Resistance to β-lactam antibiotics in Campylobacter is often associated with the production of a β-lactamase; to date the genomically encoded bla\(_{OXA-61}\) and the closely related cj0299 are the only described β-lactamase genes of Campylobacter. Cj0299 of C. jejuni NCTC11168 was assigned a novel oxacillinase number OXA-193. Previously, a novel β-lactamase (CjBla2)was identified in two bla\(_{OXA-193}\) negative isolates,P843 and P854. Southern blotting with a probe for bla\(_{OXA-193}\) confirmed that Bla2 is not the product of a mutated bla\(_{OXA-193}\) gene. A further thirteen veterinary isolates of Campylobacter have been identified that have the same phenotype as P843 and P854. Whole genome sequencing of P854 revealed four putative beta-lactamase genes, one of which, P854_1490, encodes a completely novel oxacillinase (OXA-184). PCR screening and sequencing of the other putative CjBla2 producers revealed six to contain the novel oxacillinase. A further five encode a variant of this novel OXA in which a point mutation has led to an amino acid coding change from leucine to isoleucine (OXA-185). These isolates represent a selection of flaA types and were isolated from two separate locations at different times, therefore it is unlikely that they are a clonal population. Two conjugative plasmids, each approximately 45Kb in size, have been identified from two veterinary isolates of Campylobacter. These plasmids have been shown to horizontally transfer resistance to tetracycline and to the β-lactams penicillin, ampicillin and oxacillin, between Campylobacter, a process never previously described. The two β-lactam resistance encoding plasmids thought to contain a β-lactamase gene(s) have been named pBla1 and pBla2. The role of efflux in beta-lactam resistance has also been investigated; inactivation of the efflux pump gene cmeB in the reference strain NCTC11168 resulted in increased susceptibility to a range of beta-lactams including the cephalosporins which Campylobacter are reported to be innately resistant to and have been included in some Campylobacter selective media. The work completed as part of this PhD program has furthered the understanding of beta-lactam resistance in Campylobacter and has demonstrated that it is clearly a multi-faceted mechanism incorporating various chromosomally encoded beta-lactamases, putative transferable beta-lactamases and efflux.

Mesenchymal stem cells (MSCs) are a subset of multipotent cells with a variety of trophic and immunosuppressive functions. Isolation of murine MSCs has traditionally been hampered by the presence of contaminating cells in culture. In this study, I prospectively isolate murine MSCs based on the co-expression of platelet-derived growth factor (PDGF) receptor alpha and stem cell antigen‐1 (PαS MSCs) and present novel data regarding their in vitro phenotype, karyotype and immunomodulatory functions. However, PαS MSCs undergo senescence after extended culture, resulting in a loss of function. Addition of fibroblast growth factor 2 (FGF2), PDGF‐BB or transforming growth factor-beta 1 (TGF-β1) was able to overcome senescence in MSC cultures. These factors also ‘lineage primed’ MSCs down specific fates at the genetic and phenotypic levels, with un‐supplemented MSCs primed towards bone, FGF2 or PDGF‐BB supplemented cells primed towards fat, and FGF2 supplemented cells primed towards cartilage. TGF‐β1 supplementation attenuated tri‐lineage differentiation of PαS MSCs but maintained their immunosuppressive functions. These findings were confirmed in a mouse model of inflammatory liver injury, with late‐passage TGF‐β1 MSCs improving liver injury compared to controls. In summary, these results have significant translational relevance as I reveal that culture conditions can functionally ‘prime’ MSCs down specific fates.

This thesis investigated markers of oxidative stress and immunosenescence in healthy adults. The results presented represent several novel findings which support the notion that oxidative stress and infection with micro-organisms shape our biology and can accelerate aspects of ageing. Acute exercise of high intensity was shown to cause alterations in the cellular composition of blood, which was most pronounced in lymphocyte sub-populations important for immunosurveillance. This exercise also resulted in increased markers of oxidative stress in lymphocytes, and resulted in a whole body oxidative stress, which was more pronounced and prolonged, following ultra-endurance exercise. Studies also showed that infection with a highly prevalent and asymptomatic herpes virus, Cytomegalovirus (CMV), shapes our immune biology in two significant ways. First, CMV amplified the magnitude and kinetics of lymphocyte responses to exercise, which could potentially facilitate immune surveillance, or aggravate inflammatory processes. Second, CMV was seen to drive the development of an ‘Immune Risk Profile’ in young adults, characterised by increased inflammatory activity and smaller responses to vaccination. These outcomes are associated with frailty, cognitive decline, and mortality in the elderly.

Regulated adherence of monocytes to extracellular matrix macromolecules is a prerequisite for their accumulation at sites of pulmonary infection and inflammation. To begin to assess the pathobiological importance of alterations in monocyte adherence to extracellular matrix in inflammatory lung diseases, the adherence properties of monocytes from patients with an inflammatory lung disease (bronchiectasis) and healthy subjects to a representative matrix component (fibronectin) were compared. Spontaneous adherence of monocytes from the control subjects was 20 to 25%, whereas that of the patients' cells was 2 to 3-fold higher and correlated with the severity of airway inflammation. Endotoxin (LPS) and cytokines from areas of airway disease are likely to be responsible for the observed monocyte activation since: 1) LPS was detected in plasma from all of the patients but none of the control subjects; and 2) LPS and cytokines produced dose-related increases in the adherence of normal monocytes in vitro. Monocyte adherence to fibronectin was substantially mediated by CD11/CD18 integrins, via both RGD-dependent and RGD-independent mechanisms. These data indicate that signals arising from foci of pulmonary inflammation are likely determinants of the accumulation of monocytes in the lungs of patients with chronic inflammatory lung diseases. There was a striking relationship between the adherence properties of monocytes and functions that are of biological importance at sites of inflammation. Spontaneously adherent monocytes had an "inflammatory effector" phenotype, non-adherent cells had an "immune modulatory" phenotype and monocytes that could stimulated to adhere by LPS (LPS-adherent cells) had an intermediate phenotype. In addition, only the adherent monocyte subpopulations were replete with HLE and these cells contained a substantial (10 to 11-fold) molar excess of HLE compared with the physiological inhibitor of this enzyme (a1-antitrypsin). Maturation in vitro increased the accumulation of a1-antitrypsin by all of the monocyte subpopulations. In contrast, proinflammatory mediators up-regulated a1-antitrypsin accumulation by only the spontaneously adherent cells, probably by translational or post-translational mechanisms. In conclusion, these data indicate that monocytes are heterogeneous in their ability to accumulate at sites of infection and inflammation. In addition, the capacity of monocytes to adhere to fibronectin is related to monocyte functions that are of biological importance at sites of infection and inflammation. Furthermore, LPS released from foci of infection, may induce the accumulation of monocytes with an inflammatory effector phenotype, and may thereby promote resolution of tissue infection. Alternatively, LPS may promote the recruitment of monocytes with capacity to contribute to HLE-mediated tissue injury.

Although it is evident that physiological ageing is accompanied by marked alterations in the function of innate immune cells, little is known regarding the underlying mechanism(s). Furthermore, the effect of age on many novel aspects of innate immunity is unknown. This thesis has identified the mechanism(s) behind the well-documented age-related decline in natural killer (NK) cytotoxicity (NKCC) and demonstrated for the first time that human ageing is accompanied by a significant reduction in the generation of neutrophil extracellular traps (NETs). Following target cell recognition, it was found that NK cells from older adults secreted into the immunological synapse (IS) significantly lower levels of perforin, a pore-forming protein that plays a non-redundant role in NKCC. This impairment led to reduced perforin binding to the target cell surface, an event that correlated strongly with NKCC. Underlying the reduction in perforin secretion was defective polarisation of lytic granules to the IS, which was associated with delayed activation of extracellular signal-regulated kinase 1/2. Whilst no age-related difference was observed in NET production triggered by phorbol 12-myristate 13-acetate (PMA), neutrophils from older adults generated significantly fewer NETs when challenged with interleukin-8 or lipopolysaccharide, which was accompanied by a reduction in reactive oxygen species generation. As PMA activates cells independent of membrane receptors, aberrant intracellular signalling proximal to the neutrophil membrane may underlie the age-related impairment in NET production.

Sepsis remains a common reason for hospital admission and is associated with a high mortality especially in the elderly. Neutrophils are one of the primary immune cells involved in the elimination of pathogens; however also contribute to the pathogenesis of multi-organ failure in sepsis. Previous studies have demonstrated an age-related decline in neutrophil migration and phagocytosis suggesting this as a mechanism for the poorer outcomes observed from sepsis in the elderly. Observational studies suggest that HMG-CoA reductase inhibitors (statins) are associated with improved outcomes from sepsis and potentially modulate neutrophil function. This thesis demonstrates that high dose (80mg) simvastatin improved the migration of neutrophils in the healthy elderly without affecting other key neutrophil functions, such as phagocytosis and reactive oxygen species production (ROS). Studies in patients with sepsis, demonstrated that circulating neutrophils displayed features of immune-paresis and failed to migrate, but were activated with increased phagocytosis and ROS. This was accompanied by reduced neutrophil extracellular trap (NET) formation and delayed late apoptosis. The use of in-vitro simvastatin failed to modulate migration, whilst ROS and NET production was reduced by simvastatin. Simvastatin remains a potential immune-modulating drug for treating early infection in the elderly.

Physiological ageing is accompanied with an increase in systemic inflammatory mediators (inflammageing), a functional decline of the immune system (immunesenescence), altered endocrine function (adrenopause) and reduced physical activity which predisposes the elderly to increased risk of disease. Little is known about the interplay between physical activity, inflammageing, adrenopause and immunesenescence and what impact interventions may have in the elderly. This thesis identified the consequences of inflammageing and its association with immunesenescence and the impact physical activity plays on limiting the severity of inflammageing. Cytomegalovirus drives immunesenescence but was not associated with inflammageing. Instead inflammageing was associated with reduced physical activity and increased body fat. Furthermore, inflammageing and adrenopause was associated with increased frailty and mortality risk over a ten-year period. Accelerometer defined physical activity levels in healthy elders revealed a reduced inflammatory profile and improved neutrophil migration towards interleukin-8. Acute exercise revealed an enhanced inflammatory profile indicative of positive tissue adaptation. Furthermore there was a reduced ratio of cortisol to dehydroepiandrosterone-sulphate which was accompanied by enhanced neutrophil and monocyte bactericidal function. Subsequently ten-weeks of high-intensity interval training, which was more than half the time commitment of regular aerobic training, revealed similar reduced inflammation and improved neutrophil and monocyte bactericidal capacity.

Until recently, CD4\(^+\)THelper (T\(_H\)) cells were thought to be permanently committed to a single lineage (e.g. T\(_H\)1, T\(_H\)17, T\(_H\)2 etc.). However there is now increasing evidence that T\(_H\) cells are plastic in nature and can gain phenotypic feature of other TH cells. Within this study I have investigated the overlap and plasticity of T\(_H\)1 cells and their presence in health and in the inflammatory setting of multiple sclerosis. Although CCR6 is considered a T\(_H\)17 marker there are other T\(_H\)cell subsets that express CCR6. I have identified a novel subset of T\(_H\)1 cells that express functional CCR6. CCR6\(^+\)T\(_H\)1 cells transcriptionally express 'T\(_H\)17'-related genes (e.g. RORC, IL-23R and IL4I1) but are distinct from IFN\(\gamma\)\(^+\)IL-17\(^+\) cells that also express CCR6, RORC and T-bet. Additionally I have identified candidate miRNAs that may play a role in controlling phenotypic features of these cells. T\(_H\)17 cells have been implicated in the pathogenesis of multiple sclerosis and enter the cerebrospinal fluid through CCR6-dependent migration. CCR6\(^+\)IFN\(\gamma\)\(^+\) cells were increased within the cerebrospinal fluid of patients with multiple sclerosis, suggesting a possible role in disease pathogenesis.

The interaction between the nervous and immune system is well documented, although is still not fully understood - particularly the impact of neurotransmitter receptors on immune cell function. 5-HT\(_3\)A receptor expression was identified on activated regulatory T cells (Treg) but not on effector T cells. Incubation of human peripheral blood mononuclear cells (PBMC) with the 5-HT\(_3\) receptor agonist DDP733 and the positive allosteric modulator 5-chloroindole increased the percentage of CD25+FoxP3+ lymphoyctes (Treg phenotype). Proliferation of PBMC was inhibited by DDP733 plus 5-chloroindole indicating functional impact by the 5-HT\(_3\) receptor. The GPR55 receptor was also expressed by human T cells. The GPR55 agonist lysophosphatidylinositol increased cell viability by preventing apoptosis. However, the induced response was not blocked by the GPR55 receptor antagonist cannabidiol casting doubt over the GPR55 receptor mediating the response. Cannabidiol was demonstrated to have a pro-apoptotic effect in its own right, although whether this effect is mediated by GPR55 or the CB2 cannabinoid receptor is unknown. Further experiments are required to elucidate the role of the 5-HT\(_3\)A receptor in lymphocyte function and the mechanism responsible for the immunoprotective role of lysophosphatidylinositol.

Hypoxia and hypoxia-reoxygenation (H-R) are pathogenic factors in many liver diseases and lead to hepatocyte death as a result of reactive oxygen species (ROS) accumulation. Activation of the Tumour Necrosis Factor-a (TNFa) super-family member CD40 by its cognate ligand CD 154 can induce hepatocyte apoptosis via induction of autocrine/paracrine F as Ligand/CD178 expression but the relationship between CD40 activation, ROS generation and hepatocyte cell death is poorly understood. Therefore, human hepatocytes were isolated from liver tissue and exposed to an in vitro model of hypoxia and H-R in the presence or absence of CD154 and/or various inhibitors. Hepatocyte ROS production, apoptosis, necrosis and autophagy were determined by a four-colour reporter flow cytometry assay. The in vivo regulation of liver injury by CD40 and CD 154 was determined using a murine model of partial liver ischaemia. Exposure of human hepatocytes to recombinant CD 154 or platelet-derived soluble CD 154 augmented ROS accumulation during H-R resulting in NADPH oxidase-dependent apoptosis and necrosis. The cyto-protectivc mechanism of autophagy limited apoptotic cell death during hypoxia and H-R. CD40 and CD 154 knockout mice but not wild type mice were protected from ischaemic liver injury. Hence, CD40:CD154 mediate hepatocytes cell death in vitro and in vivo during hypoxia and H-R.