Dynamic changes in and time sequence of ultraviolet B-induced apoptosis in rat corneal epithelial cells / Alterações dinâmicas na sequência temporal da apoptose induzida por radiação ultravioleta B em células epiteliais da córnea de ratos

ABSTRACT Purpose: To systematically examine the dynamic changes and time sequence in corneal epithelial cell apoptosis after excessive ultraviolet B irradiation. Methods: Ultraviolet B (144 mJ/cm2) was used to irradiate rat corneal epithelial cells for 2 h. Cell morphology was observed on differential interference contrast microscopy, and the numbers of the different kinds of apoptotic cells were counted using the ImageJ software. Cell viability was measured with the 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide method. Cell apoptotic rate and loss of mitochondrial membrane potential were detected using flow cytometric analyses. The expression levels of 3 apoptotic genes were measured with real-time quantitative polymerase chain reaction at different time points within 0-24 h after irradiation. Results: After 144-mJ/cm2 ultraviolet B irradiation for 2 h, the expression levels of caspase-8 and Bax were highest at 0 h; furthermore, the mitochondrial membrane potential decreased at 0 h and remained constant for 6 h in a subsequent culture. At 6 h, caspase-3 was activated. The decrease in cell viability and increase in apoptotic rate peaked at 6 h. The caspase-3 expression level decreased within 12-24 h, which led to a decline in apoptotic rate and change in apoptotic stage. Conclusions: The corneal epithelial cells exhibited rapid apoptosis after ultraviolet B irradiation, which was associated with both extrinsic and intrinsic pathways.

RESUMO Objetivos: Explorar sistematicamente as mudanças dinâmicas e a sequência temporal no processo de apoptose de células epiteliais corneanas após excesso de irradiação com ultravioleta B. Métodos: A radiação ultravioleta B (144 mJ/cm2) foi utilizada para irradiar células epiteliais da córnea de rato durante 2h. A morfologia celular foi observada por meio de microscópio de contraste de interferência diferencial, e os números de diferentes tipos de células apoptóticas foram contados e registrados pelo software ImageJ. A viabilidade celular foi medida pelo método brometo de 3- (4, 5-dimetil-2-tiazolil) -2, 5-difenil-2-H-tetrazólio. A taxa apoptótica celular e a perda do potencial da membrana mitocondrial foram detectadas por meio de análises citométricas de fluxo. Os níveis de expressão de três genes apoptóticos foram medidos por reação em cadeia da polimerase quantitativa em tempo real em diferentes momentos dentro de 0-24 h após a irradiação. Resultados: Após 144 mJ/cm2 de irradiação com ultravioleta B por 2h, os níveis de expressão de caspase-8 e Bax foram maiores em 0h; o potencial da membrana mitocondrial diminuiu a 0h e permaneceu constante por 6h na cultura subsequente. Às 6h, a caspase-3 foi ativada. A diminuição da viabilidade celular e o aumento da taxa apoptótica atingiu o pico em 6h. A expressão de caspase-3 diminuiu dentro de 12 - 24 h, levando a um declínio na taxa apoptótica e alteração no estágio apoptótico. Conclusões: As células epiteliais da córnea apresentaram uma apoptose rápida após excesso de irradiação com ultravioleta B, e esse processo foi associado tanto à via extrínseca como à via intrínseca.

Endoscopic observation of different repair patterns in human traumatic tympanic membrane perforations / Observação endoscópica de diferentes padrões de reparo em perfurações humanas traumáticas da membrana timpânica

Abstract Introduction: In the last decade, there has been an increasing use of biomaterial patches in the regeneration of traumatic tympanic membrane perforations. The major advantages of biomaterial patches are to provisionally restore the physiological function of the middle ear, thereby immediately improving ear symptoms, and act as a scaffold for epithelium migration. However, whether there are additional biological effects on eardrum regeneration is unclear for biological material patching in the clinic. Objective: This study evaluated the healing response for different repair patterns in human traumatic tympanic membrane perforations by endoscopic observation. Methods: In total, 114 patients with traumatic tympanic membrane perforations were allocated sequentially to two groups: the spontaneous healing group (n = 57) and Gelfoam patch-treated group (n = 57). The closure rate, closure time, and rate of otorrhea were compared between the groups at 3 months. Results: Ultimately, 107 patients were analyzed in the two groups (52 patients in the spontaneous healing group vs. 55 patients in the Gelfoam patch-treated group). The overall closure rate at the end of the 3 month follow-up period was 90.4% in the spontaneous healing group and 94.5% in the Gelfoam patch-treated group; the difference was not statistically significant (p > 0.05). However, the total average closure time was significantly different between the two groups (26.8 ± 9.1 days in the spontaneous healing group vs. 14.7 ± 9.1 days in the Gelfoam patch-treated group, p < 0.01). In addition, the closure rate was not significantly different between the spontaneous healing group and Gelfoam patch-treated group regardless of the perforation size. The closure time in the Gelfoam patch-treated group was significantly shorter than that in the spontaneous healing group regardless of the perforation size (small perforations: 7.1 ± 1.6 days vs. 12.6 ± 3.9, medium-sized perforations: 13.3 ± 2.2 days vs. 21.8 ± 4.2 days, and large perforations: 21.2 ± 4.7 days vs. 38.4 ± 5.7 days; p < 0.01). Conclusion: In the regeneration of traumatic tympanic membrane perforations, Gelfoam patching not only plays a scaffolding role for epithelial migration, it also promotes edema and hyperplasia of granulation tissue at the edges of the perforation and accelerates eardrum healing.

Resumo Introdução: Na última década, houve um uso crescente de placas biomateriais na regeneração de perfurações traumáticas da membrana timpânica. As principais vantagens das placas de biomateriais são restaurar provisoriamente a função fisiológica da orelha média, assim melhoram imediatamente os sintomas da orelha e atuam como um suporte para a migração do epitélio. No entanto, não se sabe se há efeitos clínicos adicionais na regeneração do tímpano em relação ao fragmento de material biológico. Objetivo: Avaliar a resposta de cicatrização para diferentes padrões de reparo em perfurações de membrana timpânica traumáticas humanas por meio de observação endoscópica. Método: Foram alocados 114 pacientes com perfurações de membrana timpânica traumáticas sequencialmente para dois grupos: o de cicatrização espontânea (n = 57) e o tratado com esponja de Gelfoam (n = 57). A velocidade de fechamento, o tempo de fechamento e a taxa de otorreia foram comparados entre os grupos aos três meses. Resultados: Foram analisados 107 pacientes nos dois grupos (52 no de cicatrização espontânea e 55 no tratado com esponja de Gelfoam). A velocidade global de fechamento no fim do período de seguimento de três meses foi de 90,4% no grupo de cicatrização espontânea e de 94,5% no grupo tratado com esponja de Gelfoam; a diferença não foi estatisticamente significativa (p > 0,05). No entanto, o tempo total médio de fechamento foi significativamente diferente entre os dois grupos (26,8 ± 9,1 dias no de cicatrização espontânea versus 14,7 ± 9,1 dias no tratado com esponja de Gelfoam, p < 0,01). Além disso, a velocidade de fechamento não foi significativamente diferente entre o grupo de cicatrização espontânea e o grupo tratado com esponja de Gelfoam, independentemente do tamanho da perfuração. O tempo de fechamento no grupo tratado com esponjas de Gelfoam foi significativamente menor do que no grupo de cicatrização espontânea, independentemente do tamanho da perfuração (pequenas perfurações: 7,1 ± 1,6 dias vs. 12,6 ± 3,9, perfurações de tamanho médio: 13,3 ± 2,2 dias vs. 21,8 ± 4,2 dias e grandes perfurações: 21,2 ± 4,7 dias vs. 38,4 ± 5,7 dias; p < 0,01). Conclusão: Na regeneração de PMT traumáticas, a esponja de Gelfoam não só desempenha um papel de estrutura para a migração epitelial, mas também promove edema e hiperplasia de tecido de granulação nas bordas da perfuração e acelera a cicatrização do tímpano.

Endoscopic observation of different repair patterns in human traumatic tympanic membrane perforations.

INTRODUCTION: In the last decade, there has been an increasing use of biomaterial patches in the regeneration of traumatic tympanic membrane perforations. The major advantages of biomaterial patches are to provisionally restore the physiological function of the middle ear, thereby immediately improving ear symptoms, and act as a scaffold for epithelium migration. However, whether there are additional biological effects on eardrum regeneration is unclear for biological material patching in the clinic. OBJECTIVE: This study evaluated the healing response for different repair patterns in human traumatic tympanic membrane perforations by endoscopic observation. METHODS: In total, 114 patients with traumatic tympanic membrane perforations were allocated sequentially to two groups: the spontaneous healing group (n=57) and Gelfoam patch-treated group (n=57). The closure rate, closure time, and rate of otorrhea were compared between the groups at 3 months. RESULTS: Ultimately, 107 patients were analyzed in the two groups (52 patients in the spontaneous healing group vs. 55 patients in the Gelfoam patch-treated group). The overall closure rate at the end of the 3 month follow-up period was 90.4% in the spontaneous healing group and 94.5% in the Gelfoam patch-treated group; the difference was not statistically significant (p>0.05). However, the total average closure time was significantly different between the two groups (26.8±9.1 days in the spontaneous healing group vs. 14.7±9.1 days in the Gelfoam patch-treated group, p<0.01). In addition, the closure rate was not significantly different between the spontaneous healing group and Gelfoam patch-treated group regardless of the perforation size. The closure time in the Gelfoam patch-treated group was significantly shorter than that in the spontaneous healing group regardless of the perforation size (small perforations: 7.1±1.6 days vs. 12.6±3.9, medium-sized perforations: 13.3±2.2 days vs. 21.8±4.2 days, and large perforations: 21.2±4.7 days vs. 38.4±5.7 days; p<0.01). CONCLUSION: In the regeneration of traumatic tympanic membrane perforations, Gelfoam patching not only plays a scaffolding role for epithelial migration, it also promotes edema and hyperplasia of granulation tissue at the edges of the perforation and accelerates eardrum healing.

Terapias oncoespecíficas en pacientes con virus del herpes simple bucal / Oncospecific therapies in patients with herpes simplex virus

Se efectuó un estudio descriptivo y transversal de 25 pacientes con cáncer infectados por el virus del herpes simple bucal, quienes tenían el sistema inmunológico deteriorado debido al tratamiento oncoespecífico, atendidos en la consulta estomatológica del Policlínico de Especialidades del Hospital Provincial Docente Clinicoquirúrgico Saturnino Lora Torres de Santiago de Cuba, desde enero de 2014 hasta igual mes de 2016, a fin de evaluar los resultados del diagnóstico clínico y citopatológico de esta afección. En la casuística prevalecieron el sexo masculino, el dolor como sintomatología clínica, el bermellón del labio inferior como el sitio de mayor incidencia y la quimioterapia como la modalidad terapéutica de mayor reactivación de este agente viral. La citología exfoliativa confirmó la presencia de células epiteliales gigantes multinucleadas con cuerpos de inclusiones intranucleares y necrosis de células infectadas, respectivamente, siendo estos los hallazgos microscópicos más significativos(AU)

A descriptive and cross-sectional study of 25 patients with cancer infected by the oral herpes simplex virus was carried out. They had the immunologic system damaged due to oncospecific treatment, assisted in the stomatological service of the Specialties Polyclinic of Saturnino Lora Torres Teaching Clinical Surgical Provincial Hospital in Santiago de Cuba, from January, 2014 to the same month in 2016, in order to evaluate the results of the clinical and cytopathologic diagnosis of this disorder. In the case material the male sex and pain as clinical symptomatology, the vermilion of the lower lip as the place of more incidence and chemotherapy as the therapeutic modality of this viral agent higher reactivation prevailed. The exfoliative cytology confirmed the presence of multinucleous giant epithelial cells with bodies of intranuclear inclusions and infected cells necrosis, respectively, being these the most significant microscopic findings(AU)

Terapias oncoespecíficas en pacientes con virus del herpes simple bucal / Oncospecific therapies in patients with herpes simplex virus

Se efectuó un estudio descriptivo y transversal de 25 pacientes con cáncer infectados por el virus del herpes simple bucal, quienes tenían el sistema inmunológico deteriorado debido al tratamiento oncoespecífico, atendidos en la consulta estomatológica del Policlínico de Especialidades del Hospital Provincial Docente Clinicoquirúrgico Saturnino Lora Torres de Santiago de Cuba, desde enero de 2014 hasta igual mes de 2016, a fin de evaluar los resultados del diagnóstico clínico y citopatológico de esta afección. En la casuística prevalecieron el sexo masculino, el dolor como sintomatología clínica, el bermellón del labio inferior como el sitio de mayor incidencia y la quimioterapia como la modalidad terapéutica de mayor reactivación de este agente viral. La citología exfoliativa confirmó la presencia de células epiteliales gigantes multinucleadas con cuerpos de inclusiones intranucleares y necrosis de células infectadas, respectivamente, siendo estos los hallazgos microscópicos más significativos

A descriptive and cross-sectional study of 25 patients with cancer infected by the oral herpes simplex virus was carried out. They had the immunologic system damaged due to oncospecific treatment, assisted in the stomatological service of the Specialties Polyclinic of Saturnino Lora Torres Teaching Clinical Surgical Provincial Hospital in Santiago de Cuba, from January, 2014 to the same month in 2016, in order to evaluate the results of the clinical and cytopathologic diagnosis of this disorder. In the case material the male sex and pain as clinical symptomatology, the vermilion of the lower lip as the place of more incidence and chemotherapy as the therapeutic modality of this viral agent higher reactivation prevailed. The exfoliative cytology confirmed the presence of multinucleous giant epithelial cells with bodies of intranuclear inclusions and infected cells necrosis, respectively, being these the most significant microscopic findings

Tejido linfático asociado a mucosas: Células presentadoras de antígenos / Mucose associated lymphoid tissue: Antigen presenting cells

Se estudiaron al microscopio electrónico biopsias de mucosas normales y patológicas (cavidad bucal y cuello uterino), con especial atención a los sistemas de defensa existentes en las células epiteliales (CE) y en las células dendríticas (CD). Las CE, cuando están activadas, muestran su capacidad de fagocitar y procesar antígenos con la finalidad de presentarlos luego a las CD; los elementos implicados en esta función son vesículas de micropinocitosis, cuerpos multivesiculares, lisosomas, fagosomas, vesículas recubiertas por clatrina, gránulos de contenido denso recubiertos por una unidad de membrana, gránulos en cuyo interior se aprecian láminas que simulan hojas de cebolla, microcuerpos y gránulos con actividad de fosfatasa ácida. Las CD que recién han ingresado al interior del epitelio son de baja densidad electrónica y poseen grandes prolongaciones citoplasmáticas, que luego se reducen de tamaño, a la vez que aumenta la densidad de su citoplasma. Muestran vesículas de micropinocitosis, algunas recubiertas por clatrina, lisosomas y corpúsculos de Birberk. En este momento son reconocidas como células de Langerhans. Tanto en las CE como en las CD existen abundantes “pliegues marginales o de superficie“ (surface folds), conteniendo numerosas vesículas de micropinocitosis. Entre la CE y la CD se establecen íntimos contactos a través de los cuales las primeras presentan los antígenos fagocitados y tratados a las CD donde son terminados de procesar y se unen a las moléculas del complejo principal de histocompatibilidad y/o a moléculas con función similar (CD1). Las CD migran a los ganglios linfáticos donde presentan los antígenos a los linfocitos T y empieza el proceso de activación de estos, que conduce a la defensa frente a las noxas que han ingresado al organismo. De esta manera tanto las CD como las CE son un lazo de unión entre los sistemas de defensa innata y la adquirida.

We studied samples of normal and abnormal human mucosae, including oral tissue and uterine cervix, using electron microscopy. Special attention was given to the functions and mechanisms of defense carried out by the epithelial (EC) and dendritic cells (DC). Activated epithelial cells posses the capacity to uptake and process antigens, in order to present them, subsequently, to the dendritic cells. The structures and elements of the cells intervening on this function are: micropinocytic vesicles, multivesicular bodies, lysosomes, phagosomes, clathrin-covered vesicles, dense granules covered by a unit membrane, granules with onion likes leaves, microbodies, and dense granules with acid phosphatase activity. When they first arrive within the epithelial layers, the DC are clear with long cytoplasmic projections, which later become short, and the density of their cytoplasm increases. They possess mycropinocytic vesicles, some clathrine-covered vesicles, lysososmes and Birbeck granules. At this moment, they are known as Langerhans cells. EC and DC present many surface folds rich in micropynocytic vesicles. Between EC and DC there are many contacts (close junctions or tight junctions), through which antigens, phagocitized and processed by the EC, are given to the DC. These cells join them to major histocompatibility complex molecules or to other molecules with similar functions (CD1). Then the Langerhans cells travel to the lymphatic node to activate T cells and continue the immunologic task. So, in this way, both the EC and the DC are a link between the natural and the acquired immunological mechanisms.

Mitochondria in the midgut epithelial cells of sugarcane borer parasitized by Cotesia flavipes (Cameron, 1891) / Mitocôndria nas Células Epiteliais do Intestino Médio da Broca-da-cana Parasitada por Cotesia flavipes

The sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae) has been controlled by Cotesia flavipes (Hymenoptera: Braconidae); however, very little is known about the effect of the parasitism in the host organs, including the midgut. This work aims to verify mitochondrial alteration in the different midgut epithelial cells of D. saccharalis parasitized by C. flavipes. Midgut fragments (anterior and posterior region) of both non-parasitized and parasitized larvae were processed for transmission electron microscopy. The mitochondria of midgut epithelial cell in the parasitized larvae exhibit morphological alteration, represented by matrix rarefaction and vacuolisation. These mitochondrial alterations are more pronounced in the anterior midgut region during the parasitism process, mainly in the columnar cell.

Diatraea saccharalis (Lepidoptera: Crambidae), broca da cana-de-açúcar, tem sido controlada por Cotesia flavipes (Hymenoptera: Braconidae); pouco se sabe sobre o efeito do parasitismo nos diferentes órgãos do inseto hospedeiro, principalmente no intestino médio. O objetivo desse trabalho foi verificar as alterações mitocondriais das diferentes células epiteliais do intestino médio de larvas de D. saccharalis parasitadas por C. flavipes. Fragmentos do intestino médio (regiões anterior e posterior) de larvas de D. saccharalis não-parasitadas e parasitadas foram processados para microscopia eletrônica de transmissão. As mitocôndrias das células epiteliais do intestino médio de larvas parasitadas exibem alterações, especialmente rarefação e vacuolização da matriz, que foram mais pronunciadas nas células epiteliais da região anterior do intestino médio na vigência do parasitismo, em especial nas células colunares.

Bleomicina: un modelo de fibrosis pulmonar / Bleomycin: a lung fibrosis animal model

La bleomicina es un glicopéptido utilizado para el tratamiento del cáncer cuyo potencial terapéutico está limitado por su toxicidad pulmonar. El efecto citotóxico depende de la dosis e involucra el desarrollo de neumonitis que progresa a fibrosis; las células epiteliales alveolares son el blanco principal del daño inducido por la bleomicina. Se considera que la muerte de células epiteliales alveolares por apoptosis es un evento clave en el inicio y la progresión de la fibrosis pulmonar (FP) que se caracteriza por el depósito excesivo de moléculas de la matriz extracelular, principalmente de colágenas fibrilares en el parénquima pulmonar. En la investigación básica de la FP, la bleomicina se ha utilizado como el principal agente fibrogénico en modelos animales. Durante los últimos años, el modelo de bleomicina desarrollado en ratones trasgénicos se ha empleado para elucidar in vivo el papel de un gran número de biomoléculas involucradas en la FP.

Bleomycin is a glycopeptide used for cancer treatment, but the therapeutic potencial of this drug is limited by its lung toxicity. The cytotoxic effect of bleomycin is dose-dependent and involves pneumonitis that proceeds to lung fibrosis (LF). Alveolar epithelial cells are the main target of bleomycin induced injury. Alveolar epithelial cell death by apoptosis is considered as a key event in the initiation and progression of LF, that is characterized by excessive deposition of extracellular matrix, mainly fibrilar collagens in the lung parenchyma. Bleomycin has been used as the main fibrogenic agent in animal models in LF basic research; in recent years, a bleomycin model developed in transgenic mice has been used to elucidate the in vivo role of a great number of biomolecules involved in LF.

Polymorphonuclear leukocyte migration across intestinal epithelium induces epithelial cytokine expression

La migración de neutrófilos polimorfonucleares es un rasgo común de la inflamación activa, que precede la formación de abscesos. La contribución relativa de células epiteliales como fuente de quimioquinas en la infiltración de leucocitos durante la inflamación intestinal no ha sido estudiada. Para evaluar esta contribución nosotros diseñamos un modelo heterólogo de migración transepitelial, “in vitro”, haciendo uso de PMN de rata y células epiteliales de origen humano. Nosotros demostramos que neutrófilos polimorfonucleares luego de su activación quimiotáctica, inducen el incremento en los niveles de ARNm de IL-1ß, IL-8 en las células epiteliales de intestino, mientras que no afecta al ARNm de ENA-78. Estos resultados sugieren que quimiocinas y citoquinas sintetizados por la célula epitelial podrían jugar un papel en el mantenimiento de la respuesta inflamatoria.

Polymorphonuclear neutrophil migration is a common feature of active inflammation that precedes the formation of abscesses. The relative contribution of epithelial cells as a source of chemokines in the recruitment of leukocytes during intestinal inflammation has not been studied. To evaluate this contribution, we have designed a heterologous “in vitro” model for transepithelial PMN migration, based on the use of rat neutrophils and human epithelial cells. We show that polymorphonuclear neutrophil, upon chemotactic activation, induces an increase in IL-1ß, IL-8 levels in intestinal epithelial cells, while not changed ENA-78 mRNA. These results suggest that chemokines and cytokines synthesized by intestinal epithelial cells could play a role in the maintenance of the inflammatory response.

Morphometric study of the midgut epithelium in larvae of Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae) / Estudo morfométrico do epitélio do intestino médio em larvas de Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae)

A larva de Diatraea saccharalis Fabricius (broca-da-cana) tem grande interesse econômico, pois afeta o cultivo e aproveitamento industrial da cana-de-açúcar. Entretanto, poucos são os estudos sobre a morfologia interna desse inseto. O objetivo deste trabalho foi estudar, morfometricamente, o seu epitélio intestinal, ao longo de seu comprimento, visando caracterizar regiões estruturalmente diferentes. O intestino médio de larvas no último instar foi subdividido em três regiões: proximal, mediana e distal e os fragmentos foram processados para observação em microscopia de luz. Os cortes histológicos foram analisados em sistema computadorizado de análise de imagens para medir comprimento, largura e área do epitélio, das diferentes células epiteliais, dos seus respectivos núcleos e do lúmen intestinal. Os dados obtidos foram submetidos ao teste estatístico de Kruskal-Wallis e à análise multivariada. Nossos resultados mostraram que o intestino médio apresentou-se constituído, morfometricamente, por duas diferentes regiões, proximal e distal; a região mediana apresentou valores coincidentes tanto com a região proximal quanto com a distal, sugerindo ser região intermediária. As células epiteliais (colunares, caliciformes e regenerativas), quando avaliadas pela análise estatística multivariada, não apresentaram diferença morfométrica nas diferentes regiões do intestino médio. Entretanto, a análise de variância, realizada para variáveis isoladas, mostrou que as células regenerativas apresentaram maior variabilidade morfométrica.

The sugarcane borer, Diatraea saccharalis Fabricius, has great economical interest as it affects the culture and industrial use of the sugarcane. However, there are few studies concerning the internal morphology of this insect. This work aims to study morphometrically the midgut and the epithelium along their lenght, trying to characterize different regions. Midgut of last instar larvae wasdivided in three regions: anterior, middle and posterior, and the fragments were processed for light microscopic observation. Histological sections were analyzed in a computerized system concerning the length, width and area of the epithelium, their cells, and the midgut lumen. The obtained data werestatistically analyzed by the Kruskal-Wallis test and by multivariate analysis. Our results showed that the midgut has two different regions, the anterior and the posterior; the middle region presents values that are coincident with the ones of either the anterior and the posterior portions, suggesting that thereis an intermediate region between the other two ones. The epithelial cells (columnar, goblet and regenerative cells), when evaluated by multivariate analysis, do not present significant morphometric differences in the different midgut regions. However, the analysis of variance for separate variables show that the regenerative cells present wide morphometric variability along the midgut.