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<p>Includes Bibliographical references (leaves 83- 94).Forty-three male athymic nude mice were implanted with human vaginal mucosal cysts under general anaesthesia with Ketamine [25mg/kg] and Medetomidine [0.5mg/kg]. Cysts in 37 mice were recovered after 9 weeks of growth. twenty three cyst linings had retained the original structure of the vaginal epithelium. No marked deifference was present between the thickness of 9 week old linings and donor vaginal epithelium. The contaminants isolated from the skin of mice before implantation were mainly normal commercals of healthy experimental animals. There was no distinct difference in the number of cases with intact cyst formation between the terramycin/vitamin cocktaik group. The frequency of poor wound healing and/ or murine epidermis ingrowth was three times higher in animals stitched with silk sutures that in those cases where nylon sutures were used.</p>

Leukocyte exit from peripheral tissues is fundamental to host defense, yet little is known about the role of adhesive molecules in this process. In my thesis I ask the question “can an integrin regulate leukocyte exit from inflamed peripheral tissues” and specifically investigate the leukocyte integrin α1β1. This is an important question because leukocyte exit, or persistence, at an inflammatory lesion can have a profound effect on the immune response. In addition, I present special in situ staining techniques which had to be developed in order to assay endogenous leukocyte migration in a murine model. The introductory sections review functional differences between myeloid and lymphoid leukocyte subsets, the leukocyte adhesion cascade, integrins, chemokine receptors and the essential concepts of signaling and the relationship between chemokines and integrin activation. I also discuss the pro-migratory paradigm of leukocyte integrins, in other words that integrin adhesion is equated with leukocyte migration. The current literature regarding what is known about integrin function in peripheral tissues and leukocyte migration is also discussed. Chapter 2 characterizes my inflammatory model and implicates α1β1integrin and macrophages as important molecular and cellular entities respectively, involved in sustaining the inflammatory response. Chapter 3 develops endogenous in-situ labeling in the blood compartment, establishing the fundamentals of my in-situ approach. Chapter 4 extends this and establishes in-situ pulse labeling (ISPL) to label endogenous leukocytes in peripheral tissues. Chapter 4 then goes on to combine the technological advances and conceptual framework established in the previous chapters to elucidate a role for α1β1integrin in the exit of macrophages from inflamed peripheral tissues. Finally, in Chapter 5 I discuss the implications of my results in the context of the host defense, how it might impact the immune response and future directions for this research.

Nineteen yearling Quarter Horses were utilized in a randomized, complete block design to evaluate systemic cytokine gene expression and circulating leukocyte population in young horses following an intra-articular lipopolysaccharide (LPS) challenge. Horses were administered an injection of 0.25 ng (n = 7) or 0.50 ng (n = 6) of LPS or lactated Ringer?s solution (n = 6; control). Blood was collected via jugular catheter at pre-injection h 0 and at 2, 6, 12, and 24 h following aseptic injection of the left radiocarpal joint. Aseptic arthrocentesis was performed at the same times to sample synovial fluid for a companion study. Total RNA was isolated from leukocytes using a commercially available kit and real-time PCR was used to determine relative gene expression of the cytokines; interleukin (IL)-1beta (beta), IL-6, IL-8, IL-10, and tumor necrosis factor-alpha (TNF-alpha). Determination of total leukocyte subpopulations and differentials was performed by Texas Veterinary Medical Diagnostic Laboratory. Data were analyzed using the PROC MIX procedure of SAS. Gene expression of all cytokines analyzed was unaffected by treatment. However, changes over time were observed in some cytokines. Interleukin-1? was increased above baseline at 6, 12, and 24 h (P = 0.04), IL-6 was decreased slightly at 6 and 12 h and then increased at 24 h (P = 0.002), and TNF-alpha was increased at 6 and 12 h (P = 0.01). Only IL-8 exceeded a 2-fold change in expression (P = 0.01), peaking at 12 h and indicating greater responsiveness to arthrocentesis than was observed in the other cytokines. No treatment effects on the leukocyte population were observed; however, total circulating leukocytes increased over time (P = 0.04), peaking at 6 h post-injection. Similarly, an increase over time was observed in monocytes (P = 0.002) and in platelets (P = 0.01) at 24 h post-injection. The results indicate that regardless of treatment, a mild immune response was elicited, likely due to repeated arthrocentesis. Future experiments should consider the effects of arthrocentesis and potential systemic inflammatory response, even in control animals, when administering intra-articular LPS to young horses.

Idiopathic endogenous posterior uveitis encompasses a spectrum of chronic intraocular inflammatory disorders which are thought to be autoimmune in nature. The animal model experimental autoimmune uveoretinitis (EAU) is mediated by CD4+ T-lymphocytes, and has proved invaluable in the study of the underlying immunopathogenesis of uveitis and alternative immunosuppressive therapies, for example, oral tolerance induction. This thesis describes, in a model of retinal-extract induced EAU, the effects of intranasal administration of retinal antigens prior to induction of EAU with retinal extract. The thesis has demonstrated that immunisation with emulsified retinal extract and CFA (without pertussis) induces a dose-dependent intraocular inflammation, which at high doses leads ultimately to total loss of rod photoreceptor outer segments and retinal necrosis. A course of intranasal inoculations with retinal extract (tolerance induction), prior to immunisation with antigen suppresses the histological and clinical response of EAU. Animals which were tolerised with microgram quantities of antigen showed evidence of mild inflammation of the ciliary body and inner retinal vessels (vasculitis) but no evidence of direct photoreceptor damage when compared to controls. Intranasal inoculation with retinal extract suppressed S-Ag-induced EAU but not vice versa, despite the ability of S-Ag intranasal inoculations to suppress S-Ag induced disease. Tolerised animals demonstrated normal antibody responses to S-Ag, IRBP and retinal extract, and exhibited a significantly suppressed delayed hypersensitivity response to retinal extract but normal response to a non-specific antigen, PPD. Adoptive transfer of splenocytes from tolerised animals suppressed the induction of EAU in some naive recipients. These findings suggest that active suppressor mechanisms are involved in the induction of tolerance, which concurs with other findings of CD8+ T-lymphocyte mediated suppression in oral tolerance models. In order to study the future application of 'tolerance therapy', we attempted to suppress sensitised animals by intranasal tolerance which resulted in an incomplete suppression of EAU.

Chronic posterior uveitis is a relatively common clinical disorder and an importance cause of visual impairment in young adults. Experimental autoimmune uveitis (EAU) and its associated experimental autoimmune pinealitis (EAP) induced by retinal autoantigens are predominantly CD4+ T cell mediated (auto) immune disease of the retina and uveal tract of the eye and the pineal gland respectively. EAU bears a close clinical and pathological resemblance to chronic posterior uveitis in humans and seves as a good animal model for the study of posterior uveitis. The EAU model was used to study means of modulating the host's immune response to suppress or inhibit the onset of uveitis. The onset of retinal S-antigen induced EAU could be successfully inhibited by pre-treating Lewis rats with a retinal S-antigen (carboxy terminus) specific monoclonal antibody called S2.4.C5. This however did not suppress the associated EAP indicating that the monoclonal antibody acted via the efferent arc of the immune mediated response. This prompted a study of the 'Blood-retinal and Blood-pineal barrier sites' during the active stages of EAU and EAP. Transmission electron microscopy of the vascular endothelium revealed changes resembling 'High endothelial venules' of lymph nodes in the retinal and pineal vasculature. In an attempt to identify one or more immunodominant epitopes of S-antigen that may be relevant to tolerance induction, an in-vitro and in-vivo study using enzyme digested preparations of S-antigen was carried out. This revealed that digestion of S-antigen by a protease derived from staphylococcus aureus V8 strain, not only inhibited the binding of the monoclonal S2.4.C5 in-vitro but was also associated with an in-vivo attenuation of the pathogenic response to S-antigen indicating that a dominant immunogenic epitope of S-antigen was located at the C-terminus of the molecule.

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TNF is a proinflammatory cytokine whose pleiotropic biological properties are signaled through the receptor TNFR1. Activation of this signaling pathway has been implicated in a broad range of biological functions, including host defense, inflammation, apoptosis, autoimmunity, and cancer. TRADD is an adaptor protein that is recruited to TNFR1 upon receptor engagement. Using a Tradd-deficient murine model, we demonstrated that TRADD is essential for both TNF-mediated apoptosis and inflammatory responses. In addition to refining the role of TRADD in TNFR1 signaling, we have also identified a novel function of TRADD in TLR3 and TLR4 pathways, which are key drivers of the innate immune response. We showed that TRADD is involved in NF-κB activation upon TLR3 and TLR4 stimulation, and Tradd-deficient macrophages showed impaired inflammatory cytokine production in response to TLR ligands in vitro. These data reveal the multifaceted functions of TRADD in immune signaling pathways. Beyond its role in the immune response, TNF has also been shown to play a crucial, cell-non-autonomous role in driving tumor growth in various models of cancer. We initially sought to determine whether TRADD is essential for this aspect of TNF function by employing the use of a chemical induced skin carcinogenesis model in which the tumor-promoting role of TNF is very well established. In this model, H-Ras is the major driving oncogene. We found that Tradd deficiency accelerated tumor formation in mouse skin, in strong contrast to what was observed in Tnfr1-deficient mice. Further in vitro analyses revealed that upon expression of oncogenic H-Ras, Tradd-deficient murine fibroblasts displayed both reduced cell cycle arrest and repression of Ras induced cellular senescence. Importantly, the level of p19Arf induced by H-Ras expression was reduced in Tradd-deficient fibroblasts in a post-translational manner. Our biochemical evidence suggests that TRADD can shuttle dynamically between the cytoplasm and the nucleus; in doing so, nuclear TRADD interacts with ULF, a newly identified E3 ubiquitin ligase for p19Arf. Interaction between nuclear TRADD and ULF sequesters ULF away from p19Arf, leading to p19Arf stabilization and tumor suppression. Together, these data demonstrate the functional diversity of TRADD in different compartments of the cell.

Activation of nociceptors causes them to secrete neuropeptides, such as substance P (SP) and calcitonin-gene related peptide (CGRP). By reacting with receptors on blood vessels these peptides contribute to inflammation by evoking vasodilation and increasing vascular permeability that allows proteins and fluid to leave the blood vessels (plasma extravasation, PE). These substances can also lead to the sensitization of nociceptors and the resulting positive feedback thereby prolongs inflammation and pain. Thus, blocking the release of neuropeptides may have important therapeutic value in pain conditions where neuropeptides have been implicated. Therefore, the aim of this study was to define the time course of changes in vascular permeability and to test the ability of novel agents to modulate this response. PE and vasodilation was evoked by stimulating the saphenous nerve or by injecting the chemical irritant capsaicin into the rat hindpaw. Changes in blood flow were evaluated with a laser Doppler and digitized image analysis was used to measure changes in reflectance of the skin due to accumulation of extravasated (EB) dye. Analysis of the change in pixel intensity in the digitized images revealed that the magnitude of PE was dependent on the stimulus pulse number. Moreover, the time course of enhanced vascular permeability produced by electrical stimulation was a transient event compared to a much longer response with capsaicin. It was also demonstrated that sumatriptan, (a 5-HT1B/D receptor agonist) and botulinum neurotoxin type-A were effective treatments for capsaicin and saphenous nerve induced vasodilation and PE. Neither drug interfered with activation of the SP or CGRP receptor, which may suggest that both drugs work by inhibiting neuropeptide release. Therefore, this study has described the time course of vascular permeability evoked by two different stimuli and has demonstrated the ability of two novel agents to attenuate these responses.