The role of apoptosis in the pathogenesis of dermatitis herpetiformis

Apoptosis is a form of cell death that is claimed to be involved in a number of chronic inflammatory and malignant skin diseases. The aim of this study was to investigate whether apoptosis may contribute to the pathogenesis of epidermal changes in dermatitis herpetiformis (DH) and, in particular, whether certain apoptosis-related markers such as Bax, Bcl-2, Fas and Fas ligand (FasL) take part in this process. For the detection of apoptotic nuclei, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling technique (TUNEL) was employed on cryostat sections. Skin lesions from six and perilesional skin from four DH patients were stained with monoclonal antibodies to Bax, Bcl-2, Fas and FasL. The same evaluation was also performed on three patients affected by bullous pemphigoid (BP) and in two healthy donors. Using TUNEL technique, a remarkable increase in the apoptotic rate within the epidermal compartment was observed in DH and BP patients in comparison with normal controls. In our immunohistochemical analysis, Bax/Bcl-2 ratio was almost the same in the epidermis of perilesional/lesional DH, BP and healthy skin specimens. In DH and BP specimens both Bax and Bcl-2 proteins were increased in the dermal perivascular compartment. Fas showed a prevalently epidermal staining, both in DH and BP lesions, while FasL was distributed in perivascular and subjunctional dermis; some FasL+ cells infiltrated the DEJ and the basal layer of epidermis. This study allowed us to highlight conspicuous apoptotic phenomena in basal and suprabasal keratinocytes within lesional and perilesional skin of DH. We conclude that in DH, as well as in BP, apoptosis plays a role in the pathogenesis of cutaneous lesions in concert with other pathogenetic mechanisms.     

Pathology Produced by Sulfur Mustard in Human Skin Grafts on Athymic Nude Mice. II. Ultrastructural Changes

Abstract

The human skin graft/nude mouse model was used to study sulfur mustard (SM) -induced pathology of skin at the ultrastructural level. The electron microscope confirms and extends the histopathology previously noted under light microscopy. SM caused extensive injury to the basal keratinocytes, the severity of which was dependent on both time and dose. Ultrastructurally visible damage took several hours to develop. The following sequence of morphological changes was observed: (a) Extensive condensation and margination of heterochromatin and loss of euchromatin, (b) blebbing of the nuclear membrane, (c) appearance of perinuclear vacuoles, (d) swelling of the endoplasmic reticulum, (e) progressive dissociation of rosettes of free polyribosomes, (f) formation of cytoplasmic vacuoles, (g) loss of the integrity of the basal cell plasma membrane, (h) leakage of cell contents and debris into the lamina lucida, (i) disruption of the anchoring filaments of hemidesmosomes with separation of the plasma membrane from the lamina densa, and accumulation of edema fluid in the lamina lucida, and (j) infiltration of phagocytes into areas of major damage. The damage to the basal cell layer eventually resulted in the separation of the epidermis from the dermis and the formation of a subepidermal microblister. Changes in the dermis were also noted, but these did not become evident until after basal cell injury was already underway. The dermal changes consisted of edema with separated but otherwise intact collagen bundles, and of damage to the fibroblasts and endothelial cells which were in close proximity to the necrotic basal cell layer. The pathology observed at the ultrastructural level strongly supports the view that the SM-induced injury to human skin begins at the basal cell layer.     

小型香猪复方壳多糖组织工程皮肤的构建及组织学特点

目的评价制备的小型香猪复方壳多糖组织工程皮肤在组织学方面的特性,为其修复皮肤缺损创面提供实验依据。方法采用传代培养的角质形成细胞和成纤维细胞作为种子细胞,I型胶原作为真皮基质。采用气—液界面方式进行培养,通过苏木精—伊红(HE)染色、比较小型香猪的体外构建组织工程皮肤与正常皮肤的组织学特征,包括表皮、真皮结构。结果制备的复方壳多糖组织工程皮肤表皮细胞增殖活跃,分层分化良好,厚约150μm,真皮层细胞生长正常,排列有序,与机体皮肤结构相似。结论复方壳多糖组织工程皮肤组织结构良好,符合新型皮肤替代物在治疗皮肤缺损时的组织学要求。     

Effects of nicotinamide on biochemical changes and microblistering induced by sulfur mustard in human skin organ cultures.

Blister formation due to sulfur mustard (HD) exposure was studied in an ex vivo human skin model. Pieces of fresh human skin were exposed to HD vapor and subsequently incubated in medium for 48 hr. During this culture period cellular NAD+ levels and uptake of glucose from the medium decreased relative to the duration of the exposure to HD. In light microscopic sections of skin that were exposed to HD for 6 min. clefts of variable size could be clearly observed between the epidermis and the dermis after a 48-hr culture period. Immunohistochemical staining of collagen type IV demonstrated that separation occurred between the basal membrane and the basal keratinocytes. The described ex vivo human skin model mimicked the in vivo process quite well. Since it was reported that nicotinamide could protect cellular NAD+ levels after HD exposure, HD-treated skin pieces were incubated in medium containing 10 mM nicotinamide. Although this incubation caused an elevation of cellular NAD+ levels and of glucose uptake from the medium compared to control values, it did not result in a substantial reduction of cell death or microblister formation as observed by light microscopy in tissue sections. It was concluded that depletion of cellular NAD+ levels is not the only cause of HD-induced cell death.     

Benzylamide derivative compound attenuates the ultraviolet B-induced hyperpigmentation in the brownish guinea pig skin

This study evaluated the effects of synthetic benzylamide compound I (2,6-dimethoxy-N-phenylbenzamide) on the ultraviolet B (UV B)-induced hyperpigmentation of the skin. UV B-induced hyperpigmentation was elicited on brownish guinea pig skin according to the method reported by Hideya et al. [Arch Dermatol Res 290 (1998) 375] with minor modifications. A lightening effect was observed following the topical application of compound I on UV-stimulated hyperpigmentation. The skin returned to its original color after treatment with compound I. Fontana-Masson staining indicated that melanin level in the hyperpigmented area was significantly decreased in the compound I-treated animals. However, the number of melanocytes were not changed in the compound I-treated groups using the S-100 stain, which is an immunohistochemical method. In vitro experiments using the cultured melanoma cells showed a 31.7% inhibition of melanin production by compound I at 100 microM. In addition, this compound had no effect on the tyrosinase enzyme function. However, it exhibited a catalyzing effect on the dopachrome transformation into 5,6-dihydroxyindole-2-carboxylic acid. Overall, the pigment-lightening effects of the compound I may due to the dopachrome tautomerase stimulation.     

Adipose-Derived Stem Cells Improve Efficacy of Melanocyte Transplantation in Animal Skin

Vitiligo is a pigmentary disorder induced by a loss of melanocytes. In addition to replacement of pure melanocytes, cocultures of melanocytes with keratinocytes have been used to improve the repigmentation outcome in vitiligo treatment. We previously identified by in vitro studies, that adipose-derived stem cells (ADSCs) could be a potential substitute for keratinocytes in cocultures with melanocytes. In this study, the efficacy of pigmentation including durability of grafted melanocytes and short-term safety was examined in the nude mouse and Sprague-Dawley rat after grafting of primary cultured human melanocytes, with or without different ratios of primary cultured human ADSCs. Simultaneous grafting of melanocytes and ADSCs, which were separately cultured and mixed on grafting at the ratios of 1:1, 1:2, or 1:3, showed better efficacy than that of pure melanocytes. Grafting of melanocytes cocultured with ADSCs resulted in a similar outcome as the grafting of cell mixtures. Skin pigmentation by melanocytes : ADSCs at the ratios of 1:1 and 1:2 was better than at 1:3. No significant difference was observed between the 1-week and 2-week durations in coculturing. Time-course microscopic examination showed that the grafted melanocytes remained a little longer than 6-week post-grafting. No inflammatory cell infiltration was observed in the grafted skin and no melanocytes were detectable in other organs. Collectively, grafting of melanocytes and ADSCs was equally safe and more effective than grafting of melanocytes alone. Despite the absence of significant differences in efficacy between the group of 1:1 and that of 1:2 ratio, 1:2 ratio for 1-week coculturing may be better for clinical use from the cost-benefit viewpoint.     

Involvement of Tryptase and Proteinase-Activated Receptor-2 in Spontaneous Itch-Associated Response in Mice With Atopy-like Dermatitis

This study investigated the involvement of tryptase and proteinase-activated receptor (PAR) subtypes in spontaneous scratching, an itch-associated behavior, in NC mice. This strain of mice showed chronic atopy-like dermatitis and severe spontaneous scratching, when kept a long time in a conventional environment. The trypsin-like serine proteinase inhibitor nafamostat mesilate (1 – 10 mg/kg) dose-dependently inhibited spontaneous scratching in mice with dermatitis. The activity of tryptase was increased in the lesional skin, which was inhibited by nafamostat at a dose inhibiting spontaneous scratching. Enzyme histochemistry revealed the marked increase of toluidine blue–stained cells, probably mast cells, with tryptase activity in the dermis of the lesional skin. Intravenous injection of anti-PAR₂ antibody suppressed spontaneous scratching of mice with dermatitis. Intradermal injection of the PAR₂-activating peptide SLI-GRL-NH₂, but not PAR₁, ₃, ₄-activating peptides, elicited scratching at doses of 10 – 100 nmol/site in healthy mice. PAR₂-immunoreactivity was observed in the epidermal keratinocytes in healthy and dermatitis mice. These results suggest that PAR₂ and serine proteinase(s), mainly tryptase, are involved in the itch of chronic dermatitis.     

Lentigo maligna occurring in a patient with the past history of laser therapy

A 70-year-old Japanese woman visited our clinic with a pigmented patch on her face from her upper lip to under her nose following laser therapy 15 years ago. Physical examination revealed an asymmetrical dark brown macule with a clear border along with irregular black dots measuring 20 mm. A biopsy specimen showed some irregular-sized atypical melanocytes with deep-colored nuclei on staining. There were observed on the basal layer and a few of them in the prickle-cell layer only in the epidermis. We diagnosed this case as lentigo maligna (LM). Total resection and reconstruction with the Abbe flap were carried out. We searched previous literature for reports on laser therapies resulting in LM and determined the following: (1) there were no reports indicating that laser therapy is one of the causes of LM, (2) judging from invalidity of treatment or recurrence of the condition, laser therapies were considered ineffective for LM treatment, and (3) the numbers of patients undergoing laser therapies, who were not diagnosed with LM, were increasing.     

Use of IN VITRO Skin Recombinants to Evaluate Cutaneous Toxicity: A Preliminary Study

Abstract

The cytotoxicity of surfactants (N cetyl-trimethylammonium bromide, sodium dodecyl sulfate, and sorbitan monooleate), formalin, and two non-water-soluble formulations was tested using a skin recombinant made of human keratinocytes cultured on dead de-epidermized dermis. Histologic observations and biochemical parameters of cytotoxicity (LDH release and MTT activity) are closely related. Moreover, as in the in vivo situation, cationic surfactant is more toxic than anionic and nonionic. Relative cytotoxicity of surfactants and formalin was markedly reduced on reconstructed epidermis in comparison with monolayer culture of human keratinocytes, emphasizing the toxicologic importance of the stratum corneum barrier. Histologic observations show that salicyclic acid, a well-known keratolytic agent in vivo, induced a reduction of the horny layer thickness of the skin recombinant. These preliminary results suggest that the skin recombinant on de-epidermized dermis may prove to be a useful in vitro model for the evaluation of cutaneous toxicity of topically applied substances.     

Melanocortin MC₁ receptor in human genetics and model systems

The melanocortin MC(1) receptor is a G-protein coupled receptor expressed in the melanocytes of the skin and hair and is known for its key role in the regulation of human pigmentation. Melanocortin MC(1) receptor activation after ultraviolet radiation exposure results in a switch from the red/yellow pheomelanin to the brown/black eumelanin pigment synthesis within cutaneous melanocytes; this pigment is then transferred to the surrounding keratinocytes of the skin. The increase in melanin maturation and uptake results in tanning of the skin, providing a physical protection of skin cells from ultraviolet radiation induced DNA damage. Melanocortin MC(1) receptor polymorphism is widespread within the Caucasian population and some variant alleles are associated with red hair colour, fair skin, poor tanning and increased risk of skin cancer. Here we will discuss the use of mouse coat colour models, human genetic association studies, and in vitro cell culture studies to determine the complex functions of the melanocortin MC(1) receptor and the molecular mechanisms underlying the association between melanocortin MC(1) receptor variant alleles and the red hair colour phenotype. Recent research indicates that melanocortin MC(1) receptor has many non-pigmentary functions, and that the increased risk of skin cancer conferred by melanocortin MC(1) receptor variant alleles is to some extent independent of pigmentation phenotypes. The use of new transgenic mouse models, the study of novel melanocortin MC(1) receptor response genes and the use of more advanced human skin models such as 3D skin reconstruction may provide key elements in understanding the pharmacogenetics of human melanocortin MC(1) receptor polymorphism.     

Melanocytes and keratinocytes have distinct and shared responses to ultraviolet radiation and arsenic

The rise of melanoma incidence in the United States is a growing public health concern. A limited number of epidemiology studies suggest an association between arsenic levels and melanoma risk. Arsenic acts as a co-carcinogen with ultraviolet radiation (UVR) for the development of squamous cell carcinoma and proposed mechanisms include generation of oxidative stress by arsenic and UVR and inhibition of UVR-induced DNA repair by arsenic. In this study, we investigate similarities and differences in response to arsenic and UVR in keratinocytes and melanocytes. Normal melanocytes are markedly more resistant to UVR-induced cytotoxicity than normal keratinocytes, but both cell types are equally sensitive to arsenite. Melanocytes were more resistant to arsenite and UVR stimulation of superoxide production than keratinocytes, but the concentration of arsenite necessary to inhibit the activity of the DNA repair protein poly(ADP-ribose)polymerase and enhance retention of UVR-induced DNA damage was essentially equivalent in both cell types. These findings suggest that although melanocytes are less sensitive than keratinocytes to initial UVR-mediated DNA damage, both of these important target cells in the skin share a mechanism related to arsenic inhibition of DNA repair. These findings suggest that concurrent chronic arsenic exposure could promote retention of unrepaired DNA damage in melanocytes and act as a co-carcinogen in melanoma.     

Metallothionein isoform 3 expression in human skin,related cancers and human skin derived cell cultures

Human skin is a well known target site of inorganic arsenic with effects ranging from hyperkeratosis to dermal malignancies. The current study characterizes the expression of a protein known to bind inorganic, As³⁺, metallothionein 3 (MT-3). Expression of this protein was assessed immunohistochemically with a specific MT-3 antibody on human formalin-fixed, paraffin-embedded biopsy specimens in normal skin, squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and melanoma. Assessment in normal skin using nine normal specimens showed moderate to intense MT-3 staining in epidermal karatinocytes with staining extending into the basal cells and moderate to intense staining in melanocytes of nevi. MT-3 immunoexpression was shown to be moderate to intense in 12 of 13 of SCC, low to moderate in 8 of 10 BCC, and moderate to intense in 12 melanoma samples. MT-3 expression in cell culture models (normal human epidermal keratinocytes, normal human melanocytes, and HaCaT cells) showed only trace expression of MT-3, while exposures to the histone deacytalase inhibitor, MS-275, partially restored expression levels. These results indicate that the epidermis of human skin and resulting malignancies express high level of MT-3 and potentially impact on the known association of arsenic exposure and the development of skin disorders and related cancers.     

ICAM-1系统在异位性皮炎发病中的意义

为了解ＩＣＡＭ－１系统在异位性皮炎（ＡＤ）发病中的作用,采用ＥＬＩＳＡ和免疫组化技术对ＡＤ患者的血清、皮损、外周血ＰＢＭＣ的ｓＩＣＡＭ－１含量及ＩＣＡＭ－１表达进行了综合研究。结果显示：①ＡＤ患者血清ｓＩＣＡＭ－１含量显著高于对照组,而每１００个ＰＢＭＣ中ＩＣＡＭ－１表达阳性细胞数两组间无明显差异;②１１例皮损中ＩＣＡＭ－１表达呈强阳性者共６例,主要位于基底层角蛋白细胞和真皮浸润的炎症细胞表面,９例对照标本仅２例基底角蛋白细胞呈弱阳性。提示ＡＤ发病与ＩＣＡＭ－１系统有密切的关系。     

Estimation of Skin Target Site Acyclovir Concentrations Following Controlled (Trans)dermal Drug Delivery in Topical and Systemic Treatment of Cutaneous HSV-1 Infections in Hairless Mice

The use of controlled transdermal delivery of acyclovir (AC V) in the treatment of cutaneous herpes simplex virus type 1 infections in hairless mice was investigated. Using an in vivoanimal model (A. Gonsho, et al. Int. J. Pharm. 65:183–194 (1990)) made it possible to quantify both, the topical and the systemic antiviral efficacy of ACV transdermal patches as a function of the drug delivery rate of the patches. Drug delivery rates required to attain systemic efficacy were found to be higher than the rates required to attain the same magnitude of topical efficacy. The ACV concentrations in the basal cell layer of the epidermis for 50% topical efficacy and 50% systemic efficacy were estimated. The basal epidermis layer was considered to be the site of antiviral drug activity (skin target site). Systemic plasma levels were obtained from pharmacokinetic studies and were used to estimate the ACV concentration achieved systemically in the basal epidermis layer. A computational model for drug permeation across skin was employed to estimate the ACV concentration achieved topically in the basal epidermis layer. Equal topical and systemic efficacies were found to correspond to equal drug concentrations at the site of antiviral activity. The length of the effective diffusion pathway of drug molecules in the dermis prior to entering the blood circulation was assumed to be approximately equal to 1/20 of the anatomical dermis thickness because of dermis vascularization.     

Melanocortin MC1 receptor in human genetics and model systems

The melanocortin MC₁ receptor is a G-protein coupled receptor expressed in the melanocytes of the skin and hair and is known for its key role in the regulation of human pigmentation. Melanocortin MC₁ receptor activation after ultraviolet radiation exposure results in a switch from the red/yellow pheomelanin to the brown/black eumelanin pigment synthesis within cutaneous melanocytes; this pigment is then transferred to the surrounding keratinocytes of the skin. The increase in melanin maturation and uptake results in tanning of the skin, providing a physical protection of skin cells from ultraviolet radiation induced DNA damage. Melanocortin MC₁ receptor polymorphism is widespread within the Caucasian population and some variant alleles are associated with red hair colour, fair skin, poor tanning and increased risk of skin cancer. Here we will discuss the use of mouse coat colour models, human genetic association studies, and in vitro cell culture studies to determine the complex functions of the melanocortin MC₁ receptor and the molecular mechanisms underlying the association between melanocortin MC₁ receptor variant alleles and the red hair colour phenotype. Recent research indicates that melanocortin MC₁ receptor has many non-pigmentary functions, and that the increased risk of skin cancer conferred by melanocortin MC₁ receptor variant alleles is to some extent independent of pigmentation phenotypes. The use of new transgenic mouse models, the study of novel melanocortin MC₁ receptor response genes and the use of more advanced human skin models such as 3D skin reconstruction may provide key elements in understanding the pharmacogenetics of human melanocortin MC₁ receptor polymorphism.     

10趾甲扁平苔藓一例

患者,女性,71岁,10趾甲出现甲板增厚,呈污黄色,有纵嵴,表面粗糙不平,甲周皮肤水肿性红斑六月余,组织病理示：表皮轻度角化过度,颗粒层楔形增厚,棘层肥厚,基底细胞液化变性。并可见表皮下裂缝。真皮浅层扩张的血管周围淋巴细胞呈带状浸润。诊断：甲扁平苔藓。     

Employment of cytology for in vitro skin irritation test using a reconstructed human epidermis model,Keraskin™

Skin irritation tests using reconstructed human epidermis (RhE) employ viability as an endpoint, but color interference or borderline results are often problematic. We examined whether the cytology of cells from treated RhE could determine skin irritancy. Six chemicals (three irritants; DnP, 1-B, PH, three non-irritants; DP, APA, HS) were evaluated in a RhE, Keraskin™. DP, HS, and PH were clearly classified with viability, but DnP, 1-B, and APA were often falsely determined, due to borderline values falling near the cutoff, 50%. In histology, the tissues treated with DnP, 1-B, and PH showed erosion of the stratum corneum, vacuolization, and necrosis in the basal layer. DP- and HS-treated tissues showed relatively normal morphology but APA induced necrosis similar to irritants. Cytology revealed that DnP, 1-B or PH depleted cells and induced irregular and abnormal cell shapes. In contrast, relatively regular and normal shapes and clear distinction between the nucleus and cytoplasm was observed for DP, APA and HS. To further confirm it, additional 10 substances, including false positives from OECD TG 439, were tested. Overall (16 substances in total), cytology: total area predicted the skin irritancy of test chemicals with the highest accuracy (87.5%) followed by cytology: cell count (81.3%), histology (75%) and viability (68.8%), confirming the utility of cytology as an alternative endpoint in the skin irritation test using RhE.     

The Role of Adiponectin in the Skin

Adiponectin (Ad), a 30 kDa molecule, is an anti-diabetic adipokine; although derived from adipose tissue, it performs numerous activities in various other tissues. It binds to its own receptors, namely adiponectin receptor 1(AdipoR1), adiponectin receptor 2 (AdipoR2), and T-cadherin (CDH13). Ad plays several roles, especially as a regulator. It modulates lipid and glucose metabolism and promotes insulin sensitivity. This demonstrates that Ad has a robust correlation with fat metabolism. Furthermore, although Ad is not in direct contact with other tissues, including the skin, it can be delivered to them by diffusion or secretion via the endocrine system. Recently it has been reported that Ad can impact skin cell biology, underscoring its potential as a therapeutic biomarker of skin diseases. In the present review, we have discussed the association between skin cell biology and Ad. To elaborate further, we described the involvement of Ad in the biology of various types of cells in the skin, such as keratinocytes, fibroblasts, melanocytes, and immune cells. Additionally, we postulated that Ad could be employed as a therapeutic target to maintain skin homeostasis.     

组织工程皮肤替代物构建的实验研究

目的探索组织工程皮肤替代物的构建方法。方法采用传代培养的角质形成细胞和成纤维细胞作为种子细胞,I型胶原作为真皮基质,采用气—液界面方式进行培养,用HE染色观察培养物的结构。结果培养的组织工程皮肤替代物具有正常皮肤的形态结构,具有结构致密的真皮和分化良好的表皮。结论本方法可用于活性皮肤替代物的制备。