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Ocular herpes simplex virus (HSV) infection results in an immune-mediated inflammation of the corneal stroma known as herpetic stromal keratitis (HSK). Recurrent HSK is a common cause of virus-induced corneal blindness in humans. The role of CD4(+) and CD8(+) T cell subsets in the disease pathogenesis is ill defined and varies with the virus strain and host genetic background. To examine the contribution of T cell subsets to corneal disease, we studied the development of recurrent HSK in CD4 or CD8 gene knockout (KO) mice ocularly infected with HSV-1 McKrae strain. Following UV-B induced viral reactivation, corneal opacity in latently infected BALB/c (HSV sensitive) CD4 and CD8 KO mice was reduced compared to infected BALB/c mice with normal genotype. In contrast, opacity in C57BL/6 (HSV resistant) CD4 and CD8 KO latent mice did not differ from genetically normal latent mice. Virus-induced corneal opacity was not demonstrable in C57BL/6 CD4/CD8 double KO mice. Increased viral shedding, measured by reactivation rate, days shedding or viral titers, occurred in CD4 KO mice of both strains. Our findings indicate that both CD4(+) and CD8(+) cells play a role in the immunopathogenesis of recurrent HSK, and their role is dependent upon the host genetic profile.

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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.

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PURPOSE: To compare methods of disinfecting Goldmann tonometer tips inoculated with hepatitis C virus. METHODS: Hepatitis C virus was placed on Goldmann tonometer tips, air dried, and then disinfected by dry gauze wipes, isopropyl alcohol wipes, cold water washes, povidone iodine 10% wipes, and hydrogen peroxide or isopropyl alcohol soaks followed by a cold water wash and dry. Hydrogen peroxide and isopropyl alcohol disinfection techniques followed the Centers for Disease Control and Prevention guidelines for prevention of possible transmission of human immunodeficiency virus (HIV). After disinfection, samples from tonometer tips were amplified by polymerase chain reaction to quantitate the amount of hepatitis C virus RNA remaining. RESULTS: Percentage of hepatitis C virus RNA remaining after disinfection: dry gauze wipes 95.65%, isopropyl alcohol 5-second wipes 88.91%, cold water wash 4.78%, povidone iodine 10% 5-second wipes 0.72%, hydrogen peroxide soak with cold water wash 0.07%, and isopropyl alcohol soak and cold water wash 0.02%. CONCLUSIONS: After inoculation of Goldmann tonometer tips with hepatitis C virus, a 5-minute soak in 3% hydrogen peroxide or 70% isopropyl alcohol followed by washing in cold water resulted in the greatest reduction in hepatitis C virus RNA.

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PURPOSE: Recurrent herpetic stromal keratitis (HSK) is a potentially blinding, immune-mediated disease. To better understand the immunopathology of recurrent HSK, we examined the cytokine profile of mouse corneas with the condition. METHODS: The eyes of latently infected mice were examined for corneal pathology and cytokine content following UV-B-stimulated herpes simplex virus (HSV) reactivation. RESULTS: Peak HSV-induced corneal disease, manifested by stromal opacification, occurred 7-14 days after viral reactivation in latently infected mice. In qualitative RT-PCR analyses, IFNgamma, IL-10, IL-4, and IL-12 p40 mRNA were simultaneously expressed before and during recurrent HSK. Competitive, semi-quantitative RT-PCR evaluation of cytokine mRNA revealed highest IFNgamma expression before and during clinical disease with a decline thereafter. IL-4 levels peaked and declined before day 14, while IL-10 peaked on days 7 or 14 and paralleled IFNgamma at lower levels. Small amounts of IL-12 p40 mRNA were detected late in the disease course. ELISA evaluation of corneal extracts demonstrated similar results, featuring early expression of Th2 cytokines relative to disease. CONCLUSIONS: The presence of Th2 cytokines during early stages of recurrent herpetic corneal lesions indicate the presence of a mixed Th1 and Th2 cell infiltrate, which is likely associated with a memory response to viral antigens. These data suggest that disease resolution in corneas with recurrent HSK may depend upon the balance between destructive and protective cytokines at individual sites of viral recurrence.

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BACKGROUND: Corneal endothelial cell transplantation has been an intriguing concept as an alternative to full-thickness penetrating keratoplasty. Using a murine corneal transplantation model, we sought to establish the optimal conditions to repopulate, ex vivo, denuded murine Descemet's membrane with life-extended cell cultures of murine corneal endothelial cells. These ex vivo repopulated corneas were used as donor corneas in a murine orthotopic corneal transplantation model to assess, in vivo, the function of the transplanted, life-extended murine corneal endothelial cells (MCEC). METHODS: Mouse corneas were surgically trephined and the native corneal endothelium was removed mechanically using a sterile cotton swab. Cultured murine corneal endothelial cells (life extended by expression of the SV40 large T antigen) were added onto the denuded Descemet's membrane and allowed to attach in culture at 37 degree C. Evidence of corneal cell attachment to Descemet's membrane was determined between 1 and 8 h by scanning and transmission electron microscopy. Donor life-extended corneal endothelial cells were labeled with a fluorescent dye to allow tracking of the donor cells following seeding onto denuded Descemet's membrane. In four independent experiments, the Descemet's repopulated corneas were placed into syngeneic mice and evaluated for corneal clarity after 6 weeks. RESULTS: We could detect attachment of the life-extended murine CEC by scanning and transmission electron microscopy to denuded Descemet's membrane. The optimal time for adherence was 2 h and these repopulated corneas were used as donors in a murine model of penetrating keratoplasty. Of 20 mice evaluated after 6 weeks, 4 displayed corneal clarity, and fluorescent evaluation demonstrated that only the donor corneal endothelial cells were present. CONCLUSIONS: This experimental protocol establishes that "life-extended" MCEC can bind to Descemet's membrane ex vivo and form a distinct monolayer. The repopulated Descemet's membrane allowed us to directly test the hypothesis that cultured life-extended corneal endothelial cells are functional when reintroduced into an in vivo milieu and provides evidence that specific corneal endothelial cell transplantation may be a viable alternative to pentrating keratoplasty.

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BACKGROUND: The relative contribution of CD4+ or CD8+ T cells in allograft rejection remains to be fully characterized. Some reports indicate that there is an absolute requirement for CD4+ T cells in allogeneic rejection, whereas others report that CD4-depleted mice are capable of rejecting certain types of allografts. METHODS: We compared the ability of CD4- knockout (KO), CD8- KO, and normal CD4+/CD8+ mice to reject allogeneic corneal or skin grafts. We also examined delayed-type hypersensitivity and CTL responses to donor alloantigens. RESULTS: Engraftment of C57BL/6 corneas to C.B6-(n5-7) CD4-KO mice resulted in significantly higher rates of acceptance (>85%) than either C.B6-(n5-7) CD8- KO (30%) or normal BALB/c mice (40%). Likewise, mean survival times for B6 skin grafts placed on C.B6-(n5-7) CD4- KO mice (29.2 +/- 3.5 days) were significantly increased over those of normal BALB/c mice (13.2 +/- 1 days), although most CD4- KO mice (70%) eventually reject their grafts. C.B6-(n5-7) CD4- KO mice that reject allogeneic grafts fail to develop a delayed-type hypersensitivity response, but they did demonstrate significantly greater cytotoxic T lymphocyte precursor (CTLp) frequencies than did CD4- KO mice that accepted such grafts or that were not grafted. CONCLUSIONS: This study indicates that mice lacking CD4+ T cells have a significantly impaired ability to reject corneal allografts, but are able, in most cases, to reject allogeneic skin grafts. Thus, in the absence of CD4+ T cells, the likely mechanism for rejection appears to involve the generation of CD8+ CTLs.

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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.

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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.

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PURPOSE: To determine whether routine office techniques used to disinfect tonometer prisms and trial contact lenses are sufficient to prevent transmission of ocular infections. METHOD: We reviewed the current literature on the efficacy of certain disinfection protocols against commonly encountered viral, bacterial, and fungal pathogens as well as Acanthamoeba. RESULTS: Some commonly used disinfecting solutions and techniques may be inadequate for disinfection of viruses such as hepatitis C virus and organisms such as Acanthamoeba. When used in accordance with guidelines published by the United States Centers for Disease Control and Prevention (CDC) and the American Academy of Ophthalmology (AAO), 3% hydrogen peroxide is a very effective disinfectant against a wide variety of microorganisms. Specifically, tonometer prisms disinfected by a 5-minute soak in 3% hydrogen peroxide (or 70% isopropyl alcohol or a 1:10 dilution of sodium hypochlorite) are adequately disinfected against most ocular pathogens, with the exception of Acanthamoeba. Trial contact lenses that are disinfected with a 2-hour soak in 3% hydrogen peroxide are effectively rid of all pathogens of concern. After disinfection, rigid lenses should be stored dry, and soft lenses should be stored in a sterile, preserved solution. Repeat disinfection should be routinely performed at 1-month intervals to prevent regrowth of organisms. CONCLUSION: A safe office environment can be maintained by following current CDC recommendations for disinfection, as well as instituting some additional procedures.

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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.

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PURPOSE: To investigate the role of heredity in determining corneal shape, axial length, and overall refractive error. METHODS: Twenty monozygotic and 19 dizygotic twin pairs, age 12 to 73 years, were enrolled in the study. Zygosity was determined by physical similarity and by responses to questions adapted from surveys. Two twin pairs were excluded because of undetermined zygosity and one pair because of keratoconus (both siblings). Refractive error was determined by an automated refractor. Manifest refraction was also recorded, as well as cycloplegic refraction in subjects under age 18 years. Corneal topography data and manual keratometer readings were also obtained. Axial lengths were determined by A-scan ultrasound. Data were analyzed by Student t tests only in the right eye. Left-eye data were comparable for all variables. RESULTS: Mean intrapair difference in refractive error (spherical equivalent) was less for monozygotic than for dizygotic twins (RE: 0.41 vs 1.53; P = .001). Mean intrapair difference in axial length was less for monozygotic twins (RE: 0.39 vs 0.76 mm; P = .031). Corneal topography data (power and meridian) in all zones (3, 5, and 7 mm) also showed smaller mean differences among monozygotic pairs than dizygotic, but the difference was statistically significant only for the 5-mm zone. In addition, most Holladay Diagnostic Summary variables that were studied did not show any statistically significant differences. CONCLUSIONS: Axial length and overall refractive error have a significant genetic basis. Corneal topography data appear to have other overriding determining factors for several of the variables studied.

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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.

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BACKGROUND: Cytomegalovirus retinitis remains a serious problem in AIDS patients, and the species specificity of human cytomegalovirus (HCMV) has hindered the development of animal models suitable for testing new therapeutic agents. Having previously described an in vivo model of HCMV retinal infection, we investigated its ability to reproduce the antiviral effects of the established anti-HCMV agent ganciclovir in order to determine the model's potential for evaluating novel agents. METHODS: Athymic rats had human fetal retinal tissue implanted in both anterior chambers. At 14 or 28 days post implantation, a suspension of a beta-galactosidase (lacZ+) mutant of HCMV was injected into each anterior chamber. Commencing 3 days prior to the injection of virus, rats in the treatment group received twice-daily intraperitoneal injections of ganciclovir (identical to a total of 100 mg/kg per day) for the duration of the study. The control rats received no drug. Twenty days after virus injection, the eyes of all rats were removed, sectioned and developed with X-gal substrate to detect any beta-galactosidase expression in the human tissue implants. RESULTS: Blue-staining foci of infection were detected in the implanted retinal tissue in 8 of 10 eyes from untreated control rats, but no beta-galactosidase expression was found in any of 12 eyes from animals which had received ganciclovir treatment. CONCLUSION: Intraperitoneal administration of ganciclovir successfully prevented HCMV replication in the intraocular retinal implants. This model of HCMV retinal infection is therefore suitable for preliminary evaluation of systemically administered antiviral agents.

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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.

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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.

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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.

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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.

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