The long-term implications of deregulation of glucose metabolism, manifesting itself as diabetes is significant. Whilst treatments for the disease are progressing, often they require multiple daily insulin injections. This may be difficult for diabetics in poorer nations. This thesis examines the possibility of utilising keratinocytes and melanocytes transfected with either normal or furin-modified proinsulin constructs, to process and secrete mature insulin via secreted protein pathways. The advantage of this approach is that it may allow normalisation of basal glucose levels, which in turn may prevent or delay the onset of many of the complications associated with diabetes. The results confirmed that keratinocytes and melanocytes are able to process and secrete mature insulin from a furin-modified proinsulin construct, thus validating this approach and paving the way for skin grafts of genetically modified cells to be used as a possible treatment for diabetes. Whilst the results regarding the ability of these cells to process a normal proinsulin construct are inconclusive, they highlight areas of interest that could help to resolve this issue / Master of Science (Hons.)

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The accumulation of leukocytes in non-lymphoid tissues and their structural organization into tertiary lymphoid structures (TLS), a process known as ectopic lymphoid neogenesis (ELN), is observed in response to chronic inflammation and in the target organ of several autoimmune diseases. TLS strongly resemble secondary lymphoid organs with specialised high-endothelial venules (HEV), segregated B/T cell areas and presence of follicular dendritic cells (FDC) networks promoting in situ affinity maturation of the antibody response. TLS have been associated with a growing number of autoimmune conditions and usually their presence is prognostic for undesirable disease progression. In Sjögren’s syndrome (SS), an autoimmune disease affecting the salivary and lachrymal glands leading to exocrine dysfunction, TLS develop in the salivary glands (SG) of around one-third of the patients. The immunobiology of the SG and the pathogenesis of SS have been poorly clarified and to date a robust and reproducible inducible animal model of SS and TLS in the SG is still absent. In my PhD, I developed and validated a novel inducible model of ELN in murine SG that also reproduces several features of SS. The retrograde administration of a replication-deficient adenovirus (AdV) in the SGs of wild-type C57Bl/6 mice was able to induce within three weeks fully formed TLS that displayed B/T cell segregation, FDC networks, HEVs and were positive for markers of germinal centres. Moreover, the AdV-treated mice showed a significant reduction of salivary flow and in 75% of the cases development of anti-nuclear antibodies.

Sjögren's syndrome (SS) and rheumatoid arthritis (RA) are characterised by breach of self-tolerance with high affinity circulating autoantibodies and peripheral B cell disturbances in the naïve and memory B cell compartments. In addition, both SS and RA develop functional ectopic B cell follicles in the respective target organs, i.e. the salivary glands and the joint synovium, whereby autoreactive B cell undergo antigen selection and affinity maturation. However, the exact stage at which errors in B cell tolerance checkpoints accumulate is unknown. In this PhD project, I amplified and sequenced Ig VH and VL gene transcripts from single B cells which were FACS sorted either from the peripheral blood of SS patients or from the RA synovium. Healthy donors (HD) were used as controls. Subsequently, I cloned and expressed recombinant monoclonal antibodies displaying identical antigenic specificity of the original B cells. Finally, I tested the poly- and autoreactivity profile of these antibodies against SS and RA-associated autoantigens. In SS, I analysed 353 VH and 293 VL sequences and obtained 114 recombinant antibodies from circulating naïve (n=66) and memory (n=48) B cells of 4 SS patients and compared their autoreactive and polyreactive profile to 45 naïve clones from 2 HD. Analysis of the VH and VL gene usage showed no significant differences between SS and HD. Conversely, I observed accumulation of circulating autoreactive naïve B cells in SS as demonstrated by Hep-2 cells, ENA, Ro/SSA and/or La/SSB reactivity. The elevated frequency of autoreactive naïve B cells in the circulation of SS patients supports the existence of early defects in B cell tolerance checkpoints in this condition In RA, I analysed the Ig gene repertoire and the VH gene somatic mutation rate of 139 VH and 175 VL sequences of synovial CD19+ B cells which demonstrated evidence of antigen selection and hypermutated alpha > gamma > mu VH chains with presence of intra-synovial clonal diversification. Recombinant antibodies from synovial B cell clones were then screened for reactivity towards citrullinated antigens with a plan for a wider analysis using autoantigen microarrays. Overall, these results highlighted the existence of B cell abnormalities and loss of tolerance for self-antigens both in the peripheral and/or lesional compartment of SS and RA. Further analysis of the fine specificity and pathogenicity of recombinant antibodies from autoreactive B cells will be invaluable in order to dissect the mechanisms and the antigens driving the development and the persistence of autoimmunity in RA and SS.

Whilst there has been a recent increase in interest in using positive reinforcement training for laboratory-housed primates, there remains a reluctance to put into practice training programmes. Much of this reticence seems to stem from lack of expertise in the running of training programmes, and a perception that training requires a large time investment, with concurrent staff costs. The aim of this thesis was to provide practical recommendations for the use of training programmes in laboratories, providing primate users and carestaff with background information needed to successfully implement training programmes whilst improving the welfare of the animals in their care. Training was carried out with two species, cynomolgus macaques (Macaca fascicularis) and common marmosets (Callithrix jacchus) in three different research laboratories to ensure practicability was as wide ranging as possible. Training success and the time investment required were closely related to the primate's temperament, most notably an individual's willingness to interact with humans, in both common marmosets and cynomolgus macaques. Age and sex however had no effect on an individual's trainability. The training of common marmosets was more successful than that with cynomolgus macaques, possibly due to differences in early experience and socialisation. Positive reinforcement training helped both species to cope with the stress of cage change or cleaning, with the monkeys showing less anxiety-related behaviour following the training programme than before. Involving two trainers in the training process did not affect the speed at which common marmosets learned to cooperate with transport box training, but behavioural observations showed that initial training sessions with a new trainer led to animals experiencing some anxiety. This however was relatively transient. Whilst the training of common marmosets to cooperate with hand capture was possible, there seemed little benefit in doing so as the monkeys did not show a reduced behavioural or physiological stress response to trained capture as compared to hand capture prior to training. However strong evidence was found that following both training and positive human interactions the marmosets coped better with capture and stress was reduced. It is recommended that an increased use of early socialisation would benefit laboratory-housed primates, and would also help improve the success of training. Further, the time investment required shows that training is practicable in the laboratory for both species, and that positive reinforcement training is an important way of improving their welfare likely through reducing boredom and fear.