Yoichiro Sano

Immunosuppressive properties of human amniotic membrane for mixed lymphocyte reaction

The combination of allograft limbal transplantation (ALT) and amniotic membrane transplantation (AMT) has been applied in the treatment of severe ocular surface diseases. The beneficial effect of this combination has been thought to result from possible immunosuppressive ability of amniotic membrane (AM). However, the mechanisms of any such ability remain unknown. In this study, we investigated whether human AM has the ability to suppress allo‐reactive T cell responses in vitro. For mixed lymphocyte reaction (MLR), lymphocytes isolated from lymph nodes of C57BL/6 mice (Mls1b, Vβ6+) were cultured with irradiated splenocytes from DBA/2 mice (Mls1a, Vβ6−) with or without human AM. For carboxyfluorescein diacetate succinimidyl ester (CFSE) experiments, responder lymph node cells were labelled with a stable intracellular fluorescent dye and cultured with irradiated stimulator cells. The ratio of responder Vβ6+ T cells was then determined by FACS analysis, and the division profiles of responder Vβ6+ T cells were analysed by CFSE content. Furthermore, Th1 and Th2 cytokine synthesis by allo‐reactive T cells in MLR culture supernatants was determined by enzyme‐linked immunosorbent assay (ELISA). Addition of AM to the MLR culture resulted in the significant inhibition of thymidine incorporation compared with control culture lacking AM. The population of responder CD4+Vβ6+ T cells was significantly reduced in the AM‐treated culture in comparison to control. CFSE analysis revealed less division and lower proliferation of responder CD4+Vβ6+ T cells in cultures with AM than without. In addition, allo‐rective T cell synthesis of both Th1 (IL‐2 and IFNγ) and Th2 (IL‐6 and IL‐10) type cytokine was significantly decreased in the presence of AM. These results indicate that human AM has the ability to suppress allo‐reactive T cells in vitro. This inhibitory effect likely contributes to the success of the ALT‐AMT combination.

Successful regrafting of cultivated corneal epithelium using amniotic membrane as a carrier in severe ocular surface disease

Purpose. Our group performed cultivated allogeneic corneal epithelial transplantation in 13 eyes from 11 patients with severe ocular surface disorders. After the clinical application of this new surgical treatment, some patients experienced epithelial and subepithelial opacities. We applied our procedure again in these patients to achieve successful ocular surface reconstruction. Methods. The corneal limbal epithelial cells from donor corneas were cultivated for 4 weeks on denuded amniotic membrane (AM) carrier, with 3T3 fibroblast coculture and airlifting. The study subjects consisted of 3 patients. At 3 and 12 months after the first operation, the failed epithelial graft with AM was replaced with new allogeneic corneal epithelium cultivated on AM. Results. At 48 hours after transplantation, the corneal surfaces of the 3 eyes were clear and smooth; the entire corneal surfaces were evenly covered with the transplanted cultivated corneal epithelium, which did not stain with fluorescein. The ocular surface epithelia of these patients are all stable without epithelial defects. Conclusions. We have shown that, in cases where the initially transplanted cultivated epithelium becomes opaque, it is possible to repeat the transplantation process with new cultivated epithelium on AM.

Methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis infections in the cornea.

Purpose To describe the incidence and clinical management of corneal infections with methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-resistant Staphylococcus epidermidis (MRSE). Methods The incidence of methicillin-resistant Stuphylococcus (MRS) at the Department of Ophthalmology, Kyoto Prefectural University of Medicine, was reviewed during the 5-year period from January 1996 to December 2000. Clinical aspects of MRS colonization or infection in the eye were investigated. Results Methicillin-resistant S. aureus or MRSE was detected from 30 eyes with ocular diseases; post-keratoplasty (11 eyes), ocular surface disorders without operation (9 eyes), and others (10 eyes). Among the 30 eyes, 12 manifested keratitis. Eight cases (8 eyes) occurred after keratoplasty, including four postoperative cases in patients with Stevens-Johnson syndrome, and two bilateral cases (4 eyes) in patients with acute-phase Stevens-Johnson syndrome. The degree of MRS keratitis was classified into 4 groups: asymptomatic carrier or conjunctivitis, intraepithelial infiltrations, superficial keratitis, and severe keratitis leading to corneal perforation. All cases of keratitis were treated successfully with topical ofloxacin (OFLX), vancomycin (VCM), or arbekacin (ABK). Conclusion Factors associated with ocular MRS colonization were long-term use of antibiotics and/or steroids, and hospitalization. Patients who had undergone keratoplasty or who had Stevens-Johnson syndrome were at increased risk of MRS keratitis. Superficial stromal infiltrations, minimal melting, and minimal stromal scarring are characteristic of MRS keratitis. Therapy for MRS keratitis is summarized. Ofloxacin, VCM, and ABK are effective in the treatment of MRS keratitis. Vancomycin eye ointment is effective as the final choice in serious cases.

Conjunctival inflammation induces Langerhans cell migration into the cornea.

PURPOSE The virtual absence of Langerhans cells (LC) in donor or recipient corneal epithelium is known to be an important factor in the acceptance of orthotopic corneal allografts. Though it is well known that various types of stimulation to the cornea induce LC migration into the corneal epithelium, resulting in poor graft survival, the influence of conjunctival inflammation on LC migration into the cornea has not been elucidated. Therefore we examined whether LCs migrate into the cornea in the presence of conjunctival inflammation. METHODS Sixteen BALB/c mice were divided into four groups. Group A: 4 mice with corneal inflammation induced by two 9-0 silk interrupted sutures in the central cornea (positive control); Group B: 4 mice with conjunctival inflammation induced by two 9-0 silk interrupted sutures in the temporal and nasal bulbar conjunctiva 1 mm from the limbus; Group C: 4 mice with conjunctival inflammation by two 10-0 nylon interrupted sutures in the temporal and nasal bulbar conjunctiva 1 mm from the limbus; and Group N: 4 mice with no inflammation (untreated, naive control). Fourteen days after suturing, the mice were sacrificed and LCs migrated into the corneal epithelium were counted by immunofluorescence assay using anti-Ia antibody. RESULTS In Group A, Ia(+) cells in the cornea totaled 29.4 +/- 3.8 cells/mm(2); in Group B, 7.9 +/- 1.2 cells/mm(2); in Group C, 7.8 +/- 0.7 cells/mm(2); and in Group N, 1. 6 +/- 0.5 cells/mm(2). Significantly greater numbers of Ia(+) cells were detected in Groups A, B and C than in Group N (p < 0.005). CONCLUSIONS Conjunctival inflammation caused by sutures in the bulbar conjunctiva induced LC migration into the cornea. These results indicate that conjunctival inflammation influences the corneal immunological environment, and may affect the fate of orthotopic corneal allografts.

Ocular surface inflammation induced by Propionibacterium acnes.

Purpose. In the eye, Propionibacterium acnes is commonly isolated in the lid, conjunctiva, and meibomian gland secretion. Well known as a causative bacterium of granulomatous endophthalmitis and a potent inflammatory stimulus, P. acnes reportedly induces a delayed-type hypersensitivity (DTH) response and forms granulomas in the liver and lung in animal models. In this study, we examined whether P. acnes can induce a DTH response in the cornea. Methods. Six- to 8-week-old female Lewis rats were immunized with heat-killed suspension of P. acnes and assessed as to DTH response via ear challenge at 2 weeks after immunization. At 3 weeks after immunization, P. acnes suspension was injected in the rat corneal stroma, which was then observed biomicroscopically at 6, 24, and 48 hours after injection. Phenol-killed Staphylococcus aureus suspension was also used for the comparison. Histological examination was also performed on the corneal tissues, using hematoxylin and eosin staining as well as immunohistochemical staining against CD4 and CD8 T cells. Results. Rats immunized with P. acnes suspension showed significantly higher ear swelling values at both the 24- and 48-hour measurements than did the naïve controls (p < 0.005). Massive cellular infiltration with stromal edema was observed biomicroscopically at 48 hours after injection of P. acnes suspension in the corneal stroma. Histological study showed that the cell infiltration pattern was similar to that of DTH in the skin, i.e., neutrophils infiltrated at 6 hours, followed by mononuclear cells that, including macrophages and lymphocytes, increased and mixed with neutrophils, accompanied by stromal edema at 48 hours. Immunohistochemical study revealed that CD4 T-cell infiltration in the corneal stroma appeared to predominate over CD8 T-cell infiltration. Conclusions. These results indicate that P. acnes can induce a DTH response in the cornea and may be a causative bacterium of ocular surface inflammation.

Soluble Fas ligand expression in the ocular fluids of uveitis patients

Purpose. The expression of Fas ligand (FasL) in ocular tissues is thought to play a critical role in maintaining immune privilege in the eye. In this study, to clarify the involvement of the Fas-FasL system in inflammatory processes of the eye,we examined soluble FasL (sFasL) in ocular inflammation. Methods. Using ELISA systems recently developed, sFasL concentrations in aqueous humor (AH) and/or vitreous fluid (VF) were measured. AH was obtained from 17 eyes of 17 uveitis patients and from 12 eyes of 12 non-uveitis (cataract) patients. VF was obtained from 22 eyes of 22 uveitis patients and 7 eyes of 7 non-uveitis (macular hole) patients. Serum levels of sFasL were also determined. Results. sFasL in AH and VF was below the detection limit of the ELISA systems in all non-uveitis eyes. On the other hand, sFasL was detected in AH from uveitis patients where it measured 367.0 ± 154.7 pg/ml (mean ± SEM). sFasL was also detected in VF from uveitis patients where it measured 1132.2 ± 281.7 pg/ml. None of the sera from patients with or without uveitis contained a detectable level of sFas L. Conclusions. sFasL levels in AH and VF are elevated in the eye during ocular inflammation. Fas-FasL mediated apoptosis may play an important role in the regulation of inflammation during uveitis.

Peripheral lamellar keratoplasty for corneoscleral cyst: three case reports.

PURPOSE To examine whether peripheral lamellar keratoplasty (LKP) using preserved cornea was effective for the treatment of corneoscleral cysts. METHODS Three patients with corneoscleral cysts underwent peripheral lamellar keratoplasty. Two patients had no history of trauma or ocular surgery and were considered to have congenital cysts. The other patient had a history of strabismus surgery that had been performed 7 years previously. The anterior wall of the cysts was removed by trephination, and the epithelial membrane lining the posterior wall was peeled off. Lamellar corneal buttons obtained from preserved corneas then were put in place and secured with 8-10 interrupted sutures. In one case, because the cyst was large and extended to the pupillary axis, peripheral LKP was performed for removal of the scleral and peripheral corneal cyst, and the inner wall of the central corneal cyst was removed with vigorous irrigation and a spatula. RESULTS Histologic examination showed that all of the cysts were lined with nonkeratinizing epithelial cells. In all three cases, cysts have not reformed after a 1-5-year follow-up. CONCLUSIONS The cysts were lined in epithelial cells, and removal of these epithelial cells was considered to be important for the prevention of recurrence. Peripheral LKP is effective for the treatment of corneoscleral cysts, since this procedure removes displaced epithelial cells and reconstructs the thin part of the cornea and sclera.

Herpes simplex keratitis after ophthalmic surgery

PurposeWe report 6 cases of herpes simplex keratitis after ophthalmic surgery, in eyes without clinical history of herpes simplex keratitis.CasesThese cases comprised 6 patients examined at our hospital between April 1992 and November 2001. Past operations were keratoplasty in 5 eyes and cataract surgery in 1 eye. Clinical findings and predisposing factors were evaluated retrospectively. The period between herpetic epithelial keratitis onset and ophthalmic surgery ranged from 1.5 to 79 months. Predisposing factors included corticosteroid therapy and operative wound. The herpetic epithelial lesions were dendritic ulcers in 2 eyes, geographic ulcer in 1 eye, and atypical epithelial lesions in 3 eyes; in all cases, herpes simplex virus (HSV)-DNA was detected by polymerase chain reaction (PCR) in tear fluid. All herpetic epithelial lesions healed with oral and topical acyclovir.ConclusionsWhen corticosteroids are used following ophthalmic surgery, physicians should be alert to the possibility of herpetic epithelial keratitis, even in patients with no clinical history of herpes simplex keratitis. PCR detection in tear fluid is helpful in diagnosing this disease. Nippon Ganka Gakkai Zasshi (J Jpn Ophthalmol Soc 107:538–542, 2003)

Corneal endothelial transplantation: results of a clinical series using deep lamellar endothelial keratoplasty (DLEK).

Purpose We present the results of our clinical series replacing posterior stroma and endothelium only by deep lamellar endothelial keratoplasty (DLEK) in patients with corneal endothelial diseases. Methods Through a 9.0-mm superior scleral incision, a deep stromal pocket was created across the cornea. A 7.5-mm posterior lamellar disc of recipient tissue was excised and replaced by same-size donor posterior disc without suture fixation. Three cases were followed for 12 months after DLEK. Best spectacle-corrected visual acuity (BSCVA), endothelial cell density, and corneal thickness were examined. Results At 12 months after surgery, all transplants were clear and in position. In the 3 cases, BSCVA at 12 months was 20/200 (hand motion before operation), 20/50 (6/200 before operation), and 20/60 (20/250 before operation), respectively. In one patient, postoperative endothelial cell density was 533 cells/mm2 with a very thin donor disc. In the other two patients, postoperative endothelial cell density was >2000 cells/mm2. Corneal thickness (±SD) averaged 0.51 ± 0.06 mm. Conclusions DLEK in the setting of corneal endothelial diseases is an effective surgical procedure without corneal surface incisions and sutures.

A case of infectious crystalline keratopathy occurring long after penetrating keratoplasty

Purpose To report a 64-year-old womans case of infectious crystalline keratopathy (ICK) that occurred 9 years after the penetrating keratoplasty (PKP). Methods This study includes clinical history and histopathologic findings of the case. Results As historic background, the patient underwent PKP for lattice dystrophy and later, persistent epithelial defect occurred on the graft. Topical steroid treatment and a therapeutic soft contact lens were applied; however, ICK developed and second PKP was performed. In the ICK region, yeasts were cultured and histologically, degenerated keratocytes containing yeast particles surrounded the intralamellar plaques of yeasts without accompanying inflammatory cells. Conclusion This case is possibly the first to develop ICK long after PKP. Histologically, ICK presumably occurred based on the immunologically incompetent condition, and, by verifying the previous reports, the neurotrophic background after PKP may partially be involved in the development of ICK.