Tadahiko Tsuru

Role of Fas-Fas ligand interactions in the immunorejection of allogeneic mouse corneal transplants.

BACKGROUND The expression of Fas ligand (FasL) in the eye has been proposed to be an important component of ocular immune privilege. Since the unusually favorable outcome of corneal transplantation is thought to result from the immune privilege of the eye, examination of the function of FasL on corneal allografts would be a test of that hypothesis. METHODS To investigate the role of Fas-FasL interaction in corneal allografts, orthotopic corneal transplantation was performed using C57BL/6 (B6, FasL+) and B6-gld (FasL-) mice as cornea donors and BALB/c mice as recipients. The rejection rate of B6-gld grafts (FasL- group) was compared with that of normal B6 control corneas. RESULTS The rejection rate at the final observation (8 weeks) in the FasL- group (89%) was significantly higher than in the FasL+ control group (47%). FasL expression was found on the corneal endothelium by staining with anti-FasL monoclonal antibodies. The TdT-mediated dUTP nick-end labeling assay revealed that apoptotic cells were attached to the endothelium in the control group but not in the FasL- groups. CONCLUSIONS Apoptosis of infiltrating cells on the corneal endothelium resulting from Fas-FasL interaction plays an important role in the high success rate of corneal transplantation.

Draining lymph nodes play an essential role in alloimmunity generated in response to high-risk corneal transplantation.

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-&ggr;)– 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-&ggr;– 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.

Simultaneous corneal inlay implantation and laser in situ keratomileusis for presbyopia in patients with hyperopia, myopia, or emmetropia: Six-month results

PURPOSE: To evaluate the safety and efficacy of simultaneous Kamra corneal inlay implantation and laser in situ keratomileusis (LASIK) for the treatment of presbyopia in emmetropic, hyperopic, or myopic patients. SETTING: Private center, Tokyo, Japan. DESIGN: Cohort study. METHODS: Patients had bilateral LASIK with simultaneous implantation of a corneal inlay in the nondominant eye to treat presbyopia and ametropia between September 2009 and April 2010. The efficacy and safety were determined by the spherical equivalent (SE) in the eye with the inlay. RESULTS: The study enrolled 360 eyes of 180 patients with a mean age of 52.4 years ± 5.1 (SD) (range 41 to 65 years). Sixty‐four patients were available for the 6‐month postoperative examination. The mean logMAR uncorrected near visual acuity in the eye with the inlay improved 7 lines in hyperopic eyes, 6 lines in emmetropic eyes, and 2 lines in myopic eyes. The mean logMAR uncorrected distance visual acuity improved by 3 lines, 1 line, and 10 lines, respectively. CONCLUSIONS: Simultaneous intracorneal inlay implantation and LASIK to treat presbyopia with emmetropia, hyperopia, or myopia was clinically safe and effective, yielding improvement in distance and near visual acuity. Patients were satisfied with decreased dependence on reading glasses regardless of the preoperative SE range. However, postoperative symptoms, such as dry eyes, halo, glare, or night‐vision disturbances, occurred occasionally. Financial Disclosure: Dr. Waring has a financial interest in and is world surgical monitor for Acufocus. No other author has a financial or proprietary interest in any material or method mentioned.

Long-Term Effects of Topical Cyclosporine A Treatment after Penetrating Keratoplasty

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.

A 10-Year Review of Penetrating Keratoplasty

PURPOSE To survey the changes in indications for penetrating keratoplasty (PKP) and re-evaluate the risk factors for allograft rejection and graft failure. METHODS We evaluated the records of 396 eyes of 335 patients who had undergone PKP at the Tokyo University Hospital between 1987 and 1997. Clinical results were analyzed by the Kaplan-Meier life table method and the log-rank test. RESULTS The overall rates of graft survival and rejection-free graft survival at 10 years were 72.2% and 76.8%, respectively. The rates of graft survival and rejection-free graft survival were 98.8% and 86.6% in keratoconus, 87.0% and 56.5% in herpetic keratitis, 76.9% and 73.1% in corneal dystrophy and degeneration, 69.4% and 80.6% in nonherpetic keratitis, 62.5% and 75.0% in chemical burns, 61.8% and 72.1% in regrafting, and 51.1% and 79.8% in bullous keratopathy, respectively. The graft survival rates were statistically higher in the PKP alone group than in the combined operation group. The graft survival and rejection-free graft survival rates were statistically higher in the first operation group than in the regrafted group, and in the avascular cornea group than in the vascular cornea group. CONCLUSIONS We recognized changes in indications for PKP. Combined operation, reoperation, and vascularization of recipient cornea were risk factors for graft failure.

Long-term outcome of systemic cyclosporine treatment following penetrating keratoplasty.

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.

Cytokine profiles of aqueous humor and graft in orthotopic mouse corneal transplantation.

BACKGROUND Cytokine profile is a key in understanding the mechanisms of allograft rejection. Cytokine expression in the aqueous humor and the correlation between the aqueous humor cells and corneal infiltrating cells are not fully understood in corneal transplantation. METHODS Orthotopic mouse corneal transplantation was performed using BALB/c (H2d) mice as recipients, and C3H/He (H2k) and BALB/c mice as donors for allografts and isografts, respectively. Immunocytochemistry was performed on aqueous humor cells. Corneal graft was studied immunohistochemically. Cytokine gene expressions of the cells infiltrating the aqueous humor and corneal grafts were determined by the semiquantitative reverse transcription and polymerase chain reaction method. RESULTS Interferon-gamma, interleukin (IL)-2, IL-4, and IL-10 were detected in the cells infiltrating the aqueous humor and corneal grafts at both the protein and gene expression levels. T helper 1 (Th1) cytokine expressions at the protein level, however, were consistently predominant in the rejected allografts compared to those of Th2 cytokines. The cytokine and surface marker profiles of the cells in the aqueous humor corresponded well to those of the cells infiltrating the corneal grafts. Cytokine protein and mRNA expression levels in the aqueous humor decreased rapidly. CONCLUSIONS Allorejection in corneal transplantation is Th1 cytokine-predominant. Infiltrating cells do not express Th2 cytokine so much in allograft rejection, as compared with Th1 cytokine. The cell infiltration patterns of the aqueous humor were well correlated with those of the cornea.

Electrical conductivity of tear fluid in healthy persons and keratoconjunctivitis sicca patients measured by a flexible conductimetric sensor

Abstract• Background: It is known that the osmolarity of tears increases in keratoconjunctivitis sicca (KCS) patients and therefore could be a sensitive and specific indicator for the diagnosis of KCS. However, owing to the difficulties in using the current methods of tear fluid measurement, these procedures have not been employed in clinical practice. A newly devised flexible conductimetric sensor fabricated using microelectronic techniques is small and flexible enough to be placed on the ocular surface to measure the electrical conductivity of tear fluid in vivo. Fluid conductivity can be considered as an indirect function of electrolyte activity, or osmolarity. Therefore, we applied this new sensor to measure the tear fluid conductivity in KCS patients and healthy volunteers. • Methods: A flexible conductimetric sensor, consisting of a hydrophilic polytetrafluoroethylene membrane placed between two gold-coated layers, was placed directly into the temporal conjunctival cul-de-sac. The tear fluid conductivity was monitored graphically on a computer display. The sodium chloride concentration of tear fluids was calculated from the calibration curve and converted to the equivalent electrolyte concentration. • Results: The electrolyte concentrations were 324.8±41.0 mEq/1 in KCS patients (29 samples obtained from 16 KCS patients) and 296.4±30.1 mEq/l in healthy persons (33 samples obtained from 17 healthy persons). The difference was significant (P<0.01). A positive correlation was found between the electrolyte concentrations in KCS and the rose bengal score (coefficient=0.36). • Conclusion: The tear fluid conductivity in healthy persons and KCS patients could be monitored without ocular damage, and the measured values were consistent with previous reports. This method will be a new diagnostic tool for detecting tear abnormalities.

Suppression of allograft rejection with anti-αβ T cell receptor antibody in rat corneal transplantation

Background. Anti-ab 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-g, 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 R73treated group were significantly lower than those of the naive and untreated control group. Conclusions. ab 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-g, IL-2, and IL-10, and high IL-4 expression. Human corneal allografts, unlike grafts of the other vascularized organs, are not rejected in most recipients. Allograft rejection, however, is a major problem in corneal transplantation in patients with previous graft failure and/or prevascularized cornea (1‐3). Therefore, more effective immunosuppressive therapy is needed for successful corneal transplants in patients with high-risk eyes. To prevent corneal allograft rejection, various immunosuppressive strategies have been performed in mouse and rat models. In the mouse model, the effectiveness of anti-adhesion molecule monoclonal antibody (mAb*) treatment ( 4‐ 9) and anti-CD4 mAb therapies (10) has been reported. Among these therapies, combined transient use of anti-very late antigen-4 mAb and anti-leukocyte function-associated antigen-1 mAb increased the indefinite graft survival rate to 75% (7). However, in a rat corneal allograft model using various strain combinations, a high acceptance rate was not achieved by postoperative short-term administration of such local and systemic immunosuppressants as anti-CD4 mAb (11, 12) and tacrolimus (13‐15). Anti-ab T cell receptor mAb (R73) has been reported to suppress autoimmune encephalomyelitis (16) and adjuvant arthritis (17, 18). Moreover, R73 has been shown to prevent allograft rejection in the heart (19 ‐22), skin (23), small bowels (24), and kidney (25). The immunosuppressive effects of R73 on corneal transplantation, however, have not been examined. In this study, we evaluated the suppressive effects of R73 on allograft rejection in a rat corneal transplantation model, using two-strain combinations of complete major and minor histocompatibility (H) antigen disparity and minor H antigen disparity alone. We further examined the local and systemic T helper (Th)1 and Th2 cytokine expression patterns by R73 therapy, mainly using a complete major and minor H antigen disparity model.

Corneal wound healing from the perspective of keratoplasty specimens with special reference to the function of the Bowman layer and Descemet membrane.

Purpose: To review corneal wound healing with special reference to the function of the Bowman layer and Descemet membrane. Methods: Corneal specimens were obtained from keratoplasties, including regrafted cases. Recipient corneal buttons were evaluated histopathologically with attention to 5 layers of corneal structure: 3 cellular layers consisting of epithelial cells, keratocytes, and endothelial cells and 2 acellular layers consisting of the Bowman layer and Descemet membrane. Results: Subepithelial fibrosis was found in advanced bullous keratopathy. The possible source of subepithelial fibrosis was either conjunctival stroma or corneal stroma through disruption of the Bowman layer. Subepithelial fibrosis was observed in the area of the Bowman layer disruption at the host-graft junction in regrafted cases. The Bowman layer was disrupted in eyes with not only keratoconus but also corneal dystrophy such as macular dystrophy and gelatinous drop-like dystrophy. Newly formed, thin Descemet membrane was found in keratoconic eyes of patients with acute hydrops. Retrocorneal membranes were observed in eyes with advanced bullous keratopathy and graft failure. Abnormal wound healing of Descemet membrane such as override and separation was found in the host-graft interface of regrafted eyes, causing stromal overgrowth. Conclusions: The Bowman layer and Descemet membrane seem to serve as barriers to separate 3 cellular layers of epithelium, stroma, and endothelium. Disruption of the Bowman layer forms a new epithelial-stromal interaction and may cause cellular proliferative response. Separation of Descemet membrane can provide the trigger for emanating stromal tissue from the wound edge.