Signalling by senescent melanocytes hyperactivates hair growth.

Niche signals maintain stem cells in a prolonged quiescence or transiently activate them for proper regeneration1. Altering balanced niche signalling can lead to regenerative disorders. Melanocytic skin nevi in human often display excessive hair growth, suggesting hair stem cell hyperactivity. Here, using genetic mouse models of nevi2,3, we show that dermal clusters of senescent melanocytes drive epithelial hair stem cells to exit quiescence and change their transcriptome and composition, potently enhancing hair renewal. Nevus melanocytes activate a distinct secretome, enriched for signalling factors. Osteopontin, the leading nevus signalling factor, is both necessary and sufficient to induce hair growth. Injection of osteopontin or its genetic overexpression is sufficient to induce robust hair growth in mice, whereas germline and conditional deletions of either osteopontin or CD44, its cognate receptor on epithelial hair cells, rescue enhanced hair growth induced by dermal nevus melanocytes. Osteopontin is overexpressed in human hairy nevi, and it stimulates new growth of human hair follicles. Although broad accumulation of senescent cells, such as upon ageing or genotoxic stress, is detrimental for the regenerative capacity of tissue4, we show that signalling by senescent cell clusters can potently enhance the activity of adjacent intact stem cells and stimulate tissue renewal. This finding identifies senescent cells and their secretome as an attractive therapeutic target in regenerative disorders.

Allergen desensitization reduces the severity of relapsed alopecia areata in dust-mite allergic patients.

Atopy may be a facilitating factor in some alopecia areata (AA) patients with early disease onset and more severe/extensive AA. The underlying immune mechanisms are unknown, but allergen responses may support a pro-inflammatory environment that indirectly promotes AA. To investigate the long-term effect of allergen immunotherapy (AIT) against house dust mite (HDM) allergy on disease severity and prognosis for AA patients. An observational comparative effectiveness study was conducted on 69 AA patients with HDM allergy. 34 patients received conventional/traditional AA treatment (TrAA) plus AIT (AIT-TrAA), and 35 patients received TrAA alone. Serum total immunoglobulin E (tIgE), HDM specific IgE (sIgE), HDM specific IgG4 (sIgG4) and cytokines (IL-4, IL-5, IL-10, IL-12, IL-13, IL-33, IFNγ) were quantified in these patients, together with 58 non-allergic AA patients and 40 healthy controls. At the end of the 3-year desensitization course, the AIT-TrAA group presented with lower SALT scores than the TrAA group, especially in non-alopecia totalis/universalis (AT/U) patients and pre-adolescent AT/U patients (age ≤ 14). In patients with elevated tIgE levels before AIT, a decrease in tIgE was correlated to reduced extent of AA on completion of the AIT course. After desensitization, elevation of IL-5 and decrease of IL-33 were observed in HDM allergic-AA patients. Desensitization to HDM in allergic AA patients reduces the severity of relapse-related hair loss over the 3-year AIT treatment course, possibly via opposing Th2 dominance. This adjunctive treatment may help reduce disease severity and curtail the disease process in allergic patients with AA.

Hair follicle immune privilege and its collapse in alopecia areata.

Anagen stage hair follicles (HFs) exhibit "immune privilege (IP)" from the level of the bulge downwards to the bulb. Both passive and active IP mechanisms protect HFs from physiologically undesired immune responses and limit immune surveillance. IP is relative, not absolute, and is primarily based on absent, or greatly reduced, intra-follicular antigen presentation via MHC class I and II molecules, along with prominent expression of "no danger" signals like CD200 and the creation of an immunoinhibitory signalling milieu generated by the secretory activities of HFs. Perifollicular mast cells, Tregs and other immunocytes may also contribute to HF IP maintenance in healthy human skin. Collapse of anagen hair bulb IP is an essential prerequisite for the development of alopecia areata (AA). In AA, lesional HFs are rapidly infiltrated by NKG2D + T cells and natural killer (NK) cells, while perifollicular mast cells acquire a profoundly pro-inflammatory phenotype and interact with autoreactive CD8+ T cells. Using animal models, significant functional evidence has accumulated that demonstrates the dominance of the immune system in AA pathogenesis. Purified CD8+T-cell and NK cell populations alone, which secrete fγ, suffice to induce the AA phenotype, while CD4+T-cells aggravate it, and Tregs and iNKT cells may provide relative protection against AA development. While IP collapse may be induced by exogenous agents, inherent IP deficiencies might confer increased susceptibility to AA for some individuals. Thus, a key goal for effective AA management is the re-establishment of a functional HF IP, which will also provide superior protection from disease relapse.

Nonsurgical Induction of Alopecia Areata in C3H/HeJ Mice via Adoptive Transfer of Cultured Lymphoid Cells.

Surgical induction of alopecia areata (AA) via full-thickness grafting of spontaneous AA-affected C3H/HeJ mouse skin to naïve recipients has been a primary method of transferring the AA disease model phenotype. However, this method is associated with the need to perform an invasive procedure that could negatively impact animal wellbeing. Therefore, a rodent model that rapidly develops AA at a predictable rate and without the need to perform invasive surgical procedures on the mice is essential for studying the pathogenesis of AA. Here we describe a cell injection technique using cultured skin-draining lymph node cells (LNCs) injected intradermally into naïve recipients to induce rapid AA development. The cultured LNCs can reach ~ten fold expansion after 6 days with specific cytokine stimulation. The LNCs derived from a single AA affected mouse donor can induce AA development in more than 80 naïve mice within 2-18 weeks. For comparative control studies, mice receiving cultured LNCs from normal donors remain normally haired. The method enables the production of large numbers of AA mice for use in research and treatment development studies while avoiding the use of surgical procedures. We anticipate that the protocol can also be adapted for use in other mouse autoimmune disease models.

Growth factor concentrations in platelet-rich plasma for androgenetic alopecia: An intra-subject, randomized, blinded, placebo-controlled, pilot study.

BACKGROUND: Platelet-rich plasma (PRP), processed from autologous peripheral blood, is used to treat androgenetic alopecia (AGA). OBJECTIVE: To determine the efficacy of PRP for hair growth promotion in AGA patients in a randomized, blinded, placebo-controlled, pilot clinical trial (NCT02074943). METHODS: The efficacy of an 8 week, five session, PRP treatment course was determined by measuring hair density and hair caliber changes in 10 AGA affected patients. For each PRP sample, the concentrations of selected growth factors were determined using a multiplex assay system. The clinical results were then correlated with the growth factor concentrations in PRP. RESULTS: At 16 weeks, 8 weeks after the last PRP injection, treated areas exhibited increased mean hair density (+12.76%) over baseline compared to placebo (+0.99%). Mean hair caliber decreased in both treated and placebo regions (-16.22% and -19.46%, respectively). Serial analysis of PRP significant variability in concentrations between patients. Overall, there was a positive correlation between GDNF concentration and hair density (P = .004). Trends, though not statistically significant, were also observed for FGF2 and VEGF. LIMITATIONS: Small sample size and lack of comparative cohorts receiving protocol variations limit confidence in the study data. CONCLUSIONS: This small pilot clinical trial suggests PRP treatment may be beneficial for AGA. However, the variable hair growth responses between patients indicate there is a significant opportunity to improve PRP therapy protocols for hair growth promotion. The variability in growth factor concentration in PRP suggests standardization of growth factors postprocessing might improve hair growth responses.

A "hair-raising" history of alopecia areata.

A 3500-year-old papyrus from ancient Egypt provides a list of treatments for many diseases including "bite hair loss," most likely alopecia areata (AA). The treatment of AA remained largely unchanged for over 1500 years. In 30 CE, Celsus described AA presenting as scalp alopecia in spots or the "windings of a snake" and suggested treatment with caustic compounds and scarification. The first "modern" description of AA came in 1813, though treatment still largely employed caustic agents. From the mid-19th century onwards, various hypotheses of AA development were put forward including infectious microbes (1843), nerve defects (1858), physical trauma and psychological stress (1881), focal inflammation (1891), diseased teeth (1902), toxins (1912) and endocrine disorders (1913). The 1950s brought new treatment developments with the first use of corticosteroid compounds (1952), and the first suggestion that AA was an autoimmune disease (1958). Research progressively shifted towards identifying hair follicle-specific autoantibodies (1995). The potential role of lymphocytes in AA was made implicit with immunohistological studies (1980s). However, studies confirming their functional role were not published until the development of rodent models (1990s). Genetic studies, particularly genome-wide association studies, have now come to the forefront and open up a new era of AA investigation (2000s). Today, AA research is actively focused on genetics, the microbiome, dietary modulators, the role of atopy, immune cell types in AA pathogenesis, primary antigenic targets, mechanisms by which immune cells influence hair growth, and of course the development of new treatments based on these discoveries.

Autologous Cell Therapy for Aged Human Skin: A Randomized, Placebo-Controlled, Phase-I Study.

INTRODUCTION: Skin ageing involves senescent fibroblast accumulation, disturbance in extracellular matrix (ECM) homeostasis, and decreased collagen synthesis. OBJECTIVE: to assess a cell therapy product for aged skin (RCS-01; verum) consisting of ~25 × 106 cultured, autologous cells derived from anagen hair follicle non-bulbar dermal sheath (NBDS). METHODS: For each subject in the verum group, 4 areas of buttock skin were injected intradermally 1 or 3 times at monthly intervals with RCS-01, cryomedium, or needle penetration without injection; in the placebo group RCS-01 was replaced by cryomedium. The primary endpoint was assessment of local adverse event profiles. As secondary endpoints, expression of genes related to ECM homeostasis was assessed in biopsies from randomly selected volunteers in the RCS-01 group taken 4 weeks after the last injection. -Results: Injections were well tolerated with no severe adverse events reported 1 year after the first injection. When compared with placebo-treated skin, a single treatment with RCS-01 resulted in a significant upregulation of TGFß1, CTGF, COL1A1, COL1A2, COL3A1, and lumican mRNA expression. LIMITATIONS: The cohort size was insufficient for dose -ranging evaluation and subgroup analyses of efficacy. CONCLUSIONS: RCS-01 therapy is well tolerated and associated with a gene expression response consistent with an improvement of ECM homeostasis.

Sequential cyclic changes of hair roots revealed by dermoscopy demonstrate a progressive mechanism of diffuse alopecia areata over time.

BACKGROUND: Diffuse alopecia areata (DAA) often leads to a complete hair shedding within a few months. OBJECTIVE: To explore features and mechanisms underlying DAA. MATERIALS AND METHODS: Scalp and hair root dermoscopy were conducted on 23 DAA patients throughout the disease process, 20 patchy Alopecia areata patients, 23 acute telogen effluvium (ATE) patients and 10 normal controls. Histopathology was also evaluated. RESULTS: We found almost all hair roots were anagen in early stage DAA in 18 patients (18/23, 78.3%) within the first 4-8 weeks after hair loss onset. Anagen effluvium (~4 weeks) was followed by catagen (~4 weeks) and then telogen/exogen (~8 weeks) effluvium with overlap. Hair root and proximal hair shaft depigmentation was more prominent in later DAA disease stages. Black dots, exclamation mark hairs and inconsistent thickness of hair shafts were found more often in early than later DAA (Ps < 0.01). Early DAA histopathology revealed more prominent inflammation and hair follicle regression than that observed in the later stages. Patchy alopecia areata patients showed mixed anagen, catagen and telogen hair roots while ATE patients showed increased exogen and mildly decreased hair root pigmentation. CONCLUSION: Sequential cyclic staging of shed hairs in DAA indicates the insult may be hair-cycle specific. We suggest that DAA is initially an anagen effluvium disease involving an intense inflammatory insult, later progressing to a brief catagen effluvium, and then to telogen effluvium with premature exogen, in later stages of DAA.

Serum level of IL-4 predicts response to topical immunotherapy with diphenylcyclopropenone in alopecia areata.

BACKGROUND: This study investigated predictors of response to topical diphenylyclopropenone (DPCP) immunotherapy in patients with alopecia areata (AA). OBJECTIVE: To identify predictors of response, or resistance, to treatment for AA through clinical observations and serum tests. METHODS: Eighty four AA patients were treated with DPCP. Serum cytokine levels were measured in 33 AA patients pre- and post-treatment, and in 18 healthy controls, using ELISA assays. RESULTS: Of patients, 56.1% responded to DPCP with satisfactory hair regrowth; the response rate was negatively correlated with hair loss extent. Before DPCP treatment, higher serum IFN-γ and IL-12 cytokine levels were observed in AA patients compared to healthy controls. Non-responders to DPCP had significantly elevated serum IL-4 pre-treatment (3.07 fold higher) and lower IL-12 levels compared with responders. After DPCP treatment, non-responders had persistently high IL-4, increased IL-12, negligible decrease in IFN-γ and decreased IL-10. Post-treatment DPCP responders exhibited significantly decreased IFN-γ and IL-12, and increased IL-4 and IL-10. Development of adverse side-effects was significantly associated with higher pre-treatment serum IgE levels. LIMITATIONS: A small number of subjects were evaluated. CONCLUSIONS: Potentially, elevated pre-treatment serum levels of IL-4 and IL-12 can be used as unfavorable and favorable predictors of DPCP therapeutic effect, respectively. In addition, pre-treatment elevated serum total IgE may predict increased risk for severe adverse side-effects to DPCP application. Whether serum cytokine expression levels can be used as predictors of response to other forms of treatment is unknown, but it may warrant investigation in the development of personalized treatments for AA.

Allergy promotes alopecia areata in a subset of patients.

In this commentary, we focus on allergy as a facilitating factor in the pathogenesis of alopecia areata (AA). From previous studies on AA, it is well known that subsets of patients can have one or more of; seasonal relapse, comorbid atopic rhinitis, asthma and dermatitis, lesion infiltrating eosinophils and plasma cells, high levels of total IgE, specific IgE for house dust mites (HDMs), and/or disrupted skin barrier function by the evaluation of filaggrin. Allergy and AA share a similar genetic background; both contributing to an immune reaction imbalance. Furthermore, adjunctive treatment with antihistamines, or desensitization for HDM, can reduce the severity of alopecia in atopic AA patients. Therefore, allergies may contribute to the onset and relapse of AA. Identification of an allergic or atopic immune component in AA patient subsets may indicate adjunctive treatment intervention measures against allergies should be taken which may improve the success of conventional AA treatment.

Increased expression of TLR7 and TLR9 in alopecia areata.

Alopecia areata (AA) is thought to be an autoimmune process. In other autoimmune diseases, the innate immune system and Toll-like receptors (TLRs) can play a significant role. Expression of TLR7, TLR9 and associated inducible genes was evaluated by quantitative PCR in peripheral blood mononuclear cells (PBMCs) from 10 healthy individuals and 19 AA patients, categorized according to disease duration, activity and hair loss extent. Microdissected scalp biopsies from five patients and four controls were also assessed by quantitative PCR and immunohistology. TLR9 was significantly upregulated 2.37 fold in AA PBMCs. Notably, TLR9 was most significantly upregulated in patients with active AA, as shown by a positive hair pull test, compared to stable AA patients. In hair follicle bulbs from AA patients, IFNG and TLR7 exhibited statistically significant 3.85 and 2.70 fold increases in mRNA, respectively. Immunohistology revealed TLR7 present in lesional follicles, while TLR9 positive cells were primarily observed peri-bulbar to AA affected hair follicles. The increased expression of TLR7 and TLR9 suggest components of the innate immune system may be active in AA pathogenesis.

Fibroblast cell-based therapy prevents induction of alopecia areata in an experimental model.

Alopecia areata (AA) is an autoimmune hair loss disease with infiltration of proinflammatory cells into hair follicles. Current therapeutic regimens are unsatisfactory mainly because of the potential for side effects and/or limited efficacy. Here we report that cultured, transduced fibroblasts, which express the immunomodulatory molecule indoleamine 2,3-dioxygenase (IDO), can be applied to prevent hair loss in an experimental AA model. A single intraperitoneal (IP) injection of IDO-expressing primary dermal fibroblasts was given to C3H/HeJ mice at the time of AA induction. While 60-70% of mice that received either control fibroblasts or vehicle injections developed extensive AA, none of the IDO-expressing fibroblast-treated mice showed new hair loss up to 20 weeks post injection. IDO cell therapy significantly reduced infiltration of CD4+ and CD8+ T cells into hair follicles and resulted in decreased expression of TNF-α, IFN-γ and IL-17 in the skin. Skin draining lymph nodes of IDO fibroblast-treated mice were significantly smaller, with more CD4+ CD25+ FoxP3+ regulatory T cells and fewer Th17 cells than those of control fibroblast and vehicle-injected mice. These findings indicate that IP injected IDO-expressing dermal fibroblasts can control inflammation and thereby prevent AA hair loss.

Alopecia Areata is Associated with Increased Expression of Heart Disease Biomarker Cardiac Troponin I.

The development of androgenetic alopecia is associated with a risk of developing cardiovascular diseases, but the association of alopecia areata with cardiovascular diseases in humans is largely unexplored. We measured the plasma level of two common cardiovascular disease markers, cardiac troponin I and C-reactive protein, in alopecia areata and androgenetic alopecia affected subjects. Also, we investigated the possible presence of pro-apoptotic factors in the plasma of hair loss subjects. The mean plasma cardiac troponin I level was highest in alopecia areata subjects, moderately higher in androgenetic alopecia subjects, and lowest in subjects without hair loss (p <0.05). Alopecia areata subjects not receiving treatments had highest levels of cTnI (p <0.05). Alopecia areata plasma samples with high cardiac troponin I levels also induced significantly higher rates of cardiomyocyte apoptosis in cell culture assays. The results suggest the potential for increased heart remodelling. Close monitoring of cardiovascular health in alopecia areata subjects, as well as subsets of androgenetic alopecia patients, may be appropriate.

Experimental and early investigational drugs for androgenetic alopecia.

INTRODUCTION: Treatments for androgenetic alopecia constitute a multi-billion-dollar industry, however, currently available therapeutic options have variable efficacy. Consequently, in recent years small biotechnology companies and academic research laboratories have begun to investigate new or improved treatment methods. Research and development approaches include improved formulations and modes of application for current drugs, new drug development, development of cell-based treatments, and medical devices for modulation of hair growth. Areas covered: Here we review the essential pathways of androgenetic alopecia pathogenesis and collate the current and emerging therapeutic strategies using journal publications databases and clinical trials databases to gather information about active research on new treatments. Expert opinion: We propose that topically applied medications, or intra-dermal injected or implanted materials, are preferable treatment modalities, minimizing side effect risks as compared to systemically applied treatments. Evidence in support of new treatments is limited. However, we suggest therapeutics which reverse the androgen-driven inhibition of hair follicle signaling pathways, such as prostaglandin analogs and antagonists, platelet-rich plasma (PRP), promotion of skin angiogenesis and perfusion, introduction of progenitor cells for hair regeneration, and more effective ways of transplanting hair, are the likely near future direction of androgenetic alopecia treatment development.

Notch signaling is significantly suppressed in basal cell carcinomas and activation induces basal cell carcinoma cell apoptosis.

A subset of basal cell carcinomas (BCCs) are directly derived from hair follicles (HFs). In some respects, HFs can be defined as 'ordered' skin appendage growths, while BCCs can be regarded as 'disordered' skin appendage growths. The aim of the present study was to examine HFs and BCCs to define the expression of common and unique signaling pathways in each skin appendage. Human nodular BCCs, along with HFs and non­follicular skin epithelium from normal individuals, were examined using microarrays, qPCR, and immunohistochemistry. Subsequently, BCC cells and root sheath keratinocyte cells from HFs were cultured and treated with Notch signaling peptide Jagged1 (JAG1). Gene expression, protein levels, and cell apoptosis susceptibility were assessed using qPCR, immunoblotting, and flow cytometry, respectively. Specific molecular mechanisms were found to be involved in the process of cell self­renewal in the HFs and BCCs, including Notch and Hedgehog signaling pathways. However, several key Notch signaling factors showed significant differential expression in BCCs compared with HFs. Stimulating Notch signaling with JAG1 induced apoptosis of BCC cells by increasing Fas ligand expression and downstream caspase-8 activation. The present study showed that Notch signaling pathway activity is suppressed in BCCs, and is highly expressed in HFs. Elements of the Notch pathway could, therefore, represent targets for the treatment of BCCs and potentially in hair follicle engineering.

Identification of Autoantigen Epitopes in Alopecia Areata.

Alopecia areata (AA) is believed to be a cell-mediated autoimmune hair loss disease. Both CD4 and cytotoxic CD8 T cells (CTLs) are important for the onset and progression of AA. Hair follicle (HF) keratinocyte and/or melanocyte antigen epitopes are suspected potential targets of autoreactive CTLs, but the specific epitopes have not yet been identified. We investigated the potential for a panel of known epitopes, expressed by HF keratinocytes and melanocytes, to induce activation of CTL populations in peripheral blood mononuclear cells. Specific synthetic epitopes derived from HF antigens trichohyalin and tyrosinase-related protein-2 induced significantly higher frequencies of response in AA CTLs compared with healthy controls (IFN-gamma secretion). Apoptosis assays revealed conditioned media from AA peripheral blood mononuclear cells stimulated with trichohyalin peptides elevated the expression of apoptosis markers in primary HF keratinocytes. A cytokine array revealed higher expression of IL-13 and chemokine ligand 5 (CCL5, RANTES) from AA peripheral blood mononuclear cells stimulated with trichohyalin peptides compared with controls. The data indicate that AA affected subjects present with an increased frequency of CTLs responsive to epitopes originating from keratinocytes and melanocytes; the activated CTLs secreted soluble factors that induced apoptosis in HF keratinocytes. Potentially, CTL response to self-antigen epitopes, particularly trichohyalin epitopes, could be a prognostic marker for human AA.

Animal Models for Alopecia Areata: What and Where?

Disease is not limited to humans. Rather, humans are but another mammal in a continuum, and as such, often share similar if not identical diseases with other mammalian species. Alopecia areata (AA) is such a disease. Natural disease occurs in humans, nonhuman primates, many domestic animals, and laboratory rodents. However, to be useful as models of human disease, affected animals need to be readily available to the research community, closely resemble the human disease, be easy to work with, and provide reproducible data. To date, the laboratory mouse (most if not all of the C3H substrains) and the Dundee experimental bald rat fit these criteria. Manipulations using full-thickness skin grafts or specific immune cell transfers have improved the models. New mouse models that carry a variety of genetic-based immunodeficiencies can now be used to recapitulate the human immune system and allow for human full-thickness skin grafts onto mice to investigate human-specific mechanistic and therapeutic questions. These models are summarized here including where they can currently be obtained from public access repositories.

The role of lymphocytes in the development and treatment of alopecia areata.

Alopecia areata (AA) development is associated with both innate and adaptive immune cell activation, migration to peri- and intra-follicular regions, and hair follicle disruption. Both CD4(+) and CD8(+) lymphocytes are abundant in AA lesions; however, CD8(+) cytotoxic T lymphocytes are more likely to enter inside hair follicles, circumstantially suggesting that they have a significant role to play in AA development. Several rodent models recapitulate important features of the human autoimmune disease and demonstrate that CD8(+) cytotoxic T lymphocytes are fundamentally required for AA induction and perpetuation. However, the initiating events, the self-antigens involved, and the molecular signaling pathways, all need further exploration. Studying CD8(+) cytotoxic T lymphocytes and their fate decisions in AA development may reveal new and improved treatment approaches.

High LIFr expression stimulates melanoma cell migration and is associated with unfavorable prognosis in melanoma.

Increased or decreased expression of LIF receptor (LIFr) has been reported in several human cancers, including skin cancer, but its role in melanoma is unknown. In this study, we investigated the expression pattern of LIFr in melanoma and assessed its prognostic value. Using tissue microarrays consisting of 441 melanomas and 96 nevi, we found that no normal nevi showed high LIFr expression. LIFr staining was significantly increased in primary melanoma compared to dysplastic nevi (P = 0.0003) and further increased in metastatic melanoma (P = 0.0000). Kaplan-Meier survival curve and univariate Cox regression analyses showed that increased expression of LIFr was correlated with poorer 5-year patient survival (overall survival, P = 0.0000; disease-specific survival, P = 0.0000). Multivariate Cox regression analyses indicated that increased LIFr expression was an independent prognostic marker for primary melanoma (P = 0.036). LIFr knockdown inhibited melanoma cell migration in wound healing assays and reduced stress fiber formation. LIFr knockdown correlated with STAT3 suppression, but not YAP, suggesting that LIFr activation might stimulate melanoma cell migration through the STAT3 pathway. Our data indicate that strong LIFr expression identifies potentially highly malignant melanocytic lesions at an early stage and LIFr may be a potential target for the development of early intervention therapeutics.