Guidelines for clinical trials of frontal fibrosing alopecia: consensus recommendations from the International FFA Cooperative Group (IFFACG).

BACKGROUND: Frontal fibrosing alopecia (FFA) has become one of the most common causes of cicatricial alopecia worldwide. However, there is a lack of clear aetiology and robust clinical trial evidence for the efficacy and safety of agents currently used for treatment. OBJECTIVES: To enable data to be collected worldwide on FFA using common criteria and assessment methods. METHODS: A multicentre, international group of experts in hair loss was convened by email to create consensus recommendations for clinical trials. Consensus was defined at > 90% agreement on each recommended part of these guidelines. RESULTS: Standardized diagnostic criteria, severity rating, staging, and investigator and patient assessment of scalp hair loss and other clinical features of FFA were created. CONCLUSIONS: These guidelines should allow the collection of reliable aggregate data on FFA and advance efforts in both clinical and basic research to close knowledge gaps in this condition.

What causes alopecia areata?

The pathobiology of alopecia areata (AA), one of the most frequent autoimmune diseases and a major unsolved clinical problem, has intrigued dermatologists, hair biologists and immunologists for decades. Simultaneously, both affected patients and the physicians who take care of them are increasingly frustrated that there is still no fully satisfactory treatment. Much of this frustration results from the fact that the pathobiology of AA remains unclear, and no single AA pathogenesis concept can claim to be universally accepted. In fact, some investigators still harbour doubts whether this even is an autoimmune disease, and the relative importance of CD8(+) T cells, CD4(+) T cells and NKGD2(+) NK or NKT cells and the exact role of genetic factors in AA pathogenesis remain bones of contention. Also, is AA one disease, a spectrum of distinct disease entities or only a response pattern of normal hair follicles to immunologically mediated damage? During the past decade, substantial progress has been made in basic AA-related research, in the development of new models for translationally relevant AA research and in the identification of new therapeutic agents and targets for future AA management. This calls for a re-evaluation and public debate of currently prevalent AA pathobiology concepts. The present Controversies feature takes on this challenge, hoping to attract more skin biologists, immunologists and professional autoimmunity experts to this biologically fascinating and clinically important model disease.

Increased expression of cell adhesion molecule 1 by mast cells as a cause of enhanced nerve-mast cell interaction in a hapten-induced mouse model of atopic dermatitis.

BACKGROUND: Neuroimmunological disorders are involved in the pathogenesis of atopic dermatitis (AD), partly through enhanced sensory nerve-skin mast cell interaction. Cell adhesion molecule 1 (CADM1) is a mast-cell adhesion molecule that mediates the adhesion to, and communication with, sympathetic nerves. OBJECTIVES: To investigate the role of mast cell CADM1 in the pathogenesis of AD, CADM1 expression levels by comparing between lesional and nonlesional skin mast cells of an AD mouse model, which was developed by repeated application of trinitrochlorobenzene, and to examine, in cocultures, how the alterations in CADM1 detected in lesional mast cells might affect the sensory nerve-mast cell interaction. METHODS: AD-like lesional and nonlesional skin mast cells were collected separately by laser capture microdissection. CADM1 expression was examined by reverse transcription-polymerase chain reaction and CADM1 immunohistochemistry. In cocultures, adhesion between dorsal root ganglion (DRG) neurites and IC2 mast cells was analysed by loading a femtosecond laser-induced impulsive force on neurite-attendant IC2 cells, while cellular communication was monitored as the IC2 cellular response ([Ca(2+)]i increase) after nerve-specific stimulant-induced DRG activation. RESULTS: AD-like lesional mast cells expressed three-fold more CADM1 transcripts than nonlesional cells. This was supported at the protein level, shown by immunohistochemistry. In coculture, CADM1 overexpression in IC2 cells strengthened DRG neurite-IC2 cell adhesion and doubled the population of IC2 cells responding to DRG activation. A function-blocking anti-CADM1 antibody abolished these effects in a dose-dependent manner. CONCLUSIONS: Increased expression of CADM1 in mast cells appeared to be a cause of enhanced sensory nerve-mast cell interaction in a hapten-induced mouse model of AD.

Coudability hairs: a revisited sign of alopecia areata assessed by trichoscopy.

BACKGROUND: We have previously reported several trichoscopic (dermatoscopic) characteristics, such as black dots, 'exclamation-mark' hairs, broken hairs, yellow dots and clustered short vellus hairs as being useful clinical indicators for alopecia areata (AA). 'Coudability hairs', which are normal-looking hairs tapered at the proximal end, have been previously reported as another sign of AA. AIMS: To use trichoscopy to evaluate coudability hairs as a clinical indicator for the disease activity of AA and a substitute-marker for the hair-pull test. METHODS: Trichoscopic examinations of hair loss and perilesional areas on the scalps of 100 East Asian patients with AA were performed using a dermatoscope. Using Spearman's rank-order correlation coefficient by rank test, we examined the correlations of scores between coudability and AA disease activity, severity or duration and other trichoscopic features, and then evaluated the coudability score as a surrogate-marker for the hair-pull test. RESULTS: Coudability scores correlated positively with AA disease activity, hair-pull tests, short duration, black dots and exclamation-mark hairs, and correlated negatively with short vellus hairs. CONCLUSIONS: Coudability hairs, more closely perceived by trichoscopy, are useful-markers for disease activity in AA and provide a surrogate-marker for the hair-pull test.

Limb and skin abnormalities in mice lacking IKKalpha.

The gene encoding inhibitor of kappa B (IkappaB) kinase alpha (IKKalpha; also called IKK1) was disrupted by gene targeting. IKKalpha-deficient mice died perinatally. In IKKalpha-deficient fetuses, limb outgrowth was severely impaired despite unaffected skeletal development. The epidermal cells in IKKalpha-deficient fetuses were highly proliferative with dysregulated epidermal differentiation. In the basal layer, degradation of IkappaB and nuclear localization of nuclear factor kappa B (NF-kappaB) were not observed. Thus, IKKalpha is essential for NF-kappaB activation in the limb and skin during embryogenesis. In contrast, there was no impairment of NF-kappaB activation induced by either interleukin-1 or tumor necrosis factor-alpha in IKKalpha-deficient embryonic fibroblasts and thymocytes, indicating that IKKalpha is not essential for cytokine-induced activation of NF-kappaB.

Akt activation induces epidermal hyperplasia and proliferation of epidermal progenitors.

Various common signaling pathways maintain tissue stem cells, including Notch and Wnt/beta-catenin signals. Phosphoinositide-3 kinase (PI3K)/Akt signaling regulates the 'stemness' of several stem cells in culture, specifically in maintaining embryonic stem and neural stem cells, and in deriving embryonic germ cells from primordial germ cells. We examined the effect of Akt signaling in epidermal cells in transgenic mice expressing an Akt-Mer fusion protein whose kinase activity was conditionally activated by treatment with 4-hydroxytamoxifen (4OHT). The topical application of 4OHT to adult skin of the transgenic mice induced new hair growth in resting phase follicles. In addition, the mice showed hyperplasia in interfollicular epidermis (IFE) and hair follicles, which was presumably caused by the extensive proliferation of keratinocytes in basal layer of IFE and outer root sheath of hair follicles, respectively. The progenitor cell population increased consistently in 4OHT-treated transgenic mice. Our results show that PI3K/Akt signaling induces epidermal hyperplasia and proliferation of epidermal progenitors.

Serial transplantation to nude mice of an androgen-dependent pilosebaceous tumor developed in Suncus murinus.

The pilosebaceous tumor spontaneously developed on the sidegland of an old male Suncus murinus was serially transplanted into male nude athymic (BALB/c-nu/nu) mice. By the 18th generation, tumor growth occurred in about 70% of them. The transplantability of the tumor maintained in these male mice to female or castrated male nude mice was less than 35%. The histological features of the tumor did not differ by the hormonal status of hosts. Tumor growth started later and was slower in females than in males. This androgen dependency seems to be restricted to an initial period of growth, since the transplantability to males was decreased by castration within a week after grafting. The tumors grown in males possessed both cytoplasmic and nuclear androgen receptors and those in females cytoplasmic androgen receptor only. The latter tumors were found androgen-sensitive, because treatment with testosterone propionate significantly increased tumor size. In addition, after [3H]-testosterone administration, bound radioactivity was detected in the nuclear fraction from female as well as male hosts. Although the mechanism responsible for the development of these androgen-independent sensitive cells is unknown, the variance in transplantability to female hosts suggests that the original tumor was composed of androgen-dependent and androgen-independent cells.

5 alpha-reductase activity in cultured human dermal papilla cells from beard compared with reticular dermal fibroblasts.

The activity of 5 alpha-reductase was assessed in cultured human beard dermal papilla cells and reticular dermal fibroblasts to elucidate the mechanism of androgen action in promoting the growth of beards in men. The monolayer was incubated with 50 nM of [1,2-3H]-testosterone. Steroids were extracted from the medium and analyzed by thin layer chromatography. The major metabolite in the beard dermal papilla cells was dihydrotestosterone (DHT), the most potent androgen in the androgen target tissue. By contrast, the amount of DHT formed was similar to that of androstenedione in reticular dermal fibroblasts. The 5 alpha-reductase activity in beard dermal papilla cells was three to five times as high as that in the reticular dermal fibroblasts from the same skin sample. The apparant Michaelis constant of 5 alpha-reductase in the beard dermal papilla cells was 1.0 X 10(-6) M, which was virtually equivalent to that of genital skin fibroblasts, typical androgen target cells. It was 4.0 X 10(-5) M in reticular dermal fibroblasts. By contrast, the activities of 5 alpha-reductase in dermal papilla cells from occipital scalp hair follicles were similar to those of reticular dermal fibroblasts of the same skin samples. These results strongly suggest that the beard dermal papilla cell is an androgen target cell, and that DHT plays a role in the growth of beards in men.

Activity of testosterone 5 alpha-reductase in various tissues of human skin.

In order to know the distribution of testosterone 5 alpha-reductase activity in human skin, we developed a micro-method, in which we used 20-50 micrograms of various tissues microdissected from freeze-dried sections. The characteristics of this enzyme in the sebaceous gland are briefly described, as follows: the identified 5 alpha-reduced metabolites are 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 beta, 17 beta-diol and 5 alpha-androstanedione; the optimal pH is about 7.5; and the apparent Km is approximately 2.4 x 10(-5) M. The measurement of 5 alpha-reductase activity of various components of the skin obtained from 7 men and 5 women revealed that the sweat gland (probably apocrine) in the axillary skin possessed the highest activity of 5 alpha-reductase: the value was nearly 400 pmoles/mg dry weight/hr in the standardized condition. The sebaceous gland also showed a high activity of 85-261 pmoles/mg/hr. The hair follicles exhibited a significantly lower activity than the sebaceous gland. The enzyme activity was negligible in the epidermis, while it was detected in the dermis though the values determined were variable probably because of contamination with other components such as sweat glands and hair follicles. Thus, the present study demonstrates that the 5 alpha-reductase activity is mainly located in the apocrine sweat gland and sebaceous gland. This suggests that 5 alpha-reduction of testosterone is an important step in mediating the action of androgens in these tissues.

Epidermal cell culture using Sephadex beads coated with denatured collagen (cytodex 3).

Epidermal cell culture using microcarriers of Sephadex beads coated with denatured collagen (cytodex 3) was performed. Epidermal basal cells (above 95%) obtained from human skin by trypsinization were cultivated statically on the beads in 96-well culture plates. Proliferation was rapid and great in synchronous waves during 2-7 days after inoculation. The growth rate depended on the inoculation cell population densities. When cells were inoculated at 7.75 X 10(4)/well, the maximum increase was 3.6-fold and at 1.69 X 10(4)/well and 0.32 X 10(4)/well, 2.5-fold and 2.1-fold, respectively. Differentiation was assessed visually on a hemocytometer. The percentage of basal cells of the total cells present in each well was reduced from 98% (on inoculation) to approximately 25% in a week and thereafter. In 1 month after inoculation, cells with keratohyaline-like granules were observed at 12%. The attachment of cells to the beads was rather loose. Cells were supposed to be attached to denatured collagen on the beads via fibronectin contained in the serum of the medium, because denatured collagen had the property to bind strongly to fibronectin. Loose attachment made it possible to harvest intact cells without the use of trypsin. This cell culture system with such new characteristics will be a useful tool for studying epidermal cell biology and biochemistry.

Characterization of 5 alpha-reductase in cultured human dermal papilla cells from beard and occipital scalp hair.

In order to gain a deeper insight into the role of 5 alpha-reductase in the growth of beards in men, we studied some kinetic properties of the enzyme in cell homogenates of cultured human dermal papilla cells from beard and occipital scalp hair. When cell homogenates were incubated with [3H]-testosterone, the 5 alpha-reductase of beard dermal papilla cells exhibited an optimum activity at pH 5.5, whereas the enzyme of dermal papilla cells from occipital scalp hair showed a broad and low plateau between pH 6.0 and 9.0, without a sharp peak. The apparent Michaelis constant of 5 alpha-reductase was 3.3 x 10(-7) M in dermal papilla cells from beard and 2.4 x 10(-5) M in those cells from occipital scalp hair. The apparent Km of 5 alpha-reductase for NADPH was 2.8 x 10(-5) M and 7.6 x 10(-4) M in beard and occipital scalp hair dermal papilla cells, respectively. There were no significant differences in the substrate specificity between these two types of cells. The 5 alpha-reductase activity was recovered mainly in the nuclear fraction of beard dermal papilla cells. By contrast, it was widely distributed among the individual subcellular fractions of dermal papilla cells from occipital scalp hair. These results strongly suggest that these two kinds of dermal papilla cells have different types of 5 alpha-reductase, and that the enzyme in beard dermal papilla cells is similar in characteristics to that in the androgen target organs such as prostate.

Human genital melanocytes as androgen target cells.

As some parts of human skin - such as genital and areolar skin - become pigmented after puberty, melanocytes in these regions are thought to be sex hormone target cells. We immunohistochemically localized androgen receptors in the nuclei of cultured human genital melanocytes by monoclonal and polyclonal antibodies. When these cells were incubated with [1,2-3H]-testosterone, the major metabolite in the medium was dihydrotestosterone and 5alpha-reduction predominated over 17beta-oxidation. Androgen receptor and type I 5alpha-reductase mRNAs could be detected in genital melanocytes by reverse transcriptase polymerase chain reaction. The tyrosinase activity was stimulated by the addition of androgen. This stimulation was antagonized by cyproterone acetate, whereas tyrosinase mRNA expression was not affected by androgen. These results indicate that human genital melanocytes are androgen target cells, and that androgen plays a role for pigmentation in the specific regional skin after puberty.

Vitamin C abrogates the deleterious effects of UVB radiation on cutaneous immunity by a mechanism that does not depend on TNF-alpha.

Acute low-dose treatment of murine skin with ultra violet B (UVB) light impairs induction of contact hypersensitivity (CH) to dinitrofluorobenzene (DNFB) in certain inbred strains of mice (termed UVB-susceptible), but not in others (termed UVB-resistant), and promotes tolerance. These deleterious effects of ultraviolet radiation (UVR) are mediated in part by TNF-alpha, which is released from UVR-exposed epidermal and dermal cells. Because UVR damage to skin has also been ascribed in part to the generation of reactive oxygen intermediates (ROIs) such as superoxide anion (O2-), hydrogen peroxide (H2O2), hydroxyl radical (OH-), and singlet oxygen ((1)O2), we investigated whether vitamin C (ascorbic acid), which can nullify ROIs, prevents the deleterious effects of UVR on the cutaneous immune system. We found that epicutaneous application of vitamin C (10% L-ascorbic acid solution) abrogated the deleterious effects of acute low-dose UVR on induction of CH and prevented the induction of tolerance. Vitamin C, however, did not reverse the effects of TNF-alpha on CH induction and tolerance. These results indicate that (i) ROIs generated intracutaneously by UVR contribute to the impaired ability of exposed skin to support the induction of CH and to promote the induction of tolerance and (ii) these effects are not dependent on TNF-alpha.

Forskolin activates adenylate cyclase activity and inhibits mitosis in in vitro in pig epidermis.

The novel adenylate cyclase activator forskolin caused rapid and high intracellular accumulation of cyclic AMP in a floating skin (epidermal) slice system. Increased cAMP levels were also detected in the media. Addition of a phosphodiesterase inhibitor to forskolin-containing medium caused only a slight increase in the intracellular cAMP level and forskolin itself did not inhibit phosphodiesterase activity. Ka of forskolin for epidermal adenylate cyclase was about 2-3 X 10(-5) M. This forskolin activation was rapidly reversed after washing. The forskolin stimulation (Ka 5 X 10(-5) M) was also found when tested with an epidermal membrane preparation which contained the catalytic unit of adenylate cyclase but lacked either the GTP or receptor stimulation. With the epidermal slice system, the combination of forskolin and epinephrine (or histamine) stimulated adenylate cyclase synergistically. The data suggest that forskolin activates not only the catalytic unit but also the nucleotide regulatory protein or the receptor-regulatory protein complex of the adenylate cyclase system. The cAMP accumulation caused by forskolin produced a dose-dependent mitotic inhibition of epidermal cells in an in vitro outgrowth system. This inhibitory effect was reversible 48 h after washing out the forskolin.

Calcium-activated, phospholipid-dependent protein kinase in pig epidermis.

We investigated calcium-activated, phospholipid-dependent protein kinase in pig epidermis. Pig epidermal homogenates were centrifuged at 30,000 g for 30 min, and the supernatant was applied on a DEAE-cellulose column for purification. The partially purified enzyme was stimulated by simultaneous addition of Ca2+ and phospholipid. Successive addition of small amounts of diolein further activated the enzyme activity. The calcium-activated phospholipid-dependent protein kinase preferentially phosphorylated serine residues and its endogenous substrate protein in the pig epidermis has a molecular weight of about 97,000.

A cell-spreading inhibitor exists in serum and in epidermal basal cells.

A unique monoclonal antibody was obtained by immunizing mice with complement-inactivated fetal bovine serum (FBS). This antibody, named SI-1, stained epidermal basal cells of humans, pig, guinea pig, and rat by an indirect immunofluorescence technique after pretreatment of cryostat sections with alkali buffer (pH 9.6). After dissociating pig epidermal cells by trypsin, the SI-1 antibody stained exclusively and strongly one type of uniquely shaped cells. They were small and hanging-bell or columnar in shape with one convoluted side on the base, consisting of less than 2.8% of the dissociated epidermal cell population. The antigen contained in FBS was partially purified by affinity chromatography using the SI-1 antibody. The affinity-purified antigen inhibited the spreading of PAM cells, a spontaneously transformed murine keratinocyte line, in serum-free medium in a dose-dependent manner at concentrations of 10(-5) to 10 ng/ml. The antigen also inhibited the spreading of trypsinized pig epidermal cells in the range of 10(-2) to 10(3) ng/ml in the presence of 0.05% FBS. Although there have been a few reports indicating that serum inhibited both spreading and attachment, a specific factor in serum has not been purified before. This is, to our knowledge, the first presentation of a cell-spreading inhibitor contained in serum.

Impaired responses of peripheral blood mononuclear cells to staphylococcal superantigen in patients with severe atopic dermatitis: a role of T cell apoptosis.

Staphylococcus aureus colonization is an almost universal feature of atopic dermatitis. In order to investigate the role of staphylococcal enterotoxin B in the pathogenesis of atopic dermatitis, we assessed the correlation between clinical disease severity and proliferative response of peripheral blood mononuclear cells to staphylococcal enterotoxin B in patients with atopic dermatitis. Peripheral blood mononuclear cells from patients with mild atopic dermatitis showed significantly increased proliferative responses to staphylococcal enterotoxin B compared to controls. In contrast, peripheral blood mononuclear cells from patients with severe atopic dermatitis showed markedly suppressed proliferative responses. Additionally, longitudinal evaluation of peripheral blood mononuclear cell samples from the same patient demonstrated that proliferative responses were suppressed only at times of severe disease exacerbation. Mixing experiments, using autologous T cells and antigen presenting cells that were isolated at different time points from the same patient, demonstrated that T cells of severe atopic dermatitis patients were dysfunctional, but their antigen presenting cell function remained intact. We found no significant differences of interleukin-2 levels in the culture supernatants between healthy controls and atopic dermatitis groups. Fluorescence-activated cell sorter analysis for APO2.7 antigen, an early apoptosis cell marker, demonstrated that approximately 60% of staphylococcal-enterotoxin-B-stimulated T cells expressed APO2.7 antigen in severe atopic dermatitis cases. By contrast, 5%-20% of T cells expressed APO2.7 after staphylococcal enterotoxin B stimulation in cases of mild atopic dermatitis and in healthy controls. Nuclear staining with Hoechst 33258 also showed approximately 40% apoptotic cells in the CD19-CD16-PBMC of severe atopic dermatitis patients, compared with only 5%-10% in the mild atopic dermatitis group and in healthy controls. Blocking monoclonal antibody to Fas ligand partially prevented the staphylococcal-enterotoxin-B-induced apoptosis detected by APO2.7 expression and Hoechst 33258 staining. Suppressed proliferation of peripheral blood mononuclear cells in severe atopic dermatitis patients may be secondary to T cell death by apoptosis. These results suggest that an infection of S. aureus producing staphylococcal enterotoxin B may play a role in aggravation of atopic dermatitis by inducing apoptosis in T cells.

Regulation of keratin 9 in nonpalmoplantar keratinocytes by palmoplantar fibroblasts through epithelial-mesenchymal interactions.

Palms and soles differ from other body sites in terms of clinical and histologic appearance, response to mechanical stress, and the distribution of keratin 9. Because keratin 9 is exclusively expressed in the palmoplantar suprabasal keratinocyte layers, it is considered a differentiation marker of palms and soles. We studied palmoplantar mesenchymal influences on keratin 9 induction in nonpalmoplantar epidermis. Although palmoplantar keratinocytes when cultured alone continued to express keratin 9 mRNA in 12 (100%) of 12 cultures, nonpalmoplantar keratinocytes did not express it in 16 of 17 cultures. Although nonpalmoplantar keratinocytes did not express keratin 9 mRNA when cultured with nonpalmoplantar fibroblasts, they did express it within 2 h in cocultures with palmoplantar fibroblasts derived from papillary dermis. Grafting of these coculture sheets on severe combined immunodeficient mice resulted in an epidermis, which histologically showed hyperkeratosis and acanthosis and immunohistochemically expressed keratin 9. Furthermore, pure epidermal sheets from nonpalmoplantar skin grafted on the human sole wounds due to burn, injury, and the resection of acral lentiginous melanoma, demonstrated adoption of palmoplantar phenotype and expressed keratin 9. Our report indicates extrinsic keratin 9 regulation by signals from dermal fibroblasts. This is also the first to suggest the possibility of treating palmoplantar wounds with nonpalmoplantar epidermis, which is much easier to obtain and harvest.

Mechanism of action of androgen in hair follicles.

In order to investigate the mode of action of testosterone (T) on human hair follicles we studied the metabolism of T and localization of androgen receptors in outer root sheath cells (ORSC) and dermal papilla cells (DPC) from different body sites. T was principally metabolized to androstenedione (delta 4) even in beard ORSC as well as epidermal keratinocytes (EK), and the ratio of apparent 5 alpha-reductase (5 alpha-R) to 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) did not differ between these two kinds of cells. The 5 alpha-R activity in beard DPC was 3 times as high as that in occipital scalp and axillary DPC. The 5 alpha-R of beard DPC exhibited a narrow optimum pH of 5.5, which is characteristic of type 2 enzyme present in androgen target cells. In contrast, 5 alpha-R of DPC from axillary and occipital scalp hair showed a broad optimum pH range between 6.5-9.0 corresponding to type 1 5 alpha-R. Androgen receptors were detected in the DPC of beard and axillary hair follicles, but not in those of occipital scalp hair follicles using immunohistochemical staining with polyclonal anti-androgen receptor antibody. Epithelial cells of the hair bulb were not stained by the antibody. Androgen receptors were also detected in the nuclei of cultured beard and axillary DPC, but the DPC from occipital scalp hair follicles showed little staining with the antibody. We also examined the effects of T on the DNA synthesis and proliferation of cultured ORSC and DPC. T did not have a proliferative effect on either type of cell when cultured alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Testosterone metabolism by cultured human beard outer root sheath cells in comparison with epidermal keratinocytes.

In order to elucidate the mechanism of action of androgen in human hair follicles, we studied testosterone metabolism by cultured beard outer root sheath cells in comparison with that of epidermal keratinocytes. When cells were incubated as a monolayer in serum-free keratinocyte growth medium with physiological concentrations (25 nM) of [3H]testosterone, the major metabolite was androstenedione in either type of cells. Small amounts of androstanedione, dihydrotestosterone and androsterone were also formed. The ratio of apparent 5 alpha-reductase to 17 beta-hydroxy-steroid dehydrogenase activities ranged from 0.11 to 0.81 and did not differ between these two kinds of cells. After these cells were cultured in the presence of 10% fetal calf serum for 10 days, the activities of both metabolic pathways markedly increased without significant changes of the ratio of activities of these two enzymes. Thus, testosterone was rather converted to weak androgens even in beard outer root sheath cells under the experimental conditions.