Document Type

Thesis - University Access Only

Award Date

2007

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology

Abstract

Transmissible Spongiform Encephalopathies (TSEs) are chronic neurological illnesses characterized by the progressive conversion of a normal cellular protein (PrPc) to a conformationally different form, the scrapie prion protein (PrP8c). TSEs have proven difficult to study, due to extended asymptomatic-but in many species, infectious incubation periods. The recent appearance of new cases of variant Creutzfeldt – Jakob disease (vCJD) transmitted via blood transfusions clearly identifies a need to characterize blood-borne sources of infectivity in these diseases. Since the hallmark of TSE diseases is the alteration of the self prion protein (PrPc), no specific immune responses have been detected which is likely due to natura self tolerance. Nonetheless, the immune system may participate in the transport and replication of prions in peripheral lymphoid organs following natural infection (Tylor et al., 1996, Mohan et al., 2004). This study is among the first to investigate the effect of acute scrapie infection on afferent lymphocyte subsets and resulting changes in migration to the draining subcutaneous lymph node. We utilized the well characterized canulation method of sheep pseudo afferent lymph to demonstrate that acute scrapie infection has no effect on the kinetics and magnitude of the afferent lymphocytes migrating into the afferent lymph. There was an increase in the representation of CD 11 c positive-CC98 positive DC in the afferent lymph associated with upregulation of PrPc expression on their surface following scrapie infection. We further observed a significant increase in the traffic of CD 11 b positive B cells, previously demonstrated to exhibit significantly lower levels of PrPc in normal and scrapie infected sheep. Most importantly, we directly observed the appearance of cell- associated PrP8c in afferent lymph beginning 8-16 hours post inoculation and continued to 24 hours post inoculation. These studies add important new information on the mechanism by which scrapie is delivered to the draining lymph node during the earliest stages of the disease. In the next series of experiments, we studied the kinetics and magnitude of changes in the exit of efferent lymphocytes from acutely scrapie-infected subcutaneous lymph nodes. Following acute scrapie-infection of SCLN there were no changes in the output of CD4 T-cells, but there were marked increases in the proportion of CD 11 b+ B cells in parallel with significant decrease in the output of both CD2 l + B cells and y <> T draining the acutely infected node. Furthermore, while no PrPsc was detected in the cellfree lymph plasma, PrP8c was found to be specifically associated with efferent lymph leukocytes drained from the scrapie injected lymph node, beginning approximately 5 days post inoculation and apparent until at least 10 days post inoculation. This experiment clearly demonstrates that the migratory leukocytes are capable of transporting PrP8c from the affected lymph node and thus may contribute to the distant hematogenic dissemination of the disease. In the final series of experiments, we developed an in vitro model of germinal center function to examine the molecular events occurring in within scrapie-affected lymph nodes. Recent data has clearly implicated germinal center cells, particular follicular dendritic cells (FDCs) in the propagation of prion diseases. We developed a method to establish stable FDC cell lines from sheep expressing prion protein gene (Pmp) genotype susceptible for scrapie. These cell lines were characterized phenotypically based on their morphological similarity to germinal center FDCs, and their expression of FDC-associated cell surface molecules. Co-culture experiments with isolated peripheral blood B cells clearly demonstrated their ability to support B cell proliferation, consistent with an FDC phenotype. Finally, we demonstrated that these cells were able to maintain PrPsc derived from scrapie brain homogenates for up to 4 passages as evidenced by recovery of cell associated PrPsc. These data clearly demonstrate that we successfully established a methodology for the isolation and culture ofFDC lines that effectively mimic in vivo events occurring in sheep with scrapie infection. This system represents an in vitro germinal center model. Furthermore, our data would indicate that FDCs are not merely playing a passive role during prion pathogenesis, and may offer options for development of novel diagnostic assays. Overall, this work has provided important new insights into the role of migratory leukocytes in the early pathogenesis ofTSEs. Based on our work, both the afferent and efferent leukocytes participate in the active transport of scrapie infection to the draining LN (afferent) and ultimately to the systemic lymphoid apparatus. It appears likely that CDl lc positive-CC98 positive DC in the afferent lymph may be involved in transport of scrapie material to germinal centers, associated with upregulation of PrPc expression. Later in the response, there is a decrease in efferent lymph in the yc> T-cells together with a shift in B cells populations towards the low expressing PrPc B cells which may indicate an immune response devised to minimize systemic dissemination of infection. Within the lymph node, the FDCs were found to be capable of maintaining the PrPsc , therefore these cells may dispense the infection to the B cells during their intimate contact in the GC during immune responses, participating in progression of the disease. In addition to the novel insights provided by these results, this role ofFDCs may offer options for development of novel diagnostic assays.

Library of Congress Subject Headings

Scrapie -- Pathogenesis
Prion diseases
Leucocytes

Format

application/pdf

Number of Pages

164

Publisher

South Dakota State University

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