RESUMEN
Chronic wasting disease (CWD) continues to spread or be recognized in the United States, Canada, and Europe. CWD is diagnosed by demonstration of the causative misfolded prion protein (PrPCWD) in either brain or lymphoid tissue using immunodetection methods, with immunohistochemistry (IHC) recognized as the gold standard. In recent years, in vitro amplification assays have been developed that can detect CWD prion seeding activity in tissues, excreta, and body fluids of affected cervids. These methods potentially offer earlier and more facile detection of CWD, both pre- and post-mortem. Here we provide a longitudinal profile of CWD infection progression, as assessed by both real-time quaking-induced conversion (RT-QuIC) and IHC on serial biopsies of mucosal lymphoid tissues of white-tailed deer orally exposed to low doses of CWD prions. We report that detection of CWD infection by RT-QuIC preceded that by IHC in both tonsil and recto-anal lymphoid tissue (RAMALT) in 14 of 19 deer (74%). Of the 322 biopsy samples collected in post-exposure longitudinal monitoring, positive RT-QuIC results were obtained for 146 samples, 91 of which (62%) were concurrently also IHC-positive. The lower frequency of IHC positivity was manifest most in the earlier post-exposure periods and in biopsies in which lymphoid follicles were not detected. For all deer in which RT-QuIC seeding activity was detected in a tonsil or RAMALT biopsy, PrPCWD was subsequently or concurrently detected by IHC. Overall, this study (a) provides a longitudinal profile of CWD infection in deer after low yet infectious oral prion exposure; (b) illustrates the value of RT-QuIC for sensitive detection of CWD; and (c) demonstrates an ultimate high degree of correlation between RT-QuIC and IHC positivity as CWD infection progresses.
Asunto(s)
Inmunohistoquímica , Técnicas de Amplificación de Ácido Nucleico/métodos , Enfermedad Debilitante Crónica/patología , Administración Oral , Animales , Ciervos , Progresión de la Enfermedad , Estudios Longitudinales , Tejido Linfoide/metabolismo , Tejido Linfoide/patología , Tonsila Palatina/metabolismo , Tonsila Palatina/patología , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Priones/administración & dosificación , Enfermedad Debilitante Crónica/metabolismoRESUMEN
Chronic wasting disease (CWD) is a prion disease of free-ranging and farmed cervids that is highly contagious because of extensive prion shedding and prion persistence in the environment. Previously, cellulose ether compounds (CEs) have been shown to significantly extend the survival of mice inoculated with mouse-adapted prion strains. In this study, we used CEs, TC-5RW, and 60SH-50, in vitro and in vivo to assess their efficacy to interfere with CWD prion propagation. In vitro, CEs inhibited CWD prion amplification in a dose-dependent manner. Transgenic mice over-expressing elk PrPC (tgElk) were injected subcutaneously with a single dose of either of the CEs, followed by intracerebral inoculation with different CWD isolates from white tailed deer, mule deer, or elk. All treated groups showed a prolonged survival of up to more than 30 % when compared to the control group regardless of the CWD isolate used for infection. The extended survival in the treated groups correlated with reduced proteinase K resistance of prions. Remarkably, passage of brain homogenates from treated or untreated animals in tgElk mice resulted in a prolonged life span of mice inoculated with homogenates from CE-treated mice (of + 17%) even in the absence of further treatment. Besides the delayed disease onset upon passage in TgElk mice, the reduced proteinase K resistance was maintained but less pronounced. Therefore, these compounds can be very useful in limiting the spread of CWD in captive and wild-ranging cervids.
Asunto(s)
Celulosa/administración & dosificación , Éter/administración & dosificación , Péptido Hidrolasas/metabolismo , Priones/metabolismo , Enfermedad Debilitante Crónica/metabolismo , Enfermedad Debilitante Crónica/prevención & control , Animales , Química Encefálica , Ciervos , Expresión Génica , Ratones , Ratones Transgénicos , Proteínas PrPSc/química , Proteínas Priónicas/química , Proteínas Priónicas/genética , Priones/administración & dosificación , Priones/efectos de los fármacos , Conformación Proteica , Proteínas RecombinantesRESUMEN
In multiple system atrophy (MSA), progressive neurodegeneration results from the protein α-synuclein misfolding into a self-templating prion conformation that spreads throughout the brain. MSA prions are transmissible to transgenic (Tg) mice expressing mutated human α-synuclein (TgM83+/-), inducing neurological disease following intracranial inoculation with brain homogenate from deceased patient samples. Noting the similarities between α-synuclein prions and PrP scrapie (PrPSc) prions responsible for Creutzfeldt-Jakob disease (CJD), we investigated MSA transmission under conditions known to result in PrPSc transmission. When peripherally exposed to MSA via the peritoneal cavity, hind leg muscle, and tongue, TgM83+/- mice developed neurological signs accompanied by α-synuclein prions in the brain. Iatrogenic CJD, resulting from PrPSc prion adherence to surgical steel instruments, has been investigated by incubating steel sutures in contaminated brain homogenate before implantation into mouse brain. Mice studied using this model for MSA developed disease, whereas wire incubated in control homogenate had no effect on the animals. Notably, formalin fixation did not inactivate α-synuclein prions. Formalin-fixed MSA patient samples also transmitted disease to TgM83+/- mice, even after incubating in fixative for 244 months. Finally, at least 10% sarkosyl was found to be the concentration necessary to partially inactivate MSA prions. These results demonstrate the robustness of α-synuclein prions to denaturation. Moreover, they establish the parallel characteristics between PrPSc and α-synuclein prions, arguing that clinicians should exercise caution when working with materials that might contain α-synuclein prions to prevent disease.
Asunto(s)
Atrofia de Múltiples Sistemas/metabolismo , Priones/metabolismo , Animales , Transporte Biológico , Encéfalo/metabolismo , Encéfalo/patología , Detergentes/farmacología , Modelos Animales de Enfermedad , Fijadores , Formaldehído , Células HEK293 , Humanos , Ratones Transgénicos , Atrofia de Múltiples Sistemas/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Mutación , Priones/administración & dosificación , Agregado de Proteínas , Estabilidad Proteica/efectos de los fármacos , Sarcosina/análogos & derivados , Sarcosina/farmacología , Acero Inoxidable , alfa-Sinucleína/administración & dosificación , alfa-Sinucleína/efectos adversos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoAsunto(s)
Enfermedad de Alzheimer/etiología , Modelos Biológicos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Amiloide/administración & dosificación , Amiloide/efectos adversos , Amiloide/química , Amiloide/metabolismo , Péptidos beta-Amiloides/administración & dosificación , Péptidos beta-Amiloides/efectos adversos , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Animales , Síndrome de Creutzfeldt-Jakob/epidemiología , Síndrome de Creutzfeldt-Jakob/etiología , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patología , Contaminación de Medicamentos , Hormona del Crecimiento/administración & dosificación , Humanos , Ratones , Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Priones/administración & dosificación , Priones/efectos adversos , Priones/química , Priones/metabolismo , Reino Unido/epidemiología , alfa-Sinucleína/administración & dosificación , alfa-Sinucleína/efectos adversos , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMEN
More than two hundred individuals developed Creutzfeldt-Jakob disease (CJD) worldwide as a result of treatment, typically in childhood, with human cadaveric pituitary-derived growth hormone contaminated with prions. Although such treatment ceased in 1985, iatrogenic CJD (iCJD) continues to emerge because of the prolonged incubation periods seen in human prion infections. Unexpectedly, in an autopsy study of eight individuals with iCJD, aged 36-51 years, in four we found moderate to severe grey matter and vascular amyloid-ß (Aß) pathology. The Aß deposition in the grey matter was typical of that seen in Alzheimer's disease and Aß in the blood vessel walls was characteristic of cerebral amyloid angiopathy and did not co-localize with prion protein deposition. None of these patients had pathogenic mutations, APOE ε4 or other high-risk alleles associated with early-onset Alzheimer's disease. Examination of a series of 116 patients with other prion diseases from a prospective observational cohort study showed minimal or no Aß pathology in cases of similar age range, or a decade older, without APOE ε4 risk alleles. We also analysed pituitary glands from individuals with Aß pathology and found marked Aß deposition in multiple cases. Experimental seeding of Aß pathology has been previously demonstrated in primates and transgenic mice by central nervous system or peripheral inoculation with Alzheimer's disease brain homogenate. The marked deposition of parenchymal and vascular Aß in these relatively young patients with iCJD, in contrast with other prion disease patients and population controls, is consistent with iatrogenic transmission of Aß pathology in addition to CJD and suggests that healthy exposed individuals may also be at risk of iatrogenic Alzheimer's disease and cerebral amyloid angiopathy. These findings should also prompt investigation of whether other known iatrogenic routes of prion transmission may also be relevant to Aß and other proteopathic seeds associated with neurodegenerative and other human diseases.
Asunto(s)
Enfermedad de Alzheimer/etiología , Péptidos beta-Amiloides/metabolismo , Angiopatía Amiloide Cerebral/etiología , Síndrome de Creutzfeldt-Jakob/etiología , Contaminación de Medicamentos , Hormona de Crecimiento Humana/administración & dosificación , Enfermedad Iatrogénica , Adulto , Alelos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/administración & dosificación , Péptidos beta-Amiloides/análisis , Autopsia , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Estudios de Casos y Controles , Angiopatía Amiloide Cerebral/metabolismo , Angiopatía Amiloide Cerebral/patología , Síndrome de Creutzfeldt-Jakob/complicaciones , Síndrome de Creutzfeldt-Jakob/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Sustancia Gris/metabolismo , Sustancia Gris/patología , Humanos , Persona de Mediana Edad , Priones/administración & dosificación , Priones/metabolismo , Factores de RiesgoRESUMEN
The prion hypothesis postulates that the infectious agent in transmissible spongiform encephalopathies (TSEs) is an unorthodox protein conformation based agent. Recent successes in generating mammalian prions in vitro with bacterially expressed recombinant prion protein provide strong support for the hypothesis. However, whether the pathogenic properties of synthetically generated prion (rec-Prion) recapitulate those of naturally occurring prions remains unresolved. Using end-point titration assay, we showed that the in vitro prepared rec-Prions have infectious titers of around 104 LD50/µg. In addition, intraperitoneal (i.p.) inoculation of wild-type mice with rec-Prion caused prion disease with an average survival time of 210-220 days post inoculation. Detailed pathological analyses revealed that the nature of rec-Prion induced lesions, including spongiform change, disease specific prion protein accumulation (PrP-d) and the PrP-d dissemination amongst lymphoid and peripheral nervous system tissues, the route and mechanisms of neuroinvasion were all typical of classical rodent prions. Our results revealed that, similar to naturally occurring prions, the rec-Prion has a titratable infectivity and is capable of causing prion disease via routes other than direct intra-cerebral challenge. More importantly, our results established that the rec-Prion caused disease is pathogenically and pathologically identical to naturally occurring contagious TSEs, supporting the concept that a conformationally altered protein agent is responsible for the infectivity in TSEs.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedades por Prión/patología , Priones/patogenicidad , Proteínas Recombinantes/administración & dosificación , Animales , Inmunohistoquímica , Inyecciones Intraperitoneales , Ratones , Priones/administración & dosificación , Proteínas Recombinantes/síntesis químicaRESUMEN
Infectious prions traverse epithelial barriers to gain access to the circulatory system, yet the temporal parameters of transepithelial transport and persistence in the blood over time remain unknown. We used whole-blood real-time quaking-induced conversion (wbRT-QuIC) to analyze whole blood collected from transmissible spongiform encephalopathy (TSE)-inoculated deer and hamsters throughout the incubation period for the presence of common prion protein-conversion competent amyloid (PrPCCCA). We observed PrPC-CCA in the blood of TSE-inoculated hosts throughout the disease course from minutes postexposure to terminal disease.
Asunto(s)
Priones/administración & dosificación , Priones/sangre , Administración Intranasal , Administración Oral , Animales , Análisis Químico de la Sangre , Ciervos , Inyecciones Intravenosas , Masculino , Mesocricetus , Factores de TiempoRESUMEN
Prion disease is a unique category of illness, affecting both animals and humans, in which the underlying pathogenesis is related to a conformational change of a normal, self-protein called PrP(C) (C for cellular) to a pathological and infectious conformer known as PrP(Sc) (Sc for scrapie). Bovine spongiform encephalopathy (BSE), a prion disease believed to have arisen from feeding cattle with prion contaminated meat and bone meal products, crossed the species barrier to infect humans. Chronic wasting disease (CWD) infects large numbers of deer and elk, with the potential to infect humans. Currently no prionosis has an effective treatment. Previously, we have demonstrated we could prevent transmission of prions in a proportion of susceptible mice with a mucosal vaccine. In the current study, white-tailed deer were orally inoculated with attenuated Salmonella expressing PrP, while control deer were orally inoculated with vehicle attenuated Salmonella. Once a mucosal response was established, the vaccinated animals were boosted orally and locally by application of polymerized recombinant PrP onto the tonsils and rectal mucosa. The vaccinated and control animals were then challenged orally with CWD-infected brain homogenate. Three years post CWD oral challenge all control deer developed clinical CWD (median survival 602 days), while among the vaccinated there was a significant prolongation of the incubation period (median survival 909 days; p=0.012 by Weibull regression analysis) and one deer has remained CWD free both clinically and by RAMALT and tonsil biopsies. This negative vaccinate has the highest titers of IgA in saliva and systemic IgG against PrP. Western blots showed that immunoglobulins from this vaccinate react to PrP(CWD). We document the first partially successful vaccination for a prion disease in a species naturally at risk.
Asunto(s)
Ciervos , Priones/administración & dosificación , Priones/inmunología , Vacunas contra la Salmonella/administración & dosificación , Enfermedad Debilitante Crónica/prevención & control , Administración a través de la Mucosa , Animales , Sangre/inmunología , Inmunoglobulina A/análisis , Inmunoglobulina G/sangre , Priones/genética , Saliva/inmunología , Vacunas contra la Salmonella/genética , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Enfermedad Debilitante Crónica/inmunologíaRESUMEN
Mouse-adapted transmissible spongiform encephalopathy (TSE) strains are routinely distinguished based on reproducible disease characteristics in a given mouse line following inoculation via a consistent route. We investigated whether different administration routes (oral, intragastric (i.g.) and intracerebral (i.c.)) can alter the disease characteristics in IM mice after serial dilution of a stabilized mouse-adapted bovine spongiform encephalopathy (BSE) strain (301V). In addition, the infectivity of distal ileum and mesenteric lymph nodes (ln) sampled at three time points (35 days postinoculation (dpi), 70 dpi and terminal disease) after i.g. inoculation of 301V strain was assessed in mice by i.c. challenge. Strain characteristics were assessed according to standard methodology and PrP(Sc) immunohistochemistry deposition patterns. Mean incubation periods were prolonged following oral or i.g. inoculations compared to the i.c. route. Lesion profiles following i.c. challenges were elevated compared to i.g. and oral routes although vacuolation in the dorsal medulla was consistently high irrespective of the route of administration. Nevertheless, the same PrP(Sc) deposition pattern was associated with each route of administration. Distal and mesenteric ln infectivity was detected as early as 35 dpi and displayed consistent lesion profiles and PrP(Sc) deposition patterns. Our data suggest that although 301V retained its properties, some phenotypic parameters were affected by the route of inoculation. We conclude that bioassay data should be interpreted carefully and should be standardized for route of inoculation.
Asunto(s)
Encefalopatía Espongiforme Bovina/patología , Encefalopatía Espongiforme Bovina/transmisión , Priones/administración & dosificación , Priones/patogenicidad , Animales , Encéfalo/metabolismo , Encéfalo/patología , Bovinos , Modelos Animales de Enfermedad , Encefalopatía Espongiforme Bovina/metabolismo , Íleon/metabolismo , Íleon/patología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Ratones , Ratones Endogámicos , Proteínas PrPSc/metabolismo , Factores de TiempoRESUMEN
To date, all clinical variant Creutzfeldt-Jakob disease (vCJD) patients are homozygous for methionine at polymorphic codon 129 (129M/M) of the prion protein (PrP) gene. However, the appearance of asymptomatic secondary vCJD infection in individuals with a PRNP codon 129 genotype other than M/M and transmission studies using animal models have raised the concern that all humans might be susceptible to vCJD prions, especially via secondary infection. To reevaluate this possibility and to analyze in detail the transmission properties of vCJD prions to transgenic animals carrying distinct codon 129 genotype, we performed intracerebral inoculation of vCJD prions to humanized knock-in mice carrying all possible codon 129 genotypes (129M/M, 129M/V, or 129V/V). All humanized knock-in mouse lines were susceptible to vCJD infection, although the attack rate gradually decreased from 129M/M to 129M/V and to 129V/V. The amount of PrP deposition including florid/amyloid plaques in the brain also gradually decreased from 129M/M to 129M/V and to 129V/V. The biochemical properties of protease-resistant abnormal PrP in the brain and transmissibility of these humanized mouse-passaged vCJD prions upon subpassage into knock-in mice expressing bovine PrP were not affected by the codon 129 genotype. These results indicate that individuals with the 129V/V genotype may be more susceptible to secondary vCJD infection than expected and may lack the neuropathological characteristics observed in vCJD patients with the 129M/M genotype. Besides the molecular typing of protease-resistant PrP in the brain, transmission studies using knock-in mice carrying bovine PrP may aid the differential diagnosis of secondary vCJD infection, especially in individuals with the 129V/V genotype.
Asunto(s)
Encéfalo/metabolismo , Codón/genética , Síndrome de Creutzfeldt-Jakob/genética , Priones/genética , Animales , Western Blotting , Encéfalo/patología , Bovinos , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/transmisión , Predisposición Genética a la Enfermedad/genética , Genotipo , Humanos , Inmunohistoquímica , Inyecciones Intraventriculares , Ratones , Ratones Transgénicos , Placa Amiloide/metabolismo , Placa Amiloide/patología , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo , Priones/administración & dosificación , Priones/metabolismoRESUMEN
Chronic wasting disease (CWD), the only known prion disease endemic in wildlife, is a persistent problem in both wild and captive North American cervid populations. This disease continues to spread and cases are found in new areas each year. Indirect transmission can occur via the environment and is thought to occur by the oral and/or intranasal route. Oral transmission has been experimentally demonstrated and although intranasal transmission has been postulated, it has not been tested in a natural host until recently. Prions have been shown to adsorb strongly to clay particles and upon oral inoculation the prion/clay combination exhibits increased infectivity in rodent models. Deer and elk undoubtedly and chronically inhale dust particles routinely while living in the landscape while foraging and rutting. We therefore hypothesized that dust represents a viable vehicle for intranasal CWD prion exposure. To test this hypothesis, CWD-positive brain homogenate was mixed with montmorillonite clay (Mte), lyophilized, pulverized and inoculated intranasally into white-tailed deer once a week for 6 weeks. Deer were euthanized at 95, 105, 120 and 175 days post final inoculation and tissues examined for CWD-associated prion proteins by immunohistochemistry. Our results demonstrate that CWD can be efficiently transmitted utilizing Mte particles as a prion carrier and intranasal exposure.
Asunto(s)
Animales Salvajes/metabolismo , Ciervos/metabolismo , Priones/metabolismo , Enfermedad Debilitante Crónica/metabolismo , Administración Intranasal , Silicatos de Aluminio/metabolismo , Animales , Bentonita/metabolismo , Arcilla , Femenino , Liofilización , Genotipo , Inmunohistoquímica , Tejido Linfoide/metabolismo , Masculino , Priones/administración & dosificación , Priones/genética , Factores de Tiempo , Enfermedad Debilitante Crónica/genética , Enfermedad Debilitante Crónica/transmisiónRESUMEN
AIMS: The misfolding and the aggregation of specific proteins are key features of neurodegenerative diseases, specifically Transmissible Spongiform Encephalopathies (TSEs). In TSEs, neuronal loss and inflammation are associated with the accumulation of the misfolded isoform (PrP(sc)) of the cellular prion protein (PrP(c)). Therefore we tested the hypothesis that augmenting a natural anti-inflammatory pathway mediated by epoxygenated fatty acids (EpFAs) will delay lethality. EpFAs are highly potent but enzymatically labile molecules produced by the actions of a number of cytochrome P450 enzymes. Stabilization of these bioactive lipids by inhibiting their degradation mediated by the soluble epoxide hydrolase (sEH) results in potent anti-inflammatory effects in multiple disease models. MAIN METHODS: Mice were infected with the mouse-adapted RML strain of scrapie by intracerebral or intraperitoneal routes. Animals received the sEH inhibitor, by oral route, administrated in drinking water or vehicle (PEG400). Infected mice were euthanized at a standard clinical end point. Histopathological, immunohistochemical and Western blot analyses of brain tissue confirmed the presence of pathology related to prion infection. KEY FINDINGS: Oral administration of the sEHI did not affect the very short survival time of the intracerebral prion infection group. However, mice infected by intraperitoneal route and treated with t-AUCB survived significantly longer than the control group mice (p<0.001). SIGNIFICANCE: These findings support the idea that inhibition of sEH or augmentation of the natural EpFA signaling in the brain offers a potential and different route to understand prion diseases and may become a therapeutic strategy for diseases involving neuroinflammation.
Asunto(s)
Epóxido Hidrolasas/antagonistas & inhibidores , Scrapie/tratamiento farmacológico , Animales , Benzoatos/farmacología , Benzoatos/uso terapéutico , Western Blotting , Encéfalo/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Epóxido Hidrolasas/fisiología , Femenino , Inyecciones Intraperitoneales , Inyecciones Intraventriculares , Ratones , Priones/administración & dosificación , Priones/efectos de los fármacos , Scrapie/patología , Urea/análogos & derivados , Urea/farmacología , Urea/uso terapéuticoRESUMEN
Transmissible spongiform encephalopathies are neurodegenerative diseases, which despite fervent research remain incurable. Immunization approaches have shown great potential at providing protection, however tolerance effects hamper active immunization protocols. In this study we evaluated the antigenic potential of various forms of recombinant murine prion protein and estimated their protective efficacy in a mouse model of prion diseases. One of the forms tested provided a significant elongation of survival interval. The elongation was mediated via an acute depletion of mature follicular dendritic cells, which are associated with propagation of the prion infectious agent in the periphery and in part to the development of humoral immunity against prion protein. This unprecedented result could offer new strategies for protection against transmissible encephalopathies as well as other diseases associated with follicular dendritic cells.
Asunto(s)
Células Dendríticas Foliculares/efectos de los fármacos , Inmunización , Enfermedades por Prión/prevención & control , Priones/inmunología , Animales , Células Dendríticas Foliculares/inmunología , Células Dendríticas Foliculares/patología , Modelos Animales de Enfermedad , Femenino , Inmunidad Humoral/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Enfermedades por Prión/inmunología , Enfermedades por Prión/patología , Priones/administración & dosificación , Priones/genética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Análisis de SupervivenciaRESUMEN
BACKGROUND: To provide information on dose-response and aid in modelling the exposure dynamics of the BSE epidemic in the United Kingdom groups of cattle were exposed orally to a range of different doses of brainstem homogenate of known infectious titre from clinical cases of classical bovine spongiform encephalopathy (BSE). Interim data from this study was published in 2007. This communication documents additional BSE cases, which occurred subsequently, examines possible influence of the bovine prion protein gene on disease incidence and revises estimates of effective oral exposure. FINDINGS: Following interim published results, two further cattle, one dosed with 100 mg and culled at 127 months post exposure and the other dosed with 10 mg and culled at 110 months post exposure, developed BSE. Both had a similar pathological phenotype to previous cases. Based on attack rate and incubation period distribution according to dose, the dose estimate at which 50% of confirmed cases would be clinically affected was revised to 0.15 g of the brain homogenate used in the experiment, with a 95% confidence interval of 0.03-0.79 g. Neither the full open reading frame nor the promoter region of the prion protein gene of dosed cattle appeared to influence susceptibility to BSE, but this may be due to the sample size. CONCLUSIONS: Oral exposure of cattle to a large range of doses of a BSE brainstem homogenate produced disease in all dose groups. The pathological presentation resembled natural disease. The attack rate and incubation period were dependent on the dose.
Asunto(s)
Tronco Encefálico/metabolismo , Encefalopatía Espongiforme Bovina/metabolismo , Periodo de Incubación de Enfermedades Infecciosas , Priones/administración & dosificación , Extractos de Tejidos/administración & dosificación , Administración Oral , Animales , Tronco Encefálico/patología , Bovinos , Modelos Animales de Enfermedad , Encefalopatía Espongiforme Bovina/patología , Encefalopatía Espongiforme Bovina/transmisión , Genotipo , Modelos Biológicos , Sistemas de Lectura Abierta , Fenotipo , Priones/genética , Priones/metabolismo , Regiones Promotoras Genéticas , Medición de Riesgo , Factores de Tiempo , Extractos de Tejidos/metabolismoRESUMEN
Prion infection and pathogenesis are dependent on the agent crossing an epithelial barrier to gain access to the recipient nervous system. Several routes of infection have been identified, but the mechanism(s) and timing of in vivo prion transport across an epithelium have not been determined. The hamster model of nasal cavity infection was used to determine the temporal and spatial parameters of prion-infected brain homogenate uptake following inhalation and to test the hypothesis that prions cross the nasal mucosa via M cells. A small drop of infected or uninfected brain homogenate was placed below each nostril, where it was immediately inhaled into the nasal cavity. Regularly spaced tissue sections through the entire extent of the nasal cavity were processed immunohistochemically to identify brain homogenate and the disease-associated isoform of the prion protein (PrP(d)). Infected or uninfected brain homogenate was identified adhering to M cells, passing between cells of the nasal mucosa, and within lymphatic vessels of the nasal cavity at all time points examined. PrP(d) was identified within a limited number of M cells 15 to 180 min following inoculation, but not in the adjacent nasal mucosa-associated lymphoid tissue (NALT). While these results support M cell transport of prions, larger amounts of infected brain homogenate were transported paracellularly across the respiratory, olfactory, and follicle-associated epithelia of the nasal cavity. These results indicate that prions can immediately cross the nasal mucosa via multiple routes and quickly enter lymphatics, where they can spread systemically via lymph draining the nasal cavity.
Asunto(s)
Mucosa Nasal/metabolismo , Enfermedades por Prión/fisiopatología , Priones/metabolismo , Migración Transendotelial y Transepitelial/fisiología , Administración por Inhalación , Animales , Cricetinae , Inmunohistoquímica , Mucosa Nasal/citología , Priones/administración & dosificación , Transporte de Proteínas/fisiologíaRESUMEN
Due to the aging of the population, the incidence of neurodegenerative diseases, such as Parkinson's and Alzheimer's, is expected to grow and, hence, the demand for adequate treatment modalities. However, the delivery of medicines into the brain for the treatment of brain-related diseases is hampered by the presence of a tight layer of endothelial cells that forms the blood-brain barrier (BBB). Furthermore, most conventional drugs lack stability and/or bioavailability. These obstacles can be overcome by the application of nanocarriers, in which the therapeutic entity has been incorporated, provided that they are effectively targeted to the brain endothelial cell layer. Drug nanocarriers decorated with targeting ligands that bind BBB receptors may accumulate efficiently at/in brain microvascular endothelium and hence represent a promising tool for brain drug delivery. Following the accumulation of drug nanocarriers at the brain vasculature, the drug needs to be transported across the brain endothelial cells into the brain. Transport across brain endothelial cells can occur via passive diffusion, transport proteins, and the vesicular transport pathways of receptor-mediated and adsorptive-mediated transcytosis. When a small lipophilic drug is released from its carrier at the brain vasculature, it may enter the brain via passive diffusion. On the other hand, the passage of intact nanocarriers, which is necessary for the delivery of larger and more hydrophilic drugs into brain, may occur via active transport by means of transcytosis. In previous work we identified GM1 ganglioside and prion protein as potential transcytotic receptors at the BBB. GM1 is a glycosphingolipid that is ubiquitously present on the endothelial surface and capable of acting as the transcytotic receptor for cholera toxin B. Likewise, prion protein has been shown to have transcytotic capacity at brain endothelial cells. Here we determine the transcytotic potential of polymersome nanocarriers functionalized with GM1- and prion-targeting peptides (G23, P50 and P9), that were identified by phage display, in an in vitro BBB model. In addition, the biodistribution of polymersomes functionalized with either the prion-targeting peptide P50 or the GM1-targeting peptide G23 is determined following intravenous injection in mice. We show that the prion-targeting peptides do not induce efficient transcytosis of polymersomes across the BBB in vitro nor induce accumulation of polymersomes in the brain in vivo. In contrast, the G23 peptide is shown to have transcytotic capacity in brain endothelial cells in vitro, as well as a brain-targeting potential in vivo, as reflected by the accumulation of G23-polymersomes in the brain in vivo at a level comparable to that of RI7217-polymersomes, which are targeted toward the transferrin receptor. Thus the G23 peptide seems to serve both of the requirements that are needed for efficient brain drug delivery of nanocarriers. An unexpected finding was the efficient accumulation of G23-polymersomes in lung. In conclusion, because of its combined brain-targeting and transcytotic capacity, the G23 peptide could be useful in the development of targeted nanocarriers for drug delivery into the brain, but appears especially attractive for specific drug delivery to the lung.
Asunto(s)
Gangliósido G(M1)/administración & dosificación , Gangliósido G(M1)/farmacocinética , Priones/administración & dosificación , Priones/farmacocinética , Administración Intravenosa , Animales , Barrera Hematoencefálica/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
We orally inoculated white-tailed deer (Odocoileus virginianus) and mule deer (Odocoileus hemionus) with a standardized, conspecific prion dose and collected biologic samples throughout the disease course. Mule deer (PRNP genotype 225SS) and PRNP genotype 96GG white-tailed deer succumbed along similar trajectories, but 96GS- and 96SS-genotype individuals tended to survive longer.
Asunto(s)
Ciervos/genética , Priones/administración & dosificación , Priones/patogenicidad , Enfermedad Debilitante Crónica/mortalidad , Animales , Predisposición Genética a la Enfermedad , Genotipo , Análisis de SupervivenciaRESUMEN
We compared prion infection rates among mule deer (Odocoileus hemionus) receiving pentosan polysulfate, tannic acid, tetracycline HCl, or no treatment 14 days before to 14 days after (dpi) oral inoculation with tonsil tissue homogenate. All deer were infected, but the rapid disease course (230-603 dpi) suggested our challenge was overwhelming.
Asunto(s)
Ciervos , Poliéster Pentosan Sulfúrico/uso terapéutico , Priones/patogenicidad , Taninos/uso terapéutico , Tetraciclina/uso terapéutico , Enfermedad Debilitante Crónica/tratamiento farmacológico , Animales , Priones/administración & dosificación , Análisis de Supervivencia , Factores de Tiempo , Enfermedad Debilitante Crónica/mortalidadRESUMEN
Prion diseases exhibit different disease phenotypes in their natural hosts and when transmitted to rodents, and this variability is regarded as indicative of prion strain diversity. Phenotypic characterization of scrapie strains in sheep can be attempted by histological, immunohistochemical and biochemical approaches, but it is widely considered that strain confirmation and characterization requires rodent bioassay. Examples of scrapie strains obtained from original sheep isolates by serial passage in mice include ME7, 79A, 22A and 87V. In order to address aspects of prion strain stability across the species barrier, we transmitted the above murine strains to sheep of different breeds and susceptible Prnp genotypes. The experiment included 40 sheep dosed by the oral route alone and 36 sheep challenged by combined subcutaneous and intracerebral routes. Overall, the combined route produced higher attack rates (~100%) than the oral route (~50%) and 2-4 times shorter incubation periods. Uniquely, 87V given orally was unable to infect any sheep. Overall, scrapie strains adapted and cloned in mice produce distinct but variable disease phenotypes in sheep depending on breed or Prnp genotype. Further re-isolation experiments in mice are in progress in order to determine whether the original cloned murine disease phenotype will reemerge.
Asunto(s)
Química Encefálica , Priones/genética , Scrapie/clasificación , Administración Oral , Animales , Mapeo Epitopo , Glicoproteínas/química , Glicosilación , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Fenotipo , Priones/administración & dosificación , Priones/metabolismo , Isoformas de Proteínas/química , Scrapie/genética , Scrapie/patología , Scrapie/transmisión , Ovinos , Especificidad de la Especie , Nervio Vago/química , Nervio Vago/patologíaRESUMEN
Prions, the causative agents of prion diseases, are immunologically tolerated because their major component, prion protein (PrP), is a host-encoded molecule. Therefore, no effective prion vaccines have been developed. We previously showed that heterologous bovine and sheep PrP immunizations of mice overcame tolerance by an antigenic mimicry mechanism to efficiently induce anti-PrP auto-antibodies (Abs), significantly prolonging incubation times in mice subsequently infected with the mouse-adapted Fukuoka-1 prion. These results prompted us to investigate if non-mammal derived molecules able to antigenically mimic anti-prion epitopes, could act as prion vaccines. We show here that immunization of mice with recombinant succinylarginine dihydrolase, a bacterial enzyme with a peptide sequence similar to an anti-prion epitope, induced anti-PrP auto-Abs with anti-prion activity and significantly retarded survival times of the mice subsequently infected with Fukuoka-1 prions. These results might open a way for development of a new type of antigenic mimicry-based prion vaccine.