Identification of the gene responsible for gelatinous drop-like corneal dystrophy.

Gelatinous drop-like corneal dystrophy (GDLD; OMIM 204870) is an autosomal recessive disorder characterized by severe corneal amyloidosis leading to blindness, with an incidence of 1 in 300,000 in Japan. Our previous genetic linkage study localized the gene responsible to a 2.6-cM interval on chromosome 1p. Clinical manifestations, which appear in the first decade of life, include blurred vision, photophobia and foreign-body sensation. By the third decade, raised, yellowish-grey, gelatinous masses severely impair visual acuity, and lamellar keratoplasty is required for most patients. Here we report DNA sequencing, cDNA cloning and mutational analyses of four deleterious mutations (Q118X, 632delA, Q207X and S170X) in M1S1 (formerly TROP2 and GA733-1), encoding a gastrointestinal tumour-associated antigen. The Q118X mutation was the most common alteration in the GDLD patients examined, accounting for 33 of 40 (82.5%) disease alleles in our panel of families. Protein expression analysis revealed aggregation of the mutated, truncated protein in the perinuclear region, whereas the normal protein was distributed diffusely in the cytoplasm with a homogenous or fine granular pattern. Our successful identification of the gene that is defective in GDLD should facilitate genetic diagnosis and potentially treatment of the disease, and enhance general understanding of the mechanisms of amyloidosis.

Differential expression of MUC16 in human oral mucosal epithelium and cultivated epithelial sheets.

Cultivated oral mucosal epithelial sheet transplantation is a new surgical strategy to treat severe ocular surface disorders such as chemical burns, ocular cicatricial pemphigoid, and Stevens-Johnson syndrome. MUC16 is thought to be the most important membrane-associated mucin on the ocular surface because it forms a protective barrier on the epithelial cell surface. In this study, we studied MUC16 expression in mRNA and protein levels and compared the expression patterns between cultivated oral mucosal epithelial cell sheet and oral mucosal tissue. Specimens (5x5 mm) of oral mucosal tissue harvested from healthy volunteers were used. The oral mucosal epithelial cells were cultured on temperature-responsive culture dishes to generate stratified cell sheets. Cultivated oral mucosal epithelial cells formed three- to five-cell thick stratified sheets for 2 weeks. Scanning electron micrographs revealed that the apical surfaces of the oral mucosal tissue and the oral mucosal sheets were covered with dense microvilli/microplicae. Real-time PCR showed significantly more MUC16 transcripts in the cultivated oral mucosal sheets and corneal epithelial sheets than in the oral mucosal tissue (P=0.023 and 0.008, respectively, Mann-Whitney rank sum test). These findings were confirmed by immunohistochemical examination using an MUC16 antibody to the protein. MUC16 protein was localized to the apical cells of the oral mucosal sheets, but the human oral mucosal tissue did not express MUC16 protein in any cell layers. In this study, interestingly, the expression of membrane-associated mucin MUC16 differs between human oral mucosal epithelia and cultivated epithelial sheets. MUC16 expressed in the oral mucosal sheets may contribute to ocular surface reconstruction after oral mucosal sheet transplantation.

Laboratory investigation of microelectronics-based stimulators for large-scale suprachoroidal transretinal stimulation (STS).

This paper describes the technological developments underlying the realization of a reliable and reproducible microchip-based stimulator with a large number of stimulus electrodes. A microchip-based stimulator with over 500 electrodes for suprachoroidal transretinal stimulation (STS) is proposed in this paper, and an example is presented. To enhance reliability and reproducibility for such a large array, we introduce a flip-chip bonding technique and place microchips on the reverse side of a substrate. A square microchip of size 600 microm was fabricated using 0.35 microm standard CMOS process technology. Twelve microchips were flip-chip bonded on a polyimide substrate through Au bumps. To evaluate the feasibility of the proposed device, we successfully fabricated a stimulator with 12 microchips and 118 electrodes made of Pt/Au bumps, and demonstrated their operation in a saline solution for 2 weeks. Also, to evaluate the device operation in vivo, a stimulator with one active IrO(x) electrode was implanted into the scleral pocket of a rabbit and electrical evoked potential (EEP) signals with a threshold of 100 microA were obtained. We also fabricated a simulator with 64 microchips that has 576 electrodes (9 electrodes in a microchip times 64 microchips).

Pars plana vitrectomy with peripheral retinotomy after injection of preoperative intravitreal tissue plasminogen activator: a modified procedure to drain massive subretinal haemorrhage.

AIMS: To report outcome of a modified procedure for draining massive subretinal haemorrhages (SRHs). METHODS: The charts of eight consecutive eyes from eight patients with massive SRHs extending to the periphery and involving two or more quadrants with haemorrhagic and bullous retinal detachment were reviewed. Tissue plasminogen activator (tPA) was injected intravitreally 12-24 h preoperatively; vitrectomy was carried out with peripheral retinotomy, drainage of the SRH from the retinotomy using perfluorocarbon liquid and gas tamponade with prone positioning postoperatively. RESULTS: The preoperative visual acuities ranged from light perception to 20/200. Most of the subretinal haematomas moved postoperatively to the vitreous cavity through the peripheral retinotomy using perfluorocarbon liquid. Residual SRHs were drained from the anterior chamber at the bedside after prone positioning overnight. SRH recurred in one eye 14 months postoperatively and was successfully retreated. No other serious complications developed. The final visual acuity improved in seven eyes (range 20/1000-20/60). Polypoidal lesions in choroidal vasculatures were present in three of seven patients. CONCLUSIONS: The technique seems safe and effective for treating massive SRH. However, visual recovery is limited by the underlying macular pathology. Polypoidal choroidal vasculopathy, other than age-related macular degeneration, may be another cause of massive SRHs.

Intravitreal injection of bevacizumab for choroidal neovascularisation associated with pathological myopia.

AIM: To assess the efficacy and safety of an intravitreal injection of bevacizumab (Avastin(R)) for myopic choroidal neovascularisation (mCNV). METHODS: Intravitreal bevacizumab (1 mg) was injected into eight eyes of eight patients with mCNV in this non-randomised, interventional case series. The best-corrected visual acuity (BCVA) was measured and the optical coherence tomography (OCT) and fluorescein angiography findings were examined before and after treatment. The minimum follow-up time was 3 months. RESULTS: The mean BCVA was 0.26 before treatment and 0.51 at the last visit (p = 0.009). The BCVA improved to two or more lines in six eyes (75%) and remained the same in two eyes (25%). Leakage from the mCNV on fluorescein angiography decreased in seven eyes (87.5%). The choroidal neovascularisation area on fluorescein angiography (p = 0.049) and the foveal thickness on OCT images decreased significantly (p = 0.027) after the treatment. No major complications developed. CONCLUSION: Intravitreal injection of bevacizumab seems to be an effective and safe treatment for mCNV.

Induction of hepatocyte growth factor in fibroblasts by tumor-derived factors affects invasive growth of tumor cells: in vitro analysis of tumor-stromal interactions.

Invasive and metastatic potentials of several types of carcinoma cells are regulated through interactions with host stromal cells, e.g., tumor-stromal interactions. Because hepatocyte growth factor (HGF), a ligand for the c-Met proto-oncogene product, is a mesenchymal- or stromal-derived factor that induces mitogenic, motogenic, and morphogenic responses, we examined the mechanisms involved in tumor-stromal interactions in vitro. The c-Met/HGF receptor was expressed in A431 human epidermoid carcinoma cells, A549 human non-small cell lung cancer cells, HuCC-T1 human cholangiocellular carcinoma cells, and SBC-3 human small cell lung carcinoma cells. HGF stimulated cell growth, scattering, and invasion of these cells. Although these cells did not produce biologically significant levels of HGF, these cells did secrete soluble factors that potently stimulated HGF production in human skin fibroblasts. These carcinoma cell-derived HGF inducers proved to be interleukin-1 (IL-1) in A431 cells, IL-1 plus basic fibroblast growth factor (bFGF) in A549 and HuCC-T1 cells, and bFGF plus platelet-derived growth factor in SBC-3 cells. When these carcinoma cells were cocultured with fibroblasts, HGF levels in the coculture system were much higher than the levels in fibroblasts alone, without cocultured carcinoma cells. Together with the increase in HGF levels, the number of invasive cells increased, but in vitro invasion of carcinoma cells in the coculture system was strongly inhibited by anti-HGF antibodies. Thus, there are mutual interactions between carcinoma cells and fibroblasts: IL-1, bFGF, and platelet-derived growth factor derived from tumor cells play a role in inducing HGF expression in stromal fibroblasts, whereas fibroblast-derived HGF, in turn, leads to invasive growth in carcinoma cells. The mutual interactions, as mediated by HGF and HGF inducers, may play a significant role in the occurrence of invasion and metastasis of carcinoma cells.

Direct penetration of spin-triplet superconductivity into a ferromagnet in Au/SrRuO3/Sr2RuO4 junctions.

Efforts have been ongoing to establish superconducting spintronics utilizing ferromagnet/superconductor heterostructures. Previously reported devices are based on spin-singlet superconductors (SSCs), where the spin degree of freedom is lost. Spin-polarized supercurrent induction in ferromagnetic metals (FMs) is achieved even with SSCs, but only with the aid of interfacial complex magnetic structures, which severely affect information imprinted to the electron spin. Use of spin-triplet superconductors (TSCs) with spin-polarizable Cooper pairs potentially overcomes this difficulty and further leads to novel functionalities. Here, we report spin-triplet superconductivity induction into a FM SrRuO3 from a leading TSC candidate Sr2RuO4, by fabricating microscopic devices using an epitaxial SrRuO3/Sr2RuO4 hybrid. The differential conductance, exhibiting Andreev-reflection features with multiple energy scales up to around half tesla, indicates the penetration of superconductivity over a considerable distance of 15 nm across the SrRuO3 layer without help of interfacial complex magnetism. This demonstrates potential utility of FM/TSC devices for superspintronics.

Molecular cloning of a novel membrane glycoprotein, pal, specifically expressed in photoreceptor cells of the retina and containing leucine-rich repeat.

We have isolated a novel retina-specific gene in a screen for genes of which expression is not apparent neonatally in rat retina but is abundant postnatally on day 14 (P14). This gene, named Pal, encodes a putative type I transmembrane protein containing five leucine-rich repeats (LRRs), a single C2-type Ig-like domain, and a single fibronectin type III domain and is considered to be a new member of the LRR and Ig superfamily. No expression of Pal was found in rat retina at P1, but it was detected at P7 and markedly increased with subsequent development. These expression patterns of Pal appeared to be correlated with the development of the photoreceptor outer segments, because in the adult rat retina it was specifically localized in these segments. Ultrastructually, Pal immunoreactivity was distributed diffusely on the disk membrane in the lamellar regions. On the basis of its structural features and localization pattern, Pal may act as a receptor for a certain trophic factor or for an adhesion molecule participating in morphogenesis. The human homolog of Pal was mapped to chromosome 10q23.2-23.3 using fluorescence in situ hybridization.

Choroidal neovascularisation in pathological myopia: an update in management.

Choroidal neovascularisation (CNV) secondary to pathological myopia is an important cause of significant visual impairment in young and middle aged adults globally and is particularly prevalent in Asian populations. In the past few years, there have been rapid advancements in the different treatments for myopic CNV. The purpose of this perspective is to give an overview of the natural history of myopic CNV and the various treatment options including laser photocoagulation, photodynamic therapy, sub-macular surgery, and macular translocation surgery. Future directions in the management of myopic CNV are also discussed.

Comparison of two methods for evaluating the quality of stored drinking water in Abidjan, Côte d'lvoire, and review of other comparisons in the literature.

Membrane filtration, multiple tube fermentation (the standard methods) and Colilert are techniques available for assessing drinking water quality, but there are no published comparisons of Colilert to standard methods in a developing country laboratory. We reviewed the published literature on Colilert and standard methods and conducted a study to compare Colilert with membrane filtration for the detection and enumeration of total coliforms and fecal coliforms (Escherichia coli bacteria) using 35 stored drinking water samples from households in Abidjan, Côte d'lvoire. Our study results are consistent with previous published studies conducted in developed countries. Results from Colilert and membrane filtration correlated for both total coliforms (r2 = 0.81) and E. coli (r2 = 0.93). Colilert is an acceptable method to measure the presence and quantity of coliforms in water samples in a developing country setting.

Isolation of a cDNA encoding a photoreceptor cell-specific actin-bundling protein: retinal fascin.

We have isolated a novel retina-specific gene, retinal fascin, encoding a new member of actin-bundling protein gene family, from a bovine retina cDNA library. The cDNA encodes a 492 amino acid protein which shows 36-57% amino acid identity with three vertebrate fascins, echinoid fascin and Drosophila singed gene. Northern blot analysis revealed that retinal fascin mRNA was exclusively expressed in the eye and not seen in other tissues examined. In situ hybridization analysis indicated that retinal fascin mRNA signals were found only in the inner segment of the photoreceptor layer and outer nuclear layer, indicating that retinal fascin was specifically expressed in photoreceptor cells. As fascins are actin-bundling proteins important for constructing several intracellular structures, retinal fascin might play a pivotal role in photoreceptor cell-specific events, such as disk morphogenesis.

Specific detections of the early process of the glycation reaction by fructose and glucose in diabetic rat lens.

The glycation reaction by fructose, as well as that by glucose, in control and diabetic rat lens was analyzed by using antibodies which specifically recognize adducts of lysine with fructose and with glucose. Levels of fructose adducts in diabetic rat lens were 2.5 times that of the control, and correlated with sorbitol levels. This was mainly due to enhanced glycation of beta- and gamma-crystallins by fructose under diabetic conditions. These data suggest that glycation by fructose may also play a role in cataract formation under conditions of diabetes and aging.

Rapid detection of M1S1 mutations by the protein truncation test.

PURPOSE: To determine a method of rapid detection of M1S1 gene mutations in patients with gelatinous droplike corneal dystrophy. METHODS: Forty-one patients from 35 families with gelatinous drop-like corneal dystrophy were studied. The entire coding region of the M1S1 gene was screened using the protein truncation test (PYT), with a polymerase chain reaction fragment amplified from genomic DNA serving as a template of in vitro translation. RESULTS: Homozygous or compound heterozygous mutations were detected in all patients by a single reaction of the PTT. This result matched those obtained using the polymerase chain reaction-restriction fragment length polymorphism and direct sequence analyses. The Q118X mutation was present in 63 of the 70 alleles, accounting for 90% of the disease-associated chromosomes in Japanese patients. CONCLUSIONS: The PTT is useful for detecting mutations in the M1S1 gene. This technique showed that the Q118X mutation is a founder mutation in Japanese patients with gelatinous droplike corneal dystrophy, and it reflects the linkage disequilibrium reported previously.

Inhibitory effects of neurocan and phosphacan on neurite outgrowth from retinal ganglion cells in culture.

PURPOSE: Neurocan and phosphacan are nervous tissue-specific chondroitin sulfate proteoglycans (CSPGs) that are highly expressed in postnatal rat retina. To elucidate potential roles of neurocan and phosphacan on neurite outgrowth from retinal ganglion cells (RGCs), in vitro experiments were conducted with purified RGCs. METHODS: Neurocan and phosphacan were purified from postnatal rat brain by DEAE-column chromatography and subsequent gel chromatography. RGCs were obtained from postnatal rat retinas by a two-step immunopanning procedure using an anti-Thy 1,1 antibody and an anti-macrophage antibody. Neurite outgrowth from RGCs was examined on poly-L-lysine (PLL)-conditioned plates, and PLL-conditioned plates treated with neurocan or phosphacan. RESULTS: Compared with PLL-conditioned plates, neurocan and phosphacan inhibited neurite outgrowth from RGCs at 48 and 72 hours after seeding. When chondroitin sulfate side chains linked to the core proteins were digested by chondroitinase ABC, the inhibitory effect remained, indicating that the core proteins are related to the effect. Furthermore, the digestion of chondroitin sulfate side chains linked to phosphacan core protein significantly promoted the inhibitory effect of phosphacan on neurite outgrowth from RGCs. CONCLUSIONS: Neurocan and phosphacan, which are highly expressed in postnatal rat retina, inhibit neurite outgrowth from postnatal rat RGCs, indicating that these proteoglycans may be inhibitory factors against neurite outgrowth from RGCs during retinal development.

Mutations in the 11-cis retinol dehydrogenase gene in Japanese patients with Fundus albipunctatus.

PURPOSE: To detect mutations in the RDH5 gene encoding 11-cis retinol dehydrogenase in patients from Japan with fundus albipunctatus. METHODS: Polymerase chain reaction and direct genomic sequencing techniques were used to detect mutations of the RDH5 coding exons (exons 2-5) in two unrelated patients with fundus albipunctatus. Selected alleles that altered the coding region or intron splice sites were evaluated further through segregation analysis in the families of the index cases. RESULTS: Two novel RDH5 mutations were identified. One of these was a missense mutation Val264Gly in exon 5, and the other was an in-frame insertion of 3 bp in exon 5. CONCLUSIONS: The data indicate that mutations in RDH5 are the primary cause of fundus albipunctatus.

Retinal changes after retinal translocation surgery with scleral imbrication in dog eyes.

PURPOSE: To examine retinal changes induced by scleral imbrication during retinal translocation surgery in dog eyes. METHODS: Fifteen dogs were anesthetized and underwent retinal translocation surgery. After lensectomy and vitrectomy, an intentional retinal detachment was created, and the upper temporal sclera around the equator was imbricated with five mattress sutures. Translocated distances were calculated by pre- and postoperative photographs. At 1, 2, and 4 weeks after the surgery, the retina was studied by TdT-dNTP terminal nick-end labeling (TUNEL) and immunohistochemistry of peanut agglutinin (PNA) lectin and glial fibrillary acidic protein (GFAP). RESULTS: The retina was translocated by a mean distance of 0.53 +/- 0.30 disc diameters or 959 +/- 543 micrometer. Retinal folds were created around the optic disc in all eyes. Histologic examination of the retinal folds 1 week after the surgery showed many TUNEL-positive cells in the outer nuclear layer, loss of photoreceptor cells, and shortening of the outer and inner segments. A strong immunoreactivity to GFAP was detected in the folds of the retina. CONCLUSIONS: . The results demonstrated that retinal translocation surgery by scleral imbrication inevitably caused retinal folds as a postoperative complication, and the retina within the folds showed extensive loss of photoreceptor cells. It is recommended that the foveal translocation surgery be planned to avoid involving the fovea in the retinal folds.

Correlation of anterior chamber-associated immune deviation with suppression of corneal epithelial rejection in mice.

PURPOSE: The authors investigated the effect of anterior chamber corneal (AC) inoculation of genetically graft-identical antigen on T-cell immunity and the suppression of alloepithelial rejection in mice. METHODS: Antigen-specific suppression of delayed-type hypersensitivity (DTH) and suppression transferability were tested in BALB/c mice injected with irradiated allogeneic B10.D2 splenocytes into AC. Other groups of BALB/c mice received irradiated B10.D2, BALB/c, or C3H/He splenocytes in the AC of the right eye. Seven days later, B10.D2 or C3H/He corneal lenticules were grafted at the limbus of the left eye (keratoepithelioplasty). Alloepithelial rejection of each grafted eye was evaluated according to clinical findings. The DTH response of the keratoepithelioplasty recipients against B10.D2 minor antigen was tested at the end of clinical observation (4 months after grafting). Also examined was spleen component transfer from BALB/c mice with AC inoculation of B10.D2 splenocytes to syngeneic acceptors and its effect on suppression of epithelial rejection against B10.D2 antigen. RESULTS: Inoculation of B10.D2 splenocytes into BALB/c AC induced antigen-specific DTH suppression, which suppression was transferable. During the 4-month observation period, AC inoculation of B10.D2 minor antigen significantly enhanced the survival of B10.D2-derived epithelium, but not of C3H/He-derived epithelium, in BALB/c mice. However, AC inoculation of BALB/c or C3H/ He splenocytes did not enhance B10.D2 epithelial survival in BALB/c mice. Incapability of antigen-specific DTH response generation was observed in the BALB/c mice with B10.D2 splenocytes in the right AC and B10.D2-derived epithelium in the left eye. Single transfer of spleen components from BALB/c mice with AC inoculation of B10.D2 splenocytes to syngeneic acceptors only delayed B10.D2 minor antigen-stimulated epithelial rejection, whereas supplementary transfers of the identical spleen components at different time intervals showed more significant effect in rejection delay. CONCLUSIONS: The results showed that AC inoculation of B10.D2 splenocytes in BALB/c mice induced antigen-specific suppression of DTH response, in a phenomenon termed anterior chamber-associated immune deviation (ACAID). It also was shown definitely that ACAID can suppress alloepithelial rejection in a murine keratoepithelioplasty model. Adoptive transfer of splenocytes from ACAID-induced mice merely affords short-term suppression of epithelial rejection, suggesting that an additional mechanism may be involved in ACAID maintenance.

The antigen-bearing eye and the spleen are indispensable in maintaining anterior chamber-associated immune deviation.

PURPOSE: To investigate the role of the eye and the spleen in maintaining suppression of delayed-type hypersensitivity (DTH) after anterior chamber (AC) inoculation of allogeneic splenocytes. METHODS: Suppression of DTH response was tested in BALB/c mice after AC inoculation of allogeneic B10.D2 splenocytes. Seven days after AC injection, the antigen-inoculated eyes were enucleated or the spleens were removed. After enculeation or splenectomy at different time intervals, DTH responses in groups of the BALB/c mice were examined. Spleen components obtained from BALB/c mice that had been primed by B10.D2 splenocytes in the AC 7 days earlier were transferred intravenously to groups of naive syngeneic acceptors. At various intervals after adoptive transfer, variations of DTH responses were tested. RESULTS: Inoculation of B10.D2 splenocytes to the AC of BALB/c mice induced antigen-specific suppression of DTH. Either enucleation of the antigen-inoculated eyes or splenectomy weakened the DTH-suppressive effect within 5 weeks and abolished it within 9 weeks, whereas the mice retaining both antigen-inoculated eyes and spleens maintained longstanding DTH suppression. Adoptive transfer of spleen components to syngeneic acceptors demonstrated DTH suppression for only 3 weeks. CONCLUSIONS: The antigen-inoculated eye and spleen are required for long-standing suppression of DTH after AC inoculation of allogeneic splenocytes.

Ocular resurfacing and alloepithelial rejection in a murine keratoepithelioplasty model.

PURPOSE: To determine definitively the epithelial origin of corneal resurfacing and to elucidate the immunologic mechanisms of epithelial rejection in a murine keratoepithelioplasty (KEP) model. METHODS: After corneal epithelial removal and peritomy, donor corneal lenticules were grafted around the limbus (KEP procedure). The process of corneal reepithelialization was observed with 0.25% methylene blue staining. The origin of the renewed epithelium was determined by immunofluorescence. Syngeneic corneal lenticules were grafted to BALB/c mice. C3H/He, C57BL/6, BALB.K, DBA/2, and B10.D2 allogeneic corneal lenticules were grafted to BALB/c mice, and A.SW and A.TL allogeneic corneal lenticules were grafted to A.TH mice. Alloepithelial rejection was evaluated on the basis of clinical findings and histologic changes in grafted corneas. RESULTS: All KEP grafts were reepithelialized entirely at 3 days after surgery. The renewed epithelium proved to be derived from the lenticules in BALB/c eyes receiving C3H/He lenticules. In syngeneic grafts 5 days after KEP, the cornea recovered clarity and smoothness, which persisted to the end of the study. After complete reepithelialization, all allogeneic grafts also experienced a short duration of clear cornea. Then followed four characteristic phases of inflammatory epithelial response: initial phase, acute phase, chronic phase, and rejected phase. Histologic examination confirmed the progress and severity of inflammatory response. The mean onset times of initial phase in assorted grafts with mismatched histocompatibility antigens were: 7.9 +/- 1.8 days for both major and minor disparity grafts, 9.5 +/- 3.8 days for major disparity grafts, 18.2 +/- 5.5 days for major histocompatibility class I disparity grafts, 25.6 +/- 7.2 days for major histocompatibility class II disparity grafts, and 9.2 +/- 2.2 days for multiple minor disparity grafts. CONCLUSIONS: In donor corneal lenticule grafting to host eyes with corneal epithelium removed and conjunctival peritomy, the ocular surface was reepithelialized by lenticule-derived epithelium. Alloepithelial rejection in this model displayed characteristic manifestations and well-defined processes, enabling easy and precise evaluation of onset and intensity of graft rejection. Both major and minor histocompatibility antigens are related to corneal epithelial rejection.

An SV40-immortalized human corneal epithelial cell line and its characterization.

PURPOSE: The authors attempted to immortalize human corneal epithelial cells; it is difficult to propagate primary human corneal epithelial cells because of scarcity of available tissue. However, cell immortalization by virus is always accompanied by shedding of free virus. The current study was performed to establish a cell line that produces no free viral particle. METHODS: Primary cultured human corneal epithelial cells were infected with a recombinant sv40-adenovirus vector and were cloned three times to obtain a continuously growing cell line. Morphologic, cytologic, and biochemical characteristics of this cell line were analyzed. RESULTS: This cell line continued to grow for more than 400 generations, exhibiting a cobblestone-like appearance similar to normal corneal epithelial cells in culture. Transmission electron microscopy showed the evidence for the characteristic features of epithelial cells, including desmosome formation and development of microvilli. It expressed cornea-specific, 64-kD cytokeratin in addition to five major insoluble proteins. By enzymatic analysis using NADP as a coenzyme and a gas chromatograph mass spectrometer, this cell line was found to possess 8.71 IU/mg protein of aldehydedehydrogenase activity. When this cell line was grown at air-liquid interface on collagen type I gel, it differentiated in a multilayered fashion. CONCLUSIONS: The authors have established an SV40-immortalized human corneal epithelial cell line with properties similar to normal corneal epithelial cells.