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Melanogenesis is regulated by melanocytes, which synthesize the pigment melanin inside melanosomes; these melanosomes are exported through dendritic extensions to adjacent keratinocytes and result in skin coloration. Chemically modified tetracyclines (CMTs) are nonantimicrobial tetracyclines that retain the capacity to inhibit matrix metalloproteinases (MMPs) and have shown several biological benefits; in particular, CMT-3 [(4-dedimethylamino sancycline (SAN)] has emerged as a candidate for therapeutic benefits in our previous studies. However, to date, studies of the effects of CMT-3 or SAN on melanogenesis are lacking. We have previously reported the anti-melanogenic activity of CMT-308 (the 9-amino derivative of CMT-3). Herein, we have compared the three tetracycline analogs, doxycycline (DOX), SAN, and CMT-3, for their effects on melanogenesis using B16F10 mouse melanoma cells and have validated results in primary human melanocytes (HEMn-DP). DOX did not show any significant effects on intracellular melanin or melanosome export in DP cells while SAN was cytotoxic at high doses but without effects on melanogenesis at lower doses. However, CMT-3 showed a robust suppression of dendricity parameters (dendrite number, dendrite length, and proportion of dendritic cells) in DP cells which was associated, at least in part, with a significant reduction of intracellular tyrosinase activity. In spite of its inhibition of tyrosinase activity, CMT-3 had no significant effects on intracellular melanin levels, suggesting that it selectively targets melanosome export. Our results demonstrate a unique structure-activity relationship (SAR) for the effects of these compounds on melanogenesis and support the conclusion that removal of the 4-dimethylamino moiety confers the selective capacity to suppress melanosome export. Collectively, these results indicate that CMT-3 might be a candidate for diminishing hyperpigmentation skin disorders.

Skin color is determined by the number of melanin granules produced by melanocytes that are transferred to keratinocytes. Melanin synthesis and the distribution of melanosomes to keratinocytes within the epidermal melanin unit (EMU) within the skin of vitiligo patients have been poorly studied. The ultrastructure and distribution of melanosomes in melanocytes and surrounding keratinocytes in perilesional vitiligo and normal skin were investigated using transmission electron microscopy (TEM). Furthermore, we performed a quantitative analysis of melanosome distribution within the EMUs with scatter plot. Melanosome count within keratinocytes increased significantly compared with melanocytes in perilesional stable vitiligo (P

Auranofin (AF) is an organogold FDA-approved drug for treating rheumatism and has been repurposed for several pharmacological applications based on its anti-bacterial, anti-fungal and anti-inflammatory activities. To the best of our knowledge, there has been no study on effects of AF on melanogenesis yet. Hence, in this work, we studied the effect of AF on melanogenesis using B16F10 mouse melanoma cells and validated results in MNT-1 human melanoma cells. Melanogenesis assay was conducted with concentrations of AF determined to be nontoxic in B16F10 cells as well as HaCaT human epidermal cell line for a duration of 48 h, followed by various assays to delineate mechanisms of melanogenesis inhibition. Ultrastructural analysis was conducted to study further if AF affected melanosome maturation and protein levels of a key melanogenic protein, tyrosinase, and the maturation signaling molecule, cyclic adenosine monophosphate (cAMP), was estimated. Our results demonstrate that AF at nontoxic concentrations of 0.25–1 µM significantly inhibited melanin synthesis in a dose-dependent manner with significant inhibition of 32.85% at 1 µM. The study of mechanisms of melanogenesis inhibition revealed that AF inhibited tyrosinase activity in lysates of B16F10 cells but did not show a direct effect on purified mushroom tyrosinase activity or on copper chelation in a cell-free system, nor did it affect levels of B16F10 tyrosinase protein levels. However, AF significantly down-regulated cAMP levels, inhibited cellular ROS and increased number of melanosomes in immature stages, and also exhibited anti-melanogenic activity in B16F10–HaCaT cocultures. Furthermore, AF showed anti-melanogenic efficacy in MNT-1 cell monocultures and cocultures with an inhibition of intracellular tyrosinase activity. In summary, our results demonstrate a proof-of-principle for AF as a depigmenting agent for hyperpigmentation disorders and adjuvant for melanoma therapeutics.

The process of melanin biosynthesis and its distribution throughout the skin is regulated by complex processes involving several enzymes in melanocytes. Recently, Diwakar et al. demonstrated that cytidine—a sialyltransferase inhibitor, 6′-sialyllactose (6′-SL) and 3′-sialyllactose (3′-SL) inhibited melanogenesis and melanosome transfer process. In this study, we have furthered this research, considering cytidine as a commercially viable and safe option over 6′-SL and 3′-SL. The efficacy of 2% w/v cytidine was studied in MelanoDerm™ skin equivalents in comparison with the positive control 1% w/v kojic acid and the vehicle control. Both the positive control and cytidine demonstrated a significant reduction in melanin content relative to the vehicle control. These experiments conclude that cytidine can effectively reduce melanin content in a skin equivalence assay and suggests that cytidine may be a good candidate for a skin lightening agent for human skin.

Melanosomes are synthesized in melanocytes and transferred to neighboring keratinocytes. However, the associations of melanosome uptake with the proliferation and differentiation of keratinocytes are not fully understood. We examined the associations of melanosome uptake with keratinocyte differentiation and proliferation. SV40T-transformed human epidermal keratinocytes (SV-HEKs) were treated with isolated melanosomes. The effects of melanosome uptake on the proliferation and differentiation of the keratinocytes were analyzed by Western blotting and flow cytometry. The relationship between melanosome uptake and keratinocyte differentiation status was verified by determining the melanin content in the cells. Melanosomes reduced the proliferation of SV-HEKs in a dose-dependent manner, but did not induce differentiation. Melanosome uptake was higher in differentiating keratinocytes compared to non-differentiating keratinocytes, and inhibited significantly by PAR-2 inhibitor. Melanosomes inhibit keratinocyte proliferation. Moreover, melanosome uptake is influenced by keratinocyte differentiation status, being highest in mid-stage differentiating keratinocytes in a PAR-2 dependent manner.

Hydrogen peroxide (H2O2) may have a biphasic effect on melanin synthesis and melanosome transfer. High H2O2 concentrations are involved in impaired melanosome transfer in vitiligo. However, low H2O2 concentration promotes the beneficial proliferation and migration of melanocytes. The aim of this study was to explore low H2O2 and its mechanism in melanosome transfer, protease-activated receptor-2 (PAR-2) expression and calcium balance. Melanosomes were fluorescein-labeled for clear visualization of their transfer. The expression of protease-activated receptor-2 (PAR-2) in keratinocytes was determined by western blot analysis. Flow cytometry was employed to evaluate the effects of H2O2 on calcium levels in keratinocytes. Fluorescence microscopy showed the upregulation of melanosome transfer into keratinocytes following 0.3 mM H2O2 treatment in the co-cultures rather than in the untreated control groups, which was associated with higher expression of PAR-2 protein and increased calcium concentration. The addition of a PAR-2 antagonist inhibited the positive activity of H2O2 and calcium flow in keratinocytes. When calcium flow was blocked by a calcium chelator, the addition of H2O2 did not increase the PAR-2 expression level in keratinocytes, therefore, inhibiting dendrite formation and melanosome transfer. Low H2O2 concentration promotes melanosome transfer with increased PAR-2 expression level and calcium concentration in keratinocytes. In addition, the interaction between melanocytes and keratinocytes is more beneficial to enhance calcium levels in keratinocytes which mediate melanin transfer. Moreover, low H2O2 concentration promotes dendrite formation, in which extracellular calcium and Par-2 were involved.

The C60-fullerene derivatives are expected, as novel and potent anti-oxidants, to more effectively protect skin cells against oxidative stress. UVA-induced oxidative stress is considered to promote melanogenesis and serious skin damage. The effect of any fullerene derivatives on UVA-induced melanogenesis is still unknown. Here, we evaluated effects of a water-soluble polyvinylpyrrolidone (PVP)-wrapped fullerene derivative (named “Radical Sponge®” because of its anti-oxidant ability) on melanogenesis, which was promoted by UVA-irradiation to human melanocytes and skin tissues. Radical Sponge® markedly scavenged UVA-induced reactive oxygen species (ROS) inside human melanocytes as shown by fluorometry using the redox indicator CDCFH-DA. After treatment with Radical Sponge® or other agents, human melanocytes and skin tissues were irradiated by UVA. Then, cellular melanin content, tyrosinase activity and the ultrastructural change of skin melanosomes were examined. Radical Sponge® showed to significantly inhibit UVA-promoted melanogenesis in normal human epidermis melanocytes (NHEM) and human melanoma HMV-II cells within a non-cytotoxicity dose range. As compared with two whitening agents, arbutin and l-ascorbic acid, Radical Sponge® demonstrated the stronger anti-melanogenic potential according to spectrophotometric quantification for extracted melanin. In human skin cultures also, UVA-promoted melanin contents were repressed by Radical Sponge® according to Fontana–Masson stain, suggesting its ability to repress UVA-induced tanning. Transmission electron microscopic ultrastructural images also proved that UVA-increased melanosomes in human skin tissue were obviously reduced by Radical Sponge®. The UVA-enhanced tyrosinase enzymatic activity in NHEM melanocytes was inhibited by Radical Sponge® more markedly than by arbutin and l-ascorbic acid. The UVA-enhanced tyrosinase protein expression, together with cell-size fatness and dendrite-formation, was also inhibited more markedly by Radical Sponge® according to immunostain and flow cytometry using anti-tyrosinase antibody. Thus the depigmentating action of Radical Sponge® might be due to its down-regulating effect on the tyrosinase expression, which is initiated by UVA-caused ROS generation.

The antimelanogenic activity of six hydrocoumarins and alpha-tocopherol (alpha-Toc) in normal human melanocytes was evaluated in both cell culture systems and cell homogenates. The inhibitory effects of hydrocoumarins depended upon their substituent groups. alpha-Toc and some of the hydrocoumarins inhibited melanogenesis in cultured normal human melanocytes, although they did not influence melanin synthesis in enzyme solution prepared as cell homogenates. In addition, alpha-Toc and the hydrocoumarins stimulated intracellular glutathione (GSH) synthesis. In particular, 7-allyl-6-hydroxy-4,4,5,8-tetramethylhydrocoumarin strongly inhibited melanogenesis and intracellular GSH synthesis in normal human melanocytes, more so than alpha-Toc. Furthermore, hydrocoumarins exhibited higher scavenging and quenching activities against with tert-butyl peroxyl radicals and singlet oxygen species. These results suggest that 7-allyl-6-hydroxy-4,4,5,8-tetramethyl hydrocoumarin would be useful as an antimelanogenic agent for the prevention or improvement of skin pigmentation induced by reactive oxygen compounds and free radicals, and may inhibit melanogenesis, including tyrosinase transfer and melanosome differentiation, by interrupting melanization by increasing the intracellular GSH content.

Because outer root sheath (ORS) cells are valuable substitutes for interfollicular epidermal keratinocytes, we wanted to determine whether epidermal equivalents generated from ORS cells and containing cultured melanocytes can serve as an in vitro model for skin pigmentation. In such epidermal equivalents prepared with ORS cells and melanocytes from donors of phototypes II, III and VI, a stratified epithelium resembling normal epidermis developed within 14 days, as documented by histological, ultrastructural (e.g. basement membrane-like structure, keratohyalin granules, keratinosomes) and immunohistochemical (e.g. keratins, integrins, gp80, involucrin, filaggrin) criteria. The melanocytes were localized in the basal layer and accounted for 10% of the total cell number. Heavily pigmented melanocytes from black donors contained regular melanosomes in all stages of maturation, whereas melanocytes derived from white donors contained predominantly melanosomes of stages I and II. Melanosome-laden dendrites were readily detected extending from the heavily pigmented melanocytes, while they were less conspicuous in melanocytes from white donors. The extent of melanosome transfer was independent of the racial origin of the ORS cells. Melanosomes could also be transferred "through racial barriers". Melanosomes, mainly of stages III and IV, were detected in the ORS cells, being distributed either as single or compound melanosomes, again irrespective of the racial origin of the ORS cells. In conclusion, pigmented epidermal equivalents generated from ORS cells offer practical advantages over other in vitro pigmentation models: (1) the ORS cells are easily and repeatedly available from any donor regardless of age; (2) primary cultures of ORS cells are free of contaminating melanocytes, a bias if using interfollicular epidermal keratinocytes; (3) a high degree of epidermal differentiation is maintained for 3 weeks in fully defined medium, enabling labelling and stimulation experiments to be performed and compounds interfering with melanin pigmentation to be tested.

Normal melanosome biogenesis requires the association of structural proteins with tyrosinase. 3T3 Swiss fibroblasts transfected with mouse tyrosinase cDNA (line 13.4, clone c) are a unique system in which melanogenesis takes place in the absence of melanosomal structural proteins. Our study confirmed that transfected fibroblasts displayed tyrosinase activity and some of them produced pigment granules. In the absence of melanosomal structural proteins the granules failed both to show a typical ultrastructure and to undergo the usual melanosome ontogenesis. The differentiating agent--dimethyl sulfoxide--increased phaeomelanin production. Pigment was localized in membrane-bound vesicles which were identified as lysosomes by means of immunogold electron microscopy. Cell line 13.4 had higher levels of lysosomal enzymes (beta-hexosaminidase, alpha-mannosidase) than both parental 3T3 cells and clone pKG4 (fibroblasts transfected with the G418 resistance plasmid). Melanosomal proteins act as scavengers of toxic products of melanogenesis, and our results suggest that in their absence cells may employ an alternative mechanism to sequester injurious products.

We previously reported that treatment of B16 melanotic melanoma cells with reduced glutathione (GSH) converts them to amelanotic cells without any significant down-regulation of tyrosinase activity. To characterize the cellular mechanism(s) involved, we determined the intracellular distribution of melanocyte-specific proteins, especially in melanin synthesis-specific organelles, termed melanosomes by subcellular fractionation followed by Western blotting and confocal laser microscopy (CFLM). In the melanosome-rich large granule fraction and in highly purified melanosome fractions, while GSH-induced amelanotic B16 cells have significantly diminished levels of protein/activity of tyrosinase and tyrosinase-related protein-1 compared with control melanized B16 cells, there was substantially no difference in the distribution and levels of dopachrome tautomerase and the processed isoform of Pmel17 (HMB45) between control melanized and GSH-induced amelanotic B16 cells. Analysis of merged images obtained by CFLM revealed that whereas tyrosinase, Pmel17 and dopachrome tautomerase colocalize with each other in the control melanized B16 cells, tyrosinase does not colocalize with Pmel17 or its processed isoform and with dopachrome tautomerase in GSH-induced amelanotic B16 cells. The sum of these findings suggests that reduced glutathione selectively disrupts the intracellular trafficking of tyrosinase and tyrosinase-related protein-1 but not dopachrome tautomerase and Pmel17 to melanosomes, which results in the attenuation of melanization, probably serving as a putative model for oculocutaneous albinism type 4.

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.

A case study was undertaken to determine whether or not the same dermal histiocytes could phagocytose both melanin and hemosiderin simultaneously. A biopsy specimen was taken from a pigmented lesion of the lower leg of a 57-year-old woman with stasis dermatitis. The specimen was processed for histology, conventional transmission electron microscopy and electron-probe X-ray microanalysis. Histologically, numerous histiocytes with their cytoplasm packed with either Prussian blue-positive granules or Fontana-Masson-positive granules were distributed almost equally in the dermis. Electron microscopically, the dermis had many histiocytes with their cytoplasm containing solitary or compound electron-dense substances. The electron-dense substances were classified into three types according to their degree of electron density. By electron-probe X-ray microanalysis, these electron-dense substances were classified into iron-containing and non-iron-containing substances. Both substances were seen in the cytoplasm of the same histiocytes and even in the same compound electron-dense substance. The former were siderosomes and the latter were probably melanosomes. These results show that the same dermal histiocytes probably phagocytose non-selectively both hemosiderin and melanin granules.

Little is known about the biologic behaviors of cultured melanocytes in relation to donor age. To in- vestigate age-dependent differences, neonatal and adult melanocytes were isolated from the same anatom- ical site, the foreskin, and cultured in the same growth medium supplemented with cAMP inducers (cholera- toxin and 3-isobutyl-methylxanthine). The morphology, melanin content, pattern of melanosome distribution, and growth rate were then compared. Neonatal melano- cytes were bipolar in appearance, whereas adult melanocytes were highly dendritic in appearance. Im- age analysis showed that adult melanocytes were larger and longer, and had a greater number of dendrites than neonatal melanocytes. When the growth medium was replaced by a medium without cAMP inducers, adult melanocytes showed a change in their morphol- ogy from dendritic to spindle-shaped, while the mor- phology of neonatal melanocytes remained unchanged. Melanosomes of adult melanocytes were distributed singly along the dendrites, and extracellular secretion of melanosomes was also found. In contrast, melano- somes of neonatal melanocytes were aggregated near the nuclei. No age-dependent differences in melanin content and growth rate were noted in the donor site- matched cultured melanocytes. These results suggest that donor age is one of the factors involved in deter- mining melanocyte dendricity and melanosome distri- bution, and that increased dendricity of adult melano- cytes is due to increased sensitivity to cAMP inducers. In addition, the adult melanocytes established in our culture system, which resembled dendritic melanocytes in vivo, could be considered a desirable model for study- ing the mechanisms of adult-onset hyperpigmentary disorders and melanogenesis.

In tyrosinase-negative (type IA) oculocutaneous albinism (tyr(-) OCA) fetuses the development of melanocytes has not been fully elucidated. We analysed the distribution of melanocytes in skin samples from a fetus with tyr(-) OCA and from four normal fetuses. Skin samples obtained from 12 different body sites of each fetus were examined by transmission electron microscopy, an electron microscopic DOPA reaction test and immunohistochemistry. No S100 protein-positive cells were detected in any sample. There were fewer HMB-45-positive melanocytes in the skin of the tyr(-) OCA fetus than in the skin of normal fetuses from all body sites sampled. The greatest number of HMB-45-positive melanocytes was present in samples from the scalp of the normal fetuses and in those from the lower limbs of the tyr(-) OCA fetus. Very few melanocytes were detected immunohistochemically in samples from the soles and palms, though their presence was confirmed by transmission electron microscopy. The electron microscopic DOPA reaction test enhanced the melanization of melanocytes in samples from the normal fetuses but not in those from the tyr(-) OCA fetus. Postembedding immunogold electron microscopy using the HMB-45 antibody revealed that the HMB-45 antigen was localized mainly on stages I and II melanosomes. The presence of epidermal melanocytes in samples from all fetal body areas obtained at 17-21 weeks of gestation justifies the use of the electron microscopic DOPA reaction test in the prenatal diagnosis of tyr(-) OCA.

A clear cell sarcoma (CCS) cell line, designated as NCS-1, was established in monolayer culture from a xenograft line originating from a metastatic CCS. Marked karyotypic aberrations and tumorigenicity in nude mice revealed the malignant derivation of the NCS-1 cell line. These cells contained abundant glycogen and were amelanotic by light microscopy. By electron microscopy, however, melanosomes in various developmental stages were seen, and some of them were partially melanized. The electron microscopic dopa reaction revealed the presence of tyrosinase activity. Enzyme-linked immunoadsorbent assay revealed that NCS-1 cells expressed a 75-kDa glycoprotein which was identified as a marker of highly differentiated melanoma cells. From these results, NCS-1 cells were found to retain both cytochemical and morphological properties of CCS. Application of NCS-1 cells to a panel of monoclonal antibodies recognizing melanocytic differentiation antigens showed that they corresponded approximately to highly differentiated melanoma cells. In conclusion, the present study strongly supports the close relationship between CCS and malignant melanoma.

In congenital naevocytic naevi (CNN), the ultrastructure of melanosomes has been demonstrated only in a few case reports. In the present study, five defined skin levels were systematically compared by electron microscopy in five giant and seven medium-sized CNN. The ages of the patients ranged from 5 days to 29 years. Six common acquired naevocytic naevi (ANN) served as controls. Melanosomes were classified morphologically into several groups. Epidermal melanosomes were normal in all cases. In 10 of the 12 patients, the morphology and size of dermal melanosomes corresponded well with those in ANN, although in four young children (up to 4-years-old) a few enlarged or atypical forms were found. On the other hand, two new-born babies showed rather different, grossly enlarged dermal melanosomes of various shapes and some unusual internal features. They were seen focally even in lower dermal layers and in naevus cells in subepidermal sweat ducts. We conclude that dermal naevus cells in CNN of new-borns, at least in some cases, may contain melanosomes with unusual morphological variations. This does not necessarily indicate malignant transformation.

In contrast to other ultraviolet (UV) wavelengths, UV-A can induce long-term or "true" pigmentation rapidly with little or no latency. The response cannot be clearly separated from immediate pigment darkening and is too rapid in onset to be explained by neomelanogenesis. In order to investigate possible mechanisms for this phenomenon, UV-irradiated skin was examined microscopically and ultrastructurally 18 h postirradiation. Specimens from skin sites tanned by exposure to melanogenic doses of UV-A showed a paradoxical reduction in the degree of basal melanization by light microscopy compared to unirradiated skin. Ultrastructurally, there was migration and dispersion of packaged melanosomes within keratinocytes from their normal, aggregated location around the nucleus towards the periphery of the cell. These changes were not observed in specimens exposed to melanogenic doses of UV-B. We propose that UV-A wavelengths can selectively cause redistribution of melanin-laden organelles within human keratinocytes in vivo and that this phenomenon accounts for the visually observed hyperpigmentation that develops soon after single exposures to these wavelengths. Dispersion of melanosomal complexes may be another mechanism by which UV-radiation (UVR) can induce tanning in human skin.

Pigmentation in a Causasian male, resulting from bleomycin therapy for Hodgkin's disease, has been studied ultrastructurally. The melanocytes, though present in normal numbers, showed several abnormalities; the endoplasmic reticulum and the Golgi apparatus were were well developed and the mitochondria were enlarged. Lipid inclusions in the endoplasmic reticulum and numerous autophagocytic vacuoles, some containing lipids were observed. Transfer of melanosomes to keratinocytes appeared to be increased. The melanosomes, which measured less than 0.55 mu were dispersed in the cytoplasm and did not form complexes, as has been observed with nitrogen mustard. The increase of melanocytic activity and the disturbance of melanosome transfer are discussed.

Hyperpigmented skin from a 10-year-old white boy with ichthyosis nigricans has been studied. Histological and ultrastructural studies reveal that the hyperpigmentation is related to both epidermal and dermal hypermelanosis. Melanocytes are hyperactive. The different stages of melanosome synthesis and melanisation appear to be normal. Increased dermal pigmentation probably results from a dischargement of the melanin granules into the dermis secondary to melanocyte alterations. Ichthyosis nigricans is a typical example of melanin pigmentary disturbances of the skin, resulting from disturbances in the normal interactions between melanocytes and keratinocytes.

Mouse S91 melanoma cells which grow as a monolayer in culture were studied by transmission and scanning electron microscopy after fixation in situ. The cells were spindle-shaped and tended to grow in dense colonies. Melanosomes at all stages of melanization (stages I-IV) were observed. Other cell organelles reflected high cellular activity. Both A-type and C-type viral particles were abundantly observed. A-type particles were 60-90 nm in diameter. They had an electron-lucent center surrounded by a double shell. They were abundantly observed in the cisternae of the endoplasmic reticulum but never extracellularly. C-type retrovirus particles were larger, 80-110 nm in diameter. They had an electron-dense core surrounded by an electron-lucent area and a limiting membrane. They were seen to bud from the plasma membrane into intracellular vacuoles or into the intercellular space.

Unusual changes in the melanin pigmentary system were observed on a warty papule biopsy taken from a patient with xeroderma pigmentosum (XP). Degenerated melanocytes full of lipids were observed from the basal layer up to the middle of the epidermis. The melanosomes were polymorphous, variable in size and shape with very strange aspects, such as spider-like and whirling configurations. The structure of the macromelanosomes suggests that they are large autophagosomes. These findings are discussed and compared with previous studies.

Hypomelanotic macules of the skin in tuberous sclerosis (TS) are caused by a complex process, involving the synthesis and maturation of melanosomes. A quantitative method was developed to investigate the maturation of the melanosomes at the level of the melanocytes. The method was tested on biopsies of hypomelanotic and adjacent normomelanotic skin from six patients with definite TS, and compared with other methods. In all patients, a statistically significant lower number of pigmented melanosomes was found per cross-sectioned melanocytic perikaryon and dendritic cell process in the hypomelanotic as compared to the normomelanotic skin. The relative numbers of stage III and IV melanosomes were decreased, and of stage II melanosomes increased in the hypomelanotic macule (also statistically significant). This indicates that it is possible to characterize the hypomelanosis in TS reliably, which may have diagnostic importance. In our hands, assessment of the size of the melanosomes did not show a statistically significant difference between hypo- and normomelanotic skin; the difference found with the DOPA technique was more evident, although not statistically significant.

Isolation of melanosomes from hair or feathers is difficult due to the need to break keratin structures using a strong chemical treatment. All recently published isolation approaches were examined in the isolation of melanosomes from human and dog hair. The best results were achieved from immediate separation of melanosomes by centrifugation after controlled NaOH hydrolysis of the hair. The crucial factor for successful separation is the choice of the optimal amount of time required for disintegration of the hair: if the time chosen is too long, melanosomes are damaged; if it is too short, contamination by residual keratin ensues. High temperatures must be avoided. Attempts to break hair structure by a number of milder reagents have failed.

To facilitate the removal of dermal melanin which is located in melanophages of the skin, we studied the effect of anti-macrophage antiserum on the elimination of dermal melanin in incontinentia pigmenti histologica. Although a pronounced inflammatory reaction was produced at the sites injected with the antiserum, no significant decrease in the amounts of radioactive melanosomes was observed at the injected sites. The failure of the intradermal injection of the antiserum to remove melanin pigment could have been due to the very low mobility of the newly recruited macrophages.

To facilitate the mobilization of dermal melanin from the skin, we studied the role of macrophages in the elimination of dermal melanin in incontinentia pigmenti. The protein and melanin moieties of mouse-melanoma melanosomes were labeled with 14C and 3H, respectively. Labeled melanosomes were injected intracutaneously into the backs of guinea pigs. Carrageenan, a macrophage toxic agent, showed an inhibitory effect on the degradation and elimination of protein moiety of melanosomes from the dermis, when carrageenan was injected i.p. at the time of inoculation of melanosomes. Delayed-type inflammatory infiltrates in contact dermatitis showed some increase in the elimination of melanin moiety of melanosomes from the dermis.

UV-A-induced skin pigmentation was investigated morphologically in semithin and thin sections from 11 volunteers, using different irradiation modalities (single doses of 10, 50 and 100 J/cm2). Visible skin pigmentation was observed in all but two probands, and erythema in two; pronounced pigmentation was present after repeated irradiation only. Contralateral non-irradiated, UV-B-irradiated and suntanned skin specimens UV-B-irradiated and suntanned skin specimens were used as controls. There was an increase in the number of clear cells in the basal layer (x1.6) and particularly of large clear cells (x1.7) after repeated irradiation. Also, the number of melanosomes in melanocytic dendrites (x2.8) increased after repeated irradiation. The number, size and shape of the melanosome complexes in both basal and suprabasal keratinocytes remained unchanged, even when a distinction was made between central and peripheral location. In contrast, suntanned skin showed an increase in melanosome complexes in basal (x5.8) and suprabasal (x3.7) keratinocytes. It is concluded that UV-A-induced skin pigmentation differs in some ways from UV-B or sun-induced melanogenesis, and that the clinical grade of tanning cannot accurately be determined by ultrastructural methods.

Cafe au lait spots appear to represent one of the cutaneous features of Ataxia-telangiectasia (A.T.). At the cellular level, they are characterized by an epidermal hypermelanosis with a normal number of melanocytes. At the subcellular level, two basic abnormalities are observed: (1) an increase in the synthesis of melanosomes; (2) a modification of their distribution within the epidermal keratinocytes. Numerous pendulous melanocytes project into the upper dermis. Altered melanocytes are also observed. These show mitochondrial dilatation, melanosomal autophagic vacuoles, as well as lipid droplets. These abnormalities are not observed in the normal surrounding skin. The significance of these observations is discussed and stress is placed on the difficulty in establishing a differential diagnosis of cafe au lait spots, based on their histoenzymological and ultrastructural features.

The ultrastruct of melanosomes in mammalian epidermis has been studied using the freeze-fracture technique. The internal structure of the melanosome, as seen in cross-fractured granules, consists of a very fine particulate matter. There is no evidence of an internal membranous structure in melanosomes, indicating that the internal matrix which is characteristic of ultra-thin sectioned material most likely represents cross-linked protein fibers. Fracture "en-face' of the melaosome limiting membrane reveals a random distribution of intramembrane particles on both the P- and E-faces of the membrane. There is a significantly higher density of IMPs on the P-face. The IMPs of the melanosome membrane may be involved in (a) selective passage of ions through the membrane, (b) membrane transducing events, and/or (c) anchoring sites for cytoplasmic fibrils and microfilaments.