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Tribbles (Trib) protein was first described in Drosophila as a regulator of proliferation, later being implicated as a G2/M modulator. In mammalian systems, three Trib gene family members have been identified, which share a conserved motif similar to the catalytic domain of serine/threonine kinases. However, they lack several conserved residues in the ATP-binding pocket and the core motif of the catalytic domain necessary for catalytic function. Tribbles 1 (Trib1) is involved in inflammation through its ability to regulate MAPK, NF-κB and the CCAAT Enhancer Binding Protein (C/EBP). Moreover, Trib1 is associated with human disease, such as atherosclerosis and acute myeloid leukaemia. In this thesis, I investigated the functional role of Trib1 in Toll-like Receptor (TLR)-induced inflammatory responses together with pro- or anti-inflammatory cytokines. The RAW264.7 myeloid cell line was stimulated with TLR2/9 ligands in the presence or absence of IFN-γ or IL-10. I observed a high level of Trib1 expression in the presence of IFN-γ and TLR2 ligands, but weak Trib1 expression following treatment with IL-10 and TLR9 ligands. In gene knock-down experiments using small interfering RNAs (siRNA) to reduce Trib1 expression, C/EBPβ was up-regulated in both stimulated (by IFN-γ and TLR2 ligands) and resting macrophage populations. TNF-α production was increased following Trib1 knockdown after treatment with IFN-γ and/or TLR2 ligands but IL-6 secretion remained unchanged. Furthermore, ERK1/2 expression was reduced in Trib1 siRNA-treated cells and failed to induce chemokinesis in macrophages. Finally, Trib1 was demonstrated to act as a modulator of cell cycle (G2/M) transition and displays a delayed apoptotic phenotype. The work in this thesis demonstrates that mammalian Trib1 contributes to the pro-inflammatory response and functions as a regulator of the ERK1/2 and C/EBPβ pathways following TLR ligand-mediated activation. Its novel functions include acting as a modulator of G2/M arrest and suppressing macrophage migration.

Diplostomum metacercariae were collected from three sources (a) the lenses of sticklebacks from Culter Compensation Dam. (b) the lenses of rainbow trout from Mill of Cantray trout farm. (c) the humours of rainbow trout from Mill of Cantray trout farm. The metacercariae from the lenses of Culter Dam sticklebacks produced infections lasting 12-15 weeks with an egg production of 6,000-10,000 eggs per day, in gulls. The metacercariae from Mill of Cantray trout however, produced short Infections of only 3-4 days with an egg production of less than 1,000 eggs per day, in the same bird hosts. All metacercariae from all sources appeared morphologically identical at the light microscope level. However, the body length and width dimensions were significantly different between all 3 types of metacercariae. Using the available taxonomic keys, all three sources of metacercariae were identified as D. spathaceum. The life cycle of D. spathaceum was established in the laboratory using metacercariae from Culter Dam sticklebacks and maintained throughout the study. Parasite egg production was recorded from herring gulls four days after infection with metacercariae from Culter Dam sticklebacks. Egg output rose to a maximum of 10,200 eggs per day after 4 weeks and then oscillated between 6,000 and 10,000 eggs per day for the. following 9 weeks, after which egg production rapidly declined. 7. Mlracidia hatched from eggs incubated at 29°C after 8-11 days. 8. At 14°C, 29% of the Initial number o£ cercarlae successfully established In the lenses of exposed rainbow trout. 9. An epidemiological survey of D. spathaceum was carried out on rainbow trout from Mill of Cantray trout farm and 3-spined sticklebacks from Culter Dam over a 30-month period. 10. The Infection period at both sites was normally between May and September each year. Transmission from snail to fish did not occur when temperatures were below 10°C. 11. A low snail prevalence of patent infections in L. pereger (0-8%) was recorded at both sites throughout the survey. 12. During the summer of 1982 rainbow trout in Raceway 3 at Mill of Cantray became infected with 139 (humour) and 70 (lens) metacercariae. In April, 1983 the raceway was cleaned and the entire length treated with copper sulphate. This resulted in a 60% reduction in the numbers of metacercariae infecting trout during the following summer. 13. The prevalence of D. spathaceum metacercariae declined from 100% to less than 20% and the abundance of metacercariae per fish declined from 12 to less than 2 during the survey period. Despite this decline a pattern of seasonal variation in prevalence and abundance was observed in both 1982 and 1983. 14. Significant correlation coefficients between abundance of metacercariae per fish and length and weight of sticklebacks Indicated that abundance Increases with size. 15. Within the confines of a flume In which temperature, flow rate, and cercarlal concentration could be manipulated Independently, It was shown that (a) It is possible to control the Infection rate of fingerllng rainbow trout by the manipulation of flow rate. (b) the relationship between mean abundance of metacercariae per fish and cercarlal concentration is linear, with a regression coefficient of 0.99 (c) that no metacercariae are found in rainbow trout infected and maintained below 10°C. (d) that it is migration of cercariae which is inhibited at low temperature and not penetration or attachment. 16. Rainbow and brown trout do not produce circulating antibody at detectable titres in response to infection with D. spathaceum cercariae. 17. A significant difference occurred in the rate of infection of rainbow trout given weekly infections of D. spathaceum cercariae in winter and summer. 18. Rainbow trout injected with a suspension of dead cercariae acquired significantly fewer metacercariae when exposed to a challenge infection.

Recognition of sugar moieties on the surface of microorganisms is one of the ways the body distinguishes potential pathogens from self-cells. The sugarbinding proteins, lectins, mediate this recognition role of the first line of defence against infections, preceding the antibody-mediated (adaptive) immune response. Collectins are calcium-dependent carbohydrate-binding proteins that have been implicated in innate immunity. Bovine conglutinin (BC) and lung surfactant protein-D (SP-D), belong to the family of 'collectins' which are characterised by four domains: an N-terminal cysteine-rich region, a collagenlike region linked with the carbohydrate recognition domain (CRD) via an ahelical neck region. BC and SP-D show remarkable similarity in their amino acid sequence (79% identity), function and biological characteristics. They have been shown to mediate microbial clearance either by directly binding to bacteria leading to phagocytosis or interacting with complement system components. The present study aims to elucidate the biological function of these proteins more precisely. Recombinant fragments (r) of BC and SP-D consisting of their CRDs and neck regions have been cloned in pET-21a and pMal-c2 vectors respectively, for expression in Escherichia coli. Recombinant conglutinin was expressed in BL21(DE3)pLysS and isolated by a denaturation-renaturing procedure. Binding of rBC(N/CRD) to mannan and complement component, iC3b, was assessed in real-time by BIAcore. The dissociation constants were calculated by Scatchard analysis. The carbohydrate structures present on the surface of the microorganisms play an important role in mediating the interactions with the immune cells. The recombinant molecules showed calcium-dependent binding to lipopolysaccharides (LPS) from gram-negative bacteria Pseudomonas aeruginosa, Klebsiella pnuemonia and Salmonella typhosa, which was inhibited in presence of sugars. rBC(N/CRD) also bound to whole bacteria as assessed by ELISA and retained its capacity to recognise various complement system components and the carbohydrate moieties on the surface of various pathogenic microorganisms. The recombinant protein retained its ability to bind various sugar residues, although with lower affinity than that of the native molecule. rBC(N/CRD) is able to bind and aggregate bacteria and cause agglutination of bacterial cell suspensions. A novel model has been used to describe the interactions of the collectins at the molecular level based on specificity of carbohydrate-recognition by the collectins. The pyocin mutant strain 1291 series of Neisseria gonorrhoeae has sequential deletions of the terminal sugars in their lipooligosaccharides (LOS). Conglutinin showed a preferential high affinity binding to 1291a mutant that expresses GlcNAc as the terminal hexose, in comparison to other mutants. This provides a unique system to understand the specific cell-surface interactions in relevance to a particular lectin. Further elucidation of the function of CRD and neck region at a structural level is in progress, using X-ray crystallography. Since the submission of the thesis, the structure of the monomeric CRD has been solved, which revealed a remarkable similarity to the SP-D and MBL structure. Trials are underway to get the structure of the trimeric CRDs. These studies aim to provide a better understanding of the collectinpathogen interaction at the biological and structural levels. The ultimate aim is to determine if the recombinant forms of these proteins can be used therapeutically to enhance the uptake and killing of pathogens.

The properties of the cell surface of Aeromonas salmonicida were studied, with particular emphasis on the additional surface layer (A-layer), found on virulent strains. This was identified by electron microscopy, as having a tetragonal subunit morphology; and by + ejectrophoresis of membrane components as a 51 kdal protein on A strains. A and A- strains, (the latter isolated by growth at elevated temperature), were compared biochemically and their interactions with various cell types investigated. Strains of A. salmonicida possessing the A-layer were shown to be more hydrophobic than those devoid of this protein. The influence of culture age, medium composition and subculture on hydrophobicity were investigated and hydrophobicity related to culture characteristics. No difference in enzyme susceptibility between A+ and A A. salmonicida was found and both phenotypes showed similar tolerance to other environmental conditions. The interactions between A. salmonicida and cells in vitro were studied using adhesion and association assays involving rädiolabelled bacteria, viable count determinations, haemagglutination and chemiluminescence. A+ A. salmonicida were found to adhere to a greater extent than A bacteria to tissue culture cell lines and to isolated fish tissue by non-specific hydrophobic interactions. Adhesion was maximal to a fish epithelial cell line and the effects of various environmental conditions on adhesion were determined. A- bacteria more commonly exhibited haemagglutination which was inhibited by specific sugars. A+ bacteria associated more than A- organisms with mouse peritoneal macrophages and rainbow trout phagocytes when in salts solutions. The effects of opsonization on A. salmonicida were strain dependent. Incubation in a variety of sera resulted in a decrease in A+ surface hydrophobicity, often accompanied by abolition of characteristic autoagglutinability, whereas opsonized A cells became more hydrophobic. These properties directly influenced the chemiluminescence response of trout macrophages.

Interleukin-8 (IL-8), a member of the C-X-C chemokine subfamily, is an important chemoattractant and cellular activator. This study was conducted to determine the role of IL-8 in the immunopathogenesis of HIV-I disease and tuberculosis. The first section involved determining the effect of infection with HIV-1, Mycobacterium tuberculosis and co-infection with both of these organisms on IL-8 j_ roduction in vivo. This was monitored by the determination of levels of serum or plasma EL-8 and peripheral cell-associated IL-8, assessing peripheral mononuclear (PBMC) and polymorphonuclear (PMN) cell capacity to produce IL-8 spontaneously or in response to various stimuli, and the detection of constitutive IL-8 mKNA expression in purified subsets of mononuclear cells. Results show that whereas there is evidence of detectable levels of cell-associated EL-8 (mKNA and protein) in peripheral cells of healthy individuals, this is largely lost in the disease states studied. Coupled with this was significantly increased circulating levels of EL-8 in serum and plasma found in HIV-1 infected individuals with or without concomitant pulmonary TB. On the other hand, the capacity of PBMC to produce IL-8 spontaneously ex vivo was enhanced in HIV-1 and TB patients and many of the HFV/TB group, but their corresponding capacities to respond to various stimuli was significantly diminished when compared to that of the normal donors. The release of IL-8 from PMN in the presence of an agonist was diminished mainly in individuals with pulmonary TB, which was further exacerbated by the presence of HIV-1 infection. HIV-1-infected individuals have an increased incidence of bacterial infections which could be related to defective functioning of PMN. The second section was aimed at detecting PMN abnormalities in HIV and I-HV/TB patients by monitoring EL-8-induced p-glucuronidase release and PMN chemotaxis in response to IL-8. IL-8-induced (I-glucuronidase release from PMN of normal individuals and TB patients occurred in a dose-dependent manner. In contrast, PMN from HTV-1 infected individuals, whether co-infected with M tuberculosis or not, showed a reciprocal response in that increasing IL-8 concentrations resulted in decreased enzyme release. This reciprocal slope of the IL-8 dose-response curve was altered for the majority of HIV-1 positive individuals tested irrespective of their CD4+ cell counts. In addition, PMN chemotaxis in response to IL-8 was also found to be significantly impaired in a group of HIV-1 infected patients coinfected w ithM tuberculosis when compared to healthy individuals. The third section of the study involved analysing the expression of the PMN cell surface markers, FcyRIII (CD 16), and the two human IL-8 receptors, designated IL -8RA and 1L-8RB. FcyRIII (CD 16) expression on the surface of PMN was significantly reduced in HIV-1 seropositive patients with pulmonary tuberculosis when compared to those individuals with either disease alone or healthy blood donors. A significant reduction in the percentage of PMN expressing IL-8RA and IL-8RB and in their respective fluorescence intensities was found in TB, HIV, and HTV/TB groups when compared to that obtained for the ND group. IL-8RA intensity of fluorescence was significantly decreased in the HTV/TB group when compared to the TB and HIV groups indicating a further down-regulation of IL-8RA expression owing to dual infection. On the other hand, IL-8RB fluorescence intensity was substantially reduced on PMN from patients with pulmonary TB and to a greater degree in those patients co-infected with HIV-1 and M. tuberculosis. Having found a reduction in the expression of both IL-8 receptors on PMN in all the infection groups, cellular events following the binding of IL-8 to IL-8 receptors on PMN isolated from dually infected patients, the group which showed the greatest reduction in IL-8 expression was analysed. Results indicated that the impairment of DL-8-dependent PMN functions such as degranulation and chemotaxis was associated with the reduced expression of IL-8 receptors on these cells. Increased circulating levels of IL-8 in HIV-1 infection and a diminished cellular capacity to produce IL-8 as shown in this study may have important implications for antimicrobial defences and normal immune processes. A dysregulated production of IL-8 in vivo is likely to play a role in the pathogenesis of HIV-1 disease, pulmonary tuberculosis, and dual infections with both organisms. In addition, cellular responses dependent on specific receptor engagement and the subsequent translation of signal transducing events that lead to phagocyte effector functions are clearly impaired in IL-8 receptor deficient phagocytes. Abnormal PMN functioning in HTV-1 infected individuals, as shown here by defective degranulation and chemotactic responses, have important implications in the pathogenesis of HIV-1 infection in terms of their ability to clear secondary microbial infections. Future attempts should be aimed at defining the mechanisms that bring about these changes in order to contribute to a greater understanding of the mechanisms that lead to an enhanced risk of superinfections in immunosuppressed individuals.