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We have investigated the presence of disease related prion protein (PrPSc) in appendix samples obtained at necropsy from four neuropathologically confirmed cases of variant Creutzfeldt-Jakob disease (vCJD). PrPSc was detected in only one vCJD appendix, at a level lower than found in a diagnostic tonsil biopsy sample obtained from the same patient. The single PrPSc positive appendix, but not the other samples, also showed abnormal prion protein immunohistochemistry. The finding that appendix samples from three of four cases of vCJD are devoid of detectable PrPSc questions the utility of screening archival appendicectomy tissues to estimate the prevalence of pre-clinical vCJD infection within the UK population.
The appearance of a novel human prion disease, variant Creutzfeldt-Jakob disease (vCJD), in the United Kingdom from 1995 onwards, and the experimental confirmation that this is caused by the same prion strain as that causing BSE in cattle, has raised the possibility that a major epidemic of vCJD will occur in the United Kingdom and other countries as a result of dietary or other exposure to BSE prions.1 The pathogenesis of vCJD differs significantly from that of other forms of CJD. Disease associated prion protein (PrPSc) is readily detectable in lymphoreticular tissues in vCJD and not in classic CJD.1–3 High levels of PrPSc are uniformly found in the central nervous system and lymphoreticular system of vCJD patients.2,3 The highest levels of PrPSc seen outside the central nervous system in vCJD are in tonsil (about 10% of that found in brain)2,3 and tonsil biopsy is used for ante-mortem diagnosis of vCJD.1–3 To date, positive prion protein immunohistochemistry has been reported in only a single appendix sample, although, importantly, this was removed from the patient before the onset of overt features of vCJD.4 While the stage at which lymphoreticular infection occurs in vCJD is unknown, PrPSc accumulation is detectable in the lymphoreticular system in natural sheep scrapie and in experimental rodent models of scrapie at a very early stage of the incubation period, long before the clinical phase of the disease. Based upon these data it has been suggested that large scale screening of surgical tonsillectomy and appendicectomy tissues for PrPSc could provide early warning of a high level of vCJD prion infection and several such studies are in progress.2,5
Recently we reported our concern after finding that PrPSc was undetectable in appendix samples obtained at necropsy from two neuropathologically confirmed vCJD cases.3 While we were not able to examine these samples using immunohistochemical methods, we have now had the opportunity to investigate appendixes from two further vCJD cases by both high sensitivity western blotting and immunohistochemistry.
Methods
Tissue samples
Tissues were collected at necropsy with consent of relatives from two patients with clinical presentations consistent with vCJD. Definite diagnoses of vCJD were confirmed by neuropathological examination and the demonstration of type 4t PrPSc in tonsil.2,3 Appendix samples from these vCJD cases, and appendixes from single neuropathologically confirmed cases of either sporadic CJD or inherited prion disease (144 base-pair insertion), were divided and prepared as either 10% homogenates in phosphate buffered saline (PBS) or fixed in 10% formal saline.
Immunohistochemistry
Tonsil tissue was fixed in 10% buffered formal saline and inactivation of prion infectivity was accomplished by incubation in 98% formic acid for one hour. After further washing for 24 hours in 10% buffered formal saline, tissue samples were processed and paraffin wax embedded. Sections were cut at a nominal thickness of 4 μm, treated with 98% formic acid for five minutes and then boiled in EDTA-TRIS-citrate buffer pH 7.8 for 20 minutes. Immunohistochemical staining was performed with anti-PrP monoclonal antibody 12F10 on a Ventana automated immunohistochemical staining machine using a basic diaminobenzidine detection system according to the manufacturers instructions (Ventana Medical Systems, Tucson, Arizona).
Detection of PrPSc
Sodium phosphotungstic acid precipitation of PrPSc from 0.5 ml 10% tissue homogenates and western blotting using high sensitivity enhanced chemiluminescence was performed as described previously.3
Results
Recently we reported that appendix samples obtained at necropsy from two neuropathologically confirmed vCJD cases contained undetectable levels of PrPSc.3 We have now examined appendix samples from two further neuropathologically confirmed vCJD cases and have detected PrPSc in only one vCJD appendix (fig 1A). The level of PrPSc present in this appendix was compared directly with the level of PrPSc present in a diagnostic tonsil biopsy sample obtained from the same patient. After proteinase K treatment of equivalent aliquots (20 μl) of 10% appendix homogenate or 10% tonsil biopsy homogenate, we observed clear detection of PrPSc in biopsy tonsil homogenate, but not in appendix homogenate (fig 1B). Similarly, PrPSc was readily detectable in necropsy tonsil obtained from the vCJD patient with PrPSc negative appendix (fig 1C). The background single immunoreactive band seen in appendix is also seen in normal tonsil and is attributable to weak cross reactivity of the secondary antibody with an immunoglobulin fragment. While this band is consistently observed after high sensitivity enhanced chemiluminescence of total lymphoreticular homogenate, it is not recovered after sodium phosphotungstic acid precipitation. We determined that the level of PrPSc present in the brain of the vCJD patient with PrPSc positive appendix patient is approximately 15-fold lower than the maximum level we have observed in vCJD brain3 (fig 1D). Based upon these findings we estimate that biopsy tonsil and appendix, contain levels of PrPSc of about 4% and about 0.5%, respectively, of that found in the brain of the same vCJD patient (see legend to fig 1).
(A–D) Western blots of tissue homogenates with anti-PrP monoclonal antibody 3F4. Western blots were analysed by high sensitivity ECL.3 The positions of molecular mass markers are indicated in kilodaltons (kDa). (A) Proteinase K (PK) digestion products from a sodium phosphotungstic acid pellet from 0.5 ml 10% normal human tonsil homogenate spiked with a control level of 10% vCJD brain homogenate (C) is compared with PK digestion products from sodium phosphotungstic acid pellets from 0.5 ml 10% appendix homogenates from vCJD (V), sporadic CJD (S) or inherited prion disease (I) cases. (B) PK digestion products from 20 μl 10% normal human tonsil spiked with a control level of 10% vCJD brain homogenate (C) is compared with PK digestion products from 20 μl of 10% appendix or 10% biopsy tonsil homogenate from the vCJD case with PrPSc positive appendix. (C) Proteinase K digestion products from 20 μl 10% normal human tonsil (NT) is compared with PK digestion products from 20 μl 10% necropsy tonsil homogenate from the vCJD case with PrPSc negative appendix. (D) The western blot shows PK digestion products from 10 μl 10% normal human brain homogenate obtained in the absence of spike (NB) or after spiking with either a control level of 10% vCJD brain homogenate (C) or 0.05, 0.1, 0.25, 0.5, or 1 μl of 10% brain homogenate from the vCJD case with PrPSc positive appendix. The control level of spike of 10% vCJD brain homogenate (C) shown in panels A, B, D is equivalent to 50 nl 10% brain homogenate from a vCJD case previously reported to have a maximal level of PrPSc in brain3; we estimate that an equivalent level of PrPSc is present in about 0.75 μl 10 % brain homogenate from the vCJD case with PrPSc positive appendix. (E) Photomicrograph of PrPSc positive appendix. Immunoreactivity for PrPSc in a lymphatic follicle of the appendix (anti-PrP monoclonal antibody 12F10). The immunostaining pattern is similar to that reported in tonsils in vCJD patients2 and suggests deposition mainly in dendritic cells.
Importantly, we were able to correlate the detection of PrPSc by western blotting in vCJD appendix with the detection of abnormal prion protein staining by immunohistochemistry. Abnormal prion protein deposits were clearly observed on sections from the PrPSc positive vCJD appendix (fig 1E), while prion protein immunoreactivity was unremarkable on sections from the PrPSc negative vCJD appendix or on sections of appendix from the sporadic CJD or inherited prion disease cases (data not shown).
Discussion
Our findings, together with our previously reported inability to detect PrPSc in two other vCJD appendixes,3 indicate that appendix does not reliably report vCJD infection even at the end stage of the disease. This observation must be considered when estimating the possible prevalence of vCJD based upon the analysis of archival appendicectomy tissues.5 Although only a minority of appendixes in vCJD may contain detectable levels of PrPSc, surgical instruments used for appendicectomy should remain a cause of concern for potential iatrogenic transmission of vCJD prions.
Footnotes
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Competing interests: none declared