Loss-of-function mutations in the filaggrin gene(FLG) have recently been shown to be strongly associated with atopic dermatitis (AD). The overall aim of this study was to explore the role of filaggrin in skin barrier function and AD. There were two main focuses in this study. The first was a functional study whose primary objective was to determine if FLG mutations correlated with skin barrier dysfunction in AD. Fifty-five mild to moderate AD individuals were recruited, genotyped and had their skin barrier assessed using three different measures - transepidermal water loss (TEWL), skin capacitance and the number of tape strips required to abrogate skin barrier. A secondary aim of this functional study was to test the hypothesis that corneocytes were less adherent to one another in filaggrin-related AD compared to wild-type AD skin. The second main focus of this thesis was a structural study aimed at interrogating the structure-function relationship of filaggrin. Filaggrin protein was extracted and purified from a total of 21 AD and non-AD subjects and analysed using mass spectrometric techniques. Specifically, matrix assisted laser desorption/ionisation time-of-flight(MALDI-TOF)mass spectrometry (MS) and nano liquid chromatography tandem MS(LC-MS/MS) were utilised. Part of this structural study also involved developing and optimising the extraction and purification of filaggrin protein, including a novel way of extracting filaggrin from skin using tape stripping. In addition, novel filaggrin-specific enzyme-linked immunosorbant assay (ELISA) was also developed, which could serve as a useful screening test for the protein. In this study, FLG mutations were found to correlate with higher TEWL and fewer number of tape strips required to abrogate skin barrier, but not with skin capacitance. FLG mutations were also not shown to correlate with AD severity. The mean amount of protein extracted from filaggrin-related AD skin was also significantly higher compared to wild-type AD skin, supporting the hypothesis that corneocytes were less adherent to one another (and therefore, densely packed) in filaggrin-related AD skin. MS analysis of filaggrin confirmed the heterogeneic nature of filaggrin protein, even within a single individual. Interestingly, this structural study also showed that filaggrin was only minimally expressed in the skin of all the AD individuals studied, whether or not they possessed any FLG mutation. Due to the limited amount of filaggrin extracted from AD skin, it was not possible to conduct comparative structural analysis between filaggrin from AD and non-AD skin.

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Atopic dermatitis, AD (synonym eczema) is a chronic inflammatory skin disease. It affects between 10 to 20% of children and 1 to 3% of adults worldwide. It is an important cause of morbidity and is estimated to cost £465 million per annum to the UK. AD is part of a family of Th-2 driven diseases and is often the first of these atopic diseases to manifest. The development of AD is often followed by asthma and allergic rhinitis later in life (a phenomenon known as the ‘atopic march’). Up to 50% of moderate to severe AD cases have been associated with genetic mutations affecting the epidermal barrier protein filaggrin. Filaggrin aggregates keratin filaments during terminal keratinocyte differentiation, allowing normal epidermal stratification. The role of filaggrin in maintaining a functional skin barrier is further supported by a clinical study conducted by ourselves. This is the first clinical study on a European cohort (58 participants) which showed that FLG mutations were associated with experimentally demonstrable defects of skin barrier function (increased baseline transepidermal water loss), more so following exposure to a chemical irritant. However, the majority of patients with AD, especially the milder cases, do not have FLG mutations. Some of the wild-type patients in our study were noticed to have accumulation of the large filaggrin proprotein and a lack of filaggrin monomers, indicating defective proteolysis of profilaggrin into the functional monomers. Our study also found disproportionately raised protease inhibitory activities amongst the AD participants. This inappropriately raised protease inhibition may interfere with profilaggrin proteolysis, leading to the development of AD in some wild-type patients. Having demonstrated that deficiency of filaggrin monomers is associated with a defective skin barrier, we focused on the function of filaggrin in the skin and attempted to improve the skin barrier function. In addition to keratin aggregation, filaggrin constitutes the natural moisturizing factors in the epidermis following its natural breakdown into amino acids. We note that filaggrin is disproportionately rich in amino acid histidine, implying that this amino acid may have a particular significance in maintaining a functional epidermal barrier. Using an in-house skin-equivalent model, we have shown that by increasing the histidine content in the cell culture media, we could increase the expression of filaggrin monomers and reduce the penetration of a fluorescent dye into the skin-equivalents. The latter indicates improved barrier function. Finally, we conducted a pilot human study which showed that histidine, when applied to mechanically damaged skin in AD and healthy participants, was associated with a faster recovery of the skin barrier function. These studies suggest that histidine is of therapeutic benefits in AD. A histidine-based treatment may be developed as an alternative to current anti-inflammatory and immunosuppressive agents used to treat AD.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Author: Denisa Kubátová Supervisor: PharmDr. Andrej Kováčik, Ph.D. Consultant: PharmDr. Lukáš Opálka, Ph.D. Title of diploma thesis: Study of sphingosine, dihydrosphingosine and phytosphingosine in skin barrier models The stratum corneum (SC), the uppermost layer of the skin, localized in the uppermost part of the epidermis, represents the skin barrier of the organism. SC is composed of corneocytes and an intercellular lipid matrix, which is formed by ceramides (Cer), free fatty acids (FFA), and cholesterol (Chol) in an equimolar ratio. Substances from the group of sphingolipids - Cer, are sphingoid bases (for example, sphingosine (S), dihydrosphingosine (dS), phytosphingosine (P)) acylated with a fatty acid (for example, lignoceric acid (LIG)). In the lipid matrix, the metabolic products of Cer (free sphingoid bases) are also present, but their role in SC barrier functions is not clear. Some studies show that Cer with different sphingoid bases, and increased presence of free sphingoid bases, can lead to a change in the permeability of the skin barrier. This work aimed to study the effect of permeability of sphingoid bases on the model membrane permeability. Nine types of membranes were prepared; they...

Aims and objectives: The research aims and objectives of this study were: Firstly to determine the positive effects and possible disadvantages of three types of barrier creams on skin barrier function by determining skin barrier function by measuring stratum corneum hydration transepidermal water loss (TEWL) and skin surface pH. Secondly to compare different racial skin types (African skin to Caucasian skin) by determining the effects of barrier cream on skin barrier function. Finally to compare the effect of the three different barrier creams on four different anatomical areas. Methods: Thirty eight non-smoking male test subjects took part in this study where three different types of barrier creams were tested on their arms and hands in a controlled laboratory environment. The thirty eight test subjects consisted of nineteen African and nineteen Caucasian test subjects. Three parameters were measured namely TEWL, stratum corneum hydration and pH condition of the skin. TEWL was measured using a Vapometer (Delfin Technology Ltd. Finland). The Multi probe Adapter system (MPA) (Courage and Khazaka, Germany) was used with a temperature and humidity sensor and with the following probes all from Courage and Khazaka, Germany: a Corneometer measuring skin hydration and a pH-Meter measuring skin surface pH. The measurements were repeated on each of the four sampling areas (forearm, wrist, back of hand and palm) with a reasonable time interval between each measurement. After the baseline measurement the barrier cream was applied by the researcher on the test subjects’ dominant arm. The long term effects were determined after the baseline measurement in intervals of 2 hours. Directly after each measurement the barrier cream was reapplied. Results: Gloves In A Bottle™ increased stratum corneum hydration, had no effect on TEWL and increased skin surface pH, whereas Reinol™ increased stratum corneum hydration and decreased TEWL and had no effect on pH values. Travabon™ decreased stratum corneum hydration and TEWL and had no effect on skin surface pH. The results indicated that there were significant differences between Caucasian and African test subjects with the use of barrier creams, because of the baseline differences and the reaction to barrier creams showed different results. There were also statistically significant differences in the four different anatomical areas where the barrier creams were applied. Conclusion: Barrier creams are beneficial in the workplace, although it should be taken into consideration that different ethnicities react differently to barrier creams under different workplace situations and therefore this should be taken into account when selecting a barrier cream. / Thesis (MSc (Occupational Hygiene))--North-West University, Potchefstroom Campus, 2013

Aims and objectives: The research aims and objectives of this study were: Firstly to determine the positive effects and possible disadvantages of three types of barrier creams on skin barrier function by determining skin barrier function by measuring stratum corneum hydration transepidermal water loss (TEWL) and skin surface pH. Secondly to compare different racial skin types (African skin to Caucasian skin) by determining the effects of barrier cream on skin barrier function. Finally to compare the effect of the three different barrier creams on four different anatomical areas. Methods: Thirty eight non-smoking male test subjects took part in this study where three different types of barrier creams were tested on their arms and hands in a controlled laboratory environment. The thirty eight test subjects consisted of nineteen African and nineteen Caucasian test subjects. Three parameters were measured namely TEWL, stratum corneum hydration and pH condition of the skin. TEWL was measured using a Vapometer (Delfin Technology Ltd. Finland). The Multi probe Adapter system (MPA) (Courage and Khazaka, Germany) was used with a temperature and humidity sensor and with the following probes all from Courage and Khazaka, Germany: a Corneometer measuring skin hydration and a pH-Meter measuring skin surface pH. The measurements were repeated on each of the four sampling areas (forearm, wrist, back of hand and palm) with a reasonable time interval between each measurement. After the baseline measurement the barrier cream was applied by the researcher on the test subjects’ dominant arm. The long term effects were determined after the baseline measurement in intervals of 2 hours. Directly after each measurement the barrier cream was reapplied. Results: Gloves In A Bottle™ increased stratum corneum hydration, had no effect on TEWL and increased skin surface pH, whereas Reinol™ increased stratum corneum hydration and decreased TEWL and had no effect on pH values. Travabon™ decreased stratum corneum hydration and TEWL and had no effect on skin surface pH. The results indicated that there were significant differences between Caucasian and African test subjects with the use of barrier creams, because of the baseline differences and the reaction to barrier creams showed different results. There were also statistically significant differences in the four different anatomical areas where the barrier creams were applied. Conclusion: Barrier creams are beneficial in the workplace, although it should be taken into consideration that different ethnicities react differently to barrier creams under different workplace situations and therefore this should be taken into account when selecting a barrier cream. / Thesis (MSc (Occupational Hygiene))--North-West University, Potchefstroom Campus, 2013

Cholesterol is a substance of a steroidal nature that has a number of functions in the human body. One of them is also an irreplaceable role in the proper functioning of the skin barrier. Cholesterol is an integral part of the lipid matrix, together with ceramides and free fatty acids in an equimolar ratio (1: 1: 1), and 5% cholesterol sulfate, which fills the intercellular space between stratum corneum cells and is responsible for the barrier properties of the skin. Cholesterol is therefore necessary for epidermal homeostasis, but its role in SC permeability is unknown. The aim of this work was to study the influence of cholesterol concentration on the permeability and microstructure of model lipid membranes of the skin barrier. Eight sets of model membranes with decreasing cholesterol concentrations (100%, 80%, 70%, 60%, 40%, 20%, 0%) and cholesterol alone were studied for which permeability and microstructure were monitored.The study of permeability of membranes was carried out in the Franz diffusion cells by measuring four permeation parameters: water loss, electrical impedance measurement, and the cumulative amount of two model drugs (theophylline and indomethacin). The microstructure of these model membranes was verified by X-ray powder diffraction. X-ray powder diffraction measurements...

Title of the Master thesis: Synthesis and evaluation of human 6-hydroxyceramides Candidate: Adam Majcher Supervisor: PharmDr. Lukáš Opálka, Ph.D. Consultant: PharmDr. Andrej Kováčik, Ph.D. Charles University, Faculty of Pharmacy in Hradec Králové Skin Barrier Research Group, Department of Organic and Bioorganic Chemistry Ceramides (Cer), the members of sphingolipid family, occur in all human cells and play an important role in cell signaling. In high concentrations, Cer can also be found in the uppermost layer of epidermis called stratum corneum, along with free fatty acids and cholesterol (in equimolar ratio), where they form the intercellular multi-lamellar lipid matrix. The key function of stratum corneum is to ensure a permeability barrier, thus, to provide water and electrolyte homeostasis, and to prevent entry of harmful substances into the organism. Cer are composed of a sphingoid base and an acyl part derived from a long-chain fatty acid. Cer based on 6-hydroxysphingosine (H) are amongst the most unusual sphingolipids. In contrast to sphingosine-based Cer, 6-hydroxysphingosine-based Cer (H-Cer) are unique for the epidermis and, in addition, H-Cer are not typical for all mammals. Moreover, the function and biosynthesis of H-Cer in the skin is still not completely understood. Several...

Charles University, Faculty of Pharmacy in Hradec Králové Department of Organic and Bioorganic Chemistry Pavla Audrlická Supervisor: prof. PharmDr. Kateřina Vávrová, Ph.D. Title of diploma thesis: Study of cholesterol depletion in human skin barrier Barrier function of human skin barrier dwells in intercellular lipid membranes of the uppermost skin layer, the stratum corneum (SC), composed of equimolar mixture of ceramides (Cer), free fatty acids (FFA) and cholesterol (Chol).1 Chol is required for proper lipid organization of SC, however, it stays unclear, why is it present in an amount so high that it separates from other lipids.2 Experiments using synthetic model membranes with decreased Chol content suggested that molar ratio of Cer:FFA:Chol 1:1:0,4 is sufficient for lipid barrier formation and its complex functionality.3 The aim of this work was to manipulate Chol content directly in human SC and to study the effects of decreased Chol content on the SC permeability and microstructure. Ex vivo SC obtained from healthy donors was extracted by methyl-β-cyclodextrin (MCD) to reduce natural Chol content. The extracted SC did not show significant changes in Cer or FFA whilst the amount of Chol was lowered to 78 %. SC barrier properties were evaluated by measurements of transepidermal water loss...

Charles University Faculty of Pharmacy in Hradec Králové Department of pharmeceutical technology Candidate: Oksana Yanok Supervisor: Pharm.Dr. Andrej Kováčik, Ph.D. Title of Diploma Thesis: The effects of glucosyl sphingosine on barrier function of skin and complex skin model. The skin barrier, which provides protection from water loss and harmful environmental influences is located in the stratum corneum. The dominant group of lipids within the stratum corneum are ceramides (Cer), which also have the most important role in ensuring the barrier properties of the skin. The enzymes sphingomyelin deacylase and glucosylceramide deacylase hydrolyze the amide bond of Cer precursors, which leads to highly polar metabolites, called lysolipids. The increased activity of these enzymes is considered to be one of the major factors leading to the development of a number of skin diseases characterized by a skin barrier disorder (for example atopic dermatitis). In this study we prepared model membranes mimicking a healthy skin barrier as an equimolar mixtures of human Cer, cholesterol, free fatty acids with the addition of 5 % cholesterol sulfate. We also prepared models in which the amount of Cer was gradually reduced and replaced by the hydrophilic lysolipid glucosylsphingosine. The permeability was measured...