Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 47
Filtrar
Más filtros

Banco de datos
País/Región como asunto
Tipo del documento
Intervalo de año de publicación
1.
Br J Haematol ; 200(3): 367-376, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36221231

RESUMEN

Hydroxyurea (HU) (hydroxycarbamide) is used as a therapeutic option in ß-thalassaemia to increase fetal haemoglobin, which results in a reduced requirement for blood transfusion. However, a potential serious adverse effect of HU is neutropenia. Abnormal neutrophil maturation and function in ß-thalassaemia/HbE patients are well documented. This raises questions about the effect of the drug with regards to the immune response these patients. This study investigated the effects of HU treatment on both innate and adaptive immunity in a cross-sectional study of 28 ß-thalassaemia/HbE patients who had received HU treatment (BE+HU) as compared with 22 ß-thalassaemia/HbE patients who had not received HU (BE-HU) and 26 normal subjects. The expression of PU.1 and C/EBPß, transcription factors, which are associated with neutrophil maturation, was significantly reduced in BE+HU patients as compared with BE-HU patients and normal subjects. Interestingly, C3bR expression on neutrophils and their oxidative burst activity in BE+HU were restored to close to normal levels when compared with BE-HU. There was no observed effect of HU on monocytes, myeloid derived suppressor cells (both granulocytic and monocytic subsets), CD4+ T cells, CD8+ T cells, complement levels and serum immunoglobulin levels in this study. The full immunophenotyping analysis in this study indicates that HU therapy in ß-thalassaemia/HbE patients does not significantly compromise the immune response.


Asunto(s)
Hidroxiurea , Talasemia beta , Humanos , Hidroxiurea/efectos adversos , Linfocitos T CD8-positivos , Estudios Transversales , Inmunofenotipificación , Inmunidad
2.
Mol Ther ; 29(9): 2841-2853, 2021 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-33940155

RESUMEN

A primary challenge in lentiviral gene therapy of ß-hemoglobinopathies is to maintain low vector copy numbers to avoid genotoxicity while being reliably therapeutic for all genotypes. We designed a high-titer lentiviral vector, LVß-shα2, that allows coordinated expression of the therapeutic ßA-T87Q-globin gene and of an intron-embedded miR-30-based short hairpin RNA (shRNA) selectively targeting the α2-globin mRNA. Our approach was guided by the knowledge that moderate reduction of α-globin chain synthesis ameliorates disease severity in ß-thalassemia. We demonstrate that LVß-shα2 reduces α2-globin mRNA expression in erythroid cells while keeping α1-globin mRNA levels unchanged and ßA-T87Q-globin gene expression identical to the parent vector. Compared with the first ßA-T87Q-globin lentiviral vector that has received conditional marketing authorization, BB305, LVß-shα2 shows 1.7-fold greater potency to improve α/ß ratios. It may thus result in greater therapeutic efficacy and reliability for the most severe types of ß-thalassemia and provide an improved benefit/risk ratio regardless of the ß-thalassemia genotype.


Asunto(s)
Vectores Genéticos/administración & dosificación , ARN Interferente Pequeño/genética , Globinas alfa/genética , Globinas beta/genética , Talasemia beta/genética , Línea Celular , Células Cultivadas , Regulación hacia Abajo , Células Eritroides/citología , Células Eritroides/metabolismo , Genotipo , Humanos , Células K562 , Lentivirus/genética , Lentivirus/fisiología , MicroARNs/antagonistas & inhibidores , Cultivo Primario de Células , Carga Viral , Talasemia beta/terapia
3.
Br J Haematol ; 194(1): 200-210, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33942901

RESUMEN

Beta-thalassaemia is an inherited blood disorder characterised by ineffective erythropoiesis and anaemia. Consequently, hepcidin expression is reduced resulting in increased iron absorption and primary iron overload. Hepcidin is under the negative control of transmembrane serine protease 6 (TMPRSS6) via cleavage of haemojuvelin (HJV), a co-receptor for the bone morphogenetic protein (BMP)-mothers against decapentaplegic homologue (SMAD) signalling pathway. Considering the central role of the TMPRSS6/HJV/hepcidin axis in iron homeostasis, the inhibition of TMPRSS6 expression represents a promising therapeutic strategy to increase hepcidin production and ameliorate anaemia and iron overload in ß-thalassaemia. In the present study, we investigated a small interfering RNA (siRNA) conjugate optimised for hepatic targeting of Tmprss6 (SLN124) in ß-thalassaemia mice (Hbbth3/+ ). Two subcutaneous injections of SLN124 (3 mg/kg) were sufficient to normalise hepcidin expression and reduce anaemia. We also observed a significant improvement in erythroid maturation, which was associated with a significant reduction in splenomegaly. Treatment with the iron chelator, deferiprone (DFP), did not impact any of the erythroid parameters. However, the combination of SLN124 with DFP was more effective in reducing hepatic iron overload than either treatment alone. Collectively, we show that the combination therapy can ameliorate several disease symptoms associated with chronic anaemia and iron overload, and therefore represents a promising pharmacological modality for the treatment of ß-thalassaemia and related disorders.


Asunto(s)
Deferiprona/uso terapéutico , Eritropoyesis/efectos de los fármacos , Hepcidinas/biosíntesis , Quelantes del Hierro/uso terapéutico , Sobrecarga de Hierro/prevención & control , Proteínas de la Membrana/antagonistas & inhibidores , ARN Interferente Pequeño/uso terapéutico , Talasemia beta/tratamiento farmacológico , Acetilgalactosamina/administración & dosificación , Animales , Deferiprona/administración & dosificación , Modelos Animales de Enfermedad , Quimioterapia Combinada , Femenino , Perfilación de la Expresión Génica , Hepcidinas/genética , Humanos , Hierro/sangre , Quelantes del Hierro/administración & dosificación , Sobrecarga de Hierro/etiología , Hígado/metabolismo , Magnesio/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Especies Reactivas de Oxígeno , Serina Endopeptidasas/genética , Bazo/metabolismo , Bazo/ultraestructura , Zinc/metabolismo , Talasemia beta/complicaciones , Talasemia beta/metabolismo , Talasemia beta/fisiopatología
4.
Br J Haematol ; 189(1): 187-198, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31884679

RESUMEN

Severe bacterial infection is a major complication causing morbidity and mortality in ß-thalassaemia/HbE patients. Innate immunity constitutes the first line of defence against bacterial infection. This study aimed to comprehensively investigate the innate immune phenotype and function related to factors predisposing to infection in non-transfusion-dependent (NTD) ß°-thalassaemia/HbE patients. Twenty-six patients and 17 healthy subjects were recruited to determine complement activity (C3, C4, mannose-binding lectin and CH50) and surface receptor expression including markers of phagocytosis (CD11b, CD16 and C3bR), inflammation (C5aR) and migration (CD11b, CXCR1 and CXCR2) on neutrophils and monocytes. In addition, phagocytosis and oxidative burst activity of neutrophils and monocytes against Escherichia coli and neutrophil migration were examined. Decreased C3 and surface expression of CD11b and C3bR on neutrophils were found in patients. However, phagocytosis of neutrophils in patients was still in the normal range. Interestingly, patients displayed a significant reduction of surface expression of CXCR2 [1705 ± 217 mean fluorescent intensity (MFI)] on neutrophils, leading to impaired neutrophil migration (9·2 ± 7·7%) when compared to neutrophils from healthy subjects (2261 ± 627 MFI and 27·8 ± 9% respectively). Moreover, surface expression of CXCR2 on neutrophils was associated with splenectomy status, serum ferritin and haemoglobin levels. Therefore, impaired neutrophil migration could contribute to the increased susceptibility to infection seen in NTD ß°-thalassaemia/HbE patients.


Asunto(s)
Movimiento Celular , Ferritinas/sangre , Regulación de la Expresión Génica , Hemoglobina E/metabolismo , Neutrófilos/metabolismo , Receptores de Interleucina-8B/sangre , Talasemia beta/sangre , Adolescente , Adulto , Proteínas del Sistema Complemento/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Monocitos/patología , Neutrófilos/patología , Fagocitosis , Esplenectomía , Talasemia beta/patología , Talasemia beta/cirugía
5.
Blood ; 129(23): 3087-3099, 2017 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-28325862

RESUMEN

ß-Thalassemia is associated with several abnormalities of the innate immune system. Neutrophils in particular are defective, predisposing patients to life-threatening bacterial infections. The molecular and cellular mechanisms involved in impaired neutrophil function remain incompletely defined. We used the Hbbth3/+ ß-thalassemia mouse and hemoglobin E (HbE)/ß-thalassemia patients to investigate dysregulated neutrophil activity. Mature neutrophils from Hbbth3/+ mice displayed a significant reduction in chemotaxis, opsonophagocytosis, and production of reactive oxygen species, closely mimicking the defective immune functions observed in ß-thalassemia patients. In Hbbth3/+ mice, the expression of neutrophil CXCR2, CD11b, and reduced NAD phosphate oxidase components (p22phox, p67phox, and gp91phox) were significantly reduced. Morphological analysis of Hbbth3/+ neutrophils showed that a large percentage of mature phenotype neutrophils (Ly6GhiLy6Clow) appeared as band form cells, and a striking expansion of immature (Ly6GlowLy6Clow) hyposegmented neutrophils, consisting mainly of myelocytes and metamyelocytes, was noted. Intriguingly, expression of an essential mediator of neutrophil terminal differentiation, the ets transcription factor PU.1, was significantly decreased in Hbbth3/+ neutrophils. In addition, in vivo infection with Streptococcus pneumoniae failed to induce PU.1 expression or upregulate neutrophil effector functions in Hbbth3/+ mice. Similar changes to neutrophil morphology and PU.1 expression were observed in splenectomized and nonsplenectomized HbE/ß-thalassemia patients. This study provides a mechanistic insight into defective neutrophil maturation in ß-thalassemia patients, which contributes to deficiencies in neutrophil effector functions.


Asunto(s)
Neutrófilos/inmunología , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Talasemia beta/genética , Talasemia beta/inmunología , Adulto , Animales , Antígeno CD11b/metabolismo , Estudios de Casos y Controles , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Quimiotaxis de Leucocito , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Persona de Mediana Edad , Activación Neutrófila , Neutrófilos/metabolismo , Neutrófilos/patología , Infecciones Neumocócicas/genética , Infecciones Neumocócicas/inmunología , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/inmunología , Especies Reactivas de Oxígeno/metabolismo , Receptores de Interleucina-8B/metabolismo , Transactivadores/deficiencia , Transactivadores/inmunología , Adulto Joven , Talasemia beta/patología
6.
Am J Physiol Endocrinol Metab ; 311(1): E214-23, 2016 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-27245334

RESUMEN

Previously, ß-thalassemia, an inherited anemic disorder with iron overload caused by loss-of-function mutation of ß-globin gene, has been reported to induce osteopenia and impaired whole body calcium metabolism, but the pathogenesis of aberrant calcium homeostasis remains elusive. Herein, we investigated how ß-thalassemia impaired intestinal calcium absorption and whether it could be restored by administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or hepcidin, the latter of which was the liver-derived antagonist of intestinal iron absorption. The results showed that, in hemizygous ß-globin knockout (BKO) mice, the duodenal calcium transport was lower than that in wild-type littermates, and severity was especially pronounced in female mice. Both active and passive duodenal calcium fluxes in BKO mice were found to be less than those in normal mice. This impaired calcium transport could be restored by 7-day 1,25(OH)2D3 treatment. The 1,25(OH)2D3-induced calcium transport was diminished by inhibitors of calcium transporters, e.g., L-type calcium channel, NCX1, and PMCA1b, as well as vesicular transport inhibitors. Interestingly, the duodenal calcium transport exhibited an inverse correlation with transepithelial iron transport, which was markedly enhanced in thalassemic mice. Thus, 3-day subcutaneous hepcidin injection and acute direct hepcidin exposure in the Ussing chamber were capable of restoring the thalassemia-associated impairment of calcium transport; however, the positive effect of hepcidin on calcium transport was completely blocked by proteasome inhibitors MG132 and bortezomib. In conclusion, both 1,25(OH)2D3 and hepcidin could be used to alleviate the ß-thalassemia-associated impairment of calcium absorption. Therefore, our study has shed light on the development of a treatment strategy to rescue calcium dysregulation in ß-thalassemia.


Asunto(s)
Calcitriol/farmacología , Calcio/metabolismo , Duodeno/efectos de los fármacos , Hepcidinas/farmacología , Absorción Intestinal/efectos de los fármacos , Hierro/metabolismo , Talasemia beta/metabolismo , Animales , Bortezomib/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Duodeno/metabolismo , Femenino , Hemicigoto , Leupeptinas/farmacología , Masculino , Ratones , Ratones Noqueados , ATPasas Transportadoras de Calcio de la Membrana Plasmática/antagonistas & inhibidores , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Proteínas de Transporte Vesicular/antagonistas & inhibidores , Globinas beta/genética , Talasemia beta/genética
8.
FASEB J ; 28(4): 1610-20, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24371119

RESUMEN

The clinical symptoms of hemoglobin disorders such as ß-thalassemia and sickle cell anemia are significantly ameliorated by the persistent expression of γ-globin after birth. This knowledge has driven the discovery of important regulators that silence γ-globin postnatally. Improved understanding of the γ- to ß-globin switching mechanism holds the key to devising targeted therapies for ß-hemoglobinopathies. To further investigate this mechanism, we used the murine erythroleukemic (MEL) cell line containing an intact 183-kb human ß-globin locus, in which the (G)γ- and ß-globin genes are replaced by DsRed and eGFP fluorescent reporters, respectively. Following RNA interference (RNAi)-mediated knockdown of two key transcriptional regulators, Myb and BCL11A, we observed a derepression of γ-globin, measured by DsRed fluorescence and qRT-PCR (P<0.001). Interestingly, double knockdown of Myb and DNA methyltransferase 1 (DNMT1) resulted in a robust induction of ε-globin, (up to 20% of total ß-like globin species) compared to single knockdowns (P<0.001). Conversely, double knockdowns of BCL11A and DNMT1 enhanced γ-globin expression (up to 90% of total ß-like globin species) compared to single knockdowns (P<0.001). Moreover, following RNAi treatment, expression of human ß-like globin genes mirrored the expression levels of their endogenous murine counterparts. These results demonstrate that Myb and BCL11A cooperate with DNMT1 to achieve developmental repression of embryonic and fetal ß-like globin genes in the adult erythroid environment.


Asunto(s)
Proteínas Portadoras/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Hemoglobina Fetal/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-myb/genética , Interferencia de ARN , Animales , Western Blotting , Proteínas Portadoras/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteínas de Unión al ADN , Eritropoyesis/genética , Hemoglobina Fetal/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Proteínas Represoras , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transgenes/genética , Globinas beta/genética , Globinas beta/metabolismo , Globinas épsilon/genética , Globinas épsilon/metabolismo , gamma-Globinas/genética , gamma-Globinas/metabolismo
9.
FASEB J ; 28(5): 2306-17, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24443374

RESUMEN

Expression of fetal γ-globin in adulthood ameliorates symptoms of ß-hemoglobinopathies by compensating for the mutant ß-globin. Reactivation of the silenced γ-globin gene is therefore of substantial clinical interest. To study the regulation of γ-globin expression, we created the GG mice, which carry an intact 183-kb human ß-globin locus modified to express enhanced green fluorescent protein (eGFP) from the Gγ-globin promoter. GG embryos express eGFP first in the yolk sac blood islands and then in the aorta-gonad mesonephros and the fetal liver, the sites of normal embryonic hematopoiesis. eGFP expression in erythroid cells peaks at E9.5 and then is rapidly silenced (>95%) and maintained at low levels into adulthood, demonstrating appropriate developmental regulation of the human ß-globin locus. In vitro knockdown of the epigenetic regulator DNA methyltransferase-1 in GG primary erythroid cells increases the proportion of eGFP(+) cells in culture from 41.9 to 74.1%. Furthermore, eGFP fluorescence is induced >3-fold after treatment of erythroid precursors with epigenetic drugs known to induce γ-globin expression, demonstrating the suitability of the Gγ-globin eGFP reporter for evaluation of γ-globin inducers. The GG mouse model is therefore a valuable model system for genetic and pharmacologic studies of the regulation of the ß-globin locus and for discovery of novel therapies for the ß-hemoglobinopathies.


Asunto(s)
Eritropoyesis/fisiología , Regulación de la Expresión Génica , gamma-Globinas/metabolismo , Animales , Metilasas de Modificación del ADN/metabolismo , Modelos Animales de Enfermedad , Epigénesis Genética , Células Eritroides/metabolismo , Femenino , Hemoglobina Fetal/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Regiones Promotoras Genéticas , Interferencia de ARN , Factores de Tiempo , Transgenes , Globinas beta/metabolismo
10.
Hemasphere ; 8(5): e78, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38752170

RESUMEN

α-Thalassemia represents one of the most important genetic modulators of ß-hemoglobinopathies. During this last decade, the ongoing interest in characterizing genotype-phenotype relationships has yielded incredible insights into α-globin gene regulation and its impact on ß-hemoglobinopathies. In this review, we provide a holistic update on α-globin gene expression stemming from DNA to RNA to protein, as well as epigenetic mechanisms that can impact gene expression and potentially influence phenotypic outcomes. Here, we highlight defined α-globin targeted strategies and rationalize the use of distinct molecular targets based on the restoration of balanced α/ß-like globin chain synthesis. Considering the therapies that either increase ß-globin synthesis or reactivate γ-globin gene expression, the modulation of α-globin chains as a disease modifier for ß-hemoglobinopathies still remains largely uncharted in clinical studies.

11.
Sci Rep ; 14(1): 10054, 2024 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-38698053

RESUMEN

ß-Thalassaemia is one of the most common genetic diseases worldwide. During the past few decades, life expectancy of patients has increased significantly owing to advance in medical treatments. Cognitive impairment, once has been neglected, has gradually become more documented. Cognitive impairment in ß-thalassaemia patients is associated with natural history of the disease and socioeconomic factors. Herein, to determined effect of ß-thalassaemia intrinsic factors, 22-month-old ß-thalassaemia mouse was used as a model to assess cognitive impairment and to investigate any aberrant brain pathology in ß-thalassaemia. Open field test showed that ß-thalassaemia mice had decreased motor function. However, no difference of neuronal degeneration in primary motor cortex, layer 2/3 area was found. Interestingly, impaired learning and memory function accessed by a Morris water maze test was observed and correlated with a reduced number of living pyramidal neurons in hippocampus at the CA3 region in ß-thalassaemia mice. Cognitive impairment in ß-thalassaemia mice was significantly correlated with several intrinsic ß-thalassaemic factors including iron overload, anaemia, damaged red blood cells (RBCs), phosphatidylserine (PS)-exposed RBC large extracellular vesicles (EVs) and PS-exposed medium EVs. This highlights the importance of blood transfusion and iron chelation in ß-thalassaemia patients. In addition, to improve patients' quality of life, assessment of cognitive functions should become part of routine follow-up.


Asunto(s)
Disfunción Cognitiva , Modelos Animales de Enfermedad , Hipocampo , Talasemia beta , Animales , Talasemia beta/patología , Talasemia beta/complicaciones , Talasemia beta/genética , Disfunción Cognitiva/etiología , Disfunción Cognitiva/patología , Ratones , Hipocampo/patología , Hipocampo/metabolismo , Masculino , Neuronas/metabolismo , Neuronas/patología , Sobrecarga de Hierro/patología , Sobrecarga de Hierro/metabolismo , Sobrecarga de Hierro/complicaciones , Vesículas Extracelulares/metabolismo , Eritrocitos/metabolismo , Eritrocitos/patología , Células Piramidales/metabolismo , Células Piramidales/patología , Aprendizaje por Laberinto
12.
Blood Cells Mol Dis ; 50(2): 86-92, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23040355

RESUMEN

Forward genetic screens have been performed in many species to identify phenotypes in specific organ systems. We have undertaken a large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screen to identify dominant mutations that perturb erythropoiesis in mice. Mutant mice that displayed an erythrocyte mean cell volume (MCV) greater than three standard deviations from the population mean were identified. Two of these lines, RBC13 and RBC14, displayed a hypochromic, microcytic anemia, accompanied by a marked reticulocytosis, splenomegaly and diminished red cell survival. Timed pregnancies from heterozygous intercrosses revealed that a quarter of the embryos displayed severe anemia and did not survive beyond embryonic day (E) 18.5, consistent with homozygous ß-thalassemia. Genetic complementation studies with a ß-thalassemia mouse line reproduced the embryonic lethality in compound heterozygotes and a genomic custom capture array and massively parallel sequencing of the ß-globin locus identified the causative mutations. The RBC13 line displayed a nonsense mutation at codon 40 in exon 2 of the ß-major gene, invoking parallels with the common ß(0)39 thalassemia mutation seen in humans. The RBC14 line exhibited a mutation at the polyadenylation signal of the ß-major gene, exactly replicating a human ß-thalassemia mutation. The RBC13 and RBC14 lines are the first ß-thalassemia mouse models that reproduce human ß-thalassemia at the genomic level, and as such highlight the power of ENU mutagenesis screens in generating mouse models of human disease.


Asunto(s)
Modelos Animales de Enfermedad , Mutagénesis , Globinas beta/genética , Talasemia beta/genética , Animales , Codón/genética , Codón sin Sentido , Índices de Eritrocitos , Etilnitrosourea , Exones/genética , Femenino , Muerte Fetal/genética , Genes Dominantes , Genes Letales , Prueba de Complementación Genética , Genotipo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mutágenos , Poliadenilación/genética , Embarazo , Bazo/patología , Talasemia beta/sangre , Talasemia beta/embriología , Talasemia beta/patología
13.
FASEB J ; 26(4): 1736-44, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22267339

RESUMEN

A greater understanding of the regulatory mechanisms that govern γ-globin expression in humans, especially the switching from γ- to ß-globin, which occurs after birth, would help to identify new therapeutic targets for patients with ß-hemoglobinopathy. To further elucidate the mechanisms involved in γ-globin expression, a novel fluorescent-based cellular reporter assay system was developed. Using homologous recombination, two reporter genes, DsRed and EGFP, were inserted into a 183-kb intact human ß-globin locus under the control of (G)γ- or (A)γ-globin promoter and ß-globin promoter, respectively. The modified constructs were stably transfected into adult murine erythroleukaemic (MEL) cells and human embryonic or fetal erythroleukemic (K562) cells, allowing for rapid and simultaneous analysis of fetal and adult globin gene expression according to their developmental stage-specific expression. To demonstrate the utility of this system, we performed RNA interference (RNAi)-mediated knockdown of BCL11A in the presence or absence of known fetal hemoglobin inducers and demonstrated functional derepression of a γ-globin-linked reporter in an adult erythroid environment. Our results demonstrate that the cellular assay system represents a promising approach to perform genetic and functional genomic studies to identify and evaluate key factors associated with γ-globin gene suppression.


Asunto(s)
Regulación de la Expresión Génica , Genes Reporteros , Globinas beta/genética , gamma-Globinas/genética , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Hemoglobina Fetal/genética , Colorantes Fluorescentes/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/genética , Humanos , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferencia de ARN , Proteínas Represoras , Transgenes
14.
Ann Hematol ; 92(3): 379-86, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23079892

RESUMEN

Thalassemia is an inherited disorder of hemoglobin molecules that is characterized by an imbalance of α- and ß-globin chain synthesis. Accumulation of unbound α-globin chains in erythroid cells is the major cause of pathology in ß-thalassemia. Stimulation of γ-globin production can ameliorate disease severity as it combines with the α-globin to form fetal hemoglobin. We examined γ-globin-inducing effect of curcuminoids extracted from Curcuma longa L. and their metabolite reduced forms in erythroid leukemia K562 and human primary erythroid precursor cells. The results showed that curcuminoid compounds, especially bisdemethoxycurcumin are potential γ-globin enhancers. We also demonstrated that its reduced analog, hexahydrobisdemethoxycurcumin (HHBDMC), is most effective and leads to induction of γ-globin mRNA and HbF in primary erythroid precursor cells for 3.6 ± 0.4- and 2.0 ± 0.4-folds, respectively. This suggested that HHBDMC is the potential agent to be developed as a new therapeutic drug for ß-thalassemia and related ß-hemoglobinopathies.


Asunto(s)
Curcumina/análogos & derivados , Curcumina/farmacología , Hemoglobina Fetal/biosíntesis , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Curcumina/química , Diarilheptanoides , Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismo , Hemoglobina Fetal/agonistas , Humanos , Células K562 , gamma-Globinas/agonistas , gamma-Globinas/biosíntesis
15.
Nat Methods ; 6(9): 659-62, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19701193

RESUMEN

A human embryonic stem cell (hESC) line that enabled globin-expressing cells to be easily recognized would facilitate optimization of erythroid differentiation in vitro and aid in the identification of hESC-derived erythroid cells in transplanted animals. We describe a genetically modified hESC line, ErythRED, in which expression of RFP, controlled by regulatory sequences from the human beta-globin locus control region, is restricted to maturing erythroid cells.


Asunto(s)
Células Madre Embrionarias/citología , Células Eritroides/citología , Animales , Diferenciación Celular , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Células Eritroides/metabolismo , Regulación de la Expresión Génica , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Globinas beta/genética , Globinas beta/metabolismo
16.
Front Cell Dev Biol ; 10: 891173, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35774227

RESUMEN

Gene editing in male germline stem (GS) cells is a potent tool to study spermatogenesis and to create transgenic mice. Various engineered nucleases already demonstrated the ability to modify the genome of GS cells. However, current systems are limited by technical complexity diminishing application options. To establish an easier method to mediate gene editing, we tested the lipofection of site-specific Cas9:gRNA ribonucleoprotein (RNP) complexes to knockout the enhanced green fluorescent protein (Egfp) in mouse EGFP-GS cells via non-homologous end joining. To monitor whether gene conversion through homology-directed repair events occurred, single-stranded oligodeoxynucleotides were co-lipofected to deliver a Bfp donor sequence. Results showed Egfp knockout in up to 22% of GS cells, which retained their undifferentiated status following transfection, while only less than 0.7% EGFP to BFP conversion was detected in gated GS cells. These data show that CRISPR/Cas9 RNP-based lipofection is a promising system to simply and effectively knock out genes in mouse GS cells. Understanding the genes involved in spermatogenesis could expand therapeutic opportunities for men suffering from infertility.

17.
Sci Rep ; 12(1): 1967, 2022 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-35121800

RESUMEN

Neutrophil dysfunction contributes to a high susceptibility to severe bacterial infection which is a leading cause of morbidity and mortality in ß-thalassaemia/HbE, especially in splenectomised patients. This study demonstrated another abnormality of neutrophil function, namely neutrophil extracellular trap (NET) formation in splenectomised and non-splenectomised ß-thalassaemia/HbE patients who had iron overload. A classification system of morphological NET formation using confocal microscopy was developed, and samples were categorized into early and late phases which were subdivided into web-like and non-web structures. At baseline, neutrophils from non-splenectomised patients (58 ± 4%) and splenectomised patients (65 ± 3%) had higher early phase NETs than those from normal subjects (33 ± 1%). As a mimic of iron overload and infection, haemin/PMA/LPS treatment led to a significant reduction of early NETs and an increase of late NETs in neutrophils from normal and non-splenectomised patients. Interestingly, neutrophils from splenectomised patients had impaired development of late NETs. This suggests that during infection bacteria might not be trapped and may spread from the site of infection resulting in higher susceptibility to severe bacterial infection in splenectomised patients.


Asunto(s)
Infecciones Bacterianas/genética , Trampas Extracelulares/genética , Neutrófilos/microbiología , Talasemia beta/genética , Infecciones Bacterianas/microbiología , Infecciones Bacterianas/patología , Trampas Extracelulares/microbiología , Humanos , Inmunidad Innata/genética , Hierro/metabolismo , Sobrecarga de Hierro/genética , Sobrecarga de Hierro/microbiología , Sobrecarga de Hierro/patología , Neutrófilos/patología , Esplenectomía , Talasemia beta/microbiología , Talasemia beta/patología
18.
Sci Rep ; 12(1): 18628, 2022 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-36329049

RESUMEN

ß-Thalassaemia results from defects in ß-globin chain production, leading to ineffective erythropoiesis and subsequently to severe anaemia and other complications. Apoptosis and autophagy are the main pathways that regulate the balance between cell survival and cell death in response to diverse cellular stresses. Herein, the death of erythroid lineage cells in the bone marrow from both ßIVS2-654-thalassaemic mice and ß-thalassaemia/HbE patients was investigated. Phosphatidylserine (PS)-bearing basophilic erythroblasts and polychromatophilic erythroblasts were significantly increased in ß-thalassaemia as compared to controls. However, the activation of caspase 8, caspase 9 and caspase 3 was minimal and not different from control in both murine and human thalassaemic erythroblasts. Interestingly, bone marrow erythroblasts from both ß-thalassaemic mice and ß-thalassaemia/HbE patients had significantly increased autophagy as shown by increased autophagosomes and increased co-localization between LC3 and LAMP-1. Inhibition of autophagy by chloroquine caused significantly increased erythroblast apoptosis. We have demonstrated increased autophagy which led to minimal apoptosis in ß-thalassaemic erythroblasts. However, increased PS exposure occurring through other mechanisms in thalassaemic erythroblasts might cause rapid phagocytic removal by macrophages and consequently ineffective erythropoiesis in ß-thalassaemia.


Asunto(s)
Eritropoyesis , Talasemia beta , Humanos , Ratones , Animales , Talasemia beta/metabolismo , Eritroblastos , Autofagia , Apoptosis
19.
Sci Rep ; 11(1): 8552, 2021 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-33879818

RESUMEN

The reactivation of γ-globin chain synthesis to combine with excess free α-globin chains and form fetal hemoglobin (HbF) is an important alternative treatment for ß-thalassemia. We had reported HbF induction property of natural curcuminoids, curcumin (Cur), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), in erythroid progenitors. Herein, the HbF induction property of trienone analogs of the three curcuminoids in erythroleukemic K562 cell lines and primary human erythroid progenitor cells from ß-thalassemia/HbE patients was examined. All three trienone analogs could induce HbF synthesis. The most potent HbF inducer in K562 cells was trienone analog of BDMC (T-BDMC) with 2.4 ± 0.2 fold increase. In addition, DNA methylation at CpG - 53, - 50 and + 6 of Gγ-globin gene promoter in K562 cells treated with the compounds including T-BDMC (9.3 ± 1.7%, 7.3 ± 1.7% and 5.3 ± 0.5%, respectively) was significantly lower than those obtained from the control cells (30.7 ± 3.8%, 25.0 ± 2.9% and 7.7 ± 0.9%, respectively P < 0.05). The trienone compounds also significantly induced HbF synthesis in ß-thalassemia/HbE erythroid progenitor cells with significantly reduction in DNA methylation at CpG + 6 of Gγ-globin gene promoter. These results suggested that the curcuminoids and their three trienone analogs induced HbF synthesis by decreased DNA methylation at Gγ-globin promoter region, without effect on Aγ-globin promoter region.


Asunto(s)
Diarilheptanoides/farmacología , Hemoglobina Fetal/biosíntesis , Globinas alfa/metabolismo , Talasemia beta/tratamiento farmacológico , gamma-Globinas/genética , Diferenciación Celular/fisiología , Línea Celular Tumoral , Desmetilación , Diarilheptanoides/análogos & derivados , Células Precursoras Eritroides/metabolismo , Humanos , Regiones Promotoras Genéticas , Talasemia beta/genética , Talasemia beta/metabolismo , Talasemia beta/patología , gamma-Globinas/química , gamma-Globinas/metabolismo
20.
J Nat Prod ; 73(4): 724-8, 2010 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-20196571

RESUMEN

Three new labdane diterpenes, curcucomosins A-C (1-3), four known labdane diterpenes, 4-7, and a known diarylheptanoid, 8, were isolated from the aerial parts of Curcuma comosa. The structures of the new diterpenes were elucidated by spectroscopic data analysis. The fetal hemoglobin (Hb F) induction potency of the isolated compounds was examined using a K562 reporter cell line harboring the enhanced green fluorescence protein (EGFP) gene under the control of a (G)gamma-globin promoter. Compound 6, isocoronarin D, exhibited the highest Hb F induction effect of 1.6-fold at 20 microM.


Asunto(s)
Curcuma/química , Diterpenos/aislamiento & purificación , Diterpenos/farmacología , Hemoglobina Fetal/efectos de los fármacos , Diterpenos/química , Células Eritroides/efectos de los fármacos , Hemoglobina Fetal/metabolismo , Proteínas Fluorescentes Verdes/genética , Humanos , Células K562 , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Tailandia
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA