Hélène Boisjoly

Reconstructed Human Cornea Produced in vitro by Tissue Engineering

The aim of the present study was to produce a reconstructed human cornea in vitro by tissue engineering and to characterize the expression of integrins and basement membrane proteins in this reconstructed cornea. Epithelial cells and fibroblasts were isolated from human corneas (limbus or centre) and cultured on plastic substrates in vitro. Reconstructed human corneas were obtained by culturing epithelial cells on collagen gels containing fibroblasts. Histological (Masson’s trichrome staining) and immunohistological (laminin, type VII collagen, fibronectin as well as β1, α3, α4, α5, and α6 integrin subunits) studies were performed. Human corneal epithelial cells from the limbus yielded colonies of small fast-growing cells when cultured on plastic substrates. They could be subcultured for several passages in contrast to central corneal cells. In reconstructed cornea, the epithelium had 4–5 cell layers by the third day of culture; basal cells were cuboidal. The basement membrane components were already detected after 3 days of culture. Integrin stainings, except for the α4 integrin, were also positive after 3 days. They were mostly detected at the epithelium-stroma junction. Such in vitro tissue-engineered human cornea, which shows appropriate histology and expression of basement membrane components and integrins, provides tools for further physiological, toxicological and pharmacological studies as well as being an attractive model for gene expression studies.

Risk Factors of Corneal Graft Failure

PURPOSE To measure the association between potential risk factors and corneal graft failure. Two failure outcomes are compared: those with and those without a prior immune allograft reaction. METHODS Based on a single-center observational study design, 539 adult recipients of a corneal graft were followed for a median time of 30 months. Survival analysis was carried out. RESULTS Eighty-two graft failures were recorded. Of 82 failures, 53 (65%) were not preceded by an immune allograft reaction. Presence of blood vessels in the recipient cornea was associated with a twofold increase in risk for both failure outcomes. Three factors increased the risk of failure without an immune reaction: prior glaucoma or uveitis (adjusted relative risk estimate = 3.1), vitreous surgery with the graft (adjusted relative risk estimate = 2.0), and a repeat graft in the study eye (adjusted relative risk estimate = 2.0). Conversely, large graft wound size (adjusted relative risk estimate = 2.0). Conversely, large graft wound size (adjusted relative risk estimate = 2.9) and human leukocyte antigen (HLA)-A, -B incompatibility (adjusted relative risk estimate = 2.2) were associated with failures that followed an immune reaction. CONCLUSION In this study, the authors support the clinical impression that corneal graft failures with and without a prior immune reaction are distinct phenomena. Enhanced surveillance in recipients with glaucoma and early intensive treatment of allograft reactions are recommended to improve the outcome of corneal grafts.

Corneal endothelial cell density in glaucoma.

Purpose We studied corneal endothelial cell density in patients with glaucoma. Methods One hundred two patients with glaucoma were compared with 52 patients without glaucoma of the same age group. Exclusion criteria included history of either corneal disease, ocular inflammation, trauma, or surgery other than peripheral iridectomy. The following data were extracted from the patient files: glaucoma type and duration, laser treatments, glaucoma medications, and documented intraocular pressure (IOP) measurements. Specular microscopies were performed on central corneas, endothelial images were analyzed by computerized planimetry, and cell counts were calculated. Results Corneal endothelial cell counts were significantly lower in patients with glaucoma (2,154 ± 419 cells/mm2) than in controls (2,560 ± 306 cells/mm2; t test, p < 0.0001). In the glaucoma group, cell counts were inversely proportional to the means of IOPs. Patients receiving three or four glaucoma medications had lower cell counts than those receiving one or two medications. Cell counts were significantly lower both in primary angle-closure glaucoma and in primary open-angle glaucoma. Conclusion This study suggests that patients with glaucoma may have lower corneal endothelial cell density than those without glaucoma of the same age group. The proposed mechanisms are direct damage from IOP, congenital alteration of the corneal endothelium in patients with glaucoma, glaucoma medication toxicity, or a combination of these.

Effect of factors unrelated to tissue matching on corneal transplant endothelial rejection.

We examined 348 consecutive adult recipients of a corneal transplant for clinical signs of an endothelial rejection episode in a single-center follow-up study. The variables studied included primary diagnosis, number of previous corneal transplants, previous transplant failures from rejection episodes, transplant size, recipient corneal vascularization, donor age, recipient age and sex, past blood transfusions, and number of pregnancies. Five important risk factors were identified: primary diagnosis of herpetic, interstitial, or traumatic keratitis; transplant size 8 mm and larger; more than one previous corneal transplant; recipients younger than 60 years of age; and the presence of recipient corneal vascularization. This information will serve eventually for analyzing the effect of donor recipient tissue matching on corneal transplant rejection.

HLA-A B and DR Matching in Corneal Transplantation

One hundred eighty-five consecutive corneal transplants were performed in recipients selected on the basis of the best available HLA-A,B and DR match. Endothelial rejection-free transplant survival in this group was compared to a retrospective historical control group of 199 consecutive transplants performed in recipients selected on the basis of age and longest wait criteria. The two groups were comparable with regards to primary diagnosis, preoperative corneal vascularization, donor and recipient age, and operative techniques. Thirty-eight transplants in the study group and 28 transplants in the control group were at high risk for endothelial transplant rejection. At 12 months, the estimated rejection-free survival (Kaplan-Meier method) of the high-risk study group transplants was 87% compared to 74% for the high-risk historical control group and transplants. This difference did not reach the significant level of 0.05 with the log-rank test. The 12-month estimated rejection-free survival of low-risk study group and historical control group transplants were similar. In the study group, the 12-month estimated rejection-free survival of well-matched transplants was 95% compared to 83% for poorly matched transplants (log rank, P less than 0.02). These findings suggest that a relationship exists between HLA-A,B and DR compatibility of donor and recipient and the corneal rejection-free transplant survival.

Association between Corneal Allograft Reactions and HLA Compatibility

The purpose of this follow-up study is to measure the association between corneal allograft reactions and donor-recipient HLA-A and HLA-B compatibility. Four hundred thirty-eight consecutive adult recipients of corneal grafts with known donor-recipient HLA matching were observed for allograft reactions and failures. Most of the recipients under observation (91%) were well matched for HLA-DR. Of 438 recipients, 158 (36%) completed a 3-year follow-up. Three factors were associated with endothelial allograft reactions: 2 to 4+ corneal vascularization (relative risk, 2.2; P = 0.0006), two mismatched antigens at either the HLA-A or HLA-B locus (relative risk, 2.1; P = 0.0009), and recipient wound size of 8 mm or greater (relative risk, 1.5; P = 0.05). Unexpectedly, a strong association between endothelial allograft reactions and HLA-A or HLA-B incompatibility was found in low-risk recipients defined as unvascularized recipients of a small graft (relative risk, 3.2; P = 0.004). A larger sample size is required to determine if HLA matching offers a solution for recipients with corneal vascularization.

Rate of Endophthalmitis After Cataract Surgery in Quebec, Canada, 1996-2005

OBJECTIVE To estimate the annual incidence of endophthalmitis after cataract surgery from January 1, 1996, through December 31, 2005, in Quebec, Canada. METHODS Deidentified data were obtained from an outpatient physician billing database (Quebec State Control for Health Insurance [RAMQ]) with regard to all cataract surgical procedures performed from January 1, 1996, through December 31, 2005, in Quebec. For this cohort, records of an International Classification of Diseases, Ninth Revision (ICD-9) code for endophthalmitis during the same years were requested from 2 sources: the RAMQ outpatient database and an inpatient hospital discharge database (Maintenance and Exploitation of Data for the Study of Hospitalized Patients [MED-ECHO]). Endophthalmitis after cataract surgery was assumed if it occurred within 90 days of the surgery. Risk factors were examined using chi(2) tests and logistic regression. RESULTS After exclusions, 490 690 cataract surgical procedures were performed from January 1, 1996, through December 31, 2005. A total of 754 cases of endophthalmitis occurred within 90 days after surgery for an overall incidence rate of 1.5 per 1000 surgical procedures (95% confidence interval [CI], 1.4-1.7). Factors associated with endophthalmitis included age of 85 years or older (odds ratio [OR], 1.34; 95% CI, 1.06-1.70), male sex (1.44; 1.24-1.66), later year of surgery (0.94; 0.92-0.97), and region of cataract surgery, because regions 6 (2.21; 1.91-2.55) and 9 (4.00; 2.48-6.43) had higher rates compared with all other regions. CONCLUSION Reasons that explain the apparent decrease in endophthalmitis, especially in 2005, should be explored and further research performed to understand why certain patients and regions have higher risks of endophthalmitis after cataract surgery.

PRETRANSPLANT AND POSTTRANSPLANT ANTIBODIES IN HUMAN CORNEAL TRANSPLANTATION

The purpose of this study was to measure the association between antibody formation and endothelial corneal allograft reactions in 533 consecutive corneal graft recipients. The median follow-up time of these recipients was 732 days. Pretransplant panel-reactive antibodies were not found to be associated with endothelial corneal allograft reactions. Out of 533 recipients, 239 developed posttransplant antibodies during the course of this study. The formation of posttransplant antibodies was frequent in recipients with pretransplant antibodies and in HLA-A,-B-incompatible recipients. Posttransplant antibodies most often appeared within the first six months after transplantation whereas endothelial allograft reactions most often occurred later. Out of 65 recipients who developed PPRA and underwent an allograft reaction, 53 had a PPRA peak prior to, or at about the time of, the allograft reaction. Corneal allograft reaction events diagnosed during the second and third year after surgery were correlated with PPRA formation during the first year after grafting. The 36-month reaction-free survival rate of transplants was estimated at 72% in recipients with PPRA compared with 86% in recipients without PPRA (log rank P value = 0.002). Furthermore, posttransplant antibody formation altered the outcome of corneal allografts in both HLA-A and -B—compatible and -incompatible recipients. These findings suggest that posttransplant antibody development represents a high risk of endothelial corneal allograft reactions.

Association between unilateral quiescent stromal herpetic keratitis and bilateral dry eyes.

Purpose: According to our clinical observation, patients with quiescent herpes simplex virus (HSV) stromal keratitis often seem to present with signs of dry eye in the contralateral eye. Our goal was to compare dry eye signs and symptoms in both eyes of patients with quiescent HSV stromal keratitis with those of age- and sex-matched control subjects with healthy corneas. Methods: A case-control study with 24 subjects per group. Results: The average age of 10 men and 14 women in each group was 58 years. The HSV eye of cases was first compared with the contralateral eye with a healthy cornea. As expected, the HSV eye had a significantly lower corneal sensation threshold (P = 0.001); no significant difference was however found for Schirmer tests done with anesthesia (basal tear secretion) and without anesthesia, tear breakup time, mucus and debris in the tear film, and eyelid margin redness or swelling. Then, the HSV eye of cases was compared with the right eye of controls, whereas the healthy eye of cases was compared with the left eye of controls. Patients with unilateral quiescent HSV stromal keratitis had significantly lower bilateral Schirmer tests both with anesthesia (P = 0.001) and without anesthesia (P = 0.02). Dry eye symptoms of the healthy cornea of cases and those of controls did not differ significantly. Conclusions: Both eyes of patients with quiescent HSV stromal keratitis in our population were dry even if many patients with HSV stromal keratitis did not have symptoms in their fellow eye.

ROLE OF PRESENSITIZATION AND DONOR-RECIPIENT CROSSMATCHING IN CORNEAL GRAFT OUTCOME

PURPOSE A positive donor-recipient crossmatch (CM) due to preexisting recipient lymphocytotoxic antibodies is known to be an important factor in allograft failure in the majority of organ transplantations. However, the effect of positive CM on corneal graft outcome is less known. METHOD Between 1982 and 1994, CM was performed by the microlymphocytotoxicity method using donor lymphocytes and recipient pretransplant serum in 759 consecutive corneal transplantations (maximal follow-up, 36 months). Patients were evaluated regarding the type of allospecificity of antibodies involved and their role on corneal graft outcome (rejection and failure). RESULTS A positive CM was found in 61 patients (8%) and a negative CM in 698 patients (92%). The positive and negative CM groups had similar graft rejection rates at 36 months. Patients with a positive CM due to antibodies directed against donor human leukocyte antigen (HLA) (as defined on the basis of private and public or CREG HLA allele specificities) did not have an increased risk of rejection. However, patients with positive CM and presensitization (previous graft or rejection history) had a statistically significant increase in risk of corneal endothelial rejection. CONCLUSION This study shows that donor-recipient CM could be a useful procedure for the selection of recipients for corneal transplantation in patients presensitized by anterior graft or previous corneal rejection.