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1.
Cytometry A ; 95(8): 917-924, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31120635

RESUMEN

The use of high-throughput flow cytometry to characterize nanoparticles has received increased interest in recent years. However, to fully realize the potential of flow cytometry for the characterization of nanometer-sized objects, suitable calibrators for size estimation must be developed and the sensitivity of conventional flow cytometers has to be advanced. Based on the scattered signal, silica and plastic beads have often been used as flow cytometric size calibrators to evaluate the size of extracellular vesicles and artificial vesicles (liposomes). However, several studies have shown that these beads are unable to accurately correlate scatter intensity to vesicle size. In this work, we present a novel method to estimate the size of individual liposomes in flow cytometry based on liposomal size calibrators prepared by fluorescence-activated cell sorting (FACS), here coined fluorescence-activated nanoparticle sorting (FANS). These calibration liposomes exhibit sizes, structures, and refractive indexes identical to the particles being studied and thus can serve as unique calibrators. First, a sample of polydisperse fluorophore-labeled unilamellar liposomes was prepared and analyzed by flow cytometry. Next, different fractions of the polydisperse liposomes were FANS-sorted according to their fluorescence intensity. Thereafter, we employed nanoparticle tracking analysis (NTA) to evaluate the liposome sizes of the FANS-sorted liposome fractions. Finally, we correlated the flow cytometric readouts (side scatter and fluorescence intensity) of the FANS-sorted liposome fractions with their corresponding size obtained by NTA. This procedure enabled us to translate the liposome fluorescence intensity to the liposome size in nanometers for all detected individual liposomes. We validated the size distribution of our polydisperse liposome sample obtained from flow cytometry in combination with our FANS-calibrators against standard methods for sizing nanoparticles, including NTA and cryo-transmission electron microscopy. This work also highlights the limitation of using the flow cytometric side scattering readout to determine the size of small (30-300 nm) artificial vesicles. © 2019 International Society for Advancement of Cytometry.


Asunto(s)
Calibración , Citometría de Flujo/métodos , Colorantes Fluorescentes/farmacología , Nanopartículas/química , Vesículas Extracelulares/química , Fluorescencia , Colorantes Fluorescentes/química , Humanos , Liposomas/química , Liposomas/farmacología , Nanopartículas/ultraestructura
2.
J Immunol ; 197(5): 1989-99, 2016 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-27481851

RESUMEN

Emergency granulopoiesis refers to the increased production of neutrophils in bone marrow and their release into circulation induced by severe infection. Several studies point to a critical role for G-CSF as the main mediator of emergency granulopoiesis. However, the consequences of G-CSF stimulation on the transcriptome of neutrophils and their precursors have not yet been investigated in humans. In this work, we examine the changes in mRNA expression induced by administration of G-CSF in vivo, as a model of emergency granulopoiesis in humans. Blood samples were collected from healthy individuals after 5 d of G-CSF administration. Neutrophil precursors were sorted into discrete stages of maturation by flow cytometry, and RNA was subjected to microarray analysis. mRNA levels were compared with previously published expression levels in corresponding populations of neutrophil precursors isolated from bone marrow of untreated, healthy individuals. One thousand one hundred and ten mRNAs were differentially expressed >2-fold throughout terminal granulopoiesis. Major changes were seen in pathways involved in apoptosis, cytokine signaling, and TLR pathways. In addition, G-CSF treatment reduced the levels of four of five measured granule proteins in mature neutrophils, including the proantibacterial protein hCAP-18, which was completely deficient in neutrophils from G-CSF-treated donors. These results indicate that multiple biological processes are altered to satisfy the increased demand for neutrophils during G-CSF-induced emergency granulopoiesis in humans.


Asunto(s)
Expresión Génica , Factor Estimulante de Colonias de Granulocitos/farmacología , Leucopoyesis/genética , Neutrófilos/fisiología , Péptidos Catiónicos Antimicrobianos/deficiencia , Péptidos Catiónicos Antimicrobianos/genética , Apoptosis/inmunología , Movimiento Celular , Citocinas/inmunología , Citocinas/metabolismo , Voluntarios Sanos , Humanos , Análisis por Micromatrices , Neutrófilos/efectos de los fármacos , Proteínas Recombinantes/inmunología , Catelicidinas
4.
Nat Commun ; 13(1): 3595, 2022 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-35739121

RESUMEN

Differentiation of multipotent stem cells into mature cells is fundamental for development and homeostasis of mammalian tissues, and requires the coordinated induction of lineage-specific transcriptional programs and cell cycle withdrawal. To understand the underlying regulatory mechanisms of this fundamental process, we investigated how the tissue-specific transcription factors, CEBPA and CEBPE, coordinate cell cycle exit and lineage-specification in vivo during granulocytic differentiation. We demonstrate that CEBPA promotes lineage-specification by launching an enhancer-primed differentiation program and direct activation of CEBPE expression. Subsequently, CEBPE confers promoter-driven cell cycle exit by sequential repression of MYC target gene expression at the G1/S transition and E2F-meditated G2/M gene expression, as well as by the up-regulation of Cdk1/2/4 inhibitors. Following cell cycle exit, CEBPE unleashes the CEBPA-primed differentiation program to generate mature granulocytes. These findings highlight how tissue-specific transcription factors coordinate cell cycle exit with differentiation through the use of distinct gene regulatory elements.


Asunto(s)
Regulación de la Expresión Génica , Factores de Transcripción , Animales , Ciclo Celular , Diferenciación Celular/genética , Granulocitos/metabolismo , Mamíferos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
5.
Metabolism ; 63(9): 1188-97, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24996265

RESUMEN

OBJECTIVE: Obesity is associated with low-grade inflammation and the infiltration of immune cells in insulin-sensitive tissues, leading to metabolic impairment. Epigenetic mechanisms control immune cell lineage determination, function and migration and are implicated in obesity and type 2 diabetes (T2D). The aim of this study was to determine the global DNA methylation profile of immune cells in obese and T2D individuals in a cell type-specific manner. MATERIAL AND METHODS: Fourteen obese subjects and 11 age-matched lean subjects, as well as 12 T2D obese subjects and 7 age-matched lean subjects were recruited. Global DNA methylation levels were measured in a cell type-specific manner by flow cytometry. We validated the assay against mass spectrometry measures of the total 5-methylcytosine content in cultured cells treated with the hypomethylation agent decitabine (r=0.97, p<0.001). RESULTS: Global DNA methylation in peripheral blood mononuclear cells, monocytes, lymphocytes or T cells was not altered in obese or T2D subjects. However, analysis of blood fractions from lean, obese, and T2D subjects showed increased methylation levels in B cells from obese and T2D subjects and in natural killer cells from T2D patients. In these cell types, DNA methylation levels were positively correlated with insulin resistance, suggesting an association between DNA methylation changes, immune function and metabolic dysfunction. CONCLUSIONS: Both obesity and T2D are associated with an altered epigenetic signature of the immune system in a cell type-specific manner. These changes could contribute to the altered immune functions associated with obesity and insulin resistance.


Asunto(s)
Linfocitos B/metabolismo , Metilación de ADN , Diabetes Mellitus Tipo 2/metabolismo , Células Asesinas Naturales/metabolismo , Obesidad/metabolismo , Regulación hacia Arriba , Adolescente , Adulto , Linfocitos B/patología , Índice de Masa Corporal , Células Cultivadas , Estudios de Cohortes , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/inmunología , Epigénesis Genética , Humanos , Resistencia a la Insulina , Células Asesinas Naturales/patología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Monocitos/patología , Obesidad/inmunología , Linfocitos T/metabolismo , Linfocitos T/patología , Adulto Joven
6.
J Leukoc Biol ; 90(3): 629-34, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21653237

RESUMEN

The current study reports a flow cytometry-based protocol for the prospective purification of human BM populations representing six successive stages of terminal neutrophil differentiation, including early promyelocytes and late promyelocytes, myelocytes, metamyelocytes, band cells, and PMN neutrophilic granulocytes. Validation experiments revealed a high purity of each bone marrow population and biological meaningful expression profiles for marker genes of neutrophil differentiation at a hitherto unprecedented resolution. Hence, the present protocol should be useful for studying neutrophil differentiation in vivo in the human setting and constitutes an important alternative to models that are based on in vitro differentiation of myeloid cell lines and HPCs.


Asunto(s)
Diferenciación Celular , Inmunofenotipificación , Neutrófilos/citología , Neutrófilos/inmunología , Western Blotting , Médula Ósea/metabolismo , Línea Celular , Separación Celular , Citometría de Flujo , Células Precursoras de Granulocitos/citología , Células Precursoras de Granulocitos/inmunología , Células Precursoras de Granulocitos/metabolismo , Granulocitos/citología , Granulocitos/inmunología , Granulocitos/metabolismo , Humanos , Técnicas para Inmunoenzimas , Células Mieloides/citología , Células Mieloides/inmunología , Células Mieloides/metabolismo , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/inmunología , Células Progenitoras Mieloides/metabolismo , Neutrófilos/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
7.
Mycorrhiza ; 15(2): 143-8, 2005 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15221578

RESUMEN

Forest soil from an experimental Norway spruce forest with four levels of wood ash addition (0, 1, 3 and 6 tonnes ha(-1)) was used to inoculate pine (Pinus sylvestris) seedlings with indigenous ectomycorrhizal (EM) fungi. Uptake of 32P and 86Rb in a root bioassay was used to estimate the demand for P and K by seedlings grown in the different soils. Utilisation of P from apatite was tested in a laboratory system where uptake by the ectomycorrhizal mycelium was separated from uptake by roots. The demand for P and K in the seedlings was similar regardless of the ash treatment. Variation in EM levels, estimated as fungal biomass (ergosterol) in roots, was large in the different soils, but not related to ash addition. Uptake of P from apatite was, on average, 23% of total seedling P and was not related to EM levels. It was concluded that the improved P uptake from apatite by EM fungi found in earlier studies is probably not a general phenomenon among EM fungi. The small effect of ash addition on EM levels and P uptake suggests that addition of granulated wood ash is a forest management treatment that will have only minor influence on ectomycorrhizal symbiosis.


Asunto(s)
Micorrizas/fisiología , Pinus sylvestris/microbiología , Raíces de Plantas/microbiología , Árboles/microbiología , Apatitas/metabolismo , Biomasa , Fósforo/metabolismo , Pinus sylvestris/fisiología , Raíces de Plantas/fisiología , Plantones/microbiología , Plantones/fisiología , Microbiología del Suelo , Árboles/fisiología , Madera
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