Tentatively diagnosed with variant Creutzfeldt-Jakob's disease (vCJD), a 22-year-old woman was back in the news last week. A resident of Florida, she is the first vCJD case reported in the U.S. Her likely diagnosis with the human version of mad cow disease (bovine spongiform encephalopathy, or BSE) stems from her childhood in her native Great Britain before moving with her parents some years ago to the U.S.
She first sought medical attention just a year ago, complaining of depression and memory loss. Her symptoms rapidly worsened and at present she is still alive but bedridden and unable to communicate with her parents.
Since the mid-1990s, some 130 people in the UK, plus a handful in France, Ireland and Italy, have died or are dying from the fatal brain disorder. They were presumed to have contracted BSE after eating meat from cows infected with the infectious form of prions. Herds of British cattle with mad cow infection have been slaughtered to curtail the infection.
Also very much in the news these days is the threat of an American equivalent to British mad cow disease among deer and elk. It's called "chronic wasting disease" - CWD. As neuroscientist Jiri Safar told BioWorld Today, "CWD in Midwestern states is becoming a major problem. It's a new disease entity, probably a new strain of prions. It was first recognized 20 years ago in herds of deer and elk, kept on open farms for human consumption."
Safar continued, "It's very difficult to transmit CWD to the laboratory strains of mice or hamsters for prion analysis. Therefore, we have very limited knowledge about the biological character of those CWD prions. They may represent threats similar to BSE in the UK or Europe. The CWD prions may transmit the infection to cattle or sheep, even to people. It's a perfectly plausible threat, also because in many cases the CWD has been detected in wild deer and elk. Many of those species are sharing the same environment with sheep and cattle. So there's a legitimate worry," Safar warned, "that CWD may jump the species barrier into some host such as cattle, sheep or even people."
He is associate adjunct professor of neurology at the University of California at San Francisco and a member of the university's Institute for Neurodegenerative Diseases, which is directed by Nobelist Stanley Prusiner, who discovered prions two decades ago.
Prusiner, Safar Bookend New Test Disclosure
Safar is lead author of a paper released online in Nature Biotechnology, dated Oct. 21, 2002, and titled: "Measuring prions causing bovine spongiform encephalopathy or chronic wasting disease by immunoassays and transgenic mice." Prusiner is its senior author.
"Now in our transgenic system - meaning the mice that are expressing multiple copies of bovine PrP [non-infectious] genes - [the mice] have a sensitivity even higher than that of natural cows because we insert multiple copies of bovine PrP genes," Safar said. "And the sensitivity of these is tenfold higher than previous prion assays in cows. So taking all those data together, we have a transgenic mouse that is 10,000 times more sensitive in detection of BSE prions than the original wild-type mice, which were used for establishing all those public safety measures in the late 1980s and early '90s. So the implication is that all those decisions or conclusions have to be revised in the fastest possible way.
"The significance of this work," Safar explained, "is that we have a system which is immensely more sensitive than previous ones. And on top of it, the incubation time in those transgenic mice is many-fold shorter than the incubation time in cows. In murine transgenics, the minimum incubation time for BSE prions ranges from about 220 days to a maximum of 400 days. That compares with about 26 months' minimum incubation time in cows up to the age of 9 years. The many-fold shorter incubation time in our mouse transgenics means we can perform many experiments in a significantly shorter time and, as a result, accumulate a lot of important data very quickly.
"The bottom line," Safar observed, "is a totally new principle we call conformation-dependant immunoassay [CDI]'. We described it for the first time in 1998, when we discovered that we can use immunoassays to distinguish and detect specific conformation of the prion protein. In prions, the basic problem is to distinguish the normal PrP protein - which is ubiquitous, noninfectious and necessary for normal brain cell function - from the abnormal infectious one, which replicates, has toxic function in the cells and causes neurodegeneration, as in Alzheimer's disease. Those two proteins," he added, "differ only in their conformation, the 3-dimensional shape of the molecule.
"It took us four years to transfer this first principle into practical application - to distinguish these two different shapes, using antibodies - which in this case is detection of the BSE prions. We had to develop antibodies with a sufficiently high affinity and, therefore, sensitivity in this immunological assay system. We wanted antibodies against specific domains of the protein, which undergo this conformation transition from normal to abnormal. They didn't exist at that time. We inserted the antibody DNA sequence into the vector, which allowed us to express these antibodies in Escherichia coli, so we could generate large quantities of identical recombinant antibody fragments. Then when we selected those that gave us the maximum sensitivity, we started validation. In the first stage, we developed a manual bench protocol for lab technicians. In the second step, we scaled up and automated the system, using a robotic station supplied by the multinational Swiss automation firm Tecan AG."
Perfect Test Score At Regulatory Agency
"We applied to the European agency for test validation and tested 200 samples in our lab. We got a perfect score - 100 percent sensitivity and 100 percent specificity. That meant we correctly identified all the PrPSc (infectious) samples as BSE and all the normal samples as normal.
"We are working very hard on the next step - to develop a diagnostic test for human vCJD, based on either lymph-node testing for tonsils, blood or blood fractions. Everything described until now was based on testing brain tissues, for which you had to have a sample," Safar concluded, "usually post-mortem."