Melanocyte destruction and repigmentation in vitiligo: A model for nerve cell damage and regrowth

Melanocytes (MCs) are melanin-producing cells of the skin that are derived from neural crest cells. Vitiligo vulgaris is a common depigmentation disorder resulting from the destruction of functional MCs in the affected skin. The three prevailing pathomechanisms of vitiligo are the immune hypothesis, the neural hypothesis and the autocytotoxic hypothesis. None of these mechanisms has been conclusively proven. Melanoblasts (MBs) in the outer root sheath of the hair follicles are the reservoir cells for repigmentation. Recovery from vitiligo is initiated by activation and proliferation of these MBs, followed by upward migration to the nearby epidermis that forms perifollicular pigmentation islands. Migration, proliferation and differentiation of MCs and MBs are regulated by keratinocyte-derived factors and some coat color genes. Any therapy for vitiligo must explain not only the repopulation of MCs but also their functional development. In patients with vitiligo, MCs are destroyed in the skin, the eyes, and possibly the ears. However, the concept of vitiligo as a systemic disease will be clearly established only when the mechanisms involved in vitiligo are identified. Recent advances in the fields of neural crest cell culture and molecular genetics have opened new perspectives in the understanding of vitiligo. Not only will this result in better treatments for vitiligo patients, but possibly will also provide a key to triggering nerve cell regrowth in other nervous diseases.     

Immune responses in a mouse model of vitiligo with spontaneous epidermal de‐ and repigmentation

To generate a mouse model of spontaneous epidermal depigmentation, parental h3TA2 mice, expressing both a human‐derived, tyrosinase‐reactive T‐cell receptor on T cells and the matching HLA‐A2 transgene, were crossed to keratin 14‐promoter driven, stem cell factor transgenic (K14‐SCF) mice with intra‐epidermal melanocytes. In resulting Vitesse mice, spontaneous skin depigmentation precedes symmetrical and sharply demarcated patches of graying hair. Whereas the SCF transgene alone dictates a greater retinoic acid receptor‐related orphan receptor gamma (RORγt)⁺ T‐cell compartment, these cells displayed markedly increased IL‐17 expression within Vitesse mice. Similar to patient skin, regulatory T cells were less abundant compared with K14‐SCF mice, with the exception of gradually appearing patches of repigmenting skin. The subtle repigmentation observed likely reflects resilient melanocytes that coexist with skin‐infiltrating, melanocyte‐reactive T cells. Similar repigmenting lesions were found in a different TCR transgenic model of vitiligo developed on an SCF transgenic background, supporting a role for SCF in repigmentation.     

Successful treatment of non-segmental vitiligo: systemic therapy with sex hormone-thyroid powder mixture

We previously reported a patient with generalized vitiligo improved by oral administration of the drug for menopausal syndrome (sex hormone-thyroid powder mixture). In this study, we reevaluated the efficiency of this drug for vitiligo, and examined its pharmacological action in melanogenesis.     

Protective effect of epigallocatechin gallate and esculetin on oxidative DNA damage induced by psoralen plus ultraviolet-A therapy

We examined antioxidants exhibiting no effects on DNA cross-linking, which is the basis of psoralen and ultraviolet-A therapy for skin diseases, and suppressing oxidative DNA damage incidental to the therapy. Epigallocatechin gallate and esculetin effectively suppressed oxidative DNA damage with little effect on the formation of DNA cross-linking. These antioxidants might be useful in suppressing the adverse reaction induced by this therapy.     

Spectroscopic study of the dark interaction and of the photoreaction between a new monofunctional psoralen : 3-carbethoxy psoralen, and DNA

3-carbethoxypsoralen (3-CPs) is a new linear psoralen derivative. Its dark interaction and photoreaction with DNA has been studied and compared with that of a well known bifunctional psoralen: 8-methoxypsoralen (8-MOP). 3-CPs is able to form in the dark a non covalent complex with native DNA. After irradiation of this complex with UV-A light (365 nm) 3-CPs is able to link covalently to DNA. Heat denaturation and renaturation patterns of treated DNA clearly show that, in contrast to 8-MOP, 3-CPs does not form DNA interstrand cross links. Fluorescence studies show that the photobinding of 3-CPs gives rise to the formation of monoadducts involving the 4′,5′ double bond of this molecule.     

Psoralea corylifolia extracts stimulate cholinergic-like psoralen receptors of tadpole-tail melanophores,leading to skin darkening

The present work was carried out to determine the effects of lyophilized seed extracts of Psoralea corylifolia along with pure psoralen, its active ingredient on the isolated tail-piece melanophores of Bufo melanostictus, a type of disguised smooth muscle cells, which offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. In the present study, it was found that lyophilized extract of P. corylifolia and its active ingredient psoralen induced powerful, dose-dependent, physiologically significant melanin dispersal effects in the isolated tail melanophores of B. melanostictus, which were completely blocked by atropine as well as hyoscine. The per se melanin dispersal effects of lyophilized extracts of P. corylifolia and its active ingredient psoralen were highly potentiated by neostigmine. It appears that the melanin dispersal effects of the extracts of P. corylifolia and psoralen are mediated by cholino-muscarinic or cholino-psoralen like receptors having similar properties that need to be studied further.     

Photochemical treatment with S-59 psoralen and ultraviolet A light to control the fate of naive or primed T lymphocytes in vivo after allogeneic bone marrow transplantation.

Donor leukocyte infusions after allogeneic bone marrow transplantation can provide a curative graft-vs-leukemia (GVL) effect, but there is a significant risk of graft-vs-host (GVH) disease. A simple and effective method for controlling the fate of naive or primed T-lymphocytes in vivo without eliminating their beneficial properties is needed. In this report, photochemical treatment (PCT) ex vivo with a synthetic psoralen (S-59) and UVA light was evaluated as a pharmacological approach to limiting the proliferation and GVH potential of naive and primed donor T cells in vivo. S-59 rapidly intercalates into and cross-links DNA on UVA illumination. The effects of PCT on T cells were found to be both S-59 and UVA dose dependent. With selected PCT regimens, treated T cells still expressed activation markers (CD25 and CD69) and secreted IL-2 on activation, but they showed limited proliferative capacity in vitro and in vivo. Clonal expansion of CTL in MLR was reduced after PCT, but short term lytic activity of primed CTL was not affected. In a murine model of MHC-mismatched bone marrow transplantation, the addition of PCT-treated T cells to T-depleted bone marrow facilitated donor engraftment and complete chimerism without causing acute or chronic graft-vs-host disease. Allospecific GVL reactivity was reduced but not eliminated after PCT treatment. In an MHC-matched model using host-presensitized donor T cells, PCT significantly reduced GVH-associated mortality without eliminating GVL reactivity. Thus, PCT ex vivo offers a simple, rapid, and inexpensive method by which to control the fate of naive and primed T cells in vivo.     

The pathogen reduction treatment of platelets with S-59 HCl (Amotosalen) plus ultraviolet A light: Genotoxicity profile and hazard assessment

Despite restrictive donor criteria and screening procedures, infections resulting from the transfusion of bacterially contaminated platelet products continue to occur. Pathogen reduction technologies targeting nucleic acids have been developed. However, concerns about the safety of these procedures exist; the main concern being the possible mutagenic and carcinogenic effects of the pathogen-inactivated preparation in the recipient. This report reviews the genotoxicity profile of the S-59 (Amotosalen) plus long wavelength ultraviolet light (UVA) pathogen reduction technology, and assesses the mutagenic and carcinogenic hazards in recipients of treated platelets. S-59, a synthetic heterocyclic psoralen, non-covalently intercalates into the nucleic acids of pathogens and forms crosslinks when UVA photoactivated. Before clinical use, the levels of residual S-59 and free photoproducts are greatly reduced using a ‘compound adsorption device’ (CAD). In vitro, S-59 is mutagenic in Salmonella typhimurium and mouse lymphoma L5178Y TK^+/− cells, and is clastogenic in CHO cells. There is reduced activity (Salmonella, CHO cells) or no activity (mouse lymphoma cells) with metabolic activation (S9 mix). When tested up to toxic dose levels, S-59 was negative in the mouse bone marrow micronucleus assay and the rat hepatocyte unscheduled DNA synthesis (UDS) test. Based on comparative studies conducted with S-59 plus UVA-treated platelets (up to 25 times without CAD), any genotoxic effects can be attributed to residual S-59. Considering (1) the known genotoxic mechanism of action for S-59, (2) the negative in vivo studies for S-59 at multiples >40,000× over clinical peak plasma levels, and (3) the fact that the positive in vitro genotoxicity effects for the end product seem due to residual S-59, any mutagenic hazard to a recipient of S-59 plus UVA-treated platelets is negligible and there is no concern about a carcinogenic potential as a consequence of a mutagenic activity. This conclusion is supported by a negative p53^+/− mouse carcinogenicity study.     

The pathogen reduction treatment of platelets with S-59 HCl (Amotosalen) plus ultraviolet A light: genotoxicity profile and hazard assessment

Despite restrictive donor criteria and screening procedures, infections resulting from the transfusion of bacterially contaminated platelet products continue to occur. Pathogen reduction technologies targeting nucleic acids have been developed. However, concerns about the safety of these procedures exist; the main concern being the possible mutagenic and carcinogenic effects of the pathogen-inactivated preparation in the recipient. This report reviews the genotoxicity profile of the S-59 (Amotosalen) plus long wavelength ultraviolet light (UVA) pathogen reduction technology, and assesses the mutagenic and carcinogenic hazards in recipients of treated platelets. S-59, a synthetic heterocyclic psoralen, non-covalently intercalates into the nucleic acids of pathogens and forms crosslinks when UVA photoactivated. Before clinical use, the levels of residual S-59 and free photoproducts are greatly reduced using a 'compound adsorption device' (CAD). In vitro, S-59 is mutagenic in Salmonella typhimurium and mouse lymphoma L5178Y TK(+/-) cells, and is clastogenic in CHO cells. There is reduced activity (Salmonella, CHO cells) or no activity (mouse lymphoma cells) with metabolic activation (S9 mix). When tested up to toxic dose levels, S-59 was negative in the mouse bone marrow micronucleus assay and the rat hepatocyte unscheduled DNA synthesis (UDS) test. Based on comparative studies conducted with S-59 plus UVA-treated platelets (up to 25 times without CAD), any genotoxic effects can be attributed to residual S-59. Considering (1) the known genotoxic mechanism of action for S-59, (2) the negative in vivo studies for S-59 at multiples >40,000x over clinical peak plasma levels, and (3) the fact that the positive in vitro genotoxicity effects for the end product seem due to residual S-59, any mutagenic hazard to a recipient of S-59 plus UVA-treated platelets is negligible and there is no concern about a carcinogenic potential as a consequence of a mutagenic activity. This conclusion is supported by a negative p53(+/-) mouse carcinogenicity study.     

Childhood exposure to ultraviolet radiation and harmful skin effects: epidemiological evidence

We review the general amount and patterns of exposure to solar ultraviolet (UV) radiation that children and teenagers experience and the spectrum of UV-related skin damage that can occur as a result. Data about the amount of solar UV received by children and teenagers are relatively few but suggest that around 40–50% of total UV to age 60 occurs before age 20. Among white children, those with the palest complexions suffer the most damage. Comparisons of prevalence and incidence of outcomes in children and teenagers sharing common ancestry, but living at different latitudes, show that prevalence rates of photoaging and melanocytic naevi are higher in Australian compared with British children, and similarly for melanoma. Genetic risk for the majority of the melanomas in teens is a function of genes controlling naevus propensity and pigmentation in the skin. High numbers of naevi and freckles, red hair, blue eyes, inability to tan, as well as a family history are the primary determinants of melanoma among adolescents. Beyond the signs of skin damage seen in children are the latent effects observed later in adulthood. Childhood is believed to be a susceptible window for long-term harmful effects of UV, as evidenced by clear differences in skin cancer risk between child and adult migrants from high to low latitudes. Effective UV radiation protection from childhood is necessary to control both immediate and long-term harmful effects on children’s skin.     

Approaches to increasing skin melanin with MSH analogs and synthetic melanins.

Increased public awareness of ultraviolet (UV) radiation-induced skin cancers has lead to new interest in technologies for protection from sun exposure. Although many sun protection formulations are available, few of them attempt to achieve that provided by melanin itself, i.e., wide-spectrum absorbance of radiant energy coupled with anti-oxidant activity from a single product. In that regard, technologies in two separate areas are at or near the commercialization stage: 1) hormonal enhancement of natural skin melanin content, and 2) inclusion of natural and synthetic melanins in cosmetic formulations to impart melanin-like color to the skin. In this article, these approaches are briefly summarized using as examples Melanotans I and II, superpotent analogs of the melanin-stimulating hormone melanocortin (MSH), and Melasyn, a group of plant-derived synthetic melanins that have successfully been incorporated into cosmetic formulations for use as sun protectants and as cover-ups for problems resulting from uneven pigmentation, such as seen in vitiligo.     

In vivo vitiligo induction and therapy model: double-blind, randomized clinical trial

In this study, we developed an in vivo vitiligo induction model to explore the underlying mechanisms leading to Koebner's phenomenon and to evaluate the efficacy of therapeutic strategies. The model consisted of 12 pigmented test regions on the back of generalized vitiligo patients that were exposed to three Koebner induction methods: cryotherapy, 755 nm laser therapy, and epidermal abrasion. In addition, four cream treatments (pimecrolimus, tacrolimus, steroid and placebo) were randomly applied. Koebnerization was efficiently induced by all three induction methods. In general, cryotherapy was the best method of Koebner induction, followed by 755 nm laser therapy and epidermal abrasion. Reproducible results were obtained, which showed enhanced depigmented surface areas and higher amounts of T lymphocytes in placebo-treated test zones compared to active treated areas. Tacrolimus and local steroids were better inhibitors of Koebner's process (P < 0.05) compared to pimecrolimus. Our in vivo vitiligo induction model is very informative to investigate vitiligo induction and to determine the efficacy of topical treatments in vitiligo. This proof of concept confirms the efficient comparison of head-to-head therapeutic strategies intra-individually in a standardized, specific and better timed way.     

Regulation of human skin pigmentation and responses to ultraviolet radiation

Pigmentation of human skin is closely involved in protection against environmental stresses, in particular exposure to ultraviolet (UV) radiation. It is well known that darker skin is significantly more resistant to the damaging effects of UV, such as photocarcinogenesis and photoaging, than is lighter skin. Constitutive skin pigmentation depends on the amount of melanin and its distribution in that tissue. Melanin is significantly photoprotective and epidermal cells in darker skin incur less DNA damage than do those in lighter skin. This review summarizes current understanding of the regulation of constitutive human skin pigmentation and responses to UV radiation, with emphasis on physiological factors that influence those processes. Further research is needed to characterize the role of skin pigmentation to reduce photocarcinogenesis and to develop effective strategies to minimize such risks.     

Time-course evaluation and treatment of skin inflammatory immune response after ultraviolet B irradiation

Skin exposure to high doses of ultraviolet B (UVB) radiation generates a severe inflammatory skin response. In the present study we aim to investigate, using in vitro and in vivo models, the time-course of the inflammatory skin immune response after an acute exposure to UVB irradiation, as well as its modulation by a topical non-steroidal anti-inflammatory drug (NSAID) treatment, naproxen. PGE2 production and TNF-alpha levels increase in a post-irradiation time-dependent manner both in vivo and in vitro. This production pattern is also reflected in the iNOS expression levels in vivo and in the IL-6 levels in vitro. Changes observed in these mediators are correlated with histological alterations and dermal infiltration after the acute UVB irradiation. Naproxen treatment notably reduces PGE2 production and iNOS expression, reflecting the COX-NOS crosstalk already reported, although it causes an important increment in TNF-alpha synthesis in the epidermis of irradiated mice. Taken together, our data indicates that the epidermis is severely damaged by UVB radiation but then it is able to fully recover, and that the immune response is modulated by the NSAID treatment, since it is able to reduce the levels of some mediators as well as it can increase others.     

A new locus for generalized epilepsy with febrile seizures plus maps to chromosome 2

Generalized epilepsy with febrile seizures plus (GEFS+) is a recently recognized but relatively common form of inherited childhood-onset epilepsy with heterogeneous epilepsy phenotypes. We genotyped 41 family members, including 21 affected individuals, to localize the gene causing epilepsy in a large family segregating an autosomal dominant form of GEFS+. A genomewide search examining 197 markers identified linkage of GEFS+ to chromosome 2, on the basis of an initial positive LOD score for marker D2S294 (Z=4.4, recombination fraction [straight theta] = 0). A total of 24 markers were tested on chromosome 2q, to define the smallest candidate region for GEFS+. The highest two-point LOD score (Zmax=5.29; straight theta=0) was obtained with marker D2S324. Critical recombination events mapped the GEFS+ gene to a 29-cM region flanked by markers D2S156 and D2S311, with the idiopathic generalized epilepsy locus thereby assigned to chromosome 2q23-q31. The existence of the heterogeneous epilepsy phenotypes in this kindred suggests that seizure predisposition determined by the GEFS+ gene on chromosome 2q could be modified by other genes and/or by environmental factors, to produce the different seizure types observed.