Hair cycle control by leptin as a new anagen inducer

Our purpose is to clarify the physiological role of leptin in hair cycle as leptin reportedly causes activation of Stat3, which is indispensable for hair cycling. While hair follicles in dorsal skin of 5‐week‐old C57/BL6 mice had progressed to late anagen phase, those in dorsal skin of 5‐week‐old leptin receptor deficient db/db mice remained in the first telogen and later entered the anagen at postnatal day 40, indicating that deficiency in leptin receptor signalling delayed the second hair cycle progression. Next, we shaved dorsal hairs on wild‐type mice at postnatal 7 weeks and injected skin with mouse leptin or a mock. After 20 days, although mock injection showed no effect, hair growth occurred around leptin injection area. Human leptin fragment (aa22–56) had similar effects. Although the hair cycle of ob/ob mice was similar to that of wild‐type mice, injection of mouse leptin on ob/ob mice at postnatal 7 weeks induced anagen transition. Immunohistochemically, leptin is expressed in hair follicles from catagen to early anagen in wild‐type mice, suggesting that leptin is an anagen inducer in vivo. Phosphorylation of Erk, Jak2 and Stat3 in human keratinocytes was stimulated by leptin and leptin fragment. In addition, RT‐PCR and ELISA showed that the production of leptin by human dermal papilla cells increased under hypoxic condition, suggesting that hypoxia in catagen/telogen phase promotes leptin production, preparing for entry into the next anagen. In conclusion, leptin, a well‐known adipokine, acts as an anagen inducer and represents a new player in hair biology.     

Para‐phenylenediamine‐specific lymphocyte activation test: a sensitive in vitro assay to detect para‐phenylenediamine sensitization in patients with severe allergic reactions

Please cite this paper as: Para‐phenylenediamine‐specific lymphocyte activation test: a sensitive in vitro assay to detect para‐phenylenediamine sensitization in patients with severe allergic reactions. Experimental Dermatology 2010; 19: 435–441. Abstract: Patients sensitized to para‐phenylenediamine (PPD) by semi‐permanent tattoos increasingly develop threatening allergic reactions in response to black hair dye. The gold standard to diagnose allergic contact dermatitis is to perform epicutaneous patch tests, however, iatrogenic sensitizations and severe patch test reactions to PPD have been described, the latter especially in patients with severe allergic reactions. We examined nine patients with severe allergic reactions in response to permanent hair dyes. Patch tests using the standard concentration of 1% or 0.5% PPD resulted in severe and sometimes even bullous reactions in all patients responsive to PPD. Titration revealed that at 1% of the standard concentration (0.01% PPD), patch test sensitivity decreased and only 50% of patients responded. Consequently, we established an in vitro assay to diagnose PPD allergy. Freshly isolated peripheral blood mononuclear cells (PBMC) were cultured with titrated concentrations of PPD with or without IL‐2 supplementation, and cell proliferation was determined by [3H]‐thymidine incorporation. Lymphocyte activation test (LAT ) detected PBMC cell proliferation specific to PPD, with at least 3.5‐fold increase in [3H]‐thymidine uptake in all PPD allergic patients. Most importantly, PPD – LAT without IL‐2 supplementation remained negative in three out of eight PPD allergic patients. Thus, PPD‐LAT with IL‐2 supplementation demonstrated a sensitivity of 100%, remained unresponsive in controls not sensitized to PPD, and in one patient sensitive to other p‐amino compounds. These data demonstrate that LAT with PPD can be used to detect PPD sensitization as a possible alternative to patch testing at least in patients with severe allergic reactions to PPD.     

Efficacy of quantifying melanosome transfer with flow cytometry in a human melanocyte–HaCaT keratinocyte co‐culture system in vitro

Please cite this paper as: Efficacy quantifying melanosome transfer with flow cytometry in a human melanocyte–HaCaT keratinocyte co‐culture system in vitro. Experimental Dermatology 2010; 19 : e282–e285. Abstract: In this study, we describe a simple, specific, reproducible and quantitative assay system to assess melanosome transfer. We first established a co‐culture model of normal human epidermal melanocytes and HaCaT keratinocytes. The cells were co‐cultured for 72 h in a serum‐free keratinocyte growth media and double labelled with Fluorescein isothiocyanate (FITC)‐conjugated antibody against the melanosome‐specific protein gp100, and with Phycoerythrin (PE)‐conjugated antibody against the keratinocyte‐specific marker cytokeratin. Then, the cells were examined using co‐focal microscope and flow cytometry. The increased melanosome transfer from melanocytes to HaCaT keratinocytes was observed in a time‐dependent manner. To verify the accessibility of this method, two known melanosome transfer inhibitors and two known melanosome transfer stimulators were applied. Consistent with previous investigation, soybean trypsin inhibitor (STI), niacinamide inhibited melanosome transfer, α‐melanocyte stimulating hormone (α‐MSH) and keratinocyte growth factor (KGF) increased melanosome transfer, respectively, in a dose‐dependent manner. The model used in this study could thus represent a rapid and reliable tool to identify modulators of human melanosome transfer.     

In vitro diagnostic assays are effective during the acute phase of delayed‐type drug hypersensitivity reactions

Background Previous reports have suggested that drug‐specific lymphocyte proliferation assays (LPA) can be used retrospectively to confirm the culprit drug following delayed‐type drug hypersensitivity reactions (DHR). However, only limited evidence supports their use in aiding acute clinical management. The aim of this study was to compare the LPA against combination cytokine assays for potential use in the acute setting. Methods A total of 43 patients with DHR (19 during the acute reaction, 20 after recovery, four during acute and after recovery) and 14 control subjects without DHR were investigated using ex vivo analysis of drug‐specific proliferation, and interferon (IFN)‐γ and interleukin (IL)‐4 production. Results Healthy controls showed negative drug‐specific proliferation and cytokine release in contrast to individuals with a known sensitivity (P < 0·0001). The assays demonstrated a test specificity of 95% (LPA), 83% (IFN‐γ) and 92% (IL‐4). The sensitivity of combined measurement of drug‐specific IFN‐γ and IL‐4 cytokines during acute DHR was better than LPA (82% vs. 50%), but all assays were less sensitive during the recovery phase. The correlation between LPA and IFN‐γ assays was strong (r = 0·7, P < 0·0001), whereas the IL‐4 assay did not correlate as well with either of these assays. In contrast to LPA, drug enzyme‐linked immunosorbent spot assays showed positive responses in patients concurrently taking immunosuppressive medication. Conclusions In vitro assays of drug‐specific IFN‐γ and IL‐4 production offer potential for use as rapid diagnostic tests. Cytokine detection offers distinct advantages over the LPA, including a shorter assay time, a greater sensitivity and effectiveness in testing immunosuppressed patients.     

A somewhat unexpected result from the deconvolution of DSC curves for human hair: There is no apparent relation between cortical cell fractions and hair curliness

A deconvolution process has been developed for curves obtained by differential scanning calorimetry in water for Merino wool and the main ethnic hair types. This enables estimation of the fractions of ortho‐ and para‐type cell groups. The results also indicate that hair may contain a further, low‐sulphur subgroup of ortho‐type cells. The sizes of the major cell fractions are in line with expectations from microscopical investigations. The fractions are comparable for hair types, and no consistent association between cell‐type fractions and hair curvature is observed.     

Homeobox transcription factor DLX4 is not necessary for skin development and homeostasis

Dlx4 is a member of a family of homeobox genes with homology to Drosophila distal‐less (dll) gene. We show that Dlx4 expression pattern partially overlaps with its cis‐linked gene Dlx3 during mouse development as well as in neonatal and adult skin. In mice, Dlx4 is expressed in the branchial arches, embryonic limbs, digits, nose, hair follicle and in the basal and suprabasal layers of mouse interfollicular epidermis. We show that inactivation of Dlx4 in mice did not result in any overtly gross pathology. Skin development, homeostasis and response to TPA treatment were similar in mice with loss of Dlx4 compared to wild‐type counterparts.     

Patch testing with hydroxyethyl-p-phenylenediamine sulfate - cross-reactivity with p-phenylenediamine

Background. Adverse reactions to permanent hair dyes are frequent, and primarily result from sensitization to p‐phenylenediamine (PPD). Objectives. To investigate the degree of cross‐reactivity to a chemically similar dye, hydroxyethyl‐p‐phenylenediamine sulfate (HPPS), and whether this might be a dyeing alternative for patients who are sensitive to PPD. Method. HPPS was patch tested in two concentrations in a total of 216 patients suspected of having contact dermatitis caused by hair dyes and/or hair cosmetics. A regular use test with a hair dye containing HPPS was suggested to every patient who had had an adverse reaction to a PPD hair dye in the past. Results. Forty of 216 (19.9%) patients reacted to 1% PPD, whereas only 2/216 (0.9%) showed a positive reaction to 1% HPPS. Reactivity to 2% HPPS was only slightly higher (5/216, 2.3%). On the basis of the 43 PPD‐positive patients, the reactivity to 2% HPPS amounted to 12%; the corresponding figure for toluene‐2,5‐diamine was 15% (5/33). In a use test on two PPD‐positive patients with a hair dye containing HPPS, no adverse reaction was seen, even after several years of regular dyeing. Conclusions. HPPS may be an alternative hair dye for individuals not tolerating PPD‐containing dyes. However, cross‐reactivity with PPD and other aromatic amines may occur. HPPS is also a known sensitizer, and the risk of de novo sensitization can only be assessed by a controlled study on a large panel and under regular use conditions.     

Gadolinium toxicity: epidermis thickness measurement by magnetic resonance imaging at 500 MHz

Purpose: Regional epidermal thickening and hair follicle width measurement by delayed gadolinium contrast magnetic resonance imaging (MRI) may assess the contrast agent gadolinium toxicity on mice skin. Materials and methods: Delayed contrast in vivo MRI was performed in mice. Six mice skin samples were removed and exposed to a gadolinium contrast agent at different times after 2, 4, 6 and 8 h. The relaxation constants of each skin structure were measured. The thickness of the epidermis and hair follicle on follow‐up ex vivo delayed‐contrast MRI served as an index of gadolinium toxicity on the skin. Results: In vivo MRI by fast low‐angle shot imaging technique showed distinct skin layers. High‐resolution gradient echo T1‐weighted and multislice multiecho proton density‐weighted MRI intensities in the epidermis and hair follicle showed a positive correlation with delayed gadolinium‐enhanced MRI hyperintensities (Pearson's correlation coefficient r²=0.81, P<0.0001) in the excised mice skin tissues. Delayed contrast‐enhanced mice skin MRI after 2–4 h showed epidermis swelling and hair follicle regions with a size measurement accuracy of 65%, a sensitivity of 95%, a specificity of 25%, a positive predictive value of 65% and a negative predictive value of 65%. Areas under the receiver operating characteristic curves by MRI were 0.92–0.94 for hair and epidermis as good discriminators. MRI visualized distinct relaxation constants of the epidermis, sebaceous gland, skin papillary and reticular dermis layers and hair follicle. Conclusion: Gadolinium contrast‐enhanced MRI may visualize the thickening of the epidermis wall and hair follicle as an index of viable mice skin. Gadolinium enhanced the MRI visibility of skin structures. Gadolinium treatment showed skin toxicity as epidermis thickening the first time due to the undesirable use of high concentrations of gadolinium in microimaging.     

Inhibition of cyclin‐dependent kinase activity exacerbates H2O2‐induced DNA damage in Kindler syndrome keratinocytes

Kindler syndrome (KS) is an autosomal recessive skin disorder characterized by skin blistering and photosensitivity. KS is caused by loss of function mutations in FERMT1, which encodes Kindlin‐1. Kindlin‐1 is a FERM domain containing adaptor protein that is found predominantly at cell‐extracellular matrix adhesions where it binds to integrin β subunits and is required for efficient integrin activation. Using keratinocytes derived from a patient with KS, into which wild‐type Kindlin‐1 (Kin1WT) has been expressed, we show that Kindlin‐1 binds to cyclin‐dependent kinase (CDK)1 and CDK2. CDK1 and CDK2 are key regulators of cell cycle progression, however, cell cycle analysis showed only small differences between the KS and KS‐Kin1WT keratinocytes. In contrast, G2/M cell cycle arrest in response to oxidative stress induced by hydrogen peroxide (H₂O₂) was enhanced in KS keratinocytes but not KS‐Kin1WT cells, following inhibition of CDK activity. Furthermore, KS keratinocytes were more sensitive to DNA damage in response to H₂O₂ and this was exacerbated by treatment with the CDK inhibitor roscovitine. Thus, in Kindlin‐1 deficient keratinocytes, CDK activity can further regulate oxidative damage induced cell cycle arrest and DNA damage. This provides further insight into the key pathways that control sensitivity to oxidative stress in KS patients.     

Leptin controls hair follicle cycling

Leptin is a cytokine well known for its ability to control body weight and energy metabolism. Several lines of evidence have recently revealed that leptin also plays an important role in wound healing and immune modulation in skin. Sumikawa et al. Exp Dermatol 2014 evaluated the effect of leptin on hair follicle cycling using mutant and wild‐type mice. They report that leptin is produced in dermal papilla cells in hair follicles and that leptin receptor–deficient db/db mice show an abnormality in hair follicle cycling. Moreover, leptin injection induced the transition into the growth stage of the hair cycle (anagen). On this basis, it now deserves exploration whether leptin‐mediated signalling is a key stimulus for anagen induction and whether this may be targeted to manage human hair disorders with defect in the control of hair follicle cycling.     

Ultrastructural findings in progressive macular hypomelanosis indicate decreased melanin production

Background The pathogenesis of progressive macular hypomelanosis (PMH) is unknown. Recently, Westerhof et al. (Arch Dermatol 2004; 140: 210–214) hypothesized that Propionibacterium acnes produces a depigmenting factor that interferes with melanogenesis in the skin, resulting in hypopigmented spots. The purpose of the study is to gain an insight into the pathogenesis of PMH. Materials and methods We took a biopsy of 2‐mm diameter from normal and lesional skin in eight PMH patients. Using electron microscopy, we compared melanization of melanosomes, melanosome transfer and amount of epidermal melanin in normal and lesional skin. Result Compared to non‐lesional skin, we observed a decrease of epidermal melanin and less melanized melanosomes in lesional skin of all patients. When comparing normal and lesional skin of patients with skin type V and VI, we observed a difference in melanosome size and maturation and a switch of transferred melanosomes from single stage IV transferred melanosomes to aggregated stage I, II and III transferred melanosomes, as seen in healthy skin of skin type I to IV. Conclusion Hypopigmentation in PMH seems to be the result of an altered melanogenesis based on a decrease in melanin formation and a change in the distribution of melanosomes. In lesional skin of PMH patients with skin type V and VI less melanized, aggregated melanosomes in stead of single, mature melanosomes are transferred from melanocytes to keratinocytes. This results in a decrease of epidermal melanin. Further investigations are needed to determine the precise role of Propionibacterium acnes in this alteration of melanogenesis.     

Physiological changes in scalp, facial and body hair after the menopause: a cross-sectional population-based study of subjective changes

Background Significant changes in scalp, facial and body hair occur after the menopause. These can have a significant negative impact on self‐esteem and are also potential markers of endocrine or metabolic diseases. Knowledge of postmenopausal hair changes is important for clinicians to distinguish between normal physiological change and those that require further medical investigation. Objectives To assess the subjective experience of scalp, facial and body hair change in a large cohort of normal postmenopausal females. Methods Postmenopausal females aged 45 years or over of northern European origin completed a questionnaire detailing scalp, facial and body hair changes following the menopause. Women with a history of thyroid disease, oophorectomy or premature menopause were excluded from the study. The Mann–Whitney U‐test and the χ² test were used to assess the correlation between scalp, facial and body hair changes with age. Results Diffuse generalized hair loss was the most common form of scalp hair loss, reported by 26% of women. Frontal hair loss was reported by 9% of women. Facial hair gain was cited by 39% of females with the chin being the most frequent site for new growth (32% of women). Body hair loss was significantly correlated with older age (P < 0·001) and was most frequent at androgen‐sensitive sites. We noted two patterns: (i) diffuse hair loss in which diffuse generalized scalp hair loss was significantly correlated with body hair loss and increasing age (P < 0·05); and (ii) frontal hair loss which was associated with higher facial hair scores and relatively younger age (P < 0·05) compared with women with diffuse hair loss. Conclusions This is the first comprehensive study of the subjective hair changes in postmenopausal women. This study demonstrates two distinct patterns of hair change relating to age, which may reflect different underlying pathophysiological mechanisms and are of relevance to the medical management of these women as well as being possible predictors of health outcomes.     

Functional studies for the TRAF6 mutation associated with hypohidrotic ectodermal dysplasia

Background Hypohidrotic ectodermal dysplasia (HED) is a rare condition characterized by hypotrichosis, hypohidrosis and hypodontia. A de novo heterozygous mutation in the tumour necrosis factor receptor‐associated factor 6 gene (TRAF6) was recently identified in a patient with HED, while functional consequences resulting from the mutation remained unknown. Objectives To determine the mechanism by which the TRAF6 mutation results in HED. Methods We performed coimmunoprecipitation (co‐IP) studies to determine whether the mutation would affect the interaction of TRAF6 with transforming growth factor β‐activated kinase 1 (TAK1), TAK1‐binding protein 2 (TAB 2) and ectodysplasin‐A receptor‐associated death domain protein (EDARADD). We then performed co‐IP and glutathione S‐transferase‐pulldown assays to determine the TRAF6 binding sequences in EDARADD. In addition, we analysed the effect of the mutant TRAF6 protein on the affinity between wild‐type TRAF6 and EDARADD, as well as on EDARADD‐mediated nuclear factor (NF)‐κB activation. Results The mutant TRAF6 protein was capable of forming a complex with TAK1 and TAB 2 in a similar way to wild‐type TRAF6. However, the mutant TRAF6 protein completely lost the affinity to EDARADD, while the wild‐type TRAF6 bound to the N‐terminal domain of EDARADD. Furthermore, the mutant TRAF6 inhibited the interaction between the wild‐type TRAF6 and EDARADD, and also potentially reduced the EDARADD‐mediated NF‐κB activity. Conclusions We conclude that the mutant TRAF6 protein shows a dominant negative effect against the wild‐type TRAF6 protein, which is predicted to affect the EDARADD‐mediated activation of NF‐κB during the development of ectoderm‐derived organs, and to lead to the HED phenotype.     

A pilot study evaluating the efficacy of topically applied niacin derivatives for treatment of female pattern alopecia

Background Female pattern alopecia is a common dermatologic condition that manifests after puberty. The only approved drug treatment for this condition is 2% minoxidil for topical application. Aims This pilot study examined the effect of topical application of two niacin derivatives, octyl nicotinate and tetradecyl nicotinate, on hair fullness in female alopecia. Patients/methods Sixty female subjects with Ludwig types I–III female pattern hair loss were evaluated in a double‐blinded, placebo‐controlled (40 active, 20 placebo) design using standardized 35‐mm photographic analyses for assessment of efficacy after 6 months of application. Results The niacin derivatives demonstrated a statistically significant increase in hair fullness (P = 0.04 compared to the placebo). Conclusion Whereas evaluation of hair growth in women is challenging, this 6‐month pilot study demonstrated statistically significant increase in hair fullness on blinded 35‐mm photographic analysis. Long‐term topical application of nicotinic acid derivatives offers promise for providing benefit in female alopecia and warrants further study.     

Could azathioprine be considered as a therapeutic alternative in the treatment of alopecia areata? A pilot study

Alopecia areata is an autoimmune disease resulting in partial or total nonscarring hair loss and the treatment of severe alopecia areata is difficult. The aim of this study was to evaluate the efficacy and safety of azathioprine as a systemic monotherapy for moderate to severe alopecia areata. A total of 20 patients [14 men (70%) and six women (30%)] with minimum 6 months history of alopecia areata were included. The extent of scalp hair regrowth during and after the completion of the 6 months treatment was evaluated by the Severity of Alopecia Tool (the SALT score). The daily drug intake was calculated as 2 mg/kg of body weight. Mean duration of current episode of scalp hair loss was 26.4 (26.4 ± 17) months. Mean regrowth percentage was 52.3% (52.3 ± 38.4). Mean hair loss percentage before treatment was 72.7% (72.7 ± 28.3) compared with 33.5% (33.5 ± 30.7) after 6 months of azathioprine treatment. This showed a highly significant statistical difference (Paired t‐test, CI 95% = 21.5–54.1). Mean hair loss score (S₀–S₅) before treatment was 3.9 (3.9 ± 1.6) and after 6 months of azathioprine treatment was 1.8 (1.8 ± 1.3). Assessment showed significant difference from baseline score (sign test, P < 0.0001). No significant statistical difference was observed with respect to gender before and after azathioprine treatment. Treatment with azathioprine as a systemic monotherapy clinically produces relevant improvement in moderate‐to‐severe alopecia areata. Generally azathioprine is a low‐cost and well‐tolerated drug and with controlled studies on larger number of patients, long‐term efficacy and safety of this treatment should be investigated.     

Cxcr4 is transiently expressed in both epithelial and mesenchymal compartments of nascent hair follicles but is not required for follicle formation

Hair follicle (HF) morphogenesis relies on the coordinated exchange of signals between mesenchymal and epithelial compartments of embryonic skin. Chemokine receptor Cxcr4 expression was recently identified in dermal condensates (DCs) of nascent HFs, but its role in promoting HF morphogenesis remains unknown. Our analyses confirmed Cxcr4 expression in condensate cells, and additionally revealed transient Cxcr4 expression in incipient epithelial hair placodes. Placodal Cxcr4 appeared prior to detection in DCs, representing a switch of expression between epithelial and mesenchymal compartments. To explore the functional role of this receptor in both compartments for early HF formation, we conditionally ablated Cxcr4 with condensate‐targeting Tbx18^cre knock‐in and epidermis‐targeting Krt14‐cre transgenic mice. Conditional knockouts for both crosses were viable throughout embryogenesis and into adulthood. Morphological and biochemical marker analyses revealed comparable numbers of HFs forming in knockout embryos compared to wild‐type littermate controls in both cases, suggesting that neither dermal nor epithelial Cxcr4 expression is required for early HF morphogenesis. We conclude that Cxcr4 expression and chemokine signaling through this receptor in embryonic mouse skin is dispensable for HF formation.     

tert-butyl hydroperoxide, an organic peroxide, causes temporary delay in hair growth in a neonatal rat model

tert‐Butyl hydroperoxide (tBHP), an organic peroxide, has been shown to cause irreversible damage to keratinocytes in vitro with prolonged administration at high concentrations, and reversible damage with short‐term administration at low concentrations. To investigate the effects of tBHP on keratinocytes in vivo, we analysed hair growth in tBHP‐treated neonatal rats. Sprague–Dawley and Long–Evans rat pups were injected subcutaneously with tBHP or vehicle once daily for 6 days, and hair growth was monitored. The tBHP‐treated rats had a significant delay in hair growth. However, this delay reversed within days, and the hair coats, including hair pigmentation, of tBHP‐treated and sham‐treated rats were indistinguishable 2 weeks later. Histological analysis and BrdU labelling of S phase cells confirmed the delay in hair‐follicle growth and its reversal in tBHP‐treated rats. Our results indicated that the changes incurred in hair follicles by short‐term use of high‐dose oxidants in vivo are temporary and reversible.     

CD109, a novel TGF‐β antagonist,decreases fibrotic responses in a hypoxic wound model

Excessive extracellular matrix deposition that occurs in many fibrotic skin disorders such as hypertrophic scarring and scleroderma is often associated with hypoxia. CD109 is a novel TGF‐β co‐receptor and TGF‐β antagonist shown to inhibit TGF‐β‐induced extracellular matrix protein production in vitro. We examined whether CD109 is able to regulate extracellular matrix deposition under low oxygen tension in vivo using transgenic mice overexpressing CD109 in the epidermis. By creating dorsal bipedicle skin flaps with centrally located excisional wounds in these mice and their wild‐type littermates, we generated a novel murine hypoxic wound model. Mice were sacrificed on 7 or 14 days post‐wounding, and tissues were harvested for histological and biochemical analysis. Hypoxic wounds in both transgenic and wild‐type mice showed increased levels of HIF‐1α and delayed wound closure, validating this model in mice. Hypoxic wounds in CD109 transgenic mice demonstrated decreased collagen type 1 and fibronectin expression, and reduced dermal thickness on day 7 post‐wounding as compared to those in wild‐type mice and to non‐hypoxic control wounds. These results suggest that CD109 decreases extracellular matrix production and fibrotic responses during hypoxic wound healing. Manipulating CD109 levels may have potential therapeutic value for the treatment of fibrotic skin disorders associated with poor oxygen delivery.