Cryopreservation of lymphocytes for immunological studies in horses / Criopreservação de linfócitos para estudos imunológicos em equinos

The use of frozen cells allows studies on diseases and other immunological assays, since it facilitates the logistics of collecting and transporting, including laboratories located in different cities or other countries. The objectives of this study were to verify if the storage in the refrigerator after collection at different times changes the viability of total leukocytes after months of freezing and the ratio of CD4/CD8 is affected by the freezing process. Venous blood of 15 healthy horses was used and the experiment was divided into 2 stages. In the first, the viability of the leukocytes before and after freezing was verified, as well as different storage times in the refrigerator (fresh blood, stored for 24 and 48 hours) before the freezing process. In the second part, the immunophenotyping of the T lymphocytes was performed, in order to observe if after thawing the relationship between LT CD4 and LT CD8 undergoes change. There was no difference between the amounts of viable leucocytes from frozen fresh blood compared to fresh blood before freezing, nor difference between the viability of blood left in the refrigerator (4°C) for 24 hours and fresh blood and fresh frozen blood. There was a decrease in viability of frozen leukocytes after 48 hours left in the freezer for other samples; however, the recovery was 107x cells. Regarding the immunophenotyping of CD2CD4+ and CD2CD8+ double-labeled T lymphocytes in the blood stored in the refrigerator for 24 hours before freezing, no difference was observed between before and after 6 months of freezing. It is concluded that cryopreservation of equine total leukocytes is possible and, although there was a difference between freezing times, even in the less viable sample, sufficient numbers of cells were recovered for other immunological assays.(AU)

A utilização de células congeladas possibilita estudos sobre doenças e outros ensaios imunológicos, pois facilita a logística de coleta e transporte, inclusive para laboratórios localizados em cidades diferentes ou outros países. Os objetivos desse estudo foram verificar se o armazenamento sobre refrigeração em diferentes tempos e a criopreservação alteram a viabilidade de leucócitos totais e se a relação entre LT CD4/CD8 é afetada pelo processo de congelamento. Utilizou-se sangue venoso de 15 cavalos hígidos e o experimento foi dividido em 2 etapas. Na primeira foi analisado se houve alteração na viabilidade dos leucócitos provenientes de amostras de sangue armazenadas em diferentes tempos em geladeira antes e depois de 6 meses de congelamento a -80°C. Na segunda parte, realizou-se a imunofenotipagem dos linfócitos T, com a finalidade de observar se após o descongelamento a relação entre LT CD4 e LT CD8 sofre alteração. Não houve diferença entre a quantidade de leucócitos viáveis da amostra de sangue fresco descongelado em relação ao sangue fresco antes do congelamento, nem diferença entre a viabilidade do sangue deixado em congelador (4°C) por 24 horas e do sangue fresco. Houve uma diminuição da viabilidade dos leucócitos, após o descongelamento de 6 meses (-80°C), das amostras de sangue deixado em geladeira por 48 horas antes do congelamento em relação às outras amostras, porém, a recuperação foi de células x107. Quanto à imunofenotipagem de linfócitos T com dupla marcação CD2CD4+ e CD2CD8+, no sangue armazenado em geladeira por 24 horas antes do congelamento, e não foi observada diferença entre antes ou depois de 6 meses de congelamento. Conclui-se que a criopreservação de leucócitos totais de equinos é possível e, embora tenha havido diferença entre os tempos de congelamento, mesmo na amostra menos viável, houve recuperação de uma quantidade de células suficientes para outros ensaios imunológicos.(AU)

Cryopreservation of lymphocytes for immunological studies in horses / Criopreservação de linfócitos para estudos imunológicos em equinos

The use of frozen cells allows studies on diseases and other immunological assays, since it facilitates the logistics of collecting and transporting, including laboratories located in different cities or other countries. The objectives of this study were to verify if the storage in the refrigerator after collection at different times changes the viability of total leukocytes after months of freezing and the ratio of CD4/CD8 is affected by the freezing process. Venous blood of 15 healthy horses was used and the experiment was divided into 2 stages. In the first, the viability of the leukocytes before and after freezing was verified, as well as different storage times in the refrigerator (fresh blood, stored for 24 and 48 hours) before the freezing process. In the second part, the immunophenotyping of the T lymphocytes was performed, in order to observe if after thawing the relationship between LT CD4 and LT CD8 undergoes change. There was no difference between the amounts of viable leucocytes from frozen fresh blood compared to fresh blood before freezing, nor difference between the viability of blood left in the refrigerator (4°C) for 24 hours and fresh blood and fresh frozen blood. There was a decrease in viability of frozen leukocytes after 48 hours left in the freezer for other samples; however, the recovery was 107x cells. Regarding the immunophenotyping of CD2CD4+ and CD2CD8+ double-labeled T lymphocytes in the blood stored in the refrigerator for 24 hours before freezing, no difference was observed between before and after 6 months of freezing. It is concluded that cryopreservation of equine total leukocytes is possible and, although there was a difference between freezing times, even in the less viable sample, sufficient numbers of cells were recovered for other immunological assays.(AU)

A utilização de células congeladas possibilita estudos sobre doenças e outros ensaios imunológicos, pois facilita a logística de coleta e transporte, inclusive para laboratórios localizados em cidades diferentes ou outros países. Os objetivos desse estudo foram verificar se o armazenamento sobre refrigeração em diferentes tempos e a criopreservação alteram a viabilidade de leucócitos totais e se a relação entre LT CD4/CD8 é afetada pelo processo de congelamento. Utilizou-se sangue venoso de 15 cavalos hígidos e o experimento foi dividido em 2 etapas. Na primeira foi analisado se houve alteração na viabilidade dos leucócitos provenientes de amostras de sangue armazenadas em diferentes tempos em geladeira antes e depois de 6 meses de congelamento a -80°C. Na segunda parte, realizou-se a imunofenotipagem dos linfócitos T, com a finalidade de observar se após o descongelamento a relação entre LT CD4 e LT CD8 sofre alteração. Não houve diferença entre a quantidade de leucócitos viáveis da amostra de sangue fresco descongelado em relação ao sangue fresco antes do congelamento, nem diferença entre a viabilidade do sangue deixado em congelador (4°C) por 24 horas e do sangue fresco. Houve uma diminuição da viabilidade dos leucócitos, após o descongelamento de 6 meses (-80°C), das amostras de sangue deixado em geladeira por 48 horas antes do congelamento em relação às outras amostras, porém, a recuperação foi de células x107. Quanto à imunofenotipagem de linfócitos T com dupla marcação CD2CD4+ e CD2CD8+, no sangue armazenado em geladeira por 24 horas antes do congelamento, e não foi observada diferença entre antes ou depois de 6 meses de congelamento. Conclui-se que a criopreservação de leucócitos totais de equinos é possível e, embora tenha havido diferença entre os tempos de congelamento, mesmo na amostra menos viável, houve recuperação de uma quantidade de células suficientes para outros ensaios imunológicos.(AU)

Protocol for collection and separation of bone marrow mononuclear cells in Chlorocebus aethiops / Protocolo para colheita e isolamento de células mononucleares de medula óssea em Chlorocebus aethiops

Chlorocebus aethiops is a species of non-human primate frequently used in biomedical research. Some research involves this species as an experimental model for various diseases and possible treatment with stem cells. The bone marrow is one of the main sources of these cells and provides easy access. The aim of this study was to standardize the protocol of collection and separation of bone marrow in C. aethiops. Ten animals were submitted to puncture of bone marrow with access to the iliac crest and cell separation by density gradient. The bone marrow of C. aethiops had an average of 97% viability. From the results achieved, we can conclude that C. aethiops is an excellent model to obtain and isolate mononuclear cells from bone marrow, fostering several studies in the field of cell therapy.(AU)

Chlorocebus aethiops é uma espécie de primata não humano frequentemente utilizados em pesquisa biomédica. Algumas pesquisas envolve esta espécie como modelo experimental para várias doenças e possível tratamento com células-tronco. A medula óssea é uma das principais fontes destas células e proporciona fácil acesso. O objetivo deste estudo foi o de padronizar o protocolo de coleta e separação de medula óssea em C. aethiops. Dez animais foram submetidos a punção de medula óssea com acesso à crista ilíaca e separação de células por gradiente de densidade. A medula óssea de C. aethiops tinha uma média de 97% de viabilidade. A partir dos resultados obtidos, podemos concluir que C. aethiops é um excelente modelo para obter e isolar células mononucleares da medula óssea, promovendo vários estudos no campo da terapia celular.(AU)

Perfil da expressão de ISG15 no sangue periférico durante o período de reconhecimento materno da gestação em bovinos

O período de reconhecimento materno da gestação (RMG) em ruminantes é um evento complexo, envolvendo diversos mecanismos celulares. O período de RMG é caracterizado pela secreção de interferon tau (IFNT) pelas células do trofloblasto do embrião, com maior intensidade aos 18 dias de gestação. A principal função dessa proteína é manter o corpo lúteo em plena atividade, evitando a luteólise. A ação direta do IFNT em tecidos extrauterinos eleva a expressão de genes estimulados por interferon (ISGs destacando-se o gene estimulado por interferon 15 (ISG15). As células polimorfonucleares do sangue periférico (PMN) são mais sensíveis ao estimulo do IFNT comparadas às células mononucleares do sangue periférico (PBMC). Os objetivos deste estudo são: (1) determinar o tempo mais precoce possível para identificar a expressão de ISG15 em novilhas de corte gestantes; (2) estabelecer a fração celular sanguínea para determinar o diagnóstico precoce de gestação com alta acurácia em novilhas de corte; e, (3) demonstrar o perfil da expressão de ISG15 nos dias 16 e 18 após a inseminação artificial (IA). A sincronização do ciclo estral foi realizada em 28 novilhas. O diagnóstico de gestação foi realizado nos dias 29 e 60 após IA e resultou em 10 novilhas gestantes, 14 novilhas não gestantes, e 4 novilhas não inseminadas (grupo controle negativo). O sangue foi coletado da veia coccígea após a IA (dias 16 e 18). As frações celulares de PBMC e PMN foram coletadas e armazenadas a -80ºC até a posterior manipulação de RNA, cDNA e qPCR. A expressão relativa de ISG15 diferiu entre a fração celular de PMN e PBMC de novilhas gestantes (p < 0,0001). A expressão do ISG15em células PMN no dia 16 diferiu de novilhas gestantes e novilhas não inseminadas (p < 0,04), bem como no dia 18 após IA (p < 0,01). No entanto, a expressão do ISG15 em células PMN no dia 16 não diferiu entre novilhas gestantes e novilhas não gestantes. Em PBMC, a expressão do ISG15 no dia 16 não diferiu entre as novilhas gestantes, não gestantes e não inseminadas. No entanto, no dia 18 após AI, há uma diferença na expressão de ISG15 entre novilhas gestantes e não gestantes (p < 0,02). A expressão de ISG15 nas células PMN aos 16 e 18 de gestação é ao menos 30 vezes maior do que a expressão em PBMC. Além disso, a expressão de ISG15 em PMN pode ser usada para identificação da sinalização embrionária aos 16 e 18 dias após a inseminação em novilhas de corte. A expressão de ISG15 em PMN no dia 18 após a IA resultou em 93% de acurácia para o diagnóstico de gestação. Sugerimos que ambas as frações celulares (PMN e PBMC) podem ser utilizadas como ferramentas para identificar a sinalização embrionária aos 18 dias de gestação em novilhas de corte.

The maternal recognition of pregnancy (MRP) in ruminants is a complex event, involving several cellular mechanisms. It is at that moment that the highest embryonic mortality occurs in cattle. The MRP period is characterized by the secretion of interferon tau (IFNT) by the embryo. IFNT is produced and secreted by the trofloblast cells of the embryo, with greater intensity at 18 days of pregnancy. The main function of this protein is to keep the corpus luteum fully active, avoiding luteolysis. Direct action of IFNT on extrauterine tissues raises the expression of interferon-stimulated genes (ISGs). Among the ISGs stimulated by IFNT, the gene stimulated by interferon 15 (ISG15) stands out. Studies have demonstrated endocrine action of IFNT. An elevation in ISG expression levels in blood and luteal cells was observed shortly after signaling by IFNT at the start of gestation in ruminants. Peripheral blood polymorphonuclear cells (PMN) are more sensitive to the IFNT stimulus compared to peripheral blood mononuclear cells (PBMCs). The objectives of this study are: (1) to determine the earliest possible time to identify ISG15 expression in pregnant heifers; (2) establish the cell fraction to determine the early diagnosis of pregnancy with high accuracy in cut heifers; (3) demonstrate the expression profile of ISG15 on days 16 and 18 after artificial insemination (AI). Synchronization of the estrous cycle was performed in 28 heifers. The pregnancy diagnosis was performed on days 29 and 60 after AI and resulted in 10 pregnant heifers, 14 non-pregnant heifers, and 4 non-inseminated heifers (negative control group). Blood was collected from the coccygeal vein after AI (Days 16 and 18). Cellular fractions of PBMC and PMN were collected and stored at -80 ° C until further extraction of RNA, cDNA and qPCR, respectively. The relative expression of ISG15 differed between the PMN and PBMC cell fraction of pregnant heifers (p <0.0001). ISG15 expression in PMN cells on Day 16 differed from heifers and non-inseminated heifers (p <0.04), as well as on Day 18 after AI (p <0.01). However, ISG15 expression in PMN cells on Day 16 did not differ from pregnant heifers and non-pregnant heifers. In PBMC cells, ISG15 expression on Day 16 did not differ from pregnant, non-pregnant and non-inseminated heifers. However, on Day 18 after AI, there is a difference in ISG15 expression of pregnant and non-pregnant heifers (p <0.02). Expression of ISG15 in PMN cells on Days 16 and 18 pregnancy is greater 30 times than expression in PBMC. In addition, the expression of ISG15 in PMN can be used to identify embryonic signaling on Day 16 after insemination in beef heifers. Expression of ISG15 in PMN on Day 18 after AI resulted in 93% accuracy for the diagnosis of pregnancy. We suggest that both cellular fractions (PMN and PBMC) can be used as a tool to identify embryonic signaling on Day 18 of pregnancy in beef heifers.

Fagocitose intensificada de Corynebacterium pseudotuberculosis por células da série monócito-macrófago de caprinos naturalmente infectados pelo vírus da artrite encefalite / Enhanced phagocytosis of Corynebacterium pseudotuberculosis by monocyte-macrophage cells from goats naturally infected with caprine arthritis encephalitis vírus

A Artrite Encefalite Caprina (AEC) e a Linfadenite Caseosa (LC) possuem alta incidência e transmissibilidade em pequenos ruminantes. Como ambas possuem tropismo por monócitos-macrófagos e afetam mecanismos da resposta inata do hospedeiro, acredita-se que a AEC predispõe o animal a infecções por Corynebacteruim pseudotuberculosis, agente etiológico da LC. Para confirmar esta hipótese, avaliou-se a fagocitose de células da série monócito-macrófago de cabras naturalmente infectadas pelo vírus da AEC (VAEC). Para tanto, foram utilizadas 30 cabras da raça Saanen, alocadas em dois grupos distintos, com 15 animais cada, conforme a sororreatividade de anticorpos séricos antivírus da AEC. Células mononucleares de sangue periférico foram isoladas por gradiente de densidade e plaqueadas para isolamento de células da série monócito-macrófago. Posteriormente, o ensaio de fagocitose de C. pseudotuberculosis foi realizado, após incubação por duas horas a 37ºC a 5% de CO2, e a visualização da fagocitose foi identificada por microscopia óptica. O presente estudo não encontrou diferença na porcentagem de monócito-macrófagos que realizaram fagocitose entre os diferentes grupos (P = 0,41). Todavia, a análise quantitativa de bactérias fagocitadas, demonstrou maior capacidade fagocítica pelos macrófagos-monócitos do grupo sororreagente ao vírus da AEC. Correlação entre monócitos fagocitando e macrófagos que fagocitaram mais de 12 bactérias foi observado neste grupo (r = 0,488; P = 0,006), não sendo o mesmo encontrado no grupo de animais sorroreagentes negativos. Os dados demonstram aumento na intensidade da fagocitose de macrófagos de animais infectados com o vírus da AEC.(AU)

Caprine arthritis encephalitis (CAE) and caseous lymphadenitis (CL) have high incidence and transmissibility in small ruminants. Since both virus have tropism for macrophages and monocytes and affect the innate immune response, it is believed that CAE can predispose the animal to infection by Corynebacteruim pseudotuberculosis, the etiological agent of CL. To confirm this hypothesis, we evaluated phagocytosis from the monocyte-macrophage cells from 30 Saanen goats. Goats were uniformly divided in two groups according to results of agar gel immunodiffusion test for CAE virus (CAEV). Peripheral blood mononuclear cells were isolated by density gradient centrifugation and the monocyte-macrophage cells were isolated from the mononuclear cells by their adhesion properties in plaques. Afterwards, phagocytosis of C. psudotuberculosis was performed for two hours at 37ºC, 5% of CO2, and assessed by microscopic visualization. There was no difference in the percentage of monocyte-macrophage cells that phagocytozed C. bovis between groups (P=0.41). However, when phagocytosis rates were classified according to the number of C. pseudotuberculosis phagocyted, the percentage of monocyte-macrophage cells that internalized more than 12 bacteria were higher in serologically CAEV positive animals compared to the serologically negative ones (P<0.001). Furthermore, a positive and significant correlation (r = 0.488; P = 0.006) between the percentage of monocyte-macrophage cells that internalized more than 12 bacteria and the percentage of monocyte that were carrying out phagocytosis was also encountered in serologically CAEV positive goats, however the same were not observed in serologically negative ones. These results demonstrated an alteration in the intensity of C. pseudotuberculosis phagocytosis by monocytes-macrophages from goats infected by CAEV. Thus, these results indicated that goats infected with CAEV may be more susceptible to CL.(AU)

Identification and biological activity of potential probiotic bacterium isolated from the stomach mucus of breast-fed lamb

The lactic acid bacterium E isolated from the stomach mucus of breast-fed lamb was identified by sequencing of 16S rDNA fragment and species-specific PCR as Lactobacillus reuteri. Its potential antimicrobial activity and ability to modulate immune system in vitro and in vivo was determined. The growth inhibition of potential pathogens decreased from Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica ser. Minnesota to Escherichia coli. The lowest inhibition activity was observed in the case of Candida albicans. The ability of L. reuteri E to modulate biological activities of human and mouse mononuclear cells was estimated in vitro and in vivo, respectively. The production of IL-1 by monocytes in vitro was significantly induced by L. reuteri E (relative activity 2.47). The ability to modulate biological activities of mononuclear cells by living L. reuteri E cells in vitro in comparison to disintegrated L. reuteri E cells in vivo differed. For example lysozyme activity in vitro was inhibited while in vivo was stimulated (relative activities 0.30 and 1.83, respectively). The peroxidase activity in vitro was stimulated while in vivo was inhibited (relative activities 1.53 and 0.17, respectively). Obtained results indicate that L. reuteri E is potential candidate to be used in probiotic preparations for animals and/or human.

Identification and biological activity of potential probiotic bacterium isolated from the stomach mucus of breast-fed lamb

The lactic acid bacterium E isolated from the stomach mucus of breast-fed lamb was identified by sequencing of 16S rDNA fragment and species-specific PCR as Lactobacillus reuteri. Its potential antimicrobial activity and ability to modulate immune system in vitro and in vivo was determined. The growth inhibition of potential pathogens decreased from Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica ser. Minnesota to Escherichia coli. The lowest inhibition activity was observed in the case of Candida albicans. The ability of L. reuteri E to modulate biological activities of human and mouse mononuclear cells was estimated in vitro and in vivo, respectively. The production of IL-1 by monocytes in vitro was significantly induced by L. reuteri E (relative activity 2.47). The ability to modulate biological activities of mononuclear cells by living L. reuteri E cells in vitro in comparison to disintegrated L. reuteri E cells in vivo differed. For example lysozyme activity in vitro was inhibited while in vivo was stimulated (relative activities 0.30 and 1.83, respectively). The peroxidase activity in vitro was stimulated while in vivo was inhibited (relative activities 1.53 and 0.17, respectively). Obtained results indicate that L. reuteri E is potential candidate to be used in probiotic preparations for animals and/or human.

UTILIZAÇÃO DE CÉLULAS MONONUCLEARES DE MEDULA ÓSSEA PARA O TRATAMENTO DE SEQUELAS NEUROLÓGICAS DE CINOMOSE CANINA CURITIBA

A cinomose é uma doença infecciosa causada por um Morbillivirus pertencente a família Paramyxoviridae. Até 30% dos cães infectados com o vírus da cinomose apresentam complicações neurológicas e aproximadamente 10% morrem por leucoencefalite aguda. Os cães que recuperam podem apresentar sequelas permanentes. Até o momento, não existe nenhum tratamento específico para animais com sinais neurológicos da cinomose, assim, são necessárias novas intervenções terapêuticas. O transplante de células-tronco tem emergido como um novo e promissor modelo de terapia. Células mononucleares derivadas da medula óssea (CMNMO) podem ser isoladas e aplicadas como estratégia terapêutica em estudos clínicos e pré-clínicos. Porém, existem alguns desafios associados a essa abordagem terapêutica. Um deles é a falta de estudos sobre a caracterização das CMNMO dos cães. Heparina e citrato-fosfato-dextrose-adenina-1 (CPDA-1) são comumente utilizados para coletar medula óssea. Porém, o efeito destes anticoagulantes em termos de isolamento dessas células também não é conhecido. Outro ponto que precisa ser mais estudado é a via de administração para ação eficiente das células transplantadas. A via intravenosa (IV) tem sido utilizada porque é o método mais fácil e menos invasivo de transplante de células. A marc

Canine distemper is an infectious disease caused by a Morbillivirus belonging to Paramyxoviridae family. Up to 30% of dogs infected with canine distemper virus showed neurological complications and approximately 10% die from acute leukoencephalitis. In addition, dogs that recover may have permanent sequels. There is no specific therapy for animals showing distemper neurological signs, and new therapeutic interventions are necessary, and cell transplantation has emerged as a promising new therapy model. Bone marrow mononuclear cells (BMMNC) can be easily isolated and broadly applied as a therapeutic strategy in a large number of preclinical and clinical studies. However, there are some challenges associated with this therapeutic approach. One of them is the lack of studies on the characterization of canine BMMNC. Heparin and citrate-phosphate-dextrose-adenine-1 (CPDA-1) are commonly used to harvest bone marrow. However, the comparison of effect of anticoagulants at harvest on terms of stem cell yield has not been studied. Another point that needs to be further studied is the route of administration for efficient cell delivery. Intravenous (IV) delivery has been used because it is easier and less invasive method of cell transplantation. The labelling and tracking of cells allow to evaluate if IV transplanted cells are attracted to the site of injury. The PKH26 is a fluorescent labeling molecule, noncytotoxic and is stable for long time period. Because of these characteristics, this dye seems to be ideal for cells tracking. The characterization of subsets of BMMNC and a better understanding of the functions of these cells may contribute to the knowledge of their potential and thus increase the possibilities to their use for clinical trials in veterinary medicine. The BMMMC administered intravenously have a lung passage 30 times larger than the mesenchymal stem cells (MSC), facilitating the migration of these cells to the injured tissues. However, the migration of these cells to the central nervous system (CNS), after intravenous administration, in animals with canine distemper sequels has not been studied. The PKH is a fluorescent labelling molecule, which is incorporated in the lipid bilayer of the cytoplasmic membrane, which has been used for the identification of cell migration. After incorporated into the cells in vitro, the PKH is not transferred to the medium or to unmarked cells, and has no toxic or immunogenic effect. BMMNC can be labeled with PKH and used for transplant intravenously, so this dye appears to be ideal for monitoring the migration of these cells. The aims of this study were the comparison of the yield of bone marrow-derived mononuclear cells harvested from dogs with two different anticoagulants, evaluate cell migration after allogeneic BMMNC transplantation in 10 animals with neurological complications of canine distemper, characterize phenotypically canine BMMNC and, evaluate the safety and efficacy of allogeneic BMMNC transplantation for treatment of neurological sequels of canine distemper in others 23 dogs. For comparison of the anticoagulants, the bone marrow from five dogs was harvest in heparin or CPDA-1, isolated in a density gradient, and stained for CD9 and CD44 for characterization by flow cytometry. The means were compared using Students paired t-test. Samples harvested with CPDA-1 yielded an average of 5.16 x 106 (±1.76 x 106) to 20.20 x 106 (±1.55 x 106) mononuclear cells/mL, whereas the yield of samples harvested with heparin varied between 4.56 x 106 (±0.69 x 106) and 24.30 x 106 (±2.12 x 106) mononuclear cells/mL. By flow cytometry, the mean percentage of double-stained cells varied from 1.96% (±0.64%) to 5.01 % (±0.73%) for CPDA-1 and from 2.23% (±0.70%) to 7.27 % (±0.97%) for heparin. No significant statistical differences were observed on yield or CD9 and CD44 expression. Bone marrow was harvested from eight donors and BMMNC were isolated, labeled with PKH26 dye and IV transplanted into the patients, to assess the labelling and tracking of cells. The cell migration was assessed in the cerebrospinal fluid (CSF) of patients at different periods of time and the percentage of labeled cells with PKH 26 dye was evaluated quantitatively by flow cytometry and qualitatively by fluorescence microscope. In the quantitative analysis by flow cytometer, it was observed a wide variation in the percentage of labeled cells in different CSF collection times. Also, there was an increase on the percentage of PKH26-labeled cells in the CSF, with highest percentage between 4 and 5 ½ hours after infusion with subsequent decrease. In the qualitative analysis, it was showed the presence of labeled BMMNC recruited by central nervous system on CSF. In order to evaluate the safety and efficacy of allogeneic BMMNC transplantation for treatment of neurological sequels of canine distemper, it was used a single blind randomized controlled trial in 46 dogs divided into treatment group and control group with weekly follow-up for 35 days. Bone marrow was harvested from 23 healthy donors and BMMNC were isolated by density gradient centrifugation. BMMNC from four donors were labeled with anti-CD8a, CD9, CD14, CD29, CD34, CD44, CD45, and CD90 for phenotypic characterization. Dogs from the treatment group received 1 x 108 BMMNC intravenously. Regarding the safety of cell transplantation, no serious adverse events related to the procedure were recorded during the 35 days of the study. Functional recovery was evaluated according to the Olby scoring system with some modifications. For the treatment group, the median and interquartile range of the functional score, with 0, 7, 14, 21, 28 and 35 days after injection were 7 (4-13), 9 (5-14), 12 (6-15), 14 (6-15), 14 (6-16), and 14 (6-16) respectively, whereas for the placebo group they were 6 (4-12), 7 (4- 13), 7 (4-12), 7 (4-12), 6 (3-12), and 6 (3-12). The differences observed were statistically significant (p < 0.05), showing the effectiveness of BMMNC transplantation in dogs with distemper leukoencephalitis. In conclusion, this study demonstrates that BMMNC can be efficiently labeled with PKH26 dye and these cells can be monitored after IV transplantation in animals with neurological complications of canine distemper.

Colheita de medula óssea em cães: modelo para obtenção da fração total de células mononucleares / Bone marrow harvest in dogs: model for acquisition of the total fraction of mononuclears cells

No presente trabalho foi elaborada uma técnica para protocolo de colheita de medula óssea (MO) (10ml. kg-1), do osso femoral, para isolamento, quantificação e viabilidade da fração total de células mononucleares (CM). Para tanto, 40 cães machos ou fêmeas, sem raça definida, com idade aproximada de dois anos, pesando em torno de 10kg, foram submetidos a procedimento asséptico em ambiente cirúrgico para colheita de MO. Para a obtenção de uma quantidade suficiente de CM, durante o procedimento foi utilizada a agulha tipo Steis anatômica, que favoreceu a colheita de volume sangüíneo em menor espaço de tempo e não danificou a viabilidade celular. Também foi utilizado o Kit Bone Marrow collection, que teve a finalidade de filtrar as espículas ósseas, mantendo a integridade das CM colhidas durante o período decorrido para o acondiconamento do sangue. Durante o período da colheita de MO, os animais foram submetidos à collheita de sangue periférico (pré, trans e pós-operatório) para avaliações hematológicas e sofreram autotransfusão sangüínea para suprir a queda acentuada de hemoglobina ocorrida nos primeiros momentos da coletaheita. O total de MO colhida e filtrada foi colocado lentamente sob gradiente de densidade Histopaque (1.077g ml-1). O material foi centrifugado a 440 x g por 30 minutos e o anel de células foi colhido, lavado e centrifugado três vezes em meio contendo solução salina 0,9 por cento, DMEM e soro sangüíneo autólogo estéril. Foi realizada a contagem do anel celular em câmara de Neubauer e foi verificada sua viabilidade utilizando corante vital. Neste estudo foi verificado que no volume de MO colhido foi possível obter a média de 2,57 x 10(6) (± 1,56) CM kg-1 e a viabilidade celular foi superior a 90 por cento (96,72 ± 2,9 por cento). Conclui-se que a técnica de colheita de MO com agulha Steis com lavagem celular no meio contendo soro autólogo e Kit Bone Marrow e agulha Steis com lavagem celular no meio contendo...(AU)

In the present research a new protocol to harvest 10ml kg-1 femoral bone marrow (BM) was developed to allow isolation, quantification and to test the mononuclear cell (MC) fraction viability. Forty male or female stray dogs, aging and weighting around two years old and 10kg respectively, were submitted to aseptic bone marrow harvest in a surgical environment. To achieve an ideal cell count of MC, an anatomical Steis needle was used during the procedure, which favored the indicated volume harvest in a shorter period of time without interfering cellular viability. A bone marrow collection kit was also used to filter bone fragments while maintaining harvested MC integrity during blood packaging. Meanwhile BM harvesting was conducted, animals peripheral blood collection was performed (pre, trans and post-operatory) to hematological evaluations and autologous blood transfusion was made to overcome the increased hemoglobin fall that takes place in the initial harvesting moments. The harvested and filtered BM was slowly placed over a Histopaque density gradient (1.077g ml-1). The material was centrifuged at 440g x for 30 minutes. The cellular ring was harvested, washed and three times centrifuged in saline 0.9 percent, DMEM and autologous sterile serum. Cellular ring count was conducted in neubauer chamber and its viability was performed with vital dye. In this study was possible to notice that with the harvested BM volume an average of 2.57 x 10(6) (± 1.56) MC kg-1 was obtained and the cell viability was over 90 percent (96.72 ± 2.9 percent). It was concluded that the bone marrow kit and Steis needle with autologous serum cellular wash BM harvesting technique allow an ideal MC number isolation which can be administered in tissue lesions to enhance the regeneration process.(AU)