No description available.

Streptococcus pneumoniae is a commensal human pathogen and the causative agent of invasive pneumococcal disease. Carriage of the pneumococcus in the nasopharynx of humans is thought to be mediated by biofilm formation. Isogenic populations of S. pneumoniae grown under biofilm conditions frequently give rise to morphological colony variants, including small colony variant (SCV) phenotypes. This work employs phenotypic characterisation and whole genome sequencing coupled with ultra-pure liquid chromatography mass spectrophotometry (UPLC/MSE) of biofilm-derived S. pneumoniae serotype 22F pneumococcal colony morphology variants to investigate the diversification during biofilm formation. Phenotypic profiling revealed that SCVs exhibit reduced growth rates, reduced capsule expression, altered metabolic profiles and increased biofilm formation compared to the parent strain. Whole genome sequencing of 12 SCVs from independent biofilm experiments revealed that all SCVs studied had mutations within the DNA-directed RNA polymerase delta subunit (RpoE). Mutations included four large-scale deletions ranging from 51-264 basepairs (bp), one insertion resulting in a coding frameshift and seven nonsense single nucleotide substitutions that result in a truncated gene product. UPLC/MSE of the SCVs revealed up-regulation of a common sub-set of stress-inducible proteins which are part of an interaction network consisting of the 60 kDa chaperonin, chaperone protein DnaK, cell division protein FtsZ and manganese superoxide dismutase. This work links mutations in the rpoE gene to SCV formation and enhanced biofilm development in S. pneumoniae, with important implications for colonisation, carriage and persistence of the organism. Furthermore, consistent mutation within the pneumococcal rpoE gene presents an unprecedented level of parallel evolution in pneumococcal biofilm development. This work has given insight into the genetic diversity which may arise during pneumococcal colonisation which in turn may help inform future drug and vaccine design.

Asthma, a chronic TH2-mediated inflammatory disease of the airways, is the most common form of allergy in the Western world, affecting 300 million people worldwide. Epidemiological studies have shown that asthma is associated with mood disorders, such as anxiety and depression, and numerous experiments have reported that asthma induces functional changes in neuronal fibres of the peripheral nervous system (PNS), which innervate the brain. It is unknown, however, how allergic lung inflammation impacts on the central nervous system (CNS). The ability for peripheral inflammation to impact on the brain, altering behaviour and neuronal activity in the CNS, is a well-recognised and physiological phenomenon, known as immune to brain communication, but has, until now, only focused on how innate pro-inflammatory and TH1, but not TH2, type immune responses impact on the brain. Critically, immunomodulatory therapeutics, which involve stimulation of an innate pro-inflammatory immune response, are currently being developed for the treatment of asthma, highlighting the importance of understanding the effect of allergic lung inflammation and its treatment on the brain. Consequently, using acute and chronic localised TH2 models of inflammation, we investigated how allergic lung inflammation impacted on the CNS and subsequently determined the secondary impact of immunomodulation with the Toll-like receptor 7 (TLR7) agonist resiquimod. Acute TH2 inflammation in the peritoneum and lung was found to communicate with the brain, via a vagal route of communication. Crucially, it led to a distinct pattern of neuronal activity, with no changes in sickness behaviour or CNS inflammation, changes widely different to those known to occur following systemic TH1 inflammation. At chronic stages of lung inflammation, changes in genes associated with synaptic plasticity in the brainstem and altered expression of the GABAB receptor and brain-derived neurotrophic factor in the hippocampus were observed, firstly providing a CNS-dependent biological explanation for airway hyperresponsiveness, a critical pathological symptom of asthma, and secondly offering a biological justification for the prevalence of mood disorders in asthmatic patients. Resiquimod treatment in allergic animals was associated with attenuated central inflammatory responses, as compared to treatment in healthy animals, encouraging and reassuring in terms of patient well-being and, critically, also insinuating that safety of therapeutics differs in diseased, as opposed to healthy individuals. The results in this thesis are some of the first to identify that physiological inflammatory diseases impact on the CNS, highlighting the importance of immune to brain communication on pathological and psychopathological symptoms of a disease, and additionally demonstrating how inflammatory conditions can modify the off-target effects of a drug. Not only do these results provide a foundation for the future of immune to brain communication research, namely understanding how physiological inflammatory diseases impact on the CNS, but also have the potential to be translational and emulated in a clinical setting.

The collectins surfactant protein A (SP-A) and SP-D are found in the lining fluid of the lung and form an important component of innate pulmonary defenses; they consist of a N-terminal region, a collagenous region, a neck region and a carbohydrate recognition domain (CRD). SP-A and SP-D bind to and have antiviral properties in vivo against the enveloped viruses respiratory syncytial virus (RSV) and influenza A virus (IAV). A recombinant fragment of SP-D (rfhSP-D) has also been developed and is composed of the neck, CRD and a short collagenous region of SP-D. The rfhSP-D retains the ability to bind to RSV and IAV. Human rhinovirus (HRV) is a non-enveloped virus and is the predominant cause of the common cold. HRV can also cause exacerbations in patients with chronic airway disease. Based on the known antiviral effects of SP-A and SP-D, and their localisation in the upper and lower airways, the present thesis sought to investigate the research question 'Can SP-A, SP-D and rfhSP-D bind to and inhibit infectivity of HRV?' Methods to purify and detect HRV were established and used in experiments with SP-A and SP-D purified from bronchoalveolar lavage and rfhSP-D expressed in Escherichia coli. A novel interaction between SP-D and rfhSP-D with HRV was identified using co-irnmunoprecipitation and surface plasmon resonance; no such interaction was detected with SP-A. Binding was calcium dependent and inhibited by the presence of sugars, indicating that binding is via the CRD. Monolayers of HeLa cells, 16HBE cells or undifferentiated primary nasal epithelial cells were infected with HRV coincubated with collectins and the infection level was assessed using flow cytometry. The presence of collectins did not significantly alter the infection level. This is the first report of a component of the innate immune system binding to the surface of HRV.

No description available.

In order to track cell migration, the project used a transgenic mouse system which ubiquitously expressed the fluorescent protein kaede that undergoes photo-conversion when exposed to violet or ultraviolet (UV) light. By inducing inflammation in peripheral tissues (e.g. the ear pinna on the hind footpad) and subsequently exposing the tissue to UV light, it was possible to spatiotemporally track the fate of the cells at these tissue sites. The study revealed that tissue resident cells do not constitute the majority of the migratory population and that immune cells must first be recruited to the site of challenge prior to migration. Migration from the site of challenge to the draining lymph node occurs within the first 48 hours post injection and returns to baseline over the following 24 hours. By combining the above approach with the YAe/Eα system to track antigen presentation, the study has also shown that antigen presentation persists for the first 24-36 hours and the majority of cells presenting antigen are CD103+CD11b+ dendritic cells. In collaboration with UCB, CyTOF profiling was performed on the migratory population to identify the diversity of these cells, identifying CD4+ T cells, CD8+ T cells, γδ T cells, B cell and neutrophils in the draining lymph node originating from the injection site. Therefore, it is suggested that the migratory cells work in tandem with one another to control the initiation of adaptive immunity and determine the nature and the magnitude of the immune response. Additionally, the nature of an inflammatory stimulus can alter the magnitude and composition of the migratory population. This study highlights that our current knowledge regarding the initiation of adaptive immunity, in particular, the kinetics and phenotype of the migratory cells, remains limited. Further developing our understanding of the migratory population, antigen presentation and the kinetics of antigen presentation may help to identify new targets for immunomodulation for the treatment of inflammatory disorders or the development of new and improved vaccine adjuvants.

In order to combat harmful pathogens the innate recognition part of the immune system uses a variety of receptors including CD14, TLR4, TLR2 and SR (MARCO). These receptors are expressed on most human monocytes and macrophages. CD14 recognizes and bind lipopolysaccharide (LPS), the main causative agents of sepsis and endotoxic shock. Signaling from CD14-LPS and TLR4/MD-2 complex activate the nuclear factor kappa B (NF-κB) family of transcription factors and JNK, a member of MAP kinase (MAPK) family. This signaling results in the production of the proinflammatory cytokines such as TNF-α and IL-1β among others. In addition, LPS also activates the DNA-repair and protein modifying enzyme poly (ADP-ribose) polymerase-1 (PARP-1). Consequently, the effect of PJ-34, a potent inhibitor of PARP on PARP-1 activation induced by LPS was studied on THP-1 cells. In response to LPS, PJ-34 reduced the PAR formation and down regulates the expression of CD14, TLR2 and TLR4. However, MARCO expression was up regulated. It was also observed that PJ-34 reduced the production of TNF-α, IL-1β and No in response to LPS. PJ-34 was also involved in a reduction of the activation of NF-κB in response to LPS but, did not have an effect in JNK activation. The physical association of CD14 and MARCO receptors was examined in response to LPS. It was found that PJ-34 reduced the colocalisation of these pair of receptors and regulates their expression at gene level. Furthermore, PJ-34 regulates expression of a number of proteins on the THP-1 cells in response to LPS.

Cystic fibrosis (CF) lung disease typically results in pulmonary infections and inflammation that produces progressive respiratory failure. The most common pathogen in adult patients with CF is Pseudomonas aeruginosa (PA), which in the majority of patients chronically colonises the airways and is associated with a neutrophil-dominated host response. This neutrophilic response fails to clear the infection but contributes to progressive lung injury. Prior to the establishment of chronic PA colonisation, patients experience intermittent pulmonary PA infections that can be cleared either by host responses or antibiotic eradication therapy. Recent attention has focused on the role of the cytokines interleukin-17 (IL-17) produced by T helper 17 (Th17) cells, in anti-microbial defences. IL-17 plays a critical role in the generation and recruitment of neutrophils to sites of infection and has been implicated in responses to PA as well as other pulmonary pathogens. Clinical data has also suggested a potential role for IL-17, Th17 cells, as well as other cellular sources of IL-17 in the pathogenesis of human CF pulmonary disease. An additional Th17-related cytokine IL-22 has previously been shown to hold a critical role in defence against acute pulmonary infections, as well as reparative functions in pulmonary disease. However, the functions of this cytokine are not clear cut, since pro-inflammatory effects are also seen depending on the local environment in which it acts. Given the pivotal role of IL-17 in neutrophilic immune responses, in this thesis I hypothesised that IL-17A is beneficial in early CF lung disease where it aids clearance of PA and delays the onset of chronic PA colonisation. However, as the organism adapts to the hostile environment and/or the lung environment becomes more conducive to bacterial growth then chronic PA infection inevitably develops; in this setting I proposed that IL-17A becomes pathogenic by driving persistent, damaging neutrophilic inflammation. In addition, I hypothesised that Th17 cells are a source of IL-17A in PA infection and that, due to persistent antigenic-stimulation in CF patients the Th17 response may be altered to that seen in healthy controls. I further proposed that IL-22 may have a reparative and protective role in persistent pulmonary PA infection, but that the cytokine's reparative effects become overwhelmed or IL-22 becomes pro-inflammatory and pathogenic in later CF lung disease with chronic PA colonisation. These hypotheses were addressed via the use of both clinical samples from patients with CF and a murine model of persistent PA infection.

The human body defends itself from pathogen infections through very complicated processes. They include host innate, adaptive and cellular intrinsic immunities. Host innate and adaptive immunities have been studied extensively in past decades, and more detail is being continuously revealed to us. Cellular intrinsic immunity has been recognised in more recent decades and has attracted great interest among researchers. Many cellular proteins have been identified to repress viral infections such as HIV, Influenza and Herpes Viruses. One aspect of intrinsic immunity, or intrinsic resistance, to herpes virus infections was demonstrated to involve nuclear structures known as ND10 (nuclear domain 10). With accumulating evidence about ND10, many components of ND10 that are involved in intrinsic immunity were identified. In the early stage of infection, HSV-1 and HCMV genomes become associated with ND10 components, which include PML, Sp100 and the hDaxx-ATRX complex. All these proteins were identified to repress HSV-1 and HCMV infection and replication. Viral proteins that have been identified to interact with PML, Sp100 and the hDaxx-ATRX complex and that counteract their anti-viral activities include ICP0 from HSV-1 and IE1 and pp71 from HCMV. Furthermore, recently studies on EBV proteins identified BNRF1 to interact with the hDaxx-ATRX complex, while EBNA1 appears to down regulate PML and EBNA-LP is involved in Sp100 dispersion from ND10. The evidence above raises the possibility that ND10 interacting proteins from different herpes viruses may be functionally interchangeable. IE1 interacts with ND10 to de-sumoylate and disperse PML and Sp100. There is also evidence of interaction between IE1 and hDaxx complex. pp71 was demonstrated to interact with and promote the degradation of hDaxx, and to disrupt the hDaxx-ATRX complex to stimulate HCMV infection and replication. For HSV-1, ICP0 was identified to degrade PML and Sp100, and counteract the effects of the hDaxx-ATRX complex on viral infection repression. Therefore, IE1 and pp71 were analysed for their ability to complement ICP0 null HSV-1, and they were demonstrated to stimulate ICP0 null HSV-1 infection to a large extent. In this study, the reverse investigation was analysed. To extend the study, EBV proteins that interact with ND10 were also analysed in stimulating HSV-1 and HCMV mutant viruses. The major findings in this thesis are summarised as follows: 1. IE1 and pp71 can separately and cooperatively stimulate ICP0 null HSV-1 infection in HepaRG cells and human fibroblasts. 2. The expression of IE1 and ICP0 in human fibroblasts can increase wt HCMV infection. 3. ICP0 can complement pp71 null HCMV infection but not IE1 null HCMV. 4. Two sequences that are essential for IE1 functions were identified and one IE1 sequence was identified to be HCMV specific on promoting viral infection and replication. 5. EBV proteins that interact with ND10 can stimulate ICP0 null HSV-1 and pp71 null HCMV infection. The data in this thesis demonstrated that the combination of IE1 and pp71 is interchangeable with ICP0 in HSV-1 infection and EBV proteins that interact with ND10 partially complement ICP0 null HSV-1 infection. The results indicated that the integrity of ND10 is an important part of cellular intrinsic resistance to HSV-1 infection. The observation on ICP0 complementing IE1 deletion HCMV indicated the HCMV specific role of IE1. ICP0, BNRF1 and pp71 are all involved in counteracting the hDaxx-ATRX complex repression on viral infection and replication. These three proteins were demonstrated to complement ICP0 null HSV-1 and pp71 null HCMV infection, which indicates the importance of the hDaxx-ATRX complex on repressing herpes viruses infection. Even though solid data in this thesis has confirmed the ND10 repression on herpes virus infection and the herpes viruses from different subfamilies counteracting this repression through different viral proteins, more detail about this intrinsic resistance mechanisms still require extensive investigation.

African trypanosomiasis is caused by a unicellular eukaryote that parasitizes multicellular organisms and causes medically and economically important diseases in humans (Human African Trypanosomiasis) and their domestic animals (African Animal Trypanosomiasis). Incidence is currently declining due to the application of present chemotherapy, although the drugs are old, toxic, difficult to administer and in some cases expensive, and of diminishing efficacy due to resistance. However, this trend needs to be sustained with the discovery of new compounds active against the resistant strains. These new treatment options must meet the current pharmacological requirements, must be parasite specific and must be relatively cheap to produce. Pharmacological manipulation of phosphodiesterases (PDEs), which hydrolyse cyclic Adenosine Monophosphate (cAMP), have been extensively studied in humans and found to have great therapeutic effect. Kinetoplastid genomes code for the same set of cyclic nucleotide-specific class 1-type phosphodiesterases, with catalytic domains similar to those of human PDEs. The locus of Trypanosoma brucei PDEB1/2 was found to be essential, by either genetic manipulation or the use of the pharmacological inhibitor CpdA, but therapeutic exploitation of TbPDEB1 has so far been hampered by its catalytic domain similarity to human PDEs. However, investigating the unique downstream cAMP signalling cascade, which includes the recently identified cAMP Response Proteins (CARPs), could reveal potentially new trypanosome-specific therapeutic targets. In this study we show that single knockout (sKO) of the CARP genes causes a decreased susceptibility to CpdA, and that null mutants of CARP2-4 display significantly increased intracellular and extracellular cAMP levels. A double knockout (dKO) of CARP2 also shows a significant growth defect. Conversely, overexpression of CARP3 causes a growth delay in both WT s427 and CpdA resistant (R0.8) cells, when exposed to CpdA, and were more sensitive to CpdA compared to other CARP overexpressing cells as well as the WT s427 and R0.8 controls. These cells also have significantly higher intracellular and extracellular cAMP levels relative to the control lines and the other CARP overexpressors. In the cells overexpressing CARP3, whether WT s427 or R0.8, the cellular content of both CARP3 messenger Ribonucleic Acid (mRNA) and protein decreases extensively within 6 h of CpdA exposure. The CARP3 protein has domains that are indicative of a role in protein-protein interaction, signalling, regulation and degradation and probably undergo acylation. Some experimental confirmation of these traits was obtained, using Co-immunoprecipitation (Co-IP) and Mass Spectrometry (MS), with the identification of Adenylyl cyclase (AC) GRESAG4s (also found through RNAinterference Target Sequencing (RITseq) and confirmed by quantitative Reverse Transcription PCR), and proteasome regulatory proteins (PRNs) in addition to membrane and flagellar binding proteins, as potential interactors. Preliminary Immunofluorescence (IF) microscopy showed that CARP3 localizes to plasma membrane ad the flagellum, CARP2 to specific bodies/organelles in the cytosol and CARP1 in the cytosol. RNA sequencing of overexpressing CARP3 reveals differentially expressed proteins involved in cell cycle and cytokinesis as well as transport proteins with several transmembrane domains, consistent with the proposed acylated membrane localisation, and with interaction of CARP3 with membrane proteins and ACs (GRESAG4 isoforms).Thus CARP3 has the domains, interactions and localization consistent with a regulatory role in cAMP metabolism. Thus the CARPs and especially CARP3 are interesting biological molecules providing key new insights into signalling and the cell biology of the trypanosome.