Plasma rico em plaquetas e células tronco no tratamento de desmopatia dos ligamentos colaterais distais em equino / Platelet-rich plasma and stem cells in the treatment of desmopathy of the distal collateral ligaments in equine

Os equinos são animais muito utilizados em eventos esportivos, sendo, desta forma, propensos a sofrer acidentes que podem levar a lesões articulares e ósseas. Na rotina clínica, artifícios como o uso de raio X e ultrassonografia são de grande importância para que o correto diagnóstico seja feito e a conduta clínica possa ser melhor planejada. Além disso, alguns biomateriais, como o plasma rico em plaquetas (PRP) e células-tronco mesenquimais (CTMs), têm se inserido nos protocolos terapêuticos, demonstrando grande efetividade no tratamento desse tipo de lesão, devido à grande quantidade de fatores de crescimento presentes em ambos os biomateriais. O PRP é um derivado sanguíneo, caracterizado pela alta concentração plaquetária. Foi produzido a partir de duas centrifugações, a primeira para separar o plasma das hemácias e a segunda para concentrar as plaquetas. As CTMs foram isoladas de tecido adiposo, cultivadas, transportadas e aplicadas de forma autóloga, assim como o PRP. No presente relato, um equino, fêmea, Brasileiro de Hipismo com 8 anos, foi diagnosticado com desmopatia nos ligamentos colaterais da articulação interfalângica distal e foi tratado com PRP e CTMs, de forma associada e sequencial. Foi encontrada uma melhora do quadro clínico, significativa, em comparação aos dados encontrados na literatura, demonstrando grande potencialidade do uso associado de PRP e CTMs no tratamento de lesões ligamentares.

Horses are animals widely used in sporting events and are therefore prone to accidents that can lead to joint and bone injuries. In the clinical routine, devices such as the use of X-ray and ultrasound are of great importance for the correct diagnosis to be made and the clinical conduct can be better planned. In addition, some biomaterials such as platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) have been included in the therapeutic protocols, demonstrating great effectiveness in the treatment of this type of injury due to the large amount of growth factors present in both biomaterials. PRP is a blood derivative, characterized by high platelet concentration. It is produced from two centrifugations, the first to separate plasma from red blood cells and the second to concentrate platelets. MSCs were isolated from adipose tissue, cultured, transported and applied autologously, as well as PRP. In the present report, an 8-year-old female Brazilian equestrian horse was diagnosed with desmopathy in the collateral ligaments of the distal interphalangeal joint and was treated with PRP and MSCs in an associated and sequential manner. A significant improvement in the clinical picture was found in comparison to the data found in the literature, demonstrating great potential of the associated use of PRP and MSCs in the treatment of ligament injuries.

Maturação in vitro de oócitos ovinos submetidos à congelação lenta / In vitro maturation of cryopreserved ovine oocytes subjected to slow freezing

A maturação in vitro de oócitos submetidos ao processo de criopreservação, ainda compreende um desafio para o sucesso da reprodução assistida na medicina veterinária. Devido a isso, estudos são desenvolvidos a fim de identificar, amenizar e superar as limitações encontradas. Nesse sentido, objetivou-se realizar a avaliação da maturação in vitro de oócitos ovinos após criopreservação pelo método de congelação lenta. Para tanto, foram colhidos 204 ovários oriundos de 102 ovelhas púberes (SPRD) pertencentes a abatedouros localizados no município de Teresina, Piauí. Os ovários foram transportados para o laboratório e, posteriormente, foram aspirados por meio de um aspirador cirúrgicoadaptado. Um total de 180 oócitos foram desnudados e, então, submetidos à congelação lenta em sistema automatizado (TK 3000®). Posteriormente foram descongelados, e submetidos à maturação in vitro(MIV). Em seguida, procedeu-se a avaliação da maturação nuclear. Os resultados foram avaliados por meio do teste de Qui-quadrado de Pearson (P ≤ 0,05). Após descongelação, 22,8% dos oócitos na avaliação em estereomicroscópio (45x) apresentavam lesões de zona pelúcida e de oolema. Dos 139 oócitos submetidos a MIV, oito maturaram (5,75%). Conclui-se que a congelação lenta de oócitos ovinos pode influenciar a maturação in vitro, devido a lesões de membrana plasmática e zona pelúcida.(AU)

The in vitro maturation of oocytes submitted to the cryopreservation process, still comprises a challenge for the success of assisted reproduction in veterinary medicine. Due to this, studies are developed in order to identify, ameliorate and overcome the limitations found. The objective of this study was to evaluate the in vitro maturation of ovine oocytes after cryopreservation by the slow freezing method. For that, 204 ovaries from 102 pubertal sheep (SPRD) belonging to slaughterhouses located in the city of Teresina, Piauí, were collected. The ovaries were transported to the laboratory and subsequently aspirated by means of an adapted surgical aspirator. A total of 180 CCO's were obtained, which were stripped and then subjected to slow freezing in an automated system (TK 3000®). Later they were thawed and submitted to in vitro maturation (IVM). Next, the nuclear maturation was evaluated. Results were evaluated using Pearson's chi-square test (P ≤ 0.05). After thawing, 22.8% of the oocytes in the stereomicroscope (45x) evaluation presented lesions of the zona pellucida and oolema. Of the 139 oocytes submitted to IVM, eight maturated (5.75%). It is concluded that slow freezing of sheep oocytesmay influence in vitro maturation due to plasma membrane and zona pellucida lesions.(AU)

Maturação in vitro de oócitos ovinos submetidos à congelação lenta / In vitro maturation of cryopreserved ovine oocytes subjected to slow freezing

A maturação in vitro de oócitos submetidos ao processo de criopreservação, ainda compreende um desafio para o sucesso da reprodução assistida na medicina veterinária. Devido a isso, estudos são desenvolvidos a fim de identificar, amenizar e superar as limitações encontradas. Nesse sentido, objetivou-se realizar a avaliação da maturação in vitro de oócitos ovinos após criopreservação pelo método de congelação lenta. Para tanto, foram colhidos 204 ovários oriundos de 102 ovelhas púberes (SPRD) pertencentes a abatedouros localizados no município de Teresina, Piauí. Os ovários foram transportados para o laboratório e, posteriormente, foram aspirados por meio de um aspirador cirúrgicoadaptado. Um total de 180 oócitos foram desnudados e, então, submetidos à congelação lenta em sistema automatizado (TK 3000®). Posteriormente foram descongelados, e submetidos à maturação in vitro(MIV). Em seguida, procedeu-se a avaliação da maturação nuclear. Os resultados foram avaliados por meio do teste de Qui-quadrado de Pearson (P ≤ 0,05). Após descongelação, 22,8% dos oócitos na avaliação em estereomicroscópio (45x) apresentavam lesões de zona pelúcida e de oolema. Dos 139 oócitos submetidos a MIV, oito maturaram (5,75%). Conclui-se que a congelação lenta de oócitos ovinos pode influenciar a maturação in vitro, devido a lesões de membrana plasmática e zona pelúcida.

The in vitro maturation of oocytes submitted to the cryopreservation process, still comprises a challenge for the success of assisted reproduction in veterinary medicine. Due to this, studies are developed in order to identify, ameliorate and overcome the limitations found. The objective of this study was to evaluate the in vitro maturation of ovine oocytes after cryopreservation by the slow freezing method. For that, 204 ovaries from 102 pubertal sheep (SPRD) belonging to slaughterhouses located in the city of Teresina, Piauí, were collected. The ovaries were transported to the laboratory and subsequently aspirated by means of an adapted surgical aspirator. A total of 180 CCO's were obtained, which were stripped and then subjected to slow freezing in an automated system (TK 3000®). Later they were thawed and submitted to in vitro maturation (IVM). Next, the nuclear maturation was evaluated. Results were evaluated using Pearson's chi-square test (P ≤ 0.05). After thawing, 22.8% of the oocytes in the stereomicroscope (45x) evaluation presented lesions of the zona pellucida and oolema. Of the 139 oocytes submitted to IVM, eight maturated (5.75%). It is concluded that slow freezing of sheep oocytesmay influence in vitro maturation due to plasma membrane and zona pellucida lesions.

Production of recombinant therapeutic proteins in genetically engineered animals: the dawn of a new era / Produção de proteínas terapêuticas recombinantes em animais geneticamente modificados: o surgimento de uma nova era

Background: The production of transgenic animals has been envisioned as a viable strategy to improve food quality, animal yield, and for the production of bioproducts that can be used for the benefit of the human and animal population. Transgenic animals have been used to improve production traits, to add value to animal products, to minimize the impact on the environment, to promote disease resistance, and most notably, to produce recombinant proteins in natural fluids, such as milk, that can be collected, purified and used as biomedical products (biopharming). This review aims to discuss past and recent technological advances in animal transgenesis, and the perspective for biopharming in Brazil.Review: Since the production of recombinant human insulin from Escherichia coli in the 1970s, continuous development of new platforms has allowed a significant expansion in the biopharmaceutical market. The animal platform has been shown to be highly competitive by adding value as low cost implementation, production and scale up, as well as high productivity of synthesized proteins. The expression of recombinant proteins in milk represents the most developed system for production of biopharmaceutical drugs in animals, with two approved biopharmaceuticals for human use: Atryn®, a recombinant antithrombin produced in the milk of goats, approved in 2006 by European Medicines Agency (EMA) and in 2009 by US Food and Drug Administration (FDA), and more recently, Ruconest®, a recombinant human C1 esterase inhibitor protein (C1INH) produced in the milk of rabbits, first approved by EMA in 2012, followed by the FDA approval in 2014. Transgenic animals have been produced by many strategies that have gradually evolved over the decades, including the use of embryo microinjection, viral vectors and transposable elements, sperm-mediated gene transfer, and cloning by somatic cell nuclear transfer (SCNT).[...](AU)

Production of recombinant therapeutic proteins in genetically engineered animals: the dawn of a new era / Produção de proteínas terapêuticas recombinantes em animais geneticamente modificados: o surgimento de uma nova era

Background: The production of transgenic animals has been envisioned as a viable strategy to improve food quality, animal yield, and for the production of bioproducts that can be used for the benefit of the human and animal population. Transgenic animals have been used to improve production traits, to add value to animal products, to minimize the impact on the environment, to promote disease resistance, and most notably, to produce recombinant proteins in natural fluids, such as milk, that can be collected, purified and used as biomedical products (biopharming). This review aims to discuss past and recent technological advances in animal transgenesis, and the perspective for biopharming in Brazil.Review: Since the production of recombinant human insulin from Escherichia coli in the 1970s, continuous development of new platforms has allowed a significant expansion in the biopharmaceutical market. The animal platform has been shown to be highly competitive by adding value as low cost implementation, production and scale up, as well as high productivity of synthesized proteins. The expression of recombinant proteins in milk represents the most developed system for production of biopharmaceutical drugs in animals, with two approved biopharmaceuticals for human use: Atryn®, a recombinant antithrombin produced in the milk of goats, approved in 2006 by European Medicines Agency (EMA) and in 2009 by US Food and Drug Administration (FDA), and more recently, Ruconest®, a recombinant human C1 esterase inhibitor protein (C1INH) produced in the milk of rabbits, first approved by EMA in 2012, followed by the FDA approval in 2014. Transgenic animals have been produced by many strategies that have gradually evolved over the decades, including the use of embryo microinjection, viral vectors and transposable elements, sperm-mediated gene transfer, and cloning by somatic cell nuclear transfer (SCNT).[...]

Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specific corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specific mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most efficient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specific and unique siRNA sequences (Stealth RNaiTM). Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (172 bp in exon 2; and 108 bp in exon 6; NM003401) genes were chosen to generate dsRNA for subsequent "Dicing" to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80ºC. Alternatively, chemically synthesized Stealth siRNAs were designed and generated to match two very specific gene sequence regions for each target gene of interest (Ku70 and Xrcc4). HCT116 cells were plated at 30% confluence in 24- or 6-well culture plates. The next day, cells were transfected by lipofection with either Diced or Stealth siRNAs for Ku70 or Xrcc4, in duplicate, at various doses, with blank and sham transfections used as controls. Cells were harvested at 0, 24, 48, 72 and 96 h post-transfection for protein determination. The knockdown of specific targeted gene products was quantified by Western blot using GAPDH as control. Transfection of gene-specific siRNA to either Ku70 or Xrcc4 with both Diced and Stealth siRNAs resulted in a down regulation of the targeted proteins to approximately 10 to 20% of control levels 48 h after transfection, with recovery to pre-treatment levels by 96 h. Discussion: By transfecting cells with Diced or chemically synthesized Stealth siRNAs, Ku70 and Xrcc4, two highly expressed proteins in cells, were effectively attenuated, demonstrating the great potential for the use of both siRNA production strategies as tools to perform loss of function experiments in mammalian cells. In fact, down-regulation of Ku70 and Xrcc4 has been shown to reduce the activity of the non-homologous end joining DNA pathway, a very desirable approach for the use of homologous recombination technology for gene targeting or knockout studies. Stealth RNAiTM was developed to achieve high specificity and greater stability when compared with mixtures of enzymatically-produced (Diced) siRNA fragments. In this study, both siRNA approaches inhibited the expression of Ku70 and Xrcc4 gene products, with no detectable toxic effects to the cells in culture. However, similar knockdown effects using Diced siRNAs were only attained at concentrations 10-fold higher than with Stealth siRNAs. The application of RNAi technology will expand and continue to provide new insights into gene regulation and as potential applications for new therapies, transgenic animal production and basic research.

Clonagem animal: a sobrevivência dos mais aptos / Animal cloning: survival of the fittest

A clonagem animal por transferência nuclear de célula somática (TNCS) apresenta inúmeras aplicações científicas e comerciais, incluindo a produção de animais transgênicos, a preservação de animais de genética desejável, rara ou em extinção, ou mesmo a aplicação para o estudo de aspectos básicos em biologia molecular, celular e do desenvolvimento. Não obstante, a clonagem por TNCS ainda é ineficiente, com menos de 5% dos embriões clones produzidos resultando em animais nascidos vivos. O sucesso na clonagem exige o exímio domínio técnico e científico de várias disciplinas e áreas de conhecimento, havendo pelo menos cinco etapas críticas no processo associadas a falhas de desenvolvimento, desde a produção in vitro dos embriões até o nascimento de um animal viável. A identificação de fatores associados às falhas em cada etapa, em especial aqueles relacionados ao oócito receptor (citoplasto), à célula doadora (carioplasto) e aos procedimentos técnicos per se de produção de embriões clones, além da observação cuidadosa dos sinais de anormalidades subsequentes à transferência dos embriões para fêmeas receptoras, é essencial para a optimização de todos os procedimentos para a obtenção, em seu final, de um animal clonado viável e que sobreviva até a vida adulta. Esta revisão visa descrever alguns eventos técnicos e biológicos associados ao sucesso e/ou insucesso da clonagem animal.

Animal cloning by somatic cell nuclear transfer (SCNT) has numerous scientific and commercial applications, including the production of transgenic animals, preservation of animals from desirable or rare gene pools, and animals in risk of extinction, or even for the study of basic aspects in molecular, cell and developmental biology. Nevertheless, cloning by SCNT is still inefficient, with less than 5% of cloned embryos resulting in liveborn animals. The cloning success depends on a proficient technical and scientific know-how of a number of disciplines and areas of knowledge, with at least five critical steps in the process associated with developmental failures, from the in vitro production of cloned embryos through the birth of a viable animal. The identification of factors associated with failures in each step, in special to those related to the recipient oocyte (cytoplast), to the nucleus donor cell (karyoplast), and to the technical procedures for the production of cloned embryos per se, along with the careful observation of signs of abnormalities following the transfer of embryos to recipient females, is essential for the optimization of procedures that, ultimately, may result in a cloned animal that survives to adulthood. This review aims to discuss some technical and biological events associated with success and/or failure in animal cloning.

Clonagem animal: a sobrevivência dos mais aptos / Animal cloning: survival of the fittest

A clonagem animal por transferência nuclear de célula somática (TNCS) apresenta inúmeras aplicações científicas e comerciais, incluindo a produção de animais transgênicos, a preservação de animais de genética desejável, rara ou em extinção, ou mesmo a aplicação para o estudo de aspectos básicos em biologia molecular, celular e do desenvolvimento. Não obstante, a clonagem por TNCS ainda é ineficiente, com menos de 5% dos embriões clones produzidos resultando em animais nascidos vivos. O sucesso na clonagem exige o exímio domínio técnico e científico de várias disciplinas e áreas de conhecimento, havendo pelo menos cinco etapas críticas no processo associadas a falhas de desenvolvimento, desde a produção in vitro dos embriões até o nascimento de um animal viável. A identificação de fatores associados às falhas em cada etapa, em especial aqueles relacionados ao oócito receptor (citoplasto), à célula doadora (carioplasto) e aos procedimentos técnicos per se de produção de embriões clones, além da observação cuidadosa dos sinais de anormalidades subsequentes à transferência dos embriões para fêmeas receptoras, é essencial para a optimização de todos os procedimentos para a obtenção, em seu final, de um animal clonado viável e que sobreviva até a vida adulta. Esta revisão visa descrever alguns eventos técnicos e biológicos associados ao sucesso e/ou insucesso da clonagem animal.(AU)

Animal cloning by somatic cell nuclear transfer (SCNT) has numerous scientific and commercial applications, including the production of transgenic animals, preservation of animals from desirable or rare gene pools, and animals in risk of extinction, or even for the study of basic aspects in molecular, cell and developmental biology. Nevertheless, cloning by SCNT is still inefficient, with less than 5% of cloned embryos resulting in liveborn animals. The cloning success depends on a proficient technical and scientific know-how of a number of disciplines and areas of knowledge, with at least five critical steps in the process associated with developmental failures, from the in vitro production of cloned embryos through the birth of a viable animal. The identification of factors associated with failures in each step, in special to those related to the recipient oocyte (cytoplast), to the nucleus donor cell (karyoplast), and to the technical procedures for the production of cloned embryos per se, along with the careful observation of signs of abnormalities following the transfer of embryos to recipient females, is essential for the optimization of procedures that, ultimately, may result in a cloned animal that survives to adulthood. This review aims to discuss some technical and biological events associated with success and/or failure in animal cloning.(AU)