Detection Of PrPSc Research Articles

Longitudinal detection of prion infection in preclinical sheep blood samples compared using 3 assays

AbstractVariant Creutzfeldt-Jakob disease (vCJD) is a devastating disease caused by transmission of bovine spongiform encephalopathy to humans. Although vCJD cases are now rare, evidence from appendix surveys suggests that a small proportion of the United Kingdom population may be infected without showing signs of disease. These “silent” carriers could present a risk of iatrogenic vCJD transmission through medical procedures or blood/organ donation, and currently there are no validated tests to identify infected asymptomatic individuals using easily accessible samples. To address this issue, we evaluated the performance of 3 blood-based assays in a blinded study, using longitudinal sample series from a well-established large animal model of vCJD. The assays rely on amplification of misfolded prion protein (PrPSc; a marker of prion infection) and include real-time quaking-induced conversion (RT-QuIC), and 2 versions of protein misfolding cyclic amplification (PMCA). Although diagnostic sensitivity was higher for both PMCA assays (100%) than RT-QuIC (61%), all 3 assays detected prion infection in blood samples collected 26 months before the onset of clinical signs and gave no false-positive results. Parallel estimation of blood prion infectivity titers in a sensitive transgenic mouse line showed positive correlation of infectivity with PrPSc detection by the assays, suggesting that they are suitable for detection of asymptomatic vCJD infection in the human population. This study represents, to our knowledge, the largest comparison to date of preclinical prion detection in blood samples from a relevant animal model. The outcomes will guide efforts to improve early detection of prion disease and reduce infection risks in humans.

RT-QuIC detection of chronic wasting disease prion in platelet samples of white-tailed deer

BackgroundChronic wasting disease (CWD) is a prion disease of captive and free-ranging cervids. Currently, a definitive diagnosis of CWD relies on immunohistochemistry detection of PrPSc in the obex and retropharyngeal lymph node (RPLN) of the affected cervids. For high-throughput screening of CWD in wild cervids, RPLN samples are tested by ELISA followed by IHC confirmation of positive results. Recently, real-time quacking-induced conversion (RT-QuIC) has been used to detect CWD positivity in various types of samples. To develop a blood RT-QuIC assay suitable for CWD diagnosis, this study evaluated the assay sensitivity and specificity with and without ASR1-based preanalytical enrichment and NaI as the main ionic component in assay buffer.ResultsA total of 23 platelet samples derived from CWD-positive deer (ELISA + /IHC +) and 30 platelet samples from CWD-negative (ELISA-) deer were tested. The diagnostic sensitivity was 43.48% (NaCl), 65.22% (NaI), 60.87% (NaCl-ASR1) or 82.61% (NaI-ASR1). The diagnostic specificity was 96.67% (NaCl), 100% (NaI), 100% (NaCl-ASR1), or 96.67% (NaI-ASR1). The probability of detecting CWD prion in platelet samples derived from CWD-positive deer was 0.924 (95% CRI: 0.714, 0.989) under NaI-ASR1 experimental condition and 0.530 (95% CRI: 0.156, 0.890) under NaCl alone condition. The rate of amyloid formation (RFA) was greatest under the NaI-ASR1 condition at 10–2 (0.01491, 95% CRI: 0.00675, 0.03384) and 10–3 (0.00629, 95% CRI: 0.00283, 0.01410) sample dilution levels.ConclusionsIncorporation of ASR1-based preanalytical enrichment and NaI as the main ionic component significantly improved the sensitivity of CWD RT-QuIC on deer platelet samples. Blood test by the improved RT-QuIC assay may be used for antemortem and postmortem diagnosis of CWD.

Development of a sensitive real-time quaking-induced conversion (RT-QuIC) assay for application in prion-infected blood.

Efforts to prevent human-to-human transmission of variant Creutzfeldt-Jakob disease (vCJD) by contaminated blood would be aided by the development of a sensitive diagnostic test that could be routinely used to screen blood donations. As blood samples from vCJD patients are extremely rare, here we describe the optimisation of real-time quaking-induced conversion (RT-QuIC) for detection of PrPSc (misfolded prion protein, a marker of prion infection) in blood samples from an established large animal model of vCJD, sheep experimentally infected with bovine spongiform encephalopathy (BSE). Comparative endpoint titration experiments with RT-QuIC, miniaturized bead protein misfolding cyclic amplification (mb-PMCA) and intracerebral inoculation of a transgenic mouse line expressing sheep PrP (tgOvARQ), demonstrated highly sensitive detection of PrPSc by RT-QuIC in a reference sheep brain homogenate. Upon addition of a capture step with iron oxide beads, the RT-QuIC assay was able to detect PrPSc in whole blood samples from BSE-infected sheep up to two years before disease onset. Both RT-QuIC and mb-PMCA also demonstrated sensitive detection of PrPSc in a reference vCJD-infected human brain homogenate, suggesting that either assay may be suitable for application to human blood samples. Our results support the further development and evaluation of RT-QuIC as a diagnostic or screening test for vCJD.

Synthesis and Characterization of Hydroxyethylamino- and Pyridyl-Substituted 2-Vinyl Chromone Derivatives for Detection of Cerebral Abnormal Prion Protein Deposits.

Prion diseases are fatal neurodegenerative diseases characterized by the deposition of abnormal prion protein aggregates (PrPSc) in the brain. In this study, we developed hydroxyethylamino-substituted styrylchromone (SC) and 2-(2-(pyridin-3-yl)vinyl)-4H-chromen-4-one (VPC) derivatives for single-photon emission computed tomography (SPECT) imaging of PrPSc deposits in the brain. The binding affinity of these compounds was evaluated using recombinant mouse prion protein (rMoPrP) aggregates, which resulted in the inhibition constant (Ki) value of 61.5 and 88.0 nM for hydroxyethyl derivative, (E)-2-(4-((2-hydroxyethyl)amino)styryl)-6-iodo-4H-chromen-4-one (SC-NHEtOH) and (E)-2-(4-((2-hydroxyethyl)(methyl)amino)styryl)-6-iodo-4H-chromen-4-one (SC-NMeEtOH), respectively. However, none of the VPC derivatives showed binding affinity for the rMoPrP aggregates. Fluorescent imaging demonstrated that the accumulation pattern of SC-NHEtOH matched with the presence of PrPSc in the brain slices from mouse-adapted bovine spongiform encephalopathy-infected mice. A biodistribution study of normal mice indicated low initial brain uptake of [125I]SC-NHEtOH (0.88% injected dose/g (% ID/g) at 2 min) despite favorable washout from the brain (0.26% ID/g, at 180 min) was displayed. [125I]SC-NHEtOH exhibited binding affinities to both artificial prion aggregates as well as prion deposits in the brain. However, significant improvement in the binding affinity for PrPSc and blood-brain barrier permeability is necessary for the development of successful in vivo imaging probes for the detection of cerebral PrPSc in the brain.

Validation and Application of Skin RT-QuIC to Patients in China with Probable CJD.

The definite diagnosis of human sporadic Creutzfeldt–Jakob disease (sCJD) largely depends on postmortem neuropathology and PrPSc detection in the brain. The development of real-time quaking-induced conversion (RT-QuIC) of cerebrospinal fluid (CSF) samples makes it possible for premortem diagnosis for sCJD. To test the diagnostic potential of RT-QuIC of skin specimens for probable sCJD, we collected the paired skin and CSF samples from 51 recruited living patients referred to the Chinese CJD surveillance center, including 34 probable sCJD, 14 non-CJD, and 3 genetic prion disease (gPrD). The samples were subjected to RT-QuIC assays using recombinant hamster PrP protein rHaPrP90-231 as the substrate. Using skin RT-QuIC assay, 91.2% (31/34) probable sCJD patients, and 1 T188K genetic CJD (gCJD) cases showed positive prion-seeding activity, while 85.7% (12/14) non-CJD patients were negative. CSF RT-QuIC positive seeding activity was only observed in 14 probable sCJD patients. Analysis of the reactivity of 38 positive skin RT-QuIC tests revealed that the positive rates in the preparations of 10−2, 10−3 and 10−4 diluted skin samples were 88.6% (39/44), 63.6% (28/44), and 25.0% (11/44), respectively. Eleven probable sCJD patients donated two skin specimens collected at different sites simultaneously. Although 95.5% (21/22) skin RT-QuIC elicited positive reaction, the reactivity varied. Our preliminary data indicate high sensitivity and specificity of skin RT-QuIC in prion detection for Chinese probable sCJD and highlight that skin prion-seeding activity is a reliable biomarker for premortem diagnosis of human prion disease.

Adaptive selection of a prion strain conformer corresponding to established North American CWD during propagation of novel emergent Norwegian strains in mice expressing elk or deer prion protein

Prions are infectious proteins causing fatal, transmissible neurodegenerative diseases of animals and humans. Replication involves template-directed refolding of host encoded prion protein, PrPC, by its infectious conformation, PrPSc. Following its discovery in captive Colorado deer in 1967, uncontrollable contagious transmission of chronic wasting disease (CWD) led to an expanded geographic range in increasing numbers of free-ranging and captive North American (NA) cervids. Some five decades later, detection of PrPSc in free-ranging Norwegian (NO) reindeer and moose marked the first indication of CWD in Europe. To assess the properties of these emergent NO prions and compare them with NA CWD we used transgenic (Tg) and gene targeted (Gt) mice expressing PrP with glutamine (Q) or glutamate (E) at residue 226, a variation in wild type cervid PrP which influences prion strain selection in NA deer and elk. Transmissions of NO moose and reindeer prions to Tg and Gt mice recapitulated the characteristic features of CWD in natural hosts, revealing novel prion strains with disease kinetics, neuropathological profiles, and capacities to infect lymphoid tissues and cultured cells that were distinct from those causing NA CWD. In support of strain variation, PrPSc conformers comprising emergent NO moose and reindeer CWD were subject to selective effects imposed by variation at residue 226 that were different from those controlling established NA CWD. Transmission of particular NO moose CWD prions in mice expressing E at 226 resulted in selection of a kinetically optimized conformer, subsequent transmission of which revealed properties consistent with NA CWD. These findings illustrate the potential for adaptive selection of strain conformers with improved fitness during propagation of unstable NO prions. Their potential for contagious transmission has implications for risk analyses and management of emergent European CWD. Finally, we found that Gt mice expressing physiologically controlled PrP levels recapitulated the lymphotropic properties of naturally occurring CWD strains resulting in improved susceptibilities to emergent NO reindeer prions compared with over-expressing Tg counterparts. These findings underscore the refined advantages of Gt models for exploring the mechanisms and impacts of strain selection in peripheral compartments during natural prion transmission.

Place de la biologie dans les maladies de Creutzfeldt-Jakob et apparentées

AbstractPrion diseases are fatal neurodegenerative disorders which are characterized by aggregations of a misfolded protein (called PrPsc) coming from its cellular isoform PrPc, mainly in brain tissue. Due to the transmissibility of this agent, every suspected case has to be monitored and reported to health authorities. Prion diseases diagnosis is a major public health issue, while the definite diagnostic is still based on post-mortem neuropathological examination. CSF biomarkers are an integral part of the diagnostic criteria and are important for the disease surveillance. The 14-3-3 detection is the first CSF surrogate biomarker helping in probable CJD classification. Despite many efforts for specific PrPsc detection, only recent technological development of assays based on protein amplification allowed to detect it in the CSF. However, reference centers have to manage this innovative technology in high security facilities (level 3). Then, nowadays, these protein amplification assays need to be optimized and standardized before their use in current practice.

Cerebrospinal Fluid and Plasma Small Extracellular Vesicles and miRNAs as Biomarkers for Prion Diseases.

Diagnosis of transmissible spongiform encephalopathies (TSEs), or prion diseases, is based on the detection of proteinase K (PK)-resistant PrPSc in post-mortem tissues as indication of infection and disease. Since PrPSc detection is not considered a reliable method for in vivo diagnosis in most TSEs, it is of crucial importance to identify an alternative source of biomarkers to provide useful alternatives for current diagnostic methodology. Ovine scrapie is the prototype of TSEs and has been known for a long time. Using this natural model of TSE, we investigated the presence of PrPSc in exosomes derived from plasma and cerebrospinal fluid (CSF) by protein misfolding cyclic amplification (PMCA) and the levels of candidate microRNAs (miRNAs) by quantitative PCR (qPCR). Significant scrapie-associated increase was found for miR-21-5p in plasma-derived but not in CSF-derived exosomes. However, miR-342-3p, miR-146a-5p, miR-128-3p and miR-21-5p displayed higher levels in total CSF from scrapie-infected sheep. The analysis of overexpressed miRNAs in this biofluid, together with plasma exosomal miR-21-5p, could help in scrapie diagnosis once the presence of the disease is suspected. In addition, we found the presence of PrPSc in most CSF-derived exosomes from clinically affected sheep, which may facilitate in vivo diagnosis of prion diseases, at least during the clinical stage.

Evaluation of the application for new alternative biodiesel production process for rendered fat including Category 1 animal by-products (BDI-RepCat® process, AT).

A new alternative method for the production of biodiesel from rendered fat, including animal by‐product (ABP) Category 1 tallow, was evaluated. The method consists of a conversion phase, based on esterification and transesterification in a single step (at temperature ≥ 200°C, pressure ≥ 70 bar with a retention time ≥ 15 min), using MgO as a catalyst and in the presence of methanol (10–15%), followed by vacuum distillation (at ≥ 150°C, ≤ 10 mbar) of the end‐product, biodiesel and the co‐product, glycerine. Prions (PrPS c), which are abnormal isoforms of the prion protein, were considered by the applicant to be the most resistant hazard. In accordance with previous EFSA Opinions and current expert evaluation, a reduction in prion infectivity, or detectable PrPS c, of at least 6 log10 should be achieved for the process to be considered equivalent to the processing method laid down in the Regulation (EU) No 142/2011. Published data from an experimental replication of the conversion step of the biodiesel production process under consideration were provided, which showed an at least 6 log10 reduction in detectable PrPS c, by Western blot, in tallow that had been spiked with murine and human prion strains. In addition, it was demonstrated that the presence of methanol does not affect the recovery or detection of PrPS c from a biodiesel substrate. Based on scientific literature, the vacuum distillation step has been shown to be capable of achieving an additional 3 log10 reduction in PrPS c. Therefore, the proposed alternative method is considered to be at least equivalent to the processing method laid down in the legislation for the production of biodiesel from raw materials including Category 1 ABP.

Real-Time Quaking-Induced Conversion Detection of PrPSc in Fecal Samples From Chronic Wasting Disease Infected White-Tailed Deer Using Bank Vole Substrate.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) that is fatal to free-range and captive cervids. CWD has been reported in the United States, Canada, South Korea, Norway, Finland, and Sweden, and the case numbers in both wild and farmed cervids are increasing rapidly. Studies indicate that lateral transmission of cervids likely occurs through the shedding of infectious prions in saliva, feces, urine, and blood into the environment. Therefore, the detection of CWD early in the incubation time is advantageous for disease management. In this study, we adapt real-time quacking-induced conversion (RT-QuIC) assays to detect the seeding activity of CWD prions in feces samples from clinical and preclinical white-tailed deer. By optimizing reaction conditions for temperature as well as the salt and salt concentration, prion seeding activity from both clinical and preclinical animals were detected by RT-QuIC. More specifically, all fecal samples collected from 6 to 30 months post inoculation showed seeding activity under the conditions of study. The combination of a highly sensitive detection tool paired with a sample type that may be collected non-invasively allows a useful tool to support CWD surveillance in wild and captive cervids.

Preclinical transmission of prions by blood transfusion is influenced by donor genotype and route of infection.

Variant Creutzfeldt-Jakob disease (vCJD) is a human prion disease resulting from zoonotic transmission of bovine spongiform encephalopathy (BSE). Documented cases of vCJD transmission by blood transfusion necessitate on-going risk reduction measures to protect blood supplies, such as leucodepletion (removal of white blood cells, WBCs). This study set out to determine the risks of prion transmission by transfusion of labile blood components (red blood cells, platelets, plasma) commonly used in human medicine, and the effectiveness of leucodepletion in preventing infection, using BSE-infected sheep as a model. All components were capable of transmitting prion disease when donors were in the preclinical phase of infection, with the highest rates of infection in recipients of whole blood and buffy coat, and the lowest in recipients of plasma. Leucodepletion of components (<106 WBCs/unit) resulted in significantly lower transmission rates, but did not completely prevent transmission by any component. Donor PRNP genotype at codon 141, which is associated with variation in incubation period, also had a significant effect on transfusion transmission rates. A sensitive protein misfolding cyclic amplification (PMCA) assay, applied to longitudinal series of blood samples, identified infected sheep from 4 months post infection. However, in donor sheep (orally infected), the onset of detection of PrPSc in blood was much more variable, and generally later, compared to recipients (intravenous infection). This shows that the route and method of infection may profoundly affect the period during which an individual is infectious, and the test sensitivity required for reliable preclinical diagnosis, both of which have important implications for disease control. Our results emphasize that blood transfusion can be a highly efficient route of transmission for prion diseases. Given current uncertainties over the prevalence of asymptomatic vCJD carriers, this argues for the maintenance and improvement of current measures to reduce the risk of transmission by blood products.

Magnetic microparticle-based multimer detection system for the electrochemical detection of prion oligomers in sheep using a recyclable BDD electrode

There is a strong need of sensitive and accurate diagnostic tests of infectious prion proteins (PrPSc) in pre-symptomatic individuals and animals. This work associates, for the first time, the magnetic microparticle multimer detection system (MDS) with a recyclable boron doped diamond (BDD) electrode for the electrochemical detection of PrPSc in plasma samples from scrapie-infected sheep with clinical signs. The electrochemical results are in agreement with the chemiluminescence results. The main advantages of the electrochemical fluidic system are its recyclability and the use of low quantity of functional magnetic beads.

Fundamentals of Chemical Reactor Engineering: A Multi-Scale Approach.

There is a strong need of sensitive and accurate diagnostic tests of infectious prion proteins (PrPSc) in pre-symptomatic individuals and animals. This work associates, for the first time, the magnetic microparticle multimer detection system (MDS) with a recyclable boron doped diamond (BDD) electrode for the electrochemical detection of PrPSc in plasma samples from scrapie-infected sheep with clinical signs. The electrochemical results are in agreement with the chemiluminescence results. The main advantages of the electrochemical fluidic system are its recyclability and the use of low quantity of functional magnetic beads.

Challenges and Advances in Antemortem Diagnosis of Human Transmissible Spongiform Encephalopathies.

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, arise from the structural conversion of the monomeric, cellular prion protein (PrPC) into its multimeric scrapie form (PrPSc). These pathologies comprise a group of intractable, rapidly evolving neurodegenerative diseases. Currently, a definitive diagnosis of TSE relies on the detection of PrPSc and/or the identification of pathognomonic histological features in brain tissue samples, which are usually obtained postmortem or, in rare cases, by brain biopsy (antemortem). Over the past two decades, several paraclinical tests for antemortem diagnosis have been developed to preclude the need for brain samples. Some of these alternative methods have been validated and can provide a probable diagnosis when combined with clinical evaluation. Paraclinical tests include in vitro cell-free conversion techniques, such as the real-time quaking-induced conversion (RT-QuIC), as well as immunoassays, electroencephalography (EEG), and brain bioimaging methods, such as magnetic resonance imaging (MRI), whose importance has increased over the years. PrPSc is the main biomarker in TSEs, and the RT-QuIC assay stands out for its ability to detect PrPSc in cerebrospinal fluid (CSF), olfactory mucosa, and dermatome skin samples with high sensitivity and specificity. Other biochemical biomarkers are the proteins 14-3-3, tau, neuron-specific enolase (NSE), astroglial protein S100B, α-synuclein, and neurofilament light chain protein (NFL), but they are not specific for TSEs. This paper reviews the techniques employed for definite diagnosis, as well as the clinical and paraclinical methods for possible and probable diagnosis, both those in use currently and those no longer employed. We also discuss current criteria, challenges, and perspectives for TSE diagnosis. An early and accurate diagnosis may allow earlier implementation of strategies to delay or stop disease progression.

Preparation of PrP-specific Polyclonal Antibody via Immunization of PRNP-knockout Mice with Recombinant Human PrP Protein

ObjectiveThe definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrPSc in the brain tissues of suspected cases. Thus, developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification. MethodsWe prepared a PrP-specific polyclonal antibody (pAb P54) in a PRNP-knockout mouse model via immunization with recombinant full-length human PrP protein residues 23–231. Thereafter, we verified that pAb in Western blot, immunohistochemistry (IHC), and immunofluorescent (IFA) assays. ResultsWestern blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein, normal brain PrPC from healthy rodents and humans, and pathological PrPSc in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases. The electrophoretic patterns of brain PrPC and PrPSc observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies. Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54. IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139A-infected mice and 263K-infected hamsters. IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei. ConclusionThe newly prepared pAb P54 demonstrated reliable specificity and sensitivity and, thus, may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.

Evaluation of an alternative method for production of biodiesel from processed fats derived from Category 1, 2 and 3 animal by-products (submitted by College Proteins).

An alternative method for the production of biodiesel from processed fats derived from Category 1, 2 and 3 animal by‐products was assessed. The method is based on a pre‐cleaning process, acidic esterification/transesterification of tallow using 1.5% methanesulfonic acid w/w; 140°C; 5.5 bar absolute pressure (bara); 4 h, followed by fractional distillation. The application focuses on the capacity of the alternative method to inactivate prions. Given the limitations that biodiesel presents for direct measurement of prion infectivity, the BIOHAZ Panel considered, based on the outcome of previous EFSA Opinions and current expert evaluation, that a reduction of 6 log10 in detectable PrPS c signal would be necessary to consider the process at least equivalent to previously approved methods for Category 1 animal by‐products. This is in addition to the inactivation achieved by the pressure sterilisation method applied before the application of any biodiesel production method. Experimental data were provided via ad hoc studies commissioned to quantify the reduction in detectable PrPS c in material spiked with scrapie hamster strain 263K, as measured by western blot, for the first two steps, with distillation assumed to provide at least an additional 3 log10 reduction, based on published data. Despite the intrinsic methodological caveats of the detection of PrPS c in laboratory studies, the BIOHAZ Panel considers that the alternative method, including the final fractional distillation, is capable of achieving the required 6 log10 reduction of the strain 263K PrPS c signal. Therefore, the method under assessment can be considered at least equivalent to the processing methods previously approved for the production of biodiesel from all categories of animal by‐product raw materials. It is recommended to check the feasibility of the proposed HACCP plan by recording the main processing parameters for a certain time period under real industrial conditions.

A probable role of copper in the comorbidity in Wilson\u2019s and Creutzfeldt-Jakob\u2019s Diseases: a case report

BackgroundTo the best of our knowledgedd, there is currently no case in the literature reporting the comorbidity of Wilson’s and Creutzfeldt-Jakob disease (CJD), linked through copper.Case presentationA 44-year-old male with a history of inherited Wilson’s disease (hepatolenticular degeneration), which manifested as mild liver injury and psychiatric symptoms, was admitted to our department due to speech and cognitive disturbances. Upon his admission, he had motor aphasia as well as psychomotor retardation with an otherwise normal neurological examination. Laboratory tests, including liver enzymes, copper and serum ammonia were all within normal range. The brain MRI showed increased T2 signal in the caudate nuclei, attributed to copper deposition in the context of Wilson’s disease. In the electroencephalogram, periodic sharp discharges were eminent, initially unilateral and then generalized. The positive 14–3-3 protein in the cerebrospinal fluid (CSF) and the new brain MRI, that demonstrated elevated DWI signal not only in the basal ganglia but also in parts of the cerebral cortex (cortical ribbon sign), all supportive of a possible CJD diagnosis. The detection of PrPSc in the patient’s CSF, using the RT-QuIC method, which has a 99.4–100% specificity for CJD, made the diagnosis of CJD highly probable.ConclusionThis is the first report of Wilson’s and Creutzfeldt-Jakob diseases co-morbidity in the literature, which could evoke a possible role of copper in the pathogenesis of CJD.

Synthesis of Fe₃C@C from Pyrolysis of Fe₃O₄-Lignin Clusters and Its Application for Quick and Sensitive Detection of PrPSc through a Sandwich SPR Detection Assay.

The prion protein (PrPSc) has drawn widespread attention due to its pathological potential to cause prion diseases. Herein, we successfully synthesized Fe3C@C by carbonizing Fe3O4-lignin clusters, which were prepared through a facile hydrogen bonding interaction between ≡Fe-OH and hydroxyl groups of lignin. Our in-depth investigation confirmed that the composites were Fe3C@C core/shell particles. We constructed a novel sandwich surface plasmon resonance (SPR) detection assay for sensitive PrPSc detection, utilizing bare gold surface and aptamer-modified Fe3C@C (Fe3C@C-aptamer). Due to the highly specific affinity of Fe3C@C-aptamer towards PrPSc, the sandwich type SPR sensor exhibited excellent analytical performance towards the discrimination and quantitation of PrPSc. A good linear relationship was obtained between the SPR responses and the logarithm of PrPSc concentrations over a range of 0.1–200 ng/mL. The detection sensitivity for PrPSc was improved by ~10 fold compared with the SPR direct detection format. The required detection time was only 20 min. The specificity of the present biosensor was also confirmed by PrPC and other reagents as controls. This proposed approach could also be used to isolate and detect other highly pathogenic biomolecules with similar structural characteristics by altering the corresponding aptamer in the Fe3C@C conjugates.

Clinical and neuropathological phenotype associated with the novel V189I mutation in the prion protein gene

Prion diseases are neurodegenerative disorders which are caused by an accumulation of the abnormal, misfolded prion protein known as scrapie prion protein (PrPSc). These disorders are unique as they occur as sporadic, genetic and acquired forms. Sporadic Creutzfeldt-Jakob Disease (CJD) is the most common human prion disease, accounting for approximately 85–90% of cases, whereas autosomal dominant genetic forms, due to mutations in the prion protein gene (PRNP), account for 10–15% of cases. Genetic forms show a striking variability in their clinical and neuropathological picture and can sometimes mimic other neurodegenerative diseases.We report a novel PRNP mutation (V189I) in four CJD patients from three unrelated pedigrees. In three patients, the clinical features were typical for CJD and the diagnosis was pathologically confirmed, while the fourth patient presented with a complex phenotype including rapidly progressive dementia, behavioral abnormalities, ataxia and extrapyramidal features, and the diagnosis was probable CJD by current criteria, on the basis of PrPSc detection in CSF by Real Time Quaking-Induced Conversion assay. In all the three patients with autopsy findings, the neuropathological analysis revealed diffuse synaptic type deposition of proteinase K-resistant prion protein (PrPres), and type 1 PrPres was identified in the brain by western blot analysis. So, the histopathological and biochemical profile associated with the V189I mutation was indistinguishable from the MM1/MV1 subtype of sporadic CJD.Our findings support a pathogenic role for the V189I PRNP variant, confirm the heterogeneity of the clinical phenotypes associated to PRNP mutations and highlight the importance of PrPSc detection assays as diagnostic tools to unveil prion diseases presenting with atypical phenotypes.

Evaluation of the effect of various main elements on the PrPSc detection by real-time quaking-induced conversion assay.

In order to definitively diagnosis sporadic Creutzfeldt-Jakob disease (sCJD), brain tissue is currently required. Therefore, there is a great need for tests that can detect sCJD in body fluids or other types of tissues. Different variables, including the amount of recombinant celluar prion protein (rPrPC), salt, cleaning surfactants and thioflavin T (ThT), in human cerebrospinal fluid (CSF) were evaluated. The reagent concentrations of 1X PBS, 170 mM NaCl, 1 mM EDTA, 0.01 mM ThT and 0.001% SDS, and the amounts of 10 µg rPrPC and 10 µl CSF were considered to be optimal for the real-time quaking-induced conversion (RT-QuIC) assay. Using these conditions, the RT-QuIC assay for prion protein (PrPSc) detection was observed to be sensitive to 10−8 diluted brain homogenates of hamsters infected with the 263K scrapie strain. Furthermore, CSF samples from 70 probable sCJD cases and 48 non-CJD cases were preliminarily screened. A substantial proportion of sCJD samples (57.14%) tested positive by RT-QuIC, with a short lag phase (<50 h post-reaction) and high peak ThT values (>25,000 relative fluorescence units). By contrast, only a small number of non-CJD samples displayed weakly positive results, and these were detected at a later stage (>50 h post-reaction) and had much lower ThT values. In conclusion, the RT-QuIC assay in CSF samples reported in the present study may provide a useful pre-mortem tool for the diagnosis of sCJD, particularly in China where postmortem examination is rarely conducted.