Nobuyuki Ebihara

A Randomized, Placebo-Controlled Clinical Trial of Tacrolimus Ophthalmic Suspension 0.1% in Severe Allergic Conjunctivitis

AIMS To examine the efficacy of tacrolimus ophthalmic suspension 0.1% in treating severe allergic conjunctivitis. METHODS This was a multicenter, randomized, double-masked, placebo-controlled clinical trial. Fifty-six patients with severe allergic conjunctivitis in whom topical antiallergic agents and corticosteroids had been ineffective were randomized to tacrolimus or placebo treatment. Patients were treated either with tacrolimus or placebo twice-daily for 4 weeks. Severity of objective signs in palpebral and bulbar conjunctiva, limbus, and corneal involvement was assessed using 4 grades. Seven subjective symptoms were evaluated by visual analog scale (VAS) assessment. The primary efficacy endpoint was change in the total score of objective signs at the end of treatment. The secondary efficacy endpoints included change in the score for each objective sign and change in the VAS for each subjective symptom. Safety was assessed based on the severity and the incidence of adverse events. RESULTS Mean change from baseline in total score for objective signs was significantly greater in the tacrolimus (-5.6 + or - 5.1) than in the placebo group (-0.1 + or - 4.5; P < 0.001). Tacrolimus significantly improved giant papillae (P = 0.001) and corneal involvement (P = 0.005). Five subjective symptoms (itching, discharge, hyperemia, lacrimation, and foreign body sensation) were significantly better in the tacrolimus than in the placebo group. The most frequent treatment-related adverse event in the tacrolimus group was mild ocular irritation upon topical instillation, which was well-tolerated. CONCLUSION Tacrolimus ophthalmic suspension 0.1% is effective in treating severe allergic conjunctivitis.

A Large Prospective Observational Study of Novel Cyclosporine 0.1% Aqueous Ophthalmic Solution in the Treatment of Severe Allergic Conjunctivitis

PURPOSE To evaluate the effectiveness and safety of a novel cyclosporine 0.1% aqueous ophthalmic solution in a large population with vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC). METHODS A prospective observational postmarketing study was initiated in Japan. A total of 594 patients with VKC or AKC were started on this drug within 1 year after market launch (January 2006) and completed a 6-month follow-up. These patients were observed clinically, and subjective ocular symptoms (itching, discharge, tearing, photophobia, foreign body sensation, and pain), objective signs (hyperemia, swelling, follicle, papillae, and giant papillae for the tarsal conjunctiva; hyperemia and edema for the bulbar conjunctiva; Trantas dots and swelling for the limbus; and corneal involvement), and adverse events were recorded. RESULTS All scores for symptoms and signs significantly decreased from Month 1 through Month 6 of treatment in both VKC and AKC. Median total symptom scores at baseline, Month 1, and Month 6 were 6, 2, and 1, respectively, for VKC, and 7, 3, and 2, respectively, for AKC. Similarly, median total sign scores were 12, 7, and 5, respectively, for VKC, and 14, 10, and 7, respectively, for AKC. The percentage of patients able to complete topical cyclosporine 0.1% therapy within 6 months due to alleviation of symptoms was higher for VKC (44.4%) than for AKC (21.9%). In both VKC and AKC, approximately 30% of steroid users were able to discontinue topical steroids. Adverse drug reactions (ADRs) were found in 12.0% of patients, and the most common ADR was eye irritation (4.4%). Infectious corneal complications were observed in five AKC patients, including two cases of bacterial corneal ulcer and three cases of herpetic keratitis; all of these patients were concomitantly using topical steroids. CONCLUSIONS Topical cyclosporine 0.1% is an effective and safe treatment for VKC and AKC.

Adhesion, migration, and proliferation of cultured human corneal endothelial cells by laminin-5.

PURPOSE To investigate the expression of laminin-5 (LM5) and its receptors by human corneal endothelial cells (HCECs) and whether recombinant human LM5 influences adhesion, proliferation, and migration of cultured HCECs. METHODS The expression of LM5 and its receptors was examined in human donor corneas by immunohistochemistry, reverse transcription-polymerase chain reaction, and flow cytometry. HCECs cultured under serum-free conditions were used for analysis of the biological effects of LM5. Changes in HCEC adhesion and proliferation due to LM5 were evaluated by counting the number of cells. HCEC migration was assessed by quantifying the percentage of wound closure in the wound-healing assay with an image-processing and -analysis software program. RESULTS Adult HCECs expressed the LM5 receptor α3β1 integrin, but not LM5 itself. Significantly more cells became adherent to recombinant LM5 (1.0 μg/mL)-coated dishes than to uncoated dishes in the cell adhesion assay. The proliferation of cultured HCECs was moderately promoted by LM5 (1.0 μg/mL) and soluble LM5 (20 ng/mL and 50 ng/mL) in the cell proliferation assay. A significantly higher percentage of wound closure was obtained with medium containing soluble LM5 than with control medium in the wound-healing assay. CONCLUSIONS HCECs express the LM5 receptor α3β1 integrin. Recombinant LM5 promotes adhesion, migration, and moderate proliferation of cultured HCECs. It may be a critical factor in promoting HCEC culture and may contribute to the practical use of tissue-engineered HCECs.

Therapeutic effects of 0.1% tacrolimus eye drops for refractory allergic ocular diseases with proliferative lesion or corneal involvement

Background The objective of this study was to investigate the efficacy of topical 0.1% tacrolimus in treating refractory allergic conjunctivitis with proliferative lesions and/or corneal involvement. Methods This prospective observational study included 1436 patients with refractory allergic conjunctivitis whose condition had responded poorly to conventional antiallergic drugs and/or topical steroids and/or topical cyclosporine. All patients received tacrolimus eye drops twice daily during the study period. Ten clinical signs and six clinical symptoms were rated on a four-grade scale. The primary endpoint was the change from baseline in total clinical signs and symptoms score at the last observation or following 6 months of treatment. Results Total signs and symptoms score significantly decreased after 1 month of treatment (p<0.001). Giant papillae and corneal lesions were also reduced by tacrolimus eye drop use (p<0.001). The drug proved effective in patients whose condition did not respond well to topical cyclosporine therapy. About 50% of all patients using topical steroids were weaned. The most common adverse reaction was a transient burning sensation (3.20%). Conclusions Tacrolimus eye drops are highly effective in treating refractory allergic conjunctivitis with proliferative lesions and/or corneal involvement, and may reduce or replace topical steroid use. Trial registration number UMIN 000008640.

Distinct Functions between Toll-Like Receptors 3 and 9 in Retinal Pigment Epithelial Cells

Retinal pigment epithelial cells (RPE cells) are key players in the first-line defense against invading organisms such as viruses and bacteria. The interaction between RPE cells and viral or bacterial components is very important for clearance of these organisms. Toll-like receptors are a family of recognition receptors involved in innate immunity. Each TLR acts as a primary sensor of conserved microbial components and drives the induction of specific biological responses. TLR 3 is involved in the recognition of viral components, such as double-stranded RNA (dsRNA) and poly(I:C), while TLR 9 recognizes viral or bacterial DNA without methylation at CpG motifs. In the present study, we investigated the expression and function of TLR 3 and 9 in RPE cells. PCR analysis revealed expression of genes for TLR 3 and 9 in RPE cells. Expression of TLR 3 and 9 protein was detected in RPE cells by flow cytometry. TLR 3 and 9 showed strong intracellular expression. To detect angiogenetic factors produced by RPE cells, culture supernatant was examined with the Human Angiogenesis Antibody Array, which can simultaneously detect 20 different angiogenetic factors including cytokines, chemokines, soluble cytokine receptors, and growth factors. RPE cells showed high production of interleukin-8 (IL-8) and monocyte chemotactic protein-I (MCP-I). Furthermore, stimulation of RPE cells with the dsRNA analogue poly(I:C) enhanced the secretion of IL-8 and MCP-I, as well as enhancing the expression of junctional adhesion molecule-I (Jam-I) and intracellular adhesion molecule-I (ICAM-I), and promoted the adhesion of monocyte to these cells. In contrast, stimulation with the CpG-DNA motif only enhanced the secretion of IL-8. However, CpG-DNA motif enhanced phagocytosis in RPE cells. These results may indicate that TLR 3 and 9 play a distinct role in the inflammatory response that clears viruses from the retina.

Bone marrow-derived cells in mouse and human cornea

Recently published experimental data on the distribution of bone marrow (BM)-derived cells in human and mouse corneas in comparison with in human skin/oral mucosa are reviewed. In mouse corneal epithelium, major histocompatibility complex (MHC) class II-negative dendritic cells (DC) are present. Immature MHC class II-negative and mature MHC class II-positive DC are present in the center and periphery of the anterior corneal stroma, respectively. Monocyte (Mo)/macrophage (MΦ) lineage cells including the MΦ marker F4/80-expressing cells reside in the posterior stroma. In human cornea, MHC class II (HLA-DR)-positive immature myeloid DC (CD11c+CD16−, CD11c+CD16+, and CD11c+CD1c+) and Mo/MΦ lineage cells are detectable in the corneal epithelium and stroma, respectively. Distribution of Mo/MΦ lineage cells (HLA.DR+CD11b+CD11c+CD14+) is predominant in the anterior stroma of the central cornea and all layers of the peripheral cornea. Both the phenotypes and distribution pattern of these cells in human cornea are different from those of human skin and nasal mucosa. These findings suggest that BM-derived cells in normal human cornea are present in situ in preparation for foreign antigen and pathogens and have critical roles in innate and acquired immunity of the ocular surface.

Mast Cell Chymase Decreases the Barrier Function and Inhibits the Migration of Corneal Epithelial Cells

Purpose: We investigated the in vitro effects of human mast cell chymase on corneal epithelial cells. Methods: Human corneal epithelial cells were incubated with human chymase at activity levels that were likely to exist in the tears of patients with vernal keratoconjunctivitis. Results: Incubation of chymase resulted in a decrease of barrier function of corneal epithelium. Occludin protein was cleaved by chymase. In the wound assay, incubation with chymase resulted in an inhibition of cell migration. Conclusion: Human chymase causes the proteolysis of occludin and fibronectin, resulting in a decrease of barrier function and inhibition of the migration of corneal epithelial cells.

Infectious keratitis outbreak after laser in situ keratomileusis at a single laser center in Japan

PURPOSE: To evaluate an outbreak of infectious keratitis after laser in situ keratomileusis (LASIK) at a single laser center in Japan. SETTING: Tokyo Dental College, Chiba, Japan. DESIGN: Case series. METHODS: Relevant demographic and clinical data were obtained from case records using a standardized multicenter questionnaire at 12 major hospitals. The clinical manifestations, management, and outcomes were analyzed. RESULTS: Thirty‐nine eyes of 30 patients developed infectious keratitis after LASIK at the specified clinic. Cases of infection continued to appear over a 5‐month period. The most common interval between LASIK and the onset of infection was within 2 weeks (36 eyes, 92.3%). Slitlamp manifestation included granular opacity beneath flap (71.8%), multiple infiltration (66.7%), and epithelial defect (30.8%). Mycobacterium was identified as the causative organism in 9 eyes (23.1%). In most cases, topical amikacin, arbekacin, and erythromycin in addition to fourth‐generation fluoroquinolones were effective. Flap amputation was necessary in 10 eyes (25.6%) of 10 patients. Decimal visual acuity at initial presentation was worse than 0.10 in 14 eyes (35.9%), 0.15 to 0.50 in 8 eyes (20.5%), and 0.60 to 0.90 in 7 eyes (17.9%) and was better than 1.00 in 10 eyes (25.6%). Final decimal visual acuity was worse than 0.10 in 2 eyes (5.1%), 0.15 to 0.50 in 5 eyes (12.8%), and 0.60 to 0.90 in 11 eyes (28.2%) and was better than 1.00 in 21 eyes (53.8%). CONCLUSIONS: Mycobacterium was identified as the causative organism of this outbreak. This study shows the possibility of an epidemic of post‐LASIK infectious keratitis. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned.

Mast cells in pterygium : Number and phenotype

PURPOSE To investigate the pathogenesis of pterygium. METHODS The number and phenotype of mast cells were examined in excised tissue from 35 pterygia patients and compared with those in normal conjunctival specimens obtained during cataract or other intraocular surgery. RESULTS Toluidine blue staining showed that the mean number of mast cells in the pterygia specimens was twice as high as that in the normal conjunctival tissues. Immunohistochemistry with a primary antibody to tryptase, specific for mast cells, also revealed a twofold increase in the mast cell number in the pterygia specimens compared with the normal conjunctival tissues. In the pterygia, more than 94% of the tryptase-positive mast cells were found to express chymase and c-kit. Almost all mast cells in the pterygia were tryptase-positive, chymase-positive mast cells (MC(TC)S). There was no phenotypic difference between the mast cells in the pterygia and those in the normal conjunctival tissues. CONCLUSIONS The MC(TC)S appear not to be immune system-related and to have functions in angiogenesis and tissue remodeling. The increase in the number of mast cells caused by nonallergic stimulation may contribute to the pathogenesis of pterygium.

Expression of Stem Cell Factor in Pterygium

PURPOSE To investigate the possible role of stem cell factor (SCF) in the pathogenesis of pterygium. METHODS The localization of SCF was examined immunohistochemically in excised tissue from 4 primary pterygia and 5 normal conjunctival specimens. RESULTS Three of the four pterygia showed strong immunoreactivity of SCF in the subepithelial connective tissue at the cap area. This immunoreactivity was completely blocked by using a primary antibody preincubated with recombinant SCF. The SCF-positive cells were identified as a population of fibroblasts by immunostaining for vimentin and prolyl 4-hydroxylase in adjacent sections. No apparent immunoreactivity of SCF was observed in the subepithelial connective tissues in the head and body of the pterygia and in the normal conjunctiva. CONCLUSION Stem cell factor is overexpressed in fibroblasts at the cap area of most pterygia.