Early preclinical detection of prions in the skin of prion-infected animals.

A definitive pre-mortem diagnosis of prion disease depends on brain biopsy for prion detection currently and no validated alternative preclinical diagnostic tests have been reported to date. To determine the feasibility of using skin for preclinical diagnosis, here we report ultrasensitive serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays of skin samples from hamsters and humanized transgenic mice (Tg40h) at different time points after intracerebral inoculation with 263K and sCJDMM1 prions, respectively. sPMCA detects skin PrPSc as early as 2 weeks post inoculation (wpi) in hamsters and 4 wpi in Tg40h mice; RT-QuIC assay reveals earliest skin prion-seeding activity at 3 wpi in hamsters and 20 wpi in Tg40h mice. Unlike 263K-inoculated animals, mock-inoculated animals show detectable skin/brain PrPSc only after long cohabitation periods with scrapie-infected animals. Our study provides the proof-of-concept evidence that skin prions could be a biomarker for preclinical diagnosis of prion disease.

Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment.

Prion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrP(Sc)). PrP(Sc) is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrP(C)) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrP(Sc) (PrP(Sc)-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrP(Sc) (PrP(Sc)-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrP(Sc) can be directly detected from cells persistently infected with prions using anti-PrP monoclonal antibody (mAb) 132 that recognizes epitope consisting of mouse PrP amino acids 119-127. The novel cell-based ELISA could distinguish prion-infected cells from prion-uninfected cells without cell lysis and PK treatment. MAb 132 could detect both PrP(Sc)-sen and PrP(Sc)-res even if all PrP(Sc) molecules were not detected. The analytical dynamic range for PrP(Sc) detection was approximately 1 log. The coefficient of variation and signal-to-background ratio were 7%-11% and 2.5-3.3, respectively, demonstrating the reproducibility of this assay. The addition of a cytotoxicity assay immediately before PrP(Sc) detection did not affect the following PrP(Sc) detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrP(Sc) detection were completed in the same plate. The simplicity and non-requirement for cell lysis or PK treatment are advantages for the high throughput screening of anti-prion compounds.

Scrapie diagnosis in a goat and four Santa Inês sheep from the same herd in Brazil / Diagnóstico de scrapie em um caprino e quatro ovinos Santa Inês de um mesmo rebanho no Brasil

Scrapie is a fatal and progressive transmissible spongiform encephalopathy (TSE) of natural occurrence in sheep and goats. The suspicion of scrapie may be based on clinical signs; however, the detection of pathological features of the prionic protein (PrP) in target tissues is necessary to diagnose the disease. The presence of an abnormal protein form (PrPSc) in lymphoreticular and nervous tissues is an important characteristic in diagnosis. This paper reports a case of scrapie in a flock of 55 Suffolk crossbred sheep, 19 Santa Inês sheep and 21 goats in the Mato Grosso state, midwestern Brazil. The animals were euthanized after the confirmation of a scrapie case with clinical signs in a Suffolk sheep in the same farm...

Scrapie é uma encefalopatia espongiforme transmissível (EET) progressiva e fatal de ocorrência natural em ovinos e caprinos. A suspeita de scrapie é baseada nos sinais clínicos, porém a manifestação patológica da proteína priônica (PrP) nos tecidos-alvo é necessária para a confirmação da doença. A presença de uma forma anormal da proteína (PrPSc) em tecido linforreticular e tecido nervoso constitui uma característica importante para o diagnóstico. Este trabalho é o relato de um foco de scrapie ocorrido em rebanho com 55 ovinos mistos Suffolk, 21 caprinos e 19 ovinos Santa Inês, na região Centro-Oeste do Brasil. Os animais foram eutanasiados após a confirmação de um caso de scrapie com sinais clínicos em um ovino Suffolk nessa propriedade...

Transcriptome analysis of CNS immediately before and after the detection of PrP(Sc) in SSBP/1 sheep scrapie.

Sheep scrapie is a transmissible spongiform encephalopathy (TSE), progressive and fatal neurodegenerative diseases of the central nervous system (CNS) linked to the accumulation of misfolded prion protein, PrP(Sc). New Zealand Cheviot sheep, homozygous for the VRQ genotype of the PRNP gene are most susceptible with an incubation period of 193 days with SSBP/1 scrapie. However, the earliest time point that PrP(Sc) can be detected in the CNS is 125 days (D125). The aim of this study was to quantify changes to the transcriptome of the thalamus and obex (medulla) at times immediately before (D75) and after (D125) PrP(Sc) was detected. Affymetrix gene arrays were used to quantify gene expression in the thalamus and Illumina DGE-tag profiling for obex. Ingenuity Pathway Analysis was used to help describe the biological processes of scrapie pathology. Neurological disease and Cancer were common Bio Functions in each tissue at D75; inflammation and cell death were major processes at D125. Several neurological receptors were significantly increased at D75 (e.g. CHRNA6, GRM1, HCN2), which might be clues to the molecular basis of psychiatric changes associated with TSEs. No genes were significantly differentially expressed at both D75 and D125 and there was no progression of events from earlier to later time points. This implies that there is no simple linear progression of pathological or molecular events. There seems to be a step-change between D75 and D125, correlating with the detection of PrP(Sc), resulting in the involvement of different pathological processes in later TSE disease.

Anti-prion activity of an RNA aptamer and its structural basis.

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrP(C)) of PrP into an abnormal form (PrP(Sc)) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrP(C) is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrP(Sc) level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrP(C), resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to prion diseases.

Heparin enhances the cell-protein misfolding cyclic amplification efficiency of variant Creutzfeldt-Jakob disease.

Highly sensitive in vitro screening tests are required to prevent the iatrogenic spread of variant Creutzfeldt-Jakob disease (vCJD). Protein misfolding cyclic amplification (PMCA) is a candidate for such a test, but the sensitivity of this method is insufficient. Polyanions were reported to enhance PMCA efficiency, but their effects on vCJD are unclear. We developed a cell-PMCA of vCJD, wherein cell lysate containing exogenously expressed human PrP was used as substrates, to investigate the effects of various sulfated polysaccharides on amplification efficiency. PrP(res) amounts after cell-PMCA were analyzed by western blotting. Heparin, dermatan sulfate, and dextran sulfate (average molecular weight [MW] 1400kDa) enhanced efficiency, but dextran sulfate (average MW 8kDa) and a heparin pentasaccharide analog had no effect. Pentosan polysulfate inhibited cell-PMCA reaction. The amplification efficiency of cell-PMCA of vCJD increased to >100-fold per round with heparin. The enhancing effects of heparin on cell-PMCA were seed dependent: it was high for vCJD, low for sporadic Creutzfeldt-Jakob disease, and low to negligible for hamster-adapted scrapie-derived 263K. In multi-round PMCA, signals were detected at earlier rounds with heparin than without heparin, and PrP(Sc) in 10(-10) diluted vCJD brain was detected by the sixth round. Heparin-assisted cell-PMCA of vCJD represents a significant step toward detecting very minute amounts of PrP(Sc) in the body fluids of asymptomatic vCJD patients.

Disease-associated prion protein in the dental tissue of mice infected with scrapie.

Transmissible spongiform encephalopathies (TSEs) induce fatal neurodegenerative diseases in man and animals. The present study demonstrates immunohistochemically the presence of disease-associated prion protein (PrP(Sc)) in the epithelial cell rests of Malassez (ERM) of mice experimentally infected with ME7 scrapie by the intracerebral route. Mouse bioassay of scrapie-infected dental tissue revealed prolonged incubation periods, suggesting that there are relatively low amounts of infectious agent in dental tissue compared with the brain. These findings indicate that PrP(Sc) may spread from the brain to the ERM along the cranial nerves via the trigeminal ganglion that innervates the dental tissues. Dental tissue might therefore be a potential source of PrP(Sc) for horizontal transmission of TSEs.

Variant CJD infection in the spleen of a neurologically asymptomatic UK adult patient with haemophilia.

SUMMARY: All UK patients with bleeding disorders treated with any UK-sourced pooled factor concentrates between 1980 and 2001 have been informed that they may be at an increased risk of infection with variant Creutzfeldt-Jakob disease (vCJD). We describe a study to detect disease-associated, protease-resistant prion protein (PrP(res)) in 17 neurologically aymptomatic patients with haemophilia considered to be at increased risk of vCJD. Materials from 11 autopsy and seven biopsy cases were analysed for PrP(res). The tissues available from each case were variable, ranging from a single biopsy sample to a wide range of autopsy tissues. A single specimen from the spleen of one autopsy case gave a strong positive result on repeated testing for PrP(res) by Western blot analysis. This tissue came from a 73-year-old male patient with no history of neurological disease, who was heterozygous (methionine/valine) at codon 129 in the prion protein gene. He had received over 9000 units of factor VIII concentrate prepared from plasma pools known to include donations from a vCJD-infected donor, and some 400,000 units not known to include donations from vCJD-infected donors. He had also received 14 units of red blood cells and had undergone several surgical and invasive endoscopic procedures. Estimates of the relative risks of exposure through diet, surgery, endoscopy, blood transfusion and receipt of UK-sourced plasma products suggest that by far the most likely route of infection in this patient was receipt of UK plasma products.

Continuous quinacrine treatment results in the formation of drug-resistant prions.

Quinacrine is a potent antiprion compound in cell culture models of prion disease but has failed to show efficacy in animal bioassays and human clinical trials. Previous studies demonstrated that quinacrine inefficiently penetrates the blood-brain barrier (BBB), which could contribute to its lack of efficacy in vivo. As quinacrine is known to be a substrate for P-glycoprotein multi-drug resistance (MDR) transporters, we circumvented its poor BBB permeability by utilizing MDR(0/0) mice that are deficient in mdr1a and mdr1b genes. Mice treated with 40 mg/kg/day of quinacrine accumulated up to 100 microM of quinacrine in their brains without acute toxicity. PrP(Sc) levels in the brains of prion-inoculated MDR(0/0) mice diminished upon the initiation of quinacrine treatment. However, this reduction was transient and PrP(Sc) levels recovered despite the continuous administration of quinacrine. Treatment with quinacrine did not prolong the survival times of prion-inoculated, wild-type or MDR(0/0) mice compared to untreated mice. A similar phenomenon was observed in cultured differentiated prion-infected neuroblastoma cells: PrP(Sc) levels initially decreased after quinacrine treatment then rapidly recovered after 3 d of continuous treatment. Biochemical characterization of PrP(Sc) that persisted in the brains of quinacrine-treated mice had a lower conformational stability and different immunoaffinities compared to that found in the brains of untreated controls. These physical properties were not maintained upon passage in MDR(0/0) mice. From these data, we propose that quinacrine eliminates a specific subset of PrP(Sc) conformers, resulting in the survival of drug-resistant prion conformations. Transient accumulation of this drug-resistant prion population provides a possible explanation for the lack of in vivo efficacy of quinacrine and other antiprion drugs.

Non-invasive, ultra-sensitive, high-throughput assays to quantify rare biomarkers in the blood.

Many diseases are easier to treat and control when detected at an early stage of disease progression. Often, disease-related antigens or biomarkers are shed from the primary site and present in the blood. Unfortunately, there are very few tests capable of detecting these rare biomarkers in the blood. A blood test would be very useful to diagnose the disease earlier, monitor effectiveness of treatments, predict recurrence, and monitor recurrence. There is certainly a need to develop assays that are ultra-sensitive, non-invasive, and high-throughput. Here we describe several highly sensitive immunological assays we have developed to detect rare serum antigens. Initially we created an assay named immuno-detection amplified by T7 RNA polymerase (IDAT). To enhance the effectiveness and streamline the procedure, this assay was amended to the facile amplification system termed fluorescent amplification catalyzed by T7 polymerase technique (FACTT). These assays have been used to analyze the tumor antigen HER2 and the prion protein PrPSc. They can also be applied to other tumor markers or antigens from a variety of diseases such as cardiovascular disease, rheumatoid arthritis, Alzheimer's disease, Parkinson's disease, and hepatitis. These tests are not limited to testing only serum, but may also be applicable to detecting biomarkers in tissue, saliva, urine, cerebrospinal fluid, etc. Clearly, the FACTT-based technology represents an important step in the detection of rare molecules in fluids or tissues for a variety of diseases.

Disease-associated prion protein is not detectable in human systemic amyloid deposits.

Cerebral and cardiac amyloid deposits have been reported after scrapie infection in transgenic mice expressing variant prion protein (PrP(C)) lacking the glycophosphatidylinositol anchor. The amyloid fibril protein in the systemic amyloid deposits was not characterized, and there is no clinical or pathological association between prion diseases and systemic amyloidosis in humans. Nevertheless, in view of the potential clinical significance of these murine observations, we tested both human amyloidotic tissues and isolated amyloid fibrils for the presence of PrP(Sc), the prion protein conformation associated with transmissible spongiform encephalopathy (TSE). We also sequenced the complete prion protein gene, PRNP, in amyloidosis patients. No specific immunohistochemical staining for PrP(Sc) was obtained in the amyloidotic cardiac and other visceral tissues of patients with different types of systemic amyloidosis. No protease-resistant prion protein, PrP(res), was detectable by Western blotting of amyloid fibrils isolated from cardiac and other systemic amyloid deposits. Only the complete normal wild-type PRNP gene sequence was identified, including the usual distribution of codon 129 polymorphisms. These reassuringly negative results do not support the idea that there is any relationship of prions or TSE with human systemic amyloidosis, including cardiac amyloid deposition.

PrPc does not mediate internalization of PrPSc but is required at an early stage for de novo prion infection of Rov cells.

We have studied the interactions of exogenous prions with an epithelial cell line inducibly expressing PrPc protein and permissive to infection by a sheep scrapie agent. We demonstrate that abnormal PrP (PrPSc) and prion infectivity are efficiently internalized in Rov cells, whether or not PrPc is expressed. At odds with earlier studies implicating cellular heparan sulfates in PrPSc internalization, we failed to find any involvement of such molecules in Rov cells, indicating that prions can enter target cells by several routes. We further show that PrPSc taken up in the absence of PrPc was unable to promote efficient prion multiplication once PrPc expression was restored in the cells. This observation argues that interaction of PrPSc with PrPc has to occur early, in a specific subcellular compartment(s), and is consistent with the view that the first prion multiplication events may occur at the cell surface.

Development of a monoclonal antibody-based immunohistochemistry method for BSE surveillance in China.

Five hybridoma cell lines secreting anti-PrP antibodies were established from the fusion between mouse myeloma Sp2/0 and spleen cells from mice immunized with recombinant Chinese Luxi yellow cattle (Bos taurus, Luxi) PrP (24-234) or recombinant Chinese small-tailed Han sheep PrP (94-227). According to their Western blot reactivity, five monoclonal antibodies (mAbs) could be divided into two groups. Group A, mAbs 1H2, 4C6, and 4C11 recognized re-PrP, PrP(C), and PrP(Sc) from both bovine and sheep. Group B, mAbs 2H3 and 4H10 only recognized re-PrP and PrP(Sc) of sheep, and especially, these two mAbs could not recognize PrP(C) from both bovine and sheep. In immunohistochemistry (IHC) test, mAb 4C11 immunostained the PrP(Sc) accumulation in tissue sections from BSE cattle and Scrapie sheep, and compared with mAb 6H4, it had the same immunohistochemical pattern. An IHC method based on mAb 4C11 for the detection of BSE was established and had been applied for the long-term surveillance of BSE in China. From 2001 to 2004, 12,692 samples from the whole country had been tested and all had negative results.

Detection of PrP(CWD) in postmortem rectal lymphoid tissues in Rocky Mountain elk (Cervus elaphus nelsoni) infected with chronic wasting disease.

Preclinical diagnostic tests for transmissible spongiform encephalopathies have been described for mule deer (Odocoileus hemionus), using biopsy tissues of palatine tonsil, and for sheep, using lymphoid tissues from palatine tonsil, third eyelid, and rectal mucosa. The utility of examining the rectal mucosal lymphoid tissues to detect chronic wasting disease (CWD) was investigated in Rocky Mountain elk (Cervus elaphus nelsoni), a species for which there is not a live-animal diagnostic test. Postmortem rectal mucosal sections were examined from 308 elk from two privately owned herds that were depopulated. The results of the postmortem rectal mucosal sections were compared to immunohistochemical staining of the brainstem, retropharyngeal lymph nodes, and palatine tonsil. Seven elk were found positive using the brainstem (dorsal motor nucleus of the vagus nerve), retropharyngeal lymph nodes, and palatine tonsil. Six of these elk were also found positive using postmortem rectal mucosal sections. The remaining 301 elk in which CWD-associated abnormal isoform of the prion protein (PrP(CWD)) was not detected in the brainstem and cranial lymphoid tissues were also found to be free of PrP(CWD) when postmortem rectal mucosal sections were examined. The use of rectal mucosal lymphoid tissues may be suitable for a live-animal diagnostic test as part of an integrated management strategy to limit CWD in elk.

Prion-induced amyloid heart disease with high blood infectivity in transgenic mice.

We investigated extraneural manifestations in scrapie-infected transgenic mice expressing prion protein lacking the glycophosphatydylinositol membrane anchor. In the brain, blood, and heart, both abnormal protease-resistant prion protein (PrPres) and prion infectivity were readily detected by immunoblot and by inoculation into nontransgenic recipients. The titer of infectious scrapie in blood plasma exceeded 10(7) 50% infectious doses per milliliter. The hearts of these transgenic mice contained PrPres-positive amyloid deposits that led to myocardial stiffness and cardiac disease.

Clusterin expression in follicular dendritic cells associated with prion protein accumulation.

Peripheral accumulation of abnormal prion protein (PrP) in variant Creutzfeldt-Jakob disease and some animal models of transmissible spongiform encephalopathies (TSEs) may occur in the lymphoreticular system. Within the lymphoid tissues, abnormal PrP accumulation occurs on follicular dendritic cells (FDCs). Clusterin (apolipoprotein J) has been recognized as one of the molecules associated with PrP in TSEs, and clusterin expression is increased in the central nervous system where abnormal PrP deposition has occurred. We therefore examined peripheral clusterin expression in the context of PrP accumulation on FDCs in a range of human and experimental TSEs. PrP was detected immunohistochemically on tissue sections using a novel highly sensitive method involving detergent autoclaving pretreatment. A dendritic network pattern of clusterin immunoreactivity in lymphoid follicles was observed in association with the abnormal PrP on FDCs. The increased clusterin immunoreactivity appeared to correlate with the extent of PrP deposition, irrespective of the pathogen strains, host mouse strains or various immune modifications. The observed co-localization and correlative expression of these proteins suggested that clusterin might be directly associated with abnormal PrP. Indeed, clusterin immunoreactivity in association with PrP was retained after FDC depletion. Together these data suggest that clusterin may act as a chaperone-like molecule for PrP and play an important role in TSE pathogenesis.

Short-term study of the uptake of PrP(Sc) by the Peyer's patches in hamsters after oral exposure to scrapie.

The disease-associated prion protein (PrP(Sc)) has been detected in the ileal Peyer's patches of lambs as early as one week after oral exposure to scrapie. In hamsters, the earliest reported time of PrP(Sc) detection in the Peyer's patches after oral exposure to scrapie is 69 days post-infection. To evaluate the acute uptake of inoculum and to investigate whether the Peyer's patches constitute the primary site of entry for scrapie after oral exposure, hamsters were each exposed orally to 1 ml of a 10% brain homogenate from hamsters in the terminal stage of infection with the 263 K strain of the scrapie agent. PrP(Sc) was demonstrated in the Peyer's patches only a few days after exposure, i.e., much earlier than previously reported. This study supports the view that the Peyer's patches constitute at least one of the primary entry sites of PrP(Sc) after oral exposure to scrapie.

Analysis of prion strains by PrPSc profiling in sporadic Creutzfeldt-Jakob disease.

BACKGROUND: Prion diseases are a group of invariably fatal neurodegenerative disorders affecting humans and a wide range of mammals. An essential part of the infectious agent, termed the prion, is composed of an abnormal isoform (PrPSc) of a host-encoded normal cellular protein (PrPC). The conversion of PrPC to PrPSc is thought to play a crucial role in the development of prion diseases and leads to PrPSc deposition, mainly in the central nervous system. Sporadic Creutzfeldt-Jakob disease (sCJD), the most common form of human prion disease, presents with a marked clinical heterogeneity. This diversity is accompanied by a molecular signature which can be defined by histological, biochemical, and genetic means. The molecular classification of sCJD is an important tool to aid in the understanding of underlying disease mechanisms and the development of therapy protocols. Comparability of classifications is hampered by disparity of applied methods and inter-observer variability. METHODS AND FINDINGS: To overcome these difficulties, we developed a new quantification protocol for PrPSc by using internal standards on each Western blot, which allows for generation and direct comparison of individual PrPSc profiles. By studying PrPSc profiles and PrPSc type expression within nine defined central nervous system areas of 50 patients with sCJD, we were able to show distinct PrPSc distribution patterns in diverse subtypes of sCJD. Furthermore, we were able to demonstrate the co-existence of more than one PrPSc type in individuals with sCJD in about 20% of all patients and in more than 50% of patients heterozygous for a polymorphism on codon 129 of the gene encoding the prion protein (PRNP). CONCLUSION: PrPSc profiling represents a valuable tool for the molecular classification of human prion diseases and has important implications for their diagnosis by brain biopsy. Our results show that the co-existence of more than one PrPSc type might be influenced by genetic and brain region-specific determinants. These findings provide valuable insights into the generation of distinct PrPSc types.

Rapid presymptomatic detection of PrPSc via conformationally responsive palindromic PrP peptides.

Structurally unique, synthetic prion peptides provide the basis of a simple assay to serve as both a detection and signal amplification system that distinguishes the normal prion protein, PrPC, from the misfolded prion protein, PrPSc, that is associated with the occurrence of transmissible spongiform encephalopathies (TSE). Proof-of-principle has been shown on brain samples from an experimental scrapie hamster model. The assay demonstrates very sensitive detection of PrPSc in animal brain tissue with potential application for early presymptomatic detection in animal screening. Furthermore, the sensitivity of the assay could enable blood tests for this TSE disease as well as other amyloid and/or misfolded protein diseases.

[Bovine spongiform encephalopathy]. / L'encéphalopathie spongiforme bovine.

The identification of variant Creutzfeldt-Jakob disease (vCJD) in human strongly reinforced the perception of risks associated with the infectious agent involved in Bovine Spongiform Encephalopathy (BSE). The development of rapid tests for the diagnosis of BSE by the detection of the abnormal prion protein allowed a huge increase in surveillance of the cattle disease. This first revealed a higher prevalence of the infection than previously believed. However, food safety measures, mainly based on the ban of the use of meat and bone meal in ruminants and the elimination of specified risk materials from the food chain, already allowed significant progress in the control of the cattle disease, especially in the United Kingdom. Nevertheless, the diagnosis can still not be obtained in the live animal, while the disease only appears following a several years incubation period. Another major issue is the identification of the BSE agent when it has been transmitted to another species. This question not only arises in veterinary medicine, with the major question of a possible infection of small ruminants by the BSE agent, but also in human in which the existence of other forms of the disease linked to the BSE agent but possibly differing from Creutzfeldt-Jakob disease cannot be excluded.