Management of the corneal flap in laser in situ keratomileusis after previous radial keratotomy.

PURPOSE: To report a method of managing the corneal flap in patients having laser in situ keratomileusis for the treatment of residual refractive errors after radial keratotomy. DESIGN: Retrospective case series. METHODS: Intraoperative dehiscence of radial keratotomy wounds occurred in 7 eyes of 6 patients treated with laser in situ keratomileusis for residual myopia or astigmatism or hyperopia 5 to 15 years after radial keratotomy. To minimize extension of these tears, the flap was initially rolled like a carpet toward the hinge before the ablation and then rolled away from the hinge to its original position after the ablation. RESULTS: Using this method of managing the laser in situ keratomileusis flap in patients with previous radial keratotomy, all eyes had successful laser in situ keratomileusis, with 1-year postoperative uncorrected visual acuity ranging between 20/16 and 20/25. No eye had loss of spectacle-corrected vision or interface epithelial ingrowth. CONCLUSIONS: Laser in situ keratomileusis has proved to be an effective treatment for correction of residual refractive errors after radial keratotomy. The surgical technique used in these cases was targeted at minimizing shearing forces in lifting the corneal flap to avoid extension of radial keratotomy wound dehiscence, which could lead to epithelial ingrowth and loss of best-corrected vision.

IL-1 and TNF-alpha are important factors in the pathogenesis of murine recurrent herpetic stromal keratitis.

PURPOSE: To better understand the role of interleukin (IL)-1 and tumor necrosis factor (NF)alpha in recurrent herpetic stromal keratitis (HSK), the cytokine content and the effects of anti-cytokine antibodies on mouse corneas with the disease were examined. METHODS: Competitive reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent analyses of IL-1alpha and TNF-alpha content were performed on corneas removed 3, 5, 7, 10, 14, and 21 days after latently infected NIH mice were irradiated with UV-B light to reactivate herpes simplex virus (HSV). In separate experiments, mice were injected with anti-IL-1 or anti-TNF-a antibodies 1 day before and 7 days after reactivation. RESULTS: UV-B irradiation stimulated an increase in corneal IL-la mRNA in reactivated (virus shedding) mice. This increase persisted longer and was higher than in UV-B irradiated uninfected control animals. IL-1alpha and TNF-alpha protein in corneas of reactivated mice was significantly elevated on days 3 to 10 compared with day 0 levels, and exceeded levels in control corneas on the same days. Anti-IL-1 and anti-TNF-alpha antibody administration both resulted in significantly decreased virus-induced corneal opacity between 7 and 21 days after UV-B exposure. CONCLUSIONS: IL-1alpha and TNF-alpha are upregulated in corneas in mice experiencing recurrent HSK. Abrogation of virus-induced corneal disease by anti-cytokine antibodies suggests that these cytokines play important roles in the pathogenesis of recurrent disease. Therefore, neutralization of specific proinflammatory cytokines may have potential therapeutic value.

Stimulation of quiescent corneal endothelial cells by direct delivery of the SV40 large T-antigen protein.

PURPOSE: To determine whether the delivery of the SV40 large T-antigen is a feasible method for transiently inducing proliferation of corneal endothelial cells. METHODS: Liposome-mediated delivery of proteins into bovine corneal endothelial cells (BCEC) was utilized in this study. Initially, beta-galactosidase was used as a marker protein for cell delivery and cells were assayed colorimetrically for beta-galactosidase activity. Subsequently, SV40 large T-antigen protein was introduced into BCEC and positive cells were identified by immunohistochemistry 24 hours after liposome-protein treatment. Quiescent BCECs were double-labeled using BrdU as a measure of de novo DNA synthesis and the SV40 large T-antigen was detected by standard immunohistochemical methods. RESULTS: Beta-galactosidase or SV40 large T antigen were introduced into BCECs using liposome transfer methods. The transfer efficiency of beta-galactosidase was > 30% of the cells. SV40 large T antigen was successfully introduced and was localized to the nuclei of BCECs. The treatment of quiescent BCECs with large T antigen caused an increase in BrdU incorporation. Co-labeling confirmed that only cells containing SV40 large T antigen were positive for de novo DNA synthesis. CONCLUSIONS: This study demonstrates that proteins can be inserted directly into corneal endothelial cells. In the case of the SV40 large T-antigen, the protein localized to the nucleus and maintained its bioactivity by inducing DNA synthesis. This finding suggests that liposome-mediated delivery of transforming proteins could be a method to transiently induce corneal endothelial cell proliferation.

Corneal iron deposits after laser in situ keratomileusis.

PURPOSE: To report the occurrence and features of corneal iron line deposition after laser in situ keratomileusis (LASIK). METHODS: We evaluated 83 eyes undergoing LASIK. Corneal iron line deposition was analyzed with respect to preoperative spherical equivalent, attempted correction, and postoperative time interval. RESULTS: Thirty-five (42.2%) of 83 eyes displayed a distinctive brown-colored corneal iron line of variable density in a ring or patch configuration near the margin of the ablated zone in the overlying corneal flap epithelium. The appearance of this iron line correlated positively with time after surgery (>3 months) and preoperative spherical equivalent (>-4.5 diopters). CONCLUSIONS: Corneal iron line deposition in a ring or patch can be associated with previous LASIK surgery. This iron deposition within the margin of the ablated zone may offer insights into the dynamics of epithelial cell hyperplasia as well as basal cell proliferation, differentiation, and migration after LASIK.

Human cytomegalovirus infection in a retinoblastoma cell line in vitro.

BACKGROUND: The focus of these studies was to determine whether the Y79 human retinoblastoma cell line could function as a good in vitro model system for studying human cytomegalovirus (HCMV) infection. METHODS: Y79 cells were exposed to an HCMV mutant carrying a LacZ gene, and the resulting beta-galactosidase expression in infected cells was assessed by flow cytometry. The extent to which the three classes of viral gene products immediate early, early, and late proteins - were expressed was analyzed by immunohistochemical staining and Western blotting. Infected Y79 cells were also co-cultivated on human foreskin fibroblast (SF cell) cultures to recover virus. RESULTS: Infection of Y79 cells with the virus resulted in beta-galactosidase expression as detected by flow-cytometric analysis. Immunohistochemical staining revealed that a portion of Y79 cells expressed antigens reactive to monoclonal antibodies against immediate early, early, and late HCMV proteins. The 43-kDa early gene product was also detected by Western blotting. Infected Y79 cells co-cultivated on SF cell cultures yielded infectious foci, which turned blue following X-gal staining, demonstrating productive HCMV infection in the Y79 cells. CONCLUSION: These results demonstrate that while HCMV can productively infect Y79 cultures, it does so in a highly inefficient manner, leading these authors to conclude that this cell line does not provide a particularly good model system to study HCMV infection.

Neural network computer program to determine photorefractive keratectomy nomograms.

PURPOSE: To evaluate a commercially available neural network program for calculation of photorefractive keratectomy treatment nomograms. SETTING: University referral refractive surgery clinic. METHODS: PRK/LASIK Brain, a commercial neural network computer program, was trained using the demographics, preoperative clinical data, surgical parameters, and 1 year postoperative clinical data of 44 patients treated with a Summit Technology excimer laser using a 5.0 mm optical zone. The neural-network derived nomogram was compared with the standard treatment nomogram for each patient. The relative contribution of age, sex, keratometry, and intraocular pressure (IOP) to the predicted nomograms was also assessed. RESULTS: Nomograms produced by the neural network were qualitatively similar to the standard nomogram. The sequence of data entry during training affected the network's predictions. Entry ordered by outcome (as opposed to entry by chronological order) yielded a nomogram that was more consistent with the standard nomogram. However, both outcome- and chronologically ordered network-derived nomograms diverged from the standard nomogram in individual patients, including a subset for whom use of the standard nomogram yielded desired refractive results (within 0.25 diopter of emmetropia). Further analysis of the neural-network-derived nomograms revealed marked sensitivity to sex, age, keratometry readings, and IOP. CONCLUSIONS: Neural networks offer a potential means of individualizing treatment nomograms, to account for patient demographics, preoperative examination, surgeon style, and equipment bias. However, a data set of 44 patients was not sufficient to train the PRK/LASIK Brain network to accurately predict treatment parameters in individual cases in the training set. A larger training set or a different learning algorithm may be required to improve the neural network's performance.

Postexposure vaccination with a virion host shutoff defective mutant reduces UV-B radiation-induced ocular herpes simplex virus shedding in mice.

A herpes simplex virus type 1 (HSV-1) recombinant (UL41NHB) deficient in the virion host shutoff (vhs) function was tested as a therapeutic vaccine in an ultraviolet (UV) light-induced mouse ocular reactivation model. Mice were infected with HSV-1 via the cornea. Following the establishment of latency by HSV-1 the mice were subsequently vaccinated intraperitoneally with one dose of UL41NHB or with uninfected cell extract. Mice were subsequently UV-irradiated to induce viral reactivation and during the 7 days post-UV irradiation, numbers of mice shedding virus were reduced from 13/23 (57%) to 3/25 (12%), and numbers of virus-positive eye swabs were reduced from 40/161 (25%) to 6/175 (3%) by the vaccine (P < 0.001). These data suggest that deletion of vhs may be a useful strategy in the development of attenuated therapeutic HSV vaccines.

Screening of myopic photorefractive keratectomy in eye bank eyes by computerized videokeratography.

BACKGROUND: In contrast to incisional keratotomy, corneas that have undergone photorefractive keratectomy may be difficult to detect by inspection with slitlamp biomicroscopy alone. Eye bank corneas that have undergone high myopic refractive surgical correction could potentially result in substantial postoperative hyperopic correction if used as donor tissue for corneal transplantation. Surface irregularities or displacement of the treated optical zone within the graft in relation to the entrance pupil of the recipient could result in significant induced astigmatism and distortion. This study examines computerized videokeratographic screening of eye bank globes as a strategy for detecting myopic photorefractive keratectomy. METHODS: Preoperative and postoperative corneal topographic maps of freshly enucleated human and rabbit eyes that have undergone myopic photorefractive keratectomy with an excimer laser were placed in a globe-fixating device and analyzed using a vertically oriented videokeratoscope. The same system was applied in an actual eye bank setting, and potentially transplantable globes from donors without a history of corneal surgery were analyzed. RESULTS: Computerized videokeratography using a vertically mounted system reliably detected photorefractive keratectomy in 12 of 12 human eye bank corneas treated by excimer photorefractive keratectomy in a range between -1.5 to -6.0 diopters. This method also detected similar changes on lased rabbit corneas enucleated 6 weeks after excimer surgery. Data processed with the tangential mode yielded a "bull's-eye" topography pattern reflecting central corneal flattening that was more sensitive in detecting myopic corrections than the conventional axial formula-based color maps. False-positive results were not detected in 96 cadaver globes sequentially screened in the eye bank. CONCLUSIONS: Computerized videokeratography represents a feasible method to screen donor globes for myopic photorefractive keratectomy as shown by the in vitro and rabbit models. However, only whole globes and not corneoscleral sections are amenable to processing with this technique. Tangential maps provided greater sensitivity in detecting low myopic corrections than the axial formula-based color maps.

Efficacy and safety of the ProTek (Vifilcon A) therapeutic soft contact lens after photorefractive keratectomy.

PURPOSE: To test the ProTek (Vifilcon A) therapeutic soft contact lens in the alleviation of post-photorefractive keratectomy pain, its effect on epithelial healing, and its safety. METHODS: Forty-seven consecutive eligible patients undergoing unilateral excimer laser photorefractive keratectomy for myopia were randomly assigned to receive standard postoperative care with or without the use of a ProTek soft contact lens. Patients prospectively graded a self-administered 5-point scale for pain and a 4-point scale for abnormal sensations at 4, 8, 12, 16, and 20 hours after surgery. They also recorded the type and dose of all medications taken during that time period. All patients were examined on the first and third days after surgery. The lenses were worn continuously for 3 days. RESULTS: The soft contact lens group (n = 24) disclosed a statistically significant (P < .05) reduction in pain intensity and abnormal sensations that was greatest at 8, 12, 16, and 20 hours postoperatively. Compared with control patients (n = 23), the soft contact lens group showed significant decreased dependence on most pain medications after the 12th hour (P < .05) and faster epithelial healing (P = .03). However, one case of bacterial keratitis, two cases of subepithelial infiltrates, and seven cases of contact lens intolerance were present in the soft contact lens group. CONCLUSIONS: The ProTek therapeutic soft contact lenses were effective in decreasing pain and other related abnormal sensations after excimer photorefractive keratectomy. They decreased dependence on pain medications and hastened epithelial healing but were not well tolerated in some patients.

Efficacy of a recombinant glycoprotein D subunit vaccine on the development of primary and recurrent ocular infection with herpes simplex virus type 1 in mice.

The protective efficacy of a glycoprotein D subunit vaccine (gD2 SB AS4) was evaluated in a mouse model of human recurrent herpetic stromal keratitis (HSK). When administered before primary infection, gD2 SB AS4 protected mice against corneal pathology, mortality, and latency resulting from ocular viral challenge with herpes simplex virus type 1 (HSV-1) McKrae strain. In addition, gD2 SB AS4 significantly decreased postreactivation corneal disease. A control vaccine, gD2 alum, protected against acute ocular infection only. When administered after primary infection, gD2 SB AS4 vaccination decreased postreactivation ocular shedding but had no other significant effects. Vaccination with gD2 SB AS4 was associated with high anti-gD antibody responses and low delayed-type hypersensitivity responses. These results have identified a prophylactic vaccine, gD2 SB AS4, with activity against acute and recurrent HSK in mice and emphasize the need for vaccine evaluation in both primary and recurrent ocular herpetic disease models.

Differential effects of HSV-1 and HCMV infection on adhesion molecule expression on human corneal keratocytes.

PURPOSE: Previous studies have shown that keratoplasty buttons obtained at surgery from patients with herpes simplex virus-1 (HSV-1) keratitis have elevated localized expression of the adhesion molecule ICAM-1, which plays a critical role in the initiation and amplification of an immune response. We performed studies to determine whether changes in expression of ICAM-1 and HLA class I are direct effects of productive infection of human corneal fibroblasts with HSV-1. METHODS: Immunocytologic and flow cytometric analyses were performed to analyze the ability of HSV-1 to induce ICAM-1 and HLA class I expression in a primary cornea-derived keratocyte cell line, E-2. Positive controls for these experiments were E-2 cells infected with human cytomegalovirus (HCMV), which has been shown to increase ICAM-1 expression in selected cells, and E-2 cells treated with IFN-gamma, which upregulates both ICAM-1 and HLA class I expression in most cell types. RESULTS: Kinetic cytometric analysis indicated decreased ICAM-1 expression 3 hours following HSV-1 infection of E-2 cells. In contrast, HCMV led to detectable increases in ICAM-1 expression starting 6 hours after infection. Infections with either HSV-1 or HCMV resulted in reduced HLA class I expression on E-2 and SF cells. CONCLUSIONS: These studies suggest that increased ICAM-1 expression seen on corneal stromal cells during clinical HSV-1 infection is not a direct result of productive viral infection, but of other mechanisms such as cytokine release by infiltrating mononuclear cells.

A critical evaluation of hepatitis C testing of cadaveric corneal donors.

PURPOSE: Enzyme immunoassays (EIAs) for hepatitis C virus (HCV) antibodies used to screen corneal donors are optimized for testing premortem sera. This study evaluated their efficiency when screening cadaveric sera. METHODS: Abbott HCV EIA 2.0 was used to rescreen 101 cadaveric sera, 70 of which had tested positive and 31 negative by EIA 1.0. Matrix-HCV recombinant immunoblot assay was used as a reference standard. Antibody titers and reactivities were compared in premortem and cadaveric sera. Selected sera from confirmed seropositive donors were screened for virus by polymerase chain reaction (PCR). RESULTS: HCV EIA 2.0 was 100% sensitive and 92.7% specific. EIA and Matrix-HCV gave similar end-point titers with pre- and postmortem sera. Viral RNA was detected in only three of 15 sera from seropositive donors. CONCLUSIONS: EIA 2.0 and Matrix-HCV efficiently screen cadaveric sera. However, HCV seropositivity does not necessarily indicate the presence of viral genomes in sera and tissues.

Retreatment of decentered excimer photorefractive keratectomy ablations.

PURPOSE: To present two cases of excimer photorefractive keratectomy decentration associated with visual distortion secondary to irregular astigmatism. METHOD: Patients were retreated with a repeat photorefractive keratectomy using a technique whereby the circle of adherent epithelium overlying the decentered ablation served as a mask. RESULTS: After retreatment, there was significant improvement in the patients' visual symptoms, decreased astigmatism on refraction, and better centration on corneal topography. CONCLUSION: This method appears to offer an effective means to treat photorefractive keratectomy ablation zone decentration by improving the abnormalities introduced by the initial decentration.

Screening potential corneal donors for HIV-1 by polymerase chain reaction and a colorimetric microwell hybridization assay.

PURPOSE: Current screening of potential corneal donors for human immunodeficiency virus type 1 (HIV-1) involves serologic detection of antibodies to the virus. However, this approach cannot detect infection during the seronegative window period of the disease. We therefore evaluated the polymerase chain reaction (PCR) assay for viral nucleic acid as a possible alternative to screening cadaveric blood for HIV-1. METHODS: Blood specimens from cadavers diagnosed at autopsy with acquired immunodeficiency syndrome (AIDS) (n = 21), at high risk for HIV-1 infection (n = 47), and at no known risk (n = 350) were screened by PCR for HIV-1 proviral DNA and human leukocyte antigen (HLA)-DQ alpha sequences, and for HIV antibodies. RESULTS: All AIDS group samples were seropositive; of these, 18 (86%) and 20 (95%) of 21 were positive for HIV by PCR of proteinase K- and Chelex-extracted pellets, respectively. The seropositive samples negative by PCR testing were shown to inhibit PCR amplification. Nine (19%) of 47 high-risk specimens were HIV-positive. The no-known-risk group yielded negative results. The overall sensitivities for PCR in the proteinase K- and Chelex-treated groups were 90% and 97%, respectively, compared with Western blot reactivity. If PCR-inhibitory samples and HLA-DQ alpha-negative samples had been eliminated, sensitivity would have been 100%. Specificity was 100% for each group. CONCLUSIONS: Screening cadaveric blood by PCR may be feasible, but further refinement of the assay and blood specimen collection practices will be necessary for it to become routine. Future studies should focus on optimizing specimen procurement and preparation to reduce or eliminate specimens that inhibit PCR.

A comparison of recurrent and primary herpes simplex keratitis in NIH inbred mice.

Herpes simplex virus (HSV) infection is one of the leading causes of corneal blindness. This study compared the clinical, virologic, and immunopathologic features of primary and recurrent murine models of herpes simplex keratitis (HSK) in the National Institutes of Health (NIH) inbred mouse strain. Primary infection resulted in multiple epithelial dendrites, followed by diffuse stromal opacification, symptoms that do not mimic what is seen in human HSK. In contrast, recurrent infection presented clinical features that included microdendrites, focal stromal opacities, disciform endotheliitis, and corneal neovascularization, which were similar to those observed in human disease. Immunohistochemical characterizations indicated that the number and duration of T cells and macrophages in recurrent HSK resemble those observed in primary disease. Results also indicated that the amount of infectious virus detected in the cornea during primary and recurrent disease was similar. However, when corneas were stained for HSV-1 antigens, mice with primary HSK displayed diffuse HSV antigen expression throughout the cornea, whereas HSV antigens were more focally distributed in recurrent disease. These data suggest that the clinical differences between the recurrent and primary herpetic keratitis may, in part, reflect the different distribution of HSV-1 antigens within the cornea.

Cystic hydrops and spontaneous perforation in Fuchs' superficial marginal keratitis.

PURPOSE: We studied a case of Fuchs' superficial marginal keratitis in which cystic corneal hydrops developed in one eye, and spontaneous corneal perforation occurred in the fellow eye. METHODS: The patient underwent bilateral annular corneoscleral lamellar patch grafts for tectonic support. RESULTS: The patient had an uncomplicated postoperative course and visual acuity of 20/25 bilaterally. CONCLUSION: Stromal loss in Fuchs' superficial marginal keratitis may be severe enough to result in cystic corneal hydrops. Patients with this disorder should be counseled regarding the possibility of corneal perforation.