bluuebunny/crossref_metadata_2025 huggingface dataset:
Human dermal fibroblasts do not exhibit directional migration on collagen I in direct‐current electric fields of physiological strength
Authors: Sillman Amy L. , Quang Dung My , Farboud Benhom , Fang Kathy S. , Nuccitelli Richard , Isseroff R. Rivkah
Published: July 2003

Abstract: Endogenous electric fields are generated lateral to skin wounds, with the cathodal pole of the field residing in the center of the wound. These fields are thought to be an important mechanism in guiding the migration of keratinocytes and other cells into wounds to effect healing. In this work, human dermal fibroblasts were exposed to direct current electric fields of physiological strength, and their migrational behavior was quantitated. Only random migration of human dermal fibroblasts was observed in direct‐current electric fields under conditions that support the directional migration of human epidermal keratinocytes. Additionally, neither the presence of serum nor serum plus additional Mg++ in the experimental medium supported directional migration. Migratory rates of fibroblasts varied depending on the experimental medium used: in serum‐containing medium the average velocity was as low as 0.23 µm/min, while in serum‐free keratinocyte medium the average velocity was as high as 0.36 µm/min. These studies suggest that dermal fibroblasts do not respond to the endogenous electric field of a wound, and use other migratory cues to direct their movement into the wound bed.
Supernatants of Pseudomonas aeruginosa induce the Pseudomonas‐specific antibiotic elafin in human keratinocytes
Authors: Meyer‐Hoffert Ulf , Wichmann Nils , Schwichtenberg Lars , White Paul C. , Wiedow Oliver
Published: July 2003

Abstract: Elafin is a skin‐derived serine‐protease inhibitor. It is thought to be important to prevent human leukocyte elastase‐mediated tissue damage and might play an important role in maintaining the integrity of the human epidermis. Recent studies have provided evidence for an antimicrobial activity of elafin against P. aeruginosa. As gram‐negative infections typically occur in barrier‐disrupted skin we were interested to determine whether supernatants of the gram‐negative bacteria P. aeruginosa and Escherichia coli were capable of inducing elafin expression.

Supernatants of various P. aeruginosa strains stimulated elafin mRNA‐expression and protein release, whereas supernatants of E. coli did not induce elafin expression. In non‐differentiated cells the relative increase of elafin mRNA was much higher (100‐fold) than in differentiated cells (sixfold), although the latter exhibited higher constitutive mRNA‐expression (150‐fold). However, concentrations of secreted elafin were similar in differentiated and non‐differentiated cells after stimulation. We could not confirm a bactericidal effect against P. aeruginosa as described previously but observed that its growth was inhibited as demonstrated for different strains in liquid cultures. Growth of E. coli was not affected by elafin.

In conclusion, the data presented in this paper suggest that elafin represents an innate immune response factor induced by secreted products of P. aeruginosa. Besides its elastase inhibitory potency elafin is an antimicrobial agent against P. aeruginosa.
Adalimumab, a fully human anti‐TNF‐α monoclonal antibody, treatment does not influence experimental UV response in the skin of rheumatoid arthritis patients
Authors: Tjioe M. , Gerritsen M. J. P. , Den Broeder A. A. , Van Hooijdonk C. A. E. M. , Kroot E. J. A. , Van Riel P. L. C. M. , Barrera P. , Van De Kerkhof P. C. M.
Published: July 2003

Abstract: TNF‐α is known to play an important role in UV‐induced immunomodulation and photodamage. It plays a role in UVB‐mediated induction of apoptosis and is a strong inducer of the c‐Jun N‐terminal kinase (JNK) pathway, which eventually leads to the loss of dermal collagen and elastin content. Recently chimeric anti‐TNF‐α has been introduced as a therapy for rheumatoid arthritis.

The aim of the present study was to investigate the effect of anti‐TNF‐α treatment on UV‐induced DNA damage, apoptosis, and induction of matrix metallo proteinases.

Twelve patients with rheumatoid arthritis were included and irradiated with 2 MED broadband UVB before and after administration of 0.5 mg/kg anti‐TNF‐α monoclonal antibody. Twenty‐four hours after irradiation biopsies were taken. Frozen and paraffin sections were stained for p53, c‐Jun, phosphorylated c‐Jun, sunburn cells and MMP‐1.

No significant changes were observed in the expression of p53 and sunburn cells and MMP‐1 content after treatment with anti‐TNF‐alpha, whereas a slight but significant decrease in c‐Jun and phosphorylated c‐Jun expression was noted (P = 0.0250 and P = 0.0431, respectively).

Our results showed no influence of anti‐TNF‐α on UV response at therapeutic doses in patients with rheumatoid arthritis.
Urokinase plasminogen activator mRNA is induced by IL‐1α and TNF‐α in in vitro acantholysis
Authors: Feliciani Claudio , Toto Paola , Wang Binghe , Sauder Daniel N. , Amerio Pierluigi , Tulli Antonio
Published: July 2003

Abstract: The role of urokinase type plasminogen activator (uPA) has been well documented in the pathogenesis of pemphigus vulgaris (PV). Activation of plasminogen into active serine protease plasmin initiates extracellular proteolysis leading to acantholysis but the mechanisms underlying this process are not clearly understood. We have previously shown that keratinocyte derived cytokines IL‐1α and TNF‐α are involved in PV‐induced acantholysis. In the present study we sought to examine whether keratinocyte‐derived IL‐1α and TNF‐α are correlated with uPA induction in keratinocytes during acantholysis. Normal human keratinocytes were incubated with diluted PV serum. mRNAs for IL‐1α, TNF‐α and uPA were examined with RT‐PCR at various time points and acantholysis was measured. IL‐1α, TNF‐α and uPA mRNAs were all induced in keratinocytes following PV serum stimulation; IL‐1α/TNF‐α mRNAs' expression was earlier than the expression of uPA mRNA. To further examine the role of IL‐1α, TNF‐α and uPA in acantholysis, we performed antibody blocking studies. Anti‐IL‐1α, anti‐TNF‐α and anti‐uPA antibodies suppressed acantholysis by 76%, 80% and 90%, respectively. In addition, anti‐IL‐1α and anti‐TNF‐α antibodies inhibited uPA mRNA induction, whereas anti‐uPA antibodies did not alter IL‐1α/TNF‐α mRNAs' expression. Our results confirm the role of uPA in acantholysis and suggest an involvement of IL‐1α/TNF‐α in uPA induction.
Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex
Authors: Sørensen Charlotte B. , Andresen Brage S. , Jensen Uffe B. , Jensen Thomas G. , Jensen Peter K. A. , Gregersen Niels , Bolund Lars
Published: July 2003

Abstract: Epidermolysis bullosa simplex (EBS) is a group of autosomal dominantly inherited skin disorders characterized by the development of intra‐epidermal skin blisters on mild mechanical trauma. The three major clinical subtypes (Weber‐Cockayne, Koebner and Dowling‐Meara) are all caused by mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene.

Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3′ end of the KRT14 cDNA sequences. The expression vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN).

No effect on the keratin cytoskeleton was observed upon transfection of NHK with the various K14 constructs neither with nor without a subsequently induced heat‐stress. In contrast, all constructs, including wild‐type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated with the most severe form of the disease, EBS Dowling‐Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P < 0.001). Thus, although a background KFN perturbance was observed upon transfection with the wild‐type K14 construct, the mutant protein associated with the most severe form of EBS worsened the KFN perturbation significantly compared with the mutant proteins associated with the milder forms of the disease and the normal K14 protein. This shows that the clinical severity of disease‐associated mutations identified in patients can be tested using this expression system, although it can not at present be used to discriminate between the milder forms.

Assessment of the endogenous K14 protein expression in NHK and HaCaT cells indicated that the higher level of endogenous keratin expression in NHK might make these cells more resistant to perturbation of the keratin cytoskeleton by overexpressed K14 protein than HaCaT cells.
Nickel‐induced keratinocyte proliferation and up‐modulation of the keratinocyte growth factor receptor expression
Authors: Marchese Cinzia , Visco Vincenzo , Aimati Laura , Cardinali Giorgia , Kovacs Daniela , Buttari Brigitta , Bellocci Marinella , Torrisi Maria Rosaria , Picardo Mauro
Published: July 2003

Abstract: Keratinocytes play a key role in the pathogenesis of allergic contact dermatitis (ADC) induced by the sensitizing agent nickel. We analyzed here the effects of treatment with nickel and of the pretreatment with zinc on HaCaT cells and primary human keratinocytes. Cell counting, 5‐bromo‐2′‐deoxyuridine incorporation assay and adenosine triphosphate (ATP) bioluminescence detection showed that treatment with NiSO4 induced DNA synthesis and cell proliferation and that pretreatment with ZnSO4 was able to abrogate this proliferative effect. This nickel‐induced cell growth appeared enhanced when primary human keratinocytes were co‐cultured with fibroblasts. Western blot analysis demonstrated that nickel ions induced up‐modulation of the expression of the keratinocyte growth factor receptors (KGFR) without affecting the keratinocyte differentiation, whereas the protein levels of the epidermal growth factor receptor (EGFR) and of its ligand transforming growth factor‐alpha (TGF‐α) appeared unmodified by the treatment. Double immunofluorescence showed that the effect of nickel on DNA synthesis was mainly exerted on KGFR expressing cells, suggesting that KGFR up‐modulation could be required for the nickel‐induced cell proliferation. These results indicate that KGFR and its ligands may play a role in the mechanism of action of nickel ions and in the protective effect of zinc pretreatment.
A Crohn's disease‐associated insertion polymorphism (3020insC) in the NOD2 gene is not associated with psoriasis vulgaris, palmo‐plantar pustular psoriasis or guttate psoriasis
Authors: Young C. , Allen M. H. , Cuthbert A. , Ameen M. , Veal C. , Leman J. , Burden A. D. , Kirby B. , Griffiths C. E. M. , Trembath R. C. , Mathew C. G. , Barker J. N. W. N.
Published: July 2003

Abstract: A C‐insertion polymorphism in the NOD2 gene (3020insC) on chromosome 16 is a rare mutation associated with Crohn's disease. Crohn's disease and psoriasis are more commonly observed together than expected by chance. Furthermore a susceptibility locus for psoriasis has been identified on chromosome 16q which overlaps the recently identified susceptibility locus for Crohn's disease. Thus, NOD2 may potentially be important as a candidate susceptibility gene for psoriasis. We tested this hypothesis by genotyping psoriasis patients for the C‐insertion polymorphism using the Taqman ABI 7700 sequencing system. No statistically significant differences were observed between psoriasis vulgaris (n = 216), palmo‐plantar pustular psoriasis (PPP) (n = 100), guttate psoriasis (n = 118) and the control group (n = 283). In both patient and control groups, no mutant homozygotes were observed and approximately 4% were heterozygotes. This particular insertion mutation in the NOD2 gene does not appear to contribute to the genetic susceptibility of psoriasis vulgaris, PPP or guttate psoriasis. However, other mutations exist in the NOD2 gene, which may potentially have a role in psoriasis susceptibility.
Mutations in the ED1 gene in Japanese families with X‐linked hypohidrotic ectodermal dysplasia
Authors: Hashiguchi Takaki , Yotsumoto Shinichi , Kanzaki Tamotsu
Published: July 2003

Abstract: X‐linked hypohidrotic ectodermal dysplasia (XLHED; OMIM 305100) is characterized by sparse hair, abnormal teeth and decreased sweating as a result of abnormal development of the sweat glands. Mutations in the ED1 gene, which encodes ectodysplasin‐A (EDA), are responsible for XLHED. Ectodysplasin‐A, a ligand for the EDA receptor, plays an important role in epidermal morphogenesis. We identified ED1 mutations including three novel mutations by sequencing genomic DNAs from eight unrelated Japanese XLHED families. Data from all reported mutations revealed that codon 156 in the furin subdomain is the most frequent site of change in EDA.
Photoperiod modulates melanoma growth in C57BL/6 mice
Authors: Lang Roland , Hintner Helmut , Hermann Anton , Brandstaetter Roland
Published: July 2003

Abstract: Seasonal variations can be found in almost any parameter of an organism's biochemistry, physiology, endocrinology, and behaviour. This phenomenon, generally called photoperiodism, results from one of the major functions of the circadian system, i.e. the translation of environmental information into rhythmic intraorganismic signals, which then regulate or influence physiology and pathology. We induced melanoma in three groups of syngeneic C57BL/6 mice synchronised to different photoperiods (8, 12, or 18 h of light within 24‐h days) by subcutaneous injections of HFH18 melanoma cell suspensions. All animals from all three photoperiodic groups developed exponentially growing tumors. The average tumor volume on day 31 post injection was significantly smaller in animals exposed to light/dark conditions (LD) 8 : 16 h as compared with animals held in LD 18 : 6 h and intermediate in animals from the equinox group. These results indicate that C57BL/6 mice react to photoperiod, which can exert a significant effect on tumor growth.
Intracellular calcium pool emptying induces DNA synthesis in HaCaT keratinocytes
Authors: Gniadecki R. , Gajkowska B.
Published: July 2003

Abstract:  Calcium signaling provides a central control mechanism for growth, differentiation and apoptosis of epidermal keratinocytes. Moreover, calcium signaling is important for carcinogenesis in view of the observations suggesting that emptying of intracellular stores in keratinocytes [e.g. by a selective blocker of calcium pump in the endoplasmic reticulum (ER), thapsigargin] facilitates skin cancer development. In this work, we analyzed whether calcium content in the intracellular stores is linked to HaCaT keratinocyte growth and apoptosis control. Treatment with thapsigargin caused calcium release from the intracellular pool and permanent pool depletion (up to 24 h) could be achieved using a high dose (1 µM) of this inhibitor. HaCaT cells cultured in these conditions exhibited an increased rate of DNA synthesis, assessed by the BrdU incorporation assay. Moreover, a weak stimulation of involucrin (terminal differentiation marker) was observed. Studies where intracellular free calcium (Cai2+) was chelated with BAPTA [1,2‐bis(o‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid] revealed that abrogation of thapsigargin‐induced Cai2+elevation did not counteract its effects on DNA synthesis, but blocked thapsigargin‐induced involucrin expression. Apoptosis was readily achieved by extracellular calcium chelation using EGTA [ethylene glycol‐bis(beta‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid], but was not observed after thapsigargin or BAPTA alone or in combination. In conclusion, depletion of intracellular calcium stores causes stimulation of keratinocyte proliferation independently of the elevation of Cai2+.
Involvement of the SREBP pathway in the mode of action of androgens in sebaceous glands in vivo
Authors: Rosignoli C. , Nicolas J. C. , Jomard A. , Michel S.
Published: July 2003

Abstract:  Androgens have profound effects on the physiology of the sebaceous gland. Using the hamster ear sebaceous gland model, we performed a detailed kinetic study to clarify the mechanism of androgen action on sebaceous gland function. We demonstrated that the growth of sebaceous glands observed after androgen treatment was due to both an increase in sebocyte proliferation and a parallel induction of sebocyte terminal differentiation, as evidenced by the induction of the synthesis of specific sebaceous lipids such as cholesterol esters, triglycerides, and squalene. Accordingly, the effect of androgen treatment on the mRNA expression of several key enzymes involved in the synthesis of sebaceous lipids has been studied using semi‐quantitative RT‐PCR. Up‐regulation by androgens of mRNA expression of HMG coenzyme A synthase and reductase, acetyl coenzyme A carboxylase (ACC), glycerol 3‐phosphate acyl transferase (GPAT), and FAR‐17c (stearoyl coenzyme A desaturase homologous), was demonstrated. Because sterol‐response element(s) (SREs) are known to be present in the promoters of these genes, we analyzed the expression by RT‐PCR and the activation of the transcription factor sterol regulatory element binding protein (SREBP) using immunoblotting experiments. Our results showed that SREBP‐1 was up‐regulated and rapidly activated after androgen treatment. Altogether, these results demonstrate for the first time that in sebaceous glands, in vivo, androgen regulates the synthesis of sebum lipids through the SREBP pathway.
A nonsense mutation in the desmoglein 1 gene underlies striate keratoderma
Authors: Kljuic Ana , Gilead Leon , Martinez‐Mir Amalia , Frank Jorge , Christiano Angela M. , Zlotogorski Abraham
Published: July 2003

Abstract:  Striate keratodermas (PPKS) (OMIM 148700) are a rare group of autosomal dominant genodermatoses characterized by palmoplantar keratoderma typified by streaking hyperkeratosis along each finger and extending onto the palm of the hand. We report a four‐generation kindred originating from Iran‐Syria in which three members were affected with PPKS. Clinically, these patients present with hyperkeratotic palms and plantar plaques. Direct DNA sequencing analysis revealed a heterozygous C‐to‐A transversion at nt 395 of the DSG1 gene. This mutation converted a serine residue (TCA) in exon 5 to a nonsense mutation (TAA) designated S132X. The mutation identified in this study is a novel mutation in the DSG1 gene and extends the body of evidence implicating the desmoglein gene family in the pathogenesis of human skin disorders.
Validation of a morphometric method for evaluating fibroblast numbers in normal and pathologic tissues
Authors: Miller C. C. , Godeau G. , Lebreton‐DeCoster C. , Desmoulière A. , Pellat B. , Dubertret L. , Coulomb B.
Published: July 2003

Abstract:  The aim of this work was to validate an image analysis method, based on cell nuclei form factor determination, for counting fibroblasts within human dermis. We first used reconstructed dermal equivalents in which fibroblasts can also be counted directly after lysis of the collagen matrix. We found a good correlation between the results of direct counting and those of image analysis from day 10 to day 28 of culture. When applied to young normal donors' skin biopsies fixed in Bouin's solution and embedded in paraffin, the image analysis method yielded mid‐dermis fibroblast counts of between 2100 and 4100 per mm3 of fresh tissue. A nuclear form factor (FF) comprised between 0.35 and 0.84 was found to be a biologic marker of fibroblasts. This was confirmed after fibroblast discrimination from other cell types, which had rounder nuclei (FF ≥ 0.85) and were identified either by their location (e.g. endothelial cells) or by labeling with specific antibodies (e.g. lymphocytes and monocytes/macrophages). Similar results were obtained with seven healthy donors' skin biopsies that had been frozen in nitrogen liquid and cryostat‐sectioned, showing that this counting method is independent of the histologic procedure. Finally, analysis of samples of hypertrophic scars from two patients revealed that fibroblast density in some parts of the dermis was more than twice the value found in other parts presenting a fibroblast density almost normal, showing that this cell counting method can also be used to assess fibroblast heterogeneity within a given tissue.
Association scan of the novel psoriasis susceptibility region on chromosome 19: evidence for both susceptible and protective loci
Authors: Hensen P. , Windemuth C. , Hüffmeier U. , Rüschendorf F. , Stadelmann A. , Hoppe V. , Fenneker D. , Ständer M. , Schmitt‐Egenolf M. , Wienker T. F. , Traupe H. , Reis A.
Published: July 2003

Abstract:  To follow up the novel psoriasis susceptibility region on chromosome 19 (PSORS6), we performed an association scan for psoriasis vulgaris using 45 evenly spaced DNA microsatellite markers. For this study, a new independent sample of 210 nuclear psoriasis families (trio design) from Northern Germany was recruited. We used the family based association test (FBAT) for an association scan over the chromosome 19 region encompassing 50.8 cM. We obtained a positive association for the markers D19S922 (allele 5, P = 0.008) and D19S916 (allele 13, P = 0.016), which correspond to the peak of the region identified in a previously performed scan. We identified two novel regions by a single marker, each showing negative association at D19S917 on 19p13.1 (allele 8, P = 0.0034) and at D19S425 (allele 9, P = 0.0005), compatible with the hypothesis of protective loci. These two novel regions were explored in more detail using novel microsatellite markers at an average distance of 100 kb. A separate analysis distinguishing between familial (n = 137) and sporadic (n = 73) psoriasis families showed that the familial trios contribute strongly in the region around D19S425 (P = 0.004), while the comparably small subset of 73 sporadic trios has a stronger effect at the locus around D19S917 (P = 0.026). These studies confirm the existence of a psoriasis susceptibility locus on chromosome 19 and give first evidence for the existence of both susceptible and protective loci in this region. Analysis of a dense marker set from these refined regions will eventually allow identification of the underlying susceptibility alleles.
Polymorphisms in the SEEK1 and SPR1 genes on 6p21.3 associate with psoriasis in the Swedish population
Authors: Holm Sofia J. , Carlén Lina M. , Mallbris Lotus , Ståhle‐Bäckdahl Mona , O'Brien Kevin P.
Published: July 2003

Abstract:  Psoriasis is a chronic skin disease that results in red and scaly lesions. Several psoriasis susceptibility loci have been identified across the genome, of which PSORS1 on 6p21.3 is predominant. There is an ongoing debate regarding whether the HLA‐C allele, Cw*0602, can be considered the major predisposing factor in this region. Investigation of other genes in the PSORS1 region with regard to psoriasis may provide alternate candidates to HLA‐C. We have characterized two overlapping genes, SEEK1 and SPR1. SEEK1 encodes two putative protein isoforms: the first being one of 152 amino acids from the full‐length splice‐isoform (exon 1–6), and the second being one of 100 amino acids from an alternate splice‐isoform (exon 1 and 6). SPR1 encodes a highly conserved protein of 134 amino acids, and in addition to characterization of human SPR1 we report the cloning of its orthologs in mouse and pig. Both SEEK1 and SPR1 are expressed in normal and psoriasis skin. In a case–control study, five of the nine single nucleotide polymorphisms (SNPs) found in SEEK1 were associated with psoriasis, while one of the four SNPs found in SPR1 showed association. Testing the Cw*0602 confounding status revealed that two of the SEEK1 SNPs showed Cw*0602‐independent association, while the SPR1 SNP showed Cw*0602‐dependent association. The second exon of SEEK1, containing the two Cw*0602‐independent SNPs, showed the highest concentration of the psoriasis‐associating SNPs, but did not appear to be translated.
Expression of angiogenic factors in neurofibromas
Authors: Kawachi Yasuhiro , Xu Xuezhu , Ichikawa Eiko , Imakado Sumihisa , Otsuka Fujio
Published: July 2003

Abstract:  We studied the expression of angiogenic factors (vascular endothelial growth factor, basic fibroblast growth factor, platelet‐derived growth factor and hepatocyte growth factor) in cutaneous neurofibroma samples from patients with neurofibromatosis‐1. Immunohistochemical staining and the reverse transcribed polymerase chain reaction (RT‐PCR) method demonstrated that vascular endothelial and basic fibroblast growths factor are highly expressed in neurofibroma cells at both the protein and mRNA level. These data suggest that vascular endothelial and basic fibroblast growth factors may contribute to both the angiogenesis and hypervascularity of neurofibromas.
Parathyroid hormone‐related peptide modulates signal pathways in skin and hair follicle cells
Authors: Thomson M. , McCarroll J. , Bond J. , Gordon‐Thomson C. , D. Williams E. , Moore G. P. M.
Published: July 2003

Abstract:  Parathyroid hormone‐related protein (PTHrP) is secreted by skin epithelial cells and is thought to play an important role in the development and function of the hair follicle. It was hypothesized that PTHrP binds to receptors in dermal papilla cells and modulates intracellular signaling systems in these cells. We tested the effects of PTHrP on protein synthesis, protein kinase A (PKA) and protein kinase C (PKC) activities as well as tyrosine phosphorylation in rat vibrissa dermal papilla and capsular fibroblast cells. Cells were cultured in the presence or absence of the N‐terminal peptide PTHrP1‐34 for 48 h and detergent extracts prepared. Proteins were separated by electrophoresis. Phosphotyrosine and the PTH/PTHrP receptor immunoreactivity was identified by Western blot analysis. PKC and PKA activities in the cells were measured using colorimetric enzyme assays. Extracts of both dermal papilla cells and capsular fibroblasts displayed immunoreactivity to the PTH/PTHrP receptor. Electrophoresis showed that PTHrP treatment reduced the density of a 50‐kDa protein in dermal papilla cells but not in capsular fibroblasts. Media conditioned by the cells showed similar changes, indicating that the PTHrP‐modulated 50‐kDa protein was secreted. Furthermore, 2‐D gel electrophoresis indicated that the protein had a number of phosphorylation sites. Western analysis with antiphosphotyrosine antibodies confirmed a significant decrease in the intensity of a phosphorylated 50‐kDa protein in papilla cells and papilla cell‐conditioned medium. PKC and PKA activities of papilla cells were unaffected by PTHrP. However, activities of PKC were increased and PKA reduced in capsular fibroblasts following peptide treatment. These cell‐specific effects showed that endogenous PTHrP may activate different intracellular pathways in mesenchymal cells of skin and elicit changes in levels of locally secreted proteins that specifically modulate normal follicular function.
Time course of ultraviolet B‐induced erythema in people with red hair harbouring homozygous melanocortin 1 receptor mutations
Authors: Ha Thomas K. K. , Waterston Karen , Bisset Yvonne , Ray Amanda , Rees Jonathan L.
Published: July 2003

Abstract:  It has previously been reported that the time course of erythema may be delayed in those with sun‐sensitive skin types and those with skin cancer. One molecular explanation for this putative phenotype would be that it is caused by mutations of the melanocortin 1 receptor (MC1R). In the present study of 20 persons, 10 of whom were MC1R homozygous, we measured erythema over a 21‐day period in response to a range of ultraviolet B doses using methods that improved on previous studies. We could detect no consistent differences in ultraviolet radiation‐induced erythema between the groups studied. The pharmacological mechanisms underpinning such prolonged inflammatory responses merit further investigation.
Role of the IGF‐II receptor in mediating acute, non‐genomic effects of retinoids and IGF‐II on keratinocyte cell death
Authors: Louafi F. , Stewart C. E. H. , Perks C. M. , Thomas M. G. , Holly J. M. P.
Published: July 2003

Abstract:  In this study, we have examined the effects of retinoic acid (RA) on the human immortalized keratinocyte cell line (HaCaT). A significant twofold (P < 0.01) increase in apoptotic cell death compared with the control was found within 24 h of treatment with 10−5 M of RA. Apoptosis was confirmed by flow cytometry. Cycloheximide did not inhibit this acute RA‐induced apoptosis. Interestingly, insulin‐like growth factor‐II (IGF‐II, 50 ng/ml) was able to significantly (67.3%; P < 0.05) reduce RA effects, whereas IGF‐I (50 ng/ml) and insulin (75 ng/ml) were without effect. Furthermore, analogues of IGF‐II [leu27 IGF‐II and Des(1‐6) IGF‐II], with altered affinities for the IGF‐I receptor and IGF‐binding proteins (IGFBPs), but retained affinities for the IGF‐II receptor, also completely inhibited (100%; P < 0.01) RA‐induced apoptosis, while an IGF‐I receptor antagonist did not reduce the survival effects of IGF‐II. Insulin pretreatment negates the survival effect of IGF‐II. In contrast, mannose 6 phosphate (M6P) did not alter RA or IGF‐II actions. These results indicate that rapid induction of cell death by RA is independent of production or secretion of new proteins. The inhibition of RA action by IGF‐II was independent of its ability to signal through the IGF‐I receptor or to interact with IGFBPs.
Aberrant signalling and transcription factor activation as an explanation for the defective growth control and differentiation of keratinocytes in psoriasis: a hypothesis
Authors: McKenzie R. C. , Sabin E.
Published: July 2003

Abstract:  Psoriasis is a chronic inflammatory skin disease characterized by the accumulation of red, scaly plaques on the skin. The plaques result from hyperproliferation and incomplete differentiation of keratinocytes (KC) in a process that seems to be driven, in part by skin‐infiltrating leucocytes. We believe that the KC have inherent defects in intracellular signalling which could be usefully targeted to allow the development of more effective therapies. We suggest that there are defects in the regulation of the transcription factors: signal transducer and activator of transcription (STAT‐1α), interferon regulated factor‐1 (IRF‐1) and NFκB which lead to loss of growth and differentiation control when the cells are subjected to physico‐chemical and immunological stress. We also highlight recent studies that suggest that peroxisome proliferator‐activated receptors, the notch receptor and defects in calcium and other ion transporting proteins may contribute to impairment in the ability of psoriatic KC to differentiate. The role of these systems in the development of the psoriatic phenotype and tests of these hypotheses are proposed.
Involvement of EGF receptor activation in the induction of cyclooxygenase‐2 in HaCaT keratinocytes after UVB
Authors: Ashida M. , Bito T. , Budiyanto A. , Ichihashi M. , Ueda M.
Published: July 2003

Abstract:  Because selective inhibition of cyclooxygenase‐2 (COX‐2) suppressed the induction of skin tumors in mice by UV and as UV has been shown to induce expression of COX‐2 in skin and cells, COX‐2 may be crucial for photocarcinogenesis of the skin. We studied the mechanism of UVB‐induced expression of COX‐2 focusing on the signal transduction pathway involved. Hydrogen peroxide (H2O2) treatment of HaCaT cells induced expression of COX‐2 and pretreatment with the antioxidant N‐acetylcysteine (NAC) partly inhibited the UVB‐induced expression of COX‐2 protein in HaCaT cells, suggesting that oxidative stress contributes to COX‐2 induction. To examine the signaling pathways involved in the UVB‐induced expression of COX‐2 in HaCaT cells, we analysed the expression of COX‐2 protein after treatment with various inhibitors of signaling molecules. Inhibition of EGFR by a specific inhibitor and by a neutralizing antibody suppressed the induction of COX‐2 expression by UV. Although a neutralizing antibody to transforming growth factor‐α (TGF‐α) suppressed COX‐2 expression induced by TGF‐α, it did not suppress COX‐2 expression by UV, indicating that a direct activation of EGFR is involved. Treatment of cells at low temperature (4°C) inhibited UVB‐induced JNK activation, but it did not inhibit COX‐2 expression by UV. Inhibitors of MEK, p38 MAP kinase and PI3‐kinase, suppressed the induction of COX‐2 expression by UV. In contrast, an erbB‐2 inhibitor augmented the UVB‐induced increase of COX‐2 protein. These data indicate that oxidative stress in association with activation of EGFR, ERK, p38 MAP kinase, and PI3‐kinase plays crucial roles in the UVB induction of expression of COX‐2.
In vitro reconstructed mucosa‐integrating Langerhans' cells
Authors: Sivard P. , Dezutter‐Dambuyant C. , Kanitakis J. , Mosnier J.‐F. , Hamzeh H. , Bechetoille N. , Berthier O. , Sabido O. , Schmitt D. , Genin C. , Misery L.
Published: July 2003

Abstract:  All three‐dimensional in vitro mucosal models constructed, thus far, have only been reconstituted by epithelial cells. We have developed a reconstructed oral and vaginal epithelium that integrates Langerhans' cells (LC), the dendritic cells (DC) of malpighian epithelia. The epithelium was composed of gingival or vaginal keratinocytes seeded on a de‐epidermized dermis (DED) and grown in submerged culture for 2 weeks. LC precursors, obtained after differentiation of cord blood‐derived CD34+ hematopoietic progenitor cells (CD34+HPC) by granulocyte macrophage‐colony stimulating factor (GM‐CSF), tumor necrosis factor‐α (TNF‐α), transforming growth factor‐β (TGF‐β) and Flt3‐ligand (Flt3‐L), were introduced after 6–8 days of culture into the reconstituted epithelium. The in vitro reconstituted mucosal epithelium formed a multilayered, well‐differentiated epithelial structure, confirmed by the immunohistochemical expression of cytokeratins 4, 6, 10, 13, 14, 16 and involucrin. LC were identified in the basal and suprabasal epithelial layers by CD1a antigen, S100 protein and Langerin/CD207 expression, and by transmission electron microscopy. Type IV collagen was expressed at the chorio–epithelial junction, and most ultrastructural features of this junction were visualized by electron microscopy. This in vitro reconstructed gingiva or vagina integrating LC represents interesting models very similar to native tissues. Because LC play an important role in the mucosal immune system, our models could be useful for conducting studies on interactions with pathogenic agents (viruses, bacteria etc.), as well as in pharmacological, toxicological and clinical research.
Melanin Inhibits Cytotoxic Effects of Doxorubicin and Daunorubicin in MOLT 4 Cells
Authors: Svensson Samuel P. S. , Lindgren Sofi , Powell Wendy , Green Henrik
Published: July 2003

In the present study we have developed a simple method to elucidate the melanin binding ability of different chemotherapeutic agents. The anthracyclines, doxorubicin and daunorubicin, or the alkylating agent cisplatin were preincubated with melanin (Sepia). Melanin and free drug was then separated through centrifugation and the cytotoxic effects of corresponding drug were evaluated in a MTT (3‐(4,5‐dimetyltiazol‐2‐yl)‐2,5‐difenyl‐tetrazoliumbromide) assay using MOLT‐4 cells. Our results show that melanin pretreatment shifted the IC50 value for doxorubicin from 0.06 to 0.97 μM and for daunorubicin from 0.04 to 0.80 μM. In contrast, the IC50 values of cisplatin was not influenced by melanin pre‐treatment indicating that cisplatin does not bind to melanin. By comparing equi‐active concentrations from concentration–response curves with or without melanin pretreatment an approximate binding capacity of melanin could be estimated. Our results show that melanin binds about 900 nmol/mg doxorubicin and 760 nmol/mg daunorubicin. Chloroquine, which is known to bind to melanin with high affinity, was found to inhibit melanin binding of both daunorubicin and doxorubicin, thereby leading to an increased sensitivity of the anthracyclines. The clinical implications of melanin binding regarding unwanted accumulation of anthracyclines in the skin as well as chemoprotective effects against chemotherapy are discussed.
Microphthalmia‐Associated Transcription Factor (MITF) is Required but is Not Sufficient to Induce the Expression of Melanogenic Genes
Authors: Gaggioli Cédric , Buscà Roser , Abbe Patricia , Ortonne Jean‐Paul , Ballotti Robert
Published: July 2003

Microphthalmia‐associated transcription factor (MITF) plays a pivotal role in melanocyte survival and differentiation. Nevertheless, until now it has not been possible to show that MITF regulates the expression of the endogenous tyrosinase or Tyrp1. Further, a direct involvement of MITF in the regulation of melanin synthesis, a key parameter of melanocyte differentiation, remains to be demonstrated. In the present report, using recombinant adenovirus encoding the wild‐type or a dominant negative form of MITF, as well as stable cell lines expressing tetracycline inducible wild‐type MITF, we reassessed the role of MITF in melanocyte differentiation and in the regulation of melanin synthesis. Immunofluorescence studies, as well as Western blot analyses, show that infection of B16 mouse melanoma cells or human melanocytes with adenovirus encoding wild‐type MITF does not increase the expression of the endogenous melanogenic enzymes. However, infection with the MITF dominant negative mutant inhibits the expression of endogenous tyrosinase and Tyrp1 proteins and blocks cAMP‐induced melanin synthesis. Thus, MITF is required but does not seem to be sufficient to induce the expression of melanogenic enzymes and we show for the first time a direct involvement of MITF in the regulation of melanin pigment synthesis. As a whole, our data point to the existence of still unknown regulatory mechanisms that co‐operate or synergize with MITF to control melanogenic gene expression and melanin synthesis. The identification of such mechanisms will greatly improve our understanding of the melanocyte differentiation processes.
The ‘Abtropfung Phenomenon’ Revisited: Dermal Nevus Cells from Congenital Nevi Cannot Activate Matrix Metalloproteinase 2 (MMP‐2)
Authors: Gontier Etienne , Cario‐André Muriel , Vergnes Pierre , Bizik Jozef , Surlève‐Bazeille Jean‐Etienne , Taïeb Alain
Published: July 2003

Since Unna's Abtropfung hypothesis, the process of migration of nevus cells in the dermis remains unknown. To investigate its mechanisms, we studied the role of gelatinases in dermal nevus cells obtained from congenital pigmented nevi, which are major actors in the remodeling of basement membrane proteins. Our previous studies have shown that dermal nevus cells express pro‐matrix metalloproteinase (MMP)‐2 exclusively and cannot return to the dermis when seeded together with keratinocytes on top of the dermis in a skin reconstruction model. To examine why MMP‐2 was not in its active form, we used Western blot to study the expression of members of the MMP‐2 activation pathway (membrane type 1‐MMP and tissue inhibitor of metalloproteinase‐2), which proved to be normally expressed. To induce the dermal passage of nevus cells artificially, we also tried to activate gelatinases with phorbol‐12‐myristate‐13‐acetate and epidermal growth factor, using epidermis reconstructed with nevus cells. No migration in the dermis could be triggered. We conclude that the absence of active MMP‐2 is due to a functional blockade of its activation pathway and may prevent dermal nevus cells from reaching the dermal compartment in skin reconstructs. Furthermore, our findings reinforce the concept that dermal nevus cells originating from congenital nevi are in a quiescent status.
Comparison of the Structural and Physical Properties of Human Hair Eumelanin Following Enzymatic or Acid/Base Extraction
Authors: Liu Yan , Kempf Valerie R. , Brian Nofsinger J. , Weinert Emily E. , Rudnicki Mark , Wakamatsu Kazumasa , Ito Shosuke , Simon John D.
Published: July 2003

Eumelanin was isolated from a sample of black, Indonesian human hair using three different published procedures: two different acid/base extractions and an enzymatic extraction. The morphology and spectroscopic properties of the isolated pigments differ significantly. The acid/base procedures both yield an amorphous material, while enzymatic extraction yields ellipsoidal melanosomes. Amino acid analysis shows that there is protein associated with the isolated pigments, accounting for 52, 40 and 14% of the total mass for the two acid/base extractions and the enzymatic extraction, respectively. The amino acid compositions do not correlate with those of keratin or tyrosinase. Metal elemental analysis shows that the acid/base extraction removes a majority of many metal ions bound to the pigment. Chemical degradation analysis by KMnO4/H+ and H2O2/OH− indicates significant differences between the pigments isolated by acid/base and enzymatic extraction. After correction for the protein mass in the two pigments, the lower yields of both pyrrole‐2,3,5‐tricarboxylic acid and pyrrole‐2,3‐dicarboxylic acid, eumelanin degradation products, indicate acid/base extraction modifies the chemical structure of the melanin, consistent with the result of Soluene solubilization assay. While the optical absorption spectra of the bulk pigments are similar, the spectra of the molecular weight less than 1000 mass fractions differ significantly. The data clearly indicate that pigment obtained from human hair by acid/base extraction contains significant protein, exhibits destruction of the melanosome, and possesses altered molecular structure. The acid/base extracted hair melanin is not representative of the natural material and is a poor model system for studying the physical and biological properties of melanins. The enzymatically extracted hair melanin, on the contrary, retains the morphology of intact melanosomes and is an excellent source of human melanin.
Genetic Determinants of Sable and Umbrous Coat Color Phenotypes in Mice
Authors: Suto Jun‐ichi , Sekikawa Kenji
Published: July 2003

The dorsal fur in yellow F1mice (F1‐Ay) between C3H/HeJ and C57BL/6J‐Ayis darker than that in C57BL/6J‐Ay. Moreover, yellow F2mice (F2‐Ay) exhibit a wide spectrum of coat color phenotypes in terms of lightness and darkness. Quantitative trait locus (QTL) analysis on F2‐Ayidentified three significant modifier loci that accounted for darkening of the coat color on chromosomes 1 (Dmyaq1andDmyaq2) and 15 (Dmyaq3), and the C3H/HeJ allele at these loci increased the darkness. Because agouti F2mice (F2‐A) also exhibited a spectrum of coat color phenotypes, the question of whether these QTLs had any effects on F2‐Awas examined.Dmyaq1andDmyaq2were shown to increase the darkness in F2‐A, whereasDmyaq3did not. The results showed thatDmyaq1–Dmyaq3were parts of determinants responsible for the sable (darker modification of yellow) coat color phenotype, and thatDmyaq1andDmyaq2were parts of determinants responsible for the umbrous (darker modification of agouti) coat color phenotype. It is, thus, demonstrated that both the sable and the umbrous phenotypes resulted from multigenic contributions, and that they shared genetic bases, as had been implied for several decades.
Biogenesis of Melanosomes in Kupffer Cells of Proteus anguinus (Urodela, Amphibia)
Authors: Prelovšek Petra‐Maja , Bulog Boris
Published: July 2003

The ultrastructural characteristics of melanosomes and premelanosomes observed during the biogenesis of melanosomes in liver pigment cells of the neotenic cave salamander Proteus anguinus (Proteidae) are described. It is well known that amphibian liver pigment cells, also known as Kupffer cells (KC), contain melanosomes and are able to synthesize melanin. Liver pigment cells of P. anguinus contain numerous siderosomes and melanosomes. The melanosomes are grouped together within single‐membrane‐bounded bodies, named as ‘clusters of melanosomes’ or ‘melanosomogenesis centers’. Inside such clusters, different structures are present: (1) filament‐like structures, characteristic of the initial stage of melanosome biogenesis, (2) medium electron‐dense melanosomes in different stages of melanization, (3) melanosomes with an electron‐dense cortical area and a less electron‐dense medullar area, and (4) uniformly highly electron‐dense mature melanosomes or melanin granules. Histochemical and cytochemical dihydroxyphenylalanine (DOPA) oxidase reactions in pigment cells were positive. Our results confirm the ability of amphibian KC to synthesize melanin and contribute to this little known subject.
Polymorphisms in the Melanocortin‐1 Receptor (MC1R) and Agouti Signaling Protein (ASIP) Genes in Korean Vitiligo Patients
Authors: Na Gun Yoen , Lee Kuk Hyeong , Kim Moon Kyu , Lee Seok Jong , Kim Do Won , Kim Jung Chul
Published: July 2003

We have examined the frequency of SNP polymorphisms within the melanocortin‐1 receptor (MC1R) and agouti signaling protein (ASIP) genes in 114 Korean vitiligo patients and 111 normal controls to assess the association of these loci with vitiligo risk. Using direct sequencing techniques, we found the following five MC1R coding region SNPs: Arg67Gln (G200A), Val92Met (G274A), Ile120Thr (T359C), Arg160Arg (C478A), and Gln163Arg (A488G). Of these, the most common were Val92Met at 14% in patients vs. 9% in controls (P = 0.17) and Gln163Arg at 17% in patients vs. 17% in controls (P = 0.84). Presence of the A allele of Val92Met (G274A) was higher in vitiligo patients {P = 0.12, odds ratio (OR) [95% confidence interval (CI)] = 1.68 (0.86–3.25)}. The other three variants showed a frequency <5% of both patients and controls. The ASIP 3′UTR genotype (g.8818A‐G) was also assessed in the same subjects. The frequency of the G allele of 3′UTR in ASIP was 17% in vitiligo and 12% in controls [P = 0.14, OR (95% CI) = 1.49 (0.87–2.54)]. Carriage of the G allele was higher in vitiligo patients [P = 0.17, OR (95% CI) = 1.50 (0.83–2.72)], and those who also carried MC1R Val92Met were more prone to vitiligo [eight of 111 patients vs. four of 111 in controls, P = 0.14, OR (95% CI) = 2.75 (0.71–8.69)]. None of these associations, however, reached statistical significance.