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Purpose: To identify primary cilia in human corneal endothelial cells (CECs) obtained from patients with bullous keratopathy (BK)., Methods: This study involved CEC specimens obtained from 10 eyes of 10 consecutive patients (three males and seven females; mean age: 74.5 years, range: 68-90 years) with BK who underwent Descemet's stripping automated endothelial keratoplasty at Baptist Eye Institute, Kyoto, Japan between August 2019 and September 2020. Three corneal buttons obtained from 3 patients who underwent penetrating keratoplasty for keratoconus were used as 'non-BK' controls. All specimens were evaluated with immunofluorescence staining using an antibody against acetylated α-tubulin., Results: Ciliary expression was observed in six of the 10 CEC specimens; i.e. in two specimens obtained from BK patients after glaucoma surgery (trabeculectomy), in two specimens obtained from patients with Fuchs endothelial corneal dystrophy, and in two specimens obtained from a patient with BK after laser iridotomy for primary angle closure. There was acetylated α-tubulin staining but no hair-like structures in two specimens, and ciliary expression was unknown in two specimens due to the absence of cells. The length of the primary cilia varied between all specimens. In contrast, no primary cilia were observed in the corneal buttons obtained from the three keratoconus patients., Conclusion: The findings in this study clearly demonstrate the expression of primary cilia in the CECs of patients afflicted with BK.

Purpose: To evaluate the toxicity of Amphotericin B (AmB) in Optisol™-GS Corneal Storage Media (Bausch & Lomb) on corneal epithelial cell (CEC) morphology and migration ability., Methods: Sclerocorneal strips were removed from male Japanese white rabbits, and then stored at 4 °C in Optisol™-GS containing 0 µg/ml of AmB (control group) and 2.5, 5, 25, and 50 µg/ml of AmB (AmB groups; four eyes per group). After 7 days of storage, CEC morphology was evaluated by hematoxylin-eosin staining, immunohistochemical staining (ZO-1), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Moreover, to evaluate CEC migration ability, three corneal blocks (6-8 × 3 mm each) from one preserved cornea were cultured for 24 h, and the area of CEC migration (2 mm at the central region) onto the stromal surface was then measured., Results: At 5, 25, and 50 µg/ml of AmB, deformation and vacuolation of CECs were observed in all preserved corneas. ZO-1 expression was significantly reduced in corneas preserved at AmB concentrations of 25 and 50 µg/ml. TUNEL Labeling Index was significantly increased at AmB concentrations of ≥5 µg/ml. CEC migration was inhibited in a dose-dependent manner at AmB concentrations of 25 and 50 µg/ml compared to the control group., Conclusions: The addition of AmB to Optisol™-GS can be toxic to CECs and inhibit their migration at a concentration of ≥5 µg/ml. AmB at a concentration of 2.5 µg/ml can be considered safe for the preservation of donor corneal tissue used in corneal epithelial transplantation surgery.

Background: To investigate the influence of age on prediction error (PE) after cataract surgery in very elderly (VE) patients aged more than 90 years., Methods: We retrospectively analyzed 66 eyes of patients aged ≥90 years (VE group) who underwent phacoemulsification and intraocular lens (IOL) implantation. As the control group (CG), we investigated 121 eyes of patients aged 70-89 years who underwent the same surgery. PE was calculated 1 month post-surgery as the actual postoperative spherical equivalent minus the target diopter, which was calculated using the Sanders-Retzlaff-Kraff/T formula. The absolute and arithmetic PE were compared between the two groups. The factors affecting absolute PE outside ±0.5 diopter (D) and ±1.0 D were determined through logistic regression analysis with the variables age, sex, axial length (AL), average corneal power, preoperative best-corrected visual acuity, target diopter, and coexisting pseudoexfoliation syndrome., Results: The absolute PE was significantly larger in the VE group than that in the CG (0.60 ± 0.52 D and 0.34 ± 0.25 D, respectively; P < .001). There was no significant difference in terms of arithmetic PE between the two groups (-0.06 ± 0.79 D and -0.07 ± 0.42 D, respectively; P = .653). In the logistic regression analysis, age was significantly associated with absolute PE outside ±0.50 D (Odds ratio [OR]: 1.05). Age and AL were significantly associated with absolute PE outside ±1.0 D (OR: 1.24 and 0.20, respectively)., Conclusions: Absolute PE tended to increase in the cataract surgery of VE patients.

Purpose: Collagen fibrils in the corneal stroma in macular corneal dystrophy, on average, are more closely spaced than in the normal cornea. This study was conducted to investigate if this occurs uniformly across the stroma or is more prevalent at certain stromal depths., Methods: Microbeam synchrotron X-ray fiber diffraction patterns were obtained in 25 microm steps across the whole thickness of a thin strip of a macular corneal dystrophy cornea obtained at keratoplasty. Data were analyzed for mean collagen interfibrillar spacing at all positions. Serum was analyzed immunochemically to determine immunophenotype, and transmission electron microscopy was carried out to visualize stromal ultrastructure., Results: Keratan sulphate was not detectable in blood serum, classifying the disease as macular corneal dystrophy type I. Collagen interfibrillar spacing dropped linearly with stromal depth from the anterior to posterior cornea, measuring 5-10% less in the posterior 100 microm of the MCD stroma compared to the anterior 100 microm (p < 0.001). Isolated pockets of collagen fibrils with unusually large diameters were identified in the deep stroma., Conclusions: Collagen fibril spacing is reduced and large-diameter collagen fibrils are seen in macular corneal dystrophy type I, with the deep stroma affected more. We speculate that the ultrastructural abnormalities are more prevalent in the posterior stroma because the structural influence of sulphated keratan sulphate glycosaminoglycans/proteoglycans is high in this region of the cornea.

The levels of IGF-I in the vitreous body and the intraocular fluid of patients with proliferative diabetic retinopathy (PDR) were determined using radioimmunoassay (RIA). Eleven vitreous specimens were obtained from the intraocular fluid of eyes of patients with PDR who underwent surgery during the operation. Eleven intraocular fluids from the same patients during reoperations were compared with controls. The expression of IGF-I mRNA in cultured human Muller glial cells was evaluated using reverse transcription-polymerase chain reaction (RT-PCR). The mean IGF-I level in the vitreous samples during initial PDR surgery and reoperation was significantly higher than that found in the vitreous of the control (p < 0.05). The level of IGF-I increased in 6 of the 11 cases. Cultured human Muller cells expressed IGF-I mRNA. The results indicate increased levels of IGF-I both in the initial vitreous and ocular fluid at post-operative re-proliferation. Muller cell is suggested as an origin of local IGF-I production.

Purpose: To investigate whether or not human Müller cells synthesize interleukin (IL)-6., Methods: Using RT-PCR, we first confirmed whether cultured human Müller cells express IL-6 mRNA. Then, to determine Müller cell IL-6 production after stimulation, cultured Müller cells were exposed to various concentrations of IL-1beta (0.2 ng/ml, 2 ng/ml, 20 ng/ml) or lipopolysaccharide (LPS) (0.001 microg/ml, 0.1 microg/ml, 10 microg/ml) in 24 hr assays. In addition, to determine Müller cell time-dependent induction of IL-6 production, cultured Müller cells were exposed to IL-1beta (0.02 ng/ml, 2 ng/ml) or LPS (10 microg/ml) for 6, 12, 24, 36 hr. IL-6 production in supernatants was quantified by ELISA:, Results: IL-6 mRNA was expressed in cultured human Müller cells, which produced IL-6 after stimulation with either IL-1beta or LPS for 24 hours. IL-1beta was a significantly more potent stimulator of IL-6 production than was LPS. Exposure of cultured human Müller cells to either IL-1beta or LPS stimulated IL-6 production in a time-dependent fashion., Conclusions: Our findings indicate that human Müller cells can produce IL-6 when stimulated by IL-1beta or LPS. Müller cell IL-6 production may have an important role in various conditions involving ocular inflammation

Purpose: Previously we reported the occurrence of estrogen receptor alpha (ER beta), estrogen receptor beta (ER beta) and androgen receptor (AR) in mouse corneas. The present study was designed to investigate the occurrence of various sex steroid hormone receptors, including ER alpha, progesterone receptor (PR) and AR, in human corneas., Methods: We used reverse transcription-polymerase chain reaction (RT-PCR) to look for sex hormone receptor mRNAs (ER alpha, PR and AR) in human corneal epithelial cells obtained at autopsy. Next, using an immunocytochemical technique, we localized these receptors in donor human corneas., Results: mRNAs encoding all receptors tested for were found in corneal epithelial cells obtained from male and female donor eyes. Immunocytochemical examination revealed that the receptors were located in the nuclei of corneal epithelial, stromal, and endothelial cells., Conclusions: Since receptors for both male and female sex hormones are present in human corneas of both genders, we postulate that the receptors may influence the biological functions of corneal cells through direct interaction with specific hormones.

Purpose: To investigate the expression of growth factor mRNA and the level of growth factor protein in preserved human amniotic membrane (AM)., Methods: RT-PCR was used to examine the expression of mRNA for eight growth factors (EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2, -beta3) and two growth factor receptors (KGFR and HGFR) in human AM preserved at -80 degrees C for one month. In addition, ELISAs were used to measure the protein concentrations of seven growth factors (EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2) in preserved human corneas and in AM both with and without amniotic epithelium., Results: RT-PCR revealed that human AM expresses mRNA for EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2, -beta3, KGFR and HGFR, while ELISAs showed that it contains EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2. AM without amniotic epithelium also contains all seven growth factors examined, however, in this tissue the protein levels of EGF, KGF, HGF and bFGF were found to be significantly lower than in native AM., Conclusions: Preserved human AM expresses mRNAs for a number of growth factors and contains several growth factor proteins that might benefit epithelialization after AM transplantation. High levels of EGF, KGF, HGF and bFGF in AM with amniotic epithelium as compared to AM without amniotic epithelium suggest an epithelial origin for these growth factors. We feel that EGF, KGF and HGF in particular might play important roles in ocular surface wound healing after AM transplantation.

Purpose: The topical administration of 1alpha,25-dihydroxy-vitamin D(3) [1alpha,25(OH)(2)D(3) ] inhibits Langerhans cell (LC) migration and corneal neovascularization in mice. Since the cytokines that induce LC migration [e.g. interleukin-1 (IL-1)] and corneal neovascularization [e.g. interleukin-8 (IL-8)] are produced by human corneal epithelial cells, we investigated the inhibitory effects of 1alpha,25(OH)(2)D( 3) on cytokine production by these cells in vitro., Methods: In this experiment, human corneal epithelial cells, cultured in DMEM-FBS until confluence, were then switched to serum-free DMEM containing insulin, transferrin, and sodium selenite (DMEM-ITS) for 48 hours. Next, they were cultured with DMEM-ITS containing 1alpha,25(OH)(2)D(3 ) at concentrations of 10(-7) M, 10(-11) M, or 10( -15) M, and vehicle only (0.1% ethanol). After 6 or 12 hours in this culture, the supernatants were collected and concentrations of IL-1alpha, IL-1b, and IL-8 were quantified by ELISA., Results: Significantly lower levels of IL-1alpha and IL-1b were detected in supernatants from cells cultured with 1alpha, 25(OH)(2)D( 3) (10(-7) M, 10(-11) M, and 10(-15) M), compared to cells cultured with vehicle only. This was true at 6 and 12 hours after the addition of 1alpha,25(OH)(2)D(3) (p < 0.05). IL-8 production inhibition by 1alpha,25(OH)(2)D(3), on the other hand, was detected at 6 hours (p < 0.0005) but not at 12 hours (p> 0.1)., Conclusions: 1alpha,25(OH)(2)D(3) inhibits cytokine (IL-1alpha, IL-1b, and IL-8) production by human corneal epithelial cells in vitro. We suspect that 1alpha,25(OH)(2)D(3) can inhibit LC migration and corneal neovascularization, as is seen in ocular surface inflammation.

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

Purpose: To elucidate inflammatory cytokine levels in normal human tears, we investigated the concentrations of interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and interleukin-8 (IL-8) both in basal tears and reflex tears of normal healthy eyes., Methods: Tear samples were collected from 270 healthy, normal volunteers with no past history of ocular surface disease; the total number of eyes was 295. All tear samples were collected in graduated disposable microcapillaries from the conjunctival cul-de-sac. Reflex tear stimulation was carried out by inserting an applicator into each volunteer's nose. Cytokine levels in basal tears and reflex tears were determined by ELISA. Tears were not diluted, and pooled samples were used for the cytokine assays., Results: In basal tears, the concentrations of IL-1alpha, -1beta, -6 and -8 were 10.9 +/- 1.2, 12.9 +/- 2.3, 226.2 +/- 29.6 and 731.4 +/- 116.2 pg/ml, respectively. In reflex tears, IL-1beta was not detectable, and the concentrations of IL-1alpha, -6 and -8 were 9.3 +/- 2.7, 11.6 +/- 1.6 and 276.1 +/- 47.5 pg/ml respectively. IL-1beta, -6 and -8 levels in reflex tears were significantly lower than those in basal tears, except for IL-1alpha., Conclusions: In the present study, the normal concentration range of inflammatory cytokines in human basal tears was clarified, as a baseline for further studies to examine the levels of these cytokines in tears during disease states.

Purpose: To help test the hypothesis that transforming growth factor beta (TGF-beta) may serve an autocrine function in the retina, we asked whether human Müller (glial) cells in culture express TGF-beta receptors, contain transcripts for various isoforms of this cytokine, and release TGF-beta s into the medium., Methods: Using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique with specific primers for TGF-beta 1, -beta 2 and -beta 3 precursors and for TGF-beta type I and type II receptors, we searched for mRNA transcripts expressed by cultured human Müller cells. Also, an ELISA assay allowed quantification of the levels of various TGF-beta s in medium exposed to these glial cells., Results: Human Müller cells in culture express transcripts for both type I and type II TGF-beta receptors and also for TGF-beta 1 and TGF-beta 2. In conditioned medium, the concentration of TGF-beta 1 in the mature form was below detectable levels, and the total TGF-beta 1 was relatively low (mean = 252 pg/ml in confluent cultures). In contrast, the mean levels of mature (55 pg/ml) and total (2530 pg/ml) TGF-beta 2 were markedly higher., Conclusions: Our observations that cultured Müller cells contain mRNA coding for the TGF-beta 2 precursor, release TGF-beta 2 into the medium and express transcripts for both type I and type II TGF-beta receptors are consistent with the idea that this cytokine serves an autocrine function for these glia in the retina.

Purpose: This study investigated the cytokine expression profile in alkali-burned mouse corneas, in order to elucidate the mechanisms of corneal damage and repair., Methods: The cytokines expressed in alkali-burned corneas were identified by polymerase chain reaction (RT-PCR), then quantitated using ELISA. Based on the ELISA results, immunohistochemical analyses were performed to localize cytokine expression., Results: Among the ten cytokines examined, IL-1 (IL-1 beta), IL-6, IL-10 and TNF-alpha mRNA were expressed in alkali-burned corneas. Quantitation revealed that IL-alpha and IL-6 were strongly induced in the early stages of alkali burn, peak production of IL-1 alpha (53.2 pg/cornea) and IL-6 (23.6 pg/cornea) occurring at days 3 and 7, respectively. The production of IL-10 and TNF-alpha was not significantly elevated during the 42 day period after burn. Immunohistochemical analyses revealed that both IL-1 alpha and IL-6 were mainly localized in regenerating epithelial basal cells., Conclusions: IL-1 alpha and IL-6 levels in the cornea are dramatically elevated in the regenerated epithelium during the early stages of alkali burn, and may play an important role in associated corneal damage and repair.

Mature and total TGF-beta 2 levels were determined in normal human tears, from 37 volunteers, using a quantitative "sandwich' enzyme immunoassay technique. Since this ELISA method recognizes only mature TGF-beta 2, total TGF-beta 2 levels in tears were measured after acid activation. Aqueous humor samples from 9 patients with cataract were also tested as controls. Mature and total TGF-beta 2 in aqueous humor (AH) averaged 182 pg/ml and 737 pg/ml, respectively. The levels of mature TGF-beta 2 in normal human tears ranged from 36 to 71 pg/ml (mean: 55 pg/ml). And the levels of total TGF-beta 2 ranged from 3446 to 14910 pg/ml (mean: 7854 Pg/ml), a very high level. Percentage of mature TGF-beta 2 ranged 0.4 to 1.3% (mean: 0.9%). It is reported for the first time that there are very high levels of total TGF-beta 2 in normal human tears.

The expression of TGF-beta 1, TGF-beta 2 and TGF-beta 3 precursors mRNA transcripts in in vivo human corneal cells were studied. Complementary DNA (cDNA) was generated from poly A+RNA extracted from in vivo human corneal epithelial cells, stromal keratocytes and endothelial cells. With the cDNAs as template, polymerase chain reaction (PCR) was carried out using specific primers of TGF-beta 1, TGF-beta 2 and TGF-beta 3 precursors, synthesized by choosing specific nucleotide sequences in the latency-associated peptide region of each precursor. Southern blot analysis of the PCR products was carried out. In corneal epithelial cells, TGF-beta 2 mRNA transcript was strongly expressed; TGF-beta 1 and -beta 3 mRNA transcripts were also expressed; stromal keratocyte samples expressed TGF-beta 1 and -beta 2 mRNA transcripts but not TGF-beta 3 mRNA transcript. Endothelial cells expressed all three transcripts. The present study, together with the authors' previous immunohistochemical study demonstrates that both protein and mRNA of TGF-beta 2-LAP are present in human corneal epithelium, indicating that TGF-beta 2 may play a crucial role in corneal epithelial cell layers.