experimental studies in mice were approved by the institutional animal care and use committee of the american red cross holland laboratory . ten inbred , 7-week - old sjl / olahsd ( harlan , bicester , uk ) female mice closely related to the sjl / j strain , which develops spontaneous b - cell lymphomas at > 8 months of age ( 8,9 ) , were intracerebrally injected under isoflurane anesthesia with 1% vcjd human brain homogenate ( world health organization reference material ) ( 10 ) diluted in physiologic saline , while 4 control animals received physiologic saline only . approximately 6 months after infection , visible tumors developed in the neck areas of 5 mice , 4 with vcjd and 1 control . two of the vcjd animals were euthanized on day 199 because of rapid tumor growth ( table ) . the remaining mice in the vcjd group , including 2 other animals with tumors , were later euthanized or died ( range 222386 days ) without noticeable signs of neurologic disease . the animal with tumors was euthanized on day 321 , and the 3 other animals without tumors were euthanized on day 405 . the autopsy of all mice , infected or not , revealed hepatomegaly and splenomegaly , with various degrees of white , nodular infiltrations of the spleen . mice with visible tumors also had massive neoplastic nodular involvement of intestinal , mesenteric , cervical , and axillary lymph nodes and thymus . brains and spleens were removed from all mice , and neoplastic tissues involving lymph nodes were removed from 4 infected and 1 uninfected mouse , and the thymus was removed from 2 infected mice ( table ) . prp was extracted from brains by using high - speed centrifugation , from spleens by using methanol precipitation according to previously described methods ( 5 ) , and from tumors with the procedure applied to the brain . western blotting ( wb ) was performed by using prp - specific monoclonal antibody 6h4 ( prionics , schlieren , switzerland ) or 6d11 as previously described ( 5 ) . in the vcjd group , prp was identified in the brains and spleens of all 10 mice . in 4 mice with tumors , prp was found in neoplastic tissues of lymph nodes and also in the neoplastic thymus of 2 of the mice ( table ) . the figure shows wb analysis of prp extracted from the brain of a vcjd patient and representative tissues of a vcjd mouse with tumors . the glycosylation pattern of prp in mouse tissues was typical of vcjd ; diglycosylated isoforms predominated over monoglycosylated and unglycosylated isoforms , with the unglycosylated isoform corresponding to a 19-kda fragment . on the basis of wb band intensity , we observed that the concentration of prp in neoplastic lymphoreticular tissues ( lanes 35 ) was similar to that seen in the human ( lane 1 ) and mouse ( lane 2 ) brains . among the control mice , prp was not detected in the brain and spleen of any animal or in neoplastic tissues of the single affected animal ( table ) . immunoblot for protease - resistant prion protein ( prp ) from tissues of sjl / olahsd mouse infected with human variant creutzfeld - jakob disease ( vcjd ) . lanes 15 show representative pattern of extracted prp after digestion with proteinase k ( 100 g / ml ) . lane 1 , brain tissue of vcjd patient ( world health organization reference sample ) . lanes 25 , samples from vcjd mouse in which spontaneous lymphoreticular system tumors developed : lane 2 , brain ; lane 3 , spleen with nodular tumors ; lane 4 , tissue from neoplastic lymph nodes ; lane 5 , neoplastic thymus . samples were denatured by boiling for 10 min in laemmli buffer containing 2% -mercaptoethanol , resolved on nupage 12% bis - tris gel ( invitrogen life technologies , carlsbad , ca , usa ) , transferred to nitrocellulose membrane , and probed with anti - prp monoclonal antibody 6d11 ( dilution 1:5,000 ) . major glycoforms of prp are present as 3 bands corresponding to diglycosylated , monoglycosylated , and unglycosylated molecules . using immunohistochemical ( ihc ) tests , hilton and colleagues ( 1 ) showed widespread prp accumulation in the lymphoreticular system of 54 vcjd patients but not in 56 patients with familial or sporadic cjd . in contrast , when sodium phosphotungstate concentration for prp was used to increase the sensitivity of the wb , prp was detected in spleens of 30% of patients with sporadic cjd ( 11 ) . a similar high - sensitivity detection method was used to screen 2,000 tonsils from the general population in a recently reported prospective study , with a negative result ( 12 ) . the same method did not show prp in the tonsils and 1 lymph node of an 83-year - old person who died from nonneurologic disease but who , 5 years before death , received a blood transfusion from a person in whom vcjd later developed ( 3 , r. will , pers . comm . ) . however , another cervical lymph node of this person tested positive for prp by ihc test , although the appendix tested negative . this observation suggests that large retrospective and prospective studies based on screening of appendixes and tonsils with wb may not detect persons who have prp in their lymph nodes . estimates of prevalence of persons infected with the vcjd agent in the uk population may have been biased as a consequence of specimen selection from mostly younger participants . a retrospective study of > 8,000 specimens of appendixes and tonsils included 70% from persons 2029 years of age ( 2 ) , and in a prospective study , approximately half the tonsillectomy samples came from children < 9 years of age ( 12 ) . our observation of the widespread presence of prp in neoplastic lymph nodes of mice infected with the vcjd agent , and its absence in an uninfected mouse , provides experimental evidence that such tissues could be a valuable source for screening for vcjd in humans . the finding of unusually high amounts of prp in neoplastic lymphoreticular tissues of vcjd mice , in a range comparable to that of the human and mouse brain , suggests that rapidly growing lymphoreticular tumors accumulate prp at a high rate . this finding is of particular importance because a recent uk study of samples collected before 1986 , the years preceding the vcjd epidemic , found no prp in lymph nodes from 58 patients with reactive conditions and 21 patients with lymphomas and carcinomas ( 1 ) , which indicates that prp does not spontaneously accumulate in tumors of uninfected persons . whether prp starts to accumulate in lymph nodes before it appears in spleens , appendixes , or tonsils of persons infected with the vcjd agent we observed prp in the brain and neoplastic spleens and lymph nodes during at least half of the incubation period ( 199 days ) when compared to mice with the longest survival time ( > 380 days ) . on the basis of our findings , we propose that screening of lymph node tissues from persons with reactive and neoplastic conditions and patients with various cancers with metastases in lymphoreticular organs could provide additional information , especially regarding older persons , on the prevalence of vcjd in the united kingdom and other european countries .
we report protease - resistant prion protein ( prpres ) in spontaneous lymphoreticular tumors of mice infected with the agent of variant creutzfeldt - jakob disease ( vcjd ) . 
 prpres may accumulate in lymphoreticular system tumors of asymptomatic persons with vcjd . 
 the statistical power of estimates of vcjd prevalence might be increased by expanding screening to include samples of lymphoreticular neoplasms .