Browsing by Subject "Corneal Transplantation"
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Item Destruction of Corneal Nerves Promotes Corneal Allograft Rejection(2014-06-11) Paunicka, Kathryn Joy; Street, Nancy E.; Niederkorn, Jerry Y.; Gruchalla, Rebecca S.; Gill, Michelle A.The human corneal endothelium has very little regenerative capacities and cannot fully heal in response to infection or trauma. Evolutionarily, the eye developed a mechanism to retain visual acuity by protecting the eye from inflammatory damage, referred to as immune privilege. The mechanisms that protect the eye from inflammation-induced injury are 1.) the presence of immunosuppressive molecules within the aqueous humor; 2.) the expression of pro-apoptotic factors on corneal cells ; and 3.) the induction of a form of immune tolerance called anterior chamber-associated immune deviation (ACAID). Immune privilege contributes to the 90% success rate of corneal allografts without the need for histocompatibility matching and use of systemic immunosuppressive therapy. However, when one or more parameters that contribute to immune privilege are broken, the cornel allograft becomes vulnerable to the immune system, resulting in corneal allograft failure. Patients that elect to have a corneal allograft replaced are the population at the highest risk of immune rejection and have only a 70% success rate with the second corneal allograft, and the rate of rejection continues to increase with each successive graft. Why subsequent corneal allografts have an increased incidence of rejection is unknown. Due to the high success rate of corneal allografts, the donor tissues are not tissue matched to the recipients. With the limited documentation on the histocompatibility gene loci expressed by the corneal tissue donors, it is unknown if the rejection of the initial corneal allograft sensitizes the corneal allograft recipient. This study provides evidence that the sensitization of the corneal transplant recipient towards alloantigens also expressed on the subsequent corneal allografts is not a requisite for the high incidence of graft rejection. Furthermore, the enhanced incidence of graft rejection is an immune response directed towards alloantigens expressed on the subsequent corneal transplant. The aim of this study is to determine why the corneal transplantation procedure enhances the rejection of subsequent corneal allografts in both eyes. Experimental evidence demonstrates that the destruction of the corneal nerves in one eye fundamentally alters the expression of the immunoregulatory neuropeptides in the contralateral eye. The altered expression of these neuropeptides inhibits both the induction and maintenance of immune privilege. The alteration in the microenvironment mediated a quick and prolonged loss of immune privilege, which could be reversed by blocking the activity of the pro-inflammatory neuropeptide substance P (SP). The survival of the corneal allograft requires the participation of antigen-specific T regulatory cells. Neuropeptides within the ocular environment are important for immune privilege through the induction of tolerance. Our results demonstrate the destruction of the corneal nerves and the release of the pro-inflammatory neuropeptide SP inhibits both the generation and function of T regulatory cells, which ultimately leads to corneal allograft rejection.Item Reconstitution of a Multi-layered, Differentiated Cornea by HTERT-Immortalized Corneal Epithelial Cells Transduced with Thymidine Kinase Transplanted onto Denuded Mouse Corneas(2011-10-25) Kalangara, Jerry P.; Cavanagh, H. DwightOBJECTIVE: To develop an animal model for the implementation of human telomerase enzyme reverse transcriptase (hTERT)-immortalized human corneal epithelial cell line (hTCEpi) transduced with the hygromycin-thymidine kinase gene (HyTK) as a viable cell source for the reconstruction of the corneal surface. METHODS: HyTK cells were cultured in KGM-2 serum-free culture media under hygromycin B selection. Limbal stem cell deficiency (LSCD) was established in athymic nude mice (n=75) using ethylenediaminetetraacetic acid (EDTA) and mitomycin C treatment followed by mechanical debridement of corneal and limbal epithelium. Immunofluorescence (IF) using Anti-Laminin and Propidium iodide (PI) was used to assess presence of basement membrane after epithelial removal. Cultured HyTK cells were stained with Cell Tracker Green CMFDA (5-chloromethylfluorescein diacetate) and transplanted onto the right eye after epithelial removal; the left eye served as a control. Transplanted cells were evaluated at 4 hours, 1 day, and 7 days post-transplantation using laser scanning confocal microscopy (LSCM). At 4 hours and 1 day, corneas were imaged for the presence of CMFDA. At day 7, corneas were stained using antibodies to Keratin 3, Ki-67, and the fluorescent probes – PI, and Phalloidin. Cytotoxicity of ganciclovir was assessed at concentrations of 0.1, 0.5, 1.0, 5.0, and 10.0 μM using Live-Dead Assay. RESULTS: IF confirmed an intact corneal basement membrane following epithelial removal. At 4 hours and 1 day post-transplantation, CMFDA staining demonstrated that transplanted cells were dispersed throughout the corneal surface. After 7 days, HyTK cells showed stratification and IF confirmed a differentiated corneal epithelial phenotype. Incubation in ganciclovir induced a cytotoxic effect on HyTK cells in vitro and had no significant effect on the hTCEpi control cell line. This effect was significant at 0.5 µM (p < 0.05). CONCLUSIONS: These studies demonstrate the first reconstitution of a multi-layered, differentiated corneal epithelium by HyTK cells in the nude mouse model; further, proliferating HyTK cells can be killed with ganciclovir treatment in vitro, which may reduce the potential for risk of oncogenic transformation in vivo.Item Role of Allergic Conjunctivitis in Inducing Corneal Transplant Rejection(2013-01-17) Reyes, Nancy Janet; Niederkorn, Jerry Y.Corneal allografts are the most commonly transplanted solid organs in humans and have a success rate of over 90%. This low incidence of graft rejection is largely due to the unique properties of the eye that decrease the likelihood of mounting an immune response, a phenomenon called "immune privilege". Despite this characteristic, immune rejection remains the leading cause of corneal graft failure, indicating that immune privilege can be abolished. We used a model of corneal transplantation in which C57BL/6 corneal allografts transplanted to BALB/c mice experienced a 50% survival rate. This is in sharp contrast to the 100% rejection that occurs with other organ grafts exchanged between these two mouse strains. The 50% survival rate decreased to 0% in mice with ongoing allergic conjunctivitis. We set out to delineate the mechanisms by which allergic conjunctivitis abolishes immune privilege and results in corneal allograft rejection. Anterior chamber-associated immune deviation (ACAID) is initiated when foreign antigens, such as histocompatibility antigens sloughed from the cornea, enter the anterior chamber of the eye and induce a systemic down-regulation of immune responses. Studies have shown that abolishing ACAID increases the incidence of corneal allograft rejection. The hypothesis that allergic conjunctivitis abolished the induction of ACAID was tested and results showed that allergic conjunctivitis did not. Moreover, allergic conjunctivitis did not cause a qualitative or quantitative difference in cell-mediated immune responses to the donor’s alloantigens. Regulatory T cells (Tregs) have also been shown to be important in corneal allograft survival. Our results indicated that allergic conjunctivitis altered the Tregs that support corneal allograft survival. Th2 cytokines, namely IL-4, generated during allergic conjunctivitis, render effector cells resistant to the suppressive ability of Tregs. Furthermore, allergic conjunctivitis did not exacerbate corneal allograft rejection unless the host’s effector T cells were able to respond to IL-4. Moreover, corneal immune privilege was restored in short ragweed (SRW) pollen-sensitized, allergic mice if they were isolated from SRW pollen for 14 days after receiving a corneal allograft. These results suggest that the exacerbation of corneal allografts in allergic hosts is due to the production of IL-4, which renders effector T cells resistant to Treg suppression.Item Role of IL-17A and Acaid in the Maintenance of Corneal Immune Privilege(2011-08-10) Cunnusamy, Khrishen; Niederkorn, Jerry Y.The human cornea, specifically the endothelial cell layer, has little to no regenerative potential. Accordingly, the eye has evolved multifactorial mechanisms that limit local immunogenic inflammation and trauma to the corneal endothelium. This local immune deviation is achieved through multiple anatomical, physiological, and immunoregulatory features intrinsic to the ocular environment that allow the cornea to enjoy an immune privileged status. Corneal immune privilege allows first time recipients of corneal allografts to enjoy a 90% success rate in the absence of systemic immunosuppressive drugs and without prior tissue typing. The myriad of immunoregulatory components that conspire to maintain the immune privilege of the cornea can be assigned to one of two extensively studied subsets. They include: (a) immunosuppressive molecules and membrane molecules present in the aqueous humor and on the surface of corneal cells layers, and (b) cell-mediated regulatory mechanisms such as anterior chamber-associated immune deviation (ACAID) that suppress systemic immune responses. This dissertation proposal investigates the role of a newly identified T cell subset, the Th17 CD4+ T cell lineage and its production of the cytokine, IL-17A in modulating corneal immune privilege and the outcome of keratoplasty. Previously, the Th17 T cell subset and its pro-inflammatory cytokine, IL-17A had been associated with the pathogenesis of several autoimmune diseases and had been implicated in cardiac, lung, and renal allograft rejections. Interestingly, in contrast to the classical paradigms that suggest that CD4+ T cells mediate graft rejection, the IL-17A produced by CD4+ T cells appears to be necessary for corneal allograft survival. My investigations led me to two distinct mechanisms via which IL-17A promotes corneal immune privilege. The results indicate that IL-17A is required for inhibiting the generation of an allospecific Th2 CD4+T cell subset that can independently exacerbate corneal allograft rejection. Simultaneously, IL-17A is also required for the functioning of CD4+CD25+ T regulatory cells (Tregs) that suppress alloimmune responses directed against the corneal transplant. Finally, by comparing and contrasting the cellular and cytokine requirements of corneal immune privilege and ACAID, these investigations unveil the independent mechanisms operating in the establishment of these respective phenomena. These findings redefine the role of IL-17A as a cytokine essential for the maintenance of corneal immune privilege and establish a new paradigm whereby interplay between IL-17A and CD4+CD25+ Tregs is necessary for survival of corneal allografts.