Metabolic signatures of tear extracellular vesicles caused by herpes simplex keratitis.

PURPOSE: Herpes simplex keratitis (HSK), caused by type 1 herpes simplex virus (HSV) reactivation, is a severe infectious disease that leads to vision loss. HSV can trigger metabolic reprogramming in the host cell and change the extracellular vesicles (EV) cargos; however, little is known about the EV metabolic signatures during ocular HSV infection. Here, we aimed to depict the EV-associated metabolic landscape in HSK patients' tears. METHODS: We collected 82 samples from 41 participants with unilateral HSK (contralateral unaffected tears were set as negative control), including subtype cohorts of 13 epithelial, 20 stromal, and 8 endothelial HSK. We isolated tear EVs via our previously established platform and conducted metabolic analysis using LC-MS/MS. The metabolic signatures for recognizing HSK and subtypes were assessed through differential analysis and machine learning algorithms. RESULTS: Hypopsia and increased extracellular CD63 levels were observed in affected eyes. We identified 339 metabolites based on sEVs isolated from tears. Differential analysis revealed alterations in energy and amino acid metabolism, as well as the infectious microenvironment. Furthermore, we observed dysregulated metabolite such as methyldopa, which is associated with inappropriate neovascularization and corneal sensation loss, contributing to the HSK severity particularly in the stromal subtype. Moreover, machine learning classification also suggested a set of EV metabolic signatures that have potential for pan-keratitis detection. CONCLUSIONS: Our findings demonstrate that tear EV metabolites can serve as valuable indicators for comprehending the underlying pathological mechanisms. This knowledge is expected to facilitate the development of liquid biopsy means and therapeutic target discovery.

Discovering the Secret of Diseases by Incorporated Tear Exosomes Analysis via Rapid-Isolation System: iTEARS.

Nanoscale small extracellular vesicles (sEVs, exosomes) in tears allow us to investigate the multisignatures of diseases. However, the translations of tear sEVs for biomarker discovery and clinical diagnostics are practically limited by low recovery, long processing time, and small sample volume. Here, we report an incorporated tear-exosomes analysis via rapid-isolation system (iTEARS) via nanotechnology to discover the secrets of ocular disorders and systemic diseases. We isolate exosomes rapidly with high yield and purity from a few teardrops (∼10 µL) within 5 min via nanoporous membrane-based resonators for the quantitative detection and biomarker discovery through proteomic and transcriptomic analysis. We have identified 904 proteins, among which 228 proteins are discovered, 426 proteins are detected from exosomes of dry eye disease, and demonstrate CALML5, KRT6A, and S100P for the classification of dry eye disease. We have also investigated 484 miRNAs in tear exosomes and show miR-145-5p, miR-214-3p, miR-218-5p, and miR-9-5p are dysregulated during diabetic retinopathy development. We believe iTEARS can be used for improving molecular diagnostics via tears to identify ocular disorders, systemic diseases, and numerous other neurodegenerative diseases and cancer.

Detection of Tear Components Using Matrix-Assisted Laser Desorption Ionization/Time-of-Flight Mass Spectrometry for Rapid Dry Eye Diagnosis.

The tear is a biological fluid that has the diagnostic potential for ocular diseases. Extracellular vesicles (EVs), widly detected in various biofluids including tears, are nanoparticles released by living cells and considered as promising detection sources for noninvasive liquid biopsy. Understanding the roles of tears and tear-EVs in ocular diseases such as dry eye can facilitate the studies of clinical diagnosis, which usually entails detecting such liquid objects with a rapid and effective method. In this study, we used a mass spectrometry-based strategy to analyze peptidome/proteome profiles of tears and EVs for rapid dry eye diagnosis. Nanosized EVs were isolated from tears of both healthy control (HC) individuals and dry eye syndrome (DES) patients, and the tear compositions were further analyzed by tracking their fingerprints with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. The fingerprints of tear-EVs could be observed in a dose-dependent manner and tears, allowing for comparison of the discriminant peaks between tears and EVs. By analyzing these peaks, the fingerprints of both tear and tear-EVs were showed to have the capability of distinguishing patients with DES from HC donors and providing an efficient way for screening potential DES biomarkers. The proposed tear and EV fingerprinting approach is expected to be a potential tool in the rapid diagnosis of ocular diseases and in-depth research on pathogenesis. Data are available via ProteomeXchange with identifier PXD020217.

Thymic Stromal Lymphopoietin-Related Allergic Pathway in Patients With Vernal Keratoconjunctivitis.

PURPOSE: To investigate the expression of thymic stromal lymphopoietin (TSLP) and the relevant signaling pathways in the giant papillae obtained from patients with vernal keratoconjunctivitis (VKC) and to study the potential functional role and molecular mechanism of TSLP. METHODS: Giant papillae from VKC patients and control samples were used to perform immunohistochemical staining and analyze the mRNA expression of TSLP and related pathway by real-time polymerase chain reaction. RESULTS: TSLP was markedly expressed in the epithelial cells and some inflammatory cells of giant papillae, but not in the control conjunctival tissue. TSLP mRNA expression in the giant papillae of VKC was increased by 9.63 ± 0.99 (mean ± SD) fold compared with controls (P < 0.01). CD11c and OX40L immunoreactive cells largely infiltrated the giant papillae as observed by immunohistochemical staining. CD4Th2 cell infiltration was observed through high immunoreactivity of CD4. Th2 cytokines (IL-4, IL-5 and IL-13) and OX40 in the VKC specimens showed increased expression. Augmented gene expression levels of CD4 (6.88 ± 1.84), OX40L (7.60 ± 1.79), OX40 (7.25 ± 1.38), IL-4 (6.89 ± 1.46), IL-5 (8.42 ± 1.55), and IL-13 (9.69 ± 1.94) were significantly different from controls (P < 0.05). CONCLUSIONS: Our observations provide strong evidence that TSLP may be a crucial factor that contributes to the development and progression of allergic conjunctivitis. The results also demonstrated that TSLP activates dendritic cells to prime CD4T cells to differentiate into Th2 type and triggers Th2-dominant allergic inflammation through the TSLP/OX40L/OX40 signaling as part of immunopathogenesis of VKC.

Deep anterior lamellar keratoplasty using acellular corneal tissue for prevention of allograft rejection in high-risk corneas.

PURPOSE: To determine whether deep anterior lamellar keratoplasty (DALK) using acellular glycerol-cryopreserved corneal tissue (GCCT) could prevent allograft rejection in high-risk corneas. DESIGN: Prospective, randomized, comparative study. SETTINGS: The Eye Hospital, Wenzhou Medical College, Zhejiang, China. STUDY POPULATION: All patients with herpes simplex virus keratitis, bacterial keratitis, fungal keratitis, or ocular burn, who were eligible as per study design, were invited to participate. OBSERVATION PROCEDURES: According to randomized block design, all patients received either GCCT or fresh corneal tissue (FCT) during DALK. Best-corrected visual acuity (BCVA), slit-lamp microscopy, and in vivo confocal microscopy examinations at 1 week and 1, 3, 6, 12, and 24 months after surgery were analyzed. Kaplan-Meier survival analysis was used to evaluate graft survival rate. MAIN OUTCOME MEASURES: Therapeutic success, 2-year rejection-free graft survival rate and 2-year graft survival rate, in vivo confocal microscopy results, BCVA, and endothelial cell density. RESULTS: Postoperative BCVA of 20/40 or better at the last follow-up visit was achieved in 57.6% (19/33) of eyes in the GCCT group and in 54.8% (17/31) of the FCT group. No graft rejection occurred in the GCCT group, while in the FCT group 10 episodes of stromal rejection developed in 7 eyes. Overall, the rejection-free graft survival rate at 2 years was significantly higher in the GCCT group as compared with the FCT group (100.0%, 78.8% respectively, P = .006). CONCLUSIONS: Deep anterior lamellar keratoplasty using acellular glycerol-preserved cornea could prevent allograft rejection and promote graft survival rate in high-risk corneas.

Comparison of fresh corneal tissue versus glycerin-cryopreserved corneal tissue in deep anterior lamellar keratoplasty.

PURPOSE: To compare the microstructural differences in fresh corneal tissue (FCT) with glycerin-cryopreserved corneal tissue (GCCT) used during deep anterior lamellar keratoplasty (DALK). METHODS: The medical records of 48 consecutive patients who underwent DALK for stromal opacity without endothelial abnormalities were retrospectively reviewed. Patients were divided into two groups: an FCT group (n = 22) and a GCCT group (n = 26), according to the corneal tissue used. The best corrected visual acuity (BCVA), slit lamp, corneal topography, pachymetry, and laser scanning in vivo confocal microscopy examinations at 2 weeks and 1, 3, 6, 12, and 24 months after surgery were analyzed. RESULTS: No graft rejection developed in the GCCT group, whereas stromal rejection developed in one eye in the FCT group. There were no significant differences in spherical equivalent (P = 0.37), astigmatism (P = 0.26), BCVA (P = 0.64), central corneal thickness (P = 0.73), or endothelial cell density (P = 0.49) between the two groups at 24 months. Confocal microscopy showed that GCCT was acellular, whereas dendritelike cells and keratocytes were found in the FCT group 2 weeks after surgery. The keratocyte density improved significantly in the GCCT group at 3 months after surgery, whereas it decreased significantly after surgery in the FCT group during follow-up. No significant difference in regeneration of nerve fibers was found in the subbasal layer and anterior stroma between the two groups at 24 months. CONCLUSIONS: DALK using GCCT provides clinical results comparable to FCT. GCCT can be used safely and effectively for DALK and may minimize stromal rejection after surgery.