Plasmodium falciparum is an obligate intracellular parasite, which causes 95% of worldwide malaria cases annually. Malarial symptoms occur during replication of parasites inside erythrocytes. Multiple cycles of host cell invasion, replication inside a parasitophorous vacuole (PV) and escape from the host cell result in gradually increasing parasitaemia. Escape from the host cell (egress) is regulated by proteases and may involve perforin-like proteins. PfSUB1, a subtilisin-like serine protease, is essential to P. falciparum blood stage development and egress. Just before cell rupture, the protease is discharged into the PV, where it is processes multiple parasite surface proteins and PV proteins. The main aim of this project was to analyse the function of PfSUB1 by three approaches which relied on in vitro biochemical analyses and P. falciparum transfections. Firstly, a conditional knockdown approach was used to analyse the function of PfSUB1 using the FKBP regulatable system. Two complementary strategies were used: down-regulation of PfSUB1 levels using a C-terminal FKBP domain and inhibition of PfSUB1 activity using an N-terminal FKBP fusion with the PfSUB1 prodomain (a potent inhibitor of recombinant PfSUB1). Expression of recombinant PfSUB1-FKBP in Sf9 insect cells demonstrated that FKBP does not interfere with PfSUB1 activity, FKBP was successfully integrated into the endogenous pfsub1 gene. In the second approach, in vitro studies showed that recombinant E. coli-derived FKBP-prodomain fusion protein inhibits recombinant PfSUB1. Strong evidence was obtained which indicates that episomal expression of a non-regulatable prodomain in P. falciparum is not tolerated by the parasite. Secondly, to further characterise the enzyme, an in silico approach was used to predict new SUB1 substrates, and a proteomic approach was taken to validate substrates in vitro. Several putative new substrates were identified, which suggest that PfSUB1 is a multifunctional enzyme with numerous roles in invasion and egress. Finally, attempts were made to establish a PfSUB1-sensitive FRET-based system to monitor PfSUB1 activity in vivo. A recombinant FRET reporter was expressed in E. coli; this was shown to exhibit FRET and to be PfSUB1-sensitive in vitro. Preliminary in vivo data are presented, which suggest that protease-sensitive FRET is possible in P. falciparum.

During haematopoiesis multipotent stem cells generate all cellular components of the blood including lymphocytes. Despite great progress in the isolation of lineage restricted progenitors, the exact precursor-product relationship of these subsets remains poorly understood. In particular the exact branch point of T- and B-cell development in the bone marrow has not been unequivocally mapped. The aim of my project was to investigate the developmental relationship of various progenitor subsets in normal mice and during an acute inflammation. In order to permanently identify all cells which emanated from early lymphoid compartments we generated a mouse model in which a Cre recombinase was inserted into the Rag1 locus and functional Cre activity would result in activation of an eYFP reporter. Expression of the reporter was found in all T- and B-cells and in a significant subset of NK-cells and dendritic cells. Furthermore this model allowed the prospective isolation of an ‘ELP analogue’ and two subsets of CLPs on the basis of their reporter expression. Functional analysis of these subsets in vivo demonstrated comparable developmental properties with slightly different kinetics. Furthermore, in vitro analysis of isolated progenitors established that reporter-positive CLPs were significantly more advanced in their commitment to the B-cell lineage. We extended our studies by investigating the impact of an acute systemic inflammation on the size and composition of early haemato-lymphoid subsets. Administration of LPS or heat-inactivated E. coli to mice in vivo resulted in a complete halt of bone marrow lymphopoiesis. In addition, a marked decrease in the number of myeloid progenitors accompanied by upregulation of Sca-1 on haematopoietic progenitors was observed. These inflammation-induced changes were found to be mainly caused by IFNγ and to a lesser extend by TNFα, thus identifying these cytokines as key mediators for the infection-induced regulation of haematopoiesis.

Patients with systemic Juvenile Idiopathic Arthritis (sJIA) have elevated serum levels of inflammatory cytokines. Treatment with interleukin-1 (IL-1) receptor antagonist (Anakinra) shows remarkable improvement in some sJIA patients. The hypothesis of this thesis is that genetic variations in IL-1 family genes contribute to disease pathogenesis. To investigate this, a two-stage case-control association study of 20 candidate genes was performed. Selected tagging SNPs were tested for association in 130 sJIA patients and 146 controls in stage-1 of the study. SNPs at significantly different frequencies in the cohorts were genotyped in an additional 105 sJIA patients and 184 controls, and stratified metaanalysis of the two-stage data performed. Analysis was also performed with 4,671 controls from the Wellcome Trust Case Control Consortium (WTCCC). No associations were found with caspase-1, cryopyrin, or IL-18. Significant disease associations were identified with SNPs in the ligand IL1A, the receptor antagonist IL1RN, and a two-SNP haplotype in the IL-18 antagonist IL18BP. Associations were also identified in the decoy receptor IL1R2, and the co-receptor IL1RAP, although these were not confirmed when re-analysed with the WTCCC controls. Transient transfection assays with haplotype constructs, performed by Dr Wen, showed that the IL18BP haplotype affected gene transcription levels in vitro. This effect was not however reproduced using PBMCs from healthy individuals. Allele specific binding to one of the haplotype SNPs was predicted in silico, but no evidence for this was seen in EMSA experiments. Further functional studies are required to corroborate involvement of this haplotype in disease. In summary, this study has identified genetic associations for susceptibility to sJIA with a number of IL1 family members. These results indicate that there may be aberrant control of IL-1 activity in patients with sJIA. Further work is required to determine how these associated SNPs affect IL-1 activity, and thereby the inflammatory response in sJIA.

The interleukin-1 gene family in humans comprises eleven members (IL-1F1-F11) that act as either functional agonists or antagonists of inflammation. Prior to this project, only two members of the IL-1 family had been identified and characterized in the chicken. The aim of this project was to identify, clone and characterise novel IL-1 family members in this species. EST sequences representing IL-1F5 (IL-36RN) and the secretory and intracellular structural variants of IL-1 receptor antagonist (IL-1RN) were identified by their similarity with chicken IL-1β. Chicken IL-1RN (chIL-1RN) cDNAs were isolated from LPS-stimulated HD11 cells. Two further putative splice variants (SVs) of both chIL-1RN structural variants were also isolated. Both full length variants of chIL-1RN exhibited biological activity resembling that of their mammalian orthologues. The four SVs, however, were not bioactive. ChIL-1RN was constitutively expressed in lymphoid and non-lymphoid tissues as well as several cell subsets. In response to bacterial and viral infection, chIL-1RN expression was inducible. Chicken IL-36RN was cloned from a liver cDNA. In mammals, this cytokine is an IL-1RL2 receptor antagonist and downregulates LPS-mediated inflammation. IL-1RL2 agonist ligands have not been identified in the chicken; therefore, an alternative bioassay to establish its function was attempted. Using a macrophage cell line, chIL-36RN did not inhibit endotoxin-mediated inflammatory effects. Constitutive IL-36RN expression was found in all tissues and cell subsets examined. In response to viral infection, chIL-36RN expression was significantly downregulated. The eleven human IL-1 genes are encoded at three separate loci. Nine of these genes, including IL-1β, IL-1RN and IL-36RN, are present at a single locus. In the chicken genome, the equivalent locus contains only IL-1β. Neither IL-1RN nor IL-36RN were identifiable anywhere in the chicken genome. Future work will seek to determine the true extent of the repertoire of chicken IL-1 family genes.

The elimination of a wide variety of infections requires the production of high affinity antibodies specific for the invading pathogen. The generation of these high affinity antibodies depends on the ability of B cells to recognise and internalise antigens from the surface of antigen-presenting cells (APCs) in an affinity-dependent manner. B cells expressing high affinity B cell receptors (BCRs) internalise, process and present more antigen, obtain better T cell help, and are selectively expanded over lower affinity equivalents. However, the molecular mechanisms by which B cells extract antigens for presentation remain unclear. Using a new fluid and flexible membrane substrate to mimic APCs, we show that B cells acquire antigen by dynamic myosin IIA-mediated contractions that pull out and invaginate the presenting membranes. Invaginations containing high affinity BCR-antigen microclusters were able to withstand the force of these contractions and recruit clathrin resulting in endocytosis. In contrast, low affinity BCR-antigen bonds quickly ruptured aborting internalisation. Thus we conclude that coupling contractility to endocytosis permits B cells to discriminate between antigen affinities, which provides a mechanism for the selective advantage seen for high affinity B cell clones in vivo. Disruptions in BCR-antigen internalisation has been linked to the growth of malignant B cells and the development of autoimmunity. Therefore, ultimately, our results could contribute to the effective design of future therapeutics for B cell diseases.

TCR gene transfer can generate tumour antigen-specific T cells for adoptive immunotherapy. Following TCR gene transfer, transduced T cells usually display the same functional avidity as the parental clone from which the TCR was isolated. However, tumour-antigen specific T cells typically recognize over-expressed self-antigen and are often of low/moderate avidity. It is known that optimal recognition of target cells by CTL requires binding of the cognate peptide MHC class I complex (MHCI) by both TCR and the CD8 co-receptor. Some CD8β chain mutations have been shown to increase CD8 binding affinity with peptide/MHCI and enhance T cell effector function. Murine CD8β chain mutants were generated affecting MHC binding sites (L58R, S53L, S54V and L58R/I25A) or glycosylation sites (T120A, T121A, T124A, and T120A/T121A/T124A). The mutated CD8β molecules were introduced into murine splenocytes using retroviral vectors together with tumour antigen-specific TCRs. The CD8β mutants or control CD8β wild type (WT) chains were first introduced into CD8aa T cells obtained from CD8β knockout mice. All T cells were co-transduced to express the murine F5-TCR which recognizes the model tumour antigen, influenza A nucleoprotein (NP366) presented by H2-Db. The L58R MHC binding CD8 co-receptor mutant (L58R) demonstrated better IFN-γ and IL-2 production in response to relevant peptide while the CD8 glycosylation mutant (T120A/T121A/T124A) mutant demonstrated the opposite effect. The in vitro function of CD4+ T cells transduced with F5-TCR showed that IL-2 and IFN-γ production was enhanced with CD8 co-receptor. In addition, introducing a L58R mutation in the CD8 co-receptor could further increase this effect. The effects of the human CD8 co-receptor with a homologous mutation (I59R) was also investigated in human CD4+ T-cells with a CMV-specific TCR. In vivo studies showed that introducing the F5-TCR alone did not endow CD4+ T cells with significant protection against injected lymphoma cells expressing NP366. However adding CD8 co-receptor to the CD4+ T cells enhanced tumour protection. The genetically modified CD4+ T cells persisted for greater than three months in surviving mice and when re-challenged with antigen the CD4+ T cells with both F5- TCR and CD8 co-receptor had greater proliferative capacity and had more central memory phenotype cells.

No description available.

Background: Chronic pain is a major health problem due to both its prevalence and the difficulties associated with its adequate diagnosis and treatment. The field of medical physiology has offered possible answers to those difficulties, namely; physiological pressure algometry and electro-physiological neuromodulation. Aims: To explore the diagnostic potential of a physiological pressure algometry device in patient populations suffering from hyperalgesia due to chronic pain; and to use this device to, both, assess and quantify the therapeutic response of patients undergoing various electrophysiological neuromodulation therapies for chronic pain. Methods: Five clinical phases, I–V, conducted on a total of two hundred and seven patients suffering from chronic pain. Results: Phase I; there was a highly significant negative correlation between the pressure pain threshold (PPT) values and each of the visual analogue scale (VAS) and numerical rating scale (NRS) scores; for PPT and VAS; ρ= -0.453 and for PPT and NRS; ρ= -0.413 (p<0.0005 in both cases). Phase II; there were clinically and statistically significant improvements in each of the VAS and NRS scores, and PPT following the activation of the spinal cord stimulation equipment (p<0.0005 in all three cases). Phase III; for the active percutaneous electrical nerve stimulation (PENS) treatments, there were clinically and statistically significant improvements in the NRS scores and PPT after therapy (p<0.0005 in both cases). For the sham treatments, there were neither clinically nor statistically significant changes (p=0.317 and p=0.055 respectively). Phase IV; there were clinically and statistically significant reductions in the median NRS scores; from 7.25 before PENS therapy to 3 after therapy (p<0.05). Phase V; there were clinically significant improvements in the PPT and NRS/VAS scores following the activation of the peripheral nerve stimulation equipment. Key Words: chronic pain, medical physiology, physiological pressure algometry, electrophysiological neuromodulation.

Cronobacter spp. (previously known as Enterobacter sakazakii) is a diverse bacterial genus consisting of opportunistic food-borne pathogens affecting all age groups, with particularly severe clinical complications such as meningitis and necrotising enterocolitis in neonates and infants. In this study, a multilocus sequence typing (MLST) approach has been established to span the entire Cronobacter genus, by employing the alleles of 7 housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB and ppsA, total length 3036 bp). The 325 Cronobacter spp. strains used in the study included isolates from the highly publicised Cronobacter cases from USA in December 2011. The scheme identified 115 sequence types (ST) across the seven Cronobacter species. Multilocus sequence analysis (MLSA) revealed considerable diversity in the genus, with intraspecific variation ranging from low diversity in C. sakazakii to extensive diversity within some species such as C. muytjensii and C. dublinensis including evidence of recombination events between species. An evolutionary analysis revealed the Cronobacter genus to have evolved 45-68 million years ago, during the period of evolution of flowering plants. The MLSA was also used in a polyphasic study for the formal recognition of two new species – C. universalis and C. condimenti. The MLST scheme also revealed the high level of clonality in the species C. sakazakii and C. malonaticus. ST4 was found to be a highly stable clone of C. sakazakii, and a strong association was established between the C. sakazakii ST4 clonal complex with neonatal meningitis cases. The curated MLST database is hosted with open access at: http://www.pubmlst.org/cronobacter. The diversity and virulence study of the organism was then extended to a whole genomic level by analysing eleven high quality draft Cronobacter spp. genomes spanning the seven species, including a representative of the C. sakazakii ST4 lineage, together with two publicly available genomes. Genome comparison revealed that pair-wise DNA sequence identity varies between 89 and 97% in the seven Cronobacter species. The number of annotated genes per genome varied between 3,700 and 4,200. The dataset revealed a pan-genome of more than 6000 genes, 32% of which was found to be conserved across the genus. Genes encoding adhesins, type six secretion systems, metal resistance genes as well as prophages were found in only subsets of genomes and have contributed considerably to the variation of genomic content. Sets of universal core genes and accessory genes unique to each species were identified, which can be used for designing genus/species specific detection assays. The C. sakazakii species was found to be unique in the Cronobacter genus in encoding genes enabling the utilization of exogenous sialic acid which may have considerable clinical significance. The C. sakazakii ST4 genome did not reveal any unique lineage-specific virulence related genes, suggesting the role of selective gene expression and increased host exposure in the pathogenicity of the organism.

Obesity and Periodontitis are two common chronic inflammatory diseases. Based upon the hypothesis that obesity associated systemic inflammation may affect susceptibility to chronic infectious diseases like periodontitis, the aim of this PhD programme was to investigate the association between overweight/obesity and periodontitis. Five studies were conducted: 1) a systematic review to summarise the current evidence on the association including a quantitative meta-analysis of odds ratios (ORs) for having periodontitis in overweight or obese individuals 2) a secondary analysis of individual patient data (n=333) ascertaining the association between overweight/obesity and the extent/severity and treatment response (2 months) of individuals with severe periodontitis 3) a case control analysis of 286 age-matched individuals to assess the odds of periodontitis diagnosis based on overweight or obese status, 4) a prospective cohort study (n=115) investigating the relationship between obesity and periodontal treatment clinical response, 5) a mechanistic study of twenty gingival specimens assessed for differential miRNAs expression between obese and normal weight individuals. Study 1 demonstrated a statistically significant association between overweight and obesity with diagnosis of periodontitis (ORs range= 1.8-2.3). In Study 2, obesity and overweight were statistically significant predictors of clinical periodontal response at 2 months (p<0.05) independently of dental plaque levels. Results from the Study 3 confirmed increased odds of diagnosis of periodontitis in overweight (OR=2.56) and obesity (OR=3.11) after adjusting for known confounders. Study 4 demonstrated that measures of body composition were predictors of poorer non-surgical periodontal treatment response (p<0.05). Study 5 confirmed statistically significant different miRNA signature profiles of gingival tissues between normal weight and obese individuals. In conclusion, this PhD programme provides evidence of a robust association between overweight/obesity and periodontitis prevalence, extent and severity, and treatment response. The results of this thesis support the classification of obesity as a risk indicator for periodontitis.