RESUMEN
BACKGROUND: Recently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8(+)CD25(+) Treg cells and the impact of microRNAs on molecules associated with immune regulation. METHODS: We purified human natural CD8(+) Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA 'signature' for CD8(+)CD25(+)FOXP3(+)CTLA-4(+) natural Treg cells. We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3'-UTR of important Treg cell-associated genes. RESULTS: The human CD8(+)CD25(+) natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo. CONCLUSIONS: We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.
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Linfocitos T CD8-positivos/metabolismo , Perfilación de la Expresión Génica , MicroARNs/genética , Linfocitos T Reguladores/metabolismo , Regiones no Traducidas 3'/genética , Antígenos CD/metabolismo , Antígeno CTLA-4/metabolismo , Proliferación Celular , Separación Celular , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Lentivirus/metabolismo , Proteínas de la Membrana/metabolismo , MicroARNs/metabolismo , Transcripción Genética , Transducción GenéticaRESUMEN
Cardiovascular disease linked to atherosclerosis is the leading cause of death worldwide. Atherosclerosis is mainly linked to dysfunction in vascular endothelial cells and subendothelial accumulation of oxidized forms of LDL. In the present study, we investigated the role of myeloperoxidase oxidized LDL (Mox-LDL) in endothelial cell dysfunction. We studied the effect of proinflammatory Mox-LDL treatment on endothelial cell motility, a parameter essential for normal vascular processes such as angiogenesis and blood vessel repair. This is particularly important in the context of an atheroma plaque, where vascular wall integrity is affected and interference with its repair could contribute to progression of the disease. We investigated in vitro the effect of Mox-LDL on endothelial cells angiogenic properties and we also studied the signalling pathways that could be affected by analysing Mox-LDL effect on the expression of angiogenesis-related genes. We report that Mox-LDL inhibits endothelial cell motility and tubulogenesis through an increase in miR-22 and heme oxygenase 1 expression. Our in vitro data indicate that Mox-LDL interferes with parameters associated with angiogenesis. They suggest that high LDL levels in patients would impair their endothelial cell capacity to cope with a damaged endothelium contributing negatively to the progression of the atheroma plaque.
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Células Endoteliales/citología , Endotelio Vascular/metabolismo , Hemo-Oxigenasa 1/metabolismo , Lipoproteínas LDL/metabolismo , MicroARNs/metabolismo , Peroxidasa/metabolismo , Animales , Células CHO , Movimiento Celular , Cricetinae , Cricetulus , Progresión de la Enfermedad , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Neovascularización Patológica , Placa Aterosclerótica/metabolismo , Transducción de Señal , Lesiones del Sistema Vascular/metabolismo , Cicatrización de HeridasRESUMEN
Regulatory T cells (Tregs) are characterized by a high expression of IL-2 receptor α chain (CD25) and of forkhead box P3 (FOXP3), the latter being essential for their development and function. Another major player in the regulatory function is the cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) that inhibits cytotoxic responses. However, the regulation of CTLA-4 expression remains less well explored. We therefore studied the microRNA signature of circulating CD4(+) Tregs isolated from adult healthy donors and identified a signature composed of 15 differentially expressed microRNAs. Among those, miR-24, miR-145, and miR-210 were down-regulated in Tregs compared with controls and were found to have potential target sites in the 3'-UTR of FOXP3 and CTLA-4; miR-24 and miR-210 negatively regulated FOXP3 expression by directly binding to their two target sites in its 3'-UTR. On the other hand, miR-95, which is highly expressed in adult peripheral blood Tregs, positively regulated FOXP3 expression via an indirect mechanism yet to be identified. Finally, we showed that miR-145 negatively regulated CTLA-4 expression in human CD4(+) adult peripheral blood Tregs by binding to its target site in CTLA-4 transcript 3'-UTR. To our knowledge, this is the first identification of a human adult peripheral blood CD4(+) Treg microRNA signature. Moreover, unveiling one mechanism regulating CTLA-4 expression is novel and may lead to a better understanding of the regulation of this crucial gene.
Asunto(s)
Regiones no Traducidas 3'/fisiología , Antígeno CTLA-4/biosíntesis , Factores de Transcripción Forkhead/biosíntesis , Regulación de la Expresión Génica/fisiología , MicroARNs/biosíntesis , Linfocitos T Reguladores/metabolismo , Adulto , Antígeno CTLA-4/genética , Femenino , Factores de Transcripción Forkhead/genética , Perfilación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Masculino , MicroARNs/genéticaRESUMEN
BACKGROUND: MicroRNAs (miRNAs) are small (19-22-nt) single-stranded noncoding RNA molecules whose deregulation of expression can contribute to human disease including the multistep processes of carcinogenesis in human. Circulating miRNAs are emerging biomarkers in many diseases and cancers such as type 2 diabetes, pulmonary disease, colorectal cancer, and gastric cancer among others; however, defining a plasma miRNA signature in acute myeloblastic leukemia (AML) that could serve as a biomarker for diagnosis or in the follow-up has not been done yet. METHODS: TaqMan miRNA microarray was performed to identify deregulated miRNAs in the plasma of AML patients. Quantitative real-time RT-PCR was used to validate the results. Receiver-operator characteristic (ROC) curve analysis was conducted to evaluate the diagnostic accuracy of the highly and significantly identified deregulated miRNA(s) as potential candidate biomarker(s). RESULTS: The plasma expression level of let-7d, miR-150, miR-339, and miR-342 was down-regulated whilst that of let-7b, and miR-523 was up-regulated in the AML group at diagnosis compared to healthy controls. ROC curve analyses revealed an AUC (the areas under the ROC curve) of 0.835 (95% CI: 0.7119- 0.9581; P<0.0001) and 0.8125 (95% CI: 0.6796-0.9454; P=0.0005) for miR-150, and miR-342 respectively. Combined ROC analyses using these 2 miRNAs revealed an elevated AUC of 0.86 (95% CI: 0.7819-0.94; P<0.0001) indicating the additive effect in the diagnostic value of these 2 miRNAs. QRT-PCR results showed that the expression level of these two miRs in complete remission AML patients resembled that of healthy controls. CONCLUSIONS: Our findings indicated that plasma miR-150 and miR-342 are novel important promising biomarkers in the diagnosis of AML. These novel and promising markers warrant validation in larger prospective studies.
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Biomarcadores de Tumor/sangre , Leucemia Mieloide Aguda/sangre , MicroARNs/sangre , Área Bajo la Curva , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Perfilación de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/genética , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Curva ROC , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Intermittent dosing of dasatinib with a once daily regimen has been shown to reduce side effects while preserving clinical efficacy in early and advanced phase chronic myeloid leukemia (CML). Yet, hematologic toxicity and fluid retention demand a dose modification or treatment discontinuation in selected patients. Patients resistant or intolerant to imatinib were retrospectively evaluated based on the toxicity-guided administration of a dose-reduced dasatinib regimen. Patients were treated with an on/off regimen (3 to 5 days on, 2 to 4 days off) to allow regression of dasatinib-dependent off-target toxicity. Patients were followed up by routine hematologic and cytogenetic assessment and molecular monitoring to safeguard clinical response to the altered drug schedule. Thirty-three CML patients primarily in chronic phase with imatinib intolerance (n = 11) or resistance (n = 22) were investigated. Nonexclusive reasons for dose reduction were hematologic toxicity (17/33, 51%) and pleural effusions (18/33, 55%). On/off treatment with a weekend drug holiday significantly reduced pleural effusions and hematologic toxicity. Eighteen of 31 (58%) patients showed effective disease control despite reduced total weekly dasatinib doses, either demonstrated by achieving an improved response level (12/31) or keeping the response level achieved by conventional continuous dosing (6/31). Of note, 10/12 patients with subsequently improved response have been treated for a minimum of 6 months with continuous dosing dasatinib regimens without having achieved the response level achieved after allowing drug holiday. Weekend treatment interruption of dasatinib allows continuation of dasatinib treatment for patients suffering from side effects. These data mandate prospective investigation of alternative intermittent targeting regimens.
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Benzamidas/uso terapéutico , Leucemia Mieloide de Fase Crónica/tratamiento farmacológico , Piperazinas/uso terapéutico , Pirimidinas/administración & dosificación , Pirimidinas/uso terapéutico , Tiazoles/administración & dosificación , Tiazoles/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Benzamidas/administración & dosificación , Dasatinib , Esquema de Medicación , Resistencia a Antineoplásicos , Femenino , Humanos , Mesilato de Imatinib , Masculino , Persona de Mediana Edad , Piperazinas/administración & dosificación , Estudios RetrospectivosRESUMEN
Since the introduction of tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML), the management of this disease has completely changed. The aim has been first to bring to the patient a maximal response and to identify at different time-points what could be considered an optimal response (which is to render the progression free survival as important as possible). To achieve this, new molecular tools were needed, the most important being the real time quantitative PCR (RT-qPCR), to measure the number of remaining transcripts after several period of treatment. The second important tool was the sequencing of the BCR-ABL kinase domain to identify potential mutations giving rise to resistance to imatinib first and next to second generation TKIs. This technique, much more sensitive than cytogenetics, has allowed the definition of important levels of transcripts (the major molecular response i.e. a three log reduction and the complete molecular response i.e. a 4.5 log reduction) the first ensuring a long term PFS on treatment, the second allowing the birth of studies looking at whether it would be possible to discontinue the treatment in this group of patients.
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Proteínas de Fusión bcr-abl/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Monitoreo Fisiológico/métodos , Inhibidores de Proteínas Quinasas/uso terapéutico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Supervivencia sin Enfermedad , Proteínas de Fusión bcr-abl/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/mortalidad , Tasa de SupervivenciaRESUMEN
Regulatory T cells (Tregs) play a key role in immune system homeostasis and tolerance to antigens, thereby preventing autoimmunity, and may be partly responsible for the lack of an appropriate immune response against tumor cells. Although not sufficient, a high expression of forkhead box P3 (FOXP3) is necessary for their suppressive function. Recent reports have shown that histones deacetylase inhibitors increased FOXP3 expression in T cells. We therefore decided to investigate in non-Tregs CD4-positive cells, the mechanisms by which an aspecific opening of the chromatin could lead to an increased FOXP3 expression. We focused on binding of potentially activating transcription factors to the promoter region of FOXP3 and on modifications in the five miRs constituting the Tregs signature. Valproate treatment induced binding of Ets-1 and Ets-2 to the FOXP3 promoter and acted positively on its expression, by increasing the acetylation of histone H4 lysines. Valproate treatment also induced the acquisition of the miRs Tregs signature. To elucidate whether the changes in the miRs expression could be due to the increased FOXP3 expression, we transduced these non-Tregs with a FOXP3 lentiviral expression vector, and found no changes in miRs expression. Therefore, the modification in their miRs expression profile is not due to an increased expression of FOXP3 but directly results from histones deacetylase inhibition. Rather, the increased FOXP3 expression results from the additive effects of Ets factors binding and the change in expression level of miR-21 and miR-31. We conclude that valproate treatment of human non-Tregs confers on them a molecular profile similar to that of their regulatory counterpart.
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Linfocitos T CD4-Positivos/fisiología , Sangre Fetal/inmunología , Factores de Transcripción Forkhead/genética , Histona Desacetilasas/genética , Ácido Valproico/farmacología , Regiones no Traducidas 5'/genética , Linfocitos T CD4-Positivos/efectos de los fármacos , Células Cultivadas , Secuencia Conservada , Cartilla de ADN , Histona Desacetilasas/inmunología , Humanos , Recién Nacido , MicroARNs/genética , Plásmidos , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , TransfecciónRESUMEN
BACKGROUND: The differentiation of CD8+ T lymphocytes following priming of naïve cells is central in the establishment of the adaptive immune response. Yet, the molecular events underlying this process are not fully understood. MicroRNAs have been recently shown to play a key role in the regulation of haematopoiesis in mouse, but their implication in peripheral lymphocyte differentiation in humans remains largely unknown. METHODS: In order to explore the potential implication of microRNAs in CD8+ T cell differentiation in humans, microRNA expression profiles were analysed using microarrays and quantitative PCR in several human CD8+ T cell subsets defining the major steps of the T cell differentiation pathway. RESULTS: We found expression of a limited set of microRNAs, including the miR-17~92 cluster. Moreover, we reveal the existence of differentiation-associated regulation of specific microRNAs. When compared to naive cells, miR-21 and miR-155 were indeed found upregulated upon differentiation to effector cells, while expression of the miR-17~92 cluster tended to concomitantly decrease. CONCLUSIONS: This study establishes for the first time in a large panel of individuals the existence of differentiation associated regulation of microRNA expression in human CD8+ T lymphocytes in vivo, which is likely to impact on specific cellular functions.
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Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Regulación de la Expresión Génica , MicroARNs/genética , Humanos , Subgrupos de Linfocitos TRESUMEN
Aberrant expression of microRNAs has been recently associated with chronic lymphocytic leukemia (CLL) outcome. Although disease evolution can be predicted by several prognostic factors, a better outcome individualization in a given patient is still of utmost interest. Here, we showed that miR-29c and miR-223 expression levels decreased significantly with progression from Binet stage A to C were significantly lower in poor prognostic subgroups (defined by several prognostic factors) and could significantly predict treatment-free survival (TFS) and overall survival (OS). Furthermore, we developed a quantitative real-time polymerase chain reaction (qPCR) score combining miR-29c, miR-223, ZAP70, and LPL (from 0 to 4 poor prognostic markers) to stratify treatment and death risk in a cohort of 110 patients with a median follow-up of 72 months (range, 2-312). Patients with a score of 0/4, 1/4, 2/4, 3/4, and 4/4 had a median TFS of greater than 312, of 129, 80, 36, and 19 months, respectively (hazard ratio, HR(0/4 < 1/4 < 2/4 < 3/4 < 4/4) = 17.00, P < .001). Patients with a score of 0-1/4, 2-3/4, and 4/4 had a median OS of greater than 312, of 183 and 106 months, respectively (HR(0/4 < 1/4 < 2/4 < 3/4 < 4/4) = 13.69, P = .001). This score will help to identify, among the good and poor prognosis subgroups, patients who will need early therapy and thus will require a closer follow-up.
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Regulación hacia Abajo/genética , Leucemia Linfocítica Crónica de Células B/diagnóstico , MicroARNs/genética , Adulto , Anciano , Anciano de 80 o más Años , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Lipoproteína Lipasa/genética , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Pronóstico , ARN Mensajero/análisis , Medición de Riesgo , Índice de Severidad de la Enfermedad , Proteína Tirosina Quinasa ZAP-70/genéticaRESUMEN
Imatinib mesylate (imatinib) has been shown to be highly efficacious in the treatment of chronic myeloid leukemia (CML). Continuous and adequate dosing is essential for optimal outcomes and with imatinib treatment possibly being lifelong, patient adherence is critical. The ADAGIO (Adherence Assessment with Glivec: Indicators and Outcomes) study aimed to assess prospectively over a 90-day period the prevalence of imatinib nonadherence in patients with CML; to develop a multivariate canonical correlation model of how various determinants may be associated with various measures of nonadherence; and to examine whether treatment response is associated with adherence levels. A total of 202 patients were recruited from 34 centers in Belgium, of whom 169 were evaluable. One-third of patients were considered to be nonadherent. Only 14.2% of patients were perfectly adherent with 100% of prescribed imatinib taken. On average, patients with suboptimal response had significantly higher mean percentages of imatinib not taken (23.2%, standard deviation [SD] = 23.8) than did those with optimal response (7.3%, SD = 19.3, P = .005; percentages calculated as proportions x 100). Nonadherence is more prevalent than patients, physicians, and family members believe it is, and therefore should be assessed routinely. It is associated with poorer response to imatinib. Several determinants may serve as alert signals, many of which are clinically modifiable.
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Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Cooperación del Paciente , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Benzamidas , Estudios de Casos y Controles , Femenino , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/epidemiología , Leucemia Mielógena Crónica BCR-ABL Positiva/psicología , Masculino , Persona de Mediana Edad , Cooperación del Paciente/estadística & datos numéricos , Piperazinas/efectos adversos , Prevalencia , Pirimidinas/efectos adversos , Estudios Retrospectivos , Factores de Riesgo , Resultado del Tratamiento , Adulto JovenRESUMEN
Treg are the main mediators of dominant tolerance. Their mechanisms of action and applications are subjects of considerable debate currently. However, a human microRNA (miR) Treg signature has not been described yet. We investigated human natural Treg and identified a signature composed of five miR (21, 31, 125a, 181c and 374). Among those, two were considerably under-expressed (miR-31 and miR-125a). We identified a functional target sequence for miR-31 in the 3' untranslated region (3' UTR) of FOXP3 mRNA. Using lentiviral transduction of fresh cord blood T cells, we demonstrated that miR-31 and miR-21 had an effect on FOXP3 expression levels. We showed that miR-31 negatively regulates FOXP3 expression by binding directly to its potential target site in the 3' UTR of FOXP3 mRNA. We next demonstrated that miR-21 acted as a positive, though indirect, regulator of FOXP3 expression. Transduction of the remaining three miR had no direct effect on FOXP3 expression or on the phenotype and will remain the subject of future investigations. In conclusion, not only have we identified and validated a miR signature for human natural Treg, but also unveiled some of the mechanisms by which this signature was related to the control of FOXP3 expression in these cells.
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Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , MicroARNs/genética , Linfocitos T Reguladores/metabolismo , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Sangre Fetal/citología , Expresión Génica/genética , Perfilación de la Expresión Génica , Células HeLa , Humanos , Lentivirus/genética , MicroARNs/antagonistas & inhibidores , MicroARNs/metabolismo , Oligonucleótidos Antisentido/genética , Subgrupos de Linfocitos T/metabolismo , Transducción GenéticaRESUMEN
BACKGROUND: Zeta-associated protein 70 (ZAP70) is a widely recognized prognostic factor in chronic lymphocytic leukemia, but mechanisms by which its higher expression leads to a poor outcome must still be fully explained. DESIGN AND METHODS: In an attempt to unveil unfavorable cellular properties linked to high ZAP70 expression, we used gene expression profiling to identify genes associated with disparities in B cells from chronic lymphocytic leukemia patients expressing high versus low ZAP70 mRNA, measured by quantitative real-time PCR. Two groups of 7 patients were compared, selected on the basis of either high or low ZAP70 mRNA expression. RESULTS: Twenty-seven genes were differentially expressed with an FDR<10%, and several genes were significant predictors of treatment-free survival (TFS) and/or overall survival; PDE8A and FCRL family genes (down-regulated in ZAP70(+) patients) could predict TFS and overall survival; ITGA4 mRNA (up-regulated in ZAP70(+) patients) could significantly predict overall survival. Importantly, gene set enrichment analysis revealed overrepresentation of adhesion/migration genes. We therefore investigated in vitro adhesion/migration capacity of chronic lymphocytic leukemia cells into a stromal microenvironment or in response to conditioned medium. We showed that ZAP70(+) cells had better adhesion/migration capacities and only ZAP70(+) patient cells responded to microenvironment contact by CXCR4 downregulation. CONCLUSIONS: We concluded that several prognostic factors are the reflection of microenvironment interactions and that the increased adhesion/migratory capacity of ZAP70(+) cells in their microenvironment can explain their better survival and thus the aggressiveness of the disease.
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Perfilación de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/genética , ARN Mensajero/metabolismo , Proteína Tirosina Quinasa ZAP-70/genética , Anciano , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/metabolismo , Linfocitos B/metabolismo , Linfocitos B/patología , Adhesión Celular , Movimiento Celular , Análisis por Conglomerados , Femenino , Citometría de Flujo , Humanos , Lectinas Tipo C , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Células Madre Mesenquimatosas/patología , Persona de Mediana Edad , Pronóstico , ARN Mensajero/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/patología , Análisis de Supervivencia , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
BACKGROUND AIMS: The transmigratory capacity of bone marrow (BM) mesenchymal stromal cells (MSC) through the endothelial cell barrier into various tissues and their differentiation potential makes them ideal candidates for cell therapy. Nevertheless, the mechanisms and agents promoting their migration are not fully understood. We evaluated the effects of several inflammatory cytokines on the migration of BM MSC and matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) production. METHODS: The migratory potential of BM MSC was evaluated using a Boyden chamber coated with Matrigel in the presence and absence of stromal cell-derived (SDF)-1alpha, platelet-derived growth factor (PDGF)bb, insulin-like growth factor (IGF)-I and interleukin (IL)-6. The ability of inflammatory cytokines to induce MSC migration was tested in presence of their respective Ab or blocking peptide. We used immunofluorescence to check the expression of cytokine receptors, and MMP/TIMP production was analyzed at the protein (human cytokine array, enzyme-linked immunosorbent assay (ELISA), gelatine zymography and Western blot) and mRNA quantitative real-time polymerase chain reaction (qRT-PCR) levels. RESULTS: We have demonstrated that inflammatory cytokines promote the migratory capacity of BM MSC according to the expression of their respective receptors. Higher migration through Matrigel was observed in response to IL-6 and PDGFbb. qRT-PCR and cytokine array revealed that migration was the result of the variable level of MMP/TIMP in response to inflammatory stimuli. CONCLUSIONS: Our observations suggest that chemokines and cytokines involved in the regulation of the immunity or inflammatory process promote the migration of MSC into BM or damaged tissues. One of the mechanisms used by MSC to promote their migration though the extracellular matrix is modulation of the production of MMP-1, MMP-2, MMP-13, TIMP-1 and TIMP-2.
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Movimiento Celular/efectos de los fármacos , Citocinas/farmacología , Mediadores de Inflamación/farmacología , Metaloproteinasas de la Matriz/biosíntesis , Células Madre Mesenquimatosas/citología , Células del Estroma/enzimología , Inhibidores Tisulares de Metaloproteinasas/biosíntesis , Células de la Médula Ósea/citología , Proliferación Celular/efectos de los fármacos , Quimiocinas/farmacología , Colágeno/metabolismo , Combinación de Medicamentos , Citometría de Flujo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/patología , Laminina/metabolismo , Metaloproteinasas de la Matriz/genética , Fenotipo , Proteoglicanos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Quimiocina/metabolismo , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Inhibidores Tisulares de Metaloproteinasas/genéticaRESUMEN
BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have been shown to possess immunomodulatory functions and proposed as a tool for managing or preventing graft-versus-host disease (GvHD) as well as promoting clinical transplantation tolerance. We investigated the capacity of human bone marrow (BM) MSC to modulate the proliferation of T cells obtained from peripheral blood (PB) and umbilical cord blood (CB). We addressed the importance of the MSC:T-cell ratio, requirement for cell contact and impact of soluble factors on the MSC-mediated effects. We also analyzed whether regulatory T cells could be modulated by MSC in co-cultures. METHODS: The effect of different MSC concentrations on T-cell proliferation induced by allogeneic, mitogenic or CD3/CD28 stimulation was analyzed using bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate-succinimidyl ester (CFDA-SE) labeling. The level of regulatory T cells was assessed using quantitative real-time polymerase chain reaction (PCR) and flow cytometry analysis. RESULTS: MSC induced a dose- and contact-dependent inhibition of T-cell proliferation but lymphocytes from CB and PB were differentially affected. At low concentrations, MSC supported both CB and PB T-cell proliferation, rather than inhibiting their proliferation. This supportive effect was contact independent and soluble factors such interleukin-6 (IL-6) appeared to be involved. Interestingly, among the expanded T-cell population in both CB and PB, regulatory T cells were increased and were a part of the new cells promoted by MSC at low doses. CONCLUSIONS: MSC represent an attractive tool for reducing the lymphocyte response by inhibiting T-cell activation and proliferation as well as promoting tolerance by maintaining and promoting the expansion of regulatory cells. Nevertheless, the dual ability of MSC to either sustain or suppress T-cell proliferation according to conditions should be considered in the context of clinical applications.
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Sangre Fetal/citología , Interleucina-6/metabolismo , Células Madre Mesenquimatosas/citología , Células del Estroma/citología , Linfocitos T/citología , ADP-Ribosil Ciclasa 1/metabolismo , Adulto , Antígenos CD4/metabolismo , Recuento de Células , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Ensayo de Unidades Formadoras de Colonias , Medios de Cultivo Condicionados/farmacología , Humanos , Tolerancia Inmunológica , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Activación de Linfocitos/efectos de los fármacos , Mitógenos/farmacología , Células del Estroma/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Adulto JovenRESUMEN
OBJECTIVE: This study examines risk recall and evolution of patients' anxiety after transmission of life-threatening risk information in an informed consent procedure for experimental HSCT. METHODS: Informed consent interviews were audio-recorded and transcribed. Patient risk recall was obtained through comparing information provided in the interview to information recalled by patients following the interview. The evolution of patients' anxiety was assessed through comparing patients' post- to patients' pre-interview anxiety using the STAI-State. Physicians' communication skills and risk framing were analyzed. RESULTS: Twenty patients were included. Patients recalled on average 4 risks (S.D.=1.6) out of 9 different risks transmitted (S.D.=2) which corresponds to a recall rate of 44% (S.D.=15.5). Patients' anxiety remained on average stable (Mean=0.4; S.D.=9.1). Linear regression analysis showed that risk recall was predicted positively by the number of risks transmitted (B=.30; P=.032) and by patients' problem-focused coping (B=.21; P=.008). The evolution of anxiety was predicted positively by the number of times benefits were transmitted (B=.83; P=.003) and negatively by the level of anxiety before the interview (B=-.50; P=.001). CONCLUSION: Results show the limits of patients' risk recall in the context of informed consent for a life-threatening procedure. PRACTICE IMPLICATIONS: This study highlights the necessity to develop strategies allowing tailoring of risk transmission to every patient's needs.
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Ansiedad/psicología , Trasplante de Células Madre Hematopoyéticas/psicología , Consentimiento Informado/psicología , Recuerdo Mental , Relaciones Médico-Paciente , Adulto , Bélgica , Femenino , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , RiesgoRESUMEN
BACKGROUND: Neuronal tissue has limited potential to self-renew or repair after neurological diseases. Cellular therapies using stem cells are promising approaches for the treatment of neurological diseases. However, the clinical use of embryonic stem cells or foetal tissues is limited by ethical considerations and other scientific problems. Thus, bone marrow mesenchymal stomal cells (BM-MSC) could represent an alternative source of stem cells for cell replacement therapies. Indeed, many studies have demonstrated that MSC can give rise to neuronal cells as well as many tissue-specific cell phenotypes. METHODS: BM-MSC were differentiated in neuron-like cells under specific induction (NPBM + cAMP + IBMX + NGF + Insulin). By day ten, differentiated cells presented an expression profile of real neurons. Functionality of these differentiated cells was evaluated by calcium influx through glutamate receptor AMPA3. RESULTS: Using microarray analysis, we compared gene expression profile of these different samples, before and after neurogenic differentiation. Among the 1943 genes differentially expressed, genes down-regulated are involved in osteogenesis, chondrogenesis, adipogenesis, myogenesis and extracellular matrix component (tuftelin, AGC1, FADS3, tropomyosin, fibronectin, ECM2, HAPLN1, vimentin). Interestingly, genes implicated in neurogenesis are increased. Most of them are involved in the synaptic transmission and long term potentialisation as cortactin, CASK, SYNCRIP, SYNTL4 and STX1. Other genes are involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuronal receptor (FK506, ARHGAP6, CDKRAP2, PMCH, GFPT2, GRIA3, MCT6, BDNF, PENK, amphiregulin, neurofilament 3, Epha4, synaptotagmin). Using real time RT-PCR, we confirmed the expression of selected neuronal genes: NEGR1, GRIA3 (AMPA3), NEF3, PENK and Epha4. Functionality of these neuron-like cells was demonstrated by Ca2+ influx through glutamate receptor channel (AMPA3) in the presence of two agonist glutamate, AMPA or CNQX antagonist. CONCLUSION: Our results demonstrate that BM-MSC have the potential to differentiate in neuronal cells with specific gene expression and functional properties. BM-MSC are thus promising candidates for cell-based therapy of neurodegenerative diseases.
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Células de la Médula Ósea/metabolismo , Perfilación de la Expresión Génica , Células Madre Mesenquimatosas/metabolismo , Enfermedades Neurodegenerativas/terapia , Neuronas/metabolismo , Trasplante de Células Madre/métodos , Células del Estroma/metabolismo , Diferenciación Celular/fisiología , Humanos , Neuronas/citología , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Although an activating mutation of Ras is commonly observed in myelodysplastic syndrome (MDS), the role of Ras in the natural history of MDS remains largely unknown. We prospectively studied efficiency and tolerance of lonafarnib, a compound able to inhibit Ras signalling pathway through an inhibition of farnesyl transferase, in patients with MDS or secondary acute myeloid leukaemia (sAML). Lonafarnib was administered orally at a dose of 200 mg twice daily for three courses of 4 weeks (separated by 1 to 4 weeks without treatment). Sixteen patients were included: FAB/RAEB (n = 10), RAEB-T (n = 2), sAML (n = 2) and chronic myelomonocytic leukaemia (CMML; n = 2); WHO/RAEB-1 (n = 4), RAEB-2 (n = 5), AML (n = 5), CMML (n = 2). Median age was 70 (53-77) years. The karyotype was complex or intermediate in 11 patients, and the International Prognostic Scoring Systems (IPSS) risk groups were low in two patients, INT-1 in one patient, INT-2 in four patients and high in six patients (unknown or not applicable in three patients). Among the 14 patients tested, five had Ras mutations in codons 12, 13 or 61 of N-Ras, K-Ras or H-Ras. One patient was excluded of the analysis for protocol violation, and 15 patients were assessable for tolerance. Gastrointestinal toxicities (diarrhoea, nausea and anorexia) and myelosuppression were the major side effects. Other toxicities included infections, fatigue, increase of liver enzymes, arrhythmia and skin rash. One patient died of infection, and the treatment was stopped in one other who developed atrial fibrillation. Doses were reduced in all but one patient treated with more than one course of farnesyl transferase inhibitor. Responses were assessable in 12 patients. A partial response in one sAML patient and a very transient decrease of blast cell count with normalisation of karyotype in one MDS patient were observed. No relation between improvement of marrow parameters and detected Ras mutations was observed. Lonafarnib alone, administered following our schedule, has shown limited activity in patients with MDS or secondary AML. Gastrointestinal and haematological toxicities appear the limiting toxicity in this population of patients.
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Farnesiltransferasa/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Síndromes Mielodisplásicos/tratamiento farmacológico , Piperidinas/efectos adversos , Piridinas/efectos adversos , Anciano , Femenino , Enfermedades Gastrointestinales/inducido químicamente , Genes ras/genética , Humanos , Masculino , Persona de Mediana EdadRESUMEN
OBJECTIVE: Chronic lymphocytic leukemia (CLL) cells develop chemoresistance over time associated with defects in apoptosis pathway. Novel treatment strategies are required to overcome resistance of cells to commonly used agents. The effects of valproic acid (VPA), an antiepileptic drug with histone deacetylase inhibitory activity, on mononuclear cells isolated from 40 CLL patients were evaluated. METHODS: CLL cells were treated with increasing doses of VPA (0.5, 1, 2, and 5 mM). The mode of cytotoxic drug action was determined by annexin binding, DNA fragmentation, and caspase activation. RESULTS: Exposure of CLL cells to VPA resulted in dose-dependent cytotoxicity and apoptosis in the 40 CLL patients. VPA treatment induced apoptotic changes in CLL cells including phosphatidylserine externalization and DNA fragmentation. The mean apoptotic rates were similar between IgV(H) mutated and unmutated patients, the latter presenting a more aggressive clinical course. VPA induced apoptosis via the extrinsic pathway involving engagement of the caspase-8-dependent cascade. Although CLL cells are commonly resistant to death receptor-induced apoptosis, VPA significantly increased sensitivity of leukemic cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and led to downregulation of c-FLIP (L) expression. VPA caused no potentialization of TRAIL-induced apoptosis on normal B cells. In addition, VPA overcame the prosurvival effects of bone marrow stromal cells. CONCLUSION: These findings point out that the combination of TRAIL and VPA, at clinically relevant concentration, may be valuable in the treatment of CLL.
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Anticonvulsivantes/farmacología , Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Proteínas de Neoplasias/metabolismo , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ácido Valproico/farmacología , Anciano , Anciano de 80 o más Años , Apoptosis/genética , Linfocitos B/metabolismo , Linfocitos B/patología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/farmacología , Femenino , Regulación Leucémica de la Expresión Génica/genética , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/genética , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Persona de Mediana Edad , Mutación , Proteínas de Neoplasias/genética , Fosfatidilserinas/metabolismo , Transducción de Señal/genética , Células Tumorales Cultivadas , Ácido Valproico/uso terapéuticoRESUMEN
BACKGROUND: During malignant progression, tumor cells need to acquire novel characteristics that lead to uncontrolled growth and reduced immunogenicity. In the Bovine Leukemia Virus-induced ovine leukemia model, silencing of viral gene expression has been proposed as a mechanism leading to immune evasion. However, whether proviral expression in tumors is completely suppressed in vivo was not conclusively demonstrated. Therefore, we studied viral expression in two selected experimentally-infected sheep, the virus or the disease of which had features that made it possible to distinguish tumor cells from their nontransformed counterparts. RESULTS: In the first animal, we observed the emergence of a genetically modified provirus simultaneously with leukemia onset. We found a Tax-mutated (TaxK303) replication-deficient provirus in the malignant B-cell clone while functional provirus (TaxE303) had been consistently monitored over the 17-month aleukemic period. In the second case, both non-transformed and transformed BLV-infected cells were present at the same time, but at distinct sites. While there was potentially-active provirus in the non-leukemic blood B-cell population, as demonstrated by ex-vivo culture and injection into naïve sheep, virus expression was completely suppressed in the malignant B-cells isolated from the lymphoid tumors despite the absence of genetic alterations in the proviral genome. These observations suggest that silencing of viral genes, including the oncoprotein Tax, is associated with tumor onset. CONCLUSION: Our findings suggest that silencing is critical for tumor progression and identify two distinct mechanisms-genetic and epigenetic-involved in the complete suppression of virus and Tax expression. We demonstrate that, in contrast to systems that require sustained oncogene expression, the major viral transforming protein Tax can be turned-off without reversing the transformed phenotype. We propose that suppression of viral gene expression is a contributory factor in the impairment of immune surveillance and the uncontrolled proliferation of the BLV-infected tumor cell.