The use of amniotic membrane (AM) is a widespread clinical practice for eye surgeries and the treatment of an increasing number of ocular surface pathologies. Here we describe the AM collection methods and donor selection criteria adopted by our tissue bank to distribute 5349 amniotic membrane patches over the last 12 years for the treatment of several ocular pathologies. Specific quality control measures are described and the long term results attained using the reported procedure are presented. A case of AM utilized to treat severe ocular ulceration is also described as an example of AM transplantation. Collective data for the total amniotic membrane patches deployed to treat various ocular diseases are discussed and success rates for AM transplantations are reported. An extensive follow-up is illustrated. The results suggest that the procedures and protocols used by the Treviso Tissue Bank Foundation and Veneto Eye Bank Foundation for collection, preservation, distribution and follow-up are of an optimal standard. Accordingly, the authors conclude that the safety and efficiency of the proposed procedure for the therapeutic use of AM to treat various ocular pathologies are reproducible, with additional evidence favoring the use of AM as an alternative to conventional medical treatment for certain ocular conditions.

Background and Purpose: The connexin 43 (Cx43) mimetic peptide Gap27 was designed to transiently block the function of this gap junction. This study was undertaken to investigate the effect of Gap27 on corneal healing, inflammation and neovascularization.Experimental Approach: The effect of Gap27 on wound healing, inflammation and vascularization was assessed in primary human corneal epithelial cells (HCEC)in vitroand whole human corneasex vivo, and in anin vivorat wound healing model.Key Results: Gap27 enhanced the wound closure of HCECin vitroand accelerated wound closure and stratification of epithelium in human corneasex vivo, but did not suppress the corneal release of inflammatory mediators IL‐6 or TNF‐αin vivo. In human corneasex vivo, F4/80 positive macrophages were observed around the wound site.In vivo, topical Gap27 treatment enhanced the speed and density of early granulocyte infiltration into rat corneas. After 7 days, the expressions of TNF‐α and TGFβ1 were elevated and correlated with inflammatory cell accumulation in the tissue. Additionally, Gap27 did not suppress VEGF release in organotypic culture, nor did it suppress early or late VEGFA expression or neovascularizationin vivo.Conclusions and Implications: Gap27 can be effective in promoting the healing of superficial epithelial wounds, but in deep stromal wounds it has the potential to promote inflammatory cell migration and accumulation in the tissue and does not suppress the subsequent neovascularization response. These results support the proposal that Gap27 acts as a healing agent in the transient, early stages of corneal epithelial wounding.

Introduction: The management of hospitalized COVID‐19 patients depends largely on controlling the intensified inflammatory response known as the cytokine storm. Candidate inflammatory cytokines can serve as new biomarkers for the management of hospitalized COVID‐19 patients.Methods: Patients (80) were recruited into three groups: room air (RA), oxygen (OX) and mechanical ventilator (MV). Blood analysis was performed for RBC, WBC, Hb, Platelets, serum albumin and creatinine, INR, PTT, and hematocrit. ELISA was used to quantify a panel of inflammatory mediators including GM‐SCF, IFN‐α, IFNγ, IL‐1β, IL‐1R, IL‐2, IL‐2Ra, IL‐6, IL‐8, IL‐10, IL‐12p70, IL‐13, MCP‐1, MIP‐1a, and TNF‐α. Correlations between laboratory results and the levels of circulating inflammation mediators were investigated.Results: Patients on MV had low RBC, Hb, albumin, and HCT and high WBC count, PTT, and INR when compared to RA and OX groups. A statistical positive correlation was found between WBC and the levels of IL‐6 and MCP‐1. RBCs correlated negatively with IL‐6 and IL‐10 and positively with IL‐8. Higher TNF‐α correlated with lower platelet counts while higher levels of IL‐1Rα and IL‐10 were associated with lower Hb levels. Increases in IFN‐γ and TNF‐α were indicative of compromised kidney functions as creatinine levels increased significantly. Most significant correlations were found between IL‐6 and lab results, showing positive correlation with WBC and INR, and negative correlation with RBC, albumin, and HCT.Conclusions: Having the most significant correlations, IL‐6 high levels in mechanically ventilated patients were shown to affect laboratory results, and, therefore, is suggested as a severity biomarker of COVID‐19.

The transmission of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) can occur through an airborne route, in addition to contaminated surfaces and objects. In hospitals, it has been confirmed by several studies that SARS‐CoV‐2 can contaminate surfaces and medical equipment especially in hospitals dedicated to coronavirus disease 2019 (COVID‐19) patients. The aim of this study was to detect the contamination of hands, objects, and surfaces in isolation rooms and also in outpatients' clinics in hospitals and polyclinics. Environmental contamination of public high‐touch surfaces in public facilities was also investigated during an active COVID‐19 pandemic. Random swabs were also taken from public shops, pharmacies, bakeries, groceries, banknotes, and automated teller machines (ATMs). Samples were analyzed for SARS‐CoV‐2 positivity using real‐time polymerase chain reaction. In the COVID‐19 regional reference hospital, only 3 out of 20 samples were positive for SARS‐CoV‐2 RNA. Hand swabs from SARS‐CoV‐2‐positive patients in isolation rooms were occasionally positive for viral RNA. In outpatients' clinics, door handles were the most contaminated surfaces. Dental chairs, sinks, keyboards, ophthalmoscopes, and laboratory equipment were also contaminated. Although no positive swabs were found in shops and public facilities, random ATM swabs returned a positive result for SARS‐CoV‐2. Although there is no longer a focus on COVID‐19 wards and isolation hospitals, more attention is required to decontaminate frequently touched surfaces in health‐care facilities used by patients not diagnosed with COVID‐19. Additionally, high‐touch public surfaces such as ATMs require further disinfection procedures to limit the transmission of the infection.

Corneal graft rejection is a major problem in chronic herpetic keratitis (HK) patients with latent infection. A new class of antiviral agents targeting latent and active forms of herpes simplex virus type 1 (HSV-1) is importantly required. Meganucleases are sequence-specific homing endonucleases capable of inducing DNA double-strand breaks. A proof-of-concept experiment has shown that tailor-made meganucleases are efficient against HSV-1 in vitro. To take this work a step forward, we hypothesized that the pre-treatment of human corneas in eye banks using meganuclease-encoding vectors will allow HK patients to receive a medicated cornea to resist the recurrence of the infection and the common graft rejection problem. However, this strategy requires efficient gene delivery to human corneal endothelium. Using recombinant adeno-associated virus, serotype 2/1 (rAAV2/1), efficient gene delivery of a reporter gene was demonstrated in human corneas ex vivo. The optimum viral dose was 3.7 × 10¹¹ VG with an exposure time of 1 day, followed by 6 days incubation in de-swelling medium. In addition, 12 days incubation can result in transgene expression in excess of 70%. Using similar transduction conditions, meganuclease transgene expression was detected in 39.4% of the endothelial cells after 2 weeks in culture. Reduction of the total viral load in the media and the endothelial cells of corneas infected with HSV-1 was shown. Collectively, this work provides information about the optimum conditions to deliver genetic material to the cornea, and demonstrates for the first time the expression of meganuclease in human corneas ex vivo and its antiviral activity. In conclusion, we demonstrate that the treatment of human corneas in eye banks before transplantation is a new approach to address the unmet clinical needs in corneal diseases.

Ocular gene therapy is rapidly becoming a reality. By November 2012, approximately 28 clinical trials were approved to assess novel gene therapy agents. Viral infections such as herpetic keratitis caused by herpes simplex virus 1 (HSV-1) can cause serious complications that may lead to blindness. Recurrence of the disease is likely and cornea transplantation, therefore, might not be the ideal therapeutic solution. This paper will focus on the current situation of ocular gene therapy research against herpetic keratitis, including the use of viral and nonviral vectors, routes of delivery of therapeutic genes, new techniques, and key research strategies. Whereas the correction of inherited diseases was the initial goal of the field of gene therapy, here we discuss transgene expression, gene replacement, silencing, or clipping. Gene therapy of herpetic keratitis previously reported in the literature is screened emphasizing candidate gene therapy targets. Commonly adopted strategies are discussed to assess the relative advantages of the protective therapy using antiviral drugs and the common gene therapy against long-term HSV-1 ocular infections signs, inflammation and neovascularization. Successful gene therapy can provide innovative physiological and pharmaceutical solutions against herpetic keratitis.