bluuebunny/crossref_metadata_2025 huggingface dataset:
Wnt10b Deficiency Promotes Coexpression of Myogenic and Adipogenic Programs in Myoblasts
Authors: Vertino Anthony M. , Taylor-Jones Jane M. , Longo Kenneth A. , Bearden Edward D. , Lane Timothy F. , McGehee Robert E. , MacDougald Ormond A. , Peterson Charlotte A.
Published: January 2005

Adult myoblasts retain plasticity in developmental potential and can be induced to undergo myogenic, adipogenic, or osteoblastogenic differentiation in vitro. In this report, we show that the balance between myogenic and adipogenic potential in myoblasts is controlled by Wnt signaling. Furthermore, this balance is altered during aging such that aspects of both differentiation programs are coexpressed in myoblasts due to decreased Wnt10b abundance. Mimicking Wnt signaling in aged myoblasts through inhibition of glycogen synthase kinase or through overexpression of Wnt10b resulted in inhibition of adipogenic gene expression and sustained or enhanced myogenic differentiation. On the other hand, myoblasts isolated from Wnt10b null mice showed increased adipogenic potential, likely contributing to excessive lipid accumulation in actively regenerating myofibers in vivo in Wnt10b-/- mice. Whereas Wnt10b deficiency contributed to increased adipogenic potential in myoblasts, the augmented myogenic differentiation potential observed is likely the result of a compensatory increase in Wnt7b during differentiation of Wnt10b-/- myoblasts. No such compensation was apparent in aged myoblasts and in fact, both Wnt5b and Wnt10b were down-regulated. Thus, alteration in Wnt signaling in myoblasts with age may contribute to impaired muscle regenerative capacity and to increased muscle adiposity, both characteristic of aged muscle.
The Carboxyl Terminus of VEGFR-2 Is Required for PKC-mediated Down-Regulation
Authors: Singh Amrik J. , Meyer Rosana D. , Band Hamid , Rahimi Nader
Published: January 2005

Vascular endothelial growth factor receptor-2 (VEGFR-2/Flk-1) is a receptor tyrosine kinase (RTK) whose activation regulates angiogenesis. The regulatory mechanisms that attenuate VEGFR-2 signal relay are largely unknown. Our study shows that VEGFR-2 promotes phosphorylation of c-Cbl, but activation, ubiquitylation, and down-regulation of VEGFR-2 are not influenced by c-Cbl activity. A structure-function analysis of VEGFR-2 and pharmacological approach revealed that down-regulation of VEGFR-2 is mediated by a distinct mechanism involving PKC. A tyrosine mutant VEGFR-2, defective in PLC-γ1 activation underwent down-regulation efficiently in response to ligand stimulation, suggesting that activation of classical PKCs are not involved in VEGFR-2 down-regulation. Further studies showed that the ectodomain of VEGFR-2 is dispensable for PKC-dependent down-regulation. Progressive deletion of the carboxyl-terminal domain showed that at least 39 amino acids within the carboxyl-terminal domain, immediately C-terminal to the kinase domain, is required for efficient PKC-mediated down-regulation of VEGFR-2. Mutation of serine sites at 1188 and 1191, within this 39 amino acid region, compromised the ability of VEGFR-2 to undergo efficient ligand-dependent down-regulation. Altogether the results show that the regulatory mechanisms involved in the attenuation of VEGFR-2 activation is mediated by nonclassical PKCs and the presence of serine sites in the carboxyl terminal of VEGFR-2.
Identification ofXenopusCENP-A and an Associated Centromeric DNA Repeat
Authors: Edwards Nathaniel S. , Murray Andrew W.
Published: January 2005

Kinetochores are the proteinaceous complexes that assemble on centromeric DNA and direct eukaryotic chromosome segregation. The mechanisms by which higher eukaryotic cells define centromeres are poorly understood. Possible molecular contributors to centromere specification include the underlying DNA sequences and epigenetic factors such as binding of the centromeric histone centromere protein A (CENP-A). Frog egg extracts are an attractive system for studying centromere definition and kinetochore assembly. To facilitate such studies, we cloned a Xenopus laevis homologue of CENP-A (XCENP-A). We identified centromere-associated DNA sequences by cloning fragments of DNA that copurified with XCENP-A by chromatin immunoprecipitation. XCENP-A associates with frog centromeric repeat 1 (Fcr1), a 174-base pair repeat containing a possible CENP-B box. Southern blots of partially digested genomic DNA revealed large ordered arrays of Fcr1 in the genome. Fluorescent in situ hybridization with Fcr1 probes stained most centromeres in cultured cells. By staining lampbrush chromosomes, we specifically identified the 11 (of 18) chromosomes that stain consistently with Fcr1 probes.
N-linked Oligosaccharides Affect the Enzymatic Activity of CD39: Diverse Interactions between SevenN-linked Glycosylation Sites
Authors: Wu James J. , Choi Lisa E. , Guidotti Guido
Published: January 2005

Rat CD39, a membrane-bound ectonucleoside triphosphate diphosphohydrolase that hydrolyzes extracellular nucleoside tri- and diphosphates, has seven potential N-glycosylation sites at asparagine residues 73, 226, 291, 333, 375, 429, and 458. To determine their roles in the structure and function of CD39, we mutated these sites individually or in combination by replacing asparagine with serine or glutamine and analyzed the surface expression and the enzymatic activity of the mutants. The results indicate that rat CD39 can be glycosylated at all seven sites when expressed in COS7 cells. Glycosylation sites 73 at the N terminus, 333 in the middle, and 429 and 458 at the C terminus were principally required for cell surface appearance of enzymatically active CD39. Whereas deletion of these sites individually had modest effects on surface ATPase activity, some double deletions of these sites had major effects on both surface activity and expression. The importance of these N-glycosylation sites is recognizable in other members of the ectonucleoside triphosphate diphosphohydrolase family.
Frat is dispensable for canonical Wnt signaling in mammals
Authors: van Amerongen Renée , Nawijn Martijn , Franca-Koh Jonathan , Zevenhoven John , van der Gulden Hanneke , Jonkers Jos , Berns Anton
Published: January 2005

Wnt-signal transduction through β-catenin is thought to require the inhibition of GSK3 by Frat/GBP. To investigate the role of Frat in mammalian development, we have generated mice with targeted mutations in all three murine Frat homologs. We show that Frat is normally expressed at sites of active Wnt signaling. Surprisingly, Frat-deficient mice do not display gross abnormalities. Moreover, canonical Wnt signaling in primary cells is unaffected by the loss of Frat. These studies show that Frat is not an essential component of the canonical Wnt pathway in higher organisms, despite the strict requirement of Frat/GBP for maternal Wnt signaling inXenopus.
The porcine snout – an in vitro model for human lips?
Authors: Jacobi U. , Toll R. , Audring H. , Sterry W. , Lademann J.
Published: January 2005

Abstract:  The morphology and histology of test sites commonly used to study the penetration of lip products differ significantly from those of the human lip itself. The aim of this study was to investigate whether the porcine snout could serve as an equivalent in vitro model for human lips. The lips of human test subjects and biopsies of porcine snout tissue were compared using histological and microscopic techniques. Using a dermatological laser scanning microscope, the penetration of topically applied fluorescent sodium fluorescein was investigated in vivo on human lips and in vitro on the porcine snout. Biopsies from the in vitro experiments were studied using fluorescence microscopy. Some parts of the porcine snout show a similar morphology and histology as human lips. The stratum corneum (SC) and the epidermis of the porcine snout are thicker than those of human tissue. Both in vivo and in vitro, the topically applied fluorescent dye was detected only on the skin surface and within the uppermost SC layer. These results indicate that porcine snout can be used as an in vitro model for human lips in penetration studies. Both human and porcine tissues exhibit an efficient barrier against the penetration of topically applied substances.
Induction of cytokine (interleukin‐1α and tumor necrosis factor‐α) and chemokine (CCL20, CCL27, and CXCL8) alarm signals after allergen and irritant exposure
Authors: Spiekstra S. W. , Toebak M. J. , Sampat‐Sardjoepersad S. , Van Beek P. J. , Boorsma D. M. , Stoof T. J. , Von Blomberg B. M. E. , Scheper R. J. , Bruynzeel D. P. , Rustemeyer T. , Gibbs S.
Published: January 2005

Abstract:  The immune system is called into action by alarm signals generated from injured tissues. We examined the nature of these alarm signals after exposure of skin residential cells to contact allergens (nickel sulfate and potassium dichromate) and a contact irritant [sodium dodecyl sulfate (SDS)].

Nickel sulfate, potassium dichromate, and SDS were applied topically to the stratum corneum of human skin equivalents. A similar concentration‐dependent increase in chemokine (CCL20, CCL27, and CXCL8) secretion was observed for all three chemicals. Exposure to nickel sulfate and SDS was investigated in more detail: similar to chemokine secretion, no difference was observed in the time‐ and concentration‐dependent increase in pro‐inflammatory cytokine [interleukin‐1α (IL‐1α) and tumor necrosis factor‐α (TNF‐α)] secretion. Maximal increase in IL‐1α secretion occurred within 2 h after exposure to both nickel sulfate and SDS and prior to increased chemokine secretion. TNF‐α secretion was detectable 8 h after chemical exposure. After allergen or irritant exposure, increased CCL20 and CXCL8, but not CCL27, secretion was inhibited by neutralizing human antibodies to either IL‐1α or TNF‐α.

Our data show that alarm signals consist of primary and secondary signals. IL‐1α and TNF‐α are released as primary alarm signals, which trigger the release of secondary chemokine (CCL20 and CXCL8) alarm signals. However, some chemokines, for example, CCL27 can be secreted in an IL‐1α and TNF‐α independent manner. Our data suggest that skin residential cells respond to both allergen and irritant exposure by releasing mediators that initiate infiltration of immune responsive cells into the skin.
Ultraviolet A and B affect human melanocytes and keratinocytes differently. A study of oxidative alterations and apoptosis
Authors: Larsson Petra , Andersson Eva , Johansson Uno , Öllinger Karin , Rosdahl Inger
Published: January 2005

Abstract:  Ultraviolet (UV) radiation is an etiologic agent for malignant melanoma and non‐melanoma skin cancer, but the spectral range responsible for tumor induction is still to be elucidated. In this study, we compared effects of UVA and UVB irradiation on normal human melanocytes (MCs) and keratinocytes (KCs) in vitro. We demonstrate that UVA irradiation induces immediate loss of reduced glutathione (GSH) in both MCs and KCs. Exposure to UVA also causes reduced plasma membrane stability, in both cell types, as estimated by fluorescein diacetate retention and flow cytometry. Furthermore, we noted reduction in proliferation and higher apoptosis frequency 24 h after UVA irradiation. UVB irradiation of KCs caused instant reduction of reduced GSH and impaired plasma membrane stability. We also found decline in proliferation and increased apoptosis after 24 h. In MCs, on the other hand, UVB had no effect on GSH level or plasma membrane stability, although increased apoptotic cell death and reduced proliferation was detected. In summary, MCs and KCs showed similar response towards UVA, while UVB had more pronounced effects on KCs as compared to MCs. These results might have implications for the induction of malignant melanoma and non‐melanoma skin cancer.
Immunohistochemical analysis of keratinocyte growth factor and fibroblast growth factor 10 expression in psoriasis
Authors: Kovacs D. , Falchi M. , Cardinali G. , Raffa S. , Carducci M. , Cota C. , Amantea A. , Torrisi M. R. , Picardo M.
Published: January 2005

Abstract:  The pathogenic mechanism underlying the hyperproliferation of keratinocytes in psoriasis is still not completely clarified. The production of cytokines released by activated T lymphocytes infiltrating the upper dermis probably has a crucial role. Even dermal fibroblasts can participate in the process through the secretion of growth factors, and some studies have reported an increased expression of the insulin‐like growth factor 1. Few studies, however, have focused on the possible involvement of the keratinocyte growth factor (KGF/FGF‐7) and the fibroblast growth factor 10 (FGF‐10/KGF‐2), which are secreted by fibroblasts and stimulate keratinocyte proliferation acting through a receptor specifically expressed by epithelial cells. The aim of this study was to investigate the expression of KGF and FGF‐10 on the skin of patients with psoriasis by immunohistochemical analysis and to evaluate the correlation with the lymphocyte infiltrate and the epidermal proliferation. Immunostaining for KGF and FGF‐10 showed that both the growth factors are upregulated in the upper dermis of psoriatic skin, and that the expression is correlated with the presence of T‐cell infiltrate and with keratinocyte proliferation. Our data suggest that in psoriatic lesions activated lymphocytes can stimulate fibroblasts to produce KGF and FGF‐10, which in turn contribute to sustain the hyperproliferative status of the keratinocytes.
UVB‐induced leucocyte trafficking in the epidermis of photosensitive lupus erythematosus patients: Normal depletion of Langerhans cells
Authors: Janssens Artiena Soe , Lashley Eileen E. L. O. , Out‐Luiting Coby J. , Willemze Rein , Pavel Stan , De Gruijl Frank R.
Published: January 2005

Background: The pathogenic mechanisms of UV‐induced skin lesions of lupus erythematosus (LE) are unknown. In a recent study of pathogenic mechanisms of polymorphic light eruption (PLE), significantly more Langerhans cells (LCs) persisted in the epidermis after UVB overexposure than in healthy individuals. Interestingly, the same phenomenon was observed in one subacute cutaneous lupus erythematosus (SCLE) patient. It could therefore be hypothesized that both photodermatoses share a common pathogenic mechanism of photosensitivity. In the present study, we tested this hypothesis by investigating leucocyte trafficking in the initial phase of cutaneous LE after intense UVB exposure. Methods: In 22 photosensitive LE patients (12 chronic discoid lupus erythematosus, seven systemic lupus erythematosus and three SCLE) and nine age/sex‐matched controls, uninvolved buttock skin was exposed to six minimal erythemal dose (MED) UVB radiation. Subsequently, biopsies were taken after 24, 48 and 72 h, and one control biopsy was taken from unirradiated skin. Skin sections were stained for the presence of LCs, neutrophils and macrophages. Areal percentages of positively stained cells within the epidermis were quantified and compared between the patients and controls. Results: A gradual decrease of epidermal LCs and a gradual increase of epidermal neutrophils and macrophages at several timepoints after six MED irradiation was observed equally in both LE patients and controls. Conclusion: Immunohistopathology of irradiated uninvolved skin of photosensitive LE patients did not reveal the same pathologic trafficking of LCs and neutrophils as described for PLE patients. We conclude that different mechanisms are operative in the pathogenesis of PLE and photosensitive LE.
UV‐exposition and vitamin‐D: How much sunlight do we need?
Authors: Reichrath Jörg , Girndt Matthias , Tilgen Wolfgang , Querings Kerstin
Published: January 2005

UV‐exposition is considered as the main reason for the development of cancers of the skin. However, 90 to 100% of the Vitamin‐D reqirement is formed within the skin through the action of sunlight. Considering the results of epidemiological studies, that have detected an association of Vitamin‐D deficiency with various types of cancer (e.g. colon‐, prostate‐ and breast cancer), this is a real dilema. The cancer protective effect of vitamin‐D is contributed to the extra renal, local production of 1α,25(OH)2D3 by the 25‐hydroxyvitamin D‐1α‐hydroxylase, which has been detected in various tissues. In respect of the novel functions of vitamin‐D and the risk of adverse consequences in case of deficiency we have screened sun deprived risk groups (A: patients with genodermatoses connected with defects in sun‐induced DNA repair: n = 4: 3 patients with xeroderma pigmentosum and 1 patient with basal cell nevus syndrome; and B: non vitamin‐D substituted renal transplant recipients under immunosuppressants: n = 33) for their vitamin‐D status. As measure of the vitamin‐D store and as substrate for the 25‐hydroxyvitamin D‐1α‐hydroxylase basal 25(OH)D3 serum levels (Nichols Institute Diagnostika GmbH, Bad Nauheim, Germany) have been analysed. In both groups decreased basal 25(OH)D3 serum levels were detected. Therefore we demand a monitoring of vitamin‐D status in patients practising sun protection, in case of vitamin‐D deficiency an oral substitution should be recommended.
UVB‐induced conversion of 7‐dehydrocholesterol to 1α,25‐dihydroxyvitamin D3(calcitriol) in cultured keratinocytes is upregulated by tumor necrosis factor‐α
Authors: Abraham S. , Lehmann B. , Knuschke P. , Meurer M.
Published: January 2005

UVB irradiation of cultured human keratinocytes induces both the conversion of 7‐dehydrocholesterol (7‐DHC) to calcitriol (1α,25(OH)2D3) and the release of tumor necrosis factor‐a (TNF‐α) in these cells. Calcitriol synthesis in human keratinocytes was reduced in the presence of a neutralizing polyclonal antibody directed against human TNF‐α. On the other hand, we found a 1.7‐fold higher stimulatory effect of UVB on liberation of TNF‐α in cultured keratinocytes enriched with 7‐DHC compared with irradiated cell cultures in absence of 7‐DHC. These observations argue in favor of a synergetic relationship between generation of TNF‐α and calcitriol in UVB irradiated keratinocytes. In addition, we found that TNF‐α potently increases the conversion rate of vitamin D3(cholecalciferol) to calcitriol this cell system. The UVB‐triggered formation of both TNF‐α and calcitriol in cultured keratinocytes as wavelength‐, time‐ and dose‐dependent. Maximum formation of TNF‐α and calcitriol was found at 300 nm and UVB doses of 30 mJ/cm2. The enhancement of both, the formation of TNF‐α and calcitriol in keratinocytes by UVB may be of relevance for regulation of growth and apoptosis in light‐exposed epidermal cells and, in addition, may play a role in the UVB treatment of deseased skin, including psoriasis.
Stimulation of keratinocyte differentiation – a new role for the vanilloid receptor subtype 1 (VR1/TRPV1)?
Authors: Ständer Sonja , Moormann Corinna , Schumacher Mark , Metze Dieter , Luger Thomas A. , Steinhoff Martin
Published: January 2005

Vanilloids and endogenous cannabinoids mediate their actions via the vanilloid receptor subtype 1 (VR1/TRPV1), a non‐selective cation channel, which is widely distributed in the central and peripheral nervous system. Only recently, VR1 has been shown to be expressed in keratinocytes in vitro and in vivo. However, a precise description of VR1 localization in epithelial cells was missing. To determine this, we investigated VR1‐immunoreactivity as well as mRNA and protein expression in a series of biopsies from normal, diseased, and capsaicin‐treated human skin. VR1 was found in epidermal keratinocytes, the inner root sheet and the infundibulum of hair follicles, differentiated sebocytes, sweat gland ducts, and the secretory portion of eccrine sweat glands upon immunohistochemistry, RT‐PCR and Western blot analysis. Interestingly, in diseased skin such as prurigo nodularis, psoriasis vulgaris, and atopic dermatitis, VR1 expression in keratinocytes correlated with the degree of epidermal differentiation. Enhanced VR1 immunoreactivity and protein content was found in prurigo nodularis in which epidermal keratinocytes are highly differentiated. Under effective capsaicin therapy of prurigo nodularis, the epidermis thinned and the distribution pattern of VR1 on epidermal keratinocytes normalized. In psoriasis vulgaris, a disease with disturbed epidermal differentiation, less intense immunostaining for VR1 was observed. This could be confirmed by western blot analysis showing less VR1 protein amount in comparison to prurigo nodularis although histologically both showed a thickened epidermis. In atopic dermatitis, which is characterized by a moderate epidermal hyperplasia only and regular differentiated keratinocytes, VR1 immunoreactivity was unchanged in comparison to normal skin. These findings suggest that VR1 may contribute to regular differentiation of keratinocytes. VR1 activation opens non‐selective cation channels with high permeability to calcium, a ion that is crucially important for the synthesis of cornification proteins such as involucrin, fillagrin and loricrin. The role of VR1 in other epithelial cells of appendage structures remains to be determined. In summary, VR1 is widely distributed in the skin suggesting a central role for this receptor not only in nociception but also maturation and function of epithelial cells.
Alpha 9 acetylcholine receptors are essential for epidermal differentiation
Authors: Kurzen Hjalmar , Berger Hans , Jäger Claudia , Hartschuh Wolfgang , Maas‐Szabowski Nicole
Published: January 2005

Extraneuronally, acetylcholine (Ach) is synthesized, stored and secreted in the tegumental cells covering the inner and outer surfaces of the body. It acts via nicotinic (nAchR) and muscarinic (mAchR) receptors in a paracrine and autocrine fashion influencing various keratinocyte functions. Using in vitro studies, we present evidence that effects of cholinergic agonists and antagonists on cutaneous biology are dependent upon specific Ach‐R, localised in the different compartments of the skin. Using immunohistochemistry on frozen section of normal skin we could previously demonstrate a differential distribution of alpha 3*, alpha 7, alpha 9 and beta 1 nAchR and m1, m3–5 mAchR in human epidermis. Here we show the functional significance of AchR blockage using organotypical keratinocytes cultures. Cholinergic agonists and antagonists were added on the day of the lift to the air‐liquid interface. After 5 days, blockage of all AchR using mecamylamine and atropine resulted in complete inhibition of terminal differentiation. Mecamylamine and atropine alone had only a retarding effect suggesting an additive mechanism of nAchR and mAchR blockage. Specific inhibition of α9 homooligomers with strychnine produced the strongest effects leaving behind only a keratinocytes monolayer. Stimulation of AchR with either nicotine or muscarine in short term organotypic cultures did not produce dramatic changes of epidermal morphology. We conclude that alpha 9 AchR that are located in the basal and lower suprabasal layers of the epidermis, are crucially involved in terminal differentiation of keratinocytes. The functional significance of other nAchR expressed in the epidermis remains unclear, due to the lack of specific agonists and antagonists.
Human epidermal acetylcholinesterase (AchE) is regulated by hydrogen peroxide (H2O2)
Authors: Schallreuter K.U. , Elwary S. , Gibbons N. , Rokos H. , Wood J.M.
Published: January 2005

Previously it has been demonstrated that the human epidermis synthesizes and degrades acetylcholine and expresses both muscarinic and nicotinic receptors. These cholinergic systems have been implicated in the development of the epidermal calcium gradient and differentiation in normal healthy skin. In vitiligo severe oxidative stress occurs in the epidermis of these patients with accumulation of H2O2 in the 10−3M range together with a decrease in catalase expression/activity due to deactivation of the enzyme active site. It was also shown that the entire recycling of the essential cofactor (6R)‐L‐erytho 5, 6, 7, 8 tetrahydrobiopterin via pterin‐4a‐carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) is affected by H2O2 oxidation of Trp/Met residues in the enzyme structure leading to deactivation of these proteins. Using fluorescence immunohistochemistry we now show that epidermal H2O2 in vitiligo patients yields also almost absent epidermal acetylcholinesterase (AchE) in association with accumulation of epidermal acetylcholine. This result was confirmed by Fluorescence excitation spectroscopy following the Trp fluorescence at λmax 280 nm. A kinetic analysis using pure recombinant human AchE revealed that low concentrations of H2O2(10−6M) activate this enzyme by increasing the Vmax > 2 fold, meanwhile high concentrations of H2O2(10−3M) deactivate the enzyme with a significant decrease in Vmax. Molecular modelling based on the established 3D structure of human AchE supported that H2O2‐mediated oxidation of Trp432, Trp435 and Met436 moves and disorients the active site His440 of the enzyme, thus explaining the deactivation of the protein. To our knowledge these results identified for the first time H2O2 regulation of AchE. Moreover, it was shown that H2O2‐mediated oxidation of AchE contributes significantly to the well established oxidative stress in vitiligo.
Perspectives of estrogen treatment in skin aging
Authors: Schmidt Jolanta B
Published: January 2005

Clinical evidence of correlations between menopause and endogenous skin aging gave input to various studies investigating the relevance of estrogens for skin functions that are associated with skin aging and their possible therapeutic effects. Skin thickness and bone density are significantly decreased already six months after menopause and are increased after the same period of hormone replacement (HRT). Fibroblast and keratinocyte function is stimulated by estrogen application, and among other effects significant increases of collagen fibres have been demonstrated six months after the onset of HRT (1). Topical use of estrogen compounds was found to diminish skin aging symptoms. The effects of conjugated estrogens (0,625% Premarin) were studied in 60 postmenopausal women (2). In another study the effects of estradiol 0,01% and estriol 0,3% were compared (3). Both studies documented significant reductions of wrinkles without any systemic side effects of the treatments. Recently significant increases of epidermal thickness during estradiol have been described (4). For cosmetic purposes phytoestrogens seem a promising alternative to the medical treatment. Isoflavone containing cosmetical creams were shown to improve skin dryness and wrinkles (5). In various studies mainly beneficial effects of systemic HRT on skin aging parameters have been documented. Although skin aging is certainly no indication for systemic hormone supplementation the beneficial action of such treatment on aging symptoms of the skin are a positive side aspect of such treatment.
Control of androgen receptor expression in human keratinocytes and in a reconstituted human epidermis model with selective antisense oligonucleotides
Authors: Fimmel S , Bonté F , Kurfürst R , Zouboulis ChC
Published: January 2005

Association of locally increased androgen activity and skin disorders is obvious in acne and androgenetic alopecia in males. In addition, testosterone was unexpectedly found to perturb the epidermal barrier. Blockade of androgen action via androgen receptor (AR) antagonism accelerates wound healing in aged individuals. Androgen activity on skin can classically be inhibited by systemic administration of compounds, which have strong affinity for AR and antagonize androgen binding to AR molecules. In this study we applied a new technology to realize the same purpose: We tested the activity of antisense oligonucleotides against the AR in primary human foreskin keratinocytes, human non‐foreskin keratinocytes from young (30 y) and older (60 y) female donors, and reconstituted human epidermis (SkinEthic model). Reconstituted human epidermis is similar to in vivo human epidermis and features a functional permeability barrier. To transfer the antisense oligonucleotides into human keratinocytes an optimum liposome‐mediated transfection system with Poly‐L‐ornithine (12 μg/ml) over 4 h was used. The transfection efficiency was assessed using FITC‐labeled (ACTG)5 random oligonucleotides, which were localized in cytoplasmic structures of the keratinocytes. AR expression on the protein level was investigated by Western blotting. Transient transfection of foreskin keratinocytes with phosphorothioate antisense oligonucleotides (PTO) revealed a reduction of AR expression (≈25%) compared to native keratinocytes after 14 h recovery time. The AR knock down in epidermal keratinocytes of the compared women was stronger in the older, more differentiated keratinocytes. After 24 h, AR expression level have returned back to the level of non‐transfected cells. The effect could be reestablished by repetition of transfection. PTO and 2?O‐methylribosyl (MRO) antisense oligonucleotides decreased AR expression at levels varying between 46% and 70% in the air‐lifted reconstituted human epidermis after 18 h recovery time. The successful inhibition of AR expression in human keratinocytes and reconstituted human epidermis is the first step to develop topically efficient compounds with oligonucleotides.
Effects and side effects of 2%‐progesterone cream on the skin of peri‐ and postmenopausal women: Results from a double blind, vehicle‐controlled, randomized study
Authors: Holzer Gregor , Riegler Elisabeth , Hönigsmann Herbert , Schmidt Jolanta
Published: January 2005

Background: Since many years topical progesterone has been prescribed by gynecologists as an anti‐aging and skin firming treatment, without any clinical scientific evidence of its effects, tolerability and its safety when topically applied to skin. Objective: The objective of this study was to evaluate the influence of 2%‐progesterone cream on the functions and texture of the skin in the peri‐ and postmenopause. Methods: A double blind, randomized, vehicle controlled study was conducted in 40 subjects. Clinical monitoring, subjective self assessment, objective measurement methods of skin elasticity, epidermal hydration and skin surface lipids and the determination of blood hormone levels (LH, FSH, estrogen, and progesterone) were used to determine effects and side effects of this treatment at four visits during 24 weeks. Results: The study demonstrated consistent superiority of 2%‐progesterone cream over vehicle in counteracting different signs of aging in the skin of peri‐ and postmenopausal women. Clinical monitoring observed higher reductions in wrinkle counts (−29,10% vs. 16,50%) and wrinkle depth (−9,72 vs. –7,35%) around the right eye, higher decrease in nasolabial wrinkle depth (−9,72% vs. –6,62%) and a significantly higher (p = 0,031) improvement of skin firmness (23,61% vs.13,24%) in the treatment group. 2%‐progesterone cream also yielded significant improvements (p < 0,05) in the objective measurement of different parameters of skin viscoelasticity and elasticity compared to the vehicle group. Epidermal hydration and skin surface lipids did not change significantly in both groups during the study. Progesterone was well absorbed in the systemic circulation, mean blood levels rose minimally, but statistically significant (p = 0,001) by 0,53 ng/nl. No serious side effects of the treatment were observed. Conclusions: The results of this study demonstrate that topical 2%‐progesterone acts primarily in a skin firming and elasticity improving manner on hormone deficient skin of the peri‐ and postmenopause. These effects in combination with good tolerability make progesterone a possible treatment agent in slowing down the aging process of the female skin after onset of menopause.
Expression of melanocortin receptors on cutaneous fibroblastic cells – collagen and beyond
Authors: Böhm M. , Eickelmann M. , Ständer S. , Vogt A. , Blume‐Peytavi U. , Tobin D. , Luger T.A.
Published: January 2005

Melanocortin receptors (MC‐Rs) mediate the biological actions of the members of melanocortin family, e.g. of α‐melanocyte‐stimulating hormone (α‐MSH) and adrenocorticotropin. We recently reported that human dermal fibroblasts derived from newborn foreskin express MC‐1R. In these cells and in a newborn mouse model of cutaneous fibrosis, α‐MSH suppressed collagen synthesis induced by the profibrotic cytokine transforming growth factor‐β1(Böhm et al., J. Biol. Chem. 2004). Here, we show that MC‐1R expression is maintained in various fibroblastic skin cell types established from adult human skin. In vitro, expression of MC‐1R at the RNA level was detected by RT‐PCR analysis in dermal fibroblasts, dermal papilla cells and connective tissue sheath fibroblasts of the hair follicle, as well as in HT1080 fibrosarcoma cells. In all of the latter cell types except in HT1080 cells, MC‐1R immunoreactivity could be visualized on the cell surface as demonstrated by immunofluorescence studies using an antibody against the amino acids 2–18 of the N‐terminal domain of human MC‐1R. In situ, MC‐1R expression was detectable in interfollicular dermal fibroblasts only by immune electron microscopy. In contrast, MC‐1R expression was detectable in dermal papilla cells and connective tissue sheath fibroblasts of the hair follicle by conventional immunohistochemistry. To further assess the relevance of MC‐1R being expressed in fibroblastic cells of the skin we treated human dermal fibroblasts in vitro with the proinflammatory cytokine interferon‐γ(IFN‐γ). α‐MSH significantly suppressed the upregulating effect of this cytokine on the expression of intercellular adhesion molecule‐1 (ICAM‐1), an adhesion molecule crucially involved in recruitment of activated leukocytes into the hair follicle. In summary, our findings form a basis upon which MC‐1R expression can be investigated in inflammatory disorders affecting the connective tissue compartment of the skin. Moreover, our preliminary data on the modulation of IFN‐γ‐driven upregulation of ICAM‐1 by α‐MSH suggest additional functions of melancortins in fibroblasts beyond collagen synthesis.
Expression and functional relevance of melatonin receptors in hair follicle biology
Authors: Kobayashi H. , Paus R.
Published: January 2005

The prototypic pineal hormone, melatonin reputedly exerts many functional effects on mammalian skin and/or its isolated cell populations in culture (e.g., melanogenesis inhibition, melanocyte growth inhibition, regulation of seasonal changes in the pelage), and is recognized as a potent free radical scavenger. In mammals, two types of high‐affinity membrane melatonin receptors, MT1 and MT2 have been identified, which inhibit adenylate cyclase activity to decrease the intracellular level of cAMP. Low‐affinity membrane receptor MT3/QR2 have also been identified, though the mechanism has not been cleared yet. Melatonin is also a natural ligand of nuclear transcription factor ROR(α and β), which is suggested to regulate cell cycle negatively via target gene such as p21WAF/CIP1. Due to its lipophilic structure, melatonin also enters through both the plasma and nuclear membrane, and acts as a potent free radical scavenger to protect macromolecules, in particular DNA. Melatonin demonstrates differential – and often still confusing seemingly contradictory– effects on cell activity in many different systems, which may be explained by this multitude of signaling pathways that are modulated by melatonin bioactivity. Recently, cultured epidermal and follicular melanocytes, keratinocytes, and fibroblasts, have also been found to display the enzymatic activity of arylalkylamine N‐acetyltransferase and hydroxyindole ‐O‐ methyltransferase for melatonin synthesis. However, little is known about the cutaneous expression and regulation of melatonin and its receptors in situ, and the functional role of melatonin in normal skin and hair follicle biology is still obscure. In order to study whether murine hair follicles in situ are indeed direct peripheral melatonin targets, the follicular expression of MT1, MT2 and/or RORα are investigated. Immunohistochemistry revealed that C57BL/6 mouse hair follicle keratinocytes in situ show prominent MT1‐like immunoreactivity (IR), which changed substantially in a hair cycle‐dependent manner. RORα‐like IR was also detected in murine hair follicles, and also displayed hair cycle dependence. RT‐PCR of MT1 and MT2, and real time PCR for MT1, MT2 and RORα on C57BL/6 mice skin cDNA revealed that all three genes are transcribed in normal mouse skin, and demonstrated that their expression/transcription is hair cycle‐dependent. In conclusion, normal murine hair follicles are indeed a prominent, direct target for melatonin bioregulation, through MT1, MT2 and RORα melatonin receptors and that at least some of these regulators are functionally active in situ. The observed hair cycle dependence of melatonin receptor expression suggests a role of melatonin in hair cycle control.
The human hair follicle has established a fully functional peripheral equivalent of the hypothamalic‐pituitary‐adrenal‐axis (HPA)
Authors: Ito N. , Ito T. , Paus R.
Published: January 2005

After the initial discovery that, in vivo, mammalian skin both transcribes and translates the proopiomelanocortin (POMC) gene, and processes its product into melanocortins (Slominski et al., Experientia 1992), it has become increasingly appreciated that the hair follicle – including the human one – is a prime source and target not only of POMC‐derived “pituitary” hormones, e.g. alpha‐MSH, ACTH and ß‐endorphin, but also expresses the most proximal control element of the hypothalamic‐pituitary‐adrenal (HPA) axis, corticotropin‐releasing hormone (CRH) and its receptor (e.g. Roloff et al. FASEB J 1998, Ito et al. J Invest Dermatol 2004). However, while all proximal elements of the HPA are expressed in both murine and human hair follicles (CRH, CHR‐R, POMC, ACTH and ACTH‐R), it has neither been shown that these are functionally linked (i.e., is CRH actually capable of modulating intrafollicular POMC gene expression and ACTH production?), nor has it been known whether the most distal HPA component – cortisol synthesis – is also present in the hair follicle. Therefore, we have investigated whether the stimulation of microdissected, organ‐cultured human hair follicles with CRH or ACTH elicits responses inside this peripheral miniorgan that imitate a functional HPA – in the absence of any systemic or neural connections and under serum‐free culture conditions. Here, we show that CRH stimulation of organ‐cultured human scalp hair follicles in the anagen VI stage of the hair cycle indeed results in significant upregulation of POMC transcription, and of alpha‐MSH, ACTH, MC1, MC2 and glucocorticoid receptor (GR) immunoreactivity in situ (immunofluorescence). ACTH stimulation, in turn, significantly up‐regulates the – already constitutively present!– cortisol‐immunoreactivity as well as cortisol secretion into the culture medium. This represents the first available evidence that normal human skin (more precisely: the hair follicle) can actually synthesize the “adrenal” steroid hormone cortisol in situ, and that this acticity is regulated by the same “hypothalamic” and “pituitary” hormones that operate as key controls of adrenal cortisol synthesis. Moreover, we show that cortisol stimulation exerts classical feedback responses inside the human anagen hair follicle recognized for the central HPA: cortisol up‐regulates GR, while it down‐regulates CRH expression. Given that the HPA operates as the major system for coordinating stress‐responses of the mammalian organism and for integrating them into changing metabolic demands and neuro‐endocrine‐immune signaling circuits, it has fascinating implications (e.g. for general skin physiology and dermatological therapy), and raises most intriguing questions, that human hair follicles are utilizing a fully functional peripheral equivalent of the central HPA.
Effect of granulocyte‐macrophage colony‐stimulating factor on post‐weaning multisystemic wasting syndrome in porcine circovirus type‐2‐transfected piglets
Authors: Loizel Christophe , Blanchard Philippe , Grasland Béatrice , Dory Daniel , Oger Aurélie , Nignol Anne‐Cécile , Cariolet Roland , Jestin André
Published: January 2005

Post‐weaning multisystemic wasting syndrome (PMWS) is a complex disease syndrome in swine, affecting nursery and fattening pigs. Although ongoing evidence suggests that porcine circovirus type‐2 (PCV2) is the causal agent of PMWS, the host immune system appears to have a crucial role in the PMWS pathogenesis of PCV2‐affected pigs. Owing to difficulties in producing a biologically pure form of PCV2 devoid of the other viral agents commonly present in swine tissues, we decided to use a tandem‐cloned PCV2 DNA providing highly pure grade reagent in order to monitor the virulence of PCV2 alone or with an immunostimulating co‐factor, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). A single intramuscular injection of tandem‐cloned PCV2 DNA into 5‐week‐old piglets produced plasmid to viral genome progeny and infectious particles as early as 8 days post‐injection in all the organs tested (the lung, the tonsil and the inguinal, mesenteric, bronchial and upper‐right axial lymph nodes). The initial plasmid load was not detected with the help of primers designed to specifically detect the acceptor plasmid, thus confirming the replication of the viral genome. Despite the presence of a high level of PCV2 genome copies in the lymphoid organs – the tonsil and the lung – and the presence of infectious particles, no detectable clinical manifestations or pathological lesions were observed in the transfected pigs over the period of observation, regardless of whether they had been co‐injected with plasmid containing GM‐CSF DNA or had received plasmid containing PCV2 DNA alone. GM‐CSF encoding DNA injection had no significant effect on viral replication or on the production of viral particles and appearance of the disease.
Ultrastructural evaluation of the radioprotective effects of melatonin against X‐ray‐induced skin damage in Albino rats
Authors: Hussein Mahmoud R. , Abu‐Dief Eman E. , Abd el‐Reheem Mohammad H. , Abd‐Elrahman Ali
Published: January 2005

Our knowledge about the radioprotective effects of melatonin against X‐ray‐induced skin damage is still lacking. To examine these effects, an animal model of 60 Albino rats was used. The animals were divided into five groups: Group 1, nonirradiated; Group 2, X‐ray irradiated (XRI, 8 Gy); Group 3, XRI pretreated with solvent (ethanol and phosphate‐buffered saline); Group 4, nonirradiated group treated with melatonin; and Group 5, XRI pretreated with melatonin. The skin was evaluated for ultrastructural changes using transmission electron microscopy (TEM). When compared to the nonirradiated skin (Groups 1 and 4), XRI skin (Groups 2 and 3) showed features of both cell injury and increased metabolic activity. The former included changes such as condensation of the nuclei, vacuolization of the cytoplasm, dilatation of the rough endoplasmic reticulum, swelling of the mitochondria with cristolysis, destruction of the ribosomes and intermediate filaments, fragmentation of the keratohyaline granules and loss of the irregularity of the basal cell borders. The central cells of the sebaceous gland alveoli had larger irregular nuclei and few lipid droplets in their cytoplasm. The hair follicle cells had heterochromatic nuclei and less electron dense cytoplasm containing few complements of the organelles. The features of increased metabolic activity included increased euchromatin, irregularity of the nuclear membrane and increased branching of the melanocytes. Also, an increased number of the Birbeck granules were seen in the Langerhans cells. When compared to the irradiated skin (Groups 2 and 3), these changes were mild or absent in the skin of XRI animals pretreated with melatonin (Group 5). The ability of melatonin to minimize the injurious effects of XRI suggests a radioprotective role. The clinical ramifications of these observations warrant further studies.
Sibling relationships in adolescence: Learning and growing together
Authors: Noller Patricia
Published: January 2005

In this article, I discuss the reasons for my interest in sibling relationships, and showcase studies on sibling relationships in adolescence carried out with my colleagues and students, in the context of the broader literature on sibling relationships. Our studies have focused on a number of important issues concerned with sibling relationships. First, I report on the associations between sibling relationships and other family relationships and the ways that the various family relationships affect each other. Second, I report a study of sibling relationships in the context of parental separation and divorce and show that sibling relationships in these families are more likely to be high in both warmth and hostility than is true for relationships in 2‐parent families. Third, I report on several data sets showing an association between the quality of sibling relationships and adolescent adjustment and the link between differential parenting, adolescent adjustment, and the quality of the sibling relationship. Fourth, I report on a study of comparison and competition in sibling relationships and the associations between sibling relationship quality and reactions to being outperformed by a sibling. Finally, I discuss possible future directions for research on sibling relationships, including the importance of multimethod studies and a longitudinal perspective.
Conflict and support interactions in marriage: An analysis of couples’ interactive behavior and on‐line cognition
Authors: Verhofstadt Lesley L. , Buysse Ann , Ickes William , De Clercq Armand , Peene Olivier J.
Published: January 2005

The present study examined the similarities and differences in couples’ interactive behavior and interaction‐based cognition that emerged in comparisons of conflict and support interactions in marriage. In a laboratory experiment, 53 couples were randomly assigned to the conditions of a 2 (type of interaction: conflict vs. support) × 2 (initiator of interaction: man vs. woman) factorial design. Partners provided questionnaire data and participated in a joint interaction and video review task. The data revealed substantial behavioral similarities (i.e., some classes of validation/facilitation behaviors and neutral problem‐solving behaviors) as well as behavioral differences (i.e., some classes of invalidation/oppositional behaviors) between conflict and support interactions, controlling for levels of marital satisfaction. Partners’ interaction‐based cognition (e.g., feeling understood, satisfied) was especially affected by classes of validation/facilitation behaviors and was consistently related to marital satisfaction. In broad terms, the impact of a particular behavior on partners’ ongoing cognition did not depend on the interaction domain (conflict vs. support) in which the behavior occurred.
Reasons for, and consequences of, revealing personal secrets in close relationships: A longitudinal study
Authors: Caughlin John P. , Afifi Walid A. , Carpenter‐Theune Katy E. , Miller Laura E.
Published: January 2005

This investigation examines whether individuals’ reasons for keeping secrets predict whether they eventually reveal those secrets and whether individuals can accurately anticipate the outcomes of revealing. Respondents (n= 342) first reported on a secret they were keeping and then returned 2 months later to report whether they had revealed it and, if so, what happened when they did. Findings indicated that participants’ reasons for keeping a secret predicted whether they revealed it. The results also indicated both accuracy and inaccuracy in secret tellers’ expectations of the outcomes of revealing a secret. Finally, despite some demonstrable inaccuracies in the forecasted outcomes, participants’ retrospective accounts after revealing suggested that participants typically believed that they had accurately predicted the consequences of revealing.