detection of the protein 14-3-3 in the CSF has been shown to be a
reliable and sensitive marker for sporadic Creutzfeldt-Jakob disease
(CJD). Other brain-specific proteins such as neuron specific enolase
(NSE), S-100b, and tau protein have also been reported to be increased
in the CSF of patients with sporadic CJD. In 1996a variant of CJD
(vCJD) was described which is likely to be causally linked to the
bovine spongiform encephalopathy agent. This study reports and compares
the findings of CSF brain specific protein analysis in 45 patients with
vCJD and in 34 control patients.
METHODS—The CSF from
45 patients with vCJD and 34 controls were investigated for the
presence of 14-3-3 by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) and western blotting with chemiluminescent detection. Tau
protein, S-100b, and NSE concentrations in CSF were measured using
14-3-3 was detected in the CSF of 22/45 patients with vCJD and in
3/34 controls. The mean concentrations of NSE, S-100b, and tau protein
in CSF were significantly raised in patients with vCJD compared with
controls. The positive predictive value of CSF 14-3-3 was 86% and
the negative predictive value was 63%. These values are lower than
those reported for sporadic CJD. An increased CSF tau had a positive
predictive value of 93% and a negative predictive value of 81%. The
combination of CSF 14-3-3 and/or increased CSF tau had a positive
predictive value of 91% and a negative predictive value of 84%.
protein 14-3-3 is not as useful a marker for vCJD as it is for
sporadic CJD. Increased concentration of CSF tau was found to be a
sensitive marker of vCJD but as concentrations may be increased in many
forms of non-CJD dementia, this may limit its usefulness as a
Brain derived proteins such as 14-3-3, neuron-specific enolase (NSE), S 100b, tau, phosphorylated tau and Aβ1–42 were found to be altered in the cerebrospinal fluid (CSF) in Creutzfeldt-Jakob disease (CJD) patients. The pathogenic mechanisms leading to these abnormalities are not known, but a relation to rapid neuronal damage is assumed. No systematic analysis on brain-derived proteins in the CSF and neuropathological lesion profiles has been performed.
CSF protein levels of brain-derived proteins and the degree of spongiform changes, neuronal loss and gliosis in various brain areas were analyzed in 57 CJD patients.
We observed three different patterns of CSF alteration associated with the degree of cortical and subcortical changes. NSE levels increased with lesion severity of subcortical areas. Tau and 14-3-3 levels increased with minor pathological changes, a negative correlation was observed with severity of cortical lesions. Levels of the physiological form of the prion protein (PrPc) and Aβ1–42 levels correlated negatively with cortical pathology, most clearly with temporal and occipital lesions.
Our results indicate that the alteration of levels of brain-derived proteins in the CSF does not only reflect the degree of neuronal damage, but it is also modified by the localization on the brain pathology. Brain specific lesion patterns have to be considered when analyzing CSF neuronal proteins.
analysis of markers in the cerebrospinal
fluid (CSF) is useful in
the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD). However,
the time at which the study
of these markers is most sensitive
To assess the influence of time of
sampling on the value of CSF tests
in the diagnosis of sCJD.
In the framework of a multinational
European study, we studied
the results of 14-3-3, S100b, neurone
specific enolase (NSE) and
tau protein in 833 CSF samples
from sCJD patients at different
stages of disease and in 66
sequentially repeated lumbar
and tau protein tended to increase
in sensitivity from onset (88%,
81%) to the advanced stage (91%,
90%). This was significant only in
the methionine-valine (MV) heterozygous
group of patients at
codon 129. The absolute levels of
S100b (p < 0.05), NSE and tau
protein increased in the last stage
of disease. High levels of tau
protein, NSE and S100b were
associated with shorter survival
times (p < 0.01). Sixty-six sCJD
patients underwent repeated LP.
These sCJD patients were younger,
had longer disease durations and
were more frequently MV at
codon 129 (p < 0.001) than the
whole group. 14-3-3 sensitivity
increased from 64% to 82% in the
second LP (p = 0.025) and 88%
sCJD patients had at least one
and absolute levels of CJD
markers increased with disease
progression and were modulated
by the codon 129 genotype. Early
negative results should be inter-preted with caution, especially in
young patients or those who are
MV at codon 129.
disease; prion; 14-3-3; tau; S100b; NSE; biological markers; repeated lumbar punctures; prognosis
To better characterize the value of cerebrospinal fluid (CSF) proteins as diagnostic markers in a clinical population of subacute encephalopathy patients with relatively low prevalence of sporadic Creutzfeldt-Jakob disease (sCJD), we studied the diagnostic accuracies of several such markers (14-3-3, tau and S100B) in 1000 prospectively and sequentially recruited Canadian patients with clinically suspected sCJD.
The study included 127 patients with autopsy-confirmed sCJD (prevalence = 12.7%) and 873 with probable non-CJD diagnoses. Standard statistical measures of diagnostic accuracy were employed, including sensitivity (Se), specificity (Sp), predictive values (PVs), likelihood ratios (LRs), and Receiver Operating Characteristic (ROC) analysis.
At optimal cutoff thresholds (empirically selected for 14-3-3, assayed by immunoblot; 976 pg/mL for tau and 2.5 ng/mL for S100B, both assayed by ELISA), Se and Sp respectively were 0.88 (95% CI, 0.81-0.93) and 0.72 (0.69-0.75) for 14-3-3; 0.91 (0.84-0.95) and 0.88 (0.85-0.90) for tau; and 0.87 (0.80-0.92) and 0.87 (0.84-0.89) for S100B. The observed differences in Sp between 14-3-3 and either of the other 2 markers were statistically significant. Positive LRs were 3.1 (2.8-3.6) for 14-3-3; 7.4 (6.9-7.8) for tau; and 6.6 (6.1-7.1) for S100B. Negative LRs were 0.16 (0.10-0.26) for 14-3-3; 0.10 (0.06-0.20) for tau; and 0.15 (0.09-0.20) for S100B. Estimates of areas under ROC curves were 0.947 (0.931-0.961) for tau and 0.908 (0.888-0.926) for S100B. Use of interval LRs (iLRs) significantly enhanced accuracy for patient subsets [e.g., 41/120 (34.2%) of tested sCJD patients displayed tau levels > 10,000 pg/mL, with an iLR of 56.4 (22.8-140.0)], as did combining tau and S100B [e.g., for tau > 976 pg/mL and S100B > 2.5 ng/mL, positive LR = 18.0 (12.9-25.0) and negative LR = 0.02 (0.01-0.09)].
CSF 14-3-3, tau and S100B proteins are useful diagnostic markers of sCJD even in a low-prevalence clinical population. CSF tau showed better overall diagnostic accuracy than 14-3-3 or S100B. Reporting of quantitative assay results and combining tau with S100B could enhance case definitions used in diagnosis and surveillance of sCJD.
The gamma-isoform of the 14-3-3 protein (14-3-3 gamma) is expressed in neurons, and could be a specific marker for neuronal damage. This protein has been reported as a detectable biomarker, especially in the cerebrospinal fluid (CSF) of Creutzfeldt-Jakob disease (CJD) patients by Western blotting (WB) or enzyme-linked immunosorbent assays (ELISAs). Western blotting for 14-3-3 gamma is not sensitive, and the reported data are conflicting among publications. An ELISA specific for 14-3-3 gamma is not available.
CJD patients (n = 114 sporadic CJD patients, 7 genetic CJD, and 3 iatrogenic CJD) and 99 patients with other neurodegenerative diseases were examined in this study. The CSF samples obtained were analyzed by Western blotting for 14-3-3 gamma, and by ELISA for total tau protein. We evaluated the sensitivity and specificity of the newly developed sandwich ELISA for 14-3-3 gamma.
The cut-off value of the 14-3-3 gamma ELISA was > 1, 683 AU/ml; and sensitivity was 95.2%, with 72.7% specificity. This specificity was the same for the total tau protein ELISA. Seven CJD cases were negative by WB but positive using the 14-3-3 gamma ELISA, indicating that the ELISA is more sensitive. All 21 cases of early stage CJD could be diagnosed using a combination of the 14-3-3γ ELISA and diffusion weighted MR imaging (DWI-MRI).
The 14-3-3 gamma ELISA was more sensitive than conventional WB, and was useful for laboratory diagnosis of CJD, similar to the ELISA for the tau protein. Using DWI-MRI and these ELISA tests on CSF, diagnosis of CJD will be possible even at early stages of the disease.
CJD; CSF; ELISA; prion disease; 14-3-3 protein; tau protein
Human prion diseases, such as Creutzfeldt-Jakob disease (CJD), are neurodegenerative and fatal. Sporadic CJD (sCJD) can be transmitted between humans through medical procedures involving highly infected organs, such as the central nervous system. However, in variant CJD (vCJD), which is due to human contamination with the bovine spongiform encephalopathy (BSE) agent, lymphoreticular tissue also harbors the transmissible spongiform encephalopathy-associated prion protein (PrPTSE), which poses a particularly acute risk for iatrogenic transmission. Two blood transfusion-related cases are already documented. In addition, the recent observation of PrPTSE in spleen and muscle in sCJD raised the possibility that peripheral PrPTSE is not limited to vCJD cases. We aimed to clarify the peripheral pathogenesis of human TSEs by using a nonhuman primate model which mimics human diseases. A highly sensitive enzyme-linked immunosorbent assay was adapted to the detection of extraneural PrPTSE. We show that affected organs can be divided into two groups. The first is peripheral organs accumulating large amounts of PrPTSE, which represent a high risk of iatrogenic transmission. This category comprises only lymphoreticular organs in the vCJD/BSE model. The second is organs with small amounts of PrPTSE associated with nervous structures. These are the muscles, adrenal glands, and enteric nervous system in the sporadic, iatrogenic, and variant CJD models. In contrast to the first set of organs, this low level of tissue contamination is not strain restricted and seems to be linked to secondary centrifugal spread of the agent through nerves. It might represent a risk for iatrogenic transmission, formerly underestimated despite previous reports of low rates of transmission from peripheral organs of humans to nonhuman primates (5, 10). This study provides an additional experimental basis for the classification of human organs into different risk categories and a rational re-evaluation of current risk management measures.
Prion diseases or transmissible spongiform encephalopathies (TSEs) are infectious neurodegenerative disorders leading to death. These include Cresutzfeldt-Jakob disease (CJD), familial, sporadic and variant CJD and kuru in humans; and animal TSEs include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) of mule deer and elk, and transmissible mink encephalopathy. All these TSEs share common pathological features such as accumulation of mis-folded prion proteins in the central nervous system leading to cellular dysfunction and cell death. It is important to characterize the molecular pathways and events leading to prion induced neurodegeneration. Here we discuss the impact of the functional genomics approaches including microarrays, subtractive hybridization and microRNA profiling in elucidating transcriptional cascades at different stages of disease. Many of these transcriptional changes have been observed in multiple neurodegenerative diseases which may aid in identification of biomarkers for disease. A comprehensive characterization of expression profiles implicated in neurodegenerative disorders will undoubtedly advance our understanding on neuropathology and dysfunction during prion disease and other neurodegenerative disorders. We also present an outlook on the future work which may focus on analysis of structural genetic variation, genome and transcriptome sequencing using next generation sequencing with an integrated approach on animal and human TSE related studies.
Prion; TSEs; gene expression; functional candiate genes; PRNP; microarray.
Transmissible spongiform encephalopathy (TSE) or prion diseases are fatal rare neurodegenerative disorders affecting man and animals and caused by a transmissible infectious agent. TSE diseases are characterized by spongiform brain lesions with neuronal loss and the abnormal deposition in the CNS, and to less extent in other tissues, of an insoluble and protease resistant form of the cellular prion protein (PrPC), named PrPTSE. In man, TSE diseases affect usually people over 60 years of age with no evident disease-associated risk factors. In some cases, however, TSE diseases are unequivocally linked to infectious episodes related to the use of prion-contaminated medicines, medical devices, or meat products as in the variant Creutzfeldt-Jakob disease (CJD). Clinical signs occur months or years after infection, and during this silent period PrPTSE, the only reliable marker of infection, is not easily measurable in blood or other accessible tissues or body fluids causing public health concerns. To overcome the limit of PrPTSE detection, several highly sensitive assays have been developed, but attempts to apply these techniques to blood of infected hosts have been unsuccessful or not yet validated. An update on the latest advances for the detection of misfolded prion protein in body fluids is provided.
Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) of cattle and was first detected in 1986 in the United Kingdom. It is the most likely cause of variant Creutzfeldt-Jakob disease (CJD) in humans. The origin of BSE remains an enigma. Here we report an H-type BSE case associated with the novel mutation E211K within the prion protein gene (Prnp). Sequence analysis revealed that the animal with H-type BSE was heterozygous at Prnp nucleotides 631 through 633. An identical pathogenic mutation at the homologous codon position (E200K) in the human Prnp has been described as the most common cause of genetic CJD. This finding represents the first report of a confirmed case of BSE with a potential pathogenic mutation within the bovine Prnp gene. A recent epidemiological study revealed that the K211 allele was not detected in 6062 cattle from commercial beef processing plants and 42 cattle breeds, indicating an extremely low prevalence of the E211K variant (less than 1 in 2000) in cattle.
Bovine spongiform encephalopathy (BSE or Mad Cow Disease), a transmissible spongiform encephalopathy (TSE) or prion disease of cattle, was first discovered in the United Kingdom in 1986. BSE is most likely the cause of a human prion disease known as variant Creutzfeldt Jakob Disease (vCJD). In this study, we identified a novel mutation in the bovine prion protein gene (Prnp), called E211K, of a confirmed BSE positive cow from Alabama, United States of America. This mutation is identical to the E200K pathogenic mutation found in humans with a genetic form of CJD. This finding represents the first report of a confirmed case of BSE with a potential pathogenic mutation within the bovine Prnp gene. We hypothesize that the bovine Prnp E211K mutation most likely has caused BSE in “the approximately 10-year-old cow” carrying the E221K mutation.
Squirrel monkeys were experimentally infected with the classical bovine spongiform encephalopathy (BSE) agent. Two to four years later, six of the monkeys developed alterations in interactive behavior, cognition and other neurological signs typical of transmissible spongiform encephalopathy (TSE). At necropsy, all brains showed pathological changes similar to those described in variant Creutzfeldt-Jakob disease (vCJD) of humans, except that the squirrel monkey brains contained no PrP amyloid plaques typical of that disease. Constant neuropathological features included spongiform degeneration, gliosis, deposition of abnormal prion protein (PrPTSE) and many deposits of abnormally phosphorylated tau protein (p-Tau) in several areas of the cerebrum and cerebellum. Western blots showed large amounts of proteinase-K-resistant prion protein in the central nervous system. The striking absence of PrP plaques (prominent in brains of cynomolgus macaques with both experimental BSE and vCJD and in patients with vCJD) reinforces the conclusion that the host plays a major role in determining the neuropathology of TSEs. Results of this study suggest that p-Tau, found in the brains of all BSE-infected monkeys, might play a role in the pathogenesis of TSEs. Whether p-Tau contributes to development of disease or appears as a secondary change late in the course of illness remains to be determined.
bovine spongiform encephalopathy; prion disease; prion protein; tau protein
The Transmissible Spongiform Encephalopathies (TSEs) are a group of rare neurodegenerative diseases, which can be transmitted between members of the same species and possibly across different species. The link between the emergence of Bovine Spongiform Encephalopathy (BSE) and the new variant form of Creutzfedlt Jakob Disease (vCJD) has been the cause of much public concern. vCJD is the most widely known of the human TSEs but by no means the most common; inherited and sporadic forms are much more prevalent. The agent responsible for these diseases is a conformationally altered form of a normal cell surface glycoprotein, called the prion protein (PrP). The normal isoform must be present for the disease to progress, and disease incubation time decreases with increased PrP expression. There is still no cure for any of these diseases but recent advances in the understanding of how prion protein expression is regulated at the genetic level, and of exogenous factors modulating expression levels, may provide new insights into potential therapeutic targets for disease management by down regulation of cellular PrP levels.
prion; prnp; promoter; TSE
The definitive diagnosis of genetic prion diseases (gPrD) requires pathological confirmation. To date, diagnosis has relied upon the finding of the biomarkers 14-3-3 protein and total tau (t-tau) protein in the cerebrospinal fluid (CSF), but many researchers have reported that these markers are not sufficiently elevated in gPrD, especially in Gerstmann-Sträussler-Scheinker syndrome (GSS). We recently developed a new in vitro amplification technology, designated “real-time quaking-induced conversion (RT-QUIC)”, to detect the abnormal form of prion protein in CSF from sporadic Creutzfeldt-Jakob disease (sCJD) patients. In the present study, we aimed to investigate the presence of biomarkers and evaluate RT-QUIC assay in patients with gPrD, as the utility of RT-QUIC as a diagnostic tool in gPrD has yet to be determined.
56 CSF samples were obtained from gPrD patients, including 20 cases of GSS with P102L mutation, 12 cases of fatal familial insomnia (FFI; D178N), and 24 cases of genetic CJD (gCJD), comprising 22 cases with E200K mutation and 2 with V203I mutation. We subjected all CSF samples to RT-QUIC assay, analyzed 14-3-3 protein by Western blotting, and measured t-tau protein using an ELISA kit. The detection sensitivities of RT-QUIC were as follows: GSS (78%), FFI (100%), gCJD E200K (87%), and gCJD V203I (100%). On the other hand the detection sensitivities of biomarkers were considerably lower: GSS (11%), FFI (0%), gCJD E200K (73%), and gCJD V203I (67%). Thus, RT-QUIC had a much higher detection sensitivity compared with testing for biomarkers, especially in patients with GSS and FFI.
RT-QUIC assay is more sensitive than testing for biomarkers in gPrD patients. RT-QUIC method would thus be useful as a diagnostic tool when the patient or the patient's family does not agree to genetic testing, or to confirm the diagnosis in the presence of a positive result for genetic testing.
Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative disorders that can be transmitted by natural infection or inoculation. TSEs include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans. The emergence of a variant form of CJD (vCJD), which has been associated with BSE, produced strong pressure to search for effective treatments with new drugs. Up to now, however, TSEs have proved incurable, although many efforts have been made both in vitro and in vivo to search for potent therapeutic and prophylactic compounds. For this purpose, we analyzed a compound library consisting of 10,000 compounds with a cell-based high-throughput screening assay dealing with scrapie-infected scrapie mouse brain and ScN2A cells and identified a new class of inhibitors consisting of 3,5-diphenylpyrazole (DPP) derivatives. The most effective DPP derivative showed half-maximal inhibition of PrPSc formation at concentrations (IC50) of 0.6 and 1.2 μM, respectively. This compound was subsequently subjected to a number of animal experiments using scrapie-infected wild-type C57BL/6 and transgenic Tga20 mice. The DPP derivative induced a significant increase of incubation time both in therapeutic and prophylactic experiments. The onset of the prion disease was delayed by 37 days after intraperitoneal and 42 days after oral application, respectively. In summary, we demonstrate a high in vitro efficiency of DPP derivatives against prion infections that was substantiated in vivo for one of these compounds. These results indicate that the novel class of DPP compounds should comprise excellent candidates for future therapeutic studies.
Sporadic Creutzfeldt-Jakob-disease (sCJD) is a fatal neurodegenerative condition that escapes detection until autopsy. Recently, brain iron dyshomeostasis accompanied by increased transferrin (Tf) was reported in sCJD cases. The consequence of this abnormality on cerebrospinal-fluid (CSF) levels of Tf is uncertain. We evaluated the accuracy of CSF Tf, a ‘new’ biomarker, as a pre-mortem diagnostic test for sCJD when used alone or in combination with the ‘current’ biomarker total-tau (T-tau). Levels of total-Tf (T-Tf), isoforms of Tf (Tf-1 and Tf-β2), and iron saturation of Tf were quantified in CSF collected 0.3–36 months before death (duration) from 99 autopsy confirmed sCJD (CJD+) and 75 confirmed cases of dementia of non-CJD origin (CJD-). Diagnostic accuracy was estimated by non-parametric tests, logistic regression, and receiver operating characteristic (ROC) analysis. Area under the ROC curve (AUC), sensitivity, specificity, positive and negative predictive values (PV), and likelihood ratios (LR) of each biomarker and biomarker combination were calculated. We report that relative to CJD-, CJD+ cases had lower median CSF T-Tf (125,7093 vs. 217,7893) and higher T-tau (11530 vs. 1266) values. AUC was 0.90 (95% confidence interval (CI), 0.85–0.94) for T-Tf, and 0.93 (95% CI, 0.89–0.97) for T-Tf combined with T-tau. With cut-offs defined to achieve a sensitivity of ∼85%, T-Tf identified CJD+ cases with a specificity of 71.6% (95% CI, 59.1–81.7), positive LR of 3.0 (95% CI, 2.1–4.5), negative LR of 0.2 (95% CI, 0.1–0.3), and accuracy of 80.1%. The effect of patient age and duration was insignificant. T-Tf combined with T-tau identified CJD+ with improved specificity of 87.5% (95%CI, 76.3–94.1), positive LR of 6.8 (95% CI, 3.5–13.1), negative LR of 0.2 (95% CI, 0.1–0.3), positive-PV of 91.0%, negative-PV of 80.0%, and accuracy of 86.2%. Thus, CSF T-Tf, a new biomarker, when combined with the current biomarker T-tau, is a reliable pre-mortem diagnostic test for sCJD.
Candidate cell substrates neither accumulated abnormal prion protein nor propagated infectivity.
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 (PrPTSE) and TSE infectivity by assay in rodents and nonhuman primates. No PrPTSE 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.
prion; prion protein; BSE; bovine spongiform encephalopathy; Creutzfeldt-Jakob disease; CJD; vaccines; TSE; transmissible spongiform encepthalopathy
Transmissible spongiform encephalopathies (TSE) are neurodegenerative diseases caused by an infectious agent with viral properties. Host prion protein (PrP), a marker of late stage TSE pathology, is linked to a similar protein called Shadoo (Sho). Sho is reduced in mice infected with the RML scrapie agent, but has not been investigated in other TSEs. Although PrP is required for infection by TSE agents, it is not known if Sho is similarly required. Presumably Sho protects cells from toxic effects of misfolded PrP. We compared Sho and PrP changes after infection by very distinct TSE agents including sporadic CJD, Asiatic CJD, New Guinea kuru, vCJD (the UK epidemic bovine agent) and 22L sheep scrapie, all passaged in standard mice. We found that Sho reductions were agent-specific. Variable Sho reductions in standard mice could be partly explained by agent-specific differences in regional neuropathology. However, Sho did not follow PrP misfolding in any quantitative or consistent way. Tga20 mice with high murine PrP levels revealed additional agent-specific differences. Sho was unaffected by Asiatic CJD yet was markedly reduced by the kuru agent in Tga20 mice; in standard mice both agents induced the same Sho reductions. Analyses of neural GT1 cells demonstrated that Sho was not essential for TSE infections. Furthermore, because all infected GT1 cells appeared as healthy as uninfected controls, Sho was not needed to protect infected cells from their “toxic” burden of abundant abnormal PrP and intracellular amyloid.
CJD; kuru; BSE; neurodegeneration; prion protein; tissue culture; toxicity; amyloid
Objective: To investigate various cerebrospinal fluid (CSF) markers that could assist in the clinical diagnosis of Creutzfeldt–Jakob disease (CJD).
Methods: CSF samples were analysed for the presence of 14-3-3 protein, microtubule associated protein tau, and ß amyloid in 250 patients with possible CJD. Densitometric analysis was used to quantify the level of 14-3-3 in all patients.
Results: Analysis of the clinical data showed that cerebellar signs or myoclonus combined with progressive dementia were the main features leading to a clinical suspicion of CJD. While 14-3-3 detection had a sensitivity of 100% and a specificity of 92%, tau determination using a threshold of 1300 pg/ml had a sensitivity of 87% and a specificity of 97%. If the protocol for the analysis of 14-3-3 was modified (using densitometric analysis) a higher specificity (97%) could be obtained, but with a lower sensitivity (96%). Maximum sensitivity, specificity, and positive predictive value were obtained with a combination of 14-3-3 and ß amyloid determinations. The concentrations of 14-3-3 and tau in the CSF were reduced in CJD patients with a long duration of disease (more than one year; p < 0.05). The concentrations of 14-3-3 or tau were lowest at the onset or at the end stage of the disease, while the ß amyloid concentration remained low throughout the course of the disease.
Conclusions: Both 14-3-3 and tau protein are sensitive and specific biomarkers for CJD. The combination of 14-3-3 and ß amyloid analysis resulted in the maximum sensitivity, specificity, and positive predictive value. When these biomarkers are used in the diagnosis of CJD, the phase of the disease in which the CSF sample was obtained should be taken into account. Disease duration, dependent on the PrP genotype, also has a significant influence on the level of 14-3-3 and tau in the CSF.
We recently developed a new in vitro amplification technology, designated “real-time quaking-induced conversion (RT-QUIC),” for detection of the abnormal form of prion protein (PrPSc) in easily accessible specimens such as cerebrospinal fluid (CSF). After assessment of more than 200 CSF specimens from Japanese and Australian patients, we found no instance of a false positive, and more than 80% accuracy for the correct diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD). Furthermore, the RT-QUIC can be applied to other prion diseases, including scrapie, chronic wasting disease (CWD) and bovine spongiform encephalopathy (BSE), and is able to quantify prion seeding activity when combined with an end-point dilution of samples. These results indicate that the RT-QUIC, with its high sensitivity and specificity, will be of great use as an early, rapid and specific assay for prion diseases.
RT-QUIC; real-time quaking-induced conversion; prion; CJD; Creutzfeldt-Jakob disease; CSF; cerebrospinal fluid
Transmissible spongiform encephalopathies (TSEs), a group of neurodegenerative diseases, are thought to be caused by an abnormal isoform of a naturally occurring protein known as cellular prion protein, PrPC. The abnormal form of prion protein, PrPSc accumulates in the brain of affected individuals. Both isoforms are encoded by the same prion protein gene (PRNP), and the structural changes occur post-translationally. Certain mutations in the PRNP gene result in genetic TSEs or increased susceptibility to TSEs.
A 70 year old woman was admitted to the hospital with severe confusion and inability to walk. Relatives recognized memory loss, gait and behavioral disturbances over a six month period prior to hospitalization. Neurological examination revealed Creutzfeldt-Jakob disease (CJD) related symptoms such as incontinence, Babinski sign and myoclonus. EEG showed periodic sharp waves typical of sporadic CJD and cerebrospinal fluid analysis (CSF) was positive for the presence of the 14-3-3-protein. As the disease progressed the patient developed akinetic mutism and died in the tenth month after onset of the disease symptoms. Unfortunately, no autopsy material was available. PRNP sequencing showed the occurrence of a point mutation on one allele at codon 193, which is altered from ACC, coding for a threonine, to ATC, encoding an isoleucine (T193I).
Here we report a novel mutation of the PRNP gene found in an elderly female patient resulting in heterozygosity for isoleucine and threonine at codon 193, in which normally homozygosity for threonine is expected (T193). The patient presented typical clinical symptoms of CJD. EEG findings and the presence of the 14-3-3 protein in the CSF, contributed to CJD diagnosis, allowing the classification of this case as a probable CJD according to the World Health Organization (WHO) accepted criteria.
Recently, pathological prion protein PrPSc, the putative key constituent of infectious agents causing transmissible spongiform encephalopathies (TSEs), was found in muscles of rodents experimentally infected with scrapie and in patients with Creutzfeldt-Jakob disease (CJD). For the assessment of risk scenarios originating from these findings (e.g., alimentary transmission of pathogens associated with bovine spongiform encephalopathy [BSE] and chronic wasting disease [CWD] via tainted beef and game or iatrogenic dissemination of CJD agent through contaminated surgical instruments) more detailed information about the time course of PrPSc accumulation in muscles at preclinical and clinical stages of incubation is needed. Here we show that PrPSc in muscles of hamsters fed with scrapie can be detected prior to the onset of clinical symptoms, but that the bulk of PrPSc was deposited late in clinical disease. Additionally, regarding the question of how muscles become invaded, we report on the intramuscular location of PrPSc and substantial indications for centrifugal spread of infection from spinal motor neurons to myofibers. Our findings in a well-established animal model for TSEs contribute to a better assessment of the risks for public health emanating from “Prions in skeletal muscle” and provide new insights into the pathophysiological spread of TSE agents through the body.
Infections with variant Creutzfeldt-Jakob disease (vCJD) have almost exclusively occurred in young patients, but the reasons for this age distribution are uncertain. Our data suggest that the pathogenesis of many peripherally acquired transmissible spongiform encephalopathy (TSE) agents is less efficient in aged individuals. Four vCJD cases linked to transfusion of vCJD-contaminated blood or blood products have been described. Three cases occurred in elderly patients, implying that intravenous exposure is more efficient in aged individuals than other peripheral routes. To test this hypothesis, young (6 to 8 weeks old) and aged (600 days old) mice were injected intravenously with a TSE agent. In aged and young mice, the intravenous route was more efficient than other peripheral routes of TSE agent exposure. However, in aged mice, disease pathogenesis was significantly reduced. Although most aged mice failed to develop clinical disease during their life spans, many showed histopathological signs of TSE disease in their brains. Thus, the effects of age on intravenous TSE pathogenesis may lead to significant levels of subclinical disease in the population. After peripheral exposure, many TSE agents accumulate upon follicular dendritic cells (FDCs) in lymphoid tissues before they infect the brain. In aged spleens, PrPC expression and TSE agent accumulation upon FDCs were reduced. Furthermore, the splenic marginal zone microarchitecture was substantially disturbed, adversely affecting the delivery of immune complexes to FDCs. This study is the first to suggest that the effects of aging on the microarchitecture and the function of the splenic marginal zone significantly influence the pathogenesis of an important pathogen.
Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder in humans included in the group of Transmissible Spongiform Encephalopathies or prion diseases. The vast majority of sCJD cases are molecularly classified according to the abnormal prion protein (PrPSc) conformations along with polymorphism of codon 129 of the PRNP gene. Recently, a novel human disease, termed "protease-sensitive prionopathy", has been described. This disease shows a distinct clinical and neuropathological phenotype and it is associated to an abnormal prion protein more sensitive to protease digestion.
We report the case of a 75-year-old-man who developed a clinical course and presented pathologic lesions compatible with sporadic Creutzfeldt-Jakob disease, and biochemical findings reminiscent of "protease-sensitive prionopathy". Neuropathological examinations revealed spongiform change mainly affecting the cerebral cortex, putamen/globus pallidus and thalamus, accompanied by mild astrocytosis and microgliosis, with slight involvement of the cerebellum. Confluent vacuoles were absent. Diffuse synaptic PrP deposits in these regions were largely removed following proteinase treatment. PrP deposition, as revealed with 3F4 and 1E4 antibodies, was markedly sensitive to pre-treatment with proteinase K. Molecular analysis of PrPSc showed an abnormal prion protein more sensitive to proteinase K digestion, with a five-band pattern of 28, 24, 21, 19, and 16 kDa, and three aglycosylated isoforms of 19, 16 and 6 kDa. This PrPSc was estimated to be 80% susceptible to digestion while the pathogenic prion protein associated with classical forms of sporadic Creutzfeldt-Jakob disease were only 2% (type VV2) and 23% (type MM1) susceptible. No mutations in the PRNP gene were found and genotype for codon 129 was heterozygous methionine/valine.
A novel form of human disease with abnormal prion protein sensitive to protease and MV at codon 129 was described. Although clinical signs were compatible with sporadic Creutzfeldt-Jakob disease, the molecular subtype with the abnormal prion protein isoforms showing enhanced protease sensitivity was reminiscent of the "protease-sensitive prionopathy". It remains to be established whether the differences found between the latter and this case are due to the polymorphism at codon 129. Different degrees of proteinase K susceptibility were easily determined with the chemical polymer detection system which could help to detect proteinase-susceptible pathologic prion protein in diseases other than the classical ones.
Transmissible spongiform encephalopathy diseases are untreatable, uniformly fatal degenerative syndromes of the central nervous system that can be transmitted both within as well as between species. The bovine spongiform encephalopathy (BSE) epidemic and the emergence of a new human variant of Creutzfeldt-Jakob disease (vCJD), have profoundly influenced beef production processes as well as blood donation and surgical procedures. Simple, robust and cost effective diagnostic screening and surveillance tools are needed for both the preclinical and clinical stages of TSE disease in order to minimize both the economic costs and zoonotic risk of BSE and to further reduce the risk of secondary vCJD.
Urine is well suited as the matrix for an ante-mortem test for TSE diseases because it would permit non-invasive and repeated sampling. In this study urine samples collected from BSE infected and age matched control cattle were screened for the presence of individual proteins that exhibited disease specific changes in abundance in response to BSE infection that might form the basis of such an ante-mortem test.
Two-dimensional differential gel electrophoresis (2D-DIGE) was used to identify proteins exhibiting differential abundance in two sets of cattle. The known set consisted of BSE infected steers and age matched controls throughout the course of the disease. The blinded unknown set was composed of BSE infected and control samples of both genders, a wide range of ages and two different breeds. Multivariate analyses of individual protein abundance data generated classifiers comprised of the proteins best able to discriminate between the samples based on disease state, breed, age and gender.
Despite the presence of confounding factors, the disease specific changes in abundance exhibited by a panel of urine proteins permitted the creation of classifiers able to discriminate between control and infected cattle with a high degree of accuracy.
The diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) is based on typical clinical findings and is supported by a positive 14-3-3 Western blot of cerebrospinal fluid. However, it is not clear whether 14-3-3 indicates general neuronal damage or is of pathophysiological relevance in CJD. The fact that the 14-3-3 isoform spectrum in cerebrospinal fluid does not correspond to that found in the brain points to a regulated process. To investigate a possible role of 14-3-3 proteins in transmissible spongiform diseases, we generated a 14-3-3γ-deficient mutant mouse line by using a classical knockout strategy. The anatomy and cage behavior of the mutant mice were normal. Western blot analyses of brain homogenates revealed no changes in the protein expression of other 14-3-3 isoforms (ɛ, β, ζ, and η). Proteomic analyses of mouse brains by two-dimensional differential gel electrophoresis showed that several proteins, including growth hormone, 1-Cys peroxiredoxin, CCT-zeta, glucose-6-phosphate isomerase, GRP170 precursor, and α-SNAP, were differentially expressed. Mutant and wild-type mice were inoculated either intracerebrally or intraperitoneally with the Rocky Mountain Laboratory strain of scrapie, but no differences were detected in the postinoculation survival rates. These results indicate that 14-3-3γ is unlikely to play a causal role in CJD and related diseases.
The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimer's disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent naïve sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry.