Improved sample preparation for MALDI-MSI of endogenous compounds in skin tissue sections and mapping of exogenous active compounds subsequent to ex-vivo skin penetration.

Localization of endogenous and exogenous compounds directly in tissue sections is a challenging task in skin research. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a powerful label-free technique that enables determination of the distribution of a large range of biomolecules directly in tissue sections. Nevertheless, its application in this field is limited in large part by the low adhesion of skin tissue sections to indium-tin oxide-coated (ITO) glass slides. For the first time corona discharge (CD) treatment was used to modify the glass slide surface for improved adhesion. Localization of endogenous cholesterol sulfate was performed directly in human skin tissue sections. A spatial resolution of approximately 30 µm was sufficient for assignment of mass signals to skin structure morphology. Furthermore, imaging of an exogenous model compound, Nile red, was performed directly in skin tissue sections after ex-vivo penetration into porcine skin, enabling determination of the pathway and depth of penetration. Finally, the ion density map of Nile red was compared with its high resolution fluorescence micrograph. This work provides new insights into the application of MALDI-MSI in skin research.

Stiffening of human skin fibroblasts with age.

Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ∼60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix.

A circadian clock in HaCaT keratinocytes.

To anticipate daily environmental changes, most organisms developed endogenous timing systems, the so-called circadian (∼24 hours) clocks. Circadian clocks exist in most peripheral tissues and govern a huge variety of cellular, metabolic, and physiological processes. Recent studies have suggested daytime-dependent variations in epidermal functions such as barrier recovery and pH homeostasis. However, a local circadian clock in epidermal keratinocytes has not been reported yet, and as such the molecular link between the circadian system and epidermal physiology remains elusive. In this study we describe a functional cell autonomous circadian clock in human adult low calcium temperature (HaCaT) keratinocytes. Using live-cell bioluminescence imaging and mRNA expression time series, we show robust circadian transcription of canonical clock genes in synchronized HaCaT keratinocytes. Genetic and pharmacological perturbation experiments as well as the phase relations between clock gene rhythms confirm that the molecular makeup of the HaCaT keratinocyte clock is very similar to that of other peripheral clocks. Furthermore, temperature was identified to be a potent time cue (Zeitgeber) for the epidermal oscillator. Temperature cycles entrain HaCaT keratinocytes, leading to the identification of rhythmic expression of several genes involved in epidermal physiology such as cholesterol homeostasis and differentiation. Thus, we present HaCaT keratinocytes as an excellent model to study the regulation of keratinocyte physiology by the circadian clock in a simple yet robust in vitro system.

In vitro detection of contact allergens: development of an optimized protocol using human peripheral blood monocyte-derived dendritic cells.

Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with relevant social and economic impacts. Animal experiments (e.g. the local lymph node assay) are still supplying most of the data used to assess the sensitization potential of new chemicals. However, the 7th amendment to the EU Cosmetic Directive will introduce a testing ban for cosmetic ingredients after 2013. In vitro alternative methods are thus being actively developed. Although promising results have been obtained with cell lines, their reduced functionality and inherent genomic instability led us to reinvestigate the use of peripheral blood monocyte-derived dendritic cells (PBMDCs) for the establishment of a reliable in vitro sensitization test. To solve the issues associated with the use of primary cells, the culture and exposure conditions (cytokine concentrations, incubation time, readout, pooled vs. single donors and cytotoxicity) were re-assessed and optimized. Here we propose a stable and reproducible protocol based on PBMDCs. This should allow a wider acceptance of PBMDCs as a reliable test system for the detection of human skin sensitizers and the inclusion of this protocol in an integrated testing strategy.

Stiffening of human skin fibroblasts with age.

Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ∼60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix.

Phase behavior of liquid-crystalline emulsion systems.

The phase behavior of a mixture containing a surfactant, fatty alcohols and water has been analyzed. Depending on the amount of surfactant, i.e. N-(3-dimethylaminopropyl) octadecanamide, the emulsion-like system forms different microstructures. With increasing surfactant content the formulation evolves from a system with lyotropic lamellar phases to a system with crystal layer phases. (13)C-CPMAS NMR studies carried out at varying surfactant levels showed significant differences in the behavior of the system. Using (2)H and (13)C-CPMAS NMR, X-ray scattering, DSC and polarization microscopy a phase diagram of this system could be derived. Additionally, ultrasonic velocity measurements showed that the ripening process of the emulsions can take up to 2 weeks and longer.

Real-time monitoring of membrane cholesterol reveals new insights into epidermal differentiation.

Cholesterol is organized in distinctive liquid-ordered micro-domains within biological membranes called lipid rafts. These micro-domains direct multiple physiological functions in mammalian cells by modulating signaling processes. Recent findings suggest a role for lipid rafts in cellular processes in human keratinocytes such as early differentiation and apoptosis. However, research of lipid rafts is hindered by technological limitations in visualizing dynamic cholesterol organization in plasma membranes. This study addresses a real-time, non-invasive method for the long-term observation of cholesterol reorganization in plasma membranes. In addition, this study also addresses the dynamic process of cholesterol depletion and repletion in primary human keratinocytes. Cholesterol reorganization was measured by observed changes in cellular impedance. Disruption of lipid rafts with low concentrations of methyl-beta-cyclodextrin (MbetaCD) resulted in an increase in the proliferative capacity of keratinocytes, which was assessed using real-time proliferation curves and adenosine triphosphate (ATP)-based proliferation assays. Quantitative PCR showed a concomitant decrease in messenger RNA (mRNA) expression of the early differentiation markers keratins 1 and 10. Conversely, specific cholesterol reintegration led to a 4.5-fold increase in keratin 2 mRNA expression, a marker for late keratinocyte differentiation, whereas depletion resulted in a significant downregulation. These findings imply a strictly controlled mechanism for the regulation of membrane cholesterol composition in both early and terminal keratinocyte differentiation. The impedance-based method that this study addresses further enhances our understanding of how physiological processes in keratinocytes are controlled by membrane cholesterol.

A novel in vitro method for the detection and characterization of photosensitizers.

Photoactivation and binding of photoactive chemicals to proteins is a known prerequisite for the formation of immunogenic photoantigens and the induction of photoallergy. The intensive use of products and the availability of new chemicals, along with an increasing exposure to sun light contribute to the risk of photosensitizing adverse reactions. Dendritic cells (DC) play a pivotal role in the induction of allergic contact dermatitis. Human peripheral blood monocyte derived dendritic cells (PBMDC) were thus perceived as an obvious choice for the development of a novel in vitro photosensitization assay using the modulation of cell surface protein expression in response to photosensitizing agents. In this new protocol, known chemicals with photosensitizing, allergenic or non-allergenic potential were pre-incubated with PBMDCs prior to UVA irradiation (1 J/cm(2)). Following a 48 h incubation, the expression of the cell surface molecules CD86, HLA-DR and CD83 was measured by flow cytometry. All tested photosensitizers induced a significant and dose-dependent increase of CD86 expression after irradiation compared to non-irradiated controls. Moreover, the phototoxicity of the chemicals could also be determined. In contrast, (i) CD86 expression was not affected by the chosen irradiation conditions, (ii) increased CD86 expression induced by allergens was independent of irradiation and (iii) no PBMDC activation was observed with the non-allergenic control. The assay proposed here for the evaluation of the photoallergenic potential of chemicals includes the assessment of their allergenic, phototoxic and toxic potential in a single and robust test system and is filling a gap in the in vitro photoallergenicity test battery.

Modification of vimentin: a general mechanism of nonenzymatic glycation in human skin.

In a recent study, we were able to show that the intermediate filament protein vimentin aggregates in human dermal fibroblasts because of modification by the advanced glycation endproduct carboxymethyllysine (CML). In this work, we investigated the formation of intracellular CML in relation to the concentration of glucose in the culture medium. The natural degradation product of glucose, methylglyoxal, was able to induce the aggregation of vimentin. This dicarbonyl leads to the formation of the modifications MG-H1 and carboxyethyllysine (CEL) as a result of the reaction with arginine and lysine residues of proteins. Furthermore, we found that the protein vimentin was modified, not only by CML and CEL, but also by pentosidine and pyrraline. These findings underline the special position of vimentin as a preferential target of the Maillard reaction in human skin.

Non-invasive monitoring of oxidative skin stress by ultraweak photon emission (UPE)-measurement. I: mechanisms of UPE of biological materials.

BACKGROUND/PURPOSE: Oxidation of proteins and amino acids is associated with generation of ultraweak photon emission (UPE), which may be used to assess oxidative processes in the skin in a non-invasive way. This first part of a series of reports addresses the physicochemical basis of oxidation-induced UPE in the skin, with a special focus on the contribution of amino acid oxidation. METHODS: UPE of biological samples and protein/amino acid solutions following oxidation with H(2)O(2) in the presence of Fe(2+) was recorded using a sensitive photomultiplier system. Signals were analyzed with regard to overall signal intensity and spectral distribution. RESULTS: Increasing concentrations of H(2)O(2) in aqueous bovine serum albumin solutions induced linearly correlated UPE and protein carbonyl compounds, with a substantially higher sensitivity for the measurement of UPE. In single amino acid solutions, strong UPE signals were generated by oxidation from Phe, Trp, His, and Cys, and weak signals from Lys and Thr. Analysis of reaction products by MS revealed high oxidative material turnover for Cys and His, whereas barely detectable oxidative material turnover seems to be sufficient to generate a UPE signal of similar strength from Trp and Phe. Combination of different amino acids did not result in a simple addition of individual oxidation-induced UPE signals, but in interactions ranging from antagonism to clear synergism. Synergism was evident between Trp- and UPE-generating amino acids such as Thr, Cys, and His, with the strongest synergism by far observed between Trp and His. The strikingly different individual UPE spectra of His and Trp, despite being of comparable overall strength, were congruent with a pure Trp UPE spectrum after combining His with Trp in solution, indicating energy transfer between both amino acids. Combination of Trp and DNA, which also gives UPE signals following oxidation, did not result in a synergistically enhanced or antagonized overall UPE signal, but in a simple addition of individual UPE signals. CONCLUSION: Measurement of UPE could be proven to be a highly sensitive method to assess oxidative processes in biological molecules. The reported data indicate that UPE generated by oxidation stressed skin is mainly due to non-fluorescent photon emission via Trp, whereby Trp acts as an energy receptor from other excited species of oxidation-modified amino acids.

Non-invasive monitoring of oxidative skin stress by ultraweak photon emission measurement. II: biological validation on ultraviolet A-stressed skin.

BACKGROUND/PURPOSE: Several physical or chemical environmental stressors generate reactive oxygen species, which trigger oxidation reactions of cells or tissues and thereby induce a correlated ultraweak photon emission (UPE) signal. The present study was designed to qualify and validate UPE measurement following ultraviolet (UV) excitation of porcine and human skin as an analytical method to assess the potency of topical antioxidants in vivo. METHODS: UPE of porcine skin in vitro and human skin in vivo following excitation with UVA was recorded using sensitive photomultiplier systems. For validation purposes, the effects of variation of extrinsic and intrinsic parameters encompassing skin thickness, humidity, temperature, pH, and composition of the surrounding atmosphere were assessed. Signals were analyzed with regard to overall signal intensity and spectral distribution. In two clinical trials enrolling 20 volunteers each, the effects of topical antioxidant treatment on UVA-induced UPE were validated. RESULTS: Different stressors encompassing exposition to ozone, UVA irradiation, or even cigarette smoke induced UPE of skin. Critical parameters affecting the quality and quantity of the UPE signal were the spectral composition of the exciting UV light, skin temperature, skin humidity, and the O(2) concentration of the surrounding atmosphere. Generally, UVA-induced UPE decreased with increasing temperature, humidity, and O(2) concentration. Skin pH had no significant effect on UPE with regard to signal quality and quantity over a pH range of 2.8-8.2. In a clinical study UPE measurement following UVA excitation could precisely reflect a dose-dependent antioxidant effect of topically applied vitamin C and alpha-glucosylrutin. CONCLUSION: Our data indicate that UVA irradiation induces UPE especially in deeper (living) skin layers, where antioxidants must be active in order to interfere with accelerated skin ageing. Based on the clinical data, and with knowledge of modulating external variables, UPE measurement following UV excitation can be qualified as a reliable and valid method for the non-invasive measurement of antioxidant efficacy on the skin.

A strategy for correlative microscopy of large skin samples: towards a holistic view of axillary skin complexity.

Knowledge about the structural elements of skin and its appendices is an essential prerequisite for understanding their complex functions and interactions. The hence necessary morphological description across several orders of scale not only requires the investigation at the light microscopic level but also ultrastructural investigation, ideally on the identical sample. For a correlative and multimodal observation one unique preparation protocol is mandatory. As a compromise between sample sizes of >500 microm in diameter on the one hand and optimal preservation of antigenicity and morphology on the other, we developed a new preparation protocol that allows (i) 3D reconstruction of the resin-embedded sample by confocal light microscopy prior to (ii) direct immunolocalization of target proteins within selected sample planes by light and fluorescence microscopy or transmission electron microscopy. Alternatively, (iii) serial cryosections of the frozen sample can be taken for characterizing the sample in toto. With this unique approach we were able to fully demonstrate the structural complexity of axillary skin samples, increasing the structural resolution from 3D reconstruction of the whole gland up to ultrastructural investigations at the subcellular level. We could demonstrate that axillary sweat glands are not separately distributed, as has been assumed to date; instead, they seem to be intricately twisted into one another. This promotes the concept of a complex axillary sweat gland organ instead of single sweat gland entities.

Inhibition of cytosolic and mitochondrial creatine kinase by siRNA in HaCaT- and HeLaS3-cells affects cell viability and mitochondrial morphology.

The creatine kinase (CK) system is essential for cellular energetics in tissues or cells with high and fluctuating energy requirements. Creatine itself is known to protect cells from stress-induced injury. By using an siRNA approach to silence the CK isoenzymes in human keratinocyte HaCaT cells, expressing low levels of cytoplasmic CK and high levels of mitochondrial CK, as well as HeLa cancer cells, expressing high levels of cytoplasmic CK and low levels of mitochondrial CK, we successfully lowered the respective CK expression levels and studied the effects of either abolishing cytosolic brain-type BB-CK or ubiquitous mitochondrial uMi-CK in these cells. In both cell lines, targeting the dominant CK isoform by the respective siRNAs had the strongest effect on overall CK activity. However, irrespective of the expression level in both cell lines, inhibition of the mitochondrial CK isoform generally caused the strongest decline in cell viability and cell proliferation. These findings are congruent with electron microscopic data showing substantial alteration of mitochondrial morphology as well as mitochondrial membrane topology after targeting uMi-CK in both cell lines. Only for the rate of apoptosis, it was the least expressed CK present in each of the cell lines whose inhibition led to the highest proportion of apoptotic cells, i.e., downregulation of uMi-CK in case of HeLaS3 and BB-CK in case of HaCaT cells. We conclude from these data that a major phenotype is linked to reduction of mitochondrial CK alone or in combination with cytosolic CK, and that this effect is independent of the relative expression levels of Mi-CK in the cell type considered. The mitochondrial CK isoform appears to play the most crucial role in maintaining cell viability by stabilizing contact sites between inner and outer mitochondrial membranes and maintaining local metabolite channeling, thus avoiding transition pore opening which eventually results in activation of caspase cell-death pathways.

Vimentin is the specific target in skin glycation. Structural prerequisites, functional consequences, and role in skin aging.

Until now, the glycation reaction was considered to be a nonspecific reaction between reducing sugars and amino groups of random proteins. We were able to identify the intermediate filament vimentin as the major target for the AGE modification N(epsilon)-(carboxymethyl)lysine (CML) in primary human fibroblasts. This glycation of vimentin is neither based on a slow turnover of this protein nor on an extremely high intracellular expression level, but remarkably it is based on structural properties of this protein. Glycation of vimentin was predominantly detected at lysine residues located at the linker regions using nanoLC-ESI-MS/MS. This modification results in a rigorous redistribution of vimentin into a perinuclear aggregate, which is accompanied by the loss of contractile capacity of human skin fibroblasts. CML-induced rearrangement of vimentin was identified as an aggresome. This is the first evidence that CML-vimentin represents a damaged protein inside the aggresome, linking the glycation reaction directly to aggresome formation. Strikingly, we were able to prove that the accumulation of modified vimentin can be found in skin fibroblasts of elderly donors in vivo, bringing AGE modifications in human tissues such as skin into strong relationship with loss of organ contractile functions.

Quantitative 13C NMR spectroscopic studies on the equilibrium of formaldehyde with its releasing cosmetic preservatives.

The aim of our study was to show that NMR spectroscopy is an excellent method to obtain reliable information about the equilibrium between free formaldehyde and its formaldehyde releasers. For this purpose, we compared several O- and N-formal-based formaldehyde releasers used in industrial and consumer products. The underlying chemical structures as well as the release of formaldehyde were followed quantitatively as a function of the pH and dilution. It was shown that only the amide-based N-formals are a reservoir for formaldehyde in the concentrations normally used in cosmetic products, whereas O-formals and the amine-based N-formals decompose completely. Since NMR spectroscopy does not affect the equilibrium between free and bound formaldehyde, we think that it is the only method for unequivocal determination of free formaldehyde. Measurements on finished products showed that free formaldehyde can be quantified down to concentrations as low as approximately 0.002 wt % in an acceptable measuring time.

Ultrasonic velocity measurements as a method for investigating phase transitions of monoglyceride emulsifier systems in pearlescent cosmetic creams.

The phase transitions of monoglyceride emulsifier systems and pearlescent effects in cosmetic creams are investigated using ultrasonic velocity measurements. The transitions between the different phases of monoglyceride emulsifier systems--the coagel, liquid-crystalline lamellar phase, and gel phase--are detected in creams by changes in the ultrasonic velocity with variation of the temperature. The phase transition temperatures correspond to DSC results. Furthermore, the slope of the ultrasound velocity curve correlates with the amount of bound water in the different phases. These insights into the ultrasonic velocity properties of the monoglyceride emulsifier system of creams make it possible to more closely study the pearlescent effect of the coagel. The temperature domain of the short time reversibility of the pearlescence as well as the back-formation time of the coagel can be determined with this method, which enable the optimization of the formulation of pearlescent creams.

Quantitative determination of cationic modified polysaccharides on hair using LC-MS and LC-MS-MS.

Cationic polysaccharides containing N-hydroxypropyl-N,N,N-trimethylammonium substituents are widely used as conditioning agents for hair-care products. A sensitive method has been developed for the quantitation of these polymers. After acidic extraction from hair the polysaccharides are hydrolyzed using trifluoroacetic acid. The cationic monoglycosides are determined using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The developed method is independent of hair treatment. Even hair cut from test persons after customary hair wash can be analyzed. After treatment of natural and bleached hair tresses using a real-life treatment procedure 180 microg and 300 microg of polymer per gram hair were quantified, respectively. Additionally the fragmentation mechanism of the cationic N-hydroxypropyl-N,N,N-trimethylammonium group during electrospray ionization was investigated. A mass loss of 60 Da in combination with loss of a single charge is observed and associated with cleavage of trimethylamine and a proton. It is assumed that this process is promoted by the anionic counter-ion which might be hydroxide in an aqueous environment.

The creatine kinase system in human skin: protective effects of creatine against oxidative and UV damage in vitro and in vivo.

Cutaneous aging is characterized by a decline in cellular energy metabolism, which is mainly caused by detrimental changes in mitochondrial function. The processes involved seem to be predominantly mediated by free radicals known to be generated by exogenous noxes, e.g., solar ultraviolet (UV) radiation. Basically, skin cells try to compensate any loss of mitochondrial energetic capacity by extra-mitochondrial pathways such as glycolysis or the creatine kinase (CK) system. Recent studies reported the presence of cytosolic and mitochondrial isoenzymes of CK, as well as a creatine transporter in human skin. In this study, we analyzed the cutaneous CK system, focusing on those cellular stressors known to play an important role in the process of skin aging. According to our results, a stress-induced decline in mitochondrial energy supply in human epidermal cells correlated with a decrease in mitochondrial CK activity. In addition, we investigated the effects of creatine supplementation on human epidermal cells as a potential mechanism to reinforce the endogenous energy supply in skin. Exogenous creatine was taken up by keratinocytes and increased CK activity, mitochondrial function and protected against free oxygen radical stress. Finally, our new data clearly indicate that human skin cells that are energetically recharged with the naturally occurring energy precursor, creatine, are markedly protected against a variety of cellular stress conditions, like oxidative and UV damage in vitro and in vivo. This may have further implications in modulating processes, which are involved in premature skin aging and skin damage.

Noninvasive measurement of cell volume changes by negative staining.

To maintain the intracellular concentration of ions and small molecules on osmotic challenges, nature has developed highly sophisticated transport systems for regulating water and ion content. An ideal measurement technique for volume changes of cells during osmotic challenges has to fulfil two requirements: it has to be osmotically inert, and it should allow online monitoring of cell volume changes. Here, a simple fluorescence microscopy-based approach is presented. Using fluorescein as a negative stain, it is possible to monitor cell volume changes without affecting the functionality of cell membranes and cell osmolarity. Measurement of Madine-Darby canine kidney (MDCK) cells after hypo- and hyperosmotic challenges reveals the main advantages of this approach: besides providing precise and reproducible quantitative data on reversible cell volume changes, the viability of the cells can be assessed directly by the appearance of stain in the cytoplasm. This becomes evident especially after hypo-osmotic challenge of glutaraldehyde-treated cells, which become leaky after fixation, followed by a massive volume change. This new approach represents a very sensitive measurement technique for cell volume changes resulting from water or ion flux, and thus seems to be an ideal tool for studying cell volume regulatory processes.