Gefarnate stimulates mucin-like glycoprotein secretion in conjunctival tissue and ameliorates corneal epithelial damage in animal dry-eye models.

PURPOSE: The aim of this study was to evaluate the effect of gefarnate on mucin-like glycoprotein secretion in isolated rabbit conjunctival tissue, and on corneal epithelial damage in rabbit and cat dry-eye models. METHODS: Conjunctival tissue isolated from rabbits was treated with gefarnate. Mucin-like glycoprotein was detected in the culture supernatant by an enzyme-linked lectin assay. Gefarnate ointment was topically applied to eyes once daily for 7 days in the rabbit dry-eye model, in which the lacrimal glands, Harderian gland, and nictitating membrane were removed, or for 4 weeks in the cat dry-eye model, in which the lacrimal gland and nictitating membrane were removed. Corneal epithelial damage was evaluated by measurement of corneal permeability by rose bengal in the rabbit model or by fluorescein staining in the cat model. RESULTS: Gefarnate stimulated mucin-like glycoprotein secretion in conjunctival tissue in a dose-dependent manner. In the rabbit dry-eye model, application of gefarnate ointment to the eyes resulted in a dose-dependent decrease in rose bengal permeability in the cornea, with the effect being significant at concentrations of ≥0.3%. In the cat dry-eye model, application of gefarnate ointment resulted in a significant decrease in the corneal fluorescein staining score. CONCLUSION: These results suggest that gefarnate stimulates in vitro secretion of mucin-like glycoprotein in conjunctival tissue and ameliorates corneal epithelial damage in animal dry-eye models. Gefarnate may therefore be effective for treating dry eye.

[Optimization of benzalkonium chloride concentration in 0.0015% tafluprost ophthalmic solution from the points of ocular surface safety and preservative efficacy].

Optimization of benzalkonium chloride (alkyl dimethylbenzylammonium chloride: BAK) concentration as preservative in 0.0015% tafluprost ophthalmic solution (Tapros 0.0015% ophthalmic solution), an anti-glaucoma medicine, was examined from the points of ocular surface safety and preservative efficacy. BAKC(12), which is dodecyl dimethylbenzylammonium chloride, and BAKmix, which is the mixture of dodecyl, tetradecyl and hexadecyl dimethylbenzylammonium chloride were used in this study. The effects of BAKC(12) concentrations and the BAK types, BAKC(12) and BAKmix, in tafluprost ophthalmic solution on ocular surface safety were evaluated using the in vitro SV 40-immobilized human corneal epithelium cell line (HCE-T). Following treatments of Tafluprost ophthalmic solutions with BAKC(12), its concentration dependency was observed on cell viability of HCE-T. The cell viability of HCE-T after treatment of these solutions with 0.001% to 0.003% BAKC(12) for 5 minutes were the same level as that after treatment of the solution without BAK. Tafluprost ophthalmic solution with 0.01% BAKC(12) was safer for the ocular surface than the same solution with 0.01% BAKmix. Preservatives-effectiveness tests of tafluprost ophthalmic solutions with various concentrations of BAKC(12) were performed according to the Japanese Pharmacopoeia (JP), and solutions with more than 0.0005% BAKC(12) conformed to JP criteria. It was concluded that 0.0005% to 0.003% of BAKC(12) in tafluprost ophthalmic solution was optimal, namely, well-balanced from the points of ocular surface safety and preservative efficacy.

Dissection of B cell differentiation during primary immune responses in mice with altered CD40 signals.

CD40 is essential for efficient humoral immune responses. CD40 has two cytoplasmic domains required for binding of tumor necrosis factor receptor-associated factors (TRAF). The TRAF6-binding site is within the membrane proximal cytoplasmic (Cmp) region, while a PXQXT motif in the membrane distal cytoplasmic (Cmd) region needs to engage TRAF2/3/5. To dissect CD40 signals necessary for B cell differentiation, we generated transgenic mice expressing wild-type and mutant human CD40 (hCD40) molecules in a mouse CD40-deficient (mCD40(-/-)) background. The B cell-specific expression of hCD40 in mCD40(-/-) mice resulted in T-dependent antibody responses including germinal center (GC) formation. Mutant hCD40 molecules that carry either a point mutation of the TRAF2/3/5-binding site or a deletion of the Cmd region rescued extrafollicular B cell differentiation but not GC formation. A mutant hCD40 that comprises of only the TRAF2/3/5-binding site in the cytoplasmic region also rescued low but significant titers of antigen-specific IgG1 without GC formation. These results demonstrated that two distinct signals either from the Cmp or from the Cmd region induced the extrafollicular B cell differentiation and Ig class switching; however, GC formation required both. We conclude that combinations of these two signals determine which of the extrafollicular or the follicular (GC) differentiation pathway B cells enter.