Regulation of the cellular redox state and the expression of DNA methyltransferase-1 in peripheral blood mononuclear cells from patients with Graves' disease.

BACKGROUND: Graves' disease is an autoimmune disorder characterised by excessive production of thyroid hormones, which induces increased cellular metabolism in most tissues and increased production of reactive oxygen species (ROS). The aim of this work was to analyse the effect of ROS on cell viability and the expression of catalase (CAT), glutathione peroxidase-1 (GPx-1), superoxide dismutase (SOD-1) and DNA methyltransferase-1 (DNMT-1) in peripheral blood mononuclear cells (PBMC) from patients with newly diagnosed Graves' disease or treated with methimazole. PATIENTS AND METHODS: For this study, women patients with newly diagnosed Graves' disease (n=18), treated with methimazole (n=6) and healthy subjects (n=15) were recruited. ROS were evaluated by flow cytometry, and the viability/apoptosis of PBMC was analysed by flow cytometry and fluorescence microscopy. Genomic expression of CAT, GPx-1, SOD-1 and DNMT-1 was quantified by real-time PCR. RESULTS: We found high levels of ROS and increased expression of CAT, GPx-1, SOD-1 and DNMT-1 in PBMC from patients with newly diagnosed Graves' disease. Methimazole treatment reversed these parameters. Cell viability was similar in all study groups. CONCLUSIONS: ROS induces the expression of CAT, GPx-1, and SOD-1. The activity of these enzymes may contribute to the protection of PBMC from the harmful effect of free radicals on cell viability. Increased expression of DNMT-1 may be associated with aberrant methylation patterns in immunoregulatory genes contributing to autoimmunity in Graves' disease.

Caracterización clínica epidemiológica de los pacientes con enfermedad de la Peyronie infiltrados con células mononucleares / Clinical Epidemiological Characterization of Peyronie's Disease Patients Infiltrated With Mononuclear Cells

Introducción: La enfermedad de la Peyronie resulta poco común e involucra a hombres de mediana edad. Objetivo: Caracterizar los aspectos clínicos y epidemiológicos de los pacientes con enfermedad de la Peyronie infiltrados con células mononucleares. Métodos: Se realizó un estudio observacional, analítico, longitudinal prospectivo en el Hospital General Docente "Comandante Pinares" desde junio de 2015 hasta mayo de 2018, con una muestra de 159 pacientes. Se controlaron las variables de edad, color de la piel, factores etiológicos, curvatura y desviación del pene además de síntomas y signos. Resultados: El mayor número de pacientes correspondieron a la edad de 50-59 años, de piel blanca, grados de curvatura entre 20o y 39o. Después del tratamiento 115 pacientes se encontraron con curvatura menor a 20o y desviación dorsal. Las causas más frecuentes de los microtraumas a nivel de pene resultan durante al acto sexual o en estado flácido del pene (105/159). La diabetes mellitus y la hipertensión arterial, con 59,7 y 30,8 por ciento, respectivamente resultan los factores etiológicos más frecuentes; los síntomas y signos fueron el dolor y la curvatura, 115 pacientes presentaron ausencia de dolor al hacer la comparación de la media al concluir el tratamiento, resultando significativo (p= 0,0000). Conclusiones: La enfermedad de la Peyronie resulta frecuente en pacientes de la quinta década de la vida, con color de piel blanca. La causa más frecuente son los microtraumas en la actividad sexual, la diabetes mellitus y la hipertensión arterial como antecedentes patológicos personales. Existe mejoría de la sintomatología en los pacientes infiltrados con células mononucleares(AU)

Introduction: Peyronie's disease is rare and involves middle-aged men. Objective: To characterize the clinical and epidemiological aspects of patients with Peyronie's disease infiltrated with mononuclear cells. Methods: An observational, analytical, prospective longitudinal study was carried out at the "Comandante Pinares" General Teaching Hospital from June 2015 to May 2018, with a sample of 159 patients. The variables of age, skin color, etiological factors, curvature and deviation of the penis, as well as symptoms and signs, were controlled. Results: The largest number of patients corresponded to the age of 50-59 years, white skin, degrees of curvature between 20o and 39o. After treatment, 115 patients were found to have curvature less than 20o and dorsal deviation. The most frequent cause of penile microtrauma is during sexual intercourse or in the flaccid state of the penis (105/159). Diabetes mellitus and arterial hypertension, with 59.7 and 30.8 percent, respectively, are the most frequent etiological factors; the symptoms and signs were pain and curvature, 115 patients presented absence of pain when comparing the mean at the end of the treatment, being significant (p= 0.0000). Conclusions: Peyronie's disease is frequent in patients of the fifth decade of life, with white skin color. The most frequent cause are microtraumas in sexual activity, diabetes mellitus and arterial hypertension as personal pathological antecedents. There is improvement of the symptoms in patients infiltrated with mononuclear cells(AU)

Terapia periodontal regenerativa con hemocomponentes en Santiago de Cuba desde lo social y formativo

RESUMEN Las periodontitis constituyen la primera causa de pérdida dentaria en la edad adulta y su tratamiento depende, casi por completo, del empleo de la implantación de sustitutos estructurales para lograr el potencial reparador necesario. El uso en los últimos años de la medicina celular regenerativa con células mononucleares, plaquetas y lisado plaquetario, han acelerado el proceso de cicatrización de los tejidos blandos y la regeneración ósea. Los resultados que se presentan derivan de una estrategia de superación profesional dirigida al periodoncista para el mejoramiento del desempeño en la aplicación de esta nueva terapéutica, como proyecto de investigación, en el Hospital Juan Bruno Zayas Alfonso de Santiago de Cuba, desde septiembre a diciembre del 2020. Su objetivo consiste en analizar la terapia periodontal regenerativa con hemocomponentes en Santiago de Cuba desde lo social y lo formativo.

ABSTRACT Periodontitis is the first cause of tooth loss in adulthood and its treatment depends, almost entirely, on the use of implantation of structural substitutes to achieve the necessary restorative potential. The use in recent years of regenerative cell medicine with mononuclear cells, platelets and platelet lysate, has accelerated the process of soft tissue healing and bone regeneration. The results presented are derived from a professional improvement strategy aimed at the periodontist to improve performance in the application of this new therapy, as a research project, at the Juan Bruno Zayas Alfonso Hospital in Santiago de Cuba, from September to December 2020. Its objective is to analyze regenerative periodontal therapy with blood components in Santiago de Cuba from a social and educational perspective.

Distancia de claudicación e índice tobillo-brazo en pacientes con insuficiencia arterial tratados con células madre / Claudication distance and ankle-arm index in patients with arterial insufficiency treated with stem cells

Introducción: La enfermedad arterial periférica de miembros inferiores constituye un problema de salud por ser responsable de más del 40 % de las amputaciones no traumáticas en diabéticos y no diabético. Las células madre pudieran contribuir positivamente a su tratamiento, al participar en el proceso angiogénico. Objetivo: Describir la distancia de claudicación y el índice de presiones tobillo-brazo en pacientes con insuficiencia arterial tratados con células madre. Métodos: Se realizó un estudio cuasi experimental, prospectivo y analítico en 36 pacientes con el diagnóstico de insuficiencia arterial, atendidos en el servicio de Angiología del Hospital Militar Central "Dr. Carlos J. Finlay". Los pacientes fueron tratados con células mononucleares autólogas obtenidas de sangre periférica, aplicadas por vía intramuscular. Las variables estudiadas resultaron: edad, sexo, factores de riesgo, patrón oclusivo, distancia de claudicación e índice de presiones tobillo-brazo. Resultados: Predominaron el sexo masculino (63,9 por ciento), el grupo de edades de 65 y más años (44,4 percent), y el color de piel blanco (61,1 percent)). Los factores de riesgo más frecuentes fueron el tabaquismo (72,2 percent)) y la hipertensión arterial (69,4 percent)). En el 77,7 percent) desapareció el dolor a la marcha (p < 0,01). La distancia de claudicación aumentó (p < 0) al concluir el sexto mes (397,2 ± 118,8 metros: IC 95 percent): 357; 437,4). Hubo un incremento altamente significativo (p < 0) del índice de presiones tobillo-brazo de la arteria pedia. Conclusiones: Las células mononucleares autólogas resultan eficaces en el tratamiento de la insuficiencia arterial, por eliminar el dolor ante el ejercicio, aumentar la distancia a la marcha y elevar los índices de presiones tobillo-brazo(AU)

ABSTRACT Introduction: Lower limb´s peripheral artery disease is a health problem as being the cause of more than 40% of non-traumatic amputations in diabetic and non-diabetic patients. Stem cells could contribute positively to their treatment by participating in the angiogenic process. Objective: Describe claudication distance and ankle-arm´s pressure index in patients with arterial insufficiency treated with stem cells. Methods: A quasi-experimental, prospective and analytical study was carried out in 36 patients diagnosed with arterial insufficiency, attended in the Angiology service of "Dr. Carlos J. Finlay" Central Military Hospital. Patients were treated with autologous mononuclear cells obtained from peripheral blood, and they were used intramuscularly. The studied variables were: age, sex, risk factors, occlusive pattern, claudication distance and ankle-arm´s pressure index. Results: Male sex (63.9%), age group of 65 years and older (44.4%), and white skin (61.1%) predominated. The most common risk factors were: smoking habit (72.2%), and high blood pressure (69.4%). In 77.7% of the cases, the pain while walking disappeared (p < 0.01). The claudication distance increased (p< 0) at the end of the sixth month (397.2 ± 118.8 meters: IC 95 %: 357; 437.4). There was a highly significant increase (p< 0) of the ankle-arm´s pressure index of the dorsalis pedis artery. Conclusions: Autologous mononuclear cells are effective in treating arterial insufficiency as they eliminate pain during exercise, increase distance while walking, and increase ankle-arm´s pressure rates.

Gene expression of FOXP3, indoleamine 2,3-dioxygenase (IDO), IL10 and CSF1 in the uterus of cows that received an intrauterine infusion of maternal and paternal antigens / Expressão gênica de FOXP3, indoleamina 2,3-dioxigenase (IDO), IL10 e CSF1 em útero de vacas que receberam infusão intrauterina de antígenos paternos e maternos

Sensitization with conceptus antigens has been shown to be useful for improving reproductive performance facilitating maternal acceptance of an allogeneic embryo through the induction of cytokines and immunoregulatory cells in the uterine microenvironment. As FOXP3, IDO, IL10 and CSF1 in the uterus are important on the recognition and development of embryos during early pregnancy, this study aimed to determine whether simultaneous or isolated administration of paternal (semen) and maternal (PBMCs) antigens in the uterus of cow, on the day of estrus, influence the gene expression of these cytokines. Forty crossbred cows were divided into four treatments: T0: Control; T1: Semen; T2: PBMCs (peripheral blood mononuclear cells) from another cow and T3: PBMCs+Semen. Antigens were administered into the uterine body on the estrus day (D0). Uterine biopsies designed for molecular analysis of gene expression were collected in vivo seven (D7) and fourteen (D14) days after immunostimulation. Transcripts from FOXP3, IDO, IL-10 and CSF-1 were detected in all RNA samples extracted from uterine biopsies. The semiquantitative analysis showed that none of the treatments caused significant increase in the expression of these genes. Furthermore, on D14 all treatments led to a decline in the number of CSF-1 transcripts; moreover, treatment with PBMCs+Semen also led to a drop in the abundance of IL-10 transcripts. Such results suggest that isolated or simultaneous administration of both antigens would not increase maternal tolerance to embryo alloantigens, nor would it create favorable conditions to its growth and pre-implantation development, at least regarding the effects mediated by these genes on D7 and D14 of the estrous cycle.

A sensibilização intrauterina com antígenos do concepto tem-se mostrado útil para melhorar o desempenho reprodutivo, facilitando a aceitação materna de um embrião alogênico por meio da indução de citocinas e células imunorreguladoras no microambiente uterino. Como FOXP3, IDO, IL10 e CSF1 no útero são importantes no reconhecimento e desenvolvimento de embriões durante a gestação inicial, este estudo teve como objetivo determinar se a administração simultânea ou isolada de antígenos paterno (sêmen) e materno (PBMCs) no útero de vacas, no dia do estro, influenciam a expressão gênica dessas citocinas. Quarenta vacas mestiças foram divididas em quatro tratamentos: T0: Controle; T1: Sêmen; T2: PBMCs (células mononucleares do sangue periférico) e T3: PBMCs + Sêmen. Os antígenos foram administrados no corpo do útero no dia do estro (D0). Biópsias uterinas projetadas para análise molecular da expressão gênica foram coletadas in vivo sete (D7) e catorze (D14) dias após imunoestimulação. Transcritos de FOXP3, IDO, IL-10 e CSF-1 foram detetados em todas as amostras de RNA extraídas de biópsias uterinas. A análise semiquantitativa mostrou que nenhum dos tratamentos causou um aumento significativo na expressão desses genes. Além disso, no D14, todos os tratamentos levaram a um declínio na quantidade de transcritos do CSF-1; Além disso, o tratamento com PBMCs + sêmen também levou a uma queda na abundância de transcritos de IL-10. Estes resultados sugerem que a administração isolada ou simultânea de ambos os antígenos não deve aumentar a tolerância materna aos aloantígenos do embrião, nem deve criar condições favoráveis ao seu crescimento e desenvolvimento pré-implantação, pelo menos em relação aos efeitos mediados por esses genes nos dia sete e catorze do ciclo estral.

Caracterización de los pacientes con enfermedad arterial periférica, tratados con células mononucleares autólogas / Characterization of patients with peripheral arterial disease treated with autologous mononuclear cells

Introducción: La enfermedad arterial oclusiva de miembros inferiores se asocia con un alto índice de amputaciones y riesgo de muerte. Al respecto, la medicina regenerativa ha mostrado resultados satisfactorios. Objetivo: Caracterizar a los pacientes con enfermedad arterial periférica de los miembros inferiores, tratados con células mononucleares autólogas. Métodos: Se realizó una investigación longitudinal prospectiva en el Hospital Universitario "Arnaldo Milián Castro", durante el período desde enero de 2015 hasta diciembre de 2017. De una población de 61 pacientes se seleccionó una muestra de 52 de forma intencional por criterios. Resultados: La edad promedio resultó de 66,9 ± 8,2 años y el 69,2 por ciento representó al sexo masculino. Los principales factores de riesgo vascular fueron el tabaquismo, la hipercolesteronemia y la hipertensión arterial. El nivel de oclusión mostró predominio fémoro poplíteo y el tractus de salida malo constituyó la principal causa de no revascularización. La viabilidad celular fue elevada y se logró cambio significativo en los estadios de Fontaine. El inicio de la mejoría clínica ocurrió entre el primer y el segundo mes en la mayoría de los casos. En una parte de los pacientes se abrieron posibilidades posquirúrgicas y se obtuvo muy buena reperfusión tisular. Conclusiones: El implante de células mononucleares autólogas en pacientes con enfermedad arterial periférica de los miembros inferiores constituye una nueva estrategia de angiogénesis terapéutica muy útil y efectiva, que abre nuevas perspectivas de tratamiento(AU)

Introduction: Low limbs´ arterial occlusive disease is associated with a high rate of amputations and risk of death. Regarding that, regenerative medicine has proven satisfactory results. Objective: To characterize patients with peripheral arterial disease in the low limbs which have been treated with autologous mononuclear cells. Methods: It was carried out a prospective longitudinal research in "Arnaldo Milián Castro" Hospital in the period from January, 2015 to December, 2017. From a population of 61 patients, it was intentionally selected by criteria a sample of 52 individuals. Results: The average age was 66,9 ± 8,2 and 69,2 percent were men. The main vascular risk factors were smoking habit, hypercholesterolemia and arterial hypertension. The level of occlusion showed predominance of femoro-popliteal and the bad output tractus was the main cause of non-revascularization. Cell viability was high and it was achieved a significant change in Fontaine stages. The beginning of a clinical improvement happened between the first and the second month in most of the cases. In some patients were open post-surgical possibilities and it was obtained great tisular reperfusion. Conclusions: The implant of autologous mononuclear cells in patients with peripheral arterial disease of low limbs represents an useful and effective new strategy of therapeutic angiogenesis which opens new treatment perspectives(AU)

Caracterización morfológica de las células de sangre de cordón umbilical / Morphological characterization of umbilical cord blood cells

Introducción: El cordón umbilical se ha convertido en un elemento de interés para la medicina regenerativa en los últimos años, pues constituye una fuente importante de células madres y progenitores hematopoyéticos. Objetivo: Caracterizar morfológicamente la sangre del cordón umbilical para terapia regenerativa en recién nacidos del Hospital Universitario Ginecobstétrico Ana Betancourt de Mora. Métodos: Estudio observacional, analítico y transversal realizado en el Centro de Inmunología y Productos Biológicos de Camagüey, entre enero y diciembre de 2017. Se evaluaron 35 muestras de sangre del cordón umbilical obtenidas de recién nacidos, que fueron partos eutócicos y sus madres no tuvieron procesos de enfermedades infecciosas. Resultados: El promedio mayor de células mononucleares correspondió a los linfocitos. En el conteo diferencial los polimorfonucleares neutrófilos ocuparon el primer lugar, seguido de los linfocitos, con medias de 0,50 y 0,46 x 109/L, respectivamente. De las células presentes en el frotis del botón, fueron más frecuente los linfocitos con 0.59 x 109/L; se observó un promedio de monocitos de 0,00-0,07 x 109/L. Conclusiones: La obtención de células mononucleares viables a través de la vena umbilical, constituye una técnica promisoria en las investigaciones biomédicas. Entre las células mononucleares predominaron los linfocitos, tanto en la sangre del cordón umbilical como en el botón celular aislado(AU)

Introduction: In recent years, the umbilical cord has become an element of interest for regenerative medicine, based on its importance as a source of stem cells and hematopoietic progenitors. Objective: To characterize the morphology of umbilical cord blood for regenerative therapy in newborns from Ana Betancourt de Mora Gyneco-obstetric University Hospital. Methods: Observational, analytical and cross-sectional study carried out at the Center of Immunology and Biological Products in Camagüey, between January and December 2017. We evaluated 35 samples of umbilical cord blood obtained from newborns who were eutocic deliveries and whose mothers did not have infectious disease processes. Results: The highest average of mononuclear cells corresponded to lymphocytes. In the differential count, neutrophil polymorphonuclear cells ranked first, followed by lymphocytes, with averages of 0.50x109 and 0.46x109 per liter, respectively. Of the cells present in the button cell smear, lymphocytes were more frequent, with 0.59x109 per L; an average of monocytes was observed, with 0.00-0.07x109 per L was observed. Conclusions: Obtaining viable mononuclear cells through the umbilical vein is a promising technique in biomedical research. Among the mononuclear cells, lymphocytes predominated, both in the cord blood and in the isolated cell button(AU)

Incremento de la viabilidad, producción de especies reactivas de oxígeno, IL-1 y TNF-α en células mononucleares de sangre periférica humana tratadas con fucoidan de Lessonia trabeculata

Las algas pardas constituyen una fuente de alto contenido de polisacáridos como los fucoidanos que poseen importantes propiedades inmunomoduladoras. El objetivo fue determinar la viabilidad de células mononucleares de sangre periférica humana (CMSPh), producción de óxido nítrico (NO), especies reactivas de oxígeno (ROS) y de las citoquinas proinflamatorias IL-1α, IL-6, TNF-α e IFN-γ en cultivos tratados con fucoidan de Lessonia trabeculata. Se empleó fucoidan de Lessonia trabeculata proveniente de la bahía San Nicolás de Marcona-Ica. Las CMSPh se aislaron empleando Ficoll-Hypaque, se distribuyeron a una concentración de 1x105 células/pocillo en medio RPMI-1640 completo y se trataron con diferentes concentraciones de fucoidan durante 24 y 48 h. La actividad citotóxica se determinó por la reducción de MTT, la producción de NO por la reacción de Griess y las ROS por la reducción del NBT. La producción de citoquinas se cuantificó por ELISA. El fucoidan de L. trabeculata estimuló la proliferación de CMSPh y produjo el incremento de ROS a concentraciones de 100-2000 μg/mL respecto al control (p<0.001), la reacción para nitritos resultó negativa. El fucoidan incrementó la producción de IL-1α y TNF-α a concentraciones de 100 y 10 μg/mL respectivamente, mientras que la producción de IL-6 e IFN-γ no mostró diferencias significativas. Se concluye que el fucoidan de L. trabeculata estimula la proliferación de CMSPh, producción de especies reactivas de oxígeno y las citoquinas proinflamatorias IL-1α y TNF-α que poseen importantes propiedades inmunomoduladoras.

Brown algae are a source of high content of polysaccharides such as fucoidans that have important immunomodulatory properties. The aim was to determine the viability of human peripheral blood mononuclear cells (hPBMC), production of nitric oxide (NO), reactive oxygen species (ROS) and the proinflammatory cytokines IL-1α, IL-6, TNF-α and IFN -γ in cultures treated with fucoidan from Lessonia trabeculata. Fucoidan from Lessonia trabeculata from San Nicolás de Marcona-Ica Bay was used. The hPBMC were isolated using Ficoll-Hypaque, distributed at a concentration of 1x105 cells/well in complete RPMI-1640 medium and treated with different concentrations of fucoidan for 24 and 48 h. The cytotoxic activity was determined by the reduction of MTT, NO production by the Griess reaction and ROS by the reduction of NBT. The production of cytokines was quantified by ELISA. The fucoidan of L. trabeculata stimulated the proliferation of hPBMC and produced the increase of ROS at concentrations of 100-2000 μg/mL with respect to the control (p <0.001), the reaction for nitrites was negative. Fucoidan increased the production of IL-1α and TNF-α at concentrations of 100 and 10 μg/mL respectively, while the production of IL-6 and IFN-γ did not show significant differences. It is concluded that the fucoidan of L. trabeculata stimulates the proliferation of hPBMC, production of reactive oxygen species and the proinflammatory cytokines IL-1α and TNF-α that possess important immunomodulatory properties.

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)

Células mononucleares autólogas en la reparación de defectos óseos provocados por la periodontitis crónica / Autologous mononuclear cells in the repair of bone defects caused by chronic periodontitis

Introducción: la periodontitis crónica es un proceso inflamatorio de origen bacteriano que afecta a los tejidos del periodonto y provoca la destrucción de los tejidos de soporte del diente. La terapia celular con células mononucleares autólogas constituye una nueva opción terapéutica para lograr la regeneración ósea. Objetivo: evaluar la efectividad del tratamiento con células mononucleares autólogas implantadas en defectos óseos provocados por la periodontitis crónica. Método: estudio cuasiexperimental que se realizó en la Clínica Provincial Docente Antonio Briones Montoto de Pinar del Río, en el periodo comprendido entre enero de 2012 hasta agosto de 2015. A los nueve pacientes del grupo de estudio, se le realizó la perfusión de células mononucleares siete días después de ser intervenidos quirúrgicamente (colgajo periodontal). La movilización a sangre periférica de células mononucleares autólogas se realizó con factor estimulante del crecimiento granulocítico Leukocim (FEC-G). Variables del estudio: dientes afectados, presencia de sangrado al sondeo, bolsas periodontales, movilidad dentaria, pérdida de inserción y evidencia radiográfica. Resultados: posterior a la terapia celular se constató que las encías presentaron características de normalidad a los 7 días de implantados, a los 12 meses se observó hueso de neoformación y aumento de la densidad ósea. Conclusiones: la terapia mostró ser un método factible, simple y seguro en la reparación de defectos óseos provocados por la enfermedad, evidenciando mejoría de los parámetros clínicos y radiográficos(AU)

Introduction: Periodontitis is an inflammatory process of bacterial origin that affects the tissues of the periodontium and causes the destruction of the tissues supporting the tooth. Cell therapy could be an effective therapeutic option to achieve bone regeneration. Objective: To evaluate the effectiveness of treatment with implanted autologous mononuclear cells in bone defects caused by periodontal disease. Methods: A quasiexperimental study was carried out in the Provincial Teaching Clinic Antonio Briones Montoto, Pinar del Río, in the period from January 2012 to August 2015. The nine patients in the study group underwent perfusion of mononuclear cells seven days after surgery (periodontal flap). The mobilization to peripheral blood of autologous mononuclear cells was made with granulocytic leukocyte growth stimulating factor (FEC-G). Study variables: affected teeth, presence of bleeding on probing, periodontal pockets, tooth mobility, loss of insertion and radiographic evidence. Results: After the cell therapy, it was found that the gums showed normality characteristics after 7 days of implantation, after 12 months neoformation bone and increase in bone density was observed. Conclusions: The therapy showed to be a feasible, simple and safe method in the repair of bone defects caused by the disease, evidencing improvement of the clinical and radiographic parameters(AU)

Células mononucleares autólogas en la reparación de defectos óseos provocados por la periodontitis crónica / Autologous mononuclear cells in the repair of bone defects caused by chronic periodontitis

Introducción: la periodontitis crónica es un proceso inflamatorio de origen bacteriano que afecta a los tejidos del periodonto y provoca la destrucción de los tejidos de soporte del diente. La terapia celular con células mononucleares autólogas constituye una nueva opción terapéutica para lograr la regeneración ósea. Objetivo: evaluar la efectividad del tratamiento con células mononucleares autólogas implantadas en defectos óseos provocados por la periodontitis crónica. Método: estudio cuasiexperimental que se realizó en la Clínica Provincial Docente Antonio Briones Montoto de Pinar del Río, en el periodo comprendido entre enero de 2012 hasta agosto de 2015. A los nueve pacientes del grupo de estudio, se le realizó la perfusión de células mononucleares siete días después de ser intervenidos quirúrgicamente (colgajo periodontal). La movilización a sangre periférica de células mononucleares autólogas se realizó con factor estimulante del crecimiento granulocítico Leukocim (FEC-G). Variables del estudio: dientes afectados, presencia de sangrado al sondeo, bolsas periodontales, movilidad dentaria, pérdida de inserción y evidencia radiográfica. Resultados: posterior a la terapia celular se constató que las encías presentaron características de normalidad a los 7 días de implantados, a los 12 meses se observó hueso de neoformación y aumento de la densidad ósea. Conclusiones: la terapia mostró ser un método factible, simple y seguro en la reparación de defectos óseos provocados por la enfermedad, evidenciando mejoría de los parámetros clínicos y radiográficos(AU)

Introduction: Periodontitis is an inflammatory process of bacterial origin that affects the tissues of the periodontium and causes the destruction of the tissues supporting the tooth. Cell therapy could be an effective therapeutic option to achieve bone regeneration. Objective: To evaluate the effectiveness of treatment with implanted autologous mononuclear cells in bone defects caused by periodontal disease. Methods: A quasiexperimental study was carried out in the Provincial Teaching Clinic Antonio Briones Montoto, Pinar del Río, in the period from January 2012 to August 2015. The nine patients in the study group underwent perfusion of mononuclear cells seven days after surgery (periodontal flap). The mobilization to peripheral blood of autologous mononuclear cells was made with granulocytic leukocyte growth stimulating factor (FEC-G). Study variables: affected teeth, presence of bleeding on probing, periodontal pockets, tooth mobility, loss of insertion and radiographic evidence. Results: After the cell therapy, it was found that the gums showed normality characteristics after 7 days of implantation, after 12 months neoformation bone and increase in bone density was observed. Conclusions: The therapy showed to be a feasible, simple and safe method in the repair of bone defects caused by the disease, evidencing improvement of the clinical and radiographic parameters(AU)

Lisado plaquetario y células mononucleares autólogas de la periodontitis crónica. Presentación de un caso / Autologous platelet lysate and mononuclear stem cell of chronic periodontitis. Case presentation

Introducción: El tratamiento de la periodontitis crónica se prolonga por persistencia de la inflamación en tejidos afectados. La medicina regenerativa muestra resultados alentadores. Es la primera vez en Cuba, según bibliografía revisada, que se usa la combinación de lisado plaquetario y células mononucleares autólogas en este tratamiento.Objetivo: Mostrar el efecto de la combinación del lisado plaquetario y células mononucleares autólogas en el tratamiento de la periodontitis.Presentación de un caso: Paciente femenina de 44 años de edad, con diagnóstico de periodontitis, quien desde hace 1 año lleva tratamiento, sin efectuarse procedimientos quirúrgicos por la inflamación persistente. Es remitida a la consulta de periodoncia del Hospital Enrique Cabrera para recibir tratamiento con medicina regenerativa. Se consideró tratar con lisado de plaquetas autólogas, una vez concluido el raspado y alisador radicular, se prefundieron las encías afectadas con el lisado plaquetario. A los 7 días de aplicado el lisado se constatan las encías sin signos clínicos de inflamación, y disminución ostensible del número de dientes afectados y la profundidad o eliminación de las bolsas periodontales y movilidad dentaria. Al mes se trataron quirúrgicamente los defectos óseos con implante de células mononucleares. Al evaluar a los 6 meses, se hallaron de forma variable, signos radiográficos de organización del trabeculado, definición de la cortical y formación ósea en zona de defectos óseos.Conclusiones: El tratamiento combinado del lisado plaquetario autólogo y células mononucleares muestra una evaluación satisfactoria en el tratamiento de la periodontitis crónica y reduce el tiempo de tratamiento(AU)

Introduction: Treatment of chronic periodontitis is prolonged by persistent inflammation in affected tissues. Regenerative medicine shows encouraging results. It is the first time in Cuba, according to the reviewed literature, that the combination of platelet lysate and autologous mononuclear cells is used in this treatment.Objective: to show the effect of the combination of platelet lysate and autologous mononuclear cells in the treatment of periodontitis.Presentation of the case: A 44-year-old female patient diagnosed with periodontitis who had been treated for 1 year without surgical procedures due to persistent inflammation. She is referred to the periodontics consultation of the "Dr. Enrique Cabrera" General Hospital to receive treatment with regenerative medicine. Treatment with autologous platelet lysate was considered, once the scaling and root planer had been completed, the affected gingivae were prefixed with the platelet lysate. At 7 days after the lysate is applied, the gingiva shows no clinical signs of inflammation, and a noticeable reduction in the number of affected teeth and the depth or elimination of periodontal pockets and tooth mobility. Bone defects with a mononuclear cell implant were surgically treated one month later. When evaluating at 6 months, radiographic signs of trabecular organization, definition of the cortical bone, and bone formation in the area of ​​bone defects were found variably.Conclusions: The combined treatment of autologous platelet lysate and mononuclear cells shows a satisfactory evaluation in the treatment of chronic periodontitis and reduces treatment time.Keywords: Regenerative medicine, autologous platelet lysate, chronic periodontitis, autologous mononuclear stem cell(AU)

Lisado plaquetario y células mononucleares autólogas de la periodontitis crónica. Presentación de un caso / Autologous platelet lysate and mononuclear stem cell of chronic periodontitis. Case presentation

Introducción: El tratamiento de la periodontitis crónica se prolonga por persistencia de la inflamación en tejidos afectados. La medicina regenerativa muestra resultados alentadores. Es la primera vez en Cuba, según bibliografía revisada, que se usa la combinación de lisado plaquetario y células mononucleares autólogas en este tratamiento. Objetivo: Mostrar el efecto de la combinación del lisado plaquetario y células mononucleares autólogas en el tratamiento de la periodontitis. Presentación de un caso: Paciente femenina de 44 años de edad, con diagnóstico de periodontitis, quien desde hace 1 año lleva tratamiento, sin efectuarse procedimientos quirúrgicos por la inflamación persistente. Es remitida a la consulta de periodoncia del Hospital Enrique Cabrera para recibir tratamiento con medicina regenerativa. Se consideró tratar con lisado de plaquetas autólogas, una vez concluido el raspado y alisador radicular, se prefundieron las encías afectadas con el lisado plaquetario. A los 7 días de aplicado el lisado se constatan las encías sin signos clínicos de inflamación, y disminución ostensible del número de dientes afectados y la profundidad o eliminación de las bolsas periodontales y movilidad dentaria. Al mes se trataron quirúrgicamente los defectos óseos con implante de células mononucleares. Al evaluar a los 6 meses, se hallaron de forma variable, signos radiográficos de organización del trabeculado, definición de la cortical y formación ósea en zona de defectos óseos. Conclusiones: El tratamiento combinado del lisado plaquetario autólogo y células mononucleares muestra una evaluación satisfactoria en el tratamiento de la periodontitis crónica y reduce el tiempo de tratamiento(AU)

Introduction: Treatment of chronic periodontitis is prolonged by persistent inflammation in affected tissues. Regenerative medicine shows encouraging results. It is the first time in Cuba, according to the reviewed literature, that the combination of platelet lysate and autologous mononuclear cells is used in this treatment. Objective: to show the effect of the combination of platelet lysate and autologous mononuclear cells in the treatment of periodontitis. Presentation of the case: A 44-year-old female patient diagnosed with periodontitis who had been treated for 1 year without surgical procedures due to persistent inflammation. She is referred to the periodontics consultation of the Dr. Enrique Cabrera General Hospital to receive treatment with regenerative medicine. Treatment with autologous platelet lysate was considered, once the scaling and root planer had been completed, the affected gingivae were prefixed with the platelet lysate. At 7 days after the lysate is applied, the gingiva shows no clinical signs of inflammation, and a noticeable reduction in the number of affected teeth and the depth or elimination of periodontal pockets and tooth mobility. Bone defects with a mononuclear cell implant were surgically treated one month later. When evaluating at 6 months, radiographic signs of trabecular organization, definition of the cortical bone, and bone formation in the area of bone defects were found variably. Conclusions: The combined treatment of autologous platelet lysate and mononuclear cells shows a satisfactory evaluation in the treatment of chronic periodontitis and reduces treatment time(AU)

Comparação de dois métodos de colheita de medula óssea de equinos e de dois meios de diluição para o isolamento de células mononucleares / Comparison of two methods of equine bone marrow aspiration and two solutions for mononuclear cell isolation

O objetivo do presente estudo foi avaliar a concentração e viabilidade da fração de células mononucleares (FCM) a partir de diferentes técnicas de colheita e processamento de medula óssea (MO) em equinos. Foram avaliados cinco equinos adultos, hígidos e sem raça definida. Obtiveram-se frações de medula óssea (MO) do osso esterno, de acordo com dois protocolos: na colheita A, utilizou-se 10mL de solução de heparina dentro da seringa e em seguida, aspirou-se a MO; na colheita B, 10mL de solução de heparina foi injetada na MO e a aspiração foi realizada após 20 segundos. Todos os animais foram submetidos aos dois protocolos de colheitas, realizadas em sequência, sem intervalo entre os dois procedimentos. Após isolamento da fração de células mononucleares (FCM), das amostras de MO obtidas nas colheitas A e B, cada amostra foi dividida em dois tubos, um contendo solução de DMEM e outro contendo PBS. Assim, alternando-se o tipo de colheita e a solução diluidora, obteve-se quatro tubos de amostras por animal. Os tubos foram centrifugados e os sedimentos foram homogeneizados nos respectivos meios obtendo-se o volume final de 100μL. Realizou-se determinação da concentração e viabilidade celular, obtendo-se as concentrações médias de FCM. Para ambos os meios de diluição, a colheita B apresentou valor numérico maior em comparação à colheita A, porém não foi significativo (p>0,05). Atribui-se tal tendência à menor ocorrência de coagulação da MO no momento da colheita B, sugerindo-se melhor aproveitamento da FCM. Não houve diferença (p>0,05) entre os meios DMEM ou PBS, indicando que os mesmos não alteraram a viabilidade celular. Os protocolos utilizados para colheita de MO e separação da FCM se mostraram eficientes, para o uso em terapia celular em equinos.

The aim of this study was to evaluate mononuclear cells fraction (MCF) concentration and viability from different techniques of bone marrow (BM) aspiration and processing in horses. Five adult horses, healthy and of unknown breed were evaluated. BM was obtained from sternum bone, according two protocols: in aspiration A, 10mL of heparin solution was used inside the syringe and BM was aspirated; in aspiration B, 10mL of heparin solution was injected into the BM, and aspiration was done after 20 seconds. All the animals were submitted by both protocols realized in sequence, without a gap between the procedures. After MCF isolation, of BM samples obtained from A and B aspiration, each sample was divided into two tubes; one contained DMEM solution and the other with PBS solution. Therefore, interchanging the aspiration protocol and the dilution solution, four sample tubes were obtained for each horse. The tubes were centrifuged and the pellet was homogenized with the respectively solution to obtain the final volume of 100μL. Cellular concentration and viability were determined to obtain the FCM medium concentration. For both solutions, the aspiration B had higher numeric values comparing with aspiration A; however, it was not significant (p>0.05). This tendency is attribute for the less BM coagulation observed in the aspiration B, suggesting greater improvement of MCF. No difference (p>0.05) was found between DMEM and PBS solution, indicating that both do not alter the cell viability. The protocols used for BM aspiration and MCF isolation were efficient for application in equine cellular therapy.

Comparação de dois métodos de colheita de medula óssea de equinos e de dois meios de diluição para o isolamento de células mononucleares / Comparison of two methods of equine bone marrow aspiration and two solutions for mononuclear cell isolation

O objetivo do presente estudo foi avaliar a concentração e viabilidade da fração de células mononucleares (FCM) a partir de diferentes técnicas de colheita e processamento de medula óssea (MO) em equinos. Foram avaliados cinco equinos adultos, hígidos e sem raça definida. Obtiveram-se frações de medula óssea (MO) do osso esterno, de acordo com dois protocolos: na colheita A, utilizou-se 10mL de solução de heparina dentro da seringa e em seguida, aspirou-se a MO; na colheita B, 10mL de solução de heparina foi injetada na MO e a aspiração foi realizada após 20 segundos. Todos os animais foram submetidos aos dois protocolos de colheitas, realizadas em sequência, sem intervalo entre os dois procedimentos. Após isolamento da fração de células mononucleares (FCM), das amostras de MO obtidas nas colheitas A e B, cada amostra foi dividida em dois tubos, um contendo solução de DMEM e outro contendo PBS. Assim, alternando-se o tipo de colheita e a solução diluidora, obteve-se quatro tubos de amostras por animal. Os tubos foram centrifugados e os sedimentos foram homogeneizados nos respectivos meios obtendo-se o volume final de 100μL. Realizou-se determinação da concentração e viabilidade celular, obtendo-se as concentrações médias de FCM. Para ambos os meios de diluição, a colheita B apresentou valor numérico maior em comparação à colheita A, porém não foi significativo (p>0,05). Atribui-se tal tendência à menor ocorrência de coagulação da MO no momento da colheita B, sugerindo-se melhor aproveitamento da FCM. Não houve diferença (p>0,05) entre os meios DMEM ou PBS, indicando que os mesmos não alteraram a viabilidade celular. Os protocolos utilizados para colheita de MO e separação da FCM se mostraram eficientes, para o uso em terapia celular em equinos.(AU)

The aim of this study was to evaluate mononuclear cells fraction (MCF) concentration and viability from different techniques of bone marrow (BM) aspiration and processing in horses. Five adult horses, healthy and of unknown breed were evaluated. BM was obtained from sternum bone, according two protocols: in aspiration A, 10mL of heparin solution was used inside the syringe and BM was aspirated; in aspiration B, 10mL of heparin solution was injected into the BM, and aspiration was done after 20 seconds. All the animals were submitted by both protocols realized in sequence, without a gap between the procedures. After MCF isolation, of BM samples obtained from A and B aspiration, each sample was divided into two tubes; one contained DMEM solution and the other with PBS solution. Therefore, interchanging the aspiration protocol and the dilution solution, four sample tubes were obtained for each horse. The tubes were centrifuged and the pellet was homogenized with the respectively solution to obtain the final volume of 100μL. Cellular concentration and viability were determined to obtain the FCM medium concentration. For both solutions, the aspiration B had higher numeric values comparing with aspiration A; however, it was not significant (p>0.05). This tendency is attribute for the less BM coagulation observed in the aspiration B, suggesting greater improvement of MCF. No difference (p>0.05) was found between DMEM and PBS solution, indicating that both do not alter the cell viability. The protocols used for BM aspiration and MCF isolation were efficient for application in equine cellular therapy.(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.

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.

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)

Produção de IL-7 canina no sistema baculovírus-células de inseto / Canine IL-7 production in insect baculovirus-cell system

INTRODUÇÃO. A interleucina 7 (IL-7) é uma citocina pleiotrópica produzida principalmente por células estromais da medula óssea, células epiteliais tímicas e intestinais. IL-7 e que promove o desenvolvimento de linfócitos T e B e diferenciação de células T memória, especialmente CD4+. Por isso, diversos autores têm estudado a capacidade de IL-7 promover a restauração da população de linfócitos em situações de linfopenia e resposta imune de memória. Cães que desenvolvem leishmaniose visceral e recebem tratamento especfico apresentam cura clínica. No entanto, após a interrupção do tratamento, a maioria dos animais exibe recaída da doença, mesmo na ausência de reinfecção. É possível que esse fenômeno esteja associado a uma dificuldade de estabelecimento de linfócitos T de memória específicos para Leishmania em cães. Em nosso laboratório, tentativas prévias foram realizadas para produzir a IL-7 canina em Escherichia coli, no entanto, o rendimento da proteína biologicamente ativa foi pequeno. OBJETIVO. Por isso, resolveu-e avaliar a viabilidade de produzir IL-7 do sistema baculovírus-células de inseto. MATERIAL E MÉTODOS. No presente trabalho, uma construção de DNA foi concebida contendo uma sequência de nucleotídeos que codificam o peptídeo sinal da proteína GP64 do baculovírus da Autographa californica (AcMNPV), a IL-7 canina (proteína madura) com códons otimizados para a tradução em Trichoplusia ni, um espaçador com 23 aminoácidos e uma cauda de seis histidinas. A construção de DNA elaborada foi sintetizada quimicamente, inserida em um plasmídeo de clonagem (pUC57-GP64caIL-7), subclonada em um plasmídeo carreador (pFastBac1-GP64caIL-7), adequado para o sistema baculovirus-célula de inseto, e transferida para o cromossoma artificial do baculovírus recombinante (bacmídeo). RESULTADOS. A produção de IL-7 recombinante canina (rcaIL-7) em células de inseto infectadas com baculovírus recombinante foi otimizada. Em seguida, a rcaIL-7 foi produzida em células High-five cultivadas em suspensão utilizando multiplicidade de infecção (MOI) de 5 e tempo de infecção (TOI) de 48 h. A proteína recombinantes foi purificada por cromatografia de afinidade em Sepharose-níquel,obtendo-se um rendimento médio de 5,5 mg por litro de cultivo. CONCLUSÃO. RcaIL-7 purificada, avaliada a 40 ng/mL,mostrou-se capaz de promover a proliferação de células mononucleares de sangue periférico de cães sadios, sem estímulo prévio ou concomitante, indicando que a proteína foi produzida biologicamente ativa. Futuros estudos serão realizados para avaliar a capacidade imunomoduladora da rcaIL-7 em cães e determinar a utilidade dessa citocinas no tratamento de enfermidades caninas, como por exemplo, na leishmaniose visceral canina.

INTRODUCTION. The interleukin 7 is a pleiotropic cytokine produced mainly by bone marrow stromal cells, thymus and intestinal epithelial cells. IL-7 promotes the development of T and B limphocytes and differentiation T cells to memory cells, especially T CD4+ cells. For these reasons, several authors have studied the ability of IL7 to promote restoration of the lymphocyte population in cases of lymphopenia and memory imune responses. Dogs which develop visceral leishmaniasis and are treated with specific drugs develop only transient clinical cure. After treatment withdrawal, most animals show disease relapse, even in the absence of reinfection. One possible explanation for the lack of control of the infection after treatment in dogs is a difficulty to develop/maintain specific memory T cells. Therefore, it is possible that effective treatments for canine visceral leishmaniasis be developed by manipulating the immune system. Previously, in our laboratory, it was attemped to produce recombinant canine IL7 (rcaIL-7) in Escherichia coli, however there was a low yield of biologically active protein. OBJECTIVE. In the current project, the baculovírus-insect cell system was evaluated to produce rcaIL-7. MATERIAL AND METHODS. In this study, a construction of DNA was designed to encode nucleotides of Autographa californica GP64 signal peptide, canine IL-7 (mature protein) with optimized codons for Trichoplusia ni, a 23-amino acid spacer and a six histidine tail. The DNA construct was chemically synthesized, inserted into a cloning plasmid (pUC57-GP64caIL-7), subcloned into a carrier plasmid (pFastBac1-GP64caIL-7), suited to the baculovirus-insect cell expression system, and transfered to an artificial chromosome of the recombinant baculovirus (bacmid)...

Eficacia del implante de células mononucleares autólogas en el tratamiento de la periodontitis crónica / Effectiveness of autologous mononuclear cells implant in patients with chronic periodontitis

Introducción: los buenos resultados en la regeneración de tejidos mediante la implantación de células mononucleares en distintas especialidades médicas en el mundo y en Cuba, ha motivado a considerar el periodonto como candidato para esta terapia.Objetivo: determinar la eficacia del implante de células mononucleares autólogas en el tratamiento de la periodontitis crónica a 48 meses de evolución. Material y Métodos: se realizó un ensayo clínico fase III, aleatorizado y controlado en pacientes con periodontitis crónica durante los años 2008 a 2014. De ese grupo de pacientes se evaluaron 30 que tenían 48 ó más meses de recibir tratamiento en la consulta de Periodoncia del Hospital Enrique Cabrera en el período comprendido de febrero a mayo de 2015; 15 habían recibido tratamiento con células mononucleares (grupo estudio) y 15 terapia convencional (grupo control). La movilización a la sangre periférica de las células mononucleares se realizó mediante la aplicación del factor estimulador de colonias de granulocitos (FEC-G). Resultados: se aprecia una disminución del promedio de todas las variables clínicas, que es marcada en cada uno de los períodos evolutivos con respecto al estado inicial de los pacientes a los 6, 12, 36 y 48 meses posteriores al tratamiento en el grupo de estudio, mientras que en el control las manifestaciones de la enfermedad fueron reapareciendo paulatinamente a medida que avanzaba el tiempo post-tratamiento. Estos resultados fueron significativos. Conclusiones: la terapia celular regenerativa con células mononucleares autólogas es eficaz para el tratamiento de pacientes con periodontitis y su efecto es prolongado(AU)

Introduction: over the last years, in Cuba and other countries, encouraging results have been obtained in different medical specialties in the field of tissue regeneration by implanting stem cells; it has motivated us to consider periodontal tissue as a target for this therapy. Objective: To determine the efficacy of the implant of mononuclear autologous cells for the treatment of 48 months evolution chronic periodontitis. Material and Methods: 30 patients attending the Periodontology Clinic of the "Enrique Cabrera" hospital for over48 months during years 2008 to 2014 where included in the study, divided into two groups 15 patients each: group A were treated with autologous mononuclear cells and group B, who underwent classic therapy. The peripheral blood autologous mononuclear cells (MNC-SP) were mobilized with granulocyte colony- stimulating factor (G-CSF). Results: group A patients showed a significant average decrease in all clinical variables, more remarkable in every of the evolution periods as compared to the initial stage of every patient at 6, 12, 24 and 48 months after starting the treatment. In Group B patients, signs of the disease gradually reappeared as the post-treatment period proceeded.Conclusions: regenerative cell therapy with MNC-SP is effective and long lasting in patients with periodontitis(AU)