Background: Hepatitis C virus infection affects more than 170 million people worldwide and is frequently associated with chronic liver disease and hepatocellular carcinoma. No protective vaccine is yet available and the current standard of care, consisting of pegylated interferon alpha and ribavirin, has limited efficacy. Ribavirin is a key component of any effective anti-HCV regimen. However, accumulation of ribavirin in the red cell compartment not only reduces drug efficacy as a result of diversion to extra-hepatic sites but also produces haemolytic anaemia which can lead to dose reduction or discontinuation of treatment. Lipid or polymer based nanoparticles can be used to deliver therapeutic agents, such as drugs or small interfering RNAs (siRNAs) directly to their site of action. We therefore elected to develop new antiviral strategies based on the targeted delivery of ribavirin to hepatocytes, coupled with the identification of new therapeutic targets. In order to inform the rational use of direct intracellular delivery of ribavirin, we enquired whether variation in expression of the ribavirin transporter may determine drug uptake and permit the identification of individuals who would benefit from these alternative approaches to treatment. Aims: The aims of this study were to: • identify host proteins involved in virus replication • demonstrate reduction of viral replication by modulation of host gene expression • develop and test a nanoparticle based system for the delivery of therapeutic molecules, including siRNAs either alone or in combination with ribavirin. • assess the relationship between ribavirin uptake by primary human hepatocytes and expression of ribavirin receptors Methods: A subgenomic HCV replicon system was established to study the virus-host relationship and identify host proteins supporting viral replication by using stealth siRNA. Viral RNAs were in vitro transcribed and transfected into Huh7 cells and expression assessed using engineered GFP as a reporter gene. siRNAs were co-transfected with viral RNAs using a nucleofector. Modulation of host gene expression was measured by both quantitative RT-PCR and protein blotting. Liposomal nanoparticles containing ApoB-100 duplexes were supplied by Lipoxen. Primary human hepatocytes were isolated by a modified two step collagenase perfusion method and cultured on collagen coated plates. HPLC and real time PCR conditions were used to measure and correlate drug uptake and receptor expression respectively. Equilibrative nucleoside transporter (ENT1) gene was analysed by direct sequencing. Results: A JFH1 (HCV genotype 2a) virus based subgenomic replicon system was successfully established. Using this model system, host proteins VAP-A and STAT3 were shown to positively regulate virus replication while ACTN1 had no effect. Liposomes failed to deliver either siRNA targeted at apoB-100 or ribavirin and this was found to be due to structural instability of the delivery vehicle. In contrast, fluorescently labelled liposomes were stable and could be taken up by human hepatocyte cell lines under optimised conditions. A protocol capable of efficient isolation and culture of hepatocytes from human donor was validated. Data from primary human hepatocytes show that ENT1 expression was highly variable in different sets of primary livers and correlated strongly with ribavirin uptake. Strikingly, Huh7 cells did not take up ribavirin despite expressing wild type ENT1. It was also found that interferon alpha does not modulate ENT1 expression and therefore ribavirin uptake, suggesting it to be a highly unlikely mode of synergism between the two drugs. Conclusion: Modulation of host proteins VAP-A and STAT3 inhibited viral replication, confirming that host genes can be used as a potential target to inhibit viral replication. Liposomes used in this study were, however, found to be ineffective vehicles for the delivery of ribavirin or siRNA, as the majority of drug leaked before cellular uptake. Polymer based nanoparticles are currently being assessed for antiviral drug delivery. Variation in ENT1 expression may account for differences in response rate in patients receiving anti-HCV therapy. Results in the Huh 7 cell line suggest that, while ENT1 is necessary, other factors are also required to mediate ribavirin uptake.

Pneumonia is a common diagnosis in general practice and is associated with significant morbidity and mortality. Current estimates of pneumonia incidence in the UK are based on studies more than a decade ago and little is known about longer term outcomes in pneumonia patients. Though much is known about the aetiology of pneumonia and predictors of mortality, an emerging area for research is the relationship between commonly prescribed drugs in general practice and pneumonia. The aims of this thesis were first, to determine overall incidence and mortality for pneumonia and how these vary by socio-demographic characteristics like age, sex, deprivation; and second, to investigate whether statins, angiotensin converting enzyme inhibitors (ACEIs) and gastric acid suppressants like proton pump inhibitors (PPIs) and histamine 2 receptor antagonists (H2RAs) modify the risk of acquiring pneumonia and its prognosis. This study used data from The Health Improvement Network (THIN) database, a longitudinal database of anonymised computerised medical patient records from 330 United Kingdom (UK) general practices at the time of data extraction in 2006. A cohort design was used to determine pneumonia incidence and mortality in the UK. Case-control, case-series and cohort study designs were used to investigate associations between the various drug exposures and pneumonia. The overall incidence of pneumonia was 237 per 100,000 person-years (95 % confidence interval (CI): 235 to 239) and this rate was stable between 1991 and 2003. Pneumonia was more common in men and in children under the age of four years and adults over the age of 65 years. There was an increased incidence of pneumonia with higher levels of socioeconomic disadvantage. Pneumonia cases showed much higher all-cause mortality as compared to the general population, both in the short and long-term and this increase was independent of underlying comorbidity. After adjusting for potential confounders, current prescriptions for statins and ACE inhibitors were associated with a significant reduction in the risk of acquiring pneumonia. Current prescriptions for PPIs were associated with an increased risk of pneumonia. With regards the impact on mortality: the use of statins was associated with a lower risk of short and long-term mortality following pneumonia whereas the use of ACEIs was associated with a decreased mortality risk only in the short-term. No relationship was observed between prescriptions for PPIs, H2RAs and pneumonia mortality. This study shows that caution must be exercised while prescribing proton pump inhibitors especially in patients known to be at high risk of pneumonia. There is also a potential role for statins in preventing pneumonia in at-risk patients and improving pneumonia outcomes but this will necessitate clinical trials to determine adequate dose, duration and safety profiles before any prescribing policy recommendations are made.

There are many surgical implanted devices in current use and all are prone to biomaterial-related infections (BRI) associated with staphylococcal biofilm formation. BRI are usually associated with S. epidermidis or S. Aureus and are characterized by treatment failure and chronicity resulting in reoperation, removal of the implant, and loss of function or death. Staphylococcal small colony variants (SCVs) may be generated by exposure to sublethal concentrations of antibiotics or nutrient limitation which may occur in biofilms. Although the characteristics of S. aureus SCVs have been well studied, little information on SCVs of S. epidermidis and their potential role in BRI is currently available. This study was designed to investigate the biochemical and phenotypic characteristics of S. epidermidis SCVs to further identify characteristics which may contribute to their ability to cause these increasingly important infections. Exposure to two to four times the gentamicin MIC led to the emergence of stable S. epidermidis SCVs, and the ability to produce SCVs was strain dependent. These variants were isogenic by PFGE and less immunogenic by western blotting, and SDS-PAGE analysis of whole cell preparations and cell wall fractions showed altered protein profiles when compared to wild type strains. S epidermidis SCVs were resistant to aminoglycosides such as amikacin and/or netilmicin and they were thiamine and/or menadione auxotrophs. Chemiluminescence assays showed a decreased ATP content reflecting the deficiency in electron transport systems which results in a growth rate – all characteristics similar to those of S. aureus SCVs. Analysis of virulence factor production indicated that S. epidermidis SCVs showed increased lipolytic and proteolytic activity when compared to those of S. aureus. Some S. epidermidis SCVs showed phase variation in exopolysaccharide production which enabled them to be more adherent to uncoated plastic -a property that may also be important for the later stages of development of biofilms. Invasion assays demonstrated that some S. epidermidis and S. aureus SCVs were internalised by HUVECs by a receptor-mediated mechanism which differed from that of the wild type strains. Interaction of staphylococci with HUVECs induced cytokine production but SCVs stimulated production of IL1, IL-6 and IL-8 at lower concentrations than their related wild type parents in the first 6 hours of co-incubation. SCVs were also less damaging to the HUVEC cell line after 24 hours when compared to wild type strains. This study supports the suggestion that a switch to the S. epidermidis SCV phenotype could be a mechanism exploited by the wild type strains to facilitate their survival inside the host. The chronicity and increased antibiotic resistance associated with BRI could in part, be explained by the characteristics of SCVs identified in this study. In particular the ability to survive intracellularly combined with reduced immunogenicity and resulting decreased cytokine production, may contribute to persistence of infection. Although SCVs are resistant to some antibiotics, surviving intracellularly may further protect staphylococci from other drugs which are unable to enter mammalian cells. Resistance may be further enhanced for some strains in biofilms where enhanced polysaccharide production may also limit antibiotic access.

AIMS: The aim of the current project was to determine if lycopene could modulate T-cell function and activity. METHODS: 1) Peripheral blood mononuclear cells (PBMC) were isolated from healthy adults and incubated with lycopene-enriched liposomes (0.0-1.2 μg/ml) then activated with or without the mitogens Con A, anti-CD3 and LPS and cultured for various lengths of time before measuring CD3, CD4, CD8, CD69, CD11a and CD25 expression.  the production of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, TGF-β and TNF-α were also measured along with the percentage of FoxP3 and IL-10 positive T-regulatory cells.  2) <i>Ex-vivo</i> expression of CD11a, CD49d, CD54, CD3 and CD69, as well as Con A stimulated proliferation were measured in subjects before and after a 12 week tomato-food and lycopene intervention study. RESULTS: Lycopene reduced PBMC proliferation and CD69 expression irrespective of the type of T-cell considered.  Lycopene reduced IL-2, IL-10, IL-17 and IFN-γ but increased IL-1β and TNF-α production.  lycopene also reduced the number of CD4⁺/CD25⁺ cells present as well as those positive for IL-10. Other parameters were not affected. CONCLUSION: Lycopene reduces T-cell activity by mechanisms dependent on early cell activation but this modulation is not specific to T-cell types.  the data support a potential beneficial effect of lycopene in the reduction of atherosclerosis through the modulation of T-cell function.

Fish diseases endanger the aquaculture industry worldwide. Approaches to combat fish diseases are hampered by the relative poor knowledge of immune relevant genes in fish. This thesis describes the cloning and expression of several immune relevant genes in rainbow trout (Oncorhynchus mykiss). The full-length cDNA and genomic DNA of the inducible nitric oxide synthase (iNOS) gene have been cloned and sequenced. The gene structure was the first to be determined outwith the mammals. The expression of the iNOS gene is induced by virus (VHSV) infection, but its detection by RT-PCR might be impaired by the secondary structure formed in the iNOS mRNA. Screening of a genomic library resulted in the isolation of a second allele of IL-1b1 gene with a large intron III. This allele appears to have resulted from a recent retroposition of a HpaI SINE into intron III. Two other retroposition events have also been recognised in the same intron. The survival mechanism of this SINE is discussed and a model of trout IL-1b gene formation proposed. Both alleles of the IL-1b1 gene are constitutively expressed and induced by LPS and recombinant trout IL-1b1 in heterozygous RTS-11 cells. The promoter of the IL-1b1 allele S gene was isolated and the transcription start site was determined. A series of promoter-reportor gene constructs were transfected into RTG cells to study their activities under different conditions. The first intron is an important part of the promoter of IL-1b1 allele S and NFkB is a transcription factor required for IL-1b1 expression. The cytokine receptor common gamma chain (gC) was also isolated from rainbow trout, the first outwith mammals. Its expression can be detected in blood, gill, kidney, brain and liver, and in macrophage cultures. The expression of gC in macrophage cell cultures is upregulated by recombinant IL-1b1 and LPS stimulation. The expression of gC is also detectable in RTG-2 cells with an increased transcript level after stimulation with recombinant IL-1b1.

Monoamine oxidase (MAO) is an oxidative enzyme that deaminates a variety of amine substrates, including the neurotransmitter dopamine. The enzymatic reaction requires molecular oxygen and produces hydrogen peroxide as a by-product. MAO is localised in the outer mitochondrial membrane and exists as two isoforms, MAO-A and MAO-B, which are differentially expressed in the body and differ in their substrate and inhibitor specificities. Previous studies have suggested that MAO-generated reactive oxygen species (ROS) contribute to oxidative stress in the cell and can directly inhibit electron transport, cause damage to mitochondrial DNA and enhance cell death signalling. In this study the role of MAO in cell death was investigated in dopaminergic neuroblastoma (SH-SY5Y) cells, in three diverse models of mitochondrially-mediated apoptosis. The relevance of MAO in cell death signalling was confirmed with the use of two unrelated MAO inhibitors and the creation of stable SH-SY5Y cell lines that either over express MAO-A or have reduced levels of MAO-A. The study is the first to over express MAO-A using recombinant technology and to use miRNA to stably knock-down MAO-A expression in human neuronal cells. Results confirm that MAO-A is involved in modulating cell death but the mechanism and extent of the involvement depends on the apoptotic inducer. In classical apoptosis induced by staurosporine (STS), cells undergo rapid morphological and biochemical changes indicative of mitochondrially-mediated apoptosis, which is partly dependent on ROS production by MAO-A and induction of mitogen-activated protein kinase (MAPK) signalling cascades. MAO-A protein and catalytic activity are increased in this model, however the mechanism by which this occurs is unknown and is not a result of increased gene transcription. In death induced by growth factor withdrawal, the MAO-A gene is up regulated via p38 and JNK MAPK pathways, which occurs downstream of caspase activation. In both the STS and growth factor withdrawal models, MAO inhibition reduced apoptosis. Most significantly reduced levels of MAO-A expression in 'knock down' cells protected against cell death induced by the complex I inhibitor rotenone, suggesting that MAO has an important role in mitochondrial function. Over expression of MAO-A resulted in stress and apoptosis, followed by a period of cellular senescence and eventually death by necrosis. These data compliment the effects of chronic exposure to oxidative stress in ageing and neurodegeneration. For the first time this work has shown that the MAO-A isoform is an important regulator of STS-induced apoptosis, that MAO-A gene expression is regulated by JNK signalling, and that MAO-A is significantly involved in mitochondrial dysfunction induced by complex I inhibition. These data raise important questions regarding predisposition to the development of neurodegenerative diseases such as Parkinson's disease and to approaches used for their treatment.

A recent study showed that a polymorphism ~35kb upstream of the HLA-C gene (-35 SNP) correlates with host control of HIV-1 in Caucasians, with the minor allele (C) associating with significantly lower set point viral loads than the major allele (T). A link between viral load and HLA-C is suggested by linkage of the two SNP alleles with different HLA-C alleles and by the fact that HLA-C, in contrast to HLA-A and -B, is not down-regulated by HIV-1 Nef protein. In addition, the -35C variant has been shown to associate with higher HLA-C messenger RNA expression in EBV-transformed B cell lines. We initially propose that increased surface expression of HLA-C in subjects with the protective SNP leads to increased breadth and magnitude of HLA-C restricted T cell responses, explaining the decrease in viral load in these subjects. This study initially investigates whether the -35 SNP correlates with the surface level of HLA-C using the monoclonal antibodies DT9, which recognises both HLA-C and HLA-E, and 3D12, which is specific for HLA-E. The lymphocytes from -35 CC subjects expressed a significantly higher level of surface HLA-C when compared to those from -35 TT subjects, but this difference in HLA-C expression can be attributed primarily to the very low expression of a single allelic product, HLA-Cw*07. Increased surface HLA-C should translate to functional differences between CC and TT subjects. This study confirmed that HLA-C restricted CD8 T cell responses against HIV-1 do exist, even for HLA-Cw*07, but represent a minority of total T cell responses. They were detected in all -35 SNP genotypes but there were no functional differences, making it unlikely that the protective effect of this SNP on viral load set point could be accounted for solely by HLA-C restricted T cell responses. Finally, a viral suppression assay was used to investigate the capacity of CD8 T cells to suppress HIV-1 replication in Caucasian and African subjects. We provide evidence that the -35 SNP effect on viral load is indeed T cell mediated. However, we suggest that the protective effect of the -35 SNP on viral load set point manifests as a result of linkage disequilibrium of this polymorphism with both favourable and unfavourable HLA-B and HLA-C alleles.

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