Simultaneous application of BrdU and WST-1 measurements for detection of the proliferation and viability of airway smooth muscle cells

BACKGROUND: BrdU is a commonly used reagent in cell proliferation assays, and WST-1 measurement is widely used to detect cell viability. However, no previous study has formally reported the combination of the two assays, which may be used to detect the proliferation and viability simultaneously. In this study, we examined the effect of adding BrdU 2 h prior to the WST-1 assay and tried to test the possibility of the combined detection using rat airway smooth muscle cells. RESULTS: The WST-1 measurements obtained from the combined detection were consistent with those obtained from the separate detection, which suggested that the addition of BrdU 2 h prior to the WST-1 analysis did not affect the WST-1 results. The BrdU measurements obtained from the combined detection also demonstrated the same trend as that obtained from the separate detection, and dosages of 200, 400 and 800 ng/ml testing reagent significantly inhibited the proliferation of rat airway smooth muscle cells. CONCLUSIONS: Our study suggests that the BrdU and WST-1 measurements can be applied simultaneously without mutual interference, which may increase the efficacy and consistency of these measurements to a certain extent.

Citologia dos lavados traqueobrônquico (LTB) e broncoalveolar (LBA) de bezerros holandeses sadios durante o primeiro mês de vida / Cytology of tracheobronchial and bronchoalveolar lavage in healthy Holteins calves during the first month of life

O período neonatal dos bezerros é um momento crítico para adaptação do recém-nascido à vida extra uterina e o sistema respiratório, um dos mais exigidos funcionalmente, é frequentemente afetado por enfermidades, redundando no prejuízo direto da sua função e acarretando perdas econômicas importantes na pecuária. O ponto básico para reduzir estas perdas, é representado pela adequada avaliação clínica dos neonatos, todavia o diagnóstico baseado exclusivamente no exame físico é muito difícil de ser estabelecido. O uso de exames complementares como a citologia do trato respiratório torna-se uma ferramenta diagnóstica importante nestes casos, porém faz-se necessário, padronizar seus achados frente às diferentes técnicas empregadas para a sua obtenção. Assim, o presente estudo propôs-se acompanhar as variações dos constituintes celulares da região traqueobrônquica e broncoalveolar obtidos por lavados respiratórios pelos métodos de traqueocentese e por colheita nasotraqueal respectivamente, durante o primeiro mês de vida de bezerros sadios. Observou-se alteração no quadro citológico ao longo do tempo, quando a região traqueobrônquica foi lavada, expresso por diminuição da porcentagem de macrófagos alveolares, com aumento de neutrófilos, possivelmente, por maior irritação local provocada pela técnica, que se repetiu sequencialmente e/ou por maior estimulo de microorganismos inalados depositados nesta região. Na região broncoalveolar, não encontraram-se variações nos constituintes celulares em função do tempo. Os resultados permitiram a conclusão que a população celular da região traqueobrônquica modificou-se ao longo das semanas de vida dos bezerros, possivelmente pela técnica empregada e/ou fisiologia normal da região, sendo representadas por maiores magnitudes de neutrófilos. De modo diverso, na região broncolaveolar, as células evidenciaram um comportamento estável durante o primeiro mês de vida dos bezerros neonatos, apresentando predomínio numérico dos macrófagos alveolares.

The neonatal calf is a critical moment for adaptation of the newborn to extra uterine life. The respiratory tract is functionally very demanded and often affected by disease, resulting in direct loss of their function and causing serious economic losses in livestock. The basic point to reduce these losses is appropriate clinical evaluation of neonates; but the diagnosis based solely in physical examination is very difficult to establish. The use of complementary analysis such cytology of the respiratory tract becomes an important diagnostic tool; however their findings must be standardized in the face of different techniques employed. This research studied the dynamics of the cellularity of the bronchoalveolar and tracheobronchial region obtained through lung lavage harvested by nasotracheal catheterization technique and tracheocenthesis respectively, during the first month of life of healthy calves. The tracheobronchial cytology was influenced by the time, showing decreased number of alveolar macrophages and greater number of neutrophils, possibly increased by local irritation caused by the technique, which was repeated sequentially, and/or through greater stimulation of inhaled microorganisms deposited in this region. In the bronchoalveolar region no variation in the cellular constituents in function of time was found. The results allowed the conclusion the cell population of the tracheobronchial region has changed over the week-old calves, possibly due to the technique used and/or to the normal region physiology, represented by higher magnitudes of neutrophils. Otherwise, the cells of the broncholaveolar region showed a stable behavior during the first month of life of newborn calves, presenting numerical predominance of alveolar macrophages.

Microscopic anatomy of the lower respiratory system of the African giant pouched rat (Cricetomys gambianus, Waterhouse 1840) / Anatomía microscópica del sistema respiratorio inferior de la rata gigante Africana (Cricetomys gambianus, Waterhouse 1840)

A qualitative and quantitative study, by light microscopy, was undertaken on the lower respiratory system of the African Giant pouched rat. Specifically, the trachea, bronchi and lungs were stained with Haematoxylin and eosin, Alcian blue at a pH of 2.5 and Periodic Acid-Schiff stains. Three cell types were identified in saggital sections of the trachea: the ciliated cells, basal cells and mucous cells. Fibers of the trachealis muscles in the laminar propria separated the underlying cartilages from the basal cells. Mucous cells were visible only in the membranous portion of the trachea and they were predominant in the rostral and caudal portion of the trachea. Lobar bronchi consisted of cuboidal epithelium and a layer of one or two smooth muscle cells and opened into segmental bronchi and respiratory bronchiole. Some tracheal cartilaginous rims stained blue with AB while most glandular cells stained red with PAS. The diameter of respiratory bronchiole, alveoli duct and alveoli were 24.93 µm (+/- 1.27), 21.14 um (+/- 0.66) and 12.95 um (+/- 0.21), respectively. These and other findings were compared with similar report in other rodents.

Se realizó un estudio cualitativo y cuantitativo, mediante microscopía de luz, en el sistema respiratorio inferior de la rata gigante Africana. La tráquea, los bronquios y los pulmones fueron teñidos con hematoxilina y eosina, azul Alcián a pH de 2,5 y ácido periódico de Schiff. Tres tipos de células fueron identificadas en las secciones sagitales de la tráquea: células ciliadas, basales y mucosas. Las fibras del músculo traqueal en la propia laminar separados los cartílagos subyacente de las células basales. las células mucosas son visibles sólo en la porción membranosa de la tráquea y predominan en la parte rostral de la porción caudal de la tráquea. Los bronquios lobares consistían en epitelio cúbico y una capa de una o dos células de músculo liso y abierto en los bronquios y bronquiolos segmentarios respiratorias. Algunos bordes azules cartilaginoso traqueal manchada con AB, mientras que la mayoría de las células glandulares teñido de rojo con PAS. El diámetro de los bronquiolos respiratorios, conductos alveolares y los alvéolos fueron 24,93 m (+/- 1,27), 21,14 m (+/- 0,66) y 12,95 m (+/- 0,21), respectivamente. Estos y otros resultados se compararon con el informe similar en otros roedores.

beta-catenin/Tcf signaling in squamous differentiation of porcine airway epithelial cells / 华中科技大学学报（医学）（英德文版）

For a preliminary study of the role of beta-catenin/Tcf signaling in squamous differentiation of airway (tracheobronchial) epithelial cells, a stable mutant of beta-catenin was transfected into primarily cultured porcine airway epithelial cells. Western blotting revealed that exogenous protein was observed in large quantity in cytoplasm and nucleus. When co-transfected with Tcf luciferase reporter plasmids, beta-catenin mutant increased the reporter's transcriptional activities. However, mRNA expression of a squamous differentiation marker, small proline-rich protein (SPRP), was not elevated, as shown by reverse transcription-polymerase chain reaction. These findings suggest that beta-catenin/Tcf signaling may not be directly involved in the squamous differentiation of porcine airway epithelial cells.

TNF-alpha-induced up-regulation of intercellular adhesion molecule-1 is regulated by a Rac-ROS-dependent cascade in human airway epithelial cells

Up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the lung airway epithelium is associated with the epithelium-leukocyte interaction, critical for the pathogenesis of various lung airway inflammatory diseases such as asthma. However, little is known about how ICAM-1 is up-regulated in human airway epithelial cells. In this study, we show that tumor TNF-alpha induces monocyte adhesion to A549 human lung airway epithelium and also up-regulation of ICAM-1 expression. These effects were significantly diminished by pre-treatment with diphenyliodonium (DPI), an inhibitor of NADPH oxidase-like flavoenzyme. In addition, the level of reactive oxygen species (ROS) was increased in response to TNF-alpha in A549 cells, suggesting a potential role of ROS in the TNF-alpha-induced signaling to ICAM-1 expression and monocyte adhesion to airway epithelium. Further, we found out that expression of Rac(N17), a dominant negative mutant of Rac1, suppressed TNF-alpha-induced ROS generation, ICAM-1 expression, and monocyte adhesion to airway epithelium. These findings suggest that Rac1 lies upstream of ROS generation in the TNF-alpha-induced signaling to ICAM-1 expression in airway epithelium. Finally, pretreatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, reduced TNF-alpha-induced ICAM-1 expression and both DPI and Rac(N17) significantly diminished NF-kappaB activation in response to TNF-alpha. Together, we propose that Rac1-ROS-linked cascade mediate TNF-alpha-induced ICAM-1 up-regulation in the airway epithelium via NF-kappaB-dependent manner.

Effect of sensitization on membrane ion fluxes & intracellular calcium in guineapigs.

BACKGROUND & OBJECTIVES: The biochemical mechanisms underlying the development of sensitization-induced airway hyperresponsiveness (AHR) in asthma are poorly defined. Alterations in the regulation of intracellular calcium may play an important role in its pathogenesis. We carried out this study to see the effect of sensitization with ovalbumin on membrane ion fluxes and intracellular calcium in a guinea pig model. METHODS: Airway reactivity to inhaled histamine was measured initially and after sensitization with ovalbumin in 28 guineapigs. Intracellular calcium [Ca(2+)]i was measured in tracheal smooth muscle cells and peripheral leukocytes using fluorescent dye FURA 2AM. Calcium and sodium ion influx across the cell membrane was measured in leukocytes. Ouabain-sensitive Rubidium ((86)Rb) influx was measured in tracheal smooth muscles cells. The activities of Na(+), K(+) ATPase and Ca(2+) ATPase were measured in tracheal smooth muscle cells. Lipid peroxides were measured in plasma. RESULTS: Airway responsiveness was significantly (P<0.001) increased after sensitization along with an increase in [Ca2+]i levels in leukocytes and tracheal smooth muscle cells, higher rates of (45)Ca and (22)Na influx in leukocytes and higher (86)Rb influx rates in tracheal smooth muscle cells, and increased levels of lipid peroxides in plasma. INTERPRETATION & CONCLUSION: In guineapig model of asthma sensitization to allergen increased the membrane permeability to calcium and sodium, and intracellular calcium levels. These alterations may play a role in the pathogenesis of airway hyper-responsiveness following sensitization.

Mucin secretion in the rat tracheal epithelial cells by epidermal growth factor and Pseudomonas aeruginosa extracts

BACKGROUND: Hypersecretion of mucin due to goblet cell hyperplasia is frequently encountered in many chronic airway diseases, such as chronic bronchitis, bronchiectasis, bronchial asthma and cystic fibrosis. Even in normal individuals, viral infection or bacterial pneumonia frequently provoke huge amounts of bronchial secretions which may cause airway obstruction. The production of mucin was regulated by epidermal growth factor (EGF) in vitro. To know whether this EGF system regulates mucin secretion in vivo and Pseudomonas also stimulates the mucin secretion by the same pathway, we studied these relationships in the cultured rat tracheal epithelial cells. METHODS: Rat tracheal epithelial cells were obtained by pronase dissociation from the male Fisher 344 rats. When cells became confluent, they were divided into 6 groups and stimulated with either EGF for 24 hours or Pseudomonas extracts for 12 hours with or without selective EGF-R tyrosine kinase inhibitor tyrphostin AG1478. RESULTS: We found that both EGF and Pseudomonas extracts phosphorylated the tyrosine residue in the EGF receptor from the rat tracheal epithelial cells and this tyrosine phosphorylation was nearly completely blocked by selective EGF-R tyrosine kinase inhibitor tyrphostin AG1478. The mucin secretion was also stimulated by either EGF or Pseudomonas extracts but more strong secretion of mucin and MUC5AC gene expression in the rat tracheal epithelial cell was done by Pseudomonas extracts. CONCLUSION: These data suggest that Pseudomonas secretes the mucin by way of the EGF receptor and MUC5AC gene expression and the inhibitors of EGF receptor tyrosine phosphorylation would be useful to prevent the huge production of mucin due to Pseudomonas aeruginosa lung infection.