylation. This antiviral activity requires high levels of ISG15 to be present in the cells before RSV infection. Finally, ISG15 is also upregulated in human respiratory pseudostratified epithelia and in nasopharyngeal washes from infants infected with RSV, pointing to a possible antiviral role of the molecule in vivo. These results advance our understanding of the innate immune response elicited by RSV and open new possibilities to control infections by the virus. IMPORTANCE At present, no vaccine or effective treatment for human respiratory syncytial virus (RSV) is available. This study shows that interferon-stimulated gene 15 (ISG15) lowers RSV growth through protein ISGylation. In addition, ISG15 accumulation highly correlates with the RSV load in nasopharyngeal washes from children, indicating that ISG15 may also have an antiviral role in vivo. These results improve our understanding of the innate immune response to RSV and identify ISG15 as a potential target for virus control.

Phosphodiesterase-4 inhibition improves corticosteroid insensitivity in pulmonary endothelial cells under oxidative stress

Several clinical studies have shown that smoking in asthmatics and chronic obstructive pulmonary disease patients is closely associated with corticosteroid refractoriness. In this work, we have analyzed glucocorticoid insensitivity in human pulmonary artery endothelial cells (HPAECs) under cigarette smoke extract (CSE) exposure as well as the possible additive effects of the combination therapy with a phosphodiesterase (PDE)‐4 inhibitor.

Nasal Ciliary Beat Frequency and Beat Pattern in Retinal Ciliopathies

PURPOSEnThe cilium in photoreceptors appears ultrastructurally very similar to the nasal ciliated epithelium. The purpose of this study was to evaluate the nasal ciliary beat frequency and beat pattern in patients with retinitis pigmentosa (RP) and Usher syndrome type II and compare it with that of healthy control subjects.nnnMETHODSnA prospective, comparative control study. Fresh samples of nasal mucosa were obtained from 13 patients with typical forms of RP, and from 4 patients with Usher syndrome type II. The nasal ciliary beat frequency (CBF) and beat pattern were determined using high-resolution digital high-speed video imaging (DHSV). The control group included 32 fresh nasal mucosa samples from 32 healthy volunteers without any other confounding diseases.nnnRESULTSnThe nasal CBF was lower in patients with Usher syndrome than in control subjects (Mann-Whitney U test, P = 0.01). The nasal CBF was 9.28 ± 0.4 (mean ± SD) Hz in patients with Usher syndrome and 10.82 ± 1.39 Hz in patients of the control group. No significant difference was observed in the nasal CBF between the RP (10.59 ± 1.54 Hz) and control group (Mann-Whitney U test, P = 0.64). Normal ciliary beat pattern was observed in all the patients and healthy volunteers.nnnCONCLUSIONSnThe nasal CBF is diminished in patients with Usher syndrome type II, whereas it remains normal in simplex RP patients. These results add evidence to the fact that Usher syndrome could be a primary ciliary disorder.

Estudio de marcadores de inflamación en el aire exhalado de pacientes con bronquiectasias no asociadas a fibrosis quística

INTRODUCTIONnThe aim of the study was to analyse the relationship between the intensity of the respiratory tract inflammation, expressed by oxidative stress markers, and the severity of the disease in patients with bronchiectasis unassociated with cystic fibrosis.nnnPATIENTS AND METHODSnThe study included 25 patients with stable bronchiectasis (15 females and 10 males). As determining factors of severity, the following parameters were collected: degree of dyspnoea, number of exacerbations/admissions in the last year, mean daily sputum volume, sputum colour (graduated colour scale), bacterial colonisation, respiratory function tests, quality of life (St. George questionnaire) and radiological extension of the lesions (Bhalla scale). Inflammation was analysed using the measurement of nitric oxide, pH and concentration of nitrites, nitrates and isoprostane in the exhaled air condensate. The C reactive protein and erythrocyte sedimentation rate were also determined in peripheral blood.nnnRESULTSnThere were no significant relationships between the markers in the exhaled air condensate and the clinical, radiological and functional involvement or the quality of life of the patients. Only bacterial colonisation (16 cases) was associated with higher values of nitrates in exhaled air (mean+/-standard deviation: 18+/-4 compared to 7+/-2microM; r(2)=0.6) and a higher number of exacerbations (3.1+/-1.9 compared to 1.7+/-1.9; r(2)=0.3).nnnCONCLUSIONSnIn our study, the measurement of inflammation markers in exhaled air is only associated with some parameters of severity in patients with bacterial bronchiectasis.

Inflammation Markers in the Exhaled Air of Patients with Bronchiectasis Unassociated with Cystic Fibrosis

Abstract Introduction The aim of the study was to analyse the relationship between the intensity of the respiratory tract inflammation, expressed by oxidative stress markers, and the severity of the disease in patients with bronchiectasis unassociated with cystic fibrosis. Patients and methods The study included 25 patients with stable bronchiectasis (15 females and 10 males). As determining factors of severity, the following parameters were collected: degree of dyspnoea, number of exacerbations/admissions in the last year, mean daily sputum volume, sputum colour (graduated colour scale), bacterial colonisation, respiratory function tests, quality of life (St. George questionnaire) and radiological extension of the lesions (Bhalla scale). Inflammation was analysed using the measurement of nitric oxide, pH and concentration of nitrites, nitrates and isoprostane in the exhaled air condensate. The C reactive protein and erythrocyte sedimentation rate were also determined in peripheral blood. Results There were no significant relationships between the markers in the exhaled air condensate and the clinical, radiological and functional involvement or the quality of life of the patients. Only bacterial colonisation (16 cases) was associated with higher values of nitrates in exhaled air (mean ± standard deviation: 18 ± 4 compared to 7 ± 2 μM; r2 = 0.6) and a higher number of exacerbations (3.1 ± 1.9 compared to 1.7 ± 1.9; r2 = 0.3). Conclusions In our study, the measurement of inflammation markers in exhaled air is only associated with some parameters of severity in patients with bacterial bronchiectasis.

CALCIO Y CONTRACTILIDAD DIAFRAGMÁTICA. MODIFICACIÓN POR LAS METILXANTINAS

En el presente estudio se han investigado las fuentes de Ca 2+ utilizadas en la contractilidad diafragmatica asi como la modificacion producida por teofilina (TE) y cafeina (CA) (5x10 -5 M a 5x10 -4 M). Para ellos, utilizamos tiras de musculo diafragmatico de cobayo (0.5x15 mm) estimulado electricamente mediante pulsos simples de 0,2 ms, 1 Hz, aplicados 3 veces por minuto. La respuesta contractil o twitch tension (TT) evocada por el estimulo electrico fue de 84 ± 7 mg F ( X ± EEM) y no se modifico en presencia de neostigmina (10 -5 M), pancuronio (10 -5 M) o tetrodotoxina (10 -6 g/ml), descartando asi cualquier accion indirecta del estimulo electrico sobre la transmision nerviosa. Cuando se utilizaron soluciones nutricias sin Ca 2+ , la contractilidad solo disminuyo un 19 ± 3% y no se modifico en presencia de verapamil 10 -5 M. Por el contrario, el dantroleno sodico produjo una disminucion en la TT del 73 ± 3%. El tratamiento con TE y CA produjo incrementos significativos en la TT del 65 ± 10% y 105 ± 13%, respectivamente. Estos incrementos tampoco se vieron modificados en presencia de verapamil 10 -5 M o solucion libre de Ca 2+ . Asimismo, TE y CA no solo no revirtieron los efectos inhibidores del dantroleno sino que potenciaron la TT en un 50 y un 72%, respectivamente. Los resultados obtenidos en nuestro modelo experimental in vitro nos indican que: a) la contractilidad diafragmatica, al igual que el resto de los musculos esqueleticos, solo dependen parcialmente del Ca 2+ extracelular y b) las metilxantinas ejercen un efecto potenciador de la fuerza contractil diafragmatica, no dependiente del Ca 2+ extracelular y probablemente relacionado con la liberacion intracelular del Ca 2+ .

132 Characterization of 2 Epithelial Cell Air-Liquid Interface (ALI) Culture Models for Human Healthy Nasal Mucosa and Nasal Polyps

Background Primary human airway epithelial cells, when submerged in culture, undergo a dedifferentiation with loss of many features of the in vivo airway epithelium. However, when cultured in an air-liquid interface (ALI), cells develop a well-differentiated, polarized, and pseudostratified epithelium. The aim of the current study was to characterize the mucociliary differentiation of human nasal mucosa and polyp epithelial cells cultured using an ALI system. Methods Nasal mucosa (NM, n = 3) and nasal polyps (NP, n = 3) were obtained from patients undergoing nasal corrective surgery and endoscopic sinus surgery, respectively. Epithelial cells were obtained from the explant method, and differentiated in ALI culture during 28 days. Cultures were studied at different time points (0, 7, 14, 21, and 28 days): tissue ultrastructure by scanning electron microscopy (SEM) and transmission electron microscopy (TEM); mucous (MUC5AC, MUC5B) and serous (lactoferrin) cell secretion by ELISA; and cytokeratin 18 (epithelial marker), &bgr;-tubulin IV (cilia marker), MUC5AC (goblet cell marker), and p63 (basal cell marker) expression by immunocytochemistry. Results In both NM and NP ALI cultures and at days 14 and 28, a pseudostratified epithelium with ciliated, mucus-secreting and basal cells was observed, and expression of cytokeratin 18, b-tubulin IV, MUC5AC and p63 was detected. In NP cultures, both MUC5AC (day 14: 2.2 ± 0.1-folds; day 28: 3.6-fold ± 0.7-fold) and MUC5B (day 14: 3.2-fold ± 0.6-fold; day 28: 3.1-fold ± 1-fold) increased over time compared to day 0 (P < 0.05). In NM cultures, only MUC5B (day 14: 3.9-fold ± 0.9-fold; day 28: 3.4-fold ± 0.4-fold; P < 0.05) but not MUC5AC increased over time compared to day 0 (P < 0.05). Secretion of lactoferrin was present but showed no changes over time in either NM or NP ALI cultures. Conclusions Epithelial cell ALI cultures provide a well-differentiated human nasal mucosa and polyp tissues that may be used as an in vitro model to study mucin regulation, inflammatory mechanisms of upper airways, and their regulation by antiinflammatory drugs.

Estudio de la contractilidad diafragmática en un modelo de asma experimental. Efectos de las metilxantinas

El objetivo del presente trabajo ha sido investigar las alteraciones producidas en la contractilidad muscular de tiras de diafragma procedentes de cobayos sensibilizados (CS) activamente con albumina serica bovina y su modificacion tras el tratamiento con teofilina (TE) y cafeina (CA). La respuesta contractil obtenida fue significativamente mayor en las tiras procedentes de CS (122 ± 13 mg F) que en las de cobayos normales (CN) (84 ± 7 mg F). Esta respuesta no se vio modificada en ninguno de los dos preparados por la presencia de verapamil y se redujo de forma similar en ambos (72% y 73%) cuando utilizamos dantroleno sodico. En ausencia de Ca 2+ libre extracelular, la contractilidad diafragmatica disminuyo un 44% en los CS frente a un 19% en los CN. El tratamiento con TE produjo aumentos similares de la fuerza contractil en CN (65 ± 10%) y CS (71 ± 13%) mientras que fue significativamente mayor en las tiras de CS (145 ± 22 vs 105 ± 13) tratadas con CA. Estas respuestas unicamente disminuyeron en los CS en ausencia de Ca 2+ extracelular. Concluimos: 1) el modelo de sensibilizacion utilizado produce una hiperreactividad del diafragma, dependiente en parte del Ca 2+ extracelular; 2) la sensibilizacion aumenta el efecto de CA pero no de TE sobre la contractilidad diafragmatica; y 3) el aumento de contractilidad inducido por TE y CA, presenta una mayor dependencia del Ca 2+ extracelular en los preparados sensibilizados. Arch Bronconeumol 1991: 27:249-253