Journal of General Virology

Migrating intestinal dendritic cells transport PrP^Sc from the gut

Fang-Ping Huang,¹† Christine F. Farquhar,² Neil A. Mabbott,² Moira E. Bruce² and G. Gordon MacPherson¹

¹ Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, UK
² Institute for Animal Health, Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh EH9 3JF, UK

Bovine spongiform encephalopathy, variant Creutzfeldt–Jakob disease (vCJD) and possibly also sheep scrapie are orally acquired transmissible spongiform encephalopathies (TSEs). TSE agents usually replicate in lymphoid tissues before they spread into the central nervous system. In mouse TSE models PrP^c-expressing follicular dendritic cells (FDCs) resident in lymphoid germinal centres are essential for replication, and in their absence neuroinvasion is impaired. Disease associated forms of PrP (PrP^Sc), a biochemical marker for TSE infection, also accumulate on FDCs in the lymphoid tissues of patients with vCJD and sheep with natural scrapie. TSE transport mechanisms between gut lumen and germinal centres are unknown. Migratory bone marrow-derived dendritic cells (DCs), entering the intestinal wall from blood, sample antigens from the gut lumen and carry them to mesenteric lymph nodes. Here we show that DCs acquire PrP^Sc in vitro, and transport intestinally administered PrP^Sc directly into lymphoid tissues in vivo. These studies suggest that DCs are a cellular bridge between the gut lumen and the lymphoid TSE replicative machinery.

The transmissible spongiform encephalopathies (TSEs), or 'prion' diseases, are neurodegenerative disorders which include Creutzfeldt–Jakob disease (CJD) and kuru in humans, bovine spongiform encephalopathy (BSE), transmissible mink encephalopathy, chronic wasting disease (CWD) in mule deer and elk, and scrapie in sheep and goats. Replication of the infectious TSE agent depends critically on the host prion protein (PrP^c), which accumulates as an abnormal, detergent-insoluble, relatively proteinase-resistant isoform, PrP^Sc, in diseased tissues (Bolton et al., 1982; Bueler et al., 1992). The precise nature of the infectious agent is uncertain, but PrP^Sc co-purifies with infectivity and is considered to be a major component (Farquhar et al., 1998; Prusiner et al., 1982).

The consumption of BSE-contaminated meat products is the most likely cause of variant (v) CJD in humans (Bruce et al., 1997; Hill et al., 1997), and ingestion has been implicated in the transmission of other TSE diseases. The timing of events in TSE pathogenesis, as determined by tracking PrP^Sc accumulation, varies depending on agent strain, host genotype and the route of infection (Farquhar et al., 1994, 1996). However, soon after experimental intragastric or oral exposure of rodents with scrapie, infectivity and PrP^Sc accumulate first in Peyer's patches, gut-associated lymphoid tissues and ganglia of the enteric nervous system (Beekes & McBride, 2000; Kimberlin & Walker, 1989), long before their detection in the central nervous system (CNS). Likewise, following experimental oral exposure of mule deer fawns with CWD, PrP^Sc is also detected first in lymphoid tissues draining the gastro-intestinal tract (Sigurdson et al., 1999). How and when sheep become infected with natural scrapie is not known, but the detection of PrP^Sc in Peyer's patches and gut-associated lymphoid tissues (Andréoletti et al., 2000; Heggebø et al., 2000) prior to detection within the CNS (van Keulen et al., 1999) suggests that this disease is also acquired orally.

Early PrP^Sc accumulation takes place on follicular dendritic cells (FDCs) within germinal centres in lymphoid tissues of patients with vCJD (Hill et al., 1999), sheep with natural scrapie (van Keulen et al., 1996) and rodents inoculated with scrapie by peripheral routes (Brown et al., 1999; Mabbott et al., 2000b; McBride et al., 1992). In mouse scrapie models, mature FDCs are critical for scrapie replication and PrP^Sc accumulation in lymphoid tissues, and in their absence neuroinvasion following peripheral challenge is significantly impaired (Brown et al., 1999; Mabbott et al., 2000a, b; Montrasio et al., 2000).

The transport mechanisms by which TSE agents reach the germinal centres from the gut lumen are not known. Migratory bone marrow-derived dendritic cells (DCs) are centrally involved in transport of proteins both within Peyer's patches and on into mesenteric lymph nodes (Banchereau et al., 2000). These cells are a distinct lineage from FDCs, which are tissue-resident and are not considered to be of haemopoietic origin (Endres et al., 1999; Kapasi et al., 1993). DCs enter the intestinal wall from the bloodstream, sample antigens from the gut lumen, and then migrate via lymph to mesenteric lymph nodes (Liu & MacPherson, 1993). These observations suggested to us that migrating DCs might provide a cellular bridge between the gut lumen and the secondary lymphoid tissues in which TSE agents replicate.

To test the hypothesis that DCs can acquire TSE agents, we first investigated the uptake of PrP^Sc by DCs in vitro. Rat bone marrow-derived DCs (BMDCs) were prepared as previously described (Huang et al., 2000) and cultured at 1x10⁶ cells/ml in RPMI 1640 medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 40 ng/ml murine GM-CSF and 1500 U/ml rat IL-4. Medium and cytokines were replaced every 72 h, and by day 10 to 12 of culture 90 % of cells had characteristic DC morphology and expressed MHC class II, B7 and CD11c. Scrapie-associated fibrils (SAF), highly infective fibrillar aggregates of PrP^Sc, were prepared from the brains of mice terminally affected with the mouse-passaged ME7 strain as previously described (Hope et al., 1986), sonicated in PBS, and a suspension equivalent to 10 mg infected brain tissue (wet weight) was added to each BMDC culture for the times indicated. Following incubation, culture medium was aspirated and cells lysed with 0.1 % N-laurylsarcosine. Lysates were treated in the presence or absence of 50 µg proteinase K for 30 min at 37 °C, subjected to electrophoresis through 12 % SDS–polyacrylamide gels (Bio-Rad) and proteins transferred to polyvinylidine difluoride membranes (Bio-Rad). PrP was detected with rabbit polyclonal antiserum 1B3 specific for PrP (Farquhar et al., 1989) and bound antibody visualized by enhanced chemiluminescence (Amersham).

Detergent-insoluble, relatively proteinase K-resistant PrP^Sc accumulations were detected in BMDC lysates within 3 h of culture with SAF, peaking at around 6 h of culture (Fig. 1). No PrP^Sc accumulations were detected at any time in lysates from BMDCs treated with PBS (Fig. 1) or SAF equivalent preparation from normal uninfected brain (data not shown). Neither was uptake identified when B or T lymphocytes were incubated with SAF in vitro (data not shown). After antigen acquisition by DCs, a large proportion is degraded in endosomal/lysosomal compartments for presentation to T lymphocytes on MHC class II (Banchereau et al., 2000). We have shown, however, that DCs, unlike macrophages, can retain some protein antigens in native, non-degraded form for at least 36 h (Wykes et al., 1998). After 24 h of culture of BMDCs with SAF, the level of PrP^Sc detected had declined moderately (Fig. 1), implying that BMDCs acquire PrP^Sc, some of which is subsequently catabolized but a considerable proportion of which is retained intact. An increased proteinase K-sensitive PrP signal was also detected after 24 h incubation (Fig. 1), which may also represent the break-up of SAF aggregates within the DC and the revealing of more epitopes as the PrP is digested.

Fig. 1. BMDCs acquire PrP^Sc following in vitro culture with SAF. BMDCs (1x10⁶ cells) were cultured in the absence (BMDC alone) or presence of SAF (equivalent to 10 mg infected brain tissue) for the times indicated. Immunoblots show the accumulation of detergent-insoluble, relatively proteinase K-resistant PrP^Sc within BMDC lysates. Treatment of lysates in the presence (+) or absence (–) of proteinase K (PK) is indicated. SAF (equivalent to 10 mg infected brain tissue) was incubated in medium alone as a control. Following PK treatment, a typical three-band pattern was observed between molecular mass values of 20 and 30 kDa, representing unglycosylated, monoglycosylated and diglycosylated isomers of PrP (in order of increasing molecular mass). SAF equivalent to 50 µg infected brain tissue and/or BMDCs equivalent to 10⁴ cells were loaded per lane.

We next sought to demonstrate whether DCs can acquire and transport PrP^Sc in vivo to mesenteric lymph nodes after delivery of SAF by intra-intestinal injection. PVG (RT1^c) rats bred and maintained under specific-pathogen-free conditions were mesenteric lymphadenectomized as previously described (Liu et al., 1998; Pugh et al., 1983). Six weeks later, when the afferent lymphatics (lacteals) draining the intestine had joined the efferent mesenteric lymphatics, SAF (equivalent to 10 mg infected brain tissue per rat) or PBS (as a control) was injected into the jejunum. Cells that would normally have been trapped in the mesenteric lymph nodes in intact animals were then collected by thoracic duct cannulation over 8 to 16 h. Lymph DCs (>90 % pure) were isolated by a combined density centrifugation and magnetic antibody cell sorting protocol as previously described (Huang et al., 2000), while T and B lymphocytes (>99 % pure) were isolated by magnetic antibody cell sorting alone.

Immunocytochemical analysis showed that after intra-intestinal SAF exposure, large amounts of PrP were present as conspicuous cytoplasmic inclusions in 4 to 5 % of lymph DCs (Fig. 2a). No such deposits were identified within T or B lymphocyte populations (Fig. 2b, c, respectively). Much weaker PrP staining was seen around lymph DCs (Fig. 2d) and B lymphocytes (data not shown) from PBS-treated controls, indicative of membrane-associated endogenously expressed rat PrP^c. Immunoblot analysis confirmed the presence of detergent-insoluble, relatively proteinase K-resistant PrP^Sc in lymph DC lysates selected from SAF-injected rats (Fig. 3, lane 4). However, the characteristic molecular mass shift in the three-band PrP^Sc signature after proteinase K digestion was not seen (Fig. 3, lane 6; Hope et al., 1986). The multiple bands detected may indicate a difference in PrP processing either in the intestine or within DCs. No PrP was detected in lysates of T or B lymphocytes selected from SAF-injected rats (Fig. 3, lanes 1, 2, respectively). Neither was PrP detected in lysates from DCs, T or B lymphocytes from mesenteric lymphadenectomized rats treated with PBS or an SAF equivalent preparation from normal uninfected brain as a control (data not shown).

Fig. 2. DCs transport intestinally injected SAF to mesenteric nodes via lymph. Lymph was collected 8 to 16 h after intestinal injection of SAF and strong cytoplasmic inclusions of PrP were detected by immunocytochemistry in a small proportion of DCs (a) but not B (b) or T (c) lymphocytes in the thoracic duct pseudo-lymph of mesenteric lymphadenectomized rats. Only endogenous PrP was detected in DCs from PBS-injected control animals (d). Magnification x1000. In all panels, PrP was detected using the PrP-specific polyclonal antiserum 1B3 (Farquhar et al., 1989).

Fig. 3. Immunoblot analysis of pooled cell lysates (1x10⁶ cells per lane) from SAF-treated rats confirmed the presence of PrP^Sc in lymph DCs (lane 4) but not in T or B lymphocytes. SAF equivalent to 2 or 4 µg of infected brain tissue was loaded in lanes 5 and 6, respectively. Treatment of samples in the presence (+) or absence (–) of proteinase K (PK) before electrophoresis is indicated. PrP dimers are seen at approximately 60 kDa in lanes 3 and 4.

We next attempted to estimate scrapie infectivity levels in cell populations by animal bioassays. Pooled cell lysates were prepared from DCs, T lymphocytes or B lymphocytes from SAF-treated rats and injected intracerebrally into groups of 12 assay mice (approximately 2.5x10⁵ cells per mouse). Despite the detection of PrP^Sc in lymph DCs by immunoblot (Fig. 3) and immunocytochemical (Fig. 2a) analysis, infectivity levels were below the level detectable by bioassay. This most probably reflects the sensitivity of the assay given that only a small number of cells were available for injection per assay mouse. Of those, only a small subset of the DCs had acquired PrP^Sc (approximately 1x10⁴ cells per mouse). This small number of cells represents the maximum we could collect; each assay mouse (usually 12 per group) receiving DCs from two cannulated rats. As infectivity bioassays are more sensitive than PrP immunoblots, the failure to detect scrapie infectivity in DC lysates despite positive detection of PrP^Sc by immunoblot is most likely because greater numbers of cells were analysed in the immunoblot study (1x10⁶ cells per lane). All other cell populations and concentrated cell-free lymph plasma (x75 using Microcon concentrators, Amicon) from SAF-injected rats were also negative.

In this study we show that DCs can acquire PrP^Sc in vitro and that a small sub-population of migrating DCs can take up and transport PrP^Sc from the gut lumen through the lymphatics to lymphoid tissue. We have also shown that the uptake of PrP^Sc from the gut lumen is restricted to DCs, as no PrP^Sc was detected in other lymph cells or cell-free lymph plasma. The small numbers of cells involved, perhaps in addition to partial intracellular degradative mechanisms, may explain the longer incubation periods, and reduced efficiency of infection, following oral exposure in comparison with other peripheral routes. Within lymphoid tissue FDCs play a critical role in the amplification of TSE infectivity outside the CNS (Brown et al., 1999; Mabbott et al., 2000a, b; Montrasio et al., 2000). Our findings suggest that following infection via the gastro-intestinal tract, DCs act as a cellular bridge between the gut lumen and the lymphoid TSE replicative machinery.

Within the intestine, DCs have been described in the lamina propria (Maric et al., 1996), and in Peyer's patches where they form a dense layer of cells in the subepithelial dome, just beneath the follicle-associated epithelium and in close contact with M cells (Kelsall & Strober, 1996). Following oral challenge of rodents with scrapie, heavy pathological PrP accumulations are detected within cells of the follicle-associated epithelium with morphology consistent with M cells (Beekes & McBride, 2000), which have the potential to transcytose infectivity in vitro (Heppner et al., 2001). However, further studies are necessary to determine whether DCs acquire PrP^Sc after it has been internalized by M cells, or by direct uptake across the mucosal epithelium as recently shown for the transport of apoptotic intestinal epithelial cells (Huang et al., 2000) or bacteria (Rescigno et al., 2001). In addition, our studies do not exclude the possibility of direct uptake into PrP-expressing enteric nerves (Shmakov et al., 2000).

The detection of infectivity within lymphoid tissues (Bruce et al., 2001) and PrP accumulation upon FDCs of patients with vCJD (Hill et al., 1999) suggest that this disease shares a similar pathogenesis to rodent TSE models. Immunomodulation alters susceptibility to TSEs in rodent models (Mabbott et al., 1998) and gut inflammation markedly stimulates DC traffic from the intestine (MacPherson et al., 1995). Our studies suggest that it will be important to investigate where TSEs are taken up in the human gastro-intestinal tract, and whether this can be exacerbated by inflammatory conditions that stimulate DC migration.

We thank Chris Jenkins (Sir William Dunn School of Pathology, Oxford, UK) and staff in the animal facility at the Institute for Animal Health (Edinburgh, UK) for excellent technical support. This work was supported by funding from the Biotechnology and Biological Sciences Research Council (grant no. BS308133).

† Present address: Department of Pathology, Queen Mary Hospital, University of Hong Kong, Pokfulam Road, Hong Kong, China.

Andréoletti, O., Berthon, P., Marc, D., Sarradin, P., Grosclaude, J., van Keulen, L., Schelcher, F., Elsen, J.-M. & Lantier, F. (2000). Early accumulation of PrP^Sc in gut-associated lymphoid and nervous tissues of susceptible sheep from a Romanov flock with natural scrapie. Journal of General Virology 81, 3115–3126.

Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y. J., Pulendran, B. & Palucka, K. (2000). Immunobiology of dendritic cells. Annual Review of Immunology 18, 767–811.

Beekes, M. & McBride, P. A. (2000). Early accumulation of pathological PrP in the enteric nervous system and gut-associated lymphoid tissue of hamsters orally infected with scrapie. Neuroscience Letters 278, 181–184.

Bolton, D. C., McKinley, M. P. & Prusiner, S. B. (1982). Identification of a protein that purifies with the scrapie prion. Science 218, 1309.

Brown, K. L., Stewart, K., Ritchie, D., Mabbott, N. A., Williams, A., Fraser, H., Morrison, W. I. & Bruce, M. E. (1999). Scrapie replication in lymphoid tissues depends on PrP-expressing follicular dendritic cells. Nature Medicine 5, 1308–1312.

Bruce, M. E., Will, R. G., Ironside, J. W., McConnell, I., Drummond, D., Suttie, A., McCardle, L., Chree, A., Hope, J., Birkett, C., Cousens, S., Fraser, H. & Bostock, C. J. (1997). Transmissions to mice indicate that 'new variant' CJD is caused by the BSE agent. Nature 389, 498–501.

Bruce, M. E., McConnell, I., Will, R. G. & Ironside, J. W. (2001). Detection of variant Creutzfeldt–Jakob disease (vCJD) infectivity in extraneural tissues. Lancet 357, 208–209.

Bueler, H., Fischer, M., Lang, Y., Bluethmann, H., Lipp, H.-P., DeArmond, S. J., Prusiner, S. B., Aguet, M. & Weissmann, C. (1992). Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 356, 577–582.

Endres, R., Alimzhanov, M. B., Plitz, T., Futterer, A., Kosco-Vilbois, M. H., Nedospasov, S. A., Rajewsky, K. & Pfeffer, K. (1999). Mature follicular dendritic cell networks depend on expression of lymphotoxin receptor by radioresistant stromal cells and of lymphotoxin and tumour necrosis factor by B cells. Journal of Experimental Medicine 189, 159–168.

Farquhar, C. F., Somerville, R. A. & Ritchie, L. A. (1989). Post-mortem immunodiagnosis of scrapie and bovine spongiform encephalopathy. Journal of Virological Methods 24, 215–222.

Farquhar, C. F., Dornan, J., Somerville, R. A., Tunstall, A. M. & Hope, J. (1994). Effect of Sinc genotype, agent isolate and route of infection on the accumulation of protease-resistant PrP in non-central nervous system tissues during the development of murine scrapie. Journal of General Virology 75, 495–504.

Farquhar, C. F., Dornan, J., Moore, R. C., Somerville, R. A., Tunstall, A. M. & Hope, J. (1996). Protease-resistant PrP deposition in brain and non-central nervous system tissues of a murine model of bovine spongiform encephalopathy. Journal of General Virology 77, 1941–1946.

Farquhar, C. F., Somerville, R. A. & Bruce, M. E. (1998). Straining the prion hypothesis. Nature 391, 345–346.

Heggebø, R., Press, C. McL., Gunnes, G., Lie, K. I., Tranulis, M. A., Ulvund, M., Groschup, M. H. & Landsverk, T. (2000). Distribution of prion protein in the ileal Peyer's patch of scrapie-free lambs and lambs naturally and experimentally exposed to the scrapie agent. Journal of General Virology 81, 2327–2337.

Heppner, F. L., Christ, A. D., Klein, M. A., Prinz, M., Fried, M., Krahenbuhl, J.-P. & Aguzzi, A. (2001). Transepithelial prion transport by M cells. Nature Medicine 7, 976–977.

Hill, A. F., Desbruslais, M., Joiner, S., Sidle, K. C. L., Gowland, I. & Collinge, J. (1997). The same prion strain causes vCJD and BSE. Nature 389, 448–450.

Hill, A. F., Butterworth, R. J., Joiner, S., Jackson, G., Rossor, M. N., Thomas, D. J., Frosh, A., Tolley, N., Bell, J. E., Spencer, M., King, A., Al-Sarraj, S., Ironside, J. W., Lantos, P. L. & Collinge, J. (1999). Investigation of variant Creutzfeldt–Jakob disease and other prion diseases with tonsil biopsy samples. Lancet 353, 183–189.

Hope, J., Morton, L. J. D., Farquhar, C. F., Multhaup, G., Beyreuther, K. & Kimberlin, R. H. (1986). The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein (PrP). EMBO Journal 5, 2591–2597.

Huang, F.-P., Platt, N., Wykes, M., Major, J. R., Powell, T. J., Jenkins, C. D. & MacPherson, G. G. (2000). A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes. Journal of Experimental Medicine 191, 435–443.

Kapasi, Z. F., Burton, G. F., Schultz, L. D., Tew, J. G. & Szakal, A. K. (1993). Induction of functional follicular dendritic cell development in severe combined immunodeficiency mice. Journal of Immunology 150, 2648–2658.

Kelsall, B. L. & Strober, W. (1996). Distinct populations of dendritic cells are present in the subepthelial dome and T cell regions of the murine Peyer's patch. Journal of Experimental Medicine 183, 237–247.

Kimberlin, R. H. & Walker, C. A. (1989). Pathogenesis of scrapie in mice after intragastric infection. Virus Research 12, 213–220.

Liu, M. & MacPherson, G. G. (1993). Antigen acquisition by dendritic cells: intestinal dendritic cells acquire antigen administered orally and can prime naive T cells in vivo. Journal of Experimental Medicine 177, 1299–1307.

Liu, L., Zhang, M., Jenkins, C. & MacPherson, G. G. (1998). Dendritic cell heterogeneity in vivo: two functionally different dendritic cell populations in rat intestinal lymph can be distinguished by CD4 expression. Journal of Immunology 161, 1146–1155.

Mabbott, N. A., Farquhar, C. F., Brown, K. L. & Bruce, M. E. (1998). Involvement of the immune system in TSE pathogenesis. Immunology Today 19, 201–203.

Mabbott, N. A., Mackay, F., Minns, F. & Bruce, M. E. (2000a). Temporary inactivation of follicular dendritic cells delays neuroinvasion of scrapie. Nature Medicine 6, 719–720.

Mabbott, N. A., Williams, A., Farquhar, C. F., Pasparakis, M., Kollias, G. & Bruce, M. E. (2000b). Tumor necrosis factor-alpha-deficient, but not interleukin-6-deficient, mice resist peripheral infection with scrapie. Journal of Virology 74, 3338–3344.

McBride, P., Eikelenboom, P., Kraal, G., Fraser, H. & Bruce, M. E. (1992). PrP protein is associated with follicular dendritic cells of spleens and lymph nodes in uninfected and scrapie-infected mice. Journal of Pathology 168, 413–418.

MacPherson, G. G., Jenkins, C. D., Stein, M. J. & Edwards, C. (1995). Endotoxin-mediated dendritic cell release from the intestine. Journal of Immunology 154, 1317–1322.

Maric, I., Holt, P. G., Perdue, M. H. & Bienstock, J. (1996). Class II MHC antigen (Ia)-bearing dendritic cells in the epithelium of the rat intestine. Journal of Immunology 156, 1408–1414.

Montrasio, F., Frigg, R., Glatzel, M., Klein, M. A., Mackay, F., Aguzzi, A. & Weissmann, C. (2000). Impaired prion replication in spleens of mice lacking functional follicular dendritic cells. Science 288, 1257–1259.

Prusiner, S. B., Bolton, D. C., Groth, D. F., Bowman, K. A., Cochran, S. P. & McKinley, M. P. (1982). Further purification and characterisation of scrapie prions. Biochemistry 21, 6942–6950.

Pugh, C. W., MacPherson, G. G. & Steer, H. W. (1983). Characterization of non-lymphoid cells derived from rat peripheral lymph. Journal of Experimental Medicine 157, 1758–1779.

Rescigno, M., Urbano, M., Valzasina, B., Francolini, M., Rotta, G., Bonasio, R., Granucci, F., Kraehenbuhl, J.-P. & Ricciardi-Castagnoli, P. (2001). Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nature Immunology 2, 361–367.

Shmakov, A. N., McLennan, N. F., McBride, P., Farquhar, C. F., Bode, J., Rennison, K. A. & Ghosh, S. (2000). Cellular prion protein is expressed in the human enteric nervous system. Nature Medicine 6, 840–841.

Sigurdson, C. J., Williams, E. S., Miller, M. W., Spraker, T. R., O'Rourke, K. I. & Hoover, E. A. (1999). Oral transmission and early lymphoid tropism of chronic wasting disease PrP^res in mule deer fawns (Odocoileus hemionus). Journal of General Virology 80, 2757–2764.

van Keulen, L. J. M., Schreuder, B. E. C., Meloen, R. H., Mooij-Harkes, G., Vromans, M. E. W. & Langeveld, J. P. M. (1996). Immunohistological detection of prion protein in lymphoid tissues of sheep with natural scrapie. Journal of Clinical Microbiology 34, 1228–1231.

van Keulen, L. J. M., Schreuder, B. E. G., Vromans, M. E. W., Langeveld, J. P. M. & Smits, M. A. (1999). Scrapie-associated prion protein in the gastro-intestinal tract of sheep with scrapie. Journal of Comparative Pathology 121, 55–63.

Wykes, M., Pombo, A., Jenkins, C. & MacPherson, G. G. (1998). Dendritic cells interact directly with naive B lymphocytes to transfer antigen and initiate class switching in a primary T-dependent response. Journal of Immunology 161, 1313–1319.

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