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.

Novel treatments for dry eye syndrome.

Dry eye syndrome (DES) is a prevalent and multifactorial disease that leads to a self-perpetuating cycle of inflammation and damage to the ocular surface. This results in symptoms such as redness, burning, and blurred vision, which can negatively affect a patient's quality of life. While treatments are available to manage DES, they only temporarily relieve symptoms. Furthermore, long-term use of certain medications can cause harm to the ocular surface. Therefore, there is a need for safer and effective treatments for DES. This review highlights the latest advancements in DES therapy, providing valuable insights into ongoing efforts to improve patient outcomes.

Neurotrophic Keratopathy After Slow Coagulation Transscleral Cyclophotocoagulation.

PURPOSE: Decreased corneal sensation and subsequent neurotrophic keratopathy (NK) is an uncommon complication after transscleral cyclophotocoagulation (TSCPC). Post-TSCPC NK has been rarely reported in the literature, predominantly after traditional, "pop technique" continuous-wave TSCPC or micropulse CPC. The authors report the first case series of NK after slow-coagulation TSCPC (SC-TSCPC). METHODS: This was a respective chart review of patients who developed NK after SC-TSCPC. The collected data included demographic data, type of glaucoma, risk factors for corneal anesthesia in addition to the number of laser spots, and the extent of the treated area. RESULTS: Four eyes experienced NK after SC-TSCPC. The median time for the development of NK was 4 weeks. At the final visit, 2 patients had a resolution of NK, 1 had a persistent corneal ulcer, and 1 had worsening NK and corneal perforation. CONCLUSIONS: NK is a rare but a vision-threatening complication that can develop after SC-TSCPC in patients with risk factors for decreased corneal sensation. Early diagnosis and proper management are crucial to reducing the risk of vision loss and improving the prognosis of these cases.

Living with Dry Eye Disease and its Effects on Quality of Life: Patient, Optometrist, and Ophthalmologist Perspectives.

Dry eye disease is a very common condition, especially among aging women. People often think of it as a very mild and non-harmful issue, but the reality is that it has a huge deleterious effect on patients' quality of life. Most publications usually focus on the scientific aspects of this pathology: its epidemiology, diagnosis, or management. However, in this article we highlight the patient's perspective and the challenges of living with dry eye disease. With prior informed consent, we interviewed a patient whose life has drastically changed since she first got the diagnosis. We also asked healthcare professionals based in Miami who were involved in this patient's care for their opinions. We hope that the messages and commentaries resonate with patients and physicians involved in the care of dry eye disease worldwide.

The inflammasome pathway: A key player in ocular surface and anterior segment diseases.

Inflammasomes are multicomplex molecular regulators with an emerging importance in regulating ocular surface and anterior segment health and disease. Key components found in the eye include NF-κB, NLRP3, NLRC4, NLRP6, ASC, IL-1ß, IL-18, and caspase-1. The role of NLRP1, NLRC4, AIM2, and NLRP3 inflammasomes in the pathogenesis of infectious ulcers, DED, uveitis, glaucoma, corneal edema, and other diseases is being studied with many developments. Attenuation of these diseases has been explored by blocking various molecules along the inflammasome pathway with agents like NAC, polydatin, calcitriol, glyburide, YVAD, and disulfiram. We provide a background on the inflammasome pathway as it relates to the ocular surface and anterior segment of the eye, discuss the role of inflammasomes in the above diseases in animals and humans, investigate new therapeutic targets, and explore the efficacy of new anti-inflammasome therapies.

Establishment of a bi-layered tissue engineered conjunctiva using a 3D-printed melt electrowritten poly-(ε-caprolactone) scaffold.

PURPOSE: To utilize melt electrowriting (MEW) technology using poly-(ε-caprolactone) (PCL) coupled with a 2-step co-culturing strategy for the development of a conjunctival bi-layer synthetic construct. METHODS: Melt electrowritten scaffolds using PCL were fabricated using an in-house-built MEW printer. Human conjunctival stromal cells (CjSCs) and epithelial cells (CjECs) were isolated from donor tissue. A 2-step co-culture method was done by first seeding the CjSCs and culturing for 4 weeks to establish a stromal layer, followed by CjECs and co-culturing for 2 more weeks. Cultured cells were each characterized by morphology and marker expression on immunofluorescence and qPCR. The produced construct was assessed for cellular proliferation using viability assays. The bi-layer morphology was assessed using scanning electron microscopy (SEM), confocal microscopy, and immunofluorescence imaging. The expression of extracellular matrix components and TGF-b was evaluated using qPCR. RESULTS: CjSCs were spindle-shaped and vimentin + while CjECs were polygonal and CK13 + . CjSCs showed consistent proliferation and optimal adherence with the scaffold at the 4-week culture mark. A 2-layered construct consisting of a CjSC-composed stromal layer and a CjEC-composed epithelial layer was appreciated on confocal microscopy, SEM, and immunofluorescence. CjSCs secreted collagens (types I, V, VI) but at differing amounts from natural tissue while TGF-b production was comparable. CONCLUSION: The 3D-printed melt electrowritten PCL scaffold paired with the 2-step co-culturing conditions of the scaffold allowed for the first approximation of a bi-layered stromal and epithelial reconstruction of the conjunctiva that can potentially improve the therapeutic arsenal in ocular surface reconstruction.

Comparison of Transcriptomic Analysis of the Conjunctiva in Glaucoma-Treated Eyes with Dry Eyes and Healthy Controls.

Ocular surface disease (OSD) associated with topical glaucoma drugs is a common issue impacting treatment adherence. We aimed to identify conjunctival transcriptomic changes in glaucoma and dry eye patients, comparing them to healthy controls. Bulbar conjunctival specimens were collected via impression cytology from 33 patients treated for glaucoma, 9 patients with dry eye, and 14 healthy controls. RNA extraction and bulk RNA sequencing were performed, followed by bioinformatics analysis to detect gene dysregulation. Ingenuity pathways analysis (IPA) identified pathways and biological processes associated with these transcriptomic changes. Sequencing analysis revealed 200 modified genes in glaucoma patients compared to healthy individuals, 233 differentially expressed genes in dry eye patients versus controls, and 650 genes in treated versus dry eye samples. In glaucoma patients, 79% of altered pathways were related to host defense, while dry eye patients showed a 39% involvement of host response, 15% in cellular proliferation and integrity, and 16% of mitochondrial dysfunction. These findings were validated through qRT-PCR. Glaucoma patients showed an intensified conjunctival immune response as a potential cause of OSD, whereas in dry eye patients, in addition to the immune response, other mechanisms such as mitochondrial dysfunction or reduced cellular proliferation were observed.

Brief incubation of corneal grafts in activated platelet rich plasma enhances corneal endothelial cell survival and regeneration.

Corneal transplantation is the most frequent organ transplantation worldwide. Unfortunately, corneal graft failure is common and endothelial decompensation is considered the major cause. Corneal endothelial cells (CECs) lack the capacity to reproduce, and perioperative and postoperative endothelial cell loss remains a significant challenge associated with corneal graft viability. Therefore, strategies to preserve CEC density are critical to extend graft survival. Activated platelet rich plasma (aPRP), a product extracted from autologous blood, has both antioxidant and regenerative properties. aPRP eye drops have shown effectiveness in the treatment of corneal pathologies such as ulcers, dry eye, and burns. Our purpose is to determine the protective and regenerative effect of aPRP on corneal grafts by evaluating aPRP's effect on the survival and proliferation of human CECs. Human corneal grafts were incubated in aPRP for 15 min to assess the activation of the CEC pAkt survival pathway as measured by ELISA. Evaluation of the protective effect of aPRP was made using an apoptotic model, which simulated oxidative stress conditions. Expression of apoptotic markers was measured using ELISA and endothelial cell viability was determined by optical microscopy. The CEC proliferation rate was measured in vitro with Ki-67 staining. Corneal graft gross structure was evaluated by Hematoxylin & Eosin and Masson trichrome staining. Our results indicate that a short incubation of human corneal grafts in aPRP protects CECs from apoptosis by upregulating the pAkt survival pathway and promoting CEC proliferation. Additionally, aPRP incubation does not induce histological changes in the grafts. A brief pre-treatment of human corneal grafts in aPRP may be beneficial for transplant longevity, as it protects CECs from apoptosis by upregulating intracellular survival pathways and promoting proliferation. In addition, this approach appears to be safe and has the potential to improve surgical outcomes following corneal transplantation.

A multicenter report of the use of plasma rich in growth factors (PRGF) for the treatment of patients with ocular surface diseases in North America.

PURPOSE: To investigate the efficacy and safety of plasma rich in growth factors (PRGF) eyedrops in the management of patients with ocular surface diseases in North America. METHODS: Multicenter interventional case series of patients using PRGF eyedrops for the first time. A cohort of patients was analyzed for corneal staining score at initial visit and at 3 months of therapy with PRGF. Another cohort responded to a 10-item questionnaire that evaluated patients' satisfaction and safety, which included the symptom assessment questionnaire in dry eye (SANDE) score, after 6 months of PRGF treatment. RESULTS: A total of 153 patients were analyzed. Of these, 102 were reviewed for corneal epitheliopathy and 99 patients responded to the questionnaire. The mean (±SD) age of the population was 63.7 ± 17 years and 72.5% were female. The clinical indications for PRGF usage were dry eye (60%), neurotrophic keratopathy (15%), dormant corneal ulcers (12%), limbal stem cell deficiency (10%), and cicatrizing conjunctivitis (4%). At the final visit, 74.3% of patients showed an improvement of their corneal staining. Those who had punctate epithelial erosions or epithelial defects were reduced from 76.5% to 47% and 23.5% to 7.8% respectively (p < 0.0001). Symptoms, measured via SANDE score, significantly decreased from a median of 90 to 34.6 out of 100 points on follow-up (p < 0.0001). Only one patient (0.98%) complained of ocular burning sensation as a side effect. CONCLUSIONS: This multicentric study demonstrates the safety and efficacy of the use of PRGF for treating signs and symptoms in patients with significant ocular surface diseases.

Role of Caspase-1 as a Biomarker of Ocular Surface Damage.

PURPOSE: To examine the potential of caspase-1 as a biomarker for ocular surface damage. DESIGN: Cross-sectional study. METHODS: A total of 113 tear samples (64 subjects) were analyzed. Sixty-one samples were from individuals with dry eye disease (DED), defined as Ocular Surface Disease Index (OSDI) ≥13 and/or corneal staining (CS) ≥3; 32 were from individuals who used glaucoma medication, irrespective of DED metrics; and 20 were from controls (CS <3 and OSDI <13). All individuals completed a medical history form and underwent an ocular surface assessment. Protein levels of caspase-1 were determined by enzyme-linked immunosorbent assay off Schirmer's strips. The primary analysis compared caspase-1 levels in individuals with signs of ocular surface damage (CS ≥3) in both case groups and controls. Secondary correlational analyses were conducted to examine relationships between caspase-1 levels and ocular signs and symptoms. Finally, area under the curve (AUC) analyses were performed to examine relationships between inflammatory markers and CS. RESULTS: The mean age of the population was 58±18 years; 70% were female. Tear samples from individuals with ocular surface damage presented higher caspase-1 levels than the control group. Caspase-1 levels showed a moderate positive correlation with CS (Spearman r = 0.31; P = .001) and eye redness (Spearman r = 0.39; P = .004), and a negative correlation with Schirmer's (Spearman r = -0.46; P < .001) and tear break-up time (Spearman r = -0.33; P = .0006). Caspase-1 showed higher sensitivity and AUC for detecting ocular surface damage than InflammaDry, and its expression was not affected by anti-inflammatory agents. CONCLUSION: Caspase-1 levels were higher in the tears of individuals with ocular surface damage, suggesting its potential to be used as a biomarker and/or therapeutic target.

Risk and Impact of Severe Acute Respiratory Syndrome Coronavirus 2 Infection on Corneal Transplantation: A Case-Control Study.

PURPOSE: The purpose of this study was to evaluate the risk of symptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection after corneal transplantation surgery, with cataract surgeries as controls, and the impact of the novel coronavirus disease pandemic in the clinical and surgical complications of corneal transplantation and cataract surgeries. METHODS: A retrospective matched case-control study of 480 consecutive individuals who underwent surgery at the Bascom Palmer Eye Institute between May 2020 and November 2020. A total of 240 patients who underwent corneal transplantation with tissue obtained from the Florida Lions Eye Bank were age, race, ethnicity, and sex matched with 240 patients who underwent cataract surgery during the same day and by the same surgical team. Only the first corneal transplant or cataract surgery during this period was considered for each individual. All donors and recipients were deemed SARS-CoV-2 negative by a nasopharyngeal polymerase chain reaction test before surgery. Postoperative SARS-CoV-2 infections were defined as previously SARS-CoV-2(-) individuals who developed symptoms or had a positive SARS-CoV-2 polymerase chain reaction test during the first postoperative month. RESULTS: Mean age, sex, race, and ethnicity were similar between groups. There were no differences between the corneal transplant and cataract groups in the rates of SARS-CoV-2 infection before (5.8% vs. 7.5%, P= 0.6) or after surgery (2.9% vs. 2.9%, P = 1). The rates of postoperative complications did not increase during the pandemic, compared with previously reported ranges. CONCLUSIONS: In this study, postoperative SARS-CoV-2 infection was similar for individuals undergoing corneal transplantation or cataract surgery. Further research is required to evaluate the transmission of SARS-CoV-2 through corneal tissue.

Effects of Mitomycin-C and 5-Fluorouracil on Ocular Adnexal Sebaceous Carcinoma Cells.

PURPOSE: To investigate the effects of mitomycin-C (MMC) and 5-fluorouracil (5-FU) on the viability, proliferation, and migratory capacity of cultured ocular adnexal sebaceous carcinoma (SC) cells. DESIGN: Laboratory investigation. METHODS: Human SC cell lines (Bascom Palmer 50 and 52 [BP50 and BP52]) and human limbal stem cells (LSCs) were treated with various concentrations of MMC and 5-FU. Cytotoxicity was assessed with the tetrazolium MTT colorimetric viability assay on normal corneal vs tumor cells. Growth curves and scratch assays were performed to characterize the effects of these chemotherapeutic agents on SC proliferation and migration, respectively. RESULTS: MMC decreased BP52 cell viability in a dose-dependent manner with a half-maximal effective dose (EC50) of 11.8 µM after 72 hours. SC viability decreased >50% at 80 mM 5-FU after 72 hours. MMC reduced LSC viability in a dose-dependent manner with an EC50 value of 3.24 µM, and 5-FU decreased LSC viability >50% at 160 µM. MMC decreased SC cell proliferation and migration in a dose-dependent manner. 5-FU displayed antiproliferative effects but did not affect cell migration at concentrations below 1000 µM. CONCLUSIONS: Our in vitro data corroborate clinical observations that MMC is efficacious for treating ocular adnexal SC, albeit at the expense of LSC viability. Our findings also demonstrate that topical 5-FU exhibits antiproliferative effects that supersede its cancer-killing and antimigratory effects on cultured SC cells.

Tissue engineering of corneal stroma via melt electrowriting.

The cornea serves as the main refractive component of the eye with the corneal stroma constituting the thickest component in a stratified layered system of epithelia, stroma, and endothelium. Current treatment options for patients suffering from corneal diseases are limited to transplantation of a human donor cornea (keratoplasty) or to implantation of an artificial cornea (keratoprosthesis). Nevertheless, donor shortage and failure of artificial corneas to integrate with local tissue constitute important problems that have not been yet circumvented. Recent advances in biofabrication have made great progress toward the manufacture of tailored biomaterial templates with the potential of guiding partially or totally the regeneration process of the native cornea. However, the role of the corneal stroma on current tissue engineering strategies is often neglected. Here, we achieved a tissue-engineered corneal stroma substitute culturing primary keratocytes on scaffolds prepared via melt electrowriting (MEW). Scaffolds were designed to contain highly organized micrometric fibers to ensure transparency and encourage primary human keratocytes to self-orchestrate their own extracellular matrix deposition and remodeling. Results demonstrated reliable cell attachment and growth over a period of 5 weeks and confirmed the formation of a dense and highly organized de novo tissue containing collagen I, V, and VI as well as Keratocan, which resembled very closely the native corneal stoma. In summary, MEW brings us closer to the biofabrication of a viable corneal stroma substitute.

Tumor necrosis factor-alpha and interferon-gamma induce inflammasome-mediated corneal endothelial cell death.

PURPOSE: Chronic corneal endothelial cell (CEC) loss results in corneal edema and vision loss in conditions such as pseudophakic bullous keratopathy (PBK), Fuchs' dystrophy, and corneal graft failure. Low CEC density has been associated with an elevation of intraocular pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interferon (INF)-γ. These cytokines are capable of triggering pyroptosis, a programmed cell death mechanism mediated by the inflammasome, prompting the activation of the pro-inflammatory cytokine interleukin (IL)-1ß, the perpetuation of inflammation, and subsequent damage of corneal endothelial tissue. Therefore, the purpose of this study was to determine the deleterious contribution of the inflammasome and pyroptosis to CEC loss. METHODS: CECs from human donor corneas were treated ex vivo with TNF-α and IFN-γ for 48 h. Levels of caspase-1 and IL-1ß were then assayed by ELISA, and the expression of caspase-1 and gasdermin-D (GSDM-D) were confirmed by immunofluorescence. Endothelial cell damage was analyzed by a lactate dehydrogenase (LDH) release assay, and oxidative stress was determined by measuring the levels of reactive oxygen species (ROS) in the culture media. RESULTS: Inflammasome activation and oxidative stress were elevated in CECs following exposure to TNF-α and IFN-γ, which resulted in cell death by pyroptosis as determined by LDH release which was inhibited by the caspase-1 inhibitor Ac-YVAD-cmk. CONCLUSION: CEC death is induced by the pro-inflammatory cytokines TNF-α and IFN-γ, which contribute to inflammasome activation. Moreover, the inflammasome is a promising therapeutic target for the treatment of chronic CEC loss.

Dry eye: why artificial tears are not always the answer.

Dry eye disease (DED) is a multifactorial disease that manifests in patients with a variety of symptoms and signs such as ocular pain, visual issues, rapid tear evaporation and/or decreased tear production. It is a global health problem and is the leading cause of optometry and ophthalmology clinic visits. The mainstay therapy for DED is artificial tears (ATs), which mimics tears and improves tear stability and properties. ATs have been found to improve symptoms and signs of disease in all DED subtypes, including aqueous deficient DED and evaporative DED. However, given the heterogeneity of DED, it is not surprising that ATs are not effective in all patients. When AT fails to relieve symptoms and/or signs of DED, it is critical to identify the underlying contributors to disease and escalate therapy appropriately. This includes underlying systemic diseases, meibomian gland dysfunction, anatomical abnormalities and neuropathic dysfunction. Thus, this review will discuss the benefits and limitations of ATs and review conditions when escalation of therapy should be considered in DED.