Macular corneal dystrophy and associated corneal thinning.

PURPOSE: To identify the molecular genetic cause of macular corneal dystrophy (MCD) in four probands, and characterize phenotypic similarities between MCD and keratoconus. METHODS: We performed ophthalmological examination, Scheimpflug imaging (Pentacam, Oculus Inc.), histopathological examination of excised corneal buttons, and direct sequencing of the CHST6 coding region. RESULTS: Pentacam measurements were taken in six eyes of three probands. All showed diffuse corneal thinning with paracentral steepening of the anterior corneal surface that was graded as keratoconus by the integrated software, but without associated ectasia of the posterior corneal surface or regional thinning. Homozygous or compound heterozygous CHST6 mutations were identified in all cases, including two novel mutations, c.13C>T; p.(Arg5Cys) and c.289C>T; p.(Arg97Cys). DISCUSSION: Localized elevation of the anterior corneal curvature can occur in MCD in the absence of other features of keratoconus. The identification of a further two Czech probands with the compound allele c.[484C>G; 599T>G] supports the enrichment of this allele in the study population.

Wnt signalling in an in vitro niche model for conjunctival progenitor cells.

Mimicking an environment in vitro that is more similar to the stem cell niche in vivo, by co-culture of mitotically active conjunctival fibroblasts (HCF) with human conjunctival epithelial cells (HCECs), improves the maintenance of epithelial cells with progenitor cell characteristics during in vitro expansion. However, little is known about the pathways controlling the fate of the epithelial progenitor cells during in vitro culture. In this study, differences in gene expression between this in vitro 'niche' model and standard culture conditions, in which growth-arrested 3 T3 feeder cells and fetal calf serum are used, were explored using a genome level microarray platform, quantitative (q)RT-PCR and western blot. The microarray analysis revealed significant alterations of biological processes involved in cell proliferation, differentiation and cell death. The analysis of stem cell-related pathways indicated changes in expression of genes involved in the Wnt signalling pathway, and further investigation by qPCR revealed significant downregulation of the Wnt ligands Wnt3, Wnt4, Wnt7B and Wnt10A, Wnt receptor proteins FZD1, LRP5, LRP6, ß-catenin and TCF7L1 and important Wnt target genes, such as CCND1, also confirmed by western blot and immunocytochemistry. The results indicate that epithelial cell expansion in the HCEC-HCF co-culture system is accompanied by significant changes in expression of genes involved in the Wnt signalling pathway. This altered pathway activation might be involved in the enhanced maintenance of epithelial progenitor cells in this in vitro 'niche' model.

The pathogenesis of keratoconus.

Keratoconus (KC) is a common degenerative condition that frequently results in visual loss with an onset typically in early adulthood. It is the single most common reason for keratoplasty in the developed world. The cause and underlying pathological mechanism are unknown, but both environmental and genetic factors are thought to contribute to the development of the disease. Various strategies have been employed to address the gap in our understanding of this complex disease, with the expectation that over time more sophisticated therapies will be developed. In this review we summarise our current knowledge of the aetiology and risk factors associated with KC.

Severe Meesmann's epithelial corneal dystrophy phenotype due to a missense mutation in the helix-initiation motif of keratin 12.

PURPOSE: To describe a severe phenotype of Meesmann's epithelial corneal dystrophy (MECD) and to determine the underlying molecular cause. METHODS: We identified a 30-member family affected by MECD and examined 11 of the 14 affected individuals. Excised corneal tissue from one affected individual was examined histologically. We used PCR and direct sequencing to identify mutation of the coding regions of the KRT3 and KRT12 genes. RESULTS: Cases had an unusually severe phenotype with large numbers of intraepithelial cysts present from infancy and they developed subepithelial fibrosis in the second to third decade. In some individuals, the cornea became superficially vascularized, a change accompanied by the loss of clinically obvious epithelial cysts. Visual loss from amblyopia or corneal opacity was common and four individuals were visually impaired (≤6/24 bilaterally) and one was blind (<6/60 bilaterally). In all affected family members, there was a heterozygous missense mutation c. 395T>C (p. L132P) in exon 1 of the KRT12 gene, which codes for the helix-initiation motif of the K12 polypeptide. This sequence change was not found in unaffected family members or in 100 unaffected controls. CONCLUSIONS: The Leu132Pro missense mutation is within the helix-initiation motif of the keratin and is predicted to result in a significant structural change of the K12 protein. The clinical effects are markedly more severe than the phenotype usually associated with the Arg135Thr mutation within this motif, most frequently seen in European patients with MECD.

Surgical rehabilitation following severe ocular burns.

Chemical and thermal burns can cause devastating injuries to the anterior segment. The consequences of alkali injuries are notoriously severe due to the rapid penetration of these agents into the ocular tissues. Denaturation of tissue, inflammation, and scarring leads to loss of function. An understanding of the pathogenesis of tissue damage has lead to a rational approach to treatment. Emergency irrigation of the eye is essential and there is a 'window of opportunity' during the first 7-10 days after injury when medical treatment can significantly limit the potentially blinding consequences. The acute injury is followed by early and late reparative phases during which the prognosis can be further improved by surgical intervention. Early surgical intervention is targeted at protecting the ocular surface and encouraging re-epithelisation. Later, surgical treatments are directed at ocular surface reconstruction and restoration of vision. However, before any attempt is made at surface reconstruction, the ocular surface environment must be optimised by division of symblepharon, and correction of lid deformity and trichiasis. If there is conjunctivalisation of the corneal surface, limbal stem cell transplantation can restore a corneal epithelial cell phenotype, and transplantation of in vitroamplified corneal epithelial stem cells has been developed as an alternative to keratolimbal transfer techniques. Keratoplasty and cataract surgery may then be necessary to clear the visual axis. Finally, keratoprosthesis is an option for the most severely damaged eyes.

Fungal keratitis in the United Kingdom 2003-2005.

PURPOSE: To describe the incidence and current management of fungal keratitis in the United Kingdom. METHODS: Cases were identified prospectively through the British Ophthalmologic Surveillance Unit (BOSU) from December 2003 to November 2005. Questionnaire data were requested at diagnosis and at 6 months follow-up. Inclusion criteria were a positive culture or microsopic proof from a scraping or biopsy, and a normal residence in the United Kingdom. RESULTS: Data were available on 39 confirmed cases at diagnosis and 34 cases at follow-up. The minimum average annualised incidence was 0.32 (95% CI, 0.24-0.44) cases per million individuals. In 22 cases (56%), only Candida was isolated and 14 of these (63%) had prior ocular surface disease treated with topical steroid. A filamentary fungus infection was more common in male patients (P=0.02), often following trauma, and the differences in risk factors between types of fungal infection was statistically significant (P<0.001). One case had a mixed yeast and filamentary fungus infection. The most frequent initial topical therapies were amphotericin B (38%) or econazole (28%). In addition, oral fluconazole was used in 11 (31%) patients and oral itraconazole in six (15%). At follow-up, the vision in 15 eyes (44%) was <6/60 including three eyes eviscerated. CONCLUSIONS: This study provides data on the minimum incidence of fungal keratitis in the United Kingdom. It provides evidence of frequent delay in diagnosis after presentation to eye departments, inconsistent management, and poor outcome. Issues that can now be addressed.

Sequencing of the CHST6 gene in Czech macular corneal dystrophy patients supports the evidence of a founder mutation.

AIMS: To characterise the role of the carbohydrate sulfotransferase gene (CHST6) in macular corneal dystrophy (MCD) in Czech patients. METHODS: The coding region of the CHST6 gene was directly sequenced in 10 affected and five unaffected members from eight apparently unrelated MCD families. The type of MCD was determined by enzyme-linked immunosorbent assay of antigenic keratan sulfate (KS) in serum and by immunohistochemical staining of corneas with monoclonal anti-KS antibody. RESULTS: The following changes in the coding sequence of the CHST6 gene were observed; homozygous mutation of c.1A>T (p.M1?); homozygous mutation c.599T>G (p.L200R); compound heterozygosity for c.599T>G and c.614G>A (p.R205Q); compound heterozygosity for c.494G>A (p.C165Y) and c.599T>G; heterozygous c.599T>G mutation and no other change in the coding sequence. One proband exhibited no changes. The pathogenic mutation c.599T>G (p.L200R) was in allelic association with the c.484C>G (p.R162G) polymorphism. Nine patients from seven families were of MCD type I including the subtype IA. CONCLUSION: Four different CHST6 missense mutations, of which p.C165Y is novel, were identified. Allelic association of the c.[484C>G; 599T>G] in six probands out of eight, as well as occurrence of this particular allele in a heterozygous state in one healthy control individual, supports a common founder effect for MCD in the Czech Republic.

Matrix metalloproteinase distribution during early corneal wound healing.

AIM: To compare matrix metalloproteinase (MMP) localisation in anterior keratectomy (AK) and lamellar keratectomy (LK) wounds. METHODS: Wounds were produced in one eye of 24 rabbits. The AK wounds were made to approximately 120 microm in depth and then allowed to re-epithelialise. The LK wounds were of similar depth, but the anterior stroma and epithelium were replaced after a second deeper keratectomy had been performed. Immunohistochemistry was used to localise the MMP-1, -2, -3, and -9 at intervals from 4 h to 14 days following surgery. The contralateral eyes acted as controls. RESULTS: After an AK wound MMP-1 was present at the leading edge of migrating epithelium after 18 h, while MMP-2 and -9 were localised behind the advancing epithelial edge. The presence of these enzymes rapidly fell to low levels after epithelial closure. There was only faint MMP-3 localisation between days 3 and 7. After an LK wound, MMP-1, -3, and -9 were not detected in the stromal interface, but MMP-2 was present at all time points. CONCLUSIONS: This study suggests that after an AK wound, MMP-1 is a key mediator of epithelial migration, while MMP-2 and -9, and to a lesser extent MMP-3, may participate in the remodelling of corneal stroma and the reformation of epithelial basement membrane. In contrast, an LK wound results in a much lower stimulus for MMP activation. The action of MMP-2 in stromal repair is thus partly independent of epithelial injury.

Systemic cyclosporin A in high risk penetrating keratoplasties: a case-control study.

AIM: To examine the efficacy of systemic cyclosporin A (CSA) in preventing rejection and graft failure in high risk keratoplasty (PK). METHODS: A retrospective case-control study with 49 patients in both the CSA group and the control group. The patients receiving CSA were at high risk of graft rejection and failure. Controls were identified from surgical audit books and had high risk characteristics. RESULTS: There was no statistical difference in preoperative risk factors and the use of postoperative topical steroids between the two groups. The median follow up in the CSA group was 22 months and 27 months in the control group. One or more rejection episodes occurred in 18 out of 49 (36.7%) cases in the CSA group and 26 out of 49 (53.1%) in the control group. Graft failure from all causes occurred in 16 (32.7%) CSA patients and 18 (36.7%) control patients. Four (8.2%) of the CSA group compared to eight (16.3%) in the control group failed because of rejection. 22 (44.9%) out of 49 patients in the CSA group had side effects. In five (10.2%) patients, CSA was stopped because of the side effects; eight patients had elevated serum urea and creatinine and four developed hypertension. Minor side effects reported include gum hyperplasia, increased sweating, backache, nausea, feeling unwell, oral candidiasis, cramps, and paraesthesia of the extremities. CONCLUSION: These results suggest that the benefit of CSA over conventional therapy in preventing rejection episodes and subsequent graft failure is only moderate and did not reach statistically significant levels in this study. Considering the high frequency of side effects and the cost of CSA, a randomised control trial may be necessary to determine the true value of CSA in high risk penetrating keratoplasty.

Outcome of cataract surgery in patients with retinitis pigmentosa.

AIM: To determine the visual benefit of cataract extraction in patients with retinitis pigmentosa and to identify risk factors for poor outcome. METHODS: A retrospective analysis was undertaken of a continuous series of 142 eyes of 89 patients with retinitis pigmentosa undergoing cataract surgery between 1985 and 1997. RESULTS: Mean age at surgery was 47.5 years (range 24-81 years). In 100 eyes there was posterior subcapsular lens opacity alone, 37 eyes also had moderate nuclear sclerosis, and five had only nuclear sclerosis. All patients had central visual fields of <10 degrees. Overall, mean visual acuity improved from 1.05 (SD 0.38) preoperatively to 0.63 (SD 0.49) postoperatively on the logMAR scale. Significant postoperative capsular opacification occurred in 88/139 eyes (63%) and 45.1% required capsulotomy. Anterior capsulotomy was undertaken in 5/52 (9.6%) eyes undergoing phacoemulsification. Postoperative macular oedema was noted in 20 (14%) eyes. Visual acuity improved in 109 eyes (77%), was unchanged in 29 eyes (20.5%), and worsened after surgery in four eyes (2.5%). 86/89 patients reported major improvement of visual function. CONCLUSIONS: Cataract surgery for relatively minor lens opacities is beneficial in patients with retinitis pigmentosa, and most report subjective improvement of visual symptoms. The incidence of capsular opacification is high and anterior capsular contraction may occur. The number of eyes with poor vision due to macular oedema was unexpectedly low.

Early decentration of plate-haptic silicone intraocular lenses.

We report 4 eyes of a consecutive series of 1299 that developed early decentration of a 10.5 mm diameter plate-haptic silicone intraocular lens (IOL) after uneventful phacoemulsification. All eyes had an intact continuous curvilinear capsulorhexis (CCC) with the IOL placed in the capsular bag. After an initial period of good vision, patients noted the onset of glare or monocular diplopia between 1 and 5 weeks after surgery. On examination, there was no significant anterior capsule contraction; however, the edge of the IOL optic was visible in the undilated pupil. There was adhesion between the anterior and posterior capsules at the margin of the CCC that maintained the IOL decentration. Decentration recurred in 1 eye after the IOL was rotated 90 degrees and recentered. Symptoms resolved in 3 eyes after the IOL was removed and replaced with a rigid IOL with a larger diameter optic.

Corneal stem cells in review.

The cornea provides the eye with protection and the refractive properties essential for visual acuity. The transparent epithelium is highly specialized with basal and stratified squamous cells that are renewed throughout life from a stem cell population. The stem cells are thought to reside at the corneal limbus and may be maintained by a variety of intrinsic and extrinsic factors such as the local environment, survival factors, and cytokines. A number of markers have been localized to the limbus in an attempt to identify stem cells; however, definite stem cell identification remains elusive. During homeostasis and following injury to the corneal epithelium, the limbal stem cells divide to produce daughter transient amplifying cells that proliferate, migrate, and differentiate to replace lost cells. However, this cannot occur if the stem cell population is depleted. Limbal stem cell deficiency then results in corneal re-epithelialization by the neighboring conjunctiva, causing pain, poor vision, and even blindness. This review will focus on corneal epithelial stem cells in ocular surface repair and regeneration. The current knowledge of stem cell biology in the corneal epithelium, clinical consequences of stem cell deficiency, and therapeutic strategies aimed at reversing stem cell deficiency will be discussed.

Assessment of the effect of oral clarithromycin on visual outcome following presumed bacterial endophthalmitis.

PURPOSE: The use of clarithromycin was assessed as a biofilm reducing agent in the management of bacterial endophthalmitis. METHODS: 84 eyes of 83 patients presenting with clinical signs highly suggestive of bacterial endophthalmitis were treated using a standard regimen of intraocular vancomycin, amikacin and systemic steroids, which in addition included oral clarithromycin. Ocular penetration of oral clarithromycin in healthy and inflamed eyes was also assessed. RESULTS: Comparing visual acuities at presentation and 6 months, 66% of patients demonstrated an improvement. Intraocular samples were culture positive in 58% of eyes. As compared to culture positive cases, more culture negative cases achieved a visual acuity of 6/12 or better (p = 0.0047). As compared to patients receiving the standard protocol but without clarithromycin, a greater number of culture negative cases demonstrated an improvement in vision of > or = + 6 Snellen lines (p = 0.023). The ocular penetration of clarithromycin into the anterior chamber of inflamed eyes appears sufficient to allow anti-biofilm activity against bacteria at the basic pH encountered in eyes with endophthalmitis. CONCLUSIONS: The ocular penetration of clarithromycin appears adequate for anti-biofilm activity in inflamed eyes. The beneficial effects of oral clarithromycin on visual outcome has been demonstrated in culture negative eyes with clinical signs highly suggestive of bacterial endophthalmitis. The final visual outcome for culture positive cases remains poor.

Effect of age on visual outcome following cataract extraction.

AIM: To determine the effect of age on final corrected visual acuity following cataract extraction. METHODS: A case series of 880 patients aged 60 years and older undergoing cataract extraction between 1996 and 1999 was studied. The best corrected visual acuity was assessed at discharge from the service and the proportion of patients who achieved a postoperative acuity of > or = 6/12 was determined for different age groups. Analysis was also performed after exclusion of patients identified preoperatively as having ocular comorbidity that was thought to limit their final corrected acuity. The odds ratios for visual outcome were calculated for age using multiple logistic regression analysis to adjust for other prognostic factors. RESULTS: A significant age effect was observed, with the proportion of patients who had no ocular comorbidity identified preoperatively and who achieved a visual acuity of > or = 6/12 at discharge decreasing with age (p<0.001). In patients with no comorbidity the odds of achieving an acuity of > or = 6/12 were 4.6 times higher in the 60-69 year age group than in the oldest age group (80+ years). CONCLUSIONS: Age is a significant determinant of visual outcome. This has implications if a points system incorporating an assessment of visual acuity or if visual acuity alone is used to determine the threshold for eligibility for cataract surgery.

In vitro antibiotic resistance in bacterial keratitis in London.

AIM: To document changes in the profile of bacterial isolates from cases of keratitis and changes in their susceptibility to first line antibiotic therapies. METHODS: A retrospective review was performed of all bacterial isolates from cases of keratitis seen between 1984 and 1999. In vitro laboratory susceptibilities to antibiotics were determined by the Kirby-Bauer disc diffusion method. The number of isolates, changes in the proportion of bacterial types, and the number that were fully resistant to monotherapy (ofloxacin), dual therapy (gentamicin and cefuroxime), and prophylactic treatment (chloramphenicol) were calculated. RESULTS: There were 1312 bacterial isolates over 16 years. Gram positive bacteria accounted for 54.7% of isolates and Staphylococcus species (33.4%) were the most frequently isolated organisms. During the study period there has been an increase in the proportion of Pseudomonas species isolates but no overall increase in the proportion of Gram negative isolates. There has not been an increase in the proportion of isolates resistant to ofloxacin since 1995 or an increase in resistance to the combination of gentamicin and cefuroxime. However, since 1984 there has been a significant increase in proportion of Gram negative organisms resistant to chloramphenicol (p=0.0019). CONCLUSIONS: An increase in the in vitro resistance of organisms to first line therapies for bacterial keratitis has not been observed. An increased resistance to chloramphenicol indicates that this drug is unlikely to provide prophylactic cover when Gram negative infection is a risk. Continued monitoring for the emergence of antibiotic resistance is recommended.

Delayed dislocation of foldable plate-haptic silicone lenses after Nd:YAG laser anterior capsulotomy.

PURPOSE: To report two eyes (two patients) that had plate haptic silicone intraocular lenses that dislocated 4 weeks after Nd:YAG laser anterior capsulotomy to relieve capsular contraction. METHODS: Case reports. The clinical records of two patients were reviewed. RESULTS: After uncomplicated phacoemulsification with implantation of plate haptic silicone lenses in the capsular bag, two eyes of two patients developed visually notable early contraction of the capsulorhexis. Three radial-relieving incisions approximately 1 mm long were made with a Nd:YAG laser in each eye to enlarge the capsulotomy. Although vision improved, both patients experienced sudden further reduction of vision after approximately 4 weeks. Upon examination of both patients, we noted an extension of an anterior radial capsulotomy incision peripherally, and the intraocular lenses had dislocated from the capsular bag into the ciliary sulcus. CONCLUSIONS: Tears may extend after radial-relieving incisions have been used to enlarge a contracted anterior capsulotomy, possibly because of continued capsular fibrosis. This may allow extrusion of a foldable intraocular lens from the capsular bag.