Increased circulating microRNAs miR-342-3p and miR-21-5p in natural sheep prion disease.

Scrapie is a transmissible spongiform encephalopathy (TSE), or prion disease, of sheep and goats. As no simple diagnostic tests are yet available to detect TSEs in vivo, easily accessible biomarkers could facilitate the eradication of scrapie agents from the food chain. To this end, we analysed by quantitative reverse transcription PCR a selected set of candidate microRNAs (miRNAs) from circulating blood plasma of naturally infected, classical scrapie sheep that demonstrated clear scrapie symptoms and pathology. Significant scrapie-associated increase was repeatedly found for miR-342-3p and miR-21-5p. This is the first demonstration, to our knowledge, of circulating miRNA alterations in any animal suffering from TSE. Genome-wide expression studies are warranted to investigate the true depth of miRNA alterations in naturally occurring TSEs, especially in presymptomatic animals, as the presented study demonstrates the potential feasibility of miRNAs as circulating TSE biomarkers.

Mononucleated Blood Cell Populations Display Different Abilities To Transmit Prion Disease by the Transfusion Route.

UNLABELLED: Previous experiments carried out in a sheep scrapie model demonstrated that the transfusion of 200 µl of prion-infected whole blood has an apparent 100% efficacy for disease transmission. These experiments also indicated that, despite the apparent low infectious titer, the intravenous administration of white blood cells (WBC) resulted in efficient disease transmission. In the study presented here, using the same transmissible spongiform encephalopathy (TSE) animal model, our aim was to determine the minimal number of white blood cells and the specific abilities of mononucleated cell populations to transmit scrapie by the transfusion route. Our results confirmed that the transfusion of 100 µl, but not 10 µl, of fresh whole blood collected in asymptomatic scrapie-infected donor sheep can transmit the disease. The data also show that the intravenous administration of 10(5) WBCs is sufficient to cause scrapie in recipient sheep. Cell-sorted CD45R(+) (predominantly B lymphocytes), CD4(+)/CD8(+) (T lymphocytes), and CD14(+) (monocytes/macrophages) blood cell subpopulations all were shown to contain prion infectivity by bioassays in ovine PrP transgenic mice. However, while the intravenous administration of 10(6) CD45(+) or CD4(+)/8(+) living cells was able to transmit the disease, similar numbers of CD14(+) cells failed to infect the recipients. These data support the contention that mononucleated blood cell populations display different abilities to transmit TSE by the transfusion route. They also represent an important input for the risk assessment of blood-borne prion disease transmission and for refining the target performance of leukoreduction processes that currently are applied to mitigate the transmission risk in transfusion medicine. IMPORTANCE: Interindividual variant Creutzfeldt-Jakob disease (vCJD) transmission through blood and blood-derived products is considered a major public health issue in transfusion medicine. Over the last decade, TSE in sheep has emerged as a relevant model for assessing the blood-borne vCJD transmission risk. In this study, using a sheep TSE model, we characterized the ability of different peripheral blood mononucleated cell populations to infect TSE-free recipients by the transfusion route. Our results indicate that as little as 10(5) WBC and 100 µl of blood collected from asymptomatic scrapie infected animals can transmit the disease. They also demonstrate unambiguously that peripheral blood mononuclear cell subpopulations display dramatically different abilities to transmit the disease. These data represent an important input for the risk assessment of blood-borne prion disease transmission and for refining the target performance of leukoreduction processes that currently are applied to mitigate the transmission risk in transfusion medicine.

Bioassay of prion-infected blood plasma in PrP transgenic Drosophila.

In pursuit of a tractable bioassay to assess blood prion infectivity, we have generated prion protein (PrP) transgenic Drosophila, which show a neurotoxic phenotype in adulthood after exposure to exogenous prions at the larval stage. Here, we determined the sensitivity of ovine PrP transgenic Drosophila to ovine prion infectivity by exposure of these flies to a dilution series of scrapie-infected sheep brain homogenate. Ovine PrP transgenic Drosophila showed a significant neurotoxic response to dilutions of 10-2 to 10-10 of the original scrapie-infected sheep brain homogenate. Significantly, we determined that this prion-induced neurotoxic response in ovine PrP transgenic Drosophila was transmissible to ovine PrP transgenic mice, which is indicative of authentic mammalian prion detection by these flies. As a consequence, we considered that PrP transgenic Drosophila were sufficiently sensitive to exogenous mammalian prions to be capable of detecting prion infectivity in the blood of scrapie-infected sheep. To test this hypothesis, we exposed ovine PrP transgenic Drosophila to scrapie-infected plasma, a blood fraction notoriously difficult to assess by conventional prion bioassays. Notably, pre-clinical plasma from scrapie-infected sheep induced neurotoxicity in PrP transgenic Drosophila and this effect was more pronounced after exposure to samples collected at the clinical phase of disease. The neurotoxic phenotype in ovine PrP transgenic Drosophila induced by plasma from scrapie-infected sheep was transmissible since head homogenate from these flies caused neurotoxicity in recipient flies during fly-to-fly transmission. Our data show that PrP transgenic Drosophila can be used successfully to bioassay prion infectivity in blood from a prion-diseased mammalian host.

Persistent residual contamination in endoscope channels; a fluorescence epimicroscopy study.

BACKGROUND AND STUDY AIMS: The increasing demand for endoscopic procedures poses new contamination challenges, given developing antimicrobial resistance worldwide and potential viral or prion diseases in populations at risk. We examined working channels from reusable luminal endoscopes used in recent years. METHODS: Very sensitive fluorescence epimicroscopy was used to examine working channels from 6 decommissioned and 6 factory-new channels, as received, or following spiking and washing in the laboratory. RESULTS: After a single contamination and wash test cycle, new channels retained approximately 75 pg/mm(2) of proteins; through 7 subsequent cycles residual proteins fluctuated between 25 and 75 pg/mm(2). Decommissioned channels harbored 1 - 4 µg of proteins each, except in one gastroscope (33 µg), including up to 2 % amyloid proteins except in one gastroscope and one sigmoidoscope (with over 80 %); lumens showed wearing with established abraded biofilms in 3 cases. After spiking with scrapie-infected blood components and washing, residual protein levels in new channels varied following standard (17.23 pg/mm(2)), duplicated (2.39 pg/mm(2)) or extended (11.3 pg/mm(2)) washing; no changes were measured among the long-established contamination in old channels. CONCLUSIONS: Our observations suggest that wear effects in endoscope lumens may contribute to the adsorption of proteins, thus facilitating retention and survival of bacteria. As demonstrated by recent outbreaks worldwide despite recommended reprocessing, the development of antimicrobial-resistant bacterial strains, and the estimated prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the UK particularly, combined with increasing demand for endoscopic procedures, call for sustained precautions and improved methods for the reprocessing of nonautoclavable, reusable surgical instruments.

Classical scrapie prions are associated with peripheral blood monocytes and T-lymphocytes from naturally infected sheep.

BACKGROUND: Classical scrapie is a transmissible spongiform encephalopathy (TSE) that affects sheep and goats. Our previous bioassay studies in lambs revealed that scrapie prions could be detected in association with peripheral blood monocular cells (PBMC), B lymphocytes and platelet-rich plasma fractions. In the present study, bioassay in lambs was again used to determine if scrapie prions are associated with the other two subsets of PBMC, monocytes and T lymphocytes. RESULTS: PBMC, monocytes and T lymphocytes were isolated from two preclinically affected VRQ/VRQ sheep naturally infected with classical ovine scrapie and intravenously transfused into VRQ/VRQ lambs post-weaning. As determined using standard immunohistochemistry for scrapie, abnormal isoforms of prion protein were detected in lymphoid tissues of lambs inoculated with PBMC (4/4 recipient lambs), monocytes (2/5) and T lymphocytes (1/4). Prion protein misfolding activity was detected by serial protein misfolding cyclic amplification (sPMCA) in PBMC from monocyte and T lymphocyte recipient sheep in agreement with antemortem rectal biopsy results, but such prion protein misfolding activity was not detected from other recipients. CONCLUSIONS: These findings show that scrapie prions are associated with monocytes and T lymphocytes circulating in the peripheral blood of sheep naturally infected with classical scrapie. Combined with our previous findings, we can now conclude that all three major subsets of PBMC can harbor prions during preclinical disease and thus, present logical targets for development of a sensitive assay to detect scrapie prions. In this regard, we have also demonstrated that sPMCA can be used to detect scrapie prions associated with PBMC.

Detection of cellular prion protein in exosomes derived from ovine plasma.

Prion protein (PrP) is present at extremely low levels in the blood of animals and its detection is complicated by the poor sensitivity of current standard methodologies. Interesting results have been obtained with recent advanced technologies that are able to detect minute amounts of the pathological PrP (PrPSc), but their efficiency is reduced by various factors present in blood. In this study, we were able to extract cellular PrP (PrPC) from plasma-derived exosomes by a simple, fast method without the use of differential ultracentrifugation and to visualize it by Western blotting, reducing the presence of most plasma proteins. This result confirms that blood is capable of releasing PrP in association with exosomes and could be useful to better study its role in the pathogenesis of transmissible spongiform encephalopathies.

Evaluation of efficacy of prion reduction filters using blood from an endogenously infected 263K scrapie hamster model.

BACKGROUND: The P-Capt prion reduction filter (MacoPharma) removes prion infectivity in model systems. This independent evaluation assesses prion removal from endogenously infected animal blood, using CE-marked P-Capt filters, and replicates the proposed use of the filter within the UK Blood Services. STUDY DESIGN AND METHODS: Two units of blood, generated from 263K scrapie-infected hamsters, were processed using leukoreduction filters (LXT-quadruple, MacoPharma). Approximately 100 mL of the removed plasma was added back to the red blood cells (RBCs) and the blood was filtered through a P-Capt filter. Samples of unfiltered whole blood, the prion filter input (RBCs plus plasma and SAGM [RBCPS]), and prion-filtered leukoreduced blood (PFB) were injected intracranially into hamsters. Clinical symptoms were monitored for 500 ± 1 day, and brains were assessed for spongiosis and prion protein deposit. RESULTS: In Filtration Run 1, none of the 50 challenged animals were diagnosed with scrapie after inoculation with the RBCPS fraction, while two of 190 hamsters injected with PFB were infected. In Filtration Run 2, one of 49 animals injected with RBCPS and two of 193 hamsters injected with PFB were infected. Run 1 reduced the infectious dose (ID) by 1.467 log (>1.187 log and <0.280 log for leukoreduction and prion filtration, respectively). Run 2 reduced prion infectivity by 1.424 log (1.127 and 0.297 log, respectively). Residual infectivity was estimated at 0.212 ± 0.149 IDs/mL (Run 1) and 0.208 ± 0.147 IDs/mL (Run 2). CONCLUSION: Leukoreduction removed the majority of infectivity from 263K scrapie hamster blood. The P-Capt filter removed a proportion of the remaining infectivity, but residual infectivity was observed in two independent processes.

Development of a bifunctional filter for prion protein and leukoreduction of red blood cell components.

BACKGROUND: Leukofiltration of blood components is currently implemented worldwide as a precautionary measure against white blood cell-associated adverse effects and the potential transmission of variant Creutzfeldt-Jakob disease (vCJD). A newly developed bifunctional filter (Sepacell Prima, Asahi Kasei Medical) was assessed for prion removal, leukoreduction (LR), and whether the filter significantly affected red blood cells (RBCs). STUDY DESIGN AND METHODS: Sepacell Prima's postfiltration effects on RBCs, including hemolysis, complement activation, and RBC chemistry, were compared with those of a conventional LR filter (Sepacell Pure RC). Prion removal was measured by Western blot after spiking RBCs with microsomal fractions derived from scrapie-infected hamster brain homogenate. Serially diluted exogenous prion solutions (0.05 mL), with or without filtration, were injected intracerebrally into Golden Syrian hamsters. RESULTS: LR efficiency of 4.44 log with the Sepacell Prima was comparable to 4.11 log with the conventional LR filter. There were no significant differences between the two filters in hemoglobin loss, hemolysis, complement activation, and RBC biomarkers. In vitro reduction of exogenously spiked prions by the filter exceeded 3 log. The titer, 6.63 (log ID50 /mL), of prefiltration infectivity of healthy hamsters was reduced to 2.52 (log ID50 /mL) after filtration. The reduction factor was calculated as 4.20 (log ID50 ). CONCLUSION: With confirmed removal efficacy for exogenous prion protein, this new bifunctional prion and LR filter should reduce the residual risk of vCJD transmission through blood transfusion without adding complexity to component processing.

Estimating prion concentration in fluids and tissues by quantitative PMCA.

Prions, the proteinaceous infectious agent responsible for prion diseases, can be detected with high sensitivity by protein misfolding cyclic amplification (PMCA) technology. Here we describe a quantitative PMCA procedure to calculate the concentration of very low levels of prions in biological samples. Using this procedure, we determined the quantities of misfolded prion protein (PrP(Sc)) in brain, spleen, blood and urine of scrapie-affected hamsters.

Evaluation of a test for its suitability in the diagnosis of variant Creutzfeldt-Jakob disease.

BACKGROUND AND OBJECTIVES: Evaluation of variant Creutzfeldt-Jakob disease (vCJD) diagnostic/donor screening tests is made complicated by the very limited supply of blood samples from clinically confirmed cases of vCJD. To determine appropriate access for test developers to rare Creutzfeldt-Jakob disease (CJD) blood samples, the oversight committee of the NIBSC CJD Resource Centre has developed a process and protocols detailing minimum requirements for both test sensitivity and specificity. This protocol is broadly similar to that outlined in the common technical specification (European Directive 98/79/EC). MATERIALS AND METHODS: Tests are subjected to a stepwise evaluation (step 1). vCJD tissue homogenates spiked into pooled human plasma (step 2). Blood samples from animals known to be incubating (Transmissible spongiform encephalopathy) TSE disease (scrapie/Bovine Spongiform encephalopathy (BSE)-infected sheep, BSE-infected primates) and appropriate controls (step 3). Fresh or frozen plasma from normal UK blood donors and (step 4). Plasma samples from individuals with confirmed clinical stage variant CJD (transfusion transmission) or sporadic CJD (no evidence of blood transmission). RESULTS: The assay evaluated performed with good sensitivity with vCJD-spiked tissue homogenates, poor sensitivity for ovine TSE-infected blood samples and failed with plasma from BSE-infected non-human primates and with true vCJD clinical samples. CONCLUSIONS: The test evaluated here is currently unsuitable for use in blood donor screening or diagnosis using blood.

Estimation of variant Creutzfeldt-Jakob disease infectivity titers in human blood.

BACKGROUND: Blood of individuals with variant Creutzfeldt-Jakob disease (vCJD) is infectious but the titer is unknown. Current estimates of possible vCJD infectivity titers in blood have largely relied on an assumption that the titers of vCJD agent in human blood are likely to be similar to those in blood of rodents infected with model transmissible spongiform encephalopathy agents, assayed by intracerebral inoculations of rodents of the same species. STUDY DESIGN AND METHODS: We analyzed published descriptions of experimental transfusion-transmitted (TT) bovine spongiform encephalopathy and scrapie in sheep and reports of TTvCJD in humans, applying statistical approaches to estimate the probable number of intravenous infectious doses (ID(iv) ) per unit of transfused blood (ID(iv) /unit). For humans, ID(iv) /unit of nonleukoreduced red blood cells (NLR-RBCs) were estimated by two statistical models. RESULTS: Sheep blood collected at or near onset of clinical illness contained a mean of 0.80 ID(iv) /unit. Estimates of infectivity in NLR-RBCs from donors incubating vCJD indicated a probable mean infectivity of 0.29 ID(iv) /unit (Model 1) and 0.75 ID(iv) /unit (Model 2). The analysis predicted a mean of 21 vCJD-infected recipients expected in a cohort transfused with vCJD-implicated NLR-RBCs in the United Kingdom. CONCLUSION: Our analysis suggested that, while less than one ID(iv ) is likely to be present in a given unit of NLR-RBCs collected from a donor incubating vCJD, there is a high probability of TT infection among recipients of vCJD-implicated blood components. The analysis supports continuing measures currently recommended to reduce the risk of TTvCJD.

Classical scrapie prions in ovine blood are associated with B lymphocytes and platelet-rich plasma.

BACKGROUND: Classical scrapie is a naturally occurring transmissible spongiform encephalopathy of sheep and goats characterized by cellular accumulation of abnormal isoforms of prion protein (PrPSc) in the central nervous system and the follicles of peripheral lymphoid tissues. Previous studies have shown that the whole blood and buffy coat blood fraction of scrapie infected sheep harbor prion infectivity. Although PrPSc has been detected in peripheral blood mononuclear cells (PBMCs), plasma, and more recently within a subpopulation of B lymphocytes, the infectivity status of these cells and plasma in sheep remains unknown. Therefore, the objective of this study was to determine whether circulating PBMCs, B lymphocytes and platelets from classical scrapie infected sheep harbor prion infectivity using a sheep bioassay. RESULTS: Serial rectal mucosal biopsy and immunohistochemistry were used to detect preclinical infection in lambs transfused with whole blood or blood cell fractions from preclinical or clinical scrapie infected sheep. PrPSc immunolabeling was detected in antemortem rectal and postmortem lymphoid tissues from recipient lambs receiving PBMCs (15/15), CD72+ B lymphocytes (3/3), CD21+ B lymphocytes (3/3) or platelet-rich plasma (2/3) fractions. As expected, whole blood (11/13) and buffy coat (5/5) recipients showed positive PrPSc labeling in lymphoid follicles. However, at 549 days post-transfusion, PrPSc was not detected in rectal or other lymphoid tissues in three sheep receiving platelet-poor plasma fraction. CONCLUSIONS: Prion infectivity was detected in circulating PBMCs, CD72+ pan B lymphocytes, the CD21+ subpopulation of B lymphocytes and platelet-rich plasma of classical scrapie infected sheep using a sheep bioassay. Combining platelets with B lymphocytes might enhance PrPSc detection levels in blood samples.

A novel method for preclinical detection of PrPSc in blood.

In this study, we demonstrate that a moderate amount of protein misfolding cyclic amplification (PMCA) coupled to a novel surround optical fibre immunoassay (SOFIA) detection scheme can be used to detect the disease-associated form of the prion protein (PrP(Sc)) in protease-untreated plasma from preclinical and clinical scrapie sheep, and white-tailed deer with chronic wasting disease, following natural and experimental infection. PrP(Sc), resulting from a conformational change of the normal (cellular) form of prion protein (PrP(C)), is considered central to neuropathogenesis and serves as the only reliable molecular marker for prion disease diagnosis. While the highest levels of PrP(Sc) are present in the central nervous system, the development of a reasonable diagnostic assay requires the use of body fluids that characteristically contain exceedingly low levels of PrP(Sc). PrP(Sc) has been detected in the blood of sick animals by means of PMCA technology. However, repeated cycling over several days, which is necessary for PMCA of blood material, has been reported to result in decreased specificity (false positives). To generate an assay for PrP(Sc) in blood that is both highly sensitive and specific, we have utilized limited serial PMCA (sPMCA) with SOFIA. We did not find any enhancement of sPMCA with the addition of polyadenylic acid nor was it necessary to match the genotypes of the PrP(C) and PrP(Sc) sources for efficient amplification.

Differentiating blood samples from scrapie infected and non-infected hamsters by detecting disease-associated prion proteins using Multimer Detection System.

This communication describes the application of a modified sandwich enzyme-linked immunosorbent assay (ELISA), termed Multimer Detection System (MDS) for the detection of disease-associated multimeric forms of the prion protein (PrPd) in hamster blood. PrPd was detected in plasma of prion-affected hamsters while MDS revealed no PrPd in identically-treated plasma of healthy animals. This is the first report of a single ELISA- based immune detection of PrPd from blood samples.

Prion diseases are efficiently transmitted by blood transfusion in sheep.

The emergence of variant Creutzfeld-Jakob disease, following on from the bovine spongiform encephalopathy (BSE) epidemic, led to concerns about the potential risk of iatrogenic transmission of disease by blood transfusion and the introduction of costly control measures to protect blood supplies. We previously reported preliminary data demonstrating the transmission of BSE and natural scrapie by blood transfusion in sheep. The final results of this experiment, reported here, give unexpectedly high transmission rates by transfusion of 36% for BSE and 43% for scrapie. A proportion of BSE-infected transfusion recipients (3 of 8) survived for up to 7 years without showing clinical signs of disease. The majority of transmissions resulted from blood collected from donors at more than 50% of the estimated incubation period. The high transmission rates and relatively short and consistent incubation periods in clinically positive recipients suggest that infectivity titers in blood were substantial and/or that blood transfusion is an efficient method of transmission. This experiment has established the value of using sheep as a model for studying transmission of variant Creutzfeld-Jakob disease by blood products in humans.

Ovine serum biomarkers of early and late phase scrapie.

BACKGROUND: Transmissible spongiform encephalopathies are fatal neurodegenerative disease occurring in animals and humans for which no ante-mortem diagnostic test in biological fluids is available. In such pathologies, detection of the pathological form of the prion protein (i.e., the causative factor) in blood is difficult and therefore identification of new biomarkers implicated in the pathway of prion infection is relevant. METHODS: In this study we used the SELDI-TOF MS technology to analyze a large number of serum samples from control sheep and animals with early phase or late phase scrapie. A few potential low molecular weight biomarkers were selected by statistical methods and, after a training analysis, a protein signature pattern, which discriminates between early phase scrapie samples and control sera was identified. RESULTS: The combination of early phase biomarkers showed a sensitivity of 87% and specificity of 90% for all studied sheep in the early stage of the disease. One of these potential biomarkers was identified and validated in a SELDI-TOF MS kinetic study of sera from Syrian hamsters infected by scrapie, by western blot analysis and ELISA quantitation. CONCLUSIONS: Differential protein expression profiling allows establishing a TSE diagnostic in scrapie sheep, in the early phase of the disease. Some proteic differences observed in scrapie sheep exist in infected hamsters. Further studies are being performed to identify all the discriminant biomarkers of interest and to test our potential markers in a new cohort of animals.

Blood chimerism confounds genetic relative susceptibility testing for classical scrapie in sheep.

Classical scrapie disease is a transmissible spongiform encephalopathy of sheep that is enzootic in the United States. Susceptibility of sheep to classical scrapie is linked to single nucleotide polymorphisms in the prion protein gene (PRNP), forming the basis for genetic testing strategies used by national efforts to eradicate scrapie. Such efforts are occasionally hampered by inconclusive results stemming from the detection of "complex" genotypes. Naturally occurring cases of ovine chimerism are thought to account for some of these instances. In the current report, 4 naturally occurring ovine chimeras are documented through cytogenetic and molecular analyses. All 4 of these sheep had chimeric cells circulating in their blood. Blood and alternate tissue samples of ear punch and hair bulbs from one of these chimeras was submitted in batch with similar samples from control sheep for routine scrapie genetic relative susceptibility testing. A complex PRNP genotype was detected in the blood of the chimeric female but not in the alternate tissue samples or in the control sheep samples. The results demonstrate that naturally occurring blood chimerism can confound current testing efforts. The potential impacts of undetected chimeras on current scrapie eradication efforts are discussed.

Neurofilaments in blood is a new promising preclinical biomarker for the screening of natural scrapie in sheep.

Scrapie is a fatal neurodegenerative disease of sheep and goats belonging to the group of Transmissible Spongiform Encephalopathy or prion diseases. The EU has adopted mandatory measures for scrapie surveillance to safeguard public and animal health because it is highly contagious and might decimate all genetic susceptible animals in affected flocks. Definite diagnosis of scrapie relies on the detection of the pathological prion protein in brain tissues and there are still no blood biomarkers available for making diagnosis in living animals that can be used for the screening of sheep in scrapie-affected flocks. Neurofilament light (NfL) protein, a valid biomarker for neuronal and axonal damages, can now be easily measured in blood by the ultra-sensitive single molecule array (Simoa) technology. Recent work reported that serum NfL is increased in neurodegenerative diseases, including human prion diseases, but no data are available for scrapie or other animal prion diseases. Here, we found that the median serum NfL concentration in scrapie animals (56.2, IQR 42.2-84.8, n = 9) was more than 15 times higher (p = 0.00084) than that found in control samples (3.4, IQR 3.0-26.3, n = 11). Moreover, serum NfL concentration in scrapie sheep with clinical signs (n = 2; 75.3, 15.7 pg/ml) did not significantly (p = 0.541; t-test) differ from scrapie animals without clinical signs (n = 7; 61.0, 10.7 pg/ml). The receiver operating characteristic (ROC) curve analysis estimated the cut-off value of 31 pg/ml serum NfL for distinguishing scrapie-infected sheep from controls. The application of this cut-off value gives an accuracy of the test of 95% (percent error of 5.23%). These data indicate that the Simoa test for serum NfL might be a useful screening method for detecting preclinical scrapie in living sheep. Finally, the preliminary data reported here need confirmation in large and more structured studies.

Analysis of the Glycosylation Profile of Disease-Associated Water-Soluble Prion Protein Using Lectins.

The disease-associated water-soluble form of hamster prion protein (ws-PrPSc) has recently been found to be less stable than classical PrPSc. Since the stability of PrP to degradation correlates with its glycosylation level, the aim of this study was to investigate whether there are differences between the glycosylation of ws-PrPSc and classical PrPSc of hamster which might account for the ws-PrPSc minor stability compared with that of the classical PrPSc. Thus, ws-PrP and classical PrP were captured from noninfected or scrapie-infected hamster brain homogenate [high-speed supernatant (SHS) and high-speed pellet (PHS)] and blood plasma by anti-PrP antibodies (3F4 and 6H4) and subjected to screening for glycans by lectins under denaturing or nondenaturing procedures in a sandwich lectin-ELISA. Glycans have been found in minor quantities and differently exposed on ws-PrPSc from SHS and plasma compared with classical PrPSc from PHS. These differences have been shown to be potentially responsible for the instability of ws-PrPSc. Treatment of infected blood with GdnHCl significantly (P<0.01) increased the detection of ws-PrPSc in ELISA, reflecting an increase in its stability, and showed efficacy in removing high-abundance proteins in silver-stained gels. This increase in ws-PrPSc stability is due to an interaction of GdnHCl not only with high-abundance proteins but also with the ws-PrPSc glycosylation with particular regard to the mannose sugar. Analysis of lectins immunoreactivity toward total proteins from plasma collected before and at different time points after infection revealed that mannose might exert a stabilizing effect toward all of hamster blood glycoproteins, regardless of scrapie infection. Since low levels of ws-PrPSc/soluble-infectivity have been estimated both in blood and brain of hamster, this glycosylation-related instability may have negatively influenced the propensity of ws-PrPC to convert to ws-PrPSc both in blood and the brain. Therefore, PrPC glycosylation characteristics may provide a tool for the determination risk of prion transmissibility.

Effect of scrapie incubation on the concentrations of plasma amino acids and L-lactate in infected lambs.

Three groups of two weeks old growing lambs differing in PrP genotype were orally inoculated with scrapie and maintained under defined conditions until disease endpoint. Plasma concentrations of free alanine and serine, but not L-lactate increased during the final 6 months of the disease. At the same time, plasma concentrations of several essential and non-essential free amino acids decreased linearly, indicating reduced feed intake and are consistent with, but occurring before establishment, of cachexia. These observations are consistent with those reported previously from studies on cattle infected with BSE and with the hypothesis that scrapie may effect peripheral tissue metabolism.