Validation of preloaded DMEK donor tissues: a laboratory-based study on endothelial cell viability and comparison of two F-mark inks.

OBJECTIVE: To investigate endothelial cell loss (ECL) associated with Descemet membrane endothelial keratoplasty (DMEK) donor tissues preloaded in the DMEK RAPID transport system after 1 and 5 days and to compare prestamping with 2 different F-mark inks. METHODS: DMEK donor tissues were stripped, marked with gentian violet dye applied as an F-mark, trephined, stained with trypan blue, and then preloaded into the DMEK RAPID transport system by an eye bank technician. Preloaded DMEK tissues were then unfolded and stained with calcein AM after 1 or 5 days of storage. Tissues were imaged, analyzed for total tissue ECL, and immunostained for corneal endothelium markers zonular occludens-1 and xCD166. Additionally, ECL and the intensity of an F-mark caused by 2 different inks were quantified. RESULTS: Preloaded DMEK tissues displayed an average ECL of 11.9% ± 4.5% (n = 8) at 1 day and 9.9% ± 4.2% (n = 9) at 5 days. No difference was found between the 2 groups. Zonular occludens-1 and activated leukocyte cell adhesion molecule (ALCAM; also know as CD166) staining showed that the corneal endothelial monolayer remained intact on preloaded tissues. On 5-day preloaded DMEK tissues, the average ECL and mean grayscale caused by the Keir Surgical ink F-mark and the Cardinal Health ink F-mark were 4.3% ± 0.8% and 158.5 ± 13.9% and 5.0% ± 1.1% and 142.9% ± 20.0%, respectively. No difference was found between the F-mark inks. CONCLUSION: Preloaded DMEK donor tissues resulted in an acceptable ECL range after 1 and 5 days of storage and were deemed suitable for transplantation. Both F-mark inks are acceptable for prestamping preloaded DMEK tissues prior to surgical transplantation with comparable ECL and intensities.

Tissue and Cell Donation: Recommendations From an International Consensus Forum.

Organ, tissue, and cell donation and transplantation legislation and policies vary substantially worldwide, as do performance outcomes in various jurisdictions. Our objective was to create expert, consensus guidance that links evidence and ethical concepts to legislative and policy reform for tissue and cell donation and transplantation systems. Methods: We identified topic areas and recommendations through consensus, using nominal group technique. The proposed framework was informed by narrative literature reviews and vetted by the project's scientific committee. The framework was presented publicly at a hybrid virtual and in-person meeting in October 2021 in Montréal, Canada, where feedback provided by the broader Forum participants was incorporated into the final manuscript. Results: This report has 13 recommendations regarding critical aspects affecting the donation and use of human tissues and cells that need to be addressed internationally to protect donors and recipients. They address measures to foster self-sufficiency, ensure the respect of robust ethical principles, guarantee the quality and safety of tissues and cells for human use, and encourage the development of safe and effective innovative therapeutic options in not-for-profit settings. Conclusions: The implementation of these recommendations, in total or in part, by legislators and governments would benefit tissue transplantation programs by ensuring access to safe, effective, and ethical tissue- and cell-based therapies for all patients in need.

The Effect of COVID-19 on Corneal Donor Volumes and Eye Bank Processes: An Analysis From the Eye Bank of Canada (Ontario Division).

PURPOSE: With the rise in COVID-19 cases, the Eye Bank of Canada (Ontario Division), the largest eye bank in Canada, was faced with challenges related to ocular donor suitability which resulted in tissue shortages after the first wave of COVID-19 cases in Ontario, Canada. This article aims to analyze the impact of COVID-19 on ocular tissue donation and transplant surgeries. METHODS: Trends in ocular donations in 2020 and the transplant rates were compared with the data from the previous year, as a benchmark of normal eye bank activity. RESULTS: Ocular donor volumes decreased during the first wave of the COVID-19 pandemic (March-June 2020) by 65% as compared to the same period in 2019. By the end of the year 2020, this had resulted in a total reduction of 29% of ocular donor volumes as compared to 2019. The ocular transplant surgery volumes in the year 2020 decreased by 32% compared to the previous year, mostly secondary to elective surgery shutdown during the first wave. Because of tissue shortages, the Eye Bank of Canada (Ontario Division) had to import 24 corneas from the United States and cancel 7 surgeries in the year 2020. CONCLUSIONS: The decline in ocular tissue donor volumes and transplant surgery was a result of an interplay of causes related to the COVID-19 pandemic. Most importantly, ruling out of COVID-19 carriers, lockdown measures affecting tissue retrieval processes, and shutdown of elective surgery were the 3 major factors accounting for tissue shortages and surgical volume reductions.

Canadian demand and access to corneal transplantation: a provincial comparison.

To gather information from stakeholders involved in corneal donation and transplantation to inform discussion at the "National Consensus Forum on Improving Cornea Donation and Transplantation Access in Canada" held in February 2020, survey questions were posed to eye banks, transplanting ophthalmologists and organ donation organizations across Canada to learn more about demand, wait times, and access to tissue for transplant. The survey response rate was one hundred percent (100%) for eye banks and organ donation organizations while 64 percent (64%) of transplant ophthalmologists provided feedback. A number of opportunities for improvement were identified including: demand forecasting; infrastructure and strategies to align supply with demand; data collection and benchmarking of wait times for assessment and transplant to support consistency, equitability and transparency in access; and national collaboration in the development of a data strategy to accurately measure demand and access to cornea transplants in a consistent manner across all provinces to facilitate equity in access nationally.

A National Consensus Forum on improving cornea donation and transplantation access in Canada.

A consensus meeting was held in Toronto on February 9-10, 2020 to discuss ways to improve cornea donation and transplantation access in Canada. The meeting brought together eye and tissue bank representatives, health authority and hospital leadership, transplant ophthalmologists, organ donation organizations, transplant recipients, donor families and several national organizations. Through facilitated discussions in multidisciplinary, gender-balanced, and geographically balanced small groups, participants identified opportunities for improvement in the Canadian cornea donation and transplantation system. Discussion occurred around broad themes of donor tissue demand, supply, access, utilization, interprovincial sharing and cost recovery, interprovincial knowledge sharing and research. This event marked the first time in 10 years in which the Canadian cornea transplantation community came together.

Nomogram to Predict Graft Thickness in Descemet Stripping Automated Endothelial Keratoplasty: An Eye Bank Study.

PURPOSE: The purpose of this study was to develop a nomogram to predict postcut thickness of corneal grafts prepared at an eye bank for Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: Retrospective chart review was performed of DSAEK graft preparations by 3 experienced technicians from April 2012 to May 2017 at the Eye Bank of Canada-Ontario Division. Variables collected included the following: donor demographics, death-to-preservation time, death-to-processing time, precut tissue thickness, postcut tissue thickness, microkeratome head size, endothelial cell count, cut technician, and rate of perforation. Linear regression models were generated for each microkeratome head size (300 and 350 µm). RESULTS: A total of 780 grafts were processed during the study period. Twelve preparation attempts resulted in perforation (1.5%) and were excluded. Mean precut tissue thickness was 510 ± 49 µm (range: 363-670 µm). Mean postcut tissue thickness was 114 ± 22 µm (range: 57-193 µm). Seventy-nine percent (608/768) of grafts were ≤130 µm. The linear regression models included precut thickness and donor age, which were able to predict the thickness to within 25 µm 80% of the time. CONCLUSIONS: We report a nomogram to predict thickness of DSAEK corneal grafts prepared in an eye bank setting, which was accurate to within 25 µm 80% of the time. Other eye banks could consider performing similar analyses.

Evolutionary routes from a prebiotic ANA-world.

Recent experimental support has been generated for a model of prebiotic development that postulates a role for Amyloid-Nucleic Acid (ANA)-fibers as the earliest replicating entities capable of undergoing Darwinian evolution. Here, this new model is compared with existing RNA-world models with a particular focus on trajectories that lead to evolutionary-beneficial interactions between nucleic acid, protein and lipid components. This analysis suggests a number of new areas for fruitful experimental studies.

Amyloid-associated nucleic acid hybridisation.

Nucleic acids promote amyloid formation in diseases including Alzheimer's and Creutzfeldt-Jakob disease. However, it remains unclear whether the close interactions between amyloid and nucleic acid allow nucleic acid secondary structure to play a role in modulating amyloid structure and function. Here we have used a simplified system of short basic peptides with alternating hydrophobic and hydrophilic amino acid residues to study nucleic acid - amyloid interactions. Employing biophysical techniques including X-ray fibre diffraction, circular dichroism spectroscopy and electron microscopy we show that the polymerized charges of nucleic acids concentrate and enhance the formation of amyloid from short basic peptides, many of which would not otherwise form fibres. In turn, the amyloid component binds nucleic acids and promotes their hybridisation at concentrations below their solution K(d), as shown by time-resolved FRET studies. The self-reinforcing interactions between peptides and nucleic acids lead to the formation of amyloid nucleic acid (ANA) fibres whose properties are distinct from their component polymers. In addition to their importance in disease and potential in engineering, ANA fibres formed from prebiotically-produced peptides and nucleic acids may have played a role in early evolution, constituting the first entities subject to Darwinian evolution.

An activation-defective mutant of the human cytomegalovirus IE2p86 protein inhibits NF-kappaB-mediated stimulation of the human interleukin-6 promoter.

The IE2p86 protein of human cytomegalovirus is an essential activator of early- and late-phase viral gene expression. Whilst IE2p86 activates expression of a number of cellular genes, it also represses certain cellular genes, particularly those activated by nuclear factor kappaB (NF-kappaB). As the interleukin-6 (IL-6) promoter can be activated by both NF-kappaB and IE2p86, it was examined whether there is competition between these two factors. Here, it is reported that both wild-type and mutant IE2p86 can block activation of the IL-6 promoter in response to interleukin-1beta. By using an artificial activator in which the activation domain of NF-kappaB is directed to the promoter by the GAL4 DNA-binding domain, it is shown that the mutant form of IE2p86 can inhibit NF-kappaB-mediated activation at a step subsequent to promoter recruitment. These data therefore suggest a novel mechanism for inhibition of NF-kappaB by IE2p86.

Posttranscriptional suppression of interleukin-6 production by human cytomegalovirus.

Human cytomegalovirus (HCMV) has evolved multiple strategies for suppression of the antiviral response of the infected cell. DNA array technology has revealed that HCMV clearly regulates host gene expression during the course of a productive infection by enhancing, sustaining, or suppressing steady-state levels of cellular transcripts. Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a central role in the immune response to infection. Here we report a detailed study of the effects of HCMV infection on IL-6 expression by human fibroblasts. UV-inactivated virus was found to induce high levels of IL-6 mRNA and protein expression, and IL-6 mRNA remained abundant in cells 16 h after inoculation even though the level of ongoing IL-6 transcription was not significantly enhanced. In lytic HCMV infections, the onset of viral gene expression resulted in two apparently antagonistic effects on IL-6 expression: (i) transcriptional activation, mediated at least in part by the IE2p86 protein, and (ii) posttranscriptional suppression mediated by destabilization of IL-6 mRNA. Transcriptional activation was outweighed by the suppressive effect, such that cells undergoing productive infection produced less IL-6 than cells challenged with inactivated virus. Suppression of IL-6 expression was independent of the viral IL-10 homologue, cmvIL-10. Destabilization of IL-6 mRNA was observed to coincide with the enhanced expression and aberrant intracellular localization of HuR, an mRNA-binding protein known to interact with IL-6 and other mRNAs containing 3' AU-rich elements. Our data suggest a novel mechanism for gene regulation by HCMV at the posttranscriptional level.