Identification of basement membrane components in eosinophilic globules in a case of Spitz's nevus.

A case of Spitz's nevus with eosinophilic globules was examined using antibodies for several components of the basement membrane. Aggregated tumor cells revealed the same characteristics as normal nevocytic nevi, that is, they were surrounded by laminin and type-IV collagen, whereas type-VII collagen was absent. All of these components of basement membranes, including type-VII collagen, were also found in eosinophilic globules, which were densely stained by these antibodies. It is assumed that these eosinophilic globules are essentially composed of basement membrane components, which are probably synthesized by epidermal and possibly also by melanocytic tumor cells.

Pigmentary system of the adult alpine salamander Salamandra atra aurorae (Trevisan, 1982).

The pigmentary system of skin from adult specimens of the amphibian urodele Salamandra atra aurorae was investigated by light microscope, electron microscope, and biochemical studies. Yellow (dorsum and head) and black (flank and belly) skin was tested. Three chromatophore types are present in yellow skin: xanthophores, iridophores, and melanophores. Xanthophores are located in the epidermis whereas iridophores and melanophores are found in the dermis. Xanthophores contain types I, II, and III pterinosomes. Some pterinosomes are very electron-dense. Black skin has a single type of chromatophore: the melanophores. Some melanophores are located in the epidermis. In contrast to the dermal melanophores, these present, in addition to typical melanosomes, organelles with different morphology and vesicles having a limiting membrane and containing little amorphous material. Both skin types present some pteridines and flavins, though they are qualitatively and quantitatively more abundant in yellow skin extracts.

Observations on pigment granules in the bones of silky fowls.

The distribution of melanin pigment-containing cells in the bones of both young and adult silky fowls was observed. Melanin pigment was detected not only in melanocytes which were mainly distributed in the periosteum, but also in all the other types of cells in the periosteum and bone. The continuity of the number of pigment granules in melanocytes and that in the other pigment-containing cells could not be recognized because the granules in the latter cells were much fewer than those in the former. In young fowls, the pigment-containing cells were distributed in all layers of the periosteum and bone, but their number was low. On the other hand, in aged fowls, most of the cells in the periosteum had pigment granules. In the bone, however, pigment granules were observed only in osteocyte situated near the surface. These findings suggest that the pigment granules which are observed in osteocytes have been transferred from melanocytes to osteogenic cells or osteoblasts before they differentiate to osteocytes, where they are presumed to be digested.

Tubulin expression in melanocytic skin tumors. An immunohistochemical study.

The microtubulus system as a part of the cellular cytoskeleton contributes to cell movement. Microtubulus assembly and disassembly is considered to be essential for tumor invasion and serves as a target for tumor chemotherapy. Using immunohistochemical methods, we investigated the distribution of tubulin in normal skin and 34 melanocytic skin tumors. In normal skin, tubulin was strongly expressed in dermal nerves, melanocytes, fibroblasts within the papillary dermis and in myoepithelial cells. In melanocytic skin tumors, nevus cells and melanoma cells stained positive, particularly at the periphery of the lesions, where there were single cells and small nests. The main difference between benign and malignant melanocytic tumors was found in the stromal cells: In melanocytic nevi, the stromal fibroblasts were entirely tubulin negative; whereas, adjacent to the invasive edge in primary and metastatic malignant melanoma, the stroma fibroblasts were strongly positive. Our results show that tubulin is regularly expressed in melanocytic skin tumors and may serve as a prerequisite for cell movement. The pronounced expression of tubulin in fibroblasts surrounding malignant melanocytic skin lesions reflects a stromal alteration that might contribute to tumor invasion.

[Cholinergic innervation of vasopressin-containing cells of the blood vessels of the brain in man]. / Kholinergicheskaia innervatsiia vazopressinsoderzhashchikh kletok krovenosnykh sosudov golovnogo mozga cheloveka.

By means of immunochemical method the presence of vasopressin in the melanocytes of the man's brain arteries was found. The terminals of the cholinergic axons come to an end on the surface of these cells. It is suggested that the melanocytes take part in the regulation of the brain arteries mobility.

Complementary expression of melanosomal antigens and constant expression of pigment-independent antigen during the evolution of melanocytic tumours.

We have generated monoclonal antibodies (MoAbs) against melanosomal proteins (MoAb 1C11 and MoAb HMSA-1) and a cytoplasmic protein strongly synthesized in neoplastic melanocytes but not associated with melanogenesis (MoAb 7H11). An immunohistochemical study of paraffin sections showed that nearly 90% of epidermal neoplastic melanocytes, including melanomas, expressed 1C11 antigen, whereas this antigen was poorly preserved in dermal melanocytic cells except melanomas. HMSA-1 antigen was expressed in a complementary manner to 1C11 antigen, being found in dermal naevus cells but not generally in the epidermal regions, except for dysplastic naevi and melanomas. In contrast, 7H11 antigen was distributed in nearly 90% of melanocytic tumours except solar lentigo and lentigo maligna lesions. The failure of MoAb 1C11 to react with dermal melanocytes may reflect a subtle alteration in melanogenesis during tumour evolution. Overall, the combined use of MoAbs serves as an accurate diagnosis of melanocytic tumours, the pigment-independent MoAb 7H11 being particularly useful for amelanotic and metastatic lesions.

Reversible dedifferentiation and redifferentiation of a melanized cell line from a goldfish tumor.

We reported previously the isolation of a melanized cell line that can undergo reversible dedifferentiation and redifferentiation. A heavily pigmented cell line, designated as P15, originally isolated by fish serum-induced melanization of some GEM 81 cells, cloned and serially passaged in fish serum medium, became noticeably less pigmented after several months in fetal calf serum medium and completely unpigmented after another year in the same medium. Addition of fish serum to the medium of this dedifferentiated cell line, designated P15D, induced pigmentation within a week. This re-induced pigmented cell line, designated as P15DI, became unpigmented when cultured in fetal calf serum medium for one month. We report here that the dedifferentiation of P15 occurs in two stages. One week after withdrawal of fish serum, the specific activity of tyrosinase of the culture dropped by approximately 70% and remained at this reduced level for at least one month. After one year, the specific activity of tyrosinase had dropped to a barely detectable level and the culture became completely unpigmented (P15D). Electron microscopic studies showed that the P15D cells have no melanosomes, probably no large vesicles for melanosome formation, but some dopa-positive trans-Golgi network (TGN). Addition of fish serum to the growth medium of P15 cultures led to a steady increase in the specific activity of tyrosinase, detectable after one day. There was also an increase in the amount of dopa-positive TGN within one day. Melanosomes first appeared after three days and became numerous after one week. Upon removal of fish serum, these re-induced cells (P15D1) underwent a rapid decrease in the specific activity of tyrosinase, reaching, after eight days, the basal level seen in P15D cells. We also report that a protein designated as p75 (Mr approximately 75,000), previously shown to be associated with melanosomes in two melanized cell types of goldfish origin, is present in all melanized cell lines, including P15 and P15DI but absent in unmelanized cell lines, including P15D.

Separation of pigmented and albino melanocytes and the concomitant evaluation of endogenous peroxide content using flow cytometry.

Flow cytometry (FCM) has been used extensively to analyze various biological properties of the cell. In this report, we describe a method by which FCM was used to determine the light scattering profile of a mixed population of pigmented and non-pigmented melanocytes, plus its subsequent use for the sorting and separation of the two cell types. In addition, the relative peroxide content in pigmented and non-pigmented melanocytes was compared by flow cytometry. Cultured avian melanocytes from a pigmented control and from three genetically distinct albino sources were studied. FCM analysis of forward versus side light scatter within a mixed suspension of pigmented and amelanotic melanocytes distinguished two overlapping populations of cells. Sorting of these two populations demonstrated that the population exhibiting much side and minimal forward light scatter was primarily pigmented melanocytes, while conversely the population exhibiting less side and more forward scatter was principally non-pigmented cells. These two melanocyte types also demonstrated differences in levels of endogenous peroxides. The intracellular content of peroxide in the two subpopulations of cells was measured utilizing the nonfluorescent compound, 2',7'-dichlorofluorescein diacetate (DCFH-DA), which within the cell is oxidized by intracellular peroxides to a fluorescent dichlorofluorescein (DCF). Non-pigmented albino melanocytes had the highest quantity of endogenous peroxides, while heavily pigmented cells had considerably less peroxide-related fluorescence. The amount of this DCF fluorescence could be enhanced by increasing concentrations of DCF used in the assay. These flow cytometric methods are useful for isolating and culturing subpopulations of melanocytes expressing various pigment levels and to investigate the relationship between melanin and its precursors with hydrogen and lipid peroxides in melanocytes.

Type IV collagen and laminin staining patterns in benign and malignant cutaneous lesions.

The presence of both laminin and type IV collagen was sought at the dermo-epidermal junction and in the dermis adjacent to benign melanocytic naevi of the junctional, compound, and intradermal types; dysplastic naevi; and both primary and secondary melanoma. In all, 154 lesions were studied, using antibodies to laminin and type IV collagen and an indirect immunoperoxidase technique. The staining patterns seen with the two antibodies were virtually identical, although that of laminin was generally fainter. Breaks in and thinning of the normally continuous line of type IV collagen and laminin at the dermo-epidermal junction were seen in association with the junctional activity of benign naevi, and in malignant melanomas in association with invasive tumour cells. Both benign and malignant cells of the melanocyte series showed relatively light pericellular staining around individual cells and clusters of cells in the papillary dermis. This staining pattern was much stronger in the deeper reticular dermis. It is concluded that the pattern of staining of these two antibodies and in particular the presence of breaks in type IV collagen and laminin at the dermo-epidermal junction are not specific for either benign or malignant melanocytic lesions and cannot be used as a diagnostic marker of invasive malignancy.

Breast carcinoma with positive results for melanoma marker (HMB-45). HMB-45 immunoreactivity in normal and neoplastic breast.

HMB-45 (melanocytic cell-specific monoclonal antibody) immunoreactivity has been detected by the immunoperoxidase technique in the cytoplasm of 2 of 100 breast carcinomas. In addition, normal breast lobules and ducts were occasionally found to have positive results in 6 of 100 cases. These findings indicate the need to exercise caution in the interpretation of HMB-45 immunoreactivity for immunohistochemical characterization of neoplasms.

Presence of cytoadhesins (IIb-IIIa-like glycoproteins) on human metastatic melanomas but not on benign melanocytes.

Glycoproteins IIb and IIIa, a heterodimer complex, play a vital role in blood platelet aggregation and are members of a wide family of membrane receptors known as integrins or cytoadhesins. Cellular interaction to extracellular matrix (ECM) adhesive proteins is mediated by integrins. Certain tumor cells are known to interact with ECM and blood platelets in the process of metastasis. However, it is not known if tumor cells, compared with their normal counterparts, acquire IIb-IIIa-like receptors to help them in their metastatic spread. In this study, monoclonal antibodies directed against the IIb-IIIa platelet glycoprotein complex were used on frozen biopsies of normal and various tumor tissues to detect the presence of these integrins. These studies demonstrate the presence of IIb-IIIa-like glycoproteins on the cells of metastatic malignant melanoma but not on benign melanocytes and rarely on other tumors. The presence of integrins on melanomas may help explain their propensity for frequent metastasis.

Nonmelanized macromelanosomes in a cellular blue nevus. Light and electron microscopic observations.

We noted nonmelanized and partially melanized macromelanosomes in a cellular blue nevus and studied their light microscopic and ultrastructural features. Numerous intracytoplasmic eosinophilic inclusions were found in the lesion; individual cells contained up to nine, although most cells demonstrated two or three. The "macromelanosomelike" inclusions ranged in size from 1 to 15 microns. Most of them were partially melanized, and some were nonmelanized. These globules were periodic acid-Schiff positive and diastase resistant, with a centrally melanized core, best seen with the Fontana-Masson technique. S100 protein stained positively the cytoplasm of some nevus cells but not the inclusions. Electron microscopy confirmed the centrifugal melanization of these structures and their targetlike morphologic characteristics. Nine other cellular blue nevi from our files failed to show similar intracytoplasmic inclusions.

[Immunochemical identification of vasopressin in neurons and in granule-containing cells of the human cerebral blood vessels]. / Immunokhimicheskaia identifikatsiia vazopressina v neironakh i granulosoderzhashchikh kletkakh krovenosnykh sosudov golovnogo mozga cheloveka.

By means of indirect immunofluorescent method vasopressin localization has been defined in neurons of the locus coeruleus, in terminals, converging to the substantia nigra cells and the sylvian aqueduct area, as well as in the granule-containing cells of the human cerebral blood vessels. The granule-containing cells are identified as melanocytes, when investigated by comparative light-optic and electron microscopical technique. The melanocytes of the cerebral vessels have various size and form in accordance to their different functional state. In some melanocytes peptide vesicles are revealed. This demonstrates a possible synthesis of vasopressin. It is supposed that local endocrine regulation of the cerebral hemocirculation is performed by cooperation of granule-containing cells, producing hormones with oppositely++ directed constrictor and dilatator action to the vessels.

Effect of wavelength on cutaneous pigment using pulsed irradiation.

Several reports have been published over the last two decades describing the successful removal of benign cutaneous pigmented lesions such as lentigines, café au lait macules' nevi, nevus of Ota, and lentigo maligna by a variety of lasers such as the excimer (351 nm), argon (488,514 nm), ruby (694 nm), Nd:YAG (1060 nm), and CO2 (10,600 nm). Laser treatment has been applied to lesions with a range of pigment depths from superficial lentigines in the epidermis to the nevus of Ota in the reticular dermis. Widely divergent laser parameters of wavelength, pulse duration, energy density, and spotsizes have been used, but the laser parameters used to treat this range of lesions have been arbitrary, with little effort focused on defining optimal laser parameters for removal of each type. In this study, miniature black pig skin was exposed to five wavelengths (504, 590, 694, 720, and 750 nm) covering the absorption spectrum of melanin. At each wavelength, a range of energy densities was examined. Skin biopsies taken from laser-exposed sites were examined histologically in an attempt to establish whether optimal laser parameters exist for destroying pigment cells in skin. Of the five wavelengths examined, 504 nm produced the most pigment specific injury; this specificity being maintained even at the highest energy density of 7.0 J/cm2. Thus, for the destruction of melanin-containing cells in the epidermal compartment, 504 nm wavelength appears optimal.

Neural crest cell differentiation and carcinogenesis: capability of goldfish erythrophoroma cells for multiple differentiation and clonal polymorphism in their melanogenic variants.

Multiple differentiation shown by a single cell line (GEM 81) of goldfish erythrophoroma (tumors of integumental erythrophores) cells after administration of chemical induction in vitro includes 1) melanogenesis, 2) formation of reflecting platelets, 3) synthesis of pteridines heterogeneous to this species, 4) formation of dermal skeletons such as teeth and fin rays, 5) production of neuronal characters, and 6) genesis of lentoid bodies. Melanogenic cells, highest in inducibility, also show remarkable phenotypic diversification in their cell morphology, pigmentation, and physiologic response. In this paper, the following findings are presented; a) multiple differentiation shown by erythrophoroma cells occurs on a clonal basis, making whole component cells of a given induced colony strikingly similar in their cell characters, and b) induced melanogenic clones manifest a remarkable polymorphism in their melanosome ultrastructure and receptor composition associated with motile response. The divergence covers concentric lamellar, multivesicular, fibrillar, and macroglobular types for the former, and a varying combination of receptors for epinephrine, melanin concentrating hormone (MCH), and melatonin for the latter. Because a spectrum of phenotypes expressed by differentiation-induced erythrophoroma cells is restricted to those of neural crest origin (except lentoid bodies) and polymorphism in induced melanized cells is composed mostly of a collection of a variety of known melanogenic characters, it is presumed that erythrophoroma cells are capable of multiple differentiation within the commitment as neural crest cells.

MACG1, a mouse monoclonal antibody detecting a monosialoganglioside expressed in tumor-infiltrating macrophages.

MacG1 is a mouse monoclonal antibody (mAb) directed against a ganglioside, which is differentially expressed by macrophages infiltrating malignant melanomas and benign melanocytic lesions. mAb MacG1 was obtained by immunization with liposomes containing a mixture of gangliosides extracted from malignant melanoma. The antibody was selected for binding to melanoma gangliosides and for reactivity with frozen tissue sections of malignant melanoma. mAb MacG1 showed reactivity in 25 of 46 melanomas examined but in only 1 of 51 nevi tested. The mAb did not react with melanoma cells but did with cytoplasmic granules and deposits associated with large dispersed cells, which were also found in some nonmelanomatous tumors and in some lymphoid tissues. Using mAbs directed against differentiation antigens these cells were identified as macrophages. In nearly all reactive tissues MacG1-positive macrophages accounted for a minority of the total macrophages. The difference in reactivity between malignant melanomas and nevi could not be explained by the variable numbers of total macrophages in these lesions. It is suggested that mAb MacG1 may define a functionally distinct subpopulation of tumor-infiltrating macrophages. Staining of cells other than macrophages was observed in some normal and malignant neural tissues. MacG1 bound to a monosialoganglioside extracted from melanoma and reacted only with NeuAc alpha 2-3Gal beta 1-4Glc-Cer when tested with a panel of ganglioside standards.

Melanocytic atypia in dysplastic nevi. Immunohistochemical and cytophotometrical analysis.

In a double-blind study a correlation was found between the histologically assessed degree of nevomelanocytic atypia in 58 dysplastic nevi (DN) and the presence of two markers associated with malignant transformation. The markers included a marked expression of histocompatibility locus Class I antigens on nevomelanocytes (P less than 0.01) and abnormalities in the nuclear DNA content as measured by DNA cytophotometry (P = 0.01). Both markers were present in most of the markedly atypical DN, in about half of the moderately atypical DN, and in less than 30% of the mildly atypical DN. These findings suggest that a DN with marked or moderate melanocytic atypia indicates a premalignant condition and identifies a patient at risk for melanoma.

Identification of a secreted Mr 95,000 glycoprotein in human melanocytes and melanomas by a melanocyte specific monoclonal antibody.

We have isolated a monoclonal antibody, designated HMB-50, that is highly specific for melanomas and melanocyte derived lesions. The antibody recognizes melanomas, neonatal melanocytes, and junctional nevi but does not react with adult melanocytes, dermal nevi, or a variety of non-melanocyte derived neoplasms. In tissue culture, the antibody reacts with five of seven human melanoma lines and neonatal foreskin melanocytes but fails to recognize fibroblasts and a number of different carcinomas. HMB-50 identifies a Mr 95,000 glycoprotein that is released into the growth medium by melanoma cells and neonatal melanocytes in vitro. This molecule is unrelated to antigens recognized by a variety of antimelanoma monoclonal antibodies isolated in other laboratories. The Mr 95,000 glycoprotein has been purified by antibody affinity chromatography and a polyclonal rabbit antiserum raised that exhibits identical specificity to the monoclonal antibody. The Mr 95,000 glycoprotein is rapidly released by melanoma cells (within 60 min) and one line produces relatively large quantities of the molecule (1 microgram/10(6) cells/24 h). The molecule in normal melanocytes differs slightly in electrophoretic mobility compared to its counterpart in melanomas and this difference appears to result from posttranslational modification.

Human nevocellular nevus cells are surrounded by basement membrane components. Immunohistologic studies of human nevus cells and melanocytes in vivo and in vitro.

Dermal nevus cells in the skin are surrounded by electron-dense deposits resembling a basement membrane (BM), and epidermal melanocytes rest on the epidermal BM. Using antibodies directed against various BM components, we have determined that the BM-like structure surrounding nevus cells in vivo contains type IV collagen, laminin, and BM-1 proteoglycan, analogous to BM throughout the body, but not bullous pemphigoid antigen or epidermolysis bullosa acquisita antigen that are keratinocyte-associated proteins present in the epidermal BM. Moreover, in vitro, both nevus cells and melanocytes derived from adult donors display intracellular and extracellular fibronectin, BM-1 proteoglycan, type IV collagen, and laminin. In contrast, newborn melanocytes maintained under identical culture conditions display none of these BM components, emphasizing the influence of donor age on cell behavior. The data suggest that dermal nevus cells manufacture a BM in vivo, as do certain other neural crest-derived cells. The apparent shared ability of cultured nevus cells and melanocytes to synthesize BM components, coupled with other previously noted behavioral and morphologic similarities in vitro, suggests that these cell types are very closely related; and that morphologic or histochemical differences present in vivo are the result of environmental influences rather than intrinsic differences.