RESUMEN
Nonalcoholic steatohepatitis (NASH) is an inflammatory and fibrotic liver disease that has reached epidemic proportions and has no approved pharmacologic therapies. Research and drug development efforts are hampered by inadequate preclinical models. This research describes a three-dimensional bioprinted liver tissue model of NASH built using primary human hepatocytes and nonparenchymal liver cells (hepatic stellate cells, liver sinusoidal endothelial cells, and Kupffer cells) from either healthy or NASH donors. Three-dimensional tissues bioprinted with cells sourced from diseased patients showed a NASH phenotype, including fibrosis. More importantly, this NASH phenotype occurred without the addition of disease-inducing agents. Bioprinted tissues composed entirely of healthy cells exhibited significantly less evidence of disease. The role of individual cell types in driving the NASH phenotype was examined by producing chimeric bioprinted tissues composed of healthy cells together with the addition of one or more diseased nonparenchymal cell types. These experiments reveal a role for both hepatic stellate and liver sinusoidal endothelial cells in the disease process. This model represents a fully human system with potential to detect clinically active targets and eventually therapies.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Humanos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Células Endoteliales/metabolismo , Hígado/metabolismo , Hepatocitos/metabolismo , Macrófagos del Hígado/metabolismo , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/patologíaRESUMEN
Verinurad (RDEA3170) is a second generation selective uric acid reabsorption inhibitor for the treatment of gout and asymptomatic hyperuricemia. Following a single oral solution of 10-mg dose of [14C]verinurad (500 µCi), verinurad was rapidly absorbed with a median time to occurrence of maximum observed concentration (Tmax) of 0.5 hours and terminal half-life of 15 hours. In plasma, verinurad constituted 21% of total radioactivity. Recovery of radioactivity in urine and feces was 97.1%. Unchanged verinurad was the predominant component in the feces (29.9%), whereas levels were low in the urine (1.2% excreted). Acylglucuronide metabolites M1 (direct glucuronidation) and M8 (glucuronidation of N-oxide) were formed rapidly after absorption of verinurad with terminal half-life values of approximately 13 and 18 hours, respectively. M1 and M8 constituted 32% and 31% of total radioactivity in plasma and were equimolar to verinurad on the basis of AUC ratios. M1 and M8 formed in the liver were biliary cleared with complete hydrolysis in the GI tract, as metabolites were not detected in the feces and/or efflux across the sinusoidal membrane; M1 and M8 accounted for 29.2% and 32.5% of the radioactive dose in urine, respectively. In vitro studies demonstrated that CYP3A4 mediated the formation of the N-oxide metabolite (M4), which was further metabolized by glucuronyl transferases (UGTs) to form M8, as M4 was absent in plasma and only trace levels were present in the urine. Several UGTs mediated the formation of M1, which could also be further metabolized by CYP2C8. Overall, the major clearance route of verinurad is metabolism via UGTs and CYP3A4 and CYP2C8.
Asunto(s)
Ácido Úrico/metabolismo , Uricosúricos/metabolismo , Radioisótopos de Carbono/metabolismo , Citocromo P-450 CYP2C8/metabolismo , Citocromo P-450 CYP3A/metabolismo , Heces , Tracto Gastrointestinal/metabolismo , Glucurónidos/metabolismo , Glucuronosiltransferasa/metabolismo , Gota/tratamiento farmacológico , Gota/metabolismo , Semivida , Humanos , Hidrólisis/efectos de los fármacos , Hiperuricemia/tratamiento farmacológico , Hiperuricemia/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Tasa de Depuración Metabólica/efectos de los fármacos , Tasa de Depuración Metabólica/fisiología , Uricosúricos/uso terapéuticoRESUMEN
Gout is caused by elevated serum urate levels, which can be treated using inhibitors of the uric acid transporter, URAT1. We exploited affinity differences between the human and rat transporters to map inhibitor binding sites in URAT1. Human-rat transporter chimeras revealed that human URAT1 serine-35, phenylalanine-365 and isoleucine-481 are necessary and sufficient to provide up to a 100-fold increase in affinity for inhibitors. Moreover, serine-35 and phenylalanine-365 are important for high-affinity interaction with the substrate urate. A novel URAT1 binding assay provides support for direct interaction with these amino acids; thus, current clinically important URAT1 inhibitors likely bind the same site in URAT1. A structural model suggests that these three URAT1 residues are in close proximity potentially projecting within the channel. Our results indicate that amino acids from several transmembrane segments functionally cooperate to form a high-affinity URAT1 inhibitor binding site that, when occupied, prevents substrate interactions.
Asunto(s)
Transportadores de Anión Orgánico/antagonistas & inhibidores , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Sustitución de Aminoácidos , Animales , Proteínas de Transporte de Anión/antagonistas & inhibidores , Proteínas de Transporte de Anión/química , Proteínas de Transporte de Anión/genética , Sitios de Unión/genética , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteína 1 de Transporte de Anión Orgánico/química , Transportadores de Anión Orgánico/química , Transportadores de Anión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/química , Proteínas de Transporte de Catión Orgánico/genética , Dominios y Motivos de Interacción de Proteínas , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ácido Úrico/metabolismoRESUMEN
Using a flow cytometry-based screen of commercial antibodies, we have identified cell-surface markers for the separation of pancreatic cell types derived from human embryonic stem (hES) cells. We show enrichment of pancreatic endoderm cells using CD142 and of endocrine cells using CD200 and CD318. After transplantation into mice, enriched pancreatic endoderm cells give rise to all the pancreatic lineages, including functional insulin-producing cells, demonstrating that they are pancreatic progenitors. In contrast, implanted, enriched polyhormonal endocrine cells principally give rise to glucagon cells. These antibodies will aid investigations that use pancreatic cells generated from pluripotent stem cells to study diabetes and pancreas biology.
Asunto(s)
Antígenos CD/metabolismo , Biomarcadores/metabolismo , Separación Celular/métodos , Células Madre Embrionarias/citología , Páncreas/citología , Animales , Anticuerpos/metabolismo , Células Cultivadas , Células Madre Embrionarias/metabolismo , Endodermo/citología , Citometría de Flujo , Humanos , Ratones , Ratones SCID , Microscopía Fluorescente , Trasplante HeterólogoRESUMEN
In a companion paper (D. Ostertag, T. M. Hoblitzell-Ostertag, and J. Perrault, J. Virol. 81:492-502, 2007), we provided indirect evidence that cell-type-specific growth restriction of the vesicular stomatitis virus (VSV) polR mutants may be due to enhanced production of double-stranded RNA (dsRNA). We show here that polR growth in mouse L-929 cells was rescued by vaccinia virus coinfection and that sole expression of the vaccinia virus dsRNA-binding E3L protein, via coinfection with an engineered VSV minigenome, also restored polR growth. Expression of dsRNA-binding protein NS1A or NS1B from influenza virus, but not C protein from Sendai virus, which does not bind dsRNA, likewise effected polR rescue. The N-terminal dsRNA-binding domain of NS1A, only 73 amino acids in length, but not a full-size mutant NS1A lacking dsRNA-binding activity, restored polR growth. Both key aspects of polR growth restriction, namely inhibition of genome replication and release of low-infectivity virus particles, were countered by expression of the dsRNA-binding proteins. We tested the effects of overproducing dsRNA in wild-type VSV infections by coinfecting cells with a VSV recombinant expressing the sense strand of the enhanced green fluorescent protein gene (VSV-GFP) and one expressing the antisense strand (VSV-PFG). These coinfections mimicked all aspects of polR restriction, including host range, lack of effect on transcription, reduced virus particle infectivity, and insensitivity to inhibition of host gene transcription or dsRNA-activated protein kinase activity. We conclude that, for some cell types, overproduction of dsRNA during VSV infection triggers an immediate and constitutive host cell antiviral effector response independent of interferon induction or signaling.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Bicatenario/metabolismo , Regulación hacia Arriba , Virus de la Estomatitis Vesicular Indiana/patogenicidad , Animales , Antivirales/metabolismo , Línea Celular , Cricetinae , ARN Polimerasas Dirigidas por ADN/genética , Células L , Ratones , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ratas , Especificidad de la Especie , Virus Vaccinia/crecimiento & desarrollo , Virus Vaccinia/patogenicidad , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/crecimiento & desarrollo , Virus de la Estomatitis Vesicular Indiana/metabolismo , Replicación ViralRESUMEN
Vesicular stomatitis virus polR mutants synthesize defective RNA replication products in vitro and display growth restriction in some cultured cells (J. L. Chuang, R. L. Jackson, and J. Perrault, Virology 229:57-67, 1997). We show here that a recombinant virus carrying the polR N protein mutation (R179H) yielded approximately 100-fold- and approximately 40-fold-lower amounts of infectious virus than the wild type in mouse L-929 and rat 3Y1 cells, respectively, but only approximately 3-fold less in hamster BHK cells. Virus genome accumulation was inhibited 6- to 10-fold in restricting cells, but transcription was not affected. No defect in encapsidation of replication products was detected, but virus protein accumulation was reduced two- to threefold in both restricting and nonrestricting cells. polR virus particles released from the latter were 5- to 10-fold less infectious than the wild type but showed no difference in protein composition. Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha) was enhanced approximately 3-fold in polR versus wild-type virus-infected L-929 cells, but neither inhibition of host gene transcription nor inhibition of double-stranded RNA (dsRNA)-activated protein kinase showed significant effects on restriction. Conditioned medium studies revealed no evidence for secretion of antiviral factors from restricting cells. We conclude that the block in polR growth is due to the combined effect of reduced genome replication and lower infectivity of released virus particles and may be due to overproduction of dsRNA. An accompanying paper (D. Ostertag, T. M. Hoblitzell-Ostertag, and J. Perrault, J. Virol. 81:503-513, 2007) provides compelling evidence for the role of dsRNA in this unique restriction phenomenon.