Varicella-zoster virus in actively spreading segmental vitiligo skin: Pathological, immunochemical, and ultrastructural findings (a first and preliminary study).

Segmental vitiligo (SV) is a unilateral subtype of vitiligo which is clinically characterized by a cutaneous depigmentation and histologically by a melanocyte loss from the epidermis and hair follicle reservoirs. To date, its pathogenesis remains a mystery. In many cases, this skin depigmentation shares several clinical features and dysfunctions with herpes zoster (HZ). So, for the first time, we examined whether any nucleus and cell fusion associated with a positive immunolabelling of varicella-zoster virus (VZV) and VZV mature virions could be found in SV skin samples as in herpes zoster (HZ). A total of 40 SV samples were used for histological and immunochemical studies. Control samples were obtained from three HZ, and 10 generalized vitiligo lesions. For ultrastructural study, three recent SV and one HZ as controls were recruited. Here, we report that nuclear fusion in epidermal cells were statistically associated with recent SV (p < .001), whereas syncytia formation was associated with long-lasting SV (p = .001). A positive detection of VZV antigen was statistically associated in the epidermis with recent SV and in the dermis with long-lasting SV (p = .001). Finally, the discovery of mature virions in 3/3 recent SV samples provides additional arguments for our viral hypothesis.

Critical role of Aquaporin-1 and telocytes in infantile hemangioma response to propranolol beta blockade.

Propranolol, a nonselective ß-adrenergic receptor (ADRB) antagonist, is the first-line therapy for severe infantile hemangiomas (IH). Since the incidental discovery of propranolol efficacy in IH, preclinical and clinical investigations have shown evidence of adjuvant propranolol response in some malignant tumors. However, the mechanism for propranolol antitumor effect is still largely unknown, owing to the absence of a tumor model responsive to propranolol at nontoxic concentrations. Immunodeficient mice engrafted with different human tumor cell lines were treated with anti-VEGF bevacizumab to create a model sensitive to propranolol. Proteomics analysis was used to reveal propranolol-mediated protein alteration correlating with tumor growth inhibition, and Aquaporin-1 (AQP1), a water channel modulated in tumor cell migration and invasion, was identified. IH tissues and cells were then functionally investigated. Our functional protein association networks analysis and knockdown of ADRB2 and AQP1 indicated that propranolol treatment and AQP1 down-regulation trigger the same pathway, suggesting that AQP1 is a major driver of beta-blocker antitumor response. Examining AQP1 in human hemangioma samples, we found it exclusively in a perivascular layer, so far unrecognized in IH, made of telocytes (TCs). Functional in vitro studies showed that AQP1-positive TCs play a critical role in IH response to propranolol and that modulation of AQP1 in IH-TC by propranolol or shAQP1 decreases capillary-like tube formation in a Matrigel-based angiogenesis assay. We conclude that IH sensitivity to propranolol may rely, at least in part, on a cross talk between lesional vascular cells and stromal TCs.

Tacrolimus (FK506) ointment combined with Nb-UVB could activate both hair follicle (HF) and dermal melanocyte precursors in vitiligo: the first histopathological and clinical study.

Topical Tacrolimus, especially when combined with Nb-UVB, has been proven clinically to be effective in the treatment of vitiligo. However, no histological study has evaluated the repigmentation mechanism of tacrolimus ointment in combination therapy with Nb-UVB. In this study, the histological findings in patients receiving Nb-UVB were compared with those receiving topical tacrolimus combined with Nb-UVB. Twenty patients were recruited and received Nb-UVB treatment. The first ten patients were selected for the combination therapy and instructed to apply tacrolimus 0.1% ointment twice daily on the specified lesion of interest. The remaining ten patients did not receive any other topical treatments. Skin biopsy was performed at baseline from the depigmented area and 2-3 months post-treatment from the repigmented area. Biopsy specimens were stained with haematoxylin-eosin-safran (HES), Fontana Masson, HMB45, Melan A, MITF, SOX10 and Nestin. Clinically, in the combination therapy group, interfollicular repigmentation in addition to the perifollicular and marginal pattern was observed. Histologically, in the combination therapy group, besides the migration of melanocytes from the bulge of the hair follicle seen in the monotherapy group, for the first time, we observed dermal melanocyte precursors located in mid- and superficial dermis.

Pigmentation abnormalities in nucleotide excision repair disorders: Evidence and hypotheses.

Skin pigmentation abnormalities are manifested in several disorders associated with deficient DNA repair mechanisms such as nucleotide excision repair (NER) and double-strand break (DSB) diseases, a topic that has not received much attention up to now. Hereditary disorders associated with defective DNA repair are valuable models for understanding mechanisms that lead to hypo- and hyperpigmentation. Owing to the UV-associated nature of abnormal pigmentary manifestations, the outcome of the activated DNA damage response (DDR) network could be the effector signal for alterations in pigmentation, ultimately manifesting as pigmentary abnormalities in repair-deficient disorders. In this review, the role of the DDR network in the manifestation of pigmentary abnormalities in NER and DSB disorders is discussed with a special emphasis on NER disorders.

Analysis of CCN Expression by Immunofluorescence on Skin Cells, Skin, and Reconstructed Epidermis.

During a long time, immunofluorescence has been neglected to benefit of molecular biology especially genetics, transcriptomics, and proteomics analyses. These techniques give good results on cell culture but for organs that are made of numerous cells with several compartments, various states of differentiation as in epidermis, immunohistochemistry is always relevant. Double (triple) staining by immunofluorescence allows positive cells identification in complex cell structure (for example, pericytes and endothelial cells in vessels) and subcellular localizations. In order to, due to improvement of antibodies avoiding especially species cross-reactions, microscopy and specific softwares, quality of staining, and acquired images have been upgraded. Consequently, this technique permits, as molecular biology analyses, quantification of the level of expression as intensity of fluorescence can be measured in each cells and each compartments (nuclear, cytoplasmic). In order to immunofluorescence on cells and tissue needs few materials and gives at the same times qualitative and quantitative results and must be used more widely especially when a mutation was associated to a disease.

Follicular vitiligo: A report of 8 cases.

BACKGROUND: Follicular vitiligo, a recently proposed new subtype of vitiligo, has primary involvement of the hair follicle melanocytic reservoir. OBJECTIVE: We sought to characterize follicular vitiligo through a case series of 8 patients. METHODS: Patients with features of follicular vitiligo who were seen at the vitiligo clinic in the National Center for Rare Skin Disorders in Bordeaux, France, were recruited. A retrospective review of case records and clinical photographs was carried out. RESULTS: There were 8 male patients with a mean age of 48 years. All patients reported significant whitening of their body and, in some, scalp hairs before cutaneous depigmentation. Examination revealed classic generalized depigmented lesions of vitiligo and an impressive presence of leukotrichia, not only in the vitiliginous areas, but also in areas with clinically normal-appearing skin. Punch biopsy specimen of the leukotrichia and vitiligo lesions demonstrated loss of melanocytes and precursors in the basal epidermis and hair follicle. LIMITATIONS: This was a cross-sectional study based on a single-center experience. CONCLUSION: Follicular vitiligo is a distinct entity within the spectrum of vitiligo. This entity may serve as the missing link between alopecia areata and vitiligo, with probable physiopathological similarities between these conditions.

Altered E-Cadherin Levels and Distribution in Melanocytes Precede Clinical Manifestations of Vitiligo.

Vitiligo is the most common depigmenting disorder resulting from the loss of melanocytes from the basal epidermal layer. The pathogenesis of the disease is likely multifactorial and involves autoimmune causes, as well as oxidative and mechanical stress. It is important to identify early events in vitiligo to clarify pathogenesis, improve diagnosis, and inform therapy. Here, we show that E-cadherin (Ecad), which mediates the adhesion between melanocytes and keratinocytes in the epidermis, is absent from or discontinuously distributed across melanocyte membranes of vitiligo patients long before clinical lesions appear. This abnormality is associated with the detachment of the melanocytes from the basal to the suprabasal layers in the epidermis. Using human epidermal reconstructed skin and mouse models with normal or defective Ecad expression in melanocytes, we demonstrated that Ecad is required for melanocyte adhesiveness to the basal layer under oxidative and mechanical stress, establishing a link between silent/preclinical, cell-autonomous defects in vitiligo melanocytes and known environmental stressors accelerating disease expression. Our results implicate a primary predisposing skin defect affecting melanocyte adhesiveness that, under stress conditions, leads to disappearance of melanocytes and clinical vitiligo. Melanocyte adhesiveness is thus a potential target for therapy aiming at disease stabilization.

A miR-34a-SIRT6 axis in the squamous cell differentiation network.

Squamous cell carcinomas (SCCs) are highly heterogeneous tumours, resulting from deranged expression of genes involved in squamous cell differentiation. Here we report that microRNA-34a (miR-34a) functions as a novel node in the squamous cell differentiation network, with SIRT6 as a critical target. miR-34a expression increases with keratinocyte differentiation, while it is suppressed in skin and oral SCCs, SCC cell lines, and aberrantly differentiating primary human keratinocytes (HKCs). Expression of this miRNA is restored in SCC cells, in parallel with differentiation, by reversion of genomic DNA methylation or wild-type p53 expression. In normal HKCs, the pro-differentiation effects of increased p53 activity or UVB exposure are miR-34a-dependent, and increased miR-34a levels are sufficient to induce differentiation of these cells both in vitro and in vivo. SIRT6, a sirtuin family member not previously connected with miR-34a function, is a direct target of this miRNA in HKCs, and SIRT6 down-modulation is sufficient to reproduce the miR-34a pro-differentiation effects. The findings are of likely biological significance, as SIRT6 is oppositely expressed to miR-34a in normal keratinocytes and keratinocyte-derived tumours.

Development and validation of the K-VSCOR for scoring Koebner's phenomenon in vitiligo/non-segmental vitiligo.

The relation of vitiligo/non-segmental vitiligo (NSV) to Koebner's phenomenon is variably appreciated. Our objective was to develop and validate a simple clinical score for Koebner's phenomenon (KP) in patients with vitiligo/NSV. The study population was composed of 351 individuals in the development sample and 285 patients in the validation sample. Seven variables were independently associated with the presence of KP: disease duration of more than 3 yr, forehead + scalp areas, eyelids, wrists, genital + belt areas, knees and tibial crests. The score computed by the weighted sum of the rounded coefficients of these seven variables ranged from 0 to 56 (mean 38.39 ± 22.93). The probability of having KP was computed as follows: exp (-2.37 + 0.1*score)/exp [1 + (-2.37 + 0.1*score)]. When applying the score to each patient in the validation and the development sample, the score maintained adequate discrimination and calibration (AUC-ROC = 0.78), arguing that KP can be adequately predicted using our score. Further studies should evaluate KP assessed by the K-VSCOR in clinical practice with the aim to determine its association with clinical profile, course and treatment response of vitiligo.

In vitro research on vitiligo: strategies, principles, methodological options and common pitfalls.

Understanding the cellular and molecular mechanisms leading to melanocyte loss in vitiligo is a mandatory step in improving the overall management of vitiligo patients. Until now, the study of vitiligo was characterised by a fragmented approach, and it is very hard to share and compare the data obtained by the different teams. The scenario mirrors the pathogenic puzzle, but it delays a true productive focus on the disease. The in vitro research is based on different models, ranging from monolayer cell culture of epidermal and dermal cells or 3D reconstructed skin to histological data, gene expression, computer simulation profile. For each model, several different (biochemical, phenotypic, immunological) aspects have been considered, increasing the mass of data difficult to be merged. Our purpose was to provide a practical synopsis of consolidated and advanced possibilities in the study of vitiligo, showing how data have been poorly shared until now. Following a short overview of the background of the disease, the approaches, ranging from basic cell biology to molecular and 'omics' studies, are summarised. New fluorescent probes and techniques open new possibilities for functional studies. Next, intracellular and superficial markers of the melanocytes, the main involved cells, are listed. Moving the focus from the epidermal level to the systemic and subcellular ones, this review aims to propose innovative multidisciplinary options for the vitiligo understanding. This paper focuses on the major practical and theoretical questions to be solved. It may be the basis for a more coordinated and productive approach to the biological question.

Study of CCN3 (NOV) and DDR1 in normal melanocytes and vitiligo skin.

We have hypothesised that melanocytes disappear in vitiligo because they are weakly attached to the epidermal basal membrane (melanocytorrhagy). In the epidermis, attachment of melanocytes to collagen IV is mediated through DDR1, which is under the control of CCN3. DDR1 genetic variants have been associated with vitiligo in patients of different ethnic origin. In vitro studies have shown that inhibition of CCN3 induces the detachment of melanocytes. We have studied in parallel the expression of CCN3 and DDR1 in lesional and perilesional skin of patients with vitiligo and the impact of the silencing of CCN3 and DDR1 in normal human melanocytes on their behaviour in epidermal reconstructs. Our in vivo study provides evidence of a dysregulation of the DDR1-CCN3 interaction in vitiligo skin as melanocytes remaining in perilesional skin did not express CCN3. Expression of DDR1 was decreased in lesional versus perilesional vitiligo skin in the majority of patients, and the expression of collagen IV was found decreased in all patients. Silencing of CCN3 in melanocytes induced a significant inhibition of cell adhesion to collagen IV whereas melanocytes transduced with shDDR1 still adhered well on collagen IV and did not increase melanocyte loss in epidermal reconstructs as compared with normal melanocytes. Melanocyte detachment was observed but not in all reconstructs using CCN3 silenced melanocytes. Overall, our study confirms that a downregulation of CCN3 is implicated in melanocyte adhesion in part through DDR1. In vitiligo skin, the interaction of CCN3 with other molecules, such as TGFß and CCN2, needs to be addressed.

Halo nevi association in nonsegmental vitiligo affects age at onset and depigmentation pattern.

OBJECTIVE: To compare factors associated with halo nevi with nonsegmental vitiligo (NSV) vs NSV alone. DESIGN: Prospective observational study in 553 patients with a confirmed diagnosis of NSV attending a vitiligo clinic between January 1, 2006, and July 1, 2010. SETTING: Vitiligo Clinic at the Department of Dermatology, University Hospital Center of Bordeaux, Bordeaux, France. PATIENTS: The Vitiligo European Task Force questionnaire was informed for each patient attending the clinic with a confirmed diagnosis of NSV after the exclusion of other forms of vitiligo (focal, mucosal, and not classifiable). Thyroid function and antithyroid antibodies were screened if not obtained in the previous year. MAIN OUTCOME MEASURES: Extent of disease and markers of autoimmunity or autoinflammation. RESULTS: Of the 553 patients, 130 had halo nevi-NSV and 423 had NSV. Family history of premature hair graying (odds ratio, 1.74; P < .01) was positively associated with halo nevi-NSV by univariate analysis. Using multivariate analysis, age at onset younger than 18 years, phototype, total body area, localization on the trunk, involvement of hands and feet, and total staging were found to be independent factors. Age at onset younger than 18 years; phototypes I, II, and III; trunk involvement; and staging were positively associated with halo nevi-NSV, whereas this association was negative for total affected area and involvement of hands and feet. CONCLUSIONS: Halo nevi association in NSV affects age at onset and depigmentation pattern and has a stronger link with familial premature hair graying, suggesting that premature hair graying may involve, at least partly, an autoimmune pathway.

Loss of epidermal hypoxia-inducible factor-1α accelerates epidermal aging and affects re-epithelialization in human and mouse.

In mouse and human skin, HIF-1α is constitutively expressed in the epidermis, mainly in the basal layer. HIF-1α has been shown to have crucial systemic functions: regulation of kidney erythropoietin production in mice with constitutive HIF-1α epidermal deletion, and hypervascularity following epidermal HIF-1α overexpression. However, its local role in keratinocyte physiology has not been clearly defined. To address the function of HIF-1α in the epidermis, we used the mouse model of HIF-1α knockout targeted to keratinocytes (K14-Cre/Hif1a(flox/flox)). These mice had a delayed skin phenotype characterized by skin atrophy and pruritic inflammation, partly mediated by basement membrane disturbances involving laminin-332 (Ln-332) and integrins. We also investigated the relevance of results of studies in mice to human skin using reconstructed epidermis and showed that HIF-1α knockdown in human keratinocytes impairs the formation of a viable reconstructed epidermis. A diminution of keratinocyte growth potential, following HIF-1α silencing, was associated with a decreased expression of Ln-322 and α6 integrin and ß1 integrin. Overall, these results indicate a role of HIF-1α in skin homeostasis especially during epidermal aging.

New clinico-genetic classification of trichothiodystrophy.

Trichothiodystrophy (TTD) is a congenital hair dysplasia with autosomal recessive transmission. Cross banding pattern under polarized light plus trichoschisis and a low sulfur content of hair shafts define the disorder, which is associated with variable and neuroectodermal symptoms. So-called photosensitive forms of TTD (with low level of in vitro UV-induced DNA repair, not constantly associated with marked clinical photosensitivity) are caused by mutations in genes encoding subunits of the transcription/repair factor IIH (TFIIH). Ten percentage of nonphotosensitive patients are known to have TTDN1 mutations, the specific role of which is unknown. We studied nine patients recruited at our institution and reviewed 79 with molecular analysis out of 122 TTD patients reported in literature with the aim to collect systematically the clinical findings in TTD patients and establish genotype-phenotype correlations. The frequency of congenital ichthyosis, collodion-baby type, was significantly higher in the TFIIH mutated group. Hypogonadism was significantly more frequent in the non-photosensitive group. There was no statistical difference regarding osseous anomalies. Mutations in TFIIH sub-units leading to abnormal expression in genes involved in epidermal differentiation could explain the particular dermatological changes seen in photosensitive cases of TTD. We suggest a new clinico-genetic classification of TTD, which may help clinicians confused by the current acronyms used (IBIDS, PIBIDS...). Understanding the TTD ichthyotic phenotype could lead to therapeutic advances in the management of TTD and other types of ichthyoses.

Segmental vitiligo as the possible expression of cutaneous somatic mosaicism: implications for common non-segmental vitiligo.

Clinical findings in vitiligo challenge the widely accepted organ specific autoimmune pathomechanisms. We draw the attention to the fact that the distribution of segmental vitiligo (SV) fits in at least a subset of patients a pattern usually associated with cutaneous mosaicism. The association of SV to non-segmental vitiligo (NSV) now confirmed by several observations indicates a continuum between the two subsets with shared predisposing genetic factors, including genes operating specifically in the skin. Some pedigrees associating SV and NSV further suggest a mechanism of loss of heterozygosity for a dominant gene controlling part of the cutaneous phenotype. The mosaic hypothesis applies only to SV and to the rare SV-NSV association, but suggests that predisposing genetic factors in common NSV should also be searched directly in the skin. SV would be a good candidate disease to explore as a proof of principle of a new gene discovery strategy useful for multigenic disorders with organ specificity, applicable in priority to chronic inflammatory skin disorders.

Differential epigenetic regulation of Aiolos expression in human tumoral cell lines and primary cells.

In order to investigate the epigenetic component of Aiolos regulation, we analyzed the methylation status of its 5' CpG island in relation to histone modifications. Inhibition of CpG methylation restores Aiolos expression, as well as euchromatin-associated markers, in U937 and 1106 mel cell lines. DNA methylation and low levels of euchromatin-associated signatures are observed in U937 and 1106 mel cell lines, while the opposite characterizes Daudi, Jurkat, T and B cells. CpG methylation is not necessary to repress transcription in monocytes and melanocytes where silencing mechanism involves heterochromatin-associated signature. We show that DNA methylation directs Aiolos silencing and chromatin status in tumor cell lines, while in primary cells is mainly regulated by histone modifications.