No description available.

Before embarking on the work presented here I showed that certain activating signals, such as IFN-gamma and TNF-alpha programmed macrophages to develop distinct sets of properties in vitro, which included unresponsiveness to other types of activation. This raised the question whether macrophage programming occurs in passive and active renal inflammation and whether the macrophage programme could be biased by systemic administration of cytokines. The data presented here shows that macrophages infiltrating acutely inflamed glomeruli of rats with nephrotoxic nephritis display programmed behaviour: operationally they behave as though programmed by IFN-gamma, and maintain these characteristics despite systematic administration of anti-inflammatory cytokines such as IL-4 or TGF-beta. This triggered further studies using a model of mesangioproliferative nephritis that can be adapted to induce resolving or progressive glomerular injury. These show that glomerular localisation does not always induce macrophage programming and that whether macrophages become programmed or not depends on the nature of the injury. Furthermore the data shows that macrophages become committed to a particular programme shortly after entering a programming environment. These observations raise question about the factors that induce macrophage programming at early stages of inflammatory disease and its consequences for its outcome. It provides an important mechanistic insight into how macrophage functional development is influenced by the underlying disease process.

No description available.

Ischaemia-reperfusion (IR) injury contributes to tissue damage that occurs in myocardial infarction and stroke, and limits the outcome of current reperfusion strategies. Ischaemic preconditioning (IPC) is an innate mechanism that protects tissues from injury during ischaemia and subsequent reperfusion. IPC has systemic effects that protect tissues remote for those undergoing preconditioning (remote IPC RIPC). RIPC might enable the clinical utility of ischaemic preconditioning to be tested, as protection of vital tissues against IR injury could be achieved by remotely preconditioning a non-vital tissue. This thesis sought to determine the optimal conditions for inducing RIPC in humans. The protective effects of RIPC were investigated in healthy volunteers, using an in vivo model of IR injury to the vascular endothelium of the brachial artery. RIPC of the limb protected against IR-induced endothelial dysfunction and the degree of protection depended on the characteristics of the preconditioning stimulus. Two phases of protection by RIPC were evident an early relatively short phase, active immediately, and a second window of protection, which is more prolonged lasting for up to 48 hours following the application of the RIPC stimulus. Repeated application of the RIPC stimulus caused tissue protection for up to 7 days. RIPC was shown to be dependent on intact autonomic function and the opening of ATP-sensitive potassium channels. The protective effects of RIPC were investigated in paediatric patients undergoing renal transplantation. Data from this clinical study are consistent with a beneficial effect of RIPC against IR injury to the renal graft and suggest that RIPC may have therapeutic potential in clinical IR syndromes.

Major injury leads to host immune dysregulation and increased susceptibility to infectious challenge. Toll-like receptors (TLRs) are archetypical pattern-recognition receptors that, in addition to a role in mediating innate immune responses to components of Gram-positive and Gram-negative pathogens, have been implicated in the recognition of endogenous mediators, released during host tissue injury. A murine model of thermal injury was employed to examine the impact of injury on TLR-mediated immune cell responses. Lymph node and spleen cell suspensions were prepared from wild type, TLR4-/- and IL-1RI-/- mice at 24 hours or 7 days after injury/sham injury, cultured for 48 hours with lipid A (LA), lipopolysaccharide (LPS), lipoteichoic acid (LTA) or peptidoglycan (PGN) and production of IL-1<span style='font-family:Symbol'>b, IL-6, IL-10 and TNF<span style='font-family:Symbol'>a measured by ELISA.  Cell subset localisation of cytokine production was assessed by intracellular cytokine detection and immunomagnetic bead T-cell depletion.  TLR4/MD2 cell surface expression was measured by flow cytometry and TLR gene induction by Real Time RT-PCR. Injury caused augmented wild type splenocyte production of IL-1<span style='font-family:Symbol'>b and TNF<span style='font-family: Symbol'>a at 24 hours and of IL-1<span style='font-family:Symbol'>b and TNF<span style='font-family:Symbol'>a at 7 days in response to all stimuli. Cytokine production was localised to macrophages and dendritic cells and the injury-augmented reactivity was independent of T-cells. Responses to LA, LPS and LTA required TLR4 whereas PGN responses were TLR4-independent.  TLR4 was not required for the <i>in vivo</i> establishment of injury-augmented proinflammatory responses. Injury did not substantially change TLR gene expression assessed by Real Time RT-PCR or TLR4/MD2 cell surface expression. IL-1 signalling was not essential for the injury-augmented proinflammatory response but was required for injury-augmented production of IL-6 and IL-10 and therefore may be important for the development of anti-inflammatory responses.

The research presented in this thesis focuses on the molecular design of an oxidation-sensitive nanocarrier and its enzyme conjugate with a view of their application in the field of biomaterials. I have polarised our attention on a specific class of polymers, the polysulfides, for their environmental responsiveness (towards oxidising substances, a condition often associated with inflammatory reactions), interesting physico-chemical properties, ease of the preparation and multiple possibilities for further modifications and bioconjugations, which are perfectly suitable for the development as systems for drug delivery applications. In this work we firstly have focused on the synthesis of amphiphilic poly(propylene sulfide)-poly(ethylene glycol) (PPS-PEG) block copolymers by employing vinyl sulfone as the functional group to link the blocks and modify the end of the PEG. This study was followed by an investigation of the macromolecular interchange and payload exchange of the formed polymeric micelles to understand the 'co-formulation' events, employing fluorophores (dansyl groups) and quenchers (dabsyl groups) either as terminal groups in macroamphiphiles or as encapsulated hydrophobic payloads. In another part of the work, I have developed a micellar system with which simultaneously to two of the most important ROS: superoxide and hydrogen peroxide, for inflammation-responsive drug release. The system is composed of superoxide dismutase (SOD) conjugated to oxidation-sensitive amphiphilic polysulfide/PEG block copolymers; the conjugate combines the SOD reactivity towards superoxide with that of hydrophobic thioethers towards hydrogen peroxide. Specifically, here we have demonstrated how this hybrid system can efficiently convert superoxide into hydrogen peroxide, which is then 'mopped-up' by the polysulfides. This mode of operation is functionally analogous to the SOD/catalase combination, with the advantage of being based on a single and more stable system.