A key regulator of translation is the mammalian target of rapamycin (mTOR), a protein kinase member of the family of phosphatidylinositol kinase (PIK)-related kinases. mTOR is dually regulated by growth factors and nutrient availability, though the precise mechanisms by which mTOR is regulated are not well understood. The C-terminal of the mTOR catalytic domain has been of regulatory interest by the identification of the insulin stimulated and nutrient sensitive S2448 phosphorylation site. The functional significance of S2448 phosphorylation on the mTOR downstream targets p70 S6 kinase (S6K1) and eIF4E-binding protein 1 (4E-BP1) are unclear. A novel nutrient responsive mTOR phosphorylation site has been identified at T2446. In contrast to S2448 phosphorylation, T2446 is dephosphorylated when CHO-IR cells are insulin stimulated and phosphorylated when cells are nutrient deprived. Studies show that activation of AMP-activated kinase (AMPK) is concomitant with an increase in mTOR T2446 phosphorylation, paralleled by a decrease in S6K1 phosphorylation. Regulation of T2446 phosphorylation may involve AMPK. Phosphorylation at T2446 and S2448 is mutually exclusive. The functional significance of phosphorylation at T2446 and S2448 on the downstream target S6K1 was investigated by a mutational strategy where each site was substituted with non-phosphorylatable alanine or phospho-mimic glutamic acid. Evidence indicates that although phosphorylation of T2446 and S2448 is mutually exclusive in response to growth factors and nutrients, their individual phosphorylation may not be enough to have a direct effect on downstream S6K1 activity. Additionally, the tuberous sclerosis complex (TSC) may have positive regulatory effects on insulin signalling. Loss of TSC2 impairs insulin signalling by down- regulating the turnover of insulin receptor substrate-1 (IRS-1) protein, affecting associated class 1a phosphoinositide 3-kinase (PI3K) activity and downstream signalling; including suppression of PKB activation and mTOR S2448 phosphorylation.

Epstein-Barr virus (EBV) specific T cells control EBV-driven proliferation of infected B cells in vivo. Deficient EBV immunity predisposes to post-transplant lymphoproliferative disease (PTLD) and EBV-positive Hodgkin's disease (HD). Treatment for PTLD is reduction in immunosuppression (RIS) allowing EBV immune recovery. Current methods of monitoring RIS include immunophenotyping for increases in MHC Class IICD8 T cells, but this is not a specific measure of EBV immunity. A method was developed for enumerating functional EBV-specific T cells using an intracellular cytokine staining (ICS) assay to measure interferon-gamma (IFN-gamma) production after brief in vitro stimulation with EBV antigens. Analysis of EBV-positive PTLD patients showed a temporal relationship between emergence of EBV-specific IFN-gamma'CD8 T cells and PTLD regression in response to RIS. The increase paralleled the rise in MHC class IICD84 T cells. Analysis of tumour infiltrating lymphocytes (TILs) from EBV-positive HD patients showed presence of EBV-specific IFN-gamma+CD4 and IFN-gamma4CD8+ T cells. Although this result indicates EBV-positive HD is not a consequence of deficient EBV immunity, the observed accumulation of CD4+ T cells may represent immune suppressive cells. The ICS assay was demonstrated as a rapid and reliable method for detecting functional EBV immunity, but requirement for several million PBMCs can limit utility. Quantitative RT-PCR (qRT-PCR) was used to measure IFN-gamma gene transcription by T cells stimulated with EBV antigen and showed comparable results to ICS using fewer cells. Clinical assessment of EBV status of tumours is hampered by lack of a single monoclonal antibody able to detect all EBV antigens. A novel monoclonal antibody, RFD3, was used to stain EBV-positive tissue and detected all forms of EBV latency. Results correlated with current diagnostic techniques. In summary, this thesis describes development and evaluation of novel approaches for detecting EBV and measuring EBV-specific T cell immunity that have potential for clinical and research applications.

My main aim was to investigate human Ab responses to RAP1. For this study, I have developed nine recombinant RAP1 proteins (rRAP1) representing almost the entire sequence of mature RAP1 that have been expressed in <I>Escherichia coli</I> as soluble hexa-histidine or GST fusions. The antigenicity of the rRAP1 proteins was assessed by immunogenicity tests in mice and rabbits, and by <I>P. falciparum </I>RAP1-specific mAbs recognising a defined linear epitope. Antisera to seven of the rRAP1 proteins specifically reacted with parasites in immunofluorescence as well as parasite-derived RAP1 protein (<I>Pf</I>RAP1) in immunoblotting. These results indicate that these rRAP1 proteins bear antigenic similarity to <I>P. falciparum</I> RAP1. Affinity purified Abs produced in rabbits against three rRAP1 proteins block invasion of red blood cells by merozoites, and this suggests that the proteins contain protective epitopes. Analysis of serum Abs of residents of malaria endemic regions by ELISA shows that RAP1 is antigenic during naturally transmitted malaria infection. The recombinant proteins are specifically recognised by IgG Abs, with detectable Abs directed mostly towards fragments containing N-terminal sequences of mature <I>Pf</I>RAP1. By contrast, only few individuals had Abs to the C-terminus. Abs from malaria patients do not complete for a linear epitope recognised by an inhibitory anti-RAP1 mAb. This indicates that Abs from malaria patients bind mostly to epitopes different from that recognised by the inhibitory mAb. In a longitudinal study of individuals conducted over a period of 4 years in a region of seasonal and unstable malaria transmission, I have found that Abs to RAP1 are produced only after a documented clinical malaria.

No description available.

The unfolded protein response (UPR) detects the presence of misfolded proteins in the endoplasmic reticulum (ER) and subsequently relieves ER stress by increasing the folding capacity of the ER. The secretory pathway substrate HLA-B27 is highly associated with the chronic inflammatory disease ankylosing spondylitis (AS) and has a tendency to misfold in the ER. Here, we show that overexpression of HLA-B27 and non-disease associated HLA-B7 in immortalised cell lines leads to heavy chain misoxidation, which is accompanied by upregulation of BiP and splicing of XBP1, a key step in the IRE1 pathway of the UPR which is increasingly being linked with intestinal inflammation. We also demonstrate that different cell lines respond to different ER stress stimuli in distinct ways. We establish that HT1080 cells inefficiently induce a UPR in response to tunicamycin and that this has consequences for cell survival. However, inefficient activation of the UPR in HT1080 cells can be overcome by secondary signals, since co-administration of the tyrosine kinase inhibitor genistein leads to activation of XBP1. Furthermore, we show that genistein can inhibit UPR induction of BiP in response to a range of ER stresses indicating that the cancer drug genistein can inhibit or activate the UPR depending on the environment and cell type. This has implications for inflammatory disease since regulation of the UPR is important in determining a cell’s tendency towards apoptosis.

NK cell function is regulated by Killer-cell Immunoglobulin-like Receptors (KIR) some of which recognise class I Major Histocompatibility Complex molecules. KIRs have been shown to exhibit a high degree of functional diversity which is generated at several levels. However, the functional relevance of this diversity remains largely unknown. This thesis describes our approach towards elucidating the functional relevance of KIR diversity. To study this we first compiled all known KIR sequences into a database. We developed bioinformatics tools to facilitate the study of these sequences and have made both the tools and database publicly accessible online. Subsequent efforts were directed towards investigating the structural impact of KIR polymorphism by means of molecular modelling software. The results that were generated by this approach have provided information with regards to the ligand binding properties of most activating KIR proteins. In addition, we have also developed a KIR gene typing system capable of detecting all known KIR genes as well as the alleles of five of the KIR proteins for which a ligand has been described. We have implemented this KIR typing system to three different sample panels: a reference panel of more than 100 B-lymphoblastoid cell lines (BLCL), a family based KIR haplotype segregation study and a cohort of 141 unrelated donor (UD) haematopoietic stem cell transplant pairs. Our investigations have allowed us to generate the largest KIR typing reference panel, to characterise the KIR profile of a Mexican Mestizo population and to investigate the clinical relevance of KIRs in UD-Haematopoietic Stem Cell Transplantation (HSCT). Our results demonstrate that the beneficial effect of NK alloreactivity in the Graft-versus-Host direction as predicted by Ruggeri's algorithm cannot be applied to the UD-HSCT setting. In addition, I describe our findings relating to the clinical role of KIR genes and alleles in the UD-HSCT cohort.

Throughout evolution, mammals have co-existed with many harmless organisms such as saprophytic mycobacteria. These "Old Friends" have helped the hosts' immune system to evolve immunoregulatory mechanisms that prevent inappropriate immune responses. Exposure, once a common occurrence has now become increasingly rare. A revised version of the hygiene hypothesis proposes that this reduced exposure may be a contributing factor to the recent increase in allergic diseases in developed countries. In models of allergic pulmonary inflammation, treatment with M. vaccae inhibits airway hyperreactivity and induces allergen specific regulatory T cells (Tregs), which secrete IL-10 and depend upon production of IL-10 and TGF-p in vivo. Since Treg-induction is dependent on antigen presenting cells (APCs) such as CD11c+ cells, this thesis addresses the effects that M. vaccae has on CD11c+ APCs, in a mouse model of allergic pulmonary inflammation. M. vaccae treatment reduces pulmonary allergic inflammation by decreasing type-2 responses such as eosinophilia and IL-4 expression. Rather than an increase in type-1 cytokines, IL-10 is elevated in the lungs, both at the protein and message level. Characterization of pulmonary CD11c+ APCs by ELISA, FACS and real time RT-PCR, shows an immunoregulatory cytokine profile with increased expression of IL-10, TGF-fJ and IFN-oc. In passive transfer experiments, CD11c+ cells appear not play a role as regulatory cells themselves, but may be involved in the induction of Treg through their cytokine release. In vitro studies show these CD11c+ APCs are capable of inducing naive T cells to become Tregs, as measured by increased production of IL-10 and Foxp3 expression. M. vaccae has been used in clinical trials of asthma and eczema, with encouraging results. This thesis goes some way to understanding one mechanism behind a potentially valuable form of immunotherapy.

Fc-fusions proteins are a growing class of bio-therapeutics, widely used in the clinic as well as off label applications. In this work, the ability of the Fc region of IgG to act as an immunological adjuvant when fused to antigen was investigated. Fc-fusions were constructed, produced and purified, wherein the malarial antigen, MSP119, was fused to the Fc region of either mouse IgG2a or human IgG1. These monomeric Fc-fusion proteins were found to bind to Fc gamma receptors (FcyRs) in a similar fashion to the antibodies they were derived from. Immunisation of mice with these fusions resulted in the production of MSP119-specific antibodies, predominantly murine IgGl, with lower levels of IgG2a and IgG2b. However, these MSP119-specific antibodies had no influence on the course of a subsequent malarial challenge. In order to improve the immunogenicity of these monomeric Fc-fusion proteins, critical residues from IgM (a polymeric class of antibody) were introduced in an attempt to polymerise these fusions. The modifications described here, resulted in the formation of dimeric and barrel shaped hexameric IgGl based fusions, whilst failing to induce polymerisation of the mlgG2a based fusion proteins. In immunisation studies, these hexameric Fc-fusions were found to be less immunogenic than their monomeric counterparts, and again, failed to protect from challenge. Analysis of hexameric Fc-fusion binding to FcyRs revealed that the inclusion of the fusion partner, MSP119, significantly reduced the ability of the fusions to bind FcyRs. The ability of the hexameric Fc scaffold alone to act as a replacement for intravenous immunoglobulins (IVIG) in the treatment of immune thrombocytopenia (ITP) was also investigated. At the dose used in this work, under 2% of the commonly used dose of IVIG, the hexameric IgGl scaffold offered no amelioration of experimental ITP in mice. This work described here forms the foundation for the future use of stable well defined barrel shaped hexameric Fc-fusions, both as a platform for use in vaccination, and as a therapeutic reagent.

No description available.

The ageing population in Europe is increasing, presenting society with potential medical and economic challenges. Immunosenescence may be defined as deterioration of the immune system with age and is a possible causative factor for many age related illnesses. The colonic microbiota undergoes certain age related changes that may affect health. For example, above the age of 55-65 years, populations of bifidobacteria have been observed to decrease markedly. Evidence shows that bifidobacteria can inhibit pathogens thus a decrease in their activities may increase susceptibility to infections. To date, research into the immunostimulating influences of prebiotics on the elderly is limited. A recent study has however shown promising results with prebiotic B-GOS (Vulevic et aI., 2008). Confirmation of these findings and the metabolic effects of B-GOS in the elderly remain to be elucidated. Hence, this study was primarily aimed at investigating the effect of B-GOS on the gut microbiota composition, immune function and metabolites in an elderly cohort. Here, 40 elderly volunteers (n= 25 females, n=15 males) aged 65 - 80 yrs completed a randomised, double-blind, placebo controlled, cross-over study. 2.75g of B-GOS was consumed daily for a period of 10 weeks, followed by a 4 week washout period and then 10 weeks of placebo (or vice versa). Results showed that B-GOS greatly improved the gut microbial composition with significant increases in Bifidobacterium spp. 'H-NMR analysis revealed that this bifidogenic response led to increased lactate in faecal waters. Immunity was enhanced through increased NK cell activity and a reduced pro-inflammatory IL-6 cytokine response. The anti-pathogenic potential of lactate produced by Bifidobacterium spp. (identified in faecal waters) may well provide some explanation for the reduction in pro-inflammation and general improvement to Immune function. This work confirms that prebiotic B-GOS exerts both bifidogenic and Immune boosting effects in the elderly and has the potential to delay the condition of immunosenescence in such individuals.