The increasing spread of antimicrobial resistant bacteria and the decline in the development of novel antibiotics have incited exploration of other avenues for antimicrobial therapy, such as the use of antibiotics that enhance the host’s defenses to infection. This study explores the influence of antibiotics on the innate immune responses to bacteria. The aims were to investigate antibiotic effects on bacterial viability, innate immune cells in response to bacteria and interactions between bacteria and the host. Five exemplar antibiotics at maximum serum concentration (C\(_m\)\(_a\)\(_x\)) and minimum inhibitory concentrations (MIC) were tested. \(Salmonella\) Typhimurium SL1344 was chosen as the model pathogen. Following incubation of SL1344 with C\(_m\)\(_a\)\(_x\) concentrations of ceftriaxone bacterial viability was undetectable. When SL1344 was incubated with ceftriaxone and ciprofloxacin treated neutrophils, there was reduced bacterial viability. Ciprofloxacin pre-treated neutrophils had reduced ability to phagocytose bacteria, while oxidative burst was increased following exposure to ceftriaxone. Adhesion of SL1344 to J774 macrophages pre-exposed to both concentrations of ciprofloxacin and ceftriaxone was increased, but only C\(_m\)\(_a\)\(_x\) of azithromycin and streptomycin. Expression of IL-1β and TNFα mRNA was greater in SL1344 infected macrophages pre-exposed to ciprofloxacin or ceftriaxone, than in macrophages exposed to antibiotics alone or SL1344 alone. In conclusion, it was found that clinical relevant concentrations of certain antibiotics enhance the response of immune cells and their interaction with bacteria, by increasing phagocytosis and killing in neutrophils, increasing bacterial adhesion to macrophages and increased cytokine production. These immunomodulatory potentials of antibiotics can be harnessed and exploited for broader therapeutic use.

Epidemiology has linked preeclampsia (PET) to vitamin D deficiency. To date, studies have focused upon serum 25-hydroxyvitamin D3 (25(0H. )D3) alone as the marker of vitamin D status. We provide strong evidence comprehensive analysis of vitamin D metabolites in pregnancy is highly informative, particularly within the context of PET. Uniquely, analysis of maternal urinary metabolites provides a novel insight into vitamin D and the kidney, with lower 25(0H)D3 and 24,25(0H)2D3 excretion early indicators of a predisposition towards PET. Since vitamin D is a potent regulator of immune function, and the decidua appears a key extra-renal site for vitamin D metabolism, we investigated effects of 1 ,25(0H)2D3 upon decidual uterine natural killer cells and macrophages. We show both express a functional vitamin-D system and demonstrate differential sensitivity to 1 ,25(0H)2D3 compared to their peripheral counterparts. To understand the functional impact of vitamin D, whole transcriptomic analysis of 1,25(0H)2D3-mediated effects upon uNK and macrophages was performed. We show the actions of vitamin D extend far beyond simple immuno-regulation, targeting major cellular functions including migration, adhesion and apoptosis. In particular, our data support effects highly relevant to decidualisation. We anticipate these findings to be highly relevant within the context of vitamin D deficiency, mal placentation and PET.

In multiple sclerosis (MS), clonally-expanded brain-resident B cells may sustain chronic disease, however their relative contributions versus recently recruited B cells is unclear. Furthermore, pro-inflammatory CD20+ T cells may also be involved in MS pathogenesis. This study aimed to characterise the cerebrospinal fluid (CSF) B cell response in MS and investigate the features of CD20+ T cells. CSF B cells and antibody-secreting cells (ASC) displayed an activated phenotype and were identified in MS CSF at a higher frequency than controls. In contrast to the periphery, CSF ASC almost exclusively expressed IgG and were strongly lgK-biased, whereas memory B cells displayed similar immunoglobulin expression profiles in both compartments. MS CSF antibodies were frequently reactive towards EBNA-1, which preferentially induced an lgK-biased antibody response. Finally, CD20+ T cells displayed a highly activated effector phenotype and were present in the CSF, although their frequencies were no different between MS and OND groups. These findings suggest that most CSF B cells result from non-specific recruitment, whereas ASC are involved in a persistent lgK-biased antigen-driven immune response, which may primarily be directed towards EBNA-1. Despite their highly activated phenotype, a role for CD20+ T cells in MS pathogenesis, if any, remains to be determined.

The Epstein-Barr virus (EBV)-encoded Latent membrane proteins (LMP) 2A and LMP2B are frequently expressed in a number of EBV-associated malignancies. Although the true function of these proteins remains to be elucidated, LMP2A appears to function as a surrogate B-cell receptor in B-cells, preventing BCR signalling and ensuring viral latency. Much less is known about the function of these proteins in epithelial cells, although LMP2A has been shown to modulate Wnt/\(\beta\)-catenin, MAPK and PI3K/Akt signalling, whilst LMP2A and LMP2B have been shown to promote cell motility and to alter the turnover of certain classes of immune receptors. Viral infection of the host cell triggers the innate immune system to promote and sustain the initiation of an anti-viral state. Viruses have developed immune evasion strategies to counteract the effects of this immune response and to prevent recognition of viral antigens by the adaptive immune response. Here novel functions for LMP2A and LMP2B are described with respect to modulation of innate immunity, findings which have implications for the role of these proteins in EBV-driven oncogenesis. The first wave of the innate immune response is mediated by Toll-like receptors (TLRs), a family of PAMP receptors that transduce signals to activate the type I interferon response. Findings presented here show that LMP2A attenuates signalling from a number of these receptors in response to TLR agonist stimulation. These data were observed only in LMP2A expressing cells while those expressing LMP2B showed no alteration of TLR signalling, indicating that the amino-terminal signalling domain of LMP2A controls these functions. Preliminary findings have also identified a role for LMP2A and LMP2B in the control of vesicular trafficking, with both proteins increasing endosomal-lysosomal trafficking and lysosomal acidification, an effect that important for degradation and turnover of receptors. These data present novel mechanisms by which EBV may evade immune responses and in doing so, contribute to the progression of the EBV-associated epithelial cell malignancy, NPC.

Thiomarinol is a novel hybrid antibiotic produced by \(Pseudoalteromonas\) \(rava\) sp. nov. SANK 73390. It is structurally similar to a clinically significant antibiotic mupirocin, but includes an additional pyrrothine moiety joined to the mupirocin-like marinolic acid via an amide bond. Thiomarinol has been shown to be more potent against a wider range of microorganisms. This potency was hypothesised to be due to either an increase in inhibition of its target enzyme, isoleucyl-tRNA synthetase (IleS) or an increase in antibiotic uptake and/or inefficient efflux by bacterial cells or a combination of both. This thesis describes experiments that investigate the basis for this increase in potency.

Kaposi’s sarcoma-associated herpes virus (KSHV) encodes genes with immunomodulatory potential, one of which is vIRF-2 that shares homology to cellular interferon regulatory factors. The innate antiviral mechanism mediating the type I interferons is an essential host cell defence mechanism limiting viral replication. The aim of this study was to determine the range and type of cellular gene sets and associated biological pathways whose expression is deregulated by vIRF-2. HEK 293-derived cell clones were engineered to express doxycycline-inducible vIRF-2. Interferon (IFN) responses were induced with recombinant (r) IFN-α and measured by an IFN stimulated response elements (ISRE) luciferase reporter gene assay. The effects of vIRF-2 on cell transcriptome profile in response to rIFN-α were determined by DNA microarray analysis and confirmed by immunoblot assay. vIRF-2 protein inhibited activation of ISRE-luc by over 50% and significantly (p<0.05) down-regulated the expression of 57/78 (73%) of rIFN-α regulated genes. The DAVID and GSEA software packages revealed vIRF-2 down-regulates the RIG-I-like receptor, JAK-STAT and Ubiquitin ligase pathways and many gene sets involved in antiviral response, transcriptional regulation and apoptosis. Immunoblot assays demonstrated reduced levels of RIG-I/DDX58, TBK-1, p-38, STAT1, pSTAT1, IRF-9 and OAS3. The biological significance of the vIRF-2 anti-IFN property was demonstrated by the rescue of encephalomyocarditis virus (EMCV) replication in vIRF-2 expressing cells treated with rIFN-α; EMCV was titred by plaque assay on L929 cells. These data confirm the role of KSHV vIRF-2 in negative regulation of the IFN-α/β innate immune response by a mechanism dependent on negative regulation of RIG-I/DDX58, STAT1, IRF-9 and OAS3.

The role of serum autoantibodies, soluble adhesion molecules, plasma and mucosal cytokines were studied following surgery for ulcerative colitis (UC). A high prevalence of ANCA was found in UC but not in Crohn’s disease. ANCA positive UC sera were found to recognise a variety of antigens, namely lactoferrin, cathepsin G, enolase and elastase. Confocal microscopy revealed maximum immunofluorescence for P-ANCA to emanate from the intranuclear and not the extra-(peri) nuclear portion of the neutrophil, as currently believed. AECA, like ANCA, are also found in UC, suggesting immunological similarities with systemic vasculitis. Perhaps, UC represents a gut limited vasculitis. Persistance of ANCA, AECA, anti-EPI and antitropomyosin antibodies in the sera, several years following total colectomy, suggests that immunological mechanisms are not halted, by total colectomy. Soluble intercellular cell adhesion molecules, ICAM-1 and E-Selectin, but not VCAM-1, were found to be useful markers of disease activity in UC. Plasma levels of cytokines do not reflect disease activity, however, there is similar quantitative expression of pro-inflammatory cytokines by isolated mucosal mononuclear cells in both active UC and pouchitis, but not in nonspecific proctitis, which seems to suggest that pouchitis is not merely a complication of ileoanal pouch surgery but, that it represents reactivation of UC in ileal mucosa, which has undergone villous atrophy and colonic metaplasia.

The eye is a glucocorticoid target tissue which orchestrates expression of target genes through the glucocorticoid receptor (GR). The classical function of GR involves its interaction with glucocorticoid to influence transcription of genes involved in numerous physiological processes which include inflammation. The first line of defence in the ocular tissues includes the mucosal barrier and expression of receptors that recognise pathogen. These mechanisms activate the innate immune response during inflammation, however, in the ‘normal’ eye, immunomodulatory components exist to promote immune privilege. 11beta hydroxysteroid dehydrogenase 1 (11β-HSD1) regulates cortisol locally in tissues and has already been localised to to some ocular surface and intraocular tissues. The aim of this thesis is to evaluate the functional role of 11β-HSD1 in the eye and the orbit and whether the 11β-HSD1 can be targeted to modify various disease processes in the eye. An animal model was used to characterise the pre-receptor regulation of glucocorticoids and this was further characterised in human ocular and orbital tissues and cells. The results showed that 11β-HSD1 is functional in certain ocular (corneal epithelial, fibroblast and conjunctival fibroblast) and orbital (orbital preadipocyte) cells. The data therefore emphasises the putative role of 11β-HSDs in the ocular and orbital microenvronment.

Known for over a hundred years, the human fungal pathogen, Cryptococcus neoformans causes cryptococcosis, a life threatening disease. Infection is acquired through inhalation of spores or dried yeast cells into the lungs from which the fungus can potentially transmit to all body parts of which the brain is the most affected organ. Once in the brain, the yeast C. neoformans causes meningoencephalitis, a fatal condition even with optimal treatment. The mechanism by which C. neoformans penetrates the normally impermeable blood brain barrier (BBB) to cause brain infection is not understood. This thesis presents two aspects of investigation: 1) the extent to which binding and uptake of cryptococci by brain microvascular endothelial cells (BMEC) explains transcytosis as a mechanism for cryptococcal traversal of the BBB and 2) the relationship between Cryptococcus – macrophage interaction and Cryptococcal meningoencaphalitis (CM) disease. We show that adherence and internalization of cryptococci by brain microvascular endothelial cells is a rare event characterized by a small number of cryptococci, an indication that C. neoformans most likely uses multiple routes to traverse the BBB. Secondly, by studying clinical isolates from cerebral spinal fluid (CSF) of HIV- associated CM patients, we demonstrate that high rate of cryptococci uptake by macrophages is associated with patient fungal burden whilst the intracellular proliferation rate is inversely associated with TNF- \(\alpha\) levels in the patient CSF. Interestingly, the high uptake – high fungal burden isolates were less encapsulated but more rapid melanin formers, traits known to modulate phagocytosis and protection from host-induced oxidative stress respectively. We therefore hypothesize that highly phagocytosed C. neoformans strains use phagocytes to disseminate faster to the brain resulting in high fungal burden.

Streptococcus pneumoniae is a major cause of morbidity and mortality in HIV infected individuals. Mortality rates remain high despite modern antibiotics, therefore, successful vaccination is key to preventing infection. Vaccination strategies against pneumococcus include a pure polysaccharide vaccine, Pneumovax® (PPV-23), and a polysaccharide-conjugate vaccine, Prevenar-13 ®(PCV-13). PCV-13 is given as part of three vaccine series in infants but is currently recommended only as a single vaccine in adults. The evidence base for either vaccine in adults is limited and guidelines vary. In 2009, national guidelines recommended PPV-23 vaccination but a PCV-13+PPV-23 schedule is now recommended (BHIVA, 2008 & 2015). The Assessment of Immune Responses to Routine Immunisations (AIR) is an observational study that has examined the immune response to UK recommended vaccinations in adults with HIV. Firstly, the AIR study went on to examine the impact of HIV-infection on pneumococcal vaccination with PPV-23, as assessed by pre- and post- vaccine IgG antibodies against 12 pneumococcal (Pn) serotypes (Pn 1, 3, 4, 5, 6B, 7F, 9V, 14,18C, 19A, 19F, and 23F at the WHO (World Health Organization) protective threshold 0.35μg/mL in ≥ 8/12 serotypes threshold using a 19-plex Luminex-based assay. HIV-infected patients responded poorly to a single dose of PPV-23 compared to HIV-negative controls. AIR then established that PCV-13 could increase the percentage of patients that reach WHO protective thresholds compared to a single dose of PPV-23 and that immune responses to PCV-13 could be maintained for a longer period compared to PPV-23. Response rates could be further improved by booster doses of PCV-13. Low antigen-specific IgG concentrations are associated with impaired opsonophagocytic killing against pneumococcus, thus a novel opsonophagocytic assay was developed in order to further understand the relationship between the quantity and opsonic functionality of Pn-specific antibody. Furthermore, assessing responses to pneumococcal vaccination is important in determining immunogenicity. Thus, this thesis also explored the whole vaccine and serotype IgG subclass (IgG1-IgG4) response to PPV-23 and PCV-13 by developing novel Pn-specific IgG subclass assays. Lastly, HIV-infection is characterised by a dysregulated humoral system, therefore, we have examined the impact on different B cell populations at baseline and relationships with total and Pn- specific antibody post-vaccination with PCV-13 are described. In summary, this study aimed to examine the impact of HIV-infection on pneumococcal vaccination by investigating the quantity and quality of the Pn-specific IgG response, IgG subclass responses, and the effects of a dysfunctional humoral system on total and Pn-specific antibody. Findings would be informative in developing vaccination strategies in HIV-infected adults in the UK.