The essential structures of ISP-I that influence serine palmitoyltransferase inhibition in Chinese hamster ovary cells.

We investigated the structure-activity relationship between various ISP-I (myriocin, thermozymocidin) analogous which has sphingosine-like structure and serine palmitoyltransferase (SPT) in Chinese hamster ovary (CHO) cells utilizing sphingolipid production as a marker. Our data suggest that the double bond and/or ketone group within the alkyl chain as well as the alkyl chain are necessary for ISP-I to inhibit SPT. In addition, a serine structure is necessary for SPT inhibitory activity, which confirms previous findings.

Effects of serine palmitoyltransferase inhibitor ISP-I on the stratum corneum of intact mouse skin.

Serine palmitoyltransferase (SPT) is involved in the ceramide synthesis pathway. We investigated the effects of ISP-I, a potent inhibitor of SPT, on the stratum corneum (SC) of hairless mouse skin. Application of ISP-I for one week resulted in a significant decrease in the amount of ceramide, which was associated with a decrease in SC hydration. However, there was an increase in the number of SC layers and less transepidermal water loss than control. Transmission Electron Microscopy observation revealed that the number of desmosome-like structures in the layers immediately above the stratum granulosum (SG) was significantly increased in ISP-I-treated skin compared to vehicle-treated skin. The activity of serine protease-an enzyme associated with the process of desquamation-was lower in the SC of ISP-I-treated skin than control. Furthermore, immunoelectronmicroscopy revealed that glucosylceramide and corneodesmosin tended to remain in corneocytes and were not secreted into the intercellular spaces of the SC in the ISP-I-treated skin. These results indicate that the application of ISP-I decreases ceramide and skin hydration, while at the same time increases the number of SC layers. The accumulation of corneocyte layers may originate from an aberrant desquamation process related to the decrease in the serine protease activity as well as an alteration in the transport of desquamation-related proteases by lamellar bodies.

The effects of serine palmitoyltransferase inhibitor, ISP-I, on UV-induced barrier disruption in the stratum corneum.

We examined the effects of ISP-I (myriocin, thermozymocidin) - a potent inhibitor of serine palmitoyltransferase (SPT) which is involved in the ceramide synthetic pathway-on skin barrier function in post-UVB-irradiated hairless mouse skin. Disruption of the skin barrier function after UVB irradiation as represented by the increase in transepidermal water loss (TEWL) was significantly suppressed with ISP-I treatment. In the ISP-I-treated skin, the peak of cell proliferation was observed 24 h earlier than in vehicle-treated skin. In addition, the number of apoptotic cells in ISP-I-treated skin showed a sharp decrease at 48 and 72 h post-irradiation. The number of stratum corneum cell layers was increased in ISP-I-treated skin at 72 h after UVB irradiation; at this time, TEWL in ISP-I-treated skin was lower than that in the vehicle-treated skin. We suggest ISP-I treatment altered cell proliferation and apoptosis after UVB exposure by modulating ceramide synthesis in epidermal cells, resulting in an increase of stratum corneum layers which lessened the effects of irradiation-induced barrier disruption.

Effects of vitamin C on dark circles of the lower eyelids: quantitative evaluation using image analysis and echogram.

BACKGROUND/AIMS: The pathogenesis of dark circles of the lower eyelid (DCLE) has been considered to involve stasis and hyperpigmentation of the eyelids. We have already reported that dermal thickness of lower eyelid skin may represent another factor that affects the appearance of DCLE. The aim of this study was to evaluate the efficacy of vitamin C, which is known to increase collagen, on DCLE through a clinical trial. METHODS: Fourteen subjects with DCLE applied either 10% sodium ascorbate (ANa) or ascorbic acid glucoside (AG) lotion in split-face fashion (opposite side: vehicle only) for 6 months. Melanin index (MI), erythema index (EI), thickness and echogenicity of the dermis at bilateral lower eyelids was measured during this trial. RESULTS: Change in EI was significantly smaller on the ANa-treated side than on the vehicle-treated side. Dermal thickness tended to be thicker for the ANa-treated side than for the vehicle-treated side, although no significant difference was seen. Both EI and dermal thickness tended to change in parallel manner. On the other hand, no significant differences in changes of EI, MI, and dermal thickness were found between AG- and vehicle-treated sides. CONCLUSION: ANa may improve DCLE by thickening the eyelid dermis and concealing dark coloration due to congested blood.

Objective evaluation of the efficacy of daily topical applications of cosmetics bases using the hairless mouse model of atopic dermatitis.

BACKGROUND/AIMS: Dry skin (xerosis) is a characteristic change associated with atopic dermatitis (AD) and has often been treated with topical petrolatum applications despite its unfavorable feel. Recently, various therapeutically effective skin-care products with better feel have been introduced. To elucidate the mechanisms underlying the clinical effectiveness of these newer treatments, we used our recently established hairless mouse model of AD. METHODS: We produced AD-like skin lesions in hairless mice with repeated applications of 2,4,6-trinitro-1-chrolobenzene (TNCB) in acetone for 36 days as reported previously. Groups of five mice with AD-like skin were treated once daily with an emollient-type cream containing petrolatum, a moisturizer-type cream containing 10% glycerin, a solution of 0.01% dexamethasone in acetone, or were left untreated. Over the duration of these treatments, we conducted non-invasive measurements of skin surface condition with biophysical instruments and electron microscopic evaluation of the surface area size and density of rear surface villi of superficial corneocytes. We also obtained skin biopsy samples and blood samples at each time point for histopathological evaluation and to assess serum IgE levels, respectively. RESULTS: After cessation of topical TNCB applications, AD-like skin underwent spontaneous resolution with normalization of skin appearance. A similar reduction in skin fold thickness was observed in the cream-treated mice and in the untreated mouse group, whereas a significant decrease in skin thickness was observed in the dexamethasone-treated mice. Transepidermal water loss, a measure of stratum corneum barrier function, rapidly normalized in all groups, without any statistical differences noted among groups. In comparison with untreated skin, skin surface hydration markedly improved after repeated applications of the moisturizer-type cream, whereas it consistently remained low in dexamethasone-treated skin. The skin treated with emollient-type cream appeared similar to skin that received no treatment. Reduced corneocyte surface area size resulting from repeated applications of TNCB returned to control size with cream treatments, while the corneocyte surface area size became much larger following dexamethasone treatment. In addition, the density of villi on the rear surface of corneocytes decreased with application of the creams or dexamethasone. By contrast, no changes were observed in the number of leukocytes in the epidermis or in serum IgE levels among the different treatment groups. In all treatment groups, even after 32 days of treatment, reapplication of TNCB resulted in early-stage skin swelling, but only in the steroid-treated animals did this swelling show a remarkably prolonged time course. CONCLUSIONS: Our present results indicate that the efficacy of skin-care products containing no active ingredients in treating atopic xerosis can be objectively evaluated using the hairless mouse model of AD.

Establishment of an atopic dermatitis-like skin model in a hairless mouse by repeated elicitation of contact hypersensitivity that enables to conduct functional analyses of the stratum corneum with various non-invasive biophysical instruments.

BACKGROUND/AIMS: Pathogenesis of atopic dermatitis (AD) has been studied in animal models such as the NC/Nga mouse strain or Balb/C mice that are repeatedly treated with 2,4,6-trinitro-1-chrolobenzene (TNCB). These mice exhibit features of chronic contact dermatitis, including an intensified early type skin reaction, increased number of mast cells and elevated serum IgE levels with a shift of cutaneous cytokine expression from a type 1 to type 2 profile. However, it is difficult to investigate the unique skin changes of AD such as dry skin, barrier dysfunction, and increased turnover of the stratum corneum (SC) in these animals with biophysical instruments because of the presence of their fur coats. In this study, we succeeded in establishing a mouse model of AD in hairless mice that are suitable for various functional analyses of the SC as well as for examining the immunological characteristics of human AD by treating TNCB-contact sensitized hairless mice with 1% TNCB every other day for 36 days. METHODS: In hairless animals treated with TNCB every 2 days for 36 days, we measured time courses of skin swelling induced by contact hypersensitivity reaction on days 0, 6, 20 and 36. During the time course, non-invasive measurements for skin surface condition with biophysical instruments were conducted, and the area size and the rear surface villi of corneocytes obtained were measured. Also skin samples and blood samples were taken at each time point for histology and measurement of serum IgE level. RESULTS: A gradual intensification of an early type contact hypersensitivity reaction was observed over the treatment period. These mice exhibited reduced SC hydration, heightened trans-epidermal water loss, and increased skin thickness. These mice also showed a decrease in the surface area size of each corneocytes and marked villus formation on their rear surface. Histologically, there was an increase in the number of CD4 and CD8 positive T cells in the epidermis. Also observed was a marked increase in the number of dermal mast cells and eosinophils, which correlated with elevated serum IgE levels induced by TNCB treatments. CONCLUSIONS: From the results obtained we conclude that repeated treatments of TNCB-sensitized hairless mice with TNCB provides a useful means by which to study the pathological characteristics of AD skin lesions as well as their immunological characteristics.

Study of active substances involved in skin dysfunction induced by crowding stress. I. Effect of crowding and isolation on some physiological variables, skin function and skin blood perfusion in hairless mice.

The effects of five levels of population density on various organs, the neuroendocrine system, skin function, skin blood perfusion, and blood parameters were studied in the hairless mouse. Skin barrier recovery was evaluated by measuring transepidermal water loss after tape stripping. Blood perfusion was measured by means of a laser Doppler imaging technique. The effect of a parasympathetic nerve stimulator, carpronium chloride, on skin function in the crowded animal model was also examined. A 7 d crowding (10, 15, 20 mice/cage) significantly increased the levels of corticosterone, catecholamines (norepinephrine, epinephrine and dopamine), glucose and serum lactate dehydrogenase activity in circulating blood, induced atrophy of kidney, ovary and thymus and hypertrophy of adrenal glands, and decreased body weight gain in comparison with the control (5 mice/cage). Crowding also increased epidermal thickness and epidermal proliferative activity, and decreased corneocyte size, rate of barrier recovery and skin blood perfusion. Most of these changes became more marked with increasing population density and/or longer exposure to a crowded environment. Isolation (1 mouse/cage) increased the level of norepinephrine and rate of skin blood perfusion, and significantly delayed barrier recovery. Repeated topical applications of carpronium chloride for 7 d improved the changes in skin blood perfusion, barrier recovery, kidney and ovary, and epidermal morphology induced by crowding. The crowded animal model could be useful for quantifying objectively the influence of crowded environment-induced stress on cutaneous function and blood perfusion.