Although the interleukin (IL)-6 related cytokine Oncostatin M (OSM) affects a variety of inflammatory events associated with disease progression, the function of OSM in the face of an inflammatory challenge remains unclear. In this thesis a peritoneal model of inflammation, in association with in vitro studies using human primary cell lines, has been used to define the influence of OSM on chemokine-mediated leukocyte recruitment. When compared to wild type mice (WT) the induction of peritoneal inflammation in Oncostatin M receptor-beta deficient mice (OSMR-KO) resulted in enhanced monocytic cell trafficking, with no differences in neutrophil or lymphocyte recruitment observed, suggesting that OSM control of leukocyte recruitment is functionally distinct from that of IL-6. Subsequent in vitro studies and an in vivo appraisal of inflammatory chemokine expression following peritoneal inflammation inferred that OSM regulation of CCL5 might account for the observed difference in monocytic cell trafficking. The OSM-mediated control of CCL5 is clearly distinct from the actions of IL-6, which acts as a more prominent in vivo regulator of CCL2 expression than OSM. Mechanistically, these studies inferred a hitherto unidentified interplay between OSM-mediated STAT signalling and NF-kappaB activation. In this respect, EMSA analysis of nuclear extracts from peritoneal membranes isolated during course of the inflammatory response showed that OSMR-KO mice display an enhanced profile of NF-kappaB activation as compared to WT mice. Initial in vivo appraisal of the role of OSMRg-mediated signalling in repeated episodes of inflammation and associated tissue damage suggest that OSM continues to regulate monocytic cell trafficking throughout recurrent inflammatory episodes and does not play a significant role in inflammation-associated peritoneal tissue damage, again a finding clearly distinct from the observed effects of IL-6 in tissue injury. These findings suggest that activation of gp 130 by IL-6 and OSM trigger distinct inflammatory responses to affect individual aspects of leukocyte trafficking.

Environmental biofilms are abundant and represent the most prevalent growth mode of microorganisms. In humans, biofilms are frequently encountered and responsible for numerous infections which can be difficult to treat since biofilms tend to be more resistant to antimicrobial agents and host immune defences. The focus of this study was to characterise two biofilms associated with specific human infections, namely oral candidosis and chronic wound infection. In vitro biofilms using microorganisms from these infections were generated using microtitre plates, a constant depth film fermenter (CDFF) and reconstituted human epithelial tissue (RHE). Confocal laser scanning microscopy of Candida biofilms on RHE showed strain-dependent tissue invasion, with differences also evident in surface colonisation and Candida morphology. Hyphal elements invaded epithelial cells and exhibited budding within the tissues. A relationship between high Candidal tissue invasion and consistent expression of secreted aspartyl proteinase (SAP) genes 4-6 was found. There was no correlation between level of invasion and expression of phospholipase and agglutinin-like sequence (ALS) genes. These results demonstrate strain variation by Candida which relate to pathogenic potential. Biofilms of the chronic wound bacteria, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus oralis and Micrococcus luteus, showed species variation occurred in the extent of biofilm formation with antagonism evident in mixed species biofilms. Importantly, the pathogenic species of P. aeruginosa and S. aureus often inhibited biofilm formation by the commensal species (M. luteus and S. oralis). Susceptibility of wound bacterial biofilms to povidone iodine was reduced compared with planktonic cells. Up-regulation of biofilm genes by P. aeruginosa (alginate genes) and S. aureus (ica genes) was evident in the biofilm models. Extrapolation of results to chronic wound environments does require additional research on wound tissue, but the ability of the pathogenic species to antagonise commensal species could explain biofilm succession within a wound, possibly promoting a chronic wound phenotype.

Human parainfluenza viruses (PIV) 1, 2, 3 and 4 (A and B) cause approximately 39-40% of all acute respiratory infections in infants and children for which there is no effective therapy. Firstly, this thesis was aimed at ascertaining a suitable guinea pig model of PIV-3 infection by determining the most efficient route of virus application. Secondly, to ascertain if a temporal association may be established during the time course of infection (Day 1-40) by measuring the following parameters: body weight, rectal temperature, airway function (sGaw), airways reactivity to inhaled histamine, inflammatory cell infiltration to the lungs measured by bronchoalveolar lavage, wet lung weights, inflammatory markers (nitric oxide and total protein levels), lung histological analysis and recovery of the virus from bronchoalveolar lavage fluid and lung tissue. Further to this, experiments were also designed to determine the impact of the glucocorticoid, dexsamethasone (in vivo and in vitro) and the phosphodiesterase inhibitor, rolipram (in vivo). Lastly, this study ascertained the effect of pre-sensitisation with antigen on the responses of antigen in PIV-3 infected guinea pigs. These investigations have shown guinea pigs to be a suitable model for PIV-3 infection and intranasal inoculation is the most efficient route of virus application. In addition, these investigations also established a temporal association between the time course of PIV-3 infection including airway reactivity to histamine, airway function (sGBW), airways reactivity to inhaled histamine, inflammatory cell infiltration, wet lung weights, inflammatory markers (nitric oxide and total protein levels), pulmonary histopathology and recovery of the virus from bronchoalveolar lavage fluid and lung tissue. Pre-treatment of PIV-3 infected guinea pigs with the glucocorticoid, dexamethasone and the phosphodiesterase inhibitor, rolipram (in vitro) ameliorated the inflammatory response and airway hyperreactivity. Unlike rolipram, dexamethasone also reduced viral titre (in vivo and in vitro), supporting a role for the anti-viral effects of dexamethasone. The anti-inflammatory effects of dexamethasone are well known and it has been speculated by other authors which maybe caused by decreased viral receptors on the epithelial cells via inhibition of intracellular adhesion molecule-1 expression. Finally, in this study PTV-3 infection, enhanced the effect of pre-allergen sensitisation, which may arise from increased permeability of the airway mucosa to allergens, due to damage of the respiratory epithelium and increased recruitment of dendritic cells. In summary, this thesis has established a clearly defined efficacy of dexamethasone use in the treatment of PIV-3 infection.

No description available.

Over the last two decades, research into the imaging of inflammation has been thoroughly investigated as it presents a promising therapeutic target for many diseases, including neurodegenerative and cardiovascular diseases. There are only a few radiopharmaceuticals which are currently in general use for imaging inflammation, e.g. 18F-FDG, nuclear labelled autologous white blood cells, 67Ga-citrate etc. However, none is specific for inflammation thus there is a need to investigate novel imaging agents for distinguishing sterile inflammation from infection. Formyl peptide receptors (FPRs) were originally identified as chemoattractant receptors, which have been shown to be implicated with potential therapeutic benefits for inflammatory diseases. Highly specific hexapeptides, cFLFLFK (cinnamoyl-Phe-D-Leu-Phe-D-Leu-Phe-Lys-NH2) and WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH2), which are selective antagonist and agonist for FPR1 and FPR2 respectively, have been modified by conjugating to a cyclen based chelate for metal ions, allowing for specific imaging of inflammation using non-invasive imaging techniques such as MRI, PET and SPECT. This thesis depicts the synthesis of a bifunctional chelate, DOTAGA containing five acetate arms. Ln.DOTAGA-conjugates were prepared by coupling the Ln(III) complexes of DOTAGA onto the desired peptides without a protection step as the four pendant acetate arms were involved in coordination. Their binding affinities and biological functions were assessed by various in vitro assays such as radioligand binding assays, chemotaxis and TNF-α induced cytokine release. In vivo imaging of inflammation using the synthesised compounds and a mu-rine model of inflammation was performed. Further to this, the DOTAGA was further modified to an anhydride analogue, which allows for post radiolabelling after conjugation to the peptides. Subsequent complexation with Cu(II) and Ga(III) ions indicated their potential use in nuclear imaging. Two organic chromophores based on Cy5 bearing two orthogonal functional groups have been synthesised. Successive conjugation with cFLFLFK and confocal microscopy indicated their potential use in optical imaging. Multimodal imaging agents featuring a Cy5 chromophore and a DOTAGA for inflammation have been designed and synthesised. Subsequent complexation with Gd(III) and Tb(III) resulted in MRI/optical and dual luminescent imaging agents, respectively.

Most adults are infected by Epstein-Barr virus (EBV), which establishes persistent infection in B cells. EBV nuclear antigen 3C (EBNA3C) is a latent viral protein that co-regulates many cellular genes, but little is known about the mechanisms by which EBNA3C acts as repressor or activator of gene expression. This study aimed to further explore molecular mechanisms that underlie EBNA3C host gene regulation. For this, four EBNA3C target genes - three repressed (COBLL1, ADAM28 and ADAMDEC1) and one induced (AICDA) - were selected based on a previous exon microarray on EBV-recombinant lymphoblastoid cell lines (LCL) carrying conditional EBNA3C (3CHT). Using EBV recombinants to infect primary B cells, EBNA3C was shown to be the main regulator of all four genes. Surprisingly, regulation by EBNA3C over orders of magnitude followed highly exponential activation or repression profiles and required ~30 days after infection. This could be reproduced efficiently in the 3CHT LCLs by activating EBNA3C, proving the utility of these cells to replicate EBNA3C gene regulation. Analysis of chromatin immunoprecipitation (ChIP) coupled to deep-sequencing identified EBNA3C binding to distal regulatory elements at all four genes and detailed ChIP analysis of histone modifications and cellular factors was performed. Unexpectedly, recruitment and/or stabilisation of the DNA-sequence binding factor RBPJ was observed at the EBNA3C binding sites only when EBNA3C was functional. This challenges existing models of how RBPJ functions in EBV-regulated gene expression. EBNA3C failed to regulate all four genes when it is unable to bind to RBPJ. Recruitment of a polycomb protein, generally linked to gene repression, was observed at all four genes irrespective of whether repression or activation by EBNA3C occurred. Changes to histone modifications at these loci correlated well with gene expression and indicate a two-step mechanism for EBNA3C-mediated repression and suggest the activation is linked to a very complex pattern of chromatin looping.

No description available.

No description available.

No description available.

No description available.