Wide-field optical coherence tomography: imaging of biological tissues.

We describe a two-dimensional optical coherence tomography technique with which we were able to obtain multiple longitudinal slices of a biological sample directly in a single Z scan. The system is based on a femtosecond Cr4+:forsterite laser and an infrared camera for wide-field imaging of the sample with a depth resolution of 5 microm. With this imaging apparatus we were able to investigate human skin and mouse ear samples and to observe the different constitutive tissues.

An ex vivo study of congenital pigmented nevi in epidermal reconstructs.

In order to study morphologic and functional characteristics of pigment cells in congenital pigmented nevi, autologous or heterologous reconstructs have been made using normal keratinocytes and nevus cells from the dermal-epidermal junction or from the dermis. All these cells, keratinocytes and nevus cells, were used as cell suspensions immediately after dissociation from the tissues or after subsequent brief cultivation in a serum-free medium. Reconstructed epidermis were cultured for 15 days at the air-liquid interface with or without ultraviolet (UV) B exposure. The reconstructs were examined macroscopically (formation of hyperpigmented macules), histologically (pigment cell nesting) and ultrastructurally (pigment structure and transfer). Typical nesting of nevus cells was observed in the dermal-epidermal junction or in the superficial dermis associated with macroscopically detectable small pigmented macules. UVB exposure induced an upward migration of nevus cells in the suprabasal layers of the epidermis. This tissue model can be considered as an excellent system for the ex vivo reproduction of pigmented nevi and as an assay of the sensitivity of nevus cells towards UVB irradiation.

Chimeric human epidermal reconstructs to study the role of melanocytes and keratinocytes in pigmentation and photoprotection.

Chimeric epidermal reconstructs made with Negroid melanocytes and Caucasoid keratinocytes (or vice versa) were studied before and after UVB irradiation to understand the respective roles of these cells in tanning and photoprotection, especially lipoperoxidation and enzymatic defences against free radicals. Using this approach, we have confirmed overall the theory of the epidermal melanin unit. We have also shown that melanocytes of poorly tanning Caucasoids, which have a comparatively higher content of unsaturated fatty acids in their cell membrane, are more prone to the peroxidative effects of UV light, and that keratinocytes participate in photoprotection via phototype-dependent antioxidant enzyme activities, especially for catalase.

Quantification of eumelanin and pheomelanin: stereologic image analysis method.

OBJECTIVE: To develop an ultrastructural stereologic image analysis method allowing quantification of intracellular melanization. STUDY DESIGN: First, in the field of image analysis, a newly elaborated segmentation method, SEM2, was compared with a previously described method based on gray level histograms. Only SEM2 allows the segmentation of melanin in micrographs of poorly melanized melanocytes. Second, in the field of stereology, estimation of cell volume remains problematic in the case of mixed cell populations. This problem is approached by the comparison of stereologic alternatives and cytochemistry (L-3, 4-dihydroxyphenylalanine reaction) in epidermal melanocytes and melanoma cells from several in vitro experiments. The cytochemical approach was found to be the best choice. RESULTS: Concerning the quantification of eumelanin and pheomelanin, the alkali elution method, permitting the specific dissolution of pheomelanin on ultrathin sections, was validated in normal human follicular melanocytes. CONCLUSION: These results allow us to envisage the stereologic quantification of eumelanin and pheomelanin at the ultrastructural level. At present our method is undergoing evaluation by comparison with the present method of reference based on high-performance liquid chromatography.

Comparison of HPLC and stereologic image analysis for the quantitation of eu- and pheomelanins in nevus cells and stimulated melanoma cells.

The aim of the study was to compare two methods of quantitating eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission electron microscopy, and stereology. In a previous study, we showed good correlations between both methods for total melanin but not for eumelanins or pheomelanins. We describe here the same comparison in more pigmented cells (nevus cells and stimulated HBL melanoma cells). Transmission electron microscopy micrographs were image analyzed to generate several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanin content, and the number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results showed a good correlation between both methods for total melanin, eumelanins, and pheomelanins with an r equal to 0.99, 0.91, and 0.93, respectively, when all the points were used in the linear regression analyses. In the melanoma cell group (HBL cells cultured in media of different compositions), the chemical and morphometric estimations were not parallel in the case of eumelanins and pheomelanins. In addition, the stereologic and high performance liquid chromatography pheomelanins to eumelanins ratios were still not correlated. These results demonstrate the relevancy of the stereologic method, but the low level of melanization, the possible lack of specificity of melanogenesis in melanoma cells, and a problem of sensitivity of the stereologic method in this context seem to be obstacles in obtaining better results. The utilization of normal human melanocytes could give some answers to our hypotheses.

A variable monilethrix phenotype associated with a novel mutation, Glu402Lys, in the helix termination motif of the type II hair keratin hHb1.

Monilethrix is a rare human hair disorder with autosomal dominant transmission that can be caused by mutations in hair keratins. Up until now, pathogenic mutations in the type II hair cortex keratins hHb6 and hHb1 were restricted to a highly conserved glutamic acid residue Glu413 (Glu117 of the 2B subdomains) in the EIATYRRLLEGEE helix termination motif of the two keratins. The critical glutamic acid residue was substituted either by a lysine or, less frequently, by an aspartic acid residue. Here we report a novel mutation in a French monilethrix family, which again consists of a lysine substitution of another highly conserved glutamic acid residue, Glu402 (Glu106 of the 2B subdomain), in the EIATYRRLLEGEE motif of hHb1. Family members bearing the hHb1 Glu402Lys mutation exhibit a particularly variable disease phenotype. The pedigree comprises two infant members, one with pronounced dystrophic alopecia, follicular keratosis, and clear-cut moniliform hair, and one with no hair loss at all and moniliform hair detectable only by electron microscopy, as well as an adult individual without any clinically or electron microscopically detectable symptoms, but with clear historical proof of the disease.

Comparison of high performance liquid chromatography and stereological image analysis for the quantitation of eumelanins and pheomelanins in melanoma cells.

The aim of the study was to compare two methods quantifying eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography (HPLC) quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission electron microscopy (TEM), and stereology. This study was carried out in cultured human melanoma cells and for each line, melanins were measured by HPLC and cells were fixed and embedded as pellets for TEM. Ultrathin sections were treated or not by the alkali elution method allowing the elimination of pheomelanins. The obtained micrographs were analyzed with our image analysis program permitting the estimation of several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanins content, and number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results show a good correlation between both methods for total melanin, particularly when using the cytoplasmic volume density of melanin (r=0.93). Moreover, we report that the number of melanized melanosomes per cell and not the melanosomal maturation is responsible for the differences in total melanin content observed between the different cell lines. However, none of the stereological melanization parameters was correlated in the case of eumelanins or pheomelanins. In order to demonstrate the utter relevancy of this stereological approach, utilization of more pigmented melanoma cells, comparative study of HPLC and stereology, in normal epidermal melanocytes and a new evaluation of the alkali elution method in appropriate animal models would help us to explain the present results.

Stereological image analysis of cultured human melanocytes observed by transmission electron microscopy.

The aims of the study were to write an image analysis (IA) program allowing the stereological quantification of human epidermal melanocyte melanization at the ultrastructural level and to specify the suitable preparative methods, in keeping with IA limits and stereological principles. Micrographs of cultured human melanocytes obtained in transmission electron microscopy were digitized with a scanner. The key step of the designed IA program is a thresholding based on the gray levels. Hence, gray level histograms (pixel frequency as a function of gray level) of melanocyte images exhibit a peak specific to melanin. The gray level thresholding used consists in isolating the melanin pixels that form profiles on a binary image and in storing the numerical data produced for a given melanocyte profile. These primary data are used to calculate numerous parameters via stereology with melanocyte cytoplasm and melanized melanosome as main reference spaces. The most important stereological parameters obtained are v(mi,cy) (melanin volume per average cell), v(mi,m) (melanin volume per average melanized melanosome), and nm (number of melanized melanosomes per average cell), and their validity is discussed. Melanocytes embedded in situ were abandoned for stereological reasons but pelleted melanocytes were found suitable. Using this computerized tool and stereology, we are able to perform quantitative studies producing varied data even from small cell samples. To our knowledge, this is the first stereological approach for quantifying intracellular melanization. A quantitative comparison of spectrophotometrical results (melanin assay) with stereological results obtained in ultraviolet B-irradiated Caucasian epidermal melanocytes will be performed in order to appraise this method.

Epidermal reconstructs in vitiligo: an extrinsic factor is needed to trigger the disease.

The primary cellular or molecular targets accounting for melanocytes loss in vitiligo are not clearly identified. To study a putative latent epidermal defect in the epidermis of vitiligo patients, we performed in vitro studies using cultured vitiligo melanocytes and keratinocytes transplanted on to a dead de-epidermized dermis according to a variant of Pruniéras' technique. Control autologous constructs were made with keratinocytes and melanocytes of normal adult epidermis and vitiligo epidermis from perilesional skin. For heterologous reconstructs we combined vitiligo-derived melanocytes or keratinocytes with their normal phototype-matched counterpart. After 15 days of culture at the air-liquid interface, epidermal reconstructs were studied macroscopically and microscopically. Immunohistochemistry was performed using antibodies to TRP-1 and NKI-beteb. All heterologous and autologous reconstructs made with melanocytes and keratinocytes from vitiligo patients had a normal histology and ultrastructure. For vitiligo melanocytes or normal melanocytes submitted to the influence of vitiligo keratinocytes, immunophenotype and function (pigment production and transfer) were similar to normal controls. So, without additional noxious stimuli, we could not discriminate between melanocytes and keratinocytes as inducers of the disease. Our data suggest that the basic abnormality in vitiligo vulgaris needs extrinsic factors to be macroscopically revealed or requires a longer period of culture to develop. Our model will allow analysis of the various pathophysiological mechanisms of vitiligo, e.g. autoantibodies or oxidative stress, at the cellular, biochemical or molecular level.

Ex vivo study of skin phototypes.

We studied skin phototypes ex vivo to validate a model of epidermal reconstruction with melanocytes. We made autologous epidermal reconstructs with keratinocytes and melanocytes of healthy donors of skin phototypes I to VI. Keratinocytes and melanocytes were seeded on a dead de-epidermized dermis (Pruniéras type) at a 1:20 melanocyte/keratinocyte ratio. Reconstructed epidermis was grown for 15 d at the air-liquid interface with or without ultraviolet B irradiation. A macroscopic, chromometric. histologic, and ultrastructural evaluation was performed. Reconstructs reproduced the initial phototype with few modifications. The intensity of melanin transfer correlated with the in vivo situation and was stimulated after ultraviolet B irradiation in reconstructs of all categories of skin phototypes.

Gluteal pigmentation after intramuscular iron injections.

We report a case of gluteal pigmentation after intramuscular iron injections. A study in light microscopy, electron microscopy and X-ray micro-analysis by energy selection confirmed iron deposition. These cutaneous pigmentations with iron deposits are infrequent because such injections are now rarely used.

Ex vivo reconstruction of the epidermis with melanocytes and the influence of UVB.

To study pigmentation, we have reconstructed an epidermis ex vivo with keratinocytes and melanocytes. Keratinocytes and melanocytes were grown first in primary cocultures and separately in secondary cultures, then seeded on a dead deepidermized dermis (Pruniéras type) at a 1:20 melanocyte/keratinocyte ratio. Reconstructed epidermis were grown in a special medium enriched with calcium and fetal bovine serum lifted for 15 days at the air-liquid interface. Using histology, immunohistochemistry and electron microscopy we have shown an excellent level of differentiation of the reconstructed epidermis and a physiologic distribution of dendritic melanocytes in the basal layer capable of melanosome transfer to keratinocytes. UVB irradiation 0.15 J/cm2 x 5 consecutive days increased melanocyte numbers and stimulated pigmentation as evidenced macroscopically and microscopically and at the biochemical level. Following UVB irradiation melanosome transfer was markedly increased and isolated or clumps of melanosomes were seen in the basal layers as well as in the stratum corneum. This model allows the study of the physiology of pigmentation ex vivo.

Growth and differentiation of normal human melanocytes in a TPA-free, cholera toxin-free, low-serum medium and influence of keratinocytes.

Melanocyte cultures were obtained from a modification of the keratinocyte culture system MCDB153. Either promelanocytes or mature melanocytes were selected from epidermal cell primary cultures. Pure subcultures of actively dividing melanocytes of both types were grown in a low-serum medium totally deprived of TPA and cholera toxin called melanocyte growth medium (MGM). Early passaged cells from MGM primary cocultures were similar to normal adult human melanocytes in vivo, exhibiting numerous melanosomes, strong dopa positivity and a high dendricity. The ability of MGM to support melanocyte growth was mainly a consequence of its basic composition, combined with a low serum concentration. Bovine pituitary extract significantly enhanced melanocyte growth. Using complete MGM, in the absence of mitogens and keratinocytes, cell growth was maintained, but the differentiation of melanocytes decreased. The presence of keratinocytes was found to promote melanocyte growth. The coculture system used strongly suggests the action of soluble keratinocyte-derived factors. Keratinocyte contact was necessary to sustain melanocyte dendricity and melanization. Melanization and dendricity behaved mostly as independent features when keratinocyte influence was withheld. Our results underline the essential role of keratinocytes in the regulation of melanocyte growth and differentiation in a physiological culture system.

Production of epidermal sheets in a serum free culture system: a further appraisal of the role of extracellular calcium.

Rhenwald and Green's technique is currently the standard method for growing stratifying epidermal cell cultures. The serum free system developed in Ham's laboratory (MCDB 153) was designed to grow keratinocyte monolayers in clonogenic conditions. Our aim was to optimize conditions in serum-free MCDB 153 for culturing epidermal sheets from adult normal skin, and to assess the effect of extracellular calcium and temperature on proliferation and differentiation of cultured keratinocytes. Sixteen strains derived from plastic surgery specimens (mean age of donors 37 years; range 5-89) were used. Primary cultures were seeded at an optimal density of 8 x 10(4) cells/cm2 in primary cultures and 10(4) cells/cm2 in secondary cultures in complete medium including EGF, insulin, hydrocortisone and bovine pituitary extract, supplemented with isoleucine, tyrosine, methionine, phenylalanine, tryptophane and histidine. Amino acid (AA) supplementation allows a 5.8-fold increase in cell counts at confluency and monolayers with densely packed cells are obtained. In AA supplemented cultures, confluency is obtained in 16 +/- 3 days in primary cultures and in 13 +/- 0.5 days at first passages. Switches to 1.1 mM calcium at first or second passages resulted in a significant increase in cell counts (P less than 0.001), when compared with AA supplemented low calcium cultures. Low temperature/low calcium cultures resulted in a 50% decrease in cell counts. Low temperature/high calcium cultures gave similar cell counts as the 37 degrees C controls. AA and calcium supplemented cultures were evaluated for differentiation markers: involucrin expression was increased, keratins 5, 6, 14, 17 were expressed, and the sheets were 6-10 layers thick by electron microscopy, with keratohyalin granules and cornified envelopes appearing at layers 3-6 (from basal layer). Dispase treatment allowed an easy detachment of these sheets. These results show that the culture medium MCDB 153 can be adapted without serum supplementation to batch culture of human adult keratinocytes to produce epidermal sheets suitable for grafting. They also indicate that extracellular calcium in physiological range of concentration is not a sufficient signal for growth arrest when other growth conditions are optimized.

Autologous grafting with noncultured melanocytes: a simplified method for treatment of depigmented lesions.

BACKGROUND: Achromic lesions on the trunk and the extremities often do not respond to treatment and little improvement is obtained in cases of segmental vitiligo. OBJECTIVE: Transplantation of autologous noncultured melanocytes was performed to obtain a successful repigmentation. METHODS: The grafting method is carried out in two steps: production of blisters on the depigmented lesions by freezing with liquid nitrogen and injection in each blister of a suspension of epidermal cells (mainly keratinocytes and melanocytes). The cellular suspension was obtained from samples of skin of the hair scalp after trypsinization. RESULTS: Repigmentation was evident within 25 to 30 days. Coalescence of the pigmented areas was spontaneously observed or obtained after UVA stimulation. Patients with two types of leukoderma-vitiligo or nevus depigmentosus had successful repigmentation after transplantation of autologous noncultured melanocytes. CONCLUSION: This technique appears to be an effective and simple method for treating patients with achromic areas lacking melanocytes.

Inflammatory familial palmoplantar keratoderma: Greither's disease?

A case of a familial palmoplantar inflammatory keratoderma with autosomal dominant inheritance is reported. Associated clinical features included vasomotor troubles and hyperhidrosis consistent with a diagnosis of Greither's disease. Light microscopy was nonspecific. Electron microscopy showed aggregated tonofilaments around the nucleus, without true clumps. Desmosomes were numerous and cell-cell junctions showed an imbricated pattern, well demonstrated in the stratum corneum. The diagnosis of Greither's keratoderma is discussed.

Linear IgA dermatosis of childhood: case report with an immunoelectron microscopic study.

A 7-year-old girl developed a cutaneous bullous eruption with genital and nasal mucous membrane involvement. Direct immunofluorescence revealed linear deposits of IgA and IgM at the basement membrane zone. No circulating antibasement membrane zone antibodies were detected. Small bowel biopsies showed a partial villous atrophy. The clinical, histologic, and immunopathologic findings were consistent with a diagnosis of linear IgA dermatosis of childhood. Immunoelectron microscopy revealed IgA deposits in the lamina lucida in association with hemidesmosomes, confirming results of two recent studies.

[Gingival hyperpigmentation caused by dental restorations: the mechanism of detoxification using selenium]. / Hyperpigmentations gingivales issues de reconstitutions dentaires: mécanisme de détoxication par le sélénium.

X-ray microanalysis and electron diffraction made on granular metallic deposits of the gingival lamina propria, inducing partial periodontal tattoos, demonstrated that these deposits consisted of crystalline particles combining silver, sulphur and selenium. The role of selenium precipitating of silver and other metals as well as its role in detoxication are discussed.

Progressive macular hypomelanosis of the trunk: primary acquired hypopigmentation.

Dermatological practice in Martinique frequently encounters a bizarre skin condition presenting as a progressive and extensive hypomelanosis on the back. The course of this disorder is highly characteristic: it occurs mainly in females from 18-25 years of age, with a progressive development of round, pale, coalescent macules on the back and sometimes on the abdomen. This disease, which does not respond to therapy, spontaneously regresses within 3 to 4 years. Decreased epidermal melanin is the only histological feature. Ultrastructural examination of two cases found that the macular lesions were characterized by a switch from Stage IV single melanosomes (negroid) to small Type I-III aggregated melanosomes (caucasoid). It may thus be stated that the variation in skin coloration in these patients was due to a variation in melanosome size and distribution.