Hyperalgesia is a symptom of tissue damage and inflammation. In primary hyperalgesia (within the area of injury), there is increased responsiveness to both heat and mechanical stimuli, whereas in secondarY hyperalgesia (outside the area of injury), it is generally reported that responses are only enhanced to mechanical, not heat stimuli. However, recent work from this laboratory has demonstrated that secondary hyperalgesia is A-nociceptor-dependent, rather than being stimulus-modality dependent, explaining the lack of heat but the presence of mechanical hyperalgesia in secondary hyperalgesia. Inflammation leads to changes in the number or affinity of opioid receptors in spinal cord. The segregated expression of delta and mu receptors (DOR & MOR) on A- and C-nociceptors has been recently reported, and is hypothesised to differentially modulate mechanical and thermal nociceptive behaviours respectively. Therefore, the overall aim of work described in this thesis was to investigate how the spinal dorsal horn processes the inputs from different subsets of primary afferent nociceptors in primary and secondary hyperalgesia, and the role of opioid receptors in the regulation of nociceptive processing in the spinal cord. Primary and secondary inflammatOlY hyperalgesia were induced by injection of Freund's Complete Adjuvant into dorsal hindpaw and knee joint respectively. The immediate early gene product Fos was used as a neuronal marker of nociceptive processing in the spinal cord. Using an experimental approach to preferentially activate A- or C-fibre nociceptors, it was found that C-fibre-evoked withdrawal reflexes were facilitated in primary hyperalgesia, whereas A-fibre-evoked withdrawal reflexes were facilitated in secondary hyperalgesia. Additionally, both A- and C-nociceptor stimulations evoked more Fos-like immunoreactivity (FU) in superficial but not deep dorsal horn in the animal model of primary hyperalgesia, whereas FU in response to Abut not C-nociceptor stimulation was increased in the superficial and deep dorsal horn in the animal model of secondary hyperalgesia. Spinal DOR and MOR activation inhibited both C- and A-nociceptor- evoked reflexes in both naives and inflamed animals. The DOR agonist had a greater effect on A-fibre-withdrawal thresholds in secondary hyperalgesia than naives, and a lesser effect on A-fibre-nociception in primary hyperalgesia, suggesting that DOR has a greater contribution to the modulation of A-nociceptor inputs in thermal primary and secondary hyperalgesia than MOR. Spinal DOR activation also had greater anti nociceptive effect on mechanical stimuli than MOR, but these anti nociceptive effects were equivalent in all animals. Spinal MOR activation produced antinociception in mechanical nociception in only secondary hyperalgesia, suggesting the more critical role ofMOR in modulating mechanical pain in secondary hyperalgesia. This study indicates the impOliant role of A-nociceptors in mediating secondary hyperalgesia and the different neuronal mechanisms between primary and secondary hyperalgesia. Such distinctions may be useful in treating clinical syndromes associated with pathophysiological pain.

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The aim of this thesis was to investigate the molecular mechanisms underlying control of macrophage phagocytosis of apoptotic cells. We have demonstrated that exposure of peripheral blood monocytes to the synthetic glucocorticoid dexamethasone “reprograms” monocyte/macrophage differentiation resulting in a macrophage phenotype with a marked augmentation in the phagocytosis of both apoptotic granulocytes and particles opsonized with low levels of IgG. Increase in phagocytic potential was not mediated by increased expression of putative “phagocytic receptors” proposed to be involved in apoptotic cell clearance. In addition dexamethasone augmentation of apoptotic cell uptake could not be inhibited by blockade of receptor function with either soluble competitive ligands or monoclonal antibodies. Dexamethasone treated macrophages were found to have altered morphology and actin organisation. In particular, loss of “podosome” structures was observed, possibly due to decreased recruitment of adhesion signalling molecules pyk2 and paxillin to focal contacts, and decreased expression of p130cas, a key adaptor molecule in integrin signalling. In addition, glucocorticoid treated cells showed increased activity of the Rho-family GTPase Rac, which has been previously shown to be important for phagocytosis and lamellipodia formation. Expression of p130cas and activation of the mitogen activated protein kinase, ERK are required for migration in a number of different cell types. Basal ERK activity was reduced by dexamethasone-treated monocyte/macrophages. We developed an <i>in vitro</i> “wounding” assay and found that despite the absence of basal ERK activity or p130cas expression in dexamethasone-treated macrophages, these cells were able to migrate.

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The production of high affinity antibodies is critical for providing effective protection against microbial agents. During the T cell dependent response to antigen, secondary diversification of the B cell receptor occurs. This provides a varied pool of B cells which can be selected and expanded resulting in affinity maturation of the response. This diversification is currently ascribed to somatic hypermutation. Both the molecular basis and the triggers for hypermutation have been studied in vitro. Recently the possibility that another process, receptor revision, might provide an additional means of diversification has emerged. Receptor revision involves the secondary rearrangement of the B cell receptor in the periphery, as yet no purpose for this process has been revealed. Experiments are described which investigate a potential contribution of receptor revision to affinity maturation. A pure population of transgenic B cells are adoptively transferred into chimaeric hosts and the affinity maturation of these cells is monitored in isolation. The development of the transfer system is documented and results presented which show no evidence for a role for receptor revision in affinity maturation.