Longitudinal Changes to Tight Junction Expression and Endothelial Cell Integrity in a Mouse Model of Sterile Corneal Inflammation

PURPOSE: We previously reported that applying toll-like receptor (TLR) ligands to an injured cornea induces corneal edema at 24 hours, which subsides by 1 week. We tested the hypotheses that endothelial expression of the tight-junction protein, zonula occludens-1 (ZO-1), would be altered during experimental sterile corneal inflammation and that endothelial cell density (ECD) would remain unaffected. METHODS: Anesthetized C57BL/6J mice received central 1-mm corneal abrasions followed by topical application of saline or cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN, TLR-9 agonist). At 24 hours, 1 week and 4 weeks post treatment, spectral-domain optical coherence tomography images were captured. Eyes were enucleated and processed for zonula occludens-1 (ZO-1) immunofluorescent staining. Corneal flatmounts were analyzed for endothelial ZO-1 expression, cell density, polymegethism, and polymorphism. Corneal stromal inflammatory cell infiltration was evaluated at 4 weeks by immunostaining for CD45. RESULTS: Central corneal thickness (CCT) was increased in CpG-ODN treated eyes at 24 hours, had normalized by 1 week, but was again thickened by 4 weeks. In eyes with CpG-ODN, endothelial cell ZO-1 expression was reduced at 24 hours but returned to normal levels by 1 week. Endothelial cell density was not altered at 24 hours or 1 week. By 4 weeks, only CpG-ODN eyes showed relatively reduced ECD, as well as large numbers of CD45+ cells in the stroma. Changes to ECD correlated with CCT (r = −0.53, P < 0.01). Compared with naïve controls, more saline- and CpG-ODN–treated eyes exhibited polymegethism. CONCLUSIONS: This study provides novel insights into the interplay between endothelial cell integrity, corneal edema, and chronic stromal leukocyte activation during sterile corneal inflammation in mice

Corneal cross-linking versus standard care in children with keratoconus - a randomised, multicentre, observer-masked trial of efficacy and safety (KERALINK): a statistical analysis plan

Background: The KERALINK trial tests the hypothesis that corneal cross-linking (CXL) treatment reduces the progression of keratoconus in comparison to standard care in patients aged 10–16 years. This article describes the statistical analysis plan for this trial as an update to the published protocol. It is written before the end of the patient follow-up, while the outcome of the trial is still unknown. Design and methods: KERALINK is a randomised controlled, observer-masked, multicentre trial in progressive keratoconus comparing epithelium-off CXL with standard care, including spectacles or contact lenses as necessary for best-corrected acuity. Keratoconus is a disorder of the shape of the cornea in which the normally round domeshaped clear front window of the eye (cornea) thins progressively leading to a cone-like bulge. This impairs the ability of the eye to focus properly, causing reduced vision which requires spectacle or contact lens wear or, in a minority of patients, eventually corneal replacement by a transplant for best vision. The primary outcome measure is the between-group difference in K2 at 18 months adjusted for K2 at baseline examination. K2 is the value of the steepest corneal meridian as measured on Pentacam topography. Secondary outcomes are keratoconus progression, time to keratoconus progression, visual acuity, refraction, apical corneal thickness and adverse events. Patient-reported effects will be explored by questionnaires. We describe in detail the statistical aspects of KERALINK: the outcome measures, the sample size calculation, general analysis principles, the planned descriptive statistics and statistical models, and planned subgroup and sensitivity analyses. Discussion: The KERALINK statistical analysis will provide comprehensive and precise information on the relative effectiveness of the two treatments. The plan will be implemented in May 2020 when follow-up for the trial is completed. Trial registration: EudraCT, 2016-001460-11. Registered on 19 May 201

Characteristics of endothelial corneal transplant rejection following immunisation with SARS-CoV-2 messenger RNA vaccine.

AIM: We report two cases of endothelial corneal allograft rejection following immunisation with SARS-CoV-2 messenger RNA (mRNA) vaccine BNT162b2 and describe the implications for management of transplant recipients postvaccination for COVID-19. METHODS: A 66-year-old woman with Fuchs endothelial corneal dystrophy (FECD) and a unilateral Descemet's membrane endothelial keratoplasty (DMEK) transplant received COVID-19 mRNA vaccine BNT162b2 14 days post-transplant. Seven days later, she presented with symptoms and signs of endothelial graft rejection. An 83-year-old woman with bilateral DMEK transplants for FECD 3 and 6 years earlier developed simultaneous acute endothelial rejection in both eyes, 3 weeks post second dose of COVID-19 mRNA vaccine BNT162b2. Rejection in both cases was treated successfully with topical corticosteroids. CONCLUSIONS: We believe this is the first report of temporal association between corneal transplant rejection following immunisation against COVID-19 and the first report of DMEK rejection following any immunisation. We hypothesise that the allogeneic response may have been initiated by the host antibody response following vaccination. Clinicians and patients should be aware of the potential of corneal graft rejection associated with vaccine administration and may wish to consider vaccination in advance of planned non-urgent keratoplasties. Patients should be counselled on the symptoms and signs that require urgent review to allow early treatment of any confirmed rejection episode

A randomised, controlled, observer-masked trial of corneal cross-linking for progressive keratoconus in children: the KERALINK protocol

INTRODUCTION: The KERALINK trial tests the hypothesis that corneal cross-linking (CXL) treatment reduces the progression of keratoconus in comparison to standard care in patients under 17 years old. KERALINK is a randomised controlled, observer-masked, multicentre trial in progressive keratoconus comparing epithelium-off CXL with standard care, including spectacles or contact lenses as necessary for best-corrected acuity. METHODS AND ANALYSIS: A total of 30 participants will be randomised per group. Eligible participants aged 10-16 years with progressive keratoconus in one or both eyes will be recruited. Following randomisation, participants will be followed up 3-monthly for 18 months. The effect on progression will be determined by K2 on corneal topography. The primary outcome measure is between-group difference in K2 at 18 months adjusted for K2 at baseline examination. Secondary outcomes are the effect of CXL on (1) keratoconus progression, (2) time to keratoconus progression, (3) visual acuity, (4) refraction, (5) apical corneal thickness and (6) adverse events. Patient-reported effects will be explored by questionnaires. ETHICS AND DISSEMINATION: Research Ethics Committee Approval was obtained on 30 June 2016 (ref: 14/LO/1937). Current protocol: V.5.0 (08/11/2017). Study findings will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: European Union clinial trials register (EudraCT) 2016-001460-11

Identification of Therapeutic Targets of Inflammatory Monocyte Recruitment to Modulate the Allogeneic Injury to Donor Cornea

Purpose: We sought to test the hypothesis that monocytes contribute to the immunopathogenesis of corneal allograft rejection and identify therapeutic targets to inhibit monocyte recruitment. Methods: Monocytes and proinflammatory mediators within anterior chamber samples during corneal graft rejection were quantified by flow cytometry and multiplex protein assays. Lipopolysaccharide or IFN-γ stimulation of monocyte-derived macrophages (MDMs) was used to generate inflammatory conditioned media (CoM). Corneal endothelial viability was tested by nuclear counting, connexin 43, and propidium iodide staining. Chemokine and chemokine receptor expression in monocytes and MDMs was assessed in microarray transcriptomic data. The role of chemokine pathways in monocyte migration across microvascular endothelium was tested in vitro by chemokine depletion or chemokine receptor inhibitors. Results: Inflammatory monocytes were significantly enriched in anterior chamber samples within 1 week of the onset of symptoms of corneal graft rejection. The MDM inflammatory CoM was cytopathic to transformed human corneal endothelia. This effect was also evident in endothelium of excised human cornea and increased in the presence of monocytes. Gene expression microarrays identified monocyte chemokine receptors and cognate chemokines in MDM inflammatory responses, which were also enriched in anterior chamber samples. Depletion of selected chemokines in MDM inflammatory CoM had no effect on monocyte transmigration across an endothelial blood–eye barrier, but selective chemokine receptor inhibition reduced monocyte recruitment significantly. Conclusions: We propose a role for inflammatory monocytes in endothelial cytotoxicity in corneal graft rejection. Therefore, targeting monocyte recruitment offers a putative novel strategy to reduce donor endothelial cell injury in survival of human corneal allografts

Effect of Corneal Cross-linking versus Standard Care on Keratoconus Progression in Young Patients: The KERALINK Randomized Controlled Trial

PURPOSE: To examine the efficacy and safety of corneal cross-linking (CXL) for stabilization of progressive keratoconus. DESIGN: Observer-masked, randomized, controlled, parallel-group superiority trial. PARTICIPANTS: Sixty participants 10 to 16 years of age with progressive keratoconus, one eye of each deemed the study eye. METHODS: The study eye was randomized to either CXL plus standard care or standard care alone, with spectacle or contact lens correction as necessary for vision. MAIN OUTCOME MEASURES: The primary outcome was steep keratometry (K2) in the study eye as a measure of the steepness of the cornea at 18 months. Secondary outcomes included keratoconus progression defined as a 1.5-diopter (D) increase in K2, visual acuity, keratoconus apex corneal thickness, and quality of life. RESULTS: Of 60 participants, 30 were randomized to CXL and standard care groups. Of these, 30 patients in the CXL group and 28 patients in the standard care group were analyzed. Mean K2 in the study eye 18 months after randomization was 49.7 D (standard deviation [SD], 3.8 D) in the CXL group and 53.4 D (SD, 5.8 D) in the standard care group. The adjusted mean difference in K2 in the study eye was -3.0 D (95% confidence interval [CI], -4.9 to -1.1 D; P = 0.002), favoring CXL. Adjusted differences between groups in uncorrected and corrected vision favored eyes receiving CXL: -0.31 logarithm of the minimum angle of resolution (logMAR; 95% CI, -0.50 to -0.11 logMAR; P = 0.002) and -0.51 logMAR (95% CI, -1.37 to 0.35 logMAR; P = 0.002). Keratoconus progression in the study eye occurred in 2 patients (7%) randomized to CXL compared with 12 patients (43%) randomized to standard care. The unadjusted odds ratio suggests that on average, patients in the CXL arm had 90% (odds ratio, 0.1; 95% CI, 0.02-0.48; P = 0.004) lower odds of experiencing progression compared with those receiving standard care. CONCLUSIONS: CXL arrests progression of keratoconus in the majority of young patients. CXL should be considered as a first-line treatment in progressive disease. If the arrest of keratoconus progression induced by CXL is sustained in longer follow-up, particular benefit may be derived from avoiding a later requirement for contact lens wear or corneal transplantation

Epithelium-off corneal cross-linking surgery compared with standard care in 10- to 16-year-olds with progressive keratoconus: the KERALINK RCT

Background: Keratoconus is a disease of the cornea affecting vision that is usually first diagnosed in the first three decades. The abnormality of corneal shape and thickness tends to progress until the patient reaches approximately 30 years of age. Epithelium-off corneal cross-linking is a procedure that has been demonstrated to be effective in randomised trials in adults and observational studies in young patients. // Objectives: The KERALINK trial examined the efficacy and safety of epithelium-off corneal cross-linking, compared with standard care by spectacle or contact lens correction, for stabilisation of progressive keratoconus. // Design: In this observer-masked, randomised, controlled, parallel-group superiority trial, 60 participants aged 10–16 years with progressive keratoconus were randomised; 58 participants completed the study. Progression was defined as a 1.5 D increase in corneal power measured by maximum or mean power (K2) in the steepest corneal meridian in the study eye, measured by corneal tomography. // Setting: Referral clinics in four UK hospitals. // Interventions: Participants were randomised to corneal cross-linking plus standard care or standard care alone, with spectacle or contact lens correction as necessary for vision, and were monitored for 18 months. // Main outcome measures: The primary outcome was K2 in the study eye as a measure of the steepness of the cornea at 18 months post randomisation. Secondary outcomes included keratoconus progression, visual acuity, keratoconus apex corneal thickness and quality of life. // Results: Of 60 participants, 30 were randomised to the corneal cross-linking and standard-care groups. Of these, 30 patients in the corneal cross-linking group and 28 patients in the standard-care group were analysed. The mean (standard deviation) K2 in the study eye at 18 months post randomisation was 49.7 D (3.8 D) in the corneal cross-linking group and 53.4 D (5.8 D) in the standard-care group. The adjusted mean difference in K2 in the study eye was –3.0 D (95% confidence interval –4.93 D to –1.08 D; p = 0.002), favouring corneal cross-linking. Uncorrected and corrected differences in logMAR vision at 18 months were better in eyes receiving corneal cross-linking: –0.31 (95% confidence interval –0.50 to –0.11; p = 0.002) and –0.30 (95% confidence interval –0.48 to –0.11; p = 0.002). Keratoconus progression in the study eye occurred in two patients (7%) randomised to corneal cross-linking compared with 12 (43%) patients randomised to standard care. The unadjusted odds ratio suggests that, on average, patients in the corneal cross-linking group had 90% (odds ratio 0.1, 95% confidence interval 0.02 to 0.48; p = 0.004) lower odds of experiencing progression than those receiving standard care. Quality-of-life outcomes were similar in both groups. No adverse events were attributable to corneal cross-linking. // Limitations: Measurements of K2 in those eyes with the most significant progression were in some cases indicated as suspect by corneal topography device software. // Conclusions: Corneal cross-linking arrests progression of keratoconus in the great majority of young patients. These data support a consideration of a change in practice, such that corneal cross-linking could be considered as first-line treatment in progressive disease. If the arrest of keratoconus progression induced by corneal cross-linking is sustained in longer follow-up, there may be particular benefit in avoiding the later requirement for contact lens wear or corneal transplantation. However, keratoconus does not continue to progress in all patients receiving standard care. For future work, the most important questions to be answered are whether or not (1) the arrest of keratoconus progression induced by corneal cross-linking is maintained in the long term and (2) the proportion of those receiving standard care who show significant progression increases with time