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• W2008575392 abstract "Transmissible spongiform encephalopathies such as scrapie in sheep, Creutzfeldt-Jakob disease (CJD) in humans, and bovine sporadic encephalopathy in cattle are characterized by the accumulation of a misfolded protein: the pathological prion protein. Ever since bovine sporadic encephalopathy was discovered as the likely cause of the new variant of CJD in humans, parallels between human and animal transmissible spongiform encephalopathies must be viewed under the aspect of a disease risk for humans. In our study we have compared prion characteristics of different forms of sheep scrapie with those of different phenotypes of sporadic CJD. The disease characteristics of sporadic CJD depend considerably on the prion type 1 or 2. Our results show that there are obvious parallels between sporadic CJD type 1 and the so-called atypical/Nor98 scrapie. These parelleles apply to the deposition form of pathological prion protein in the brain, detected by the paraffin-embedded-tissue blot and the prion aggregate stability with regard to denaturation by the chaotropic salt guanidine hydrochloride. The same applies to sporadic CJD type 2 and classical scrapie. The observed parallels between types of sporadic CJD and types of sheep scrapie demonstrate that distinct groups of prion disease exist in different species. This should be taken into consideration when discussing interspecies transmission. Transmissible spongiform encephalopathies such as scrapie in sheep, Creutzfeldt-Jakob disease (CJD) in humans, and bovine sporadic encephalopathy in cattle are characterized by the accumulation of a misfolded protein: the pathological prion protein. Ever since bovine sporadic encephalopathy was discovered as the likely cause of the new variant of CJD in humans, parallels between human and animal transmissible spongiform encephalopathies must be viewed under the aspect of a disease risk for humans. In our study we have compared prion characteristics of different forms of sheep scrapie with those of different phenotypes of sporadic CJD. The disease characteristics of sporadic CJD depend considerably on the prion type 1 or 2. Our results show that there are obvious parallels between sporadic CJD type 1 and the so-called atypical/Nor98 scrapie. These parelleles apply to the deposition form of pathological prion protein in the brain, detected by the paraffin-embedded-tissue blot and the prion aggregate stability with regard to denaturation by the chaotropic salt guanidine hydrochloride. The same applies to sporadic CJD type 2 and classical scrapie. The observed parallels between types of sporadic CJD and types of sheep scrapie demonstrate that distinct groups of prion disease exist in different species. This should be taken into consideration when discussing interspecies transmission. Transmissible spongiform encephalopathies (TSEs) are characterized by aggregates of a partly protease-resistant, self-replicating protein called a “proteinacous infectious particle” (hereafter referred to as “prion”) in the central nervous system (CNS). According to the prion hypothesis, the disease-associated prion protein (PrPSc) is the principal or only constituent of the infectious agent.1Prusiner SB Novel proteinaceous infectious particles cause scrapie.Science. 1982; 216: 136-144Crossref PubMed Scopus (4072) Google Scholar The physiological isoform, a cell surface protein (PrPc), is expressed not only in the CNS, but also in a number of non-neuronal tissues. A long incubation period followed by a short clinical disease course after experimental transmission to animals, led in the early 50s of the last century to the concept of a “slow virus disease.”2Hadlow WJ Scrapie and Kuru.Lancet. 1959; 2: 289-290Abstract Scopus (290) Google Scholar Serial passages of scrapie isolates in inbred mice and hamsters revealed different incubation periods and vacuolation patterns defining strains, which was in line with the virus hypothesis,3Fraser H Dickinson AG Scrapie in mice: agent-strain differences in the distribution and intensity of grey matter vacuolation.J Comp Pathol. 1973; 83: 29-40Crossref PubMed Scopus (277) Google Scholar although a causative virus was not found.4Kellings K Meyer N Mirenda C Prusiner SB Riesner D Further analysis of nucleic acids in purified scrapie prion preparations by improved return refocusing gel electrophoresis.J Gen Virol. 1992; 73: 1025-1029Crossref PubMed Scopus (108) Google Scholar Prion strains are defined by their different incubation times and lesion profiles on inoculation in a new host species with an identical genetic background of the prion protein gene (PRNP).5Aguzzi A Heikenwalder M Polymenidou M Insights into prion strains and neurotoxicity.Nat Rev Mol Cell Biol. 2007; 8: 552-561Crossref PubMed Scopus (257) Google Scholar, 6Bessen RA Marsh RF Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters.J Gen Virol. 1992; 73: 329-334Crossref PubMed Scopus (227) Google Scholar For human sporadic Creutzfeldt-Jakob disease (CJD) no animal model that propagates PrPSc characteristics of all known clinical phenotypes is currently available. The most promising attempt to find out whether clinical phenotypes of sporadic CJD represent strains in a new host seems to be the bank vole model.7Nonno R Di Bari MA Cardone F Vaccari G Fazzi P Dell'Omo G Cartoni C Ingrosso L Boyle A Galeno R Sbriccoli M Lipp HP Bruce M Pocchiari M Agrimi U Efficient transmission and characterization of Creutzfeldt-Jakob disease strains in bank voles.PLoS Pathog. 2006; 2: e12Crossref PubMed Scopus (182) Google Scholar However, at least two biochemically distinguishable PrPSc types of sporadic CJD have been identified in the original species, termed type 1 and type 2. It has been shown in human sporadic CJD that, besides the methionine/valine polymorphism at codon 129 of the PRNP, the prion type is responsible for the clinical disease course as well as the pathological lesion profile.8Parchi P Giese A Capellari S Brown P Schulz-Schaeffer W Windl O Zerr I Budka H Kopp N Piccardo P Poser S Rojiani A Streichemberger N Julien J Vital C Ghetti B Gambetti P Kretzschmar H Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects.Ann Neurol. 1999; 46: 224-233Crossref PubMed Scopus (1191) Google Scholar It is widely accepted that human prion types 1 and 2 result from different pathological protein conformations, leading to a difference in the proteinase K-cleavage sites despite identical primary sequences.9Parchi P Zou W Wang W Brown P Capellari S Ghetti B Kopp N Schulz-Schaeffer WJ Kretzschmar HA Head MW Ironside JW Gambetti P Chen SG Genetic influence on the structural variations of the abnormal prion protein.Proc Natl Acad Sci USA. 2000; 97: 10168-10172Crossref PubMed Scopus (266) Google Scholar If all clinically and neuropathologically distinguishable sporadic CJD phenotypes (resulting from the combination of PrPSc types and relevant PRNP polymorphisms) belong to different strains, each of the human prion types could contain several strains. Applying this notion to the numerous scrapie strains that have been isolated in inbred mouse lines after the transmission of ovine scrapie samples,10Bruce ME Boyle A Cousens S McConnell I Foster J Goldmann W Fraser H Strain characterization of natural sheep scrapie and comparison with BSE.J Gen Virol. 2002; 83: 695-704Crossref PubMed Scopus (189) Google Scholar classical sheep scrapie may represent only one prion type11Buschmann A Biacabe AG Ziegler U Bencsik A Madec JY Erhardt G Luhken G Baron T Groschup MH Atypical scrapie cases in Germany and France are identified by discrepant reaction patterns in BSE rapid tests.J Virol Methods. 2004; 117: 27-36Crossref PubMed Scopus (171) Google Scholar harboring a certain heterogeneity.12Arsac JN Andreoletti O Bilheude JM Lacroux C Benestad SL Baron T Similar biochemical signatures and prion protein genotypes in atypical scrapie and Nor98 cases: France and Norway.Emerg Infect Dis. 2007; 13: 58-65Crossref PubMed Scopus (87) Google Scholar The recently identified new forms in sheep scrapie13Benestad SL Sarradin P Thu B Schonheit J Tranulis MA Bratberg B Cases of scrapie with unusual features in Norway and designation of a new type: Nor98.Vet Rec. 2003; 153: 202-208Crossref PubMed Scopus (375) Google Scholar and bovine spongiform encephalopathy (BSE)14Biacabe AG Laplanche JL Ryder S Baron T Distinct molecular phenotypes in bovine prion diseases.EMBO Rep. 2004; 5: 110-115Crossref PubMed Scopus (265) Google Scholar, 15Casalone C Zanusso G Acutis P Ferrari S Capucci L Tagliavini F Monaco S Caramelli M Identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic Creutzfeldt-Jakob disease.Proc Natl Acad Sci USA. 2004; 101: 3065-3070Crossref PubMed Scopus (384) Google Scholar differ from the previously described “classical” forms in several aspects such as the Western blot profile, histopathological lesion profile, and epidemiology. Potential parallels between animal and human prion diseases can be detected15Casalone C Zanusso G Acutis P Ferrari S Capucci L Tagliavini F Monaco S Caramelli M Identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic Creutzfeldt-Jakob disease.Proc Natl Acad Sci USA. 2004; 101: 3065-3070Crossref PubMed Scopus (384) Google Scholar which might be of some relevance regarding the transmissibility to humans. Parallels between human and animal TSEs have received increasing attention ever since classical BSE was discovered to be the likely cause of human variant CJD.16Bruce ME Will RG Ironside JW McConnell I Drummond D Suttie A McCardle L Chree A Hope J Birkett C Cousens S Fraser H Bostock CJ Transmissions to mice indicate that “new variant” CJD is caused by the BSE agent.Nature. 1997; 389: 498-501Crossref PubMed Scopus (1718) Google Scholar This study compares the deposition characteristics and aggregate stability of naturally occurring atypical/Nor98 and classical sheep scrapie with those of human PrPSc type 1 and type 2 of sporadic CJD. We hypothesize that similarities between distinct prion diseases in sheep and groups of clinical phenotypes in sporadic CJD characterize interspecies groups of prion diseases. This concept suggests that PrPSc types are the major determinant of prion disease forms. On the basis of these prion types, prion strains may be caused by additional factors that manifest themselves as strain characteristics on inoculation into a new host. Consequently, we will use the term “prion strain” only if incubation time and lesion profile have been defined in a different host species. In contrast, the term “prion type” will apply to a group within a prion disease that is markedly set apart by biochemical characteristics such as the Western blot profile and the aggregate stability and distinct forms of PrPSc deposition. Thus, it is conceivable that within one prion type more than one strain may be found, but not vice versa. CNS and lymphatic tissue were taken from 19 German sheep (15 ALRQ/ALRQ, two ALRQ/VLRQ, and two VLRQ/ALRH; letters represent amino acids at codon 136, 141, 154, and 171, respectively), five Norwegian sheep (four ALRQ/VLRQ and one VLRQ/ALRH), and nine French sheep (three ALRQ/ALRQ, three ALRQ/VLRQ, and three VLRQ/VLRQ) diagnosed with classical scrapie as well as 25 Norwegian Nor98 cases (one ALRQ/ALRQ, one ALHQ/ALRQ, six ALHQ/ALHQ, one ALHQ/ALRH, two ALHQ/AFRQ, five AFRQ/AFRQ, four ALHQ/ALRR, two ALRR/AFRQ, one ALRR/ALRR, and one unknown) and one German atypical case (ALHQ/ALRQ). Tissues from six clinically healthy sheep (four ALRR/ALRR, one ALRR/ALRH, and one ALRQ/ALRQ) originating from scrapie-free flocks were used as negative controls. The PrP genotypes were determined either by PCR and melting curve analysis17Schutz E Scharfenstein M Brenig B Genotyping of ovine prion protein gene (PRNP) variants by PCR with melting curve analysis.Clin Chem. 2006; 52: 1426-1429PubMed Google Scholar or by automated sequencing as described previously.13Benestad SL Sarradin P Thu B Schonheit J Tranulis MA Bratberg B Cases of scrapie with unusual features in Norway and designation of a new type: Nor98.Vet Rec. 2003; 153: 202-208Crossref PubMed Scopus (375) Google Scholar CNS tissues came from patients with a diagnosis of sporadic CJD, who were included in the National CJD Surveillance Study in Germany (approved by the ethics committee of the medical faculty of the University of Goettingen). The methionine (M)/valine (V) polymorphism at codon 129 of the PRNP was determined by using a standard protocol according to Windl et al.18Windl O Giese A Schulz-Schaeffer W Zerr I Skworc K Arendt S Oberdieck C Bodemer M Poser S Kretzschmar HA Molecular genetics of human prion diseases in Germany.Hum Genet. 1999; 105: 244-252Crossref PubMed Scopus (159) Google Scholar Tissues from frontal, temporal, and parietal cortices as well as cerebellum were used to determine the PrPSc type according to the Parchi classification, which was chosen due to its reproducibility in different laboratories. Alternative classifications do not take into account possible inconsistent pH-conditions during proteinase K-digestion, which result in band shifting during PrPSc typing.19Notari S Capellari S Giese A Westner I Baruzzi A Ghetti B Gambetti P Kretzschmar HA Parchi P Effects of different experimental conditions on the PrPSc core generated by protease digestion: implications for strain typing and molecular classification of CJD.J Biol Chem. 2004; 279: 16797-16804Crossref PubMed Scopus (122) Google Scholar Cases with mixed PrPSc types or PRNP-mutations were excluded from this study. The final setting included tissue from 10 patients with PrPSc type 1 (four MM1, three MV1, and three VV1) and 10 patients with PrPSc type 2 (three MM2, three MV2, and four VV2). CNS and lymphatic tissues were either snap frozen and stored at −80°C or fixed in 4% buffered formalin and embedded in paraffin. A decontamination step with formic acid (98%) was applied to the majority of fixed tissue blocks. The paraffin-embedded-tissue (PET) blot method was performed as described elsewhere.20Schulz-Schaeffer WJ Tschoke S Kranefuss N Drose W Hause-Reitner D Giese A Groschup MH Kretzschmar HA The paraffin-embedded tissue blot detects PrP(Sc) early in the incubation time in prion diseases.Am J Pathol. 2000; 156: 51-56Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 21Wemheuer WM Benestad SL Wrede A Wemheuer WE Brenig B Bratberg B Schulz-Schaeffer WJ Detection of classical and atypical/Nor98 scrapie by the paraffin-embedded tissue blot method.Vet Rec. 2009; 164: 677-681Crossref PubMed Scopus (13) Google Scholar Primary antibodies were mAb L42, mAb P4 (both from R-Biopharm AG, Darmstadt, Germany), or F89/160.1.5 (Veterinary Medical Research and Development, Pullman, WA) for ovine tissues and mAb 12F10 (kindly provided by W. Bodemer and D. Motzkus, German Primate Center, Goettingen, Germany) for human sections (each at a dilution of 1:5000). An alkaline phosphatase-coupled goat anti-mouse antibody (Dako, Glostrup, Denmark) and NBT/BCIP subtrate were used to visualize the detection of PrPSc. Tissue sections of all samples were also put on glass slides and stained with H&E or Luxol Fast Blue/PAS to aid the identification of neuroanatomical structures in the PET blots. A total of 94 gray and white matter structures were assessed for intensity (quantified in 0.5 grades ranging from 0 to 4) and PrPSc deposition patterns, which were verified by immunohistochemistry. Intensity profiles using 17 representative neuroanatomic sites of the sheep brain were created by Sigma Plot software (Systat Software Inc., San Jose, CA). Immunohistochemistry was performed as described before.21Wemheuer WM Benestad SL Wrede A Wemheuer WE Brenig B Bratberg B Schulz-Schaeffer WJ Detection of classical and atypical/Nor98 scrapie by the paraffin-embedded tissue blot method.Vet Rec. 2009; 164: 677-681Crossref PubMed Scopus (13) Google Scholar The primary mAbs P4, L42, F89/160.1.5, and 12F10 were used 1:500 in combination with an alkaline phosphatase-coupled goat anti-mouse antibody (Dako) and neufuchsine-chromogen-subtrate. Alternatively, a commercially available kit from Dako (Envision AEC) was applied by using mAb F89/160.1.5 at a dilution of 1:2000 in combination with the mAb 2G11 (1:200). Ten percent tissue homogenates (w/v) were prepared in PBS containing 0.5% deoxycholic acid sodium salt using glass grinding tubes and pestles or 20% homogenates were obtained by the standard sampling procedure of the TeSeE Western blot Kit (Bio-Rad, Hercules, CA). Some ovine homogenates were processed by using the TeSeE sheep/goat Western blot Kit (Bio-Rad) according to the manufacturer’s instructions. P4 was added at a dilution of 1:1000 to the primary antibody of the kit. All other homogenates were subjected to a different protocol by using 15% acrylamid gels, a 0.45-μm nitrocellulose membrane (Bio-Rad) for semidry blotting, the primary antibodies P4 (1:2000) for ovine samples, and 3F4 (1:3000; kindly provided by M. Beekes, Robert-Koch Institute, Berlin, Germany) for human samples. Further primary antibodies used for the epitope mapping were L42, 12F10, and 3B5 (kindly provided by W. Bodemer and D. Motzkus). A horseradish peroxidase-conjugated goat anti-mouse (Dako, Carpinteria, CA) and Super Signal Femto West Maximum Sensitivity Substrate (Perbio, Erembodegem, Belgium) were used to visualize the result on X-ray film. The molecular size of PrPSc was compared within one system only. The membrane was treated with 4M guanidine isothiocyanate (GdnSCN, 30 minutes) and 0.2% Casein (30 minutes) before antibodies were applied. Deglycosylation was performed after homogenates were digested with proteinase K (20 μg/ml for ovine tissue and 50 μg/ml for human tissue), by using either 0.5U N-Glycosidase F (Roche, Indianapolis, IN) per mg brain wet weight (12 hours, 37°C) or a deglycosylation kit (New England Biolabs, Ipswich, NJ) according to the manufacturer’s instructions. Ten percent tissue homogenates (in PBS, 0.1% deoxycholic acid sodium salt) were incubated with DNase 1 (Applichem, Darmstadt, Germany) using 500 μg/ml for 30 minutes (37°C). On treatment with proteinase K (see above) samples were diluted in Tris-buffered saline and subjected to a commercially available slot blot/dot blot device (Bio-Rad), run by a diaphragm pump. Samples were drawn through a nitrocellulose membrane (0.45 μm, Bio-Rad), following the assay by Winklhofer et al22Winklhofer KF Hartl FU Tatzelt J A sensitive filter retention assay for the detection of PrP(Sc) and the screening of anti-prion compounds.FEBS Lett. 2001; 503: 41-45Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar with modifications. During suction, proteins adsorb to the membrane, which was prepared with 10% Roti Block (Roth, Karlsruhe, Germany). The slots/dots were rinsed with 200 μl 0.1% deoxycholic acid sodium salt before and after the samples were applied and the membrane was treated as described above with an additional blocking step (0.3% H2O2, 20 minutes). Samples were incubated with suitable guanidine hydrochloride (GdnHCl) concentrations to achieve end concentrations of 1, 1.5, 2, 2.5, 3, 3.5, and 4M GdnHCl (Roth, Karlsruhe, Germany) for 1.5 hours (room temperature). Afterward, the samples were diluted in Tris-buffered saline containing 0.1% Brij (pH7.8) so that tissue concentrations of 1:1000 were obtained in all samples and proteinase K was added (see above). GdnHCl concentrations were only 0.1M in each sample in order not to interfere with proteinase K activity.23Peretz D Scott MR Groth D Williamson RA Burton DR Cohen FE Prusiner SB Strain-specified relative conformational stability of the scrapie prion protein.Protein Sci. 2001; 10: 854-863Crossref PubMed Scopus (213) Google Scholar Two hundred micrograms of brain wet weight were applied to each dot. The membrane was treated as described above. Proteinase K-digestion and Western blot analysis in all classical scrapie cases revealed the characteristic triplet band pattern of PrPSc comprising the di-, mono-, and unglycosylated band in the range of 18 to 30 kDa (Figure 1A). In contrast, all atypical/Nor98 scrapie cases displayed the same multiple band pattern with a small fragment at 11 to 12 kDa (Figure 1B),24Benestad SL Arsac JN Goldmann W Noremark M Atypical/Nor98 scrapie: properties of the agent, genetics, and epidemiology.Vet Res. 2008; 39: 19Crossref PubMed Scopus (199) Google Scholar also characterized as migrating below 15 kDa.25EFSA Opinion of the Scientific Panel on Biological Hazards on classification of atypical transmissible spongiform encephalopathy (TSE) cases in small ruminants.EFSA J. 2005; 276: 1-30Google Scholar Epitope mapping (Figure 1F) demonstrated that classical scrapie samples were detected by antibodies against ovine epitopes in the region of the octarepeats (mAb 3B5 [AA 62 to 91]), the proteinase K cleavage site (mAb P4 [AA 89 to 104]; Figure 1A), and the helix 1 region (mAb L42 [AA 145 to 162], mAb 12F10 [AA 153 to 163], and ICSM18 [AA 146 to 156]). Atypical/Nor98 scrapie samples were only detected by mAb P4 and mAb L42, which is consistent with previous reports.26Gretzschel A Buschmann A Langeveld J Groschup MH Immunological characterization of abnormal prion protein from atypical scrapie cases in sheep using a panel of monoclonal antibodies.J Gen Virol. 2006; 87: 3715-3722Crossref PubMed Scopus (40) Google Scholar No differences were detected in the size of the unglycosylated fragment of approximately 19 kDa after proteinase K-digestion and deglycosylation in classical scrapie cases of different genotypes (13 ALRQ/ALRQ, two ALRH/VLRQ, and two VLRQ/ALRQ) (Figure 1D). We are nevertheless aware that the analyzed samples might not be representative of the whole diversity found in classical scrapie isolates. Three fragments at approximately 25, 20, and 11 to 12 kDa were observed on digestion of atypical/Nor98 scrapie homogenates with proteinase K and PNGase F (Figure 1E). These are considered corresponding to the PrPSc fragments of approximately 23, 18, and 11 kDa, as described by Arsac et al12Arsac JN Andreoletti O Bilheude JM Lacroux C Benestad SL Baron T Similar biochemical signatures and prion protein genotypes in atypical scrapie and Nor98 cases: France and Norway.Emerg Infect Dis. 2007; 13: 58-65Crossref PubMed Scopus (87) Google Scholar since small differences in size may well be due to different protocols of sample preparation. The PET blot was used in this study since it is known to provide a high sensitivity and specificity in the topographic detection of disease-associated prion aggregates.20Schulz-Schaeffer WJ Tschoke S Kranefuss N Drose W Hause-Reitner D Giese A Groschup MH Kretzschmar HA The paraffin-embedded tissue blot detects PrP(Sc) early in the incubation time in prion diseases.Am J Pathol. 2000; 156: 51-56Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 21Wemheuer WM Benestad SL Wrede A Wemheuer WE Brenig B Bratberg B Schulz-Schaeffer WJ Detection of classical and atypical/Nor98 scrapie by the paraffin-embedded tissue blot method.Vet Rec. 2009; 164: 677-681Crossref PubMed Scopus (13) Google Scholar PrPSc deposits were detected in all scrapie sheep but not in control animals. The observed PrPSc deposits were present in many forms including reticular/synaptic, granular, fine punctate, perivacuolar, intra- and perineuronal, and plaque-like and ramified (i.e., glia-associated27Gonzalez L Martin S Begara-McGorum I Hunter N Houston F Simmons M Jeffrey M Effects of agent strain and host genotype on PrP accumulation in the brain of sheep naturally and experimentally affected with scrapie.J Comp Pathol. 2002; 126: 17-29Crossref PubMed Scopus (190) Google Scholar) deposits. The following results are based on the PET blot method, but PrPSc deposition patterns were also confirmed by conventional immunohistochemistry. Classical and atypical/Nor98 scrapie were easily distinguished by their forms of PrPSc deposition. In atypical/Nor98 scrapie cases predominantly reticular/synaptic PrPSc deposits in the gray matter were observed, complemented by granular PrPSc deposits in a number of white matter tracts. In contrast, in sheep affected by classical scrapie a variety of PrPSc deposition forms were seen in the CNS, creating a complex deposition pattern that particularly involved intraneuronal, perineuronal, and glia-associated PrPSc deposits. Classical and atypical/Nor98 sheep scrapie also differed clearly in the neuroanatomical PrPSc distribution. The deposition of PrPSc in gray and white matter structures were scored28Gonzalez L Martin S Jeffrey M Distinct profiles of PrP(d) immunoreactivity in the brain of scrapie- and BSE-infected sheep: implications for differential cell targeting and PrP processing.J Gen Virol. 2003; 84: 1339-1350Crossref PubMed Scopus (110) Google Scholar on a scale from zero to four, as illustrated in Figure 2. A more rostral localization of PrPSc in atypical/Nor98 scrapie sheep was observed, contrasting strongly with the PrPSc profile of classical scrapie in sheep. In the latter, the spread from the brainstem to the cerebellum and cerebrum29van Keulen LJ Schreuder BE Vromans ME Langeveld JP Smits MA Pathogenesis of natural scrapie in sheep.Arch Virol Suppl. 2000; : 57-71PubMed Google Scholar is mirrored by the more caudal accumulation of PrPSc. Classical scrapie sheep also showed large amounts of PrPSc in a number of gray matter structures that were devoid of PrPSc in all atypical/Nor98 scrapie sheep (Figure 2). These structures include the dorsal motor nucleus of the vagus nerve, the tegmental nucleus (rostral brainstem), and the central gray matter of the midbrain. The cerebral cortex of all atypical/Nor98 scrapie sheep displayed disease-associated PrPSc deposition in all cortical layers, resembling synaptic staining (Figure 3, D and E). Subcortical fibers also contained large amounts of PrPSc. In contrast, in classical sheep scrapie cortical deposits were mainly localized beneath the pia mater and in the deeper cortical layers (Figure 3, A and B), while staining of the white matter was usually glia-associated and never linked to subcortical fibers. Perivascular PrPSc deposition proved to be a prominent pattern in classical scrapie, but was not observed in atypical/Nor98 cases. Staining of the cerebellar cortex of classical scrapie showed that the majority of PrPSc accumulation was found as intra- and extracellular deposits in the granular layer and surrounding the Purkinje cells (Figure 4A). In most atypical/Nor98 cases, however, the molecular layer was most intensely stained (Figure 4C). The deposition form in all atypical/Nor98 cases displayed reticular/synaptic PrPSc aggregates in the molecular and granular layer.Figure 3Obvious parallels in disease-associated PrPSc deposition forms between ovine and human prion types. A reticular/synaptic deposition pattern is observable in the cortices of atypical/Nor98 scrapie sheep (D and E) and sporadic CJD type 1 (F; codon 129 methionine homozygote), while a complex deposition pattern, which is mainly localized in the deep cortical layers, can be seen in classical sheep scrapie (A and B) and sporadic CJD type 2 (C; codon 129 valine homozygote). Ovine tissue: PET blot mAb P4 1:5000 (A and D) and immunohistochemistry mAb P4 1:500 (B and E); human tissue: PET blot 12F10 1:5000 (C and F); scale bar = 300 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Similarities in the cerebellar cortex between ovine and human prion types regarding the deposition pattern of PrPSc. Synaptically located PrPSc deposits were characteristic for sporadic CJD type 1 (D; codon 129 methionine homozygote) and atypical/Nor98 sheep scrapie (C). In sporadic CJD type 2 (B; codon 129 methione/valine heterozygote) and classical sheep scrapie (A) more complex PrPSc aggregates were visible. Human PET blots: 12F10 1:5000 (B and D); ovine PET blots: mAb L42 (C) and P4 1:5000 (A); scale bar = 300 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the majority of classical scrapie cases (29 out of 31) where lymphatic tissue was available, PrPSc deposits were visible in lymphatic tissue, predominantly restricted to the follicles. By contrast, no PrPSc was detected in the lymphatic tissue of 15 of the atypical/Nor98 scrapie animals of which tonsils and/or lymph nodes were available. Using a membrane adsorption assay, PrPSc was reliably detected from an amount of 20-μg brain wet weight in the CNS from 15 examined classical scrapie cases (12 ALRQ/ALRQ, 2 VLRQ/ALRH, and 1 VLRQ/ALRQ) and six atypical/Nor98 scrapie cases (two ALHQ/ALHQ, one ALHQ/AFRQ, one ALHQ/ALRQ, one ALHQ/ALRH, and one AFRQ/AFRQ). To check the stability of the protein conformation, PrPSc of classical and atypical/Nor98, scrapie was first denatured by exposing brain tissue lysates to ascending concentrations of GdnHCl and then digested with proteinase K.23Peretz D Scott MR Groth D Williamson RA Burton DR Cohen FE Prusiner SB Strain-specified relative conformational stability of the scrapie prion protein.Protein Sci. 2001; 10: 854-863Crossref PubMed Scopus (213) Google Scholar To each dot 200 μg of brain wet weight were applied. PrPSc of atypical/Nor98 cases was only detectable after mild denaturation with GdnHCl: PrPSc was detected after denaturation with 1.5M GdnHCl but not 2M GdnHCl (Figure 5), although slightly different amounts" @default.
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• W2008575392 date "2009-12-01" @default.
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• W2008575392 title "Similarities between Forms of Sheep Scrapie and Creutzfeldt-Jakob Disease Are Encoded by Distinct Prion Types" @default.
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