Imaging of melanin distribution using multiphoton autofluorescence decay curves

Background/purpose: Multiphoton fluorescence lifetime imaging (FLIM) is a technique that produces an image based on differences in the decay rate of fluorescence from a sample. Based on this method, the DermaInspect was developed to observe human skin components non-invasively. In this study, we used the DermaInspect to study melanin in skin.
Methods: A human three-dimensional skin model containing melanocytes was embedded in an OCT compound, frozen and sectioned at 10 μm. The melanin distribution in each section was visualized by the DermaInspect using time-resolved single-photon counting and near-infrared femtosecond laser pulse excitation. The melanin distribution of the same sections was then visualized using the Fontana-Masson staining method.
Results: High-resolution images were generated from the ratio of a ₁/ a ₂ ( a ₁e^{− t /120}+ a ₂e^{− t /1100} was chosen to express the exponential fluorescent decay curve) obtained using the DermaInspect. Granules with a high a ₁/ a ₂ ratio, approximately 1 μm in diameter, were observed. Fontana-Masson staining identified these granules as melanin. This new technique was then applied for in vivo observation of melanin in human skin. 'Melanin caps' were visualized in the basal cell layer around the nuclei in images derived from the a ₁/ a ₂ ratio.
Conclusion: Our study confirms that FLIM can non-invasively provide data of the melanin distribution with almost the same quality as the conventional Fontana-Masson staining method, and demonstrates that FLIM is useful for in vivo observation of melanin granules in human skin.     

Tenascin is overexpressed in vitiligo lesional skin and inhibits melanocyte adhesion

The aetiology of vitiligo remains obscure. In this study, the role of integrins in the observed inability of melanocytes to repopulate lesional skin was investigated. Antibodies directed to α₂, α₃, α₅, α_v, α₆, β₁ and β₃ integrin subunits were used. Immunohistology revealed no marked differences in the overall levels of expression of integrins between control, non-lesional, perilesional or lesional skin. Moreover, no differences were noted in the level of expression of integrins or the adhesive capacity between cultured control cells derived from three separate donors and vitiligo-derived melanocytes from two donors. Rather, it was clearly observed that towards the lesion, vitiligo skin contains increasing amounts of tenascin in the basal membrane and papillary dermis in five patients employing T2H5 antihuman tenascin antibody. The anti-adhesive effect observed in vitro for this extracellular matrix molecule using normal melanocytes may contribute to loss of pigment cells in vitiligo or to ineffective repopulation of the lesions.     

Investigation of micronized titanium dioxide penetration in human skin xenografts and its effect on cellular functions of human skin-derived cells

Abstract:  Titanium dioxide (TiO₂) nanoparticles are ubiquitously used materials in everyday life (e.g. paints, household products and plastic goods). However, despite the wide array of common applications, their pathogenetic role was also suggested under certain conditions (e.g. pulmonary neoplasias and lung fibrosis). From a dermatological point of view, it is also of great importance that TiO₂ also serves as a physical photoprotective agent in sunscreens and is widely used in various cosmetic products. However, the effect of TiO₂ on human cutaneous functions is still unknown. Therefore, in the current study, we investigated the in vivo penetration of TiO₂ via human skin transplanted to immunodeficient mice and, furthermore, we measured the in vitro effects of nanoparticles on various functional properties of numerous epidermal and dermal cells in culture. Hereby, using various nuclear microscopy methods, we provide the first evidence that TiO₂ nanoparticles in vivo do not penetrate through the intact epidermal barrier. However, we also report that TiO₂, when exposed directly to cell cultures in vitro , exerts significant and cell-type dependent effects on such cellular functions as viability, proliferation, apoptosis and differentiation. Therefore, our novel findings will hopefully inspire one to systemically explore in future, clinically oriented trials whether there is indeed a risk from micronized TiO₂-containing products on skin with an impaired stratum corneum barrier function.     

Characterization of angiotensin-converting enzyme expression during epidermis morphogenesis in humans: a potential marker for epidermal stem cells

Background  Recent evidence has revealed that angiotensin-converting enzyme (ACE) participates in cutaneous wound healing and contributes to the pathophysiological process of some skin diseases. However, little is known about the role of ACE in epidermis morphogenesis during development.
Objective  To clarify the expression pattern of ACE during embryonic development of human skin.
Methods  Skin samples were obtained from aborted fetuses at different gestational ages and from healthy individuals. Localization of ACE, together with β₁-integrin, keratin 19 (K19) and p63 was examined by immunofluorescence and immunohistochemical staining.
Results  In human fetal skin, at 11–13 weeks of gestation, ACE-positive cells were observed in the primitive epidermis. As the fetuses developed, ACE-positive cells appeared in all the epidermal layers. From 21 weeks of gestation, ACE expression was largely restricted to the basal layer of the fetal epidermis. In contrast, ACE-positive cells were found only in the adult skin basal layer which harbours epidermal stem cells. To explore the possible link between ACE and epidermal stem cells, we further examined the expression of β₁-integrin, K19 and p63, the putative markers for epidermal stem cells. Consistent with the results of ACE expression, from 21 weeks of gestation, the expression of β₁-integrin, K19 and p63 was mainly confined to the basal layer. Immunofluorescent double labelling revealed that ACE-positive cells substantially overlapped with β₁-integrin-, K19- and p63-positive cells.
Conclusions  Our results suggest that ACE may play a role in human epidermis morphogenesis during fetal life and serve as an unrecognized marker for keratinocyte progenitor cells.     

HLA and β2-microglobulin Expression in Basal and Squamous Cell Carcinomas of the Skin

Abstract: Histologic sections of seven squamous cell carcinomas (SCC), 13 basal cell carcinomas (BCC), and a Bowenoid actinic keratoses were examined for expression of HLA class 1 antigens (HLA-ABC) using a monoclonal antibody and an immunoperoxidase technique. Expression of β₂-microglobulin was examined with a polyclonal antibody method. Neither cell marker was detected within the Bowenoid actinic keratoses. Squamous cell carcinomas and basal cell carcinomas exhibited decreased expression of both HLA-ABC and β₂-microglobulin and often did not express these antigens at all. HLA-ABC was present in only two of 13 basal cell carcinomas and four of seven squamous cell carcinomas. β₂-microglobulin was present in one of 13 basal cell carcinomas and two of seven squamous cell carcinomas. When present, these antigens often were present in a few areas of the tumor, but absent in others. In both SCC and BCC. both antigens were usually lost simultaneously. In all tumors with β₂-microglobulin, HLA-ABC a/so was retained. There was no apparent relationship of anatomic site or type of tumor with retention of surface antigens. Since some tumors or portions of tumors retained HLA-ABC and β₂-microglobulin on their surfaces, the absence of these antigens is not an absolute marker for malignancy.     

Book Review

Book reviewed in this article:
Neonatal Dermatology . L.M.S olomon AND N.B.E sterley . Volume IX in the series— Major Problems in Clinical Pediatrics . A lexander J.S chaffer (Ed.) (1973).
Handbuch der Speziellen pathologischen Anatomie und Histologie . Vol. ₇. Weibliche Geschlechtsorgane . F.U ehlinger (Ed.) Part ₄. Vulva, Vagina, Uretra . G.D allenbach -H ellweg , B.E gloff , H.G.H illemanns , J.J aeger , G.F.K lostermann , H.L imburg , K.S taffeldt and P.S toll (1972).
Rheumatology, An Annual Review , Vol. ₄, Oculocutaneous Manifestations of Rheumatic Diseases . G.E.E hrlich (Volume Ed.)(1973).
Human Melanin . E ugene F ollmann (1973).     

Cytokeratins in human basal and squamous cell carcinomas: biochemical, immunohistological findings and comparisons with normal epithelia

The nature of epithelial cell cytokeratins from epidermal basal cell carcinomas (BCC) (8 cases) and squamous cell carcinomas (SCC) (5 cases) was investigated by biochemical and immunological analysis. Cytokeratin proteins were extracted with high salt buffer and triton X 100 and were comparatively analyzed by SDS (sodium dodecyl sulphate) polyacrylamide gel electrophoresis. Both types of tumor showed either an absence or a very low amount (5% of the total protein) of the major protein band (MW 67000) present in normal human epidermis. This correlated well with results of immunolabelling showing that 67000 keratin antisera, only reacted with some dyskeratotic cells in sections of these tumors. Gel electrophoresis showed in BCC and SCC, three distinct groups of predominant polypeptide bands of apparent relative MW: (1) 60–62000 (2) 54–56000 and (3) 49000, representing respectively about 43.0%, 31.0% and 20.4% of the total proteins.
Antibodies raised in animals against polypeptide bands C₁ (MW 62000), C₂ (MW 56000) and C₃ (MW 49000) from SCC, strongly labelled (indirect immunofluorescence) all malignant cells present in the 2 kinds of tumors. These antisera showed a preferential reaction with the basal epithelial cells, in sections of human and animal epidermis and mucosa thus, suggesting numerous common antigenic determinants between epithelial cells from diverse origins. On the other hand, strong differences between mucosal and epidermal upper layers were noted with C₁ C₂, C₃, and 67000 antisera. These results are further evidence for the existence of different pathways of keratinization in epidermis and mucosa.     

Hemidesmosomal variants of epidermolysis bullosa

Abstract: Epidermolysis bullosa (EB), a heterogeneous group of genodermatoses, is characterized by fragility and blistering of the skin, associated with characteristic extracutaneous manifestations. Based on clinical severity, constellation of the phenotypic manifestations, and the level of tissue separation within the cutaneous basement membrane zone, EB has been divided into distinct subcategories. Traditionally, these include the simplex, junctional and dystrophic variants of EB. Recent attention has been drawn to variants of EB demonstrating tissue separation at the level of hemidesmosomes, ultrastructurally recognizable adhesion complexes within the cutaneous basement membrane zone. Clinically, these hemidesmosomal variants manifest either as generalized atrophic benign epidermolysis bullosa (GABEB), EB with pyloric atresia, or EB with late-onset muscular dystrophy, Elucidation of basement membrane zone components by molecular cloning and development of mutation detection strategies have revealed that the hemidesmosomal variants of EB result from mutations in the genes encoding the subunit polypeptides of the 180-kD bullous pemphigoid antigen/type XVII collagen, the α₆β₄ integrin, or plectin, respectively. Collectively, these data add to the understanding of the molecular complexity of the cutaneous basement membrane zone in EB, as attested by the fact that mutations in 10 different genes can underlie different variants of EB. Elucidation of mutations in different forms of EB has direct application to genetic counseling and DNA-based prenatal testing in families with EB.     

A numerical-experimental method to characterize the non-linear mechanical behaviour of human skin

Background/aims: Human skin is a complex tissue consisting of several distinct layers. Each layer consists of various components with a specific structure. To gain a better insight into the overall mechanical behaviour of the skin, we wish to study the mechanical properties of the different layers. A numerical-experimental method was developed to characterize the non-linear mechanical behaviour of human dermis.
Methods: Suction measurements at varying pressures were performed on the volar forearm skin of 10 subjects aged 19–24 years old. Deformation of dermis and fat during suction was measured using ultrasound. The experiment was simulated by a finite element model exhibiting extended Mooney material behaviour to account for the non-linear stress–strain relationship. An identification method is used to compare the experimental and numerical results to identify the parameters of the material model.
Results: C _{10, dermis} was found to be 9.4 ± 3.6 kPa and C _{11, dermis} to be 82 ± 60 kPa. A first rough estimate of C _{10, fat} was 0.02 kPa.
Conclusions: The resulting finite element model demonstrated its ability to describe the response of the skin to suction at various pressures. In the future, this method can be used to characterize the mechanical behaviour of different skin layers using various aperture sizes and to characterize the skin behaviour under various loading conditions.     

COX-2在皮肤鳞状细胞癌中的检测

目的探讨环氧化酶-2(COX-2)在皮肤鳞状细胞癌中的水平及与增殖指标K i67的关系。方法应用免疫组化SP染色法对57例皮肤鳞状细胞癌皮损及20例正常皮肤组织石蜡切片进行COX-2,K i67染色,在光镜下观察并计数其阳性细胞。结果①COX-2与K i67在正常皮肤组织中均无阳性染色,在皮肤鳞状细胞癌皮损中染色强度显著增高(P<0.05);②分化程度不同的皮肤鳞状细胞癌皮损中COX-2蛋白强弱差异无显著性(P=0.756),而K i67有显著性差异(P<0.01);③COX-2与K i67在皮肤鳞状细胞癌皮损中的水平呈显著正相关(r=0.767,P=0.05)。结论在皮肤鳞状细胞癌中COX-2,K i67表达异常增高,二者呈正相关;过度表达的COX-2蛋白可能参与肿瘤细胞的增殖过程。     

Allergen-sensitive atopic dermatitis is improved by injections of allergen combined with F(ab')2 fragments of specific antibodies

Summary Symptoms of atopic dermatitis (AD) can be provoked by exposure to airborne allergens. We have previously shown that patients hypersensitive to D. pteronyssinus (Dpt) allergens were improved by administration of complexes composed of specific antibodies and allergen, which reduce the allergen-specific immune response. We now report that similar results can be achieved by using F(ab')₂ fragments of specific antibodies instead of whole antibody molecules. Eight adult patients with severe AD were included in a single-blind study. During the first 11 months patients were maintained on injections of carrier buffer alone, in an effort to evaluate the extent of spontaneous improvement. They were then treated with intradermal injections of allergen-F(ab')₂ complexes made from autologous specific antibodies and Dpt allergens. The majority of the patients improved spontaneously during the summer months, with an average 30% reduction of symptoms. However, a much more pronounced improvement was observed after 3 months on active therapy, corresponding to a cumulative amount of 60 μg F(ab')₂ and 15 μg allergens. The patients continued to improve over the next 5 months, showing an average 83% reduction of severity scores. The use of F(ab')₂ antibody fragments reduces the risk of inducing an anti-allotypic immune response, and raises the possibility of adding adjuvants to allergen-antibody complexes and/or using specific antibodies isolated from pooled gammaglobulins.     

MORPHOLOGICAL AND BIOCHEMICAL STUDIES ON THE DEVELOPMENT OF FOETAL MOUSE SKIN

SUMMARY.— It has been noted that biochemical and morphological changes occurred simultaneously during the embryogenesis of mouse skin. The onset of histological maturation of collagen fibres coincided with a change in the pattern of the in vitro incorporation of both ³⁵SO₄ into the sulphated mucopoly-saccharide and ¹⁴C-proline into the collagen of whole skin. Simultaneously, an increase in thickness and differentiation of the epidermis and changes in the basement membrane were observed. Thus, by applying a variety of techniques it has been demonstrated that a critical period of skin maturation involving dermis and epidermis is reached on the eighteenth day of embryogenesis in the mouse.     

Skin irritants of the sun spurge (Euphorbia helioscopia L)

Euphorbia helioscopia is a common herbaceous weed found in the British Isles and parts of Europe. It has been responsible for poisoning of livestock resulting in severe inflammation particularly of mucous membranes and the eyes. Four esters of 12-deoxyphorbol were isolated from the fresh aerial parts of the plant and their irritant doses 50% (I.D.50) ascertained using female L.A.C.A. mice. 12-Deoxyphorbol-13-phenylacetale-20-acetate was found to be the major component of the toxic fraction and occurred in yields of 0.0012 % w/w. This compound was also the most irritant substance isolated and exhibited an I.D.₅₀ of 0.038μg, and is accordingly considered to be responsible for the major part of the toxicity of E. helioscopia. A high molecular weight aliphatic ester, 12-deoxyphorbol-13-dodec-dienoate-20-acetate, was also obtained in low yields. This compound exhibited an T.D-no of 0.12μg. The final two esters obtained from the toxic fraction of the plant were the low molecular weight homologues, 12-deoxyphorbol-13-[2-methyl-cis-2-butenoate].-20-acetate and 12-deoxyphorbol-13-[2-methyl-cis-2-butenoate]. These compounds exhibited I.D.₅0 of 3.09 and 0.72 μg, respectively and were the least irritant of the series of compounds isolated.     

Characterization of irritant patch test reactions to topical D vitamins and all-trans retinoic acid in comparison with sodium lauryl sulphate. Evaluation by clinical scoring and multiparametric non-invasive measuring techniques

The study was a single-centre, double-blind randomized, placebo-controlled within-subject comparison of 42 healthy volunteers. Occlusive patch test for 48 h was performed with solutions of 1α,25(OH)₂D₃ (calcitriol), two vitamin D analogues (calcipotriol and KH 1060 (lexacalcitol)), all-trans retinoic acid and sodium lauryl sulphate (SLS) as reference irritant. Solution vehicles and an empty chamber was also included. Test evaluation was performed at day 2, day 3 and again on day 7. Test evaluation was based both on clinical scoring and on various non-invasive measuring methods. 1α,25(OH)₂D₃, calcipotriol and KH 1060 all showed mild irritation in the concentrations tested. The number and severity of test reactions was found to be dose dependent based both on clinical scoring and on non-invasive measurements. Irritation of the vitamin D analogues mainly affected the vasculature with vasodilation and increased cutaneous blood flow. All-trans retinoic acid showed irritant reactions with some similarity to the tested vitamin D analogues; however, the reactions were more prolonged. Transepidermal water loss (TEWL) was affected neither after application of vitamin D analogues nor after application of all-trans retinoic acid and it was thus concluded that these substances are non-corrosive. SLS showed the known irritant mechanism with corrosion and increase in TEWL as the primary event.     

Sodium tetrachloropalladate (Na2[PdCl4]) as an improved test salt for palladium allergy patch testing

Background:  In the last decades, palladium is widely used in dentistry. Allergic reactions to palladium are rarely diagnosed with patch testing, even when positive results would be expected. Palladium tends to cross-react with nickel, which should give rise to more positive reactions to palladium dichloride (standard test salt).
Objective:  The aim of the study was to test whether or not mono-nuclear sodium tetrachloropalladate (Na₂[PdCl₄]) in petrolatum is a better test salt for diagnosing palladium allergy. Positive reactions to the investigated test salt are compared not only with PdCl_2(aq.), but also to NiSO_4(aq.) and NiSO_4(pet.).
Patients/Methods:  Concentration series of Na₂[PdCl₄] were carried out. 164 consecutive patients were patch tested.
Results:  3% of Na₂[PdCl₄]_(pet.) was found to be the highest non-irritative concentration. The results show ( n  = 164) that Na₂[PdCl₄] covers all reactions to PdCl₂ (1.8%) and provokes more positive reactions (14%). From the 164 patients, 18.3% reacted positively to at least 1 of the nickel salts.
Conclusion:  The sensitivity of patch testing with Na₂[PdCl₄] is increased compared with the PdCl₂ salt. Therefore, it can be concluded that Na₂[PdCl₄] is to be a better test salt for diagnosing palladium allergy with patch testing.     

Effects of a ceramide‐containing water‐in‐oil ointment on skin barrier function and allergen penetration in an IL‐31 treated 3D model of the disrupted skin barrier

Atopic dermatitis (AD) is a chronically relapsing, pruritic inflammation of the skin with dryness and disturbed skin barrier function. Recently, we established that IL‐31 treatment of human 3D skin models resulted in a disrupted skin barrier phenotype resembling AD. In this model, we found that IL‐31 interferes with the differentiation of keratinocytes and inhibits the expression of terminal differentiation markers. In the present study, we investigated the effects of a ceramide‐containing water‐in‐oil skin care ointment on the physical skin barrier structure and function in disrupted skin barrier models, generated either by using primary normal human epidermal keratinocytes (NHEK) or HaCaT cells. We observed that the physical skin barrier of the models recovered after daily topical treatment with the ceramide‐containing ointment. Topical application of the ointment prevented downregulation of filaggrin and disorganization of other differentiation markers, such as keratin 10 and β4‐integrin, as demonstrated by immunohistological analysis. The expression of Ki67 was also upregulated in response to the ointment. Furthermore, functional studies revealed that local application of the ointment diminished the increased uptake of fluorescently labelled recombinant allergens of timothy grass (phl p1) in our model. In conclusion, our data revealed that topical application of a ceramide‐containing skin care ointment reduced IL‐31 induced impairments of the physical skin barrier and skin barrier function in an in vitro model of the disrupted skin barrier. This standardized model can be utilized in the future to monitor ex vivo effects of various topical therapies on skin morphology, physiology, and gene expression.     

Pathophysiology of scleroderma: an update

Objectives To review the pathophysiological background of systemic sclerosis in relation to the main, components involved: microvascular system, immunological system and fibroblasts of the connective tissue.
Background Although many particular aspects of the pathophysiology of systemic sclerosis have been investigated in recent years, the complexity of the pathogenesis and the important links between the components involved remain unclear.
Methods Literature review.
Results and conclusion Scleroderma is a connective tissue disorder resulting in a progressive fibrosis of skin and internal organs. The genetic background is not clear. The microvascular system is one of the first targets involved (damage of capillaries, enhanced expression of adhesion molecules interacting with lymphocytes, perivascular infiltrates as starting points for tissue fibrosis). The immune system is unbalanced (selection of T-cell subpopulations, elevated serum levels of several cytokines, occurrence of autoantigens to topoisomerase I, centromeric proteins and RNA polymerases). As far as autoimmunity is concerned the triggering autoantigen is still unknown. Development of connective tissue fibrosis is prominent (sub-populations of fibroblasts with disturbed regulation of collagen turnover by TGF- β , CTGF and collagen receptors (α₁β₁, α₂β₂)). Investigation of pathophysiology of scleroderma is effected by monitoring the serum levels for soluble mediators, by cell culture studies of affected and non-affected fibroblasts and EC, by studying environmentally induced forms of scleroderma and by studies using animal models.