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Diagnosis and Reporting of Creutzfeldt-Jakob Disease Singeltary, Sr et al. JAMA.2001; 285: 733-734. http://jama.ama-assn.org/cgi/content/full/285/6/733 Full Text Tue, 13 Feb 2001 JAMA Vol. 285 No. 6, February 14, 2001 Letters Diagnosis and Reporting of Creutzfeldt-Jakob Disease To the Editor: In their Research Letter in JAMA. 2000;284:2322-2323, Dr Gibbons and colleagues1 reported that the annual US death rate due to Creutzfeldt-Jakob disease (CJD) has been stable since 1985. These estimates, however, are based only on reported cases, and do not include misdiagnosed or preclinical cases. It seems to me that misdiagnosis alone would drastically change these figures. An unknown number of persons with a diagnosis of Alzheimer disease in fact may have CJD, although only a small number of these patients receive the postmortem examination necessary to make this diagnosis. Furthermore, only a few states have made CJD reportable. Human and animal transmissible spongiform encephalopathies should be reportable nationwide and internationally. Terry S. Singeltary, Sr Bacliff, Tex To the Editor: At the time of my mother's death, various diagnoses were advanced such as "rapid progressive Alzheimer disease," psychosis, and dementia. Had I not persisted and personally sought and arranged a brain autopsy, her death certificate would have read cardiac failure and not CJD. Through CJD Voice1 I have corresponded with hundreds of grief-stricken families who are so devastated by this horrific disease that brain autopsy is the furthest thing from their minds. In my experience, very few physicians suggest it to the family. After the death and when families reflect that they never were sure what killed their loved one it is too late to find the true cause of death. In the years since my mother died I think that the increasing awareness of the nature of CJD has only resulted in fewer pathologists being willing to perform an autopsy in a suspected case of CJD. People with CJD may die with incorrect diagnoses of dementia, psychosis, Alzheimer disease, and myriad other neurological diseases. The true cause of death will only be known if brain autopsies are suggested to the families. Too often the physician's comment is, "Well, it could be CJD but that is so rare it isn't likely." Until CJD is required to be reported to state health departments, as other diseases are, there will be no accurate count of CJD deaths in the United States and thus no way to know if the number of deaths is decreasing, stable, or increasing as it has recently in the United Kingdom. Dorothy E. Kraemer Stillwater, Okla In Reply: Mr Singeltary and Ms Kraemer express an underlying concern that our recently reported mortality surveillance estimate of about 1 CJD case per million population per year in the United States since 1985 may greatly underestimate the true incidence of this disease. Based on evidence from epidemiologic investigations both within and outside the United States, we believe that these national estimates are reasonably accurate. Even during the 1990s in the United Kingdom, where much attention and public health resources have been devoted to prion disease surveillance, the reported incidence of classic CJD is similar to that reported in the United States. In addition, in 1996, active US surveillance for CJD and new variant (nv) CJD in 5 sites detected no evidence of the occurrence of nvCJD and showed that 86% of the CJD cases in these sites were identifiable through routinely collected mortality data. Our report provides additional evidence against the occurrence of nvCJD in the United States based on national mortality data analyses and enhanced surveillance. It specifically mentions a new center for improved pathology surveillance. We hope that the described enhancements along with the observations of Singeltary and Kraemer will encourage medical care providers to suggest brain autopsies for more suspected CJD cases to facilitate the identification of potentially misdiagnosed CJD cases and to help monitor the possible occurrence of nvCJD. Creutzfeldt-Jakob disease is not on the list of nationally notifiable diseases. In those states where surveillance personnel indicate that making this disease officially notifiable would meaningfully facilitate collection of data that are needed to monitor the incidence of CJD and nvCJD, including the obtaining of brain autopsy results, we encourage such a change. However, adding CJD to the notifiable diseases surveillance system may lead to potentially wasteful, duplicative reporting because the vast majority of the diagnosed cases would also be reported through the mortality surveillance system. Furthermore, making CJD a notifiable disease may not necessarily help identify undiagnosed CJD cases. The unique characteristics of CJD make mortality data a useful surrogate for ongoing surveillance. Unlike many other neurologic diseases, CJD is invariably fatal and in most cases rapidly progressive and distinguishable clinically from other neurologic diseases. Because CJD is least accurately diagnosed early in the course of the illness, notifiable disease surveillance of CJD could be less accurate than mortality surveillance of CJD. In addition, because death as a condition is more completely and consistently reported, mortality surveillance has the advantage of being ongoing and readily available. The absence of CJD and nvCJD from the list of nationally notifiable diseases should not be interpreted to mean that they are not important to public health; this list does not include all such diseases. We encourage medical caregivers to report to or consult with appropriate public health authorities about any diagnosed case of a transmissible disease for which a special public health response may be needed, including nvCJD, and any patient in whom iatrogenic transmission of CJD may be suspected. Robert V. Gibbons, MD, MPH Robert C. Holman, MS Ermias D. Belay, MD Lawrence B. Schonberger, MD, MPH Division of Viral and Rickettsial Diseases National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Ga http://jama.amaassn.org/cgi/content/full/285/6/733maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=dignosing+and+reporting+creutzfeldt+jakob+disease&searchid=1048865596978_1528&stored_search=&FIRSTINDEX=0&journalcode=jama Singeltary, Sr et al. JAMA Neurology 2003;60:176-181 Reply to Singletary RE-Monitoring the occurrence of emerging forms of Creutzfeldt-Jakob disease in the United States -------------------------------------------------------------------------------- Send Post-Publication Peer Review to journal: disease in the United States I lost my mother to hvCJD (Heidenhain Variant CJD). I would like to comment on the CDC's attempts to monitor the occurrence of emerging forms of CJD. Asante, Collinge et al [1] have reported that BSE transmission to the 129-methionine genotype can lead to an alternate phenotype that is indistinguishable from type 2 PrPSc, the commonest sporadic CJD. However, CJD and all human TSEs are not reportable nationally. CJD and all human TSEs must be made reportable in every state and internationally. I hope that the CDC does not continue to expect us to still believe that the 85%+ of all CJD cases which are sporadic are all spontaneous, without route/source. We have many TSEs in the USA in both animal and man. CWD in deer/elk is spreading rapidly and CWD does transmit to mink, ferret, cattle, and squirrel monkey by intracerebral inoculation. With the known incubation periods in other TSEs, oral transmission studies of CWD may take much longer. Every victim/family of CJD/TSEs should be asked about route and source of this agent. To prolong this will only spread the agent and needlessly expose others. In light of the findings of Asante and Collinge et al, there should be drastic measures to safeguard the medical and surgical arena from sporadic CJDs and all human TSEs. I only ponder how many sporadic CJDs in the USA are type 2 PrPSc? http://www.neurology.org/cgi/eletters/60/2/176#535 My name is Terry S Singeltary Sr, and I live in Bacliff, Texas. I lost my mom to hvCJD (Heidenhain variant CJD) and have been searching for answers ever since. What I have found is that we have not been told the truth. CWD in deer and elk is a small portion of a much bigger problem. largely unsatisfied after being told that a close relative died from a rapidly progressive dementia compatible with spontaneous Creutzfeldt-Jakob disease (CJD). So he decided to gather hundreds of documents on transmissible spongiform encephalopathies (TSE) and realised that if Britons could get variant CJD from bovine spongiform encephalopathy (BSE), Americans might get a similar disorder from chronic wasting disease (CWD) the relative of mad cow disease seen among deer and elk in the USA. Although his feverish search did not lead him to the smoking gun linking CWD to a similar disease in North American people, it did uncover a largely disappointing situation. occurrence of CJD and CWD in the USA. Only a few states have made CJD reportable. Human and animal TSEs should be reportable nationwide and internationally, he complained in a letter to the Journal of the American Medical Association (JAMA 2003; 285: 733). I hope that the CDC does not continue to expect us to still believe that the 85% plus of all CJD cases which are sporadic are all spontaneous, without route or source. region in Colorado. But since early 2002, it has been reported in other areas, including Wisconsin, South Dakota, and the Canadian province of Saskatchewan. Indeed, the occurrence of CWD in states that were not endemic previously increased concern about a widespread outbreak and possible transmission to people and cattle. transmitted to cattle by intracerebral inoculation and that it can cross the mucous membranes of the digestive tract to initiate infection in lymphoid tissue before invasion of the central nervous system. Yet the plausibility of CWD spreading to people has remained elusive. is only reported in those areas known to be endemic foci of CWD. Moreover, US authorities have been criticised for not having performed enough prionic tests in farm deer and elk. issued a directive to state public-health and agriculture officials prohibiting material from CWD-positive animals from being used as an ingredient in feed for any animal species, epidemiological control and research in the USA has been quite different from the situation in the UK and Europe regarding BSE. teeth, Singeltary argues. You get it when they want you to have it, and only what they want you to have. University of Michigan (Ann Arbor, MI, USA), says that current surveillance of prion disease in people in the USA is inadequate to detect whether CWD is occurring in human beings; adding that, the cases that we know about are reassuring, because they do not suggest the appearance of a new variant of CJD in the USA or atypical features in patients that might be exposed to CWD. However, until we establish a system that identifies and analyses a high proportion of suspected prion disease cases we will not know for sure . The USA should develop a system modelled on that established in the UK, he points out. Ali Samii, a neurologist at Seattle VA Medical Center who recently reported the cases of three hunters two of whom were friends who died from pathologically confirmed CJD, says that at present there are insufficient data to claim transmission of CWD into humans; adding that [only] by asking [the questions of venison consumption and deer/elk hunting] in every case can we collect suspect cases and look into the plausibility of transmission further. Samii argues that by making both doctors and hunters more aware of the possibility of prions spreading through eating venison, doctors treating hunters with dementia can consider a possible prion disease, and doctors treating CJD patients will know to ask whether they ate venison. the [Samii] cases because there is no evidence that the men ate CWD-infected meat. He notes that although the likelihood of CWD jumping the species barrier to infect humans cannot be ruled out 100% and that [we] cannot be 100% sure that CWD does not exist in humans& the data seeking evidence of CWD transmission to humans have been very limited. BRITISH MEDICAL JOURNAL SOMETHING TO CHEW ON BMJ CWD is just a small piece of a very big puzzle. I have seen while deer hunting, deer, squirrels and birds, eating from cattle feed troughs where they feed cattle, the high protein cattle by products, at least up until Aug. 4, 1997. So why would it be so hard to believe that this is how they might become infected with a TSE. Or, even by potentially infected land. It's been well documented that it could be possible, from scrapie. Cats becoming infected with a TSE. Have you ever read the ingredients on the labels of cat and dog food? But, they do not put these tissues from these animals in pharmaceuticals, cosmetics, nutritional supplements, hGH, hPG, blood products, heart valves, and the many more products that come from bovine, ovine, or porcine tissues and organs. So, as I said, this CWD would be a small piece of a very big puzzle. But, it is here, and it most likely has killed. You see, greed is what caused this catastrophe, rendering and feeding practices. But, once Pandora's box was opened, the potential routes of infection became endless. No BSE in the U.S.A.? I would not be so sure of that considering that since 1990; Since 1990 the U.S. has raised 1,250,880,700 cattle; Since 1990 the U.S. has ONLY checked 8,881 cattle brains for BSE, as of Oct. 4, 1999; There are apprx. 100,000 DOWNER cattle annually in the U.S., that up until Aug. 4, 1997 went to the renders for feed; Scrapie running rampant for years in the U.S., 950 infected FLOCKS, as of Aug. 1999; Our feeding and rendering practices have mirrored that of the U.K. for years, some say it was worse. Everything from the downer cattle, to those scrapie infected sheep, to any roadkill, including the city police horse and the circus elephant went to the renders for feed and other products for consumption. Then they only implemented a partial feed ban on Aug. 4, 1997, but pigs, chickens, dogs, and cats, and humans were exempt from that ban. So they can still feed pigs and chickens those potentially TSE tainted by-products, and then they can still feed those by-products back to the cows. I believe it was Dr. Joe Gibbs, that said, the prion protein, can survive the digestinal track. So you have stopped nothing. It was proven in Oprah Winfrey's trial, that Cactus Cattle feeders, sent neurologically ill cattle, some with encephalopathy stamped on the dead slips, were picked up and sent to the renders, along with sheep carcasses. Speaking of autopsies, I have a stack of them, from CJD victims. You would be surprised of the number of them, who ate cow brains, elk brains, deer brains, or hog brains. I believe all these TSE's are going to be related, and originally caused by the same greedy Industries, and they will be many. Not just the Renders, but you now see, that they are re-using medical devices that were meant for disposal. Some medical institutions do not follow proper auto- claving procedures (even Olympus has put out a medical warning on their endescopes about CJD, and the fact you cannot properly clean these instruments from TSE's), and this is just one product. Another route of infection. Regardless what the Federal Government in the U.S. says. It's here, I have seen it, and the longer they keep sweeping it under the rug and denying the fact that we have a serious problem, one that could surpass aids (not now, but in the years to come, due to the incubation period), they will be responsible for the continued spreading of this deadly disease. It's their move, it's CHECK, but once CHECKMATE has been called, how many thousands or millions, will be at risk or infected or even dead. You can't play around with these TSE's. I cannot stress that enough. They are only looking at body bags, and the fact the count is so low. But, then you have to look at the fact it is not a reportable disease in most states, mis-diagnosis, no autopsies performed. The fact that their one-in-a- million theory is a crude survey done about 5 years ago, that's a joke, under the above circumstances. A bad joke indeed........ The truth will come, but how many more have to die such a hideous death. It's the Government's call, and they need to make a serious move, soon. This problem, potential epidemic, is not going away, by itself. Terry S. Singeltary Sr. BMJ In reading your short article about 'Scientist warn of CJD epidemic' news in brief Jan. 1, 2000. I find the findings in the PNAS old news, made famous again. Why is the U.S. still sitting on their butts, ignoring the facts? We have the beginning of a CJD epidemic in the U.S., and the U.S. Gov. is doing everything in it's power to conceal it. The exact same recipe for B.S.E. existed in the U.S. for years and years. In reading over the Qualitative Analysis of BSE Risk Factors-1, this is a 25 page report by the USDA:APHIS:VS. It could have been done in one page. The first page, fourth paragraph says it all; "Similarities exist in the two countries usage of continuous rendering technology and the lack of usage of solvents, however, large differences still remain with other risk factors which greatly reduce the potential risk at the national level." Then, the next 24 pages tries to down-play the high risks of B.S.E. in the U.S., with nothing more than the cattle to sheep ratio count, and the geographical locations of herds and flocks. That's all the evidence they can come up with, in the next 24 pages. Something else I find odd, page 16; "In the United Kingdom there is much concern for a specific continuous rendering technology which uses lower temperatures and accounts for 25 percent of total output. This technology was _originally_ designed and imported from the United States. However, the specific application in the production process is _believed_ to be different in the two countries." A few more factors to consider, page 15; "Figure 26 compares animal protein production for the two countries. The calculations are based on slaughter numbers, fallen stock estimates, and product yield coefficients. This approach is used due to variation of up to 80 percent from different reported sources. At 3.6 million tons, the United States produces 8 times more animal rendered product than the United Kingdom." "The risk of introducing the BSE agent through sheep meat and bone meal is more acute in both relative and absolute terms in the United Kingdom (Figures 27 and 28). Note that sheep meat and bone meal accounts for 14 percent, or 61 thousand tons, in the United Kingdom versus 0.6 percent or 22 thousand tons in the United States. For sheep greater than 1 year, this is less than one-tenth of one percent of the United States supply." "The potential risk of amplification of the BSE agent through cattle meat and bone meal is much greater in the United States where it accounts for 59 percent of total product or almost 5 times more than the total amount of rendered product in the United Kingdom." Considering, it would only take _one_ scrapie infected sheep to contaminate the feed. Considering Scrapie has run rampant in the U.S. for years, as of Aug. 1999, 950 scrapie infected flocks. Also, Considering only one quarter spoonful of scrapie infected material is lethal to a cow. Considering all this, the sheep to cow ration is meaningless. As I said, it's 24 pages of B.S.e. To be continued... Terry S. Singeltary Sr. P.O. Box 42 Bacliff, Texas USA http://www.bmj.com/cgi/eletters/320/7226/8/b#EL1 HUMAN TSE USA 2005 Animal Prion Diseases Relevant to Humans (unknown types?) About Human Prion Diseases / Bovine Spongiform Encephalopathy (BSE) is a prion disease of cattle. Since 1986, when BSE was recognized, over 180,000 cattle in the UK have developed the disease, and approximately one to three million are likely to have been infected with the BSE agent, most of which were slaughtered for human consumption before developing signs of the disease. The origin of the first case of BSE is unknown, but the epidemic was caused by the recycling of processed waste parts of cattle, some of which were infected with the BSE agent and given to other cattle in feed. Control measures have resulted in the consistent decline of the epidemic in the UK since 1992. Infected cattle and feed exported from the UK have resulted in smaller epidemics in other European countries, where control measures were applied later. Compelling evidence indicates that BSE can be transmitted to humans through the consumption of prion contaminated meat. BSE-infected individuals eventually develop vCJD with an incubation time believed to be on average 10 years. As of November 2004, three cases of BSE have been reported in North America. One had been imported to Canada from the UK, one was grown in Canada, and one discovered in the USA but of Canadian origin. There has been only one case of vCJD reported in the USA, but the patient most likely acquired the disease in the United Kingdom. If current control measures intended to protect public and animal health are well enforced, the cattle epidemic should be largely under control and any remaining risk to humans through beef consumption should be very small. (For more details see Smith et al. British Medical Bulletin, 66: 185. 2003.) Chronic Wasting Disease (CWD) is a prion disease of elk and deer, both free range and in captivity. CWD is endemic in areas of Colorado, Wyoming, and Nebraska, but new foci of this disease have been detected in Nebraska, South Dakota, New Mexico, Wisconsin, Mississippi Kansas, Oklahoma, Minnesota, Montana, and Canada. Since there are an estimated 22 million elk and deer in the USA and a large number of hunters who consume elk and deer meat, there is the possibility that CWD can be transmitted from elk and deer to humans. As of November 2004, the NPDPSC has examined 26 hunters with a suspected prion disease. However, all of them appeared to have either typical sporadic or familial forms of the disease. The NPDPSC coordinates with the Centers for Disease Control and state health departments to monitor cases from CWD-endemic areas. Furthermore, it is doing experimental research on CWD transmissibility using animal models. (For details see Sigurdson et al. British Medical Bulletin. 66: 199. 2003 and Belay et al. Emerging Infectious Diseases. 10(6): 977. 2004.) http://www.cjdsurveillance.com/abouthpd-animal.html SEE STEADY INCREASE IN SPORADIC CJD IN THE USA FROM 1997 TO 2004. SPORADIC CJD CASES TRIPLED, and that is with a human TSE surveillance system that is terrible flawed. in 1997 cases of the _reported_ cases of cjd were at 54, to 163 _reported_ cases in 2004. see stats here; p.s. please note the 47 PENDING CASES to Sept. 2005 p.s. please note the 2005 Prion D. total 120(8) 8=includes 51 type pending, 1 TYPE UNKNOWN ??? p.s. please note sporadic CJD 2002(1) 1=3 TYPE UNKNOWN??? p.s. please note 2004 prion disease (6) 6=7 TYPE UNKNOWN??? http://www.cjdsurveillance.com/resources-casereport.html CWD TO HUMANS = sCJD ??? AS implied in the Inset 25 we must not _ASSUME_ that snip... http://www.bseinquiry.gov.uk/files/yb/1991/01/04004001.pdf ATYPICAL TSEs in USA CATTLE AND SHEEP ? http://www.bseinquiry.gov.uk/files/sc/seac17/tab03.pdf Infected and Source Flocks As of August 31, 2005, there were 115 scrapie infected and source flocks (figure 3). There were 3 new infected and source flocks reported in August (Figure 4) with a total of 148 flocks reported for FY 2005 (Figure 5). The total infected and source flocks that have been released in FY 2005 are 102 (Figure 6), with 5 flocks released in August. The ratio of infected and source flocks released to newly infected and source flocks for FY 2005 = 0.69 : snip... full text ; http://www.aphis.usda.gov/vs/nahps/scrapie/monthly_report/monthly-report.html AS of July 31, 2005, there were 120 scrapie infected soure flocks (figure 3). There were 16 new infected and source flocks reorted in July (Figure 4) with a total of 143 flocks reported for FY 2005 (Figure 5). The total infected and source flocks that have been released in FY 2005 are 89 (Figure 6), with 8 flocks released in July. The ratio of infected and source flocks released to newly infected and source flocks for FY = 0.62 : 1. IN addition, as of July 31, 2005, 524 scrapie cases have been confirmed and reported by the National Veterinary Services Laboratories (NVSL), of which 116 were RSSS cases (Figure 7). This includes 76 newly confirmed cases in July 2005 (Figure 8). Fifteen cases of scrapie in goats have been reported since 1990 (Figure 9). The last goat case was reported in May 2005. ........... snip... http://www.aphis.usda.gov/vs/nahps/scrapie/monthly_report/monthly-report.html SCRAPIE USA MONTHLY REPORT 2005 AS of March 31, 2005, there were 70 scrapie infected source flocks (Figure FULL TEXT ; http://www.aphis.usda.gov/vs/nahps/scrapie/monthly_report/monthly-report.html Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0502296102 A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes ( sheep prion | transgenic mice ) Annick Le Dur *, Vincent Béringue *, Olivier Andréoletti , Fabienne Reine *, Thanh Lan Laď *, Thierry Baron , Bjřrn Bratberg ¶, Jean-Luc Vilotte ||, Pierre Sarradin **, Sylvie L. Benestad ¶, and Hubert Laude * Scrapie in small ruminants belongs to transmissible spongiform encephalopathies (TSEs), or prion diseases, a family of fatal neurodegenerative disorders that affect humans and animals and can transmit within and between species by ingestion or inoculation. Conversion of the host-encoded prion protein (PrP), normal cellular PrP (PrPc), into a misfolded form, abnormal PrP (PrPSc), plays a key role in TSE transmission and pathogenesis. The intensified surveillance of scrapie in the European Union, together with the improvement of PrPSc detection techniques, has led to the discovery of a growing number of so-called atypical scrapie cases. These include clinical Nor98 cases first identified in Norwegian sheep on the basis of unusual pathological and PrPSc molecular features and "cases" that produced discordant responses in the rapid tests currently applied to the large-scale random screening of slaughtered or fallen animals. Worryingly, a substantial proportion of such cases involved sheep with PrP genotypes known until now to confer natural resistance to conventional scrapie. Here we report that both Nor98 and discordant cases, including three sheep homozygous for the resistant PrPARR allele (A136R154R171), efficiently transmitted the disease to transgenic mice expressing ovine PrP, and that they shared unique biological and biochemical features upon propagation in mice. These observations support the view that a truly infectious TSE agent, unrecognized until recently, infects sheep and goat flocks and may have important implications in terms of scrapie control and public health. -------------------------------------------------------------------------------- Author contributions: H.L. designed research; A.L.D., V.B., O.A., F.R., T.L.L., J.-L.V., and H.L. performed research; T.B., B.B., P.S., and S.L.B. contributed new reagents/analytic tools; V.B., O.A., and H.L. analyzed data; and H.L. wrote the paper. A.L.D. and V.B. contributed equally to this work. To whom correspondence should be addressed. Hubert Laude, E-mail: laude@jouy.inra.fr www.pnas.org/cgi/doi/10.1073/pnas.0502296102 From: TSS () 0022-538X/05/$08.00+0 doi:10.1128/JVI.79.21.13794-13796.2005 Interspecies Transmission of Chronic Wasting Disease Prions to Squirrel Monkeys (Saimiri sciureus) Received 3 May 2005/ Accepted 10 August 2005 Chronic wasting disease (CWD) is an emerging prion disease of deer and elk. The risk of CWD transmission to humans following exposure to CWD-infected tissues is unknown. To assess the susceptibility of nonhuman primates to CWD, two squirrel monkeys were inoculated with brain tissue from a CWD-infected mule deer. The CWD-inoculated squirrel monkeys developed a progressive neurodegenerative disease and were euthanized at 31 and 34 months postinfection. Brain tissue from the CWD-infected squirrel monkeys contained the abnormal isoform of the prion protein, PrP-res, and displayed spongiform degeneration. This is the first reported transmission of CWD to primates. * Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178. Phone: (402) 280-1811. Fax: (402) 280-1875. E-mail: jbartz@creighton.edu . Deceased. Journal of Virology, November 2005, p. 13794-13796, Vol. 79, No. 21 Title: Experimental Second Passage of Chronic Wasting Disease (Cwd-Mule Deer) Agent to Cattle Hamir, Amirali http://www.ars.usda.gov/research/publications/publications.htm?seq_no_115=178318 Lancet Neurology 2005; 4:805-814 DOI:10.1016/S1474-4422(05)70225-8 Coexistence of multiple PrPSc types in individuals with Creutzfeldt-Jakob disease Magdalini Polymenidou a, Katharina Stoeck a, Markus Glatzel a b, Martin Vey c, Anne Bellon c and Adriano Aguzzi a Summary Methods Findings Interpretation Affiliations a Institute of Neuropathology, University Hospital Zurich, Switzerland Correspondence to: Dr Adriano Aguzzi, Institute of Neuropathology, University Hospital of Zürich, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland http://www.thelancet.com/journals/laneur/article/PIIS1474442205702258/abstract DOI:10.1016/S1474-4422(05)70225-8 Coexistence of multiple PrPSc types in individuals with Creutzfeldt-Jakob disease Magdalini Polymenidou a, Katharina Stoeck a, Markus Glatzel a b, Martin Vey c, Anne Bellon c and Adriano Aguzzi a Introduction Prion diseases are invariably fatal neurodegenerative disorders of infectious, sporadic, or genetic origin, that affect human beings and many species of animals. Biochemically, these diseases are characterised by the accumulation of a pathological protein, called the scrapie prion protein (PrPSc). This protein is a conformational isoform of a cellular protein, PrPC, and is thought to be the infectious agent. Conversion of PrPC into its pathological isoform involves a structural modification that results in an increase in -sheet content,1–3 aggregation,4,5 and partial resistance to proteolytic cleavage.6 Incubation of a prion-infected brain homogenate with proteinase K (PK) under defined conditions results in complete degradation of PrPC, but not PrPSc. PrPSc loses approximately 65 N-terminal amino acids, but maintains its resistant core, often referred to as PrP27–30 which indicates the altered molecular weight range of the partially digested molecule.6,7 This truncated molecule of PrPSc serves as a well-established marker of prion infection. Prion strains are defined as infectious prion isolates that show distinct disease phenotypes, such as incubation times and histopathological lesion profiles, which persist on serial transmission.8–10 The existence of prion strains was originally taken as evidence against the protein-only hypothesis, since it was difficult to imagine how the distinct information of each strain could be captured in a protein-only infectious agent, without any contribution by nucleic acids. Experiments with two strains of transmissible mink encephalopathy suggested that the prion diversity could indeed be conferred by a single protein, and that the information could be enciphered within distinct protein structures.11,12 Although distinct prion strains can only be identified by bioassays with confirmatory transmission of the strain characteristics to new hosts, variations in the banding pattern of PK-digested PrPSc can serve as a biochemical prion strain indicator.13 Differences in the level of glycosylation, as well as in the size of the PK-digested PrPSc, are widely used as surrogates of prion strain typing (eg, to distinguish sporadic from variant Creutzfeldt-Jakob disease [CJD]).14 However, the molecular basis for these differences and their relation to disease characteristics remain unknown. The differences in the size of PK-digested PrPSc molecules are thought to result from strain-specific conformational states that, in turn, lead to exposure of distinct cleavage sites for the enzyme.11 The latter phenomenon is thought to produce the observed variation in PrPSc from CJD patients. These biochemically distinguishable types of sporadic CJD (sCJD) are believed to represent distinct human prion strains, although they formally do not qualify as such, unless serial passage in experimental animals is done and persistence of strain characteristics in new hosts is shown. Since transmission is not practical for each case, the biochemical appearance of PrPSc serves as a surrogate strain-typing marker for CJD, which allows molecular classification of CJD types. In addition, the host genotype at the polymorphic codon 129 (which can be either methionine [M] or valine [V]) of the prion protein gene PRNP influences the course of disease and the CJD type.15–18 However, investigators deviate in their nomenclature, and to date at least two different CJD classifications have been proposed19,20 (figure 1). In the classification proposed by Gambetti and colleagues,20 two distinct PrPSc were identified after PK digestion: one Published online October 31, 2005 DOI:10.1016/S1474-4422(05) 70225-8 Institute of Neuropathology, University Hospital Zurich, Switzerland (M Polymenidou PhD, K Stoeck MD, M Glatzel MD, A Aguzzi PhD); present address: Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany (M Glatzel MD), and ZLB Behring, Marburg, Germany (M Vey PhD, A Bellon PhD) Correspondence to: Dr Adriano Aguzzi, Institute of Neuropathology, University Hospital of Zürich, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland adriano@pathol.unizh.ch http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8 1 Coexistence of multiple PrPSc types in individuals with Creutzfeldt-Jakob disease Magdalini Polymenidou, Katharina Stoeck, Markus Glatzel, Martin Vey, Anne Bellon, and Adriano Aguzzi Summary Background The molecular typing of sporadic Creutzfeldt-Jakob disease (CJD) is based on the size and glycoform ratio of protease-resistant prion protein (PrPSc), and on PRNP haplotype. On digestion with proteinase K, type 1 and type 2 PrPSc display unglycosylated core fragments of 21 kDa and 19 kDa, resulting from cleavage around amino acids 82 and 97, respectively. Methods We generated anti-PrP monoclonal antibodies to epitopes immediately preceding the differential proteinase K cleavage sites. These antibodies, which were designated POM2 and POM12, recognise type 1, but not type 2, PrPSc. Findings We studied 114 brain samples from 70 patients with sporadic CJD and three patients with variant CJD. Every patient classified as CJD type 2, and all variant CJD patients, showed POM2/POM12 reactivity in the cerebellum and other PrPSc-rich brain areas, with a typical PrPSc type 1 migration pattern. Interpretation The regular coexistence of multiple PrPSc types in patients with CJD casts doubts on the validity of electrophoretic PrPSc mobilities as surrogates for prion strains, and questions the rational basis of current CJD classifications. Articles with an unglycosylated fragment migrating at 21 kDa, named PrPSc type 1, and the second, with unglycosylated fragment of 19 kDa, named PrPSc type 2. Protein sequencing revealed that PrPSc type 1 results from PK cleavage at position 82, whereas PrPSc type 2 is generated by cleavage at amino acid 97.7 Hill and colleagues19 subclassified PrPSc type 1 into two subgroups with core fragment sizes differing by less than 1 kDa. These cases were classified as CJD type 1 and CJD type 2 (with slightly lower molecular weight). Moreover, another type, observed in only one case, was reported, with a molecular weight slightly higher than type 1 (CJD type 6).19 PrPSc type 2 in Gambetti’s classification represents CJD type 3 according to Hill and colleagues,19 who have distinguished two further subtypes within this group. Both are detectable with unglycosylated fragments at 19 kDa, which intensity in comparison with the monoglycosylated band is either equally strong (in MV patients) or weaker (in subtype VV or MM individuals). Herein, we will refer to CJD type 1 and 2 as defined by the Gambetti classification,20 unless indicated otherwise. The controversy is possibly due to very small differences in PK-digested unglycosylated PrPSc fragments and to deviations of their apparent molecular weight due to diverse gel and western blotting systems. In addition to the main PK digestion sites at positions 82 and 97, several others have been identified, possibly accounting for the disparity in the classification of PrPSc type 1 cases.7 Dependence of PK digestion site on the concentration of metal ions,21 or on the pH of brain homogenates,22,23 may also contribute to the discrepancies. The recent identification of CJD cases with apparent co-occurrence of multiple PrPSc types indicates that CJD types can combine to increase disease diversity.24–26 Some studies have suggested that monoclonal antibodies with epitopes directed against the sequence of PrP that is differentially cleaved by PK in different strains can be used for strain typing. For example, an octarepeat-specific antibody was shown to discriminate sheep scrapie from bovine spongiform encephalopathy (BSE).27,28 Here, we have produced and characterised by epitope mapping several anti-PrP monoclonal antibodies with specificities that span the entire prion protein. Two of these, named POM2 and POM12, were found to detect repetitive epitopes located exactly at the boundary that is differentially cleaved by PK on PrPSc types 1 and 2. POM2 and POM12 specifically recognise PrPSc species that are cleaved at any site N-proximal of amino acid 82, but not after this position. Comparative western blot analysis of brain samples from 70 patients with sCJD and three with variant CJD (vCJD) showed that all patients classified as CJD type 2 or vCJD, on the basis of conventional strain typing from cortical samples, showed POM2/POM12 reactivity and PrPSc type 1 migration pattern in cerebellum and other PrPSc-rich brain areas. Methods Generation of monoclonal antibodies Prnpo/o mice were immunised with recombinant mouse PrP23–230 (rmPrP23–230), produced and purified as described previously.29 For the initial injection, 10 µg of protein were emulsified in complete Freund’s adjuvant. Boosting injections were done over a period of 65 days according to the following schedule: subcutaneous injection of 10 µg of mPrP23–230 plus complete Freund’s adjuvant on day 1; subcutaneous injection of 10 µg of mPrP23–230 on day 22, and of 20 µg of mPrP23–230 on day 43; intravenous injection of 10 µg of mPrP23–230on day 64, on day 65, and on day 66. At day 66, the mice were sacrificed, and splenocytes were fused to the myeloma cell line Sp2/0-Ag14 with polyethylene glycol by use of a standard protocol. Fused cells were selected with hypoxanthine, aminopterin, and thymidine medium. Clones consisting of small numbers of cells were transferred into fresh wells and cultured. Based on a first screen of 55 positive clones, 19 clones were selected for further characterisation. The clones are hereafter termed POM1 to POM19. Antibody purification and labelling Hybridoma cells were cultured to a log-phase state. During early scale-up, standard fetal calf serum was gradually replaced with ultralow-IgG fetal calf serum while cells were closely monitored for proliferation and viability. Cells were transferred into production flasks at high density and kept in static culture. Supernatant was harvested, centrifuged, and filtered. Antibodies were purified by affinity chromatography on protein G columns, eluted with glycine buffer (pH 3·0), and dialysed against phosphate-buffered saline (PBS; 2 http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8 1 2 3 4 1 2 2b MM CJD type Codon 129 MM MM MV or VV MM, MV or VV MM, MV or VV MM, MV or VV A B Figure 1: Classifications of Creutzfeldt-Jakob disease (CJD) . (A) In the classification proposed by Hill and colleagues, 19 three distinct banding patterns are identified for sporadic CJD, designated CJD types 1 to 3. Type 4 represents variant CJD, with a diglycosylated band. Polymorphisms at codon 129 of PRNP are indicated under each type. (B) According to Gambetti’s classification, 20 two main PrPSc types are identified: type 1, with an apparent molecular weight of approximately 21 kDa, and type 2, of approximately 19 kDa. Type 2b is associated to variant CJD . Its electrophoretic migration pattern is similar to type 2, despite their distinct glycosylation patterns. Grey boxes represent unglycosylated fragments. Articles pH 7·2–7·4). Purity was assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and protein concentration was determined by the Lowry assay. 1 mg of each purified POM1 and POM2 antibodies was used for labelling with cyanine 2 (Cy2) and cyanine 3 (Cy3), respectively. Conjugation of dyes to antibodies was done using FluoroLink-Ab labelling kits (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK), according to the manufacturer’s instructions. Patients All CJD cases included in this study were derived from an unselected series of patients with clinically, genetically, and neuropathologically proven sCJD, with the exception of vCJD samples, which were a kind gift of Dr J W Ironside (National CJD Surveillance Unit, University of Edinburgh, Edinburgh, UK). For sCJD, tissue specimens were retrieved from the tissue bank of the Swiss National Reference Centre of Prion Diseases (Zurich, Switzerland). For all tissue specimens, consent to examine necropsy material had been obtained from each patient or legal guardian. Clinical data and PRNP haplotypes were available for all cases. Frozen brain tissue was stored at –80şC, and samples were taken from the following brain regions: frontal, parietal, occipital, and temporal cortices, putamen, thalamus, midbrain, medulla oblongata, and cerebellum. A neuropathologically proven sCJD case (codon 129 MM; PrPSc type 2, thalamus region) was chosen as the positive control, and a randomly selected necropsied brain from a nondemented patient (age 79 years, female) and the brain from a patient with Alzheimer’s disease were used as negative controls. Sample preparation and western blotting Brain homogenates of CJD or control patients were prepared in PBS, 0·5% (w/v) sodium deoxycholate, and 0·5% (v/v) Nonidet P-40. Total protein concentration was estimated using a standard colorimetric method based on bicinchoninic acid (Pierce Biotechnology, Rockford, IL, USA). 100 µg of protein were incubated with PK (25 µg/mL, 20 units/mg; Sigma-Aldrich, St Louis, MO, USA) at 37şC for 30 min. The digestion was stopped by adding 3 mM phenylmethyl-sulphonyl- fluoride. For the PrPSc spiking experiment, homogenates of solely PrPSc type 1 and PrPSc type 2 brain samples were mixed in percentage ratios of 50:50, 60:40, 70:30, 80:20, and 90:10, according to their protein content, and digested with PK. For deglycosylation, 50 µg of PK-digested protein was precipitated with trichloroacetic acid (Sigma-Aldrich), using a standard protein precipitation protocol. Proteins were then boiled in denaturation buffer, according to the manufacturer’s protocol (New England Biolabs, Ipswich, MA, USA), and incubated with 5000 units of N-Glycosidase F (PNGaseF; New England Biolabs) for 2 h at 37şC. Bacterially expressed human prion proteins (a kind gift from Dr K Wuthrich, Institute for Molecular Biology and Biophysics, Swiss Federal Institute of Technology, Zurich, Switzerland) were produced and purified as described previously.29,30 40 µg of total protein (digested or not with PK), or bacterially expressed human prion protein (100 ng), were mixed with loading buffer (NuPAGE 4 lithium dodecyl sulphate sample buffer; Invitrogen, Carlsbad, CA, USA) and run on 12% NuPAGE gels (MES running buffer; Invitrogen), transferred onto nitrocellulose membranes, incubated first with hybridoma cell supernatants or purified monoclonal anti-PrP antibodies, and then with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (gamma) antibody (Zymed, Invitrogen). Blots were developed by using HRP substrate (enhanced chemiluminescent substrate; Pierce), and signals were acquired with photosensitive film (Kodak, Rochester, NT, USA) or with a Versadoc 3000 imaging system (Bio-Rad, Hercules, CA, USA). Alternatively, membranes were incubated with fluorescentlyconjugated antibodies (POM1-Cy2 and POM2-Cy3, as described above) for 3 h at room temperature, or overnight at 4şC. In such cases, fluorescence was scanned with a gel and blot imager (Typhoon 9400; Amersham Biosciences). Densitometric and statistical analysis Densitometric analysis used for the triplot representation was done with a one-dimensional software analysis program (Quantity One; Bio-Rad). We used SPSS version 11 for Macintosh (SPSS Inc, Chicago, IL, USA) for statistical analysis. To compare type 1 and type 2 sample groups, we used paired t test for each glycoform, detected with either non-discriminatory 3F4/POM1 or the type 1-specific POM2/POM12. Screening and isotyping of clones by ELISA against recombinant mPrP (rmPrP) Plates were coated overnight at 4şC with 5 µg/mL of rmPrP23–230 or rmPrP121-230 in PBS and washed with PBS containing 0·1% (v/v) Tween-20, blocked with 5% (w/v) bovine serum albumin (BSA), and incubated for 2 h at room temperature with 30 µL of two-fold serially diluted hybridoma cell supernatant (1:10 prediluted) in PBS-Tween containing 1–5% BSA. Plates were washed with PBS-Tween and probed with a HRPconjugated anti-mouse antibody (total IgG, or isotypespecific antibodies IgM, IgG1, IgG2a, IgG2b, and IgG3, all from Zymed, used at 1:1000 dilution in PBS-Tween-BSA). Plates were developed with 2,2- azinodiethyl-benzothiazoline-sulphonate, and optical density was measured at 405 nm. Titres were defined as the highest dilution showing an optical density more than twice the background, which was calculated as the average density of uncoated wells and wells incubated without serum. http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8 3 Articles Epitope mapping by peptide competition A PrP peptide library of 100 dodecamers was synthesised (JPT Peptide Technologies GmbH, Berlin, Germany). The first peptide of the library represented amino acids 23–34 of the mature mouse prion protein. All other peptides sequences were shifted by two amino acids of PrP sequence (eg, 25–36, 27–38, …, 219–230, and 221–232). 384-well plates were coated with 300 ng/mL of mouse PrP23–230 overnight at 4şC. Plates were washed with PBS containing 0·1% Tween-20, and blocked with 5% BSA for 2 h at room temperature. After washing, plates were incubated with 30 µL of two-fold serially diluted hybridoma cell supernatant (prediluted 1:1000–1:5000) in PBS-Tween containing 1% BSA, with or without peptides in a final concentration of 0·8–8·0 ng/mL. After 2 h at room temperature, plates were washed and probed with HRP-conjugated rabbit anti-mouse IgG (1:1000 dilution; Zymed) for 1 h at room temperature. The assay was developed with 2,2-azinodiethyl-benzothiazolinesulphonate. Optical density was measured at 405 nm. Surface plasmon resonance measurements All experiments were performed on a Biacore 3000 (Biacore International AB, Uppsala, Sweden). For pairwise antibody mapping, monoclonal antibodies (POMs) or pooled IgG1 (isotype control) were immobilised on CM-5 chips (Biacore) after activation with N,N-(3- dimethylaminopropyl)-N’-ethyl-carbodiimide hydrochloride and N-hydroxysuccinimide at a flow rate of 5 µL/min. A 5 µL aliquot of a 50 µg/mL antibody solution in acetate buffer (pH 4·5) was used to immobilise approximately 2500–5000 response units of covalently bound antibody. After inactivation with ethanolamine, the surface was washed twice with 20 µL of 0·5% (w/v) SDS to remove any non-covalently bound antibody. The system was primed and a new sensogram was started with HBS-EP (0.01M Hepes pH 7·4, 0·15M NaCl, 3mM EDTA, 0·005% (v/v) Surfactant P20; Biacore) as running buffer. 20 µL of HBS-EP were used at a constant flow rate (5 µL/min) for all injections. Proteins (BSA or PrP) or antibodies were injected at a concentration of 50 µg/mL diluted in HBS-EP buffer. For chip regeneration, several injections of 0·5% SDS were done until the baseline returned to zero. For control, injections were done in flow cells 1 and 2, or 3 and 4, for which the first flow cell was coated with pooled IgG1 (Zymed), and the second with the relevant antibody. Analysis of peptide competition for N-terminal POMs by surface plasmon resonance The coating and washing of the flow cells with control IgG or POM antibodies were performed as described above. A volume of 20 µL was used for all injections at a constant flow rate of 5 µL/min. rmPrP121–230 (negative control) and rmPrP23–230 were diluted in HBS-EP buffer at a concentration of 10 µg/mL. Injection of POM antibodies (40 µg/mL in HBS-EP) was done with or without individual peptides (20 µg) from a library of 100 partially overlapping dodecapeptides, each shifted by two amino acids and covering the entire mature mouse PrP sequence. For selected epitopes, mutant undecameric peptides with scanning single amino acid deletions were used (Jerini AG, Berlin, Germany). Role of the funding source The sponsors of this study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit it for publication. Results By immunising Prnpo/o mice with bacterially produced rmPrP, we induced potent immune responses towards various PrP epitopes. By screening with western blot and ELISA (data not shown), 19 clones were selected for further characterisation and named POM1 to POM19. Immunoglobulin-isotype-specific ELISA showed that most POM hybridomas secreted IgG1 (table 1). In ELISA assays against immobilised rmPrP23–230 and rmPrP121–230, POM2, POM3, POM11, POM12, POM14, and POM18 were found to bind full-length PrP, but not N-terminally truncated PrP, indicating that their epitopes were situated N-proximally of position 121 (table 1). By contrast, POM1, POM4–POM10, POM13, POM15–POM17, and POM19 reacted with both rmPrP23–230 and rmPrP121–230, indicating that their epitopes were situated in the globular PrP domain (amino acids 121–230). For the purposes of this study, we focused on 4 http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8 Isotype Amino-acid position on murine PrP (epitope sequence) MAbs against unstructured PrP POM2 IgG1 58–64, 66–72, 74–80, 82–88 (QPXXGG/SW) POM3 IgG1 95–100 (HNQWNK) POM11 IgG1 64–72, 72–80 (GQPHGGSW) POM12 IgG1 58–64, 66–72, 74–80, 82–88 (QPXGGG/SW) POM14 IgG1 53–64, 61–72, 69–80, 77–88 (GGTWGQPHGGG/SW) POM18 IgG1 .. MAbs against the globular PrP domain POM1 IgG1 121–230 POM4 IgG1 121–134, 218–221 POM5 IgG1 168–174 POM6 IgG2a 121–230 POM7 IgG1/IgG2a 121–230 POM8 IgG1 121–230 POM9 IgG2a 121–230 POM10 IgG1 121–134, 218–221 POM13 IgG1 121–230 POM15 IgG1 121–230 POM16 IgG2b 121–230 POM17 IgG1 121–230 POM19 IgG1 121–134, 218–221 MAbs=monoclonal antibodies; ..=not done. Table 1: Isotypes and specificity of novel anti-PrP antibodies Figure 2: Discrimination of PrPSc from different Creutzfeldt-Jakob disease (CJD) types by POM2 and POM12 (A) Representative replica blots containing proteinase-K digested CJD samples, classified as type 1 or 2, probed with five different monoclonal antibodies (as indicated in the right side of each panel). POM2 and POM12 recognise the samples classified as CJD type 1 only, whereas POM1 and 3F4 detect both. POM7 exclusively detects the unglycosylated fragments. The red star indicates a type 2 cortical sample with trace amounts of type 1 probed with both POM2 and POM12. (B) Diagram representing the molecular basis of POM2 and POM12 specificity towards PK-digested PrPSc type 1 CJD. Their epitopes are cleaved by PK on CJD type 2 samples. The dashed and black boxes represent -helices and -sheets, respectively. The round markers represent the glycan chains. Numbers correspond to the amino acid sequence of human PrP. (C) Scheme showing the localisation of POM2 and POM12 epitopes with regard to predicted PK cleavage sites on human PrP. Any PrPSc digested at position 82 or upstream will be detected by POM2 and POM12; digestion at any residue downstream of this position will disrupt the POM2/12 epitope. The red filled boxes indicate the minimum sequence required for POM2 and POM12 binding on the human PrP sequence. The blue box indicates the 3FA epitope. (D) Analysis of the antibodies specificity by dual-colour western blot of two different variants of rhPrP. The two arrows indicate PrP degradation products including POM1 epitope but not POM2 (upper arrow), or POM2 epitope but not POM1 (lower arrow). The low intensity band running approximately at 25 kDa in the hPrP96–230 preparation detected by POM1 only, represents a PrP dimer, which is often detected in non-reducing conditions . the C-proximal POM1 and POM7 epitopes, and the N-proximal POM2 and POM12. The epitopes of POM1 and POM7 were defined by surface plasmon resonance experiments, which showed that they both competed for binding with monoclonal antibody 6H4 (Prionics, Schlieren, Switzerland). This indicates that their epitopes are located on alpha helix 1 of the structured domain of PrP (data not shown). The POM7 epitope is sterically hindered by the PrP sugar chains. Hence POM7 binds preferentially to the unglycosylated PrP subpopulation. The epitopes recognised by POM2 and POM12 were characterised using a library of 100 synthetic dodecapeptides, each shifted by two amino acids, which spanned the entire PrP sequence (webappendix, webfigures 1 and 2). Our analysis shows that POM2 and POM12 recognise similar but not identical epitopes. The POM12 epitope is QPXGGGW, whereas the POM2 epitope is QPXXGGW. By western blotting, we explored the reactivity of POM hybridomas to human brain samples from patients with CJD and controls. All patients were classified according to cortical PrPSc electrophoretic migration using Articles monoclonal antibody 3F4 and codon 129 polymorphism. 20 Replica western blots were probed with five different antibodies: 3F4 or POM1, which bind indistinguishably to all PrPSc types; POM7, which only interacts with the unglycosylated PrP band; octarepeatspecific POM2; and POM12. POM1 and 3F4 showed the typical migration pattern of CJD type 1 and 2 (Gambetti classification), or type 1, type 2 (MV2 and VV2), and type 3 (MM2 and MV2), according to Hill and colleagues’ classification (figure 2).19 In addition, POM1 recognised a shorter PK-resistant PrPSc C-terminal fragment, running at about 16–17 kDa, as described previously for other C-terminal specific antibodies.31 When PK-digested PrPSc from CJD samples were stained with POM2 and POM12, only PrPSc type 1 samples were detected, whereas replica blots incubated with 3F4 or POM1 identified all four samples with typical migration patterns of the respective types (figure 2). The POM2 and POM12 epitopes are preserved by PK cleavage at position 82 or upstream, but destroyed by digestion at any residue downstream of this position. In the human PrP sequence, the POM2 and POM12 epitopes of CJD type 2 samples were destroyed by PK (figure 2). Full-length and truncated recombinant human PrP (rhPrP23–230 and rhPrP96–230) were co-incubated with Cy2-labelled POM1 and Cy3- labelled POM2. Two-colour fluorescence detection confirmed that POM2 detects epitopes upstream of position 96, whereas POM1 recognizes both PrP variants (figure 2). We then screened brain samples from 70 sCJD patients (41 subtype MM1, six MV1, three MM2, 11 MV2, and nine VV2) and three vCJD patients (all subtype MM), whose PrPSc had been previously typed by 3F4 western blot at necropsy. As expected, POM2 and POM12 recognised PK-digested PrPSc from all CJD type 1 samples. However, in 11 of 23 type 2 cortical samples, low-intensity POM2 and POM12 reactivity was observed. The latter signal always showed a typical type 1 migration pattern, indicating that low amounts of PrPSc type 1 coexist in the cortex of approximately 50% of the CJD type 2 patients (figure 2, table 2). Samples from the cerebellum and other PrPSc-rich areas (as determined by Schoch and colleagues32) were then probed with POM2/POM12, 3F4, and POM1. Surprisingly, each of the patients classified as type 2 showed obvious POM2/POM12 reactivity with PrPSc type 1 electrophoretic migration pattern, either in cerebellum or in other brain areas (figure 3, table 3). Although all type 2 cerebellar samples would have been identified as such based on the 3F4 or POM1 blots, when probed with POM2 they appeared positive with a PrPSc type 1 migration pattern. Moreover, brain samples from frontal cortex of the three vCJD patients were also found to be positive for POM2 on western blot with a type 1-like PrPSc electrophoretic migration (figure 3). Case 59 was atypical, and was diagnosed as type 2 according to 3F4 western blot analysis of a cortical sample. However, cerebellar sample analyses with POM1, 3F4, POM2, and POM12 identified a typical type 1 pattern without any obvious type 2 fragments. PrPSc glycoform ratios have been reported to diverge in various CJD types.19 We therefore analysed PrPSc glycoform ratios of all cerebellar type 2 samples, according to 3F4/POM1 western blot and compared them to replica blots incubated with POM2/POM12. Moreover, we selected 10 type 1 cortical samples and did the same type of analysis, using either 3F4/POM1 or POM2/12 (webfigure 3). Statistical analysis of PrPSc glycoform ratios detected by 3F4/POM1 and 6 http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8 3F4/POM1 POM2/POM12 (positive/total) (positive/total) CJD type 1 MM1 41/41 41/41 MV1 6/6 6/6 CJD type 2 MM2 3/3 2/3 MV2 11/11 5/11 VV2 9/9 4/9 Samples typed according to the Gambetti classification.20 Table 2: Summary of cortical samples by Creutzfeldt-Jakob disease (CJD) subtype screened with POM1/3F4 and POM2/POM12 monoclonal antibodies MM1 VV2 MM1 PK Codon 129 37 kDa– 25 kDa– 20 kDa– 15 kDa– PK Codon 129 37 kDa– 25 kDa– 20 kDa– 15 kDa– 3F4 POM1 POM2 POM1 POM2 VV VV MV MV MM1 VV2 MM1 VV VV MV MV MM1 VV2 MM1 VV VV MV MV Cortex Type 2/cerebellum Cortex Type 2/cerebellum Cortex Type 2/cerebellum MM MM MM MM MV MV VV VV Cortex Type 1 Type 2 vCJD Cortex Cerebellum MM MM MM MM MV MV VV VV Cortex Type 1 Type 2 vCJD Cortex Cerebellum A B Figure 3: Co-existence of Creutzfeldt-Jakob disease (CJD) types (A) Three replica blots containing conventionally classified CJD type 2 samples (as indicated) blotted with either 3F4 and POM1, which recognise all PrPSc species, or POM2, which only binds to proteinase K (PK)-digested type 1 PrPSc. POM1 also bound to some C-terminal PrP fragments in the non-PK-digested samples that did not react with either 3F4 or POM2. When probed with POM2, a type 1 PrPSc pattern is detected. (B) The same phenomenon is shown in cortical samples of three vCJD patients; when probed with POM2, they show type 1 PrPSc migration pattern. Articles POM2/POM12 in the same group of samples did not show any significant variations within the analysed groups (webtable). We investigated the concentration of PK to find the best conditions for our analysis. It has been proposed that a PK concentration of 100 µg/mL should be used for the analysis of CJD brain samples.22 However, for optimum evaluation, the PK concentration for reliable prion disease diagnosis should be the minimum concentration that will completely digest PrPC. Any concentration higher than this will potentially decrease the sensitivity of the assay and, in the worst-case scenario, may lead to false negatives due to excessive digestion of PrPSc. It has been long known than PrPSc is only partially resistant to PK: it can be degraded by high PK concentrations or by long incubation times (figure 4). Any lower concentration is also suboptimum and may result in false positives due to residual PrPC. In our study, 25 µg/mL of PK for 30 min at 37şC were sufficient to degrade PrPC from a healthy control or an Alzheimer’s disease brain sample (figure 4). Furthermore, this digestion protocol allows to positively diagnose CJD from necropsies of patients with very low PrPSc load. The same patients would have been falsely diagnosed as negative if a higher PK concentration (100 µg/mL) had been used. The differences in signal intensity observed in the blots were due to the variable PrPSc content in brain samples, as indicated by probing those samples, before PK digestion, with control anti-actin antibodies (figure 4). We also investigated whether differences in pH of the total homogenate at the time of PK digestion might interfere with our results, since it has been shown that pH may affect the size of the PrPSc fragments generated.22 We found that there was no effect of pH on the POM2/POM12-positive intrinsic type 1 PrPSc detected in CJD type 2 brain samples (figure 4). We then reasoned that analytical limitations intrinsic to the western blot detection method may have prevented the detection of intrinsic type 1 PrPSc within type 2 brain samples in the past. By mixing brain homogenates that contain solely type 1 or type 2 PrPSc in various ratios, we determined that western blots using non-discriminatory antibodies, such as POM1 (or 3F4), consistently fail to detect type 1 PrPSc whenever it represents less than 40% of total PrPSc (figure 5). We went on to determine quantitively the type 1 PrPSc content within a cerebellum sample of a CJD type 2 patient. To achieve this, we used POM2- positive PrPSc band-intensity of a bona fide type 1 sample serially diluted within a single type 2 sample in various ratios to obtain a standard curve of type 1 content (figure 5). We found that PrPSc type 1 in the CJD type 2 cerebellar sample (VV) was 6·9%, which was well below detectability of any non-discriminatory antibody. These results support our notion that, without a type-specific antibody, the intrinsic type 1 content in every type 2 sample would have been missed due to low sensitivity. Deglycosylation allows the detection of different PrPSc types in the same sample, even with non-discriminatory antibodies, such as 3F4.24 We investigated whether our observations using type 1 specific antibodies could have been discovered by deglycosylation. We found that despite elimination of alternative glycoforms, POM1 still fails to detect intrinsic type 1 content within the type 2 sample, whereas it is clearly detected by POM2 (figure 5). Moreover, when the latter blot was reincubated with biotinylated POM1, the presence of type 2 PrPSc was revealed. As an alternative to deglycosylation, we also used POM7 antibody, which is specific for the unglycosylated PrP band. It also failed to detect intrinsic type 1 content within the type 2 sample in these conditions (figure 5). Discussion The discovery of heritable polymorphic PK cleavage sites in PrPSc has been used for the molecular classification of CJD cases.11,13,14,17 In concert with the codon 129 PRNP haplotype, the different PrPSc types correlate with distinct disease phenotypes. Most patients with the MM1 or MV1 subtype present with so-called classic CJD, and show rapid progressive dementia, early myoclonus, visual disturbances including cortical blindness, and a disease duration of See Lancet Online for webfigure 3 and webtable http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8 7TSS Frontal cortex Cerebellum Other (PrPSc rich) 3F4/POM1 POM2/POM12 3F4/POM1 POM2/POM12 3F4/POM1 POM2/POM12 CJD type MM2 59 130 .. .. 221 .. .. CJD type MV2 98 145 154 155 166 172 214 228 .. .. 242 .. .. 247 248 .. .. CJD type VV2 78 111 .. .. 116 .. .. 129 137 142 .. .. 161 .. .. .. .. 197 233 =weak; =moderate; =high; =no PrP signal; ..=not done. Table 3: Antibody reactivity of samples from patients classified as type 2 Creutzfeldt-Jakob disease (CJD) . Figure 4: Effect of proteinase K (PK) concentration and pH on prion diagnosis (A) Schematic representation of PrPC and PrPSc degradation curves. The optimum concentration for prion diagnosis is the minimum concentration that suffices to digest PrPSc completely. (B) Western blot showing that 25 g/mL of PK lead to complete digestion of PrPC from a healthy control (indicated by “C”) or an Alzheimer’s disease (AD) brain sample. (C) Western blots showing that 100 g/mL of PK treatment can lead to false negatives. Upper panel shows CJD diagnosis of cerebellum samples from MM1 and MV1 patients with low PrPSc load when 25 g/mL of PK were used; middle panel shows apparent PrPSc negativity when 100 g/mL PK were used; lower panel shows the actin control blot of the same samples before PK digestion. (D) Lack of effect of pH on prion diagnosis. Cortical or cerebellar samples were digested with 25 g/mL of PK in a pH buffer 7 or 8 (as indicated). approximately 4 months. By contrast, patients with the MV2 or VV2 subtype show an atypical disease course, with a longer disease duration (6–18 months), early ataxia, predominant extrapyramidal symptoms, and late-onset dementia.18 Because of the limited resolution of conventional western blot systems, the coexistence of PK-digested PrPSc bands at 19 kDa and 21 kDa is not recognisable by conventional antibodies binding to both PrPSc types, including POM1. By mixing brain homogenates that contain solely type 1 or type 2 PrPSc, we have found that 3F4-based or POM1-based western blots consistently fail to detect type 1 PrPSc whenever it represents less than 30–40% of total PrPSc. We addressed these problems by developing a set of antibodies binding Nproximal epitopes of PrP, which recognise PK-digested type 1 PrPSc (cleaved at amino acid 82 or upstream), but not type 2 PrPSc (cleaved at amino acids 97 or 86). 8 http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8TSS On investigation of 70 sporadic CJD cases, we determined that 50% of the patients formerly classified as CJD type 2 had low but detectable amounts of PrPSc type 1 in their cerebral cortex. Investigations of additional brain areas revealed that significant amounts of PrPSc type 1 coexist with type 2 in at least some areas of all patients classified as having type 2 disease. Co-occurrence of CJD types in the same brain has been previously reported in approximately 30% of the tested cases.24 Using unambiguous analytical tools, we have established that type 2 PrPSc does not exist independently from type 1 PrPSc, at least not in the Swiss CJD patients included in our study. Furthermore, using our type 1-specific antibodies, we found type 1 PrPSc content in three cortical samples of vCJD patients. Glycotyping of blots incubated with 3F4/POM1 versus POM2/POM12 showed no significant differences in glycoform ratios, neither for the same groups incubated with different antibodies, nor for the two PrPSc groups, in accordance with previously published data.33 The homogeneity of glycoforms suggests that our collective is comparable to those investigated in other countries, despite the peculiarities of Swiss CJD epidemiology.34 The results presented here do not question the validity of the established correlations between PrPSc types and clinical findings. The coincident presence of PrPSc type 1 may not alter the expected clinical outcome of patients with predominant PrPSc type 2 deposition. However, the existence of heritable PrPSc types implies that strainspecific characteristics of prions are enshrined in the conformation of PrPSc, and that the different core fragment sizes of PK-digested PrPSc may be used as surrogates of such conformational variations. In the light of the data presented here, the strength of these arguments becomes somewhat questionable. Why did the co-existence of type 1 and type 2 PrPSc go often undetected in the past, despite its ubiquitous presence? Maybe the 21 kDa band characteristic of type 1 PrPSc is easily obfuscated by the simultaneous occurrence of the neighbouring 19 kDa type 2 band and of the monoglycosylated PrPSc band. We tested this hypothesis by ascertaining the detectability threshold of type 1 PrPSc in mixtures of pure type 1 and type 2 samples. Indeed, we found that type 1 PrPSc becomes undiscernible as soon as 40% or more type 2 PrPSc is present. This observation provides a plausible explanation for the failure to appreciate the invariable coexistence of PrPSc types in previous reports. To determine whether a similar phenomenon occurs in patients classified as type 1 would be interesting. However, this issue is impossible to address at present, as there is no CJD type-2-specific antibody. The fact that the type 1 coexistence is also apparent in cortical samples from CJD patients, indicates that the currently described phenomenon might be a more general one. The above results set the existing CJD classifications into debate and introduce interesting questions about human CJD types. For example, do human prion types exist in a dynamic equilibrium in the brains of affected individuals? Do they coexist in most or even all CJD cases? Is the biochemically identified PrPSc type simply the dominant type, and not the only PrPSc species? http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8 9TSS % type 1 % type 2 25 kDa– 20 kDa– 15 kDa– 25 kDa– 20 kDa– 15 kDa– 25 kDa– 20 kDa– 15 kDa– 25 kDa– 20 kDa– 15 kDa– 25 kDa– 20 kDa– 15 kDa– 25 kDa– 20 kDa– 15 kDa– % type 1 100 50 40 30 20 0 PrPSc band-intensity 10 20 30 40 50 60 y=0·5084x R2=0·783 % of type 1 CJD 30 25 20 15 10 5 0 X 0 100 0 0 100 50 50 60 40 70 30 80 20 90 10 10 100 0 0 100 50 50 60 40 70 30 80 20 90 10 POM1 POM2 POM2 POM1 POM1 POM2 POM1 POM7 POM1 POM2 POM2 Type 1 Type 2 MV VV MM Type 1 Type 2 MV VV MM Type 1 Type 2 MV VV VV VV MM Type 1 Type 2 MV MV MM VV VV Type 1 Type 2 MV MV MM Codon 129 A B C E F D Figure 5: Non-discriminatory antibodies fail to detect coexistance of PrPSc types Homogenised brain samples containing pure type 1 and type 2 PrPSc were mixed in various ratios (as indicated). Replica blots with sample mixtures were probed with (A) non-discriminatory antibody POM1, or with (B) type 1- specific antibody POM2. Type 2 PrPSc is detected when lanes contain 50% or more of the type 2 brain sample, but it is not detected in lanes with a contribution of 40% or less. In the blots probed with POM2 (type 1 specific antibody), type 1 PrPSc is detected in all cases , even if it corresponds to 10% of the total sample. (C) Comparison of mixed type 1 and type 2 samples in various ratios (as indicated) with a cerebellar sample of a Creutzfeldt-Jakob disease (CJD) type 2 patient (VV, indicated as X) when probed with POM2 (upper panel) and biotinylated POM1 (lower panel). (D) Standard curve of type 1 content , obtained according to the band-intensities of the serially diluted “bona fide” type 1 in C (upper blot). (E) Replica blots analysing brain homogenates from two type 1 (cortex) and one type 2 (cerebellum) patients treated with PNGase and proteinase K (PK). Despite elimination of alternative glycoforms, POM1 fails to detect type 1 content within the type 2 sample (left panel), whereas it is clearly detected by POM2 (middle panel). When the same blot is reincubated with biotinylated POM1, the type 2 PrPSc band is revealed (right panel). (F) Replica blots probed with POM1 and POM7. POM7 specifically detects the unglycosilated fragment. However, it does not detect type 1 content within type 2 samples. POM1 detection pattern is shown here for comparison. Articles Acknowledgments We thank all referring physicians, Mike Scott, Ralph Schlapbach, and the Functional Genomics Center of Zurich for valuable help with the surface plasmon resonance experiments and development of the fluorescent blot, and Dieter Zimmerman for genotyping. We thank Burkhardt Seifert (Department of Biostatistics, University of Zurich) for advice on the statistical analysis. We also thank Wiegand Lang for contributing to the production of hybridomas, and Albrecht Gröner for discussions. MP is supported by a PhD fellowship of the Zentrum für Neurowissenschaften Zürich, by UBS grant BA29 AKRB-DZZ (675/B), and by a career award from the Foundation of Research at the Medical Faculty, University of Zurich. This study was supported by a grant of the Swiss Federal Office of Health to AA. Authors’ contributions MP contributed in the production of the antibodies, performed their characterisation, contributed in sample analysis, and drafted the manuscript. KS analysed the CJD samples and contributed in writing the manuscript. MG obtained the cases and handled samples. MV and AB contributed in the production of the antibodies. AA supervised this study and contributed to writing and finalising the manuscript. MP, KS, MG, and AA contributed in the design of the study. MP and KS contributed equally to this work. 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