RESUMEN
BACKGROUND: Systemic pentavalent antimonials, mainly meglumine antimoniate, continue to be the first-choice drugs for treatment of cutaneous leishmaniasis (CL) despite their toxicity, difficulty of administration and high cost. In the search for therapeutic alternatives, combining two treatment interventions has emerged as a potential alternative to either reduce the use of antimonials with the associated toxicities, or to increase efficacy. Here, we report the results of a recently completed trial assessing the efficacy and safety of a combination of thermotherapy (TT) plus a short course of miltefosine (MLT) for the treatment of uncomplicated CL in Colombia and Peru. METHODS: A multicenter, randomized, evaluator-blinded, phase II, controled clinical trial was conducted. Adult volunteers with a parasitologically confirmed diagnosis of uncomplicated CL were randomly allocated to receive either a single session of TT or a combination of TT plus a short course of MLT (3 weeks). Therapeutic response outcomes and safety were assessed. RESULTS: 130 subjects were included in the study, of whom 64 were randomly assigned to the TT arm and 66 to the TT + MLT arm. Cure at 3 months' follow-up was achieved in 57.8% (n = 37) and 80.3% (n = 53) in the TT and TT + MLT groups, respectively, in the intention to treat analysis. The TT + MLT regimen was better that TT alone (p = 0.0055). The presence of vesicles at the site of heat application was the most common adverse event reported associated with the use of TT; while vomiting (31.8%) and elevation of liver enzymes (28.8%) were the most frequent adverse events reported associated with the use of MLT. CONCLUSION: The combination of TT plus a short course of MLT was shown to be significantly better than TT alone for the treatment of uncomplicated CL in the New World. TRIAL REGISTRATION: Registered in clinicaltrials.gov NCT02687971.
Asunto(s)
Antiprotozoarios , Hipertermia Inducida , Leishmaniasis Cutánea , Compuestos Organometálicos , Adulto , Antiprotozoarios/efectos adversos , Humanos , Hipertermia Inducida/efectos adversos , Leishmaniasis Cutánea/tratamiento farmacológico , Leishmaniasis Cutánea/etiología , Meglumina/uso terapéutico , Antimoniato de Meglumina/uso terapéutico , Compuestos Organometálicos/uso terapéutico , Fosforilcolina/análogos & derivados , Resultado del TratamientoRESUMEN
We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a 40 years old female patient homozygous for the mutation c.535 G > A p.G179S on the KCNQ1 gene, causing a severe form of autosomal recessive Long QT Syndrome type 1 (AR-LQT1). The hiPSCs, generated using classical approach of the four retroviruses enconding the reprogramming factors OCT4, SOX2, cMYC and KLF4, display pluripotent stem cell characteristics, and differentiate into cell lineages of all three germ layers: endoderm, mesoderm and ectoderm.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Síndrome de QT Prolongado/genética , Adulto , Diferenciación Celular , Línea Celular , Femenino , Humanos , Factor 4 Similar a KruppelRESUMEN
We generated PSMi001-A and PSMi008-A hiPSC lines from two individuals belonging to a South African (SA) founder population in which the malignant KCNQ1-A341V mutation cosegregates with the Long QT Syndrome (LQTS) phenotype. PSMi001-A was derived from an asymptomatic KCNQ1-A341V mutation carrier, whereas PSMi008-A was derived from a healthy non-mutation carrier, heterozygous for the minor variant rs16847548 on the NOS1AP gene, associated with QT prolongation in the general population, and with a greater risk for cardiac arrest in the affected members of the SA founder population. The hiPSCs, generated using the Yamanaka's retroviruses, display pluripotent stem cell features and trilineage differentiation potential.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Síndrome de QT Prolongado/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Paro Cardíaco/genética , Paro Cardíaco/metabolismo , Humanos , Inmunohistoquímica , Cariotipificación , Mutación/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/citología , SudáfricaRESUMEN
We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a male carrier of the heterozygous mutation c.1781â¯Gâ¯>â¯A p.R594Q on the KCNQ1 gene. hiPSCs, generated using four retroviruses each encoding for OCT4, SOX2, KLF4 and cMYC, display pluripotent stem cell characteristics, and can be differentiated into spontaneously beating cardiomyocytes (hiPSC-CMs).
Asunto(s)
Diferenciación Celular , Fibroblastos/patología , Células Madre Pluripotentes Inducidas/patología , Canal de Potasio KCNQ1/genética , Mutación , Miocitos Cardíacos/patología , Síndrome de Romano-Ward/genética , Adulto , Células Cultivadas , Reprogramación Celular , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Masculino , Miocitos Cardíacos/metabolismo , Fenotipo , Síndrome de Romano-Ward/patologíaRESUMEN
We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a woman carrier of the heterozygous mutation c.568Câ¯>â¯T p.R190W on the KCNQ1 gene. hiPSCs, obtained using four retroviruses enconding the reprogramming factors OCT4, SOX2, cMYC and KLF4, display pluripotent stem cell characteristics, and can be differentiated into spontaneously beating cardiomyocytes (hiPSC-CMs).
Asunto(s)
Diferenciación Celular , Reprogramación Celular , Fibroblastos/patología , Células Madre Pluripotentes Inducidas/patología , Canal de Potasio KCNQ1/genética , Mutación , Síndrome de Romano-Ward/genética , Adulto , Células Cultivadas , Femenino , Fibroblastos/metabolismo , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Fenotipo , Síndrome de Romano-Ward/patologíaRESUMEN
We generated human induced pluripotent stem cells (hiPSCs) from a symptomatic Long QT Syndrome (LQTS) type 1 patient, belonging to a South African (SA) founder population segregating the heterozygous mutation c.1022Câ¯>â¯T p.A341V on the KCNQ1 gene. The patient is also homozygous for the two minor variants rs4657139 and rs16847548 on the NOS1AP gene, associated with greater risk for cardiac arrest and sudden death in LQTS mutation carriers of the founder population. hiPSCs, obtained using four retroviruses encoding the reprogramming factors OCT4, SOX2, cMYC and KLF4, display pluripotent stem cell characteristics, and can be differentiated into spontaneously beating cardiomyocytes (hiPSC-CMs).
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular , Células Madre Pluripotentes Inducidas , Canal de Potasio KCNQ1/genética , Síndrome de Romano-Ward/genética , Diferenciación Celular , Técnicas de Reprogramación Celular , Análisis Mutacional de ADN , Femenino , Heterocigoto , Homocigoto , Humanos , Cariotipo , Factor 4 Similar a Kruppel , Persona de Mediana EdadRESUMEN
OBJECTIVES: There is accumulating evidence that yoga and mindfulness meditation can alleviate symptoms of anxiety, although the mechanisms by which this occurs remain unclear. The purpose of this study was to examine the relationship between yoga practice and self-reported anxiety as well as the potential mediating roles of mindfulness and emotional avoidance. METHODS: Using a cross-sectional design, 367 participants were recruited online and completed measures of anxiety, avoidance, and mindfulness. RESULTS: Results showed that length of yoga practice was significantly correlated with lower anxiety in yoga practitioners. Avoidance and mindfulness mediated the relationship between length of yoga practice and anxiety, shedding light on possible mechanisms by which these practices reduce anxiety. CONCLUSIONS: Future experimental and longitudinal research is needed to examine the causal role of mindfulness and avoidance in the relationship between yoga practice and anxiety, and whether yoga is a useful adjunct to cognitive behaviour therapy for anxiety disorders.
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Ansiedad/terapia , Reacción de Prevención/fisiología , Atención Plena , Yoga , Adulto , Anciano , Estudios Transversales , Femenino , Humanos , Masculino , Meditación , Persona de Mediana Edad , Adulto JovenRESUMEN
We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a 51years old female patient homozygous for the mutation c.535 G>A p.G179S on the KCNQ1 gene, causing a severe form of autosomal recessive Long QT Syndrome type 1 (AR-LQT1), not associated with deafness. The hiPSCs, generated using four retroviruses each encoding for a reprogramming factor OCT4, SOX2, KLF4, cMYC, are pluripotent and can differentiate into spontaneously beating cardiomyocytes (hiPSC-CMs).
Asunto(s)
Técnicas de Reprogramación Celular , Genes Recesivos , Células Madre Pluripotentes Inducidas , Síndrome de Romano-Ward , Línea Celular , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Factor 4 Similar a Kruppel , Persona de Mediana Edad , Síndrome de Romano-Ward/genética , Síndrome de Romano-Ward/metabolismo , Síndrome de Romano-Ward/patología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
We report the generation of human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a female patient carrier of the two compound heterozygous mutations c.568 C>T p.R190W (maternal allele), and c.1781 G>A p.R594Q (paternal allele) on the KCNQ1 gene, causing Jervell and Lange-Nielsen Syndrome (JLNS). To obtain hiPSCs, we used the classical approach of the four retroviruses each encoding for a reprogramming factor OCT4, SOX2, KLF4, cMYC. The obtained hiPSC clones display pluripotent stem cell characteristics, and differentiate into spontaneously beating cardiomyocytes (hiPSC-CMs).
Asunto(s)
Heterocigoto , Células Madre Pluripotentes Inducidas , Síndrome de Jervell-Lange Nielsen , Canal de Potasio KCNQ1/genética , Mutación Missense , Sustitución de Aminoácidos , Línea Celular , Niño , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Síndrome de Jervell-Lange Nielsen/genética , Síndrome de Jervell-Lange Nielsen/metabolismo , Síndrome de Jervell-Lange Nielsen/patología , Factor 4 Similar a KruppelRESUMEN
INTRODUCTION: Progress with the treatment of cutaneous leishmaniasis (CL) has been hampered by inconsistent methodologies used to assess treatment effects. A sizable number of trials conducted over the years has generated only weak evidence backing current treatment recommendations, as shown by systematic reviews on old-world and new-world CL (OWCL and NWCL). MATERIALS AND METHODS: Using a previously published guidance paper on CL treatment trial methodology as the reference, consensus was sought on key parameters including core eligibility and outcome measures, among OWCL (7 countries, 10 trial sites) and NWCL (7 countries, 11 trial sites) during two separate meetings. RESULTS: Findings and level of consensus within and between OWCL and NWCL sites are presented and discussed. In addition, CL trial site characteristics and capacities are summarized. CONCLUSIONS: The consensus reached allows standardization of future clinical research across OWCL and NWCL sites. We encourage CL researchers to adopt and adapt as required the proposed parameters and outcomes in their future trials and provide feedback on their experience. The expertise afforded between the two sets of clinical sites provides the basis for a powerful consortium with potential for extensive, standardized assessment of interventions for CL and faster approval of candidate treatments.
Asunto(s)
Antiprotozoarios/uso terapéutico , Ensayos Clínicos como Asunto/normas , Leishmaniasis Cutánea/tratamiento farmacológico , Humanos , Resultado del TratamientoRESUMEN
Splenic hematopoiesis is a major feature in the course of myelofibrosis (MF). In fact, the spleen of patients with MF contains malignant hematopoietic stem cells retaining a complete differentiation program, suggesting both a pivotal role of the spleen in maintaining the disease and a tight regulation of hematopoiesis by the splenic microenvironment, in particular by mesenchymal stromal cells (MSCs). Little is known about splenic MSCs (Sp-MSCs), both in normal and in pathological context. In this work, we have in vitro expanded and characterized Sp-MSCs from 25 patients with MF and 13 healthy subjects (HS). They shared similar phenotype, growth kinetics, and differentiation capacity. However, MF Sp-MSCs expressed significant lower levels of nestin, and favored megakaryocyte (Mk) differentiation in vitro at a larger extent than their normal counterpart. Moreover, they showed a significant upregulation of matrix metalloprotease 2 (MMP2) and fibronectin 1 (FN1) genes both at mRNA expression and at protein level, and, finally, developed genetic abnormalities which were never detected in HS-derived Sp-MSCs. Our data point toward the existence of a defective splenic niche in patients with MF that could be responsible of some pathological features of the disease, including the increased trafficking of CD34+ cells and the expansion of the megakaryocytic lineage.
Asunto(s)
Células Madre Mesenquimatosas/patología , Mielofibrosis Primaria/patología , Bazo/patología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD34 , Estudios de Casos y Controles , Movimiento Celular , Proliferación Celular , Femenino , Fibronectinas/metabolismo , Hematopoyesis , Humanos , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Megacariocitos/patología , Persona de Mediana Edad , Nestina/metabolismo , Adulto JovenRESUMEN
hTERT component is the key regulator of telomerase. Alternatively spliced variants of hTERT generate different telomerase activity. The goal of the study was to determine the role of different hTERT isoforms in the regulation of telomerase expression in AML patients. Among the 97 studied patients, 45 had a complex karyotype and 52 a normal karyotype. hTERT isoforms expression was determined in bone marrow samples by q-RT-PCR, using SYBR Green I. hTERT expression was lower in AML patients than controls (median 2.5 vs. 10.1, p = .003), though no difference was observed between the complex and normal karyotype (median 3.2 vs. 2.3, p = .37). High trans-dominant negative isoform expression increased the response rate by two. High expression of inactive product (-α - ß) was shown to increase the risk of relapse by about three times. In conclusion, our data suggest an intriguing link between the control of hTERT isoforms expression and AML outcome.
Asunto(s)
Empalme Alternativo , Médula Ósea/patología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Leucemia Mieloide Aguda/genética , Recurrencia Local de Neoplasia/genética , Telomerasa/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Médula Ósea/metabolismo , Estudios de Casos y Controles , Aberraciones Cromosómicas , Femenino , Estudios de Seguimiento , Humanos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/enzimología , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/terapia , Pronóstico , Tasa de Supervivencia , Telomerasa/metabolismoRESUMEN
INTRODUCTION: The association between congenital pulmonary airway malformations (CPAM) and malignancy is reported in the literature. Interactions between the tumor, immune, and mesenchymal stromal/stem cells (MSCs) have been recognized as crucial for understanding tumorigenesis. We characterized MSCs isolated from CPAM lesions in order to define potential malignancy risks. METHODS: CPAM II pulmonary tissue was used for MSC expansion; a "healthy" lung section from the same child was used as a comparator. Morphology, immunophenotype, differentiation and immunological capacity, proliferative growth, gene signature telomerase activity, and in vivo tumorigenicity in nude mice were evaluated. RESULTS: MSCs were successfully isolated and propagated from CPAM tissue. CPAM-MSCs presented the typical MSC morphology and phenotype, while exhibiting high proliferative capacity, reaching confluence at a median time of 5 days as well as differentiation capabilities. CPAM-MSCs at early passages were not neoplastic and chromosomally normal, even though unbalanced chromosomal rearrangements were noted by molecular karyotype. CONCLUSIONS: CPAM-MSCs exhibited specific features similar to tumor derived MSCs. Whilst there was no evidence of malignant transformation in the cystic tissue, our results provide evidence that this abnormal tissue has malignant potential. MSCs are considered important players in the tumor microenvironment and they have been closely linked to regulation of tumor survival, growth, and progression. Thus, early lesion resection also in asymptomatic patients might be indicated to exclude that the microenvironment may be potentially permissive to cancer development.
Asunto(s)
Pulmón/anomalías , Pulmón/citología , Células Madre Mesenquimatosas/citología , Anomalías del Sistema Respiratorio , Animales , Diferenciación Celular , Proliferación Celular , Transformación Celular Neoplásica , Humanos , Lactante , Leucocitos Mononucleares/citología , Masculino , Ratones , Ratones Desnudos , Fenotipo , RiesgoRESUMEN
A novel series of feruloyl-donepezil hybrid compounds were designed, synthesized and evaluated as multitarget drug candidates for the treatment of Alzheimer's Disease (AD). In vitro results revealed potent acetylcholinesterase (AChE) inhibitory activity for some of these compounds and all of them showed moderate antioxidant properties. Compounds 12a, 12b and 12c were the most potent AChE inhibitors, highlighting 12a with IC50 = 0.46 µM. In addition, these three most promising compounds exhibited significant in vivo anti-inflammatory activity in the mice paw edema, pleurisy and formalin-induced hyperalgesy models, in vitro metal chelator activity for Cu2+ and Fe2+, and neuroprotection of human neuronal cells against oxidative damage. Molecular docking studies corroborated the in vitro inhibitory mode of interaction of these active compounds on AChE. Based on these data, compound 12a was identified as a novel promising drug prototype candidate for the treatment of AD with innovative structural feature and multitarget effects.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Indanos/farmacología , Terapia Molecular Dirigida/métodos , Piperidinas/farmacología , Acrilatos/química , Acrilatos/farmacología , Animales , Antiinflamatorios , Antioxidantes , Línea Celular , Células Cultivadas , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Donepezilo , Diseño de Fármacos , Humanos , Indanos/química , Masculino , Ratones , Simulación del Acoplamiento Molecular , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Piperidinas/química , Relación Estructura-ActividadRESUMEN
In myelodysplatic syndromes and acute myeloid leukemia (MDS/AML) deletion of the 11q14 region is a rare chromosomal defect (incidence: 0.6-1.0%), included within the intermediate risk criteria by the International Prognostic Scoring System. No fluorescence in situ hybridization (FISH) study has yet been performed to identify a common breakpoint region (CBR). In our study through FISH with bacterial artificial chromosomes and commercial probes, we analyzed seven patients with MDS/AML harboring 11q14 deletion on conventional cytogenetic analysis. FISH revealed deletions in five patients and amplifications in two. Three patients with deletion carried a CBR, two had a deletion involving a more centromeric breakpoint. These five patients exhibited multilineage dysplasia, blast cells with large round nuclei, loose chromatin, small and abundant nucleoli, and vacuolated cytoplasm with very thin Auer bodies. In conclusion, the morphological features which occur independently of the extent of the deletion are of multilineage dysplasia in MDS and leukemic blasts strongly reactive to peroxidase in AML; despite the variable size of the deleted area, some patients harbor a CBR.
Asunto(s)
Puntos de Rotura del Cromosoma , Deleción Cromosómica , Cromosomas Humanos Par 11/genética , Leucemia Mieloide/genética , Síndromes Mielodisplásicos/genética , Enfermedad Aguda , Adulto , Anciano , Femenino , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Persona de Mediana EdadRESUMEN
Cardanol is a phenolic lipid component of cashew nut shell liquid (CNSL), obtained as the byproduct of cashew nut food processing. Being a waste product, it has attracted much attention as a precursor for the production of high-value chemicals, including drugs. On the basis of these findings and in connection with our previous studies on cardanol derivatives as acetylcholinesterase (AChE) inhibitors, we designed a novel series of analogues by including a protonable amino moiety belonging to different systems. Properly addressed docking studies suggested that the proposed structural modifications would allow the new molecules to interact with both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE, thus being able to act as dual binding inhibitors. To disclose whether the new molecules showed the desired profile, they were first tested for their cholinesterase inhibitory activity towards EeAChE and eqBuChE. Compound 26, bearing an N-ethyl-N-(2-methoxybenzyl)amine moiety, showed the highest inhibitory activity against EeAChE, with a promising IC50 of 6.6 µM, and a similar inhibition profile of the human isoform (IC50 = 5.7 µM). As another positive feature, most of the derivatives did not show appreciable toxicity against HT-29 cells, up to a concentration of 100 µM, which indicates drug-conform behavior. Also, compound 26 is capable of crossing the blood-brain barrier (BBB), as predicted by a PAMPA-BBB assay. Collectively, the data suggest that the approach to obtain potential anti-Alzheimer drugs from CNSL is worth of further pursuit and development.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Inhibidores de la Colinesterasa/farmacología , Colinesterasas/metabolismo , Fenoles/farmacología , Enfermedad de Alzheimer/enzimología , Sitios de Unión/efectos de los fármacos , Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/química , Relación Dosis-Respuesta a Droga , Células HT29 , Humanos , Estructura Molecular , Fenoles/síntesis química , Fenoles/química , Relación Estructura-ActividadRESUMEN
A tight relationship between the acute myeloid leukemia (AML) population and the bone marrow (BM) microenvironment has been convincingly established. The AML clone contains leukemic stem cells (LSCs) that compete with normal hematopoietic stem cells (HSCs) for niche occupancy and remodel the niche; whereas, the BM microenvironment might promote AML development and progression not only through hypoxia and homing/adhesion molecules, but also through genetic defects. Although it is still unknown whether the niche influences treatment results or contains any potential target for treatment, this dynamic AML-niche interaction might be a promising therapeutic objective to significantly improve the AML cure rate.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Leucemia Mieloide Aguda/terapia , Terapia Molecular Dirigida , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Nicho de Células Madre/efectos de los fármacos , Animales , Bencilaminas , Médula Ósea/patología , Hipoxia de la Célula/efectos de los fármacos , Linaje de la Célula , Quimiocina CXCL12/antagonistas & inhibidores , Quimiocina CXCL12/inmunología , Quimiocina CXCL12/fisiología , Ensayos Clínicos como Asunto , Terapia Combinada , Trasplante de Células Madre de Sangre del Cordón Umbilical , Ciclamas , Difosfonatos/uso terapéutico , Modelos Animales de Enfermedad , Compuestos Heterocíclicos/uso terapéutico , Humanos , Péptidos y Proteínas de Señalización Intercelular/fisiología , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Ratones , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/fisiología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/inmunología , Receptores CXCR4/fisiología , Células del Estroma/clasificación , Células del Estroma/efectos de los fármacos , Células del Estroma/patología , Trasplante Heterólogo , Microambiente Tumoral/efectos de los fármacosRESUMEN
Fanconi anaemia (FA) is an inherited disorder characterized by pancytopenia, congenital malformations and a predisposition to develop malignancies. Alterations in the haematopoietic microenvironment of FA patients have been reported, but little is known regarding the components of their bone marrow (BM) stroma. We characterized mesenchymal stromal cells (MSCs) isolated from BM of 18 FA patients both before and after allogeneic haematopoietic stem cell transplantation (HSCT). Morphology, fibroblast colony-forming unit (CFU-F) ability, proliferative capacity, immunophenotype, differentiation potential, ability to support long-term haematopoiesis and immunomodulatory properties of FA-MSCs were analysed and compared with those of MSCs expanded from 15 age-matched healthy donors (HD-MSCs). FA-MSCs were genetically characterized through conventional karyotyping, diepoxybutane-test and array-comparative genomic hybridization. FA-MSCs generated before and after HSCT were compared. Morphology, immunophenotype, differentiation potential, ability in vitro to inhibit mitogen-induced T-cell proliferation and to support long-term haematopoiesis did not differ between FA-MSCs and HD-MSCs. CFU-F ability and proliferative capacity of FA-MSCs isolated after HSCT were significantly lower than those of HD-MSCs. FA-MSCs reached senescence significantly earlier than HD-MSCs and showed spontaneous chromosome fragility. Our findings indicate that FA-MSCs are defective in their ability to survive in vitro and display spontaneous chromosome breakages; whether these defects are involved in pathophysiology of BM failure syndromes deserves further investigation.
Asunto(s)
Anemia de Fanconi/metabolismo , Células Madre Mesenquimatosas/metabolismo , Antígenos de Superficie/metabolismo , Estudios de Casos y Controles , Técnicas de Cultivo de Célula , Ciclo Celular/genética , Diferenciación Celular , Proliferación Celular , Senescencia Celular/genética , Niño , Preescolar , Ensayo de Unidades Formadoras de Colonias , Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Femenino , Genotipo , Hematopoyesis , Humanos , Inmunofenotipificación , Lactante , Cariotipo , Masculino , Repeticiones de Microsatélite/genéticaRESUMEN
Transcribed gene fusions are key biomarkers in many hematologic and solid tumors, often representing the primary oncogenic driver mutation. Here, we report an experimental and computational pipeline for detecting fusion transcripts using single-molecule RNA FISH and unbiased correlation analysis (FuseFISH). We constructed a genome-wide database of optimal oligonucleotide sequences, enabling quick design of FuseFISH probes against known and novel fusions. We implemented FuseFISH in cell lines, tissue sections, and purified RNA, reliably detecting one BCR-ABL1 positive in 10,000 negative cells. In 34 hematologic samples, we detected BCR-ABL1 transcripts with high specificity and sensitivity. Finally, we measured BCR-ABL1 expression heterogeneity and dynamics in single CML cells exposed to the kinase inhibitor Nilotinib. Our resource and methods are ideal for streamlined validation of fusions newly identified by next-generation sequencing, and they pave the way to studying the impact of fusion expression variability on clinical outcome.