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Purpose: To further evaluate the role of draining cervical lymph nodes (CLN) in high-risk (HR) corneal allografts performed in vascularized beds. Recently, we have shown that CLN are critical for promotion of allograft rejection in normal-risk corneal allografts., Methods: Fully mismatched HR orthotopic corneal transplantation was performed in BALB/c hosts that had their CLN excised before transplantation (CLN-). Graft rejection and allospecific delayed-type hypersensitivity (DTH) were used as measures of alloreactivity. Numbers of interferon gamma (IFN-gamma)- and interleukin 2 (IL-2)-expressing cells were compared among hosts that retained their native CLN (CLN+) and CLN-hosts. Additionally, splenectomized mice (Sp-), and hosts without either CLN or spleen (CLN-Sp-) were evaluated., Results: Within 5 weeks, 100% of HR grafts among CLN+ hosts were rejected, and hosts of these grafts uniformly demonstrated allospecific delayed-type hypersensitivity (DTH). In contrast, 92% of CLN-hosts accepted their HR allografts, and demonstrated suppressed allospecific DTH response. Moreover, significantly lower numbers of IFN-gamma- and IL-2-expressing cells were infiltrating corneal grafts in CLN-group. All Sp-hosts rejected corneal allografts in an accelerated manner, whereas 86% of CLN-Sp-hosts accepted their allografts indefinitely., Conclusions: Draining CLN play a critical role in alloimmunity and rejection of HR corneal grafts, suggesting the essential function of CLN in corneal alloimmunization regardless of the degree of preoperative risk. Modulation of factors that regulate access of alloantigens or antigen-laden antigen-presenting cells to draining CLN may offer novel strategies in controlling induction of alloimmunity in corneal transplantation.

The purpose of this study was to investigate the role of CD80 and CD86 costimulatory molecules in corneal allograft rejection. Anti-CD80 and anti-CD86 monoclonal antibodies (mAbs) were administered after orthotopic corneal allograft transplantation. Graft rejection was observed by biomicroscopy. Population and localization of CD80(+)and CD86(+)cells in the cornea, cervical lymph nodes, and spleen were examined by flow cytometry and immunohistochemistry. The combined use of anti-CD80 and anti-CD86 mAbs was effective in prolonging corneal allograft survival. In the untreated mice bearing rejected graft, many CD86(+)and CD80(+)cells were found around the host-graft junctional area in the cornea, and CD86(high)cells were found in the cervical lymph node and spleen. In contrast, few CD86(+)or CD80(+)cells were observed in the cornea, cervical lymph node, and spleen from the mice treated with anti-CD80/CD86 mAbs. These results demonstrated a significant role of CD80 and CD86 costimulatory molecules in corneal allograft rejection., (Copyright 2002 Elsevier Science Ltd.)

Purpose: To perform a retrospective study to evaluate the long-term outcome of systemic cyclosporine treatment as an adjunct to topical corticosteroid treatment after penetrating keratoplasty (PKP)., Methods: Twenty-six high-risk patients (27 eyes) who received systemic cyclosporine following PKP for an average of 5.4 months were compared with another series of 57 patients (57 eyes) who did not receive cyclosporine after PKP., Results: Endothelial rejection developed in 2 cases during cyclosporine treatment and in 6 cases after discontinuation. The rate of rejection-free graft survival was similar between the treated and the control groups. The control group showed a significantly higher rate of graft survival than the treated group. As side effects in the treatment group, transient elevation in blood urea nitrogen or creatine developed in 7 cases. Increase in glutamate oxaloacetate transaminase (GOT) or glutamate pyruvate transaminase (GPT) developed in 4 cases. Severe side effects were absent throughout the series in both groups of patients., Conclusion: Systemic cyclosporine treatment for several months did not reduce the incidence of rejection nor improve the rate of graft clarity in the long term in high-risk patients after PKP.

Purpose: To evaluate risk factors for graft failure and allograft rejection after penetrating keratoplasty (PK)., Methods: We retrospectively studied clinical results of PKs in terms of graft survival and rejection-free graft survival rates. PKs were done on 271 eyes between 1987 and 1997. Clinical results were analyzed by Kaplan-Meier's life table method and the log-rank test. Relative risks and adjusted survival probabilities for each value of the factor were compared with the risk for a specified reference value., Results: The overall rates of graft survival and rejection-free graft survival in 10 years after PK were 79.3% and 77.9%, respectively. Higher relative risk of graft failure was associated with corneal vascularization (relative risk for within one quadrant = 1.67, two quadrants = 2.37, three or more quadrants = 3.39), regraft (relative risk for one failed previously graft = 2.08, two or more failed previously graft = 2.65), aphakia (relative risk = 2.17) or pseudophakia (relative risk = 3.02), presence of anterior synechia (relative risk = 2.91), presence of posterior synechia (relative risk = 2.56), long (more than 85 minutes) operation time (relative risk = 2.20), and older (more than 50 years) recipient age (relative risk = 2.38). Higher relative risk of rejection was associated with corneal vascularization (relative risk for within one quadrant = 2.35, two quadrants = 2.03, three or more quadrants = 2.63), long (more than 85 minutes) operation time (relative risk = 1.47), and younger (less than 60 years) donor age (relative risk = 2.10). There was no association between graft failure or allograft rejection and graft size or suture technique, respectively., Conclusion: The risk factors for graft failure after PK were corneal vascularization, regraft, aphakia or pseudophakia, presence of anterior synechia, presence of posterior synechia, long operation time, and older recipient age. The risk factors after PK for allograft rejection were corneal vascularization, long operation time, and younger donor age.

To determine whether preservation of the donor cornea prevents allograft rejection, orthotopic corneal transplantation was performed using corneas preserved in storage medium (Optisol-GS((R))). Donor corneas harvested from C3H/He (H-2(k)) mice and B10.D2 (H-2(d)) mice were preserved in storage medium for 0, 3, 7 and 14 days, and then transplanted into the corneal beds of the recipient BALB/c (H-2(d)) mice. Graft survival was determined clinically and histologically. The expression of major histocompatibility complex (MHC) molecules in the preserved corneas was analysed by immunohistochemistry and Western blotting. Donor-specific cytotoxic T lymphocyte (CTL) and delayed-type hypersensitivity (DTH) responses were assessed 3 weeks after grafting. Active suppression of DTH in the recipient mice was also examined 3 weeks after grafting. The survival of 14 day preserved allografts was significantly higher than that of the non-preserved allografts in both MHC and minor histocompatibility (H) antigens, and minor H only disparate combination. The recipients of the preserved allografts failed to induce both CTL and DTH. The active suppression of DTH was not acquired in these recipients. The expression of donor-derived MHC class I antigens was markedly reduced in the corneas after preservation. Preservation of the donor cornea had a remarkable effect on the prevention of corneal allograft rejection. Since the preserved allografts failed to induce donor-specific CTL and DTH, and active suppression of DTH was not acquired in the recipients, the prevention of allo-rejection is due to a failure of allo-sensitization. These results indicate that the reduction of MHC class I antigens and minor H antigens expression in the preserved grafts induces a failure of allo-sensitization and leads to the high rate of acceptance in corneal allografts., (Copyright 2000 Academic Press.)

Purpose: To evaluate the long-term outcome of topical 2% cyclosporine A (CsA) treatment as an adjunct to topical corticosteroid treatment of patients after penetrating keratoplasty (PKP)., Methods: We reviewed the records of 83 patients (86 eyes) who had undergone PKP and received topical CsA treatment postoperatively; also the records of 95 PKP patients (97 eyes) who received the same treatment, except for the 2% CsA eyedrops, and served as controls. The patients were also subdivided into high-risk and low-risk groups. The clinical outcome of PKP was evaluated by the rates of graft survival and rejection-free graft survival, using the Kaplan-Meier method, and compared with the log-rank test., Results: In the high-risk patients, the rejection-free graft survival rate was 69.7% in the CsA group and 45.4% in the control group (P =.030), but there was no significant difference in the graft survival rate between the two groups., Conclusion: Topical cyclosporine treatment is effective in reducing the risk of allograft rejection in high-risk patients.

Purpose: To assess the effects of histocompatibility Y (H-Y) antigen matching on the rate of corneal allograft rejection after penetrating keratoplasty (PKP)., Methods: We retrospectively investigated the graft survival rate and rejection-free graft survival rate after PKP in 396 eyes. The compatible combinations of H-Y antigen included male donors and male recipients (n = 135), female donors and male recipients (n = 107), and female donors and female recipients (n = 60). Incompatible combination was from male donors and female recipients (n = 94). The eyes were classified into two groups--high-risk (168 eyes) and low-risk (228 eyes)--depending on the degree of vascularisation in the recipient corneas or a history of previous allograft rejection. Data were analysed using the Kaplan-Meier life table method, the log-rank test and the Cox proportional hazards model., Results: In both the high-risk and low-risk groups, the graft survival and rejection-free graft survival rates were not affected by the H-Y compatibility. The graft survival (p < 0.001) and rejection-free graft survival (p < 0.001) rates were higher in the low-risk group than in the high-risk group. High-risk PKP was associated with greater risk of graft failure (risk ratio, 2.33) and rejection (risk ratio, 2.05) than low-risk PKP., Conclusion: H-Y antigen matching does not influence the rate of allograft rejection after PKP.

Purpose: To evaluate effects of ABO antigen blood-group matching on the rates of corneal allograft rejection after penetrating keratoplasty., Methods: We retrospectively studied clinical results of penetrating keratoplasties in terms of graft survival rates and rejection-free graft survival rates. Penetrating keratoplasties were done on 95 eyes between 1993 and 1997. Clinical results were analyzed using the Kaplan-Meier life table method and log-rank test. The corneal transplantations were classified into 2 groups, high-risk (35 eyes) and low-risk (60 eyes) transplantations. High-risk transplantation was defined as significant vascularization in the recipient corneas or a history of graft failure. The remaining transplantations were defined as low-risk., Results: The respective graft survival (p = 0.031) and rejection-free graft survival (p = 0.0097) rates were higher in the low-risk than in the high-risk group. In the high-risk group, the rejection-free graft survival was 68.9% for the ABO-compatible subgroup and 36.4% for the ABO-incompatible subgroup (p = 0.007)., Conclusion: ABO matching is effective in reducing the risk of allograft rejection in high-risk corneal transplantations.

Purpose: To evaluate the immunologic effect of anterior synechiae (AS) in a murine model of corneal transplantation., Methods: Orthotopic penetrating keratoplasty with 12 interrupted sutures was performed on C57BL/6 donor mice and BALB/c recipient mice without AS (AS- group). In contrast to suturing in the AS- group, 3 of the 12 sutures were placed to create AS (AS+ group). The average graft opacity scores and rejection rates of both groups were compared. Cytotoxic T-lymphocyte (CTL) reactions and delayed hypersensitivity (DH) were evaluated 3 weeks after transplantation. Corneal cytokine expression was evaluated., Results: The opacity scores of the AS+ group were consistently greater than those of the AS- group, and the rejection rate of the AS+ group was significantly greater than that of the AS- group (86% versus 54%, P = 0.03). The AS+ group had significantly higher CTL activity compared with the AS- group. There was no significant difference in DH between the two groups. The cytokine expression pattern in the AS+ group became similar to that of the AS- group in which the grafts were rejected., Conclusions: These findings indicate that AS impairs ocular immune privilege by mediating CTL activity, but without intensifying the DH response. Therefore, AS is a critical risk factor in allograft rejection in a murine model of corneal transplantation.

Background: Anti-alphabeta T cell receptor monoclonal antibody (R73) has been reported to be a potent immunosuppressant. The suppressive effects of this antibody on allograft rejection after corneal transplantation are unknown., Methods: Orthotopic rat penetrating keratoplasty was performed using Lewis rats as recipients and Brown Norway and Fisher rats as donors. The treated groups received R73 intraperitoneally until day 12 after the transplantation. In grafted rats with or without R73 treatment, cytokine expression of the aqueous humor, corneal-infiltrating cells, draining lymph nodes, and splenocytes was determined. Delayed-type hypersensitivity (DTH) responses were compared., Results: All allografts in the untreated controls of Fisher-to-Lewis or BN-to-Lewis rat combinations were rejected within 14 days. In contrast, indefinite survival rates of the postoperative R73-treated group increased to 86% in the Fisher-to-Lewis and 23% in the Brown Norway-to-Lewis combinations, respectively. Interferon-y, interleukin (IL)-2 (T helper [Th]1), and IL-10 (Th2), but not IL-4 (Th2), expression of the eye and DTH responses in the control group were suppressed in the R73-treated group. Both IL-2 and IL-10 expression after mixed lymphocyte culture in the R73-treated group were significantly lower than those of the naive and untreated control group., Conclusions: alphabeta T cell receptor-targeted therapy prevents allograft rejection in rat corneal transplantation as evidenced by suppression of DTH responses. The cytokine profile after R73 treatment was characterized by low interferon-gamma, IL-2, and IL-10, and high IL-4 expression.

It has been reported that allograft rejection is mediated by a variety of adhesion molecules. Using a corneal allograft model in mice, we studied the role of very late antigen (VLA)-4 and leukocyte function-associated antigen (LFA)-1 adhesion molecules in corneal allograft rejection and the effects of monoclonal antibodies (mAbs) to them in suppressing corneal rejection. C3H/He donor corneas were transplanted into BALB/c corneal beds. The allografted mice were treated with a control mAb (M18/2), mAbs to VLA-4, or LFA-1 or their combination by i.p. injection until day 7. The expression of VLA-4, LFA-1, major histocompatibility complex (MHC) class II antigens, interleukin (IL)-2, IL-2 receptor and interferon gamma (IFNgamma) in the grafted cornea were studied immunohistochemically. Cytotoxic T lymphocyte (CTL) responses to donor alloantigens were assessed. The skins from a syngeneic donor or a third-part strain were transplanted 8 weeks after the initial keratoplasty onto the mice treated with anti-LFA-1 plus anti-VLA-4 mAbs. Fourteen of 16 allografts in non-treated mice and control mAb-treated mice became opaque by 2 weeks after transplantation. At 2 weeks, non-treated allografts showed expression of MHC class II antigens on keratocytes and mononuclear cells at the host-graft junction. Also, mononuclear cells expressing VLA-4, LFA-1, IL-2, IL-2 receptor and IFNgammawere present in the stroma at the host-graft junction. The allografts treated with either anti-VLA-4 or anti-LFA-1 alone, or anti-VLA-4 plus anti-LFA-1 remained transparent for more than 2 weeks, and the survival rates at 14 weeks was 0%, 16.7%, and 75.0%, respectively. The combined use of anti-VLA-4 and anti-LFA-1 mAbs prolonged graft survival significantly (P<0.05) at 14 weeks as compared with anti-LFA-1 mAb alone. At 3 weeks, CTL responses to donor alloantigens were depressed in mice treated with either anti-LFA-1 alone or anti-LFA-1 plus anti-VLA-4. Specific prolongation of donor-syngeneic skin was observed after treatment with the combination of these two mAbs. These results indicate that VLA-4 and LFA-1 have important roles in rejection process of corneal allografts, and that the combined use of mAbs to these molecules has remarkable effects on inducing alloantigen-specific immunosuppression in corneal transplantation.

We clarified the preoperative risk factors for graft failure in penetrating keratoplasty with fresh donor corneas. This analysis covered 698 consecutive keratoplasty cases in a single center between 1971 and 1992, and was designed to assess the risk factors. The risk factors in penetrating keratoplasty were determined by the Cox multiple regression model which considers the follow-up periods and multiple factors simultaneously. The factors which worsen the prognosis of keratoplasty significantly were found to be preoperative endothelial dysfunction (hazard ratio = 2.3), prior glaucoma/ocular hypertension (hazard ratio = 2.0), preoperative corneal vascularization (hazard ratio for within one quadrant = 1.3, hazard ratio for 2 or more quadrants = 1.6), anterior synechiae of iris (hazard ratio = 1.5), aphakia or pseudophakia (hazard ratio = 1.4), and older donor age (hazard ratio = 1.3 in proportion to the increase of each 10-year period of age). Surgeons must take these risk factors into consideration to obtain a better prognosis for keratoplasty.

Background: It has been reported that adhesion molecules play an important role in immunological rejection after organ transplantation. In the present study, we examined the role of ICAM-1/ LFA-1 adhesion molecules in corneal allograft rejection and evaluated the immunological specificity of monoclonal antibodies (mAbs) in preventing allograft rejection in mice., Methods: The allografted mice were intraperitoneally injected with 100 micrograms/day of the following mAbs: a control mAb, anti-ICAM-1 mAb, anti-LFA-1 mAb, or a mixture of anti-ICAM-1 and anti-LFA-1 mAbs from 1 day before to 7 days after surgery. The expression of ICAM-1 and LFA-1 molecules in the grafted cornea was studied immunohistochemically. The corneas from a syngeneic donor or a third-party strain were transplanted 4 weeks after the initial keratoplasty onto the mice treated with both anti-ICAM-1 and anti-LFA-1 mAbs., Results: The allografts treated with anti-LFA-1 mAb alone or both anti-ICAM-1 and anti-LFA-1 mAbs remained transparent for more than 2 weeks, and the survival rate at 8 weeks was 40% in both groups. ICAM-1 was expressed on the mononuclear cells, keratocytes and endothelial cells in the allografts without treatment. The second corneal grafts syngeneic to the initial donor remained transparent at 2 weeks, whereas those from the third party were rejected., Conclusions: ICAM-1 and LFA-1 adhesion molecules play a crucial role in the pathophysiology of corneal transplant rejection. The immunosuppressive effects of anti-ICAM-1 and anti-LFA-1 mAbs are highly allospecific. The administration of mAbs to the adhesion molecules represents a new means of suppressing allograft rejection after penetrating keratoplasty.

In order to evaluate preoperative risk factors that affect the incidence of endothelial rejection after penetrating keratoplasty, we performed statistical analysis on 571 consecutive cases who had been followed at the University of Tokyo Hospital for at least one year. We applied a logistic regression model (multiple variate analysis) to determine the significance of the association between the preoperative factors and the incidence of rejection. We identified four preoperative risk factors of endothelial rejection in a year as follows: the extent of corneal vascularization, failed previous graft continuous sutures, and preoperative corneal endothelial damage. When the rejection cases were divided into an early group that suffered rejection within 3 months and a late group that suffered rejection after 6 to 12 months, young patients were likely to suffer rejection in the early postoperative period.