Modulation of Contact Inhibition by ZO-1/ZONAB Gene Transfer-A New Strategy to Increase the Endothelial Cell Density of Corneal Grafts.

Purpose: Endothelial cell density (ECD) is the principal factor determining the success of corneal transplants. Here we explored a strategy to increase corneal ECD in human explants via modulation of the ZO-1/ZONAB pathway. In multiple cell types, ZO-1 maintains G1 cell cycle arrest via cytoplasmic sequestration of the mitosis-inducing transcription factor ZONAB. In this study, we assessed the effects of lentiviral vector-mediated downregulation of ZO-1 or overexpression of ZONAB upon ECD and the integrity of the endothelial monolayer. Methods: HIV-based lentiviral vectors were used to deliver either constitutively expressed ZONAB (LNT-ZONAB), or a small hairpin RNA targeting ZO-1 (LNT-shZO1). Human corneal specimens were bisected and each half was exposed to either treatment or control vector. After 1 week in ex vivo culture, effects were assessed by quantitative RT-PCR, immunohistochemistry, and ECD assessment. Results: LNT-shZO1 achieved an ∼45% knockdown of ZO-1 mRNA in corneal endothelial cells cultured ex vivo, reduced ZO-1 staining, and did not affect morphologic endothelial monolayer integrity. The proliferative effect of LNT-shZO1 correlated with control ECD but not with donor age. Within a low-ECD cohort an ∼30% increase in ECD was observed. LNT-ZONAB achieved a >200-fold overexpression of ZONAB mRNA, which led to an ∼25% increase in ECD. Conclusions: ZO-1 downregulation or ZONAB upregulation increases corneal ECD via interference with contact inhibition and cell cycle control. With further development, such approaches might provide a means for improving ECD in donor corneas before transplantation.

Sustained and Widespread Gene Delivery to the Corneal Epithelium via In Situ Transduction of Limbal Epithelial Stem Cells, Using Lentiviral and Adeno-Associated Viral Vectors.

Corneal epithelial dystrophies are typically characterized by symptoms such as pain, light sensitivity, and corneal opacification leading to impaired vision. The development of gene therapy for such conditions has been hindered by an inability to achieve sustained and extensive gene transfer, as the epithelium is highly replicative and has evolved to exclude foreign material. We undertook a comprehensive study in mice aiming to overcome these impediments. Direct injection of lentiviral vector within the stem cell niche resulted in centripetal streaks of epithelial transgene expression sustained for >1 year, indicating limbal epithelial stem cell transduction in situ. The extent of transgene expression varied markedly but at maximum covered 26% of the corneal surface. After intrastromal injection, adeno-associated viral (AAV) vectors were found to penetrate Bowman's membrane and mediate widespread, but transient (12-16 days), epithelial transgene expression. This was sufficient, when applied within a Cre/lox system, to result in recombined epithelium covering up to approximately 80% of the corneal surface. Lastly, systemic delivery of AAV2/9 in neonatal mice resulted in extensive corneal transduction, despite the relative avascularity of the tissue. These findings provide the foundations of a gene therapy toolkit for the corneal epithelium, which might be applied to correction of inherited epithelial dystrophies.

Papilloma development and long-term ciclosporin use in chronic ocular allergy with associated keratoconus.

PURPOSE: Conjunctival papillomata are squamous epithelial tumors with a strong association with human papilloma virus (HPV) types 6 and 11. They are benign conjunctival tumors that can be treated by surgical excision. We report a case where topical immunosuppressive therapy modified the local T-cell immunity in the conjunctiva resulting in papilloma development in a patient with keratoconus and a strong atopic history. METHODS: A case report of a 44-year-old man with a history of severe ocular and generalized atopy is presented. We present the problems encountered in management of his severe ocular allergy and how these impeded the management of his keratoconus. RESULTS: Conventional antiallergy topical medication was not producing symptom relief in this patient, and so topical immunosuppression was commenced using ciclosporin ointment 0.2%. This therapy modified the local T-cell immunity in the conjunctiva resulting in the development of papillomata which contributed to the intolerance of contact lens wear for visual rehabilitation of the keratoconus in the patient. These lesions were surgically removed but typically recurred and required further surgical excision. Adjunct cryotherapy was also performed at the time of the surgery to try to stem the recurrence of the papillomas. CONCLUSIONS: To the best of our knowledge and following a review of the published literature using key databases that include Medline and PubMed, this is the first report confirming the development of conjunctival papillomas secondary to HPV type 6 in a ciclosporin-treated patient.

Immune privilege of corneal allografts.

Corneal transplantation has been performed successfully for over 100 years. Normally, HLA typing and systemic immunosuppressive drugs are not utilized, yet 90% of corneal allografts survive. In rodents, corneal allografts representing maximal histoincompatibility enjoy >50% survival even without immunosuppressive drugs. By contrast, other categories of transplants are invariably rejected in such donor/host combinations. The acceptance of corneal allografts compared to other categories of allografts is called immune privilege. The cornea expresses factors that contribute to immune privilege by preventing the induction and expression of immune responses to histocompatibility antigens on the corneal allograft. Among these are soluble and cell membrane molecules that block immune effector elements and also apoptosis of T lymphocytes. However, some conditions rob the corneal allograft of its immune privilege and promote rejection, which remains the leading cause of corneal allograft failure. Recent studies have examined new strategies for restoring immune privilege to such high-risk hosts.

Penetrating and deep anterior lamellar keratoplasty for keratoconus: a comparison of graft outcomes in the United kingdom.

PURPOSE: To compare outcomes after penetrating keratoplasty (PK) and deep anterior lamellar keratoplasty (DALK) for keratoconus in the United Kingdom. METHODS: Patient outcome data were collected at the time of transplantation and at 1, 2, and 5 years after surgery. Data were analyzed by Kaplan-Meier survival curves, Cox regression, and binary logistic regression to determine the influence of surgical procedure on graft survival and visual outcome. RESULTS: The risk of graft failure for DALK was almost twice that for PK (P = 0.02). Nineteen percent of the DALK failures occurred in the first 30 postoperative days compared with only 2% of PK failures. When these early failures were excluded, there was little difference between the 3-year graft survivals for DALK (92%; 95% confidence interval [CI], 85%-95%) and PK (94%; 95% CI, 92%-95%) (P = 0.8). Although the mean best corrected visual acuity (BCVA) was similar for the two procedures (P = 0.7), 33% of patients who underwent PK achieved a BCVA of 6/6 or better at 2 years compared with only 22% of those who underwent DALK (P < 0.001). Those with DALK were also likely to be more myopic (< -3 D) but there was little difference in scalar cylinder. CONCLUSIONS: DALK had a higher overall failure rate than PK. The difference was largely accounted for by early failures, which appeared to be related to the surgeon's experience. DALK recipients were less likely to achieve BCVA of 6/6 than were PK recipients and were more likely to have -3 D or worse myopia.

Expression of the chemokine antagonist vMIP II using a non-viral vector can prolong corneal allograft survival.

BACKGROUND: The expression of chemokines is central to the recruitment of inflammatory cells for graft rejection, and modulation of chemokine action is of potential in preventing graft rejection. We have examined chemokine expression in a murine model of corneal allograft rejection, and also determined the effect of expressing a broad acting chemokine antagonist, viral macrophage inflammatory protein II (vMIP II), on graft survival. METHOD: The expression of chemokines in a murine model of corneal transplantation was determined by real time RT-PCR and, in the case of regulated on activation normal T-cell expressed and secreted, by ELISA. The plasmid encoding the virally derived chemokine antagonist, vMIP II, was introduced into the corneal endothelial cells using a non-viral vector consisting of liposomes and transferrin. The expression and activity of vMIP II was determined by ELISA and functional assays, and the effect on graft survival noted. RESULTS: After allotransplantation, there was up-regulation of all 11 chemokines examined. After gene delivery, there was expression of active vMIP II for more than 14 days and considerable prolongation of graft survival. This was associated with a decrease in leukocyte infiltration of the stroma of the cells. CONCLUSION: As expected there was considerable up-regulation of chemokines during allograft rejection. The expression of vMIP II showed considerable prolongation of graft survival. This is the first time we have observed prolongation of graft survival after a non-viral (as opposed to viral) means of gene delivery and indicates the potential of interfering with chemokine action to prevent corneal graft failure.

Comparison of HIV-1 and EIAV-based lentiviral vectors in corneal transduction.

In this study we compare the ability of self-inactivating Human Immunodeficiency Virus 1 (HIV-1) and Equine Infectious Anaemia Virus (EIAV)-based vectors to mediate gene transfer to rabbit and human corneas and to a murine corneal endothelial cell line. Both vectors were pseudotyped with vesicular stomatitis virus-G (VSV-G) envelope and contained marker transgenes under the control of an internal CMV promoter. For specificity of action, the heterologous promoter in the EIAV-vector was exchanged for an inducible E-Selectin promoter, previously shown to regulate gene-expression in a plasmid system. We show that EIAV is more efficient than HIV in transducing human and rabbit corneal endothelial cells. Rabbit corneal endothelial cells are transduced in higher quantity than human corneal endothelial cells. In the inducible system, however, we detected impairment between the vector and its internal E-Selectin promoter. Instead of controlled transgene expression or silencing of promoter activity, the U3-modified long-terminal-repeats (LTR) impaired the conditional activity of the E-Selectin promoter. Significant transgene expression was seen without stimulation of the inducible promoter. We show efficient transduction by lentiviruses of a corneal endothelial cell line and of full thickness corneas from different species, confirming that those vectors would be appropriate tools for gene therapy of selected corneal diseases. However, the modification within the U3-LTR did not adequately allow regulated transgene expression. These findings have important implications for vector design for diagnostic or therapeutic opportunities.

Standardized arcuate keratotomy for postkeratoplasty astigmatism.

PURPOSE: To assess the effect of standardized, paired arcuate keratotomy (AK) on the change in astigmatism in postkeratoplasty eyes. SETTING: Moorfields Eye Hospital, London, United Kingdom. METHODS: A retrospective review was conducted of 20 eyes of 19 patients having the same AK procedure regardless of the magnitude of the preoperative astigmatism. Each eye had a pair of 60-degree arc length incisions placed in the corneal stroma. The incisions were 600 mum deep and 6.0 mm apart. The preoperative and postoperative refractions and complications were analyzed. Astigmatic change was analyzed without regard to axis, as surgically induced refractive change, and using a modified polar plot of change in astigmatism. RESULTS: The mean cylinder was reduced from -10.99 diopters (D) +/- 4.26 (SD) to -3.33 +/- 2.18 D. There was no significant change in the mean spherical equivalent. There was a strong correlation between the magnitude of the preoperative cylinder and the magnitude of the change in astigmatism (R2 = 0.76). In 3 eyes, the surgically induced axis of astigmatism was more than 15 degrees from that expected. CONCLUSIONS: In postkeratoplasty eyes, the change in the magnitude of astigmatism induced by standardized AK was proportional to the preoperative magnitude of astigmatism. Arcuate nomograms for congenital astigmatism have no role in the management of astigmatism in postkeratoplasty eyes.

Modulation of human dendritic-cell function following transduction with viral vectors: implications for gene therapy.

Genetic modification of dendritic-cell (DC) function is an attractive approach to treat disease, either using mature DCs (mDCs) to immunize patients, or immature DCs (iDCs) to induce tolerance. Viral vectors are efficient at transducing DCs, and we have investigated the effect of transduction with a variety of viral vectors on the phenotype and function of DCs. Adenovirus (Ad), human immunodeficiency virus (HIV), equine anemia virus (EIAV), and Moloney murine leukemia virus (MMLV) all up-regulate costimulatory molecules and major histocompatibility complex (MHC) class II expression on DCs, as well as, in the case of Ad and lentiviral vectors, inducing production of Th1 and proinflammatory cytokines. Following transduction there is activation of double-stranded (ds) RNA-triggered pathways resulting in interferon (IFN) alpha/beta production. In addition, the function of virally infected DCs is altered; iDCs have an increased, and mDCs a decreased, ability to stimulate a mixed lymphocyte reaction (MLR). Viral transduction of mDCs results in up-regulation of the indoleamine 2,3-dioxygenase (IDO) enzyme, which down-regulates T-cell responsiveness. Inhibition of IDO restores the ability of mDCs to stimulate an MLR, indicating that IDO is responsible for the modulation of mDC function. These data have important implications for the use of viral vectors in the transduction of DCs.

Acanthamoeba sclerokeratitis: treatment with systemic immunosuppression.

OBJECTIVE: This study describes the clinical features, management, and outcome of 19 patients who had severe Acanthamoeba sclerokeratitis (ASK) unresponsive to conventional management, requiring systemic immunosuppression to control disease. DESIGN: Retrospective, non-comparative, interventional case series. PARTICIPANTS: Records of all patients with Acanthamoeba keratitis treated at Moorfields Eye Hospital between 1989 and 2000 were reviewed. From more than 200 patients, 19 who developed ASK treated with systemic immunosuppression were identified. MAIN OUTCOME MEASURES: Visual acuity, level of pain, and degree of inflammation were recorded after immunosuppressive treatment. RESULTS: ASK requiring immunosuppression occurred in 20 eyes of 19 patients (11 males and 8 females). The mean age (mean +/- standard deviation) at onset was 38.6 +/- 13.2 years. On presentation, best-corrected visual acuity was counting fingers or worse in 11 eyes (55%), 6/18 to 6/60 in 5 eyes (25%), and 6/12 or better in 4 eyes (20%). The mean time between onset of initial symptoms of Acanthamoeba keratitis and commencement of systemic immunosuppression was 4.8 +/- 3.5 months. The mean duration of immunosuppression required to control inflammation was 7.2 +/- 3.9 months. Severe scleritic pain remained uncontrolled in two patients and resulted in enucleation. Best-corrected visual acuity at final follow-up was counting fingers or worse in eight eyes (40%), 6/18 to 6/60 in six eyes (30%), and 20/40 or better in six eyes (30%). The mean follow-up period after resolution of inflammation was 24.3 +/- 20.9 months (range, 0.2-59.7 months). CONCLUSIONS: ASK is an uncommon complication of Acanthamoeba keratitis. The scleritis associated with this infection seems to be an immune-mediated response. After topical amebicidal treatment, systemic immunosuppression may be required to control the pain and tissue destruction associated with ASK.

Effect of administration of CTLA4-Ig as protein or cDNA on corneal allograft survival.

PURPOSE: To examine the role of the CD28-CD80-CD86 pathway of T-lymphocyte costimulation in corneal allograft rejection and the effect of blockade of that pathway on graft survival. METHODS: Kinetics of CD80 and CD86 expression in the cornea and draining lymph nodes were examined by RT-PCR and immunohistochemistry in untreated allograft recipients in a high-responder rat model. The effect of blockade of CD28-mediated costimulation was first examined by ex vivo incubation of excised Brown Norway rat donor cornea with the inhibitory protein CTLA4-Ig or an adenovirus vector (AdCTLA) expressing CTLA4-Ig, before grafting into Lewis rat recipients. A second group of graft recipients received systemic posttransplantation treatment with either CTLA4-Ig or AdCTLA. RESULTS: Expression of CD80 mRNA was increased in both donor and recipient cornea 16 hours after transplantation, whereas CD86 was detected constitutively, with no significant early increase. Immunohistochemistry on day 5 after transplantation demonstrated major histocompatibility complex (MHC) class II expression, no CD80, and only a trace of CD86 in corneal allografts. In lymph nodes strong MHC class II, weak CD80, and moderate CD86 expression was noted. Both donor cornea and recipient treatment with CTLA4-Ig resulted in prolonged allograft survival. AdCTLA was found to induce sustained secretion of bioactive CTLA4-Ig from corneas infected ex vivo. Survival of corneal allografts incubated with AdCTLA was marginally prolonged, and systemic treatment with AdCTLA significantly prolonged survival. CONCLUSIONS: Protein- or gene-based administration of CTLA4-Ig prolongs allograft survival by treatment of either the recipient or the donor tissue ex vivo before grafting.