Peripheral Tissue Involvement in Sporadic, Iatrogenic, and Variant Creutzfeldt-Jakob Disease
2004; Elsevier BV; Volume: 164; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)63105-7
ISSN1525-2191
AutoresMark Head, Diane Ritchie, Nadine Smith, Victoria McLoughlin, William H. Nailon, Sazia Samad, Steven Masson, Matthew Bishop, Linda McCardle, James W. Ironside,
Tópico(s)Prion Diseases and Protein Misfolding
ResumoHuman prion diseases are rare fatal neurodegenerative conditions that occur as acquired, familial, or idiopathic disorders. A key event in their pathogenesis is the accumulation of an altered form of the prion protein, termed PrPSc, in the central nervous system. A novel acquired human prion disease, variant Creutzfeldt-Jakob disease, is thought to result from oral exposure to the bovine spongiform encephalopathy agent. This disease differs from other human prion diseases in its neurological, neuropathological, and biochemical phenotype. We have used immunohistochemistry and Western blot techniques to analyze the tissue distribution and biochemical properties of PrPSc in peripheral tissues in a unique series of nine cases of variant Creutzfeldt-Jakob disease. We have compared this with the distribution and biochemical forms found in all of the major subtypes of sporadic Creutzfeldt-Jakob disease and in a case of iatrogenic Creutzfeldt-Jakob disease associated with growth hormone therapy. The results show that involvement of the lymphoreticular system is a defining feature of variant Creutzfeldt-Jakob disease, but that the biochemical isoform of PrPSc found is influenced by the cell type in which it accumulates. Human prion diseases are rare fatal neurodegenerative conditions that occur as acquired, familial, or idiopathic disorders. A key event in their pathogenesis is the accumulation of an altered form of the prion protein, termed PrPSc, in the central nervous system. A novel acquired human prion disease, variant Creutzfeldt-Jakob disease, is thought to result from oral exposure to the bovine spongiform encephalopathy agent. This disease differs from other human prion diseases in its neurological, neuropathological, and biochemical phenotype. We have used immunohistochemistry and Western blot techniques to analyze the tissue distribution and biochemical properties of PrPSc in peripheral tissues in a unique series of nine cases of variant Creutzfeldt-Jakob disease. We have compared this with the distribution and biochemical forms found in all of the major subtypes of sporadic Creutzfeldt-Jakob disease and in a case of iatrogenic Creutzfeldt-Jakob disease associated with growth hormone therapy. The results show that involvement of the lymphoreticular system is a defining feature of variant Creutzfeldt-Jakob disease, but that the biochemical isoform of PrPSc found is influenced by the cell type in which it accumulates. The human prion diseases or transmissible spongiform encephalopathies are unique in a number of important respects. Firstly, they occur as familial, acquired, or sporadic forms, each of which appears to be transmissible under certain circumstances. Secondly, the responsible agent is notoriously resistant to standard forms of decontamination. Thirdly, they are closely associated with accumulation in the central nervous system of an abnormally folded isoform of a host-encoded glycoprotein, termed PrPSc, which the prion hypothesis equates with the infectious agent.1Prusiner SB Prions.Proc Natl Acad Sci USA. 1998; 95: 13363-13383Crossref PubMed Scopus (5168) Google Scholar PrPSc can be operationally defined by its partial resistance to proteases and as such, it is referred to as PrPres. The normal cellular form of the protein (PrPC) is susceptible to protease degradation and termed PrPsens. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common human prion disease occurring world wide with a frequency of approximately one per million of the population per annum. Although the cause is unknown, a random stochastic event resulting in the chance conversion of normal cellular PrP (PrPC) to the pathogenic isoform (PrPSc) and its subsequent self-propagation is one possibility. Familial forms of human prion disease are all tightly associated with mutations in the coding sequence of the gene encoding PrP (PRNP), resulting in the formation of a mutant protein that is hypothesized to make conversion to the abnormal isoform considerably more likely. Human prion disease can also be acquired, orally through endocannibalism (kuru), or by medical exposure to contaminated human tissues or derived products (iatrogenic Creutzfeldt-Jakob disease or iCJD) by intracranial, intraocular, or subcutaneous and intramuscular routes.2Brown P Preece M Brandel J-P 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 (469) Google Scholar Susceptibility, and in some instances disease phenotype, in acquired, familial, and sporadic human prion diseases are substantially influenced by the methionine/valine polymorphism at codon 129 of the PRNP gene.3Deslys JP Marce D Dormont D Similar genetic susceptibility in iatrogenic and sporadic Creutzfeldt-Jakob disease.J Gen Virol. 1994; 1: 23-27Crossref Scopus (101) Google Scholar, 4Monari L Chen SG Brown P Parchi P Petersen RB Mikol J Gray F Cortelli P Montagna P Ghetti B Goldfarb LG Gajdusek DC Lugaresi E Gambetti P Autilio-Gambetti L Fatal familial insomnia and familial Creutzfeldt-Jakob disease: different prion proteins determined by a DNA polymorphism.Proc Natl Acad Sci USA. 1994; 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47: 575-582Crossref PubMed Scopus (378) Google Scholar and a highly stereotyped neuropathology.9Ironside JW Head MW Bell JE McCardle L Will RG Laboratory diagnosis of variant Creutzfeldt-Jakob disease.Histopathology. 2000; 37: 1-9Crossref PubMed Scopus (161) Google Scholar All available evidence points to this disease being the first documented zoonotic human prion disease resulting from exposure (probably oral) to the bovine spongiform encephalopathy (BSE) agent.7Will RG Ironside JW Zeidler M Cousens SN Estibeiro K Alperovitch A Poser S Pocchiari M Hofman M Smith PG A new variant of Creutzfeldt-Jakob disease in the UK.Lancet. 1996; 347: 921-925Abstract PubMed Scopus (1877) Google Scholar, 10Collinge J Sidle KC Meads J Ironside J Hill AF Molecular analysis of prion strain variation and the aetiology of 'new variant' CJD.Nature. 1996; 383: 685-690Crossref PubMed Scopus (1594) Google Scholar, 11Bruce 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; 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294: 1729-1731Crossref PubMed Scopus (85) Google Scholar, 16Andrews NJ Farrington CP Ward H Cousins SN Smith PG Molesworth AM Knight R Ironside JW Will RG Deaths from variant Creutzfeldt-Jakob disease in the UK.Lancet. 2003; 361: 751-752Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Peripheral tissue involvement in prion disease, as judged by PrPSc accumulation in elements of the lymphoreticular system, is common in sheep scrapie17Van Keulen LJM Schreuder BEC Meloen RH Mooij-Harkes G Vromans MEW Langeveld JPM Immunohistochemical detection of prion protein in lymphoid tissues of sheep with natural scrapie.J Clin Microbiol. 1996; 34: 1228-1231PubMed Google Scholar and in rodent models;18McBride PA Eikelenbloom P Kraal G Fraser H Bruce ME PrP protein is associated with follicular dendritic cells of spleens and lymph nodes in uninfected and scrapie-infected mice.J Pathol. 1992; 168: 413-418Crossref PubMed Scopus (212) Google Scholar however, this has not been a consistently reported feature of sporadic or familial human prion diseases.19Kitamoto T Mohri S Tateishi J Organ distribution of proteinase-resistant prion protein in human and mice Creutzfeldt-Jakob diseases.J Gen Virol. 1989; 70: 3371-3397Crossref PubMed Scopus (51) Google Scholar, 20Hainfellner JA Budka H Disease associated prion protein may deposit in the peripheral nervous system in human transmissible spongiform encephalopathies.Acta Neuropathol. 1999; 98: 458-460Crossref PubMed Scopus (60) Google Scholar In contrast PrPSc accumulates in the tonsil, spleen, lymph node, and appendix in vCJD21Hill AF Butterworth RJ Joiner S Jackson G Rossor MN Thomas DJ Frosh A Tolley N Bell JE Spencer M King A Al-Sarraj S Ironside JW Lantos PL Collinge J Investigation of variant Creutzfeldt-Jakob disease and other human prion diseases with tonsil biopsy samples.Lancet. 1999; 353: 183-189Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar, 22Joiner S Linehan J Brandner S Wadsworth JDF Collinge J Irregular presence of abnormal prion protein in appendix in variant Creutzfeldt-Jakob disease.J Neurol Neurosurg Psychiatry. 2002; 73: 597-598Crossref PubMed Scopus (29) Google Scholar and this accumulation, in the case of appendix may precede the clinical onset by several years.23Hilton DA Fathers E Edwards P Ironside JW Zajicek J Prion immunoreactivity in appendix before clinical onset of variant Creutzfeldt-Jakob disease.Lancet. 1998; 352: 703-704Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar The presence of PrPSc in peripheral tissues in vCJD may offer diagnostic possibilities.21Hill AF Butterworth RJ Joiner S Jackson G Rossor MN Thomas DJ Frosh A Tolley N Bell JE Spencer M King A Al-Sarraj S Ironside JW Lantos PL Collinge J Investigation of variant Creutzfeldt-Jakob disease and other human prion diseases with tonsil biopsy samples.Lancet. 1999; 353: 183-189Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar Furthermore, because the tonsil and spleen from these cases contain measurable infectivity in mouse bioassay,24Bruce 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 (245) Google Scholar peripheral tissues from those incubating vCJD may also present a risk of iatrogenic transmission. Understanding peripheral pathogenesis is also important in identifying potential therapeutic targets such as peripheral replication, which could present an attractive route to prophylactic treatments.25Farquhar C Dickinson A Bruce M Prophylactic potential of pentosan polysulphate in transmissible spongiform encephalopathies.Lancet. 1999; 353: 117Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar We have examined a wide variety of tissues (n = 14) in a series of sCJD (n = 7) and vCJD (n = 9) cases using a combination of Western blot and immunohistochemistry to detect the presence of the disease-associated form of PrP. Our data shows a consistent pattern of peripheral involvement in vCJD that distinguishes it from both sCJD and, interestingly, peripherally acquired iCJD. Cases of CJD were selected for this study on the basis of the availability of fixed and frozen tissue specimens from a wide range of organs retained at autopsy and the existence of consent for tissue retention and research. Local ethical approval for the use of the material for research has been obtained. 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 reached by established criteria.9Ironside JW Head MW Bell JE McCardle L Will RG Laboratory diagnosis of variant Creutzfeldt-Jakob disease.Histopathology. 2000; 37: 1-9Crossref PubMed Scopus (161) Google Scholar, 26Budka H Aguzzi A Brown P Brucher JM Bugiani O Gullotta F Haltia M Haw JJ Ironside JW Jellinger K Kretzschmar HA Lantos PL Masullo C Schlote W Tateishi J Weller RO Neuropathological diagnostic criteria for Creutzfeldt-Jakob disease (CJD) and other human spongiform encephalopathies (prion diseases).Brain Pathol. 1995; 5: 459-466Crossref PubMed Scopus (392) Google Scholar The case of iCJD was associated with growth hormone therapy. The protease-resistant prion protein (PrPres) isotype found in brain was classified type 1, 2A, or 2B as previously described9Ironside JW Head MW Bell JE McCardle L Will RG Laboratory diagnosis of variant Creutzfeldt-Jakob disease.Histopathology. 2000; 37: 1-9Crossref PubMed Scopus (161) Google Scholar according to accepted nomenclature of Parchi and colleagues5Parchi P Castellani R Capellari S Ghetti B Young K Chen SG Farlow M Dickson DW Sima AAF Trojanowski JQ Petersen RB Gambetti P Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease.Ann Neurol. 1996; 39: 767-778Crossref PubMed Scopus (727) Google Scholar, 27Parchi 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 (1220) Google Scholar, 28Parchi 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 (268) Google Scholar A case of clinically possible CJD that was given a final diagnosis of Lewy body dementia was included as a control for neurodegenerative disease without prion involvement and the absence of PrPres from the brain. The polymorphic status of codon 129 of the prion protein gene PRNP of each case was determined by restriction fragment length polymorphism.8Will RG Zeidler M Stewart GE Macleod MA Ironside JW Cousens SN Mackenzie J Estibeiro K Green AJE Knight RSG Diagnosis of new variant Creutzfeldt-Jakob disease.Ann Neurol. 2000; 47: 575-582Crossref PubMed Scopus (378) Google Scholar The biochemical and genetic data for each case is summarized in Table 1. All possible codon 129 genotype and PrPres isotype combinations are represented in the sCJD cases chosen (MM1, MV1, MM2A, MV2A, VV2A) except the very rare VV1 class. All vCJD cases were methionine homozygotes and had a type 2B PrPres isotype in brain tissue. The tonsil biopsy material was from a case of vCJD diagnosed outside the UK but thought likely to have resulted from a British exposure to BSE and has been described previously.29Kay R Lau WY Ng HK Chan YL Lyon DJ van Hasselt CA Variant Creutzfeldt-Jakob disease in Hong Kong.Hong Kong Med J. 2001; 7: 296-298PubMed Google ScholarTable 1Key to the Diagnostic Classification of the Cases AnalyzedCase identifierDiagnosisPRNP codon 129 genotypeBrain PrPres isotypeC1Lewy body dementiaMM0S1Sporadic CJDMV1S2Sporadic CJDMM2AS3Sporadic CJDMM1S4Sporadic CJDMV2AS5Sporadic CJDVV2AS6Sporadic CJDMM1S7Sporadic CJDMM1V1Variant CJDMM2BV2Variant CJDMM2BV3Variant CJDMM2BV4Variant CJDMM2BV5Variant CJDMM2BV6Variant CJDMM2BV7Variant CJDMM2BV8Variant CJDMM2BV9Variant CJDMM2BI1Iatrogenic CJDMV2A Open table in a new tab PrP was localized in sections of formalin-fixed or periodate-lysine paraformaldehyde-fixed, formic acid-treated tissue by immunohistochemistry. Five-μm paraffin sections were mounted on Superfrost plus slides. PrP immunohistochemistry was performed using the mouse monoclonal antibodies, KG9 (IAH, Compton, UK) and 3F4 (DAKO, Ely, UK) in a protocol that distinguishes PrPSc from the normal cellular form of PrP. Before immunolabeling, sections were taken to water and formalin pigment removed with saturated picric acid. Endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 30 minutes. Sections were pretreated by autoclaving at 121°C in distilled water for 10 minutes, followed by immersion in 96% formic acid for 5 minutes and digestion with proteinase K, diluted to 10 μg/ml in phosphate-buffered saline for 5 minutes. After blocking with normal rabbit serum, nonspecific binding of avidin and biotin was blocked using an avidin/biotin blocking kit (Vector SP-2001; Vector Laboratories, Peterborough, UK), before incubating with the primary antibody KG9 (KG9: 1/20,000 in normal rabbit serum). Selected cases were also immunostained using the same protocol but substituting the monoclonal antibody 3F4 (1:2000 in normal rabbit serum) for KG9. Immunolabeling was completed using the catalyzed signal amplification (CSA) amplification system (DAKO) that is superior in terms of sensitivity to most other immunohistochemical detection systems.30Sabattini E Bisgaard K Ascani S Poggi S Piccioli M Ceccarelli C Pieri F Fraternali-Orcioni G Pileri SA The En Vision++ system: a new immunohistochemical method for diagnostics and research. Critical comparison with the APAAP, ChemMate CSA, LABC and SABC techniques.J Clin Pathol. 1998; 51: 506-511Crossref PubMed Scopus (406) Google Scholar Labeling was visualized using diaminobenzidine and lightly counterstained with hematoxylin. CR2 (the receptor for the C3d complement C3 fragment) was visualized in certain of these same tissues using the monoclonal antibody CD21 (DAKO). For this antigen, sections were taken to water, endogenous peroxidase activity blocked with 3% H2O2 in methanol, and the material partially digested with trypsin. Subsequent detection used the Vectastain elite avidin/biotin kit (Vector Laboratories). For the quantitative assessment of PrP immunostaining, adjacent sections of positive lymphoid follicles from selected organs were cut and stained by immunohistochemistry for KG9 and CD21. Sections were analyzed on a Leica DMR microscope (Leica Microscopy Systems, Milton Keynes, UK) fitted with a Sony 3CCD color video camera (model XC-003P) having a 6.00 mm × 4.96 mm sensing area CCD and 752(H) × 582(V) effective picture elements. A motorized stage (Prior Scientific Instruments Ltd., Cambridge, UK) was fitted to the microscope to provide software control of position and focus. Software developed using Leica Advanced QUIPS, a macro programming facility, was used for automatic image capture, processing, and analysis. For each section, 25 overlapping images were acquired and aligned to form a composite view of the characteristic neuropathology. From this view, four component images were selected at random and those stained for CD21 used to count the number of lymphoid follicles containing follicular dendritic cells. Component images from the adjacent section stained for PrPres using the KG9 antibody were then examined to count the number of follicles that also stained positively for PrPres. All counting was performed by computer-controlled image analysis. The data obtained for each organ was grouped to give an average of the number of stained follicles per section examined. Frozen tissues were analyzed by a variation of the Western blotting protocol described previously.9Ironside JW Head MW Bell JE McCardle L Will RG Laboratory diagnosis of variant Creutzfeldt-Jakob disease.Histopathology. 2000; 37: 1-9Crossref PubMed Scopus (161) Google Scholar, 31Head MW Tissingh G Uitdehaag BMJ Barkhof F Bunn TJR 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 Briefly, 10% w/v extracts were made in nondenaturing conditions by homogenization. The cleared lysate was digested with 50 μg/ml of proteinase K (VWR, Poole, UK) for 1 hour at 37°C. Brain samples were run as twofold serial dilutions of 10% extracts. Proteinase K-treated peripheral tissue extracts, usually 200 μl in the first instance were then concentrated by centrifugation at 21,000 × g for 1 hour at 4°C.32Lee DC Stenland CJ Hartwell RC Ford EK Cai K Miller JLC Gilligan KJ Rubenstein R Fournel M Petteway SR Monitoring plasma processing steps with a sensitive Western blot assay for the detection of the prion protein.J Virol Methods. 2000; 84: 77-89Crossref PubMed Scopus (116) Google Scholar The resultant pellet was resuspended in denaturing buffer and analyzed by Western blotting using the monoclonal antibody 3F4 (DAKO), enhanced chemiluminescence, and exposure to X-ray film.33Head MW Northcott V Rennison K Ritchie D McCardle L Bunn TJR McLennan N Ironside JW Tullo A Bonshek RE Prion protein accumulation in eyes of patients with sporadic and variant Creutzfeldt-Jakob disease.Invest Ophthalmol Vis Sci. 2003; 44: 342-346Crossref PubMed Scopus (83) Google Scholar Where peripheral tissue samples were found to give too strong a signal after concentration, the volume of extract used was scaled down appropriately. In preliminary experiments 10% brain extracts were separated by centrifugation into a supernatant and pellet fraction before proteolytic digestion. The pellet was resuspended in the original starting volume of extraction buffer and equivalent amounts of supernatant and pelleted material digested with proteinase K. In separate experiments the efficiency of recovery of PrPres was also investigated by diluting 5 μl of proteinase K-treated 10% brain extract into 200 μl of extraction buffer and recovery by centrifugation using the centrifugation protocol described above. Protease-resistance and aggregation are features of PrPSc, which distinguish it from the normal cellular form, PrPC. To test whether centrifugation separates protease-sensitive PrPC (PrPsen) and protease-resistant PrPSc (PrPres) from human brain we centrifuged extracts of frontal cortex from sCJD and vCJD. The supernatant and pellet fractions were analyzed separately with and without digestion with proteinase K (Figure 1). Western blot analysis shows the presence of PrP in both the supernatant and pellet fractions. Treatment with proteinase K shows an absence of detectable PrP in the supernatant fraction. In contrast the pelleted material contains readily detectable protease-resistant PrP. Because these Western blots were loaded with equivalent amounts of tissue extracts (∼500 μg of brain) the results suggest that a simple centrifugation at 21,000 × g effects a quantitative separation of PrPres from PrPsen. PrPres found in the cases of sCJD and vCJD used here differ both in the fragment size and glycosylation ratio (PrPres isotype 1 and 2B, respectively). The efficiency of centrifugal recovery and the conservation of the PrPres isotype were determined by analysis of sCJD and vCJD brain extracts that had been diluted by a factor of 40 in extraction buffer and then concentrated by centrifugation. The pellets were resuspended in the starting volume of extraction buffer then compared with the original extract. Loading of Western blots with equivalent amounts of starting brain extracts (∼500 μg) demonstrates that centrifugal recovery is efficient (Figure 1). Moreover the procedure does not alter the mobility of the fragments. Neither does there seem to be any selectivity for the recovery of any one of the three glycoforms present. Centrifugal concentration therefore offers a simple, efficient method for the concentrated of PrPres from tissues in which it may be present at low abundance and allows isotype analysis of the concentrated material. Western blot analysis of representative brain and tonsil samples show that PrP is abundant in all samples of frontal cortex (Figure 2A) but that seen in Lewy body dementia is sensitive to protease digestion (Figure 2B). A significant proportion of the signal remains in frontal cortex samples from each case of CJD indicating the presence of abundant PrPres in the brain (Figure 2B). The signal from untreated tonsil samples is considerably lower than that seen in brain (Figure 2C) and the proportion that appears resistant to protease digestion (Figure 2D) is less abundant than that seen in the corresponding brain samples (Figure 2B). Note that the same amount of brain and tonsil tissue (∼1 mg) was loaded in each lane but that the exposure time for brain samples (Figure 2, A and B) was reduced (by a factor of 36) to the minimum possible (5 seconds) in an attempt to avoid signal saturation. These data suggest a gross discrepancy between the amounts of PrP in brain and tonsil and the proportion of that PrP that is resistant to proteolytic degradation. Positive signals from tonsil are most obvious in the cases of vCJD but faint bands of the same mobility are also present in all other lanes (Figure 2D). To distinguish between genuine aggregated protease-resistant PrPSc and potentially artifactual signals seen in tonsil, protease-treated tonsil extracts were enriched for PrPres by centrifugal concentration of 80 μl of a 10% extract and this was compared with 10 μl of unconcentrated 10% extract (Figure 3). The three PrPres glycoforms with similar mobilities to those found in brain samples were seen in samples of vCJD tonsil but no signals were seen in tonsil samples from sCJD and iCJD (Figure 3B). Centrifugal concentration, usually of 200 μl was then routinely used to analyze all other available peripheral tissue samples. Where possible semiquantitative estimates were made of the amount of PrPres present in peripheral tissue extracts compared to that found in a standard sample of vCJD frontal cortex. The principle is illustrated in Figure 4, in which 200 μl of concentrated tonsil extract from vCJD case V7 was analyzed alongside a twofold serial dilution series (starting with 5 μl) of brain extract from the same case. Visual inspection indicates that the signal intensity from the tonsil lies between the fourth (1:16) and fifth (1:32) serial dilution of brain. Hence, 20 mg tissue equivalents of tonsil gave a signal intermediate between 31 μg and 15 μg tissue equivalents of brain, indicating that the abundance of PrPres in the tonsil sample was between 0.08% and 0.16% of that found in the frontal cortex sample from the same case.Figure 3Western blot analysis of PrPres in tonsil samples from cases C1 (Lewy body dementia), S5, S6, S7 (sCJD) V4, V5, V7 (vCJD), and I1 (iCJD) before (A) and after (B) centrifugal enrichment for PrPres from an eightfold larger volume of extract. +, Indicates a lane loaded with a one-tenth volume of the frontal cortex from vCJD case V7. Both blots were processed together and exposed for the same length of time.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Western blot analysis of a dilution series of cerebral cortex (CC) and concentrated tonsil (To) extract from a case of vCJD (V7). The serial dilution was by a factor of two, starting with a loading of 5 μl of a 10% extract of frontal cortex in the first lane. The lane of tonsil represents 200 μl of a 10% extract enriched (×40) by centrifugal concentration for PrPres.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fourteen different tissues from 18 cases were selected for study. The complete set of tissues was not available in the form of both fixed and frozen tissue for each individual case; however, all available tissues for this data set were analyzed resulting in 158 tissues examined by Western blot and the same number examined by immunohistochemistry. The results of the combined Western blot and immunohistochemical analysis of these tissues are shown in Table 2. The pattern of PrPres accumulation falls into discrete groups:Table 2Summary of the Results of Immunohistochemical (IHC) and Western Blot (WB) Analysis of Prpres in Cerebral Cortex (CC), Trigeminal Ganglion (TG), Dorsal Root Ganglion (DRG), Peripheral Nerve (PN), Tonsil (To), Spleen (Sp), Cervical Lymph Node (LN), Appendix (Ap), Adrenal Gland (Ad), Kidney (Ki), Liver (Li), Lung (Lu), Heart (He), and
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