B. Delbosc

Homozygous Nonsense Mutation in the FOXE3 Gene as a Cause of Congenital Primary Aphakia in Humans

Congenital primary aphakia (CPA) is a rare developmental disorder characterized by the absence of lens, the development of which is normally induced during the 4th-5th wk of human embryogenesis. This original failure leads, in turn, to complete aplasia of the anterior segment of the eye, which is the diagnostic histological criterion for CPA. So far, the genetic basis for this human condition has remained unclear. Here, we present the analysis of a consanguineous family with three siblings who had bilateral aphakia, microphthalmia, and complete agenesis of the ocular anterior segment. We show that a null mutation in the FOXE3 gene segregates and, in the homozygous state, produces the mutant phenotype in this family. Therefore, this study identifies--to our knowledge, for the first time--a causative gene for CPA in humans. Furthermore, it indicates a possible critical role for FOXE3 very early in the lens developmental program, perhaps earlier than any role recognized elsewhere for this gene.

Value of two mortality assessment techniques for organ cultured corneal endothelium: trypan blue versus TUNEL technique

Background/aim: It is known that trypan blue staining is not a good predictor of loss of corneal endothelial cells (ECs) during organ culture. As it is primarily an indicator of membrane integrity, it would also not be expected to identify ECs undergoing apoptosis. The aim of this study was to determine the ability of the in situ TdT dUTP mediated nick end labelling (TUNEL) technique to detect cell death in the corneal endothelium caused by apoptosis during organ culture, compared with conventional vital staining with trypan blue. Methods: 31 human corneas were organ cultured at 31°C for 3–35 days. Staurosporine was used to induce apoptosis in five control corneas. The endothelium was assessed by trypan blue and by the in situ TUNEL technique. The percentages of trypan and TUNEL positive ECs were compared. Their links with sex, donor age, time from donor death and organ culture, initial and final EC density and cell loss were studied. Results: TUNEL stained ECs were observed in all corneas. TUNEL positive ECs were mostly located either in corneal folds or at the periphery of corneal folds showing central shedding. The mean percentage of cell death at the end of storage, assessed by the trypan blue technique, was 1.47% (SD 2.63, range 0.03–12); assessed by the TUNEL technique it was 12.7% (SD 16.4 range 0.6–65.5). There was a significant correlation between the two techniques (r = 0.7, p<0.001). The percentage of TUNEL stained ECs was correlated negatively with EC density at the end of storage (r = −0.47, p <0.005) and positively with percentage EC loss during storage (r = 0.46, p < 0.05). Conclusion: This study demonstrates that organ cultured corneas systematically carry non-viable ECs that are implicated in cell death by apoptosis and go undetected when trypan blue staining is used. Because the in situ TUNEL assay detects earlier events in the cell death process than does trypan blue, it should be used to quantify endothelial viability, especially for experiments with new storage media.

Morphology, Histology, and Ultrastructure of Human C31 Organ-Cultured Corneas

Our objective was to evaluate corneal structural modifications induced by corneal preservation in a C31 organ culture medium. Twenty-four postmortem human corneas preserved in C31 medium (CRTS, Besanc,on, France) for 2-21 days and 12 fresh postmortem human corneas were studied and evaluated by means of light microscopy, morphometry, and transmission electron microscopy (TEM). Endothelial cell loss during preservation averaged 9.2% (± 7.2%). Endothelial morphology displayed significantly more moderate alterations in the group of fresh corneas than in the group of preserved corneas. Endothelial morphometry resulted in a higher coefficient of variation (p=0.002) in the preserved group but showed no difference for the average figure coefficient and average cell area. The histological study showed corneal swelling and epithelial sloughing in the preserved group. TEM displayed moderate preservation injuries, such as numerous vacuoles, mitochondrial swelling, and increased cell thickness. These results are consistent with previous findings that indicated that C31 medium induces moderate preservation injuries.

Influencing factors on the suitability of organ-cultured corneas.

PurposeTo determine the factors related to donor and tissue retrieval, which influence the suitability of organ-cultured corneas for transplantation.Patients and methodsWe retrospectively analysed 2596 donor corneas. Polytomic logistic regression analysis was used to assess the influence of various factors (that is, donor age, cause of death, death-to-tissue retrieval time, tissue retrieval-to-reception time, and tissue retrieval method) on the suitability of grafts for transplantation. Positive predictive values (PPVs) were computed.ResultsForty-three percent (1118/2596) of corneas were discarded. The leading cause for discarding corneas was poor endothelial quality (21.5%). Corneas from donors older than 80 years were more likely to be discarded because of endothelial insufficiency (OR=2.37, P=0.001). Longer time between death and tissue retrieval was associated with increased risk of positive serology (OR=1.43, P=0.02). Increased time between tissue retrieval and reception was associated with increased risk of contamination (OR=1.57, P=0.03). PPV increased from 38.5% for corneas retrieved from donors older than 80 years featuring a death-to-tissue retrieval time of more than 6 h and a tissue retrieval-to-reception time of more than 24 h to 64.7% for corneas retrieved from donors younger than 80 years featuring a death-to tissue retrieval time shorter than 6 h and a tissue retrieval-to-reception time shorter than 24 h.ConclusionThe percentage of discarded corneas can be reduced by including donors aged 80 years or less, using a time from donors death to tissue retrieval shorter than 6 h, and a tissue retrieval-to-reception time shorter than 24 h.

Reliability of cone counts using an adaptive optics retinal camera

To assess the reproducibility and repeatability of cone imaging in healthy human eyes, using the RTx‐1 Adaptive Optics Retinal Camera and its proprietary cone‐counting software.

Quantification of cone loss after surgery for retinal detachment involving the macula using adaptive optics

Aims To image the cones in eyes with anatomically successful repair of retinal detachment (RD) involving the macula and in healthy fellow eyes using an adaptive optics (AO) camera and to correlate the results to clinical outcomes. Methods Twenty-one patients (42 eyes) operated for macula-off RD were imaged 6 weeks after surgery using an AO camera (RTX 1, Imagine Eyes, Orsay, France). Cone density (cells/mm2), spacing between cells (µm) and the percentage of cones with six neighbours were measured. Best-corrected visual acuity (BCVA) and thickness of the inner segment ellipsoid (ISe) band imaged by SD-optical coherence tomography were also measured. Results The parafoveal cone density was decreased in eyes operated for RD (mean±SD 14 576±4035/mm2) compared with fellow eyes (20 589±2350/mm2) (p=0.0001). There was also an increase in cone spacing (10.3±2.6 vs 8.0±1.0.9 µm, respectively, p<0.0001). The nearest-neighbour analysis revealed a reduction in the percentage of cones with six neighbours (36.5±4.2 vs 42.7±4.6%, p=0.0003). The ISe thickness, thinner in the operated eyes, was correlated to the cone density (r=0.62, p<0.0001). BCVA was significantly correlated to cone density (r=0.8, p<0.001). Conclusions There was a decrease in the cone density after RD with an estimated loss of one-third of the cones. Postoperative visual acuity was highly correlated with the cone density. AO may be a valuable prognostic tool after RD surgery.

Sutureless intrascleral intraocular lens implantation after ocular trauma

Purpose To report the results and safety of sutureless intrascleral haptic fixation in traumatized eyes and to compare this procedure with retropupillary iris‐claw intraocular lens (IOL) fixation. Setting University Hospital of Strasbourg, Strasbourg, France. Design Interventional case series. Methods Patients with traumatic cataract and severely damaged capsular bags were divided into 2 groups (Group 1: intrascleral IOLs [Acrysof MN60 AC]; Group 2: retropupillary iris‐claw IOLs [Verisyse]). The main outcome was the final visual acuity. The surgically induced astigmatism (SIA) was calculated by the vectorial method. Results Twenty‐six eyes of 23 patients were studied, 8 eyes in Group 1 and 18 eyes in Group 2. The mean follow‐up was 14 months. There was no difference in corrected distance visual acuity (CDVA) at the time of the surgery (P>.05). The mean CDVA (logMAR) was 1.68 ± 1.15 (SD) preoperatively and 0.55 ± 0.9 postoperatively in Group 1 (P = .03) and 1.11 ± 1.13 and 0.32 ± 0.47, respectively, in Group 2 (P = .003). The final CDVA was not different between groups (P>.05). The mean SIA was 1.91 ± 1.66 diopters (D) in Group 1 and 2.74 ± 1.92 D in Group 2 (P>.05). No intraoperative complications occurred in Group 2; a haptic broke in Group 1. Macular edema occurred in both groups. Conclusions Sutureless intrascleral IOLs corrected posttraumatic aphakia. The SIA was comparable between groups. This procedure should be considered after trauma when other implantation techniques are not possible. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.

Cancer transmission through corneal transplantation.

Purpose. A retrospective study to demonstrate the safety of corneas from donors affected by systemic malignancies in a view of keratoplasty. Methods. Using the data of the cancer registry department in a district of 500,000 residents, we analyzed retrospectively 143 patients followed up in the same region and transplanted between 1987 and 1995. We compared the incidence of cancer in recipients of cornea coming from donors with malignancies with recipients of cornea from donors without malignancies. The rate of cancer in this population was also compared with the incidence of cancer in the general population at the same age. Results. Eleven patients (7%) were excluded from the study, eight of them for the presence of cancer in their medical history before surgery and the three others because of lack of information about their follow-up after surgery. Forty patients received corneas from cancerous donors and 103 from donors without cancer. Six patients developed malignancies between 1 and 4 years after transplantation, and only one of them received a cornea from a donor with a systemic malignancy. This recipient developed a different type of cancer from that of the donor. The five other patients received corneas from donors without systemic malignancies. By comparing theses results, there was no relationship between the occurrence of malignancies and transplantation of corneas from cancerous donors (relative risk = 0.49, 95% confidence interval = 0.01–13.62). Conclusion. There was no increased incidence of cancer in our patient population compared with reference population. Based on this study, there is no statistical or clinical evidence to suggest the transmission of cancer from donors with malignancies via corneal transplantation, according to the accepted criteria of donor selection.

Modulation of HLA-DR and CD1a expression on human cornea with low-dose UVB irradiation

PURPOSE To evaluate the effects of low-dose UVB irradiation of HLA and CD1a expression and the toxic effects of UVB on human corneas. METHODS 24 pairs of human corneas from 24 donors were studied. One cornea from each pair was randomly irradiated with UVB (100 mJ/cm2) after enucleation. All corneas were then organ-cultured for 2, 7, 14 or 21 days. Endothelium was studied after enucleation and organ culture. Following preservation, corneas were evaluated by means of light microscopy, morphometry and TEM. HLA and CD1a staining was performed using an immuno-alkaline-phosphatase technique. RESULTS Endothelial cell loss during organ culture averaged 9.1% in the UVB group and 9.2% in the control group (NS). The number of rosette and reformation figures (p = 0.004) and the coefficient of variation (p = 0.014) were higher in the control group. Epithelial sloughing was more accentuated in the UVB group. We observed the same moderate ultrastructural injuries in both groups. In the epithelium, the average number of HLA-DR+ cells per field was 0.12 in the UVB group and 0.42 in the control group (p = 0.035). In the stroma, these figures were respectively 1.04 and 1.34 (p = 0.026). In the epithelium, the average number of CD1a + cells was respectively 0. 025 and 0.078 (p = 0.019). In the preservation mediums, the average percentage of CD1a + cells was 0.07% in the UVB group and 0.27% in the control group (p = 0.014). CONCLUSIONS Low-dose UVB (100 mJ/cm2) decreases HLA-DR and CD1a expression of organ-cultured human corneas and induces moderate corneal injuries. Low-dose UVB might be useful for preventing allograft rejection.

Danger of systemic cyclosporine for corneal graft.

Purpose. To report a case of posttransplant lymphoproliferative disorder (PTLD) in a patient receiving oral cyclosporine (CS) for immunosuppression in a high-risk keratoplasty. Methods. A systemic CS therapy was given to prevent graft rejection of a keratoplasty. Risk rejection was high in regard to a previous graft rejection and persistence of a corneal vascularization. One year after the keratoplasty, the patient developed a gastric Epstein-Barr virus (EBV)-induced B cell lymphoma. The outcome was favorable after chemotherapy. Conclusions. This unique case of lymphoma (PTLD) in the course of corneal graft management questions the indications and the follow-up of patients with CS therapy and raises the issue of topical CS treatment.