Low Levels Of PrP Research Articles

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans(9,18). The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrP(C)) and the infectious protein (PrP(Sc)), an abnormally-folded isoform of PrP(C 8). One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation(13). This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrP(Sc) are poorly characterized due to their unusual conformation and aggregation states. PrP(Sc) can seed the conversion of PrP(C) to PrP(Sc) in vitro(14). PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrP(Sc), at an accelerated rate, from a system containing excess amounts of PrP(C) and minute amounts of the PrP(Sc) seed(19). This technique has proven to effectively recapitulate the species and strain specificity of PrP(Sc) conversion from PrP(C), to emulate prion strain interference, and to amplify very low levels of PrP(Sc) from infected tissues, fluids, and environmental samples(6,7,16,23) . This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Lymphoid follicles of the ileal Peyer's patch of lambs express low levels of PrP, as demonstrated by quantitative real-time RT-PCR on microdissected tissue compartments, in situ hybridization and immunohistochemistry

The expression level of normal cellular prion protein (PrP(C)) is thought to influence the transmission of transmissible spongiform encephalopathies (TSEs) from the peripheral entry site to the site of pathological changes in the central nervous system. In many TSEs, the clinical disease is preceded by a period in which the agent accumulates in lymphoid organs, particularly in association with follicular dendritic cells of lymphoid follicles. As the probable route of entry of the TSE agent is via the gut, the expression profile of PrP was examined in well-developed gut-associated lymphoid tissue of lambs, the ileal Peyer's patch, by laser microdissection and real-time RT-PCR. Lymphoid follicles were found to have very low levels of expression, whilst highest levels were detected in the outer submucosa and the muscular layer. These findings were supported by in situ hybridization and immunohistochemistry, which showed specific labelling in nerve cells in ganglia of the submucosal (Meissner's) and myenteric (Auerbach's) plexi of the enteric nervous system. Based on the assumption that potential sites for conversion to the scrapie-related prion protein (PrP(Sc)) should display high levels of expression of PrP(C), this study suggests that the accumulation of PrP(Sc) in the lymphoid follicles of the Peyer's patch is not preceded by PrP conversion in the same tissue compartment.

The Paraffin-Embedded Tissue Blot Detects PrP Sc Early in the Incubation Time in Prion Diseases

With the appearance of bovine spongiform encephalopathy (BSE) and a new variant of Creutzfeldt-Jakob disease (nvCJD) that seems to be caused by BSE, there is an increased need for improvement of diagnostic techniques and recognition of all variants of prion diseases in humans and animals. Publications on the immunohistochemical identification of PrP(Sc) in the tonsils and appendix in the incubation period of nvCJD indicate that new and more sensitive techniques for the detection of PrP(Sc) in various tissues may be a valuable tool for early diagnosis in prion diseases. We developed a new and sensitive technique to detect PrP(Sc) in formalin-fixed and paraffin-embedded tissue, the paraffin-embedded tissue blot (PET blot), and reinvestigated archival brain material from CJD as well as BSE and scrapie. In addition, C57/Bl6 mice experimentally infected with the ME7 strain were investigated sequentially during the incubation time to compare this new technique with conventional methodologies. The PET blot detects PrP(Sc) in idiopathic (sporadic) and acquired prion diseases, even in cases with equivocal or negative immunohistochemistry, and is more sensitive than the conventional Western blot and histoblot techniques. The PET blot makes possible the detection of PrP(Sc) during the incubation period long before the onset of clinical disease and in prion disease variants with very low levels of PrP(Sc). In mice experimentally infected with the ME7 strain, the PET blot detects PrP(Sc) in the brain 30 days after intracerebral inoculation-145 days before the onset of clinical signs. Its anatomical resolution is superior to that of the histoblot technique. It may therefore be of particular interest in biopsy diagnosis. Thus it complements other tissue-based techniques for the diagnosis of prion diseases in humans and animals.