Ocular Gene Therapy in a Patient with Dystrophic Epidermolysis Bullosa.

Dystrophic epidermolysis bullosa is a rare genetic disease caused by damaging variants in COL7A1, which encodes type VII collagen. Blistering and scarring of the ocular surface develop, potentially leading to blindness. Beremagene geperpavec (B-VEC) is a replication-deficient herpes simplex virus type 1-based gene therapy engineered to deliver functional human type VII collagen. Here, we report the case of a patient with cicatrizing conjunctivitis in both eyes caused by dystrophic epidermolysis bullosa who received ophthalmic administration of B-VEC, which was associated with improved visual acuity after surgery.

Cellular and molecular assessment of rose bengal photodynamic antimicrobial therapy on keratocytes, corneal endothelium and limbal stem cell niche.

Rose Bengal Photodynamic Antimicrobial Therapy (RB-PDAT) is a novel potential treatment for progressive infectious keratitis. The principle behind this therapy is using Rose Bengal as a photosensitizer that can be activated by green light and results in the production of oxygen free radicals which in turn eradicate the microorganism. Given RB-PDAT's mechanism of action and the potential cytotoxic effects, concerns regarding the safety of this technique have arisen. The purpose of this study was to evaluate the effect of RB-PDAT on keratocytes, while focusing on the safety profile that the photo-chemical reaction has on the limbal stem cell (LSC) niche and endothelial cell layer of the treated cornea. To perform RB-PDAT, Rose Bengal solution (0.1% RB in BSS) was applied to the right cornea of rabbits for 30 min and then irradiated by a custom-made green LED light source (525 nm, 6 mW/cm2) for 15 min (5.4 J/cm2). Three rabbits were sacrificed and enucleated after 24 h for evaluation. TUNEL assay and immunohistochemistry for endothelium and limbal stem cell viability were performed on whole mounts and frozen sections in treated and control eyes. LSC of both eyes were isolated and cultured to perform MTT viability and proliferation, and scratch wound healing assays under time-lapse microscopy. Interestingly, while Rose Bengal dye penetration was superficial, yet associated cellular apoptosis was evidenced in up to 1/3 of the stromal thickness on frozen sections. TUNEL assay on whole mounts showed no endothelial cell death following treatment. Immunohistochemistry on frozen sections of LSC displayed no structural difference between treated and non-treated eyes. There was no difference in LSC proliferation rates and scratch wound healing assay demonstrated adequate cell migration from treated and non-treated eyes. The current study suggests that even though penetration of the RB dye has been shown to be limited, oxidative stress produced by RB-PDAT can reach deeper into the corneal stroma. Nevertheless, our results show that performing RB-PDAT is safe on the corneal endothelium and has no effect on LSC viability or function.

Risks factors and regression model for risk calculation of anesthesiologic intervention in topical and intracameral cataract surgery.

PURPOSE: To determine risk factors for anesthesiologist intervention during routine cataract surgery performed with topical and intracameral anesthesia and establish a regression model to identify high-risk patients. SETTING: Department of Ophthalmology, Clínica Universidad de Navarra, Pamplona, Spain. DESIGN: Prospective case series. METHODS: After cataract surgery at an ambulatory surgical center, anesthesia personnel completed a questionnaire to determine adverse medical events and risk factors related to anesthesiologist intervention. A Poisson regression model was used to calculate the interventional risks. Bootstrapping was performed for internal model validation. RESULTS: Of the 1010 cases, 50 (4.95%) required anesthesiologist intervention. Univariate analysis identified an association between anesthesiologist intervention and hypertension (P<.001), psychiatric history (P=.002), initial systolic blood pressure (P<.001), surgical duration (P=.001), and diabetes (P=.018). Scores were obtained using the following proposed regression model equation: (-8.68 + 0.33 × sex [men, 0; women, 1] + -0.02 × age [years] + 0.68 × hypertensive history [no, 0; yes, 1] + 1.18 × psychiatric background [no, 0; yes, 1] + 0.04 × initial systolic blood pressure [mm Hg]). The area under the receiver-operating curve was 0.803 (95% confidence interval [CI], 0.721-0.886). The area under the curve found in the validation method was 0.813 (95% CI, 0.727-0.887). CONCLUSION: Hypertension was the main risk factor for anesthesiologist intervention. The regression model discriminated between patients at lower and higher risk for intraoperative intervention for monitored anesthesia care. The probability of anesthesiologist intervention was 11.7 times higher when the model obtained a high score. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

A novel method of isolation, preservation, and expansion of human corneal endothelial cells.

PURPOSE: To explore new strategies for effective isolation, preservation, and expansion of human corneal endothelial cells (HCECs). METHODS: Human corneal Descemet's membrane and corneal endothelial cells were digested with collagenase A or Dispase II in supplemented hormonal epithelial medium (SHEM) for 1.5 to 16 hours. HCEC aggregates derived from collagenase A digestion were preserved in serum-free medium with low or high calcium for up to 3 weeks. Cryosections of HCEC aggregates were subjected to immunostaining with ZO-1, connexin 43, type IV collagen, laminin-5, and perlecan, and apoptosis was determined by TUNEL or cell-viability assay. For expansion, HCEC aggregates were seeded directly or after brief treatment with trypsin/EDTA in SHEM, with or without additional bovine pituitary extract (BPE), nerve growth factor (NGF), or basic fibroblast growth factor (bFGF). The resultant HCECs were immunostained with ZO-1, connexin 43, and Ki67. RESULTS: Digestion with collagenase A, but not Dispase, of the stripped Descemet's membrane generated HCEC aggregates, which preserved cell-cell junctions and basement membrane components. High cell viability of HCEC aggregates was preservable in a serum-free, high-calcium, but not low-calcium, medium for at least 3 weeks. Brief treatment of HCEC aggregates with trypsin/EDTA resulted in a higher proliferation rate than without, when cultured in SHEM, and the resultant confluent monolayer of hexagonal cells retained cell-cell junctions. However, additional BPE, NGF, or bFGF did not increase cell proliferation, whereas additional BPE or bFGF disrupted cell-cell junctions. CONCLUSIONS: Collagenase A digestion successfully harvested aggregates with viable HCECs that were preservable for at least 3 weeks in a serum-free, high-calcium medium and, with brief trypsin/EDTA treatment, expanded in the SHEM into a monolayer with hexagonal cells that exhibited characteristic cell junctions.