A Bispecific, Tetravalent Antibody Targeting Inflammatory and Pruritogenic Pathways in Atopic Dermatitis.

Inhibition of IL-4/IL-13 signaling has dramatically improved the treatment of atopic dermatitis (AD). However, in many patients, clinical responses are slow to develop and remain modest. Indeed, some symptoms of AD are dependent on IL-31, which is only partially reduced by IL-4/IL-13 inhibition. Thus, there is an unmet need for AD treatments that concomitantly block IL-4/IL-13 and IL-31 pathways. We engineered NM26-2198, a bispecific tetravalent antibody designed to accomplish this task. In reporter cell lines, NM26-2198 concomitantly inhibited IL-4/IL-13 and IL-31 signaling with a potency comparable with that of the combination of an anti-IL-4Rα antibody (dupilumab) and an anti-IL-31 antibody (BMS-981164). In human PBMCs, NM26-2198 inhibited IL-4-induced upregulation of CD23, demonstrating functional binding to FcγRII (CD32). NM26-2198 also inhibited the secretion of the AD biomarker thymus and activation-regulated chemokine (TARC) in blood samples from healthy human donors. In male cynomolgus monkeys, NM26-2198 exhibited favorable pharmacokinetics and significantly inhibited IL-31-induced scratching at a dose of 30 mg/kg. In a repeat-dose, good laboratory practice toxicology study in cynomolgus monkeys, no adverse effects of NM26-2198 were observed at a weekly dose of 125 mg/kg. Together, these results justify the clinical investigation of NM26-2198 as a treatment for moderate-to-severe AD.

'Speed-ageing' of human skin in serum-free organ culture ex vivo: An instructive novel assay for preclinical human skin ageing research demonstrates senolytic effects of caffeine and 2,5-dimethylpyrazine.

Preclinical human skin ageing research has been limited by the paucity of instructive and clinically relevant models. In this pilot study, we report that healthy human skin of different age groups undergoes extremely accelerated ageing within only 3 days, if organ-cultured in a defined serum-free medium. Quantitative (immuno-)histomorphometry documented this unexpected ex vivo phenotype on the basis of ageing-associated biomarkers: the epidermis showed significantly reduced rete ridges and keratinocyte proliferation, sirtuin-1, MTCO1 and collagen 17a1 protein levels; this contrasted with significantly increased expression of the DNA-damage marker, γH2A.X. In the dermis, collagen 1 and 3 and hyaluronic acid content were significantly reduced compared to Day 0 skin. qRT-PCR of whole skin RNA extracts also showed up-regulated mRNA levels of several (inflamm-) ageing biomarkers (MMP-1, -2, -3, -9; IL6, IL8, CXCL10 and CDKN1). Caffeine, a methylxanthine with recognized anti-ageing properties, counteracted the dermal collagen 1 and 3 reduction, the epidermal accumulation of γH2A.X, and the up-regulation of CXCL10, IL6, IL8, MMP2 and CDKN1. Finally, we present novel anti-ageing effects of topical 2,5-dimethylpyrazine, a natural pheromone TRPM5 ion channel activator. Thus, this instructive, clinically relevant "speed-ageing" assay provides a simple, but powerful new research tool for dissecting skin ageing and rejuvenation, and is well-suited to identify novel anti-ageing actives directly in the human target organ.

Mechanical epilation exerts complex biological effects on human hair follicles and perifollicular skin: An ex vivo study approach.

OBJECTIVE: Electrical epilation of unwanted hair is a widely used hair removal method, but it is largely unknown how this affects the biology of human hair follicles (HF) and perifollicular skin. Here, we have begun to explore how mechanical epilation changes selected key biological read-out parameters ex vivo within and around the pilosebaceous unit. METHODS: Human full-thickness scalp skin samples were epilated ex vivo using an electro-mechanical device, organ-cultured for up to 6 days in serum-free, supplemented medium, and assessed at different time points by quantitative (immuno-)histomorphometry for selected relevant read-out parameters in epilated and sham-epilated control samples. RESULTS: Epilation removed most of the hair shafts, often together with fragments of the outer and inner root sheath and hair matrix. This was associated with persistent focal thinning of the HF basal membrane, decreased melanin content of the residual HF epithelium, and increased HF keratinocyte apoptosis, including in the bulge, yet without affecting the number of cytokeratin 15+ HF epithelial stem cells. Sebocyte apoptosis in the peripheral zone was increased, albeit without visibly altering sebum production. Epilation transiently perturbed HF immune privilege, and increased the expression of ICAM-1 in the bulge and bulb mesenchyme, and the number of perifollicular MHC class II+ cells as well as mast cells around the distal epithelium and promoted mast cell degranulation around the suprabulbar and bulbar area. Moreover, compared to controls, several key players of neurogenic skin inflammation, itch, and/or thermosensation (TRPV1, TRPA1, NGF, and NKR1) were differentially expressed in post-epilation skin. CONCLUSION: These data generated in denervated, organ-cultured human scalp skin demonstrate that epilation-induced mechanical HF trauma elicits surprisingly complex biological responses. These may contribute to the delayed re-growth of thinner and lighter hair shafts post-epilation and temporary post-epilation discomfort. Our findings also provide pointers regarding the development of topically applicable agents that minimize undesirable sequelae of epilation.

OBJECTIF: L'épilation électrique des poils indésirables est une méthode d'épilation largement utilisée, mais on ne connaît pas l'ampleur de son effet sur la biologie des follicules pileux humains (FP) et de la peau périfolliculaire. Dans cette étude, nous avons commencé à explorer comment l'épilation mécanique modifie certains paramètres de mesures biologiques clés ex vivo à l'intérieur et autour de l'unité pilo­sébacée. MÉTHODES: Des échantillons de peau du cuir chevelu humain de pleine épaisseur ont été épilés ex vivo à l'aide d'un dispositif électromécanique, cultivés biologiquement pendant un maximum de 6 jours dans un milieu complet sans sérum, et évalués à différents moments par (immuno­)histomorphométrie quantitative pour certains paramètres de mesures pertinents dans des échantillons avec épilation et des échantillons témoins avec épilation simulée. RÉSULTATS: L'épilation a enlevé la plupart des poils, souvent avec des fragments de la gaine de la racine externe et de la matrice pileuse. Cela a été associé à un amincissement focal persistant de la membrane basale du FP, à une diminution de la teneur en mélanine de l'épithélium résiduel du FP et à une augmentation de l'apoptose des kératinocytes du FP, y compris dans la surface arrondie, mais sans affecter le nombre de cellules souches épithéliales du FP positives pour la cytokératine 15. L'apoptose des sébocytes de la zone périphérique était augmentée, sans pour autant altérer visiblement la production de sébum. L'épilation a temporairement perturbé l'immunoprivilège du FP et a augmenté l'expression de l'ICAM­1 dans la surface arrondie et le mésenchyme du bulbe, ainsi que le nombre de cellules périfolliculaires du CMH de classe II et des mastocytes autour de l'épithélium distal, et a favorisé la dégranulation des mastocytes autour de la zone supra­bulbaire et bulbaire. En outre, par rapport aux échantillons témoins, plusieurs acteurs clés de l'inflammation neurogène cutanée, de la démangeaison et/ou de la thermosensation (TRPV1, TRPA1, NGF et NKR1) ont été exprimés de manière différentielle dans la peau après l'épilation. CONCLUSION: Ces données générées dans la peau du cuir chevelu humain dénervée et cultivée biologiquement démontrent que le traumatisme du FP induit par l'épilation mécanique provoque des réponses biologiques étonnamment complexes. Celles­ci peuvent contribuer à retarder la repousse des poils plus fins et plus clairs après l'épilation, et à provoquer une gêne temporaire après l'épilation. Nos résultats fournissent également des pistes concernant le développement d'agents applicables par voie topique qui minimisent les séquelles indésirables de l'épilation.

Management of the human hair follicle microbiome by a synthetic odorant.

BACKGROUND: Human scalp hair follicles (HFs) engage in olfactory receptor (OR)-dependent chemosensation. Activation of olfactory receptor family 2 subfamily AT member 4 (OR2AT4) by the synthetic, sandalwood-like odorant Sandalore® up-regulated HF antimicrobial peptide expression of dermcidin (DCD), which had previously been thought to be produced exclusively by sweat and sebaceous glands. OBJECTIVES: To understand if intrafollicular DCD production can be stimulated by a commonly used cosmetic odorant, thus altering human HF microbiome composition in a clinically beneficial manner. METHODS: DCD expression was compared between fresh-frozen scalp biopsies and microdissected, full-length scalp HFs, organ-cultured in the presence/absence of the OR2AT4 agonist, Sandalore® and/or antibiotics and/or the competitive OR2AT4 antagonist, Phenirat®. Amplicon-based sequencing and microbial growth assays were performed to assess how this treatment affected the HF microbiome. RESULTS: Synthetic odorant treatment upregulated epithelial DCD expression and exerted antimicrobial activity in human HFs ex vivo. Combined antibiotic and odorant treatment, during an ex vivo dysbiosis event, prevented HF tissue damage and favoured a more physiological microbiome composition. Sandalore®-conditioned medium, containing higher DCD content, favoured Staphylococcus epidermidis and Malassezia restricta over S. aureus and M. globosa, while exhibiting antimicrobial activity against Cutibacterium acnes. These effects were reversed by co-administration of Phenirat®. CONCLUSIONS: We provide the first proof-of-principle that a cosmetic odorant impacts the human HF microbiome by up-regulating antimicrobial peptide production in an olfactory receptor-dependent manner. Specifically, a synthetic sandalwood-like odorant stimulates intrafollicular DCD production, likely via OR2AT4, and thereby controls microbial overgrowth. Thus, deserving further exploration as an adjuvant therapeutic principle in the management of folliculitis and dysbiosis-associated hair diseases.

Dominant frontonasal dysplasia with ectodermal defects results from increased activity of ALX4.

Frontonasal dysplasia (FND) refers to a group of rare developmental disorders characterized by abnormal morphology of the craniofacial region. We studied a family manifesting with clinical features typical for FND2 including neurobehavioral abnormalities, hypotrichosis, hypodontia, and facial dysmorphism. Whole-exome sequencing analysis identified a novel heterozygous frameshift insertion in ALX4 (c.985_986insGTGC, p.Pro329Argfs*115), encoding aristaless homeobox 4. This and a previously reported dominant FND2-causing variant are predicted to result in the formation of a similar abnormally elongated protein tail domain. Using a reporter assay, we showed that the elongated ALX4 displays increased activity. ALX4 negatively regulates the Wnt/ß-catenin pathway and accordingly, patient keratinocytes showed altered expression of genes associated with the WNT/ß-catenin pathway, which in turn may underlie ectodermal manifestations in FND2. In conclusion, dominant FND2 with ectodermal dysplasia results from frameshift variants in ALX4 exerting a gain-of-function effect.

Preliminary evidence that Merkel cells exert chemosensory functions in human epidermis.

The mechanotransduction of light-touch sensory stimuli is considered to be the main physiological function of epidermal Merkel cells (MCs). Recently, however, MCs have been demonstrated to be also thermo-sensitive, suggesting that their role in skin physiologically extends well beyond mechanosensation. Here, we demonstrate that in healthy human skin epidermal MCs express functional olfactory receptors, namely OR2AT4, just like neighbouring keratinocytes. Selective stimulation of OR2AT4 by topical application of the synthetic odorant, Sandalore®, significantly increased Piccolo protein expression in MCs, as assessed by quantitative immunohistomorphometry, indicating increased vesicle trafficking and recycling, and significantly reduced nerve growth factor (NGF) immunoreactivity within MCs, possibly indicating increased neurotrophin release upon OR2AT4 activation. Live-cell imaging showed that Sandalore® rapidly induces a loss of FFN206-dependent fluorescence in MCs, suggesting OR2AT4-dependent MC depolarization and subsequent vesicle secretion. Yet, in contrast to keratinocytes, OR2AT4 stimulation by Sandalore® altered neither the number nor the proliferation status of MCs. These preliminary ex vivo findings demonstrate that epidermal MCs also exert OR-dependent chemosensory functions in human skin, and invite one to explore whether these newly identified properties are dysregulated in selected skin disorders, for example, in pruritic dermatoses, and if these novel MC functions can be therapeutically targeted to maintain/promote skin health.

Differential regulation of IL-17A and IL-17F via STAT5 contributes to psoriatic disease.

BACKGROUND: IL-17A plays a pivotal pathogenic role in several immune-mediated inflammatory diseases. Despite sharing 50% sequence homology with IL-17A, the role of IL-17F remains less clear. Clinical findings suggest that dual inhibition of IL-17A and IL-17F in psoriatic disease is more efficacious than IL-17A inhibition alone, positing a pathogenic role for IL-17F. OBJECTIVE: We characterized the regulation of IL-17A and IL-17F in psoriatic disease. METHODS: Using both in vitro systems and lesional skin tissue from patients, we interrogated the chromosomal, transcriptional, and protein expression landscape of IL-17A+ and IL-17F+ TH17 cells. Alongside established assays such as single-cell RNA sequencing, we developed a novel cytokine-capture technique that was combined with chromatin immunoprecipitation sequencing and RNA sequencing. RESULTS: We confirm a preferential elevation of IL-17F over IL-17A in psoriatic disease and show that expression of each isoform predominantly occurs in distinct cell populations. The expression of both IL-17A and IL-17F exhibited a high degree of plasticity, with the balance between the 2 isoforms influenced by proinflammatory signaling and by anti-inflammatory drugs such as methylprednisolone. This plasticity was reflected in a broad H3K4me3 region at the IL17A-F locus, while opposing effects of STAT5/IL-2 signaling were observed for each of the 2 genes. Functionally, higher IL17F expression was linked to greater cell proliferation. CONCLUSION: There are key differences in the regulation of IL-17A and IL-17F in psoriatic disease, leading to distinct inflammatory cell populations. As such, we propose that both IL-17A and IL-17F neutralization may be required to maximally inhibit IL-17-driven pathology.

Involvement of ILC1-like innate lymphocytes in human autoimmunity, lessons from alopecia areata.

Here, we have explored the involvement of innate lymphoid cells-type 1 (ILC1) in the pathogenesis of alopecia areata (AA), because we found them to be significantly increased around lesional and non-lesional HFs of AA patients. To further explore these unexpected findings, we first co-cultured autologous circulating ILC1-like cells (ILC1lc) with healthy, but stressed, organ-cultured human scalp hair follicles (HFs). ILClc induced all hallmarks of AA ex vivo: they significantly promoted premature, apoptosis-driven HF regression (catagen), HF cytotoxicity/dystrophy, and most important for AA pathogenesis, the collapse of the HFs physiological immune privilege. NKG2D-blocking or IFNγ-neutralizing antibodies antagonized this. In vivo, intradermal injection of autologous activated, NKG2D+/IFNγ-secreting ILC1lc into healthy human scalp skin xenotransplanted onto SCID/beige mice sufficed to rapidly induce characteristic AA lesions. This provides the first evidence that ILC1lc, which are positive for the ILC1 phenotype and negative for the classical NK markers, suffice to induce AA in previously healthy human HFs ex vivo and in vivo, and further questions the conventional wisdom that AA is always an autoantigen-dependent, CD8 +T cell-driven autoimmune disease.

Autologous Th2-polarized lymphocytes induce atopic dermatitis lesions in non-atopic human skin xenotransplants.

BACKGROUND: The key signals that suffice to induce atopic dermatitis (AD) in human skin remain incompletely understood. Also, current mouse models reflect human AD only unsatisfactorily. Therefore, we have asked whether a humanized AD mouse model can be developed that reflects human AD more faithfully and permit to identify key signals that suffice to induce AD lesions in previously healthy human skin in vivo. METHODS: Healthy human skin from non-atopic donors was transplanted onto SCID/beige mice. After xenotransplant reinnervation by mouse sensory nerve fibers had occurred, mixed autologous human Th2 CD4+ and Tc2 CD8+ T cells that had been pretreated in vitro with IL-2, IL-4, and LPS were injected intradermally into the xenotransplants without skin barrier disruption. RESULTS: Injected non-atopic xenotransplants rapidly developed a morphological, functional, and immunological phenocopy of human AD lesions regarding skin barrier defects, immunopathology including intraepidermal eosinophils, mast cell activation, increase of thymic stromal lymphopoietin, eotaxin-1 and type 2 cytokine circuits, and even showed characteristic neuroimmunological abnormalities such as ß2-adrenergic receptor downregulation. The experimentally induced AD lesions in human skin responded to standard AD therapy (topical dexamethasone or tacrolimus; systemic anti-IL-4Rα antibody [dupilumab]), and relapsed when neurogenic skin inflammation was induced by exposing mice to perceived stress. CONCLUSIONS: This new animal model uniquely mimics the complexity of human AD and its clinical response to standard therapy and psychoemotional stressors in vivo, and shows that Th2-polarized lymphocytes associated with excessive IL-4Rα-mediated signaling suffice to induce human AD skin lesions, while atopy and epidermal barrier disruption are dispensable.

Application of Topical Sandalore® Increases Epidermal Dermcidin Synthesis in Organ-Cultured Human Skin ex vivo.

INTRODUCTION: Several olfactory receptors (ORs) are expressed in human skin, where they regulate skin pigmentation, barrier function, wound healing, and hair growth. Previously, we found that the selective activation of OR family 2 subfamily AT member 4 (OR2AT4) by the synthetic, sandalwood-like odorant Sandalore® differentially stimulates the expression of antimicrobial peptides (AMPs) in human scalp hair follicle epithelium ex vivo. As OR2AT4 is also expressed by epidermal keratinocytes, we hypothesized that it may modulate intraepidermal AMP synthesis, thereby contributing to skin microbiome management. METHODS: We investigated this hypothesis in organ-cultured human skin in the presence of Sandalore® and antibiotics and evaluated epidermal production of two AMPs, LL37 (cathelicidin) and dermcidin (DCD), as well as OR2AT4, by quantitative immunohistomorphometry. Moreover, we quantified DCD secretion into the culture medium by ELISA and studied the effect of culture medium on selected bacterial and fungal strains. RESULTS: Topical application of Sandalore®to organ-cultured human skin increased OR2AT4 protein expression, the number of DCD-positive intraepidermal cells, and DCD secretion into culture media, without significantly affecting epidermal LL37 expression. In line with the significantly increased secretion of DCD into the culture medium, we demonstrated, in a spectrophotometric assay, that application of conditioned media from Sandalore®-treated skin promotes Staphylococcus epidermidis, Malassezia restricta, and, minimally, Cutibacterium acnes and inhibits Staphylococcus aureus growth. CONCLUSION: In addition to demonstrating for the first time that DCD can be expressed by epidermal keratinocytes, our pilot study suggests that topical treatment of human skin with a cosmetic odorant (Sandalore®) has the potential to alter the composition of the human skin microbiome through the selective upregulation of DCD. If confirmed, Sandalore® could become an attractive adjuvant, nondrug treatment for dermatoses characterized by dysbiosis due to overgrowth of S. aureus and Malassezia, such as atopic dermatitis and seborrheic dermatitis.

Intermediate Hair Follicles from Patients with Female Pattern Hair Loss Are Associated with Nutrient Insufficiency and a Quiescent Metabolic Phenotype.

Female pattern hair loss (FPHL) is a non-scarring alopecia resulting from the progressive conversion of the terminal (t) scalp hair follicles (HFs) into intermediate/miniaturized (i/m) HFs. Although data supporting nutrient deficiency in FPHL HFs are lacking, therapeutic strategies are often associated with nutritional supplementation. Here, we show by metabolic analysis that selected nutrients important for hair growth such as essential amino acids and vitamins are indeed decreased in affected iHFs compared to tHFs in FPHL scalp skin, confirming nutrient insufficiency. iHFs also displayed a more quiescent metabolic phenotype, as indicated by altered metabolite abundance in freshly collected HFs and release/consumption during organ culture of products/substrates of TCA cycle, aerobic glycolysis, and glutaminolysis. Yet, as assessed by exogenous nutrient supplementation ex vivo, nutrient uptake mechanisms are not impaired in affected FPHL iHFs. Moreover, blood vessel density is not diminished in iHFs versus tHFs, despite differences in tHFs from different FPHL scalp locations or versus healthy scalp or changes in the expression of angiogenesis-associated growth factors. Thus, our data reveal that affected iHFs in FPHL display a relative nutrient insufficiency and dormant metabolism, but are still capable of absorbing nutrients, supporting the potential of nutritional supplementation as an adjunct therapy for FPHL.

Human organ rejuvenation by VEGF-A: Lessons from the skin.

Transplanting aged human skin onto young SCID/beige mice morphologically rejuvenates the xenotransplants. This is accompanied by angiogenesis, epidermal repigmentation, and substantial improvements in key aging-associated biomarkers, including ß-galactosidase, p16ink4a, SIRT1, PGC1α, collagen 17A, and MMP1. Angiogenesis- and hypoxia-related pathways, namely, vascular endothelial growth factor A (VEGF-A) and HIF1A, are most up-regulated in rejuvenated human skin. This rejuvenation cascade, which can be prevented by VEGF-A-neutralizing antibodies, appears to be initiated by murine VEGF-A, which then up-regulates VEGF-A expression/secretion within aged human skin. While intradermally injected VEGF-loaded nanoparticles suffice to induce a molecular rejuvenation signature in aged human skin on old mice, VEGF-A treatment improves key aging parameters also in isolated, organ-cultured aged human skin, i.e., in the absence of functional skin vasculature, neural, or murine host inputs. This identifies VEGF-A as the first pharmacologically pliable master pathway for human organ rejuvenation in vivo and demonstrates the potential of our humanized mouse model for clinically relevant aging research.

An extract of Leontopodium alpinum inhibits catagen development ex vivo and increases hair density in vivo.

OBJECTIVES: Hair loss and reduction in hair volume are hallmarks of hair disorders, such as telogen effluvium, or male or female pattern hair loss, and hair ageing, which can cause severe distress in both men and women. Common anti-hair loss drugs carry some side effects; therefore, novel, safer approaches targeting milder phenotypes are highly advocated. In this context, we investigated an extract of the alpine plant Edelweiss, Leontopodium alpinum var. Helvetia, for its ability to modulate hair follicle (HF) growth ex vivo and inhibit hair loss while increasing hair regeneration in vivo. METHODS: Human amputated HFs were microdissected from three donors, two women and one man, and cultured ex vivo for 6 days. After treatment with 0.001% Edelweiss extract (EWDE), we investigated hair shaft production and anagen/catagen conversion, and measured known parameters associated with hair growth, that is hair matrix keratinocyte proliferation and apoptosis, dermal papilla inductivity, and growth factors, by quantitative (immuno)histomorphometry. To assess the anti-hair loss potential of the alpine plant compound, we performed a randomized, placebo-controlled human study enrolling Caucasian women and men, aged 18 to 65 years, with normal hair loss. After 5 months' daily use of an extract containing leave-on serum, we analysed hair density and anagen-to-catagen/telogen ratio by the Trichogram analysis. RESULTS: Our results revealed a significant prolongation in the anagen phase in HFs treated with 0.001% Edelweiss, as indicated by an increase in HFs remaining in anagen and a significant decrease in hair cycle score. In line with this effect, EWDE significantly stimulated hair matrix (HM) keratinocyte proliferation, and dermal papilla inductivity, as shown by a significant up-regulation of versican expression and alkaline phosphatase activity, and a tendential increase in FGF7 immunoreactivity in the dermal papilla of all HFs or only anagen VI HFs. Corroborating the ex vivo results, we observed a significant increase in growing hair shaft numbers (hair density) after treatment with Edelweiss extract formulation, and a tendential up-regulation in the anagen-to-catagen/telogen ratio. CONCLUSIONS: We show here, through several lines of evidence, that the selected extract of the alpine plant Leontopodium alpinum var Helvetia (Edelweiss) inhibits premature catagen induction, possibly by stimulating dermal papilla inductivity. It is therefore worth exploiting this extract clinically as an anti-hair loss agent, both for preventing ageing-associated hair shedding and as an adjuvant therapy for hair loss disorders.

OBJECTIFS: La perte de cheveux et la réduction du volume des cheveux sont caractéristiques des troubles capillaires, tels que l'effluvium télogène, ou la calvitie chez l'homme ou la femme, et le vieillissement des cheveux, qui peuvent causer une certaine détresse chez les hommes et les femmes. Les médicaments courants contre la chute des cheveux ont des effets secondaires, par conséquent, de nouvelles approches plus sûres ciblant des phénotypes légers sont fortement recommandées. Dans ce contexte, nous avons étudié un extrait de la plante alpine Edelweiss, Leontopodium alpinum var. Helvetia, pour sa capacité à stimuler la croissance du follicule pileux (HF) ex vivo et à inhiber la chute des cheveux tout en augmentant la régénération des fibres capillaires in vivo. MÉTHODES: Les follicules pileux (HF) humains prélevés ont été microdisséqués chez trois donneurs, deux femmes et un homme, et cultivés ex vivo pendant 6 jours. Après le traitement avec l'extrait d'Edelweiss à 0,001 % (EWDE), nous avons étudié la production de fibre capillaire et la conversion anagène/catagène, ainsi que mesuré les paramètres connus associés à la croissance des cheveux, à savoir, la prolifération des kératinocytes dans la matrice capillaire et l'apoptose, l'induction des papilles dermiques, et des facteurs de croissance, par (immuno-)histomorphométrie quantitative. Pour évaluer le potentiel des propriétés anti-chute du cheveu de l'extrait de plante alpine, nous avons réalisé une étude clinique aléatoire avec placebo, sur des femmes et des hommes de type caucasien âgés de 18 à 65 ans présentant une perte de cheveux normale. Après cinq mois d'utilisation quotidienne d'un sérum sans rinçage contenant l'extrait de plante, nous avons analysé la densité capillaire et le rapport anagène à catagène/télogène par trichogramme. RÉSULTATS: Nos résultats ont révélé une prolongation significative de la phase anagène dans les HF traités avec 0,001% d'Edelweiss, comme l'indique une augmentation des HF restant en phase anagène et une diminution significative du « hair cycle score ¼. En ligne avec cet effet, EWDE a stimulé de façon significative la matrice du cheveux (HM), la prolifération des kératinocytes, et l'induction de la papille dermique, comme le montre une augmentation significative de l'expression du versican et de l'activité de la phosphatase alcaline, et une augmentation tendancielle de l'immunoréactivité FGF7 dans la papille dermique de tous les HF ou seulement des HF anagènes VI. Corroborant les résultats ex vivo, nous avons observé une augmentation significative du nombre de fibres capillaires (densité de cheveux) après le traitement avec la formulation d'extrait d'Edelweiss, et une augmentation tendancielle dans le rapport anagène à catagène/télogène. CONCLUSIONS: Nous montrons ici, à travers plusieurs éléments de preuve, que l'extrait sélectionné de la plante alpine Leontopodium alpinum var Helvetia (Edelweiss) inhibe l'induction prématurée de la phase catagène, en stimulant la papille dermique. Il est donc possible d'utiliser cet extrait comme un agent anti-chute, à la fois pour prévenir la chute des cheveux associée au vieillissement mais aussi comme une thérapie complémentaire pour les troubles liés à la perte des cheveux.

Human hair follicles operate an internal Cori cycle and modulate their growth via glycogen phosphorylase.

Hair follicles (HFs) are unique, multi-compartment, mini-organs that cycle through phases of active hair growth and pigmentation (anagen), apoptosis-driven regression (catagen) and relative quiescence (telogen). Anagen HFs have high demands for energy and biosynthesis precursors mainly fulfilled by aerobic glycolysis. Histochemistry reports the outer root sheath (ORS) contains high levels of glycogen. To investigate a functional role for glycogen in the HF we quantified glycogen by Periodic-Acid Schiff (PAS) histomorphometry and colorimetric quantitative assay showing ORS of anagen VI HFs contained high levels of glycogen that decreased in catagen. qPCR and immunofluorescence microscopy showed the ORS expressed all enzymes for glycogen synthesis and metabolism. Using human ORS keratinocytes (ORS-KC) and ex vivo human HF organ culture we showed active glycogen metabolism by nutrient starvation and use of a specific glycogen phosphorylase (PYGL) inhibitor. Glycogen in ORS-KC was significantly increased by incubation with lactate demonstrating a functional Cori cycle. Inhibition of PYGL significantly stimulated the ex vivo growth of HFs and delayed onset of catagen. This study defines translationally relevant and therapeutically targetable new features of HF metabolism showing that human scalp HFs operate an internal Cori cycle, synthesize glycogen in the presence of lactate and modulate their growth via PYGL activity.

Evidence for a functional interaction of WNT10A and EBF1 in male-pattern baldness.

More than 300 genetic risk loci have been identified for male pattern baldness (MPB) but little is known about the exact molecular mechanisms through which the associated variants exert their effects on MPB pathophysiology. Here, we aimed at further elucidating the regulatory architecture of the MPB risk locus on chromosome (chr.) 2q35, where we have previously reported a regulatory effect of the MPB lead variant on the expression of WNT10A. A HaploReg database research for regulatory annotations revealed that the association signal at 2q35 maps to a binding site for the transcription factor EBF1, whose gene is located at a second MPB risk locus on chr. 5q33.3. To investigate a potential interaction between EBF1 and WNT10A during MPB development, we performed in vitro luciferase reporter assays as well as expression analyses and immunofluorescence co-stainings in microdissected human hair follicles. Our experiments confirm that EBF1 activates the WNT10A promoter and that the WNT10A/EBF1 interaction is impacted by the allelic expression of the MPB risk allele at 2q35. Expression analyses across different hair cycle phases and immunhistochemical (co)stainings against WNT10A and EBF1 suggest a predominant relevance of EBF1/WNT10A interaction for hair shaft formation during anagen. Based on these findings we suggest a functional mechanism at the 2q35 risk locus for MPB, where an MPB-risk allele associated reduction in WNT10A promoter activation via EBF1 results in a decrease in WNT10A expression that eventually results in anagen shortening, that is frequently observed in MPB affected hair follicles. To our knowledge, this study is the first follow-up study on MPB that proves functional interaction between two MPB risk loci and sheds light on the underlying pathophysiological mechanism at these loci.

Resident human dermal γδT-cells operate as stress-sentinels: Lessons from the hair follicle.

Murine γδT-cells have stress-surveillance functions and are implicated in autoimmunity. Yet, whether human γδT-cells are also stress sentinels and directly promote autoimmune responses in the skin is unknown. Using a novel (mini-)organ assay, we tested if human dermis resident γδT-cells can recognize stressed human scalp hair follicles (HFs) to promote an alopecia areata (AA)-like autoimmune response. Accordingly, we show that γδT-cells from healthy human scalp skin are activated (CD69+), up-regulate the expression of NKG2D and IFN-γ, and become cytotoxic when co-cultured with autologous stressed HFs ex vivo. These autologous γδT-cells induce HF immune privilege collapse, dystrophy, and premature catagen, i.e. three hallmarks of the human autoimmune HF disorder, AA. This is mediated by CXCL12, MICA, and in part by IFN-γ and CD1d. In conclusion, human dermal γδT-cells exert physiological stress-sentinel functions in human skin, where their excessive activity can promote autoimmunity towards stressed HFs that overexpress CD1d, CXCL12, and/or MICA.

Topical odorant application of the specific olfactory receptor OR2AT4 agonist, Sandalore® , improves telogen effluvium-associated parameters.

BACKGROUND: Human hair follicles (HFs) express the olfactory receptor (OR)2AT4, which is selectively stimulated by the synthetic sandalwood-like odorant, Sandalore® . In organ-cultured, human scalp HFs, Sandalore® prolongs anagen and suppresses apoptosis by up-regulating intrafollicular IGF-1 mediated signaling. AIMS: The objective of this study is to demonstrate whether effects of Sandalore® observed ex vivo translate into a clinically relevant effect in patients with telogen effluvium. PATIENTS/METHODS: In a randomized, double-blinded, placebo-controlled, clinical trial, 60 female volunteers (18-65 years) affected by telogen effluvium received over a period of 24 weeks treatment with either 1% Sandalore® solution (n = 30) or placebo (identically smelling, but non-OR2AT4 activating sandalwood oil n = 30). The study read-out parameters were the degree of hair shedding, hair volume, terminal/vellus hair ratio, anagen/catagen-telogen ratio, and patient self-assessment. RESULTS: Sandalore® 1% ameliorated clinical signs of telogen effluvium, namely it reduced hair shedding, and increased hair volume and the percentage of anagen HFs, the latter two parameters significantly more than placebo when changes were calculated to baseline. Sandalore® also increased the ratio of terminal/vellus hairs at week 8. Most of the anti-hair shedding effects were seen after 8 weeks and maintained at week 24. Patient questionnaire showed that verum group patients were more satisfied than the placebo group in regard to the overall results. CONCLUSION: This clinical trial supports previous findings of anagen-prolonging effects of Sandalore® ex vivo with similar results now reproduced in clinical practice. It also provides proof-of-principle that a topically applied cosmetic odorant acting through HF olfactory receptors can be a therapeutic alternative to treat hair loss disorders characterized by excessive hair shedding such as telogen effluvium.

Pro-inflammatory Vδ1+T-cells infiltrates are present in and around the hair bulbs of non-lesional and lesional alopecia areata hair follicles.

BACKGROUND: It is widely accepted that NKG2D+cells are critically involved in alopecia areata (AA) pathogenesis. However, besides being expressed in CD8+T-cells and NK cells, NKG2D is also found in human γδT-cells. AA lesional hair follicles (HFs) overexpress NKG2D and γδTCR activating ligands, e.g. MICA and CD1d, and chemoattractants for γδT-cells, such as CXCL10. OBJECTIVE: To investigate whether abnormal activities of γδT-cells may be involved in AA pathogenesis. METHODS: We analyzed the number and activation status of γδT-cells in human healthy, lesional and non-lesional AA scalp biopsies by FACS and/or quantitative (immuno-)histomorphometry. RESULTS: In healthy human scalp skin, the few skin-resident γδT-cells were found to be mostly Vδ1+, non-activated (CD69-NKG2Ddim) and positive for CXCL10, and CXCL12 receptors. These Vδ1+T-cells predominantly localized in/around the HF infundibulum. In striking contrast, the number of Vδ1+T-cells was significantly higher around and even inside the proximal (suprabulbar and bulbar) epithelium of lesional AA HFs. These cells also showed a pro-inflammatory phenotype, i.e. higher NKG2D, and IFN-γ and lower CD200R expression. Importantly, more pro-inflammatory Vδ1+T-cells were seen also around non-lesional AA HFs. Lesional AA HFs also showed significantly higher expression of CXCL12. CONCLUSION: Our pilot study introduces skin-resident γδT-cells as a previously overlooked, but potentially important, mostly (auto-)antigen-independent, new innate immunity protagonist in AA pathobiology. The HF infiltration of these activated, IFN-γ-releasing cells already around non-lesional AA HFs suggest that Vδ1+T-cells are involved in the early stages of human AA pathobiology, and may thus deserve therapeutic targeting for optimal AA management.