RESUMEN
Betacoronaviruses encode a conserved accessory gene within the +1 open reading frame (ORF) of nucleocapsid called the internal N gene. This gene is referred to as "I" for mouse hepatitis virus (MHV), ORF9b for severe acute respiratory CoV (SARS-CoV) and SARS-CoV-2, and ORF8b for Middle East respiratory syndrome CoV (MERS-CoV). Previous studies have shown ORF8b and ORF9b have immunoevasive properties, while the only known information for MHV I is its localization within the virion of the hepatotropic/neurotropic A59 strain of MHV. Whether MHV I is an innate immune antagonist or has other functions has not been evaluated. In this report, we show that the I protein of the neurotropic JHM strain of MHV (JHMV) lacks a N terminal domain present in other MHV strains, has immunoevasive properties, and is a component of the virion. Genetic deletion of JHMV I (rJHMVIΔ57-137) resulted in a highly attenuated virus both in vitro and in vivo that displayed a post RNA replication/transcription defect that ultimately resulted in fewer infectious virions packaged compared with wild-type virus. This phenotype was only seen for rJHMVIΔ57-137, suggesting the structural changes predicted for A59 I altered its function, as genetic deletion of A59 I did not change viral replication or pathogenicity. Together, these data show that JHMV I both acts as a mild innate immune antagonist and aids in viral assembly and infectious virus production, and suggest that the internal N proteins from different betacoronaviruses have both common and virus strain-specific properties.IMPORTANCECoV accessory genes are largely studied in overexpression assays and have been identified as innate immune antagonists. However, functions identified after overexpression are often not confirmed in the infected animal host. Furthermore, some accessory proteins are components of the CoV virion, but their role in viral replication and release remains unclear. Here, we utilized reverse genetics to abrogate expression of a conserved CoV accessory gene, the internal N ("I") gene, of the neurotropic JHMV strain of MHV and found that loss of the I gene resulted in a post replication defect that reduced virion assembly and ultimately infectious virus production, while also increasing some inflammatory molecule expression. Thus, the JHMV I protein has roles in virion assembly that were previously underappreciated and in immunoevasion.
Asunto(s)
Virus de la Hepatitis Murina , Proteínas Virales , Replicación Viral , Virus de la Hepatitis Murina/genética , Virus de la Hepatitis Murina/patogenicidad , Virus de la Hepatitis Murina/inmunología , Virus de la Hepatitis Murina/fisiología , Animales , Ratones , Virulencia , Proteínas Virales/metabolismo , Proteínas Virales/genética , Virión/metabolismo , Inmunidad Innata , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/inmunología , Línea Celular , Sistemas de Lectura Abierta , HumanosRESUMEN
BACKGROUND: Amniocentesis is a common procedure, the primary purpose of which is to collect cells from the fetus to allow testing for abnormal chromosomes, altered chromosomal copy number, or a small number of genes that have small single- to multibase defects. Here we demonstrate the feasibility of generating an accurate whole-genome sequence of a fetus from either the cellular or cell-free DNA (cfDNA) of an amniotic sample. METHODS: cfDNA and DNA isolated from the cell pellet of 31 amniocenteses were sequenced to approximately 50× genome coverage by use of the Complete Genomics nanoarray platform. In a subset of the samples, long fragment read libraries were generated from DNA isolated from cells and sequenced to approximately 100× genome coverage. RESULTS: Concordance of variant calls between the 2 DNA sources and with parental libraries was >96%. Two fetal genomes were found to harbor potentially detrimental variants in chromodomain helicase DNA binding protein 8 (CHD8) and LDL receptor-related protein 1 (LRP1), variations of which have been associated with autism spectrum disorder and keratosis pilaris atrophicans, respectively. We also discovered drug sensitivities and carrier information of fetuses for a variety of diseases. CONCLUSIONS: We were able to elucidate the complete genome sequence of 31 fetuses from amniotic fluid and demonstrate that the cfDNA or DNA from the cell pellet can be analyzed with little difference in quality. We believe that current technologies could analyze this material in a highly accurate and complete manner and that analyses like these should be considered for addition to current amniocentesis procedures.
Asunto(s)
Líquido Amniótico/metabolismo , Feto/metabolismo , Genoma Humano , Secuenciación Completa del Genoma , Anomalías Múltiples/genética , Adulto , Amniocentesis , Trastorno del Espectro Autista/genética , Estudios de Cohortes , Variaciones en el Número de Copia de ADN , Enfermedad de Darier/genética , Cejas/anomalías , Estudios de Factibilidad , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , MutaciónRESUMEN
The aims of this study were two-fold; first to report on the use of the novel InteliSite Companion device to deliver material to the colon, and second to use this new technology to assess the potential of mucoadhesive polymers to be retained in the large intestine. In this three-way crossover study in beagle dogs, two mucoadhesive polymers and a non-mucoadhesive polymer were remotely delivered in powder form to the colon. The retention of 150mg doses of the radiolabelled mucoadhesive polymers Carbopol 980 and polycarbophil AA-1, and the retention of ethylcellulose (control) in the colon of three canines was examined using gamma scintigraphy. The InteliSite Companion device had a mean gastric emptying time of 1.0+/-0.8h and a mean caecal arrival time of 2.3+/-1.0h. The device was remotely activated to expel the polymers at the caecum. Although incomplete release was noted with all polymers, Carbopol 980 was found to have increased retention in the proximal colon of all three dogs. The mean retention time within the proximal colon for Carbopol 980 (15.3+/-1.4h) was significantly higher than that of polycarbophil AA-1 (10.0+/-5.7h) and the control (7.1+/-1.4h) (p<0.05). The increased colon retention time demonstrated by Carbopol 980 may be suggestive of a mucoadhesive effect.
Asunto(s)
Membrana Mucosa , Polímeros/administración & dosificación , Adhesivos Tisulares , Animales , Estudios Cruzados , Perros , MasculinoRESUMEN
PURPOSE: To develop a robust radiolabeling technique to enable evaluation of difficult to radiolabel gastric retentive formulations using gamma scintigraphy. The use of a successful radiolabel will allow accurate assessment of the gastric residence time of the formulations. MATERIALS AND METHODS: The retention of two radionuclides, indium ((111)In) and samarium ((153)Sm), with and without further processing to improve radiolabel performance were evaluated in simulated gastric pH in vitro. The most successful formulation from the in vitro screening was further evaluated in preclinical and clinical studies. RESULTS: In vitro evaluation revealed significant radionuclide leakage at pH 1.5 for most radiolabeling attempts. Radionuclide leakage at pH 4.5 was less pronounced. The most successful radiolabel was formulated by adsorbing indium chloride onto activated charcoal, followed by entrapment in a cellulose acetate polymer melt. This provided the best radiolabel retention under both pH conditions in vitro. The radiolabel also proved to be successful during preclinical and clinical evaluations, allowing evaluation of gastric retention performance as well as complete gastrointestinal transit. CONCLUSION: A simple, yet robust radiolabel was developed for gastric retentive formulations to be evaluated pre-clinically or in a clinical setting by entrapping the radionuclide in an insoluble polymer through a simple polymer melt process.
Asunto(s)
Vaciamiento Gástrico , Indio/administración & dosificación , Marcaje Isotópico/métodos , Óxidos/administración & dosificación , Radiofármacos/administración & dosificación , Samario/administración & dosificación , Estómago/diagnóstico por imagen , Tecnología Farmacéutica/métodos , Administración Oral , Adulto , Animales , Celulosa/análogos & derivados , Celulosa/química , Carbón Orgánico/química , Química Farmacéutica , Estudios Cruzados , Preparaciones de Acción Retardada , Perros , Composición de Medicamentos , Estabilidad de Medicamentos , Cámaras gamma , Tránsito Gastrointestinal , Semivida , Humanos , Concentración de Iones de Hidrógeno , Indio/química , Masculino , Persona de Mediana Edad , Óxidos/química , Radioisótopos/administración & dosificación , Cintigrafía , Radiofármacos/química , Samario/química , Solubilidad , Factores de TiempoRESUMEN
Previous work has shown that polyethylene glycol 400 (PEG 400) has an accelerating effect on gastrointestinal transit and a modulating influence on drug absorption in humans. The aim of this study was to assess the impact of various excipients, PEG 400, propylene glycol, d-alpha-tocopheryl-polyethylene glycol-1000 succinate (TPGS) and Labrasol on gastrointestinal transit and drug absorption in four beagle dogs using scintigraphy. Each dog received, on five separate occasions, water (control) or a dose of excipient equivalent to 1 g PEG 400, 2 g propylene glycol, 1 g TPGS or 2 g Labrasol dissolved in water and administered in the form of two capsules. The model drugs ampicillin (200mg) and antipyrine (100mg) were co-administered in the capsules. The capsule solutions were radiolabelled with technetium-99m to follow their transit using a dual-headed gamma camera, and blood samples were collected to determine drug pharmacokinetics. On a separate occasion, the drugs were dissolved in saline and given intravenously. The capsules rapidly disintegrated in the stomach liberating their liquid contents. The mean small intestinal transit times for the different treatments (control, PEG 400, propylene glycol, TPGS and Labarasol) were 183, 179, 195, 168 and 154 min, respectively. The corresponding mean absolute oral bioavailability figures were 36, 32, 39, 42 and 32% for ampicillin and 76, 74, 85, 73 and 74% for antipyrine, respectively. The transit and bioavailability data for the excipient treatments were not significantly different from the control. In summary, these excipients, at the doses administered, have limited influence on gastrointestinal transit and drug in beagle dogs.
Asunto(s)
Excipientes/farmacología , Vaciamiento Gástrico/efectos de los fármacos , Absorción Intestinal/efectos de los fármacos , Ampicilina/farmacocinética , Animales , Antipirina/farmacocinética , Disponibilidad Biológica , Perros , Femenino , Glicéridos , Masculino , Compuestos Orgánicos/farmacología , Polietilenglicoles/farmacología , Propilenglicol/farmacología , Succinatos/farmacología , Vitamina E/análogos & derivados , Vitamina E/farmacologíaRESUMEN
In this blinded randomized placebo-controlled trial, 20 dogs with atopic dermatitis (AD) were given placebo (8 dogs) or misoprostol (12 dogs) at 5 micro g kg-1, orally, three times daily for 3 weeks. Administration of the active drug, but not of placebo, led to a significant decrease in lesional and pruritus scores. The median reduction from baseline of both scores was approximately 30%. Misoprostol therapy did not lead to decreases of dermal cell counts or skin tumour necrosis factor (TNF)alpha mRNA copy numbers that were significantly different from those of placebo. Skin TNFalpha protein production, assessed using indirect immunofluorescence, decreased or remained unchanged in dogs receiving misoprostol. In contrast, post treatment TNFalpha fluorescence scores were higher in all but two dogs given placebo. The changes from baseline of TNFalpha fluorescence scores did not correlate significantly with those of lesional or pruritus indices. These observations confirm the modest efficacy of misoprostol for treatment of canine AD and suggest that its mild anti-allergic effects are not associated with either inhibition of inflammatory cell emigration or TNFalpha production.
Résumé Dans cette étude randomisée en aveugle, 20 chiens présentant une dermatite atopique ont reçu un placebo (8 chiens) ou du misoprostol (12 chiens) à la posologie de 5 µg kg−1 , par voie orale, trois fois par jour pendant 3 semaines. L'administration du misoprostol mais pas du placebo a permis une diminution significative des scores de prurit et lésionnels. La réduction moyenne des 2 scores était d'environ 30%. Le misoprostol n'a pas diminué le nombre de cellules dans le derme ou le nombre de copies d'ARNm du TNF qui étaient significativement différentes du placebo. La production de TNF, determinée par immunofluorescence indirecte a diminué ou n'a pas évolué chez les chiens recevant le misoprostol. Au contraire, les scores de fluorescence pour le TNF étaient plus élevés chez tous les chiens recevant le placebo sauf deux. Les modifications des scores de fluorescence du TNF nétaient pas corrélées aux scores de prurit ou lésionnel. Ces observations confirment l'efficacité modeste du misoprostol pour le traitement de la dermatite atopique canine et suggèrent que ses effets antiallergiques modérés ne sont pas associés à une inhibition de la migration des cellules inflammatoires ou de la production de TNF.
Resumen En este ensayo ciego, al azar y controlado con placebo, a veinte perros con DA se les administró oralmente placebo (8 perros) o misoprostol (12 perros) a una dosis de 5 µg kg−1 tres veces al día durante tres semanas. La administración de la droga activa, pero no la del placebo, dio lugar a una disminución del índice de lesiones y prurito. La reducción media en ambos índices fue de aproximadamente el 30%. La terapia de misoprostol no produjo una disminución del recuento de células dérmicas o del número de copias del ARNm del FNT-α (TNFα), los cuales eran significativamente diferentes del grupo con placebo. La producción de la proteína FNTα, determinada por un método indirecto de immunofluorescencia, disminuyó o permaneció igual en perros que recibieron misoprostol. En cambio, el nivel de fluorescencia del FNTα fue más elevado en el postratamiento en todos los perros, con excepción de dos de ellos en el grupo con placebo. Los cambios en el nivel de fluorescencia, con respecto a la línea basal, no se correlacionaron significativamente con los índices de lesión o prurito. Estas observaciones confirman la modesta eficacia del misoprostol para el tratamiento de las DA caninas y sugiere que sus efectos mínimos antialérgicos no están asociados con la inhibición de la emigración de células inflamatorias o la producción del FNTα.