Avaliação da toxicidade de partículas da exaustão do diesel em explantes de traqueia e cultura de células do epitélio respiratório: estudo da expressão gênica, citotoxicidade e sinalização celular / Toxicity tests on tracheal explants and respiratory epithelial cells exposed to diesel exhaust particles: a study on gene expression, cytotoxicity, and cell signaling

Partículas da exaustão de motores a diesel (DEP) têm propriedades toxicológicas, devido às características físico-químicas. O DEP é capaz de ativar as vias de sinalização intracelular e induzir alterações metabólicas em células e tecidos do sistema respiratório. O objetivo desta tese foi: 1) avaliar o perfil das mucinas e alterações epiteliais em explantes de traqueia de camundongo BALB/c expostos ao DEP e DEP tratado com ácido nítrico e solventes orgânicos; e 2) em cultura de células de epitélio brônquico humano (BEAS-2B) expostas ao DEP e DEP tratado com hexano (DEP/HEX) para avaliar ativação de MAPK (ERK e JNK), citotoxidade, integridade de citoesqueleto, viscoelasticidade celular e expressão gênica de enzimas envolvidas no estresse oxidativo e apoptose. Os resultados mostraram que, em explantes de traqueia, o DEP causa aumento significativo em relação ao grupo controle na quantidade de muco ácido (p= 0,001), diminuição no muco neutro (p=0,001), diminuição de muco misto (p= 0,001), aumento de vacuolização (p= 0,001), aumento de apoptose (p=0,001), ora com aumento de pERK e diminuição de pJNK, e vice-versa. Os explantes submetidos à exposição ao DEP e DEP/MET aumentaram significativamente o muco ácido (p=0,01) e DEP/HEX provocou aumento da extrusão do muco (p=0,007), provavelmente devido à ação do enriquecimento inorgânico. Para as células BEAS-2B, nos resultados obtidos com células epiteliais expostas ao DEP e DEP/HEX, foram observadas alterações na membrana citoplasmática, mitocôndrias e citoesqueleto. As células expostas apenas ao DEP em baixas concentrações (15ug/mL) apresentaram alterações na expressão de genes envolvidos no apoptose (BCL-2 e Caspase-3 (p=0,05 e p=0,01) e estresse oxidativo [(SOD1 e SOD2 e GPx. p=0,01 )], e CYP1A1 ((p=0,01).

Diesel exhaust particles (DEPs) from diesel engines have toxic properties that result from their physical and chemical characteristics. DEPs are able to activate intracellular signaling pathways and induce metabolic changes to cells and tissues of the human respiratory system. This dissertation sought to evaluate: 1) the profile of mucins and the epithelial changes to the tracheal explants of BALB/c mice exposed to both DEP and DEP treated with nitric acid and organic solvents (50 and 100 ug/mL; and 2) human bronchial epithelial cells (BEAS-2B) in culture after their exposure to both DEP and DEP treated with hexane (DEP/HEX) at 100 ug/mL in order to determine MAPK (ERK/JNK) activation, cytotoxicity, cytoskeletal integrity, cell viscoelasticity and gene expression of the enzymes involved in oxidative stress and apoptosis. The results show that, in tracheal explants, DEP causes a significant increase (compared to the control) in the quantity of acidic mucus (p=0.001), a decrease in alkaline mucus (p=0.001), a decrease in mixed mucus (p=0.001), an increase in vacuolization (p=0.001), an increase in apoptosis (p=0.001), along with an increase in pERK and a decrease in pJNK, and vice versa. The explants that were exposed to DEP and DEP/MET were found to have significantly higher quantities of acidic mucus (p=0.01), and DEP/HEX caused an increase in mucus extrusion (p=0.007), which was likely due to inorganic enrichment. In the case of BEAS-2B cells, the results obtained from epithelial cells exposted to DEP and DEP/HEX revealed alterations in the cytoplasmic membrane, the mitochondria, and the cytoskeleton. The cells exposed to DEP alone at low concentrations (15 ug/mL) experienced alterations in the genes involved in apoptosis (BCL-2 and Caspase-3; p=0.05 and p=0.01, respectively), as well as oxidative stress [(SOD1, SOD2, and GPx; p=0.01 )], and changes to CYP1A1 (p=0.01).

Airborne agricultural particulate matter induces inflammatory cytokine secretion by respiratory epithelial cells: Mechanisms of regulation by eicosanoid lipid signal mediators.

The purpose of this study was to elucidate the mechanism of the airborne poultry dust (particulate matter, PM)-induced respiratory tract inflammation, a common symptom in agricultural respiratory diseases. The study was based on the hypothesis that poultry PM would induce the release of inflammatory cytokine interleukin-8 (IL-8) by respiratory epithelial cells under the upstream regulation by cytosolic phospholipase A2 (cPLA2) activation and subsequent formation of cyclooxygenase (COX)- and lipoxygenase (LOX)-catalyzed arachidonic acid (AA) metabolites (eicosanoids). Human lung epithelial cells (A549) in culture were treated with the poultry PM (0.1-1.0 mg) for different lengths of time, following which PLA2 activity, release of eicosanoids and secretion of IL-8 in cells were determined. Poultry PM (1.0 mg/ml) caused a significant activation of PLA2 in a time-dependent manner (15-60 min), which was significantly attenuated by the calcium-chelating agents, cPLA2-specific inhibitor (AACOCF3) and antioxidant (vitamin C) in A549 cells. Poultry PM also significantly induced the release of COX- and LOX-catalyzed eicosanoids (prostaglandins, thromboxane A2 and leukotrienes B4 and C4) and upstream activation of AA LOX in the cells. Poultry PM also significantly induced release of IL-8 by the cells in a dose- and time-dependent manner, which was significantly attenuated by the calcium chelating agents, antioxidants and COX- and LOX-specific inhibitors. The current study for the first time revealed that the poultry PM-induced IL-8 release from the respiratory epithelial cells was regulated upstream by reactive oxygen species, cPLA2-, COX- and LOX-derived eicosanoid lipid signal mediators.

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.

Sulforaphane Induces Antioxidative and Antiproliferative Responses by Generating Reactive Oxygen Species in Human Bronchial Epithelial BEAS-2B Cells

Sulforaphane (SFN) is a naturally occurring compound which is known to induce the phase II antioxidant genes via Nrf2 activation, although the underlying mechanism has not been fully elucidated. In this study, we investigated Nrf2 induction in response to SFN in human bronchial epithelial BEAS-2B cells and determined the signaling pathways involved in this process. SFN treatment reduced cell viability. Prior to cell death, intracellular reactive oxygen species (ROS) were generated at a high rate within a minute of commencing SFN treatment. Pretreatment with antioxidant N-acetylcysteine (NAC) blocked SFN-induced decrease in cell growth. Erk1/2 was activated within 30 min of SFN addition, whereas Akt phosphorylation did not significantly change until the first 8 hr after SFN treatment but then became substantially low until 48 hr. Inhibition of Erk1/2 phosphorylation attenuated SFN-induced loss of cell viability. Nrf2 protein levels in both nuclear and whole cell lysates were increased by SFN treatment, which was dependent on ROS production. Knockdown of Nrf2 with siRNA attenuated SFN-induced heme oxygenase-1 (HO-1) up-regulation. Induction of the Nrf2/HO-1 after SFN treatment was potently suppressed by pretreatment with NAC. Overall, our results indicate that SFN mediates antioxidative and antiproliferative responses by generating ROS in BEAS-2B cells.

Infrared spectroscopy characterization of normal and lung cancer cells originated from epithelium

The vibrational spectral differences of normal and lung cancer cells were studied for the development of effective cancer cell screening by means of attenuated total reflection infrared spectroscopy. The phosphate monoester symmetric stretching nus(PO3(2-)) band intensity at ~970 cm-1 and the phosphodiester symmetric stretching nus(PO2-) band intensity at ~1,085 cm-1 in nucleic acids and phospholipids appeared to be significantly strengthened in lung cancer cells with respect to the other vibrational bands compared to normal cells. This finding suggests that more extensive phosphorylation occur in cancer cells. These results demonstrate that lung cancer cells may be prescreened using infrared spectroscopy tools.

Identification of potential lung cancer biomarkers using an in vitro carcinogenesis model

Lung cancer is one of the deadliest and commonly diagnosed neoplasms. Early diagnosis of this disease is critical for improving clinical outcome and prognosis. Because the early stages of lung cancer often produce no symptoms, it is necessary to identify biomarkers for early detection, prognostic evaluation, and recurrence monitoring of the cancer. To identify potential lung cancer biomarkers, we analyzed the differential protein secretion from transformed bronchial epithelial cells (1198 and 1170-I) as compared to immortalized normal bronchial epithelial cells (BEAS-2B) and non-transformed cells (1799) all of which are derived from BEAS-2B and represent multistage bronchial epithelial carcinogenesis. The proteins recovered from the conditioned media of the cells were separated on two-dimensional gels. There was little difference between the secretome of the BEAS-2B and 1799 cells, whereas the patterns between the transformed 1198 and 1170-I cells and non-transformed 1799 cells were significantly different. Using mass spectrometry and database search, we identified 20 proteins including protein gene product 9.5 (PGP9.5), translationally controlled tumor protein (TCTP), tissue inhibitors of metalloproteinases-2 (TIMP-2), and triosephosphate isomerase (TPI), that were either increased or decreased simultaneously in conditioned media of both 1198 and 1170-I cells. Furthermore, levels of PGP9.5, TCTP, TIMP-2, and TPI were significantly increased not only in the conditioned media of both transformed cell lines when compared to those of BEAS-2B and 1799 cells, but also in plasmas and tissues from lung cancer patients when compared to those in normal controls. We suggest the PGP9.5, TCTP, TIMP-2, and TPI as promising candidates for lung cancer serum biomarkers.

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.

Growth of respiratory syncytial virus in mink lung epithelial cells.

Mink lung epithelial cells (Mv-1-Lu) were tested for their ability to support the growth and serial passage of respiratory syncytial virus (RSV) in vitro. Indian isolates of RSV induced distinctive cytopathic effect with typical rounding of cells followed by detachment with more than 50 per cent cells showing bright fluorescence using anti-RSV monoclonal antibodies in immunofluorescence test. Serial passage of RSV was possible in Mv-1-Lu cells without loss of sensitivity of the cells for virus growth. Titration of cell associated virus and virus released in the supernatant indicated that 60 per cent of the virus was released in the supernatant, and 40 per cent remained cell associated. Transmission electron microscopic studies of negatively stained RSV particles and ultra-thin sections of RSV infected Mv-1-Lu cells showed roughly spherical particles with club shaped projections, budding from the cytoplasmic membrane. These results indicate that Mv-1-Lu cell line is suitable for the growth and propagation of RSV.

Diferencias en la celularidad de expectoración en pacientes con asma estable y asma en crisis / Differences in the cellularity of stable and critical asthma patient sputum

Introducción: En el estudio clínico del paciente asmático, no contamos con herramientas diagnósticas prácticas para conocer el grado y tipo de inflamación de la vía aérea. En la expectoración, síntoma frecuente de esta enfermedad, es posible realizar diversos estudios, sin riesgo y en múltiples ocasiones. El objetivo del presente trabajo fue conocer el número y tipo de células inflamatorias presentes en la expectoración espontánea de pacientes con asma, correlacionando estos datos con el estado clínico y funcional, y con el uso de diferentes antiinflamatorios esteroideos.Material y métodos: El trabajo fue observacional, prospectivo, transversal y comparativo. Se estudiaron 77 pacientes adultos que fueron divididos en dos grupos: Estables (n = 55), edad promedio de 38 años y en crisis (n = 22), edad promedio de 52 años, a quienes se les realizó espirometría y citología en expectoración con cuenta diferencial. En el grupo estable, 29 pacientes no usaban esteroides, 22 utilizaban beclometasona y 4 prednisona; en el de crisis, 4 pacientes utilizaban dosis altas de prednisona y 18 metilprednisolona.Resultados: Se encontró un incremento en el número total de células en expectoración en el grupo en crisis (2,353,611 células/mL) al compararlo con el estable (1,830,084 células/mL); asimismo, los eosinófilos se observaron cuantitativamente elevados en ambos grupos, especialmente en crisis, estos datos no mostraron significancia estadística. Por otra parte, se determinó una significante correlación negativa entre el VEF1y el porcentaje de eosinófilos (p<0.05). Al analizar estos datos con el tipo de esteroides empleados, se encontró una menor población de eosinófilos en el grupo que utilizaba beclometasona (p NS).Conclusión: El estudio de la celularidad en la expectoración de los pacientes asmáticos puede ser un método útil para la evaluación no invasiva de la inflamación bronquial y servir como parámetro de seguimiento, principalmente en países en donde los recursos económicos son limitados.

Beta agonist regulation of sodium transport in fetal lung epithelium: roles of cell volume, cytosolic chloride and protein tyrosine kinase

1) A beta agonist stimulated Na+ transport and decreased the intracellular Cl concentration ([Cl]c) associated with cell shrinkage via an increase in cytosolic cAMP level by activating adenylate cyclase in rat fetal distal lung epithelial (FDLE) cells. 2) Lowering [Cl-]c activated a 28-pS nonselective cation (NSC) channel by elongating the open time of the channel. 3) cAMP signals were converted to a protein tyrosine kinase (PTK)-mediated signal. 4) The PTK-mediated signal was involved in the cAMP-stimulated Na+ transport in rat FDLE cells.

Unexpected effects of pathogens on epithelial Na+ channels

No abstract available.