Recolección de células hematopoyéticas periféricas para trasplante alogénico con una dosis intermedia de filgrastim

Antecedentes: las células hematopoyéticas de sangre periférica son un recurso para combatir diversas enfermedades que requieren un trasplante alogénico para su curación. El método internacionalmente aceptado para obtener estas células es la administración de 2 a 24 μg/kg de peso de un factor estimulante de colonias de granulocitos y realizar la aféresis al cuarto, quinto o sexto días de iniciada la estimulación del donador. Objetivo: determinar la dosis adecuada del factor estimulante de colonias de granulocitos y el día óptimo para realizar una sola aféresis. Pacientes y método: se incluyeron 11 donadores que recibieron factor estimulante de colonias de granulocitos en dosis diarias de 8 μg/kg de peso, durante cuatro días, y se realizaron dos aféresis: una en el cuarto y otra en el quinto día de estimulación. Se analizaron retrospectivamente los procedimientos de recolección de células hematopoyéticas de sangre periférica para trasplante alogénico realizados entre febrero del 2003 y mayo del 2005 en el Hospital Universitario Dr. José Eleuterio González de la UANL. Resultados: en 63% de los pacientes, la cantidad de células CD34+ recolectada fue significativamente mayor en el quinto día que en el cuarto (3.10 × 106 vs 2.9 × 106), ya que el volumen plasmático promedio procesado por aféresis fue menor en la primera ocasión que en la segunda (8,800 vs 14,080 mL). Conclusión: una dosis de 8 μg/kg de peso del factor estimulante de colonias de granulocitos es efectiva para favorecer la obtención de células hematopoyéticas periféricas. Una aféresis al quinto día de iniciada la estimulación es suficiente para obtener la cantidad necesaria de células CD34+ que aseguren la recuperación hematológica del paciente trasplantado. Este procedimiento reduce los efectos colaterales del factor y de la aféresis, así como el costo final del trasplante.

Análise da estabilidade genética de células-tronco mesenquimais humanas

Human multipotent mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have become an important and attractive therapeutic tool since they are easily isolated and cultured, have in vitro expansion potential, substantial plasticity and secrete bioactive molecules that exert trophic effects. The human umbilical cord as a cell source for cell therapy will help to avoid several ethical, political, religious and technical issues. One of the main issues with SC lines from different sources, mainly those of embryonic origin, is the possibility of chromosomal alterations and genomic instability during in vitro expansion. Cells isolated from one umbilical cord exhibited a rare balanced paracentric inversion, likely a cytogenetic constitutional alteration, karyotype: 46,XY,inv(3)(p13p25~26). Important genes related to cancer predisposition and others involved in DNA repair are located in 3p25~26. Titanium is an excellent biomaterial for bone-implant integration; however, the use can result in the generation of particulate debris that can accumulate in the tissues adjacent to the prosthesis, in the local bone marrow, in the lymph nodes, liver and spleen. Subsequently may elicit important biological responses that aren´t well studied. In this work, we have studied the genetic stability of MSC isolated from the umbilical cord vein during in vitro expansion, after the cryopreservation, and under different concentrations and time of exposition to titanium microparticles. Cells were isolated, in vitro expanded, demonstrated capacity for osteogenic, adipogenic and chondrogenic differentiation and were evaluated using flow cytometry, so they met the minimum requirements for characterization as MSCs. The cells were expanded under different concentrations and time of exposition to titanium microparticles. The genetic stability of MSCs was assessed by cytogenetic analysis, fluorescence in situ hybridization (FISH) and analysis of micronucleus and other nuclear alterations (CBMN). The cells were able to internalize the titanium microparticles, but MSCs preserve their morphology, differentiation capacity and surface marker expression profiles. Furthermore, there was an increase in the genomic instability after long time of in vitro expansion, and this instability was greater when cells were exposed to high doses of titanium microparticles that induced oxidative stress. It is necessary always assess the risks/ benefits of using titanium in tissue therapy involving MSCs, considering the biosafety of the use of bone regeneration using titanium and MSCs. Even without using titanium, it is important that the therapeutic use of such cells is based on analyzes that ensure quality, security and cellular stability, with the standardization of quality control programs appropriate. In conclusion, it is suggested that cytogenetic analysis, FISH analysis and the micronucleus and other nuclear alterations are carried out in CTMH before implanting in a patient

Produção e caracterização de quitooligossacarídeos produzidos pelo fungo Metarhizium anisopliae e avaliação da citotoxicidade em células tumorais

Chitosan is a natural polymer, biodegradable, nontoxic, high molecular weight derived from marine animals, insects and microorganisms. Oligomers of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) have interesting biological activities, including antitumor effects, antimicrobial activity, antioxidant and others. The alternative proposed by this work was to study the viability of producing chitooligosaccharides using a crude enzymes extract produced by the fungus Metarhizium anisopliae. Hydrolysis of chitosan was carried out at different times, from 10 to 60 minutes to produce chitooligosaccharides with detection and quantification performed by High Performace Liquid Chromatography (HPLC). The evaluation of cytotoxicity of chitosan oligomers was carried out in tumor cells (HepG2 and HeLa) and non-tumor (3T3). The cells were treated for 72 hours with the oligomers and cell viability investigated using the method of MTT. The production of chitosan oligomers was higher for 10 minutes of hydrolysis, with pentamers concentration of 0.15 mg/mL, but the hexamers, the molecules showing greater interest in biological properties, were observed only with 30 minutes of hydrolysis with a concentration of 0.004 mg/mL. A study to evaluate the biological activities of COS including cytotoxicity in tumor and normal cells and various tests in vitro antioxidant activity of pure chitosan oligomers and the mixture of oligomers produced by the crude enzyme was performed. Moreover, the compound with the highest cytotoxicity among the oligomers was pure glucosamine, with IC50 values of 0.30; 0.49; 0.44 mg/mL for HepG2 cells, HeLa and 3T3, respectively. Superoxide anion scavenging was the mainly antioxidant activity showed by the COS and oligomers. This activity was also depending on the oligomer composition in the chitosan hydrolysates. The oligomers produced by hydrolysis for 20 minutes was analyzed for the ability to inhibit tumor cells showing inhibition of proliferation only in HeLa cells, did not show any effect in HepG2 cells and fibroblast cells (3T3)

Transcriptoma diferencial entre células-tronco mesenquimais humanas jovens e senescentes

Human mesenchymal stem cells (MSC) are powerful sources for cell therapy in regenerative medicine. The long time cultivation can result in replicative senescence or can be related to the emergence of chromosomal alterations responsible for the acquisition of tumorigenesis features in vitro. In this study, for the first time, the expression profile of MSC with a paracentric chromosomal inversion (MSC/inv) was compared to normal karyotype (MSC/n) in early and late passages. Furthermore, we compared the transcriptome of each MSC in early passages with late passages. MSC used in this study were obtained from the umbilical vein of three donors, two MSC/n and one MSC/inv. After their cryopreservation, they have been expanded in vitro until reached senescence. Total RNA was extracted using the RNeasy mini kit (Qiagen) and marked with the GeneChip ® 3 IVT Express Kit (Affymetrix Inc.). Subsequently, the fragmented aRNA was hybridized on the microarranjo Affymetrix Human Genome U133 Plus 2.0 arrays (Affymetrix Inc.). The statistical analysis of differential gene expression was performed between groups MSC by the Partek Genomic Suite software, version 6.4 (Partek Inc.). Was considered statistically significant differences in expression to p-value Bonferroni correction ˂.01. Only signals with fold change ˃ 3.0 were included in the list of differentially expressed. Differences in gene expression data obtained from microarrays were confirmed by Real Time RT-PCR. For the interpretation of biological expression data were used: IPA (Ingenuity Systems) for analysis enrichment functions, the STRING 9.0 for construction of network interactions; Cytoscape 2.8 to the network visualization and analysis bottlenecks with the aid of the GraphPad Prism 5.0 software. BiNGO Cytoscape pluggin was used to access overrepresentation of Gene Ontology categories in Biological Networks. The comparison between senescent and young at each group of MSC has shown that there is a difference in the expression parttern, being higher in the senescent MSC/inv group. The results also showed difference in expression profiles between the MSC/inv versus MSC/n, being greater when they are senescent. New networks were identified for genes related to the response of two of MSC over cultivation time. Were also identified genes that can coordinate functional categories over represented at networks, such as CXCL12, SFRP1, xvi EGF, SPP1, MMP1 e THBS1. The biological interpretation of these data suggests that the population of MSC/inv has different constitutional characteristics, related to their potential for differentiation, proliferation and response to stimuli, responsible for a distinct process of replicative senescence in MSC/inv compared to MSC/n. The genes identified in this study are candidates for biomarkers of cellular senescence in MSC, but their functional relevance in this process should be evaluated in additional in vitro and/or in vivo assays

Efeito do tratamento térmico do titânio sobre a proliferação de células pré-osteoblásticas

Titanium is a biomaterial widely employed in biomedical applications (implants, prostheses, valves, stents). Several heat treatments are usually used in order to obtain physical properties required to different applications. This work studied the influence of the heat treatment on microstructure of commercial pure titanium, and their consequences in growth and proliferation of MC3T3-E1 cells. Discs of titanium were treated in different temperatures, and characterized by optical microscopy, image analysis, wettabillity, roughness, hardness and X-ray diffraction. After the heat treatment, significant modifications in these properties were observed. Pattern images of titanium, before and after the cell culture, were compared by overlapping to analyze the influence of microstructure in microstructure and preferences guidance cells. However, in general, titanium discs that showed a higher residual strength also presented an increase of cells numbers on surface

Células madre intratecales autólogas en niños con daño cerebral hipoxico/isquémico

La parálisis cerebral (PC) se puede presentar como consecuencia de una hipoxia perinatal severa, la cual deja una daño cerebral permanente en 50% de los recién nacidos prematuros que sobreviven este evento. La terapia celular es una opción terapéutica potencial para la PC, la cual se cree que funciona a través diversos mecanismos, los cuales incluyen la inmunomodulación a través de citocinas (C) y de la secreción de factores de crecimiento.Se incluyeron 18 pacientes con PC con el fin de evaluar la seguridad de la aplicación intratecal (IT) e intravenosa (IV) de células nucleadas totales (CNT) derivadas de médula ósea (MO) autóloga después de la estimulación con factor estimulador de colonias de granulocitos (FEC-G). Se evaluó clínicamente las áreas motora gruesa y fina, cognitiva, de comunicación, personal-social y adaptativa por medio de una prueba validada llamada Inventario del Desarrollo de Battelle (IDB). Se realizaron tres evaluaciones en cada paciente, la primera previa al procedimiento, y después a los 30 y a los 180 días. También se llevaron a cabo estudios de resonancia magnética nuclear (RMN) antes del procedimiento y uno de control 6 meses después del tratamiento.

Processamento e análise de imagens na caracterização da superfície do titânio submetido a um ensaio de cultura de células

This work aims to develop a methodology for analysis of images using overlapping, which assists in identification of microstructural features in areas of titanium, which may be associated with its biological response. That way, surfaces of titanium heat treated for 08 (eight) different ways have been subjected to a test culture of cells. It was a relationship between the grain, texture and shape of grains of surface of titanium (attacked) trying to relate to the process of proliferation and adhesion. We used an open source software for cell counting adhered to the surface of titanium. The juxtaposition of images before and after cell culture was obtained with the aid of micro-hardness of impressions made on the surface of samples. From this image where there is overlap, it is possible to study a possible relationship between cell growth with microstructural characteristics of the surface of titanium. This methodology was efficient to describe a set of procedures that are useful in the analysis of surfaces of titanium subjected to a culture of cells

Inmunoterapia autóloga con células dendríticas en modelos caninos con tumor venéreo transmisible

La inmunoterapia autóloga utiliza células del mismo cuerpo para estimular y restaurar las defensas naturales del sistema inmunológico. Algunas de las células que han sido utilizado en años recientes son: linfocitos infiltrantes de tumor, linfocitos T citotóxicos, células asesinas activadas por linfocinas y células dendríticas (CD). Las CD son células especializadas y su función es el capturar, procesar y presentar antígenos a los linfocitos para iniciar una respuesta inmune. El tumor venéreo transmisible (TVT), también conocido como sarcoma infeccioso o tumor de Sticker, es un cáncer transmisible en perros que afecta mayormente los genitales externos y se transmite durante el coito. En este trabajo se implementó por primera vez un modelo de TVT en el órgano genital (vagina) de los pacientes y se les administró la inmunoterapia autóloga con CD específicas contra el tumor. Para estudiar esta terapia se utilizaron tres grupos experimentales: el tumor sin tratamiento, el tumor tratado con sangre completa autóloga, y el tumor tratado con CD autólogas específicas para el TVT. Por último se evaluó la capacidad inmunológica del extracto tumoral total (ETT) del tumor como método de prevención in vivo. Para el tratamiento autólogo con las CD, se esperó que el tumor midiese 3cm, se realizó un cultivo primario de las células de TVT y se les extrajo el 4% de su peso corporal de sangre a los pacientes para realizar una diferenciación de los monocitos a CD. Para evaluar el efecto de la inmunoterapia autóloga con CD se observaron los efectos secundarios, el tamaño tumoral, las poblaciones de linfocitos, los niveles de IFN-γ en sangre y los linfocitos infiltrantes de tumor por histopatología. Los monocitos se diferenciaron a CD y se les realizó un análisis fenotípico mediante citometría de flujo. Los monocitos mostraron una expresión de CD14+ de 80.1%, CD80+ de 15.6%, CD83+ de 0.4% y un DLA II de1.8%. En las CD se obtuvo una expresión de 8.7% para CD14+, 80.3% para CD80+, 76.4% para CD83+ y 86.5% para DLA II y 62% en la prueba de fagocitosis. La terapia no mostró ningún efecto secundario en nuestro grupo experimental y hubo una regresión tumoral del 100% para la semana doce. Se encontró un aumento de expresión celular en sangre de CD4+ de 29%, de CD8+ de 34% y de IFN-γ de 120 pg/mL. Nuestros resultados demuestran que la inmunoterapia autóloga con CD específicas inducen una regresión del TVT en caninos.

Sinterização de Ce1-XEuXO2-(X/2) para aplicação como eletrólito sólido em células a combustível de temperaturas intermediárias

Given the environmental concern over global warming that occurs mainly by emission of CO2 from the combustion of petroleum, coal and natural gas research focused on alternative and clean energy generation has been intensified. Among these, the highlight the solid oxide fuel cell intermediate temperature (IT-SOFC). For application as electrolyte of the devices doped based CeO2 with rare earth ions (TR+ 3) have been quite promising because they have good ionic conductivity and operate at relatively low temperatures (500-800 ° C). In this work, studied the Ce1-xEuxO2-δ (x = 0,1, 0,2 and 0,3), solid solutions synthesized by the polymeric precursor method to be used as solid electrolyte. It was also studied the processing steps of these powders (milling, compaction and two step sintering) in order to obtain dense sintered pellets with reduced grain size and homogeneous microstructure. For this, the powders were characterized by thermal analysis, X-ray diffraction, particle size distribution and scanning electrons microscopy, since the sintered samples were characterized by dilatometry, scanning electrons microscopy, density and grain size measurements. By x-ray diffraction, it was verified the formation of the solid solution for all compositions. Crystallites in the nanometric scale were found for both sintering routes but the two step sintering presented significant reduction in the average grain size

Efecto citotóxico de hipoclorito de sodio y EDTA sobre células madre de pulpa dental

Introducción. La presente investigación surge de la importancia que implica la irrigación en relación a la regeneración del complejo dentino-pulpar. En la actualidad, una gran cantidad de artículos han enfocado sus objetivos en el efecto antibacteriano de soluciones irrigantes, sin embargo, son pocas las investigaciones que evalúan el efecto de dichas soluciones sobre las células madre presentes en la pulpa dental. Objetivo. Evaluar el efecto citotóxico de hipoclorito de sodio y EDTA sobre células madre de pulpa dental in vitro. Materiales y métodos. Se utilizaron Células madre de pulpa dental cortesía de la Unidad de Odontología Integral y Especialidades del Centro de Investigación y Desarrollo en Ciencias de la Salud. Para la evaluación de la citotoxicidad, se utilizó la solución de EDTA a concentraciones de 1.7%, 1%, 0.5% y 0.1% y NaOCl al 0.25%, 0.15%, 0.1% y 0.5% por triplicado. Ambas moléculas (EDTA y NaOCl) se incubaron con las células a 37° C, 5% de CO2 en una atmosfera a 95% de humedad durante 3 y 24 horas. Los controles negativos fueron incubados con PBS. Resultados. En los resultados se observó una alta toxicidad del NaOCl frente a las DPSCs a 3 y 24 horas de exposición. Así mismo se observó que la combinación del NaOCl y el EDTA no disminuyó el efecto citotóxico sobre las células, a excepción de la combinación de EDTA .1% + NaOCl .05% la cual presentó un ligero aumento en la viabilidad celular. Por otra parte, todas las concentraciones evaluadas de la solución de EDTA fueron menos toxicas cuando se incubaron durante 3 y 24 horas frente a las células. Conclusiones. De acuerdo a los resultados observados en el presente estudio podemos concluir lo siguiente: el NaOCl presentó alta citotoxicidad en todas sus concentraciones a las 3 y 24 horas de exposición, el EDTA presentó una mayor viabilidad a las 3 y 24 horas cuando se utilizó al 0.5% y 0.10%, la combinación del EDTA y NaOCl resultó menos citotóxica cuando se utilizó al 0.1% y 0.5% respectivamente a las 3 horas de exposición.

Silenciamento do canal de potássio Eag1 potencializa efeitos da temozolomida em células U-87 MG de glioblastoma multiforme

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2015.