Characterization of vernix caseosa: water content, morphology, and elemental analysis.

Recent studies have prompted interest in the use of epidermal barrier creams as protective biofilms for very low birthweight preterm infants. The key to understanding the role of epidermal barrier films is an elucidation of their interaction with water and a basic knowledge of their composition. In this study, we investigated the morphologic properties and elemental composition of the naturally occurring biofilm, vernix caseosa. This biofilm is typically lacking in preterm infants and its production coincides in utero with terminal differentiation of the epidermis and formation of the stratum corneum. Significantly, vernix (80.5+/-1.0% H2O) had a much higher water content than other barrier creams (Eucerin: 17.1+/-0.6%, Aquaphor: 0.33+/-0.03%, Ilex: 0.19+/-0.02%, petrolatum: 0.03+/-0.01%; all p<0.05). Phase contrast microscopy of vernix showed multiple cellular elements with nucleic "ghosts" embedded in a putative lipid matrix. Transmission electron microscopy revealed flattened structures approximately 1-2 microm in thickness with distinct cellular envelopes indicative of differentiated corneocytes. Compared with mature corneocytes in adult stratum corneum, vernix corneocytes appeared swollen, the density of the keratin filaments was less, and there was a relative lack of tonofilament orientation. Cryofractured specimens were examined by cryoscanning electron microscopy with subsequent elemental localization by X-ray beam analysis. The findings indicate the high water content of vernix is largely compartmentalized within fetal corneocytes. These results are consistent with the novel view of vernix as a "fluid phase" stratum corneum consisting of a hydrophobic lipid matrix with embedded fetal corneocytes possessing unique biomechanical and water-binding properties.

Water disrupts stratum corneum lipid lamellae: damage is similar to surfactants.

Using electron microscopy, we investigated the effect of (i) a dilute surfactant and of water alone on the ultrastructure of stratum corneum lipids in pig skin exposed in vitro at 46 degrees C, and (ii) of water alone on human skin exposed in vivo at ambient temperature. For pig skin, the surfactant sodium dodecyl sulfate disrupts stratum corneum intercellular lamellar bilayers, leading to bilayer delamination and "roll-up" in a water milieu after 1 h, extensive bilayer disruption after 6 h, and nearly complete dissociation of corneocytes after 24 h. Corneodesmosomes show progressive degradation with exposure time. Water alone also disrupts the stratum corneum, but with a slower onset. Alterations in intercellular lamellar bilayers, but not intercellular lamellar bilayer roll-up, are detected after 2 h. Intercellular lamellar bilayer roll-up occurs after 6 h. Extensive dissociation of corneocytes occurs after 24 h of water exposure. Unlike sodium dodecyl sulfate, water exposure results in the formation of amorphous intercellular lipid. Corneodesmosome degradation parallels intercellular lamellar bilayer disruption; calcium appears to offer some protection. Similar disruption of intercellular lamellar bilayers occurs in human skin in vivo at ambient temperature. Our studies show that water can directly disrupt the barrier lipids and are consistent with surfactant-induced intercellular lamellar bilayer disruption being due at least in part to the deleterious action of water. Intercellular lamellar bilayer disruption by water would be expected to enhance permeability and susceptibility to irritants; accordingly, increased attention should be given to the potential dangers of prolonged water contact. For common in vitro procedures, such as skin permeation studies or isolation of stratum corneum sheets, exposure to water should also be minimized.

Neurofibromin, the neurofibromatosis type 1 Ras-GAP, is required for appropriate P0 expression and myelination.

The neurofibromatosis type 1 (NF1) gene product, neurofibromin, regulates activation of the Ras intracellular signaling pathway in Schwann cells. Schwann cells purified from mouse embryos with null mutations in the Nf1 gene increase expression of the major myelin glycoprotein P0. v-Ras expression in cultured Schwann cells partially mimics loss of Nf1, suggesting a role for Ras in upregulation of P0 expression in Nf1-deficient cells. We tested whether loss of Nf1 alters the ability of Schwann cells to form myelin. No significant changes in myelin formation resulted when Nf1-deficient or v-Ras-expressing Schwann cells were cultured with normal neurons. Yet, in organotypic cultures of neurons, Schwann cells, and fibroblasts without neurofibromin, myelination was dramatically reduced. We suggest that Nf1-dependent signaling cascades in neurons and/or fibroblasts, as well as Schwann cells, are required for normal myelination.

Ocular pathology in mice with a transgenic insertion at the microphthalmia locus.

A DNA insertional mutation at the microphthalmia locus (mi) in a transgenic mouse has been developed and given the allele symbol of mitg (Krakowsky et al., 1993). Mice homozygous for this transgene have eyes markedly reduced in size and relatively unpigmented. In this study, we examined the morphology of these eyes using light and electron microscopy. Transgenic homozygous (mitg/mitg) animals have a structurally normal choroid which lacks melanocytes but contains occasional leukocytes. The elastica of Bruch's membrane is absent except in an occasional site. The retinal pigmented epithelium (RPE) appears dramatically abnormal. It displays cellular heterogeneity, residual basal infoldings and apical microvilli, rare and immature melanosomes, and numerous cilia-like structures. Occasionally, cells of the RPE appear to have extruded into or from the choroid. The photoreceptor cells are devoid completely of outer segments and partially of inner segments. Numerous active macrophages are present between the amelanotic RPE and neuro-retina and also within the vitreous body. The anterior uveal tract is underdeveloped and hypomelanotic. This new microphthalmia model exhibits ocular pathology with similarities and differences to other mutations and the mi (microphthalmia) locus.

Chronic growth stimulation of human adult melanocytes by inflammatory mediators in vitro: implications for nevus formation and initial steps in melanocyte oncogenesis.

In the human epidermis, melanocytes are distributed at a distance from each other. In contrast, melanocytes in nevi, which are considered benign neoplasms of melanocytes, are grouped in nests. Although still not well defined, environmental factors are thought to play an important role in the development of nevi. We found that chronic growth stimulation by leukotriene C4, a compound found in increased amounts in inflamed skin, induced pleiotropic modifications in the normal melanocyte phenotype. These changes include loss of contact inhibition and formation of structures resembling tumor spheroids. In parallel with these changes, there was a constitutive expression of Fos protein. Switching these cultures to medium supplemented with phorbol ester sustained growth with reversion of the altered phenotype. In contrast, a cAMP stimulator, cholera toxin, induced features of terminal differentiation. Our findings suggest a role for inflammatory mediators in human epidermal melanocytes. This observation provides insight into melanocyte growth alterations which may have relevance in early stages of melanocyte oncogenesis.

Neurofibromin-deficient fibroblasts fail to form perineurium in vitro.

To identify cell type(s) that might contribute to nerve sheath tumors (neurofibromas) in patients with neurofibromatosis type 1, we generated cell cultures containing neurons. Schwann cells and fibroblasts from transgenic mouse embryos in which the type 1 neurofibromatosis gene was disrupted by homologous recombination (Brannan et al. (1994) Genes Development, 8,1019-1029). Normal fascicle formation by perineurial cells failed to occur in the absence of neurofibromin. Fascicles were reduced in number and showed abnormal morphology when normal neurons and Schwann cells were cultured up to 37 days with fibroblasts lacking neurofibromin. Proliferation was increased in a majority of fibroblast cell strains analyzed from embryos lacking neurofibromin. These observations suggest that mutations in the neurofibromatosis type I gene affect fibroblast behavior that might contribute to neurofibroma formation in patients with neurofibromatosis type 1.

Misrouting of tyrosinase with a truncated cytoplasmic tail as a result of the murine platinum (cp) mutation.

Mice homozygous for the platinum (cp) allele at the albino locus manifest severe oculocutaneous albinism despite the presence in vitro of tyrosinase activity at 25% wild-type levels. We demonstrate that the cp allele results from an A-->T substitution, changing a lysine residue at position 489 to a termination codon, with truncation of tyrosinase's cytoplasmic tail. In choroidal melanocytes of neonatal mutant mice, tyrosinase activity could be detected in the trans Golgi network, but was absent from melanosomes. Instead, it was detected in vesicles in the cell periphery and dendrites, and on the extracellular surface. In the retinal pigment epithelium, activity was present on the extracellular apical and basolateral surfaces. Our results demonstrate misrouting of a mutant tyrosinase lacking its cytoplasmic tail, providing an explanation for the severe effect of this mutation on ocular and cutaneous pigmentation.

On the analysis of the pathophysiology of Chediak-Higashi syndrome. Defects expressed by cultured melanocytes.

BACKGROUND: The Chediak-Higashi syndrome (CHS) is a disorder that affects the synthesis and/or maintenance of storage/secretory granules in various types of cells. Lysosomes of leukocytes and fibroblasts, dense bodies of platelets, azurophilic granules of neutrophils and melanosomes of melanocytes are generally larger in size and irregular in morphology, indicating that a common pathway in storage organellogenesis is affected in patients with CHS. EXPERIMENTAL DESIGN: A pure line of melanocytes has been established using a 2 cm2 shave biopsy from a child with CHS. This 4-week-old male patient had oculocutaneous albinism and expressed neutropenia, impaired platelet function, and no natural killer cell activity. The cultured CHS melanocytes were analyzed for cell biological and biochemical aberrancies. RESULTS: Cultured melanocytes demonstrated some large and/or complexed melanosomes that resembled those observed in melanocytes from ultrastructural sections of the biopsy. Cytoplasmic localization of tyrosinase, tyrosinase-related protein-1 and granulophysin (a 40 kilodalton membrane protein originally identified as a component in dense bodies of platelets) demonstrated a prominent perinuclear accumulation. The basal synthesis of melanin and the activity levels of tyrosine hydroxylase, dihydroxyphenylalanine (DOPA) oxidase, or DOPAchrome tautomerase were comparable to control Caucasian melanocytes in culture. However, melanin synthesis as well as the catalytic activities of tyrosinase were not dramatically upregulated in CHS melanocytes by the addition of isobutyl methylxanthine and cholera toxin in the growth medium when parameters were assayed in cell lysates. In contrast, when assays were performed using live cells, tyrosine hydroxylase demonstrated dramatic upregulation. Medium conditioned by CHS melanocytes demonstrated phenylthiourea-inhibitable tyrosinase activity. Melanocyte lysates and conditioned medium analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DOPA staining showed an extra, approximately 100 kilodalton soluble protein band with DOPA positivity and tyrosinase immunoreactivity. In addition to tyrosinase, one of three lysosomal enzymes assayed (beta-glucuronidase) was aberrantly secreted into the medium. CONCLUSIONS: These results demonstrate that melanocytes cultured from CHS express a defect in the structure and/or function of the melanosome and abnormal trafficking of some cellular proteins.

Smyth chicken melanocyte autoantibodies: cross-species recognition, in vivo binding, and plasma membrane reactivity of the antiserum.

Smyth line (SL) chickens, which develop a depigmenting disorder similar to human vitiligo, produce circulating anti-melanocyte antibodies (Austin, L.M. et al., (1992) The detection of melanocyte autoantibodies in the Smyth chicken model for vitiligo. Clin. Immunol. Immunopathol., 64:112-120). In order to characterize these autoantibodies, we studied the reactivity of cultured chicken, mouse, and human melanocytes, as well as frozen sections of chicken feather follicles and embryonic eyes, against SL serum, employing indirect immunofluorescence. Light Brown Leghorn (LBL) serum was used as a negative control. Chicken (SL and LBL), mouse, and human melanocytes exhibited greater fluorescence with SL serum than with LBL serum (up to a 1:60,000 dilution). The fluorescent pattern was predominant along the perimeter of the cells, suggesting plasma membrane staining. Fluorescence-activated flow cytometry analysis and immunocytochemical localization at the ultrastructural level using intact chicken cells supported this hypothesis. Melanocytes were readily stained in cryosections of regenerating feather follicles and embryonic eyes incubated with SL, but not LBL, serum. In addition, amelanotic melanocytes in albino chicken feathers reacted with SL serum. SL serum also preferentially stained cells emigrating from cultured avian neural tubes and within the dermis of the proliferative germ of regenerating feather follicles suggesting that melanoblasts express the antigens. We conclude that Smyth line serum contains melanocyte autoantibodies that cross-react with mouse and human melanocytes, are able to bind to pigment cells within tissues, and recognize antigens expressed in the cytoplasm and on the surface of melanocytes and melanoblasts.

Mutation in and lack of expression of tyrosinase-related protein-1 (TRP-1) in melanocytes from an individual with brown oculocutaneous albinism: a new subtype of albinism classified as "OCA3".

Most types of human oculocutaneous albinism (OCA) result from mutations in the gene for tyrosinase (OCA1) or the P protein (OCA2), although other types of OCA have been described but have not been mapped to specific loci. Melanocytes were cultured from an African-American with OCA, who exhibited the phenotype of Brown OCA, and his normal fraternal twin. Melanocytes cultured from the patient with OCA and the normal twin appeared brown versus black, respectively. Melanocytes from both the patient with OCA and the normal twin demonstrated equal amounts of NP-40-soluble melanin; however, melanocytes from the patient with OCA contained only 7% of the amount of insoluble melanin found from the normal twin. Tyrosinase- related protein-1 (TRP-1) was not detected in the OCA melanocytes by use of various anti-TRP-1 probes. Furthermore, transcripts for TRP-1 were absent in cultured OCA melanocytes. The affected twin was homozygous for a single-bp deletion in exon 6, removing an A in codon 368 and leading to a premature stop at codon 384. Tyrosine hydroxylase activity of the OCA melanocytes was comparable to controls when assayed in cell lysates but was only 30% of controls when assayed in intact cells. We conclude that this mutation of the human TRP-1 gene affects its interaction with tyrosinase, resulting in dysregulation of tyrosinase activity, promotes the synthesis of brown versus black melanin, and is responsible for a third genetic type of OCA in humans, which we classify as "OCA3."