Analysis of p63 and cytokeratin expression in a cultivated limbal autograft used in the treatment of limbal stem cell deficiency.

AIM: To investigate the expression of p63 and cytokeratins throughout the course of producing a cultivated autograft of limbal epithelial cells. METHODS: A 75 year old male with a severe alkali burn to his right eye received two cultivated autografts of limbal epithelial cells on amniotic membrane followed by a corneal allograft. Immunostaining for p63 and cytokeratins was performed during ex vivo expansion with 3T3 fibroblasts, following subcultivation on amniotic membrane, and on the excised corneal button. RESULTS: Cultures grown in the presence of 3T3 fibroblasts or on amniotic membrane displayed positive staining for keratins 14 and 19, and p63, but poor staining for keratin 3 (K3). The excised corneal button possessed a stratified epithelium of K3 positive cells residing on amniotic membrane. CONCLUSIONS: Our results document for the first time the co-expression of cytokeratins 14 and 19 with p63 in a cultivated limbal graft. These data support the conclusion that cultivated grafts of limbal epithelium contain predominantly undifferentiated cells with the potential to regenerate a normal corneal epithelium.

Phenotypic analyses of limbal epithelial cell cultures derived from donor corneoscleral rims.

Grafted cultures of limbal epithelial cells aid repair of the corneal epithelium, but their phenotype is unclear. In this study, the phenotype of cultures that were similar in age to those used clinically were analysed. Limbal epithelial cells were isolated from donor corneoscleral rims and grown in various media, including those designed for keratinocytes. Successful cultures in each medium developed predominantly small (10 microm) tightly packed cells. Immunocytochemistry and western blotting revealed expression of keratins 3, 14 and 19. Expression of these keratins in situ was confirmed by immunohistochemistry. Basal limbal epithelial cells were positive for keratins 14 and 19, and suprabasal cells were positive for keratin 3. However intense staining for keratin 14 was also observed at the inner cut edge of corneoscleral rims. These findings demonstrate the potential importance of keratins 14 and 19 as markers of epithelial cell differentiation in the human cornea.

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.

Perfluorodecalin corneal toxicity: five case reports.

Perfluorodecalin is a perfluorocarbon liquid used intraoperatively in retinal detachment repair. It is usually removed at the end of the procedure; however, residual amounts may be retained when poor corneal clarity or intraocular hemorrhage obscures the view. No clinical reports exist on the consequences of retained perfluorodecalin in the anterior segment. We report five cases in which perfluorodecalin was in prolonged contact with the cornea. The period of time for corneal pathology to occur and the role perfluorodecalin played in the etiology of such changes is discussed. A total of 348 patients with retinal detachments in one retinal practice underwent repair using pars plana vitrectomy combined with intraoperative perfluorodecalin between January 1992 and May 1994. Postoperatively, residual perfluorodecalin was observed in the anterior chamber in contact with the corneal endothelium in five patients. The patients were followed clinically for a period of up to 18 months. Four of five patients developed corneal changes from prolonged contact with perfluorodecalin. Corneal edema developed in the area perfluorodecalin-endothelial contact in three of five eyes. The period of perfluorodecalin-endothelial contact before corneal decompensation occurred ranged from 4 to 13 weeks. Two eyes required penetrating keratoplasties for progressive corneal edema. Corneal edema was reversed in one eye after removal of perfluorodecalin from the anterior chamber via multiple paracentesis. One of the remaining eyes developed deep corneal vascularization without edema in the area of perfluorodecalin contact after 12 months. These observations suggest that corneal toxicity may be induced by intraocular perfluorodecalin if it is allowed direct contact with the corneal endothelium for periods as short as 1 month. Some of these changes may be reversible if perfluorodecalin is aspirated from the anterior chamber. Further investigations are required to examine perfluorodecalin-induced corneal toxicity.

Local efficacy of cyclosporine in corneal transplant therapy.

Studies were conducted to evaluate the efficacy of direct injections of cyclosporine (CsA) into the anterior chamber for the prevention of corneal allograft rejection in Dutch Belted rabbits. The mean survival time (MST) of grafts progressively increased from 50 to 89 days as the CsA concentration in the dose was increased from 1 to 10 mg/mL. Injection of 30 microL of 20 mg/mL CsA in olive oil prolonged graft survival to beyond 125 days without any signs of rejection. By comparison, the MST of allografts in control animals which received no therapy was 32 +/- 5 days, and the MST in animals administered a placebo of olive oil only was 31 +/- 4 days. The observed concentration dependence of the MST on CsA concentration is likely related to the time over which the drug delivery rate provides sufficient drug to achieve a therapeutic concentration in the aqueous humor; these studies suggest that the minimum delivery rate to the anterior chamber is between 200 and 325 ng/day. The efficacy of CsA was due to local delivery, and was likely not a systemic effect, because CsA was not detected in the systemic circulation at any time. This indicates that direct delivery of CsA to the eye can be useful in prolonging corneal graft survival, while minimizing systemic side effects. Separate experiments revealed that episodes of advanced rejection could not be reversed by a 30 microL dose of 20 mg/mL CsA to the anterior chamber, indicating the importance of avoiding long periods of subtherapeutic dosing.