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From: TSS ()
Subject: Re: Detection of prions in blood Joaquín Castilla1, Paula Saá1, 2 & Claudio Soto1
Date: August 30, 2005 at 12:12 pm PST

In Reply to: Detection of prions in blood Joaquín Castilla1, Paula Saá1, 2 & Claudio Soto1 posted by TSS on August 28, 2005 at 3:53 pm:

30 August 2005

An Easy Assay for Prions?

An innovative new technique could finally lead to an early diagnostic test for diseases such as Bovine Spongiform Encephalitis (BSE), or "mad cow", and its human counterpart, variant Creutzfeldt-Jacob Disease (vCJD). Researchers say the results in lab animals, while encouraging, need to be replicated and note that competing tests are also under development.

Batty bovines. Cows with bovine spongiform encephalitis have holes in their grey matter, which give their brains a spongy look.
BSE, vCJD, and several other neurodegenerative diseases are believed to be caused by so-called prion proteins, which can occur in two forms: the normal variety, called PrPc, and a misfolded variant that forms clumps in the brain (PrPSc). The diseases are infectious, most researchers think, because PrPSc has the peculiar ability to impose its abnormal folding on PrPc. So far, scientists can only detect prion diseases in the brain, after death. The problem is that levels of prions circulating in blood plasma are extremely low, so any test would need to be exquisitely sensitive.

In previous studies, Claudio Soto and colleagues at the University of Texas Medical Branch in Galveston had shown that they could amplify these minute amounts of prions in a cyclical process reminiscent of the way the polymerase chain reaction amplifies DNA (ScienceNOW, 21 April). The researchers added normal prion proteins to a sample in the test tube, let the misfolded proteins "convert" the normal ones, broke up the resulting clumps with a sound pulse, and then repeated the process over and over until the PrPSc became detectable with ordinary methods. In a new study published online this week in Nature Medicine, Soto and his colleagues show that a vastly optimized and automated version of their test correctly identified 16 out of 18 prion-infected hamsters, without false positives in a control group of 12 uninfected hamsters.

Perluigi Gambetti, who directs the National Prion Disease Pathology Surveillance Center at Case Western Reserve University in Cleveland, Ohio, has a few criticisms: The hamsters were infected by injecting the protein directly into the brain, for instance, which is "like creating a stab wound" through which protein may have entered the blood stream. Still, "this is a breakthrough," he says.

Replication is needed, says Paul Brown, a retired prion researcher at the National Institute of Neurological Disorders and Stroke in Bethesda, Maryland. Other labs have had trouble achieving the prion amplification levels reported by Soto's group in previous papers, he says: "There's some nervousness about that." Soto acknowledges there were difficulties with replication at first but says these have largely been overcome.

Several competing tests are in the works as well, adds Jean-Philippe Deslys, who heads the Prions Research Group at the Atomic Energy Commission in France. Deslys says he has seen data on two promising ones that will be presented at an upcoming meeting. "It's really too early to say which test will be the best one," he says.


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The report
More on prion diseases

21 April 2005

Proof Positive for Prions

A new study has taken another step toward proving the "prion hypothesis"—the idea that rogue proteins are responsible for a host of fatal neurological diseases, including Mad Cow Disease and scrapie.

Prion factory. A schematic representation of the cyclic process by which a molecule of PrPc (left side of circle) interacts with and is converted into PrPSc (right side of circle). PrPSc damages normal brain tissue (left panel) by forming long fibers (right panel).
CREDIT: Eric Smith, Harvard University
According to the prion hypothesis, the disease-causing protein, PrPSc, makes copies of itself inside the brain, multiplying until it forms fibers that destroy neurons and eventually kill the animal. PrPSc has the same amino acid sequence as a normal brain protein called PrPc, but folds into a different three-dimensional shape. It replicates by corrupting any PrPc it encounters, twisting it into a new PrPSc, which in turn transforms more PrPc. Although recent research has overwhelmingly supported the prion hypothesis [ScienceNOW 15 October, 2003], one vital experiment was missing: No one had induced a prion disease in a healthy animal using pure PrPSc made in a test tube.

In work reported in the 22 April Cell, neurobiologist Claudio Soto of the University of Texas Medical Branch in Galveston and colleagues come close to achieving that goal. To generate high levels of PrPSc, Soto's group performed 20 rounds of “infection” in a test tube. In the first round, the group mixed ground up healthy brain with brain material from a scrapie-infected animal. Just as in an intact animal, the PrPc in the healthy brain material was converted to PrPSc. In subsequent rounds, they used material from the previous round to infect fresh healthy brain material. The procedure yielded billions of molecules of PrPSc from just a few starting molecules. Although some of the original scrapie brain material was carried through the early rounds of the experiment, it eventually became diluted to the point where it disappeared from the sample. Nonetheless, the material was very infectious, efficiently causing scrapie when injected into the brains of hamsters.
The caveat, UCSF neurobiologist Giuseppe Legname notes, is that because the researchers started with scrapie brain, not pure PrPSc, something else that contributes to scrapie may have been amplified along with PrPSc. However, Legname says, this system could be used to detect the low levels of prions present in the blood of infected animals. Such a test is "desperately needed," he says, to reduce the chances that prions will be spread through food or medical blood products.


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January 2004

NIAID Research on Prion Diseases

Prion diseases are a related group of rare, fatal brain diseases that affect animals, including humans. Also known as transmissible spongiform encephalopathies (TSE), they include bovine spongiform encephalopathy (BSE, or "mad cow" disease) in cattle; Creutzfeldt-Jakob disease (CJD) in humans; scrapie in sheep; and chronic wasting disease in deer and elk.

Much about TSE diseases remains to be discovered. The diseases are characterized by certain misshapen protein molecules that appear in brain tissue. Normal forms of these prion protein molecules reside on the surface of many types of cells, including brain cells, but scientists do not understand what normal prion protein does. On the other hand, scientists believe that abnormal prion protein, which clumps together and accumulates in brain tissue, is the likely cause of the brain damage that occurs in TSE diseases. Scientists do not have a good understanding of what causes the normal prion protein to take on the misshapen abnormal form.

The National Institute of Allergy and Infectious Diseases (NIAID) conducts TSE disease research in its Rocky Mountain Laboratories (RML) in Hamilton, MT, and also funds prion disease research in university labs. Two other National Institutes of Health Institutes also fund prion disease research — the National Institute of Neurological Disorders and Stroke and the National Institute on Aging.

Variant Creutzfeldt-Jakob Disease

In some cases, TSE diseases appear to be infectious. For example, there is good evidence that a newly discovered TSE disease — variant CJD (vCJD) — was first spread to humans from cattle infected with BSE, although that type of transmission is believed to be very inefficient. Identifying the transmissible agent is still an ongoing area of research. A leading hypothesis about the origin of vCJD is that abnormal forms of prion protein ingested through diseased meat sicken people by causing normal human prion protein to form incorrectly. This change from normal to abnormal prion protein spreads to the brain, where the misshapen protein aggregates in the spaces between brain cells and produces disease. An alternative hypothesis is that the disease is transmitted by a virus, which subsequently triggers the process of forming abnormal prion protein.

Health officials in the United Kingdom have responded to the possibility that beef infected with BSE may have spread vCJD to humans. Since the 1980s, when the mad cow epidemic began in the United Kingdom, millions of cattle in Europe have been destroyed. Worldwide, there have been 153 reported cases of vCJD, according to the Centers for Disease Control and Prevention (CDC). All of these cases were among people who ate beef in a country with a BSE outbreak, and nearly all — 143 of the cases — were in the United Kingdom.

The first North American case of mad cow disease was found in Canada in May 2003. Discovery of the first U.S. case of mad cow disease followed in December 2003 in a Washington state cow. Although no cases of endemic vCJD have been reported in the United States, more research is needed to confirm whether vCJD is spread to humans from cattle with mad cow disease.

An older human TSE, Creutzfeldt-Jakob disease, is similar to vCJD but progresses much more quickly and affects older people. Medical experts believe it can be both inherited and transmitted through infection, although some cases occur sporadically without a known cause.

Chronic Wasting Disease

A different TSE disease called chronic wasting disease has been detected in U.S. deer and elk populations. So far, scientists have uncovered no evidence that deer or elk with chronic wasting disease might transmit some form of TSE disease to people who eat deer or elk or have close contact with them. More research must be done to determine with certainty whether chronic wasting disease poses any risk to humans, particularly because it is spreading over a wider geographical area in the United States.

Basic Research

NIAID scientists at RML are examining how abnormal prion protein molecules cause TSE diseases. RML is one of the world's premiere laboratories for studying TSE diseases: scientists there co-discovered and were among the first to clone the prion protein gene. NIAID scientists also discovered that abnormal prion protein can convert normal prion protein to the abnormal form. This also may account for the disease process in the brain.

RML scientists are working to insert genes into mice that would allow the mice to carry the prion protein of deer and elk. These mice would react to a prion protein infection as a deer or an elk would. Thus, scientists can study the disease in these mice rather than in the larger animals, which are much more expensive and labor-intensive to maintain.

At Colorado State University, NIAID has established an emerging diseases research center focused on studying chronic wasting disease. This center is investigating the mechanics of chronic wasting disease infection in deer and elk, especially in the lymph nodes of their immune systems. Such studies underlie the search for improved diagnostics and therapies. The researchers also will seek to better understand the entire spectrum of disease transmission and under what circumstances chronic wasting disease might "jump" to other species. In addition, scientists at the center are working on a possible vaccine to prevent the spread of chronic wasting disease in deer and elk.

To assist investigators in obtaining needed materials to study TSE diseases, NIAID has created a TSE repository with joint funding from the National Institute of Neurological Disorders and Stroke. The repository will help researchers obtain standard biochemical reagents and animals with genetic modifications for use in prion protein research.

Cross-Species Transmission

At RML, studies are ongoing to understand the mechanisms by which TSE infections cross species. Experiments have demonstrated that species once thought to be resistant to certain TSE strains can be life-long carriers of the infection without ever becoming sick.

RML scientists are also investigating whether chronic wasting disease can be transmitted from deer or elk to monkeys if monkeys eat brain matter from deer or elk that are infected with chronic wasting disease. That knowledge would provide valuable insight into whether chronic wasting disease could be transmitted to humans.

Therapeutic Approaches

Although there are no known ways to treat TSE diseases, scientists around the world are working to develop treatments. Using infected tissue culture cells for fast initial screening, NIAID researchers have tested thousands of compounds and identified hundreds of molecules that inhibit the formation of the abnormal form of prion protein. Further testing of the most potent of these inhibitors has revealed several that can prolong the lives of rodents if treatments are begun near the time of infection. Researchers at RML, and at Utah State University through a contract with NIAID, are testing these compounds in animals. Other groups are further testing two of the inhibitors in Creutzfeldt-Jakob disease patients. However, no compounds have proven to have therapeutic effects after the onset of clinical signs of disease.

RML researchers have also identified antibodies and short synthetic protein molecules (fragments of prion protein) that can block the conversion of normal prion protein to the abnormal form. If successful, these investigations will lead to safe and effective methods to prevent prion infections as well as therapies that work in either the pre-symptomatic or symptomatic phases of disease.


Improved diagnostic tests for TSE diseases are critical for the success of both therapeutic strategies and eradication programs. NIAID scientists are exploring various approaches to creating new tests. In addition, NIAID has provided funds to the CDC for better monitoring of TSE disease incidence and to improve collection of tissue specimens for TSE disease-related diagnostics.

Related Information

Other federal agencies work on prion issues from standpoints of research, public health, food supply safety, and animal health. The following links provide more information.


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