Tranexamic acid blocks the thrombin-mediated delay of epidermal permeability barrier recovery induced by the cedar pollen allergen, Cry j1.

We previously demonstrated that Cry j1, the major pollen allergen of Cryptomeria japonica (Japanese cedar), transiently increases protease activity and intracellular Ca2+ concentration in cultured human keratinocytes, and delays recovery after stratum corneum barrier disruption in human skin ex vivo. Topical application of tranexamic acid or trypsin-type serine protease inhibitors accelerates barrier recovery. We hypothesized that tranexamic acid might prevent the transient protease activity increase and the barrier recovery delay induced by Cry j1. Here, we tested this hypothesis and examined the mechanism involved. In cultured human keratinocytes, knock-down of protease-activated receptor 1 (PAR-1) reduced the transient increase of calcium induced by Cry j1, whereas knock-down of PAR-2 did not. Knock-down of thrombin significantly reduced the transient increases of calcium concentration and protease activity. Tranexamic acid, soybean trypsin inhibitor, or bivalirudin (a thrombin inhibitor) also reduced the calcium elevation induced by Cry j1 and/or thrombin. Co-application of tranexamic acid or bivalirudin with Cry j1 to human skin ex vivo blocked the delay of barrier recovery. These results suggest that thrombin and PAR-1 or PAR-1-like receptor might mediate the adverse effects of Cry j1 on human epidermal keratinocytes, and could open up a new strategy for treating inflammatory skin diseases.

Sex difference in human fingertip recognition of micron-level randomness as unpleasant.

We investigated sex difference in evaluation, using the human fingertip, of the tactile impressions of three different micron-scale patterns laser-engraved on plastic plates. There were two ordered (periodical) patterns consisting of ripples on a scale of a few micrometres and one pseudo-random (non-periodical) pattern; these patterns were considered to mimic the surface geometry of healthy and damaged human hair, respectively. In the first experiment, 10 women and 10 men ran a fingertip over each surface and determined which of the three plates felt most unpleasant. All 10 female participants reported the random pattern, but not the ordered patterns, as unpleasant, whereas the majority of the male participants did not. In the second experiment, 9 of 10 female participants continued to report the pseudo-random pattern as unpleasant even after their fingertip had been coated with a collodion membrane. In the third experiment, participants were asked to evaluate the magnitude of the tactile impression for each pattern. The results again indicated that female participants tend to report a greater magnitude of unpleasantness than male participants. Our findings indicate that the female participants could readily detect microgeometric surface characteristics and that they evaluated the random pattern as more unpleasant. Possible physical and perceptual mechanisms involved are discussed.

Effect of endothelial nitric oxide synthase on epidermal permeability barrier recovery after disruption.

BACKGROUND: We previously demonstrated that neuronal nitric oxide synthase (nNOS) in epidermal keratinocytes is associated with epidermal permeability barrier homeostasis. OBJECTIVES: In the present study, we examined the contributions of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) to epidermal permeability barrier homeostasis. METHODS: We measured the barrier recovery rate after tape stripping of the epidermis of iNOS and eNOS knockout mice, and carried out electron-microscopic observation of the epidermis after acetone treatment. RESULTS: The barrier recovery rate of eNOS knockout mice was significantly faster than that of the wild-type control, while no significant difference was observed between iNOS knockout mice and wild-type mice. Electron-microscopic observation at 1 h after acetone treatment indicated that barrier recovery of both nNOS and eNOS mice was faster than that of wild-type mice, and lamellar body secretion was accelerated in both types of knockout mice. CONCLUSIONS: These results suggested that both nNOS and eNOS play roles in epidermal barrier homeostasis and lamellar body secretion.

Acceleration of permeability barrier recovery by exposure of skin to 10-30 kHz sound.

BACKGROUND: Previous reports show that ultrasound can influence human brain electrical activity and systemic hormone levels in various parts of the body, other than the ear, so there may be an unknown ultrasound-responsive system in humans. OBJECTIVES: In the present study, we examined the effects of sound on skin permeability barrier homeostasis. METHODS: We broke the skin barrier of hairless mice by tape stripping, and then exposed the skin to sound for 1 h to evaluate the effect on barrier recovery rate. RESULTS: Exposure of skin to sound at frequencies of 10, 20 and 30 kHz for 1 h accelerated barrier recovery, and 20 kHz sound induced the fastest recovery. Application of 5 kHz sound had no effect on barrier recovery rate. Significant acceleration was observed even when the sound source was located 3 cm away from the skin surface. The recovery rate depended on the sound pressure. An electron-microscopic study indicated that lamellar body secretion between stratum corneum and stratum granulosum was increased by exposure to sound at 20 kHz. CONCLUSIONS: These results suggest that epidermal keratinocytes might be influenced by ultrasound in a manner that results in modulation of epidermal permeability barrier homeostasis.

Methodology to improve epidermal barrier homeostasis: how to accelerate the barrier recovery?

Good water-impermeable barrier function is vital for healthy skin. Abnormality of the barrier function is observed in a variety of skin diseases, such as atopic dermatitis, psoriasis and contact dermatitis. Moreover, repeated barrier disruption induces epidermal hyperplasia and inflammation. On the other hand, acceleration of the barrier recovery prevents epidermal hyperplasia induced by barrier disruption in a dry environment. Thus, methods to improve the barrier function are very important for clinical dermatology. Recently, we have been searching for new reagents and/or new materials to improve barrier homeostasis. In this review, I will describe our recent findings and show how they provide the basis for a new perspective for clinical dermatology.

Function of oleic acid on epidermal barrier and calcium influx into keratinocytes is associated with N-methyl D-aspartate-type glutamate receptors.

BACKGROUND: Unsaturated fatty acids from sebum affect calcium dynamics in epidermal keratinocytes, disrupt the barrier function and induce abnormal keratinization. However, the mechanisms of these effects have not been clarified. OBJECTIVES: To investigate the function of unsaturated fatty acids in epidermis. METHODS: Antagonists of calcium channel receptors were applied to mouse skin together with oleic acid. Measurements were made of transepidermal water loss (TEWL), and hyperproliferation was assessed. The effects of the antagonists on calcium influx into cultured normal human keratinocytes and on cytokine production were also evaluated. RESULTS: N-methyl-d-aspartate (NMDA) receptor antagonists such as MK801 and D-AP5 specifically inhibited the increase in TEWL caused by oleic acid, and suppressed keratinocyte hyperproliferation. These compounds also inhibited the increase in the intracellular concentration of calcium ions induced by oleic acid. MK801 suppressed the production of interleukin-1alpha by keratinocytes induced by oleic acid. CONCLUSIONS: Unsaturated fatty acids such as oleic acid might function via NMDA receptors.

Potassium channel openers accelerate epidermal barrier recovery.

BACKGROUND: Maintenance of a competent permeability barrier in the face of external and internal stressors requires signals between the stratum corneum interface and the metabolic machinery in the underlying nucleated layers. For example, reductions in the ion gradients for Ca2+ after acute barrier disruption stimulate lamellar body (LB) secretion, a response required to restore barrier homeostasis. Although alterations in external K+ levels also regulate barrier recovery after acute insults, the mechanisms whereby K+ regulates barrier function remain unknown. OBJECTIVES: To evaluate effects of regulators of K+ channels on barrier homeostasis in hairless mice. METHODS: We tested a number of chemically different drugs that alter intracellular K+ levels. Results Single applications of either K+ channel openers (i.e. 1-EBIO, minoxidil, diazoxide) or the K+ ionophore, valinomycin, accelerated barrier recovery after acute insults to murine skin, paralleled by a reduction in intracellular K+ levels in cultured human keratinocytes. In contrast, applications of K+ channel blockers (i.e. gilbenclamide, dequalinium) delayed barrier recovery. Alterations in intracellular K+ regulated barrier homeostasis by either stimulating (reduced K+) or inhibiting (elevated K+) LB secretion. Finally, development of epidermal hyperplasia, a downstream consequence of barrier disruption, was also inhibited by agents that reduce intracellular K+ levels. CONCLUSIONS: These results demonstrate that changes in K+ levels that can be presumed to occur after barrier disruption signal metabolic responses, i.e. LB secretion, which accelerates normalization of barrier function.

Paradoxical effects of beta-estradiol on epidermal permeability barrier homeostasis.

BACKGROUND: Previous studies have demonstrated that sex hormones modulate epidermal permeability barrier homeostasis, and when the balance of these hormones is altered at menopause or during the menstrual cycle, skin sensitivity or barrier function is changed. OBJECTIVES: To observe the direct effects of sex hormones on epidermal homeostasis. METHODS: We examined the effects of topical application of sex hormones on permeability barrier recovery after tape stripping in the hairless mouse. To avoid the influence of systemic hormonal alteration, we employed male animals. RESULTS: Application of androgen (testosterone or androsterone) delayed the barrier recovery, and the delay was overcome by co-application of beta-estradiol. Progesterone also delayed the barrier recovery, but in this case the delay was enhanced by beta-estradiol. CONCLUSIONS: These results suggest that changes in sex hormone balance might be associated with the skin dysfunction that often occurs during menopause, and at certain points during the menstrual cycle.

Topical application of ionic polymers affects skin permeability barrier homeostasis.

We previously demonstrated that the external electric potential affected skin barrier homeostasis. On the other hand, topical application of an ionic polymer formed a diffusion electric double layer on the surface of the skin. Thus, we evaluated effects of topical application of ionic polymers on the damaged skin barrier. Application of a nonionic polymer did not affect barrier recovery. Application of sodium salts of anionic polymers accelerated barrier recovery, while that of cationic polymers delayed it. Topical application of a sodium-exchange resin accelerated barrier recovery, but application of a calcium-exchange resin had no effect even when the resins had the same structure. Application of a chloride-exchange resin delayed barrier recovery. Topical application of ionic polymers influenced skin barrier homeostasis.

Barium sulphate with a negative zeta potential accelerates skin permeability barrier recovery and prevents epidermal hyperplasia induced by barrier disruption.

BACKGROUND: Barium sulphate, a stable inorganic material, has been used in contrast media and cosmetic products because of its stability. As a negative external electric potential accelerates the skin barrier repair after barrier disruption, we hypothesized that topical application of barium sulphate may affect the skin barrier recovery rate depending on its zeta potential. OBJECTIVES: To investigate whether barium sulphate particles in aqueous solution have different zeta potentials depending on their surface structure, and to investigate the possible relation between zeta potential and skin barrier recovery rate. METHODS: Mice were subjected to tape stripping to disrupt barrier function, or were treated with acetone and kept in a dry environment to induce epidermal hyperplasia. They were then treated with different forms of barium sulphate, and barrier recovery was monitored by measurements of transepidermal water loss. RESULTS: There was a significant correlation between the barrier recovery rate and zeta potential of barium sulphate applied topically. Barium sulphate with a negative zeta potential significantly accelerated barrier recovery, but barium sulphate with a positive zeta potential did not accelerate or even delayed barrier repair. Barium sulphate with a negative zeta potential had an X-ray diffraction pattern different from that with a positive potential. The distribution of calcium in the epidermis was also influenced by the polarity of zeta potential. CONCLUSIONS: These findings suggest a new pharmacological approach towards altering barrier function or epidermal hyperplasia with inorganic particles in healthy and diseased skin.

Dry environment increases mast cell number and histamine content in dermis in hairless mice.

BACKGROUND: A variety of skin diseases, such as atopic dermatitis, senile xerosis or psoriasis tends to worsen during the dry winter season. These skin diseases are also characterized by itch sensation. OBJECTIVES: To evaluate the generation of histamine in the dermis of hairless mice kept under various conditions of environmental humidity. MATERIALS AND METHODS: We carried out the quantification of mast cell population and evaluated the histamine content in the epidermis, dermis and serum of the mice kept under various conditions of environmental humidity. RESULTS: Histamine content in the dermis of the mice kept in a low environmental humidity (relative humidity < 10%) for 3 and 5 days was significantly higher than that of the mice kept in a high environmental humidity (relative humidity > 80%) for the same period of time. No significant difference was observed on the histamine content in the epidermis between the two humidity groups. The number of mast cells in the dermis of the mice kept in the low environmental humidity was significantly higher than that of the mice kept in the high environmental humidity. Topical application of petrolatum reduced the level of histamine in the dermis of the mice under the low environmental humidity. CONCLUSIONS: These results suggest that a low environmental humidity increases mast cell number and dermal histamine content.

Epidermal proliferative response induced by sodium dodecyl sulphate varies with environmental humidity.

BACKGROUND: Previous studies have suggested that susceptibility of skin to external agents increases in the dry winter season. OBJECTIVES: To test the hypothesis that environmental humidity affects skin sensitivity to irritants. METHODS: The epidermal hyperplasia induced by sodium dodecyl sulphate (SDS) under various humidity conditions was evaluated on the skin of hairless mice. RESULTS: Mice kept under low humidity for 2 days showed more obvious epidermal proliferation 24 h after topical application of SDS than those kept under high or normal humidity for 2 days. In contrast, mice kept under high humidity for 2 weeks showed more obvious epidermal proliferation 24 h after topical application of SDS than those kept under low or normal humidity. The transepidermal water loss was altered significantly in the animals kept under high humidity for 2 weeks, although it was not altered during the first 7 days under either low or high humidity. CONCLUSIONS: These results suggest that environmental humidity influences the sensitivity of skin to topical application of SDS and that increased sensitivity is not always associated with alteration of the water impermeability of the stratum corneum.

Immunoreactivity of VR1 on epidermal keratinocyte of human skin.

We demonstrated the immunoreactivity of the receptor proteins, VR1, ion channels associated with pain sensation, on the epidermis of the human skin. Immunohistochemistry using antiserum against VR1 derived peptide showed immunoreactivity on the keratinocytes cell membrane of the human epidermis and cultured keratinocytes. The blocking peptide of the antiserum reduced the immunoreactivity on the epidermis. RT-PCR assay of cultured human keratinocyte also showed expression of VR1 mRNA. These results suggest the existence of VR1-like protein in epidermal keratinocytes of human skin.

Skin surface electric potential induced by ion-flux through epidermal cell layers.

The skin surface electric potential has been widely used for psychological studies because it is sensitive to emotional conditions. We measured the electric potential on the surface of hairless mice skin in organ culture with several physiological factors. Disruption of mitochondrial function and inhibition of ATPase reduced the skin surface potential 50-70%. Calcium, potassium, and sodium channel blockers also reduced the potential. A calcium-specific and potassium ionophore reduced the potential, but the calcium and magnesium ionophore increased it. EDTA decreased the potential but EGTA had no effect. Skin surface barrier disruption reduced the potential and calcium and potassium channel blockers partially prevented the decrease. Substance P and corticotropin-releasing factor decreased the potential, and antagonists blocked the decreases. These results suggest that the ion flux in the nucleated layer of the epidermis induce the skin surface potential and it is influenced by environmental and neuroendocrinological factors.

A Brassica oleracea gene expressed in a variety-specific manner may encode a novel plant transmembrane receptor.

The species Brassica oleracea includes several agricultural varieties characterized by the proliferation of different types of meristems. Using a combination of subtractive hybridization and PCR (polymerase chain reaction) techniques we have identified several genes which are expressed in the reproductive meristems of the cauliflower curd (B. oleracea var. botrytis) but not in the vegetative meristems of Brussels sprouts (B. oleracea var. gemmifera) axillary buds. One of the cloned genes, termed CCE1 (CAULIFLOWER CURD EXPRESSION 1) shows specific expression in the botrytis variety. Preferential expression takes place in this variety in the meristems of the curd and in the stem throughout the vegetative and reproductive stages of plant growth. CCE1 transcripts are not detected in any of the organs of other B. oleracea varieties analyzed. Based on the nucleotide sequence of a cDNA encompassing the complete coding region, we predict that this gene encodes a transmembrane protein, with three transmembrane domains. The deduced amino acid sequence includes motifs conserved in G-protein-coupled receptors (GPCRs) from yeast and animal species. Our results suggest that the cloned gene encodes a protein belonging to a new, so far unidentified, family of transmembrane receptors in plants. The expression pattern of the gene suggests that the receptor may be involved in the control of meristem development/arrest that takes place in cauliflower.

Epidermal interleukin-1 alpha generation is amplified at low humidity: implications for the pathogenesis of inflammatory dermatoses.

BACKGROUND: We have previously reported that low humidity amplifies the hyperproliferative and inflammatory response to barrier disruption. Other reports suggest that epidermal interleukin (IL)-1 alpha is stimulated by various factors related to epidermal inflammation and that it may induce other proinflammatory molecules. OBJECTIVES: To evaluate the generation of IL-1 alpha in the skin of hairless mice kept under various conditions of environmental humidity. METHODS: We carried out an immunohistochemical study, and evaluated epidermal IL-1 alpha mRNA and protein levels, and release of IL-1 alpha from skin after tape stripping, in hairless mice kept under low or high humidity. RESULTS: The immunohistochemical study showed that the amount of IL-1 alpha in the epidermis was higher in animals kept in a low-humidity environment than in a high-humidity one. The epidermal IL-1 alpha mRNA and protein levels increased significantly when the animals were kept under low humidity. Moreover, the release of IL-1 alpha from skin immediately after tape stripping was significantly higher in animals kept in a low-humidity environment than in a high-humidity one. CONCLUSIONS: These results suggest that IL-1 alpha is an important factor in mediating the relationship between environmental humidity and epidermal pathology.

Identification of immature cornified envelopes in the barrier-impaired epidermis by characterization of their hydrophobicity and antigenicities of the components.

Cornified envelopes (CEs), rigid and insoluble structures in the stratum corneum, which are assembled by crosslinking of several precursor proteins by transglutaminases, provide a hydrophobic foundation for barrier function; omega-hydroxyceramides are covalently attached to the outer surface of CE components, and onto this hydrophobic assembly, lamellar layers of intercellular lipids are organized. Morphologically irregular, fragile CEs are found in the deep layer of the stratum corneum or in certain disorders, such as psoriasis, whereas most CEs from healthy subjects are rigid and polygonal. We have established a staining method to characterize such fragile CEs as immature and less hydrophobic CEs, and employed it to examine regional differences in the properties of CEs, especially in relation to the barrier function of the skin. CEs from the outermost stratum corneum of the trunk and extremities of healthy subjects were relatively uniform in morphology with larger shape, and were homogeneous in hydrophobicity as judged from the use of an environment-sensitive fluorescent dye, Nile red. However, CEs from the face were strikingly heterogeneous, and consisted of both rigid and fragile CEs. Rigid CEs were Nile red-positive and little stained by anti-involucrin. In contrast, fragile CEs were Nile red-negative but strongly stained with anti-involucrin, as detected by indirect immunofluorescence. Thus, CEs from the face were stained with Nile red or involucrin in a mutually exclusive manner. Fragile CEs were stained with antibodies against other CE components, including loricrin, envoplakin, filaggrin, and isopeptides. Such fragile, involucrin-positive CEs were detected not only in the face, but also in the deep layer of the stratum corneum of the arm. In addition, experimental barrier disruption resulted in the appearance of involucrin-positive CEs in the outermost stratum corneum. These results suggest that involucrin-positive, fragile CEs are immature and less hydrophobic, and that their occurrence is closely related to impairment of the barrier function of the skin.

Histamine H1 and H2 receptor antagonists accelerate skin barrier repair and prevent epidermal hyperplasia induced by barrier disruption in a dry environment.

Keratinocytes have histamine H1 and H2 receptors, but their functions are poorly understood. To clarify the role of histamine receptors in the epidermis, we examined the effects of histamine receptor antagonists and agonists applied epicutaneously on the recovery of skin barrier function disrupted by tape stripping in hairless mice. Histamine H2 receptor antagonists famotidine and cimetidine accelerated the recovery of skin barrier function, but histamine and histamine H2 receptor agonist dimaprit delayed the barrier repair. Application of compound 48/80, a histamine releaser, also delayed the recovery. Imidazole, an analog of histamine, had no effect. The histamine H1 receptor antagonists diphenhydramine and tripelennamine accelerated the recovery. Histamine H3 receptor agonist Nalpha-methylhistamine and antagonist thioperamide had no effect. In addition, topical application of famotidine or diphenhydramine prevented epidermal hyperplasia in mice with skin barrier disrupted by acetone treatment in a dry environment (humidity < 10%) for 4 d. In conclusion, both the histamine H1 and H2 receptors in the epidermis are involved in skin barrier function and the cutaneous condition of epidermal hyperplasia.