Tyrosinase and acid phosphatase activities in melanocytes from avian albinos.

Two forms of cutaneous albinism in the chicken were investigated for the presence and distribution of tyrosinase and acid phosphatase in melanocytes in situ and in culture. In sex-linked recessive tyrosinase-positive albinism, sal, melanocytes in regenerating feathers and neural tube-derived cultures contained morphologically normal and abnormal premelanosomes. Tyrosinase was localized primarily to the abnormal premelanosomes and probably not to the normal ones. The cells possessed, in addition, vacuoles with membranous inclusions, located in the dendrites, and capped by dopa-positive vesicles (capping vesicles). Acid phosphatase colocalized with tyrosinase in the abnormal premelanosomes and capping vesicles. Tyrosinase activity in extracts of cultured sal melanocytes equalled that of e+ control melanocytes. A tyrosinase antiserum, raised against hamster tyrosinase (Pomerantz), precipitated 2 proteins, 68 kD and 82 kD, which had a precursor-product relationship. The amount of immunoprecipitate was the same in sal and control extracts, but in sal extracts the lower-molecular-weight protein was twice as abundant as the higher-molecular-weight protein. Melanocytes in regenerating feathers from an autosomal recessive, tyrosinase-negative albino, ca, also contained morphologically normal and abnormal premelanosomes. In culture, ca melanocytes had no formal premelanosomes but only dopa-negative multivesicular bodies with wispy filamentous material. Tyrosinase activity and immunoprecipitable tyrosinase were absent. These results suggest that: the tyrosinase-positive albino, sal, has an aberration in both its tyrosinase and acid phosphatase profiles and the tyrosinase-negative albino, ca, lacks functionally and antigenically normal tyrosinase.

Transplantation of human melanocytes.

Recent advances in the culturing of pigment cells from human beings have made it possible to begin the transplantation of autologous melanocytes into areas of skin that are hypopigmented. In a patient with piebaldism we were able to take pigment cells from a shave biopsy of the normally pigmented skin of the back, expand the cells in culture, and return them to an area devoid of pigment cells and get a perfect take. To grow the cells in culture we used 12-O-tetradecanoyl-phorbol-13-acetate (TPA) as well as cholera toxin and isobutylmethyl xanthine. At this time, one can substitute basic fibroblast growth factor for TPA. The procedure of using autologous pigment cell cultures opens the door for further advances in the treatment of patients who do not have melanocytes in certain areas of the skin, as seen in patients with vitiligo or piebaldism, or as a consequence of severe mechanical or thermal trauma.

Reconstitution of structure and cell function in human skin grafts derived from cryopreserved allogeneic dermis and autologous cultured keratinocytes.

Grafts of allogeneic dermis plus autologous epidermal cell cultures were used to replace extensively burned skin. Cryopreserved split-thickness cadaveric skin was grafted onto debrided burn wound, and autologous keratinocytes were cultured from uninjured donor sites. Several weeks later, allograft epidermis was abraded and replaced with the keratinocyte cultures. The final grafts were thus composites of autologous cultured epidermis and allogeneic dermis. In a case with 28 months follow-up, reconstitution of the dermal-epidermal (BMZ.1) and microvascular (BMZ.2) basement membrane zones was studied immunohistochemically and ultrastructurally. Immediately before grafting, thawed cryopreserved skin reacted with antibodies against laminin and type IV collagen in normal patterns. Twenty-nine days after grafting, BMZ.1 reacted weakly with both antibodies, and anticollagen type IV reactivity was absent from BMZ.2. Antilaminin reactivity of BMZ.2, however, was moderately intense, consistent with recent neovascularization. On day 29, the allograft epidermis was replaced with autologous keratinocyte cultures. Twenty-five days later (54 d after allografting), staining of both BMZs was intense with both antibodies. Ultrastructurally, at day 76 (47 d after culture placement) BMZ.1 revealed only small hemidesmosomes, few incipient anchoring fibrils, and a discontinuous lamina densa. BMZ.2, however, was fully reconstituted. By 124 d, both BMZs appeared normal. Observations in the dermis at 76 d included the presence of lymphocytes, organellar debris, and hyperactive collagen fibrillogenesis, all indicative of dermal remodelling. The microvasculature was well differentiated, but no elastic fibers or nerves were found. In the epidermis, melanocytes and evidence of melanosome transfer were seen at 5, 47, and 95 d after grafting of keratinocyte cultures. We conclude that the composite procedure reconstitutes skin with excellent textural and histologic qualities.

A mouse model for vitiligo.

As the result of a long search for a depigmenting mouse that could serve as a model for the study of vitiligo, we have located a strain that arose from the C57BL/6J. Its provisional genetic designation is C57BL/6J Ler-vit/vit. This vitiligo mouse has congenital dorsal and ventral white spots (piebaldism) as well as progressive replacement of pigmented hairs by white hairs with each spontaneous molt or after plucking. The lack of pigment is due to the absence of melanocytes from the amelanotic hair follicles and epidermis. As in human beings and the Smyth chicken model, there is also diminution of ocular pigment. Reciprocal skin transplants between C57BL/6J and vitiligo mice, and transplants into nude mice, suggest a programmed pigment cell death in the vitiligo mice. Like human beings with vitiligo, maximally depigmented vitiligo mice have a decreased contact sensitivity response in comparison to age-matched C57BL/6J controls. The resistance to injected B16 melanomas is lowered. Vitiligo mice show no signs of premature aging. Already at this early stage in the study of this new animal model, there are findings that open a range of new approaches to the study and treatment of patients with vitiligo and melanomas.

Morphology of melanocytes in hair bulbs and eyes of vitiligo mice.

The vitiligo mouse C57BL/6J Ler-vit/vit is a new, murine model for vitiligo in humans. It was studied with respect to morphology and fine structure of melanocytes in hair and eyes before and during depigmentation. The coat of vitiligo mice lightens progressively with age because of an increase in the ratio of white to pigmented hairs with each molt. The bulbs of white hairs are devoid of pigment, and they lack melanocytes. In other respects the epithelium is morphologically normal as determined by light and electron microscopy. The bulbs of pigmented hairs are histologically normal. By electron microscopy, however, some of the melanocytes are shown to have undergone degenerative changes. In addition, disruption of the basement membrane underlying the melanocytes and herniation of melanocytes into dermal papillae were observed at various stages of hair growth. Papillary melanophages are prominent in pigmented as well as in white hair bulbs. Newborn vitiligo mice have no uveal pigment. Pigment appears in the iris and ciliary body by Day 4 and in the choroid by Week 3. On Day 4, along with pigmentation, conspicuous spherical amelanotic cells appear over the anterior border of the iris. These cells become numerous in the ensuing weeks and gradually acquire large melanophagosomes. They occur also in the stroma of the iris and the ciliary body, associated with necrotic melanocytes. The spherical cells are identical to the clump cells of Koganei and are far more numerous in vitiligo mice than in controls. Macroscopically, no progressive decrease in iridial pigment is apparent for the life of the vitiligo mouse. In the choroid, an amelanotic patch surrounds the optic nerve. In the pigmented areas, melanocytes show compartmentalization of melanosomes and degeneration. The retinal pigment epithelium generally appeared continuous. In older animals some epithelial cells contained large fat bodies or were devoid of melanin.

Anti-tumor activity of class II MHC antigen-restricted cloned autoreactive T cells. I. Destruction of B16 melanoma cells mediated by bystander cytolysis in vitro.

Two Lyt-1+, L3T4a+ autoreactive T cell clones specific for self-class II major histocompatibility complex (MHC) gene products were established from lymph node cells and spleen cells of C57BL/6J mice, respectively, by different methods. They were stimulated to proliferate in culture in response to I-Ab antigen-bearing syngeneic spleen cells in a class II MHC-restricted manner. This stimulation was inhibited completely by the addition of anti-L3T4a (GK1.5) or anti-I-Ab (3JP) monoclonal antibodies. The autoreactive T cell clones lysed syngeneic I-Ab+ target cells such as lipopolysaccharide (LPS) blasts. They also lysed I-A- bystander cells such as Cloudman and B16 melanoma and lymphoid tumor cells in the presence of I-Ab+ stimulator cells but not I-Ad+ cells. This bystander killing was most likely mediated by soluble factors released from the autoreactive T cells in response to I-Ab antigens, because culture supernatants from activated autoreactive T cells inhibited the proliferation of B16 melanoma cells in vitro and also had significant cytolytic activity. Both lymphotoxin and interferon-gamma were released from activated autoreactive T cells, suggesting that these cytotoxic lymphokines were responsible for autoreactive T cell-mediated cytolysis. The finding that the two clones, established independently and by different methods, show self-class II MHC antigen-restricted cytolysis, and bystander cytolysis suggests that these properties are not restricted to a unique population of autoreactive T cells. These results favor the concept that in vivo, autoreactive T cells may express not only regulatory activity in regard to antibody responses, but also anti-tumor activity via bystander cytolysis.