Early Clinical Outcomes of the First Commercialized Human Autologous Ex Vivo Cultivated Oral Mucosal Epithelial Cell Transplantation for Limbal Stem Cell Deficiency: Two Case Reports and Literature Review.

The first product in the world for ex vivo cultivated oral mucosal epithelial cell transplantation (COMET) to treat limbal stem cell deficiency (LSCD), named Ocural®, was launched in June 2021 in Japan. COMET was performed on two patients, including the first case in the post-marketing phase of Ocural®. Pathological and immunohistochemical examinations were also carried out using specimens obtained before and after COMET and the spare cell sheet. In case 1, the ocular surface remained free from epithelial defects for approximately six months. In case 2, although defect of the cornea-like epithelia was observed after COMET for one month, it was resolved after the insertion of lacrimal punctal plugs. In case 1, adjuvant treatment was interrupted due to an accident during the second month after COMET, resulting in conjunctival ingrowth and corneal opacity. Eventually, a lamellar keratoplasty was required at six months after COMET. Immunohistochemistry revealed the presence of markers for stem cells (p63, p75), proliferation (Ki-67), and differentiation (Keratin-3, -4, and -13) in both the cornea-like tissue after COMET and a cultivated oral mucosal epithelial cell sheet. In conclusion, Ocural® can be accomplished without major complications, and the stem cells derived from oral mucosa might be successfully engrafted.

Contributions of Interleukin-33 and TSLP in a papain-soaked contact lens-induced mouse conjunctival inflammation model.

INTRODUCTION: Pathological changes of severe chronic allergic conjunctivitis are driven not only via acquired immunity but also via innate immunity. Type 2 immune response-initiating cytokines may play some roles as innate immunity-dependent components of the ocular surface inflammation. To investigate the involvement of type 2 immune response-initiating cytokines in innate immunity-dependent, papain-induced conjunctival inflammation model using IL-25-, IL-33-, and TSLP receptor (TSLPR)-knockout (KO) mice with reference to basophils and ILC2. METHODS: Papain-soaked contact lenses (papain-CLs) were installed in the conjunctival sacs of C57BL/6-IL-25 KO, IL-33 KO, TSLPR KO, Rag2 KO, Bas-TRECK, and wild-type mice and their eyes were sampled at day 5. The eosinophil and basophil infiltration in papain-CL model was evaluated histologically and cytokine expression was examined. To clarify the roles of basophils and ILC2, basophil/ILC2-depletion experiments were carried out. RESULTS: Papain-induced conjunctival inflammation exhibited eosinophil infiltration and upregulation of Th2 cytokine expression. Reduction of eosinophil and basophil infiltration and attenuated Th2 cytokine expression were observed in the papain-CL model using IL-33 KO and TSLPR KO mice. Depletion of basophils or ILC2s in the conjunctivae of the papain-CL model reduced eosinophil infiltration. CONCLUSIONS: Innate immunity-driven type 2 immune responses of the ocular surface are dependent on IL-33, TSLP, basophils, and ILC2. These components may be possible therapeutic targets for refractory allergic keratoconjunctivitis.

Roles of Epithelial Cell-Derived Type 2-Initiating Cytokines in Experimental Allergic Conjunctivitis.

PURPOSE: To clarify the possible involvement of the type 2-initiating cytokines interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP) in the pathophysiology of allergic conjunctivitis, we evaluated ragweed (RW)-induced experimental allergic conjunctivitis (EAC) models by using IL-25 knockout (KO), IL-33 KO, and TSLP receptor (TSLPR) KO mice. METHODS: Interleukin-25 KO, IL-33 KO, TSLPR KO, and BALB/c wild-type mice were sensitized twice with RW in alum and then challenged with RW in eye drops. Clinical scores and eosinophil infiltration were evaluated. Expression levels of serum immunoglobulin E (IgE) and cytokines in the conjunctival tissues were quantified and immunohistochemical analysis was carried out. RESULTS: Significant reductions in clinical scores and numbers of infiltrating eosinophils were observed in the RW-EAC model using IL-33 KO mice. There were no significant differences in clinical scores and numbers of infiltrating eosinophils among IL-25KO, TSLPR KO, and wild-type mice. Serum IgE concentration was upregulated after RW challenges, and there were no differences among the mouse genotypes. Expression levels of of il4, il5, il13, and ccl5 mRNA were diminished in the conjunctivae of the RW-EAC model using IL-33 KO mice compared to those in wild-type mice. Interleukin-33 expression was upregulated as early as 1 hour after RW eye-drop challenge. The number of infiltrating basophils in the conjunctivae of the RW-EAC model using IL-33 KO mice was diminished compared to that in wild-type mice. CONCLUSIONS: Among the type 2-initiating cytokines, IL-33 may play a major role in conjunctival inflammation in an RW-EAC model.

DNA Methylation Analysis of Human Trabecular Meshwork Cells During Dexamethasone Stimulation.

PURPOSE: We investigated the changes in the DNA methylation status of cultured human trabecular meshwork (TM) cells during glucocorticoid exposure, and evaluated the effect of epigenetic modification on the gene expression of TM cells. METHODS: Three batches of primary culture TM cells were treated with and without 100 nM dexamethasone (DEX) for 14 days. Genome-wide methylation analysis was done using Illumina 450 K methylation chips. The gene expression profile of the TM cells also was examined. The epigenetic effect of DEX stimulation on gene expression in TM cells was further verified by treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) and subsequent real-time PCR analysis. RESULTS: After DEX stimulation, we found demethylated cytosine-phosphate-guanine (CpG) sites within the FKBP5, ZBTB16, and SCNN1A gene promoter regions with increases of corresponding gene expression for all three TM batches, and methylated CpG sites within the ARSI, HIC1, GREM2, and MATN2 gene promoter regions with decreases of corresponding of gene expression for all three batches. Inhibition of DNA methylation by 5-aza-dC treatment induced a further increase of FKBP5 and SCNN1A mRNA expression under DEX stimulation. CONCLUSIONS: The DEX stimulation induces alteration of the DNA methylation status in human TM cells. Epigenetic modification could affect the TM gene expression profile.