Chemokines play a fundamental role in regulating phagocyte responses during an infection. To test a role of the unusual β chemokine CCL6 in vivo, a mouse model was generated through germline mutagenesis of CCL6. Increased number of bone-marrow cells, myelopoietic activity and enhanced phagocyte recruitment in mutants indicated a regulatory, anti-inflammatory role for CCL6 in vivo, which was suppported by increased chemotaxis of mutant neutrophils to the CCR1 agonist, CCL3/MIP-1α in vitro. Microbiological analysis revealed controlled Listeria monocytogenes infection in both control and mutants. The data presented here is a report on CCL6 deficiency in mice of a mixed background (129xC57BL/6) and 129 congenic background. The importance of background genetics in characterising the role of CCL6 in LPS-induced inflammation became apparent in an in vivo assay for endotoxemia. Upon LPS challenge, CCL6-deficient F2 hybrids produced 30-fold increase in serum TNFα than wild types and succumbed to shock. However, the 129 congenic mice showed increased susceptibility to LPS. A lower expression of CCL6 and linkage to a set of LPS response genes affecting TNFα production and PMN recruitment accounts for the hypersensitivity observed in this background. Despite the effect of background genetics, a contribution of CCL6 in LPS-induced responses was supported by enhanced production of proinflammatory cytokines upon LPS stimulation by macrophages in vitro. Generated by phagocytes themselves, CCL6 regulated hematopoiesis in vivo and also helped sustain the recruitment threshold of phagocytes and CCR1-mediated neutrophil migration via feedback inhibiton. Taken together, the data provides a genetic evidence of the immunomodulatory role of CCL6 in homeostasis and inflammation. Moreover, it provides an in vivo model, the first of its kind, through which the physiological role of the human C6 chemokines may be elucidated.

The Red Queen hypothesis elegantly reflects virus-host interactions whereby alternate switching in selective advantage occurs between host and parasite. HIV has a prolific ability to generate genetic diversity, contributing to viral persistence. This property presents serious challenges to sustained treatment with antiviral drugs on a global scale. In resource poor settings where viral monitoring is not currently feasible, the possibility of more widespread resistance exists. In this thesis I demonstrate using meta-analysis that lack of viral load monitoring in the first year of antiretroviral treatment is associated with higher prevalence of resistance in patients with viral failure. A second meta-analysis suggests that use of boosted protease inhibitors as first line therapy is associated with lower prevalence of resistance mutations compared to non-nucleoside reverse transcriptase inhibitors, therefore potentially limiting the emergence of resistant HIV strains. The use of Pis in such areas might, however, be complicated by our findings that certain viruses circulating in resource poor regions (not previously exposed to PIs) have reduced PI susceptibility due to gag polymorphism, highlighting the plasticity of HIV. The continual evolution of drug resistance means that further research into alternative targets is required. Such targets may include host factor-virus interactions. The mammalian restriction factor BST-2/tetherin is counteracted by HIV-1 using its Vpu protein. We show that tetherin has been under positive selection in primates. Furthermore tetherin can be rendered almost insensitive to Vpu by a single positively selected substitution, revealing this as a potentially important drug target, and suggesting tetherin's evolution has been shaped by viruses. Intriguingly, most SIV do not encode Vpu, and I show that the envelope protein of SIVtan is able to counteract tetherin by intracellular sequestration. Pharmacological blockade of Vpu-tetherin interactions should be investigated, although we have highlighted a potential mechanism for virus escape through gain of function by envelope.

Background: An increasing men's studies movement attempts to understand how different masculine norms affect men's health and behaviour and how behaviours such as alcohol use and violence act as ways of coping both with the pressure to fulfil masculine norms and with emotional distress. However, the vast majority of this work has been in western contexts. This study sought to extend this fairly western-centric work by examining the relationships between gender norms, emotional distress and men's alcohol use, perpetration of intimate partner violence (IPV) and sexual behaviour in a low-income area of Mumbai, India. Methods: Secondary quantitative data (n=2,381) from a survey of men in three low-income districts in Mumbai were analysed in order to identify psychosocial factors associated with men's perpetration of IPV, alcohol use and extramarital sex. A period of fieldwork was undertaken in Dharavi, Mumbai in 2009-10 which included in-depth interviews with 29 married men, aged 21-52. Results Quantitative analyses found evidence for associations between men's ability to fulfil masculine norms and perpetration of IPV as well as psychological distress. Qualitative interviews highlighted the range of norms men were exposed to, defended and contested. Many men struggled to fulfil dominant notions of masculinity. In addition, many men had poor emotional and social support, frequently dealing with distress on their own. Men reported using behaviour such as alcohol use, violence and extramarital sex as ways of dealing with difficult emotions, social isolation, as well as a range of difficulties in their marital relationships. Conclusion: Norms around masculinity and the effects these have upon men emotionally are important in understanding men's involvement in these behaviours in this context. Understanding men as gendered as well as emotional beings is important in engaging with a wide variety of men in order to bring about lasting social as well as behavioural change.

Matching for HLA alleles between patients and their potential donors is critical for the success of haematopoietic stem cell transplantation. Current genotyping methods identify HLA alleles but not the haplotypes in which they reside, although both are important for transplantation outcome. The aim of this thesis was to identify HLA specific polymorphisms and tagSNPs that could identify 3 common HLA haplotypes present in population of Northern European (NE) Caucasian ethnicity, HLA-A*01-B*08-DRB1*03, HLA-A*02-B*44-DRB1*04, and HLA-A*03-B*07-DRB1*15. Phylogenetic and bioinformatics methods were used to select polymorphisms for each of the nine HLA alleles that were unique, or only found at low frequency in other HLA alleles. PCR -sequence specific priming (SSP) was used to validate the nine selected polymorphisms, which were then incorporated into a multiplex SSP-PCR assay. However, this method does not differentiate between homozygous and heterozygous samples positive for the HLA alleles and therefore an allele specific primer extension (ASPE)-Luminex assay was developed to allow the simultaneous detection of the selected HLA polymorphisms. In order to determine the HLA haplotypes, sets of tagSNPs exhibiting high specificity for the common haplotypes were then identified. This was achieved by using British Birth Control Cohort (~2,000 samples) SNP genotyping data generated from Illumina and Affymetrix gene chips. Eight tagSNPs were identified; rs2187668 and rs2734986 for HLA-A*01-B*08-DRB1*03; rs2844821, rs2596477 and rs660895 for HLA-A*02-B*44-DRB1*04 and rs3094170, rs3129860 and rs3130933 for HLA-A*03-B*07-DRB1*15 haplotypes. These tagSNPs capture the three haplotypes with a specificity ranging from 96 to 100 %. Luminex-ASPE multiplex assays were developed to validate the eight tagSNPs using 59 homozygous and 70 heterozygous samples previously typed at high resolution level by sequence-based typing. A further validation was then performed using a blind cohort of 93 previously HLA typed samples. This Luminex-ASPE tagSNP multiplex method correctly identified the three most common HLA haplotypes present in the Northern European population. In summary, this is the first time when a novel tagSNPs HLA genotyping assay has been developed and successfully applied to detect three common HLA haplotypes in a blinded retrospective study.

Our body is under constant attack by pathogens such as viruses or bacteria. The immune system has evolved to efficiently counteract these attacks using an array of cells and soluble proteins. In particular, B cells support the immune system by producing high-affinity antibodies, which target pathogens for destruction by recognition of antigenic structures. B cells furthermore contribute to immunological memory to quickly mount responses to secondary challenges by the same pathogen. In order to produce antibodies, B cells need to get activated in secondary lymphoid organs by a two-step process: First, B cells sense antigen molecules on the surface of antigen-presenting cells using their B cell receptor (BCR), which leads to intracellular signalling and antigen acquisition. B cells then present processed antigen molecules to specific helper T cells. Both BCR ligation and adequate T cell help lead to full B cell activation and the production of antigenspecific antibodies. This thesis investigates the early molecular events of B cell activation. Using a combination of genetics, lipid bilayers and high-resolution microscopy we identify a role for the adaptors Grb2 and Dok-3 alongside the ubiquitin ligase Cbl in antigen gathering by B cells. Moreover, we show recruitment of Grb2, Dok-3 and Cbl to the signalling BCR and establish Grb2 as a central downstream adaptor. We further assess the role of several motor proteins in antigen gathering and find that dynein is recruited to the signalling BCR driving antigen gathering by the movement of antigen-containing BCR microclusters on the underlying microtubule network. Interestingly, recruitment of dynein to BCR microclusters is impaired in B cells lacking Grb2, Dok-3 or Cbl. As the amount of antigen acquired by B cells directly correlates with the extent of T cell help, the data presented significantly contributes to our understanding of the early molecular events underlying B cell activation.

Dendritic cells (DC) respond to danger signals from tissue injury by amplifying their immune-inducing capacity. In the cancer context, this may lead to in vivo anti-tumour synergism between DC and DNA-damaging chemotherapeutic agents. Neither the interaction between DC and dying tumour cells, nor whether different ways of inducing cell injury can deliver danger signals of different strength to DC, has been studied rigorously. Furthermore, the physical interactions of DC with injured tumour cell, and whether physical contact is required for sensing danger signals, have not been clearly defined. Here we report that co-culture of immature DC with tumour cells treated with the alkylating agents Melphalan and Chlorambucil leads to enhanced autologous and allogeneic T-cell activation, up-regulation of surface expression of MHC and costimulatory molecules, and increased interleukin (IL)-12 secretion. However, exposure of DC to tumour cells killed by Cytarabine or by freeze-thaw (primary necrosis) resulted in significantly less T-cell proliferation and IL-12 production, indicating that DC are able to sense and respond differentially to the mode of cell death. Exposure of DC to DNA purified from tumour cells treated with alkylating agents also increased their T-cell-­stimulating capacity, expression of CD86, and IL-12 secretion, supporting the hypothesis that the activating effects of tumour cells are linked to the nature of the DNA damage. Furthermore, physical contact between DC and injured tumour cells is necessary for DC to sense and respond to danger signals. DC physically intereract with tumour cells via actin­and microtubule-dependent phagocytic mechanisms. This is the first study that shows that DC respond differentially to killed tumour cells, depending upon the mechanism of DNA damage and consequent T-cell death.

Immunity declines during ageing, however the mechanisms involved are not known. In this study we show that the cutaneous delayed type hypersensitivity response (DTH) to recall antigens is significantly decreased in old individuals and that this was unrelated to CCR4, CLA or CD11a expression or physical capacity for migration of CD4+ T cells. Instead, there was defective activation of dermal blood vessels of these subjects that resulted from decreased TNF-α secretion by macrophages after antigen-challenge in vivo. However, isolated skin macrophages from these subjects could be induced to secrete TNF-α after stimulation with TLR 1/2 or TLR 4 ligands in vitro, indicating that the defect is reversible. The decreased conditioning of tissue microenvironments by macrophage-derived cytokines may therefore lead to defective immunosurveillance by memory T cells. This may be a predisposing factor for the development of malignancy and infection in the skin during ageing.

Retroviruses can establish persistent infection despite induction of a multipartite antiviral immune response. Whether collective failure of all parts of the immune response or selective deficiency in one crucial part underlies the inability of the host to clear retroviral infections is currently uncertain. In this study, the contribution of virus-specific CD4+ T cells in resistance against Friend virus (FV) infection in the murine host is examined. The results show that the magnitude and duration of the FV-specific CD4+ T cell response is directly proportional to resistance against acute FV infection and subsequent disease. Notably, significant protection against FV-induced disease is afforded by FV-specific CD4+ T cells in the absence of a virus-specific CD8+ T cell or B cell response. Enhanced spread of FV infection in hosts with increased genetic susceptibility causes a proportional increase in the number of FV-specific CD4+ T cells required to control FV-induced disease. Thus, these results suggest that FV-specific CD4+ T cells provide significant direct protection against acute FV infection, the extent of which critically depends on the ratio of FV-infected cells to FV-specific CD4+ T cells. In addition to precursor frequency, the T cell receptor (TCR) affinity for antigen is also considered an important contributor to CD4+ T cell expansion and effector function. Study of polyclonal TCRβ-transgenic FV-specific CD4+ T cells has previously revealed a distinct pattern of clonal evolution during FV infection. In the current study, immunisation regimens, including adjuvanted peptide and replication-attenuated retrovirus vaccination, are assessed for the overall TCR affinity of the CD4+ T cell population they elicit. The implications for vaccine design and vaccine-induced CD4+ T cell-mediated protection are discussed.

Lentiviral vector delivery of anti-HIV elements could provide the basis of alternative therapies against HIV, potentially providing long term protection after a single intervention. Some primate species have evolved restriction factors formed by the fusion of TRIM5α and Cyclophilin A (TRIM5Cyp) following retrotransposition of CypA cDNA into the TRIM5 gene, which provide potent resistance against certain lentiviruses. We have designed humanised versions of these proteins combining both TRIM5 and TRIM21 with CypA, and investigated their potential for use in gene therapy against HIV-1. Both TRIM5- and TRIM21-Cyp fusion proteins provided strong restriction of HIV-1 in all of the systems tested, including primary human T cells. However, TRIM5Cyp was shown to disrupt the antiretroviral effect of endogenous TRIM5α and rescue murine retrovirus infection, whereas TRIM21Cyp caused no interference. In contrast, neither TRIM5CypA nor TRIM21CypA expression affected the antiviral activity of endogenous TRIM21. In addition to TRIMCyp restriction factors, a second anti-HIV strategy was investigated using zinc finger nucleases (ZFNs) to knockout the HIV-1 co-receptor, CCR5. ZFNs introduce a double stranded break into the CCR5 gene, which can be restored by homology directed repair. Provision of a green fluorescent protein (GFP) or TRIM21Cyp donor template exploits this repair mechanism to allow site specific integration at the CCR5 locus, although at low efficiency. Using integrating vectors, we have shown that TRIMCyp mediated restriction is so potent that no additional inhibition was conferred by CCR5 knockout. In conclusion, delivery of TRIMCyp genes using lentiviral vectors could form the basis of an intracellular vaccination strategy against HIV-1, with TRIM21Cyp having benefits by maintaining endogenous TRIM function. With further optimisation to improve efficiency, this could be combined with ZFNs for site specific integration of the transgene and knockout of CCR5 to provide a dual method of HIV-1 inhibition.

Infections are one of the most common reasons for patients to consult their doctor. Growing concern about antibiotic resistance has led to pressure on General Practitioners to reduce prescribing. There is a clear need to understand the epidemiology and management of infections in primary care. Routine datasets contain valuable information on infections, their management, hospitalisations and antimicrobial resistance but have been underutilised . In chapter one I give an overview of the history of antibiotics, the development of antimicrobial resistance and measures used to reduce prescribing in primary care. In chapter two I describe a systematic literature review showing how community antibiotic prescribing leads to antibiotic resistance. In chapter three I describe a data-linkage study showing strong relationships between recent GP antibiotic prescribing and the risk of antibiotic resistance in UTIs. In chapter four I describe analyses of the GPRD showing the burden of infection in primary care, frequent over-prescribing for minor respiratory tract infections, and high levels of consultation and prescribing in children, women and those living in socially deprived areas. In chapter five I describe an analysis of the GPRD showing that over 4000 patients with URTI (URTI), sore throat or otitis media need to be treated with antibiotics to prevent one serious complication. Only around 40 elderly people with chest infections, however, need treating to prevent one case of pneumonia. In chapter six I describe concerns about the emergence or community acquired MRSA and analyses of national HES data showing marked increases in hospitalisations for community onset staphylococcal disease. In chapter seven I summarise the conclusions from the research, reflect on the advantages and disadvantages of using large routinely available datasets for infectious disease research, describe the clinical and public health implications of the research and outline further research needs.