Inhibition of T-cell activity in alopecia areata: recent developments and new directions.

Alopecia areata (AA) is an autoimmune disease that has a complex underlying immunopathogenesis characterized by nonscarring hair loss ranging from small bald patches to complete loss of scalp, face, and/or body hair. Although the etiopathogenesis of AA has not yet been fully characterized, immune privilege collapse at the hair follicle (HF) followed by T-cell receptor recognition of exposed HF autoantigens by autoreactive cytotoxic CD8+ T cells is now understood to play a central role. Few treatment options are available, with the Janus kinase (JAK) 1/2 inhibitor baricitinib (2022) and the selective JAK3/tyrosine kinase expressed in hepatocellular carcinoma (TEC) inhibitor ritlecitinib (2023) being the only US Food and Drug Administration-approved systemic medications thus far for severe AA. Several other treatments are used off-label with limited efficacy and/or suboptimal safety and tolerability. With an increased understanding of the T-cell-mediated autoimmune and inflammatory pathogenesis of AA, additional therapeutic pathways beyond JAK inhibition are currently under investigation for the development of AA therapies. This narrative review presents a detailed overview about the role of T cells and T-cell-signaling pathways in the pathogenesis of AA, with a focus on those pathways targeted by drugs in clinical development for the treatment of AA. A detailed summary of new drugs targeting these pathways with expert commentary on future directions for AA drug development and the importance of targeting multiple T-cell-signaling pathways is also provided in this review.

Beyond the Epidermal-Melanin-Unit: The Human Scalp Anagen Hair Bulb Is Home to Multiple Melanocyte Subpopulations of Variable Melanogenic Capacity.

The visual appearance of humans is derived significantly from our skin and hair color. While melanin from epidermal melanocytes protects our skin from the damaging effects of ultraviolet radiation, the biological value of pigmentation in the hair follicle, particularly on the scalp, is less clear. In this study, we explore the heterogeneity of pigment cells in the human scalp anagen hair follicle bulb, a site conventionally viewed to be focused solely on pigment production for transfer to the hair shaft. Using c-KIT/CD117 microbeads, we isolated bulbar c-KIT-positive and c-KIT-negative melanocytes. While both subpopulations expressed MITF, only the c-KIT-positive fraction expressed SOX10. We further localized bulbar melanocyte subpopulations (expressing c-KIT, SOX10, MITF, and DCT) that exhibited distinct/variable expression of downstream differentiation-associated melanosome markers (e.g., gp100 and Melan-A). The localization of a second 'immature' SOX10 negative melanocyte population, which was c-KIT/MITF double-positive, was identified outside of the melanogenic zone in the most peripheral/proximal matrix. This study describes an approach to purifying human scalp anagen hair bulb melanocytes, allowing us to identify unexpected levels of melanocyte heterogeneity. The function of the more immature melanocytes in this part of the hair follicle remains to be elucidated. Could they be in-transit migratory cells ultimately destined to synthesize melanin, or could they contribute to the hair follicle in non-melanogenic ways?

Highlighting nuances of blue light phototherapy: Mechanisms and safety considerations.

The efficacy of blue light therapy in dermatology relies on numerous clinical studies. The safety remains a topic of controversy, where potentially deleterious effects were derived from in vitro rather than in vivo experiments. The objectives of this work were (1) to highlight the nuances behind "colors" of blue light, light propagation in tissue and the plurality of modes of action; and (2) to rigorously analyze studies on humans reporting both clinical and histological data from skin biopsies with focus on DNA damage, proliferation, apoptosis, oxidative stress, impact on collagen, elastin, immune cells, and pigmentation. We conclude that blue light therapy is safe for human skin. It induces intriguing skin pigmentation, in part mediated by photoreceptor Opsin-3, which might have a photoprotective effect against ultraviolet irradiation. Future research needs to unravel photochemical reactions and the most effective and safe parameters of blue light in dermatology.

Autoantigen Discovery in the Hair Loss Disorder, Alopecia Areata: Implication of Post-Translational Modifications.

Alopecia areata (AA) is a chronic, multifactorial, polygenic, and heterogeneous disorder affecting growing hair follicles in susceptible individuals, which results in a non-scarring and reversible hair loss with a highly unpredictable course. Despite very considerable research effort, the nature of the precipitating factor(s) responsible for initiating AA in any given hair follicle remains unclear, due largely to significant gaps in our knowledge of the precise sequence of the etiopathogenic events in this dermatosis. However, disease-related changes in the immune-competence of the lower growing hair follicle, together with an active immune response (humoral and cellular) to hair follicle-associated antigens, are key associated phenomena. Confirmation of the hair follicle antigen(s) implicated in AA disease onset has remained stubbornly elusive. While it may be considered somewhat philosophical by some, it is also unclear whether immune-mediated hair loss in AA results from a) an ectopic (i.e., in an abnormal location) immune response to native (unmodified) self-antigens expressed by the healthy hair follicle, b) a normal immune response against modified self-antigens (or neoantigens), or c) a normal immune response against self-antigens (modified/non-modified) that were not previously visible to the immune system (because they were conformationally-hidden or sequestered) but become exposed and presentable in an MHC-I/-II molecule-restricted manner. While some candidate hair follicle antigen target(s) in AA are beginning to emerge, with a potential role for trichohyalin, it is not yet clear whether this represents the initial and immunodominant antigenic focus in AA or is simply one of an expanding repertoire of exposed hair follicle tissue damage-associated antigens that are secondary to the disease. Confirmation of autoantigen identity is essential for our understanding of AA etiopathogenesis, and consequently for developing a more informed therapeutic strategy. Major strides have been made in autoantigen discovery in other autoimmune conditions. In particular, some of these conditions may provide insights into how post-translational modifications (e.g., citrullination, deamidation, etc.) of hair follicle-restricted proteins may increase their antigenicity and so help drive the anti-hair follicle immune attack in AA.

Loss of 'Epidermal Melanin Unit' Integrity in Human Skin During Melanoma-Genesis.

Cutaneous melanoma can be a most challenging neoplasm of high lethality, in part due to its extreme heterogeneity and characteristic aggressive and invasive nature. Indeed, its moniker 'the great masquerader' reflects that not all melanomas are created equal in terms of their originating cellular contexts, but also that melanoma cells in the malignant tumor can adopt a wide range of different cell states and variable organotropism. In this review, we focus on the early phases of melanomagenesis by discussing how the originating pigment cell of the melanocyte lineage can be influenced to embark on a wide range of tumor fates with distinctive microanatomical pathways. In particular, we assess how cells of the melanocyte lineage can differ by maturation status (stem cell; melanoblast; transiently amplifying cell; differentiated; post-mitotic; terminally-differentiated) as well as by micro-environmental niche (in the stratum basale of the epidermis; within skin appendages like hair follicle, eccrine gland, etc). We discuss how the above variable contexts may influence the susceptibility of the epidermal-melanin unit (EMU) to become unstable, which may presage cutaneous melanoma development. We also assess how unique features of follicular-melanin unit(s) (FMUs) can, by contrast, protect melanocytes from melanomagenesis. Lastly, we postulate how variable melanocyte fates in vitiligo, albinism, psoriasis, and alopecia areata may provide new insights into immune-/non immune-mediated outcomes for melanocytes in cutaneous melanin units.

Uveal Melanoma Cell Line Proliferation Is Inhibited by Ricolinostat, a Histone Deacetylase Inhibitor.

Metastatic uveal melanoma (MUM) is characterized by poor patient survival. Unfortunately, current treatment options demonstrate limited benefits. In this study, we evaluate the efficacy of ACY-1215, a histone deacetylase inhibitor (HDACi), to attenuate growth of primary ocular UM cell lines and, in particular, a liver MUM cell line in vitro and in vivo, and elucidate the underlying molecular mechanisms. A significant (p = 0.0001) dose-dependent reduction in surviving clones of the primary ocular UM cells, Mel270, was observed upon treatment with increasing doses of ACY-1215. Treatment of OMM2.5 MUM cells with ACY-1215 resulted in a significant (p = 0.0001), dose-dependent reduction in cell survival and proliferation in vitro, and in vivo attenuation of primary OMM2.5 xenografts in zebrafish larvae. Furthermore, flow cytometry revealed that ACY-1215 significantly arrested the OMM2.5 cell cycle in S phase (p = 0.0001) following 24 h of treatment, and significant apoptosis was triggered in a time- and dose-dependent manner (p < 0.0001). Additionally, ACY-1215 treatment resulted in a significant reduction in OMM2.5 p-ERK expression levels. Through proteome profiling, the attenuation of the microphthalmia-associated transcription factor (MITF) signaling pathway was linked to the observed anti-cancer effects of ACY-1215. In agreement, pharmacological inhibition of MITF signaling with ML329 significantly reduced OMM2.5 cell survival and viability in vitro (p = 0.0001) and reduced OMM2.5 cells in vivo (p = 0.0006). Our findings provide evidence that ACY-1215 and ML329 are efficacious against growth and survival of OMM2.5 MUM cells.

Learning from disease registries during a pandemic: Moving toward an international federation of patient registries.

High-quality dermatology patient registries often require considerable time to develop and produce meaningful data. Development time is influenced by registry complexity and regulatory hurdles that vary significantly nationally and institutionally. The rapid emergence of the coronavirus disease 2019 (COVID-19) global pandemic has challenged health services in an unprecedented manner. Mobilization of the dermatology community in response has included rapid development and deployment of multiple, partially harmonized, international patient registries, reinventing established patient registry timelines. Partnership with patient organizations has demonstrated the critical nature of inclusive patient involvement. This global effort has demonstrated the value, capacity, and necessity for the dermatology community to adopt a more cohesive approach to patient registry development and data sharing that can lead to myriad benefits. These include improved utilization of limited resources, increased data interoperability, improved ability to rapidly collect meaningful data, and shortened response times to generate real-world evidence. We call on the global dermatology community to support the development of an international federation of patient registries to consolidate and operationalize the lessons learned during this pandemic. This will provide an enduring means of applying this knowledge to the maintenance and development of sustainable, coherent, and impactful patient registries of benefit now and in the future.

Insights into the mechanics of solid conical microneedle array insertion into skin using the finite element method.

In order to develop optimum microneedle designs, researchers must first develop robust, repeatable and adaptable test methods which are representative of in vivo conditions. However, there is a lack of experimental tools which can accurately comparatively interrogate functional microneedle penetration of tissue. In this study, we seek to develop a state of the art finite element model of microneedle insertion into and penetration of human skin. The developed model employs a 3D hyperelastic, anisotropic pre-stressed multi-layered material which more accurately reflects in vivo skin conditions, while the microneedle is modeled as an array, which can capture the influence of adjacent microneedles on the overall response. Using the developed finite element model, we highlight the importance of accurate computational modeling which can decipher the mechanics of microneedle insertion, including the influence of its position within an array and how it correlates well with experimental observations. In particular, we have concluded that, for our model microneedle array, increasing skin pretension from 0 to 10% strain reduces the penetration force by 13%, ultimate local deformation about the microneedle by 22% and the ultimate penetration efficiency by 15%. We have also concluded that the presence of a base plate limits the penetration efficiency by up to 24%, while the penetration efficiency across a 5 × 1 microneedle array may vary by 27%. This model elucidates, for the first time, the combined effects of skin tension and needle geometry on accurately predicting microneedle penetration efficiency. STATEMENT OF SIGNIFICANCE: Microneedles arrays (MNAs) are medical devices with microscale protrusions, typically designed to penetrate the outermost layer of the skin, that upon optimisation, could lead to disruptive minimally-invasive disease management. However, the mechanics of MNA insertion are complex, due in part to a 'bed of nails' effect, and difficult to elucidate experimentally. Therefore, comparisons between designs, functional assessment of production batches and ultimately the likelihood of clinical translation are challenging to predict. Here, we have develop the most sophisticated in silico model of MNA insertion into pre-tensioned human skin to predict the extent of MNA penetration and therefore the likelihood of successful therapeutic delivery. Researchers can customise this model to predict the penetration efficiency of any MNA design.

Hidradenitis suppurativa: A folliculotropic disease of innate immune barrier dysfunction?

The innate immune system of human skin consists of a multi-layered barrier consisting of cells and soluble effector molecules charged with maintaining homeostasis and responding to insults and infections. It has become increasingly clear that these barrier layers become compromised in skin diseases, especially in disorders of an (auto)inflammatory nature. In the case of hidradenitis suppurativa, great strides have been made in recent years in characterizing the underlying breakdown in homeostatic innate immunity, including an increasing understanding of the central role of the hair follicle in this process. This breakdown appears to occur at multiple levels: the pilosebaceous unit, associated epithelium, the cutaneous microbiome, alteration of immune cell function and local molecular events such as complement activation. This review seeks to summarize, contextualize and analyse critically our current understanding of how these innate immune barriers become dysregulated in the early stage(s) of hidradenitis suppurativa, and to speculate on where potential hidradenitis suppurativa research could be most fruitful.

Quantitative mapping of human hair greying and reversal in relation to life stress.

Background: Hair greying is a hallmark of aging generally believed to be irreversible and linked to psychological stress. Methods: Here, we develop an approach to profile hair pigmentation patterns (HPPs) along individual human hair shafts, producing quantifiable physical timescales of rapid greying transitions. Results: Using this method, we show white/grey hairs that naturally regain pigmentation across sex, ethnicities, ages, and body regions, thereby quantitatively defining the reversibility of greying in humans. Molecularly, grey hairs upregulate proteins related to energy metabolism, mitochondria, and antioxidant defenses. Combining HPP profiling and proteomics on single hairs, we also report hair greying and reversal that can occur in parallel with psychological stressors. To generalize these observations, we develop a computational simulation, which suggests a threshold-based mechanism for the temporary reversibility of greying. Conclusions: Overall, this new method to quantitatively map recent life history in HPPs provides an opportunity to longitudinally examine the influence of recent life exposures on human biology. Funding: This work was supported by the Wharton Fund and NIH grants GM119793, MH119336, and AG066828 (MP).

Hair greying is a visible sign of aging that affects everyone. The loss of hair color is due to the loss of melanin, a pigment found in the skin, eyes and hair. Research in mice suggests stress may accelerate hair greying, but there is no definitive research on this in humans. This is because there are no research tools to precisely map stress and hair color over time. But, just like tree rings hold information about past decades, and rocks hold information about past centuries, hairs hold information about past months and years. Hair growth is an active process that happens under the skin inside hair follicles. It demands lots of energy, supplied by structures inside cells called mitochondria. While hairs are growing, cells receive chemical and electrical signals from inside the body, including stress hormones. It is possible that these exposures change proteins and other molecules laid down in the growing hair shaft. As the hair grows out of the scalp, it hardens, preserving these molecules into a stable form. This preservation is visible as patterns of pigmentation. Examining single-hairs and matching the patterns to life events could allow researchers to look back in time through a person's biological history. Rosenberg et al. report a new way to digitize and measure small changes in color along single human hairs. This method revealed that some white hairs naturally regain their color, something that had not been reported in a cohort of healthy individuals before. Aligning the hair pigmentation patterns with recent reports of stress in the hair donors' lives showed striking associations. When one donor reported an increase in stress, a hair lost its pigment. When the donor reported a reduction in stress, the same hair regained its pigment. Rosenberg et al. mapped hundreds of proteins inside the hairs to show that white hairs contained more proteins linked to mitochondria and energy use. This suggests that metabolism and mitochondria may play a role in hair greying. To explore these observations in more detail Rosenberg et al. developed a mathematical model that simulates the greying of a whole head of hair over a lifetime, an experiment impossible to do with living people. The model suggested that there might be a threshold for temporary greying; if hairs are about to go grey anyway, a stressful event might trigger that change earlier. And when the stressful event ends, if a hair is just above the threshold, then it could revert back to dark. The new method for measuring small changes in hair coloring opens up the possibility of using hair pigmentation patterns like tree rings. This could track the influence of past life events on human biology. In the future, monitoring hair pigmentation patterns could provide a way to trace the effectiveness of treatments aimed at reducing stress or slowing the aging process. Understanding how 'old' white hairs regain their 'young' pigmented state could also reveal new information about the malleability of human aging more generally.

Visible light and human skin pigmentation: The importance of skin phototype.

Melanin is synthesised within melanocytes and transferred to keratinocytes in human skin, thereby regulating skin colour and protecting skin cells against UVR-induced damage. We commonly divide human skin into six phototypes (SPT)-I to -VI (Fitzpatrick scale) according to the skin's tanning response to UVR. In this pilot study, we investigated the impact of UVR (maximum 311nm), blue (peak 450nm) and green visible light (peak 530nm) on melanin production and type in healthy human skin histocultures (SPT-I, -II and -III). UVR, blue and green light stimulated a surface tanning response in SPT-II and -III, but not SPT-I. Using the Warthin-Starry stain for sensitive melanin detection, all three light treatments induced melanogenesis in SPT-II and -III skin. Surprisingly, blue and green light (but not UVR) stimulated melanin synthesis in SPT-I skin. Moreover, melanin synthesis induced by blue and green visible light in SPT-I, SPT-II, and SPT-III skin was not associated with a detectable increase in DNA damage or cell apoptosis. By contrast, both responses were detected after UVR. These data suggest that blue and green visible light can stimulate melanin production in fair-skinned individuals without, at least some of, the harmful consequences of UVR-induced pigmentation. We are currently examining the molecular basis of UVR-independent melanogenesis in fair skin.

Shedding light on therapeutics in alopecia and their relevance to COVID-19.

As of July 9, 2020, there were more than 12 million confirmed cases of coronavirus disease 2019 (COVID-19) across the globe, with more than 550,000 deaths. Many European countries, including Belgium, the United Kingdom, Italy, and Spain, have had the highest numbers of fatalities per capita. This indicates the potential for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus to overwhelm even the most advanced health care systems despite extreme societal interventions. Since its emergence, SARS-CoV-2 has disseminated across the globe, affecting the structure of global societies, infrastructure, and economies. Patients with alopecia are a diverse group who, for various indications, are prescribed a number of antimicrobials and antiandrogen treatments in addition to immunomodulatory therapies such as hydroxychloroquine, oral corticosteroids, and a range of broad immunosuppressants. These drugs are being scrutinized for their capacity to potentially affect SARS-CoV-2 outcomes. We examine these treatments and highlight the critical role that patient registries will play in generating real-world evidence to assess their impact on COVID-19 outcomes.

Melanin Distribution in Human Skin: Influence of Cytoskeletal, Polarity, and Centrosome-Related Machinery of Stratum basale Keratinocytes.

Melanin granules cluster within supra-nuclear caps in basal keratinocytes (KCs) of the human epidermis, where they protect KC genomic DNA against ultraviolet radiation (UVR) damage. While much is known about melanogenesis in melanocytes (MCs) and a moderate amount about melanin transfer from MC to KC, we know little about the fate of melanin once inside KCs. We recently reported that melanin fate in progenitor KCs is regulated by rare asymmetric organelle movement during mitosis. Here, we explore the role of actin, microtubules, and centrosome-associated machinery in distributing melanin within KCs. Short-term cultures of human skin explants were treated with cytochalasin-B and nocodazole to target actin filaments and microtubules, respectively. Treatment effects on melanin distribution were assessed by the Warthin-Starry stain, on centrosome-associated proteins by immunofluorescence microscopy, and on co-localisation with melanin granules by brightfield microscopy. Cytochalasin-B treatment disassembled supra-nuclear melanin caps, while nocodazole treatment moved melanin from the apical to basal KC domain. Centrosome and centriolar satellite-associated proteins showed a high degree of co-localisation with melanin. Thus, once melanin granules are transferred to KCs, their preferred apical distribution appears to be facilitated by coordinated movement of centrosomes and centriolar satellites. This mechanism may control melanin's strategic position within UVR-exposed KCs.

Dermal fibroblasts cultured from donors with type 2 diabetes mellitus retain an epigenetic memory associated with poor wound healing responses.

The prevalence of Type 2 diabetes mellitus (T2DM) is escalating globally. Patients suffer from multiple complications including the development of chronic wounds that can lead to amputation. These wounds are characterised by an inflammatory environment including elevated tumour necrosis factor alpha (TNF-α). Dermal fibroblasts (DF) are critical for effective wound healing, so we sought to establish whether there were any differences in DF cultured from T2DM donors or those without diabetes (ND-DF). ND- and T2DM-DF when cultured similarly in vitro secreted comparable concentrations of TNF-α. Functionally, pre-treatment with TNF-α reduced the proliferation of ND-DF and transiently altered ND-DF morphology; however, T2DM-DF were resistant to these TNF-α induced changes. In contrast, TNF-α inhibited ND- and T2DM-DF migration and matrix metalloprotease expression to the same degree, although T2DM-DF expressed significantly higher levels of tissue inhibitor of metalloproteases (TIMP)-2. Finally, TNF-α significantly increased the secretion of pro-inflammatory cytokines (including CCL2, CXCL1 and SERPINE1) in ND-DF, whilst this effect in T2DM-DF was blunted, presumably due to the tendency to higher baseline pro-inflammatory cytokine expression observed in this cell type. Collectively, these data demonstrate that T2DM-DF exhibit a selective loss of responsiveness to TNF-α, particularly regarding proliferative and secretory functions. This highlights important phenotypic changes in T2DM-DF that may explain the susceptibility to chronic wounds in these patients.

The biology of human hair greying.

Hair greying (canities) is one of the earliest, most visible ageing-associated phenomena, whose modulation by genetic, psychoemotional, oxidative, senescence-associated, metabolic and nutritional factors has long attracted skin biologists, dermatologists, and industry. Greying is of profound psychological and commercial relevance in increasingly ageing populations. In addition, the onset and perpetuation of defective melanin production in the human anagen hair follicle pigmentary unit (HFPU) provides a superb model for interrogating the molecular mechanisms of ageing in a complex human mini-organ, and greying-associated defects in bulge melanocyte stem cells (MSCs) represent an intriguing system of neural crest-derived stem cell senescence. Here, we emphasize that human greying invariably begins with the gradual decline in melanogenesis, including reduced tyrosinase activity, defective melanosome transfer and apoptosis of HFPU melanocytes, and is thus a primary event of the anagen hair bulb, not the bulge. Eventually, the bulge MSC pool becomes depleted as well, at which stage greying becomes largely irreversible. There is still no universally accepted model of human hair greying, and the extent of genetic contributions to greying remains unclear. However, oxidative damage likely is a crucial driver of greying via its disruption of HFPU melanocyte survival, MSC maintenance, and of the enzymatic apparatus of melanogenesis itself. While neuroendocrine factors [e.g. alpha melanocyte-stimulating hormone (α-MSH), adrenocorticotropic hormone (ACTH), ß-endorphin, corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH)], and micropthalmia-associated transcription factor (MITF) are well-known regulators of human hair follicle melanocytes and melanogenesis, how exactly these and other factors [e.g. thyroid hormones, hepatocyte growth factor (HGF), P-cadherin, peripheral clock activity] modulate greying requires more detailed study. Other important open questions include how HFPU melanocytes age intrinsically, how psychoemotional stress impacts this process, and how current insights into the gerontobiology of the human HFPU can best be translated into retardation or reversal of greying.

Circulating Melanoma-Derived Extracellular Vesicles: Impact on Melanoma Diagnosis, Progression Monitoring, and Treatment Response.

Malignant melanoma, one of the most aggressive human malignancies, is responsible for 80% of skin cancer deaths. Whilst early detection of disease progression or metastasis can improve patient survival, this remains a challenge due to the lack of reliable biomarkers. Importantly, these clinical challenges are not unique to humans, as melanoma affects many other species, including companion animals, such as the dog and horse. Extracellular vesicles (EVs) are tiny nanoparticles involved in cell-to-cell communication. Several protein and genomic EV markers have been described in the literature, as well as a wide variety of methods for isolating EVs from body fluids. As such, they may be valuable biomarkers in cancer and may address some clinical challenges in the management melanoma. This review aimed to explore the translational applications of EVs as biomarkers in melanoma, as well as their role in the clinical setting in humans and animals. A summary of melanoma-specific protein and genomic EV markers is presented, followed by a discussion of the role EVs in monitoring disease progression and treatment response. Finally, herein, we reviewed the advantages and disadvantages of methods utilised to isolate EVs from bodily fluids in melanoma patients (human and animals) and describe some of the challenges that will need to be addressed before EVs can be introduced in the clinical setting.

Stress-sensing in the human greying hair follicle: Ataxia Telangiectasia Mutated (ATM) depletion in hair bulb melanocytes in canities-prone scalp.

Canities (or hair greying) is an age-linked loss of the natural pigment called melanin from hair. While the specific cause(s) underlying the loss of melanogenically-active melanocytes from the anagen hair bulbs of affected human scalp remains unclear, oxidative stress sensing appears to be a key factor involved. In this study, we examined the follicular melanin unit in variably pigmented follicles from the aging human scalp of healthy individuals (22-70 years). Over 20 markers were selected within the following categories: melanocyte-specific, apoptosis, cell cycle, DNA repair/damage, senescence and oxidative stress. As expected, a reduction in melanocyte-specific markers in proportion to the extent of canities was observed. A major finding of our study was the intense and highly specific nuclear expression of Ataxia Telangiectasia Mutated (ATM) protein within melanocytes in anagen hair follicle bulbs. ATM is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks and functions as an important sensor of reactive oxygen species (ROS) in human cells. The incidence and expression level of ATM correlated with pigmentary status in canities-affected hair follicles. Moreover, increased staining of the redox-associated markers 8-OHdG, GADD45 and GP-1 were also detected within isolated bulbar melanocytes, although this change was not clearly associated with donor age or canities extent. Surprisingly, we were unable to detect any specific change in the expression of other markers of oxidative stress, senescence or DNA damage/repair in the canities-affected melanocytes compared to surrounding bulbar keratinocytes. By contrast, several markers showed distinct expression of markers for oxidative stress and apoptosis/differentiation in the inner root sheath (IRS) as well as other parts of the hair follicle. Using our in vitro model of primary human scalp hair follicle melanocytes, we showed that ATM expression increased after incubation with the pro-oxidant hydrogen peroxide (H2O2). In addition, this ATM increase was prevented by pre-incubation of cells with antioxidants. The relationship between ATM and redox stress sensing was further evidenced as we observed that the inhibition of ATM expression by chemical inhibition promoted the loss of melanocyte viability induced by oxidative stress. Taken together these new findings illustrate the key role of ATM in the protection of human hair follicle melanocytes from oxidative stress/damage within the human scalp hair bulb. In conclusion, these results highlight the remarkable complexity and role of redox sensing in the status of human hair follicle growth, differentiation and pigmentation.

Ewastools: Infinium Human Methylation BeadChip pipeline for population epigenetics integrated into Galaxy.

BACKGROUND: Infinium Human Methylation BeadChip is an array platform for complex evaluation of DNA methylation at an individual CpG locus in the human genome based on Illumina's bead technology and is one of the most common techniques used in epigenome-wide association studies. Finding associations between epigenetic variation and phenotype is a significant challenge in biomedical research. The newest version, HumanMethylationEPIC, quantifies the DNA methylation level of 850,000 CpG sites, while the previous versions, HumanMethylation450 and HumanMethylation27, measured >450,000 and 27,000 loci, respectively. Although a number of bioinformatics tools have been developed to analyse this assay, they require some programming skills and experience in order to be usable. RESULTS: We have developed a pipeline for the Galaxy platform for those without experience aimed at DNA methylation analysis using the Infinium Human Methylation BeadChip. Our tool is integrated into Galaxy (http://galaxyproject.org), a web-based platform. This allows users to analyse data from the Infinium Human Methylation BeadChip in the easiest possible way. CONCLUSIONS: The pipeline provides a group of integrated analytical methods wrapped into an easy-to-use interface. Our tool is available from the Galaxy ToolShed, GitHub repository, and also as a Docker image. The aim of this project is to make Infinium Human Methylation BeadChip analysis more flexible and accessible to everyone.