Explore the antiproliferative phytocompounds from ethanolic extracts of Citrus paradisi against liver cancer cell line by chemical analysis using TLC and FT-IR spectroscopy

Abstract The aim of the present study was to evaluate the in vitro antiproliferative activity of ethanolic extract of leaves and fruits Citrus paradisi plant on HepG-2 liver cell lines by MTT (3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-terazolium bromide) assay and to isolate and characterize the antiproliferative compounds by TLC (Thin layer chromatography) and FT-IR (Fourier transforms Infrared) spectroscopy. Qualitative phytochemical screening tests were performed to detect phytochemicals compounds from the crude extracts. Antioxidant activity of the plant extracts were characterized by using DPPH (2,2-Diphenyl-1-picrylhydrazyl) free radical scavenging method. The results showed that antioxidant activity using DPPH were found to be increased in a concentration dependent manner and decreased cell viability and cell growth inhibition in a dose dependent manner. The findings from this study indicated that fruit extract exhibited good antiproliferation and antioxidant potential. The seven functional groups of phytocompounds such as carboxylic acid, amine salt, aromatic compounds, cyclic alkene, aldehyde, fluoro compounds and alkene were detected by FT-IR which indicated that fruit extracts of Citrus paradisi possessed vast potential as a medicinal drug especially in liver cancer treatment.

Resumo O objetivo do presente estudo foi avaliar a atividade antiproliferativa in vitro do extrato etanólico de folhas e frutos da planta Citrus paradisi em linhagens de células hepáticas HepG-2 por MTT (3- (4, 5-dimetil-2-tiazolil) -2, Ensaio de brometo de 5-difenil-2H-terazólio) e isolar e caracterizar os compostos antiproliferativos por espectroscopia de TLC (cromatografia de camada fina) e FT-IR (infravermelho com transformadas de Fourier). Testes qualitativos de triagem fitoquímica foram realizados para detectar compostos fitoquímicos nos extratos brutos. A atividade antioxidante dos extratos vegetais foi caracterizada pelo método de eliminação de radicais livres DPPH (2,2-difenil-1-picrilhidrazil). Os resultados mostraram que a atividade antioxidante usando DPPH aumentou de uma maneira dependente da concentração e diminuiu a viabilidade celular e a inibição do crescimento celular de uma maneira dependente da dose. Os resultados deste estudo indicaram que o extrato de fruta exibiu bom potencial antiproliferação e antioxidante. Os sete grupos funcionais de fitocompostos, como ácido carboxílico, sal de amina, compostos aromáticos, alceno cíclico, aldeído, compostos de flúor e alceno, foram detectados por FT-IR, o que indicou que extratos de frutas de Citrus paradisi possuíam vasto potencial como medicamento, especialmente no tratamento de câncer do fígado.

Explore the antiproliferative phytocompounds from ethanolic extracts of Citrus paradisi against liver cancer cell line by chemical analysis using TLC and FT-IR spectroscopy / Explorar os fitocompostos antiproliferativos de extratos etanólicos de Citrus paradisi contra linhagem de células de câncer de fígado por análise química usando espectroscopia de TLC e FT-IR

The aim of the present study was to evaluate the in vitro antiproliferative activity of ethanolic extract of leaves and fruits Citrus paradisi plant on HepG-2 liver cell lines by MTT (3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Hterazolium bromide) assay and to isolate and characterize the antiproliferative compounds by TLC (Thin layer chromatography) and FT-IR (Fourier transforms Infrared) spectroscopy. Qualitative phytochemical screening tests were performed to detect phytochemicals compounds from the crude extracts. Antioxidant activity of the plant extracts were characterized by using DPPH (2,2-Diphenyl-1-picrylhydrazyl) free radical scavenging method. The results showed that antioxidant activity using DPPH were found to be increased in a concentration dependent manner and decreased cell viability and cell growth inhibition in a dose dependent manner. The findings from this study indicated that fruit extract exhibited good antiproliferation and antioxidant potential. The seven functional groups of phytocompounds such as carboxylic acid, amine salt, aromatic compounds, cyclic alkene, aldehyde, fluoro compounds and alkene were detected by FT-IR which indicated that fruit extracts of Citrus paradisi possessed vast potential as a medicinal drug especially in liver cancer treatment.

O objetivo do presente estudo foi avaliar a atividade antiproliferativa in vitro do extrato etanólico de folhas e frutos da planta Citrus paradisi em linhagens de células hepáticas HepG-2 por MTT (3- (4, 5-dimetil-2-tiazolil) -2, Ensaio de brometo de 5-difenil-2H-terazólio) e isolar e caracterizar os compostos antiproliferativos por espectroscopia de TLC (cromatografia de camada fina) e FT-IR (infravermelho com transformadas de Fourier). Testes qualitativos de triagem fitoquímica foram realizados para detectar compostos fitoquímicos nos extratos brutos. A atividade antioxidante dos extratos vegetais foi caracterizada pelo método de eliminação de radicais livres DPPH (2,2-difenil-1-picrilhidrazil). Os resultados mostraram que a atividade antioxidante usando DPPH aumentou de uma maneira dependente da concentração e diminuiu a viabilidade celular e a inibição do crescimento celular de uma maneira dependente da dose. Os resultados deste estudo indicaram que o extrato de fruta exibiu bom potencial antiproliferação e antioxidante. Os sete grupos funcionais de fitocompostos, como ácido carboxílico, sal de amina, compostos aromáticos, alceno cíclico, aldeído, compostos de flúor e alceno, foram detectados por FT-IR, o que indicou que extratos de frutas de Citrus paradisi possuíam vasto potencial como medicamento, especialmente no tratamento de câncer do fígado.

Alternatives for obtaining a continuous cell line from Apis mellifera / Alternativas para a obtenção de uma linhagem celular contínua de abelhas Apis mellifera

In worldwide there are reports of a significant decrease in colonies of the species Apis mellifera, caused by several factors, including viral infections. In order to study and diagnose illnesses caused by viruses, in vitro cell culture is used as a valuable tool. Yet, there are still no immortalized cell lines of honey bee Apis mellifera. Primary cell cultures are promising for this purpose and can supply the lack of continuous strains, but their establishment is difficult and laborious, which often makes them unfeasible for many research centers. Through the use of cell immortalization techniques, it is possible to develop continuous cell lines and thus benefit, in different ways, research related to different species of bees. The choice of technique is challenging, since in addition to the ability to remain viable for countless passages, cells must keep the genotype and phenotype similar or identical to the original tissue. This review intends to present methodologies that can be used to immortalize Apis mellifera cells, aiming to establish a cell line. The genotypic and phenotypic implications of each technique are evaluated, and the purpose of the cell line to be developed.

Ao redor do mundo há relatos da diminuição significativa de colônias da espécie Apis mellifera, causada por diversos fatores, incluindo infecções virais. Para estudo e diagnóstico de enfermidades causadas por vírus utiliza-se, como uma ferramenta valiosa, o cultivo celular in vitro. Contudo, ainda não existem linhagens celulares imortalizadas de abelhas Apis mellifera. Os cultivos celulares primários são promissores para este fim e podem suprir a falta de linhagens contínuas, porém seu estabelecimento é difícil e laborioso o que, muitas vezes, os torna inviáveis para muitos centros de pesquisa. Através do uso de técnicas de imortalização celular é possível desenvolver linhagens contínuas de células e assim beneficiar, de diversas formas, as pesquisas relacionadas às diferentes espécies de abelhas. A escolha da técnica é desafiadora, visto que, além da capacidade de permanecer viável por inúmeras passagens, as células devem manter o genótipo e fenótipo semelhante ou idêntico ao tecido original. O objetivo deste trabalho é apresentar metodologias que podem ser utilizadas para imortalização de células de Apis mellifera, visando o estabelecimento de uma linhagem celular. São avaliadas as implicações genotípicas e fenotípicas de cada técnica, e a finalidade da linhagem celular a ser desenvolvida.

Alternatives for obtaining a continuous cell line from Apis mellifera / Alternativas para a obtenção de uma linhagem celular contínua de abelhas Apis mellifera

ABSTRACT: In worldwide there are reports of a significant decrease in colonies of the species Apis mellifera, caused by several factors, including viral infections. In order to study and diagnose illnesses caused by viruses, in vitro cell culture is used as a valuable tool. Yet, there are still no immortalized cell lines of honey bee Apis mellifera. Primary cell cultures are promising for this purpose and can supply the lack of continuous strains, but their establishment is difficult and laborious, which often makes them unfeasible for many research centers. Through the use of cell immortalization techniques, it is possible to develop continuous cell lines and thus benefit, in different ways, research related to different species of bees. The choice of technique is challenging, since in addition to the ability to remain viable for countless passages, cells must keep the genotype and phenotype similar or identical to the original tissue. This review intends to present methodologies that can be used to immortalize Apis mellifera cells, aiming to establish a cell line. The genotypic and phenotypic implications of each technique are evaluated, and the purpose of the cell line to be developed.

RESUMO: Ao redor do mundo há relatos da diminuição significativa de colônias da espécie Apis mellifera, causada por diversos fatores, incluindo infecções virais. Para estudo e diagnóstico de enfermidades causadas por vírus utiliza-se, como uma ferramenta valiosa, o cultivo celular in vitro. Contudo, ainda não existem linhagens celulares imortalizadas de abelhas Apis mellifera. Os cultivos celulares primários são promissores para este fim e podem suprir a falta de linhagens contínuas, porém seu estabelecimento é difícil e laborioso o que, muitas vezes, os torna inviáveis para muitos centros de pesquisa. Através do uso de técnicas de imortalização celular é possível desenvolver linhagens contínuas de células e assim beneficiar, de diversas formas, as pesquisas relacionadas às diferentes espécies de abelhas. A escolha da técnica é desafiadora, visto que, além da capacidade de permanecer viável por inúmeras passagens, as células devem manter o genótipo e fenótipo semelhante ou idêntico ao tecido original. O objetivo deste trabalho é apresentar metodologias que podem ser utilizadas para imortalização de células de Apis mellifera, visando o estabelecimento de uma linhagem celular. São avaliadas as implicações genotípicas e fenotípicas de cada técnica, e a finalidade da linhagem celular a ser desenvolvida.

Alternatives for obtaining a continuous cell line from Apis mellifera / Alternativas para a obtenção de uma linhagem celular contínua de abelhas Apis mellifera

In worldwide there are reports of a significant decrease in colonies of the species Apis mellifera, caused by several factors, including viral infections. In order to study and diagnose illnesses caused by viruses, in vitro cell culture is used as a valuable tool. Yet, there are still no immortalized cell lines of honey bee Apis mellifera. Primary cell cultures are promising for this purpose and can supply the lack of continuous strains, but their establishment is difficult and laborious, which often makes them unfeasible for many research centers. Through the use of cell immortalization techniques, it is possible to develop continuous cell lines and thus benefit, in different ways, research related to different species of bees. The choice of technique is challenging, since in addition to the ability to remain viable for countless passages, cells must keep the genotype and phenotype similar or identical to the original tissue. This review intends to present methodologies that can be used to immortalize Apis mellifera cells, aiming to establish a cell line. The genotypic and phenotypic implications of each technique are evaluated, and the purpose of the cell line to be developed.(AU)

Ao redor do mundo há relatos da diminuição significativa de colônias da espécie Apis mellifera, causada por diversos fatores, incluindo infecções virais. Para estudo e diagnóstico de enfermidades causadas por vírus utiliza-se, como uma ferramenta valiosa, o cultivo celular in vitro. Contudo, ainda não existem linhagens celulares imortalizadas de abelhas Apis mellifera. Os cultivos celulares primários são promissores para este fim e podem suprir a falta de linhagens contínuas, porém seu estabelecimento é difícil e laborioso o que, muitas vezes, os torna inviáveis para muitos centros de pesquisa. Através do uso de técnicas de imortalização celular é possível desenvolver linhagens contínuas de células e assim beneficiar, de diversas formas, as pesquisas relacionadas às diferentes espécies de abelhas. A escolha da técnica é desafiadora, visto que, além da capacidade de permanecer viável por inúmeras passagens, as células devem manter o genótipo e fenótipo semelhante ou idêntico ao tecido original. O objetivo deste trabalho é apresentar metodologias que podem ser utilizadas para imortalização de células de Apis mellifera, visando o estabelecimento de uma linhagem celular. São avaliadas as implicações genotípicas e fenotípicas de cada técnica, e a finalidade da linhagem celular a ser desenvolvida.(AU)

Adhesion of pathogenic bacteria to polystyrene, skin and gut mucus of gilthead seabream, infectious capacity and antibiotics susceptibility / Adesão de bactérias patogênicas a poliestireno, pele e muco intestinal de gilthead seabream, capacidade infecciosa e susceptibilidade a antibióticos

Linking proprieties of adhesion, infectious capacities and antibiotic resistance of pathogen bacteria could help to treat fish diseases. Adhesions of ten fish pathogenic bacteria were tested in microtiter plates vacant, coated with skin or gut mucus, fixed with methanol, stained with 2% crystal-violet and revealed by colorimetric method. Infectious capacity was performed by exposing gilthead seabream fibroblast cell line (SAF-1) to 107 -108 CFUmL-1 of pathogen bacteria. Cell viability was measured 3h, 9h and 20 hours post-infection. The sensitivity to antibiotics was executed by disk diffusion. Data showed that all the bacteria tested adhere to polystyrene. For skin mucus, Vibrio harveyi, Vibrio alginolyticus, Halomonas venusta, and Aeromonas bivalvium were moderately adherent. Dietzia maris was strongly adherent. For gut mucus, 60% of tested bacteria were weakly adherent and 40% were non adherent. For infection, D. maris, V. harveyi and A. bivalvium decreased the cells viability to 89% after only 3h. After 20h, the viability percentage ranged between 1% and 32%. All isolates presented resistance to 1000 mg ml-1 of sulphonamide, 60% were resistant to sulfonamide and penicillin G. Present findings could be relevant in fish aquaculture and underscore the importance of the linkage between adhesion, infectious capacity, and antibiotic susceptibility of pathogen bacteria to avoid fish diseases.

Estudar o link entre propriedades de adesão, capacidades infecciosas e resistência a antibióticos de bactérias patogênicas pode ajudar a tratar doenças de peixes. A adesão de dez bactérias patogênicas foi testada em placas de microtitulação vazias, revestidas com muco da pele ou do intestino, fixadas com metanol, coradas com 2% de violeta cristal e reveladas pelo modo colorimétrico. A capacidade infecciosa foi realizada expondo a linha celular de fibroblasto de dourada (SAF-1) a 107-108 CFUmL-1 de bactérias patogénicas. A viabilidade celular foi medida 3h, 9h e 20 horas após a infecção. A sensibilidade aos antibióticos foi executada por difusão em disco. Os dados mostram que todas as bactérias testadas aderem ao poliestireno. Para o muco cutâneo, Vibrio harveyi, Vibrio alginolyticus, Halomonas venusta e Aeromonas bivalvium foram moderadamente aderentes. Dietzia maris foi fortemente aderente. Para o muco intestinal, 60% das bactérias testadas eram fracamente aderentes e 40% não aderentes. Para infecção, D. maris, V. harveyi e A. bivalvium diminuíram a viabilidade celular para 89% após apenas 3h. Após 20h, o percentual de viabilidade variou entre 1% e 32%. Todos os isolados apresentaram resistência a 1000 mg mL-1 de sulfonamida, 60% foram resistentes à sulfonamida e à penicilina G. Os achados atuais podem ser relevantes na aqüicultura e ressaltam a importância da ligação entre adesão, capacidade infecciosa e suscetibilidade a antibióticos de bactérias patogênicas para evitar doenças em peixes.

Adhesion of pathogenic bacteria to polystyrene, skin and gut mucus of gilthead seabream, infectious capacity and antibiotics susceptibility / Adesão de bactérias patogênicas a poliestireno, pele e muco intestinal de gilthead seabream, capacidade infecciosa e susceptibilidade a antibióticos

Linking proprieties of adhesion, infectious capacities and antibiotic resistance of pathogen bacteria could help to treat fish diseases. Adhesions of ten fish pathogenic bacteria were tested in microtiter plates vacant, coated with skin or gut mucus, fixed with methanol, stained with 2% crystal-violet and revealed by colorimetric method. Infectious capacity was performed by exposing gilthead seabream fibroblast cell line (SAF-1) to 107 -108 CFUmL-1 of pathogen bacteria. Cell viability was measured 3h, 9h and 20 hours post-infection. The sensitivity to antibiotics was executed by disk diffusion. Data showed that all the bacteria tested adhere to polystyrene. For skin mucus, Vibrio harveyi, Vibrio alginolyticus, Halomonas venusta, and Aeromonas bivalvium were moderately adherent. Dietzia maris was strongly adherent. For gut mucus, 60% of tested bacteria were weakly adherent and 40% were non adherent. For infection, D. maris, V. harveyi and A. bivalvium decreased the cells viability to 89% after only 3h. After 20h, the viability percentage ranged between 1% and 32%. All isolates presented resistance to 1000 mg ml-1 of sulphonamide, 60% were resistant to sulfonamide and penicillin G. Present findings could be relevant in fish aquaculture and underscore the importance of the linkage between adhesion, infectious capacity, and antibiotic susceptibility of pathogen bacteria to avoid fish diseases.(AU)

Estudar o link entre propriedades de adesão, capacidades infecciosas e resistência a antibióticos de bactérias patogênicas pode ajudar a tratar doenças de peixes. A adesão de dez bactérias patogênicas foi testada em placas de microtitulação vazias, revestidas com muco da pele ou do intestino, fixadas com metanol, coradas com 2% de violeta cristal e reveladas pelo modo colorimétrico. A capacidade infecciosa foi realizada expondo a linha celular de fibroblasto de dourada (SAF-1) a 107-108 CFUmL-1 de bactérias patogénicas. A viabilidade celular foi medida 3h, 9h e 20 horas após a infecção. A sensibilidade aos antibióticos foi executada por difusão em disco. Os dados mostram que todas as bactérias testadas aderem ao poliestireno. Para o muco cutâneo, Vibrio harveyi, Vibrio alginolyticus, Halomonas venusta e Aeromonas bivalvium foram moderadamente aderentes. Dietzia maris foi fortemente aderente. Para o muco intestinal, 60% das bactérias testadas eram fracamente aderentes e 40% não aderentes. Para infecção, D. maris, V. harveyi e A. bivalvium diminuíram a viabilidade celular para 89% após apenas 3h. Após 20h, o percentual de viabilidade variou entre 1% e 32%. Todos os isolados apresentaram resistência a 1000 mg mL-1 de sulfonamida, 60% foram resistentes à sulfonamida e à penicilina G. Os achados atuais podem ser relevantes na aqüicultura e ressaltam a importância da ligação entre adesão, capacidade infecciosa e suscetibilidade a antibióticos de bactérias patogênicas para evitar doenças em peixes.(AU)

Estabelecimento e caracterização de linhagens celulares embrionárias de Rhipicephalus (Boophilus) microplus (RBME-6) e Amblyomma sculptum (ASE-14) (Acari: Ixodida) do Brasil

Os carrapatos são artrópodes de grande importância médica e veterinária, sendo responsáveis pela transmissão de vírus, bactérias e protozoários aos seus hospedeiros. Alguns dos patógenos transmitidos por carrapatos são difíceis de crescer in vitro e não crescem em meios artificiais ou outros substratos. As linhagens de células de carrapatos representam uma ferramenta útil para estudar muitos aspectos da pesquisa de carrapatos e patógenos transmitidos por eles. Aqui, estabelecemos e caracterizamos duas linhagens celulares RBME-6 e ASE-14 derivadas de embriões de Rhipicephalus microplus e Amblyomma sculptum, respectivamente, ambos coletados no Brasil. Culturas primárias de células foram preparadas a partir de ovos embrionados das duas espécies de carrapatos, em meio L-15B, e mantidas à 30 °C. Quando as culturas atingiram uma densidade celular adequada, foram subcultivadas e criopreservadas em várias passagens. As análises citológicas foram feitas usando microscopia de contraste de fase, com células que foram submetidas à citocentrifugação e coradas com Giemsa, enquanto as técnicas de coloração com ácido periódico de Schiff e azul de bromofenol foram usadas para detectar polissacarídeos e proteinas, respectivamente. Não foi detectado o DNA de Anaplasma spp., Anaplasma marginale, Babesia/Theileria spp, Bartonella spp., Coxiella spp., Ehrlichia canis, Rickettsia spp. ou Mycoplasma spp. nas células através de ensaios de PCR. Além disso, realizamos a caracterização cromossômica das linhagens celulares de ambas espécies de carrapatos e confirmamos a origem dessas linhagens por meio de PCR convencional e sequenciamento de um fragmento do gene mitocondrial 16S rRNA. Em conclusão, duas novas linhagens celulares derivadas dos embriões de carrapatos foram geradas e caracterizadas neste estudo. Essas linhagens celulares poderão ser usadas em estudos futuros sobre diferentes aspectos, como as relações entre carrapatos e patógenos transmitidos por eles, expressão de proteínas em células infectadas e não infectadas e obtenção de antígenos. Além disso, é importante ter um painel maior de linhagens de células de carrapatos, pois elas podem servir como ferramentas eficientes para o avanço de pesquisas em várias áreas da virologia, bacteriologia, biologia e controle desses carrapatos.

Ticks are arthropods of great medical and veterinary importance, being responsible for the transmission of viruses, bacteria, and protozoa to their hosts. Some of the tick-borne pathogens are difficult to grow in vitro, and do not grow on artificial media or other substrates. Tick cell lines represent a useful tool for studying many aspects of tick and tick-borne pathogen research. Here, we established and characterized two cell lines RBME-6 and ASE-14 derived from embryos of Rhipicephalus microplus and Amblyomma sculptum, respectively, both from Brazil. Primary tick cell cultures were prepared in L-15B at 30°C.When they reached an adequate density, the cells were subcultured and cryopreserved in several passages. Cytological analysis were performed using phase contrast microscopy and cytocentrifuge smears stained with Giemsa, while periodic acid-Schiff and bromophenol blue staining techniques were used to detect total polysaccharides and the total protein, respectively. Anaplasma spp., Anaplasma marginale, Babesia/Theileria spp., Coxiella spp., Ehrichia canis, Mycoplasma spp. and Rickettsia sp. were not detected in the cells through PCR assays. In addition, we performed chromosomal characterization of the tick cell line and confirmed the origin of the cell line through conventional PCR and sequencing of the 16S rRNA gene mitochondrial fragment. In conclusion, two new cell lines derived from embryos of ticks were generated and characterized in this study. These cell lines can be used in future studies on different aspects, such as the relationship between ticks and pathogens transmitted by them, expression of proteins in infected and non-infected cells and obtaining antigens. In addition, it is important to have a larger panel of tick cell lines, as they can serve as efficient tools for advancing research in various areas of virology, bacteriology, biology and control of these ticks.

AVALIAÇÃO DAS CONDIÇÕES DE CULTIVO E CRIOPRESERVAÇÃO SOBRE O ESTABELECIMENTO DE LINHAGENS FIBROBLÁSTICAS DE ONÇAS-PINTADAS, Panthera onca (LINNAEUS, 1758)

O quantitativo populacional cada vez mais reduzido de onças-pintadas, associado à sua importância ecológica, tem resultado no desenvolvimento de estratégias que promovam a sua conservação. Nesse sentido, células somáticas derivadas da pele destes animais podem ser empregadas para essa finalidade, seja na produção de embriões clones, na obtenção de células induzidas à pluripotência e nos estudos genéticos da espécie. Para tanto, o estabelecimento adequado destas amostras é etapa inicial para essas aplicações. Portanto, o objetivo do presente estudo foi avaliar os danos gerados pelas condições de cultivo in vitro e criopreservação sobre o estabelecimento de cinco linhagens fibroblásticas derivadas de onças-pintadas adultas. Assim, fragmentos da pele da região auricular apical de uma fêmea e quatro machos foram cultivados in vitro para a obtenção das células somáticas. Inicialmente, para a identificação do tipo celular, células foram confirmadas como fibroblastos após análise morfológica e de imunofluorescência, e as cinco linhagens (um animal/uma linhagem) foram submetidas a dois experimentos. No primeiro experimento, células foram avaliadas em diferentes passagens do cultivo (primeira, terceira décima) para viabilidade, metabolismo e atividade proliferativa. No segundo experimento, células criopreservadas foram avaliadas quanto a viabilidade, metabolismo, atividade proliferativa, níveis de espécies reativas de oxigênio (EROs), potencial de membrana mitocondrial (m) e apoptose após descongelação em uma, três e dez passagens de cultivo. Células não criopreservadas foram utilizadas como controle. O cultivo in vitro após a primeira (26,1 h ± 4,9), terceira (22,9 h ± 1,6) e décima passagem (22,8 h ± 3,7) e criopreservação (30,0 h ± 1,4) não afetaram a atividade proliferativa. Além disso, nenhuma diferença foi observada para a viabilidade após a primeira (98,9% ± 0,8), terceira (92,5% ± 6,2), décima (95,7% ± 1,4) passagem e criopreservação (73,2% ± 9,8). Contudo, células cultivadas até a décima passagem (49,0% ± 3,3) e criopreservadas (32,7% ± 2,8) reduziram seu metabolismo. Adicionalmente, células criopreservadas mostraram em unidades de fluorescência arbitrárias altos níveis de EROs (1,4 ± 0,1) e m alterado (0,9 ± 0,0), quando comparados às células não criopreservadas (1,0 ± 0,1 e 1,0 ± 0,2). Finalmente, células criopreservadas e cultivadas após dez passagens reduziram a atividade proliferativa reduzida (45,1% ± 12,0) e número de células viáveis (30,4% ± 5,7), quando comparadas as células criopreservadas e cultivadas após uma e três passagens. Em conclusão, fibroblastos viáveis podem ser estabelecidos a partir da orelha de onças-pintadas e que embora essas células não tenham mostrado alterações na viabilidade e atividade proliferativa, elas sofrem danos durante um longo cultivo e criopreservação nas condições estudadas.

The increasingly small population quantity of jaguars, associated with their ecological importance, has resulted in the development of strategies that promote their conservation. In this sense, somatic cells derived from the skin of these animals can be used for this purpose, either in the production of clone embryos, in obtaining cells induced to pluripotency and in genetic studies of the species. Thus, the proper establishment of these samples is an initial step for these applications. Therefore, the aim of the present study was to assess the damage caused by in vitro culture and cryopreservation conditions on the establishment of five fibroblast lines derived from adult jaguars. Thus, fragments of skin from the apical auricular region of one female and four males were cultured in vitro to obtain somatic cells. Initially, to identify the cell type, cells were confirmed as fibroblasts after morphological and immunofluorescence analysis, and the five strains (one animal/one line) were subjected to two experiments. In the first experiment, cells were evaluated in different culture passages (first, third, tenth) for viability, metabolism and proliferative activity. In the second experiment, cryopreserved cells were evaluated for viability, metabolism, proliferative activity, levels of reactive oxygen species (ROSs), mitochondrial membrane potential (m) and apoptosis after thawing and one, three and ten culture passages. Non-cryopreserved cells were used as controls. The in vitro culture after the first (26.1 h ± 4.9), third (22.9 h ± 1.6) and tenth passage (22.8 h ± 3.7) and cryopreservation (30.0 h ± 1.4) did not affect proliferative activity. Moreover, no difference was observed for viability after the first (98.9% ± 0.8), third (92.5% ± 6.2), tenth (95.7% ± 1.4) passage and cryopreservation (73.2% ± 9.8). Nevertheless, cells cultured to the tenth passage (49.0% ± 3.3) and cryopreserved (32.7% ± 2.8) reduced their metabolism. Additionally, cryopreserved cells showed high levels of ROS (1.4 ± 0.1) and altered m (0.9 ± 0.0) in arbitrary fluorescence units, when compared to non-cryopreserved cells (1.0 ± 0.1 and 1.0 ± 0.2). Finally, cryopreserved cells and cultured after ten passages reduced the reduced proliferative activity (45.1% ± 12.0) and number of viable cells (30.4% ± 5.7), when compared to cryopreserved and cultured cells after one and three passages. In conclusion, viable fibroblasts can be established from jaguars ears and that although these cells have not shown changes in viability and proliferative activity, they suffer damage during a long culture and cryopreservation under the studied conditions.

Development of experimental in vitro burn model

To propose an experimental burn model in NIH-3T3 cell line. Induction of thermal injury in cultures of mouse fibroblast - NIH-3T3- cell line and determination of cell viability by MTT and imunofluorescence. The heating of the Petri dish increased proportionally to the temperature of the base and the time of exposure to microwave. In this in vitro burn model, using the cell line NIH-3T3 was observed drastic cellular injury with significant changes in cell viability and activity. It showed drastically modified cell morphology with altered membrane, cytoskeleton and nucleus, and low cellularity compared to the control group. The burn model in vitro using the cell line NIH-3T3 was reproductive and efficient. This burn model was possible to determine significant changes in cell activity and decreased viability, with drastic change in morphology, cell lysis and death.(AU)

Potentiation of a novel palladium (II) complex lethality with bee venom on the human T-cell acute lymphoblastic leukemia cell line (MOLT-4)

Background : Although honeybee venom (BV) has been reported to induce apoptosis in different types of cancerous cells, its synergistic effects with customary anti-cancer drugs remain largely unknown. In the present study, we evaluated the cytotoxic effect of BV alone (as a natural product) and the synergistic cytological effects of this component in combination with [Pd (bpy) (Pi-Pydtc)]NO3 - a novel palladium complex on human T-cell lymphoblastic leukemia cells. To investigate the cytotoxic effect of the BV alone and in combination with palladium complex on MOLT-4 cells MTT assay was performed. In order to determine the apoptotic effects of BV separately and in combination with Pd (II) complex on these cells and its ability to induce apoptosis, morphological examination, flowcytometric analysis and caspase-3 colorimetric assay were done. Results : We found that BV induced morphological changes, namely nuclear shrinkage, and inhibited MOLT-4 cell proliferation; both effects were dose- and time-dependent. Flow cytometry by Annexin-V antibody demonstrated that BV induced apoptosis in MOLT-4 cells. Furthermore, BV induced apoptosis independently of caspase-3 in these cells. In addition, we proved a clear synergistic effect of BV on [Pd (bpy) (Pi-Pydtc)]NO3. The apoptotic pathway activated by BV in combination with Pd complex was caspase-3-dependent. Conclusions : These observations provide an explanation for the anti-proliferative properties of BV, and suggest that this agent may be useful for treating lymphoblastic leukemia alone or in combination with chemotherapy drugs pending further investigations on animal models as preclinical tests.(AU)

Potentiation of a novel palladium (II) complex lethality with bee venom on the human T-cell acute lymphoblastic leukemia cell line (MOLT-4)

Background : Although honeybee venom (BV) has been reported to induce apoptosis in different types of cancerous cells, its synergistic effects with customary anti-cancer drugs remain largely unknown. In the present study, we evaluated the cytotoxic effect of BV alone (as a natural product) and the synergistic cytological effects of this component in combination with [Pd (bpy) (Pi-Pydtc)]NO3 - a novel palladium complex on human T-cell lymphoblastic leukemia cells. To investigate the cytotoxic effect of the BV alone and in combination with palladium complex on MOLT-4 cells MTT assay was performed. In order to determine the apoptotic effects of BV separately and in combination with Pd (II) complex on these cells and its ability to induce apoptosis, morphological examination, flowcytometric analysis and caspase-3 colorimetric assay were done. Results : We found that BV induced morphological changes, namely nuclear shrinkage, and inhibited MOLT-4 cell proliferation; both effects were dose- and time-dependent. Flow cytometry by Annexin-V antibody demonstrated that BV induced apoptosis in MOLT-4 cells. Furthermore, BV induced apoptosis independently of caspase-3 in these cells. In addition, we proved a clear synergistic effect of BV on [Pd (bpy) (Pi-Pydtc)]NO3. The apoptotic pathway activated by BV in combination with Pd complex was caspase-3-dependent. Conclusions : These observations provide an explanation for the anti-proliferative properties of BV, and suggest that this agent may be useful for treating lymphoblastic leukemia alone or in combination with chemotherapy drugs pending further investigations on animal models as preclinical tests.

Potentiation of a novel palladium (II) complex lethality with bee venom on the human T-cell acute lymphoblastic leukemia cell line (MOLT-4)

Background : Although honeybee venom (BV) has been reported to induce apoptosis in different types of cancerous cells, its synergistic effects with customary anti-cancer drugs remain largely unknown. In the present study, we evaluated the cytotoxic effect of BV alone (as a natural product) and the synergistic cytological effects of this component in combination with [Pd (bpy) (Pi-Pydtc)]NO3 - a novel palladium complex on human T-cell lymphoblastic leukemia cells. To investigate the cytotoxic effect of the BV alone and in combination with palladium complex on MOLT-4 cells MTT assay was performed. In order to determine the apoptotic effects of BV separately and in combination with Pd (II) complex on these cells and its ability to induce apoptosis, morphological examination, flowcytometric analysis and caspase-3 colorimetric assay were done. Results : We found that BV induced morphological changes, namely nuclear shrinkage, and inhibited MOLT-4 cell proliferation; both effects were dose- and time-dependent. Flow cytometry by Annexin-V antibody demonstrated that BV induced apoptosis in MOLT-4 cells. Furthermore, BV induced apoptosis independently of caspase-3 in these cells. In addition, we proved a clear synergistic effect of BV on [Pd (bpy) (Pi-Pydtc)]NO3. The apoptotic pathway activated by BV in combination with Pd complex was caspase-3-dependent. Conclusions : These observations provide an explanation for the anti-proliferative properties of BV, and suggest that this agent may be useful for treating lymphoblastic leukemia alone or in combination with chemotherapy drugs pending further investigations on animal models as preclinical tests.

Differential susceptibilities of human lung, breast and skin cancer cell lines to killing by five sea anemone venoms

Although sea anemones are well known for being rich sources of toxins, including cytolysins and neurotoxins, their venoms and toxins have been poorly studied. In the present study, the venoms from five sea anemones (Heteractis crispa, Heteractis magnifica, Heteractis malu, Cryptodendrum adhaesivum and Entacmaea quadricolor) were obtained by the milking technique, and the potential of these venoms to kill cancer cells was tested on three cell lines (A549 lung cancer, T47D breast cancer and A431 skin cancer). The total protein level in the crude extract was determined by the bicinchoninic acid (BCA) protein assay. The cytotoxicity on different cell lines was assayed using the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay which measures survival based on the detection of mitochondrial activity and by the crystal violet assay, which measures survival based on the ability of cells to remain adherent to microplates. The results indicate that the sea anemone venom is cytotoxic to human cancer cells. The A549 cell line was the most sensitive of the cell lines tested with a significant reduction in viability observed at 40 µg/mL. H. malu, C. adhaesivum and E. quadricolor had a significant inhibitory effect on A431 cells. Furthermore, H. malu and C. adhaesivum had a significant inhibitory effect on T47D cell line at 40 µg/mL. In conclusion, the sea anemone venoms tested have the potential to be developed as anticancer agents.(AU)

Differential susceptibilities of human lung, breast and skin cancer cell lines to killing by five sea anemone venoms

Although sea anemones are well known for being rich sources of toxins, including cytolysins and neurotoxins, their venoms and toxins have been poorly studied. In the present study, the venoms from five sea anemones (Heteractis crispa, Heteractis magnifica, Heteractis malu, Cryptodendrum adhaesivum and Entacmaea quadricolor) were obtained by the milking technique, and the potential of these venoms to kill cancer cells was tested on three cell lines (A549 lung cancer, T47D breast cancer and A431 skin cancer). The total protein level in the crude extract was determined by the bicinchoninic acid (BCA) protein assay. The cytotoxicity on different cell lines was assayed using the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay which measures survival based on the detection of mitochondrial activity and by the crystal violet assay, which measures survival based on the ability of cells to remain adherent to microplates. The results indicate that the sea anemone venom is cytotoxic to human cancer cells. The A549 cell line was the most sensitive of the cell lines tested with a significant reduction in viability observed at 40 µg/mL. H. malu, C. adhaesivum and E. quadricolor had a significant inhibitory effect on A431 cells. Furthermore, H. malu and C. adhaesivum had a significant inhibitory effect on T47D cell line at 40 µg/mL. In conclusion, the sea anemone venoms tested have the potential to be developed as anticancer agents.(AU)

PROPRIEDADES CITOTÓXICAS DA LAPACHONA EM CÉLULAS DE OSTEOSSARCOMA IN VITRO

O osteossarcoma é o principal tumor ósseo primário, com prognóstico desfavorável, alta mortalidade e elevada incidência de metástases. O tratamento de escolha é remoção do tumor associada à quimioterapia combinada, cujos efeitos adversos aludem à necessidade crescente de desenvolver novos medicamentos. As plantas constituem grandes reservas naturais de compostos que possuem propriedades medicinais e terapêuticas, como o lapachol e seu derivado, a lapachona. O objetivo desse estudo foi avaliar os efeitos citotóxicos da lapachona em células de osteossarcoma cultivadas in vitro. As células foram cultivadas e tratadas com a lapachona em diferentes concentrações e tempos de exposição. Foram realizados os métodos de exclusão com azul de Tripan, redução do tetrazólio e ensaio de sobrevivência celular para avaliar os efeitos do composto sobre as células. As células tratadas com 0,1M de lapachona apresentaram menor citotoxicidade inicial, no tempo de 24h, enquanto aquelas submetidas a 1,0M apresentaram menor viabilidade após 72h de tratamento. A citotoxicidade aumentou de acordo com o aumento da concentração e tempo de exposição. O menor IC50 (0,148M) foi observado nas células tratadas por 72h. O crescimento celular após o tratamento foi menor grupo sob concentração de 1,0µl após 72h e o maior crescimento celular foi observado sob concentração de 0,1µl após 24h. Não houve diferença entre grupos quanto à proliferação celular após o tratamento tendo a fração de sobrevivência das células sido menor após 72h de exposição. Concluiu-se que lapachona apresenta efeitos citotóxicos em células de osteossarcoma cultivadas in vitro.

Osteosarcoma is the main primary bone tumor, with unfavorable prognosis, high mortality and high incidence of metastases. The treatment of choice is the removal of the tumor associated with combined chemotherapy, whose adverse effects allude to the increasing need to develop new drugs. The plants constitute a large natural reserve of compounds that have medicinal and therapeutic properties, such as lapachol and its derivative, -lapachone. The aim of this study was to evaluate the cytotoxic effects of -lapachone in osteosarcoma cells cultured in vitro. Cells were cultured and treated with -lapachone at different concentrations and times of exposure. Tripan blue exclusion, tetrazolium reduction and cell survival assay methods were performed to evaluate the effects of the compound on the cells. Cells treated with 0,1M -lapachone showed lower initial cytotoxicity in the 24h time, whereas those submitted to 1,0M showed less viability after 72h of treatment. Cytotoxicity increased as the concentration and time of exposure increased. The lowest IC50 (0,148M) was observed in treated cells for 72h. Cell growth after treatment was lower in the 1.0l group after 72h and the highest cell growth was observed under a concentration of 0.1l after 24h. There was no difference between groups for cell proliferation after treatment, and the cell survival fraction was lower after 72h of exposure. It was concluded that -lapachone has cytotoxic effects on osteosarcoma cells cultured in vitro.

Mouse embryonic stem cells: The establishment of the system to produce differentiated cell types in vitro

During the last few years, embryonic stem (ES) cells have been a new tool in cell biology which is very promising for the scientific community to develop new cell therapies. ES cells are the only cell type that can differentiate into derivates of the three primary germ layers, not only in vivo but also, and most important, in vitro. This so-called pluripotency has resulted in the field of stem cell technology going into overdrive, and the establishment of many protocols for optimal maintenance, culture, genetic transfection and in vitro differentiation. The first pluripotent cells had been derived from teratocarcinomas, maligne tumors, and showed some disadvantages. Therefore later embryonic stem cells, and now adult stem cells are getting special attention from the scientists. In this study, we established for the first time in our country, the prolonged culture of undifferentiated ES cells in vitro and the pointed induction of cell differentiation into specific cell types. It is the result of an international collaboration program supported by Brazil and Germany, CAPES and DAAD (PROBRAL). The well-established routine should be clearly demonstrated by the continuous culture and propagation of several mouse ES lines in vitro under specific culture conditions preventing differentiation. On the other hand, these ES cells were exposed to defined differentiation induction systems t

Mouse embryonic stem cells: The establishment of the system to produce differentiated cell types in vitro

During the last few years, embryonic stem (ES) cells have been a new tool in cell biology which is very promising for the scientific community to develop new cell therapies. ES cells are the only cell type that can differentiate into derivates of the three primary germ layers, not only in vivo but also, and most important, in vitro. This so-called pluripotency has resulted in the field of stem cell technology going into overdrive, and the establishment of many protocols for optimal maintenance, culture, genetic transfection and in vitro differentiation. The first pluripotent cells had been derived from teratocarcinomas, maligne tumors, and showed some disadvantages. Therefore later embryonic stem cells, and now adult stem cells are getting special attention from the scientists. In this study, we established for the first time in our country, the prolonged culture of undifferentiated ES cells in vitro and the pointed induction of cell differentiation into specific cell types. It is the result of an international collaboration program supported by Brazil and Germany, CAPES and DAAD (PROBRAL). The well-established routine should be clearly demonstrated by the continuous culture and propagation of several mouse ES lines in vitro under specific culture conditions preventing differentiation. On the other hand, these ES cells were exposed to defined differentiation induction systems t

Mouse embryonic stem cells: The establishment of the system to produce differentiated cell types in vitro

During the last few years, embryonic stem (ES) cells have been a new tool in cell biology which is very promising for the scientific community to develop new cell therapies. ES cells are the only cell type that can differentiate into derivates of the three primary germ layers, not only in vivo but also, and most important, in vitro. This so-called pluripotency has resulted in the field of stem cell technology going into overdrive, and the establishment of many protocols for optimal maintenance, culture, genetic transfection and in vitro differentiation. The first pluripotent cells had been derived from teratocarcinomas, maligne tumors, and showed some disadvantages. Therefore later embryonic stem cells, and now adult stem cells are getting special attention from the scientists. In this study, we established for the first time in our country, the prolonged culture of undifferentiated ES cells in vitro and the pointed induction of cell differentiation into specific cell types. It is the result of an international collaboration program supported by Brazil and Germany, CAPES and DAAD (PROBRAL). The well-established routine should be clearly demonstrated by the continuous culture and propagation of several mouse ES lines in vitro under specific culture conditions preventing differentiation. On the other hand, these ES cells were exposed to defined differentiation induction systems t

Molecular characterization and pathology of field isolates of foot-and-mouth virus in Swiss albino mice / Caracterização molecular e patologia de isolados de campo do vírus da febre aftosa em camundongos albinos suíços

Foot-and-mouth disease is responsible for severe economic losses to the livestock industry of Pakistan. This study aimed to use Swiss albino mice as a cost-effective experimental animal model to study different immunological and histopathological aspects of FMDV instead of natural targeted species like cattle. After isolation of field isolates FMDV on BHK-21 cell line, biological titer of the virus and mice infectious dose50 was calculated. Virus was injected in 45 Swiss albino mice (group A) through intraperitoneal route. The gross, histopathological and immunopathological lesions in heart, trachea and lungs were recorded at different day's intervals. Histopathologically, the heart showed congestion, hemorrhages and necrosis of cardiac muscles. Trachea showed deciliated epithelium and lungs showed hemorrhages, bronchial edema and alveolar emphysema. Immunohistochemical studies revealed the presence of virus in cardiac muscles, tracheal and bronchial epithelium and alveolar lumen. The findings evoked a thought that laboratory animals could be an alternative to large animals to meet budget limitations for further research on foot-and-mouth-disease.

A febre aftosa (FMD) é responsável por graves perdas econômicas para a indústria pecuária do Paquistão. Este estudo teve como objetivo usar camundongos albinos suíços como um modelo animal experimental de baixo custo para estudar diferentes aspectos imunológicos e histopatológicos do FMDV em vez de espécies naturais como o gado. Após o isolamento dos isolados de campo do FMDV na linhagem celular BHK-21, calculou-se o título biológico do vírus e a dose infecciosa dos camundongos50. O vírus foi injetado em 45 camundongos albinos suíços (grupo A) por via intraperitoneal. As lesões macroscópicas, histopatológicas e imunopatológicas no coração, traqueia e pulmões foram registradas em diferentes intervalos de dias. Histopatologicamente, o coração apresentava congestão, hemorragias e necrose dos músculos cardíacos. A traqueia apresentava epitélio deciliado e os pulmões apresentavam hemorragias, edema brônquico e enfisema alveolar. Estudos imuno-histoquímicos revelaram a presença de vírus em músculos cardíacos, epitélio traqueal e orçamentárias para pesquisas adicionais sobre a febre aftosa