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An Interview with Professor Manuel Varela: Mad Cows and an Englishman?

Aug 12, 2018 by

At first glance, a “mad cow” might be envisaged unpretentiously as a cow who is angry. However, the popular phrase actually refers to the abnormal ...
Glow in cattle’s eyes may be a sign of mad cow disease

Michael F Shaughnessy –

1) Professor Varela, we have all heard the term “mad cow” but what exactly does it mean?

At first glance, a “mad cow” might be envisaged unpretentiously as a cow who is angry. However, the popular phrase actually refers to the abnormal behavior of cows as observed in England, where being mad may often be regarded as being out of one’s mind—crazy, if you will. In this case, the mad cow phenomenon is now generally recognized as an infectious or a genetic disease.

The incident started in 1984 at Pitsham Farm, located in South Downs, England. The affected British cattle were thought to have just simply gone mad. Shortly after the slowly progressive onset of the mad behavior occurred, the cows quickly succumbed to the ailment and died. When necropsied, the brain matter of the dead cattle showed a sponge-like lesion throughout the neural tissue.  The brains appeared to have been converted into sponges. The less colloquial but more modern scientific term for the mad cow phenomenon is referred to as the bovine spongiform encephalopathy (BSE). The disease, however, is not limited to just the cows.  In fact, a wide variety of animals can succumb to the affliction, too.  For example, goats and sheep can suffer from the so-called scrapie. Deer, mules, and elk may acquire the chronic wasting disease. Minks can get the transmissible mink encephalopathy.

Even humans may be susceptible, coming down perhaps with the Creutzfeldt-Jakob disease (CJD), the variant CJD (vCJD), the Gerstmann-Sträussler-Scheinker (GSS) syndrome, fatal familial insomnia, the sporadic fatal insomnia, or the kuru.  The CJD condition can be genetic in its onset. The disease may be infectious, as well.  Remarkably, the kuru disease was discovered in individuals who were members of the Fore tribe from New Guinea.  In the Fore people, the kuru might have made its appearance in victims who participated in ritualistic cannibalism during funeral ceremonies of their dead relatives. 

In general, the term spongiform encephalopathy has been used to collectively describe these maladies, independent of the species of the suffering victims. Others have referred to the constellation of the ailments as the prion diseases.  This latter term refers to the nature of the infectious agent, the so-called prion. The term prion arises out of the fact that the infectious agent is a protein.
2) What causes it and what are the symptoms?

There are two main known causes of the spongiform encephalopathy syndrome. The first is genetic in nature, and the second is infectious in its mode of acquisition. A third cause of the prion diseases remains poorly understood, manifesting itself only sporadically in its epidemiological occurrences, with no known connection genetically or in its infectious mode of transmission. In the genetic prion diseases, the affected prion protein is called PrP, and the prion-encoding gene is called PRNP or prnp.  Known modes of transmission of the infectious agent for the CJD in humans include injection or transplantation of infected tissue, exposure to contaminated medical implant devices, and consumption of food that is contaminated with the prion agent.

The prion-associated spongiform encephalopathies are characterized by a slow onset of neurodegeneration in affected individuals followed by a rapid progression to death. The course of the CJD ailments seems to follow about five distinct stages or phases. The first phase is called the incubation period, and it starts at the moment that the prion agent is acquired and can range from a year to as long as 30 years. The second stage is called the prodrome, lasting about 3 to 18 months. Symptoms of the prodrome stage include extreme fatigue in the patients and trouble in maintaining their focus and concentration. The next stage in the disease is called progressive neurological decline, lasting about a year and involving staggering, trembling in the hands, trouble maintaining balance, difficulty with memory, and extreme pain in the lower extremities. The fourth stage of the disease, called the late neurologic phase, includes loss of mental status and memory, plus inability to walk on their own. The last phase, the terminal stage, is death. 

3) Can there be inter-species transmission of this?

Indeed, yes. There are several documented occurrences of prion transmission from one species to another distinctive species.

One such early studied instance of this type of transmission involved the transfer of the prion infectious agent from sheep to cows in the U.K. during the mid-1980s. In this case, the prion agent that had been present in scrapie-diseased sheep carcasses had survived the rendering process. The resulting offal material had then been fed to healthy cattle.  The newly fed cattle eventually acquired the BSE mad cow disease and then promptly died.

Another instance of scientifically documented inter-species prion transmission involved cattle to humans. In the 1990s, about 140 human patients developed the vCJD version of the spongiform encephalopathy and succumbed to its lethal effects. These victims were all strongly suspected of having eaten prion-contaminated meat from prion-infected cattle. Subsequent epidemiological and molecular studies supported the association between the mad cows and the vCJD in the human patients. The cattle-to-human BSE transmission has been widely accepted; in fact, biomedical scientists consider BSE (cows) and vCJD (humans) to be the same disease.

4) Now, who was Stanley B. Prusiner? And what did he have to do with mad cow and prions?

Stanley Benjamin Prusiner is an American physician, biomedical scientist, and 1997 co-Nobel Laureate in physiology or medicine. He shared the prize with Dr. Carlton Gajdusek, who first demonstrated that the kuru disease was infectious in nature.  Prusiner’s Nobel work involved discovering the prion protein and showing that the infectious agent was primarily proteinaceous in its molecular constitution.

Prusiner was born in Des Moines, Iowa, in the U.S., on the 28th day of March in 1942 to parents Lawrence, his father, and Miriam, his mother. Prusiner’s middle name Benjamin originates with his father’s father, who was a Russian immigrant to the U.S. in 1896. Stanley spent his early childhood years first in Des Moines, then in Cincinnati, Ohio, where he had enrolled in Walnut Hills High School and where he credits his acquisition of Latin. After high school graduation, Prusiner attended the University of Pennsylvania, concentrating in the field of chemistry.  In 1964, Prusiner earned his A.B. undergraduate degree, with honors.

At that time, Prusiner entered medical school at the University of Pennsylvania and took his M.D. degree in 1968. It is during the years he spent in his medical studies that he acquired an interest in the biomedical sciences field of research. Dr. Prusiner then moved to San Francisco where he became a medical intern at the University of California (UCSF) until 1969, when he then moved to Bethesda, Maryland, for advanced studies in bacterial enzymes at the National Institutes of Health. In 1972, Dr. Prusiner became a medical resident specializing in the field of neurology at UCSF.

Dr. Prusiner then became a faculty member at UCSF in 1974. Then, in 1979, he moved to the University of California at Berkeley, where he became an assistant professor of virology.  In 1980 he became associate professor of neurology at UCSF.  Starting in 1984, Dr. Prusiner was promoted to full-professor of neurology at UCSF and virology at Berkeley. Then in 1988 he became professor of biochemistry at UCSF. As of this writing, Dr. Prusiner is the director in charge of the Institute for Neurodegenerative Diseases in San Francisco, CA.

Dr. Prusiner’s entry into his Nobel Prize-winning prion work started during his residency-training while at UCSF, in 1972. Two months into his new medical position as a resident, Dr. Prusiner encountered a female patient presenting with a deficiency in her memory recall and in her ability to perform several normal tasks, exhibiting what is now considered the classic neurodegeneration signs and symptoms of CJD. 

Not having admitted a CJD patient before, Dr. Prusiner studied the scientific literature at the medical library.  Dr. Prusiner then learned about a controversy regarding an association between the so-called slow viruses and the spongiform encephalopathies. Furthermore, he learned about a set of studies conducted by Drs. Tikvah Alper and John Stanley Griffith who had demonstrated that infected scrapie material survived harsh treatment with ultraviolet light, a method that would readily destroy any nucleic acid component of a slow virus, leaving behind only the protein part of such a virus. Since the infectious agent was able to mediate its effects without RNA or DNA, then it meant that the protein aspect of the agent was infectious.  Dr. Prusiner reasoned that the causative agent of the infectious scrapie, therefore, might be protein, devoid of RNA and DNA.

Whatever the case, whether a slow-virus or a protein, Dr. Prusiner first set out to purify the causative agent of the scrapie. The laboratory isolation of the infectious element, however, turned out to be a formidable task, taking almost a decade.  Some of the setback was due to lack of grant funding for the work. Thinking at first that the purified agent might actually be a virus, Dr. Prusiner was astonished to find out that they could not detect any sort of nucleic acid.  You see, viruses are composed of a nucleic acid genome and an outer covering composed of protein, sometimes with sugar and sometimes with a lipid membrane called an envelope. However, Dr. Prusiner’s agent contained no nucleic acid. Conducting a series of control experiments, Dr. Prusiner and his laboratory colleagues tried to destroy all vestiges of any nucleic acid, no matter how minute, while purifying the prion protein. They gathered data that showed the results of a pure protein in their preparations, while using several distinctive conventional biochemical means for protein detection and without detecting a trace of nucleic acid.

Prusiner published the work as a single author in the journal Science, on April 9, 1982. In this seminal paper, he presented a series experiments, all of which were meant to demonstrate how the infectious agent consisted of protein that was absent of RNA and DNA. Eventually, Dr. Prusiner’s laboratory was able to show that a candidate agent that they had purified and mixed with neural tissue from the brains of laboratory hamsters conferred the scrapie disease. It is in this paper that Prusiner coined the term “prion,” for proteinaceous infectious particle.

Almost immediately, the publication was met with widespread disbelief, especially amongst the virologists, who were sure that a slow virus was responsible for the scrapie and insisted that such a slow viral particle simply had yet to be found.  Prusiner’s work continued to be harshly criticized.  Prusiner himself called it a “firestorm” in his Nobel lecture. He spoke of scornful personal attacks and of his detractors being “very vicious.”

Meanwhile a series of supporting evidence had emerged not only from the Prusiner laboratory, but from other independent laboratories, as well. First, Dr. Leroy Hood, a molecular biologist and inventor of rapid sequencing methodology managed to determine the amino acid sequence of the prion protein.  Then, Prusiner’s research laboratory had been successful in cloning the DNA gene segment that encoded the prion protein, now called PrP. Importantly, the lab was able to produce antibody proteins, which could potently bind and detect the prion protein, further allowing a relatively pure preparation to be obtained for the causative agent of the scrapie.

Finally, an experiment was conducted that managed to tip the balance in favor of the prion hypothesis versus the slow virus hypothesis for causing the scrapie. Genetically engineered laboratory mice were produced in which the PRNP gene was “knocked-out” meaning that the gene was mutated beyond repair, being deleted from the mouse genome. These knock-out mice could not acquire the scrapie, even if injected with the prion agent. Then, in a coup de grâce, a mutation was found the gene that encoded the PrP agent, and it was shown to confer scrapie when introduced into laboratory animals. The scrapie-inducing version of the prion protein was now called PrPSC (SC for scrapie) while the corresponding non-pathogenic form of the prion protein was denoted as PrPC, for the normal cellular form.

5) What is the current state in terms of “mad cow” disease, and how do prions fit in?

Presently, the protein-infection means of causing the mad cow, scrapie, and other spongiform encephalopathies is firmly established and widely accepted, even amongst the great majority of the virologists. While the slow viruses may be involved in causing infection for a different set of diseases, the prions by themselves do indeed cause ailments such as the mad cow, scrapie, CJD, etc.

The progress of the prion infection starts with exposure to the PrPSC, the pathogenic scrapie-form of the prion protein.  This usually occurs by eating food that is contaminated with infected animal neural tissue, or perhaps PrPSC entry through the patient’s skin that is broken, or by direct contact of the PrPSC with the patient’s brain or spinal cord by some sort of trauma like a medical procedure. After eating the PrPSC-infected brain tissue, the prion proteins climb aboard mobile cells in the circulation and move to the patient’s central nervous system (CNS).

Next, the pathogenesis mechanism proceeds in a cyclic manner to manifest the prion disease.  A model for this pathogenic system has been proposed. It is known as the template-mediated protein refolding mechanism. The model is described as follows.

First in the cycle is the aggregation of the PrPSC molecules, forming an abnormal clump structure called an aggregate. Second, the aggregate then binds the neuronal cells. Third, the PrPSC molecules in the aggregate convert a normal PrPC that is present on the cell surface into another PrPSC molecule!  Fourth, as the cell continues to normally produce more of the PrPC molecules, they, too, are converted to the PrPSC form. Fifth, the newly formed PrPSC molecules form a growing chain. Sixth, PrPSC molecules at the end of the chain break off, which then go on to start the aggregation step, repeating the cycle.

The PrPC differs from the PrPSC in their structural conformations and in their degrees of susceptibility to acids, chemicals, UV light, proteases, heat, etc. Anyhow, the PrPSC molecules build up as they are produced, forming vacuoles and somehow resulting in the sponge character in the diseased brain tissue.

There is currently no treatment for any of the prion diseases. Furthermore, there is no vaccine available.

6 ) Should I avoid cows and farms– especially if I am overseas?

The prion disease outbreak that occurred in the U.K. resulted in new legislation banning the use of dead animal material as food for live farm animals. Furthermore, the U.K. more closely monitors cattle than had been conducted prior to the 1984 mad cow outbreak.  Meat products that may have been contaminated with nerve tissue during butchering should be promptly discarded. It is also likely a good idea to avoid the funeral practices involving the cannibalistic rituals of the Fore people, of course.

Furthermore, it is recommended that medical instruments be thoroughly sterilized by autoclaving for at least an hour, as opposed to the normal 15 – 20 minutes that is frequently conventionally followed.  Alternatively, medical instruments should be exposed to solutions of strong bleach or concentrated sodium hydroxide to achieve proper sterilization. Such decontamination procedures of medical instruments will help to prevent or reduce iatrogenic transmission (i.e., caused by a medical procedure or treatment) of infectious prion proteins to unsuspecting individuals.

7) What have I neglected to ask?

Although we have a clear idea of the insidiousness of the PrPSC scrapie-causing form of the prion protein, for a long time it was unclear what the purpose was of the normal PrPC molecule. Recent evidence proposes that the normal cellular prion protein molecule functions in living beings to help development of the neurons, suggesting that the PrPC molecule plays a role in fine-tuning the differentiation process of a neuron during CNS development. Alternatively, it has been suggested that the PrPC molecule is required for coaxing either an undifferentiated stem cell or a neuronal progenitor to become a mature neuronal cell.

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