The Japan Cornea Society Survey of the Current Status of Corneal and Conjunctival Disorders Due to Systemic Antitumor Drugs.

Purpose: To investigate the current status of corneal and conjunctival disorders due to antitumor drugs in Japan. Methods: Questionnaires on corneal and conjunctival disorders due to antitumor drugs were sent to members of the Japan Cornea Society, and data on patients' background, clinical findings, treatment and prognosis of cases between January 2009 and December 2011 were collected and analyzed. Results: Out of all 221 cases from 66 facilities, TS-1Ⓡ had been administered in 210 cases (95.0%). Corneal findings were noted in 192 cases (86.9%), including 161cases (72.9%) of superficial punctate keratopathy, 55 cases (24.9%) of epithelial crack line, 38 cases (17.2%) of sheet-like epithelial abnormality, and 15 cases (6.8%) of corneal erosion. Conjunctival and ciliary findings were observed in 49 cases (22.2%). Lacrimal obstruction and constriction were found in 81cases (36.7%). Logistic regression analyses revealed the discontinuation and switching of antitumor drugs as the significant factor of good prognosis of clinical signs and visual acuity in cases with TS-1Ⓡ administration. Conclusions: Although corneal and conjunctival disorders due to antitumor drugs, especially TS-1Ⓡ, are important adverse effects, the only effective treatment at this time is the discontinuation and switching of antitumor drugs. Future prospective studies are needed to elucidate pathogenesis, aiming to the prediction and prevention of the occurrence.

Rapid Corneal Calcification Following Endothelial Keratoplasty: A Case Report.

Corneal calcification typically progresses slowly but can occasionally advance rapidly. This report details severe stromal calcification following repeat Descemet's stripping automated endothelial keratoplasty (DSAEK) in a 75-year-old patient with diabetes, hypertension, and prior ocular surgeries, including cataract surgery, intraocular lens extraction with suturing, and trabeculectomy. Persistent epithelial defects after the surgery led to rapid central stromal calcification within four weeks, significantly reducing visual acuity. Management included switching from betamethasone sodium phosphate to fluorometholone, facilitating complete epithelial recovery within two months. However, persistent stromal opacity necessitated a subsequent penetrating keratoplasty. Infrared absorption spectrophotometry identified calcium phosphate as the primary component of the calcification. This case highlights the importance of vigilant monitoring and proactive management of epithelial defects to prevent rapid calcification following endothelial keratoplasty.

Blocking Air in the Filtering Bleb: Effects on Graft Adhesion After Descemet's Stripping Automated Endothelial Keratoplasty.

BACKGROUND: To evaluate the outcome of Descemet's stripping automated endothelial keratoplasty (DSAEK) with "blocking air" in the filtering bleb in patients with previous trabeculectomy. METHODS: In total, 299 eyes in 283 patients who underwent DSAEK were retrospectively reviewed. Endothelial graft adhesion, intraocular pressure (IOP), and air volume in the anterior chamber with (group A) or without (group B) a filtering bleb were compared between the groups. Group A was divided into two subgroups according to the presence (group A1) or absence (group A2) of air in the filtering bleb; the same three factors were compared between the subgroups. RESULTS: The graft detachment rate was significantly higher in group A (14.3%) than in group B (6.5%) (p = 0.04). IOP was significantly lower in group A than in group B before surgery (p = 0.01), at the end of surgery (p = 0.04), at three hours (p < 0.001), and one week postoperatively (p = 0.02). The graft detachment rate did not significantly differ between groups A1 and A2. There were no differences in IOP at each follow-up time, whereas there was a statistically significant increase in IOP from the preoperative measurement to the end of surgery in group A1 (21.0±7.0 mmHg) compared with group A2 (14.2±8.6 mmHg) (p = 0.02). CONCLUSIONS: The presence of blocking air in the filtering bleb helps ensure increased IOP during the early postoperative period but had no significant effect on graft detachment rates.

Matrix metalloproteinase and cytokine expression in Tenon fibroblasts during scar formation after glaucoma filtration or implant surgery in rats.

Failure of surgery for glaucoma is usually due to post-surgical scarring (fibrosis), a process in which fibroblasts play a prominent role. We investigated the molecular mechanisms of such scarring by examining the expression of matrix metalloproteinases and cytokines in Tenon fibroblasts isolated from rats after glaucoma surgery. Filtration surgery was performed in one eye and implant surgery in the other; and Tenon fibroblasts were isolated from the tissue surrounding the bleb after surgery. The cells were cultured and examined for the expression of matrix metalloproteinases (MMPs) by reverse transcription-polymerase chain reaction, immunoblot and gelatin zymographic analyses. Culture supernatants were also assayed for cytokines with a multiplex array. The amounts of MMP-1 and MMP-3 mRNAs and proteins were greater in cells isolated after implant surgery than in those isolated after filtration surgery, with the progression of scar formation being more complete after the former surgery. The secretion of interleukin-6 (IL-6) by cells isolated after filtration surgery was greater than that for cells isolated after implant surgery. Depletion of IL-6 by RNA interference in cells isolated after filtration surgery increased the expression of MMP-1 and MMP-3 in these cells. These results thus suggest that the expression of MMP-1 and MMP-3 in Tenon fibroblasts is regulated by IL-6 during, and may play an important role in, scar formation after glaucoma surgery.

Refractory Fungal Keratitis Caused by Acrophialophora fusispora: A Case Report and Literature Review.

Acrophialophora fusispora is a filamentous fungus that is found in soil and rarely infects humans. We herein report the first case of fungal keratitis caused by A. fusispora in Japan and present a review of the literature on human infections with Acrophialophora species. A 62-year-old Japanese male on immunosuppressive therapy developed fungal keratitis after the removal of a corneal foreign body from his left eye. Voriconazole eye drops and systemic therapy for post-traumatic fungal keratitis did not resolve the infection, and the patient required a therapeutic corneal transplant. The isolate was identified as A. fusispora based on the nucleotide sequence of the internal transcribed spacer region. In a drug susceptibility test, the minimum inhibitory concentration of voriconazole was 0.5 µg/mL. Based on this case and previous cases from the literature review, fungal keratitis caused by A. fusispora is often refractory.

Usefulness of smear microscopy for therapeutic decision-making in patients with infectious keratitis.

PURPOSE: To determine the usefulness of smears in the diagnosis of infectious keratitis by comparing smears and 2 different culture methods. STUDY DESIGN: Retrospective, observational study. METHODS: The foci of 73 patients diagnosed with infectious keratitis at Hiroshima University Hospital between July 2011 and September 2015 were abraded, and smear microscopy and culturing were performed. The microorganism detection rates and other parameters were compared. RESULTS: Microorganisms were detected in 47 of 73 specimens. Microorganisms were identified in 32 of 69 cases cultured on plain medium (detection rate, 46.4%) compared with 22 of 61 cases cultured on swab transport medium (detection rate, 36.1%). There was no significant difference in the microbial detection rate between the plain medium method and the swab transport medium method (P = 0.23). Smear microscopy and culture findings were concordant in 21 (28.8%) cases, and different microorganisms were detected in 9 cases. In 17 cases, the culture was negative, despite the presence of microorganisms on smear microscopy, and in 7 cases, the culture was positive, despite the absence of microorganisms on smear microscopy. The positivity rate of microbial detection was significantly higher when no antimicrobial agents had been administered previously (odds ratio 7.50, P = 0.017). CONCLUSION: Smear microscopy of abrasions from lesions is useful for the initiation of treatment for infectious keratitis. However, culture studies should be conducted at the same time to confirm antimicrobial sensitivity. If possible, smear microscopy should be performed before the initiation of antimicrobial therapy.

Corneal Copper Deposition Secondary to Monoclonal Gammopathy in a Patient With Chronic Lymphocytic Leukemia: A Case Report.

Hypercupremia-induced corneal copper deposition secondary to monoclonal gammopathy is rare and shows a characteristic corneal opacity quite different from other causes of hypercupremia, such as Wilson's disease. This report describes a case of corneal copper deposition in a patient with monoclonal gammopathy associated with chronic lymphocytic leukemia. An 84-year-old man with slowly progressive corneal opacity was referred to our hospital. The corneal opacity was present at least five years ago. The patient's best-corrected visual acuity was 20/25 OU (in both eyes) at the initial visit to our hospital. Slit-lamp examination and anterior segment optical coherence tomography revealed bilateral brown-colored opacity localized to deep layers of the central cornea. In vivo confocal microscopy (IVCM) showed indistinct corneal stromal cells in the deep layer and endothelial cells. The possible differential diagnoses were corneal dystrophy and Wilson's disease, but the color, shape, or site of corneal opacity was inconsistent with the disease. As the patient had a history of chronic lymphocytic leukemia, which is often associated with monoclonal gammopathy, we suspected that the corneal opacity was copper deposition in association with the hematologic diseases. Laboratory examinations showed elevated serum copper and normal ceruloplasmin. Serum protein electrophoresis revealed significantly high IgG levels with depression of IgA, IgE, and IgM. These results supported our diagnosis. Followingly, we consulted the patient's attending hematologist, and the doctor initiated treatment for hypercupremia. In conclusion, hypercupremia secondary to monoclonal gammopathy should be considered a possible cause of central brown-colored corneal opacity.

Lumican is required for neutrophil extravasation following corneal injury and wound healing.

An important aspect of wound healing is the recruitment of neutrophils to the site of infection or tissue injury. Lumican, an extracellular matrix component belonging to the small leucine rich proteoglycan (SLRP) family, is one of the major keratan sulfate proteoglycans (KSPGs) within the corneal stroma. Increasing evidence indicates that lumican can serve as a regulatory molecule for several cellular processes, including cell proliferation and migration. In the present study, we addressed the role of lumican in the process of extravasation of polymorphonuclear leukocytes (PMNs) during the early inflammatory phase present in the healing of the corneal epithelium following debridement. We used Lum(-/-) mice and a novel transgenic mouse, Lum(-/-),Kera-Lum, which expresses lumican only in the corneal stroma, to assess the role of lumican in PMN extravasation into injured corneas. Our results showed that PMNs did not readily invade injured corneas of Lum(-/-) mice and this defect was rescued by the expression of lumican in the corneas of Lum(-/-),Kera-Lum mice. The presence of lumican in situ facilitates PMN infiltration into the peritoneal cavity in casein-induced inflammation. Our findings are consistent with the notion that in addition to regulating the collagen fibril architecture, lumican acts to aid neutrophil recruitment and invasion following corneal damage and inflammation.

Up-regulation of matrix metalloproteinase-1 and interleukin-6 expression in cocultures of corneal fibroblasts and neural cells.

The cornea is the most sensitive tissue in the human body, with the dense nerve endings of the cornea being derived from the first division of the ophthalmic nerve. The existence of such organized nerve fibers reflects the role of neural regulation in corneal homeostasis, with the proper distribution and function of these nerve fibers thus being required for maintenance of a healthy cornea. We recently established an in vitro model, based on the coculture of human corneal epithelial cells and fibroblasts on opposite sides of a collagen vitrigel membrane. We have now examined the role of neural cells in corneal homeostasis with the use of a similar coculture system. Reverse transcription-polymerase chain reaction and immunoblot analyses showed that the presence of neural cells (differentiated PC12 cells) increased the expression of matrix metalloproteinase-1 (MMP-1) in human corneal fibroblasts at both the mRNA and protein levels. The expression of MMP-2 and MMP-9 in corneal fibroblasts was not affected by PC12 cells. Furthermore, a multiplex assay showed that, among various cytokines assayed, only the release of interleukin-6 in cocultures of the two cell types was markedly greater than that in cultures of corneal fibroblasts alone. These results thus suggest that factors released from neural cells may play an important role in regulation of the function of corneal fibroblasts and thereby contribute to the maintenance of corneal structure and function.

Differential expression of semaphorin 3A and its receptors during mouse retinal development.

Semaphorins not only function in axon guidance during development but also contribute to various other biological processes. We have now examined the expression of semaphorin 3A (Sema3A) and its receptor components neuropilin 1 (Npn1) and plexin A (PlxA) during development of the mouse retina. Immunohistofluorescence analysis revealed that the expression patterns of Sema3A and Npn1 were similar during embryonic and postnatal development. The expression pattern of PlxA was also similar to those of Sema3A and Npn1 during embryonic and early postnatal (before eye opening) developments. However, the pattern of PlxA expression changed markedly after eye opening, with the expression disappearing from the optic nerve and increasing in intensity in the retinal pigment epithelium. Immunoprecipitation analysis showed that Sema3A interacted with PlxA in the retinal pigment epithelial cell line ARPE19 but not in the retinal ganglion cell line RGC5, whereas the opposite pattern of association was apparent for Sema3A and Npn1. Given that atmospheric oxygen is thought to play a role in the differentiation and maintenance of various ocular cell types, our results suggest that Sema3A-PlxA signalling activated by an effect of ambient oxygen on PlxA expression may contribute to differentiation of the retinal pigment epithelium.

[A case of anterior segment dysgenesis showing change in anterior eye features].

PURPOSE: Using an ultrasound biomicroscope (UBM) and an endoscope just after birth, We observed the morphological changes in the anterior segment in a case of anterior segment dysgenesis. CASE: The patient was a 9-day-old baby girl born with central opacity and high intraocular pressure. The central cornea was thin and the opacity was ring-shaped. Corneal vascularization was associated with the increase in the central corneal opacity, and finally progressed to fatty degeneration. Just after birth, UBM showed a double anterior chamber in one part of the cornea. The space in the cornea was filled with solid material, and the corneal thickness worsened. Surgical endoscopy showed a red membrane on the inner side of the cornea. The red color faded with time. CONCLUSION: We reasoned that an immature corneal stroma developed after birth, and that this secondary stroma filled the space between the retrocorneal membrane and the corneal stroma. We also reasoned that the red membrane of the posterior cornea might be caused by bleeding from the corneal neovascular vessels, or that the vascular membrane of the posterior cornea encouraged proliferation of collagen fibers. Later on the vessels and bleeding regressed.

[Outcome of conjunctival autograft with amniotic membrane transplantation for recurrent pterygium].

PURPOSE: To investigate the outcome of conjunctival autograft with amniotic membrane transplantation without the use of mitomycin C for cases of recurrent pterygium. CASES AND METHODS: Thirty-nine eyes of 35 patients (aged 27 to 76 years) who underwent the surgical procedure for recurrent pterygium at Yamaguchi University Hospital from November 1998 to July 2007 were evaluated retrospectively from their medical records. Patients who did not undergo postoperative follow-up examination for at least 6 months were excluded. The mean number of prior surgeries was 2.9 (range, 2 to 10), and the mean +/- SD follow-up time was 42.0 +/- 29.6 months. RESULTS: Twenty seven of 29 eyes (93.1%) with recurrent grade 3 pterygium showed a reduction in the size of the new growth after surgery. The symblepharon improved postoperatively in 5 of 15 eyes (33.3%) and restriction of ocular movement improved in 11 of 24 eyes (45.8%). Most recurrences occurred within 6 months after surgery, although one case did not show a recurrence until 2 years postsurgery. CONCLUSION: Conjunctival autograft with amniotic membrane transplantation without the use of mitomycin C reduced the size of the recurrent pterygium. However, 6 of the 16 eyes (37.5%) required subsequent surgery, suggesting that the procedure has limitations. Mitomycin C should therefore possibly be considered for cases with severe symblepharon or restriction of ocular movement. In choosing the surgical procedure for recurrent pterygium, diplopia can be an important indicator of the severity of a symblepharon or restriction of ocular movement.

Expression of semaphorin 3A and its receptors during mouse corneal development.

Semaphorin 3A (Sema3A) functions to guide the growth of neurons during development, with its effects being mediated by receptor complexes composed of neuropilin (Npn) and plexin (Plx) proteins. We have now examined the expression of Sema3A and its receptor components Npn1 and PlxA during development of the mouse cornea. Sema3A and Npn1 showed similar patterns of expression by immunohistofluorescence analysis, with such expression being prominent in the corneal epithelium during both embryonic and postnatal development. In contrast, PlxA was not expressed in the corneal epithelium until after eye opening between postnatal days 12 and 14. Laser capture microdissection followed by reverse transcription and polymerase chain reaction analysis also showed that the abundance of PlxA mRNA in corneal epithelial cells increased significantly during postnatal development, again in association with eye opening. Given that atmospheric oxygen is thought to play a role in corneal epithelial differentiation and maintenance, our results suggest that the up-regulation of PlxA expression in the corneal epithelium during postnatal development is triggered by exposure of the cornea to the atmosphere. Furthermore, the newly expressed PlxA may contribute to the differentiation of corneal epithelial cells by mediating Sema3A signaling.

Sequence specificity of the PHSRN peptide from fibronectin on corneal epithelial migration.

The Pro-His-Ser-Arg-Asn (PHSRN) sequence in fibronectin is a second cell-binding site that synergistically affects with Arg-Gly-Asp. The PHSRN peptide also induces cell invasion and accelerates wound healing. Here, we examined the sequence specificity of PHSRN on corneal epithelial migration using various synthetic peptides. Elongation and deletion analyses of Ac-PHSRN-NH(2) suggest that the five amino acid length was a minimum and essential sequence for promotion of rabbit corneal epithelial migration ex vivo. Additionally, alanine substituted analysis indicated that the Ser- and Arg-residues are critical for the biological activities. The Ser-Arg motif is involved in various biologically active peptides, suggesting that the unique sequence interacts with cellular receptor(s) and regulates biological functions. Further, the N-acetyl and C-amide of Ac-PHSRN-NH(2) contributed effectively for the chemical stability in tears. The Ac-PHSRN-NH(2) peptide has potential to use as a therapeutic reagent especially for corneal wound healing.

In vivo observation of Langerhans cells by laser confocal microscopy in Thygeson's superficial punctate keratitis.

PURPOSE: To characterize the cornea of individuals with Thygeson's superficial punctate keratitis (TSPK) at the cellular level by laser confocal biomicroscopy. METHODS: Both corneas of three patients with TSPK referred to Yamaguchi University Hospital were imaged with a laser confocal biomicroscope. Morphological changes were evaluated for each layer of the cornea. RESULTS: The number of Langerhans cells was greatly increased in the basal cell layer of the focal corneal epithelium and in Bowman's layer in the four eyes affected by TSPK. Aggregates of these cells were associated with the subepithelial nerve plexus. Langerhans cells were also evident in the unaffected eyes of the two patients with unilateral TSPK, although their numbers were much smaller than those in the affected eyes. Topical treatment with betamethasone phosphate resulted in the virtual disappearance of Langerhans cells from the affected eyes. CONCLUSION: The prominent association of Langerhans cells with TSPK suggests that the activation of these cells by inflammatory conditions might contribute to the pathogenesis of this disorder.

Noninvasive direct detection of ocular mucositis by in vivo confocal microscopy in patients treated with S-1.

PURPOSE: S-1 is an oral antineoplastic agent that contains a prodrug of 5-fluorouracil and has adverse effects on skin, alimentary tract mucosa, and the ocular surface. We investigated the effects of S-1 on the corneal epithelium by in vivo confocal microscopy and histopathologic analysis. METHODS: Twelve patients with eye problems related to S-1 treatment participated in the study. Twenty eyes of ten subjects were evaluated by laser-scanning confocal microscopy. Corneal epithelial debridement as a diagnostic therapy and histopathologic analysis were performed for five eyes of three subjects affected in the pupillary zone of the cornea. RESULTS: Slitlamp examination revealed a local limbal abnormality characterized by epithelial invasion toward the center of the cornea in all 24 eyes. In vivo confocal microscopy revealed an altered structure of the corneal epithelium with abnormal epithelial cells and inflammation. One of five specimens subjected to cytologic diagnosis showed moderate dysplasia. Hematoxylin and eosin staining showed that each abnormal epithelial sheet lacked the stratified structure of the normal corneal epithelium. Immunofluorescence analysis revealed the presence of cells positive for one, both, or neither of cytokeratins 12 and 4 in each lesion. CONCLUSIONS: S-1 can induce ocular mucositis with dysplasia, likely affecting cellular functions, including differentiation, of the corneal epithelium. In vivo confocal microscopy allowed the noninvasive detection of cellular changes in the cornea as an adverse effect of S-1 administration.

Double mutation (R124H, N544S) of TGFBI in two sisters with combined expression of Avellino and lattice corneal dystrophies.

PURPOSE: The R124H mutation of the keratoepithelin gene (TGFBI) causes Avellino corneal dystrophy whereas the N544S mutation of this same gene gives rise to lattice corneal dystrophy. We now report two cases with both R124H and N544S mutations of TGFBI. METHODS: Genomic DNA and cDNA were isolated from the proband and family members and were subjected to polymerase chain reaction-mediated amplification of exons 1-17 of TGFBI. The amplification products were directly sequenced. Allele-specific cloning and sequencing were applied to evaluate the compound heterozygous mutation. RESULTS: Molecular genetic analysis revealed that the proband and one sister harbored both a heterozygous CGC-->CAC (Arg-->His) mutation at codon 124 and a heterozygous AAT-->AGT (Asn-->Ser) mutation at codon 544 of TGFBI. Slit-lamp examination revealed multiple granular regions of opacity and lattice lines in the corneal stroma of the proband and her sister with the double mutation. Allele-specific cloning and sequencing revealed that the R124H and N544S mutations are on different chromosomes. CONCLUSIONS: As far as we are aware, this is the first report of a patient with a double mutation (R124H, N544S) of TGFBI causing an autosomal dominant form of corneal dystrophy. The clinical manifestations of the two cases with both R124H and N544S mutations appeared to be a summation of Avellino and lattice corneal dystrophies.

[Effect of preservative-free betamethasone sodium phosphate eyedrops on the development of corneal epithelial disorders after penetrating keratoplasty].

PURPOSE: To investigate the effects of preservative-free eyedrops containing betamethasone sodium phosphate (BSP) on the development of corneal epithelial disorders after penetrating keratoplasty (PKP). METHODS: The records of 48 patients who underwent unilateral PKP and were followed for 6 months postoperatively were reviewed retrospectively. Twenty-four patients were treated with BSP eyedrops containing preservatives and the other 24 patients with preservative-free BSP eyedrops four times daily. Patients in the two groups were matched for age and sex. Corneal epithelial disorders and other complications were examined with a slitlamp biomicroscope at 1, 3, and 6 months after surgery. RESULTS: None of the patients developed corneal erosion or corneal epithelial defects. The incidence of superficial punctate keratopathy in the preservative-free group (2 of 24 eyes) was significantly reduced compared with that in the preservative group (9 of 24 eyes) 1 month after surgery. The SPK score in the preservative-free group (0.17 +/- 0.56 points) was also significantly smaller than that in the preservative group (1.04 +/- 1.46 points) at 1 month. There was no significant difference in other postoperative complications between the two groups. CONCLUSIONS: Preservative-free BSP eyedrops are effective for preventing the development of corneal epithelial disorders after PKP.

Induction by latanoprost of collagen gel contraction mediated by human tenon fibroblasts: role of intracellular signaling molecules.

PURPOSE: The outcome of glaucoma filtration surgery is affected by subconjunctival wound healing. The effects of the antiglaucoma drug latanoprost on the contractility of human Tenon fibroblasts (HTFs) cultured in a three-dimensional collagen gel were investigated. METHODS: HTFs were cultured in a type I collagen gel with latanoprost or various inhibitors of intracellular signaling. Collagen gel contraction was evaluated by measurement of gel diameter, and collagen degradation was determined by measurement of the amount of hydroxyproline generated by acid-heat hydrolysis of culture supernatants. Phosphorylation of mitogen-activated protein kinases (MAPKs), focal adhesion kinase (FAK), and myosin light chain (MLC) was assessed by immunoblot analysis, and the formation of actin stress fibers was examined by laser confocal microscopy. RESULTS: HTF-mediated collagen gel contraction was stimulated by latanoprost in a concentration- and time-dependent manner. Latanoprost had no effect on collagen degradation by HTFs. Latanoprost induced phosphorylation of MAPKs (ERK, p38, and JNK) and FAK, as well as the formation of stress fibers in HTFs. Furthermore, latanoprost-induced collagen gel contraction was reduced by inhibitors of ERK (PD98059 and ERK inhibitor II), p38 (SB203580), JNK (JNK inhibitor II), Rho-associated kinase (Y27632), phospholipase C (U73122), and MLC kinase (ML-7). CONCLUSIONS: Latanoprost induced collagen gel contraction mediated by HTFs. This action of latanoprost appeared to depend on the formation of stress fibers and the activation of MAPKs, FAK, Rho-associated kinase, phospholipase C, and MLC kinase in HTFs. Latanoprost may therefore influence subconjunctival wound healing by affecting the contractility of Tenon fibroblasts.

Upregulation of tight-junctional proteins in corneal epithelial cells by corneal fibroblasts in collagen vitrigel cultures.

PURPOSE: To investigate the effects of corneal fibroblasts on the differentiation of corneal epithelial cells in a coculture system based on a collagen vitrigel membrane. METHODS: Simian virus 40-transformed human corneal epithelial (HCE) cells and human corneal fibroblasts were cultured on opposite sides of a collagen vitrigel membrane. The distribution of HCE cells and corneal fibroblasts on the collagen membrane was determined by immunofluorescence staining and immunoblot analysis of marker proteins. Expression of the tight-junctional proteins ZO-1, occludin, and claudin and of the adherens-junctional proteins E- and N-cadherin in HCE cells was determined at the mRNA and protein levels by reverse transcription-polymerase chain reaction analysis and immunoblot analysis, respectively. RESULTS: The abundance of ZO-1, occludin, and claudin mRNA and proteins in HCE cells was markedly increased by coculture with corneal fibroblasts. The expression of E- or N-cadherin did not differ between HCE cells cultured with corneal fibroblasts and those cultured without them. PD98059, a specific inhibitor of signaling by extracellular signal regulated kinase (ERK), prevented the upregulation of tight-junctional proteins in HCE cells by corneal fibroblasts. CONCLUSIONS: Human corneal fibroblasts regulated the expression of tight-junctional proteins in HCE cells, suggesting that corneal fibroblasts may play an important role in the differentiation of corneal epithelial cells.