Evaluation of non-immunoaffinity methods for isolation of cellular prion protein from bovine brain.

Transmissible spongiform encephalopathies (TSEs) are progressive neurodegenerative diseases that affect the central nervous system of many animals, including humans. Research suggests that TSEs are caused by conversion of the cellular prion protein (PrP(C)), which is encoded in many tissues, especially brain, to the pathological form (PrP(Sc)). This conversion affects PrP(Sc) structure, conferring different biochemical properties, such as the increased resistance to proteinase K, that have been widely used for its purification. By contrast, PrP(C) is less resistant and its isolation is more challenging. Here, we propose a purification strategy to efficiently recover PrP(C) from healthy bovine brain using conventional non-immunoaffinity methods. The applicability of extraction using detergents, size exclusion chromatography, diafiltration with molecular weight cutoff (MWCO) filters, and immobilized metal affinity chromatography (IMAC) using Western blot (WB) analysis to detect the presence of PrP(C) is discussed in detail.

Isolation of soluble and insoluble PrP oligomers in the normal human brain.

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrP(C) into its pathogenic isoform PrP(Sc 1). PrP(C) is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrP(Sc) forms detergent-insoluble aggregates and is partially resistant to PK(2-6). The conversion of PrP(C) to PrP(Sc) is known to involve a conformational transition of α-helical to ß-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrP(Sc), intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain(7). Using a combination of biophysical and biochemical approaches, we identified insoluble PrP(C) aggregates (designated iPrP(C)) from uninfected mammalian brains and cultured neuronal cells(8, 9). Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms(10), and PK-treatment. The combination of these approaches isolates not only insoluble PrP(Sc) and PrP(C) aggregates but also soluble PrP(C) oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrP(Sc) from infected brains and iPrP(C) from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrP(C) are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrP(Sc) that shares the immunoreactive behavior and fragmentation with iPrP(C 11, 12). Moreover, we recently demonstrated that iPrP(C) is the main species that interacts with amyloid-ß protein in Alzheimer disease(13). In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease(13), suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

Correlation of polydispersed prion protein and characteristic pathology in the thalamus in variant Creutzfeldt-Jakob disease: implication of small oligomeric species.

The vacuolation, neuronal loss and gliosis that characterize human prion disease pathology are accompanied by the accumulation of an aggregated, insoluble and protease-resistant form (termed PrP(Sc)) of the host-encoded normal cellular prion protein (PrP(C)). In variant Creutzfeldt-Jakob disease the frontal cortex and cerebellum exhibit intense vacuolation and the accumulation of PrP(Sc) in the form of amyloid plaques and plaque-like structures. In contrast the posterior thalamus is characterized by intense gliosis and neuronal loss, but PrP(Sc) plaques are rare and vacuolation is patchy. We have used sucrose density gradient centrifugation coupled with conformation dependent immunoassay to examine the biochemical properties of the PrP(Sc) that accumulates in these different brain regions. The results show a greater degree of PrP(Sc) polydisperal in thalamus compared with frontal cortex or cerebellum, including a subpopulation PrP(Sc) molecules in the thalamus that have sedimentation properties resembling those of PrP(C). Much effort has focused on identifying aspects of PrP(Sc) biochemistry that distinguish between different forms of human prion disease and contribute to differential diagnosis. Here we show that PrP(Sc) sedimentation properties, which can depend on aggregation state, correlate with, and may underlie the distinct neurodegenerative processes occurring in different regions of the variant Creutzfeldt-Jakob disease brain.

A new method for the characterization of strain-specific conformational stability of protease-sensitive and protease-resistant PrPSc.

Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrP(Sc), a disease-associated isoform of the host-encoded cellular protein (PrP(C)). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrP(Sc). However, PrP(Sc) is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrP(Sc) aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrP(C) and PrP(Sc) by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrP(Sc) solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl](1/2) values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl](1/2) values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M), followed by sheep scrapie (2.2 M) and by MM2 sCJD (1.6 M). In order to test the ability of CSSA to characterise protease-sensitive PrP(Sc), we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrP(Sc) aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrP(Sc) conformational stabilities of protease-resistant and protease-sensitive PrP(Sc) and that it is a valuable tool for strain typing in natural hosts, such as humans and sheep.

Intraspecies transmission of L-type-like Bovine Spongiform Encephalopathy detected in Japan.

It has been assumed that the agent causing BSE in cattle is a uniform strain (classical BSE); however, different neuropathological and molecular phenotypes of BSE (atypical BSE) have been recently reported. We demonstrated the successful transmission of L-type-like atypical BSE detected in Japan (BSE/JP24 isolate) to cattle. Based on the incubation period, neuropathological hallmarks, and molecular properties of the abnormal host prion protein, the characteristics of BSE/JP24 prion were apparently distinguishable from the classical BSE prion and closely resemble those of bovine amyloidotic spongiform encephalopathy prion detected in Italy.

Semisynthesis of membrane-attached prion proteins.

Conversion of cellular prion protein (PrP(C)) into the pathological conformer (PrP(Sc)) is the hallmark of prion diseases and has been studied extensively by using recombinantly expressed PrP (rPrP). Because of the inherent difficulties of expressing and purifying posttranslationally modified rPrP variants only a limited amount of data is available for membrane-associated PrP and its behavior in vitro and in vivo. Protein semisynthesis provides two alternative routes to access multimilligram amounts of membrane-attached rPrP, which are described in detail here. In both cases, rPrP fused to a C-terminal extension comprising either the Mycobacterium xenopi GyrA mini-intein or the Synechocystis sp. DnaE N-terminal split intein is expressed in E. coli. Protein purification was followed by reaction with chemically synthesized palmitoylated membrane anchor peptides to yield rPrP(Palm) or with a chemically synthesized glycosylphosphatidylinositol (GPI) anchor to give rPrP(GPI). Solubility problems encountered with synthetic membrane anchors were overcome by either incorporating a polyethylene glycol-based C-terminal tag (removable by specific proteolysis) or by direct incorporation into liposomes. The new rPrP(Palm) variants studied by a variety of in vitro methods exhibited a high affinity to liposomes and an increased lag phase during aggregation when compared to rPrP. Similar results were obtained for rPrP(GPI), in which only one alkyl chain is sufficient for quantitative membrane attachment. In vivo studies demonstrated that double lipidated rPrP(Palm) is efficiently taken up into the membranes of mouse neuronal and human epithelial kidney cells.

Production and characterization of a panel of monoclonal antibodies against native human cellular prion protein.

The human prion diseases, such as variant Creutzfeldt-Jakob disease (vCJD), are characterized by the conversion of the normal cellular prion protein (PrP(C)) into an abnormal disease associated form (PrP(Sc)). Monoclonal antibodies (MAbs) that recognize these different PrP isoforms are valuable reagents both in the diagnosis of these diseases and in prion disease research in general but we know of no attempts to raise MAbs against native human PrP(C). We immunized prion protein gene ablated (PrP(-/-)) mice with native human PrP(C) purified from platelets (pHuPrP) generating a predominantly IgG isotype anti-pHuPrP polyclonal antibody response in all mice. Following fusion of splenocytes from the immunized mice with SP2/0 myeloma cells, we were able to identify single cell clone and cryopreserve 14 stable hybridoma cell lines producing MAbs that reacted with pHuPrP. The properties of these MAbs (such as isotype, binding to native/denatured pHuPrP, and HuPrP epitopes recognized) are described. Furthermore, several of these MAbs showed a selectivity in their ability to immunoprecipitate disease associated PrP(Sc) and its corresponding protease resistant core (PrP(res)).

The polybasic N-terminal region of the prion protein controls the physical properties of both the cellular and fibrillar forms of PrP.

Individual variations in structure and morphology of amyloid fibrils produced from a single polypeptide are likely to underlie the molecular origin of prion strains and control the efficiency of the species barrier in the transmission of prions. Previously, we observed that the shape of amyloid fibrils produced from full-length prion protein (PrP 23-231) varied substantially for different batches of purified recombinant PrP. Variations in fibril morphology were also observed for different fractions that corresponded to the highly pure PrP peak collected at the last step of purification. A series of biochemical experiments revealed that the variation in fibril morphology was attributable to the presence of miniscule amounts of N-terminally truncated PrPs, where a PrP encompassing residue 31-231 was the most abundant of the truncated polypeptides. Subsequent experiments showed that the presence of small amounts of recombinant PrP 31-231 (0.1-1%) in mixtures with full-length PrP 23-231 had a dramatic impact on fibril morphology and conformation. Furthermore, the deletion of the short polybasic N-terminal region 23-30 was found to reduce the folding efficiency to the native alpha-helical forms and the conformational stability of alpha-PrP. These findings are very surprising considering that residues 23-30 are very distant from the C-terminal globular folded domain in alpha-PrP and from the prion folding domain in the fibrillar form. However, our studies suggest that the N-terminal polybasic region 23-30 is essential for effective folding of PrP to its native cellular conformation. This work also suggests that this region could regulate diversity of prion strains or subtypes despite its remote location from the prion folding domain.

Amplification of purified prions in vitro.

The infectious agents of prion diseases are unorthodox, and they seem to be composed primarily of a misfolded glycoprotein called the prion protein (PrP). Replication of prion infectivity is associated with the conversion of PrP from its normal, cellular form (PrP(C)) into a pathogenic form (PrP(Sc)), which is characterized biochemically by relative detergent insolubility and protease resistance. Several techniques have been developed in which PrP(C) molecules can be converted into the PrP(Sc) conformation in vitro. These biochemical techniques recapitulate several specific aspects of in vivo prion propagation, and one method, the protein misfolding cyclic amplification technique, also has been shown to amplify infectivity. In this chapter, we describe a method for amplifying PrP(Sc) molecules from hamster prions in vitro using purified substrates. Specific protocols for substrate preparation, reaction mixture, and product detection are explained. Purified PrP(Sc) amplification assays are currently being used to study the biochemical mechanism of prion formation.

Expression and purification of full-length recombinant PrP of high purity.

Certain applications in the prion field require recombinant prion protein (PrP) of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian prion protein. This protocol has been proved to yield PrP of extremely high purity that lacks PrP adducts, which are normally generated as a result of spontaneous oxidation or degradation.

Intraspecies transmission of BASE induces clinical dullness and amyotrophic changes.

The disease phenotype of bovine spongiform encephalopathy (BSE) and the molecular/ biological properties of its prion strain, including the host range and the characteristics of BSE-related disorders, have been extensively studied since its discovery in 1986. In recent years, systematic testing of the brains of cattle coming to slaughter resulted in the identification of at least two atypical forms of BSE. These emerging disorders are characterized by novel conformers of the bovine pathological prion protein (PrP(TSE)), named high-type (BSE-H) and low-type (BSE-L). We recently reported two Italian atypical cases with a PrP(TSE) type identical to BSE-L, pathologically characterized by PrP amyloid plaques and known as bovine amyloidotic spongiform encephalopathy (BASE). Several lines of evidence suggest that BASE is highly virulent and easily transmissible to a wide host range. Experimental transmission to transgenic mice overexpressing bovine PrP (Tgbov XV) suggested that BASE is caused by a prion strain distinct from the BSE isolate. In the present study, we experimentally infected Friesian and Alpine brown cattle with Italian BSE and BASE isolates via the intracerebral route. BASE-infected cattle developed amyotrophic changes accompanied by mental dullness. The molecular and neuropathological profiles, including PrP deposition pattern, closely matched those observed in the original cases. This study provides clear evidence of BASE as a distinct prion isolate and discloses a novel disease phenotype in cattle.

Native, amyloid fibrils and beta-oligomers of the C-terminal domain of human prion protein display differential activation of complement and bind C1q, factor H and C4b-binding protein directly.

Prion protein (PrP) is an endogenous protein involved in the pathogenesis of bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Murine PrP has been reported to bind C1q and activate the classical pathway of complement in a copper-dependent manner. Here we show that various conformational isoforms (native, amyloid fibrils, and beta-oligomers) of recombinant human PrP (90-231 and 121-231) bind C1q and activate complement. PrP binds both the globular head and collagenous stalk domains of C1q. Native, beta-oligomeric and amyloid fibrils of PrP all activate the classical and alternative pathways of complement to different extent. However, they do not trigger the lectin pathway. Of the tested PrP conformational isoforms we find that beta-oligomers bind C1q and activate complement most strongly. Membrane attack complex formation initiated by PrP is subdued in comparison to deposition of early complement components. This is most likely attributed to the interaction between human PrP and complement inhibitors factor H and C4b-binding protein. Accordingly, PrP-triggered complement activation in the terminal pathway was increased in serum lacking C4b-binding protein. Taken together the present study indicates that complement activation may be an important factor in human prion diseases, suggesting that complement induced activities may prove relevant therapeutic targets.

The elk PRNP codon 132 polymorphism controls cervid and scrapie prion propagation.

The elk prion protein gene (PRNP) encodes either methionine (M) or leucine (L) at codon 132, the L132 allele apparently affording protection against chronic wasting disease (CWD). The corresponding human codon 129 polymorphism influences the host range of bovine spongiform encephalopathy (BSE) prions. To fully address the influence of this cervid polymorphism on CWD pathogenesis, we created transgenic (Tg) mice expressing cervid PrPC with L at residue 132, referred to as CerPrPC-L132, and compared the transmissibility of CWD prions from elk of defined PRNP genotypes, namely homozygous M/M or L/L or heterozygous M/L, in these Tg mice with previously described Tg mice expressing CerPrPC-M132, referred to as Tg(CerPrP) mice. While Tg(CerPrP) mice were consistently susceptible to CWD prions from elk of all three genotypes, Tg(CerPrP-L132) mice uniformly failed to develop disease following challenge with CWD prions. In contrast, SSBP/1 sheep scrapie prions transmitted efficiently to both Tg(CerPrP) and Tg(CerPrP-L132) mice. Our findings suggest that the elk 132 polymorphism controls prion susceptibility at the level of prion strain selection and that cervid PrP L132 severely restricts propagation of CWD prions. We speculate that the L132 polymorphism results in less efficient conversion of CerPrPC-L132 by CWD prions, an effect that is overcome by the SSBP/1 strain. Our studies show the accumulation of subclinical levels of CerPrPSc in aged asymptomatic CWD-inoculated Tg(CerPrP-L132) mice and also suggests the establishment of a latent infection state in apparently healthy elk expressing this seemingly protective allele.

A general model of prion strains and their pathogenicity.

Prions are lethal mammalian pathogens composed of aggregated conformational isomers of a host-encoded glycoprotein and which appear to lack nucleic acids. Their unique biology, allied with the public-health risks posed by prion zoonoses such as bovine spongiform encephalopathy, has focused much attention on the molecular basis of prion propagation and the "species barrier" that controls cross-species transmission. Both are intimately linked to understanding how multiple prion "strains" are encoded by a protein-only agent. The underlying mechanisms are clearly of much wider importance, and analogous protein-based inheritance mechanisms are recognized in yeast and fungi. Recent advances suggest that prions themselves are not directly neurotoxic, but rather their propagation involves production of toxic species, which may be uncoupled from infectivity.

Discriminating between cellular and misfolded prion protein by using affinity to 9-aminoacridine compounds.

Quinacrine and related 9-aminoacridine compounds are effective in eliminating the alternatively folded prion protein, termed PrP(Sc), from scrapie-infected cultured cells. Clinical evaluations of quinacrine for the treatment of human prion diseases are progressing in the absence of a clear understanding of the molecular mechanism by which prion replication is blocked. Here, insight into the mode of action of 9-aminoacridine compounds was sought by using a chemical proteomics approach to target identification. Cellular macromolecules that bind 9-aminoacridine ligands were affinity-purified from tissue lysates by using a 9-aminoacridine-functionalized solid-phase matrix. Although the 9-aminoacridine matrix was conformationally selective for PrP(Sc), it was inefficient: approximately 5 % of PrP(Sc) was bound under conditions that did not support binding of the cellular isoform, PrP(C). Our findings suggest that 9-aminoacridine compounds may reduce the PrP(Sc) burden either by occluding epitopes necessary for templating on the surface of PrP(Sc) or by altering the stability of PrP(Sc) oligomers, where a one-to-one stoichiometry is not necessary.

Conversion of the BASE prion strain into the BSE strain: the origin of BSE?

Atypical neuropathological and molecular phenotypes of bovine spongiform encephalopathy (BSE) have recently been identified in different countries. One of these phenotypes, named bovine "amyloidotic" spongiform encephalopathy (BASE), differs from classical BSE for the occurrence of a distinct type of the disease-associated prion protein (PrP), termed PrP(Sc), and the presence of PrP amyloid plaques. Here, we show that the agents responsible for BSE and BASE possess different biological properties upon transmission to transgenic mice expressing bovine PrP and inbred lines of nontransgenic mice. Strikingly, serial passages of the BASE strain to nontransgenic mice induced a neuropathological and molecular disease phenotype indistinguishable from that of BSE-infected mice. The existence of more than one agent associated with prion disease in cattle and the ability of the BASE strain to convert into the BSE strain may have important implications with respect to the origin of BSE and spongiform encephalopathies in other species, including humans.

Packaging of prions into exosomes is associated with a novel pathway of PrP processing.

Prion diseases are fatal, transmissible neurodegenerative disorders associated with conversion of the host-encoded prion protein (PrP(C)) into an abnormal pathogenic isoform (PrP(Sc)). Following exposure to the infectious agent (PrP(Sc)) in acquired disease, infection is propagated in lymphoid tissues prior to neuroinvasion and spread within the central nervous system. The mechanism of prion dissemination is perplexing due to the lack of plausible PrP(Sc)-containing mobile cells that could account for prion spread between infected and uninfected tissues. Evidence exists to demonstrate that the culture media of prion-infected neuronal cells contain PrP(Sc) and infectivity but the nature of the infectivity remains unknown. In this study we have identified PrP(C) and PrP(Sc) in association with endogenously expressing PrP neuronal cell-derived exosomes. The exosomes from our prion-infected neuronal cell line were efficient initiators of prion propagation in uninfected recipient cells and to non-neuronal cells. Moreover, our neuronal cell line was susceptible to infection by non-neuronal cell-derived exosome PrP(Sc). Importantly, these exosomes produced prion disease when inoculated into mice. Exosome-associated PrP is packaged via a novel processing pathway that involves the N-terminal modification of PrP and selection of distinct PrP glycoforms for incorporation into these vesicles. These data extend our understanding of the relationship between PrP and exosomes by showing that exosomes can establish infection in both neighbouring and distant cell types and highlight the potential contribution of differentially processed forms of PrP in disease distribution. These data suggest that exosomes represent a potent pool of prion infectivity and provide a mechanism for studying prion spread and PrP processing in cells endogenously expressing PrP.

Prion protein in milk.

BACKGROUND: Prions are known to cause transmissible spongiform encephalopathies (TSE) after accumulation in the central nervous system. There is increasing evidence that prions are also present in body fluids and that prion infection by blood transmission is possible. The low concentration of the proteinaceous agent in body fluids and its long incubation time complicate epidemiologic analysis and estimation of spreading and thus the risk of human infection. This situation is particularly unsatisfactory for food and pharmaceutical industries, given the lack of sensitive tools for monitoring the infectious agent. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an adsorption matrix, Alicon PrioTrap, which binds with high affinity and specificity to prion proteins. Thus we were able to identify prion protein (PrP(C))--the precursor of prions (PrP(Sc))--in milk from humans, cows, sheep, and goats. The absolute amount of PrP(C) differs between the species (from microg/l range in sheep to ng/l range in human milk). PrP(C) is also found in homogenised and pasteurised off-the-shelf milk, and even ultrahigh temperature treatment only partially diminishes endogenous PrP(C) concentration. CONCLUSIONS/SIGNIFICANCE: In view of a recent study showing evidence of prion replication occurring in the mammary gland of scrapie infected sheep suffering from mastitis, the appearance of PrP(C) in milk implies the possibility that milk of TSE-infected animals serves as source for PrP(Sc).

Passage of chronic wasting disease prion into transgenic mice expressing Rocky Mountain elk (Cervus elaphus nelsoni) PrPC.

Chronic wasting disease (CWD) of elk (Cervus elaphus nelsoni) and mule deer (Odocoileus hemionus) is one of three naturally occurring forms of prion disease, the others being Creutzfeldt-Jakob disease in humans and scrapie in sheep. In the last few decades, CWD has spread among captive and free-ranging cervids in 13 US states, two Canadian provinces and recently in Korea. The origin of the CWD agent(s) in cervids is not known. This study describes the development of a transgenic mouse line (TgElk) homozygous for a transgene array encoding the elk prion protein (PrP(C)) and its use in propagating and simulating CWD in mice. Intracerebral injection of one mule deer and three elk CWD isolates into TgElk mice led to disease with incubation periods of 127 and 95 days, respectively. Upon secondary passage, the incubation time was reduced to 108 and 90 days, respectively. Upon passage into TgElk mice, CWD prions (PrP(Sc)) maintained the characteristic Western blot profiles seen in CWD-affected mule deer and elk and produced histopathological modifications consistent with those observed in the natural disease. The short incubation time observed on passage from cervid to mouse with both mule deer and elk CWD brain homogenates and the demonstrated capacity of the animals to propagate (mouse to mouse) CWD agents make the TgElk line a valuable model to study CWD agents in cervid populations. In addition, these results with this new transgenic line suggest the intriguing hypothesis that there could be more than one strain of CWD agent in cervids.

Lymphoid follicles of the ileal Peyer's patch of lambs express low levels of PrP, as demonstrated by quantitative real-time RT-PCR on microdissected tissue compartments, in situ hybridization and immunohistochemistry.

The expression level of normal cellular prion protein (PrP(C)) is thought to influence the transmission of transmissible spongiform encephalopathies (TSEs) from the peripheral entry site to the site of pathological changes in the central nervous system. In many TSEs, the clinical disease is preceded by a period in which the agent accumulates in lymphoid organs, particularly in association with follicular dendritic cells of lymphoid follicles. As the probable route of entry of the TSE agent is via the gut, the expression profile of PrP was examined in well-developed gut-associated lymphoid tissue of lambs, the ileal Peyer's patch, by laser microdissection and real-time RT-PCR. Lymphoid follicles were found to have very low levels of expression, whilst highest levels were detected in the outer submucosa and the muscular layer. These findings were supported by in situ hybridization and immunohistochemistry, which showed specific labelling in nerve cells in ganglia of the submucosal (Meissner's) and myenteric (Auerbach's) plexi of the enteric nervous system. Based on the assumption that potential sites for conversion to the scrapie-related prion protein (PrP(Sc)) should display high levels of expression of PrP(C), this study suggests that the accumulation of PrP(Sc) in the lymphoid follicles of the Peyer's patch is not preceded by PrP conversion in the same tissue compartment.