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The Role of Calcium and pH in Epidermal physiology and disease: Ionic Homeostasis observed through Fluorescence lifetime imaging

Final Activity Report Summary - CA2+ AND PH IN SKIN (The Role of Calcium and pH in Epidermal physiology and Disease: Ionic Homeostasis Observed Through Fluorescence Lifetime Imaging)

The skin is composed of three main layers, subcutaneous fat for mechanical and temperature isolation, the dermis for mechanical resistance and the epidermis as a physical, chemical and immunological barrier to the external environment.

In order to properly form this physicochemical barrier, epidermis displays two unique and characteristic electrolytic gradients. Firstly, calcium (Ca2+), which regulates cell-physiologic processes including cell adhesion, growth, differentiation and apoptosis has a steep gradient towards the Stratum granulosum (SG), which controls Lamellar body (LB) secretion and thereby permeability barrier function. Secondly, an acidic pH of the Stratum corneum (SC), the functional endpoint in epidermal differentiation, is also required for normal permeability barrier function and regulates desquamation.

To better understand the role of ion fluxes, changes in ionic gradients, the functional significance of their localisation patterns within the epidermis and SC and their generation and influence on related proteins and structures, such as ion-transporters and tight junctions, Dr Behne and his colleagues pioneered the use of Fluorescence lifetime imaging microscopy (FLIM), applied to ex-vivo biopsies of intact skin for pH and Ca2+ measurements. Most descriptions of ionic concentrations and localisations by the time of the project elaboration were still based on outdated methodologies applied to both rodent and human skin, while the use of more advanced technology was limited to highly specialised laboratories, generally outside the medical field, and not applied to basic physiologic and biochemical, let alone clinical, questions.

At the outset of this proposal, a method for epidermal pH measurements via FLIM was established in collaboration at the Laboratory for Fluorescence Dynamics (LFD), then in Urbana-Champaign, Illinois, United States of America. Due to the technical complexities and the financial and personal investments that were necessary, this method still appeared, to my knowledge, not to be established neither there nor in any other laboratory working in epidermal research by the time of this proposal. Therefore, one major goal of this grant was to establish a lifetime-capable instrument in Hamburg and then use this instrument to continue the initiated work in collaboration between Dr Behne's prior research groups in San Francisco and Urbana, as mentioned in the first two research objectives.

An additional aim was to establish methods for the investigation of epidermal tight junctions in knockout mice, which was a prerequisite for the elucidation of the interaction of TJ, pH and Ca gradients, as mentioned in research objective three. Finally, several skin diseases and skin ageing were investigated with lifetime technology, as noted by research objective four. Localisation and dynamics of TJ were investigated in order to prepare these experiments. In addition, the almost completed clinical training of Dr Behne would be very helpful to include the appropriate patients for these studies.

In the course of this grant, Dr Behne managed to acquire funds so that a high-end research microscope was set up in Hamburg, equipped for lifetime experiments and high-speed ratiometric imaging. This was a very important achievement, even though it was not necessarily strictly scientific. In addition, he continued to develop calcium-FLIM as part of the continuing collaboration with LFD. Furthermore, he was given the opportunity to present these results to the national and international research communities which were the recipients for this outcome. He gave presentations at the German dermatological research society (ADF), the European Society for Dermatological Research (ESDR), the American Society for Dermatological Research (SID) and was offered, on each occasion, the opportunity of an oral presentation indicating the significant interest his research was generating. He was finally accepted for participation at the Gordon Conference on epithelial differentiation and keratinisation, which was the most reputable Gordon Conference in the field and had very limited capacity, thus completing the range of audiences for publication. At the closing of this grant, two manuscripts were close to completion, a number of abstracts and posters were presented and several related manuscripts were published.