Plasmodium falciparum is responsible for most malaria-related morbidity and mortality, mostly affecting young children, non-immune adults and pregnant women. A characteristic feature of the pathogenesis of infection caused by P. falciparum is the cytoadherence of infected erythrocytes to the endothelial cells lining the microvessels of host organs. This phenomenon, termed ''sequestration'', mainly results from the adhesive interactions between P. falciparum erythrocyte membrane protein-1 (PfEMP1) proteins on the surface of infected erythrocytes and various host endothelial receptors such intercellular adhesion molecule 1 (ICAM-1), which is hypothesised to have a role in cerebral malaria. PfEMP1 molecules consist of several Duffy binding-like (DBL) and cysteine rich interdomain region (CIDR) domains that have different cytoadhesive functions. The second class of DBL domains, DBLβ, has been associated with adhesion to ICAM-1 receptors. In the present study, we selected four recombinant PfEMP1ICAM-1-DBLβ domains for mouse immunisations. Thirteen monoclonal (mAbs) and polyclonal antibodies (pAbs) were raised to three recombinant domains (DBL13, DBL31 and DBL41). All mouse mAbs and pAbs comprised IgM antibodies that recognised homologous and heterologous DBLβ domains. Most mAbs and pAbs labelled the surface of erythrocytes infected by P. falciparum isolates, with an IgM labelling capacity ranging from 10.1% to 67.6% of total IEs. Mouse antibodies showed similar patterns of reactivity with ICAM-1-binding and non-binding isolates, and reacted with a parasite isolate from a different genome (3D7). Surprisingly, we detected a remarkable reduction in IE population after incubation with mouse mAbs and pAbs, and this was mainly observed with antibodies that strongly labelled the surface of IEs. We demonstrated that this haemolysis was resulted from an immunological interaction between mouse IgMs and a parasite-derived component on the surface of live IEs. Antibodies raised to DBL41 were the most effective in all assays. Of these, three antibodies (pAb B5, mAb B4, mAb G6) and an anti-DBL31 mAb (E7) significantly blocked IE adhesion to purified proteins (ICAM-1 and CD36) under static and flow conditions. These antibodies also blocked parasite adhesion to HUVEC under conditions of blood flow. In a separate work, we characterised the immune response of eight semi-immune serum samples obtained from female adults living in Kilifi County, Kenya. Our results indicated that semi-immune sera specifically recognised five recombinant DBLβICAM-1 domains and a VAR2CSA DBL domain, and recognised the surface of erythrocytes infected by diverse parasite isolates with variable levels of reactivity. Some sera, particularly JA225 and JA235, significantly inhibited IE adhesion to ICAM-1 under both static and flow conditions. To our knowledge, this is the first study to examine the use of PfEMP1ICAM-1-DBLβ domains for the development of mouse mAbs and pAbs that recognise homologous and heterologous parasite isolates and block IE adhesion. However, further work is required to identify the surface ligand(s) involved in interaction with mouse IgM and to investigate the mechanisms of IgM-mediated IE lysis.

Immunotherapy for renal cell carcinoma (RCC) has yielded some clinical responses. However this approach frequently fails, possibly due to inefficient migration of T-cells to tumour tissue or immunosuppressive mechanisms within the tumour environment. To aid development of T-cell therapy for RCC I investigated how T-cells are recruited to this tumour, which T-cell subsets infiltrate, and how they function. Analysis of the expression of all 19 chemokine receptors on matched TIL and PBMC demonstrated that CCR5, CXCR3 and CXCR6 were expressed at significantly higher levels on tumour-infiltrating T-cells than memory T-cells in PBMC, suggesting a role for these receptors in recruitment to RCC. Immunohistochemistry showed the corresponding ligands were present in RCC, and transwell assays confirmed the ligands induce migration of TIL. I demonstrated Foxp3\(^+\)CD25\(^{hi}\)CD127\(^{low}\) Tregs were enriched within the tumour, and also expressed high levels of CCR5, CXCR3 and CXCR6, as well as CCR6. They lacked expression of IL-2 and IFN-\(\gamma\) post-stimulation, consistent with a regulatory phenotype. Functional characterisation of Foxp3\(^-\) TIL demonstrated they can function ex vivo, however their high expression of the inhibitory molecule PD-1 may indicate exhaustion in vivo. Double positive CD4\(^+\)CD8\(^+\) T-cells were also enriched in TIL and had a similar functional profile to CD8 T-cells.

Gene directed enzyme prodrug therapy using E.coli the enzyme nitroreductase (NR) to activate the prodrug CB1954, is being developed as an attractive targeted chemotherapy for eradication of localized tumours. In addition to direct killing of NR-expressing tumour cells and potentially also their immediate neighbours via local spread of the activated prodrug, the consequent release of tumour antigens from dying tumour cells has the potential to induce antitumour immune responses. The present study investigates the capacity of NR/CB1954-mediated tumour cell death to activate CD8\(^+\) T cell responses using ovalbumin (OVA), as a model tumour antigen. The transgenic adenocarcinoma mouse prostate tumour cell line (Tramp-C1) was modified to stably express the therapeutic NR gene together OVA. These modified tumour cells were used to seed tumours in mice and OVA-specific T cell responses to gene therapy were investigated. Treatment of mice bearing NR-expressing tumours with CB1954 enhanced expansion of endogenous OVA-specific CD8\(^+\) T cells and marginally enhanced OVA-specific cytotoxic T lymphocyte (CTL) activity, however long-term CD8\(^+\) T cell dependent immunity was insignificant. The possibility of enhancing NR/CB1954-mediated long-term antitumour immune responses by combining with other immunogene therapies namely, 4-1BB costimulatory ligand (4-1BBL) or granulocyte macrophage colony stimulation factor (GM-CSF) was further explored. These combined therapies notably increased the frequency of memory OVA-specific CD8\(^+\) T cell and CTL response in some lymphoid tissue relative to NR/CB1954 monotherapy. One of the obstacles to cancer immunotherapy is the development of T cell anergy early in the course of tumour progression, therefore it was of interest to investigate the potential of NR/CB1954 and 4-1BBL combined tumour therapy to reverse CD8\(^+\) T cells anergy in vivo. This study describes preliminary results showing the effect of this combined therapy on the proliferative and functional responsiveness of anergic CD8\(^+\) T cells. In conclusion, these findings indicate that NR/CB1954-mediated tumour cell death is a weakly immunogenic process that facilitates short-term antitumour CD8\(^+\) T cell responses. Combining NR/CB1954 with intratumoural GM-CSF or 4-1BBL immunotherapy can enhance the frequency and effector function of memory tumour antigen-specific CD8\(^+\) T cells; and thus has the potential to provide long-term antitumour immunity.

Myeloid and lymphoid malignancies are potentially curable through a graft versus leukaemia (GvL) effect following allogeneic haematopoietic stem cell transplantation. Whilst donor T cell are thought to be the main mediators of GvL, the effect of donor NK cells within HLA matched T cell depleted transplant setting is more unclear. Patient blood samples were analysed during the first month post-transplant, with higher reconstitution of NK cells at two weeks conferring a relapse protection association. Donor stem cell graft samples, from which NK cells within the patient at two weeks are thought to be derived, similarly displayed a strong association between high NK cell dose and protection from disease relapse. CD56dimDNAM+ NK cells were found to be the population with the most significant association. The ability of NK cells to kill AML blasts in a DNAM dependent manner was shown indicating that direct killing of residual tumour cells may be a valid mechanism of GvL. These findings suggest that optimising the number of NK cells within stem cell grafts should be considered as a means to prevent disease relapse.

Dr James A. Thomson reached a milestone discovery in 1998, as he managed to isolate and in vitro expand embryonic stem cells originating from human blastocysts. Since then, human embryonic stem (hES) cells have served as excellent tools for the understanding of a plethora of events that take place during embryogenesis. A full and comprehensive analysis of the molecular mechanisms that regulate both pluripotency and differentiation procedures will ultimately allow these cells to be utilised for therapeutic purposes. The first part of the present thesis is dedicated to investigating the implication of ADP-Ribosylation Factor 6 (ARF6) in TGFβ signalling. ARF6 is a low molecular weight GTPase involved in various cellular functions. Our preliminary data indicate that ARF6 interacts with SMAD4. Building on that, we uncover novel interactions of ARF6 with proteins SMAD2/3 and the interconnection between nucleotide status and downstream signalling events. The connection between ARF6 and TGFβ signalling led us to hypothesize a role for the GTPase in hES cells. In that system, we characterise the effects of ARF6 activation or knockout on both Activin A and BMP4 signalling. In addition, we uncover a novel role for the GTPase during mesendoderm specification. In the last part of the thesis, we utilise a broad transcriptomic approach to reveal novel candidates that are implicated in early differentiation of hES cells to mesendoderm. The assay has been carried out using a novel culture system, based on the ability of Activin A to preserve pluripotency and BMP4 to initiate differentiation.

Nontyphoidal Salmonella (NTS) are a major cause of fatal bacteremia in Africa. We investigated the role of bactericidal antibody in complement-mediated killing of NTS. Immunised mice serum lacked such activity due to weak complement activity. Mouse anti-Salmonella antibodies were able to effect killing when given a source of human complement. Human serum bactericidal assays showed that the serum-susceptibility of an African clinical isolate varied based on growth conditions. In vitro kinetics of serum-killing, phagocytosis and antibody and complement deposition indicated that a proportion of Salmonellae are phagocytised before serum-killing occurs and this may explain how the protective effects of anti-Salmonella antibodies are undermined in IFN\(\gamma\) deficiency. We studied targets of bactericidal antibodies using an optimised serum-adsorption procedure and a range of different NTS strains and serovars as well as LPS mutants. Antibodies against the immuno-dominant O-antigen (OAg) were a major target of bactericidal antibodies against NTS in human serum. These data support development of an OAg based vaccine against NTS. Finally, using electron microscopy, we showed the physical effects of serum-killing on Salmonellae and also demonstrated that a major difference between inhibitory and bactericidal serum was the quantity of complement deposited on Salmonellae.