Incidence and Mitigation of Corneal Pseudomicrocysts Induced by Antibody-Drug Conjugates (ADCs).

Purpose of Review: This study is to highlight the incidence of corneal pseudomicrocysts in FDA-approved antibody-drug conjugates (ADCs), and success of preventive therapies for pseudomicrocysts and related ocular surface adverse events (AEs). Recent Findings: ADCs are an emerging class of selective cancer therapies that consist of a potent cytotoxin connected to a monoclonal antibody (mAb) that targets antigens expressed on malignant cells. Currently, there are 11 FDA-approved ADCs with over 164 in clinical trials. Various AEs have been attributed to ADCs, including ocular surface AEs (keratitis/keratopathy, dry eye, conjunctivitis, blurred vision, corneal pseudomicrocysts). While the severity and prevalence of ADC-induced ocular surface AEs are well reported, the reporting of corneal pseudomicrocysts is limited, complicating the development of therapies to prevent or treat ADC-related ocular surface toxicity. Summary: Three of 11 FDA-approved ADCs have been implicated with corneal pseudomicrocysts, with incidence ranging from 41 to 100% of patients. Of the six ADCs that reported ocular surface AEs, only three had ocular substudies to investigate the benefit of preventive therapies including topical steroids, vasoconstrictors, and preservative-free lubricants. Current preventive therapies demonstrate limited efficacy at mitigating pseudomicrocysts and other ocular surface AEs.

Characterization of Infectious Keratitis in Opioid Users in a County Hospital Setting.

Purpose: To determine risk factors and clinical course of corneal ulcers in the setting of opioid use. Methods: A retrospective cohort study was performed of patients presenting with bacterial or fungal keratitis at a county hospital from 2010-2021. Subjects were separated into three groups: opioid drug users (heroin, methadone, fentanyl), non-opioid drug users, and non-drug users. 24 opioid users, 77 non-opioid drug users, and 38 non-drug users were included in the study. Chi-square and t-tests were used to compare hospitalization for corneal ulcer treatment; length of hospitalization; loss to follow-up; final best corrected visual acuity (BCVA); medication noncompliance; time to ulcer resolution; and visual disability (defined either by the legal limit for driving in California or the federal limit for blindness). Results: Opioid users had higher rates of unemployment (p=0.002), homelessness (p=0.018), and psychiatric conditions (p=0.024) compared with non-opioid and non-drug users. They had more severe presentations, with worse initial BCVA of the affected eye (p=0.003), larger ulcer size (p=0.023), and higher rates of individuals below the legal vision thresholds for driving (p=0.009) and blindness (p=0.033) at initial presentation. Opioid use was associated with increased rate of hospitalization (p<0.001), higher fortified antibiotic use (p=0.009), worse final BCVA of the affected eye (p=0.020), and increased rates of BCVA worse than the legal vision thresholds for driving (p=0.043) and blindness (p<0.001) on final presentation. Conclusions: Infectious keratitis associated with opioid use is associated with more severe presentations and poorer outcomes, including higher rates of visual disability.

Novel Mechanisms Guide Innovative Molecular-Based Therapeutic Strategies for Fuchs Endothelial Corneal Dystrophy.

ABSTRACT: Major advances in genomics have dramatically increased our understanding of Fuchs endothelial corneal dystrophy (FECD) and identified diverse genetic causes and associations. Biomarkers derived from these studies have the potential to inform both clinical treatment and yield novel therapeutics for this corneal dystrophy.

Risk Factors Predicting Loss to Follow-Up, Medication Noncompliance, and Poor Visual Outcomes Among Patients With Infectious Keratitis at a Public County Hospital.

PURPOSE: Infectious keratitis is a vision-threatening condition requiring close follow-up and disciplined eye drop administration to achieve resolution. Although patients presenting to county hospitals often have more severe presentations, there is a paucity of risk and outcomes data in this setting. This study investigates risk factors predicting loss to follow-up (LTFU), medication noncompliance, and poor outcomes for infectious keratitis in the county hospital setting. METHODS: This was a retrospective case-control study at Zuckerberg San Francisco General Hospital and Trauma Center. Inclusion criteria were patients who had corneal cultures for suspected infectious bacterial or fungal keratitis between 2010 and 2021. Exclusion criteria were patients with viral keratitis only. Multivariable logistic regression was used to analyze the relationship of social and medical risk factors with LTFU, medication noncompliance, worsened visual acuity (VA), and delayed resolution time. RESULTS: Of 174 patients with infectious keratitis in this analysis, 69 (40.0%) had LTFU. Unemployment was associated with increased risk of LTFU (odds ratio 2.58, P = 0.049) and worse final VA ( P = 0.001). Noncompliance trended toward an association with homelessness (odds ratio 3.48, P = 0.095). Increasing age correlated with longer resolution time, with each 1-year increase associated with delayed resolution by 0.549 days ( P = 0.042). CONCLUSIONS: Patients experiencing unemployment, homelessness, or increased age demonstrate higher risk for treatment barriers including loss to follow-up and medication noncompliance, resulting in worse VA and delayed time to resolution. These risk factors should be considered when determining the need for more deliberate follow-up measures in patients with infectious keratitis.

Characterization of Polymicrobial and Antibiotic-Resistant Infectious Keratitis in a County Hospital Setting.

Purpose: Infectious keratitis is a serious cause of visual impairment, particularly in low-income communities. This study examines the associations between social risk factors and polymicrobial keratitis, multidrug resistance, pathogen spectrum, and outcomes at a county hospital. Methods: We performed a retrospective study of Zuckerberg San Francisco General Hospital patients treated for infectious keratitis from 2010-2021. Multivariable regression was performed to analyze the relationships between social, medical, and psychiatric risk factors with polymicrobial growth, multidrug resistance, and clinical outcomes. Results: Of 174 patients with infectious keratitis, 44 (25%) had polymicrobial growth. Six patients (14%) with polymicrobial growth had multidrug-resistant organisms. Homeless patients were more likely to present with polymicrobial infection (OR 3.4, p = 0.023), and polymicrobial infections were associated with multidrug-resistant organisms (p = 0.018). Smoking, drug use, HIV positivity, prior corneal pathology, and contact lens use were not associated with an increased risk of polymicrobial infection. Eleven patients (6.3%) were started on topical antibiotics prior to presentation; of these, none developed polymicrobial infections or multidrug-resistant organisms. Polymicrobial infections increased the likelihood to initiation of fortified antibiotics (OR 2.9, p = 0.011) but did not impact ulcer size, final visual acuity, time to resolution, or likelihood of emergent procedures. Conclusions: Homelessness correlates with an increased risk of polymicrobial keratitis and subsequent multidrug resistance, supporting initiation of broad antibiotic coverage in this population. Prior topical antibiotics did not increase risk of polymicrobial infection. Polymicrobial infection did not significantly worsen clinical outcomes.

Disrupting autorepression circuitry generates "open-loop lethality" to yield escape-resistant antiviral agents.

Across biological scales, gene-regulatory networks employ autorepression (negative feedback) to maintain homeostasis and minimize failure from aberrant expression. Here, we present a proof of concept that disrupting transcriptional negative feedback dysregulates viral gene expression to therapeutically inhibit replication and confers a high evolutionary barrier to resistance. We find that nucleic-acid decoys mimicking cis-regulatory sites act as "feedback disruptors," break homeostasis, and increase viral transcription factors to cytotoxic levels (termed "open-loop lethality"). Feedback disruptors against herpesviruses reduced viral replication >2-logs without activating innate immunity, showed sub-nM IC50, synergized with standard-of-care antivirals, and inhibited virus replication in mice. In contrast to approved antivirals where resistance rapidly emerged, no feedback-disruptor escape mutants evolved in long-term cultures. For SARS-CoV-2, disruption of a putative feedback circuit also generated open-loop lethality, reducing viral titers by >1-log. These results demonstrate that generating open-loop lethality, via negative-feedback disruption, may yield a class of antimicrobials with a high genetic barrier to resistance.

Characterization of Recruited Mononuclear Phagocytes following Corneal Chemical Injury.

Mononuclear phagocytes (MP) have central importance in innate immunity, inflammation, and fibrosis. Recruited MPs, such as macrophages, are plastic cells and can switch from an inflammatory to a restorative phenotype during the healing process. However, the role of the MPs in corneal wound healing is not completely understood. The purpose of this study is to characterize the kinetics of recruited MPs and evaluate the role of macrophage metalloelastase (MMP12) in the healing process, using an in vivo corneal chemical injury model. Unwounded and wounded corneas of wild-type (WT) and Mmp12-/- mice were collected at 1, 3, and 6 days after chemical injury and processed for flow cytometry analysis. Corneal MP phenotype significantly changed over time with recruited Ly6Chigh (proinflammatory) cells being most abundant at 1 day post-injury. Ly6Cint cells were highly expressed at 3 days post-injury and Ly6Cneg (patrolling) cells became the predominant cell type at 6 days post-injury. CD11c+ dendritic cells were abundant in corneas from Mmp12-/- mice at 6 days post-injury. These findings show the temporal phenotypic plasticity of recruited MPs and provide valuable insight into the role of the MPs in the corneal repair response, which may help guide the future development of MP-targeted therapies.

Spontaneous hyphema in the setting of COVID-19 pneumonia.

PURPOSE: To report a challenging case of spontaneous hyphema in the setting of prone positioning for COVID-19 pneumonia. OBSERVATIONS: A previously healthy patient was concomitantly diagnosed with acute myelogenous leukemia (AML) and COVID-19 infection. During his hospitalization he required intubation and prone positioning. Following change from prone to supine positioning, he was noted to have developed a large unilateral spontaneous hyphema. CONCLUSIONS AND IMPORTANCE: We present a challenging case of spontaneous hyphema due to a hematologic malignancy in the setting of prone positioning for COVID-19 pneumonia.

Corneal Repair Models in Mice: Epithelial/Mechanical Versus Stromal/Chemical Injuries.

The tissue response to injury is a complex process. The cornea is an excellent model for studying wound repair processes because of its simple anatomy, easy accessibility, and normal avascular state. Here, we describe two corneal repair models in mice: an epithelial/mechanical injury model and a stromal/chemical injury model. The two models induce different repair responses, and consequently enable the study of independent repair processes. Here, we describe how these two wound models may be used to study basic cellular and molecular mechanisms of corneal repair.

Author Correction: Aberrant DNA methylation of miRNAs in Fuchs endothelial corneal dystrophy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Aberrant DNA methylation of miRNAs in Fuchs endothelial corneal dystrophy.

Homeostatic maintenance of corneal endothelial cells is essential for maintenance of corneal deturgescence and transparency. In Fuchs endothelial corneal dystrophy (FECD), an accelerated loss and dysfunction of endothelial cells leads to progressively severe visual impairment. An abnormal accumulation of extracellular matrix (ECM) is a distinctive hallmark of the disease, however the molecular pathogenic mechanisms underlying this phenomenon are not fully understood. Here, we investigate genome-wide and sequence-specific DNA methylation changes of miRNA genes in corneal endothelial samples from FECD patients. We discover that miRNA gene promoters are frequent targets of aberrant DNA methylation in FECD. More specifically, miR-199B is extensively hypermethylated and its mature transcript miR-199b-5p was previously found to be almost completely silenced in FECD. Furthermore, we find that miR-199b-5p directly and negatively regulates Snai1 and ZEB1, two zinc finger transcription factors that lead to increased ECM deposition in FECD. Taken together, these findings suggest a novel epigenetic regulatory mechanism of matrix protein production by corneal endothelial cells in which miR-199B hypermethylation leads to miR-199b-5p downregulation and thereby the increased expression of its target genes, including Snai1 and ZEB1. Our results support miR-199b-5p as a potential therapeutic target to prevent or slow down the progression of FECD disease.

MMP12 Inhibits Corneal Neovascularization and Inflammation through Regulation of CCL2.

Following corneal injury, coordinated cellular and protein interactions occur at the wound site to restore tissue homeostasis. Regulation of this response is required to prevent the development of chronic inflammation, abnormal neovascularization, and fibrosis. The chemokine CCL2 and its primary receptor CCR2 are key regulators of the inflammatory and neovascular responses to injury. In this study, we investigated the role of macrophage-associated matrix metalloproteinase 12 (MMP12) in the regulation of CCL2 and CCR2 after corneal wounding. Using two corneal injury models, we examined the temporal and spatial expression of CCL2 and CCR2 in Mmp12-/- and wild-type (WT) mice. Our data showed that MMP12 downregulated CCL2 and CCR2 expression in a manner dependent on the timing and mechanism of injury. We also examined the effect of CCL2 on the injury response in Mmp12-/- and WT corneas. We found that macrophage infiltration and neovascularization following CCL2 blockade was significantly reduced in Mmp12-/- corneas as compared with WT corneas. These findings indicate that MMP12 inhibits corneal inflammation and neovascularization after injury through its regulation of CCL2.

Overexpression of MMPs in Corneas Requiring Penetrating and Deep Anterior Lamellar Keratoplasty.

Purpose: Matrix metalloproteinases (MMPs) comprise a family of zinc-dependent endopeptidases involved in wound healing processes, including neovascularization and fibrosis. We assessed MMP protein expression levels in diseased corneas of patients requiring penetrating and deep anterior lamellar keratoplasty. The purpose of this study was to test the hypothesis that upregulation of MMPs in diseased corneas is positively associated with clinical levels of corneal neovascularization and fibrosis. Methods: Protein expression levels of nine individual MMPs were quantified simultaneously in human corneal lysates by using the Bio-Plex Pro Human MMP 9-Plex Panel and the MAGPIX technology. Measurements of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP12, and MMP13 were performed on diseased specimens from 21 patients undergoing corneal transplantation (17 for penetrating keratoplasty and 4 for deep anterior lamellar keratoplasty) and 6 normal control corneas. Results: Luminex-based expression analysis revealed a significant overexpression of four of the nine MMPs tested (MMP2, MMP8, MMP12, and MMP13) in patient samples compared to control. Significant overexpression of MMP1, MMP2, MMP8, MMP12, and MMP13 was observed in diseased corneas with neovascularization compared with diseased corneas without neovascularization. Overexpression of MMP1, MMP2, MMP8, MMP12, and MMP13 also corresponded with the levels of corneal fibrosis. Finally, reduced expression of MMP3 was detected in keratoconus patients. Conclusions: Multiple MMPs are expressed in the corneas of patients with chronic disease requiring keratoplasty even when the pathologic process appears to be clinically inactive. In particular, the expression of several MMPs (MMP2, MMP8, MMP12, and MMP13) is positively associated with increased levels corneal fibrosis and neovascularization.

Epidemiology of uveitis in a US population-based study.

BACKGROUND: The purpose of this study is to assess the relationship between self-reported uveitis and purported demographic and clinical risk factors, using an American adult population extracted from the National Health and Nutrition Examination Survey (NHANES) for the years 2009 and 2010. This is a cross-sectional, population-based study using a sample of 5106 subjects between 20 and 69 years old. The main outcome for our study was the self-report of a diagnosis of uveitis. The demographic analysis included age, gender, and ethnicity. Potential predictors were having a diagnosis of ankylosing spondylitis (AS), ulcerative colitis (UC), or Crohn's disease (CD); a history of cigarette smoking; vitamin D deficiency; and different mental health measures. Univariate and multivariate analyses were conducted using RStudio. RESULTS: Of the 5106 participants, 27 had reported a diagnosis of uveitis, showing an adjusted prevalence of 5.4 per 1000 subjects (95% CI 3.4-8.5/1000). Increased age was associated with higher uveitis prevalence in the multivariate analysis (odds ratio [OR] = 1.04, 95% CI 1.01-1.07; p = 0.02). Positive smoking history was reported in 59.2% of patients. Multivariate analysis comparing smoking with the presence of uveitis showed an OR of 3.18 (95% CI 1.59-6.37; p = 0.003), adjusting for age and gender. Moreover, 11.1% of the participants from the uveitis group self-reported a diagnosis of AS and 11.7% informed a diagnosis of UC and 7.1% of CD. The ORs were of 16.64 (95% CI 3.64-76.09; p = 0.001), 11.34 (95% CI 2.69-47.88; p = 0.003), and 22.16 (95% CI 2.64-186.17; p = 0.007), respectively when compared with the non-uveitis group in the multivariate analysis. CONCLUSIONS: Age, cigarette smoking, AS, UC, and CD are positively associated with self-reported uveitis. There is previous evidence that smoking and female gender are positive risk factors for uveitis, as well as evidence that HLA-B27-positive spondyloarthritides have the highest association with non-infectious uveitis in the adult population in North America and Europe. However, there are no prior studies that have utilized a representative US population-based sample to validate these findings. The present study supports smoking as a risk factor, which has clinical relevance since this is a modifiable habit.

Comprehensive characterization of DNA methylation changes in Fuchs endothelial corneal dystrophy.

Transparency of the human cornea is necessary for vision. Fuchs Endothelial Corneal Dystrophy (FECD) is a bilateral, heritable degeneration of the corneal endothelium, and a leading indication for corneal transplantation in developed countries. While the early onset, and rarer, form of FECD has been linked to COL8A2 mutations, the more common, late onset form of FECD has genetic mutations linked to only a minority of cases. Epigenetic modifications that occur in FECD are unknown. Here, we report on and compare the DNA methylation landscape of normal human corneal endothelial (CE) tissue and CE from FECD patients using the Illumina Infinium HumanMethylation450 (HM450) DNA methylation array. We show that DNA methylation profiles are distinct between control and FECD samples. Differentially methylated probes (10,961) were identified in the FECD samples compared with the control samples, with the majority of probes being hypermethylated in the FECD samples. Genes containing differentially methylated sites were disproportionately annotated to ontological categories involving cytoskeletal organization, ion transport, hematopoetic cell differentiation, and cellular metabolism. Our results suggest that altered DNA methylation patterns may contribute to loss of corneal transparency in FECD through a global accumulation of sporadic DNA methylation changes in genes critical to basic CE biological processes.

Effects of MMP12 on cell motility and inflammation during corneal epithelial repair.

Corneal epithelial defects are a common cause of ocular morbidity and can result in corneal scarring if they do not heal properly. Matrix metalloproteinases (MMPs) are extracellular matrix proteinases that regulate multiple aspects of corneal repair. We have previously shown that MMP12 has a protective effect on corneal fibrosis through its regulation of neutrophil and macrophage infiltration and angiogenesis in a chemical injury model involving full thickness damage to the cornea. However, the role of MMP12 in injuries limited to the corneal epithelium is relatively unknown. This study investigates the reparative effects of MMP12 following isolated corneal epithelial injury. Using a corneal epithelial debridement injury model performed on corneas of wild-type (WT) mice, we show that Mmp12 is expressed early following corneal epithelial injury with highest expression levels at 8 h after injury and lower expression levels at 4 and 8 days after injury. We investigated whether MMP12 has an effect on the rate of epithelial repair and cell migration using in vivo and in vitro scratch assays performed on WT and Mmp12-/- mice. We found that loss of MMP12 results in a slower scratch wound repair rate both in vivo and in vitro. We also found that corneas of Mmp12-/- mice have decreased neutrophil infiltration following injury. Loss of MMP12, however, does not affect cell proliferation in the center of the wounds. These data support a role of MMP12 in promoting early repair processes following corneal epithelial injury by enhancing epithelial cell migration and neutrophil infiltration.

sFlt Multivalent Conjugates Inhibit Angiogenesis and Improve Half-Life In Vivo.

Current anti-VEGF drugs for patients with diabetic retinopathy suffer from short residence time in the vitreous of the eye. In order to maintain biologically effective doses of drug for inhibiting retinal neovascularization, patients are required to receive regular monthly injections of drug, which often results in low patient compliance and progression of the disease. To improve the intravitreal residence time of anti-VEGF drugs, we have synthesized multivalent bioconjugates of an anti-VEGF protein, soluble fms-like tyrosine kinase-1 (sFlt) that is covalently grafted to chains of hyaluronic acid (HyA), conjugates that are termed mvsFlt. Using a mouse corneal angiogenesis assay, we demonstrate that covalent conjugation to HyA chains does not decrease the bioactivity of sFlt and that mvsFlt is equivalent to sFlt at inhibiting corneal angiogenesis. In a rat vitreous model, we observed that mvsFlt had significantly increased intravitreal residence time compared to the unconjugated sFlt after 2 days. The calculated intravitreal half-lives for sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, we show that mvsFlt is more effective than the unconjugated form at inhibiting retinal neovascularization in an oxygen-induced retinopathy model, an effect that is most likely due to the longer half-life of mvsFlt in the vitreous. Taken together, our results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF and this conjugation significantly improves drug half-life. These in vivo results suggest that our strategy of multivalent conjugation could substantially improve upon drug half-life, and thus the efficacy of currently available drugs that are used in diseases such as diabetic retinopathy, thereby improving patient quality of life.

Metalloproteinases: a Functional Pathway for Myeloid Cells.

Myeloid cells have diverse roles in regulating immunity, inflammation, and extracellular matrix turnover. To accomplish these tasks, myeloid cells carry an arsenal of metalloproteinases, which include the matrix metalloproteinases and the adamalysins. These enzymes have diverse substrate repertoires, and are thus involved in mediating proteolytic cascades, cell migration, and cell signaling. Dysregulation of metalloproteinases contributes to pathogenic processes, including inflammation, fibrosis, and cancer. Metalloproteinases also have important nonproteolytic functions in controlling cytoskeletal dynamics during macrophage fusion and enhancing transcription to promote antiviral immunity. This review highlights the diverse contributions of metalloproteinases to myeloid cell functions.