From early in life humans come into contact with a wide variety of infectious agents including bacteria, fungi and viruses. However, despite the vast array of potential pathogens, only a minority have the capacity to cause disease in a human host. This is largely due to the efficacy of the human innate and adaptive immune systems. The innate immune system consists of a variety of components including mechanical barriers, secretions, cells, excreted proteins, and serum proteins including the complement components and mannose binding lectin (MBL). MBL is a liver derived, acute phase, circulating serum protein that acts as a pattern recognition molecule. It is able to bind to a range of sugars presented in particular conformations on the surface of microbes and, having bound to its target, it can activate the lectin complement pathway and enhance complement-independent opsonophagocytosis. Over the last decade the biological importance of MBL has become increasingly apparent as indicated by the clinical consequences of the MBL deficient state. This has mainly been addressed by looking at the effect that MBL deficiency has on the susceptibility to a wide range of infections. More recently, interest has focussed on the role that MBL may play in the modulation of inflammation. It is well recognised that individuals differ in their susceptibility to, and severity of, both infectious and non infectious diseases. This raises the question, could the role of inherited factors governing host response to infection and inflammation be important The work described in this thesis investigates the role of MBL in two diseases that both involve an infective and inflammatory component, namely children with the chronic disease cystic fibrosis (CF) and those with acute inflammation/sepsis in intensive care. In clinical studies I show that MBL deficiency is associated with worse pulmonary function tests in adults with CF and an increased risk of death and/or lung transplantation in children with CF. The reasons for this were explored by examining MBL binding to bacterial pathogens seen in CF and by examining bronchoalveolar lavage (BAL) fluid from children with and without CF. Here I show that MBL can be detected in the BALs of children with both acute and chronic respiratory disease but not in the controls. In another study of 142 children admitted to paediatric intensive care, I demonstrate an association between MBL deficiency and the development of the systemic inflammatory response syndrome and an increased severity of sepsis. This effect of MBL is independent of age, sex, ethnicity, polymorphisms in the genes for TNF-a, IL-6, IL-10, angiotensin converting enzyme (ACE), plasma activator inhibitor 1 (PAI-1) and levels of antibodies to endotoxin. The work presented here highlights the importance of MBL in susceptibility to infection and may demonstrate a role for MBL in modifying the inflammatory response. These data may assist in furthering the understanding of mechanisms in disease pathogenesis as well as paving the way for exploratory research into MBL as a potential therapeutic agent.

In 2010 there were an estimated 219 million cases of malaria worldwide, accounting for approximately 0.66 million deaths. With growing resistance to current antimalarial agents a challenge is placed on the scientific community to provide efficacious and cost effective methods for the diagnosis and treatment of malaria. As protein phosphorylation regulates most aspects of cell life, understanding the function of protein kinases in malaria parasites has the potential to uncover novel drug targets. To this end, this work focused on the study of three essential Plasmodium falciparum kinases using two different chemical genetic approaches: γ-modified ATP analogues for the investigation of PfCK2; and covalent complementarity for the study of PfCLK1 and PfCLK3. The work presented here highlights the instability of the P-N bond of ATP phosphoramidates during the acidic conditions required for analysis by MALDI-TOF MS and seeks to overcome this through the development of alternative linkers to the γ-phosphate of ATP. Apparent IC[subscript 50] values recorded provide evidence of PfCK2α tolerating modification of the γ-phosphoryl group of ATP; however, no evidence was found to support GTP serving as an alternative co-substrate of PfCK2α. The gatekeeper mutant kinases PfCLK1F630C and PfCLK3F444C were successfully produced. It was found that this mutation rendered PfCLK3 inactive and was detrimental to the activity of PfCLK1. Under the conditions used PfCLK1F630C was not inhibited by a panel of 23 electrophilic inhibitors.

Properdin or complement factor P is a conserved serum glycoprotein of the immune defence. It plays a role in strengthening the activation of complement, a system of proteins essential in the first line defence against infection. Properdin is the only positive regulator and plays a major role in regulating the alternative pathway of the complement system, an effector system of the innate immune response, by binding and stabilising two specific converting enzyme complexes, which are normally labile (C3bBb and C3bBbC3b). Mouse models have shown that complement, in particular complement receptor 3 (CR3) and complement 5 (C5), contributes to survival of infection with Listeria monocytogenes. The purpose of the project was to characterise the contribution of properdin in the response to L. monocytogenes (EGD-e), a Gram-positive, intracellular pathogen, which can cause severe infectious disease in human and animals, by using in vitro and in vivo methods. In vitro assays for the first time point to the significant role of properdin in infection with L. monocytogenes: using dendritic cells and macrophages derived from the bone marrows of properdin-deficient (KO) and wild type mice (WT), cells from WT mice showed greater intracellular load of viable L. monocytogenes at an early time point. Cells from KO mice produced less IFN-γ and nitric oxide compared to cells from WT mice and showed less surface expression of CD40. In addition properdin is found to react as a hypoxia sensitive gene; its expression in WT macrophages was also significantly decreased after infection compared to uninfected cells. In vivo experiments demonstrated for the first time that properdin is necessary in the survival of acute murine listeriosis: properdin-deficient mice were more susceptible to intravenous infection with L. monocytogenes compared to wild type mice, and had a greater systemic IFN-gamma and splenic IL-17A response and greater disease severity with impaired M1 type activation. In conclusion, these findings show that properdin is essential in survival of murine listeriosis and in sustaining a cellular response to the intracellular pathogen L. monocytogenes.

No description available.

Diagnosis of schistosomiasis is still widely reliant on traditional parasitological methods, i.e. the Kato-Katz faecal smear for Schistosoma mansoni and urine filtration for S. haematobium. Since these methods are insensitive, relatively laborious and expensive to perform, much effort has been expended into developing alternative ways of diagnosing the disease. Antibody-detection is the best method for diagnosis in areas of low endemicity. It has the merit of high sensitivity and is likely to be useful for schistosomiasis control as programmes are expanded and accelerated towards meeting the WHO's 2020 goals for neglected tropical diseases (NTDs). A rapid diagnostic test (RDT) for use at the point-of-care (POC) is much more likely to be useful in low-middle income countries than the current assays that are available for antibody-detection. Work has therefore begun towards developing such a test that incorporates S. mansoni cercarial transformation fluid (SmCTF) for the detection of anti -schistosome antibodies in human blood. Here it is demonstrated that SmCTF performs equivalently to S. mansoni soluble egg antigens (SmSEA) in an enzyme-linked immunosorbent assay (ELlSA) format for the detection of anti -So mansoni, anti-So haematobium and anti-So japonicum antibodies.

Trichinella spiralis infection induces chronic small intestinal inflammation and around the time of worm expulsion, the following occur: villus atrophy, crypt hyperplasia, increase in Paneth, goblet and mast cell numbers and increases in mucosal expression of all three isoforms of transforming growth factor beta (TGF-[Beta]). However the in vivo role of TGF-[Beta] and Paneth cell-secreted antimicrobial peptides in chronic intestinal inflammation remain to be fully characterised. The main aim of these studies was to investigate the small intestinal epithelial responses to TGF-[Beta] and Paneth cell-secreted antimicrobial peptides in T. spiralis infection. The first experiment, the investigation of the role of TGF-[beta] was undertaken using a mouse model with inactivation of the transforming growth factor beta receptor II gene in intestinal epithelial cells (Vil-Cre; TGFBR2FIOxIFIOX). These studies showed there was delayed worm expulsion in Vil-Cre;TGFBR2Flox/Flox mice compared to controls. Histological analysis showed a significant reduction in intestinal mast cell counts in Vil-Cre;TGFBR2Flox/Flox mice when compared to wild type mice.

Human parechovirus (HPeV) is a specIes, containing 16 types (HPeVI-I6), that belongs to the genus Parechovirus, a member of the Picornaviridae family. There is growing evidence that HPeVs are medically significant. They cause mild respiratory tract infection and gastroenteritis (mainly HPeV1 strains), or more serious illness such as eNS infection (mainly HPe V3 strains). This thesis describes work designed to improve our understanding of HPe V virulence, by analyzing the !RES (internal ribosome entry site) within the 5'UTR, and also cell receptor tropism. The IRES study included a comprehensive bioinformatic analysis of all picornaviruses that have a type II IRES and revealed novel relationships between structural domains in different viruses. The Human parechovirus 5'UTR was studied in detail and HPeV3-specific features were identified. A bicistronic construct was used to identify the significance of !RES domains in HPe V translation, and also showed that the IRES was not active in all cells lines used. Receptor tropism of several HPeV isolates was investigated by using blocking antibodies against integrins, previously reported to be receptors for the prototype HPeVI strain Harris. All the HPeVI isolates tested were blocked efficiently by the avB6 antibody, suggesting this is the key receptor for most HPe V isolates, although some blocking by the avB3 antibody was observed and this molecule could also be important. Two isolates were identified which probably use heparan sulphate as a coreceptor, as infection was blocked by heparin, and sequencing of these and non-blocked isolates identified an amino acid difference in VP1. This is probably the key amino acid involved in binding. Overall, the work has given new insights into HPeV molecular biology, which may ultimately allow us to understand how these viruses infect cells and cause disease.

Staphylococcus aureus is a leading cause of nosocomial infections. Acanthamoeba is a protozoan pathogen that is remarkably similar to macrophages, particularly in their cellular structure and phagocytosis. The interactions of methicillin-resistant S. aureus (MRSA), methicillin-sensitive S. aureus (MSSA) and S. epidermidis (SE), clinical isolates, with A. castellanii and macrophage-like cell line (ThP 1) revealed similarities in their binding/association, invasion and survival. Additionally, it was shown that bacteria survive and multiply inside A. castellanii during the encystment process as evidenced by bacterial recovery from mature cysts and EM. Using an environmental isolate of S. aureus, BHICC, and mutants lacking major autolysin gene (atl), polysaccharide intercellular adhesion gene (ica) and fibronectin binding proteins A and B (FnBPAB), this study demonstrated the ability and importance of atl gene, ica gene and FnBP AB in attachment, invasion and survival within pathogenic Acanthamoeba T4 genotype, non-pathogenic Acanthamoeba T7 genotype and ThPl macrophage-like cells. Subsequently, this study investigated the effectiveness of five disinfectants currently in use in the UK hospitals, namely, 10% actichlor, 70% ethanol, 1% virkon, 5% biocleanse and hand sanitizer, on MRSA, MSSA and SE survival within trophozoites and cysts of pathogenic A. castellanii T4 genotype and non-pathogenic A. astronyxis T7 genotype. The fmdings revealed that the multi-drug resistant intracellular MRSA withstands exposure to these disinfectants more readily than the intracellular, antibiotic-sensitive MSSA, or the avirulent SE. The next aspect studied was the effectiveness of 5-ALA mediated photodynamic therapy (PDT) on MRSA, MSSA, SE, Acanthamoeba and internalized bacteria. The results showed that 5-ALA mediated PDT reduces Acanthamoeba trophozoites and intracellular bacterial viability as compared with controls, which were maintained in the dark. Interestingly, in percentage terms, the number ofMRSA, MSSA and SE that survived within trophozoites, mirrored the percentage determined for bacteria cultured under axenic conditions.

HT61 is a novel, quinoline-derived antimicrobial that has shown potent activity against both multiplying and non-multiplying Gram positive bacteria, specifically MRSA. It has been previously suggested, but not investigated, that HT61 damages the cytoplasmic membrane of susceptible bacteria. In this study, the putative membrane-activity of HT61 in both Gram positive and Gram negative bacteria was investigated using various techniques, including the BacLight, membrane potential, ATP release and protoplast assays. It was shown that HT61 disrupts both Gram positive and Gram negative membranes resulting in increased permeability and depolarisation of the membrane, leading to leakage of intracellular components and lysis of Staphylococcus aureus protoplasts. Biomimetic bacterial cytoplasmic membrane models were developed using synthetic lipids to investigate the putative disruptive effects of HT61 on the lipid portion of bacterial membranes. Langmuir monolayer studies showed that HT61 primarily interacts electrostatically with model systems c6ntaining anionic lipids, followed by penetration into the monolayer. In the presence of salt, HT61 was also able to penetrate into zwitterionic lipid monolayers due to increased hydrophobicity. HT61 causes enough disruption to bilayers containing anionic lipids to elicit release of dye from liposomes, whilst also allowing access of a quencher to the aqueous core of the liposome. Monitoring the size and number of liposomes using nanoparticle tracking analysis indicated that following HT61 exposure the number of liposomes decreased and their size distribution became more polydisperse. The use of neutron reflectivity confirmed that HT61 interacts with the lipid bilayer, without penetrating through the membrane, by non-specifically targeting anionic lipids, eventually causing loss of material from the bilayer. It is therefore proposed that HT61 interacts with the headgroups of anionic lipids, inserting itself within the bilayer, causing lipid packing disruption, and at concentrations above a threshold, result in lysis of the bilayer and mixed micelle formation.

Pro- and anti-inflammatory cytokines operate as highly complex network systems featuring synergism, antagonism and functional redundancy whose deregulations feature prominently in the systemic inflammation vyhich characterises sepsis. Most studies conducted to date have focussed on single or small groups of cytokines as biomarkers of sepsis with mixed results, possibly due to a lack of recognition of their effects as a network output rather than as individual mediators. Severe sepsis has also proven to be a difficult condition to manage because of a lack of available therapeutic interventions to control its wayward inflammation. In this respect, simvastatin has recently been shown to alter cytokines in a number of different models of sepsis, making this an attractive candidate warranting fuller investigation. This study therefore aimed (i) to investigate a larger panel of cytokines (including some novel mediators) in order to assess their role(s) in the pathophysiology of sepsis and the mechanism(s) governing the regulation of their production, and (ii) to assess the purported anti-inflammatory effects of simvastatin on cytokine production.