Different populations of melanocytes are present in hair follicles and epidermis

Melanocytes in human skin reside both in the epidermis and in the matrix and outer root sheath of anagen hair follicles. Comparative study of melanocytes in these different locations has been difficult as hair follicle melanocytes could not be cultured . In this study we used a recently described method of growing hair follicle melanocytes to characterize and compare hair follicle and epidermal melanocytes in the scalp of the same individual. Three morphologically and antigenically distinct types of melanocytes were observed in primary culture. These included (1) moderately pigmented and polydendritic melanocytes derived from epidermis; (2) small, bipolar, amelanotic melanocytes; and (3) large, intensely pigmented melanocytes; the latter two were derived from hair follicles. The three sub-populations of cells all reacted with melanocyte-specific monoclonal antibody. Epidermal and amelanotic hair follicle melanocytes proliferated well in culture, whereas the intensely pigmented hair follicle melanocytes did not. Amelanotic hair follicle melanocytes differed from epidermal melanocytes in being less differentiated, and they expressed less mature melanosome antigens. In addition, hair follicle melanocytes expressed some antigens associated with alopecia areata, but not antigens associated with vitiligo, whereas the reverse was true for epidermal melanocytes. Thus antigenically different populations of melanocytes are present in epidermis and hair follicle. This could account for the preferential destruction of hair follicle melanocytes in alopecia areata and of epidermal melanocytes in vitiligo.     

The Notch signalling pathway in hair growth

The Notch signalling pathway is an important mediator of cell fate selection whose involvement in epidermal appendage formation is now becoming recognised. Hair follicle development and hair formation involve the co-ordinated differentiation of several different cell types in which Notch appears to have a role. We report intricate expression patterns for the Notch-1 receptor and three ligands, Delta-1, Jagged-1 and Jagged-2 in the hair follicle. Notch-1 is expressed in ectodermal-derived cells of the follicle, in the inner cells of the embryonic placode and the follicle bulb, and in the suprabasal cells of the mature outer root sheath. Delta-1 is only expressed during embryonic follicle development and is exclusive to the mesenchymal cells of the pre-papilla located beneath the follicle placode. Expression of Jagged-1 or Jagged-2 overlaps Notch-1 expression at all stages. In mature follicles, Jagged-1 and Jagged-2 are expressed in complementary patterns in the follicle bulb and outer root sheath, Jagged-1 in suprabasal cells and Jagged-2 predominantly in basal cells. In the follicle bulb, Jagged-2 is localised to the inner (basal) bulb cells next to the dermal papilla which do not express Notch-1, whereas Jagged-1 expression in the upper follicle bulb overlaps Notch-1 expression and correlates with bulb cell differentiation into hair shaft cortical and cuticle keratinocytes.     

Effect of alendronate treatment on the osteoclastogenic potential of bone marrow cells in mice

The expression of cytokeratins (CK), involucrin, vimentin, CD34, and alpha-smooth-muscle actin was studied in fetal and adult hair follicles. The first stage of the developing hair follicle is characterized by palisaded, elongated epithelial cells budding from the epidermal basal layer. These cells express CK5/6, CK14, CK17, CK19, and vimentin. During the following weeks of gestation, different structures in the developing hair follicle can be identified and characterized. The matrical cells display only CK19. The keratinocytes of the outer root sheath express CK5/6, CK14, CK17, CK19, and involucrin; those of the inner root sheath, CK4, CK18, and involucrin; those of the isthmus, the same profile as the ORS. In the infundibulum, the basal-layer keratinocytes express CK5/6, CK14, CK17, and CK19, whereas in the suprabasal layers CK1, CK4, CK10, CK14, and CK17 are seen. The adult hair follicle in anagen fails to express CK19 in the matrical cells and isthmus and both CK17 and CK19 in the infundibulum. These profiles of intermediate filaments and other markers appear to be potentially useful in categorizing neoplasms with apparent follicular differentiation.     

Distinct patterns of NCAM expression are associated with defined stages of murine hair follicle morphogenesis and regression

Hair follicle development, growth (anagen), and regression (catagen) largely result from bidirectional epithelial-mesenchymal interactions whose molecular basis is still unclear. Because adhesion molecules are critically involved in pattern formation and because the fundamental importance of neural cell adhesion molecule (NCAM) for feather development has been demonstrated, we studied the protein expression patterns of NCAM during hair follicle development and regression in the C57BL/6 mouse model. During murine hair follicle development, NCAM immunoreactivity (IR) was first detected on epithelial hair placodes and later on selected keratinocytes in the distal outer root sheath. Mesenchymal NCAM immunoreactivity (IR) was noted on fibroblasts of the future dermal papilla (DP) and the perifollicular connective tissue sheath. Fetal hair follicle elongation coincided with strong, ubiquitous dermal NCAM IR, which remained strong until the follicles entered into their first neonatal catagen. At this time, the strong interfollicular dermal NCAM IR decreased substantially. During consecutive hair cycles, mesenchymal NCAM IR was seen exclusively on DP and perifollicular connective tissue sheath fibroblasts and on the trailing cells of regressing catagen hair follicles. These highly restricted and developmentally controlled expression patterns suggest an important role for NCAM in hair follicle topobiology during morphogenesis and cyclic remodeling of this miniorgan.     

Fasciola hepatica development in the experimentally infected black rat Rattus rattus

Coat colour changes in polar animals are related to seasonal variation in photic inputs. The present work was performed to study the photoresponses of hair follicular melanocytes in human skin. The melanocytes, being photosensitive cells, can function as UV biosensors, since dendrites extend towards the source of UV light. Fifty-one skin biopsies from the margin of vitiligo were subjected to whole skin organ cultures. These were exposed to a pulse of UV light to study hair bulb melanocytes in vitiligo. It is observed that the melanocytes are seen within the anagen matrix. These melanocytes are poorly dendritic in control and dark-incubated cultures. On UV exposure, they become highly dendritic, the dendrites extending towards the hair shaft in 93.5%. They show prominent catechol oxidase and noradrenaline positivity, all features of UV responsiveness. The melanocytes within the hair follicle are not directly exposed to UV light. The melanocyte dendricity and the alignment of dendrites towards the shaft on UV exposure indicate that the columns of the cells in the hair shaft act as an efficient fibre-optic system, transmitting UV light. Morphologically, the keratinocytes in the hair shaft are arranged in compressed linear columns which resemble the coaxial bundles of commercial fibre-optic strands as is observed in plants. Keratinocytes in the inner and outer sheaths do not show this arrangement. Thus the hair follicle functions as a specialised UV receptor in the skin responding to nuances of photic inputs in human skin. This is reflected in coat colour changes in animals exposed to large variations in day-night cycles.     

Transforming growth factor-beta1 inhibits basal melanogenesis in B16/F10 mouse melanoma cells by increasing the rate of degradation of tyrosinase and tyrosinase-related protein-1

Current evidence suggests that melanogenesis is controlled by epidermal paracrine modulators. We have analyzed the effects of transforming growth factor-beta1 (TGF-beta1) on the basal melanogenic activities of B16/F10 mouse melanoma cells. TGF-beta1 treatment (48 h) elicited a concentration-dependent decrease in basal tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (Dopa) oxidase activities, to less than 30% of the control values but had no effect on dopachrome tautomerase activity (TRP-2). The inhibition affected to similar extents the Dopa oxidase activity associated to tyrosinase-related protein-1 (TRP-1) and tyrosinase. This inhibition was noticeable between 1 and 3 h after the addition of the cytokine, and maximal after 6 h of treatment. The decrease in the enzymatic activity was paralleled by a decrease in the abundance of the TRP-1 and tyrosinase proteins. TGF-beta1 mediated this effect by increasing the rate of degradation of tyrosinase and TRP-1. Conversely, after 48 h of treatment, the expression of the tyrosinase gene decreased only slightly, while TRP-1 and TRP-2 gene expression was not affected. An increased rate of proteolytic degradation of TRP-1 and tyrosinase seems the main mechanism accounting for the inhibitory effect of TGF-beta1 on the melanogenic activity of B16/F10 cells.     

TNF-alpha induces apoptosis in rat fetal brown adipocytes in primary culture

The balding (bal) mutation of the mouse is an autosomal recessive mutation that causes alopecia and immunologic anomalies. A new allele was identified by allelism testing after using an interspecific backcross to localize the mutation to the centromeric end of mouse chromosome 18. We investigated the skin and hair histologic lesions of two alleles (bal(J) and bal(Pas)) at this locus and analyzed the expression of several keratinocyte markers and the production of autoantibodies by immunofluorescence on frozen skin sections. The lesions observed included separation of the inner and outer root sheath in anagen follicles resulting in the hair fiber being very easily plucked from the follicle. Vesicles on the ventral tongue, mucocutaneous junction of the eyelid, foot pads, and rarely in skin were also evident. Separation occurred between the basal and suprabasilar cells forming an empty cleft, resembling that observed in human pemphigus vulgaris. Immunofluorescence studies did not reveal the presence of tissue-bound or circulating autoantibodies. Expression of keratinocyte markers in hair follicles was normal. Keratin 6-positive cells were found on either side of the follicular separation suggesting a molecular defect in adhesion molecules between the inner layer of the outer root sheath cells to layers on either sides. This hypothesis has been confirmed by another group who demonstrated that the bal(J) mutation is due to the insertion of a thymidine in the desmoglein 3 gene, resulting in a premature stop codon.     

Generation of polyclonal rabbit antisera to mouse melanoma associated antigens using gene gun immunization

Lymphocytes from patients with melanoma have been used to clone melanoma associated antigens which are, for the most part, nonmutated melanocyte tissue differentiation antigens. To establish a mouse model for the use of these 'self' antigens as targets for anti-tumor immune responses, we have employed the mouse homologues of the human melanoma antigens Tyrosinase, Tyrosinase Related Protein-1 (TRP-1), gp100, and MART-1. We sought to generate antisera against these proteins for use in the construction of experimental recombinant and synthetic anti-cancer vaccines, and for use in biologic studies. Using genes cloned from the B16 mouse melanoma or from murine melanocytes, we immunized rabbits with plasmid DNAs coated onto microscopic gold beads that were then delivered using a hand-held, helium-driven 'gene gun'. This strategy enabled us to generate polyclonal rabbit sera containing antibodies that specifically recognized each antigen, as measured by immunostaining of vaccinia virus infected cells. The sera that we generated specifically for TRP-1, gp100, and MART-1 recognized extracts of the spontaneous murine melanoma, B16. The identities of the recognized proteins was confirmed by Western blot analysis. The titers and specificities of these antisera were determined using ELISA. Interestingly, serum samples generated against murine MART-1 and gp100 developed antibodies that were cross-reactive with the corresponding human homologues. Recognition of human gp100 and murine Tyrosinase appeared to be dependent upon conformational epitopes since specificity was lost upon denaturation of the antigens. These antisera may be useful in the detection, purification and characterization of the mouse homologues of recently cloned human tumor associated antigens and may enable the establishment of an animal model of the immune consequences of vaccination against 'self antigens.     

Laparoscopic surgery combined with preservation of the spleen for distal pancreatic tumors

BACKGROUND: Hair follicle preservation for the purpose of delayed application would help us to transplant hair follicles more efficiently. METHODS: Isolated single hair follicles were preserved at 4 degrees C in four different solutions. Viability of preserved follicles was judged by organ culture and cell culture. In addition, a small number of hair follicles were transplanted into athymic mice. RESULTS. By cell culture, both dermal papilla and outer root sheath cells could be cultivated after 7 days of preservation. Hair follicles preserved for 48 hours showed a significant increase of hair shafts in organ culture. Those preserved for 7 days regrew well when transplanted into athymic mice. CONCLUSION: Preservation of hair follicles at 4 degrees C could be one option to prepare many follicular units at one time for transplantation.     

Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles

The erbB-2 proto-oncogene belongs to a receptor tyrosine kinase family that includes the epidermal growth factor receptor, erbB-2, erbB-3, and erbB-4. erbB-2 is expressed in basal cells of the squamous epithelia and the outer root sheath of the hair follicles, but its function in epidermal development has not been well studied. To investigate its role in the skin, we created transgenic mice harboring an activated erbB-2 oncogene under the control of the human keratin 14 promoter. The keratin 14 promoter directed its expression to cells in which erbB-2 is normally expressed, whereas the activated receptor gene ensured increased signaling. All transgenic founder mice exhibited extensive and striking skin phenotype, including epidermal hyperplasia, preneoplasia, papilloma, hyperkeratosis, and dyskeratosis. The majority of the hair follicles were replaced by bizarre hyperproliferative intradermal squamous invaginations, whereas the rest of the follicles exhibited severe hyperplasia and disorganization. All but one of the transgenic mice died before or shortly after birth, probably as a consequence of defects in the skin and esophagus. These observations demonstrate that the skin is sensitive to erbB-2 signaling, suggesting an important role for this receptor tyrosine kinase in epidermal growth, differentiation, and hair follicle morphogenesis.     

Phylogenetic analysis of the Bunocotylinae dollfus, 1950 (Digenea: Hemiuridae)

We investigated the functional determinants of the cutaneous expression of elements of the hypothalamic-pituitary-adrenal axis. In the present work, the presence of adrenocorticotropin (ACTH) peptide in skin of C57/BL6 mouse was demonstrated by reversed-phase HPLC analysis combined with specific radioimmunoassay. ACTH concentration that was low in telogen, increased during anagen in two steps: a rapid phase in anagen I, and a slower rise that reached its peak in anagen VI. Immunofluorescence localized the ACTH antigen to the basal layer of epidermis, outer root sheath of hair follicle and subcutaneous muscle of anagen VI skin. At physiological plasma concentration (10-9 M), ACTH selectively stimulated DNA synthesis in dermis, while pharmacological doses (10-7-10-6 M) inhibited DNA synthesis in both dermis and epidermis. In conclusion, we suggest that local production of ACTH may represent a regulatory element in the control of skin functions including hair growth.     

Antibodies to hair follicles in alopecia areata

Although alopecia areata is suspected to be an autoimmune disease, no direct evidence of an altered immune response to components of the hair follicle has been reported. We studied whether antibodies to normal human anagen scalp hair follicles are present in individuals with alopecia areata. Thirty-nine alopecia areata sera and 27 control sera were tested by Western immunoblotting for antibodies to 6 M urea-extractable proteins of normal anagen scalp hair follicles. At serum diluted 1:80, all alopecia areata subjects (100%), but only 44% of control individuals, had antibodies directed to one or more antigens of approximately 57, 52, 50, 47, or 44 kD. The incidence of antibodies to individual hair follicle antigens in alopecia areata was up to seven times more frequent than in control sera and their level up to 13 times greater and was statistically significant for all five antigens. Tissue specificity analysis indicated that these antigens were selectively expressed in hair follicles. These findings indicate that individuals with alopecia areata have abnormal antibodies directed to hair follicle antigens, and support the hypothesis that alopecia areata is an autoimmune disease.     

Multiple warty dyskeratomas of the scalp

Two patients presented with the unusual condition of multiple warty dyskeratomas on the scalp. Biopsies of affected skin stained positive with human keratin monoclonal antibodies HKN-6 and -7, specific for cortex and inner root sheath of normal human hair, respectively. Multiple warty dyskeratomas are a rare occurrence and their aetiopathogenesis remains elusive. Positive immunohistochemical staining of a lesion with antikeratin antibodies HKN-6 and -7, specific for human hair keratin, suggests a follicular origin for warty dyskeratoma.     

Analysis of apoptosis during hair follicle regression (catagen)

Keratinocyte apoptosis is a central element in the regulation of hair follicle regression (catagen), yet the exact location and the control of follicular keratinocyte apoptosis remain obscure. To generate an "apoptomap" of the hair follicle, we have studied selected apoptosis-associated parameters in the C57BL/6 mouse model for hair research during normal and pharmacologically manipulated, pathological catagen development. As assessed by terminal deoxynucleotide transferase dUTP fluorescein nick end-labeling (TUNEL) stain, apoptotic cells not only appeared in the regressing proximal follicle epithelium but, surprisingly, were also seen in the central inner root sheath, in the bulge/isthmus region, and in the secondary germ, but never in the dermal papilla. These apoptosis hot spots during catagen development correlated largely with a down-regulation of the Bcl-2/Bax ratio but only poorly with the expression patterns of interleukin-1beta converting enzyme, p55TNFR, and Fas/Apo-1 immunoreactivity. Instead, a higher correlation was found with p75NTR expression. During cyclophosphamide-induced follicle dystrophy and alopecia, massive keratinocyte apoptosis occurred in the entire proximal hair bulb, except in the dermal papilla, despite a strong up-regulation of Bax and p75NTR immunoreactivity. Selected receptors of the tumor necrosis factor/nerve growth factor family and members of the Bcl-2 family may also play a key role in the control of follicular keratinocyte apoptosis in situ.