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Marlena Zuber

I wish I had a money tree in my backyard. The chances of that happening, however, would be better if I were a biochemist, a virologist, or a protein researcher investigating prions, those now-infamous high-molecular-weight proteins (see box, “A prion primer”). Last October, the U.S. Department of Agriculture Agricultural Research Service (USDA–ARS) was slated to receive some fruit from the money tree in the form of $5 million appropriated by Congress for bovine spongiform encephalopathy (BSE) research—the first research of this kind to be performed in the United States. As of this writing, the full House of Representatives had approved the funding, as had all Senate committees (1). The funding was awaiting a vote from the entire Senate membership when terrorists attacked the Pentagon and World Trade Center on September 11.

Floyd Horn, administrator of ARS, expects even higher appropriations for BSE research in the coming years. His deputy, Associate Administrator Edward Knipling, speculated that congressional support would continue for BSE research to avoid any tampering with our food supply.

“There’s already support for it because of its human health threat and threat to the animal industry, but in light of the terrorist activities we’ve experienced, one could speculate that there will be an enhanced awareness of all potential forms of terrorism, including bioterrorism with pathogens that would affect both humans and the food supply. Among the types of programs that the budget is enhancing, [BSE research] would be among those likely to receive continued high-priority attention,” Knipling said.

After the events of September 11, however, everything is uncertain. There may be some scaling back on funding, Knipling said. Prior to the terrorist attacks, when a steadily tightening U.S. economy threatened much scientific research, USDA was focusing some of its efforts on a long-running research project: breeding the elusive money tree.

“We’re trying to develop some new breeds of plants that grow money, but so far we haven’t been successful,” said Doug las Moore, safety and environmental officer for USDA’s Plum Island Animal Disease Center off the coast of Long Island (New York). Moore has been hoping for more money to further explore the causes and spread of BSE, more commonly known as “mad cow disease”. Moore added, “There is no work being done on BSE in the United States now.”

The funding will be shared among the National Animal Disease Center in Ames, IA, the Animal Diseases Research Unit in Pullman, WA, and the Plum Island center, all associated with ARS.

The research that they hope to fund using the $5 million “is actually just the beginning of what we think will be a bigger understanding of what this global disease is. . . . We have no real understanding of the mode of action of the prion—the infectious agent”, Horn said. Because the mode of action is not understood, he explains, “There is strict agreement on how that money would be spent. . . . The only restriction is that we not work with infectious material here.” Research using deactivated agents will be done in the United States, and active agents will be studied abroad.

The initial cash outlay will not be enough to cover all the research Horn proposes. That research concerns modes of action and rapid detection (particularly in live animals), prion precursors and degradation products, and the predisposition of individuals or groups of animals to prion infection.

Moore explains the problem further: “Prions are metabolites. They are components of animal cell activity. No one knows what that protein does, although one can infer that if you have a protein, it’s doing something. So the interest is: What happens in normal prion proteins? What happens when it’s finished doing that? Why is the scrapie form of the prion protein different from the one in BSE, and what is it’s metabolic pathway? There are clearly some pathway problems.”

The link to eating habits
BSE is caused by cannibalistic practices, such as feeding swill that contains cattle parts to other cattle (more on this below). The theory of how prion-induced diseases are spread derives from studies on the Fore tribespeople in Papua New Guinea who died of kuru, a disease that caused brains to be “riddled with gaping holes and strange plaques—flower-shaped waxy buildups of protein called amyloid. Kuru brains had holes where neurons used to be, accompanied by enlarged astrocytes, the star-shaped cells that attach themselves to blood vessels inside the brain,” wrote Sheldon Rampton and John Stauber in Mad Cow U.S.A. (3).

Scientists researching the disease among tribespeople discovered the Fore’s practices of eating the brains of their enemies or of family or community members who had died. The practice, the researchers determined, spread the disease to the extent that the numbers of women, children, and elderly were decreasing rapidly. Kuru, which was later found to be a prion disease, is always fatal. Because women, children, and the elderly have low rank in the tribe, they ate the internal organs and the brain. The men ate the prime portions of human bodies, which is why the disease was seen less commonly in men, according to Rampton and Stauber.

The outbreak of BSE in England also has been traced to a type of cannibalism. Specifically, the disease was spread when cattle were fed protein supplements derived from other cows. The practice of feeding cows back to cows created an amplification loop that enabled what would have otherwise been an extremely rare disease to proliferate into a huge epidemic, Rampton explained.

BSE spreads easily and consistently from cattle to humans when humans eat infected beef. Estimates of people expected to die from a type of BSE found in humans go as high as 500,000 for England alone over a span of about 40 years, according to Rampton.

Several countries throughout Europe have reported related cases, and some have taken dramatic actions. Among them is Germany. In June, Renate Künast, Federal Minister of Consumer Protection, Food and Agriculture, initiated a shift in farming subsidies away from traditional factory farming and toward organic farming. The plan is to increase organic farming in Germany to 20% from the current 2%.

The effort is intended as a precaution to prevent BSE and foot and mouth disease from spreading further there. Germany also is not accepting shipments of animal feed from the United Kingdom that contain ground cattle, even if the country certifies the feed does not contain any parts of cattle found to have BSE.

New variations
Scientists determined that younger people exhibiting CJD actually had what became known as new-variant CJD (nvCJD), a prion-induced disease like CJD and BSE. The first case of nvCJD in Japan surfaced earlier this year. England, France, Germany, and Slovakia have also reported cases, according to the Centers for Disease Control and Prevention (Atlanta). Between 1995 and 2000, 83 cases of nvCJD were reported worldwide, and the total had risen to 107 as of September 28, 2001 (4).

Researchers have determined that BSE is passed to humans as nvCJD when they eat infected beef. Humans can also contract nvCJD by eating meat from animals infected with other prion diseases such as wasting disease or scrapie. This accounts for the increase in occurrences of CJD and nvCJD observed in younger human populations. CJD and nvCJD can take up to 40 years to incubate before symptoms show, making short-term research especially difficult. Until recently, the only way to confirm the disease was to perform an autopsy on the brain after death. Because a prion is simply a protein, it is immune to any of the conventional treatments used to treat viruses or bacteria.

Fortunately, BSE, CJD, and nvCJD are not easy to transmit, said Rampton in an interview. “The only proven route of transmission is actually eating the flesh and especially the brain tissue of an infected animal,” he said, also noting that the disease also can be passed on genetically. “We still don’t know how many more people are going to die because this disease has a very long invisible incubation period. And virtually everyone in England has eaten infected beef,” he explained.

The antibody approach
David Peretz, associate professor of neurology at the Institute for Neurodegenerative Diseases and a professor of neurology, biochemistry, and biophysics at the University of California, San Francisco, is working on the targeted attack method. Peretz and his colleagues at the institute published their research on ways of inhibiting prion propagation in August 2001 (5). Specifically, the team examined the ability of several recombinant antibody antigen-binding fragments (Fabs) to inhibit prion propagation in cultured mouse neuroblastoma cells (ScN2a) infected with pathogenic prion protein (PrP^Sc). The research shows that antibodies binding cell- surface endogenous cellular prion protein (PrP^C) inhibit PrP^Sc formation in a dose-dependent manner. In the report, the authors found the most potent antibody, Fab D18, suggested a cure for established infection by prions.

“Our observations support the use of antibodies in the prevention and treatment of prion diseases and identify a region of PrP^C for drug targeting,” the researchers wrote. For the study, they used recombinant prion protein Fabs to study inhibition of prion protein propagation by antibodies.

Human clinical trials begin
Until recently, research into prion diseases in the United States was restricted to other transmissible spongiform encephalopathies (TSEs), such as scrapie in sheep; wasting disease in elk, mules, and white face deer; and a type of spongiform encephalopathy found in mink. “BSE and scrapie are representative of TSEs but are not the same agent,” wrote Keith Baker, a veterinary surgeon and 1998–1999 president of the British Veterinary Association, in an online interview. The agents, he said, are tremendously difficult to work with because they have such long incubation periods and require large numbers of laboratory animals for the work.

In early 2000, researchers from the National Institute of Allergy and Infectious Diseases (Bethesda, MD) found that cyclic tetrapyrroles, a class of compounds that includes drugs currently used in cancer therapy, can slow the development of TSE in mice (6, 7). TSE occurs when normal prion protein converts to an altered, disease-causing form. Cyclic tetrapyrroles have been shown to block that conversion. If this line of research is extended to trials on humans, these compounds may benefit not only patients with CJD and nvCJD, but also patients with nonprion protein aggregation diseases including Alzheimer’s disease and type 2 diabetes.

Although USDA’s new funding applies only to animals, clinical trials on humans began earlier this year in England and the United States to investigate chemical treatments for nvCJD. The U.S. Food and Drug Administration (FDA) allowed neurologist Bruce Miller of the University of California, San Francisco (UCSF), to conduct tests on humans using two known drugs that may prevent the disease, or at least slow its progression. Miller is applying some previous research (8) under FDA’s “compassionate treatment” provision, which allows doctors and scientists to treat people off of protocol when a life-threatening situation exists. He is working with Fred Cohen of UCSF to test quinacrine and chlorpromazine, which have been used for many years to control malaria and schizophrenia, respectively, to determine whether they reduce nvCJD symptoms.

Two patients with nvCJD are receiving either quinacrine or chlorpromazine. Miller is reserving his opinion on the drugs’ effectiveness in curtailing nvCJD symptoms in humans, but in August, a London tabloid reported that the condition of one of the patients improved during the experiment (9).

Quinacrine is commonly referred to as atabrine or mepacrine, and it was used widely during World War II as an antimalarial agent. Quinacrine was recently banned in India (10), where it was used by unethical doctors as a female sterilization agent (it works by scarring the uterus and blocking the fallopian tubes). Chlorpromazine (marketed as Thorazine) has been used to treat schizophrenia and other psychotic conditions since the 1950s. Cohen thinks the drugs will work on humans because recently published findings define a new class of antiprion compounds (11). Those compounds consist of a tricyclic scaffold with an aliphatic side chain extending from the middle ring moiety. The researchers suggest that quinacrine and chlorpromazine are immediate candidates for treating CJD and other prion diseases because they fit the structural criteria and are known to pass the blood–brain barrier.

Cohen said he and Miller want to recruit as many research subjects as possible. At least 30 patients will be needed to answer certain statistical questions. The pool to choose from is small, however. Not only that, but “there is a limited number of doctors who see patients with nvCJD,” Cohen explained.

Several scientists have postulated various enzymatic functions of prion proteins, but that research has not been confirmed, Cohen said. Also, some work on antibodies that can eliminate prion infection in cultured cells has shown promise, he said, adding “but that’s a long way from you and me.”

“There are a couple of logical ways to think about interfering with prion disease. One is large-molecule–like antibodies, or protein therapeutics. Another is the more traditional approach using small molecules to either come up with a new one or find a new use for an existing one,” Cohen said.

Overreacting?
Fred Cohen still eats beef, even though he knows more than most people about prion diseases, nvCJD in particular. So does Gavin Meerdink, beef extension veterinarian for the University of Illinois at Urbana–Champaign, and a BSE expert.

“I don’t think there’s any nvCJD in the United States,” said Meerdink, who refers to incidents such as Oprah Winfrey’s well-publicized beef boycott as “mass hysteria” (12). “In the big scheme of things, this is not a huge epidemic by any means. We know how to control it to a large degree—by eradicating the animals.” Burning the animals, he said, is the only known chemical process that is effective against BSE because it destroys the chemical substrate containing the prion proteins. “To do that, you’re going to destroy all the other proteins that are there, unless you can find some very specific enzymatic treatment technology that surgically attacks the particular protein you’re after,” Meerdink said.

References

1. Legislative Updates, 107th Congress. Animal Disease Risk Assessment, Prevention, and Control Act of 2001, Public Law 107-9 (S.700); http://olpa.od.nih.gov/Legislation/legislative_updates_107th.htm.
2. www.cmpharm.ucsf.edu/~wallacea/nobel.html.
3. Rampton, S.; Stauber, J. Mad Cow U.S.A.: Could the Nightmare Happen Here? Common Courage Press: Monroe, ME, 1997; p 48.
4. U.K. Department of Health. Monthly Creutzfeldt-Jakob Disease Statistics; www.doh.gov.uk/cjd/stats/oct01.htm.
5. Peretz, D.; Williamson, R. A.; Kaneko, K.; et al. Nature 2001, 412, 739–743.
6. National Institute of Allergy and Infectious Diseases, National Institutes of Health. NAID Researchers Identify New Drugs to Treat “Mad Cow”-like Disease in Mice. Press release, Feb 24, 2000; http://www.niaid.nih.gov/newsroom/releases/prionmice.htm.
7. Priola, S. A.; Raines, A.; Caughey, W. S. Science 2000, 287, 1503–1506.
8. Korth, C.; May, B.C. H.; Cohen, F. E.; Prusiner, S. B. Proc. Nat. Acad. Sci. 2001, 98 (17), 9836–9841.
9. Hamilton, D. P.; Regalado, A. FDA Approves Human Trials of Drugs Targeting Variant of Mad-Cow Disease. The Wall Street Journal, Aug 13, 2001, p A6.
10. Khan, S. Y. Women Are Guinea Pigs in Contraceptive Trial. The Sunday Times, Mumbai, India; www.africa2000.com/GNDX/quinban.html.
11. Borman, S. Chem. Eng. News 2001, 79 (34), 11.
12. Oprah Winfrey Faces Mad Cow Libel Suit. BBC News, Jan 20, 1998; http://news6.thdo.bbc.co.uk/hi/english/world/newsid_48000/48964.stm.

Note: All of the URLs were accessed in December 2001.

[Editor’s note: A brief news item on this topic appeared in the October 2001 issue of Chemical Innovation.]

Debra A. Schwartz is a freelance writer and editor in the Washington, DC, area (debinmld@aol.com). She is working toward a doctorate in journalism at the University of Maryland, College Park.