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Cell Metab ; 29(3): 745-754.e4, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30449685


Identification of cell-surface markers specific to human pancreatic ß cells would allow in vivo analysis and imaging. Here we introduce a biomarker, ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase3), that is expressed on the cell surface of essentially all adult human ß cells, including those from individuals with type 1 or type 2 diabetes. NTPDase3 is expressed dynamically during postnatal human pancreas development, appearing first in acinar cells at birth, but several months later its expression declines in acinar cells while concurrently emerging in islet ß cells. Given its specificity and membrane localization, we utilized an NTPDase3 antibody for purification of live human ß cells as confirmed by transcriptional profiling, and, in addition, for in vivo imaging of transplanted human ß cells. Thus, NTPDase3 is a cell-surface biomarker of adult human ß cells, and the antibody directed to this protein should be a useful new reagent for ß cell sorting, in vivo imaging, and targeting.

Cancer Res ; 75(1): 181-93, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25398437


Therapeutics that induce cancer cell senescence can block cell proliferation and promote immune rejection. However, the risk of tumor relapse due to senescence escape may remain high due to the long lifespan of senescent cells that are not cleared. Here, we show how combining a senescence-inducing inhibitor of the mitotic kinase Aurora A (AURKA) with an MDM2 antagonist activates p53 in senescent tumors harboring wild-type 53. In the model studied, this effect is accompanied by proliferation arrest, mitochondrial depolarization, apoptosis, and immune clearance of cancer cells by antitumor leukocytes in a manner reliant upon Ccl5, Ccl1, and Cxcl9. The AURKA/MDM2 combination therapy shows adequate bioavailability and low toxicity to the host. Moreover, the prominent response of patient-derived melanoma tumors to coadministered MDM2 and AURKA inhibitors offers a sound rationale for clinical evaluation. Taken together, our work provides a preclinical proof of concept for a combination treatment that leverages both senescence and immune surveillance to therapeutic ends.

Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Aurora Quinase A/antagonistas & inibidores , Melanoma Experimental/tratamento farmacológico , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Aurora Quinase A/metabolismo , Azepinas/administração & dosagem , Azepinas/farmacologia , Proliferação de Células/efeitos dos fármacos , Humanos , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Melanoma/metabolismo , Melanoma/patologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia
Stem Cell Reports ; 1(6): 532-44, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24371808


Heterogeneity within pluripotent stem cell (PSC) populations is indicative of dynamic changes that occur when cells drift between different states. Although the role of metastability in PSCs is unclear, it appears to reflect heterogeneity in cell signaling. Using the Fucci cell-cycle indicator system, we show that elevated expression of developmental regulators in G1 is a major determinant of heterogeneity in human embryonic stem cells. Although signaling pathways remain active throughout the cell cycle, their contribution to heterogeneous gene expression is restricted to G1. Surprisingly, we identify dramatic changes in the levels of global 5-hydroxymethylcytosine, an unanticipated source of epigenetic heterogeneity that is tightly linked to cell-cycle progression and the expression of developmental regulators. When we evaluated gene expression in differentiating cells, we found that cell-cycle regulation of developmental regulators was maintained during lineage specification. Cell-cycle regulation of developmentally regulated transcription factors is therefore an inherent feature of the mechanisms underpinning differentiation.

Pontos de Checagem do Ciclo Celular/genética , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes/citologia , 5-Metilcitosina/análogos & derivados , Técnicas de Cultura de Células , Diferenciação Celular/genética , Citosina/análogos & derivados , Citosina/metabolismo , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética
Am J Respir Cell Mol Biol ; 49(2): 180-9, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23492192


Although the antibody-based recognition of cell-surface markers has been widely used for the identification of immune cells, overlap in the expression of markers by different cell types and the inconsistent use of antibody panels have resulted in a lack of clearly defined signatures for myeloid cell subsets. We developed a 10-fluorochrome flow cytometry panel for the identification and quantitation of myeloid cells in the lungs, including pulmonary monocytes, myeloid dendritic cells, alveolar and interstitial macrophages, and neutrophils. After the initial sorting of viable CD45(+) leukocytes, we detected three leukocyte subpopulations based on CD68 expression: CD68(-), CD68(low), and CD68(hi). Further characterization of the CD68(hi) population revealed CD45(+)/CD68(hi)/F4/80(+)/CD11b(-)/CD11c(+)/Gr1(-) alveolar macrophages and CD45(+)/CD68(hi)/F4/80(-)/CD11c(+)/Gr1(-)/CD103(+)/major histocompatibility complex (MHC) class II(hi) dendritic cells. The CD68(low) population contained primarily CD45(+)/CD68(low)/F4/80(+)/CD11b(+)/CD11c(+)/Gr1(-)/CD14(low) interstitial macrophages and CD45(+)/CD68(low)/F4/80(+)/CD11b(+)/CD11c(-)/Gr1(low)/CD14(hi) monocytes, whereas the CD68(-) population contained neutrophils (CD45(+)/CD68(-)/F4/80(-)/CD11b(+)/Gr1(hi)). The validity of cellular signatures was confirmed by a morphological analysis of FACS-sorted cells, functional studies, and the depletion of specific macrophage subpopulations using liposomal clodronate. We believe our approach provides an accurate and reproducible method for the isolation, quantification, and characterization of myeloid cell subsets in the lungs, which may be useful for studying the roles of myeloid cells during various pathological processes.

Células Dendríticas/citologia , Citometria de Fluxo , Pulmão/citologia , Macrófagos Alveolares/citologia , Monócitos/citologia , Animais , Conservadores da Densidade Óssea/farmacologia , Ácido Clodrônico/farmacologia , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Pulmão/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/fisiologia , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Transgênicos , Monócitos/metabolismo
Nat Med ; 17(2): 195-9, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21131958


Pandemic influenza viruses often cause severe disease in middle-aged adults without preexisting comorbidities. The mechanism of illness associated with severe disease in this age group is not well understood. Here we find preexisting serum antibodies that cross-react with, but do not protect against, 2009 H1N1 influenza virus in middle-aged adults. Nonprotective antibody is associated with immune complex-mediated disease after infection. We detected high titers of serum antibody of low avidity for H1-2009 antigen, and low-avidity pulmonary immune complexes against the same protein, in severely ill individuals. Moreover, C4d deposition--a marker of complement activation mediated by immune complexes--was present in lung sections of fatal cases. Archived lung sections from middle-aged adults with confirmed fatal influenza 1957 H2N2 infection revealed a similar mechanism of illness. These observations provide a previously unknown biological mechanism for the unusual age distribution of severe cases during influenza pandemics.

Complexo Antígeno-Anticorpo/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Adolescente , Adulto , Fatores Etários , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Complemento C3/análise , Reações Cruzadas/imunologia , Citocinas/sangue , Humanos , Influenza Humana/sangue , Influenza Humana/patologia , Influenza Humana/virologia , Interferon-alfa/sangue , Interferon beta/sangue , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Pessoa de Meia-Idade , Adulto Jovem
Hum Mol Genet ; 19(22): 4353-72, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20739296


Abnormalities in the development of enteric neural crest-derived progenitors (ENPs) that generate the enteric nervous system (ENS) can lead to aganglionosis in a variable portion of the distal gastrointestinal tract. Cumulative evidence suggests that variation of aganglionosis is due to gene interactions that modulate the ability of ENPs to populate the intestine; however, the developmental processes underlying this effect are unknown. We hypothesized that differences in enteric ganglion deficits could be attributable to the effects of genetic background on early developmental processes, including migration, proliferation, or lineage divergence. Developmental processes were investigated in congenic Sox10(Dom) mice, an established Hirschsprung disease (HSCR) model, on distinct inbred backgrounds, C57BL/6J (B6) and C3HeB/FeJ (C3Fe). Immuno-staining on whole-mount fetal gut tissue and dissociated cell suspensions was used to assess migration and proliferation. Flow cytometry utilizing the cell surface markers p75 and HNK-1 was used to isolate live ENPs for analysis of developmental potential. Frequency of ENPs was reduced in Sox10(Dom) embryos relative to wild-type embryos, but was unaffected by genetic background. Both migration and developmental potential of ENPs in Sox10(Dom) embryos were altered by inbred strain background with the most highly significant differences seen for developmental potential between strains and genotypes. In vivo imaging of fetal ENPs and postnatal ganglia demonstrates that altered lineage divergence impacts ganglia in the proximal intestine. Our analysis demonstrates that genetic background alters early ENS development and suggests that abnormalities in lineage diversification can shift the proportions of ENP populations and thus may contribute to ENS deficiencies in vivo.

Sistema Nervoso Entérico/embriologia , Doença de Hirschsprung/genética , Crista Neural/citologia , Fatores de Transcrição SOXE/genética , Células-Tronco/citologia , Animais , Antígenos CD57/metabolismo , Modelos Animais de Doenças , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/metabolismo , Gânglios/embriologia , Gânglios/patologia , Doença de Hirschsprung/embriologia , Doença de Hirschsprung/metabolismo , Humanos , Imuno-Histoquímica , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Intestinos/citologia , Intestinos/embriologia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mutação , Crista Neural/embriologia , Especificidade da Espécie