BSE and vCJD: Their biology and management
BSE and vCJD: Their biology and management

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BSE and vCJD: Their biology and management

2 The biology of prions

The increasing interest in kuru during the 1950s and 1960s had the effect of stimulating research into TSEs in humans and other animals.

Question 6

Summarise, in general terms, the possible causes of disease in animals.

Answer

A disease might have a genetic basis. Alternatively, it might be caused by a harmful agent of some kind entering the animal's body through its lungs, in its food or drink or by penetrating its skin. Such an agent might be chemical or biological.

A genetic mutation in the DNA of either all the cells in the animal's body (i.e. a congenital disease) or in some of them (e.g. in many cancers) may result in the production of protein that is either non-functional or does not function properly. Such a protein might be a key enzyme in a biochemical process or it might regulate the expression of other genes. Among the biological agents that cause diseases are viruses, bacteria, fungi, protoctists and small animals such as parasitic worms and insects.

Stanley Prusiner of the University of California at San Francisco started to research the biology of TSEs following the death of a CJD patient in 1972. Ten years later, he published a key scientific research paper in the prestigious peer-reviewed journal Science (see Box 1) [C].

Box 1: Referencing sources of information

The author(s) of a scientific research paper (or other scholarly work) conventionally acknowledge any non-original information or ideas they use - at least, any that are not so well established as to be included in standard textbooks - by briefly citing the source in their paper and then giving full details in a list of references at the end. Thus, the author(s) might write something like 'Prusiner (1982) claimed …' or 'It has been claimed (Prusiner, 1982) …' and then in the references section of their paper give the following details of Prusiner's paper:

Prusiner, S. B. (1982) Novel proteinaceous infectious particles cause scrapie, Science, 216, pp.136-144.

This gives (in order): the author's name (plus initials), the date of publication, the title of the paper, the name of the journal in which it was published (in italics), the volume number of the journal (in bold) and the paper's page numbers. This should be sufficient information to locate it.

The details of a book used as a source may be provided in the following format:

Ridley, R. M. and Baker, H. F. (1998) Fatal Protein: The story of CJD, BSE and other prion diseases, Oxford, Oxford University Press.

This gives (in order): the authors' names, the date of publication, the title of the book, the place of publication and the name of the publisher.

In fact, for referencing both scientific papers and books, several alternative formats are commonly used. The important thing is that the author(s) should select an appropriate format for (say) scientific papers and then use it consistently.

The journal Science in the USA (as well as Nature in the UK) is often described as a 'prestigious' journal. This suggests that other journals may be less prestigious. What accounts for this difference? Science and Nature are both read by large numbers of scientists, but more importantly these scientists are drawn from diverse scientific disciplines. This is not true of, for instance, The Veterinary Record, which has a much more specialist readership. Although influential in a more general sense, popular science magazines such as New Scientist do not publish research papers and do not involve peer review. It is the publication in Science and Nature of detailed research papers that may well be read by specialists in many other scientific disciplines - and the recognition and kudos that this can bring to their authors - that makes it so desirable to have one's work appear in these journals. [C]

In his 1982 paper, Prusiner proposed - extremely controversially and based on relatively limited experimental evidence - that both scrapie and CJD-like diseases were caused by an infectious agent consisting of a protein molecule but no genetic material. Previously, it had been believed that any infectious agent had to contain genetic information stored in nucleic acid (either DNA or RNA). Prusiner named this protein molecule 'prion', shorthand for 'proteinaceous infectious particle' (although, logically, he should have called it 'proin'!). [C]

Despite his paper having been peer-reviewed for publication in a prestigious journal - which should have established for it a very high level of credibility among both scientists and media professionals reporting science - the initial reaction to Prusiner's hypothesis among some fellow biologists has been described as ranging 'from scepticism to outrage' (although others welcomed its explanatory power). After the American scientific magazine Discover roundly criticised what it perceived as his promotion of the prion hypothesis in a 1986 article tellingly entitled 'The Name of the Game is Fame: But is it Science?', Prusiner resolved not to talk to journalists while he and his colleagues concentrated on their research into the biology of prions. In 1997 he was awarded the Nobel Prize in Physiology or Medicine for his discovery of 'Prions - a new biological principle of infection'. A few experts (including Gajdusek, the recipient of the first Nobel Prize awarded for work on TSEs) remained unconvinced by the 'protein-only hypothesis' of the cause of TSEs. Nevertheless, TSEs are now often called prion diseases and increasing numbers of scientists refer to themselves as prion researchers.

Activity 2

Timing: 0 hours 15 minutes

The previous three paragraphs contain several hints that science may not always be conducted in the disinterested, dispassionate way in which it has traditionally been portrayed. Re-read the paragraphs carefully, looking for evidence of controversy and how this might have affected the debate. Write two or three brief paragraphs summarising this evidence and then compare these with the commentary below.

Discussion

If the reactions of other scientists really did include 'outrage' at the ideas presented in a peer-reviewed scientific paper (rather than to, say, any courting of the media that Prusiner might have engaged in), then this suggests the paper challenged really deeply held ideas. The same could be said of the article in Discover magazine, even though its title suggests it was primarily concerned about the amount of publicity given to a concept supported by limited experimental evidence and to the concept's originator. The concept itself - that TSEs are caused by an infectious agent which contained no genetic material - was certainly controversial. Prusiner's self-imposed ban on talking to the media suggests that he might have been hurt by this criticism - but not so severely as to stop working in the area. Indeed, he was presumably convinced that he was right. We have to assume Gajdusek's unwillingness to accept Prusiner's 'protein-only' hypothesis is attributable to genuine scientific scepticism. Of course, the eventual award of a Nobel Prize for his work gave Prusiner tremendous prestige both within and beyond the scientific community. Among the many scientists who now accept Prusiner's explanation of TSEs, and readily use his newly coined word 'prion', are presumably some of those who were originally 'outraged' by his ideas.

Although it is now generally accepted that all TSEs are caused by prion proteins as proposed by Prusiner - and that this hypothesis explains the essential features of these diseases - this was certainly not the situation when BSE arose in the mid-1980s or earlier when researchers were trying to understand how diseases such as CJD, kuru and scrapie were transmitted.

A major problem facing TSE researchers was that there seemed to be a genetic basis to some TSEs (e.g. familial CJD, GSS and FFI), whilst a biological agent of some kind seemed to be responsible for others (e.g. kuru). To complicate matters further, in yet other TSEs there seemed to be both a genetic and an infectious component (e.g. although scrapie was widely believed to be spread through sheep grazing contaminated pasture, some breeds seem to be quite susceptible to the disease whilst others seem to be relatively immune). In addition, the relatively long incubation periods of some TSEs made it difficult to identify their initial cause(s) or to study them. This resulted in considerable confusion and contest between different research teams and certainly no consensus on the underlying biology of TSEs.

At an early stage in his work on TSEs, Prusiner deliberately infected mice with scrapie to use them as animal 'models' of the disease (see Box 2). He then showed that extracts from the brains of these mice:

  • caused scrapie in other mice when injected into their brains;

  • contained high concentrations of a particular protein with a particular three-dimensional shape, or conformation.

Furthermore, these brain extracts lost their infectiveness when they were exposed to treatments that destroyed proteins, e.g. protein-digesting enzymes (proteases) or short wavelength ultraviolet light, but not when they were exposed to treatments that destroyed nucleic acids, e.g. nucleic acid-digesting enzymes (nucleases) or longer wavelength ultraviolet light. At least some of this protein was the nucleic acid-free biological agent that Prusiner had called a 'proteinaceous infectious particle' or 'prion'. He went on to isolate a particular conformation of a protein that appeared to be unique to scrapie-infected brains. Because this protein was relatively resistant to protease enzymes, which readily degrade most proteins, he called it a protease-resistant protein or PrP. Prusiner surmised that PrP protein and prion were one and the same thing.

Box 2: The use of animal 'models' to study diseases

There are several reasons why there had been rather limited progress over the years in studying scrapie in sheep. Sheep are quite large animals that normally have to be kept in fields, where they are exposed to all sorts of uncontrolled aspects of the physical and biological environment which might have a bearing on whether or not they develop scrapie. They also have relatively long generation times and normally produce only one or two offspring at a time. Thus, working with sheep is both slow and comparatively expensive. It is also difficult to achieve adequate replication and sufficient control over potentially relevant variables in experiments with these animals.

Smaller animals (such as mice, rats or hamsters) breed faster and more prolifically than sheep. Large numbers can be kept conveniently and relatively cheaply in controlled conditions in laboratories. Furthermore, after many generations of inbreeding these laboratory animals are genetically uniform, thus eliminating a potential source of variability. Thus, many of the problems of working with sheep could be by-passed by artificially infecting small laboratory animals with scrapie so that they served as experimental 'models' for scrapie-infected sheep.

Of course, Prusiner was not especially interested in scrapie for its own sake. For him, scrapie was effectively a model of the human TSE (CJD) that he was studying. However, the sort of experiments he was doing could not possibly be carried out on either human patients or volunteers even if they were fully informed of any risks. Informed consent is a legal requirement in such circumstances in the UK and most other countries.

Some people would object as a matter of principle to artificially infecting any animal with a fatal disease, even if the purpose was to understand and eventually cure that disease or a similar one in humans. In this instance, it would be the means and not the purpose to which they objected. Others might question the appropriateness of mice, rats or hamsters as 'models' for either sheep or humans. However, the facts are that through experiments like these, Prusiner and others made enormous strides in developing our understanding of TSEs. These days, laboratory animals can be genetically engineered to produce particular proteins of other species (such as sheep, cattle or humans) in their brains instead of their own versions of these proteins. These genetically modified animals are assumed to be even more appropriate as 'models' of other species.

We now go on to discuss TSEs in terms of molecular biology. This course assumes you are already familiar with basic molecular biology from previous studies. In case you are not confident about the terminology, Box 3 provides a brief outline. In order to adequately understand the biology of prions, you may have to study or revise basic molecular biology more thoroughly.

Box 3: Revision of basic molecular biology

In eukaryotic organisms (whose cells have nuclei, in contrast to prokaryotes such as bacteria which don't), most of the genetic information is stored in chromosomes in the nucleus. Each chromosome consists of a DNA (deoxyribonucleic acid) molecule and various proteins. DNA molecules exist as two extremely long intertwined strands of subunits called nucleotides, each consisting of a molecule of the sugar deoxyribose, a phosphate group and a nitrogenous base. Since there are just four types of base (adenine, cytosine, guanine and thymine, or A, C, G and T for short), there are four types of nucleotide. Many (but not all) genes code for proteins, such as enzymes. Proteins consist of relatively long strands of about 20 different amino acid subunits. Each individual amino acid is coded for by three consecutive nucleotides (a triplet) in one of the strands (the coding strand) of the DNA molecule. The first stage in the production of a protein molecule (transcription) involves part of the coding strand of a DNA molecule (a gene) being copied as single-stranded mRNA (messenger ribonucleic acid) molecules (Figure 5). The mRNA molecules leave the nucleus and enter the cell's cytoplasm. There, organelles called ribosomes attach themselves to the mRNA molecules and effectively 'read' them. Ribosomes - together with transfer RNA molecules (tRNAs) and enzymes - add amino acids to the growing protein chains according to the sequence of triplets encountered in the mRNA. This process (translation) is completed when the ribosome 'reads' a particular RNA triplet that is always interpreted as 'stop'.

Figure 5
Figure 5 Transcription of DNA into mRNA and translation of mRNA into simple protein molecules

Scientists established the amino acid sequence in the PrP protein, worked out the DNA sequence that gives rise to PrP and searched for that DNA sequence among the genes of mice and, in due course, people. In fact, the PrP gene (see Box 4 on the names of genes) has been found in every species of mammal so far investigated. When the PrP gene is switched on in the nucleus of a cell, PrP protein is synthesised at ribosomes in the cell's cytoplasm. Although the PrP gene is present in every nucleated cell of the body, it is switched on mainly in brain cells. Brain cells therefore produce lots of PrP protein. This suggests that PrP protein must play an important - but, so far, poorly understood - role in the brain. On the other hand, mice in which the PrP gene is 'knocked out' (i.e. rendered non-functional by genetic engineering) before birth seem to be normal apart from having problems with their daily (circadian) rhythms of sleeping, eating, etc.

Box 4 The names of genes

Nice though it would be simply to use one of the 26 letters of the alphabet (A, B, C, … Z) to refer succinctly to each gene, this is not possible. Humans alone have between 20 000 and 25 000 genes distributed around our 46 chromosomes. Considering all living organisms, there are huge numbers of different genes. The PrP protein is coded for by the gene known as PrP (note that, conventionally, the names of genes are italicised). The names of various other genes (e.g. Hb and CPEB) are used later in this course. In addition to distinguishing between different genes, it is often necessary to distinguish between different alleles (or 'versions') of the same gene. Thus, two alleles of the human haemoglobin gene are referred to as HbA and HbS. [C]

As soon as proteins are synthesised, they fold spontaneously into complex 3-D shapes or conformations (Figure 5). The precise conformation into which a protein folds depends largely on the sequence of its amino acids. Moreover, a protein's behaviour within a cell is strongly influenced by its conformation. This is most clearly seen in enzymes, in which the molecules' conformation determines which reactants can be brought into contact with one another and therefore which product(s) can be produced - in other words, which reactions can be catalysed. This is the so-called 'lock-and-key' hypothesis of enzyme action which you may have met in previous studies.

Prusiner realised that without any change in their amino acid sequence PrP proteins exist in (at least) two conformations. He called the 'normal' conformation PrPC (for cellular PrP) and the abnormal conformation PrPSc (for scrapie-causing PrP). Compared to PrPSc, more of the PrPC molecule folds into helices (the α-helix structure) and less folds into pleated sheets (the β-sheet structure) (Figure 6). Crucially, whilst PrPC is soluble in cells, PrPSc molecules collect together into insoluble deposits. Cells containing such deposits no longer function normally and eventually die. The loss of these cells leads to holes in brain tissue, the 'spongy' effect typical of all these diseases (see Figure 4).

Figure 6
Figure 6 Structure of part of a 'normal' human PrP, i.e. PrPC, protein molecule. Note the preponderance of α-helices (shown in red). The two short sections of β-sheet are shown in pale blue. (White denotes unstructured regions.) The yellow areas within the α-helical regions are amino acid positions at which mutations associated with TSEs have been found

Figure 7a shows how PrP protein acts within a cell in normal circumstances. Within the nucleus, the PrP gene is transcribed into mRNA. The mRNA migrates out of the nucleus into the cytoplasm. At ribosomes, the mRNA is translated into a sequence of amino acids corresponding to the sequence of nucleotide triplets in the PrP gene. Even as it is synthesised, the growing amino acid chain folds spontaneously into the characteristic conformation of PrPC protein (i.e. largely comprising α-helices). The PrPC protein is then transported to the cell membrane, where it becomes attached to the cell's external surface.

Figure 7
Figure 7 (a) How PrP protein acts within a cell in normal circumstances. (b) Prusiner's explanation for how a cell with 'normal' PrP genes becomes infected with PrPSc protein.

Figure 7b summarises Prusiner's explanation for how a cell becomes infected with PrPSc protein. PrPC protein is synthesised in the cell as described above. However, in this case one or more PrPSc molecules have entered the cell from elsewhere and interact in some way with newly synthesised PrPC molecules in the cytoplasm. These interactions cause the PrPC molecules to become PrPSc molecules by changing their conformation (i.e. by increasing the proportion of β-sheet structure compared to α-helical regions in the molecules). Not only can these newly created PrPSc molecules then clump together and disrupt the cell's normal functioning, they themselves can also interact with PrPC molecules, causing the production of yet more PrPSc molecules. (Note that, unlike PrPC, PrPSc does not appear on outside of the cell membrane.) It can readily be appreciated that this is a 'chain reaction' in which more and more PrPSc molecules accumulate in infected cells. Furthermore, any of these PrPSc molecules released from an infected cell (e.g. upon its death) become available to infect other cells. As more and more cells - particularly brain cells - become infected with PrPSc, the animal develops symptoms of the TSE and eventually dies. Mice in which the PrP gene has been 'knocked out' experimentally - and which therefore do not synthesise PrP protein - do not develop TSE diseases.

Where might the PrPSc molecules that infect 'normal' individuals have come from? Clearly, in Prusiner's mice experiments they were injected into the recipient animal in the brain extracts from animals that already had scrapie. In the case of kuru, abnormal PrP molecules are presumed to have been present in the tissue - particularly the brain tissue - of people whose bodies were eaten. It is likely that kuru started from a sporadic case of CJD and became established as a relatively common disease within the Foré tribe through some of those who participated in these mortuary feasts becoming infected with kuru in this way and then themselves being eaten after death and so on. The fact that the disease has now almost disappeared some five decades after the cessation of cannibalism supports this explanation.

Activity 3

Timing: 0 hours 10 minutes

Go back over Sections 1 and 2, locate the places where you noted down the letter E, and compare your choices with those suggested below, where short explanations are provided. How do these explanations compare with your own notes?

From now until the end of Section 6, continue to note down the letter E when you identify material that you consider to be particularly relevant to ethical issues. However, this time write more detailed explanations of the way(s) in which the material is relevant to this theme. You will be asked to compare these explanations with our 'Comments' in Activity 4 at the end of Section 6.

Discussion

As noted in the text, although cattle displaying symptoms of BSE are usually killed immediately (see Section 1.2), this is not a policy applied to humans who contract fatal diseases.

Ethical issues may arise from medical use of biological materials (such as growth hormone or corneas) derived from the bodies of deceased people (presumably with their explicit consent) even if their undiagnosed CJD had not caused iatrogenic CJD in the patients (see Section 1.3)

Cannibalism is one of the great taboos in most cultures and societies. Routine cannibalism among the Foré until the mid-20th century (Section 1.3) would therefore certainly be regarded an ethical issue in a pejorative sense by many UK citizens. On the other hand, the Foré presumably engaged in this activity as a mark of great respect for their dead, and from their point of view not to engage in cannibalism would be an ethical issue. This illustrates how value systems from different cultures and societies influence how ethical issues are defined.

As discussed in Box 2 and elsewhere, the deliberate infection of animal 'models' with disease - and, indeed, the use of non-human animals in experiments generally - is an ethical issue because of the suffering and possible deaths of these animals. Whilst this is manifestly so for people who object on principle to all or most such experimentation, this should also be true for all responsible citizens and especially for those engaged in designing or carrying out experiments involving animals.

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