Detection and Localization of PrPSc in the Skeletal Muscle of Patients with Variant, Iatrogenic, and Sporadic Forms of Creutzfeldt-Jakob Disease
2006; Elsevier BV; Volume: 168; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2006.050788
ISSN1525-2191
AutoresAlexander Peden, Diane Ritchie, Mark Head, James W. Ironside,
Tópico(s)Prion Diseases and Protein Misfolding
ResumoVariant Creutzfeldt-Jakob disease (vCJD) differs from other human prion diseases in that the pathogenic prion protein PrPSc can be detected to a greater extent at extraneuronal sites throughout the body, principally within lymphoid tissues. However, a recent study using a high-sensitivity Western blotting technique revealed low levels of PrPSc in skeletal muscle from a quarter of Swiss patients with sporadic CJD (sCJD). This posed the question of whether PrPSc in muscle could also be detected in vCJD, sCJD, and iatrogenic (iCJD) patients from other populations. Therefore, we have used the same high-sensitivity Western blotting technique, in combination with paraffin-embedded tissue blotting, to screen for PrPSc in muscle tissue specimens taken at autopsy from 49 CJD patients in the United Kingdom. These techniques identified muscle PrPSc in 8 of 17 vCJD, 7 of 26 sCJD, and 2 of 5 iCJD patients. Paraffin-embedded tissue blotting analysis showed PrPSc in skeletal muscle in localized anatomical structures that had the morphological and immunohistochemical characteristics of nerve fibers. The detection of PrPSc in muscle tissue from all forms of CJD indicates the possible presence of infectivity in these tissues, suggesting important implications for assessing the potential risk of iatrogenic spread via contaminated surgical instruments. Variant Creutzfeldt-Jakob disease (vCJD) differs from other human prion diseases in that the pathogenic prion protein PrPSc can be detected to a greater extent at extraneuronal sites throughout the body, principally within lymphoid tissues. However, a recent study using a high-sensitivity Western blotting technique revealed low levels of PrPSc in skeletal muscle from a quarter of Swiss patients with sporadic CJD (sCJD). This posed the question of whether PrPSc in muscle could also be detected in vCJD, sCJD, and iatrogenic (iCJD) patients from other populations. Therefore, we have used the same high-sensitivity Western blotting technique, in combination with paraffin-embedded tissue blotting, to screen for PrPSc in muscle tissue specimens taken at autopsy from 49 CJD patients in the United Kingdom. These techniques identified muscle PrPSc in 8 of 17 vCJD, 7 of 26 sCJD, and 2 of 5 iCJD patients. Paraffin-embedded tissue blotting analysis showed PrPSc in skeletal muscle in localized anatomical structures that had the morphological and immunohistochemical characteristics of nerve fibers. The detection of PrPSc in muscle tissue from all forms of CJD indicates the possible presence of infectivity in these tissues, suggesting important implications for assessing the potential risk of iatrogenic spread via contaminated surgical instruments. Creutzfeldt-Jakob disease (CJD) is a member of the human prion diseases or transmissible spongiform encephalopathies, a group of fatal degenerative diseases of the central nervous system (CNS). A key event in the pathogenesis of prion diseases is the conversion of the cellular prion protein PrPC to the abnormal disease-associated form PrPSc. According to the prion hypothesis, PrPSc is the principle or sole component of the infectious agent, and the accumulation of PrPSc within the CNS has been proposed to be the primary event leading to neurodegeneration.1Prusiner SB Prions.Proc Natl Acad Sci USA. 1998; 95: 13363-13383Crossref PubMed Scopus (5131) Google Scholar Prion diseases occur in idiopathic, inherited, and acquired forms. The most common human prion disease is sporadic CJD (sCJD), which occurs worldwide with a frequency of approximately one per million of the population per annum.2Holman RC Khan AS Kent J Strine TW Schonberger LB Epidemiology of Creutzfeldt-Jakob disease in the United States, 1979–1990: analysis of national mortality data.Neuroepidemiology. 1995; 14: 174-181Crossref PubMed Scopus (51) Google Scholar, 3Ladogana A Puopolo M Croes EA Budka H Jarius C Collins S Klug GM Sutcliffe T Giulivi A Alperovitch A Delasnerie-Laupretre N Brandel JP Poser S Kretzschmar H Rietveld I Mitrova E Cuesta JP Martinez-Martin P Glatzel M Aguzzi A Knight R Ward H Pocchiari M van Duijn CM Will RG Zerr I Mortality from Creutzfeldt-Jakob disease and related disorders in Europe, Australia, and Canada.Neurology. 2005; 64: 1586-1591Crossref PubMed Scopus (265) Google Scholar, 4Nakamura Y Yanagawa H Hoshi K Yoshino H Urata J Sato T Incidence rate of Creutzfeldt-Jakob disease in Japan.Int J Epidemiol. 1999; 28: 130-134Crossref PubMed Scopus (35) Google Scholar The cause of this disease is unknown, although a random stochastic event resulting in a conversion of PrPC to PrPSc and the subsequent propagation of this process is one possibility.5Hsiao K Meiner Z Kahana E Cass C Kahana I Avrahami D Scarlato G Abramsky O Prusiner SB Gabizon R Mutation of the prion protein in Libyan Jews with Creutzfeldt-Jakob disease.N Engl J Med. 1991; 324: 1091-1097Crossref PubMed Scopus (225) Google Scholar CJD can also be acquired by iatrogenic transmission (iCJD) as a result of certain neurosurgical procedures, dura mater or corneal transplantation, and through treatment with cadaveric human growth hormone as reviewed by Brown and colleagues.6Brown P Preece M Brandel JP Sato T McShane L Zerr I Fletcher A Will RG Pocchiari M Cashman NR d'Aignaux JH Cervenakova L Fradkin J Schonberger LB Collins SJ Iatrogenic Creutzfeldt-Jakob disease at the millennium.Neurology. 2000; 55: 1075-1081Crossref PubMed Scopus (464) Google Scholar A novel acquired human prion disease, variant Creutzfeldt-Jakob disease (vCJD), is thought to result from oral exposure to the bovine spongiform encephalopathy agent.7Will RG Ironside JW Zeidler M Cousens SN Estibeiro K Alperovitch A Poser S Pocchiari M Hofman A Smith PG A new variant of Creutzfeldt-Jakob disease in the UK.Lancet. 1996; 347: 921-925Abstract PubMed Scopus (1867) Google Scholar vCJD is clearly distinct from other forms of CJD in its neurological, neuropathological, and biochemical phenotype. PrPSc differs from PrPC in its relative resistance to proteases and is often referred to as PrPres after partial proteolytic degradation. Although PrPSc accumulation occurs primarily in the brain, PrPres has also been detected in the peripheral tissues of CJD patients.8Glatzel M Abela E Maissen M Aguzzi A Extraneural pathologic prion protein in sporadic Creutzfeldt-Jakob disease.N Engl J Med. 2003; 349: 1812-1820Crossref PubMed Scopus (291) Google Scholar, 9Head MW Ritchie D Smith N McLoughlin V Nailon W Samad S Masson S Bishop M McCardle L Ironside JW Peripheral tissue involvement in sporadic, iatrogenic, and variant Creutzfeldt-Jakob disease: an immunohistochemical, quantitative, and biochemical study.Am J Pathol. 2004; 164: 143-153Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 10Wadsworth JD Joiner S Hill AF Campbell TA Desbruslais M Luthert PJ Collinge J Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay.Lancet. 2001; 358: 171-180Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar This is particularly the case for vCJD in which PrPres is readily detected in the lymphoid tissues including, spleen, tonsil, lymph nodes, and Peyer's patches using standard Western blotting and immunohistochemistry techniques.9Head MW Ritchie D Smith N McLoughlin V Nailon W Samad S Masson S Bishop M McCardle L Ironside JW Peripheral tissue involvement in sporadic, iatrogenic, and variant Creutzfeldt-Jakob disease: an immunohistochemical, quantitative, and biochemical study.Am J Pathol. 2004; 164: 143-153Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 10Wadsworth JD Joiner S Hill AF Campbell TA Desbruslais M Luthert PJ Collinge J Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay.Lancet. 2001; 358: 171-180Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar In contrast, in sCJD PrPres accumulation (and by implication infectivity) was thought to be primarily confined to the CNS.9Head MW Ritchie D Smith N McLoughlin V Nailon W Samad S Masson S Bishop M McCardle L Ironside JW Peripheral tissue involvement in sporadic, iatrogenic, and variant Creutzfeldt-Jakob disease: an immunohistochemical, quantitative, and biochemical study.Am J Pathol. 2004; 164: 143-153Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 10Wadsworth JD Joiner S Hill AF Campbell TA Desbruslais M Luthert PJ Collinge J Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay.Lancet. 2001; 358: 171-180Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 11Bruce ME McConnell I Will RG Ironside JW Detection of variant Creutzfeldt-Jakob disease infectivity in extraneural tissues.Lancet. 2001; 358: 208-209Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar However, earlier transmission studies had demonstrated infectivity in extraneural tissues, including spleen, lung, liver, and kidney, but not skeletal muscle or peripheral nerve.12Brown P Gibbs Jr, CJ Rodgers-Johnson P Asher DM Sulima MP Bacote A Goldfarb LG Gajdusek DC Human spongiform encephalopathy: the National Institutes of Health series of 300 cases of experimentally transmitted disease.Ann Neurol. 1994; 35: 513-529Crossref PubMed Scopus (727) Google Scholar Also, there was always the possibility that the detection of low levels of PrPres in peripheral organs in all forms of CJD may be limited by the sensitivity of the assays used. This was highlighted recently by Glatzel and colleagues8Glatzel M Abela E Maissen M Aguzzi A Extraneural pathologic prion protein in sporadic Creutzfeldt-Jakob disease.N Engl J Med. 2003; 349: 1812-1820Crossref PubMed Scopus (291) Google Scholar who were able to detect PrPres in autopsy specimens of spleen and muscle in a proportion of sCJD patients in Switzerland using a high-sensitivity Western blotting technique involving selective precipitation of PrPres with sodium phosphotungstic acid (NaPTA). The detection of PrPres in the muscle of Swiss sCJD patients presents the worrying prospect of the transmission of prion infection via instruments used in ordinary surgical procedures. Indeed, a collaborative case control study performed on sCJD patients in Europe showed that a past history of surgery was a risk factor.13Ward HJ Everington D Croes EA Alperovitch A Delasnerie-Laupretre N Zerr I Poser S van Duijn CM Sporadic Creutzfeldt-Jakob disease and surgery: a case-control study using community controls.Neurology. 2002; 59: 543-548Crossref PubMed Scopus (70) Google Scholar The study by Glatzel and colleagues8Glatzel M Abela E Maissen M Aguzzi A Extraneural pathologic prion protein in sporadic Creutzfeldt-Jakob disease.N Engl J Med. 2003; 349: 1812-1820Crossref PubMed Scopus (291) Google Scholar requires confirmation in sCJD patients from other populations. The study was purely biochemical in nature and did not investigate the localization of PrPres within the muscle tissue. Lastly, the work of Glatzel and colleagues8Glatzel M Abela E Maissen M Aguzzi A Extraneural pathologic prion protein in sporadic Creutzfeldt-Jakob disease.N Engl J Med. 2003; 349: 1812-1820Crossref PubMed Scopus (291) Google Scholar raises the question of whether PrPres is present in the muscle of patients with other forms of CJD, most notably vCJD, where the extent of peripheral PrPres tends to be greater.9Head MW Ritchie D Smith N McLoughlin V Nailon W Samad S Masson S Bishop M McCardle L Ironside JW Peripheral tissue involvement in sporadic, iatrogenic, and variant Creutzfeldt-Jakob disease: an immunohistochemical, quantitative, and biochemical study.Am J Pathol. 2004; 164: 143-153Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 10Wadsworth JD Joiner S Hill AF Campbell TA Desbruslais M Luthert PJ Collinge J Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay.Lancet. 2001; 358: 171-180Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar Because of the oral route of infection in vCJD and the presumed long incubation period,14Will R Variant Creutzfeldt-Jakob disease.Folia Neuropathol. 2004; 42: 77-83PubMed Google Scholar infectious prions may be present in the peripheral tissues for a considerable period of time before the onset of clinical symptoms. For this reason vCJD may pose an increased risk for the iatrogenic transmission of the disease. This risk had been highlighted recently by two cases of the secondary transmission of vCJD infection by blood transfusion from donors who only developed vCJD after donation.15Llewelyn CA Hewitt PE Knight RS Amar K Cousens S Mackenzie J Will RG Possible transmission of variant Creutzfeldt-Jakob disease by blood transfusion.Lancet. 2004; 363: 417-421Abstract Full Text Full Text PDF PubMed Scopus (940) Google Scholar, 16Peden AH Head MW Ritchie DL Bell JE Ironside JW Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient.Lancet. 2004; 364: 527-529Abstract Full Text Full Text PDF PubMed Scopus (770) Google Scholar To address the above issues, we have used the same high-sensitivity NaPTA precipitation/Western blotting technique to screen skeletal and heart muscle samples from UK patients with vCJD, iCJD, as well as sCJD. We show that PrPres is indeed present in a significant proportion of autopsy skeletal muscle samples taken from vCJD, iCJD, and sCJD patients, and we go on to use the paraffin-embedded tissue (PET) blotting technique to determine the localization of PrPres within human muscle tissue. Seventeen vCJD patients, twenty-six sCJD cases, and five iCJD cases (associated with human growth hormone therapy) were analyzed in this study by a combination of Western blotting and PET blotting. Twelve cases of clinically suspected CJD that were given an alternative final pathological diagnosis were included as negative controls because they would be expected to lack PrPres in the brain and peripheral tissues. Cases were selected for this study on the basis of the availability of fixed and frozen tissue specimens retained at autopsy and the existence of consent for tissue retention and research use. Ethical approval for the acquisition and use of autopsy material for research on transmissible spongiform encephalopathies in the National CJD Surveillance Unit brain bank is covered by LREC 2000/4/157 (Prof. J.W. Ironside). All autopsy cases were of UK origin. The brain from each case had previously been examined histologically and biochemically, and a definite diagnosis of variant, sporadic, or iatrogenic CJD (or non-CJD) had been reached by established criteria.17Budka H Aguzzi A Brown P Brucher JM Bugiani O Gullotta F Haltia M Hauw JJ Ironside JW Jellinger K Neuropathological diagnostic criteria for Creutzfeldt-Jakob disease (CJD) and other human spongiform encephalopathies (prion diseases).Brain Pathol. 1995; 5: 459-466Crossref PubMed Scopus (388) Google Scholar, 18Ironside JW Head MW Bell JE McCardle L Will RG Laboratory diagnosis of variant Creutzfeldt-Jakob disease.Histopathology. 2000; 37: 1-9Crossref PubMed Scopus (157) Google Scholar The protease-resistant prion protein (PrPres) isotype found in brain was classified as type 1, 2A, or 2B as previously described according to the nomenclature of Parchi and colleagues.19Parchi P Castellani R Capellari S Ghetti B Young K Chen SG Farlow M Dickson DW Sima AA Trojanowski JQ Petersen RB Gambetti P Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease.Ann Neurol. 1996; 39: 767-778Crossref PubMed Scopus (721) Google Scholar, 20Parchi 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 (1202) Google Scholar, 21Parchi 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, 22Head MW Bunn TJ Bishop MT McLoughlin V Lowrie S McKimmie CS Williams MC McCardle L Mackenzie J Knight R Will RG Ironside JW Prion protein heterogeneity in sporadic but not variant Creutzfeldt-Jakob disease: U.K. cases 1991–2002.Ann Neurol. 2004; 55: 851-859Crossref PubMed Scopus (131) Google Scholar The polymorphic status of codon 129 of the prion protein gene PRNP of each case was determined by restriction fragment length polymorphism.22Head MW Bunn TJ Bishop MT McLoughlin V Lowrie S McKimmie CS Williams MC McCardle L Mackenzie J Knight R Will RG Ironside JW Prion protein heterogeneity in sporadic but not variant Creutzfeldt-Jakob disease: U.K. cases 1991–2002.Ann Neurol. 2004; 55: 851-859Crossref PubMed Scopus (131) Google Scholar The skeletal muscle taken at autopsy was from quadriceps, sternomastoid, or intercostal muscles. Frozen skeletal and cardiac muscle tissues from CJD and non-CJD control patients were analyzed by the high-sensitivity Western blotting protocol described by Glatzel and colleagues8Glatzel M Abela E Maissen M Aguzzi A Extraneural pathologic prion protein in sporadic Creutzfeldt-Jakob disease.N Engl J Med. 2003; 349: 1812-1820Crossref PubMed Scopus (291) Google Scholar and Wadsworth and colleagues10Wadsworth JD Joiner S Hill AF Campbell TA Desbruslais M Luthert PJ Collinge J Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay.Lancet. 2001; 358: 171-180Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar previously, with a few modifications. Briefly, 10% w/v extracts were made of 65- to 100-mg samples of tissue by homogenizing in an appropriate volume of ice-cold 2% sarkosyl/phosphate-buffered saline (PBS), pH 7.4, using the FastPrep instrument (Qbiogene, Cambridge, UK). The samples were then cleared by centrifugation at 5200 × g for 5 minutes at 4°C. Samples (0.5 ml) of the cleared lysates were diluted with a further 0.5 ml of 2% sarkosyl/PBS and incubated for 10 minutes at 37°C. Benzonase (Sigma, Poole, UK) and MgCl2 were added at final concentrations of 50 U/ml and 1 mmol/L, respectively, and incubation at 37°C was continued for a further 30 minutes. Eighty-one μl of a stock solution of 4% w/v NaPTA and 170 mmol/L MgCl2, pH 7.4, was added (final concentration of NaPTA, 0.3% w/v), and precipitation was allowed to occur for 30 minutes at 37°C. The samples were centrifuged at 20,800 × g for 30 minutes at 37°C. The resultant supernatant was discarded and the pellets were resuspended in 20 μl of 0.1% w/v sarkosyl in PBS, pH 7.4, and digested with 50 μg/ml proteinase K for 30 minutes. Digestion was terminated by the addition of 1 mmol/L PefaBloc SC (Roche, Lewes, UK). Electrophoresis was performed using the NuPAGE Novex gel system (Invitrogen, Paisley, UK). Before electrophoresis, NuPAGE LDS sample buffer was added to each of the samples to a final concentration of 1×. The samples were boiled for 10 minutes and separated on 10% Bis-Tris NuPAGE gels. The separated proteins were then transferred onto immunoblot polyvinylidene difluoride membrane (Bio-Rad, Hertfordshire, UK). For immunodetection, the anti-PrP monoclonal antibody 3F4 (DakoCytomation, Cambridgeshire, UK) was used at a final concentration of 50 ng/ml IgG for 1 hour. Horseradish peroxidase-conjugated anti-mouse IgG F(ab′)2 fragment (Amersham Biosciences, Amersham, UK) was used at a dilution of 1 in 40,000 for 1 hour. The detection reagent used was SuperSignal West fempto maximum sensitivity substrate (Pierce, Rockford, IL). The immunoblots were exposed to HyperFilm ECL (Amersham Biosciences) for periods of 10 seconds to 30 minutes. The molecular weight of PrPres was estimated by reference to IgG-binding MagicMark XP Western protein standards (Invitrogen), and standard PrPres samples from vCJD brain were run on all blots throughout the study. These standards were prepared by PK digestion using the conventional method described previously.18Ironside JW Head MW Bell JE McCardle L Will RG Laboratory diagnosis of variant Creutzfeldt-Jakob disease.Histopathology. 2000; 37: 1-9Crossref PubMed Scopus (157) Google Scholar, 23Head MW Tissingh G Uitdehaag BM Barkhof F Bunn TJ Ironside JW Kamphorst W Scheltens P Sporadic Creutzfeldt-Jakob disease in a young Dutch valine homozygote: atypical molecular phenotype.Ann Neurol. 2001; 50: 258-261Crossref PubMed Scopus (29) Google Scholar Frozen muscle samples from nine non-CJD neurological patients were available for use as negative controls in the Western blots in this study. As a positive control in these experiments, 10 μl of 1% w/v vCJD (type MM2B) brain homogenate was diluted into 0.5 ml of 10% w/v muscle tissue homogenate from one of the non-CJD control patients. This spiked muscle homogenate was then diluted with a further 0.5 ml of 2% sarkosyl as described above. Precipitation with NaPTA and PK digestion were performed as described for the test samples. Samples of CJD patient muscle tissue were assessed by comparison with positive and negative control samples run either on the same gel or on a separate gel run in parallel at the same time using exactly the same conditions. Our laboratory and others have shown tissue-specific effects on the apparent glycosylation profile of PrPres that can lead to an underrepresentation of one of the three characteristic bands observed on the Western blots.9Head MW Ritchie D Smith N McLoughlin V Nailon W Samad S Masson S Bishop M McCardle L Ironside JW Peripheral tissue involvement in sporadic, iatrogenic, and variant Creutzfeldt-Jakob disease: an immunohistochemical, quantitative, and biochemical study.Am J Pathol. 2004; 164: 143-153Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 10Wadsworth JD Joiner S Hill AF Campbell TA Desbruslais M Luthert PJ Collinge J Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay.Lancet. 2001; 358: 171-180Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar Therefore, the following criteria were established before interpreting the results. A positive result was assigned if three or at least two bands were observed to co-migrate with any of the corresponding PrPres bands in the positive control and no bands or smears were seen in the lane containing non-CJD control muscle sample, after maximum exposure to HyperFilm ECL. A negative result was assigned if less than two bands were present in the correct region in the lane containing the test sample on a blot where the positive control was detected. The localization of PrPres in paraffin sections of cardiac and skeletal muscles was investigated using the highly sensitive PET blot method.24Ritchie DL Head MW Ironside JW Advances in the detection of prion protein in peripheral tissues of variant Creutzfeldt-Jakob disease patients using paraffin-embedded tissue blotting.Neuropathol Appl Neurobiol. 2004; 30: 360-368Crossref PubMed Scopus (43) Google Scholar, 25Schulz-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 (190) Google Scholar To establish the cellular location of PrPres deposits, conventional immunohistochemistry was performed to compare the localization of a number of cellular markers with the distribution of PrPres. Serial 5-μm sections from both formalin-fixed and periodate-lysine paraformaldehyde-fixed, formic acid-treated sections were mounted on Superfrost plus slides (VWR, Poole, UK) for immunohistochemistry and on 0.45-μm nitrocellulose membrane for PET blot analysis. Slides and membranes were incubated overnight at 55°C before use. PET blot analysis was performed as described by us previously24Ritchie DL Head MW Ironside JW Advances in the detection of prion protein in peripheral tissues of variant Creutzfeldt-Jakob disease patients using paraffin-embedded tissue blotting.Neuropathol Appl Neurobiol. 2004; 30: 360-368Crossref PubMed Scopus (43) Google Scholar using a modified version of the method of Schulz-Schaeffer and colleagues25Schulz-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 (190) Google Scholar Briefly, sections mounted on nitrocellulose were dewaxed before an overnight digestion in 25 μg/ml of proteinase K. Membranes were washed in Tris-buffered saline containing 0.1% Tween-20 before treatment with 3 mol/L guanidine isothiocyanate for 10 minutes. After a further wash the membranes were blocked with casein and incubated for 2 hours with 3F4 antibody (1:500 dilution). Labeling was completed using a Vectastain ABC-AmP detection system (Vector Laboratories, Peterborough, UK) and visualized using the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate chromogen system. Sections from sCJD brain were included with each PET blot run as a positive control for the primary antibody. Labeling was observed and photographed using a stereomicroscope. Investigations on the cellular location of PrPres in muscle tissue were performed using antibodies raised against neurofilament protein, neuron-specific enolase, synaptophysin, and S-100 protein (all from DakoCytomation). Before immunolabeling sections were dewaxed and immersed in picric acid to remove formalin pigment. After washing in water all sections were pretreated by microwaving in 10 mmol/L citrate buffer, pH 6.0, for 15 minutes. Immunolabeling was then completed using the Envision Plus HRP kit (DakoCytomation) for the anti-neuron-specific enolase antibody of Vectastain Elite ABC kit (Vector Laboratories) for the other antibodies. Staining was visualized using diaminobenzidine. We applied our modified Western blotting protocol to assess muscle tissue samples from a total of 49 CJD patients and 9 non-CJD neurological disease patients. Seventeen patients with a definite diagnosis of vCJD (numbered V1 to V17) were analyzed for PrPres in their skeletal muscle, and eight positives were identified (Figure 1) using the criteria described above. The immunoblot for patient V9 shows that samples taken from these patients contained varying amounts of PrPres (Figure 1, top). Samples assigned as positive from the remaining seven vCJD patients are shown together in the bottom panel of Figure 1. Only a proportion of samples taken from these patients contained detectable PrPres (Table 1), suggesting that PrPres has a heterogeneous distribution within the muscle tissue. The samples that were positive contained PrPres at levels approximately equal to the positive control, which contained 100 μg of vCJD brain homogenate. Because each sample is the NaPTA precipitate from 50 mg of wet weight of tissue, we estimate that the levels of PrPres in these positive muscle samples were ∼1:500 that of vCJD brain.Table 1Pathological, Genetic, and Clinical Profiles of CJD Patients with Muscle Shown to Be Positive for PrPres by Western Blotting (WB) and PET BlottingCaseWBPETCodon 129Brain PrPres typevCJD patients (17 analyzed) V2(1/4)(0/1)MM2B V5(3/7)(0/1)MM2B V6(1/2)(0/1)MM2B V8(1/7)(0/1)MM2B V9(2/4)(0/1)MM2B V12(1/13)(0/1)MM2B V13(1/3)(0/1)MM2BsCJD patients (26 analyzed) S7(1/2)(1/1)MV2A (+1) S8(1/2)(0/1)MM1 + 2A S10(3/3)(1/1)MV1 S11(2/7)(0/1)MM1 S14(0/3)(1/1)MV2A S24(1/2)(0/1)MM1 S25(1/2)(0/1)MV2AiCJD patients (5 analyzed) I4(4/5)(0/1)VV2A I5(0/3)(1/1)MV2AThe number of samples tested (no. positive for PrPSc/total) is indicated in parentheses. The positive scores are indicated in bold type. Open table in a new tab The number of samples tested (no. positive for PrPSc/total) is indicated in parentheses. The positive scores are indicated in bold type. Using this same Western blotting technique, 6 of 26 patients with definite sCJD were identified as being positive for skeletal muscle PrPres (Figure 2). Two of these patients, S7 and S10, were also shown to be positive by PET blotting (see below). Patient S10 had a particularly strong signal for PrPres in all three samples taken. Interestingly, the unglycosylated PrPres fragment was underrepresented in the muscle from patient S10 when compared with PrPres from the CNS (spinal cord) of this patient (Figure 2). The bands identified as being PrPres in the other positive samples were often diffuse. However, the signals obtained were distinct from the results obtained from an analysis of skeletal muscle from nine patients with non-CJD neurological disease. A total of 22 muscle samples were analyzed from this cohort of nine patients, and no bands were observed even on extended exposures, as exemplified by the control samples shown in Figure 1, Figure 2, Figure 3.Figure 3Western blots of iCJD patient muscle samples positive for PrPres. Three samples of muscle from an iCJD patient (I4) are compared with 100 μg of vCJD brain homogenate diluted in non-CJD control muscle homogenate, NaPTA precipitated, and digested with PK (vCJD spike). Also shown are samples of vCJD brain homogenate before (−) and after (+) digestion with PK (50 μg and 200 μg, respectively) without NaPTA precipitation. Nonspiked, non-CJD muscle homogenate, digested with PK, is shown
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