Investigating the effects of preclinical prion disease on blood protein composition and infectivity
Since 1996 there have been 226 cases of variant Creutzfeldt-Jakob disease (vCJD) in the UK following dietary exposure to bovine spongiform encephalopathy (BSE) in the late 80s to early 90s. A recent study on archived appendix tissue has suggested that up to 1/2000 of the UK population may be silent carriers of infection. This is a significant portion of the UK population who may be incubating the disease and thus potentially passing it on to others via blood transfusion. As there is currently no effective screening method to test donated blood this is a major public health concern. The aim of this project was to utilize archived blood samples from transfusion experiments performed with BSE infected sheep, identify protein expression differences between uninfected and infected blood and discover potential diagnostic markers besides the abnormal prion protein (PrPSc). Infected samples were taken from sheep 10 months after oral inoculation with BSE, when their blood was proven to be infectious but the sheep lacked clinical signs. Uninfected samples were taken from the same sheep prior to BSE exposure. Samples from 9 sheep were pooled to control for genetic and metabolic differences. The plasma was processed using ProteoMiner columns to deplete highly abundant proteins while simultaneously enriching for low abundant proteins. Subsequently, isotopic labelling was used to distinguish between the two samples and levels of protein expression were compared by mass spectrometry. Initially 153 proteins were identified based on one unique peptide match. The extent of differential expression was assessed by filtering the median H/L (heavy/light isotopes) and proteins between >1.5 and <0.666 were retained, leaving a dataset of 44 proteins. Elimination of the most common proteins reduced this to 37 proteins and after considering the current literature a candidate shortlist of 8 proteins were chosen to validate by western blotting and LI-COR imaging. The buffy coat fraction of blood from the same 9 sheep were similarly processed. The original dataset of 1148 proteins was cut down to 124 and Ingenuity Pathway Analysis (IPA) employed. This highlighted 191 biological processes of potential interest, including LXR/RXR activation, acute phase response signalling, leukocyte extraversion signalling and mitochondrial dysfunction. A shortlist of 8 candidate proteins were then determined for validation. These findings are the basis for a proposed biomarker panel for an effective preclinical diagnostic blood test distinct from other neurodegenerative disorders such as Alzheimer’s or Parkinson’s disease. Additionally, in order to investigate how cells pass infection by the human blood transfusion route, blood components from the natural scrapie flock were utilised to infect the Rov9 cell line. The aim of this work was to develop a system to study the processes by which live white blood cells (WBCs) transfer infection to other cells. A cell culture model to study infectivity from scrapie infected WBCs of the Roslin Scrapie Flock was achieved. Further work is needed to examine whether membrane based cellular interactions are essential for efficient propagation of infection and thus spread of disease.