Effects of SV40 T antigen transduction on human corneal endothelial cell wound healing in vitro.

OBJECTIVE: The aim of this study was to investigate the endothelial wound healing effects of SV40 large T and small t antigen transduction on cultured human corneal endothelial cells (HCEC). METHODS: Human corneal endothelial cells were infected with either mock solution, Ad green fluorescent protein (GFP), or Ad SV40 T/t antigen/GFP, then mechanically wounded 48 h later. The endothelial wound healing rate was quantified by an analysis of the photographs taken every 12 h after wounding. The characteristics of Ad SV40 T/t Ag/GFP-infected human corneal endothelial cells were evaluated with cell morphology, cell density, contact inhibition, and cytoskeletal features using rhodamine phalloidin to stain F-actin. DNA synthesis was assessed using 5-Bromo-2'-deoxy-uridine (BrdU) labeling. RESULTS: Wound healing rates in the first 12 and 24 h after wounding were significantly faster in the Ad SV40 T/t antigen/GFP-infected group than the other two groups. In all three groups, the morphology, cell density, and cytoskeletal features of cells at confluency was similar and contact inhibition retained. There were no differences in the pattern of F-actin and endothelial cell density 4 d after wound closure. However, during the process of wound healing, prominent stress fibers in migrating cells near the wound edge were noted in normal cells at 36 h after wounding, whereas the Ad SV40 T/t Ag/GFP-infected cells showed similar changes as early as 12 h after wounding. BrdU staining results revealed that the Ad SV40 T/t antigen/GFP-infected group had labeled cells showing DNA synthesis in the wound area at 12 h after wounding, while no labeled cells were found in the other two groups. CONCLUSIONS: In an in vitro model, transduction of human corneal endothelial cells using a recombinant adenoviral vector expressing SV40 T/t antigen enhanced both the wound healing rate and proliferative capacity, especially in the first 12 h after wounding, and the characteristic morphologic features of the infected cells were maintained.

Twenty percent alcohol toxicity on rabbit corneal epithelial cells: electron microscopic study.

PURPOSE: To evaluate 20% ethanol toxicity on the rabbit corneal epithelium, ethanol-treated rabbit corneas were examined with electron microscopy. METHODS: Rabbit corneas (24 eyes) were treated with 20% ethanol for 30 seconds, 1 minute, and 2 minutes by using LASEK (laser-assisted subepithelial keratectomy) instruments and then washed with sterile water. Zero time, 1, 3, 5 days after ethanol treatment, corneas were excised and examined with scanning and transmission electron microscopy (SEM and TEM). RESULTS: Widespread partial or total damage of microvilli, focal breaks of intercellular junction, and cellular edema was observed. The damage was more severe in corneas with longer ethanol treatment. In corneas with ethanol treatment more than 1 minute, slough of superficial corneal epithelium occurred and progressed with time. Two-minute ethanol treatment resulted in complete destruction of microvilli and significant separation of intercellular junction. These pathologic changes persisted 5 days after ethanol treatment. CONCLUSIONS: From these results, increasing exposure time to ethanol more than 1 minute results in significant damage to superficial corneal epithelium and prolongs its normal recovery time.

Elevated soluble Fas in aqueous humor of patients with Behcet's uveitis: correlation with uveitis severity.

PURPOSE: To examine the role of soluble Fas (sFas) in patients with Behcet's uveitis. METHODS: We measured the sFas levels in both sera and aqueous humor (AH) of patients (n = 40) with uveitis and of non-uveitis controls (n = 27) using an enzyme-linked immunosorbent assay. The patients with uveitis comprised 24 with Behcet's disease, 6 pan-uveitis, 5 anterior uveitis, 2 lens-induced uveitis, 1 Vogt-Koyanagi-Harada disease, 1 sarcoidosis, and 1 retinal vasculitis. The severity of uveitis was determined by the Hogan grading method (0--4 grade) at the time of sampling. RESULTS: The concentration of aqueous sFas in uveitis patients was significantly higher than that in non-uveitis controls, while there was no difference in the serum concentration of sFas between the two groups. In the paired samples of serum and AH, obtained simultaneously, the aqueous sFas levels were higher than serum Fas levels in patients with uveitis, whereas the non-uveitis controls displayed significantly lower sFas levels in AH than in the serum. The sFas levels in AH or serum were not different between Behcet's uveitis patients and non-Behcet's uveitis patients. However, in patients with Behcet's uveitis, circulating sFas strongly correlated with aqueous sFas, which was not so in those with non-Behcet's uveitis. Patients (n = 29) with more active (grade greater-than-or-equal 2) uveitis had significantly higher levels of aqueous sFas than those (n = 11) with less active (grade < 2) uveitis. After treatment with steroids and/or immunosuppressive agents, aqueous sFas levels decreased in parallel with a reduction in the number of inflammatory cells. CONCLUSIONS: The levels of sFas were elevated in patients with Behcet's uveitis and correlated well with the uveitis severity in these patients.