Desarrollo y estandarización de un banco de sangre de cordón umbilical

Tesis de la Universidad Complutense de Madrid, Facultad de Medicina, Departamento de Medicina, leída el 16-05-2000El objetivo principal de este estudio ha sido la evaluación y estandarización de protocolos para la creación de un Banco de Sangre de Cordón Umbilical(SCU), habiendo sido desglosado en dos objetivos parciales: la caracterización de la SCU mediante estudio experimental de las propiedades hematopoyéticas e inmunológicas de la misma, y la puesta a punto del procesamiento de SCU para su validación como unidades trasplantables. Se ha realizado un análisis fenotípico y de clonogenicidad de células hematopoyéticas, así como un estudio fenotípico y funcional de poblaciones linfocitarias de SCU, comparándose con muestras control de médula ósea(MO) y sangre periférica de adultos(SPA). Respecto al segundo objetivo del trabajo, se ha hecho un estudio comparativo de diferentes protocolos de obtención, fracionamiento y congelación con el fin de preservar la máxima celularidad y viabilidad en las unidades procesadas. Asimismo, para su validación definitiva, se han definido criterios normalizados que incluyen la tipificación del complejo HLA con técnicas de alta resolución y la detección de posibles enfermedades transmisibles que garantice la seguridad en los eventuales receptores de trasplante. Las conclusiones globales de este estudio son:1) la proporción de células progenitoras hematopoyéticas es superior en SCU que en MO y SPA movilizada, lo que sugiere un mayor potencial de repoblación hematopoyética;2) los linfocitos de SCU muestran una relativa inmadurez fenotípica y funcional que permite predecir un menor potencial de enfermedad injerto contra huesped asociado a la SCU;3) la combinación de venopunción y perfusión de la placenta enriquece significativamente la celularidad de las unidades; 4) la SCU conservada a 4º C más de 12 horas sufre una reducción importante en la clonogenicidad y viabilidad celular;5) se ha optimizado la estandarización de métodos de procesamiento basada en el empleo de hidroxietil almidón para el fraccionamiento, bolsas de congelación de 24 ml. Inmersión en nitrógeno líquido y solución de albúmina/dextrano en la descongelación de las unidades, con los que se consigue preservar casi la totalidad de su potencial hematopoyético; 6) los criterios aplicados en la validación garantizan su idoneidad y una óptima selección de parejas donante/receptor. Como consecuencia de este estudio,se ha establecido un Banco de SCU en el Hospital 12 de Octubre de Madrid con la inclusión, hasta el momento, de 139 unidades validadas y potencialmente elegibles, en el contexto del REDMO, para su utilización como fuente de células progenitoras hematopoyéticas para trasplante.Fac. de MedicinaTRUEpu

Dexamethasone sensitizes to ferroptosis by glucocorticoid receptor-induced dipeptidase-1 expression and glutathione depletion

Dexamethasone is widely used as an immunosuppressive therapy and recently as COVID-19 treatment. Here, we demonstrate that dexamethasone sensitizes to ferroptosis, a form of iron-catalyzed necrosis, previously suggested to contribute to diseases such as acute kidney injury, myocardial infarction, and stroke, all of which are triggered by glutathione (GSH) depletion. GSH levels were significantly decreased by dexamethasone. Mechanistically, we identified that dexamethasone up-regulated the GSH metabolism regulating protein dipeptidase-1 (DPEP1) in a glucocorticoid receptor (GR)-dependent manner. DPEP1 knockdown reversed the phenotype of dexamethasone-induced ferroptosis sensitization. Ferroptosis inhibitors, the DPEP1 inhibitor cilastatin, or genetic DPEP1 inactivation reversed the dexamethasone-induced increase in tubular necrosis in freshly isolated renal tubules. Our data indicate that dexamethasone sensitizes to ferroptosis by a GR-mediated increase in DPEP1 expression and GSH depletion. Together, we identified a previously unknown mechanism of glucocorticoid-mediated sensitization to ferroptosis bearing clinical and therapeutic implications

CSL–MAML-dependent Notch1 signaling controls T lineage–specific IL-7Rα gene expression in early human thymopoiesis and leukemia

Notch1 activation is essential for T-lineage specification of lymphomyeloid progenitors seeding the thymus. Progression along the T cell lineage further requires cooperative signaling provided by the interleukin 7 receptor (IL-7R), but the molecular mechanisms responsible for the dynamic and lineage-specific regulation of IL-7R during thymopoiesis are unknown. We show that active Notch1 binds to a conserved CSL-binding site in the human IL7R gene promoter and critically regulates IL7R transcription and IL-7R α chain (IL-7Rα) expression via the CSL–MAML complex. Defective Notch1 signaling selectively impaired IL-7Rα expression in T-lineage cells, but not B-lineage cells, and resulted in a compromised expansion of early human developing thymocytes, which was rescued upon ectopic IL-7Rα expression. The pathological implications of these findings are demonstrated by the regulation of IL-7Rα expression downstream of Notch1 in T cell leukemias. Thus, Notch1 controls early T cell development, in part by regulating the stage- and lineage-specific expression of IL-7Rα

A Novel, Non-Apoptotic Role for Scythe/BAT3: A Functional Switch between the Pro- and Anti-Proliferative Roles of p21 during the Cell Cycle

BACKGROUND: Scythe/BAT3 is a member of the BAG protein family whose role in apoptosis has been extensively studied. However, since the developmental defects observed in Bat3-null mouse embryos cannot be explained solely by defects in apoptosis, we investigated whether BAT3 is also involved in cell-cycle progression. METHODS/PRINCIPAL FINDINGS: Using a stable-inducible Bat3-knockdown cellular system, we demonstrated that reduced BAT3 protein level causes a delay in both G1/S transition and G2/M progression. Concurrent with these changes in cell-cycle progression, we observed a reduction in the turnover and phosphorylation of the CDK inhibitor p21, which is best known as an inhibitor of DNA replication; however, phosphorylated p21 has also been shown to promote G2/M progression. Our findings indicate that in Bat3-knockdown cells, p21 continues to be synthesized during cell-cycle phases that do not normally require p21, resulting in p21 protein accumulation and a subsequent delay in cell-cycle progression. Finally, we showed that BAT3 co-localizes with p21 during the cell cycle and is required for the translocation of p21 from the cytoplasm to the nucleus during the G1/S transition and G2/M progression. CONCLUSION: Our study reveals a novel, non-apoptotic role for BAT3 in cell-cycle regulation. By maintaining a low p21 protein level during the G1/S transition, BAT3 counteracts the inhibitory effect of p21 on DNA replication and thus enables the cells to progress from G1 to S phase. Conversely, during G2/M progression, BAT3 facilitates p21 phosphorylation by cyclin A/Cdk2, an event required for G2/M progression. BAT3 modulates these pro- and anti-proliferative roles of p21 at least in part by regulating cyclin A abundance, as well as p21 translocation between the cytoplasm and the nucleus to ensure that it functions in the appropriate intracellular compartment during each phase of the cell cycle.Dissertatio

Desarrollo y estandarización de un banco de sangre de cordón umbilical

El objetivo principal de este estudio ha sido la evaluación y estandarización de protocolos para la creación de un Banco de Sangre de Cordón Umbilical(SCU), habiendo sido desglosado en dos objetivos parciales: la caracterización de la SCU mediante estudio experimental de las propiedades hematopoyéticas e inmunológicas de la misma, y la puesta a punto del procesamiento de SCU para su validación como unidades trasplantables. Se ha realizado un análisis fenotípico y de clonogenicidad de células hematopoyéticas, así como un estudio fenotípico y funcional de poblaciones linfocitarias de SCU, comparándose con muestras control de médula ósea(MO) y sangre periférica de adultos(SPA). Respecto al segundo objetivo del trabajo, se ha hecho un estudio comparativo de diferentes protocolos de obtención, fracionamiento y congelación con el fin de preservar la máxima celularidad y viabilidad en las unidades procesadas. Asimismo, para su validación definitiva, se han definido criterios normalizados que incluyen la tipificación del complejo HLA con técnicas de alta resolución y la detección de posibles enfermedades transmisibles que garantice la seguridad en los eventuales receptores de trasplante. Las conclusiones globales de este estudio son:1) la proporción de células progenitoras hematopoyéticas es superior en SCU que en MO y SPA movilizada, lo que sugiere un mayor potencial de repoblación hematopoyética;2) los linfocitos de SCU muestran una relativa inmadurez fenotípica y funcional que permite predecir un menor potencial de enfermedad injerto contra huesped asociado a la SCU;3) la combinación de venopunción y perfusión de la placenta enriquece significativamente la celularidad de las unidades; 4) la SCU conservada a 4º C más de 12 horas sufre una reducción importante en la clonogenicidad y viabilidad celular;5) se ha optimizado la estandarización de métodos de procesamiento basada en el empleo de hidroxietil almidón para el fraccionamiento, bolsas de congelación de 24 ml. Inmersión en nitrógeno líquido y solución de albúmina/dextrano en la descongelación de las unidades, con los que se consigue preservar casi la totalidad de su potencial hematopoyético; 6) los criterios aplicados en la validación garantizan su idoneidad y una óptima selección de parejas donante/receptor. Como consecuencia de este estudio,se ha establecido un Banco de SCU en el Hospital 12 de Octubre de Madrid con la inclusión, hasta el momento, de 139 unidades validadas y potencialmente elegibles, en el contexto del REDMO, para su utilización como fuente de células progenitoras hematopoyéticas para trasplante

Comparative analysis of the pathogenic mechanisms associated with the G8363A and A8296G mutations in the mitochondrial tRNA(Lys) gene

6 pages, 4 figures, 1 table.-- et al.Two mutations (G8363A and A8296G) in the mtDNA (mitochondrial DNA) tRNA(Lys) gene have been associated with severe mitochondrial diseases in a number of reports. Their functional significance, however, remains unknown. We have already shown that homoplasmic cybrids harbouring the A8296G mutation display normal oxidative phosphorylation, although the possibility of a subtle change in mitochondrial respiratory capacity remains an open issue. We have now investigated the pathogenic mechanism of another mutation in the tRNA(Lys) gene (G8363A) by repopulating an mtDNA-less human osteosarcoma cell line with mitochondria harbouring either this genetic variant alone or an unusual combination of the two mutations (A8296G+G8363A). Cybrids homoplasmic for the single G8363A or the A8296G+G8363A mutations have defective respiratory-chain enzyme activities and low oxygen consumption, indicating a severe impairment of the oxidative phosphorylation system. Generation of G8363A cybrids within a wild-type or the A8296G mtDNA genetic backgrounds resulted in an important alteration in the conformation of the tRNA(Lys), not affecting tRNA steady-state levels. Moreover, mutant cybrids have an important decrease in the proportion of amino-acylated tRNA(Lys) and, consequently, mitochondrial protein synthesis is greatly decreased. Our results demonstrate that the pathogenicity of the G8363A mutation is due to a change in the conformation of the tRNA that severely impairs aminoacylation in the absence of changes in tRNA stability. The only effect detected in the A8296G mutation is a moderate decrease in the aminoacylation capacity, which does not affect mitochondrial protein biosynthesis.This work was supported by grants from Ministerio de Educación y Ciencia of Spain, Comunidad de Madrid and Instituto de Salud Carlos III, Redes de centros RCMN (C03/08) and Temáticas (G03/011).Peer reviewe

Mitochondrial gene expression and respiratory enzyme activities in cardiac diseases

Respiratory enzyme activities and steady-state level of two mitochondrial-encoded transcripts were quantified in heart muscle biopsies from patients suffering various types of cardiomyopathies unrelated to mitochondrial primary disorders. We have found that although the mitochondrial DNA copy number and the concentration of COI and ND4 transcripts remain fairly constant, there is an important increase (up to 6-fold) in respiratory enzyme activities affecting to several oxidative phosphorylation complexes. Idiopathic dilated cardiomyopathy shows the greatest increase, followed by ischemic heart and ventricular hypertrophy due to aortic stenosis. The results suggest an energetic compensatory mechanism in the heart muscle, in the absence of mitochondrial proliferation or activation of mitochondrial gene expression.This paper was supported by grants of the Fondo de Investigaciones Sanitarias de la Seguridad Social FISss 93/0339 and 95/0658 and Comunidad de Madrid 07/043/96.Peer Reviewe

A novel E2 box-GATA element modulates Cdc6 transcription during human cells polyploidization

Cdc6 is a key regulator of the strict alternation of S and M phases during the mitotic cell cycle. In mammalian and plant cells that physiologically become polyploid, cdc6 is transcriptionally and post-translationally regulated. We have recently reported that Cdc6 levels are maintained in megakaryoblastic HEL cells, but severely downregulated by ectopic expression of transcriptional repressor Drosophila melanogaster escargot. Here, we show that cdc6 promoter activity is upregulated during megakaryocytic differentiation of HEL endoreplicating cells, and that Escargot interferes with such activation. Transactivation experiments showed that a 1.7 kb region located at 2800 upstream cdc6 transcription initiation site behaved as a potent enhancer in endoreplicating cells only. This activity was mainly dependent on a novel cis-regulatory element composed by an E2 box overlapping a GATA motif. Ectopic Escargot could bind this regulatory element in vitro and endogenous GATA-1 and E2A formed specific complexes in megakaryoblastic cells as well as in primary megakaryocytes. Chromatin Immunoprecipitation analysis revealed that both transcription factors were occupying the E2 box/GATA site in vivo. Altogether, these data suggest that cdc6 expression could be actively maintained during megakaryocytic differentiation through transcriptional mechanisms involving specific cis- and trans-regulatory elements