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1.
Proc Natl Acad Sci U S A ; 117(5): 2570-2578, 2020 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-31964813

RESUMEN

The thymus generates cells of the T cell lineage that seed the lymphatic and blood systems. Transcription factor regulatory networks control the lineage programming and maturation of thymic precursor cells. Whether extrathymic antigenic events, such as the microbial colonization of the mucosal tract also shape the thymic T cell repertoire is unclear. We show here that intestinal microbes influence the thymic homeostasis of PLZF-expressing cells in early life. Impaired thymic development of PLZF+ innate lymphocytes in germ-free (GF) neonatal mice is restored by colonization with a human commensal, Bacteroides fragilis, but not with a polysaccharide A (PSA) deficient isogenic strain. Plasmacytoid dendritic cells influenced by microbes migrate from the colon to the thymus in early life to regulate PLZF+ cell homeostasis. Importantly, perturbations in thymic PLZF+ cells brought about by alterations in early gut microbiota persist into adulthood and are associated with increased susceptibility to experimental colitis. Our studies identify a pathway of communication between intestinal microbes and thymic lymphocytes in the neonatal period that can modulate host susceptibility to immune-mediated diseases later in life.


Asunto(s)
Microbioma Gastrointestinal , Linfocitos/inmunología , Timo/crecimiento & desarrollo , Animales , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Bacteroides fragilis/fisiología , Diferenciación Celular , Colitis/genética , Colitis/inmunología , Colitis/microbiología , Colon/microbiología , Humanos , Linfocitos/citología , Ratones , Ratones Endogámicos C57BL , Proteína de la Leucemia Promielocítica con Dedos de Zinc/genética , Proteína de la Leucemia Promielocítica con Dedos de Zinc/inmunología , Timo/citología , Timo/inmunología
2.
Nat Immunol ; 11(8): 743-50, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20581830

RESUMEN

Viral infection induces type I interferons (IFN-alpha and IFN-beta) that recruit unexposed cells in a self-amplifying response. We report that the transcription factor MafB thwarts auto-amplification by a metastable switch activity. MafB acted as a weak positive basal regulator of transcription at the IFNB1 promoter through activity at transcription factor AP-1-like sites. Interferon elicitors recruited the transcription factor IRF3 to the promoter, whereupon MafB acted as a transcriptional antagonist, impairing the interaction of coactivators with IRF3. Mathematical modeling supported the view that prepositioning of MafB on the promoter allows the system to respond rapidly to fluctuations in IRF3 activity. Higher expression of MafB in human pancreatic islet beta cells might increase cellular vulnerability to viral infections associated with the etiology of type 1 diabetes.


Asunto(s)
Factor 3 Regulador del Interferón/inmunología , Interferón Tipo I/inmunología , Factor de Transcripción MafB/inmunología , Virosis/inmunología , Animales , Línea Celular Tumoral , Humanos , Factor 3 Regulador del Interferón/antagonistas & inhibidores , Interferón Tipo I/biosíntesis , Interferón beta/genética , Factor de Transcripción MafB/genética , Ratones , Ratones Noqueados , Modelos Inmunológicos , Regiones Promotoras Genéticas , Factor de Transcripción AP-1/inmunología , Transcripción Genética
3.
Proc Natl Acad Sci U S A ; 113(1): 122-7, 2016 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-26699463

RESUMEN

Analysis of embryonic fibroblasts from GFP reporter mice indicates that the fibroblast cell type harbors a large collection of developmentally and phenotypically heterogeneous subtypes. Some of these cells exhibit multipotency, whereas others do not. Multiparameter flow cytometry analysis shows that a large number of distinct populations of fibroblast-like cells can be found in cultures initiated from different embryonic organs, and cells sorted according to their surface phenotype typically retain their characteristics on continued propagation in culture. Similarly, surface phenotypes of individual cloned fibroblast-like cells exhibit significant variation. The fibroblast cell class appears to contain a very large number of denumerable subtypes.


Asunto(s)
Embrión de Mamíferos/citología , Fibroblastos/fisiología , Animales , Línea Celular , Separación Celular , Fibroblastos/metabolismo , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Ratones , Ratones Transgénicos , Pericitos/citología , Pericitos/fisiología , Fenotipo , Análisis de la Célula Individual , Transcriptoma , Factor A de Crecimiento Endotelial Vascular/genética
4.
J Cell Sci ; 127(Pt 18): 3916-27, 2014 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-24849652

RESUMEN

GPR107 is a type III integral membrane protein that was initially predicted to be a member of the family of G-protein-coupled receptors. This report shows that deletion of Gpr107 leads to an embryonic lethal phenotype that is characterized by a reduction in cubilin transcript abundance and a decrease in the representation of multiple genes implicated in the cubilin-megalin endocytic receptor complex (megalin is also known as LRP2). Gpr107-null fibroblast cells exhibit reduced transferrin internalization, decreased uptake of low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) cargo and resistance to toxins. Colocalization studies and proteomic analyses suggest that GPR107 associates with clathrin and the retromer protein VPS35 and that GPR107 might be responsible for the return of receptors to the plasma membrane from endocytic compartments. The highly selective deficits observed in Gpr107-null cells indicate that GPR107 interacts directly or indirectly with a limited subset of surface receptors.


Asunto(s)
Endocitosis , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/genética , Animales , Membrana Celular/genética , Membrana Celular/metabolismo , Femenino , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica , Receptores de Superficie Celular/genética , Receptores Acoplados a Proteínas G/deficiencia , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
5.
PLoS Genet ; 9(11): e1003950, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24278032

RESUMEN

Epoxyeicosatrienoic acids (EETs) confer vasoactive and cardioprotective functions. Genetic analysis of the contributions of these short-lived mediators to pathophysiology has been confounded to date by the allelic expansion in rodents of the portion of the genome syntenic to human CYP2J2, a gene encoding one of the principle cytochrome P450 epoxygenases responsible for the formation of EETs in humans. Mice have eight potentially functional genes that could direct the synthesis of epoxygenases with properties similar to those of CYP2J2. As an initial step towards understanding the role of the murine Cyp2j locus, we have created mice bearing a 626-kb deletion spanning the entire region syntenic to CYP2J2, using a combination of homologous and site-directed recombination strategies. A mouse strain in which the locus deletion was complemented by transgenic delivery of BAC sequences encoding human CYP2J2 was also created. Systemic and pulmonary hemodynamic measurements did not differ in wild-type, null, and complemented mice at baseline. However, hypoxic pulmonary vasoconstriction (HPV) during left mainstem bronchus occlusion was impaired and associated with reduced systemic oxygenation in null mice, but not in null mice bearing the human transgene. Administration of an epoxygenase inhibitor to wild-type mice also impaired HPV. These findings demonstrate that Cyp2j gene products regulate the pulmonary vascular response to hypoxia.


Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Hipoxia/patología , Pulmón/patología , Vasoconstricción/genética , Animales , Animales Modificados Genéticamente , Citocromo P-450 CYP2J2 , Sistema Enzimático del Citocromo P-450/metabolismo , Recombinación Homóloga , Humanos , Hipoxia/genética , Pulmón/metabolismo , Ratones , Oxidación-Reducción , Eliminación de Secuencia
6.
BMC Biochem ; 15: 14, 2014 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-25007711

RESUMEN

BACKGROUND: Secreted luciferases are highly useful bioluminescent reporters for cell-based assays and drug discovery. A variety of secreted luciferases from marine organisms have been described that harbor an N-terminal signal peptide for release along the classical secretory pathway. Here, we have characterized the secretion of Gaussia luciferase in more detail. RESULTS: We describe three basic mechanisms by which GLUC can be released from cells: first, classical secretion by virtue of the N-terminal signal peptide; second, internal signal peptide-mediated secretion and third, non-conventional secretion in the absence of an N-terminal signal peptide. Non-conventional release of dNGLUC is not stress-induced, does not require autophagy and can be enhanced by growth factor stimulation. Furthermore, we have identified the golgi-associated, gamma adaptin ear containing, ARF binding protein 1 (GGA1) as a suppressor of release of dNGLUC. CONCLUSIONS: Due to its secretion via multiple secretion pathways GLUC can find multiple applications as a research tool to study classical and non-conventional secretion. As GLUC can also be released from a reporter construct by internal signal peptide-mediated secretion it can be incorporated in a novel bicistronic secretion system.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Bioquímica/métodos , Luciferasas de Luciérnaga/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Bacterianas/genética , Secreciones Corporales , Genes/genética , Genes Reporteros/genética , Células HEK293 , Humanos , Luciferasas de Luciérnaga/genética , Señales de Clasificación de Proteína/genética
7.
J Immunol ; 189(6): 2879-89, 2012 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-22875804

RESUMEN

Influenza is a major cause of morbidity and mortality in the United States. Studies have shown that excessive T cell activity can mediate pneumonitis in the setting of influenza infection, and data from the 2009 H1N1 pandemic indicate that critical illness and respiratory failure postinfection were associated with greater infiltration of the lungs with CD8+ T cells. T cell Ig and mucin domain 3 (Tim3) is a negative regulator of Th1/Tc1-type immune responses. Activation of Tim3 on effector T cells has been shown to downregulate proliferation, cell-mediated cytotoxicity, and IFN-γ production, as well as induce apoptosis. In this article, we demonstrate that deletion of the terminal cytoplasmic domain of the Tim3 gene potentiates its ability to downregulate Tc1 inflammation, and that this enhanced Tim3 activity is associated with decreased phosphorylation of the TCR-CD3ζ-chain. We then show that mice with this Tim3 mutation infected with influenza are protected from morbidity and mortality without impairment in viral clearance or functional heterotypic immunity. This protection is associated with decreased CD8+ T cell proliferation and decreased production of inflammatory cytokines, including IFN-γ. Furthermore, the Tim3 mutation was protective against mortality in a CD8+ T cell-specific model of pneumonitis. These data suggest that Tim3 could be targeted to prevent immunopathology during influenza infection and demonstrate a potentially novel signaling mechanism used by Tim3 to downregulate the Tc1 response.


Asunto(s)
Infecciones por Orthomyxoviridae/inmunología , Receptores Virales/metabolismo , Regulación hacia Arriba/inmunología , Animales , Complejo CD3/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Citotoxicidad Inmunológica/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Receptor 2 Celular del Virus de la Hepatitis A , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/mortalidad , Fosforilación/genética , Fosforilación/inmunología , Receptores Virales/genética , Receptores Virales/fisiología , Eliminación de Secuencia/genética , Eliminación de Secuencia/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Análisis de Supervivencia , Regulación hacia Arriba/genética
8.
Nucleic Acids Res ; 40(Database issue): D1144-9, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22086960

RESUMEN

Optimization of primer sequences for polymerase chain reaction (PCR) and quantitative PCR (qPCR) and reaction conditions remains an experimental challenge. We have developed a resource, PrimerBank, which contains primers that can be used for PCR and qPCR under stringent and allele-invariant amplification conditions. A distinguishing feature of PrimerBank is the experimental validation of primer pairs covering most known mouse genes. Here, we describe a major update of PrimerBank that includes the design of new primers covering 17,076 and 18,086 genes for the human and mouse species, respectively. As a result of this update, PrimerBank contains 497,156 primers (an increase of 62% from the previous version) that cover 36,928 human and mouse genes, corresponding to around 94% of all known protein-coding gene sequences. An updated algorithm based on our previous approach was used to design new primers using current genomic information available from the National Center for Biotechnology Information (NCBI). PrimerBank primers work under uniform PCR conditions, and can be used for high-throughput or genome-wide qPCR. Because of their broader linear dynamic range and greater sensitivity, qPCR approaches are used to reanalyze changes in expression suggested by exploratory technologies such as microarrays and RNA-Seq. The primers and all experimental validation data can be freely accessed from the PrimerBank website, http://pga.mgh.harvard.edu/primerbank/.


Asunto(s)
Cartilla de ADN/química , Bases de Datos de Ácidos Nucleicos , Perfilación de la Expresión Génica , Reacción en Cadena de la Polimerasa , Algoritmos , Animales , Expresión Génica , Humanos , Internet , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa
9.
Nat Genet ; 36(9): 921-4, 2004 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15340423

RESUMEN

Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain.


Asunto(s)
Ratones Noqueados , Creación de Embriones para Investigación , Alelos , Animales , Investigación Genética , Ratones , Fenotipo , Creación de Embriones para Investigación/economía
10.
Bioorg Med Chem ; 20(2): 750-8, 2012 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-22217933

RESUMEN

The STAT6 (signal transducer and activator of transcription 6) protein facilitates T-helper cell 2 (Th2) mediated responses that control IgE-mediated atopic diseases such as asthma. We have identified compounds that bind to STAT6 and inhibit STAT6 tyrosine phosphorylation induced by IL-4. In the bronchial epithelial cell line BEAS-2B, compound (R)-84 inhibits the secretion of eotaxin-3, a chemokine eliciting eosinophil infiltration. (R)-84 appears to prevent STAT6 from assuming the active dimer configuration by directly binding the protein and inhibiting tyrosine phosphorylation.


Asunto(s)
Quimiocinas CC/metabolismo , Células Epiteliales/metabolismo , Indoles/química , Piridinas/química , Factor de Transcripción STAT6/antagonistas & inhibidores , Línea Celular , Quimiocina CCL26 , Dimerización , Humanos , Indoles/síntesis química , Indoles/farmacología , Interleucina-4/farmacología , Fosforilación , Piridinas/síntesis química , Piridinas/farmacología , Receptores de Interleucina-4/metabolismo , Factor de Transcripción STAT6/metabolismo , Transducción de Señal/efectos de los fármacos , Estereoisomerismo
11.
Nucleic Acids Res ; 38(Database issue): D792-9, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19906719

RESUMEN

PrimerBank (http://pga.mgh.harvard.edu/primerbank/) is a public resource for the retrieval of human and mouse primer pairs for gene expression analysis by PCR and Quantitative PCR (QPCR). A total of 306,800 primers covering most known human and mouse genes can be accessed from the PrimerBank database, together with information on these primers such as T(m), location on the transcript and amplicon size. For each gene, at least one primer pair has been designed and in many cases alternative primer pairs exist. Primers have been designed to work under the same PCR conditions, thus facilitating high-throughput QPCR. There are several ways to search for primers for the gene(s) of interest, such as by: GenBank accession number, NCBI protein accession number, NCBI gene ID, PrimerBank ID, NCBI gene symbol or gene description (keyword). In all, 26,855 primer pairs covering most known mouse genes have been experimentally validated by QPCR, agarose gel analysis, sequencing and BLAST, and all validation data can be freely accessed from the PrimerBank web site.


Asunto(s)
Biología Computacional/métodos , Cartilla de ADN/genética , Bases de Datos Genéticas , Bases de Datos de Ácidos Nucleicos , Perfilación de la Expresión Génica , Reacción en Cadena de la Polimerasa/métodos , Animales , Secuencia de Bases , Biología Computacional/tendencias , Bases de Datos de Proteínas , Humanos , Almacenamiento y Recuperación de la Información/métodos , Internet , Ratones , Datos de Secuencia Molecular , Programas Informáticos
12.
J Exp Med ; 201(12): 1891-7, 2005 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-15955840

RESUMEN

Eosinophil lineage-committed progenitors (EoPs) are phenotypically isolatable in the steady-state murine bone marrow. Purified granulocyte/monocyte progenitors (GMPs) gave rise to eosinophils as well as neutrophils and monocytes at the single cell level. Within the short-term culture of GMPs, the eosinophil potential was found exclusively in cells activating the transgenic reporter for GATA-1, a transcription factor capable of instructing eosinophil lineage commitment. These GATA-1-activating cells possessed an IL-5Ralpha(+)CD34(+)c-Kit(lo) phenotype. Normal bone marrow cells also contained IL-5Ralpha(+)CD34(+)c-Kit(lo) EoPs that gave rise exclusively to eosinophils. EoPs significantly increased in number in response to helminth infection, suggesting that the EoP stage is physiologically involved in eosinophil production in vivo. EoPs expressed eosinophil-related genes, such as the eosinophil peroxidase and the major basic protein, but did not express basophil/mast cell-related mast cell proteases. The enforced retroviral expression of IL-5Ralpha in GMPs did not enhance the frequency of eosinophil lineage read-outs, whereas IL-5Ralpha(+) GMPs displayed normal neutrophil/monocyte differentiation in the presence of IL-5 alone. Thus, IL-5Ralpha might be expressed specifically at the EoP stage as a result of commitment into the eosinophil lineage. The newly identified EoPs could be the cellular target in the treatment of a variety of disorders mediated by eosinophils.


Asunto(s)
Células de la Médula Ósea/citología , Linaje de la Célula/fisiología , Eosinófilos/citología , Células Precursoras de Granulocitos/citología , Animales , Antígenos CD34/metabolismo , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Citometría de Flujo , Factor de Transcripción GATA1 , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-5 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismo , Transducción Genética
13.
FASEB J ; 24(5): 1572-82, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20056716

RESUMEN

We describe for the first time fluorescent virus-like particles decorated with biologically active mono- and multisubunit immune receptors of choice and the basic application of such fluorosomes (FSs) to visualize and target immune receptor-ligand interactions. For that purpose, human embryonic kidney (HEK)-293 cells were stably transfected with Moloney murine leukemia virus (MoMLV) matrix protein (MA) GFP fusion constructs. To produce FSs, interleukins (ILs), IL-receptors (IL-Rs), and costimulatory molecules were fused to the glycosyl phosphatidyl inositol anchor acceptor sequence of CD16b and coexpressed along with MoMLV group-specific antigen-polymerase (gag-pol) in MA::GFP(+) HEK-293 cells. We show that IL-2 decorated but not control-decorated FSs specifically identify normal and malignant IL-2 receptor-positive (IL-2R(+)) lymphocytes by flow cytometry. In addition to cytokines and costimulatory molecules, FSs were also successfully decorated with the heterotrimeric IL-2Rs, allowing identification of IL-2(+) target cells. Specificity of binding was proven by complete inhibition with nonlabeled, soluble ligands. Moreover, IL-2R FSs efficiently neutralized soluble IL-2 and thus induced unresponsiveness of T cells receiving full activation stimuli via T-cell antigen receptor and CD28. FSs are technically simple, multivalent tools for assessing and blocking mono- and multisubunit immune receptor-ligand interactions with natural constituents in a plasma membrane context.


Asunto(s)
Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/química , Interleucina-2/inmunología , Receptores de Interleucina-2/inmunología , Proteínas Recombinantes/química , Virión/química , Línea Celular Transformada , Fluorescencia , Proteínas Ligadas a GPI , Proteínas Fluorescentes Verdes/genética , Humanos , Interleucina-2/análisis , Ligandos , Linfoma de Células B/diagnóstico , Linfoma de Células B/inmunología , Microscopía Confocal , Virus de la Leucemia Murina de Moloney , Receptores de IgG , Receptores de Interleucina-2/análisis , Proteínas Recombinantes/genética , Linfocitos T/inmunología , Virión/genética
14.
Pharmacol Res ; 63(4): 284-93, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21215314

RESUMEN

Sodium glucose co-transporter 2 (SGLT2) is a renal type III integral membrane protein that co-transports sodium and glucose from filtrate to epithelium in the proximal tubule. Human subjects with homozygous or compound heterozygous mutations in SLC5A2 exhibit glucosuria without hypoglycemia or other obvious morbidity, suggesting that blockade of SGLT2 has the potential to promote normalization of blood glucose without hypoglycemia in the setting of type 2 diabetes. This report presents the in vitro and in vivo pharmacological activities of EGT1442, a recently discovered SGLT2 inhibitor in the C-aryl glucoside class. The inhibitory effects of EGT1442 for human SGLT1 and SGLT2 were evaluated in an AMG uptake assay and the in vivo efficacy of treatment with EGT1442 was investigated in rats and dogs after a single dose and in db/db mice after chronic administration. The effect of EGT1442 on median survival of SHRSP rats was also evaluated. The IC(50) values for EGT1442 against human SGLT1 and SGLT2 are 5.6µM and 2nM, respectively. In normal rats and dogs a saturable urinary glucose excretion was produced with an ED(50) of 0.38 and 0.09mg/kg, respectively. Following chronic administration to db/db mice, EGT1442 dose-dependently reduced HbA(1c) and blood glucose concentration without affecting body mass or insulin level. Additionally, EGT1442 significantly prolonged the median survival of SHRSP rats. EGT1442 showed favorable properties both in vitro and in vivo and could be beneficial to the management of type 2 diabetic patients.


Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hemoglobina Glucada/metabolismo , Hipertensión/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Animales , Glucemia/metabolismo , Perros , Electrólitos/orina , Prueba de Tolerancia a la Glucosa , Glucosuria/tratamiento farmacológico , Humanos , Hipoglucemiantes/farmacología , Masculino , Ratones , Ratas , Ratas Endogámicas SHR , Ratas Sprague-Dawley , Transportador 1 de Sodio-Glucosa/metabolismo , Transportador 2 de Sodio-Glucosa/metabolismo
15.
Bioorg Med Chem Lett ; 21(15): 4465-70, 2011 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-21737266

RESUMEN

A series of C-aryl glucosides with various substituents at the 4'-position of the distal aryl ring have been synthesized and evaluated for inhibition of hSGLT1 and hSGLT2. Introduction of alkyl or alkoxy substituents at the 4'-position was found to improve SGLT2 potency, whereas introduction of a hydrophilic group at this position was deleterious. Compounds with alkoxy-, cycloalkoxy- or cycloalkenyloxy-ethoxy scaffolds exhibited good inhibitory activity and high selectivity toward SGLT2. Selected compounds were investigated for in vivo efficacy.


Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Glucósidos/química , Hipoglucemiantes/química , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Glucósidos/síntesis química , Glucósidos/uso terapéutico , Humanos , Hipoglucemiantes/síntesis química , Hipoglucemiantes/uso terapéutico , Transportador 1 de Sodio-Glucosa/antagonistas & inhibidores , Transportador 1 de Sodio-Glucosa/metabolismo , Transportador 2 de Sodio-Glucosa/metabolismo , Relación Estructura-Actividad
16.
Nat Med ; 9(6): 796-800, 2003 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12754503

RESUMEN

The content and structure of collagen is essential in governing the delivery of therapeutic molecules in tumors. Thus, simple histological staining of tumor tissue biopsies for collagen could be used to assess the accessibility of molecular therapeutics in tumors. Here we show that it is possible to optically image fibrillar collagen in tumors growing in mice using second-harmonic generation (SHG). Using this noninvasive technique, we estimated relative diffusive hindrance, quantified the dynamics of collagen modification after pharmacologic intervention and provided mechanistic insight into improved diffusive transport induced by the hormone relaxin. This technology could offer basic scientists and clinicians an enhanced ability to estimate the relative penetrabilities of molecular therapeutics.


Asunto(s)
Colágenos Fibrilares/metabolismo , Microscopía Confocal/métodos , Neoplasias/metabolismo , Neoplasias/patología , Animales , Colagenasas/metabolismo , Elastina/metabolismo , Colágenos Fibrilares/química , Humanos , Ratones , Trasplante de Neoplasias , Relaxina/metabolismo , Células Tumorales Cultivadas
17.
Lab Invest ; 90(9): 1357-64, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20548287

RESUMEN

Histiocytic sarcoma (HS), a rare hematological malignancy, is an aggressive neoplasm that responds poorly to therapy. The molecular etiology and pathology of this disease remain unclear, hampering the development of an effective therapy, and there remains a need for more, and more realistic, animal models. HS cells typically show a histiocytic (ie, tissue macrophage-like) morphology and express histiocyte/macrophage markers in the absence of lymphocyte markers. In this study, we report that Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice develop HS, but do not exhibit elevated incidence of other types of tumors. These mutant mice showed earlier mortality than wild-type (WT) or the other mutant mice, and this mortality was associated with HS. In total, 17 of 21 tumor-bearing Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice necropsied at 25-66 weeks of age showed multiple organ spread, with osteolytic lesions and orthotopic invasion from the bone marrow to skeletal muscle. Tumors from the mice were transplantable. In addition, all Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice, but only a small proportion of Dok-3(-/-) mice and no Dok-1(-/-)Dok-2(-/-) mice, exhibited abnormal accumulation of macrophages in the lung on necropsy at 8-12 weeks of age. Macrophages derived from Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice displayed an exaggerated proliferative response to macrophage colony-stimulating factor (M-CSF) or granulocyte- macrophage colony-stimulating factor (GM-CSF) compared with WT and mutant controls. Together, these findings indicate that Dok-1, Dok-2, and Dok-3 cooperatively suppress aggressive HS, and commend Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice as a useful model for the study of this neoplasia.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Unión al ADN/genética , Sarcoma Histiocítico/genética , Pulmón/patología , Macrófagos/patología , Fosfoproteínas/genética , Proteínas de Unión al ARN/genética , Animales , Factores Estimulantes de Colonias/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Sarcoma Histiocítico/patología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Organismos Libres de Patógenos Específicos
18.
Bioorg Med Chem ; 18(12): 4422-32, 2010 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-20576578

RESUMEN

A series of 2-substituted C-aryl glucosides have been synthesized and evaluated for inhibition of hSGLT1 and hSGLT2. Introduction of an appropriate ortho substituent at the proximal phenyl ring adjacent to the glycosidic bond was found to improve SGLT2 inhibitory activity and dramatically increase selectivity for hSGLT2 over hSGLT1. Selected compounds were investigated for in vivo efficacy.


Asunto(s)
Glucósidos/química , Hipoglucemiantes/química , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Línea Celular , Glucósidos/síntesis química , Glucósidos/farmacología , Humanos , Hipoglucemiantes/síntesis química , Hipoglucemiantes/farmacología , Transportador 1 de Sodio-Glucosa/antagonistas & inhibidores , Transportador 1 de Sodio-Glucosa/metabolismo , Transportador 2 de Sodio-Glucosa/metabolismo , Relación Estructura-Actividad
19.
J Allergy Clin Immunol ; 124(1): 121-8, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19500826

RESUMEN

BACKGROUND: T(H)2 lymphocytes play an important role in the induction and maintenance phase of type I allergy. Modulation of the responses of T(H)2 lymphocytes by novel forms of antigen-presenting platforms may help shape the immune response to allergen and palliate allergic diseases. OBJECTIVE: To present HLA class II/allergen-peptide complexes on virus-like particles (VLPs) and to evaluate their potential to modulate allergen-specific T-cell responses. METHODS: Virus-like particles that express the immunodominant T-cell epitope Art v 1(25-34) of the major mugwort pollen allergen in the context of HLA-DR1 and costimulatory molecules were produced by transfection of 293 cells. The effect of VLPs on IL-2 promoter activity, proliferation, and cytokine production of allergen-specific T cells derived from donors with and without mugwort pollen allergy was determined. RESULTS: Flow-cytometric analyses showed that HLA class II molecules, invariant chain::Art v 1 fusion proteins, and costimulatory molecules were expressed on 293 cells. Biochemical analyses confirmed that these molecules were efficiently targeted to VLPs. The engineered VLPs activated Art v 1-specific T cells in a costimulation-dependent manner. VLPs lacking costimulators induced T-cell unresponsiveness, which was overcome by addition of exogenous IL-2. Costimulation could be provided by CD80, CD86, or CD58 and induced distinct cytokine profiles in allergen-specific T cells. Unlike the other costimulatory molecules, CD58 induced IL-10/IFN-gamma-secreting T cells. CONCLUSION: Virus-like particles represent a novel, modular, acellular antigen-presenting system able to modulate the responses of allergen-specific T cells in a costimulator-dependent fashion. Allergen-specific VLPs show promise as tools for specific immunotherapy of allergic diseases.


Asunto(s)
Antígenos HLA-DR/metabolismo , Antígenos HLA-DR/farmacología , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunización/métodos , Linfocitos T/inmunología , Línea Celular , Clonación Molecular , Citocinas/inmunología , Vectores Genéticos/genética , Antígenos HLA-A/genética , Antígenos HLA-A/metabolismo , Antígenos HLA-A/farmacología , Antígenos HLA-DR/genética , Cadenas HLA-DRB1 , Humanos , Células Jurkat , Riñón/citología , Linfocitos T/efectos de los fármacos , Virión/genética
20.
J Cell Physiol ; 219(2): 430-7, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19170073

RESUMEN

Lymphatic vessels play a key role in maintaining tissue-fluid homeostasis, immune surveillance and metastasis. The hyaluronan receptor, LYVE-1, is widely used as a molecular marker for adult and embryonic lymphatic endothelium, but its physiological functions have not yet been established in vivo. In agreement with a recent report, LYVE-1(-/-) mice, which are healthy and fertile, do not display any defects related to congenital abnormalities of the lymphatic system. One hypothesis for the absence of a phenotype in LYVE-1 null mice is that other hyaluronan receptors, such as CD44, may compensate for LYVE-1. To test this hypothesis, we created LYVE-1/CD44 double knockout mice with appropriate littermate controls. Lymphatic vessel structure and function, as determined by histological methods and intravital microscopy, show that LYVE-1(-/-), CD44(-/-) and LYVE-1(-/-)/CD44(-/-) mice are indistinguishable from wild-type mice under normal conditions. Furthermore, resolution of carrageenan-induced paw edema is comparable in all genotypes. However, LYVE-1(-/-)/CD44(-/-) mice exhibit increased edema formation in a carrageenan-induced paw inflammation model compared to wild-type mice, but not to LYVE(-/-) or CD44(-/-) mice. These data suggest that LYVE-1 and CD44 are not required for the formation or function of lymphatics, but do not rule out a role for LYVE-1 in inflammation.


Asunto(s)
Glicoproteínas/metabolismo , Receptores de Hialuranos/metabolismo , Vasos Linfáticos , Ratones Noqueados , Animales , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Femenino , Genotipo , Glicoproteínas/genética , Receptores de Hialuranos/genética , Vasos Linfáticos/anatomía & histología , Vasos Linfáticos/fisiología , Masculino , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos C57BL , Fenotipo
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