Assessing the presence of BSE and scrapie in slaughterhouse wastewater.

AIMS: This paper describes a procedure for evaluating the presence and the stability of the proteinase K-resistant form of the prion protein (PrP(res)) in slaughterhouse wastewater. METHODS AND RESULTS: Wastewater samples were spiked with either scrapie or bovine spongiform encephalopathy agents and PrP(res) was concentrated and detected by western blotting. The detection limit was estimated to be 2-4 microg of either scrapie or BSE-infected brain tissue in 15 ml of sewage. Wastewater samples from three abattoirs were analysed, two of which had processed BSE-infected animals. No PrP(res) was detected. The effect of sewage on the inoculum and the persistence of transmissible spongiform encephalopathy agents in wastewater were also considered. CONCLUSIONS: The results of the assay suggest that wastewaters from abattoirs where one positive BSE case has been identified would contain titres lower than 0.6-26 x 10(-4) cattle oral ID(50) per litre resulting from specified risk material tissue contamination. Moreover, the effect of abattoir wastewaters is to reduce the persistence of PrP(res). SIGNIFICANCE AND IMPACT OF THE STUDY: The assay may be a useful tool for risk assessment studies and for reducing the potential risk of contamination with BSE via sewage sludge fertilizer procedures.

Efficacy of adjuvant chemotherapy according to Prion protein expression in patients with estrogen receptor-negative breast cancer.

BACKGROUND: Prion protein (PrPc) has been previously reported to be associated with resistance to proapoptotic stimuli. We evaluated whether the expression of PrPc was associated with the resistance to adjuvant chemotherapy in patients with estrogen receptor (ER) -negative breast cancer. PATIENTS AND METHODS: The expression of PrPc by primary tumors was assessed by immunohistochemistry in a series of 756 patients included in two randomized trials that compared anthracycline-based chemotherapy to no chemotherapy. The PrPc expression was correlated with ER expression and the benefit of adjuvant chemotherapy was assessed according to PrPc expression in patients with ER-negative tumors. RESULTS: Immunostaining analysis showed that PrPc was mainly expressed by myoepithelial cells in normal breast tissue. Tissue microarray analysis from 756 breast tumors showed that PrPc was associated with ER-negative breast cancer subsets (P < 0.001). Adjuvant chemotherapy was not associated with a significant risk reduction for death in patients with ER-negative/PrPc-positive disease [adjusted hazard ratio (HR) for death = 0.98, 95% confidence interval (CI) 0.45-2.1, P = 0.95], while it decreased the risk for death (HR = 0.39, 95% CI 0.2-0.74, P = 0.004) in patients with ER-negative/PrPc-negative tumors. CONCLUSION: These data indicate that ER-negative/PrPc-negative phenotype is associated with a high sensitivity to adjuvant chemotherapy.

Discrimination of sheep susceptible and resistant to transmissible spongiform encephalopathies by an haplotype specific monoclonal antibody.

In the present report, the selective detection of sheep PrP haplotypes by monoclonal antibody 2A11 is described. It is showed that the substitution of glutamine by arginine but not by histidine at ovine PrP position 171 abolishes completely the recognition of either PrP(c) or PrP(d) by mAb 2A11, in such a way that the application of this antibody allows the unambiguous discrimination of R(171) homozygotes. On the basis of the high resistance to classical scrapie and bovine spongiform encephalophaty (BSE) infection associated to the R(171) PrP haplotype, animals bearing the ARR allele are currently selected within the scrapie national plan initiated in Great Britain. A 2A11-based immuno enzymatic test have been developed and evaluated using a panel of plasma and sera from sheep of different PrP genotypes and breeds. The test allows the efficient discrimination of R(171) homozygotes, R(171) heterozygotes and non-R(171) carriers, therefore offering a rapid, cheap and easy to use alternative method to select sheep for their resistance to scrapie.

Immunoreactivity enhancement with chelators for increasing the detection sensitivity of human PrPSc by Western blotting.

Prion diseases are neurodegenerative disorders affecting humans as Creutzfeldt-Jakob disease. The host-encoded prion protein (PrP(C)) will be converted into a structurally altered isoform (PrP(Sc)). PrP(Sc) differ in sizes and glycoform patterns and can be identified using molecular typing with Western blotting. The electrophoretic mobility of PrP(Sc) changes on treatment with metal ions or chelators prior to digestion with proteases. The effects of chelators applied to PrP(Sc) after protease digestion had not been examined in detail, we investigated these effects in this study. Application of EDTA, NTA and DTPA, and to a lesser extent EGTA, significantly enhanced PrP(Sc) signals in immunoblots. PrP(Sc) intensities increased two- to three-fold compared with untreated PrP(Sc). Since the immunoblot method is highly specific, sensitivity is the limiting factor. Enhancing sensitivity might be important in the determination of PrP(Sc) at levels close to or just below the limits of detection. It is to be expected that application of chelators to digested protein samples will increase the sensitivity of PrP(Sc) detection using the Western blot technique.

Immunohistochemical features of PrP(d) accumulation in natural and experimental goat transmissible spongiform encephalopathies.

Scrapie is a transmissible spongiform encephalopathy (TSE) or prion disease, which naturally affects sheep and goats. Immunohistochemical epitope mapping of abnormal PrP accumulations (PrP(d)) in brain can help in characterizing sheep TSE sources or strains and in identifying potential bovine spongiform encephalopathy (BSE) infections of sheep. Natural and experimental TSE infections of goats were examined to determine whether the epitope mapping approach could also be applied to aid recognition of BSE infection in goats. Goats experimentally infected with the SSBP/1 or CH1641 sheep scrapie strains or with cattle BSE, together with four field cases of natural TSE in goats, were examined immunohistochemically with six different antibodies. CH1641 and SSBP/1 infections in goats, as in sheep, showed PrP(d) accumulations which were mainly intracellular. Some differences in targeting, particularly of Purkinje cells, was evident in inter-species comparisons of CH1641 and SSBP/1. PrP(d) labelling of goat BSE experimental cases showed extensive intracellular and extracellular accumulations, also similar to those in sheep BSE. Intra-neuronal PrP(d) in both goat and sheep BSE was labelled only by antibodies recognizing epitopes located C-terminally of residue His99, whereas in natural sheep TSE sources, and in sheep and goat SSBP/1, PrP(d) was also detected by antibodies to epitopes located between residues Trp93 and His99. Testing of four natural goat TSE samples showed one case in which epitope mapping characteristics and the overall patterns of PrP(d) accumulation was identical with those of experimental goat BSE. The four natural goat scrapie cases examined showed some degree of immunohistochemical phenotype variability, suggesting that multiple strains exist within the relatively small UK goat population.

[Screening tests for animal TSE: present and future]. / Tests de dépistage des ESST animales: présent et futur.

In 1999, three rapid tests (Prionics, Bio-Rad, Enfer) have been validated by the European Commission for the post-mortem diagnosis of BSE in cattle. They are now used on a large scale over the entire Europe. In absence of antibodies specifically recognizing the native conformation PrPres, its selective determination is based on the biochemical properties of this abnormal form (PK resistance, aggregation in presence of detergents). In addition, all these tests include a denaturation step so that PrP can be detected by appropriate antibodies. When applied on "risk populations" or on "healthy animals" entering into the human food chain, these rapid tests have provided a better estimation of the epizootic and allowed an efficient removal of animals bearing a risk for human consumption. Since 2002, they have also been used for the post-mortem diagnosis of scrapie in sheep and goat. Five new tests have been recently evaluated (ID-Lelystad; Perkin-elmer, Prionics Check LIA, UCSF, Imperial college) but it is too early to know which place they will take in the field. Current tests allow a preclinical diagnosis of TSE, especially in sheep and goats for which a very early detection is possible in peripheral lymphoid tissues. However, to date, no test on living animal has been validated. Taking into account the important number of research teams now involved on this topic one may expect spectacular progress in the forthcoming years.

Abnormal prion protein in genetically resistant sheep from a scrapie-infected flock.

The central molecular event in transmissible spongiform encephalopathies, such as scrapie in sheep, is the accumulation in tissues of an abnormal isoform of the cellular prion protein. A previous investigation of 26 sheep showed that the accumulation of PrP(res) in brain correlated more with the prnp genotype than with the severity of the clinical disease. Here, the ability of a sandwich ELISA to detect PrP(res) distribution in the brain was demonstrated. Immunohistochemistry also strongly supported the hypothesis that the dorsal motor nucleus of the vagus nerve is the possible entry site in the brain for the scrapie agent. Remarkably, three asymptomatic (or possibly asymptomatic for scrapie) sheep carrying an allele known to be associated with clinical scrapie resistance (ARR), which were negative for the detection of PrP(res) by Western blotting and immunohistochemistry, were positive for the presence of PrP(res) by ELISA, raising the possibility of carriers resistant to the disease and possibly contributing to the persistence of scrapie in certain flocks.

PrPSc accumulation in myocytes from sheep incubating natural scrapie.

Because variant Creutzfeldt-Jakob disease (vCJD) in humans probably results from consumption of products contaminated with tissue from animals with bovine spongiform encephalopathy, whether infectious prion protein is present in ruminant muscles is a crucial question. Here we show that experimentally and naturally scrapie-affected sheep accumulate the prion protein PrP(Sc) in a myocyte subset. In naturally infected sheep, PrP(Sc) is detectable in muscle several months before clinical disease onset. The relative amounts of PrP(Sc) suggest a 5,000-fold lower infectivity for muscle as compared to brain.

A general method allowing the design of oligonucleotide primers to amplify the variable regions from immunoglobulin cDNA.

The amplification of variable regions of immunoglobulins by reverse transcription polymerase chain reaction (RT-PCR) has become an invaluable technique either for the cloning of monoclonal antibodies (mAbs), or for the building of single-chain fragment variable (ScFv) libraries. Numerous applications have been described either for studying the antigen-antibody interactions or for medical purposes, with the recent development of recombinant antibodies for therapeutic use. Several publications by different groups have reported primer sequences to perform such amplification, but the strategy used to design these primers, and particularly the way of performing the necessary alignments, generally appear poorly detailed. In the present work, we propose a rational method of designing primers in order to amplify the variable region of heavy chain (VH) and variable region of light chain (VL) domains for framework 1 (FR1) of immunoglobulins. The described sets of primers have been designed to hybridize with the entire VH and VL mouse repertory without modification of amino acids since amino acids of framework 1 play a role in the folding, and thus in the functionality, of recombinant antibody. These primers have been applied to the cloning of monoclonal antibodies previously produced in the laboratory. This approach can be extended to other species or members of the immunoglobulin superfamily.

Overexpression of PrPc triggers caspase 3 activation: potentiation by proteasome inhibitors and blockade by anti-PrP antibodies.

We examined the influence of cellular prion protein (PrPc) in the control of cell death in stably transfected HEK293 cell line and in the PrPc-inducible Rov9 cells. PrPc expression in stably transfected HEK293 human cells did not modify basal apoptotic tonus but drastically potentiated staurosporine-stimulated cellular toxicity and DNA fragmentation as well as caspase 3-like activity and immunoreactivity. An identical staurosporine-induced caspase 3 activation was observed after doxycycline in the PrPc-inducible Rov9 cell line. Interestingly, proteasome inhibitors increase PrPc-like immunoreactivity and unmasked a basal caspase 3 activation. Conversely, we show that anti-PrPc antibodies sequestrate PrPc at the cell surface and drastically lower PrPc-dependent caspase activation. We suggest that intracellular PrPc could sensitize human cells to pro-apoptotic phenotype and that blockade of PrPc internalization could be a track to prevent intracellular toxicity associated with PrPc overexpression.

A marked disparity between the expression of prion protein and its message by neurones of the CNS.

Expression of the normal cellular form of prion protein is both necessary and rate-limiting in the spread of prion disease, yet its cellular expression in vivo is poorly understood. To optimise immunohistochemical labelling of this protein in mouse brain, we have developed novel antibodies that recognise cellular prion protein in glutaraldehyde-fixed tissue. Expression was found to be predominantly neuronal, and to differ between different classes of neurone. Thus, neurones immunoreactive for GABA expressed very high levels of normal prion protein; most projection neurones expressed much lower levels, particularly on their axons in the major fibre tracts, and some neurones (e.g. those positive for dopamine) displayed no detectable prion protein. In marked contrast, all neurones, even those that were immunonegative, expressed high levels of message for prion protein, shown by non-radioactive in situ hybridisation. Glia expressed very low levels of message, and undetectable levels of prion protein. We conclude that the steady-state level of prion protein, which differs so markedly between different neuronal types, is primarily controlled post-transcriptionally, possibly by differences in protein trafficking or degradation. These marked differences in the way different neurones produce and/or degrade their normal cellular prion protein may influence the selective spread and neurotoxic targeting of prion diseases within the CNS.

Rapid test for the preclinical postmortem diagnosis of BSE in central nervous system tissue.

The efficacy of a rapid test for detecting PrP(Sc) in central nervous system tissue was evaluated for the postmortem diagnosis of BSE at different times during the course of the disease. One hundred and six samples of brain, at the level of the medulla oblongata, and spinal cord, derived from the experimental study of the pathogenesis of BSE carried out in Great Britain between 1991 and 1995, were examined. PrP(Sc) was detected in the samples from most of the exposed animals killed 32 months or more after they had been exposed to the agent, and before the onset of clinical signs which were first recorded at 35 months. Comparisons with the results of histology, fibril detection, PrP immunohistochemistry and mouse bioassay indicated that the rapid test is at least as sensitive as these conventional confirmatory diagnostic methods and its result can be obtained more quickly.

Direct determination of phosphorylated intracellular anabolites of stavudine (d4T) by liquid chromatography/tandem mass spectrometry.

The objective was to develop and validate a routine assay for active intracellular anabolites of stavudine (d4T), a nucleoside reverse transcriptase inhibitor in human PBMC, applicable to pharmacokinetic studies and treatment monitoring. This was achieved using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), which theoretically allies optimum sensitivity, specificity and high sample throughput. After cellular lysis in a Tris/methanol buffer, the extract spiked with 2[H(8)]-ATP (internal standard) is directly injected into the LC/MS/MS system. Phosphorylated metabolites of d4T as well as deoxythymidine-triphosphate, the competitor on the reverse transcriptase, are separated from d4T on a reverse-phase microbore column with ion pairing. The detection is performed in the multiple reaction monitoring (MRM) mode after drug ionisation in negative mode electrospray. The limit of quantitation for d4T-TP was 138 fmol per 7 mL blood (9.8 fmol per 10(6) cells) and CV% for repeatability and intermediate precision were lower than 15%. Stability of compounds was checked before and during the process of isolation of PBMC. Cellular samples from several d4T-treated patients were successfully analysed using this method and d4T-triphosphate and deoxythymidine triphosphate were recovered. In conclusion, we have developed and validated a routine LC/MS/MS method that allows the simultaneous determination of mono-, di- and triphosphorylated anabolites of d4T in PBMC as well as the natural corresponding triphosphate in one analysis. For the first time, the chain terminator ratio (d4T-TP/dT-TP) could be directly measured. This method can be used simply and routinely on more than 35 samples per day. Extension to other nucleoside analogues is under development.

The disintegrins ADAM10 and TACE contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein.

We showed previously that PrPc undergoes constitutive and phorbol ester-regulated cleavage inside the 106-126 toxic domain of the protein, leading to the production of a fragment referred to as N1. Here we show by a pharmacological approach that o-phenanthroline, a general zinc-metalloprotease inhibitors, as well as BB3103 and TAPI, the inhibitors of metalloenzymes ADAM10 (A disintegrin and metalloprotease); and TACE, tumor necrosis factor alpha-converting enzyme; ADAM17), respectively, drastically reduce N1 formation. We set up stable human embryonic kidney 293 transfectants overexpressing human ADAM10 and TACE, and we demonstrate that ADAM10 contributes to constitutive N1 production whereas TACE mainly participates in regulated N1 formation. Furthermore, constitutive N1 secretion is drastically reduced in fibroblasts deficient for ADAM10 whereas phorbol 12,13-dibutyrate-regulated N1 production is fully abolished in TACE-deficient cells. Altogether, our data demonstrate for the first time that disintegrins could participate in the catabolism of glycosyl phosphoinositide-anchored proteins such as PrPc. Second, our study identifies ADAM10 and ADAM17 as the protease candidates responsible for normal cleavage of PrPc. Therefore, these disintegrins could be seen as putative cellular targets of a therapeutic strategy aimed at increasing normal PrPc breakdown and thereby depleting cells of the putative 106-126 "toxic" domain of PrPc.

The production and evaluation of antibodies for enzyme immunoassay of AZTTP.

We describe the development of the first enzyme immunoassay for quantifying AZTTP that does not use of radioactive labeling. Anti-AZTTP antibodies were raised in rabbits by immunizing with an AZTTP-kelhoyle limpet hemocyanin (KLH) conjugate. Competitive immunoassays indicated a nanomolar sensitivity to AZTTP. One of the antisera produced was specific for AZTTP.

Pharmacodynamic and receptor binding changes during chronic lorazepam administration.

To assess pharmacodynamic and neurochemical aspects of tolerance, lorazepam (2 mg/kg/day), or vehicle was administered chronically to male Crl: CD-1(ICR)BR mice via implantable osmotic pump. Open-field behavior, benzodiazepine receptor binding in vitro, receptor autoradiography, and muscimol-stimulated chloride uptake were examined at both 1 and 14 days. Open-field activity was depressed in lorazepam-treated animals on Day 1. On Day 14, open-field parameters were indistinguishable from those of vehicle-treated animals, indicating behavioral tolerance. Benzodiazepine binding, as determined by the specific binding of [125I]diazepam, was also decreased in cortex on Day 14. Hippocampal binding was unchanged following chronic lorazepam exposure. Apparent affinity in cortical membrane preparations was unchanged, indicating that altered ligand uptake was due to decreased receptor number. Muscimol-stimulated chloride uptake into cortical synaptoneurosomes from lorazepam-treated animals was not significantly different on Day 1 or Day 14 compared to vehicle-treated animals. These results confirm that down-regulation of benzodiazepine receptor binding is closely associated with behavioral tolerance to benzodiazepines. These observed changes in binding are not necessarily associated with robust changes in receptor function.

Use of free radical chemistry in an immunometric assay for 17 beta-estradiol.

BACKGROUND: We wished to develop an enzyme immunometric assay for 17 beta-estradiol (E2) in human serum using solid-phase immobilized epitope immunoassay (SPIE-IA) technology and free radical chemistry. METHODS: We used an anti-estradiol monoclonal antibody as capture antibody and Fenton-like reagents to cross-link it to E2. The same antibody, labeled with acetylcholinesterase, was used for detection. Serum was diluted 10-fold before assay. RESULTS: After correction by the dilution factor, the detection limit was 5 ng/L for human serum and intra- and interassay CVs were <7% and 15%, respectively, at concentrations of 169-2845 ng/L. No cross-reactivity was seen with other natural steroids. In comparison with a competitive commercial RIA performed on 88 undiluted human sera, the slope (SD) of the regression line was 1.05 (+/- 0.02) and the intercept was 47 (+/-27) ng/L (S(y/x) = 186 ng/L) at concentrations of 20-5000 ng/L (r(2) = 0.97). CONCLUSIONS: The use of Fenton-like chemistry in SPIE-IA technology allows a sensitive measurement of E2 in human serum and could be a new approach for the development of sensitive immunoassays.

Pharmacological in vitro evaluation of new substance P-cyclodextrin derivatives designed to drug targeting towards NK1-receptor bearing cells.

Some biological properties of new bifunctional conjugates designed for drug targeting were evaluated through in vitro experiments. Eight peptidylcyclodextrin compounds were used, which correspond to modified beta- or gamma-cyclodextrin (CD) grafted on neuropeptide substance P (SP) or a shorter derivative (SP(4-11)). Using anti-SP and anti-CD antibodies as molecular probes, we showed that the main structural features of the two moieties of these adducts were preserved. Binding experiments, using CHO cells expressing the human SP-specific NK1 receptor, demonstrated the functionality of all peptidylcyclodextrin derivatives, which exhibited IC50 values in a 10(-9)-10(-7) M range. All compounds were able to induce a pharmacological response, triggering phosphatidylinositol turnover with EC50 values in the same range as the natural ligand. Moreover, autoradiography analysis of rat spinal corn sections proved that [125I]SP binding was dose-dependently displaced by one selected compound (a gamma-CD-SP), showing a similar affinity of this adduct for the rat neurokinin 1 receptor. Our observations demonstrate that these peptidylcyclodextrins efficiently target NK1 receptor-expressing cells.

A monoclonal antibody directed against the neurokinin-1 receptor contains a peptide sequence with similar hydropathy and functional properties to substance P, the natural ligand for the receptor.

Monoclonal antibody (mAb) PS12, obtained using the complementary peptide methodology, mimics the neuropeptide substance P (SP) in recognizing the SP-binding domain of the neurokinin-1 receptor (NK1R) and eliciting production of polyclonal antibodies cross-reacting with SP with a high affinity (Déry et al., 1997. J. Neuroimmunol. 76, 1-9). The aim of the present study was to investigate which structural features of mAb PS12 might account for this molecular mimicry. Cloning and sequencing of variable regions of both light (VL) and heavy (VH) chains of this 'SP-like' antibody did not indicate any primary sequence homology between SP and any antibody region. Instead, they revealed a striking similarity between the hydropathic profile of SP and that of an 11-amino-acid region in the light chain encompassing the second complementarity determining region (CDR2). When applied to CHO cells expressing the human NK1R, a synthetic extended 17-amino-acid peptide (denoted CDR2L) corresponding to this VL region inhibited the high-affinity binding of radiolabeled SP and antagonized the SP-induced inositol phosphate production. Moreover, a re-examination of the sequences of several antibodies that previously served in the design of CDR-derived bioactive peptides indicated that these antibodies also carried the hydropathic image of the respective ligands that they mimic. In agreement with previous observations on artificial synthetic peptides, our data thus suggest that the molecular mimicry between natural proteins (i.e. antibody and hormone, for example) could be understood on a structural level directly related, at least in part, to hydropathic homology. These results could then guide the search for bioactive paratope-derived peptides of potential pharmacological interest. We also observed inverse hydropathy between multiple CDRs of mAb PS12 (including CDR3H and CDR3L) and the peptide epitope, confirming the importance of hydropathic complementarity in antigen-antibody interactions.