Three-dimensional echocardiography improves accuracy and compensates for sonographer inexperience in assessment of left ventricular ejection fraction.

This study was performed to determine whether 3-dimensional echocardiography (3DE) with a magnetic tracking system for image plane localization, which unlike standard 2-dimensional echocardiography (2DE), does not require acquisition of specific image planes or "standard views" for quantitative measurement of left ventricular volume and ejection fraction (EF), could compensate for sonographer inexperience. Eight adults underwent magnetic resonance imaging (MRI) scanning; they also had 2DE and 3DE performed by 2 experienced and 3 novice sonographers. Data were analyzed by a single expert reader blinded to patient and sonographer identity. Linear regression of MRI EF (reference standard) against echocardiographic EF yielded the following results, where RD indicates the residual difference between measured MRI values and those predicted using echocardiographic results: expert 3DE: r = 0.97, RD = 2.4%, and r = 0.96, RD = 2.8%; novice 3DE: r = 0. 83, RD = 5.1%, to r = 0.95, RD = 4.8%; expert 2DE: r = 0.85, RD = 4. 8%, and r = 0.86, RD = 4.9%; and novice 2DE: r = 0.34, RD = 11.7%, to r = 0.69, RD = 6.6%. Comparison of error variances indicated that novices who used 3DE equaled the performance of experts who used 2DE, although experts were always more accurate than novices when both used the same echocardiographic method (3DE vs 3DE, 2DE vs 2DE). In a comparison of methods, 3DE was always superior to 2DE, regardless of sonographer experience. Three-dimensional echocardiography allows even novice sonographers to obtain diagnostic-quality data sets, which they were unable to accomplish with 2DE. These results suggest that scanning with 3DE, combined with remote expert interpretation, may be useful in providing echocardiographic services in regions where they are presently unavailable.

Vitiligo: repigmentation with cultured melanocytes after cryostorage.

We have developed a technique through which we can multiply melanocytes in culture from a small specimen of normally pigmented buttock skin and reimplant them into depigmented sites of vitiligo. To date, 90 patients have benefited in our hands from such autologous transplantation of pigment cells. Often we have an excess of cells which we would like to store for later use in the event a patient requires further treatment. We report here on 4 cases in which we have cryostored cultured melanocytes for 6-12 months, reimplanted them into vitiliginous sites of the donor after one week of reculture, and obtained optimal repigmentation. We now routinely freeze melanocytes left over after treatment.

Pigment cell transplantation for treatment of vitiligo: a progress report.

BACKGROUND: Because available treatments for vitiligo generally provide unsatisfactory results, the search for viable therapeutic alternatives continues. OBJECTIVE: Our purpose was to evaluate several transplantation procedures with cultured autologous melanocytes for their practicality in treating patients with vitiligo. METHODS: Twenty-seven patients with stable or active vitiligo were treated after superficial dermabrasion with application of suspensions of autologous cultured melanocytes, melanocyte-keratinocyte mixtures, or epidermal sheets established in vitro. RESULTS: Regardless of disease activity, use of each method resulted in repigmentation to a similar degree and without scarring in all patients. Melanocyte suspensions offer several advantages: They are easily prepared, can be applied in a controlled manner, permit coverage of large areas, and produce a homogeneous skin color that affords the best cosmetic restoration. The ultrastructure of transplant sites resembled that of uninvolved skin, with one exception: the melanocytes were located slightly higher than in uninvolved skin. CONCLUSION: Application of cultured autologous melanocytes to lightly abraded skin is an advantageous addition to the treatments available for patients with vitiligo.

Two rare manifestations of melanomas: generalized cutaneous melanosis and rapid solar induction of showers of small pigmented lesions. A critical review of the literature and presentation of two additional cases.

Two unusual patients with metastatic melanoma are presented. One had generalized melanosis, and the other a rapid induction of multiple small pigmented lesions within a day of exposure to the sun. From a variety of clinical observations on these 2 patients and ones reported in the literature, and from recent advances in basic biomedical knowledge, we conclude that in both conditions the anomalous presence of growth factors that stimulate the proliferation and melanogenic differentiation of normal and malignant melanocytes played a major role in producing the clinical events.

The effect of vitiligo on sexual relationships.

To study the effect of vitiligo on interference with sexual relationships, we surveyed 158 patients by questionnaire. Although a majority of patients reported a negative impact on sexual relationships, most patients felt embarrassment when showing their body or meeting strangers. The majority of patients who reported a negative impact on sexual relationships attributed the problems to their embarrassment. Those who were particularly affected were those with low self-esteem, men, those to whom appearance is important, and single persons. Dermatologists should be especially alert to the effects of disfigurement and should attempt to assist patients with this problem.

Action of light on frog pigment cells in culture.

Solar radiation induces numerous biologic effects in skin but the mechanism underlying these responses is poorly understood. To study the etiology of these phenomena, we investigated the effect of light on cultured Xenopus laevis melanophores. Visible light stimulated a marked increase in intracellular cAMP levels within the first minute of irradiation. This light-induced elevation in cAMP was blocked by melatonin and was not seen in fibroblasts irradiated in a similar manner. These data show that the photoresponse of pigment cells from amphibian skin can be mediated by a cAMP-dependent mechanisms and suggest that a unique member of the rhodopsin family is involved in this process.

Tyrosinases of murine melanocytes with mutations at the albino locus.

Tyrosinase (monophenol monooxygenase; monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1) is a key enzyme in the synthesis of melanin. Reduced levels of tyrosinase play an important role in albinism. The data described here show differences in the expression and characteristics of tyrosinase in cutaneous murine melanocytes grown in culture from normal wild-type strains (C/C); from three albino locus mutants: himalayan (ch/ch), chinchilla (cch/cch), and albino (c/c); and from the double-mutant heterozygous pink-eyed chinchilla (cchp/cp). Our results suggest that the diminished pigmentation in all mutants is due to abnormal posttranslational modification of the enzyme: the levels of mRNA for tyrosinase in wild-type, himalayan, and pink-eyed chinchilla melanocytes are similar; the himalayan mutation confers a deficiency in N-linked glycosylation, which results in an extremely unstable enzyme that is also temperature sensitive; the chinchilla and albino mutations confer susceptibility to proteolytic cleavage; the pink-eye dilution confers a reduction in the levels of immunoprecipitable tyrosinase, and what little enzyme there is fails to be translocated from the trans-Golgi network to melanosomes. The kinetics of activation and inhibition of the enzyme by the cofactor dopa are unique for the mutants tested and differ from those of tyrosinase from wild-type melanocytes. The findings support the conclusion that the albino locus in mice encodes the structural gene of 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.

Normal murine melanocytes in culture.

A major obstacle to applying the techniques of molecular biology to the genetics and cell biology of pigmentation has been our inability to grow normal murine melanocytes in culture. We report here the establishment and characterization of continuously proliferating cultures of cutaneous pigment cells from seven strains of mice. Melanocytes were grown from the dermis of newborn mice in medium containing 12-0-tetradecanoyl-13-phorbol-acetate; a substance, such as melanotropin, that raises intracellular levels of cyclic AMP; and an extract made from human placenta.

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. II. Novel immunotherapy of B16 melanomas by local and systemic adoptive transfer.

Lyt-1+, L3T4a+ autoreactive cloned T cells, producing lymphotoxin (LT) and interferon-gamma (IFN-gamma) in response to self-class II major histocompatibility complex antigen in vitro were examined for their anti-tumor effect in vivo against B16 melanomas. Without the aid of exogenous interleukin 2, the autoreactive T cells, when injected immediately and at an equal cell number into the site of s.c. inoculated B16 melanoma cells inhibited tumor growth in sublethally irradiated and nonirradiated syngeneic mice. The autoreactive T cells also induced regression of tumors established 3 days earlier. Normal spleen cells or class II-restricted cloned T cells specific for chicken gamma-globulin (CGG) had no inhibitory effect on tumor growth. A single injection of autoreactive T cells delayed tumor growth and prolonged the survival of mice that had received a lethal dose of B16 melanoma cells. The autoreactive T cells caused extensive necrosis at the injection site. A treatment regime consisting of two successive injections of anti-I-Ab monoclonal antibody 3JP prevented the inhibition of tumor growth, supporting the hypothesis that the autoreactive T cells inhibited the growth of melanomas by releasing LT and IFN-gamma upon recognition of I-A antigen-bearing cells at the injection site. The CGG-specific control T cells did not cause necrosis and survived within the nests of uninhibited tumor cells. Autoreactive T cells administered i.v. immediately after i.v. injection of B16 melanoma cells markedly reduced pulmonary metastases, whereas CGG-specific T cells did not. These results indicate that autoreactive T cells can function in vivo as inhibitors of tumor growth.

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.

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.