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Summary Background Androgenetic (male-type) alopecia (AGA) is caused by genetic and androgenetic effects. The progression of baldness results in smaller hair papillae, thinner hair and a shortened hair cycle. Alopecia occurs mainly in the frontal region and, to a lesser extent, in the occipital region. Objectives The morphological differences in the hair follicular units between the alopecic frontal scalp and the vertex and occipital regions were compared using cross-sectional histology and three-dimensional reconstruction. Methods Skin specimens were obtained from the frontal, vertex and occipital regions of 24 male human cadavers with fully progressed AGA, and from the frontal region of 32 normal cadaveric scalps. These specimens were fixed, processed using routine histological methods, serially sectioned at a thickness of 10 μm and then stained with Masson's trichrome. The serial sections were reconstructed three-dimensionally using ‘Reconstruct’ software. Results The ratios between the numbers of terminal and vellus hairs in the frontal and occipital regions in the AGA scalps were 0·2 : 1 and 3·5 : 1, respectively. Almost all of the hair follicles in the frontal region were vellus hair follicles. The sebaceous gland and arrector pili muscle were larger in the frontal region than in the occipital region. Conclusions The morphology of the AGA scalp has been characterized. The terminal-to-vellus hair ratio in the occipital (normal) region was different from that in the frontal (alopecic) region. Moreover, sebaceous glands were larger in the frontal alopecic region than in the occipital region. These larger glands may be associated with other dermatological pathologies, such as seborrhoeic dermatitis.

Summary Background Blue and red light have been reported to have beneficial effects on acne. However, there has been no double-blind, randomized study of acne treatment for combined blue and red light-emitting diode (LED) devices, and the associated molecular mechanisms have rarely been investigated. Objectives To evaluate the efficacy, safety and histological changes of combined blue and red LED phototherapy for acne vulgaris. Methods Thirty-five patients with mild-to-moderate acne were randomly assigned to either a home-use irradiation group using an LED device, or a control group using a sham device. The treatment group was instructed to serially irradiate their forehead and cheeks with 420-nm blue light and 660-nm red light for 2·5 min twice daily for 4 weeks. Results At the final visit at 12 weeks, both inflammatory and noninflammatory acne lesions had decreased significantly, by 77% and 54%, respectively, in the treatment group. No significant difference was observed in the control group. In the treatment group, sebum output reduction, attenuated inflammatory cell infiltrations and a decreased size of the sebaceous gland were found. The immunostaining intensities for interleukin (IL)-8, IL-1α, matrix metalloproteinase-9, toll-like receptor-2, nuclear factor-κB, insulin-like growth factor-1 receptor and sterol response element binding protein (SREBP)-1 were reduced concomitantly. Messenger RNA expression of SREBP-1c was also decreased. No severe adverse reactions were reported. Conclusions This LED phototherapy was safe and effective for treating not only inflammatory but also noninflammatory acne lesions, with good compliance. The experimental results correlated well with clinical results, partly elucidating the related molecular mechanisms.

Recent work has focused on the hair follicle as the main source of multipotent stem cells in the skin. The hair follicle bulge contains multipotent stem cells that can form the epidermis, hair follicles and sebaceous glands and help in repopulation of the epidermis after injury. The localization of these stem cells to the bulge area may explain why some types of inflammatory alopecia cause permanent loss of hair (cicatricial alopecia) (such as lichen planopilaris and discoid lupus erythematosus), while others (such as alopecia areata) are reversible (noncicatricial alopecia). The lack of distinctive bulge morphology in human hair follicles has hampered studies of bulge cells. To date, the best marker for bulge stem cells in human hair is cytokeratin (CK) 15; human bulge cells have been reported to express CK15 selectively throughout all stages of the hair cycle in different types of follicles. There is direct evidence in the mouse, and indirect evidence in the human, that compromising the integrity of the sebaceous gland and/or bulge is important in the development of alopecia. Several interesting studies have been done in the last few years to investigate the role of stem cells in alopecia, especially nonscarring types. This is a review about the role of stem cells in the pathogenesis of alopecia (scarring and nonscarring).

Background Isotretinoin’s (13-cis RA) full mechanism of action in treating acne is unknown. 13-cis RA induces key genes in sebocytes that are involved in apoptosis. In this study, we report that 13-cis RA up-regulates expression of Tumor necrosis factor Related Apoptosis Inducing Ligand (TRAIL) within SEB-1 sebocytes.

Summary Background The pathogenesis of hidradenitis suppurativa (HS) is not clearly understood. The nomenclature suggests an important role for the apocrine glands but recent evidence implicates the pilosebaceous unit as a more likely candidate to play a central role in the pathogenesis. Objectives Our aim was to estimate the volume of the follicular epithelium, the follicular lumen and the sebaceous glands of patients with HS and healthy controls by means of stereology. Methods Four-millimetre punch biopsies were taken from 21 patients with HS and nine healthy controls, fixed in formalin, embedded in paraffin and stained with haematoxylin and eosin prior to volume estimation using the Cavalieri principle. Results Sebaceous gland tissue could be visualized in only 10 of 15 suitable hair follicle biopsies from patients with HS but was present in all biopsies from healthy controls (P = 0·05) and the mean sebaceous gland volume per follicle was one-seventh of that of healthy controls (P = 0·03). There was no significant difference between patients with HS and healthy controls with regard to follicular epithelium and follicle lumen volume. Conclusions Our results suggest that the absence or reduced volume of the sebaceous gland may play a role in the pathogenesis of HS. The presence of fibrosis suggests that sebaceous glands are obliterated early in the pathogenesis of HS.

Skin is the largest human organ, accounting for approximately 12-15% of body weight.1 Volatile organic compounds (VOCs) emanating from skin contribute to a person’s body odour, and may convey important information about metabolic processes. VOCs from skin derive from eccrine, sebaceous and apocrine gland secretions and their interactions with resident skin bacteria.2,3 These glands are distributed differently across the body; hence different regions of the body have different VOC profiles, and thus different odours. Eccrine glands are found throughout the skin, but are especially concentrated in palms of hands, soles of feet, and the forehead. Eccrine sweat is mostly water, but contains glycoproteins (notably interleukin 1), lactic acid, sugars, amino acids and electrolytes.4 Sebaceous glands are concentrated on the upper part of the body.3 The upper chest, back, scalp, face and forehead may have as many as 400-900 sebaceous glands cm-2. Sebaceous gland secretions are rich in lipid materials such as cholesterol, cholesterol esters, long-chain fatty acids, squalene and triglycerides.3 These lipids provide substrate for growth and metabolism of skin microorganisms. Apocrine glands are concentrated in the axillae, pubic area and areolas.2,4 Apocrine secretions are the chief source of underarm odorants (commonly known as ‘body odour’) and play a role in chemical signalling (for a review see Wysocki and Preti.5) Many previous studies have focused on VOCs emanating from the axillae, which reflect some contribution from all skin glands located in the axillae.6-10 VOCs from nonaxillary skin secretions have been studied as potential mosquito attractants,11-13 indicators of seasonal changes14 and ageing,15 and moderators of fragrances.16-19 It was recently demonstrated that skin emanations could be collected via rolling a stir-bar coated with polydimethylsiloxane across the arm with subsequent desorption and analysis by gas chromatography/mass spectrometry (GC/MS).20 Bernier et al.11-13 reported that hundreds of compounds canbe volatilized from skin secretions collected from the palms and backs of hands. Most of these compounds have been documented to be organic acids ranging in carbon size from C2 to C20. However, the most abundant (75-80%) organic acids found on skin are C16 and C18 saturated, monounsaturated and diunsaturated acids,12 which are not volatile at body temperatures. In contrast, collection of skin VOCs using solid-phase microextraction (SPME) will collect low molecular weight compounds that are volatile at body temperature. SPME-GC/MS analyses of hand/wrist VOCs sampled in both winter and spring revealed 35 organic compounds.14 VOCs were reported to be more abundant in winter samples; however, the relative ratios of many (but not all) of the compounds did not vary between seasons. This observation led the authors to speculate that the moist spring air allowed the skin to harbour more bacteria that hydrolysed and decomposed some of the VOCs. A study of male Japanese subjects used T-shirts worn for 3 days to collect skin odours. VOCs emanating from rectangular pieces cut from the backs of these T-shirts were studied.15 The authors suggested that skin secretions in men older than age 39 years contain larger amounts of unsaturated aldehydes than secretions from younger men. These compounds, particularly 2-nonenal, were reported to impart an unpleasant ‘ageing odour’ to older Japanese men. Most acids, alcohols and aldehydes found in skin secretions apparently originate from the interactions between sebaceous gland secretions and cutaneous bacteria.12,21 Anaerobic bacteria living in the hair follicle/sebaceous gland duct use lipases to liberate long-chain acids from triglycerides, which are further metabolized by aerobic bacteria into longer, saturated and unsaturated acids and smaller volatile acids, aldehydes and alcohols.21 Other potential sources of skin volatiles are putative odorants carried by the apocrine secretion odour-binding protein (ASOB) 1.22 This protein might bind odorous acids that can be liberated by interaction with cutaneous bacteria. A previous study demonstrated that this protein does carry odorants in the underarm, and is present in sebaceous-rich perspiration from the forehead.22 Tracking dogs trained to recognize an individual’s scent on garments from a particular body part (e.g. chest, arm, leg) are not reliably able to generalize the individual’s odour to other body parts, suggesting that a different odorant milieu may characterize the back and forearm (for a review see Jenkins23). In this study, we performed a comprehensive analysis of VOCs from the upper back and forearm of 25 healthy subjects using two complementary sampling techniques, SPME and solvent extraction. Skin secretions from the back of each subject also were analysed for the presence of the potential VOC carrier ASOB1. The goals of the study were: (i) to identify VOCs emanating from the upper back and forearm skin; (ii) to determine whether VOCs vary in a qualitative or quantitative manner; and (iii) to determine if VOCs vary in relative abundance with gender, age or locus, i.e. back and forearm. The upper back and forearm were chosen because they are easily accessible with minimal inconvenience to subjects. In addition, these body regions differ with respect to density of sebaceous glands (upper back > forearm) and skin bacteria, as well as exposure to sun, environment and consumer products.3,21

New models of the structural relationship between the arrector pili (AP) muscle and the sebaceous gland (SG) have been proposed recently.The purpose of the present study was to establish the actual morphological relationship between components of the follicular unit (FU) including the hair follicles, AP muscle and SG using 3D reconstruction of serially sectioned specimens so as to expand previous explanations of the secretory mechanism of the SG and to suggest other possible mechanisms based on newly proposed model.Scalp skin specimens were processed using routine histological procedures, with serially sectioned tissue slides being stained with Masson's trichrome. 'Reconstruct' software was used to align, assemble and reconstruct the sections, with observations of the 3D-reconstructed FU [including hair follicles (HFs), AP muscle and SG].Fifty FUs were reconstructed. The AP muscle was curved and concave as it supported the basal portion of the sebaceous lobules in the perifolliculum. Sebaceous lobules were located between the AP muscle and HFs (angular area) and some sebaceous lobules located in the opposite (counter-angular) area.We propose that the concave part of the AP muscle pushes up the basal portion of the sebaceous lobule between the HFs and AP muscle during AP muscle contraction and hair erection. In addition, the sebaceous lobule located at the counter-angular position is squeezed by the HF during AP muscle relaxation and hair repositioning. Combined with the previous mechanism of SG secretion, this newly established mechanism based on the 3D structure of the FU will improve our understanding of AP muscle function and SG secretion.

BACKGROUND Despite the clinical evidence that androgens stimulate sebaceous lipids, androgens in vitro have shown no similar effects. This contradiction led to the assumption that cofactors may be required for lipid regulation and peroxisome proliferator-activated receptor (PPAR) ligands were suggested to be adequate candidates. OBJECTIVES The influence of testosterone and linoleic acid, a PPAR ligand, as single agents and in combination with of LY191704, a 5alpha-reductase type I inhibitor, was examined on 5alpha-dihydrotestosterone (5alpha-DHT) synthesis and lipid content in human SZ95 sebocytes. METHODS Cell proliferation and viability were measured by the 4-methylumbelliferyl heptanoate fluorescence assay and by the Boehringer Lactate Dehydrogenase Assay kit, respectively. 5alpha-DHT enzyme-linked immunosorbent assay was used for the detection of 5alpha-DHT synthesis in cell supernatants after treatment, whereas lipid production was documented by means of the Nile red lipid microassay and fluorescence microscopy. RESULTS Testosterone promoted 5alpha-DHT synthesis (P < 0.001), whereas linoleic acid increased sebaceous lipids (P < 0.001). The combination of testosterone and linoleic acid exhibited a synergistic effect on the synthesis of 5alpha-DHT (P < 0.01 vs. testosterone) and sebaceous lipids (P < 0.01 vs. linoleic acid). Furthermore, LY191704 reduced 5alpha-DHT and sebaceous lipid levels (P < 0.01 and P < 0.001 in comparison with testosterone/linoleic acid, respectively). Cell proliferation and viability remained unchanged under treatment with all compounds tested. CONCLUSIONS These data suggest a catalytic effect of PPAR ligands on cellular testosterone activation by 5alpha-reduction and the importance of the latter for the regulation of sebaceous lipids.

Summary Background Retinoids and active vitamin D3 analogues regulate the proliferation and differentiation of keratinocytes and are effective in the treatment of psoriasis. Retinoids are known to be effective against acne vulgaris through comedolysis. However, the comedolytic effect of active vitamin D3 analogues has not been reported. Objectives To investigate whether maxacalcitol, one of the active vitamin D3 analogues, has a comedolytic effect by using spontaneously comedogenic rhino mice. Methods Rhino mice were treated topically with tretinoin and maxacalcitol once daily for 2 and 4 weeks, respectively. The dermal side of the epidermal sheet was observed to determine the size of the utricle. Haematoxylin and eosin-stained vertical sections were used to measure utricle diameter and density and to evaluate histological changes. Results Maxacalcitol (25 μg g−1) and tretinoin (0·1%) significantly decreased the size and the diameter of the utricle after 1 week of treatment. However, maxacalcitol did not affect the density while tretinoin did have an effect. Histopathologically, maxacalcitol and tretinoin markedly induced epidermal hyperplasia accompanied by a minor accumulation of inflammatory cells in the dermis, with and without hypercornification, respectively. Conclusions These results indicate that maxacalcitol has a prominent effect on comedolysis and that its mechanism of action may be different from that of retinoids.

Summary We have previously developed methods for the isolation and maintenance of human sebaceous glands and hair follicles. However, in long-term culture the maintenance of both is suboptimal. This may be due to a lack of stem cells, which are thought to be located in the bulge area of the hair follicle, and this region is not present in either model. Isolation of the entire pilosebaceous unit would retain this region, and may lead to improved maintenance of both structures. We describe a method for the isolation of viable, individual, pilosebaceous units by microdissection from human scalp face-lift skin. The viability of isolated pilosebaceous units has been determined by light microscopy, patterns of DNA synthesis by [methyl-3H] thymidine autoradiography, and lipogenesis by [U-14C] acetate uptake into lipids. When maintained for 7 days in supplemented Williams E medium, isolated pilosebaceous units showed a significant increase in length. This was due to the production of a keratinized hair fibre which grew at the in vivo rate of 0.3 mm/day. Light microscopy and [methyl-3H] thymidine autoradiography confirmed that after 7 days maintenance the hair follicle retained apparently normal morphology and patterns of DNA synthesis. However, the morphology of the sebaceous gland on maintenance was more variable, generally showing luminal keratinization. Moreover [methyl-3H] thymidine autoradiography of sebaceous glands showed a marked reduction on maintenance. The rates and patterns of lipogenesis by the whole pilosebaceous unit were, respectively, lower and different from those seen with isolated human sebaceous glands: this indicates that the bulk of pilosebaceous lipogenesis is derived from the hair follicle. Rates of recovery of [14C] from 2 mM-[U-14C] sodium acetate into thin-layer chromatography plates after 7 days maintenance decreased, although this was not statistically significant, indicating that rates of lipogenesis may fall on maintenance. Pilosebaceous units were maintained for 7 days on Gelfoam (an absorbable gelatin sponge) at the media-air interface. Initial results show a marked improvement in sebaceous gland morphology. It is possible, therefore, to obtain viable human pilosebaceous units by microdissection, and to maintain them in vitro for up to 7 days, with apparently full retention of hair follicle function, but only partial retention of sebaceous gland function.

Summary Background It is generally believed that the sebaceous gland is an intracrine organ which synthesizes its own active hormones to meet its local needs. Objectives To understand further the mechanisms of sex steroid action in mouse sebaceous glands. Methods We have used immunocytochemistry to examine the expression of oestrogen receptor α (ERα) and androgen receptor (AR) in mouse sebaceous glands. Results In intact males AR is exclusively localized in the nuclei of basal and mature sebocytes, while in females it is present at a lower level in both the nuclei and the cytoplasm. Three weeks following gonadectomy (GDX), a marked decrease of AR labelling is observed in male sebocytes, while no change occurs in female sebocytes. Treatment of GDX animals with dihydrotestosterone (DHT) or dehydroepiandrosterone (DHEA) increases AR expression, while 17β-estradiol (E2) decreases the stimulatory effect of DHT and DHEA. ERα is detected only in basal sebocytes of intact females but not in males. Following treatment with E2, ERα expression becomes visible in GDX males while DHT and DHEA inhibit the effect of E2. Conclusions The present data show gender differences and demonstrate that DHT, E2 and DHEA exert specific effects on the expression of AR and ERα in mouse sebocytes.

Although diabetes mellitus is known to induce many pathophysiological changes in the skin, none of the earlier studies has focused on the state of the stratum corneum (SC) in patients with diabetes. In our previous report on a diabetic mouse model, we described a decreased SC hydration state and abnormalities in the function of the sebaceous gland together with a normally retained barrier function of the SC.To examine the functional changes of the SC in patients with diabetes.The following values were measured in 49 patients with diabetes: fasting plasma glucose (FPG) and haemoglobin A1c (HbA1c); skin surface lipid level on the forehead; and high-frequency conductance (HFC) and transepidermal water loss (TEWL) of the SC of the volar forearm and extensor surface of the lower leg. These functions of the SC were compared in two pairs of patient groups, i.e. between groups with high and low FPG, and between groups with high and low HbA1c.The HFC levels on the extensor leg and volar forearm were significantly lower in the group with high FPG (FPG110 mg dL(-1)) than in the group with low FPG (FPG110 mg dL(-1)), although TEWL values were almost the same in these two groups. The skin surface lipids on the forehead were significantly lower in the former than in the latter. The HFC did not differ significantly between the group with high HbA1c (HbA1c5.8%) and the group with low HbA1c (HbA1c5.8%), whereas the TEWL of the volar forearm was slightly lower in the former than in the latter.These new results as well as our earlier results with diabetic mice suggest that patients with diabetes mellitus tend to show a reduced hydration state of the SC together with decreased sebaceous gland activity, without any impairment of the SC barrier function.

Summary Background The skin and its appendages support aerobic glycolytic and glutaminolytic metabolism. Their major fuels are glucose and glutamine, which are, however, largely catabolized anaerobically. Objectives For the human sebaceous gland it has been reported that glucose, lactate and acetate provide good lipogenic substrates but that glutamine does not. Therefore, we have investigated the intermediary metabolism in vitro of freshly isolated human sebaceous glands to determine if their metabolism of glutamine is anomalous relative to the rest of the skin. Methods Glycolytic rate, glucose and glutamine oxidation, and glucose metabolism by the pentose phosphate pathway were determined in freshly isolated human chest sebaceous glands. Further, sebaceous intermediary metabolites were analysed using spectrophotometry and high-performance liquid chromatography. Moreover, glands were maintained in vitro as whole organs to investigate the effects of precursors and inhibitors of polyamine synthesis on rates and patterns of lipogenesis and DNA synthesis. Results We confirm that the human sebaceous gland is a glycolytic and glutaminolytic tissue. Glucose is mainly converted to lactate, with only 6% of glucose being oxidized to CO2. Glutamine is largely converted to glutamate, alanine, serine, glycine, aspartate, threonine, lactate and ammonia, with only 12% being oxidized. We have also shown that exogenous glutamine is required for cellular proliferation and lipogenesis by human sebaceous glands. However, in its absence spermidine could fully restore rates of DNA synthesis and lipogenesis. Conclusions Although glutamine is a poor substrate for sebaceous lipogenesis, this cannot be attributed to its lack of catabolism. We have shown that glutamine is an essential fuel, but that it can be replaced by exogenous spermidine. Therefore, we suggest that in sebocytes both glutamine and spermidine may act as essential purine and pyrimidine precursors.

Summary Background Although hair greying is a very common phenomenon characterized by loss of pigment in the hair shaft, the events that cause and control natural hair whitening with age in humans are still unclear. Objectives To decipher the origin of natural hair whitening. Methods Human hair melanocytes were immunohistochemically characterized at different stages of whitening. Results Loss of hair shaft melanin was found to be associated with a decrease in both bulb melanin content and bulb melanocyte population. Although few melanocytes were present in the bulbs of grey hair, they still expressed tyrosinase and tyrosinase-related protein-1, synthesized and transferred melanins to cortical keratinocytes as seen by the presence of melanin granules. In white hair bulbs, no melanocytes could be detected either with pMel-17 or vimentin labelling. Pigmented hair follicles are known to contain inactive melanocytes in the outer root sheath (ORS), and grey and white hairs were also found to contain some of these quiescent melanocytes. However, their population was decreased compared with pigmented hair follicles, ranging from small to nil. This depletion of melanocytes in the different areas of white hairs was detected throughout the hair cycle, namely at telogen and early anagen stages. In contrast, the infundibulum and sebaceous gland of both pigmented and white hairs showed a similar distribution of melanocytes. Furthermore, other distinct cell populations located in the ORS, namely putative stem cells, Merkel cells and Langerhans cells were equivalently identified in pigmented and white hairs. Conclusions Thus, hair greying appears to be a consequence of an overall and specific depletion of bulb and ORS melanocytes of human hair.

SummaryBackground Acne lesions spontaneously remit, but the mechanism of this remission has not been elaborated. It is known, however, that the remission is associated with a de-differentiation of sebocytes, causing a cessation of sebum secretion specific to that particular pilosebaceous unit. We have previously described the cytokines that will promote in vitro the lesions of acne. Objectives To show that those same cytokines may also promote a de-differentiation of sebocytes analogous to that seen during remission of some lesions. Methods Human chest sebaceous glands were maintained in vitro as whole organs. We then chronicled the effects of the appropriate cytokines and growth factors on the glandular rates of (i) lipogenesis and (ii) DNA synthesis, as well as on (iii) glandular morphology, (iv) the expression patterns of the proliferation marker Ki-67, (v) keratinocyte-specific markers, and (vi) the sebocyte marker epithelial membrane antigen. Results We have shown that the same cytokines that promote comedogenesis (interleukin-1α), expression of infundibular intercellular adhesion molecule-1 and human leucocyte-associated antigen-DR (tumour necrosis factor-α and interferon-γ), and infundibular disruption (epidermal growth factor/transforming growth factor-α) in human infundibula in vitro, will also inhibit sebaceous lipogenesis in vitro and will also induce, histologically, a de-differentiation of human sebocytes into a keratinocyte-like phenotype. Conclusions These results confirm our hypothesis that the cytokines that induce the infundibular changes in acne may also inhibit the secretion of lipid from the sebaceous gland and thus, on diffusing down to the gland, contribute to the remission of the individual lesions. These findings help to explain the known natural history of the disease.

Background The fact that Pityrosporum ovale plays a part in seborrhoeic dermatitis is well established but the mechanism of this relationship has not been established. Objectives To compare the number and type of inflammatory cells and mediators in skin biopsies from normal and lesional skin from the trunk and scalp in patients with seborrhoeic dermatitis, Pityrosporum (Malassezia) folliculitis and in normal skin from healthy controls. Methods The skin biopsies were stained using the labelled Streptavidin-biotin method. The following markers were studied: CD4, CD8, CD68, HLA-DR, NK1, CD16, Clq, C3c, IgG, CD54 (ICAM-1), interleukin (IL) -1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, tumour necrosis factor-a and interferon-γ. Results HLA-DR+ cells were seen in the highest number, and were higher in lesional skin compared with normal skin from both patients and healthy volunteers, ICAM-1 expression was also increased in lesional skin. Clq and the interleukins showed an increased cellular and intercellular staining in patients compared with healthy controls and the intercellular staining was often more intense in lesions compared with non-lesional skin. Staining was often more intense when Malassezia (Pityrosporum ovale) yeast cells were present. Conclusions An increase in NK1+ and CD16+ cells in combination with complement activation indicates that an irritant non-immunogenic stimulation of the immune system is important. The result with the interleukins showed both an increase in the production of inflammatory interleukins as well as in the regulatory interleukins for both T H 1 and T H 2 cells. Similarities to the immune response described for Candida albicans infections indicate the role of Malassezia in the skin response in seborrhoeic dermatitis and Pityrosporum folliculitis.

p21WAF is a cyclin-dependent kinase inhibitor which is widely expressed in epidermal structures. Using a combination of double immunocytochemical staining and combined in situ hybridization, we show that there is a striking exclusivity between the expression of Ki67 and p21WAF in the hair matrix. Some cells that are Ki67-positive also express p53, but as they exit the cell cycle they assume p21WAF-positive/p53-negative status. By contrast, cells in the interfollicular epidermis of psoriatic lesions, in the sebaceous gland, and in the outer root sheath are p21WAF-positive/p53-positive but Ki67-negative. These results suggest that in some anatomical parts of the epidermis, p21WAF expression can accompany p53 expression, whereas in other parts, the expression of these markers is reciprocal, suggesting that other pathways may be controlling p21WAF expression. In order to define, functionally, the presence of p53-independent p21WAF expression in skin, we examined lesions of Bowen's disease in which both alleles of p53 were inactivated. p21WAF expression was still observed, confirming a role for p53-independent expression of p21WAF in human skin in vivo.

Darier disease (DD; Darier―White disease; OMIM 124200) is an autosomal dominant inherited disorder. 1 Clinically, it is characterized by recurrent and multiple hyperkeratotic papules or nodules affecting the trunk and flexural aspects of the extremities. 1 Characteristic histopathological features are dyskeratotic cells in the form of corps ronds and grains, suprabasal acantholysis forming suprabasal lacunae and irregular upward proliferation into the lacunae of papillae lined with a single layer of basal cells, the so-called villi. 2 The causative gene is ATP2A2 (OMIM 108740) on chromosome 12, which encodes the sarco/endoplasmic reticulum calcium pump ATPase (SERCA2). 2 Clinical variants include the hypertrophic, vesiculobullous, hypopigmented, cornifying, zosteriform and linear subtypes, and the rare subtype comedonal Darier disease (CDD). 1,3―6 CDD tends to appear in seborrhoeic areas. The characteristic morphological features are prominent follicular involvement, sometimes associated with keratotic plugs, and the presence of greatly elongated dermal villi and papillary projections. 4 There have been no conclusive reports on the aetiology of CDD and it is still controversial as to whether or not CDD is a variant of DD, and if it is caused by ATP2A2 gene mutations, although a combination of CDD and classic DD was reported in one patient. 7 The present study identifies a previously unreported three-base deletion mutation in ATP2A2 in a patient with CDD.

High levels of 5alpha-reductase activity have been detected in human apocrine glands, and the concentration of dihydrotestosterone has been found to be higher than that of testosterone in the nuclear fraction of the skin of patients who suffer from excessive or abnormal odour derived from apocrine sweat (osmidrosis). Although these results suggest that 5alpha-reductase may play a central role in the action of androgens in the apocrine gland, the isozyme responsible is not known. We therefore assayed 5alpha-reductase type I and type II activity and mRNA expression in isolated apocrine glands from four patients with osmidrosis. When we incubated gland homogenates with [3H]testosterone, we found that the biochemical properties of the apocrine gland enzyme were consistent with those of type I 5alpha-reductase: at substrate concentrations of both 50 nmol/L and 1 micromol/L, the optimum pH was in the range 6.0-7.5, and the apparent Km was 21.1 micromol/L. The apocrine gland enzyme was inhibited by MK386, a specific inhibitor of type I 5alpha-reductase, in a dose-dependent manner, but it was hardly affected by finasteride, a specific inhibitor of type II isozyme, in that a nanomolar concentration of finasteride produced only a slight inhibition. Reverse transcriptase-polymerase chain reaction showed that the apocrine gland expressed type I 5alpha-reductase mRNA exclusively, except for a faint band of type II isozyme in a few preparations. These data indicate that the type I isozyme is the predominant form of 5alpha-reductase in the apocrine gland and may play a central role in the anabolic activity of androgens, as reported for the sebaceous gland. In addition, a small amount of type II isozyme may be expressed by mesenchymal cells that surround the apocrine glands and also contribute to their development.

To explore the function of the enzyme transglutaminase 1 (TGase 1), its distribution was analysed by immunofluorescence microscopy, postembedding immunoelectron microscopy and in situ hybridization. TGase 1 was expressed in the outer root sheath (ORS) cells in the distal portion of the isthmus and infundibulum of human hair follicles. In the level of the proximal and middle portion of the isthmus, TGase 1 was observed in the keratinized area of the inner root sheath (IRS) and ORS cells. In the bulbar and suprabulbar portions, TGase 1 was present in the ORS and IRS cells. The cortex and medulla cells in these regions also contained TGase 1. A high level of fluorescence was observed at the cuticle of the cortex. Sebaceous and sweat gland cells contained abundant TGase 1. Possible functions of TGase 1 in these epidermal appendages are discussed.

Muir-Torre syndrome (MTS) is an autosomal dominant disorder characterized by the presence of at least one sebaceous gland tumour and a minimum of one visceral malignant tumour. Recently, microsatellite instability (MSI) has been detected in the tumours of patients with MTS and germline mutations of the hMSH2 and hMLH1 mismatch repair genes have been detected in some patients with this syndrome. To determine if the tumours of patients with MTS have widespread genomic instability and whether loss of heterozygosity (LOH) in the chromosomal regions containing hMSH2 and hMLH1 is detectable, MSI and LOH were examined at 10 dinucleotide repeats on chromosomes 2p, 3p, 5q, 9p, 17p and 18q. Data were obtained from six sebaceous gland tumours and two adenocarcinomas of the colon from three patients of two Muir-Torre families. MSI was detected at more than half of the loci tested in all sebaceous tumours examined. In addition, there was LOH at D2S119 in one sebaceoma and one sebaceous carcinoma from one patient. The colon carcinomas from two patients showed MSI at five of the 10 loci analysed. These results show that widespread MSI is a feature of tumours in patients with MTS. In addition, the finding of LOH at D2S119, a marker located in the vicinity of hMSH2, in sebaceous tumours of one patient indicates that this gene may have a pathogenetic role in this patient.

The enzyme 5 alpha-reductase converts testosterone (T) to dihydrotestosterone (DHT). Although this enzyme has been localized to various regions of the pilosebaceous unit, its activity has not been studied in the follicular portion of either vellus or sebaceous follicles. The goal of our study was to determine the relative activities of 5 alpha-reductase within various regions of these follicles with particular emphasis on the infrainfundibulum. A finding of increased 5 alpha-reductase activity in upper follicles compared to epidermis might support the hypothesis that increased follicular production of DHT is involved in the hyperkeratinization observed in this region of the follicle in acne vulgaris. 5 alpha-reductase activity was determined at pH 5 (optimal for the type 2 isozyme) and pH 7 (optimal for the type 1 isozyme) in isolated infrainfundibular segments from sebaceous and vellus follicles, homogenized epidermis from various anatomical areas and in microdissected segments of the pilosebaceous unit from breast skin of normal subjects. Enzyme activity was also determined at pH 7 in cultured infrainfundibular keratinocytes and in interfollicular epidermal keratinocytes. Homogenates of infrainfundibular segments demonstrated significantly greater activity at pH 7 compared to pH 5 (P < 0.001), confirming activity of the type 1 5 alpha-reductase in this region. Activity of 5 alpha-reductase was much lower in homogenized epidermis and did not demonstrate a clear pH preference. Keratinocytes cultured from the infrainfundibulum demonstrated significantly greater 5 alpha-reductase activity compared to keratinocytes from interfollicular epidermis (P = 0.04). In the dissected segments of pilosebaceous units from breast skin, 5 alpha-reductase activity was greatest in the sebaceous gland followed by the sebaceous duct, infrainfundibulum, whole skin and epidermis. These data indicate that 5 alpha-reductase activity varies within regions of the pilosebaceous unit and compared with interfollicular epidermal cells, infrainfundibular keratinocytes have an increased capacity for producing androgens which may play a role in the follicular hyperkeratinization seen in acne.

Pigmented follicular cyst is a rare disorder which typically presents as a pigmented papule on the head or neck and which, histologically, exhibits terminally differentiated, pigmented hair shafts in an epidermoid cyst. We report a 22-year-old man with the multiple variant of this disorder. Clinically he had numerous brown-blue to flesh-coloured, domed-shaped papules, on the anterior chest and abdomen, of 10 years duration. Histologically, hybrid cysts exhibiting trichilemmal and epidermoid keratinization were seen. The cysts contained numerous pigmented, terminally differentiated hair shafts and, embedded in the wall of one cyst, was a sebaceous gland. The condition of multiple pigmented follicular cysts, is thought to represent a distinct subtype within the spectrum of multiple pilosebaceous cystic disorders.

This study compared the pattern of keratin expression in pilosebaceous follicles in uninvolved trunk skin of acne patients, comedones, and normal control skin by immunohistochemistry, using both immunofluorescent (IF) and immunoperoxidase (IP) techniques. The shape, size and gross morphology of truncal follicles varied greatly. There was no difference in keratin expression between normal skin and uninvolved skin of patients with acne. The upper part of the pilosebaceous duct expressed keratins K1, K5, K10 and K14, whereas the lower duct expressed keratins K5, K6, K14, K16, K17 and K19. The sebaceous gland showed considerable heterogeneity in keratin expression, with some lobules expressing keratins K1, K5, K7, K10, K14 and K17. The comedone wall showed a pattern of keratin expression similar to that of the upper follicle, except that there was, in addition, expression of keratins K6 and K16 suprabasally, and panepithelial expression of K17 in the comedone wall. IF techniques were found to be less sensitive than the IP method in the detection of individual keratins, but the use of multiple antibodies and many different transverse and longitudinal sections of follicles permitted full conclusions to be made. The increased expression of keratins K6, K16 and K17 in the comedone wall is likely to represent a secondary effect of increased cell turnover due to the primary underlying mechanism of comedogenesis, which is as yet unknown.

Adherence of Malassezia yeast cells to human keratinocytes was assessed by a novel technique using double-sided Sellotape. Although adherence using double-sided Sellotape is still merely a model for in vivo adherence, it approximates to the conditions found on the skin surface. There were no differences in adhesive properties to human keratinocytes between Malassezia strains originating from HIV-positive and HIV-negative patients with seborrhoeic dermatitis, nor was there a relationship between the severity of seborrhoeic dermatitis and in vitro adherence to human keratinocytes.

BP180 is a 180kDa hemidesmosomal protein recognized by bullous pemphigoid (BP) and pemphigoid gestationis (PG) autoantibodies. Recent cloning and sequence analysis performed by our laboratory have revealed that BP180 is a transmembrane protein with a long extracellular collagen-like region. A rabbit polyclonal antibody has been generated against a recombinant protein, designated GST-N delta 1, containing a segment of the BP180 ectodomain. The resulting antiserum, RN delta 1A, was shown to specifically react with BP180 on immunoblot, and labelled the extracellular region of the epidermal hemidesmosome on immunoelectron microscopy. A panel of normal and neoplastic human tissues were analysed by indirect immunofluorescence (IF) and RN delta 1A, to determine the distribution of BP180. A total of nine basal cell carcinomas (BCCs) and four squamous cell carcinomas (SCCs) of the skin were also studied. Intense IF staining was seen along the basement membrane zone (BMZ) of the epidermis, hair follicles, and the periphery of sebaceous gland lobules. The sebaceous lobules showed more intense staining in areas close to the duct. The epithelial BMZ of the following tissues also reacted with RN delta 1A: cornea, ocular conjunctiva, buccal mucosa, upper oesophagus, placenta (amnion placentum), umbilical cord and transitional epithelium of the bladder. The epithelium of the jejunum and ovary failed to react with RN delta 1A. Staining of the BCCs and SCCs was variable. Five of six nodular BCCs showed some anti-BP180 staining at the tumour-stromal interface, although the level of staining was less intense than that observed in the overlying normal epidermis. All three morphoeic BCCs analysed in this investigation did not show any staining with RN delta 1A. Three of four SCCs showed weak staining at the tumour-stromal interface. Thus, the tissue distribution of BP180 paralleled that of hemidesmosomes, and expression of this protein was found to be decreased or absent in cutaneous neoplasms.

The aim of this study was to investigate the effects of 13-cis retinoic acid treatment on cellular retinoic acid binding protein II (CRABP II) mRNA expression in sebaceous follicles from acne patients, using in situ hybridization. Biopsies were taken from uninvolved skin areas in close juxtaposition to inflamed comedos before therapy, and at 2-4 or 14-16 weeks of treatment. Paraffin sections were used for in situ hybridization study with riboprobes transcribed from human CRABP II cDNA. After oral treatment with 13-cis retinoic acid, sebaceous glands were reduced in size and atrophic, and the ratio of sebum-free to fully differentiated (sebum-producing) sebocytes was dramatically increased. The CRABP II expression in the sebaceous gland, and to some extent in infundibular structures, was strongly increased compared with the level of expression in the epidermis. The maximum signal was always found in layers of suprabasal sebocytes lacking lipid droplets, but never in the basal layers. These findings indicate a selective activity of 13-cis retinoic acid on CRABP II mRNA expression in the sebaceous glands of acne patients.

Summary We report two cases of Apert's syndrome, each of whom developed the severe acne in adolescence which is a feature of this disorder. Both responded to isotretinoin therapy. Immunohistochemical techniques, using a mouse monoclonal antibody, were employed to stain sebocyte androgen receptors in the two patients, and in five controls. This showed no difference in the number of cells with androgen receptor expression between the patients with Apert's syndrome and controls. These results support the concept that the underlying problem in Apert's syndrome is an abnormal sensitivity to normal circulating levels of androgens, and not an excess number of androgen receptors.

Summary For unknown reasons, the pilosebaceous unit displays prominent alkaline phosphatase (AP) activity, and alterations in AP activity are seen in alopecia areata. The role of AP in hair biology and pathology has been obscured by contradictory reports on the localization and activity of AP during the hair cycle, and by a paucity of instructive models for studying AP functions. Using the C57 BL-6 mouse model for hair research, we have characterized endogenous AP with a simple histochemical developing solution routinely employed for AP immunohistology. This method was selective for AP, and revealed distinctive hair cycle-dependent changes in AP activity and localization. Although the dermal papilla displays unusually strong AP activity during the entire hair cycle, the outer root sheath is AP-positive only during late anagen and early catagen. Strong, rather homogeneous AP activity is seen in the sebaceous gland (SG) only during catagen and telogen. This AP staining pattern indicates hair cycle-dependent changes in SG functions, and differs to some extent from the previously reported AP activity during the hair cycle of various species. We propose a simple and effective technique for follicle classification based on the AP histochemistry of dermal papilla and sebaceous gland, and discuss uses of the C57 BL-6 mouse model for functional AP studies.

The Dundee experimental bald rat (DEBR) undergoes hair loss associated with perifollicular infiltrates of mononuclear cells (MNC), a pathological characteristic of human alopecia areata (AA). To investigate further the pathogenesis of the disease in this animal model, we have studied the development, composition and extent of the perifollicular MNC infiltration in young (6-week-old), prelesional (3-month-old), active lesional, and established lesional DEBR rats, using 6-week- and 6-month-old Wistar rats as normal controls. The proportions of hair follicles showing infiltration by MNC and their main subsets were determined using immunohistochemical staining of serial cryostat sections of flank skin biopsies. There was a good correlation between the degree of leucocyte (OX-1+) infiltration of anagen hair follicles and the development of hair loss. In 6-week-old DEBR skin, there were few perifollicular cells expressing MHC class II, with positively stained dendritic cells in the dermis above the sebaceous gland. There was a sparse perifollicular distribution of CD4+ cells (W3/25) and macrophages (ED-1+). No CD8+ cells (OX-8+) were seen associated with DEBR hair follicles, and only small numbers were present in Wistar rats. In prelesional DEBR rats there was an increased perifollicular presence of MHC class II+ cells, macrophages, and particularly of CD8+ cells, with little change in CD4+ cells. Active and established lesional rats, i.e. animals with overt loss of hair, showed a significant increase in the degree of MNC infiltration and the proportion of infiltrated follicles, the majority of which were in dystrophic anagen. In the perifollicular infiltrate the CD4+:CD8+ ratio was approximately 2:1. An intrafollicular infiltrate was prominent, and was composed of CD8+ cells and macrophages, with bulbar and suprabulbar keratinocytes expressing MHC class II antigens. CD4+ cells were not detected in follicular epithelium. ICAM-1 expression correlated with MNC infiltration. These results show marked similarities to lesional human AA. They also focus on a possible active role for CD8+ cells in the pathogenesis of hair loss in the DEBR rat.

Darier's disease is an inherited disorder with well-recognized patterns of presentation. Lesions commonly affect the trunk and flexures. The diagnosis is based on the typical clinical appearance and histology showing acantholytic dyskeratosis. We report two unusual cases with prominent nodular, comedonal lesions on the face and scalp.

As neonates have a high sebum production compared with 6-month-old babies, we wished to investigate the relationship of sebum production in mother and neonate. The sebum excretion rate (SER) was therefore measured in 10 mothers prenatally, and in each mother and baby as soon after birth as possible, and again 5-12 weeks postnatally. There was a significant correlation between the maternal and neonatal SER perinatally. The perinatal SER in the babies was markedly higher than the postnatal sample. These observations suggest an important role for the maternal hormonal environment on the infant sebaceous glands. There is animal evidence which suggests that the endocrine environment of the neonate influences the sebaceous gland development in puberty, but it is not known whether babies with a high SER are more prone to seborrhoea and acne in later life.

Summary Background Cultured hair follicles lacking sebaceous glands do not appear to degrade the inner root sheath (IRS), suggesting that the gland may be involved in this process. Objectives To examine this supposition in cultured hair follicles. Methods Pilosebaceous units were isolated from hair follicles cultured in vitro, and IRS degradation was studied by histology. Results When grown in culture, the fibres of follicles lacking sebaceous glands were encased in a layer of translucent tissue. During hair growth in vitro this tissue remained intact at the distal end of the follicle but disappeared further down towards the bulb and then reappeared towards the proximal end. Transection within the region lacking this tissue resulted in the release of a naked hair fibre and the production of hair with no attached tissue upon subsequent hair growth. The translucent tissue represented the IRS, thereby demonstrating that this tissue is indeed degraded in vitro. Histological comparison with freshly isolated pilosebaceous units indicated that IRS degradation in vitro strongly resembled the process that occurs in vivo. Conclusions These data suggest that the sebaceous gland does not itself participate in IRS degradation. Indeed, this phenomenon appears to be a function of the follicle itself and is probably intimately linked with the processes of cellular proliferation, differentiation and death that occur during hair biogenesis.

Senile sebaceous gland hyperplasia has been studied in 18 subjects in an attempt to shed some light on its pathogenesis. The sizes of different segments of the pilosebaceous unit were compared in senile sebaceous gland hyperplasia (SGH) and in control skin samples, using stereological techniques. The whole follicle was found to be significantly increased in volume compared with follicular structures in the control specimens. The size of differentiated sebaceous gland cells was found to be reduced compared with controls (490 +/- 72 microns 2 and 554 +/- 98 microns 2, respectively), but not significantly so. The mean tritiated thymidine autoradiographic labelling index determined in vitro was found to be decreased (5.42 +/- 1.79) compared with controls (7.48 +/- 2.44), but again, not significantly so. In addition the degree of solar elastotic degenerative change was found to be similar in SGH bearing skin compared with control samples. It is of interest that SGH and prostatic hypertrophy are exceptions to the usual atrophy of ageing and that both tissues are androgen dependent.

SUMMARY The proliferative activity of sebaceous glands of uninvolved skin from acne patients was analysed by means of 3H-thymidine autoradiography and by planimetry. Twelve specimens of normal skin from the back (with thirty-five glands) and two specimens from the face (with six glands) were analysed. Labelling indices and planimetric values were determined separately for the two portions within each gland: the differentiating cell pool with germinative cells and large lipid cells; and the undiffer-entiated cell pool with multilayered cells. The germinative cells of the differentiating cell pool on the back and face of patients with acne showed a significantly greater labelling index than in control subjects. The undifferentiated cell pool, although having a higher labelling index than the differentiating cell pool, was similar both in acne patients and in controls. Planimetry confirmed that the sebaceous glands of patients are much larger than those of normal subjects. Therefore, the known increase in sebum excretion in acne patients is a result of two factors—an increase in lipid cell production and an increase in sebaceous gland size.

The activity of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) was assayed in various tissues microdissected from the freeze-dried human skin of fourteen subjects. The apocrine sweat gland, sebaceous gland and hair follicle possessed a high activity of 17 beta-HSD. The enzyme activity was negligible in the epidermis, except that the scalp epidermis showed much the same activity as the hair follicle. The demis showed variable activity because of contamination with other components.

SUMMARY By screening 1500 sera obtained from patients undergoing a bone marrow graft, five sera have been selected which contained antibodies to the basal cell layer (B. C. L.) of the outer root sheath (O. R. S.) of hair follicles. It was demonstrated that these sera reacted with the B. C. L. of the O. R. S. but not with the B. C. L. of the adjacent epidermis. The change in antigenic properties was situated at the orifice of the sebaceous gland canal; thus basal cells below this orifice express an antigen distinct from those expressed by the epidermal basal cells. This is a novel example of tissue specific differentiation antigens that are linked to the type of keratinocyte differentiation.

A method of assay alpha-galactosidase and acid phosphatase activities in single hair roots is described. Enzyme histochemical studies show that the distribution of acid phosphatase in the human hair root matches that of alpha-galactosidase. Histochemically, the main activity is located in the upper part of the sheath near the orifice of the duct of the sebaceous gland. This is confirmed by enzyme assays on different parts of the hair root after dissection. The variation in the values found in individual hair roots is improved by relating alpha-galactosidase to acid phosphatase activities. Storage experiments indicate a remarkable stability of both alpha-galactosidase and acid phosphatase in human hair roots.

SUMMARY Follicular and sebaceous gland cell differentiation was investigated during treatment of acne patients with isotretinoin. Sebaceous glands were significantly reduced in volume and showed decreased metabolic activity as measured by glucose-6-phosphate dehydrogenase and succinic dehydrogenase enzyme activities. There was no measurable change in the volume of follicular duct epithelium or in its metabolic activity during treatment. Metabolic activity was also unchanged in the interfollicular epidermis, contrasting with the effects of another retinoid, etretinate. These findings provide no support for the proposition that alteration in the process of follicular keratinization forms a substantial part of the mode of action of isotretinoin.

The effect of castration on the size of sebaceous glands of male hairless mice and male hairless hamsters was studied over a period of 4 weeks. Statistically significant decreases in the size of sebaceous glands were observed. The effects of intracutaneous injections of micronized crystalline suspensions of testosterone, testosterone propionate, testosterone phenylpropionate, testosterone decanoate and nandrolone phenylpropionate on the sebaceous glands of castrated hairless mice, castrated hairless hamsters and intact female hairless hamsters were studied over a period of one week. Highly significant increases in sebaceous gland volume were observed 6-7 days after treatment. The effects of subcutaneous and intracutaneous injections of micronized crystalline suspensions of testosterone on the sebaceous glands of intact male and female hairless hamsters were examined at sites local and distal to the injection. Increases in sebaceous gland volume were limited to local sites in the male but increases were observed locally and distally in the female. There was no difference between subcutaneous and intracutaneous injections.

SUMMARY A modified photometric technique has shown considerable differences between the refatting kinetics of the scalp and forehead. The refatting on the forehead occurs rapidly, probably due to the large follicular reservoir. The follicular reservoir of the scalp, which bears a large terminal hair, is much smaller and so the refatting time is longer. Thus, sebum excretion and sebum production rates probably correlate better on the scalp than on the forehead, and we suggest that the scalp may be a better site than the forehead for investigating sebaceous gland physiology.

SUMMARY Incorporation of 3H-thymidine into different squamous epithelia and other tissues was studied in mice bearing the Hewitt keratinizing epidermal carcinoma with a raised epidermal chalone content in the body. The results showed that DNA synthesis in the tumour-bearing mice was inhibited strongly in the epithelium of the ear (c. 80%), moderately in the epithelium of the back skin and the foot (c. 55%), and weakly in the epithelium of the tongue and the cornea (c. 25%). These inhibitions appear to be specific in nature, because cell proliferation was not detectably affected in the sebaceous gland, intestinal epithelium, bone marrow, and thymus lymphocytes. Thymus size, however, was reduced in the tumour-bearing mice compared with controls, and 3H-thymidine uptake was depressed in the spleen and to some extent also in the lymph nodes, possibly owing to non-specific stress. Labelling index of human psoriatic epidermis was suppressed in diffusion-chamber cultures carried by the tumour-bearing host mice compared with controls. However, this reaction was probably not specific in nature, because the labelling index of mouse bone-marrow cells was also decreased when grown in tumour-bearing animals.