Transmissible Spongiform Encephalopathies

Prion diseases

Prion diseases are infectious diseases of the brain and occur both in animals and people. The disease is given a different name according to the species

Humans:

CJD (Creutzfeldt Jakob Disease)


GSS (Gerstmann Sträussler Syndrome)

Cattle:
BSE (Bovine Spongiforme Encephalopathy)
Sheep:
CWD (Chronic Waisting Disease)
The abbreviation TSE (Transmissible Spongiform Encephalopathy) is used as a general term.
Prion diseases are transmissible between species and bring about the slow degeneration of the central nervous system, which inevitably leads to death. A very long period of time elapses between infection and the appearance of the first clinical symptoms: typically 2-4 years in sheep, 3-6 years in cattle and more than 10 years with humans. As a general rule, once the symptoms have appeared, the disease leads to death within only a few months.
 
The Prion
The pathogen causing prion diseases, the so-called prion, is a newly discovered type which differs considerably from currently known pathogens like bacteria or viruses. Prions are extremely resistant to heat and chemicals. Even heating to 100°C can not inactivate prions adequately and many of the usual disinfectants have hardly any effect at all. Prions are also very difficult to decompose biologically – in soil they survive for many years.
Until today it has not been possible to completely resolve the composition of prions and the ways in which they propagate.

A disease-specific protein can be identified in the infected brain and other organs. This protein, the "scrapie prion protein", PrPSc in short (also called "BSE prion protein" PrPBSE in the case of BSE), is derived from the normal form of the prion protein, PrPC, occurring in the normal body.

The two proteins, the disease-specific PrPSc and the normal PrPC , differ through their spatial structures and the fact that PrPSc is resistant to destruction through digestive enzymes (proteins which digest food in the human stomach), whereas PrPC is completely destroyed when treated with digestive enzymes.

As a result of many studies it has been possible for scientists to come to the conclusion that PrPSc is a component of the pathogen of the prion diseases. It is even speculated that PrPSc represents the complete pathogen. According to this theory, an infection brought about by the penetrating PrPSc causes PrPC to be converted into PrPSc. The newly formed PrPSc can now, for its part, bring about the conversion of more PrPC into PrPSc. This leads to the disastrous chain reaction where the PrPSc production increases exponentially and the brain is damaged irrevocably. Notably, the increase in PrPSc during the course of the disease correlates with the increase in infectivity.

BSE

BSE (Bovine Spongyform Encaphalopathy) was first described in the UK in 1985. In 1996, novel scientific findings indicated that BSE is transmissible to man and causes a variant of CJD.

After the infection of cattle with BSE, 3-6 years pass on average before clinical symptoms (such as the decline in milk performance, shaking and timidness) develop. This incubation time can be widely variable and spans from 20 months (youngest) to more than 15 years. As a result of this incubation time, products from infected but not visibly ill animals end up in the human food chain.

In a first phase after infection, the infectivity of an affected animal is at a very low level. Officials therefore assume that BSE-infected animals do not pose a health-risk to humans in this phase. Nevertheless, it should be stated here, that the possibility of potential risks can not be completely ruled out.

In a second phase, before the occurrence of clinical symptoms, the infectious agent is found to be highly concentrated especially in the brain and in the spinal cord. It is this phase which represents the main risk factor for public health: an animal in this phase of infection poses the same risk to the consumer like a visibly ill animal, but is not detected due to the lack of signs of disease. The duration of the second phase is thought to be at least 6 months.

In a third phase clinical symptoms occur followed by death. The age of animals in the third phase of the disease ranges from 20 months to 16 years. The average and most frequent age is between 3 to 5 years

Creutzfeldt-Jakob Disease

1. Classical Creutzfeldt-Jakob Disease (``CJD'')

CJD is a rare and fatal neurodegenerative condition that occurs in humans worldwide. Like other TSEs,CJD can be transmitted (in a laboratory) to other species.Three forms of classical CJD have been recognised, none of which have been shown to be caused by animal TSEs:

Sporadic CJD, the most common subtype (approximately 85 per cent. of cases), usually affects individuals between the age of 50 and 75, and is characterised by a rapidly progressive dementia.The cause of sporadic CJD remains unknown. Epidemiological studies indicate a worldwide occurrence of sporadic CJD with an incidence of approximately 1 case per million per year
Hereditary CJD (including Fatal Familial Insomnia and Familial CJD) (approximately 10-15 percent. of cases) is an inherited disease associated with mutations in the PrP gene.


Iatrogenic CJD (less than 5 per cent. of cases) results from transmission of the causative agent via medication (e.g. growth hormone treatment) or surgery using contaminated instruments ormaterials. This is due to the unusual resistance of Pathologic PrP to the currently used methods ofsterilisation.

2. Variant Creutzfeldt-Jakob Disease (`"vCJD'')
Variant CJD was first identified in 1996. It is generally thought to have been caused by theconsumption of BSE infected products. Symptoms of the disease include depression, involuntary musclecontractions and impaired muscular co-ordination. In contrast to typical cases of classical CJD, vCJD seems to affect predominantly young patients. By October 1999, XX cases of vCJD have been reported in the UK and one case in France. The first ten cases of nvCJD were observed in 1996, approximately ten years after the outbreak of the BSE epidemic in the UK.

3. Other human prion diseases
 

Gerstmann-Sträussler-Scheinker Disease ("GSS'')
GSS is a rare hereditary disease characterised by impaired muscular co-ordination and dementia, whichis associated with a mutation in the gene encoding for the human prion protein. Death follows the firstsymptoms within 2 to 6 years. Approximately 50 families with GSS mutations have been identified to date.

Kuru
Kuru is a prion disease in humans which was shown to be transmitted through certain cannibalisticrituals amongst the Fore-people in Papua New Guinea involving the handling and eating of the brains of the deceased. Following an incubation time of up to 30 years, the disease progresses very rapidly. Death occurswithin 3-12 months after observation of the first symptoms. Following the abolition of cannibalism in PapuaNew Guinea, Kuru has virtually disappeared

Scrapie

Scrapie is a prion disease affecting sheep and goats. It is thought to be widespread in Europe, the US and the Middle East, but it is today notably absent in Australia and New Zealand.

The earliest records on scrapie date back to the early 18th century. The name scrapie comes from the tendency of the diseased sheep to scrape off their wool. Other clinical symptoms include difficulties in walking and loss of coordination, which is reflected in the names 'Traberkrankheit' (trotting disease) and 'la tremblante' (the tremble), names that were originally used in Germany and in France, respectively.

CWD

Chronic Waisting Disease (CWD) is a prion disease which occurs in elks and deer in North America. It was first observed in the late 1960. In some regions in the USA, up to 5% of hunter harvested animals are affected with the disease. The most common symptoms of CWD are emaciation, excessive salivation and behavioural changes

Health Risks

The primary health risk for human beings is through food products derived from BSE-infected cattle. Our current knowledge suggests that the transmission risk to human beings increases with the increasing infectiousness of cattle. Therefore the aim of any preventative intervention is to remove, as far as possible, all the animals with high levels of infectivity from the food chain.

The authorities of the countries concerned have taken various measures to reduce the risk of transmission. BSE animals with recognisable symptoms are withdrawn from circulation in all countries. In Great Britain at the present point in time the so-called specified bovine offal (SBO; i.e. brain, eyes, small intestine, spleen, spinal column and tubular bones) is banned from the food chain. Moreover, all cattle over 30 months old are not used for consumption. In Switzerland the SBO from all cattle must be destroyed. At present the authorities view these measures as adequate to prevent the transmission of BSE. However, now that a specific test has become available it is possible to remove from the food chain not only visibly infected animals but also symptom-free animals with similarly serious levels of infectivity. The largest German Federal State, North Rhine Westphalia has decided to use the Prionics-Check on a broad basis to document and monitor its freedom from BSE.

A further transmission risk is a possible infection through human blood and plasma products, which originated from donators infected with prions. After the appearance of a new form of Creutzfeldt-Jakob Disease (nvCJD, believed to be caused by BSE), the British Ministry of Health announced that no blood from English donors is to be used for pharmaceutical products

Diagnostics

Diagnostic methods for prion diseases encompass clinical examination (analysis of visible disease symptoms),  identification of the infectious agent, histogical examinations (examination of brain slices for disease-specific signs of brain damages), and, in the case of classical CJD, Electroencephalography (EEG).

The diagnostic tools used for BSE are outlined below:
 

Clinical symptoms
Early clinical symptoms are often unspecific and include, in the case of the cow, reduced milk production. Later clinical symptoms include timidness and mobility disturbances. Before the introduction of the diagnostic BSE test Prionics-Check, only BSE-cases with clinical symptoms were detected. Today, the majority of Swiss BSE cases are detected with the Prionics-Check test, which is used as a surveillance tool to test approx. 15'000 animals annually.

Detection of the BSE-specific prion proteins

Prionics-Checkcan be used to identify the prion proteins specific to the disease within only a few hours. As a result, a reliable method which can be routinely carried out on slaughtered animals is now available

Examination of cross-sections of the brain: The Reference Methods in BSE-Diagnostics

Various regions of the diseased brain reveal small holes, the number of which increases as the disease progresses and which make the brain spongy in appearance, characteristic for prion diseases. Apart from the spongiform changes, there are deposits of the disease-specific prion proteins which, when suitably stained, are visible under a microscope (so-called Immunohistochemistry). Cross-sections of the brain are usually examined for a conclusive BSE diagnosis in animals which have been emergency-slaughtered after the occurrence of clinical symptoms.Immunohistochemistry is a precise and sensitive method; however, it takes days to weeks to complete and can therefore not be used as a rapid BSE-surveillance tool.

 
Evidence of infectivity
The most accurate method of prion disease diagnosis is the detection of infectivity. In this case the infectious material is injected directly into the brain of laboratory animals. After a long incubation period (for BSE approx. 8-12 month) the animals become ill and die. An examination of the brain shows the characteristic spongiform changes and reveals large quantities of the disease-specific prion protein. However, such transmission experiments are limited to research experiments and are not suitable for routine diagnostics.
Prionics-Check
Prionics-Checkis an immunological test for the detection of prions in brain- and spinal cord tissue. The test is mainly used for BSE and scrapie diagnostics. Prionics-Check is carried out as a service from the Prionics diagnostic laboratory and is also available as a kit for partner laboratories. The kit contains the essential reagents for the test.
Prionics-Check is based on the so-called Western Blot technique and uses a novel antibody developed by Prionics. The brain tissue of slaughtered animals is removed, liquefied and examined to determine the presence of the disease-specific prion protein, PrPSc. The results of the tests are available approx. 6-7 hours after the specimen arrives in the laboratory.
Using the Prionics-Check it is possible to recognize PrPSc accumulations in the brain of the animal before it shows recognizable symptoms of Bovine Spongiform Encephalopathy (BSE or Mad Cow Disease). Its reliability is the same as previous reference methods (immunohistochemistry) but results are achieved in a considerably shorter time and with less expense. According to current knowledge. PrPSc accumulation in the brain of cattle begins at least 6 months before the occurrence of the typical BSE symptoms. As a result, the test makes earlier recognition of BSE possible and, therefore, makes a significant contribution to reducing the risk of transmitting BSE pathogens in animals through the food chain to humans.

The primarily areas of application for the test:


Prion Reviev

1. Transmissible Spongiform Encephalopathies

Transmissible spongiform encephalopathies (TSEs) encompass a group of fatal neurodegenerative diseases in animals and man which can be transmitted experimentally. The etiology of naturally occurring TSEs seems to comprise horizontal and vertical transmission as well as genetic predisposition, yet for the majority of cases the etiology is unclear. The onset of clinical illness is preceded by a prolonged incubation period of months to decades. Clinical symptoms of TSEs include dementia and loss of movement coordination. Neuropathological examination typically reveals gliosis and spongiform changes, sometimes accompanied by the formation of amyloid deposits (amyloid plaques). In the 1980s it was established that a common hallmark of TSEs was the accumulation of an abnormal isoform of the host-encoded prion protein in the brains of affected animals and humans.

The earliest records on TSEs date back to observations made in the early 18th century on scrapie in sheep (reviewed by M'Gowan, 1914). The name scrapie comes from the tendency of the diseased sheep to scrape off their wool. Other clinical symptoms include difficulties in walking and loss of coordination, which is reflected in the names 'Traberkrankheit' (trotting disease) and 'la tremblante' (the tremble), names that were originally used in Germany and in France, respectively. In 1936, Cuillé and Chelle demonstrated transmissibility of scrapie by intraocular injection of healthy sheep with spinal cord from sheep affected with scrapie (Cuillé and Chelle, 1936). The incubation periods observed in these experiments were unusually long - 14 to 22 months. Experimental transmissions of scrapie to goats, mice, rats and hamster (Cuillé and Chelle, 1939; Chandler, 1961; Chandler, 1962; Chandler, 1963; Chandler and Fisher, 1963; Zlotnik, 1963) demonstrated the transmissibility of the disease to other species and confirmed the long incubation periods as a distinct feature of scrapie. In 1959, Hadlow pointed out the remarking similarities between scrapie and a then newly described human disorder (Hadlow, 1959). This disease had become endemic among the Fore natives in the Eastern Highlands of New Guinea, and was termed kuru, meaning 'tremor' in the Fore language (Gajdusek and Zigas, 1957; Zigas and Gajdusek, 1957; Gajdusek and Zigas, 1959). Hadlow speculated that scrapie and kuru might be related and therefore transmissible, and suggested transmission experiments of kuru to animals in order to evaluate the claim. In the following years, Gajdusek and his colleagues succeeded in transmitting and passaging kuru to chimpanzees (Gajdusek et al., 1966; Gajdusek et al., 1967). A second human disorder, Creutzfeldt-Jacob disease (CJD), with a neuropathology similar to that of kuru (Klatzo et al., 1959) was transmitted to animals shortly thereafter (Gibbs et al., 1968). Subsequently, a number of other spongiform encephalopathies were found to be transmissible and were classified as TSEs: Gerstmann-Sträussler-Scheinker syndrome (GSS) in humans (Tateishi et al., 1979; Masters et al., 1981a; Tateishi et al., 1988a; Tateishi et al., 1990), transmissible mink encephalopathy (TME) in farmed mink (Burger and Hartsough, 1965), and chronic wasting disease (CWD) in mule deer and elks (Williams and Young, 1980; Williams and Young, 1982). during the last decade, novel forms of TSEs have been discovered in domestic cattle (bovine spongiform encephalopathy, BSE; Wells et al., 1987; Bassett and Sheridan, 1989), domestic cat (Wyatt et al., 1990) and in several other species like nyala, gemsbok, eland, Arabian oryx and greater kudu (Jeffrey and Wells, 1988; Fleetwood and Furley, 1990; Kirkwood et al., 1990).

2. Discovery of Prion Proteins

Purified preparations of the infectious agent (also denominated 'prion'; Prusiner, 1982) were found to contain a 27-30 kDa protease-resistant protein, termed PrP 27-30 (from prion protein) which accumulated in the brain during the disease (Bolton et al., 1982; McKinley et al., 1983; Prusiner et al., 1984; Gabizon et al., 1988). Surprisingly, PrP 27-30 turned out to be the protease-resistant core of an abnormal isoform of a host protein (Oesch et al., 1985; Basler et al., 1986; Borchelt et al., 1990). This abnormal isoform was denominated PrPSc (the scrapie-specific isoform of PrP) to distinguish it from its normal, protease-sensitive cellular isoform, PrPC.

PrPSc and PrPC have an apparent molecular weight of 33-35 kDa on SDS-polyacrylamid gels and are glycosylated at two asparagine residues (Oesch et al., 1985; Bolton et al., 1985; Multhaup et al., 1985; Sklaviadis et al., 1986; Haraguchi et al., 1989). After proteinase K treatment, PrPSc is shortened to a 27-30 kDa fragment while PrPC is digested (Bolton et al., 1982; Oesch et al., 1985). Studies on the synthesis and localization of the two PrP isoforms in cultured cells have shown that PrPC is attached to the cell surface by a glycosyl phosphatidylinositol (GPI) anchor while PrPSc accumulates intracellularly within cytoplasmic vesicles (Stahl et al., 1987; Safar et al., 1990a; Taraboulos et al., 1990; Caughey et al., 1991; McKinley et al., 1991; Borchelt et al., 1992; Taraboulos et al., 1992b). The observed kinetics of PrPC and PrPSc synthesis in cultured cells suggests that PrPSc is generated from PrPC in a post-translational process (Caughey et al., 1989; Borchelt et al., 1990; Caughey and Raymond, 1991; Borchelt et al., 1992; Taraboulos et al., 1992b). The conversion of PrPC into PrPSc involves a reduction of alpha helix structures and an increase in beta sheet structures in the protein (Pan et al., 1993). So far, no chemical differences between the two isoforms have been observed (Turk et al., 1988; Pan et al., 1993; Stahl et al., 1993).

The lack of a PrP gene in infectious preparations suggests that PrPC and PrPSc are encoded by the same single-copy host gene (Oesch et al., 1985, Basler et al., 1986). Prion protein genes have been identified in a number of mammalian species including human, monkey, hamster, mouse, rat, mink and cattle (reviewed by Oesch et al., 1991; Goldmann, 1993; Schätzl et al., 1995), as well as in chicken (Harris et al., 1991; Gabriel et al., 1992). The human PrP gene, PRNP, and the mouse gene, Prn-p, are located on chromosomes 20 and 2 of the respective genome (Liao et al., 1986; Robakis et al., 1986; Sparkes et al., 1986). The entire open reading frame of all known PrP genes is encoded by a single exon and consists of 253 (human) to 257 (mink) codons (Basler et al., 1986; Westaway et al., 1987; Gabriel et al., 1992). The primary translation product is shortened to approximately 210 amino acids in the mature protein by the cleavage of the N-terminal signal sequence (Hope et al., 1986; Bolton et al., 1987; Safar et al., 1990b), and by the removal of a C-terminal peptide when the GPI anchor is added (Stahl et al., 1987).
 

3. Genetic Linkage of TSE Traits to the Prion Protein Gene

The demonstrated transmissibility of TSEs suggested a contagious spread of the disease; however, some forms of TSEs like GSS and a subset of CJD appeared to be associated with families, resembling a genetic disease. In 1989, Hsiao and coworkers reported the identification of a mutation in the PrP gene which was linked to the occurrence of GSS in an autosomal dominant pattern (Hsiao et al., 1989). Since then, several more mutations in the PrP gene were shown to be linked to the occurrence of GSS or familial CJD (reviewed by Kitamoto and Tatieishi, 1994). When the GSS-associated mutation described by Hsiao and co-workers in 1989 was introduced into transgenic mice, these mice subsequently developed a neurodegenerative disease with detectable, albeit low production of infectivity (Hsiao et al., 1990; Hsiao et al., 1994). Taken together, the above results suggest that certain mutations in the PrP gene are able to trigger a spongiform encephalopathy.

Subsequently, mice homozygous for an ablated PrP gene were shown to be resistant to scrapie (Büeler et al., 1993) establishing that an intact PrP gene is a prerequisite for infection and/or prion propagation.

Genetic linkage to the PrP gene was also observed for the so-called species barrier for infection. Normally, experimental transmission between different species requires high doses of infectivity and results in long incubation times (Pattison, 1965, 1966). Once transmission to a new species has been established, however, subsequent passaging within this species can be done with lower doses and results in shorter incubation times. The infectious agent is therefore specific for the host species it is produced in. When transgenic mice expressing hamster PrP in addition to their own mouse PrP were inoculated with infectious material from hamster, they exhibited much shorter incubation times than wild type mice and produced hamster-specific infectivity (Scott et al., 1989; Prusiner et al., 1990). Subsequent studies established that the species specificity resided within the coding sequence of the PrP gene (Scott et al., 1993).

Differences in scrapie incubation times between different mouse strains have been shown to be linked to the Sinc/Prn-i gene, whose genetic localization has been mapped closely to the Prn-p gene (Dickinson and Meikle, 1971; Carlson et al., 1986, 1993; Bruce and Dickinson, 1987; Hunter et al., 1987). It has not been formally proven, however, that Sinc/Prn-i is identical to Prn-p.

The genetic traits mapped to the PrP gene locus may also be linked to other, unknown genes at the same locus. Goldgaber (1991) and Hewinson and co-workers (1991) provided findings implicating the existence of an 'anti-PrP' gene on the DNA strand opposite to the PrP coding sequence. This 'anti-PrP' was suggested to be responsible for properties formerly ascribed to PrP. A subsequent study, presented in this thesis, provided evidence against the conjectured 'anti-PrP' expression (Moser et al., 1993) refuting the hypothesis that genetic traits mapped to the Prn-p locus may represent inherent properties of an 'anti-PrP' gene.
 

4. Hypotheses about the Nature of the Infectious Agent

The transmissibility of spongiform encephalopathies implies that the various forms of human and animal TSEs are caused by a common infectious agent, yet the nature of this agent is still controversial. Attempts to determine the size of the infectious particle based on the target size for ionizing radiation have led to conflicting conclusions. While some authors calculated target sizes which were clearly smaller than that of any known viruses or viroids (Alper et al., 1966; Bellinger-Kawahara et al., 1988) others claimed that the infectious particle might have the size of a small virus (Rohwer, 1984, 1991). Conflicting results were also reached with regard to the chemical composition of the infectious agent. On one hand, the agent existed in a variety of distinct mutable strains (reviewed by Bruce, 1993) which seemed to imply the existence of genetic information in a nucleic acid of the infectious particle; on the other hand, infectivity was resistant to treatments destroying or modifying nucleic acids, such as nucleases, hydroxylamine and psoralens, while it was sensitive to treatments denaturing or modifying proteins (summarized by Prusiner, 1982, 1987a). It was therefore suggested that TSEs were caused by an unconventional virus or virino which could resist treatments modifying nucleic acids (reviewed by Dickinson and Outram, 1988; Rohwer, 1991; Pocciari, 1994), or by a novel pathogen lacking nucleic acids (Alper et al., 1967; Griffith, 1967; Pattison and Jones, 1967b).

In the virus hypothesis, the genetic information defining the properties of the various scrapie strains is encoded by a nucleic acid. The important role of PrP in disease might be explained in that PrP plays an essential role in the infection and spreading of the virus in the host i. e. PrPC could be a host cell receptor for the virus thereby controlling susceptibility to the disease (Rohwer, 1991). Mutations in PrPC would alter susceptibility to the disease. The species barrier for infection could be caused by a reduced affinity of a given virus to the PrPC molecule of a different host species. Subsequent passage in the new host species would be more efficient because the infectious agent would contain viruses which would have adapted to the new host by mutation. Conversion of PrPC into PrPSc would be brought about by the interaction of the virus with PrPC. This conversion my or may not be essential for the propagation of the infectious particle or for causing pathology. The virus hypothesis readily explains the existence of distinct scrapie strains; however, it is challenged by the claim that in preparations of highly enriched infectivity, the average size of a nucleic acid per infectious unit is not larger than 80 nucleotides (Kellings et al., 1992). This claim is supported by experiments evaluating the target size for ionizing radiation (Alper et al., 1966; Bellinger-Kawahara et al., 1988; but see Rohwer, 1991, and Pocchiari, 1994, for alternative interpretations).

As a modification of the virus hypothesis, the virino hypothesis postulates that the infectious particle consists of a small nucleic acid coated by a host protein (Dickinson and Outram, 1988). Variations in the nucleic acid sequence could account for the existence of distinct strains, as for example, in viroids (Schnölzer et al., 1985). The size of the nucleic acid could be very small since it does not have to encode for any proteins. The unusual resistance to treatments destroying or modifying nucleic acids could result from a protective effect of the coat. The most likely candidate for the host protein forming the coat of the virino is PrPSc since an association of infectivity with PrPSc (or with a very similar molecule) is suggested by the copurification of PrPSc with infectivity (Bolton et al., 1982; McKinley et al., 1983; Prusiner et al., 1984) and, conversely, by the copurification of infectivity with PrPSc on an immunoaffinity column (Gabizon et al., 1988). Standard preparations of purified infectivity usually contain approximately 105 PrPSc molecules per infectious unit (Bolton et al., 1991); however, this ratio has been reported to be subject to considerable variations depending on the experimental settings used for its determination (Manuelidis et al., 1987b; Aiken et al. 1989; Sakaguchi et al., 1993; Xi et al., 1992). Within the framework of the virino theory, these variations can be explained by the assumption that the ratio of correctly assembled virinos to the total amount of PrPSc need not be constant in different experimental settings. Alternatively, infectivity might be associated to a subpopulation of PrPSc molecules, denominated PrP* (Weissmann, 1991a), which may have escaped detection because it is extremely rare or difficult to distinguish from the rest of PrPSc.

In the ‘protein only’ hypothesis (Alper et al., 1967; Griffith, 1967; Pattison and Jones, 1967b; Prusiner, 1982), the information for strain specificity is carried by an infectious protein. To explain the mechanism of propagation of such an unusual infectious agent, it was proposed that the agent consisted of a modified host protein which was able to convert the host protein into a likeness of itself (Griffith, 1967; Prusiner, 1982). In order to account for the strain specificities, the modified host protein would have to exist in various different isoforms. The most likely candidate for an infectious protein causing TSEs is PrPSc (or PrP*) because it represents a disease-associated posttranslational modification of a host-encoded protein (Bolton et al., 1982; Oesch et al., 1985; Basler et al., 1986; Borchelt et al., 1990) and because of its alleged association with infectivity (see above).

Two ‘protein only’ models for the molecular mechanism of the conversion of PrPC into PrPSc have been proposed, the ‘refolding’ and the ‘nucleation-dependent polymerization’ models (originally outlined by Griffith, 1967; refined by Prusiner, 1982; Jarrett and Lansbury, 1993; and Weissmann, 1995). Both models suggest a direct interaction between PrPSc and PrPC. In the ‘refolding’ model, the rate-determining step is the conformational conversion of PrPC into PrPSc, a process which is catalyzed by the formation of a PrPC - PrPSc heterodimer. Upon infection, exogenous PrPSc would initiate conversion of PrPC into PrPSc, leading to a cascade of subsequent conversions induced by newly formed PrPSc. In the ‘nucleation’ model, the rate-determining step is the formation of a nucleus of polymerized PrPSc, which, once formed, promotes further polymerization of PrPSc. Such nuclei would act as polymerization seeds in an infected host thereby rapidly driving host PrP into the aggregated state.

The association of certain mutations in the PrP gene with the occurrence of familial TSEs can be explained on the basis of assumed mutation-induced alterations in the thermodynamic properties of PrP. In the ‘refolding’ model, mutated PrPC might be more likely than normal PrPC to convert spontaneously into PrPSc thereby initiating a cascade of PrPSc -catalyzed PrP conversions, or it might be more susceptible to conversion by low levels of ubiquitous PrPSc. Sporadic occurrence of TSEs, which accounts for the majority of human TSE cases, could be caused by somatic mutations in the PrP gene. In the ‘nucleation’ model, mutations in the prion protein might alter the PrPC / PrPSc equilibrium resulting in shortened nucleation times. In both models, the species barrier for transmission can be explained by the assumption that interactions of homologous PrP molecules are favored over interactions between PrP molecules which differ in their primary amino acid sequence. This hypothesis was supported by the finding that the species barrier for transmission of hamster prions to mice could be overcome by the expression of hamster PrP in mice (Prusiner et al., 1990). When transgenic mice expressing hamster PrP in addition to their own mouse PrP were inoculated with infectious material from hamster, they produced hamster specific prions but they produced mouse specific prions when inoculated with infectious material from mouse (Prusiner et al., 1990). Hence the inoculum dictated which prions were synthesized. This finding is most readily explained by an assumed direct species-specific interaction between PrPSc in the inoculum and the homologous PrPC in the host. Additional support for this hypothesis comes from the observation that in familial prion diseases, PrPSc molecules apparently originate from the mutant rather than from the wild type allele (Kitamoto et al., 1993a; Tagliavini et al., 1994). Furthermore, homozygosity at codon 129 seems to predispose to both sporadic and iatrogenic CJD in the human Caucasian population (Collinge et al., 1991; Palmer et al., 1991; Brown et al., 1994b; Deslys et al., 1994) and homozygosity for glutamine at codon 171 predisposes to natural scrapie in sheep (Westaway et al., 1994). These findings support the hypothesis that PrPSc interacts with homologous PrPC, although it should be noted that in the Japanese population no similar predisposition of codon 129 homozygosity could be observed (Kitamoto and Tateishi, 1994). Interestingly, PrPSc molecules containing a valine at codon 129 were found to differ from those containing a methionine both in the size of the protease-resistant core and in the relative abundance of differently glycosylated forms (Monari et al., 1994).

Within the framework of the ‘protein only’ hypothesis, the molecular basis for the existence of distinct scrapie strains could be explained by means of variations in the conformation of PrPSc (or PrP*). Interestingly, strain specificity in two strains of hamster adapted transmissible mink encephalopathy is associated with differences in the biochemical properties of PrPSc (Bessen and Marsh, 1994). PrPSc molecules associated with ‘hyper’ and ‘drowsy’ strains are cleaved at different amino-terminal sites by proteinase K resulting in PrP molecules which differ by 1 kDa in their molecular weight. This finding is in agreement with the hypothesis that strain-specificity may be mediated by stable variations in the conformation of PrPSc molecules rather than by a nucleic acid. Using in vitro conversion of radiolabeled PrPC to protease-resistant PrP by incubation with PrPSc preparations of either strain, Bessen et al. (1995) could confer the respective properties to the newly converted protease-resistant PrP. While these and other reports of PrP conversion in a cell free system (Kocisko et al., 1994, 1995) were interpreted in favor of the 'protein only' hypothesis, it cannot be excluded that unidentified components of the infectious preparations were required for the generation of protease-resistant PrP. Furthermore, it could not be determined whether the formation of protease-resistant PrP was accompanied by a respective increase of infectivity, since newly formed protease-resistant PrP in the reaction mix was much less abundant than the initially added PrPSc, so that a possible small increase in infectivity would have been undetectable.

The following alternative ways for reconciling the concept of an infectious prion protein with the existence of distinct scrapie strains have been presented. In the 'unified theory', Weissmann (1991b) proposes that PrPSc (or PrP*) may be associated with a host derived episomal nucleic acid which encoded for strain-specificity but was not essential for infection. This hypothesis unifies the views of the virino hypothesis which explains the existence of distinct, mutable strains of the infectious agent on the basis of nucleic acid encoded information with the view of the 'protein only' hypothesis which accounts for the resistance of the infectious agent to treatments which modify or destroy nucleic acids by postulating that PrPSc (or PrP*) in the infectious preparation is sufficient for infection. The 'targeting theory' (Hecker et al., 1992) is based on the observation of strain-specific differences in the histological accumulation pattern of PrPSc. It proposes that strain specificity is determined by the cell subtype in which PrPSc molecules are propagated. In this model, each of these cell subtypes confers upon the prion protein a specific posttranslational modification which targets the PrPSc molecules to the same subtype of cells in the following transmission. This hypothesis may seem less favorable after the recent report of species-specific PrP conversion in an cell-free system (Bessen et al., 1995, see above), although the possibility has to be considered that cell type-specific components conferring strain-specificity might have been brought into the cell-free system via the PrPSc preparations.

To date, none of the above models for the propagation of the scrapie agent has been experimentally confirmed or ruled out. The ‘protein only’ model has gained wide acceptance because a scrapie-specific nucleic acid has not been found despite massive efforts (Oesch et al., 1988; Aiken and Marsh, 1990; Meyer et al., 1991). The observation of strain-specific variations in the biochemical properties of PrPSc opens up the exciting possibility that the most daring prediction of the ‘protein only’ hypothesis namely the storage of inheritable information in the conformation of the prion protein might in fact be true.

5. Transmissible Spongiform Encephalopathies

Most pathological changes observed with transmissible spongiform encephalopathies are confined to the brain; however, scrapie-induced disorders of the pancreas have also been described (Carp et al., 1989; Ye et al., 1994). Neuropathology may result in a dramatic spongiform disruption of the tissue (Spielmeyer, 1922) but may also be subtle and non-characteristic, even at terminal stages of the disease (Collinge et al., 1990). In the latter cases, diagnosis has to rely on features like clinical signs, transmissibility, detection of PrPSc and identification of mutations in the PrP gene. Pathological changes are generally bilaterally symmetrical although asymmetrical pathology has also been documented both in experimental scrapie and in sporadic CJD (Bruce and Fraser, 1982; Yamanouchi et al., 1986). Sometimes, an increase in the number of macrophages and activated microglia and an association of microglia with amyloid plaques is observed in the infected brain (Wisniewski et al., 1981; McKinley et al., 1989; Barcikowska et al., 1993; Guiroy et al., 1994; Williams et al., 1994); however, there is a lack of a classical immunological and inflammatory response usually observed in encephalitis (McFarlin et al., 1971; Kasper et al 1982).

The earliest visible change in the course of the disease is pronounced astrogliosis. Astrocytes respond with hypertrophy of end-plates attached to blood vessels, followed by a general hypertrophy and upregulation of glial fibrillary acidic protein (GFAP) production (Eklund et al., 1963; Pattison and Jones, 1967a; Masters et al., 1984; Manuelidis et al., 1987a; Diedrich et al., 1991). Increased GFAP production is easily observed by immunohistochemistry at the light microscopic level, or by Western or Northern blot analysis, and has therefore provided a helpful criterion in the diagnosis of TSEs. The role of this upregulation in astrogliosis is not clear since it was shown with GFAP-deficient mice that GFAP is neither required for the development of gliosis nor for the production of prions (Gomi et al., 1995). A second pathological change mostly observed in TSEs is a spongiform degeneration which is caused by the formation of vacuoles in neuronal processes, astrocytes and possibly oligodendrocytes (Landis et al., 1981; Kim and Manuelidis, 1983; Roikhel et al., 1983; Jeffrey et al., 1991; Jeffrey et al., 1992b). These vacuolations may result in a severe spongiform disruption of the tissue, described as 'status spongiosus' (Spielmeyer, 1922). Other ultrastructural changes include a loss of dendritic spines and synaptic contacts, axonal degeneration (Landis et al., 1981) and nerve cell loss (Masters and Richardson, 1978). Some investigators find increased permeability of the blood brain barrier in brain and spinal cord of scrapie infected mice (Wisniewski et al., 1983; Lossinsky et al., 1987) while others describe the blood brain barrier as being essentially intact (Eikelenboom et al., 1987).

Pathological changes at late stages of the disease often include the appearance of amyloid plaques containing PrPSc (Bendheim et al., 1984; DeArmond et al., 1985; Kitamoto et al., 1986; Roberts et al., 1988; Tagliavini et al., 1991). Amyloid plaques stained with Congo red show a characteristic green-gold birefringence under polarized light. Their morphology ranges from a diffuse-type appearance in GSS to a small compact appearance in kuru (Roberts et al., 1988). Plaques are usually surrounded by (re)active astrocytes (Masters et al., 1981a; Masters et al., 1981b; Roberts et al., 1988). In a study on 300 cases of human TSEs, microscopically visible amyloid plaques were found in 5% of patients with Creutzfeldt-Jakob disease, 75% of those with kuru, and 100% of those with Gerstmann-Sträussler syndrome (Brown et al., 1994c). In CJD as well as in experimental scrapie, amyloid plaques occur mostly in cases with long duration of incubation time and illness (Masters et al., 1981a; Bruce et al., 1976). The formation of plaques therefore seems to be a slow process unrelated to general pathogenesis.
 

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Sporadic Creutzfeldt-Jakob Disease Presenting as Major Depression
Tianrong Tim Jiang, MD, PhD, Howard Moses, MD, Helen Gordon, MD, Eugene Obah, MD, From the Department of Medicine, Greater Baltimore Medical Center, Baltimore, Md.
[South Med J 92(8):807-808, 1999. © 1999 Southern Medical Association]

Introduction
Common causes of dementia include Alzheimer's disease and vascular dementia. Creutzfeldt-Jakob disease (CJD), a rare cause of dementia, is one of the four human prion diseases. Prion, a proteinaceous particle devoid of nucleic acid, is a newly recognized extraordinary infectious agent.[1] Although early presentation with psychiatric symptoms is one striking feature of the new variant CJD[2,3] (nv CJD) in the United Kingdom, such a presentation is unusual in sporadic CJD.[4]

Case Report
A 68-year-old white woman was initially seen by a psychiatrist for depressed mood and apathy 6 weeks before her admission to Greater Baltimore Medical Center (GBMC). Evaluation revealed apparent major depression, with depressed mood, anhedonia, weight loss, insomnia, and fatigue, without suicidal thoughts or delusion. Neurologic evaluations including magnetic resonance imaging (MRI) were unremarkable; grasp reflexes and paratonia were absent; and an electroencephalogram (EEG) was not obtained at that time. She was subsequently admitted to a psychiatric hospital (Sheppard Pratt Hospital, Baltimore) where she had electroconvulsive therapy. When psychiatric treatment failed, and confusion, disorientation, memory loss, myoclonic jerks, ataxia, and seizures developed, the patient was transferred to GBMC for evaluation. She had no history of exposure to human pituitary-derived hormones, neurosurgical procedures, or tissue grafting. Her medical history was unremarkable. There was no family history of dementia or other neurologic illness.
General physical examination was unremarkable. Neurologic examination revealed dementia, myoclonic jerks, bilateral spasticity, hyperreflexia, Babinski signs, and ataxia.

An EEG showed diffuse bilateral cerebral hemisphere epileptic foci. Computed tomography of the head with contrast and MRI both showed mild cerebral atrophy. The cerebrospinal fluid (CSF) revealed elevated protein of 47 and no pleocytosis. Cerebrospinal fluid cultures were negative, and CSF immunoassay was remarkable for the presence of the 14-3-3 marker protein.

Left frontal brain biopsy revealed many round empty vacuoles, consistent with so-called spongiform change (Figure). Immunohistochemical staining using anti-prion protein monoclonal antibodies revealed positive staining of the abnormal isoform of the prion protein (PrPSC).

The patient continued to decline rapidly, became mute, and lapsed into unresponsiveness with bilateral decorticate posturing; her myoclonus continued. She was discharged to a nursing home for comfort care.
 

Discussion
Creutzfeldt-Jakob disease presenting with solely psychiatric symptoms may lead to an erroneous initial diagnosis of psychosis,[5] depressive pseudodementia,[6] or hysteria.[7] In such an atypical presentation, recognition of CJD depends on the development of classic neurologic signs such as rapidly progressive dementia, ataxia, and myoclonus. The diagnosis of CJD should then be confirmed by brain biopsy.

In our patient's case, the brain biopsy not only showed spongiform changes, but also positive staining of the abnormal isoform of prion protein. Also supporting the diagnosis was the presence of the highly sensitive 14-3-3 marker protein in CSF. Although this patient had unusual initial psychiatric symptoms as seen in nv CJD, it did not have the other features of nv CJD such as the early and prominent sensor y deficit, young age at onset (mean, 27 years), and prolonged duration of illness (median, 14 months).

Creutzfeldt-Jakob disease is rare, with an estimated incidence of one case/million. However, recent data suggest that some cases of CJD may be clinically unrecognized. Bruton et al[8] reviewed all cases of dementia (more than 1,000) collected in the Runwell Hospital Brain Archive between 1964 and 1990. They found that only 60% of CJD cases with pathologically typical spongiform changes were identified clinically during life and suggested that human prion disease might be more common than previously supposed.

The spongiform encephalopathies[9] occur in both humans (CJD) and animals (bovine spongiform encephalopathy or mad cow disease). They are due to the aggregation of the abnormal isoform of the prion protein (PrPSC) forming amyloid deposits, which presumably are toxic to neurons.[1,9]

Sporadic CJD may present as a psychiatric syndrome leading to a wrong diagnosis if subsequent organic features are not pursued.

Acknowledgment

We thank Clarence J. Gibbs, MD, Chief of Laboratory of Central Nervous System Studies, National Institute of Neurological Diseases and Stroke, National Institutes of Health, for CSF 14-3-3 marker protein and brain immunohistochemical staining studies.


Prionics AG and PrioSense Join Forces in the Development of
the First Diagnostic Live Test for Mad Cow and Creutzfeldt-Jakob Disease

ZURICH, Switzerland and JERUSALEM, April 30 /PRNewswire/ -- Prionics AG, the world leader in prion diagnostics, and PrioSense, a newly founded spin-off company of Hadasit at the Hadassah University Medical Centre in Jerusalem, have started the joint development of a live test for prion diseases.  The test is based on the recent discovery of disease associated prion protein in the urine of both animals infected with BSE and humans infected with Creutzfeldt-Jakob Disease (CJD).

"Current diagnostic screening tests can only be used for post mortem diagnosis of the disease as they require brain tissue for analysis," explains Dr. Ruth Gabizon who is directing the research at PrioSense. "The new urine based test provides reliable early indicators of the prion disease."  It will for the first time facilitate screening of live animals and humans for prion diseases.

The joint research project of Prionics and the Israeli scientists is based on a previous co-operation in an EU project. In this project the research unit at Hadassah University led by Ruth Gabizon had succeeded in demonstrating for the first time the existence of disease associated prion protein in urine of humans and cattle.

"We are very excited about the new range of applications for prion testing, particularly in the field of human medicine", says Dr. Bruno Oesch, CEO and Head of Research at Prionics. "For years we thought that live tests might not be possible based on the direct detection of prion proteins in body fluids, but might require testing of less reliable third markers. The prion protein correlates 100% with disease and therefore facilitates the highest accuracy in diagnosis of BSE and CJD."

"We are delighted to formally partner with Prionics," said Dr. Raphael Hofstein, managing Director of Hadasit and Director at PrioSense. "It is an honour to work with a company that is internationally recognised for its competence in prion diagnostics as well as for its technological and marketing skills."

About Prionics

The Swiss company Prionics AG is the global leader in the early diagnosis of prion diseases. The company is renowned for revealing the extent of the mad cow disease spread in Europe with their BSE tests. The company has evolved into the worlds leading centre of competence for prion diagnostics. Research and Development projects in the fields of neurology and prion diseases are the business fields in which Prionics is active. Prionics' research team is part of a network of prion specialists and leading neuroscientists from all over the world.

About PrioSense

The seminal work of Dr. Ruth Gabizon at Hadassah University has led to the foundation of the spin-off company PrioSense. In 2001, the investment company Wolfson-Clore-Mayer provided Hadasit Ltd. (see below) with the initial investment to establish the company, with the express purpose of pursuing the research, development and marketing of innovative diagnostic and therapeutic products for the control and treatment of neurodegenerative diseases.

About Hadasit Ltd.

Hadasit Medical Research Services and Development Ltd. promotes and markets the intellectual property generated by the Hadassah Medical Organisation (HMO), a leading medical and research institution in Israel. Hadasit utilises the expertise, technologies and patents produced by Hadassah research teams in the fields of medical biotechnology, medical devices and medical diagnostics for commercial purposes. Hadasit has already established more than a dozen start-up companies, obtained patents on over 200 new concepts and is actively involved in preparing numerous research projects for presentation to the marketplace.


Challenging the Clinical Utility of the 14-3-3 Protein for the Diagnosis of Sporadic Creutzfeldt-Jakob Disease

Michael D. Geschwind, MD, PhD; Jennifer Martindale, BS; Deborah Miller, MD; Stephen J. DeArmond, MD, PhD; Jane Uyehara-Lock, MD; David Gaskin, MD; Joel H. Kramer, PhD; Nicholas M. Barbaro, MD; Bruce L. Miller, MD

Arch Neurol. 2003;60:813-816.

Background  Creutzfeldt-Jakob disease (CJD) is a rapidly progressive and fatal neurodegenerative disorder for which there is no noninvasive and disease-specific test for premortem diagnosis. Previous studies have suggested that, in the proper clinical context, the 14-3-3 protein in cerebrospinal fluid is a reliable marker for sporadic CJD.

Objective  To assess the sensitivity of the cerebrospinal fluid 14-3-3 protein test among patients with definite sporadic CJD.

Design and Setting  We reviewed cases of sporadic CJD referred to our institution that were ultimately proved by pathological examination and on which cerebrospinal fluid 14-3-3 testing had been performed.

Participants  Patients with CJD referred to our institution for clinical and/or pathological evaluation (biopsy- or autopsy-confirmed diagnosis) from January 1, 1998, through July 15, 2002, and on whom 14-3-3 testing had been performed. Thirty-two such patients with definite sporadic CJD were identified.

Main Outcome Measure  The 14-3-3 test results, from various laboratories, in these 32 patients.

Results  Seventeen of the 32 patients had a positive result for the 14-3-3 test, yielding a sensitivity of only 53%. A positive 14-3-3 result was significantly correlated with a shorter time between disease onset and the lumbar puncture for the 14-3-3 test.

Conclusions  Testing for the 14-3-3 protein is only modestly sensitive to sporadic CJD, and we caution against ruling out a diagnosis of the disease on the basis of a negative 14-3-3 result.
 

From the Departments of Neurology (Drs Geschwind, D. Miller, Kramer, and B. L. Miller and Ms Martindale), Pathology (Drs DeArmond, Uyehara-Lock, and Gaskin), and Neurosurgery (Dr Barbaro), University of California, San Francisco Medical Center, San Francisco.
 

Vol. 60 No. 6, June 2003
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