Utilização da morfometria celular no microambiente neoplásico e sua relação com a imunoexpressão da interleucina-4 / Use of cellular morphometry in the neoplastic microenvironment and its relationship with the immunoexpression of interleukin-4

A morfometria celular é um tipo de análise quantitativa que utiliza as medidas geométricas para obter informações acerca da morfologia nuclear, citoplasmática e outras medidas gerais das células, sendo que estes parâmetros podem estar alterados devido a processos fisiológicos e patológicos que modificam a morfologia celular normal, sendo uma análise relevante no prognóstico de diversas lesões. Os ceratinócitos possuem morfologia poliédrica com núcleos ovoides, mas podem sofrer alterações com eventos patológicos e fisiológicos, por exemplo, no núcleo. Essas alterações podem estar presentes em neoplasias malignas, como, no carcinoma epidermoide. A interleucina-4 (IL-4) é uma citocina secretada por diversos tipos de células, estando envolvida no desenvolvimento e diferenciação de células Th2, atuando na resposta antiinflamatória e sua expressão parece estar relacionada com o desenvolvimento de algumas neoplasias, incluindo o câncer oral. O objetivo da pesquisa foi realizar análise morfométrica celular (compartimentos nuclear e citoplasmático), identificação de irregularidades nucleares de ceratinócitos malignos e comparar com a imunoexpressão da IL-4 e profundidade de invasão nos casos de Carcinoma Epidermoide de Lábio Inferior (CELI) e Língua Oral (CELO). Foram analisados 30 casos de cada lesão. Para morfometria celular foram analisados 16 ceratinócitos malignos por caso; a imunoexpressão de IL-4 foi analisada no parênquima e estroma das lesões, utilizando softwares de análises de imagens. Foram aplicados os testes estatísticos de Análise de Variância, Kruskal-Wallis e Correlação de Spearman. A imunoexpressão de IL-4 no parênquima foi maior nos casos de CELI em todos os campos analisados (p<0,05); Houve correlação positiva entre área celular total e profundidade de invasão (p=0,038) e negativa entre imunoexpressão da IL-4 no parênquima superficial e perímetro nuclear superficial (p=0,007). Portanto, sugere-se que exista uma relação protetora da imunoexpressão IL-4 com as lesões analisadas, bem como alterações morfométricas dos ceratinócitos malignos com a imunoexpressão da IL-4 nessas lesões (AU).

Cell morphometry is a type of quantitative analysis that uses geometric measurements of cells to obtain information about the nuclear, cytoplasmic and general morphology of cells. Such parameters may be altered due to physiological and pathological processes that modify normal cell morphology, being a relevant analysis in the prognosis of several injuries. Keratinocytes have a polyhedral morphology with ovoid nuclei, but may undergo changes with pathological and physiological events, for example, in the nucleus. These changes may be present in malignant neoplasms, such as squamous cell carcinoma. Interleukin-4 (IL-4) is a cytokine secreted by several types of cells, being involved in the development and differentiation of Th2 cells, acting in the anti-inflammatory response and its expression seems to be related to the development of some neoplasms, including the oral cancer. The objective of the research was to perform cellular morphometric analysis (nuclear and cytoplasmic compartments), identification of nuclear irregularities of malignant keratinocytes and compare with the immunoexpression of IL-4 and depth of invasion in cases of Epidermoid Carcinoma of the Lower Lip (CELI) and Oral Tongue. (CELLO). Thirty cases of each lesion were analyzed. For cell morphometry, 16 malignant keratinocytes were analyzed per case; IL-4 immunoexpression was analyzed in the parenchyma and stroma of the lesions, using image analysis software. The statistical tests of Analysis of Variance, Kruskal-Wallis and Spearman's Correlation were applied. The immunoexpression of IL-4 in the parenchyma was higher in cases of CELI in all analyzed fields (p<0,05); There was a positive correlation between total cell area and depth of invasion (p=0.038) and a negative correlation between IL-4 immunoexpression in the superficial parenchyma and superficial nuclear perimeter (p=0.007). Therefore, it is suggested that there is a protective relationship between IL-4 immunoexpression and the lesions analyzed, as well as morphometric alterations of malignant keratinocytes with IL-4 immunoexpression in these lesions (AU).

Morphology and morphometry of feline bone marrow-derived mesenchymal stem cells in culture / Morfologia e morfometria das células-tronco mesenquimais isoladas de medula óssea de gato

Mesenchymal stem cells (MSC) are increasingly being proposed as a therapeutic option for treatment of a variety of different diseases in human and veterinary medicine. Stem cells have been isolated from feline bone marrow, however, very few data exist about the morphology of these cells and no data were found about the morphometry of feline bone marrow-derived MSCs (BM-MSCs). The objectives of this study were the isolation, growth evaluation, differentiation potential and characterization of feline BM-MSCs by their morphological and morphometric characteristics. in vitro differentiation assays were conducted to confirm the multipotency of feline MSC, as assessed by their ability to differentiate into three cell lineages (osteoblasts, chondrocytes, and adipocytes). To evaluate morphological and morphometric characteristics the cells are maintained in culture. Cells were observed with light microscope, with association of dyes, and they were measured at 24, 48, 72 and 120h of culture (P1 and P3). The non-parametric ANOVA test for independent samples was performed and the means were compared by Tukey's test. On average, the number of mononuclear cells obtained was 12.29 (±6.05x106) cells/mL of bone marrow. Morphologically, BM-MSCs were long and fusiforms, and squamous with abundant cytoplasm. In the morphometric study of the cells, it was observed a significant increase in average length of cells during the first passage. The cell lengths were 106.97±38.16µm and 177.91±71.61µm, respectively, at first and third passages (24 h). The cell widths were 30.79±16.75 µm and 40.18±20.46µm, respectively, at first and third passages (24 h).The nucleus length of the feline BM-MSCs at P1 increased from 16.28µm (24h) to 21.29µm (120h). However, at P3, the nucleus length was 26.35µm (24h) and 25.22µm (120h). This information could be important for future application and use of feline BM-MSCs.(AU)

As células tronco mesenquimais são utilizadas na terapia de várias doenças na medicina humana e veterinária. As células tronco foram isoladas da medula óssea de gato, entretanto, existem poucos dados referentes a morfologia e não existem informações sobre a morfometria das células tronco isoladas da medula óssea. Os objetivos do presente estudo foram o isolamento, avaliação do crescimento, potencial de diferenciação e caracterização morfológica e morfométrica das células mesenquimais de gato isoladas de medula óssea. A diferenciação in vitro foi realizada para confirmar a multipotencialidade das células mesenquimais de gato (diferenciação em osteoblastos, condrócitos, adipócitos). As células mesenquimais foram mantidas em cultivo para avaliações morfológica e morfométrica. As células foram coradas e observadas em microscopia ótica. As mensurações foram realizadas com 24, 48, 72 e 120h de cultura (primeira e terceira passagens). O teste não paramétrico ANOVA foi utilizado e as médias foram comparadas pelo teste de Tukey. O número médio de células mononucleares obtido foi de 12,29 (±6,05x106) células/mL de medula óssea. As células mesenquimais são longas e fusiformes, e escamosas com citoplasma abundante. No estudo morfométrico, observou-se aumento no comprimento médio das células durante a primeira passagem. As medidas de comprimento das células foram: 106,97±38,16µm e 177,91±71,61µm, respectivamente, na primeira e terceira passagens (24 horas). As medidas de largura das células foram: 30,79±16,75 µm e 40,18±20,46 µm, respectivamente, na primeira e terceira passagens (24 horas). O comprimento do núcleo na primeira passagem aumentou de 16,28µm (24h) para 21,29µm (120h) e na terceira passagem foi de 26,35µm (24h) para 25,22µm (120h). As informações são importantes para futuras aplicações e uso da célula mesenquimal de gato.(AU)

Estudio de cultivos celulares primarios derivados de Lucilia sericata (Diptera: Calliphoridae) / Study of primary cell cultures derived from Lucilia sericata (Diptera: Calliphoridae)

El propósito principal de la investigación aquí presentada fue obtener cultivos celulares primarios derivados de Lucilia sericata (Diptera: Calliphoridae). Esta mosca necrófaga es utilizada para determinar el intervalo post-mortem y en terapia larval. A partir de huevos embrionados, se realizaron explantes en diversos medios de cultivo (Grace, Schneider, MM/VP12, DMEM, Grace/L-15 y L-15), suplementados con 20% de suero fetal bovino. La esterilización del material biológico se efectuó mediante la aplicación de soluciones de formaldehido e hipoclorito de sodio. El crecimiento celular se inicio en los medios L-15, MM/VP12, Grace/L-15 y Schneider, en un tiempo promedio de 10 días después de efectuadas las siembras de tejidos embrionarios, mediante la proliferación de grupos de colonias dispersas en la superficies de los frascos de cultivo y a partir de las terminaciones de los fragmentos larvales. La evolución del crecimiento celular hasta la formación de la monocapa semiconfluente fue relativamente rápida, se alcanzo a las tres semanas post-explantes. La morfología de las células en los cultivos fue heterogénea, se destacaron formas epitelioides, similares a nerviosas, gigantes e irregulares. La comparación de las características de crecimiento de los cultivos celulares de L. sericata con los obtenidos de otras especies de dípteros mostro mayor favorabilidad en la evolución, en razón a que las células se adaptaron mejor a las condiciones fisico-quimicas de varios medios de cultivo. Este es el primer informe de cultivos celulares de una mosca de la familia Calliphoridae.

The main purpose of this study was to obtain primary cell cultures derived from Lucilia sericata (Diptera: Calliphoridae). Necrophagous this fly is used for determination of post-mortem interval and larval therapy. Since explants embryonated eggs were performed in various culture media (Grace Schneider, MM/VP12, DMEM, Grace/L-15 and L-15), supplemented with 20% fetal serum. Sterilization of the biological material was carried out by immersing it in formaldehyde and sodium hypochlorite solutions. The cell growth was initiated in the L-15, MM/VP12, and Schneider Grace/L-15 in an average time of 10 days after completion of planting by the proliferation of groups of colonies scattered on the surface of the boxes crops and also from the endings of larval fragments. The evolution of cell growth to the formation of monolayer semi-confluent was relatively fast, reaching at 3 weeks post-explant. Cellular morphology in cultured cells was heterogeneous, especially epithelioid forms, similar to nerve, giant and irregular. Comparison of the growth characteristics of these cell cultures with those obtained from other species of flies was more favorable in the evolution of those obtained from L. sericata, on the grounds that the cells are better adapted to the physical-chemical conditions of several culture media. This is the first report of a cell culture-fly family Calliphoridae.