Biomedicine and Health: Prions and Koch's Postulates
Biomedicine and Health: Prions and Koch's Postulates
Biomedicine and Health: Prions and Koch's Postulates
Prions, whose name stems from the designation “proteinaceous infectious particles” (abbreviated PrPs), are proteins that are by themselves infectious.
Prion diseases are a group of rare and invariably fatal brain disorders that occur in both animals and humans. They are unusual in that the infective agent is neither a virus nor a bacterium, but an abnormal form of the prion protein (PrP) that is normally found in the brain. Prion disease leads to the development of tiny holes within brain tissue, giving it a characteristic “spongiform” appearance at post-mortem. Hence, prion diseases are also known as the transmissible spongiform encephalopathies (TSEs).
The discovery of prions and confirmation of their infectious nature was revolutionary and controversial in microbiology. This is because it overturned a long-held dogma that infections were caused by intact organisms, particularly microorganisms such as bacteria, fungi, parasites or viruses, which contained genetic material in the form of either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Because proteins lack genetic material, the prevailing attitude was that any protein alone was incapable of causing disease.
This article will discuss Koch's postulates of disease and the role they play in the laboratory identification of infectious agents. Prions, protein-based disease agents thought to be the infectious agents that transmit madcow disease and its human form, variant Creutzfeldt—Jakob disease (vCJD), may be exceptions to Koch's rules. The biochemistry of prions, the debate over the protein-only hypothesis, and the economic and medical effects of prion-based diseases will also be explained.
Historical Background and Scientific Foundations
Robert Koch and His Postulates
Nobel Prize winner Robert Koch (1843–1910) was a German physician and provincial medical officer who did some of the first groundbreaking work on pathogenic (disease-causing) organisms, conducting experiments in his home laboratory. While studying anthrax and tuberculosis bacilli between 1884 and 1890, he formulated a set of criteria about bacteria and their ability to cause disease known as Koch's postulates. Today, these rules of laboratory practice must generally be satisfied before it can be proven that a certain microorganism causes a particular disease.
During his research to identify the tubercle bacillus (the cause of tuberculosis), Koch employed precisely regimented laboratory procedures that later allowed him to formulate his postulates. Using microbes that he cultured from different sources, he injected them into guinea pigs and monitored the results. Once he induced tuberculosis in these animals, he proved that the original microbe was the infectious agent by identifying it in new cultures from the test subjects' tissues.
From these experiments, Koch, in a series of later papers about anthrax, established his postulates, which set standards for pathological microbiology (the study of microbes and their role in disease) and epidemiology (the study of the transmission and spread of disease in populations). The postulates are:
- The bacteria must be present in every case of the disease.
- The bacteria must be isolated from the host with the disease and grown in pure culture.
- The specific disease must be reproduced when a pure culture of the bacteria is inoculated into a healthy susceptible host (in a laboratory animal).
- The bacteria must be recoverable from the experimentally infected host.
Koch's postulates hold true for the vast majority of infectious diseases, but they do have limitations. The particular microbe may not be able to be grown in pure culture or in a laboratory animal. Other exceptions include viruses, parasites, rickettsias, and chlamydia. The latter two infect humans only, making it difficult if not impossible to verify Koch's third postulate: Since the disease cannot be grown in an animal host, a healthy human would have to be infected.
Prions and the Protein-Only Hypothesis
For many of the same reasons, such related diseases as bovine spongiform encephalopathy (BSE or mad cow disease), scrapie in sheep, and variant Creutzfeldt—Jakob disease (vCJD) in humans cannot be proven by Koch's postulates. The American neurologist Stanley Prusiner (1942–) coined the term prion in 1981 to identify the cause of these brain diseases. Prusiner's work, for which he won the Nobel Prize in 1997, showed that unlike bacteria or viruses, prions lack either DNA or RNA. Prions are composed of pure proteins called PrPSc; these have been isolated from tissues infected with all known prion diseases.
PrPSc appears to be an abnormal form of a normal brain protein called PrP, in which a change to its genes (and amino acid sequence) causes it to fold incorrectly. Genes code for chemicals called amino acids. There are twenty different types of amino acids with different chemical behaviors. (For instance, some amino acids like to interact with water, others do not, and others have more capability for hydrogen bonding.) These differing properties mean that longer chains of amino acids interact with themselves and fold into the three-dimensional structures of proteins. The protein's structure determines its function and chemical behavior. In other words, different amino acid sequences produce proteins with different conformations and functions. As genes determine amino acid sequences, mutations or changes in the gene change the amino acid sequence and therefore, the way the protein works.
In prion diseases, the protein-only hypothesis states that the abnormal protein itself causes the disease. In an in vitro (test tube) model, abnormal PrPSc proteins can bind to normal PrP molecules and convert them to the abnormal form. The converted proteins then apparently interlink. In the brain, abnormal proteins are thought to form long chains that pierce brain tissue, producing its spongiform appearance and creating a lack of coordination and dementia, eventually killing the host.
The hypothesis concerning prions' mode of disease transmission is controversial, because it completely bypasses the more usual bacterial or viral method of infection and seems to violate Koch's postulates. In addition, there is a long incubation period after an animal or human host is infected with abnormal prions before there are any outward signs of disease, and there is no immune response. In addition, unlike any other form of life, prions lack genetic material, which many scientists believe is the only way an organism can reproduce and multiply.
More disturbingly, traditional means of treating these types of brain diseases are ineffective. If they are caused by proteins, the only known means to denature or destroy protein structures and render them innocuous is incineration. In recent experiments by Giovanna Mallucci at the Medical Research Council Prion Unit in London, mice that have been genetically engineered to stop producing normal prion proteins at around 10 weeks of age have not developed any signs of disease after being injected by abnormal prion proteins. While this raises the hope of future gene or pharmaceutical therapy to prevent prions from forming, as of 2007 there is no known cure or treatment for the 161 reported cases of vCJD in the United Kingdom. Epidemiologists are thus naturally keen to understand its means of infection and transmission.
Modern Cultural Connections
Current evidence indicates that BSE arose in British cattle in the mid-1980s, most likely caused by cattle eating feed that contained the remains of sheep infected with scrapie. BSE therefore, was a species jump from sheep to cattle that apparently spread as these infected cattle carcasses were themselves processed into animal feed for other cows. Transmission into humans in the form of vCJD can apparently occur by eating meat from cattle infected with BSE, particularly if that meat contains brain or spinal cord tissue infected with abnormal prion proteins. This was a further indication that prions could jump the species barrier.
Furthermore, as of December 2006, a total of 66 people in the United Kingdom are known to have received tainted blood transfusions from people infected with vJCD. Eight have died of the disease, three tested positive, and 24 others are at high risk of developing it. Acquiring vJCD through infected blood seems to produce symptoms much faster (six years) than eating infected beef, in which disease latency can last 12 to 50 years.
The British government responded to the initial vCJD threat with a huge cull of cattle in 1996, destroying 4.7 million older cows to prevent the disease from spreading further. There was a European-wide ban on all British beef products for three years from 1996–1999, costing the industry 2.4 billion U.S. dollars (£1.5 billion). Extensive media coverage about BSE and its spread into the human population also affected high-profile celebrities, such as American talk show host Oprah Winfrey (1954–) who devoted a segment of her popular talk show in 1995 to mad cow disease. Ms. Winfrey's comments that “cows should not be eating other cows” and that learning this had stopped her “cold from eating another burger” led to a defamation lawsuit against her by a group of Texas cattle producers.
Though Ms. Winfrey was found not guilty of making “false or disparaging statements,” the British and American beef industries did experience an economic downturn from which they have only slowly recovered. Tighter guidelines on cattle feed and regular testing of cattle have partially restored public confidence in the U.K. beef industry, although recent discovery of BSE in Canadian cattle has the potential to hinder North American trade.
The biotechnology industry has also responded to the vCJD threat. Hematech, a Midwestern American firm, has developed a cow with no prions in its brain tissue. As many cow products, such as cow blood serum, are used in laboratory cell cultures, and cow gelatin and collagen are utilized in cosmetics, Hematech offers a cow guaranteed to be without mad cow disease. Much in the same vein as Mallucci's genetically engineered mice, cows without prions have no PrP proteins that can be transformed to abnormal disease-causing molecules. Hematech's president and chief scientific officer claims these prion-free cows do not appear to be affected in their behavior or health. Although stricter cattle feeding and handling rules have seemingly confined the disease in the United States to levels of one case per million cattle (this is according to the U.S. Department of Agriculture), pharmaceuticals and laboratories have welcomed Hematech's prion-free animals and their products.
New Evidence about Prions
Though the agricultural, biotechnological, and research communities have responded to the prion threat, their role in disease transmission is by no means completely clear. Some scientists have challenged the prion and the protein-only hypothesis. Laura Manuelidis at Yale University has hypothesized that that tiny virus-like particles observed in infected brains are the true infectious agents for diseases like vCJD. Such particles have in the past been dismissed as microscopic artifacts in degenerating brain tissue. Looking at infected neural cells however, Manuelidis noted that these particles were in regular patterns, and that cells with more virus-like particles were better at infecting other cell cultures. She also found that when the viruses were stopped from replicating, they were unable to infect other cells. She has stated that the presence of prions seemed largely irrelevant to the spread of the disease. However, another leading prion researcher, Adriano Aguzzi of the University Hospital of Zurich in Switzerland, says that Manuelidis must isolate the disease agent and prove that her virus-like particles cause spongiform encephalopathies—i.e., follow Koch's postulates—before her work will be accepted.
Primary Source Connection
The potential for acquiring diseases through blood transfusion has been well-known for years, but more recently, with the discovery of prions, new diseases such as variant Creutzfeldt Jakob disease (vCJD) have created a need for new filtering systems, like those described in the article below by Roxanne Khamsi for New Scientist.
PRIONS THREATEN NOT ONLY THE FOOD SUPPLY, BUT THE BLOOD SUPPLY
A new filtering device can remove dangerous “mad cow disease” proteins from blood. Scientists say the device could prevent the spread of deadly prion diseases via blood transfusions.
At least three people have died in the UK after receiving blood contaminated with variant Creutzfeldt Jakob disease (vCJD)—the human equivalent of mad cow disease. There is currently no test available to screen blood for the misfolded “prion” proteins that cause the illness.
The new “P-Capt” filter designed by Robert Rohwer of the VA Medical Center in Baltimore, Maryland, US, and colleagues aims to make blood safer by removing these proteins. Their tests show the filter can prevent infections in hamsters and can remove the vCJD prions from human blood.
The biggest challenge in filtering the prion proteins from blood is that they are present at extremely low concentrations. To tackle this, the researchers screened roughly 8 million compounds to find one with an extremely high affinity for the hamster and human prions.
The compound they found, dubbed L–13, was then coated onto tiny plastic beads and placed in a small pouch to form the filter. When blood passes through the filter, the misfolded and normal prion proteins both attach to the beads.
To test how well the device works, scientists conducted animal tests using blood from hamsters with late-stage prion disease. As a control, the researchers injected the extract into the brains of 50 healthy hamsters, half of which subsequently died of prion disease.
Ripe Old Age
Next, the team removed all of the white blood cells from the extract, a common practice in countries such as the UK, which saw outbreaks of vCJD in the last decade. Studies suggest removing these cells halves the infectivity of blood. Out of the 99 hamsters that received injections of this extract, 15% died of prion disease.
Rohwer's team also passed some of the extract without white blood cells through the P-Capt filter. Not one of the 196 animals that received these blood injections became sick with prion disease. Instead, they lived well into hamster old age—about 540 days.
The team also showed that L—13 absorbs both the normal human prions and the misfolded prions associated with vCJD.
The work is the first time that an animal test has shown a filter to be effective against prion disease, according to Rowher. He says the technology will “definitely make it safer” for people to have blood transfusions.
“I would regard it as a big development,” says Marc Turner, clinical director of the Scottish National Blood Transfusion Service in Edinburgh, UK. He says that his team will be “very keen to evaluate” the new filter in the next year as there is “continuing concern that vCJD is being transmitted” through transfusions.
In September the P-Capt filter received European regulatory approval for use. Representatives from the Quebec-based company ProMetic Life Sciences, which along with the American Red Cross helped fund the device's development, expect the filter will be available by late 2007 in countries such as the UK and France.
khamsi, roxanne. “filter removes mad cow proteins from blood.” new scientist. (december 22, 2006). http://www.newscientist.com/channel/health/bse/dn10854-filter-removes-mad-cow-proteins-from-blood.html (accessed december 31, 2007).
See Also Biomedicine and Health: Antibiotics and Antiseptics; Biomedicine and Health: Bacteriology; Biomedicine and Health: Galen and Humoral Theory; Biomedicine and Health: Hormonal Regulation of the Body; Biomedicine and Health: Immunity and the Immune System; Biomedicine and Health: The Germ Theory of Disease; Biomedicine and Health: Virology; Physics: Microscopy.
“Blood Risk of vCJD Highlighted.” New Scientist 2582 (December 16, 2006): 7.
Grimes, D. Jay. “Koch's Postulates: Then and Now.” Microbe Magazine (May 2006).
Soto, Claudio, and Joaquin Castilla. “The Controversial Protein-Only Hypothesis of Prion Propagation.” Nature Medicine 10 (2004): S63–S67.
CNN. “Oprah Accused of Whipping up Anti-Beef ‘Lynch Mob’.” January 21, 1998. http://www.cnn.com/US/9801/21/oprah.beef/ (accessed June 13, 2007).
Khamsi, Roxanne. “Filter Removes Mad Cow Proteins from Blood.” New Scientist. (December 22, 2006). http://www.newscientist.com/channel/health/bse/dn10854-filter-removes-mad-cow-proteins-from-blood.html (accessed December 31, 2007).
New Scientist. “Protein Blocking Reverses ‘Mad Cow Disease’ in Mice.” January 31, 2007. http://www.newscientist.com/article.ns?id=dn11062 (accessed June 13, 2007).
New Scientist. “Virus in the Frame for Prion Diseases.” February 12, 2007. http://www.newscientist.com/article/dn11168-virus-in-the-frame-for-prion-diseases.html (accessed June 13, 2007).
Nova Online: The Brain Eater. “Do Prions Exist?” http://www.pbs.org/wgbh/nova/madcow/prions.html (accessed June 13, 2007).
Radio Iowa. “Company Develops BSE-Resistant Cow.” January 11, 2007. http://www.radioiowa.com/gestalt/go.cfm?objectid=1283953D-05E2-72D7-F12F035DB8F8F069 (accessed June 13, 2007).
Anna Marie Eleanor Roos