Myoblots: dystrophin quantification by in-cell western assay for a streamlined development of Duchenne muscular dystrophy (DMD) treatments.

AIMS: New therapies for neuromuscular disorders are often mutation specific and require to be studied in patient's cell cultures. In Duchenne muscular dystrophy (DMD) dystrophin restoration drugs are being developed but as muscle cell cultures from DMD patients are scarce and do not grow or differentiate well, only a limited number of candidate drugs are tested. Moreover, dystrophin quantification by western blotting requires a large number of cultured cells; so fewer compounds are as thoroughly screened as is desirable. We aimed to develop a quantitative assessment tool using fewer cells to contribute in the study of dystrophin and to identify better drug candidates. METHODS: An 'in-cell western' assay is a quantitative immunofluorescence assay performed in cell culture microplates that allows protein quantification directly in culture, allowing a higher number of experimental repeats and throughput. We have optimized the assay ('myoblot') to be applied to the study of differentiated myoblast cultures. RESULTS: After an exhaustive optimization of the technique to adapt it to the growth and differentiation rates of our cultures and the low intrinsic expression of our proteins of interests, our myoblot protocol allows the quantification of dystrophin and other muscle-associated proteins in muscle cell cultures. We are able to distinguish accurately between the different sets of patients based on their dystrophin expression and detect dystrophin restoration after treatment. CONCLUSIONS: We expect that this new tool to quantify muscle proteins in DMD and other muscle disorders will aid in their diagnosis and in the development of new therapies.

Valoración del daño cerebral tras la administración de surfactante / Brain damage assesment after surfactant administration

Objetivo: La terapia de reemplazo de surfactante se asocia frecuentemente a fluctuaciones del flujo sanguíneo cerebral (FSC). Presentamos la administración de surfactante nebulizado para evitar las fluctuaciones del FSC. Métodos: El estudio se llevó a cabo en muestras cerebrales (congeladas y fijadas) de corderos prematuros que recibieron surfactante instilado (SFinstil, administración clásica) o surfactante nebulizado (SFneb). Se analizaron el FSC regional, la actividad de las enzimas antioxidantes, el Factor de Necrosis Tumoral (TNFα) y el número de células apoptóticas (TUNEL). También se realizó una valoración semi-cuantitativa del daño cerebral por un anátomo- patólogo. Se analizaron zonas corticales (corteza frontal y occipital), zonas internas (tálamo, estriado e hipocampo), el cerebelo y el bulbo cefalorraquídeo. Resultados: La administración de surfactante nebulizado produjo una respuesta hemodinámica cerebral diferente a la instilación intratraqueal, especialmente en las zonas internas donde a los cinco minutos el FSC registrado resultó ser significativamente superior en el grupo SFinstil. No se registraron diferencias significativas en la actividad de las enzimas antioxidantes. El porcentaje de células positivas para TNFα y el número de células TUNEL positivas en las zonas internas fue significativamente superior en el grupo SFinstil (p<0.05). La valoración histológica determinó un mayor grado de necrosis neuronal (p<0.05) en el tálamo en el grupo SFinstil. Conclusión: La administración de surfactante en forma de aerosol debería tenerse en cuenta como una alternativa menos agresiva a la instilación intratraqueal (AU)

Objective: Surfactant replacement therapy has been associated with cerebral blood flow (CBF) fluctuations. We propose the administration of aerosolized surfactant to prevent those fluctuations. Methods: Brain samples (frozen and paraffin-fixed) of preterm lambs received instilled surfactant (SFinstil, common administration) or aerosolized surfactant (SFneb). Regional CBF, the activity of antioxidant enzymes, the number of TNFα positive cells and the number of apoptotic cells (TUNEL) were determined. In addition, a semi-quantitative histological evaluation was performed by an expert pathologist. Cortical zones (frontal and occipital), inner zones (thalamus, striatum and hippocampus), cerebellum and the brain stem were analyzed. Results: Surfactant delivered as an aerosol produced a different cerebral hemodynamic response than surfactant instillation, especially towards the inner zones, where already five minutes after the start of the therapy the regional CBF was significantly higher in the SFinstil group. There were no differences between groups in the activity of antioxidant enzymes. The percentage of TNFα positive cells and the number of TUNEL positive cells in the inner zones was significantly higher in the SFinstil group. The histological score also showed a significantly higher necrosis in the SFinstil group compared to the SFneb group. Conclusion: Surfactant delivered as an aerosol should be considered as a less harmful method of surfactant administration (AU)

Bronchoalveolar lavage versus bolus administration of lucinactant, a synthetic surfactant in meconium aspiration in newborn lambs.

This study was designed to study effects of lung lavage versus the classical bolus instillation with a peptide-based synthetic surfactant (lucinactant) in a model of Meconium Aspiration Syndrome (MAS). Eighteen newborn lambs received meconium and were randomized to: the experimental meconium installation (eMAS) group-lambs with eMAS kept on conventional mechanical ventilation (control); the SF-Bolus group-eMAS receiving a lucinactant bolus (30 mg/ml); or the D-SF-Lavage group-eMAS treated with dilute lucinactant bronchoalveolar lavage (10 mg/ml). Systemic and pulmonary arterial pressures, blood gases, and pulmonary mechanics were recorded for 180 min. In addition, the intrapulmonary distribution of the lucinactant was determined using dye-labeled microspheres. Following meconium instillation, severe hypoxia, hypercapnia, acidosis, and pulmonary hypertension developed, and dynamic compliance decreased (50% from baseline). After lung lavage with dilute lucinactant, gas exchange significantly improved versus bolus instillation (P < 0.05). Further, only in the lavage group did pulmonary arterial pressure return to basal values and dynamic compliance significantly increased. Both lung lavage and bolus techniques for the administration of lucinactant resulted in a non-uniform lung distribution. In conclusion, in newborn lambs with respiratory failure and pulmonary hypertension induced by meconium, lung lavage with dilute lucinactant seems to be an effective and safe alternative for treatment for MAS.