Our laboratory has identified an additional Rev binding site in the 5’ UTR of HIV-1 RNA. This site is referred to as Loop A and is found on Stem Loop 1 of the packaging signal in the 5’ untranslated region. We tested the effect of Rev on translation of HIV-1 RNA to dissect the roles of the RRE and Loop A. <i>In vitro</i>, Rev stimulates translation of HIV-1 RNAs at intermediate concentrations and inhibits translation non-specifically at high concentrations. Stimulation appears to be dependent on the presence of Loop A rather than the RRE. Rev does not stimulate the translation of non-HIV RNAs and does not affect the stability or splicing of target RNAs. Using a luciferase assay reporter system, we show that Rev is also able to enhance the translation of Loop A containing receptor RNAs in COS-1 cells. The effect of Rev is most striking in cellular systems and published work indicates that Rev may influence the cellular localisation of target RNAs. Accordingly, we developed a system for the detection of Rev and genomic RNA molecules in cells. COS-1 cells were transfected with a packaging-deficient proviral plasmid. Rev protein was detected via immunofluorescence. Genomic RNA was detected using a biotinylated complementary RNA probe, which was in turn detected by a fluorophore-conjugated streptavidin molecule. This system was optimised for the detection of both targets through a number of approaches. However, the predominant nuclear localisation of Rev proved to be prohibitive for the investigation of Rev-RNA interactions in the cytoplasm. We propose that during the early phase of viral gene expression, small amounts of Rev stimulate translation but as levels increase and the focus shifts to particle production, high levels of Gag and Rev inhibit translation, contributing to Rev’s tight control of the early to late shift in viral gene expression.

I have demonstrated that the net growth rate of the virulent <i>S. enterica</i> serovar Typhimurium C5 strain can be increased by the presence of the attenuated <i>aroA S. </i>Typhimurium SL3261 vaccine strain in the same tissue. The growth acceleration is dependent upon the release of the immunosuppressive cytokine IL-10. This work illustrates that IL-10 production in response to <i>S. enterica</i> requires TLR4 and occurs <i>via</i> signalling pathways involving both TRIF and MyD88 adaptor molecules. Acceleration of the growth of C5 <i>Salmonella </i>does not require simultaneous co-injection of the attenuated bacteria. This indicates that intravenous administration of an <i>S. enterica</i> vaccine strain could potentially exacerbate an established infection with wild-type bacteria. These data could also suggest that the severity of an infection with a virulent <i>S. enterica </i>strain can be increased by the prior administration of a live attenuated vaccine strain. Secondly, the bacterial distribution of <i>Salmonella</i> <i>enterica</i> within host cells was investigated to try to understand the role of host and bacterial mechanisms in determining bacterial load <i>per</i> host cell. During infections of hosts lacking components of the innate immune system it was found that the bacteria had an increased net growth rate and increased numbers of bacteria <i>per </i>cell; this is in qualitative agreement with the current model of <i>Salmonella in vivo</i> distribution. Temperature sensitive SPI-1 and SPI-2 <i>Salmonella</i> mutants were used to examine the role of bacterial effectors and division in determining bacterial distribution within host cells. This thesis shows that temperature sensitive <i>Salmonella</i> that are unable to replicate at host body temperature reach lower bacterial numbers <i>per </i>host cell. The SPI-2 mutant exhibited a surprising distribution within the host, reaching very high bacterial loads in non-nucleated micro colonies. SPI-2 effectors may promote host cell lysis <i>in vivo.</i>

No description available.

Chemostat studies were undertaken to validate the correlation between bacterial growth rate and 16S ribosomal RNA content for intestinal isolates, and also to identify potential 'signature' cellular fatty acids (CFA) for use as markers of bacterial community structure. These two methods were then used, in conjunction with traditional techniques, to investigate changes in the human colonic microflora associated with carbohydrate metabolism, antibiotic administration, and the ageing process. Short chain carbohydrates (SCC) were used to manipulate the composition and activities of the intestinal microbiota. They showed considerable potential as prophylactic agents in the prevention <I>C. difficile</I> infection. Viable count and 16S rRNA methodologies indicated that these carbohydrates predominantly stimulated bifidobacteria, although the metabolism of other bacterial genera could be affected, and the suppressive effect they induced against <I>C. difficile</I> was not limited to a specific species or genus. However, administration of SCC to patients undergoing antibiotic therapy could result in further impairment of colonisation resistance, with more fermentable carbohydrates, and broad spectrum antibiotics being of highest risk. Bifidobacteria were found to exhibit SCC substrate preferences when growing as part of the normal colonic microflora, which could have important consequences if a particular bacterial strain is to be targeted, such as during probiotic or prebiotic therapy. In conclusion, CFA analysis was effective in detecting differences between environmental samples, although careful consideration is needed to attribute these changes to an altered bacterial community structure rather than modification of the composition of bacterial membranes in stable populations. Viable count methodology proved advantageous for analysing inhibition of bacterial populations, whilst 16S rRNA analysis was the technique of choice for investigating metabolically active samples since it precludes the subjective nature of bacterial identification.

No description available.

This thesis describes the development of models designed to capture the dynamics of infections taking place within mixing networks. These models, formulated as systems of differential equations using pair-wise moment closure techniques, are investigated for a wide variety of situations and shown to be highly flexible and often very different from traditional approaches. When compared to full stochastic simulations on computer-generated networks they are remarkably accurate, giving excellent agreement both to the initial growth and the equilibrium behaviour of epidemics. The models are used to investigate a range of interventions, including targeted control measures and contact tracing. Contact tracing, which attempts to identify contacts of infected individuals, is an intrinsically network-based intervention that the pair-wise models developed here are well suited to capturing. It is shown to be a naturally targeted and powerful control that can reduce prevalence by automatically concentrating efforts on high-risk subpopulations. Network methods are particularly applicable to sexually transmitted diseases (STDs), for which networks are relatively easy to define and frequently measured. To better capture the behaviour of STDs in the general population, a monogamous network model is developed, including partnership turnover within a serially monogamous society. The influence of turnover rates is apparent, and the reduced impact of high mixing groups within such populations has ramifications for the design of control policies. The presence of partnership dynamics within monogamous networks introduces a second time-scale that allows the existence of multiple pathogen strains whereas, in a polygamous environment, only one would be able to persist. The coexistence of otherwise mutually exclusive strains has implications for disease evolution, and demonstrates the importance of population mixing patterns and behaviour.

The two subtypes of the Human Immunodeficiency Virus, HIV-1 and HIV-2, differ in the way that they package their genomic RNA with the structural Gag protein. HIV-1 packages its genomic RNA predominantly in a <i>trans</i> manner, and can cross-package HIV-2 RNA in co-transfections. Conversely, HIV-2 is not capable of packaging HIV-1 RNA reciprocally, and packages its own RNA using a co-translational mechanism. Chimeric viruses where the RNA-binding domain of HIV-1 is substituted into the structural of Gag protein of HIV-2 appear to transfer the <i>trans</i> packaging phenotype, but this function is only maximally present when the adjacent p2 peptide is also included. The aim of the work presented here was to investigate possible direct and indirect effects of the p2 domain in RNA-protein capture in HIV-1 and HIV-2. Chimeric Gag proteins were expressed using bacterial systems and their interaction with <i>in vitro</i> transcribed RNA of the HIV leader sequences investigated using GST-Pulldown assays, UV-crosslinking and mobility shift assays. Evidence is presented that is consistent with a complex and possibly cooperative binding interaction occurring between Gag and the packaged RNA, which correlates to some extent with the presence of the p2 domain. The subcellular location of the Gag protein was investigated using transfection with Gag-expressing and viral constructs, following by confocal microscopy of the immunostained cells, and no difference was found that could be attributed to the p2 domain. Additionally, the replication characteristics of the chimeric viruses were examined in short and long term culture. All chimeric viruses were defective in short term culture, but one culture did produce a replication-competent strain on long-term passage. This strain had wild type replication kinetics, and sequencing of the revertant virus revealed a single-base substitution in the highly basic region of the MA domain of Gag.

No description available.

<I>Chlamydia trachomatis</I> (CT) is an obligate intracellular bacteria and is a common cause of ocular and genital infections in human populations. Murine models of CT infection have shown that Natural Killer cells (NK cells) are recruited into the genital tract early after infection, and are an important source of the cytokine IFN-γ. NK cells are part of the innate defences against infection through cytokine production (IFN-γ, TNF-α and GM-CSF) and cytolytic activity. The hypothesis investigated in this thesis was that CT-infection of target cells leads to alterations in the target cell that are recognized by NK cells, activating one or more of their effector functions. Cervical epithelial tumour lines (HeLa and SiHa) were infected with CT and used as targets in cytotoxicity assays, with polyclonal NK cell lines from healthy individuals as the effector cells. It was found that the level of lysis was significantly raised when CT-infected targets were compared to mock-infected controls. UV-inactivated CT did not induce this increase in lysis, and inhibiting CT-protein synthesis also abolished the effect of live infection. Primary human fibroblasts were not lysed significantly following infection. Investigation of the expression NK cell ligands following CT infection revealed reductions in Classical Class I MHC and HLA-E levels on infected cells. This reduction was seen on both the epithelial cell lines and the primary fibroblasts. The different susceptibility to NK cells is likely to be the consequence of expression of different activatory ligands by the different target cells. Cytokine production by the NK cells following exposure to supernatants from CT-infected cells was assessed and it was shown that supernatants from CT-infected HeLa cells could, in conjunction with rhIL-12, induce NK cell IFN-γ production. Supernatants from CT-infected dendritic cells (DCs) were found to induce NK cell IFN-γ production in conjunction with rhIL-18. This thesis provides evidence for interactions between CT-infected cells and Human NK cells; both cell contact-dependent and soluble mediator-dependent interactions have been demonstrated. Thus, mechanisms have been elucidated via which human NK cells could play an important role in the innate defences against CT infection.

Objective: To test the hypothesis that the viraemia pattern of common latent and persistent viruses is ubiquitous to immunodeficiency in general, and can therefore serve as surrogate marker of immunocompetence. Design: A cross-sectional case-control design was used. The elderly, HIV and blood donor cohorts were recruited from Kumasi Teaching Hospital, Ghana, and the autoimmune archived samples from Addenbrooke’s hospital, UK. Results: Common viraemia profiles were shared between HIV-1 and age-related immunodeficiency. EBV had significant OR of reactivation in the elderly, and all stages of HIV, while CMV was only relevant to the elderly and AIDS patients. Detection in either cellular or plasma fraction was informative of reactivation of EBV only. Although no viraemia was detected for VZV, there was a significant increase in seroprevalence suggestive of reactivation in all HIV stages and in the elderly. B19V viraemia was specific to the elderly, while HBV (OBI) and GBV-C viraemia and indirect evidence of possible HHV-8 reactivation (increased seroprevalence) were specific only to HIV. In the autoimmune cohorts there was a complete absence of viraemia, and no significant change in seroprevalence for any virus screened. Although below significance there was a trend of anti-VZV antibody loss in the ANCA-associated small vessel vasculitis, additionally past-exposure to GBV-C was only observed in Microscopic Polyangitis. Conclusions: While evidence for the hypothesis of generality of reactivation of persistent viruses was apparently unfounded, as GBV-C and HBV were specific for HIV and B19V specific for the elderly, there was however some evidence for a general reactivation profile (in Ghana) of the latent herpesviruses, with EBV and CMB showing significant reactivation (viraemia) and potential VZV reactivation suggested by increased seroprevalence.