Typescript (photocopy) / 132 leaves, [2] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1982

Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1982. / Typescript (photocopy).

Transmissible spongiform encephalopathies (TSEs) or prion diseases, are subacute neurodegenerative diseases that infect humans and animals. Many of these diseases are acquired by peripheral exposure (e.g. orally). After oral exposure prion replication within the Peyer's patches (PP) in the small intestine is necessary for the efficient spread of the disease to the brain. Within the intestine, bacteria and pathogenic microorganisms can affect the status of the gut associated lymphoid tissue (GALT). GALT consists of PP and isolated lymphoid follicles (ILF) that maintain homeostasis and protect from infections. Therefore, factors which modify GALT status, might dramatically affect oral prion disease pathogenesis by influencing the uptake of prions from the gut lumen or expanding their distribution within the host. Chronic intestinal helminth infections are common in animals and in man, and can cause significant pathology within the intestine. Little is known of the effects that intestinal helminth infections may have on oral prion diseases susceptibility. Therefore, in this study the influence that co-infection with Heligmosomoides polygyrus (a natural pathogen of the mouse small intestine) may have on oral prion disease pathogenesis and susceptibility was determined. The studies consisted of groups of 4 (for H. polygyrus characterization and for early prion detection) and 8 (for H. polygyrus-prion co-infection to terminal stage) mice infected with H. polygyrus (orally) alone or subsequently infected with ME7 scrapie prions (orally) at different time-points after parasitic infection. The effects of the H. polygyrus infection alone, and on oral prion disease pathogenesis and susceptibility were then determined. Initially the characterization of H. polygyrus infection on the host intestine revealed that this parasite caused significant pathology in the small intestine and affected the GALT microarchitecture. In the PP follicles, H. polygyrus infection increased the area of follicular dendritic cell expression, altered the positioning of mononuclear phagocytes and increased M cell density. H. polygyrus infection also reduced the number of ILF in both the small and large intestines. Additional studies in mice co-infected with a low dose of prions, revealed that these pathological changes affected the survival time and disease susceptibility. Data also show that the extent of the effects on prion disease pathogenesis and susceptibility were dependent on the stage of the helminth infection at which the mice were orally-exposed to prions. Data demonstrate that co-infection with the gastrointestinal helminth H. polygyrus can influence oral prion disease pathogenesis and susceptibility. Helminth infections can significantly modify the microarchitecture of the gut and the GALT. Data presented suggest the pathological changes that pathogens such as small intestinal helminths cause, may also influence the uptake of prions from the gut lumen after oral exposure.

Le tropisme cellulaire des prions infectieux diffère selon lespèce animale, celui-ci est corrélé à la souche infectieuse et à des facteurs spécifiques de lhôte. Par exemple, certains prions infectieux sont lymphotropiques, notamment en cas de scrapie chez les moutons et de variant de la maladie de Creutzfeldt-Jakob (vMCJ) chez lhomme. Par opposition, certains prions se caractérisent par un neurotropisme comme observé chez des patients Creutzfeldt-Jakob atteints de la forme sporadique ou chez des bovins atteints dencéphalopathies spongiformes bovines (ESB). Lhypothèse de notre travail repose sur les observations suivantes : dans le cas du variant de la maladie de Creutzfeldt-Jakob et des encéphalopathies spongiformes bovines, lagent responsable est identique, la voie dinoculation et les lésions neurologiques le sont également, seul le tropisme de cette souche pour les organes lymphoïdes diffère. En effet, les amygdales, la rate et lappendice sont infectieux chez lhomme. Par contre, linfectiosité est surtout confinée au niveau du système nerveux chez le bovin. Lors dune inoculation expérimentale par voie orale de lagent responsable de lESB chez les bovins, les plaques de Peyer iléales sont les seuls tissus lymphoïdes infectieux. Notre hypothèse de travail est que des propriétés de lhôte interviennent dans le tropisme de lagent infectieux. Deux axes de recherche ont été envisagés afin de vérifier cette hypothèse :  Lanalyse de la distribution des fibres nerveuses au sein des tissus lymphoïdes associés aux muqueuses (MALT) des espèces bovine et humaine  Létude de lexpression de PrPc et de ses isoformes au sein des tissus lymphoïdes et nerveux des espèces bovine et humaine. Pour atteindre au mieux nos objectifs, il nous manquait un outil essentiel permettant la caractérisation spécifique des FDC bovines. En effet, aucun marqueur spécifique de ces cellules nétait commercialisé. Nous avons donc produit, en collaboration avec le Centre dEconomie Rural de Marloie, un anticorps monoclonal spécifiquement dirigé contre les cellules folliculaires dendritiques (FDC) bovines. Cet anticorps nous a permis détudier la distribution des FDC au sein des organes lymphoïdes bovins. Une attention particulière a été portée aux FDC isolées à partir des plaques de Peyer jéjunales (PPJ) et iléales (PPI). Lapparente différence dinfectivité de ces tissus lymphoïdes chez des bovins atteints expérimentalement dESB nous a conduit à comparer les capacités fonctionnelles des FDC isolées à partir de PPJ et de PPI. Ces observations sont décrites et discutées dans le chapitre 1. Dans le chapitre 2, nous avons établi une cartographie des fibres nerveuses au sein des amygdales, des plaques de Peyer iléales et jéjunales bovines de plusieurs catégories dâge et ensuite comparé ce pattern dinnervation à celui des amygdales humaines; ceci permettra de pister les voies potentielles de neuro-invasion. Une attention particulière a été portée à linterface cellules folliculaires dendritiques fibres nerveuses. En effet, les FDC matures jouent un rôle prépondérant dans la pathogenèse des maladies à prion puisquen leur absence, une infection périphérique na pas lieu. De plus, la proximité entre fibres nerveuses et FDC est un paramètre intervenant dans la neuro-invasion; nous avons dès lors aussi analysé les contacts entre les FDC et les éléments nerveux. Lexpression de la PrPc est une condition sine qua non pour la formation de PrPres. Cette protéine cellulaire sert probablement de récepteur pour son homologue infectieux mais sert surtout de substrat pour lamplification de PrPres ; toute modification au niveau de sa synthèse pourrait entraîner un changement de la cinétique dinfection et pourrait expliquer lapparente absence dinfectivité constatée au niveau du système immunitaire chez les bovins. Lexpression tissulaire et cellulaire spécifique disoformes de la PrPc représente un facteur de lhôte potentiellement capable dinfluencer le tropisme cellulaire de lagent infectieux chez lhumain et le bovin. Cette expression a été étudiée dans les systèmes MALT bovins et humains. Pour affiner notre étude, nous avons analysé, par des techniques de western-blotting, le glycopattern de la PrPc ainsi que lexpression de ses formes tronquées dans les tissus lymphoïdes humains et bovins mais également dans des populations cellulaires spécifiques, les lymphocytes et les FDC. Afin de vérifier si les isoformes de PrPc sont spécifiques aux tissus lymphoïdes, nous avons effectué une étude comparative du pattern de glycosylation et du ratio des formes clivées de PrPc, exprimés au sein de différentes régions du système nerveux central bovin et humain. Les résultats de ces travaux sont repris dans le chapitre 3.

8 ABSTRACT Historically pig is allocated to a group of animals which use certain parts of their small intestine to acquire a fully developed primary B cell reperoire. Development of such primary repertoire is independent on the antigen presence and resembles the primary lymphopoietic activity of avian bursa of Fabricius. However, some findings concernig the pig's alignment in the above mentioned group suggest otherwise. This graduation thesis is focused on the investigtion of lymphocyte populations and subpopulations in the small intestine of germ-free and conventional piglets. The aim is to determine whether the percentage amounts of lymphocyte populations is dependent on the intestinal colonization. Using Flow Cytometry the significant differences between individual samples were assesed allowing us to conclude which parts of the small intestine could possibly be used for the development of B cell repertoire. Moreover, the status of isotype switching of B lymphocytes isolated from different intestinal parts was determined by the means of PCR analysis. Our data suggest that the small intestine colonization has a crucial role in development of all the main lymphocyte populations as well as some of their subpopulations. The greatest influence of colonization was observed concerning B lymphocytes and their...

Crohn's disease, a chronic inflammation of the bowel, is a multi-factorial condition where uncontrolled immune responses to luminal bacteria occur in genetically predisposed individuals. The first observable clinical signs are small ulcers that form at a specialised form of epithelium, follicle-associated epithelium (FAB). The FAB covers immune inductive sites, Peyer's patches, which function primarily as sensory areas that sample the externaI gut environment. Dendritic cells are one of the key cells that are involved in sensing luminal contents and orchestrating the gut immune system. The main aim of this thesis was to determine whether the barrier of the FAB is breached in Crohn's disease and if dysfunctional immune regulators, namely dendritic cells, playaroIe in initiating and/or maintaining the chronic intestinal inflammation. Using biopsies and surgical specimens, we were able to show that in Crohn's disease, there was an increased transmucosaI transport of Escherichia coli compared to specimens from ulcerative colitis and non-inflammatory bowel disease (IBD) controIs. Dendritic cells internalised a higher percentage of bacteria that had translocated across the FAB in the Crohn's samples. Furthermore, significantly higher concentrations of TNF-u was released upon bacterial stimulation by tissues from patients with Crohn's disease than in controIs. We went on to characterise the dendritic cells present in the Peyer's patches of patients with Crohn's disease. We found an accumulation of both immature and mature dendritic cells beneath the FAB, in the sub-epithelial dome (SED). Normally, mature dendritic cells migrate towards T cell-rich areas. However, we observed mature dendritic cells accumulating in the SED because they lacked the CCR7 migratory receptor. Furthermore, they were more prone to take-up bacteria, and produced TNF-α. To study the function of mucosal dendritic cells, we performed isolation experiments and mixed Iymphocyte reactions. Dendritic cells from both the ileum and blood of patients with active Crohn's had reduced capacity for inducing T cell proliferation than non-IBD controIs. Blood dendritic cells of patients in remission had normalised function that was similar to dendritic cells from healthy controls. The SAMPl/YitFc mice, considered an appropriate murine model for Crohn's disease, had an inherent permeability defect that increased with the chronicity of intestinaI inflammation. However unlike in human Crohn's disease, dendritic cells did not seem to playaroIe in murine ileitis. This thesis highlights the accumulation of the actively surveying dendritic cells that are prone to bacterial internalisation, and points to their possible different functional roles in active versus in-active disease; thereby confirming dendritic cells as one ofthe key components in the pathogenesis ofCrohn's disease.

Thesis summary The process of B cell lymphogenesis in swine remains uncertain. Some reports indicate that pigs belong to a group of animal that use ileal Peyers's patches (IPP) for the generation of B cells while others point to the possibility that the bone marrow is functional throughout life. The functional subpopulations of B cells in swine are also unknown. Together with other ruminants, and also birds, γδ T cells in swine may account for >70% of all T cells which is in apparent contrast with humans and mice. The purpose of this thesis was to address these discrepancies and unresolved issues. The results disprove the existing paradigm that the IPP is primary lymphoid tissue and that B cells develop in IPP in an antigen-independent manner. On the other hand, it shows that bone marrow is fully capable of B cell lymphogenesis and remains active at least for the same period of time as it had been speculated for the IPP. This thesis also identified functionally different subsets of porcine peripheral B cells, and shows that CD21 molecules can be expressed in differential forms. Finally, this thesis identifies two lineages of γδ T cells that differ in many functional and phenotype features. This finding may explain why γδ T cells constitute of minority of lymphocytes in circulation of humans and mice.