Characterization of doxycycline-dependent inducible Simian Virus 40 large T antigen immortalized human conjunctival epithelial cell line.

PURPOSE: To present the properties of a newly developed immortalized human conjunctival epithelial cell (iHCjEC) line. METHODS: iHCjECs were developed to induce Simian Virus 40 large T-antigen (SV40LT) by incorporating lentivirus in a tetracycline (Tet)-regulated gene-expression system into primary cultures of human conjunctival epithelial cells. The population doubling time and morphology of the iHCjECs were analyzed. The expressions of CK13, CK19, CK12, and MUC1, MUC4, MUC16, and MUC5AC were determined by real time PCR and immunohistochemically under different culture conditions. The organotypic culture model in which iHCjECs were cultured on rabbit conjunctival fibroblast-embedded collagen gel was used to characterize the iHCjECs. RESULTS: The iHCjECs cultured with doxycycline (Dox) continued to proliferate for at least 20 passages and had a cobblestone-like appearance. The expressions of CK13 and CK19 but not CK12 were detected in the iHCjECs, and the expression of CK13 increased in culture media lacking Dox (Dox-). The expressions of MUC1, MUC4, MUC16, and MUC5AC were detected in iHCjECs, and a relatively strong immunostaining of MUC5AC was detected with Dox(-) added 5% FBS. Stratified iHCjECs were observed in organotypic culture at 5 days. CONCLUSION: The iHCjECs had high proliferation rates and abilities to control the differentiation potency to control the expression of SV40 LT-antigen with Tet-regulated gene-expression system. They are able to express the mucin gene repertoire of their native epithelia. The iHCjECs can be a useful experimental cell line to study conjunctival epithelial cell characteristics and for pathophysiological and toxicological studies.

Combined effect of coffee ingestion and repeated bouts of low-intensity exercise on fat oxidation.

We investigated the effect of the combination of coffee ingestion and repeated bouts of low-intensity exercise on fat oxidation. Subjects were seven young, healthy male adults. They performed four trials: a single 30-min bout of exercise following ingestion of plain hot water (WS) or coffee (CS); a trial with three 10-min bouts of exercise separated by 10-min periods of rest following ingestion of plain hot water (WR) or coffee (CR). The coffee contained 5 mg kg-1 of caffeine. All trials were performed on a cycle ergometer at 40% maximal oxygen uptake for each subject an hour after beverage ingestion. Oxygen uptake in the CS and CR trials was higher compared with the WS and WR trials at 90 min after exercise (P<0·05). Respiratory exchange ratio (RER) in the CS and CR trials was decreased during the whole recovery period compared with baseline (P<0·05), whereas no significant decreases were observed in either the WS or WR trials. Moreover, RER was significantly lower at 30 min after exercise in the CR trial than in either the WS or WR trials (P<0·05 each). Similarly, it is notable that fat oxidation rate in the CR trial was significantly higher at 30 min after exercise compared to that in the WS and WR trials (P<0·05). These results suggest that the combination of coffee intake and repeated bouts of low-intensity exercise enhances fat oxidation in the period after exercise.

Horizontal intracorneal swirling water migration indicative of corneal endothelial function.

PURPOSE: To test our hypothesis about whether there is water migration in the horizontal corneal plane and investigate its developmental mechanism. METHODS: A fluorescein solution was intrastromally injected into normal and edematous corneas of rabbits, and the movement of the fluorescein solution was observed and recorded over time. RESULTS: In normal corneas, the water flow was characterized by a swirling movement from the center to the periphery in the stroma. The fluorescein solution ultimately spread and occupied the entire cornea, indicating horizontal intracorneal swirling of water. In contrast, when the corneal endothelia were injured by intracameral injection of a preservative to create corneal edema, no water migration occurred, suggesting that the integrity of the corneal endothelial function is essential for water migration. The water migration stopped with injection of a sodium-potassium pump inhibitor, indicating that the enzyme is necessary for physiologic water migration in the cornea. With recovery of corneal endothelial function, the water migration began, and focal edema remained in the periphery with no water migration in this edematous area. CONCLUSIONS: We report for the first time the presence of horizontal water migration in the cornea in a swirling pattern (i.e., intracorneal swirling migration of water, generated by the pump function in the corneal endothelial cells), which may supplement the conventional concept of development of corneal edema in the vertical plane. This dynamic water circulatory system may be involved in increasing the efficiency of the water transfer in the entire cornea.