Threitol Ceramide (ThrCer) is a truncated, non-glycosidic, acyclic analogue of the prototypical CD1d agonist, α-GalCer, and has potential therapeutic application. Synthesis and biological assessment of a series of deoxy ThrCer analogues showed that all three hydroxyl groups in the sugar head-group are necessary for effective iNKT cell activation. Postulating that the increased conformational flexibility of the acyclic head-group in ThrCer accounts for its lower biological activity compared to α-GalCer, analogues were prepared in which the threitol unit is contained within a carbocycle. Biological results indicate that incorporating the threitol head group into a six- or seven-membered ring can restore activity to similar levels to that displayed by α-GalCer. Routes to double bond-containing carbocyclic analogues have also been explored. In contrast to acquired immunity, innate immunity can act immediately to recruit phagocytes to a site of infection, which can then engulf and disable invading pathogens. Pathogen uptake is usually through pattern recognition receptors, which recognise specific pathogen-associated molecular patterns. Macrophage-inducible C-type lectin (Mincle) is one such receptor which recognises mycobacterial trehalose-6,6’-dimycolate (TDM). Synthesis and biological assessment of TDM analogues revealed that the length of the acyl chain can modulate Mincle stimulation, although the optimal chain length has not yet been determined.

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.

Introduction: Alloimmunity is a major contributor to chronic allograft injury. There are currently no routine clinical cell-based assays that allow quantification of the recipients' alloimmune response towards a graft. Previous work from our group identified indirect alloimmune responses to non-polymorphic regions of HLA Class 1. The aim of this thesis was to assess the alloimmune response in renal transplant recipients (RTRs) by using synthetic peptides to non­polymorphic regions ofHLA Class 2. Methods: Responses to newly synthesized HLA Class 2 peptides were tested in RTRs via any­ interferon ELISPOT assay. Cell surface staining techniques and Luminex technology were used to identify the T-cell subsets driving the immune responses and subsequent cytokine production respectively. Results: Increased responses to HLA Class 2 derived peptides were detected in renal transplant recipients compared to healthy controls. The activated effector memory subset ofT-cells was expanded in RTRs compared to healthy controls and generated these responses. T effector memory cell dependent TNF-a and IL-2 and T regulatory dependent IL-10 synthesis in the presence of specific peptide antigen was detected. Conclusion: A potential reproducible assay ofT cell alloreactivity has been identified to help stratify RTRs at risk of an ongoing alloimmune response but needs further testing in a larger multicentre study.

Immunity to Salmonella enterica serovar Typhimurium (STm) is complex and requires both cell mediated and humoral immunity at different stages of infection. In infants in sub-Saharan Africa infection with non-typhoidal Salmonella (NTS), such as STm, can commonly cause fatal invasive disease. Evidence indicates that disease may be preventable by antibody, which makes vaccine development against these devastating infections a promising option. This work has explored the cell-mediated and humoral response to STm and its component antigens, their intrinsic properties, and capacity to act as protective immunogens in a mouse model. In particular, responses to surface exposed structures such as the outer membrane proteins (Omps) and the flagellar protein FliC, which are potent, immunodominant antigens and frequent targets of antibody, that may offer potential as vaccine candidates have been examined. Immunisation with soluble flagellin (sFliC) induces a potent Th2 response. Despite this, immunisation with sFliC results in accelerated clearance of STm after the first week of infection in an antibody independent, but T-bet-regulated manner. This suggests that the Th2 responses to flagellin are flexible since they can promote Th1 mediated clearance of STm. This moderate protection conferred by sFliC contrasts with the potent benefit conferred by porins. These proteins induce, and can mediate protection through a T-independent B1b cell population. In particular, antibody to OmpD is key for this protection. These results suggest that vaccines that induce protective antibody to STm may be more effective than vaccines that induce T cell-mediated protection, since they reduce bacterial numbers at the earliest stages of infection. Lastly, experiments using N. brasiliensis show that infectious history can impact on the host’s ability to control primary STm infection and the efficacy of antibody-mediated protection against infection. These projects further our understanding of the relationship between host and pathogen and the mechanisms used to control infection, but also identify the need to consider the impact of infectious history on the host’s capacity to implement protective immunity.

The Pet Autotransporter protein was engineered and recently proposed as recombinant protein production (RPP) system. This system allows targeting the protein of interest in the culture supernatant fraction. The reduction of diversity and quantity of process impurities and size and number of downstream steps required, increase the overall process robustness and speed-up the process development time for RPP. In the context of this study the platform was investigated for the production of a “difficult” E.coli protein with commercial relevance, C1275. Pet autotransporter was suitable for the production and one step-purification of a protein with comparable purity, thermal stability and immunogenicity to that produced using conventional technology. Additionally, the autotransporter platform was tested for the first time in scaled-up production conditions. The system was compatible with fermentation and the scaled-up conditions resulted in high yield of antigen production even though a further system optimization would be required for a one step-purification process.

Mammalian cell entry (MCE) domains are so called due to the reported ability of an Escherichia coli strain harbouring the mce1 gene from Mycobacterium tuberculosis to invade mammalian cells. Bioinformatic analyses presented here demonstrated that proteins containing a single MCE domain are widespread in bacteria and that proteins containing multiple MCE domains are specific to and have evolved within Proteobacteria. Gene neighbourhood analyses revealed that MCE domain containing proteins are components of transporters and that multi MCE domain containing proteins constitute a novel type of transporter. E. coli was shown to harbour three MCE proteins: the single MCE domain protein MlaD and two multi-domain proteins PqiB and YebT. All three proteins were shown to locate to the inner membrane and bind phospholipids. Phenotypic studies revealed that their functions overlap but are distinct. Infection studies with Salmonella showed that the proteins are important for systemic infection but are not required for mammalian cell entry. Phospholipid growth experiments with Salmonella demonstrated that they are important for phospholipid uptake. These findings suggest that MCE domain containing proteins in Proteobacteria are not directly involved in mammalian cell entry and instead play a role in other aspects of mammalian infection related to phospholipid trafficking.

Salmonella typhimurium is one of the most common causes of bacteraemia in children in sub-Saharan Africa and is prevalent in HIV-infected individuals. However, symptoms of this systemic infection are unclear, and while fatalities are frequent, how infection kills is unknown. Here we use a mouse model of systemic (but resolving) infection to investigate physiological and immunological aspects of the host response to infection. The liver is colonised during systemic infection, and in the model used, bacterial numbers peak at day 7 and are largely resolved within a month. Inflammatory lesions, consisting of multiple leukocyte populations, develop within the liver. These persist and are more severe once bacterial clearance is established. Whilst lesions can develop in the absence of T and B cells, these cells contribute to the regulation of inflammatory foci. In the absence of interferon-γ, lesions do not develop and inflammation in the liver is largely absent. In parallel, extensive platelet thrombosis occurs in the liver venous system and the shared kinetics with lesion formation suggest these phenotypes may be co-regulated. Here we describe how parenchymal and vascular inflammation are anchored by inflammatory up-regulation of podoplanin expression in the liver. Thrombosis is substantially abrogated in the absence of C-like lectin-type receptor-2 (CLEC-2) expression on platelets and we show that podoplanin (the physiological ligand for CLEC-2) expression on clodronate-sensitive myeloid populations is necessary for thrombus development. Therefore, the parallel association between inflammation and platelet activation could be the basis for developing novel treatments for systemic bacterial infections in humans.

The ability of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) to immunomodulate offers therapeutic potential in liver injury but the inherent heterogeneity of unsorted MSC populations may explain varied/reduced function as well as posing regulatory challenges. Thus, we aimed to evaluate the therapeutic potential of purified CD362+ MSC infusion in murine models of acute liver injury. UC-MSCs were injected intravenously into mice injured by single dose of Carbon tetrachloride (CCl4) & OVA-BIL mice. MSC used were either unsorted or sorted CD362+. The extent of liver damage was determined by liver histology, serum analysis, gene expression and FACS analysis 3 or 5 days after cell infusion. Homing and bio-distribution of stem cells was determined by whole mouse cryo-imaging of Q-dot labelled MSC following infusion of UC-MSC into injured mice. CD362+ MSC were as effective as unsorted MSC in ameliorating liver injury, with reductions in serum ALT seen in both models. In contrast heat-inactivated MSC had no effect on liver injury. MSC also led to a reduction in CD45+staining on liver sections in both models of liver injury corroborated by an accompanying reduction in hepatic CD45+ cells in (FACS analysis of liver digest). In addition, there was a significant reduction in hepatic CD19+ B cells in digested liver in CCl4 injury. CD362+ MSCs were found to have the ability to reduce the level of adhesion molecules (ICAM and VCAM) in Ova-Bil mice. Cryo-imaging of time-course in both animal models indicated that MSC had migrated to the lung within 1 hour and were then cleared rapidly, although there was a liverspecific increase in MSC 2-3 day in Ova-Bil mice. CD362+ human MSC exert potent anti-inflammatory activity in toxic and immune-mediated murine liver injury with demonstrable reductions in infiltrating inflammatory leucocytes and B cells.

Arboviruses (Arthropod-borne viruses) cause acute diseases that are increasingly affecting both human and animal health. Currently, there is a critical lack of understanding about the nature of arbovirus-host interactions in the lymph nodes (LNs), the place where the adaptive immune response is initiated and shaped. In this study, we used bluetongue virus (BTV) and its natural sheep host, to characterise the early events of an arbovirus infection with particular focus on the LNs. Our findings reveal a previously uncharacterized mechanism used by an arbovirus to manipulate host immunity. This study shows that BTV, similarly to other antigens delivered through the skin, is transported rapidly via the lymph to the peripheral lymph nodes. Here, BTV infects and disrupts the stromal network of marginal reticular cells and follicular dendritic cells composing the scaffolding of the follicular area. These cells contribute to antigen presentation and affinity maturation of B-cells for the production of antibodies. Consequently, we observed a loss of germinal centre structure, which hinders B-cell proliferation. This process results in a delayed production of high affinity and virus neutralizing antibodies that is directly related to the virulence of the BTV strain used and the severity of disease. Moreover the humoral immune response to a different antigen is also hampered in BTV-infected animals. Our data show that an arbovirus can evade the host antiviral responses by inducing an acute immunosuppression. Although transient, this immunosuppression occurs at the critical early stages of infection when a delayed host humoral immune response likely affects virus systemic dissemination and the clinical outcome of disease.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor family plays a key role in the regulation of the immune response and the transcriptional response to infection through transcriptional activation of genes involved in those processes. The NF-κB response is regulated in the nucleus by the balance between ubiquitination and deubiquitination processes. Ubiquitination of the p65 subunit of NF-κB terminates the NF-κB response by targeting p65 for proteasomal degradation. Nevertheless, the ubiquitin molecules can be removed from targeted proteins by the action of deubiquitinating enzymes (DUBs). Ubiquitin-specific protease 7 (USP7) is a deubiquitinase enzyme from the ubiquitin-specific protease (USP) family which deubiquitinates p65. Besides p65 deubiquitination, USP7 is involved in a huge variety of biological processes due to stabilisation or localisation of proteins involved in those processes. USP7 is a multidomain protein formed by an N-terminal Meprin and tumour necrosis factor receptor-associated factor homology (MATH) / tumour necrosis factor receptor-associated factor (TRAF) domain, a catalytic domain (CD) and five ubiquitin-like domains (UBLs) in the C-terminal region. p65 recognition by USP7 takes place through the C-terminal region, but the molecular determinants involved in the interaction are still unknown. New therapeutic compound design strategies are based on interrupting the interaction interface between both proteins involved in the interaction. Therefore, in order to design a specific inhibitor of the deubiquitinase activity of USP7 on p65 we performed a peptide array and subsequent alanine scan followed by site directed mutagenesis experiments. We concluded that UBL2 of USP7 is necessary for the interaction with p65. UBL2 deletion completely abolishes the interaction and the deubiquitinase activity of USP7 on p65. Specificity of this mutant was tested by immunoprecipitation assays with different USP7 substrates. In silico modelling revealed a putative binding pocket in USP7 UBL2 that may be targeted to inhibit the interaction with p65. Together our data suggest that a binding pocket present on UBL2 composed by amino-acids 627-ARSNGTK-633, 736-EEVKPNLTER-745 and 757-LDELMDGD-764 directs the interaction with p65, besides UBL2 when deleted inhibits the interaction with p65 and subsequently its deubiquitination in a substrate specific manner.