RESUMEN
We evaluated the impact of recipient and cord blood unit (CBU) genetic polymorphisms related to immune response on outcomes after unrelated cord blood transplantations (CBTs). Pretransplant DNA samples from 696 CBUs with malignant diseases were genotyped for NLRP1, NLRP2, NLRP3, TIRAP/Mal, IL10, REL, TNFRSF1B, and CTLA4. HLA compatibility was 6 of 6 in 10%, 5 of 6 in 39%, and ≥4 of 6 in 51% of transplants. Myeloablative conditioning was used in 80%, and in vivo T-cell depletion in 81%, of cases. The median number of total nucleated cells infused was 3.4 × 107/kg. In multivariable analysis, patients receiving CBUs with GG-CTLA4 genotype had poorer neutrophil recovery (hazard ratio [HR], 1.33; P = .02), increased nonrelapse mortality (NRM) (HR, 1.50; P < .01), and inferior disease-free survival (HR, 1.41; P = .02). We performed the same analysis in a more homogeneous subset of cohort 1 (cohort 2, n = 305) of patients who received transplants for acute leukemia, all given a myeloablative conditioning regimen, and with available allele HLA typing (HLA-A, -B, -C, and -DRB1). In this more homogeneous but smaller cohort, we were able to demonstrate that GG-CTLA4-CBU was associated with increased NRM (HR, 1.85; P = .01). Use of GG-CTLA4-CBU was associated with higher mortality after CBT, which may be a useful criterion for CBU selection, when multiple CBUs are available.
Asunto(s)
Antígeno CTLA-4/inmunología , Trasplante de Células Madre de Sangre del Cordón Umbilical , Antígenos HLA/inmunología , Neoplasias Hematológicas/genética , Polimorfismo Genético , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Adolescente , Adulto , Alelos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Antígeno CTLA-4/genética , Niño , Preescolar , Supervivencia sin Enfermedad , Femenino , Sangre Fetal/citología , Sangre Fetal/inmunología , Sangre Fetal/trasplante , Expresión Génica , Genotipo , Antígenos HLA/genética , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/patología , Neoplasias Hematológicas/terapia , Prueba de Histocompatibilidad , Humanos , Lactante , Masculino , Persona de Mediana Edad , Agonistas Mieloablativos/uso terapéutico , Proteínas NLR , Modelos de Riesgos Proporcionales , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Estudios Retrospectivos , Acondicionamiento Pretrasplante , Donante no EmparentadoRESUMEN
Recent evidence has shown that deregulated expression of members of the microRNA-29 (miR-29) family may play a critical role in human cancer, including hematological malignancies. However, the roles of miR-29 in the molecular pathophysiology of T-cell acute lymphoblastic leukemia (T-ALL) has not been investigated. Here, we show that lower levels of miR-29a were significantly associated with higher blast counts in the bone marrow and with increased disease-free survival in T-ALL patients. Furthermore, miR-29a levels are extremely reduced in T-ALL cells compared to normal T cells. Microarray analysis following introduction of synthetic miR-29a mimics into Jurkat cells revealed the downregulation of several predicted targets (CDK6, PXDN, MCL1, PIK3R1, and CXXC6), including targets with roles in active and passive DNA demethylation (such as DNMT3a, DNMT3b, and members of the TET family and TDG). Restoring miR-29a levels in Jurkat and Molt-4 T-ALL cells led to the demethylation of many genes commonly methylated in T-ALL. Overall, our results suggest that reduced miR-29a levels may contribute to the altered epigenetic status of T-ALL, highlighting its relevance in the physiopathology of this disease.
Asunto(s)
Metilación de ADN/genética , Epigénesis Genética/genética , Regulación Leucémica de la Expresión Génica/genética , MicroARNs/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Antígenos de Neoplasias/biosíntesis , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Fosfatidilinositol 3-Quinasa Clase Ia , Quinasa 6 Dependiente de la Ciclina/biosíntesis , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A , Proteínas de Unión al ADN/biosíntesis , Daunorrubicina/farmacología , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos/genética , Humanos , Células Jurkat , Oxigenasas de Función Mixta , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Peroxidasas , Fosfatidilinositol 3-Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Receptores de Interleucina-1/biosíntesis , ADN Metiltransferasa 3BRESUMEN
Autologous haematopoietic stem-cell transplantation (AHSCT) has been experimented as a treatment in patients affected by severe forms of multiple sclerosis (MS) who failed to respond to standard immunotherapy. The rationale of AHSCT is to 'reboot' the immune system and reconstitute a new adaptive immunity. The aim of our study was to identify, through a robust and unbiased transcriptomic analysis, any changes of gene expression in T-cells potentially underlying the treatment effect in patients who underwent non-myeloablative AHSCT for treatment of MS. We evaluated by microarray DNA-chip technology the gene expression of peripheral CD4+ and CD8+ T-cell subsets sorted from patients with MS patients before AHSCT, at 6 months, 1 year and 2 years after AHSCT and from healthy control subjects. Hierarchical clustering analysis revealed that reconstituted CD8+ T-cells of MS patients at 2 years post-transplantation, aggregated together with healthy controls, suggesting a normalization of gene expression in CD8+ cells post-therapy. When we compared the gene expression in MS patients before and after therapy, we detected a large number of differentially expressed genes (DEG) in both CD8+ and CD4+ T-cell subsets at all time points after transplantation. We catalogued the biological function of DEG and we selected 27 genes known to be involved in immune function for accurate quantification of gene expression by real-time PCR. The analysis confirmed and extended with quantitative data, a number of significant changes in both the CD4+ and CD8+ T-cells subsets from MS post-transplant. Notably, CD8+ T-cells revealed more extensive changes in the expression of genes involved in effector immune responses.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Esclerosis Múltiple/terapia , Inmunidad Adaptativa/genética , Adulto , Linfocitos T CD4-Positivos , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunologíaRESUMEN
Pemphigus foliaceus (PF) is a complex autoimmune disease characterized by bullous skin lesions and the presence of antibodies against desmoglein 1. In this study we sought to contribute to a better understanding of the molecular processes in endemic PF, as the identification of factors that participate in the pathogenesis is a prerequisite for understanding its biological basis and may lead to novel therapeutic interventions. CD4+ T lymphocytes are central to the development of the disease. Therefore, we compared genome-wide gene expression profiles of peripheral CD4+ T cells of various PF patient subgroups with each other and with that of healthy individuals. The patient sample was subdivided into three groups: untreated patients with the generalized form of the disease, patients submitted to immunosuppressive treatment, and patients with the localized form of the disease. Comparisons between different subgroups resulted in 135, 54 and 64 genes differentially expressed. These genes are mainly related to lymphocyte adhesion and migration, apoptosis, cellular proliferation, cytotoxicity and antigen presentation. Several of these genes were differentially expressed when comparing lesional and uninvolved skin from the same patient. The chromosomal regions 19q13 and 12p13 concentrate differentially expressed genes and are candidate regions for PF susceptibility genes and disease markers. Our results reveal genes involved in disease severity, potential therapeutic targets and previously unsuspected processes involved in the pathogenesis. Besides, this study adds original information that will contribute to the understanding of PF's pathogenesis and of the still poorly defined in vivo functions of most of these genes.
Asunto(s)
Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Pénfigo/genética , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Análisis por Conglomerados , Femenino , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Masculino , Persona de Mediana Edad , Pénfigo/inmunología , Pénfigo/metabolismo , Transducción de Señal , Adulto JovenRESUMEN
Recent studies have demonstrated the role of adenosine (ADO) in sickle-cell anemia (SCA). ADO is produced by CD39 and CD73 and converted to inosine by adenosine deaminase (ADA). We evaluated the effects of hydroxycarbamide (HU) treatment on the modulation of adenosine levels in SCA patients. The expressions of CD39, CD73, and CD26 were evaluated by flow cytometry on blood cells in 15 HU-treated and 17 untreated patients and 10 healthy individuals. RNA was extracted from monocytes, and ADA gene expression was quantified by real-time PCR. ADA activity was also evaluated. We found that ADA transcripts were two times higher in monocytes of HU-treated patients, compared with untreated (P = 0.039). Monocytes of HU-treated patients expressed CD26, while monocytes of controls and untreated patients did not (P = 0.023). In treated patients, a lower percentage of T lymphocytes expressed CD39 compared with untreated (P = 0.003), and the percentage of T regulatory (Treg) cells was reduced in the treated group compared with untreated (P = 0.017) and controls (P = 0.0009). Besides, HU-treated patients displayed increased ADA activity, compared with untreated. Our results indicate a novel mechanism of action of HU mediated by the reduction of adenosine levels and its effects on pathophysiological processes in SCA.
Asunto(s)
Adenosina/metabolismo , Anemia de Células Falciformes/metabolismo , Antidrepanocíticos/farmacología , Células Sanguíneas/efectos de los fármacos , Células Sanguíneas/metabolismo , Hidroxiurea/farmacología , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Adolescente , Adulto , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Antidrepanocíticos/uso terapéutico , Apirasa/genética , Apirasa/metabolismo , Células Sanguíneas/patología , Estudios de Casos y Controles , Niño , Dipeptidil Peptidasa 4/genética , Dipeptidil Peptidasa 4/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Hidroxiurea/uso terapéutico , Redes y Vías Metabólicas/efectos de los fármacos , Redes y Vías Metabólicas/genética , Persona de Mediana Edad , Adulto JovenRESUMEN
Mesenchymal stem cells (MSCs) are known to induce the conversion of activated T cells into regulatory T cells in vitro. The marker CD69 is a target of canonical nuclear factor kappa-B (NF-κB) signalling and is transiently expressed upon activation; however, stable CD69 expression defines cells with immunoregulatory properties. Given its enormous therapeutic potential, we explored the molecular mechanisms underlying the induction of regulatory cells by MSCs. Peripheral blood CD3(+) T cells were activated and cultured in the presence or absence of MSCs. CD4(+) cell mRNA expression was then characterized by microarray analysis. The drug BAY11-7082 (BAY) and a siRNA against v-rel reticuloendotheliosis viral oncogene homolog B (RELB) were used to explore the differential roles of canonical and non-canonical NF-κB signalling, respectively. Flow cytometry and real-time PCR were used for analyses. Genes with immunoregulatory functions, CD69 and non-canonical NF-κB subunits (RELB and NFKB2) were all expressed at higher levels in lymphocytes co-cultured with MSCs. The frequency of CD69(+) cells among lymphocytes cultured alone progressively decreased after activation. In contrast, the frequency of CD69(+) cells increased significantly following activation in lymphocytes co-cultured with MSCs. Inhibition of canonical NF-κB signalling by BAY immediately following activation blocked the induction of CD69; however, inhibition of canonical NF-κB signalling on the third day further induced the expression of CD69. Furthermore, late expression of CD69 was inhibited by RELB siRNA. These results indicate that the canonical NF-κB pathway controls the early expression of CD69 after activation; however, in an immunoregulatory context, late and sustained CD69 expression is promoted by the non-canonical pathway and is inhibited by canonical NF-κB signalling.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Lectinas Tipo C/metabolismo , Activación de Linfocitos/inmunología , Células Madre Mesenquimatosas/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Linfocitos T Reguladores/inmunología , Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos T/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular , Células Cultivadas , Perfilación de la Expresión Génica , Humanos , Lectinas Tipo C/genética , Análisis por Micromatrices , FN-kappa B/genética , Nitrilos , Trasplante de Células Madre de Sangre Periférica/métodos , Fenotipo , Reacción en Cadena en Tiempo Real de la Polimerasa , Sulfonas , Linfocitos T Reguladores/metabolismo , Factor de Transcripción ReIB/genética , Factor de Transcripción ReIB/metabolismoRESUMEN
Temporal lobe epilepsy (TLE) is the most common form of partial epilepsy and affects 40% of the patients. Seizures arising from the mesial temporal lobe structures (i.e., amygdala and hippocampus) are common, whereas neocortical seizures are rare. In recent years, many studies aimed to identify the pattern of gene expression of neurotransmitters involved in molecular mechanisms of epilepsy. We used real-time PCR to quantify the expression of GABA(A) (subunits α1, ß1, ß2) and NMDA (subunits NR1, NR2A, and NR2B) receptor genes in amygdalae of 27 patients with TLE and 14 amygdalae from autopsy controls. The NR1 subunit was increased in patients with epilepsy when compared with controls. No differences were found in expression of NMDA subunits NR2A and NR2B or in α1, ß1, and ß2 subunits of GABA(A) receptors. Our results suggest that the NR1 subunit of NMDA receptors is involved in the amygdala hyperexcitability in some of the patients with TLE.
Asunto(s)
Amígdala del Cerebelo/fisiopatología , Epilepsia del Lóbulo Temporal/genética , Epilepsia del Lóbulo Temporal/fisiopatología , Predisposición Genética a la Enfermedad/genética , Receptores de GABA-A/genética , Receptores de N-Metil-D-Aspartato/genética , Adulto , Anciano , Amígdala del Cerebelo/metabolismo , Epilepsia del Lóbulo Temporal/metabolismo , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Masculino , Persona de Mediana Edad , Subunidades de Proteína/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Regulación hacia Arriba/genética , Adulto JovenRESUMEN
To identify novel genes involved in the molecular pathogenesis of chronic lymphocytic leukemia (CLL) we performed a serial analysis of gene expression (SAGE) in CLL cells, and compared this with healthy B cells (nCD19(+)). We found a high level of similarity among CLL subtypes, but a comparison of CLL versus nCD19(+) libraries revealed 55 genes that were over-represented and 49 genes that were down-regulated in CLL. A gene ontology analysis revealed that TOSO, which plays a functional role upstream of Fas extrinsic apoptosis pathway, was over-expressed in CLL cells. This finding was confirmed by real-time reverse transcription-polymerase chain reaction in 78 CLL and 12 nCD19(+) cases (P < .001). We validated expression using flow cytometry and tissue microarray and demonstrated a 5.6-fold increase of TOSO protein in circulating CLL cells (P = .013) and lymph nodes (P = .006). Our SAGE results have demonstrated that TOSO is a novel over-expressed antiapoptotic gene in CLL.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/etiología , Proteínas de la Membrana/genética , Receptor fas , Proteínas Reguladoras de la Apoptosis/fisiología , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Proteínas de la Membrana/fisiologíaRESUMEN
OBJECTIVE: The relationship of multipotent mesenchymal stromal cells (MSC) with pericytes and fibroblasts has not been established thus far, although they share many markers of primitive marrow stromal cells and the osteogenic, adipogenic, and chondrogenic differentiation potentials. MATERIALS AND METHODS: We compared MSCs from adult or fetal tissues, MSC differentiated in vitro, fibroblasts and cultures of retinal pericytes obtained either by separation with anti-CD146 or adhesion. The characterizations included morphological, immunophenotypic, gene-expression profile, and differentiation potential. RESULTS: Osteogenic, adipocytic, and chondrocytic differentiation was demonstrated for MSC, retinal perivascular cells, and fibroblasts. Cell morphology and the phenotypes defined by 22 markers were very similar. Analysis of the global gene expression obtained by serial analysis of gene expression for 17 libraries and by reverse transcription polymerase chain reaction of 39 selected genes from 31 different cell cultures, revealed similarities among MSC, retinal perivascular cells, and hepatic stellate cells. Despite this overall similarity, there was a heterogeneous expression of genes related to angiogenesis, in MSC derived from veins, artery, perivascular cells, and fibroblasts. Evaluation of typical pericyte and MSC transcripts, such as NG2, CD146, CD271, and CD140B on CD146 selected perivascular cells and MSC by real-time polymerase chain reaction confirm the relationship between these two cell types. Furthermore, the inverse correlation between fibroblast-specific protein-1 and CD146 transcripts observed on pericytes, MSC, and fibroblasts highlight their potential use as markers of this differentiation pathway. CONCLUSION: Our results indicate that human MSC and pericytes are similar cells located in the wall of the vasculature, where they function as cell sources for repair and tissue maintenance, whereas fibroblasts are more differentiated cells with more restricted differentiation potential.
Asunto(s)
Antígeno CD146/genética , Fibroblastos/citología , Perfilación de la Expresión Génica , Células Madre Mesenquimatosas/citología , Pericitos/citología , Cordón Umbilical/citología , Antígeno CD146/fisiología , Diferenciación Celular/fisiología , Separación Celular/métodos , Células Cultivadas , Análisis por Conglomerados , Fibroblastos/fisiología , Citometría de Flujo , Humanos , Inmunofenotipificación , Células Madre Mesenquimatosas/fisiología , Pericitos/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/genética , Cordón Umbilical/fisiologíaRESUMEN
TP73 encodes for two proteins: full-length TAp73 and DeltaNp73, which have little transcriptional activity and exert dominant-negative function towards TP53 and TAp73. We compared TATP73 and DeltaNTP73 expression in acute myeloid leukaemia (AML) samples and normal CD34(+) progenitors. Both forms were more highly expressed in leukaemic cells. Amongst AML blasts, TATP73 was more expressed in AML harbouring the recurrent genetic abnormalities (RGA): PML-RARA, RUNX1-RUNX1T1 and CBFB-MYH11, whereas higher DeltaNTP73 expression was detected in non-RGA cases. TP53 expression did not vary according to DeltaNTP73/TATP73 expression ratio. Leukaemic cells with higher DeltaNTP73/TATP73 ratios were significantly more resistant to cytarabine-induced apoptosis.
Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Citarabina/uso terapéutico , Proteínas de Unión al ADN/genética , Genes p53/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Proteínas Supresoras de Tumor/genética , Apoptosis/efectos de los fármacos , Aberraciones Cromosómicas , Resistencia a Antineoplásicos , Expresión Génica , Reordenamiento Génico , Humanos , Reacción en Cadena de la Polimerasa , RecurrenciaRESUMEN
Jacobsen syndrome (JBS) is a contiguous gene deletion syndrome involving terminal chromosome 11q. The haploinsufficiency of multiple genes contributes to the overall clinical phenotype, which can include the variant Paris-Trousseau syndrome, a transient thrombocytopenia related to FLI1 hemizygous deletion. We investigated a boy with features of JBS using classic cytogenetic methods, FISH and high-resolution array CGH. The proband was found to have a mosaic ring chromosome 11 resulting in a hemizygous 11q terminal deletion of 8.6 Mb, leading to a copy number loss of 52 genes. The patient had a hemizygous deletion in the FLI1 gene region without apparent thrombocytopenia, and he developed diabetes mellitus type I, which has not previously been described in the spectrum of disorders associated with JBS. The relationship of some of the genes within the context of the phenotype caused by a partial deletion of 11q has provided insights concerning the developmental anomalies presented in this patient with atypical features of JBS.
RESUMEN
The preferentially expressed antigen in melanoma (PRAME) gene is aberrantly expressed in chronic lymphoproliferative disorders (CLD). We produced and characterized an anti-PRAME monoclonal antibody (MoAb), which was then applied in a quantitative flow cytometric (QFC) method to evaluate PRAME expression in leukemic cells from the peripheral blood (PB) of 47 patients with chronic lymphocytic leukemia and seven with mantle cell lymphoma as well as in the PB mononuclear cells (PBMCs) and B lymphocytes from 15 healthy subjects. Approximately 90% of CLD, but none of the normal samples, presented more than 20% of PRAME+ lymphocytes. Moreover, the intensity of PRAME expression was significantly higher in CLD cells compared to normal B lymphocytes and PBMCs. By immunofluorescence microscopy and by permeabilized flow cytometry we demonstrated that PRAME is a membrane antigen and a cytoplasmic protein aberrantly expressed in malignant CLD. Our results suggest that the analysis of PRAME protein may contribute for the distinction between normal and leukemic cells in CLD, and that PRAME may be a potential target for therapy.
Asunto(s)
Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/genética , Linfoma de Células del Manto/genética , Trastornos Linfoproliferativos/genética , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Citometría de Flujo/métodos , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Hibridación Fluorescente in Situ/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
BACKGROUND: MicroRNAs (miRNAs or miRs) are post-transcriptional regulators of eukaryotic cells and knowledge of differences in miR levels may provide new approaches to diagnosis and therapy. METHODS: The present study measured the levels of nine miRs in head and neck squamous cell carcinomas (HNSCC) and determined whether clinical pathological features are associated with differences in miR levels. SET (I2PP2A) and PTEN protein levels were also measured, since their levels can be regulated by miR-199b and miR-21, respectively. Nine miRs (miR-15a, miR-21, miR-29b, miR-34c, miR-100, miR-125b, miR-137, miR-133b and miR-199b) were measured by real time qRT-PCR in HNSCC samples from 32 patients and eight resection margins. SET (I2PP2A) and PTEN protein levels were estimated by immunohistochemistry in paired HNSCC tissues and their matched resection margins. RESULTS: In HNSCC, the presence of lymph node invasion was associated with low miR-15a, miR-34c and miR-199b levels, whereas the presence of perineural invasion was associated with low miR-199b levels. In addition, miR-21 levels were high whereas miR-100 and miR-125b levels were low in HNSCC compared to the resection margins. When HNSCC line HN12, with or without knockdown of SET, were transfected with miR-34c inhibitor or miR-34c mimic, the miR-34c inhibitor increased cell invasion capacity while miR-34c mimic decreased the cell invasion. CONCLUSIONS: We showed that the levels of specific miRs in tumor tissue can provide insight into the maintenance and progression of HNSCC. GENERAL SIGNIFICANCE: MiRNAs are up- or down-regulated during cancer development and progression; they can be prognosis markers and therapeutic targets in HNSCC.
RESUMEN
BACKGROUND: Halofuginone (HF) is a low-molecular-weight alkaloid that has been demonstrated to interfere with Metalloproteinase-2 (MMP-2) and Tumor Growth Factor-ß (TGF-ß) function and, to present antiangiogenic, antiproliferative and proapoptotic properties in several solid tumor models. Based on the fact that high levels of Vascular Endothelial Growth Factor (VEGF) and increased angiogenesis have been described in acute myeloid leukemia and associated with disease progression, we studied the in vivo effects of HF using an Acute Promyelocytic Leukemia (APL) mouse model. METHODS: NOD/SCID mice were transplanted with leukemic cells from hCG-PML/RARA transgenic mice (TM) and treated with HF 150 µg/kg/day for 21 days. The leukemic infiltration and the percentage of VEGF+ cells were evaluated by morphology and flow cytometry. The effect of HF on the gene expression of several pro- and antiangiogenic factors, phosphorylation of SMAD2 and VEGF secretion was assessed in vitro using NB4 and HUVEC cells. RESULTS: HF treatment resulted in hematological remission with decreased accumulation of immature cell and lower amounts of VEGF in BM of leukemic mice. In vitro, HF modulated gene expression of several pro- and antiangiogenic factors, reduced VEGF secretion and phosphorylation of SMAD2, blocking TGF-ß-signaling. CONCLUSION: Taken together, our results demonstrate that HF inhibits SMAD2 signaling and reduces leukemia growth and angiogenesis.
Asunto(s)
Leucemia Promielocítica Aguda/metabolismo , Piperidinas/metabolismo , Quinazolinonas/metabolismo , Proteína Smad2/genética , Animales , Modelos Animales de Enfermedad , Humanos , Inmunofenotipificación , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Neovascularización Patológica , Fosforilación , Proteína Smad2/metabolismo , Células Tumorales CultivadasRESUMEN
Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system, due to an immune reaction against myelin proteins. Multipotent mesenchymal stromal cells (MSCs) present immunosuppressive effects and have been used for the treatment of autoimmune diseases. In our study, gene expression profile and in vitro immunomodulatory function tests were used to compare bone marrow-derived MSCs obtained from MS patients, at pre- and postautologous hematopoietic stem cell transplantation (AHSCT) with those from healthy donors. Patient MSCs comparatively exhibited i) senescence in culture; ii) similar osteogenic and adipogenic differentiation potential; iii) decreased expression of CD105, CD73, CD44, and HLA-A/B/C molecules; iv) distinct transcription at pre-AHSCT compared with control MSCs, yielding 618 differentially expressed genes, including the downregulation of TGFB1 and HGF genes and modulation of the FGF and HGF signaling pathways; v) reduced antiproliferative effects when pre-AHSCT MSCs were cocultured with allogeneic T-lymphocytes; vi) decreased secretion of IL-10 and TGF-ß in supernatants of both cocultures (pre- and post-AHSCT MSCs); and vii) similar percentages of regulatory cells recovered after MSC cocultures. The transcriptional profile of patient MSCs isolated 6 months posttransplantation was closer to pre-AHSCT samples than from healthy MSCs. Considering that patient MSCs exhibited phenotypic changes, distinct transcriptional profile and functional defects implicated in MSC immunomodulatory and immunosuppressive activity, we suggest that further MS clinical studies should be conducted using allogeneic bone marrow MSCs derived from healthy donors. We also demonstrated that treatment of MS patients with AHSCT does not reverse the transcriptional and functional alterations observed in patient MSCs.
Asunto(s)
Células de la Médula Ósea/citología , Células Madre Mesenquimatosas/metabolismo , Esclerosis Múltiple/patología , Transcriptoma , Adulto , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Análisis por Conglomerados , Técnicas de Cocultivo , Citocinas/análisis , Femenino , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Persona de Mediana Edad , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/terapia , Transducción de Señal , Linfocitos T/citología , Linfocitos T/inmunología , Adulto JovenRESUMEN
Human T lymphotropic virus type I (HTLV-1) is the etiological agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). CD8+ T cells may contribute to the protection or development of HAM/TSP. In this study we used SAGE methodology to screen for differentially expressed genes in CD8+ T cells isolated from HTLV-1 asymptomatic carriers (HAC) and from HAM/TSP patients to identify genes involved in HAM/TSP development. SAGE analysis was conducted by pooling samples according to clinical status. The comparison of gene expression profiles between HAC and HAM/TSP libraries identified 285 differentially expressed tags. We focus on cytotoxicity and cytokine-related genes due to their potential biological role in HTLV-1 infection. Our results showed that patients with HAM/TSP have high expression levels of degranulation-related genes, namely GZMH and PRF1, and of the cytoskeletal adaptor PXN. We found that GZMB and ZAP70 were overexpressed in HTLV-infected patients compared to the noninfected group. We also detected that CCL5 was higher in the HAM/TSP group compared to the HAC and CT groups. Our findings showed that CD8+ T cells of HAM/TSP patients have an inflammatory and active profile. PXN and ZAP70 overexpression in HTLV-1-infected patients was described for the first time here and reinforces this concept. However, although active and abundant, CD8+ T cells are not able to completely eliminate infected cells and prevent the development of HAM/TSP and, moreover, these cells might contribute to the pathogenesis of the disease by migrating to the central nervous system (CNS). These results should be further tested with biological functional assays to increase our understanding on the role of these molecules in the development of HTLV-1-related diseases.
Asunto(s)
Linfocitos T CD8-positivos/virología , Infecciones por HTLV-I/inmunología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Paraparesia Espástica Tropical/virología , Adulto , Anciano , Infecciones Asintomáticas , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/fisiología , Citocinas/genética , Citocinas/metabolismo , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica/genética , Regulación Viral de la Expresión Génica/fisiología , Granzimas/genética , Granzimas/metabolismo , Infecciones por HTLV-I/virología , Humanos , Masculino , Persona de Mediana Edad , Paraparesia Espástica Tropical/inmunología , Paraparesia Espástica Tropical/metabolismo , Paxillin/genética , Paxillin/metabolismo , Perforina , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto Joven , Proteína Tirosina Quinasa ZAP-70/genética , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
Mesenchymal stromal cells (MSCs) suppress T cell responses through mechanisms not completely understood. Adenosine is a strong immunosuppressant that acts mainly through its receptor A(2a) (ADORA2A). Extracellular adenosine levels are a net result of its production (mediated by CD39 and CD73), and of its conversion into inosine by Adenosine Deaminase (ADA). Here we investigated the involvement of ADO in the immunomodulation promoted by MSCs. Human T lymphocytes were activated and cultured with or without MSCs. Compared to lymphocytes cultured without MSCs, co-cultured lymphocytes were suppressed and expressed higher levels of ADORA2A and lower levels of ADA. In co-cultures, the percentage of MSCs expressing CD39, and of T lymphocytes expressing CD73, increased significantly and adenosine levels were higher. Incubation of MSCs with media conditioned by activated T lymphocytes induced the production of adenosine to levels similar to those observed in co-cultures, indicating that adenosine production was mainly derived from MSCs. Finally, blocking ADORA2A signaling raised lymphocyte proliferation significantly. Our results suggest that some of the immunomodulatory properties of MSCs may, in part, be mediated through the modulation of components related to adenosine signaling. These findings may open new avenues for the development of new treatments for GVHD and other inflammatory diseases.
Asunto(s)
Adenosina/biosíntesis , Antígenos CD/biosíntesis , Apirasa/biosíntesis , Células Madre Mesenquimatosas/inmunología , Células del Estroma/inmunología , Linfocitos T/inmunología , Adenosina/inmunología , Adenosina Desaminasa/inmunología , Antígenos CD/inmunología , Apirasa/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/inmunología , Procesos de Crecimiento Celular/inmunología , Humanos , Activación de Linfocitos , Células Madre Mesenquimatosas/citología , Receptor de Adenosina A2A/inmunología , Transducción de Señal/inmunología , Células del Estroma/citología , Linfocitos T Reguladores/inmunología , Regulación hacia ArribaRESUMEN
Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) expression in acute promyelocytic leukemia (APL) impairs transforming growth factor beta (TGFß) signaling, leading to cell growth advantage. Halofuginone (HF), a low-molecular-weight alkaloid that modulates TGFß signaling, was used to treat APL cell lines and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice subjected to transplantation with leukemic cells from human chorionic gonadotrophin-PML-RARα transgenic mice (TG). Cell cycle analysis using incorporated bromodeoxyuridine and 7-amino-actinomycin D showed that, in NB4 and NB4-R2 APL cell lines, HF inhibited cellular proliferation (P<0.001) and induced apoptosis (Pâ=â0.002) after a 24-hour incubation. Addition of TGFß revealed that NB4 cells were resistant to its growth-suppressive effects and that HF induced these effects in the presence or absence of the cytokine. Cell growth inhibition was associated with up-regulation of TGFß target genes involved in cell cycle regulation (TGFB, TGFBRI, SMAD3, p15, and p21) and down-regulation of MYC. Additionally, TGFß protein levels were decreased in leukemic TG animals and HF in vivo could restore TGFß values to normal. To test the in vivo anti-leukemic activity of HF, we transplanted NOD/SCID mice with TG leukemic cells and treated them with HF for 21 days. HF induced partial hematological remission in the peripheral blood, bone marrow, and spleen. Together, these results suggest that HF has anti-proliferative and anti-leukemic effects by reversing the TGFß blockade in APL. Since loss of the TGFß response in leukemic cells may be an important second oncogenic hit, modulation of TGFß signaling may be of therapeutic interest.
Asunto(s)
Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Piperidinas/farmacología , Quinazolinonas/farmacología , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Animales , Recuento de Células Sanguíneas , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia Promielocítica Aguda/sangre , Leucemia Promielocítica Aguda/genética , Ratones , Ratones SCID , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Considering that the importance of cancer/testis (CT) antigens in multiple myeloma (MM) biology is still under investigation, the present study aimed to: (1) identify genes differentially expressed in MM using microarray analysis of plasma cell samples, separated according to the number of expressed CTs; (2) examine possible pathways related to MM pathogenesis; (3) validate the expression of candidate genes by quantitative real-time PCR (RQ-PCR). Three samples predominantly positive (>6 expressed), including the U266 cell line, and three samples predominantly negative (0 or 1 expressed CT for the 13 analyzed CT antigens), were submitted for microarray analysis. Validation by RQ-PCR from 24 MM samples showed that the ITGA5 gene was downregulated in predominantly positive (>6 expressed CTs, p = 0.0030) and in tumor versus normal plasma cells (p = 0.0182). The RhoD gene was overexpressed in tumor plasma cells when compared to normal plasma cells (p = 0.0339). Results of the microarray analysis corroborate the hypothesis that MM could be separated into predominantly positive and predominantly negative expression. The differential expression of ITGA5 and RhoD suggests disruption of the focal adhesion pathway in MM and offers a new target field to be explored in this disease.
Asunto(s)
Antígenos de Neoplasias/genética , Biomarcadores de Tumor/genética , Adhesiones Focales/fisiología , Mieloma Múltiple/genética , Transducción de Señal , Testículo/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Integrina alfa5/genética , Integrina alfa5/metabolismo , Masculino , Persona de Mediana Edad , Mieloma Múltiple/metabolismo , Mieloma Múltiple/terapia , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Plasmáticas/metabolismo , Células Plasmáticas/patología , Pronóstico , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
BACKGROUND: Pleiotrophin (PTN) is a secreted cytokine with several properties related with tumor development, including differentiation, angiogenesis, invasion, apoptosis and metastasis. There is evidence that PTN has also a relevant role in primary brain neoplasms and its inactivation could be important to treatment response. Astrocytic and oligodendroglial tumors are the most frequent primary brain neoplasms. Astrocytic tumors are classified as pilocytic astrocytoma (PA), diffuse astrocytoma (DA), anaplastic astrocytoma (AA) and glioblastoma (GBM). Oligodendroglial tumors are classified as oligodendroglioma (O) and anaplastic oligodendroglioma (AO). The aim of the present study was to compare PTN expression, in astrocytomas and oligodendrogliomas and its association with the histological diagnosis, microvascular density, proliferate potential and clinical outcome. METHODS: Seventy-eight central nervous system tumors were analyzed. The histological diagnosis in accordance with WHO classification was: 13PA, 18DA, 8AA, 15GBM, 16O and 8AO. Immunohistochemistry was realized with these specific antibodies: pleiotrophin, CD31 to microvascular density and Ki-67 to cell proliferation. RESULTS: PTN expression was significantly higher in GBM and AA when compared to PA and higher in GBM compared to DA. PTN expression did not differ between O and AO. Proliferate index and microvascular density were evaluated only in high grade tumors (AA, GBM and AO) divided in three groups according to PTN expression (low, intermediate and high). These results showed no statistical difference between PTN expression and index of cellular proliferation and neither to PTN expression and microvascular density. Overall survival (OS) analysis (months) showed similar results in high grade gliomas with different levels of PTN expression. CONCLUSIONS: Our results suggest that PTN expression is associated with histopathological grade of astrocytomas. Proliferation rate, microvascular density and overall survival do not seem to be associated with PTN expression.