Christopher R. Birkett

Molecular analysis of ovine prion protein identifies similarities between BSE and an experimental isolate of natural scrapie, CH1641

New variant Creutzfeldt-Jakob disease (vCJD) and bovine spongiform encephalopathy (BSE) are caused by the same strain of pathogen and, as sheep can develop experimental BSE, this has raised concern that humans may be at risk from eating mutton if BSE has naturally transmitted to sheep. Biochemical typing of abnormal prion proteins (PrPsc) has been suggested to detect BSE in sheep. Although this approach is ingenuous, we can now report biochemical evidence of strain variation in contemporary and archival brain tissue from cases of experimental BSE or experimental and natural scrapie in sheep. Interestingly, we found at least one isolate of natural scrapie (CH 1641) with a very similar, but not identical, PrPsc profile to BSE but which differs from BSE in its transmission characteristics to mice.

Scrapie strains maintain biological phenotypes on propagation in a cell line in culture.

Bovine spongiform encephalopathy (BSE) and its human equivalent, variant Creutzfeldt–Jakob disease (vCJD), are caused by the same strain of infectious agent, which is similar to, but distinct from, >20 strains of their sheep scrapie homologue. A better understanding of the molecular strain determinants could be obtained from cells in monoculture than from whole animal studies where different cell targeting is commonly a strain‐related feature. Although a few cell types can be infected with different strains, the phenotypes of the emergent strains have not been studied. We have cured the scrapie‐infected, clonal SMB cell line with pentosan sulfate, stably re‐infected it with a different strain of scrapie and shown that biological properties and prion protein profiles characteristic of each original strain are propagated faithfully in this single non‐neuronal cell type. These findings attest to the fact that scrapie strain determinants are stable and host‐independent in isolated cells.

Transfer of scrapie prion infectivity by cell contact in culture.

BACKGROUND When a cell is infected with scrapie prions, newly synthesized molecules of the prion protein PrP(C) are expressed at the cell surface and may subsequently be converted to the abnormal form PrP(Sc). In an experimental scrapie infection of an animal, the initial innoculum of PrP(Sc) is cleared relatively rapidly, and the subsequent propagation of the infection depends on the ability of infected cells to convert uninfected target cells to stable production of PrP(Sc). The mechanism of such cell-based infection is not understood. RESULTS We have established a system in dissociated cell culture in which scrapie-infected mouse SMB cells are able to stably convert genetically marked target cells by coculture. After coculture and rigorous removal of SMB cells, the target cells express PrP(Sc) and also incorporate [35S]methionine into PrP(Sc). The extent of conversion was sensitive to the ratio of the two cell types, and conversion by live SMB required 2500-fold less PrP(Sc) than conversion by a cell-free prion preparation. The conversion activity of SMB cells is not detectable in conditioned medium and apparently depends on close proximity or contact, as evidenced by culturing the SMB and target cells on neighboring but separate surfaces. SMB cells were killed by fixation in aldehydes, followed by washing, and were found to retain significant activity at conversion of target cells. CONCLUSIONS Cell-mediated infection of target cells in this culture system is effective and requires significantly less PrP(Sc) than infection by a prion preparation. Several lines of evidence indicate that it depends on cell contact, in particular, the activity of aldehyde-fixed infected cells.

Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes.

In chickens, single functional immunoglobulin variable and joining gene segments at each of the heavy and light chain loci undergo V(D)J rearrangement. Diversity is subsequently introduced by conversions templated by upstream pseudo V region genes in such a way that practically all V regions in mature B cells have identical ends. This greatly simplifies the representative amplification of V region genes. Furthermore, the entire naive repertoire of the adult chicken is produced in the bursa of Fabricius of the young bird. These special properties of the generation of immunoglobulin diversity in chickens have been exploited in the development of procedures to produce large libraries of diverse antibody combining sites derived from chicken Ig genes and expressed on filamentous bacteriophage. The utility of this library was assessed by selection of specifically binding phage using three solid phase-bound protein antigens, hen egg white lysozyme, bovine thyroglobulin and bovine serum albumin. The sequences of the V region genes thus isolated demonstrated that selection was specific and that the library contained useful diversity of binding sites. This library provides access to a repertoire whose diversity is based on a mechanism different from that underlying previously available libraries. The demonstrated feasibility of generating chicken phage antibodies may lead to the production of monoclonal reagents from immunised chickens, and the derivation of reagents for studying immunoglobulin mediated selection in avian B cell development.

Screening Congo Red and its analogues for their ability to prevent the formation of PrP-res in scrapie-infected cells.

Transmissible spongiform encephalopathies (TSEs) are incurable, fatal diseases. The dye Congo Red (CR) can cure cells infected with agents of the sheep TSE, scrapie, but is not used as a therapeutic or prophylactic agent in vivo, as its effects are small, possibly due to low blood-brain barrier permeability, and complicated by its intrinsic carcinogenicity. In this paper, the development is described of a structure-activity profile for CR by testing a series of analogues of this dye for their ability to inhibit the formation of the protease-resistant prion protein, PrP-res, a molecular marker for the infectious agent, in the scrapie-infected, SMB cell line. It was found that the central benzidine unit in CR, which gives the molecule potential carcinogenicity, can be replaced by other, less toxic moieties and that the sulphonate groups on the core molecule can be replaced by carboxylic acids, which should improve the brain permeability of these compounds. However, detailed dose-response curves were generated for several derivatives and they revealed that, while some compounds showed inhibition of PrP-res accumulation at high concentrations, at low concentrations they actually stimulated levels of PrP-res above control values.

Immunodetection of PrPSc in spleens of some scrapie-infected sheep but not BSE-infected cows

The development of diagnostic tools for transmissible spongiform encephalopathies (TSEs) would greatly assist their study and may provide assistance in controlling the disease. The detection of an abnormal form of the host protein PrP in noncentral nervous system tissues may form the basis for diagnosis of TSEs. Using a new antibody reagent to PrP produced in chickens, PrP can be readily detected in crude tissue extracts. PrP from uninfected spleen had a lower molecular mass range than PrP from brain, suggesting a lower degree of glycosylation. A simple method for detecting the abnormal form of the protein, PrPSc, in ruminant brain and spleen has been developed. PrPSc was detected in sheep spleen extracts from a flock affected by natural scrapie and was also found in spleens from some, but not all, experimental TSE cases. In spleens from cattle with bovine spongiform encephalopathy (BSE) no PrPSc was detected. It is therefore suggested that there is differential targeting of PrPSc deposition between organs in these different types of TSE infection which, with other factors, depends on strain of infecting agent.

Distribution of cell‐associated prion protein in normal adult blood determined by flow cytometry

Leucocyte subpopulations from normally healthy individuals were identified by recognized combinations of fluorochrome‐conjugated antibodies to CD markers and stained by different monoclonal antibodies (MAb) to normal cellular prion protein (PrPC), including the 3F4 MAb. Cell preparations were examined by three‐colour flow cytometry. All mononuclear leucocyte subpopulations and platelets expressed PrPC, but polymorphonuclear leucocytes and red blood cells expressed little or no PrPC. The amounts of PrPC expressed by the different cells were calculated by comparison to bead standards. Mononuclear leucocytes expressed 3000–4000 molecules of antibody‐reactive PrPC per cell. Resting platelets expressed around 1400 molecules of PrPC per cell, whereas activated platelets expressed around 4800 molecules of PrPC per cell. Extrapolation of these values to the amounts of the various cells in whole blood showed that platelet PrPC accounted for at least 96% of cell‐expressed PrPC in blood. The PrPC on mononuclear cells and platelets was sensitive to enzymatic treatment of cells by proteinase k and phosphatidylinositol‐specific phospholipase C. Certain anti‐PrPC MAbs which showed equivalent intensity of staining to MAb 3F4 on fresh cells showed relative reductions of staining compared to MAb 3F4 on stored cells, indicating possible structural alterations of PrPC under these conditions.

A monoclonal antibody that enables specific immunohistological detection of prion protein in bovine spongiform encephalopathy cases.

The specificity of a monoclonal antibody (mAB) raised against recombinant bovine prion protein (PrP) for the immunohistological detection of PrP accumulation in the medulla oblongata of bovine spongiform encephalopathy (BSE) and ovine scrapie cases was investigated. mAB KG9 showed a diffuse low intensity reaction with the cytoplasm of neurones in normal cattle and sheep sections. In BSE sections the mAB detected widespread granular deposits of PrP associated with neurones and the neuropil. Although scrapie sections showed similar levels of granular deposits with another antibody to PrP these were not detected by KG9 which did however detect diffuse staining in neuronal cytoplasm. Possible explanations for the specificity of binding of KG9 are discussed.