Isolamento, expansão e caracterização de células-tronco do tecido adiposo de equinos / Isolation, expansion, and characterization of equine adipose-derived stem cells

Devido aos excelentes resultados obtidos em pesquisas com células-tronco e suas aplicabilidades promissoras, a terapia com estas células encontra-se em momento de máxima ascensão, estimulando assim, inúmeras pesquisas. A fim de padronizar o cultivo e avaliar a viabilidade de células-tronco mesenquimais do tecido adiposo de equinos visando a terapia celular nesta espécie, conduziu-se um estudo de isolamento, cultivo e caracterização de tais células. Para tal, foi realizada coleta de tecido adiposo de cinco equinos sem raça definida, com aproximadamente cinco anos. Em seguida, o tecido foi refrigerado e transportado ao laboratório, onde foi realizado o protocolo de cultivo celular durante aproximadamente 14 dias. Após o término deste procedimento, uma alíquota de células foi encaminhada para a realização do teste de quantificação e viabilidade celular com Azul de Tripan 1%, sendo outra porção destinada à confirmação da linhagem de célula tronco mesenquimal por meio de imunofenotipagem com os anticorpos monoclonais anti-CD11b, anti-CD45, anti-CD90 e anti-CD105, e de diferenciação celular em linhagem condrogênica, adipogênica e osteogênica. Houve diferenciação celular e marcação positiva dos antígenos de superfície CD44 e MHCI e negativa dos CD45 e CD11b, confirmando-se a origem mesenquimal e não hematopoiética. A viabilidade celular média obtida das células-tronco mesenquimais foi de 92,83% considerada satisfatória. Portanto, a utilização deste protocolo para obtenção de células-tronco mesenquimais do tecido adiposo se mostrou uma opção confiável e viável para utilização na terapia celular em equinos.(AU)

Due to the excellent results of stem cell research and its promising applicability, the therapy with these cells is undergoing a period of maximum visibility and numerous studies. In order to standardize the culture and to evaluate the viability of mesenchymal stem cells from adipose tissue of horses, aiming at cell therapy in this species, a study of isolation, expansion and characterization was conducted. For this, adipose tissue was collected from five crossbred horses aged approximately five years old. Subsequently, the tissue was immediately refrigerated and transported to the laboratory where the isolation and culture protocol, lasting approximately 14 days, was performed. After the end of this procedure, a cell pellet was obtained and resuspended in PBS. A portion of material obtained by centrifugation was collected and the cells were stained with 1% Trypan Blue for quantification and assessment of cell viability. Another sample was collected to confirm the lineage of stem cells by flow cytometry using monoclonal against the surface markers CD11b, CD45, CD90 and CD105; and by cellular differentiation assays to chondrogenic, adipogenic and osteogenic lineages. The isolated cells differentiated in the three lineages and showed positive staining for CD90 and CD105 and negative staining for CD11b and CD45, confirming the isolation of stem cells of mesenchymal origin. The average cell viability obtained was 92.83% and thus satisfactory. Therefore, the use of this protocol to obtain mesenchymal stem cells from adipose tissue was deemed to be a promising option for cell therapy in horses.(AU)

Isolamento, expansão e caracterização de células-tronco do tecido adiposo de equinos / Isolation, expansion, and characterization of equine adipose-derived stem cells

Devido aos excelentes resultados obtidos em pesquisas com células-tronco e suas aplicabilidades promissoras, a terapia com estas células encontra-se em momento de máxima ascensão, estimulando assim, inúmeras pesquisas. A fim de padronizar o cultivo e avaliar a viabilidade de células-tronco mesenquimais do tecido adiposo de equinos visando a terapia celular nesta espécie, conduziu-se um estudo de isolamento, cultivo e caracterização de tais células. Para tal, foi realizada coleta de tecido adiposo de cinco equinos sem raça definida, com aproximadamente cinco anos. Em seguida, o tecido foi refrigerado e transportado ao laboratório, onde foi realizado o protocolo de cultivo celular durante aproximadamente 14 dias. Após o término deste procedimento, uma alíquota de células foi encaminhada para a realização do teste de quantificação e viabilidade celular com Azul de Tripan 1%, sendo outra porção destinada à confirmação da linhagem de célula tronco mesenquimal por meio de imunofenotipagem com os anticorpos monoclonais anti-CD11b, anti-CD45, anti-CD90 e anti-CD105, e de diferenciação celular em linhagem condrogênica, adipogênica e osteogênica. Houve diferenciação celular e marcação positiva dos antígenos de superfície CD44 e MHCI e negativa dos CD45 e CD11b, confirmando-se a origem mesenquimal e não hematopoiética. A viabilidade celular média obtida das células-tronco mesenquimais foi de 92,83% considerada satisfatória. Portanto, a utilização deste protocolo para obtenção de células-tronco mesenquimais do tecido adiposo se mostrou uma opção confiável e viável para utilização na terapia celular em equinos.

Due to the excellent results of stem cell research and its promising applicability, the therapy with these cells is undergoing a period of maximum visibility and numerous studies. In order to standardize the culture and to evaluate the viability of mesenchymal stem cells from adipose tissue of horses, aiming at cell therapy in this species, a study of isolation, expansion and characterization was conducted. For this, adipose tissue was collected from five crossbred horses aged approximately five years old. Subsequently, the tissue was immediately refrigerated and transported to the laboratory where the isolation and culture protocol, lasting approximately 14 days, was performed. After the end of this procedure, a cell pellet was obtained and resuspended in PBS. A portion of material obtained by centrifugation was collected and the cells were stained with 1% Trypan Blue for quantification and assessment of cell viability. Another sample was collected to confirm the lineage of stem cells by flow cytometry using monoclonal against the surface markers CD11b, CD45, CD90 and CD105; and by cellular differentiation assays to chondrogenic, adipogenic and osteogenic lineages. The isolated cells differentiated in the three lineages and showed positive staining for CD90 and CD105 and negative staining for CD11b and CD45, confirming the isolation of stem cells of mesenchymal origin. The average cell viability obtained was 92.83% and thus satisfactory. Therefore, the use of this protocol to obtain mesenchymal stem cells from adipose tissue was deemed to be a promising option for cell therapy in horses.

ISOLATION, EXPANSION AND CHARACTERIZATION OF EQUINE ADIPOSE TISSUE DERIVED STEM CELLS / Isolamento, expansão e caracterização de células-tronco do tecido adiposo de equinos

Devido aos excelentes resultados obtidos em pesquisas com células-tronco e suas aplicabilidades promissoras, a terapia com estas células encontra-se em momento de máxima ascensão, estimulando assim, inúmeras pesquisas. A fim de padronizar o cultivo e avaliar a viabilidade de células-tronco mesenquimais do tecido adiposo de equinos visando a terapia celular nesta espécie, conduziu-se um estudo de isolamento, cultivo e caracterização de tais células. Para tal, foi realizada coleta de tecido adiposo de cinco equinos sem raça definida, com aproximadamente cinco anos. Em seguida, o tecido foi refrigerado e transportado ao laboratório, onde foi realizado o protocolo de cultivo celular durante aproximadamente 14 dias. Após o término deste procedimento,  uma alíquota de células foi encaminhada para a realização do teste de quantificação e viabilidade celular com Azul de Tripan 1%,  sendo outra porção  destinada à confirmação da linhagem de célula tronco mesenquimal  por meio de imunofenotipagem com os anticorpos monoclonais anti-CD11b, anti-CD45, anti-CD90 e anti-CD105,

ISOLATION, EXPANSION AND CHARACTERIZATION OF EQUINE ADIPOSE TISSUE DERIVED STEM CELLS / Isolamento, expansão e caracterização de células-tronco do tecido adiposo de equinos.

Devido aos excelentes resultados obtidos em pesquisas com células-tronco e suas aplicabilidades promissoras, a terapia com estas células encontra-se em momento de máxima ascensão, estimulando assim, inúmeras pesquisas. A fim de padronizar o cultivo e avaliar a viabilidade de células-tronco mesenquimais do tecido adiposo de equinos visando a terapia celular nesta espécie, conduziu-se um estudo de isolamento, cultivo e caracterização de tais células. Para tal, foi realizada coleta de tecido adiposo de cinco equinos sem raça definida, com aproximadamente cinco anos. Em seguida, o tecido foi refrigerado e transportado ao laboratório, onde foi realizado o protocolo de cultivo celular durante aproximadamente 14 dias. Após o término deste procedimento,  uma alíquota de células foi encaminhada para a realização do teste de quantificação e viabilidade celular com Azul de Tripan 1%,  sendo outra porção  destinada à confirmação da linhagem de célula tronco mesenquimal  por meio de imunofenotipagem com os anticorpos monoclonais anti-CD11b, anti-CD45, anti-CD90 e anti-CD105,

Parâmetros eletrocardiográficos de equinos desclassificados por exaustão em competições de enduro / Electrocardiographic parameters of horses disqualified from exhaustion in endurance competitions

Foram examinados, por meio de eletrocardiografia, 14 equinos Puro Sangue Árabe, 12 machos e duas fêmeas, desclassificados por exaustão em provas de enduro entre 60 e 160km. Foi observado predomínio de taquicardia sinusal, seguido de arritmia sinusal e complexo atrial prematuro com a frequência cardíaca variando de 48 a 78bpm e 93 a 111bpm, respectivamente. Ocorreu aumento da amplitude das ondas P, R, S e T e redução nas suas durações, bem como redução nos intervalos e segmentos, porém o complexo QRS quase não se alterou. O alongamento observado do QTc indicou fadiga miocárdica moderada em resposta ao exercício, e o supradesnível ST foi indicativo de hipovolemia. O eixo elétrico no plano frontal apresentou desvio à direita, aumento de câmara e hipertrofia secundários ao treinamento.

By using electrocardiographic examination, 14 Purebred Arabian horses, 12 males and two females, disqualified due to exhaustion in endurance races from 60 and 160km were evaluated. Predominance of sinus tachycardia, followed by sinus arrhythmia and atrial premature complex, with the heart rate ranging from 48 to 78bpm, and 93 to 111bpm, respectively, were observed. There was increase in the amplitude of waves P, R, S, and T, and decrease in their durations, as well as reduction in the intervals and segments; nevertheless, the QRS complex was not almost altered. The QTc lengthening pointed to a moderate myocardial fatigue in response to exercise, and the elevation of ST was indicative of hypovolemia. The electrical axis in frontal plane presented deviation to the right, chamber increase, and hypertrophy secondary to training.

Parâmetros eletrocardiográficos de equinos desclassificados por exaustão em competições de enduro / Electrocardiographic parameters of horses disqualified from exhaustion in endurance competitions

Foram examinados, por meio de eletrocardiografia, 14 equinos Puro Sangue Árabe, 12 machos e duas fêmeas, desclassificados por exaustão em provas de enduro entre 60 e 160km. Foi observado predomínio de taquicardia sinusal, seguido de arritmia sinusal e complexo atrial prematuro com a frequência cardíaca variando de 48 a 78bpm e 93 a 111bpm, respectivamente. Ocorreu aumento da amplitude das ondas P, R, S e T e redução nas suas durações, bem como redução nos intervalos e segmentos, porém o complexo QRS quase não se alterou. O alongamento observado do QTc indicou fadiga miocárdica moderada em resposta ao exercício, e o supradesnível ST foi indicativo de hipovolemia. O eixo elétrico no plano frontal apresentou desvio à direita, aumento de câmara e hipertrofia secundários ao treinamento.(AU)

By using electrocardiographic examination, 14 Purebred Arabian horses, 12 males and two females, disqualified due to exhaustion in endurance races from 60 and 160km were evaluated. Predominance of sinus tachycardia, followed by sinus arrhythmia and atrial premature complex, with the heart rate ranging from 48 to 78bpm, and 93 to 111bpm, respectively, were observed. There was increase in the amplitude of waves P, R, S, and T, and decrease in their durations, as well as reduction in the intervals and segments; nevertheless, the QRS complex was not almost altered. The QTc lengthening pointed to a moderate myocardial fatigue in response to exercise, and the elevation of ST was indicative of hypovolemia. The electrical axis in frontal plane presented deviation to the right, chamber increase, and hypertrophy secondary to training.(AU)

Analysis of Model for End-Stage Liver Disease (MELD) score in a liver transplantation waiting list.

BACKGROUND: The Model for End-Stage Liver Disease (MELD) was introduced in 1999 to quantify the 3-month prognosis of cirrhotic patients after a transjugular intrahepatic portosystemic shunt (TIPS). Because of the imbalance between organ donors and patients on the waiting list, the MELD was adopted by the United States in 2002 to allocate liver grafts for transplantation. Preliminary results have indicated a reduction in waiting list deaths and an increase in transplantation rates for candidates. Seeking to find a new model to predict death on the waiting list and after liver transplantation, retrospective studies have examined MELD scores in waiting list patients. The aim of this study was to analyze the MELD scores of patients on the liver waiting list for comparisons between transplanted patients. PATIENTS AND METHODS: A retrospective study was performed analyzing 131 registrations of 127 orthotopic liver transplant (OLT) patients (4 underwent retransplantation) grafted between November 2000 and January 2006, excluding 24 patients: 2 had urgent retransplantations due to hepatic artery thrombosis and 22 had incomplete data. These patients were divided into 3 groups: group I (transplanted patients)-53 patients underwent 55 OLT; group II-29 patients who died on the waiting list; group III-patients on the waiting list including 23 patients still waiting as of the date of the study. RESULTS: The main indication for OLT was hepatitis C virus cirrhosis (50.50%), followed by alcoholic liver cirrhosis (23.30%), cryptogenic cirrhosis (12.60%), autoimmune hepatitis (5.80%), hepatitis B virus cirrhosis (4.85%), and primary biliary cirrhosis (2.91%). Group I: MELD score 15.62 (range, 6-39) on admission to the list, and 18.87 (range, 7-39) at transplantation. The mean waiting time for OLT was 478.39 days (range, 2-1270 days). The 38 patients who survived underwent 39 OLT (1 retransplantation). The MELD score at entrance to the list was 14.62 (range, 7-30) and at transplantation, 17.70 (range, 7-39). The mean time between admission to the list and transplantation was 505.37 days (range, 6-1270 days). The 15 patients who died had received 16 OLT (1 retransplantation). Their MELD scores were 17.80 (range, 6-39) and 21.81 (range, 9-39) at admission to the list and at transplantation, respectively, with a mean time on the waiting list of 417.93 days (range, 2-872 days). Group II: 29 patients died before OLT, at a mean age of 52.60 years (range, 22-67 years). Their MELD score was 19.24 (range, 7-45), and the interval between admission to the waiting list and death was 249.55 days (range, 3-1247 days). Group III: 23 patients still active on the OLT waiting list at the time of study displayed a mean MELD score of 13.65 (range, 6-28) and 354.30 days (range, 2-905 days) waiting until the moment. In conclusion, MELD score at the time of admission to the waiting list was higher among those patients who died either awaiting a liver graft (19.24) or after OLT (17.80) compared with those who survived after OLT (14.60) or are still awaiting OLT (13.65).

Thyroid peroxidase activity is inhibited by amino acids.

Normal in vitro thyroid peroxidase (TPO) iodide oxidation activity was completely inhibited by a hydrolyzed TPO preparation (0.15 mg/ml) or hydrolyzed bovine serum albumin (BSA, 0.2 mg/ml). A pancreatic hydrolysate of casein (trypticase peptone, 0.1 mg/ml) and some amino acids (cysteine, tryptophan and methionine, 50 microM each) also inhibited the TPO iodide oxidation reaction completely, whereas casamino acids (0.1 mg/ml), and tyrosine, phenylalanine and histidine (50 microM each) inhibited the TPO reaction by 54% or less. A pancreatic digest of gelatin (0.1 mg/ml) or any other amino acid (50 microM) tested did not significantly decrease TPO activity. The amino acids that impair iodide oxidation also inhibit the TPO albumin iodination activity. The inhibitory amino acids contain side chains with either sulfur atoms (cysteine and methionine) or aromatic rings (tyrosine, tryptophan, histidine and phenylalanine). Among the amino acids tested, only cysteine affected the TPO guaiacol oxidation reaction, producing a transient inhibition at 25 or 50 microM. The iodide oxidation inhibitory activity of cysteine, methionine and tryptophan was reversed by increasing iodide concentrations from 12 to 18 mM, while no such effect was observed when the cofactor (H2O2) concentration was increased. The inhibitory substances might interfere with the enzyme activity by competing with its normal substrates for their binding sites, binding to the free substrates or reducing their oxidized form.

Thyroid peroxidase activity is inhibited by amino acids

Normal in vitro thyroid peroxidase (TPO) iodide oxidation activity was completely inhibited by a hydrolyzed TPO preparation (0.15 mg/ml) or hydrolyzed bovine serum albumin (BSA, 0.2 mg/ml). A pancreatic hydrolysate of casein (trypticase peptone, 0.1 mg/ml) and some amino acids (cysteine, tryptophan and methionine, 50 µM each) also inhibited the TPO iodide oxidation reaction completely, whereas casamino acids (0.1 mg/ml), and tyrosine, phenylalanine and histidine (50 µM each) inhibited the TPO reaction by 54 per cent or less. A pancreatic digest of gelatin (0.1 mg/ml) or any other amino acid (50 µM) tested did not significantly decrease TPO activity. The amino acids that impair iodide oxidation also inhibit the TPO albumin iodination activity. The inhibitory amino acids contain side chains with either sulfur atoms (cysteine and methionine) or aromatic rings (tyrosine, tryptophan, histidine and phenylalanine). Among the amino acids tested, only cysteine affected the TPO guaiacol oxidation reaction, producing a transient inhibition at 25 or 50 µM. The iodide oxidation inhibitory activity of cysteine, methionine and tryptophan was reversed by increasing iodide concentrations from 12 to 18 mM, while no such effect was observed when the cofactor (H2O2) concentration was increased. The inhibitory substances might interfere with the enzyme activity by competing with its normal substrates for their binding sites, binding to the free substrates or reducing their oxidized form.

Hydrograph separation of the amazon river using 18o as an isotopic tracer

The 18O content of rain and river waters was used as an isotopic tracer in order to carry out the hydrograph separation of the Amazon river, during the 1973-1974 hydrological years, and to estimate the contributions of the surface runoff (event water) and baseflow (pre-event water) components to the total river flow. The average surface runoff and baseflow contributions were 30.3 and 69.7% respectively. At peak discharge, the mean contribution of the baseflow was about 57%. The results of the isotopic separation model were compared with the filter-separation autoregressive method, showing similar behavior and magnitude.

Os teores de 18O em águas de chuva e amostras de água de rio foram utilizados como traçadores num modelo isotópico de separação de hidrógrafas do rio Amazonas durante o período 1973-1974, com o intuito de se estimar as contribuições do escoamento superficial rápido e escoamento de base para o escoamento total da bacia de drenagem. As contribuições médias desses reservatórios foram de 30,3 e 69,7% respectivamente. Durante o período de pico de cheia, o fluxo de base contribuiu com cerca de 57% do volume total de água escoada. Os resultados do modelo de separação isotópico foram comparados com o método estatístico de filtros auto-recursivos, mostrando comportamentos similares.

HYDROGRAPH SEPARATION OF THE AMAZON RIVER USING 18O AS AN ISOTOPIC TRACER

The 18O content of rain and river waters was used as an isotopic tracer in order to carry out the hydrograph separation of the Amazon river, during the 1973-1974 hydrological years, and to estimate the contributions of the surface runoff (event water) and baseflow (pre-event water) components to the total river flow. The average surface runoff and baseflow contributions were 30.3 and 69.7% respectively. At peak discharge, the mean contribution of the baseflow was about 57%. The results of the isotopic separation model were compared with the filter-separation autoregressive method, showing similar behavior and magnitude.

Os teores de 18O em águas de chuva e amostras de água de rio foram utilizados como traçadores num modelo isotópico de separação de hidrógrafas do rio Amazonas durante o período 1973-1974, com o intuito de se estimar as contribuições do escoamento superficial rápido e escoamento de base para o escoamento total da bacia de drenagem. As contribuições médias desses reservatórios foram de 30,3 e 69,7% respectivamente. Durante o período de pico de cheia, o fluxo de base contribuiu com cerca de 57% do volume total de água escoada. Os resultados do modelo de separação isotópico foram comparados com o método estatístico de filtros auto-recursivos, mostrando comportamentos similares.