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Advanced Drug Delivery Reviews 56 (2004) 915–920

Mechanism of intestinal entry of infectious prion protein in the

pathogenesis of variant Creutzfeldt–Jakob disease

Subrata Ghosh*

Department of Gastroenterology, Faculty of Medicine, Imperial College London, Hammersmith Hospital, London, UK

Received 3 October 2003; accepted 3 November 2003

Abstract

The pathogenesis of variant Creutzfeldt–Jakob disease (vCJD) is most likely to be dependent on intestinal entry of orally

ingested infectious prion proteins, though tonsils or other oral portals of entry are possible. The exact route of entry of infectious

prion proteins is uncertain but receptors for prion proteins such as laminin receptor precursor (LRP) may be expressed on

intestinal brush border. Cellular prion protein (PrPc) is expressed on intestinal enteric nervous system and is separated by a

single layer of epithelial cells from ingested infectious prion proteins. Intestinal M cells in the Peyer’s patches may also

transcytose prion proteins which may be transported to the lymphatic system by migrating dendritic cells underlying the M

cells. The relative importance of the several potential portals of intestinal entry of infectious prion proteins is uncertain but may

determine susceptibility of the population and also potential preventive strategies.

D 2004 Published by Elsevier B.V.

Keywords: Variant Creutzfeldt–Jakob disease; Laminin receptor precursor; Infectious prion protein; Intestinal M cells; Enteric nervous system

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 916

1.1. Prions and transmissible spongiform encephalopathies . . . . . . . . . . . . . . . . . . . . . . . . . . . 916

1.2. Variant Creutzfeldt–Jakob disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 916

2. Intestinal entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 916

2.1. Prion receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 916

2.2. Enteric nervous system expression of cellular prion protein . . . . . . . . . . . . . . . . . . . . . . . . 917

2.3. M-cell transcytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 917

2.4. Presence of PrPSc in intestinal tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 918

3. Infectivity of extra-neural tissues from vCJD victims . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 919

4. Therapeutic aspects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 919

5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 919

References

0169-409X/$ - see front matter D 2004 Published by Elsevier B.V.

doi:10.1016/j.addr.2003.10.035

* Tel.: +44-208-383-3266; fax: +44-208-749-3436.

E-mail address: s.ghosh@imperial.co.uk (S. Ghosh).

S. Ghosh / Advanced Drug Delivery Reviews 56 (2004) 915–920916

Table 2

Differences between PrPc and PrPSc

PrPc PrPSc

40% a-helix 20% a-helix

Little h-sheet 50% h-sheetSoluble Insoluble

Protease sensitive Protease resistant

Constitutively expressed in

brain and other tissues

Post-translationally

converted from PrPc

1. Introduction

1.1. Prions and transmissible spongiform

encephalopathies

Prions are small infectious proteins without nucleic

acid. Prusiner first coined the term, and also hypoth-

esized that prions were responsible for transmissible

spongiform encephalopathies (TSE) [1]. A number of

TSEs are recognized in animals and humans, and the

human diseases are listed in Table 1. A key event in

the pathogenesis of TSEs is the conversion of host

cellular prion protein (PrPc) to its infectious isoform

(PrPSc) [2]. Human PrPc is coded by a single gene

(PrP) on the short arm of chromosome 20 [3]. It is a

copper-binding glycoprotein consisting of 253 amino

acids. Structurally it contains 5 amino terminal octa-

peptide repeats, 2 glycosylated sites and 1 disulfide

bridge. During processing, two signal sequences in

the amino and carboxy terminal ends are removed and

the prion protein is attached to the outer surface of the

cell membrane by glycosyl-phosphatidyl-inositol

(GPI) anchor [4].

Infectious PrPSc converts host PrPc post-transla-

tionally to further PrPSc. This conformational change

is the principal biochemical change in prion diseases.

Table 2 shows the differences between PrPc and PrPSc

[5]. PrPSc is resistant to protease digestion and hence

can survive degradation in the gastrointestinal tract.

1.2. Variant Creutzfeldt–Jakob disease

Variant Creutzfeldt–Jakob disease (vCJD) was

first reported in 1996 [6], and enormous media inter-

est and public consternation resulted from its link to

bovine spongiform encephalopathy (BSE). BSE itself

was described in 1986 [7] and the source of BSE was

Table 1

Human TSE

Creutzfeldt– Jakob disease (CJD)

Sporadic

Familial

Iatrogenic

Variant CJD (vCJD)

Kuru

Gerstmann–Straussler syndrome (GSS)

Fatal familial insomnia (FFI)

the meat and bone meal feed contaminated by infec-

tious prions.

The magnitude of the vCJD ‘epidemic’ remains

uncertain. Statistical modeling has suggested that

between 63 and 136,000 people might be affected

[8] but these figures might have to be revised based on

new information. Already 135 cases have been

reported to the National CJD Surveillance Unit in

Edinburgh (May 2003) and the incidence of the

disease has been steadily rising each year. vCJD

victims have also been reported in France and Ireland.

In this era of travel and immigration, potential risk of

transmission is present worldwide, irrespective of the

geography of BSE affection. The incidence in UK is

higher in the North of England and Scotland com-

pared to the South [9]. This is most likely to be due to

dietary factors and socioeconomic deprivation. Forty

percent of the population have the methionine codon

at codon 129 of the PrP gene and are considered to be

genetically susceptible [8], though whether other

genotypes may simply have longer incubation periods

is uncertain. Recent data from the Communicable

Disease Surveillance Center in London suggested that

the previously increasing trend in mortality may have

slowed down, but future trends are uncertain [10].

2. Intestinal entry

2.1. Prion receptors

The critical step in vCJD pathogenesis is the

conformational change of PrPc to protease resistant

h-sheet rich PrPSc, and host constitutive expression of

PrPc is an essential step in the pathogenesis [11].

Accumulation of the aberrant prion, PrPSc, in the

central nervous system is a hallmark of vCJD. PrPSc

has been found in the enteric nervous system (ENS) of

S. Ghosh / Advanced Drug Delivery Reviews 56 (2004) 915–920 917

orally challenged rodent models of TSE, suggesting

that this might be the route of entry via the intestinal

mucosa [12]. A number of putative receptors for

prions have been reported, but most interest has

focussed on the 37-kDa laminin receptor precursor

(LRP) [13]. LRP is incorporated into the 67-kDa

mature laminin receptor (LR). We have described

intestinal brush border expression of the 67-kDa LR

in 40% of human subjects [14], using the monoclonal

antibody MLuC5 for immunohistochemistry on par-

affin sections.

2.2. Enteric nervous system expression of cellular

prion protein

PrPc is expressed within different components of

the ENS. The monoclonal antibody 3F4 was used on

frozen sections of endoscopic biopsies and ileal re-

section specimens. PrPc was co-localized with neural

filament protein within the enteric nerve fibers [15].

PrPc was also co-localized with glial fibrillary acidic

protein in glial cells within the ileal myenteric ganglia.

In situ hybridization confirmed the presence of PrP

mRNA within nerve cell bodies and glial cells in the

enteric ganglia. PrPc positive nerve endings in the

ENS were intimately associated with intestinal epi-

thelial cells. The 67-kDa prion binding LR incorpo-

rating the 37-kDa LRP was found to be expressed in

40% of small intestinal specimens and only a thin

layer of epithelium stands between the ingested BSE

agent and host PrPc in the ENS.

A postulated mechanism for entry of infectious

PrPSc after ingestion might be initial binding to the

LRP followed by internalization. The internalized

PrPSc will then convert host PrPc expressed on ENS

to PrPSc, providing a route for the spread of ‘infection’

eventually to the central nervous system. Direct proof

of this hypothesis is, however, lacking. We have

recently shown uptake of recombinant bovine prion

protein (rBoPrP) by human colonic epithelium main-

tained in organ culture (unpublished data).

2.3. M-cell transcytosis

In vitro evidence of intestinal M cells as the portal

of entry of infectious prion proteins has come from the

Caco-2 monolayer model co-cultured with Raji B

cells. Such a co-culture model shows functional and

morphological features of transformation to M-cell

phenotype (reviewed by Ghosh [16]). This is demon-

strated functionally by active, temperature dependent

transport of inert FITC-conjugated latex beads. Scra-

pie strain prions (5 or 3 log LD50) were used to assay

for prion transport in the intestinal M cell model with

infectivity determined by bio-assay in tga20 mice

which overexpress a Prnp transgene and develop

scrapie rapidly after infection [17]. In the in vitro

model M-cell differentiation was necessary and suffi-

cient for active transepithelial prion transport. There

was no significant prion transport, however, in Caco-2

cultures without M-cells. The immune cells underlie

the M-cells in intestinal Peyer’s patches and therefore

M-cell dependent transcytosis may enable prions to

gain access to the intestinal immune cells, especially

dendritic cells. Dendritic cells form a dense layer of

cells in the subepithelial dome in the Peyer’s patch

region just beneath the follicle associated epithelium

and in close contact with M cells. It has been shown

that dendritic cells can acquire PrPSc in vitro and that

a small sub-population of migrating dendritic cells can

take up and transport PrPSc from the gut lumen

through the lymphatics to the lymphoid tissue [18].

It was also shown that the uptake of PrPSc from the

gut lumen is restricted to dendritic cells as no PrPSc

was detected in other lymph cells or in cell free lymph

plasma. Follicular dendritic cells express host prion

protein PrPc and are critical to replication in lymphoid

tissues and neuroinvasion. Temporary de-differentia-

tion of follicular dendritic cells by lymphotoxin hreceptor immunoglobulin fusion protein (LT h R-Ig)

before intraperitoneal scrapie inoculation blocked the

early accumulation of infectivity and disease specific

PrPSc within the spleen and substantially reduced

disease susceptibility [19]. Treatment with LT h R-

Ig before oral scrapie inoculation also blocked PrPSc

accumulation in Peyer’s patches and mesenteric

lymph nodes and prevented neuroinvasion [19]. Lym-

phoreticular requirements for enteric and for intraper-

itoneal uptake of prions differ from each other.

Peyer’s patches of h7� /� mice are normal in number

but are atrophic and almost entirely devoid of B

lymphocytes, as B lymphocytes depend on a4h7

expression binding to MADCAM-1 expressed on

intestinal microvasculature. Minimal infectious dose

and disease incubation after oral exposure to prion

inoculum were similar in h7� /� and wild-type mice

S. Ghosh / Advanced Drug Delivery Reviews 56 (2004) 915–920918

[20]. Despite significant reduction in B lymphocytes,

M cells were present in h7� /� mice. Mice in which

numbers of Peyer’s patches are reduced (TNFa� /��LTa� /�; RAG-1� /�; AMT) were highly resistant to

oral challenge, and their intestines were devoid of

prion infectivity at all times after challenge [20].

2.4. Presence of PrPSc in intestinal tissue

Recently, a high sensitivity immunoblotting (upto

5 nl 10% vCJD brain homogenate) has shown the

presence of PrPSc in the tonsil, spleen and lymph

nodes of four vCJD victims. The concentration of

PrPSc in these extra-neural tissues ranged from 0.1%

to 15% of vCJD brain. Very low concentrations were

detected in the rectum, adrenal and thymus from one

Fig. 1. Hypothesis regarding routes of entry of PrPS

vCJD victim [21]. A previous study using less sensi-

tive immunohistochemistry had shown the presence of

PrPSc of follicular dendritic cells of tonsils and Peyer’s

patches, appendix, spleen and lymph nodes of vCJD

victims [22]. A patient had appendicectomy 8 months

prior to onset of symptoms of vCJD and PrPSc was

detected in the removed appendix upon lookback [23].

Appendix, however, does not reliably report cCJD

infection even at the end stage of the disease [24]. The

presence of PrPSc in the follicular dendritic cells of the

Peyer’s patches and the lymphoreticular spread sug-

gests M-cell dependent uptake. Therefore, both M-cell

dependent (lymphoreticular) and independent path-

ways (ENS) are possible, based on circumstantial

evidence, but direct proof of the existence of these

pathways requires further experimentation.

c via the intestinal tract and outcome of entry.

S. Ghosh / Advanced Drug Delivery Reviews 56 (2004) 915–920 919

The two processes hypothesized for intestinal prion

entry are summarized in Fig. 1.

3. Infectivity of extra-neural tissues from vCJD

victims

Recent bio-assay using mice intracerebral inocula-

tion of homogenized vCJD affected tissues has shown

that spleen and tonsil were infective, but 100–1000

times less so than brain. These tissues transmitted to

only a proportion of mice and had a longer incubation

period than brain tissue [25]. Buffy coat and plasma

failed to transmit. Second passage amongst humans is,

however, likely to be at least 500 times more efficient

than inter-species transmission experiments.

4. Therapeutic aspects

Understanding of the mechanism of transmission

of the BSE agent has led to certain pragmatic recom-

mendations regarding reducing the risks of iatrogenic

transmission by endoscopy (#). In wild-type mice, the

expression of PrPc rendered soluble and dimeric by

fusion to immunoglobulin Fc gamma {PrP-Fc(2)}

delays PrPSc accumulation, agent replication and

onset of disease following inoculation with infective

prions [26].

5. Summary

After oral challenge with the BSE agent, the

human ENS might be one of the early targets, pro-

viding the route of spread to the central nervous

system as well as a site for initial generation of

infectivity and PrPSc. The presence of the prion

receptor LRP at the luminal surface of the intestinal

epithelium of approximately 40% of subjects may

represent a risk factor for susceptibility to infectious

prions, enabling transfer of the infectious agent across

the human intestinal epithelium to the adjacent PrPc

positive enteric nerve endings. These findings may

have implications both for susceptibility to vCJD after

ingestion of BSE agent as well as theoretical risk of

transmission via endoscopic procedures. For a de-

tailed review of the association of PrPSc with intestinal

tissues the reader may also read the review by

Shmakov and Ghosh [27].

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