Atypical spleen hemophagocytic histiocytic sarcoma in a dog

Background: The histiocytic sarcoma (HS) complex is a set of malignant neoplasms originating from interstitial dendritic cells or macrophages. When it involves macrophages of the splenic red pulp and bone marrow, it is referred to as hemophagocytic histiocytic sarcoma (HHS). HHS behaves more aggressively than HS and is usually fatal. HHS can be diagnosed by cytological and histopathological examination of neoplastic tissue. HHS is confirmed by immunohistochemistry using an anti-CD11d antibody. This neoplasm is often confused with immune-mediated hemolytic anemia or Evans syndrome due to erythrophagocytosis and platelet consumption. The clinical presentation of the animals progresses with evident anemia and thrombocytopenia, leading to signs such as prostration, inappetence, and pale mucosa, making diagnosis challenging and often late. This study aimed to report the clinic-pathological aspects of a canine with atypical hemophagocytic splenic HS. Case: A 4-year-old male Shih-Tzu canine was referred to the Veterinary Hospital with a history of prostration and anorexia. Pale mucous membranes were observed on physical examination. Blood tests revealed non-regenerative anemia, leukopenia, and thrombocytopenia. Serum protein levels were below the reference values for the species in biochemical examinations. Hemoparasitosis was suspected; however, the result of the polymerase chain reaction was negative. Abdominal ultrasound revealed a splenomegaly with heterogeneous parenchyma and a slightly irregular surface, but no visible mass in the spleen. Due to the difficulty of stabilizing the patient, even after successive transfusions, the animal underwent exploratory laparotomy with medial access and posterior splenectomy. Subsequently, the spleen was surgically removed, fixed in 10% buffered formalin, and processed routinely. Macroscopically, it had an irregular reddish-brown capsular surface. Histopathological examination of the spleen revealed a densely cellular neoplasm composed of round to spindle cells (histiocytes) arranged haphazardly in variably sized sheets separating the pre-existing spleen stroma. These histopathological findings were consistent with a histiocytic malignant neoplasm. Immunohistochemical analysis was performed to better define the origin of the histiocytic neoplasm. Neoplastic cells showed positive immunostaining of more than 80% of tumor cells for the CD11d antibody and weak immunostaining for CD11c and lysozyme. The patient survived for less than 30 days after the first hospital visit. Discussion: The diagnosis of HHS was based on the histological characteristics and positive immunostaining of more than 80% of the tumor cells for the CD11d antibody. HHS is an extremely aggressive and rare tumor that affects elderly dogs of any breed. In this study, HHS had atypical histologic characteristics, in which erythrophagocytosis and hemosiderin were not observed within macrophages. HHSs arise from macrophages of the red pulp of the spleen or bone marrow and express the b2 integrin, CD11d, and have low expression of CD1 and CD11c, which are predominantly expressed by non-hemophagocytic HS. The hematological and biochemical changes observed in this case were similar to those described in other dogs with HHS. Treatment of HHS is only palliative. Erlichia ewingii, E. canis, Anaplasma phagocytophilum, A. platys, Borrelia burgdorferi, Dirofilaria immitis, Leishmania infantum and immune-mediated hemolytic anemia are the main differential diagnoses because they cause anemia and thrombocytopenia accompanied by splenomegaly.

Markers and antibodies for characterization of goat mammary tissue and the derived primary epithelial cell cultures

A selection of commercially available antibodies, targeted against markers employed in studies of mammary gland biology, was tested to determine their reactivity in goat mammary tissue and the derived tissue cultures. Expression of the markers smooth muscle actin (SMA), selected keratins (KRT) 5, 14, 18, and 19, CD24 molecule (CD24), epithelial cell adhesion molecule (EPCAM), mucin 1 (MUC1), integrin subunit alpha 6 (ITGA6; CD49F), integrin subunit beta 1 (ITGB1; CD29), cyclin dependent kinase inhibitor 1A (CDKN1A; p21), membrane metalloendopeptidase (MME; CD10), progesterone receptor (PGR), estrogen receptor 1 (ESR1), and vimentin (VIM) was first assessed on mRNA level, using reverse transcription PCR (RT-PCR). The reactivity of the antibodies in the tissue sections and the derived tissue cultures was determined using immunofluorescence. The result of this study is a list of commercially available antibodies, raised mostly against human antigens, which also recognize orthologous goat antigens and are useful for characterization of different mammary cell types. Additionally, primers that are functional in detecting expression of mammary lineage markers in goat mammary mRNA isolates were validated. The suggested antibodies, PCR primers, and the described methods are of practical value for researchers interested in characterization and isolation of cell types comprising mammary tissue of goats and probably other ruminants.(AU)

Association of polypropylene scaffolds and mesenchymal stem cells for use in tissue engineering / Associação de arcabouço de polipropileno e células tronco mesenquimais para uso em engenharia de tecido

Tissue engineering replaces injured tissues by manipulating cells, making scaffolds, and using molecules that stimulate the tissue. Mesenchymal stem cells (MSCs) are good candidates for tissue engineering, as this is one of the cell types which are recruited to repair injured tissues. Scaffolds are structural devices that allow cell fixation and migration, with polypropylene meshes being an example. This study aims to evaluate the culture of adipose tissue-derived mesenchymal stem cells (ADSCs), isolated from C57Bl/6 GFP + mice, in two types of polypropylene meshes (macroporous and microporous) in conventional culture plates and plates coated with methacrylate, over a period of fifteen days. The objective was to obtain the best interaction protocol between the mesh and the cells. The choice of the best method was based on adherence, maintenance of adherence and viability during culture. The amount of ADSCs adhering was checked daily by counting in a Neubauer Chamber and by using a growth curve performed with the MTT assay. The ADSCs adhering to the meshes were visualized with DAPI, panotic, hematoxylin and eosin, immunohistochemistry (integrin), and immunofluorescence (actin). ADSCs adhere to all forms of culture and to the two types of polypropylene mesh. ADSCs adhered more to the microporous mesh, within the seven day period of culture and in the plates without methacrylate. Thus, polypropylene meshes offer a good scaffold for ADSCs to adhere to.

A engenharia de tecidos substitui tecidos danificados com a manipulação de células, confecção de arcabouços e a utilização de moléculas que estimulem o tecido. As células-tronco mesenquimais (MSCs) são boas candidatas para engenharia de tecido, pois são um dos tipos celulares recrutadas para a reparação de tecidos lesionados. O arcabouço deve ser um dispositivo estrutural que forneça uma estrutura para o crescimento e a diferenciação celular no sítio, sendo a tela de polipropileno um exemplo. O objetivo deste estudo foi avaliar o cultivo de células-tronco mesenquimais de tecido de adiposo (ADSCs), isoladas de camundongos C57Bl/6 GFP+, em dois tipos de telas de polipropileno (macroporosa e microporosa) em placas de cultura convencionais e revestidas com metacrilato, durante quinze dias, para obter o melhor protocolo de interação entre a tela e as células. A escolha do melhor método foi baseada na adesão, manutenção da adesão e viabilidade durante cultivo. A quantidade de ADSCs aderidas foi verificada diariamente em contagem em Câmara de Neubauer e através de uma curva de crescimento realizada através de ensaio de MTT. As ADSCs aderidas nas telas foram visualizadas com a marcação de DAPI, panótico, hematoxilina e eosina, imumo-histoquímica (integrina) e imunofluorescência (actina). Nas duas formas de cultivo e nos dois tipos de telas de polipropileno houve aderência das ADSCs. Houve maior aderência na tela microporosa, no período de sete dias de cultivo e em placas sem metacrilato. Conclui-se que a tela de polipropileno oferece um bom arcabouço para as ADSCs se aderirem.

Associação de arcabouço de polipropileno e células tronco mesenqimais para uso em engenharia de tecido / Association of polypropylene scaffolds and mesenchymal stem cells for use in tissue engineering

A engenharia de tecidos tem como objetivo substituir tecidos danificados, manipulando células, confecção de arcabouços e a utilização de moléculas que estimulem o tecido. A proposta deste estudo foi avaliar duas técnicas de cultivo de células-tronco mesenquimais (MSC) em diferentes placas de cultura, utilizando dois tipos de telas de polipropileno (macroporoso e microporoso), para obter as melhores condições de interação entre a tela e as células, e definir uma proposta de protético para engenharia de tecidos. As telas de polipropileno foram cultivadas com células-tronco mesenquimais de tecido adiposo (ADSCs) isoladas de camundongos C57B1/6 GFP+ durante quinze dias em placas revestidas com metacrilato ou não revestidas com metacrilato. A quantidade de ADSCs aderidas foram verificadas diariamente em Câmara de Neubauer e através de uma curva de crescimento realizada pelo ensaio MTT. As ADSCs aderidas às malhas foram visualizadas com marcação de DAPI, panóticas, hematoxilina e eosina imuno-histoquímica e imunofluorescência. O melhor protocolo foi na tela microporosa, no o período de sete dias de cultivo e em placas sem metacrilato. Conclui-se que a tela de polipropileno fornece um bom suporte para as ADSCs se aderirem podendo ser utilizada na engenharia de tecidos.(AU)

Tissue engineering replaces injured tissues by manipulating cells, making scaffolds, and using molecules that stimulate the tissue. Mesenchymal stem cells (MSCs) are good candidates for tissue engineering, as this is one of the cell types which are recruited to repair injured tissues. Scaffolds are structural devices that allow cell fixation and migration, with polypropylene meshes being an example. This study aims to evaluate the culture of adipose tissue-derived mesenchymal stem cells (ADSCs), isolated from C57Bl/6 GFP + mice, in two types of polypropylene meshes (macroporous and microporous) in conventional culture plates and plates coated with methacrylate, over a period of fifteen days. The objective was to obtain the best interaction protocol between the mesh and the cells. The choice of the best method was based on adherence, maintenance of adherence and viability during culture. The amount of ADSCs adhering was checked daily by counting in a Neubauer Chamber and by using a growth curve performed with the MTT assay. The ADSCs adhering to the meshes were visualized with DAPI, panotic, hematoxylin and eosin, immunohistochemistry (integrin), and immunofluorescence (actin). ADSCs adhere to all forms of culture and to the two types of polypropylene mesh. ADSCs adhered more to the microporous mesh, within the seven day period of culture and in the plates without methacrylate. Thus, polypropylene meshes offer a good scaffold for ADSCs to adhere to.(AU)

Associação de arcabouço de polipropileno e células tronco mesenqimais para uso em engenharia de tecido / Association of polypropylene scaffolds and mesenchymal stem cells for use in tissue engineering

A engenharia de tecidos tem como objetivo substituir tecidos danificados, manipulando células, confecção de arcabouços e a utilização de moléculas que estimulem o tecido. A proposta deste estudo foi avaliar duas técnicas de cultivo de células-tronco mesenquimais (MSC) em diferentes placas de cultura, utilizando dois tipos de telas de polipropileno (macroporoso e microporoso), para obter as melhores condições de interação entre a tela e as células, e definir uma proposta de protético para engenharia de tecidos. As telas de polipropileno foram cultivadas com células-tronco mesenquimais de tecido adiposo (ADSCs) isoladas de camundongos C57B1/6 GFP+ durante quinze dias em placas revestidas com metacrilato ou não revestidas com metacrilato. A quantidade de ADSCs aderidas foram verificadas diariamente em Câmara de Neubauer e através de uma curva de crescimento realizada pelo ensaio MTT. As ADSCs aderidas às malhas foram visualizadas com marcação de DAPI, panóticas, hematoxilina e eosina imuno-histoquímica e imunofluorescência. O melhor protocolo foi na tela microporosa, no o período de sete dias de cultivo e em placas sem metacrilato. Conclui-se que a tela de polipropileno fornece um bom suporte para as ADSCs se aderirem podendo ser utilizada na engenharia de tecidos.

Tissue engineering replaces injured tissues by manipulating cells, making scaffolds, and using molecules that stimulate the tissue. Mesenchymal stem cells (MSCs) are good candidates for tissue engineering, as this is one of the cell types which are recruited to repair injured tissues. Scaffolds are structural devices that allow cell fixation and migration, with polypropylene meshes being an example. This study aims to evaluate the culture of adipose tissue-derived mesenchymal stem cells (ADSCs), isolated from C57Bl/6 GFP + mice, in two types of polypropylene meshes (macroporous and microporous) in conventional culture plates and plates coated with methacrylate, over a period of fifteen days. The objective was to obtain the best interaction protocol between the mesh and the cells. The choice of the best method was based on adherence, maintenance of adherence and viability during culture. The amount of ADSCs adhering was checked daily by counting in a Neubauer Chamber and by using a growth curve performed with the MTT assay. The ADSCs adhering to the meshes were visualized with DAPI, panotic, hematoxylin and eosin, immunohistochemistry (integrin), and immunofluorescence (actin). ADSCs adhere to all forms of culture and to the two types of polypropylene mesh. ADSCs adhered more to the microporous mesh, within the seven day period of culture and in the plates without methacrylate. Thus, polypropylene meshes offer a good scaffold for ADSCs to adhere to.

Alternagin-C binding to 21 integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells

Background Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of 21 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an 21 integrin. Herein, we used ALT-C as a 21 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration. Methods ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The 21, integrin binding properties of ALT-C, its dissociation constant (Kd) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting. Results Our data demonstrate that ALT-C, after binding to 21 integrin...(AU)

Alternagin-C binding to 21 integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells

Background Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of 21 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an 21 integrin. Herein, we used ALT-C as a 21 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration. Methods ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The 21, integrin binding properties of ALT-C, its dissociation constant (Kd) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting. Results Our data demonstrate that ALT-C, after binding to 21 integrin...

Cell migration inhibition activity of a non-RGD disintegrin from Crotalus durissus collilineatus venom

In recent decades, snake venom disintegrins have received special attention due to their potential use in anticancer therapy. Disintegrins are small and cysteine-rich proteins present in snake venoms and can interact with specific integrins to inhibit their activities in cell-cell and cell-ECM interactions. These molecules, known to inhibit platelet aggregation, are also capable of interacting with certain cancer-related integrins, and may interfere in important processes involved in carcinogenesis. Therefore, disintegrin from Crotalus durissus collilineatus venom was isolated, structurally characterized and evaluated for its toxicity and ability to interfere with cell proliferation and migration in MDA-MB-231, a human breast cancer cell line. Methods: Based on previous studies, disintegrin was isolated by FPLC, through two chromatographic steps, both on reversed phase C-18 columns. The isolated disintegrin was structurally characterized by Tris-TricineSDS-PAGE, mass spectrometry and N-terminal sequencing. For the functional assays, MTT and wound-healing assays were performed in order to investigate cytotoxicity and effect on cell migration in vitro, respectively. Results: Disintegrin presented a molecular mass of 7287.4 Da and its amino acid sequence shared similarity with the disintegrin domain of P-II metalloproteases. Using functional assays, the disintegrin showed low cytotoxicity (15% and 17%, at 3 and 6 µg/mL, respectively) after 24 h of incubation and in the wound-healing assay, the disintegrin (3 µg/mL) was able to significantly inhibit cell migration (24%, p < 0.05), compared to negative control. Conclusion: Thus, our results demonstrate that non-RGD disintegrin from C. d. collilineatus induces low cytotoxicity and inhibits migration of human breast cancer cells. Therefore, it may be a very useful molecular tool for understanding ECM-cell interaction cancer-related mechanisms involved in an important integrin family that highlights molecular aspects of tumorigenesis. Also, non-RGD disintegrin has potential to serve as an agent in anticancer therapy or adjuvant component combined with other anticancer drugs.(AU)

Effects of blocking av b3 integrin by a recombinant RGD disintegrin on remodeling of wound healing after induction of incisional hernia in rats

PURPOSE: To investigate the changes induced by DisBa-01 on repair of wound healing after induced incisional hernia (IH) in rats. METHODS: Thirty two male albino rats were submitted to IH and divided into four experimental groups: G1, placebo control; G2, DisBa-01-treated; G3, anti-αv β3 antibodies-treated and G4, anti-α2 antibodies-treated. Histological, biochemical and extracellular matrix remodeling analysis of abdominal wall were evaluated. RESULTS: After 14 days, 100% of the G2 did not present hernia, and the hernia ring was closed by a thin membrane. In contrast, all groups maintained incisional hernia. DisBa-01 also increased the number macrophages and fibroblasts and induced the formation of new vessels. Additionally, MMP-2 was strongly activated only in G2 (p<0.05). Anti- αv β3 -integrin antibodies produced similar results than DisBa-01 but not anti-α2 integrin blocking antibodies. CONCLUSION: DisBa-01 has an important role in the control of wound healing and the blocking of this integrin may be an interesting therapeutically strategy in incisional hernia.(AU)

Effects of Blocking avb3 integrin by a recombinant RGD disintegrin on remodeling of wound healing after induction of incisional hernia in rats

Incisional hernia (IH) is characterized by defective wound healing process. Disba-01, a αvb3 integrin blocker has shown to control the rate of wound repair and therefore it could be a target for new wound healing therapies.The objective of the study was to determine the changes induced by Disba-01 on repair of wound healing after induced IH in rats. Thirty two male albino rats were submitted to IH and divided into 4 experimental groups: G1, placebo control; G2, DisBa-01-treated; G3, anti-αvβ3 antibodies-treated and G4, anti-α2 antibodies-treated. Histological. biochemical and extracellular matrix remodeling analysis of abdominal wall were evaluated. After 14 days, 100% of the G2 did not present hernia, and the hernia ring was closed by a thin membrane. In contrast, all groups maintained incisional hernia. DisBa-01 also increased the number macrophages and fibroblasts and induced the formation of new vessels. Additionally, MMP-2 was strongly activated only in G2 (P<0.05). Anti- αvβ3-integrin antibodies produced similar results than Disba-01 but not anti-α2 integrin blocking antibodies. These results strongly indicate that Disba-01 has an important role in the control of wound healing and the blocking of this integrin may be an interesting therapeutical strategy in IH.(AU)

SNPs E MICROSSATÉLITES NA CARACTERIZAÇÃO RACIAL E DE RESISTÊNCIA A ENFERMIDADES EM BOVINOS DA RAÇA CURRALEIRO PÉ-DURO

A raça bovina local brasileira Curraleiro Pé-Duro é patrimônio genético importante que tem como características rusticidade, adaptabilidade às condições ambientais e resistência a doenças e intoxicações. Objetivou-se com esse estudo observar a variabilidade genética em populações de Curraleiro Pé-Duro, verificando a ocorrência de polimorfismos em marcadores SNP e microssatélites para caracterização racial e resistência a infeções por Brucella abortus, Leptospira spp, Neospora caninum, e pelos vírus da leucose, rinotraqueite infecciosa e diarreia viral bovina. Foram utilizadas 1.182 amostras de soro e sangue de bovinos Curraleiro Pé-Duro, oriundos dos estados de Goiás, Tocantins e Piauí, pertencentes ao banco de dados da Rede Pró-Centro-Oeste. O DNA foi obtido por método de extração salting out modificado e as reações em cadeia da polimerase (PCR) foram realizadas em sistema multiplex. Foram utilizados 27 marcadores de caracterização racial indicados pela FAO e 13 marcadores relacionados aos genes do complexo antígeno leucocitário bovino (BoLA) e ao SLC11A1. Os produtos de PCR foram separados por eletroforese em sequenciador automático ABI3500 e genotipados com auxílio do software GeneMapper. Foram investigados 24 polimorfismos de base única (SNP) nas mesmas amostras, detectados por meio da técnica de KASP (Kompetitive Allele-Specific PCR). Os SNPs foram pesquisados em genes relacionados à expressão de citocinas (integrina, interleucina, toll like receptor e nod like receptors), comumente associados à resistência ou suscetibilidade a enfermidades parasitárias, bacterianas e virais. A análise Bayesiana agrupou os animais em dois clusters em ambas análises de microssatélites, indicando a presença de uma subpopulação. Os valores de heterozigosidade observada foram inferiores à esperada na maioria dos locos e os Fst indicaram pouca diferenciação entre os clusters, sugerindo homozigotia aumentada. Foi realizada análise univariada para associação dos genótipos com os fenótipos, seguida de regressão logística considerando nível de significância de 5% ao teste de Fisher. Foram identificados 42 alelos significativos (p<0,05) para as infecções, indicando ter importante papel na resposta imune do hospedeiro. Todos os microssatélites analisados mostraram-se informativos. Os haplótipos dos SNPs rs55617325, rs55617286, rs55617437 e rs43702941, presentes no cromossomo 6, foram associados a animais negativos contra Neospora caninum, podendo ser indicativos de uma resistência inata ao patógeno. Os alelos identificados podem ser utilizados em pesquisas futuras sobre a resistência bovina.

The Brazilian cattle breed Curraleiro Pé-Duro is an important genetic resource by the characteristics of rusticity, adaptability to environmental conditions and resistance to diseases and intoxications. The aim of this study was to analyse the genetic diversity of Curraleiro Pé-Duro populations and verify the presence of SNPs and microsatellite polymorphisms for breed characterization and disease resistance to Brucella abortus, Leptospira spp, Neospora caninum, leukosis virus, herpesvirus and diarrhea viral infections. A total of 1.182 serum and blood samples of Curraleiro Pé-Duro cattle, from Goiás, Tocantins and Piauí states, belonging to the Pro-Centro-Oeste Network database were used. DNA was obtained by modified salting out extraction method and polymerase chain reactions (PCR) were performed in a multiplex system. A panel of 27 markers of breed characterization indicated by FAO and 13 markers related to the bovine leukocyte antigen (BoLA) and SLC11A gene were used. PCR products were separated by eletroforesis in an ABI3500 automatic sequencer and genotyped on GeneMapper software. Twenty-four single-base polymorphisms (SNPs) were investigated in the same samples, detected by KASP (Kompetitive Allele-Specific PCR). SNPs were investigated in genes related to the expression of cytokines (integrin, interleukin, toll like receptor and nod-like receptors), commonly associated with resistance or susceptibility to parasitic, bacterial and viral diseases. Bayesian analysis grouped the animals into two clusters in both microsatellite analyzes, indicating the presence of a subpopulation. The observed heterozygosity were lower than expected in most loci and and Fst values indicated little differentiation between clusters and increased homozygosity. Univariate analysis was performed for the association of genotypes with phenotypes, followed by logistic regression considering a significance level of 5% for the Fisher test. We identified 42 significant alleles (p <0.05) for infections that play an important role in the host immune response. All microsatellites were informative. SNP haplotypes rs55617325, rs55617286, rs55617437 e rs43702941, present on chromosome 6, were associated with negative animals against Neospora caninum and could be indicative of an innate resistance to the pathogen. The identified alleles can be used in future research on bovine resistance.

ESTRATÉGIAS PARA O ESTUDO DE MARCADORES MOLECULARES DA FERTILIDADE EM MODELOS ANIMAIS: PERFIL METABOLÔMICO DO PLASMA SEMINAL EM TOUROS E EXPRESSÃO DA INTEGRINA SUBUNIDADE BETA 5 NO ESPERMATOZOIDE, OÓCITO E EMBRIÃO BOVINO

Os metabólitos e as proteínas, incluindo as integrinas, são importantes na fisiologia reprodutiva de animais e seres humanos. Entretanto, ainda são desconhecidos os metabólitos do plasma seminal que estão associados com a fertilidade de touros e ainda não se sabe os possíveis papéis da integrina subunidade beta 5 (ITG5) na fecundação e no desenvolvimento embrionário de bovinos. Assim sendo, os objetivos do presente trabalho foram: (1) determinar o metaboloma do plasma seminal de touros com diferentes escores de fertilidades, (2) identificar metabólitos que demonstram ser potenciais marcadores de fertilidade em touros, (3) avaliar a expressão da ITG5 em espermatozoides, oócitos e embriões bovinos em fases iniciais de desenvolvimento e, (4) verificar a conservação evolutiva da ITG5 por meio de ferramentas de bioinformática. No estudo 1 foi utilizada a técnica de cromatografia gasosa associada à espectrometria de massas (CG-EM) para determinar o metaboloma do plasma seminal de 16 touros com alta e baixa fertilidade. Em seguida, os dados foram submetidos a análise estatística multivariada e univariada. A partir destes resultados, análises de bioinformática foram realizadas a fim de identificar as vias metabólicas associadas com o metaboloma do plasma seminal desses touros. Sessenta e três metabólitos foram identificados no plasma seminal de touros com diferentes escores de fertilidade. A frutose foi detectada como o metabólito mais abundante, seguido pelo ácido cítrico, ácido lático, ureia e ácido fosfórico. Já androstenediona, 4-cetoglicose, D-xilofuranose, ácido 2-oxoglutárico e o ácido eritrônico foram os metabólitos menos abundantes do fluído seminal de touros. A Análise Discriminante por Mínimos Quadrados Parciais (PLS-DA) revelou uma separação distinta entre os touros de alta e de baixa fertilidade. Os metabólitos com as maiores pontuações de Importância da Variável na Projeção (VIP > 2) foram o ácido 2-oxoglutárico e a frutose. A análise do mapa de calor (heatmap), com base na pontuação de VIP, e as análises univariadas indicaram que o ácido 2-oxoglutárico foi significativamente menor (P = 0,02) e a frutose foi significativamente maior (P = 0,02) em touros de alta fertilidade comparados aos touros de baixa fertilidade. O presente estudo foi o primeiro a identificar o perfil metabolômico do plasma seminal de touros empregando a CG-EM e o primeiro a mostrar o ácido 2-oxoglutárico e a frutose como potenciais biomarcadores de fertilidade em touros. No estudo 2, os níveis de expressão da proteína ITG5 em espermatozoide de touros foi avaliada pela técnica de western blotting. A reação de transcrição reversa seguida da reação em cadeia da polimerase quantitativa em tempo real (RT-qPCR) foi empregada para avaliar os níveis de expressão de transcritos da ITG5 em oócitos e embriões bovinos. Além disso, ferramentas de bioinformática foram utilizadas com o intuito de determinar a conservação evolutiva da proteína ITG5 entre várias espécies. Os resultados do western blotting confirmaram a presença da proteína ITG5 em espermatozoides de touros e, com base nas análises de RT-qPCR, foi observado que a ITG5 está presente nos óocitos e que os níveis dela são significativamente mais elevados nos embriões bovinos de 2 células, seguidos pelos embriões de 8-16 células. As análises de bioinformática mostraram que a ITG5 é conservada em várias espécies. Desta forma, nossos resultados demonstram a presença da ITG5 em espermatozoides, bem como em oócitos e embriões bovinos em estágios iniciais se desenvolvimento, sugerindo um importante papel da ITG5 na fecundação e no desenvolvimento embrionário inicial desta espécie.

Metabolites and proteins including integrins are important in the reproductive physiology of animals and humans. However, seminal plasma metabolites that are associated with bull fertility are still unknown and the possible roles of the integrin subunit beta 5 (ITG5) in fertilization and embryonic development of bovine are not yet known. Therefore, the objectives of the present study were (1) to determine comprehensive metabolome of seminal plasma from Holstein bulls with differing fertility scores, and (2) to ascertain potential metabolites as biomarkers associated with bull fertility, (3) to evaluate expression of ITG5 in sperm, oocytes and early bovine embryos, and (4) to ascertain the evolutionary conservation of ITG5. In the study 1, chromatography-mass spectrometry (GC-MS) was employed to determine seminal plasma metabolome from 16 Holstein bulls with two fertility scores. Soon after, data were evaluated using multivariate and univariate analyses. Bioinformatics analyzes were performed in order to identify metabolic pathways associated with bulls seminal plasma metabolites. Our results showed that 63 metabolites were identified in bull seminal plasma. Fructose was the most abundant metabolite detected followed by citric acid, lactic acid, urea, and phosphoric acid. Androstenedione, 4-ketoglucose, D-xylofuranose, 2-oxoglutaric acid and erythronic acid were the least predominant metabolites found in bulls seminal fluid. PLS-DA showed a distinct separation between high and low fertility bulls as regard to scores plot. Metabolites with the greatest VIP score (VIP > 2) were 2-oxoglutaric acid and fructose. Heat-map analysis based on VIP score and univariate analysis indicated that 2-oxoglutaric acid was less (P = 0.02) and fructose was significantly greater (P = 0.02) in high-fertility bulls as compared to low-fertility bulls. To the best of our knowledge the current study is the first to identify the comprehensive metabolomic profiling of bull seminal plasma with differing fertility score and to present the potential of metabolites such as 2-oxoglutaric acid and fructose as biomarkers of bull fertility using GC-MS. In the study 2, expression levels of ITG5 protein in bull sperm were evaluated by western blotting. Real time-qPCR was carried out to evaluate the expression levels of ITG5 transcripts in oocytes and embryos from bovine species. In addition, bioinformatic tools were performed to determine to evolutionary conservation of ITG5 across species. Western blotting results showed the presence of ITG5 on bull spermatozoa. Moreover, RT-qPCR results demonstrated that ITG5 is present in bovine oocytes and that ITG5 levels were significantly higher in 2-cell embryos and 8-16 cell embryos. Bioinformatics analyses showed that ITG5 is conserved across species. Therefore, our results showed the presence of ITG5 in spermatozoa as well as oocytes and embryos in early stages of development, suggesting an important role of ITG5 upon fecundation and early embryo development in the bovine species.

ALT-C, a disintegrin-like Cys-rich protein from Bothrops alternatus, increases skeletal myoblast viability

ALT-C, an ECD motif (glutamic acid, cysteine, aspartic acid) disintegrin from Bothrops alternatus snake venom, induces 21 integrin-mediated signaling and neutrophil chemotaxis. In vitro, in human umbilical vein endothelial cells (HUVEC), ALT-C induces cell proliferation, thus showing an interesting potential for tissue regeneration studies. This work aimed to evaluate the influence of ALT-C in myoblast viability and differentiation. Myoblasts were obtained from hind limb muscles of 3 to 4-day old Wistar rats. The cells were incubated with ALT-C at different concentrations and incubation periods were followed by total RNA isolation. cDNA synthesis and real time polymerase chain reaction (PCR) were performed with primers of myoD as well as of both (slow and fast) myosin heavy chain isoforms (MHC). ECD-disintegrin increased myoblast viability in a dose-dependent way, mostly with 50 to 100 nM concentrations, and such effect was more prevalent after 48 hours. No changes in gene expression of both MHC isoforms were observed in ALT-C-treated cells. MyoD expression was not detected, which suggests that myoblasts were in mature stages. Protease activity and cytokine array tested in a medium of 50 nM ALT-C-treated cells after 48 hours were not different from controls. In conclusion, it was shown that myoblats are sensitive to ALT-C indicating an integrin-mediated intracellular signaling that increases cell viability.

Influência da expressão da alfa-6 integrina na produção de células-tronco espermatogoniais murinas transgênicas / Influence expression of integrin alpha-6 in the production of transgenic murine spermatogonial stem cells

Ao longo da vida reprodutiva dos machos adultos, espermatozoides são formados pelas células-tronco espermatogoniais (SSCs, do inglês spermatogonial stem cells) por um processo conhecido como espermatogênese. O cultivo in vitro de SSCs abriu novas possibilidades para a transgenia, no entanto os protocolos requerem a adição de fatores de crescimento, o que encarece a manutenção dessas células por um tempo prolongado, fazendo da criopreservação de SSCs uma alternativa para esse problema. O fenótipo molecular de células-tronco espermatogoniais tem sido objeto de estudo nas últimas décadas. Uma das moléculas mais estudadas como marcador na caracterização de espermatogônias é a alfa-6 integrina. Baseado nestas informações, a seguinte hipótese foi proposta: SSCs que apresentam maior expressão de alfa-6 integrina são mais eficientes para modificações genéticas e colonização de túbulos seminíferos. Para testar essa hipótese, as SSCs murinas foram dissociadas de testículos de camundongos com 8 a 11 dias de idade. Essas células foram cultivadas in vitro, caracterizadas quanto sua morfologia, congeladas e incubadas com anticorpo anti-alfa-6 integrina para a purificação das SSCs por separação celular ativada por fluorescência (FACS). Células testiculares foram geneticamente modificadas com a inserção do gene marcador LacZ e o transplante através dos ductos eferentes foi padronizado. As Células germinativas testiculares foram dissociadas e cultivadas in vitro, porém a viabilidade foi aquém da desejada, inviabilizando as etapas de transformação, transplante e posterior avaliação histológica. Usando o marcador molecular alfa-6 integrina foi possível separar populações de células germinativas testiculares por FACS e a expressão gênica de ITGα6, GFRα1, Oct-4 e Thy-1 foi detectada por RT-PCR quantitativo. Em conclusão, não foi possível comprovar a eficiência de transdução e colonização em túbulos seminíferos por células-tronco espermatogoniais selecionadas com alfa-6 integrina (AU)

Throughout the reproductive life of adult males, spermatozoa are formed from spermatogonial stem cells (SSCs) by a process known as spermatogenesis. The in vitro culture of SSCs created new possibilities for transgenesis, however the protocols require addition of growth factors, which increases the maintenance costs of these cells for a prolonged time, making of the cryopreservation of SSCs an alternative for this problem. The molecular phenotype of spermatogonial stem cells have been target of studies in recent decades. One of the most studied marker molecule in the characterization of spermatogonia is integrin alpha-6. Based on these informations, the following hypothesis was proposed: SSCs that show increased expression of integrin alpha-6 are more efficient for genetic modification and colonization of seminiferous tubules. In order to test this hypotesis, murine SSCs were dissociated from testes of 8 to 11 days old mice. These cells were in vitro cultured, characterized based on its morphology, frozen and incubated with anti-integrin alpha-6 antibody for the purification of SSCs by activated cell sorting fluorescence (FACS). Testicular cells were genetically modified with the insertion of the marker gene LacZ and transplantation through the efferent ducts was standardized. The testicular germ cells were dissociated and in vitro cultured, however viability was below expected, precluding the steps of transformation, transplantation and subsequent histological evaluation. Using molecular marker integrin alpha-6 it was possible to separate testicular germ cell populations by FACS and gene expression of ITGα6, GFRα1, Oct-4 and Thy-1was detected by quantitative RT-PCR. In conclusion, it was not possible to prove the efficiency of transduction and colonization in the seminiferous tubules by spermatogonial stem cells selected with alpha 6 integrin (AU)

Influência da expressão da alfa-6 integrina na produção de células-tronco espermatogoniais murinas transgênicas / Influence expression of integrin alpha-6 in the production of transgenic murine spermatogonial stem cells

Ao longo da vida reprodutiva dos machos adultos, espermatozoides são formados pelas células-tronco espermatogoniais (SSCs, do inglês spermatogonial stem cells) por um processo conhecido como espermatogênese. O cultivo in vitro de SSCs abriu novas possibilidades para a transgenia, no entanto os protocolos requerem a adição de fatores de crescimento, o que encarece a manutenção dessas células por um tempo prolongado, fazendo da criopreservação de SSCs uma alternativa para esse problema. O fenótipo molecular de células-tronco espermatogoniais tem sido objeto de estudo nas últimas décadas. Uma das moléculas mais estudadas como marcador na caracterização de espermatogônias é a alfa-6 integrina. Baseado nestas informações, a seguinte hipótese foi proposta: SSCs que apresentam maior expressão de alfa-6 integrina são mais eficientes para modificações genéticas e colonização de túbulos seminíferos. Para testar essa hipótese, as SSCs murinas foram dissociadas de testículos de camundongos com 8 a 11 dias de idade. Essas células foram cultivadas in vitro, caracterizadas quanto sua morfologia, congeladas e incubadas com anticorpo anti-alfa-6 integrina para a purificação das SSCs por separação celular ativada por fluorescência (FACS). Células testiculares foram geneticamente modificadas com a inserção do gene marcador LacZ e o transplante através dos ductos eferentes foi padronizado. As Células germinativas testiculares foram dissociadas e cultivadas in vitro, porém a viabilidade foi aquém da desejada, inviabilizando as etapas de transformação, transplante e posterior avaliação histológica. Usando o marcador molecular alfa-6 integrina foi possível separar populações de células germinativas testiculares por FACS e a expressão gênica de ITGα6, GFRα1, Oct-4 e Thy-1 foi detectada por RT-PCR quantitativo. Em conclusão, não foi possível comprovar a eficiência de transdução e colonização em túbulos seminíferos por células-tronco espermatogoniais selecionadas com alfa-6 integrina

Throughout the reproductive life of adult males, spermatozoa are formed from spermatogonial stem cells (SSCs) by a process known as spermatogenesis. The in vitro culture of SSCs created new possibilities for transgenesis, however the protocols require addition of growth factors, which increases the maintenance costs of these cells for a prolonged time, making of the cryopreservation of SSCs an alternative for this problem. The molecular phenotype of spermatogonial stem cells have been target of studies in recent decades. One of the most studied marker molecule in the characterization of spermatogonia is integrin alpha-6. Based on these informations, the following hypothesis was proposed: SSCs that show increased expression of integrin alpha-6 are more efficient for genetic modification and colonization of seminiferous tubules. In order to test this hypotesis, murine SSCs were dissociated from testes of 8 to 11 days old mice. These cells were in vitro cultured, characterized based on its morphology, frozen and incubated with anti-integrin alpha-6 antibody for the purification of SSCs by activated cell sorting fluorescence (FACS). Testicular cells were genetically modified with the insertion of the marker gene LacZ and transplantation through the efferent ducts was standardized. The testicular germ cells were dissociated and in vitro cultured, however viability was below expected, precluding the steps of transformation, transplantation and subsequent histological evaluation. Using molecular marker integrin alpha-6 it was possible to separate testicular germ cell populations by FACS and gene expression of ITGα6, GFRα1, Oct-4 and Thy-1was detected by quantitative RT-PCR. In conclusion, it was not possible to prove the efficiency of transduction and colonization in the seminiferous tubules by spermatogonial stem cells selected with alpha 6 integrin

Nódulos fibro-histiocíticos no baço de uma cadela: relato de caso / Splenic fibroistiocytic nodules in a bitch: case report

Descreve-se a ocorrência de múltiplos nódulos fibro-histiocíticos no baço de uma cadela Rottweiler, de sete anos. O diagnóstico foi feito por histopatologia e confirmado por imunoistoquímica positiva para ED1, CD18, vimentina e lisozima. O animal foi sacrificado três meses após o diagnóstico, por severo agravamento dos sinais clínicos.(AU)

Multiple fibrohistiocytic nodules in the spleen of a 7-year-old female Rottweiler were reported. The diagnosis was made by histology and immunohistochemistry; cells were positive for ED1, CD18, vimentin and lysozyme. The dog was euthanatized three months after the tumor was diagnosed, due to severe clinical signs.(AU)

Integrins, cancer and snake toxins (mini-review)

Integrins encompass a family of transmembrane heterodimeric proteins of adhesion that maintain cells attached to other cells and to the extracellular matrix (ECM). Integrins work as bi-directional mechanotransducers, conveying mechanical signal from outside to inside the cell through a cascade of phosphorylation signals. On the other hand, the signal from inside to outside controls the strength and affinity of integrin adhesion. As proteins of focal contact, integrins are involved in diverse cell functions, such as cell activation, migration, growth, and survival. In the development of neoplastic disease and metastatic tumor, integrins can influence cancer invasiveness and progression, as well as mediate the formation of new blood vessels (angiogenesis). Diverse snake venom toxins have the ability to interact with multiple integrins, what results in inhibition of cell attachment, inhibition of angiogenesis, and induction of apoptotic death of tumor and vascular endothelial cells. The aim of this review is to present data about snake venom toxins that bind to integrins and evoke antiangiogenesis and antitumoral effects.

Interação celular na placenta de gestações de bovinos clonados com especial ênfase às integrinas / Cell-cell interactions in the bovine placenta from pregnancies with cloned fetuses with special respect to integrins and their receptors

Integrinas são glicoproteínas transmembrânicas envolvidas na adesão célula-célula e célula-proteina da matriz extracellular (ECM) que participam da migração e fusão de células trofoblásticas gigantes (TGC) com células do epitélio materno no placentoma bovino e interagem com inibidores teciduais de metalloproteinases (TIMPs), considerados importantes reguladores das metaloproteinases de matriz (MMPs), que são reconhecidas como passo limitande para ação de enzimas que atuam no remodelamento da ECM na implantação e na gestação. Uma vez que as integrinas são consideradas responsáveis pela manutenção da arquitetura tecidual e que as MMPs podem regular a degradação e a reconstituição da ECM necessária para a invasão do trofoblasto, temos como objetivo verificar uma possível relação das integrinas com alterações placentárias comumente observadas em gestações de animais clonados (SCNT). Para avaliar a funcionalidade placentária, a expressão do lactogênio placentário (PL) e do antígeno Ki-67 foram também verificadas. Placentomas bovinos foram coletados e divididos em 3 grupos: 1) início (n = 6) e 2) final (n = 3) da gestação de animais derivados de monta natural (n=9) e final da gestação de animais clonados (n=8), clones machos (n=4) e clones fêmeas (n=4). As proteínas foram localizadas por imuno-histoquímica indireta, exceto as subunidades de integrina α6, β1, αV e β3 que foram localizadas por imunofluorescência. A especificidade dos anticorpos e expressão protéica foi confirmada por Western blot e a expressão do RNAm foi avaliada por meio de RT-PCR e PCR em tempo real quantitativo. As células positivas para a laminina, TIMP-2, Ki-67 e lactogênio placentário (PL) foram quantificadas e para comparação entre os grupos. A proteína das subunidades α6 do receptor de integrina e β1 foi observada nos animais clonados e não clonados nos estromas materno e fetal e porção basal dos epitélios materno e fetal e foi co-localizada com a laminina. A proteína das subunidades αV e β3 do receptor de integrina foi observado nos estromas materno e fetal e foi co-localizado com a fibronectina. O TIMP-2 foi exclusivamente e especificamente localizado nas TGCs no início e final da gestação de animais clonados e não clonados, mas não houve diferença significativa em sua expressão. O PL apresentou a mesma localização do TIMP-2 em todos os grupos analisados. Houve diferença significativa (p<0,05) entre 270d e clones ao se comprar a expressão relativa do RNAm da subunidade de integrina β1 e do lactogênio placentário e ao se comparar a expressão protéica por western blot da laminina e do TIMP-2. Foi possível observar diferença significativa (p<0,05) entre o grupo controle e o grupo de animais clonados ao se quantificar o número de TGCs positivas para a laminina e para o lactogênio placentário. Apesar de algumas diferenças terem sido observadas na expressão de integrinas e de proteínas da ECM, sugere-se que a interação das integrinas com seus ligantes da ECM a termo não é um determinante da sobrevivência neonatal de bovinos clonados. Contudo, outros estudos são necessários para esclarecer se estas proteínas representam elementos chave na placentação de bovinos clonados em início de gestação. As diferenças observadas na expressão de integrinas e principalmente do PL entre clones fêmeas e machos sugerem possível influência de genes associados ao cromossomo sexual ou imprinting

Integrins are transmembrane glycoproteins involved in cell-cell and cell-extracellular matrix (ECM) adhesion and signal transduction. They participate in the migration and fusion of trophoblast giant cells (TGC) with uterine epithelial cells in bovine placentomes, and interact with tissue inhibitors of metalloproteinases (TIMPs), considered important regulators of matrix metalloproteinases (MMPs). MMPs are rate-limiting enzymes degrading ECM proteins during tissue remodeling around embryo implantation, pregnancy and parturition. Since integrins are considered to be responsible for the maintenance of the architecture within tissues and MMPs may also regulate degradation and reconstitution of ECM required for trophoblast invasion, we aimed to verify a potential relation of integrins with common placental alterations observed in cloned animals (SCNT). To verify the placental functionality, expression of placental lactogen (PL) and Ki-67 antigen was also assessed. Bovine placentomes were collected and divided into 3 groups: 1) early and 2) late gestation derived from natural mating (n=9) and 3) late gestational bovine clones (n=8), also divided into male clones (n=4) and female clones (n=4). All proteins were localized by indirect immunohistochemistry except for integrin subunits α6, β1, αV and β3 that were shown by immunofluorescence. The antibody specificity and protein expression were confirmed by Western blot. Expression of mRNA was evaluated using RT-PCR and quantitative Real Time PCR. Positive cells for laminin, TIMP-2, Ki-67 and placental lactogen were quantified for comparisons among groups. The protein of integrin receptor subunits α6 and β1 was observed in both cloned and non-cloned animals in the fetal and maternal stroma and at the basal membrane of maternal and fetal epithelial cells, co-localized with laminin and with collagen IV. The integrin receptor subunits αV and β3 were observed in the fetal and maternal stroma, co-localized with fibronectin. TIMP-2 was specifically and exclusively localized in TGC in the early and term gestation in both cloned and non-cloned animals. PL has shown the same localization of TIMP-2 in all analyzed samples. Statistically significant differences (p<0,05) between term gestation of non-cloned and cloned animals were observed comparing the mRNA expression of integrin β1 subunit and placental lactogen and the protein expression of laminin and TIMP-2. There were also statistically significant differences (p<0,05) between non-cloned and cloned animals in the number of laminin and placental lactogen positive cells. Despite some differences in integrin and ECM proteins expression, our results suggest that the interaction between integrins and their ECM ligands in term gestation is not a determinant for the survival rate of newborn cloned calves. However further studies are necessary to elucidate if integrins represent key elements in the early placentation of SCNT bovines. The differences found in the integrin and principally in the placental lactogen expression between female and male cloned calves suggests the influence of sex-chromosome associated genes or imprinting

Uso de matriz extracelular (Matrigel®) para estabelecimento de cultivo de células-tronco embrionárias de suínos e caracterização da expressão de moléculas associadas à pluripotência / Use of extracellular matrix (Matrigel®) for establishment of porcine embryonic stem cells and expression characterization of plurpotency related molecules

O estabelecimento de cultivo de células-tronco embrionárias (ESC) ainda não foi realizado com sucesso. Verificação de marcadores de pluripotência e diferenciação nos três folhetos germinativos são necessárias para validação de uma linhagem celular pluripotente. O objetivo deste estudo foi estabelecer e caracterizar o cultivo de ESC suínas usando Matrigel e comparar a expressão dos marcadores de pluripotência Oct-4, CD9 e α6-integrina em embriões. Blastocistos in vitro ou in vivo foram submetidos à imunocirurgia para cultura da massa celular interna, fixados para imunocitoquímica ou extração de RNA total para RT-PCR. Nenhuma colônia de ESC foi obtida usando co-cultivo em fibroblastos embrionários murinos (MEF) ou em Matrigel. Expressão de Oct-4, CD9 e α6-integrina foi detectada por PCR. Os produtos de PCR de CD9 e α6-integrina tiveram suas sequências nucleotídicas determinadas e comparadas com bases de dados públicas. O produto de CD9 foi idêntico à seqüência do CD9 suíno e o produto de α66integrina foi similar à humana e eqüina. Reação de Imunocitoquímica revelou a presença de Oct-4 no citoplasma de células da massa celular interna e do trofoblasto. CD9 e α6-integrina foram observados preferencialmente em células do trofoblasto. Não foi possível comparar a expressão dos marcadores de pluripotência entre ESC e embriões em suínos. Porém, este estudo descreve pela primeira vez a expressão de CD9 e α6-integrina em blastocistos suínos, os quais podem não estar relacionados com células pluripotentes embrionárias suínas

Establishment of embryonic stem cell (ESC) culture in pigs has not been achieved. Verification of pluripotency markers and differentiation in the three embryonic layers are necessary for validation of a pluripotent cell line. The objective of this study was to establish and characterize porcine ESC culture using Matrigel and compare the expression of pluripotency markers Oct-4, CD9 and α6-integrin with embryos. In vitro or in vivo porcine blastocysts were submitted to immunosurgery for culture of inner cell mass, fixation for immunocytochemistry or total RNA extraction for RT-PCR. No ESC colonies were obtained using co-culture on mouse embryonic fibroblasts (MEF) or on Matrigel. Expression of Oct-4, CD9 and α6-integrin was detected by PCR. CD9 and α6-integrin PCR products had their nucleotide sequence assessed and compared with public nucleotide database. CD9 product was identical to CD9 porcine sequences and α6-integrin product was similar to human and equine α6-integrin. Immunocytochemistry revealed Oct-4 expression in cytoplasm of the inner cell mass and trophoblast cells. CD9 and α6-integrin were observed preferentially on trophoblast cells. It was not possible to compare expression of pluripotency markers between porcine ESC and embryos. However, this study describes for the first time expression of CD9 and α6-integrin in porcine blastocysts, which may not be related to pluripotent porcine embryonic cells