Maria Fideliz de la Paz

Clinical utility of objective tests for dry eye disease: variability over time and implications for clinical trials and disease management.

Purpose: To evaluate the efficacy of commonly used biomarkers in dry eye disease management in a longitudinal observational case series study followed by an interventional study in a subset of subjects treated with cyclosporine A (0.05%). Methods: Bilateral tear osmolarity, Schirmer, tear film breakup time (TBUT), staining, meibomian grading, and Ocular Surface Disease Index were measured for a period of 3 consecutive months in participants recruited from a clinic-based population at 2 study sites. Fifty-two subjects completed the study (n = 16 mild/moderate, n = 36 severe; age, 47.1 ± 16.1 years). After the 3-month observation period, severe dry eye patients were prescribed topical cyclosporine A and evaluated for an additional 3 months. Results: Tear osmolarity (8.7 ± 6.3%) exhibited significantly less variability over a 3-month period than corneal staining (12.2 ± 8.8%, P = 0.040), conjunctival staining (14.8 ± 8.9%, P = 0.002), and meibomian grading (14.3 ± 8.8%, P < 0.0001) across the entire patient population. Osmolarity also demonstrated less variation than TBUT (11.7 ± 9.0%, P = 0.059), Schirmer tests (10.7 ± 9.2%, P = 0.67), and Ocular Surface Disease Index (9.3 ± 7.8%, P = 0.94), although the differences were not significant. Variation in osmolarity was less for mild dry eye patients (5.9 ± 3.1%) than severe dry eye patients (10.0 ± 6.9%, P = 0.038). After treatment, average osmolarity and variability were lowered from 341 ± 18 mOsm/L to 307 ± 8 mOsm/L (P < 0.0001, n = 10). A downward trend in symptoms followed changes in osmolarity, declining from 44 ± 17 mOsm/L to 38 ± 18 mOsm/L (P = 0.35). None of the other signs demonstrated a change after treatment. Conclusions: Over a 3-month period, tear film osmolarity was found to have the lowest variability among commonly used signs of dry eye disease. Reductions in osmolarity preceded changes in symptoms during therapy.

Impact of Clinical Factors on the Long-Term Functional and Anatomic Outcomes of Osteo-odonto-keratoprosthesis and Tibial Bone Keratoprosthesis

PURPOSE To report the long-term functional and anatomic outcomes of osteo-odonto-keratoprosthesis and tibial bone keratoprosthesis; to analyze the influence of clinical factors, such as surgical technique, primary diagnosis, age, and postoperative complications, on the final outcome. DESIGN Retrospective cohort study. METHODS setting: Centro de Oftalmología Barraquer, between 1974 and 2005. PARTICIPANTS Two hundred twenty-seven patients. intervention: Biological keratoprosthesis using osteo-odonto-keratoprosthesis or tibial bone keratoprosthesis. main outcome measures: Functional survival with success defined as best-corrected visual acuity ≥0.05; anatomic survival with success defined as retention of the keratoprosthesis lamina. RESULTS Osteo-odonto-keratoprosthesis and tibial bone keratoprosthesis have comparable anatomic survival at 5 and 10 years of follow-up, but osteo-odonto-keratoprosthesis has a significantly better functional success than tibial bone keratoprosthesis at the same time periods. Among the primary diagnoses, Stevens-Johnson syndrome, chemical burn, and trachoma have generally good functional and anatomic outcomes and the least favorable prognosis is for ocular cicatricial pemphigoid. Younger patients fared better than those in older age groups. The most frequent complications were extrusion (28%), retinal detachment (16%), and uncontrolled glaucoma (11%). The glaucoma group had the best anatomic success but the worst functional results, only exceeded by the retinal detachment group in terms of functional outcome. CONCLUSION Clinical factors, such as surgical technique, primary diagnosis, age, and postoperative complications, can affect the long-term anatomic and functional successes of biological keratoprosthesis. Knowledge about the impact of each of these factors on survival can help surgeons determine the best approach in every particular case.

Comparison of the Histology, Gene Expression Profile, and Phenotype of Cultured Human Limbal Epithelial Cells from Different Limbal Regions

PURPOSE To investigate whether human limbal epithelial cells (HLECs) derived from various regions of the limbus exhibit differences in gene expression and epithelial characteristics. METHODS HLECs were derived from explants taken from the superior, nasal, inferior, and temporal limbus and cultured for 21 days. Whole genome transcript profiling was performed with a gene microarray. The microarray results were validated by using RT-PCR. Epithelial morphology was studied with light microscopy and transmission electron microscopy, and phenotype was evaluated by immunohistochemistry. RESULTS Epithelial outgrowth was present in most cultures of superior origin (88%) in contrast to cultures of temporal origin (38%). The epithelial thickness and number of cell layers were significantly greater in cultures of superior origin than in cultures from inferior and temporal areas. TRIM36, OSR2, and RHOU, which are involved in morphogenesis, were significantly differentially expressed in the superior region, compared with the other regions. Proposed limbal stem cell, progenitor, and differentiation markers were not differentially expressed. The uniform gene expression of ocular surface markers correlated with homogeneous immunostaining of corresponding protein markers in HLEC cultures from all regions, demonstrating an undifferentiated phenotype (p63(+), DeltaNp63alpha(+), ABCG2(+), K19(+), vimentin(+), integrin beta1(+), nestin(-), K3(-), K5(+), and E-cadherin(+)). CONCLUSIONS No major transcriptional or phenotypic differences were observed in cultured HLECs derived from different regions of the limbus. However, explants of superior origin demonstrated the highest outgrowth success rate and generated epithelia with greater epithelial thickness and number of cell layers, which may prove useful for transplantation purposes.

Long‐term visual prognosis of corneal and ocular surface surgery in patients with congenital aniridia

Purpose:  To evaluate the long‐term visual prognosis of corneal and ocular surface surgery in patients with congenital aniridia.

Neural contrast sensitivity calculated from measured total contrast sensitivity and modulation transfer function

Purpose:  To test the feasibility of calculating neural contrast sensitivity function (neural CSF) from conventionally measured total contrast sensitivity function (total CSF) and measured modulation transfer function (MTF). Neural CSF considers the retina and the brain, whereas total CSF considers the optical eye media, the retina and the brain together.

The role of visual evoked potential and electroretinography in the preoperative assessment of osteo-keratoprosthesis or osteo-odonto-keratoprosthesis surgery

Purpose:  To determine the value of electroretinography (ERG) and visual evoked potential (VEP) in predicting visual outcome in patients undergoing osteo‐keratoprosthesis (OKP) or osteo‐odonto‐keratoprosthesis (OOKP) surgery.

Influence of primary diagnosis and complications on visual outcome in patients receiving a Boston type 1 keratoprosthesis.

Purpose: To analyse how primary diagnosis and complications affect the evolution of post-operative visual acuity (VA). Methods: We performed retrospective chart analysis on 59 eyes in 57 patients with various diagnoses, most of which were non-standard indications for Boston type 1 keratoprosthesis (Kpro) implantation. The follow-up period was at least 3 months. Patients were classified based on the evolution of post-operative VA: group A demonstrated stable VA improvement, group B lost VA improvement and group C no significant VA improvement. Results: We assigned 46% of our cases to group A with stable VA improvement, 32% to group B with lost VA improvement, and 22% to group C with no VA improvement. The number of graft failures before Kpro implantation did not influence VA outcome. Except for the relatively good VA outcome in chemical burn and radiation injury patients, there seems to be no association between primary diagnosis and positive or negative VA outcome. Only 9% of patients with posterior segment complications and 20% with infections and associated pathologies were assigned to group A. Conclusion: Most cases (78%) showed improvement in VA after Boston type 1 Kpro (groups A and B). Posterior segment complications and infections mostly resulted in persistent loss of vision. These complications should be prevented and carefully treated.

Serum-Free and Xenobiotic-Free Preservation of Cultured Human Limbal Epithelial Cells

Aim/Purpose of the Study To develop a one-week storage method, without serum and xenobiotics, that would maintain cell viability, morphology, and phenotype of cultured human limbal epithelial sheets. Materials and Methods Human limbal explants were cultured on intact human amniotic membranes for two weeks. The sheets were stored in a hermetically sealed container at 23°C in either a serum-free medium with selected animal serum-derived compounds (Quantum 286) or a xenobiotic-free medium (Minimal Essential Medium) for 4 and 7 days. Stored and non-stored cultures were analyzed for cell viability, amniotic membrane and epithelial sheet thickness, and a panel of immunohistochemical markers for immature cells (ΔNp63α, p63, Bmi-1, C/EBP∂, ABCG2 and K19), differentiated cells (K3 and Cx43), proliferation (PCNA), and apoptosis (Caspase-3). Results The cell viability of the cultures was 98 ± 1% and remained high after storage. Mean central thickness of non-stored limbal epithelial sheets was 23 ± 3 μm, and no substantial loss of cells was observed after storage. The non-stored epithelial sheets expressed a predominantly immature phenotype with ΔNp63α positivity of more than 3% in 9 of 13 cultures. After storage, the expression of ABCG2 and C/EBP∂ was reduced for the 7 day Quantum 286-storage group; (P = 0.04), and Bmi-1 was reduced after 4 day Quantum 286-storage; (P = 0.02). No other markers varied significantly. The expression of differentiation markers was unrelated to the thickness of the epithelia and amniotic membrane, apart from ABCG2, which correlated negatively with thickness of limbal epithelia (R = -0.69, P = 0.01) and ΔNp63α, which correlated negatively with amniotic membrane thickness (R = -0.59, P = 0.03). Conclusion Limbal epithelial cells cultured from explants on amniotic membrane can be stored at 23°C in both serum-free and xenobiotic-free media, with sustained cell viability, ultrastructure, and ΔNp63α-positivity after both 4 and 7 days.

Histopathology Findings of Corneal Buttons in Congenital Aniridia Patients

Purpose: To evaluate the corneal button of primary penetrating keratoplasty of patients diagnosed with congenital aniridia. The study took place at the Instituto Universitario Barraquer and the Centro de Oftalmología Barraquer. Methods: A retrospective analysis of cases diagnosed with congenital aniridia was carried out. We analyzed 13 corneal buttons of 11 eyes with congenital aniridia. We only included those patients who underwent penetrating keratoplasty for the first time. The corneal buttons were analyzed for histological characteristics of the presence of vascularization, the presence or not of Bowmans layer, the thickness of the stroma and Descemets membrane, and endothelium layer alterations. Results: We found alterations in the epithelium and stroma in all patients, although this loss of architecture was not seen in Descemets membrane and the endothelial population. Conclusion: Patients with advanced congenital aniridic keratopathy may be good candidates for deep or superficial anterior lamellar keratoplasty for the preservation of normal endothelium and Descemets membrane, along with limbal stem cell transplantation, to address epithelial and stromal pathology.

Transcriptome Analysis of Cultured Limbal Epithelial Cells on an Intact Amniotic Membrane following Hypothermic Storage in Optisol-GS

The aim of the present study was to investigate the molecular mechanisms underlying activation of cell death pathways using genome-wide transcriptional analysis in human limbal epithelial cell (HLEC) cultures following conventional hypothermic storage in Optisol-GS. Three-week HLEC cultures were stored in Optisol-GS for 2, 4, and 7 days at 4 °C. Partek Genomics Suite software v.6.15.0422, (Partec Inc., St. Louis, MO, USA) was used to identify genes that showed significantly different (P < 0.05) levels of expression following hypothermic storage compared to non-stored cell sheets. There were few changes in gene expression after 2 days of storage, but several genes were differently regulated following 4 and 7 days of storage. The histone-coding genes HIST1H3A and HIST4H4 were among the most upregulated genes following 4 and 7 days of hypothermic storage. Bioinformatic analysis suggested that these two genes are involved in a functional network highly associated with cell death, necrosis, and transcription of RNA. HDAC1, encoding histone deacetylase 1, was the most downregulated gene after 7 days of storage. Together with other downregulated genes, it is suggested that HDAC1 is involved in a regulating network significantly associated with cellular function and maintenance, differentiation of cells, and DNA repair. Our data suggest that the upregulated expression of histone-coding genes together with downregulated genes affecting cell differentiation and DNA repair may be responsible for increased cell death following hypothermic storage of cultured HLEC. In summary, our results demonstrated that a higher number of genes changed with increasing storage time. Moreover, in general, larger differences in absolute gene expression values were observed with increasing storage time. Further understanding of these molecular mechanisms is important for optimization of storage technology for limbal epithelial sheets.