Suppression of the allogeneic response by the anti-allergy drug N-(3,4-dimethoxycinnamonyl) anthranilic acid results from T-cell cycle arrest.

Previously we have shown that indoleamine 2,3-dioxygenase (IDO) and the tryptophan metabolite, 3-hydroxykynurenine (3HK) can prolong corneal allograft survival. IDO modulates the immune response by depletion of the essential amino acid tryptophan by breakdown to kynurenines, which themselves act directly on T lymphocytes. The tryptophan metabolite analogue N-(3,4-dimethoxycinnamonyl) anthranilic acid (DAA, 'Tranilast') shares the anthranilic acid core with 3HK. Systemic administration of DAA to mice receiving a fully MHC-mismatched allograft of cornea or skin resulted in significant delay in rejection (median survival of controls 12 days, 13 days for cornea and skin grafts, respectively, and of treated mice 24 days (P < 0.0001) and 17 days (P < 0.03), respectively). We provide evidence that DAA-induced suppression of the allogeneic response, in contrast to that induced by tryptophan metabolites, was a result of cell cycle arrest rather than T-cell death. Cell cycle arrest was mediated by up-regulation of the cell cycle-specific inhibitors p21 and p15, and associated with a significant reduction in interleukin-2 production, allowing us to characterize a novel mechanism for DAA-induced T-cell anergy. Currently licensed as an anti-allergy drug, the oral bioavailability and safe therapeutic profile of DAA make it a candidate for the prevention of rejection of transplanted cornea and other tissues.

Arginine depletion as a mechanism for the immune privilege of corneal allografts.

The cornea is an immune privileged tissue. Since arginase has been found to modulate T-cell function by depleting arginine, we investigated the expression of arginase in the cornea and its possible role in immune privilege using a murine transplant model. We found that both the endothelium and epithelium of murine corneas express functional arginase I, capable of down-regulating T-cell proliferation in an in vitro culture system. The administration of the specific arginase inhibitor N-hydroxy-nor-L-Arg to recipient mice resulted in an accelerated rejection of allogeneic C57BL/6 (B6) corneal grafts. In contrast, in vivo blockade of arginase activity had no effect in altering the course of rejection of primary skin grafts that express little, if any, arginase. In addition, the inhibition of arginase did not alter systemic T-cell proliferation. These data show that arginase is functional in the cornea and contributes to the immune privilege of the eye, and that modulation of arginase contributes to graft survival.

Twenty years of limbal epithelial therapy: an update on managing limbal stem cell deficiency.

Limbal stem cell damage after chemical injury, autoimmune disorders or iatrogenic trauma leads to corneal conjunctivalisation with new vessel formation, epithelium instability and visual loss. Limbal stem cell transplantation includes reconstructive surgical procedures to restore a corneal epithelium. The recognised options are: conjunctival limbal autograft, in which stem cells are taken from the patient's healthy eye; conjunctival limbal allograft, in which stem cells are taken from a living, related or dead donor and the keratolimbal allograft. Each of these procedures has some drawbacks; in particular, the conjunctival limbal autograft needs a relatively healthy fellow eye and needs a relatively large amount of donor tissue from the healthy eye (about one-third of the healthy limbal stem cell tissue) with potential risks to the donor eye. In the case of keratolimbal allograft transplants, the recipient needs an immunosuppressive treatment to reduce the risk of rejection with the associate possible side effects. More modern treatment options are reviewed. Cultivated oral mucosa epithelial transplantation success rate can vary between 50% and 70% at 3-4 years of follow-up. Simple limbal epithelial transplantation results show a success rate from 75.2% to 83.8% after 1 year of follow-up. Inclusion criteria for autologous cultivated limbal epithelial transplantation as approved by the National Institute of Health and Care Excellence are also shown in this paper. On the basis of these more contemporary treatment options, a stepladder approach to evaluate which procedure is most appropriate and personalised to the patient's conditions is proposed.

Herpes simplex keratitis misdiagnosed as rheumatoid arthritis-related peripheral ulcerative keratitis.

PURPOSE: To report 2 cases of herpes simplex keratitis misdiagnosed as rheumatoid arthritis (RA)-related peripheral ulcerative keratitis (PUK), where isolation of the herpes simplex virus (HSV) led to a complete modification in management. METHODS: This is a case report. RESULTS: Two patients with RA presented with painful red right eyes. Ocular examination in both revealed an ulcer involving the peripheral cornea. The adjacent conjunctiva was infected, and the underlying sclera appeared inflamed. A diagnosis of corneal PUK secondary to RA was therefore made. The first patient had corneal scrapes taken for routine microbiological examination, which included polymerase chain reaction (PCR) for HSV. In the second patient, despite systemic immunosuppressive therapy, the ulcer progressed to involve deeper stroma and more central cornea. The conjunctiva adjacent to the ulcer was resected, and healthy conjunctival tissue was mobilized to cover the peripheral corneal ulcer. Resected conjunctival and corneal tissue was histopathologically assessed. In the first patient, PCR for HSV yielded a positive result. This prompted treatment with immediate systemic and topical acyclovir. The ulcer responded well to treatment. In the second patient, histopathological assessment and electron microscopy identified HSV. Treatment with topical trifluorothymidine and steroids was started, and a good recovery was made. CONCLUSIONS: Treatment of PUK is with systemic immunosuppressive therapy, and such therapies have serious side effects. PUK may have an occult cause in RA, and a search for a secondary agent may be beneficial. In particular, occult HSV infection must be ruled out before commencing immunosuppressive therapy.

Identification of Therapeutic Targets of Inflammatory Monocyte Recruitment to Modulate the Allogeneic Injury to Donor Cornea.

PURPOSE: We sought to test the hypothesis that monocytes contribute to the immunopathogenesis of corneal allograft rejection and identify therapeutic targets to inhibit monocyte recruitment. METHODS: Monocytes and proinflammatory mediators within anterior chamber samples during corneal graft rejection were quantified by flow cytometry and multiplex protein assays. Lipopolysaccharide or IFN-γ stimulation of monocyte-derived macrophages (MDMs) was used to generate inflammatory conditioned media (CoM). Corneal endothelial viability was tested by nuclear counting, connexin 43, and propidium iodide staining. Chemokine and chemokine receptor expression in monocytes and MDMs was assessed in microarray transcriptomic data. The role of chemokine pathways in monocyte migration across microvascular endothelium was tested in vitro by chemokine depletion or chemokine receptor inhibitors. RESULTS: Inflammatory monocytes were significantly enriched in anterior chamber samples within 1 week of the onset of symptoms of corneal graft rejection. The MDM inflammatory CoM was cytopathic to transformed human corneal endothelia. This effect was also evident in endothelium of excised human cornea and increased in the presence of monocytes. Gene expression microarrays identified monocyte chemokine receptors and cognate chemokines in MDM inflammatory responses, which were also enriched in anterior chamber samples. Depletion of selected chemokines in MDM inflammatory CoM had no effect on monocyte transmigration across an endothelial blood-eye barrier, but selective chemokine receptor inhibition reduced monocyte recruitment significantly. CONCLUSIONS: We propose a role for inflammatory monocytes in endothelial cytotoxicity in corneal graft rejection. Therefore, targeting monocyte recruitment offers a putative novel strategy to reduce donor endothelial cell injury in survival of human corneal allografts.

3-hydroxykynurenine suppresses CD4+ T-cell proliferation, induces T-regulatory-cell development, and prolongs corneal allograft survival.

PURPOSE: IDO (indoleamine 2,3-dioxygenase) modulates the immune response by depletion of the essential amino acid tryptophan, and IDO overexpression has been shown to prolong corneal allograft survival. This study was conducted to examine the effect of kynurenines, the products of tryptophan breakdown and known to act directly on T lymphocytes, on corneal graft survival. METHODS: The effects of kynurenines on T-cell proliferation and death, T-regulatory-cell development, and dendritic cell function, phenotype, and viability were analyzed in vitro. The effect of topical and systemic administration of 3-hydroxykynurenine (3HK) on orthotopic murine corneal allograft survival was examined. RESULTS: T-lymphocyte proliferation was inhibited by two of the four different kynurenines: 3HK and 3-hydroxyanthranilic acid (3HAA). This effect was accompanied by significant T-cell death. Neither 3HK nor 3HAA altered dendritic cell function, nor did they induce apoptosis or pathogenicity to corneal endothelial cells. Administration of systemic and topical 3HK to mice receiving a fully mismatched corneal graft resulted in significant prolongation of graft survival (median survival of control grafts, 12 days; of treated, 19 and 15 days, respectively; P < 0.0003). While systemic administration of 3HK was associated with a significant depletion of CD4(+) T, CD8(+) T, and B lymphocytes in peripheral blood, no depletion was found after topical administration. CONCLUSIONS: The production of kynurenines, in particular 3HK and 3HAA, may be one mechanism (in addition to tryptophan depletion) by which IDO prolongs graft survival. These molecules have potential as specific agents for preventing allograft rejection in patients at high rejection risk.