Frailty, the increased vulnerability of an individual to stressors, and sarcopenia, the loss of muscle mass with age, share many of the same clinical outcomes, associations and suggested pathophysiology. The pathophysiology of both conditions is incompletely characterised but it is postulated the immune system is central to development and propagation. 40 healthy young, 40 healthy older, and 37 frail older adults were recruited to three groups. A further 73 healthy young adults were recruited for ultrasound assessment of muscle. Ultrasound was reviewed as a diagnostic technique in the identification of sarcopenia using a simple scanning protocol to produce the bilateral anterior thigh thickness (BATT). The BATT was measured in a reference population, 113 in total, and proposed criteria for the identification of low muscle mass in older adults was based on this reference population. Neutrophils exhibit a frailty related decline in migratory accuracy towards chemoattractants; this was both independent of age and associated with physical and cognitive parameters of frailty. Incubation of neutrophils from frail older adults with PI3kinase inhibitors class 1A \(\delta\) and class lB y restored migratory accuracy and this presents a novel therapeutic target for management of frailty.

No description available.

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.

Macrophages are key players in both regulatory and inflammatory immune responses. They are implicated in the pathogenesis of rheumatoid arthritis (RA) where they accumulate in the synovium and produce pro-inflammatory cytokines including TNFα and IL-6. The rheumatoid synovium is metabolically distinctive, with low oxygen perfusion and high concentrations of lactate and reactive oxygen species (ROS). Macrophages are known to respond to metabolic signals, therefore we wanted to explore whether metabolic phenotypes of differentiated macrophages could play a role in the persistence of RA. We used an in vitro model of pro-inflammatory “classically activated” and “alternatively activated” macrophages to study macrophage behaviour using metabolomic and transcriptomic techniques. Differentiation with GMCSF and M-CSF produced macrophages with distinctive profiles. GM-CSF macrophages were metabolically active, metabolising glucose, glutamine and fatty acids, while M-CSF macrophages utilised fatty acid β-oxidation alone. Activation of macrophages with LPS, LPS+IFNγ or IL-4 produced metabolic changes, however, differences between MCSF groups were modest. LPS activation of GM-CSF macrophages drove both depletion of intracellular metabolites and transcriptional downregulation. In contrast, IL-4 activation of M-CSF macrophages was metabolically activating. We propose that the metabolic adaptability of GM-CSF macrophages may put them at an energetic advantage in the hypoxic, ROS-enriched rheumatoid synovium.

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.

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.

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.

Tetraspanins are a superfamily of glycoproteins that function as ‘organisers’ of membranes by clustering with each other to form tetraspanin-enriched microdomains, into which certain other receptors and signalling proteins are recruited and regulated. Tetraspanin microdomains have been implicated in a range of biological processes including cell signalling, adhesion, intracellular trafficking, cell-cell fusion and viral entry. The tetraspanin CD37 was recently shown to negatively regulate the C-type lectin-like receptor dectin-1, which is essential for innate immune responses to fungal pathogens. The aim of this thesis was to firstly develop a cell line model system to investigate the mechanism by which tetraspanins inhibit dectin-1, and to secondly extend this work to the dectin-1-related CLEC-2, which is essential for platelet thrombus formation and stability. Using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay in the Jurkat T-cell line, transient over-expression of CD37 was found to powerfully inhibit dectin-1 signalling following stimulation with its ligand, β-glucan. Over-expression of other tetraspanins also inhibited dectin-1 signalling, but did not globally inhibit receptor signalling because the platelet collagen receptor, GPVI, was unaffected. Similar to dectin-1, CLEC-2 signalling in response to its ligand, the snake venom toxin rhodocytin, was also abrogated following tetraspanin over-expression. However, stable tetraspanin over-expression only partially reduced signalling. Moreover, knockdown of the major Jurkat cell tetraspanin, CD81, and deletion of the major platelet tetraspanin, CD9, did not affect dectin-1 and CLEC-2 signalling, respectively. In summary, the importance of transient tetraspanin over-expression for dectin-1 and CLEC-2 inhibition, and the fact that any tetraspanin can inhibit, suggests that tetraspanin microdomains are disrupted by the presence of one over-expressed tetraspanin. This leads to a failure of dectin-1 and CLEC-2 signalling by a mechanism that is not clear, but suggests that tetraspanin microdomains are important for signalling by these C-type lectin-like receptors.

Chronic Lymphocytic Leukaemia (CLL) is associated with significant immunosuppression, with infection being the predominant cause of death. The immunosuppression is multifactorial and includes hypogammaglobulinaemia and T cell dysfunction. Expanded populations of effector memory T cells which are oligoclonal are postulated to arise in response to common antigenic stimulation. Increased expression of the inhibitory receptor programmed-death 1 (PD-1) has also been reported on the total CD4+ and CD8+ T cells in patients with CLL. Cytomegalovirus is a ubiquitous herpes virus which contributes to the oligoclonal populations of CD4+ and CD8+ T cells described. In the healthy elderly, CMV is associated with an immune risk phenotype and leads to an earlier death. But the impact of CMV on clinical outcome measures in CLL is unknown. Using 60 CMV class I tetramer and for the first time 15 class II tetramer responses, I have characterised the phenotype and function of CMV-specific T cells in patients with CLL. Interestingly, increased expression of PD-1, was observed on CD4+ but not CD8+ CMV-specific T cells which remained constant over time and was not a result of recent T cell activation. Cytokine production of both CD4+ and CD8+ CMV-specific T cells was shown to be impaired in patients with CLL and PD-1 expression on CMV-specific CD4+ T cells contributed to this. Telomere lengths were also greatly reduced in CMV-specific T cells. I have also used droplet digital PCR to successfully measure latent CMV viral load and found in advanced stage disease an increased CMV viral load was detectable. This most likely arises as a result of the increased immunosuppression and T cell dysfunction observed. Despite these findings, this work reports no evidence that CMV infection impacts on clinical outcomes including time to first treatment or overall survival in two large independent cohorts of patients with CLL.