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The DNA assisted solid-phase proximity ligation assay (SP-PLA) provides a unique opportunity to specifically detect prion protein (PrP) aggregates by investigating the collocation of 3 or more copies of the specific protein. We have developed an SP-PLA that can detect PrP aggregates in brain homogenates from infected hamsters even after a 10(7)-fold dilution. In contrast, brain homogenate from uninfected animals did not generate a detectable signal at 100-fold higher concentration. Using either of the 2 monoclonal anti-PrP antibodies, 3F4 and 6H4, we successfully detected low concentrations of aggregated PrP. The presented results provide a proof of concept that this method might be an interesting tool in the development of diagnostic approaches of prion diseases.

Prion diseases are a group of neurodegenerative disorders affecting humans as well as captive and wild animals. The mechanisms and routes governing the natural spread of prions are not completely understood and several hypotheses have been proposed. In this study, we analyzed the effect of gender in prion incubation period, as well as the possibility of prion transmission by sexual and parental contact using 263K infected hamsters as a model. Our results show that males have significantly longer incubation periods compared with females when exposed to the same quantity of infectious material. Importantly, no evidence of sexual or parental prion transmission was found, even 500 d after sexual contact or birth, respectively. Western blotting and PMCA were unable to detect sub-clinical levels of PrP(Sc) in experimental subjects, suggesting a complete absence of prion transmission by these routes. Our results show that sexual and parental transmission of prions does not occur in this model. It remains to be studied whether this conclusion is valid also for other prion strains and species.

Background: The manufacturing processes of plasma products include steps that can remove prions. The efficacy of these steps is measured in validation studies using animal brain‐derived prion materials called spikes. Because the nature of the prion agent in blood is not known, the relevance of these spikes, particularly with steps that are based on retention mechanisms such as nanofiltration, is important to investigate. Study Design and Methods: The aggregation and sizes of PrPres assemblies of microsomal fractions (MFs) extracted from 263K‐infected hamster brains were analyzed using velocity gradients. The separated gradient fractions were either inoculated to Tg7 mice expressing hamster‐PrPc to measure infectivity or used in Protein Misfolding Cyclic Amplification for measuring seeding activity. The collected data allowed for reanalyzing results from previous nanofiltration validation studies. Results: A significant portion of MFs was found to be composed of small PrPres assemblies, estimated to have a size ≤24 mers (~22–528 kDa), and to contain a minimum of 20% of total prion infectivity. With this data we could calculate reductions of 4.10 log (15 N), 2.53 log (35 N), and 1.77 log (35 N) from validation studies specifically for these small PrPres objects. Conclusion: Our gradient data provided evidence that nanofilters can remove the majority of the smallest PrPres entities within microsomes spikes, estimated to be in a size below 24 mers, giving insight about the fact that, in our conditions, size exclusion may not be the only mechanism for retention nanofiltration. [ABSTRACT FROM AUTHOR]

The most prominent hallmark of prion diseases is prion protein conversion and the subsequent deposition of the altered prions, PrP, at the pathological sites of affected individuals, particularly in the brain. A previous study has demonstrated that the N-terminus of the pathogenic prion isoform (PrP) is modified with advanced glycation end products (AGEs), most likely at one or more of the three Lys residues (positions 23, 24, and 27) in the N-terminus (23KKRPKP28). The current study investigated whether N-(carboxymethyl)lysine (CML), a major AGE form specific to Lys residues produced by nonenzymatic glycation, is an AGE adduct of the N-terminus of PrP. We show that CML is linked to at least one Lys residue at the N-terminus of PrP in 263K prion-infected hamster brains and at least one of the eight Lys residues (positions 101, 104, 106, 110, 185, 194, 204, and 220) in the proteinase K (PK)-resistant core region of PrP. The nonenzymatic glycation of the Lys residue(s) of PrP with CML likely occurs in the widespread prion-deposit areas within infected brains, particularly in some of the numerous tyrosine hydroxylase-positive thalamic and hypothalamic nuclei. CML glycation does not occur in PrP but is seen in the pathologic PrP isoform. Furthermore, the modification of PrP with CML may be closely involved in prion propagation and deposition in pathological brain areas. [ABSTRACT FROM AUTHOR]

Highlights: [•] We identified sub-infectious presence of prions in brains and spleens after peripheral administration. [•] We report the persistence of this material for up to 9days after challenge in both, spleen and brain. [•] We describe a new methodology to detect infectious prions in tissues at early stages of in vivo replication. [ABSTRACT FROM AUTHOR]