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1.
Genes Dev ; 24(15): 1620-33, 2010 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-20679398

RESUMEN

The bicistronic microRNA (miRNA) locus miR-144/451 is highly expressed during erythrocyte development, although its physiological roles are poorly understood. We show that miR-144/451 ablation in mice causes mild erythrocyte instability and increased susceptibility to damage after exposure to oxidant drugs. This phenotype is deeply conserved, as miR-451 depletion synergizes with oxidant stress to cause profound anemia in zebrafish embryos. At least some protective activities of miR-451 stem from its ability to directly suppress production of 14-3-3zeta, a phospho-serine/threonine-binding protein that inhibits nuclear accumulation of transcription factor FoxO3, a positive regulator of erythroid anti-oxidant genes. Thus, in miR-144/451(-/-) erythroblasts, 14-3-3zeta accumulates, causing partial relocalization of FoxO3 from nucleus to cytoplasm with dampening of its transcriptional program, including anti-oxidant-encoding genes Cat and Gpx1. Supporting this mechanism, overexpression of 14-3-3zeta in erythroid cells and fibroblasts inhibits nuclear localization and activity of FoxO3. Moreover, shRNA suppression of 14-3-3zeta protects miR-144/451(-/-) erythrocytes against peroxide-induced destruction, and restores catalase activity. Our findings define a novel miRNA-regulated pathway that protects erythrocytes against oxidant stress, and, more generally, illustrate how a miRNA can influence gene expression by altering the activity of a key transcription factor.


Asunto(s)
Proteínas 14-3-3/metabolismo , Células Eritroides/metabolismo , Regulación del Desarrollo de la Expresión Génica , MicroARNs/metabolismo , Estrés Oxidativo , Proteínas 14-3-3/genética , Transporte Activo de Núcleo Celular , Animales , Secuencia de Bases , Catalasa/metabolismo , Células Eritroides/enzimología , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/metabolismo , Técnicas de Silenciamiento del Gen , Ratones , Ratones Noqueados , MicroARNs/genética , Alineación de Secuencia , Eliminación de Secuencia/genética , Pez Cebra/genética , Pez Cebra/metabolismo
2.
J Immunol ; 186(1): 73-82, 2011 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-21106852

RESUMEN

Exosomes are nano-sized membrane vesicles released from a wide variety of cells, formed in endosomes by inward budding of the endosomal limiting membrane. They have immune stimulatory-, inhibitory-, or tolerance-inducing effects, depending on their cellular origin, which is why they are investigated for use in vaccine and immune therapeutic strategies. In this study, we explored whether exosomes of different origins and functions can selectively target different immune cells in human peripheral blood. Flow cytometry, confocal laser scanning microscopy, and multispectral imaging flow cytometry (ImageStream) revealed that exosomes derived from human monocyte-derived dendritic cells and breast milk preferably associated with monocytes. In contrast, exosomes from an EBV-transformed B cell line (LCL1) preferentially targeted B cells. This was not observed for an EBV(-) B cell line (BJAB). Electron microscopy, size-distribution analysis (NanoSight), and a cord blood transformation assay excluded the presence of virions in our LCL1 exosome preparations. The interaction between LCL1-derived exosomes and peripheral blood B cells could be blocked efficiently by anti-CD21 or anti-gp350, indicating an interaction between CD21 on B cells and the EBV glycoprotein gp350 on exosomes. The targeting of LCL1-derived exosomes through gp350-CD21 interaction strongly inhibited EBV infection in B cells isolated from umbilical cord blood, suggesting a protective role for exosomes in regulating EBV infection. Our finding also suggests that exosome-based vaccines can be engineered for specific B cell targeting by inducing gp350 expression.


Asunto(s)
Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/virología , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/prevención & control , Exosomas/inmunología , Herpesvirus Humano 4/inmunología , Glicoproteínas de Membrana/metabolismo , Receptores de Complemento 3d/fisiología , Proteínas de la Matriz Viral/metabolismo , Subgrupos de Linfocitos B/metabolismo , Línea Celular Transformada , Membrana Celular/inmunología , Membrana Celular/metabolismo , Membrana Celular/virología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/virología , Exosomas/metabolismo , Exosomas/virología , Humanos , Lactancia , Leche Humana/inmunología , Leche Humana/metabolismo , Leche Humana/virología , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/virología , Unión Proteica/inmunología , Receptores de Complemento 3d/biosíntesis , Proteínas Estructurales Virales/metabolismo
3.
PLoS One ; 7(6): e38258, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22675532

RESUMEN

Subcapsular sinus macrophages (SSMs) in lymph nodes are rapidly exposed to antigens arriving in afferent lymph and have a role in their capture and display to B cells. In tissue sections SSMs exhibit long cellular processes and express high amounts of CD169. Here, we show that many of the cells present in lymph node cell suspensions that stain for CD169 are not macrophages but lymphocytes that have acquired SSM-derived membrane blebs. The CD169 bleb(+) lymphocytes are enriched for IL-17 committed IL-7Rα(hi)CCR6(+) T cells and NK cells. In addition, the CD169 staining detected on small numbers of CD11c(hi) dendritic cells is frequently associated with membrane blebs. Counter intuitively the CD169 bleb(+) lymphocytes are mostly CD4 and CD8 negative whereas many SSMs express CD4. In situ, many IL-7Rα(hi) cells are present at the subcapsular sinus and interfollicular regions and migrate in close association with CD169(+) macrophages. These findings suggest SSMs undergo fragmentation during tissue preparation and release blebs that are acquired by closely associated cells. They also suggest an intimate crosstalk between SSMs and IL-17 committed innate-like lymphocytes that may help provide early protection of the lymph node against lymph-borne invaders.


Asunto(s)
Extensiones de la Superficie Celular/metabolismo , Interleucina-17/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Linfocitos/inmunología , Macrófagos/inmunología , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Comunicación Celular , Fraccionamiento Celular , Linaje de la Célula , Citometría de Flujo , Interleucina-17/biosíntesis , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Linfocitos/citología , Macrófagos/citología , Ratones , Receptores CCR6/metabolismo , Receptores de Interleucina-7 , Lectina 1 Similar a Ig de Unión al Ácido Siálico , Coloración y Etiquetado
4.
J Vis Exp ; (64): e3884, 2012 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-22710268

RESUMEN

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed. Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStream(X) system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.


Asunto(s)
Citometría de Flujo/métodos , Citometría de Imagen/métodos , Macrófagos/metabolismo , Macrófagos/microbiología , Nanopartículas/química , Polianhídridos/metabolismo , Salmonella enterica/metabolismo , Actinas/análisis , Actinas/metabolismo , Animales , Línea Celular , Citocalasina D/química , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Ratones , Fagocitosis , Polianhídridos/química , Salmonella enterica/química , Salmonella enterica/genética , Transformación Bacteriana
5.
J Immunol Methods ; 347(1-2): 79-86, 2009 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-19524586

RESUMEN

Activation of T lymphocytes by antigen-presenting cells (APC) results in the formation of an immunological synapse. Following contact with the target cell, key signaling and adhesion molecules polarize within minutes to hours to the T cell-APC interface. Multispectral imaging flow cytometry, a new technology which combines flow cytometry with imaging, was used to visualize and quantify the recruitment of the CD3epsilon and Lck signaling molecules during the evolution of an immune synapse. Using this technology, thousands of T cell/macrophage conjugates could be analyzed for each experimental time point. Following Ca++ triggered T cell activation, the dynamics of Lck and CD3epsilon recruitment to the synapse, analyzed by two independent methods, were comparable. However, CD3epsilon exhibited longer residence times (>8 min) at the synapse than Lck.


Asunto(s)
Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Citometría de Flujo , Citometría de Imagen , Sinapsis Inmunológicas , Activación de Linfocitos , Macrófagos/inmunología , Transducción de Señal , Complejo CD3/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Humanos , Procesamiento de Imagen Asistido por Computador , Cinética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Transporte de Proteínas
6.
Syst Biol Reprod Med ; 55(5-6): 244-51, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19938959

RESUMEN

Spermatid specific thioredoxin-3 protein (SPTRX-3) accumulates in the superfluous cytoplasm of defective human spermatozoa. Novel ImageStream technology combining flow cytometry with cell imaging was used for parallel quantification and visualization of SPTRX-3 protein in defective spermatozoa of five men from infertile couples. The majority of the SPTRX-3 containing cells were overwhelmingly spermatozoa with a variety of morphological defects, detectable in the ImageStream recorded images. Quantitative parameters of relative SPTRX-3 induced fluorescence measured by ImageStream correlated closely with conventional flow cytometric measurements of the same sample set and reflected the results of clinical semen evaluation. Image Stream quantification of SPTRX-3 combines and surpasses the informative value of both conventional flow cytometry and light microscopic semen evaluation. The observed patterns of the retention of SPTRX-3 in the sperm samples from infertility patients support the view that SPTRX3 is a biomarker of male infertility.


Asunto(s)
Biomarcadores/análisis , Citometría de Flujo/instrumentación , Espermatozoides/ultraestructura , Tiorredoxinas/análisis , Adulto , Citometría de Flujo/métodos , Humanos , Infertilidad Masculina/diagnóstico , Masculino
7.
J Exp Med ; 205(12): 2899-913, 2008 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-19015308

RESUMEN

Hem1 (Hematopoietic protein 1) is a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins. Orthologues of Hem1 in Dictyostelium discoideum, Drosophila melanogaster, and Caenorhabditis elegans are essential for cytoskeletal reorganization, embryonic cell migration, and morphogenesis. However, the in vivo functions of mammalian Hem1 are not known. Using a chemical mutagenesis strategy in mice to identify novel genes involved in immune cell functions, we positionally cloned a nonsense mutation in the Hem1 gene. Hem1 deficiency results in defective F-actin polymerization and actin capping in lymphocytes and neutrophils caused by loss of the Rac-controlled actin-regulatory WAVE protein complex. T cell development is disrupted in Hem1-deficient mice at the CD4(-)CD8(-) (double negative) to CD4(+)CD8(+) (double positive) cell stages, whereas T cell activation and adhesion are impaired. Hem1-deficient neutrophils fail to migrate in response to chemotactic agents and are deficient in their ability to phagocytose bacteria. Remarkably, some Rac-dependent functions, such as Th1 differentiation and nuclear factor kappaB (NF-kappaB)-dependent transcription of proinflammatory cytokines proceed normally in Hem1-deficient mice, whereas the production of Th17 cells are enhanced. These results demonstrate that Hem1 is essential for hematopoietic cell development, function, and homeostasis by controlling a distinct pathway leading to cytoskeletal reorganization, whereas NF-kappaB-dependent transcription proceeds independently of Hem1 and F-actin polymerization.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Inmunidad Innata/fisiología , Linfopoyesis/fisiología , Proteínas de la Membrana , Mutación Puntual , Actinas/metabolismo , Anemia/inmunología , Animales , Linfocitos B/citología , Linfocitos B/fisiología , Movimiento Celular/fisiología , Análisis Mutacional de ADN , Células Madre Hematopoyéticas/fisiología , Sistema Hematopoyético/citología , Sistema Hematopoyético/fisiología , Interferón gamma/inmunología , Interleucina-17/metabolismo , Interleucina-2/inmunología , Activación de Linfocitos , Linfopenia/inmunología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/citología , Neutrófilos/inmunología , Neutrófilos/fisiología , Fagocitosis/fisiología , Linfocitos T/citología , Linfocitos T/fisiología , Quimera por Trasplante
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