Apocrine glands are bystanders in hidradenitis suppurativa and their involvement is gender specific.

BACKGROUND: Apocrine glands have been long considered as the initial targeted skin compartment in hidradenitis suppurativa/acne inversa (HS). OBJECTIVE: Detection of apocrine gland involvement in HS. METHODS: Apocrine glands were isolated from skin biopsies of involved and uninvolved skin of HS patients (n = 16, females : males 1 : 1) by laser capture microscopy and studied by whole transcriptome profiling. Dysregulated genes were detected by comparing lesional and non-lesional skin obtained from female and male HS patients using the Agilent array platform. RESULTS: SULF1 was the only gene, whose expression levels were found upregulated in apocrine glands of HS lesions of the entire group. Further dysregulated genes associated with vascular functions (FGF1, IL17D and S100A9) were detected. Genes, which are characteristic for glandular epithelia, confirmed the glandular origin of the studied tissue. The gene upregulation profile of female apocrine glands included several genes (MRO, DYRK3, SDK2, GLB1L, CATSPERB and PRPS2), which are specifically transcribed during testis differentiation and/or regulated by androgens. Genes related to lipid metabolism (AGPAT3, GAL, ELOVL3, THRSP, DGAT2L3, OLAH, THRSP, FADS1, NR2F2, FADS2, PTGDS and HAO2) were mostly downregulated in the apocrine glands of male patients. The levels of RECK and PCSK5, which are upstream genes of metalloproteinase-9 and -1, and of S100A9, which encodes calgranulin B, were commonly increased in the apocrine glands of female and male patients, respectively, and in our previous whole skin study. CONCLUSION: Our findings indicate that apocrine glands are bystanders in HS. Inflammatory signalling is not prominent but a gender-specific response was detected, which is mostly associated with androgen-responsive genes in females and alterations of lipid metabolism in males.

Characterisation of epidermal regeneration in vivo: a 60-day follow-up study.

OBJECTIVE: To characterise the long-term course of epidermal regeneration in a suction-blister wound model in healthy humans. METHOD: A single-centre, prospective cohort study was conducted. Suction blister wounds of 8mm diameter were created on the volar forearms of healthy volunteers. Planimetry was used to measure the wound surface area. Transepidermal water loss was estimated to characterise the skin barrier function. Skin brightness was measured using the chromametric luminance L* parameter and skin (visco)elastic properties were measured by a controlled suction device. RESULTS: Thirty-two subjects (mean age 28.6 years) participated. Epithelisation was nearly completed after 8 days, but it took approximately 3 weeks for complete skin barrier restoration. Post-inflammatory hyperpigmentation was observed at the end of day 60 in the majority of skin areas. Elastic and viscoelastic deformation and recovery at the end of the follow-up period did not reach baseline values. CONCLUSION: Newly formed epidermis requires considerable time before reaching complete recovery of the skin barrier function. Up to 2 months after the injury, regenerated epidermis in junction with the reticular dermis is stiffer compared to before. Under mechanical loading increased local stiffness might increase the risk for subsequent injuries at the same or adjacent skin areas. Due to its increased vulnerability it is recommended to protect or to offload the epidermal tissue as long as possible to support the structural long-term regeneration. Artificial suction blister wounds are well standardised and controlled models for a wide range of clinical studies and they offer advantages over uncontrolled patient conditions in wound healing studies. DECLARATION OF INTEREST: This study was funded by La Roche-Posay Laboratoire Pharmaceutique (Asnieres, France). The sponsor had no influence on the design, conduct, and analysis and presentation of the data and on the content of this manuscript. The authors have no financial interest in this article.

Involvement of the corticotropin-releasing hormone system in the pathogenesis of acne vulgaris.

BACKGROUND: The sebaceous gland exhibits an independent peripheral endocrine function and expresses receptors for neuropeptides. Previous reports have confirmed the presence of a complete corticotropin-releasing hormone (CRH) system in human sebocytes in vitro. The capability of hypothalamic CRH to induce lipid synthesis, induce steroidogenesis and interact with testosterone and growth hormone implicates a possibility of its involvement in the clinical development of acne. OBJECTIVES: The purpose of the study was to detect expression changes of CRH/CRH binding protein (CRHBP)/CRH receptors (CRHRs) in acne-involved skin, especially in the sebaceous glands. METHODS: Expression of CRH/CRHBP/CRHRs was analysed by immunohistochemistry in biopsies from facial skin of 33 patients with acne, noninvolved thigh skin of the same patients and normal skin of eight age-matched healthy volunteers. RESULTS: Very strong positive reaction for CRH was observed in acne-involved skin in all types of sebaceous gland cells, irrespective of their differentiation stage, whereas in noninvolved and normal skin sebaceous glands exhibited a weaker CRH staining depending upon the differentiation stage of sebocytes. The strongest reaction for CRHBP in acne-involved sebaceous glands was in differentiating sebocytes. CRHR-1 and CRHR-2 exhibited the strongest expression in sweat glands and sebaceous glands, respectively. CONCLUSIONS: Expression of the complete CRH system is abundant in acne-involved skin, especially in the sebaceous glands, possibly activating pathways which affect immune and inflammatory processes leading to the development and stress-induced exacerbation of acne.

Inhibition of the androgen receptor by antisense oligonucleotides regulates the biological activity of androgens in SZ95 sebocytes.

One novel strategy for the blockade of the androgen receptor could be the selective inhibition of androgen receptor by antisense oligonucleotides or small interfering RNA molecules. Here we describe the down regulation of the androgen receptor in cultured human SZ95 sebocytes with antisense oligonucleotides modified with phosphorothioates and 2'- O-methylribosyl residues. The ability of antisense oligonucleotides to cross the cellular membrane was enhanced by establishing a transient transfection system based on cationic lipid vesicles. Both antisense oligonucleotide types administered caused assumedly translational arrest. Dose-dependent inhibition of androgen receptor protein expression was observed after SZ95 sebocyte transfection with modified phosphorothioate oligonucleotides and modified 2'- O-methylribonucleotides which were directed against the translational start of the androgen receptor mRNA. The strongest transient inhibition of androgen receptor expression was detected after 14 hours with 1.0 muM antisense 2'- O-methylribonucleotides (88+/-1.3%, p<0.001). With longer recovery times than 24 hours, androgen receptor protein expression returned to the native control levels. Inhibition of the expression of androgen receptor by antisense oligonucleotides, reduced the enhanced proliferation of SZ95 sebocytes challenged by testosterone and 5alpha-dihydrotestosterone. This administration opens new therapeutic possibilities in androgen-associated skin diseases, since we could also show androgen inhibition with these antisense oligonucleotides in a reconstituted human epidermis model (Horm Metab Res 2007; 39:157-165).

Responsiveness to androgens and effectiveness of antisense oligonucleotides against the androgen receptor on human epidermal keratinocytes is dependent on the age of the donor and the location of cell origin.

Local androgen excess has been associated with attenuation of wound healing in elderly individuals and with a decline in permeability barrier homeostasis in adult human skin. In this study we have applied specific antisense oligonucleotides, whose activity has already been investigated in SZ95 sebocytes, to inactivate transiently the androgen receptor in a reconstituted epidermis model and in primary human epidermal keratinocytes of different origin (breast, abdomen, foreskin) and donor age (females, 30- and 60-year-old). Further a possible interaction between blockage of androgen receptor and the expression of tissue inhibitors of matrix metalloproteinases was investigated. Androgen receptor levels were similar in pooled keratinocytes of the two age groups. Cell transfection with antisense oligonucleotides against the androgen receptor resulted in decreasing protein levels detected in all epidermal keratinocytes tested, whereas cells of aged donors (60-year-old) exhibited a stronger response than cells of young individuals (30-year-old). Keratinocytes from aged donors also responded to androgens with a stronger regulation of proliferation than keratinocytes of young individuals. The pattern of the androgen-induced response was dependent on the skin region of keratinocyte origin. The expression levels of tissue inhibitor of matrix metalloproteinase-1 were not age-related. Our results demonstrate an enhanced androgen sensitivity of keratinocytes from aged individuals associated with an origin-specific type of response.

Corticotropin-releasing hormone skin signaling is receptor-mediated and is predominant in the sebaceous glands.

There is increasing evidence that the sebaceous gland expresses receptors for several neuropeptides and is involved in responses to stress. Among them, corticotropin-releasing hormone (CRH) was currently found to be produced also in the skin. In this study, the distribution of CRH, CRH receptors 1 and 2 (CRH-R1 and CRH-R2), and CRH binding protein (CRH-BP) in cultured human (SZ95) sebocytes was further characterized. Moreover, the effects of CRH and CRH-like peptides on proliferation and inflammatory signaling of CRH receptor-expressing SZ95 sebocytes IN VITRO were investigated. Urocortin (Uct), urotensin and sauvagine are recently described members of the family of structurally related CRH-like peptides, whereas Uct shares a 45% homology with CRH. CRH and Uct inhibited SZ95 sebocyte proliferation with CRH also stimulating interleukin-6 (IL-6) and interleukin-8 (IL-8) release from SZ95 sebocytes. However, CRH had no effect on interleukin-1alpha and interleukin-1beta production in these cells. alpha-Helical-CRF, a CRH antagonistic peptide, annulled the CRH effect on SZ95 sebocyte proliferation and interleukin secretion, while the non-peptidic CRH-R1 selective antagonist antalarmin inhibited the increased production of neutral lipids caused by CRH. In conclusion, CRH, and to a lesser extent Uct, may be involved in signaling of stress pathophysiology in the skin. However, further investigations into the downstream effects of CRH and Uct are required to elucidate the mechanism by which these neuropeptides could establish a stress-related pathophysiological condition in the skin.

Influence of physiological androgen levels on wound healing and immune status in men.

Aging in men is associated with a progressive decline in the production of several hormones, including androgens. The extent to which an age-dependent decline in androgen levels lead to health problems or can affect quality of life remains under debate. Clinical results on replacement therapy do not yet provide a definitive clue on the benefit/risk balance. A sexual dimorphism of the immune system is well established, and the differences between female and male immune responses under normal, as well as pathological, conditions are generally attributed to the influence of estrogens, progestins, and androgens. The suppressive effects of male sex hormones on immune functions have been observed in a wide variety of disease processes and appear to be testosterone-mediated. Endogenous testosterone inhibits skin wound healing response in males and is associated with an enhanced inflammatory response. Although there are no known gender-related differences in permeability barrier function in adults, estrogens accelerates--whereas testosterone retards--barrier development in fetal skin, and male fetuses demonstrate slower barrier development than female littermates.

Cryotherapy modifies synthetic activity and differentiation of keloidal fibroblasts in vitro.

In order to obtain a persuasive explanation for the beneficial clinical effect of cryotherapy on keloids, we developed a reproducible model to apply freezing temperatures on cell cultures, and investigated their influence on proliferation, viability, synthetic activity and differentiation of dermal fibroblasts in vitro. Cell cultures were established from 13 untreated keloids and 10 healthy skin specimens matched for age and skin localization to the donors. No significant influence of cell freezing on the proliferation rates of both keloidal and normal fibroblasts was documented, but mechanical cell destruction with a wide variation in lethality rates (29% average lethal effect on keloidal fibroblasts and 41% on normal ones) was observed. When comparing specimens of keloidal and normal tissue derived from the same four donors, the keloidal fibroblasts were similar regarding their synthetic activity but presented enhanced tenascin-C expression compared with the normal fibroblasts. After cryotherapy, delayed collagen III increase was detected in both cell types (P = 0.03). The collagen II/collagen I ratio increased from 1.6 to 2.8 in the keloidal and only from 1.9 to 2.2 in the normal fibroblasts after subcultivation. Normal fibroblasts exhibited a significantly lasting increase in fibronectin synthesis after freezing (P = 0.03). The intensity of staining against tenascin-C was decreased in five of nine keloidal fibroblast cultures after cryotherapy (P < 0.05) but increased in four of five normal fibroblast cultures (P = 0.016), so that the intensity of tenascin-C staining after freezing became identical in both cell types. Immunoblot studies in four patients and two controls confirmed a temporary decrease of tenascin-C in keloidal but not in normal fibroblasts immediately after freezing. Significantly decreased staining with two markers of myogenic differentiation, myosin in keloidal fibroblasts (P = 0.002) and desmin (P = 0.007) in normal fibroblasts, could also be detected after treatment. In summary, with the help of a model for controlled cell freezing in vitro, cryotherapy was found to modify collagen synthesis and differentiation of keloidal fibroblasts.

Growth and characterization of a cell line from a human primary neuroendocrine carcinoma of the skin (Merkel cell carcinoma) in culture and as xenograft.

The primary neuroendocrine carcinoma of the skin or Merkel cell carcinoma (MCC) is a skin tumor with aggressive biological behaviour. Experimental models for investigating the biological properties of the tumor are prerequisite for developing new therapeutic approaches. In this study, we report the establishment and characterisation of a cell line derived from the lymph-node metastasis of a patient with highly aggressive MCC. Merkel carcinoma cells (MCC-1) grew as floating aggregates in suspension cultures for more than two years and over 70 subcultures. The proliferation rate in suspension cultures was rather moderate with a population doubling time of 69 h. The immunocytochemical pattern of the cultured MCC-1 was similar to that of the original tumor with expression of cytokeratin 18, neuron-specific enolase, neurofilaments, and synaptophysin. In addition, reverse transcriptase polymerase chain reaction (RT-PCR) revealed presence of chromogranin A mRNA in the MCC-1 cell line. Furthermore, electron microscopy yielded the rare finding of neuroendocrine granules in the cytoplasm of the cultured cells. The cell line MCC-1 was able to form colonies in soft agar. Nude mice developed solid tumors with similar histology to the original tumor after subcutaneous and intravenous injections of cultured MCC-1, and malignant ascites was seen after intraperitoneal injection. Also, two MCC-1 sublines were established by reculturing cells from the xenografts grown in vivo and immunocytochemistry confirmed their neuroendocrine origin. The MCC-1 line may thus serve as a model for studying the biology and the metastatic potential of Merkel cell carcinoma.

Development of efficient transient transfection systems for introducing antisense oligonucleotides into human epithelial skin cells.

Systemic treatment with antisense oligonucleotides is confounded by the dual problems of potential cytotoxicity of antisense oligonucleotides and carrier molecules such as cationic lipids. Treatment of pathologic conditions affecting the skin may avoid these problems to a large degree due to local application. The success of antisense strategies has been limited by the poor uptake of the transfection reagent and inadequate intracellular compartmentalization. Human skin epithelial cells, therefore, are attractive experimental tools for testing both in vitro and in vivo antisense therapies. In the present study, we determined commercially available liposomes which reproducibly induced a nontoxic increase of oligonucleotide uptake in cultured SZ95 sebocytes and keratinocytes. The final protocol for SZ95 sebocytes was a 4-hour incubation with DOTAP in a 2:1 (w/w) lipid/oligonucleotide ratio in serum-free medium. The fluorescein-labeled (ATCG)(5) random oligonucleotide molecules were detected within the nucleus. The optimum transfection system for primary keratinocytes was poly-L-ornithine (12 microg/ml) in a medium without bovine pituitary extract over 4 hours. The uptake of the oligonucleotide increased in the presence of the polycation and oligonucleotide molecules were localized in the cytoplasm of keratinocytes. Oligonucleotide transfection with the help of cationic lipids did not affect the expression of androgen receptor and of the house-keeping gene beta-actin. Thus, cationic lipids are useful for delivery of antisense oligonucleotides into skin cells in vitro and may be used for topical application on animal and human skin.

Desensitization of melanoma cells to autocrine TGF-beta isoforms.

Previous studies have suggested that transforming growth factor-beta 1 (TGF-beta1) acts as an autocrine growth inhibitor on normal human melanocytes, while melanoma cells may not respond to this stimulus. The role of other TGF-beta isoforms such as TGF-beta2 and TGF-beta3 remained less well characterized. In the present study, the mRNA and protein levels of all three isoforms of TGF-beta were analyzed in a panel of human melanoma cell lines and in cultures of normal human melanocytes in vitro. Northern analysis showed that the degree of TGF-beta1, -beta2, -beta3 mRNA expression varied considerably in melanoma cells, whereas TGF-beta expression was very low in melanocytes. In melanoma cells, secreted amounts of TGF-beta1 and TGF-beta3 were found increased in comparison to normal melanocytes: 615 pg/ml vs. 118 pg/ml and 193 pg/ml vs. 30 pg/ml (mean values). In addition, low levels of TGF-beta2 were detected (mean value: 28 pg/ml). Although TGF-beta secretion increased, the proliferation of melanoma cells was found to be only moderately inhibited by TGF-beta isoforms, in contrast to its strong antiproliferative effect on normal human melanocytes: - 15%, -11%, and -18% vs. -52%, -46%, and -50% average inhibition at 0.5 ng/ml TGF-beta1, -beta2, and -beta3, respectively. The different efficacy of TGF-beta on melanocyte and melanoma cells was highly significant (P<0.0001); in addition, TGF-beta-dependent growth inhibition of melanoma cells from primary tumors vs. cells from metastases showed a trend for further decreased response for the metastatic populations (P< or = 0.075). Measurements of DNA synthesis revealed even more pronounced differences between melanocytes (-86%, -78%, and -80% inhibition, respectively, for TGF-beta1, -beta2, and -beta3) and melanoma cells (no inhibition). Our data show loss of responsiveness of melanoma cells to the growth-inhibitory function of TGF-beta isoforms but not of melanocytes. Although melanoma cells are not growth-inhibited by all three TGF-beta isoforms, they secrete significantly higher levels of TGF-beta, as compared to melanocytes. The reduced response indicates their escape from TGF-beta surveillance with ongoing tumor progression.

Trace elements and carrier proteins in the aged.

Serum level of trace elements can be used as a marker for diagnosis and management of diseases. We evaluated the effects of age, sex and diurnal rhythm on serum concentration of Cu, Fe, Zn and their carrier proteins. The subjects (N=336) were participants of the randomized multidisciplinary Berlin Aging Study (BASE), stratified for age (70-103 yrs) and sex. There is no diurnal variation for the carrier proteins coeruloplasmin, transferrin and albumin. The age-related decline of these proteins is not significant. Not only serum Fe but also Zn levels undergo a progressive decrease during the day. In the aged the serum concentration of both trace elements decreased, but the most important changes are diurnal variations. To avoid mistakes in the interpretation of clinical findings, so called time-quantified reference values are recommended.

Topography of surface-exposed amino acids in the membrane protein bacteriorhodopsin determined by proteolysis and micro-sequencing.

The topography of membrane-surface-exposed amino acids in the light-driven proton pump bacteriorhodopsin (BR) was studied. By limited proteolysis of purple membrane with papain or proteinase K, domains were cleaved, separated by SDS-PAGE, and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Fragments transferred were sequenced in a gas-phase sequencer. Papain cleavage sites at Gly-65, Gly-72, and Gly-231, previously only deduced from the apparent molecular weight of the digestion fragments, could be confirmed by N-terminal micro-sequencing. By proteinase K, cleavage occurred at Gln-3, Phe-71, Gly-72, Tyr-131, Tyr-133, and Ser-226, i.e., in regions previously suggested to be surface-exposed. Additionally, proteinase-K cleavage sites at Thr-121 and Leu-127 were identified, which are sites predicted to be in the alpha-helical membrane-spanning segment D. Our results, especially that the amino acids Gly-122 to Tyr-133 are protruding into the aqueous environment, place new constraints on the amino-acid folding of BR across the purple membrane. The validity of theoretical prediction methods of the secondary structure and polypeptide folding for membrane proteins is challenged. The results on BR show that micro-sequencing of peptides separated by SDS-PAGE and blotted to PVDF can be successfully applied to the study of membrane proteins.