The Influence of Donor and Recipient Gender Incompatibility on Corneal Transplant Rejection and Failure.

In vascularized organ transplants, gender mismatches have higher rates of immunological rejection. We investigated the influence of gender incompatibility, including H-Y incompatibility, on corneal transplant graft rejection and failure. Patients were included who had undergone a first corneal transplant for keratoconus (KC), Fuchs endothelial dystrophy (FED), pseudophakic bullous keratopathy (PBK), infection and other indications. A Cox regression model was fitted for each indication to determine factors affecting graft failure and rejection at 5 years. The impact of gender, including H-Y, matching was analyzed after accounting for other factors, including known risk factors. Of 18 171 patients, 4314 had undergone a transplant for FED, 4783 for KC, 3669 for PBK, 1903 for infection and 3502 for other disorders. H-Y mismatched (male [M]→female [F]) corneas were at greater risk of graft failure or rejection. For FED, F→F were 40% less likely to fail (p < 0.0001) and 30% less likely to reject (p = 0.01); M→M were 20% less likely to fail (p = 0.04) and 30% less likely to reject (p = 0.01). For KC, M→M matched corneas were 30% less likely to fail (p = 0.05) and 20% less likely to reject (p = 0.01) compared with H-Y mismatches. H-Y antigen mismatched (M→F) patients were at greater risk of rejection or graft failure.

The impact of donor age and endothelial cell density on graft survival following penetrating keratoplasty.

PURPOSE: To determine if donor age and preoperative endothelial cell density (ECD) affect corneal endothelial failure following penetrating keratoplasty (PK). METHODS: Preoperative and postoperative data for PKs performed in the UK between April 1999 and March 2012 were analysed. Donor age was split into three groups (0-60, 61-75 and >75 years) and donor ECD was split into three groups (≤2400, 2401-2600 and >2600 cells/mm2). Cox proportional hazards regression was used to determine whether the selected subgroups of donor age and donor ECD have an impact on endothelial failure and a systematic analysis of the interaction between donor ECD and donor age was conducted. The analysis was stratified for primary corneal diagnosis (Fuchs endothelial dystrophy (FED), pseudophakic bullous keratopathy (PBK) and other) and corrected for potentially confounding factors (human leukocyte antigen matching, donor trephine diameter, deep vascularisation, the occurrence of reversible rejection episodes and receipt of systemic antiviral medication, long-term steroids or other immunosuppressive agents). RESULTS: A total of 9415 patients, from the National Health Service Blood and Transplant UK Transplant Registry, who received their first PK for visual reasons were included in this study. The overall 5-year graft survival rate due to endothelial failure was 89%. Survival rates in recipients with FED, PBK and 'all other indications' were 95%, 83% and 89%, respectively. Our analysis shows that donor ECD did not affect outcome following corneal graft within the preselected categories, irrespective of diagnosis and after allowing for any potential confounding factors. Furthermore, HRs for each level of donor ECD, relative to >2600 cells/mm2, for each combination of age group and indication, were not statistically significant. CONCLUSIONS: We were unable to detect a significant effect of donor age, up to 90 years, and preoperative donor ECD, above the lower limit of 2200 cells/mm2, on endothelial failure at 5 years following PK.

Experimental gene transfer to the corneal endothelium.

Transfer of cDNA to corneal endothelial cells has been demonstrated in cell monolayers in vitro, in endothelium of whole thickness corneas ex vivo and following intracameral injection. Studies examining the feasibility and optimal methods for gene transfer to the cornea have used viral and non-viral vectors, initially histochemical or fluorescent marker genes, and in endothelium of numerous species ranging from mouse to man. As the feasibility of genetic modification of corneal endothelial cells has been successfully demonstrated in a number of cell culture and animal models, there is significant potential for gene transfer in the treatment of human corneal endothelial disease. The two most widely studied applications of gene transfer to endothelium are (i) transduction of immunomodulatory genes to donor corneal endothelium prior to transplantation as a strategy to delay allogeneic rejection and (ii) modulation of apoptosis or induction of replication in human corneal endothelial cells to increase cell density. Although continued improvements in vectors for gene transfer will improve the efficacy and safety of gene therapy, more detailed understanding of the altered biology of endothelium in disease will be necessary to allow selection of appropriate targets for a gene-based treatment approach.

The effect of perioperative allergic conjunctivitis on corneal lymphangiogenesis after corneal transplantation.

INTRODUCTION: Perioperative allergic conjunctivitis accelerates the speed of corneal allograft rejection. This study examines the effect of allergic conjunctivitis, with and without dexamethasone treatment, on the early inflammatory response and lymphangiogenesis in the host cornea following corneal transplantation. METHODS: Allogeneic fully MHC-mismatched C57Bl/6 strain donor corneas were transplanted into naive A/J mice and into A/J mice with active allergic conjunctivitis. Further groups of allograft recipients with allergic conjunctivitis were treated post-operatively with twice daily topical dexamethasone 0.1% or phosphate-buffered saline. Mice were killed on days 2 and 6 and corneas were examined by (i) fluorescent immunohistochemistry of frozen sections using anti-CD11b, anti-F4/80 and anti-Gr-1 antibodies, or (ii) whole-mount staining with anti-LYVE-1 antibody. Lymphatic ingrowth and numbers of cells infiltrating the host cornea were compared between groups. RESULTS: There were significantly higher numbers of CD11b(+) cells and LYVE-1(+) vessels in the host cornea at day 2 in allergic compared with naive recipients, but no differences between naive and allergic recipients at day 6. In allergic eyes, dexamethasone treatment significantly inhibited LYVE-1 expression at days 2 and 6, and significantly improved allograft survival in recipients with allergic conjunctivitis if maintained for a week. CONCLUSIONS: The innate immune response to allogeneic corneal tissue is more vigorous in the presence of allergic conjunctivitis than in naive eyes and is associated with accelerated lymphatic ingrowth to host cornea. Topical dexamethasone inhibits lymphatic ingrowth and this may be one mechanism by which topical steroid enhances graft survival.

Use of ultrasonic pachymetry for measurement of changes in corneal thickness in mouse corneal transplant rejection.

BACKGROUND/AIMS: Diagnosis of rejection in the mouse model of corneal transplantation is based on subjective judgement of loss of graft transparency. The aims of this study were to (1) evaluate a pachymetry technique to measure changes in mouse corneal thickness and (2) correlate increases in transplant thickness with clinical and histological features of rejection. METHODS: Orthotopic corneal allografts (C57BL/6 strain donor) and syngeneic grafts were performed in A/J mice. Graft transparency was graded and corneal thickness measured by pachymetry on alternate days. Transverse sections of donor cornea excised from eyes representative of clinical opacity grades 1-4 were prepared, photographed, graft section thickness measured and stromal graft-infiltrating cells counted. Intraobserver and interobserver variations in pachymetry were statistically tested. RESULTS: Graft thickness, as measured by pachymetry, increased with each clinical opacity grade. Thickness for opacity grades 0, 1 and 2 was less than 300 microm in all recipients. Graft thickness for grades 3 and 4 was greater than 300 microm in all cases. For measurements up to 400 mum, there was a good correlation between thickness as measured by in vivo pachymetry and in histopathological sections. The mean interobserver bias was -11.35 microm, while the mean intraobserver bias was +3.96 microm. Stromal cellularity increased with increasing corneal thickness up to approximately 300 microm. CONCLUSION: In vivo graft pachymetry provides a new and reliable way to objectively diagnose rejection in the mouse model of corneal transplantation.

Characterization of dendritic cell phenotype in allergic conjunctiva: increased expression of Fc(epsilon)RI, the high-affinity receptor for immunoglobulin E.

PURPOSE: Dendritic cells (DCs) express the high-affinity receptor for IgE (Fc(epsilon)RI) on their surface, which may enhance their ability to capture and internalize antigens for presentation to T-lymphocytes. The aim of this study was to determine if expression of Fc(epsilon)RI(+) DCs is increased in the conjunctivae of vernal keratoconjunctivitis (VKC) patients compared with those of normal controls. METHODS: Conjunctival biopsies were obtained from non-atopic and VKC patients. Double immunohistochemical staining was carried out using antibodies against Fc(epsilon)RI and the CD1a antigen, a DC marker. The double-positive cells were counted in five representative fields of view for each conjunctival sample. RESULTS: Fc(epsilon)RI(+) CD1a(+) cells were present in significantly higher numbers in VKC conjunctivae compared with normal controls (mean cell count of 21.3 in VKC vs5.0 in controls, P<0.005). In normal patients the Fc(epsilon)RI-expressing DCs tended to be confined to the epithelial layer or the superficial substantia propria, but in the VKC samples these Fc(epsilon)RI(+) cells were mainly concentrated in the deeper substantia propria. CONCLUSIONS: Fc(epsilon)RI(+) DC numbers are elevated in the conjunctivae of VKC patients, a finding consistent with the results of other studies focusing on atopic conditions. Elevated expression of Fc(epsilon)RI on DCs would facilitate antigen presentation and enhance T-cell priming, thereby contributing to ocular symptoms.

Aqueous humor alloreactive cell phenotypes, cytokines and chemokines in corneal allograft rejection.

As biopsies are not taken at the time of human corneal allograft rejection, most information on the early cellular changes in rejection is from animal models. We examined the phenotype of alloreactive cells present in the human anterior chamber during corneal graft rejection by flow cytometry and quantified aqueous humor levels of cytokines and chemokines using cytometric bead array. Aqueous and peripheral blood samples were taken from patients with graft endothelial rejection (n = 11) and from control patients undergoing cataract surgery (n = 8). CD45(+)CD4(+), CD45(+)CD8(+) and CD45(+)CD14(+) cells were found in aqueous during rejection; no CD45(+) cells were seen in control samples. Higher proportions of CD45(+) cells found in aqueous during rejection were CD14(+), denoting monocyte/macrophage lineage, than were CD4(+) or CD8(+). Large elevations were seen in aqueous levels of IL-6, MCP-1 and IP-10 during rejection compared with controls; smaller but still statistically significant increases were seen in MIP-1alpha and eotaxin. The role of CD14(+) cells in allorejection is unclear as is the potential of these chemokines and their receptors as therapeutic targets. Aqueous humor samples offer a unique opportunity to analyze components of the allogeneic response in direct contact with donor tissue but without artifacts inherent in examination of tissue.

Upregulation of chemokine expression in murine cornea due to mechanical trauma or endotoxin.

BACKGROUND AND AIMS: Allograft rejection is the commonest cause of corneal transplant failure and is significantly higher in high-risk patients. Corneal tissue is reported to produce chemokines in response to stress/inflammation. Expression of chemokines is central to the recruitment of leucocytes during inflammatory events. This study was designed to evaluate the effects of surgical trauma or storage conditions on chemokine expression. METHODS: Murine corneas were manipulated by incubation in different conditions for up to 24 h, by the addition of endotoxin or by surgical trauma. The ex vivo production of chemokines was assessed using a real-time reverse-transcriptase PCR (RT-PCR) assay to measure mRNA encoding MIP-1alpha, MIP-1beta and MIP-1gamma, MCP1, IP-10, lymphotactin, fractalkine, RANTES, eotaxin, MIG, MIP2 and the cytokine MIF. The expression of RANTES was also determined by ELISA, and the ability of supernatant from corneas on chemotaxis of cells was also determined. Finally, we compared the survival of corneal grafts that had (or had not) been treated with endotoxin. RESULTS: We found that on incubation in corneal storage medium, expression of mRNA for the majority of these chemokines greatly increased. Upregulation of chemokine mRNA expression was also seen following the mechanical trauma of suture insertion and exposure of the cornea to endotoxin. In the case of mechanical trauma, functional activity of the chemokines was demonstrated using a chemotaxis assay. Orthotopic transplantation of LPS-treated corneas, in which chemokine expression was elevated, resulted in increased infiltration by leucocytes and more rapid rejection of allogeneic grafts. CONCLUSION: Our results indicate that ex vivo storage and manipulation of murine corneas can influence the expression of chemokines in corneas, and can result in earlier graft rejection. This may be of importance when considering procedures for manipulation and ex vivo storage of donor corneas prior to transplantation, as well as the surgical procedure itself.

Expression of indoleamine 2,3-dioxygenase (IDO) by endothelial cells: implications for the control of alloresponses.

Indoleamine 2,3-dioxygenase (IDO) is an important enzyme in the regulation of immune responses; cells that express IDO can suppress T-cell responses and promote tolerance. Because of the critical role of endothelial cells in graft rejection, we have investigated the role of IDO expression by vascular endothelial cells and its consequence on immunoregulation. We compared the expression of IDO by primary human umbilical vein endothelial cells (HUVECs), human saphenous vein endothelial cells (HSVECs) and arterially derived endothelial cells using reverse transcriptase PCR, Western blotting and assays for enzymatic activity. In HUVECs IDO is upregulated by incubation with cytokines or in mycoplasma-infected cells. On the other hand HSVECs and arterially derived endothelial cells express little IDO, which is poorly upregulated upon activation (except by mycoplasma). Inhibition of IDO activity improved the ability of HUVECs to stimulate allogeneic T-cell responses. If either HUVECs or HSVECs are transfected with the gene encoding IDO, then they are incapable of stimulating allogeneic T-cell responses and induce anergy in allospecific T cells (which can also act as regulatory cells). The variable expression of IDO in different endothelial cells is important not only in understanding the role of endothelial cells in the regulation of graft rejection, but also as a potential therapeutic strategy.

Corneal transplantation: the forgotten graft.

The most commonly performed transplant is that of the cornea, with 2292 corneal grafts performed in the UK in 2002-03, compared with 1775 renal transplants [1]. In the USA approximately 40 000 transplants are performed every year [2]. However this preponderance is not reflected in the amount of attention given to this transplanted tissue by the scientific community: for example up till now there have been no papers published in the American Journal of Transplantation that have cornea as a key or title word (as determined by a Medline search in December 2003). There are several reasons for this. The first is that corneal grafting is the province of ophthalmologists, who (with notable exceptions) are isolated from the transplant community. The second is that there is a widespread belief that, because of the existence of immune privilege, corneal grafts are not rejected and so there is no need for further research. As we will discuss later, this is incorrect. In this article we will seek to show that study of corneal transplantation is important in its own right, and also that it has lessons for those interested in other forms of allograft.

Prospects for gene therapy in corneal disease.

Transfer of cDNA to corneal cells has been accomplished using viral and nonviral vectors. Studies examining the feasibility and optimal methods for vector-mediated gene transfer to the cornea have, as in other tissues, been performed using histochemical or fluorescent marker genes. These have used corneal cells or cell lines in vitro, and whole corneas maintained in ex vivo culture. Gene-based interventions have been examined in specific corneal disorders such as allograft rejection, postexcimer laser scarring, and herpes simplex keratitis using experimental models. As the feasibility of genetic modification of corneal cells has been successfully demonstrated, there is great potential for gene therapy vectors in the treatment of human corneal disease. Continued improvements in vectors for gene transfer will improve the efficacy and safety of gene therapy. In addition to use of cDNA transfer as an alternative to drug or protein treatments in acquired corneal disorders, our expanding knowledge of the genetic basis of inherited corneal disorders will ultimately lead to the development of specific and effective gene therapies in this category of diseases.

A clinical, histopathological, and genetic study of Avellino corneal dystrophy in British families.

AIMS: To establish a clinical, histopathological, and genetic diagnosis in two unrelated British families with Avellino corneal dystrophy (ACD). METHODS: Genomic DNA was extracted from peripheral blood leucocytes of all members participating in the study. Exons 4 and 12 of the human transforming growth factor beta induced (BIGH3) gene were amplified by polymerase chain reaction. The mutation and polymorphism were identified by direct sequencing and restriction digest analysis. A review of the patients' clinical symptoms and signs was undertaken and a histopathological study on corneal specimen obtained from the proband of one family after keratoplasty was performed. RESULTS: A heterozygous G to A transition at the second nucleotide position of codon 124 of BIGH3 gene was detected in all affected members of both families. This mutation changes an arginine residue to a histidine. The clinical diagnosis for ACD was more evident with advancing age. Histopathological study revealed granular deposits in the anterior stroma and occasional positive Congo red areas of amyloid deposition in the mid to deep stroma typical of ACD. CONCLUSIONS: This is the first report of ACD families in the United Kingdom and, furthermore, of BIGH3 gene mutation in British patients with this rare type of corneal dystrophy. The results indicate that BIGH3 gene screening along with clinical and histopathological examinations is essential for the diagnosis and clinical management of corneal dystrophies.

Antibody targeted gene transfer to endothelium.

BACKGROUND: One of the drawbacks of the currently available vectors for gene therapy is the lack of selectivity in gene delivery. We have therefore investigated a strategy to generate immunoliposomes to target non-viral vectors to cell surface receptors on endothelium. MATERIALS AND METHODS: We have developed a novel method of coupling antibodies (Abs) to liposomes complexed to DNA, using mild heat treatment to aggregate the immunoglobulin G (IgG). The interaction of plasmid DNA, liposomes and Abs was measured using a gel retardation assay and a resonant mirror biosensor. The size of the transfection complex was determined by light scattering, and the binding and internalization of the complex to cells was followed using flow cytometry. The transfection ability was tested on cell lines and primary cells in vitro and human corneal or vascular tissues ex vivo. RESULTS: The interaction of antibodies with liposomes is relatively stable (t(1/2) congruent with 45 min). The size of the liposome, Ab and DNA complex was found to be around 500 nm in 4% BSA. The addition of anti-transferrin receptor Abs increased the internalization of the liposome-DNA complex into cells. Abs against both transferrin receptor and E-selectin were shown to augment transfection efficiency of liposomes to cell expressing the appropriate antigens. They are also shown to be efficient in mediating gene delivery to corneal and vascular tissues ex vivo. CONCLUSIONS: We have shown that our novel vector is capable of in vitro and ex vivo gene delivery to cells and human tissues including cornea, artery and vein. In particular, an Ab against E-selectin was effective at selectively delivering genes to activated endothelial cells expressing the adhesion molecule. Such a strategy will have applications for targeting these tissues prior to transplantation or autologous grafting, and, in the longer term, may allow in vivo targeting of gene therapy to inflammatory sites.

Modulation of hydrogen peroxide induced injury to corneal endothelium by virus mediated catalase gene transfer.

AIM: To examine the effect of catalase gene transfer on survival of corneal endothelial cells (EC) following challenge with hydrogen peroxide (H(2)O(2)) in an ex vivo model of oxidative stress. METHODS: A recombinant adenovirus vector (AdCL) was used to transfer human catalase cDNA into EC of whole thickness rabbit corneas ex vivo. The resulting catalase protein concentration was measured in corneal lysates by ELISA; catalase functional activity in lysates was determined using a H(2)O(2) activity assay. To examine the morphological effects of catalase gene transfer in modulation of H(2)O(2) induced injury, transduced corneas were maintained in ex vivo culture and challenged with H(2)O(2). Laser scanning confocal microscopy was used to image EC injury. Cell density, cell morphology, and ratios of viable to necrotic cells were determined. RESULTS: Following incubation with AdCL, catalase expression reached maximum at 5-7 days. Corneas transduced with AdCL showed increased EC cell survival following challenge with H(2)O(2) on day 3 when compared to null vector control or mock infected corneas. CONCLUSIONS: Ex vivo catalase gene transfer can protect EC from death mediated by H(2)O(2). This gene based approach to the protection of corneal endothelium from oxidative stress may have application in prevention of EC loss in pathological conditions in which H(2)O(2) is involved and in ex vivo donor corneal storage before transplantation.