Hepatitis C Virus (HCV) exists as a population of sequence variants that evolves during infection adapting to host pressures. The main targets for the immune response are the envelope glycoproteins E1 and E2, which also mediate viral cell entry. The first hypervariable region (HVR1) of E2, previously implicated in the outcome of acute infection, has been a focus of many studies. However more broadly neutralising antibodies tend to target epitopes outside this region, yet evolution of full length E1E2 heterodimer is poorly understood. The HCV transmission and window period as well as seroconversion are the evolutionary events shaping primary infection hence influencing outcome of acute infection. However, due to the asymptomatic character of the early phases of HCV infection, evolutionary data describing this interval is still lacking depth. Defining the genetic and phenotypic characteristics of HCV population of sequence variants that establish infection in a new host would aid vaccine and new therapy design. This study aimed to identify patterns of HCV envelope glycoprotein evolution upon transmission and during early phases of disease. We studied this in three settings: experimental transmission of immunocompromised mice by known inoculum; occurrence of horizontal transmission in a haemodialysis unit between hypothesised source and index case individuals; and unrelated cases of acutely infected HCV patients. The single genome amplification (SGA) approach was utilised, which allowed us to accurately assign linkage between substitutions and determine the frequency distribution of E1E2 variants in analysed viral populations. Data from the first experimental setting indicates that a selective sweep occurs upon HCV transmission, with selective amplification of envelope sequence variants that possess fitness advantage at entry level. Molecular determinants associated with this enhanced infectivity have also been identified. In further part of the project we confirmed a horizontal infection in haemodialysis unit with use of phylogenetic methods and suggested revision of current safety guidelines. Analysis of sequences from the last setting showed that indeed HVR1 might not be a good enough indicator of evolutionary events in the acute phase, as linked substitutions occur also outside this region. Seroconversion is associated with increasing population diversity indicating role of antibodies in driving HCV evolution, which is host specific.

Campylobacter jejuni is a gram-negative thermotolerant microaerobic pathogen that causes human gastroenteritis worldwide. The production of extracellular polymeric substances to create a biofilm is a mechanism by which C. jejuni can protect itself from unfavourable environments, and is a contributory factor to the survival and transmission of the organism to farms animals and into the human food chain. Bacteriophages are natural predators of bacteria that have the potential for use as targeted biocontrol agents with the advantage that they can penetrate and affect bacteria embedded in biofilms. The effects of virulent Campylobacter-specific bacteriophages CP8 and CP30 on C. jejuni biofilms formed on glass by strains NCTC 11168, PT14 and HPC5 at 37 °C under microaerobic conditions were investigated. Independent bacteriophage treatment led to 1 to 3 Log10 CFU/cm2 reductions in the viable count 24 h postinfection compared with control levels. In contrast, bacteriophage applied under these conditions effected a reduction of less than 1 Log10 CFU/ml in planktonic cells. Resistance to bacteriophage in bacteria surviving bacteriphage treatment of C. jejuni NCTC 11168 biofilms was >80%, whereas bacteriophage resistance was not found in similarly recovered C. jejuni PT14 cells. Concomitant dispersal of the biofilm matrix by bacteriophage was demonstrated by crystal violet staining and transmission electron microscopy (TEM). The resistant survivors of bacteriophage treatment of biofilms formed by HPC5 and PT14 remained closely associated with the phage but not NCTC 11168. Analysis of the DNA contents of these isolates by PFGE and Southern transfer confirmed the presence of phage genomic DNA (approximately 140 kb) leading to the conclusion that these strains represent examples of the carrier state life cycle (CSLC) reported for other bacterial species. TEMs of CSLC cultures demonstrated the association of bacteriophage particles with Campylobacter cells that were devoid of flagella. Physiological studies of the CSLC strains showed the bacteria were non-motile but able to grow at a similar rate to parental cultures until reaching the phage proliferation threshold (7 Log10 CFU/ml) when growth rate declined and the phage titre increased. Of further note the CSLC strains had a greater capacity to survive atmospheric oxygen under nutrient limited conditions. CSLC phages exhibited differences in host binding, efficiency of plating and host range. Transcriptome analyses of CSLC strains harvested from microaerobic cultures at early exponential phase prior to phage proliferation were performed using DNA microarrays to demonstrate changes in host gene expression as compared with parental cultures. Notably genes involved in metabolism and the modification of macromolecules were up-regulated and specific flagella biosynthesis functions down-regulated in the CSLC strains.

Ubiquitin conjugating (UBC) enzymes play a key role in eukaryotes during the posttranslational modification of proteins by covalent attachment of ubiquitin. A gene was identified in the double stranded DNA genome of African swine fever virus (ASFV) which was predicted to encode a protein with high homology to eukaryotic UBC enzymes. This ASFV encoded enzyme (UBCv) was expressed in E. coli and was shown to have ubiquitin conjugating activity in vitro. Antisera against recombinant UBCv were used to detect UBCv in ASFV infected cells. UBCv was shown to be a cytosolic protein present throughout the early and late stages of ASFV replication and was packaged in ASPV virions. Attempts to inhibit UBCv activity during ASFV infection using antisense oligonucleotides were unsuccessful, and a recombinant ASPV mutant with the UBCv gene disrupted by the luciferase reporter could not be isolated. However, ASFV replication was impaired late in infection in TS20 cells at a temperature which inhibits the ubiquitin conjugating pathway. No novel ubiquitinated proteins could be detected in ASFV infected cells by immunoblotting, although an unspecific increase of cellular ubiquitin conjugation was observed in early infection. However, virus factories were intensely stained late in ASFV infection by immunofluorescence using anti-ubiquitin antisera. In addition, several ubiquitinated structural proteins were detected in purified ASFV extracellular particles by both immunoblotting and immunogold electron microscopy. An 18 kDa ubiquitinated structural protein, probably localized in the virion periphery, was purified to homogeneity and the sequence of its N-terminal 10 amino acids was determined. The N-terminal sequence of this protein matched exactly the product of a gene of unknown function encoded by the ASPV genome.

Quorum sensing signalling molecules (QSSMs) are important to Pseudomonas aeruginosa virulence and biofilm development which aid establishment and persistence of these bacteria in the host. Recent progress in quorum sensing (QS) research has demonstrated that the two QSSMs, 3-oxo-C12-HSL and PQS interact with eukaryotic cells and modulate immune responses. Early research has indicated these two QSSMs are immunosuppressive, and because T cells play an important role in defending the host against the attack of P. aeruginosa (Stevenson et al., 1995), this warrants investigations into the interactions between QSSMs and T cells. Previous studies have shown 3-oxo-C12-HSL and PQS can exert differential immune-modulatory effects on mammalian immune responses, however, no studies have confirmed these activities using pure human T cells. The purpose of my PhD was to investigate for the first time, the effects of these two QSSMs on pure human T cells in a staged manner, beginning with mouse splenocytes, human peripheral blood mononuclear cells (hPBMCs) and finally pure T cells, then if successful, paving the way for gene array technology. This present work confirms inhibitory effects by QSSMs on mouse splenocytes stimulated to proliferate using the lectin concanavalin (ConA) or anti-CD3 antibody, and hPBMCs stimulated to proliferate using anti-CD3 and anti-CD28 antibody. In order to further understand interactions between QSSMs and the immune system, the effects of 3-oxo-C12-HSL and PQS on pure human T cell proliferation and cytokine production following stimulation of T cells with monoclonal antibodies directed against CD3 and CD28 were compared, using CsA as the positive control. All three compounds inhibited pure T cell proliferation. CsA and PQS were the more potent anti-proliferative compounds with IC50 values of 3.2±0.31 µM and 3.8±0.15 µM respectively compared to 19±1.62 µM for 3-oxo-C12-HSL, indicating the QSSMs ability to suppress T cell activity and therefore advantageous to P. aeruginosa. To further comprehend the mechanism of action of these two QSSMs, the effects of QSSMs on cytokine production were assessed. 3-oxo-C12-HSL significantly inhibited IL-2 release while PQS enhanced the production of IL-2 even though suppression of T cell proliferation was observed, suggesting a cytostatic effect and demonstrating PQS may in fact act proximally to the IL-2 receptor (IL-2R) or downstream of the T cell signalling pathway, whereas 3-oxo-C12-HSL acts on early T cell signalling events. 3-oxo-C12-HSL also inhibited production of IFNγ in pure T cells and although results were inconclusive for PQS in pure T cell assays, both QSSMs were shown to have an inhibitory effect on IFNγ in mouse splenocytes, suggesting suppression of T cell proliferation is via Th1. Furthermore, 3 oxo C12 HSL suppressed IL-4, IL-5, IL-10 and TNFα while PQS suppressed IL 10 release at 3.12 µM and enhanced TNFα release, indicating these QSSMs may inhibit T cell proliferation by eliminating both Th1 and Th2 response. The immune suppressive properties of 3-oxo-C12-HSL and PQS show potential as future therapeutic entities. Immunosuppressive drugs such as CsA and rapamycin are routinely used to maintain transplants and treat auto-immune disorders. However, they can be non-selective and are limited by their side effects including nephrotoxicity and neurotoxicity. Despite recent developments of new immunosuppressants, there remains an unmet need for less toxic and more widely applicable immunosuppressive agents. 3-oxo-C12-HSL and PQS are worthy of attention as possible future immunosuppressive agents used in conjunction with or in place of present immunosuppressants. In summary, this study clearly demonstrates for the first time that the two structurally diverse QSSMs, 3-oxo-C12-HSL and PQS, can exert differential modulatory effects on pure T cells, opening a path for further study into their mode of actions within the T cell signalling pathways and their effects at an RNA level.

Staphylococcus aureus is one of the leading causes of hospital acquired infections. The ability of S. aureus to acquire resistance to a diverse range of antimicrobial compounds, results in limited treatment options, particularly in methicillin-resistant S. aureus. A mechanism by which S. aureus develops reduced susceptibility to antimicrobials is through the formation of small colony variants (SCVs). Reduced antimicrobial susceptibility in S. aureus SCVs is not related to ‘classical’ mechanisms of resistance, but occurs as a direct result of the development of the SCV phenotype. S. aureus SCVs are frequently associated with defects in the bacterial electron transport chain and these defects are responsible for the characteristics associated with the SCV phenotype. This study aimed to investigate and characterise the selection of S. aureus SCVs in the presence of various antibiotics and also to examine their biofilm forming capabilities. Four members of the aminoglycoside family of antibiotics were shown to select for S. aureus SCVs. In addition, a broad range (X 0.25 MIC – X 4 MIC) of aminoglycoside concentrations were shown to select for S. aureus SCVs. Characterisation of these isolates revealed that differences in auxotrophy, biochemical profiles, carotenoid production, haemolysis, levels of intracellular ATP, mutation frequency and reversion rate were present. Members of the tetracycline family of antibiotics were also shown to select for S. aureus SCVs. Tetracycline selected S. aureus SCVs show attenuated catalase, coagulase and heamolysis activity and reduced production of extracellular DNase and lipase and reduced susceptibility to various antimicrobial agents. As SCVs have been linked to persistent and recurrent infections their ability to form biofilms was also investigated. A range of S. aureus SCVs isolated from various backgrounds were shown to form greater biofilms in comparison to parent strains, which was attributed to increased production of polysaccharide intracellular adhesin. In addition S. aureus SCV biofilms displayed a more pronounced reduction in antimicrobial susceptibility, which was attributed to a reduction in antimicrobial penetration through SCV biofilms. Limited discovery of novel antibiotics in recent years and the observation that S. aureus SCVs can be selected for by various antimicrobial compounds highlights the need for novel antimicrobial compounds. Accordingly, an investigation into the susceptibility of S. aureus to various plant compounds was undertaken. Both S. aureus SCVs and parent strains showed susceptibility to five plant antimicrobials tested, of which SCVs were more susceptible to cinnamon bark, green tea and oregano. Resistance to these plant antimicrobials could not be induced and synergistic relationships between certain plant antimicrobials and antibiotics were demonstrated. Finally, formation of SCVs in bacterial species other than S. aureus was examined. Gentamicin induced SCV selection in Escherichia coli, Pseudomonas aeruginosa and S. epidermidis as well as chloroamphenicol and ciprofloxacin in E. coli and tetracycline in S. epidermidis. SCVs from these bacterial species shared common characteristics associated with the SCV phenotype including altered growth and biochemical profiles, auxotrophy for compounds involved in electron transport, reduction in expression of virulence factors and reduced antimicrobial susceptibility. Additionally all SCVs showed an increased capacity to form biofilms. The ability of certain antibiotics to select for SCVs and their increased capacity to form biofilms suggest that SCV are an important adaptation to aid survival and persistence in times of stress. Reduced susceptibility to commonly used antibiotics in SCVs signifies that the development of new antimicrobial compounds is required. Harnessing naturally occurring plant antimicrobials and their synergistic relationship with antibiotics may offer a novel approach to treating antibiotic resistant infections whilst overcoming antibiotic selection for SCVs.

Burkholderia cepacia complex bacteria can contaminate and survive in a variety of antimicrobial and preserved industrial products. Contamination may lead to economic loss for manufacturers and also potentially pose a risk to the health of vulnerable consumers. Understanding the interaction between Bcc bacteria and preservatives, and the molecular basis for their resistance, is essential in order to better target these organisms and to facilitate the implementation of improved preservative strategies which target resistance mechanisms. In the present study, multi-locus sequence typing analysis of a collection of 67 Bcc isolates from environmentalindustrial sources was used to expand the current knowledge of Bcc species diversity within this niche and identified B. lata (n=17) and B. cenocepacia (n=11) as predominant species groups. The relationship between Bcc species diversity, isolation source and preservative susceptibility was investigated using a collection of 83 genetically diverse Bcc strains from clinical, environmental and environmental-industrial isolation sources. Susceptibility to eight preservatives was not related to Bcc taxonomy, as susceptibility profiles varied both between and within species groups. However, Bcc isolates from environmental-industrial sources had a significantly higher minimum inhibitory and minimum bactericidal concentration (MIC and MBC) for the formaldehyde releasing agent DMDM hydantoin. This suggests that for this preservative agent, susceptibility was related to source and that the selection of highly tolerant Bcc bacteria had occurred within the niche. Isothiazolone, DMDM hydantoin, phenoxyethanol and methyl paraben preservatives were observed to be highly efficacious against Bcc bacteria when evaluated in growth medium at the maximum concentration regulated for use in rinse-off personal care products in EU-regulated countries. Benzethonium chloride and sodium benzoate preservatives had the weakest anti-Bcc activity at these levels but were effective against several strains. Combinations of preservatives, and preservatives with potentiating agents, were evaluated for synergistic anti- Bcc activity. The greatest anti-Bcc activity was observed when isothiazolone preservatives were combined with EDTA or phenoxyethanol, with each combination resulting in an additive effect. The competency of Bcc bacteria to adapt to preservatives was explored via the progressive sub-culture of B. lata strain 383 in subinhibitory preservative concentrations. This genome sequence strain represented a Bcc species commonly encountered in the environmental-industrial niche. Stable adaptive-resistance to isothiazolone and benzethonium chloride preservatives was developed. Phenoxyethanol, DMDM hydantoin and methyl paraben preservatives were recalcitrant to B. lata strain 383 adaptation. The preservative and antibiotic susceptibility profiles of the adapted B. lata strain 383 derivatives differed, suggesting the induction of agent-specific adaptive-resistance mechanisms had occurred. The B. lata 383-CMIT,-BIT, derivatives (adapted respectively to chloromethylisothiazolinone and benzisothiazolinone), demonstrated cross-resistance to isothiazolone preservatives and fluoroquinolone antibiotics. Sequence analysis of the topoisomerase genes in these derivatives revealed fluoroquinolone resistance was not mediated by target modification. Preservative-induced adaptive resistance was not associated with overall increased multi-drug resistance. The molecular basis for resistance to DMDM hydantoin and isothiazolone preservatives was investigated via the random transposon mutagenesis of B. lata strain 383 using pTnModOTp’. Several genetic pathways were identified as putative preservative resistance determinants, suggesting that resistance is multi-factorial. These included the detoxification of formaldehyde via a glutathione-dependent pathway; a type II general secretory system (A3244_A3233 genes); a homologue of an ABC-type efflux system involved in resistance to organic solvents (A3512_A3517 genes); homologues of multi-drug RND-type efflux systems EmrB/QacA-Emr-TolC; and bacterial defence mechanisms against oxidative stress. A transcriptomic microarray-based approach was used to profile global gene expression of B. lata strain 383 in response to sub-MIC of 0.00162% DMDM hydantoin and 0.00001498% of a methylisothiazolinone and CMIT blend, as well as isothiazolone-induced adaptive resistance. With a 1.5-fold change and P < 0.05 confidence level criterion applied, few significant changes were observed after a single sub-MIC exposure, and the differential expression of putative resistance determinants identified by transposon mutagenesis was not induced at these concentrations. Isothiazolone-induced adaptive-resistance involved a greater number of significant gene expression changes that were stable irrespective of the presence of the priming agent, with 126 up-regulated and 90 down-regulated genes. Transcriptomic analysis suggested that isothiazolone-induced adaptive resistance was multi-factorial in nature, and identified active efflux as a putative key resistance mechanism. A novel role for a RND-type efflux system (B1004_B1006 genes) was identified, and the up-regulation of the ABC-efflux system (A3512_A3517 genes) and bacterial defence mechanisms against oxidative stress corroborated the transposon mutagenesis findings. B. lata strain 383-CMIT demonstrated a four-fold reduction in MIC for the priming preservative (2.81E-04%) in the presence of 512 mg/L of the efflux inhibitor PAβN. Resistance mechanism targeted preservative strategies such as using efflux inhibitors may work to improve preservation.

Trypanosoma brucei is a parasitic protozoan that can cause human African trypanosomiasis (HAT) and Nagana in cattle. Human African trypanosomiasis is deadly when left untreated, and thus there is an urgent need to develop new drugs against this disease. As trypanosomes are early diverged eukaryotes, it is anticipated that studying their metabolism can identify novel drug targets. The main drug currently in use against the late encephalitic stage, Eflornithine, was shown to inhibit an essential pathway in trypanosomes (Yarlett and Bacchi, 1989). In this Thesis three approaches were used to apply metabolomic and proteomic techniques for protein function identification and to investigate metabolic pathways. The genome of T. brucei has been published (Berriman et al., 2005) and data is available via databases, such as TriTrypDB, a database dedicated to the trypanosomatids (Aslett et al., 2009). An estimated 40% of the identified genes in this organism are annotated with an unknown or putative function. In 2006, Saito et al. developed a systematic method to ascertain enzyme function based on an in vitro assay, in combination with metabolite profiling. This approach was successfully applied in several other studies. Here, I investigate the use of this method for its application in a high throughput approach for unknown enzyme identification in trypanosomes. Seven putative identified enzymes were randomly selected from TriTrypDB, cloned and expressed in E. coli and a function could be attributed to at least one of the enzymes. Furthermore, the amino acid metabolism in trypanosomes was investigated; using stable isotope labelling combined with metabolomics. The flux of labelled compounds could be traced through the organism showing the active metabolic pathways of L-methionine, L-proline and L-arginine in T. brucei. Two T. b. brucei strains used in this study, GVR35 and 427, cause different forms of infections in their mammalian host. GVR35 causes a chronic infection and invades the central nervous system (CNS) with varying parasitemia in mice, whereas infection with strain 427 presents an acute form with high parasitaemia, causing high mortality, without invading the CNS. What causes this difference in the progression of infection? Secreted or excreted proteins from the parasites, referred to as the secretome, have been described as being important factor for virulence and avoiding the host immune response (Geiger et al., 2010) and Garzon et al. (2006) showed that excreted/secreted proteins can inhibit the maturation of dentritic cells and stop them from inducing a lymphocytic allogenic response. Significant differences in proteins secreted from these two strains are discussed; although the results are preliminary.

Inbred strains of C5731 and NIH nice infected with the A/S strain of Plasmodium chaubaudi usually developed high parasitaemias but infections were rarely fatal in immunocompetent mice and in most mice the parasites could be eradicated within 53 days or less. The immune response of C57B1 and NTH mice to infection with the A/S strain of P. chabaudi was studied. The principle method used in this study for investigating the immune response of the mice was to examine the immunity conferred on syngeneic mice, either X-irradiated or non-irradiated, by transferring to them lymphoid cells or serum from immune or semi-immune donors. The lymphoid cell populations examined were unfractionated spleen cells, nylon wool column enriched subpopulations of thymus-derived lymphocytes (T cells) and the so-called bursa-derived lymphocytes (B cells), bone marrow cells and phagocytic cells. In the course of these experiments observations were made on the effect of X-irradiation on the subsequent growth and multiplication of the parasite. In addition, an in vitro assay for antibody-dependent cell mediated cytotoxicity was used to investigate the activity of splenic K cells during malaria infection. K cells are lymphoid cells which may include lymphocytes of an undefined category, but possess receptors for the Fc portion of antibody on their surface and have the ability to non-specifically lyse target cells coated in antibodies. a) The adoptive transfer of immunity to P.chabaudi with immune spleen cells. Spleen cells from mice which had previously been infected with P.chabaudi were able to confer some immunity on syngeneic mice which had been irradiated with 600 or 800 rads. The protection was detected as a shortened patent parasitaemia in immune cell recipients compared to controls. The early experiments indicated the value of using irradiated recipients rather than non-irradiated recipients. In irradiated mice, a) smaller numbers of immune cells were required to promote detectable immunity than in non-irradiated mice, b) there was an amplification of the difference in the duration of primary parasitaemias in recipients of immune cells and normal cells compared to non-irradiated mice and c) as the irradiated host is immunodepressed, the protective effect of donor cells can be examined with a reduced contribution by the hosts own immune system. An initial non-specific resistance to P.chabaudi infection was observed in irradiated mice, although the infection in most of these mice was subsequently more severe than in non-irradiated mice. The non-specific resistance could be reduced or abolished by injecting lymphoid cells into mice shortly after irradiation or by infecting irradiated mice more than 15 days after irradiation. Other workers suggest that following irradiation, the reticulo-endothelial system is stimulated at the time that the non-specific resistance to P.chabaudi was observed. b) the adoptive transfer of immunity in syngeneic mice with enriched subpopulations of splenic immune T cells, B. cells, bone marrow cells and phagocytes. Immunity to P.chabaudi could be adoptively transferred with enriched spleen subpopulations of immune T cells or immune B cells in mice which had been irradiated 600 or 300 rads. The protective effects of unfractionated immune cells was, however, usually better than that of either immune T or F cell subpopulations. In most experiments enriched immune T cell recipients were more likely to suffer relapsing patent parasitaemias than either enriched immune B cell recipients or unfractionated immune cell recipients. In one experiment a comparison was made of the course of P.chabaudi infection in mice which had been irradiated with either 600 rads or 300 rads and which received injections of different immune cells. A dose of 600 rads permits the immune system of mice to recover from the effects of irradiation, but a dose of 800 rads is lethal to mice unless lymphoid cells are injected after irradiation. It was found that in recipients of enriched immune T or B cells, which had been irradiated with 600 rads, the parasitaemia became subpatent before their equivalents irradiated with 800 rads, but that there was little difference in parasitaemias between recipients of unfractionated immune cells given 600 or 800 rads. Experiments in which enriched immune T cells and B cells were recombined and injected into syngeneic mice gave inconclusive results as to whether the immune subpopulations acted synergistically. Similar experiments in which immune subpopulations of lymphoid cells were recombined with normal subpopulations of lymphoid cells demonstrated that the latter cells did not enhance the protective effect of the former cells. Bone marrow cells from immune mice were able to confer some protection on syngeneic recipients, but were not as protective as enriched immune T cells or B cells. The results obtained in adoptive transfer experiments using phagocytic cells from the spleen of immune mice depended on the length of time spleen cells were incubated in petri-dishes at 37° C before harvesting the phagocytes. Using C57B1 mice, phagocytes harvested after 15 hours incubation were as protective as unfractionated immune cells in a cell transfer experiment, but phagocytes harvested after 16 hours incubation were not protective. Examination of NIH phagocytic cells after 2.5 hours incubation at 37°C, which were as protective as unfractionated immune spleen cells in a cell transfer experiment, demonstrated that the petri-dish adherent cells may have contained B lymphocytes. c) The passive transfer of immunity with serum from P.chabaudi infected mice. The passive transfer of serum from C57B1 mice which had been previously infected with P.chabaudi to normal or irradiated syngeneic mice demonstrated that the serum recipients were initially protected from infection. Irradiated mice, however, were delayed longer in the onset of parasitaemia compared to non-irradiated mice. Using NIH mice, sera were collected from unfractionated immune spleen cell recipients, enriched immune T cell recipients and normal spleen recipients on the 11th day of a P.chabaudi infection, just after peak parasitaemia, and also on the 14th day of infection. On day 14, all immune cells recipients and most of the enriched immune T cell recipients had become subpatent but all normal cell recipients still had patent infections. Sera collected from the different spleen cell recipients on the 11th day of infection and passively transferred to irradiated mice demonstrated little protection. Sera collected on the 14th day of infect ion, however, reflected the immune status of the donors in their protective properties in mice infected with P.chabaudi. The serum from unfractionated immune cell recipients was the most protective of the 3 sera when compared to normal NIH serum and the serum from enriched immune T cell recipients was slightly protective, but the serum from normal cell recipients produced an enhanced infection in mice infected with P.chabaudi. d) Antibody-dependent cell-mediated cytotoxicity of spleen cells in P.chabaudi infected mice. In a preliminary investigation of K cell activity in the spleens of P.chabaudi infected mice, it was found that there was an increased activity of K cells collected at around peak parasitaemia compared to the activity of K cells in non-infected mice, and that this increased activity could also be found in mice which had recently become subpatent. As the target cell for antibody-dependent cell-mediated cytotoxicity employed was the thick red blood cell, it is not known whether the K cell is involved in the killing of P.chabaudi parasites. These results suggest that both T cells and B cells and antibody may be important in the immune response to P.chabaudi in mice. Primed T cells may act as helper cells in the production of malarial antibodies, but, as enriched primed T cells could confer protection on immunodepressed mice, it is possible that a cell-mediated mechanism of immunity may also exist.

Following disease or injury to the CNS, the formation of a glial scar represents a physical and molecular barrier to repair. Although some therapies have promoted axonal sprouting into the lesion site, these fibres are often tangled and disorientated. To date, there has been little evidence of regenerating fibres successfully exiting the glial scar to reform functional connections. Furthermore, remyelination after disease or injury is limited, often consisting of shorter internodes of myelin and thinner sheaths. Thus, potential therapies aimed at enhancing CNS repair should support the outgrowth of neurites, guide their exit from the glial scar and perhaps aid remyelination. Since multiple factors impede the regeneration of the CNS, a combinatorial approach to therapies including cell-transplantation may be a more promising strategy.

Clostridium difficile is a major cause of nosocomial intestinal infection. The pathogen possesses two potent toxins, Toxin A and Toxin B, both of which contribute to diarrhoea, intestinal inflammation and tissue damage. Antibiotics are effective against the disease, however around 20 % of patients on treatment relapse after the termination of antibiotic therapy. The binding of Toxin A to a receptor on human intestinal epithelial cells initiates disease: this is considered the starting point from which the toxin elicits its effect. One feature of the carboxy-terminal domain of Toxin A is the presence of repeating units of amino acids that form a series of binding sites able to recognise disaccharides and trisaccharides on glycolipid and glycoprotein receptor molecules. Antibody response against the toxin can protect against C. difficile disease and efforts to generate vaccines have focused upon the carboxy-terminal, receptor binding domain. The aims of this project were to use phage display to isolate recombinant antibodies against those features of the carboxy-terminal domain of Toxin A thought to be responsible for receptor-binding and to assess if the antibodies were capable protecting against the action of Toxin A. Using published crystallographic data that has shown the interaction of Toxin A and trisaccharide, a region of about 113 amino acids from the carboxy-terminal region of Toxin A was expressed as a fusion to maltose-binding protein. The MBP fusion protein was expressed, purified on amylose resin, and characterised. The fusion protein was then used to isolate single chain antibodies from the Tomlinson libraries of scFvs, a synthetically diversified phage display library of single scaffold human antibodies. Conventional bio-panning methods were used in which the MBP fusion protein was bound to a plastic surface and the phage display libraries were pre-mixed with native MBP to inhibit the isolation of anti-MBP antibodies. Progressive enrichment of scFvs through 3 rounds of selection was observed. Those scFvs that showed strongest reaction against the target protein in ELISA but failed to react with native MBP were sequenced, expressed as soluble antibodies and purified on nickel chelating columns. While the resulting panel of scFvs showed similarities of sequence, none were identical. All were reactive with native, full-length Toxin A and appeared to bind to conformational (nonlinear) epitopes. Cross-reaction with Toxin B from C. difficile was also evident. A panel of truncation mutants were generated from the MBP fusion protein and using these in ELISA with the scFvs, reactivity appeared to be directed to features of a long repeat sequence of Toxin A. To assay whether the isolated scFvs possessed biological activity of significance, in vitro and in vivo protection assays were established. For experiments in vitro, the action of Toxin A upon cultured Vero cells was studied. Native Toxin A triggered a conversion of the cells from stellate to rounded morphology. When cells were exposed to 100 ng of toxin, this effect was evident within 60 minutes; at a 10-fold lower dose, the minimum quantity to which a response was detectable, virtually all cells had undergone rounding within 2 h. When individual scFvs were mixed with 10 ng of Toxin A prior to addition to Vero cells, there was a consistent delay in cytopathic activity that extended to 5 h. In this assay, the percentage of cells that had retained their stellate morphology 5 h post-challenge was dependent on the scFv used. To quantify the potency of this neutralising activity, the amount of each scFv required to achieve 50% protection during a 2 h challenge period was established. This revealed 3.5-fold difference between the most and the least effefctive scFv. The most potent scFv was used in an in vivo assay in which Toxin A was administered to the ligated intestinal loops of rats. Again, protective activity was evident. Overall, phage display technology enabled the assembly of a panel of scFv antibodies against the putative receptor binding site in the carboxy-terminal domain of Toxin A from C. difficile. The scFvs were able to protect against the cytopathic activity of Toxin A in vitro and in vivo and proposals are made about how these observations could be taken forward in a model of C. difficile infection that best mimics the human disease.