RESUMEN
Biospecimen quality can vary depending on many pre- and post-collection variables. In this study, we consider a natural disaster as a post-collection variable that may have compromised the quality of frozen tissue specimens. To investigate this possible link, we compared the quality of nucleic acids, the level of antigenicity, and the preservation of histology from frozen specimens collected before and after the power outage caused by Hurricane Sandy. To analyze nucleic acid quality, we extracted both DNA and RNA and performed capillary electrophoresis to compare the quality and concentrations of the nucleic acids. To compare antigenicity, frozen sections were cut and immunostained for thyroid transcription factor 1 (TTF-1), a nuclear transcription protein commonly used as a diagnostic biomarker for multiple cancer types, including thyroid and lung cancers. Positive expression of TTF-1, as noted by homogenous nuclear staining, would demonstrate that the TTF-1 proteins could still bind antibodies and, therefore, that these proteins were not significantly degraded. Furthermore, representative frozen sections stained with hematoxylin and eosin were also assessed qualitatively by a trained pathologist to examine any possible histologic aberrations. Due to the similar quality of the tissue samples collected before and after the storm, Hurricane Sandy had no discernable effect on the quality of frozen specimens, and these specimens exposed to the natural disaster are still valuable research tools.
Asunto(s)
Criopreservación/métodos , Tormentas Ciclónicas , Desastres , Conservación de Tejido/métodos , Femenino , Humanos , MasculinoRESUMEN
The initiation and maintenance of airway immune responses in Th2 type allergic diseases such as asthma are dependent on the specific activation of local airway dendritic cells (DCs). The cytokine microenvironment, produced by local cells, influences the recruitment of specific subsets of immature DCs and their subsequent maturation. In the airway, DCs reside in close proximity to airway epithelial cells (AECs). We examined the ability of primary culture human bronchial epithelial cells (HBECs) to synthesize and secrete the recently described CC-chemokine, MIP-3alpha/CCL20. MIP-3alpha/CCL20 is the unique chemokine ligand for CCR6, a receptor with a restricted distribution. MIP-3alpha/CCL20 induces selective migration of DCs because CCR6 is expressed on some immature DCs but not on CD14+ DC precursors or mature DCs. HBECs were stimulated with pro-inflammatory cytokines tumor necrosis factor-alpha and interleukin (IL)-1beta or, because of their critical role in allergic diseases, IL-4 and IL-13. Cells were also exposed to small size-fractions of ambient particulate matter. Each of these stimuli induced MIP-3alpha/CCL20 gene and protein expression. Moreover, these agents upregulated mitogen-activated protein kinase pathways in HBECs. Inhibition of the ERK1/2 pathway or p38 reduced cytokine-induced MIP-3alpha/CCL20 expression. These data suggest a mechanism by which AEC may facilitate recruitment of DC subsets to the airway.
Asunto(s)
Contaminantes Atmosféricos/efectos adversos , Bronquios/citología , Bronquios/inmunología , Quimiocinas CC/metabolismo , Citocinas/farmacología , Células Epiteliales/metabolismo , Proteínas Inflamatorias de Macrófagos/metabolismo , Bronquios/efectos de los fármacos , Células Cultivadas , Quimiocina CCL20 , Quimiocinas CC/genética , Quimiocinas CC/inmunología , Células Dendríticas/citología , Células Dendríticas/metabolismo , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Flavonoides/farmacología , Humanos , Imidazoles/farmacología , Sistema de Señalización de MAP Quinasas , Proteínas Inflamatorias de Macrófagos/genética , Proteínas Inflamatorias de Macrófagos/inmunología , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Tamaño de la Partícula , Piridinas/farmacología , ARN Mensajero/metabolismo , Receptores CCR6 , Receptores de Quimiocina/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/metabolismoRESUMEN
Environmental pollutants, including ambient particulate matter (PM), increase respiratory morbidity. Studies of model PM particles, including residual oil fly ash and freshly generated diesel exhaust particles, have demonstrated that PM affects inflammatory airway responses. Neither of these particles completely represents ambient PM, and therefore questions remain about ambient particulates. We hypothesized that ambient PM of different size fractions collected from an urban environment (New York City air), would activate primary culture human bronchial epithelial cells (HBECs). Because of the importance of granulocyte-macrophage colony-stimulating factor (GM-CSF) on inflammatory and immunomodulatory processes, we focused our studies on this cytokine. We demonstrated that the smallest size fraction (ultrafine/fine; < 0.18 micro m) of ambient PM (11 micro g/cm(2)), upregulated GM-CSF production (2-fold increase). The absence of effect of carbon particles of similar size, and the day-to-day variation in response, suggested that the chemical composition, but not the particle itself, was necessary for GM-CSF induction. Activation of the extracellular signal-regulated kinase and the p38 mitogen-activated protein kinase was associated with, and necessary for, GM-CSF release. These studies serve to corroborate and extend those on model particles. Moreover, they emphasize the role of the smallest size ambient particles in airway epithelial cell responses.