In the African Cape buffalo, circulating serum xanthine oxidoreductase (XOR) produces trypanotoxic levels of hydrogen peroxide (H2O 2). Previous, preliminary studies suggest that mammalian serum XOR H 2O2 production rates (“specific activities”) are species- and breed-associated. The research objectives pursued here were: (1) to verify that mammalian serum XOR specific activities were truly species- and breed-associated, and if found to be so (2) to determine if the noted activity differences were a product of translational and/or post-translational mechanism(s). The ultimate intent of this work was to reveal strategies by which trypanosome tolerance might be achieved in susceptible mammals. By a novel kinetic peroxidase assay, developed and used here, it was found that the serum XOR specific activities of Cape buffalo, Holstein and Hereford heifers, Sprague-Dawley rats, and humans were significantly different from one another (p < 0.025). A concurrent, comparative analysis of XOR nucleotide/amino acid sequences and tissue-specific transcription rates in Cape buffalo and representative bovine breeds yielded no points of variability that might explain observed activity differences. To determine if such differences were, instead, a product of distinct translational and/or post-translational regulatory mechanism(s), serum XOR was isolated, quantified, and characterized. Two immunoaffinity (IA) chromatography protocols were developed to isolate serum XOR. Mouse monoclonal antibodies directed against cow milk xanthine oxidase (α-CMXO) exhibited a recognition for serum XOR that was limited to bovid species. Polyclonal rabbit α-CMXO antibodies were found to recognize serum XOR from a greater range of mammalian species. However, IA eluent resolution by SDS-PAGE (under reducing conditions) elucidated polypeptide profiles that were species-specific, suggesting the presence of co-eluting contaminants. MALDI-TOF analysis identified the putative contaminants as components of serum IgG and IgM. These immunoglobulins (Igs) were found to exist in complex with the isolated serum XOR or were recognized directly by the polyclonal α-CMXO antibodies of the IA column, in an idiotypic manner. The consequences and implications of such Ig contamination are discussed, specifically with regard to: (1) the future use of α-XOR antibodies in extracting serum XOR, and (2) the possible roles of XOR-Ig complexes in systemic inflammatory pathologies.

Dendritic cells (DCs) are regulators of the immune response and are thought to be critical in maintaining tolerance towards the intestinal microbiota. Recent data have identified distinct subsets of DCs with specific functional properties. The objective of this thesis was to further define CD103⁺ and CX3CR1⁺ DCs in the intestine and to determine how DCs and regulatory T (Treg) cell responses are influenced by the microbiota. Using multicolour flow cytometry, we identified two CD103⁺ DC subsets with differential aldehyde dehydgrogenase (ALDH) activity and two populations of CX3CR1⁺ cells. In the mesenteric lymph node CD103⁺ALDH⁺ DCs were highly mature (CD86^hi, MHCII^hi), likely migratory (CCR7⁺) and enhanced Treg cell induction compared with ALDH⁻ DCs. CX3CR1^int cells accumulated during bacterially-induced colitis suggesting a pro-inflammatory role whereas CX3CR1^hi cells were associated with the production of the anti-inflammatory cytokine IL-10 during homeostasis. We also assessed the generation of CD103⁺ DCs from bone marrow progenitors. Although only small proportions of CD103⁺ DCs were detected in culture with FLT3L or GM-CSF alone, the combination of FLT3L and GM-CSF induced CD103⁺ DCs with a phenotype similar to those found in the small intestine. Using this system we showed that TLR ligands and retinoic acid induce ALDH enzyme activity in vitro. In order to assess how DCs and Treg cells respond to changes in the microbiota we employed broad-spectrum antibiotic treatment to deplete endogenous bacteria and also analyzed the impact of colonization with the model organism Helicobacter hepaticus. Interestingly, we did not detect alterations in the proportions of different DC subsets following antibiotic treatment or H. hepaticus infection. However, using a novel FoxP3^huCD2-IL-10^GFP reporter mouse, we found that IL-10 production by Treg cells was ablated following antibiotic treatment and significantly elevated following H. hepaticus infection. Preliminary investigation of the mechanism underlying this effect suggests a role for IL-27. In summary, this thesis provides further detail on the phenotype of intestinal DCs and shows that Treg cell IL-10 production is sensitive to the composition of the microbiota.

Thesis (M.S.)--University of Wisconsin--Madison, 1968. / eContent provider-neutral record in process. Description based on print version record.

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Dendritic cells (DC) play a key role in immunity and represent a great target for modulation, because of their ability to prime T cells and direct their polarisation into effector subsets. Ticks release immunomodulatory compounds in their saliva, possibly in order to evade host immune responses during feeding. We have recently reported that Rhipicephalus appendiculatus ticks produce ‘Japanin’, a secretory lipocalin that arrests differentiation of monocytes into DC and reprogrammes maturation of DC in response to various stimuli towards a tolerogenic phenotype . Japanin was cloned and recombinantly expressed in a baculovirus system for subsequent immunological and biochemical analysis. This study was set out to further investigate the immunomodulatory activity of Japanin as well as the underlying mechanism of action. We have discovered that Japanin prevents DC-mediated proliferation and polarisation of allogeneic T cells. Experiments with labelled Japanin have demonstrated that it binds predominantly to ex vivo generated human monocyte-derived DC (moDC) and to a reduced degree to monocyte and DC populations in peripheral blood, yet to no other blood leucocytes. We have identified CD206, also known as the mannose receptor, as a Japanin-binding receptor on moDC. This identification has been achieved by crosslinking and subsequent pull-down of Japanin-receptor complexes from moDC. Affinity studies with recombinant CD206 constructs have confirmed the binding to Japanin. Moreover, the binding has been verified by specific siRNA knock-down of CD206 in moDC, which resulted in significantly decreased binding of Japanin. Unexpectedly, CD206 has appeared to be dispensable for at least most of the DC-modulatory activity of Japanin. Therefore, attempts were made to determine other factors in the mode of action of Japanin, through which we have found that IL-10 is not essentially involved. Further results have suggested that the activity of Japanin demands cell contact. Collectively, we have come to the conclusion that the mechanism of action of Japanin might require internalisation by DC, potentially enabling modulation of intracellular pathways involved in the regulation of DC maturation.