Molecular characterization of monoclonal antibodies that inhibit acetylcholinesterase by targeting the peripheral site and backdoor region.

The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis.

Impact of leucocyte depletion and prion reduction filters on TSE blood borne transmission.

The identification in the UK of 4 v-CJD infected patients thought to be due to the use of transfused Red Blood Cell units prepared from blood of donors incubating v-CJD raised major concerns in transfusion medicine. The demonstration of leucocyte associated infectivity using various animal models of TSE infection led to the implementation of systematic leuco-depletion (LD) of Red Blood cells concentrates (RBCs) in a number of countries. In the same models, plasma also demonstrated a significant level of infectivity which raised questions on the impact of LD on the v-CJD transmission risk. The recent development of filters combining LD and the capture of non-leucocyte associated prion infectivity meant a comparison of the benefits of LD alone versus LD/prion-reduction filters (LD/PR) on blood-borne TSE transmission could be made. Due to the similarity of blood/plasma volumes to human transfusion medicine an experimental TSE sheep model was used to characterize the abilities of whole blood, RBCs, plasma and buffy-coat to transmit the disease through the transfusion route. The impact of a standard RBCs LD filter and of two different RBCs LD/PR prototype filters on the disease transmission was then measured. Homologous recipients transfused with whole-blood, buffy-coat and RBCs developed the disease with 100% efficiency. Conversely, plasma, when intravenously administered resulted in an inconstant infection of the recipients and no disease transmission was observed in sheep that received cryo-precipitated fraction or supernatant obtained from infectious plasma. Despite their high efficacy, LD and LD/PR filtration of the Red Blood Cells concentrate did not provide absolute protection from infection. These results support the view that leuco-depletion strongly mitigates the v-CJD blood borne transmission risk and provide information about the relative benefits of prion reduction filters.

Highly efficient prion transmission by blood transfusion.

It is now clearly established that the transfusion of blood from variant CJD (v-CJD) infected individuals can transmit the disease. Since the number of asymptomatic infected donors remains unresolved, inter-individual v-CJD transmission through blood and blood derived products is a major public health concern. Current risk assessments for transmission of v-CJD by blood and blood derived products by transfusion rely on infectious titers measured in rodent models of Transmissible Spongiform Encephalopathies (TSE) using intra-cerebral (IC) inoculation of blood components. To address the biological relevance of this approach, we compared the efficiency of TSE transmission by blood and blood components when administrated either through transfusion in sheep or by intra-cerebral inoculation (IC) in transgenic mice (tg338) over-expressing ovine PrP. Transfusion of 200 µL of blood from asymptomatic infected donor sheep transmitted prion disease with 100% efficiency thereby displaying greater virulence than the transfusion of 200 mL of normal blood spiked with brain homogenate material containing 10³ID50 as measured by intracerebral inoculation of tg338 mice (ID50 IC in tg338). This was consistent with a whole blood titer greater than 10³·6ID50 IC in tg338 per mL. However, when the same blood samples were assayed by IC inoculation into tg338 the infectious titers were less than 32 ID per mL. Whereas the transfusion of crude plasma to sheep transmitted the disease with limited efficacy, White Blood Cells (WBC) displayed a similar ability to whole blood to infect recipients. Strikingly, fixation of WBC with paraformaldehyde did not affect the infectivity titer as measured in tg338 but dramatically impaired disease transmission by transfusion in sheep. These results demonstrate that TSE transmission by blood transfusion can be highly efficient and that this efficiency is more dependent on the viability of transfused cells than the level of infectivity measured by IC inoculation.

Prionemia and leukocyte-platelet-associated infectivity in sheep transmissible spongiform encephalopathy models.

The dynamics of the circulation and distribution of transmissible spongiform encephalopathy (TSE) agents in the blood of infected individuals remain largely unknown. This clearly limits the understanding of the role of blood in TSE pathogenesis and the development of a reliable TSE blood detection assay. Using two distinct sheep scrapie models and blood transfusion, this work demonstrates the occurrence of a very early and persistent prionemia. This ability to transmit disease by blood transfusion was correlated with the presence of infectivity in white blood cells (WBC) and peripheral blood mononucleated cells (PBMC) as detected by bioassay in mice overexpressing the ovine prion protein PrP (tg338 mice) and with the identification of abnormal PrP in WBC after using protein misfolding cyclic amplification (PMCA). Platelets and a large variety of leukocyte subpopulations also were shown to be infectious. The use of endpoint titration in tg338 mice indicated that the infectivity in WBC (per ml of blood) was 10(6.5)-fold lower than that in 1 g of posterior brainstem sample. In both WBC and brainstem, infectivity displayed similar resistance to PK digestion. The data strongly support the concept that WBC are an accurate target for reliable TSE detection by PMCA. The presence of infectivity in short-life-span blood cellular elements raises the question of the origin of prionemia.

Design, synthesis and studies of triphosphate analogues for the production of anti AZT-TP antibodies.

Triphosphates anabolites are the active chemical species of nucleosidic reverse transcriptase inhibitors in HIV-therapy. Herein, we describe (i) the design of stable triphosphate analogues of AZT using molecular modelling, (ii) their synthesis and (iii) their use for producing anti AZT-TP antibodies in the aim of developing an immunoassay for therapeutic drug monitoring.

Immunotherapeutic effect of anti-PrP monoclonal antibodies in transmissible spongiform encephalopathy mouse models: pharmacokinetic and pharmacodynamic analysis.

Prion diseases are transmissible neurodegenerative disorders for which no therapeutic or prophylactic regimens exist. Passive immunization with appropriate antibodies directed against the cellular form of the prion protein (PrPC) can delay the onset of prion disease after peripheral infection, but mechanisms and parameters determining their in vivo efficacy remain unknown. In the present study, we characterized the main pharmacokinetic properties of anti-PrP antibodies in different mouse models expressing various levels of PrPC (Prnp(0/0), C57BL/6 and tga20 mice) in correlation with therapeutic effect. Plasma levels of free antibodies, total endogenous PrPC and PrPC-antibody complexes were monitored after a single intraperitoneal monoclonal antibody (mAb) injection. Efficacy in delaying PrPSc peripheral accumulation seemed to be associated with mAb capacity to form long-lasting complexes with endogenous PrPC in the plasma. In agreement with previous observations on cellular models of transmissible spongiform encephalopathy infection, we observed that injection of anti-PrP antibodies induced a large (up to 100-fold) increase in circulating PrPC. Finally, the most efficient antibody extended the lifespan of infected animals greatly. These results allowed us to define critical characteristics of anti-PrP mAbs associated with therapeutic efficacy and could constitute a useful reference for designing optimized passive immunotherapies for prion diseases.

Peptide mass-assisted antibody cloning strategy for accurate characterization of potential therapeutic monoclonal antibodies against neurodegenerative diseases.

The development of therapeutic recombinant antibodies involves accurate characterization of immunoglobulin variable light (VL) and heavy (VH) chains. However, it has been reported that the use of subgroup or isotype-specific primers for the amplification of monoclonal antibody (mAb) variable domains introduces heterogeneities within the variable domains, or amplifies aberrant productive Ig domains. To address these issues, we have combined the rapid amplification of cDNA ends PCR (RACE-PCR) for the full-length VL and VH amplification, with peptide mass fingerprinting of the corresponding Ig chain. Using this strategy, we amplified full-length cDNA chains of SAF34 and SAF32, two potential therapeutic mAbs against neurodegenerative diseases directed to the prion protein (PrP). We report an unambiguous correlation between hybridoma cDNA sequences and protein fingerprints of the variable domains of each mAb, indicating the discrimination between mutated, pseudo-genes and functional Ig genes. As a proof of principle for this dual strategy of full-length PCR amplification of variable domains and their characterization by MALDI-TOF, we show that the corresponding scFvs recognize the native PrP and retain full capacity to bind to human PrP, as does the parental mAb. This finding addresses the need for reliable light and heavy chain characterization, a key factor for humanization of mouse antibodies and for its use in passive immunotherapy applications.

Transmissibility of atypical scrapie in ovine transgenic mice: major effects of host prion protein expression and donor prion genotype.

Atypical scrapie or Nor98 has been identified as a transmissible spongiform encephalopathy (TSE) that is clearly distinguishable from classical scrapie and BSE, notably regarding the biochemical features of the protease-resistant prion protein PrP(res) and the genetic factors involved in susceptibility to the disease. In this study we transmitted the disease from a series of 12 French atypical scrapie isolates in a transgenic mouse model (TgOvPrP4) overexpressing in the brain approximately 0.25, 1.5 or 6x the levels of the PrP(ARQ) ovine prion protein under the control of the neuron-specific enolase promoter. We used an approach based on serum PrP(c) measurements that appeared to reflect the different PrP(c) expression levels in the central nervous system. We found that transmission of atypical scrapie, much more than in classical scrapie or BSE, was strongly influenced by the PrP(c) expression levels of TgOvPrP4 inoculated mice. Whereas TgOvPrP4 mice overexpressing approximately 6x the normal PrP(c) level died after a survival periods of 400 days, those with approximately 1.5x the normal PrP(c) level died at around 700 days. The transmission of atypical scrapie in TgOvPrP4 mouse line was also strongly influenced by the prnp genotypes of the animal source of atypical scrapie. Isolates carrying the AF(141)RQ or AHQ alleles, associated with increased disease susceptibility in the natural host, showed a higher transmissibility in TgOvPrP4 mice. The biochemical analysis of PrP(res) in TgOvPrP4 mouse brains showed a fully conserved pattern, compared to that in the natural host, with three distinct PrP(res) products. Our results throw light on the transmission features of atypical scrapie and suggest that the risk of transmission is intrinsically lower than that of classical scrapie or BSE, especially in relation to the expression level of the prion protein.

Pilot pharmacokinetic study of human immunodeficiency virus-infected patients receiving tenofovir disoproxil fumarate (TDF): investigation of systemic and intracellular interactions between TDF and abacavir, lamivudine, or lopinavir-ritonavir.

Previous work has demonstrated the existence of systemic interaction between tenofovir (TFV) disoproxil fumarate (TDF) and didanosine as well as between TDF and lopinavir-ritonavir (LPV/r). Here we investigated TDF interactions with the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine (3TC) and abacavir (ABC), comparing both the concentrations of nucleoside/nucleotide reverse transcriptase inhibitors in plasma and the intracellular concentrations of their triphosphate metabolites (NRTI-TP) for human immunodeficiency virus-infected patients receiving these NRTIs with TDF and after 4 weeks of TDF interruption. We also looked at interactions between TDF-ABC and LPV/r, comparing patients receiving or not receiving LPV/r. Blood samples were taken at baseline and at 1, 2, and 4 h after dosing. Liquid chromatography-tandem mass spectrometry was used to measure NRTIs and NRTI-TPs. Statistical analyses were performed on pharmacokinetic parameters: the area under the concentration-time curve from 0 to 4 h (AUC(0-4)), the maximum concentration of the drug (C(max)), and the residual concentration of the drug at the end of the dosing interval (C(trough)) for plasma and the AUC(0-4) and C(trough) for intracellular data. Among the groups of patient discontinuing TDF, the very long intracellular half-life of elimination (150 h) of TFV-DP (the diphosphorylated metabolite of TFV, corresponding to a triphosphorylated species) was confirmed. Comparison between groups as well as the longitudinal study showed no significant systemic or intracellular interaction between TDF and ABC or 3TC. Significant differences were observed between patients receiving LVP/r and those receiving nevirapine. For ABC, plasma exposure was decreased (40%) under LVP/r, while, in contrast, plasma exposure to TFV was increased by 50% and the intracellular TFV-DP AUC(0-4) was increased by 59%. A trend for a gender effect was observed for TFV-DP at the intracellular level, with higher and C(trough) values for women.

Prions in milk from ewes incubating natural scrapie.

Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrP(Sc) accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 microg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species.

Characterization of truncated forms of abnormal prion protein in Creutzfeldt-Jakob disease.

In prion disease, the abnormal conformer of the cellular prion protein, PrP(Sc), deposits in fibrillar protein aggregates in brain and other organs. Limited exposure of PrP(Sc) to proteolytic digestion in vitro generates a core fragment of 19-21 kDa, named PrP27-30, which is also found in vivo. Recent evidence indicates that abnormal truncated fragments other than PrP27-30 may form in prion disease either in vivo or in vitro. We characterized a novel protease-resistant PrP fragment migrating 2-3 kDa faster than PrP27-30 in Creutzfeldt-Jakob disease (CJD) brains. The fragment has a size of about 18.5 kDa when associated with PrP27-30 type 1 (21 kDa) and of 17 kDa when associated with type 2 (19 kDa). Molecular mass and epitope mapping showed that the two fragments share the primary N-terminal sequence with PrP27-30 types 1 and 2, respectively, but lack a few amino acids at the very end of C terminus together with the glycosylphosphatidylinositol anchor. The amounts of the 18.5- or 17-kDa fragments and the previously described 13-kDa PrP(Sc) C-terminal fragment relatively to the PrP27-30 signal significantly differed among CJD subtypes. Furthermore, protease digestion of PrP(Sc) or PrP27-30 in partially denaturing conditions generated an additional truncated fragment of about 16 kDa only in typical sporadic CJD (i.e. MM1). These results show that the physicochemical heterogeneity of PrP(Sc) in CJD extends to abnormal truncated forms of the protein. The findings support the notion of distinct structural "conformers" of PrP(Sc) and indicate that the characterization of truncated PrP(Sc) forms may further improve molecular typing in CJD.

Prions impair bioaminergic functions through serotonin- or catecholamine-derived neurotoxins in neuronal cells.

The conversion of the cellular prion protein, PrP(C), to an abnormal isoform, PrP(Sc), is a central event leading to neurodegeneration in prion diseases. Deciphering the molecular and cellular changes imparted by PrP(Sc) accumulation remains an arduous task due to the small number of cell lines supporting prion replication. Here we introduce the 1C11 cell line as a new in vitro model to investigate prion pathogenesis. This cell line is a committed neuroectodermal progenitor able to differentiate into fully functional serotonergic or catecholaminergic neurons. 1C11 cells, which naturally express PrP(C) from the undifferentiated state, can be chronically infected with various prion strains. Prion infection does not promote any noticeable phenotypic change in the progenitor cells nor prevent the onset of the serotonergic and catecholaminergic differentiation programs. Pathogenic prions, however, deviate the overall neurotransmitter-metabolism in both pathways by decreasing bioamine synthesis, storage, and transport, and enhancing catabolism. Noteworthy, oxidized derivatives of both serotonin and catecholamines are selectively detected in the differentiated progenies of infected cells and contribute to irreversible impairment in bioamine synthesis. Finally, the level of PrP(Sc) accumulation, that of infectivity, and the extent of all prion-induced changes in infected cells appear to be correlated. The report of such specific effects of infection on neuronal functions provides a foundation for dissecting the events underlying loss of neuronal homeostasis in prion diseases.

Beyond PrP res type 1/type 2 dichotomy in Creutzfeldt-Jakob disease.

Sporadic Creutzfeldt-Jakob disease (sCJD) cases are currently subclassified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the proteinase K (PK) digested abnormal prion protein (PrPres)identified on Western blotting (type 1 or type 2). These biochemically distinct PrPres types have been considered to represent potential distinct prion strains. However, since cases of CJD show co-occurrence of type 1 and type 2 PrPres in the brain, the basis of this classification system and its relationship to agent strain are under discussion. Different brain are as from 41 sCJD and 12 iatrogenic CJD (iCJD) cases were investigated, using Western blotting for PrPres and two other biochemical assays reflecting the behaviour of the disease-associated form of the prion protein (PrPSc) under variable PK digestion conditions. In 30% of cases, both type 1 and type 2 PrPres were identified. Despite this, the other two biochemical assays found that PrPSc from an individual patient demonstrated uniform biochemical properties. Moreover, in sCJD, four distinct biochemical PrPSc subgroups were identified that correlated with the current sCJD clinico-pathological classification. In iCJD, four similar biochemical clusters were observed, but these did not correlate to any particular PRNP 129 polymorphism or western blot PrPres pattern. The identification of four different PrPSc biochemical subgroups in sCJD and iCJD, irrespective of the PRNP polymorphism at codon 129 and the PrPres isoform provides an alternative biochemical definition of PrPSc diversity and new insight in the perception of Human TSE agents variability.

Beyond PrP9res) type 1/type 2 dichotomy in Creutzfeldt-Jakob disease.

Sporadic Creutzfeldt-Jakob disease (sCJD) cases are currently subclassified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the proteinase K (PK) digested abnormal prion protein (PrP(res)) identified on Western blotting (type 1 or type 2). These biochemically distinct PrP(res) types have been considered to represent potential distinct prion strains. However, since cases of CJD show co-occurrence of type 1 and type 2 PrP(res) in the brain, the basis of this classification system and its relationship to agent strain are under discussion. Different brain areas from 41 sCJD and 12 iatrogenic CJD (iCJD) cases were investigated, using Western blotting for PrP(res) and two other biochemical assays reflecting the behaviour of the disease-associated form of the prion protein (PrP(Sc)) under variable PK digestion conditions. In 30% of cases, both type 1 and type 2 PrP(res) were identified. Despite this, the other two biochemical assays found that PrP(Sc) from an individual patient demonstrated uniform biochemical properties. Moreover, in sCJD, four distinct biochemical PrP(Sc) subgroups were identified that correlated with the current sCJD clinico-pathological classification. In iCJD, four similar biochemical clusters were observed, but these did not correlate to any particular PRNP 129 polymorphism or western blot PrP(res) pattern. The identification of four different PrP(Sc) biochemical subgroups in sCJD and iCJD, irrespective of the PRNP polymorphism at codon 129 and the PrP(res) isoform provides an alternative biochemical definition of PrP(Sc) diversity and new insight in the perception of Human TSE agents variability.

Rapid typing of transmissible spongiform encephalopathy strains with differential ELISA.

The bovine spongiform encephalopathy (BSE) agent has been transmitted to humans, leading to variant Creutzfeldt-Jakob disease. Sheep and goats can be experimentally infected by BSE and have been potentially exposed to natural BSE; however, whether BSE can be transmitted to small ruminants is not known. Based on the particular biochemical properties of the abnormal prion protein (PrPsc) associated with BSE, and particularly the increased degradation induced by proteinase K in the N terminal part of PrPsc, we have developed a rapid ELISA designed to distinguish BSE from other scrapie strains. This assay clearly discriminates experimental ovine BSE from other scrapie strains and was used to screen 260 transmissible spongiform encephalopathy (TSE)-infected small ruminant samples identified by the French active surveillance network (2002/2003). In this context, this test has helped to identify the first case of natural BSE in a goat and can be used to classify TSE isolates based on the proteinase K sensitivity of PrPsc.

Excretion of BSE and scrapie prions in stools from murine models.

Faeces from infected animals have been suggested as a potential source of contamination and transmission of prion diseases in the environment. This work describes the development of a procedure for the detection of PrP(res) in stools which is based on a detergent-based extraction and immunoprecipitation (IP). The procedure was evaluated by analyzing TSE-spiked sheep and mice faeces, and proved to be specific for PrP(res) with sensitivities of 5-10 microg of infected brain tissue. In order to analyze the shedding of prions, we studied stools from orally inoculated mice over 4-days post-inoculation and also stools from terminally sick scrapie-infected mice. PrP(res) was only detected in stools shortly after the oral ingestion of TSE agents. The procedure described could be a useful tool for studying the excretion of prions and for evaluating potential environmental contamination by prions.

High levels of zidovudine (AZT) and its intracellular phosphate metabolites in AZT- and AZT-lamivudine-treated newborns of human immunodeficiency virus-infected mothers.

Newborns from human immunodeficiency virus-infected mothers are given antiretroviral prophylaxis against mother-to-child transmission, including predominantly nucleoside reverse transcriptase inhibitors. Pharmacological monitoring of these drugs in newborns has so far been limited to plasma and cord blood. In this study, samples from newborns (up to 45 days old) treated with zidovudine (AZT) alone (n = 29) or in combination with lamivudine (3TC) (n = 20) were analyzed for both intracellular concentrations of phosphate metabolites in peripheral blood mononuclear cells and levels of parent drugs in plasma. Plasma AZT and intracellular AZT-monophosphate and AZT-triphosphate (TP) concentrations were significantly higher during the first 15 days of life (199 versus 52.7 ng/ml [P < 0.0001], 732 versus 282 fmol/10(6) cells [P < 0.0001], and 170 versus 65.1 fmol/10(6) cells [P < 0.0001], respectively) and then became comparable to those of adults. No difference in intracellular AZT metabolite concentrations was found when AZT- and AZT-3TC-treated groups were compared. Plasma 3TC levels (lower limit of quantification [LLOQ], 1,157 ng/ml; median, 412.5 ng/ml) were not associated with the newborn's age, gender, or weight. Intracellular 3TC-TP concentrations (LLOQ, 40.4 pmol/10(6) cells; median, 18.9 pmol/10(6) cells) determined for newborns receiving the AZT-3TC combination were associated with neither the age nor weight of the newborns. Concentrations in females were significantly higher (1.8-fold [P = 0.0415]) than those in males. Unexpectedly, newborns on AZT monotherapy whose mothers' treatment included 3TC displayed residual plasma 3TC and intracellular 3TC-TP levels up to 1 week after birth.

Progress and limits of TSE diagnostic tools.

Following the two "mad cow" crises of 1996 and 2000, there was an urgent need for rapid and sensitive diagnostic methods to identify animals infected with the bovine spongiform encephalopathy (BSE) agent. This stimulated research in the field of prion diagnosis and led to the establishment of numerous so-called "rapid tests" which have been in use in Europe since 2001 for monitoring at-risk populations (rendering plants) and animals slaughtered for human consumption (slaughterhouse). These rapid tests have played a critical role in the management of the mad cow crisis by allowing the removal of prion infected carcasses from the human food chain, and by allowing a precise epidemiological monitoring of the BSE epizootic. They are all based on the detection of the abnormal form of the prion protein (PrP(Sc) or PrP(res)) in brain tissues and consequently are only suitable for post-mortem diagnosis. Since it is now very clear that variant Creutzfeldt-Jakob disease (vCJD) can be transmitted by blood transfusion, the development of a blood test for the diagnosis of vCJD is a top priority. Although significant progress has been made in this direction, including the development of the protein misfolding cyclic amplification (PMCA) technology, at the time this paper was written, this objective had not yet been achieved. This is the most important challenge for the years to come in this field of prion research.

Human prion diseases: from antibody screening to a standardized fast immunodiagnosis using automation.

Demonstration of pathological prion protein accumulation in the central nervous system is required to establish the diagnosis of transmissible subacute encephalopathies. In humans, this is frequently achieved using prion protein immunohistochemistry in paraffin-embedded tissue, a technique that requires multiple epitope retrieval and denaturing pretreatments. In addition to being time-consuming, this procedure induces tissue alterations that preclude accurate morphological examination. The aim of this study was to simplify prion protein immunohistochemistry procedure in human tissue, together with increased sensitivity and specificity. We screened a panel of 50 monoclonal antibodies produced using various immunogens (human and ovine recombinant prion protein, prion protein peptides, denatured scrapie-associated fibrils from 263K-infected Syrian hamsters) and directed against different epitopes along the human prion protein sequence. A panel of different forms of genetic, infectious and sporadic transmissible subacute encephalopathies was assessed. The monoclonal 12F10 antibody provided a high specificity and fast immunodiagnosis with very limited denaturing pretreatments. A standardized and reliable fast immunostaining procedure was established using an automated diagnostic system (Nexes, Ventana Medical Systems) and allowed prion protein detection in the central nervous system and in tonsil biopsies. It was evaluated in a series of 300 patients with a suspected diagnosis of transmissible subacute encephalopathies and showed high sensitivity and specificity.

Absolute and relative quantification of sheep brain prion protein (PrP) allelic variants by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

Transmissible spongiform encephalopathies (TSEs) are characterised by the accumulation in the tissues of affected individuals of an abnormal form (PrP(Sc)) of a protein naturally produced by the host, the cellular prion protein (PrP(C)). In sheep, susceptibility to TSEs is tightly controlled by polymorphism at positions 136 (A or V), 154 (R or H) and 171 (R or Q) of the Prnp gene encoding the prion protein (PrP). Quantification of PrP variants at positions 136, 154 and 171 can be achieved by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometric analysis of the respective peptides 114-139, 152-159 and 160-171 obtained after tryptic digestion of the PrP protein. In this study we quantified the tryptic peptide 114-139 containing the first polymorphic site. Quantification was either relative, between variants of this peptide, or absolute with respect to the C-terminally (18)O-labelled peptide obtained by hydrolysing known amounts of recombinant protein with trypsin in H(2) (18)O. After purification of PrP(C) and PrP(Sc) from the brain of two heterozygous sheep carrying either the ARQ/VRQ or ARR/VRQ genotypes, the proportion of each variant was measured. In the ARQ/VRQ animal, while both variants were equally represented in the normal isoform, the VRQ variant was predominantly found in the abnormal PrP protein, suggesting dissimilar behaviour of the two variants in the pathological process. The situation was even more contrasted in the ARR/VRQ animal where PrP(Sc) was solely composed of the VRQ variant. These two examples clearly illustrate the value of MALDI-TOF analysis, combined with appropriate immunopurification techniques, in seeking a precise understanding of the influence of PrP polymorphisms on TSE pathogenesis.