DNA microarray-based gene expression profiling in porcine keratocytes and corneal endothelial cells and comparative analysis associated with xeno-related rejection.

Porcine to rat corneal xenotransplantation resulted in severe inflammation and rejection of the corneal stroma, whereas an allograft showed mainly endothelial cell-associated rejection. We, therefore, investigated and compared the gene expression between porcine keratocytes and corneal endothelial cells. RNA was isolated from primary cultured porcine or human keratocytes and porcine corneal endothelial cells. Gene expression was comparatively analyzed after normalization with microarray method using Platinum pig 13 K oligo chip (GenoCheck Co., Ltd., Ansan, Korea). Real-time polymerase chain reaction (PCR) was performed for C1R, CCL2, CXCL6, and HLA-A in porcine keratocytes and corneal endothelial cells. As a result, upregulated expression more than 2 folds was observed in 1,162 genes of porcine keratocytes versus porcine endothelial cells. Among the immune-regulatory genes, SEMA3C, CCL2, CXCL6, F3, HLA-A, CD97, IFI30, C1R, and G1P3 were highly expressed in porcine keratocytes, compared to porcine corneal endothelial cells or human keratocytes. When measured by real-time PCR, the expression of C1R, CCL2, and HLA-A was higher in porcine keratocytes compared to that in porcine corneal endothelial cells. In conclusion, the increased expression of C1R, CCL2, and HLA-A genes in porcine keratocytes might be responsible for the stromal rejection observed in a porcine to rat corneal xenotransplantation.

Acute cell-mediated rejection in orthotopic pig-to-mouse corneal xenotransplantation.

BACKGROUND: To investigate the role of T cells and natural killer (NK) cells in mediating corneal xenograft rejection in a pig-to-mouse model. METHODS: Pig corneas were orthotopically transplanted into BALB/c, C57BL/6, nude, severe combined immunodeficiency (SCID), and NOD/SCID/gammac(null) (NOG) mice. Graft survival was clinically assessed by slit-lamp biomicroscopy and median survival times (MST) were calculated. The rejected grafts were histologically evaluated using antibodies against CD4, CD8, NK1.1, and F4/80. RESULTS: The pig corneal xenografts were acutely rejected by BALB/c and C57BL/6 mice (MST 9.0 days), while nude, SCID and NOG mice rejected pig corneas in a more delayed fashion (MST 16.0, 16.4, and 16.9 days, respectively). The majority of infiltrating cells found in rejected grafts in C57BL/6 mice were macrophages and CD4(+) T cells, while CD8(+) T cells and NK cells were rarely found. The grafts in nude mice had markedly decreased inflammatory infiltration with small numbers of macrophages and CD4(+) T cells. Infiltration was even more modest in grafts in SCID and NOG mice. CONCLUSIONS: T cells play an important role in acute rejection of pig corneal xenografts in mice, although acute rejection is not solely the result of T-cell-mediated immunity. NK cells are less likely to be involved in the rejection process.

The potential of viral vector-mediated gene transfer to prolong corneal allograft survival.

The cornea is a particularly attractive target for gene therapy designed to improve the outcome of corneal transplantation. First, there is a clear and well-defined clinical need. Second, because donor corneas can be preserved for days if not weeks within an eye bank, ex vivo transduction of a donor cornea can be carried out without the urgency associated with many other forms of transplantation. Finally, the partial sequestration of the eye from the systemic circulation decreases the likelihood of spillover of vector and transgene, and the immune privileged nature of the cornea and anterior segment affords a degree of protection from immune responses directed against the vector. A wide range of vectors has been investigated for gene transfer to the cornea. A number of viral vectors, in particular, have proved to be efficient at transducing the cornea and in association with a variety of transgenes, have been used successfully to prolong corneal allograft survival significantly in animal models. The most suitable such vector for future clinical studies in corneal transplantation has yet to be determined, but the most likely include recombinant adenoviral, adeno-associated viral and lentiviral vectors. In this review, we examine the ability of these viral vectors to transduce the cornea, and summarise those studies in which gene therapy has been used to prolong experimental corneal allograft survival.

'Chimeric' grafts assembled from multiple allodisparate donors enjoy enhanced transplant survival.

Certain components of a graft that provoke alloimmunity may not be vital for graft function or critical as targets of rejection. Corneal transplantation is an example of this, because graft epithelium plays a role in allosensitization, whereas corneal graft endothelium-which shares the same alloantigens-is the critical target in allorejection. In this study, we found that exploiting this biology by replacing donor epithelium of an allograft with an allodisparate third-party epithelium yields a marked enhancement in transplant survival. Such 'chimeric' allografts consisted of a C3H/He (H-2(k)) corneal epithelium over a C57BL/6 (H-2(b)) epithelial-denuded cornea (or v.v.) and orthotopically placed on BALB/c (H-2(d)) hosts. Conventional corneal allografts (C3H/He or C57BL/6) or isografts (BALB/c) were also transplanted on BALB/c hosts. Alloreactive T-cell frequencies (CD4(+) interferon [IFN]-gamma(+)) primed to the graft endothelium were strongly diminished in chimeric hosts relative to conventionally allografted hosts. This was corroborated by a decreased T-cell infiltration (p = 0.03) and a marked enhancement of allograft survival (p = 0.001). Our results represent the first successful demonstration of chimeric tissue, epithelial-denuded allograft plus third-party allodisparate epithelium, in the promotion of allograft survival. Moreover, chimeric grafting can be readily performed clinically, whereby corneal allograft rejection remains a significant problem particularly in inflamed graft beds.

Differential roles of direct and indirect allorecognition pathways in the rejection of skin and corneal transplants.

BACKGROUND: It is generally accepted that all transplants are not rejected in the same fashion. However, the extrinsic and intrinsic factors that control the recognition and rejection of a particular allograft by the host are not well characterized. METHODS: We compared the mechanisms underlying the response with donor antigens by T cells activated after transplantation of fully allogeneic skin and corneal grafts in mice. RESULTS: In corneal-transplanted mice, the CD4+ T-cell response was exclusively mediated by T cells recognizing minor antigens in an indirect fashion and producing low levels of interleukin-2. In contrast, skin grafts elicited both direct and indirect CD4+ T-cell responses primarily directed to major histocompatibility complex antigens and characterized by high interleukin-2 levels. Although CD8+ T-cells producing gamma interferon were activated directly in both skin- and corneal-grafted mice, only CD8+ T cells from skin-transplanted mice mounted a cytotoxic response. Next, we investigated whether failure of corneal transplants to induce a CD4+ direct alloresponse is due to their poor immunogenicity or due to the site of placement (eye). We observed that corneas transplanted under the skin and splenocytes transplanted in the eye were both capable of inducing direct CD4+ T-cell alloreactivity. CONCLUSIONS: This shows that failure of orthotopic corneal allotransplants to elicit a CD4+ T-cell direct alloresponse is associated with the combination of two factors, their low immunogenicity and the immune-privileged properties of the eye.

Levels of Foxp3 in regulatory T cells reflect their functional status in transplantation.

Foxp3 expressing CD4(+)CD25(+) regulatory T cells (Tregs) have been shown to prevent allograft rejection in clinical and animal models of transplantation. However, the role of Foxp3 in regulating Treg function, and the kinetics and mechanism of action of Tregs in inducing allograft tolerance in transplantation, are still not fully understood. Thus, we investigated the kinetics and function of Tregs in a mouse model of orthotopic corneal transplantation, the most common form of tissue grafting worldwide. In this study, using in vitro functional assays and in vivo Treg adoptive transfer assays, we show that far more relevant than Treg frequency is their level of Foxp3 expression, which is directly associated with the potential of Tregs to prevent allograft rejection by producing regulatory cytokines and suppressing effector T cell activation. In addition, our data clearly demonstrate that Tregs primarily suppress the induction of alloimmunity in regional draining lymph nodes rather than suppressing the effector phase of the immune response in the periphery. These findings provide new insights on Treg dynamics in transplantation, which are crucial for designing therapeutic strategies to modulate Treg function and to optimize Treg-based cell therapies for clinical translation.

Graft rejection episodes after Descemet stripping with endothelial keratoplasty: part one: clinical signs and symptoms.

AIM: To investigate characteristics of initial immunological graft rejection after Descemet stripping with endothelial keratoplasty (DSEK). METHODS: The incidence, symptoms and clinical characteristics of initial immunological graft rejection episodes were analysed retrospectively in 598 eyes treated with primary DSEK at a single tertiary referral centre. RESULTS: Graft rejection episodes occurred in 54 eyes of 48 patients. Thirty-five per cent of the eyes were asymptomatic and were diagnosed during routine examination. Signs of immunological rejection at the initial diagnosis included keratic precipitates (69%), diffuse corneal oedema (11%) or both (20%); no endothelial rejection lines were observed. In contrast to standard full-thickness grafts, there were no epithelial immunological reactions because the epithelium and anterior stroma are not transplanted in DSEK. Most grafts cleared; four (7%) progressed to graft failure and were successfully regrafted with DSEK. CONCLUSIONS: Immunological graft rejection is an important postoperative complication after DSEK. The range of clinical findings indicative of corneal graft rejection differs in some respects between DSEK and standard penetrating keratoplasty.

Graft rejection episodes after Descemet stripping with endothelial keratoplasty: part two: the statistical analysis of probability and risk factors.

AIM: To investigate risk factors and probability of initial immunological graft rejection episodes after Descemet stripping with endothelial keratoplasty (DSEK). METHODS: Outcomes of 598 DSEK cases from a single tertiary referral centre were reviewed. Risk factors and probability of rejection were assessed by multivariate Cox proportional hazards modelling. RESULTS: Rejection episodes occurred in 54 eyes of 48 patients. Estimated probability of a rejection episode was 7.6% by 1 year and 12% by 2 years after grafting. Relative risk of rejection was five times higher for African-American patients compared with Caucasians (p = 0.0002). Eyes with pre-existing glaucoma (9%) or steroid-responsive ocular hypertension (27%) had twice the relative risk of rejection (p = 0.045) compared with eyes that did not have those problems. Patient age, sex and corneal diagnosis did not significantly influence rejection risk. Risk of rejection was not increased when fellow eyes were grafted within 1 year of the first eye (p = 0.62). CONCLUSIONS: Pre-existing glaucoma or steroid-responsive ocular hypertension and race were the two factors that independently influenced relative risk of rejection after DSEK. Rejection risk was not increased if the fellow eye was grafted within the prior year with DSEK.

DNA Microarray-Based Gene Expression Profiling in Porcine Keratocytes and Corneal Endothelial Cells and Comparative Analysis Associated with Xeno-related Rejection

Porcine to rat corneal xenotransplantation resulted in severe inflammation and rejection of the corneal stroma, whereas an allograft showed mainly endothelial cell-associated rejection. We, therefore, investigated and compared the gene expression between porcine keratocytes and corneal endothelial cells. RNA was isolated from primary cultured porcine or human keratocytes and porcine corneal endothelial cells. Gene expression was comparatively analyzed after normalization with microarray method using Platinum pig 13 K oligo chip (GenoCheck Co., Ltd., Ansan, Korea). Real-time polymerase chain reaction (PCR) was performed for C1R, CCL2, CXCL6, and HLA-A in porcine keratocytes and corneal endothelial cells. As a result, upregulated expression more than 2 folds was observed in 1,162 genes of porcine keratocytes versus porcine endothelial cells. Among the immune-regulatory genes, SEMA3C, CCL2, CXCL6, F3, HLA-A, CD97, IFI30, C1R, and G1P3 were highly expressed in porcine keratocytes, compared to porcine corneal endothelial cells or human keratocytes. When measured by real-time PCR, the expression of C1R, CCL2, and HLA-A was higher in porcine keratocytes compared to that in porcine corneal endothelial cells. In conclusion, the increased expression of C1R, CCL2, and HLA-A genes in porcine keratocytes might be responsible for the stromal rejection observed in a porcine to rat corneal xenotransplantation.

Clinical features of immune rejection after corneoscleral transplantation.

PURPOSE: To summarize the clinical features of immune rejection after corneoscleral transplantation. DESIGN: A retrospective, noncomparative, observational case series. METHODS: Patients who received corneoscleral transplantation because of whole corneal ulcer or corneal perforation at Shandong Eye Institute from July 1, 2003 through July 31, 2005 were included. Fourteen patients (14 eyes) with immune rejection but not recurrence or other complications were reviewed, including ocular vision, rejection onset time, symptoms, and characteristics. RESULTS: The average rejection time in the 14 eyes was 35 days. The rejection arose rapidly, and the mean best-corrected visual acuity decreased to counting fingers or hand movements. Circular limbal congestion and edema developed with circuitous and dilatational vessels. Whole graft edema and Descemet membrane folds were present, but no epithelial rejection line, endothelial rejection line, or keratic precipitate were observed. The average intraocular pressure (IOP) dropped from 13.6 mm Hg to 7.4 mm Hg. Seven eyes had shallow anterior chambers (AC). Retinal and choroidal edema was observed in five eyes. CONCLUSIONS: The clinical features of immune rejection after corneoscleral transplantation include rapid onset of rejection, vision decrease, circular limbal congestion and edema with circuitous and dilatational vessels, whole graft edema and shallow AC, low IOP, and no rejection line or keratic precipitate.

Immune modulation in corneal transplantation.

Allograft rejection is the most common reason for corneal transplant failure, despite the immunologic privilege of both the graft and the anterior chamber. To prevent corneal allograft rejection, various immunomodulatory strategies have been used in experimental corneal transplantation. These include (1) anti-T-cell receptor and T-cell depletion therapy; (2) manipulation of costimulatory molecule function, including both down-regulation of positive stimulatory molecules and/or up-regulation of inhibitory molecules and overproduction of tumor necrosis factor-related, apoptosis-induced ligand; (3) modulation of cytokine production by reducing proinflammatory cytokines (tumor necrosis factor alpha, interleukin [IL]-12, and IL-1) and/or increasing immunoregulatory cytokines (IL-10 and IL-4); (4) macrophage depletion; and (5) overexpression of the immunomodulatory molecule indoleamine 2,3-dioxygenase. Although these approaches appear promising in animal corneal transplantation models, there has been very little translation of these immunomodulatory approaches in human corneal transplantation.

Lack of IFN-gamma synthesis in aqueous humor during corneal graft rejection correlates with suppressed nitric oxide production by macrophages.

PURPOSE: To investigate cytokine production by leukocytes in aqueous humor (AH) during corneal graft rejection and nitric oxide (NO) production by macrophages as a potential mediator of graft damage. METHODS: Rats received corneal allotransplants and were killed during acute rejection. Leukocytes in AH that expressed tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-10 were quantified by flow cytometry. Isograft and further allograft recipients were killed, and sectioned corneas with conjunctivae were examined by histology for production of inducible nitric oxide synthase (iNOS), NO, and nitrotyrosine (NT). RESULTS: Between 80% and 90% of T cells, NK cells, and macrophages in AH expressed TNF-alpha, and at least 20% expressed IL-10. However, IFN-gamma was undetectable unless cells were first stimulated in vitro with PMA and ionomycin, which yielded IFN-gamma in 25% of cells. iNOS(+) macrophages were identified in donor cornea and AH, correlating precisely with rejection. Cells producing low levels of NO (NO(dim) cells) were found in donor stroma, but NT(+) cells were rare. Both NT(+) and NO(+) cells were rare in the anterior chamber (AC) or attached to corneal endothelium. NT(+) macrophages that were also NO(bright) were associated with sutures in allograft and isograft recipients and within conjunctivae, either scattered or in leukocyte aggregates. CONCLUSIONS: IFN-gamma synthesis is lacking in the AC during rejection, correlating with lack of NO but not of iNOS expression. NO does not appear to mediate endothelial cell death. NT and high levels of NO production are associated with nonspecific inflammatory cells.

Susceptibility of porcine keratocytes to immune-mediated damage in xeno-related rejection.

We admixed cultured porcine keratocytes or corneal endothelial cells in the presence of human sera or peripheral blood mononuclear cells (PBMCs) for 4 to 72 hours to investigate their immune-related susceptibilities to xeno-related rejection. We evaluated complement deposition at 48 hours by flow cytometry after staining with the C3 anti-goat cy3 antibody. The inhibition of proliferation of porcine corneal cells by human sera was examined using the 3-[4,5-dimethy/thiazol-2,5-dephenyl tetrazolium bromide (MTT) assay over 24 to 72 hours. The amount of 51chromium (Cr)-release was estimated after a reaction between the porcine cells and human PBMCs for 4 hours. There was greater C3 deposition in keratocytes (60.2%) than in endothelial cells (26.9%; P = .05, Mann-Whitney U test). Both keratocytes and endothelial cells showed significant levels of proliferative inhibition over a period of 72 hours. The number of 51Cr-release cells on interleukin-2 addition was significantly higher among keratocytes (88.0%) than endothelial cells (51.4%) at a 1:100 target:effector ratio (P = .04, Mann-Whitney U test). Our present data suggested that porcine keratocytes might be key target cells in xeno-related rejections when the porcine cornea is transplanted to primates.

Effect of CXCL-1/KC production in high risk vascularized corneal allografts on T cell recruitment and graft rejection.

BACKGROUND: The survival rate of corneal allografts in high-risk vascularized corneal bed recipients is poor, similar to vascularized solid organ allografts. Although the early induction of selective chemokines in solid organs is required for the optimal recruitment of T cells into rejecting allografts, little is known about the role of these chemokines in high risk corneal allografts. METHODS: Orthotopic corneal allotransplants were performed in low-risk (nonvascularized) and high-risk (vascularized) C57BL/6 (H-2b) recipients using Balb/c (H-2d) donors. Intragraft production of CXC chemokines was measured by Luminex and enzyme-linked immunosorbent assay on corneal transplant extracts at different times after surgery. Rabbit anti-KC serum was used to test its role in high risk corneal allograft survival. RESULTS: Early upregulation of CXCL1/KC occurs 3 days after transplantation in high risk allograft only. Moreover, the T-cell chemoattractants, CXCL9/Mig and CXCL10/IP10, are produced late (day 10) after surgery and their production correlates with the recruitment of CD4 T cells into the graft. Furthermore, in vivo neutralization of CXCL1/KC with anti-KC sera results in increased graft survival and decreased recruitment of T cells into high-risk allografts. CONCLUSION: We propose that a high risk vascularized cornea behaves like a vascularized solid organ transplant. The early production of CXCL1/KC is crucial to the induction of T-cell chemoattractants necessary for the recruitment of allospecific CD4 T cells into the graft. In vivo neutralization of CXCL1/KC represents a potential novel therapy that could be used to increase the survival rate of high-risk vascularized corneal allografts.

Visual outcomes and graft survival following corneal transplants: the need for an Irish National Corneal Transplant Registry.

PURPOSE: To evaluate visual outcomes and corneal graft survival at 12 months postoperatively, and to demonstrate the need for an Irish National Corneal Transplant Registry. METHODS: Retrospective, single surgeon, single center, analysis of 44 consecutive corneal transplants was performed between 2001 and 2005. RESULTS: Forty-four eyes of 41 patients underwent penetrating keratoplasty (n = 37), lamellar keratoplasty (n = 6) or limbal graft (n = 1). Indications for surgery (%) were keratoconus 45.45%; bullous keratopathy 2.7%; graft failure 9%; Fuchs endothelial dystrophy 2 4.5%; Irido corneal endothelial syndrome 2.27% and tectonic 15.9%. Visual outcome at 1 year (%) were as follows: improved by one or more Snellen lines (79.5%); disimproved by one or more Snellen lines (9%); unchanged (11.5%). Graft survival at 1 year was 93%. DISCUSSION: Although our findings compare favourably with those of the Australian Corneal Graft Registry, there is no Irish Corneal Transplant Registry with which to compare our results.

[Keratoplasty in HSV keratitis: prevention and therapy for immunological complications]. / Keratoplastik nach HSV-Keratitis: Prävention und Therapie immunologischer Komplikationen.

Herpes simplex viral (HSV) keratitis is the leading infectious cause of secondary visual loss in the western world. Corneal stromal scaring is still an important indication for penetrating keratoplasty. Over the past decades the epidemiology of HSV infection as well as treatment strategies have changed. This review will address these recent findings with a focus on specific aspects of keratoplasty subsequent to HSV keratitis.

Ly49 C/I-dependent NKT cell-derived IL-10 is required for corneal graft survival and peripheral tolerance.

Similar to their activity on NK cells, Ly49 molecules play a pivotal role in influencing how NKT cells respond. It is known that Ly49 C/I is an inhibitory receptor capable of down-modulating proliferation, IFN-gamma response, and cytotoxic activity in cells that express it. In a model of peripheral tolerance induced via the eye, we observed that Ly49 C/I-positive, invariant NKT cells were required. To test if the NK inhibitory receptor functionally contributed to tolerance development, we used blocking antibody, in vivo and in vitro, to interfere with the development of antigen-specific suppression. A result of blocking ligation of Ly49 C/I inhibitory receptor prevented NKT cell production of IL-10 and the subsequent development of tolerance. Ly49 C/I-blocking antibodies also prevented corneal graft survival, a phenomenon dependent on eye-induced tolerance. Furthermore, in the presence of TCR stimulation, cross-linking of Ly49 C/I on CD4(+) NKT cells stimulated an increase in IL-10 mRNA and a decrease in IFN-gamma. The concept of Ly49 inhibitory receptors regulating immune reactivity to self by regulating immune activity of individual cells is thus expanded to include a role for the inhibitory receptors in the more global process of peripheral tolerance to foreign antigens.