Synthesis of polysaccharide intercellular adhesin (PIA), accumulation associated protein (Aap) and extracellular matrix binding protein (Embp) are major mechanisms used by Staphylococcus epidermidisto evade immunity through biofilm formation. These evasion strategies are particularly suited for colonisation of medical devices such as heart valves, joint prostheses and central venous catheters resulting in significant patient morbidity. Two biological activities of PIA, Aap and Embp contribute to their role as an evasion molecules. Firstly their ‘barrier’ function limiting penetration of immune cells and antibiotics. Secondly, their ‘immunomodulatory’ properties which influence cytokine responses. At present little is known about these functional activities in physiological media and biological fluids. This thesis uses a cell biology approach to study the environment necessary for PLA production. Specifically in vitromodelling of biofilm formation, PIA production and S. epidermidisleukocyte co-culture experiments have been used to assess conditions that are conducive for PIA production. This thesis has identified that:• Specific cell culture media cause unique profiles of biofilm accumulation with differential production of PIA, Aap and Embp. • Fetal bovine serum and pooled human serum support S. epidermidisgrowth but differentially affect biofilm formation by PIA, Aap and Embp. • Large scale production of PIA (~20mg) can be achieved by culturing in Iscove's Modified Dulbecco's Media which has allowed streamlining of current isolation procedures. • PIA induction of cytokines, including IL-8 and TNF is dependent on being tethered to the bacterial membrane. • Macrophages can penetrate into a S. epidermidisproduced PIA ‘barrier’. • Immunosuppression of whole blood with dexamethasone unmasks the pathogenic advantage of PIA in S. epidermidis expressing PIA compared to negative controls. • Whole blood killing of S. epidermidisis dependent on CD1 lb/CD 18. • PIA induces whole blood killing dysfunction which is likely related to C5a production. • PIA can be produced in a whole blood environment.• Inocula of -1 0 colony forming unit of S. epidermidisare required to form biofilms in whole blood. This study suggests the importance of studying clinically important biofilm production mechanisms under conditions that closely resemble those in human disease.

Due to the lack of an effective cure for human immunodeficiency virus (HIV-1), with approximately twenty million new infections expected by 2031, research has moved towards optimised prevention strategies. Drug transporters, characterised extensively in the human liver, kidney and gastrointestinal tract, can be manipulated to improve the pharmacokinetic properties of orally delivered anti-retrovirals (ARV's). There is paucity in knowledge of drug transporter expression in the human cervicovaginal (CV) tract and representative pre-clinical models, where characterisation and manipulation may enable optimisation of topically applied ARVs for prevention of sexual transmission of HIV-1. In this thesis, RT-qPCR was used to characterise drug transporter profiles of CV pre-clinical models (in vitro, ex vivo and in vivo) for comparison with human CV tissue profiles, where distinguishable differences were observed, most notably in the efflux transporters P-gp and MRP2-4. This may explain the contrasting efficacy observed between pre-clinical and clinical trials of ARV-microbicides. Additionally, CV physiological factors (hormones, immunoregulatory proteins, microbes, pH) and microbicide candidate ARV's (dapivirine and darunavir) were shown to modulate expression of ARV-relevant drug transporters in CV cell lines. This highlights the potential intra-variability and inter-variability challenges associated with microbicide development and optimisation, in addition to potential drug-drug interactions in combination ARV-microbicides. Modulation of cellular pharmacokinetic properties in response to physiological pH levels and pro-inflammatory cytokines, observed within drug transport experiments, further emphasises these challenges. Molecular cloning experiments demonstrated the potential to develop a robust high throughout in vitro screening tool for the development of optimised microbicides. In conclusion, this thesis provides evidence exposing the limitations of pre-clinical CV models commonly used during ARV-microbicide screening, development and optimisation. The potential clinical implications of physiologically-induced and ARV-induced modulation of ARV accumulation in the CV tract when topically applied is highlighted within this thesis. It is imperative these key factors be incorporated into the pre-clinical screening and development of optimised microbicides.

Food allergies in children, teenagers and adults are managed by eliminating the allergenic food from the diet. Healthcare professionals and policy makers have developed guidelines for the dietary management of food allergies, but as yet there has been no assessment of how individuals with food allergies are able to adapt their behaviour to them. In order to be able to improve the diet and nutrition of children, teenagers and adults with food allergies, and thereby to increase their quality of life, it needs to be understood which processes influence food choices and management of food and eating in this population, and how their actual diet is affected by the chronic condition. This research consisted of four stages, the first three addressing food choice behaviour among age groups of children, teenagers and adults; the fourth stage evaluating the impact of food allergies on nutrient intakes of this population. A mixed-method approach has guided this research. The findings indicate that food choice behaviour is mostly affected by food allergies in adults. This is probably because personal cognitive factors play a more dominant role during food choice decisions than during childhood and adolescence, where social influences are more prevalent. Adults reported a lack of satisfaction and joy from food, had difficulties sharing meals, and felt the need to organise their eating. Teenagers struggled to widen their palate, felt secure under parental protection, and expressed the wish to eat similar foods to their friends. Children showed highest engagement with foods if the mother displayed an authoritative parenting style. Although they appeared least affected by the allergic condition in the way they were choosing food, children have been shown to be the age group making most nutritional compromises. Protein, vitamin B12, potassium, calcium, phosphorus, and iodine intakes were lower than among healthy age-matched children. This research has provided a cross-sectional survey of food choice behaviour and dietary intake among food-allergic children, teenagers and adults with many implications for practice and future research. It is recommended that dietary management of food allergies should place emphasis on dietary variety and enjoyment aspects of eating as well as the importance of social relationships that are built around food. Additionally, regular evaluations of dietary intake should be conducted, in particular for children with a cow’s milk allergy or individuals with multiple food allergies.

Peptide immunotherapy for Type 1 diabetes aims to restore tolerance to self, whilst leaving the rest of the immune system intact. Once the right peptide isdelivered to the right cell, it is important to closely monitor the effect of such atherapy, both in the regards to the immune and metabolic response. Clinical trials are designed to test the effect of a drug at the end of the trial period, which can be years later. Ex-vivo human models are not subject to extensive regulatory requirements, and can rapidly provide proof of principle on the efficacy of a treatment, which can be then translated to the clinic. I have shown that the skin organ bath culture is a useful system for studying treatment effects of variety of ex-vivo delivered agents. When used to optimise peptides delivery, it indicated a potential role of dry coated microneedles in targeting epidermal DCs, important because of their endogenous tolerogenic potential, which can be further modified by topical treatments and locally injected agents. Whether true tolerogenic potential can be achieved in such a way, is subject to further studies designed to optimise the type, dose and the duration of the treatment by the conditioning agent. My data also suggested that lymph node fine needle aspiration biopsy is a feasible non-invasive method suitable for monitoring the cellular immune responses after antigen skin delivery. Subject to confirmatory study, it has a potential to find immediate application as an efficient and reliable tool for monitoring immune response after antigen-specific immunotherapy in clinical trials. Once recognised as ‘immune responders’ in such a way, participants in clinical trials can be subjected to the monitoring of the metabolic response to the immune intervention, by measuring !-cell function via stimulated UCPCR as a non-invasive and more compliant-prone alternative to the standard MMTT.

For more than thirty years, the HIV pandemic has caused immense harm across sub-Saharan Africa. From the middle of the last decade, however, a treatment revolution has been underway, as effective antiretroviral drugs (ARVs) have become available to millions of ordinary people. This thesis examines the far-reaching consequences of this new reality in Northern Nigeria. It argues that the significance of the ARV era cannot be fully understood simply by monitoring how many patients are receiving treatment, but instead must be explained in terms of the multifaceted changes it has driven in institutions and the lives of HIV positive people. This study uses ethnographic case studies and participatory methods to understand this new historical moment from ‘below’. It provides new empirical perspectives on how the ARV era has profoundly altered the ways in which HIV positive people suffer. The difficulties of daily life when subjected to opportunistic infections, side effects from drugs, and social stigma are compounded by memories of past trauma and fears for an uncertain future. Previous studies have indicated HIV positive people often form new relationships (e.g. Rhine, 2009), but rarely have these post-HIV relationships been described. This study argues that these new relationships, often distant from conventional family supervision, have a unique character, blending traditional forms with ‘modern’ ideas about romance. After a HIV disclosure, incomes and assets (particularly those reliant on family relationships) are often reduced. Along with the cost of treatment (broadly defined to include a range of curative practices), this forces those living with HIV to adapt their livelihood strategies, often using networks of solidarity between positive people. The process of lobbying for improvements in medical care is also explored. Both doctors and NGOs advocate on behalf of HIV positive people, but do so with strikingly different tactics and results. This has important implications for continuing debates about working ‘with the grain’ (Crook and Booth, 2011) for development in patrimonial states. In summary, whilst HIV treatment has saved the lives of millions, inventing drugs and getting them to the people who need them are merely the first steps in alleviating suffering. The thesis traces the most important tasks in securing wellbeing in the ARV era – those pursued by HIV positive people themselves.

The bacteria Ureaplasma has long been associated with a wide range of adverse health implications, including preterm birth, preterm premature rupture of the membrane and lung disorders, such as bronchopulmonary dysplasia in neonatal infants, but still, little is known about the pathogenic properties of Ureaplasma and possible direct association with adverse health complications. Estimated prevalence of Ureaplasma colonisation in sexually active adults is between 40 – 80%, therefore further understanding of its pathogenic properties and its ability to initiate an immune response is crucial. Specifically selected human cell-lines were examined in vitro to determine whether an innate immune response could be activated by Ureaplasma infection. If inflammatory immune responses were detected in human cell-lines, pathogenic properties of Ureaplasma would be confirmed, and its role in pregnancy and neonatal complications could be supported. Using a range of techniques, activation of immune response pathways were examined, as too were the production of detrimental pro-inflammatory cytokines that would strengthen the suggested associations of Ureaplasma infection with the above-mentioned complications. Myeloid-derived leukocytic monocytes, human bronchial epithelial cells and human amniotic epithelial cells were examined, as these would be the most relevant cell lines to determine if Ureaplasma could induce preterm birth, preterm premature rupture of the membrane and bronchopulmonary dysplasia. All cell lines studied showed immune response and inflammatory cytokine production after stimulation with Ureaplasma. This supports that Ureaplasma is capable of causing tissue damage in neonatal respiratory tracts that may lead to bronchopulmonary dysplasia and damage to the amniotic and chorion membranes that may lead to preterm premature rupture of the membrane. Ureaplasma was detected at the cell surface of human amniotic epithelial cells (HAECs) by TLR2 and TLR2/6 heterodimers. Results suggest that Ureaplamsma multiple banded antigen (MBA) is the strong ligand for TLR2 and TLR6 and stimulation of HAECs with MBA alone caused an immune response. TLR9 was responsible for the detection of internalised Ureaplasma, which is also able to initiate an immune response and inflammatory cytokine production. v Ureaplasma stimulation results in the production of the inflammatory cytokines TNF-α, IL-8 IL-6 via the NF-κB signaling pathway. Production of the potent inflammatory cytokine IL-1β was also observed, which would suggest the formation of inflammasome complexes. NLRs were investigated to find which NLR inflammasome were activated. It was shown that genetically knocking down NLRP7 significantly reduced the amount of IL-1β that was produced after Ureaplasma stimulation, suggesting that NLRP7 inflammsones are activated by Ureaplasma. Reduction in IL-1β was also observed, but to a lesser extent, when NLRP3 was knocked down. We decided to investigate the role of NLRP7 further and found a novel immune pathway, where NH3 causes activation and formation of the NRLP7 inflammasone. NH3 is produced as a bi-product of urease activity, which an essential process for Ureaplasma. The addition of a potent urease inhibitor to HAECs being stimulated with Ureaplasma significantly reduced the production of IL-1β, strongly supporting that NH3 plays a significant role in the detection of Ureaplasma infection and is responsible for causing the tissue damage that contributes to preterm premature rupture of the membrane leading to preterm birth. This investigation strongly supports that Ureaplasma is responsible for causing preterm birth and health complications in neonates, and that more robust treatment and monitoring of Ureaplasma is required, especially in pregnant women. These undertakings will hopefully reduce the rates of preterm birth and the associated health implications, in addition to reducing rates of bronchopulmonary dysplasia in neonates.

Transforming growth factor-beta (TGF-β) drives the development of immature LC from hematopoietic progenitor cells and shapes the cells functions. Here I showed that two LC model cells, MuLC and MDLC, used exchangeably in the research, differ significantly in their phenotype and immune responses. Discrepancies between these models were specifically visible during stimulation with type-I IFN, where MuLC failed to up-regulate ISG levels. Yet both MuLC and MDLC demonstrated low susceptibility to HIV-1 infection, even in the absence of SAMHD-1. This post-entry restriction was conferred by the action of TGF-β on differentiation cells as indicated by our study. Indeed, in the absence of TGF-β supplementation, derived cells showed MDDC phenotype related to high susceptibility of the cells to HIV-1 infection during co-infection with SIV-Vpx. Additionally blocking of the TGF-β signalling, reversed the restrictive phenotype of LC. Importantly this pattern was also confirmed in skin extracted real epidermal LC versus dermal DC, suggesting that SAMHD-1-independent restriction activity operates in TGF-β derived cells. Accordingly to PCR analysis virus replication in LC is interrupted prior to integration, suggesting the role of additional restriction factors at early stages of virus infection or lack of essential viral dependency factors such as dNTPs. Interestingly maturation of MDLC with a synthetic bacterial triacylated lipopeptide or TNF-alpha significantly increased their susceptibility to HIV-1 infection, which may explain why HIV-1 acquisition is increased during co-infection with other STIs. In summary, our study strongly supports the action of SAMHD-1-independent HIV-1 restriction mechanisms in LC. A better understanding of the balance between HIV-1 restriction and propagation from LC to CD4+ T cells may help in the development of new microbicides or vaccines to curb HIV-1 infection at its earliest stages.

Multiple sclerosis (MS) is an inflammatory disease that affects the central nervous system and is considered to be a T-cell mediated autoimmune disease. The “ideal” method in treating MS would be an antigen-specific therapy that does not require generalized immunosuppression. To date there are no definitive treatments for MS but there are several licensed therapies such as -interferon. Unfortunately the effect of interferon (IFN) is reduced by the development of neutralizing antibodies (NAbs) in up to 35% of MS patients within two years of starting treatment. An immunization schedule was developed in the BALB/c mice by subcutaneous administration of recombinant human IFN, and this resulted in development of high incidence of NAbs to the protein in the BALB/c model termed “NAbs model”. The mechanism of NAbs formation in this model is believed to be similar to that observed in IFN-treated MS patients with NAbs, which is as a result of an immune response to the protein. We elected to study NAbs in the context of IFN rather than MS directly to investigate the effects of antigen-specific tolerization strategies on the outcome of NAbs and indirectly on the outcome of IFN treatment in MS disease. The depletion of the immune cells triggers a reconstitution program that leads to renewal of the immune cell repertoire. Tolerance can be induced by intravenous administration of a protein. Within this window of reconstitution following depletion, it is hoped that the immune system can be manipulated to tolerate an otherwise foreign protein (human recombinant IFN). The tolerance strategy employed in this project was immune cell depletion using antibodies and mitoxantrone, followed by intravenous re-introduction of rhIFN. Tolerance was successfully induced in the NAbs model by intravenous administration of rhIFN, and further enhanced by immune cell depletion prior to intravenous administration of rhIFN. The BALB/c “NAbs model” offers a suitable model for use in investigating induction of tolerance to rhIFN following the formation of NAbs to the protein. The antigen of interest is known and the time to NAbs formation is also known. Tolerance induction can be monitored and investigated in this model.

The aetiology of Common Variable Immunodeficiency (CVID) has fascinated immunologists since Dr. Janeway reported the first case in 1953. While the advances in molecular biology have enlightened us on the aetiology in some patients, the majority is not caused by inherited genetic disorders. A convincing mechanism accounting for the intrinsic variable and partial nature of the condition has yet been proposed. CVID separates itself from other primary antibody deficiencies by the procurement of an abnormal T-cell compartment. Data from this study support that both T-cells and B-cells are subjected to similar deficiency. Investigation of the T-cell receptor repertoire by next-generation sequencing and multi-parametric flow cytometry suggests a severe reduction in naïve T-cell output from the thymus. Similarly, the study of long-lived plasma cell generation and survival highlighted the greatest functional deficits in the naïve B-cell pool, altogether supporting an acquired arrest in lymphogenesis. Using DNA methylation as a surrogate marker for pre-VDJ clonality, this study further shows that some CVID patients exhibited clonal haematopoiesis, adjoining CVID to other clonal haematopoiesis related acquired haematological disorders. Further work is being focused on using high resolution techniques to confirm this association and mechanistically define the development of antibody deficiency in adulthood.

Filarial nematode infections remain a significant threat of morbidity in the Tropics. Collectively known as filariasis, these helminth infections can result in gross inflammatory pathology of the infected host. However, more commonly, the host remains relatively asymptomatic, exhibiting a somewhat suppressed, non-inflammatory immune response to the parasite, permitting the longevity of infection commonly seen with many helminth infections. In addition to promoting parasite survival, such immunomodulatory actions appear conducive to host health by limiting development of pathological lesions resulting from aggressive, pro-inflammatory responses. This can prove to be advantageous for humans, as although nematode infection can result in severe pathology, the majority of infected people exhibit little evidence of an inflammatory response or overt tissue destruction/disruption commonly associated with autoimmune disorders. Consistent with this, there is increasing evidence supporting an inverse relationship between worm infection and Th1/17-based inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease, type-1 diabetes and multiple sclerosis. Moreover, in the developed world, there has been an alarming increase in these inflammatory diseases, coincident with recent improvements in hygiene; a trend not observed in parasite-endemic countries. Therefore, the ”hygiene hypothesis” proposes that these trends are directly associated, by predicting that in the absence of helminth infection, there is a lack of parasite-induced immunoregulation that can result in an over-active immune response in susceptible individuals, resulting in the development of autoimmunity. Filarial nematodes are known to secrete immunomodulatory excretory-secretory products during infection, which act to modulate inflammatory host immune responses and thus, protect the parasite from elimination. ES-62, a phosphorylcholine-containing glycoprotein secreted by the rodent filarial nematode, Acanthocheilonema viteae, has previously been shown to modulate the responses of several innate cells to promote anti-inflammatory immune responses in vitro and in vivo. Indeed, this laboratory demonstrated that ES-62 exhibited anti-inflammatory properties that extended to potential therapeutic action in autoimmune inflammatory diseases such as Rheumatoid Arthritis (RA). This was evidenced by studies demonstrating that ES-62 not only reduced severity of disease in the murine model of collagen induced arthritis (CIA), but also acted to reduce pro-inflammatory cytokine and auto-antibody production from blood and synovial cultures derived from human RA patients. Perhaps rather surprisingly therefore, ES-62 was subsequently found not to offer any protection in some other models of inflammation, including the NOD mouse model of Type 1 Diabetes, the Plasmodium chabaudi model of malaria and the Toxoplasma gondii model of toxoplasma, all of which have been linked with Th1-like pathology. However, given the recent reassessment of CIA as a Th17-, rather than Th1-, mediated disease, it has been hypothesised that ES-62 exhibits therapeutic potential in models, such as CIA, which reflect Th17- rather than Th1 mediated pathology. As the previous CIA/RA studies did not address the role of the Th17 family of pro-inflammatory cytokine mediators, which have been implicated as orchestrating much of the pathology seen in CIA, the core aim of this thesis was to define whether ES-62 was mediating its therapeutic action in autoimmune inflammatory disorders by targeting this Th17 population. Indeed, in chapter 3 of this thesis, it was found that the significant inhibition of disease scores in ES-62-treated CIA mice was accompanied by a significant reduction in levels of IL-17-producing cells, as detected by flow cytometry. Moreover, such analysis revealed that ES-62 reduced IL-17 production from both CD4+ (Th17) cells and γδ cells, which were the two major IL-17-producing populations in the LN of CIA mice. By contrast, ES-62 was not found to modulate the levels of IFNγ-producing cells, either in terms of CD4 or CD8 T cells, which constitute the major cellular compartments generating this cytokine in CIA, supporting the hypothesis that ES-62 targets Th17/IL-17-, rather than Th1-associated inflammation. In addition to suppressing CIA, pilot studies had shown that prophylactic treatment with ES-62 significantly inhibited the development of glomerulonephritis and articular inflammation in MRL/lpr mice, two pathologies commonly associated with SLE in humans. The anti-inflammatory actions of ES-62 were evidenced by a reduction of proteinuria levels and footpad swelling, respectively, but were not associated with modulation of lymphadenopathy, splenomegaly or hypergammaglobulinemia occurring in tandem with autoimmune pathology in such MRL/lpr mice. Studies in chapter 4 of this thesis characterised the cytokine profile of MRL/lpr lupus prone mice, in comparison to congenic MRL/MP control mice, throughout the course of disease induction and progression. These studies showed that IL-17 production was detected in the lymph nodes and serum of MRL/lpr mice, before the onset of lupus pathology. Furthermore, γδ T cells were the major IL-17 producing cell type examined in these mice. Prophylactic treatment with ES-62 resulted in reduced cytokine (IL-17 and IFNγ) release by lymph node cells and splenocytes in response to ex vivo stimulation with the mitogen, ConA, although this did not prove to be significant. As IL-17 was one of the cytokines targeted, this suggested that, as with the CIA model, ES-62 mediated its anti-inflammatory effects through modulation of this cytokine. Consistent with this, it was also found that exposure to ES-62 in vivo reduced the levels of IL-17-producing cells, particularly the γδ T cell population at the earliest time-point examined. By contrast, again as with the CIA model, there was no reduction in the levels of IFNγ-producing cells in ES-62 treated mice, confirming that ES-62 was not acting directly on these Th1 cells. One of the hallmarks of the MRL/lpr model of lupus is the accumulation of B220+CD3+ Double Negative (DN) T cells, which account for much of the lymphadenopathy observed. These cells, which would otherwise have been deleted during development, are known to survive and accumulate due to the lpr (lymphoproliferative) mutation of the Fas gene in these mice. Despite being the predominant LN cell population following onset of disease in MRL/lpr mice, DN T cells have not generally been considered to be pathogenic in this model, but rather proposed to exhibit an anergic-like phenotype. However, some studies have suggested that they can produce cytokines, such as IFNγ and more recently it has been shown that they are a source of IL-17 [1, 2]. Moreover, DN T cells producing IL-17 have been found in the kidneys of SLE patients, suggesting they might indeed play a pathogenic role in lupus-like inflammation [3]. The data presented here established that such DN T cells are capable of producing a range of cytokines, including IL-10, IL-17 and IFNγ and indeed, the most novel finding of these studies was that DN T cells are capable of producing IL-22, a cytokine generally associated with Th17 cells. While the function of this IL-22 production is unclear, it was seen that the percentage of DN T cells producing IL-22 were significantly inhibited by exposure to ES-62 in vivo and hence, whilst ES-62 did not appear to influence the expansion of these cells, it did modulate their effector function by suppressing their capacity to produce this cytokine, although the absolute numbers of cells was not significant reduced. In summary, the novel findings presented in this thesis support the theory that parasite-derived products such as ES-62 may protect against development of the autoimmune inflammatory diseases prevalent in developed society.