The Spondyloarthritides (SpA) are a group of genetically and pathophysiologically related diseases. Ankylosing spondylitis (AS), the prototypic SpA family member, is a systemic inflammatory disease primarily affecting the axial skeleton, characterised by sacroiliitis and bone formation, promoting joint inhibition. AS is highly heritable; approximately 90% of AS susceptibility is defined by an individuals’ genetic background, to which the MHC class I molecule HLA-B27 contributes approximately 30%. This association was discovered 40 years ago, yet the pathogenic role of HLA-B27 remains elusive. Dendritic cells (DCs) belong to the myeloid lineage and, the principal antigen presenting cells (APCs) of the immune system, activate naïve T cells and contribute to the balance between activation and suppression of the immune response. If affected by HLA-B27, DCs are therefore likely to contribute to the T cell-mediated aspects of AS pathogenesis. Studies in our laboratory, using HLA-B27 transgenic (HLA-B27 TG) rats, have revealed HLA-B27-mediated effects on DC populations. The affected DCs induce abnormally high levels of IL-17 production from T cells; CCR6+ IL-17-secreting cells appear to be important in driving pathology both in the HLA-B27 TG rats and in AS patients. We therefore aimed to perform the first characterisations of the phenotype and functions of DCs and other myeloid populations purified directly from AS patients, to understand their role in AS pathogenesis. Analyses of circulating myeloid populations revealed that AS patients have a reduced proportion of the CD1c-expressing blood DCs, offset by an increase in CD14- CD16+ mononuclear cells. Interactions between CD14- CD16+ mononuclear cells and CD4+ T cells generated high levels of IL-6 secretion, required for the generation of Th17 cells. CD14- CD16+ mononuclear cells also induced T cells to express CCR6, and may therefore contribute to pathology by promoting Th17 responses. Interestingly, our data also indicate that APCs of mucosal origin may make a significant contribution to the systemic inflammation observed in AS patients. These observations give new insights into the pathogenic mechanisms in AS.

Sepsis is not only the leading cause of death in non-coronary intensive care units (ICUs) but also one of the most common causes of morbidity and mortality for all hospitalised patients. Globally, 20 to 30 million patients are estimated to be afflicted every year with an astonishing hospital mortality rates between 30 and 60%. There is no current therapy for sepsis other than anti-infectives and supportive care. These approaches only give the body time to recover, but do not treat the cause of the problem. In this study we seek to discover the effects of the virulence factor from a bacterium, Yersinia pestis V antigen on LPS-induced responses. Y. pestis, the causative agent of the three main plague pandemics has been responsible for over 200 million deaths. The bacterium has been so successful because it encodes several virulence factors, one of which is V antigen. Our results demonstrate a modulatory role of Y. pestis V antigen on bacterial infections. Using monocytic and macrophage cells we have shown that V antigen can reduce the expression of pattern recognition receptors (PRRs) of the innate immune system, causing the modulation of the cellular response directed towards LPS bacterial pathogen-associated molecular patterns (PAMPs). We also tested different fractions of Y. pestis in order to identify the functional domain of responsible for this immunomodulation. Our results demonstrated that this functional domain in found within the amino acids 135-275 of V antigen. Of a greater magnitude, our in vivo data show an impressive 80% reduction in mortality in a mouse model of sepsis when mice are treated with V antigen in comparison to those that were not. This protein should unquestionably be further investigated as a possible therapeutic intervention for sepsis.

Chronic obstructive pulmonary disease (COPD) encompasses the diseases of chronic bronchitis and emphysema, both of which can cause the main feature of this disease; airflow obstruction. COPD is the third leading cause of mortality in the world, and although cigarette smoking is the main risk factor of the disease, other environmental factors and genetic disorders can also play a role. The pathogenesis of COPD is currently still poorly documented, which results in poor diagnosis and treatment for the disease. The involvement of autoimmunity in the pathogenesis of the COPD is becoming more apparent; in recent studies, high levels of circulating autoantibodies have been detected in patients with COPD, suggesting that this lung disorder may have an autoimmune component. Therefore, research has focused on identifying a set of autoantigens known to be associated with other autoimmune disorders that may play a role in the pathogenesis of this disease. The aim of this study was to develop antigen microarrays for profiling antigen specific-autoantibodies in the serum of patients with COPD, as a diagnostic tool at the early stage of the disease. To achieve the project aim, we have employed antigen microarrays. Before applying this technique, quality control experiments were performed to optimize the procedure. This included testing different slide surface coatings, investigating numerous blocking buffers and methods to amplify the signal intensities, as well as a series of validatory tests to determine if the assay was both accurate and reproducible. The optimization results showed that a combination of amino-silaine coated slides with I Block buffer and Genisphere amplifier performed well due to the high signal detection and low background observed. Validation for optimized technique was achieved and showed no cross-reactivity occurred, an acceptable limit of the coefficient of variation (CV %), and a significant correlation between ELISA and the antigen microarray platform, with microarray giving a bigger dynamic range of signals. The results for the limit of detection (LOD) were applied and the cut off for a positive autoantibody response was taken from the 95th percentile of the healthy non-smokers control group. The results for the investigation of the different 39 autoantigens showed that there was a significant increase in reactivity for both the IgG and IgM autoantibodies in the COPD group compared to the control for CENP-B, collagen5, RNP/sm, La ssb, histone, ro-52 and SCL-70, which suggests these antigens have the potential to be used as diagnostic biomarkers for the detection of COPD. These results also showed that a number of healthy smokers produced an increased reactivity to the same autoantigens as the COPD patients, which suggests there is potential susceptibly of these smokers to develop COPD. In conclusion, this thesis developed a rapid, inexpensive, broad-spectrum antigen microarray technology, which could have a pivotal future role in the early diagnosis of COPD.

Cholera is a persistent threat to public health and is endemic in many countries. Of late, there is an emergence of antibiotic resistance in Vibrio cholerae and treatment is effective only if given early, thus there is a need for rapid and more effective treatment of cholera. One such treatment could be the use of bacteriophages. During infection, V. cholerae adheres to the surface of enterocytes but does not invade the host. They are therefore not protected from bacteriophage infection. The study presented in this dissertation evaluates the potential of bacteriophage being used as a biocontrol for V. cholerae. The aim of this project was isolation and in vitro characterisation of bacteriophages, selection of a candidate bacteriophage for biocontrol and its use in an infant rabbit model to assess its therapeutic efficacy. Seven phages were isolated in China, attempts to isolate in the UK environments were unsuccessful and five more phages were obtained from various sources. In total twelve phages were characterised for the one step growth curves following their host strain growth curves, their lytic spectra, electron microscopy, PFGE, restriction analysis and annotation of sequenced genomes. These in vitro characterisations could help in selecting the candidate bacteriophage for in vivo phage therapy trials. Amongst the phages studied, the phage Φ1 most nearly fitted the selection criteria. Its burst size was 43 ± 5.5; while the latent period was 12 ± 0.0 and it had broad host range as it could lyse 67 % of the total 91 strains; while its genome did not show any undesirable genes associated with lysogeny/antibacterial resistance or any cholera toxin genes upon genome annotation. In therapeutic trials using an infant rabbit model, Φ1 reduced the bacterial numbers significantly (4.7 log10 reduction with P < 0.001) and treated animals showed no symptoms of disease.

Background: Bacterial infections, particularly those of the respiratory tract, are a serious cause of mortality worldwide despite vaccinations and optimized treatment strategies. Prior to the introduction of antibiotics, passive immunotherapy was widely used to treat a range of bacterial infections. A successful opsonin based treatment, however, requires effective clearance by phagocytic cells. Augmented passive immunotherapy (API) is a novel treatment strategy that combines pathogen specific immunoglobulin (IVIG) and the immunomodulating peptide P4 to treat fulminant bacterial infections. Using in vivo, in vitro and ex vivo models of pneumococcal infection, this thesis aimed to describe host responses to API. Methods: The in vivo recruitment and activation of phagocytes following peptide treatment in the absence of infection was characterised using flow cytometry and electron microscope imaging. These findings were translated to murine infection models by treating pneumococcal septicemia with intravenous peptide administration and pneumococcal pneumonia with intranasal peptide administration. In vitro phagocytosis killing assays were used to determine the ability of P4 to augment bacterial killing of a range of pneumococcal serotypes and describe the involvement of phagocytic Fcγ receptors. Ex vivo assays of phagocytic function of human neutrophil and alveolar macrophages were used to translate murine and in vitro findings. Results: Peptide administration in the absence and presence of infection led to rapid recruitment of monocytes, neutrophils and macrophages and their activation as demonstrated by increased FcγR expression. Intravenous peptide administration during septicemia led to significantly increased survival rates in models of invasive and acute invasive pneumococcal diseases in young and aged mice of various genetic backgrounds. Intranasal peptide administration during pneumonia prevented the onset of septicemia and subsequent host mortality. Increased survival was associated with reduced bacterial burden in affected tissue. Peptide treatment of neutrophil cell lines, human neutrophils and alveolar macrophages modulates FcγR expression resulting in augmented phagocytic killing of opsonised pneumococci. Conclusion: Augmented passive immunotherapy is a synergistic treatment that enhances natural host immune responses to infection by opsonizing pathogens and inducing their effective phagocytic clearance.

TFH cells have been identified as a new T helper subset specialized to regulate the development of effector and memory B cells and long-lived plasma cells. The interaction between TFH and B cells leads to the activation of B cells and germinal centers (GC) formation. Considering the importance of TFH for B cell antibody response, novel vaccine adjuvants and intranasal vaccines to boost TFH number or function may be an attractive vaccination strategy to enhance vaccine efficacy in humans. Adenotonsillar tissues are major parts of nasopharyngeal associated lymphoid tissues (NALT) and they are important in response to upper respiratory tract pathogens and intranasal vaccination. This PhD project investigated the frequencies of TFH in human NALT and PBMC in children and adults. The effects of CpG-DNA and live attenuated influenza vaccine (LAIV) on TFH in human NALT and the TFH-mediated B cell immunity to influenza virus were studied. The importance of the cytokine IL-21 and plasmacytoid dendritic cells (pDC) in TFH cell-mediated B cell antibody production was also investigated. Adenotonsillar MNC and PBMC were isolated from adenotonsillar tissues and peripheral blood respectively. TFH (CD4+ CXCR5high ICOShigh) numbers and function were analysed by flowcytometry and intracellular cytokine staining. Purified TFH (CD4+ CXCR5hi) and non-TFH cells (CD4+ CXCR5-) were co-cultured with B cells in the presence of influenza virus antigen and CpG-DNA or of LAIV. Purified pDC were added to the TFH-B cell co-culture to study their importance in TFH -mediated B cell antibody production. Haemagglutinin (HA)-specific antibody production was analysed by ELISA and ELISpot assay. IL-21 receptor blocking by neutralization was used to study the importance of IL-21 in TFH-mediated B cell antibody production. A prominent number of TFH were found in human NALT which were considerably higher than in PBMC. There was an age-associated difference in TFH numbers in NALT and BPMC, i.e. the mean TFH number was higher in children than in adults. TFH in NALT were shown to express high levels of IL-4, IL-10 and IL-21 and that were important for B cell antibody production. A good correlation between the numbers of GC B cell and TFH in NALT was seen. Co-culture of purified TFH but not non-TFH with B cells promoted antibody production. Stimulation of adenotonsillar MNC by CpG-DNA significantly increased TFH number and that was correlated with HA-specific antibody production following influenza antigen stimulation. Co-incubation of TFH-B cell with pDC enhanced the CpG-DNA-mediated antibody production. We also found that stimulation with LAIV significantly increased TFH number and that was correlated with HA-specific antibody production. Blocking the IL-21R significantly reduced the number of TFH that was correlated with a significant reduction of HA-specific antibody production. Enhancing vaccine immunogenicity through modulation of TFH numbers or function in human NALT using immunological adjuvants such as CpG-DNA and through intranasal vaccination may be an effective vaccination strategy against respiratory pathogens.

Streptococcus pneumoniae (pneumococcus) is a gram-positive bacterium that can cause significant morbidity and mortality in humans especially in children and elderly. T regulatory cells (Treg) have an important role in modulation of immune responses to microbial infection. Although Th17 cells are involved in autoimmune diseases, these cells may play a protective role against pathogens. In this PhD project, Th17 and Treg cells in nasal-associated lymphoid tissue were characterised and their relationship with nasopharyngeal carriage of pneumococcus studied in children and adults. Frequencies of Th17 and Treg in tonsillar tissue and peripheral blood samples obtained from children and adults were analysed for intracellular expression of IL17A and Foxp3 by flow cytometry. Also, tonsillar MNC and PBMC were stimulated by pneumococcal culture supernatant (CCS) derived from wild type stain D39. The ratio of Th17/Treg cells in NALT was studied in both children and adults together with their association with pneumococcal carriage. Numbers of Th17 and Treg cells in in tonsillar tissues were shown to be significantly higher than in peripheral blood in both children and adults. The ratio of tonsillar Th17/Treg was shown to increase with age and tended to be higher in pneumococcal culture negative children than in culture-positive. It is suggested that the balance of Th17/Treg is a crucial determinant of pneumococcal clearance or persistence/carriage in human nasopharynx. A significant increase in numbers of Th17 and Treg cells were shown following pneumococcal CCS stimulation. CCS derived from isogenic mutant strains (i.e., Ply- and CbpA-) elicited lower numbers of Th17 and Treg cells. It is suggested that pneumococcal proteins including Ply and CbpA may activate Th17 and Treg cells in human NALT, and therefore may contribute to the regulation of pneumococcal carriage or clearance in human nasopharynx. Induction of Th17 and Treg from tonsillar MNC were studied using tonsillar MNC depleted of activated and memory T cells. Stimulation with pneumococcal CCS induced Th17 from naïve T cells in tonsillar MNC in the presence of exogenous cytokines (i.e., TGF-β/IL21/IL1-β). TGFβ was shown to be crucial in Treg induction. Thus, the induction of both Th17 and Treg in human tonsillar tissue may be common in humans especially in children during natural infection/carriage, and the balance of the two may determine the clearance or carriage of pneumococcus in nasopharynx. Pneumococcal proteins including pneumolysin (Ply), its toxoid (PdB) and choline binding proteins (CBP) were shown to activate and promote Treg and Th17 cells in tonsillar MNC, thus they may play an important part in modulation of pneumococcal carriage in human nasopharynx. Understanding the development of natural immunity to pneumococcus and to pneumococcal proteins in particular may provide important information in the development of protein-based vaccines against pneumococcal infection in humans. Key words: Pneumococcus, Tonsillar MNC, Treg cells and Th17 cells.

Streptococcus pneumoniae (pneumococcus) is a leading cause of childhood morbidity and mortality around the world. The available polysaccharide-based vaccines are limited by either low efficacy in young children or narrow serotype coverage. Recent research has focused on developing protein vaccines. Pneumococcal pilus-1 proteins play an important role in pneumococcal adherence to host respiratory epithelium and subsequent bacterial colonization or invasion; and may therefore be an effective vaccine candidate. This PhD project investigated natural immunity in humans to pneumococcal pilus-1 proteins and its association with nasopharyngeal carriage of pneumococcus. Nasopharyngeal carriage of pneumococcus was analysed by bacteriological culture of nasal swabs on blood agar. Pneumococci were identified based on their colony morphology and optochin sensitivity; and further confirmed by PCR detection of pneumolysin gene in the isolates. Pneumococcal carriage was found to be common in young children, which gradually decreased with advancing age. PCR detection of pilus islet 1 (PI-1) gene revealed that percentage of pilus-1 positivity was low among these carriage isolates. This low prevalence may be associated with the recent introduction of pneumococcal conjugate vaccination, which covers the common piliated serotypes. ELISA based measurement of serum and salivary antibodies to pilus-1 proteins detected significant antibody levels to both RrgA and RrgB, presumably developed as a part of natural immune response in children and adults. An age-dependent increase in serum antibody levels to both RrgA and RrgB was also observed, and anti-RrgA appeared to develop earlier in childhood than anti-RrgB. Moreover, higher levels of antibody, especially anti-RrgA were found in children who were culture-negative than in those who were culture-positive for pneumococcus. It suggests that these naturally developed antibodies may contribute to the protection against pneumococcal carriage in humans. Using an in vitro model of human NALT, the study revealed that adenotonsillar tissues are important induction sites for immune response to these antigens, by priming B cell memory. The induction of antibody secreting cells in NALT was enumerated by ELISpot assay; and the antibody production was measured by antigen-specific ELISA. In vitro stimulation with a wild type (TIGR4) pneumococcal culture supernatant (containing both RrgA and RrgB proteins) induced a significant memory B cell and antibody response in adenotonsillar cells. Current carriage in vivo enhanced the memory B cell response and antibody production. Flowcytometric analysis of CFSE labelled adenotonsillar MNC suggested that pneumococcal pilus-1 proteins RrgA and RrgB were capable of stimulating CD4+ T cell proliferative response in human NALT. Stimulation with pneumococcal CCS induced Th17 related cytokines production in both human adenotonsillar MNC and PBMC culture supernatant. Importantly, this in vitro production of Th17 cytokines after stimulation with TIGR4wt CCS was significantly higher than that of CCS derived from its isogenic RrgA-/- and RrgB-/- mutants, indicating a contribution from both of these proteins. The ability of these pilus-1 proteins to stimulate CD4+ T cell proliferation and Th17 response may contribute to the natural immunity to pneumococcus in humans. This study has revealed that both of these pilus-1 proteins, RrgA and RrgB are immunogenic and are capable of priming for memory B and T cell response in human NALT. These findings aid to our understanding on the naturally developed immune response to pilus-1 proteins, and may inform future vaccination strategy with intranasal immunisation containing protein antigens against pneumococcal infection.

Cells are continuously exposed to different intracellular and extracellular mutagens, which can damage several different molecules, including DNA. To ensure survival, cells have evolved various defence and repair mechanisms. Mismatch repair (MMR) is the mechanism that serves to repair mismatched bases in DNA that are missed by the proof reading activity of DNA polymerases. Besides this, MMR also corrects base mismatches formed by altered bases modified by certain chemical mutagens. Thus, MMR is important to avoid mutagenesis and maintain genome fidelity. MMR is a complex, highly conserved pathway that involves a range of proteins along with several accessory proteins. In T. brucei, as in other eukaryotes, MMR core functions are carried out by bacterial MutS and MutL homologues, working as heterodimers: MSH2α (MSH2-MSH3) and MSH2β (MSH2-MSH6), and MLH1-PMS1, respectively. To date, only MSH2 and MLH1 function have been examined and only in bloodstream form (BSF) T. brucei cells. It was observed that MMR mutants are tolerant towards methylation damage, exhibit microsatellite instability and display elevated rates of homologous recombination between imperfectly matched DNA molecules. This confirmed the role of MMR in genome maintenance in BSF T. brucei. More recently, it was observed that BSF MSH2 mutants were sensitive towards oxidative damage, though the same phenotype was not observed in MLH1 mutants. This suggests that some aspect of the MMR machinery acts to protect BSF T. brucei cells against oxidative stress, but the machinery and mode of action is unknown. In this study we have generated null mutants of MSH2 and MLH1 in procyclic form (PCF) T. brucei cells, and MSH3 and MSH6 mutants in BSF cells. Characterization of tolerance to DNA methylation damage and evaluating microsatellite stability shows that each gene acts in MMR in both the life cycle stages, with the exception of MSH3, where null mutants show no discernible phenotypes. Mutants were also analyzed for their action towards oxidative stress in both the life stages and, remarkably, we find life cycle stage differences, with MSH2 mutants displaying hydrogen peroxide sensitivity and resistance in the BSF and PCF, respectively. The same phenotypes are not seen in MLH1 mutants, and we show that resistance to hydrogen peroxide in PCF cells is due to adaptation during the loss of MSH2. We have also shown that PCF MSH2 mutants may show a decrease in microsatellite instability when subjected to oxidative stress. This leads to the hypothesis that there might be an unidentified system, apart from MMR, present in T. brucei PCF cells that works as a defence in response to oxidative stress and can assume greater prominence when MSH2 is lost. Although we have tried to explore various cellular processes that might contribute this activity, our results are inconclusive. MSH2 and MLH1 have also been epitope tagged to explore the subcellular localization of these proteins and to ask if any changes in expression levels or changes in localisation are seen when subjected to oxidative stress. These preliminary data suggest that both factors are nuclear and cytoplasmic. We have also tried to ask if MSH2 and/or MLH1 co-localize with either MSH5 or MSH4, which are MutS-like factors that act in meiosis in other eukaryotes, but whose functions have not been explored in T. brucei. However, our attempts at this analysis have been unsuccessful.

The theme of this thesis is to study the interactions of non-typable Haemophilus influenzae (NTHi) and Pseudomonas aeruginosa (PA) with intact human respiratory mucosa in vitro. Recent evidence suggests that bacteria are mainly associated with respiratory mucus during exacerbation of chronic bronchitis but penetration of antibiotics into respiratory mucus is generally poor. A study was therefore performed to evaluate the effects of 0.25 and 0.5 minimal inhibitory concentrations of amoxycillin, loracarbef (a new carbacephem) and ciprofloxacin on NTHi infection of adenoid organ cultures in an agar-embedded model in which only the intact respiratory mucosa was exposed to bacteria-containing culture medium. The results from this study may help explain the clinical efficacy of antibiotics in treatment of bronchial infection despite poor antibiotic penetration into respiratory secretions. By using the same organ culture model the effects of NTHi infection of intact human bronchial mucosa was also studied. NTHi infection of bronchial organ cultures was associated with ultrastructural damage compared with uninfected organ cultures after 24h incubation. This damage was similar to the pattern observed in adenoid organ cultures described earlier. Similar experiments using nasal turbinate tissue showed virtually no adherence of NTHi to nasal respiratory mucosa suggesting that there may be a difference in epithelial surface receptors for NTHi between adenoid and nasal turbinate mucosa. Infection of adenoid organ cultures with an air-mucosal interface by PA caused significant ultrastructural damage (mitochondrial damage, loss of cilia, cytoplasmic blebbing and extrusion of cells from the epithelial surface) and slowing of ciliary beat when assessed by transmission electron and light microscopy respectively after 8h incubation. PA was found to cause disruption of epithelial tight junctions and adhere to basement membrane collagen. A matrix-like material was probably produced by PA which bridged PA with respiratory mucosa and might therefore be a PA adhesin. PA formed bacterial biofilms on the surface of respiratory mucosa that might have hindered its removal by the mucociliary clearance mechanism. These findings might help explain the difficulty in eradicating PA from the lower respiratory tract of patients with cystic fibrosis and bronchiectasis. The organ culture model with an air-mucosal interface was also used to study the effects of a bacterial toxin on intact human respiratory mucosa. An exotoxin of PA, pyocyanin was found to cause significant mucosal damage to adenoid organ cultures when assessed by transmission electron microscopy. Moreover, by using a newly developed transmission electron microscopy method to assess orientation of central microtubules of cilia and foot processes, pyocyanin was found to cause significant disorientation of the central microtubules of respiratory cilia but not the foot processes. PA pyocyanin may therefore have a role in the pathogenesis of PA infection in vivo. By using these organ culture models of intact human respiratory mucosa, bacteria interactions with human respiratory mucosa can be studied using the transmission, scanning electron, and light microscopy methods described in this thesis. Potential virulent factors for NTHi and PA can be tested and the mechanisms of bacterial pathogenesis can be studied further to advance current understanding of bacterial interactions with the human respiratory tract mucosa that may lead to the development of novel therapies.