Combining large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers for promoting protective melanosome autophagy via the PI3K/Akt/mTOR signalling pathway for the treatment of melasma.

Melasma is a common condition of hyperpigmented facial skin. Picosecond lasers are reported to be effective for the treatment of melasma. We aimed to identify the most effective therapeutic mode and elucidate the potential molecular mechanisms of picosecond lasers for the treatment of melasma. Female Kunming mice with melasma-like conditions were treated using four different picosecond laser modes. Concurrently, in vitro experiments were conducted to assess changes in melanin and autophagy in mouse melanoma B16-F10 cells treated with these laser modes. Changes in melanin in mouse skin were detected via Fontana-Masson staining, and melanin particles were evaluated in B16-F10 cells. Real-time polymerase chain reaction and western blotting were used to analyse the expression levels of melanosome and autophagy-related messenger ribonucleic acid (mRNA) and proteins. A combination of large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers resulted insignificant decreases in melanin as well as in mRNA and protein expression of melanin-synthesizing enzymes (TYR, TRP-1 and MITF). This combination also led to increased expression of the autophagy-related proteins, Beclin1 and ATG5, with a marked decrease in p62 expression. Intervention with the PI3K activator, 740 Y-P, increased TYR, TRP-1, MITF, p-PI3K, p-AKT, p-mTOR and p62 expression but decreased the expression of LC3, ATG5 and Beclin1. A combination of large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers proved more effective and safer. It inhibits melanin production, downregulates the PI3K/AKT/mTOR pathway, enhances melanocyte autophagy and accelerates melanin metabolism, thereby reducing melanin content.

Tranexamic acid may promote melanocores clustering in keratinocytes through upregulation of Rab5b.

Tranexamic acid (TXA) is a promising therapeutic agent in melasma that can act on multiple pathophysiologic mechanisms of melasma. However, it is unclear whether TXA affects melanin in keratinocytes. To explore the effect of TXA on melanocores in keratinocytes. The melanocore-incorporated keratinocytes were constructed by co-incubating normal human epidermal keratinocytes (NHEK) with melanocores. After being treated with TXA, autophagy- and melanin-related protein expressions were detected. Then, transcriptome sequencing was used to compare the genetic changes in melanocore-incorporated keratinocytes before and after TXA treatment and further verified the differentially expressed genes. At the same time, the distribution of melanocores in human keratinocytes was observed by transmission electron microscopy. We found that TXA does not promote melanin degradation in primary keratinocytes by inducing autophagy. Protein transport and intracellular protein transport-related genes were enriched after TXA treatment, and Rab5b was significantly upregulated. Transmission electron microscopy showed that the percentage of melanocores distributed in clusters increased after treatment with TXA, which was reduced after Rab5b silencing. In addition, results suggested that melanocores could colocalize with Rab5b and lysosome-associated membrane protein1 (LAMP1). Our study found that Rab5b may be involved in the melanocore distribution in keratinocytes. TXA may promote the clustering distribution of endocytic melanocores through upregulation of Rab5b, representing a potential mechanism of TXA treatment against melasma.

Depigmenting topical therapy based on a synergistic combination of compounds targeting the key pathways involved in melasma pathophysiology.

Melasma has a complex pathophysiology with different cell types and signalling pathways involved. Paracrine factors secreted by keratinocytes, fibroblasts and endothelial cells act on melanocytes and stimulate melanogenesis. These paracrine factors are involved in the oxidative stress, inflammatory, vascular and hormonal pathways, among others. Damage of the dermoepidermal barrier also occurs and facilitates melanin deposition in the dermis, also known as dermal or mixed melasma. We used artificial intelligence tools to define the best combination of compounds for skin pigmentation inhibition. Mathematical models suggested the combination of retinol, diosmin and ferulic acid to be the most effective one. In vitro cellular tyrosinase activity assay proved that this combination had a synergistic depigmenting effect. Further assays proved that the combination could inhibit key pathways involved in melasma by downregulating ET-1 and COX-2 gene expression and IBMX-induced dendricity in human melanocytes, and upregulated the gene expression of IL-1b, TIMP3 and several endogenous antioxidant enzymes. The combination also reduced melanin levels in a phototype VI 3D epidermis model. These results indicate that the combination of retinol, diosmin and ferulic acid is an effective synergistic complex for the treatment of melasma by regulating the key molecular pathways involved in skin hyperpigmentation pathophysiology.

Paracrine roles of hormone receptors in Riehl's melanosis: A quantitative analysis of oestrogen and progesterone receptor expression patterns.

The incidence of Riehl's melanosis (RM) is most common in the fifth or sixth decade of life with a female preponderance. As the skin is regarded a non-reproductive organ on which sex steroid hormones act, a possible relationship between the pathogenesis of RM and sex steroid hormone receptors can be inferred. This study intended to evaluate the expression profile of oestrogen receptor (ER)ß and progesterone receptor (PR) in RM. Twelve lesional and perilesional normal-appearing skin samples of RM patients and the skin of 12 healthy controls were retrieved for the analysis. Real-time PCR analysis and immunohistochemical studies were conducted for ERß and PR, respectively. The lesional and perilesional normal-appearing skin of 12 patients with RM and the skin of 12 healthy controls were retrieved for the analysis. Interestingly, the dermal ERß immunostaining intensity was increased more in lesional skin than in perilesional skin. When compared to healthy controls, increased expression of ERß and PR mRNAs was observed in the lesional skin of patients with RM. Of note, epidermal and dermal ERß and dermal PR expressions showed increased staining intensities in the lesional skin of RM patients compared with healthy controls. The altered expression of ERß and PR in RM supports the possible role of these hormone receptors in the pathogenesis of RM.

Tumoral Melanosis in the Setting of Targeted Immunotherapy for Metastatic Melanoma-A Single Institutional Experience and Literature Review.

BACKGROUND: Tumoral melanosis clinically resembles metastatic melanoma, occurs in the context of regressed disease, and requires evaluation to rule out underlying melanoma and metastatic disease. Histopathology demonstrates a nodular infiltrate of melanophages in the dermis, subcutaneous tissue, deep soft tissue, or lymph nodes in the absence of viable melanocytes. Recent limited reports of tumoral melanosis in the context of immunotherapy with ipilimumab (monoclonal antibody targeting CTLA-4) as well as nivolumab and pembrolizumab (humanized monoclonal antibodies against programmed death 1 receptor) highlight a unique presentation representative of treatment-related tumor regression and an association with a favorable clinical response. OBJECTIVE: To describe our experience with tumoral melanosis in the setting of immunotherapy for metastatic melanoma and elucidate the clinical and histopathological features. METHODS: Retrospective case series from a single tertiary care institution. RESULTS: We describe 10 cases of patients with metastatic melanoma who received treatment with immunotherapy before the development of tumoral melanosis. Length of time between the initiation of therapy and the onset of tumoral melanosis ranged from 2 to 20 months with a mean time of 10 months. At the end of the follow-up period, 8 patients were classified as having a complete or partial response to treatment with immunotherapy. One patient had progression of visceral and cutaneous disease on ipilimumab despite developing tumoral melanosis, and 1 patient had yet to undergo repeat imaging. Furthermore, at the end of follow-up, 3 patients were alive with no evidence of active disease, 5 patients were alive with disease, and 1 patient was deceased, although this patient died of a cardiovascular event unrelated to his underlying melanoma. Of the patients who were classified as alive with disease, 2 patients had minimal remaining disease, and 2 patients had an almost complete response on immunotherapy with recurrence of visceral metastases after immunotherapy was discontinued. One patient developed new peritoneal and cutaneous metastases on pembrolizumab despite development of tumoral melanosis. CONCLUSIONS: The underlying biologic mechanisms and prognostic implications of tumoral melanosis in the setting of immunotherapy remain to be elucidated. Further prospective studies with a larger cohort and prolonged follow-up are necessary to better understand the incidence, prevalence, and oncologic outcomes in patients with tumoral melanosis who receive immunotherapy.

Skin pigmentation provides evidence of convergent melanism in extinct marine reptiles.

Throughout the animal kingdom, adaptive colouration serves critical functions ranging from inconspicuous camouflage to ostentatious sexual display, and can provide important information about the environment and biology of a particular organism. The most ubiquitous and abundant pigment, melanin, also has a diverse range of non-visual roles, including thermoregulation in ectotherms. However, little is known about the functional evolution of this important biochrome through deep time, owing to our limited ability to unambiguously identify traces of it in the fossil record. Here we present direct chemical evidence of pigmentation in fossilized skin, from three distantly related marine reptiles: a leatherback turtle, a mosasaur and an ichthyosaur. We demonstrate that dark traces of soft tissue in these fossils are dominated by molecularly preserved eumelanin, in intimate association with fossilized melanosomes. In addition, we suggest that contrary to the countershading of many pelagic animals, at least some ichthyosaurs were uniformly dark-coloured in life. Our analyses expand current knowledge of pigmentation in fossil integument beyond that of feathers, allowing for the reconstruction of colour over much greater ranges of extinct taxa and anatomy. In turn, our results provide evidence of convergent melanism in three disparate lineages of secondarily aquatic tetrapods. Based on extant marine analogues, we propose that the benefits of thermoregulation and/or crypsis are likely to have contributed to this melanisation, with the former having implications for the ability of each group to exploit cold environments.

Metastatic Melanoma Negative for 5 Melanocytic Markers, Complete Regressed Primary Cutaneous Melanoma, and Melanoma-Associated Leukoderma in the Same Patient.

Melanomas with complete histological regression have been seen very infrequently. On the other hand, the diagnosis of metastatic melanoma is based on the histopathology and positivity of markers such as S100, Melan-A, and HMB-45 whose sensitivity is 99%, 82%, and 76%, respectively. It is very rare that metastatic melanomas and even more primary melanoma are negative for all of these markers. In these rare cases, there is usually a known primary. We present the case of a 82-year-old woman with a erythematous mass in the left groin and a 1-cm black-bluish irregular nodule on the skin of the ipsilateral foot. This lesion was clinical and dermoscopically compatible with primary melanoma. In the histological evaluation of the skin, a dermis full of melanophages and hemosiderophages were found in a background of fibrosis, scarce lymphocytic infiltrate, and neovascularization. Any cells expressing melanocytic markers were observed. It was diagnosed as tumoral melanosis. Lymph nodes showed a proliferation of atypical epithelioid cells with eosinophilic cytoplasm. Mitosis was conspicuous. Tumoral cells were vimentin and CD99 positive, and S100, CD34, HMB-45, Melan-A, SOX 10, tyrosinase, C-KIT, CD45, and CKAE1/AE3 negative, and BRAF-V600 mutated was detected. During follow-up, atypical vitiligo-like lesions were discovered, suggesting the diagnosis of metastatic melanoma totally regressed in our patient.

Comparison of the Inflammatory Infiltrates in Tumoral Melanosis, Regressing Nevi, and Regressing Melanoma.

BACKGROUND: Tumoral melanosis (TM) is a histologic diagnosis characterized by abundant pigment-laden macrophages in the dermis. It is generally thought to represent a regressed melanoma, although it has also been reported after benign pigmented lesions as well. Determining the antecedent lesion in cases of TM is of clinical importance to accurately guide therapy and prognostication. Comparing the histopathologic and immunohistochemical (IHC) characteristics of TM, halo nevi (HN), and regressing melanoma (RM) may help predict the antecedent lesion in cases of TM. METHODS: Cases of TM, HN, and RM were selected and assessed for histopathologic (preservation of junctional melanocytic component, depth and width, solar elastosis, fibrosis, and preservation of rete ridge architecture) and IHC (SOX-10, CD138, and PD-1) parameters. PD-L1 immunostaining was also evaluated in cases of HN and RM. RESULTS: Severe solar elastosis, fibrosis, and marked rete ridge effacement were more frequent in RM than in HN. By contrast, numerous plasma cells, clusters of lymphocytes expressing PD-1, and >50% PD-L1 expression in melanocytes were more common in HN than in RM. However, the association of these variables did not reach statistical significance. DISCUSSION: Although studies with higher statistical power are needed, this study serves as an initial investigation to characterize the histopathologic and IHC characteristics, which may help better understand TM and its precursor lesions.

Evaluation of large clinically atypical vulvar pigmentation with RCM: atypical melanosis or early melanoma?

BACKGROUND: Vulvar melanosis can occasionally be clinically challenging by mimicking an early melanoma. OBJECTIVE: To report our experience of initial evaluation and follow-up in this peculiar subset of vulvar melanosis using reflectance confocal microscopy (RCM). METHODS: We retrospectively evaluated 18 consecutive cases referred for atypical vulvar pigmentation or for which melanoma was considered and that underwent both RCM examination and histopathological assessment. In 13 cases with available dermoscopic pictures, RCM classification was compared to dermoscopic diagnosis, and in all cases, the density of melanocytes was evaluated on biopsies using MelanA immunostaining. RESULTS: Among the 18 atypical pigmented lesions, 17 vulvar melanosis and one melanoma were histologically determined. RCM concluded a benign vulvar melanosis in 10 of 17 cases, whereas dermoscopy did so in three of 12 cases. RCM identified the only early malignant lentiginous melanoma. In several cases of vulvar melanosis, RCM could identify foci of melanocytic hyperplasia in an otherwise benign pattern. CONCLUSIONS: In this clinically and dermoscopically challenging subset of vulvar pigmentations, RCM appears relevant for initial extensive evaluation, especially to target initial biopsy sampling, and to perform non-invasive monitoring of foci of melanocytic hyperplasia.

Increased expression of TGF-ß protein in the lesional skins of melasma patients following treatment with platelet-rich plasma.

Background: Melasma is a common acquired facial hyperpigmented skin disorder. Platelet-rich plasma (PRP) is autologous plasma containing higher than normal platelets concentrations. Recently, PRP has been used as a therapeutic modality in melasma with significant clinical improvement, possibly due to its abundant contents of growth factors such as TGF-ß. The latter represents a natural multifunctional polypeptide that negatively regulates melanocyte differentiation and therefore reduces skin hyperpigmentation. To date, the expression pattern of TGF-ß protein in skin of melasma patients following PRP injection is unknown. Here we hypothesize that "injection of PRP in the lesional skin of melasma patients is associated with alterations of TGF-ß protein expression".Patients and Methods: The study included 20 adult patients with melasma. Autologous PRP was delivered into the lesional skin either through microneedling or as intradermal microinjections. TGF-ß protein expression was immunohistochemically examined in the perilesional and lesional skins before and after PRP treatment and in the healthy skins of nine volunteers (control group).Results: TGF-ß protein was expressed within the epidermis, dermal adnexal structures, vascular endothelium, nerves and arrector pili muscle fibers of the healthy skins (control group), perilesional and lesional skins of melasma patients before and after treatment with PRP. Before treatment with PRP, the expression ofTGF-ß protein in the lesional (1.26 ± 0.41) and perilesional (1.68 ± 0.51) skins of melasma patients were significantly lower than that in the healthy skins (2.26 ± 0.37, p value<.05). After treatment with PRP, the expression of TGF-ß protein was significantly increased in the lesional (2.15 ± 0.44) skin of melasma patients.Conclusions: Our study provides the first indication about increased TGF-ß protein expression in skin of melasma patients after PRP treatment. The alterations of TGF-ß protein in skin of melasma patients not only support its roles in the development of this condition but also have some therapeutic ramifications.

Efficacy of oral tranexemic acid in refractory melasma: A clinico-immuno-histopathological study.

Tranexamic acid (TXA), a plasmin inhibitor, is an antifibrinolytic drug widely used to prevent and treat hemorrhage. We evaluated the effects of oral TXA clinically and immunohistopathologically in patients of refractory melasma. To evaluate the efficacy of oral TXA in patients with refractory melasma and correlate histopathological and immunohistochemical changes in pretreatment and post-treatment skin biopsies in patients willing to undergo biopsy. Thirty patients with refractory melasma were treated with oral TXA 500 mg twice daily along with a sunscreen and followed up. Modified MASI score (MMASI) and melasma quality of life (MELASQOL) were noted at baseline and after treatment. In patients willing to undergo skin biopsy, a 2 mm punch biopsy was obtained for histopathology and immunohistochemistry examination both before and after treatment with TXA. Clinical, histopathological, and immunohistochemical parameters were compared and correlated. Clinical improvement in melasma correlated in a perfect linear relationship with quality of life, decrease in epidermal pigmentation and decrease in Melan A staining on immunohistochemistry. Based on our observations, TXA can be said to have an inhibitory action on melanin synthesis and melanocyte proliferation. Future studies are required to further characterize the effects of TXA on the histopathology and immunohistochemistry of melasma, to standardize dosing schedule, duration of treatment and long term outcome, of which there are no definitive guidelines at present.

Bisretinoid Photodegradation Is Likely Not a Good Thing.

Retinaldehyde adducts (bisretinoids) accumulate in retinal pigment epithelial (RPE) cells as lipofuscin. Bisretinoids are implicated in some inherited and age-related forms of macular degeneration that lead to the death of RPE cells and diminished vision. By comparing albino and black-eyed mice and by rearing mice in darkness and in cyclic light, evidence indicates that bisretinoid fluorophores undergo photodegradation in the eye (Ueda et al. Proc Natl Acad Sci 113:6904-6909, 2016). Given that the photodegradation products modify and impair cellular and extracellular molecules, these processes likely impart cumulative damage to retina.

Exploring pathways for sustained melanogenesis in facial melasma: an immunofluorescence study.

BACKGROUND: The physiopathology of epidermal hypermelanization in melasma is not completely understood. Several cytokines and growth factors are increased in skin with melasma, nevertheless, nor the pathways involved in the increased αMSH expression have been adequately evaluated, nor a model for sustained focal melanogenesis is available. OBJECTIVE: To explore stimulatory pathways for epidermal pigmentation in facial melasma related to αMSH: those linked to ultraviolet radiation, oxidative stress, inflammation, neural crest pigmentation cell differentiation and antagonism of αMSH. METHODS: Paired skin biopsies (3 mm) from 26 women with facial melasma and from normal adjacent skin (<2 cm far) were processed for immunofluorescence with markers for p53, p38, αMSH, MC1R, Melan-A, IL-1α, COX2, Wnt1, WIF-1 and ASIP. RESULTS: The fluorescence intensity in the skin from melasma was higher for MC1R, αMSH at epidermis as at melanocytes (P < 0.05). There were no differences between the sites in epidermal protein expression of COX2, IL-1α, p53, WIF-1 and ASIP (P > 0.1). P53 was expressed only in epidermis, without difference between sites (P = 0.92). WNT1 was remarkable in the epidermis of melasma (P < 0.01), but not in dermis. Positive p38 cells were prominent in the upper dermis of melasma (P < 0.01), despite no marking in epidermis. CONCLUSION: Melanogenesis in melasma involves epithelial secretion of αMSH and activation of the Wnt pathway; nevertheless, it seems to be independent of the stimulation by ultraviolet radiation/p53, IL-1α, COX2/PgE2 , WIF-1 and ASIP. Damaged cells at upper dermis suggest the role of senescence/autophagy in sustained pigmentation in melasma.

Efficacy and Safety of Tranexamic Acid in Melasma: A Meta-analysis and Systematic Review.

Tranexamic acid is a novel treatment option for melasma; however, there is no consensus on its use. This systematic review searched major databases for relevant publications to March 2016. Eleven studies with 667 participants were included. Pooled data from tranexamic acid-only observational studies with pre- and post-treatment Melasma Area and Severity Index (MASI) showed a decrease of 1.60 in MASI (95% confidence interval (CI), 1.20-2.00; p<0.001) after treat-ment with tranexamic acid. The addition of tranexamic acid to routine treatment modalities resulted in a further decrease in MASI of 0.94 (95% CI 0.10-1.79; p = 0.03). Side-effects were minor, with a few cases reporting hypo-menorrhoea, mild abdominal discomfort, and transient skin irritation. These results support the efficacy and safety of tranexamic acid, either alone or as an adjuvant to routine treatment modalities for melasma.

Cadherin 11 Involved in Basement Membrane Damage and Dermal Changes in Melasma.

Basement membrane (BM) disruption and dermal changes (elastosis, collagenolysis, vascular ectasia) have been reported in melasma. Although ultraviolet (UV) irradiation can induce these changes, UV is not always necessary for melasma development. Cadherin 11 (CDH11), which is upregulated in some melasma patients, has previously been shown to stimulate melanogenesis. Because CDH11 action requires cell-cell adhesion between fibroblasts and melanocytes, BM disruption in vivo should facilitate this. The aim of this study was to examine whether CDH11 overexpression leads to BM disruption and dermal changes, independent of UV irradiation. Immunohistochemistry/immunofluorescence, real-time PCR, Western blotting, and zymography suggested that BM disruption/dermal changes and related factors were present in the hyperpigmented skin of CDH11-upregulated melasma patients and in CDH11-overexpressing fibroblasts/keratinocytes. The opposite was seen in CDH11-knockdown cells. UV irradiation of the cultured cells did not increase CDH11 expression. Collectively, these data demonstrate that CDH11 overexpression could induce BM disruption and dermal changes in melasma, regardless of UV exposure.

Role of fibroblast-derived factors in the pathogenesis of melasma.

BACKGROUND: The hyperactive melanocytes present in melasma skin are confined to the epidermis, but epidermal ablation to treat melasma pigmentation may lead to disease recurrence and aggravation. Melanocyte function is regulated by interactions between melanocytes and neighbouring cells such as keratinocytes and fibroblasts. Because melasma skin usually shows dermal changes after exposure to sunlight, we hypothesized that sun-damaged fibroblasts might play a crucial role in the pathogenesis of melasma. AIM: In this study, the melanogenic role of primary cultured fibroblasts from human melasma skin was investigated. METHODS: We explored whether primary cultured fibroblasts from melasma tissue have a melanogenic function on cultured human epidermal melanocytes and artificial skin. The cytokine profile derived from fibroblasts and their effect on the pigmented epidermal equivalents were investigated. RESULTS: Fibroblasts from the melasma lesion and perilesional skin increased melanogenesis in cultured human epidermal melanocytes and in artificial skin. Fibroblasts from the melasma lesion and perilesional skin secreted more nerve growth factor (NGF)-ß than those in normal buttock skin, and also increased melanogenesis and the expression level of NGF-ß in cultured human epidermal melanocytes and artificial skin. CONCLUSIONS: These results suggest that fibroblasts may play a role in melanogenesis and the pathogenesis of melasma.

Efficacy and possible mechanisms of topical tranexamic acid in melasma.

BACKGROUND: Tranexamic acid (TA) has been suggested as an effective treatment for melasma. AIM: To investigate the effects and mechanism of action of topical TA in the treatment of melasma. METHODS: In this study, 23 participants with melasma applied a 2% TA formulation to the whole face for 12 weeks. Clinical effects were evaluated using the modified Melasma Area and Severity Index (mMASI) and a chromameter. Skin biopsies were obtained from 10 participants to evaluate pigmentation, vascularity and the expression levels of possible paracrine factors contributing to the effect of TA. RESULTS: Most of the participants had mild melasma, with mMASI of < 5. The mMASI scores significantly improved in 22 of 23 participants after application. The L* values were increased and the a* values were decreased in both lesional and perilesional normal skin. Fontana-Masson staining showed a significant decrease in melanin content in the epidermis. The number of CD31-positive vessels and the expression of the vascular endothelial growth factor both tended to decrease. Endothelin (ET)-1 was found to be downregulated with TA. CONCLUSIONS: Topical TA is effective for melasma. This immunohistochemical study found that suppression of ET-1 could be one of the mechanisms of action of TA on melasma.

Raman spectroscopy analysis of the skin of patients with melasma before standard treatment with topical corticosteroids, retinoic acid, and hydroquinone mixture.

BACKGROUND: Melasma is an abnormal acquired hyperpigmentation of the face of unknown origin, it is considered a single disease and very little has been found regarding its pathogenesis. It is usually assumed that melasma is due to excessive melanin production, but previous work using Raman spectroscopy showed degraded molecules of melanin in some melasma subjects, which may help to explain the success or failure of the standard therapy. METHODS: We perform Raman spectroscopy measurements on in vivo skin from melasma patients before treatment to identify the molecular structure of melanin within every melasma lesion. The Raman spectra were grouped according to the treatment response from patient, and the Raman spectra were analyzed. RESULTS: Raman spectroscopy measurements showed a different molecular structure of the patients who did not respond to treatment, those patients shows atypical Raman skin spectrum with peaks associated with melanin not well defined, which is consistent with molecular degradation and protein breakdown. CONCLUSION: Our results are consistent with our previous work in the sense that melasma patients who do not respond to treatment have an abnormal melanin. We believe it will eventually help to decide the treatment of melasma in clinical dermatology.