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From: TSS ()
Subject: Presymptomatic Detection of Prions in Blood
Date: July 6, 2006 at 1:51 pm PST

Presymptomatic Detection

of Prions in Blood

Paula Saa´ ,1,2 Joaquý´n Castilla,1 Claudio Soto1*

Prions are thought to be the proteinaceous infectious agents responsible for transmissible

spongiform encephalopathies (TSEs). PrPSc, the main component of the infectious agent, is also

the only validated surrogate marker for the disease, and its sensitive detection is critical for

minimizing the spread of the disease. We detected PrPSc biochemically in the blood of hamsters

infected with scrapie during most of the presymptomatic phase of the disease. At early stages of

the incubation period, PrPSc detected in blood was likely to be from the peripheral replication of

prions, whereas at the symptomatic phase, PrPSc in blood was more likely to have leaked from the

brain. The ability to detect prions biochemically in the blood of infected but not clinically sick

animals offers a great promise for the noninvasive early diagnosis of TSEs.

Prion diseases, also called transmissible

spongiform encephalopathies (TSEs), are

a group of fatal and infectious neurodegenerative

diseases, including Creutzfeldt-Jakob

disease (CJD) in humans and bovine spongiform

encephalopathy (BSE), scrapie, and chronic

wasting disease (CWD) in animals. Prions are

composed mainly or exclusively of the misfolded

prion protein (PrPSc) (1), which replicates in the

body, transforming the normal prion protein

(PrPC) into more of the misfolded isoform.

Although prion diseases are rare in humans,

the established link between a new variant form

of CJD (vCJD) and BSE (2–4) has raised concern

about a potential epidemic in the human

population. Over the past few years, BSE has

become a substantial health problem affecting

many countries (5), and it seems now apparent

that vCJD can be iatrogenically transmitted from

human to human by blood transfusion (6, 7).

Exacerbating this state of affairs is the lack of a

reliable test to identify individuals incubating

the disease during the long and silent period

from the onset of infection to the appearance of

clinical symptoms (8–10).

PrPSc is not only the main component of the

infectious agent and the most likely cause of

the disease, but it is also the only validated

surrogate marker for TSEs (9). However, PrPSc

concentration is high enough for routine biochemical

detection only in the brain and some

lymphoid tissues at a time close to the symptomatic

stage of the disease (9). The development

of highly sensitive presymptomatic assays

for the biochemical detection of PrPSc is critical

for minimizing the spread of the disease (9).

One important aim in prion diagnosis is the

noninvasive and presymptomatic biochemical

detection of PrPSc in biological fluids, particularly

using blood, a fluid known to contain infectivity

even before the onset of clinical signs

(6, 11, 12).

PrPSc has been detected in the blood of sick

animals by means of the protein misfolding cyclic

amplification (PMCA) technology (13).

PMCA produces accelerated prion replication,

which dramatically amplifies the quantity of

PrPSc present in a sample (14, 15). In a cyclical

process, large quantities of PrPC are converted

into the misfolded form triggered by the presence

of minute and otherwise undetectable

amounts of PrPSc. The method is highly specific

for the detection of PrPSc and leads to a severalmillion-

fold increase in sensitivity as compared

to that of standard Western blot assays (13).

In order to evaluate the application of PMCA

for the detection of prions in blood during the

presymptomatic phase, 46 hamsters were inoculated

intraperitoneally with 10% brain homogenate

of the 263K scrapie strain, and 38 control

animals were injected with phosphate-buffered

saline (PBS). At different times during the

incubation period, groups of animalswere killed,

blood was collected, and the buffy coat fraction

was separated (13). Samples of the buffy coat

were resuspended directly on healthy hamster

brain homogenate and subjected to 144 PMCA

cycles. Three different aliquots were tested from

each sample. To refresh the substrate, after a

round of PMCA cycling, samples were diluted

10-fold into normal brain homogenate, followed

by another round of 144 PMCA cycles. This

procedure was repeated seven times, because

according to our results, this enables the detection

of 20 to 50 molecules ofmonomeric hamster PrP,

which seems to correspond to a single unit of

infectious oligomeric PrPSc (16).

The first group of hamsters was killed 2

weeks after intraperitoneal inoculation. None of

the five infected or control animals showed any

detectable quantity of PrPSc in their blood (Fig.

1 and Table 1). Thus, the PrPSc present in the

inoculum disappeared to undetectable levels

during the first few days after inoculation. PrPSc

was, however, readily detectable in blood 1

week later (20 days after inoculation) in 50% of

the animals infected but in none of the controls

(Fig. 1 and Table 1). The highest percentage of

positive animals during the presymptomatic

phase was observed 40 days after intraperitoneal

inoculation, in which the sensitivity of

PrPSc detection was 60%. After 60 days, the

detection of PrPSc in blood became harder. Indeed,

only one out of five animals scored positive

at 70 days, whereas none of the five

infected hamsters had detectable PrPSc in their

blood 80 days after inoculation (Table 1). At

the symptomatic stage, which in this experiment

was at 114.2 T 5.6 days, 80% of animals

had PrPSc in their blood (Fig. 1). We never

detected a false positive result in any of the 38

control samples analyzed (Table 1).

The distribution of PrPSc detection at different

times of the incubation period showed

1George and Cynthia Mitchell Center for Alzheimer’s

Disease Research, Departments of Neurology, Neuroscience

and Cell Biology, and Biochemistry and Molecular Biology,

University of Texas Medical Branch, 301 University

Boulevard, Galveston, TX 77555–0646, USA. 2Centro de

Biologý´a Molecular, Universidad Auto´noma de Madrid,

Madrid, Spain.

*To whom correspondence should be addressed. E-mail:

clsoto@utmb.edu

7 JULY 2006 VOL 313 SCIENCE www.sciencemag.org 92

REPORTS

an interesting trend (Fig. 2). A first peak of

PrPSc detection was observed early during the

presymptomatic phase, between 20 and 60 days

after inoculation. The peripheral administration

of prions is known to result in an early

phase of replication in lymphoid tissues and

the spleen, before any infectious material

reaches the brain (17, 18). Indeed, little or no

infectivity can be detected in the brain of

animals peripherally inoculated during the first

half of the incubation period (19). Thus, it is

likely that the source of PrPSc in blood during

the early presymptomatic phase is the spleen

and other lymphoid organs. The quantity of

PrPSc in blood goes down after this initial

phase and actually disappears 80 days after

inoculation (Table 1 and Fig. 2). The rise of

PrPSc in blood during the early presymptomatic

phase appears to coincide with the

time of its exponential replication in lymphoid

organs, whereas the reduction of PrPSc in

blood occurs when infectivity in peripheral

tissues has reached a plateau and is migrating

from the periphery to the brain (17, 18). Although

the explanation for these results in

unknown, it is possible that the proportion of

circulating lymphocytes carrying PrPSc is much

higher during the exponential phase of peripheral

replication than during the stationary phase.

At the symptomatic period, PrPSc can again be

detected in the blood of most of the animals

(Fig. 2). It has been reported that large

quantities of PrPSc appear in the brain only a

few weeks before the onset of clinical signs

(19, 20). Thus, PrPSc in blood samples at the

symptomatic stage is likely to have come from

brain leakage. It is known that at the time of

symptomatic disease, TSE-affected individuals

have extensive brain degeneration in the form

of massive neuronal death, synaptic alterations,

and brain inflammation (21). These abnormalities

probably cause a disruption of the blood/

brain barrier resulting in the leakage of cerebral

proteins to the blood (22), in particular PrPSc,

which by this time is highly abundant in the

brain.

Infectivity studies have shown that the blood

carries prions in both the symptomatic and

presymptomatic stages of the disease in animals

(11, 23, 24). Upon experimental BSE infection

of sheep, infectivity can be transmitted by

blood transfusion from asymptomatic infected

animals (25), indicating that the infectious

agent is present in blood during the incubation

period. Recently, three cases of vCJD have

been associated with blood transfusion from

asymptomatic donors who subsequently died

from vCJD (6, 7). The alarmingly high proportion

of cases transmitted by blood transfusion

suggests that prions exist in relatively

elevated quantities in the blood of individuals

silently incubating vCJD. Based on studies with

animal models, it is believed that all of the human

population may be susceptible to vCJD

infection (26), although clinical cases have so

far occurred only in methionine homozygotes at

codon 129 in the human prion protein gene.

Because the incubation period may be several

decades, it is currently unknown how many

people may be in an asymptomatic phase of

Fig. 1. PrPSc detection

in the blood of scrapieinfected

hamsters by

PMCA. Blood samples

from groups of scrapieinoculated

and control

animals were taken at

different times during

the incubation period.

Three milliliters of blood

were separated in three

aliquots of 1 ml each to

prepare the buffy coat

(13). Samples were subjected

to 144 cycles of

PMCA. Ten microliters of

the sample from this first

round of amplification

were diluted into 90 ml

of normal brain homogenate,

and a new round

of 144 PMCA cycles was

performed. This process

was repeated a total of

seven times. Each panel

represents the results

obtained in the seventh

round of PMCA with the

samples from each group

of animals, which are representative of the three independent aliquots taken from each animal. Ix, samples

from hamsters infected with 263K scrapie; Cx, samples from control animals injected with PBS. All samples

were treated with proteinase K (PK) before electrophoresis, except for the normal brain homogenate (NBH), in

which no PK treatment (–PK) is indicated.

Table 1. Number of animals used and results obtained regarding the presymptomatic detection of

PrPSc in the blood.

Time

(days)

Controls

(positives/total)

Infected

(positives/total)

Sensitivity/

specificity

14 0/5 0/5 0%/100%

20 0/4 3/6 50%/100%

40 0/5 6/10 60%/100%

60 0/4 2/5 40%/100%

70 0/5 1/5 20%/100%

80 0/5 0/5 0%/100%

Symptomatic

phase

0/10 8/10 80%/100%

Fig. 2. Proportion of animals whose blood was

PrPSc positive at different times during the

incubation period. The percentage of samples

scoring positive for PrPSc in blood is represented

versus the time after inoculation at which

samples were taken. Two phases of PrPSc detectability

were observed: an early stage during

the incubation period, which probably corresponds

to the time during which peripheral prion

replication in lymphoid tissues is occurring, and

a second phase at the symptomatic stage, in

which the brain contains extensive quantities of

PrPSc. The vertical gray section indicates the

symptomatic phase.

www.sciencemag.org SCIENCE VOL 313 7 JULY 2006 93

REPORTS

vCJD infection. In addition, it is possible that

some infected patients may never develop clinical

symptoms but will remain asymptomatic

carriers who can potentially transmit the disease

to other individuals (26, 27). In the absence of

screening tests and effective therapies to treat this

disease, a formidable worldwide public health

challenge lies ahead to prevent further infections,

assess infection rates, and treat infected patients.

The ability to detect PrPSc, the major component

of infectious prions, biochemically in the blood

of infected but asymptomatic experimental

animals will hopefully lead to the development

of tests for human blood. Indeed, although technically

more challenging, the PMCA technology

has been adapted to amplify prions of human

origin (20). The ability to accurately detect

PrPSc in the presymptomatic stages of vCJD

would potentially help to reduce the risk that

many more people will be infected by this fatal

and terrible disease.

References and Notes

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(1998).

2. S. N. Cousens, E. Vynnycky, M. Zeidler, R. G. Will,

R. G. Smith, Nature 385, 197 (1997).

3. J. Collinge, Lancet 354, 317 (1999).

4. M. E. Bruce et al., Nature 389, 498 (1997).

5. R. Bradley, P. P. Liberski, Folia Neuropathol. 42 (suppl. A),

55 (2004).

6. C. A. Llewelyn et al., Lancet 363, 417 (2004).

7. A. H. Peden, M. W. Head, D. L. Ritchie, J. E. Bell,

J. W. Ironside, Lancet 364, 527 (2004).

8. Q. Schiermeier, Nature 409, 658 (2001).

9. C. Soto, Nat. Rev. Microbiol. 2, 809 (2004).

10. L. Ingrosso, V. Vetrugno, F. Cardone, M. Pocchiari, Trends

Mol. Med. 8, 273 (2002).

11. P. Brown, L. Cervenakova, H. Diringer, J. Lab. Clin. Med.

137, 5 (2001).

12. F. Houston, J. D. Foster, A. Chong, N. Hunter,

C. J. Bostock, Lancet 356, 999 (2000).

13. J. Castilla, P. Saa, C. Soto, Nat. Med. 11, 982 (2005).

14. G. P. Saborio, B. Permanne, C. Soto, Nature 411, 810

(2001).

15. C. Soto, G. P. Saborio, L. Anderes, Trends Neurosci. 25,

390 (2002).

16. J. R. Silveira et al., Nature 437, 257 (2005).

17. R. H. Kimberlin, C. A. Walker, J. Comp. Pathol. 89, 551

(1979).

18. M. Glatzel, A. Aguzzi, Microbes Infect. 2, 613 (2000).

19. R. H. Kimberlin, C. A. Walker, J. Gen. Virol. 67, 255

(1986).

20. C. Soto et al., FEBS Lett. 579, 638 (2005).

21. J. Castilla, C. Hetz, C. Soto, Curr. Mol. Med. 4, 397

(2004).

22. W. A. Banks, J. Neurovirol. 5, 538 (1999).

23. P. Brown, Vox Sang. 89, 63 (2005).

24. N. Hunter et al., J. Gen. Virol. 83, 2897 (2002).

25. N. Hunter, Br. Med. Bull. 66, 171 (2003).

26. M. T. Bishop et al., Lancet Neurol. 5, 393 (2006).

27. J. W. Ironside, Haemophilia 12, 8 (2006).

28. This research was supported in part by NIH grants

AG0224642 and NS049173.

Supporting Online Material

www.sciencemag.org/cgi/content/full/313/5783/92/DC1

Materials and Methods

References

21 April 2006; accepted 5 June 2006

10.1126/science.1129051

7 JULY 2006 VOL 313 SCIENCE www.sciencemag.org

TSS




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