Inhibition of 5α-reductase activity in human skin by zinc and azelaic acid

The effects of zinc sulphate and azelaic acid on 5 alpha-reductase activity in human skin were studied using an in vitro assay with 1,2[3H]-testosterone as substrate. When added at concentrations of 3 or 9 mmol/l, zinc was a potent inhibitor of 5 alpha-reductase activity. At high concentrations, zinc could completely inhibit the enzyme activity. Azelaic acid was also a potent inhibitor of 5 alpha-reductase; inhibition was detectable at concentrations as low as 0.2 mmol/l and was complete at 3 mmol/l. An additive effect of the two inhibitors was observed. Vitamin B6 potentiated the inhibitory effect of zinc, but not of azelaic acid, suggesting that two different mechanisms are involved. When the three substances were added together at very low concentrations which had been shown to be ineffective alone, 90% inhibition of 5 alpha-reductase activity was obtained. If this inhibition is confirmed in vivo, zinc sulphate combined with azelaic acid could be an effective agent in the treatment of androgen related pathology of human skin.     

A method for the evaluation of the local antiandrogenic action of 5α-reductase inhibitors on human skin

A method has been developed for the first time that allows the evaluation of the effect of therapeutic concentrations of 5α-reductase inhibitors in human skin, without applying radioactivity to the skin. Moreover, the method makes it possible to determine whether inhibition is found only at the application site or also in other parts of the skin.     

Immunohistochemical identification of interleukin 1α and β in human eccrine sweat-gland apparatus

Bouin-fixed paraffin sections or acetone-fixed cryostat sections were labelled with the avidin-biotin complex (ABC) or peroxidase-antiperoxidase (PAP) method using three monoclonal antibodies (MAbs) and two polyclonal antisera to human recombinant interleukin I beta (IL-I beta) and three polyclonal antisera to human recombinant interleukin I alpha (IL-I alpha). In the secretory coil both IL-alpha and beta were detected in the clear, but not in the dark cells. Both luminal and basal cells of the coiled and straight ducts expressed IL-I alpha and beta, the IL-I labelling being more intense in the luminal cells. IL-I was not usually detected in the initial portion of the intraepidermal eccrine sweat duct, whereas intense labelling was seen in the upper part including through the stratum corneum. In skin biopsies of the palm, taken after exercise, there was only faint IL-I labelling of the secretory cells, whereas the luminal cells of the dermal ducts showed intense labelling for both IL-I alpha and beta. In the acrosyringium, exercise did not alter the pattern for IL-I alpha and beta, except that in the palm, some of the antibodies to IL-I beta produced a more intense immunolabelling of the acrosyringeal cells. This study identifies a distinct and similar distribution of the two forms of IL-I throughout the eccrine sweat-gland apparatus and indicates that part of the IL-I epidermal pool originates from the sweat.     

The distribution of α6β4 integrins in lesional and non-lesional skin in bullous pemphigoid

The alpha 6 beta 4 integrin is associated ultrastructurally with the hemidesmosomes of the basal keratinocytes and with the bullous pemphigoid antigen (BPA), suggesting an important role in adhesion of epidermal cells to the basement membrane. Using an immunofluorescence technique with chain-specific monoclonal antibodies to the alpha and beta subunits we have investigated the distribution of the alpha 6 beta 4 integrin in normal skin (n = 3) and in BP skin (uninvolved, perilesional and lesional) [n = 11]). The findings have been compared with other types of subepidermal blisters and with normal skin split by chemical means (n = 2) and by suction (n = 2). The distribution of alpha 6 beta 4 integrin was compared with that of bullous pemphigoid antigen (BPA) and with other basement membrane zone (BMZ) macromolecules, laminin, collagen type IV, collagen type VII and the BM600 antigen. In uninvolved, perilesional and early pre-blistered lesional BP skin the distribution of both the alpha 6 and beta 4 integrin subunits, BPA laminin, collagen types IV and VII and the BM600 antigen was identical to normal skin, i.e. a linear band in the BMZ. Within BP blisters, both alpha 6 and beta 4 integrin subunits and BPA were absent, except in two blisters in which the integrin expression was retained in the blister roof, despite loss of BPA. The other BMZ components were expressed on the blister floor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suprabasal expression of epidermal α2β1 and α3β1 integrins in skin treated with topical retinoic acid

In normal adult human skin, expression of epidermal integrins is confined to keratinocytes in the basal layer. However, suprabasal expression of α2, α3 and β1 integrin subunits is noted in hyperproliferative epidermis in wound repair and psoriasis. In this study, we examined the effect of topical all- trans -retinoic acid (RA), known to induce epidermal hyperplasia, on expression of integrins in human epidermis. Immunostaining of vehicle-treated skin revealed expression of α2, α3 and β1, as well as α6 and β4 integrin subunits entirely on basal keratinocytes. Topical application of RA (0.1%) for 2 weeks resulted in marked suprabasal expression of α2, α3 and β1 integrin subunits, whereas α6 and β4 staining remained on basal keratinocytes. Staining for putative ligands of α2β1 and α3β1 integrins, i.e. type IV collagen, laminin-5 and fibronectin, was not detected in the epidermal layer in RA- or vehicle-treated skin. Treatment of HaCaT keratinocytes in culture with RA (1 μmol/L) enhanced α2 and β1 mRNA abundance. Furthermore, RA slightly up-regulated the expression of α2, α3 and β1 integrin subunits on primary epidermal keratinocytes and HaCaT cells in culture with no effect on cell proliferation. These results provide evidence that RA-elicited epidermal hyperplasia is associated with aberrant suprabasal expression of α2β1 and α3β1 integrins, and that this also involves direct stimulation of keratinocyte integrin expression by RA.     

Re-epithelialization of normal human excisional wounds is associated with a switch from αvβ5 to αvβ6 integrins

Summary During cutaneous wound repair, keratinocytes move laterally across the wound surface. For this lateral movement epidermal cells must disassemble their tenacious connections to the basement membrane and their neighbouring cells, and express surface receptors that permit translocation over the wound surface extracellular matrix. If the basement membrane is disrupted, the epidermis migrates over a provisional matrix that contains fibrinogen, fibronectin, vitronectin and tenascin. Although α5β1 integrin, a fibronectin receptor, is expressed by human epidermis during reepithelialization of excisional and incisional wounds, the spatial and temporal patterns of vitronectin, tenascin, and other fibronectin receptors are less clear. Other potential receptors include αvβ5 for vitronectin and αvβ6 for fibronectin and tenascin. To study provisional matrix integrin expression during human wound healing, full-thickness 4-mm punch biopsies were performed on the inner surface of the upper arm in adult volunteers. At 3, 7 and 14 days after injury wound sites were excised, bisected, quick frozen in liquid nitrogen, and examined for the expression of α5, β1, αv, β5 and β6. At 3 days, α5β1 and αvβ5, but not αvβ6, appeared around the basal and suprabasalar cells of the migrating epidermis. At 7 days, α5β1, αvβ5, and αvβ6 appeared around the perimeter of the basal cells of the migrating epidermis. By 14 days, when re-epithelialization was complete, all basal and suprabasalar cells overlying the wound expressed α5β1 and αvβ6, but not αvβ5. Thus, αv appeared to switch its heterodimeric association from β5 to β6 subunit during re-epithelialization.     

Expression of the α1–α6 collagen IV chains in the dermoepidermal junction during human foetal skin development: temporal and spatial expression of the α4 collagen IV chain in an early stage of development

To study the expression of the α1–α6 chains of type IV collagen in the dermoepidermal junction (DEJ) during human foetal skin development, human foetal (10 and 20 weeks of gestation) and adult skin was immunostained with specific monoclonal antibodies to the α1–α6 chains of type IV collagen. Intense expression of the α4 chain and weak expression of the α2 and α6 chains were observed in the DEJ of 10 weeks gestational skin. In contrast, the α1, α2, α5 and α6 chains were detected in the DEJ of 20 weeks gestational and adult skin. Preferential expression of α4 during the early phase of development (10 weeks of gestation) may suggest a chain-specific regulatory mechanism for type IV collagen expression and its potential role in DEJ formation during development.     

A case of skin hyperpigmentation due to α-MSH hypersecretion

A case is presented of generalized skin hyperpigmentation due to alpha-MSH hypersecretion from the pituitary that was most marked in the light-exposed areas. The patient also had secondary adrenal dysfunction, peripheral lymphadenopathy, streptococcal glomerulonephritis and malabsorption. Analysis of this patient's alpha-MSH using high-pressure liquid chromatography (HPLC) showed a novel acetylation profile compared to normal individuals and to patients with Cushing's disease and Nelson's syndrome. Glucocorticoid replacement therapy resulted in suppression of alpha-MSH hypersecretion and complete resolution of the illness.     

Immunohistochemical studies of amyloid P component distribution in normal human skin

The distribution of amyloid P component (AP) in normal human skin was investigated by a light and electron microscopic immunoperoxidase technique, using antibodies to serum amyloid P component (SAP). AP, or an immunologically cross-reactive protein, was found to be specifically localized to the microfibrils of papillary oxytalan fibers and to the peripheral microfibrillar mantle surrounding the elastin core of mature elastic fibers in the reticular dermis; collagen fibers were not stained with anti-SAP. AP was not detected in the dermal-epidermal basement membrane or in the basement membranes surrounding dermal papillary blood vessels and eccrine structures. These findings, which establish the detailed distribution of normal tissue AP in the skin, provide a basis for further studies of the function and behavior of this protein in health and disease.     

Structure-activity relationships in the murine local lymph node assay for skin sensitization: α,β-diketones

The biological activity of skin-sensitizing chemicals is related to their ability to react, either directly or after metabolic activation, with appropriate skin proteins. For direct acting electrophilic compounds, this ability can be modelled, using the RAI (relative alkylation index) approach, by a combination of electrophilicity and hydrophobicity parameters. Several structure-activity relationships based on this approach have been reported, but most of them cover guinea pig sensitization test data on what chemists would classify as relatively soft electrophilic chemicals. In the present work, an electrophilicity parameter based on Taft substituent constants is derived for hard electrophiles having a reactive carbonyl group, and is used to calculate RAI values for the analysis of sensitization test data obtained in the murine local lymph node assay (LLNA) for a series of alpha, beta-diketones. The sensitization potential of these reactive hard electrophilic carbonyl compounds in the LLNA shows a good correlation with the RAI. Overall, the findings reaffirm our view that physical organic chemistry is the key to understanding why some chemicals sensitize more strongly than others, while some do not sensitize at all, and provide further evidence of the value of the LLNA for SAR studies.     

Fibroblast expression of collagen integrin receptors α1β1 and α2β1 is not changed in systemic scleroderma

The skin of patients with systemic scleroderma (SSc) is characterized by excessive extracellular matrix deposition in the dermis. As collagens represent the major structural component, we used fluorescence-activated cell sorter analysis to study the levels of collagen receptors expressed at the surface of fibroblasts derived from involved skin areas. In contrast to previous reports, no differences in the expression of alpha1, alpha2 or beta1 integrin subunits, which constitute the major collagen receptors on fibroblasts, were detected on SSc fibroblasts as compared with normal control fibroblasts. Variation of cell culture conditions, e. g. passage number (from 2 to 10), seeding density, cell cycle or serum concentration, did not change this result. These observations indicate that any abnormal response of SSc fibroblasts to their matrix environment is not controlled at the level of receptor expression.     

Expression of MC-R, POMC and POMC peptides and evidence for a immunoregulatory role of α-MSH in human dermal papilla cells

Pro-opiomelanocortin (POMC)-derived peptides are well-known regulators of pigmentation and proliferation of epidermal and hair follicle-derived melanocytes. We demonstrated that human dermal papilla cells (DPCs), a distinct myofibroblastic cell population of the hair follicle, participate in the cutaneous POMC system. DPCs in vitro and in situ expressed the melanocortin receptor-1 (MC-1R) as well as MC-4R as shown by RT-PCR, immunofluorescence and immunohistochemistry. Expression of POMC but not agouti signalling protein, a natural MC-1R/MC-4R antagonist, was also detectable in DPC. Generation of POMC peptides by DPCs in vitro was demonstrated by immunofluorescence and ELISA studies revealing the expression of both adrenocorticotropin and β-endorphin. To investigate the functional relevance of MC-R expression in DPCs, we examined the effect of α-MSH on interferon-γ (IFN-γ)-induced expression of intercellular adhesion molecule-1 (ICAM-1), an adhesion molecule upregulated in inflammatory disorders of the hair follicle such as alopecia areata. α-MSH markedly suppressed the IFN-γ-mediated upregulation of ICAM-1 in DPCs as shown by real-time PCR studies, while α-MSH alone did not have any effect. Our data suggest that melanocortins such as α-MSH mediate paracrine and autocrine effect in the dermal papilla whose disruption may contribute to inflammatory diseases of the hair follicle.     

Effect of PUVA on plasma and skin immunoreactive α-melanocyte stimulating hormone concentrations

Plasma alpha-melanocyte stimulating hormone (alpha-MSH) concentrations were measured in patients receiving PUVA therapy as treatment for mycosis fungoides, and PUVA or UVB as treatment for psoriasis. Skin immunoreactive alpha-MSH was also measured in those patients who received PUVA. The mean plasma and skin alpha-MSH concentrations after 2-3 weeks of PUVA were not significantly different from pre-treatment values and showed no relationship either to skin type or to the degree of tanning that occurred in response to PUVA. Plasma alpha-MSH concentrations were also unchanged after UVB. There was also no short term change in plasma alpha-MSH concentrations in patients after receiving their first treatment with PUVA. It would appear that circulating and skin alpha-MSH levels are unaffected by UV and show no causal relationship to PUVA induced pigmentation.     

The depigmenting effect of α-tocopheryl ferulate on human melanoma cells

Oral vitamin E (alpha-tocopherol, alpha-T) supplementation has been reported to improve facial hyperpigmentation. alpha-Tocopheryl ferulate (alpha-TF) is a compound of alpha-T and ferulic acid connected by an ester bond; ferulic acid is also an antioxidant, and could scavenge free radicals induced by ultraviolet (UV) radiation, and thus maintain the long-lasting antioxidative effect of alpha-T. Our aim was to see whether alpha-TF might be useful as a whitening agent and an antioxidant to improve and prevent facial hyperpigmentation following UV exposure. In this study, the inhibitory effect of alpha-TF on melanogenesis was examined biochemically using human melanoma cells in culture. The results show that alpha-TF, solubilized in ethanol or in 0.5% lecithin, inhibited melanization significantly, as did alpha-T at a concentration of 100 microg/mL, without inhibiting cell growth. This phenotypic change was associated with inhibition of tyrosinase and 5, 6-dihydroxyindole-2-carboxylic acid polymerase activities, and the degree of inhibition was dose dependent. No significant effect on DOPAchrome tautomerase activity was observed. alpha-TF did not directly inhibit tyrosinase activity of the large granule fraction extracted from human melanoma cells, and Western blotting revealed that there were no changes in protein content or in molecular size of tyrosinase, tyrosinase-related protein (TRP)-1 or TRP-2. Therefore, the inhibition of tyrosinase activity by alpha-TF might be due to effects at the post-translational level, and possibly by a secondary molecule activated by alpha-TF. These results suggest that alpha-TF is a candidate for an efficient whitening agent which suppresses melanogenesis and inhibits biological reactions induced by reactive oxygen species.