Investigation of teratogenic effects of letrozole on fetal bone development / Investigación de los efectos teratogénicos de letrozol en el desarrollo óseo fetal

SUMMARY: Letrozole is mainly used for the treatment of unexplained infertility, breast cancer and polycystic ovarian syndrome, with secondary use in ovarian stimulation. In cases of unexpected or unknown pregnancy during the use of letrozole, letrozole may cause a teratogenic effect on the fetus. In this reason, in this study, we aimed to determine the effect of letrozole on fetal bone development. In this study, 32 pregnant Wistar albino rats were used. The rats were divided into four groups: Control (saline) and high; 0.3 mg/kg, medium; 0.03 mg/kg, low; 0.003 mg/ kg letrozole. Saline and letrozole were administered in 100 mL solutions by intraperitonaly from day 11 to day 15 of pregnancy. The skeletal system development of fetuses was examined with double skeletal staining, immunohistochemical staining methods and mineral density scanning electron microscopy. A total of 100 fetuses from female rats, 25 in each group, were included in the study. As a result of that, ossification rates were observed to decrease depending on the dose of letrozole in the forelimb limb (scapula, humerus, radius, ulna) and hindlimb (femur, tibia, fibula) limb bones. As a result of the statistical analysis, a statistically significant decrease was found in the ossification rates of all bones between the control group and low, medium, high letrozole groups (p<0.001). Exposure to letrozole during pregnancy adversely affected ossification and bone growth. However, the teratogenic effects of letrozole are unclear. Therefore, it needs to be investigated more extensively.

RESUMEN: Letrozol se usa principalmente para el tratamiento de la infertilidad inexplicable, el cáncer de mama y el síndrome de ovario poliquístico, con estimulación ovárica de uso secundario. En casos de embarazo inesperado o desconocido durante el uso de letrozol, puede causar un efecto teratogénico en el feto. Por esta razón, en este estudio, nuestro objetivo fue determinar el efecto de letrozol en el desarrollo óseo fetal. Se utilizaron 32 ratas albinas Wistar preñadas las cuales se distribuyeron en cuatro grupos: Control (solución salina) y alta; 0,3 mg/kg, medio; 0,03 mg/kg, bajo; 0,003 mg/kg de letrozol. Se administró solución salina y letrozol en soluciones de 100 mL por vía intraperitoneal desde el día 11 hasta el día 15 de la preñez. El desarrollo del sistema esquelético de los fetos se examinó con tinción esquelética doble, métodos de tinción inmunohistoquímica y microscopía electrónica de barrido de densidad mineral. Se incluyeron en el estudio un total de 100 fetos de ratas hembra, 25 en cada grupo. Como resultado, se observó que las tasas de osificación disminuían dependiendo de la dosis de letrozol en los huesos de los miembros torácicos (escápula, húmero, radio, ulna) y de las miembros pélvicos (fémur, tibia, fíbula). Se encontró una disminución estadísticamente significativa en las tasas de osificación de todos los huesos entre el grupo control y los grupos de letrozol bajo, medio y alto (p<0,001). La exposición a letrozol durante la preñez afectó negativamente la osificación y el crecimiento óseo. Sin embargo, los efectos teratogénicos del letrozol no están claros por lo que debe ser investigado más extensamente.

Identification of new human mesenchymal stem cell biomarker by aptamers / Identificação de novos biomarcadores de células tronco mesenquimais de origem humana por meio de aptâmeros

Stem cells are undifferentiated cells that can be distinguished from others by their ability to self-renew and to differentiate into new specific cell types. Mesenchymal stem cells (MSC) are adult stem cells that can be obtained from different sources, such as adipose tissue, bone marrow, dental pulp, and umbilical cord. They can either replicate, originating new identical cells, or differentiate into cells of mesodermal origin and from other germ layers. MSC have been studied as new tools for regenerative therapy. Although encouraging results have been demonstrated, MSC-based therapies still face a great barrier: the difficulty of isolating these cells from heterogeneous environments. MSC are currently characterized by immunolabelling through a set of multiple surface membrane markers, including CD29, CD73, CD90 and CD105, which are also expressed by other cell types. Hence, the present work aimed to identify new specific biomarkers for the characterization of human MSC using DNA aptamers produced by the SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technique. Our results showed that MSC from different origins bound to DNA candidate aptamers, that is, DNA or RNA oligonucleotides selected from random libraries that bind specifically to biological targets. Aptamer-bound MSC could be isolated by fluorescenceactivated cell sorting (FACS) procedures, enhancing the induction of differentiation into specific phenotypes (chondrocytes, osteocytes and adipocytes) when compared to the whole MSC population. Flow cytometry analyses revealed that candidate aptamers bound to 50% of the MSC population from dental pulp and did not present significant binding rates to human fibroblasts or lymphocytes, both used as negative control. Moreover, immunofluorescence images and confocal analyses revealed staining of MSC by aptamers localized in the surfacemembrane of these cells. The results also showed internal staining of human monocytes by our investigated aptamers. A non-specific control aptamer (CNTR APT) obtained from the random pool was then utilized to compare the specificity of the aptamers bound to the analyzed non-apoptotic cells, showing no staining for MSC. However, 40% of the monocytes bound to the CNTR APT. Normalized data based on the cells bound to candidate aptamers compared to those bound to the CNTR APT, revealed a 10 to 16-fold higher binding rate for MSC against 2-fold for monocytes. Despite its low specificity, monocyte-aptamer binding occurs probably due to the expression of shared markers with MSC, since monocytes are derived from hematopoietic stem cells and are important for the immune system ability to internalize/phagocyte external molecules. Given that, we performed a pull-down assay followed by mass spectrometry analysis to detect which MSC-specific protein or other target epitope not coexpressed by monocytes or the CNTR APT would bind to the candidate aptamer. Distinguishing between MSC and monocyte epitopes is important, as both cells are involved in immunomodulatory effects after MSC transplantations. ADAM17 was found to be a target of the APT10, emerging as a possible biomarker of MSC, since its involvement in the inhibition of the TGF signaling cascade, which is responsible for the differentiation of MSC. Thus, MSC with a higher stemness profile should overexpress the protein ADAM17, which presents a catalytic site with affinity to APT10. Another target of Apt 10 is VAMP3, belonging to a transmembrane protein complex that is involved in endocytosis and exocytosis processes during immune and inflammatory responses. Overall, proteins identified as targets of APT10 may be cell surface MSC biomarkers, with importance for MSC-based cell and immune therapies

Células tronco são células indiferenciadas que podem ser distinguidas de outros tipos celulares por meio da habilidade de se auto renovarem e de se diferenciarem em novos tipos celulares. Células tronco mesenquimais (MSC) são células tronco adultas encontradas em diferentes tecidos como tecido adiposo, polpa de dente e cordão umbilical. Estas células podem se autodividir em células idênticas ou se diferenciarem em células de origem mesodermal. Estas células têm sido estudadas em novas aplicações que envolvem terapia regenerativas. Embora resultados encorajadores tenham sido demonstrados, terapias que utilizam MSC ainda encontram uma grande barreira: a dificuldade no isolamento destas células a partir de um ambiente heterogêneo. MSC são caracterizadas por populações positivas em ensaios de imunomarcação para os epítopos membranares CD29, CD73, CD90 e CD105, presentes também em outros tipos celulares. Assim, o presente trabalho tem o objetivo de identificar novos biomarcadores de MSC de origem humana, utilizando aptâmeros de DNA produzidos pela técnica SELEX (Systematic Evolution of Ligands by EXponential Enrichment) como ferramenta. Nossos resultados mostraram que MSC de diferentes origens ligam-se a aptâmeros (oligonucleotídeos de DNA ou RNA que atuam como ligantes específicos de alvos moleculares) de DNA candidatos que atuam no isolamento de MSC por meio da técnica FACS de separação celular, promovendo uma maior indução de diferenciação em células específicas (condrócitos, osteócitos e adipócitos) comparada com a população total de MSC. Análises de citometria de fluxo mostraram que os aptâmeros candidatos se ligam a 50% das MSC de polpa de dente e não apresentam taxa de ligação significante para fibroblastos e linfócitos de origem humana - utilizados como controles negativo. Além domais, imagens de imunofluorescência e confocal mostraram ligação na superfície da membrana de MSC e a marcação interna de monócitos a estes aptâmeros. Portanto, um aptâmero controle (CNTR APT) foi utilizado para comparar a especificidade dos aptâmeros ligados a células viáveis, mostrando a não ligação deste aptâmero a MSC. Porém, 40% da população de monócitos ligou-se ao CNTR APT. Uma normalização baseada na comparação entre as taxas de ligação entre células ligadas com aptâmeros candidatos e o aptâmero controle gerou uma taxa de especificidade entre 10-16 vezes maior para MSC contra 2,5 vezes para os monócitos. Deste modo, embora os resultados tenham mostrado uma taxa de ligação entre monócitos e aptâmeros, as MSC ligadas aos aptâmeros candidatos possuem uma maior taxa de especificidade devido a uma maior presença de antígenos que são expressos em ambas as células. Um ensaio de Pull Down seguido de espectrometria de massas foi utilizado para a identificação de biomarcadores que se ligariam aos aptâmeros candidatos, e que não seriam co-expressos por monócitos e por antígenos ligados ao aptâmero controle. Deste modo, a proteína ADAM17 foi identificada nas amostras de APT10 ligadas às MSC. Tal proteína está relacionada à inibição de uma cascata de sinalização da família de proteínas TGF, responsável pela diferenciação de MSC. Assim, MSC com maior potencial tronco deveriam expressar ADAM17 em maior quantidade. Tal proteína apresenta um sítio catalítico que demonstra interagir com o APT10, de acordo com predição Docking entre proteína e DNA. Foi identificada também, a proteína VAMP3, que pertence a um complexo proteico transmembranar responsável pelos processos de endocitose e exocitose, e que podem ter um papel importante na liberação de citocinas e outras moléculas relacionadas às respostas imune e inflamatórias. Deste modo, o APT10 identificou proteínas importantes que devem estar relacionas com a melhora de imunoterapias que utilizam MSC

Using histological staining techniques to improve visualization and interpretability of tooth cementum annulation analysis / Uso de técnicas de tinción histológica para mejorar la visualización y la interpretación del análisis de la anulación del cemento dental

SUMMARY: The aim of the present study was to evaluate the effect of different staining techniques on applicability and accuracy of tooth cementum annulation (TCA) method. Nine decalcination techniques, 8 dehydration protocols and 8 different techniques were applied in 3 teeth from the persons of a known age. Black and white, and color images of histological sections were captured. An x- ray was taken of each tooth and they were photographed. Researchers were asked to observe both black/white and color images of histological sections. Researchers were divided into two groups. The first group analyzed histological images only, and the second group had photos of teeth and X-rays. In the first group of observers (without X ray) the differences in age estimation between real and observed age were significant for 2 younger patients, but not for the oldest patient, where the observed and real values matched. Of the 6 raters, the assesments of the last 3 (that used x-ray images together with histological sections) did not differ significantly from the real values. Extensive analysis and multiple repetitions performed in the present investigation revealed that the most optimal method of decalcification for TCA method was EDTA II for a period longer than 14 days at a section thickness of 2-3mm, while the most optimal protocol for dehydration was number IV. When it comes to staining, the most optimal staining protocol used for the cemental lines visualization and counting was Crocein Scarlet/Acid Fuchsin staining and Toluidine blue staining used at semithin section. Additional use of preexperimental evaluation employing x-ray of analyzed teeth decreased the errors of age estimation.

RESUMEN: El objetivo del presente estudio fue evaluar el efecto de diferentes técnicas de tinción sobre la aplicación y precisión del método de anulación de cemento dental (TCA). Se usaron nueve técnicas de descalcinación, 8 protocolos de deshidratación y 8 técnicas diferentes en 3 dientes de personas de edad conocida. Se capturaron imágenes en blanco y negro y en color de cortes histológicos. Se tomó una radiografía de cada diente y se fotografiaron. Los investigadores observaron las imágenes en blanco y negro y en color de las secciones histológicas. Los investigadores se dividieron en dos grupos; el primer grupo analizó solo imágenes histológicas y el segundo grupo tenía fotografías de los dientes y las radiografías. En el primer grupo de observadores (sin rayos X) las diferencias en la estimación de la edad entre la edad real y la edad observada fueron significativas para 2 pacientes más jóvenes, pero no para el paciente de mayor edad, donde los valores observados y reales coincidieron. De los 6 evaluadores, las valoraciones de los 3 últimos (que utilizaron imágenes de rayos X junto con cortes histológicos) no difirieron significativamente de los valores reales. El análisis exhaustivo y las múltiples repeticiones realizadas en la presente investigación revelaron que el método de descalcificación más óptimo para el método TCA fue EDTA II durante un período superior a 14 días con un grosor de sección de 2-3 mm, mientras que el protocolo óptimo para la deshidratación fue el número IV. En lo que respecta a la tinción, el protocolo de tinción más óptimo utilizado para la visualización y el recuento de las líneas de cemento fue la tinción con croceína escarlata / fucsina ácida y la tinción con azul de toluidina utilizada en la sección semifina. El uso adicional de la evaluación pre-experimental que emplea los rayos X de los dientes analizados disminuyó los errores de estimación de la edad.

Value of the toluidine blue test as an aid to determine the biopsy site in actinic prurigo cheilitis

Actinic prurigo (AP) is a type of photodermatosis that primarily affects the Latin American mestizo population. Histologically, AP cheilitis exhibits acanthosis with spongiosis and vacuolation of the basal cell layer overlying a dense lymphocytic inflammatory infiltrate that forms well-defined lymphoid follicles. Toluidine blue is a thiazide, acidophilic, and metachromatic dye used in vivo to selectively stain the acidic components of tissues such as sulfates, carboxylates, and phosphate radicals that are incorporated into DNA and RNA. It is necessary to develop a method that allows detecting, on clinical grounds the area of the lesion in which it is more feasible to find such structures. Thus to increase the sensitivity of the biopsy, in AP cheilitis to accurately identify where the lymphoid follicles reside, based on the higher concentration of DNA in such structures and thus confirm the diagnosis. In this study, staining was positive in 85% of patients with AP cheilitis, in 14 of whom 82% lymphoid follicles were observed by histopathology. One of the pathologist's problems in establishing the diagnosis of AP is that the main histopathological characteristics are not always identified in the submitted samples because it is not easy to clinically identify the most representative site of the lesion selected for performing a biopsy. Based on our results, we propose using toluidine blue as an auxiliary method to choose a tissue sample to facilitate the diagnosis and allow clinicians to make clinical correlations between the histopathological and therapeutic findings.

Histological comparison of two special methods of staining melanin in human skin / Comparación histológica de dos métodos especiales de tinción de melanina en piel humana

SUMMARY: Human skin melanin was stained using the Fontana's silver nitrate method and Schmorl method. The results showed that, in the Fontana's silver nitrate method, melanin and silver-bound cells were black and other tissues were red. When stained using the Schmorl method, effects on melanin differed based on whether the nuclei were stained. When the nucleus was stained, melanin appeared blue-black or blue-green, and other tissue structures were purple. When the nucleus was not stained, melanin was orange and other structures were pink. Comparing the two staining methods, we concluded that Fontana's silver nitrate method takes a long time; in contrast, the Schmorl method showed two different types of results depending on whether the nucleus was stained, and it takes less time than Fontana staining, so we here consider the Schmorl method more suitable for special staining of melanin than Fontana's silver nitrate method.

RESUMEN: La melanina de la piel humana se tiñó utilizando el método del nitrato de plata de Fontana y el método Schmorl. Los resultados mostraron que, en el método del nitrato de plata de Fontana, la melanina y las células unidas a plata eran negras y otros tejidos eran rojos. Cuando se tiñó con el método de Schmorl, los efectos sobre la melanina difirieron en función de si se tiñeron los núcleos. Cuando se tiñó el núcleo, la melanina apareció de color azul-negro o azul-verde, y otras estructuras de tejido fueron de color púrpura. Cuando el núcleo no estaba teñido, la melanina era naranja y otras estructuras eran rosadas. Al comparar los dos métodos de tinción, llegamos a la conclusión de que el método del nitrato de plata de Fontana lleva mucho tiempo; por el contrario, el método Schmorl mostró dos tipos diferentes de resultados dependiendo de si el núcleo estaba teñido, y lleva menos tiempo que la tinción de Fontana, por lo que aquí consideramos que el método Schmorl es más adecuado para la tinción especial de melanina que el método del nitrato de plata de Fontana.

The modified method of luxol fast blue for paraffin-embedded myelin sheath staining / El método modificado de luxol fast blue para la tinción de mielina fijada en parafina

SUMMARY: Histological techniques are the study of animal and human tissues through staining and examining them under a microscope. To demonstrate the axonal degeneration and demyelination in histological studies, the Luxol Fast Blue staining is gold standard techniques. In this study, a new histochemical method based on modified Luxol Fast Blue for the staining of the myelin sheath in sciatic nerve tissues described. The sciatic nerves of rats were removed and then the sciatic nerve was immersed in 10 % formaldehyde for one week and embedded in paraffin block. Next, thin sections (5 µm) were cut, using a microtome and stained with conventional and modified Luxol Fast Blue. Our results showed that a new method of modified Luxol Fast Blue staining can accurately identify the myelin in the sciatic nerve fibers. The current study showed that the Luxol Fast Blue combination with Light Green has a good effect on myelin coloration, and the results of this study are comparable to LFB combination with Sirius red.

RESUMEN: Las técnicas histológicas son el estudio de tejidos animales y humanos mediante tinción y examen bajo un microscopio. Para demostrar la degeneración axonal y la desmielinización en estudios histológicos, la tinción Luxol Fast Blue es una técnica estándar de oro. En este estudio, se describe un nuevo método histoquímico basado en Luxol Fast Blue modificado para la tinción de mielina en los tejidos del nervio ciático. Se seccionaron los nervios ciáticos de ratas y luego el nervio ciático se sumergió en formaldehído al 10 % durante una semana y se fijó en bloque de parafina. Posteriormente, se cortaron secciones delgadas (5 µm) usando un microtomo y se tiñeron con Luxol Fast Blue convencional y modificado. Nuestros resultados mostraron que un nuevo método de tinción Luxol Fast Blue modificado puede identificar con precisión la mielina en las fibras del nervio ciático. El estudio actual mostró que la combinación Luxol Fast Blue con Light Green es un buen efecto sobre la coloración de mielina, y los resultados de este estudio son comparables a la combinación LFB con Sirius red.

Morphological relationship between the superficial cortical and deep grey matter structures in adult human brains: a cadaveric study / Relación morfológica entre las estructuras superficiales de la sustancia gris cortical y profunda en los cerebros humanos adultos: un estudio en cadáveres

While various neurodegenerative diseases affect cortical mass differently, finding an optimal and accurate method for measuring the thickness and surface area of cerebral cortex remains a challenging problem due to highly convoluted surface of the cortex. We therefore investigated cortical thickness in a sample of cadaveric specimens at the Discipline of Clinical Anatomy, Nelson R Mandela School of Medicine, University of KwaZulu-Natal, South Africa to provide some clue as to possible variations in the parameters. Following ethical approval, 60 brain samples were uniformly sectioned (5 mm thickness) and eight slices taken from each brain across regions of interest (ROI) prepared and stained by Mulligan's technique. Thickness was measured at selected angles (0º, 45º, 90º, 135º and 180º) for both right and left cerebral hemispheres. Mulligan's stain produced good cortical differentiation and clear images that enabled manual delineation of structures. Cortical thickness ranged from 3 to 5 millimeters across the ROI. Interestingly, there was rightward hemispheric asymmetry of cortical thickness of selective slices at suggested angles which is related to structurally and functionally important brain regions. Moreover, there was no significant correlation between the surface area of superficial cortex and the deep nuclei at the same level. The superficial cortex and deep nuclei are manifested independently in normal aging, neuropsychiatric or developmental disorders. Providing accurate morphometric evaluation of cortical thickness and area based on gross staining of the brain slices could provide qualitative data that may support the study of human cerebral cortex even in disease conditions.

Si bien varias enfermedades neurodegenerativas afectan a la masa cortical de manera diferente, encontrar un método óptimo y preciso para medir el grosor y el área de la superficie de la corteza cerebral sigue siendo un problema difícil debido a la superficie altamente enrevesada de la corteza. Por lo tanto, investigamos el grosor cortical en una muestra de cadáveres del Departamento de Anatomía Clínica de la Facultad de Medicina Nelson R. Mandela de la Universidad de KwaZulu-Natal, Sudáfrica, para proporcionar alguna pista sobre posibles variaciones en dichos parámetros. Después de la aprobación ética, 60 muestras de cerebro se seccionaron uniformemente (5 mm de grosor) y se tomaron ocho cortes de cada cerebro en regiones de interés (ROI) preparadas y teñidas con la técnica de Mulligan. El espesor se midió en los ángulos seleccionados (0º, 45º, 90º, 135º y 180º) para los hemisferios cerebrales derecho e izquierdo. La tinción de Mulligan produjo una buena diferenciación cortical e imágenes claras que permitieron la delineación manual de las estructuras. El grosor cortical osciló entre 3 y 5 milímetros a través del ROI. Curiosamente, hubo una asimetría hemisférica hacia la derecha del grosor cortical de los cortes en ángulos sugeridos que se relacionan con regiones cerebrales estructural y funcionalmente importantes. Además, no hubo una correlación significativa entre el área de la superficial de la corteza superficial y los núcleos profundos en el mismo nivel. La corteza superficial y los núcleos profundos se manifiestan de manera independiente en el envejecimiento normal, en los trastornos neuropsiquiátricos o del desarrollo. Realizar una evaluación morfométrica precisa del grosorcortical y el área basada en la tinción macroscópica de los cortes del cerebro, podría proporcionar datos cualitativos que puedan respaldar el estudio de la corteza cerebral humana incluso en condiciones de enfermedad.

Comparative study of metachromatic staining methods in assessing the exfoliative cell types during oestrous cycle in Sprague-Dawley laboratory rats / Estudio comparativo de los métodos de tinción metacromática en la evaluación de los tipos de células exfoliativas durante el ciclo estral en ratas Sprague-Dawley de laboratorio

This study was aimed at comparing the commonly used metachromatic stains viz., Papanicolaou stain, WrightGiemsa, Toluidine blue and Methylene blue in the assessment of cell types of the oestrous cycle in rats. Eight female Sprague-Dawley rats aged 8-9 weeks were used for this assessment. Cotton Swabs were gently inserted in the animals vagina to obtain cells from which they were then transferred to glass slides for staining and evaluation under microscopy. The different cell types were compared for their morphological features and clarity of cellular detail under all four stains. The application, advantages and limitations of all stains were then discussed. It was concluded that the selection of the most effective stain in the assessment of vaginal cytology depends on their application to clinical or research which was based on the cellular detail of interest, time, cost and availability of each staining procedure.

El presente estudio tuvo como objetivo comparar las tinciones metacromáticas comúnmente utilizadas, Wright's-Giemsa, azul de toluidina, azul de metileno y tinción de Papanicolaou, en la evaluación de los tipos de células del ciclo estral en ratas. El estudio se realizó en ocho ratas hembras SpragueDawley, con edades entre 8 y 9 semanas, y se usaron hisopos vaginales de algodón para preparar portaobjetos. Los diferentes tipos de células se compararon por sus características morfológicas y claridad en las cuatro tinciones. La aplicación, ventajas y limitaciones de todas las tinciones fueron discutidas. Se concluye que la selección de la tinción más efectiva en la evaluación de la citología vaginal depende de su uso, es decir, clínico o de investigación, el detalle celular de interés, tiempo, costo y disponibilidad.

Evaluation of dermal collagen stained with picrosirius red and examined under polarized light microscopy

Abstract: The special picrosirius red staining highlights the natural birefringence of collagen fibers when exposed to polarized light. The results from birefringence allow to evaluate the organization of the collagen fibers in the tissues. The authors intend to elucidate all steps to obtain and capture images of histological sections stained with picrosirius red and evaluated under polarized light microscopy, as well as possible artefacts that may occur.

Evaluation of microscopic protocols for somatic cell counts in milk of dairy sheep / Avaliação de protocolos do método microscópico para contagem de células somáticas no leite de ovelhas leiteiras

The somatic cell count (SCC) is a diagnostic tool that indicates the mammary gland health and can be determined by the counting of cells in the microscope. There are discussions regarding appropriate staining method to the ewes' milk. The present study aimed to identify a methodology of microscopic SCC proper to the milk of the ovine species. Therefore, glass slides for smear were manufactured with 10 µL of ewe's milk in 1 cm2, and the fixers xylol and Carnoy's solution were tested, as well as and May-Grünwald, Broadhurst-Paley, Wrigth and Panoptic stainings. Carnoy's solution was elected, because it allowed a better fixation of the dairy film to the microscope slides, and Broadhurst-Paley staining, due to its good coloration and visualization of cells, as well as the differentiation of cytoplasmic corpuscles in ewe's milk. Broadhurst-Paley coloration is a tool applicable to the somatic cell count in ovine specie's milk.(AU)

A contagem de células somáticas é uma ferramenta de diagnóstico indicativa da saúde da glândula mamária e pode ser determinada em microscópio. Sobre ela, existem discussões quanto à coloração adequada ao leite de ovelhas. O presente estudo objetivou identificar uma metodologia de preparação de lâminas para a contagem microscópica de células somáticas do leite da espécie ovina. Para tanto, confeccionaram-se lâminas de esfregaços de 10 µL de leite de ovelha em 1 cm2, testaram-se os fixadores de xilol e solução de Carnoy e as colorações de May-Grünwald, Broadhurst-Paley, Wrigth e Panótico. Elegeram-se a solução de Carnoy, pois esta permitiu melhor fixação do filme lácteo às lâminas de microscopia, e o corante Broadhurst-Paley, que propiciou boa coloração e visualização das células, bem como a diferenciação dos corpúsculos citoplasmáticos presentes no leite ovino. A coloração é uma ferramenta aplicável à contagem de células somáticas no leite da espécie ovina.(AU)

Utilidad de tinción de tricrómico de Masson en la cuantificación de densidad media vascular en mucosa oral normal, displasia epitelial y carcinoma oral de células escamosas / Utility of Masson's trichrome stain in the quantification of mean vascular density in normal oral mucosa, epithelial dysplasia and oral squamous cell carcinoma

RESUMEN: El objetivo de este estudio fue valuar la utilidad del uso de la tinción de Tricrómico de Masson (TM) en la cuantificación de la densidad media vascular (DMV) en Mucosa Oral Normal (MON), Displasia Epitelial Oral (DEO) y Carcinoma Oral de Células Escamosas (COCE). Estudio descriptivo de serie de casos. Se analizaron 17 muestras de MON, 15 muestras de DEO y 16 de COCE, teñidas con TM. Para determinar su utilidad, se compararon con las mismas muestras analizadas con técnica de inmunohistoquímica contra CD31. La cuantificación de la DMV se realizó en las 3 áreas de mayor vascularización de cada muestra. Se determinó la DMV según diagnóstico mediante la tinción TM e inmunohistoquímica contra CD31, y se calculó la correlación entre ambos. La DMV cuantificada con TM y contra CD31 difiere según el diagnóstico, observándose un aumento de la DMV al malignizarse el diagnóstico. No se encontraron diferencias al comparar la DMV cuantificada con TM y contra CD31. La correlación de la DMV analizado por TM y contra CD31 es significativa y moderada. La cuantificación de vasos sanguíneos es posible mediante la tinción de TM en muestras de MON, DEO y COCE, con una correlación moderada con la inmunohistoquímica contra CD31.

SUMMARY. The objective of this study was to evaluate the utility of Masson's Trichrome (TM) staining in the quantification of the mean vascular density (DMV) in samples of normal oral mucosa (MON), oral epithelial dysplasia (ODE) and oral squamous cell carcinoma (COCE). The design - a descriptive study of case series. We analyzed 17 samples of MON, 15 samples of DEO and 16 samples of COCE, stained with TM. To determine usefulness, we compared and analyzed the same samples, either stained with TM or with immunohistochemical technique against CD31. Quantification of the DMV was performed in the 3 areas of greatest vascularization in each sample. DMV was determined according to diagnosis by TM staining and immunohistochemistry against CD31, and the correlation between the two was then calculated. DMV quantified with TM and against CD31 differs according to the diagnosis, with an increase in DMV upon malignant diagnosis. No differences were found when comparing DMV quantified with TM and against CD31. The correlation of the DMV analyzed by TM and against CD31 is significant and moderate. Quantification of blood vessels is possible by TM staining in samples of MON, DEO and COCE. TM staining is moderately correlated with immunohistochemistry against CD31.

Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer

ABSTRACT The spoilage of beer by bacteria is of great concern to the brewer as this can lead to turbidity and abnormal flavors. The polymerase chain reaction (PCR) method for detection of beer-spoilage bacteria is highly specific and provides results much faster than traditional microbiology techniques. However, one of the drawbacks is the inability to differentiate between live and dead cells. In this paper, the combination of propidium monoazide (PMA) pretreatment and conventional PCR had been described. The established PMA-PCR identified beer spoilage Lactobacillus brevis based not on their identity, but on the presence of horA gene which we show to be highly correlated with the ability of beer spoilage LAB to grow in beer. The results suggested that the use of 30 µg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable L. brevis cells. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. brevis cells was 2.0 µg/mL. The detection limit of PMA-PCR assay described here was found to be 10 colony forming units (CFU)/reaction for the horA gene. Moreover, the horA-specific PMA-PCR assays were subjected to 18 reference isolates, representing 100% specificity with no false positive amplification observed. Overall the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for detection of potential beer spoilage L. brevis and efficiently differentiates between viable and nonviable cells.

Importancia del control de la calidad de la baciloscopia en los laboratorios de diagnóstico de tuberculosis / Importance of quality control of baciloscopy in laboratories that perform diagnosis of tuberculosis

Introducción: La baciloscopia es la herramienta primaria en el diagnóstico de la tuberculosis (TBC) pulmonar activa, siendo esta la técnica más utilizada internacionalmente en la búsqueda de casos infecciosos. El control de calidad consiste en la relectura de las láminas por un observador altamente calificado. Objetivo: Evaluar y destacar la importancia del control de la calidad de la baciloscopia en los laboratorios provinciales encargados del diagnóstico de TBC en Cuba. Material y Métodos: Este estudio fue realizado en el Laboratorio Nacional de Referencia e Investigaciones de Tuberculosis, Lepra y Micobacterias del Instituto de Medicina Tropical "Pedro Kourí", La Habana, Cuba. Fueron evaluadas 2.676 láminas recibidas en el período de enero de 2013-diciembre de 2014, procedentes de los diferentes Centros Provinciales de Higiene, Epidemiología y Microbiología de Cuba, incluido el Municipio Especial Isla de la Juventud. Resultados: Hubo 2.664 (99,5%) láminas concordantes, la concordancia obtenida para las láminas positivas fue 96,5% y las negativas 99,8%. Se identificaron 12 errores de lectura: 7 (3,5%) falsos positivos, 5 (0,2%) falsos negativos. Se calificaron láminas con calidad de la muestra adecuada en 2.039 (76,2%), presentaron deficiencias en la realización de la extensión 1.464 (54,7%), y la tinción fue adecuada en 2.343 (87,6%). El índice de kappa fue de 0.9674. Conclusión: Aunque hubo una adecuada concordancia entre las observaciones realizadas, se recomienda mejorar la calidad del extendido, mantener programa de entrenamiento al personal que realiza esta actividad, al igual que las supervisiones periódicas por parte de especialistas, para continuar mejorando la calidad del diagnóstico.

Background: Baciloscopy is the primary tool for pulmonary tuberculosis diagnosis, being this technique the most used internationally in the search for infectious cases. Quality control is the process of the rechecking smears by a highly qualified observer. Aim: To evaluate and highlight the importance of quality control of smear microscopy in the Provincial Laboratories diagnosticians of Tuberculosis in Cuba. Methods: This study was conducted at the National Reference Laboratory and Research in Tuberculosis, Leprosy and Mycobacteria in the Institute of Tropical Medicine "Pedro Kouri", Havana, Cuba, Were evaluated 2676 smears received from January 2013 to December 2014, from Provincial Centers of Hygiene, Epidemiology and Microbiology of Cuba, including the special municipality Isla de la Juventud. Results: 2,664 (99.5%) were concordant smears, the correlation obtained for the positive smears were 96.5% and 99.8% for negative. Were identified12 reading errors: 7 (3.5%) false positive and 5 (0.2%) false negatives. Slides were classified with adequate quality of smears in 2039 (76.2%), showed difficulties in realizing the extension in 1464 (54.7%) and staining were adequate in 2343 (87.6%). The kappa index was 0.9674. Conclusion: Although there was good agreement between observations it is recommended to improve the quality of extended, maintain staff training program that performs this activity, like regular supervision by specialists, to further improve the quality of diagnosis.

The dynamic structure of Spitzenkörpers of Trichosporon asahii examined by the fluorescent probe FM4-64

Abstract The Spitzenkörper is a dynamic and specialized multicomponent cell complex present in the tips of hyphal cells. The amphiphilic styryl dye FM4-64 was found to be ideal for imaging the dynamic changes of the apical vesicle cluster within growing hyphal tips. It is widely used as a marker of endocytosis and to visualize vacuolar membranes. Here we performed uptake experiments using FM4-64 to study the dynamic of the Spitzenkörper in Trichosporon asahii. We observed that Spitzenkörpers were present at the tip of the budding site of the spore, blastospore, and the germ tube of T. asahii. We also found that Spitzenkörpers were present at the tip of the hyphae as well as the subapical regions. Cytochalasin D, an inhibitor of actin polymerization, leads to abnormal Spitzenkörper formation and loss of cell polarity.

Fluorescent membrane markers elucidate the association of Borrelia burgdorferi with tick cell lines

This study aimed to describe the association of Borrelia burgdorferi s.s. with ixodid tick cell lines by flow cytometry and fluorescence and confocal microscopy. Spirochetes were stained with a fluorescent membrane marker (PKH67 or PKH26), inoculated into 8 different tick cell lines and incubated at 30°C for 24 h. PKH efficiently stained B. burgdorferi without affecting bacterial viability or motility. Among the tick cell lines tested, the Rhipicephalus appendiculatus cell line RA243 achieved the highest percentage of association/internalization, with both high (90%) and low (10%) concentrations of BSK-H medium in tick cell culture medium. Treatment with cytochalasin D dramatically reduced the average percentage of cells with internalized spirochetes, which passed through a dramatic morphological change during their internalization by the host cell as observed in time-lapse photography. Almost all of the fluorescent bacteria were seen to be inside the tick cells. PKH labeling of borreliae proved to be a reliable and valuable tool to analyze the association of spirochetes with host cells by flow cytometry, confocal and fluorescence microscopy.

Single-walled carbon nanotubes functionalized with sodium hyaluronate enhance bone mineralization

The aim of this study was to evaluate the effects of sodium hyaluronate (HY), single-walled carbon nanotubes (SWCNTs) and HY-functionalized SWCNTs (HY-SWCNTs) on the behavior of primary osteoblasts, as well as to investigate the deposition of inorganic crystals on titanium surfaces coated with these biocomposites. Primary osteoblasts were obtained from the calvarial bones of male newborn Wistar rats (5 rats for each cell extraction). We assessed cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay and by double-staining with propidium iodide and Hoechst. We also assessed the formation of mineralized bone nodules by von Kossa staining, the mRNA expression of bone repair proteins, and the deposition of inorganic crystals on titanium surfaces coated with HY, SWCNTs, or HY-SWCNTs. The results showed that treatment with these biocomposites did not alter the viability of primary osteoblasts. Furthermore, deposition of mineralized bone nodules was significantly increased by cells treated with HY and HY-SWCNTs. This can be partly explained by an increase in the mRNA expression of type I and III collagen, osteocalcin, and bone morphogenetic proteins 2 and 4. Additionally, the titanium surface treated with HY-SWCNTs showed a significant increase in the deposition of inorganic crystals. Thus, our data indicate that HY, SWCNTs, and HY-SWCNTs are potentially useful for the development of new strategies for bone tissue engineering.

COMPARISON OF PERMANENT STAINING METHODS FOR THE LABORATORY DIAGNOSIS OF TRICHOMONIASIS

Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease (STD) in the world. The diagnosis is based on wet mount preparation and direct microscopy on fixed and stained clinical specimens. The aim of this study was to compare the performance of different fixing and staining techniques used in the detection of T. vaginalis in urine. The smears were fixed and submitted to different methods of permanent staining and then, the morphological aspects of the parasites were analyzed and compared. The Papanicolaou staining with ethanol as the fixative solution showed to be the best method of permanent staining. Our data suggest that staining techniques in association with wet mount examination of fresh specimens contribute to increase the sensitivity in the diagnosis of trichomoniasis.

Estimativa da viabilidade polínica de araçazeiro (Psidium cattleianum Sabine) através de distintos métodos de coloração / Estimation of pollen viability of strawberry guava (Psidium cattleianum Sabine) through distinct staining methods

RESUMO A análise histoquímica dos grãos de pólen através do uso de corantes permite a determinação de sua viabilidade polínica. O objetivo deste estudo foi comparar a eficiência dos corantes orceína acética 2% e reativo de Alexander modificado, para estimar a viabilidade polínica de vinte acessos de araçazeiro (Psidium cattleianum Sabine) – uma árvore de frutos muito apreciados que também é utilizada para fins medicinais. Botões florais na pré-antese foram coletados e fixados em etanol:ácido acético (3:1) por 24 horas, após os botões foram transferidos para etanol 70% e mantidos sob refrigeração. A técnica de esmagamento das anteras foi utilizada no preparo das lâminas. Para cada acesso foram preparadas 2 lâminas por corante e analisados 500 grãos de pólen por lâmina. A viabilidade polínica foi estimada através da porcentagem de grãos de pólen viáveis. De modo geral, observou-se que os grãos de pólen corados com orceína acética 2% apresentaram viabilidade alta, acima de 98,1%, e os acessos não diferiram entre si. A viabilidade polínica através da coloração com a solução de Alexander variou de 43% (acesso Silveira Martins 1) a 97% (acesso Candelária 1), mostrando que provavelmente exista variabilidade genética entre os acessos. Em apenas dois acessos não houve diferença significativa entre os valores de viabilidade encontrados através dos dois corantes testados. Conclui-se então que a orceína acética 2% superestimou a viabilidade polínica de P. cattleianum e que o reativo de Alexander é o mais preciso, devido a sua dupla coloração (verde de malaquita + fucsina ácida). Assim, indica-se o reativo de Alexander para análise de estimativa da viabilidade polínica de genótipos de araçá envolvidos em um programa de melhoramento genético.

ABSTRACT The histochemical analysis of pollen grains through the use of stains makes it possible to determine their pollen viability. The purpose of this study was to compare the efficiency of 2% acetic orcein and Alexander’s stain modified, in estimating the pollen viability of twenty strawberry guava accessions (Psidium cattleianum Sabine) – a valued fruit tree which is used for medicinal purposes. Pre-anthesis flower buds were collected and fixed in ethanol:acetic acid (3:1 v/v) for 24 hours at room temperature, subsequently they were stored in 70% ethanol under refrigeration. The anther squash technique was performed for slide preparation. In each accession two slides per stain and 500 pollen grains were analyzed. The pollen viability was estimated according to the percentage of viable pollen grains. Overall, it was observed that pollen grains stained with 2% acetic orcein evinced a high viability, higher than 98,1%, and the accessions did not differ. The pollen viability estimated with Alexander’s stain varied from 43% (accession of Silveira Martins 1) to 97% (accession of Candelária 1), indicating that there is probably genetic variability among the accessions. There was no significant difference among the levels of viability found by means of the two stains in only two accessions. In conclusion, 2% acetic orcein overestimated the pollen viability of P. cattleianum, whereas the Alexander’s stain is in turn more accurate due to its two stains (green malachite and fuchsine). Thus, it is indicated Alexander’s stain for analysis estimation of pollen viability of strawberry guava genotypes involved in a genetic improvement program.

MUC-1 expression in pleomorphic adenomas using two human milk fat globule protein membrane antibodies (HMFG-1 and HMFG-2)

Pleomorphic adenoma (PA) is the most common salivary gland tumor and its microscopic features and histogenesis are a matter of debate. Human milk fat globule protein membrane (HMFG) monoclonal antibodies (MoAbs) comprise a set of antibodies against the mucin 1 (MUC-1) protein detected in several salivary gland tumors. Objective The aim of this study was to assess the immunoexpression of the PA neoplastic cells to MUC-1 protein using HMFG-1 and HMFG-2 MoAbs, contrasting these results with those from normal salivary gland tissue. Material and Methods Immunohistochemical detection of MUC-1 protein using HMFG-1 and HMFG-2 MoAbs was made in 5 mm thick, paraffin embedded slides, and the avidin-biotin method was used. Results Positivity to HMFG-1 and HMFG-2 MoAbs was found in ductal, squamous metaplastic and neoplastic myoepithelial cells, keratin pearls and intraductal mucous material. Two kinds of myoepithelial cells were identified: classic myoepithelial cells around ducts were negative to both MoAbs, and modified myoepithelial cells were positive to both MoAbs. This last cellular group of the analyzed tumors showed similar MUC-1 immunoexpression to ductal epithelial cells using both HMFG antibodies. Intraductal mucous secretion was also HMFG-1 and HMFG-2 positive. Conclusions Our results showed there are two kinds of myoepithelial cells in PA. The first cellular group is represented by the different kinds of neoplastic myoepithelial cells and is HMFG-positive. The second one is HMFG-negative and represented by the neoplastic myoepithelial cells located around the ducts. .