Preclinical Detection of Prions in Blood of Nonhuman Primates Infected with Variant Creutzfeldt-Jakob Disease.
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ABSTRACT:
SUBMITTER: Concha-Marambio L
PROVIDER: S-EPMC6924915 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Human prion diseases are infectious and invariably fatal neurodegenerative diseases. They include sporadic Creutzfeldt-Jakob disease (sCJD), the most common form, and variant CJD (vCJD), which is caused by interspecies transmission of prions from cattle infected by bovine spongiform encephalopathy. Development of a biochemical assay for the sensitive, specific, early, and noninvasive detection of prions (PrPSc) in the blood of patients affected by prion disease is a top medical priority to increase the safety of the blood supply. vCJD has already been transmitted from human to human by blood transfusion, and the number of asymptomatic carriers of vCJD in the U.K. alone is estimated to be 1 in 2000 people. We used the protein misfolding cyclic amplification (PMCA) technique to analyze blood samples from 14 cases of vCJD and 153 controls, including patients affected by sCJD and other neurodegenerative or neurological disorders as well as healthy subjects. Our results showed that PrPSc could be detected with 100% sensitivity and specificity in blood samples from vCJD patients. Detection was possible in any of the blood fractions analyzed and could be done with as little as a few microliters of sample volume. The PrPSc concentration in blood was estimated to be ~0.5 pg/ml. Our findings suggest that PMCA may be useful for premortem noninvasive diagnosis of vCJD and to identify prion contamination of the blood supply. Further studies are needed to fully validate the technology.
| S-EPMC5538786 | biostudies-literature
Project description:Human prion diseases constitute a group of infectious and invariably fatal neurodegenerative disorders associated with misfolding of the prion protein. Variant Creutzfeldt-Jakob disease (vCJD) is a zoonotic prion disease linked to oral exposure to the infectious agent that causes bovine spongiform encephalopathy (BSE) in cattle. The most recent case of definite vCJD was heterozygous (MV) at polymorphic codon 129 of the prion protein gene PRNP while all of the previous 177 definite or probable vCJD cases who underwent genetic analysis were methionine homozygous (MM). Retrospective prevalence studies conducted on lympho-reticular tissue suggest that the number of asymptomatic vCJD carriers in the United Kingdom might be around 1 in 2000 people. In addition, there have been four known cases of the transmission of vCJD infection via blood transfusion. For these reasons, a sensitive, reliable, and fast diagnostic test is currently needed. We describe a rapid and highly sensitive seeding conversion assay that detects disease-associated prion protein in the brain and cerebrospinal fluid in vCJD after 48-96 h of amplification, with 100% sensitivity and specificity. This method can amplify prions from definite, probable, and possible vCJD cases from patients who are either MM or MV at PRNP-codon 129.
| S-EPMC5903693 | biostudies-literature
Project description:BACKGROUND:Prions, the infectious agents responsible for transmissible spongiform encephalopathies, consist mainly of the misfolded prion protein (PrP(Sc)). The unique mechanism of transmission and the appearance of a variant form of Creutzfeldt-Jakob disease, which has been linked to consumption of prion-contaminated cattle meat, have raised concerns about public health. Evidence suggests that variant Creutzfeldt-Jakob disease prions circulate in body fluids from people in whom the disease is silently incubating. METHODS:To investigate whether PrP(Sc) can be detected in the urine of patients with variant Creutzfeldt-Jakob disease, we used the protein misfolding cyclic amplification (PMCA) technique to amplify minute quantities of PrP(Sc), enabling highly sensitive detection of the protein. We analyzed urine samples from several patients with various transmissible spongiform encephalopathies (variant and sporadic Creutzfeldt-Jakob disease and genetic forms of prion disease), patients with other degenerative or nondegenerative neurologic disorders, and healthy persons. RESULTS:PrP(Sc) was detectable only in the urine of patients with variant Creutzfeldt-Jakob disease and had the typical electrophoretic profile associated with this disease. PrP(Sc) was detected in 13 of 14 urine samples obtained from patients with variant Creutzfeldt-Jakob disease and in none of the 224 urine samples obtained from patients with other neurologic diseases and from healthy controls, resulting in an estimated sensitivity of 92.9% (95% confidence interval [CI], 66.1 to 99.8) and a specificity of 100.0% (95% CI, 98.4 to 100.0). The PrP(Sc) concentration in urine calculated by means of quantitative PMCA was estimated at 1×10(-16) g per milliliter, or 3×10(-21) mol per milliliter, which extrapolates to approximately 40 to 100 oligomeric particles of PrP(Sc) per milliliter of urine. CONCLUSIONS:Urine samples obtained from patients with variant Creutzfeldt-Jakob disease contained minute quantities of PrP(Sc). (Funded by the National Institutes of Health and others.).
| S-EPMC4162740 | biostudies-literature
Project description:The emergence of variant Creutzfeldt Jakob Disease (vCJD) is considered a likely consequence of human dietary exposure to Bovine Spongiform Encephalopathy (BSE) agent. More recently, secondary vCJD cases were identified in patients transfused with blood products prepared from apparently healthy donors who later went on to develop the disease. As there is no validated assay for detection of vCJD/BSE infected individuals the prevalence of the disease in the population remains uncertain. In that context, the risk of vCJD blood borne transmission is considered as a serious concern by health authorities. In this study, appropriate conditions and substrates for highly efficient and specific in vitro amplification of vCJD/BSE agent using Protein Misfolding Cyclic Amplification (PMCA) were first identified. This showed that whatever the origin (species) of the vCJD/BSE agent, the ovine Q171 PrP substrates provided the best amplification performances. These results indicate that the homology of PrP amino-acid sequence between the seed and the substrate is not the crucial determinant of the vCJD agent propagation in vitro. The ability of this method to detect endogenous vCJD/BSE agent in the blood was then defined. In both sheep and primate models of the disease, the assay enabled the identification of infected individuals in the early preclinical stage of the incubation period. Finally, sample panels that included buffy coat from vCJD affected patients and healthy controls were tested blind. The assay identified three out of the four tested vCJD affected patients and no false positive was observed in 141 healthy controls. The negative results observed in one of the tested vCJD cases concurs with results reported by others using a different vCJD agent blood detection assay and raises the question of the potential absence of prionemia in certain patients.
| S-EPMC4055790 | biostudies-literature
Project description:The metazoan Hsp70 disaggregase protects neurons from proteotoxicity that arises from the accumulation of misfolded protein aggregates. Hsp70 and its co-chaperones disassemble and extract polypeptides from protein aggregates for refolding or degradation. The effectiveness of the chaperone system decreases with age and leads to accumulation rather than removal of neurotoxic protein aggregates. Therapeutic enhancement of the Hsp70 protein disassembly machinery is proposed to counter late-onset protein misfolding neurodegenerative disease that may arise. In the context of prion disease, it is not known whether stimulation of protein aggregate disassembly paradoxically leads to enhanced formation of seeding competent species of disease-specific proteins and acceleration of neurodegenerative disease. Here we have tested the hypothesis that modulation of Hsp70 disaggregase activity perturbs mammalian prion-induced neurotoxicity and prion seeding activity. To do so we used prion protein (PrP) transgenic Drosophila that authentically replicate mammalian prions. RNASeq identified that Hsp70, DnaJ-1 and Hsp110 gene expression was downregulated in prion-exposed PrP Drosophila. We demonstrated that RNAi knockdown of Hsp110 or DnaJ-1 gene expression in variant Creutzfeldt-Jakob disease prion-exposed human PrP Drosophila enhanced neurotoxicity, whereas overexpression mitigated toxicity. Strikingly, prion seeding activity in variant Creutzfeldt-Jakob disease prion-exposed human PrP Drosophila was ablated or reduced by Hsp110 or DnaJ-1 overexpression, respectively. Similar effects were seen in scrapie prion-exposed ovine PrP Drosophila with modified Hsp110 or DnaJ-1 gene expression. These unique observations show that the metazoan Hsp70 disaggregase facilitates the clearance of mammalian prions and that its enhanced activity is a potential therapeutic strategy for human prion disease.
| S-EPMC9473358 | biostudies-literature
Project description:Variant Creutzfeldt-Jakob disease (vCJD) may be transmissible by blood. To prevent secondary transmission through blood components, several countries have started to exclude as donors persons who have received a blood transfusion. We investigated the effectiveness of this measure by using a dynamic age-structured model. It is the first such model based on epidemiologic data: 1) blood donor activities, 2) a case-control study on CJD, 3) age distribution of recipients, and 4) death of recipients of blood transfusions. The model predicts that an infection like vCJD, which has been introduced into the population by the alimentary route, could not become endemic by transfusion alone and that only <1% of cases would be avoided by excluding from blood donation those persons who have received a transfusion.
| S-EPMC2725807 | biostudies-literature
Project description:We report the presence of infectivity in erythrocytes, leukocytes, and plasma of 1 person with variant Creutzfeldt-Jakob disease and in the plasma of 2 in 4 persons whose tests were positive for sporadic Creutzfeldt-Jakob disease. The measured infectivity levels were comparable to those reported in various animals with transmissible spongiform encephalopathies.
| S-EPMC3884712 | biostudies-literature
Project description:Molecular typing of the abnormal form of the prion protein (PrP(Sc)) has come to be regarded as a powerful tool in the investigation of the prion diseases. All evidence thus far presented indicates a single PrP(Sc) molecular type in variant Creutzfeldt-Jakob disease (termed type 2B), presumably resulting from infection with a single strain of the agent (bovine spongiform encephalopathy). Here we show for the first time that the PrP(Sc) that accumulates in the brain in variant Creutzfeldt-Jakob disease also contains a minority type 1 component. This minority type 1 PrP(Sc) was found in all 21 cases of variant Creutzfeldt-Jakob disease tested, irrespective of brain region examined, and was also present in the variant Creutzfeldt-Jakob disease tonsil. The quantitative balance between PrP(Sc) types was maintained when variant Creutzfeldt-Jakob disease was transmitted to wild-type mice and was also found in bovine spongiform encephalopathy cattle brain, indicating that the agent rather than the host specifies their relative representation. These results indicate that PrP(Sc) molecular typing is based on quantitative rather than qualitative phenomena and point to a complex relationship between prion protein biochemistry, disease phenotype and agent strain.
| S-EPMC1592651 | biostudies-literature
Project description:The biochemical and neuropathological properties of bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) prions are faithfully maintained upon transmission to guinea pigs. However, primary and secondary transmissions of BSE and vCJD in guinea pigs result in long incubation periods of ?450 and ?350 days, respectively. To determine if the incubation periods of BSE and vCJD prions could be shortened, we generated transgenic (Tg) mice expressing guinea pig prion protein (GPPrP). Inoculation of Tg(GPPrP) mice with BSE and vCJD prions resulted in mean incubation periods of 210 and 199 days, respectively, which shortened to 137 and 122 days upon serial transmission. In contrast, three different isolates of sporadic CJD prions failed to transmit disease to Tg(GPPrP) mice. Many of the strain-specified biochemical and neuropathological properties of BSE and vCJD prions, including the presence of type 2 protease-resistant PrPSc, were preserved upon propagation in Tg(GPPrP) mice. Structural modeling revealed that two residues near the N-terminal region of ?-helix 1 in GPPrP might mediate its susceptibility to BSE and vCJD prions. Our results demonstrate that expression of GPPrP in Tg mice supports the rapid propagation of BSE and vCJD prions and suggest that Tg(GPPrP) mice may serve as a useful paradigm for bioassaying these prion isolates. IMPORTANCE:Variant Creutzfeldt-Jakob disease (vCJD) and bovine spongiform encephalopathy (BSE) prions are two of the prion strains most relevant to human health. However, propagating these strains in mice expressing human or bovine prion protein has been difficult because of prolonged incubation periods or inefficient transmission. Here, we show that transgenic mice expressing guinea pig prion protein are fully susceptible to vCJD and BSE prions but not to sporadic CJD prions. Our results suggest that the guinea pig prion protein is a better, more rapid substrate than either bovine or human prion protein for propagating BSE and vCJD prions.
| S-EPMC5068510 | biostudies-literature
Project description:Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder caused by the conformational conversion of the prion protein (PrPC) into an abnormally folded form, named prion (or PrPSc). The combination of the polymorphism at codon 129 of the PrP gene (coding either methionine or valine) with the biochemical feature of the proteinase-K resistant PrP (generating either PrPSc type 1 or 2) gives rise to different PrPSc strains, which cause variable phenotypes of sCJD. The definitive diagnosis of sCJD and its classification can be achieved only post-mortem after PrPSc identification and characterization in the brain. By exploiting the Real-Time Quaking-Induced Conversion (RT-QuIC) assay, traces of PrPSc were found in the olfactory mucosa (OM) of sCJD patients, thus demonstrating that PrPSc is not confined to the brain. Here, we have optimized another technique, named protein misfolding cyclic amplification (PMCA) for detecting PrPSc in OM samples of sCJD patients. OM samples were collected from 27 sCJD and 2 genetic CJD patients (E200K). Samples from 34 patients with other neurodegenerative disorders were included as controls. Brains were collected from 26 sCJD patients and 16 of them underwent OM collection. Brain and OM samples were subjected to PMCA using the brains of transgenic mice expressing human PrPC with methionine at codon 129 as reaction substrates. The amplified products were analyzed by Western blot after proteinase K digestion. Quantitative PMCA was performed to estimate PrPSc concentration in OM. PMCA enabled the detection of prions in OM samples with 79.3% sensitivity and 100% specificity. Except for a few cases, a predominant type 1 PrPSc was generated, regardless of the tissues analyzed. Notably, all amplified PrPSc were less resistant to PK compared to the original strain. In conclusion, although the optimized PMCA did not consent to recognize sCJD subtypes from the analysis of OM collected from living patients, it enabled us to estimate for the first time the amount of prions accumulating in this biological tissue. Further assay optimizations are needed to faithfully amplify peripheral prions whose recognition could lead to a better diagnosis and selection of patients for future clinical trials.
| S-EPMC8990253 | biostudies-literature