RESUMEN
Extracellular vesicles derived from mesenchymal stem cells (MSCs) represent a novel approach for regenerative and immunosuppressive therapy. Recently, cytochalasin B-induced microvesicles (CIMVs) were shown to be effective drug delivery mediators. However, little is known about their immunological properties. We propose that the immunophenotype and molecular composition of these vesicles could contribute to the therapeutic efficacy of CIMVs. To address this issue, CIMVs were generated from murine MSC (CIMVs-MSCs) and their cytokine content and surface marker expression determined. For the first time, we show that CIMVs-MSCs retain parental MSCs phenotype (Sca-1+, CD49e+, CD44+, CD45-). Also, CIMVs-MSCs contained a cytokine repertoire reflective of the parental MSCs, including IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL-13, IL-17, CCL2, CCL3, CCL4, CCL5, CCL11, G-CSF, GM-CSF and TNF-α. Next, we evaluated the immune-modulating properties of CIMVs-MSCs in vivo using standard preclinical tests. MSCs and CIMVs-MSCs reduced serum levels of anti-sheep red blood cell antibody and have limited effects on neutrophil and peritoneal macrophage activity. We compared the immunomodulatory effect of MSCs, CIMVs and EVs. We observed no immunosuppression in mice pretreated with natural EVs, whereas MSCs and CIMVs-MSCs suppressed antibody production in vivo. Additionally, we have investigated the biodistribution of CIMVs-MSCs in vivo and demonstrated that CIMVs-MSCs localized in liver, lung, brain, heart, spleen and kidneys 48 h after intravenous injection and can be detected 14 days after subcutaneous and intramuscular injection. Collectively our data demonstrates immunomodulatory efficacy of CIMVs and supports their further preclinical testing as an effective therapeutic delivery modality.
Asunto(s)
Micropartículas Derivadas de Células/inmunología , Citocalasina B/farmacología , Citocinas/inmunología , Vesículas Extracelulares/inmunología , Inmunosupresores/farmacología , Macrófagos Peritoneales/inmunología , Células Madre Mesenquimatosas/inmunología , Animales , Micropartículas Derivadas de Células/efectos de los fármacos , Células Cultivadas , Vesículas Extracelulares/efectos de los fármacos , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , RatonesRESUMEN
BACKGROUND: Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes. METHODS: A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays. RESULTS: Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a 'methylator' phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype. CONCLUSION: Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy.
Asunto(s)
Caspasa 8/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Silenciador del Gen , Genes Supresores de Tumor , Neoplasias de Células Germinales y Embrionarias/tratamiento farmacológico , Neoplasias de Células Germinales y Embrionarias/genética , Apoptosis , Niño , Preescolar , Análisis por Conglomerados , Resistencia a Antineoplásicos , Tumor del Seno Endodérmico/tratamiento farmacológico , Tumor del Seno Endodérmico/genética , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Germinoma/tratamiento farmacológico , Germinoma/genética , Humanos , Masculino , Análisis por Micromatrices , Neoplasias de Células Germinales y Embrionarias/patología , Fenotipo , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ADN Metiltransferasa 3BRESUMEN
The Alternative Lengthening of Telomeres (ALT) mechanism is utilised by approximately 10% of human tumours and a higher proportion of some types of sarcomas. ALT+ cell lines and tumours show heterogeneous telomere length, extra-chromosomal circular and linear telomeric DNA, ALT associated promyelocytic bodies (APBs), a high frequency of post-replication exchanges in telomeres (designated as telomere-sister chromatid exchanges, T-SCE) and high instability at a GC-rich minisatellite, MS32 (D1S8). It is clear that there is a link between the minisatellite instability and the mechanism that underpins ALT, however currently the nature of this relationship is uncertain. Single molecule analysis of telomeric DNA from ALT+ cell lines and tumours has revealed complex telomere mutations that have not been seen in cell lines or tumours that express telomerase. These complex telomere mutations cannot be explained by T-SCE but must arise by another inter-molecular process. The break-induced replication (BIR) model that may explain the observed high frequency of T-SCE and the presence of complex telomere mutations is reviewed.
