RESUMEN
Few techniques are available to characterize in vivo the early cellular dynamics of long-term reconstitution of hematopoiesis after transplantation of hematopoietic stem cells (HSCs) after lethal irradiation. Using a fiber-optic imaging system, we track the early steps of in vivo recruitment and proliferation of Lin(-)Sca-1(+)c-Kit(+)CD34(-) (LSKCD34(-)) HSCs highly enriched in HSCs and transplanted into lethally irradiated mice. Recruitment of the transplanted LSKCD34(-) hematopoietic cells first occurs in the femoral head and is continuous during 24 hours. Quantification of the fluorescence emitted by the transplanted hematopoietic cells shows that proliferation of LSKCD34(-) hematopoietic cells in the femoral head was potent 3 days after transplantation. Using a development of this fiber-optic imaging system, we show that the transplanted LSKCD34(-) hematopoietic cells are associated with vascularized structures as early as 5 hours after transplantation. This early association is dependent on reactive oxygen species (ROS) partly through the regulation of vascular cell adhesion molecule-1 expression on endothelial cells and is followed by a ROS-dependent proliferation of LSKCD34(-) hematopoietic cells. This new in vivo imaging technique permits the observation of the early steps of hematopoietic reconstitution by HSCs in long bones and shows a new role of ROS in the recruitment of HSCs by bone marrow endothelial cells.
Asunto(s)
Hematopoyesis/efectos de los fármacos , Imagen Molecular/métodos , Especies Reactivas de Oxígeno/farmacología , Animales , Antígenos CD34/metabolismo , Antígenos Ly/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Proliferación Celular/efectos de los fármacos , Fémur/citología , Fémur/metabolismo , Fémur/fisiología , Tecnología de Fibra Óptica/métodos , Hematopoyesis/fisiología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/fisiología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Modelos Biológicos , Proteínas Proto-Oncogénicas c-kit/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estudios de Validación como Asunto , Irradiación Corporal TotalRESUMEN
In this study, we address generalized autonomous mobile robot exploration of unknown environments where a robotic agent learns a traversability model and builds a spatial model of the environment. The agent can benefit from the model learned online in distinguishing what terrains are easy to traverse and which should be avoided. The proposed solution enables the learning of multiple traversability models, each associated with a particular locomotion gait, a walking pattern of a multi-legged walking robot. We propose to address the simultaneous learning of the environment and traversability models by a decoupled approach. Thus, navigation waypoints are generated using the current spatial and traversability models to gain the information necessary to improve the particular model during the robot's motion in the environment. From the set of possible waypoints, the decision on where to navigate next is made based on the solution of the generalized traveling salesman problem that allows taking into account a planning horizon longer than a single myopic decision. The proposed approach has been verified in simulated scenarios and experimental deployments with a real hexapod walking robot with two locomotion gaits, suitable for different terrains. Based on the achieved results, the proposed method exploits the online learned traversability models and further supports the selection of the most appropriate locomotion gait for the particular terrain types.
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The purpose of this article is to compare two predictive methods of J integral assessments for center-cracked plates, single-edge cracked plates and double-edge cracked plates produced from X52 and X70 steels, and a longitudinally cracked pipe produced from X70 steel. The two methods examined are: the GSM method and the Js procedure of the French RCC-MR construction code, designated here as the FC method. The accuracy of J integral predictions by these methods is visualized by comparing the results obtained with the "reference" values calculated by the EPRI method. The main results showed that both methods yielded similar J integral values, although in most cases, the GSM predictions were slightly more conservative than the FC predictions. In comparison with the "reference" values of the J integral, both methods provided conservative results for most crack configurations, although the estimates for cracks of a relative length smaller than 1/8 were not found to be so conservative. The prediction of burst pressures for external longitudinal semielliptical part-through cracks in X70 steel pipe showed that the magnitudes of predicted burst pressures came very close to each other, and were conservative compared to FEM (finite element method) calculations and experimentally determined burst pressures.
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PURPOSE: To evaluate the heritability of intrinsic radiosensitivity, the induction of apoptosis in lymphocyte subpopulations was determined on samples from related individuals belonging to large kindred families. METHODS AND MATERIALS: Quiescent lymphocytes from 334 healthy individuals were gamma-irradiated in vitro. Apoptosis was determined 18 h after irradiation by eight-color flow cytometry. Radiosensitivity was quantified from dose-effect curves. Intrafamilial correlations and heritability were computed for 199 father-mother-offspring trios using the programs SOLAR (Sequential Oligogenic Linkage Analysis Routines) and SAGE (Statistical Analysis for Genetic Epidemiology). Segregation analyses were conducted using SAGE. RESULTS: Marked differential susceptibility of naive and memory T lymphocytes was demonstrated. Also, although age and gender were significant covariates, their effects only accounted for a minor part of the inter-individual variation. Parent-offspring and sib-sib correlations were significant for the radiosensitivity of B cells, T4, and T8 and of effector memory T4 and T8 subpopulations. In the T4-effector memory subpopulation, the phenotype showed correlations most consistent with dominant or additive genetic effects, and the results of the segregation analysis were consistent with the contribution of a bi-allelic dominant locus. CONCLUSIONS: Heritability was demonstrated for the susceptibility to ionizing radiation-induced apoptosis of lymphocyte populations, and the segregation of the T4-effector memory radiosensitivity phenotype was consistent with a simple mendelian transmission model involving one major gene.
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Apoptosis/genética , Linfocitos B/efectos de la radiación , Linfocitos T CD4-Positivos/efectos de la radiación , Linfocitos T CD8-positivos/efectos de la radiación , Tolerancia a Radiación/genética , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Relación Dosis-Respuesta en la Radiación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Factores Sexuales , Hermanos , EspososRESUMEN
In his program of thematic one-day workshops, the French Association of Cytometry had organized a workshop dedicated to the fluorescence compensation settings in multicolor flow cytometry. This special day was in honor of our past President Jean Luc D'Hautcourt who has been involved in the quality of the use of flow cytometry in its clinical and research purposes. Review on fluorescence phenomena, compensation rules, settings, and few observed confounding situations were presented.
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Congresos como Asunto , Citometría de Flujo/métodos , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Factores de Confusión Epidemiológicos , Fluorescencia , Colorantes Fluorescentes/análisis , Inmunofenotipificación , FotoblanqueoRESUMEN
OBJECTIVE: Clock genes are known to mediate circadian rhythms in the central nervous system and peripheral organs. Although they are expressed in mouse hematopoietic progenitor and stem cells, it is unknown if they are related to circadian rhythms in these cells. We therefore investigated the 24-hour patterns in the activity of several clock genes in the bone marrow (BM) side population (SP) primitive stem cells, and compared these 24-hour patterns to clock gene variations in the whole BM and liver. METHODS: Cells were obtained from 84 B6D2F(1) mice in three replicate experiments on the second day after release into constant darkness from a standardizing light-dark schedule. mRNA expression of clock genes was measured with quantitative reverse transcriptase polymerase chain reaction. RESULTS: mPer2 displayed circadian rhythms in SP cells, whole BM, and liver cells. mPer1 and mRev-erb alpha showed a circadian rhythm in whole BM and liver, but not SP cells. mBmal1 was not expressed rhythmically in SP cells, nor in the whole BM, contrary to rhythms observed in the liver. CONCLUSIONS: With the exception of mPer2, most clock genes studied in primitive hematopoietic SP stem cells were not oscillating in a fully organized circadian manner, which is similar to immature cells in rapidly proliferating organs, such as the testis and thymus. These findings indicate that circadian clock gene expression variations in BM are developmentally regulated.
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Relojes Biológicos/fisiología , Ritmo Circadiano/fisiología , Regulación de la Expresión Génica/fisiología , Células Madre Hematopoyéticas/fisiología , Proteínas Nucleares/biosíntesis , Factores de Transcripción/biosíntesis , Animales , Proteínas de Ciclo Celular , Femenino , Perfilación de la Expresión Génica/métodos , Células Madre Hematopoyéticas/citología , Masculino , Ratones , Proteínas Nucleares/genética , Especificidad de Órganos/fisiología , Proteínas Circadianas Period , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Transcripción/genéticaRESUMEN
PURPOSE: With the aim to evaluate intrinsic radiosensitivity, the susceptibility of lymphocyte subpopulations to radiation-induced apoptosis was determined. The investigated parameters included measurement reliability, phenotypic variance, intra- and inter-individual variability, and correlations between radiation-induced and spontaneous apoptosis. METHODS AND MATERIALS: Quiescent lymphocytes of 63 healthy volunteers, sampled up to four times over a 1-year period were gamma-irradiated in vitro. Subsequent apoptosis (annexin V) was measured for T4-, T8-, and B-lymphocyte subpopulations using 6-color flow cytometry. Spontaneous apoptosis was measured and radiosensitivity was quantified from the dose-effect curves. RESULTS: After thawing and short-term culture, both spontaneous apoptosis as well as radiation-induced apoptosis (radiosensitivity) differed among the three lymphocyte subpopulations, with T4 being most resistant, and B most sensitive. Spontaneous and radiation-induced apoptosis were correlated in all cell types, and variance between individuals was considerably higher than variance within individuals for both. A small but highly significant increase of both spontaneous and radiation-induced apoptosis was observed with age for T8, but not for T4 and B. Radiosensitivity of T8 and B proved to be sex-independent, whereas female T4 lymphocytes were less radiosensitive than those from males. T4 and T8 radiosensitivities were loosely correlated, and neither of them was related to B radiosensitivity. CONCLUSION: Tendency to spontaneous and radiation-induced apoptosis of lymphocyte subpopulations differs among individuals. In addition, depending on the cell types, age and sex are factors influencing these parameters.
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Apoptosis/fisiología , Linfocitos B/efectos de la radiación , Linfocitos T CD4-Positivos/efectos de la radiación , Linfocitos T CD8-positivos/efectos de la radiación , Tolerancia a Radiación , Linfocitos B/fisiología , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Citometría de Flujo , Humanos , Análisis de RegresiónRESUMEN
We have devised a simple method to purify mitotically active Schwann cells (SC) from peripheral nerves of adult mice. Nerves were predegenerated in vitro for 7 days and after dissociation cells were plated on poly-L-lysine/laminin coated dishes in N2 serum-free culture medium supplemented with forskolin and heregulin-beta1. Primary cultures were purified from contaminating fibroblasts by magnetic cell sorting (MACS) based on SC membrane specific expression of p75(NGFR) and enriched to about 99% of SC after MACS from 34 to 91% before sorting. After sorting, purified adult mouse SC were propagated for three passages until confluent to a total surface of 160 cm(2) per mouse (two sciatic and two trigeminal nerves). In addition, we show that this method can be used to purify tumoral SC from mouse NF2-related schwannomas.
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Citometría de Flujo/métodos , Magnetismo/instrumentación , Nervios Periféricos/citología , Células de Schwann/citología , Animales , División Celular/fisiología , Células Cultivadas , Ratones , Nervios Periféricos/metabolismo , Células de Schwann/metabolismoRESUMEN
The decision to use chemotherapy in the treatment of colon cancer patients depends on the risk of developing metastases, as estimated by clinicopathological staging combining body imaging and pathological findings. The aim of this study was to identify all chromosome arms that, when allelotyped, correlate with the metastatic process, add prognostic information to pathology, and are of relevance for predicting metachronous metastases. A 5-year follow-up survey enrolled 401 MSS (microsatellite stable) colon cancer patients who were divided into three groups. Staging was performed with and without imaging data (called tumor and patient staging, respectively). The first 192 patients were used to construct a model prognosticating metastases. The subsequent 146 patients were used to validate this model. The third group evaluated its consistency by comparing the status of the relevant chromosome arms in 63 liver metastases and primary tumors that did or did not metastasize. The first group identified three factors: tumor staging (P < 0.0001), 5q status (P = 0.003), and gender (P = 0.02). The second group confirmed 5q as a marker of metastasis occurrence (P = 0.004). Merged data predicted that, when both 5q arms are retained, metastatic risk increases 4.3-fold in stage II patients. The third group corroborated these findings, with a 5q retention rate in metastases comparable to that of primary tumors that metastasize, but significantly higher than that observed in nonmetastatic tumors (one-tail, P = 0.0005). Long arm of chromosome 5 allelotyping detects high-risk stage II tumors. This simple, easily implemented, and inexpensive test increases the power of randomized studies that evaluate chemotherapy.
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Deleción Cromosómica , Cromosomas Humanos Par 5 , Neoplasias del Colon/genética , Neoplasias Hepáticas/genética , Anciano , Neoplasias del Colon/patología , Femenino , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/secundario , Pérdida de Heterocigocidad , Masculino , Repeticiones de Microsatélite , Estadificación de Neoplasias , Valor Predictivo de las PruebasRESUMEN
Previous studies have indicated that single relative telomere lengths are defined in the zygote. In order to explore the possibility that single telomere lengths segregate in families, we compared relative telomere lengths obtained from allelic chromosome extremities transmitted from parent to child, representing a total of 31 independent meiotic events. We find a significant positive correlation of 0.65 (P=0.0004) between these telomere lengths, whereas the correlation between the non-transmitted parental homologue and the transmitted homologue in the child is not statistically significant (r=0.16; P=0.195). This study indicates that, even though there is a telomerase-mediated maintenance/elongation of telomeres in germ cells, allele-specific relative telomere lengths are preserved in the next generation.