Conservation of vCJD Strain Properties After Extraction and In Vitro Propagation of PrPSc from Archived Formalin-Fixed Brain and Appendix Tissues Using Highly Sensitive Protein Misfolding Cyclic Amplification.

Three retrospective lymphoreticular tissue studies (Appendix I, II, and III) aimed to estimate the UK prevalence of variant Creutzfeldt-Jakob disease (vCJD), following exposure of the population to the bovine spongiform encephalopathy (BSE) agent, in the late 1980s and 1990s. These studies evaluated the presence of abnormal prion protein aggregates, in archived formalin-fixed paraffin-embedded (FFPE) appendectomy samples, by immunohistochemical detection. Although there was concordance in the estimated prevalence of vCJD from these studies, the identification of positive specimens from pre- and post-BSE-exposure periods in Appendix III study has raised questions regarding the nature and origin of the detected abnormal prion protein. We applied a robust and novel approach in the extraction of disease-associated prion protein (PrPSc) present in frozen and FFPE samples of brain and appendix from a patient with pathologically confirmed vCJD. The extracted material was used to seed the highly sensitive protein misfolding cyclic amplification assay (hsPMCA) to investigate the in vitro and in vivo propagation properties of the extracted abnormal prion protein. We demonstrate that PrPSc can be successfully extracted from FFPE appendix tissue and propagated in vitro. Bioassay in wild-type and gene-targeted mouse models confirmed that the extracted and amplified product is infectious and retains strain properties consistent with vCJD. This provides a highly sensitive and reliable platform for subsequent analysis of the archived FFPE appendix tissue derived from the Appendix II and III surveys, to further evaluate the nature of the abnormal PrP detected in the positive samples.

Understanding Intra-Species and Inter-Species Prion Conversion and Zoonotic Potential Using Protein Misfolding Cyclic Amplification.

Prion diseases are fatal neurodegenerative disorders that affect humans and animals, and can also be transmitted from animals to humans. A fundamental event in prion disease pathogenesis is the conversion of normal host prion protein (PrPC) to a disease-associated misfolded form (PrPSc). Whether or not an animal prion disease can infect humans cannot be determined a priori. There is a consensus that classical bovine spongiform encephalopathy (C-type BSE) in cattle transmits to humans, and that classical sheep scrapie is of little or no risk to human health. However, the zoonotic potential of more recently identified animal prion diseases, such as atypical scrapie, H-type and L-type BSE and chronic wasting disease (CWD) in cervids, remains an open question. Important components of the zoonotic barrier are (i) physiological differences between humans and the animal in question, (ii) amino acid sequence differences of the animal and human PrPC, and (iii) the animal prion strain, enciphered in the conformation of PrPSc. Historically, the direct inoculation of experimental animals has provided essential information on the transmissibility and compatibility of prion strains. More recently, cell-free molecular conversion assays have been used to examine the molecular compatibility on prion replication and zoonotic potential. One such assay is Protein Misfolding Cyclic Amplification (PMCA), in which a small amount of infected tissue homogenate, containing PrPSc, is added as a seed to an excess of normal tissue homogenate containing PrPC, and prion conversion is accelerated by cycles of incubation and ultrasonication. PMCA has been used to measure the molecular feasibility of prion transmission in a range of scenarios using genotypically homologous and heterologous combinations of PrPSc seed and PrPC substrate. Furthermore, this method can be used to speculate on the molecular profile of PrPSc that might arise from a zoonotic transmission. We discuss the experimental approaches that have been used to model both the intra- and inter-species molecular compatibility of prions, and the factors affecting PrPc to PrPSc conversion and zoonotic potential. We conclude that cell-free prion protein conversion assays, especially PMCA, are useful, rapid and low-cost approaches for elucidating the mechanisms of prion propagation and assessing the risk of animal prions to humans.

Unusual tissue distribution of carcinin, an antibacterial crustin, in the crab, Carcinus maenas, reveals its multi-functionality.

Crustins are whey acidic four-disulphide core (WFDSC) domain-containing proteins in decapods that are widely regarded as antimicrobial agents that contribute to host defence. Whilst there have been many analyses of crustin gene expression in tissues, few studies have been made of the distribution of the natural proteins. Here we report an immunostaining investigation of carcinin, a native crustin from Carcinus maenas, in the body organs. The results show that the protein is largely confined to the haemocytes with only a weak signal detected in the heart, hepatopancreas and midgut caecum where it is restricted to the outer surfaces. Importantly, carcinin was seen to be deposited by the haemocytes on these surfaces. Higher levels of staining were detected in the gonads with carcinin particularly abundant in the capsule of ovary as well as some oocytes. Conspicuous staining was further evident in the cuticle of the eyestalk peduncles. Ablation of the eyestalks resulted in a reduction of carcinin in the maturing ovary with the mature eggs rarely displaying a strong signal for the protein. Interestingly, the degree of carcinin also strongly increased in the healing peduncle, indicating that the protein may be associated with wounding, cell damage and/or tissue regeneration.