Regulation of Endothelin-1-Induced Trabecular Meshwork Cell Contractility by Latanoprostene Bunod.

PURPOSE: Previous in vivo studies demonstrated that latanoprostene bunod (LBN), a nitric oxide (NO)-donating prostaglandin F2α receptor agonist, results in greater intraocular pressure (IOP) lowering than latanoprost. The present series of investigations compared the effects of LBN and latanoprost on primary human trabecular meshwork cell (HTMC) contractility and underlying signaling pathways to determine whether LBN might mediate this additional IOP lowering via the conventional outflow pathway. METHODS: The effect of LBN (1-100 µM) on HTMC cGMP levels was determined by ELISA with or without the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Endothelin-1 (ET-1) was used to induce HTMC contractility. To determine the effect of LBN on myosin light chain-2 (MLC-2) phosphorylation, HTMCs were pretreated with 10 to 60 µM LBN for 1 hour and then ET-1 for 5 minutes. MLC-2 phosphorylation was determined by Western blotting. Effects of LBN (30 and 45 µM) on ET-1-induced filamentous (F)-actin cytoskeletal stress fibers and the focal adhesion associated protein vinculin were determined by confocal microscopy. ET-1-induced HTMC monolayer resistance in the presence of LBN (45 µM) was determined by electrical cell substrate impedance sensing, as an indicator of cell contractility. Latanoprost and SE 175 (an NO donor which releases NO on reductive transformation within the cells) were used as comparators in all studies. RESULTS: LBN (1-100 µM) significantly increased cGMP levels in a dose-dependent manner, with a half maximal effective concentration (EC50) of 1.5 ± 1.3 µM, and with maximal effect similar to that of 100 µM SE 175. In contrast, latanoprost caused a minimal increase in cGMP levels at 100 µM only. The cGMP elevation induced by LBN or SE 175 was abolished by ODQ and was therefore sGC-dependent. The two NO donors SE 175 and LBN elicited a reduction in ET-1-induced MLC-2 phosphorylation that was significantly greater than that mediated by latanoprost in HTMCs. SE 175 (100 µM) and LBN (30 or 45 µM) caused a dramatic reduction in ET-1-induced actin stress fibers and vinculin localization at focal adhesions, whereas 45 µM latanoprost was without observable effect. SE 175 reduced ET-1-induced increases in HTMC resistance in a dose-dependent manner. A synergistic effect on reduction of HTMC resistance was observed when latanoprost and SE 175 doses were given together. LBN significantly reduced ET-1-induced HTMC monolayer resistance increases to a greater extent than latanoprost, indicating a greater reduction in cell contractility with LBN. CONCLUSIONS: LBN, SE 175, and latanoprost caused relaxation of ET-1-contracted HTMCs. The effect on HTMC relaxation observed with LBN was significantly greater in magnitude than that observed with latanoprost or SE 175. Data indicate that the NO-donating moiety of LBN mediates HTMC relaxation through activation of the cGMP signaling pathway and a subsequent reduction in MLC-2 phosphorylation. These findings suggest that increased conventional outflow facility may mediate the additional IOP-lowering effects of LBN over that of latanoprost observed in in vivo studies.

Anti-allergic effects of mapracorat, a novel selective glucocorticoid receptor agonist, in human conjunctival fibroblasts and epithelial cells.

PURPOSE: To determine the ocular anti-allergic effects of mapracorat, a novel selective glucocorticoid receptor agonist (SEGRA) in primary human conjunctival fibroblasts and epithelial cells. METHODS: Two primary human conjunctival cell types, human conjunctival epithelial cells (HConEpiC) and human conjunctival fibroblasts (HConF), were challenged with interleukin-4 (IL-4) or IL-13 plus tumor necrosis factor-alpha (TNF-α). Luminex technology was used to profile the resulting inflammatory response. The effects of mapracorat on the release of eotaxin and regulated on activation, normal T cell expressed and secreted (RANTES), two allergy-related chemokines, as well as proinflammatory cytokines and intercellular adhesion molecule 1 (ICAM-1) were then determined. Small interfering RNA was used to determine whether the effects of mapracorat were mediated via the glucocorticoid receptor (GR). Dexamethasone was used as the control. RESULTS: IL-13 or IL-4 plus TNF-α in the HConF or HConEpiC significantly increased eotaxin-1 (HConF only), eotaxin-3, RANTES, multiple proinflammatory cytokines, and ICAM-1. Synergistic effects of IL-13 or IL-4 plus TNF-α were observed in the HConEpiC for RANTES and monocyte chemoattractant protein-1, and in the HConF for eotaxin-1, eotaxin-3, and RANTES. Mapracorat significantly reduced IL-4 or IL-13 plus TNF-α-induced cytokine release and ICAM-1 protein in a dose-dependent manner in both cell types, with comparable efficacy to dexamethasone. These effects were mediated through the glucocorticoid receptor (GR), as demonstrated by the reversal of inhibitory effects after silencing of glucocorticoid receptor expression. CONCLUSIONS: Data from these in vitro models indicate that mapracorat is efficacious and potent in reducing IL-4 or IL-13 plus TNF-α-induced release of allergy-related and proinflammatory cytokines from the HConF and the HConEpiC, supporting clinical evaluation of the compound in reducing allergic and inflammatory reactions in allergic conjunctivitis.

Evaluation of online instruction to improve medical and dental students' communication and counseling skills.

Online, interactive video modules were created to demonstrate good skills in history taking, counseling, and communication. The authors evaluated the effect of the modules on students' data gathering, counseling, and communication skills with standardized patients (SPs). A student cohort without the online modules (n = 76 medical students and n = 43 dental students) was compared to a cohort of different students who were assigned the modules (n = 88 medical students and n = 39 dental students). Students were evaluated by SPs using case-specific content checklists and the Master Interview Rating Scale (MIRS). Compared to their counterparts who did not use the modules, medical and dental students who used the modules showed significantly higher performance on several outcomes. The areas that showed benefit were those that were novel to students. Student accuracy in grading others was generally unrelated to their own performance. In conclusion, the online, interactive video modules were associated with improvements in a majority of clinical skills.

Anti-inflammatory and anti-oxidative effects of the green tea polyphenol epigallocatechin gallate in human corneal epithelial cells.

PURPOSE: To determine the anti-inflammatory and anti-oxidant effects of epigallocatechin gallate (EGCG), the major polyphenol component of green tea, in human corneal epithelial cells (HCEpiC). METHODS: HCEpiC were challenged with interleukin-1ß (IL-1ß) for 18 h or hyperosmolarity (440 mOsm) for 24 h. Luminex technology was used to determine the effects of EGCG (0.3-30 µM) on IL-1ß- or hyperosmolar-induced cytokine release into the medium. Cell metabolic activity was measured using the alamarBlue assay. Effects of EGCG on mitogen-activated protein kinase (MAPK) phosphorylation were determined by cell-based enzyme-linked immunosorbent assay (ELISA) and western blotting. Effects of EGCG on nuclear factor kappa B (NFκB) and activator protein-1 (AP-1) transcriptional activity were assessed by reporter gene assay. The effects of EGCG on glucose oxidase (GO)-induced reactive oxygen species (ROS) production was determined using the ROS probe CM-H2DCFDA. RESULTS: Treatment of HCEpiC with 1 ng/ml IL-1ß for 18 h significantly increased release of the cytokines/chemokines granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1), while hyperosmolarity-induced release of IL-6 and MCP-1. When cells were treated with IL-1ß and EGCG or hyperosmolarity and EGCG there was a dose-dependent reduction in release of these cytokines/chemokines, with significant inhibition observed at 3-30 µM. There was no effect of EGCG on cell metabolic activity at any of the doses tested (0.3-30 µM). EGCG significantly inhibited phosphorylation of the MAPKs p38 and c-Jun N-terminal kinase (JNK), and NFκB and AP-1 transcriptional activities. There was a significant dose-dependent decrease in GO-induced ROS levels after treatment of HCEpiC with EGCG. CONCLUSIONS: EGCG acts as an anti-inflammatory and anti-oxidant agent in HCEpiC and therefore may have therapeutic potential for ocular inflammatory conditions such as dry eye.

Mapracorat, a novel selective glucocorticoid receptor agonist, inhibits hyperosmolar-induced cytokine release and MAPK pathways in human corneal epithelial cells.

PURPOSE: Increasing evidence suggests that tear hyperosmolarity is a central mechanism causing ocular surface inflammation and damage in dry eye disease. Mapracorat (BOL-303242-X) is a novel glucocorticoid receptor agonist currently under clinical evaluation for use in the treatment of dry eye disease. This study assessed the anti-inflammatory effects of mapracorat in an in vitro osmotic stress model which mimics some of the pathophysiological changes seen in dry eye. METHODS: Human corneal epithelial cells were cultured in normal osmolar media (317 mOsM) or 440 mOsM hyperosmolar media for 24 h. Luminex technology was used to determine the effect of mapracorat on hyperosmolar-induced cytokine release. Effects of mapracorat on mitogen-activated protein kinase (MAPK) phosphorylation were determined by cell based ELISA. Effects of mapracorat on nuclear factor kappa B (NFkappaB) and activator protein-1 (AP-1) transcriptional activity were assessed by reporter gene assay. Dexamethasone was used as a control. RESULTS: Hyperosmolar conditions induced release of the pro-inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) from cultured human corneal epithelial cells, and altered the phosphorylation state of p38 and c-Jun N-terminal kinase (JNK) and transcriptional activity of NFkappaB and AP-1. Incubation of cells with mapracorat inhibited hyperosmolar-induced cytokine release with comparable activity and potency as dexamethasone. This inhibition was reversed by the glucocorticoid receptor antagonist mifepristone (RU-486). Increased phosphorylation of p38 and JNK caused by hyperosmolarity was inhibited by mapracorat. Mapracorat also significantly decreased the hyperosmolar-induced increase in NFkappaB and AP-1 transcriptional activity. CONCLUSIONS: Mapracorat acts as a potent anti-inflammatory agent in corneal epithelial cells challenged with osmotic stress, with comparable activity to the traditional steroid dexamethasone. These in vitro data suggest that mapracorat may be efficacious in the treatment of dry eye disease.

Impact of the site specialty of a continuity practice on students' clinical skills: performance with standardized patients.

BACKGROUND: The assessment of clinical competence and the impact of training in ambulatory settings are two issues of importance in the evaluation of medical student performance. PURPOSE: This study compares the clinical skills performance of students placed in three types of community preceptors' offices (pediatrics, medicine, family medicine) on yearly clinical skills assessments with standardized patients. Our goal was to see if the site specialty impacted on clinical performance. METHODS: The students in the study were completing a 3-year continuity preceptorship at a site representing one of the disciplines. Their performance on the four clinical skills assessments was compared. RESULTS: There was no significant difference in history taking, physical exam, communication, or clinical reasoning in any year (ANOVA p< or = .05) There was a small but significant difference in performance on a measure of interpersonal and interviewing skills during Years 1 and 2. CONCLUSION: The site specialty of an early clinical experience does not have a significant impact on performance of most of the skills measured by the assessments.

Comparison of the effect of multipurpose contact lens solutions on the viability of cultured corneal epithelial cells.

PURPOSE: To determine the effect of four marketed multipurpose contact lens solutions (MPSs) on corneal epithelial cell viability. METHODS: Comparison of the effect of MPS A (Renu MultiPlus, Bausch & Lomb), MPS B (OPTI-FREE Express, Alcon), MPS C (AQuify, CibaVision), and MPS D (OPTI-FREE RepleniSH, Alcon) on cell viability was performed by quantifying cellular ATP content, resazurin reduction, and lactate dehydrogenase (LDH) release in transformed human corneal epithelial cells (HCEpiC) and primary bovine corneal epithelial cells (BCEpiC). RESULTS: Significant reductions in cellular ATP content were observed at 40% solution and above with both MPS B and MPS D, compared to at 100% only for MPS A and MPS C, and similar results were obtained in BCEpiC. Effects on resazurin reduction were also less in HCEpiC exposed to increasing doses of MPS A and MPS C than in cells exposed to MPS B and MPS D. After 15 min, HCEpiC viability measured by both resazurin reduction and cellular ATP levels was significantly lower for cells exposed to MPS B, MPS D, and MPS C, while HCEpiC exposed to MPS A were not affected. MPS B and MPS D reduced cell viability more than MPS A and MPS C over a 2-h time course in both HCEpiC and BCEpiC. CONCLUSIONS: Both MPS B and MPS D can cause large decreases in the viability of cultured corneal epithelial cells even with just a 2h exposure at multiple doses. Significant reduction in cell viability is evident at brief 15-30 min exposures. In contrast, MPS A and MPS C have significantly less effect on the cell viability of corneal epithelial cells at multiple doses, after these short exposure times.

Use of a human corneal epithelial cell line for screening the safety of contact lens care solutions in vitro.

PURPOSE: Sodium fluorescein permeability assay, alamarBlue assay, and scanning electron microscopy were performed to study the effects of various contact lens disinfecting multipurpose solutions (MPS) on the integrity of the ocular surface epithelium by using corneal epithelial cells. METHODS: The sodium fluorescein permeability and alamarBlue activity of monolayer cultures of human corneal epithelial cells were compared after exposure to ReNu MultiPlus, OPTI-FREE Express, AQuify 5 Minute, SOLO-care Plus With Aqualube, and Complete Moisture Plus contact lens care solutions for 15 minutes. Additional cell monolayers were prepared for each treatment and were analyzed with a scanning electron microscope after a 10-minute exposure. RESULTS: The sodium fluorescein permeability assay, alamarBlue assay, and scanning electron microscopy showed that OPTI-FREE Express was significantly more damaging to the human corneal epithelial cell monolayer than ReNu MultiPlus, SOLO-care Plus With Aqualube, Complete Moisture Plus, and AQuify 5 Minute contact lens solutions. Cell monolayers treated with OPTI-FREE Express were more permeable to sodium fluorescein and showed lower metabolic activity than cell monolayers treated with the other multipurpose solutions. CONCLUSIONS: This experiment shows that ReNu MultiPlus, SOLO-care Plus With Aqualube, Complete Moisture Plus, and AQuify 5 Minute contact lens solutions have a minimal effect on human corneal epithelial cells in culture, whereas OPTI-FREE Express has a higher negative effect on tight junctions, cell membranes, and overall metabolism of these human corneal epithelial cells.

Improving patient care outcomes by teaching quality improvement to medical students in community-based practices.

PURPOSE: As part of the Undergraduate Medical Education for the 21st Century (UME-21) project, the University of Connecticut School of Medicine developed and implemented a quality improvement curriculum. This study examined its impact on educational outcomes and the effect of the students' continuous quality improvement (CQI) projects on the quality of care delivered at community practice sites. METHOD: Seventy-seven second-year students working in groups of two to four conducted CQI projects on diabetes mellitus at 24 community-based primary care practices. They collected baseline data, implemented a results-specific intervention, and re-assessed quality indicators six months later. Students' knowledge, attitudes, and beliefs were evaluated using Likert-scale rated items as well as open-ended questions. RESULTS: A total of 513 charts were abstracted for the baseline sample, with 380 charts abstracted post-intervention. Attitudinal data revealed students acknowledged the benefit of outcomes measurement in clinical practice despite their frustration with the tedium of the chart-abstraction process. The rate of documentation of performances of foot and eye exams increased significantly from baseline to remeasurement (51.3% to 70.2%; p <.001 and 26.9% to 37.8%; p <.001, respectively). The mean value for glycohemoglobin dropped from 7.71% at baseline to 7.22% at remeasurement (p <.001). CONCLUSIONS: Medical student-driven CQI projects can improve the quality of care for diabetes at practices in which the students participate while introducing them and their preceptors to the process of quality measurement and improvement. Formative input from students should be used to optimize CQI experiences. Using medical students to lead CQI efforts in private practices may represent an underutilized resource to improve the care of patients in community-based practices.