Publications by authors named "Rosalind M K Stewart"

22 Publications

  • Page 1 of 1

More than a scratch: emergency setting eye evaluation during COVID-19 lockdown.

BMJ Case Rep 2021 Jan 18;14(1). Epub 2021 Jan 18.

Ophthalmology, Leeds Teaching Hospitals NHS Trust, Leeds, UK.

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January 2021

Mass Spectrometry Reveals α-2-HS-Glycoprotein as a Key Early Extracellular Matrix Protein for Conjunctival Cells.

Invest Ophthalmol Vis Sci 2020 03;61(3):44


Purpose: To determine the composition of extracellular matrix (ECM) proteins secreted by a conjunctival epithelial cell line and to identify components that aid conjunctival epithelial cell culture.

Methods: Human conjunctival epithelial cell line (HCjE-Gi) cells were cultured in serum-free media and their ECM isolated using ammonium hydroxide. Growth characteristics were evaluated for fresh HCjE-Gi cells plated onto ECMs obtained from 3- to 28-day cell cultures. Mass spectrometry was used to characterize the ECM composition over 42 culture days. Cell adhesion and growth on pre-adsorbed fibronectin and α-2-HS-glycoprotein (α-2-HS-GP) were investigated.

Results: Day 3 ECM provided the best substrate for cell growth compared to ECM obtained from 5- to 28-day cell cultures. Mass spectrometry identified a predominantly laminin 332 matrix throughout the time course, with progressive changes to matrix composition over time: proportional decreases in matrix-bound growth factors and increases in proteases. Fibronectin and α-2-HS-GP were 5- and 200-fold enriched as a proportion of the early ECM relative to the late ECM, respectively. Experiments on these proteins in isolation demonstrated that fibronectin supported rapid cell adhesion, whereas fibronectin and α-2-HS-GP both supported enhanced cell growth compared to tissue culture polystyrene.

Conclusions: These data reveal α-2-HS-GP as a candidate protein to enhance the growth of conjunctival epithelial cells and raise the possibility of exploiting these findings for targeted improvement to synthetic tissue engineered conjunctival substrates.
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March 2020

Anterior Segment Optical Coherence Tomographic Angiography Assessment of Acute Chemical Injury.

Am J Ophthalmol 2019 09 10;205:165-174. Epub 2019 May 10.

Department of External Eye Diseases, Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom; Institute of Ophthalmology, University College London, London, United Kingdom.

Purpose: To compare routine clinical examination with optical coherence tomography angiography (OCTA) for the assessment of limbal conjunctival ischemia following a chemical burn.

Setting: Validity analysis.

Methods: We assessed 10 participants (15 eyes) with an acute chemical injury. Clinical photographs were used to determine the extent of any limbal conjunctival epithelial defect and ischemia. These were compared with the extent of limbal ischemia identified on OCTA images of the ocular surface. Quantitative and longitudinal analysis using the OCTA software were also performed. Correlations with visual outcome were sought using clinical and OCTA-derived variables.

Results: The extent of clinically determined limbal ischemia was less than that identified with OCTA (2.3±3.6 clock hours vs 5.1±4.2 clock hours, P = .003), which in turn was less than the size of limbal conjunctival epithelial defect (7.3±5.1 clock hours, P = .03). Longitudinal OCTA analysis showed that mean vessel area increased by 0.2%±0.1% during the study, corresponding to a rate of vascular recovery of 0.9 mm/d. Significant correlations were found between visual outcome at 3 months and limbal conjunctival fluorescein staining (r = 0.67, P = .006), and limbal conjunctival ischemia on OCTA (r = 0.76, P = .001).

Conclusions: OCTA can objectively identify and monitor the recovery of limbal ischemia following an acute ocular chemical injury. OCTA confirms that limbal ischemia is usually more extensive than is suggested by clinical examination, and the former is highly correlated with visual outcome. OCTA therefore is a useful tool in the management of ocular chemical injury.
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September 2019

A prospective study of the incidence, associations and outcomes of ocular surface squamous neoplasia in the United Kingdom.

Eye (Lond) 2019 02 28;33(2):283-294. Epub 2018 Dec 28.

School of Clinical Sciences, University of Edinburgh, Edinburgh, UK.

Purpose: To describe the incidence, associations and outcomes of ocular surface squamous neoplasia (OSSN) in the United Kingdom.

Methods: Prospective, observational study of every new case of OSSN reported via the British Ophthalmological Surveillance Unit reporting scheme over a 12-month period. Cases were followed up for 12 months.

Results: The reported incidence of OSSN was 0.53 cases/million/year (conjunctival intraepithelial neoplasia: 0.43 cases/million/year; squamous cell carcinoma: 0.08 cases/million/year). Eighty-five per cent of affected patients were male, 97% were Caucasian, and the mean age at presentation was 67.9 (±12.8) years. Information on potential underlying risk factors was frequently unknown. The most commonly affected sites were the limbus and the nasal and temporal bulbar conjunctivae. Most patients presented with a visual acuity of 6/9 or better, without symptoms of pain or visual loss. Excision (with or without additional treatment) was the most common first-line treatment and interferon (with or without additional treatment) was the most common second-line treatment, although management varied widely. Complications of treatment were rare but occasionally severe. Recurrence within 12 months of follow-up occurred in at least 6% of patients.

Conclusion: Although subject to reporting bias, these data suggest that there has not been a significant change in the incidence of OSSN in the United Kingdom, or its demographic profile, since 1996. The broad range of management approaches identified in this study reflect a lack of consensus as to the optimal referral and treatment pathways.
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February 2019

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

Invest Ophthalmol Vis Sci 2017 09;58(11):4499-4505

Department of Eye and Vision Science, Institute of Ageing and Chronic Diseases, University of Liverpool, Liverpool, United Kingdom.

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.
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September 2017

Development of decellularized conjunctiva as a substrate for the ex vivo expansion of conjunctival epithelium.

J Tissue Eng Regen Med 2018 02 12;12(2):e973-e982. Epub 2017 Jun 12.

National Health Service Blood and Transplant Tissue Services, Speke, Liverpool, UK.

This study was performed to develop a method to decellularize human conjunctiva and to characterize the tissue in terms of its deoxyribose nucleic acid (DNA) content, tensile strength, collagen denaturation, basement membrane, extracellular matrix components and its potential to support conjunctival epithelial growth. Human conjunctival tissues were subjected to a decellularization process involving hypotonic detergent and nuclease buffers. Variations in sodium dodecyl sulfate concentration (0.05-0.5%, w/v) were tested to determine the appropriate concentration of detergent buffer. DNA quantification, collagen denaturation, cytotoxicity and tensile strength were investigated. Human conjunctival cell growth by explant culture on the decellularized tissue substrate was assessed after 28 days in culture. Samples were fixed and paraffin embedded for immunohistochemistry including conjunctival epithelial cell markers and extracellular matrix proteins. Conjunctival tissue from 20 eyes of 10 donors (age range 65-92 years) was used. Decellularization of human conjunctiva was achieved to 99% or greater DNA removal (p < 0.001) with absence of nuclear staining. This was reproducible at the lowest concentration of sodium dodecyl sulfate (0.05% w/v). No collagen denaturation (p = 0.74) and no difference in tensile strength parameters was demonstrated following decellularization. No significant difference was noted in the immunolocalization of collagen IV, laminin and fibronectin, or in the appearance of periodic acid-Schiff-stained basement membranes following decellularization. The decellularized tissue did not exhibit any cytotoxicity and explant culture resulted in the growth of stratified conjunctival epithelium. Allogeneic decellularized human conjunctiva can be successfully decellularized using the described protocol. It represents a novel substrate to support the expansion of conjunctival epithelium for ocular surface cellular replacement therapies. Copyright © 2017 John Wiley & Sons, Ltd.
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February 2018

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

Invest Ophthalmol Vis Sci 2016 Jul;57(8):3708-13

Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom 2St. Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom.

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.
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July 2016

Overestimation of Intraocular Pressure by Goldmann Applanation Tonometry Without Astigmatic Correction.

JAMA Ophthalmol 2016 Mar 10;134(3):e153691. Epub 2016 Mar 10.

St Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, England2Department of Eye and Vision Science, University of Liverpool, England.

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March 2016

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

Invest Ophthalmol Vis Sci 2015 Feb 26;56(3):2021-30. Epub 2015 Feb 26.

Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom St. Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom.

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.
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February 2015

Rheumatoid arthritis-associated corneal ulceration: mortality and graft survival.

Ophthalmology 2013 Apr 3;120(4):682-6. Epub 2013 Jan 3.

St. Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom.

Purpose: To investigate mortality and graft survival in patients undergoing penetrating keratoplasty (PKP) for rheumatoid arthritis-associated corneal ulceration (RACU), Fuchs' endothelial dystrophy (FED), and pseudophakic bullous keratopathy (PBK).

Design: Case-control study.

Participants And Controls: Patients listed on the UK Transplant Registry who had undergone a PKP for RACU, FED, or PBK between January 4, 1999, and January 4, 2006. Comparative standardized mortality ratios (SMRs) and causes of death were obtained from the Office for National Statistics.

Methods: Outcome data were collected from the UK Ocular Tissue National Transplant database and supplementary questionnaires at transplantation and at 1, 2, and 5 years. Institutional review board approval for the National Health Service Blood and Transplant to undertake the study was obtained.

Main Outcome Measures: Mortality and graft survival.

Results: A total of 3665 patients were included: RACU (117), PBK (1701), and FED (1847). Five-year survival of patients with RACU was 42% (95% confidence interval [CI], 26-56) compared with 76% (95% CI, 72-78) for FED and 55% for PBK (95% CI, 50-60; P < 0.01). The SMRs for female and male patients with RACU were 43.5 (95% CI, 19.5-63.3) and 12.2 (95% CI, 7.1-19.5), respectively, in comparison with 1.84 and 1.45 for patients with RA, respectively (P < 0.01). There were no significant differences in the causes of death among patients with RACU, FED, or PBK (P > 0.9), with infection the most common cause. The 5-year graft survival rate was 48% (95% CI, 32-62) for RACU, 59% (95% CI, 56-62) for PBK, and 84% (95% CI, 82-86) for FED (P < 0.01).

Conclusions: Mortality and ocular morbidity were significantly increased in patients with RACU. Accelerated immunosenescence should be considered in the differential diagnosis of patients presenting with RACU, and a multidisciplinary approach to management is required.
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April 2013

Ocular epithelial transplantation: current uses and future potential.

Regen Med 2011 Nov;6(6):767-82

Department of Eye & Vision Science, Institute of Ageing & Chronic Disease, University of Liverpool, Daulby Street, L69 3GA, UK.

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.
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November 2011

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

J Clin Microbiol 2011 Mar 12;49(3):993-1003. Epub 2011 Jan 12.

Institute of Infection and Global Health, University of Liverpool, Duncan Building, Daulby Street, Liverpool, L69 3GA, United Kingdom.

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.
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March 2011

Effect of glaucoma on corneal graft survival according to indication for penetrating keratoplasty.

Am J Ophthalmol 2011 Feb 18;151(2):257-62.e1. Epub 2010 Dec 18.

St Paul's Eye Unit, Royal Liverpool Hospital, Liverpool, United Kingdom.

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.
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February 2011

Conjunctival-corneal melt in association with carotid artery stenosis.

Clin Ophthalmol 2008 Sep;2(3):649-55

St Paul’s Eye Unit, Royal Liverpool University Hospital, Liverpool, UK.

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.
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September 2008

Cyclosporin-A associated malignancy.

Clin Ophthalmol 2007 Dec;1(4):421-30

St. Pauls Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom.

The use of cyclosporin is well established within the ophthalmology community, especially against sight threatening intraocular inflammation. It is well known however, that immunosuppression in general is a risk factor for the development of malignancy and numerous studies point to the risk imposed by cyclosporin. This article analyses and reviews all relevant studies with regard to the development of malignancy associated with the use of cyclosporin and extrapolates this into the ophthalmic setting. This is to enable clinicians to assess the risks in individual patients and to present a monitoring regime which can be used in patients undergoing cyclosporin treatment. The review is solely concerned with the risk of the development of malignancy following cyclosporin immunosuppression and not with any other adverse effect.
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December 2007

Vision impairment with an interstitial deletion of the short arm of chromosome 8.

Ophthalmic Genet 2007 Jun;28(2):101-4

Department of Paediatric Ophthalmology, Royal Liverpool Children's Hospital - Alder Hey, Liverpool, United Kingdom.

We describe the phenotype of a male infant with an interstitial deletion of the short arm of chromosome 8 (p. 11.2-p. 21). Visual impairment is a major feature in this case. The clinical, radiographic and electrodiagnostic findings are presented. Only four other cases have been reported in which visual problems are associated with a deletion of 8 p.
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June 2007