Epithelial dysplasia in pterygium postoperative granuloma.

Pterygium postoperative granuloma (PPG) is one of the common complications of pterygium surgery. In order to provide the structural features of PPG, and to further explore its pathogenetic mechanism, we analyzed clinical and pathological characteristics of 12 PPG cases. New blood vessels were observed under a slit lamp in PPG and peripheral conjunctival tissues. In vivo confocal imaging showed that there was extensive neovascularization in the stroma, accompanied by infiltration of dendritic cells and inflammatory cells. Dense fibrous structures were observed in some PPG tissues. H&E staining results confirmed neovascularization and inflammatory cells in PPG tissues. In addition, H&E staining exhibited epithelioid tissue covering some PPG tissues. The immunofluorescence results demonstrated that the PPG epithelium was negative for K19, K10 and Muc5AC. Compared with the normal conjunctiva and pterygium, the expression of collagen IV in PPG basement membrane decreased, the expression of pan-cytokeratin (PCK), claudin 4 and E-cadherin in PPG epithelium was significantly lower, while the expression of vimentin, α-SMA and Snail was significantly increased. Therefore, our results suggest that the expression of epithelial keratin markers and goblet cell specific mucin marker is downregulated in the PPG tissues, and it likely is associated with the occurrence of EMT in granulomatous tissues.

Effects of diesel exhaust particles on the condition of mouse ocular surface.

In order to evaluate the effects of diesel exhaust particles (DEP) on the ocular surface, different concentrations (100 and 1000 µg/ml) of DEP eye drops were administered on the mouse ocular surface for a period of 28 days. After DEP treatment, the corneal epithelial permeability to Oregon Green Dextran was studied, which increased proportionally with time. Also, the number of corneal epithelial cell layers significantly increased, which was accompanied with a high Ki67 expression. On the other hand, the number of goblet cells in the conjunctival fornix were reduced, and apoptotic cells were detected in the corneal and conjunctival epithelium by TUNEL assay in the DEP treated group, along with increased Caspase 3/8 expression. Furthermore, the number of CD4 positive cells significantly increased in the conjunctiva, while NF-κB p65 (phospho S536) expression was elevated in the cornea and also the conjunctiva. Our data revealed that the topical administration of DEP on the ocular surface in mouse disrupted the organized structure of the ocular surface and induced an inflammation of the cornea and conjunctiva.

Liposomes-Encapsulating Double-Stranded Nucleic Acid (Poly I:C) for Head and Neck Cancer Treatment.

Polyriboinosinic acid-polyribocytidylic acid (Poly I:C) serves as a synthetic mimic of viral double-stranded dsRNA, capable of inducing apoptosis in numerous cancer cells. Despite its potential, therapeutic benefits, the application of Poly I:C has been hindered by concerns regarding toxicity, stability, enzymatic degradation, and undue immune stimulation, leading to autoimmune disorders. To address these challenges, encapsulation of antitumor drugs within delivery systems such as cationic liposomes is often employed to enhance their efficacy while minimizing dosages. In this study, we investigated the potential of cationic liposomes to deliver Poly I:C into the Head and Neck 12 (HN12) cell line to induce apoptosis in the carcinoma cells and tumor model. Cationic liposomes made by the hydrodynamic focusing method surpass traditional methods by offering a continuous flow-based approach for encapsulating genes, which is ideal for efficient tumor delivery. DOTAP liposomes efficiently bind Poly I:C, confirmed by transmission electron microscopy images displaying their spherical morphology. Liposomes are easily endocytosed in HN12 cells, suggesting their potential for therapeutic gene and drug delivery in head and neck squamous carcinoma cells. Activation of apoptotic pathways involving MDA5, RIG-I, and TLR3 is evidenced by upregulated caspase-3, caspase-8, and IRF3 genes upon endocytosis of Poly(I:C)-encapsulated liposomes. Therapeutic evaluations revealed significant inhibition of tumor growth with Poly I:C liposomes, indicating the possibility of MDA5, RIG-I, and TLR3-induced apoptosis pathways via Poly I:C liposomes in HN12 xenografts in J:NU mouse models. Comparative histological analysis underscores enhanced cell death with Poly I:C liposomes, warranting further investigation into the precise mechanisms of apoptosis and inflammatory cytokine response in murine models for future research.

Study on polymethylmethacrylate ring in protecting limbal stem cells during collagen cross-linking.

OBJECTIVE: The UV rays used in the collagen cross-linking (CXL) procedure seem to cause potential damage to the limbal stem cells. This study was designed to evaluate the ability of polymethylmethacrylate (PMMA) hemiannulus as an alternative to protect corneal limbal stem cells during CXL. METHODS: Ten freshly enucleated human cadaveric eyeballs were subjected to the corneal CXL procedure. The cadaveric eye ball was divided into 2 sectors: A and B. Sector A was left unprotected, while sector B was covered by a PMMA shield. Limbal biopsies from both sectors before and after the procedure were analyzed. Each limbal tissue was placed on human amniotic membrane (HAM) to check the cultivability and was subjected to marker studies using reverse transcriptase PCR. RESULTS: Before CXL, biopsies from both sectors showed growth on HAM. After CXL, biopsies from sector A showed no growth on HAM while 2 out of the 10 from sector B covered with the PMMA ring did show growth on HAM. The putative stem-cell marker ABCG2 was negative in all the samples from sector A after CXL and was positive in 2 out of the 10 samples from sector B. CONCLUSION: Covering the limbal region with PMMA offers partial protection of the limbus from the UV rays during the CXL procedure.

Tissue engineered corneal epithelium derived from clinical-grade human embryonic stem cells.

PURPOSE: To construct tissue engineered corneal epithelium from a clinical-grade human embryonic stem cells (hESCs) and investigate the dynamic gene profile and phenotypic transition in the process of differentiation. METHODS: A stepwise protocol was applied to induce differentiation of clinical-grade hESCs Q-CTS-hESC-1 and construct tissue engineered corneal epithelium. Single cell RNA sequencing (scRNA-seq) analysis was performed to monitor gene expression and phenotypic changes at different differentiation stages. Immunostaining, real-time quantitative PCR and Western blot analysis were conducted to detect gene and protein expressions. After subcutaneous transplantation into nude mice to test the biosafety, the epithelial construct was transplanted in a rabbit corneal limbal stem cell deficiency (LSCD) model and followed up for eight weeks. RESULTS: The hESCs were successfully induced into epithelial cells. scRNA-seq analysis revealed upregulation of ocular surface epithelial cell lineage related genes such as TP63, Pax6, KRT14, and activation of Wnt, Notch, Hippo, and Hedgehog signaling pathways during the differentiation process. Tissue engineered epithelial cell sheet derived from hESCs showed stratified structure and normal corneal epithelial phenotype with presence of clonogenic progenitor cells. Eight weeks after grafting the cell sheet onto the ocular surface of LSCD rabbit model, a full-thickness continuous corneal epithelium developed to fully cover the damaged areas with normal limbal and corneal epithelial phenotype. CONCLUSION: The tissue engineered corneal epithelium generated from a clinical-grade hESCs may be feasible in the treatment of limbal stem cell deficiency.