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Arch Ophthalmol. 2001;119:298-300.

Stevens-Johnson syndrome (SJS), also known as the erythema multiform, is an acute, self-limited, inflammatory disorder of the skin and mucous membranes. Although the skin lesions are self-limited, the ocular disease gets worse and often results in bilateral blindness owing to a lack of corneal stem cells,¹ corneal scarring, and subconjunctival fibrosis. Prognosis of corneal transplantation in the acute phase is poor because of the difficulty of overcoming the severe inflammation and allograft rejection. Even in the chronic phase, it is difficult to control persistent inflammation, dry eye, and trichiasis, which induce persistent epithelial defect and allograft rejection. Although corneal stem cell transplantation and amniotic membrane (AM) transplantation, combined with dry eye treatment and strong immunosuppressive therapy, have been attempted at the scarring stage,² the management is difficult, and the visual prognosis is not satisfactory. However, if it were possible to control the severe inflammation and manage the corneal transplantation in the acute phase with less scarring change, the visual prognosis could be greatly improved. Recently, attention has focused on cultivated corneal epithelial transplantation as a new approach for ocular surface reconstruction in limbal-deficient disorders.³ We ensured the suitability of the denuded AM as a carrier for the corneal epithelial cell culture⁴ and have successfully established a surgical system for cultivated corneal limbal epithelium transplantation in rabbits using AM as a carrier.⁵ We applied this procedure in 2 patients with the acute phase of SJS, and we achieved successful ocular surface reconstruction.

Human AM was obtained at cesarean section,⁵ and the amniotic epithelium was removed by EDTA treatment.⁴ Small pieces of donor limbal cornea were cultured on acellular AM with 3T3 fibroblasts. The culture was submerged in the medium for 2 weeks, after which it was exposed to the air to promote epithelial stratification for 1 to 2 weeks. The cultivated corneal epithelium consisted of 4 to 5 layers, appeared very similar to normal corneal epithelium, and stained with antibodies specific for corneal epithelium keratin 3 (AE5) and keratin 12 (J7) (Figure 1).

View larger version (37K): [in this window] [in a new window] Figure 1. A, Cultivated corneal epithelium on amniotic stroma (asterisk) consists of 4 to 5 layers that are very similar in appearance to normal corneal epithelium (B) (hematoxylin-eosin, original magnification x100). C and D, Cultivated corneal epithelium stained with antibodies specific to corneal epithelium keratin 3 and keratin 12.

Report of Cases
The patients were a 32-year-old man (patient 1) and a 21-year-old man (patient 2), each of whom had had SJS for 3 months. Their eyes showed persistent corneal epithelial defects surrounded by inflammatory subconjunctival fibrosis that was resistant to conventional therapy (Figure 2, A and B). They showed total stem cell deficiency. After informed consent from the patients and approval from the university ethics committee were obtained, we performed the cultivated corneal epithelial transplantation on the right eye of patient 1 and in both eyes of patient 2. After removal of the conjunctival tissue on the cornea up to 3 mm outside the limbus, we treated the subconjunctival fibroblasts for 5 minutes with 0.04% mitomycin and vigorous saline washing. We secured the cultivated allocorneal epithelium on AM onto the corneal surface with 10-0 nylon sutures, then we covered it with a therapeutic soft contact lens. Postoperatively, 0.3% ofloxacin and 0.1% dexamethasone were instilled 4 times per day, and corticosteroid (1 mg/d), cyclosporin (150 mg/d), and cyclophosphamide (100 mg/d) were administered to prevent postoperative inflammation and allograft rejection. Forty-eight hours after transplantation, the corneal surfaces of the 3 eyes were clear and smooth, and the entire corneal surfaces were perfectly covered with transplanted allocorneal epithelium that did not stain with fluorescein (Figure 2, C). The transplanted corneal epithelium was surrounded by a conjunctival epithelial defect at 360°, suggesting there was no contamination of host conjunctival epithelium. Five days after transplantation, all areas of the ocular surface were covered with transparent epithelium. Shortly after the transplantation, conjunctival inflammation rapidly subsided, and visual acuity recovered to 20/20 OD in patient 1 and 20/20 OU in patient 2. After a posttransplantation observation period of 6 months, their ocular surface epithelia were stable and without defects (Figure 2, D). We are carefully monitoring the transplanted allocorneal epithelial cells to determine their longevity.

View larger version (51K): [in this window] [in a new window] Figure 2. A, Persistent corneal epithelial defects of patient 2 (right eye) surrounded by inflammatory subconjunctival fibrosis. B, Fluorescein staining clearly shows the area of epithelial defect surrounded by conjunctival invasion. C, Two days after the cultivated corneal epithelial transplantation, corneal surface is covered with transplanted corneal epithelium, which shows no fluorescein staining. D, Three months after transplantation, a clear corneal surface is maintained, and visual acuity is 20/20 OD.

Comment
The concept that corneal epithelial stem cells reside in the limbal basal epithelium was established in the 1980s.¹ Conventional stem cell transplantation has great potential as a treatment for stem cell deficiency. However, this procedure needs several weeks to cover the total corneal surface with migrated corneal epithelium from donor cornea, and the denuded corneal surface induces severe inflammation, especially in acute phase SJS. The patients we report here were in acute phase and would have been contraindicated for conventional stem cell transplantation. Our method of cultivated allocorneal epithelial transplantation allowed us to cover their ocular surfaces with clear and silent corneal epithelium during surgery, and subside their inflammation. In conclusion, cultivated corneal epithelial transplantation using AM as a carrier is an effective ocular surface reconstruction technique for acute phase SJS.

AUTHOR INFORMATION
This work was supported by the Japanese Ministry of Health and Welfare, grant 10470365 from the Japanese Ministry of Education, the Kyoto Foundation for the Promotion of Medical Science, and the Intramural Research Fund of the Kyoto Prefectural University of Medicine.

The authors thank Nigel Fullwood, PhD, for reviewing the manuscript, and Satoshi Kawasaki, MD, for immunohistochemistry.

Noriko Koizumi, MD, PhD; Tsutomu Inatomi, MD, PhD; Tomo Suzuki, MD; Chie Sotozono, MD, PhD; Shigeru Kinoshita, MD, PhD
Kyoto, Japan

Corresponding author and reprints: Shigeru Kinoshita MD, PhD, Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kawaramachi-hirokoji, Kamigyo-ku, Kyoto 602-0841, Japan (e-mail: skinoshi{at}ophth.kpu-m.ac.jp).

REFERENCES
1. Schermer A, Galvin S, Sun TT. Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol. 1986;103:49-62. FREE FULL TEXT 2. Tsubota K, Satake Y, Ohyama M, et al. Surgical reconstruction of the ocular surface in advanced ocular cicatricial pemphigoid and Stevens-Johnson syndrome. Am J Ophthalmol. 1996;122:38-52. ISI | PUBMED 3. Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R, DeLuca M. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet. 1997;349:990-993. FULL TEXT | ISI | PUBMED 4. Koizumi N, Fullwood NJ, Bairaktaris G, Inatomi T, Kinoshita S, Quantock AJ. Cultivation of corneal epithelial cells on intact and denuded human amniotic membrane. Invest Ophthalmol Vis Sci. 2000;41:2506-2513. FREE FULL TEXT 5. Koizumi N, Inatomi T, Quantock AJ, Fullwood NJ, Dota A, Kinoshita S. Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits. Cornea. 2000;19:65-71. FULL TEXT | ISI | PUBMED

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