Expression of mesenchymal stem cell phenotype in human nasal respiratory epithelial cells / Expresión del fenotipo de células troncales mesenquimales en células epiteliales nasales humanas

The respiratory epithelium is the first line of contact with the external hazards. Thus it can be damaged and need to be replaced to avoid healing by fibrosis. Tracheal tissue engineering is an alternative promising treatment modality. Mesenchymal stem cell markers are surface proteins, which are responsible for some of these cells unique properties. The objective of this study was to detect the mesenchymal stem cell phenotype among the human nasal respiratory epithelial cells via two immunophenotyping techniques. Respiratory epithelial cells were cultured using co-culture technique, fibroblasts was removed at confluence leaving respiratory epithelial cells, which were passage further to passage 4. Cells were evaluated for mesenchymal stem cell markers that were CD73, CD90, CD105 and the hematopoietic stem cell marker CD45 at passage 1 (P1) and passage 4 (P4) using Flow cytometry and Immunocytochemistry techniques. Respiratory epithelial cells expressed the mesenchymal stem cell markers at P1 and maintain the expression these markers until P4. Using both techniques, to compare the values of mesenchymal stem cell markers expression at P1 to P4 there was no significant difference. This study indicates that respiratory epithelial cells derived from nasal turbinate retain some of mesenchymal stem cells properties even after serial passages. Both methods of Immunophenotyping are comparable.

El epitelio respiratorio es la primera línea de contacto con los peligros externos. Por lo tanto, puede ser dañado y necesita ser reemplazado para evitar uan cicatrización por fibrosis. La ingeniería de tejidos traqueales es una modalidad de tratamiento alternativo prometedora. Los marcadores de células troncales mesenquimales son proteínas de superficie, que son responsables de algunas propiedades únicas de estas células. El objetivo fue detectar el fenotipo de células troncales mesenquimales entre las células epiteliales respiratorias nasales humanas a través de dos técnicas de inmunofenotipaje. Fueron cultivadas las células epiteliales respiratorias utilizando la técnica de co-cultivo; los fibroblastos se eliminaron en la confluencia dejando solo células epiteliales respiratorias, resultantes de los 4 pasajes. Las células fueron evaluadas para encontrar marcadores de células troncales mesenquimales mediante CD73, CD90, CD105 y el marcador de células troncales hematopoyéticas CD45 en el paso 1 (P1) y el paso 4 (P4), usando citometría de flujo y técnicas de inmunocitoquímica. Las células epiteliales respiratorias expresaron los marcadores de células troncales mesenquimales en P1 y mantuvieron la expresión de estos marcadores hasta P4. No hubo diferencias significativas en el uso de ambas técnicas al comparar los valores de los marcadores de células troncales mesenquimales expresadas desde P1 a P4. Este estudio indica que las células epiteliales respiratorias derivadas de la concha nasal retienen algunas de las propiedades de células troncales mesenquimales, incluso después de pases seriados. Ambos métodos de inmunofenotipificación son comparables.

Cultivo primario de células ciliadas de adenoides humanos: un modelo experimental para evaluar la actividad ciliar in vitro / Primary culture of human adenoid ciliated cells: an experimental model to evaluate ciliar activity in vitro

Introducción: El clearance mucociliar normal es el mecanismo de defensa básico de las vías respiratorias. Sin embargo, los mecanismos de control ciliar aún se desconocen. Con el fin de entenderlo mejor, se han desarrollado diferentes técnicas de cultivo de células ciliadas. Objetivos: Desarrollar un modelo experimental a partir de cultivos primarios de tejido adenoideo y cornete medio. Caracterizarla respuesta a adenosin trifosfato (ATP), agonista conocido de la frecuencia de batido ciliar (FBC). Material y método: Cultivos primarios a partir de explantes de epitelio adenoideo y cornete medio humano. Medición de FBC, con técnica de microfotodensitometría, en condición basal y en respuesta a ATP a diferentes concentraciones. Resultados: La FBC basal (promedio (X) + - desv estándar (DE)) para los cultivos de cornete medio fue 11,9 + -1,5 Hz y para tejido adenoideo fue 10,9 + - 1,9 (p >0,05). Se observó un aumento en la FBC en respuesta a ATP, dosis dependiente. No hubo diferencia significativa en la FBC basal ni en la respuesta a ATP entre cultivos de cornete medio y adenoides. Conclusión: El cultivo primario de células ciliadas nasales a partir de explantes de adenoides, es un modelo experimental reproducible, en el que es posible observar actividad ciliary una respuesta funcional concordante con lo descrito en la literatura.

Introduction. Mucociliary clearance constitutes the main defense mechanism of the airway, but the mechanisms of ciliary control are still unknown. With the aim of a better understanding of this process, many ciliated cells culture techniques have been developed. Aims. 1. To develop an experimental model based on primary cultures from adenoid and middle turbinate tissue. 2. To characterize in this model the response to ATP, a known agonist of ciliary beat frequency (CBF). Material and Method. Primary cultures derived from human adenoid tissue and middle turbinate epithelial explants were obtained. CFB was measured by microphotodensitometry, both in basal conditions and in response to ATP at different concentrations. Results. Basal CFB (average (X) +- standard deviation (SD)) for middle turbinate cultures was 11.9 +-1.5 Hz, and for adenoid tissue was 10.9 +-1.9 Hz (p< 0.05). A CBF increase was observed in response to ATP, in a dose-dependent manner. No significant difference in basal CFB or in response to ATP was found between middle turbinate and adenoid cultures. Conclusion. Primary culture of nasal ciliated cells derived from adenoid explants is a reproducible experimental model, in which it is possible to observe both ciliary activity and a functional response in accordance to what has been reported in the literature.

Genetic analysis of the G and P genes in ungulate respiratory syncytial viruses by RNase A mismatch cleavage method.

The G and P genes of bovine, ovine and caprine respiratory syncytial (RS) viruses were analyzed by RNase A one-dimensional fingerprinting, using A 51908 as the reference strain. Antisense G or P RNA probes of bovine RS virus strain A 51908 were hybridized to total RNA extracted from bovine turbinate cells infected with bovine, ovine or caprine RS virus strains. The RNA:RNA heteroduplexes were digested with RNase A and the resistant products were analyzed by gel electrophoresis. Comparative analysis of the cleavage patterns revealed heterogeneity among bovine, ovine and caprine RS virus isolates. Ovine RS virus strains generated RNA cleavage patterns more distantly related to the bovine or caprine RS virus strains, particularly in the G gene. Statistical analysis of the results obtained indicated that genetic differences between bovine and ovine viruses were larger, compared with the ones among bovine strains themselves. The same analysis also revealed a close genetic relation among bovine and caprine strains. These results are discussed in terms of ungulate RS virus genetic variation and vaccine development.

Binding of Pseudomonas aeruginosa to respiratory epithelial cells from patients with various mutations in the cystic fibrosis transmembrane regulator.

OBJECTIVE: To determine whether there is an association between mutations of the cystic fibrosis transmembrane regulator (CFTR) and the predilection of patients with cystic fibrosis (CF) for Pseudomonas aeruginosa infection. METHOD: We quantified the adherence of P. aeruginosa PA01, labeled with sulfur 35-methionine, to epithelial monolayers derived from nasal scrapings of patients with specific CFTR mutations, and of carriers and normal subjects. RESULTS: Adherence of P. aeruginosa to epithelial cells from patients with CF was significantly greater than to cells from either carriers (t = 2.94; p = 0.009) or normal subjects (t = 3.32; p = 0.004). Adherence to epithelial cells from patients with CF who were homozygous for the delta F508 mutation ranged from 12% to 35% (mean, 23.7%) of the added inoculum, which was significantly greater than the binding to cells from patients with other mutations, which ranged from 3% to 18% (mean, 9.4%; t = 3.71; p = 0.002), from heterozygote carriers (3% to 11%; mean, 7.9%; t = 4.87; p = 0.002), or from normal subjects (2% to 10%: mean, 7.0%; t = 5.21; p = 0.002). CONCLUSION: Adherence to P. aeruginosa can be correlated with homozygosity for the delta 508 mutation; CFTR dysfunction may be one of the factors involved in the pathogenesis of pulmonary infection in CF.