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IMPORTANCE Expected outcomes from endokeratoplasty may vary with surgeon experience. BACKGROUND It was explored whether a surgeon learning curve exists for Descemet stripping endothelial keratoplasties (manual or automated) performed in Australia. DESIGN This is a prospective cohort study, with various clinical settings. PARTICIPANTS There were 2139 recipients of 2615 endothelial grafts, registered by 85 surgeons between January 2006 and December 2013. METHODS Kaplan-Meier survival analyses and Cox proportional hazards regression were used to examine longitudinal graft survival. Manual and automated Descemet stripping endothelial keratoplasties were analysed together. Pearson chi-squared analyses were performed to examine differences amongst groups. Continuity correction was used for 2 × 2 tests, and statistical significance was set at P

To compare graft survival and visual outcomes for endothelial keratoplasty (EK) after a first penetrating keratoplasty (PK), with outcomes of repeat PK after a first PK.400 eyes with a second graft (65 EKs, 335 PKs) performed after failure of a primary PK were identified through the Australian Corneal Graft Registry, a national prospectively followed cohort. Grafts were performed after January 2008 (follow-up of the second graft extending to 6.75 years maximum). Kaplan-Meier graft survival plots were constructed and Cox proportional hazards regression was used to identify independent risk factors for graft failure. Best-corrected Snellen visual acuity (BCVA) at last follow-up was compared with pregraft acuity.Poor Kaplan-Meier graft survival was observed for PK-EK compared with PK-PK (log-rank=29.66, p0.001). Variables retained in multivariate analysis as significantly influencing survival of the second graft included graft type (PK-EK or PK-PK, p0.001), length of survival of the previous PK (global p=0.011), graft era (global p=0.018), occurrence of rejection in the second graft (p=0.005) and a history of raised intraocular pressure at any time (p=0.048), but not indication for the first graft. BCVA improved in the majority of surviving grafts and attainment of 6/12 vision was similar for both PK-EK and PK-PK groups.Our registry findings suggest that repeat PK may deliver a better outcome in terms of graft survival than EK after a failed PK that was performed initially for keratoconus or pseudophakic bullous keratopathy. For surviving grafts, visual outcomes appeared equivalent across groups.

The cornea is the clear window at the anterior surface of the eye. Corneal clarity is essential for vision. Corneal transplantation can restore sight when the cornea is diseased, and the cornea is the most commonly transplanted solid tissue. In penetrating corneal transplantation (penetrating keratoplasty), the whole thickness of the cornea is replaced. New surgical techniques of lamellar keratoplasty have been developed in which only the diseased layers of the cornea are replaced, to minimise the risks of graft rejection and optical distortion. Both penetrating and lamellar grafts may fail and need to be replaced. An implication for histopathologists is that the specimen sent for pathological analysis from a patient with a failed lamellar graft will differ from that from a patient with a failed penetrating graft.

Corneal transplantation is a triumph of modern ophthalmology. The possibility of corneal transplantation was first raised in 1797 but a century passed before Zirm achieved the first successful penetrating graft in 1905. Gibson reported the first corneal graft in Australia from Brisbane in 1940 and English established the first eye bank there a few years later. Corneal transplantation evolved steadily over the twentieth century. In the second half of the century, developments in microsurgery, including surgical materials such as monofilament nylon and strong topical steroid drops, accounted for improvements in outcomes. In 2013, approximately 1500 corneal transplants were done in Australia. Eye banking has evolved to cope with the rising demands for donor corneas. Australian corneal surgeons collaborated to establish and support the Australian Corneal Graft Registry in 1985. It follows the outcomes of their surgery and has become an important international resource for surgeons seeking further improvement with the procedure.

Objectives To investigate changing patterns of practice of keratoplasty in Australia, graft survival, visual outcomes, the influence of experience, and the surgeon learning curve for endothelial keratoplasty. Design Observational, prospective cohort study. Participants From a long-standing national corneal transplantation register, 13 920 penetrating keratoplasties, 858 deep anterior lamellar keratoplasties (DALKs), and 2287 endokeratoplasties performed between January 1996 and February 2013 were identified. Methods Kaplan-Meier functions were used to assess graft survival and surgeon experience, the Pearson chi-square test was used to compare visual acuities, and linear regression was used to examine learning curves. Main Outcome Measures Graft survival. Results The total number of corneal grafts performed annually is increasing steadily. More DALKs but fewer penetrating grafts are being performed for keratoconus, and more endokeratoplasties but fewer penetrating grafts are being performed for Fuchs' dystrophy and pseudophakic bullous keratopathy. In 2012, 1482 grafts were performed, compared with 955 in 2002, translating to a requirement for 264 extra corneal donors across the country in 2012. Comparing penetrating grafts and DALKs performed for keratoconus over the same era, both graft survival ( P 0.001) and visual outcomes ( P 0.001) were significantly better for penetrating grafts. Survival of endokeratoplasties performed for Fuchs' dystrophy or pseudophakic bullous keratopathy was poorer than survival of penetrating grafts for the same indications over the same era ( P 0.001). Visual outcomes were significantly better for penetrating grafts than for endokeratoplasties performed for Fuchs' dystrophy ( P 0.001), but endokeratoplasties achieved better visual outcomes than penetrating grafts for pseudophakic bullous keratopathy ( P 0.001). Experienced surgeons (>100 registered keratoplasties) achieved significantly better survival of endokeratoplasties ( P 0.001) than surgeons who had performed fewer grafts ( Conclusions More corneal transplants, especially DALKs and endokeratoplasties, are being performed in Australia than ever before. Survival of DALKs and endokeratoplasties is worse than the survival of penetrating grafts performed for the same indications over the same timeframe. Many endokeratoplasties fail early, but the evidence for a surgeon learning curve is unconvincing.

Expected outcomes from endokeratoplasty may vary with surgeon experience.It was explored whether a surgeon learning curve exists for Descemet stripping endothelial keratoplasties (manual or automated) performed in Australia.This is a prospective cohort study, with various clinical settings.There were 2139 recipients of 2615 endothelial grafts, registered by 85 surgeons between January 2006 and December 2013.Kaplan-Meier survival analyses and Cox proportional hazards regression were used to examine longitudinal graft survival. Manual and automated Descemet stripping endothelial keratoplasties were analysed together. Pearson chi-squared analyses were performed to examine differences amongst groups. Continuity correction was used for 2 × 2 tests, and statistical significance was set at P 0.05 (two-sided).The main parameter measured was endothelial graft survival.Survival of the first 56 registered grafts was significantly poorer than survival of subsequent grafts (χOur findings suggest that for less experienced or low-volume surgeons, longitudinal graft survival improved once 56 or more endokeratoplasties had been performed, indicative of a learning curve. The learning curve was less apparent for surgeons with 57 or more Descemet stripping endothelial keratoplasties and/or Descemet stripping automated endothelial keratoplasties registered during the 8-year study period. Different learning curves may be anticipated for these two groups of surgeons.

[Extract] Microsporidia are intracellular pathogens of animals andhumans. Although not hyphal, a recent study suggests theyare fungi descended from an ancestral zygomycete. Theyhave a unique method for invading susceptible tissue: an internal coiled tube everts rapidly and sporoplasm moves into the cell as if through a hypodermic needle. In the human eye, microsporidial infection presents as keratoconjunctivitis or stromal keratitis. The former has been reported in HIV+ patients because of Encephalitozoon and in immunocompetent patients because of several genera. Stromal infection is seen in immunocompetent patients and is caused mostly by Vittaforma corneae. We report a case in which keratoconjunctivitis preceded deep stromal infection in a patient with a low neutrophil count.

To examine factors affecting penetrating corneal graft survival and visual outcomes in patients aged less than 20 years.Large prospective, cohort study.Records of 14 865 followed penetrating corneal grafts in 11 929 patients were searched to identify 765 grafts in 640 patients aged younger than 20 years of age at the time of graft.Records submitted to the Australian Corneal Graft Registry by 381 ophthalmic surgeons and 253 follow-up practitioners from May 1985 to June 2009 were analyzed using Kaplan-Meier survival plots and Cox proportional hazards regression analysis.Probability of corneal graft survival and Snellen acuity at the time of most recent follow-up and at defined intervals post-graft.Infants (5 years) exhibited poorer graft survival than children aged 5 to 12 years. Adolescents (13-19 years) exhibited better corneal graft survival than other age groups; 86% of grafts in adolescents were for keratoconus. Factors significantly affecting corneal graft survival in pediatric patients included indication for graft, graft inflammation, history of intraocular surgery, vascularization, rejection episodes, post-graft operative procedures, and refractive surgery. Fourteen percent of pediatric grafts failed, of which 65% failed within 2 years post-graft. Forty-four percent of failures were due to unknown causes (18) or irreversible rejection (30).Corneal grafts for keratoconus in adolescents show excellent survival. Infants exhibit poor graft survival and visual outcomes, especially those undergoing transplantation for Peters' anomaly. Corneal graft survival and visual outcomes vary more by indication for graft than recipient age. The major reason for graft failure is irreversible rejection. Corneal transplantation improves overall bilateral vision in pediatric patients.

Bronchopulmonary dysplasia and retinopathy of prematurity affect premature infants exposed to supplemental oxygen. Susceptibility to oxygen-induced retinopathy in the rat is heritable, with inbred Dark Agouti (DA) rats being more susceptible than Fischer 344 (F344) rats. To establish if hyperoxic exposure sufficient to induce florid retinopathy would induce strain-specific lung changes, newborn DA and F344 rats were exposed to cyclic hyperoxia or room air for up to 18 days. Lung function was assessed at 18 days, and standardized morphometry and immunohistochemistry were performed at intervals. No differences in arterial blood gases or protein concentration of bronchoalveolar lavage fluid were observed amongst groups at 18 days, although lung wet-to-dry weights were significantly lower for F344 than for DA rats. Pulmonary vascularity increased in all oxygen-exposed animals compared with room air-exposed controls, but there was no significant difference between strains. The lung surface area of oxygen-exposed F344 rats was significantly increased at day 10 compared with F344 controls and oxygen-treated DA rats, but at 14 and 17 days the oxygen-exposed DA rats showed increased lung surface area compared with oxygen-exposed F344 rats. The minor morphological differences found in the lung did not affect pulmonary function, suggesting that mechanisms inducing oxygen-induced retinopathy and bronchopulmonary dysplasia are fundamentally different, and that susceptibility to bronchopulmonary dysplasia is not heritable in the rat.

Douglas G. Parker, Douglas J. Coster, Helen M. Brereton, Prue H. Hart, Rachel Koldej, Donald S. Anson and Keryn A. Williams

Different inbred strains of rat differ in their susceptibility to oxygen-induced retinopathy (OIR), an animal model of human retinopathy of prematurity. We examined gene expression in Sprague-Dawley (susceptible) and Fischer 344 (resistant) neonatal rats after 3 days exposure to cyclic hyperoxia or room air, using Affymetrix rat Genearrays. False discovery rate analysis was used to identify differentially regulated genes. Such genes were then ranked by fold change and submitted to the online database, DAVID. The Sprague-Dawley list returned the term "response to hypoxia," absent from the Fischer 344 output. Manual analysis indicated that many genes known to be upregulated by hypoxia-inducible factor-1alpha were downregulated by cyclic hyperoxia. Quantitative real-time RT-PCR analysis of Egln3, Bnip3, Slc16a3, and Hk2 confirmed the microarray results. We conclude that combined methodologies are required for adequate dissection of the pathophysiology of strain susceptibility to OIR in the rat. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12177-009-9041-7) contains supplementary material, which is available to authorized users.

Aim: Topical glucocorticosteroids are administered to virtually every corneal transplant recipient, but irreversible immunological rejection remains the leading cause of graft failure. Ex vivo gene therapy of the donor cornea has been shown to modulate graft rejection in experimental models. The efficacy of a glucocorticosteroid-inducible promoter was assessed in controlling transgene expression following lentivirus-mediated gene transfer to ovine and human corneas. Methods: A glucocorticosteroid response element (GRE5) was cloned into a lentiviral vector (LV-GRE-IL10) encoding the model transgene interleukin 10. Transgene expression by LV-GRE-IL10-transduced A549 cells, ovine corneas and human corneas cultured with or without dexamethasone was quantified by an IL10-specific enzyme-linked immunosorbent assay. Results: IL10 levels were 30–40-fold higher in supernatants from LV-GRE-IL10-transduced A549 cells cultured with dexamethasone than in controls. Dexamethasone withdrawal resulted in restoration of baseline IL10 levels. Supernatants from LV-GRE-IL10-transduced ovine and human corneas cultured in dexamethasone contained nine to 10 times more IL10 than supernatants from transduced corneas cultured without dexamethasone. Conclusion: The GRE5 promoter in a lentiviral vector drove rapid, sustained and inducible transgene expression in both ovine and human corneas in the presence of dexamethasone. A steroid-inducible promoter may be useful for controlling transgene expression in gene-modified donor corneal allografts.

Corneal transplantation has not matched the improvements in outcome seen with other clinical transplantation procedures. The therapeutic strategies, which have improved the outcomes of solid vascularised organs are not applicable to corneal transplantation. Corneal transplantation is different with respect to relevant transplantation biology and the clinical context in which it is practiced. New approaches need to be developed which provide regional rather than systemic immunosuppression. The accessibility of the cornea makes it particularly suitable for topical medication and for gene therapy approaches. Engineered antibodies, small enough to pass through the cornea, and directed at key molecules in the allograft response have been developed. Gene therapy had been developed using viral vectors to transfect the corneal endothelium with the genes for immunosuppressive lymphokines. Both approaches show promise.

The progress in antibody engineering over the last 20 years has created the tools for the development of novel antibody-based drugs and constructs, such as small antibody fragments, suitable for topical administration. In rheumatology, oncology, transplantation medicine and ophthalmology, therapeutic antibody constructs, and antibody fragments have been responsible for the clinical progress seen over the last decade. Although antibody-based therapies have become a well-established immunosuppressive option in solid organ transplantation, there are only very few reports with regard to corneal transplantation. The following review explains some of the important aspects of engineered antibody-based therapeutic agents and summarises the current use of such immunosuppressive therapies in transplantation medicine and corneal transplantation.

Single chain antibody fragment genes are commonly created by splicing together the immunoglobulin light chain (VL) and heavy chain variable (VH) genes of a monoclonal antibody produced by a hybridoma. Selective PCR amplification of the functional immunoglobulin variable gene rearrangements can be complicated by the existence of other unproductive immunoglobulin gene rearrangements in the hybridoma. Here we report the detection and preferential amplification of aberrant transcripts from two unproductive VH gene rearrangements derived from the fusion partner of a hybridoma. The functional VH gene of the monoclonal antibody was successfully amplified by selective use of primers to individual JH segments.

Corneal allotransplantation is highly successful in the short term, but much less successful in the longer term. Many corneal grafts in recipients with corneal neovascularization or the sequelae of ocular inflammation undergo irreversible rejection, despite topical immunosuppression with glucocorticosteroids. Sensitization to cornea-derived alloantigen proceeds by both direct and indirect routes, but the anatomic location of sensitization remains unclear. Multiple and redundant mechanisms operate in the effector phase of corneal graft rejection, which is largely cell-mediated rather than antibody-mediated. Human leukocyte antigen matching may improve outcomes in high-risk patients but systemic immunosuppression is frequently ineffective and is seldom used.

Recent evidence suggests that retinopathy of prematurity, a potentially blinding condition of premature human neonates, has a genetically-determined component. Different inbred strains of rat exhibit differential susceptibility to oxygen-induced retinopathy (OIR), a well-established experimental model of retinopathy of prematurity. To explore the basis for this differential susceptibility, we quantified the retinal expression of 8 angiogenesis-related genes during early post-natal retinal development in rats with OIR. Inbred Fischer 344 (F344), Dark Agouti (DA) and Sprague Dawley (SPD) rat neonates were exposed to alternating cycles of 80% oxygen in air and normoxia for up to 14 days. After 14 days of cyclic hyperoxic exposure, some rats were exposed to normoxia for a further 4 days. Retinal mRNA for vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), pigment epithelium-derived factor (PEDF), angiopoietin-2 (Ang2), Tie2, cyclooxygenase-2 (COX2), insulin-like growth factor-1 (IGF1) and erythropoietin (EPO) were quantified by real-time reverse-transcriptase polymerase chain reaction at different time-points. Time-course analysis showed that expression of mRNA for VEGF, VEGFR2 and Ang2 was significantly greater in OIR-resistant (F344) retinae than in OIR-susceptible (DA) retinae during the first 9 days of cyclic hyperoxia. However, at post-natal days 14 and 18, retinal mRNAs for VEGF, EPO, VEGFR2, Ang2, IGF1, COX2 and PEDF were expressed to a significantly greater extent in OIR-susceptible (DA, SPD) than OIR-resistant (F344) retinae. The VEGF/PEDF ratio was greater in the F344 compared with the DA strain up to day 9, but was higher in the DA than the F344 strain at days 14 and 18. Thus, we found that retinal expression of angiogenesis-related genes was significantly higher in OIR-resistant rats than in OIR-susceptible rats during early retinal development, but the pattern reversed during the proliferative phase of OIR. We conclude that susceptibility to OIR correlates with differential gene expression very early in retinal microvascular development, during periods of cyclic hyperoxic exposure rather than during subsequent sustained hypoxia.

Gene therapy of the cornea shows promise for modulating corneal transplant rejection but the most appropriate vector for gene transfer has yet to be determined. We investigated a lentiviral vector (LV) for its ability to transduce corneal endothelium. A lentivector expressing enhanced yellow fluorescent protein (eYFP) under the control of the Simian virus type 40 early promoter (LV-SV40-eYFP) transduced 80-90% of rat, ovine and human corneal endothelial cells as detected by fluorescence microscopy. The kinetics of gene expression varied among species, with ovine corneal endothelium showing a relative delay in detectable reporter gene expression compared with the rat or human corneal endothelium. Vectors containing the myeloproliferative sarcoma virus promoter or the phosphoglycerate kinase promoter were not significantly more effective than LV-SV40-eYFP. The stability of eYFP expression in rat and ovine corneas following ex vivo transduction of the donor cornea was assessed following orthotopic corneal transplantation. Following transduction ex vivo, eYFP expression was maintained in corneal endothelial cells for at least 28 days after corneal transplantation in the sheep and >60 days in the rat. Thus, rat, ovine and human corneal endothelial cells were efficiently transduced by the LV, and gene expression appeared stable over weeks in vivo.

The insertion of an intraocular lens during cataract surgery is routine but delayed opacification of hydrophilic foldable intraocular lenses (IOLs) has been increasingly noticed as a late post-operative complication. Such opacification may present with reduced visual acuity and can be diagnosed with slitlamp biomicroscopy. Lens explantation may be required. We report an 89-year-old female presenting with opacification of a Hydroview IOL and correlate the clinical findings of the lens in situ with the light microscopy of the explanted lens, as well as transmission electron microscopy (TEM), scanning electron microscopy (SEM) and elemental analysis. Pathological analysis of an explanted IOL may assist in a better understanding of the condition and may become relevant in medico-legal proceedings.

To describe the implementation and assess the efficacy of an ophthalmic nurse practitioner (ONP) emergency eye clinic.In a 13-month period, patients were assessed in an ONP emergency eye clinic in a teaching hospital setting. The ONP clinic was run on a defined scope of practice. Risk assessment was carried out in two audit sessions, 4 weeks duration each, at months 1 and 7. Patient outcomes were monitored for reattendance to the department following discharge.A total of 259 patients were assessed, 143 (55.2%) were within the scope of practice. In the two audit periods, concordance with the ophthalmologist was high, diagnosis 100% and management 95.2%. Many patients assessed (111, 42.9% of total) had minor external eye conditions--chiefly corneal foreign body or abrasion (n = 50), conjunctivitis (n = 14) and minor (non-alkali) chemical injuries (n = 11). No reattendance was noted in these patients when monitored for a mean of 12 months (range 7-19 months).This study demonstrates the safety and effectiveness of an ONP emergency eye clinic when practising within a defined scope of practice. An ONP-led emergency eye clinic is a viable addition to acute ophthalmic eye care in Australia.

Immunological rejection is the major cause of human corneal allograft failure. We hypothesized that local production of IL-4 or the p40 subunit of IL-12 (p40 IL-12) by the grafted cornea might prolong allograft survival. Replication-deficient adenoviral vectors encoding ovine IL-4 or p40 IL-12 and GFP were generated and used to infect ovine corneas ex vivo. mRNA for each cytokine was detected in infected corneas, and the presence of secreted protein in corneal supernatants was confirmed by bioassay (for IL-4) or immunoprecipitation (for p40 IL-12). Sheep received uninfected or gene-modified orthotopic corneal allografts. Postoperatively, untreated corneas (n = 13) and corneas expressing GFP (n = 6) were rejected at a median of 21 and 20 days, respectively. Corneas expressing IL-4 (n = 6) underwent rejection at 18.5 days (p > 0.05 compared with controls) and histology demonstrated the presence of eosinophils. In contrast, corneas expressing p40 IL-12 (n = 9) showed prolonged allograft survival (median day to rejection = 45 days, p = 0.003). Local intraocular production of p40 IL-12 thus prolonged corneal graft survival significantly, but local production of the prototypic immunomodulatory cytokine IL-4 induced eosinophilia, inflammation, and rejection. These findings have important implications for the development of novel strategies to improve human corneal graft survival.

Antibody fragments have been shown to penetrate into the anterior chamber when applied to the cornea. The aim of this study was to investigate whether such fragments could penetrate into the vitreous cavity after topical administration to the ocular surface of rabbits.An engineered single-chain variable-domain antibody fragment with specificity for an irrelevant rat determinant was applied as a 50 microl eye drop to the eyes of live rabbits at 20-min intervals over 12 h. Eye drops contained 0.8-1.1 mg/ml protein in a buffered salt solution supplemented with penetration and viscosity enhancers. Samples were collected by paracentesis from the vitreous cavity immediately postmortem. Antibody fragments in these samples were quantified by measuring the binding activity to specific antigen, using flow cytometry.Topically applied antibody fragments were detectable in the vitreous of rabbit eyes after 4-12 h but had cleared at 12 h following the final eye drop. Concentrations of the antibody fragment in the vitreous samples were estimated to be 50-150 ng/ml at 12 h. Penetration of the parental whole antibody into the vitreous was not observed.Antibody fragments penetrate into the vitreous chamber of the rabbit eye after topical administration to the ocular surface. Such fragments may have therapeutic potential for diseases affecting the posterior segment.

Irreversible immunological rejection is the major cause of human corneal allograft failure and occurs despite the use of topical glucocorticoid immunosuppression. Systemic pharmacological interventions have not found widespread favour in corneal transplantation because of associated morbidities and inadequate demonstration of efficacy. Gene therapy offers tantalising prospects for improving corneal allograft survival, especially in those recipients at high risk of graft rejection. Donor corneas can be gene-modified ex vivo, while in storage prior to implantation, and the relative isolation of the transplanted cornea from the circulation decreases the risk of potential systemic complications. A wide variety of vectors have been found suitable for gene transfer to the cornea. The mechanisms involved in corneal graft rejection have been placed on a relatively secure footing over the past decade and in consequence a number of transgenes with promise for modulating rejection have been identified. However, relatively few studies have thus far demonstrated significant prolongation of corneal allograft survival after gene transfer to the donor cornea. In these instances, the therapeutic protein almost certainly acted at a proximal level in the afferent immune response, within the ocular environs.

To compare the performance of keratoconus, penetrating keratoplasty (PK), and control subjects on clinical tests of contrast and glare vision, to determine whether differences in vision were independent of visual acuity (VA), and thereby establish which vision tests are the most useful for outcome studies of PK for keratoconus.All PK subjects had keratoconus before grafting and no subjects had any other eye disease. The keratoconus (n = 11, age 35.0 (SD 11.1) years), forme fruste keratoconus (n = 6, 33.0 (13.0)), PK (n = 21, 41.2 (7.9)), and control (n = 24, 33.7 (8.6)) groups were similar in age. Vision testing, conducted with optimal refractive correction in place, included low contrast visual acuity (LCVA) and Pelli-Robson contrast sensitivity (PRCS) both with and without glare, as well as VA.Normal subjects saw better than PK subjects who in turn saw better than keratoconus subjects on all raw measures. However, when adjusted for VA, the normal group only saw significantly better than the keratoconus group on LCVA (low contrast loss 0.05 (0.04) v 0.15 (0.12), F(2,48) = 6.16; p0.01, post hoc Sheffé p0.05), and the decrements to glare were no worse than for normals. The forme fruste keratoconus group were indistinguishable from normals on all measures.PK subjects have superior vision to keratoconus subjects, but not as good as normal subjects. Including mild keratoconus subjects within a keratoconus group could confound these differences in vision. While VA is an excellent test for comparing normal, keratoconus and PK groups, additional information can be provided by LCVA and PRCS, but not by glare testing. Outcomes research into keratoconus management should include a measure in the contrast domain.

The worldwide shortage of organs and tissue for transplant has led to many ethical discussions involving restrictions concerning organ and tissue donation, including living donations and payment for donation. Efforts are being made to increase the donor pool; however, it is timely to ask whether moral decisions such as the rejection of directed donations are defensible. In this Clinical Conundrum, six specialists delve into the ethical and practical issues surrounding directed donation of human organs and tissues with particular reference to its implications in the field of ophthalmology.

We describe an economical 20 litre bench-top fermenter suitable for production of recombinant antibody fragments in bacterial expression systems. The bacterial culture contained within a polycarbonate carboy is mixed (400–600 rpm) and aerated (1 vessel vol./min) by a high-shear radial flow impeller mounted on a hollow stainless steel shaft, through which pressurised air is pumped. Air is dispersed as fine bubbles into the culture medium by the turbine impeller, without the need for a porous sparger. A stainless steel baffle stabilised by a gliding counterweight increases mixing. The components can easily be disassembled for cleaning and sterilisation. Temperature (range 20–37 °C) and pH (range 7.0–7.5) are controlled manually. Using the apparatus, it proved possible to achieve Escherichia coli cell culture densities equivalent to an optical density at 600 nm (OD600) of 30–32, compared with OD600 4–6 in shake flasks. A yield of 40 mg/litre/day of a recombinant antibody fragment was obtained with the fermenter, which was 15-fold more than the yield of 2.5 mg/litre/day achieved in shake flasks. The fermenter may be particularly suited for research purposes.

Aim To investigate the site of alloantigen presentation in the rat following orthotopic corneal transplantation. Methods Adult inbred Fischer 344 rats received penetrating corneal allografts from inbred Wistar Furth donors (n=17), without lymphadenectomy. A second group (n=8) underwent bilateral removal of superficial cervical and facial lymph nodes 7 days before transplantation. A third group (n=9) underwent bilateral removal of superficial cervical, facial, internal jugular and posterior cervical nodes. Graft survival was assessed by corneal clarity and rejection was confirmed histologically. Results All allografts underwent rejection. The median time to rejection for unmodified allografts was day 15, compared with day 14.5 for minimally lymphadenectomised recipients and day 18 for more extensively lymphadenectomised recipients (p>0.05, all comparisons). The median day to rejection for the combined group of lymphadenectomised rats was day 17 (p>0.05 compared with unmodified grafts). The rejection process was similar in all recipients. Conclusions Removal of multiple lymph nodes in the neck and thorax did not significantly influence the incidence, tempo or nature of the corneal allograft response. Sensitisation and clonal expansion of corneal alloantigen-reactive cells cannot occur only in superficial cervical, facial, internal jugular and posterior cervical lymph nodes in the rat.

Background Keratoconus is an ectatic (weakening) disease of the cornea, which is the clear surface at the front of the eye. Approximately 10% to 15% of patients diagnosed with keratoconus require corneal transplantation. This may be full-thickness (penetrating) or partial-thickness (lamellar). Objectives To compare visual outcomes after deep anterior lamellar keratoplasty (DALK) and penetrating keratoplasty for keratoconus, and to compare additional outcomes relating to factors which may contribute to poor visual outcomes (e.g. astigmatism, graft rejection and failure). Search methods We searched a number of electronic databases including CENTRAL, PubMed and EMBASE without using any date or language restrictions. We last searched the electronic databases on 31 October 2013. We also handsearched the proceedings of several international ophthalmic conferences. Selection criteria We included all randomised controlled trials (RCTs) comparing the outcomes of DALK and penetrating keratoplasty in the treatment of keratoconus. Data collection and analysis Two authors assessed trial quality and extracted data independently. For dichotomous data (graft failure, rejection, achievement of functional vision) results were expressed as odds ratios (ORs) and 95% confidence intervals (CIs). For continuous data (postoperative best corrected visual acuity (BCVA), uncorrected visual acuity (UCVA), keratometric astigmatism and spherical equivalent) results were expressed as mean differences (MDs) and 95% CIs. Main results We identified two completed studies, with a total of 111 participants (n = 30 and n = 81), both conducted in Iran, that met our inclusion criteria. Participants had moderate to severe keratoconus pre-operatively and were randomly allocated to receive either DALK or penetrating keratoplasty. Only one eye of each participant was treated as part of the trials. The smaller study had 12 month follow-up data for all participants. For the larger study, four DALK surgeries had to be abandoned due to technical failure and visual and refractive outcomes were not measured in these participants. Follow-up length for the remaining 77 participants ranged from 6.8 to 36.4 months, with all 77 followed for at least three months post-suture removal. Details of the randomisation procedure were unavailable for the smaller study and so sensitivity analyses were conducted to determine if the results from this study had affected the overall results of the review. Neither of the included studies reported a difference between groups on any of the measures of post-graft visual achievement, keratometric astigmatism or spherical equivalent. A single case of graft failure in a penetrating keratoplasty was reported. No postoperative graft failures were reported in the DALK group of either study. Instances of graft rejection were reported in both groups, in both studies. The majority of these cases were successfully treated with steroids. The data, which related to all cases in each study - given that the four cases that did not go ahead as planned had already technically failed without presence of rejection - showed that rejection was less likely to occur in DALK (odds ratio (OR): 0.33, 95% confidence interval (CI) 0.14 to 0.81, GRADE rating: moderate). Results of the sensitivity analysis indicated that inclusion of the Razmju 2011 study did not bias the results with regards to rejection episodes. While sensitivity analysis showed altered results with regards to failure rates, the data available from the Javadi 2010 study alone had a very wide 95% CI, suggesting an imprecise estimate. Therefore, even after removal of the Razmju 2011 data, it is still difficult to draw conclusions regarding superiority of one technique over another with regards to graft failure. DALK was unable to be completed as planned in four cases and in a further three cases, complications during dissection required further intervention. Other adverse events, of varying severity, were reported in both intervention groups with similar frequency. For both types of surgery, these included postoperative astigmatism, steroid induced ocular hypertension and persistent epithelial defects. In recipients of DALK, one participant had interface neovascularisation (a proliferation of blood vessels where the host and donor cornea come together) and one had wrinkling of Descemet's membrane, the basement membrane separating the corneal stroma from the corneal endothelium. In the penetrating keratoplasty groups, one participant required graft resuturing and one had an atonic pupil, a condition in which the pupil dilates and is non-reactive. Overall, the quality of the evidence was rated as very low to moderate, with methodological limitations, incomplete data analysis and imprecision of findings, as well as high risk of bias in several areas for both studies. Authors' conclusions We found no evidence to support a difference in outcomes with regards to BCVA at three months post-graft or at any of the other time points analysed (GRADE rating: very low). We also found no evidence of a difference in outcomes with regards to graft survival, final UCVA or keratometric outcomes. We found some evidence that rejection is more likely to occur following penetrating keratoplasty than DALK (GRADE rating: moderate). The small number of studies included in the review and methodological issues relating to the two, mean that the overall quality of the evidence in this review is low. There is currently insufficient evidence to determine which technique may offer better overall outcomes - final visual acuity and time to attain this, keratometric stabilisation, risk of rejection or failure, or both, and risk of other adverse events - for patients with keratoconus. Large randomised trials comparing the outcomes of penetrating keratoplasty and DALK in the treatment of keratoconus are needed.

Corneal transplantation is a triumph of modern ophthalmology. The possibility of corneal transplantation was first raised in 1797 but a century passed before Zirm achieved the first successful penetrating graft in 1905. Gibson reported the first corneal graft in Australia from Brisbane in 1940 and English established the first eye bank there a few years later. Corneal transplantation evolved steadily over the twentieth century. In the second half of the century, developments in microsurgery, including surgical materials such as monofilament nylon and strong topical steroid drops, accounted for improvements in outcomes. In 2013, approximately 1500 corneal transplants were done in Australia. Eye banking has evolved to cope with the rising demands for donor corneas. Australian corneal surgeons collaborated to establish and support the Australian Corneal Graft Registry in 1985. It follows the outcomes of their surgery and has become an important international resource for surgeons seeking further improvement with the procedure.

Lamellar (component-cell) corneal transplantation is replacing penetrating keratoplasty for some corneal disorders in humans; but the relative risks of immunological graft rejection for the two procedures are uncertain. A model of component endothelial cell keratoplasty (endokeratoplasty) was developed in the outbred sheep. Clinical and histological graft outcomes after endokeratoplasty were then compared with contemporaneous penetrating corneal allografts. No topical or systemic immunosuppression was administered to any recipient sheep. Endothelial cell allografts (n = 10) took significantly longer to achieve perfect transparency following surgery than did penetrating corneal grafts (n = 7) (day 10 versus day 4; p = 0.003; two-tailed Mann- Whitney U-test). The median day to rejection of penetrating grafts was post-operative day 18; and of endothelial cell grafts was day 48 (p = 0.04; two-tailed Mann-Whitney U-test). The clinical courses of the two procedures were quite different. Penetrating grafts gained clarity quickly but exhibited rapid graft neovascularization. Clinical rejection was preceded by inflammation in the anterior segment. Endothelial cell grafts exhibited a fluctuating; more indolent course of opacification; although all did eventually fail. Histological analysis confirmed immunological rejection in all failed grafts; but with different patterns of leukocytic infiltration in endokeratoplasties compared with penetrating keratoplasties. Inflammatory cells in endothelial cell grafts were generally fewer in number and were more often found in the posterior stroma. We conclude that in the absence of immunosuppression; all endothelial cell allografts do undergo immunological rejection; albeit at a slower tempo than penetrating grafts., This work was funded by the Ophthalmic Research Institute of Australia. KAW is supported by the Australian National Health & Medical Research Council (NHMRC).

Purpose: Modification of a donor cornea by gene therapy has potential to modulate irreversible rejection, the major cause of corneal graft failure. The sheep is a useful model for the human in this respect, as ovine endothelial cells are amitotic. The aim of the study was to investigate the ability of various non-viral and viral agents to transfer a reporter gene to ovine corneal endothelium. Methods: The non-viral agents Transfectin-10, Transfectin-20, Transfectin-50, SuperFect, Effectene and CLONfectin were used to deliver the reporter gene, Escherichia coli lacZ, to ovine corneal endothelium in vitro. A Herpes simplex virus-1 and an adenoviral vector each encoding E. coli lacZ were similarly tested. Infected corneas were organ-cultured for up to 7 days in vitro to allow transfection efficiency, duration of gene expression and toxicity attributable to each vector to be compared. Results: Scattered single or clusters of endothelial cells expressing the reporter gene were observed after transfection with CLONfectin, Transfectin-10, Transfectin-20 and Transfectin-50. SuperFect and Effectene were virtually in-effective. At best, the absolute number of infected cells per endothelial monolayer after 3 or 7 days of organ culture was estimated as < 0.01%. The Herpes simplex virus-1 vector also failed to transduce ovine corneal endothelium efficiently. In contrast, transfection rates of up to 70% of endothelial cells were observed with the adenoviral vector. Conclusion: Non-viral vectors and Herpes simplex virus-1 are unlikely to be suitable for gene therapy of corneal endothelium, because the efficiency of transfection is low compared with the rates achieved with adenoviral vectors.

BACKGROUND/AIMS Limbal allotransplantation is increasingly being used for ocular surface repair in patients with limbal stem cell dysfunction. However, it is uncertain whether donor cells survive long term on the ocular surface and whether patients maintain the early benefits of the procedure. The aims of this study were to investigate the long term outcome of clinical limbal allografts and to correlate outcome with donor cell survival. METHODS Five patients who had undergone allotransplantation—four keratolimbal allografts and one tarsoconjunctival allograft—from 3–5 years previously, and for whom residual frozen donor ocular tissue was available, were reviewed. Survival of donor cells lifted from the recipient ocular surface by impression cytology was investigated by DNA fingerprinting using primers detecting variable nucleotide tandem repeat sequences. Recipient buccal cells and scleral samples from the remnant donor eye were used to genotype recipients and donors, respectively. Polymerase chain reaction products were sized by Genescan analysis. RESULTS An objective long term benefit from the procedure (improved Snellen acuity, reduced frequency of epithelial defects, reduced vascularisation, and scarring) was recorded for four patients. Some subjective benefit was also reported. However, in no instances were donor cells recovered from the ocular surface at 3–5 years post-graft. Initial experiments to examine sensitivity indicated that any surviving donor cells must have constituted less than 2.5% of cells sampled. CONCLUSION Limbal stem cell allotransplantation can provide long term benefits, as measured by objective criteria. However, such benefits do not necessarily correlate with survival of measurable numbers of donor cells on the ocular surface.

Background Modification of a donor cornea by gene therapy ex vivo has potential to modulate irreversible rejection, the major cause of corneal graft failure. Our aim was to transfer the gene encoding mammalian IL-10 to ovine donor corneas and to determine subsequent orthotopic corneal allograft survival in an outbred sheep model. Methods The replicative capacity of ovine corneal endothelium was determined by autoradiography after deliberate injury. A replication-defective adenovirus was used to deliver the lacZ reporter gene to ovine corneas and transfected corneas were organ-cultured in vitro to allow transfection efficiency, duration of reporter gene expression, and toxicity attributable to the vector to be determined. A cDNA encoding full-length ovine IL-10 was cloned into an adenoviral vector that was used to transfect donor corneas ex vivo before transplantation. Orthotopic penetrating corneal transplantation was performed in outbred sheep. Results Sheep corneal endothelium was found to be essentially amitotic. Transfection of > 70% corneal endothelial cells was achieved with the viral vector and expression was maintained for 28 days in vitro. IL-10 mRNA was detectable in transfected, organ-cultured corneas for 21 days in vitro. Donor corneas transfected with cDNA encoding IL-10 showed significantly prolonged survival after penetrating keratoplasty (median 55 days, range 19 > or =300 days) compared with control corneas (median 20.5 days, range 18-32 days, P=0.011). Conclusion Local gene therapy-mediated expression of the immunomodulatory cytokine IL-10 has the potential to reduce the incidence of corneal graft rejection and to prolong corneal allograft survival.

Aims—Corneal perfusion chambers are important tools in the development and assessment of ophthalmic drugs. The aim of this study was to design and test a modified perfusion chamber suitable for topical application of drugs to isolated corneoscleral preparations, and which allowed continuous monitoring of endothelial cell function. Methods—A polycarbonate and stainless steel perfusion chamber was designed to clamp corneas in a horizontal plane suitable for topical drug delivery. Endothelial cell function was assessed by ultrasonic pachymetry and specular microscopy during perfusion. Epithelial barrier function was assessed by penetration of fluorescein. Leakage was examined by measuring penetration of a large protein, IgG. Tissue architecture after perfusion was examined by conventional histology. Results—Corneas maintained a functionally and morphologically intact endothelial monolayer during perfusion periods of up to 14 hours. The epithelial barrier function was well preserved. The tissue clamp sealed the preparation eVectively against leakage of macromolecules. Conclusion—The new chamber device forms a reliable tool for in vitro drug penetration and toxicity studies in isolated perfused corneoscleral tissue. (Br J Ophthalmol 2001;85:450‐453)

Purpose: To determine current practices in the prevention and management of corneal allograft rejection in Australia. Methods: A questionnaire was circulated to attendees at the 1998 Eye Bank Meeting in Adelaide. Twenty-four responses were received and analysed. Results: All respondents used topical corticosteroids for routine prophylaxis and to treat established rejection episodes. Prednisolone acetate was the most frequently prescribed topical corticosteroid. Systemic non-steroidal immunosuppression was prescribed almost exclusively for high-risk grafts. Seventy-five per cent of surgeons used systemic antiviral agents for the treatment of graft rejection in patients with Herpes simplex keratitis. Conclusion: There was a wide variation amongst surgeons in the choice of therapy for routine prophylactic immunosuppression as well as for the treatment of established corneal allograft rejection.

Objective To determine the long-term effect on vision of penetrating keratoplasty performed for keratoconus. Design Retrospective noncomparative case series. Participants All patients with keratoconus who received a corneal graft and who remained in our center for follow-up and visual rehabilitation during the study period. Intervention Penetrating keratoplasty was performed in 93 eyes of 78 patients. Main outcome measures Graft survival, visual acuity, and astigmatism. Results One (1.08%) graft failure was encountered over a mean follow-up of 46 months. Mean preoperative (best corrected) and postoperative visual acuity is (best-tolerated correction) were 0.9 (20/160) and 0.24 (20/80) logMAR, respectively. Visual acuity in 86% of eyes was 0.3 logMAR (20/40) or better at the latest follow-up, with 67% of eyes being corrected with spectacles. Mean preoperative corneal power by keratometry was more than 52 diopters (D) in 83% of eyes; mean postoperative corneal power was 45 ± 2 D. No significant predictors of postgraft astigmatism were found. Mean preoperative and postoperative best-eye acuities of the better eye were 0.32 (20/40−1) and 0.18 (20/32+1) logMAR, respectively ( P Conclusions Graft survival was excellent. A corrected visual acuity of 20/40 or better was obtained in 86% of eyes. Astigmatism could not be predicted from preoperative factors. Visual acuity measured in the better eye improved by 0.14 logMAR (1.4 lines), implying an overall functional gain for the patient.

Purpose: To determine the in vitro drug susceptibility of Australian corneal isolates of Acanthamoeba and to correlate the results with patient treatment and visual outcome. Methods: Acanthamoeba isolates were obtained from a reference laboratory. Cyst suspensions were prepared from 19 strains and exposed to 10 antimicrobial agents for 7 days. The minimum drug concentrations required to inhibit excystation were determined. Inhibited cells were then plated out to determine minimum cysticidal concentrations. Results: Overall, propamidine proved to be the most active anti-Acanthamoeba agent tested. The disinfectant polyhexamethylene biguanide, either pure or in Baquacil, was also effective. Pentamidine, hexamidine, chlorhexidine and chloroxylenol had intermediate activity, while neomycin, amphotericin B and povidone-iodine had poor activity. There was no clear relationship between in vitro susceptibility and visual outcome. Conclusions: Propamidine and polyhexamethylene biguanide drops are recommended as initial choices for the treatment of Acanthamoeba keratitis. The variability in the susceptibility to any one agent suggests that individual testing of isolates is necessary to identify the most appropriate treatment. A number of factors influence visual outcome in these cases; further studies are required to resolve the importance or otherwise of in vitro susceptibility.

AIMS: To examine the hypothesis that apoptosis of infiltrating cells contributes to spontaneous resolution of uveitis in clinically relevant rodent models. METHODS: Experimental melanin induced uveitis (EMIU) was induced in Fischer 344 rats by immunisation with 250 microg bovine ocular melanin. Endotoxin induced uveitis (EIU) was induced by injection of 200 microg Escherichia coli lipopolysaccharide. Formalin fixed, paraffin embedded ocular cross sections were stained by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labelling (TUNEL) to identify apoptotic cells. Indirect immunoperoxidase staining of paraformaldehyde lysine periodate fixed tissue cross sections was used to demonstrate expression of inducible nitric oxide synthase (iNOS). RESULTS: TUNEL positive mononuclear cells were observed in the anterior uvea during both EMIU and EIU at all selected time points. However, whereas the majority of mononuclear cells appeared apoptotic from the outset of disease, neutrophils were notably TUNEL negative at all time points examined. Many infiltrating neutrophils expressed iNOS. CONCLUSION: Apoptosis occurs early in the course of rat EMIU and EIU, and may contribute to resolution of these diseases. In general, infiltrating mononuclear cells die rapidly, while neutrophils survive, producing inducible nitric oxide synthase which may contribute to disease pathogenesis.

Purpose: A case of linear IgA disease is reported to alert ophthalmologists and physicians to this unusual cause of chronic cicatrizing conjunctivitis. Methods: Clinical records of a patient suffering from linear IgA disease were reviewed. Results: A 65-year-old woman with a complicated medical history experienced rapidly progressive chronic cicatrizing conjunctivitis leading to corneal perforation. Undiagnosed gingivitis and palatal ulceration had been present for 5 years prior to the onset of ocular symptoms and vitamin C deficiency had followed the consequent dietary restrictions. A diagnosis of linear IgA disease was made on conjunctival biopsy, which demonstrated linear deposits of IgA along the epithelial basement membrane. The perforation was managed successfully with a conjunctival pediculate flap. Control of the inflammation was achieved with systemic prednisolone and cyclophosphamide but at the expense of serious systemic side-effects. Conclusions: Linear IgA disease causes progressive conjunctival cicatrization in many affected individuals. Although dapsone generally controls the inflammation, heavier systemic immunosuppression was required in this case. Involvement of skin or other mucosal surfaces may become symptomatic before the conjunctivitis, and physicians must be educated to refer patients for ophthalmological review on diagnosis. Conversely, ophthalmologists encountering ocular linear IgA disease should be aware of the possibility of other mucosal involvement requiring physician intervention.

SUMMARY Experimental melanin-induced uveitis (EMIU) is a rodent model of acute anterior uveitis which was described in 1993. We investigated strain susceptibility, and age and gender characteristics of the model, undertook histological and immunohistochemical studies to investigate underlying cellular mechanisms, and examined several treatment options. Rats were immunized with bovine ocular melanin (250 μg), and disease was followed by slit lamp examination. Lewis, Fischer 344 and Porton rats were found to be susceptible to EMIU, whereas Wistar-Furth, DA, and Hooded Wistar strains were resistant. EMIU was neither age- nor gender-dependent. In Fischer 344 rats, EMIU was characterized clinically by florid anterior segment inflammation. Histopathological findings included infiltration of ciliary body and iris with mononuclear cells and neutrophils. Both CD4+ and CD8+ T lymphocytes were prominent. Rats were then treated with intraperitoneal injections of anti-CD4, anti-CD8 or irrelevant isotype-matched MoAb on days −3, 0, 3, 6 and 9 with respect to melanin immunization. Incidence of uveitis was significantly reduced in rats treated with a non-depleting cocktail of anti-CD4 MoAbs (P = 0.007), whereas a depleting anti-CD8 antibody had no effect on the disease. Mannose-6-phosphate inhibits lymphocyte migration in some models of T cell-mediated inflammation. This simple sugar was administered to additional rats via intraperitoneal osmotic pumps for 14 days following disease induction, but did not influence the uveitis. We conclude that EMIU is controlled by CD4+ T cells, and disease may be abrogated by treatment with anti-CD4 MoAbs.

Uveal mast cells have previously been considered to be vital mediators of experimental uveitis. We extended the study of these cells to experimental melanin-induced uveitis (EMIU), a recently described clinically relevant model, and re-examined their role in endotoxin-induced uveitis (EIU) using genetically mast cell-depleted mice on a single background. EMIU was induced in Fischer 344 rats by immunisation with bovine ocular melanin (250 µg). Animals were killed immediately, and on days 1 and 3 of clinical disease. Numbers of uveal mast cells and the percentage of degranulated cells were counted in whole-mount preparations. There was no significant change in either measure across the selected time points. To induce EIU, normal and mast cell-depleted DBA/2 mice were injected with Escherichia coli lipopolysaccharide (400 µg). Cells infiltrating the eye 24 h after injection were quantified in 5 µm ocular cross-sections. Disease was not significantly reduced in the mast cell-depleted mutants. We conclude that uveal mast cells are not required for the development of EMIU or, in contrast to earlier work, EIU.

Failure of a specialized population of corneal epithelial stem cells found in the peripheral cornea and limbus results in ocular surface disease, which may be amenable to treatment by transplantation of limbal tissue. This study was designed to investigate donor limbal stem cell allograft survival in rabbits with ocular surface disease. Rabbits underwent corneal epithelial debridement and limbal ablation to induce ocular surface disease and were then treated by limbal stem cell allotransplantation, by allotransplantation plus topical steroid, or by topical steroid only (n = 7 for each group). Donors and recipients were sex mismatched. Recipients were followed for up to 5 months. Outcome was assessed by daily slit-lamp examination, weekly impression cytology and photographic record, end-point sex chromatin and fluorescent cell tracer analyses, histology, and immunohistochemistry. In no case was a completely normal ocular surface regained, but some animals that received grafts plus corticosteroids fared best by all criteria used. In the absence of immunosuppression, graft hemorrhagia (believed to be a manifestation of graft rejection) occurred within the first month, the cornea became resurfaced with conjunctiva-derived cells, and no donor cells survived centrally in the long term. Topical corticosteroids reduced the number and severity of these episodes significantly, and were associated with survival of some donor-derived cells in the central cornea of some grafted animals. Thus, rabbit limbal stem cell allografts appeared to undergo rejection, which could be modified by immunosuppression, but useful regeneration of the ocular surface occurred only where rejection was circumvented.

BACKGROUND Penetrating keratoplasty is one of the most common and successful ophthalmic procedures. However, controversy and uncertainty still call to question the significance of certain risk factors for graft failure. METHODS A consecutive series of 1819 penetrating keratoplasties at a single center was studied to determine donor and recipient risk factors for graft failure. Mean follow up was 2.3 years (range, 1 to 96 months) with 139 (7.7%) eyes lost to follow up. RESULTS Previous graft failure was the most significant risk factor for secondary failure (P = .0013). The risk of failure significantly decreased with increased postoperative time. Significant patient risk factors for secondary failures in initial grafts included race (P = .01), age (P = .004), iris color (P = .02), use of preoperative glaucoma medications (P = .0008), deep stromal vascularization (P = .002), and host horizontal diameter (P = 0.007). Significant risk factors for failures associated with immunologic allograft reactions in initial grafts included horizontal corneal diameter (P = .002), donor size (P = .05), differences between horizontal corneal diameter, and both donor size (P = .02) and recipient trephination size (P = .01). However, deep stromal vascularization was only marginally significant (P = .09). A history of preoperative glaucoma medication usage was not a significant risk factor. CONCLUSIONS The relationship of the recipient's horizontal corneal diameter to immunologic graft rejection is a new risk factor that surgeons can directly control and thereby help avoid graft failure.

AIMS--The aim of this work was to investigate different surgical options for the repair of the ocular surface, using various extensions of the procedure of limbal stem cell allotransplantation. METHODS/RESULTS--Straightforward lamellar limbal transplantation was performed in one patient with contact lens induced limbal stem cell failure. A second patient with a neoplastic corneal lesion underwent limbal allotransplantation, followed later by a second procedure in which 360 degrees of limbus and the entire ocular surface was transplanted. A third patient who had suffered extensive chemical burns was treated by penetrating keratoplasty to restore central corneal clarity, followed later by a lamellar allograft comprising a 360 degrees annulus of peripheral cornea to repair the ocular surface. A fourth patient with long standing, chronic trachomatous eye disease underwent allotransplantation of the upper lid tarsal plate and conjunctiva, with reconstruction of the fornix. Finally, a child with Goldenhar's syndrome underwent reconstruction of the medial fornix with autologous buccal mucosa, followed by a lamellar corneal and conjunctival allograft. A stable ocular surface has been achieved in each case and there have been no obvious rejection episodes. CONCLUSION--Limbal allotransplantation can be extended to engraftment of the entire superficial cornea, limbus, conjunctiva, and tarsal plate in patients with a range of pathologies. We have described the surgical management of five cases which demonstrate the potential of the technique, but which raise questions which still need to be explored.

To investigate whether expression of an anti-CD4 antibody fragment (scFv) by a lentivector-transduced donor cornea can prolong rat corneal allograft survival. Methods Inbred Fischer 344 rats received penetrating corneal allografts from Wistar-Furth donors after a 3 h transduction of the donor cornea with a lentivector carrying anti-CD4scFv cDNA (Lv-CD4scFv), a lentivector carrying the reporter gene-enhanced yellow fluorescence protein (LV-eYFP), or an adenoviral vector carrying anti-CD4 scFv cDNA (Ad-CD4scFv). Unmodified controls were also performed. Graft survival was assessed by corneal clarity, and rejection was confirmed histologically. Results In organ-cultured corneas, expression of anti-CD4 scFv was detected at 2 days post-transduction with the adenoviral vector, compared with 5 days post-transduction with the lentivector, and was 10-fold higher than the former. More inflammation was observed in Ad-CD4scFv-modified allografts than in Lv-CD4scFv-modified grafts at 15 days postsurgery (p=0.01). The median time to rejection for unmodified, LV-eYFP and Ad-CD4scFv grafts was day 17, compared with day 22 for Lv-CD4scFv grafts (p≤0.018). Conclusion Donor corneas transduced with a lentiviral vector carrying anti-CD4scFv cDNA showed a modest but significant prolongation in graft survival compared with unmodified, Lv-eYFP and Ad-CD4scFv grafts. However, rejection still occurred in all Lv-CD4scFv grafts, indicating that sensitisation may have been delayed but was not prevented., Australian National Health & Medical Research Council and the Ophthalmic Research Institute of Australia.