1 CEA, DSV/DRM, 18 route du Panorama, BP6, 92265 Fontenay aux Roses Cedex, France
2 OTR3 Sarl, 33 rue Pierre Brossolette, 94000 Créteil, France
3 Laboratoire CRETT, CNRS FRE2412, Université Paris XII-Val de Marne, avenue du Général de Gaulle, 94010 Créteil Cedex, France
Correspondence
Corinne Ida Lasmézas
lasmezas{at}cea.fr
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Published ahead of print on 11 June 2003 as DOI 10.1099/vir.0.19073-0
Present address: Ecole Nationale Vétérinaire d'Alfort, 7 avenue du Général de Gaulle, 94704 Maisons-Alfort, Cedex, France.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The appearance of variant CJD (vCJD) in Europe has been linked to the contamination of the human food chain by BSE-infected cows. Because the extent of the vCJD epidemic in forthcoming years is still uncertain (Ghani et al., 2000, 2002
), and given that no effective treatments are presently available for TSEs, the development of new therapeutic strategies is of crucial importance. One class of molecules that has shown significant efficiency in the treatment of prion diseases is the class of sulfated polyanions, such as dextran sulfate 500 (DS500), pentosane polysulfate and suramin (Farquhar & Dickinson, 1986
; Kimberlin & Walker, 1986
; Ladogana et al., 1992
; Caughey & Raymond, 1993
; Beringue et al., 2000
; Gilch et al., 2001
). However, their use is limited by their toxicity, restricted efficiency and narrow window of intervention following infection (Diringer & Ehlers, 1991
; Ladogana et al., 1992
).
Sulfated polyanions found in biological systems are heparan sulfate proteoglycans. These molecules are either secreted or membrane-bound and consist of a protein core to which variably sulfated glycosaminoglycan (GAG) chains are attached. During their biosynthesis, specific modifications of the GAG chains can generate a vast diversity of molecules, each with specific functions, ranging from mechanical support to regulation of adhesion, motility and proliferation (reviewed by Bernfield et al., 1999; Turnbull et al., 2001
). In addition to their diverse biological functions, heparan sulfates may be good candidates for therapeutic intervention in prion diseases, since they can bind to PrP (Gabizon et al., 1993
; Brimacombe et al., 1999
; Gonzalez-Iglesias et al., 2002
; Warner et al., 2002
), play an active role in the PrP endocytic pathway (Shyng et al., 1995
) and act as co-receptors for the binding of PrP to the cellular receptor LRP/LR (Hundt et al., 2001
). For these reasons, various heparan sulfate mimetics (HMs) (see Fig. 1
for structural information) containing specific structure modifications in their GAG domains, developed initially for their interesting properties in wound healing (Desgranges et al., 1999
), were tested in vitro for their activity as anti-prion drugs. Two molecules designated HM2602 and HM5004 were chosen and analysed in vivo. We report in this study that HM2602 can (i) inhibit PrPres accumulation in scrapie-infected GT1 (ScGT1) cells in a dose-dependent manner, (ii) exert a long-term effect (PrPres does not reappear up to 50 days following removal of the drug), (iii) hamper PrPres formation in the spleens of mice infected intraperitoneally with either scrapie or BSE and (iv) prolong significantly the survival of 263K scrapie-infected hamsters. In contrast, and although it was efficient in vitro, HM5004 displayed no anti-prion activity in vivo in any of the experimental models used. Structure analysis provides some clues that the hydrophobicity of the molecule may be important for its anti-prion activity. Since HMs are amenable to various chemical modifications, this study paves the way for the development of this family of compounds as anti-TSE drugs.
|
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture.
ScGT1-7 cells (GT1 hypothalamic neuronal cells chronically infected with the Chandler isolate of scrapie), generously provided by S. Lehmann (Montpellier, France), were cultured as described previously (Mange et al., 2000), except that DMEM was replaced by Opti-MEM.
Treatment of cells.
HMs and control molecules were added at various concentrations to the medium of cells seeded at a density of 10 % and cultures were incubated with the molecules for 4 days.
Analysis of cellular PrPres levels.
ScGT1-7 cells from a 75 cm2 flask were collected and lysed in a buffer containing 50 mM Tris/HCl (pH 7·4), 0·5 % sodium deoxycholate and 0·5 % Triton X-100 at 4 °C for 10 min. Samples were centrifuged at 10 000 g for 1 min and the supernatant was collected. Following this step, protein concentrations were measured using a BCA Protein Assay kit (Pierce) and all samples were normalized to equal protein concentrations, treated with proteinase K (PK) (10 µg mg-1 total protein) for 60 min at 37 °C and centrifuged at 20 000 g for 45 min at 4 °C. The pellet was resuspended in 50 µl Laemmli's sample buffer and boiled for 5 min. Proteins were separated by SDS-PAGE on a 12 % polyacrylamide gel, immunoblotted with a PrP-specific monoclonal antibody, SAF84 (kindly provided by J. Grassi, CEA, Saclay, France), and revealed using enhanced chemiluminescence (Amersham).
Kinetics of PrPres elimination.
For time-course experiments, ScGT1-7 cells were seeded to 10 % confluency in five 75 cm2 flasks. One flask was left untreated, while the other flasks were treated with 10 µg HM2602 ml-1, each flask at intervals of 24 h to obtain different durations of treatment when harvesting all flasks on the same day at the end of the experiment. The cells in each flask were lysed 96 h after the start of the treatment of the first flask, normalized for equal protein concentration and subjected to the PrPres purification protocol described above.
Analysis of spleens.
Female, 8-week-old, C57BL/6 mice (Centre d'élevage René Janvier, Le Genest-St-Isle, France) were injected intraperitoneally with 100 µl 2 % (wt/vol) brain homogenates of scrapie- (C506M3) or BSE-infected (6PB1) mice (Lasmézas et al., 1996) diluted in 5 % glucose. Following inoculation, mice were treated intraperitoneally twice a week with 25 mg HMs kg-1, diluted in 5 % glucose, for a total period of 30 days, the first treatment being given 24 h post-inoculation. Treatment with DS500, diluted in 5 % glucose, was performed with the same time-schedule and at the same dose. Control mice were treated with a 5 % glucose solution. To determine the levels of PrPres in the spleens of these mice, animals were sacrificed at 30 days post-inoculation and spleens were collected and homogenized at 10 % (w/v) using a ribolyser (Bio-Rad) in 5 % sterile glucose. PrPres was purified by centrifugation in the presence of detergents, after PK digestion, according to the scrapie-associated fibril (SAF) protocol reported previously (Lasmézas et al., 1997
). The level of PrPres in the spleen of each animal was revealed by Western blot using the PrP-specific antibodies JB007 (see Fig. 5
) and SAF60 (see Fig. 6
) (kindly provided by J. Grassi) and quantified by ELISA, according to a protocol described previously (Grassi et al., 2001
), but modified to detect murine PrP, in which the antibodies SAF53 and 11C6 were used as the capture and detection antibodies, respectively.
|
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Long-term inhibition of PrPres formation by HMs
To assess further the extent of the in vitro effect of HM2602 and HM5004, we verified whether the PrPres-free status of the cells achieved after 96 h of treatment could be maintained. For this purpose, duplicates of ScGT1-7 cells were treated with HM2602 at a concentration of 10 µg ml-1 for 4 days, then transferred into a new flask and the same treatment was repeated for an additional 4 days. This second exposure of the cells to HM2602 was performed to eliminate any contamination of the new flask with residual PrPres from the transferred cells. Cell lysates were then collected at various time-points up to day 50, day 1 corresponding to the first day after removal of HM2602. The PrPres signal did not reappear for the whole 50 day period post-treatment (corresponding approximately to 12 passages, Fig. 4). Thus, two consecutive 4 day treatments with HM2602 result in a long-term inhibition of PrPres formation. Exposure of the cells to the polyene antibiotic derivative MS-96130, chosen as a negative control as it exhibited no anti-prion activity in vivo (data not shown), did not cause a significant reduction in the formation of PrPres (Fig. 4
), demonstrating the specificity of the treatment by HM2602.
HMs hamper the development and progression of scrapie and BSE infection in animals
These encouraging results obtained in vitro prompted us to investigate whether HMs were also effective in animals. For this purpose, we used a rapid animal model developed recently for the screening of anti-prion molecules (Deslys et al., 1998). This model is based on the fact that after peripheral infection, TSE agents are directed to the spleen where they replicate with a concomitant accumulation of PrPres. Thus, to assess whether a drug interferes with prion replication, the level of PrPres in the spleens of mice treated with the compound is measured at the time that a plateau would be reached normally in untreated mice. Based on this principle, HM2602 and HM5004 were administered twice a week at a dose of 25 mg kg-1 to a group of 10 scrapie-infected mice for a period of 30 days. Following treatment, the spleen of each animal was collected and the levels of PrPres in this organ were determined (Fig. 5
). Our results showed that compared to non-treated animals, biweekly injections of HM2602 reduced significantly the accumulation of PrPres in the spleen of infected mice (Fig. 5b
), suggesting that HM2602 can slow the pace of infection. Intriguingly, although HM2602 and HM5004 were both efficient in our in vitro model, HM5004 did not exhibit any effect when used at the same dose in scrapie-infected mice (Fig. 5c
). The response of the mice to treatment was quantified using ELISA (Fig. 5a
), which showed a reduction in PrPres levels of about 70 % after exposure to HM2602 (P<0·005, Student's t-test).
As it has been shown that BSE is sometimes more resistant than scrapie to therapeutic intervention (Adjou et al., 1996), we next tested the therapeutic potential of HM2602 in a mouse BSE infection model. Moreover, because the effects of DS500 and HM2602 were indistinguishable in ScGT1-7 cells (Fig. 2c
), DS500 was tested also in this model to determine whether it had the same therapeutic potential in vivo. HM2602 exhibited potent anti-prion properties in BSE-infected mice; only faint PrPres signals were detected in the spleens of animals treated with HM2602 compared to non-treated controls (Fig. 6
). Quantification of the Western blot signals showed a 95 % reduction in the PrPres signal with HM2602 as compared to 68 % with DS500. Furthermore, we observed 10 % mortality in mice treated with DS500, demonstrating that the threshold of toxicity was attained with this molecule but not with HM2602.
We next set out to determine whether the hindrance of PrPres accumulation in the lymphoreticular system would affect the CNS and translate into a delay in the progression of disease and occurrence of clinical signs. Thus, hamsters were inoculated with the neuroinvasive 263K strain of scrapie and treated with 25 mg HM2602 or HM5004 kg-1 body weight. Treatments were performed once a week until time of death. We observed that the survival time in animals treated with HM2602 was prolonged by 24 days (196±4 days for treated animals compared to 172±2 days for control animals) (Fig. 7). Interestingly, HM5004 did not prolong significantly the survival time of infected hamsters (mean survival time 174±2 days), which is in agreement with the results obtained in the spleens of infected mice.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two molecules, HM2602 and HM5004, were examined for their capacity to interfere with PrPres formation in ScGT1-7 cells. This type of assay, where cells chronically infected with prions are used as a screening method to identify potential therapeutic molecules, has been exploited on several occasions (Caughey & Raymond, 1993; Mange et al., 2000
; Korth et al., 2001
; Peretz et al., 2001
; May et al., 2003
). It is widely accepted that a decrease in the levels of PrPres in cultures is indicative of a hindrance to prion replication (Supattapone et al., 2001
). In ScGT1-7 cells, we found that both HMs were efficient at blocking prion replication (Fig. 2
). The effect was dose dependent and at the higher dose the PrPres signal was abolished. The more efficient dose of 10 µg HMs ml-1 was, therefore, used in subsequent experiments (Figs 3 and 4
). The effect of this new class of compounds was clearly superior to the one observed using the highest non-toxic dose of the amphotericin B derivative MS-8209 (Fig. 2
), a drug known to delay significantly the incubation time of experimental scrapie in vivo (Adjou et al., 1995
).
To assess whether HMs could durably eliminate PrPres from ScGT1 cultures, we treated the cells for 4 days twice consecutively and then monitored PrPres levels over 50 days. During this whole period, PrPres was undetectable (Fig. 4). This result suggested that PrPres was eliminated from the cultures or decreased to trace amounts insufficient to act as a seed for PrPres formation. These cells were then sub-passaged further in our laboratory and, to our surprise, we observed the reappearance of PrPres at day 120. While we have no explanation for this observation, this finding suggests that minute amounts of the abnormal prion protein present in a few cells are sufficient to reinitiate the growth of infection. Furthermore, our findings underline the need, in the future, for longer monitoring periods when assessing the therapeutic potential of anti-prion molecules in vitro.
The kinetics experiment showed that the molecule decreases the PrPres level early after its application (Fig. 3, 24 h); we estimated the time of treatment needed for PrPres to become undetectable by Western blot to be around 96 h. Compared to the kinetics of action of Congo red and polyene antibiotics tested previously in the same ScGT1 cells, where a PrPres signal is still present 5 and 15 days after drug application, respectively (data not shown; Mange et al., 2000), HM2602 appears to act faster. Moreover, the time of disappearance of PrPres after HM2602 treatment (96 h) corresponds to that of PrPres catabolism described previously (Caughey et al., 1989
), suggesting that the drug does not contribute to PrPres degradation but acts by inhibiting the de novo production of PrPres by the cells. Moreover, HMs do not alter the level of expression of PrPC in cell cultures nor in the spleens of mice (data not shown), demonstrating that the block in PrPres formation does not occur by inhibiting PrPC synthesis.
Given the kinetics of elimination of PrPres in our infected cells, the heparan-binding properties of PrP (Gabizon et al., 1993; Brimacombe et al., 1999
; Warner et al., 2002
) and the role of heparan sulfates in the binding of PrP to the cellular receptor LRP/LR and in PrP endocytosis (Shyng et al., 1995
; Hundt et al., 2001
), two mechanisms of action can be proposed at this stage. HMs might interact with PrPC and hamper its conversion into PrPres through steric inhibition. Alternatively, HMs might compete with the natural cellular heparan sulfates and block the PrPLRP/LR interaction. We speculate that this latter interaction is relevant to prion propagation because (i) LRP/LR is essential to the endocytosis of PrPC (Gauczynski et al., 2001
) and/or PrPres, which, in turn, is necessary for the accumulation of PrPres in the cells and (ii) LRP/LR may be a direct co-factor for PrP conversion. These hypotheses have been underpinned recently by our demonstration that antibody blockage or expression inhibition of LRP/LR inhibits PrPres formation in cultured cells (Leucht et al., 2003
). Therefore, a blockage of the interaction of PrP with LRP/LR through HMs would prevent cell-to-cell prion propagation.
Turning to in vivo experiments, we show by two different methods that HM2602, unlike HM5004, can interfere with the progression of TSEs. The first depends on a rapid spleen assay consisting of measurement of PrPres formation in the spleen of mice 30 days after intraperitoneal inoculation. Although when a species barrier is crossed, PrPres and infectivity do not always correlate (Lasmézas et al., 1997; Hill et al., 2000
; Race et al., 2001
, 2002
), measuring PrPres levels is now generally accepted as a screening method for drugs or inactivation procedures using host-adapted TSE models (Lee et al., 2000
). By this method, we have observed a substantial decrease in PrPres levels in the spleen of HM2602-treated mice compared to controls; HM5004, on the other hand, did not exert an effect (Fig. 5
). Secondly, a survival experiment was set up in the 263K golden Syrian hamster model (Kimberlin & Walker, 1977
). The animals treated with HM2602 (injection by intraperitoneal route with 25 mg HMs kg1 body weight, once weekly) lived, on average, 24 days longer than untreated animals, corresponding to a 14 % increase in survival time. Interestingly, HM5004 did not have any effect on the survival times of these hamsters (Fig. 7
). Given the short plasma retention time of this family of compounds (Meddahi et al., 2002
), it is probable that increasing the frequency of treatment would improve substantially the observed therapeutic effect of HM2602. This is substantiated by our in vitro data showing a positive correlation between the treatment regimen and the hindrance of PrPres formation (Fig. 2
) and by a separate experiment in which scrapie-infected mice were treated with HM2602 every day for 30 days (instead of twice weekly as in Fig. 5
), resulting in a reduction in spleen PrPres below detectable levels (data not shown). This argues that in the hamster 263K model, a higher frequency of treatment would result in a further increase in animal survival time. Overall, both the survival data and the results obtained in the rapid spleen assay demonstrate the therapeutic potential of HM2602. Therefore, to assess if this drug might be useful to treat vCJD, we also tested its efficacy in a BSE-infection model. This was important since it was shown for other drugs like amphotericin B that BSE is often more resistant than scrapie to therapeutic intervention (Adjou et al., 1996
). HM2602 inhibited PrPres accumulation in the spleens of BSE-infected mice such that the protein was barely detectable at 30 days post-inoculation. HM2602 was more efficient than the reference molecule DS500 (Fig. 6
) and also less toxic, thus allowing a much larger therapeutic window.
Surprisingly, although both HMs successfully reduced the accumulation of PrPres in ScGT1-7 cells, only HM2602 impeded PrPres formation in vivo (Fig. 5). The mechanisms responsible for this discrepancy are not known. However, since the activity of anti-prion drugs has been shown to vary depending on the strain of prions (Adjou et al., 1996
), it is conceivable that the variation seen here might be due to the different strains of scrapie used in the experimental models (Chandler for the ScGT1-7 cells and C506M3/263K for the animal infections). Analysis of the chemical composition of the compounds provides clues to an alternative explanation: HM2602, due to the presence of a Bn group, exhibits a higher hydrophobicity profile than HM5004. This difference probably affects the tissue distribution of the molecules in such a way that the concentration of the less hydrophobic HM5004 in target organs is too low to exert an effect. Moreover, the conversion of PrPC into PrPres is characterized by the exposure to the surface of hydrophobic residues that are normally buried in PrPC (Safar et al., 1998
). This phenomenon, which explains partly the high propensity of PrPres to aggregate, also suggests the possibility of a stronger interaction with a more hydrophobic molecule like HM2602 and hence a better inhibition of the template-assisted conversion by PrPres. This effect might act in conjunction with the higher tissue concentration of the drug. Such a hydrophobic interaction between PrPres and anti-prion drugs like Congo red and iododoxorubicin has been demonstrated previously (Prusiner et al., 1983
; Merlini et al., 1995
; Tagliavini et al., 1997
).
In this study, we have demonstrated that HM2602 and HM5004 block PrPres accumulation in ScGT1 cells. In vivo, HM2602 was shown as a potential therapeutic agent against prion diseases. The inhibition of PrPres accumulation in the spleen observed with HM2602 was more effective than we had experienced previously with other drugs such as amphotericin B derivatives. Combined with our survival data, these results constitute a strong incentive towards the development of long-term trials using these molecules, with increased frequency of administration. Importantly, the effect was not strain-restricted, as it could be observed with both scrapie- and BSE-infected mice, suggesting a general mechanism of action. Notwithstanding the need for further drug characterization and development, our study defines HMs as a new class of drugs for the treatment of TSEs. These molecules are particularly amenable to drug design and may prove efficient even in later stages of the disease due to their properties to enhance tissue repair (Desgranges et al., 1999).
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adjou, K. T., Demaimay, R., Lasmézas, C. I., Seman, M., Deslys, J. P. & Dormont, D. (1996). Differential effects of a new amphotericin B derivative, MS-8209, on mouse BSE and scrapie: implications for the mechanism of action of polyene antibiotics. Res Virol 147, 213218.[CrossRef][Medline]
Barret, A., Tagliavini, F., Forloni, G. & 13 other authors (2003). Evaluation of quinacrine treatment for prion diseases. J Virol (in press).
Beringue, V., Adjou, K. T., Lamoury, F., Maignien, T., Deslys, J. P., Race, R. & Dormont, D. (2000). Opposite effects of dextran sulfate 500, the polyene antibiotic MS-8209, and Congo red on accumulation of the protease-resistant isoform of PrP in the spleens of mice inoculated intraperitoneally with the scrapie agent. J Virol 74, 54325440.
Bernfield, M., Gotte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J. & Zako, M. (1999). Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem 68, 729777.[CrossRef][Medline]
Bolton, D. C., McKinley, M. P. & Prusiner, S. B. (1982). Identification of a protein that purifies with the scrapie prion. Science 218, 13091311.[Medline]
Brimacombe, D. B., Bennett, A. D., Wusteman, F. S., Gill, A. G., Dann, J. C. & Bostock, C. J. (1999). Characterization and polyanion-binding properties of purified recombinant prion protein. Biochem J 342, 605613.[CrossRef][Medline]
Caughey, B. & Raymond, G. J. (1993). Sulfated polyanion inhibition of scrapie-associated PrP accumulation in cultured cells. J Virol 67, 643650.[Abstract]
Caughey, B., Race, R. E., Ernst, D., Buchmeier, M. J. & Chesebro, B. (1989). Prion protein (PrP) biosynthesis in scrapie-infected and uninfected neuroblastoma cells. J Virol 63, 175181.[Medline]
Desgranges, P., Barbaud, C., Caruelle, J. P., Barritault, D. & Gautron, J. (1999). A substituted dextran enhances muscle fiber survival and regeneration in ischemic and denervated rat EDL muscle. FASEB J 13, 761766.
Deslys, J.-P., Beringue, V. & Lasmézas, C. (1998). Method for screening substances with therapeutical action in the treatment of transmissible spongiform encephalopathy. Europe Commissariat à l'Energie Atomique, patent no. EP1998000902078.
Diringer, H. & Ehlers, B. (1991). Chemoprophylaxis of scrapie in mice. J Gen Virol 72, 457460.[Abstract]
Ehlers, B. & Diringer, H. (1984). Dextran sulphate 500 delays and prevents mouse scrapie by impairment of agent replication in spleen. J Gen Virol 65, 13251330.[Abstract]
Farquhar, C. F. & Dickinson, A. G. (1986). Prolongation of scrapie incubation period by an injection of dextran sulphate 500 within the month before or after infection. J Gen Virol 67, 463473.[Abstract]
Farquhar, C., Dickinson, A. & Bruce, M. (1999). Prophylactic potential of pentosan polysulphate in transmissible spongiform encephalopathies. Lancet 353, 117.[CrossRef][Medline]
Fraser, H. (1976). The pathology of natural and experimental scrapie. In Slow Virus Diseases of Animals and Man, pp. 267305. Edited by R. H. Kimberlin. New York: North-Holland.
Gabizon, R., Meiner, Z., Halimi, M. & Ben-Sasson, S. A. (1993). Heparin-like molecules bind differentially to prion-proteins and change their intracellular metabolic fate. J Cell Physiol 157, 319325.[Medline]
Gauczynski, S., Peyrin, J. M., Haik, S. & 8 other authors (2001). The 37-kDa/67-kDa laminin receptor acts as the cell-surface receptor for the cellular prion protein. EMBO J 20, 58635875.
Ghani, A. C., Ferguson, N. M., Donnelly, C. A. & Anderson, R. M. (2000). Predicted vCJD mortality in Great Britain. Nature 406, 583584.[CrossRef][Medline]
Ghani, A. C., Donnelly, C. A., Ferguson, N. M. & Anderson, R. M. (2002). The transmission dynamics of BSE and vCJD. C R Acad Sci III 325, 3747.[Medline]
Gilch, S., Winklhofer, K. F., Groschup, M. H. & 7 other authors (2001). Intracellular re-routing of prion protein prevents propagation of PrPSc and delays onset of prion disease. EMBO J 20, 39573966.
Gonzalez-Iglesias, R., Pajares, M. A., Ocal, C., Espinosa, J. C., Oesch, B. & Gasset, M. (2002). Prion protein interaction with glycosaminoglycan occurs with the formation of oligomeric complexes stabilized by CuII bridges. J Mol Biol 319, 527540.[CrossRef][Medline]
Grassi, J., Comoy, E., Simon, S. & 8 other authors (2001). Rapid test for the preclinical postmortem diagnosis of BSE in central nervous system tissue. Vet Rec 149, 577582.[Medline]
Hill, A. F., Joiner, S., Linehan, J., Desbruslais, M., Lantos, P. L. & Collinge, J. (2000). Species-barrier-independent prion replication in apparently resistant species. Proc Natl Acad Sci U S A 97, 1024810253.
Hundt, C., Peyrin, J. M., Haik, S. & 8 other authors (2001). Identification of interaction domains of the prion protein with its 37-kDa/67-kDa laminin receptor. EMBO J 20, 58765886.
Kimberlin, R. H. & Walker, C. A. (1977). Characteristics of a short incubation model of scrapie in the golden hamster. J Gen Virol 34, 295304.[Abstract]
Kimberlin, R. H. & Walker, C. A. (1986). Suppression of scrapie infection in mice by heteropolyanion 23, dextran sulfate, and some other polyanions. Antimicrob Agents Chemother 30, 409413.[Medline]
Korth, C., May, B. C., Cohen, F. E. & Prusiner, S. B. (2001). Acridine and phenothiazine derivatives as pharmacotherapeutics for prion disease. Proc Natl Acad Sci U S A 98, 98369841.
Ladogana, A., Casaccia, P., Ingrosso, L., Cibati, M., Salvatore, M., Xi, Y. G., Mascullo, C. & Pocchiari, M. (1992). Sulphate polyanions prolong the incubation period of scrapie-infected hamsters. J Gen Virol 73, 661665.[Abstract]
Lasmézas, C. I., Deslys, J. P., Demaimay, R., Adjou, K. T., Hauw, J. J. & Dormont, D. (1996). Strain specific and common pathogenic events in murine models of scrapie and bovine spongiform encephalopathy. J Gen Virol 77, 16011609.[Abstract]
Lasmézas, C. I., Deslys, J. P., Robain, O. & 7 other authors (1997). Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein. Science 275, 402405.
Lee, D. C., Stenland, C. J., Hartwell, R. C. & 7 other authors (2000). Monitoring plasma processing steps with a sensitive Western blot assay for the detection of the prion protein. J Virol Methods 84, 7789.[CrossRef][Medline]
Leucht, C., Simoneau, S., Rey, C., Vana, K., Rieger, R., Lasmézas, C. I. & Weiss, S. (2003). The 37 kDa/67 kDa laminin receptor is required for PrPSc propagation in scrapie-infected neuronal cells. EMBO Rep 4, 290295; erratum 4, 439.
Mange, A., Nishida, N., Milhavet, O., McMahon, H. E., Casanova, D. & Lehmann, S. (2000). Amphotericin B inhibits the generation of the scrapie isoform of the prion protein in infected cultures. J Virol 74, 31353140.
May, B. C., Fafarman, A. T., Hong, S. B., Rogers, M., Deady, L. W., Prusiner, S. B. & Cohen, F. E. (2003). Potent inhibition of scrapie prion replication in cultured cells by bis-acridines. Proc Natl Acad Sci U S A 100, 34163421.
Meddahi, A., Bree, F., Papy-Garcia, D., Gautron, J., Barritault, D. & Caruelle, J. P. (2002). Pharmacological studies of RGTA(11), a heparan sulfate mimetic polymer, efficient on muscle regeneration. J Biomed Mater Res 62, 525531.[CrossRef][Medline]
Merlini, G., Ascari, E., Amboldi, N. & other authors (1995). Interaction of the anthracycline 4'-iodo-4'-deoxydoxorubicin with amyloid fibrils: inhibition of amyloidogenesis. Proc Natl Acad Sci U S A 92, 29592963.[Abstract]
Peretz, D., Williamson, R. A., Kaneko, K. & 10 other authors (2001). Antibodies inhibit prion propagation and clear cell cultures of prion infectivity. Nature 412, 739743.[CrossRef][Medline]
Petit, E., Papy Garcia, D. & Barbier Chassefiere, V. (2002). Procédé de sulfonation de composés comprenant des groupements hydroxyle (OH) libres ou des amines primaires ou secondaires OTR3. Patent no. FR2832708.
Prusiner, S. B. (1982). Novel proteinaceous infectious particles cause scrapie. Science 216, 136144.[Medline]
Prusiner, S. B., McKinley, M. P., Bowman, K. A., Bolton, D. C., Bendheim, P. E., Groth, D. F. & Glenner, G. G. (1983). Scrapie prions aggregate to form amyloid-like birefringent rods. Cell 35, 349358.[Medline]
Race, R., Raines, A., Raymond, G. J., Caughey, B. & Chesebro, B. (2001). Long-term subclinical carrier state precedes scrapie replication and adaptation in a resistant species: analogies to bovine spongiform encephalopathy and variant CreutzfeldtJakob disease in humans. J Virol 75, 1010610112.
Race, R., Meade-White, K., Raines, A., Raymond, G. J., Caughey, B. & Chesebro, B. (2002). Subclinical scrapie infection in a resistant species: persistence, replication, and adaptation of infectivity during four passages. J Infect Dis 186 (Suppl. 2), S166S170.[CrossRef][Medline]
Safar, J., Wille, H., Itri, V., Groth, D., Serban, H., Torchia, M., Cohen, F. E. & Prusiner, S. B. (1998). Eight prion strains have PrPSc molecules with different conformations. Nat Med 4, 11571165.[CrossRef][Medline]
Schätzl, H. M., Laszlo, L., Holtzman, D. M., Tatzelt, J., DeArmond, S. J., Weiner, R. I., Mobley, W. C. & Prusiner, S. B. (1997). A hypothalamic neuronal cell line persistently infected with scrapie prions exhibits apoptosis. J Virol 71, 88218831.[Abstract]
Shyng, S.-L., Lehmann, S., Moulder, K. L. & Harris, D. A. (1995). Sulfated glycans stimulate endocytosis of the cellular isoform of the prion protein, PrPC, in cultured cells. J Biol Chem 270, 3022130229.
Supattapone, S., Wille, H., Uyechi, L., Safar, J., Tremblay, P., Szoka, F. C., Cohen, F. E., Prusiner, S. B. & Scott, M. R. (2001). Branched polyamines cure prion-infected neuroblastoma cells. J Virol 75, 34533461.
Tagliavini, F., McArthur, R. A., Canciani, B. & 18 other authors (1997). Effectiveness of anthracycline against experimental prion disease in Syrian hamsters. Science 276, 11191122.
Turnbull, J., Powell, A. & Guimond, S. (2001). Heparan sulfate: decoding a dynamic multifunctional cell regulator. Trends Cell Biol 11, 7582.[CrossRef][Medline]
Warner, R. G., Hundt, C., Weiss, S. & Turnbull, J. E. (2002). Identification of the heparan sulfate binding sites in the cellular prion protein. J Biol Chem 277, 1842118430.
Will, R. G., Ironside, J. W., Zeidler, M. & 7 other authors (1996). A new variant of CreutzfeldtJakob disease in the UK. Lancet 347, 921925.[Medline]
Received 24 December 2002;
accepted 26 May 2003.