Award details

Transmission of scrapie agent from intestine to lymph nodes

Reference	BB/D00313X/1
Principal Investigator / Supervisor	Dr Gordon MacPherson
Co-Investigators / Co-Supervisors	Dr Susan Paulin
Institution	University of Oxford
Department	Sir William Dunn Sch of Pathology
Funding type	Research
Value (£)	333,014
Status	Completed
Type	Research Grant
Start date	01/02/2006
End date	31/01/2009
Duration	36 months

Abstract

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of chronic degenerative disorders of the central nervous system (CNS), affecting both humans and animals. Examples of TSEs include bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats and Creutzfeldt-Jacob disease (CJD) in humans. Most TSEs result from peripheral exposure to these infectious agents (TSEA), as has been shown for scrapie and is suggested for BSE. However much of what happens in the periphery following exposure, and preceding the development of CNS disease, remains obscure. For example, it is not clear how TSEA cross the intestinal epithelial barrier: M cells in Peyer's patches, dendritic cell (DC) processes or transport as complexes with ferritin are current possibilities. After crossing this barrier, infectivity is initially found in local secondary lymphoid organs. Subsequently, prions can be detected in other lymph nodes, the spleen and finally the CNS. How TSEA get to lymph nodes, more specifically to follicular dendritic cells (FDC) (FDC are quite separate from DC that migrate from the intestine in lymph) is also unclear. Direct transport in lymph as free protein, in exosomes or other vesicles (perhaps even chylomicrons) or within migrating DC are all possible mechanisms. We have shown previously that migrating DC can acquire and transport TSEA from the intestine in rats, and that in contrast to macrophages, lymph DC do not degrade the scrapie form of prion protein (PrPsc) efficiently. More recently, we have shown that PrPsc taken up by DC retains it's infectivity, whereas infectivity is rapidly lost in macrophages. In the current project we will ask the following questions: 1. Do exosomes or other vesicles transport PrPsc in lymph after oral administration? We will use our well-characterised model to collect rat pseudo-afferent intestinal lymph after gavage with brain homogenates from terminally ill mice or ME7 scrapie-associated fibrils (SAF). Differential centrifugation will be used to separate exosomes (if they exist) and chylomicrons from lymph. Given that a recent study shows that PrPsc may be transported across intestinal epithelium complexed with ferritin, we will attempt to isolate ferritin-containing vesicles using auto-MACS. In collaboration with Professor Peters (Amsterdam) we will use immuno-electron microscopy to examine vesicles for PrPsc and ferritin. 2. How do DC acquire PrPsc from the gut? a) Do DC capture PrPsc directly from the ileum? Rats or mice will be orally-dosed with SAF. Cryosections from the terminal ileum will be labelled for DC markers (CD11b/c, MHC class II, CD103, CD172) and for PrPsc or ferritin. If results are promising, EM-immunolabelling will be carried out as above. b) Can PrPsc be detected in intestinal or migrating DC? Intestinal cryosections will be examined as above. Mice or rats will be treated with FLT-3 ligand to increase DC numbers in the gut and fed as above. Intestinal DC will be isolated enzymatically. Lymph DC from PrPsc-fed rats, and intestinal DC, will be examined for ferritin and PrPsc by immuno-labelling of permeabilised cells, FACS, and immuno-EM. c) Can intestinal DC acquire PrPsc in vitro? Intestinal DC will be isolated from FLT-3 ligand-treated animals, or generated from bone marrow with FLT-3 ligand or GM-CSF and IL-4, and incubated with SAF or brain homogenates. Ferritin or anti-ferritin antibodies will be used to attempt to inhibit uptake. 3. Does intestinal inflammation modulate PrPsc uptake and transport? Given that there are reports that inflammatory stimulation can increase susceptibility to scrapie, we will use intestinal infection with Salmonella typhimurium to induce inflammation, and compare transport and uptake of PrPsc as described above. Overall these experiments will give novel insights into peripheral TSE pathogenesis, and may inform future therapeutic strategies.

Summary

Transmissible Spongiform Encephalopathies (TSEs) are prolonged degenerative diseases of the central nervous system (CNS) that affect both humans and animals. Examples of TSEs include scrapie in sheep; bovine spongiform encephalopathy (BSE) in cattle and variant Creutzfeldt-Jacob disease (vCJD) in humans. TSE agents (prions, which are thought to be an abnormal form of a normal host protein) may be spread by several different mechanisms including, importantly for BSE and vCJD, via the consumption of contaminated meat products. There is much uncertainty about many stages in the development of CNS disease. After crossing the intestinal epithelial cells (themselves a tough barrier to proteins) the TSE agent does not appear to migrate directly to the CNS but accumulates and replicates on cells in the lymph nodes (glands) that drain lymph from the intestine. We do not know how the TSE agent crosses the epithelium or is transported to the particular cells in the nodes on which it replicates. We have several possibilities for both stages, and this project will use novel models and state-of-the-art immunological techniques to study early interactions between host immune cells and prion proteins. More specifically, we aim to determine the precise mechanisms by which TSE agents are transported across the intestine and on to the regional lymph nodes. The elucidation of these mechanisms will contribute to our understanding of the complex and relatively poorly understood interactions between TSE agent and host, and may lead to intervention therapies to help control the disease in man and animals. The work will make use of unique techniques that our group has developed and will benefit from the wide expertise available at the University of Oxford, and from that of other project partners undertaking TSE research in the UK and Europe. By addressing questions relating to the initial translocation of prion proteins and the interaction with immune cells, and by establishing collaborations between other groups with complementary expertise, the proposed project addresses the remit of the Agri-Food Committee for studying the control of food-borne pathogens, and in particular the priority areas of understanding the biology of transmissible spongiform encephalopathies.

Committee	Closed Committee - Animal Sciences (AS)
Research Topics	Animal Health, Immunology, TSEs (transmissible spongiform encephalopathies)
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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