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No description available.

The aim of this project was to characterise the humoral immune responses to RhD-derived synthetic peptides as a first step towards developing alternative peptide immunogens to replace RBC in immunising donors for anti-D IgG antibody production for clinical use. In the first part, 4 peptides (Dp6, Dp13, Dp17, and Dp28) containing Th-cell epitopes on the RhD protein were used to stimulate PBMC from 2 D-alloimmunised donors in vitro. A mixture of the four peptides was also utilised to boost HLA-DR15 transgenic mice previously immunised with purified RhD protein. The ability of the four peptides to boost anti-D titre significantly varied between the two donors, with a maximum 3-fold increase in anti-D titre induced by Dp17 in donor 2. The mouse antibody response to peptide boosting was also variable, with 4 of the 6 (66.7%) peptide-boosted DR15 mice demonstrating positive changes in anti-RhD antibody levels. The findings from the in vitro and mice studies demonstrated that these four peptides had the potential to boost anti-D titres in D-alloimmunised donors. In the second part of this study, an alternative approach to designing a totally synthetic peptide immunogen was investigated. An RhD mimotope (DM4) was selected from a set of previously identified RhD mimotopes, and its antigenic mimicry for the D antigen was verified. The DM4 peptide was coupled with the Dp6 peptide as a Th-cell epitope, to construct a chimeric T-B peptide immunogen. The immunogenicity of the T-B and DM4 peptides were evaluated in DR15-transgenic mice. Both peptides were able to induce significant DM4-specific IgG antibodies. The immunogenicity of the DM4 was significantly augmented by colinear coupling to the Dp6 peptide. The immunogenic mimicry of the DM4 peptide for the D antigen was examined through the ability of mouse anti-DM4 antibodies to recognise human D antigen on D-positive RBC. However, these antibodies appeared to recognise D antigen non-specifically, reacting with all tested human RBC regardless of their D phenotypes.

Secondary lymphoid structures are organised networks which support the generation of efficient immune responses by facilitating antigen presentation between an antigen presenting cell (APC) and an antigen-specific naive T cell. Professional APC such as dendritic cells (DC) are potent primers of T cells and have the capacity to determine the T-helper type 1 or 2 direction of the immune response. Lymphotoxin (LT) is a cytokine of the TNF family which has diverse biological roles, including one as a cytotoxic mediator of immunity. Moreover LT-mediated signals are required for the organogenesis and maintenance of lymphoid structures. The role of various lymphoid structures may have profound effects on the co-ordination of primary immune responses and LT-disruption has been used to examine these requirements. To date, most studies have focussed on systemic infection and here, the roles of LT and various gut-associated lymphoid tissues (GALT) were addressed in the context of enteric infection with the gut-tropic apicomplexan parasite, Eimeria vermiformis. Immunity to infection and the induction of protective gut Th1 responses were dependent upon the rapid recruitment of DC to lymphoid structures. Deficiency in lymphoid structures affected the induction of protective immunity and increased susceptibility to infection. Despite the lack of infection in the PP (E. vermiformis targets crypt enterocytes), a role for PP was established in the rapid induction of immunity. The anti-parasite response required a co-operative interaction between PP and mesenteric lymph nodes (MLN) with the PP influencing the time of induction of responses in the MLN. Examination of DC numbers and phenotypes revealed a delay in accumulation of DC subsets in PP-deficient mice and supports the role for DC traffic between these lymphoid structures in the induction of rapid gut immune responses.

The tripartite motif (TRIM) proteins are important in a variety of cellular functions including antiviral activity. We systematically analyzed mRNA expression of representative TRIMs in primary mouse macrophages, myeloid and plasmacytoid dendritic cells, and a selection of CD4+ T cell subsets. These cells have different effector functions in innate and adaptive immune responses, to a large extent due to the different patterns of cytokines that they produce. Here, we defined four clusters of TRIM genes based on their selective expression in these cell subsets. The first group of TRIMs was preferentially expressed in CD4+T cells and contained the C0S-FN3 motif.

Consumption of whole-grains (WGs) is associated with a wide range of health benefits, but clinical data regarding effects of wG consumption on blood lipids, glucose metabolism, inflammation and immune function is either conflicting or limited. There is also a lack of information on how cereal type and processing may influence the prebiotic potential and health effects of WGs. The work described in this thesis combined a human intervention study of WO and an ill vitro investigation of the effect of processing and cereal type on fermentation properties of WOs. Habitual 10w consumers of wG (< 24 g/d) participated in a randomised crossover study with a diet high in WG (> 80 g/d) or low in WG (< 16 g/d), with intervention periods of 6 weeks, separated by a 4-week washout. Adherence to the dietary regimes was achieved by specific dietary advice and provision of a range of food products and verified with food diaries and plasma alkylresorcinols (ARs). In the ill vitro studies, cereal samples and functions were fermented in anaerobic pH-controlled faecal batch cultures and bacterial concentrations and diversity and short chain fatty acid concentrations were analysed. On the WG intervention, consumption increased on average to 168 g/d (P < 0.(01), accompanied by a significant increase in ARs (P < 0.001) and fibre intake (P < 0.001), without any effect on intake of energy or macronutrients. However, there were no effects on body composition, blood pressure, blood lipids, gut microbiology, immune system or markers of inflammation or glucose metabolism. Nevertheless, during the WO period, there were trends for higher 24 h faecal weight and lower body weight and BMI, and trends for alterations in some immune, inflammatory and metabolic markers. In the ill vitro fermentation studies, all samples and fractions altered bacterial diversity, but maize and unprocessed flour were the most bifidogenic of the cereal samples and processing conditions, while refined samples resulted in greater increases in lactobacilli. Of the cereal fractions, fructan exhibited the greatest bifidogenic effect, while β-glucan resulted in the greatest increase in propionic acid. This study demonstrates that a significant increase in WG consumption in habitual low-consumers had little effect on parameters of gut, immune and metabolic health. Cereals differ significantly in their ill vitro prebiotic potential, and this is influenced by the degree and type of processing, which may underlie the lack of effect in the intervention stud y..