Immunocytochemical characterization of ex vivo cultured conjunctival explants; marker validation for the identification of squamous epithelial cells and goblet cells

Tissue-engineered products are at the cutting edge of innovation considering their potential to functionally and structurally repair various tissue defects when the body’s own regenerative capacity is exhausted. At the ocular surface, the wound healing response to extensive conjunctival damage results in tissue repair with structural alterations or permanent scar formation rather than regeneration of the physiological conjunctiva. Conjunctival tissue engineering therefore represents a promising therapeutic option to reconstruct the ocular surface in severe cicatrizing pathologies. During the rapid race to be a pioneer, it seems that one of the fundamental steps of tissue engineering has been neglected; a proper cellular characterization of the tissue-engineered equivalents, both morphologically and functionally. Currently, no consensus has been reached on an identification strategy and/or markers for the characterization of cultured squamous epithelial and goblet cells. This study therefore evaluated the accuracy of promising markers to identify differentiated conjunctival-derived cells in human primary explant cultures through immunocytochemistry, including keratins (i.e., K7, K13, and K19) and mucins (i.e., MUC1, MUC5AC, and PAS-positivity). Comparison of the in vivo and in vitro cellular profiles revealed that the widely used goblet cell marker K7 does not function adequately in an in vitro setting. The other investigated markers offer a powerful tool to distinguish cultured squamous epithelial cells (i.e., MUC1 and K13), goblet cells (i.e., MUC5AC and PAS-staining), and conjunctival-derived cells in general (i.e., K19). In conclusion, this study emphasizes the power alongside potential pitfalls of conjunctival markers to assess the clinical safety and efficacy of conjunctival tissue-engineered products

Outcomes of human leukocyte antigen-matched allogeneic cultivated limbal epithelial transplantation in aniridia-associated keratopathy - a single-center retrospective analysis

To assess the efficacy and safety of human leukocyte antigen-matched allogeneic cultivated limbal epithelial stem cell grafts in the treatment of aniridia-associated keratopathy (AAK). Methods: Six eyes of 6 patients with severe AAK received an allogeneic stem cell graft between January 2010 and March 2017. Anatomical and functional results were assessed at 6 months, 1 year, 2 years, and the final follow-up visit available. Safety analysis was performed by considering all perioperative and postoperative adverse events and additional surgeries required during the follow-up period.Results:The mean follow-up was 53.6 months (range 24-104 months). In most patients (80%), there was an early improvement of the keratopathy postoperatively, which slowly regressed during longer follow-up. At the final follow-up, 4 of the eyes were graded as failure and 1 eye was graded as partial success. Grading the sixth eye was not possible because of an adverse event. None of the patients maintained a total anatomical success in the long-term. Only 1 patient maintained a modest improvement in best-corrected visual acuity from hand motion to counting fingers. Four serious adverse events were recorded in 2 patients.Conclusions:Severe AAK remains a challenging condition to manage. Transplantation of allogenic ex vivo cultivated limbal stem cells may provide a temporary improvement in ocular surface stability, but anatomical and functional results are poor in the long-term. The eyes are prone to adverse events, and any surgical treatment should take this into consideration

Designer Descemet membranes containing PDLLA and functionalized gelatins as corneal endothelial scaffold

Corneal blindness is the fourth leading cause of visual impairment. Of specific interest is blindness due to a dysfunctional corneal endothelium which can only be treated by transplanting healthy tissue from a deceased donor. Unfortunately, corneal supply does not meet the demand with only one donor for every 70 patients. Therefore, there is a huge interest in tissue engineering of grafts consisting of an ultra-thin scaffold seeded with cultured endothelial cells. The present research describes the fabrication of such artificial Descemet membranes based on the combination of a biodegradable amorphous polyester (poly (d,l-lactic acid)) and crosslinkable gelatins. Four different crosslinkable gelatin derivatives are compared in terms of processing, membrane quality, and function, as well as biological performance in the presence of corneal endothelial cells. The membranes are fabricated through multi-step spincoating, including a sacrificial layer to allow for straightforward membrane detachment after production. As a consequence, ultrathin (90%), semi-permeable membranes could be obtained with high biological potential. The membranes supported the characteristic morphology and correct phenotype of corneal endothelial cells while exhibiting similar proliferation rates as the positive control. As a consequence, the proposed membranes prove to be a promising synthetic alternative to donor tissue

Phase 2 randomized, double-masked, vehicle-controlled trial of recombinant human nerve growth factor for neurotrophic keratitis

Purpose: To evaluate the safety and efficacy of topical recombinant human nerve growth factor (rhNGF) for treating moderate-to-severe neurotrophic keratitis (NK), a rare degenerative corneal disease resulting from impaired corneal innervation. Design: Phase 2 multicenter, randomized, double-masked, vehicle-controlled trial. Participants: Patients with stage 2 (moderate) or stage 3 (severe) NK in 1 eye. Methods: The REPARO phase 2 study assessed safety and efficacy in 156 patients randomized 1:1:1 to rhNGF 10 μg/ml, 20 μg/ml, or vehicle. Treatment was administered 6 drops per day for 8 weeks. Patients then entered a 48- or 56-week follow-up period. Safety was assessed in all patients who received study treatment, whereas efficacy was by intention to treat. Main Outcome Measures: Corneal healing (defined as <0.5-mm maximum diameter of fluorescein staining in the lesion area) was assessed by masked central readers at week 4 (primary efficacy end point) and week 8 (key secondary end point) of controlled treatment. Corneal healing was reassessed post hoc by masked central readers using a more conservative measure (0-mm staining in the lesion area and no other persistent staining). Results: At week 4 (primary end point), 19.6% of vehicle-treated patients achieved corneal healing (<0.5-mm lesion staining) versus 54.9% receiving rhNGF 10 μg/ml (+35.3%; 97.06% confidence interval [CI], 15.88–54.71; P < 0.001) and 58.0% receiving rhNGF 20 μg/ml (+38.4%; 97.06% CI, 18.96–57.83; P < 0.001). At week 8 (key secondary end point), 43.1% of vehicle-treated patients achieved less than 0.5-mm lesion staining versus 74.5% receiving rhNGF 10 μg/ml (+31.4%; 97.06% CI, 11.25–51.49; P = 0.001) and 74.0% receiving rhNGF 20 μg/ml (+30.9%; 97.06% CI, 10.60–51.13; P = 0.002). Post hoc analysis of corneal healing by the more conservative measure (0-mm lesion staining and no other persistent staining) maintained statistically significant differences between rhNGF and vehicle at weeks 4 and 8. More than 96% of patients who healed after controlled rhNGF treatment remained recurrence free during follow-up. Treatment with rhNGF was well tolerated; adverse effects were mostly local, mild, and transient. Conclusions: Topical rhNGF is safe and more effective than vehicle in promoting healing of moderate-to-severe NK

Phase I trial of recombinant human nerve growth factor for neurotrophic keratitis

Neurotrophic keratitis/keratopathy (NK), a rare degenerative corneal disease, lacks effective pharmacologic therapies.1 Because NK pathology involves trigeminal nerve damage and loss of corneal innervation, nerve growth factor (NGF) is surmised to promote healing of NK.2 Preliminary studies with murine NGF demonstrated efficacy for treating corneal neurotrophic ulcers;3 however, the complex tertiary structure of NGF has complicated the production of recombinant human NGF (rhNGF) suitable for clinical development. To this end, we developed an Escherichia coli–derived rhNGF formulation that demonstrated to be well tolerated and safe for topical ophthalmic use in a phase I study in healthy volunteers.4 We report phase I results of topical rhNGF for patients with moderate-to-severe NK

Future directions in managing aniridia-associated keratopathy

Congenital aniridia is a panocular disorder that is typically characterized by iris hypoplasia and aniridia-associated keratopathy (AAK). AAK results in the progressive loss of corneal transparency and thereby loss of vision. Currently, there is no approved therapy to delay or prevent its progression, and clinical management is challenging because of phenotypic variability and high risk of complications after interventions; however, new insights into the molecular pathogenesis of AAK may help improve its management. Here, we review the current understanding about the pathogenesis and management of AAK. We highlight the biological mechanisms involved in AAK development with the aim to develop future treatment options, including surgical, pharmacological, cell therapies, and gene therapies

Human Tears Reveal Insights into Corneal Neovascularization

Corneal neovascularization results from the encroachment of blood vessels from the surrounding conjunctiva onto the normally avascular cornea. The aim of this study is to identify factors in human tears that are involved in development and/or maintenance of corneal neovascularization in humans. This could allow development of diagnostic tools for monitoring corneal neovascularization and combination monoclonal antibody therapies for its treatment. In an observational case-control study we enrolled a total of 12 patients with corneal neovascularization and 10 healthy volunteers. Basal tears along with reflex tears from the inferior fornix, superior fornix and using a corneal bath were collected along with blood serum samples. From all patients, ocular surface photographs were taken. Concentrations of the pro-angiogenic cytokines interleukin (IL)-6, IL-8, Vascular Endothelial Growth Factor (VEGF), Monocyte Chemoattractant Protein 1 (MCP-1) and Fas Ligand (FasL) were determined in blood and tear samples using a flow cytometric multiplex assay. Our results show that the concentration of pro-angiogenic cytokines in human tears are significantly higher compared to their concentrations in serum, with highest levels found in basal tears. Interestingly, we could detect a significantly higher concentration of IL- 6, IL-8 and VEGF in localized corneal tears of patients with neovascularized corneas when compared to the control group. This is the first study of its kind demonstrating a significant difference of defined factors in tears from patients with neovascularized corneas as compared to healthy controls. These results provide the basis for future research using animal models to further substantiate the role of these cytokines in the establishment and maintenance of corneal neovascularization

DNA damage in circulating leukocytes measured with the comet assay may predict the risk of death

The comet assay or single cell gel electrophoresis, is the most common method used to measure strand breaks and a variety of other DNA lesions in human populations. To estimate the risk of overall mortality, mortality by cause, and cancer incidence associated to DNA damage, a cohort of 2,403 healthy individuals (25,978 person-years) screened in 16 laboratories using the comet assay between 1996 and 2016 was followed-up. Kaplan–Meier analysis indicated a worse overall survival in the medium and high tertile of DNA damage (p < 0.001). The effect of DNA damage on survival was modelled according to Cox proportional hazard regression model. The adjusted hazard ratio (HR) was 1.42 (1.06–1.90) for overall mortality, and 1.94 (1.04–3.59) for diseases of the circulatory system in subjects with the highest tertile of DNA damage. The findings of this study provide epidemiological evidence encouraging the implementation of the comet assay in preventive strategies for non-communicable diseases.This article has been corrected. Link to the correction: [https://farfar.pharmacy.bg.ac.rs/handle/123456789/3975

Author Correction: DNA damage in circulating leukocytes measured with the comet assay may predict the risk of death (Scientific Reports, (2021), 11, 1, (16793), 10.1038/s41598-021-95976-7)

Link to the corrected article: [https://farfar.pharmacy.bg.ac.rs/handle/123456789/3944