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Koji Hirano,1 Hidenori Tanaka,2 Kumiko Kato,3 Kaoru Araki-Sasaki4 1Department of Ophthalmology, Fujita Health University Bantane Hospital, Nagoya, Aichi, 454-8509, Japan; 2Department of Ophthalmology, Fujita Health University School of Medicine, Toyoake, Aichi, 470-1192, Japan; 3Department of Ophthalmology, Mie University Graduate School of Medicine, Tsu, Mie, 514-8507, Japan; 4Department of Ophthalmology, Kansai Medical University, Hirakata, Osaka, 573-1010, JapanCorrespondence: Koji HiranoDepartment of Ophthalmology, Fujita Health University Bantane Hospital, 6-10, Otoubashi 3-cho-me, Nakagawa-ku, Nagoya, Aichi, 454-8509, JapanTel +81-52-321-8171Fax +81-52-322-4734Email kojihira@fujita-hu.ac.jpPurpose: In clinical practice we sometimes encounter patients with severe corneal ulcers who have been treated with topical corticosteroids. This study reviewed the clinical features and visual outcomes of these patients and investigated the background of the prescription of topical corticosteroids.Patients and Methods: The medical records of patients who visited the Cornea Service at Fujita Health University Bantane Hospital and were treated for infectious keratitis from April 2016 to March 2020 were retrospectively reviewed. Patients treated with topical corticosteroids before a culture-proven diagnosis were studied in terms of demographics, best-corrected visual acuity at arrival and at last visit, the clinical course after visit, ocular history, and combination therapy by the previous ophthalmologist.Results: Out of the 200 eyes of 197 patients with infectious keratitis, 14 eyes of 14 patients were treated with topical corticosteroids before a culture-proven diagnosis. All 14 patients were referred, as they had severe keratitis that could not be cured with topical antibiotics and corticosteroids. Based on the culture results, we diagnosed Acanthamoeba keratitis (AK) in six patients, fungal keratitis (FK) in two patients, bacterial keratitis (including a suspected case) in two patients, and unknown cause in four patients. Two patients with AK, FK, and unknown keratitis had unfortunate clinical courses and poor visual outcomes. From the information in the referral letters, at least six of the 14 patients were treated with either acyclovir ocular ointment or valaciclovir tablets, along with topical corticosteroids.Conclusion: Application of topical corticosteroids for keratitis that does not respond to empirical antibiotic therapy is harmful since AK or FK is likely involved in these topical antibiotic-resistant cases. Microbiological evidence, as well as a differential diagnosis of herpetic stromal keratitis, is needed when prescribing topical corticosteroid for the treatment of suspected infectious keratitis.Keywords: culture-proven diagnosis, empiric antibiotic therapy, herpetic keratitis, infectious keratitis, topical corticosteroids

Koji Hirano,1 Hidenori Tanaka,1,2 Kumiko Kato,3 Kaoru Araki-Sasaki4 1Department of Ophthalmology, Ban Buntane Hotokukai Hospital, School of Medicine, Fujita Health University, Nagoya, Aichi 454-8509, Japan; 2Department of Ophthalmology, School of Medicine, Fujita Health University, Toyoake, Aichi 470-1192, Japan; 3Department of Ophthalmology, Mie University School of Medicine, Tsu, Mie 514-8507, Japan; 4Department of Ophthalmology, Japan Community Health Care Organization Hoshigaoka Medical Center, Hirakata, Osaka 573-8511, Japan Background: Deep anterior lamellar keratoplasty (DALK) is indicated to correct high astigmatism in patients with keratoconus (KC) and no evidence of Descemet’s membrane rupture. However, some patients with KC experience graft rejection-like inflammatory reactions within 2 months (usually in the first week) after DALK. The aim of this study was to identify the characteristics and influencing factors of these reactions by reviewing the records of patients who underwent DALK or penetrating keratoplasty (PKP) for KC or other corneal problems.Methods: We retrospectively reviewed the medical records of patients who underwent DALK for KC (DALK/KC), PKP for KC (PKP/KC), or DALK for other corneal problems (DALK/non-KC) at Ban Buntane Hotokukai Hospital between January 2006 and December 2015 and who were followed for more than 1 year. We collected data on the characteristics and incidence of severe inflammatory graft reactions in the early postoperative phase (ie, within 2 months after keratoplasty) and visual outcomes after these inflammatory reactions.Results: Postoperative inflammatory reactions characterized by persistent epithelial defects, loose suture with infiltration, and vessel invasion occurred in seven of eleven DALK/KC patients, three of 50 DALK/non-KC patients, and none of five PKP/KC patients. These reactions were nonresponsive to topical steroids, and suture removal was required. Although a clear corneal graft in the pupillary area was obtained and best-corrected visual acuity was good after the resolution of inflammation, a risk of corneal astigmatism remained.Conclusion: The incidence of these reactions in the early postoperative period is high after DALK for KC. These reactions do not respond well to topical steroids, and suture removal may be required, which may cause high astigmatism after the inflammation subsides. Lamellar keratoplasty should be considered carefully for patients with KC. Keywords: deep anterior lamellar keratoplasty, keratoconus, stromal rejection, graft rejection-like reactions, early postoperative period, secondary corneal amyloidosis

Kaoru Araki-Sasaki,1 Yasuhiro Osakabe,2 Koji Fujita,2 Kazunori Miyata,3 Koji Hirano4 1Department of Ophthalmology, Japan Community Health Care Organization, Hoshigaoka Medical Center, Hirakata, Japan; 2Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan; 3Department of Ophthalmology, Miyata Eye Hospital, Miyakonojo, Japan; 4Department of Ophthalmology, Ban Buntane Hotokukai Hospital, Fujita Health University, Nagoya, Japan Abstract: We describe the histological changes in the collagen fibers of a 50-year-old male who presented keratoconus with secondary corneal amyloidosis. Corneal tissue from the patient was obtained following a penetrating keratoplasty and was subjected to histochemical analysis using Masson’s trichrome staining, Congo red staining, anti-lactoferrin antibody, and anti-transforming growth factor-beta-induced protein (TGFBIp) antibody. A Congo red-positive region was detected in the anterior half of the stroma in the center and inferior cornea. Although hemotoxylin and eosin staining revealed irregularity in the Congo red-positive region, other parts of the stroma did not show any abnormalities. Positive staining both by anti-TGFBIp and anti-lactoferrin antibodies was observed in the Congo red-positive region. Interestingly, all the layers of the corneal stroma, including the peripheral region, were positively stained by anti-TFGBIp antibody, even in the Congo red-negative area. Masson’s trichrome staining also showed irregular staining throughout the corneal stroma, even outside of the Congo red-positive region. Additionally, Bowman’s layer, which consists of collagen type IV, was damaged. TGFBIp was strongly expressed and Masson’s trichrome staining was reduced throughout the entire keratoconic stroma. The constant qualitative changes in keratoconic collagen fibers, along with the observed abnormality in the Bowman’s membrane, might point to the pathogenesis of secondary corneal amyloidosis in keratoconus. Keywords: cornea, amyloid, stroma, TGFBIp, lactoferrin

Norihiko Tou,1 Ryohei Nejima,2 Yoshifumi Ikeda,3 Yuichi Hori,4 Kaoru Araki-Sasaki,5 Kazunori Miyata,2 Yoshitsugu Inoue,3 Akihiko Tawara1 1Department of Ophthalmology, University of Occupational and Environmental Health, Fukuoka, 2Miyata Eye Hospital, Miyazaki, 3Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, Tottori, 4Department of Ophthalmology, Toho University Sakura Medical Center, Chiba, 5Ideta Eye Hospital, Kumamoto, Japan Purpose: The purpose of this study was to evaluate the clinical utility of SG17, an ophthalmic antimicrobial susceptibility measurement plate.Design: This was a multicenter, retrospective, observational study.Patients and methods: Using clinical isolates from patients with ocular infections, drug susceptibility testing using the Clinical Laboratory and Standards Institute standards was routinely conducted at five facilities. The minimum inhibitory concentrations of the isolated strains were determined using SG17 at the Research Foundation for Microbial Diseases of Osaka University. The records of antimicrobial drugs used and the therapeutic course were evaluated for all cases. The susceptibility results from SG17 and routine methods used at each facility were compared.Results: A total of 112 bacterial strains were isolated from 92 patients. Of these cases, keratitis was the most common (52.2%), followed by conjunctivitis (21.7%) and others (26.1%). Principal signs and symptoms resolved in all patients, indicating that therapeutic effects had been achieved. With SG17, drug susceptibility was determined in 98.9% of isolates compared with 30.4% of isolates determined using conventional methods. By adapting the Clinical Laboratory and Standards Institute standards to SG17 results, we found that 91.3% of patients were susceptible and 7.6% were resistant. In five patients, drugs with a resistant evaluation were initially administered with no effect, and the patients were then switched to drugs with a susceptible evaluation with final resolution, indicating agreement of clinical results with SG17.Conclusion: SG17 can be used to determine drug susceptibility to antimicrobial agents currently used in ophthalmic practice. SG17 is useful for selecting antimicrobial drugs. Keywords: ophthalmic antimicrobial agent, drug susceptibility, minimum inhibitory concentration, Clinical Laboratory and Standards Institute

Kaoru Araki-Sasaki,1 Yasuhiro Osakabe,2 Hideki Fukuoka,3 Ryuichi Ideta,4 Koji Hirano5 1Department of Ophthalmology, Japan Community Health Care Organization, Hoshigaoka Medical Center, Hirakata, 2Department of Molecular Pathology, Tokyo Medical University, Tokyo, 3Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, 4Ideta Eye Hospital, Kumamoto, 5Department of Ophthalmology, Ban Buntane Hotokukai Hospital, School of Medicine, Fujita Health University, Nagoya, Japan Purpose: To describe observations by ultrahigh-resolution optical coherence tomography (OCT) in a secondary corneal amyloidosis (SCA) patient with histological analysis of excised tissue. A unique finding under OCT of her fellow eye is also described.Case: A 39-year-old female had suffered from trichiasis in both of her eyes for more than 30 years. Slit-lamp examination showed a milky-white soft mass on her left cornea and a linear opacity on the fellow cornea at the cilia-attached region. OCT demonstrated the presence of a mass region within a thin epithelial layer and no destruction of Bowman’s layer in her left cornea. In the fellow cornea, which exhibited a linear opacity, a high-density spot in Bowman’s layer was observed at the cilia-attached region covered by the epithelial layer, with normal thickness. Histological examination of the excised cornea showed that the mass was positive with both Congo red and antilactoferrin antibody.Conclusion: SCA, amyloid gradually accumulates above Bowman’s layer, occupying the epithelial layer, with no destruction of Bowman’s layer until the advanced stage. A high-density spot in Bowman’s layer might be the first stage of SCA. Keywords: OCT, secondary corneal amyloidosis, lactoferrin, cornea

Kaoru Araki-Sasaki,1,2 Atsuko Fukumoto,1 Yasuhiro Osakabe,3 Hideya Kimura,1 Shinichiro Kuroda1 1Nagata Eye Clinic, Nara, Japan; 2Department of Ophthalmology, Japan Community Health Care Organization, Hoshigaoka Medical Center, Osaka, Japan; 3Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan Abstract: The purpose of this study was to describe the clinical characteristics of fungal keratitis caused by Candida albicans in an eye after Descemet stripping automated endothelial keratoplasty (DSAEK). A 72-year-old male with a history of three trabeculectomies, cataract surgery, and two DSAEK procedures developed a corneal ulcer in his right eye two years after his last DSAEK. Fungal keratitis was most likely related to the immunosuppressive conditions that occurred due to the previous operations, the continuous use of steroid eye drops, and the use of disposable soft contact lenses. A smear and culture from the ulcer detected Candida albicans. Slit-lamp examination showed the characteristic feature was the presence of interface infiltrates located between the host and the graft cornea and in the enlarged area around the ulcer. Two weeks after intense antimycotic treatments with voriconazole, miconazole, and natamycin, perforation of the cornea occurred and further therapeutic penetrating keratoplasty was required. Histological analysis revealed an accumulation of infiltrated cells and fibrotic tissue. The poor prognosis for fungal keratitis that occurs in eyes after undergoing DSAEK may be related to the rapid expansion of inflammatory cells through the interface between the host and the graft. In eyes that develop fungal keratitis after DSAEK, special attention should be paid to the possibility that perforation could occur in these patients. Keywords: DSAEK, Candida albicans, fungal keratitis, keratomycosis, post-operative infection

Background : To evaluate changes in the visual field (VF) after Descemet stripping automated endothelial keratoplasty (DSAEK) in eyes with advanced glaucoma and previous trabeculectomy. Methods : Changes in VF, best-corrected visual acuity (BCVA), intraocular pressure (IOP), and number of glaucoma medications were analyzed before and after DSAEK in 19 eyes. The VFs were evaluated using the 10-2 program of the Humphrey Field Analyzer (HFA) and/or Goldmann perimetry (GP). Results : In nine eyes, the MD improved from -22.24 ± 6.5 dB to -18.36 ± 5.1 dB in HFA. In five out of nine eyes, postoperative MD improved >1 dB compared to preoperative MD. In GP testing, 10 out of 15 eyes showed an improvement, that is, greater than 20° in VF enlargement by the isopter of I-4e and/or new detection of a smaller or darker isopter. Overall, improvement in VF with the HFA and/or GP test was observed in 12/19 (63.2%) eyes after DSAEK. Postoperative BCVA improved by more than two lines in logMAR VA in 18 of 19 (94.7%) eyes. There were no significant differences between the preoperative and postoperative IOP and the number of glaucoma medications. Conclusions : DSAEK may produce subjective improvement in the visual field as well as improved visual acuity, even in advanced glaucomatous eyes.

The characteristic features of neurotrophic keratopathy have been well documented by in vivo and in vitro studies using animal models. However, case reports of neurotrophic keratopathy induced by neurosurgery are limited. We describe the clinical characteristics, anterior segment optical coherence tomography (AS-OCT) and in vivo confocal microscopy (IVCM) findings of neurotrophic keratopathy induced by surgery for intracranial lesions. This is a case series including 6 eyes of 3 patients (mean age, 69.67 ± 12.50 years) with unilateral neurotrophic keratopathy. The clinical findings of three patients were described and IVCM findings of three patients were analyzed. The duration of neuropathy ranged from 2 to 30 years (median, 22 years). Thickening of the epithelial layer and higher reflection density of the anterior stroma were observed during the healing process using AS-OCT. The mean nerve fiber density of the subepithelial plexus, as determined by IVCM, was 1943 ± 1000 μm/mm2 for neurotrophic eyes and 2242 ± 600.3 μm/mm2 for contralateral eyes (p = 0.0347). The mean respective dendritic cell densities were 30.8 ± 21.8 and 6.25 ± 5.59 cells/mm2 (p < 0.0001), while the mean basal cell sizes were 259 ± 86.5 and 185 ± 45.9 μm2 (p < 0.0001), respectively. These findings suggest that neurosurgery-induced neurotrophic keratopathy may be associated with alterations in the healing process and immune cell distribution in the cornea.

Objective: We conducted a feasibility study to verify the effectiveness of 16S ribosomal RNA (rRNA) gene analysis using the nanopore sequencer MinION for identifying causative bacteria in several types of ocular infections., Methods and Analysis: Four cases of corneal ulcers, one case of endophthalmitis and one case of a conjunctival abscess were included in this study. DNA was extracted from corneal scraping, vitreous samples and secretions from the conjunctival abscess. We conducted 16S rRNA gene amplicon sequencing using MinION and metagenomic DNA analysis. The efficacy of bacterial identification was verified by comparing the conventional culture method with smear observations., Results: 16S rRNA gene sequencing analysis with MinION identified the causative organisms promptly with high accuracy in approximately 4 hours, from ophthalmic specimens. The results of the conventional culture method and 16S rRNA gene sequencing were consistent in all cases. In four of the six cases, a greater variety of organisms was found in the 16S rRNA gene analysis than in bacterial culture., Conclusion: Using our workflow, 16S rRNA gene analysis using MinION enabled rapid and accurate identification possible in various kinds of bacterial ocular infections., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To investigate the clinical characteristics and causative fungi in patients with fungal keratitis in Japan, and to determine factors related to the prognosis., Study Design: Multicenter prospective observational study., Methods: Eligible patients were enrolled from November 2011 to October 2013 at the 1st stage and from April 2015 to March 2016 at the 2nd stage. The corneal foci were scraped, and the scrapings were cultured in potato dextrose agar. The isolated fungi were identified by gene analyses. Data were collected from the clinical records and statistically analyzed by Cox and logistic regression analyses., Results: Ninety-four fungal strains were isolated from 93 cases, including 42 yeast-like fungi and 52 filamentous fungi. The fungi affected the deep layers of the cornea in 23 cases (24.7%) and the peripheral cornea in 29 cases (31.2%). The incidences of lid swelling/redness, ciliary injection, anterior chamber cells/flare, anterior chamber fibrin, and hyphate ulcer in cases of filamentous fungi were significantly higher than in yeast-like fungi. No history of topical steroids, absence of a main lesion in the peripheral cornea, and best-corrected visual acuity (BCVA) of more than 0.04 at the first visit were related to a shorter healing time. No history of ocular surgery, absence of lesion at one-third deep stromal layer and BCVA of more than 0.04 at the first visit were correlated with BCVA at 3 months after the initial examination., Conclusion: Fungal keratitis is caused by various species of fungi and can become refractory due to poor prognosis factors., (© 2022. Japanese Ophthalmological Society.)

Purpose: To determine the effects of a combination of two antifungal drugs against causative fungi of fungal keratitis in Japan., Study Design: Multicenter prospective observational study., Methods: Eighteen isolates of yeast-like fungi and 22 isolates of filamentous fungi collected by the Multicenter Prospective Observational Study of Fungal Keratitis in Japan were studied. Specially manufactured minimum inhibitory concentration (MIC) measurement plates were used to test the effectiveness of 10 combinations of two antifungal drugs against the isolates. The combinations were pimaricin (PMR) + voriconazole (VRCZ), PMR + fluconazole (FLCZ), PMR + miconazole (MCZ), PMR + micafungin (MCFG), VRCZ + FLCZ, VRCZ + MCZ, VRCZ + MCFG, VRCZ + amphotericin-B (AMPH-B), MCZ + FLCZ, and MCZ + MCFG. The checkerboard microdilution method was used, and the fractional inhibitory concentration (FIC) index was calculated based on the guidelines of The Clinical & Laboratory Standards Institute (CLSI)., Results: In yeast-like fungi, additive effects were observed between PMR and MCFG in 77.8% of the isolates, and they were also observed between the azoles. Synergistic effects were observed on 11.1% of the isolates for MCZ and FLCZ. On the other hand, antagonistic effects were present between PMR and azoles with 88.9% between PMR and VRCZ, 72.2% between PMR and FLCZ, and 94.4% between PMR and MCZ. In filamentous fungi, additive effects were observed between PMR and MCFG in 40.9% of the isolates, and between VRCZ and MCZ in 40.9% of the isolates. Antagonistic effects were observed for PMR and the azoles., Conclusions: The combination of drugs prescribed for fungal keratitis incurs a possibility of synergistic, additive, indifferent, or antagonistic effects, depending on drug combinations and fungal strains., (© 2022. Japanese Ophthalmological Society.)

Purpose: Exposure keratopathy often progresses even with conventional various treatments and needs plastic surgery. However, plastic surgery of eye lid is often difficult in cases with poor general condition by cerebrovascular disorders. We will propose a novel method using synthetic rubber sheet to manage the exposure keratopathy under poor general conditions., Observations: We treated with synthetic rubber sheet on 9 eyes of 9 patients who suffered from refractory exposure keratopathy due to cerebrovascular disorders. Sheets cut from sterile surgical gloves made of synthetic rubber (SR sheets) were placed directly onto the ocular surface with antibiotic ointment and fixed with gauze and tape. Severity of the exposure keratopathy was scored before and after the application of SR sheet. Covered ocular surface with SR sheet could keep an adequate moist environment and exposure keratopathy was improved in all the cases with no adverse effects during an average observation period of 166.2 days., Conclusions and Importance: Sterile synthetic rubber sheet cut from surgical gloves is an effective, safe, easy, and economical material to maintain better condition of ocular surface and especially useful for refractory exposure keratopathy under poor general condition., Competing Interests: The following authors have no financial disclosures: KT, FG, AY, KS, NT, RF., (© 2021 The Authors. Published by Elsevier Inc.)

Purpose: To determine whether there is a correlation between the clinicals characteristics including various types of keratic precipitates and the copy numbers of the DNA of cytomegalovirus (CMV) in eyes with CMV corneal endotheliitis., Methods: We reviewed the medical charts of four cases of corneal endotheliitis that were CMV-positive. We have classified types of clinical phenomenon into four types: coin-shaped KPs, sectoral corneal edema with or without Khodadoust line-like KPs, mutton-fat KPs, and fine KPs and have graded their severity. We also determined the copy numbers of the DNA of CMV in the aqueous humor by real-time polymerase chain reaction before and during the treatment. We evaluated the correlation between the patterns of clinical characteristics and copy number of the DNA of CMV., Results: There were clinical improvements in all eyes following topical ganciclovir in conjunction with low dose of topical steroid treatment, with or without oral valganciclovir. The clinical characteristics and the copy numbers of the DNA of CMV varied during the treatment period. The presence of coin-shaped KPs was correlated with high copy numbers (10 5 -10 3  copies/ml) of the DNA of CMV. The copy numbers of the DNA of CMV with sectoral corneal edema with or without Khodadoust line-like KPs ranged from 10 4 to 10 2 copies/ml, and it was occasionally accompanied by high intraocular pressure. Mutton-fat KPs were observed inferiorly, sometimes together with coin-shaped KPs and sectoral corneal edema, or solely. The copy numbers in eyes with mutton-fat KPs varied and occasionally less than the cutoff level. Fine-pigmented KPs were observed after the resolution of the endotheliitis, and no DNA of CMV was detected in the aqueous humor., Conclusions: Careful observations of the clinical characteristics such as the KPs and corneal edema might be helpful in estimating the amount of the DNA of CMV in eyes with corneal endotheliitis.

Lactoferrin (LF) is an iron-binding basic glycoprotein that has an antimicrobial effect against certain microbes. The purpose of this study is to evaluate the amoebicidal effect of bovine milk LF (bLF) against Acanthamoeba clinical-isolate trophozoites, which cause severe keratitis. Most of the risk factor for Acanthamoeba keratitis is from wearing soft contact lenses (SCLs). Acanthamoeba trophozoites were incubated in bovine LF (bLF) solution, and the ratios of viability and encystment were determined with microscopic analysis of cyst formation. The amoebicidal effect of bLF was assessed by Trypan blue assay. The ratios of viable cells in the presence of iron-free bLF (apo-bLF), native-bLF, and iron-saturated bLF (Fe-bLF) at the concentration of 10 μmol/L for 60 min were 7.7% ± 4.6%, 80.7% ± 10.1%, and 97.3% ± 1.5%, respectively. Apo-bLF showed potent amoebicidal effect against Acanthamoeba trophozoites, but Fe-bLF did not have this effect. After treating with apo-bLF, most dead cells were nonglobular forms of trophozoites but not cystic forms. Encystment of Acanthamoeba was assessed by the sarkosyl-calcofluor white assay. The encystment ratios treated with 0.5% propylene glycol (positive control) and 10 μmol/L apo-bLF for 24 h were 96.12% ± 10.6% and 0.47% ± 0.5%, respectively. These results suggest that the amoebicidal effect of apo-bLF without encystment might lead to the prevention of contamination of Acanthamoeba in SCL stock cases.

Background: To determine the most effective route of administration of corticosteroids in the treatment of ocular surface disease, by characterizing the difference between oral prednisolone and topical dexamethasone administration using an animal model., Methods: Pharmacokinetic analyses determined the corticosteroid concentrations in the normal ocular tissues of rabbits after oral or topical administration of corticosteroids using LC-MS/MS. In wound healing analyses, the area of the epithelial defect created by keratectomy using a 6-mm trephine was calculated with an image analyzer using an orally or topically steroid-administrated animal model. The average size of basal epithelial cells, the frequency of mitotic basal epithelial cells, the number of squamous cells, and the number of hypertrophic stromal fibroblasts were determined in the enucleated corneal tissues after wound closure., Results: By slit lamp examination, no remarkable differences were observed between orally and topically administered groups. Pharmacokinetic analyses showed that the distribution of dexamethasone after topical administration was superior to that after oral administration in the cornea. In contrast, both concentrations of corticosteroid applied topically and orally were similar with regards to AUCs (area under the concentration-time curve) in the conjunctiva. Although the healing rate was slower in the topical group, all corneas were almost healed within 96 h in the wound healing analysis. According to the histological analyses of epithelial cells, the average basal cell size was larger, the frequency of mitotic basal cells was greater, and the number of squamous epithelial cell layers was lower in the topically administered group although all of these differences were with no statistical significance. However, the number of hypertrophic stromal fibroblasts in the topically administered group was significantly lower than that in the orally administered group., Conclusions: There are different distributions and effects between orally and topically administered corticosteroids on the ocular surface. The data may provide the useful information in selecting the appropriate route of corticosteroid application for the treatment of ocular surface disease.

Purpose: To investigate the current status of fungal keratitis in Japan., Methods: The patients with fungal keratitis were examined at 27 facilities in Japan from November 1st 2011 to October 31st 2013, concerning isolates, patient background, clinical findings, treatment and prognosis., Results: Out of 139 cases, 133 were diagnosed as fungal keratitis, of which fungi were isolated from 72 samples of 71 cases (yeast-like fungi 32 strains and filamentous fungi 40 strains). The corrected visual acuity at the first visit of 88 cases (66.2%) was less than 20/200 and 42 cases (31.6%) were involved with deep stromal lesions, indicating high proportion of severe cases in this study. Three months later, 56 cases (42.1%) were still under treatment, and corrected visual acuity of 57 cases (42.9%) was less than 20/200. In cases with yeast-like fungi, there were significantly more cases with past history of corneal diseases, ocular surgery including keratoplasty, and eye drops' use such as steroids than those with filamentous fungi. On the other hand, there were significantly more cases of filamentous fungi, with trauma on the onset and with intervention of previously attending doctors than those with yeast-like fungi. Logistic regression analyses revealed that contact lens wearing was a significant factor of good prognosis, and yeast-like fungi as one of poor outcome compared with no fungal isolation., Conclusion: Although the choice of antifungal drugs has been increasing, fungal keratitis is still severe, refractory and vision-threatening disease.

Purpose: To investigate the causative fungi of fungal keratitis in Japan and their drug susceptibility., Methods: Identification and antifungal susceptibility test for 8 drugs (micafungin, amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, miconazole and pimaricin) were performed using isolated fungi from patients with fungal keratitis treated at 27 facilities in Japan between November 1, 2011 and October 31, 2013., Results: Fungal strains were detected in 72 (50.7%) out of 142 samples. The major isolates were Fusarium spp. (18), Candida parapsilosis (12), C. albicans (11) and Alternaria spp. (6), in all, fungi of 31 species were identified by gene analysis. In the yeast-like fungi, susceptibility rates were evident for more than 80% in voriconazole, pimaricin, flucytosine, micafungin, amphotericin B and fluconazole. In filamentous fungi, the susceptibility rate was less than 50% except for PMR (90%). Fusarium spp., which were susceptible to amphotericin B and pimaricin, showed lower susceptibility rates compared with other genera., Conclusions: Although various genera and species of fungi cause fungal keratitis, the obtained drug susceptibility data in this study demonstrates the different susceptibility patterns among the major isolates (Fusarium spp., C. parapsilosis, C. albicans and other groups). This is important evidence useful for fungal keratitis treatment.

Background/aims: The novel immunochromatographic assay (ICGA) kit was recently developed to diagnose herpes simplex virus (HSV) infection. This multicentre study aimed to evaluate the value of the ICGA kit for the diagnosis of herpetic epithelial keratitis by comparing it with immunofluorescence assay (IFA) and real-time PCR., Methods: Corneal scrapings were collected from 117 patients, including 77 with herpetic keratitis as their final clinical diagnosis as well as 40 others at 21 facilities. These samples were tested by the ICGA kit, IFA and real-time PCR., Results: The positive concordance between final clinical diagnosis and ICGA was 46.7% (35/75 cases) and the negative concordance was 100% (39/39). The positive and negative concordance between real-time PCR and ICGA were 57.4% (35/61 cases) and 100% (53/53), respectively. The positive and negative concordance between IFA and ICGA were 61.1% (22/36 cases) and 83.3% (55/66), respectively. In 92 cases where anti-HSV drugs were not prescribed prior to corneal scraping, the positive and negative concordance between final clinical diagnosis and ICGA were 55.0% (33/60 cases) and 100% (32/32), respectively., Conclusions: The ICGA kit has moderate sensitivity and high specificity, indicating clinical utility in the diagnosis of herpetic epithelial keratitis.

Purpose: To classify secondary corneal amyloidosis (SCA) by its clinical appearance, to analyze the demographics of the patients, and to determine the involvement of lactoferrin., Design: Retrospective, observational, noncomparative, multicenter study., Participants: Twenty-nine eyes of 29 patients diagnosed with SCA by corneal specialists at 9 ophthalmologic institutions in Japan were studied., Methods: The clinical appearance of SCA was determined by slit-lamp biomicroscopy and was classified into 3 types. The demographics of the patients, for example, age, gender, and the duration of the basic disease (trichiasis, keratoconus, and unknown), were determined for each clinical type. Surgically excised tissues were stained with Congo red and antilactoferrin antibody. The postoperative prognosis also was determined., Main Outcome Measures: Clinical appearance of the 3 types of SCA, along with the gender, age, and duration of the basic diseases were determined., Results: Classification of SCA into 3 types based on clinical appearance found 21 cases with gelatinous drop-like dystrophy (GDLD)-like appearance (GDLD type), 3 cases with lattice corneal dystrophy (LCD)-like appearance (LCD type), and 5 cases with the combined type. Patients with the GDLD type were younger (average age: 40.9 years for the GDLD type, 74.3 years for the LCD type, and 46.8 years for the combined type), predominantly women (85.7% for the GDLD type, 33.3% for the LCD type, and 60% for the combined type), and had the basic disease over a longer time (average duration: 22.1 years for the GDLD type, 14.0 for the LCD type, and 11.4 for the combined type). The distribution of the basic diseases (trichiasis vs. keratoconus vs. unknown) was not significantly different for each type. Surgical treatments, for example, phototherapeutic keratectomy, lamellar keratoplasty, and simple keratectomy, resulted in a good resolution in all surgically treated cases. One subject dropped out of the study. Spontaneous resolution was seen in one subject after epilation of the cilia. Amorphous materials in the excised tissues showed positive staining results by Congo red and by antilactoferrin antibody., Conclusions: Secondary corneal amyloidosis can be classified into 3 clinical types based on its clinical appearance. Larger numbers of females and lactoferrin expression were seen in all 3 types., Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article., (Copyright © 2013 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: We investigated by in vivo confocal microscopy alterations in the subepithelial nerve plexus in different types of herpes simplex keratitis (HSK)., Methods: Seventeen patients (seven women and ten men, mean age 63.9 years) with a history of HSK were classified into three groups according to the classification of Herpetic Keratitis Infection Research Group. Slit-lamp examinations, corneal sensitivity measurements, and corneal in vivo confocal microscopy examinations [Rostock Corneal Module attached to the Heidelberg Retina Tomograph II (HRT II-RCM)] were performed., Results: Among the 17 cases, three were classified as epithelial type, ten as stromal type, and four as endothelial type HSK. The average corneal sensitivities were 11.41 ± 9.46 mm in the affected eyes and 43.24 ± 12.2 mm in controls. Decreases in three parameters in the affected eyes (long nerve-fiber density, nerve-branch density, nerve thickness) were statistically significant compared with controls. Decreases in the three parameters were more remarkable in the epithelial and stromal types than in the endothelial type., Conclusion: The morphology of the corneal subepithelial nerve plexus may get gradually destroyed along with recurrent episodes of epithelial and stromal HSK. However, the destruction does not seem remarkable in the endothelial type of HSK, suggesting the possibility of a different route of virus recruitment in this type.

Purpose: To report the detection of cytomegalovirus (CMV) DNA in the cornea of a CMV endotheliitis patient after penetrating keratoplasty (PKP)., Case: A 71-year-old man without immunodeficiency developed corneal endotheliitis in the right eye. The patient had previously received PKP several times. Polymerase chain reaction (PCR) detected CMV-DNA in the aqueous humor in his affected eye, and we started administration of ganciclovir. There was resolution of the inflammation; however, bullous keratopathy was subsequently noted in the cornea. Additional PKP was performed with perioperative intravenous administration of ganciclovir. The failed graft obtained during the additional PKP was subjected to PCR analysis and histopathological examination. PCR analysis showed CMV-DNA in the failed graft. Little inflammatory change was noted in either the epithelial or stromal layers of the failed graft. With continued ganciclovir treatment, the graft remained clear and no recurrence or rejection occurred until 12 months after the last PKP., Conclusion: Our PCR analysis showed the presence of CMV-DNA within the cornea of the patient with corneal endotheliitis.

Purpose: To identify the candidate genes for pterygia recurrence from a pterygia transcriptome and to analyze their transcriptional regulation and functional relationships., Methods: Transcriptional networks for pterygia recurrence were constructed using network analysis that was applied to 184 genes that showed a significant twofold change in the whole genome. Of the identified recurrence-related candidate genes in the major networks, periostin and IL-4 were analyzed for transcriptional relationships using pterygia-derived fibroblasts. Immunohistochemical analysis was used to study pterygia tissue. Effector candidate molecule for recurrence periostin was analyzed for cell adhesive function., Results: The pterygia transcriptome was divided into four major biological networks with high significance scores (P < 10(-17)). The classifier with the highest accuracy using the support vector machine algorithm was periostin, which was successfully linked to the network of cell cycle, connective tissue development and function, and cell morphology. Analyses using pterygia-derived fibroblasts showed that periostin was required for cell adhesion that was mediated by a presumed pterygia-related extracellular matrix protein, fibronectin. Periostin was found to be transcriptionally induced by IL-4. The IL-4-stimulated periostin induction was suppressed by MAP kinase/ERK kinase 1 inhibitor, indicating an involvement of the MAP kinase pathway. Pathologically, IL-4 was transcriptionally elevated in recurrent pterygia tissue and was localized to perivascular tissues and endothelial cells in the stroma of the subconjunctiva of pterygia., Conclusions: Periostin is induced by IL-4 and is involved in the fibronectin-mediated pterygia fibroblast adhesion. These findings indicate that periostin probably promotes the recurrence of pterygia.

Purpose: To examine the efficacy of a novel method of decellularizing porcine corneal stroma using N(2) gas from liquid N(2) and the feasibility of using decellularized porcine corneal stroma in a corneal transplantation model in rabbits., Methods: Porcine corneas were placed in a tube, and N(2) gas from liquid N(2) was poured into the tube to freeze the corneas and make the inside of the tube hypoxic. After fastening the cap firmly, the tube was kept at room temperature for seven days, and the porcine corneas were examined histologically. A porcine corneal stromal disk treated with the aforementioned method was inserted into a pocket of rabbit corneal stroma and observed for six months., Results: Hoechst 33342 and hematoxylin and eosin staining both showed few cellular nuclei in the porcine corneal stroma incubated in N(2) gas for one week. A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay showed many positively stained nuclei in the porcine corneal stroma incubated in N(2) gas for three days. The porcine corneal stroma that was decellularized and transplanted into a rabbit corneal stromal pocket remained clear for six months after transplantation., Conclusions: This method using N(2) gas decellularizes corneal stroma without reducing corneal transparency.

Background: The chemotactic signals regulating cell trafficking in the herpes simplex virus type 1 (HSV-1) infected cornea are well documented, however, those in the cornea-associated tissues, such as the trigeminal ganglion (TG) and draining lymph nodes (LNs), are largely unknown., Objectives: To examine chemokine expression and subsequent cell infiltration in the HSV-1 infected cornea and its associated tissues., Study Design: Eight-week-old female BALB/c mice were infected with 10 mu l HSV-1 (CHR3 strain: 5 x 106 PFU/ml) by corneal scarification. Total RNAs were extracted from the corneas, TGs, and LNs at pre-inoculation, 3 days post-inoculation (P.I.) and 7 days P.I. The mRNA for 28 different chemokines in the extracts was amplified by RT-PCR. Infiltrating cells were identified by immunohistochemistry., Result: After the HSV-1 infection, the corneal stroma became edematous by infiltrated cells under the eroded epithelium. The TG and LNs were markedly swollen. The cornea was infiltrated with granulocytes and CD11b+ cells at 3 days P.I., followed by CD4+ and CD8+ T cells at 12 days P.I. In the TG, CD11b+ cells, but no granulocytes, infiltrated throughout the observation period. T cells migrated into the TG earlier than into the cornea. Gene expressions of neutrophil-attracting chemokines (CXCL1, 2, 3, and 5) increased in the cornea, but they did not enhance in the TG or LNs. On the other hand, gene expressions of chemokines which attract CD11b+ cells such as CCL2, 8, 7, 12, CCL3, 4, and CCL5, increased in the cornea and TG with its peak at 3 days P.I. Gene expressions of chemokines those work on T cells and B cells, such as CCL19, CCL21, CXCL9, CXCL13, CXCL10, XCL1, and CXCL16, were up-regulated and peaked at 3 days P.I. in the cornea and in the TG. Thus, pattern of chemokine gene expression was similar in the cornea and in the TG. On the contrary, gene expressions of chemokines in the draining LNs affecting CD11b+ cells and T cells were temporarily down-regulated., Conclusion: Upon HSV-1 infection, dynamic gene expression of chemokines was observed not only in the inoculated cornea but also in its associated tissues.

Purpose: To describe a Pseudomonas aeruginosa corneal infection resulting from orthokeratology., Methods: Case report., Results: A 17-year-old boy wearing orthokeratology (OK) lenses was referred to our clinic because of redness in his right eye in spite of his usage of ofloxacin (OFLX) eye drops. An excavated paracentral corneal ulcer with an immune ring and hypopyon was observed. It was positioned under the paracentral steeper portion of the optic of the OK lens. Culture of the lens solution revealed P. aeruginosa. The patient was treated with topical OFLX and cefmenoxime (CMX) plus intravenous and subconjunctival injections of cefozopran (CZOP), successfully. The antibiotic susceptibility of P. aeruginosa by the disk diffusion susceptibility test was reduced under moderately hypoxic conditions. Glycocalyx slime was formed on the OK lens in vitro by P. aeruginosa isolated from the case., Conclusions: Changes in P. aeruginosa susceptibility to antibiotics under moderately hypoxic conditions and glycocalyx slime formation might affect the features of OK lens-associated infections.

Gas-permeable hard contact lenses were prescribed to 2 patients with corneal microstriae 3 months and 9 months, respectively, after laser in situ keratomileusis. The uncorrected visual acuity (UCVA) in both patients when the hard contact lenses were prescribed was 20/30 with +2.25 -1.25 x 60 and +1.50 -2.25 x 150, respectively. At 6 months in Case 1 and 3 months in Case 2, the topographic change showed corneal remodeling, with the UCVA 20/20 in both cases.

We have studied the synthesis of laminins (Ln) and determined the specific integrins mediating the adhesion of immortalized human corneal epithelial cells to mouse Ln-1, and human Lns-5 and -10. Immunofluorescence microscopy of the cells demonstrated integrin alpha(2), alpha(3), alpha(6), beta(1)and beta(4)subunits, integrins alpha(6)and beta(4)being found in a typical 'leopard-skin' like manner. Immunoprecipitation studies showed that the cells produced alpha 3, beta 3 and gamma 2 chains of Ln-5, but not Lns-1 or -10. In culture Ln-5 was found as small plaques beneath the adhering cells within 1 hr, while in 4 hr widely spread Ln-5 plaques were observed in colocalization with beta(4)integrin subunit. By using a quantitative cell adhesion assay and function-blocking monoclonal antibodies we showed that integrin beta(1)subunit plays a role in mediating corneal epithelial cell adhesion to mouse Ln-1. However, none of the available function-blocking antibodies to integrin alpha-subunits inhibited the adhesion. Integrin alpha(3)beta(1)complex mediated the adhesion of corneal epithelial cells to human Lns-5 and -10. Integrin complex alpha(3)beta(1), as well as laminin alpha(3)chain, was also shown to mediate cell adhesion to newly produced endogenous Ln-5. The present results show that integrin alpha(3)beta(1)complex mediates the adhesion of corneal epithelial cells to Lns-5 and -10, while a yet unknown integrin alpha subunit appears to play a role in the adhesion to Ln-1. The results also show that among corneal basement membrane laminins, Ln-5 is synthetized by epithelial cells while Ln-10 may be a product of keratocytes., (Copyright 2001 Academic Press.)

Although the absence of Substance P (SP), a neurotransmitter in the trigeminal nerve, has been speculated as a cause for developing neurotrophic keratitis, its exact pathogenesis is still not clarified. In a previous report, we showed with electron microscopic examination that epithelial cell attachment was weakened in denervated corneas. In this study, SV40-transformed human corneal epithelial cells (HCE-Ts) were used to explore the molecular mechanisms responsible for mediating regulation of E-cadherin expression in response to Substance P receptor stimulation. Expression of the mRNAs for specific SP receptors, neurokinin (NK)-1R, NK-2R, and NK-3R, was demonstrated with RT-PCR. The cells were treated with various concentrations of SP in vitro, and the expression of an adhesion molecule E-cadherin was analyzed by immunofluorescence, immunoblotting, and enzyme-linked immunosorbent assay (ELISA) using an anti-E-cadherin antibody. E-cadherin expression was increased by SP in a dose-dependent manner both in the cytosolic fraction and in the cell membrane fraction. This increase in E-cadherin expression was completely inhibited by Calphostin C (PKC inhibitor) and KN-62 (CaMK inhibitor), but not by H-89 (PKA inhibitor), indicating that SP-induced E-cadherin expression involves the activation of protein kinase C (PKC) and calmodulin kinase (CaMK). SP did not affect cell proliferation at all. All these findings indicate that SP induced E-cadherin expression through PKC and CaMK activation and suggest that a lack of SP may account in part for the pathogenesis of neurotrophic keratitis., (Copyright 2000 Wiley-Liss, Inc.)

Purpose: To determine the ability of norepinephrine to modulate proliferation, adhesion, and migration of SV-40 transformed human corneal epithelial cells., Methods: Assays were performed using SV-40 transformed human corneal epithelial cells. For proliferation assays, cells were plated in 96-well plates coated with fibronectin and collagen (FNC). A dose-response curve was generated for norepinephrine in concentrations of 100 nM-100 microM. The cell number in each well was evaluated using the fluorochrome Calcein AM (an intracellular esterase cleavage substrate), and fluorescence was determined using an automated fluorescent plate reader. For cell adhesion, 25 x 10(-3) cells were plated onto FNC-coated 96-well plates, incubated in 10 nM-100 microM norepinephrine for 90 min, gently irrigated, and the remaining adherent cells quantitated. Cell migration was measured using blind-well migration chambers with a 10-microm pore size and FNC-coated filters. Cells (250 x 10(3)) were added to the upper chamber, incubated for 18 h in the presence of factors, after which time the cells that had migrated through the filter were quantitated. The toxicity of norepinephrine was evaluated using a standard Live/Dead assay employing the combined fluorochromes of ethidium homodimer (to indicate dead cells) and Calcein AM (to indicate viable cells). Varying concentrations of norepinephrine were added, and the cells incubated for 3 h and the fluorometric assay performed., Results: Norepinephrine stimulated corneal epithelial cell proliferation and migration over a wide range of concentrations. It did not modulate cell adhesion and demonstrated cell toxicity only at the highest (supraphysiologic) concentration tested., Conclusions: Norepinephrine is normally found in the cornea and may be important in the maintenance of normal corneal homeostasis and in wound-healing processes.

Purpose: An immortalized human corneal epithelial cell line (HCE) was tested as a screening tool for prediction of topical ocular irritation/toxicity by pharmaceuticals, Methods: Effects of various drugs, excipients and cyclodextrins (CDs) on viability of HCE cells were evaluated using two in vitro cytotoxicity tests, 3-(4,5-dimethlthiazol-2-yl)-205-diphenyl tetrazolium bromide (MTT) dye reduction assay and propidium iodide assay., Results: Mitochondrion-based MTT test was a more sensitive indicator of cytotoxicity than the plasma membrane-based propidium iodide test. The tests revealed following cytotoxic rankings for ophthalmic drugs: dipivefrin > timolol > pilocarpine approximately equal to dexamethasone; for excipients: benzalkonium chloride (BAC) > sodium edetate (NA2 EDTA)>polyvinyl alcohol (PVA) > methylparaben; and for CDs :alpha- CD > dimethyl beta-cyclodextrin (DM-beta-CD) > sulfobutyl ether beta-cyclodextrin ((SBE)7m-beta-CD approximately equal to hydroxypropyl-beta-cyclodextrin (HP-beta-CD) > lambda CD. In consideration of the in vivo clinical situation, the short exposure time (5 min) is more relevant even though toxic effects of some test substances were seen only after longer exposure time (30 and 60 min)., Conclusions: Immortalized HCE cells are a promising tool for rapid cytotoxicity assays of ocular medications. The cell line is potentially useful in predicting the in vivo coreal toxicity of ocularly applied compounds.

Purpose: Neutrophil invasion is a primary event in the development of herpetic keratitis. It has been reported that HSV-1 infection of keratocytes induces the synthesis of IL-8, a potent neutrophil chemoattractant, while corneal epithelium does not. Nevertheless, little is known about the correlation between neutrophil migration and the production of chemotactic factors by HSV-1-infected corneal cells, especially in epithelial cells which form an initial barrier of the ocular surface. We examined whether human corneal epithelial cells as well as keratocytes could induce neutrophil chemotaxis in response to HSV-1 infection., Methods: Human corneal epithelial cells immortalized with SV40 (HCE) and human keratocytes were infected with HSV-1. The culture fluids collected at 4, 12, 24 h after infection were assayed for human neutrophil chemotaxis using a modified Boyden chamber method. IL-8 levels in these supernatants were measured using enzyme-linked immunosorbent assay (ELISA)., Results: The chemotactic activity induced by HCE and keratocytes after MP strain of HSV-1 infection peaked as early as 4 h postinfection, then declined. Chemotactic activity induced by HSV-1-infected HCE and IL-8 levels on these supernatants paralleled with the infectious virus titer. It was inhibited by monoclonal anti-IL-8 antibody. UV-inactivation of MP strain abrogated neither the induction of chemotactic activity nor IL-8 secretion of infected HCE., Conclusions: At the early phase of HSV-1 infection, corneal epithelial cells play an important role in inducing neutrophil chemotaxis, which was mediated by IL-8.

Purpose: To test whether human corneal epithelial cells (HCECs) respond to small applied electric fields (EFs) in a similar manner to bovine corneal epithelial cells (BCECs), the orientation and directed migration in small EFs of both primary cultures and of a human corneal epithelial cell line were quantified., Methods: Primary cultures of human corneal epithelial cells (PHCECs) and transformed human corneal epithelial cells (THCECs) were exposed to EFs (100 mV/mm-250 mV/mm) in different media. Cell migration was traced using an image analyser., Results: PHCECs and THCECs reoriented and migrated towards the cathode (negative pole) when cultured in small direct current (dc) EFs. Both the reorientation and directional migration were voltage- and serum-dependent, as shown previously for bovine cells. PHCECs and THCECs showed significant perpendicular orientation in EFs at 150 mV/mm in medium with serum, while at the same voltage, no significant orientation was found in serum free medium. PHCECs started to show perpendicular reorientation around 30 min after onset of EF at 150 mV/mm. They showed significant directional migration at 150 mV/mm, with directedness of 0.35 +/- 0.07 and a migration rate of 9.1 +/- 0.7 microns/h (n = 90), both significantly higher than that of cells in serum free medium. Addition of EGF-induced significant reorientation and directional migration of THCECs at 100 mV/mm. Additionally, as for BCECs, which remained viable and responsive to electric fields for at least 75 h at 150 mV/mm, THCECs also remained viable and showed responsiveness during long periods of exposure to EFs (at least 20 h)., Conclusions: Cultured human primary CECs and a human corneal epithelial cell line both responded to small EFs with perpendicular reorientation and cathodally-directed migration. Cell responses were qualitatively similar to those reported previously for bovine CECs. The endogenous EFs generated by wounded cornea may play an important role in promoting cell shape changes and directed migration of CECs during the healing process.

The effects of carbachol (CCh) on phospholipase C(PLC)-mediated phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and its modulation by isoproterenol were investigated in SV40-adenovirus transformed rabbit corneal epithelial cells (RCEC). When examined under light microscope, these cells exhibited a cobblestone-like appearance typical of the corneal epithelial cells grown in primary culture. Addition of CCh (0.1 mM) for 30 min to RCEC, prelabeled with 32Pi, decreased the radioactivity in phosphatidylinositol 4-phosphate and PIP2 by 15 and 27%, respectively, and concomitantly increased the radioactivity in phosphatidylinositol and phosphatidic acid by 14 and 38%, respectively. When the concentration of CCh was increased to 1 mM, the changes in radioactivity were even more pronounced. Addition of CCh (0.1 mM) to the cells, prelabeled with myo[3H]inositol, increased the accumulation of [3H]inositol 1,4,5-trisphosphate ([3H]InsP3) by 115%, indicating stimulation of PLC-mediated PIP2 hydrolysis. Similar increases were also observed in [3H]InsP1 and [3H]InsP2. The effects of CCh on inositol phosphate accumulation were time- and dose-dependent, and were inhibited by atropine (10 microM), suggesting that the observed effects of CCh were mediated by activation of muscarinic cholinergic receptors. The effects of CCh were antagonized more potently by 4-diphenylacetoxy N-methyl-piperidine than by pirenzepine, indicating that the muscarinic receptors involved in PLC activation are probably of M3 type. By Western immunoblotting analysis with various anti-PLC antibodies, the RCEC were shown to contain PLC gamma 1 and PLC delta 1 in the soluble fraction and PLC beta 1 in the microsomal fraction. Addition of isoproterenol to RCEC, increased cAMP both in a time- and dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

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.