The ocular microenvironment is immunosuppressive in animal models of antigen presenting cell function. My hypothesis was that in humans the normal ocular microenvironment maintains an immature dendritic cell (DC) phenotype, whereas in intraocular inflammation (uveitis) this regulation fails, permitting full DC maturation leading to the production and recruitment of pathogenic effector T cells to the eye. Using an in vitro model of DC function, I observed that non-inflammatory aqueous humour (AqH) inhibited DC maturation, with reduced MHC and CD86 expression, and reduced capacity to induce proliferation of allogeneic T cells, an effect which was cortisol and TGFβ2 dependent. In contrast, exposure to uveitis AqH generated a distinct DC profile with IFNγ dependent elevation of MHC class I, but reduced MHC class II and CD86 expression and impaired induction of T cell proliferation. Exposure to uveitis AqH from patients on topical glucocorticoid treatment caused additional suppression of CD86. Characterisation of ex vivo myeloid DC from patients with uveitis supported the findings of the in vitro model, with AqH-derived myeloid DC showing elevated MHC, but reduced CD86 expression. In summary human AqH is shown to be a powerful inhibitor of DC maturation, retaining this regulatory role during uveitis.

Thyroid-Associated Ophthalmopathy (TAO) is a debilitating inflammatory condition of the orbit occurring in 30-50% of Graves' Disease (GD) patients. It is not currently possible to predict which GD patients will develop TAO or the severity of their eventual ophthalmic manifestations. The aim of this thesis was to evaluate novel biomarkers for this purpose. I developed two immunoassays to detect serum antibodies to insulin-like growth factor-1 receptor (IGF-lR-Ab) in GD, TAO and healthy controls (HC). Assays were validated to measure commercial monoclonal IGF-lR-Ab but no study group differences, or correlation with clinical activity or severity, were noted with sera. Differential IGF-lR expression on peripheral blood CD4+ and CDS+ T lymphocyte memory subsets was observed, although without variance between groups. However, T cell differentiation was perturbed, with elevated proportions of naïve, and reduced cytokine-producing effector memory T cells, in GD and TAO compared to HC. Nuclear magnetic resonance-based serum metabolomic analysis differentiated GD and TAO subjects, and varying TAO clinical activity, with good uncorrected sensitivity and specificity. Distinguishing metabolites included lactate, isopropanol, methylguanidine and pyruvate. Collectively these data cast doubt on a simple model of IGF-lR-Ab being responsible for orbital inflammation in GD, but highlight the biomarker potential of metabolomics in TAO.

Haemopoietic Stem Cell Transplant (HSCT) is used as a treatment for a number of conditions particularly leukaemias. Following conditioning and HSCT, there is a ‘resetting’ of the immune system, which reconstitutes over a number of months. Graft-versus-Host Disease (GvHD) is a life-threatening complication of HSCT that includes severe, sight-threatening dry eye disease. In GvHD transplanted immune cells mount an immune response against the host. This thesis investigated how the immune cells of the conjunctiva are affected by HSCT and how the ocular surface leukocytes reconstitute. A non-invasive technique, ocular surface impression cytology (OSIC), was used to demonstrate that whilst there was no apparent depletion of innate immune cells in the conjunctiva, there was a marked reduction in the lymphocytes, which gradually reconstituted, returning to normal levels at the 6 months timepoint. Secondly OSIC was used to profile the leukocyte population in a cohort of patients post-HSCT with and without eye disease. In patients with dry eye disease following HSCT, the conjunctiva contained increased CD8+ lymphocytes, macrophages and neutrophils; a pattern that was distinct to that found in patients with dry eye disease following HSCT.

Ocular Mucous Membrane Pemphigoid (OcMMP) is a blinding immunobullous disease, characterised by auto-antibody driven conjunctival inflammation and scarring. My hypothesis was that progressive fibrosis in OcMMP, occurring in the apparent absence of clinical inflammation, was driven by underlying inflammatory processes. I observed that in OcMMP, progressive scarring did occur in the apparent absence of clinically identifiable inflammation and I was able to improve clinical documentation by developing and validating an objective Fornix Depth Measurer (FDM) for assessment of scarring. I optimised non-invasive Ocular Surface Impression Cytology (OSIC) combined with flow cytometry to characterise conjunctival leukocytes. I found that CD8αβ+ effector memory, cytotoxic, mucosal-homing T cells were the dominant population in health. This population was unaltered with age but CD4+ T cells, capable of producing IFN-γ, increased. In OcMMP, the conjunctiva was characterised by decreased CD8+ lymphocytes and an elevation in CD45INTCD11b+CD16+CD14- neutrophils. Although neutrophils correlated with clinical inflammation, they were even present in the absence of identifiable conjunctivitis. This elevation was associated with progression of scarring assessed by FDM, even in the clinically Non-inflamed eye. These findings confirmed my hypothesis and provide a platform for quantifying neutrophils as a biomarker of sub-clinical inflammation and their role in the scarring process.

Cataract is the opacification of the crystalline lens of the eye. Both childhood and later-onset cataracts have been linked with complex genetic factors. Cataracts vary in phenotype and exhibit genetic heterogeneity. They can appear as isolated abnormalities, or as part of a syndrome. During this project, analysis of syndromic and non-syndromic cataract families using genetic linkage studies was undertaken in order to identify the genes involved, using an autozygosity mapping and positional candidate approach. Causative mutations were identified in families with syndromes involving cataracts. The finding of a mutation in CYP27A1 in a family with Cerebrotendinous Xanthomatosis permitted clinical intervention as this is a treatable disorder. A mutation that segregated with disease status in a family with Marinesco Sjogren Syndrome was identified in SIL1. In a family with Knobloch Syndrome, a frameshift mutation in COL18A1 was detected. Analysis of families with non-syndromic autosomal recessive cataracts was also performed, identifying homozygous candidate regions, and sequencing candidate genes within these regions. The identification of a potential putative mutation in one family in CDC25A illustrated the challenges of distinguishing between rare benign variants and pathogenic mutations. Identification of novel genes involved in cataractogenesis will increase understanding of the pathways involved in cataract formation, and benefit affected families through genetic counselling, and, potentially personalised management.

Betaglycan, also known as TGFβRIII, is a component of the TGFβ receptor, being thought to act as a co-factor in signal transduction. It has proven roles in TGFβ-mediated embryogenesis and carcinogenesis, and facilitates signalling by activins and inhibins within the endocrine system. Despite the recognised importance of TGFβ in immunity, little is known about betaglycan in immune regulation. In mice, absolute deficiency of any of the three isoforms of TGFβ, or any of the individual TGFβ receptor components results in embryonic or perinatal lethality, limiting the ability to study peripheral immune responses in these models. We developed a chimeric model in which betaglycan-deficient stem cells were transferred to immunodeficient hosts, allowing study of mature animals with betaglycan deletion restricted to T and B lymphocytes. We were thus able to investigate peripheral immune responses in vivo, and assess TGFβ signalling to T lymphocytes in vitro, in the presence and absence of betaglycan. Betaglycan deficiency resulted in upregulated CD4+ and CD8+ T lymphocyte activation, with increased Th1 polarisation in peripheral lymphocyte compartments in both naïve and antigen-experienced animals. These observations confirm the involvement of betaglycan in T lymphocyte biology, providing the first evidence for its role in regulation of peripheral immune responses.

Introduction A risk factor for POAG is elevated intraocular pressure induced by reduced outflow of aqueous humour through the fibrosed trabecular meshwork, causing retinal ganglion cell death. This study aimed to: 1) create a rodent model of increased intraocular pressure by inducing trabecular meshwork fibrosis; 2) evaluate the ability of the anti-fibrotic agent, Decorin to diminish trabecular meshwork fibrosis and lower intraocular pressure; 3) correlate these findings to RCG survival and 4) investigate alternative translational methods for delivery of Decorin into the eye. Methods Fibrotic agents, kaolin or TGF-β, were intracamerally injected to induce fibrosis and intraocular pressure elevation. Following this human recombinant Decorin was administered intracamerally. Fibrosis was measured by immunohistochemistry and electron microscopy. Retinal ganglion cell counts were performed from retinal whole mounts. Results TGF-β induced fibrosis of the trabecular meshwork leading IOP elevation and retinal ganglion cell death, thus providing a reliable in vivo model of the ocular fibrosis. Decorin reversed the TGF-β induced fibrosis in the trabecular meshwork, lowered intraocular pressure and indirectly prevented retinal ganglion cell death. Decorin was successfully delivered to the anterior chamber in an eye drop formulation. Conclusion Decorin may be an effective treatment to preserving visual function in patients with POAG.

No description available.

Introduction: Transforming growth factor-β1 (TGF-β1) is a key cytokine that promotes fibrosis after injury in many adult tissues. Here, we hypothesise that delivery of human recombinant decorin (hr-decorin), a natural antagonist of TGF-β1, loaded into gellan-based biomembrane sheet dressings and fluid gel eye drops, prevents cutaneous and corneal scarring after injury. Methods: Gellan-based wound dressings loaded with hr-decorin were characterised for swelling, release profile of hr-decorin, systemic absorption of hr-decorin and human skin reactions. Topical gellan-based fluid gels loaded with hr-decorin were also tested for penetration of hr-decorin into the cornea and aqueous humour (AqH). The effect of hr-decorin on cell migration, differentiation and expression of scar-associated molecules of TGF-β1-stimulated human dermal fibroblasts (HDF) was also evaluated. Results: Gellan-based wound dressings had a high fluid uptake capacity, no adverse reactions on human skin, and sustained local release but no systemic absorption of hr-decorin after application to pig mid-dermal burns. Gellan-based fluid gel eye drops also released hr-decorin over time, which penetrated the cornea and was detected in AqH. The mRNA expression of scar-associated molecules in cultured TGF-β1-stimulated HDF was significantly decreased by hr-decorin, but no changes at the protein level were detected. Conclusion: The results suggest that delivery of Decorin through gellan-based wound dressing and fluid gel eye drop formulations have the potential for translation into therapies for cutaneous and corneal scarring.

Introduction: Multiple sclerosis is a disabling disease with impact on the social, financial and occupational life of an individual. Diagnosis of Multiple sclerosis even with the McDonald criteria, sometimes can take many years, by which time the patient has already accumulated significant disability. Hence the need for early diagnosis and prompt treatment for a better prognosis highlighted in different MS studies. Visual dysfunction in patients with Multiple sclerosis is a common finding. Asymptomatic visual loss has been highlighted in various studies. In the United Kingdom, studies have focused on genetics, environmental factors, assessment of neurological state (EDSS, MSFC) along with MRI of the brain and spinal cord. There is no single study in the UK that has looked at the visual pathway in Multiple sclerosis and correlated with the neurological outcomes. Aim: Correlation of structure and function of visual pathway in Multiple Sclerosis Methodology: We recruited 55 (110 eyes) patients with relapsing remitting MS and 25 controls (50 eyes) into this project with specific inclusion and exclusion criteria. Power calculations were done based on the parameters used for the study. Coefficient of reliability and variability was tested prior to the study in a subset of control patients. Logistic regression model with odds ratio was used to assess the ability of each test to differentiate disease from control. Subgroup analysis was done for the optic neuritis group and the group receiving disease-modifying therapy. Function of the visual pathway was assessed using Sloan contrast charts along with HRR colour chart and HVF indices. Structure of the visual pathway was mainly assessed using different protocols from Optical Coherence Tomography. Ethical approval was obtained through the North Midlands IRAS and Keele University. Results: The structure of the visual pathway correlates significantly with function of vision. This in turn correlates with neurological function. Visual tests are more sensitive than neurological testing by EDSS and hence can be used in diagnosis and to determine prognosis in RRMS. GCIP index, EDI OCT and posterior pole protocols on OCT have revealed useful and valuable information that can be translated into clinical use to manage patients with MS. Conclusion: Sloan contrast charts and HRR colour testing are sensitive measures of disease progression. Visual field test indices on the Humphrey’s field test closely relate to RNFL thickness on posterior pole OCT. GCIP and EDI-OCT show different clinico-pathological staging in MS. Visual parameters have synergistic power in evaluating the disease process in MS. Visual testing for structure and function is a valuable addition to the routine assessment of MS patients. Visual assessment helps in detection of the spectrum of disease in MS with specifics to the range of manifestations and severities of illness. Multiple sclerosis like many other diseases exhibit the iceberg phenomenon. The iceberg phenomenon describes a situation in which a large percentage of a problem is subclinical or otherwise hidden from view. Thus, only the "tip of the iceberg" is apparent to the clinician. Visual assessments help in uncovering disease below the sea level by early detection and subsequent better disease control. Visual studies in MS using the parameters in this study must be considered for routine use in our day to day practice in diagnosis and managing patients more effectively.