Rheumatoid arthritis (RA) is a chronic disease characterised by inflammatory infiltration of the synovial membrane, with concomitant destruction of adjacent cartilage and bone. Elucidation of immunoregulatory networks within the synovium offers the potential for therapeutic intervention. Two such pathways were investigated in the present study. Interleukin-15 (IL-15) is a novel pleiotropic cytokine produced by macrophages and fibroblasts, which induces T cell migration and activation and B cell maturation and immunoglobulin production. IL-15 was identified in RA synovial fluids and synovial membrane cultures and, using immunohistochemistry, its expression was localised in the RA synovial membrane to the lining layer and T lymphocyte aggregates. Enhanced proliferation and cytokine production to IL-15 was observed in RA synovial fluid (SF) T cells in comparison to matched peripheral blood (PB) T lymphocytes, which in turn, were more sensitive to IL-15 induced proliferation than PBT cells from normal controls. Following IL-15 mediated activation, PBT cells were capable of inducing TNF production from a macrophage cell line, from syngeneic PB monocytes, and from synovial macrophage/synoviocyte co-cultures, through a cell-contact dependent mechanism, which required no T cell cytokine synthesis. RASFT cells exhibited similar properties, which were IL-15 dependent . IL-15 upregulated CD69 expression on CD45ROT cells and neutralisation studies determined that such CD69 expression, in combination with LFA-1 and ICAM-1, was partly responsible for cell-contact mediated macrophage activation by T cells. Finally, in a murine model, IL-15 injection induced significant local tissue T cell invasion, confirming previous observations of its chemotactic properties. IL-15 expression in RA synovial membrane therefore provides a mechanism whereby polyclonal T cell recruitment and activation can lead to macrophage activation and TNF production, without T cell cytokine synthesis.

D6 is a novel chemokine receptor, homologous to other members of the CC-chemokine receptor family, which recognises a number of inflammatory CC-chemokines with high affinity. The aims of this thesis were to further our understanding of the biology of D6, chiefly through characterisation of immune responses in D6-deficient animals. Firstly, as described in Chapter 3, I analysed the cellular composition of lymphoid tissues of D6 KO mice. These studies revealed higher proportions of CD11c+ and F4/80+ cells in the D6 KO spleen compared with WT controls, suggesting that increased accumulation of myeloid lineage cells was occurring at this site. In Chapter 4, I examined the role of D6 in myeloid cell responses, by comparing monocyte recruitment to the inflamed peritoneum and dendritic cell development from bone-marrow (BM) cultures. I found that while the accumulation of inflammatory monocytes/macrophages appeared quantitatively similar in WT and D6 KO animals, D6 KO cells expressed greater levels of CD11c, suggesting preferential accumulation of DC-like cells in the inflamed peritoneum. D6 may influence the development and function of myeloid lineage cells. As D6 is expressed at high levels in the small and large intestine, I next investigated both tolerogenic and inflammatory intestinal responses in D6 KO animals. As detailed in Chapter 5 of this thesis, the induction of oral tolerance in response to a high dose feeding protocol was normal in D6 KO mice. However, D6 KO mice showed increased resistance to experimental colitis. As described in Chapter 6, various D6 KO populations displayed differential chemokine receptor profiles compared with their WT counterparts. The results suggest a role for D6 in the normal development of leukocytes populations, with absence of this atypical receptor leading to the dysregulated expression of other chemokine receptors. Taken together, my data suggest that the biological functions of D6 may be more complicated than previously appreciated. Indeed, I found no evidence for a decoy role of D6 in vivo, but D6-deficient animals were characterised by altered leukocyte development, aberrant chemokine receptor expression and increased resistance to experimental colitis induction.

Dental caries remains one of the most common chronic infectious childhood diseases and individuals remain susceptible to the disease throughout their lifetime. The disease continues to inflict a substantial economic burden. Moreover, dental caries demonstrates considerable socioeconomic disparities with the lowest socioeconomic groups suffering the greatest burden of disease. There is an unmet need to improve prevention and therapeutics and yet there remain fundamental gaps in the knowledge of the interrelationships between caries-associated risk factors, in particular how the immune system interacts with the evolving cariogenic biofilm in young children. This thesis sought to investigate the immune response to cariogenic biofilms. Three different approaches were used to achieve this. Firstly, the salivary immune response and development of the oral biofilm in very young children were investigated prior to the onset of caries, as part of a pilot longitudinal clinical study, using a dental public health program as a platform. Secondly, the initiation of adaptive immune responses to S. mutans exposure were investigated using a series of In vitro and In vivo studies. Thirdly, a novel S. mutans In vitro biofilm model was developed and optimised. Childsmile is a dental health improvement programme for children in Scotland and provides children with specific dental health interventions depending on need, from birth and up to 16-years of age. To achieve the first and primary aim of this thesis, plaque and saliva samples were collected from children aged one-year and again at age three-years. At follow-up, dental disease scores were also measured. Additionally, the biological mechanisms underlying the socioeconomic disparities in the dental health of young children were investigated, including the measurement of salivary cortisol as a surrogate measure of stress. Sixty-three Childsmile participants aged one-year were recruited to the study at baseline. Twenty-three children aged three-years were successfully recalled at follow-up. This work demonstrated that variables hypothesised to influence the development of carious disease can be collected and successfully quantified in children aged one- to three-years. Nonetheless, it was extremely challenging to recruit children of this age and the data were compromised by the small sample sizes. During the study period both the intensity and incidence of S. mutans colonisation increased in the dental plaque of children aged one- to three-years. Coincidentally, concentrations of salivary antimicrobial proteins increased, including lactoferrin, LL37, calprotectin, the HNPs 1-3 and sIgA antibody titres specific for oral streptococci. It could not be determined from these studies whether the increased colonisation with S. mutans or the concentrations of salivary antimicrobial proteins influenced the prevalence of dental caries. The major limitation of this study was the low recruitment rates which resulted in low power to detect statistically significant differences. As a consequence there was insufficient evidence to identify the potential biological pathways that may underlie the socioeconomic disparities of dental caries. From this pilot study a number of valuable lessons were learned regarding the recruitment of children of this age and recommendations for future clinical studies conducted within Childsmile are made. In children with high risk of developing dental caries effective salivary antibody responses are required to provide protection. The mechanisms leading to effective antibody responses remain unclear. Thus, the second aim of this thesis was to investigate the initiation of an adaptive immune response to S. mutans, in an attempt to elucidate the mechanisms that lead to effective antibody production. Using a novel system, In vitro evidence indicated that S. mutans does not elicit a robust inflammatory immune response upon colonisation of the host. Dendritic cells exposed to S. mutans were not functionally mature and failed to induce antigen-specific T cell proliferation. Furthermore, In vivo, dendritic cells failed to become activated in response to oral exposure to S. mutans. An In vitro S. mutans sucrose-dependent biofilm model was developed and optimised. Using this model an antibody fragment known as a minibody, denoted ‘SS2’ was demonstrated to inhibit S. mutans biofilm formation. This biofilm model represents an important first step for examining the potential of therapeutic molecules to inhibit S. mutans biofilm formation, prior to their application in In vivo models of dental caries and possible subsequent use in human clinical trials. Data described here indicate that S. mutans colonises the oral cavity at a time when children are immunologically immature. Increased colonisation by S. mutans coincides with the maturation of salivary immune responses. Moreover, In vitro and In vivo evidence suggest that S. mutans does not elicit a robust immune response upon colonisation of the host. Thus, early acquisition of S. mutans in a relatively immunologically immature host together with the absence of an inflammatory immune response likely aids the colonisation of S. mutans and its persistence within the oral biofilm and subsequent contribution to dental caries.

Periodontal disease is associated with an inflammatory response to a pathogenic biofilm. The host response may cause gingival inflammation, which can progress to irreversible gingival recession, alveolar bone destruction and tooth loss. Enhanced understanding of the host-biofilm relationship may inform novel therapeutic approaches. A key molecule involved in inducing and mediating pro-inflammatory responses are the IL-17 cytokine family. An in vitro model system potentially provides a platform to investigate biofilm interaction with epithelial cells. The aim of this study was to develop in vitro mono-species and multi-species biofilms and investigate the survival of biofilms in cell culture conditions, and simultaneously assess the epithelial response to the bacterial biofilms and planktonic cells with respect to viability, apoptosis and inflammatory mediators. This study also looked to determine whether IL-17A is expressed within and released from periodontal tissues and to investigate its role in the regulation of epithelial cell cytokine and chemokine production. Mono- and multi-species biofilms of P. gingivalis, F. nucleatum, A. actinomycetemcomitans and S. mitis were developed, which were assessed for survival in cell culture conditions, recovery from biofilms and morphology. Gingival tissue from patients with chronic periodontitis or healthy controls were analysed for IL-17A gene expression by qPCR. Protein expression and cellular localization was determined by immunofluorescence. Single cell suspensions of gingival tissue were stimulated in vitro and IL-17A release assessed. Epithelial response after bacterial and IL-17A co-culture was assessed. The individual bacteria survived preferentially in multi-species biofilm compared with mono-species biofilm in cell culture conditions. The viability, apoptosis and inflammatory mediator response depended on the type (pathogen or commensal) and form (planktonic or biofilm) of bacteria. Diseased gingival tissues expressed significantly higher levels of IL-17A mRNA than healthy samples. IL-17A localised to mast cells in the inflamed gingival tissue, and was released in cell culture supernatants following stimulation. Stimulation of epithelial cells with IL-17A resulted in the transcriptional regulation and release of numerous cytokines and chemokines. The initial component of the entire investigation has provided a quantitative and qualitative assessment of both mono- and multi-species biofilms that can be used to investigate how oral biofilms interact with the host epithelium. The epithelial-biofilm co-culture model has demonstrated clear differences between (i) planktonic and biofilms, (ii) pathogens and commensals, and (iii) live and dead bacterial challenge. These observations and the utility of the model will provide a platform to investigate key questions relating to pathogen and host within the oral cavity and beyond. From this study, it appears that IL-17A plays an important role in the protective periodontal immune response to bacterial pathogens. The upregulation of acute inflammatory mediators (such as IL-8) will promote neutrophil recruitment and potentiate the removal of any invading microbial threat. Therefore it is important to understand the benefits of this cytokine, before systemic therapeutic agents are used to antagonise its actions. The hope for the future is to unravel the details of the mechanisms involved and thereby identify novel therapeutic targets for inflammatory and infectious disease.

Staphylococcus aureus is the most common cause of hospital-acquired infection and contributes significantly to patient morbidity and mortality. The ability of S. aureus to switch to an alternative phenotype in the presence of antimicrobial agents is clearly favourable. One of these alternatives are small colony variants (SCVs). The novel phenotypes include changes to colony morphology, antibiotic susceptibility, haemolytic activity and many other physiological activities. It is now recognised that SCVs have a deficiency in electron transport, owing to mutations affecting its efficacy. This study investigated SCVs in various ways. In the evolution component changes (mutations) occurring sequentially in successive cycling (15 cycles), were identified. In this experiment selection was made for sequentially SCV mutants and wild type revertants. Two sequenced clinical MRSA strains COL and N315 were chosen so changes in sequence in SCVs and wild type revertants could be compared. Selection for SCVs was made independently for triclosan and gentamicin for both strains. The final SCV and WT strains isolated were compared physiologically and genetically and showed differences in frequency, biochemical profiles, pigment production, haemolysis, catalase, coagulase, levels of intracellular ATP and phage yield. The genomic sequence of the final 4 cycle isolates (SCV15) showed numerous and diverse mutations occurred COL and N315 SCVs. Over 70 mutations were found and 33 were determined as historic mutations and the rest were termed novel mutations. The novel mutations occurred during the cycling process. The historic mutations occurred prior to the experiment and these mutations were acquired during growth in laboratory culture. Only one mutation was found to be common between COL and N315 and this was in the fabI gene. These data indicate mutations occurring in ~1.3% of the genome (~ 40 Kb) can generate mutants with the SCV phenotype. Susceptibility to phage 80α and transduction of S. aureus wild type and their SCVs 1-3 was studied. Wild type strain of S. aureus and SCV3 both yielded a high number of lysogens (~68%) the remaining being resistant mutants. SCV 1 and SCV 2 provided a much lower proportion of lysogens (4-10%). There was no obvious relationship between cellular ATP levels and lysogen formation. Consequently the frequency of lysogen formation (or that of resistance mutants) cannot be related to energy status. Transduction of ciprofloxacin resistance (grlA) was observed into COL wild type at a 5-10-fold higher frequency than into SCV1. Transduction of rifampicin resistance (rpoB) into SCVs was reduced almost 10-fold. As transduction was significantly decreased into SCVs it is hypothesised this process was influenced by ATP levels. The data thus suggests that SCV strains will be less efficient in gene exchange by transduction in vivo. Three SCVs previously isolated from S. aureus COL on the basis of different growth rate were further studied. Results clearly support the hypothesis that there is a physiological diversity in SCV populations. Sensitivity of S. aureus wild type and SCVs strains to various antimicrobial was determined. The SCV strains were more sensitive to some antibiotics and heavy metals than the wild type strain.

Campylobacter is a major cause of acute bacterial gastroenteritis worldwide, with the highest number of infections being attributed to Campylobacter jejuni. C. jejuni is a Gram negative, spiral, motile bacterium that belongs to the campylobacterales order and is related to both Helicobacter spp. and Wolinella sp. It has long been established that proton pump inhibitors (PPIs) and other benzimidazole derivatives display anti-Helicobacter activity in vitro. PPIs have in the past been shown to affect Helicobacter pylori growth, survival, motility, morphology, adhesion/invasion potential and susceptibility to conventional antibiotics. PPIs are highly effective drugs that are well tolerated, safe for prolonged daily use and are therefore in high demand. Both the PPIs omeprazole and lansoprazole featured in the top ten drugs prescribed in England in 2014. In 2014 Campylobacter was also the most commonly diagnosed gastrointestinal infection in Scotland, in England and Wales and also in Europe. It has previously been generally accepted that patients who are being treated with PPIs are more susceptible to enteric infections such as Campylobacter than people not taking PPIs. The effect of PPI exposure on H. pylori has been investigated rigorously in the past. A single previous study has hinted that PPIs may also be capable of affecting the related organism C. jejuni,but investigations have been extremely limited in comparison to those investigating the effect of PPIs on H. pylori. This study has investigated the in vitro effects of direct contact with PPIs on the biology ofC. jejuni. Exposure to the PPI pantoprazole was found to affect C. jejuni growth/survival, motility, morphology, biofilm formation, invasion potential and susceptibility to some conventional antibiotics. Microarray studies showed that the cmeA and Cj0561c genes were significantly up-regulated in response to pantoprazole exposure and a CmeABC deficient mutant was found to be significantly more susceptible to killing by pantoprazole than was the parent strain. Proteomic analysis indicated that the oxidative stress response of C. jejuni was induced following exposure to sub-lethal concentrations of pantoprazole. C. jejuni gene expression was assessed using qRT-PCR and the genes encoding for thiol peroxidase and GroEL co-chaperonin (both involved in the C. jejuni oxidative stress response) were found to be around four times higher in response to exposure to sub-lethal concentrations of pantoprazole. Experiments using the oxidative stress inhibitors thiourea (a hydroxyl radical quencher) and bipyridyl (a ferrous iron chelator) showed that killing by pantoprazole was not mediated by hydroxyl radical production.

Monoclonal antibodies are a class of therapeutic that is an expanding area of the lucrative biopharmaceutical industry. These complex proteins are predominantly produced from large cultures of mammalian cells; the industry standard cell line being Chinese Hamster Ovary (CHO) cells. A number of optimisation strategies have led to antibody titres from CHO cells increasing by a hundred-fold, and it has been proposed that a further bottleneck in biosynthesis is in protein folding and assembly within the secretory pathway. To alleviate this bottleneck, a CHO-derived host cell line was generated by researchers at the pharmaceutical company UCB that stably overexpressed two critical genes: XBP1, a transcription factor capable of expanding the endoplasmic reticulum and upregulating protein chaperones; and Ero1α, an oxidase that replenishes the machinery of disulphide bond formation. This host cell line, named CHO-S XE, was confirmed to have a high yield of secreted antibody. The work presented in this thesis further characterises CHO-S XE, with the aim of using the information gained to lead the generation of novel host cell lines with more optimal characteristics than CHO-S XE. In addition to antibodies, it was found that CHO-S XE had improved production of two other secreted proteins: one with a simple tertiary structure and one complex multi-domain protein; and higher levels of a number of endogenous protein chaperones. As a more controlled system of gene expression to unravel the specific roles of XBP1 and Ero1α in the secretory properties of CHO-S XE, CHO cells with inducible overexpression of XBP1, Ero1α, or a third gene involved in the Unfolded Protein Response, GADD34, were generated. From these cell lines, it was shown that more antibody was secreted by cells with induced overexpression of XBP1; however, Ero1α and GADD34 overexpression did not improve antibody yield. Further investigation revealed that endogenous XBP1 splicing was downregulated in the presence of an abundance of the active form of XBP1. This result indicated a novel aspect of the regulation of the activity of IRE1, the stress-induced endoribonuclease responsible for XBP1 splicing. Overall, the work described in this thesis confirms that the overexpression of XBP1 has an enhancing effect on the secretory properties of CHO cells; information which could contribute to the development of host cells with a greater capacity for antibody production.

Streptococcus pneumoniae is a major human pathogen and causes a significant burden of disease in both developed and developing countries. Currently, two pneumococcal vaccines are available, a polysaccharide conjugate vaccine for children <2 years of age and an adult polysaccharide vaccine for ‘at risk’ groups such as the elderly and immunocompromised. Unfortunately, due to the vast variation and highly recombinant nature of the pneumococcus vaccine escape through serotype replacement is significantly decreasing the efficacy of pneumococcal vaccines globally. New cost-effective and protective pneumococcal vaccines are urgently required. Pneumolysin (PLY) is a 53Kd cholesterol-dependent cytolysin that is largely conserved in all strains of Streptococcus pneumoniae, making it an ideal candidate for inclusion in a broad spectrum vaccine. It has been shown that PLY is not only a protective immunogen but also has potent adjuvant properties and stimulates both IgG and IgA antibody responses to antigens genetically coupled to the toxin (Douce et al., 2010). Both systemic and mucosal responses are induced when PLY is used as an adjuvant which may prevent colonization and therefore provide non-serotype specific herd immunity to Streptococcus pneumoniae. The cytolytic activity of PLY prevents its inclusion in a human vaccine; a non-lytic deletion mutant 76PLY was created for this purpose which retains adjuvanticity, albeit slightly reduced. The aim of this study was to elucidate the mechanism(s) of PLY/Δ6PLY adjuvanticity, it will be essential to have a basic model of adjuvant activity before PLY-based vaccines can be advanced to human clinical trials. This project used a combination of high-throughput methods such as protein pulldowns and gene expression profiling to examine the abilities of PLY, 76PLY and the truncation mutants D123PLY and D4PLY to bind to and be internalized by host cells and to differentially regulate gene expression. These studies highlighted specific and direct interactions between PLY variants and the host cytoskeleton that could mediate antigen/PLY uptake; they also revealed a pattern of gene expression that is similar to those of other adjuvants and could provide the basis for a model of adjuvanticity. Finally, through the use of reporter cell lines and transgenic TLR4-/- BMDM, the relationship between PLY and TLR4 has been further defined. A novel method for preparing vehicle controls provided evidence that the ligation of TLR4 in this system is PLY-dependent and is not an artefact caused by contaminating TLR ligands such as LPS. Once this was established it was possible to further investigate the role of TLR4 in the adjuvant activity of PLY, in particular the PLYdependent production of IL-1@. Through these studies a surprising role for TLR4 in in vitro PLY-dependent cytokine production was discovered. Additionally, it was found that complement has an essential role in the PLY-dependent production of IL-1@. The role(s) of complement and IL-1@ in the adjuvant activity were further investigated using an in vivo immunization model and the biological basis for the difference in adjuvant activity of PLY and 76PLY was defined.

No description available.

Background: There are a large number of viruses spread by mosquitoes, many of which cause debilitating, often fatal, neurological disease such as acute encephalitis. In this study we have used two different neurotropic viruses: Semliki Forest virus (SFV), and West Nile virus (WNV), both of which can cause severe panencephalitis in the mouse. The influx of leukocytes into the infected tissues is mediated by chemokines and is believed to be important for virus clearance. To date, we have only limited insights into the precise nature of chemokine involvement, and an improved understanding of these important axes provides a new target for the development of novel therapies. Hypothesis: Based on previous studies investigating the role of chemokines during neuroinflammation it was hypothesised that chemokines and other cytokines are highly upregulated during viral encephalitis, and the blockade of selected chemokine receptors would lead to altered disease outcome. It was also hypothesised that chemokine receptors would present plausible targets for the treatment of viral encephalitis. Results: To test these hypotheses, the chemokine expression pattern and the kinetics of chemokine mediated leukocyte recruitment during viral encephalitis were analysed in unprecedented detail by TaqMan low density array, and flow cytometry, respectively, and key chemokine receptor were identified as therapeutic targets. Both SFV and WNV exhibited a similar pattern of chemokine upregulation, although WNV induced significantly higher fold expression. The key chemokines upregulated were CCL2, 3, 5, 7, CXCL9 and CXCL10. The upregulation of chemokines coincided with leukocyte influx into the CNS. After identifying the key chemokines upregulated during viral encephalitis, next a selected panel of chemokine receptor antagonists was utilized to evaluate the hierarchy and relative importance of distinct chemokine receptors for CNS leukocyte influx, viral clearance, neuropathogenesis and host survival. We identified the CXCR3 axis as being the key instigator of CNS inflammation in response to alphavirus infection, placing it at the top of a hierarchal cascade that is followed by CCR2 and CCR5. Critically, inhibition of both CXCR3 and CCR2 simultaneously, significantly improved host survival to otherwise lethal encephalitis. Conclusion: These data suggest that chemokine receptors represent plausible therapeutic targets for viral encephalitis.