Fatal familial insomnia

CONTINUING EDUCATION

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LEARNING OBJECTIVES

Upon completion of this article, the reader will be able to:

1. Define and discuss the relevance of prion biology in transfusion medicine.

2. Discuss laboratory testing for prion disease.

3. Discuss symptoms of various TSE clinical syndromes.

The word prion--suggested by Nobel Laureate Stanley Prusiner--is a contrived abbreviation for an infectious protein believed to be the causative agent of a family of progressive neurodegenerative diseases known as transmissible spongiform encephalopathies (TSE). The most notorious TSE is bovine spongiform encephalopathy (BSE), which is responsible for "mad cow" disease.

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Concern over BSE has heightened since it was identified that ingestion of beef can cause TSE in humans. The United Kingdom (U.K.) has experienced most of the reported cases of BSE, and citizens of the U.K. have contracted the majority of cattle-derived TSE cases referred to as variant Creutzfeldt-Jakob Disease (vCJD). At this time, references to BSE and vCJD are found regularly in the news. vCJD has captured considerable attention among politicians, government groups, and academic researchers alike with a disproportionately large rate of growth shown in publications in the medical literature (see Figure 1).

This review provides a basic understanding of the pathogenesis of prion diseases and highlights the magnitude of the current concern over BSE and vCJD with an emphasis upon transmissibility and the implications for transfusion and blood testing in both humans and animals.

Prion history

The first TSE was identified in sheep in 1759. Since afflicted animals would appear to scrape their sides along the fences of their pens, the condition was commonly referred to as scrapie. Although the experimental transmissibility was not reported until 1936, postmortem examination revealed that the brains of infected animals were sponge-like, resulting in the rapid acceptance of the term "transmissible spongiform encephalopathy" as a common and recurring characteristic of the disease in sheep and other animals.

In the 1920s, two separate reports appeared describing a progressive neurodegenerative disease in humans presenting with central nervous system dysfunctions reminiscent of those seen in scrapie. The first was reported by Hans Creutzfeldt (1920) and the second by Anton Jakob (1921). Thus, the constellation of symptoms they described became known as Creutzfeldt-Jakob disease (CJD).

In 1966, T. Alper and colleagues provided the first clue to demonstrate the unique pathogenesis associated with TSE. They showed that chemical manipulations resulting in the destruction of nucleic acids, known as the infective component of viruses, did not alter prion infectivity. It was Prusiner, however, who is credited with providing overwhelming evidence suggestive of the radical idea that a protein was the infective agent--an effort he published in 1982 and for which he was subsequently awarded a Nobel Prize.

In 1956 and 1957, another form of human TSE referred to as kuru reached epidemic proportions through the practice of cannibalism among the Fore people of Papua New Guinea. As the practice subsided, the incidence of kuru among these people decreased. The cultural practice reflected a devotion to their ancestors through the ingestion of brain matter of the deceased--predominantly by the women and children--and the consumption of their muscle by the men. Women often succumbed to kuru, but men did not. This further implicated neural tissue in the pathogenesis of TSE.

In the mid-1980s, the U.K. experienced an outbreak of TSE among cattle. The brains were affected in all afflicted cattle, and the symptoms included awkward movements and abnormal gait with exaggerated behaviors characterized by some as madness--hence, the term "mad cow disease" was coined for BSE.

In 1987, it was thought that scrapie was passed to cattle because they were fed sheep-derived meat and bone meal. In 1989, selected parts of slaughtered cattle (brain, spinal cord, intestine, thymus, tonsils, and spleen--collectively referred to as specified bovine offal, or SBO) were banned from becoming byproducts to be fed to cattle, thus preventing prions from entering the human food chain.

[FIGURE 1 OMITTED]

Recently, there has been some concern over the rising incidence of prion disease in deer and elk (called chronic wasting disease, or CWD). It is also appreciated that many animals are capable of acquiring TSEs, including cats (feline spongiform encephalopathy, or FSE), mink (transmissible mink encephalopathy, or TME), and a number of exotic zoo animals as well.

Prion biology

Since the advance of molecular biological techniques, we have gained considerable knowledge of basic prion biology beginning with the molecular structure. The molecular structure of the prion protein (PrP) is dictated by the prion gene, the human form of which is abbreviated as PRNP. PRNP encodes for a protein of 254 amino acids in length. PRNP undergoes post-translational modifications in two important ways--cleavage and glycosylation. First, the leading 22 amino acids and trailing 23 amino acids are cleaved to leave a protein with 210 amino acids. Since the average molecular weight of an amino acid is 118 daltons, multiplication by 210 produces an unglycosylated protein of about 25 kilodaltons (kd).

The protein undergoes glycosylation at three sites. The first site is a small C-terminal glycophospha-tidylinositol (GPI) moiety, which links the prion to cell membranes; this tells us that the prion protein has a cell-associated form. The other two sites are more extensive glycosylations at the asparagine amino acids in positions 181 and 197. Sulfide-containing amino acids at positions 178 and 213 form a disulfide bridge that constrains the motion of the molecule. In fact, the molecule is quite restricted in its movement for all except the first (N-terminus) 90 amino acids.

Molecular biology techniques have been extended to functional analyses of prion proteins. Mice have been raised in whom Prnp (the designation for the prion protein gene in mice) has been eliminated or knocked out of their genome. Such knock-out mice are apparently capable of surviving and reproducing normally, suggesting that prion protein does not appear to be important for these functions. Interestingly, pathogenic prion infection does not lead to disease in Prnp knockout mice. Therefore, pathogenic prion must recruit normal prion to convert to an isoform that is pathogenic. With advancing age, some Prnp knock-out mice present with symptoms of neurological abnormalities, suggesting that prions may play a role in normal nerve-impulse transmission and the production of an insulative coating around the nerve fiber that aids in electrical conduction within the length of the nerve cells. Some knock-out mice also show disturbances in sleep patterns (oddly enough, the major symptom of a human TSE called familial fatal insomnia (fFI) is sleep disturbance, specifically insomnia as the name indicates).

Prion diseases can develop spontaneously through a single nucleotide polymorphism (SNP) or replacement of one nucleotide for another in the sequence of nucleotides that comprise genetic information within DNA. Every three nucleotides encode for a particular amino acid and are referred to as a codon. If one nucleotide is replaced for another, the replaced nucleotide can result in a codon that translates into a different amino acid in the programmed sequence comprising the prion protein. Such SNPs can occur naturally; when they result in disease in humans, the disease is called sporadic CJD (sCJD).

Prion diseases are, of course, transmissible and have been shown to develop in response to biological products (human growth hormone, chorionic gonadotropin administration), through the use of grafts (cornea or dura mater), or via contaminated surgical instruments used in neurosurgery. Such prion diseases are commonly referred to as iatrogenic CJD (iCJD).

Finally, transmission can occur through ingestion within the gut, and recent data, discussed further below, show that it is possible for infected subjects to be free of symptoms for quite some time. Infected persons who become blood donors may be a source of infection in recipients of transfused blood products. Ingestion of beef from asymptomatic BSE-infected cattle has led to over 150 cases of vCJD, predominantly in the U.K. Among these, there are two suspected cases of transfusion-transmitted vCJD.

Normal prion protein is widely distributed throughout the body but has its highest concentrations in neural tissue. It is found within the central nervous system and in secondary lymph organs including lymph nodes, the spleen, and Peyer's patches in the gut. Prion protein has been found in the placenta and muscle. Another interesting observation is that noninfectious prion protein has been detected in the urine of TSE-affected animals and humans. We have already learned that through the presence of GPI linkages, prion protein has a cell-associated form. Now, with the observation that prion protein can be found in urine, which is largely free of cells, we know that prion can exist in a soluble form as well. The fact that prion can have both cell-associated and soluble forms may have implications for transmissibility, particularly as it relates to transfusion.

The Western blot assay

Before moving on to transmissibility, however, it is important to understand how prion is detected. Here, too, techniques applied within the discipline of molecular biology are important, with the Western blot being the most informative method of detection to date. The Western blot can help differentiate pathogenic from normal prion protein. The Western blot assay involves subjecting a liquid sample or tissue homogenate to the proteolytic action of the enzyme Proteinase K (PK). There are rare exceptions to the rule that PK degrades normal prion protein whereas it leaves the pathogenic form of prion largely unaffected and therefore resistant. The nonpathogenic form of prion is often referred to as [PrP.sup.C] because it is found normally in cells or [PrP.sup.sen] because it is sensitive to PK. In contrast, the pathogenic form of prion is more commonly referred to as [PrP.sup.res] because it is resistant to PK.

PK-digested sample, when placed onto a polyacrylamide gel and subjected to electrophoreses, results in the separation of the sample into three bands of proteins in most species. In the electroblotting step, the proteins are transferred with the aid of an electric current to nitrocellulose or another suitable membrane. The membrane containing the transferred protein is subjected to immersion into a solution of antibody directed against prion protein. After the membrane is washed, a secondary antibody conjugated to an enzyme is employed. Then the prion protein can be visualized by exposing the membrane to substrates for the linked enzyme, which in turn catalyzes a chemiluminescent reaction wherein the light that is given off can be captured on film. The developed images appear much like the one shown in Figure 2A.

[FIGURE 2 OMITTED]

The foregoing discussion left us with an understanding of the size of the prion protein estimated at 25 kd in the unglycosylated form, and the image shows it--along with two other bands--representing the mono- and diglycosylated forms of the prion protein. In contrast, [PrP.sup.C] (or [PrP.sup.sen]) will not appear on the Western blot because the PK-digested fragments would be so small as to cause them to migrate off the gel.

PK sensitivity or resistance reflects just one of a number of differences in the chemical properties of the normal versus the pathogenic form of the prion protein. [PrP.sup.C] is soluble in aqueous solution, but [PrP.sup.res] is not. Consequently, it has been difficult to study the three-dimensional structure of [PrP.sup.res] since many of the methodological approaches for such studies require the target compound to be present in solution. [PrP.sup.res] may have the same amino acid sequence as [PrP.sup.C], along with the same post-translational glycosylations and disulfide bond. [PrP.sup.sen], however, shows a three-dimensional structure, which contains an alpha-helical configuration along about 40% of its length, whereas only 3% is in what is known as a beta-sheet configuration. In contrast, [PrP.sup.res] appears to possess less (about 20%) alpha-helical conformation, and a major portion (over 50%) is in the beta-sheet form. Therefore, pathogenic prion would be expected to be more apparent in tissue than the soluble, innocuous counterpart, and it is not surprising that the brains of afflicted individuals and laboratory animals appear riddled with holes that look like fat globules.

The Western blot has revealed differences in the intensity and migration patterns of the three bands representing the un-, mono- and diglycosylated forms of prion. The relative intensity of the bands in the Western blot reflect different amounts of glycosylated prion and may be used to differentiate one subtype of prion disease from another. Moreover, the migration of the unglycosylated form may present in one of two positions on the gel, which co-migrate with a protein marker of either 19 kd or 21 kd--the larger and more slowly migrating band is referred to as type 1 and the faster 19 kd band as type 2. Some of the clinical syndromes can be described in terms of their homozygous or heterozygous property at codon 129 combined with a type 1 or 2 electrophoretic mobility pattern in the Western blot.

Clinical syndromes

Although symptoms characterizing different clinical syndromes vary, the brains of afflicted individuals show remarkable similarity upon gross inspection. Human prion diseases are referred to as TSEs because they may be passed from animal to animal of the same species; and the brain degenerates so visibly as to appear, upon gross inspection and histopathologic examination, like a sponge with holes. Damage is confined largely to the gray matter. This is often accompanied, as in vCJD, with abundant amyloid plaque formation. Amyloid plaque is a translucent proteinaceous substance with a waxy consistency. It is made of protein in combination with sugars (polysaccharides) and may be associated with Alzheimer's disease and other disorders.

Furthermore, it has been noted that SNPs in codon 129 of the human prion coding region [i.e., methionine (MET) homozygosity, versus valine (VAL) homozygosity, versus methionine/valine (MET/VAL) heterozygosity] along with the types of bands present on Western blot [i.e., type 1 (21 kd) versus type 2 (19 kd)] may be used in a system to characterize the clinical and histopathological manifestations of various subtypes of spontaneously occurring CJD or sCJD.

Occurring in roughly one in 1 million persons, sCJD is generally characterized by varying degrees of awareness or cognitive impairment and psychosis, along with ataxia (lack of coordination), visual field defects, and other neurological manifestations, with onset most common in the seventh decade of life. The most common subtypes are characterized by the SNPs and migration patterns on the Western blot as seen in Figure 2B. They include MET homozygosity and type 1 (19 kd) migration, referred to as MM1. Another sub-type is characterized by VAL homozygosity and type 2 (21 kd) migration, denoted as VV2. Finally, heterozygous codon 129 with type 1 migration, referred to as MV1, is the third most frequent subtype. These subtypes run their courses to a fatal outcome in a matter of four to six months, while other subtypes may proceed more gradually over 15 to 17 months on average.

Some prion diseases appear to occur on a familial basis. Familial CJD (fCJD) is very similar to sCJD in terms of its clinical and histopathological manifestations. Familial fatal insomnia is another such disorder characterized by intractable insomnia, myoclonus (muscle twitches), and autonomic dysfunction with a mean age at onset of 49 years and a duration of 11 to 23 months. Finally, a familial prion disease due to a PRNP open reading frame mutation, or SNP, is known as Gerstmann-Straussler-Scheinker syndrome (GSS). It is characterized by a gradual progression of cerebellar ataxia, dementia, spastic paraparesis (weakness of the legs), and extrapyramidal signs over five to six years beginning in the fifth decade. Extrapyramidal signs include involuntary movements of the mouth, lips, and tongue as well as tremors, restlessness, or rigidity, among other things.

Most importantly, a number of prion diseases have a recognized mode of transmission. Kuru, as discussed earlier, was one of the first to be uncovered. The Fore people, indigenous to Papua New Guinea, gave this disorder its name, which means "to shiver." Characterized by progressive and ultimately fatal cerebellar ataxia over six to nine months, onset usually occurred between the second and fourth decades.

It is possible to contract prion disease from contaminated surgical instruments as well as biological preparations, and the resulting disease is referred to as iatrogenic CJD (iCJD). Depending upon the source of infection, the onset varies among those contracting iCJD. Infection sources include neurosurgical instrumentation (with onset in 12 to 28 months), human growth hormone (50 to 450 months), corneal transplantation (16 to 320 months), dural patches (18 to 216 months), and human gonadotropin (144 to 192 months).

Of greatest concern, however, is vCJD, which occurs years after the ingestion of meat products containing traces of neural tissue from cows infected with BSE. Despite this long latency period, there has been a mean age at onset of 28 years. vCJD is manifested by psychiatric and sensory disturbances and dementia that progresses to its inexorably fatal outcome in an average of just over one year. It is believed that neuroinvasion of pathogenic prion is facilitated by white blood cells, particularly B-lymphocytes, that are present in intestinal Peyer's patches. Recently, two cases of vCJD transmitted by blood transfusion have been reported. This raises concerns about protecting the blood supply from prion diseases. Screening methods for prion detection in blood have been elusive.

Adding to the level of concern is the fact that the incidence of progressive neurological diseases, such as Alzheimer's disease, has increased dramatically in recent years, even when adjusted for the increasing population of elderly individuals. These progressive disorders are diagnosed almost exclusively based upon the clinical observation of signs and symptoms that overlap with those of CJD. Recently, histopathological examination of the brains of patients thought to have died from Alzheimer's disease revealed that 8% of patients in one group and 26% of patients in another group actually died of prion disease. Therefore, TSEs may be under-reported illnesses. If substantiated, such diagnostic uncertainty will make laboratory testing of pathogenic prion in man and animals an important addition to the testing armamentarium of laboratorians. But just how prevalent TSEs may become is related to the resistance to infection across species--the so-called species barrier.

Species barriers

There are many examples of the transmission of prion disease within a given species. There is, however, a barrier to disease transmission when the source pathogenic prion derives from a different species. The essence of the protein-only hypothesis of prion propagation is that [PrP.sup.Sc] replicates itself by recruiting [PrP.sup.c] molecules and inducing a conformational change, which results in the accumulation of more [PrP.sup.Sc]. This [PrP.sup.Sc] may, in turn, convert more of the cellular isoform to the pathogenic form. Based upon this hypothesis, it might be expected that prion diseases are transmissible between different species. Indeed, experimental transmission across mammalian species has been well established.

Interspecies transmission, as measured by the appearance of clinical signs in the host, is frequently limited by a species barrier. This barrier has been characterized by (1) longer latency periods between infection and symptom onset, which may even exceed the animal's typical lifespan, (2) atypical signs of disease in the recipient animal, and (3) a reduced rate of recipient animals succumbing to disease relative to the rate noted in the species of the source animal. Following the transmission of [PrP.sup.Sc] across a species barrier, serial passage of pathogenic prion within that same recipient species is characterized by shorter latency periods and more uniform signs of disease. The number of passages required for this to occur is one way to quantify the magnitude of the particular species barrier. Additional evidence for the species barrier has been demonstrated in transgenic mice that overexpress Syrian hamster PrP transgenes. These mice, in contrast with their wild-type littermates, show no species barrier when infected with hamster prions.

Several factors appear to contribute to the species barrier. One is the degree of variability in PrP gene sequences between that of the source animal and that of the recipient. Species barriers can also be affected by [PrP.sup.Sc] conformation, a feature that also characterizes prion strains. Different strains appear to have different transmission profiles. The form and degree of [PrP.sup.Sc] glycosylation are thought to be additional variables determining transmissibility. Finally, an as yet unknown factor termed "Protein X" has been hypothesized to have an effect on the species-barrier phenomenon. Protein X, if it exists, has not been characterized and may indeed be PrP itself.

The implications of the species barrier are that disease may be more prevalent than is recognized. This is compounded by the protracted latency from infection to manifestation of symptoms. It is thought that the central nervous system tissue of scrapie-infected sheep entered the food supply and was fed to cows, which resulted in BSE. Thus, scrapie transmission to humans represents an example of a species barrier that is bridged by cows. There are other examples of TSEs from one species affecting a second, and that species in turn becoming infectious for yet another species. Therefore, the extent to which other species may be potential sources of human TSE is not well understood. The best information we have comes from the National CJD Surveillance effort in the U.K. as shown in Figure 3. The peak of exposure in the human food chain to BSE-infected cattle occurred in 1989, and the peak in vCJD appears to have occurred in 2001, suggesting a latency of 12 years from infection to symptom onset.

It appears that some of those asymptomatic citizens of the U.K. became blood donors and, as noted previously, there are two cases of vCJD victims who received blood product from confirmed vCJD donors. There may be other species incubating TSEs with longer latencies for which humans may be susceptible; thus, the sense of urgency for developing tests for TSEs is warranted.

BSE testing

Aside from the Western blot assay, the only tests on the market are enzyme-linked immunosorbent assays (ELISA) or other immunoassays based upon the use of antibodies that do not possess specificity for pathogenic prion as opposed to normal prion protein. Like the Western blot, sample pretreatment with Proteinase K is a requirement. Table 1 provides an overview of the assays currently available and some of those known to be in development. There is a lack of antemortem assays, which would be most widely used to screen cattle for their status as satisfactory subjects for introduction into the food chain. The only available antemortem test requires a veterinarian to access the lymphoid tissue in the nictiating membrane (so-called third eyelid) of cattle. The sampling technique shows about a 60% success rate, and the assay has a sensitivity of 99% and specificity of only 70%, leaving quite a margin of opportunity for the development of better assays.

Protecting the safety of the blood supply is being approached in yet another way. There are ongoing efforts to manufacture products that can either reduce or remove prions, including pathogenic prions, that are either cell-associated or free in plasma or both.

Filtration of blood for prion removal

The recent occurrences of probable cases of transfusion-transmitted vCJD raise concerns about the safety of the blood supply. Because there is a long latency from infection to symptom onset and there is no antemortem test to screen for infectivity, aymptomatic blood donors may be compromising the safety of our nation's blood supply. Although the U.K. experience of susceptibility to vCJD suggests that codon 129 MET homozygotes may predispose individuals to disease, one of the two reported cases of transfusion-transmitted vCJD was detected in a patient who was a heterozygote MET/VAL at codon 129. The distribution of codon 129 is such that M/M and M/V together comprise about 88% of the Caucasian population of the developed nations. According to some thought leaders, these findings have major implications for results from surveillance of vCJD. In the absence of an effective antemortem test that can be used to detect the presence of infectious prion in potential blood donors who may be carrying the causative agent of vCJD, an alternative strategy must be considered to protect the safety of transfusion recipients. One such approach is to filter the blood to reduce or remove pathogenic prions.

Results from experimental models of TSE suggest that approximately half of infectious prions are found within leukocytes and the balance is found in plasma. Therefore, removal of leukocytes from blood is a prudent and necessary first step in minimizing the risk of transmission of vCJD. A recent report suggests, however, that the current generation of leukocyte-reduction filters was effective in removing only 42% of the total TSE infectivity in endogenously infected blood.

A new filtration technology that reduces both leukocytes and prions from the most often transfused blood product, packed red blood cells, is nearing commercialization. This filter reduces leukocytes and their associated prions by more than three orders of magnitude. Concerning the plasma-associated infectivity, in a preliminary study, a prototype of the filter was shown to reduce prion infectivity by approximately four orders of magnitude. Preliminary results of an endogenous infectivity study show the filter also prevents the transmission of prion disease in hamsters. Although the risk of transfusion-transmitted vCJD is not quantifiable at this time, the use of prion-reducing filters will provide a necessary measure of protection until such time as the risk is better understood either through antemortem blood testing or clinical surveillance.

[FIGURE 3 OMITTED]

Summary

Although eerily silent for many years after the recognition of scrapie in 1759, TSEs remained present within the genome of some mammals. Not since the mid-1950s when Dr. Carleton Gadjusek visited the Fore Indians of New Guinea to study kuru, however, has there been a more frenetic interest by governmental investigators. Certainly, the U.K. experience has heralded a renewed interest in TSEs due to the notoriety associated with younger subjects succumbing to a variant CJD traced to the ingestion of beef. Human TSEs and the potential for their transmission among and across species of mammals has also captured the attention of many. Yet, to date, there is no reliable antemortem test available to screen for infected animals or humans. Antibody-based assays are difficult to develop because most of them do not have specificity for the pathogenic form of prion protein.

Whether or not prion testing efforts will change dramatically depends upon the incidence of disease. Some speculate a reduction in testing, because BSE incidence is waning since the adoption of remedial steps in the U.K. in 1989. Others remind us, however, of the long latency of prion diseases and of the recent observations of two patients who succumbed to vCJD after having received blood products from donors who subsequently died of vCJD. The growing incidence of CWD, combined with the emerging observation that as many as 26% of Alzheimer's patients may have been misdiagnosed--having died instead of prion disease--maintains pressure for legislators to adhere to the precautionary principle and support blood-donor exclusionary criteria, antemortem-test development, and pathogen removal from donated blood. The laboratorian can expect to see new tests for prion disease work their way into clinical-testing practice in the near future. In addition, the adoption of newer filtration technologies holds the promise of improved protection from transfusion-transmitted prion disease.

Girolamo A. (Jerry) Ortolano, PhD, graduated from Columbia University (BS) and the University of Rhode Island (MS, PhD -- Pharmacology), completed a postdoctoral fellowship at the University of Michigan Hospital, and continued research there before joining Pall Corporation. He has authored over 60 scientific articles and abstracts and co-authored five book chapters. Samuel O. Sowemimo-Coker, PhD, is principal scientist and Jeffrey Schaffer, DVM, is New Initiatives staff scientist at Pall Medical in PortWashington, NY. Joseph S. Cervia, MD, FACP, FAAP, is medical director and senior vice president, Biomedical Division at Pall Medical in East Hills, NY, as well as professor of Clinical Medicine and Pediatrics at Albert Einstein College of Medicine in the Bronx, NY.

Suggested Reading List

1. Collins SJ, Lawson VA, Masters CL. Transmissible spongiform encephalopathies. Lancet. 2004;363:51-61.

2. Prusiner SB. Prions. Nobel lecture, December 8, 1997. Nobel Prize website. Available at: http://nobelprize.org/medicine/laureates/1997/prusiner-lecture.pdf. Accessed August 3, 2005.

3. Ironside JW, Head MW. Neuropathology and molecular biology of variant Creutzfeldt-Jakob disease. Curr Top Microbiol Immunol. 2004;284:133-159.

4. Heikenwalder M, Zeller N, Seeger H, et al. Chronic lymphocytic inflammation specifies the organ tropism of prions. Science. 2005;307:1107-1110.

5. Weissmann C, Aguzzi A. Approaches to therapy of prion diseases. Annu Rev Med. 2005;56:321-344.

6. The Inquiry into BSE and variant CJD in the United Kingdom. The BSE Inquiry Report Contents page. Available at: http://www.bseinquiry.gov.uk/report/index.htm. Accessed August 3, 2005.

7. Gregori L, McCombie N, Palmer D, et al. Effectiveness of leucoreduction for removal of infectivity of transmissible spongiform encephalopathies from blood. Lancet. 2004;364:529-531.

8. Sowemimo-Coker SO, Kim A, Zinn F, et al. Removal of infectious prion from naturally infected red blood cell concentrates, Vox Sang. 2004;87(suppl 3):10.

CE test on PRION BIOLOGY IN TRANSFUSION MEDICINE: IMPLICATIONS FOR LAB TESTING

MLO and Northern Illinois University (NIU), DeKalb, IL, are co-sponsors in offering continuing education units (CEUs) for this issue's article on PRION BIOLOGY IN TRANSFUSION MEDICINE: IMPLICATIONS FOR LAB TESTING. CEUs or contact hours are granted by the College of Health and Human Sciences at NIU, which has been approved as a provider of continuing education programs in the clinical laboratory sciences by the ASCLS P.A.C.E.[R] program (Provider No. 0001) and by the American Medical Technologists Institute for Education (Provider No. 121019; Registry No. 0061). Approval as a provider of continuing education programs has been granted by the state of Florida (Provider No. JP0000496), and for licensed clinical laboratory scientists and personnel in the state of California (Provider No. 351). Continuing education credits awarded for successful completion of this test are acceptable for the ASCP Board of Registry Continuing Competence Recognition Program. After reading the article on page 10, answer the following test questions and send your completed test form to NIU along with the nominal fee of $20. Readers who pass the test successfully (scoring 70% or higher) will receive a certificate for 1.0 contact hour of P.A.C.E.[R] credit. Participants should allow four to six weeks for receipt of certificates.

The fee for each continuing education test will be $20.

All feature articles published in MLO are peer-reviewed.

CE questions prepared by Girolamo A. Ortolano, PhD; Samuel O. Sowemimo-Coker, PhD; Jeffrey Schaffer, DVM; and Joseph S. Cervia, MD, FACP, FAAP. CE questions and learning objectives reviewed and submitted by Shirley A. Richmond, PhD, MT(ASCP), CSL(NCA), Dean, College of Health and Human Sciences, Northern Illinois University, DeKalb, IL.

1. The word PRION derives as an abbreviation for

a. pathogenic virion.

b. pervasive ions.

c. infectious proteins.

d. proteinaceous influenza.

2. The first report of TSE occurred in which century?

a. 18th century.

b. 19th century.

c. 20th century.

d. 21st century.

3. TSE is a general description of prion diseases, but the right column represents the forms of most diseases associated with specific genera. Select the correct matches.

4. To whom was the Nobel Prize awarded for identifying protein as an infectious agent of TSEs?

a. Creutzfeld.

b. Jakob.

c. Alper.

d. Prusiner.

5. Which of the features of the pathogenic prion proteins are consistent across all species?

I. They all have about 210 amino acids.

II. They all have a C-terminal glycophosphatidylinositol moiety.

III. They all have two additional sites of post-translational glycosylation.

IV. They all have sensitivity to proteinase K.

a. I and II.

b. I, II, and III.

c. I, II, and IV.

d. All of the above

6. The molecular weight of the unglycosylated prion protein is

a. 25,000 kd.

b. 2.5 d.

c. 2,500 d.

d. 25 kd.

7. Chronic wasting disease (CWD) is found in

a. goat and sheep.

b. deer and elk.

c. cattle and cat.

8. Human TSEs include

a. iCJD, sCJD, nCJD, and kuru.

b. MSE, BSE, fFI, and scrapie.

c. fFI, iCJD, sCJD, and kuru.

d. kuru, pSE, nCJD, and MSE.

9. Select the statement that is true.

a. Western blot separates proteins based upon molecular weight.

b. Western blot separates proteins based upon the extent of glycosylation.

c. Western blot separates proteins based upon the fragment sizes remaining after Proteinase K digestion.

d. All of the above.

10. The three bands most often seen in the Western blot represent (select the correct order)

a. di-, mono-, and unglycosylated pathogenic prion.

b. mono-, di-, and unglycosylated pathogenic prion.

c. di-, un-, and monoglycosylated pathogenic prion.

d. diglycosylated nonpathogenic prion.

11. The Western blot Type 1 prion fragment has an approximate size of

a. 9 kd.

b. 11 kd.

c. 19 kd.

d. 21 kd.

12. Which statement is false?

a. Antibodies with specificity for pathogenic prion are not well characterized.

b. Most assays using antibodies require proteinase K sample pretreatment.

c. There are several tests for antemortem detection of prion in blood.

d. The sampling of "third--eye" tissue requires a technically expert technician or clinician.

13. All of the following have been implicated in the transmission of prion diseases, except

a. ingestion of meat.

b. blood transfusion.

c. airborne droplet nuclei.

d. corneal transplantation.

e. growth hormone administration.

14. It is currently believed that neuroinvasion by pathogenic prion is facilitated most by

a. erythrocytes.

b. B-lymphocytes.

c. CD4+ lymphocytes.

d. astroglial cells.

e. tissue macrophages.

15. All of the following characterize both vCJD and Alzheimer's disease, except

a. absence of a reliable antemortem diagnostic laboratory test.

b. dementia.

c. amyloid plaque formation.

d. fatal outcome in approximately one year.

e. progressive neurological decline.

16. All of the following are characteristic of the prion species barrier, except

a. affords absolute protection against interspecies transmission of pathogenic prion.

b. provides longer latency between infection and symptom onset.

c. produces atypical signs of disease in the recipient animal.

d. reduces rate of recipient animals succumbing to disease relative to the species of the source animal.

17. All of the following appear to contribute to the species barrier, except

a. variability in PrP gene sequences.

b. [PrP.sup.Sc] conformation.

c. variability in protein C.

d. form and degree of [PrP.sup.Sc] glycosylation.

e. variability in Protein X.

18. The implications of the species-barrier phenomenon include all of the following, except

a. all species that may be sources of human TSEs are known.

b. there may be more prion-associated disease than is currently recognized.

c. human cases of TSEs that are acquired from other species may have relatively long latency periods between the time of infection and symptom onset.

d. TSE-infected individuals may be asymptomatic and become blood donors.

e. the full extent of widespread exposure to pathogenic prion may not be completely appreciated for a decade or more following that exposure.

19. Important implications of the observation that vCJD can be transmitted by transfusion include

a. asymptomatic infected blood donors may have already contaminated the blood supply.

b. such observations demand the development of an antemortem test to screen blood donors and the employment of pathogen removal or inactivation technologies.

c. the problem of human TSE disease may be larger than we appreciate when limiting risk assessment to the vCJD derived from BSE alone and not considering the potential for human transmission from other species like deer (CWD).

d. All of the above.

20. Which of the following statements is not likely to be true?

a. Prions represent a novel infectious agent.

b. Prion disease affects a variety of mammalian species.

c. The species barrier can be circumvented by an intermediate species.

d. Prion disease in humans will be no greater risk than the incidence of sporadic CJD.

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By Girolamo A. Ortolano, PhD; Samuel O. Sowemimo-Coker, PhD; Jeffrey Schaffer, DVM; and Joseph S. Cervia, MD, FACP, FAAP

COPYRIGHT 2005 Nelson Publishing
COPYRIGHT 2005 Gale Group