A thesis submitted to the faculty of medicine, University of the Witwatersrand, Johannesburg for the degree of Doctor of Philosophy (Medicine). Johannesburg 1991. / This thesis describes corneal ulceration in the black South African and how the pattern of the disease differs from that seen in white South Africans. Initial comparative surveys of one year each were conducted at St John Eye Hospital and the Johannesburg Hospital. These surveys showed that the main problem area in the black patient was central bacterial corneal ulcers while in the white, central viral ulcers accounted for most of the cases seen. Marginal catarrhal ulceration. in the black was commonly seen but it showed a better response to treatment and a better visual outcome. There was generally a marked male predominance in blacks and this was mainly related to predisposing factors. TIle commonest predisposing factors. in the black. patient were episodes of corneal trauma, climatic droplet keratopathy, corneal scarring, malnutrition and alcohol abuse. Pretreatment with antibiotics was found in nearly half of the patients and this was significantly associated with the absence of significant isolates in 40% of central ulcers. Single Gram-posltlve bacterlal isolates were round in 30% of-central ulcers and single Gram-negative isolates in 15%, While fungal isolates were recovered in 5.5% of central ulcers. Streptococcus pneumoniae was by far the commonest orgllnism. accounting for 42% of SIgnificant bacterial. isolates. This organism and Pseudomonas aeruginos were associated with some of the major complications (deseemetocoeles and perforations). Using fixed treatment regiments the results of treatment in mild and moderate bacterial and fungal keratitis were good, as well as most types of marginal ulcers. Vision was shown to improve significantly in the moderate bacterial infections but not in the severe and complicated bacterial cases, A numerical ranking system for the statistical analysis of visual. acuity is described and the method was found to produce consistent results. In a controlled clinical trial using topical steroids plus antibiotics versus antibiotics alone, no difference in visual outcome could be shown. Complications in the two groups were similar, however, and the steroid did not appear to retard the rate of heating. Recommendations for the incorporation of topical steroid Into a treatment regimen are made. Principles for the management of suppurative keratitis in a developing population are outlined in the conclusions. / AC2017

Modulation of corneal transplant rejection using gene therapy shows promise in experimental models but the most appropriate vector for gene transfer is yet to be determined. The overarching aim of the thesis was to evaluate the potential of a lentiviral vector for use in human corneal transplantation. Specific aims were: (i) to assess the ability of an HIV-1-based lentiviral vector to mediate expression of the enhanced yellow fluorescent protein (eYFP), and a model secreted protein interleukin-10 (IL10), in ovine and human corneal endothelium; and (ii) to examine the influence of lentivirus-mediated IL10 expression on the survival of ovine corneal allografts. Four lentiviral vectors expressing eYFP under the control of different promoters, were tested: the simian virus type-40 (SV40) early promoter, the phosphoglycerate kinase (PGK) promoter, the elongation factor-1alpha (EF) promoter, and the cytomegalovirus (CMV) promoter. Two lentiviral vectors expressing IL10 were tested: one containing the SV40 promoter and another containing a steroid-inducible promoter (GRE5). Lentivirus-mediated expression in transduced ovine and human corneal endothelium was assessed by fluorescence microscopy, real-time quantitative RT-PCR and ELISA, following alterations of transduction period duration (2–24 hr) and vector dose, as well as in the presence or absence of polybrene or dexamethasone (GRE5 vector). It was also compared to expression mediated by adenoviral vectors. Orthotopic transplantation of ex vivo transduced donor corneas was performed in outbred sheep. Allografts were reviewed daily for vascularisation and signs of immunological rejection. Lentivirus-mediated eYFP expression was delayed in ovine corneal endothelium compared to human. However, in both species the final transduction rate was greater than 80% and expression was stable for at least 14 d in vitro. Lentivirus-mediated expression in ovine and human corneal endothelium was higher with the viral promoters in comparison to the mammalian promoters. A 24 h transduction of ovine corneal endothelium with the lentiviral vector encoding IL10 resulted in expression levels which were increasing after 15 d of organ culture but logarithmically lower than those achieved by adenovirus. Shortening the lentiviral transduction period to 2 h led to a reduction in expression, but the addition of polybrene (40 micrograms / ml) to the transduction mixture restored expression to levels comparable to those attained after a 24 h transduction period. Lentivirus-mediated IL10 expression was higher and more rapid in human corneal endothelium compared to ovine corneas. Dexamethasone-responsive transgene expression was observed in both ovine and human corneal endothelium using the lentiviral vector containing the GRE5 promoter. Lentivirus-mediated expression in ovine corneal endothelium was stable for 28 d in vivo. A modest prolongation of ovine corneal allograft survival (median of 7 d) was achieved by transduction of donor corneas for 2–3 h with the lentivirus expressing IL10. Attempts to increase the expression of IL10 by the addition of polybrene (40 micrograms / ml) to the transduction mixture, resulted in a toxic effect on corneal allografts which abrogated the beneficial effect of IL10. The lentiviral vector shows potential for the stable expression of therapeutic transgenes in human corneal transplantation. However, the mechanisms underlying the species-specific differences in HIV-1-mediated transgene expression will need to be elucidated and overcome if the ovine preclinical model is to provide justification for a clinical trial.

OBJECTIVE: This case series evaluated the vision and refractive outcomes of patients who underwent Raindrop corneal inlay removal with or without concomitant LASIK treatment. METHODS: This study utilized a case series design and a retrospective data review of electronic medical records from a private ophthalmology clinic in Boston, MA. Twenty-one patients who had a Raindrop inlay placed and subsequently removed were identified. To be included in the study, patient records had to contain vision and refraction measurements from (1) before Raindrop inlay placement, (2) before Raindrop inlay removal, and (3) at least 1 month after Raindrop inlay removal. Fifteen patients with the necessary records were included in the study. Of these 15 patients, 7 had Raindrop inlays removed and the remaining 8 had Raindrop inlays removed with concomitant LASIK treatment. RESULTS: Fourteen of the 15 total patients had a best-corrected distance vision of 20/20 or better prior to inlay placement. This study found that 10 of these 15 patients experienced a loss of at least 1 line of best-corrected distance vision by the time of inlay removal. Inlay removal was performed at a mean of 523 days, or approximately 17 months, after placement. Even after inlay removal (>6 weeks), 7 patients continued to sustain a loss of 1 or more lines of best-corrected distance vision. In patients receiving LASIK at or after the time of inlay removal, the refractive outcome was close to target in only one case. CONCLUSION: After Raindrop inlay removal, many patients experience an improvement in their vision compared with pre-explantation; however, their best-corrected distance vision may not immediately return to preoperative values. Corneal haze and inflammation may continue to persist post-explantation and affect measurements of refractive error. Concomitant LASIK treatment upon inlay removal may therefore fail to achieve the intended result of monovision as an alternative treatment for presbyopia.

No description available.

Meesmann's epithelial corneal dystrophy (MEeD), which clinically presents with microcysts that can cause irritation, blurred vision or photophobia, is a genetic disorder caused by dominant-negative mutations in the KRT3 and KRT12 genes. Eradicating the mutant protein or tipping the balance strongly in favour of the wild type allele are viable options for therapeutic intervention. Here we studied two therapeutic approaches for suppression of the mutant KRTl2 allele and have developed, characterised and initiated in vivo testing using two novel KRTl2 mouse models. For a transient therapeutic approach, short interfering RNAs (siRNAs) were designed and proved capable of mutation-specific inhibition of the alleles responsible for two MEeD causative mutations (p.Leu132Pro and p.Arg135Thr; 70-90%) in vitro. No off-target issues were observed and suppression of endogenously expressed p.Leu132Pro was also shown in an ex vivo model. For a more generic, yet potentially permanent therapeutic approach, total KRTl2 was suppressed (~50%) with an siRNA expressed from a short hairpin by targeting a region homologus to both the WT and mutant mRNAs. KRT 12 was replaced with a co-expressed recoded allele made resistant to the siRNA. To further develop these potential therapeutics, two novel mouse models were generated allowing evaluation of gene modulation in vivo. (1) A humanised dominant negative mutant model that expresses K12 p.Leu132Pro revealed major changes to corneal phenotype in homozygous animals. Microcysts were observed and keratin expression patterns disrupted. Additionally, RNAseq analysis highlighted over 1600 dysregulated genes, which could feature other potential therapeutic targets for the treatment of symptomatic MEeD. Heterozygous mice presented with a subtler phenotype. (2) A Krt12 luciferase reporter mouse model was optimised and will facilitate live animal corneal imaging, thus aiding the development of topical siRNA delivery formulations. These mouse models in conjunction with our gene silencing development programme pave the way for in vivo assessment of RNA i-based therapeutics for the cornea.

No description available.

No description available.

No description available.

No description available.

No description available.