Acute anterior uveitis (AAU) is the most common form of intraocular inflammatory disease and an important cause of visual impairment. Microbial triggers to the development of AAU have been strongly implicated, however the pathogenesis and molecular mechanisms for this are unclear. Toll-like receptors (TLR) and nucleotide oligomerisation domain receptors (NOD) are pattern recognition receptors (PRR) of the innate immune system that facilitate immediate recognition and immunostimulatory responses to unique molecular patterns of microbial components, including the production of chemoattractant cytokines called chemokines. The major aim of this study was to investigate the role of PRRs, namely TLRs and NODs, in the pathogenesis of human AAU. The mRNA and protein expressions of TLR4 and NOD2 in the normal human eye were determined by RT-PCR and immunohistochemistry. Resident antigen presenting cells of the normal human uvea expressed TLR4 protein. NOD2 protein expression was highly restricted to a subpopulation of retinal cells. A selective perturbation in the expression and function of TLRs were demonstrated in active human AAU by flow cytometry and in vitro stimulation with selective TLR agonists. These changes were not due to any polymorphisms in the TLR genes. Elevated plasma levels of lipopolysaccharide (LPS), an agonist for TLR4, were not detected in patients with active AAU by the limulus amebocyte lysate assay. New information regarding the complex in vivo network of cytokines in active human AAU were provided by multiplex cytokine bead immunoassay on aqueous humor samples, including a Th1-polarised pattern of aqueous humor cytokines, the intraocular expression of multiple LPS-inducible cytokines, and the aqueous humor expression of cytokines such as IL-17 in AAU. Expressions of the respective chemokine receptors on peripheral blood leukocytes were also determined. The findings of this study provides several lines of evidence to support the hypothesis that PRRs, namely TLRs and NODs, are of pathogenic importance in the development of clinical AAU. The results of this study provide significant new information regarding the role of PRRs in ocular immunity and immune privilege, their role in the pathogenesis of AAU, and it provides a molecular mechanism whereby microbial triggers could initiate the development of uveitis.

published_or_final_version / Community Medicine / Master / Master of Public Health

Proefschrift Universiteit van Amsterdam. / Omslagtitel: Observations on clinical and experimental ocular autoimmunity. - Auteursnaam op omslag: Marc D. de Smet. Met lit. opg. - Met samenvatting in het Nederlands.

Thesis (M.P.H.)--University of Hong Kong, 2008. / Includes bibliographical references (p. 19-20).

Thesis (Ph.D.)--Aberdeen University, 2008. / Title from web page (viewed on June 15, 2009). Includes bibliographical references.

Thesis (M.S.)--University of Missouri-Columbia, 2007. / "May 2007" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Includes bibliographical references.

Inflammatory ocular disease is an important cause of blindness and uveitis accounts for 1.0% of blind patients in Canada.¹ This disease can be particularly troublesome to treat, because the nature of the causal factor or factors and mechanisms of progressi n are usually unknown. Non-specific anti - inflammatory agents have been used orally and systemically with some success to treat uveitis, ²⁻⁸ but they may produce serious side effects both locally and elsewhere in the body.⁹̛¹²̛¹⁴ With prolonged use tolerance to these drugs may develop , making them ineffective. Recently a powerful immuno suppressive agent, Cyclosporine (Cy), used orally and systemically in the treatment of uveitis has shown promising results.¹⁶⁻¹⁹̛²⁸ However, its routine use is limited because of a narrow therapeutic index and renal toxicity. Several studies have shown that subconjunctival injection of a number of antineoplastic agents enhanced ocular absorption ²⁰⁻²⁴ in a traditional pharmacological sanctuary,¹³̛¹⁴ and circumvented the associated systemic side effects. Therefore, if Cy were administered subconjunctivally it might be possible to avoid the side effects associated with the oral and systemic routes, and at the same time provide higher levels of Cy to the eye. A protocol for the administration of Cy subconjunctivally was developed in New Zealand white rabbits, to study toxicity, ocular pharmacokinetics following equidose administration subconjunctivally and systemically and the effects of Cy on an animal model of uveitis. Subconjuntival administration of 5mg of Cy in O.lcc (Sandimmune I.V.(R) 50 mg/ml) weekly was found to be the maximum tolerated dose by the rabbitsˈ eye, and was superior to intravenous injection for ocular penetration while minimizing systemic exposure. The uveitis model showed that Cy was effective in reducing the inflammatory response and the earlier the application of Cy the milder the uveitis. The results from our study support the contention that local administration of Cy would lead to higher levels of Cy absorption and circumvent the side effects of systemic administration. This may facilitate the routine use of Cy in ocular inflammatory disease. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Experimental Autoimmune Uveitis (EAU) is the murine and rat model of the equivalent chronic inflammatory condition in humans. Tolerance to EAU can be induced via a single intra-nasal administration of the retinal autoantigen interphotoreceptor retinoid-binding protein (hIRBP_1-20). Tolerance initiation to EAU has been associated with an initial elevation of Th2-type cytokines in the cervical lymph nodes and spleens of experimental animals. Moreover, tolerance initiation was enhanced in mice with inhibited expression of the myeloid cell-regulatory protein CD200. Binding of CD300/CD200R initiates a signalling cascade that ultimately results in the down-regulation of myeloid cell activation. Therefore, enhanced tolerance initiation in the CD200-deficient mice was a paradox and the main aim of this study was to examine the molecular events leading to enhanced tolerance. The first aim of this study was the identification of the exact time-point of tolerance initiation. Because of the association of Th2-type cytokines in tolerance induction, Northern Blotting detection for the <i>Stat6</i> transcript was performed. Maximal expression of the <i>Stat6</i> transcript was observed in the spleens of the CD200-deficient mice 8 h post-tolerisation. Having identified 8 h as a potentially relevant point in tolerance initiation, a GenaArray study was performed for the analysis of global gene expression in the cervical lymph nodes of IRBP-tolerised animals in respect to the sham-terrorised controls. Furthermore, the alternative molecular pathways initiated or inhibited in the cervical lymph nodes and spleens of CD20-deficient animals in respect to the WT controls were also examined. The results obtained in the microarray study were verified by Western Blotting and qRT-PCR. The results obtained in our study demonstrated that the nasal administration of autoantigen and the shift from a WT to a CD200-deficient phenotype had more diverse biological effects than previously thought.

Recently, there has been considerable interest in developing specific cell-based immunotherapies using dendritic cells (DCs). Here the mechanisms underlying the tolerogenic properties of DCs in the suppression of experimental autoimmune uveoretinitis (EAU), the mouse model of human sight threatening autoimmune uveitis, were examined. Immature DCs have the ability to promote immune tolerance to self antigens and to prevent the development of autoimmune disorders including EAU. However there is a risk that immature DCs placed in the inflammatory environment would undergo maturation and instead of tolerance promote immunity. Therefore much effort has been directed to develop protocols that stabilize the tolerogenic DC properties which would ultimately ensure safety and effectiveness of DC-based vaccines. Previous work demonstrated that activation of DCs with lipopolysaccharide (LPS) increases the ability of these cells to prevent EAU. Here, it was demonstrated that LPS promotes the activation of both TRIF and MyD88 signalling pathways in DCs which after 24 h LPS treatment secreted a significant amount of IL-10, IFN-β, IL-1β, IL-6 and TNF-α while the level of secreted IL-12 is low. It was further shown that LPSinduced enhancement of the tolerogenic properties of DCs correlates with the state of endotoxin tolerance in the DCs, rendering them refractory to further stimulation. It was hypothesised that the LPS-induced enhancement of DC tolerogenicity is due to the reduced expression of the TLR4, which subsequently disables several signalling pathways and prevent DCs from initiating adaptive T cell immunity. In summary, the presented data provides valuable insights into the mechanisms involved in the suppression of autoimmune uveitis using a DC-based therapy.

The objectives of this study were to characterize the ocular and systemic adverse effects, and systemic pharmacokinetics of topical 0.1% diclofenac. This was investigated in 8 healthy cats using a blinded, randomized, placebo-controlled, cross-over design. Drops were administered bilaterally 4 times daily for 7 days. Ocular, hepatic and renal variables were measured at various timepoints. Pharmacokinetic sampling occurred on Days 1 and 7. Treated animals were 8 times more likely to develop conjunctival hyperemia than control animals (p=0.0161). Pharmacokinetic analysis showed that accumulation occurs with repeated dosing. Topical 0.1% diclofenac treatment did not have any significant effect on hepatic or renal function, other than reduction GFR in the second phase of the study (p=0.0013). In conclusion, topical 0.1% diclofenac appears to be safe in healthy cats causing only mild ocular irritation. Careful patient selection may be indicated as systemically-absorbed diclofenac may be associated with reduction in GFR in volume-contracted states. / Ontario Veterinary College Pet Trust Fund