The influence of rejection episodes in recipients of bilateral corneal grafts.

We investigated whether a rejection episode in one graft was associated with rejection in the other graft, in recipients with bilateral corneal transplants. In a prospectively maintained, national register of 14,865 followed corneal grafts, 1476 patients with bilateral penetrating corneal grafts were identified. Occurrence of rejection was a risk factor for graft failure (p < 0.0001). Logistic regression was used to calculate the adjusted odds ratio for rejection in one eye following rejection in the other eye. In the subset of 1118 patients with bilateral grafts but no history of previous grafts or rejections in either eye, the adjusted odds ratio for a rejection episode in the first eye following rejection in the second was 3.27 (95% confidence interval, CI 1.85, 5.79; p < 0.001). The adjusted odds ratio was 2.04 (95% CI 1.07, 3.91; p = 0.03) for rejection in the second eye following rejection in the first. The median time between the first rejection episode in one eye and the first rejection episode in the other eye was 15 months. Patients with bilateral corneal grafts who suffer a graft rejection episode in one eye are at significantly greater odds of suffering a rejection episode in the other corneal transplant.

A steroid-inducible promoter for the cornea.

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.

Mechanisms of corneal allograft rejection and regional immunosuppression.

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.

Antibody-based immunosuppressive agents for corneal transplantation.

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.

Prospects for genetic modulation of corneal graft survival.

Irreversible immunological rejection is the major cause of clinical corneal graft failure. Ex vivo gene therapy directed at the donor cornea has been shown to prolong orthotopic corneal allograft survival significantly in experimental animal models including the mouse, rat, rabbit, and sheep. Transgenes effective in prolonging corneal graft survival include immunomodulatory cytokines and cytokine receptors, an inhibitor of neovascularisation, a blocker of antigen-presenting cell-T cell co-stimulation, nerve growth factor, a dominant negative regulator of apoptosis, and the enzyme indoleamine 2,3-dioxygenase. Although many viral and non-viral vectors have been shown to transduce the corneal endothelium efficiently, allograft survival has so far been prolonged only following transduction of the donor cornea with adenoviral and lentiviral vectors. The degree of graft prolongation, although promising, is still insufficient for immediate translation to the clinic. Increasing the time that the therapeutic gene is expressed in the eye with an integrative, non-immunogenic viral vector is likely to be one way to achieve long-term graft survival. Simultaneous targeting of multiple pathways of graft rejection with more than one transgene is likely to be another. We suggest that the use of an adeno-associated viral or lentiviral vector combined with multiple transgenes may provide the key to future clinical trials.

Lentivirus-mediated gene transfer to the rat, ovine and human cornea.

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.

Ophthalmic nurse practitioner led diabetic retinopathy screening. Results of a 3-month trial.

PURPOSE: To describe the design and implementation of a nurse led diabetic retinopathy screening clinic. To present the results of a 3-month trial period assessing the concordance of retinopathy grading between a nurse practitioner and an ophthalmologist. METHOD: Patients attending for annual diabetic eye review during an initial 3-month trial period were assessed in a dedicated diabetic eye clinic by an ophthalmic nurse practitioner and an ophthalmologist, with both grading the degree of diabetic retinopathy using to the Wisconsin grading system. Each was masked as to the other's findings. The concordance of retinopathy grading between ophthalmic nurse practitioner and ophthalmologist was assessed. RESULTS: A total of 95 patients (189 eyes) were assessed during the study period. A 92% concordance was achieved between the ophthalmologist and the ophthalmic nurse practitioner. In total, 72 eyes were graded as having some degree of retinopathy by the ophthalmologist. The sensitivity of the nurse practitioner for diagnosing the presence of diabetic retinopathy was 93%, and the specificity 91%. Nine eyes with severe nonproliferative diabetic retinopathy or worse, and four with clinically significant macular oedema were seen. All were correctly identified by the nurse practitioner. CONCLUSIONS: The structure and management protocols of the clinic are described. An excellent concordance between ophthalmologist and nurse practitioner was achieved in this group of patients with relatively less advanced retinopathy.

Influence of format on in vitro penetration of antibody fragments through porcine cornea.

AIM: Antibody fragments, appropriately formulated, can penetrate through the ocular surface and thus have potential as therapeutic agents. The aim was to investigate the influence of protein fragment format on the kinetics and extent of ocular penetration in vitro. METHODS: Immunoglobulin single chain variable domain fragments of a murine monoclonal antibody with specificity for rat CD4 were engineered with a 20 or 11 amino acid linker by assembly polymerase chain reaction, expressed in Escherichia coli and purified by chromatography. Fab fragments of the parental antibody were prepared by papain digestion. Antibody fragments were formulated with a penetration and a viscosity enhancer and were applied to the surface of perfused pig corneas for up to 10 hours in vitro. Penetration was quantified by flow cytometry on rat thymocytes. RESULTS: 20-mer antibody fragments formed natural monomers and dimers following purification that could be separately isolated, while 11-mer fragments were dimeric. All formats of fragment (20-mer monomers and dimers, 11-mer dimers, Fab) showed penetration through the pig cornea after 6 hours of intermittent topical administration. CONCLUSION: Antibody fragments of different shapes and sizes can penetrate the cornea after topical administration, thereby increasing the potential of this class of proteins for topical ophthalmic use.

In vitro adenovirus mediated gene transfer to the human cornea.

BACKGROUND/AIMS: Replication deficient adenovirus is an efficient vector for gene transfer to the cornea. The aim was to optimise the transduction of human corneal endothelium with adenoviral vectors and to measure transgene production from transduced corneas. METHODS: Adenoviral vectors (AdV) encoding enhanced green fluorescent protein (eGFP) or a transgenic protein (scFv) were used to transfect 34 human corneas. Reporter gene expression was assessed after 72-96 hours of organ culture. The kinetics of scFv production was monitored in vitro for 1 month by flow cytometric analysis of corneal supernatants. RESULTS: Transduction of human corneas with high doses (5 x 10(7)-3 x 10(8) pfu) of AdV caused eGFP expression in 12-100% of corneal endothelial cells. Corneas were efficiently transduced following up to 28 days in cold storage. Very high AdV doses (2 x 10(9) pfu) reduced endothelial cell densities to 98 (SD 129) nuclei/mm(2) (compared to 2114 (716) nuclei/mm(2) for all other groups). Transgenic protein production peaked at 2.4 (0.9) microg/cornea/day at 2 weeks post-transduction, and decreased to 1.2 (0.4) microg/cornea/day by 33 days, at which time endothelial cell density had decreased to 431 (685) nuclei/mm(2). CONCLUSION: Human corneas can be efficiently transduced by AdV following extended periods of cold storage, and transgene expression is maintained for at least 1 month in vitro.

Corneal graft rejection occurs despite Fas ligand expression and apoptosis of infiltrating cells.

BACKGROUND/AIMS: Constitutive expression of Fas ligand (CD95L) protects the eye against cell mediated immune responses by inducing apoptosis in infiltrating Fas bearing T cells. This study was designed to examine Fas ligand expression on acutely rejecting rat corneal grafts and to investigate the kinetics of induction of apoptosis in infiltrating leucocytes. METHODS: Orthotopic penetrating corneal transplantation was performed between genetically disparate inbred rats. Fas ligand expression and the phenotype of infiltrating leucocytes were examined by immunohistochemistry. Apoptotic nuclei were visualised in sections of normal rat cornea, rejecting allografts, and time matched isografts by terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labelling (TUNEL) and quantified by video image analysis. Staining with Hoechst dye 33258 was used to confirm the presence of apoptotic nuclei. RESULTS: Fas ligand was expressed on corneal endothelial and epithelial cells during acute corneal graft rejection. At all time points examined, including as early as the fifth postoperative day, the cells infiltrating both corneal isografts and allografts were TUNEL positive. By the 15th postoperative day, over 90% of all nuclei, many of which were T cells, were apoptotic. CONCLUSION: Expression of Fas ligand is not downregulated on the cornea during allograft rejection and infiltrating leucocytes in both isografts and allografts die rapidly in situ. Despite the death of the cells believed to be responsible for rejection, isografts survive indefinitely whereas allografts are irreparably damaged.

Topically applied antibody fragments penetrate into the back of the rabbit eye.

AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Contrast and glare testing in keratoconus and after penetrating keratoplasty.

AIM: 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. METHODS: 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. RESULTS: 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; p<0.01, post hoc Sheffé p<0.05), and the decrements to glare were no worse than for normals. The forme fruste keratoconus group were indistinguishable from normals on all measures. CONCLUSIONS: 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.

Late onset post-keratoplasty astigmatism in patients with keratoconus.

AIM: 10 eyes of 10 patients are reported where progression of keratoconus in the host cornea occurred more than 10 years after penetrating keratoplasty with resultant increase in astigmatism. The technique and results of graft refractive surgery in seven eyes are presented. METHODS: The clinical features and management of these patients were retrospectively analysed. Graft refractive surgery involved an incision at the graft-host junction adjacent to the host thinning with compressive resuturing. Astigmatic changes were calculated using vector analysis. RESULTS: There were seven men and three women with a mean age of 41.2 years. The average age when undergoing penetrating keratoplasty in the affected eye was 28.4 years and the average time after penetrating keratoplasty until keratoconus appeared in the host cornea defined by host thinning was 13.5 years. The mean cylinder power before host thinning was noted was 5.07 D (SD 2.19) and the mean after host thinning was 11.0 D (2.53). The mean vector calculated disease induced astigmatism magnitude was 7.59 D (3.09). Graft refractive surgery was performed in seven eyes. The mean cylinder power before and after graft refractive surgery was 11.28 D (2.15) and 7.09 D (5.53) respectively. The surgically induced astigmatism vector magnitude was 7.36 D (4.88). CONCLUSION: Progression of keratoconus in the host cornea late after penetrating keratoplasty is characterised by a large astigmatic change where the flat axis of astigmatism passes through an area of host thinning visible on slit lamp examination. Compressive resuturing performed in the area of host thinning resulted in satisfactory reduction of astigmatism.

Management of high-risk corneal grafts.

Corneal transplantation is not invariably successful despite the anterior chamber of the eye being an immunologically privileged site. Inflammation erodes privilege. Other than by reducing inflammation through meticulous surgery, careful postoperative surveillance, and effective topical corticosteroids in the postoperative phase, there is little that a surgeon can do to improve the outlook for the majority of patients receiving corneal transplants. For patients at appreciable risk, HLA Class I matching may help where it is available. So too will systemic immunosuppression where it can be justified. Despite these measures, the results of corneal transplantation have not shown the improvement seen in solid organ transplantation over the last 30 years. New approaches applicable to corneal transplantation are required.

Penetration of engineered antibody fragments into the eye.

Antibodies are powerful immunotherapeutic agents but their use for treating ocular disorders is limited by their poor penetration into the eye. We hypothesized that antibody fragments of relatively small size might penetrate the cornea more readily. Monovalent single chain variable region (scFv) antibody fragments and divalent miniantibodies were engineered from existing monoclonal antibodies, expressed in a bacterial expression system, and purified by metal ion affinity chromatography. Corneoscleral preparations from normal pig and cat eyes were mounted in a corneal perfusion chamber. Intact antibodies and antibody fragments were applied topically to the anterior corneal surface over 12-h periods, and samples were collected from the artificial anterior chamber. Similar experiments were performed with whole enucleated pig and human eyes. Penetration of antibodies and fragments was quantified by high-sensitivity flow cytometry on appropriate target cells. Both monovalent scFv and divalent miniantibody fragments (but not whole immunoglobulin molecules) passed through de-epithelialized and intact corneas after topical administration, and could be detected by antigen binding. Addition of 0.5% sodium caprate facilitated penetration through intact corneas. Topically-applied scFv was found to penetrate into the anterior chamber fluid of rabbit eyes in vivo. The engineered fragments were stable and resistant to ocular proteases. Monovalent and divalent antibody constructs of molecular weight 28 kD and 67 kD, respectively, can penetrate through intact corneas into the anterior chamber, with retention of appropriate antigen-binding activity. Such constructs may form novel therapeutic agents for topical ophthalmic use.

Serratia ficaria endophthalmitis.

We report a case of Serratia ficaria endophthalmitis in a 73-year-old man. The patient's ocular history included a chemical burn, glaucoma, and corneal transplantation. S. ficaria is part of the fig tree ecosystem and is rarely isolated from clinical specimens. When it has been previously implicated as an agent of disease, the patients have been treated successfully and there have been no complications. In our patient, however, the infection resulted in the loss of the infected eye. This case illustrates that S. ficaria infection in a compromised patient can have serious consequences.

Gene transfer to ovine corneal endothelium.

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-cutured 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 ineffective. 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.

Prolongation of sheep corneal allograft survival by ex vivo transfer of the gene encoding interleukin-10.

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.

Clinical evaluation of twice-daily emedastine 0.05% eye drops (Emadine eye drops) versus levocabastine 0.05% eye drops in patients with allergic conjunctivitis.

PURPOSE: The efficacy and safety of emedastine 0.05% eye drops (Emadine; Alcon Laboratories, Inc, Fort Worth, Texas), a new H(1) antagonist, were studied in comparison to levocabastine 0.05% eye drops (Livostin; Janssen-Cilag N V, Berchem, Belgium) during a twice-daily treatment schedule for 6 weeks in adult and pediatric patients with seasonal allergic conjunctivitis. METHODS: In a prospective, multicenter, randomized, double-masked, parallel group study, 222 patients with allergic conjunctivitis were randomized (221 received treatment) to either emedastine or levocabastine, instilled twice daily for 6 weeks. Patient diaries were completed four times daily (before the morning and evening instillations, at noon, and in the afternoon), and clinical examinations were conducted at regular intervals. Primary efficacy variables of ocular redness and itching and secondary efficacy variables of chemosis, eyelid swelling, patient diary data, and physician's global assessment were analyzed. RESULTS: Both emedastine and levocabastine produced a statistically significant (P =.0001) reduction in itching and redness within 5 minutes of the first instillation. All signs and symptoms improved progressively over the 6-week treatment period. After 7 days of use, and throughout the remainder of the study, emedastine was statistically superior to levocabastine (P <.006) in preventing and alleviating the signs and symptoms (itching, redness, chemosis, and eyelid swelling) of allergic conjunctivitis. CONCLUSIONS: Emedastine 0.05% eye drops administered twice daily are more efficacious than levocabastine 0.05% eye drops in the prevention and treatment of the signs and symptoms of allergic conjunctivitis in adults and children of 4 years and above. Both emedastine 0.05% eye drops and levocabastine 0.05% eye drops were well tolerated.

The long term outcome of limbal allografts: the search for surviving cells.

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.