The role of IFN in acquisition of immunity against erythrocyte forms of P.c. chabaudi AS was studied. Inbred NIH mice given the construct 7 days before malaria infection, showed a significant delay in the onset and in the level of the recrudescent parasitaemia in comparison with controls. No differences, however, were observed in the recrudescent parasitaemia between the groups. NIH mice infected with malaria 3 days after or on the same day as the administration of the IFN construct, showed a primary peak of infection similar to controls, but the resolution of this patent parasitaemia occurred 1 or 2 days earlier in the experimental mice when compared with controls. In the same experiment, mice given the construct 10 days before malaria infection had a similar course of infection as controls. Simultaneous inoculation with two S. typhimurium constructs: IFN and TNF, 8 days before malaria infection resulted in a course of parasitaemia similar to that observed in mice given the IFN construct alone. On the other hand, inoculation of 'susceptible' inbred A/J mice with S. typhimurium/IFN 3 or 8 days before malaria infection had no effect on the course of the parasitaemia when compared with controls. The immune mechanisms involved in the better control of the malaria infection of NIH mice given S. typhimurium/ IFN, seem to be independent of nitric oxide (NO) production, since increased levels of the molecule were demonstrable around the peak of the primary parasitaemia in control groups but not in experimental mice. In the latter basal levels of serum NO were observed from the period after the S. typhimurium/ IFN inoculation until up to three days after the peak of the parasitaemia.

A growing problem with drug resistance in Human African Trypanosomiasis has necessitated the implementation of screening programmes to monitor for its spread. This thesis describes the study of several factors that can influence the selection and propagation of drug resistance in T. brucei. Human African Trypanosomiasis (HAT) is caused by T. brucei gambiense and T. brucei rhodesiense. The few drugs used for the treatment of the disease are either toxic, cause severe side effects or suffer from parasite resistance. The T. brucei P2 transporter, which is encoded by the gene TbAT1, mediates uptake of melaminophenyl arsenicals and diamidines. Reduced P2 uptake is associated with drug resistance. A number of point mutations found in a laboratory derived melarsoprol resistant T. brucei stock (STIB 777R) allowed development of a PCR/RFLP based molecular method to identify resistance alleles. By 1999, 20-30% of patients treated in Omugo, NW Uganda were failing to respond to melarsoprol. PCR/RFLP analysis indicated that mutant alleles accounted for 58.5% of those in circulation. Melarsoprol was withdrawn in 2001 and by 2003 mutant TbAT1 alleles accounted for only 14% of those in circulation in NW Uganda. The current study aimed to determine the incidence of the PCR/Sfa NI TbAT1 mutant alleles in 2006, some five years after melarsoprol had been withdrawn as first-line treatment. Successful molecular analysis of 91 of 132 (68.9%) T. b. gambiense field isolates from Omugo and Moyo in NW Uganda indicated the presence of only TbAT1 wild type alleles. Mutant alleles thus appear to have disappeared. This may be the result of parasite fitness cost following the withdrawal of melarsoprol as a stage II first-line drug from Omugo health centre, Arua, since 2001. This apparent instability of TbAT1 mutants in the field may be exploited for rational or alternating use of melarsoprol and eflornithine (DFMO) to ensure a longer life for eflornithine, delaying the onset of resistance. Insight into the overall population structure of the T. b. gambiense from Omugo, Arua (N=54) and Moyo (N=17) was obtained using mini/microsatellite marker analysis. Genetic diversity was observed to be more intra than inter regional. Multilocus genotype data analysis revealed the Omugo, Arua, population was genetically distinct from the Moyo population (Nei’s genetic distance=0.176). The evidence indicated surprisingly little genetic exchange with an excess in homozygosity (Fis >0) and alleles in linkage disequilibrium (P<0.05) within the Omugo, trypanosome population. This excess in homozygosity may be due to population sub-structuring, trypanosome inbreeding, or migration of patients. The latter is likely occurring from the neighbouring T. b. gambiense endemic disease focus in Southern Sudan. The findings suggested that the T. b. gambiense from Arua is not panmictic, clonal or epidemic but there is some level of genetic exchange. The possibility that T. b. gambiense can infect animals raises the prospect that wild or domestic animals may act as a reservoir and that a veterinary link to gambiense Human African Trypanosomiasis exists. Treatment of animals for babesiosis and trypanosomes with diminazene, uptake of which is mediated through TbAT1/P2 could select for P2-defective drug resistant trypanosomes, thereby threatening control of the human disease as well. Species detection by PCR for animal and human trypanosomes in dog isolates (N=190) from the tsetse fly endemic Jos Plataeu, Nigeria did not reveal T. b. gambiense, but multiple infections with T. brucei (95%), T. vivax (89%), and subspecies T. congolense forest (54%) and savannah (50%) were detected. The dogs were also infected with other parasites, including Babesia canis (22%) and Hepatozoon canis (16%). Multiple infections can make correct diagnosis difficult and the infections are likely to be missed by the less sensitive microscopy method. The trypanocidal action of the diamidine group of trypanocides, diminazene, pentamidine and furamidine (DB75) are principally mediated through the TbAT1/P2. In addition, pentamidine is taken up by two additional T. brucei transporters called High Affinity Pentamidine Transporter (HAPT1) and the Low Affinity Pentamidine Transporter (LAPT1). DB75 also has a secondary unknown route. Loss of TbAT1/P2 leads to significant resistance to DB75 and diminazene but not pentamidine. Identification of other markers of resistance is necessary to determine if other routes of drug entry do exist apart from P2 and whether these can be exploited for the delivery of new trypanocides into the trypanosomes. Adaptation of the T. brucei tbat1 knock-out cell line to higher concentrations of diminazene by in vitro selection for resistance led to loss of HAPT1. The resultant phenotype was similar to the previously characterised pentamidine resistant clone B48, but more resistant to diminazene and DB75. The adapted line was still capable of accumulating 1 µM radiolabelled diminazene suggesting both HAPT1 and LAPT1 as possible routes for diminazene uptake. Adaptation of the T. brucei tbat1 knock-out cell line to a high concentration of DB75 over the same 6 months period did not lead to increased resistance. Overall the project has confirmed an important role for tbat1/P2 in development of resistance to melarsoprol in the field. Importantly, it appears that removal of the selection pressure of melarsoprol leads to a loss of tbat1 alleles associated with resistance in a population of trypanosomes capable of genetic exchange in NW Uganda. Although evidence for a dog reservoir for T. b. gambiense in Nigeria was lacking in this study, a risk of selecting resistance in animals must remain high on any list of consideration. I have further shown that the diamidine drug, diminazene, used in veterinary medicine also appears to enter T. brucei via the HAPT1 transporter, as well as the P2 transporter. Loss of HAPT1 through selection with diminazene leads to high level pentamidine resistance, which could indicate a further risk in selection of human infectious trypanosomes also resistant to drugs like pentamidine.

Asthma is a chronic disease characterised by variable airflow obstruction, bronchial hyperresponsiveness and airways inflammation. At an immunological level Th2 inflammation and the presence of activated eosinophils and mast cells are key features of asthma. ST2, the receptor for the novel cytokine IL-33, is expressed upon Th2 lymphocytes and mast cells but its role in clinical and experimental asthma remains unclear. IL-33 has been shown to induce local and systemic eosinophilia when administered to the peritoneum of mice. In this thesis I have set out to test the hypothesis that the activation of mast cells by IL-33 acting on cell surface ST2 plays a critical role in allergic airways inflammation. I began by studying the function of ST2 on mast cells in vitro. I found that ST2 was expressed at an early stage of development, and correlated closely with the expression of the stem cell factor receptor (c-kit), a marker present on mast cells from a progenitor stage. Despite this mast cells generated form ST2 gene deleted mice proliferated and matured normally. When mast cells were activated by IL-33, acting in an ST2-dependent manner, pro-inflammatory cytokines and chemokines were released that have potential roles in asthma, specifically IL-6, IL-13, MIP-1α and MCP-1. To extend these findings I looked at the role of ST2 in allergic airways inflammation. I first optimised and validated an ovalbumin and adjuvant based ‘short’ twelve day model of murine asthma and demonstrated that ST2 gene deletion results in attenuated eosinophilic inflammation. In addition to being ST2 dependent it is possible that this adjuvant based short model is mast cell dependent, unlike longer adjuvant based models which are mast cell and ST2 independent. Therefore I went on to study an adjuvant-free model of asthma which has been demonstrated to be mast cell dependent. In this adjuvant-free model of asthma the airway inflammation was attenuated in ST2 gene deficient mice compared with wild type mice, while AHR was unaffected. There was an associated reduction in IgE production and thoracic lymph node recall Th2 cytokine responses. I then examined the effect of ST2 activation in the lungs. When IL-33 was administered directly to the airways of naïve mice it induced the features of experimental asthma. There was extensive eosinophilic inflammation within the lung tissue and airspaces. The Th2 cytokines IL-5 and IL-13, and the eosinophil chemoattractant chemokines eotaxin-1 and eotaxin-2 were detected at increased concentrations. Significant airways hyperresponsiveness was also generated. Using ST2 gene deleted mice I demonstrated that these effects were ST2 specific. Although I have shown that mast cells are activated by IL-33 in vitro, I used mast cell deficient mice to demonstrate that the eosinophilic inflammation generated by IL-33 is unaffected by the absence of mast cells. These data show that IL-33 can induce in the lungs the cardinal pathological characteristics of asthma, and that it appears to act upstream of other important mediators such as IL-13 and the eotaxins. Furthermore the IL-33 receptor ST2 is required in an adjuvant free model of asthma, which is more akin to human disease. Placing these findings in the context of recent evidence that IL-33 is released by structural cells in response to damage or injury suggests that IL-33 may play a key role in initiating the immunological features of clinical asthma. As a consequence of this position in the hierarchy of inflammation IL-33 offers a promising direct target for novel biological therapies in asthma.

Reproduction is a crucial process, required for bringing the next generation into the world. In preparation for pregnancy, and throughout pregnancy itself, reproductive tissues recruit specific populations of immune cells that are thought to contribute in a variety of ways to successful reproduction. Pregnancy culminates in parturition, an inflammatory process characterised by an influx of inflammatory cells into reproductive tissues. Effective healing of reproductive tissues in the post-partum period is vital for continued reproductive success, and it too is thought to involve specific populations of immune cells. For leukocytes to effectively perform their functions in the reproductive system and elsewhere, migration to the right place at the right time is crucial. Key regulators of leukocyte homing are the chemokine family of chemoattractants and their G-protein coupled receptors. The chemokine network is complex and controls migration of leukocytes from the Bone Marrow (BM) into the blood and from the blood into tissues. Chemokines influence leukocyte position within tissues, and orchestrate their departure. Very little is known about the types of leukocytes present within reproductive tissues in the post-partum period, or the chemokines and receptors that could be involved in their migration. Exploring these processes is critical for an understanding of how tissues are repaired in readiness for subsequent pregnancies. In this thesis I have examined the leukocyte populations in reproductive and peripheral tissues of mice during the post-partum period and compared them to those found in Non-Pregnant (NP) mice. This analysis has encompassed a range of myeloid cell types, and also the complex populations of CD3+ cells that exist in reproductive tissues. I was also interested in how these cells are instructed to enter reproductive tissues, and in particular on the role of CC chemokine Receptor 2 (CCR2), a receptor associated with the recruitment of monocytes and T cells into tissues. My work has clearly identified cells expressing CCR2 both in reproductive tissues and elsewhere, and defined the impact of the genetic deletion of this receptor on leukocyte populations during the post-partum period. These experiments exploited a variety of standard techniques including histology, quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR), Enzyme-Linked ImmunoSorbent Assay (ELISA) and Luminex, but they also required the development of challenging multiparameter flow cytometry protocols that allowed the simultaneous analysis, and definitive identification, of several leukocyte populations in various tissues and at specific reproductive time-points. Chapter 3 describes detailed experiments that focussed on characterising the myeloid cell populations across a variety of tissues in NP, 1 Day Post Partum (DPP) and 7DPP mice. Most strikingly, this revealed a profound accumulation of several myeloid cell populations in reproductive tissues at 1DPP, including inflammatory Ly6Chigh (hi) monocytes and neutrophils. Moreover, many of these myeloid cells expressed active CCR2 and remarkably CCR2 deletion was associated with a dramatic reduction in myeloid cell abundance in the uterine horn one day after birth. Thus, CCR2 appeared to be required for myeloid cell recruitment to the post-partum uterine horn. Chapters 4 and 5 describe changes in CD3+ cell populations over the post-partum period. Interestingly, the main finding from reproductive tissues was that the large majority of CD3+ cells lacked expression of CD4 and CD8, and were thus termed CD3+ Double-Negative (DN) cells. Three main CD3+ DN cell populations were described. CD3+CD25+NK1.1+TCRβ+ DN cells, likely to be Natural Killer T (NKT) cells, which were mainly found in reproductive tissues and blood. All tissues studied were found to contain CD3+NK1.1-TCRβ+ DN cells, likely to be ‘true’ DN T cells and CD3+NK1.1-TCRβ-TCRγδ+ DN cells, which were consistent with a γδ T cell phenotype. CD3+ DN cells were also found to increase in number at 1DPP, compared to NP tissues, driven by an increase in DN T cells. In contrast to myeloid cells CCR2 was not required for this change. However, at 1DPP there was a CCR2-dependent increase in the proportion of CD3+ DN cells in the blood. Finally, in Chapter 6, hormonal influences on leukocyte populations in reproductive and peripheral tissues were considered. This work had two major components: analysing sex differences in myeloid and T cell populations and exploring the effect that lactation has on these cell subsets over the post-partum period. Females were found to have an increased proportion of eosinophils in their blood, whereas males had a higher proportion of monocytes. I also found that female and male reproductive tissues, as well as peripheral tissues, have very similar CD3+ DN cell populations, suggesting that these cells serve roles in reproductive tissues that are not unique to one sex. Finally, CD3+ cell populations in the post-partum period were found to be controlled to some extent by lactation. Collectively, this work has significantly extended our understanding of leukocytes in various tissues in the post-partum period, and revealed the importance of chemokines in the regulation of these cells. It has laid the groundwork for future investigations aimed at dissecting the functions of these cells in reproductive tissues in the post-partum period.

Cytotoxic chemotherapy remains the mainstay of treatment for patients with cancer, however immunotherapy is starting to emerge as an additional modality of treatment. Evidence suggests that chemotherapy can synergise with immunotherapy to improve responses. Although CD8 T cells have been regarded as the main anti-tumour effector cell, the role of CD4 T cells in orchestrating CD8 and other anti-tumour responses is increasingly recognised. However, the CD4 T cell population contains effector and suppressive subsets with diverse and opposing functions. This thesis describes the establishment of a murine mesothelioma model with which to study the effects of different CD4 subsets on anti-tumour immune responses, and investigate their capacity to provide cognate help to tumour antigen specific CD8 T cells. Haemagluttin (HA) specific CD4 T cells from transgenic mice were polarised in vitro into Th1, Th2, Th17 and Treg subsets and adoptively transferred alongside HA specific CD8 T cells into mice bearing HA expressing tumours derived from a mesothelioma cell line. The effects of the different CD4 subtypes on tumour growth and their capacity to provide ‘help’ to CD8 T cells was investigated in a prophylactic treatment model and in the context of treatment with gemcitabine chemotherapy. Results showed that survival and behaviour of in vitro differentiated CD4 subtypes after adoptive transfer was highly variable and that only Th1s displayed anti-tumour activity when injected prophylactically, prior to tumour inoculation. Cytotoxic chemotherapy did not provide a favourable environment for adoptive transfer of in vitro differentiated CD4 cells. No antitumour activity was seen against established tumours, which may have been due to overriding tumour induced immunosuppressive mechanisms. Successful treatment of established tumours that had been treated with chemotherapy required both the provision of HA specific CD8 cells and the prior removal of an established, endogenous regulatory CD4 T cell population.

Human cytomegalovirus (HCMV) infection in newborns and immunocompromised individuals with immature or deficient immune systems can cause life-threatening diseases. The clinical and subsequent economical burden of HCMV infection led the US Institute of Medicine designating a vaccine for HCMV as the highest level of priority. Complex virus-host interactions have developed over millions of years of co-evolution, making the understanding of the pathogenesis of HCMV disease particularly challenging. Consequently, a crucial factor in aiding the development of effective vaccinations and therapies to significantly reduce morbidity and mortality associated with HCMV infection is elucidating what immune mechanisms contribute to/impede protection against infection. For example, is the induction of immunomodulatory agents such as cytokines beneficial or harmful to the host during infection? Given the known immunosuppressive properties of one such cytokine, interleukin-10 (IL-10), in conjunction with the evolutionary acquisition by HCMV of its own IL-10 homologue, I hypothesised that mammalian- and viral-IL-10 suppress protective immunity during acute CMV infection. Utilising a mouse model of CMV infection, I revealed a surprising antiviral role for IL-10 during acute infection in vivo, which was achieved via limitation of activation-induced death of NK cells. The IL-10-related cytokine interleukin-22 (IL-22) provides critical protection against certain infectious agents and I therefore hypothesised that IL-22 provides protective immunity during acute CMV infection. Utilising the murine infection model once more, I discovered a tissue-specific antiviral role for IL-22 during acute infection in vivo and made the surprising finding that neutrophils play a protective role during infection. I also demonstrated that neutrophils can directly inhibit viral replication in vitro. Thus, novel insights into cytokine biology in the context of viral infections in vivo revealed by these studies highlighted important considerations for future research into herpesvirus infections, and has major implications for the treatment of this important infectious disease.

For the second approach, monoclonal antibodies were raised against rabbit, rat, mouse and human C6. The two most interesting antibodies were raised against human C6 and inhibited complement-mediated haemolysis in a cell-based assay. Both of these antibodies were species specific, excluding the possibility of testing their therapeutic properties in animal models of complement-mediated disease. Instead, an ex vivo model of cardiopulmonary bypass was established and used to test the ability of these antibodies to block soluble C5b-9 formation. Neither antibody inhibited soluble C5b-9 formation, suggesting that they might be interfering with the insertion of C6 into the cell membrane during MAC assembly.

Most of our understanding of immunity has been gained through studies of humans or laboratory rodents. However, such studies do not allow the immune system to be studied in the ecological context in which it has evolved and, as such, they provide a poor model for studying the variation in infectious disease resistance and immune function observed in natural settings. Studies of natural populations have provided fresh insights into the evolution and phenotypic consequences of immunogenetic variation, but have thus far concentrated almost exclusively on genes of the major histocompatibility complex (MHC). As these genes form only a fraction of the vertebrate immune repertoire, there is a need to broaden research in natural populations to include non-MHC genes, in order to gain a more comprehensive understanding of natural selection and immunity. In this thesis, the genetic diversity of a range of non-MHC immune genes was examined in a natural population of field voles (Microtus agrestis L.) in Kielder Forest, UK, which are subject to infection by a range of pathogens. Cytokine genes were the primary focus of this study as they play a central role in regulating the immune response but have rarely been studied in wild species. I examined the hypothesis that, as cytokines are crucial to immunity, variation within these genes may be under selection within populations and between species, and may mediate phenotypic differences between individuals in parasite resistance and immune function. Coding regions from nine cytokine and three other non-MHC vole genes were sequenced, yielding 6.6 Kb of sequence data and 26 SNPs (1 per 255 bp). Three cytokine genes (Il1b, Il2, and Tnf) exhibited patterns of polymorphism consistent with balancing selection maintaining genetic diversity, including an excess of intermediate frequency mutations and more even allele frequencies than one would expect under neutrality. Polymorphism within Il1b and Il2 was also consistently associated with variation in parasite resistance, providing evidence that pathogens are the selective force driving the maintenance of genetic diversity at these loci. In addition, Il1b and Il2 exhibited repeated associations with variation in host immune phenotype, while the Il12b gene was associated both with variation in pathogen resistance and with altered expression levels of Il1b and Il2. Variation in immune function, mediated through the cytokine network, is therefore likely to contribute to parasite resistance in the field vole. This work is the first to show that variation in cytokine genes of a natural population can be maintained by selection, and that this variation can lead to phenotypic variation in parasite resistance and immune function. More broadly, this thesis demonstrates that wild rodents are an excellent model to help us bridge the gap in our understanding between the mechanistic insights gained through studies on laboratory rodents and the variation in infectious disease susceptibility and immune function observed in nature.

The transcription factors STAT3 and NF-κB play key roles in inflammation, immunity and cell fate. In the liver, they are responsible for transcribing hundreds of genes in response to combinations of IL-6, TNFα and IL-1β, and so together co-ordinate the acute phase response to infection. Dysregulated STAT3 and NF-κB signalling leads to chronic inflammation and is implicated in the development of many cancers. A variety of highly context-dependent intercellular and intracellular mechanisms have been discovered which facilitate both positive and negative cross-talk between STAT3 and NF-κB. Whilst the long-term signalling dynamics of NF-κB have been characterised in single cells, and were found to be oscillatory, imaging studies on STAT3 have focused upon the short-term mechanisms of nuclear transport rather than the long-term dynamics. STAT3 has been shown to oscillate in a population of synchronised cells so it is possible that STAT3 will exhibit oscillatory spatio-temporal signalling dynamics in response to cytokine stimulation. The primary aim of this thesis was to characterise the long-term signalling dynamics of STAT3 in response to IL-6, using fluorescent fusion protein reporters for STAT3 and its inhibitor SOCS3, in conjunction with live single cell fluorescence microscopy. Towards these aims, STAT3 and SOCS3 fluorescent fusion proteins were constructed. The responses of a candidate cell line to IL-6 and TNFα were investigated, and then the fluorescent reporters were characterised in that cell line. The N-terminal tagged EGFP-STAT3 reporter was found to be the most accurate reporter of IL-6 signalling. The EGFP-STAT3 was then used to investigate the single cell spatio-temporal dynamics of STAT3 in response to differently timed lengths of IL-6 stimulation. STAT3 was found to oscillate with a period of approximately 90 min in response to continuous IL-6 stimulation, but only underwent a transient nuclear translocation in response to a 30 min IL-6 pulse. Furthermore, the patterns of gene expression were characterised for the timed IL-6 treatments. The quantified single cell dynamics were used to constrain an existing generic model of STAT:SOCS signalling; the model was able to capture the observed single cell dynamics using a minimal ordinary differential equation approach. The secondary aim of the thesis was to study cross-talk between STAT3 and NF-κB using live cell microscopy techniques. The effects of co-stimulation of NF-κB and STAT3 were investigated using combinations of TNFα and IL-6 stimuli. Combinations of single or dual transfections, and single or dual stimulation were performed as controls in order to tease apart the effects of co-expression and co-stimulation. The importance of the timing of cytokine stimulation was also investigated. Finally, the effects of IL-1β upon IL-6 induction of STAT3 were investigated, as this was shown elsewhere to inhibit STAT3 signalling and so was expected to produce interesting spatio-temporal signalling effects. This preliminary study revealed distinct subpopulations of cells with different p65 and STAT3 response patterns. The STAT3 response was knocked down or significantly delayed in many cells but a small subset exhibited atypical oscillatory dynamics. Interestingly, the p65 dynamics were also significantly perturbed by IL-6 and IL-1β co-stimulation, indicating that there are cross-talk events occurring in both directions. Consequently these studies represent a very important area for future investigation.

The Gram negative bacillus Mannheimia haemolytica is a natural inhabitant of the upper respiratory tract in ruminants and the most common secondary agent of the bovine respiratory disease complex. It is known to produce the extracellular neuraminidase NanH, which has a yet unknown biological role but is suspected to be important for bacterial adhesion to host cells, colonisation, capsule synthesis and biofilm formation. The structure of NanH is not known therefore, the functional domains of NanH, the tertiary structure and the residues involved in catalysis were predicted by sequence homology to the coordinates of other neuraminidases solved by crystallography. The catalytic domain was delimited from residues 23 to 435 and purified. The predicted catalytic residues were substituted in the recombinant NanH for confirmation of their role in hydrolysis of sialic acid. The function of the additional domains is unknown but analysis of NanH sequence and other associated genes found in the chromosome of M. haemolytica, suggest the presence of an autotransporter domain. The role of NanH in colonisation and infection is not known however, molecular characterisation is presented in this work. These data provide the basic knowledge required for future studies on using Nanh as a therapeutic and prophylactic target.