RESUMEN
Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative infections. Variant Creutzfeldt-Jakob disease (vCJD) and sporadic CJD (sCJD) are human TSEs that, in rare cases, have been transmitted by human-derived therapeutic products. There is a need for a blood test to detect infected donors, identify infected individuals in families with TSEs and monitor progression of disease in patients, especially during clinical trials. We prepared panels of blood from cynomolgus and rhesus macaques experimentally infected with vCJD, as a surrogate for human blood, to support assay development. We detected abnormal prion protein (PrPTSE) in those blood samples using the protein misfolding cyclic amplification (PMCA) assay. PrPTSE first appeared in the blood of pre-symptomatic cynomolgus macaques as early as 2 months post-inoculation (mpi). In contrast, PMCA detected PrPTSE much later in the blood of two pre-symptomatic rhesus macaques, starting at 19 and 20 mpi, and in one rhesus macaque only when symptomatic, at 38 mpi. Once blood of either species of macaque became PMCA-positive, PrPTSE persisted through terminal illness at relatively constant concentrations. Infectivity in buffy coat samples from terminally ill cynomolgus macaques as well as a sample collected 9 months before clinical onset of disease in one of the macaques was assayed in vCJD-susceptible transgenic mice. The infectivity titres varied from 2.7 to 4.3 infectious doses ml-1. We also screened macaque blood using a four-member panel of biomarkers for neurodegenerative diseases to identify potential non-PrPTSE pre-symptomatic diagnostic markers. Neurofilament light-chain protein (NfL) increased in blood before the onset of clinical vCJD. Cumulatively, these data confirmed that, while PrPTSE is the first marker to appear in blood of vCJD-infected cynomolgus and rhesus macaques, NfL might offer a useful, though less specific, marker for forthcoming neurodegeneration. These studies support the use of macaque blood panels to investigate PrPTSE and other biomarkers to predict onset of CJD in humans.
Asunto(s)
Síndrome de Creutzfeldt-Jakob , Enfermedades por Prión , Animales , Biomarcadores , Síndrome de Creutzfeldt-Jakob/diagnóstico , Síndrome de Creutzfeldt-Jakob/metabolismo , Humanos , Filamentos Intermedios/metabolismo , Macaca fascicularis , Macaca mulatta , Ratones , Enfermedades por Prión/metabolismo , Proteínas PriónicasRESUMEN
BACKGROUND: Blood donations must be tested for evidence of syphilis, a transfusion-transmitted infection. Screening blood for syphilis-related antibodies greatly reduced the risk of transfusion-transmitted syphilis (TTS). It is commonly believed that Treponema pallidum (Tp), the bacterium causing syphilis, does not survive in blood during cold storage-suggested as one reason why no cases of TTS have been recognized in the United States for many years. Some have suggested that routine syphilis screening of blood donations is no longer needed. To address the effect of storage, we investigated the survival of Tp experimentally spiked into blood and platelets stored under conventional conditions. STUDY DESIGN AND METHODS: We spiked fresh human blood products with high concentrations of Tp and inoculated samples at intervals into rabbits, a sensitive assay detecting infectious Tp. We tested whole blood (WB) stored refrigerated (1-6°C) for 9 days and platelets stored at room temperature for 7 days or refrigerated for 14 days. We assayed sera of the rabbits collected at intervals for seroconversion using two different tests and assessed orchitis. Rabbits were considered infected if one or both serological test results became positive. RESULTS: Viable Tp survived 7 days in WB and 6 days in platelets stored at both ambient and cold temperatures. DISCUSSION: Tp at concentrations much higher than those possibly present in an infected blood unit survived in cold blood products longer than previously reported and, thus, storage conditions cannot be relied upon to eliminate T. pallidum from blood or platelets. TTS remains a topic of concern for public health.
Asunto(s)
Sífilis , Treponema pallidum , Animales , Anticuerpos Antibacterianos , Donantes de Sangre , Plaquetas , Humanos , Masculino , Tamizaje Masivo , ConejosRESUMEN
US manufacturers, concerned about bovine spongiform encephalopathy (BSE), ceased marketing bovine heparin in the 1990s. Recent short supplies of safe porcine heparin suggest that reintroducing bovine heparin might benefit public health. We purified heparin from crude bovine extract spiked with BSE agent, removing substantial infectivity and abnormal prion proteins (PrPTSE).
Asunto(s)
Encefalopatía Espongiforme Bovina , Priones , Animales , Encéfalo/metabolismo , Bovinos , Encefalopatía Espongiforme Bovina/prevención & control , Heparina , Proteínas Priónicas , Priones/metabolismo , PorcinosRESUMEN
PURPOSE: In the late1990s, reacting to the outbreak of bovine spongiform encephalopathy (BSE) in the United Kingdom that caused a new variant of Creutzfeldt-Jakob disease (vCJD) in humans, manufacturers withdrew bovine heparin from the market in the United States. There have been growing concerns about the adequate supply and safety of porcine heparin. Since the BSE epidemic has been declining markedly, the US Food and Drug Administration reevaluates the vCJD risk via use of bovine heparin. METHODS: We developed a computational model to estimate the vCJD risk to patients receiving bovine heparin injections. The model incorporated information including BSE prevalence, infectivity levels in the intestines, manufacturing batch size, yield of heparin, reduction in infectivity by manufacturing process, and the dose-response relationship. RESULTS: The model estimates a median risk of vCJD infection from a single intravenous dose (10 000 USP units) of heparin made from US-sourced bovine intestines to be 6.9 × 10-9 (2.5-97.fifth percentile: 1.5 × 10-9 -4.3 × 10-8 ), a risk of 1 in 145 million, and 4.6 × 10-8 (2.5-97.fifth percentile: 1.1 × 10-8 -2.6 × 10-7 ), a risk of 1 in 22 million for Canada-sourced products. The model estimates a median risk of 1.4 × 10-7 (2.5-97.fifth percentile: 2.9 × 10-8 -9.3 × 10-7 ) and 9.6 × 10-7 (2.5-97.fifth percentile: 2.1 × 10-7 -5.6 × 10-6 ) for a typical treatment for venous thromboembolism (infusion of 2-4 doses daily per week) using US-sourced and Canada-sourced bovine heparin, respectively. CONCLUSIONS: The model estimates the vCJD risk from use of heparin when appropriately manufactured from US or Canadian cattle is likely small. The model and conclusions should not be applied to other medicinal products manufactured using bovine-derived materials.
Asunto(s)
Anticoagulantes/efectos adversos , Síndrome de Creutzfeldt-Jakob/etiología , Heparina/efectos adversos , Animales , Bovinos , Aprobación de Drogas , Encefalopatía Espongiforme Bovina/epidemiología , Humanos , Modelos Teóricos , Factores de Riesgo , Reino Unido/epidemiología , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Heparin is an anticoagulant sourced from animal tissues. In the 1990s, bovine-sourced heparin was withdrawn from the U.S. market due to a theoretical concern that the bovine spongiform encephalopathy (BSE) agent might contaminate crude heparin and spread to humans as variant Creutzfeldt-Jakob disease. Only porcine intestinal heparin is now marketed in the U.S. FDA has encouraged the reintroduction of bovine heparin. We applied a scaled-down laboratory model process to produce heparin as an active pharmaceutical ingredient (API) starting from bovine intestinal mucosa. The process consisted of two phases. To model the first phase, we applied enzymatic proteolysis, anionic resin separation and methanol precipitation of crude heparin. Bovine intestinal mucosa was spiked with BSE or scrapie agents. We assayed BSE- or scrapie-associated prion protein (PrPTSE) using the Real-Time Quaking-Induced Conversion (RT-QuIC) assay at each step. The process reduced PrPTSE by 4 log10 and 6 log10 from BSE-spiked and scrapie-spiked mucosa, respectively. To model the entire process, we spiked mucosa with scrapie agent and produced heparin API, reducing PrPTSE by 6.7 log10. The purification processes removed large amounts of PrPTSE from the final products. Heparin purification together with careful sourcing of raw materials should allow safely reintroducing bovine heparin in the U.S.
Asunto(s)
Productos Biológicos/metabolismo , Encefalopatía Espongiforme Bovina/metabolismo , Heparina/aislamiento & purificación , Mucosa Intestinal/metabolismo , Proteínas Priónicas/aislamiento & purificación , Priones/metabolismo , Animales , Anticoagulantes/aislamiento & purificación , Anticoagulantes/metabolismo , Anticoagulantes/farmacología , Bovinos , Contaminación de Medicamentos/prevención & control , Heparina/metabolismo , Heparina/farmacología , Humanos , Proteínas Priónicas/metabolismo , Medición de Riesgo/métodosRESUMEN
Transmissible spongiform encephalopathies (TSEs) are infections that are experimentally transmissible to laboratory animals. TSE agents (prions) can be serially passaged in the same animal species. The susceptibility of mice to infection with specific TSE agents can be unpredictable and must be established empirically. We challenged wild-type C57BL/6 and RIIIS/J mice and transgenic mice overexpressing bovine prion protein (TgBo110) with a human brain infected with variant Creutzfeldt-Jakob disease (vCJD) agent and pooled brains of macaques experimentally infected with human vCJD agent (first-passage macaque vCJD). The human vCJD brain yielded a wide range of infectivity titres in different mouse models; TgBo110 mice were the most sensitive. In contrast, infectivity titres of macaque vCJD brain were similar in all three murine models. The brains of RIIIS/J mice infected with both human and macaque vCJD had mild or no vacuolation, while infected C57BL/6 and TgBo110 mice had spongiform degeneration with vacuolation. Abnormal prion protein (PrPTSE) extracted from the brains of vCJD-infected TgBo110 mice displayed different glycosylation profiles and had greater resistance to denaturation by guanidine hydrochloride than PrPTSE from infected wild-type mice or from either inoculum. Those histopathological features of TSE and physical properties of PrPTSE in mice with experimental vCJD were intrinsic to the host, even though we also observed differences between wild-type mice infected with either agent, suggesting a modulatory effect of the inoculum. This study compared three widely used mouse models infected with two different vCJD inocula. The results show that the host plays a major role in manifestations of experimental TSEs.
RESUMEN
BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) has been transmitted by blood transfusion (TTvCJD). The US Food and Drug Administration (FDA) recommends deferring blood donors who resided in or traveled to 30 European countries where they may have been exposed to bovine spongiform encephalopathy (BSE) through beef consumption. Those recommendations warrant re-evaluation, because new cases of BSE and vCJD have markedly abated. STUDY DESIGN AND METHODS: The FDA developed a risk-ranking model to calculate the geographic vCJD risk using country-specific case rates and person-years of exposure of US blood donors. We used the reported country vCJD case rates, when available, or imputed vCJD case rates from reported BSE and UK beef exports during the risk period. We estimated the risk reduction and donor loss should the deferral be restricted to a few high-risk countries. We also estimated additional risk reduction by leukocyte reduction (LR) of red blood cells (RBCs). RESULTS: The United Kingdom, Ireland, and France had the greatest vCJD risk, contributing approximately 95% of the total risk. The model estimated that deferring US donors who spent extended periods of time in these three countries, combined with currently voluntary LR (95% of RBC units), would reduce the vCJD risk by 89.3%, a reduction similar to that achieved under the current policy (89.8%). Limiting deferrals to exposure in these three countries would potentially allow donations from an additional 100,000 donors who are currently deferred. CONCLUSION: Our analysis suggests that a deferral option focusing on the three highest risk countries would achieve a level of blood safety similar to that achieved by the current policy.
Asunto(s)
Donantes de Sangre , Seguridad de la Sangre , Transfusión Sanguínea , Síndrome de Creutzfeldt-Jakob/prevención & control , Selección de Donante , Animales , Bovinos , Síndrome de Creutzfeldt-Jakob/sangre , Síndrome de Creutzfeldt-Jakob/epidemiología , Femenino , Humanos , Masculino , Estados Unidos/epidemiologíaRESUMEN
UNLABELLED: Estimates for the risk of transmitting variant Creutzfeldt-Jakob disease (vCJD) via blood transfusion have relied largely on data from rodent experiments, but the relationship between dose (amount of infected blood) and response (vCJD infection) has never been well quantified. The goal of this study was to develop a dose-response model based on nonhuman primate data to better estimate the likelihood of transfusion-transmitted vCJD (TTvCJD) in humans. Our model used dose-response data from nonhuman primates inoculated intracerebrally (i.c.) with brain tissues of patients with sporadic and familial CJD. We analyzed the data statistically by using a beta-Poisson dose-response model. We further adjusted model parameters to account for the differences in infectivity between blood and brain tissue and in transmission efficiency between intravenous (i.v.) and i.c. routes to estimate dose-dependent TTvCJD infection. The model estimates a mean infection rate of 76% among recipients who receive one unit of whole blood collected from an infected donor near the end of the incubation period. The nonhuman primate model provides estimates that are more consistent with those derived from a risk analysis of transfused nonleukoreduced red blood cells in the United Kingdom than prior estimates based on rodent models. IMPORTANCE: TTvCJD was recently identified as one of three emerging infectious diseases posing the greatest immediate threat to the safety of the blood supply. Cases of TTvCJD were reported in recipients of nonleukoreduced red blood cells and coagulation factor VIII manufactured from blood of United Kingdom donors. As the quantity of abnormal prions (the causative agent of TTvCJD) varies significantly in different blood components and products, it is necessary to quantify the dose-response relationship for a wide range of doses for the vCJD agent in transfused blood and plasma derivatives. In this paper, we suggest the first mechanistic dose-response model for TTvCJD infection based on data from experiments with nonhuman primates. This new model may improve estimates of the possible risk to humans.
Asunto(s)
Síndrome de Creutzfeldt-Jakob/transmisión , Reacción a la Transfusión , Animales , Modelos Animales de Enfermedad , Humanos , Primates , Medición de Riesgo , Reino UnidoRESUMEN
BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) is a fatal neurodegenerative infection that can be transmitted by blood and blood products from donors in the latent phase of the disease. Currently, there is no validated antemortem vCJD blood screening test. Several blood tests are under development. Any useful test must be validated with disease-relevant blood reference panels. STUDY DESIGN AND METHODS: To generate blood reference materials, we infected four cynomolgus macaques with macaque-adapted vCJD brain homogenates. Blood was collected throughout the preclinical and clinical phases of infection. In parallel, equivalent blood was collected from one uninfected macaque. For each blood collection, an aliquot was stored as whole blood and the remainder was separated into components. Aliquots of plasma from terminally ill macaques were assayed for the presence of PrP(TSE) with the protein misfolding cyclic amplification (PMCA) method. Infectivity of the macaque brain homogenate used to infect macaques was titrated in C57BL/6 and RIII J/S inbred wild-type mice. RESULTS: We sampled blood 19 times from the inoculated monkeys at various stages of the disease over a period of 29 months, generating liters of vCJD-infected macaque blood. vCJD was confirmed in all inoculated macaques. After PMCA, PrP(TSE) was detected in plasma from infected monkeys, but not from uninfected animals. Both mouse models were more sensitive to infection with macaque-adapted vCJD agent than to primary human vCJD agent. CONCLUSION: The macaque vCJD blood panels generated in this study provide a unique resource to support vCJD assay development and to characterize vCJD infectivity in blood.
Asunto(s)
Síndrome de Creutzfeldt-Jakob/sangre , Priones/sangre , Secuencia de Aminoácidos , Animales , Síndrome de Creutzfeldt-Jakob/transmisión , Modelos Animales de Enfermedad , Femenino , Humanos , Macaca fascicularis , Masculino , Ratones , Datos de Secuencia Molecular , Estándares de ReferenciaRESUMEN
Proteins aggregate in several slowly progressive neurodegenerative diseases called 'proteinopathies'. Studies with cell cultures and transgenic mice overexpressing mutated proteins suggested that aggregates of one protein induced misfolding and aggregation of other proteins as well - a possible common mechanism for some neurodegenerative diseases. However, most proteinopathies are 'sporadic', without gene mutation or overexpression. Thus, proteinopathies in WT animals genetically close to humans might be informative. Squirrel monkeys infected with the classical bovine spongiform encephalopathy agent developed an encephalopathy resembling variant Creutzfeldt-Jakob disease with accumulations not only of abnormal prion protein (PrP(TSE)), but also three other proteins: hyperphosphorylated tau (p-tau), α-synuclein and ubiquitin; ß-amyloid protein (Aß) did not accumulate. Severity of brain lesions correlated with spongiform degeneration. No amyloid was detected. These results suggested that PrP(TSE) enhanced formation of p-tau and aggregation of α-synuclein and ubiquitin, but not Aß, providing a new experimental model for neurodegenerative diseases associated with complex proteinopathies.
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Encéfalo/patología , Encefalopatía Espongiforme Bovina/patología , Priones/análisis , Ubiquitina/análisis , alfa-Sinucleína/análisis , Proteínas tau/análisis , Animales , Bovinos , Modelos Animales de Enfermedad , SaimiriRESUMEN
BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) is transmitted by blood transfusion. To mitigate the risk of transfusion-transmitted vCJD (TTvCJD), the US Food and Drug Administration has recommended deferral of potential at-risk blood donors, but some risk remains. We describe a quantitative risk assessment to estimate residual, postdeferral TTvCJD risk in the United States. STUDY DESIGN AND METHODS: We assumed that certain US donors may have acquired vCJD infection through dietary exposure to the agent of bovine spongiform encephalopathy during time spent in the United Kingdom, France, and other countries in Europe. Because of uncertainties regarding the prevalence of vCJD in the United Kingdom, we used both low and high UK prevalence estimates as model inputs. The model estimated the risk of infection from a transfusion in year 2011 and the cumulative risk from 1980 through 2011. The model was validated by comparing the model predictions with reported cases of vCJD. RESULTS: Using the low UK prevalence estimate, the model predicted a mean risk of 1 in 134 million transfusions, zero TTvCJD infections acquired in the year 2011, and zero cumulative clinical TTvCJD cases for the period spanning 1980 to 2011. With the high UK prevalence estimate, the model predicted a mean risk of 1 in 480,000 transfusions, six infections for 2011, and nine cumulative clinical cases from 1980 to 2011. CONCLUSIONS: Model validation exercises indicated that predictions based on the low prevalence estimate are more consistent with clinical cases actually observed to date, implying that the risk, while highly uncertain, is likely very small.
Asunto(s)
Síndrome de Creutzfeldt-Jakob/transmisión , Transfusión de Eritrocitos/efectos adversos , Animales , Bovinos , Síndrome de Creutzfeldt-Jakob/epidemiología , Encefalopatía Espongiforme Bovina/epidemiología , Encefalopatía Espongiforme Bovina/transmisión , Humanos , Modelos Teóricos , Medición de Riesgo , Reino Unido/epidemiología , Estados UnidosRESUMEN
Transmissible spongiform encephalopathies (TSEs) or prion diseases are characterized by the accumulation in affected tissues of the abnormal prion protein PrPTSE. We previously demonstrated PrPTSE in the blood of macaques experimentally infected with variant Creutzfeldt-Jakob disease (vCJD), a human TSE, months to years prior to clinical onset. That work supported the prospect of using PrPTSE as a blood biomarker to detect vCJD and possibly other human TSEs before the onset of overt illness. However, our results also raised questions about the origin of PrPTSE detected in blood early after inoculation and the effects of dose and route on the timing of the appearance of PrPTSE. To investigate these questions, we inoculated vCJD-susceptible transgenic mice and non-infectable prion protein-knockout mice under inoculation conditions resembling those used in macaques, with additional controls. We assayed PrPTSE in mouse blood using the protein misfolding cyclic amplification (PMCA) method. PrPTSE from the inoculum cleared from the blood of all mice before 2 months post-inoculation (mpi). Mouse PrPTSE generated de novo appeared in blood after 2 mpi. These results were consistent regardless of dose or inoculation route. We also demonstrated that a commercial ELISA-like PrPTSE test detected and quantified PMCA products and provided a useful alternative to Western blots.
Asunto(s)
Síndrome de Creutzfeldt-Jakob , Enfermedades por Prión , Priones , Ratones , Humanos , Animales , Síndrome de Creutzfeldt-Jakob/metabolismo , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Ratones Transgénicos , Cinética , Enfermedades por Prión/metabolismo , Priones/metabolismo , Macaca , Ratones NoqueadosRESUMEN
Incubation periods in humans infected with transmissible spongiform encephalopathy (TSE) agents can exceed 50 years. In humans infected with bovine spongiform encephalopathy (BSE) agents, the effects of a "species barrier," often observed when TSE infections are transmitted from one species to another, would be expected to increase incubation periods compared with transmissions of same infectious agents within the same species. As part of a long-term study investigating the susceptibility to BSE of cell cultures used to produce vaccines, we inoculated squirrel monkeys (Saimiri sp., here designated SQ) with serial dilutions of a bovine brain suspension containing the BSE agent and monitored them for as long as ten years. Previously, we showed that SQ infected with the original "classical" BSE agent (SQ-BSE) developed a neurological disease resembling that seen in humans with variant CJD (vCJD). Here, we report the final characterization of the SQ-BSE model. We observed an unexpectedly marked difference in incubation times between two animals inoculated with the same dilution and volume of the same C-BSE bovine brain extract on the same day. SQ-BSE developed, in addition to spongiform changes and astrogliosis typical of TSEs, a complex proteinopathy with severe accumulations of protease-resistant prion protein (PrPTSE), hyperphosphorylated tau (p-tau), ubiquitin, and α-synuclein, but without any amyloid plaques or ß-amyloid protein (Aß) typical of Alzheimer's disease. These results suggest that PrPTSE enhanced the accumulation of several key proteins characteristically seen in human neurodegenerative diseases. The marked variation in incubation periods in the same experimental TSE should be taken into account when modeling the epidemiology of human TSEs.
RESUMEN
Transmissible spongiform encephalopathy (TSE) agents have contaminated human tissue-derived medical products, human blood components, and animal vaccines. The objective of this study was to determine the potential susceptibility to infection of 5 cell lines used or proposed for manufacture of biological products, as well as other lines. Cell lines were exposed to the infectious agents of sporadic and variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy (BSE). Exposed cultures were tested for TSE-associated prion protein (PrP(TSE)) and TSE infectivity by assay in rodents and nonhuman primates. No PrP(TSE) or infectivity has been detected in any exposed cell line under study so far. Animals inoculated with BSE brain homogenate developed typical spongiform encephalopathy. In contrast, animals inoculated with cells exposed to the BSE agent remained asymptomatic. All cell lines we studied resisted infection with 3 TSE agents, including the BSE agent.
Asunto(s)
Contaminación de Medicamentos , Enfermedades por Prión/transmisión , Vacunas/aislamiento & purificación , Animales , Bioensayo , Células CHO , Bovinos , Técnicas de Cultivo de Célula , Línea Celular , Chlorocebus aethiops , Enfermedades Transmisibles Emergentes/transmisión , Síndrome de Creutzfeldt-Jakob/transmisión , Cricetinae , Cricetulus , Modelos Animales de Enfermedad , Perros , Encefalopatía Espongiforme Bovina/transmisión , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Priones/aislamiento & purificación , Priones/patogenicidad , Saimiri , Scrapie/transmisión , Células VeroRESUMEN
BACKGROUND: The possible risk of iatrogenic transmissible spongiform encephalopathies (TSEs, prion diseases) from transplantation of marrow-derived mesenchymal stem cells (MSCs) is uncertain. While most cell lines resist infection, a few propagate TSE agents. STUDY DESIGN AND METHODS: We generated MSC-like (MSC-L) cell cultures from bone marrow (BM) of mice inoculated with the human-derived Fukuoka-1 (Fu) strain of TSE agent. Cultured cells were characterized for various markers and cellular prion protein (PrP(C) ) by fluorescence-activated cell sorting and for PrP(C) and its pathologic TSE-associated form (PrP(TSE) ) by Western blotting (WB). Cell cultures were tested for their susceptibility to infection with Fu in vitro. The infectivity of one Fu-infected cell culture was assayed in mice. RESULTS: BM cells from Fu-infected mice expressed neither PrP(C) nor PrP(TSE) after 3 days in culture as demonstrated by WB. Cells adherent to plastic and maintained under two different culture conditions became spontaneously immortalized and began to express PrP(C) at about the same time. One culture became transformed shortly after exposure to Fu in vitro and remained persistently infected, continuously generating PrP(TSE) through multiple passages; the infectivity of cultured cells was confirmed by intracerebral inoculation of lysates into mice. Both persistently TSE-infected and uninfected cells expressed a number of typical MSC markers. CONCLUSION: BM-derived MSC-L cells of mice became persistently infected with the Fu agent under certain conditions in culture-conditions that differ substantially from those currently used to develop investigational human stem cell therapies.
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Células de la Médula Ósea/patología , Células Madre Mesenquimatosas/patología , Enfermedades por Prión/patología , Priones/patogenicidad , Adipogénesis/efectos de los fármacos , Adipogénesis/fisiología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/fisiología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/fisiología , Mesodermo/efectos de los fármacos , Ratones , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Enfermedades por Prión/metabolismo , Enfermedades por Prión/transmisión , Priones/metabolismo , Priones/farmacología , Priones/fisiologíaRESUMEN
Recent studies in animal models demonstrate that certain misfolded proteins associated with neurodegenerative diseases can support templated misfolding of cognate native proteins, to propagate across neural systems, and to therefore have some of the properties of classical prion diseases like Creutzfeldt-Jakob disease. The National Institute of Aging convened a meeting to discuss the implications of these observations for research priorities. A summary of the discussion is presented here, with a focus on limitations of current knowledge, highlighting areas that appear to require further investigation in order to guide scientific practice while minimizing potential exposure or risk in the laboratory setting. The committee concluded that, based on all currently available data, although neurodegenerative disease-associated aggregates of several different non-prion proteins can be propagated from humans to experimental animals, there is currently insufficient evidence to suggest more than a negligible risk, if any, of a direct infectious etiology for the human neurodegenerative disorders defined in part by these proteins. Given the importance of this question, the potential for noninvasive human transmission of proteopathic disorders is deserving of further investigation.
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Péptidos beta-Amiloides , Enfermedades Neurodegenerativas/patología , Deficiencias en la Proteostasis/patología , Animales , Humanos , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMEN
Based on previous preclinical evaluation in mice and monkeys, the chimeric TBEV/DEN4Delta30 virus, carrying the prM and E protein genes from a highly virulent Far Eastern strain of tick-borne encephalitis virus (TBEV) on the backbone of a nonneuroinvasive dengue type 4 virus (DEN4), has been identified as a promising live attenuated virus vaccine candidate against disease caused by TBEV. However, prior to use of this vaccine candidate in humans, its neurovirulence in nonhuman primates needed to be evaluated. In the present study, we compared the neuropathogeneses of the chimeric TBEV/DEN4Delta30 virus; Langat virus (LGTV), a former live TBEV vaccine; and yellow fever 17D virus vaccine (YF 17D) in rhesus monkeys inoculated intracerebrally. TBEV/DEN4Delta30 and YF 17D demonstrated remarkably similar spatiotemporal profiles of virus replication and virus-associated histopathology in the central nervous system (CNS) that were high in cerebral hemispheres but progressively decreased toward the spinal cord. In contrast, the neurovirulence of LGTV exhibited the reverse profile, progressing from the site of inoculation toward the cerebellum and spinal cord. Analysis of the spatiotemporal distribution of viral antigens in the CNS of monkeys revealed a prominent neurotropism associated with all three attenuated viruses. Nevertheless, TBEV/DEN4Delta30 virus exhibited higher neurovirulence in monkeys than either LGTV or YF 17D, suggesting insufficient attenuation. These results provide insight into the neuropathogenesis associated with attenuated flaviviruses that may guide the design of safe vaccines.
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Infecciones por Flavivirus/virología , Flavivirus , Macaca mulatta/inmunología , Macaca mulatta/virología , Neuronas/virología , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Antígenos/inmunología , Chlorocebus aethiops , Flavivirus/efectos de los fármacos , Flavivirus/inmunología , Flavivirus/patogenicidad , Infecciones por Flavivirus/inmunología , Infecciones por Flavivirus/patología , Inmunohistoquímica , Células Vero , Proteínas Virales/inmunología , Virulencia , Replicación ViralRESUMEN
Infections of humans with intraerythrocytic parasites of the genus Babesia can be locally prevalent in diverse regions of the United States. Transfusion of blood and blood products collected from donors infected with Babesia may result in a serious illness that can be fatal. In September 2008, the Food and Drug Administration organized a public workshop to discuss the various aspects of transfusion-transmitted babesiosis in the United States including the possible strategies to identify and defer blood donors who may have been infected with Babesia. Discussions were also held on the biology, pathogenesis, and epidemiology of Babesia species. In this article, we summarize the scientific presentations and panel discussions that took place during the workshop.
Asunto(s)
Babesia/crecimiento & desarrollo , Babesiosis , Transfusión Sanguínea/estadística & datos numéricos , Reacción a la Transfusión , Babesiosis/epidemiología , Babesiosis/prevención & control , Babesiosis/transmisión , Humanos , Prevalencia , Factores de Riesgo , Estados UnidosRESUMEN
Detection of misfolded prion protein, PrPTSE, in biological samples is important to develop antemortem tests for transmissible spongiform encephalopathies (TSEs). The real-time quaking-induced conversion (RT-QuIC) assay detects PrPTSE but requires dedicated equipment and relatively long incubation times when applied to samples containing extremely low levels of PrPTSE. It was shown that a microplate shaker with heated top (Thermomixer-C) accelerated amplification of PrPTSE in brain suspensions of 263K scrapie and sporadic Creutzfeldt-Jakob disease (sCJD). We expanded the investigation to include TSE agents previously untested, including chronic wasting disease (CWD), macaque-adapted variant CJD (vCJD) and human vCJD, and we further characterized the assays conducted at 42°C and 55°C. PrPTSE from all brains containing the TSE agents were successfully amplified using a truncated hamster recombinant protein except for human vCJD which required truncated bank vole recombinant protein. We compared assays conducted at 42°C on Thermomixer-C, Thermomixer-R (without heated top) and on a fluorimeter used for RT-QuIC. QuIC on Thermomixer-R achieved in only 18 hours assay sensitivity similar to that of RT-QuIC read at 60 hours (or 48 hours with sCJD). QuIC on Thermomixer-C required 24 hours to complete and the endpoint titers of some TSEs were 10-fold lower than those obtained with RT-QuIC and Thermomixer-R. Conversely, at 55°C, the reactions with sCJD and CWD on Thermomixer-C achieved the same sensitivity as with RT-QuIC but in shorter times. Human vCJD samples tested at higher temperatures gave rise to high reactivity in wells containing normal control samples. Similarly, reactions on Thermomixer-R were unsuitable at 55°C. The main disadvantage of Thermomixers is that they cannot track formation of PrP fibrils in real time, a feature useful in some applications. The main advantages of Thermomixers are that they need shorter reaction times to detect PrPTSE, are easier to use, involve more robust equipment, and are relatively affordable. Improvements to QuIC using thermal mixers may help develop accessible antemortem TSE tests.
Asunto(s)
Encéfalo/metabolismo , Enfermedades por Prión/etiología , Enfermedades por Prión/metabolismo , Proteínas Priónicas/química , Proteínas Priónicas/metabolismo , Animales , Encéfalo/patología , Síndrome de Creutzfeldt-Jakob , Cricetinae , Modelos Animales de Enfermedad , Humanos , Macaca , Enfermedades por Prión/patología , Deficiencias en la Proteostasis , Proteínas Recombinantes , Temperatura , Enfermedad Debilitante CrónicaRESUMEN
The first of several pivotal moments leading to current understanding of human transmissible spongiform encephalopathies (TSEs) occurred in 1959 when veterinary pathologist W.J. Hadlow first recognized several similarities between scrapie-a slow infection of sheep caused by an unusual infectious agent-and kuru, a fatal exotic neurodegenerative disease affecting only people of a single language group in the remote mountainous interior of New Guinea, described two years earlier by D.C. Gajdusek and V. Zigas. Based on the knowledge of scrapie, Gajdusek, C.J. Gibbs, Jr., and M.P. Alpers soon initiated efforts to transmit kuru by inoculating kuru brain tissue into non-human primates, that-although requiring several years-ultimately proved successful. In the same year that Hadlow first proposed that kuru and scrapie might have similar etiology, I. Klatzo noted that kuru's histopathology resembled that of Creutzfeldt-Jakob disease (CJD), another progressive fatal neurodegenerative disease of unknown etiology that A.M. Jakob had first described in 1921. Gajdusek and colleagues went on to demonstrate that not only the more common sporadic form of CJD but also familial CJD and a generally similar familial brain disease (Gerstmann-Sträussler-Scheinker syndrome) were also transmissible, first to non-human primates and later to other animals. (Other investigators later transmitted an even rarer brain disease, fatal familial insomnia, to animals.) Iatrogenic CJD (spread by human pituitary-derived hormones and tissue grafts) was also transmitted to animals. Much later, in 1996, a new variant of CJD was attributed to human infection with the agent of bovine spongiform encephalopathy; vCJD itself caused an iatrogenic TSE spread by blood transfusion (and probably by a human-plasma-derived clotting factor). Starting in the 1930s, the scrapie agent was found to have a unique constellation of physical properties (marked resistance to inactivation by chemicals, heat and radiation), eventually interpreted as suggesting that it might be an unconventional self-replicating pathogen based on protein and containing no nucleic acid. The work of S.B. Prusiner led to the recognition in the early 1980s that a misfolded form of a ubiquitous normal host protein was usually if not always detectable in tissues containing TSE agents, greatly facilitating the diagnosis and TSEs and understanding their pathogenesis. Prusiner proposed that the TSE agent was likely to be composed partly if not entirely of the abnormal protein, for which he coined the term "prion" protein and "prion" for the agent. Expression of the prion protein by animals-while not essential for life-was later found to be obligatory to infect them with TSEs, and a variety of mutations in the protein clearly tracked with TSEs in families, explaining the autosomal dominant pattern of disease and confirming a central role for the protein in pathogenesis. Prusiner's terminology and the prion hypothesis came to be widely though not universally accepted. A popular corollary proposal, that prions arise by spontaneous misfolding of normal prion protein leading to sporadic cases of CJD, BSE, and scrapie, is more problematic and may serve to discourage continued search for environmental sources of exposure to TSE agents.