A Novel Ex Vivo Blinking Method for Comparing the Corneal Residence Time of New Shampoo Formulations.

In the cosmetics sector, many products such as shampoos have a probability of accidental ocular exposure during their routine use. One very specific safety parameter is the residence time of the substance on the corneal surface, as prolonged exposure may cause injury. In this study, we developed a system that simulates corneal exposure to blinking and tear flow, for comparing the corneal clearance times of viscous detergent formulations. The Ex Vivo Eye Irritation Test (EVEIT), which uses corneal explants from discarded rabbit eyes from an abattoir, was used as the basis for the new system. To simulate blinking, we developed a silicone wiping membrane to regularly move across the corneal surface, under conditions of constant addition and aspiration of fluid, to mimic tear flow. Six shampoo formulations were tested and were shown to differ widely in their corneal clearance time. Three groups could be identified according to the observed clearance times (fast, intermediate and slow); the reference shampoo had the shortest clearance time of all tested formulations. With this new system, it is now possible to investigate an important physicochemical parameter, i.e. corneal clearance time, for the consideration of ocular safety during the development of novel cosmetic formulations.

A group of cationic amphiphilic drugs activates MRGPRX2 and induces scratching behavior in mice.

BACKGROUND: Mas gene-related G protein-coupled receptors (MRGPRs) are a G protein-coupled receptor family responsive to various exogenous and endogenous agonists, playing a fundamental role in pain and itch sensation. The primate-specific family member MRGPRX2 and its murine orthologue MRGPRB2 are expressed by mast cells mediating IgE-independent signaling and pseudoallergic drug reactions. OBJECTIVES: Our aim was to increase knowledge about the function and regulation of MRGPRX2/MRGPRB2, which is of major importance in prevention of drug hypersensitivity reactions and drug-induced pruritus. METHODS: To identify novel MRGPR (ant)agonists, we screened a library of pharmacologically active compounds by utilizing a high-throughput calcium mobilization assay. The identified hit compounds were analyzed for their pseudoallergic and pruritogenic effects in mice and human. RESULTS: We found a class of commonly used drugs activating MRGPRX2 that, to a large extent, consists of antidepressants, antiallergic drugs, and antipsychotics. Three-dimensional pharmacophore modeling revealed structural similarities of the identified agonists, classifying them as cationic amphiphilic drugs. Mast cell activation was investigated by using the 3 representatively selected antidepressants clomipramine, paroxetine, and desipramine. Indeed, we were able to show a concentration-dependent activation and MRGPRX2-dependent degranulation of the human mast cell line LAD2 (Laboratory of Allergic Diseases-2). Furthermore, clomipramine, paroxetine, and desipramine were able to induce degranulation of human skin and murine peritoneal mast cells. These substances elicited dose-dependent scratching behavior following intradermal injection into C57BL/6 mice but less so in MRGPRB2-mutant mice, as well as wheal-and-flare reactions following intradermal injections in humans. CONCLUSION: Our results contribute to the characterization of structure-activity relationships and functionality of MRGPRX2 ligands and facilitate prediction of adverse reactions such as drug-induced pruritus to prevent severe drug hypersensitivity reactions.

P13-A113†The EVEIT bioreactor system as platform for artificial cornea prototypes.

PURPOSE: Corneal donor tissue is in short supply. Only a fraction of the demand is satisfied. The tissues can vary in quality and sometimes have limited use. To address the issue, the generation of artificial corneal grafts is intensively researched.Various aspects of these prototypes need to be tested, ranging from structural integrity to cellular morphology. Our Ex Vivo Eye Irritation Test (EVEIT) is based on an air-lift organ culture system, where we currently are using rabbit corneas from food industry. We constantly expanded our capabilities in quantifying various parameters concerning metabolism, structural integrity and optical properties. This also opens up the possibility of using the system as a testing platform for prototypical artificial corneal constructs. METHODS: Various ophthalmological aspects can be investigated using the EVEIT system:Self-healing of superficial injuries and morphological characteristics can be observed over several days by live-tissue staining macroscopy.Metabolic parameters are recordable via the endothelial nutrient supply mechanism.Acute changes in internal pressure can be measured in the artificial anterior chamber with high resolution.Corneal barrier functions and pharmacokinetic properties can be quantified using photometric analysis methods.Dry-Eye model and established corneal edema models can be employed to test the efficacy of potential therapeuticsAdvanced 3D design and printing methods allow us to quickly adapt the bioreactor, for example, to incorporate human corneas or to improve the mobility of the system.In order to comply with the 3Rs principle, testing of several different chemicals on one cornea is now also possible with the aid of automated multi-applicationRecent developments of the EVEIT system include the engineering of an artificial eyelid model. RESULTS: Our long experience in using and optimizing the EVEIT system led to a unique adaptability to accommodate different testing conditions and requirements. Established disease models such as corneal edema and in dry eye syndrome (in process) are involved in testing new drugs. CONCLUSION: Our established EVEIT system, in addition to its experimental capabilities, could contribute to the development of artificial corneal grafts in the future, as we have shown in previous work. The flexibility of the system allows us to adjust and improve an enormous range of test conditions and parameters.

P34-A132†Air-lift cultivation of human corneal donor tissues.

PURPOSE: Corneal donor tissue can be used for a number of different reconstructive surgical operations involving the rehabilitation of injured or degraded anterior, posterior and intermediate corneal lamellas.Potential corneal donor tissues undergo a rigorous screening process including medical evaluation of the endothelial and stromal layers. Depending on this assessment, the tissue´s scope of use is often narrowed down to few types of emergency procedures mainly due to an insufficient number of viable endothelial cells or divergent cell morphology.In addition to all these limitations, one must not ignore the sometimes critical post-preparation degeneration caused by the submerged culturing process itself, leading to epithelial debridement and stromal edema. All these factors reduce the already short supply of donor corneas. In this study, we aim to optimize this culturing process to avoid tissue degradation. METHODS: We used an organ culturing system based on our long established Ex Vivo Eye Irritation Test System (EVEIT) (Spöler et al., 2015). This bioreactor has been modified in size and shape to accommodate human-sized corneal explants. The established mechanisms for supplying the cornea with nutrients and physiologically relevant pressure conditions were adapted to support sterility. Human donor corneas that failed the initial quality protocol and which are released for research were obtained from our cornea bank and inserted in the EVEIT culture system. Corneal integrity was observed during the cultivation period of 19 days. RESULTS: The human cornea observed maintained transparency in contrast to what generally can be observed in the established European culturing system with submersion of the cornea. Final endothelial layer examinations confirmed the presence of viable endothelial cells, as documented during initial corneal bank quality control. CONCLUSION: With this proof of principle, we confirmed that we can maintain the integrity of the human donor cornea in our modified EVEIT organ culture system. Further investigation, optimization and confirmation will be pursued to meet medical regulations.

Defining corneal chemical burns: A novel exact and adjustable ocular model.

INTRODUCTION: Live-animal-free ocular toxicity models and tests are a necessity in multiple branches of medicine, industry and science. Corneal models with adjustable ranges of injury severities do not exist. In this work, a novel and precise and dose - response method to induce and observe ex vivo corneal chemical burns has been established. METHODS: The EVEIT (Ex Vivo Eye Irritation Test) is based on an ex vivo corneal organ model for rabbit corneas from food industry. Further, a highly precise three - axis workstation has been employed to apply liquid corrosive, sodium hydroxide (NaOH), droplets in a nanolitre (nL) range onto the corneal surface. Optical Coherence Tomography (OCT) has been used to observe and quantify the elicited changes in the corneal layers. RESULTS: The speed and intervals of single nanodroplet application played a crucial role in the extent of the corneal changes. Similar total volumes applied at low frequencies elicited deep and extensive changes in the corneal layers whereas high application frequencies elicited comparatively superficial changes. Increasing NaOH concentrations effected measurably increasing corneal changes. Increasing the volume of applied NaOH also showed an increase in corneal changes. CONCLUSIONS: OCT imaging proved to be effective in observing, documenting and quantifying the changes in the corneal layers. The ex vivo model, in conjunction with the novel application method was able to induce and display distinctive and consistent correlations between NaOH volume, concentration and elicited corneal changes. This ex vivo ocular chemical burn model provides a consistent in vitro basis for pharmaceutical and toxicological experiments and investigations into corneal chemical burn mechanisms and treatment.

Activating Calcium-Sensing Receptor Mutations: Prospects for Future Treatment with Calcilytics.

Activating mutations of the G protein-coupled receptor, calcium-sensing receptor (CaSR), cause autosomal dominant hypocalcemia and Bartter syndrome type 5. These mutations lower the set-point for extracellular calcium sensing, thereby causing decreased parathyroid hormone secretion and disturbed renal calcium handling with hypercalciuria. Available therapies increase serum calcium levels but raise the risk of complications in affected patients. Symptom relief and the prevention of adverse outcome is currently very difficult to achieve. Calcilytics act as CaSR antagonists that attenuate its activity, thereby correcting the molecular defect of activating CaSR proteins in vitro and elevating serum calcium in mice and humans in vivo, and have emerged as the most promising therapeutics for the treatment of these rare and difficult to treat diseases.

Heterozygous inactivating CaSR mutations causing neonatal hyperparathyroidism: function, inheritance and phenotype.

BACKGROUND: Homozygous inactivating mutations of the calcium-sensing receptor (CaSR) lead to neonatal severe hyperparathyroidism (NSHPT), whereas heterozygous inactivating mutations result in familial hypocalciuric hypercalcemia (FHH). It is unknown why in some cases heterozygous CaSR mutations cause neonatal hyperparathyroidism (NHPT) clinically similar to NSHPT but with only moderately elevated serum calcium. METHODS: A literature survey was conducted to identify patients with heterozygous CaSR mutations and NHPT. The common NHPT CaSR mutants R185Q and R227L were compared with 15 mutants causing only FHH in the heterozygous state. We studied in vitro calcium signaling including the functional consequences of co-expression of mutant and wild-type (wt) CaSR, patients' phenotype, age of disease manifestation and mode of inheritance. RESULTS: All inactivating CaSR mutants impaired calcium signaling of wt-CaSR regardless of the patients' clinical phenotype. The absolute intracellular calcium signaling response to physiologic extracellular calcium concentrations in vitro showed a high correlation with patients' serum calcium concentrations in vivo, which is similar in NHPT and FHH patients with the same genotype. Pedigrees of FHH families revealed that paternal inheritance per se does not necessarily lead to NHPT but may only cause FHH. CONCLUSIONS: There is a significant correlation between in vitro functional impairment of the CaSR at physiologic calcium concentrations and the severity of alterations in calcium homeostasis in patients. Whether a particular genotype leads to NHPT or FHH appears to depend on additional predisposing genetic or environmental factors. An individual therapeutic approach appears to be warranted for NHPT patients.