MAD COW DISEASE: A CASE FOR STUDYING LIVING ANIMALS

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MAD COW DISEASE: A CASE FOR STUDYING LIVING ANIMALS

 

By Howard B. Urnovitz, Ph.D

 

I. Introduction

 

Mad cow disease or bovine spongiform encephalopathy (BSE) is a progressive,

neurodegenerative disease in cattle. The presence of BSE cattle in the food

supply has been recognized as an urgent public health concern since 1996, when

British scientists hypothesized that some individuals exposed to BSE cows

developed a similar fatal condition, new variant Creutzfeld-Jakob Disease

(nv-CJD). No treatment exists for BSE or related illnesses and historically,

these diseases are only diagnosable after death. Once suspected BSE animals are

destroyed, their brains are examined for the architectural destruction (i.e.,

vacuolation and fibril formation) seen in the disease. Some research facilities

also test for the presence of an abnormal protein associated with BSE, a

misfolded form of the naturally occurring prion protein. (Prion protein, in both

its normal and abnormal forms, is sometimes abbreviated " PrP. " ) Investigators

have previously been unable to develop a test for BSE in live animals

because research has focused nearly exclusively on the abnormal prion protein,

which is genetically and serologically indistinguishable from the " normal " prion

protein found in healthy brain tissue.

 

The presence of BSE cows in the human food supply has significant financial, as

well as public health, implications. The goal is to eliminate BSE cows from the

food chain, and a test performed on live animals could provide a significant new

tool for protecting human health as well as guarding against massive losses in

beef-related industries by identifying BSE cows earlier (i.e., prior to

slaughter or development of symptoms). Chronix Biomedical has developed the

first test that can be performed on live animals, the " Surrogate Marker Living

Test for BSE. " Our test profiles a surrogate marker never before detected in a

professional laboratory setting: endogenous RNA in the serum of living animals.

 

During the discovery process that led to the development of the surrogate marker

living test, we made a number of unique observations about BSE cattle. Our

studies of current BSE literature suggest that, in fact, we are far from fully

understanding the genesis of BSE, its transmissibility and natural history, and

other basic facts that could prove crucial to efforts to prevent BSE in cattle

and potentially result in an effective treatment for humans who develop nv-CJD.

 

II. Backgrounder: Bovine Spongiform Encephalopathy

 

First diagnosed in Britain in 1986, BSE is a member of a group of chronic

degenerative diseases of the central nervous system called " transmissible

spongiform encephalopathies " or TSEs. These invariably fatal diseases cause

vacuolation of the brain (producing a sponge-like tissue, hence the term

" spongiform " ), and have been identified in a number of species. In humans, TSE

diseases include kuru, sporadic Creutzfeld-Jakob disease (CJD, believed to be a

familial condition), and new variant Creutzfeld-Jakob disease (nv-CJD, believed

to arise in individuals in contact with mad cows or who consume BSE-tainted beef

products). Two lesser-known TSEs also affect humans, Gerstmann-Straussler

syndrome and fatal familial insomnia.

 

TSEs also affect sheep and goats (scrapie), elk and deer (wasting disease), mink

(mink encephalopathy), and cats (feline spongiform encephalopathy).

 

The TSE diseases are believed to be caused by similar, not-yet fully

characterized transmissible agents. According to the United States Department of

Agriculture, the TSEs share certain common characteristics:

 

a prolonged incubation of months or years;

a progressive, debilitating neurological illness that is invariably fatal;

brain tissue from animals or humans affected by these diseases demonstrate

the presence of scrapie associated fibrils (SAF) under the electron microscope;

the central nervous system (CNS) alone appears to be affected;

pathological changes seen in the CNS include holes (vacuolation) in brain

stem tissue, giving it a sponge-like appearance; there is also an increase in a

type of brain cells called astrocytes;

the putative transmissible agent has not been found to elicit a specific,

detectable immune response in the host, i.e., an antibody that could be used for

diagnosis in live animals;

to date, all confirmation of a TSE diagnosis occurs after death.

 

In cattle with BSE, the animals may appear aggressive or nervous; appear clumsy

(develop abnormal posture, stumbling, and difficulty rising); have decreased

milk production; or lose body condition despite normal feeding. After developing

symptoms, affected cattle die within six months or are destroyed.

 

Although the causative agent of BSE and the other TSEs has not yet been fully

characterized, the U.S. agriculture agency offers three hypotheses about it,

alternatively:

 

An unconventional virus.

A prion or abnormal partially-proteinase K-resistant protein, devoid of

nucleic acid, capable of causing normal prion protein in the host to change and

form more abnormal protein.

A virino or " incomplete " virus composed of naked nucleic acid protected by a

host protein.

 

USDA researchers further hypothesize that the TSE agent is small, unusually

resistant to destruction by heat, radiation, light, and many disinfectants; and

capable of eluding the host immune system (that is, detectable antibodies

against it are not created). The agent has not been observed under the

microscope.

 

Some researchers strongly suggest that the most reliable marker for BSE and the

other TSEs, an abnormally-configured version of the naturally occurring prion

protein, is the cause of these diseases. While these investigators claim that

the abnormal prion protein cannot be separated from infectivity in TSEs, other

researchers have published data showing infectivity exists in the absence of

these abnormal prion proteins, an apparent conundrum we shall examine below.

 

III. BSE: The Tests

 

At the present time, the most widely used tests for BSE in slaughtered cattle

both target the abnormal prion proteins. The two most frequently used tests are

manufactured by Prionics AG (Schlieren, Switzerland) and Bio-Rad Laboratories

(Hercules, CA, USA)

 

Both are antibody-based tests conducted on brain samples from slaughtered

cattle. The Surrogate Marker Living Test for BSE developed by Chronix Biomedical

and our colleagues at the Institute of Veterinary Medicine, Georg-August

University (Göttingen, Germany), is a blood test.

 

The Prionics® -Check WESTERN uses Western-Blot technology. The first rapid BSE

test officially recognized by Swiss authorities in 1998, the company describes

it as the most frequently used quick BSE test in the world.

 

Prionics® -Check WESTERN detects a protein, PrP(sc), in affected animals' brains

and works, according to the company's web site essentially like this:

 

Bovine brain stem is removed at the slaughterhouse.

The brain stem sample is homogenized and transferred to a 96-well analysis

plate.

Homogenized brain samples are treated with a reagent mixture of digestion

enzymes and buffer solution that degrades the normal prion protein, leaving only

the abnormal BSE linked prion protein in the test sample.

The BSE related prion protein is bound with Prionics Antibody® 6H4 and other

reagents, then tested by means of an immunofluorescent assay.

 

Prionics claims 100 percent sensitivity and 100 percent specificity at the time

of its approval by the European Union in 1999. Prionics® -Check WESTERN is said

by the company to identify histologically negative, immunohistochemically

positive BSE cattle.

 

Bio-Rad's PLATELIA BSE test employs ELISA technology. On its web site, Bio-Rad

suggests a " posttranscriptional illness hypothesis " to explain BSE, which they

describe as " a protein synthesis disorder resulting in the accumulation of a

pathogenic biochemical form of the normal protein in the central nervous

system. "

 

In February 2000, Bio-Rad entered into a collaboration with the French

Commissariat a l'énergie atomique (CEA) to commercialize a test for BSE. The

resulting PLATELIA BSE test was shipped to European customers beginning in

November 2000.

 

The PLATELIA BSE test specifically detects the abnormal protein " PrPres " in

bovine brain samples. According to the company's web site, results can be

obtained in about four hours. Bio-Rad describes two stages involved in running

the test:

 

PrPres is purified using reagents that includes proteinase K, resulting in

the degradation of normal prion proteins (PrPsens), leaving only abnormal prion

protein (PrPres) immunoreactivity.

PrPres immunoreactivity is measured using the company's DAS-ELISA assay.

 

Bio-Rad asserts 100% sensitivity and 100% specificity on tested samples.

 

Both of these assays (and possibly others in use or development) appear to be

extremely accurate at detecting the presence of the BSE prion proteins.

Nevertheless, they can only be performed on brain tissue, i.e., following

slaughter. The obvious way to limit the proliferation of BSE is to diagnose the

disease in living animals so they can be immediately culled before their

carcasses enter the human food chain or are used in bovine tissue-containing

products such as gelatin, vitamins, pharmaceuticals, cosmetics, and others.

 

Our efforts in this area have resulted in the Surrogate Marker Living Test for

BSE. The discovery process that led to the development of the live BSE blood

test revealed a number of unique observations about BSE. Instead of studying the

immunochemistry of prion proteins as most other investigators in the field

currently do, we identified a completely unrelated biochemical marker in the

blood of cows with BSE and their cohorts: microvesicular associated RNA.

 

In the past it has been assumed that such microvesicles were of foreign origin

and they have been referred to as viruses. While microvesicles are found in both

healthy and diseased animals, their associated RNAs appear to differ.

 

Developed in a German-American collaboration between the laboratories of Prof.

Bertram Brenig, Director of the Institute of Veterinary Medicine, Georg-August

University (Göttingen, Germany) and Chronix, the Chronix Biomedical Surrogate

Marker Living Test for BSE is a real time PCR test for the in vitro detection of

the reaction pattern of bovine serum isolated RNA with a Chronix proprietary

target. This test is intended for use in professional laboratory settings as a

surrogate marker living test for assessing the risk of cattle to developing

bovine spongiform encephalopathy. Cattle showing repeat reactivity in this test

should be monitored closely. A second more specific test should be used to make

a conclusive diagnosis of BSE.

 

Principles of the Procedure for the Surrogate Marker Living Test for BSE:

 

RNA is present in the liquid fraction of blood samples from both healthy and

diseased cattle. The RNA is found in the blood fraction that contains primarily

microvesicles. A surrogate marker living test for BSE was developed to detect

RNA profiles in bovine serum.

 

Thus far, the Chronix Biomedical Surrogate Marker Living Test is 100% sensitive

on six out of six BSE cows confirmed with a licensed prion test, and 100%

specific on animals from known healthy herds (219 confirmed prion free).

 

IV. Organizing The Current Data On Experimental TSE Diseases

 

During the process that led to the development of the Chronix Biomedical

Surrogate Marker Living Test for BSE, we realized that the only way to truly

protect the food supply from BSE is to organize the current medical and

veterinary wisdom. Research is proceeding in a number of directions: development

of diagnostic tests, of potential treatments, and of possible vaccines. Only by

sorting the current medical wisdom into " facts " and " opinions " can we discern

the most effective way to solve the BSE problem.

 

Our investigations during the development of the surrogate marker blood test led

my colleagues and I to the realization that little is known with certainty about

the natural history of BSE and its related illnesses, the transmissible

spongiform encephalopathies (TSEs) like nv-CJD. We have adopted new terminology

for these conditions when they are studied in the laboratory: experimental

transmissible spongiform encephalopathies, or " eTSEs. "

 

eTSEs differ from BSE and other similar illnesses found in nature in that they

are never induced in the same way as a wild type disease would arise in an

animal or a herd. Most eTSEs are produced by injecting foreign biological matter

(usually diseased brain tissue emulsified, denatured, heated, or otherwise

adulterated with chemicals) directly into the brains of presumably healthy

experimental animals. (Intraperitoneal injection of infectious material is also

employed as a method of transmitting eTSEs in the laboratory, which possesses

the same relationship to the diseases' natural history as does injection of

foreign matter into the brain.) Given the lack of available systems to study the

TSEs in natural settings (i.e., determine their natural history in live

animals), we must ask, just how solid are the existing data on these devastating

neurological diseases?

 

A. What Causes Spongiform Encephalopathies? The Hypotheses

 

The Prion Hypothesis

 

Have we identified the appropriate target in spongiform encephalopathies, e.g.,

the abnormal prion proteins?

 

In 1982, Stanley B. Prusiner (at the University of California, San Francisco)

reported in Science magazine the discovery of a " novel proteinaceous infectious

particle " he hypothesized to be the cause of scrapie. (15) Later that year,

Prusiner and UCSF colleagues described a protein that appeared to co-purify with

the putative " scrapie agent " and that appeared to be required for

infectivity.(3)

 

Prusiner and colleagues named these " unusual infectious particles, " seemingly a

protein-scrapie agent complex, " prions. " Additionally, the scrapie-associated

protein was found to be resistant to digestion by proteinase K, unlike other CNS

proteins, consistent with the resistance of the scrapie agent to destruction by

heat, ultraviolet light, ionizing radiation, and disinfectants. These

investigators reported that, " Initial results suggest that the amount of this

protein correlates with the titer of the [scrapie] agent. " Prusiner and

colleagues reported finding the protein in all samples from scrapie-infected

brains, but in none of the normal brains. They hypothesized that this protein

" is a structural component of the scrapie agent. " (3)

 

In 1986, Prusiner and colleagues published a research report in Cell that

examined the strength of the correlation in scrapie-infected hamster brain

tissues between the most abundant prion protein (designated PrP 27-30) and the

putative scrapie agent. (13) " Previous work has shown that preparations from

infected hamster brains enriched for scrapie infectivity contain PrP 27-30 as

the major protein component and that denaturation with SDS or treatment of such

preparations with reagents that attack proteins abolished infectivity, " Prusiner

and colleagues reported. " …To date, we have been unsuccessful in separating

scrapie infectivity from PrP 27-30. " (13)

 

In the 1986 Cell article, Prusiner and colleagues also suggest that " one

possible model for the prion is that it is devoid of nucleic acid. " Failure to

identify a rich source of nucleic acid (i.e., viral DNA or RNA) in affected

brains was a major reason that viruses were dismissed as possible causes early

in TSE research. Prusiner's group also suggested that the pathogenic prion

protein did not elicit an immune response in affected brains because of

immunotolerance of the " normal PrP cellular homolog, " i.e., the normal prion

protein found in healthy animals' brains.(13)

 

Prusiner and colleagues summarized the Cell study by noting, " By molecular

cloning of a cDNA encoding scrapie PrP 27-30, we have shown that a single

cellular gene encodes this protein and that both normal and scrapie-infected

tissues contain PrP-related mRNA sequences. " (13)

 

Prusiner's finding that a single gene codes for the prion proteins found in both

healthy and diseased tissues raised an important question that remains

unanswered by current eTSE research: How does the abnormal prion protein

" invade " the central nervous system and cause large numbers of normal prion

proteins to become abnormal, thereby inducing disease? Although numerous

theories on how this happens have been hypothesized, the infectious mechanism of

prions remains a mystery, and some researchers in the field do not believe that

prions are the causative agents of this class of diseases.

 

Yale Medical School investigator Laura Manuelidis has shown laboratory

infectivity of Syrian hamster CJD in the absence of abnormal prion proteins

(discussed below). Unlike other investigators, Manuelidis and her group found

significant amounts of nucleic acids associated with eTSE infectivity, and they

have suggested that the true cause of these diseases is more likely a virus or a

virus-protein complex rather than the abnormal prion protein. (2,11)

 

Adriano Aguzzi at the University Hospital of Zurich (Switzerland), like

Manuelidis, questions whether abnormal prion proteins are cause or effect in

TSEs. How, he asks, can the " more-or-less accepted wisdom " assert that

TSE-causing prions consist solely of abnormal prion proteins, which have exactly

the same amino acid structure as non-pathogenic prion proteins? (1)

 

Writing in Haematologica in 2000, Aguzzi comments, " A more noncommittal way of

wording this fact would be to state that [the abnormal protein] PrPSc is the

only known surrogate marker for prion infectivity: this latter statement is

likely to be acceptable to both the proponents of the protein-only hypothesis

and to those who still believe that the infectious agent is a virus. " (1)

 

The Viral Hypothesis

 

In the mid-1990s, Manuelidis and her group at Yale University identified

" endogenous viral complexes with long RNA cosediment " in hamster CJD, and

suggested that these viral particles are required for infectivity in the absence

of abnormal prion protein, (2,11) a finding that contradicts an earlier study by

Prusiner and colleagues (12).

 

In a 1994 report in Nucleic Acids Research, Manuelidis and her colleagues

described finding intracisternal A particles (IAP) as large as approximately

5,000 contiguous bases. They argued that their data " strongly indicates specific

viral complexes cofractionate with the CJD agent " and suggested that these

infectious complexes are more likely to be the causative agent of hamster CJD

than the abnormal PrP (i.e., the abnormal prion protein). The Yale group further

suggested that PrP is a viral receptor, not the causative agent. (2) In a 1995

paper, (11) the Manuelidis group described experiments in which preparations

from hamster CJD tissues that were " devoid of host PrP " were shown nevertheless

to retain infectivity even after 99% of the starting prion protein has been

separated from the virus-like particles. " Furthermore, our accompanying

analytical data indicate that intact viral-like complexes are required for

infection, " they reported. (11)

 

What are these " virus-like particles " described by the Yale group? It is not

clear to us that the isolated RNA observed by Manuelidis' group is necessarily

that of a virus, rather than endogenous host RNA packaged in a naturally

occurring microvesicle.

 

In her latest review (9), Manuelidis elegantly summarizes the viral hypothesis

theory. Figure 4 shows a molecular strategy for identifying the RNAs associated

with TSEs. We know from experience that this strategy will not reveal the whole

story because the vast amount of molecular clones recovered will be mRNA (e.g.,

selected by its poly A+ tail, using this method). It is not clear to us that the

target molecule should be mRNA. What if regulatory genes (missed by the poly A+

selection procedure) are components of the spongiform encephalopathy disease

process? Why doesn't the experimental design (9) address the small and short

RNAs?

 

Recently, small nucleic acids–RNAi (RNA interference) and siNAs (short

interfering nucleic acids), among others–have come under increased scrutiny

(16). These small RNAs (and others still being characterized) appear to be able

to " turn off " specific genes. Biotechnologists are attempting to develop

therapeutics using small RNAs that may potentially " silence " harmful genes.

Sirna Therapeutics, for example, is currently focusing on producing

" therapeutic " RNAi to tackle a wide variety of diseases including cancer as well

as " metabolic, inflammatory, central nervous system, renal and infectious

diseases. "

 

RNAi's typically work as short RNA sequences (around 25 nucleotides in length).

(8) Biotechnology companies are increasingly pursuing similar avenues of drug

development utilizing the special properties of small RNAs.

 

Therefore, our new knowledge about RNAi and other small RNAs should be

considered as we attempt to fully characterize the TSE agent. For example, are

short nucleic acids co-purifying with prions? Do small RNAs play a role in the

disease process? If so, are they of endogenous origin? To our knowledge, such

experiments remain to be performed.

 

B. Concerns About Current Systems For Studying eTSEs

 

One major problem in eTSE research is that the appropriate positive

control–injection of healthy brain matter treated precisely like the diseased

brain tissue–is rarely employed in studies of eTSEs. A research report in the

March 28, 2000, issue of the Proceedings of the National Academy of Sciences

communicated by senior author D. Carleton Gajdusek illustrates the inherent

artificiality in much current BSE/eTSE research, as well as a lack of adequate

controls.(5)

 

Gajdusek and U.S. and French government scientists examined how exposure to

increasing amounts of heat affected the apparent infectivity of the

hamster-adapted scrapie agent (from sheep). It is difficult to isolate prion

proteins from the brain matter so that a pure preparation can be injected into

an experimental animal. These investigators followed common practice in eTSE

research and pooled " crude brain tissue " from a number of hamsters previously

infected (route of infection is not specified) with the 263K strain of

hamster-adapted scrapie agent. One-gram samples of the pooled brain matter were

exposed for either five or 15 minutes to dry heat ranging from 150 degrees C to

1000 degrees C. Those heat-treated crude brain tissue samples were then

inoculated intracerebrally into healthy hamsters. The hamsters were observed for

ten months, sacrificed, and their brains Western-blot tested for the presence of

the scrapie-associated prion protein. The objective of the study was to

determine at what temperature the prion protein was inactivated (i.e.,

presumably rendered uninfectious). They note that, at 600 degrees C (as well as

at 1000 degrees C), the brain aliquots " produced a flaming tissue combustion

that lasted several seconds and yielded a residue of glowing black ash that had

lost 98-99% of its initial weight. " Nevertheless, even after combustion at 600

degrees C, these authors noted that a " trace amount of infectivity " remained in

those brain samples. (5)

 

The unexpected finding that brain matter reduced to ash after being heated to

600 degrees C nevertheless created neurological illness when injected into the

brains of healthy animals caused Gajdusek and colleagues to hypothesize an

" inorganic template of replication. " (5)

 

" An alternative explanation [to the survival of intact scrapie-related prion

proteins] is that an inorganic replica of the necessary molecular geometry was

made, which nucleated the conformational change of the PrP precursor protein to

its infectious, beta-pleated isoform, similar to the heteronucleation by

minerals of many protein crystallizations, " Gajdusek and co-authors suggested.

The combustion of the brain matter may have caused the organic molecules (i.e.,

the prions) to form a " microporous or mesoporous replica in silica or alumina

silicates, " creating an " enzyme-mimicking inorganic material " responsible for

the transmission of scrapie into healthy hamster brains even after the

presumably infectious organic matter was reduced to ash, according to Gajdusek

and coauthors. (5)

 

Whether Gajdusek and colleague's suggested " fossil templates " of " organic

amyloid nucleants " are responsible for the transmission of scrapie after

exposing scrapie-infected brain matter to high levels of heat and even

combustion remains to be further investigated. Given that the formation of

inorganic matter from organic matter (ash from brain) was observed, and that

injection of the inorganic matter into hamster brains produced neurological

disease, wouldn't it be prudent to examine the effect of injecting other organic

and inorganic matter into hamster brains? Does an animal injected intracranially

with a heat-treated preparation of normal brain tissue develop neurological

disease? What effect does injecting inorganic ash from another biological tissue

have on the nervous systems of these animals?

 

As Gajdusek et al.'s PNAS paper illustrates, the eTSEs are created in laboratory

animals by the injection of foreign biological or inorganic matter into the

brain or gut. While experimentation with eTSEs has certainly provided important

insights into BSE and related illnesses to date, it has also provided a model of

these illnesses that bears little resemblance to the biological processes that

occur in nature.

 

C. The Question Of Transmissibility of TSEs And Its Prevention

 

Like BSE, CJD, nv-CJD, and other TSEs, Alzheimer's disease (AD) occurs in

association with misfolded or misshapen proteins. U.S. National Institutes of

Health researcher Paul Brown has studied possible drug therapies for familial

(hereditary) CJD, its " environmentally acquired " relative (nv-CJD), and related

illnesses like AD, as well as hypothesized development of a vaccine to protect

against these neurodegenerative diseases. In a 2002 review in Neurology, (6)

Brown summarized current treatment strategies for familial CJD and AD.

Reflecting the state of the field, these potential treatments all targeted the

abnormal proteins found in CJD and AD, misfolded prion proteins and amyloid

plaques, respectively. While providing an overview of numerous ways to eliminate

these abnormal proteins from the central nervous system–by interfering with

protein production, either chemically or via gene therapy, or by preventing

proteins from folding into the abnormal conformation associated with the

conditions–Brown suggested that the differences observed between CJD and AD may

hold a key to understanding both diseases.

 

In the 2002 Neurology review, Brown astutely observed, " The most fundamental

difference between [environmentally acquired] CJD and AD lies in the phenomenon

of transmissibility. Discovering ways to break the chain of events responsible

for the accumulation of amyloid will undoubtedly have useful therapeutic

consequences for both diseases, but will not explain why [environmentally

acquired] CJD is infectious and AD is not. Aggregation of peptide fragments into

amyloid fibrils is an altogether different matter than the ability of a

misfolded protein to 'seed' the production of similarly misfolded protein, and

constitutes the single most important unanswered question confronting TSE

research. " (6)

 

While it seems only prudent to resolve the question of what causes TSEs before

creating vaccines that attempt to prevent them, numerous investigators are

forging ahead with vaccine development before questions about causation have

been fully answered.

 

For example, University of Toronto neurologist Neil Cashman was senior author of

a study published in the June 1, 2003, online edition of Nature Medicine (14)

announcing the detection of an antibody specific to " the pathologically

misfolded conformation " of the prion protein. This finding, Cashman suggested in

an interview with the Canadian Press service, could result in the development of

a BSE vaccine in about one year. (4) Cashman and colleagues reported in Nature

Medicine that they identified an antibody repeat motif

(tyrosine-tyrosine-arginine) in mice that recognized the abnormally folded prion

protein but not the correctly folded form found in healthy cells. (14) The

research was primarily funded by and performed in collaboration with Caprion

Pharmaceuticals Inc. (Quebec, Canada), which owns all commercial applications of

this research.

 

Like Cashman, Brown supports vaccine development efforts as the best method of

controlling TSEs. In his 2002 Neurology review, Brown commented that " The ideal

preventive strategy would be the development and use of a vaccine " against prion

proteins. He further suggested that the creation of mice genetically engineered

to resist intraperitoneal infectious challenge with scrapie " proves the point

that with imaginative genetic manipulation, protective immunization against the

'prion' protein need not result in a generalized autoimmune reaction, an

accomplishment that invites new efforts to create a practical and potent vaccine

against the agents that cause TSE. " (6)

 

Anti-prion vaccines are not the only ones being considered by eTSE researchers.

In the Proceedings of the National Academy of Sciences USA in 1998, Manuelidis

suggested developing a " live virus " TSE vaccine against the putative virus-prion

complex she hypothesizes may cause these illnesses. (10) Manuelidis in

retrospect chose two rather unfortunate examples to argue in favor of a live

virus vaccine to protect against TSE diseases: the live, oral poliovirus vaccine

and a possible " live virus " vaccine to protect against HIV. In June 1999, the

Centers for Disease Control and Prevention (CDC) recommended a return to the

inactivated, injectible polio virus vaccine, noting that every case of polio in

the United States since 1979 had resulted from use of the live virus oral polio

vaccine.

 

Development of a vaccine against HIV, although no live virus vaccines are

currently in clinical trials, hit a major bump in the road when a recent phase

III HIV vaccine trial showed no efficacy, revealing a statistically

insignificant 3.8 percent difference in new HIV infections between vaccine and

placebo groups. (7)

 

Problems inherent in the vaccine approach seem more likely to stall than to

solve the public health issues presented by the TSE diseases.

 

The inherent risks of vaccine development also might be quite unnecessary to

assume, if Aguzzi is correct about the level of background (subclinical) CJD

infections compared to those that become symptomatic. (1) In considering the

ease of transmissibility of CJD via medical procedures (during neurosurgery, for

example), Aguzzi expresses surprise that such cases are so rare. " This is, in my

view, not totally understood, given that the frequency of subclinical CJD must

be much higher than that of manifest disease, and that most neurosurgical

instruments are not sterilized in a way that would reliably inactivate prions, "

he points out. (1) Even in a case when " several thousands " of individuals were

exposed to CJD contaminated cadaveric dura matter, fewer than 2 percent

developed a TSE disorder. " While we can rejoice about this low efficiency in the

take of infectivity, we do not fully understand the biological basis for the

apparent protection enjoyed by most subjects exposed to CJD

prions, " Aguzzi commented. (1)

 

So we have returned to the central question: In noting that there should be more

iatrogenic cases of CJD, is Aguzzi inadvertently pointing out that prions are

not the cause of CJD or other TSEs?

 

In her 2003 review article, Manuelidis also pursues the answer to that question.

With so much evidence suggesting that abnormal prion proteins are not the cause

of any TSE disease, why do both the scientific community and the public at large

believe that prions cause these neurodegenerative illnesses? " I think the casual

or expansive use of words, especially those taken out of their properly defined

scientific context, has obscured the truth of evidence. It is especially hard

then to sort out and clarify for others all the different features ascribed to

'prions', " she writes. Among them she lists the prion as cause of TSEs, as

pathologic response to the disease, as a factor in susceptibility to infection,

and as " the spontaneous self-inflicted pathology of a protein. " (9)

 

" Thus, the prion is the only proper thing to evaluate if we are to gain insight

into all meaningful mechanisms of infection and disease, " Manuelidis continued.

" But what is a prion? " Its meaning has been stretched and changed sufficiently

to have created a " muddle, " according to Manuelidis, making a true understanding

of TSE diseases difficult, if not impossible, to achieve. " It is time to look at

the actual molecules that could be part of this structure, rather than making

believe that nothing else is needed, and nothing else is there, " she concludes.

(9)

 

V. Summary

 

Our review of the literature has made it clear that the true cause of BSE has

yet to be identified. We hope that the identification of living cows with BSE

will help to focus our efforts to understand the natural mechanisms involved in

spongiform encephalopathies, as well as directing world-wide efforts to prevent

and treat the natural forms of these diseases. Scientific method should include

debate and challenge. We have great respect for the research that has been

carried out to date as well as the articulate arguments presented in favor of

the various hypotheses. Our greatest concern is that questionable theories not

form the basis of mandatory pharmaceuticals such as vaccines.

 

 

 

 

 

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long RNA cosediment with the agent of Creutzfeldt-Jakob Disease. NUCLEIC ACIDS

RESEARCH 1994;22(6):1101-1107.

 

3.Bolton D.C., McKinley M.P., Prusiner, S.B. Identification of a Protein that

Purifies with Scrapie Prion. SCIENCE 1982 December 24;218:1310.

 

4. Brautigam T. Mad cow vaccine in works. THE GLOBE AND MAIL, June 1, 2003.

 

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trial. MEDSCAPE MEDICAL NEWS 2003

 

8. Gura T. A silence that speaks volumes. NATURE 2000 April;404:804-808.

 

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11. Manuelidis L., Sklavidis T., Akowitz A., and Fritch, W. Viral particles are

required for infection in neurodegenerative Creutzfeldt-Jakob disease.

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16. Sharp, Phillip A. RNA interference--2001. GENES DEV. 2001 Mar

1;15(5):485-90. Director, McGovern Institute for Brain Research, MIT.

 

 

 

 

 

 

 

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