Purpose:The purpose of this study was to determine whether neutrophil extracellular trap (NET)-related markers are elevated in the aqueous humor of patients with glaucoma compared with healthy controls. Methods:We performed a case-control study of patients scheduled to undergo cataract extraction and/or glaucoma surgery from 2020 to 2022. Results:One hundred seventy-seven eyes were included: 127 were healthy controls, 43 were patients with glaucoma, and 7 were patients with uveitis. Pro-inflammatory markers were elevated in glaucomatous aqueous humor as compared with controls including: interleukin-1β (IL-1β; 1.31 pg/mL, interquartile range [IQR] = 1.87 vs. 1.12 pg/mL, IQR = 0.55, P < 0.0001), interferon gamma-inducible protein 10 (IP-10; 61.4 pg/mL, IQR = 106 vs. 25.9 pg/mL, IQR = 36.4, P = 0.0002), IL-8 (5.46 pg/mL, IQR = 5.10 vs. 2.52 pg/mL, IQR = 2.16, P < 0.0001), neutrophil gelatinase-associated lipocalin (NGAL; 2760 pg/mL, IQR = 4450 vs. 1690 pg/mL, IQR = 1370, P < 0.0001), and eDNA (0.0324 µg/mL, IQR = 0.0400 vs. 0.025 µg/mL, IQR = 0.0120, P < 0.0001). Furthermore, the glaucoma group had higher levels of IgG (3470 ng/mL, IQR = 4130 vs. 2370 ng/mL, IQR = 1800, P < 0.0001), IgA (188 ng/mL, IQR = 315 vs. 84.9 ng/mL, IQR = 198, P = 0.001), and IgM (3.45 ng/mL, IQR = 2.32 vs. 3.00 ng/mL, IQR = 0.880, P = 0.033) within the aqueous humor than the controls. Conclusions:Multiple levels of the inflammatory pathway are implicated in glaucoma, including inflammatory cytokines (IL-1β), T-cell mediators (IP-10), neutrophil chemoattractant (IL-8), NET-associated molecules (NGAL and eDNA), and the adaptive immune response (immunoglobulins IgM, IgG, and IgA). Findings support further investigation of immunomodulatory approaches to treatment of glaucoma. Translational Relevance:This study looks at the basic inflammatory pathways and biomarkers relevant to glaucoma.

Background/Objectives: Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in cardiovascular disease, particularly through the transfer of regulatory microRNAs. However, direct evidence of cytosolic microRNA delivery by mesenchymal stem cell (MSC)-derived EVs into cardiomyocytes remains limited. Many prior studies have inferred transfer without fully excluding endogenous microRNA induction or surface-bound artifacts. The objective of this study was to determine whether MSC-derived EV microRNA undergoes functional cytoplasmic delivery into cardiomyocytes and to identify the endocytic pathways involved. Methods: To eliminate background from endogenous microRNA expression, MSC-derived EVs were loaded with the exogenous microRNA cel-miR-39-3p. H9C2 cardiomyocytes were treated with increasing vesicle doses, and intracellular microRNA levels were quantified using real-time polymerase chain reactions. Cytosolic accessibility was assessed indirectly using a cholesterol-modified antagomir targeting cel-miR-39-3p. Pharmacologic inhibitors of macropinocytosis (5-(N-ethyl-N-isopropyl) amiloride (EIPA)) and clathrin-mediated endocytosis (chlorpromazine and ES9-17) were applied to delineate uptake pathways, with transferrin assays confirming clathrin inhibition. Results: EV treatment produced a dose-dependent increase in intracellular cel-miR-39-3p levels. Antagomir administration reduced intracellular microRNA levels by 89%, providing indirect functional evidence that transferred cel-miR-39-3p reached an antagomir-accessible cytosolic compartment. At the highest preparation dose, EIPA and ES9-17 reduced cell-associated cel-miR-39-3p by 71% and 36%, respectively, consistent with contributions from EIPA-sensitive and clathrin-associated uptake processes. Notably, chlorpromazine increased microRNA accumulation despite blocking transferrin uptake, suggesting compensatory or membrane curvature-dependent effects. Conclusions: These findings provide strong functional evidence that exogenous cel-miR-39-3p associated with MSC EV preparations reaches an antagomir-accessible cytoplasmic compartment in H9C2 cardiomyocytes and implicate EIPA-sensitive uptake consistent with macropinocytosis and clathrin-associated uptake. Additional genetic, localization, and pulse-chase studies are required to establish the precise intracellular trafficking mechanisms. This work strengthens the mechanistic foundation for EV-based therapies and informs the rational development of cell-free strategies for cardiovascular disease.

Lysosomal dysfunction and elevated lysosomal pH are hallmark features of age-related neurodegenerative diseases including age-related macular degeneration (AMD), Alzheimer's disease (AD), and Parkinson's disease (PD). Restoring lysosomal acidity is important for maintaining enzymatic degradation, preventing protein aggregation, and reducing cellular waste accumulation in degenerating tissues. Acidic nanoparticles represent a promising therapeutic strategy to normalize lysosomal pH; however, accurate monitoring of their delivery, retention, and dosage is critical for rigorous evaluation. To address this, we developed fluorescently labeled poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles conjugated with Cyanine3 amine (Cy3). Nanoparticle uptake was systematically optimized, achieving over 90% delivery to lysosomes of induced pluripotent stem cell-derived retinal pigment epithelial (iPS-RPE) cells, although uptake rates varied among adjacent cells. Once internalized, nanoparticles demonstrated remarkable stability, with no detectable change in concentration, distribution, or size for at least 28 days. iPS-RPE cells exhibited higher nanoparticle internalization compared with the ARPE-19 cell line and optic nerve head astrocytes. The capacity of the nanoparticles to restore function to stressed lysosomes was confirmed by their ability to reacidify lysosomes, restore cathepsin B activity, and increase the levels of active cathepsin D. The nanoparticles also reduced the levels of LC3II in astrocytes treated with chloroquine, indicating that they can also restore autophagy rates. In summary, this study demonstrates the value of Cy3 labeling for enhanced nanoparticle tracking to lysosomes. The findings also identify PLGA nanoparticles as powerful tools for restoring degradative lysosomal function and autophagy in cells undergoing lysosomal stress. NEW & NOTEWORTHY Tools that restore acidic pH in compromised lysosomes can enhance autophagy and waste clearance in degenerative disorders characterized by excessive accumulation. Here, we describe the synthesis of lysosome-targeted nanoparticles composed of poly(d,l-lactide-co-glycolide) (PLGA) polymers covalently bound to the fluorescent dye Cyanine3 amine (Cy3). These Cy3-PLGA nanoparticles enable precise tracking of lysosomal delivery and demonstrate sustained long-term retention within lysosomes, supporting their potential for future applications aimed at restoring lysosomal pH in aging and degenerating diseases.

Introduction: Tear-deficient dry eye disease (DED), including Sjögren’s syndrome and ocular graft-versus-host disease (oGVHD), is often refractory to therapy. We tested whether platelet-derived growth factor (PDGF) isoform imbalance, quantified as a PDGF-AB/BB: PDGF-AA ratio (“PDGF Ratio”), is associated with innate activation and corneal epitheliopathy. Methods: Tear samples from 151 participants were profiled across two independent assay runs: discovery (n = 79) and replication (n = 72). Data were harmonized as within-plate rank percentiles. Plate-adjusted mediation tested whether the PDGF Ratio mediated associations between tear-deficient status and an innate NF-κB composite (IL-18/IL-6/IL-8/TNF-α) and National Eye Institute (NEI) scale graded corneal staining. A parallel model for staining included Schirmer scores to distinguish trophic from aqueous volume deficiency. Results: The PDGF Ratio differed significantly across diagnostic groups (Kruskal-Wallis p < 0.001), driven by concurrent PDGF-AA depletion (median rank 0.69 healthy vs. 0.19 oGVHD) and PDGF-AB/BB enrichment (0.18 vs. 0.62; both p < 0.001). High ratio co-occurred with higher innate cytokine ranks and lower EGF and IFN-γ. In pooled mediation, the ratio accounted for 62.8% of the tear-deficient association with innate inflammation (indirect 0.168; 95% CI 0.118–0.225) and 34.2% of the association with corneal staining (indirect 0.117; 95% CI 0.055–0.190). The staining pathway remained significant after accounting for Schirmer (indirect 0.113; 95% CI 0.050–0.185). Conclusion: Tear-deficient DED exhibits a reproducible PDGF isoform imbalance that statistically accounts for substantial portions of innate activation and corneal staining. These findings support the PDGF Ratio as a coherent candidate biomarker and therapeutic axis for isoform-selective rebalancing.

PURPOSE:Antibody-drug conjugate (ADC)-associated ocular toxicity is an emerging, sometimes dose-limiting complication. Macropinocytosis has been implicated in off-target ADC uptake by corneal epithelial cells. This study evaluated whether ocular surface immunoglobulin (OSIG) inhibit macropinocytosis in human corneal epithelial cells (HCE-T) and characterized ocular biodistribution and tolerability following topical and subconjunctival administration. METHODS:Macropinocytosis was quantified in corneal epithelial cells after exposure to vehicle or increasing concentrations of OSIG. Ocular pharmacokinetics were evaluated in New Zealand White rabbits following single topical instillation or subconjunctival injection of 5% OSIG. OSIG concentrations were quantified in ocular tissues, ocular fluids, tears, serum, and submandibular lymph nodes using enzyme-linked immunosorbent assay (ELISA). Local tolerability was assessed after single and repeated subconjunctival injections using clinical observations, Draize scoring, and ophthalmic examinations. RESULTS:OSIG inhibited Dextran Texas Red uptake in a concentration-dependent manner, with near-complete inhibition at 5%. Topical OSIG produced only transient conjunctival exposure and did not achieve sustained corneal concentrations. In contrast, subconjunctival administration resulted in sustained ocular surface exposure, with peak corneal concentration of 11,200.8 ng/g on Day 14 and measurable conjunctival exposure through 28 days. Serum exposure was detectable but lower than ocular surface tissue exposure. Subconjunctival OSIG was generally well tolerated after single and repeated dosing, with findings limited mainly to transient conjunctival redness and chemosis. Two clinical cases of ADC-associated ocular toxicity treated with topical OSIG were also reviewed. CONCLUSIONS:OSIG inhibits macropinocytosis in HCE-T and achieves sustained corneal and conjunctival exposure following subconjunctival administration. These findings support further evaluation of subconjunctival OSIG as a local strategy to mitigate ADC-associated corneal toxicity.

Introduction: Corneal fluorescein (FL) staining is the most widely used efficacy endpoint in dry eye disease (DED) registrational trials, yet dye diffusion and variable readout timing can blur punctate staining, reducing the precision and consistency of grading. We assessed the performance of FL staining as an efficacy endpoint by analyzing endpoint success across FDA-reviewed DED trials and evaluated whether a standardized, diffusion-resistant dye could provide an alternative quantitative measure of corneal staining. Methods: We conducted a synthesis of FDA-reviewed DED registrational clinical trials through April 2025 to determine the proportion of trials that met the corneal FL staining efficacy endpoint. Clinical relevance was assessed using a minimal clinically important difference (MCID) of ≥3 units on the 0–15 National Eye Institute (NEI) scale. In a clinical cohort of 50 DED patients (97 eyes), total corneal staining was compared after sequential instillation of 5 µL of 2% FL (readout ∼2.5 min) and 1% lissamine green (LG) (readout ≤30 s). Results: Among 20 FDA-reviewed registrational trials that prespecified corneal FL staining as an efficacy endpoint, 10 (50%) failed to meet this endpoint. Of the seven trials in which FL staining served as a primary or co-primary endpoint, only one achieved MCID for corneal staining. Across all trials meeting the endpoint, treatment-vehicle differences were small (mean additional reduction 0.4–1.2 units; Cohen’s d < 0.5), indicating that FL staining yielded uniformly small effect sizes. In the clinical cohort, 1% LG produced corneal staining that was strongly associated with 2% FL for mild (ρ = 0.99), moderate (ρ = 0.93), and severe (ρ = 0.69) disease (p < 0.001). Bland-Altman analysis showed that while the mean bias was negligible, the width of the 95% limits of agreement (−1.26 to 0.85) suggests the two corneal staining dyes are not fully interchangeable at the individual-measurement level. Conclusions: Corneal FL staining demonstrates limited responsiveness as an efficacy endpoint in DED registrational trials. Because 1% LG provides corneal staining strongly associated with 2% FL while preserving discrete punctate detail and allowing standardized, early readouts, it may represent a suitable alternative vital dye for future DED efficacy and safety assessments.

Laminin N-terminus α31 (LaNt α31) is a netrin-like protein generated by alternative splicing of the laminin α3 gene. While previously shown to regulate vascular permeability in vivo, its role in epithelial tissues remains less defined. Here, we demonstrate that LaNt α31 modulates epithelial cell behavior by altering laminin 332 (LM332) organization and hemidesmosome (HD) maturation. Adenoviral driven overexpression of LaNt α31 in corneal epithelial cells led to premature HD assembly, marked by enhanced recruitment of collagen XVII and BPAG1e to β4 integrin, and reduced cell migration. LaNt α31 expression reorganized LM332 from diffuse arcs into tight clusters and co-localized with LMβ3 during matrix deposition. Notably, phenotypes were rescued by precoated extracellular matrix, indicating a matrix-dependent mechanism. Furthermore, LaNt α31 increased matrix metalloproteinase (MMP) activity and LMα3 proteolytic processing, both essential for its effects, as MMP inhibition reversed LM clustering and HD maturation. These findings identify LaNt α31 as a regulator of epithelial homeostasis through modulation of LM332 architecture and cell-matrix adhesion.

Background: Stomatitis is a common adverse event associated with targeted therapies for hormone receptor-positive, HER2-negative (HR+/HER2–) breast cancer, particularly those inhibiting the PI3K/AKT/mTOR pathway. While mTOR-inhibitor-associated stomatitis is well established, less is known about its occurrence with other kinase inhibitors in real-world settings. We performed a pharmacovigilance disproportionality analysis of the FDA Adverse Event Reporting System (FAERS) to evaluate stomatitis reports for alpelisib, capivasertib, everolimus, and palbociclib. Methods: Events were identified using four term sets—Stomatitis, Original Trial Terms (OTT), Comprehensive Trial Terms (CTT), and Stomatitis-Associated Main Terms (SAMT)—which reflect varying definitions and medical terminologies. Disproportionality analyses using reporting odds ratio (ROR), proportional reporting ratio (PRR), and Information Component (IC) were calculated with 95% confidence intervals. Results: All agents showed ROR and PRR >1, indicating higher odds and reporting proportions of stomatitis compared with other drugs. These findings were confirmed by IC analysis. Everolimus demonstrated the strongest association (ROR: 30.72 [29.61–31.88]), followed by alpelisib (ROR: 13.11 [11.79–14.58]) and palbociclib (ROR: 11.73 [11.35–12.11]). Capivasertib had the lowest reporting odds (ROR: 3.14 [1.81–5.43]), though limited by fewer reports. Differences between CTT and SAMT were minimal (~2%). Conclusions: These results support the use of the SAMT as an efficient screening tool. Furthermore, these findings underscore the need for optimized stomatitis detection and continued monitoring, particularly for PI3K and mTOR inhibitors, in both clinical trials and postmarketing surveillance.

Lysosomal pH is frequently elevated in age-dependent neurodegenerations like Age-related Macular Degeneration (AMD), Alzheimer's Disease (AD), and Parkinson's Disease (PD). Tools that restore lysosomal pH to an optimal acidic range could enhance enzymatic degradation and reduce waste accumulation. Acidic nanoparticles offer a promising strategy for restoring lysosomal function, but accurate tracking of organelle delivery and long-term retention is needed to optimize dosage. To improve detection and enhance delivery, nanoparticles were synthesized from Poly(D,L-lactide-co-glycolide) (PLGA) polymers covalently linked to the fluorescent Cyanine3 amine (Cy3) probe. Nanoparticle concentration and loading times were optimized to achieve >90% delivery to lysosomes in cultured induced pluripotent stem cell-derived retinal pigment epithelial (iPS-RPE) cells. Uptake was heterogeneous, varying between adjacent cells. Once loaded into lysosomes, the nanoparticles were stably retained, with no detectable changes in concentration, distribution, or size for at least 28 days. iPS-RPE cells internalized more nanoparticles than the ARPE-19 cell line or mouse optic nerve head astrocyte cultures. Functionally, PLGA nanoparticles restored an acidic pH and cathepsin D levels in compromised lysosomes. In summary, Cy3-PLGA nanoparticles enabled improved tracking and long-term delivery to lysosomes, supporting future in vivo applications to restore lysosomal pH in aging and degenerating tissues.

Purpose Exposure to particulate matter (PM) and air pollution has been implicated in the etiology of ocular surface diseases (OSD). The purpose of this systematic review is to evaluate and synthesize peer-reviewed literature on the impact of PM exposure on the ocular surface, integrating results from preclinical in vitro and in vivo studies with clinical findings to provide a comprehensive understanding of molecular mechanisms, physiological effects, clinical implications, and potential therapies to target acute and chronic PM-induced ocular toxicity. Methods A systematic literature search was performed using PubMed and EMBASE over the period from 2009 to 2024 following the recommendations for the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines. 102 studies were identified that met the inclusion/exclusion criteria. All studies were assessed for the risk of bias and qualitative data were analyzed. Results Preclinical studies using models of corneal and conjunctival cells found that exposure to PM and similar air pollutants resulted in apoptosis, primarily via inflammatory and oxidative stress pathways as well as allergic and immune responses. Animal models resulted in phenotypes reminiscent of that of dry eye disease, presenting with reduced tear volumes and ocular surface damage. These results were corroborated by clinical studies, which reported that patients commonly presented with symptoms of itching, burning, and irritation, and ocular surface signs correlated with a diagnosis of dry eye disease, conjunctivitis, and allergic eye disease. Conclusions This systematic review provides a comprehensive summary of our current understanding of PM exposure on the ocular surface, highlighting the correlation between exposure to PM and ocular surface dysfunction.

IntroductionThis study investigated the efficacy of pooled human immune globulins (Flebogamma® DIF) to combat the formation of neutrophil extracellular traps (NETs) and NETosis, along with neutrophil adhesion to corneal epithelial cells in response to dry eye disease relevant stimuli.MethodsHuman neutrophils were isolated by bead-based immunomagnetic depletion of non-target cells from human whole blood. NETosis was induced using phorbol 12-myristate 13-acetate (PMA) or anti-citrullinated histone 4 R3 antibody (H4R3 ACPA). Extracellular DNA was used as a surrogate biomarker of NETosis, and it was quantified using a 96-well, plate reader-based fluorescent assay and by confocal microscopy in 8-well chambers using the DNA dye, SYTOXTM Green. Neutrophils were labeled with calcein-AM and adhesion to human corneal epithelial cells was measured. The efficacy of a dose-range of pooled human immune globulin (Flebogamma® DIF, 0.01%–5%) was tested in all assays.ResultsPooled human immune globulins (Flebogamma® DIF) dose-dependently inhibited both PMA and H4R3 ACPA induced NETosis, with concentrations ≥2.5% fully preventing release of extracellular DNA over a 2–16 h time period. Similarly, Flebogamma® 5% DIF prevented NETosis against PMA (20 nM) and a dose range (0.1–10 μg/mL) of H4R3 ACPA. Both PMA and H4R3 ACPA increased adhesion of neutrophils to corneal epithelial cells by 20% and 5%, respectively. Flebogamma® DIF treatment resulted in a dose-dependent reduction of neutrophil adhesion, with Flebogamma® 5% DIF reducing adhesion to baseline levels.DiscussionThese findings show the dose-dependent efficacy of pooled human immune globulins, specifically Flebogamma® DIF against experimentally and pathologically induced NETosis and neutrophil adhesion to corneal epithelial cells, in vitro. The results from this study support the continued clinical development of Flebogamma® 5% DIF as a novel and efficacious treatment for the signs and symptoms of dry eye disease.

Purpose: To evaluate the safety and tolerability of pooled human immune globulins, Flebogamma (R) 5% DIF and Flebogamma (R) 10% DIF, administered by topical ophthalmic instillation to New Zealand White (NZW) rabbits. Methods: Male NZW rabbits were used in this study. In the acute single dose tolerability study, rabbits (n = 12) received a single topical dose of Flebogamma (R) 5% DIF. In the two-week repeated-dose tolerability study, rabbits (n = 5 for each group) were administered either Flebogamma (R) 5% DIF or Flebogamma (R) 10% DIF by topical bilateral administration four times daily (q.i.d.) between 8 am and 6 pm for a period of two weeks. Full ophthalmic examinations were conducted to evaluate ocular tolerability at baseline, Day 7, and Day 14. Results: In the acute single dose study, mild hyperaemia was observed in 1 out of 4 eyes at each 4 h and 24 h post-instillation of Flebogamma (R) 5% DIF. In the repeated dose study, no ocular signs were detected after q.i.d. topical instillation of Flebogamma (R) 5% DIF, while Flebogamma (R) 10% DIF resulted in mild hyperaemia in 8 out of 10 eyes on Day 7, and 5 out of 10 eyes on Day 14. No positive corneal fluorescein staining was detected. Schirmer tear test results were unremarkable. No other ocular signs were observed. Administration of immune globulins had no effect on intraocular pressure. Conclusions: Flebogamma (R) 5% DIF and Flebogamma (R) 10% DIF were well-tolerated by NZW rabbits following single and repeat dose topical ophthalmic administration, supporting the future development of topical pooled human immune globulins for the treatment of ocular surface disease.