Corneal Allograft Rejection Research Articles

Function of indoleamine 2,3‐dioxygenase in corneal allograft rejection and prolongation of allograft survival by over‐expression

Indoleamine 2,3-dioxygenase (IDO) suppresses T cell responses by its action in catabolising tryptophan. It is important in maintenance of immune privilege in the placenta. We investigated the activity of IDO in the cornea, following corneal transplantation and the effect of IDO over-expression in donor corneal endothelium on the survival of corneal allografts. IDO expression was analysed and functional activity was quantified in normal murine cornea and in corneas following transplantation as allografts. Low levels of IDO, at both mRNA and protein levels, was detected in the normal cornea, up-regulated by IFN-gamma and TNF. Expression of IDO in cornea was significantly increased following corneal transplantation. However, inhibition of IDO activity in vivo had no effect on graft survival. Following IDO cDNA transfer, murine corneal endothelial cells expressed functional IDO, which was effective at inhibiting allogeneic T cell proliferation. Over-expression of IDO in donor corneal allografts resulted in prolonged graft survival. While, on one hand, our data indicate that IDO may augment corneal immune privilege, up-regulated IDO activity following cytokine stimulation may serve to inhibit inflammatory cellular responses. While increasing IDO mRNA expression was found in allogeneic corneas at rejection, over-expression in donor cornea was found to significantly extend survival of allografts.

Influence of local and systemic CTLA4Ig gene transfer on corneal allograft survival

To analyse the effects of local (ex vivo) or systemic (in vivo) administration of adenovirus type 5 encoding CTLA4Ig (AdCTLA4Ig) on its influence to prolong corneal allograft survival and to study the underlying mechanisms. A MHC class I/II mismatched rat corneal transplant model was used. Recipients were randomly assigned to receive ex vivo gene-modified corneas expressing either CTLA4Ig, CTLA4Ig/IL-10 or a single intraperitoneal (i.p.) injection (1.0 x 10(9) or 1.0 x 10(10) infectious particles) of AdCTLA4Ig 1 day before transplantation and graft survival was analysed. The immunoregulatory effect of this treatment was examined by analysing intra-graft cytokine mRNA expression pattern at day 12 post-transplant. The anti-adenovirus immunity also was investigated. Ex vivo gene transfer resulted in a modest but significant prolongation of graft survival (p = 0.0036 compared to no treatment). In contrast, systemic gene therapy (1.0 x 10(9) or 1.0 x 10(10) infectious particles) significantly prolonged graft survival (p = 0.0007 and 0.0001, respectively, compared to no treatment). Systemic (1.0 x 10(10) infectious particles) therapy resulted in frequent indefinite survival of allogeneic grafts which was not observed in the other therapeutic regimens. Moreover, systemic therapy prevented the intra-graft accumulation and activation of T cells and resulted in a reduced mRNA expression of both TH1 and TH2 cytokines. The generation of anti-adenovirus antibodies was also efficiently inhibited. CTLA4Ig gene therapy is a successful strategy for the prevention of allogeneic graft rejection in corneal transplantation. Our work has further elucidated the mechanisms of corneal allograft rejection which may lead to novel therapeutic strategies.

Topical Cyclosporine A in Corneal Graft Rejection

Corneal graft rejection is now the most common cause of graft failure after penetrating keratoplasty. This study was designed to determine whether, addition of 2% topical cyclosporine (CSA) to local and systemic steroids in treatment of endothelial corneal allograft rejection, would improve the outcome. A prospective randomized treatment trial was carried out on 40 consecutive corneal graft recipients, presenting with the first episode of endothelial graft rejection in two groups. Group one (20 patients) received topical steroids eye drops and systemic prednisolone (1 mg/kg) by oral route plus placebo. Group two (20 patients) received the same topical and systemic steroid therapy plus 2% cyclosporine A (CSA) eye drop. The patients were followed up for three months and their clinical outcomes were evaluated by the rates and time of rejection reversal. In group one, 14 (70%) cases had total reversal of graft rejection episode but in CSA group, it occurred in 18 (90%) cases (P=0.21) .Improvement were started within a mean period of 3 and 1.5 days respectively (P value<0.001). Among patients who sought treatment early (<6 days), the survival rates were 85% and 100% respectively (P=0.2). In high risk patients the rejection reversal rate was 66% in CSA group and 25% in the control group (P=0.5). Our study indicates addition of 2% CSA eye drop to topical and systemic steroids in graft rejection decreases the interval between treatment intervention and improvement of clinical signs. In high risk patients it may improve the reversal rate, however it needs further studies.

Sustained rapamycin drug delivery system in prevention of high risk corneal allograft rejection and neovascularization in rabbits

To evaluate the immunosuppressive and antiangiogenesis effects of rapamycin drug delivery system (RAPA DDS) in high risk rabbit model of penetrating keratoplasty (PK). (1) RAPA DDS preparation: 50 mg of PGLC and 50 mg of RAPA were mixed as a RAPA drug delivery system. (2) High risk rabbit model: Corneal vascularization was induced in 45 New Zealand white rabbits (45 eyes) by passing 5 - 0 silk sutures in corneal stroma in each quadrant. (3) 40 rabbits with corneal neovascularization beyond three quadrants were received a unilateral 7 mm diameter central PK. The 40 were divided into four treatment groups: Group A, control group and received no therapy; Group B, 1 mg PGLC carrier was implanted in the anterior chamber; Group C, 1% RAPA eye drops was applied four times daily; Group D, 0.5 mg RAPA DDS was implanted in the anterior chamber. (4) Postoperative examination: The cornea allografts (opacity, edema and neovascularization) were examined by the slit-lamp biomicroscopy for ninety days. Rejection index (RI) and neovascularization index (NI) of these animal models were recorded. RAPA concentration in the aqueous humor was detected on 2, 4, 8 and 12 weeks in group C and D after surgery; the expressions of IL-2R, MCP-1, Fas/FasL in samples were detected with in situ hybridization; TNF-alpha and VEGF were detected with immuno-histochemical technique three weeks after the operation in all groups. Histochemical method was carried out on the procured specimens of cornea, retina, liver and kidney at ninety days. (1) Allografts rejection: Mean survival times in 4 trial groups were (16.5 +/- 2.5), (16.0 +/- 2.6), (47.1 +/- 13.2), (87.6 +/- 5.8) d respectively (P = 0.000). (2) Corneal neovascularization: Mean NI was 2.4 +/- 0.7, 2.1 +/- 0.5, 0.6 +/- 0.5, 0.3 +/- 0.5 (P = 0.000) 2 weeks after the operation, and the NI value was 3.8 +/- 0.5, 3.8 +/- 0.4, 0.8 +/- 0.7, 0.4 +/- 0.8 (P = 0.000) 12 weeks after the operation in groups A, B, C and D respectively. (3) RAPA concentration in aqueous humor: Mean RAPA concentration in aqueous humor was 10.7, 12.0, 9.2, 7.0 ng/ml in group D in the 2, 4, 8 and 12 weeks after the operation respectively. RAPA can not be detected in group C. (4) Cytokine expression: IL-2R, MCP-1, TNF-alpha and VEGF were overexpression in group A and B, and undetectable in group C and D. Fas and FasL were negative in all groups. (5) No inflammatory cell infiltration was found in retina, liver and kidney tissue ninety days after the surgery. Sustained RAPA DDS and eyedrops can prolong allograft survival and inhibit cornea neovascularization in rabbit model. However, RAPA DDS is better than eyedrops.

Comparison of FK 506, Mycophenolate Mofetil, and Aminoguanidine Effects on Delay of Corneal Allograft Rejection in an Experimental Model of Low-Risk and High-Risk Keratoplasty

The purpose of our study was to compare the effectiveness of immunosuppressive drugs on the prevention of allograft rejection in a murine model of low-risk and high-risk keratoplasty. The therapy included FK 506 (tacrolimus; 0.2mg/kg), mycophenolate mofetil (30mg/kg), aminoguanidine (0.1g/kg), and combination of FK506 + mycophenolate mofetil or FK506 + aminoguanidine. The results obtained from the Gray’s survival model stratified according to the type of subjects suggest that a major rejection risk reduction was achieved using FK506; good results also were obtained for mycophenolate mofetil. Although the point estimates of both the survival and relative risk of rejection suggest a deferred effect of the combination FK506 + mycophenolate mofetil, this finding did not prove statistically significant.

The Impact of Corneal Allograft Rejection on the Long-Term Outcome of Corneal Transplantation

To examine the influence of corneal allograft rejection on the survival of penetrating corneal transplantation, to review the status of conventional therapies to improve graft survival, and to consider prospects for alternative approaches to reduce the impact of rejection. Perspective, including prospective, observational cohort study. An examination of the literature on human corneal graft rejection and data from the Australian Corneal Graft Registry, reviewed in the context of clinical experience. Corneal graft outcome is not improving with era. The sequelae of inflammation, whether occurring before corneal transplantation or subsequently, exert a profound influence by predisposing the graft to rejection. Of the developments that have been instrumental in reducing rejection in vascularized organ transplantation, living-related donation is not an option for corneal transplantation. However, HLA matching may be beneficial and requires reassessment. The evidence base to support the use of systemic immunosuppressive agents in corneal transplantation is thin, and topical glucocorticosteroids remain the drugs of choice to prevent or reverse rejection episodes. Experimental approaches to local allospecific immunosuppression, including the use of antibody-based reagents and gene therapy, are being developed but may be difficult to translate from the laboratory bench to the clinic. Corneal allograft rejection remains a major cause of graft failure. High-level evidence to vindicate the use of a particular approach or treatment to prevent or treat corneal graft rejection is lacking. In the absence of extensive data from randomized, controlled clinical trials, corneal graft registers and extrapolation from experimental models provide some clinically useful information.

Centennial review of corneal transplantation

Abstract One hundred years ago, on 7 December 1905, Dr Eduard Zirm performed the world's first successful human corneal transplant. This significant milestone was achieved only after many decades of unsuccessful trial and error; however, it did not lead to relatively 'routine' keratoplasty success for several more decades. The idea of replacing an opaque cornea had been suggested for centuries, and had stimulated theoretical approaches to the problem by many esteemed physicians throughout history. However, little practical progress was made in the ultimate realization of the dream until the 19th century when pioneering surgeons pursued extensive studies in relation to both animal and human 'keratoplasty'. Clinical progress and scientific insight developed slowly, and it was ultimately due to parallel advances in medicine such as anaesthesia and antisepsis that Zirm's success was finally achieved. Key concepts were enshrined such as the use of fresh tissue from the same species, careful placement and handling of tissue, and the development of specialized instrumentation such as the circular trephine. In the latter half of the 20th century, many 'masters' of corneal surgery evolved significant refinements in technique and instrumentation with the development of corticosteroids, antibiotics, surgical microscopes, improved trephines, viscoelastics and suture materials, that enable this delicate procedure to be routinely performed with the prospect of success. There are still limitations to corneal transplantation, and corneal allograft rejection still poses the greatest challenge to the modern corneal surgeon. In the foreseeable future it may be in the laboratory, rather than the theatre, that further milestones will be achieved. This review aims to highlight the significant milestones in the rich history of corneal transplantation, and to pay tribute to the many inspired and dedicated individuals involved in the development of keratoplasty to a point where the procedure is now a standard tool in the repertoire of ophthalmic surgery and more than a million people have enjoyed restoration of useful sight.

Thiol Redox and Immune Regulation in Corneal Transplantation

Penetrating keratoplasty (PKP) is the most common type of clinical grafting performed in humans. Although PKP has emerged as the most successful form of transplantation, PKP in "high-risk" eyes shows high incidence of allograft rejection. The incidence of epithelial rejection after limbal transplantation (LT) is extremely higher and swifter than PKP rejection, and even intensive systemic immunosuppressive therapy is often of no avail. Because failure of corneal grafts is an important cause of blindness, developing new strategies for suppressing graft rejection is a worthy goal for research. Corneal allograft rejection is mainly mediated by the TH1-type immune response, which leads to a delayed-type hypersensitivity reaction. Because the TH2-type immune response regulates the TH1-type immune response, we have successfully elicited allograft survival after both PKP and LT by inducing systemic TH2-type immune responses. Because intracellular thiol redox status of antigen-presenting cells (APC) reportedly regulates TH1/TH2 balance via distinctive cytokine production by APC, we also investigated the effect of modulating macrophage intracellular thiol redox status on corneal allograft survival. These strategies are quite effective in major histocompatibility complex (MHC) matching in mice, although it is believed that MHC matching has no effect on corneal allograft survival according to many rodent studies. Recently, many laboratories are reconsidering HLA matching for allograft survival in human corneal transplantation. It may be possible that MHC matching improves corneal allograft survival in the context of TH1 suppression. We propose that the suppression of the TH1-type immune response and MHC matching together may promote allograft survival in humans.

AB0-Blutgruppenexpression in Hornhäuten mit Transplantatversagen

ABO blood group antigens are only expressed by the epithelial cells of normal human corneas. Since AB0 blood group antigens are also known to be expressed on stromal and endothelial cells of inflamed corneas, this study aimed to investigate the extend of ABO blood group antigen expression in corneal allograft failures. Twenty-two failed corneal allografts of 22 patients were examined. In 12 cases the patients had clinically proven corneal allograft rejection. In 10 cases there was no evident history of allograft rejection and the diagnosis graft failure due to chronic endothelial cell loss was made. Immunohistochemical staining of paraffin embedded sections was performed with monoclonal mouse antibodies to human blood group antigen A or B using the streptavidin-biotin-peroxidase complex technique. Blood group antigens A or B were expressed by stromal keratocytes in 5 out of 12 and by endothelial cells in 7 out of 12 corneas with clinically proven immunologic graft rejection. Corneal transplants with chronic endothelial cell loss expressed blood group antigens A and/or B on the endothelial cells in three out of ten cases. The results of this study show that ABO blood group antigens can be up-regulated in cases of corneal allograft failure, especially in cases of immune mediated graft rejection. This phenomenon might play a role in corneal allograft rejection.

Experimental study on the effects of rapamycin in prevention of rat corneal allograft rejection

To investigate the effects of rapamycin on prevention of corneal allograft rejection in murine corneal transplantation. The outbred strain SD and Wistar rats were used as donors and recipients, respectively. Sixty-eight Wistar rats were divided into four groups: Group A, autograft control; Group B, allograft control (the control groups were given placebo only); Group C and D, allograft groups, were treated with orally RAPA (3 mg.kg(-1).d(-1)) and CsA (10 mg.kg(-1).d(-1)), respectively. The drugs were delivered for 12 days beginning at the day of transplantation. Each animal was examined by operating microscopy. The grafts were evaluated clinically by means of Holland's scoring system and graft survival was assessed by Kaplan-Meier analysis. The neovascular indexes of rejection were compared among different groups. Histological examination on ocular tissues was performed on day 14 to confirm the clinical diagnosis of rejection. The average transplant survival time in the allogenic control (Group B) was (11.0 +/- 1.5) d. Treatment with RAPA (Group C) led to a statistically significant prolongation of transplant survival to (36.1 +/- 14.9) d (P < 0.05). Treatment with RAPA 3 mg.kg(-1).d(-1) prolonged transplant survival as compared with treatment with CsA (Group D), but the difference was not statistically significant (P > 0.05). Corneal neovascularization was induced after the surgery. In RAPA group, corneal neovascularization was markedly reduced as compared with allograft control (Group B) (P < 0.05) and CsA group (Group D) (P < 0.05). Fewer inflammatory cells were found in the corneal stroma of the RAPA group. These results show that oral immunosuppression with RAPA can prevent corneal graft rejection and corneal neovascularization.

Alpha‐1‐‐antitrypsin in aqueous humour from patients with corneal allograft rejection

To investigate the presence and concentration of alpha1-antitrypsin in aqueous humour at the time of corneal rejection and to compare results obtained from patients with reversible and irreversible rejection. Samples of aqueous humour were obtained from 17 patients with acute corneal endothelial allograft rejection. The presence of alpha1-antitrypsin in aqueous humour was confirmed by immunoblotting and measured employing a sandwich ELISA. Total protein concentrations in aqueous humour were measured using Bradford's method. The outcome of corneal rejection episodes was determined 1 month after diagnosing corneal rejection and described as reversible or irreversible rejection. alpha1-antitrypsin was detected in aqueous humour. Patients with reversible rejection had significantly higher alpha1-antitrypsin concentration than patients with irreversible rejection (p = 0.044). There was no significant difference in total protein concentrations (p = 0.745), and no correlation was found between alpha1-antitrypsin and total protein concentrations (p = 0.368). alpha1-antitrypsin in aqueous humour seems to signal a favourable outcome of corneal rejection. The possible mechanism is discussed.

Cutting Edge: Atopy Promotes Th2 Responses to Alloantigens and Increases the Incidence and Tempo of Corneal Allograft Rejection

A large body of evidence suggests that corneal allograft rejection is mediated by a type 1 Th cell response and that deviation toward type 2 immunity favors graft survival. However, clinical observations indicate that patients with severe ocular allergies have increased risk of corneal allograft rejection. We used a mouse model of atopic conjunctivitis to evaluate the effects of Th2 immune deviation on corneal allograft survival and possible mechanisms of graft rejection. Our results reveal the following novel findings: 1) atopic conjunctivitis promotes systemic Th2 immune responses to corneal graft donor alloantigens; 2) corneal allografts in atopic host eyes have an increased incidence and swifter tempo of rejection; 3) increased rejection is associated with alterations in systemic T cell-mediated responses to donor alloantigens; and 4) corneal allograft rejection in atopic hosts does not require the direct involvement of infiltrating eosinophils.

Local Gene Transfer to Modulate Rat Corneal Allograft Rejection

Allograft rejection is the leading cause of corneal graft failure. CD4(+) T cells control the allograft response and represent targets for antirejection therapy. The purpose of this study was to transfer cDNA encoding a monomeric anti-CD4 antibody fragment to donor corneal endothelium, to attempt to modulate orthotopic corneal allograft rejection in the rat. A replication-deficient adenoviral vector (AdV) encoding anti-CD4 single-chain, variable-domain antibody fragment (scFv) and enhanced green fluorescent protein (eGFP) was constructed (AdCD4GFP). AdV encoding eGFP alone (AdGFP) was used as a control. Transgenic product was detected by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, flow cytometry, and fluorescence microscopy. The alloinhibitory capacity of anti-rat CD4 scFv was measured in the one-way mixed lymphocyte reaction (MLR). The survival of Wistar-Furth corneas transduced with AdV either immediately or 3 days before orthotopic transplantation in Fischer 344 recipients was examined. ScFv and eGFP mRNAs were detected in rat corneas transduced in vitro, and active scFv secreted in corneal supernatants peaked at days 4 to 5 after transduction at 23 +/- 4 ng of protein per cornea per day. Antibody and scFv against rat CD4 blocked alloproliferation in MLR. However, transduction of corneas with AdCD4GFP ex vivo, immediately before transplantation, or in vivo, 3 days before transplantation, did not significantly prolong corneal allograft survival (P > 0.05). Anti-CD4 scFvs were capable of blocking allostimulation, but their local expression within the eye did not prolong corneal allograft survival, suggesting that sensitization may still occur.

Susceptibility of corneal allografts and xenografts to antibody-mediated rejection

The effects of passive transfer of antisera containing cytotoxic antibodies to allo- and xenoantigens on survival of corneal allografts and xenografts were evaluated in experimental models. Corneas from allogeneic B10 or xenogeneic rat Lewis donors were grafted orthotopically into BALB/c mice. Recipient mice were treated with donor-specific antisera administered at the period of grafting or at 2 weeks after transplantation. Rejection was determined by the severity of corneal opacity using a standard scoring system. Treatment of graft recipients with donor-specific antisera accelerated the onset of graft rejection and significantly shortened survival times of both corneal allografts and xenografts. Corneal xenografts, which had been accepted after treatment with anti-CD4 monoclonal antibody, were acutely rejected by the passive transfer of antiserum against xenoantigens. The results suggest that corneal grafts are vulnerable to antibody-dependent immunity and that cytotoxic antibodies against graft donor antigens can mediate rejection of both corneal allografts and xenografts.

The corneal allograft rejection features in CD4 and CD8 knock-out mice

To study the mechanism and the features of corneal allograft rejection using a model of CD4 and CD8 knock-out mice. The mice were divided into three groups with 20 mice in each group. CD4 knock-out mice, CD8 knock-out mice and C57BL/6 mice were used as recipients and BALB/c mice as corneal graft donors. Postoperative evaluation included slit-lamp biomicroscopy and immunohistological studies. The rejection times were recorded and the cytokines in the eye after the surgery were measured at 1 week, 2 weeks and 4 weeks after the transplantation. Thirty mice (each group ten mice) received skin transplantation using BALB/c as donors in the second week after penetrating keratoplasty. The time of skin grafts rejection was recorded and the cornea grafts were inspected when the skin grafts were rejected. The rejection time varied in these three groups. The corneal grafts in CD4 knock-out mice were clear and no rejection occurred > 90 days. The corneal grafts in CD8 knock out mice were rejected at (28 +/- 3) days. The grafts in the control groups were rejected at (14 +/- 2) days (F = 2034, P < 0.01). The skin grafts rejection were recorded at (14 +/- 2), (12 +/- 1), (10 +/- 1) days in CD4 knock-out mice, CD8 knock-out mice and control groups, respectively (F = 42.54, P < 0.01). CD4 and CD8 knock-out mice are useful models for studying of corneal graft rejection. The cornea allograft rejection after penetrating keratoplasty is mediated primarily by CD(4)(+) T lymphocytes. The CD(8)(+) T lymphocytes also participate in the rejection processes.

Early Results of Penetrating Keratoplasty After Cultivated Limbal Epithelium Transplantation

To describe the early results of penetrating keratoplasty (PKP) in patients who had previously undergone cultivated limbal epithelium transplantation. Medical records of patients with limbal stem cell deficiency due to chemical burns who underwent PKP after cultivated limbal epithelium transplantation were reviewed for demographics, primary etiology, type of limbal transplantation, ocular surface stability, visual acuity, graft clarity, and complications. Histopathologic features of the recipient corneal buttons were studied with special attention to epithelial status. Of the 125 patients with limbal stem cell deficiency treated with cultivated limbal epithelium transplantation, 15 underwent PKP at a mean interval of 7 months (range, 2-12 months) following cultivated limbal epithelium transplantation (autologous, n = 11; allogenic, n = 4). All 4 patients treated with allogenic cultivated limbal epithelium transplantation were undergoing immunosuppressive therapy. Fourteen (93%) of the 15 eyes had a successful corneal graft with a stable corneal epithelium. Preoperative best-corrected visual acuity was less than 20/200 in 14 of the 15 eyes. At a mean +/- SD follow-up of 8.3 +/- 5.0 months after PKP, the best-corrected visual acuity was more than 20/60 in 8 eyes, 20/200 to 20/60 in 5 eyes, and less than 20/200 in 2 eyes. Three of the 15 eyes experienced corneal allograft rejection, which was managed successfully. One eye with graft rejection also had glaucoma. None of the limbal epithelial allografts showed signs of rejection. Early results of PKP following cultivated limbal epithelium transplantation are favorable when performed after stabilizing the ocular surface. Adequate immunosuppression is essential for allogenic cultivated limbal epithelium transplantation to avoid rejection. Corneal allografts can separately reject the limbal allografts.

Ipsilateral lymphadenectomy to inhibit corneal allograft rejection in rats.

In order to investigate the ipsilateral lymphadenectomy for inhibiting rejection in rat corneal transplantation, corneal allogenic transplantation models were established in rats. Eighteen female Wister rats were used as donors, and 36 Sprague Dawley rats as recipients. After penetrating corneal transplantation, recipients were randomly divided into 3 groups: group A (control group); group B, the ipsilateral lymphadenectomy group; group C, the bilateral lymphadenectomy group. Among 12 rats in each group, the corneas of 2 rats in each group were used for pathological study at day 14 after the transplantation, and the remaining 10 rats were used for studying corneal rejection by a slit lamp. The time points when allograft rejection occurred were recorded and mean survival time (MST) was compared. The results showed that MST in groups B and C was 46.30 +/- 9. 464 days and 44.43 +/- 7. 604 days, respectively, which was significantly prolonged as compared with that in group A (10.71 +/- 1.567 days, P < 0.01). There was no significant difference in MST between groups B and C (P > 0.05). It was concluded that both bilateral and ipsilateral lymphadenectomy therapies could effectively inhibit the corneal allograft rejection. Ipsilateral lymphadenectomy is a less complex surgical procedure and is just as effective in preventing rejection.

CD4+ T-Cell–Mediated Mechanisms of Corneal Allograft Rejection: Role of Fas-Induced Apoptosis

The role of CD4(+) T cells as effector cells in corneal allograft rejection is poorly understood. We investigated the role of CD4(+) T cells as helper cells in the generation of allospecific effector macrophages in corneal graft rejection and the role of CD4(+) T cells as apoptosis-inducing effector cells. Corneal allografts were transplanted to CD4 knockout, FasL-deficient, and macrophage-depleted hosts. An Annexin-V binding assay was used to evaluate the susceptibility of corneal cells to both Fas-dependent and CD4 T-cell-mediated apoptosis in vitro. Macrophages were essential for graft rejection, but not as effector cells. Anti-BALB/c CD4(+) T cells from immunized C57BL/6 mice induced apoptosis of BALB/c corneal epithelial and endothelial cells. However, anti-BALB/c CD4(+) T cells from FasL-deficient gld/gld mice did not induce apoptosis of BALB/c corneal endothelial cells. Moreover, gld/gld mice had a reduced capacity to reject BALB/c corneal allografts. Although the initial results suggested a role for Fas-induced apoptosis in corneal graft rejection, additional experiments indicated otherwise. The incidence and tempo of immune rejection of Fas-deficient lpr/lpr corneal allografts were no different than those for corneal grafts from Fas-bearing C57BL/6 donors. Moreover, CD4(+) T-cell-mediated apoptosis of corneal cells could not be blocked with either Fas-Fc fusion protein or anti-FasL blocking antibody. The results suggest that CD4(+) T cells function directly as effector cells and not as helper cells in the rejection of corneal allografts. Although the corneal endothelium is highly susceptible to Fas-induced apoptosis, this is apparently not the primary mechanism of CD4(+) T-cell-dependent rejection.

Early Results of Penetrating Keratoplasty following Limbal Stem Cell Transplantation

To describe the early results of penetrating keratoplasty (PKP) in patients who had earlier received limbal transplantation (LT). Prospective, non-comparative interventional case series comprising of four patients with limbal stem cell deficiency (LSCD) due to chemical injury (Cases 1, 2, 4) and xeroderma pigmentosum (Case 3). Cadaveric kerato-limbal allografts or living-related conjunctival-limbal allografts were done in four eyes followed by PKP for visual rehabilitation 3-4.5 months later. The following details were noted: demographics, primary aetiology, type of limbal transplant (cadaveric or living-related), immunosuppression, vision and ocular surface stability before and after LT and PKP, surgical complications and outcome of PKP. Three eyes received living-related conjunctival-limbal allotransplantation and one received cadaveric kerato-limbal allograft. Duration of follow up after PKP ranged from 4 to 11 months. Visual acuity improved in the early postoperative period in all patients but reduced in 2 due to endothelial rejection and after trans-scleral cyclophotocoagulation for medically uncontrolled glaucoma. The ocular surface remained stable in all patients. All patients were started on immunosuppression on the first postoperative day. This was continued till the last follow-up visit. Post-PKP complications were punctate epithelial keratopathy, corneal allograft rejection and secondary glaucoma (one patient each). Satisfactory visual rehabilitation is possible after PKP following LT without compromising ocular surface stability. However, a prolonged and close follow-up is warranted to avert complications.

Rapid Corneal Allograft Rejection with TH2 Cell Involvement in a Model of Atopic Conjunctivitis

Mice injected with single or multiple doses of soluble alloantigen extracts (SAE) derived from H-2b cells failed to develop thymus dependent cell-mediated immunity (CMI) upon subsequent immunization with EL-4 (H-2b) tumor allograft. By contrast, the humoral response was only slightly affected. Nevertheless, the mice rejected the tumor allograft with no sign of enhancement. Suppression of cell-mediated immunity (SCMI) was obtained with SAE prepared from either tumor or spleen cells and was shown to be specific, i.e., SAE derived from sources histoincompatible with the allografted cells had no effect. The best conditions used to demonstrate specific SCMI were the following: (A) the alloantigen must be soluble; (B) the SAE must be administered prior to tumor inoculation; (C) multiple injections of SAE were more effective than single injection; (D) it was best observed with suboptimal tumor dose inoculums. Mice treated with a single dose of SAE showed appreciable blocking activity in their serum prior to tumor inoculation. Blocking, however, was not detected in the serum of mice injected with multiple injections of soluble alloantigen. These findings offered possible mechanisms for SCMI (afferent and/or central blocking).We conclude from these results that SAE preferentially suppress the formation of T-cytotoxic cells but not the T-helper cells. This preferential effect is discussed in view of its importance in promoting graft survival.