The Effect of an Anti-oxidative and Anti-inflammatory Functional Dietary Supplement in Dry Eye Syndrome

PURPOSE: To evaluate the effect of Optibiol(R), which is composed of multiple anti-oxidative and anti-inflammatory dietary elements, on the symptoms, clinical manifestations, and expression of various inflammatory cytokines in dry eye patients. METHODS: Patients suffering from dry eye were given Optibiol(R) for 3 months. They completed questionnaires regarding dry eye symptom and underwent slit lamp biomicroscopic examinations, tear film breakup times, Shirmer tests, and conjunctival fluorescein staining examinations on a monthly basis during the intake of Optibiol(R). Sampling of serum and tears was conducted at baseline and 3 months after taking Optibiol(R), and various inflammatory cytokines in the serum and tears were measured with multiplex bead immunoassay. RESULTS: Sixty-three patients were included in this study. After taking Optibiol(R), the dry eye symptoms, Schirmer scores, tear film breakup times, and conjunctival staining scores (Oxford scale) showed significant improvement, and the expression of most inflammatory cytokines had decreased: in particular, IL-1 beta and MIP-1 beta in serum, and IL-17 and MIP-1 beta in tears were significantly lower. CONCLUSIONS: Optibiol(R), an anti-oxidative and anti-inflammatory functional dietary supplement, is an effective dietary supplement in patients with dry eye syndrome. We posit that the decreased expression of inflammatory cytokines is an important mechanism in this effect.

Expression of Local Immunosuppressive Factor, Indoleamine 2,3-dixygenase, in Human Coreal Cells

PURPOSE: To identify the localization of indoleamine 2,3-dioxygenase (IDO) in human corneal cells and to evaluate its ability to act as a local immunosuppressive factor. METHODS: The expression profile of IDO was obtained with RT-PCR and Western blot of in a primary culture of human corneal cells (fibroblasts, epithelial cells and endothelial cells). In order to investigate the immunosuppressive function of IDO, immune cells were cultured in a human corneal cell-conditioned medium, and their prolifleration was identified by the MTT assay. Moreover, apoptotic effects of IDO in immune cells treated with IFN-gamma were also investigated with apoptosis ELISA. RESULTS: Among the three different types of human corneal cells analyzed, mRNA and protein expression of IDO was observed only in human corneal fibroblasts. Immune cells cultured in a human corneal fibroblast-conditioned medium showed inhibited proliferation. Moreover, IFN-gamma-induced expression of IDO significantly enhanced apoptotic ability in a dose-depandant manner. CONCLUSIONS: Our results suggest that human corneal fibroblasts are relatively immuno-resistant and that expression of IDO may be one of the factors involved in the immune tolerance observed in corneal grafts.

Characterization of Immortalized Human Corneal Endothelial Cell Line using HPV 16 E6/E7 on Lyophilized Human Amniotic Membrane

PURPOSE: To establish the immortalized human corneal endothelial cell line (IHCEn) by transducing human papilloma virus (HPV) 16 E6/E7 oncogenes, and to identify their characteristics when cultivated on a lyophilized human amniotic membrane (LAM). METHODS: Primary human corneal endothelial cells (PHCEn) were infected using a retroviral vector with HPV 16 E6/E7, and transformed cells were clonally selected by G418. Growth properties and characteristics of IHCEn were compared with PHCEn by cell counting and RT-PCR of VDAC3, SLC4A4, CLCN3, FGF-1, Col IV, and Na+/K+ ATPase. IHCEn were cultured on LAM. Messenger RNA expressions of VDAC3, CLCN3, and Na+/K+ ATPase, and protein expressions of Na+/K+ ATPase and Col IV in IHCEn cultivated on LAM were investigated by RT-PCR, immunofluorescence, and immunohistochemical staining, respectively. RESULTS: Successful immortalization was confirmed by stable expression of HPV 16 E6/E7 mRNA by RT-PCR, and IHCEn exhibited typical corneal endothelial morphology. Doubling time of IHCEn was 30.15+/-10.96 hrs. Both IHCEn and PHCEn expressed VDAC3, CLCN3, SLC4A4, FGF-1, Col IV, and Na+/K+ ATPase. IHCEn cultivated on LAM showed stronger expression of VDAC3, CLCN4, and Na+/K+ ATPase mRNA than on plastic culture dish. Immunohistochemical staining and immunofluorescence revealed the positive expression of Na+/K+ ATPase and Col IV. CONCLUSIONS: IHCEn were successfully established, and LAM is a good substrate for the culture of human corneal endothelial cells.

The Effect of Antiglaucoma Medication on Cultured Human Conjunctival Epithelial Cells

PURPOSE: To investigate the toxicity of a short-term application of timolol maleate, dorzolamide, and benzalkonium chloride (BAC) on human conjunctival epithelial cells in vitro. METHODS: Chang's conjunctival epithelial cell line was treated for 5 min, 15 min, 30 min, and 60 min with various concentrations of timolol, dorzloamide, or BAC, and then examined 4 hrs or 24 hrs later. Cell viabilities were assessed by MTT assay. The expressions of various cytokines by timolol maleate, dorzolamide, and BAC treatment in human conjunctival epithelial cells were evaluated using ELISPOT. RESULTS: BAC significantly decreased survival of conjunctival epithelial cells in a dose and time dependent manner compared with timolol and dorzolamide. Inflammatory cytokines, IL-6 and IL-8, were highly expressed in conjunctival epithelial cells treated with timolol, dorzolamide, and BAC. CONCLUSIONS: The present study suggests that increased expression of inflammatory markers, IL-6 and IL-8 might explain the ocular surface disorder in patients receiving antiglaucoma medication.

The Involvement of Adult Stem Cells Originated from Bone Marrow in the Pathogenesis of Pterygia

Pterygium is a proliferative disease. Recent research has reported that stem cells are involved in the pathogenesis of various proliferative diseases, including solid tumors and diabetic proliferate vitreoretinopathy. In previous literature, we hypothesized that adult stem cells originated from bone marrow were involved in the pathogenesis of pterygium. We proved this by immunohistochemical staining with various stem cell markers. The staining showed adult stem cells in the pterygium. c-kit positive cells were observed primarily in the stroma, and some cells were also found in the basal epithelium. AC133 and CD34 positive cells were primarily found in the basal epithelium and were ovoid shaped, similar to the c-kit cells. However, some cells were found in vascular endothelium. STRO-1 positive cells were found mainly in the stroma and were spindle shaped. In recurrent pterygium, cells were more scattered and the expression pattern was denser. Therefore, we suggest a new theory of pterygium pathogenesis. Inflammation caused by environmental factors triggers the abnormal production of some growth factors and cytokines in order to recover from cellular damage. If these healing signals are excessive, limbal basal cells will be changed to abnormally-altered pterygial cells. The excessive wound healing process and remnant altered cells result in recurrence using the same mechanism.