Rosalind M. K. Stewart

Genetic characterization indicates that a specific subpopulation of Pseudomonas aeruginosa is associated with keratitis infections.

ABSTRACT Pseudomonas aeruginosa is a common opportunistic bacterial pathogen that causes a variety of infections in humans. Populations of P. aeruginosa are dominated by common clones that can be isolated from diverse clinical and environmental sources. To determine whether specific clones are associated with corneal infection, we used a portable genotyping microarray system to analyze a set of 63 P. aeruginosa isolates from patients with corneal ulcers (keratitis). We then used population analysis to compare the keratitis isolates to a wider collection of P. aeruginosa from various nonocular sources. We identified various markers in a subpopulation of P. aeruginosa associated with keratitis that were in strong disequilibrium with the wider P. aeruginosa population, including oriC, exoU, katN, unmodified flagellin, and the carriage of common genomic islands. The genome sequencing of a keratitis isolate (39016; representing the dominant serotype O11), which was associated with a prolonged clinical healing time, revealed several genomic islands and prophages within the accessory genome. The PCR amplification screening of all 63 keratitis isolates, however, provided little evidence for the shared carriage of specific prophages or genomic islands between serotypes. P. aeruginosa twitching motility, due to type IV pili, is implicated in corneal virulence. We demonstrated that 46% of the O11 keratitis isolates, including 39016, carry a distinctive pilA, encoding the pilin of type IV pili. Thus, the keratitis isolates were associated with specific characteristics, indicating that a subpopulation of P. aeruginosa is adapted to cause corneal infection.

Effect of Glaucoma on Corneal Graft Survival According to Indication for Penetrating Keratoplasty

PURPOSE To determine whether the risk of graft failure in patients with glaucoma is dependent on the indication for penetrating keratoplasty (PK). DESIGN Retrospective cohort study. METHODS All patients on the United Kingdom Transplant Registry undergoing their first PK over a 7-year period with at least 1 year of follow-up were included. Data were collected on indication for PK, presence and management of glaucoma, graft diameter, recipient risk factors, and graft survival. Kaplan-Meier survival curves, a Cox regression model, and χ(2) and t tests were used in group comparisons. RESULTS A total of 6255 transplants in eyes without glaucoma and 1994 in eyes with glaucoma were analyzed. Three-year transplant survival was 86% and 72% respectively (P < .0001), and 73% in eyes with medically managed glaucoma compared to 63% in surgically managed glaucoma (P = .07). Glaucoma patients undergoing PK for pseudophakic bullous keratopathy or Fuchs dystrophy had significantly increased relative risks of graft failure (1.5 and 1.9 with topical and 2.0 and 3.1 with oral antiglaucoma medication respectively, compared to those without glaucoma). There was no equivalent significant difference for those with keratoconus, previous noncataract ocular surgery, trauma, or noninfectious ulcerative keratitis. Endothelial decompensation accounted for a significantly greater proportion of graft failure in recipients with glaucoma (topical [9%] and oral medication [13%]) than in those without glaucoma (3%) (P < .001). DISCUSSION The presence of glaucoma carries an increased risk of graft failure, in particular from endothelial decompensation. This risk is, however, also dependent on the indication for PK, with transplants undertaken for primary corneal endothelial disease carrying a higher risk.

Ocular epithelial transplantation: current uses and future potential.

Visual loss may be caused by a variety of ocular diseases and places a significant burden on society. Replacing or regenerating epithelial structures in the eye has been demonstrated to recover visual loss in a number of such diseases. Several types of cells (e.g., embryonic stem cells, adult stem/progenitor/differentiated epithelial cells and induced pluripotent cells) have generated much interest and research into their potential in restoring vision in a variety of conditions: from ocular surface disease to age-related macular degeneration. While there has been some success in clinical transplantation of conjunctival and particularly corneal epithelium utilizing ocular stem cells, in particular, from the limbus, the replacement of the retinal pigment epithelium by utilizing stem cell sources has yet to reach the clinic. Advances in our understanding of all of these cell types, their differentiation and subsequent optimization of culture conditions and development of suitable substrates for their transplantation will enable us to overcome current clinical obstacles. This article addresses the current status of knowledge concerning the biology of stem cells, their progeny and the use of differentiated epithelial cells to replace ocular epithelial cells. It will highlight the clinical outcomes to date and their potential for future clinical use.

Human Conjunctival Stem Cells are Predominantly Located in the Medial Canthal and Inferior Forniceal Areas

PURPOSE The conjunctiva plays a key role in ocular surface defence and maintenance of the tear film. Ex vivo expansion of conjunctival epithelial cells offers potential to reconstruct the ocular surface in cases of severe cicatrising disease, but requires initial biopsies rich in stem cells to ensure long-term success. The distribution of human conjunctival stem cells, however, has not been clearly elucidated. METHODS Whole human cadaveric conjunctiva was retrieved and divided into specific areas for comparison. From each donor, all areas from one specimen were cultured for colony-forming efficiency assays and immunocytochemical studies; all areas from the other specimen were fixed and paraffin embedded for immunohistochemical studies. Expression of CK19, p63, and stem cell markers ABCG2, ΔNp63, and Hsp70 were analyzed. Results were correlated to donor age and postmortem retrieval time. RESULTS Conjunctiva was retrieved from 13 donors (26 specimens). Colony-forming efficiency and expression of stem cell markers ABCG2, ΔNp63, and Hsp70 in cultures and ABCG2 in fixed tissue were all consistently demonstrated throughout the tissue but with highest levels in the medial canthal and inferior forniceal areas (P < 0.01 for each). Both increasing donor age and longer postmortem retrieval times were associated with significantly lower colony-forming efficiency, stem cell marker expression in cell cultures and ABCG2 expression in fixed tissue. CONCLUSIONS Biopsies from the medial canthus and inferior forniceal areas, from younger donors, and with short postmortem retrieval times offer the greatest potential to developing conjunctival stem cell-rich epithelial constructs for transplantation.

Insulin Resistance and Autoregulatory Dysfunction in Glaucoma and Retinal Vein Occlusion

r PHTHALMOLOGISTS RARELY ARE PERTURBED TO discover that their patients also have some or all of the following: hypertension, diabetes, hyperipidemia, and coronary artery disease. The pivotal role of nsulin resistance in producing this constellation of disorers was recognized almost 20 years ago by Reaven. Insulin resistance is the result of a complex of genetic nd environmental factors and may be defined as the mpaired ability of insulin (either endogenous or exogeous) to lower blood glucose. It is the early metabolic bnormality that precedes the development of overt type II r non–insulin-dependent diabetes mellitus (NIDDM). he inability of the pancreatic cell to increase its insulin ecretory response and to maintain chronic hyperinsulinmia (and thus normoglycemia) leads first to impaired lucose tolerance (IGT) and ultimately to NIDDM. Insuin resistance is present in most patients with IGT or IDDM and in approximately 25% of nonobese individals with normal oral glucose tolerance, the degree of nsulin resistance being of similar severity regardless of the agnitude of hyperglycemia. Insulin resistance may explain coexistent hyperlipidmia, coronary artery disease, and obesity. Additional vidence suggests that insulin resistance is the link beween diabetes and essential hypertension. Compared with geand weight-matched normotensive controls, hyperensive patients show a heightened plasma insulin response o glucose challenge. Additionally, hypertension has been roduced in normal rats by experimental induction of nsulin resistance and hyperinsulinemia, suggesting that he relationship between these variables may be a causal ne. Systemic abnormalities associated with both ocular hyertension and open-angle glaucoma include essential ypertension, diabetes, hyperlipidemia, cardiovascular disase, and obesity. Studies reporting these invariably have ollected information from patient self-reporting only. ecause it is accepted that hypertension, hyperlipidemia, nd NIDDM often may be unrecognized, formal testing is equired to clarify the true extent of these relationships. imilarly, retinal vein occlusion is associated with hyperension, hyperlipidemia, and diabetes; again, the extent as yet to be established formally.

The use of voriconazole in the treatment of Aspergillus fumigatus keratitis

There are a few reports of the use of voriconazole for the treatment of fungal keratitis.1–5 We report another case of its apparent success in Aspergillus fumigatus keratitis and discuss the dilemma of prescribing an expensive drug in the absence of defined ophthalmic therapeutic levels. A 51 year old diabetic man presented a week after being poked in his eye by a child’s finger, with a 3.0×3.5 mm central corneal ulcer overlying full thickness stromal infiltrate with associated hypopyon (fig 1). Pinhole visual acuity was 2/60 in this eye. Topical ciprofloxacin 0.3% was started. Aspergillus fumigatus was isolated after 48 hours’ incubation and topical amphotericin B 0.15%, voriconazole 1% hourly, and …

“Here’s egg in your eye”: a prospective study of blunt ocular trauma resulting from thrown eggs

Objective: To see if a public awareness campaign might be justified around Halloween with regard to the dangers of egg throwing. Method: A prospective study was carried out of all patients who attended the St Paul’s Eye Unit’s Primary Care Department with ocular injuries resulting from a thrown egg over a 14-month period from November 2004. All injuries were classified as minor, intermediate or major and patients were followed up until discharge. Results: 13 ocular injuries that were attributed to assault with a raw egg were reported. In all the 13 cases, the eggs had been thrown by strangers. 12 of the patients were men and the average age of the victims was 27.9 years. 9 patients were injured in the left eye and there were no bilateral injuries. On presentation, only 1 patient had a visual acuity of 6/6, 7 presented at 6/9, with the remainder having 6/18 or worse. All the patients had closed globe injuries. 8 injuries were classified as major injuries. 4 patients had permanent sequelae, with one suffering permanent, severe visual loss. Conclusions: Although most of our patients showed improvement in visual acuity, there were severe injuries, with the potential for severe ocular morbidity. We conclude that there is sufficient injury caused by this prank to warrant a public health message. At the least this practice should not be promoted by the press.

Conjunctival-corneal melt in association with carotid artery stenosis.

Purpose To report a case of severe conjunctival-corneal melt in association with carotid artery stenosis. Methods Observational case report. Results A 76-year-old man with a history of bilateral severe carotid artery occlusion and nonarteritic ischemic optic neuropathy developed a spontaneous bulbar conjunctival defect. Despite intensive lubrication, and attempts at surgical closure including an amniotic membrane patch graft, it progressed with subsequent adjacent corneal perforation. Thorough investigations revealed no underlying disease, except markedly delayed episcleral vessel filling on anterior segment fluorescein angiography. Conclusions Neovascularisation is a known factor in the inhibition of ulceration. In light of the findings in this report, ocular ischemia should be considered as a cause or contributing factor in the differential diagnosis of conjunctival-corneal melt.

Development of a Poly-ε-Lysine Contact Lens as a Drug Delivery Device for the Treatment of Fungal Keratitis

Purpose The purpose of this study was to develop a more efficient drug delivery device to overcome the limitations of current drop therapy for the treatment of fungal keratitis. Methods Amphotericin B (AmpB), 0 to 30 μg/mL, was associated with a poly-ε-lysine (pεK) hydrogel. Fungicidal effect against Candida albicans was assessed at 18 and 42 hours by optical density (OD600) and growth on agar. Tear film dilution effect was mimicked by storage of AmpB pεK gels in 3.4 mL sterile PBS for 24 hours prior to fungal incubation. Drug elution over 96 hours was evaluated by HPLC, and drug stability was tested while associated with the gel by OD600 up to 48 hours. Lack of cytotoxicity toward the HCE-T corneal epithelial cell line was assessed over 7 days. Results AmpB pεK gels show fungicidal activity in normal conditions (0.057 OD600, SD 0.003, P < 0.005) and in the presence of horse serum (0.048 OD600, SD 0.028 P < 0.005) at 18 hours. The drug release profile was above therapeutic levels (0.188 μg/mL) for up to 72 hours. Tear dilution had no significant effect at higher concentrations of AmpB (3 to 10 μg/mL). AmpB pεK gels were not cytotoxic to the HCE-T cell line. Conclusions We demonstrated that AmpB pεK gels confer sustained therapeutic antifungal activity for at least 48 hours without corneal epithelial cell line cytotoxicity, suggesting their potential for in vivo use as an antifungal bandage contact lens. This could avoid the need for intensive topical medication in the treatment of fungal keratitis.

Yield and Viability of Human Limbal Stem Cells From Fresh and Stored Tissue

PURPOSE We compared cell number, putative stem cell markers, and clonogenic ability in fresh uncultured human limbal epithelial cells to that obtained from stored organ-cultured tissue. METHODS Cell suspensions were formed from fresh and organ culture-stored human limbal epithelium. Expression of putative stem cell markers ΔNp63 and TrkA was performed using immunofluorescent staining before culture. Colony-forming efficiency (CFE) assays were performed at first passage. The effects of tissue storage, age, and postmortem/culture times were analyzed in a general linear model. RESULTS Limbal tissue from 94 donors (34 fresh and 60 stored) was compared. Three times more cells were obtained per eye from fresh (35.34 × 104; SD, 17.39) than stored (11.24 × 104; SD, 11.57; P < 0.01) tissue. A higher proportion of cells from fresh tissue were viable (91.9%; SD, 5.7 vs. 85%; SD, 10.8) P < 0.01. Higher total cell expression of ΔNp63 (20.19 × 104; SD, 15.5 vs. 3.28 104; SD, 4.33) and TrkA (59.24 × 104; SD, 13.21 vs. 7.65 × 104; SD, 1.05) was observed in fresh than stored tissue (P < 0.01). Colony-forming efficiency was higher for fresh (1.42; SD, 0.12) than stored (0.43; SD, 0.15; P < 0.01) cells. For stored tissue only, there was a significant inverse relationship between donor age and total number of cells isolated (R2 = 0.27, P < 0.001). CONCLUSIONS Storage of corneoscleral discs in organ culture medium leads to significant reduction in limbal epithelial cell number, expression of ΔNp63 and TrkA, and viability compared to fresh tissue. There is a smaller basal stem cell population in stored compared to fresh tissue.