The cationic host defence peptide LL-37 is an immunomodulatory agent that plays an important role in epithelial innate immunity. Previously, concentrations of LL-37 thought to represent levels present during inflammation have been shown to elicit the production of cytokines and chemokines by epithelial cells. To investigate the potential of lower concentrations of LL-37 to alter epithelial cell responses, normal primary keratinocytes and bronchial epithelial cells were treated with pro-inflammatory stimuli in the presence or absence of 1 – 3 μg/ml LL-37. Low, physiologically relevant concentrations of LL-37 synergistically increased IL-8 production by both proliferating and differentiated keratinocytes in response to IL-1β and the TLR5 agonist flagellin, and synergistically increased IL-8 production by bronchial epithelial cells in response to IL-1β, flagellin, and the TLR2/1 agonist PAM3CSK4. Treatment of bronchial epithelial cells with LL-37 and the TLR3 agonist poly(I:C) resulted in synergistic increases in IL-8 release and cytotoxicity. The synergistic increase in IL-8 production observed when keratinocytes were co-stimulated with flagellin and LL-37 was suppressed by pretreatment with inhibitors of Src-family kinase signalling and NF-κB translocation. These data suggest that low concentrations of LL-37 may alter epithelial responses to microbes in vivo. Microarray analysis of keratinocyte transcriptional responses after LL-37 treatment suggest that LL-37 may alter the expression of growth factors and a number of genes important to innate immune responses. LL-37 may thus play a more important role than previously suspected in the regulation of epithelial inflammation; an improved understanding of the mechanisms by which LL-37 alters chemokine responses could lead to the development of novel anti-infective and anti-inflammatory therapeutics.

In this thesis, the monocrotaline (MCT)-induced rat model of pulmonary arterial hypertension (PAH) was used to investigate the role of innate immunity in the pathogenesis of PAH and the mode of action of experimental therapies. The first section of this thesis is an investigation of the therapeutic mechanism of human, early and late-outgrowth endothelial progenitor cells (EPCs) in the MCT-induced, nude rat model of PAH. While late-outgrowth EPCs provided no therapeutic benefit in this model, early EPCs (E-EPCs) prevented the elevation of right ventricular systolic pressure (RVSP, P<0.001) and right ventricular (RV) hypertrophy (P<0.01). Ablation of natural killer (NK) and natural killer T cells with anti-asialo GM-1 antiserum (ASGM-1) enhanced human cell retention in the lung but abrogated the therapeutic capacity of E-EPCs. In vitro studies demonstrated that E-EPCs are similar to monocyte-derived regulatory dendritic cells (DCs) and possess the capacity to stimulate both autologous and rat NK cells in co-culture. Imatinib mesylate has been reported to reverse established PAH both clinically and in the MCT model. Imatinib can also induce NK activation through inhibition of c-kit signaling in DCs, suggesting that imatinib and the DC-like E-EPCs may prevent PAH through a similar, NK-mediated mechanism. In the second section of this thesis, imatinib prevented MCT-induced increases in RVSP (P<0.001) and RV hypertrophy (P<0.01) in immunocompetent Fisher 344 rats, but not in nude rats or Fisher rats following ablation of NK cells and T lymphocytes with ASGM-1. These data suggest that the stimulation of NK activity by imatinib is insufficient to prevent disease in the absence of T lymphocytes. Hyaluronan (HA) fragments are a potent inflammatory stimulus, capable of inducing macrophage activation and DC maturation. In the third section of this thesis, HA synthesis and degradation were investigated in the MCT model of PAH. While the early stages of disease were characterized by enhanced hyaluronidase-1 activity and a loss of high molecular weight (HMW) HA, severe disease was associated with HMW HA synthesis and HA accumulation in the lungs. The early degradation of HMW HA may drive inflammation and stimulate pathological vascular remodeling in PAH.

In this thesis, the monocrotaline (MCT)-induced rat model of pulmonary arterial hypertension (PAH) was used to investigate the role of innate immunity in the pathogenesis of PAH and the mode of action of experimental therapies. The first section of this thesis is an investigation of the therapeutic mechanism of human, early and late-outgrowth endothelial progenitor cells (EPCs) in the MCT-induced, nude rat model of PAH. While late-outgrowth EPCs provided no therapeutic benefit in this model, early EPCs (E-EPCs) prevented the elevation of right ventricular systolic pressure (RVSP, P<0.001) and right ventricular (RV) hypertrophy (P<0.01). Ablation of natural killer (NK) and natural killer T cells with anti-asialo GM-1 antiserum (ASGM-1) enhanced human cell retention in the lung but abrogated the therapeutic capacity of E-EPCs. In vitro studies demonstrated that E-EPCs are similar to monocyte-derived regulatory dendritic cells (DCs) and possess the capacity to stimulate both autologous and rat NK cells in co-culture. Imatinib mesylate has been reported to reverse established PAH both clinically and in the MCT model. Imatinib can also induce NK activation through inhibition of c-kit signaling in DCs, suggesting that imatinib and the DC-like E-EPCs may prevent PAH through a similar, NK-mediated mechanism. In the second section of this thesis, imatinib prevented MCT-induced increases in RVSP (P<0.001) and RV hypertrophy (P<0.01) in immunocompetent Fisher 344 rats, but not in nude rats or Fisher rats following ablation of NK cells and T lymphocytes with ASGM-1. These data suggest that the stimulation of NK activity by imatinib is insufficient to prevent disease in the absence of T lymphocytes. Hyaluronan (HA) fragments are a potent inflammatory stimulus, capable of inducing macrophage activation and DC maturation. In the third section of this thesis, HA synthesis and degradation were investigated in the MCT model of PAH. While the early stages of disease were characterized by enhanced hyaluronidase-1 activity and a loss of high molecular weight (HMW) HA, severe disease was associated with HMW HA synthesis and HA accumulation in the lungs. The early degradation of HMW HA may drive inflammation and stimulate pathological vascular remodeling in PAH.

Nucleotides, such as ATP and its derivatives, are released at high concentrations at sites of inflammation and modulate the immune response. When cultured in the presence of ADP or stable analogue ADP?S, DC surface expression of MHC-II and co-stimulatory molecules, CD40 and CD86 was unchanged. When DCs were pre-treated with ADP or ADP?S, there was no change in their ability to activate naïve CD4+ T cells. However, when CD4+ T cells were activated in the presence of ADP or ADP?S, activation and proliferation were significantly decreased. This correlated with a significant reduction in IL-2 secretion and CD25 surface expression, which may be due to decreased ERK and Akt phosphorylation. CD8+ T cell proliferation was unaffected by the addition of ADP or ADP?S, but secretion of IFN-? was significantly reduced. By demonstrating that ADP inhibits CD4+ T cell responses, we have identified a potential target of immune modulation by clinical intervention.

Walleye (Sander vitreus M.) are valuable in commercial and recreational fisheries and are affected by bacterial, fungal and viral disease. Pattern recognition receptors (PRRs) are germline-encoded and constitutively expressed and bind non-self or altered-self for immune recognition. Walleye were hypothesised to have circulating PRRs that were capable of binding diverse pathogens. These PRRs were hypothesised to increase with infection, be distributed in immunologically relevant tissues and to be strain and age specific. PRR binding was measured by affinity chromatography, plasma binding assays,SDS-PAGE, Western blots, ELISA, PCR, and immunohistochemistry. ELISA and affinity chromatography assays were developed in rainbow trout (Oncorhynchus mykiss) with known PRRs. Trout ladderlectin was confirmed as a PRR binding viral hemorrhagic septicemia virus (VHSV). These techniques were adapted to walleye using Flavobacterium columnare, chitin, VHSV and Sepharose resin. A 22 kDa protein bound to F. columnare, a 17 kDa protein bound to chitin and a 34 kDa protein bound to VHSV were identified as similar to bass apolipoprotein, carp C3 and rainbow trout intelectin, respectively. PCR and 3'-RACE-PCR were used to generate nucleotide sequence to confirm identity of walleye apolipoprotein and mannose-binding lectin (MBL)-like protein from the intelectin-like sequence. Two rabbit polyclonal antibodies were raised to 34 and 67 kDa MBL amino acid sequences and used to verify MBL-like protein as a PRR for VHSV. Healthy walleye MBL-like protein plasma concentration was 7.5 ng/ml. Significant differences were found between geographically distant strains of walleye. An ELISA demonstrated that MBL-like protein had significant differences in binding affinity between multiple strains of VHSV and different viruses found in Ontario. MBL-like protein plasma levels increased with initial infection of naïve fish with waterborne and IP VHSV (107 pfu) but did not change with IP reinfection. Previous infection with VHSV significantly decreased walleye mortality. IHC of walleye shows MBL-like protein is distributed in epithelial surfaces, primarily skin, oropharynx, gill, gastrointestinal system, renal nephrons, connective tissue of gonads and plasma. There was no qualitative difference in MBL-like protein tissue distribution in healthy and VHSV-infected walleye. This is the first evidence for fish lectins binding viruses.

Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that mediate host-responses to pathogens. In mammals, TLR ligands promote cellular activation and the production of cytokines. Several TLR ligands have been employed prophylactically for the control of bacterial or viral diseases in the mouse model. However, the TLR-mediated responses in chickens have not been well described. Importantly, the utility of TLR agonists for the control of viral pathogens, such as avian influenza virus (AIV), has not been fully explored in chickens. To this end, the studies described in this thesis characterized the kinetics of in vivo responses in chickens to the TLR4 ligand lipopolysaccharide (LPS) and the TLR21 ligand CpG ODN. It was demonstrated that both of these ligands induced the up-regulation of several immune system genes in the spleen, including those associated with pro-inflammatory and antiviral responses, as well antigen presentation. By harnessing the immunostimulatory properties of TLR ligands, it was also demonstrated that the prophylactic administration of either poly I:C (a TLR3 ligand), LPS or CpG ODN may confer immunity to a low pathogenic avian influenza virus, as determined by a reduction in both oropharyngeal and cloacal virus shedding in infected birds. Furthermore, transcriptional analysis of genes in the spleen and lungs identified interleukin (IL)-8, interferon (IFN)-α and IFN-γ as correlates of immunity. In conclusion, TLR ligands may modulate several aspects of the chicken immune system to induce an anti-viral state, thereby conferring immunity to AIV.

More than ever before, two imperatives, ‘the right to know’ and ‘the need to keep secret’, find themselves in a contest for a position of primacy in the contemporary legal system. The need to keep secret is antipathetic to the right to know. The Canadian Charter of Human Rights and Freedoms has entrenched a person’s right to disclosure of both exculpatory and inculpatory material in possession of the prosecution. Moreover, the common law has placed the additional responsibility on the prosecution to inquire of third parties as to the existence and production of material relevant to the defence. Despite the entrenchment of the right to disclosure the demands by the state have steadily grown for more evidence to be withheld from defendants, parties to proceedings and the public in general. The applications for in camera or ex parte hearings are common place and frequently acceded to. This thesis seeks to examine the clash of the two imperatives from the Canadian perspective. By using a comparative analysis of other jurisdictions throughout the thesis, it examines the various legislative instruments and common law employed in the Canadian Courts in respect of ‘ordinary’ criminal trials as well as trials of suspected terrorists, specifically, in respect of disclosure and the ability to withhold material from other parties and refrain from the obligation to disclose. The thesis includes a full analysis of disclosure options, public interest immunity, informer privilege, special advocates and other regimes, and claims of privilege in the interests of national security. The thesis provides a number of detailed recommendations as to how Canada can better balance rights of accused against the public interest and the needs of those who enforce the law.. The recommendations call for legal reforms, some new institutions for better accountability and new internal standards for those engaged in the investigation of crimes and national security matters. / Thesis (Ph.D, Law) -- Queen's University, 2012-04-25 15:01:59.292

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No description available.

In response to viral infection, host cells activate various antiviral defence mechanisms to inhibit virus replication. Therefore in order for a virus to replicate efficiently, it must counteract cellular antiviral defence. Promyelocytic leukemia protein (PML) is a cellular protein involved in intrinsic immunity. It inherently forms nuclear bodies (PML-NBs) that assemble at the site of viral genomes. Proteins related to epigenetic regulation are recruited to PML-NBs, and silence viral gene transcription. This study focuses on the role of two herpes simplex virus type 2 (HSV-2) proteins, ICP0 and Us3, in disrupting PML-NBs and counteracting cellular antiviral defence. En passant mutagenesis was used to create recombinant HSV-2 viruses lacking ICP0, Us3, or both ICP0 and Us3. Growth analysis of these recombinants indicates no growth defects for the ICP0Δ virus, while the Us3Δ virus grows to one log lower titres than wild type virus (WT). By contrast, the ICP0Δ virus displays a delay in PML-NB disruption, but the Us3Δ virus is as efficient as WT. However, Us3 is still important for PML-NB disruption, since the ICP0Δ/Us3Δ double mutant exhibits a greater delay than the ICP0Δ single mutant. Although PML is a mediator of the interferon (IFN) response and it was predicted that ICP0 and Us3 interfere with the IFN response through disruption of PML-NBs, my results show that only some HSV-2 Us3Δ clones are hypersensitive to the effects IFN, and others are resistant. Us3 affects more than one cellular pathway, and those cellular pathways are affected by more than one viral protein. I conclude that the activities of multiple viral proteins create a fine balance between activating cellular pathways to promote virus replication, and inhibiting cellular antiviral defence. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2014-01-23 10:55:16.715