No description available.

The primary aim of this project was to investigate whether the medically important fungal pathogen C. albicans, can be induced to undergo a programmed cell death response. Cell death in C. albicans was examined after treatment with low and high fungicidal doses of acetic acid, hydrogen peroxide and amphotericin B. Exposure of C. albicans cells to relatively low fungicidal doses of these agents produced cellular changes reminiscent of mammalian apoptosis; whilst, C. albicans cells treated at high fungicidal doses resulted in cellular changes typical of necrosis. The Ras/cAMP pathway in fungi is known to translate external signals to changes in gene expression and is involved in metabolism, proliferation and stress responses. In mammals, Ras has been demonstrated to exhibit both pro- and anti-apoptotic functions. Therefore, the role of the Ras/cAMP pathway in the programmed cell death events of C. albicans was investigated. The Ras/cAMP pathway was modulated in cells of C. albicans by genetic or pharmacological approaches. These cells were then exposed to a range of death-inducing treatments, and the temporal progression of cell death was studied. This detailed temporal analysis indicated that C. albicans Rasl is pro-apoptotic, and that the activation of the Ras/cAMP pathway accelerates the entry-rate of C. albicans cells into cell death responses. The exploitation of endogenous fungal programmed cell death responses, in terms of providing new anti-fungal agents, is a long way off. However, this research project was the first to demonstrate that a medically-important fungus can undergo programmed cell death events, and that these events can be triggered by treatment with exogenous drugs.

The prevention of post-operative wound infection is still an important aspect of the surgical care of a patient. Recent advances in surgical technique and the advent of new and more potent antibiotics have not diminished its importance. For example, Cruse (1970) noted (in 1967) that wound infection alone cost the Province of Alberta $1,000,000 mainly due to increased hospital stay. In 1973 Alexander estimated the yearly economic loss to the United States due to wound infection to be $9,400 million. In terms of morbidity Irvin and his colleagues (1977) found that the incidence of wound dehiscence and post-operative wound herniation was greater in patients whose wounds became infected. In considering the aetiology of primary infections, many variables are encountered. By operating in a sterile atmosphere such as that provided by the plastic flexible film isolator (Trexler and Reynolds, 1957) some of these variables can be eliminated. A study of the use of such an isolator in total hip arthroplasty has been carried out and forms the first part of the thesis. The results stress the importance of the skin as a possible source of wound contamination. A study of the methods available to reduce this potential source of post-operative infection is made in the second part of this thesis.

Background: Signal transducer and activator of transcription (STAT)3 activation triggers transcription of interleukin (IL)-17 which is crucial for mounting protective immune responses against fungi. Several mutations affecting the STAT3/IL-17 pathway have been reported, resulting in selective susceptibility to fungal (Candida) infection characteristic of Chronic Mucocutaneous Candidiasis (CMC). Patients with autosomal dominant (AD)-CMC have defective T helper (Th)-17 responses (Ng et al, JACI 2010) and harbour gain-of-function (GOF) STAT1 (rather than STAT3) mutations (van de Veerdonk et al NEJM 2011), leading to hyper-phosphorylation of STAT1 (Smeekens et al PLosOne 2011). How this affects STAT3 or leads to decreased IL-17 production is unknown. Objective: To assess how GOF-STAT1 mutations affect STAT3 activation, DNA-binding, gene expression, cytokine production and whether this can be epigenetically modified. Approaches: Peripheral blood mononuclear cells (PBMCs) or Epstein-Barr virus (EBV)-transformed cell lines were stimulated with various cytokines (IL-23, IL-6, IL-21, IL-27, IFN-α, IFN-γ) in the absence or presence of fludarabine (a STAT1 inhibitor) or trichostatin A (a histone deacetylase inhibitor, HDACi). Activation of STAT1 and STAT3 was measured by western blotting (WB). DNA-binding of STAT1 and/or STAT3 was evaluated using electrophoretic mobility shift assay (EMSA), TransAm STAT3 kit and chromatin immunoprecipitation (ChIP) assay. STAT3-inducible gene (RORc, IL-17, IL-22, IL-10, c-Fos, SOCS3, c-Myc) and STAT1-inducible gene (CXCL10, IRF1) expression was assessed using quantitative real time-polymerase chain reaction (qRT-PCR). Gene silencing was performed with small interfering RNA (siRNA) targeting HDAC1, 2 and 3. Candida albicans-induced cytokine (IL-17, IL-22, IL-10) production was measured by enzyme-linked immunosorbent assay (ELISA). Key Findings: GOF-STAT1 mutations lead to hyper-phosphorylation of STAT1 and delayed dephosphorylation. Mutations decrease STAT3-induced gene expression and Th-17-mediated cytokine production without affecting STAT3 phosphorylation, nuclear accumulation or DNA-binding to a STAT-consensus binding site - human sis-inducible element (hSIE). The disrupted STAT3 function can be modified either by enhancing acetylation or inhibiting STAT1 activation. Conclusion: GOF-STAT1 mutations impair STAT3 function, which can be rescued by enhancing acetylation and/or inhibiting STAT1. These findings are the likely mechanisms underlying decreased Th-17 (IL-17A and IL-22) cytokine production and susceptibility to fungal infections in CMC.

Chlamydia trachomatis is an obligate intracellular bacterium, which is the leading cause of acquired blindness and the most prevalent bacterial sexually transmitted infection worldwide. Chlamydiae exist in two distinct forms. The infectious spore-like elementary bodies (EBs) that invade host cells differentiate into non-infectious reticulate bodies (RBs) that replicate intracellularly within a modified membrane-bound vacuole called the inclusion. Under stress, Chlamydiae can enter a persistent state, in which aberrant bodies (ABs) with reduced metabolic activity are formed. Surprisingly little is known about the mechanisms employed by the bacteria to maintain and manipulate their environment within host cells. This thesis investigates the role of inorganic ions in sustaining the inclusion throughout the Chlamydia infection cycle. Potassium starvation of intracellular RBs either after specific ionophore treatment or inhibition of inward rectifying cellular potassium channels induced the formation of ABs, which no longer differentiated into infectious EBs. These data demonstrate an essential role for potassium during C.trachomatis replication. Analysis of live RBs, using a potassium sensitive fluorescent probe, illustrated that potassium is actively scavenged from the host cell. Furthermore, when bacteria undergo RB-EB differentiation accumulated potassium is released prior to inclusion lysis. Experimentally reducing potassium ion concentration at this stage caused cells to expel bacteria in bursts. This event is distinct from previously described extrusion mechanisms, where either the inclusion is released intact or the host cell is lysed. These data show that RBs actively accumulate potassium during replication, with starvation leading to persistence. Loss of potassium ions during re-differentiation into EBs suggests that potassium efflux has a role in triggering inclusion lysis or bacteria exit from the host cell.

It has long been long known that core body temperature declines with age. However, its effect on bone cell function and skeletal homeostasis have been little studied. To investigate this, bone-forming osteoblasts and bone resorbing osteoclasts were exposed to mild hypothermia (35.5oC) and severe hypothermia (34oC). Formation of 'trabecular' bone structures by osteoblasts was reduced by up to 95% in hypothermic cultures, compared to 37oC controls. In addition to reductions in osteoblast cell number, expression of osteocalcin, type I collagen, alkaline phosphatase and Runx2 were also down-regulated in hypothermia. Microarray analysis indicated an initial period of down-regulation of transcripts in hypothermia, followed surprisingly, by up-regulation as cultures progressed. In contrast, formation of osteoclasts showed up to 2-fold stimulation in hypothermia; resorption pit formation was similarly increased. Microarray analysis showed that genes involved in osteoclast formation and cold response were up-regulated in hypothermic cultures. In addition to reductions in core temperature, the elderly have a higher prevalence of type 2 diabetes and hyperlipidaemia. These conditions are independent risk factors in the pathogenesis of osteoporosis; however, drug therapies, namely PPAR agonists, have been reported to affect bone cell function. Thiazolidinediones (rosiglitazone and troglitazone) are PPAR-γ agonists used in the management of type 2 diabetes; fibrates (such as fenofibrate) are PPAR-α agonists used in the treatment of hypertriglyceridaemia. Bone formation by osteogenic cells was inhibited by up to 85% in cultures treated with rosiglitazone, and by 45% in troglitazone or fenofibrate treated cultures. Lipid formation however, increased by 40-70% in these cultures. Expression of Runx2, alkaline phosphatase, osteocalcin and type I collagen were down-regulated, whereas PPAR-γ was up-regulated, in treated cultures. Osteoclast formation and resorptive activity were diminished with fenofibrate treatment, whereas both thiazolidinediones reduced resorptive activity without affecting osteoclast number. These findings raise the possibility that hypothermia or treatment with PPARs could adversely affect bone metabolism in the elderly.

No description available.

The pathogenesis of Clostridium welchii type D epsilon toxin intoxication has been investigated in mice and sheep with special reference to nouropathology. High concentrations of epsilon toxin in the gut of sheep cause enterotoxaemia/pulpy kidney disease and may also cause a focal symmetrical encephalomalacia (FSE). Brains from sheep dead from pulpy kidney disease were examined histologically. No brain was devoid of lesions although the severity varied considerably; one third had sufficient foci of malaoia to justify a diagnosis of FSE. Similar lesions were produced experimentally by injecting lambs with epsilon toxin and it was shown that there was initially a breakdown of the blood brain barrier. In mice it was found that this breakdown of the blood brain barrier could be prevented by the prior administration of formalinised epsilon prototoxin. It was concluded that epsilon toxin causes its effect by binding to specific receptor sites and that the prototoxin competes for these sites. With isotope-labelled prototoxin it was shown that receptor sites for the toxin existed in the brain, kidneys, heart, liver and spleen of mice. Immunoperoxidase studies showed that these were situated on the luminal surfaces of vascular endothelial cells and certain renal tubules. Similar results were obtained in lambs. The effect of epsilon toxin on the lympho-reticular system was investigated. It was found that toxin killed guinea pig liquid paraffin induced peritoneal macrophages and it was also shown that the receptor sites for epsilon toxin were probably situated on the surface of the cells. It was found that epsilon toxin increased the permeability of blood vessels in guinea pig skin as do several other bacterial toxins which are known to stimulate cyclic adenocine 30, 50- monophoaphate (CAMP) production. Also plasma cAMP concentrations in mice treated with epsilon toxin rose significantly. It was concluded that Clostridium welchii type D epsilon toxin causes widespread damage, after its absorption from the gut lumen, by binding to specific receptor sites on the surface of curtain cells where it stimulates adenyl cyclone to produce increased amounts of cAMP.

No description available.

No description available.